APOIO:PROMOÇÃO/REALIZAÇÃO:

LIVRO DE

25 - 28 MAIOBALNEÁRIO CAMBORIÚ

SC/BRASIL

SCIENTIFIC COMMITTEE XXII BRAZILIAN CONGRESS OF TOXICOLOGYMarcelo Dutra Arbo - Universidade Federal do Rio Grande do Sul (UFRGS) - RS - President

Aline Franco Martins - Universidade Estadual de Campinas (UNICAMP) - SPArtur Christian Garcia da Silva - Universidade Federal de Goiás (UFG) - GOAnderson Martino Andrade - Universidade Federal do Paraná (UFPR) - PR

Carla Brigagão Pacheco da Silva - Faculdade de Medicina de Ribeirão Preto (FMRP-USP) - SPEduardo Geraldo de Campos - Universidade Estadual de Campinas (UNICAMP) - SP

Eliane Dallegrave - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS Elisa Sauer - Instituto Geral de Perícias de Santa Catarina (IGP) - SC

Fabrício Pelição - Polícia Civil - ESFlavia Thiesen - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) - RS

Fávero Reisdorfer Paula - Universidade Federal do Pampa (UNIPAMPA) - RSFlavio Manoel Rodrigues da Silva Júnior - Universidade Federal do Rio Grande (FURG) - RS

Gabriela Göethel - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RSIsarita Martins - Universidade Federal de Alfenas (UNIFAL) - MG

José Luiz da Costa - Universidade Estadual de Campinas (UNICAMP) - SPJuliano Smanioto Barin - Universidade Federal de Santa Maria (UFSM) - RS

Leonardo Costalonga Rodrigues - Universidade Estadual de Campinas (UNICAMP) - SPLuciana Grazziotin Rossato-Grando - Universidade de Passo Fundo (UPF) - RS

Marina Venzon Antunes - Universidade Feevale (FEEVALE) - RSMaurício Homem de Mello - Universidade de Brasília (UnB) - DF

Mirna Bainy Leal - Universidade Federal do Rio Grande do Sul (UFRGS) - RSOsmar Damian Prestes - Universidade Federal de Santa Maria (UFSM) - RS

Rachel Picada Bulcão - Polícia Científica - PRRafael Lanaro - Universidade Estadual de Campinas (UNICAMP) - SP

Raphael Caio Tamborelli Garcia - Universidade Federal de São Paulo (UNIFESP) - SPSandra Manoela Dias Macedo - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS

Sandro Cruz Chaves - Instituto Médico Legal André Roquette (IMLAR) - MGSarah Carobini Werner de Souza Eller - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS

Silvya Stuchi Maria-Engler - Universidade de São Paulo (USP) - SPTiago Peixe - Universidade Estadual de Londrina (UEL) - PR

Vanessa Moraes de Andrade - Universidade do Extremo Sul Catarinense (UNESC) - SC

ORGANIZING COMMITTEEJosé Roberto Santin - Universidade do Vale do Itajaí (UNIVALI) - SC - President

Alcíbia Helena de Azevedo Maia - Universidade Federal de Santa Catarina (UFSC) - SCAndré Valle de Bairros - Universidade Federal de Santa Maria (UFSM) - RS

Ernani Pinto Junior - Universidade de São Paulo (USP) - SPIsabel Daufenback Machado - Universidade Regional de Blumenau (FURB) - SCMarcelo Dutra Arbo - Universidade Federal do Rio Grande do Sul (UFRGS) - RS

Marize Campos Valadares - Universidade Federal de Goiás (UFG) - GOTiago Franco de Oliveira - Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) - RS

ORGANIZATIONAGeventos Assessoria

PUBLISHINGIsabel Kubaski - isabelkubaski@gmail.com

COVER CREATIONSilvio Lovato - silvio@lovatodesign.com.br

3

SUMMARY

01 Alternative Animal Models & Emerging in vitro models .............................................................................. 24

Adipose Tissue 3D: new tools in cell culture ........................................................................................................................25Buchele, Maria Luiza Caneiro; Mora, Tamara Dal; Saleh, Najla Adel; Silva, Adny Henrique; Monteiro, Fabíola Branco Filippin

Analysis of cytotoxicity and anti-melanogenic activity of kojic acid and glycolic acid in SK-MEL-28 and B16F10 cells .............................................................................................................................................................26

Ribeiro, Milena Mariano; Silva, Ana Cléia Cardoso; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Bioprinted and manual human epidermis reconstruction: a compared performance for irritation tests ....27Bagatin, Julia de Toledo; Camarena, Denisse Esther Mallaupoma; Osaki, Luciana Harumi; Freitas, Vanessa M.; Nold, Juliana C. Lago; Maria-Engler, Silvya Stuchi

Cellfate®Matrix: building the reality in three dimensions .............................................................................................. 28Reis, Emily Marques; Cavichion, Rafael Filipe Battisti; Colla, Guilherme; Godoi, Manuella Machado; Koepp, Janice

Cellfate®RHE: a brazilian commercial model of reconstituted human epidermis in vitro ...................................29Reis, Emily Marques; Cavichion, Rafael Filipe Battisti; Colla, Guilherme; Koepp, Janice

Characterization and analysis of the expression of the surface marker Stro-1 in human dental pulp stem cells ..................................................................................................................................................................30

Oliveira, Leandro Leal Rocha; Lima, Aliny Pereira; Farias, Evelyn Rayani Araújo; Gomes, Thaisângela Rodrigues Lopes e Silva; Macedo, Larissa Matuda; Leite, Jacqueline Alves; Valadares, Marize Campos

Characterization and applicability of a novel physiologically relevant 3D-tetraculture bronchial model for in vitro assessment of chemical respiratory allergy ................................................................ 31

Silva, Artur Christian Garcia; Mendonça, Izadora Caroline Furtado; Valadares, Marize Campos

Concomitant exposure to air particulate matter and solar radiation reduces epidermal barrier function in a reconstructed human epidermis model ........................................................................................32

Silva, Claudia Larissa Viana; Carvalho, Larissa Anastacio da Costa; Camarena, Denisse Esther Mallaupoma; Bagatin, Julia de Toledo; Assis, Silvia Romano; Maria-Engler, Silvya Stuchi; Barros, Silvia Berlanga de Moraes

Creation of a biorepository from dental stem cells: as an alternative method to the use of animals...........33Oliveira, Leandro Leal Rocha; Lima, Aliny Pereira; Farias, Evelyn Rayani Araújo; Gomes, Thaisângela Rodrigues Lopes e Silva; Macedo, Larissa Matuda; Leite, Jacqueline Alves; Valadares, Marize Campos

Decellularized extracellular matrix hydrogel derived from bovine cornea for application in 3D models ... 34Santos, Jordana Andrade; Silva, Artur Christian Garcia; Dias, Wanessa Amorim; Valadares, Marize Campos

Development of a methodology to assess key event 2 to quantify NRF2 in cutaneous allergenicity ...........35Pedralli, Bruna Cristiane Oliveira; Valadares, Marize Campos

Development of an ex vivo model to evaluate pulmonary toxicity mechanisms ..................................................36Furtuoso, Marcella Miranda Siqueira; Tavares, Kvetta Pinheiro Teixeira; Pedralli, Bruna Cristiane Oliveira; Valadares, Marize Campos

Dimethoate exposition: in vitro x in vivo study ....................................................................................................................37Andrade, Ana Rosa Brissant; Carvalho, Deoclécio Lustosa; Souza, Asley Thalia Medeiros; Kishishita, Juliana; Pimenta, Camila de Almeida Perez; Santana, Davi Pereira; Leal, Leila Bastos

Effects of Myrcia pubipetala Miq (Myrtaceae) extract on innate inflammatory response ............................... 38Pacassa, Pâmela; Benvenutti, Larissa; Echterhoff, Marcelo Rodrigo Franke; Lopes, Bruna Gonçalves; Quintão, Nara Lins Meira; Debiasi, Michele Alberton; Santin, José Roberto; Machado, Isabel Daufenback

Effects of pulmonary inhaled irritants on 3D alveolar model .......................................................................................39Furtuoso, Marcella Miranda Siqueira; Tavares, Kvetta Pinheiro Teixeira; Valadares, Marize Campos

Efficiency of the Reconstructed Human Epidermis (RHE) in the classification of biological product by the in vitro irritation and corrosion tests (OECD 439 and 431) .............................................................. 40

Bechtold, Bruna Assunção; Cianci, Julio Cesar; Coelho, Maria Paula Mancini; Costa, Larissa Gabrielli; Dakic, Vanja; De Vecchi, Rodrigo; Fava, Luis Paulo; Padua, Amanay Sousa; Santana, Thatiane Nunes; Silva, Priscilla Muniz Ribeiro;Vecina, Juliana Falcato

Embryotoxicity of triclopyr in early development of zebrafish (Danio rerio) .......................................................... 41Andrade, Ítalo Bertoni Lopes; Sales, Bianca Camargo Penteado; Peixoto, Paloma Vitória Lima; Viriato, Cristina; Pereira, Lílian Cristina

Evaluation of ocular irritation potential of raw materials presented in xampus formulations using non-animal methodology ................................................................................................................... 42

Rocha, Daniela Barbosa; Almeida, Jéssica Azarias; Andrade, Wanessa Machado

Evaluation of the cytotoxicity and the anti-melanogenic activity of Selenium and Zinc ................................... 43Silva, Ana Cléia Cardoso; Ribeiro, Milena Mariano; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Galleria mellonella as an Alternative Model for the Assessment of Permeation and Toxicity of Assets from Natural Sources ............................................................................................................................... 44

Silva, Samanta de Matos; Singulani, Junya de Lacorte; Carvalho, Angélica Romão; Migliato, Ketylin Fernanda; Giannini, Maria José Soares Mendes; Fusco-Almeida, Ana Marisa

Identification and quantification of constituents in the fetal bovine serums available in the market used in cell culture ..................................................................................................................................................45

Stival, Ana Clara Silva; Silva, Artur Christian Garcia; Valadares, Marize Campos

Implementation of SkinEthicTM Human Corneal Epithelium (HCE) in Brazil ............................................................. 46Dakic, Vanja; Mattos, Guilherme; Rigaudeau, Anne-Sophie; Garcia, Cristina; Bouez, Charbel; De Vecchi, Rodrigo

In silico evaluation of erythrinic alkaloids from Mulungu, Erythrina verna, in GABAA β-3 receptor complexed with benzamide .....................................................................................................................................47

Bairros, André Valle; Nascimento, Marcelo Henrique Santana; Paula, Fávero Reisdorfer

In silico pharmacokinetics and pharmacodynamics prediction of resveratrol and two glycosylated derivatives ............................................................................................................................................................. 48

Schimith, Lucia Emanueli; André-Miral, Corrine; Muccillo-Baisch, Ana Luiza; Hort, Mariana Appel

In vitro oral and topic absorption toxicity test standardization using 3D cell cultures and microfluidic systems .................................................................................................................................................................... 49

Ganzerla, Melissa Dibbern; Indolfo, Nathalia de Carvalho; Arroteia, Kelen Fabiola; Figueira, Ana Carolina Migliorini

In vitro strategy to assess skin irritation and sensitization potential of a commercial formulation containing the antiseptic chlorhexidine ......................................................................................................50

Kawakami, Camila Martins; Silva, Gustavo Henrique; Moura, Kézia; Pinheiro, Ana L.T.A.; Pinheiro, Adriano da Silva; Eberlin, Samara; Gaspar, Lorena Rigo; Fuzinaga, Thais; Vicente, Eduardo;Facchini, Gustavo

Inflammatory alterations triggered by respiratory sensitizers in human dendritic cells: providing evidence to support the chemical respiratory allergy mechanistic background ................................51

Mendonça, Izadora Caroline Furtado; Silva, Artur Christian Garcia; Carvalho Filho, Sérgio de Morais; Valadares, Marize Campos

Influence of a combination containing UV filters and insect repellent IR3535 in photoinduced processes ..........52Gluzezak, Ana Júlia Pasuch; Kawakami, Camila Martins; Tavares, Renata Spagolla Napoleão; Fuzinaga, Thais Yume Toriy; Maria-Engler, Silvya Stuchi; Gaspar, Lorena Rigo

Influence of extracellular matrix organ-specific in behavior and cellular functionality in HEPG-2 cell line ...............................................................................................................................................................................53

Santos, Thaís Rosa Marques; Borges, Amanda Cecília Guimarães; Santos, Jordana Andrade; Silva, Artur Christian Garcia; Marize Campos Valadares

Macroscopic evaluation of Score® cytotoxicity by the Allium cepa test.....................................................................54Massucato, Lucas Eduardo; Siqueira, Gabriella Ferreira; Motomura, Larissa Tiemi Akamine; Lini, Renata Sano; Souza-Kaneshima, Alice Maria; Mossini, Simone Aparecida Galerani

Microfibrillated cellulose and silica nanoparticles as sustainable alternatives to developing nanotechnology products: the evaluation of skin irritation....................................................................55

Cruz, Juliana Varella; Gagosian, Viviana Costa; Magalhães, Washington; Cademartori, Pedro Henrique Gonzalez; Oliveira, Danielle Palma; Leme, Daniela Moraes

Nasal and buccal absoption of dimethoate: in vitro x in vivo study ............................................................................56Andrade, Ana Rosa Brissant; Carvalho, Deoclécio Lustosa; Souza, Asley Thalia Medeiros; Kishishita, Juliana; Silva, José Wellithom Viturino; Bedor, Danilo César Galindo; Santana, Davi Pereira; Leal, Leila Bastos

Photoprotective effect of Rapanea ferruginea bark extract-loaded nanoemulgel .............................................57Cordenuzzi, Dorys Angela; Benvenutti, Larissa; Santin, José Roberto; Lucinda-Silva, Ruth Meri

Preliminary data on a newly developed biomimetic Reconstituted Human Ocular Epithelium Model using an animal free defined medium ...............................................................................................58

Bosquetti, Bruna; Catarino, Carolina Motter; Costa, Meg Cristina da Castilho; Thá, Emanoela Lundgren; Silva, Artur Christian Garcia; Schuck, Desiree Cigaran; Canavez, Andrezza Di Pietro Micali; Brohem, Carla Abdo; Valadares, Marize Campos

Safety evaluation of nanoemulsion and emulgel containing pomegranate peel extract using alternative in vitro methods. ..........................................................................................................................................59

Rudolf, Carline; Benvenutti, Larissa; Rocha, Anna Carolina Furaer; Cordenuzzi, Dorys Angela; Santin, José Roberto; Lucinda-Silva, Ruth Meri

Spectroscopic assessment of lung mucus as a tool for toxicokinetic/toxicodynamic assessment of chemical respiratory sensitizers............................................................................................................... 60

Mendonça, Izadora Caroline Furtado; Silva, Artur Christian Garcia; Mendanha, Sebastião Antônio; Valadares, Marize Campos

Study of the toxicity of NPS amphetamines and cathinones using in silico tools ................................................. 61Rodrigues, Caio Henrique Pinke; Castro, Jade Simões; Bruni, Aline Thais

Testing strategies for evaluation of eye irritation potential of agrochemical formulations as an alternative to animal testing ..........................................................................................................................................62

Choksi, Neepa; Latorre, Andreia Oliveira; Pais, Mariana Castello Novo; Murata, Rosana Zoriki Hosomi; Catalano, Shadia M.I.; Aguilera, Mariana; Pires, Janaina Aparecida Cardoso; Ogasawara, Maryanne; Habe, Priscila; Perjessy, Gisele; Allen, David

The endothelial compartment can actively participate in the adverse outcome pathway (AOP) of chemical respiratory allergy ....................................................................................................................................63

Silva, Artur Christian Garcia; Carvalho Filho, Sérgio de Morais; Valadares, Marize Campos

The Implementation of Alternative Methods to the Use of Animals for ocular toxicity: difficulties and opportunities for innovation....................................................................................................................... 64

Gimenes, Izabela; Presgrave, Octavio; Gonzalez, Marcelo; Alves, Gutemberg

The use of NAMs to evaluate the toxicity of UV filters: emphasis on aquatic toxicity and endocrine disrupting effects .....................................................................................................................................................65

Nonino, Elisa de Castro Wille; Leme, Daniela Morais; Pestana, Cynthia Bomfim

Toxicity features of agrochemical formulations related to eye and skin irritation potential that are important to build weight of evidence for integrated approach of testing strategies ......................66

Latorre, Andreia Oliveira; Pepato, Ana Claudia de Andrade; Faria, Patricia Miranda; Cazarin, Karen

Toxicological evaluation of amphetamine-likes employing in silico methodology...............................................67Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Toxicological evaluation of effects of bothropic venoms through the chorioallantoic membrane assay HET-CAM ...................................................................................................................................................... 68

Braga, Jacqueline Ramos Machado; Lima, Natália Cavalcante Barbosa; Alves, Carolina Esmeraldo Lima; Rocha, Danilo Galvão; Jorge, Roberta Jeane Bezerra; Biondi, Ilka Borges

Use of a biomolecular decellularized bovine cornea derived solution as a method of evaluating opacity and ocular irritation .................................................................................................................................69

Santos, Jordana Andrade; Dias, Wanessa Amorim; Valadares, Marize Campos

Use of CellFate®iN for a three-dimensional in vitro model of human lung epithelium ........................................70Santos, Jeniffer Farias; Reis, Emily Marques; Berti, Fernanda Vieira; Koepp, Janice; Nunes, Viviane Abreu

02 Analytical Toxicology ............................................................................................................................................. 71

Analysis of cannabinoids profile in cannabis-based products by HPLC-DAD .........................................................72Cardoso, Marilia Santoro; Oliveira, Claudete C.; Costa, José Luiz

Analysis of carbofuran and chlorpyrphos-methyl in GC-MS under different conditions of the injection port and column oven temperatures ............................................................................................................73

Rosa, Victória Gomes; Bairros, André Valle; Ugalde, Gustavo Andrade; Chimendes, Nayomi de Andrade; Santos, Lara Celestina; Pacheco, André Lucas Bezerra; Reginato, Fernanda Ziegler; Berlato, Dener Gomes; Nascimento, Marcelo Henrique Santana

Analysis of cholinesterase inhibitor pesticides by GC-MS in larvae of Lucilia cuprina (Calliphoridae) for entomotoxicological studies .................................................................................................................74

Ugalde, Gustavo Andrade; Chimendes, Nayomi de Andrade; Pacheco, André Lucas Bezerra; Santos, Lara Celestina; Reginato, Fernanda Ziegler; Berlato, Dener Gomes; Cardoso, Leonardo Corrêa; Nascimento, Marcelo Henrique Santana; Rosa, Victória Gomes; Santos, Rachel; Pereira, Alessandra de Oliveira; Monteiro, Silvia Gonzalez; Bairros, André Valle

Case report of valproic acid intoxication: the relevance of chromatographic methods for elucidating cases in hospitals ....................................................................................................................................................75

Sampaio, Amanda Mello Kasper Vaz; Reginato, Fernanda Ziegler; Bairros, André Valle; Roehrs, Miguel; Lovatel, Ivy Bauer; Ugalde, Gustavo Andrade

Detection of cobalt in human urine by liquid chromatography coupled with high resolution mass spectrometry for anti-doping control purposes ...............................................................................76

Santos, Vanessa Farelo; Carneiro, Gabriel Reis Alves; Coelho, Matheus Campelo da Costa; Machado, Sérgio de Paula; Pereira, Henrique Marcelo Gualberto

Determination of 5-fluorouracil in dried blood spots (DBS) by UPLC/MS-MS ........................................................ 77Silva, Laura Cé; Grando, Ana Paula; Hahn, Roberta Zilles; Linden, Rafael; Antunes, Marina Vezon

Determination of cocaine and metabolites applying dried spot oral fluid using well plate by HPLC-MS/MS .............................................................................................................................................................................78

Borges, Gabriela Ramos; Santos, Bruno Pereira; Gouveia, Giovanna Cristiano; Dalanhol, Carolina Silveira; Scherer, Juliana; Eller, Sarah; Oliveira, Tiago Franco

Determination of cortisol and cortisone in urine samples from patients with Parkinson’s disease by dispersive tip microextraction solid phase and ultra-efficient liquid chromatography tandem-mass spectrometry ...................................................................................................................79

Kakuda, Priscila; Souza, Israel Donizeti; Tumas, Vitor; Queiroz, Maria Eugênia Costa

Determination of Ethyl Glucuronide and Ethyl Sulfate in urine samples from brazilian truck drivers ......... 80Costa, C.D.D.; Oliveira Neto, J.R.; Oliveira, N.R.L.; Alcântara, K.C.; Cunha, L.C.

Determination of pharmaceuticals and personal care products (PPCPs) in surface water in Southern Brazil ........................................................................................................................................................................... 81

Bastiani, Marcos Frank; Hahn, Roberta Zilles; Lizot, Lilian de Lima Feltraco; Bondan, Amanda Pacheco; Castilhos, Maria Amélia; Freitas, Mariana; Favreto, Camila; Linden, Rafael

Determination of psychoactive substances in sweat samples using disposable pipette extraction (DPX) and GC-MS ..................................................................................................................................................... 82

Gomes, Nayna Cândida; De Martinis, Bruno Spinosa

Determination of synthetic cannabinoid receptor agonists (SCRAs) in infused papers and herbal materials seized in Brazilian prisons ....................................................................................................................... 83

Martins, Aline Franco; Barbosa, Ingrid Lopes; Milanez, Guilherme Paier; Costa, José Luiz

Development and validation of a method for the determination of meropenem employing micro samples of dried blood spots obtained from capillary punctures ................................................................. 84

Busato, Maria Amelia de Castilhos; Antunes, Marina Venzon; Linden, Rafael

Development and validation of analytical method for the quantification of chemical elements in whole blood with volumetric absorptive microsampling by ICP-MS .................................................85

Sousa Júnior, Wellington Tavares; Morais, Déborah Araújo; Oliveira, Silvana Ruella; Barbosa Jr, Fernando

Development of a HPLC-DAD-RF method for quantification of neurotransmitters in heads of Drosophila melanogaster .................................................................................................................................................... 86

Carriço, Murilo Ricardo Sigal; Rodrigues, Marina Diaz; Paz, Maria Elizabeth Gomes; Molina, Higor Severo; Nogueira, Caroline Lacerda; Denardin, Elton Luis Gasparotto; Roehrs, Rafael

Development of a method based on Green Analytical Toxicology to determine the active components of ayahuasca in hair ............................................................................................................................................87

Santos, Fabiana Pereira; Fabris, Andre Luis; Bruno, Vitor; Yonamine, Mauricio

Development of a method for analysis of simvastatin and simvastatin hydroxyacid by HPLC in human plasma using HF-LPME extraction .......................................................................................................... 88

Vasconcelos, Mayrla Emilia Dantas; Gaitani, Cristiane Masetto; Moraes, Natália Valadares; Demets, Mariana Barbieri Alvarez

Development of a miniaturized sample preparation method for determination of ketamine, its metabolites and analogs in oral fluid samples using Dispersive Liquid-Liquid Microextraction (DLLME) ............................................................................................................................................... 89

Kahl, Júlia M.M.; Berlinck, Débora Zorrón; Chinaglia, Kauê de Oliveira; Rodrigues, Leonardo Costalonga; Costa, Jose Luiz

Development of a simple and fast method for iodine determination in hair samples by ICP-MS after alkaline solubilization at room temperature ............................................................................................ 90

Morais, Déborah Araújo; Sousa Junior, Wellington Tavares; Oliveira, Silvana Ruella; Barbosa Junior, Fernando

Development of an analytical method based on Green Analytical Toxicology employing umbilical cord tissue for the evaluation of maternal fetal exposure to cocaine .................................................... 91

Meirelles, Gabriela de Paula; Pereira e Silva, Jefferson; Yonamine, Mauricio

Development of an extraction method for the pesticide 2,4-dichlorophenoxyacetic acid and its metabolite 2,4-dichlorophenol from Apis mellifera ...........................................................................................92

Paz, Maria Elizabeth Gomes; Carriço, Murilo Ricardo Sigal; Rodrigues, Marina Diaz; Ramborger, Bruna Piaia; Denardin, Elton Luis Gasparotto; Roehrs, Rafael

Development of dispersive solid phase microextraction (d-SPE) and HPLC-DAD for emergency toxicological analysis of toxic drugs in urine samples .............................................................................93

Luz, Heloisa Peres; Silva, Bruna Espíndola; Santos, Claudia Regina; Marchioni, Camila

Ethanol quantification in ethanol-based hand sanitizers: alert in the COVID-19 pandemic scenario ........... 94El Haddad, Lohanna Pereira; Costa, Bruno Ruiz Brandão; Bigão, Vitor Luiz Caleffo Piva; De Martinis, Bruno Spinosa

Evaluation of cork as a natural extraction phase for the determination of new psychoactive substances in blood samples .........................................................................................................................95

Birk, Letícia; Eller, Sarah; Oliveira, Tiago Franco

Evaluation of the spontaneous hydrolysis of cocaine under different pH and temperature conditions ......96Gomes, Geovana Maria de Lima; Santos, Vanessa Farelo; Carneiro, Gabriel Reis Alves; Trajano, Christian Farias; Pereira, Henrique Marcelo Gualberto

First proof of concept of a new passive sampler for marine biotoxins .....................................................................97Fitarelli, Bruna; Fabichak, Ana; Deolindo, Carolina Turnes Pasini; Kleemann, Cristian; Hoff, Rodrigo

In vitro metabolism studies of ADB-4en-PINACA, ADB-FUBIATA and BZO-CHMOXIZID using human liver microsomes ............................................................................................................................................... 98

Cunha, Kelly F.; Krotulski, Alex J.; Walton, Sara E.; Papsun, Donna M.; Costa, Jose Luiz; Logan, Barry K.

Liquid chromatography–tandem mass spectrometry method for simultaneous quantification of neurotransmitters in rat brain tissue ...................................................................................................99

Viana, Roberta Rodrigues; Pego, Ana Miguel Fonseca; Dallegrave, Eliane; Oliveira, Tiago Franco; Eller, Sarah

Metabolism study of coca leaf tea in urine by liquid chromatography coupled with high resolution mass spectrometry ................................................................................................................................................100

Gomes, Geovana Maria de Lima; Santos, Vanessa Farelo; Carneiro, Gabriel Reis Alves; Trajano, Christian Farias; Pereira, Henrique Marcelo Gualberto

New tools in analytical toxicology: analysis of Paraquat in urine with a mobile phone ................................... 101Chinaglia, Kauê de Oliveira; Lanaro, Rafael; Arantes, Ana Carolina Furiozo; Costa, José Luiz

Preliminary results of method validation for dihydropyrimidine dehydrogenase (DPD) deficiency screening and 5-fluorouracil determination in plasma by UPLC-MS/MS .........................................102

Grando, Ana Paula; Silva, Laura Cé; Hahn, Roberta Zilles; Linden, Rafael;Antunes, Marina Vezon

Safety studies of a potential cathepsin K inhibitor: 4-methoxy chalcone and its degradation products ....... 103Benvenutti, Danyela Francine; Buzzi, Fatima de Campos; Corrêa, Rogerio; Couto, Angélica Garcia; Wagner, Theodoro; Paula, Favero R.; Giovagnoli, Stefano; Vivani, Riccardo; Ricci, Maurizio; Santos, Carlos Eduardo Matos; Santin, José Roberto; Bresolin, Tania Mari Bellé

Use of design of experiment tools to enhance the recovery of synthetic cannabinoids in dried blood spots (DBS)..............................................................................................................................................................104

Berlinck, Débora Zorrón; Cunha, Kelly Francisco; Costa, José Luiz

Validation and pre-clinical evaluation of pharmacokinetic profile of antineoplasic prototype LQMF030 in rats by LC-MS/MS ..........................................................................................................................105

Pereira, I.B.; Zoghaib, I.V.J.; Gomes, S.A.; Menegatti, R.; Cunha, L.C.

Validation of a DPX-GC-MS method for simultaneous quantification of psychoactive substances and its metabolites in human breast milk ..................................................................................................106

Santos Junior, Wilson José Ramos; Gomes, Nayna Cândida; Costa, Bruno Ruiz Brandão; Bigão, Vitor Luiz Caleffo Piva; De Martinis, Bruno Spinosa

Validation of a method for plasma vancomycin therapeutic monitoring by High Performance Liquid Chromatography with UV detector ...............................................................................................107

Paula, Eliza Bianchini; Santos, Claudia Regina; Marchioni, Camila

Validation of an analytical method for the determination of psychoactive substances in an oral fluid sample using the DPX-SCX disposable tips and the GC-MS ...............................................................108

Gomes, Nayna Cândida; De Martinis, Bruno Spinosa

Validation of disposable pipette extraction method with quantification by GC-MS to determine ethylenethiourea in human urine samples ...................................................................................................109

Romoli, Jéssica Cristina Zoratto; Scanferla, Deborah Thais Palma; Aguera, Raul Gomes; Lini, Renata Sano; Castro, Juliana Cristina; Bando, Érika; Alves, Gessé de Souza; Nerilo, Samuel Botião; Mossini, Simone Aparecida Galerani; Marchioni, Camila;Machinski Junior, Miguel

03  Clinical and Laboratorial Toxicology ............................................................................................................... 110

A simple procedure for the determination of mercury in urine by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) ................................................................................................................................... 111

Sugawara, Eduardo Kinio; Silva, Graciele Machado; Paulucci, Leticia Trevisan; Ramadan, Debora R.; Tufik, Sergio; Soares, Marcela de Oliveira

Analysis of plasmatic levels of antipsychotics through an absorptive paper-based extraction followed by fast gas chromatography-mass spectrometry ...................................................................112

Gouveia, Giovanna Cristiano; Borges, Gabriela Ramos; Santos, Bruno Pereira; Sebben, Viviane Cristina; Arbo, Marcelo Dutra; Eller, Sarah; Oliveira, Tiago Franco

Assessment of mercury exposure and health of riverside populations affected by the Brumadinho disaster ....................................................................................................................................................................113

Moreira, Camila Francisco; Nolasco, Daniela; Souza, Tainá Brumate; Mendes, Michele Polyana Rocha; André, Leiliane Coelho; Paiva, Maria José Nunes

Case Report: follow-up care of a cocaine intoxication case at a Toxicological Information and Assistance Center in Fortaleza - Ceará ....................................................................................................................... 114

Salles, Gabriela Pereira; Ferreira e Silva, Hendyelle Rodrigues; Silva, Victória da Costa; Batista, José Márcio Machado; Albuquerque, Polianna Lemos Moura Moreira; Moreira, Rhubens Levy Rodrigues; Ferreira, Maria Augusta Drago

Case Report: follow-up care of a kerosene intoxication case at a Toxicological Information and Assistance Center in Fortaleza - Ceará ........................................................................................................................115

Salles, Gabriela Pereira; Ferreira e Silva, Hendyelle Rodrigues; Silva, Victória da Costa; Farias, Beatriz Valentim; Magalhães, Karla do Nascimento; Batista, José Márcio Machado; Albuquerque, Polianna Lemos Moura Moreira; Ferreira, Maria Augusta Drago

Case Report: Phencyclidine cross-reaction investigation in immunochromatographic tests for rapid drugs detection .................................................................................................................................................116

Cardoso, Leonardo Corrêa; Rosa, VictóriaGomes; Pacheco, André Lucas Bezerra; Santos, Rachel; Ugalde, Gustavo Andrade; Berlato, Dener Gomes; Reginato, Fernanda Ziegler; Chimendes, Nayomi Andrade; Santos, Lara Celestina; Nascimento, Marcelo Henrique Santana; Oliveira, Sarah Carobini Werner de Souza Eller Franco; Oliveira, Tiago Franco; Bairros, André Valle

Clinical-epidemiological profile of elapid accidents in pediatric population registered at CIATox/SC .........117Silva, Stephanie Soares; Messias, Nayara Casagrande; Bresolin, Nilzete Liberato; Silva, Denise Bousfield; Santos, Claudia Regina

Determination of pesticides in blood samples from patients after suicide attempt by micro-QuEChERS and LC-MS/MS ........................................................................................................................................... 118

Godoi, Alexandre Barcia; Silva, Mariana Cristina; Costa, José Luiz

Determination of tricyclic antidepressants in whole blood by liquid chromatography with diode diarray detector employing dispersive liquid-liquid microextraction...........................................................119

Berlato, Dener Gomes; Rosa, VictóriaGomes; Pacheco, André Lucas Bezerra; Santos, Rachel; Ugalde, Gustavo Andrade; Cardoso, Leonardo Corrêa; Reginato, Fernanda Ziegler; Chimendes, Nayomi Andrade; Santos, Lara Celestina; Nascimento, Marcelo Henrique Santana; Bairros, André Valle

Development and validation of a method to measure Tenofovir concentrations in urine of HIV-positive patients ...................................................................................................................................................................120

Santos, Rafaela Knak; Linden, Rafael

Early adverse reactions to different types of anti-poison serum in the period from 2017 to 2021: a descriptive analysis .......................................................................................................................................................121

Priedols, Gustavo Abud; Alves, Jonas Alher Meira; Oliveira, Jordana Meirelles; Girotto, Edmarlon; Guidoni, Camilo Molino

Epidemiological variables and anti-poison serotherapy: a descriptive analysis of early adverse reactions .........................................................................................................................................................................122

Alves, Jonas Alher Meira; Priedols, Gustavo Abud; Oliveira, Jordana Meirelles; Girotto, Edmarlon; Guidoni, Camilo Molino

Estimation of hematocrit values by potassium quantification in capillary dried blood spots (DBS) ..........123Silva, Laura Cé; Alves, Pedro Adolfo Pereira; Linden, Rafael; Antunes, Marina Vezon

Evaluation of cytotoxic potential of polyhydroxylated phenylchromones in human osteosarcoma in vitro models .................................................................................................................................................124

Oliveira, José Miguel P. Ferreira; Proença, Carina; Santos, Raquel; Moreira, Beatriz; Rufino, Ana T.; Freitas, Marisa; Ribeiro, Daniela; Fernandes, Eduarda

Evaluation of experimental conditions for sample preparation to identify the untargeted metabolomic profile of plasma by GC-MS. ......................................................................................................................... 125

Nolasco, Daniela M.; Pires, Sumaia Araújo; Souza, Tainá Brumate; Souza, Mirna Maciel D'Auriol; Paiva, Maria José Nunes; André, Leiliane Coelho

Exclusive therapy of N-acetylcysteine in accidental butanox intake: toxicological analysis of methyl ethyl ketone peroxide .............................................................................................................................................126

Santos, Rachel; Bairros, André V.; Saldanha, Geovane A.; Berlato, Dener G.; Moraes, Liliana S.; Gündel, Augusto R.; Carvalho, José A.M.; Habib, Isabela A.; De Carli, Diego M.; Oliveira, Tiago F.; Oliveira, Sarah C.W.S.E.F.

Fast method of quantitative analysis of serum vitamin A (retinol) using LC-MS/MS ........................................ 127Soares, Marcela de Oliveira; Sugawara, Eduardo Kinio; Mazete, Fernanda Pine S.; Ramadan, Debora R.; Tufik, Sergio

Finger-prick volumetric absorptive microsampling (VAMS) for imatinib therapeutic drug monitoring: method development and validation ..........................................................................................................128

Guterres, Fernanda S.; Krutzmann, Maria Eduarda; Kohlrausch, Ramona; Linden, Rafael; Antunes, Marina Vezon

Garlic burn injuries: a clinical-epidemiological profile of cases registered at CIATox/SC, Brazil ...................129Cordeiro, Gabriela Batista Cavalcanti; Arruda, Fernanda Wolff da Silva; Petry, Andrea; Resener, Marisete Canello; Santos, Claudia Regina

Hepatotoxicity risk assessment in suicide attempts by acetaminophen with doses between 7.5g and 10g at a reference center in Santa Catarina, Brazil......................................................................130

Fonseca, Karoline Kuhnen; Costa, Ana Carolina Conchon; Cordeiro, Gabriela Batista Cavalcanti; Arruda, Fernanda Wolff da Silva; Resener, Marisete Canello

Liver and kidney function at the pesticide’s exposure of rural workers from Casimiro de Abreu - RJ ..........131Nunes, Rafaella Ferreira Nascimento; Poça, Kátia Soares; Cabral, Yngrid dos Santos; Aguiar, Gilberto Santos; Siqueira, Janas; Geraldino, Barbara Rodrigues; Otero, Ubirani Barros; Martins, Isarita; Sarpa, Márcia Sarpa de Campos

microRNAs as serum biomarker for Senecio brasiliensis poisoning in cattle ......................................................132Winter, Evelyn; Cisilotto, Julia; Goetten, André L.F.; Veiga, Ângela; Ramos, Adriano T.; Zimermann, Francielli C.; Reck, Carolina; Creczynski-Pasa,Tânia B.

Profile of drug-associated toxicological events in women of reproductive age ..................................................133Costa, Quezia dos Santos; Priedous, Gustavo Abud; Brunello, Giovanna Cristina Spagnuolo; Alfieri, Daniela Frizon; Guidoni, Camilo Molino; Alves, Jonas Alher Meira; Girotto, Edmarlon

Profile of suicide attempts by self-poisoning in the Rio Grande Sul, Brazil, between 2010-2020 and the influence of the COVID-19 pandemic .........................................................................................................134

Santos, Bruno Pereira; Eller, Sarah; Gouveia, Giovanna Cristiano; Borges, Gabriela Ramos; Sebben, Viviane Cristina; Arbo, Marcelo Dutra; Oliveira, Tiago Franco

Services provided by the Nucleus Applied to Toxicology (NAT) during the period 2019-2021 ........................ 135Reginato, Fernanda Ziegler; Bairros, André Valle; Santos, Lara Celestina; Rosa, Victória Gomes; Chimendes, Nayomi de Andrade; Santos, Rachel; Pacheco, André Lucas Bezerra; Cardoso, Leonardo Corrêa; Ugalde, Gustavo Andrade

Suicide attempts in Santa Catarina, Brazil, before and during Covid-19 pandemic: epidemiological profile ...............................................................................................................................................................136

Costa, Ana Carolina Conchon; Taruhn, Lillian Freitas; Resener, Marisete Canello

Suicide attempts in adolescents assisted by a toxicological information and assistance center due to toxicological events ......................................................................................................................................... 137

Brunello, Giovanna Cristina Spagnuolo; Alves, Jonas Alher Meira; Costa, Quezia dos Santos; Priedous, Gustavo Abud; Alfieri, Daniela Frizon; Guidoni, Camilo Molino; Girotto, Edmarlon

Variation of cholinesterase s activity and the importance of the involved toxic agent identification on the acute intoxication evolution by an acetylcholinesterase inhibitor – Case Report .....138

Bauermann, Lauren; França, Liana Conrado; Nogueira, Janer Alves; Paula, Eliza Bianchini; Marchioni, Camila; Santos, Claudia Regina

04  Drugs of Abuse .......................................................................................................................................................139

A green dispersive liquid-liquid microextraction for quantitative analysis of synthetic cathinones in urine and postmortem blood .......................................................................................................................140

Fabris, André Luis; Yonamine, Mauricio

A novel workflow for the analysis of drugs of abuse in oral fluid samples using disposable pipette extraction technologies (DPX-XTR) ........................................................................................................................ 141

Cabrices, Oscar G.

Alcohol and alcohol combined with texting: effects on speed and braking behaviours in a closed-course section. ...............................................................................................................................................................142

Costa, Bruno Ruiz Brandão; Freitas, Bruno Toledo; Bigão, Vítor Luiz Caleffo Piva; Perdoná, Gleici da Silva Castro; De Martinis, Bruno Spinosa

Analytical evaluation of four screening devices for drug detection in Brazilian traffic enforcement ..........143Scherer, Juliana N.; Borges, Gabriela Ramos; Santos, Bruno Pereira; Dalanhol, Carolina Silveira; Gouveia, Giovanna; Viola, Patrícia Pacheco; Carlson, Renato Romera; Govoni, Bruna; Mello, Raissa; Vasconcelos, Mailton; Pechansky, Flavio

Assessment of possible neurotoxic effects of oxycodone in SH-SY5Y cell lineage ........................................... 144Lima, Luiza Siqueira; Costa, Nayara de Souza; Pereira, Meire Ellen; Almeida, William; Cestari, Marta Margarete; Irioda, Ana Carolina; Oliveira, Cláudia Sirlene

Blood alcohol levels in fatal victims of traffic accidents ...............................................................................................145Everton Boff; Daniel, Caroline; Sá, Clodoaldo Antônio; Brugnera, Débora Schmitz; Silva, Maria Isabel Gonçalves; Marcon, Scheila; Corralo, Vanessa da Silva

Determination of a comprehensive set of drugs of abuse, metabolites and human biomarkers in wastewater using passive sampling followed by UHPLC-MS/MS analysis .............................146

Hahn, Roberta Zilles; Bastiani, Marcos Frank; Lizot, Lilian de Lima Feltraco; Linden, Rafael

Determination of levamisole in cocaine samples seized in the south of Santa Catarina ................................147Bombazar, Amanda; Martins, André Bittencourt; Steiner, Bethina Trevisol; Ávila, Ricardo Andrez Machado

Developing a drug checking service in Brazil and the potential of harm reduction strategies to reduce intoxication risks ................................................................................................................................ 148

Maluf, Ana Cristhina Sampaio; Costa, José Luiz

Development and validation of a highly sensitive method for detection of Synthetic Cannabinoid JWH-018, AB-CHMINACA, and their metabolites in hair samples by LC-MS/MS .......................149

Paulo, Breno F. Pereira; Zauli, Danielle Alves Gomes Breno

Epidemiological aspects of suicide in the State of São Paulo ......................................................................................150Souza, Karla Aparecida de Oliveira; Gianvecchio, Victor Alexandre Percinio; Gianvecchio, Daniele Muñoz; Jorge, Maria Helena Prado de Mello; Costa, José Luiz

Exogenous poisoning by drug abuse in Brazil: before and during the COVID-19 pandemic ..............................151Baccule, Nicole Santos; Scanferla, Deborah Thais Palma; Lini, Renata Sano; Marchioni, Camila; Mossini, Simone Aparecida Galerani

Identification of synthetic drugs seized in the region of Balneário Camboriú using ATR-FTIR ...................... 152Costa, Karina Oliveira; Bagio, Jéssica

In silico evaluation of biological, therapeutical, and toxicological properties for five groups of NPS: amphetamines, cathinones, benzodiazepines, synthetic opioids, and synthetic cannabinoids. ............................................................................................................................................................. 153

Santos, Christiano; Bruni, Aline Thais

In silico parameters and chemometrics applied to the study of NBOMes and amphetamine-type stimulants ...............................................................................................................................................154

Mariotto, Lívia Salviano; Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Intoxication case series by N-ethylpentylone (N-ethylnorpentylone or ephylone)........................................... 155Denkena, Isadora Locilento; Cunha, Kelly Francisco; Lanaro, Rafael; Cunha, Ricardo Leal; Costa, Jose Luiz

MDA in silico toxicity assessment suggests low hepatotoxicity and calls for further pre-clinical investigations .................................................................................................................................................................. 156

Oliveira, Arthur Lima; Miranda, Raul Ghiraldelli; Dorta, Daniel Junqueira

MDMA in silico toxicity analysis reinforces need of mimicking consumption patterns towards translational reliability ............................................................................................................................................. 157

Oliveira, Arthur Lima; Miranda, Raul Ghiraldelli; Dorta, Daniel Junqueira

Profile of new psychoactive substances (NPS) in seized materials analyzed in the Rio de Janeiro State during the COVID-19 pandemic ....................................................................................................................158

Oliveira, Adriana Sousa; Almeida, Fernando G.; Bhering, Cecília de A.; Antonio, Ananda da S.; Wurzler, Gleicielle T.; Costa, Gabriela Vanini

Risk factors associated with deaths by multiple drugs of abuse intoxications ................................................... 159Messias, Nayara Casagrande; Silva, Stephanie Soares; Resener, Marisete Canello; Albino, Danielle Bibas Legat; Barotto, Adriana Mello; Santos, Claudia Regina; Marchioni, Camila

Study of NBOMes using MetadrugTM and Chemometrics ..............................................................................................160Mariotto, Lívia Salviano; Bruni, Aline Thais

Study of synthetic cannabinoids regarding their metabolites: an in silico approach ..........................................161Castro, Jade Simões; Rodrigues, Caio Henrique Pinke; Bruni, Aline Thais

Tracking methamphetamine surges in the Joinville region (Santa Catarina) from 2014 to 2021 ...................162José, Gustavo Pinheiro Coelho; Pericolo, Suellen; Parabocz, Gisele Chibinski; Costa, Karina Oliveira; Marchioni, Camila; Ramos, Silvia Aparecida

Use of psychedelics in the treatment of anxiety and depression disorder - literature review ......................163Silva, Luíza Madureira; Holanda, Wiron Pimentel; Honório Júnior, José Eduardo Ribeiro

05 Environmental Safety and Health ....................................................................................................................164

Acute toxicity of Diuron, DCA and DCPMU in the early life and adultohood of the zebrafish (Danio rerio) ................................................................................................................................................................................... 165

Sales, Bianca Camargo Penteado; Andrade, Ítalo Bertoni Lopes; Peixoto, Paloma Vitória Lima; Camargo, João Lauro Viana; Pereira, Lilian Cristina

Blood total mercury levels of preschool children from Sao Paulo, Brazil, and associated risk factors .....166Olympio, Kelly Polido Kaneshiro; Salles, Fernanda Junqueira; Pereira, Elizeu Chiodi; Oliveira, Allan Santos; Costa, Eric Augusto Caravaggio; Costa, Brenda Natasha Souza; Pereira, João Paulo

Goes; Jesus, Iracina Maura; Queiroz, Thais Karolina Lisboa; Lima, Marcelo de Oliveira; Silva, Agnes Soares; Cardoso, Maria Regina Alves

Determination of biomarkers of exposure to pythroid pesticides through wastewater-based epidemiology ..................................................................................................................................................................... 167

Lizot, Lilian de Lima Feltraco; Bastiani, Marcos Frank; Hahn, Roberta Zilles; Linden, Rafael

Embryonic exposure to genistein induces persistent anxiolytic and antisocial behaviors in zebrafish .........168Freddo, Natália; Menegasso, Aloma Santin; Soares, Suelen Mendonça; Fortuna, Milena; Maffi, Victoria Costa; Mozzato, Mateus Timbola; Barcellos, Leonardo José Gil; Rossato-Grando, Luciana Grazziotin

Energetic metabolism and antioxidant system modulation after fish chronic exposure to glyphosate and imidacloprid ...................................................................................................................................................169

Sobjak, Thaís Maylin; Zazula, Matheus Felipe; Macarini, Leanna Camila; Rizzo, Elizete; Guimarães, Ana Tereza Bittencourt⁴

Ethnic-racial disparity in exogenous intoxications in Brazil ........................................................................................170Moraes, Niely Galeão da Rosa; Silva Júnior, Flavio Manoel Rodrigues

Evaluating the levels of toxic elements and toxicity testing in water samples of Paraopeba River after the Brumadinho dam rupture ......................................................................................................171

Devóz, Paula Pícoli; Rocha, Cecília Cristina de Souza; Barbosa Jr, Fernando

Evaluation of the effect of a nutrient solution on the phytoremediation potential of a blue dye by the aquatic macrophyte Salvinia auriculata ........................................................................................................ 172

Schmitt, Maria Laura Videiro; Carriço, Murilo Ricardo Sigal; Nogueira, Caroline Lacerda; Rodrigues, Marina Diaz; Molina, Higor Severo; Denardin, Elton Luis Gasparotto; Roehrs, Rafael

New approaches to periphytic diatoms as bioindicators of contamination by iron ore tailings from the collapse of the Fundão dam in the Doce River (Brazil) ................................................................ 173

Souza, Luiz Claudio Cindra; Reis, Luciane Ayres Castro; Rangel, Lara Luiza Pimenta; Barroso, Gilberto Fonseca

Periphytic community in experimental rivers as a tool for the assessment of anthropogenic stressors in fluvial ecosystems ................................................................................................................174

Reis, Luciane Ayres Castro; Almeida, Stefano Zorzal; Barroso, Gilberto Fonseca

Urinary lead levels and factors associated with their increase in schoolchildren living in a coal mining area in the extreme south of Brazil ............................................................................................................... 175

Brum, Rodrigo de Lima; Baisch, Ana Luíza Muccillo; Silva Júnior, Flavio Manoel Rodrigues

Water quality assessment of the Rio do Peixe: a preliminary study ......................................................................... 176Gemelli, Bianca Aparecida Martins; Summy, Maria Julia; Cesaro, Humberto Luis; Alves, Rômulo Couto; Saraiva, Illyushin Zaak; Remor, Aline Pertille; Baú, Morgana; Lima, Daina; Almeida, Eduardo Alves; Müller, Gabrielle do Amaral e Silva

06 Experimental Toxicology .................................................................................................................................... 177

Acute toxicity of 2,4-dichlorophenoxyacetic acid in the early life of the zebrafish (Danio rerio) ..................178Viriato, Cristina; Andrade, Ítalo B.L.; Peixoto, Paloma V.L.; Pereira, Lílian C.

Alterations in the activity of antioxidant enzymes and NA+K+-ATPase in lead poisoning in the cerebral cortex and hippocampus of rats ................................................................................................................... 179

Ferrazza-Heitich, Magda; Lima, Daniela Delwing; Borgmann, Gabriela; Plautz, Katherine

Carvacrol and its anti-inflammatory effect under cigarette smoke-induced acute lung injury .....................180Moura, Maria Joana Nogueira; Silva, Aline Gabrielle Gomes, Santos, Caio Cesar Araújo; Pereira, Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Lima, Crystiane Calado, Golçalves, A.P.; Melo, Paolo Oliveira; Silva, Gerlane Modesto; Feitosa, Emanuel Kenedy; Borges, Cibele dos Santos

Chronic exposure to methylphenidate during juvenile period alters zebrafish behavior in adulthood ...... 181Nardi, Jessica; Freddo, Natália; Biazus, Inara Carbonera; Oliveira, Ana Paula; Soares, Suelen Mendonça; Fortuna, Milena; Pompermeier, Aline; Siqueira, Lisiane; Cole, Amanda Carolina;

Tamagno, Wagner; Amaral, Francieli Ubirajara India; Costa, Vitoria Cadore; Mozzato, Mateus Timbola; Barcellos, Leonardo José Gil; Grando, Luciana Grazziotin Rossato

Chronic plus binge ethanol feeding synergistically induces abdominal aorta dysfunction and gut dysbiosis in mice ..........................................................................................................................................................182

Silva, Carla Brigagão Pacheco; Rodrigues, Vanessa Fernandes; Costa, Bruno Ruiz Brandão; Sartori, Daniela Carlos; De Martinis, Bruno Spinosa; Tostes, Rita C.

Commercial 2,4-D and its isolated compounds, 2,4-dichlorophenoxyacetic acid and 2,4-D dimethylamine, induces changes in mitochondrial function parameters ..............................................................183

Polido, Lucas Roberto Ferreira; Rizzi, Joyce Santana; Pereira, Lílian Cristina

Could Eugenol promote the reduction of the inflammatory process in acute injuries to the lung of rats caused by cigarette smoke? ............................................................................................................................ 184

Silva, Aline Gabrielle Gomes; Moura, Maria Joana Nogueira; Santos, Caio Cesar Araújo; Pereira, Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Araujo, Bruno Vinicios Silva; Barbosa, Maria Clara de Oliveira; Melo, Paolo Oliveira; Feitosa, Emanuel Kenedy; Borges, Cibele dos Santos

Cytotoxicity assessment of potential antitumor 4-methyl coumarins ...................................................................185Göethel, Gabriela; Souza, João Pedro Silveira; Neves, Gustavo Machado; Kagami, Luciano Porto; Peruzzi, Caroline Portela; Cattani, Shanda; Garcia, Solange Cristina; Battastini, Ana Maria Oliveira; Figueiró, Fabrício; Eifler-Lima, Vera Lucia

Cytotoxicity evaluation in human hepatic cells HepG2 induced by emerging contaminants Decametilcyclopentasiloxane and Triclopyr ......................................................................................................................186

Kohori, Natália Akemi; Teodoro, João Soeiro; Palmeira, Carlos Manuel Marques; Pereira, Lilian Cristina

Different diets and physical exercises: assessment of the impact on the female reproductive system after 90 days ........................................................................................................................................187

Moura, Maria Joana Nogueira; Silva, Aline Gabrielle Gomes; Santos, Caio Cesar Araújo; Gomes, Francisca Tayná da Silva; Pereira, Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Fonseca, Ivana Alice Teixeira; Borges, Cibele dos Santos

Discreet alterations on sperm quality promoted by Aripiprazole ............................................................................ 188Moura, Maria Joana Nogueira; Felix, Renata Gleysiane de Sousa; Pereira, Artemia Kelly Holanda; Angelo, Ana Beatriz Silva; Santos, Caio Cesar Araújo; Silva, Aline Gabrielle Gomes;Silva, Ana Lucelha dos Santos; Freire, Livia Horrana Forte; Dantas, Joao Artur Diogenes; Silva, Mateus Limerio Carlos; Borges, Cibele dos Santos

Diuron herbicide-induced toxicity on Caenorhabditis elegans ..........................................................................189Lima, Thania Rios Rossi;Martins Jr., Airton Cunha; Pereira, Lílian Cristina; Aschner, Michael

Effect of inorganic selenium on triple-negative breast cancer cell lines ................................................................190Costa, Nayara de Souza; Lima, Luiza Siqueira; Oliveira, Franciele Aparecida Mendes; Galiciolli, Maria Eduarda de Andrade; Oliveira, Cláudia Sirlene; Irioda, Ana Carolina

Evaluation of irritability of Myrcia pubipetala miq. by HET-CAM methodology ....................................................191Perini, Camila Maria; Kohler, Cristofer José Weege; Wagner, Eduardo José; Machado, Isabel Daufenback

Evaluation of metabolic activity of HepG2 cells co-exposed to micro or nanoplastics and bisphenol A or S .............................................................................................................................................................................192

Rocha, Cecília Cristina de Souza; Devoz, Paula Pícoli; Antunes, Lusania Maria Greggi; Barbosa Jr, Fernando

Evaluation of the female wistar rats reproductive system submitted to different diet formulations and activity patterns during 180 days .......................................................................................................193

Dantas, Joao Artur Diogenes; Silva, Aline Gabrielle Gomes; Moura, Maria Joana Nogueira; Santos, Caio Cesar Araújo; Gomes, Francisca Tayná da Silva; Pereira, Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Fonseca, Ivana Alice Teixeira; Borges, Cibele dos Santos

Evaluation of the oil toxicity of Astrocaryum vulgare Mart. through renal and hepatic indicators of Wistar rats ............................................................................................................................................................194

Reginato, Fernanda Ziegler; Guex, Camille Gaube; Figueredo, Kássia Caroline; Graiczik, James Ramires Penteado; Cassanego, Gabriela Buzatti; Pappis, Lauren; Heck, Amanda Szymansky; Bauermann, Liliane de Freitas

evaluation of toxicity of extracts from Myrcia tijucensis in Artemia salina leach model ................................ 195Kohler, Cristofer José Weege; Perini, Camila Maria; Alberton, Michele Debiasi

Exposure of adult female rats to different doses of the antipsychotic Aripiprazole: a look at the reproductive system .......................................................................................................................................................196

Silva, Aline Gabrielle Gomes;Moura, Maria Joana Nogueira; Pereira, Artemia Kelly Holanda; Felix, Renata Gleysiane de Sousa; Angelo, Ana Beatriz Silva; Santos, Caio Cesar Araújo; Silva, Ana Lucelha dos Santos; Freire, Livia Horrana Forte; Dantas, Joao Artur Diogenes; Silva, Mateus Limerio Carlos; Borges, Cibele dos Santos

Imidacloprid-based commercial pesticide causes behavioral and biochemical impairments in Wistar rats ....................................................................................................................................................... 197

Tonietto, Bruna Ducatti; Laurentino, Ana Olívia Martins; Costa-Valle, Marina Tuerlinckx; Cestonaro, Larissa Vivan; Antunes, Bibiana Pereira; Manfio, Cleofas Sates; Santos, Nícolas Guimarães; Dallegrave, Eliane; Garcia, Solange Cristina; Leal, Mirna Bainy; Arbo, Marcelo Dutra

Immediate toxicological impairment of Lisdexamfetamine Dimesylate in male rats treated during juvenile period and periuberty ...................................................................................................................198

Stein, Julia; Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Nagaoka, Lívia Trippe; Manoel, Beatriz de Matos; Valente, Letícia Cardoso; Arena, Arielle Cristina

Investigation of the lipid-lowering and antiatherogenic effects of Plinia cauliflora bark extract in an experimental model of atherosclerosis .....................................................................................................199

Dalmagro, Mariana; Donadel, Guilherme; Pinc, Mariana Moraes; Berta, João Antonio; Borba, Maria Eduarda; Gasparotto Junior, Arquimedes; Jacomassi, Eliza; Zardeto, Giuliana; Hoscheid, Jaqueline; Ceranto, Daniela de Cassia Faglioni Boleta; Lourenço, Emerson Luiz Botelho

Levamisole, a cocaine adulterant, promotes acute and subchronic toxic effects in wistar rats .................200Laurentino, Ana Olívia Martins; Salomón, Janaína; Sebben, Viviane; Tonietto, Bruna Ducatti; Cestonaro, Larissa Vivan; Dallegrave, Eliane; Garcia, Solange; Arbo, Marcelo Dutra; Leal, Mirna Bainy

Maternal exposure to a phthalate combination associated with prostate lesions and cancer in F1 and F2 offspring ....................................................................................................................................................201

Aquino, Ariana Musa; Costa, Luiz Guilherme Alonso; Santos, Sérgio Alexandre Alcantara; Magosso, Natália; Souza, Patrick Vieira; Rocha, Vanessa Aguiar; Oliveira, Marcos Antônio Fernandes; Barbisan, Luis Fernando; Justulin Junior, Luís Antonio; Flaws, Jodi A.; Scarano, Wellerson Rodrigo

Methylmercury toxicity induces structural and cellular damage in cardiac ventricles ....................................202Krüger, Nathália Ronconi Zilli; Pinheiro, Jacqueline; Simioni, Carmen; Nazari, Evelise Maria

Mitochondrial injury induced by Diuron and its metabolites in the rat liver .........................................................203Seloto Danielle Gabriel; Lima Thania Rossi Rios Rossi; Camargo João Lauro Viana;Pereira Lilian Cristina

Paternal Origins of Health and Disease: BaP exposure via paternal germ cells causes negative reproductive consequences in female offspring (F2) in rats ...................................................................204

Jorge, Bárbara Campos; Stein, Julia; Reis, Ana Carolina Casali; Nogueira, Jéssica Bueno; Paschoalini, Beatriz Rizzo; Moreira, Suyane da Silva; Manoel, Beatriz de Matos; Kassuya, Cândida Aparecida Leite; Arena, Arielle Cristina

Pathological findings arising from continuous exposure to trace concentrations of organochlorine DDT residues in multigeneration of wistar rats ............................................................................... 205

Guimarães, Ana Tereza Bittencourt; Silva, Fernanda Coleraus; Quiozini, Nathaly de Matos; Simon, Jaqueline; Pavlak, Jaíne Luana; Azevedo, Camilla de Marchi Sanches; Rangel, Ana Lúcia Carrinho Ayroza

Production and in vitro toxicological studies of natural dyes .....................................................................................206Yli-Öyrä, Johanna; Herrala, Mikko; Albuquerque, Anjaina Fernandes; Farias, Natália Oliveira; Morales, Daniel Alexandre; Räisänen, Riikka; Freeman, Harold S.; Umbuzeiro, Gisela de Aragão; Rysä, Jaana

Reproductive repercussions in adulthood of male rats exposed to Lisdexamfetamine Dimesylate on peripuberty ..................................................................................................................................................... 207

Stein, Julia; Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Nagaoka, Lívia Trippe; Manoel, Beatriz de Matos; Valente, Letícia Cardoso; Pupo, André Sampaio; Arena, Arielle Cristina

Skeletal abnormalities caused by L-mimosine: development toxicity study in rats..........................................208Almeida, Elaine Renata Motta; Pereira, Edimar Cristiano; Hueza, Isis Machado

Study of the toxicological effects of thimerosal and aluminum hydroxide on Danio rerio’s kidney ............209Galiciolli, Maria Eduarda A.; Silva, Juliana F.; Guiloski, Izonete C.; Oliveira, Cláudia S.

Systemic toxicity and testicular damage produced by exposure to benzo(a)pyrene in low-dose from juvenile to peripuberty in male rats .................................................................................................................210

Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Stein, Julia; Paschoalini, Beatriz Rizzo; Nogueira, Jéssica Bueno; Arena, Arielle Cristina

Teratogenic and toxic effects induced by the herbicide 2,4-D (DMA® 806) in zebrafish (Danio rerio) embryos ..................................................................................................................................................................211

Viriato, Cristina; Andrade, Ítalo B.L.; Peixoto, Paloma V.L.; Pereira, Lílian C.

The relationship between oxidative system and sperm motility in wistar rats continuously exposed to organochlorine DDT residues ................................................................................................212

Silva, Fernanda Coleraus; Quiozini, Nathaly de Matos; Simon, Jaqueline; Schwengber, Heloysa Talia; Lesniewski, Isabela Ramos; Azevedo, Camilla de Marchi Sanches; Itinose, Ana Maria; Marek, Carla Brugin; Guimarães, Ana Tereza Bittencourt

The white blood cells behavior in multigenerations of wistar rats exposed to DDT organochlorine residues ............................................................................................................................................................213

Silva, Fernanda Coleraus; Quiozini, Nathaly de Matos; Simon, Jaqueline; Azevedo, Camilla de Marchi Sanches; Vieira, Bruna Todeschini; Marek, Carla Brugin; Guimarães, Ana Tereza Bittencourt

Toxicological effects of Color Index Disperse Red 1 textile azo dye in sperm of mice orally exposed .........214Tanamachi, Amanda Rodrigues; Fernandes, Fábio Henrique; Lima, Geovana Cristina Ribeiro; Mariani, Noemia Aparecida Partelli; Silva, Alan Andrew dos Santos; Silva, Erick José Ramo; Salvadori, Daisy Maria Fávero

Toxicological safety studies of plant extract of Eugenia uniflora L. at different safety doses .......................215Donadel, Guilherme; Dalmagro, Mariana; Pinc, Mariana Moraes; Berta, João Antonio; Borba, Maria Eduarda; Gasparotto Junior, Arquimedes; Zardeto, Giuliana; Hoscheid, Jaqueline; Lourenço, Emerson Luiz Botelho

Transgenerational reproductive effects (F2) in male offspring exposed to benzo(a)pyrene by paternal germ cells in rats .................................................................................................................................................216

Jorge, Bárbara Campos; Reis, Ana Carolina Casali; Stein, Julia; Paschoalini, Beatriz Rizzo; Nogueira, Jéssica Bueno; Moreira, Suyane da Silva; Manoel, Beatriz de Matos; Arena, Arielle Cristina

Triclopyr induces changes in mitochondrial parameters .............................................................................................. 217Rizzi, Joyce Santana; Seloto, Danielle Gabriel; Pereira, Lílian Cristina

Use of zebrafish as animal model for research for neurotoxicological substances: a literature review ........218Silva, Luíza Madureira; Pinheiro, Dávylla Rennia Saldanha; Braga, Ádrya Lariela Lima; Holanda, Wiron Pimentel; Honório Júnior, José Eduardo Ribeiro

Whole-mount skeletal staining in rabbits fetuses for teratogenicity evaluation (OECD 414) ........................219Matsui, Andresa;Alves, Paula Daniela Sabino de Freitas; Santana, Thatiane Nunes; Vecina, Juliana Falcato; Bechtold, Bruna Assunção; Fava, Luis Paulo

07 Food Safety ............................................................................................................................................................ 220

Aluminum levels in fruits and in fruit and vegetable juices .........................................................................................221Pinelli, Juliana Junqueira; Custódio, Flávia Beatriz

Analysis of physical chemical parameters and cyanogen residues in macaxeira flour (Manihost esculenta Crantz), produced in a municipality of Paraense .................................................................. 222

Araújo, Maria Eduarda Lima; Estumano, Saulo Braga; Oliveira, Sidney Julio Vieira; Santiago, Mayla Andra de Andrade; Costa, Eliene dos Santos da Silva; Oliveira, Cláudia Simone Baltazar

Assessment of the incidence and concentration of polycyclic aromatic hydrocarbons in cocoa-derived products ............................................................................................................................................................ 223

Silva Júnior, Clovis Reis; Almeida, Gabriela de Oliveira; Moretti, Gabriela; Jager, Alessandra Vincenzi

Caffeine content in pre-workout supplements and energy drinks marketed in Brazil: are the actual levels safe and in accordance with the labels? .........................................................................................224

Costa, Bruno Ruiz Brandão; El Haddad, Lohanna Pereira; Freitas, Bruno Toledo; Marinho, Pablo Alves; De Martinis, Bruno Spinosa

Cytotoxic activity of green banana (Musa sinensis) flour by Allium cepa test .................................................... 225Tadeu, Vitória Costa; Roehrs, Rafael; Farias, Fabiane Moreira; Kieling, Ketelin Monique Cavalheiro; Nogueira, Caroline Lacerda; Denardin, Elton Luis Gasparotto

Design of Experiments as a tool for HS-SPME-GC-MS method development applied to qualitative analysis of volatile alcohol congeners in seized whiskeys ................................................................... 226

Bigão, Vítor Luiz Caleffo Piva; Costa, Bruno Ruiz Brandão; Gomes, Nayna Cândida; Marinho, Pablo Alves; Silva, Jonas Joaquim Mangabeira; De Martinis, Bruno Spinosa

Development of a new quantitative field-testing assay for the analysis of histamine in seafood samples ......................................................................................................................................................................... 227

Cabrices, Oscar G.

Food Safety: regulatory and toxicological aspects of food packaging ....................................................................228Almeida, Giulia Forni; Pinheiro, Fabriciano

Ozone processing of peanut “milk”: degradation of aflatoxins, reduction of allergenic proteins and impact on lipid oxidation ................................................................................................................................ 229

Romero, Alessandra de Cássia; Sartori, Alan Giovanini de Oliveira; Caetano-Silva, Maria Elisa; Alencar, Severino Matias; Calori-Domingues, Maria Antonia; Augusto, Pedro Esteves Duarte

Using R language to study worldwide Lead contamination distribution on food using the GEMSFOODS(WHO) database during 1995-2020 ............................................................................................................230

Silva, Fabiano; Couto, Nilton; Almeida, Mariana

08 Forensic ...................................................................................................................................................................231

An overview of NPS in northeast Brazil: NMR-based identification and analysis of ecstasy tablets by GC-MS ......................................................................................................................................................................... 232

Cunha, Ricardo Leal; Oliveira, Celinalva da Silva Lima; Oliveira, Aline Lima; Maldaner, Adriano Otávio; Pereira, Pedro Afonso de Paula

Analysis of diglycolic acid in victims intoxicated by the consumption of beer containing diethylene glycol .......................................................................................................................................................................... 233

Goulart, Cristiano O.L.; Bordoni, Leonardo S.; Nascentes, Clésia C.; Costa, Letícia M.

Determination of phosphatidyl ethanol in dried blood spots by LC-MS/MS ........................................................234Guterres, Fernanda S.; Tegner, Mariane; Ott, Isabela Ritter; Linden, Rafael; Antunes, Marina Vezon

First Report of national database on toxicological criminal information (ToxCrim system) .......................... 235Costa, Rony Anderson Rezende; Costa, Jose Luiz

Forensic entomotoxicology: application of studies in the development and rearing of Lucilia cuprina flies (Diptera: Calliphoridae)...................................................................................................................... 236

Chimendes, Nayomi de Andrade; Bairros, André Valle; Ugalde, Gustavo Andrade; Santos, Lara Celestina; Pacheco, André Lucas Bezerra; Reginato, Fernanda Ziegler; Berlato, Dener Gomes; Rosa, Victória Gomes; Nascimento, Marcelo Henrique Santana; Pereira, Alessandra de Oliveira; Monteiro, Silvia Gonzalez

Interference of larvae matrix of flies Lucilia cuprina in the determination of Azamethiphós and Paraoxon ethyl using dispersive solid phase extraction (dSPE) ....................................................................... 237

Pacheco, André L.B.; Ugalde, Gustavo A.; Chimendes, Nayomi A.; Santos, Lara C.; Berlato, Dener G.; Nascimento, Marcelo H.S.; Reginato, Fernanda Z.; Rosa, Victória Gomes; Santos, Rachel; Cardoso, Leonardo Correa; Monteiro, Silvia Gonzalez; Stainki, Daniel Roulim; Pereira, Alessandra de Oliveira; Bairros, André V.

Nortriptyline overdose and quantitative analysis in post mortem vitreous humor – a case study ............238Cunha, Ricardo Leal; Dalbosco, Juliana Santos; Brito, Maria Carolina Santos Ribeiro; Costa, José Luiz

Optimization of extraction procedure for benzodiazepines and antidepressants analysis in vitreous humor using cork sheet ...................................................................................................................................... 239

Ossanes, Daniela Souza; Birk, Letícia; Eller, Sarah; Oliveira, Tiago Franco

Prevalence of volatile organic compounds in forensic toxicology reports of São Paulo State......................240Rodrigues, Taís B.; Medeiros, Elvis A.; Chinaglia, Kauê O.; Gianvecchio, Victor A.P.; Costa, Jose Luiz

Profile of synthetic drugs seized between January 2019 and December 2021 analyzed by the Forensic Toxicology Center of the Forensic Expertise in the State of Ceará (PEFOCE) ..............................241

Oliveira, Juliana Ribeiro Ibiapina Leitão; Magalhães, Danielle de Paula; Martins, Mayane Emanuella Melo Lopes; Almeida, Vivian Romero Santiago; Saboya, Andrea Luiza Rocha; Holanda Júnior, Wanderley Pinheiro

Profile of toxicological analysis of suicide cases reported by the Santa Catarina Scientific Police in the last 5 years ...........................................................................................................................................................242

Silva, Bruna Espíndola; Boff, Bruna de Souza; Silveira Filho, Jair; Schroeder, Samilla Driessen; Rezin, Kéttulin Zomer; Marchioni, Camila

Profile of victims of traffic accidents with blood samples collected for alcohol tests and analyzed by the Forensic Expertise of the State of Ceará (PEFOCE) from January 2020 to December 2021 .............................................................................................................................................................................243

Magalhaes, Danielle de Paula; Holanda Júnior, Wanderley Pinheiro; Saboya, Andréa Luiza Rocha; Oliveira, Juliana Ribeiro Ibiapina Leitão; Martins, Mayane Emanuela Melo Lopes; Santiago, Vivian Romero

Quantifying ethanol content in alcoholic beverages by HS-GC/FID .........................................................................244Bigão, Vítor Luiz Caleffo Piva; Costa, Bruno Ruiz Brandão; Santos Júnior, Wilson José Ramos; De Martinis, Bruno Spinosa

Selective determination of 4-aminopyrine in seized cocaine samples by a reduced graphene oxide/Prussian blue nanocomposite...............................................................................................................245

Reis, Karsonn B.; Borges, Pedro H.S.; Rocha, Raquel G.; Ramos, David L.O.; Muñoz, Rodrigo A.A.; Richter, Eduardo M.; Nossol, Edson

Sociodemographic profile of authors involved in illicit drug arrest in the city of Betim, Minas Gerais ......246Costa, Anna Carolina de Moura; Oliveira, Lara Luiza Freitas; Sales, Thais Lorenna Souza; Sanches, Cristina; Chequer, Farah Maria Drumond

Survey of drugs/adulterants found concomitantly with cocaine, in victims of violent death, in the state of Minas Gerais, in the year 2021 ...................................................................................................... 247

Corrêa, Brunna F.; Bordoni, Leonardo S.; Couto, Tauer J.G.; Goulart , Christiane G.L.; Goulart, Cristiano O.L.

The increase in the number of incarcerations in Brazil involving drug crimes and the main drugs seized over ten years (2009-2019) ..........................................................................................................................248

Bonfioli, Maysa Guilherme; Coelho, Júlia França Figueredo; Melo, Saulo Nascimento; Belo, Vinícius Silva; Chequer, Farah Maria Drumond

The mystery behind the apprehensions of the selective agonist to cannabinoid type 2 receptor BZO-HEXOXIZID (MDA-19) as a drug of abuse ................................................................................................249

Araujo, Karen Rafaela Gonçalves; Fabris, André Luis; Neves Júnior, Luiz F.; Ponce, Júlio de Carvalho; Costa, José Luiz; Yonamine, Mauricio

Toxicological findings in cases attended by Division of Postmortem Inspection of Porto Alegre in 2021 ..... 250Birk, Letícia; Barbosa, Fábio de Souza; Petry, Adriana Ubirajara Silva; Menezes, Francisco Paz; Gonzaga, Alexsandro Pinto; Eller, Sarah; Oliveira, Tiago Franco

Validation of methods for the determination of cocaine, benzoylecgonine, and cocaethylene in dried blood using the hemapen microsampling device ................................................................251

Smidt, Mariana; Bastiani, Marcos Frank; Hahn, Roberta Zilles; Linden, Rafael

09  Genotoxicity and Carcinogenesis .................................................................................................................... 252

Cellular spheroids as a platform for toxicogenomic assays in cancer.................................................................... 253Queijo, Rodrigo Gonçalves; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya Stuchi

Collaborative analysis of complex nitrosamines ............................................................................................................254Avila, Carolina Martins; Ponting, David. J.; Werner, Anne-Laure D.; Tennant, Rachael E.; Oliveira, Antonio Anax F.; Heghes, Crina

Copper (II) complex as a potential treatment to overcome BRAF inhibitor resistance and aggressive NRAS point-mutation .......................................................................................................................................... 255

Moraes Junior, Manoel Oliveira; Carvalho, Larissa Anastácio da Costa; Nunes, Cleia Justino; Ferreira, Ana Maria da Costa; Maria-Engler, Silvya Stuchi

Evaluation of photomutagenic potential of UV filters and IR3535 combination ................................................. 256Fuzinaga, Thaís Y.T.; Gluzezak, Ana J.P.; Tavares, Renata S.N.; Kawakami, Camila M.; Abe, Flávia R.; Oliveira, Danielle P.; Maria-Engler, Silvya S.; Gaspar, Lorena R.

Gliotoxin and its potential cytotoxicity against NRAS-mutated melanoma ......................................................... 257Noma, Isabella Harumi Yonehara; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya Stuchi

Strategies for overcoming toxicity and resistance in melanoma progression and in adaptative resistance to braf inhibitors using ADK modulation ................................................................................258

Silva, Julia Rezende; Oliveira, Érica Aparecida; Carvalho, Larissa Anastacio da Costa; Maria-Engler, Silvya Stuchi

The peroxiredoxin mimetic gliotoxin overcomes heterogeneity, intrinsic and acquired resistance to BRAF inhibitor in metastatic melanoma .................................................................................................. 259

Carvalho, Larissa A.C.; Noma, Isabella H.Y.; Uhera, Adriana H.; Siena, Ádamo D.D.; Harumi, Luciana; Mori, Matheus P.; Goding, Colin; Pinto, Nadja C. Souza; Freitas, Vanessa M.; Silva-Jr, Wilson A.; Smalley, Keiran S.M.; Maria-Engler, Silvya S.

10 Immunotoxicology................................................................................................................................................ 260

Analyzes of clove essential oil immunotoxicity based on in vitro and in silico studies .....................................261Etcheverry, Bibiana Frasson; Gomes, Gabriela Cristiane Mendes; Rios, Nathália Vieira; Chaves, Pamella Eduardha Espindola; Sotelo, Êmily Clori; Zuravski, Luísa; Machado, Michel Mansur

Cigarette smoke and heat-not-burn tobacco vapor exposures alter granulopoiesis and neutrophil functions during experimental arthritis ........................................................................................................ 262

Scharf, Pablo; Heluany, Cíntia; Sandri, Silvana; Schneider, Ayda Henriques; Barbim, Paula Donate; Fock, Ricardo; Cunha, Fernando; Farsky, Sandra

Cytotoxicity of Marinobufagenin in Glial Cells Chalenged Whith LPS ...................................................................... 263Farias, Evelyn Rayani Araújo; Silva, Rivia Regina Lopes; Souza, Natacha Medeiros; Marques, Ana Maria; Scavone, Cristoforo; Quintas, Luís E.M.; Leite, Jacqueline Alves

In vitro and in silico analysis of the cytotoxicity of Foeniculum vulgare essential oil in human lymphocytes ...................................................................................................................................................................264

Gomes, Gabriela Cristiane Mendes; Etcheverry, Bibiana Frasson; Chaves, Pamella Eduardha Espindola; Rios, Nathalia Vieira; Campos, Dyene Nascimento; Zuravski, Luísa; Machado, Michel Mansur

Lymphotoxicity of Brazil mint essential oil: an in vitro and in silico study ............................................................. 265Chaves, Pamella Eduardha Espindola; Rios, Nathália Vieira; Gomes, Gabriela Cristiane Mendes; Etcheverry, Bibiana Frasson; Campos, Dyene Nascimento; Zuravski, Luísa; Machado, Michel Mansur

Mitochondria as a target for imidacloprid toxicity on RAW 264.7 cells .................................................................266Cestonaro, Larissa V.; Schmitz, Felipe; Ferreira, Fernanda S.; Conte, Fernanda M.; Piton, Yasmin V.; Wyse, Angela T.S.; Garcia, Solange C.; Arbo, Marcelo D.

New pyrazoline compounds: effects on neutrophils and macrophages functions ........................................... 267Goldoni, Fernanda C.; Benvenutti, Larissa; Vaz, Milena Menegazzo; Carlos Rafael; Buzzi, Fátima C.; Quintão, Nara L.M.; Santin, José Roberto

Patchouli (Pogostemon cablin) essential oil: safe or a hidden risk? ......................................................................268Rios, Nathália Vieira; Chaves, Pamella Eduardha Espindola; Gomes, Gabriela Cristiane Mendes; Etcheverry, Bibiana Frasson; Sotelo, Êmily Clori; Zuravski, Luísa; Machado, Michel Mansur

Trilobolide-6-O-isobutyrate shows in silico and in vitro potential for antitumor activity of A549 and NCI-H460 lung cancer cell lines .........................................................................................................................269

Miranda-Sapla, Milena Menegazzo; Concato, Virginia Marcia; Gonçalves, Manoela Daiele; Matos, Ricardo Luiz Nascimento; Conchon-Costa, Ivete; Arakawa, Nilton Syogo; Pavanelli, Wander Rogério

11 Nanotoxicology ...................................................................................................................................................... 270

Cytotoxicity and effectiveness in macrophage polarization of Annexin A1-surface-functionalized metal-complex multi-wall lipid core nanocapsule ........................................................................... 271

Broering, Milena F.; Leão, Matheus C.; Scharf, Pablo; Sandri, Silvana; Uchiyama, Mayara K.; Araki, Koiti; Guterres, Silvia S.; Pohlmann, Adriana R.; Farsky, Sandra H.P.

Preclinical evaluation of short-term gold nanoparticles toxicity ............................................................................. 272Plautz, Katherine; Gastaldi, Alessandra Betina; Maia, Thayná Patachini; Pereira, Eduardo Manoel; Ferreira, Gabriela Kozuchovski; Borgmann, Gabriela; Cabral, Heloisi; Delwing-Dal Magro, Débora; Delwing-De Lima, Daniela

Predictive analysis of ocular and lung damage in cells exposed by zinc oxide or cerium dioxide nanoparticles using electrical impedance compared to MTT test ............................................................ 273

Alexandre, Angela de Oliveira; Souza, Wanderson; Dal-Cheri, Beatriz Kopke de Assis; Granjeiro, Jose Mauro; Pereira, Leonardo da Cunha Boldrini

Study of the acute and chronic toxicity of chrome III oxide nanoparticles on the marine microcrustacean Mysidopsis juniae (Silva, 1979) ............................................................................................................ 274

Plautz, Katherine; Fugazza, Jonas; Gastaldi, Alessandra Betina; Kleine, Tamila; Matias, William Gerson; Oliveira, Therezinha Maria Novais

Study of the interaction between a potential labeled nano-enabled pesticide and aquatic plant .............. 275Forini, Mariana M.L.; Antunes, Débora R.; Cavalcante, Luiz A.F.; Pontes, Montcharles S.; Santiago, Etenaldo F.; Sanches, Alex O.; Martins, Aline R.; Grillo, Renato

Survival rate of Drosophila melanogaster exposed to nanoparticles containing Naringenin and in vitro determination of inhibition kinetics against cholinergic enzymes ............................ 276

Cunha, Viviane Augusta de Medeiros Garcia; Rossi, Bruna Franzon; Leimann, Fernanda Vitória; Ineu, Rafael Porto; Foleis, Vanessa Kaplum; Gonçalves, Odinei Hess

Survival rate of Drosophila melanogaster exposed to Silymarin-loaded nanoparticles and the ex vivo inhibition of cholinergic enzymes ....................................................................................................................277

Souza, Daniela Cristina; Rossi, Bruna Franzon; Leimann, Fernanda Vitória; Gonçalves, Odinei Hess; Appelt, Patrícia; Ineu, Rafael Porto

12 Risk Assessment ................................................................................................................................................... 278

Animal-Free Safety Assessment of Cosmetics: a global education and training program ............................. 279Marigliani, Bianca; Willett, Catherine

Biomonitoring of occupational exposure to triazoles and the disruptive effects of steroidogenesis on androstenedione biosynthesis in humans .................................................................................280

Marciano, Luiz Paulo de Aguiar; Costa, Luiz Filipe; Freire, Josiane Oliveira; Feltrim, Fernando; Machado, Simone Caetani; Silvério, Alessandra Cristina Pupin; Martins, Isarita

Buccal micronucleus cytome assay as a biomarker of genotoxic effect rural workers exposed to triazole fungicides.................................................................................................................................................281

Costa, Luiz Filipe; Marciano, Luiz Paulo de Aguiar; Freire, Josiane Oliveira; Feltrim, Fernando; Silvério, Alessandra Cristina Pupin; Martins, Isarita

Changes in biomarkers of exposure and potential harms in adult smokers following 180 days of gloTM tobacco heating product use ........................................................................................................................282

Esteves, Iuri; Zebele, Patricia; Gale, Nathan; Hardie, George; McEwan, Michael; Gaca, Marianna; Goodall, Sharon

COVID-19 and susceptibility of firefighters exposed to smoke: A scope review .................................................283Silva, Rafael Araújo; Marciano, Luiz Paulo de Aguiar;Costa, Luiz Filipe; Nunes, Rafaella Ferreira Nascimento; Sakakibara, Isarita Martins

Determination of urinary triazoles as reliable biological indicator for the application in coffee growers ....284Marciano, Luiz Paulo de Aguiar; Costa, Luiz Filipe; Freire, Josiane Oliveira; Feltrim, Fernando; Machado, Simone Caetani; Silvério, Alessandra Cristina Pupin; Martins, Isarita

Dispersive liquid-liquid microextraction to determinate parabens in body cream by liquid chromatography ..........................................................................................................................................................................285

Ramos, Thalita da Silva; Barbosa, Alyne Maria da Costa; Rath, Susanne;Martins, Isarita

Evaluating the cytotoxicity of quantum dots using two different keratonocyte-cell based systems ........286Souza, Isisdoris Rodrigues; Cruz, Juliana Varella; Thá, Emanoela Lundgren; Gagosian, Viviana Stephanie Costa; Araujo-Souza, Patrícia Savio; Cestari, Marta Margarete; Faustman, Elaine M.; Leme, Daniela Morais

Exposure driven data generation strategies for dietary and non-dietary risk evaluation of crop protection products to inform safety and minimise unnecessary animal testing ................................... 287

Catalano, Shadia M.I.; Pais, Mariana Castello Novo; Latorre, Andreia Oliveira; Faria, Patricia Miranda; Soares, Daniel; Freeman, Elaine

GHS classification of chemicals frequently used in laboratories: A risk assessment .......................................288Silvério, Kérolyn Aparecida; Pinheiro, Fabriciano

How can nitrosamine risk assessments be supported by in silico systems and data sharing initiatives? ......................................................................................................................................................................289

Waechter, Fernanda; Kocks, Grace; Avila, Carolina Martins; Burns, Michael J.; Ponting, David J.; Tennant, Rachael E.; Oliveira, Antonio Anax F.; Heghes, Crina

Human health risks assessment by iron mining dam failure (VALE S.A) in Brumadinho-MG ........................290Domingos, Líllian Maria Borges; Castilhos, Zuleica Carmen

Occurrence and dietary exposure to histamine by the Brazilian population .........................................................291Diniz, Fabiana Barbosa; Braga, Douglas Evangelista; Custódio, Flávia Beatriz; Gloria, Maria Beatriz Abreu

Occurrence and exposure to aspartame from soft drinks by the Brazilian population ................................... 292Sousa, Roberto Cesar Santos; Custódio, Flávia Beatriz; Gloria, Maria Beatriz A.

Permitted Daily Exposure (PDE) from preclinical studies of ginkgo biloba dry extract ................................... 293Lamb, Liliane Weber Bolfe; Rodrigues, Gabriela Zimmermann Prado; Saraiva, Thalia Emmanoella Sebulsqui; Souza, Douglas; Berna, Gabriel da Costa; Garcia, Ana Letícia Hilário; Bertoldi, Fernando; Kayser, Juliana Machado; Ferreira, Julia Gabriele de Jesus, Veiverberg, Andriele; Marco, Mariana; Gehlen, Günter; Betti, Andresa Heemann;Mattos, Cristiane Bastos

Thyroid and sex hormonal profile and manganese exposure in pregnant women from a Brazilian birth cohort: preliminary results .........................................................................................................................294

Santos, Nathália Ribeiro; Bah, Homegnon Antonin Ferréol; Gomes-Júnior, Erival Amorim; Martinez, Victor Otero; Costa, Daisy Oliveira; Menezes-Filho, José Antonio

Total and inorganic arsenic in fish and exposure to inorganic arsenic by fish consumption in Brazil ........ 295Ramos, Vitor Serrão; Vasconcelos Neto, Milton Cabral; Custódio, Flávia Beatriz

13 Toxicologic Hazard in the Workplace .............................................................................................................. 296

Assessment of serum levels of DDT and metabolites in workers in malaria control campaigns in the state of Pará, Amazon, Brazil............................................................................................................... 297

Rocha, Thiago L.; Rocha, Cássia C.S.; Miranda, Antônio M.M.; Mendes, Rosivaldo de A.

Case Studies: challenges on implementing the GHS classification of pesticides in the reevaluation of active substances ........................................................................................................................................298

Aguiar, Larissa Muratori; Braz, Juliana Machado; Moreira, Camila Queiroz; Santos, Thiago Santana; Freitas, Daniel Roberto Coradi

Clinical and laboratorial evidenced organic damage due to the incorrect use of PPE in rural producers of tomato and onion crops in the contestado region - Santa Catarina ..................................299

Boff, Everton; Kampmann, Micheli Gabardo; Benvenutti, Régis Carlos

Evaluation of proteins and genes expression in workers exposed to crystalline silica in Southern Brazil .............................................................................................................................................................................300

Peruzzi, Caroline Portela; Göethel, Gabriela; Flesch, Ingrid; Nascimento, Sabrina; Nardi, Jessica; Arbo, Marcelo Dutra; Garcia, Solange

Evaluation of residual contamination in anticancer drug packaging using UHPLC-MS/MS ...........................301Silva, Luciana Stein; Silva, Laura Cé; Machado, Cibele da Silva Barbosa; Capp, Edison; Linden, Rafael; Ness, Sandro Luis Ribeiro; Antunes, Marina Vezon

Green tobacco sickness occurrence and evaluation of life quality among tobacco workers in Paraná countryside ................................................................................................................................................................302

Bortoli, Stella; Schamne, Tatiane; Kalv, Danielle Crystiane; Pedroso, Bruno; Vellosa, Jose Carlos Rebuglio

Pesticide poisoning in banana cultivation in Corupá city, Santa Catarina .............................................................303Borgmann, Gabriela; Plautz, Katherine; Zimmath, Michel, Tenfen, Adrielli

Skin exposure to KrCl excimer lamp emitting at 222 nm cause tissue and cellular alterations of concern................................................................................................................................................................304

Tavares, Renata Spagolla Napoleão; Girassole, Alessandra; Adamoski, Douglas; Caznok, Ana Clara; Domingues, Romênia; Leme, Adriana Paes; Carvalho, Murilo; Dias, Sandra Martha Gomes

The influence of occupational exposure to cyanide on metahemoglobinemia in cassava flour producers ............................................................................................................................................................................. 305

Estumano, Saulo Braga; Oliveira, Cláudia Simone Baltazar; Araújo, Maria Eduarda Lima; Costa, Eliene dos Santos da Silva; Chisté, Renan Campos; Lameira, Christian Neri; Amaro, Beatriz Oliveira; Pinheiro, Maria da Conceição Nascimento

Toxic metal backgrounds among waste pickers in hair sampling: a cross-sectional study in Brazil (Federal District) .........................................................................................................................................................306

Gonçalves, Michelly Rodrigues; Verpaele, Steven; Marques, Carla Pintas; Bashash, Morteza; Cruvinel, Vanessa Resende Nogueira; Santos, Vivian da Silva

14 Other ........................................................................................................................................................................ 307

Attempted suicide: temporal series and agents involved before and during the COVID-19 pandemic in Brazil .......................................................................................................................................................................308

Holanda Júnior, Wanderley Pinheiro; Magalhães, Danielle de Paula; Oliveira, Juliana Ribeiro Ibiapina Leitão

Brazil now prioritizes animal-free safety assessment of school supplies ...........................................................309Marigliani, Bianca

Early exposure to toxic metals and child neurodevelopment: proposal of a theoretical model based on Bronfenbrenner’s bioecological theory ...............................................................................................310

Bah, Homègnon Antonin Ferréol; Santos, Nathália Ribeiro; Costa, Daisy Oliveira; Carvalho, Chrissie Ferreira; Martínez, Victor Otero; Gomes-Júnior, Erival Amorim; Menezes-Filho, José Antonio

Junkie – Teaching project in comic book for the development of learning in toxicology ....................................311Bairros, André Valle; Reginato, Fernanda Ziegler; Ugalde, Gustavo Andrade; Berlato, Dener Gomes; Pacheco, André Lucas Bezerra; Nascimento, Marcelo Henrique Santana; Oliveira, Letícia Cimó; Santos, Lara Celestina; Chimendes, Nayomi de Andrade; Rosa, Victória Gomes; Dalmazzo, André Kusser; Maia, Affonso Enriques Montagner

The use of paracetamol as cause for male infertility .....................................................................................................312Silva, Luíza Madureira; Holanda, Wiron Pimentel; Capelo, Melissa Figueiredo; Honório Júnior, José Eduardo Ribeiro

Index of Authors .............................................................................................................................................................313

01 ALTERNATIVE ANIMAL MODELS & EMERGING

IN VITRO MODELS

25

Obesity is characterized by the excessive accumulation of white adipose tissue resulting from the imbalance between energy consumption and expenditure. It is considered a complex, multifactorial and heterogeneous disease with genetic, environmental, behavioural and socioeconomic origins. Worldwide, obesity has become a main public health issue responsible for 2.8 million deaths per year, according to the World Health Organization (WHO). Besides, obesity increases considerably the risk for type 2 diabetes, cardiovascular diseases and some types of cancer. The involvement of the adipose tissue, considered a complex endocrine organ, raises the importance of a better understanding of its metabolic state at a cellular level is essential. Thus, three-dimensional (3D) in vitro models could represent a new alternative to the classic models and provide new insights on the study of obesity and related diseases. Therefore, this study aimed to develop a 3D co-culture model using three different cell types that are constituents of the native adipose tissue. The cell lines used were 3T3-L1 (murine pre-adipocyte), NIH-3T3 (murine embryo fibroblasts) and J774 (murine macrophages). The 3D model was obtained through a non-adhesive 96 well plate coated with agarose 2% seeded with 1.2 × 104 cells/well (1:1:1). After 4 days of culture in standard conditions, the spheroids were submitted to adipogenic differentiation medium

(MDI), containing DMEM supplemented with 10% of fetal bovine serum (FBS), 1 µM dexamethasone, 0.5 mM IBMX, and 1.67µM insulin for 72h. After that, 50% of the supernatant was replaced by DMEM supplemented with 10% FBS and 1.67µM insulin changed every 48h until 11 days of culture. The control medium was prepared by supplementing DMEM with 10% calf serum (CS). Finally, the spheroids were stained with Nile Red, a selective fluorescent stain for intracellular lipid droplets. Spheroids submitted to the MDI medium were compared with spheroids formed in the control medium. According to the results obtained by trypan blue assay, the cell viability for both types of spheroids, formed in MDI or control medium, was around 60%. Spheroid diameter and morphology were analyzed by photomicroscopy and scanning electron microscopy (SEM), respectively. The differentiation of pre-adipocytes into adipocytes was analyzed by flow cytometry and confocal microscopy. Interestingly, the results of flow cytometry and confocal microscopy showed that spheroids formed in the control medium also presented adipogenic differentiation. In conclusion, it was possible to develop a 3D co-culture spheroid model of adipose tissue that can be used to study new metabolic pathways and new therapeutic targets, as well, toxicity effects of new drugs for the therapy of obesity and obesity-related diseases.

Adipose Tissue 3D: new tools in cell culture

Buchele, Maria luiza caneiro1; Mora, TaMara Dal1; Saleh, najla aDel1; Silva, aDny henrique1; MonTeiro, FaBíola Branco Filippin1,2

1 Programa de Pós Graduação em Farmácia, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil; 2 Departamento de Análises Clínicas, Centro de

Ciências da Saúde, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil.

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Background: Melasma is known as a hypermelanosis caused by a dysfunction in the melanogenesis process. The main factors that stimulate this dysfunction is exposure to the sun, causing an increase in the production of melanin in melanocytes, which will be deposited in keratinocytes. There are several compounds used to treat melasma. Among them are kojic and glycolic acids. Kojic acid is used as a depigmenting agent, causing a melanin reduction. Also, kojic acid has antioxidant potential and is compatible with non-ionic bases, so it can be associated with glycolic acid, which results in an exfoliative and antioxidant effect. Although these compounds are already used in the treatment of melasma in isolation, it is of scientific relevance to analyze the cytotoxicity of these compounds, as well as their anti-melanogenic potential, isolated and combined. Objective: To evaluate the cytotoxicity and anti-melanogenic effect of kojic and glycolic acid, isolated or combined in SK-MEL-28 and B16F10 cells. Methods: The research was carried out with melanoma cells (SK-MEL-28 and B16F10). The cytotoxicity of the compounds kojic acid and glycolic acid in the two lines was investigated by the MTT assay. This assay was carried out in a 48-well plate, where cells were seeded (2.5x104 cells/well). In the SK-MEL-28 cell, the compounds were tested isolated or combined at concentrations of 0, 1, 10, 30, 100, 300 and 1000 µM and in B16F10 only at a concentration of 1000 µM. The exposure period was 48h. To analyze the anti-melanogenic activity, the melanin content of the B16F10 cell was measured, in the presence or absence of the alpha-MSH hormone

(200 nM) and the compounds kojic acid and glycolic acid (1000 µM) isolated and combined. For this, the cells were cultivated in a 24-well plate, with a concentration of 4.5x104 cells/well. The exposure period was 48h. The results were analyzed by one-way ANOVA followed by Tukey’s pos hoc. The results were considered significant when p<0.05. The program used was GraphPad Prisma 6. Results: One-way ANOVA revealed no effect of kojic acid and glycolic acid, isolated and combined, on cell viability. In the presence of the compounds, in the SK-MEL-28 lineage, there was no reduction in cell viability at any of the concentrations tested. From these results, a concentration of 1000 µM was selected to assess the melanin content and cell viability in the presence of acids, isolated or combined, in the cells. It was observed that the compounds did not show cytotoxicity to the B16F10 cells. By obtaining the results of melanin content, one-way ANOVA revealed that both kojic acid and glycolic acid, in the presence or absence of alpha-MSH hormone, decreased the melanin content. Conclusion: It is concluded that the acids, isolated or combined, did not cause cytotoxic effect, in the concentrations and cells tested. Also, regarding the anti-melanogenic activity, the acids at 1000 µM concentration, in combined, obtained a more significant reduction in the melanin content when compared to them isolated. Thus, kojic acid and glycolic acid can be used in future experiments at any concentration tested in this work. Further test will be carried out to clarify the effects that the compounds have on their anti-melanogenic activity.

Analysis of cytotoxicity and anti-melanogenic activity of kojic acid and glycolic acid

in SK-MEL-28 and B16F10 cells

riBeiro, Milena Mariano1; Silva, ana cléia carDoSo2; irioDa, ana carolina2; oliveira, cláuDia Sirlene2

1 Curso de Farmácia, Faculdades Pequeno Príncipe, Curitiba, Paraná, Brazil; 2 Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, Paraná, Brazil.

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Introduction: The progress of in vitro skin models relies on increasing its human relevance and reliability as platforms to evaluate the safety of consumable products. 3D bioprinting allows the precise spatial control in the deposition of biological materials to achieve more complex and accurate skin mimetic models. To evaluate skin models for safety assessment, the Organization for Economic Co-operation and Development (OECD) guideline 439 (TG 439) provides in vitro hazard identification of irritant chemicals using validated human reconstructed epidermis (RHE) models as an alternative method for the Draize test. This guideline also describes detailed quality control and performance standards required for the validation of similar RHE models for skin irritation testing. Objective: This study aimed to compare the performance of a bioprinted human epidermis (B-RHE) to a manually reconstructed human epidermis (M-RHE) at the in vitro skin irritation test described in the OECD TG 439. Methods: To reconstruct the RHEs primary isolated human epidermal keratinocytes were seeded in transwell inserts previously coated with collagen using manual techniques or a BioEnder 3 extrusion bioprinter (BioEdTech, Brazil). The models were evaluated following OECD guidelines TG 439. Tissue morphology was verified by histology and immunofluorescence. The quality of the barrier function was determined by the inhibitory concentration that reduces viability by 50% (IC50) after 18h of exposure to SDS using the MTT method. Skin irritation testing was assessed by the exposure of the tissues to reference chemicals (3 irritants and 3 non-irritants) using the MTT viability assay. Results: RHEs developed using manual or bioprinted methodologies exhibited high quality morphology consistent with the quality control described by the OECD TG 439. Both models exhibited a well-differentiated and multiple layered epidermis

with a viable corneal layer. The epidermal barrier function performance of both models also achieved the performance standards as the validated RHE models described in TG 439, with no significative differences within the methodologies. After the achievement of quality control standards for morphology and barrier function, the RHEs performance for the in vitro irritation was evaluated. Both RHE models correctly discriminated the reference substances classified as irritant or non-irritant according to tissue viability threshold value (50%). No significative differences between the methodologies were observed, except for the higher viability of potassium hydroxide applied to bioprinted RHE. Discussion/Conclusion: 3D bioprinting is an emerging technology issued to achieve more reliable human mimetics tissues, but methodological comparison to the classical manual techniques is scarce in literature. The semi-permeable function of the corneal layer determines the pathological response against the exposure to irritant chemicals. Both epidermal models presented excellent morphology and barrier function. Therefore, the models were capable of distinguishing reference irritant chemicals from non-irritant chemicals when compared to the validated models described in TG 439. The similarity of the performance of the methodologies for the in vitro skin irritation test indicates that bioprinting can be of great contribution for the automation of reconstruction of epidermal models. Therefore, bioprinting can provide great assistance for cosmetic and pharmaceutical industries to increase the production of epidermal mimetics aiming to perform large scale in vitro trials for safety assessments of consumable topical products. Acknowledgments: This work was supported by FAPESP [grant number #2019/14527-7 and # 2017/04926-6] and CAPES [scholarship grant number #88887.363766/2019-00].

Bioprinted and manual human epidermis reconstruction: a compared

performance for irritation tests

BagaTin, julia De ToleDo1; caMarena, DeniSSe eSTher MallaupoMa1; oSaki, luciana haruMi2; FreiTaS, vaneSSa M.2; nolD, juliana c. lago3; Maria-engler, Silvya STuchi1

1 School of Pharmaceutical Sciences - University of São Paulo; 2 Biomedical Sciences Institute – University of São Paulo; 3 Natura & Co - São Paulo, Brazil.

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Recent technologies in three-dimensional (3D) cell culture have called much attention in the fields of cancer study and drug development due to the capacity of 3D cell culture mimicking aspects of in vivo microenvironments. 3D cell culture systems can provide cells with ideal structures that are much closer to tumors in vivo conditions in terms of the organization and formation of biological, histological and molecular microenvironment, when compared to standard 2D cultures. The latter offers them a homogeneous concentration of nutrients, growth factors and cytokines present in the culture media. This affects intracellular signaling and phenotypic fate, leading to unnatural interactions with soluble factors, in contrast to in vivo circumstances. Great efforts have been made to develop 3D cell culture models that have strong potential to overcome the deficiencies of 2D cell cultures. However, most of them are studies carried out at universities and research centers and are not applied in industries and companies that have screening tests routine for new drugs, for example, representing a big barrier and a challenge when it comes to changing the 2D cell culture to the 3D techniques in Brazil. The problems encountered in this routine change are due to the lack of availability of robust commercial 3D matrices, without interference, with an affordable price and defined protocols. In addition, training staff members to start using 3D tests in their screening routines is necessary, as there is no doubt that 3D culture faithfully mimics the in vivo environment, leading to much more robust data results than those found in 2D. Biocelltis became the first Brazilian company to produce and sell matrices for cell culture in 3D - the 3D CellFate® matrix. 3D CellFate® matrix is a hydrogel biomaterial made up by natural polymeric nanofibers

from 50 to 100 nm in diameter, similar to human collagen, biocompatible, sterile and ready-for-use, ideal for growing human and animal cells in research labs. This study aims to pre-validate an alternative cytotoxicity methodology using the 3D CellFate® Matrix, a candidate for replacement of traditional methods of cytotoxicity assays in a 2D environment. Cell concentration, seeding volume, adhesion time, culture time, exposure time, morphology and cell viability were evaluated parameters in some tumoral cell lines. The results obtained showed that cells in 3D CellFate® culture showed a completely different behavior and morphology when compared to 2D culture. In addition, these results corroborate with drug resistance presented by cells in 3D culture in relation to cytotoxicity found in 2D culture. Together the results herein obtained suggest that a protocol using 3D CellFate® Matrix is a great candidate for an alternative to 2D methods in vitro. The 3D CellFate® matrix proved to mimic the in vivo microenvironment, influencing cell survival, shape, migration, proliferation and differentiation in the presence of cytotoxic agents, thus leading cells to have its morphology and physiology similar to in vivo behavior. Besides that, we have developed a robust and easy - to - use protocol that can be used in high demands for in vitro assays, showing the potential use of a 3D matrix in industry. In this context, the use of 3D CellFate® matrix could avoid the over or underestimation of a specific drug in cases of drug sensitivity and resistance assays, as well as its dosage, leading to a reduction in the number of animals in subsequent phases of drug screening, making the process more exact and resources consuming. Keywords: 3dmodel, cellfate, cytotoxicityassay

Cellfate®Matrix: building the reality in three dimensions

reiS, eMily MarqueS; cavichion, raFael Filipe BaTTiSTi; colla, guilherMe; goDoi, Manuella MachaDo; koepp, janice

Biocelltis Biotecnologia S.A., Santa Catarina, Brazil.

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In 2019, normative resolution No. 18/2014 of the National Council for the Control of Animal Experimentation - CONCEA came into force in Brazil, which made the implementation of alternative methods to the use of animals mandatory, imposing on manufacturers of products that pose a risk to human health to cease the use of animals in their safety trials, especially corrosion and irritation tests, proposing as an alternative the use of in vitro reconstituted human skin (RHE). The majority of RHE models are produced by European and American countries, and its importation is not feasible due to the nature of the product. In fact, in Brazil, there is the commercialization of just one validated RHE model, where all the inputs for the construction of the model are imported, including the cells, making the physiology of the skin very different from the Brazilian one. Based on this, Biocelltis a national biomaterials industry, has been developing a commercial model of RHE, using its 3D CellFate® matrix and adapting it to the anatomical and physiological functions of Brazilian skin. Here, we present CellFate®RHE Model a solution for generating 3D epidermal skin models composed of normal human primary epidermal keratinocytes derived from skin. Numerous performance standards were evaluated, growth media and supplements, cell seeding density, passage number, as well as stratification conditions.

The results show that seeding neonatal keratinocytes in the 3D CellFate® matrix and cultured in chemically defined medium resulted in the consistent generation of 3D epidermal skin equivalents. The models produced using the optimized protocol exhibit physiologically relevant morphology, displaying a comparable number of cell layers and stratification to the human epidermis. The CellFate®RHE model developed presents a well-differentiated epidermis similar to Validated Reference Methods (VRM) and native human epidermis. Quality parameters, ie, negative control optical density, tissue integrity, and barrier function, were similar to VRM, making it ideal for evaluating preclinical and R&D effects of topical compounds on human skin. Biocelltis has developed a low-cost commercial model of reconstructed human epidermis that mimics the barrier function of the human stratum corneum as well as a viable complete epidermis. The model meets a national demand, overcoming of technical barriers and promoting the country’s technological autonomy in alternative methods to animal experimentation. Still, for the availability of the product to the national territory, intra- and inter-laboratory validation tests are undergoing to meet the OECD regulations. Keywords: reconstituted human epidermis in vitro, rhe, cellfate, 3Dmodel, invitromodel.

Cellfate®RHE: a brazilian commercial model of reconstituted human epidermis in vitro

reiS, eMily MarqueS; cavichion, raFael Filipe BaTTiSTi; colla, guilherMe; koepp, janice

Biocelltis Biotecnologia S.A., Santa Catarina, Brazil;

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Introduction: Dental pulp stem cells (DPSCs) are a promising source of cells to be applied in regenerative medicine. DPSCs are stem cells which can originate from the neural crest exhibiting neuro-ectodermal features and mesenchymal (MSC) properties. DPSCs express MSC cell surface markers, such as CD90, CD105, CD146 and Stro-1, in addition several studies have provided evidence of the differentiation capacity of these cells and they can be used in the repair of bone defects, treatment of neural tissue injuries and degenerative diseases. Traditionally, rodents are used for toxicological studies. However, the differences between primates and rodents make it difficult for rodent models to accurately or even recap human physiology and clinic. In this way, the technogolia of dental stem cells emerges as a possibility for the development of fully humanized models to better understand the action of toxicants. Objective: to characterize the culture of DPSCs, as well as the expression of Stro-1 during serial passages in the routine cell culture system, as well as the application of this technology to the development of human models for the toxicological study of new drugs. Concerning that, the naive DPSCs were obtained

from the biorepository of dental stem cells of the Laboratory of Teaching and Research in Toxicology In Vitro-Tox In- from Faculdade de Farmácia at UFG (University of Góias). Cells were cultured in DMEM/F12 medium supplemented with fetal bovine serum (10%), penicillin/streptomycin (1%). After the second and fifth passage, the cells were trypsinized and their viability determined. The morphology as well as the expression of the surface marker Stro-1 were evaluated by means of flow cytometry. Results: The partial results demonstrate that DPSC cells showed proliferative capacity after thawing, reaching confluence after 7 days in culture. We interestingly observed a reduction in Stro-1 expression with passages. Conclusion: In this way, we can partially conclude that the passages alter the expression of the MSC Stro-1 marker, which may restrict the differentiation capacity of DPSCs, however, further assays need to be performed to better understand the effect of passages on the differentiation capacity of these cells. Furthermore, the standardization of the model by our group will allow the development of human models for the toxicological study of new drugs. Keywords: stem cells, dental pulp, cell culture

Characterization and analysis of the expression of the surface marker Stro-1

in human dental pulp stem cells

oliveira, leanDro leal rocha; liMa, aliny pereira; FariaS, evelyn rayani araújo; goMeS, ThaiSângela roDrigueS lopeS e Silva; MaceDo, lariSSa MaTuDa; leiTe, jacqueline alveS; valaDareS, Marize caMpoS

Universidade Federal de Goiás - UFG.

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Introduction: Chemical Respiratory Allergy (CRA) consists of pathological conditions characterized by clinical manifestations that encompass asthma and rhinitis, which can be correlated with exposure to Low Molecular Weight (LMW) sensitizers, mainly regarding an occupational context. From a risk/toxicity assessment point of view, there are still no validated methods for evaluating the respiratory sensitization potential of chemicals once the in vivo-based models usually employed for inhalation toxicity addressment do not comprise allergenicity endpoints specifically. Besides, most of the classic cell culture-based in vitro techniques reside in submerged culture conditions, which fail to emulate toxicant exposure’s toxicokinetics within the airways. Summed up, these issues justify the urgent need for developing physiologically relevant methods that resemble the respiratory system complexity, allow the mimicking of airways toxicant exposure conditions, and the establishment of quantifiable endpoints that correlate with the allergenicity potential of chemicals. Objective: In this work, we developed, characterized, and evaluated the applicability of a 3D-tetraculture bronchial model reconstructed with the main airway cell types. Furthermore, we performed the tissue exposure to aerosols of two LMW respiratory sensitizers at an air-liquid interface, following the measurement of tissue viability and the activation of dendritic cells within the model. Methods: The co-culture model was reconstructed using four different cell lines, including bronchial epithelial cells (BEAS-2B), lung fibroblasts (MRC-5), endothelial cells (EA.hy926), and monocytic cells (THP-1). Briefly, the endothelial cells were cultivated in the basolateral side of 24-well semipermeable transwell inserts for 4 hours. Then, a collagen type I hydrogel containing lung fibroblasts was polymerized into the apical surface of the system. After 24 hours of cultivation, BEAS-2B epithelial cells were added upon the collagen matrix and drifted to an air-liquid interface. On the fifth day, the models were exposed to different concentrations of Chloramine-T and Maleic Anhydride aerosols, generated using the VitroCell Cloud 12 chamber and cultivated for additional 24 hours in the presence

of THP-1 monocytic cells in the basal compartment. Tissue viability was assessed using MTT reduction assay and dendritic cells activation/maturation biomarkers CD86 and HLA-DR were evaluated using flow cytometry. Results: The co-culture model was successfully reconstructed with the four respiratory system cell types. The morphological characterization demonstrated the presence of a continuous epithelial cell layer distributed upon a collagen matrix containing homogeneously dispersed lung fibroblasts. The bronchial epithelial cells also regularly expressed the biomarkers cytokeratin, E-cadherin and MUC-1, demonstrating suitable cohesion and function of epithelial cells. Moreover, endothelial cells constituted a continuous layer that remained integrally attached until the sixth day of cultivation in the basolateral side of the transwell inserts. The LMW sensitizers aerosols promoted a concentration-dependent decrease of tissue viability upon the 3D bronchial model, and further exposures to the CV80 (80% tissue viability concentration) led to an increase of CD86 and HLA-DR surface biomarkers expression by THP-1 cells in the basal compartment, demonstrating activation/maturation of dendritic cells led by the toxicant permeation, as well as the crosstalk between the different cell types. Discussion/Conclusion: Taken together, our results demonstrated that the 3D-tetraculture bronchial model presents hallmarks that can be correlated with the structure and function of human airways. Moreover, the integration of immune system cells to the epithelial model allowed the measurement of quantifiable endpoints that represent the proposed mode of action of LMW respiratory sensitizers, considering a physiologically-relevant air-liquid interface exposure to aerosols that mimics the human airway susceptibility to chemicals. Thus, the model is a promising tool for assessing respiratory toxicity/chemical respiratory allergy potential of inhaled toxicants. Aknowledgements: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Financiadora de Estudos e Projetos (FINEP).

Characterization and applicability of a novel physiologically relevant 3D-tetraculture

bronchial model for in vitro assessment of chemical respiratory allergy

Silva, arTur chriSTian garcia; MenDonça, izaDora caroline FurTaDo; valaDareS, Marize caMpoS

Laboratory of Research and Education in In vitro Toxicology (Tox In), Faculty of Pharmacy, Federal University of Goiás.

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Exposure to airborne particulate matter (PM) has been associated with premature skin aging and the aggravation of several skin diseases such as atopic dermatitis. Solar radiation initiates the inflammatory process in the skin and promotes DNA damage. The consequences of concomitant exposure of the skin to air PM and solar radiation as observed in the daily life are poorly understood. Reconstructed human epidermis (RHE) model was used to investigate the alterations induced by the exposure to sun light emitted by a solar simulator (5 J/cm2), and a standardized air particulate matter – NIST SRM 1648a (8,9 µg/cm2), alone or concomitantly. Histological parameters, cell viability, epidermal barrier function, inflammatory cytokines IL1-α and IL8- secretion and epidermal proteins loricrin (LOR), cytokeratin 10 (CK10), aquaporin 3 (AQP3) and NOTCH1 were evaluated. Reduction in viability in radiation-only exposed RHEs was observed when compared to non-exposed group. Interestingly, epidermis barrier function was increased in the irradiated-only group when compared to all other treatments, while the double exposure with PM

reversed this alteration. There were no changes in the secretion of IL1-α in all treatments, whereas IL-8 showed a significant reduction in expression in the double exposed group when compared to the all other groups. Immunohistochemical observations indicated no noteworthy differences. CK10 and NOCTH1 protein expression were increased in the double exposure compared to irradiated-only group followed by a significant reduction in LOR expression compared to the control and irradiated-only group. Double exposure to PM and solar radiation induced a decrease in AQP3 expression when compared to the irradiated-only group. Our findings reiterate the role of NOTCH1 in the signaling of barrier function epidermal proteins AQP3, CK10 and LOR. These observations highlight the importance of further studies with concomitant exposure for tightly controlling the release of pollutants and diminishing solar exposure to prevent more serious diseases. Supported by CNPq (Process: 130103/2019-5) Claudia L.V.da Silva MSc fellowship at the MSc Program of Pharmacy- Physiopathology and Toxicology and FAPESP 16/19963-1.

Concomitant exposure to air particulate matter and solar radiation reduces

epidermal barrier function in a reconstructed human epidermis model

Silva, clauDia lariSSa viana; carvalho, lariSSa anaSTacio Da coSTa; caMarena, DeniSSe eSTher MallaupoMa; BagaTin, julia De ToleDo; aSSiS, Silvia roMano; Maria-engler, Silvya STuchi; BarroS, Silvia Berlanga De MoraeS

Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of Sao Paulo, São Paulo, SP, Brazil.

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Introduction: Preclinical studies to screen for new drugs involve the use of animals, which is very expensive and time-consuming and, depending on the research hypothesis, may have scientific limitations, as well as little relevance to human biology. According to this, alternative models have been used to replace animals in scientific research, representing a social and economic improvement. Stem cells are generally defined as clonogenic cells capable of self-renewal and differentiation into multiple lineages, and may be of embryonic or postnatal origin. Isolating postnatal stem cells from available high-quality sources has been an important target for toxicological research into new drugs. Dental stem cells (DSCs) which are originate from the neural crest have been gaining prominence, exhibiting neuro-ectodermal features. Recent studies have shown a broad spectrum of

differentiation of DSCs in osteoblasts, adipocytes, chondrocytes, endothelial cells, muscle tissue, neural cells, among others, thus demonstrating a potential in the use of these cells for toxicological investigation, as well as drug discovery. Objective: isolation of stem cells from dental pulp to maintain a biorepository. Methodology: first step: to divide the teeth into three areas: pulp, papilla and periodontal ligament. second step: separating the cells from the selected parts using sterile materials. third step: through the enzyme collagenase, there is digestion of the extracellular matrix. fourth step: centrifugation and insertion of the explant into the cell culture vial. Results: we seek to quantify how many cells it is possible to have from a single tooth. Acknowledgments: The authors gratefully acknowledge the financial support CNPq, CAPES, FAPEG, FINEP.

Creation of a biorepository from dental stem cells: as an alternative

method to the use of animals

oliveira, leanDro leal rocha1; liMa, aliny pereira1; FariaS, evelyn rayani araújo2; goMeS, ThaiSângela roDrigueS lopeS e Silva3; MaceDo, lariSSa MaTuDa2; leiTe, jacqueline alveS2; valaDareS, Marize caMpoS1

1 Laboratório de Ensino e Pesquisa em Toxicologia In vitro, Faculdade de Farmácia, Universidade Federal de Goiás - UFG; 2 Instituto de Ciências Biológicas,

Universidade Federal de Goiás-UFG; 3 Centro universitário UNIFASAM.

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The use of 3D models has been growing in recent years, due to the demand for new methodological approaches to toxicology that can mimic microarchitecture, physiology and biochemistry of biological tissues in a better way than 2D models. The development of these models is based on the use of cells and scaffold similar to the extracellular matrix, which provides an ideal environment for cell proliferation, differentiation and functioning. The extracellular matrix is an essential component for the construction of the 3D model and is usually composed of biocompatible biomaterials from synthetic or natural sources. As it is more abundant in the extracellular matrix of mammals, the collagen type 1 is widely used. Of the commercial sources of collagen type 1, rat tail tendon stands out, which has disadvantages such as high cost, ethical issues and for being an isolated component, which does not completely mimic the original complexity of the matrix. The use of Decellularized Extracellular Matrix hydrogel from industrial waste may be a more sustainable, low-cost alternative with greater biometric potential. Based on this, the present study aims to propose obtaining a hydrogel from a natural source (bovine cornea extracellular matrix) for application in 3D corneal models. Bovine eyes were collected in a slaughterhouse in Goiania, Goias, Brazil and the corneas were isolated in the laboratory. To obtain the hydrogel, the cornea samples underwent a decellularization process with Sodium Dodecyl Sulfate (SDS), lyophilization, enzymatic digestion, neutralization and for polymerization the hydrogels were incubated at 37ºC. The decellularization process in the decellularized extracellular matrix (dECM) and in the dECM hydrogel were confirmed by histology in Hematoxylin and Eosin (HE) and by Hoechst

staining. The preservation of matrix components was qualitatively evaluated by histology with Azan Blue stains for collagen and Alcian Blue stain for glycosaminoglycans. The structure of collagen fibers was observed in scanning electron microscopy (SEM). To evaluate the amount of proteins, the BCA method was used. The applicability of the hydrogel in cell culture was evaluated in culture with HaCat cells (human keratinocyte), compared to a 3D corneal model developed in the TOXIN/UFG laboratory by Da Silva (2019), in which murine collagen type 1 was used. Cultures were maintained for 21 days and subsequently evaluated for viability using the MTT reduction method and histological evaluation with HE staining.Histological characterization showed that decellularization was effective, due to the absence of cells stained with HE and Hoechst in the dECM and hydrogel models. Collagen was preserved, however there was a slight loss of glycosaminoglycans. The collagen fibers were presented in a randomized and similar way between the native tissue, the dECM and hydrogel, confirming the presence of collagen and other proteins in large quantities. The hydrogel was able to support cell culture, and previous results showed that the culture remained stable until the twenty-first day of culture and offered conditions for cell proliferation, as observed for the 3D corneal model based on collagen type 1. The bovine cornea dECM hydrogel can be a natural biomaterial that can be used for cell culture and 3D cornea models making, and can contribute to the national production of research inputs, reduction of animal experimentation and to the growth of the area tissue engineering in the country.

Decellularized extracellular matrix hydrogel derived from bovine cornea

for application in 3D models

SanToS, jorDana anDraDe; Silva, arTur chriSTian garcia; DiaS, WaneSSa aMoriM; valaDareS, Marize caMpoS

Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.

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Introduction: when it comes to discuss about regulation, modern toxicology aims to develop several alternative methods based on human physiology in order to generate more relevant data about chemical substances when in contact with the skin. Despite the countless discoveries already made in the field of dermal sensitization being evaluated with animal tests, the data obtained with in vitro methodologies have allowed a greater recognition of the 3R’s policy (reduce, replace and refine), and consequently a significant evolution in the change of guidelines in order to analyze chemical products and substances. In addition, alternative methods may effectively complement the analysis of potential allergenicity, generating reliable data for safety and risk assessment. The mechanistic understanding of the AOP was of paramount importance for assessing skin sensitization in vitro and for the development of new OECD guidelines. This pathway is based on four key events: formation of immunogenic hapten-protein complexes or molecular initiation event; activation of keratinocytes; dendritic cell activation and T lymphocyte activation and proliferation. Among the guidelines used to assess key events, the KeratinoSensTM assay principle is the only OECD validated test guide to assess the Keap1-Nrf2-Are regulatory pathway, which is commonly activated by contact allergens. Despite being a technique validated by the OECD, the high costs can make the method’s accessibility difficult, as it requires the HaCaT strain containing the luciferase enzyme gene and specific equipment for luminescence

quantification. Therefore, the development of non-animal methodologies which assess skin allergenicity in a safe, effective and accessible way are essential. Objective: The work aims to develop a new methodology which evaluates key event 2 to quantify the transcription factor Nrf2 involved in the potential for skin allergenicity. Methodology: Substances categorized as dermal sensitizers and non-sensitizers according to the OECD were selected to assess cytotoxicity and NRF2 activation. The human keratinocyte line HaCaT without the luciferase enzyme gene was also used. To assess cytotoxicity, day 1 was chosen for plating at a density of 3x105 cells/ml and stored in a culture oven with controlled humidity and temperature for 24 hours. On day 2, exposure was performed at different concentrations. On day 3, the results were read by flow cytometry using the propidium iodide marker and the CV75 of each analyzed substance was found. To assess the activation of Nrf2, the same protocol for cytotoxicity was performed, however, using the Nrf2 antibody. Discussion/Conclusion: Preliminary results suggest that flow cytometry showed satisfactory results after exposure of substances in HaCaT cells through quantitative measurement by detection of emitted fluorescence. Non-sensitizing glycerol and lactic acid were not statistically significant, whereas sensitizing PPD and DNCB were statistically significant. This condition was verified when ‘’p’’ was lower than 0.05. All the analyzes were performed using the Graphpad Prism 8.0 software. Acknowledgments: CAPES, CNPq, Toxin e FINEP.

Development of a methodology to assess key event 2 to quantify NRF2

in cutaneous allergenicity

peDralli, Bruna criSTiane oliveira; valaDareS, Marize caMpoS

Laboratory of Research and Education in In vitro Toxicology (ToxIn) – Faculty of Pharmacy – Federal University of Goiás (UFG).

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Introduction: The main function of the respiratory system is to promote gas exchange through respiration, however, several toxic substances can be inhaled causing irritation, inflammation, and sensitization of the respiratory tract. Currently, safety tests validated by international regulatory bodies to assess inhalation toxicity are performed on animals, demanding the development of innovative techniques that can mimic the respiratory system for the replacement, reduction, and refinement of the use of animals. In vitro studies using monolayer A549, BEAS-2B and Calu-3 cell lines are under development to assess the toxicity of these substances. Furthermore, three-dimensional (3D) models of human respiratory epithelium are even better than the 2D ones, however, these approaches are limited by not presenting the complex architecture present in the human respiratory tract for mechanistic studies. On the other hand, human ex vivo models of Precision Cut Lung Slices (PCLS) contain functional cells of the lungs, extracellular matrix and mimic the cellular biological environment, however, there is limited human tissue availability, making its application difficult. Regarding this, the porcine lung, a residue of the food industry, has anatomical, physiological, and cellular features like the human, presenting a high potential of ex vivo model in PCLS for preliminary tests in the evaluation of toxicity. Objective: To

develop and characterize an ex vivo porcine model in PCLS to assess pulmonary mechanisms of toxicity. Methods: The porcine lungs were donated by the regional industry. The PCLS were obtained using the Tissue Slicer DTK-3000W (Vibratome) equipment, at a thickness of approximately 300µm to 500µm and were cultivated in an air-liquid interface for twenty days. To analyze the tissue viability, the tetrazolium reduction method (MTT) was performed. Tissues were processed and stained with hematoxylin-eosin for histological analysis. Tissue exposure to aerosolized paraformaldehyde by nebulization (Vitrocell® Cloud 12 exposure chamber) was performed on the fifth day of cultivation. The presence of ROS was measured by DCFH-DA staining and assessment of mitochondrial activity was also measured by MITOTRACKER® labeling. In addition, using indirect immunofluorescence, caspase was evaluated. Results: Preliminary results suggest that the ex vivo porcine model of PCLS in the histological and MTT assays-maintained cell viability and alveolar morphology for eight days, without signs of tissue degeneration. Tissue exposure to aerosol of paraformaldehyde, followed by assessment of ROS, mitochondrial activity, and caspase were performed to determine toxicity mechanisms. Discussion/Conclusion: The ex vivo porcine model in PCLS can be an important tool for the assessment of pulmonary toxicity. Acknowledgments: CAPES, CNPq and FINEP.

Development of an ex vivo model to evaluate pulmonary toxicity mechanisms

FurTuoSo, Marcella MiranDa Siqueira; TavareS, kveTTa pinheiro Teixeira; peDralli, Bruna criSTiane oliveira; valaDareS, Marize caMpoS

Laboratory of Education and Research in In Vitro Toxicology, Tox In, Faculty of Pharmacy, Federal University of Goiás, Goiânia, Goiás, Brazil.

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Introduction: In developing countries, such as Brazil, family farming is responsible for the income of more than 70% of Brazilians employed in rural areas. This type of agriculture is transferred between generations and is seen by workers as safe and harmless, so that often, in addition to failing to comply with health and safety standards (indiscriminate use of pesticides, without adequate protection measures), the extensive contact of workers with different classes of pesticides exposes them to greater risk. This fact is evidenced by the records in the Notifiable Diseases Information System (Sinan), where 84,206 cases of pesticide poisoning were reported in Brazil between 2007 and 2015. Objective: In view of these aspects, the objective of this study was to evaluate dermal absorption of dimethoate, using in vitro and in vivo approaches, considering the environmental conditions of work in family agriculture. Methods: In order to evaluate dermal absorption under typical working conditions, dimethoate on clothing and stratum corneum (SC) was measured in seven rural workers during application, 1 resident and 1 researcher who monitored all applications. The evaluation was made by using eight cotton patches fixed in specific places on workers’ clothing, and SC was collected in three different areas by tape stripping. The in vivo dermal exposure and SC penetration factor (PF) of dimethoate were calculated according to the OECD guideline No.9 (1997) modified. To correlate the results, the in vitro permeation test (IVPT) was performed by using rat, pig and human skin in Franz vertical diffusion cells with receptor medium of 6.0 mL, stirring at 32 ± 1°C and 1.77 cm2 of permeation area. 36 µL of the Dimexion® (600 µg/mL) was applied and at 2, 4, 6, 8, 10, 12, and 24 hours, 1

mL of the receptor fluid was collected and replaced. After 6 hours, the surfaces of the skins were cleaned without stopping the test. The retention was analysed in SC and in the rest of skin. The in vivo study and use of human skin for IVPT was approved by CEP/UFPE 5.007.282 as well as the use of rat skin (CEUA/UFPE: 007/2021). The dimethoate was analysed by validated LC-MS/MS method. Results: In this IVPT study, excluding all tapes, the calculated absorption data were 6.49 ± 0.04; 3.49 ± 0.29 and 1.79 ± 0.19 µg/cm2 from rat, pig, and human skin respectively for diluted product. The calculated absorption through the human skin was 14,75%. However, according to EFSA (2017), the default value to be used in the absence of experimental data for this commercial product is 70%. The mass balance was between 95 and 105%, demonstrating the reproducibility of the methodology. The dimethoate extracted from cotton patches in different regions of body was between 0.38 and 318,155.14 ng/cm2. The calculated dermal exposure was between 0.27 and 1,353.04mg/body region and the PF (%) was between 0.06 and 25.24 in the forearms and between 0.44 and 14.44 in the back of the neck. Discussion/Conclusion: Except for the researcher, none of the workers in the study wore the appropriate clothing as described in the product package insert. These data reveal great concern about the family farm workers who have been routinely working without the proper use and care of safety equipment specifically for “pesticide applicators”. Further studies performed with other pesticides with different characteristics will contribute to the understanding of their transport through the skin. Acknowledged: CNPq and FACEPE.

Dimethoate exposition: in vitro x in vivo study

anDraDe, ana roSa BriSSanT; carvalho, Deoclécio luSToSa; Souza, aSley Thalia MeDeiroS; kiShiShiTa, juliana; piMenTa, caMila De alMeiDa perez; SanTana, Davi pereira; leal, leila BaSToS

Universidade Federal de Pernambuco (NUDFAC-UFPE), Recife, PE, Brazil.

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Background: Myrcia pubipetala, an endemic plant present in Brazil, which has few studies, is part of the Myrtaceae family, a large group with important specimens in popular use, such as “jaboticaba”, “goiaba” and “eucalipto” and others that have reported anti-inflammatory activity. Studies on M. pubipetala are scarce, and this work is the first to report the biological activity of the M. pubipetala extract. Objective: Evaluate the innate immune response involved in the in vivo and in vitro anti-inflammatory activity of M. pubipetala Crude Hydroalcoholic Extract (CHE-MP). Methods: The CHE-MP was obtained from the maceration of dried leaves in 70% alcohol, and dried so that the biological activity could be described. The in vivo anti-inflammatory activity was evaluated by the air pouch method, which was approved by the ethics committee of Universidade Regional de Blumenau under protocol 009/20. The exudate was quantified as well as the total and differential leukocytes, nitric oxide and cytokines (interleukin – IL-1β, IL-6 and tumor necrosis factor – TNF) and a histological analysis of the tissue that lines the pouch was performed. The in vitro part used RAW 264.7 cells where nitric oxide and cytokines (IL-1β, IL-6 and TNF) were measured in the culture supernatant and cell viability and expression of adhesion molecules (CD62L [L- Selectin] and CD18 [β2-Integrin]) on the cell surface. For the toxicological analysis was

performed MTT and HET-CAM assay. Results: CHE-MP inhibited leukocyte migration as well as reduced polymorphonuclear migration at all doses (3, 30 and 300 mg/kg v.o.), which was confirmed by histology. At doses of 30 and 300 mg/kg, the reduction in exudate volume and nitric oxide concentration was highlighted. As for immunomodulation, there was a reduction in the expression of IL-1β, IL-6 and TNF. Cell viability in vitro demonstrated that CHE-MP does not show cytotoxicity at the doses tested (1, 10 and 100 µg/mL). CHE-MP inhibited the increase in nitric oxide at all doses tested, and the dose of 100 µg/mL showed a statistical difference compared to the dose of 10 µg/mL. There was a reduction of IL-1β, IL-6 and TNF in the culture supernatant, confirming the results obtained in vivo. There was lower expression in CD18 in cells treated with doses of 10 and 100 µg/mL. In the both toxicological assays, CHE-MP preserve the cell viability and preserve the integrity of the blood vessel in HET-CAM. Conclusion: The results obtained confirm that M. pubipetala has immunomodulatory activity, as well as anti-inflammatory activity in vivo and in vitro and the extract provides security to use. Acknowledgments: Fundação Universidade Regional de Blumenau – FURB and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina – FAPESC.

Effects of Myrcia pubipetala Miq (Myrtaceae) extract on innate inflammatory response

pacaSSa, pâMela1; BenvenuTTi, lariSSa2; echTerhoFF, Marcelo roDrigo Franke3; lopeS, Bruna gonçalveS3; quinTão, nara linS Meira2; DeBiaSi, Michele alBerTon1; SanTin, joSé roBerTo2; MachaDo, iSaBel DauFenBack1

1 Postgraduate Program in Biodiversity, Fundação Universidade Regional de Blumenau, Blumenau/SC, Brazil; 2 Postgraduate Program in Pharmaceutical Science,

Universidade do Vale do Itajaí, Itajaí/SC, Brazil; 3 Postgraduate Program in Chemistry, Fundação Universidade Regional de Blumenau, Blumenau/SC, Brazil.

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Introduction: Innovation on pharmaceutical fields has been developing a series of new products concerning the most diverse purposes that demand toxicological investigations. One of the main routes of exposure to chemicals and/or particles is the human respiratory tract. The vulnerability of the lung occurs due to its external connection susceptible to environmental exposure, along with systemic connection, with proximity to the brain and heart. Regulatory agencies seek to expand existing information about inhalation toxicology of products in the global market. Currently, all inhalation toxicity investigations are performed through in vivo tests. With the advancement of science and the New Approach Methodologies (NAMs), which mimics regions of the human respiratory system have been developed. According to this, our group developed an in-house 3D model, using the A549 human lung alveolar cells, that can mimic human exposure through cultivation at an air-liquid interface for toxicity assessments. Objective: To investigate the effects of exposing lung cells to pulmonary irritants directly on the tissue, using the in-house developed lung model. Methods: The lung model was developed in-house through the cultivation of the A549 cell in an air-liquid interface. The test substances were prepared according to the stablished concentrations and physicochemical properties of each compound.

The toxicants from different categories of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) were added directly on the tissue, and PBS was used as a negative control. The tissues were exposed for 3 hours to toxicants and incubated at 37°C in a humidified atmosphere of 5% CO2, after which they were evaluated by the MTT assay. Morphological evaluation of the tissue developed in-house was also carried out through histological processing. Results: The results showed that using the in-house pulmonary model developed was possible to categorize the pulmonary irritant toxicants used according to the GHS classification and also, allowed perform prediction for inhalatory LC50 concentrations in animals. Discussion: The model showed to be useful to investigate inhaled toxicants like the commercially available models. In addition, the model can predict the LC50 value in animals for inhalation toxicity tests, it can also drastically reduce the number of these tests in 80%, and it may reach 100% for tests with corrosive substances. Conclusion: Based on the results obtained, the in-house developed pulmonary biomimetic model has the potential to be a useful tool in acute inhalation toxicity tests and highlights the importance of NAMs instead of using models concerning animals. Acknowledgments: CAPES, CNPq and FINEP.

Effects of pulmonary inhaled irritants on 3D alveolar model

FurTuoSo, Marcella MiranDa Siqueira; TavareS, kveTTa pinheiro Teixeira; valaDareS, Marize caMpoS

Laboratory of Education and Research in In Vitro Toxicology, Tox In, Faculty of Pharmacy, Federal University of Goiás, Goiânia, Goiás, Brazil.

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Introduction/Objective: Following the global trend and especially Art 6 of RDC 294 (July 29, 2019), in which alternative methods must be presented for regulatory purposes, we evaluated the in vitro irritation (OECD 439) and corrosion (OECD 431) methods to classify biological products, using reconstructed human epidermis (RHE, SkinEthicTM) provided by Episkin Brasil. This model represents the in vitro target organ, that consists of normal human keratinocytes cultured to maturity of the epidermis. For this reason, it has a histological morphology comparable to in vivo tissue. Some care must be taken when handling the biological product in order to avoid cross contamination or loss of effectiveness of the product. Methods: A biological product has been tested according to OECD 439 and OECD 431. For the Irritation test (OECD 439) the tissues were firstly exposed to 16 mg or 16 µL of each item, according its physical characteristics, concurrently to negative (NC=PBS) and positive (PC=SDS, 5%) controls for 42 minutes, then washed and incubated for an additional 42 hours at 37 °C, recovery period. Following this post incubation period, the tissues were incubated with a MTT (1 mg/mL) solution for 3 hours. For the Corrosion Test (OECD 431), the tissues were firstly exposed to 20 mg or 40 µL of each item, according its physical characteristics, concurrently to negative (NC= sterile water) and positive (PC= KOH, 8N) controls for 60 and 3 minutes, then washed

and incubated with a MTT (1 mg/mL) solution for 3 hours. After 3 hours of incubation with MTT, the formed formazan crystals were solubilized with isopropanol. The optical density was evaluated at 570 nm. Results/Discussion: Viability results obtained in vitro (mean ± SD) were NC: 100 ± 3.27; PC: 1.92 ± 0.07; Biological Product: 104.05 ± 7.92 for the irritation test (OECD 439). The viability obtained were NC: 100 ± 7.90; Biological Product: 99.62 ± 1.19 for 3 minutes of exposure and NC: 100 ± 1.65; PC: 2.75 ± 0.13; Biological Product: 94.63 ± 0.46 for 60 minutes of exposure for the Corrosion test (OECD 431). Our results were within the acceptance criteria of the guides and our historical data. As expected, the positive control demonstrated a significant relative cell viability reduction when compared to the negative in both tests. This fact is explained by the cytotoxic action of SDS and KOH. In addition, according to the results obtained, the Biological Product was classified according to UN GHS Category as “No Category” and “Non-Corrosive”, corroborating with in vivo classification tests (Pubchem, 2021; Meleney and Johnson, 1949). Conclusion: In view of the results, the use of RHE for classification of biological products is valid, as long as proper precautions are taken to obtain quality results. Acknowledgments: -Episkin, Rio de Janeiro-RJ, Brazil; -Bioagri Laboratórios Ltda (Merieux NutriSciences), Piracicaba-SP, Brazil.

Efficiency of the Reconstructed Human Epidermis (RHE) in the classification of

biological product by the in vitro irritation and corrosion tests (OECD 439 and 431)

BechTolD, Bruna aSSunção1; cianci, julio ceSar1; coelho, Maria paula Mancini1; coSTa, lariSSa gaBrielli1; Dakic, vanja2; De vecchi, roDrigo2; Fava, luiS paulo1; paDua, aManay SouSa1;

SanTana, ThaTiane nuneS1; Silva, priScilla Muniz riBeiro1; vecina, juliana FalcaTo1

1 Mérieux NutriScience / Bioagri Laboratórios Ltda, Piracicaba-SP, Brazil; 2 Episkin Academy, Rio de Janeiro-RJ, Brazil

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Background: Triclopyr is an auxin-like herbicide used to control a wide range of weeds in food crops and pastures. Triclopyr is known to pollute the aquatic ecosystems. Its potential effects on the aquatic environment and human health, and the lack of monitoring of triclopyr in environmental compartments are an emerging concern. In this study, we used zebrafish as a biomarker of ecotoxicity to assess the effects induced by triclopyr at concentrations already detected in aquatic systems. Objective: The aim of the current work was to evaluate the mechanisms of the acute toxicity of triclopyr in early development of zebrafish. Methods: For all tests, we used six environmentally realistic concentrations of triclopyr: 0.5, 1, 5, 10, 50 and 100 µM. The embryotoxicity assessment was based on the Fish Embryo Acute Toxicity (FET) test (OECD TG236), extended up to 144 hours. We determined the LC50, EC50 and teratogenic index for triclopyr, based on the lethal and sublethal effects induced in embryos and larvae within 96 hours of exposure. Acetylcholinesterase activity, based on Ellman’s essay adapted to 96-well plate, was used as a neurotoxicity biomarker for triclopyr. The results were evaluated by analysis of variance (ANOVA) followed by the Dunnett test. Results with p=<0.05 were considered statistically significant. Results: In the FET test triclopyr significantly induced lethal effects (coagulated embryos and lack of heartbeat) in zebrafish embryos at all concentrations tested. The LC50 50.07 µM and LC50 47.74 µM for triclopyr was defined at 96 and 144 h, respectively. In addition, it was observed sublethal effects such as

pericardium and yolk sac edema, non-hatched eggs, and uninflated swim bladder in zebrafish embryos exposed to at all non-lethal concentrations tested of triclopyr (0.5-50 µM), up to 144 hours. At 10 and 50 µM of triclopyr was observed teratogenic effects such as scoliosis and malformation of the head and eyes. A significant decrease in larval length was observed for all tested concentrations of triclopyr, when compared to the negative control. The EC50 38.77 µM at 96 h was defined based the effects observed and the teratogenicity index of 1.29 was defined for triclopyr. Acetylcholinesterase activity did not change after exposure to triclopyr. Discussion/Conclusion: Even at relatively low concentrations, the present study has shown that triclopyr can cause embryotoxicity in early development of zebrafish by inducing both lethal and sublethal effects. Furthermore, triclopyr has proven to be a teratogenic compound for zebrafish, which can directly and indirectly impact the survival and maintenance of the fish population. Also, triclopyr showed no neurotoxic activity at the sub-lethal concentrations tested. Although further studies are needed, taken together and from an ecotoxicological perspective, our results suggest the impact on the health of other aquatic organisms. Keywords: Triclopyr; herbicide; contaminant of emerging concern; toxicity; fish. Acknowledgments: We thank the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP: Processes No. 18/00229-1, São Paulo, Brazil) for the financial support.

Embryotoxicity of triclopyr in early development of zebrafish (Danio rerio)

anDraDe, íTalo BerToni lopeS1,2; SaleS, Bianca caMargo penTeaDo1,2; peixoTo, paloMa viTória liMa1,2; viriaTo, criSTina2,3; pereira, lílian criSTina2,4

1 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM); 3 São Paulo State University (Unesp), Institute

of Biosciences, Botucatu; 4São Paulo State University (Unesp), School of Agriculture, Botucatu.

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Introduction: Cosmetic products must be safe under normal conditions or have predictable risks, remaining within an adequate safety margin. As they are freely accessible, cosmetics can accidentally permeate barriers, being partially or totally absorbed and causing possible signs of irritation such as eye irritation, so it is necessary to guarantee their safety when in contact with the eyes, and must be approved in tests of eye irritation. In the 80’s the development of alternative experimental models for the cosmetic area was started, replacing the use of laboratory animals. Among these methods, the BCOP test is an in vitro test using corneas isolated from the eyes of cattle obtained as a by-product from slaughterhouses to assess the potential ocular irritation of a test substance. Objective: This work aims to support the development of alternatives to animal experimentation by assessing the potential for ocular irritation. Methods: The substances used in this work were selected according to their frequency of occurrence on shampoo labels marked in Brazil. The four substances most commonly found in the composition of the different shampoos evaluated were cataloged according to their pharmacotechnical characteristics in shampoos, usual concentration and ocular toxicological profile. The BCOP assay was performed according to OECD 437. Results: Raw materials EDTA 20%, Cocoamidopropyl betaine 20%,

Cocoamidopropyl betaine 6% and Sodium lauryl ether sulfate 30% were classified as No prediction can be made, because IVIS was higher than 3, IVIS 10.6; 5.8 and 10.5 respectively. While EDTA 0.1%, Silicone DC 200/350 100%, Silicone DC 200/350 40% and Sodium Lauryl ether sulfate 10% were classified as No Category because they presented IVIS less than 3, IVIS 1.1; 0.7; 2.0 and 1.2 respectively. Discussion/Conclusion: The various limitations of in vivo methods in force, such as ethical issues, animal management, biological variability and the worldwide trend to replace the use of animals in experimentation demonstrates the current need for the development and implementation of alternative in vitro methodologies to enhance performing tests with greater accuracy, deadlines and lower costs than tests performed on animals. From the studies and technologies developed for the cosmetic tests and worldwide mobilization for the development of alternative methodologies, it is possible to envisage the substitution or reduction of the use of animals in scientific experimentation. Acknowledgments: This research was supported by the Laboratory of Teaching and Research in Toxicology In Vitro (Tox In) of the Faculty of Pharmacy of the Federal University of Goiás (UFG) and by the Faculty of Pharmacy of the Pontifical Catholic University of Goiás (PUC-GO).

Evaluation of ocular irritation potential of raw materials presented in xampus

formulations using non-animal methodology

rocha, Daniela BarBoSa; alMeiDa, jéSSica azariaS; anDraDe, WaneSSa MachaDo

Pontifícia Universidade Católica de Goiás - PUC GOIAS.

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Introduction: Melasma is characterized by dark spots on the skin, resulting from increased melanogenic activity that alters epidermal pigmentation. The cause of melasma is multifactorial, the most common being exposure to the sun, pregnancy, and birth control pills. There are several compounds used in the treatment of melasma, such as kojic acid and hydroquinone, which can cause side effects. In this context, the trace elements, zinc (Zn) and selenium (Se) must be explored. Zn is a component of superoxide dismutase and metallothionein, both antioxidant molecules, in addition to displacing more dangerous metal ions, which cause the formation of free radicals. Se is a component of important selenoproteins, for example, glutathione peroxidase, which has antioxidant activity. Based on the above data, it is of scientific relevance to analyze the influence of Zn and Se on the pathophysiology of melasma. Method: The research was carried out with SK-MEL-28 (human melanoma) and B16F10 (mouse melanoma) cells. The cytotoxicity of zinc chloride, sodium selenite and diphenyl diselenide was evaluated by colorimetric MTT assay. About 2.5x10⁴ cells per well were seeded in 48-well plates; then, the cells were exposed to zinc chloride (1-1000 µM), to sodium selenite (1-1000 µM) and to diphenyl diselenide (1-1000 µM) for 48h. After exposure, 10 µL of MTT (5 mg/mL) were added to the cells and incubated for 3h at 37ºC. After the supernatant was discarded and 100 μL of DMSO was added. Cells were kept shaking for 30 minutes at room temperature. Absorbance was measured at 570 nm. The melanin content of the B16F10 cell line was also verified. Cells (2 x 104/well) were plated in 24-well plates for 24h. Afterwards, the cells were incubated in the presence of α-MSH hormone (200 nM) and the compounds

sodium selenite (5 µM), diphenyl diselenite (1 µM), and zinc chloride (100 µM) for 48h. After incubation, the cells will be washed with phosphate saline buffer (PBS), dissolved in 200 µL, then 500 uL of NaOH with 10% DMSO was added, incubated at 80 °C for 1h, then the absorbance was reading was at 490 nm. Statistical analyzes were performed on GraphPad Prism 6 (version 6.01, GraphPad Software, Inc., USA). Result: One-way ANOVA revealed effect of sodium selenite, zinc chloride and diphenyl diselenide on SK-MEL-28 cell viability. Sodium selenite caused a decrease in cell viability from the concentration of 10 µM. Zinc chloride reduced cell viability from the concentration of 300 µM; and, diphenyl diselenide caused a decrease in cell viability from the concentration of 100 µM. Based on the results observed in the SK-MEL-28 cells, concentrations of 5 µM for sodium selenite, 1 µM for diphenyl diselenide and 100 µM for zinc chloride were selected to verify the absence of cell cytotoxicity in the B16F10 cells (which will be used for testing anti-melanogenic activity). One-way ANOVA revealed no effect of sodium selenite and zinc chloride, at the concentrations tested, on cell viability of the B16F10 cells; however, it revealed an effect of diphenyl diselenide. At a concentration of 1 µM, diphenyl diselenide caused a slight increase in cell viability (~18%). For the melanin content, one-way Anova revealed treatment effect. Cells exposed to zinc chloride showed a significant 18% decrease in melanin content when compared to the control. Conclusion: Among the compounds tested, zinc chloride was the most promising as an anti-melanogenic compound. It is likely that it is inhibiting the tyrosinase enzyme, further tests will be carried out to elucidate its mechanism of action.

Evaluation of the cytotoxicity and the anti-melanogenic activity of Selenium and Zinc

Silva, ana cléia carDoSo; riBeiro, Milena Mariano; irioDa, ana carolina; oliveira, cláuDia Sirlene

Programa de Pós-graduação Strictu sensu Aplicada à Saúde da Criança e do Adolescente. Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, PR, Brazil; Faculdades Pequeno Príncipe, Curitiba, PR, Brazil

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Introduction Recent advances in legislation focus on alternative methods to guarantee efficacy and safety, following the 3Rs principles (reduce, replace, and refine) of animal experimentation. Galleria mellonella is an alternative widely used and established safety and efficacy research model. This animal model is mostly studied in the injectable route in studies of pathogens-host interaction and chemical compounds toxicity. Although it is a promising methodology due to its cuticle composition of three layers, like human skin, mostly composed of lipids, proteins, and chitin, there is a lack of data that assess other administration routes, such as topical that could be used as an in vivo alternative animal model for absorption and toxicity assays. Objective To remedy this lack and contribute to the validation of G. mellonella as a model in cutaneous absorption and toxicity studies, this study aims to evaluate the absorption of gallic acid (GA), a secondary plant metabolite with several biological activities. Method The study was conducted using an in vitro and in vivo toxicity testing platform, using three alternative assays. The first uses GA concentrations in human keratinocyte (HaCat), human dermal fibroblasts (HDFa), and human liver (HepG2) cell lines in the concentration range between 7.81 to 1000 µg/ml. The second trial evaluated the toxicity of GA in C. elegans in the same concentration ranges. Finally, the third in G. mellonella using the injectable and topical routes according to the OECD recommendations (404, 2002), by the “Acute dermal irritation/corrosion in vivo method”, in concentrations of 261 to 920mg /kg.

Results The results of the in vitro assays showed that HaCaT cells were more sensitive to GA (IC50 of 138.8µg/ml), followed by HepG2 cells ( IC50 of 207.8 µg/ml), and HDFa (IC50 of 247 µg/ml). For in vivo assays, GA showed greater toxicity to C. elegans with LC50 values of 16.08 µg/ml. For G. mellonella, the data showed that this alternative animal has a high tolerance to GA with LD50 values greater than 920mg/kg both by the topical and injectable route, however, alterations were observed in the humoral immune response of the larvae, represented by an increase in melanization in concentrations of 463 to 920mg/kg for the injectable route and 695 to 920mg/kg for the topical route. In addition, characteristic histopathological findings were presented at the same concentrations for both evaluated routes of administration. Discussion/conclusion According to the literature, the tests carried out on G. mellonella showed results equivalent to those of mammals according to studies carried out in mice and rats by the same routes of administration. The literature does not point to physiological and biochemical correlations between alternative and conventional methodologies that could equate to toxicity results. However, alternative animals are proven methodologies for evaluating the toxicity of chemical substances, as well as being predictive of toxicity in mammals. In this way, the combination of more than one model guarantees an increase in the reliability of the safety of natural compounds, promoting a reduction in research costs and an improvement in ethical conduct in the world.

Galleria mellonella as an Alternative Model for the Assessment of Permeation and

Toxicity of Assets from Natural Sources

Silva, SaManTa De MaToS1,2; Singulani, junya De lacorTe1; carvalho, angélica roMão1,2; MigliaTo, keTylin FernanDa3; giannini, Maria joSé SoareS MenDeS1,2; FuSco-alMeiDa, ana MariSa1,2

1 Department of Clinical Analysis, Laboratory of Mycology, School of Pharmaceutical Sciences, São Paulo State University – UNESP, Araraquara, São Paulo, Brazil; 2 Community Service

Center – NAC - Laboratory of Alternative Methods for Bioproducts - FCF UNESP Araraquara SP, Brazil; 3 Central Paulista University Center – UNICEP, São Carlos, São Paulo, Brazil.

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Introduction: Fetal Bovine Serum (SFB) is a complex mixture of biomolecules in distinct concentration and composition. The variation imposed on the product is a result of the complexity involved from its obtention, seasonality, dietary characteristics to which the animals are exposed. The animal product is the main source of amino acids, vitamins, trace elements, hormones and factors for cell growth and maintenance in vitro. The product can present contamination, composition and concentration varied in each batch and/or brand available, ethical factors related to animal suffering and genetic characteristics affecting cell differentiation. The variation found and not routinely tracked, imposes an inconsistency in cell culture with unreproductive results in experimental studies. The lack of reproducibility of already validated methods and the disparity between the results described in the literature regarding in vitro methodologies, has been related to the use of SBF from different sources. Objective: Identify and quantify the constituents of FBS available on the market applied to cell culture. Methodology: Forty-seven biochemical constituents estimated in the composition of FBS were quantitatively evaluated. Tests performed were enzymatic, colorimetric,

bromocresol biuret, chemiluminescence, UPLC, UV kinetic and HPLC MS/MS to quantify SBF constituents. Samples from each batch were analyzed in triplicate and the results compared with those described in the literature. Results: The composition of the SFB was estimated based on literature data according to their characteristics correspond to: 69% of proteins, 10% of lipids, 10% of ions, 3% of sugars and 3% of vitamins. The preliminary quantification of the SFB components indicated a variation higher than 15%, when related to the theoretical reference. For triglycerides, albumin, and T4, the values reached 17%, 148% and 25% of difference in relation to the literature. The results showed difference between the reference values and those found in the preliminary analyses. Discussion: It is already described in the literature that inter-lot factors alter the concentration of FBS constituents. The identification and quantification of the components of FBS can demonstrate that a biological product with no standardization of the constitutions, such as FBS, can interfere in cell growth and, consequently in the cell culture results, with high impact in the reproducibility of in vitro assays. Acknowledgment: CAPES, FUNAPE, FAPEG, FINEP and CNPq

Identification and quantification of constituents in the fetal bovine serums

available in the market used in cell culture

STival, ana clara Silva; Silva, arTur chriSTian garcia; valaDareS, Marize caMpoS

Laboratório de Ensino e Pesquisa em Toxicologia in vitro, Universidade Federal de Goiás, Goiânia-GO, Brasil.

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Eye irritation data are essential for safety and efficacy evaluation of topically applied products and chemicals. In Europe, the full ban of animal testing was implemented in 2013 and validated methods like Eye irritation test (EIT, adopted into Test Guideline 492 of OECD) are highly used for chemicals classification. Brazil and South America is going along with this trend step by step, and the urge for robust alternative models is increasing in the last few years. This work describes the implementation of validated SkinEthicTM Human Corneal Epithelium (HCE) model, that closely mimics the histological, morphological, and physiological properties of the human corneal epithelium. 15 chemicals were tested and correctly classified using

tissues produced in Brazil. The highly reproducible and consistent results confirm the quality and robustness of HCE model and methods. Moreover, recent publications described the development of Time-to-Toxicity (TTL) approach based on this model, showing that the EIT is capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (no cat.), from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (cat. 1). With this update EIT replaces the animal model (Draize test), increasing its usefulness and making its availability a big priority in Brazil and Sought American countries.

Implementation of SkinEthicTM Human Corneal Epithelium (HCE) in Brazil

Dakic, vanja1,3; MaTToS, guilherMe1,3; rigauDeau, anne-Sophie3; garcia, criSTina3; Bouez, charBel4; De vecchi, roDrigo1,3

1 Episkin, Rio de Janeiro, Brazil; 2 Episkin, Lyon, France; 3 L Oréal Research and Innovation, Rio de Janeiro, Brazil; 4 L Oréal Research and Innovation, Clark, USA.

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Introduction: The mulungu, Erythrina verna, has its bark used in folk medicine as a tea to treat mild emotional disorders such as anxiety, agitation, depression, panic attacks, epilepsy, and compulsion due to its anxiolytic and hypnotic actions. Objective: Evaluate the interactions of erythrinic alkaloids present in mulungu tea with the amino acid residues of the active site of the GABAA β-3 receptor and prediction of toxicity through computer modeling. Methods: The alkaloids selected for the study were erythravine, erythrartine, 11-α-hydroxy erythravine, 11-α-hydroxy erysothrin, 11-hydroxy erythratidinone, erisorthine-n-oxide, erysortrine, erythradine, erythrartine-n-oxide and erythratidinone. For the in-silico evaluation, molecular docking methodologies were adopted using Spartan’08® software and quantum chemistry methodologies, AM1 followed by DFT B3LYP / with 6-311G data base in gas phase, for geometry optimization and conformational analysis of the erythrinic alkaloids. The iGemDOCK® software was used for molecular docking, using the 3D model of the benzamide-complexed GABAA β-3 receptor, PDB ID 4COF. The alkaloids from the group mentioned above and the diazepam molecule, used as a control, were submitted individually for docking in the BEN cavity of the 4COF model. For the prediction of toxicological risks based on chemical structure, OSIRIS Property Explorer® software was used. Parameters of mutagenic, carcinogenic, irritant potential, effects on reproduction, drug likeness and drug-score based

on molecular structure were evaluated. Results and Discussion: In the prediction of toxicity risks, none of the molecules presented a potential risk, in addition, the drug likeness and drug-score parameters obtained high ranking values, indicating a good potential of the molecules as a drug. In the docking process, the molecules were evaluated at the central molecular site of interaction BEN, using the slow calculation method with ten individual conformational poses. The pharmacological interactions of hydrogen bonding, Van der Waals and electrostatics, alkaloids and diazepam were evaluated. The interactions occur at the main amino acid residues of the receptor, the main interaction is Van der Waals. All alkaloids studied showed good interaction with the site of interaction, with energy values -109.44 kcal for erythrartine and -100.56 kcal for diazepam, the other molecules showed values between -83.09 and -94.14. Unlike the alkaloids that also showed hydrogen interactions, diazepam only has Van der Waals interactions. Conclusion: The in-silico evaluation corroborates the benzodiazepine-like action of the alkaloids, having interactions with the main amino acid residues of the GABAA β-3 receptor like those that occur with diazepam, justifying its anxiolytic and hypnotic effects. In addition, the molecules present in mulungu tea demonstrate the safety regarding the toxicological risks of the molecules according to the evaluated methodology.

In silico evaluation of erythrinic alkaloids from Mulungu, Erythrina verna, in GABAA β-3 receptor complexed with benzamide

BairroS, anDré valle1; naSciMenTo, Marcelo henrique SanTana2; paula, Fávero reiSDorFer3

1 Nucleus Applied to Toxicology (NAT) Federal University of Santa Maria (UFSM); 2 NAT – UFSM; 3 Federal University of Pampa (UNIPAMPA).

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Background/Introduction: Resveratrol (3,4’,5- trihydroxy-trans-stilbene) is a natural stilbene found mainly in grapes, peanuts, and wines. It is known for having different biological activities as antioxidant, anti-inflammatory, antibacterial, and, neuroprotective, although their poor water-solubility and limited therapeutic efficacy. Structural optimization via glycosylation of molecules appears as an alternative for this issue. Pharmacokinetic properties may be one of the main causes of new drug candidates’ failures in clinical trials. In silico prediction methodologies may impact the drug development process, becoming a crucial part of the drug and related target discovery progress. Objective: We aimed to evaluate the pharmacokinetics and pharmacodynamics profile of resveratrol and two glycosylated derivatives through in silico methods. Methods: Swiss ADME, ADMETlab, and SwissTargetPrediction online platforms were used to predict pharmacokinetics and pharmacodynamics properties of resveratrol, polydatin (resveratrol-3-O-β-ᴅ-glucoside), and resveratrol 3-α-glucoside (resveratrol-3-O-α-ᴅ-glucoside). Results: The results obtained from in silico analysis indicated that the three compounds are highly absorbed in the gastrointestinal tract when orally administered and showed a bioavailability score of 55%. Additionally, no one of them acts as a P-glycoprotein substrate, responsible to affect the bioavailability of drugs. Resveratrol is able to cross the blood-brain barrier (BBB), however, its glycosylated forms showed no BBB permeability limiting its central nervous system use. The half-

time presented by resveratrol was approximately 1.5 hours, slightly longer than polydatin and resveratrol 3-α-glucoside that exhibited a half-time of about 1.1 hours. Glycosylated forms showed no interaction with cytochrome P450 enzymes, different from resveratrol which can act as CYP1A2, CYP2C9, and CYP3A4 inhibitors and as a substrate for CYP2A9 and CYP2D6 isoforms. Druglikeness analysis was used to recognize how similar the substances are to drugs already approved. Resveratrol exhibited no violations of Lipinski’s Rule of Five, in contrast to both glycosylated derivatives that showed the number of hydrogen bond donors exceeded as a violation. A range of possible molecular targets was identified for resveratrol (69 targets), polydatin (11 targets), and resveratrol 3-α-glucoside (10 targets), including enzymes, receptors, and transporters, being able to modulate several cell signaling pathways. Discussion/Conclusion: Finally, through in silico approaches, we obtained information about the pharmacokinetics and pharmacodynamics profile of resveratrol, polydatin, and resveratrol 3-α-glucoside. Thus, our study may guide future investigations regarding the possible mechanisms of action of resveratrol and its glycosylated derivatives in order to contribute to diseases treatments. Acknowledgements: This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) [Finance Code 001]; and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [research grant 423028/2018-9].

In silico pharmacokinetics and pharmacodynamics prediction of resveratrol

and two glycosylated derivatives

SchiMiTh, lucia eManueli1; anDré-Miral, corrine2; Muccillo-BaiSch, ana luiza1; horT, Mariana appel1

1 Programa de Pós-graduação em Ciências da Saúde, Faculdade de Medicina, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil; 2 Nantes Université, Unité en

Sciences Biologiques et Biotechnologies (US2B), UMR 6286, Nantes, France.

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Organ-on Chip is a result of tissue engineer and microfluidic convergency, acting as an effective solution to pursue new methodologies for drug discovery and personalized disease treatments. The high cost of drug development demands the need to develop more predictive tissue models using human cells to determine drug efficacy and safety in advance of clinical testing. However, a more predictive model requires the integration of different tissues, dynamic cell environments and cellular communication to the expression of high-fidelity organ function. In this context, the microfluidic technology is poised to fill the gaps in drug screening by offering predictive human tissue models with methods of sophisticated tissue assembly. Here we propose a junction of three different 3D tissue engineered cultures (skin, intestine and liver) in a 3-organ-on chip microfluidic

device to propose a methodology to verify topic or oral drug administration and liver toxicity. For this, we developed models of human reconstituted skin, intestinal barrier and liver spheroids, which were deep characterized in terms of histology, morphology and functionality. Our results show that our models is functional and mimetites functions of the real organs. The Chip integration of all the tissues on the chip was well succeeded and improved viability of the 3D cultures. After treatment with known toxic substances and, we observed absorption of drugs, which caused liver injury, as expected. In conclusion, here we present a new methodology to screen liver toxicity before animal testes in two contexts, oral and topic administration of drugs. Acknowledgments – CNPEM, NATURA, MCTI

In vitro oral and topic absorption toxicity test standardization using 3D cell cultures and microfluidic systems

ganzerla, MeliSSa DiBBern1; inDolFo, naThalia De carvalho2; arroTeia, kelen FaBiola2; Figueira, ana carolina Migliorini1

1 Laboratório Nacional De Biociencias, CNPEM, Campinas, Brasil; 2 Natura Co, São Paulo, Brazil.

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Introduction: The recent scenario of COVID-19 has resulted in an increase in hand cleansing products such as soaps, synthetic detergents, antiseptic handwashes and alcohol-based hand sanitizers. Consequently, it has been observed an emerging of several skin conditions including irritant contact (ICD) and allergic contact dermatitis (ACD) due to the excessive personal hygiene measures. These skin problems may result from a cascade of events which involve cell damage, inflammatory responses, cytokines release and activation of dendritic and T-cells; the latter being a key biological event underlying skin sensitization. Chlorhexidine is an effective antiseptic agent which is widely used in cosmetic, hospital products and medicines. Although clinical studies for chlorhexidine are common, the in vitro assessment and the molecular mechanisms involved in the irritant and allergic contact dermatitis are rarely reported. Objective: Evaluate the skin irritation and skin sensitization potential of a commercial formulation containing the antiseptic chlorhexidine (1%) by in vitro methods. Methods: The skin irritation potential was evaluated according to OECD TG 439 through the determination of cell viability in ex vivo human skin fragments using the vital dye MTT. Additionally, the inflammatory cytokine IL-1α was quantified. For skin sensitization assessment, the in chemico direct peptide reactive assay (DPRA) was performed following OECD TG 442C, which evaluates the cysteine- or lysine-containing peptide depletion using high-performance liquid chromatography (HPLC). The skin sensitization was also evaluated using the human cell line activation test (h-CLAT, OECD TG 442E), which quantifies changes of cell surface marker expression (CD86 and CD54) on a human monocytic leukemia cell

line (THP-1 cells), using flow cytometry. Results: The results of skin irritation (OECD TG 439) demonstrated that the positive control LSS (5%) was considered irritant since it reduced more than 50% in cell viability when compared to non-treated control. On the other hand, the product containing chlorhexidine (1%) was classified as non-irritant, since they presented cell viability of approximately 73.55%. In addition, treatment with the product assessed did not promote a significant increase in the inflammatory cytokine IL1-α release when compared to baseline control. The in chemico DPRA assay demonstrated that, as expected, the positive control dinitrochlorobenzene (DNCB) presented positive prediction for skin sensitization. The ingredient and the product assessed also presented positive prediction for skin sensitization (low reactivity) according to OECD TG 442C. Finally, the results from h-CLAT assay showed that the positive control DNCB presented relative fluorescence intensities (RFI) ≥ 150 for CD86 and ≥ 200 for CD54, which classifies this substance as sensitizing, according to OECD TG 442E. Moreover, the product containing chlorhexidine (1%) also presented sensitizing potential since RFI for CD86 were above the acceptable value for non-sensitizing substances (on going experiment). Conclusion: The results showed that the product containing chlorhexidine (1%) did not present potential for skin irritation. In addition, it demonstrated positive prediction for skin sensitization potential, for both in chemico DPRA and in vitro h-CLAT assays. Further studies should be conducted with this hand hygiene products category to better understand the molecular mechanisms involved in the irritant and allergic contact dermatitis.

In vitro strategy to assess skin irritation and sensitization potential

of a commercial formulation containing the antiseptic chlorhexidine

kaWakaMi, caMila MarTinS1; Silva, guSTavo henrique1; Moura, kézia1; pinheiro, ana l.T.a.1; pinheiro, aDriano Da Silva1; eBerlin, SaMara1; gaSpar, lorena rigo2; Fuzinaga, ThaiS2; vicenTe, eDuarDo3; Facchini, guSTavo1

1 Kosmoscience Group, Valinhos, SP, Brazil; 2 School of Pharmaceutical Sciences of Ribeirão Preto, University of Sao Paulo, Brazil; 3 School of Science and

Engineering/Chemistry Institute, Sao Paulo State University, Brazil.

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Introduction: Chemically-induced respiratory sensitization is an immune-mediated response caused by repetitive exposures to low molecular weight (LMW) allergens and consequently the development of clinical symptoms comprising allergic asthma and rhinitis. However, there are no in vivo or in vitro methods for predicting pre-clinically the respiratory sensitization potential of LMW chemicals. One of the challenges concerning this scenario is the lack of knowledge related to the immunological mechanisms involved in respiratory sensitization. Regarding contact dermatitis, for instance, it is known that dendritic cells (DCs) induce a Th1-type response mediated by T lymphocytes. On the other hand, although it is well documented that chemical respiratory allergens can trigger a Th2 reaction even after dermal exposure, the mechanisms underlying this immunological outcome are still not fully understood. Given the mandatory role of DCs in linking the innate and adaptative immune responses, we investigated the inflammatory alterations triggered by LMW respiratory allergens in this cell type, aiming to subsidize the development of in vitro methods for predicting chemical respiratory allergy potential. Objective: We proposed using the THP-1 monocytic dendritic cells model to investigate the inflammatory profile, maturation, and activation of DCs after exposure to respiratory sensitizers. Methods: In the first stage, we evaluated the cytotoxicity of seven known respiratory sensitizers (Chloramine-T, Piperazine, Maleic Anhydride, Cyanuric Chloride, Trimellitic Anhydride Chloride, Trimellitic Anhydride, and Glutaraldehyde) using the MTT reduction assay 24h after exposure for further determination of the 80% cell viability concentration (CV80). Thus, cells were exposed to respiratory sensitizers CV80 for 24 hours, following the quantification of inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and

TNF-α) in both supernatant and lysate samples using the Cytometric Bead Array (CBA) technique. Moreover, THP-1 dendritic cells underwent flow cytometry analysis to verify the expression of surface activation biomarkers (CD86, HLA-DR and CD11c) subsequently the respiratory allergens exposure. Results: The results demonstrated that most LMW respiratory sensitizers increased the CD86 and HLA-DR activation/maturation biomarkers expression by THP-1 cell line. Moreover, the surface integrin CD11c was upregulated by all evaluated test chemicals. Regarding the inflammatory profile, five of the seven allergens triggered a significant production increment of IL-6 in both cell lysate and supernatant, meanwhile increased IL-8 expression was observed just for three evaluated chemicals. Discussion/Conclusion: The obtained results support the hypothesis that exposure to respiratory allergens can promote alterations in DCs maturation/activation status, as well as trigger pro-inflammatory changes in these cells, highlighting the increased expression of CD11c surface biomarker and IL-6. The literature previously reported that the increase in IL-6 production by pulmonary CD11c+ DCs is linked to a polarization of the immune response towards a Th2 profile, suppressing the Th1 response that is regularly observed considering dermal sensitizers exposure and contact dermatitis progression. Therefore, evidence was found to support the elucidation of chemical respiratory allergy pathogenesis, as well as give mechanistic background to allow the differentiation between dermal and respiratory sensitizers regarding the seeking of Th2 and Th1 responses, respectively. Aknowledgements: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Financiadora de Estudos e Projetos (FINEP).

Inflammatory alterations triggered by respiratory sensitizers in human dendritic cells:

providing evidence to support the chemical respiratory allergy mechanistic background

MenDonça, izaDora caroline FurTaDo; Silva, arTur chriSTian garcia; carvalho Filho, Sérgio De MoraiS; valaDareS, Marize caMpoS1

Laboratory of Research and Education in In vitro Toxicology (Tox In), Faculty of Pharmacy, Federal University of Goiás.

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Despite the fact that UV filters are considered safe to use, there are few reports about their skin sensitizing and photosensitizing potential. The majority of tests are performed in monolayers or through photopatch test reactions. Recently, OECD published TG 498 addressing in vitro phototoxicity (photoirritation) using a reconstructed skin model, but there are still no validated assays using the 3D model to assess skin sensitization and photosensitization. In recent years, the concomitant use of insect repellents, mainly DEET and IR3535, with UV filters has increased due to the high incidence of UV radiation and a higher occurrence of mosquito-borne diseases such as dengue, Zika and Chikungunya. Therefore, the purpose of this study was to evaluate the phototoxic and skin sensitizing/photosensitizing potential of the combination of UV filters (avobenzone, ethylhexyl methoxycinnamate and octocrylene) with the insect repellent IR3535 (ethyl butylacetylaminopropionate), using a reconstructed human skin model. This combination was submitted to phototoxicity tests, according to OECD TG 498 (OECD, 2021) and to skin sensitization and photosensitization tests (IL-18 analysis) in a reconstructed human skin model, exposed or not to 6 J/cm2 of UVA radiation. Among the results obtained, the combination containing UV filters and IR3535 proved to be non-phototoxic, with lower reduction in cell viability

(Δ: 3.5%) when compared to non-irradiated tissue treated with the same concentrations of substances, following the international recommendations for the assay and OECD TG 498, which indicate a cut-off of 30%. Furthermore, the combination did not present photosensitizing potential, since it did not promote a statistically significant reduction in IL-18 production after irradiation and presented a low stimulation index, IL-18 SI (Irr/non-irr): 0.9, which according to Galbiati et al. (doi.org/10.1016/j.tiv.2013.06.008) is not considered a photoallergen (cut-off: SI > 1.3). According to the ECHA (European Chemicals Agency) and HSDB (Hazardous Substances Data Bank) platforms, IR3535 has no evidence of sensitizing or photosensitizing potential, while avobenzone, alongside with other UV filters, continued to be the most common allergens that elicited photopatch test reactions. To conclude, the 3D model used in this study proved to be promising in the evaluation of phototoxicity and sensitization/photosensitization of the combination containing UV filters and insect repellent IR3535, which so far has proven to be safe for use. Acknowledgments: This work was developed within the framework “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and “Conselho Nacional de Pesquisa” (CNPq, Brazil).

Influence of a combination containing UV filters and insect repellent IR3535

in photoinduced processes

gluzezak, ana júlia paSuch1; kaWakaMi, caMila MarTinS1; TavareS, renaTa Spagolla napoleão1; Fuzinaga, ThaiS yuMe Toriy1; Maria-engler, Silvya STuchi2; gaSpar, lorena rigo1

1 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil; 2 School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

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Introduction: The number of molecules applying for new drugs discarded in large drug development studies justifies the efforts of the scientific community to develop more predictive models for liver toxicity assessment. The lack of biosimilar models capable of predicting these damages has led to the development of models on different fronts, such as bioengineering to recreate the hepatic parenchyma’s complex organization or even using liver-on a-chip to simulate the elimination flow of newly metabolized xenobiotics. As an additional strategy, the use of an organ-specific matrix to construct more predictive liver models is a strategy to stimulate the differentiation of cellular phenotype and the functionality of already established liver lines. Objective: The aims to evaluate the influence of organ-specific extracellular matrix and bioengineering resources on the functionality of HepG2 liver cells, creating a model for evaluating liver metabolism in vitro closer to the relevant functional subsets in liver biology using, for this, the complex extracellular matrix from the porcine liver of spoil from the food industry. Methods: The complex hepatic extracellular matrix was derived from the decellularization of swine liver and the chemical digestion that followed. The resulting product was characterized by biochemical and kinetic endpoints using histological and biochemical methods. The

proposed model was used using the sandwich configuration and the hepatic lineage was used HepG2. Increased liver metabolism due to the organ-specific microenvironment, cell morphology, formation of liver canaliculi, presence of MRP-2 receptors, and cytochrome p-450 activity were assessed. Results: The obtained product and the biochemical parameters defined show that obtaining the hepatic extracellular matrix derived from the porcine liver is feasible, as well as suitable for cell culture, and can be used as a scaffold for models of bioconstructed liver tissue. The cell morphology of the HepG2 line cultivated in a complex organ-specific matrix exhibits polygonal form, clear cytoplasm, and structures like stable canaliculi, constating to HepG2 line cultivated without an extracellular matrix. Discussion/Conclusion: Changings on the cellular morphology of the HepG2 scanner, when grown in an organ-specific matrix, can indicate an increase in metabolic activity compared to single-layer culture. The presence of canaliculi and elimination of specific substrate via MRP-2 are indicators of biliary activity, fundamental for metabolic activity. Furthermore, the acquisition and use of the swine liver-derived extracellular matrix is consistent with the 3R principles. Acknowledgments: CAPES, CNPq, FINEP, FUNAPE, and FAPEG.

Influence of extracellular matrix organ-specific in behavior and cellular

functionality in HEPG-2 cell line

SanToS, ThaíS roSa MarqueS1; BorgeS, aManDa cecília guiMarãeS1; SanToS, jorDana anDraDe1; Silva, arTur chriSTian garcia1; Marize caMpoS valaDareS2

1 Laboratory of Education and Research in In Vitro Toxicology, Faculty of Pharmacy, Universidade Federal de Goiás, Goiânia, Goiás State, Brazil; 2 Laboratory of Education and Research in In Vitro

Toxicology, Faculty of Pharmacy, Universidade Federal de Goiás, Goiânia, Goiás State, Brazil

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Background: The growing and excessive use of pesticides has made the Brazilian agricultural production process increasingly dependent on these products to increase food production. According to IBAMA, in 2019, 583,864.50 tons of active ingredients of pesticides were used and in 2020 there was an increase of 8.65% in commercialization in the country, totaling 634,372.03 tons. When comparing data from 2010 we can see an increase in sales of 86.82% in ten years. Constant exposure to these toxic agents can cause chronic effects such as difficulty sleeping, forgetfulness, changes in the functioning of some organs, such as the liver, kidneys, and abortions. In addition, studies point to groups of pesticides as potential carcinogens. Fungicides are a class of pesticides used to combat various fungal groups that invade plantations, they can be classified as organic, inorganic, and systemic. The most exposed population are rural workers who handle, apply these fungicides, harvest, transport, store, and often reside on the property next to the crops. Objective: To assess the cytotoxic effect of Score® by Allium cepa test. Methodology: The test with Allium cepa described in the literature was used, with some modifications to adapt the method to our laboratory. This test allowed the evaluation of the cytotoxicity from the growth of Allium cepa roots. For the evaluation, onions of the Allium cepa species were chosen and a systemic fungicide that contains difenoconazole as active ingredient and has as a trade name Score®, was acquired from a grape producer. Two concentrations of Score® recommended by the Agência de Defesa Agropecuária do Paraná were prepared. Being concentration 1

(C1)= 0.08µl/mL and concentration 2 (C2)= 0.12µl/mL. As a positive control (C+) a concentration 4 times higher than C2 was used. For the negative control (C-) distilled water was used. The tests were performed in quadruplicate. The onions were left with the bulb immersed in water for 48 hours. After this time, the onions of C1, C2, and C+ were removed from the water and submerged in the respective concentrations of pesticide. The onions were left for another 72 hours with the bulb immersed, totaling 120 hours of the test. Throughout the test, the negative control was kept in distilled water. After this period, the onions were removed from the solutions and the size of the roots was measured. Result: C1 presented an average growth of 0.5 cm, C2 an average growth of 0.4 cm, C+ average growth size of 0.3 cm and in C- there was an average growth of 2 cm. Discussion/Conclusion: From the macroscopic analysis it was possible to observe the low growth of the roots that were exposed to the fungicide compared to the negative control. This low growth may be related to the cytotoxic effect of the Score® fungicide. Comparing the growth of Allium cepa roots, between the concentrations of the fungicide and the negative control, there was a difference in growth, which may be related to the cytotoxic effects of Score®. In the future, we intend to analyze nuclear alterations in dividing cells of these same roots to observe the genotoxic effects of the fungicide Score®. Keywords: Score®, Allium cepa test, cytotoxicity. Acknowledgments: The State University of Maringá, Postgraduate Program in Biosciences and Pathophysiology (PBF/UEM), CAPES e CNPq.

Macroscopic evaluation of Score® cytotoxicity by the Allium cepa test

MaSSucaTo, lucaS eDuarDo1; Siqueira, gaBriella Ferreira1; MoToMura, lariSSa TieMi akaMine1; lini, renaTa Sano2; Souza-kaneShiMa, alice Maria3; MoSSini, SiMone apareciDa galerani1,2

1 Laboratory of Toxicology, Department of Basic Health Sciences, State University of Maringá, Paraná, Brazil; 2 Postgraduate Program in Bioscience and Physiopathology,

State University of Maringá, Paraná, Brazil; 3 Laboratory of Pathology, Department of Basic Health Sciences, State University of Maringá, Paraná, Brazil.

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Introduction: Microfibrilated cellulose (MFC) and silica nanoparticles (SiO2NP) has been proposed for several applications in industry. MFC presents a thickener property and allows interactions with polymers and nanoparticles. SiO2NP can be used to increase the time of action of chemical products, working as a controlling delivery system. Those MFC and SiO2NP properties are being used to develop a more sustainable and renewable alcohol-based hand rub and a long-term disinfectant. Considering the SARS-CoV-2 pandemic, disinfection of hands and surfaces became an important nonpharmaceutical intervention, and the increase in the usage of disinfectants and alcohol led to a lack in the source of materials to produce hygiene and disinfection products. Therefore, new alternatives are being implemented with a sustainable and renewable feature, predicting that the use of alcohol-based hand rubs and disinfectants will now become a habit to avoid other public health outbreaks. Furthermore, it is important to consider the use of more sustainable and renewable source materials for other types of nanotechnology products. However, few studies have evaluated the skin irritation potential of these nanomaterials. Objective: This work aims to verify whether MFC and SiO2NP are skin irritants after acute and repeated exposure, using a reconstructed human epidermis (RHE). Methods: The skin irritation test was performed accordingly with OECD TG 439 using an in-house RHE model constructed from neonatal keratinocytes. The model used was within the quality control, presenting the four epidermis layers, verified by hematoxylin and eosin histology. The acute

exposure of MFC (1%) and SiO2NP (0.5%) followed the SkinEthic protocol. Repeated exposure was performed from an adaptation of the exposure conditions described by the same protocol. For negative controls, RHE was exposed to phosphate buffered saline and ultrapure water, which were also used as a vehicle. For the positive control, RHE was exposed to sodium dodecyl sulfate at 1%. Cell viability was measured by enzymatic conversion of the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye into formazan salt. Results: According to OECD TG 439, an irritant response is defined by a threshold of cell viability lower than 50% compared to the negative control. Our results showed that MFC and SiO2NP are not potential irritants in both exposure conditions (acute and repeated exposure). Previous studies obtained similar findings; Rashad et al., 2019 reported that after 3 days of exposure to in vitro U937 cells, MFC did not induce inflammatory chemokine expression, and 4 days after exposure on in vivo model, a higher expression of anti-inflamatory IL-1Ra was observed. Park et al., 2012 showed that SiO2NP did not cause acute skin irritation. Although our work confirm previous findings, there is no data regarding MFC and SiO2NP repeated exposure in RHE model. Conclusions: This study showed that MFC and SiO2NP are not skin irritants even after in a condition of continuous exposure. Overall, we believe that MFC and SiO2NP may be sustainable alternatives to be used in developing nanotechnology products, such as products proposed for control SARS-CoV-2 transmission. Acknowledgments: CAPES and CNPq for the financial support.

Microfibrillated cellulose and silica nanoparticles as sustainable alternatives

to developing nanotechnology products: the evaluation of skin irritation

cruz, juliana varella1,2; gagoSian, viviana coSTa1; MagalhãeS, WaShingTon3; caDeMarTori, peDro henrique gonzalez4; oliveira, Danielle palMa2; leMe, Daniela MoraeS1

1 Department of Genetics, Federal University of Paraná, Curitiba, PR, Brazil; 2 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP,

Brazil; 3 Embrapa Florestas, Colombo, PR, Brazil; 4 Graduate Program in Engineering and Science of Materials – PIPE, Federal University of Paraná, Curitiba, PR, Brazil.

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Introduction: The impact of pesticides on human health has been related by many different researchers. In developing countries, such as Brazil, the use of agrochemicals has increased considerably over the years, and thus damage to human health resulting from exposure has become greater, mainly due to a lack of and/or incomplete use of appropriate clothing as described in the product package insert, but also to a lack of good practice in the use of pesticides, especially in family farming. Objective: Considering the above, the objective of this work was to evaluate in vitro and in vivo dimethoate absorption through oral and nasal mucous membranes. Methods: The in vitro permeation test (IVPT) was performed in side-by-side diffusion cells, 3.4 mL receptor solution, stirring at 37 ± 1 °C, permeation area of 0,65cm2 by using oral (cheek, sublingual, esophageal) and nasal mucosa. 7 collections at 0.25, 0.5, 1, 1.5, 2, 3 and 4h from receptor solution were made. Dimethoate retention was analysed in all mucosa. The in vivo study was performed by collected saliva (spit in a test tube) and collection of nasal fluid by using swabs, 30 minutes before and four hours after dimethoate application on a lemon plantation, in seven rural workers, 1 resident and 1 researcher who monitored all applications. The CEP/UFPE approval was n° 5.007.282. All dimethoate analyses were performed by validated LC-MS/MS. Results: The cumulative dimethoate permeation was 2.5 times greater through the esophageal compared to the buccal mucosa. However, the thickness of the buccal mucosa was about 3 times greater than that of the esophagus (356.0 ± 48.79 and 962.0 ± 80.44mm), justifying this result. The percentage of dimethoate

permeated was around 1, 2 and 8% of the amount applied, from the buccal, esophageal and sublingual mucosa respectively. The dimethoate permeated through the nasal mucosa was 3,683.28 ng/cm2 after the 4-hour experiment, which means 11,08 ± 2,04 % of the amount applied. For the in vivo study, at the end of 4 hours of application, the highest amount of dimethoate in saliva was found in two workers, 3 and 6 (383.76 and 1,198.27 ng/mL) corroborating the observations by the researcher (talking during the application and stopping the application to smoke). Dimethoate was found in the nasal fluid of three workers at concentrations of 6.17, 3.16 and 4.36 ng/mL. Discussion/Conclusion: According to Thredgold (2019), in agricultural settings it is reported that 90% of total pesticide exposure occurs in ways attributed to dermal absorption or ingestion, while 10% is attributed to respiratory absorption. Even so, these less important routes are also responsible for exposure of workers. The pesticide solubilized in saliva and found in nasal mucosa can be directly absorbed through the tissue. At the same time, nasal mucosa has a large contact surface and high vascularization. It is worth mentioning that a direct correlation cannot be made between the amount of dimethoate found in the participant’s saliva and nasal mucosa with the IVPT results, since these fluids do not remain static in the individual during the 4 hours of work. However, these data are worrying, given that the application of pesticides is a routine activity and can have a serious impact on workers’ health. A great effort in the area of education must be made in an attempt to reverse this situation. Acknowledged: CNPq and FACEPE.

Nasal and buccal absoption of dimethoate: in vitro x in vivo study

anDraDe, ana roSa BriSSanT; carvalho, Deoclécio luSToSa; Souza, aSley Thalia MeDeiroS; kiShiShiTa, juliana; Silva, joSé WelliThoM viTurino; BeDor, Danilo céSar galinDo; SanTana, Davi pereira; leal, leila BaSToS

Universidade Federal de Pernambuco (NUDFAC-UFPE), Recife, PE, Brazil.

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Background/Introduction: Rapanea ferruginea is a brazilian plant popularly used to treatment of itching, rashes, hives and eczema. The bark extract has shown anti-inflammatory and anti-nociceptive activities. Nanosystems, such as nanoemulsions and nanoemulgel, are dermatological carriers that improve drug release and dermal drug permeation. Nanoemulsions with the R. ferruginea extract showed improved anti-inflammatory capacity in in vitro and in vivo models. Objective: To evaluate the effects of nanemulgel containing Rapanea ferruginea bark extract on cell viability, irritative potential and photoprotection using in vitro models. Methods: The glycolic extract of R. ferruginea bark was obtained by the dissolution of the concentrated alcoholic extract in propylene glycol. The nanoemulsion was prepared by phase inversion method. The R. ferruginea bark extract-loaded nanoemulgel was prepared using nanoemulsion:carboxyvinyl gel proportion1:2. The antioxidant activity of the extract was evaluated by DPPH method. The extract cytotoxity (1, 10, 100 and 300 µg/mL) and the nanoemulsion on L929 cells was evaluated by MTT method. The irritative potential of the extract, nanoemulsion and nanoemulgel was tested on L929 cells through an agarose-overlay method. Then, the extract and the nanoemulsion were tested for their photochemoprotective (UVA and UVB) effect using L929 cells. A photoprotection study of the R. ferruginea bark extract-loaded nanoemulgel was performed in L929 cells using irradiation method and quartz plates. A commercial sunscrenn FPS 30 and the base nanoemulgel were utilized as controls. The photoprotection factor was calculated by accessing the cell viability of irradiated and non-irradiated cells. Results: The extract presents antioxidante activity in DPPH method with CE50 of 0.95 mg/mL ± 0.03. The extract and nanoemulsion preparation

did not exhibited cytotoxic effect in L929 cells. Pure R. ferruginea extract and loaded formulations did not present irritative potential using the agarose-overlay method. The extract (100 and 300 µg/mL) and the nanoemulsion show photoprotective effect on both UVA and UVB radiations, protecting the L929 cells of the cytotoxicity irradiation effect. In the photoprotection study, the nanoemulgel containing R. ferruginea extract show a photoprotection factor of 12.96 ± 2.29 and the base gel provided an insignificant photoprotection factor of 1.49 ± 0.50. Discussion: The skin is highly exposed to the deleterious effects of UV solar radiation, which has turned into a major environmental carcinogen. In vitro methods using fibroblasts showed that R. ferruginea extract did not present cytotoxic effects on L929 cells and the agarose-overlay method demonstrated that the formulations are no irritative. The extract and nanoemulgel showed capacity of protecting L929 cell of UVA/UVB irradiations. Nanoemulgel formulation containing R. ferruginea extract showed photoprotective properties, similarly as the one reported by commercial FPS15 sunscreen. The biochemical, molecular and histological changes induced by acute skin UVB exposure can lead to oxidative stress, inflammation and photoaging. This research showed that in addition to photoprotective characteristics, the extract presents antioxidant activity and literature data demonstrated that R. ferruginea preparations decrease the release of the TNF, IL-1β, and KC cytokines and MPO activity in skin inflammation. Conclusion: The results show the antioxidant and photoprotective potential of the R. ferruginea bark extract and the respective loaded nanostructured system, which can be considered a promising active in the development of new products for skin protection and care.

Photoprotective effect of Rapanea ferruginea bark extract-loaded nanoemulgel

corDenuzzi, DoryS angela; BenvenuTTi, lariSSa; SanTin, joSé roBerTo; lucinDa-Silva, ruTh Meri

Postgraduate Program in Pharmaceutical Sciences. University of Vale do Itajaí, Itajaí, SC, Brazil.

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Introduction: The physiological characteristic of the human corneal epithelium makes it more susceptible to tissue damage caused by substances or environmental conditions. As such, eye corrosion and irritation are important endpoints for the toxicological evaluation of ingredients and cosmetics. For a long time, these outcomes have been evaluated using animal models (Draize test). However, social pressure, ethical concerns and scientific motivations lead to the development of alternative methods, such as reconstituted human corneal epithelium (RhCE) models. These models have been widely used as assessment tools for scientific and regulatory purposes. Currently, there are four commercially available models validated for this purpose, which integrate the OECD Test Guideline n. 492 (EpiOcular™ EIT, SkinEthic™ HCE, LabCyte CORNEA MODEL 24 EIT and MCTT HCE™ EIT). In Brazil, due to bureaucratic and customs issues, the acquisition of these models is extremely challenging. In this context, some laboratories developed their own models, such as the Tox in Ocular model developed by Artur and collaborators (Silva, 2019). All these models are formed by a stratified epithelium nurtured by media with different compositions, most of which are based on commercial sources whose exact content is not available. The dependence on commercial media impacts the cost of tests and can limit customization (e.g.: developing a free animal content model). Furthermore, unavailability in the market and discontinuation could impair the use of a model or even require completely new development. Objective: The goal of this project is to develop and validate (interlaboratory) a new RhCE model based on the Tox in Ocular using a chemically defined culture medium. Methods: For the reconstructed tissue, HaCaT keratinocytes were seeded in 24-well inserts at a density of 5×105 cells/insert and cultured for 4h under a submerged condition for adhesion. Following that, the apical medium was removed,

and cells were maintained at an air-liquid interface for 5 to 8 days until adequate stratification with the specific media. Four media conditions were evaluated (each condition had different composition of the supplement blend), as well the presence or absence of type I collagen matrix on the inserts. The choice of the supplements for the culture medium was based on the concentration scale provided in the commercial kit. After the differentiation period, histological analyzes of the models using hematoxylin-eosin were performed. Results/Discussion: The four conditions maintained at the air-liquid interface for 5 days showed no significant histological differences. 4-5 layers of cells formed an epithelium with a thickness comparable to Tox in Ocular model and the human cornea. The morphology of the samples maintained at the air-liquid interface for 5 days was comparable to the Tox in Ocular model. The samples kept for extra 3 days at the air-liquid interface, presented the formation of many vacuoles, which resulted in a loss of homogeneity of the tissue demonstrating cellular stress. These results lead us to believe that the major impact in the model is the stratification period time and not the tested variations on media components (such as the animal components elimination) or the matrix absence. These remain to be confirmed with further tests. Conclusion: With the results obtained so far, we have demonstrated the possibility of developing a biomimetic RhCE model using a known media composition, bringing the advantage of a free animal content model. The next steps of the study are the characterization of the tissue through biomarkers and the response using reference substances. Acknowledgments: Grupo Boticário, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) e Fundação de Apoio à pesquisa do Estado de Goiás.

Preliminary data on a newly developed biomimetic Reconstituted Human Ocular Epithelium Model using an

animal free defined medium

BoSqueTTi, Bruna1; caTarino, carolina MoTTer1; coSTa, Meg criSTina Da caSTilho1; Thá, eManoela lunDgren1; Silva, arTur chriSTian garcia2; Schuck, DeSiree cigaran1; canavez,

anDrezza Di pieTro Micali1; BroheM, carla aBDo1; valaDareS, Marize caMpoS2

1 Grupo Boticário; 2 Universidade Federal de Goiás.

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Introduction: Plants rich in substances with antioxidant and depigmenting potential can be useful to prevent skin aging and the appearance of hyperchromias, being used as actives in cosmetics. Several studies indicate that the pomegranate peel extract has strong antioxidant and depigmenting potential in in vitro studies, but because they are phytocomplexes, it is necessary to evaluate the safety of these products before possible clinical tests on volunteers. Objective: To evaluate the safety of nanoemulsion and emulgel cosmetics containing pomegranate peel extract using alternative in vitro methods. Methods: The pomegranate peel extract was obtained by dynamic maceration and characterized in terms of total phenolic content, ellagic acid content by HPLC and antioxidant activity (AA) by the DPPH method. The extract was incorporated into a nanoemulsion (NE) and emulgel (EM) cosmetic base at a concentration of 0.1%. The cytotoxicity of pomegranate peel extract (1, 10, 100 μg/mL) and products was determined by cell viability assay using the MTT method on L929 murine fibroblast cell line. The skin irritation potential (Agarose overlay) of the products was determined by the agar diffusion method, using L929 cells. Extract, nanoemulsion and emulgel samples were tested at concentrations of 1, 10, 100 μg/mL. The degree of irritation was evaluated by the lysis zone, according to the classification described by the American Pharmacopoeia. The irritating potential was also analyzed by the HET-CAM model, applying the nanoemulsion and the concentrated extract directly, without dilution, and the emulgel with 1:10 previous dilution. The samples were applied on the chorio-allantoic membrane and the possible irritating effects observed after 5 minutes. The results were compared with the positive and negative controls, 0.1 M NaOH

and 0.9% NaCl, respectively. Results: The soft extract showed AA with EC50 of 300.03 ± 0.52 μg/mL, total phenolic content of 296.72 ± 6.92 mg EAG/g and ellagic acid content of 2.73 ± 0.60%. Two phycosmetic formulations containing 0.1% of the pomegranate peels soft extract were obtained, NE and EM. In the cell viability assay by the MTT method, the extract did not promote cytotoxicity after 24 hours of incubation at all concentrations evaluated (1, 10 or 100 µg/mL). In the agarose ovelay assay, the extract samples at concentrations 1, 10 or 100 µg/mL were not able to cause the formation of a toxicity halo around the sample, the nanoemulsion samples, with and without extract, showed a toxicity halo with classification 3 – moderate, and the emulgel samples, with and without extract, presented reactivity classification 2 – mild. The safety evaluation by the HET-CAM method showed that the extract and tested formulations, NE and EM, did not promote blood extravasation or even the formation of blood clots in the microvessels present. Discussion: The viability test by the MTT method demonstrated that the extract does not promote toxicity. In the agarose ovelay assay, the extract did not show reactivity, the emulgel showed mild reactivity, while the nanoemulsion showed moderate reactivity, probably due to the presence of the surfactants used with irritating potential. The safety evaluation by the HET-CAM method demonstrated that the soft extract and tested formulations were classified as safe. Conclusion: Conducting the research allowed obtaining scientific knowledge about the safety of pomegranate peel extract and phytocosmetic products containing 0.1% of the extract, as well as contributing to the development of innovative cosmetics.

Safety evaluation of nanoemulsion and emulgel containing pomegranate peel

extract using alternative in vitro methods.

ruDolF, carline; BenvenuTTi, lariSSa; rocha, anna carolina Furaer; corDenuzzi, DoryS angela; SanTin, joSé roBerTo; lucinDa-Silva, ruTh Meri

Postgraduate Program in Pharmaceutical Sciences. University of Vale do Itajaí, Itajaí, SC, Brazil.

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Introduction: Low molecular weight (LMW) respiratory sensitizers are chemical entities that trigger hypersensitivity in the lungs and airways after being inhaled, culminating in the development of allergic asthma/rhinitis after long-term exposures mainly regarding an occupational context. Contrastingly with the dermal sensitizers, known by contact dermatitis elicitation after cutaneous exposure, the mechanisms that underly the toxic response to LMW respiratory allergens are not entirely available, so up to now, there are still no validated methods for addressing the lung sensitization potential of chemicals. Besides, although it is known that these toxicants must bind covalently to proteins to be recognized by the immune system cells, it is still not clear which are the molecular targets that are involved in this haptenization process. Thus, even though some attempts to apply skin sensitization assays to evaluate respiratory sensitizers have been taken, there are significant anatomical/structural discrepancies regarding these two different biological compartments (skin versus lungs), which can impact the toxicokinetics/toxicodynamic and mode of action (MoA) of this class of compounds. Bearing in mind that mucus is a considerable physiological barrier overspread in the respiratory tract and that it has a major role in the pharmaco/toxicokinetic features of chemicals that enter this system through inhalation, in this work, we investigated the potential interactions of LMW respiratory sensitizers with the mucin, the major protein mucus component, using a spectroscopy-based method commonly used for addressing mucus interaction with inhaled drugs. Objectives: The purpose of this work was to perform an in chemico spectroscopy analysis to evaluate the potential interactions between LMW respiratory sensitizers and mucus, which could drive the formation of toxicant-mucin complexes. Methods: We evaluated seven known respiratory sensitizers (Chloramine T, Piperazine, Maleic Anhydride, Cyanuric Chloride, Trimellitic Anhydride Chloride, Trimellitic Anhydride,

and Glutaraldehyde) and two non-sensitizers (Glycerol and Lactic Acid). The test chemicals were solubilized in Phosphate-buffered Saline (PBS) in four concentrations ranging from 200 – 1,25µM and incubated with a solution of isolated mucin at 0,12 mg/mL (pH 7.4) for 0 and 24 hours at 37°C. After incubation, absorbance was measured in a spectrophotometer at the wavelengths from 200 to 400 nm (10 nm steps) to evaluate changes in the spectroscopy behavior of mucin resulting from a toxicant-mucin complex formation. Results: The results demonstrated that mucin had an absorption peak at 260 nm, as expected according to described in the literature. Furthermore, we observed a slight absorbance increase in the higher evaluated concentrations of glutaraldehyde and cyanuric chloride (hyperchromism) and a decrease in the chemical chloramine-T (hypochromism) after 24 hours of incubation. However, additional data evaluating the chemical glutaraldehyde were obtained through the Isothermal Titration Calorimetry (ITC) method, where no potential covalent interactions were detected between this chemical and isolated mucin. Nevertheless, the other chemicals did not promote significant alterations in the mucin spectroscopic profile. Discussion/Conclusion: In this work, we demonstrated that, although some minor alterations were detected, LMW respiratory sensitizers do not potentially interact with mucin, the major protein component of pulmonary mucus. These results clarified two essential aspects of human response to these toxicants: I) the lung mucus barrier does not seem to have a mandatory role in the toxicokinetic of these chemicals and II) the pulmonary mucin is not a worthwhile target for the molecular initiation event (covalent binding to proteins) of the respiratory sensitization, once no possible interactions were detected between this protein and the evaluated chemicals. Aknowledgements: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Financiadora de Estudos e Projetos (FINEP).

Spectroscopic assessment of lung mucus as a tool for toxicokinetic/toxicodynamic

assessment of chemical respiratory sensitizers

MenDonça, izaDora caroline FurTaDo1; Silva, arTur chriSTian garcia1; MenDanha, SeBaSTião anTônio2; valaDareS, Marize caMpoS1

1 Laboratory of Research and Education in In vitro Toxicology (Tox In), Faculty of Pharmacy, Federal University of Goiás; 2 Center for Research, Technological

Development and Innovation in Pharmaceuticals, Medicines and Cosmetics (FarmaTec), Faculty of Pharmacy, Federal University of Goiás.

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New psychoactive substances (NPS) have emerged in recent years to offer an alternative to classical drugs. The idea behind NPS appearance is to provide recreative effects to the users and circumvent the prohibition. These substances can yield many threats to both law enforcement and health. The most common NPS are amphetamines and cathinones, which have similar structures and stimulating effects. There is a lack in both the structural and toxicological properties of these drugs. Because of their complexity, experimental approaches can be challenging to provide results. Given these facts, in silico tools are an alternative to obtaining data about NPS. The study’s primary goal was to give an insight into the toxicological properties using in silico evaluation. We studied 42 (forty-two) pairs of homologous amphetamines and cathinones, a total of 84 (eight-four) structures. For this purpose, we calculated the toxicological properties of these compounds from different in silico software: Protox II, SwissADME, GUSAR, and TEST. The resulting data matrix contained 84 samples split into two classes (amphetamines and cathinones) and 24 (twenty-four) collected variables containing calculated values for LD50, solubility, and LogP using different models according to each software. We evaluate the influence of the variables on the samples by multivariate tools. Principal Component Analysis – PCA was used to explore the system, whereas Soft Independent Modeling of Class Analogy – SIMCA and Partial Least Square Discriminant Analysis – PLS-DA were used to evaluate the classification. PCA showed a profile in discriminating amphetamines and cathinones. This

behavior was further assessed through SIMCA and PLS-DA. In both supervised techniques, we performed a variable selection. According to their activity, SIMCA results showed around 10% of error in the classification of amphetamines and cathinones. Four principal components and twelve variables were necessary to model the classes. Solubility information was found as those variables with the most discriminating power. PLS-DA results showed that no misclassification was found. Three principal components were necessary to model the classes, containing around 93.5% of the complete information. Both internal (Q2) and external (R2) regression coefficients showed a good fit (0.83 and 0.85, respectively). Calculated LogP values were the most important variables in this case. In conclusion, we can say that in silico tools raised information about NPS amphetamines and cathinones. Multivariate analysis showed that the software could help give insight into the toxicological properties of these drugs. Solubility and LogP variables were the most important in discriminating amphetamines and cathinones. It makes sense, given that the only difference among homologous structures is a carbonyl group. Finally, it is crucial to strengthen that having values from different software and different models for the properties is essential to establish the classes. We thank the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 465450/2014-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) for financial support.

Study of the toxicity of NPS amphetamines and cathinones using in silico tools

roDrigueS, caio henrique pinke; caSTro, jaDe SiMõeS; Bruni, aline ThaiS

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

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The in vivo rabbit eye test was developed over 75 years ago to assess the irritation and corrosion potential of substances that may come into contact with the eye. Today, regulatory hazard classification and labeling systems based on United Nations Globally Harmonized System of Classification and Labeling (GHS) are still based on this method. While multiple in vitro methods have been developed as non-animal alternatives, their applicability to evaluate eye irritation potential of agrochemical formulations still need confirmation. Due to the complex nature of these products, they were not typically included as reference chemicals in test methods validation. In an attempt to identify one or more alternative methods that are reliable to evaluate eye irritation potential of end-use products, we conducted a retrospective evaluation of 158 agrochemical formulations. These agrochemical formulations had in vivo and in vitro data available and the additivity calculation of GHS was performed to determine if these non-animal alternative methods could accurately differentiate non-irritant and irritant formulations. This work is sponsored by Crop Life Brazil Alternative Methods Team (CLB_MAlt) to ILS and the data was provided by associated companies. In vivo data were available from the Draize test (OECD 405). In vitro results were available from at least one of three test methods:

bovine corneal opacity and permeability (BCOP, OECD 437); isolated chicken eye (ICE, OECD 438); and the Reconstructed Human Cornea-Like Epithelium (RhCE, OECD 492). All in vitro methods were conducted according to respective OECD test guidelines. The results herein suggest that the individual methods were highly predictive of end-use products that do not require classification for eye irritation hazard. However, even in vitro methods validated for identifying chemicals GHS category 1 (BCOP and ICE) did not reliably identify the agrochemicals that induce serious eye damage. In fact, for category 1, the major driver for classification of agrochemicals is the persistence of effects, which is not evaluated by the current in vitro methods. Nevertheless, the majority of agrochemical formulations falls into category that does not require classification (around 70%). Thus, these data suggest that the four alternative approaches can be used in different testing strategies to identify agrochemical formulations that do not require eye irritation hazard classification without using animals, which could significantly reduce the use of animals for this endpoint. Acknowledgments: Kolle S, Stinchcombe S, Inforzato M, Masinja W, Grivel A, Corvaro M and Baldassari J as representants of the international working group supporting this project.

Testing strategies for evaluation of eye irritation potential of agrochemical formulations

as an alternative to animal testing

chokSi, neepa1; laTorre, anDreia oliveira2; paiS, Mariana caSTello novo3; MuraTa, roSana zoriki hoSoMi4; caTalano, ShaDia M.i.5; aguilera, Mariana6; pireS, janaina apareciDa

carDoSo7; ogaSaWara, Maryanne8; haBe, priScila9; perjeSSy, giSele10; allen, DaviD1

1 Integrated Laboratory Systems, Research Triangle Park, NC, USA; 2 BASF SA, Sao Paulo, Brazil; 3 Syngenta Crop Protection, Sao Paulo, SP, Brazil; 4 Bayer SA, Sao Paulo, SP, Brazil; 5 Corteva

Agriscience, Barueri, SP, Brazil; 6 Ihara, Sorocaba, SP, Brazil; 7 Ourofino, Ribeirão Preto, SP, Brazil; 8 FMC, Campinas, SP, Brazil; 9 Sumitomo, Sao Paulo, SP, Brazil; 10 Crop Life Brazil, Sao Paulo, SP, Brazil.

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Introduction: Occupational asthma is a workplace-related allergic condition characterized by airway hyperresponsiveness and airflow limitation in response to different agents exposure. Specifically, the low molecular weight (LMW) respiratory sensitizers are defined as chemical entities that can cause airway hypersensitivity after inhaled, being classified by the EU REACH as Substances of Very High Concern (SVHC). For instance, this classification makes the regulatory background behind these substances as rigorous as those adopted for genotoxic/mutagenic chemicals. Although the relevance in both regulatory and human health contexts, there are still no validated methods for the risk assessment of LMW respiratory sensitizers, and one of the primary reasons for this issue resides in the fact that the Adverse Outcome Pathway (AOP), which precedes the clinical manifestations of occupational asthma, is not entirely elucidated. Bearing in mind the respiratory tract multicompartmental structure and the massive extension of the air-blood-barrier (ABB), composed primarily by pneumocytes and endothelial cells, most in vitro models for pulmonary toxicity are only based on the assessment of the epithelial compartment response to toxicant exposure. We hypothesized that the endothelial cells also contribute actively to the chemical respiratory allergy initiation/elongation processes, responding directly to respiratory sensitizers regardless of the cell crosstalk in the asthma pathological microenvironment. Objectives: This work proposed to evaluate in vitro the alterations triggered by LMW respiratory sensitizers in human endothelial cells to investigate this cell type’s participation in the mechanistic framework of chemical respiratory allergy, which clinically leads to occupational asthma. Methods: The study was conducted employing the endothelial cell line EA.hy926, so that seven known chemical respiratory allergens were evaluated (Chloramine T, Piperazine,

Maleic Anhydride, Cyanuric Chloride, Trimellitic Anhydride Chloride, Trimellitic Anhydride, and Glutaraldehyde). First, the cytotoxicity of the test materials was determined using the MTT reduction assay, following exposure for 24 hours. Thus, each substance’s cell viability 80% concentration (CV80) was defined. Posteriorly, endothelial cells were exposed to this concentration for 24 hours, and the inflammatory cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α) were measured afterward in both supernatant and cell lysate samples using the cytometric bead array (CBA) method. Moreover, the expression of the endothelial activation biomarker ICAM-1 was performed by flow cytometry analysis after exposing the EA.hy926 cells for 24 and 48 hours to the toxicants CV80. Results: The results have shown that endothelial cells exposed to five of the seven evaluated respiratory sensitizers displayed a significative increased production of IL-6 in both supernatant and lysate samples. Besides, some chemicals also prompted an increase in IL-8 and IL-1β production. Considering the expression of ICAM-1, five of the seven LMW sensitizers promoted a significant increase in this biomarker expression by endothelial cells not at 24 hours but 48 hours after exposure. Discussion/Conclusion: The results demonstrated that respiratory sensitizers can directly activate the human endothelium by releasing inflammatory mediators and increasing the adhesion molecules expression. These findings support the active participation of endothelial compartment in the mechanistic framework of chemical respiratory allergy, which might be encompassed considering the development of New Approach Methodologies (NAMs) for risk assessment of respiratory toxicants. Aknowledgements: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Financiadora de Estudos e Projetos (FINEP).

The endothelial compartment can actively participate in the adverse outcome pathway

(AOP) of chemical respiratory allergy

Silva, arTur chriSTian garcia; carvalho Filho, Sérgio De MoraiS; valaDareS, Marize caMpoS

Laboratory of Research and Education in In vitro Toxicology (Tox In), Faculty of Pharmacy, Federal University of Goiás.

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Brazil presents the fourth highest consumption of personal hygiene products, perfumery and cosmetics, on a market of around 2 billion dollars. Determining the potential for eye irritation is a key safety estimate for these products. The Normative Resolution (RN) 18/2014 brought a regulatory change that included a mandatory replacement of animal testing by alternative methods in Brazil. As a result, the interest in validated alternative methods has increased. The Bovine Cornea Opacity and Permeability Test (BCOP) is among the alternative methods accepted for regulatory purposes. Therefore, knowing the challenges and limitations of its implementation can contribute to a global diagnosis and increase its benefits and applicability in Brazil. Objective: to map the main challenges and technical limitations faced by the performers of alternative methods, with a focus on Eye Irritation and BCOP. Methods: This project was approved by the local Research Ethics Committee (CAAE: 17552519.1.0000.5243). Participants were practitioners of alternative methods in Brazil, including managers and professionals at academic laboratories, research centers (public and/or private). Volunteers were recruited through a list of 51 participants from the National Network of Alternative Methods-RENAMA/MCTI. Questionnaires with open and closed questions and focal individual interviews were performed with a discourse analysis of records and transcriptions for a qualitative synthesis. Results: The results show that 95% of the participants perform and 5% have already worked with alternative methods. Most respondents (71%) stated that the greatest difficulty faced is related to the high cost of validation/implementation, followed by the limited access to training (61.9%) and high cost of materials (61.9%), as well as purchasing reagents and equipment (42.9%). Regarding of eye irritation potential, 33% of respondents perform the BCOP test and 66.7% perform the short-term in vitro test (STE). The low availability of slaughterhouses to

obtain the bovine eye (50%) was its main limitation followed by the fact that this method provides a less assertive classification (42.9%) and a large waste of the eyeballs due to scratches and other damage (28.6%). Focal interviews (n=7) pointed out that the low availability of the eyeballs sources is related to the facilities of material disposal. Three participants described a 2-3 hours travel to the slaughterhouses, hindering the performance of the test on the same day. The test is not validated for execution after 24 hours of collection. Participants stated that preservation or cryopreservation system could solve the problem of material loss and reduce visits to collection sites. All participants addressed difficulties with the OECD Guide due to the lack of technical details, demanding other support materials and contact with experienced professionals. The need for access to more courses, practical activities and training for implementation of testes was presented, as RENAMA s actions through PremaSul were not seen as sufficient to absorb all the demand. Most participants from public laboratories reinforced the need for investments and the high cost of imported materials, indicating that the relative costs for validation in Brazil are much higher than in other countries. Discussion/Conclusion: These findings indicate several challenges to the implementation of BCOP and alternative methods, related to infrastructure, access to information and training, high costs, and technical limitations. Overcoming such limitations may demand an open innovation environment through dialogue between the Triple Helix (Companies; Scientific and Technological Institutions–ICTs and Government), with the strengthening of initiatives such as the RENAMA and the Brazilian Center for the Validation of Alternative Methods. Keywords: alternative methods, qualitative research, BCOP, eye irritation. Acknowledgements: The authors thank the RENAMA/MCTI for the technical assistance, and the financial support by Inova Fiocruz/Fundação Oswaldo Cruz.

The Implementation of Alternative Methods to the Use of Animals for ocular toxicity:

difficulties and opportunities for innovation

giMeneS, izaBela1,2; preSgrave, ocTavio1; gonzalez, Marcelo2; alveS, guTeMBerg2

1 Fundação Oswaldo Cruz-Fiocruz; 2 Universidade Federal Fluminense-UFF.

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Introduction: UV filters are widely used in the composition of cosmetics (such as sunscreen lotions) and a range of products to prevent from damage caused by the ultra-violet (UV) component of sunlight. Despite the benefits to human health, this group of substances has been drawing attention due to their possible environmental impacts when released into the aquatic environment, including endocrine disrupting effects. Objectives: Due to the restriction on the use of animals in the cosmetic industry, endpoints should be evaluated through alternative methods such as in vitro and in silico tools. which are now referred to as being New Approach Methodology (NAM) data. In order to obtain information on the endocrine disrupting effects as well as damage to aquatic environments assessed through data on the potential PBT (persistence/biodegradation, bioaccumulation and toxicity), ten organic UV filters were investigated using alternative methods. Methods: The following organic UV filters were investigated: diethylamino hydroxybenzoyl hexyl benzoate, octocrilene, ethylhexyl triazone, tris-biphenyl triazine; bis-ethylhexyloxyphenol methoxyphenyl triazine; butyl methoxydibenzoylmethane, homosalate, ethylhexyl salicylate, octyl methoxycinnamate and phenylbenzimidazole sulfonic acid. In vitro assays were retrieved from “ToxCast” (Toxicity Forecaster)”, an USEPA database available at https://comptox.epa.gov/dashboard. In silico data were retrieved from VEGA (https://www.vegahub.eu), T.E.S.T. (Toxicity Estimation Software Tool) and OECD Toolbox (version 4.3). The following endocrine receptors were investigated: androgen receptor (AR), estrogen receptor (ER1 and 2) and thyroid receptor beta (TRb). For environmental evaluation, biodegradation, bioaccumulation and fish toxicity were searched. Results: In vitro assays were available for 60% of the UV filters: three of them were active as antagonists (binding to the receptor blocking its action) to ER1 and ER2, AR and TRb receptors and inactive as

agonists for the same receptors. Two UV filters were totally inactive (phenylbenzimidazole sulfonic acid and ethylhexyl methoxycinnamate). One filter (ethylexyl salicylate) was only active to estrogen receptor antagonists. The in silico data confirmed in vitro data for all compounds, except for butyl methoxydibenzoylmethane, for which no activity was predicted in silico and therefore was considered inconclusive. From the 4 other UV filters investigated using only in silico tools, no endocrine activity was predicted for three of them. Discussion/Conclusion: By analyzing all the collected data, consistency in the results was verified for most (80%) of the compounds, since 30% were classified as endocrine disruptors (octocrilene, homosalate and ethylhexyl salicylate) and 50% as non-endocrine disruptors (bis-ethylhexyloxyphenol methoxyphenyl triazine, ethylhexyl methoxycinnamate, ethylhexyl triazone, tris-biphenyl triazine and phenylbenzimidazole sulfonic acid). Only two UV filters (diethylamino hydroxybenzoyl hexyl benzoate and butyl methoxydibenzoylmethane) could not be classified. In the environmental evaluation, although all UV filters presented high LogPow values, low bioconcentration fators were predicted in silico (categories 3 or 4). On the other hand, all compounds presented high toxicity to fishes and half of them were predicted as not readily biodegradable and/or persistent. Our results suggest that UV filters can be potentially harmful to the aquatic environment due to their possible acute toxicity and low biodegradability. The potential endocrine disrupting activity of 3 out of 10 UV filters also raises concerns, especially homosalate, which is also not readily biodegradable and toxic for fishes. Considering the environmental impact of substances depends not only on their toxic potential but also on exposure, further risk assessment analysis could provide more relevant information on those health care products. Acknowledgments: CNPq.

The use of NAMs to evaluate the toxicity of UV filters: emphasis on aquatic toxicity

and endocrine disrupting effects

nonino, eliSa De caSTro Wille; leMe, Daniela MoraiS; peSTana, cynThia BoMFiM

Department of Genetics, Federal University of Paraná, Curitiba, PR, Brazil.

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The registration of agrochemical formulations remains supported to a large extent based on animal testing to characterize eye and skin irritation potential, despite the multiple in vitro methods already developed as non-animal alternatives. Some advances toward regulatory acceptance can be granted applying Integrated Approaches to Testing and Assessment (IATA) developed by OECD. IATAs are flexible frameworks that “integrates and weights all relevant existing evidence and guides the targeted generation of new data, where required, to inform regulatory decision-making regarding potential hazard and/or risk.” An essential step of an IATA is to characterize the toxicity features of the target chemicals. In fact, considering that agrochemicals formulations are mixtures, the hazard characterization related to eye and skin irritation potential from large datasets can help building weight of evidence (WoE) and improve the integrated approach of testing strategies without animal testing to predict these endpoints. Therefore, a public dataset from the Brazilian Health and Regulatory Agency (ANVISA) of registered agrochemicals formulations until 2019 was retrospectively reviewed. This dataset provides the hazard classification based on United Nations Globally Harmonized System of Classification and Labeling (GHS) adopted by ANVISA in that year. The GHS hazard category for both endpoints, eye and skin irritation, and the type of formulation were available for a total of 1640 out of 1705 formulations. The prevalence of the GHS hazard categories was evaluated as well as the correlation between skin and eye irritation, since IATA 263 for serious eye damage and irritation considers data on skin corrosion (GHS Cat 1) in the WoE analysis to predict the eye irritation potential. The evaluation of this dataset revealed around 70% (1128/1640) of the formulations did not

require classification for eye irritation, while 19% (316/1640) and 12% (196/1640) were classified as GHS cat 2 and cat 1, respectively. Once considered only the liquid formulations water-based, the percentage of formulations not classified for eye irritation was even higher, 88.6% (444/501). For skin irritation, most formulations did not require classification as well, approximately 85% (1396/1640), while 9% (148/1640) and 5% (85/1640) were classified as GHS cat 2 and cat 3, respectively and only 1% (11/1640) were classified as skin corrosive GHS cat 1. The 11 formulations classified as skin corrosive, 46% (5/11) did not require classification for eye irritation, while 18% (2/11) and 36% (4/11) were classified as GHS cat 2 and cat 1, respectively. Considering the formulations classified as severe eye irritants (GHS cat 1), 127 out 196 formulations (65%) did not require classification for skin irritation and only 2% (4/196) were classified as skin corrosive. Overall, these data indicate that both, skin corrosion and severe eye irritation potential, should not be considered in the WoE analysis of agrochemicals formulations to predict the hazard potential of these endpoints. Nevertheless, it is likely that the negative eye irritation potential can provide evidence predicting the negative skin irritation. In this dataset, 93% of formulations (1045/1128) not classified for eye irritation were also not classified for skin irritation. In summary, the higher prevalence of formulations that did not require classification for eye and skin irritation indicates that integrated testing strategies with bottom-up approaches will be more accurate predicting human outcome. Additionally, data on skin corrosion or on eye damage should be carefully considered in WoE analysis to predict eye and skin irritation potential of agrochemical formulations. Acknowledgments: Kolle S and Stinchcombe S

Toxicity features of agrochemical formulations related to eye and skin irritation potential that

are important to build weight of evidence for integrated approach of testing strategies

laTorre, anDreia oliveira; pepaTo, ana clauDia De anDraDe; Faria, paTricia MiranDa; cazarin, karen

BASF SA, Sao Paulo, Brazil;

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From the first decade of the 2000s onwards, a world order phenomenon began to draw the attention of various agencies. This phenomenon became known as New Psychoactive Substances (NPS). They were spread as an alternative to classic drugs, mimicking their effects in a “legal” way. The main problem for individuals is the threat to health due to the absence of toxicological information. The most prominent NPS were amphetamines and cathinones. As they appeared in the market quickly, the experimental approaches classically employed faced a challenge in providing results. Given these facts, we found in silico tools a faster alternative for obtaining data on NPS. For these reasons, the study aimed to study and provide information on the toxicological properties of amphetamines and cathinones using in silico methodologies. In this work, we applied the MetadrugTM software for analyzing 21 pairs of homologous amphetamines and cathinones, a total of 42 structures. This software is part of the MetaBaseTM API to access the knowledge base behind all systems biology products of Clarivate Analytics (https://www.cortellislabs.com/page/?api=api-MB). Simulated variables are structural properties, CYP450 QSAR Models, Protein binding QSAR Models, ADME QSAR Models, predict the therapeutic activity and toxic effects. The total number of variables was 78. We used Soft Independent Modeling of Class Analogy (SIMCA) to evaluate the answers. This method considers the classification between amphetamines and cathinones and indicates the most significant parameters to define the groups. Results showed that both classes needed three principal component analysis to be modeled, and no misclassification was observed. To amphetamines, three variables presented the highest modeling power. They were i) affinity to human serum albumin, log value of the retention time (ADME QSAR Models); ii) potential to activate 5-hydroxytryptamine (serotonin) receptor 2B (Protein binding QSAR Models); and iii) Potential activity against arthritis (Prediction of therapeutic activity). On the other hand, to classify

the cathinones, the variables that result in the highest modeling power were seven. They were: i) affinity to human serum albumin, log value of the retention time (ADME QSAR Models); ii) potential to be a substrate of human P-glycoprotein transporter (Protein binding QSAR Models); iii) potential to activate 5-hydroxytryptamine (serotonin) receptor (Protein binding QSAR Models); iv) potential for inducing liver lipid accumulation (Prediction of toxic effects); v) potential to inhibit CYP2D6 (CYP450 QSAR Models); vi) potential activity against arthritis (Prediction of therapeutic activity); and vii) Potential activity against schizophrenia (Prediction of therapeutic activity). These results indicated that amphetamines are more stable in the transport protein. For cathinones, we observed that the main variables were related to the liver and correlated proteins. However, both groups showed potential to activate the serotonin receptor and activity against arthritis. These observations showed a lower toxic potential for cathinones and their greater instability when compared to amphetamines. In conclusion, we can say that in silico methodologies can provide assertive information about amphetamines and cathinones. The SIMCA analysis showed that it is possible to obtain toxicological properties that differentiate these drugs and indicate parameters for educational and health measures. The variables linked to the liver were the most important in the discrimination of amphetamines and cathinones, as cathinones are more water-soluble. It makes sense, given that the only difference between homologous structures is a carbonyl group. Because of the responses of this work, it is essential to reinforce the importance of in silico methods to assist in decision-making in the face of the rapid emergence of different available NPS. We thank the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 465450/2014-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) for financial support.

Toxicological evaluation of amphetamine-likes employing in silico methodology

roDrigueS, caio henrique pinke; Bruni, aline ThaiS

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

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Introduction: The Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) is an alternative toxicological method widely used to determine the irritation potential of a substance, by monitoring the damage caused to blood vessels, mainly in ocular toxicity assessment tests. However, few studies have investigated the usefulness of this method to evaluate the properties of snake venoms, as an alternative for hemorrhagic tests in animals. Objective: This study aimed to evaluate the coagulant and hemorrhagic effects of Bothrops leucurus (BlV) and Bothrops erythromelas (BeV) snake venoms through the HET-CAM assay. Methods: Five fertile fresh White Leghorn hen eggs (n=80) were used for each group, and the tests were performed in duplicate. In the HET-CAM assay, the eggs were incubated horizontally and rotated in a brooder for nine days (38 oC ± 5 oC, 70%). On the 9th day of incubation, the CAM was exposed and 200 µL of pool of bothropic venom diluted in saline was added (50 µg/mL; 100 µg/mL; 200 µg/mL; 1mg/ml), being the changes filmed (5 min) and photo documented (0s; 30s; 3min; 5min). As negative control, 200 µL of saline (0.9%) was used. The irritation index (IS) was calculated using Luepke’s

classification system and the data analyzed by ANOVA and Student’s t-test (p<0.05). Results: The results revealed that the BeV has severe irritation properties (IS 12.7 ± 0.5), even at low concentrations (50 µg/mL), in a time-dependent manner, since the first signs of severe effects (hemorrhage and coagulation) were detected already after 30 seconds of exposure in the CAM. However, BlV showed moderate irritation (IS 7.8 ± 0.3) from the concentration of 100 µg/mL, and severe irritation properties (IS 11.8 ± 0.6) after 60s of exposure. Conclusion: BeV was more irritating to CAM than BlV. This can be explained by the already known higher phospholipase activity of BeV, when compared to BlV. Thus, the HET-CAM test proved to be a good alternative model for toxicological analysis of non-neurotoxic venoms. However, considering that this was a pilot study used as an alternative method for evaluating the effect of bothropic venoms, we suggest that venom composition tested in the HET-CAM assay and the ontogenetic and sexual variations of the snakes must be considered when analyzing the results. Keywords: Bothrops, embryo, chicken, alternative methods.

Toxicological evaluation of effects of bothropic venoms through the

chorioallantoic membrane assay HET-CAM

Braga, jacqueline raMoS MachaDo1; liMa, naTália cavalcanTe BarBoSa2; alveS, carolina eSMeralDo liMa2; rocha, Danilo galvão2; jorge, roBerTa jeane Bezerra2; BionDi, ilka BorgeS3

1 Universidade Federal do Recôncavo da Bahia, Centro de Ciências Agrárias Ambientais e Biológicas, Laboratório de Répteis e Anfíbios. Cruz das Almas, Bahia; 2 Universidade

Federal do Ceará, Núcleo de Pesquisa e Desenvolvimento de Medicamentos, Laboratório de Farmacologia de Venenos e Toxinas. Fortaleza, Ceará; 3 Universidade Estadual de Feira de

Santana, Laboratório de Animais Peçonhentos e Herpetologia, Feira de Santana, Bahia.

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Currently, there is a need to develop new methodological approaches for the assessment of ocular toxicity, which reproduce results more similar to those found in humans and which aim to replace the in vivo Draize eye test. Ocular opacity is one of the top assessments for eye damage. This opacity is related to the interaction of the biomolecules of the corneal extracellular matrix with the substances to which it was exposed. The extracellular matrix of the cornea is composed of molecules such as proteins – mostly collagen – and glycosaminoglycans, and they have an important organizational characteristic that gives it transparency. Once this organization is disturbed, by different mechanisms such as ‘coagulation’ described as the replication/denaturation of macromolecules (particularly proteins) or ‘saponification’ described as the breakdown of lipids, opacity is observed. One of the main alternative tests for ocular assessment, Opacity and Ocular Permeability of Bovine Cornea (BCOP – OECD TG 437, uses opacity as one of the classification parameters for substances as categories at the extremes of the eye irritation spectrum. These extreme categories are classified). as ‘no categories’ and ‘category 1’ according to the Globally Harmonized System (GHS) classification. The test is already validated and well established, however, some issues involving access to raw material (bovine cornea), preservation of the same, and use of specific equipment, end up making this test less widespread. Since access to slaughterhouses may be limited depending on the locations of the laboratories, maintaining the viability and transparency of the corneas is essential, something that can be obtained by reagents that preserve the organ during transport but have a high cost. Based on this, the present study aims to propose accessibility and low-cost method, based on the decellularized extracellular matrix derived by bovine cornea for application as an in chemico test of complexation of biomolecules with substance for pre-screening and strategy for orientation of the

next steps for categorization of GHS Category 1 and uncategorized chemicals. For this, bovine corneas were collected and underwent decellularization and digestion processes until obtaining a hydrogel, which was previously characterized to confirm the preservation of its compounds, mainly collagen and glycosaminoglycans. The biomolecular solution was prepared in different concentrations, 100%, 50%, 25%, 12,5%, 6% and 3% of Hydrogel in phosphate buffer saline (PBS). The biomolecular solutions were placed in 24-well plates to identify the most suitable concentration for the method. For exposure to the substances, a device was developed by 3D printing, so that a cellulose membrane was suspended above the biomolecular solution. PBS was used as a negative control and 1-octanol (pure) and benzalkonium chloride (5%) were used as test substances. The biomolecular solutions were exposed to the substances through the membrane for 24h at room temperature, 25°. After that, the solutions were evaluated for their turbidity, by spectrophotometry, at 405nm, and compared with PBS control. Previous results showed that there was a change in turbidity in all concentrations of biomolecular solutions exposed to the test substances when compared to PBS. Among the concentrations of biomolecular solutions, those that proved to be the most suitable for evaluation in the spectrophotometer were 25% and 12.5%. This indicates that the biomolecular solution, based on natural extracellular matrix components, was able to change its initial characteristics when exposed to highly irritating chemicals with the potential to generate ocular opacity, which indicates that the test can be used pre-screening of Category 1 or unclassified substances. A cheaper test, accessible to researchers who do not have immediate access to slaughterhouses and who do not have the specific inputs to perform the BCOP test. Acknowledgments: Capes and CNPq

Use of a biomolecular decellularized bovine cornea derived solution as a method of

evaluating opacity and ocular irritation

SanToS, jorDana anDraDe; DiaS, WaneSSa aMoriM; valaDareS, Marize caMpoS

Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.

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In vitro assays have been a good tool to study the pathophysiology of respiratory diseases and to identify molecular targets for future clinical applications. Bidimensional models of lung cell cultures are widely used, but do not reflect the architecture of the lung tissue. Recently, biotechnology has been used for the establishment of three-dimensional models that mimic the in vivo tissue microenvironment and to accelerate the process of validation and efficacy of drugs used in the clinical phase. The company Biocelltis Biotecnologia S.A. recently launched CellFateiN®, the extracellular matrix biocompatible functionalized to mimic the lung tissue. To establish a functional in vitro three-dimensional model of human pulmonary epithelium cultivated on CellFate®iN, human pulmonary fibroblasts and epithelial cells (CALU-3) were cultured on the CellFate®iN for up to 21 days in an air-liquid interface (IAL) and submitted to histological analyzes. The activity of angiotensin converting enzyme 2 (ACE2), was identified by hydrolysis of

fluorogenic substrate. The Solute Carrier Organic Anion Transporter Family Member 3A1 (SLCO3A1) and e-cadherin, which are proteins expressed by pulmonary epithelial cells, was identified by qPCR. SLCO3A1 was expressed on the first day of culture in ALI, and e-cadherin at the end of the culture period (21 day). ACE2 has 4 times more activity than pulmonary fibroblasts (control), with its highest activity identified on the 14th day of IAL culture. CellFate®iN seems to be a viable platform for in vitro cultivation of pulmonary cells, and, after 21 days, these cells were organized as a pseudostratified epithelium, reproducing the pulmonary epithelium structure. Detection of proteins expressed in pulmonary epithelium suggests that a functional epithelium might be formed. These data show that this three-dimensional model of pulmonary epithelium might be used for in vitro 3D assays for a better understanding of the physiology of the respiratory epithelium. Keywords: human lung epithelium, cellfatein, 3Dmodel

Use of CellFate®iN for a three-dimensional in vitro model of human lung epithelium

SanToS, jeniFFer FariaS1; reiS, eMily MarqueS2; BerTi, FernanDa vieira2; koepp, janice2; nuneS, viviane aBreu1

1 Laboratory of Skin Physiology and Tissue Bioengineering. University of Sao Paulo. Sao Paulo, Brazil; 2 Biocelltis Biotecnologia S.A., Santa Catarina, Brazil;

02 ANALYTICAL TOXICOLOGY

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Background: More than 500 constituents have been identified in Cannabis sativa L., including cannabinoids, terpenes, flavonoids, alkaloids and others. The interest in Cannabis related studies grow even further with endocannabinoid system’s discovery, which suggested a pathophysiological modulation of this system useful for treatment of several diseases. Due to high cost of medicines and Cannabis derivatives (imported and domestic) for patients who do not have financial conditions for acquisition and / or medical prescription, domestic cultivation of its plant-based extracts occur to be the only alternative. Unfortunately, as these products are not yet subject to the minimum quality and safety tests and requirements in the production chain, such as: quantitation of cannabinoids, terpenes and contaminants (mycotoxins, residual solvents and pesticides), there is probably an inadequate exposure levels harmful to users. In this context, the main concerns and considerations of this work are associated with cultivation process and self-medication with Cannabis, which can cause health risks. In addition, pharmacological studies are also needed to evaluate plant profile constituents’ variability and its therapeutic efficacy, as they can be modified by geographic and climatic factors. Objective: To develop and validate an analytical method to determine 10 cannabinoids (cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, cannabinol, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, cannabichromene and tetrahydrocannabinolic acid) in Cannabis-based products (plant, oil and extracts) by high performance liquid chromatography technique with diode array detection (HPLC-DAD). And also analyze real samples donated from Associação De Apoio À Pesquisa E Pacientes De Cannabis Medicinal (APEPI), Brazil. Methods: For quantification of cannabinoids, a representative aliquot of the sample is diluted and analyzed by HPLC-DAD, using a Nexera X2 chromatographic system (Shimadzu, Kyoto, Japan), equipped with an C18 chromatographic column, maintained at 35 °C. Mobile phase were composed of ultra-pure water (A) containing formic acid (0.1%, v/v)

and acetonitrile (B) with flow rate of 2 mL/min and elution in isocratic mode UV absorption spectra are acquired from 200 to 400 nm, with quantification of cannabinoids at 228 nm and a total analysis time of 22 min. Samples were diluted factor range from 1:400 (for minority cannabinoids) to 1:1000 (for major cannabinoids). Plants samples (200 mg) were diluted in metanol (10 mL), and then diluted (1:150 and 1:10 to majority and minority cannabinoids, respectively). Results: The method fulfilled all Brazilian’s Health Regulatory Agency (ANVISA) recommended criteria (selectivity, accuracy, precision, linearity, limit of quantitation, ruggedness and dilution integrity), demonstrating to be adequate for all analytes. Linearity was achieved between 2 to 50 µg/mL (r > 0.99, 1/x). Precision (% RSD) and bias (%) were investigated for each analyte at three concentration levels (low, medium, and high controls). The controls were evaluated with one-way analysis of variance (ANOVA) performed in each concentration to assess potentially significant variability (p < 0.05). Precision results were below 1.8% and bias results between 93.8% and 107.9%. No carryover was observed. A total of 168 samples already were analyzed and at least two cannabinoids were identified in each sample, including: cannabidivarin (31.5% of the samples), cannabidiol (75.6%), cannabidiolic acid (16.1%), cannabigerol (58.9%), cannabinol (22.6%), cannabigerolic acid (7.1%), Δ9-tetrahydrocannabinol (85.1%), Δ8-tetrahydrocannabinol (0.0%), cannabichromene (61.3%) and tetrahydrocannabinolic acid (10.7%). The most identified substances were cannabidiol and Δ9-tetrahydrocannabinol, with quantified concentration between 0.8 – 48.2 mg/mL in Cannabis oil and 0.2 - 78.2% w/w in extract and between 0.8 – 88.6 mg/mL in oil and 0.3 – 86.4% w/w in extract, respectively. Discussion/Conclusion: The method was successfully validated fulfilling all ANVISA’s recommended criteria and will be applied for more quantification of Cannabis-based products donated from APEPI. Acknowledgments: This study was supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Analysis of cannabinoids profile in cannabis-based products by HPLC-DAD

carDoSo, Marilia SanToro1,3; oliveira, clauDeTe c.4,5; coSTa, joSé luiz2,3

1 School of Medical Sciences, University of Campinas (UNICAMP), Campinas-SP, Brazil; 2 Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas-SP, Brazil; 3 Campinas Poison Control Center, School of Medical Sciences, University of Campinas, Campinas-SP, Brazil;

4 Farmanguinhos- Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro – RJ, Brazil; 5 Associação de Apoio à Pesquisa e Pacientes de Cannabis Medicinal (APEPI), Rio de Janeiro-RJ, Brazil.

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Introduction: Brazil is one of the biggest consumers of chemical products such as carbamates and organophosphates pesticides. According to the Phytosanitary Pesticides System (Agrofit) from Brazilian Ministry of Agriculture, in 2014 there was the largest increase in the consumption or sales of pesticides in the country. Carbamates and organophosphates act as cholinesterase inhibitors, as they prevent the inactivation of acetylcholine, and they are responsible for most cases of poisoning in rural workers. Objectives: Develop a method to analyze carbofuran (carbamate) and chlorpyriphos-methyl (organophosphorate), in a gas chromatography system coupled with a single quadrupole mass spectrometer (GC-MS). Methods: It was used a gas chromatography coupled to a single quadrupole mass spectrometry (GC-MS) with an automatic injector (Shimadzu, Kyoto, Japan). The column used was a Rtx®-5 MS (30m x 0.25mm, 0.25µm film thickness) with 5% diphenyl and 95% dimethyl polysiloxane. Helium gas was used as carrier gas at a flow rate of 1.20 mL/min and 1µL was injected in splitless mode. Different temperatures at the injection port were tested: 175, 200, 225, 250, 275 and 300°C. In addition, different configurations in column oven temperature settings were tested as follows: Method 1: 50ºC (held for 1 min) → 50 °C (10 °C/min) → 300°C (held for 3 min.), totaling 29 minutes; Method 2: 50ºC (held for 4 min) → 50 °C (35°C/min) → 190°C (held for 7 min.) → 190ºC (40 °C/min) → 300ºC (held for 1 min.), totaling 15.75 minutes. Interface and ion source temperatures were kept at 300 °C. Mass spectra were recorded over 50-700 atomic mass units (amu) in the range of 0.30

scan/second. The analytes were identified by a GC-MS applying electron impact ionization (EI) at 70 eV to form the ions of interest in scan acquisition mode. For the tests, the analytical standards of carbofuran and chlorpyriphos-methyl at a concentration of 5 µg/mL in methanol were used. Results: The column oven configuration that presented the best performance for tested molecules and its thermal degradation products was the method 2, as it reduced the total analysis time per 13.25 minutes. Carbofuran was degraded at the injection port at all tested temperatures. Due to the fact that most of carbamates are thermally unstable, as is the case of carbofuran, breakage occured at the injector port or column to their phenolic and amino acid counterparts. For the best column oven configuration (method 2), carbofuran-phenol retention time was 5.134 minutes while carbofuran retention time was 8.019 minutes. Chlorpyrifos-methyl showed best chromatographic behavior in method 2 applied in the column oven and the temperature at the injection port that presented the best chromatographic performance was 175 °C. When the higher tested temperatures were applied at the injection port, greater peak areas for the thermal degradation product of chlorpyrifos-methyl (retention time of 10.388 minutes) and formation of carbofuran-phenol were obtained in a directly proportional way. Conclusion: Concluded that the analytical method used was efficient for chlropyrifos, and may be very useful for toxicological analysis while carbofuran required other strategies for its determination in GC-MS. Acknowledgements: We wish to thank PROBIC/FAPERGS-CNPq for providing financial support.

Analysis of carbofuran and chlorpyrphos-methyl in GC-MS under different conditions of the

injection port and column oven temperatures

roSa, vicTória goMeS1; BairroS, anDré valle2; ugalDe, guSTavo anDraDe2; chiMenDeS, nayoMi De anDraDe2; SanToS, lara celeSTina2; pacheco, anDré lucaS Bezerra2; reginaTo,

FernanDa ziegler2; BerlaTo, Dener goMeS2; naSciMenTo, Marcelo henrique SanTana2

1 Nucleus Applied to Toxicology (NAT); 2 NAT.

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Introduction: When human body is in an advanced state of decomposition where tissue toxicological analysis is impracticable, insect larvae can be considered an alternative option for researching toxicants. Entomotoxicological studies with cholinesterase inhibitors (carbamates and organophosphates) are scarce using this alternative matrix. Objective: The objective of this work was the development of an analytical method able to determine organophosphate and carbamates pesticides through gas chromatography coupled to mass spectrometry (GC-MS), followed by subsequent application of recovery studies in L3 stage larval matrix of Lucilia cuprina. Methods: Analyzes were performed on a GC-MS with a Rtx®-5MS column (30 m x 0.25 mm, 0.25 µm film thickness). Helium was the carrier gas at a constant flow rate of 1.20 mL/min. The chromatographic parameters used were as follows: injector temperature 175°C; 1 µL of injection volume in splitless mode; column oven setting: 50°C (held for 4 min) → 50°C (35°C/min) → 190°C (held for 7 min.) → 190°C (40°C/min) → 300°C (held for 1 min.), totaling 15.75 minutes of chromatographic execution. After obtaining the L3 stage larvae, the QuEChERS method, proposed by Anastassiades et al. (2003), with modifications and two variations in cleanup step were employed in order to test analytes recovery. The steps are summarized below: 1g of the homogenized larvae was weighed into a 15 mL falcon tube, then the analytical standards (Aldicarb, Carbaryl, Carbofuran, Methomyl, Propoxur, Carbophenothione, Chlorpyriphos Ethyl, Chlorpyriphos Methyl, Ethion, Fenitrothion, Formotion, Malathion, Malaoxon, Paraoxon Ethyl, Paraoxon Methyl, Pyrimphos Ethyl, Pyrimiphos Methyl, Triazophos) and the internal standard were spiked at a concentration of 50 ng/g were added in duplicate. Afterwards, 2 mL of

acetonitrile were added and the tubes were manually shaked for 1 minute. Then 1 g of NaCl and 4 g of MgSO4 were added. Then, two methodologies were tested as cleanup step. Method 1: After centrifugation (4,000 RPM, for 10 minutes), 1 mL of the supernatant was transferred to another 15 mL falcon tube, with 150 mg of NaCl and 25 mg of Primary Secondary Amine (PSA) previously weighed. Method 2: After centrifugation (4,000 RPM, for 10 minutes), 1 mL of the supernatant was transferred to another falcon tube, with 150 mg of NaCl, 25 mg of Secondary Primary Amine (PSA) and 25 mg of C18 sorbent. Finally, the tubes were manually shaked for another 1 minute, centrifuged at 4,000 RPM and 500 µL of the supernatant was transferred into a 2 mL chromatography vial and dried in a sample concentrator, resuspended in 50 µL of ethyl acetate and 1 µL was injected into the GC-MS. Results: Eight analytes (Ethion, Aldicarb, Methyl and Ethyl Chlorpyriphos, Malaoxon, Methyl and Ethyl Pyrimiphos and Triazophos) were successfully recovered by both cleanup methods tested, however a better recovery was obtained with the method whose cleanup step was performed with isolated PSA sorbent not associated with the C18 sorbent with the following calculated recovery values: 52.4% for Ethion, 33.1% for Aldicarb, 32.3% for Methyl Chlorpyrifos, 20% for Malaoxon, 65.7% for Methyl Pirimiphos, 32% for Ethyl Cholrpyrifos, 12% for Ethyl Pirimiphos and 9% for Triazofos. Conclusion: The proposed methodology allowed determinate eight from 19 analytes tested. This work is part of doctoral thesis and will continue to be optimized in order to involve the recovery of all tested analytes. Acknowledgements: We wish to thank PROBIC/FAPERGS-CNPq for providing financial support.

Analysis of cholinesterase inhibitor pesticides by GC-MS in larvae of Lucilia cuprina

(Calliphoridae) for entomotoxicological studies

ugalDe, guSTavo anDraDe1; chiMenDeS, nayoMi De anDraDe1; pacheco, anDré lucaS Bezerra1; SanToS, lara celeSTina1; reginaTo, FernanDa ziegler1; BerlaTo, Dener goMeS1; carDoSo, leonarDo

corrêa1; naSciMenTo, Marcelo henrique SanTana1; roSa, vicTória goMeS1; SanToS, rachel1; pereira, aleSSanDra De oliveira2; MonTeiro, Silvia gonzalez2; BairroS, anDré valle1

1 Nucleus Applied to Toxicology (NAT); 2 Parasitic Disease Laboratory (LAPAVET).

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Background/Introduction: A 15-year-old patient was referred to the university hospital with high sensory relegation, coma and under intubation. The consumption of 60 quetiapine tablets (25 mg) and the suspicion of other central nervous system depressant drugs in the 12 hours prior to hospital admission were reported. However, toxicological analysis based in immunoassay did not detect any drug. So, chromatographic techniques were employed to elucidate this case. Objective: Emphasize the importance of toxicological analysis, especially chromatographic methods in cases of intoxication arriving in hospitals. Methods: Rapid test for qualitative detection of multiple drugs of abuse by immunochromatography (Abon®) in NaF plasma and urine samples was employed in the first moment. Quetiapine analysis by liquid chromatography with ultraviolet-visible detector (LC-UV/Vis) in 500 µL NaF plasma sample in pH 10 extracted with hexane (2.5 mL). After homogenization (2 min) and centrifugation (4000 rpm / 10 min), solvent was dried, resuspended with 100 µL of mobile phase and 20 µL was injected. Mobile phase was composed by phosphate buffer pH 5.5/methanol/acetonitrile (30:30:40) in isocratic mode (1.0 mL/min) using analytical column (C18 4.6 x 150 mm x 5 µm) at 225 nm. Medazepam was used as an internal standard. Valproic acid determination in serum sample (0.5mL) was based in liquid-liquid extraction (LLE) by HCl (30 µL, 2M), after addition of ethyl acetate (1.5 mL) and mixed on the vortex (1 min). Centrifugation (4000 rpm for 5 min) and passage of 1.25 mL of solvent into a tube containing sodium sulfate. An aliquot of this extracted solvent (500 µL) is put into a vial and 1 µL is injected in the gas chromatography coupled to mass spectrometry (GC-

MS) in split mode (1:10). Initial temperature was 70°C (3 min), increasing 35°C/min to 180°C (3 – 6 min) and 50°C/min to 280°C (6 – 8 min). Final temperature fixed for 4 min. Total chromatography time was 12 min. Flow rate in the first 7 min was 1.5 mL/min. It was increased to 2.5 mL/min over 6 min and held at that rate until the chromatography was finished. Retention time of valproic acid under these conditions was 5.50 min. Mass spectrometer operated in electronic ionization (EI) mode with 70 eV applying Selected Ion Monitoring (SIM) mode [m/z = 73, 102 and 115]. Results: Immunochromatography test was undetectable for the tested drugs in urine and plasma samples. Quetiapine was detectable (limit of detection = 150 ng/mL), however it was not quantified (limit of quantification = 500 ng/mL). Valproic acid concentration in serum sample was 977.96 µg/mL, considered potentially fatal. Discussion/Conclusion: The reports from this case mentioned high self-administration of quetiapine tablets. From an analytical investigation and new information during the patient’s hospitalization, we proved that quetiapine was not involved in this case, while high concentrations of valproic acid were determined in GC-MS, confirming the intoxication of the patient. Toxicological analyzes are still rarely used in the laboratory routine at a hospital in Brazil. Chromatographic techniques, especially liquid chromatography and gas chromatography, allow the analysis of different class of drugs and other substances that immunoassay tests did not evaluate. The use of chromatographic methods in order to help hospitals for intoxications cases are essential for the diagnosis and prognosis of intoxications as well as the choice of the best treatment. Acknowledgments: UFSM

Case report of valproic acid intoxication: the relevance of chromatographic methods

for elucidating cases in hospitals

SaMpaio, aManDa Mello kaSper vaz; reginaTo, FernanDa ziegler; BairroS, anDré valle; roehrS, Miguel; lovaTel, ivy Bauer; ugalDe, guSTavo anDraDe

Núcleo Aplicado à Toxicologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria

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Introduction: The World Anti-Doping Agency (WADA) accredited laboratories must follow a List of Prohibited Substances and Methods [1], updated every year. Some analytical strategies are developed to cover most of it. However, the analysis of cobalt, which was incorporated in 2015 to the list, is yet a challenge because of its incompatibility with the classical strategies available in the anti-doping laboratories (i.e. liquid chromatography and gas chromatography coupled with mass spectrometry). The supplementation with cobalt salts has effect on stimulus of erythropoiesis via stabilization of the hypoxia-inducible factors. In 2015, Thevis et al. firstly reported products containing significant amounts of undeclared cobalt and nickel species in products advertised as erythropoiesis-stimulating agents by inductively coupled plasma mass spectrometry (ICP-MS) [2]. Objective: The project aims to develop a cost-effective approach for the detection of cobalt in athletes’ urine employing liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS). Methods and results: To allow the detection of metal ions in urine by LC-HRMS, it is necessary its complexation from the matrix with a ligand. Based on the reported formation of cobalt complex with the diethyldithiocarbamate (DDC, (C2H5)2NCSS-) ligand [3], it was the complexation strategy adopted to achieve the results necessary to show the potentiality of the proposed method. Due to the same coordination number, oxidation state and geometry of the complex, rhodium was proposed as one quality control for the analysis. The method development was guided by many stages of Design of Experiments (DoE) in both instrumental analysis and sample preparation (e.g. in the electrospray ionization, mass spectrometry conditions, extraction with organic solvent,

complexation, composition of mobile phases and pH of the synthesis). The final setup involves a liquid-liquid extraction with methyl tert-butyl ether, in which the organic layer is transferred to another tube and evaporated to dryness in a thermostatic bath under a nitrogen stream at 40°C. The samples are reconstituted with 50 µL of the mixture water pH 4 (5mM ammonium formate/formic acid) and acetonitrile (30:70). A Thermo Dionex Ultimate 3000 UHPLC system coupled to a QExactiveTM hybrid quadrupole-orbitrap mass spectrometer equipped with an electrospray ionization source is used. Separation is performed in a reversed-phase column (Syncronis – Thermo, USA, C18, 1.7 µm, 50 mm x 2.1 mm) at 40°C in a five-minutes isocratic chromatographic run (30% water pH 4 and 70% acetonitrile), at a constant flow rate of 300 µL min-1. Conclusion: As a conclusion, it was possible to detect cobalt as its DDC complex in a cost-effective and high throughput analysis by LC-HRMS. As a future perspective of the project, the method will be validated according to WADA guidelines for further application in anti-doping purposes. Acknowledgments: The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Autoridade Brasileira de Controle de Dopagem (ABCD).

REFERENCES1. World Anti-Doping Agency. Prohibited List 2022, Montreal, 2022. Available at https://www.wada-ama.org/sites/default/files/2022-01/2022list_final_en_0.pdf.2. Thevis, M, et al. Solutions advertised as erythropoiesis-stimulating products were found to contain undeclared cobalt and nickel species. Int J Sports Med. 2015;36: 82-84.3. Minakata, K, Suzuki, M, & Suzuki, O Application of electrospray ionization tandem mass spectrometry for the rapid and sensitive determination of cobalt in urine. Analytica Chimica Acta. 2008;614:161-164.

Detection of cobalt in human urine by liquid chromatography coupled with

high resolution mass spectrometry for anti-doping control purposes

SanToS, vaneSSa Farelo1,2; carneiro, gaBriel reiS alveS1; coelho, MaTheuS caMpelo Da coSTa1; MachaDo, Sérgio De paula2; pereira, henrique Marcelo gualBerTo1

1 Laboratório Brasileiro de Controle de Dopagem, Chemistry Institute, Federal University of Rio de Janeiro; 2 Laboratório de Química Inorgânica Computacional,

Chemistry Institute, Federal University of Rio de Janeiro.

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Background: 5-Fluorouracil (5-FU) is an antimetabolite drug that belongs to the class of fluoropyrimidines. It is a compound analogous to endogenous uracil, having a fluorine atom in the C-5 position. Since it was synthesized in the 1950s, 5-FU remains one of the most used chemotherapeutics to treat various types of cancer, especially gastrointestinal tumors. Among patients treated with 5-FU, only 20-30% reach the therapeutic target and its use has been associated with potentially severe toxicity, being a candidate for therapeutic drug monitoring. Considering the ease of collection and the intrinsic logistical advantages of dried samples, the availability of a finger-prick dried blood spots (DBS) assay for measuring 5-FU concentrations has the potential to allow identification of patients who needs dose adjustments. Up to date there is no report of a DBS-based method for 5-FU quantification. Objective: To develop and validate a method for determination of 5-FU in dried blood samples (DBS) by UPLC/MS-MS. Methods: 5-FU were extracted from 8 mm DBS punches with 100 µL of ultrapure water containing IS (5-clorouracil 0.3 ng mL-1) incubated for 10 minutes at 46 °C and 1000 rpm, followed by 10 minutes ultrasound incubation. After, the aqueous extracted was precipitated with 200 µL of methanol cooled at -20 ºC for 15 min. After centrifugation, 100 µL of supernatant was transferred to a new tube and diluted with 100 µL of water with 0.5% acetic acid. After homogenization the extract was transferred to vial and 1 µL injected onto UPLC/MS-MS Acquity® I-Class UPLC coupled to a Xevo®

TQS-micro triple quadrupole mass spectrometer with electrospray ionization in positive mode. Chromatographic separation occurred in an Acquity® UPLC HSS C18 (1.8 µm 2.1 x 150 mm) at 25 °C. Mobile phase was water with 0,5% acetic acid (eluent A) and acetonitrile with 0.5% acetic acid (eluent B) eluted in gradient mode from 95:5 to 10:90 (A:B, v/v) at flow rate of 0.25 mL min -1. The MRM transition for quantification were 5-FU m/z 131 → 58 and IS 5-CU m/z 147 → 74. The method was validated according to FDA, to date specificity linearity, precision/accuracy, sensitivity, selectivity and matrix effect tests have been completed. Results: Total analytical run time was 5 min, retention time was 1.94 min for 5-FU and 2.85 min for IS. The method was selective, with no interference peaks eluting at the same relation time as 5-FU. The lowest limit of quantification 100 ng mL-1 presented accuracy of 115% and 3.39 % inter assay and 2.50 % intra assay CV%. The method was linear from 100 to 2000 ng mL-1 (r2 > 0.99), with accuracy of 91-102%, precision with CV% intra-assay within 3.24-4.47%, inter-assay within 5.0-14.51%, and mean extraction yield 70%. The internal standard compensated adequately the matrix effect from +4.96 to +14.49%. Discussion/Conclusion: The method has been shown to be adequate for determining the concentrations of 5-FU in DBS samples. After completion of the validation tests regarding the impact of hematocrit on assay accuracy and extraction yield, the method will be applied in a clinical study. Acknowledgments: Financial support from Fapergs, CAPES and CNPq.

Determination of 5-fluorouracil in dried blood spots (DBS) by UPLC/MS-MS

Silva, laura cé1,2; granDo, ana paula1; hahn, roBerTa zilleS1; linDen, raFael1,2; anTuneS, Marina vezon1,2

1 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil

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Background/Introduction: Cocaine is one of the most illicit drugs used in Brazil. This substance is known for the significant risks to users and leads up to significant public health issues, compromising one’s quality of living. In this context, the development of new techniques for drug detection applied in a simple way to unconventional biological matrices, such as oral fluid, can be an important tool for toxicological analysis. Objective: This study aims to develop and validate a dried spot oral fluid using well plate sample preparation technique by LC-MS/MS for analysis of cocaine (COC), ecgonine methyl ester (EME), benzoylecgonine (BE), cocaethylene (CE) e o-hydroxy cocaine (COC-OH) in oral fluid. Methods: Oral fluid samples, collected by expectoration, were obtained from patients in different addiction treatment services in, Porto Alegre, RS. Sample Procedure: For sample processing, 24-well plates were used as support. In each well were placed four uniform layers of filter paper. Oral fluid samples were centrifuged during 5 minutes at 10000 rpm and diluted in ultrapure water in the proportion 1:1 (v/v). An aliquot of 100μL of sample was pipetted into each well and dried for 60 minutes at room temperature. Then, 250μL of a mixture of methanol and acetonitrile in the proportion 1:3 (v/v) were added. The internal standards, BE-d3 and COC-d3, were added to the solvent. The plate was shaken for 5 min at 150 rpm, the supernatant was collected and 2μL was injected in the analytical system. The extraction solvent was determined through a simplex-centroid design by the software Statistica 8.0. The drying time and the number of layers of filter paper used were also evaluated. Instrumentation: A Shimadzu liquid chromatograph mass spectrometer LCMS-8045 was used with an electrospray source operating in positive ion mode as follows: capillary voltage, 4000 V; nebulizer gas, 3L/min; drying gas, 10L/min; drying temperature, 250°C; heating block temperature, 400 °C. The chromatographic separation

was achieved on a Shim-pack GISS column (100 mm × 2.1 mm, 1.9μm), eluted with a gradient of water (solvent A) and acetonitrile (B) both fortified with 0.1% formic acid, at a 0.4mL/min flow rate and 40 °C. Results: Following the recommendations of ANSI/ASB Standard 036, the developed method has been validated for lower limit of quantitation (LLOQ), linearity, within-run and between-run precision, bias, selectivity, ionization suppression/enhancement and carryover. The LLOQ was 1 ng/mL for all of the analytes. The concentration ranges used were 1-100ng/mL for EME, COC, BE, COC-OH, CE and all analytes presented a coefficient of determination (r2) ≥ 0,99. The occurrence of heteroscedasticity was verified and the weighting factor of 1/x2 was applied. The within-run and between-run precision range between 2.2 - 12.04%, considering relative standard deviation. The bias for all the analytes varied between 90.9 – 109.6%. No significant level of interfering substances was observed. Suppression of ionization was observed for all analytes ranging between -50.6 – 71.9%. No carryover effects were detected. After the full validation, 110 samples were analyzed. Of these, 90 samples (81.8%) were positive for BE, 81 samples (73.6%) for COC, 33 (30%) for EME, 4 (3.6%) for CE and one (0.9%) for COC-OH. Discussion/Conclusion: All parameters evaluated were within the limits recommended by the guidelines. The suppression in ionization was probably caused by the phenomenon of the matrix effect, although the other validation parameters were not negatively affected. Furthermore, the sample processing has optimized, up to 24 samples can be prepared, simultaneously. The present study demonstrated that the method was successfully developed and validated for determining the presence of cocaine and its metabolites by LC-MS/MS, proving to be an analytical alternative, simplifying the process of analysis of fluid oral. Acknowledgments: FAPERGS; CAPES; CPAD/HCPA.

Determination of cocaine and metabolites applying dried spot oral fluid using

well plate by HPLC-MS/MS

BorgeS, gaBriela raMoS1,2; SanToS, Bruno pereira1,2,3; gouveia, giovanna criSTiano1,2; Dalanhol, carolina Silveira1,2,3; Scherer, juliana2; eller, Sarah1; oliveira, Tiago Franco1

1 Pharmacosciences Department, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil; 2 Center for Drug and Alcohol Research and Collaborating Center on Alcohol and Drugs, Hospital de

Clínicas de Porto Alegre (CPAD/HCPA), Brazil; 3 Federal University of Rio Grande do Sul (UFRGS), Brazil.

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Parkinson’s disease (PD) is a progressive and multifactorial neurodegenerative disease. Studies in PD patients have evidenced augmented cortisol levels, which can be related to anxiety and depression disorders and may exacerbate motor symptoms and compulsive behavior, besides contributing to negative PD progression. We have developed and validated new strategies to determine possible endocrine biomarkers, like cortisol and cortisone, in urine samples from PD patients by using a dispersive tip microextraction solid phase and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). This efficient microextraction technique consumes less organic solvent and requires small sample volume. Turbulent air bubble mixing creates a suspension of sorbent in the sample, ensuring optimal contact and highly efficient extraction. Microextraction

was carried out by using modified pipette tips filled with a HLB (hydrophilic lipophilic balanced) phase loosely accommodated between two porous filters. The variables washing solvent volume, washing organic solvent concentration, sorption equilibrium time, sample pH, sample volume, and elution solvent were evaluated. The proposed method was linear at concentrations ranging from 0.5 ng mL-1 (LLOQ) to 500 ng mL-1 (R = 0.9983) for cortisol and from 3 ng mL-1 (LLOQ) to 500 ng mL-1 (R = 0.9972) for cortisone. The precision assays gave RSD values ranging from 0.9% to 13.3% and accuracy ranging from 83.5% a 113.4%, without significant matrix effect. We successfully applied this innovative UHPLC-MS/MS method to determine cortisol and cortisone in urine samples from PD patients. Acknowledgments: CAPES - Process numbers 88887.501207/2020-00

Determination of cortisol and cortisone in urine samples from patients with Parkinson’s

disease by dispersive tip microextraction solid phase and ultra-efficient liquid

chromatography tandem-mass spectrometry

kakuDa, priScila1; Souza, iSrael DonizeTi1; TuMaS, viTor2; queiroz, Maria eugênia coSTa1

1 Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Department of Neuroscience and

Behavior, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.

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Background: According to the National Registry of Traffic Accidents and Statistics/RENAEST, in the last four years Brazil recorded about 3,190,830 traffic accidents on national highways, with the consumption of licit and illicit substances by drivers, mainly the alcohol, one of those responsible for these numbers. Objective: The aim of this study was to identify the presence of ethanol consumption markers, being Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS) in urine samples from truck drivers. Methods: A total of 321 truck drivers were interviewed at a gas station on BR-153, in West Center, between February 2014 and February 2015 and collected approximately 50 mL of urine for toxicological evaluation for the presence of alcohol metabolites. EtG and EtS measurements were performed by liquid chromatography coupled to mass spectrometry (LC-MSMS), using a reverse phase column and a gradient of acetonitrile and ammonium formiate as mobile phase. Deuterated analytes were utilized as internal standards, EtG-D5 and EtS-D5. Few volume of urine (100 µL) was used, with simple dilution, followed by centrifugation, before injecting into the chromatographic system. The lower limit of quantification (LIQ) established for both markers

was 100 ng/mL. Basics descriptive statistics were performed, including percentage frequency, median and means were processed and analyzed using the software Epi Info. To analyse the association between the variables was used multiple correspondence analysis and hierarchical cluster analysis. Results: All of them were male with a median age of 42 years, most of them were married, low scholarity. 47% related consumption moderate or high risk of alcohol and tobacco and 21% use of cocaine, cannabinoids, amphetamine, hypnotics/sedatives, opioids, inhalants. Measurement of urinary EtS and EtG was performed by LC-MS/MS and showed 34.5% (n=111) of positive results for both markers and 15% (n=48) for just EtG. Discussion/Conclusion: Markers of recent alcohol consumption were identified in the urine samples, as well as it was found that truck drivers consume tobacco, cocaine and other illicit drugs during the working day and this behaviors increase the risk of being involved in accidents on the highways. from the country. Based on this, measures are needed to improve the quality of life of drivers, as well as to prevent and treat individuals who use alcohol in traffic accidents. Acknowledgements: UNODC, UFG.

Determination of Ethyl Glucuronide and Ethyl Sulfate in urine samples

from brazilian truck drivers

coSTa, c.D.D.; oliveira neTo, j.r.; oliveira, n.r.l.; alcânTara, k.c.; cunha, l.c.

Universidade Federal de Goiás - Rua 240 - Setor Leste Universitário, 74605-170 Goiânia, GO, Brasil

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Background: In recent decades, the consumption of pharmaceuticals and personal care products (PPCPs) grew up all over the world, both because of the increased availability and the ease of access. PPCPs are chemical products, synthetic or natural, that can be found in pharmaceuticals, over-the-counter products, deodorants, repellents, fragrances, sunscreens, among others, all of which are widely used throughout the world. PPCPs are known to be released into aquatic environments through various routes, including domestic wastewater, hospital wastewater, improper disposal by the manufacturers, wastewater treatment plants, and water treatment plants. Objective: The objectives of this work were to identify and endocrine disrupting chemicals (bisphenol A, estradiol, and ethinylestradiol), antimicrobials and other pharmaceuticals such as antibiotics, anti-inflammatories, analgesics, lipid regulators, β-blockers, antidepressant, and antiepileptics in surface waters used as influents of water treatment plants located in 5 cities of the Vale do Rio dos Sinos region, the Rio Grande do Sul, Brazil. Samples are being collected fortnightly. Methods: Compounds of interest were extracted from 200 mL surface water samples using solid-phase extraction with an OASIS® HLB (3 ml/60 mg) cartridge. SPE was automatically performed using an ASPEC GX-271 device. Estradiol, ethinylestradiol, and bisphenol A were derivatized with dansyl chloride prior to mass spectrometric detection. Derivatization conditions were selected based on a statistically designed

experiment and response surface analysis. The evaluation of the experimental data was performed using Design Expert 13® (Stat-Ease, USA). The optimized derivatization conditions were incubation time of 5 minutes, incubation temperature of 60 °C, and concentration of dansyl chloride solution of 2.5 mg/mL. The extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with electrospray ionization both in positive and negative mode. Results/Discussion: The curves were linear with a correlation coefficient (r) greater than 0.99. The compounds measured in surface water and concentration ranges were the following: amoxicillin - 300 to 765 ng L-1; atenolol - 5.64 to 89.5 ng L-1; azithromycin - 3.8 to 13.6 ng L-1; bezafibrate - 0.43 to 4.35 ng L-1; carbamazepine - 4.65 to 24.1 ng L-1; caffeine - 207 to 4730 ng L-1; ciprofloxacin - 0.52 to 2.74 ng L-1; diclofenac - 9.8 to 58.2 ng L-1; fluoxetine - 1.20 to 2.27 ng L-1; ibuprofen - 14.32 to 185.8 ng L-1; propranolol - 13.95 to 77.24 ng L-1; salicylic acid - 7.55 to 20.5 ng L-1; sulfamethoxazole - 0.5 to 57.5 ng L-1; triclosan - 6.48 to 39.5 ng L-1; bisphenol A - 12.15 to 86.67 ng L-1; β-estradiol - 44.2 to 697.8 pg L-1; 17-α- ethinylestradiol - 132.15 to 819.6 pg L-1. Conclusion: Significant concentrations of various compounds were found in Sinos river and these concentrations will be used to assess ecotoxicological risks. The monitoring of PPCPs in surface waters is essential to evaluate the presence of possible ecotoxicological and human health risks. Acknowledgments: Feevale University, CAPES.

Determination of pharmaceuticals and personal care products (PPCPs) in

surface water in Southern Brazil

BaSTiani, MarcoS Frank; hahn, roBerTa zilleS; lizoT, lilian De liMa FelTraco; BonDan, aManDa pacheco; caSTilhoS, Maria aMélia; FreiTaS, Mariana; FavreTo, caMila; linDen, raFael

Feevale University

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Introduction: There is little scientific information available on the actual composition of drugs sold and consumed, which are often masked or unknown. Objective: Develop and validate a method for analyzing amphetamine, methamphetamine, MDMA, MDA, MDEA, cocaine, cocaethylene, dibutylone, n-ethylpentylone, 25E-NBOMe, 25C-NBOMe, 2C-E, 2C-C, fentanyl and carfentanyl in a sweat using the DPX-CX1 and GC-MS. Methods: 650µL of artificial sweat were applied on absorbent cellulose paper. They were fortified with the analytical standards and with the internal standards amphetamine-d11, fentanyl-d5 and cocaine-d3. They were submitted to the extraction process using the DPX-CX1 tip. This filter paper was inserted into a glass tube. 2ml of methanol was added, then vortexed and on the shaker table. Subsequently, 1mL of this solution was transferred to another glass tube and 100μL of 0.1 M H3PO4 was added. The conditioning of the DPX-CX1 tip was done with 1mL of acetonitrile. Then, 1mL of the solution containing 0.1MH3PO4 was aspirated. Afterwards, it was washed with 500μL of methanol. For the elution of the analytes, 1500μL of a solution of dichloromethane:isopropanol:ammonium hydroxide (78:20:2) was used.The eluate was evaporated under nitrogen flow, reconstituted with 40μL of ethyl acetate. It was then derivatized with 40μL of MSTFA. The separation was carried out in a HP5 fused silica capillary column (30mx0.25mmx0.25μm film thickness). The injector temperature was 280°C in splitless mode and the injection volume was 1µL. Helium was used as a carrier gas. The initial oven temperature was 90°C, remaining for 2 minutes, and later, it was raised to 220°C at a heating rate of 10°C.min-1. Then, the temperature was raised to 290°C at a heating rate of 20°C.min-1, remaining for 4 minutes, totaling 22.5 minutes of analysis.The mass spectrometer with a quadrupole mass analyzer was initially used in the full scan operation mode (80 to 490m/z) and later it was operated in the SIM mode.The ionization mode used

was electron ionization. The temperature used in the MS interface, source and quadrupole was 280, 230 and 150°C, respectively. The parameters evaluated during the validation of the method were those recommended by resolution 27/2012 and 899/2003 of ANVISA. Results: The detection limit ranged from 1ng/patch to 15ng/patch for amphetamine, MDMA, MDA, dibutylone and n-ethylpentylone analytes, 2C-E and 2C-C, respectively. The analyzed interferents did not interfere in the retention time, identification and quantification of analytes. No residual effect was observed either. The matrix effect ranged from 2.12% for fentanyl to 9.32% for MDEA. The method was considered linear in the concentration range of 2 to 400ng/adhesive for amphetamine, MDMA, MDA, dibutylone and n-ethylpentylone, from 3 to 400ng/adhesive for methamphetamine, from 5 to 400ng/adhesive for MDEA, 25C- NBOMe, fentanyl and carfentanyl, from 10 to 600ng/adhesive for cocaine, cocaethylene and 25E-NBOMe and from 30 to 1100ng/adhesive for 2C-E and 2C-C. Analyte recovery values range from 71.34% to 98.79% for the HQC of 2C-C and dibutylone, respectively. The intraday precision ranged from 1.13 to 14.79%, which correspond to the LQC and MCQ values of the 2C-E and MDEA, respectively. And the interday precision ranged from 1.23 to 18.50%, which were the LQC values of 2C-E and LLOQ of MDMA, respectively. The intraday accuracy values ranged from -9.18% to 17.86% which correspond to the MCQ and LLOQ values of amphetamine. And the intraday accuracy ranged from -10.89% for the amphetamine MQC to 19.11% for the MDMA LLOQ. The analytes were stable in both the biological sample and the standard solution. Discussion/Conclusion: The method developed and validated met all the criteria required by resolution 27/2012 and 899/2003 of ANVISA. Acknowledgments: This research was supported by CAPES – Brazil (Financing Code 001) and FAPESP 2017/18021-5.

Determination of psychoactive substances in sweat samples using disposable

pipette extraction (DPX) and GC-MS

goMeS, nayna cânDiDa1; De MarTiniS, Bruno SpinoSa2

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil; 2 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil.

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Introduction: As reported by Rodrigues et al. (2021), the number of synthetic cannabinoid receptor agonists (SCRAs) seizures in Brazilian prison systems are increasing, mostly as infused papers. A study realized in infused papers seized in Scottish prisons from June 2018 to September 2019, observed an increase over time in the number of samples in which multiple SCRAs were detected and a variation of which SCRAs became the most detected. 5F-MDMB-PINACA (5F-ADB), 5F-MDMB-PICA, 4F-MDMB-BINACA, and MDMB-4en- PINACA were the most detected at different times during the study, which reinforces the need for constant monitoring of these substances in the market. The present study described the identification and quantitation of SCRAs in 23 infused paper and in 3 herbal material samples, seized in prisons located in São Paulo state. Methods: The selected samples were previously analyzed by the São Paulo State Police, in a qualitative analysis by gas chromatography–mass spectrometry (GC–MS) using full-scan and library search for identification. Afterward, these samples were analyzed by liquid chromatography-tandem mass spectrometry in a triple quadrupole (LC-MS/MS) system operating in MRM mode for determination of the majority and the possible minority SCRAs present in the samples. For the LC-MS/MS analysis the samples were extracted with methanol and diluted in a proportion of 1:1.000 and 1:10.000. Results and discussion: All substances identified by GC-MS were also identified by LC-MS/MS, but not the opposite, showing GC-MS full-scan method employed were unable to detect those SCRAs presented in lower concentration levels. The presence of a single SCRA in the samples was observed in 11 samples (27.5%), while in 29 samples (72.5%) two or more SCRAs were identified. The SCRAs most frequently detected in

the samples were MDMB-4en-PINACA and ADB-BUTINACA, present in 72% and 70% of the samples, respectively. The concentration of SCRAs shown great variability, ranging from 0.2 µg/infused paper to 3,000 µg/infused paper. In cases with more than one SCRA detected, the minority compounds did not exceed the concentration of 50 µg/infused paper. Comparing two different samples of a single case it is possible to identify the same substances but in different concentration levels, or different compositions. The highest concentration of SCRA present in the infused papers was for the cases involving MDMB-4en-PINACA as the major compound, reaching until 3,000 µg/infused paper, but this SCRA was also found as minority in other cases, with concentrations as low as 0.9 µg/infused paper. Among all samples, 4F-MDMB-BUTICA was detected in only one case, at a concentration of 125 µg/infused paper. For the three samples of plant material, ADB-BUTINACA appears as the major compound, with the presence of other minor SCRAs ranging from 0.2 to 5 µg/mg. Conclusions: These results shows that the studied samples did not present homogeneity in the composition and concentration levels, even when comparing a single case, but it seems that some SCRAs are commonly found together. The presence of different SCRAs in the same sample may contribute to the increase in the toxicity of these compounds, which reinforces the need to obtain quantitative and qualitative data from the seized samples. Acknowledgments: The authors thank the Superintendence of the Technical Scientific Police of the State of Sao Paulo and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Projeto INSPEQT, Edital nº 16/2020, Processo 88887.516176/2020-00).

Determination of synthetic cannabinoid receptor agonists (SCRAs) in infused papers and

herbal materials seized in Brazilian prisons

MarTinS, aline Franco1,2; BarBoSa, ingriD lopeS3; Milanez, guilherMe paier3; coSTa, joSé luiz2,4

1 Faculty of Medical Sciences, University of Campinas, Campinas, SP, Brazil; 2 Campinas Poison Control Center, University of Campinas, Campinas, SP 13083-859, Brazil; 3 Superintendence

of the Technical-Scientifc Police, Institute of Criminalistics, São Paulo, SP 05507-060, Brazil; 4 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, SP 13083-871, Brazil .

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Background/Introduction: Meropenem is an antimicrobial drug characterized as bactericidal, widely used in intensive care units. The PK/PD parameter best correlated with the efficacy of carbapenems is the time in which the free fraction of the drug remains above the minimum inhibitory concentration of the bacteria. Based on preclinical models, this time should be greater than 40%. Moreover, high concentrations of meropenem are associated with nephrotoxic effects. Despite having a known therapeutic range of exposure, therapeutic monitoring of meropenem is scarcely used in our Country due to the unavailability of assays for quantification of the drug. An alternative to increase access to these tests is the use of dried blood spots (DBS), which allows facilitated collection and transport of specimens for testing. Objectives: To develop and validate an assay method for quantification of meropenem in blood microsamples obtained in the form of DBS, using LC-MS/MS. Methods: DBS samples were obtained by pipetting 50 μL of whole blood on Whatman 903 paper. After drying, 6 mm diameter punches of the paper were transferred to plastic microtubes and added with 300 μL of the extraction solution (acetonitrile: water, 70:30 v/v, containing ciprofloxacine-D8 3.3 μg/mL), followed by vortex-mixing for 15 min and sonication for 5 min. An aliquot of 50 μL of the supernatant was transferred to another tube and added with 200 μL of water and 200 μL of dichloromethane. After homogenization and centrifugation, the supernatant was injected into the

analytical system. The analytes were quantified in a liquid chromatography system associated with a tandem quadrupole mass spectrometer (LC-MS/MS), operating in positive electrospray ionization mode. The separation was performed in an Acquity C8 column (100 x 2.1 mm, 1.7 μm), with mobile phases composed of water and acetonitrile, both containing 0.1% formic acid and a flow rate of 0.3 mL/min. Calibrators were prepared in blood at the concentrations of 0.5, 1, 2,5, 5, 10, 25, and 50 μg/mL. Accuracy and precision were evaluated at the concentrations of 0.5, 0,75, 12.5, and 35 μg/mL. The method was validated according to international guidelines, including evaluation of specificity, stability, matrix effect, extraction yield, and hematocrit effect. Results: The method was linear in the range of 0.5 to 50 μg/mL. The inter-assay precision was between 5.2 and 7.5%, the intra-assays precision was between 6.4 to 7.5%, and the accuracy was between 101.2 and 101.7%. No interfering factors were identified in blank samples. Hematocrit had a limited effect on the accuracy and the extraction efficiency. The matrix effect was adequately compensated by the internal standard. Meropenem was stable for up to 24 h in DBS maintained at high temperature. Discussion/Conclusion: A method for the determination of meropenem in DBS using LC-MS/MS was developed and validated. The method presents adequate analytical performance for use in a clinical study, which is in progress. Acknowledgments: UNIMED Vale dos Sinos, CNPq, Feevale University.

Development and validation of a method for the determination of meropenem

employing micro samples of dried blood spots obtained from capillary punctures

BuSaTo, Maria aMelia De caSTilhoS; anTuneS, Marina venzon; linDen, raFael

Universidade Feevale - FEEVALE.

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Introduction: The use of biological specimens for biomonitoring is important for assessing the risk of environmental and occupational exposures to toxic elements and evaluate the deficiency of essential elements in the human body. The most common method of sampling blood is venipuncture. Alternative blood sampling techniques have been proposed, such dried blood spot (DBS). This technique consists in a collection of small volumes of whole blood on filter paper. However, a number of issues and challenges have been associated with the use of the DBS approach. Main issues are the hematocrit effect and the homogeneity of the sample. Volumetric absorptive microsampling (VAMS) is a novel sampling blood method. This device consists an absorbent tip attached to a plastic holder designed to absorb a fixed sample volume. The VAMS technique provides the same advantages of DBS: reduced blood sampling volumes, simplification of the sample collection, simplification of pre-treatment methods, reduced costs of shipment and storage. Moreover, the VAMS overcome the issues with hematocrit and homogeneity. Objective: The aim of this study was to develop and validate a method for the multielemental quantification of Li, Be, Mg, Mn, Co, Ni, Zn, Cu, As, Se, Rb, Sr, Cd, Cs, Ba, Hg, Tl, Pb e Bi in whole blood collected with VAMS, by using inductively coupled plasma mass spectrometry (ICP-MS). Methods: The analysis were performed by dilute-and-shoot procedure. Briefly, the VAMS was added in an extraction solution containing 0.2% v/v HNO3 and 0.05% (v/v) Triton X-100 in the ratio 1:50 (v/v). The resulting solution was directly injected into the ICP-MS. The VAMS of 20 μL were obtained from Neoteryx®. Reference materials of whole blood were used for method optimization and validation purposes. A pre-treatment with VAMS were evaluated: contamination from blanks analysis, absorbed volume

in the device tip and extraction conditions (vortex for 5 minutes, shaking for 60 minutes, ultrasonic bath for 30 and 60 minutes). The following validation tests were performed: limit of quantification (LOQ), limit of detection (LOD), linearity, accuracy, intra and inter assay precision. Results: The linearity of the calibration plots provided a correlation coefficient higher than 0.99 for all chemical elements. Estimated LODs and LOQs of the proposed method ranged from 0.10 to 29.2 ng L-1 and 0.35 to 88.6 ng L-1, respectively. The blank of the VAMS showed values <1 µg L-1 for Li, Mg, Ni, Zn, Cu, As, Sr and Ba while Be, Mn, Co, Se, Rb, Cd, Cs, Hg, Tl, Pb and Bi were below the limit of detection. The volume of blood absorbed in the device tip were 22,50 ± 0,80 µL with no statistical differences (p<0,05) from the manufacturer’s certificate of conformance of 22,80 µL. Shaking extraction for 60 minutes had low performance with the lowest recoveries found for Mn, Bi, Cu and Hg with values of 51.4, 54.3, 57.2 and 49.0%, respectively. Vortex and ultrasonic bath extraction exhibited good recoveries for all analytes (close to 100%). The average extraction recoveries found ranged from 96 to 106% while RSD values were <10%. Discussion/Conclusion: The proposed method were satisfactory and met the acceptance criteria of the validation guidelines. To avoid recovery issues, the extraction must be optimized. The ultrasound bath for 30 minutes is an effective method for chemical elements extraction which allows the preparation of more samples in a relatively short time. Moreover, the VAMS technique procedure could be an attractive method for human biomonitoring studies when regular monitoring of a population’s blood is required or special group with low adhesion, such as childrens. Acknowledgments: We thank for the financial support of CAPES (Finance Code 001) and CNPq (168344/2017-3).

Development and validation of analytical method for the quantification of chemical elements in whole blood with volumetric

absorptive microsampling by ICP-MS

SouSa júnior, WellingTon TavareS; MoraiS, DéBorah araújo; oliveira, Silvana ruella; BarBoSa jr, FernanDo

Analytical and System Toxicology Laboratory, Department of Clinical, Toxicological and Bromatological Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, Ribeirão Preto - SP, Brazil.

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Drosophila melanogaster is an important experimental model due to its high evolutionary conservation of many human-like genetic and physiological processes. D. melanogaster is used for studies involving cancer, metal toxicity, nanomaterials, depression and human neurodegenerative diseases such as Parkinson’s and Alzheimer’s. Clinical and experimental evidence has shown that changes in monoaminergic neurotransmitters in the nervous system are indicators of these neurodegenerative diseases. In this sense, techniques that can accurately identify and quantify these compounds, such as high-performance liquid chromatography (HPLC), can be important tools for establishing these neurotoxicity indicators in various studies. In this perspective, the present study aims to develop a chromatographic method by HPLC for the quantification of octopamine, dopamine, serotonin and tryptophan in the head of D. melanogaster. To quantify the compounds, we used high-performance liquid chromatography equipment coupled to a Young Lin (YL) 9100 diode array detector (HPLC-DAD) equipped with an Inertsil ODS-3 5 µm column (4.6 × 250 mm). The compounds were separated using the mobile phases (FM) composed of water acidified to pH 2.55 (H3PO4) (A) and methanol (B). With a gradient that consisted of FM 98:2 (A:B - flow 0.4 mL.min-1) up to 6 minutes, increasing the organic phase to 50:50 (A:B) up to 11 min returning to initial conditions in 13 min (flow 0.5 mL.min-1), and I reestablish the initial flow in 20 min and keeping it that way for up to 30 min. An injection volume of 20 µL was used, the compounds were identified by comparing their retention time and DAD spectrum with the reference standards. The analytical curve was constructed with Octopamine (198 nm), Dopamine (198 nm) and serotonin (204 nm) and consisted of 6 points at concentrations of 0.1; 0.5; 1.0; 2.0; 5.0 and 10.0 mg.L-1 for each compound. The validation criteria evaluated were selectivity, linearity, repeatability,

detection limit (LD), quantification limit (LQ). As an application of the previously validated methodology, 30 flies (age 0 - 4 days) were frozen (-80 °C) and their heads were homogenized with 400 µL of saline (0.9% NaCl and 0.5 mol.L-1 HCl - 96:4) (n=3). Then the samples were centrifuged at 10000 RPM (4°C), the supernatant was filtered on a 0.22 µm PTFE syringe filter and analyzed on HPLC-DAD. The chromatographic method proved to be selective, with the separation of the 4 compounds confirmed according to retention time and DAD spectrum, as well as lineares with R2 of 0.9996 for octopamine, 0.9993 for dopamine, 0.9993 for serotonin and 0.9996 for tryptophan. In addition, the method is repetitive and sensitive, with relative standard deviations between 0.015% and 2.9% and LD and LQ of 0.025 and 0.075 mg.L-1 for octopamine, 0.043 and 0.131 mg.L-1 for dopamine, 0.01 and 0, 03 mg.L-1 for serotonin and 0.038 and 0.151 mg.L-1 for tryptophan. For real head samples of D. melanogaster we were able to quantify 3 of the 4 compounds present in the calibration curve, octopamine with 1.01 ± 0.11 mg.L-1, dopamine with 0.68 ± 0.01 mg.L-1, and serotonin with 0.64 0.08 mg.L-1. The chromatographic method met all the validation steps within what is recommended by national and international regulatory agencies, proving to be a selective, linear, sensitive and repetitive method for the quantification of neurotransmitters present in the head of D. melanogaster. The chromatographic method also uses a low amount of organic solvents, in addition to not using derivatives or detectors that require other consumables such as the mass spectrometer (MS), which makes it an inexpensive method when compared to others in the literature. Furthermore, it can be an important tool for investigations of diseases in D. melanogaster that change the composition of these compounds. Keywords: HPLC-DAD; neurotransmitters; method validation.

Development of a HPLC-DAD-RF method for quantification of neurotransmitters

in heads of Drosophila melanogaster

carriço, Murilo ricarDo Sigal1; roDrigueS, Marina Diaz2; paz, Maria elizaBeTh goMeS1; Molina, higor Severo2; nogueira, caroline lacerDa1; DenarDin, elTon luiS gaSparoTTo3; roehrS, raFael3,4

1 Student of the Postgraduate Program in Biochemistry, Federal University of Pampa – Uruguayan campus. RS; 2 Undergraduate student in Pharmacy, Federal University of

Pampa Uruguayan campus. RS; 3 Professor at the Federal University of Pampa – Uruguayan campus. RS; 4 Professor at the Federal University of Pampa – Bagé campus. RS.

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Introduction: Ayahuasca is a hallucinogenic beverage originated in South America and obtained through the fermentation of two plants, P. viridis, containing N,N-dimethyltryptamine (DMT), and B. caapi, containing the β-carbolines (harmine, harmaline, and tetrahydro-harmine); the combined ingestion of these substances allows DMT to be absorbed through the gastrointestinal tract and exert its psychoactive effects. This beverage is commonly used recreationally, although recent research shows therapeutical applications of ayahuasca for the treatment of some psychiatric disorders. Considering the growing of ayahuasca consumption, analytical methods for detecting these analytes in conventional matrices has become popular. However, no methods so far have simultaneously detected DMT and the main β-carbolines in hair samples, which is a matrix that provides information on long-term exposure. In addition, a modern trend is the Green Analytical Toxicology (GAT) that aims at reducing the environmental impact of such methods. Objective: To develop an analytical methodology for the quantitation of ayahuasca alkaloids in hair using GAT principles. Methods: Twenty milligrams of hair samples were chemically digested with NaOH 1M and the analytes were extracted using the dispersive liquid-liquid microextraction (DLLME). Briefly, a mixture of tetrahydrofuran with chloroform (THF:CHCl3) was added to the digested samples followed by 30s of vortexing and centrifugation. Then, the organic layer was transferred to another vial containing with water and carbonate-bicarbonate buffer pH 10.8 0.1M. Next, samples were again vortexed for 30s and centrifuged so the organic layer could be transferred to another vial, dried under nitrogen flow, and resuspended with 50μL of mobile phase prior injection into the UPLC-MS/MS. Results: First, different organic solvents were tested to digest the hair samples and

a solution of NaOH was the only that fully digested hair fibers. Next, the Design of Experiment statistical tool was used to optimize both sample digestion and subsequent analyte extraction. The best condition of sample digestion associated with less degradation of the targeted analytes was 1 mL of NaOH 1M incubated for 90min at 50°C. Furthermore, once the samples were digested, different mixtures of extraction and dispersive solvents were investigated and two main candidates were obtained: THF:CHCl3 and hexane with methyl acetate (HEX:MA) however, the first mixture yielded the highest analyte recovery. Next, different buffers were tested to maximize analyte recovery and carbonate-bicarbonate pH 10.8 0.1M provided higher recovery. Finally, the method was tested with hair samples spiked at 10pg/mg and was able to successfully recovery and detect all targeted analytes. In addition, 2 authentic hair samples of ayahuasca users were analyzed to test the applicability of the method. Discussion/Conclusion: Although different solvents were assessed, only a solution of NaOH was able to fully digest hair fibers. In that context, the common organic solvents employed for that purpose (e.g. methanol) did not fully digest hair fibers, thus NaOH was chosen. Furthermore, albeit the analytes were exposed to harsh conditions during alkaline digestion and suffered partial degradation, satisfactory sensitivity was achieved. Moreover, even though HEX:MA efficiently recovered all target analytes, THF:CHCl3 was chosen due to the superior recovery paramount to achieve the intended limits of detection using only 20mg of hair samples (10pg/mg). Despite this mixture is more hazardous than HEX:MA, only 200µL was used. Finally, the method accomplished not only to recover the analytes from authentic hair samples, but also detected them at the concentration of 10pg/mg. Acknowledgements: CAPES-PROEX: 88887.499615/2020-00

Development of a method based on Green Analytical Toxicology to determine the

active components of ayahuasca in hair

SanToS, FaBiana pereira; FaBriS, anDre luiS; Bruno, viTor; yonaMine, Mauricio

Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo.

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Simvastatin is a prodrug in the lactone form (SVL), and must be hydrolyzed in a 1st pass metabolism by CYP3A4 for its main active metabolite, simvastatin hydroxyacid (SVA) to exert a pharmacological activity consisting of the inhibition of hydroxymethylglutaryl coenzyme A reductase, an enzyme that participates in the metabolic pathway of cholesterol production, thus being an important drug in the treatment of dyslipidemias, and a potential marker for CYP3A4 activity. Associated with the fact that it is a drug commonly used by obese patients or at risk of obesity, SVL can be considered a marker of choice for monitoring this enzyme in patients undergoing Roux-en-Y gastric bypass (RYGB) bariatric surgery. Despite the benefits of this surgery, there is a detriment to the patient regarding the absorption and oral bioavailability of nutrients and drugs, as well as an increase in hepatic CYP3A4 expression to compensate for the reduction in intestinal CYP3A4 activity, an overproduction that can generate long-term liver damage. This study aims to develop a method for the analysis of SVL and SVA by high performance liquid chromatography in plasma, using HF-LPME as the extraction method. Firstly, the chromatographic conditions were defined having as stationary phase a 5.0 µm reversed-phase C18 column (4.6 mm x 100 mm) from Waters coupled to a guard column (2 µm, 2.1 mm x 5 mm) and as mobile phase a mixture of methanol and ammonium acetate buffer (pH 4) in gradient mode: 0.01-5.00 min→65 MeOH: 35 Buffer/ 5.01-10.00 min→80 MeOH:20 Buffer, flow of 1.00 ml/min, temperature of 40º C, injection volume of 20 ul and wavelength for UV detection of

238 nm. Using this method with the UV detector we had a range of 100 ng/ml – 10 ug/ml for SVL and SVA in standard solution. After defining the analytical condition, the extraction of SVL and SVA in plasma samples was performed using the two-phase HF-LPME, which consists of a 15 cm MF-PP Q3/2 hollow capillary membrane impregnated in organic solvent, coupled to two tips and then immersed in a test tube with the donor solution that contains 500 ul of plasma previously centrifuged, 50 ul of standard solution (SVL and SVA) and 3.5 ml of 0.25 mol/l acetic acid solution (pH 2.0). For the chromatographic analysis under these conditions, the SVA had a retention time of 6.7 minutes and the SVL of 8.0 minutes having satisfactory resolution value between peaks (0.893). For the extraction, the solvent that had the best performance was octanol, being used in the two-phase HF-LPME, the extraction was selective because there were no interferences from the plasma sample in the analysis and the recovery of the method was 19,7% for SVL and 21,5% for SVA, it is worth mentioning that according to the literature this is a method that usually offers low recovery values, however, as long as they remain constant between analyzes and provide detection of the necessary concentration range of the analyte, there is no compromise of the method. Further studies will evaluate other solvents, improve parameters such as resolution and detection range and finally validate the analytical method making it suitable for its application in plasma samples from patients undergoing bariatric surgery.

Development of a method for analysis of simvastatin and simvastatin hydroxyacid by

HPLC in human plasma using HF-LPME extraction

vaSconceloS, Mayrla eMilia DanTaS1; gaiTani, criSTiane MaSeTTo1; MoraeS, naTália valaDareS2; DeMeTS, Mariana BarBieri alvarez1

1 Universidade de São Paulo - USP; 2 University of Florida - , UFL.

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Background/Introduction: Ketamine is a dissociative anesthetic, which present heterogeneous pharmacology, as it interacts with several receptors. This molecule is metabolized into three active metabolites, such as 6-hydroxynorketamine. Nowadays, ketamine has been found at parties (mainly electronic music parties, “raves”), being used as drug of abuse, and representing 87% of the quantity of hallucinogens seized in the last 5 years. Objective: the aim of this work was the development and validation of an analytical methodology based on dispersive liquid-liquid microextraction (DLLME) and liquid chromatography–mass spectrometry (LC-MS/MS) for simultaneous determination of ketamine, its metabolites norketamine and 6-hydroxynorketamine, and its analogs dechloroketamine and 2-fluorodeschloronorketamine (sold as New Psychoactive Substances) in oral fluid samples. Methods: The DLLME procedure was optimized during the method development. Parameters such as the extraction and dispersive solvents, and solvents volume were studied and optimized. In this stage were used drug-free blank samples, fortified with solutions of analytes at the calibrators and controls concentrations. For extraction, a 50μL aliquot of oral fluid (collected with Quantisal) was extracted using 100μL methanol and 50μL chloroform, as the disperser and extractor solvents, respectively. Subsequently

to this stage the samples are centrifuged and the extractive solvent, sedimented, containing the analyte of interest. The organic phase was transferred to another polypropylene tube and evaporated under a nitrogen stream (30°C). The residue was resuspended with 100μL of methanol and 2μL were injected into the LC-MS/MS. Results: The instrumental conditions were optimized, and the analyses were performed in a Nexera-UHPLC chromatographic system, coupled with a LCMS8060 mass spectrometer. The chromatographic separation was performed with a Raptor™ Biphenyl column (100×2.1mm, 2.7μm), maintained at 40°C. The mobile phase consisted of ultra-pure water (A) and methanol (B), both containing formic acid (0.1 %) and 2 mmol/L ammonium formate. The flow rate was set to 0.4mL/min, and the elution gradient was isocratic, with 35%B. The total run time was 4.5min. The method presents good linearity from 10 to 1000ng/mL (r> 0.995, 1/x2 weighted linear regression). Discussion/Conclusion: A reliable and accurate method was developed for all the compounds. In the next step, the method will be applied to the analysis of oral fluid samples collected from volunteers at parties and electronic music festivals. Acknowledgments: the authors thanks to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Development of a miniaturized sample preparation method for determination of

ketamine, its metabolites and analogs in oral fluid samples using Dispersive

Liquid-Liquid Microextraction (DLLME)

kahl, júlia M.M.1,2; Berlinck, DéBora zorrón2; chinaglia, kauê De oliveira1,2; roDrigueS, leonarDo coSTalonga1,2; coSTa, joSe luiz1,2

1 Campinas Poison Control Center, University of Campinas (UNICAMP), Brazil; 2 Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Brazil.

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Introduction: Iodine is an essential chemical element, which participates in the biosynthesis of thyroid hormones (tyrosine derived), thyroxine (T4) (4 iodine atoms) and triiodothyronine (T3) (3 iodine atoms). Iodine deficiency and excess consumption can lead to health problems. Thus, iodine human biomonitoring is very important, and its determination in urine is the most commonly used procedure. However, information about the consumption of iodine in a long-term period of time can be most relevant than a single point determination in urine. Hair is a promising matrix, as it accumulates the analyte excreted along the strand, registering variations in consumption over months or years, depending on the length of the sample. However, studies aiming at the determination of iodine in this matrix are still scarce, probably due to the limited number of analytical methods available in literature. Objective: The present study aims to develop and validate a fast, simple, and, low-cost sample preparation procedure for iodine determination in hair samples by ICP-MS employing alkaline solubilization with tetramethylammonium hydroxide (TMAH) at room temperature. Methods: Two different Human Hair Reference Materials (QM-H-Q1616 and QM-H-Q1725) from the Institut Nacional de Santé Publique du Quebec (INSPQ - Centre de toxicologie, Canada, Quebec) were used for method optimization and validation purposes. The concentration of TMAH (10, 25, 50, 75 and 100% (v/v)), the solubilization time (7, 10, 12 h and overnight) and the sample mass (between 15 and 20 mg and between 70 and 100 mg) were optimized. Then, high purity de-ionized water was added to the samples until maximum concentration of 1% (v/v) TMAH. Finally, samples were directly analyzed by ICP-MS (PerkinElmer, NexION® 2000 B, Waltham, EUA). Results: No statistical differences were found between the results for solubilization times of 10, 12

h, and overnight, TMAH concentrations of 50, 70, and 100% (v/v) and, sample masses (between 15 and 20 mg and between 70 and 100 mg) at 95% confidence level on applying the t-test. Good analytical iodine recoveries (p > 0.05) were obtained after solubilizing the samples at least for 10h using 50% (v/v) of TMAH (Human Hair QM-H-Q1725 reference material) and mass between 15 and 20 mg (Human Hair QM-H-Q1725 and QM-H-Q1616 reference materials), attesting the accuracy of the method. The intra (n = 4) and inter (n = 4) precision were 6.2% and 6.3%, respectively, showing good analytical repeatability (Human Hair QM-H-Q1725 and QM-H-Q1616 reference materials). Discussion/Conclusion: A new and promising analytical method for iodine determination in hair samples by ICP-MS was developed employing 50% (v/v) TMAH, overnight solubilization, and sample mass between 15 and 20 mg. This method is useful to analyze small hair segments (≤ 1 cm) since it requires low hair masses (< 20 mg). The lower TMAH concentration evaluated (50% (v/v)) provided good analytical results with the advantage of avoiding damages on the ICP-MS cones (sample, skimmer, and hyper skimmer). With the proposed procedure, at least 400 samples can be weighed, left overnight in the alkaline medium for complete solubilization at room temperature, diluted, and analyzed the next day, considerably increasing the analytical capacity in large-scale routine compared to methods that require microwave digestion procedures. Therefore, the sample preparation procedure using TMAH to hair samples solubilization, is a quick, simple, and a low-cost alternative to be applied in studies of body iodine evaluation. Acknowledgments: We thank for the financial support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001).

Development of a simple and fast method for iodine determination in hair samples by ICP-MS

after alkaline solubilization at room temperature

MoraiS, DéBorah araújo; SouSa junior, WellingTon TavareS; oliveira, Silvana ruella; BarBoSa junior, FernanDo

Analytical and System Toxicology Laboratory, Department of Clinical, Toxicological and Bromatological Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, Ribeirão Preto - SP, Brazil.

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Introduction: The use of cocaine (COC) during pregnancy has deleterious effects on both the pregnant woman and the child, in prenatal and postnatal periods, as well as obstetric complications. However, this subject is still relatively unexplored, and since the toxicological evaluation of in utero exposure is challenging, the development of new analytical methodologies for better toxicological management urges. The umbilical cord has been regarded as an alternative sample for such analyzes because of its advantages, such as its presence throughout the whole pregnancy and the fact that it is the biggest channel for fetus exposure to drugs. Therefore, this work intends to develop an ecologically friendly, simple and low cost analytical method based on Green Analytical Toxicology. Objective: This project aims to develop and validate a method for the determination of COC and its metabolites in umbilical cord through green extraction and gas chromatography coupled to mass spectrometry. Methods: For sample preparation, 500 mg of umbilical cord tissue (UCT) was placed in an eppendorf, 500 µL of 0.1 M phosphate buffer (pH 6) was added and homogenized using Ultra-Turrax for 1-2 min, followed by addiction of 1,5 mL of 0.1 M phosphate buffer. The homogenized tissue was centrifuged and the supernatant (500 µL) was transferred to another eppendorf. Then, 1,5 ml of dichloromethane: isopropanol: ammonium hydroxide (12:3:0.3) were added for the purpose of extraction. The mixture was stirred and centrifuged. The extract was collected and transferred to a vial, being dried at 40°C/10 min under nitrogen flow. Finally, it was derivatized with 30 µL of PFP and PFPA at 70°C/30 min, after being dried under nitrogen flow. It was resuspended with 50 µL of ethyl acetate and injected into the GC-MS. Results: This method was

applied in a possibly positive UTC from a mother with detection of 2478,03 ng/mL of benzoylecgonine in urine. The results have shown the determination of COC, benzoylecgonine (BZE), ecgonina methyl esther (EME), ecgonine (EC) and anydroecgonine methyl esther (AEME). Although the method worked, an optimization was performed in order to improve the extraction of the analytes. To optimize sample preparation, an experimental design was carried out by factor analysis, using the statistical tool DOE (Design of Experiment), and Response Optimizer, in Minitab® Statistical Software. An experimental model was built to evaluate 3 factors, at 2 levels, with critical influence on the extraction yield of all analytes. The model evaluated the following effects:• Volume of solvent mixture: 1,5 and 2 mL• Agitation time: 2 and 10 min;• PSA: 0 and 25 mg.Only for EC, the factors of volume of solvent mixture, PSA and the interaction between them presented significant difference, so the extraction was improved with 2 mL of solvent mixture and without PSA. In addition, an optimization of the response was performed with the prediction showed that volume of solvent mixture of 1,7828 mL, agitation for 10 min, and the absence of PSA were the best conditions for the extraction of the analytes (D=07508). Conclusions: This extraction was able to detect COC and its metabolites from the positive sample. According to the results, DLLME (dispersive liquid-liquid microextraction), without PSA, proved to be a more promising procedure than QuEChERS, with PSA. Due to the sample complexity and despite not having an especially micro volume, it can still be considered a green method.

Development of an analytical method based on Green Analytical Toxicology employing

umbilical cord tissue for the evaluation of maternal fetal exposure to cocaine

MeirelleS, gaBriela De paula; pereira e Silva, jeFFerSon; yonaMine, Mauricio

Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sao Paulo.

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2,4-dichlorophenoxyacetic acid, known as 2,4-D, which is an organochlorine herbicide used in post-emergence against broadleaf weeds. This herbicide is made from the compound 2,4-dichlorophenol (2,4-DCP), which is also its main metabolites. Both 2,4-D and 2,4-DCP are considered highly toxic, with known genotoxic and hepatotoxic potential. The intoxication of unwanted places occurs through drift, this process occurs when the product is applied in inappropriate weather conditions, being carried by the wind to other places, contaminated by non-target native organisms. Apis mellifera is an important bioindicator for this contamination, as they travel large distances daily in search of resources for the hive, they may come into contact with pesticides. Thus, the aim of this study was to develop a QuEChERS method for the extraction of the 2,4-D herbicide and its 2,4-DCP metabolite in Apis mellifera. The initial parameters of the extraction method were performed with Drosophila melanogaster (matrix), due to its greater availability. Initially, 0.5 g of frozen D. melanogaster were homogenized with 5 mL of water in Falcon tubes (15 mL) and fortified with the standard solution of 2,4-D and 2,4-DCP to reach the final concentration of 5 mg.L-1, and vortexed immediately for 1 minute. Then, we evaluated the recovery of compounds from a partition step with acetonitrile (MeCN) also evaluating the influence of different proportions of H3PO4 in MeCN (1, 2, 3, 5 and 10%) on the extraction of 2,4-D and 2,4-DCP. The tubes were again shaken and taken to an ultrasound bath (1 minute) for the subsequent Salting-out step. This step consisted of adding 2 g of MgSO4 and 0.5 g of NaCl followed by vortexing (1 minute) and centrifugation for 5 minutes at 7000 rpm. Then 1 mL of the organic phase was collected and its cleaning was done by vortexing the aliquot with 150 mg of MgSO4 and 50 mg of

octadecylsilane followed by centrifugation (7000 rpm) for 5 minutes. The supernatant was filtered on a hydrophobic PTFE syringe filter (0.22 µm). After defining the extraction phase, we applied the method to A. mellifera samples. All samples were analyzed by Young Lin high performance liquid chromatography (YL 9100) with diode array detector (HPLC-DAD). Chromatographic separation was performed isocratically with an Inertsil ODS-3 column (GL Sciences) and mobile phases acetonitrile/ultrapure water acidified to pH 3 with H3PO4 (87.5:12.5 v/v) with an analysis time of 16 minutes, flow of 0.8 mL.min-1 and 20 µL injection volume. The detection of 2,4-D (230 nm) and 2,4-DCP (220 nm) was confirmed by comparing the retention time and DAD spectra of the analytical standards obtained by the calibration curve with 6 points between 0.5 and 12 mg.L-1. The results were expressed as mean ± relative standard deviation (%RSD), considering as ideal recoveries between 70% and 120% with RSD < 20%. The extraction phase that recovered the greatest amount of compounds and met these criteria was MeCN with 5% H3PO4, which recovered 103.6% ± 8.85% of 2,4-DCP and 104.8% ± 9.9% of 2,4-D. The application of the extraction method in A. mellifera showed a recovery of 75.4% ± 3.3% for 2,4-DCP and 73.9% ± 13% for 2,4-D. Another important aspect for choosing the extraction phase was the lower extraction of interferents from the sample, as we observed that by increasing the proportion of acid, the extraction of interferents was reduced. The results presented demonstrate that the extraction method allowed the extraction of the 2,4-D herbicide and its metabolite 2,4-DCP in samples of A. mellifera reliably and can be an important tool for environmental monitoring of non-target organisms intoxicated by 2,4-D and its metabolite.

Development of an extraction method for the pesticide 2,4-dichlorophenoxyacetic

acid and its metabolite 2,4-dichlorophenol from Apis mellifera

paz, Maria elizaBeTh goMeS1; carriço, Murilo ricarDo Sigal1; roDrigueS, Marina Diaz2; raMBorger, Bruna piaia1; DenarDin, elTon luiS gaSparoTTo3; roehrS, raFael3

1 Student of the Postgraduate Program in Biochemistry, Federal University of Pampa, Campus Uruguaiana; 2 Pharmacy Student, Federal University of Pampa,

Campus Uruguaiana; 3 Professor, Federal University of Pampa.

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Introduction: Assistance and Toxicological Information Center of Santa Catarina (CIATox/SC) registered, in 2020, 5824 cases of intoxication involving toxic agents. Among the 10 drugs with the highest frequency of registration in humans are amitriptyline and diazepam, totaling 343 and 303 registered cases, respectively. Cocaine is the second drug of abuse most related in intoxication cases registered in CIATox/SC, with 221 cases. In this scenario, the emergency toxicological analysis has become relevant due to the constant and growing number of cases of acute intoxication, since it helps in the diagnosis, prognosis or support treatment. Objective: Develop a dispersive solid phase microextraction (d-SPE) and analyze by high-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD) that will be used for the identification of amitriptyline, diazepam and cocaine/ benzoylecgonine in urine samples. Methods: Analytical standards of amitriptyline, cocaine and diazepam were used at concentrations of 1μg/mL, 1μg/mL and 0.5μg/mL, respectively. The first step consisted of the evaluation of the chromatographic conditions. For the tests, the isolated standards prepared in acetonitrile or the mixture of all drug standards were dried and resuspended in 100µL of initial mobile phase (MP). Thermo Scientific® HPLC-DAD (Massachusetts, USA) and C18 column (250 mm x 4.6 mm x 5 μm) was used. The following parameters were evaluated: wavelength, composition and proportion of MP, isocratic or gradient elution, MP flow and run time. d-SPE was tested using three different sortive phases (DSCMCAX, Strata-X e Strata-X-Aw, 20 mg). 500μL de enrichment urine sample (pH 5) and 1000μL of ammonium acetate (pH 5) was mixed

with sortive phase. The system was shaken and centrifuged. Supernatant was discarded and 100μL de acetonitrile with 2% of ammonium hydroxide was added. The sample was shaken for 10 minutes and the supernatant was dried. The residue was resuspended in 100μL of MP. Results: DAD detector conditions were evaluated by scanning (190–800 nm), where the best wavelengths were: 200 nm for amitriptyline, 225 nm for cocaine, and 198 nm for diazepam. In the development of the chromatographic method, the isocratic elution presented the best results, with a MP composed of acetic acid buffer/ammonium acetate 0.25 M pH 5.5: acetonitrile (80:20, v/v) with a flow rate of 0.7mL/min, resulting in a better chromatographic resolution, where amitriptyline presented a retention time of 10.4 min, cocaine 6 min and diazepam 5.3 min. In the first d-SPE test the best phase was DSCMCAX for amitriptyline (recovery = 26.1%). For cocaine (recovery = 16.6%) and diazepam (recovery = 25,1%) Strata-X-Aw was better. Conclusion: Until now, all aspects of the chromatographic run and detection system were evaluated obtaining a fast and simple run, that minimally affected in the chromatographic system. At this stage, it was possible to perform the chromatographic analysis of 3 different substances using a run of only 14 minutes and with an adequate chromatographic resolution. The next step will be the optimization of d-SPE to obtain better recovery. With the development and validation of this method, it will be possible to apply it in the routine of emergency analysis. Keywords: Amitriptyline, Diazepam, Cocaine, HPLC-DAD, d-SPE, Clinical Toxicology. Acknowledgement: CNPq for the financial support and CIATox/SC for the partnership.

Development of dispersive solid phase microextraction (d-SPE) and HPLC-DAD

for emergency toxicological analysis of toxic drugs in urine samples

luz, heloiSa pereS1; Silva, Bruna eSpínDola2; SanToS, clauDia regina3; Marchioni, caMila3

1 Pharmacist Student, Federal University of Santa Catarina, Florianópolis, Brazil; 2 Resident pharmaceuticals, University Hospital, Federal University of Santa Catarina, Florianópolis, Brazil;

3 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

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Background: The COVID-19 pandemic sharply increased the demand for ethanol-based hand sanitizers (EBHS). To meet this great demand, regulatory health agencies worldwide have altered their regulatory guidelines, raising concern about the products quality. Objective: This study aimed to quantify ethanol content and to qualitatively assess common impurities in EBHS products marketed in Brazil. Methods: For the quantitative evaluation, 0.10 g of the EBHS were weighed in a 20 mL headspace (HS) vial, to which 5 mL of deionized water and 20 μL of isobutyl alcohol (internal standard for quantitative analysis) were added. The samples were vortexed for 10 s and incubated at 90°C for 6 min, and then 400 µL of the vapor phase was injected (on split mode 50:1) into a Gas Chromatography with Flame Ionization Detector (GC/FID) system. This quantitative method was validated according to the Anvisa Guideline on Analytical Method Validation. For the qualitative evaluation, 0.25 g of the EBHS were weighed in a 20 mL HS vial, to which 4 mL of deionized water and 5 µL of acetonitrile (internal standard for qualitative analysis) were added. The samples were vortexed for 10 s and incubated at 90 °C for 10 min, and then 400 µL of the vapor phase was injected (on split mode 10:1) into the same GC/FID system mentioned previously. Forty-eight commercial samples marketed in Brazil were analyzed. Results: The Anvisa establishes that the ethanol content in EBHS commercialized in Brazil must be at least 70% (w/w) and that the actual

quantity should not vary more than 10% in relation to the declared amount. Moreover, products with ethanol content less than 52.1 % (w/w) may not be effective against virus and bacteria. In this context, 27.1% of the analyzed products (n=13) presented unless one nonconformity regarding the amount of ethanol: seven products declared an ethanol content less than 70%, and in eight other products the actual ethanol content presented a difference superior to 10% than what was labeled (two samples presented both nonconformities). Furthermore, two samples contained ethanol levels less than the amount considered effective against microorganisms. According to the qualitative results, 12 samples (25%) presented acetaldehyde or ethyl acetate. However, the estimated levels for these substances were not toxic considering the common use of EBHS. Discussion/Conclusion: A simple and fast HS-GC/FID method to quantify ethanol in EBHS was developed, validated, and applied to commercial samples in Brazil. The huge demand for EBHS may have impacted their quality. Thus, the regulatory authorities must be more vigilant to ensure that the commercially available products meet the recommended specifications. Because concern with proper hand hygiene tends to remain an issue for a long period, more studies about quality control of hand sanitizers will be needed. Acknowledgments: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Ethanol quantification in ethanol-based hand sanitizers: alert in the

COVID-19 pandemic scenario

el haDDaD, lohanna pereira1; coSTa, Bruno ruiz BranDão2; Bigão, viTor luiz caleFFo piva2; De MarTiniS, Bruno SpinoSa1

1 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Faculdade de Ciências Farmacêuticas de

Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.

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Background: New psychoactive substances (NPS) represent a worldwide concern since distribution and synthesis of new compounds continues to increase. In this scenario, there is a need in developing methodologies that can efficiently extract these analytes from biological samples and provide a straightforward analysis. In addition, new analytical methods should be concerned with the use of environmentally friendly alternatives following the principles of “green analytical chemistry”. Therefore, the use of natural materials as biosorbents in solid phase-based techniques can be explored. Objective: This study aimed to investigate and optimize the variables of a solid phase-based extraction coupled to a 96-deepwell plate employing cork for LC-MS/MS determination of NPS in blood. Methods: An aliquot of 200 µL of blood was priorly submitted to protein precipitation with 250 µL of water, 250 µL of zinc sulfate and 50 µL of NaOH, followed by agitation and centrifugation. Afterwards, 500 µL of the supernatant was submitted to the proposed procedure, which consisted in its addition to a 96-deepwell plate containing cork. The extraction occurred in an orbital shaker at 300 rpm, followed by the removal of the aqueous phase and addition of the desorption solvent with further agitation. The organic layers were recovered and dried under nitrogen stream, reconstituted in 30 µL of methanol and analyzed. A Nexera UFLC system coupled to a LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Japan) was used for the analysis. Regarding optimization studies, blank blood samples were spiked with selected analytes (phenethylamines, cathinones and classic synthetic drugs), in a concentration of 50 ng/mL. First evaluation was the selection between cork sheet or cork powder for the procedure. Selection of extraction significant variables was performed through fractional factorial design (25-1) evaluating cork particle size, cork mass, extraction time, desorption time and desorption solvent volume. Variables

confirmed as significant were optimized by response surface methodologies and desorption solvent choice by a simplex-centroid design. Resulting data from the optimization studies was analyzed by the Statistica® software. Results: The use of cork powder was selected for the procedure since it presented higher analytical responses based on chromatographic peak areas. Significant variables for the process were cork mass, extraction time and desorption solvent volume. These were evaluated through a central composite design (CCD) for the achievement of optimum values. CCD analysis of cork mass vs. extraction time resulted in two optimum areas: (1) less cork mass and greater extraction time, (2) greater cork mass and less extraction time. These two conditions were evaluated in triplicate and shown no statistically significant difference (p > 0.05). Therefore, the use of a minor extraction time and greater mass of cork was chosen. Simplex-centroid results pointed to the use of 100% of acetonitrile as desorption solvent. Final procedure stands as follows: addition of 500 µL of the supernatant to 30 mg of cork and extraction for 15 minutes at 300 rpm, followed by the removal of the aqueous phase and addition of 1.15 mL of acetonitrile with desorption for 2 minutes at 300 rpm. LLOQs for the analytes ranged between 0.5 and 5 ng/mL. Discussion/Conclusion: Optimal responses achieved for cork powder over cork sheet can be explained by the higher contact surface between sorbent and sample. A higher mass of cork was chosen because it is a natural material with low-cost, while reduced extraction time improves the method high-throughput. In conclusion, this study demonstrates the applicability of cork as extraction phase for the determination of NPS in blood and optimizes the parameters that influence extraction efficiency. Nevertheless, the use of the 96-deepwell plate allows simultaneous extraction of multiple samples in the same batch, with possibility of automation. Acknowledgments: CAPES.

Evaluation of cork as a natural extraction phase for the determination of new

psychoactive substances in blood samples

Birk, leTícia; eller, Sarah; oliveira, Tiago Franco

Graduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil.

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Introduction: Cocaine (COC) is a stimulant of the central nervous system, and it is included World Anti-Doping Agency (WADA) Prohibited List1 to athletes. The metabolism of COC is well described and its main metabolites are ecgonine methyl ester (EME) and benzoylecgonine (BZE), the last being generated by the liver carboxylesterase or through spontaneous hydrolysis2. In the light of the new WADA regulation regarding COC cases, only the concentrations of COC and BZE are considered during analysis and results management. The project aims to evaluate the stability of COC in different pH and temperature conditions. Methods: Stability of COC was evaluated using water and urine for comparison reasons. For water matrix, COC reference material was spiked at a concentration of 100 ng/mL buffered at ranges from pH 4 to 9 and kept in ambient temperature (25 ± 5°C). Another batch, with the same composition was kept at refrigerator (4 ± 1°C) for seven days. One aliquot of 70 µL per day was collected and a “dilute-and-shoot” approach was performed with 30 µL of methanol containing the internal standard 7-propyltheophylline. For the stability study in human urine, the pH considered was intrinsic to the sample. No buffer was added. Real urine human samples from pH 5 to 8 range were used. Three samples per interval of pH was chosen according to the division: interval 1 (pH 5.0 to 5.9), interval 2 (pH 6.0 to 6.9), interval 3 (pH 7.0 to 7.9) and interval 4 (pH 8.0 to 8.9). COC reference material was spiked at 100 ng/mL in all samples, which were exposed to the same conditions of the study in water, for the same period. One aliquot of 1.00 mL per was daily collected and stored in freezer (-28 ± 2°C) until preparation by solid-phase extraction and analysis using liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS)3. Results and Discussion: In both matrices evaluated, the instability of COC was more pronounced in the pH 8 and 9, in which only small fractions of initial COC were detected after seven days of experiment in ambient temperature. In human urine

samples, COC in pH 8 decreased 78% in relation to the initial condition under ambient temperature and 34% under refrigeration. COC were still detected in all samples, even in alkaline pH. However, it could be attributed to the high sensitivity of the analytical approach adopted (LOD < 1 ng/mL). Higher instability was observed in urine samples and compared with water matrix, what suggest that other factors, endogenous to urine samples, developed a hole in the COC – BZE conversion. Water and human urine samples were considered to evaluate the difference in the spontaneous hydrolysis of COC to BZE. Conclusion: As described in the literature, samples with acid pH have shown the inhibition of the spontaneous hydrolysis. In contrast, as much alkaline the samples, more significant is the instability of COC. Hence, the influence of temperature and natural excretion pHs on stability of COC shows the possible impact when considering the sample transport conditions and mid-term storage until analysis. During results management related to antidoping cases, pharmacokinetics illations is not advisable, considering the lack of information of the original concentrations of COC and BZE at the sample collection time and the impact of temperature and pH in the spontaneous reaction observed. Excretions studies of COC administration becomes necessary to better understand the impact of the spontaneous COC - BZE conversion in antidoping cases interpretation. Acknowledgments: The authors thank Autoridade Brasileira de Controle de Dopagem (ABCD) for financial support.

REFERENCES1. World Anti-Doping Agency. Prohibited List 2022, Montreal, 2022. Available at https://www.wada-ama.org/sites/default/files/2022-01/2022list_final_en_0.pdf.2. Hoffman, R, et al. Experimental treatments for cocaine toxicity. The Journal of pharmacology and experimental therapeutics, 2013, 347. 3. Sardela VF, et al. Comprehensive analysis boy liquid chromatography Q-Orbitrap Mass Spectrometry: Fast Screening of peptides and organic molecules. J. Mass Spectom., 2018, 1-28.

Evaluation of the spontaneous hydrolysis of cocaine under different

pH and temperature conditions

goMeS, geovana Maria De liMa1; SanToS, vaneSSa Farelo1; carneiro, gaBriel reiS alveS1; Trajano, chriSTian FariaS2; pereira, henrique Marcelo gualBerTo1

1 Laboratório Brasileiro de Controle de Dopagem, Chemistry Institute, Federal University of Rio de Janeiro; 2 Comitê Olímpico Brasileiro.

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Introduction: Marine biotioxins (MBs) are toxins produced by algae. These compounds can be absorbed by edible bivalve mollusks such as oysters and mussels. The ingestion of such organism containing MBs can lead to several health damages such as diarrhea, paralysis, and even death. To assure the seafood safety to the consumers, the production of oyster and mussels in Santa Catarina state is strictly regulated, with weekly laboratorial monitoring of MBs. However, these compounds can be precociously detected in the water near to the sea farms. Thus, the use of direct detection or the use of passive samplers can be very useful to detect MBs before the seafood contamination. Objective: To perform a proof of concept of an innovative model of passive sampler, using a small scale prototype and okadaic acid and domoic acid as MBs models. Methods: The passive sampler is composed by a floating basis constructed with high density expanded polystyrene containing six wells with small holes. At the bottom of each well, a pellet with adsorbent was inserted. A test solution composed of ultrapure water was fortified with analytical standards of domoic acid (DA, 44.3 µg mL-1) and okadaic acid (OA, 16 µg mL-1), producing a solution with 17.72 ng mL-1 (DA) and 6.4 ng mL-1 (OA). This test solution (TS) was placed into a beaker. After that, the floating prototype was placed in the TS with the following adsorbent pellets: i) three pellets of bamboo activated carbon (BAC) with approximately 4-5 mm of diameter; ii) two pellets of cork with the same dimensions used for carbon; iii) one pellet of silicon rubber coated with cork powder. The beaker was covered and sealed. A halogen lamp was placed near to simulate solar heating. The experiment was carried out during 68 hours. Three aliquots of TS was taken at zero time and at the end of the experiment. After the experiment period, the pellets were removed and transferred to a polypropylene tube of 15-mL. To

each tube, 5 mL of methanol was added. Then, the tubes were mixed submitted to ultrasound-assisted extraction during 20 minutes. After that, samples were filtered and the solution was evaporated in a water bath (40 °C). The dry residue was reconstituted and submitted to LC-MS/MS analysis according to the official method (Molognoni et al, 2018). Results: The TS analysis showed a decreased in the MB levels from the 0 h until 68 h later of 27.7% and 3.3%, respectively. Pellet analysis showed that BAC is effective to adsorb both MBs. For OA, the initial calculated concentration was 6.1 ng mL-1. At the end of the experiment, the OA level was calculated as 4.8 6.1 ng mL-1. The OA mass estimated as adsorbed by the activated carbon pellet was 1.82 ng. The cork pellet was not able to adsorb detectable amounts of OA. However, a relevant peak of DA was identified. For DA, results closer to the OA was obtained using BAC. However, calibration curves were performed just for OA, not allowing the estimation of DA in terms of mass. A signal comparison shows an intense peak corresponding to DA and confirmed using three m/z transitions. Discussion/Conclusion: A proof of concept was performed for a new passive sampler to MBs detection in sea water. DA and OA was effectively adsorbed using BAC, showing that the concept of the floating passive sampler is valid. Further studies will be carried out to optimize the main parameters of the model in order to achieve a prototype able to be evaluated under real filed condition in large term studies.

REFERENCESMolognoni L, Dos Santos JN, Kleemann CR, Costa ACO, Hoff RB, Daguer H. Cost-Effective and High-Reliability Analytical Approach for Multitoxin Screening in Bivalve Mollusks by Liquid Chromatography Coupled to Tandem Mass Spectrometry. J Agric Food Chem. 2019 Mar 6;67(9):2691-2699. doi: 10.1021/acs.jafc.8b06600.

First proof of concept of a new passive sampler for marine biotoxins

FiTarelli, Bruna1; FaBichak, ana2; DeolinDo, carolina TurneS paSini3; kleeMann, criSTian3; hoFF, roDrigo3

1 Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil; 2 Departmento de Agricultura, Biodiversidade e Florestas,

Universidade Federal de Santa Catarina, Curitibanos, SC, Brazil; 3 Ministério da Agricultura, Pecuária e Abastecimento, Laboratório Federal de Defesa Agropecuária, São José, SC, Brazil

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Background/Introduction: The synthetic cannabinoids (SC) was the largest group (209) of new psychoactive substances monitored by the EU Early Warning System in 2020, in which 11 had being reported for the first time that year (1). Occasionally, the SC consumption has led to fatal intoxications worldwide. Considering their prevalence in the USA, ADB-4en-PINACA and ADB-FUBIATA have been strongly recommended to be incorporated into the SC scope analysis performed by toxicology laboratories; while BZO-CHMOXIZID is also recommended to be included after a slightly increasing trend (2). Objective: The aim of this work was to identify the main phase I metabolites of 3 SC (ADB-4en-PINACA, ADB-FUBIATA and BZO-CHMOXIZID) obtained by in vitro metabolism assay using human liver microsomes (HLMs) and analyzing by liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Methods: HLMs incubation was performed following previously published protocol (3). To 520 µL phosphate buffer 100 mM (pH = 7.4) was added 5 µL phosphate buffer/acetonitrile mixture solution (1:1, v/v) containing the analyte at 1,000 µg/mL, 50 µL NADPH aqueous solution (10 mM) and 25 µL pooled HLMs. The tubes were maintained at water bath (37 °C) under shaking during 2h, and the reaction was stopped with 500 µL acetonitrile. After centrifuged, samples were dried down to reduce volume and centrifuged (10,000 rpm/5 min) again using a tube filter. The extracts were transferred to vials and injected into an UHPLC Nexera XR (Shimadzu, Japan) coupled to a HRMS TripleTOF® 5600+ (Sciex, Canada), using electrospray in positive mode. Acquisition was performed using Information Dependent Acquisition (IDA). Diazepam was used as positive reaction control. Results: During the ADB-4en-PINACA ([M+H]+ at m/z 343.2129) in vitro assay was produced and identified 11 phase I metabolites. Major metabolites were obtained through hydroxylation combined with internal hydrolysis (m/z 377.2183), hydroxylation (m/z 359.2078) and amide hydrolysis combined with hydroxylation (m/z 360.1915). For ADB-FUBIATA ([M+H]+ at m/z 396.2082), 6 metabolites were identified. The most intense metabolites were formed by hydroxylation (m/z 412.2038), followed by N-dealkylation combined with loss of NH and dehydrogenation (m/z 271.1444) and amide hydrolysis combined with dehydrogenation (m/z 395.1774). Four metabolites had been identified related to BZO-CHMOXIZID ([M+H]+ at m/z 362.1863). The most prevalent identified metabolic pathways were hydroxylation (m/z 378.1812), dihydroxylation (m/z 394.1763) and ketone formation (m/z 376.1657), respectively. Discussion/Conclusion: Our work provides the identification of several metabolites related to 3 SC of interest currently. There are no other published works related to in vitro metabolism studies

of the emerging “OXIZID” generation of SC, as well regarding ADB-FUBIATA. Due to structural similarities, part of ADB-4en-PINACA metabolic pathway is compared with those reported for MDMB-4en-PINACA during in vitro metabolism study and confirmed in in vivo authentic samples (4), with metabolites formed via hydroxylation, dihydroxylation, N-dealkylation and double bond oxidation. Nine metabolites for ADB-4en-PINACA were formed when incubation was performed using human hepatocyte (5), in which 7 were also identified on this present work. Analysis of one authentic femoral blood and urine samples from a postmortem case kindly provided by the NMS Labs (Willow Grove, PA, USA) corroborated with 7 of 11 ADB-4en-PINACA metabolites identified during our in vitro assay. Metabolites formed by hydroxylation, hydroxylation combined with internal hydrolysis, dihydroxylation and/or double bond oxidation combined with hydroxylation are suggested to be used as biological marker. Although authentic ADB-FUBIATA and BZO-CHMOXIZID positive-biological samples have not been identified yet, the detection of these analytes in drug material recently suggests that they should be investigated and the potential metabolites identification during our in vitro studies could help in the development of future analysis methodology. Acknowledgments: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

REFERENCES1. EMCDDA. European Drug Report 2021: Trends and Developments. Publications Office of the European Union. 2021. p. 60. https://www.emcdda.europa.eu/system/files/publications/13838/TDAT21001ENN.pdf. Accessed Feb 2022. 2. Discovery N. Recommended Scope for NPS Testing in the United States - Q4 2021. 2022. https://www.npsdiscovery.org/wp-content/uploads/2022/02/Q4-2021-NPS-Scope-Recommendations_NPS-Discovery_021722.pdf. Accessed Feb 2022.3. Krotulski AJ, Mohr ALA, Papsun DM, Logan BK. Metabolism of novel opioid agonists U-47700 and U-49900 using human liver microsomes with confirmation in authentic urine specimens from drug users. Drug Test Anal. 2018;10(1):127–36. 4. Erol Ozturk Y, Yeter O. In Vitro Phase I Metabolism of the Recently Emerged Synthetic MDMB-4en-PINACA and Its Detection in Human Urine Samples. J Anal Toxicol. 2021;44(9):976–84. 5. Kronstrand R, Norman C, Vikingsson S, Biemans A, Crespo BV, Edwards D, et al. The metabolism of the synthetic cannabinoids ADB‐BUTINACA and ADB‐4en‐PINACA and their detection in forensic toxicology casework and infused papers seized in prisons. Drug Test Anal. 2021;

In vitro metabolism studies of ADB-4en-PINACA, ADB-FUBIATA and BZO-CHMOXIZID

using human liver microsomes

cunha, kelly F.1,2; kroTulSki, alex j.2; WalTon, Sara e.2; papSun, Donna M.3; coSTa, joSe luiz4; logan, Barry k.2,3

1 Faculty of Medical Sciences, University of Campinas, Campinas, SP, Brazil; 2 Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, 2300 Stratford

Ave, Willow Grove, PA 19090, USA; 3 NMS Labs, 3701 Welsh Rd, Willow Grove, PA 19090, USA; 4 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, SP, Brazil

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Background: The combination of advanced analytical techniques makes it possible to determine the concentrations of neurotransmitters (NTs) in different biological matrices, providing a complex and comprehensive study of the effects of psychoactive substances. Therefore, methods must be specific, sensitive, and robust to simultaneously monitor different NTs, which can then be used to evaluate the neuronal modulation caused by the effects of xenobiotics. Objective: The present study aimed to develop and validate a fast and simple analytical method for the determination of NTs acetylcholine, serotonin, γ-aminobutyric acid (GABA), glutamate, dopamine, and metabolites 3-4-dyhydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA) in rats brain tissue by liquid chromatography coupled to tandem mass spectrometry. Methods: An aliquot of rats brain tissue was homogenized in type 1 water containing 2% formic acid to a final concentration of 100 mg/mL. The brain homogenate (50 μL) and 20 μL of dopamine-d4 (internal standard, 250 ng/mL) were added to plastic tubes, followed by the addition of 930 μL of acetone prior to agitation. The samples were then centrifuged, and the supernatant was collected and dried under nitrogen stream. After cooling down, the residue was reconstituted in 50 μL of type 1 water, transferred to an auto‐sampler vial and 20 μL were injected into the analytical system. A Nexera-i LC-2040C Plus system coupled to a LCMS-8045 triple quadrupole mass spectrometer was used for analysis. The chromatographic separation was conducted using a Shim-pack CLC ODS 4.6 mm x 25 cm column eluted with water (solvent A) and methanol (solvent B) both fortified with 0.1% formic acid with a flow rate of 0.5 mL/min. Results: The optimization of the best extraction solvent was performed through a simplex-centroid design by testing the solvents

methanol, acetonitrile, and acetone. The best results were obtained using acetone as an extraction solvent (100%) as it yielded the region with the best response. Moreover, the mathematical model exhibited satisfactory results with a coefficient of determination (R2) of 0.9059, which indicates that the experimental results can be well explained with this modeling strategy. The proposed method was validated providing determination coefficients higher than 0.99 for all analytes; the LLOQ values were 0.001 ng/mg for dopamine, serotonin, DOPAC and HVA and 0.1 ng/mg for acetylcholine, GABA, and glutamate; intra-run precision ranged from 0.47 up to 11.52%; inter-run precision from 0.68 to 17.54% and bias from 89.10 to 109.60%. Discussion/Conclusion: The NTs are considered external and independent markers, capable of quantitatively demonstrating the effects of neuropsychiatric conditions and the consumption of psychoactive substances on the signal transmission of neuronal synapses, having wide applicability in metabolomics studies. However, current techniques described in the literature show the need for multiple sample preparation steps, as well as considerable volumes of sample and organic solvents, which results in time-consuming analysis, the need for high sample volume, in addition to having a greater negative impact on the environment. The analytical strategy developed in this study was not only able to overcame the afore-mentioned issues but it has also proven to be efficient for the simultaneous determination of NTs at basal levels in rats brain tissue homogenate. In conclusion, this analytical tool can be used to support the study of changes in the neurochemical profile for the characterization of the mechanism of action of psychoactive substances, and both neurologic and psychiatric diseases.

Liquid chromatography–tandem mass spectrometry method for simultaneous quantification of

neurotransmitters in rat brain tissue

viana, roBerTa roDrigueS1; pego, ana Miguel FonSeca2; Dallegrave, eliane1; oliveira, Tiago Franco1; eller, Sarah1

1 Pharmacosciences Department, Federal University of Health Sciences of Porto Alegre, Porto Alegre, RS 90050-170, Brazil; 2 John Jay College of Criminal Justice,

City University of New York, 524 W 59th St, New York, NY 10019, USA.

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Introduction: Cocaine (COC) is the main alkaloid present in Coca leaf (Erythroxylum coca), and it is included in the Prohibited List of World Anti-Doping Agency (WADA). However, in the antidoping context, the use of cocaine is prohibited only in-competition. Currently, in-competition period shall in principle be the period commencing just before midnight (at 11:59 p.m.) on the day before a competition in which the athlete is scheduled to participate until the end of the competition and the sample collection process. WADA accredited laboratories findings for COC must be reported as an Adverse Analytical Finding (AAF) if, and only if, either (or both) of follow conditions is met: i) The estimated concentration of the COC is above 10 ng/mL, and/or the estimated concentration of the metabolite benzoylecgonine (BZE) is above 50 ng/mL. The aim of the project was to evaluate the COC and BZE level in urine from an excretion study from coca tea administration and evaluated these levels under the light of WADA rules to trigger AAFs. In addition, the impact of the pH in the spontaneous hydrolysis of COC in BZE was also evaluated. Methods: Coca tea was administrated to 12 volunteers distributed in two groups, one in the morning and other in the afternoon. Volunteers were 6 females and 6 males of different ages between 22 and 50. A commercial tea bag of coca leaf was prepared by infusion for 5 minutes in 100mL of hot water. An aliquot of 5mL was collected for analysis. A single administration of tea was made to each volunteer and aliquots of all urine of the firsts 48h and first urine in the morning after 48h until the fifth day after administration. Also, a fraction of each urine aliquoted was transferred to a Falcon tube containing a buffer solution of formic acid/ammonium formate 2.0M in pH4 to control the sample pH. Samples were kept under refrigeration (-305°C) until preparation by solid-phase extraction

and analysis using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Results and Discussion: Preliminary results showed COC and BZE concentration values much higher than necessary for the configuration of an AAF during the firsts 24h after administration. COC concentrations decreased fast, reaching the LOD of the method (1ng/mL). The interval of Cmax of COC excreted among the volunteers was of 2.4-87.1ng/mL and BZE was off 630.9-2432.1ng/mL, showing a large inter-individual variation. Buffered samples showed the inhibition of spontaneous hydrolyses of COC in volunteers’ urine. The impact of pH and the inter-variability of the excretion profiles observed among the volunteers hamper the pharmacokinetics approach in results interpretation. Continuously analysis will be done to verify if the spontaneous reaction o hydrolysis can influence the result management of COC and BZE in doping samples. Conclusion: Excretion of coca tea has shown high concentrations of COC and BZE in urine samples, exceeding the established values for doping control several hours after the administration. An in-competition interval discussion is interesting to evaluate if a consumption outside of this range could be seen within the criteria currently adopted for an adverse analytical result. The inter-variability of the excretion profiles and the wide range observed in pH values in real urine samples make the interpretation of results very challenge related to the estimative of the consumption time. Samples with buffer addition will be compared with the samples without pH control to further evaluation of the impact of spontaneous hydrolysis of COC. Athletes should be aware about the potential to an AAF from the consumption of coca tea even out of completion period. Acknowledgments: The authors thank Autoridade Brasileira de Controle de Dopagem (ABCD) for financial support.

Metabolism study of coca leaf tea in urine by liquid chromatography coupled with

high resolution mass spectrometry

goMeS, geovana Maria De liMa1; SanToS, vaneSSa Farelo1; carneiro, gaBriel reiS alveS1; Trajano, chriSTian FariaS2; pereira, henrique Marcelo gualBerTo1

1 Laboratório Brasileiro de Controle de Dopagem, Chemistry Institute, Federal University of Rio de Janeiro; 2 Comitê Olímpico Brasileiro

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Background/Introduction: Paraquat is a widely used herbicide which use is prohibited by Brazilian’s Health Regulatory Agency (ANVISA). Its toxic effects relate to respiratory oxidative stress, which affects liver, heart, and mainly lungs, causing hemorrhages, fibrosis, edema, severe lungs failure and death. Paraquat’s high toxicity and the absence of a specific antidote leads to treatment difficulties, intensifying the need for a fast intoxication diagnostic. Considering that, a rapid identification for paraquat’s acute intoxication can be realized through a colorimetric assay using sodium dithionite, which changes the urine color to blue in the pesticide’s presence. The color change can be measured using PhotoMetrix, a free application for smartphones developed for in situ chemical analysis that decompose, and process digital images obtained by the phone camera. It can be used for univariate (calibration) and multivariate analysis (principal component analysis [PCA]). Considering that the application works with signal intensity, it can be employed to toxicological colorimetric assays, as in paraquat in urine, measuring the concentration through the difference in color and signal intensity, emerging as solution for identification and quantification of paraquat in acute intoxication. Objective: Development of an analytical method for the quantification of paraquat in urine using the mobile phone application PhotoMetrix and colorimetric reaction with sodium dithionite. Methods: For sample preparation, 2 mL of basic buffer solution (pH 9.0) were transferred to a cell culture plate containing 6 flat-bottomed wells, followed by 1 mL of urine with paraquat and 1 mL of solution of sodium dithionite at

0.1 g/mL. PhotoMetrix application was installed on a mobile phone Xiaomi Redmi Note 7 with a camera of 48 Mpx and it was calibrated with 6 points from 10 to 150 µg/mL on univariate analysis, Vector RGB mode. The region of interest chosen was 96x96, Flash mode On, Exposure 0, Focus Mode on infinity, auto White-Balance and resolution of 640x480. A ring light was also used to improve illumination. Then, samples were analyzed on a spectrophotometer DU-8200 Drawell® set at 600 nm to compare the correlation of absorbance and concentration values. Results: The developed method was able to quantify paraquat in urine with linearity from 10 to 150 (r = 0.98). The application generated the calibration curve equation y = 1.893x + 10.439 while the plotted absorbance values from the spectrophotometer generated y = 0.0126x + 0.0546 (r = 0.99). Urine sample from an intoxicated patient was analyzed and a concentration of 152.23 ng/mL was estimated with the PhotoMetrix while on the spectrophotometer the calculated concentration for the same sample was 169.62 ng/mL. Discussion/Conclusion: PhotoMetrix presented satisfactory results for the analysis of paraquat in urine showing good correlation with the spectrophotometry technique. The application emerges as good alternative due to its low cost, practicality and efficiency, being an accessible technique to provide reliable information about estimated concentration after paraquat’s intoxication. Acknowledgments: Materials and reagents used for this research were financed by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

New tools in analytical toxicology: analysis of Paraquat in urine with a mobile phone

chinaglia, kauê De oliveira1,2; lanaro, raFael2; aranTeS, ana carolina Furiozo1,2; coSTa, joSé luiz1,2

1 Faculty of Medical Sciences, University of Campinas, Campinas, SP, 13083887, Brazil; 2 Campinas Poison Control Center, Faculty of Medical

Sciences, University of Campinas, Campinas, SP, 13083859, Brazil.

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Background: 5-Fluorouracil (5-FU) is an antimetabolic drug widely used for the treatment of gastrointestinal tumors, analogous to the endogenous uracil. Dihydropyrimidine dehydrogenase (DPD) deficiency is responsible for approximately 80% of severe toxicities related to fluoropyrimidines. DPD also converts endogenous uracil (U) to dihydrouracil (UH2), with U levels > 16 ng/ml and the ratio U/UH2 <4.0 predictors of DPD deficiency, allowing the identification of patients at risk for 5-FU severe toxicity even before the first chemotherapy cycle. During chemotherapy, plasma area under the curve concentrations of 5-FU in the range of 20 to 30 mg.h/L have been associated with better outcomes. A method for determination of 5-FU and endogenous U and UH2 in plasma would help to individualize and improve cancer treatment. Objective: To develop and validate a method for the simultaneous determination of 5-FU, U and UH2 in plasma by UPLC/MS-MS. Methods: As U and UH2 are endogenous compound, the calibration and control samples were prepared with the isotopes uracil (U 2-13C15N2) and dihydrouracil (UH2-13C15N2) standards. The analytes were extracted from 200 µL of plasma after addition of 150 µL ammonium sulfate 1 M, 100 µL of internal standard (UH2-D4 200 ng/ml and clorouracil 5-CU 4 µg/ml) and 2.1 mL of solvent ethyl acetate:isopropanol (85:15, v/v). Samples were homogeneized by rotation for 10 minutes and centrifuged at 3000 rpm for 10 minutes. The organic layer was transferred to a clean tube and concentrated in an Eppendorf vacuum concentrator at 60 °C for 1 hour. The extract was recovered with 200 µL of acetic acid 0.5% in water and after the lipid content was cleaned out with the addition of 50 µL of dichloromethane. After homogeneization and centrifugation, the

aqueous phase was transferred to a vial and 1 µL injected onto UPLC/MS-MS Acquity® I-Class UPLC coupled to a Xevo® TQS-micro triple quadrupole mass spectrometer with electrospray ionization in positive mode. Chromatographic separation occurred in an Acquity® UPLC HSS C18 (1.8 µm 2.1 x 150 mm) at 25 °C. Mobile phase was water with 0,5% acetic acid (eluent A) and acetonitrile with 0.5% acetic acid (eluent B) eluted in gradient mode from 100:0 to linear gradient of 10:90 (A:B, v/v) at 4.7 mi, maintained for 1 min, with flow rate of 0.25 mL min -1. The MRM transition for quantification were 5-FU m/z 131 → 58 and it’s IS 5-CU m/z 147 → 74, and UH2-13C15N2 m/z 118.1→ 70, U 2-13C15N2 m/z 116.1→ 71 and their IS UH2-D4 m/z 119→ 70. The method was validated according to FDA, to date specificity linearity, precision/accuracy, sensitivity and selectivity were performed. Results: Total analytical run time was 8 min, retention time was 2.68 for U-13C15N2, UH2-D4 and UH2-13C15N2, 2.97 min for 5-FU and 3.8 min for 5-CU. The method was selective, with no interference peaks eluting at the same relation time as the analytes with areas above 20% of those from the lower limit of quantification. The method was linear from 2.5 to 250 ng mL-1 for U-13C15N2, 5 to 500 ng mL-1 for UH2-13C15N2 and 50 to 2000 for 5-FU (r2 > 0.99 for 1/x fit for all analytes), with accuracy of 89-105%. Precision with CV% intra-assay within 1.90-9.87%, inter-assay within 4.9-13.5% for all analytes. Discussion/Conclusion: The method has been shown to be adequate for determining the concentrations of 5-FU and U in plasma samples. After conclusion of the validation tests for matrix effect and extraction yield, the method will be applied in a clinical study. Acknowledgments: Financial support from Fapergs, CAPES and CNPq.

Preliminary results of method validation for dihydropyrimidine dehydrogenase (DPD)

deficiency screening and 5-fluorouracil determination in plasma by UPLC-MS/MS

granDo, ana paula1; Silva, laura cé1,2; hahn, roBerTa zilleS1; linDen, raFael1,2;anTuneS, Marina vezon1,2

1 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

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Background: The chalcone (2E)-1-phenyl-3-(4-methoxy-phenyl)-2-propen-1-one, called 4-MC, has demonstrated important anti-inflammatory and bone regeneration activities, being a potential inhibitor of cathepsin K. However, little is known about its toxicity. Studies with cathepsin K inhibitor drugs have shown important toxic effects, increasing the risk of stroke, adverse dermatological effects, and others. Objective: This work aims to provide a toxicological analysis through in silico, in vitro techniques, in addition to the in silico toxicology of major degradation products. Methods: The molecule was synthesized, purified and characterized as to its solubility, crystalline habit, and purity. Stability was addressed by in silico and in vitro models (photostability), monitored by HPLC-UV and UPLC-MS/MS. Results: The 4-MC has a crystalline form, without polymorphism, being practically insoluble in water. The in silico analysis suggested the hydrogens attached to the carbon located at the C6 position of both rings of 4-MC as the most probable positions for auto-oxidation. The 4-MC showed photo instability especially against visible light (loss of 22.1 and 56.4% at 1.2 and 2.4 million lux/h, respectively). The UPLC-MS/MS analysis revealed the appearance

of seven major degradation products, in agreement with in silico prediction. The probable degradation products are isomers and dimers of 4-MC, formed by loss of double bond and rotation of the molecule followed by the addition process. In silico toxicological analysis suggest that chalcone 4-MC, and its major degradation products, showed potential mutagenicity or skin sensibility being moderately toxic for liver tissue. However, 4-MC did not show inflammatory angiogenic or hemorrhagic activity, suggestive of biocompatibility with mucous membranes in the HET-CAM test. Discussion: Thus, these results enabled the understanding of the intrinsic lability and pointed out the potential toxicity profile of 4-MC, which must be proven by in vitro and in vivo studies, aiming to compare with other drug candidates, previously reproved in clinical trials. Keywords: in silico toxicology; in silico stability; HET-CAM test; photostability. Acknowledgments: CAPES (PVE, grant 88887.116106/2016- 00), CNPq (J.R.S. -process # 429505/2018–3;310326/2020–6 and T.M.B.B. -process # 305484/2018–4 are researcher professor granted by CNPq with the respective processes and also to Edital Universal (grant 88887.122964/2016-00).

Safety studies of a potential cathepsin K inhibitor: 4-methoxy chalcone

and its degradation products

BenvenuTTi, Danyela Francine1; Buzzi, FaTiMa De caMpoS1; corrêa, rogerio1; couTo, angélica garcia1; Wagner, TheoDoro1; paula, Favero r.2; giovagnoli, STeFano3; vivani, riccarDo3; ricci, Maurizio3;

SanToS, carloS eDuarDo MaToS4; SanTin, joSé roBerTo1; BreSolin, Tania Mari Bellé1

1 Pharmaceutical Sciences Graduate Program, Universidade do Vale do Itajaí (UNIVALI), Itajaí, SC, Brazil; 2 Pharmaceutical Sciences Graduate Program, Universidade Federal

do Pampa, Uruguaiana, RS, Brazil; 3 Department of Pharmaceutical Sciences Università degli Studi di Perugia, Perugia, Italy; 4 Altox Lab, São Paulo, SP, Brazil.

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Background: The concept of statistical analysis during the planning stages of research was introduced by Sir Ronald Fisher in the beginning of the 20th century. This approach presents among its benefits the rationalization of experiments and process optimization. The pharmaceutical industry has traditionally used the one-factor-at-a-time (OFAT) model to study process optimization. Despite the recent introduction of new and potentially better statistical models, the pharmaceutical industry has continued to use the previous system. This is potentially due to lack of studies regarding new models. Conversely, others such as chemical and food industries have already introduced new models such as Design of Experiments (DoE) and Principal Component Analysis (PCA) in their routine. Among such recent mathematical modeling models, DoE has been shown to better understand the effects of multidimensional and interactions of input factors on the output responses of pharmaceutical products and analytical methods. Objective: The aim of this study was to access the effect of the implementation of DoE on the extraction efficiency (recovery) of synthetic cannabinoids in dried blood spots (DBS) samples. Further objectives were to further analyze 17 of synthetic cannabinoids’ analytes by liquid chromatography–mass spectrometry (LC-MS/MS) method. Methods: The Plackett-Burman method was be used for the screening variables, followed by central composite designs (CCD) to determine the best conditions for the extraction of synthetic cannabinoids. Other statistical parameters used were: analysis of variance (ANOVA), regression significance, analysis of residuals, determination coefficients (R2

and R2-adj) and lack-of-fit of regression model. Six

variables were used as input factors: resuspension volume, material used to resuspend extract, rotation type, rotation time, vortex time and material for extraction. The STATISTICA 10.0® software package was used for analyses. Results: Of the six input factors studied, two achieved statistical significance (p<0.05) and hence taken to the next step of the process (i.e. resuspension volume and rotation time). From the four remaining factors, one was not significant for any responses and three were qualitative factors. For the next step, four different levels and respective centre points were used for each factor. Resuspension volumes were set as 40 µL and 110 µL for the “star” points and 50 µL and 100 µL for the factorial design; and rotation time as 6 and 34 minutes and 10 and 30 minutes. The optimal condition found after the experiments was a resuspension with 50 µL mobile phase and rotation time of 34 minutes. In these conditions, the results obtained varied between 70 and 185% for the increment of analyte recovery, when compared to the other conditions. Discussion/Conclusion: The determination of the optimal conditions for the extraction of synthetic cannabinoids in DBS samples is essential bearing in mind the limited amount of sample available for analysis. With this study, the interaction of two factors was taken into account in order to determine the optimal conditions, what cannot be achieved with the OFAT model. Our results show that the use of DoE in the optimization process is crucial for obtaining the best possible results. This is of essence especially for large scale experiments used in industry, as it contributes to lower costs and greater efficiency. In addition, it optimizes the use of limited samples, a common scenario in forensic toxicology.

Use of design of experiment tools to enhance the recovery of synthetic

cannabinoids in dried blood spots (DBS)

Berlinck, DéBora zorrón2; cunha, kelly FranciSco1; coSTa, joSé luiz1,2

1 Toxicology Assistance and Information Center (CIATox), University of Campinas, Campinas, Brazil; 2 Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Brazil.

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Introduction: LQFM030 was obtained by molecular simplification of nutlins and having demonstrated excellent in vitro antineoplastic activity. Therefore, a preclinical pharmacokinetic study was planned and a rapid, sensitive, and selective LC/MS-MS bioanalytical method was developed and validated to quantify LQFM030 plasmatic concentrations in rats. Methods: Chromatographic analysis was performed using a 3200 QTRAP LC-MS/MS System and ACE® C18 analytical column (5 µm 100 x 4.6 mm) and 4 minutes total run time. An (50:50) methanol and 2 mM ammonium acetate with 0.025% formic acid was used as mobile phase. A multiple reaction monitoring (MRM) method transitions were used m/z = 319.162 / 191.20 and m/z = 426.032 / 175.20 for LQFM030 and internal standard respectively. Post validation was performed after administration of LQFM030 100 mg/kg by gavage and 1.0 mL blood samples were collected at intervals 0-8 h. The kinetic parameters were calculated by WinNonlin 5.0 software (Pharsight™). Results: Calibration curve was linear

over the concentration range examined 10-15000 ng/mL with r> 0.99 and 10 ng/mL limit of quantitation. Intra-assay precision was 0.6 to 5.5% and accuracy from 95.5 to 111.3%. Inter-assay precision was 1.8 to 6.7%, and accuracy was 99.0 to 107.0%. The average recovery was 74.1% and the matrix effect from -7.9 to 1.5%. The prototypes were considered stable in the biological matrix and the solution proposed tests. Pharmacokinetic results (mean ± SD): half-life (t1/2) 3.61 ± 0.68 h, total clearance (CLT) 36.49 ± 2.23 mL/min/kg, volume of distribution (Vd) 11,40 ± 1.58 L/kg. Conclusion: The validated analytical method proved to be adequate to quantify low plasma concentrations of LQFM030, allowing to establish the relationship between dose and its pharmacological effects. As proof of concept, pharmacokinetic parameters of LQFM030 were calculated in experimental animals. Keywords: LQFM030. LC-MS/MS. Preclinical pharmacokinetics. Acknowledgments: Instituto de Ciências Farmacêuticas - ICF; FAPEG, CNPQ, CAPES.

Validation and pre-clinical evaluation of pharmacokinetic profile of antineoplasic prototype LQMF030 in rats by LC-MS/MS

pereira, i.B.1; zoghaiB, i.v.j.1; goMeS, S.a.1; MenegaTTi, r.2; cunha, l.c.1

1 Núcleo de Estudos e Pesquisas Toxico-Farmacológicas (NEPET), Faculty of Pharmacy, Federal University of Goiás (UFG), Goiânia, Brazil; 2 Laboratório de Química Farmacêutica Medicinal

(LQFM) - ), Faculty of Pharmacy, Federal University of Goiás (UFG), Goiânia, Brazil.

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Introduction: Due to physicochemical properties, several psychoactive licit and illicit compounds consumed by a lactating woman can be transferred from her bloodstream into human breast milk (HMB), which can cause damage in newborn’s short and long term development. Previous research show the presence of drugs of abuse in breast milk samples from surveys’ volunteers or milk bank donations, such as marijuana (THC) and cocaine. In this context, a method based on Disposable Pipette Extraction (DPX) with analysis by gas-chromatography coupled to mass spectrometry (GC-MS) was developed and validated for the quantification of several of psychoactive substances in HBM. Objectives: To validate a DPX-GC-MS method for simultaneous quantification of psychoactive substances and its metabolites in human breast milk. Methods: An HBM pool was spiked with the analytes at 100ng.mL-1 (amphetamine, methamphetamine, methylenedioxymethamphetamine, nicotine, cotinine, methadone, morphine, cocaine, cocaethylene and benzoylecgonine) and deuterated internal standards at 200ng.mL-1. The sample preparation was previously optimized by design of experiments and consisted of adding 2.5 mL of acetonitrile and 100 µL of phosphoric acid to 1 mL of HBM. Then, the samples were agitated using a horizontal shaker (200 rpm for 10 min) and centrifuged (2000 rpm for 10 min). After the protein precipitation, DPX tips of cationic exchange phase were used for extraction and pre-concentration: 0,5 mL of acetonitrile for solid phase conditioning (30 seconds); 0,5 mL of methanol/water 70% (v/v) for washing (10 seconds); 875 µL of extraction solution (dichloromethane: isopropanol: ammonium hydroxide 78:20:2 v/v/v, repeated three times for 30 seconds each). The extraction solution was vaporized at 30 °C

using N2 and then reconstituted with 35 µL of ethyl acetate. Finally, 2 µL of the sample were injected in splitless mode in the GC-MS. The method was validated based on ANVISA and SWGTOX Guidelines on Bioanalytical Method Validation. Results and Discussion: The limits of detection (LODs) reached from 2,0 to 5,0 ng.mL-1 and the lower limit of quantification (LLOQ) was 5,0 ng.mL-1 for all analytes. Calibration curves were linear between 5.0 and 500 ng.mL-1 with correlation coefficient values higher than 0.99 using weighted linear regression (1/x2). Recovery values ranged from 15.0 to 86.0%, intra-day precision 2,0-19,0% (CV%), inter-day precision 4.0-19.0% (CV%) and accuracy of 5.0-13.0% (RSE%). Quality controls (QCs) samples were stable in long-term stability, post-processing and thawing cycle tests. No carryover effect was observed. Conclusion: The method validation indicated accurate sensibility and reproducibility on evaluated parameters. A large volume of acetonitrile had to be used during protein precipitation, although in DPX procedure was possible to reduce solvent consumption after statistical optimization. DPX-GC-MS demonstrated to be a suitable technique for complex matrices analysis for forensic toxicology purposes, such as HBM, with quick and less laborious steps and also meeting Green Analytical Chemistry principles. The validated method will be applied for analysis of HBM collected at milk banks in the city of Ribeirão Preto, São Paulo, Brazil. Acknowledgements: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Pro-Forenses 25/2014) and Fundação de Amparo à Pesquisa de Estado do São Paulo (FAPESP – 2017/18021-5).

Validation of a DPX-GC-MS method for simultaneous quantification of

psychoactive substances and its metabolites in human breast milk

SanToS junior, WilSon joSé raMoS1; goMeS, nayna cânDiDa1; coSTa, Bruno ruiz BranDão1; Bigão, viTor luiz caleFFo piva1; De MarTiniS, Bruno SpinoSa2

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Faculdade de Filosofia, Ciências e Letras de

Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.

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Introduction: Vancomycin is a potent hospital antibiotic used in the treatment of gram positive bacterial infections including methicillin-resistant Staphylococcus aureus (MRSA). The recommended plasma concentration is 15 to 20 µg/mL. Lower values cause development of resistant strains and can lead to therapeutic failure. However, plasma values above 20 ug/mL can cause nephrotoxicity and ototoxicity. Therefore, a narrow therapeutic window is observed for the treatment, besides, it’s known that to be a drug with high intra individual kinetic variation, hepatic and renal insufficiency patients have different plasma concentrations with the same dose applied. Consequently, patients’ therapeutic monitoring becomes vitally important to prevent the adverse effects caused by the high concentration and also avoid the bacterial resistance development. Objective: Validate an analytical method capable of determining plasma vancomycin, using High Performance Liquid Chromatography with UV detector (HPLC-UV). Methodology: For plasma vancomycin quantification, a method was developed using Shimadzu HPLC-UV system, reverse C18 column (4.6 mm x 15 cm x 5 µm) adopting as mobile phase a two-phase system constituted by acetic buffer pH 4.0 (A) and methanol: acetonitrile in proportion to 70:30 (v/v) (B). The gradient starts 90A:10B (v/v), at 4 minutes the race reaches a concentration of 40A:60B (v/v), at 9 minutes 70A:30B (v/v) and back to 90A:10B (v/v) up to 12 minutes, maintaining this ratio up to 22 minutes. The

vancomycin retention time obtained was 11.5 minutes. As an Internal Standard, it was used imidacloprid (50 µg/mL), which obtained a 14.2 minutes retention time. The method analytical validation was performed based on the standards of the National Health Surveillance Agency of Brazil (ANVISA). Results: The method presented a Limit of Detection (LOD) of 2 µg/mL and a Limit of Quantification (LOQ) of 5 µg/mL. Linearity was determined in the range of 5 to 100 µg/mL. The intraday and interday precision were approved with a Coefficient of Variation (CV) at 9.27%. The intra and interday accuracy was also adequate with a Relative Standard Deviation (RSD) at 10.52%. No residual effect was observed. As for selectivity, there were no significant peaks in the same retention time of interest analytes. There was also no matrix effect (p > 0.05). For stability, the samples were stable for 24h at room temperature, with up to 3 freeze and thaw cycles, post-processed for 24h and long term (3 and a half months to date). Conclusion: The chromatographic method for vancomycin quantification proved to be sensitive, precise, accurate, with no residual or matrix effect. It has been proven yet vancomycin stability in biological matrix in freeze/thaw cycles, post-processing and short/long duration. In conclusion, the validated method complies with the requirements to be used in hospital routine for necessary dose adjustments in patients on antibiotic treatment. Keywords: Vancomycin, Therapeutic monitoring, Plasma, Validation, HPLC-UV.

Validation of a method for plasma vancomycin therapeutic monitoring by High Performance

Liquid Chromatography with UV detector

paula, eliza Bianchini1; SanToS, clauDia regina2; Marchioni, caMila2

1 Federal University of Santa Catarina (UFSC), Brazil; 2 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

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Introduction: The emergence and dissemination of new psychoactive substances around the world represent a worrying public health problem. Objective: Develop and validate a method for the analysis of amphetamine, methamphetamine, MDMA, MDA, MDEA, cocaine, cocaethylene, anhydroecgonine methyl ester, dibutylone, n-ethylpentylone, 25E-NBOMe, 25C-NBOMe, 2C-E, 2C-C, fentanyl and carfentanyl in oral fluid sample using the GC-MS. Method: In a glass tube were pipetted: 500 µL of oral fluid, the analytical standards and the internal standards amphetamine-d11, fentanyl-d5 and cocaine-d3. 2 mL of methanol was added, vortexed and shaker table, followed by centrifugation. 2 mL of this solution was transferred to another glass tube and 100 μL of 0.1 M H3PO4 was added. The conditioning of the DPX-SCX tip was done with 1mL of acetonitrile. 2mL of solution containing 0.1 M H3PO4 were aspirated. Washing was performed using 500 μL of methanol and, for elution, 1500 μL of dichloromethane: isopropanol: ammonium hydroxide (78:20:2) solution was used. The eluate was evaporated, reconstituted with 40 μL of ethyl acetate and derivatized with 40 μL of MSTFA. HP 5 fused silica capillary column (30 m x 0.25 mm x 0.25 μm film thickness) was used. The injector temperature was 280 °C (splitless mode), the injection volume was 1µL and the carrier gas used was helium. The initial oven temperature was 90 °C for 2 minutes. This was raised to 220 °C at a heating rate of 10 °C.min-1. Then, elevation to 290 °C with a heating rate of 20 °C.min-1 for 4 minutes. The temperature used in the MS interface, source and quadrupole was 280, 230 and

150 °C, respectively. The parameters evaluated in the validation of the method were those recommended by ANVISA resolution 27/2012 and 899/2003. Results: The detection limit ranged from 1 to 15 ng/mL. and no residual effect was observed. The matrix effect ranged from 1.62% for dibutylone to 10.45% for 2C-E. The method was considered linear in the concentration range from 2 to 400 ng/mL for amphetamine, MDMA, MDA, dibutylone and n-ethylpentylone, from 3 to 400 ng/mL for methamphetamine, from 5 to 400 ng/mL for MDEA, 25C-NBOMe, fentanyl and carfentanyl, from 10 to 600 ng/mL for cocaine, cocaethylene, anhydroecgonine methyl ester and 25E-NBOMe and from 30 to 1100 ng/mL for 2C-E and 2C-C. The analyte recovery values range from 74.45% to 97.38% for the LLQ of 2C-E and n-ethylpentylone, respectively. The intraday precision ranged from 0.32 to 14.03% for the LQC and LLQ values of dibutylone and 25C-NBOMe, respectively. The interday precision varied from 1.15 to 13.93%, which were the LLQ values of 25E-NBOMe and HQC of 2C-C, respectively. The intraday accuracy values ranged from -12.98% to 15.79% for the LLQ and MQC values of amphetamine and 2C-C, respectively. The intraday accuracy ranged from -15.60% for the amphetamine LLQ to 12.86% for the carfentanyl LLQ. Analytes were stable in oral fluid and standard solutions. Discussion/Conclusion: The method developed and validated met all the criteria required by resolution 27/2012 and 899/2003 of ANVISA. Acknowledgments: This research was supported by CAPES – Brazil (Financing Code 001) and FAPESP 2017/18021-5.

Validation of an analytical method for the determination of psychoactive substances

in an oral fluid sample using the DPX-SCX disposable tips and the GC-MS

goMeS, nayna cânDiDa1; De MarTiniS, Bruno SpinoSa2

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil. 2 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Ribeirão Preto, São Paulo, Brasil.

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Introduction: Ethylenethiourea (ETU), biotransformation product of dithiocarbamates (DTCs) fungicides, has been suggested as a biomarker of occupational exposure, requiring the development and validation of methods for its quantification. Some researchers have been used analysis by Gas Chromatography-Mass Spectrometry (GC-MS) and sample preparation by classic methods, like liquid-liquid extraction. The Disposable Pipette Extraction (DPX) technique is a miniaturization of solid phase extraction that allows rapid and simple extraction of analytes due to sorption equilibrium. Objective: Validate a methodology for quantification of ETU in urine samples by DPX-GC-MS. Methods: A 1 mL tip containing 40 mg of silica gel freely accommodated between two porous polymer filters was developed for use in the DPX procedure. With the aid of a syringe device, HCl 6N (100 µL) was aspirated as a conditioning solvent from the extractor phase. Afterwards, the urine sample (200 µL) was inserted into the pipette followed by aspiration of air (turbulent air bubbles) for 1 min, to create a sorbent suspension in the sample, which was subsequently discharged. Then, the sorbent was washed (cleaning step) with 100 µL of hexane:dichloromethane (95:5, v/v) with a draw/eject cycle. Finally, the ETU was eluted with 200 µL of hexane:acetone (50:50, v/v) by aspirating air for 1 min and the eluate was dried in a water bath at 40 °C. The dried extract was reconstituted with 50 µL of derivatizing solution (ethyl acetate: BSTFA: t-BuMe2Si-Cl (5:4:1, v/v/v)) and incubated at 60°C/30 minutes, modified from Fustinoni et al. (2005). The extract was analyzed by GC-MS, quadratic high-quadratic ISQ TRACE 1300, coupled to a mass spectrometer (ThermoScientific®). Capillary column of Rxi®-5ms capillary column (30m x 0.25 mm x 0.25 µm) was used and helium was a carrier gas at 1.0 mL/min. Injections were made in the pulsed splitless mode. The injector

liner (250 °C) injected 1 µL of the sample into the chromatographic column. The oven temperature was kept at 150 °C for 1 min, then the temperature was increased to 240 °C at the rate of 20 °C min-1. The oven was kept at 240 ºC for 5 min. The total run time was 11.5 min. Thus, the method was validated, evaluated linear range, limit of quantification, precision, accuracy, matrix effect, carryover, selectivity and stability. Validation followed current guidelines published by the European Medicines Agency (EMA), Food and Drug Administration (FDA) and National Health Surveillance Agency (ANVISA) Results: Two ions were selected form ETU monitoring and confirmation, m/z 159 and m/z 231. The linear range was 100 to 1000 ng/mL (r=0.999). Values of precision had ranged from 0.6 to 10.8% and accuracy was in the range -13.2 to 13.4%. The limit of quantification was 100 ng/mL. Carryover was not observed according to acceptance criterion. It was verified that there was a matrix effect (r<0.001), being of the rotational type. Therefore, analysis by standard addition will be used to circumvent this effect, since tests in sample preparation were performed and satisfactory results were not obtained. Stability was not obtained for the freezing/thawing (two cycles at -20°C) and post-process (-20°C/96h) tests, however, long-term stability studies are being carried out. Discussion/Conclusion: Even though the method DPX-GC-MS should a matrix effect, the validation was effective, once it is possible for the analysis of samples to be performed by standard addition. The method developed is innovative, cheap, simple, fast and requires a small amount of biological sample and organic solvent, making it a good option for application in rural workers in order to assess occupational exposure. Keywords: Ethylenethiourea, gas chromatography, DPX, validation. Acknowledgements: CNPq, CAPES and COMCAP-UEM for their support.

Validation of disposable pipette extraction method with quantification

by GC-MS to determine ethylenethiourea in human urine samples

roMoli, jéSSica criSTina zoraTTo1; ScanFerla, DeBorah ThaiS palMa2; aguera, raul goMeS2; lini, renaTa Sano2; caSTro, juliana criSTina3; BanDo, érika4; alveS, geSSé De Souza4; nerilo, SaMuel

BoTião4,5; MoSSini, SiMone apareciDa galerani2,4; Marchioni, caMila6; MachinSki junior, Miguel1,4

1 Postgraduate Programin Health Sciences, State University of Maringá, Paraná, Brazil; 2 Postgraduate Program in Bioscience and Physiopathology, State University of Maringá,

Paraná, Brazil; 3 Postgraduate Program in Pharmaceutical Sciences, State University of Maringá, Paraná, Brazil; 4 Laboratoty of Toxicology, Departament f Basic Health Sciences,

State University of Maringá, Paraná, Brazil; 5 University Center Ingá, Maringá, Paraná, Brazil; 6 Department of Pathology, Federal University of Santa Catarina, Florianopolis, Brazil.

03  CLINICAL AND

LABORATORIAL TOXICOLOGY

111

Background: The material (Hg) is distributed in different media, circulating in different states, being found in different states: gaseous, liquid and solid. Different degrees of forms, exhibiting varying degrees of toxicity. H can cause kidney damage, central nervous system disorders, intellectual disorders and even death. Therefore, an efficient assessment of suspected intoxication through the determination of Hg levels in various biological matrices, mainly blood and urine, is of vital importance. The aim of the study was to validate an easy and suitable for analysis of both organic and inorganic Hg in urine. Methods: Validation was performed using a Inductively Coupled Plasma Mass Spectrometry, 7850 ICP-MS. Analytical analysis is ensured using the most abundant isotope 200Hg as monitoring. The procedure involves a small amount of 500 µL of urine, followed by dilution with 500 µL of internal standard at concentration of 1.0 µg/L more 4000 µL of hydrochloric acid and 2% nitric acid. The internal standard was purchased from Agilent at a concentration of 10.0 µg/mL. After preparation the samples are followed by aspiration into the SPS 4 model. A cleaning solution used was hydrochloric acid and 2% nitric acid at a rate of 0.1 rps. Quantitation is achieved by the comparison of the

responses from a given sample with the responses of calibrators with known concentrations prepared through Multi Element Cabibration at concentration of 10.0 µg/mL of Hg. Linearity was observed in the expected concentration range from 0.1 to 1.6 µg/L and urine samples were evaluated at six different concentrations and six times each at the same time of the study. For validation, the same extraction diluent was used and to evaluate the matrix effect, tests were performed with a white urine sample. Standards acquired by Agilent. Results: The linearity coefficient of determination (R) was 0.9931. The method showed 100% selectivity and no residual effect interference. In order to determine the average inter-assay CV%, three different concentrations were analyzed over three days and the results for each low, medium, and high concentration level were 7.64, 8.08 and 5.82%, respectively. The accuracy of the method was cheeked by analyzing samples of known concentration and expressed as a percentage. A time total analysis time was 3.5 min. Conclusion: The method was fast and efficient for the determination of urine Hg. The efficiency and selectivity combined with the technical robustness can be used in the diagnosis of intoxication and monitoring also in cases of toxicity.

A simple procedure for the determination of mercury in urine by Inductively Coupled

Plasma Mass Spectrometry (ICP-MS)

SugaWara, eDuarDo kinio; Silva, graciele MachaDo; paulucci, leTicia TreviSan; raMaDan, DeBora r.; TuFik, Sergio; SoareS, Marcela De oliveira

Associação Fundo de Incentivo à Pesquisa - AFIP.

112

Background/Introduction: Antipsychotics are commonly used in the treatment of patients with psychic disorders symptoms. In 2021, the cases of antipsychotic exposure represented almost 20% of all medicine intoxication cases attended by the Toxicological Information Center of Rio Grande do Sul (CIT/RS). Therefore, the development of quick, easy and efficient methodologies for the diagnosis of these intoxications is essential for clinical practice. Objective: The aim of this study was to develop and validate an absorptive paper-based extraction for fast gas chromatography-mass spectrometry (fast-GC/MS) analysis of chlorpromazine, haloperidol, levomepromazine, and promethazine in human plasma samples. Methods: The samples were obtained from emergency cases attended by the Toxicological Analysis Center at the CIT/RS. Sample procedure: To perform this technique 24-well plates were used with two discs (16 mm) of Whatman® qualitative filter paper Grade in each well. An aliquot of 50 μL of plasma was transferred into the plate and dried for 10 min at room temperature. Then, 500 μL of a mixture of methanol and acetonitrile in the proportion 1:1 (v/v), and 15 μL of internal standard (chlorpromazine-d4) was added to the well. The plate is placed on the orbital shaker for 5 min at 200 rpm. After that, aliquots of 450 μL of the solvent of extraction were collected and dried under a nitrogen stream. The residue was reconstituted with 20 μL of methanol followed of 2 μL injection with a split ratio of 1:5 into the analytical system. Optimization: The solvents were optimized using the simplex-centroid design and the volumes of sample and extraction solvent were determined using the Doehlert design, both by software Statistica 8.0. Instrumentation: The chromatographic analysis was performed in a GC-MS-QP 2010 Plus gas chromatography coupled to mass spectrometry and autosampler model AOC-20i (Shimadzu, Kyoto, Japan). Chromatographic

separation was carried out in a Rxi fused silica capillary column model RH-Rix-5MS (20 m × 0.18 mm × 0.18 μm). The ultrapure helium was used at a constant flow of 1.42 mL.min−1. The injector temperature was set at 250 °C and the initial oven temperature was set at 200 °C, increasing to 310 °C, and held for 0.6 min, then increased to 330 °C and holding for 1.78 min, totalizing 4.0 min of chromatographic run. The pressure was set at 297 kPa. The quantification of the analytes was performed in selected ion monitoring (SIM) mode using the highest intensity m/z ratio for each substance. Results: The method validation was conducted according to European Medicines Agency guidelines on bioanalytical method validation. The developed method has been validated for the lower limit of quantitation (LLOQ), linearity, selectivity, and carryover. The LLOQ was 50 ng/mL for all the analytes. The concentration ranges used were 50-800 ng/mL for chlorpromazine, haloperidol, levomepromazine, and promethazine. All analytes presented a coefficient of determination (r2) ≥ 0,99. The heteroscedasticity was calculated, and the correction factor was applied. No carryover effects were observed. Total of 28 samples were analyzed. Six samples were found to be positive for promethazine (21%), including one case reaching a toxic concentration and three with concentrations higher than the therapeutic recommendation. Fourteen samples were positive for chlorpromazine (50%) and nine were above the therapeutic range. Discussion/Conclusion: The technique proposed in this work presents a quick analysis considering the extraction and chromatography procedure. Therefore, the implementation of this methodology in toxicological centers can optimize the analytical procedure supporting the decision-making and, consequently, more effective management, and treatment, for the intoxicated patient. The method will still be completed with all the parameters required by the guideline. Acknowledgments: CAPES; FAPERGS.

Analysis of plasmatic levels of antipsychotics through an absorptive

paper-based extraction followed by fast gas chromatography-mass spectrometry

gouveia, giovanna criSTiano1; BorgeS, gaBriela raMoS1; SanToS, Bruno pereira1,3; SeBBen, viviane criSTina2; arBo, Marcelo DuTra3; eller, Sarah1; oliveira, Tiago Franco1

1 Graduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil; 2 Toxicological Information Center of Rio Grande do

Sul (CIT/RS), Brazil; 3 Federal University of Rio Grande do Sul (UFRGS), Brazil.

113

Introduction: On January 25, 2019, dam I at the Córrego do Feijão mine in Brumadinho, Minas Gerais, collapsed releasing more than 10 million cubic meters of tailings into the environmental and water body of Paraopeba river. This occurrence had several social, economic, political, environmental and health impacts on people living in the municipality and also in nearby communities. However, the magnitude of these effects on the population needs further investigation, especially regarding environmental expousure to contaminants. The research addressed here portays the assessment of the health of comunities close to the Paraopeba river throught the toxicology of metal and metalloids, being the first work of this nature in these locations. Objective: The purposes were to evaluate the environmental exposure to mercury, as well as the health of the population residing in the communities near the Paraopeba River. Methods: 121 volunteers from the municipalities of Mário Campos, Juatuba e São Joaquim de Bicas participated in this cross-sectional study. Data were obtained through self-repot in a pre-collection interview, blood and urine samples Blood were collected for analysis of: blood count, renal and hepatic function biomarkers by biochemical measurements and metals and metalloids by DMA (Direct Mercury Analysis). Results: Volunteers reported hypertension (47), dermatites, allergies and skin irritation (26), diabetes (17) and mental health conditions (11). The blood count demonstrated probable incidences of erythrocytosis

(23,3%), erythropenia (8,6%), hemoglobin deficiency (6.9%), reduction in RDW-CV (68.1%), leukocytosis (12.1%) and eosinophilia (9.5%). The measurement of liver and kidney function biomarkers showed an increase in: alkaline phosphatase (23.5%), AST/TGO (47.3%) and serum urea (75%). In addition, 13 volunteers had an AST/ ALT ratio greater than 2 and 4 albumin results were above 30 mg g -1 creatinine. The validated methodology for mercury analysis was satisfactory, there were 15 levels above the detection limit of the method (0,1212 ng; 0,606 µg L-1), 2 of them above the quantification limit (0,2108 ng; 1,054 µg L-1), in whole blood. The multivariate analysis indicates similarity between the populations evaluated in terms of global health parameters accessed Discussion and Conclusion: The data presented in this work show important hematological and biochemical alterations in individuals living in communities along the Paraopeba River after the Brumadinho disaster. These events may be associated with exposure to metals and metalloids in the region, however, mercury levels were similar to those found in other populations not occupationally exposed. Other potentially toxic elements have been measured by ICP-MS to help understand changes in tests performed and the magnitude of impacts generated in these locations. Acknowledgment: We thank the Ezequiel Dias Foundation partners for their collaboration and enabling important stages of this work.

Assessment of mercury exposure and health of riverside populations affected

by the Brumadinho disaster

Moreira, caMila FranciSco; nolaSco, Daniela; Souza, Tainá BruMaTe; MenDeS, Michele polyana rocha; anDré, leiliane coelho; paiva, Maria joSé nuneS

Toxicological Analysis Laboratory, Department of Clinical and Toxicologic Analysis, College of Pharmacy - Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

114

Introduction: Cocaine is a tropane alkaloid derived from the leaves of the plant species Erytroxylum coca and used worldwide as a drug of abuse. According to the Single Convention on Narcotic Drugs of 1961 and to the United States Convention against Illicit Traffic in Narcotic Drugs and Psychotropic Substances of 1988, the distribution, import, export, manufacture and production of this drug or derivative compounds are illegal in most countries. To evade detection and apprehension by law enforcement agencies, traffickers employ a vast and diverse array techniques, notably the swallowing of drug packets containing cocaine. Objective: To describe patient care, trying to assess the therapeutic procedures adopted by the health team in handling a case of cocaine poisoning related to the ingestion of drug packets. Methods: All data pertinent to this case report were elicited from the health team of the Toxicological Information and Assistance Center located in the city of Fortaleza, state of Ceará. A thorough review of the specialized literature on the subject was also carried out. Results: On the day of 03/10/2019, a 34-year-old male was forwarded to the Toxicological Information and Assistance Center about twelve hours after having–according to the patient himself–ingested twenty cocaine packets. By the time the Toxicological Center was called upon to assist in treatment, the patient, having already excreted nine intact packets and manifesting hematemesis, had a blood pressure of 123/69 mmHg (120/80 mmHg) and a cardiac frequency of 103 beats per minute (60-64 beats/min). Initial management consisted of intravenous hydrations to avoid hypotension, and of the administration of mineral oil to facilitate the excretion of the remaining drug packets; also, an echocardiogram (ECHO) and other biochemical tests were requested. The latter’s results, which were issued only in the morning of the next day (04/10/2019), revealed high levels of AST: 41 U/L (0 - 35 U/L); CPK:1291 U/L (32 - 294U/L); and of

CKMB: 32 ng/mL (< 5 ng/mL). Hemoglobin (Hb) level was below normal: 12,9 g/dL (14 - 17 g/dL). Tests performed in the afternoon of 04/10/2019 detected a higher-than-normal lactate dehydrogenase (LDH) level–that is, 485 IU/L (115 - 225 IU/L)–and a further elevation of the CPK level–to 4.371 U/L (32 - 294 U/L). In view of such results, the medical staff decided to keep the patient intravenously hydrated and to request an abdominal x-ray to learn if the “packets” had been eliminated. On 05/10/19, the patient fled the hospital. Discussion/Conclusion: Depending on its clinical manifestations, cocaine poisoning treatment consists initially of procedures aiming ventilatory and hemodynamic stabilization; additionally, vital signs and body temperature should be carefully monitored. Given that cocaine may cause an increase of catecholamines release, cardiac frequency elevation was to be expected. Considering that the use of laxatives, by stimulating peristalsis, may cause the drug packets to rupture, the administration of mineral oil, in general, preferable in facilitating excretion. The patient’s low Hb count may be related to the episodes of hematemesis he reported. According to the specialized literature, the concomitant rise of CPF, CKMB and LDH levels within 24 hours after exposure is indicative of heart damage and/or distress consistent with cocaine poisoning. As this Center does not perform toxicological analyses, the adoption of therapeutic procedures was based solely on the signs and symptoms manifested, laboratory test results and on information provided by the patient himself. This case-study reveals that the transportation of drugs inside the gastrointestinal tract represents an enormous health hazard. In addition, it was observed that the therapeutic procedures adopted by the health team were in accordance with what up-to-date scientific literature recommends. Keywords: Intoxication, Drug trafficking, Cocaine, Toxicological Information and Assistance Center

Case Report: follow-up care of a cocaine intoxication case at a Toxicological Information

and Assistance Center in Fortaleza - Ceará

SalleS, gaBriela pereira1; Ferreira e Silva, henDyelle roDrigueS4; Silva, vicTória Da coSTa2; BaTiSTa, joSé Márcio MachaDo3; alBuquerque, polianna leMoS Moura Moreira3;

Moreira, rhuBenS levy roDrigueS4; Ferreira, Maria auguSTa Drago1

1 University of São Paulo, Ribeirão Preto – SP, Brazil; 2 Federal University of Ceará, Fortaleza – CE, Brazil; 3 Dr. José Frota Institute, Toxicological Information and Assistance Center, Fortaleza-

CE, Brazil; 4 Toxicology Study Center of Federal University of Ceará, Fortaleza-CE, Brazil.

115

Introduction: a mixture of hydrocarbons distilled from petroleum, kerosene was very much present in the domestic environment as it fueled oil lamps, heaters and stoves before the development of electricity as a source of power. In our times, the substance is used mainly as a solvent, degreaser, or lubricant in transport vehicles. Although nowadays less frequent, intoxications caused by this substance still occur and generally due to improper handling or storage. Objective: To describe patient care, trying to assess the therapeutic procedures adopted by the health team in handling a case of kerosene poisoning. Methods: All data pertinent to this case report was elicited from the health team of the Toxicological Information and Assistance Center located in the city of Fortaleza, state of Ceará. A thorough review of the specialized literature on the subject was also carried out. Results: On the day of 11/09/2019, a 1 year and 3 months old male patient was forwarded to the Toxicological Information and Assistance Center by an Emergency Care Unit just four hours after having accidently ingested kerosene while in his family home. Medicated with a corticosteroid, a gastric protector and dipyrone by the emergency healthcare team, by the time the Toxicological Center was called upon to assist in treatment, the patient, tachypneic and tachycardic, presented a cardiac frequency of 149 beats per minute (80-130 beats per min.) and a respiratory rate of 80 breaths per minute (24-40 breaths per min.). Thoracic x-ray, complete blood count and a set of biochemicals tests (Prothrombin Time, Activated Partial Thromboplastin Time, Urea, Creatinine, Potassium, Chlorine, Magnesium, Sodium, Amylase, Lipase) were requested and performed, revealing, however, no significant abnormalities. The initial management, using corticosteroids and a gastric protector, was maintained. On the very next day (11/10/2019), the diagnose of pneumonitis was confirmed by a new thoracic x-ray. In view of the result,

the medical staff recommended the administration of the intravenous antibiotic Cefepime (2g), and the patient was subsequently discharged. Discussion/Conclusion: Given its low rate of gastrointestinal absorption, kerosene, when ingested, usually does not to produce significant systemic toxic effects. However, the substance, an irritant, may cause inflammation of GI tract tissues and, consequently, vomiting, which, in turn, if kerosene is aspired along with other gastric contents, may cause pneumonitis–that is precisely why the induction of emesis is not recommended as a method of decontamination in such cases. Regarding the therapeutic procedures adopted by the health team in this specific case, the use of gastric protectors, as to avoid possible damage to the stomach mucosa, and of dipyrone, for relieving pain, were in accordance with the most up-to-date scientific literature recommendations, as was antibiotic prophylaxis–to prevent secondary infections or other respiratory complications. On the other hand, the use corticosteroids in the face of possible kerosene pneumonitis is no longer advised, studies showing it has uncertain results in improving patient outcome. Being physically and mentally immature, given to curious and exploratory behavior, children are particularly vulnerable to accidental poisoning by household products–which is a fairly common toxicological pediatric emergency. Additionally, factors such as the inadequate storage of these products, inadequate parental supervision, economic and educational problems within the family, contribute to the occurrence of these intoxications. Accidents such as the one pertaining to this case study, though deemed unforeseen, might be avoided by promoting public awareness on the toxicity and possible health hazard posed by household available chemicals. Keywords: Intoxication, hydrocarbons, kerosene, children, Toxicological Information and Assistance Center

Case Report: follow-up care of a kerosene intoxication case at a Toxicological Information

and Assistance Center in Fortaleza - Ceará

SalleS, gaBriela pereira1; Ferreira e Silva, henDyelle roDrigueS4; Silva, vicTória Da coSTa2; FariaS, BeaTriz valenTiM2; MagalhãeS, karla Do naSciMenTo3; BaTiSTa, joSé Márcio MachaDo3;

alBuquerque, polianna leMoS Moura Moreira3; Ferreira, Maria auguSTa Drago2

1 University of São Paulo, Ribeirão Preto – SP, Brazil; 2 Federal University of Ceará, Fortaleza - CE, Brazil; 3 Dr. José Frota Institute, Toxicological Information and Assistance Center, Fortaleza-

CE, Brazil; 4 Toxicology Study Center of Federal University of Ceará, Fortaleza-CE, Brazil.

116

Introduction: According to recent data from the National System of Toxic-Pharmacological Information (SINITOX), Brazil recorded more than 27.000 cases of poisoning during 2017. This includes poisonings caused by drugs, precursors, household products, industrial agents, cosmetics, metals, drugs of abuse, plants, food, venomous animals and unknown agents whose diagnosis indicates the action of a xenobiotic (SINITOX, 2017). Drugs and drugs of abuse account for more than 33% of intoxication cases in the Rio Grande do Sul (SINITOX, 2017; CIT, 2019), however the report of cases of intoxications by drugs and medicines that are not common to be found in the state are important, especially considering the analytical screening methodologies such as immunoassays. The case report relates a 50-year-old male patient who attempted suicide in his residence through massive ingestion of prescribed drugs. Toxicological analysis was performed with an immunochromatography test and detected phencyclidine (an uncommon drug in Brazil). Considering the patient’s history, the greatest suspicion was a cross-reaction not studied by the scientific literature. Objective: To evaluate whether the suspected substances the patient had contact with are likely to cause cross-reactions with the immunochromatographic kit. Determine possible drugs and medicines in the patient’s urine and blood samples through confirmatory equipment such as liquid chromatography coupled to mass spectrometry and gas chromatography coupled to mass spectrometry (LC-MS and GC-MS). Methodology: The urine and blood samples of the patient were stored at

- 8ºC and used for the investigation of the molecules suspected by the cross-reactions identified on the day of the analysis by GC-MS and LC-MS. In addition, a chronological evaluation of the patient’s case demonstrated the clinical evolution of the patient, the main results of laboratory tests and the treatment used. Results: The results obtained were:- Immunogromatography test:

* Serum: positive for TCA.* Urine: positive for PCP, BZO and TCA.

- GC-MS: No traces found in urine or serum;- LC-MS:

*Serum: positive for Midazolam, Fentanyl and Lidocaine

*Urine: positive for Midazolam, Fentanyl and Lidocaine

Discussion and Conclusion: The results obtained in the analysis of LC-MS showed the presence of midazolam, fentanyl and lidocaine, the first two being used by the hospital team at the patient’s entrance. Lidocaine, however, was not used by the hospital staff or by the patient. Lidocaine has two degradation products (o-toluidine and 2.6 dimethylaniline) that have structural similarities to phencyclidine and may result in a false-positive immunoassay. The analysis of the Tanimoto index and LC-TOF analysis will be made to confirm this cross-reaction not studied in the literature. Both analyses will be done before the presentation of this work and will be on the final poster. Acknowledgments: HUSM/UFSM and DACT/UFSM

Case Report: Phencyclidine cross-reaction investigation in immunochromatographic

tests for rapid drugs detection

carDoSo, leonarDo corrêa1; roSa, vicTória goMeS2; pacheco, anDré lucaS Bezerra2; SanToS, rachel2; ugalDe, guSTavo anDraDe1; BerlaTo, Dener goMeS1; reginaTo, FernanDa ziegler1; chiMenDeS, nayoMi

anDraDe2; SanToS, lara celeSTina2; naSciMenTo, Marcelo henrique SanTana2; oliveira, Sarah caroBini Werner De Souza eller Franco3; oliveira, Tiago Franco3; BairroS, anDré valle1

1 Nucleus Applied to Toxicology, Postgraduate Program in Pharmaceutical Sciences, UFSM; 2 Nucleus Applied to Toxicology, Course of Pharmacy, UFSM; 3 Federal

University of Health Sciences Foundation of Porto Alegre, UFCSPA.

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Introduction: Snakes of the genus Micrurus are the main representatives of the Elapidae family and are distributed throughout the Brazilian territory. Previous studies demonstrate that accidents involving coral snakes are responsible for about 1 to 2% of all snakebites in Brazil. Although relatively rare due to the non-aggressive behavior of these snakes, the ophidian accidents caused by Micrurus genus have great medical importance and are potentially serious because of the neurotoxic effects of the venom on the neuromuscular junction. However, the incidence of these accidents and the behavior of elapid venom among the pediatric population is still poorly described in the literature. Objective: Describe the epidemiological and clinical characteristics of pediatric victims of Micrurus snakebites in the state of Santa Catarina. Methods: A retrospective, descriptive, cross-sectional, observational study was conducted using data recorded by the Center for Information and Toxicological Assistance of Santa Catarina (CIATox-SC) of patients aged 0 to 18 years old who were victims of accidents with venomous snakes in the state of Santa Catarina during the period of 2014 to 2020. Results: During the period of study, 41 cases involving snakes of the genus Micrurus were registered in children and adolescents, which corresponds to 7.1% of the total number of ophidic accidents reported in the pediatric population. The snake species was identified in 27 cases (18 M. corallinus and 9 M. altirostris) and in 14 cases only the genus Micrurus spp. was identified. Most accidents occurred in the months of December, February, March and April (63.4%), in males (63.4%), at the habitual residence (68.2%) and the mean age affected was 7 years old. The macro-regions of Santa Catarina with the highest number of elapid accidents

were Grande Oeste (31.7%), followed by Vale do Itajaí (19.5%) and Grande Florianópolis (19.5%). The most frequently affected body site was the right foot (29.2%). The average time elapsed between the event and the first medical assistance was 1.25 hours. There were reported 32 mild cases, 5 moderate cases, and 4 severe cases. Among the severe cases, 2 of them required orotracheal intubation due to respiratory failure and occurred in children under 1 year of age. The main clinical manifestation observed was local pain (39%). The symptoms of neurotoxicity, such as drowsiness, paresthesia, altered olfaction/ palate, ptosis, paralysis, occurred in 7 cases (17%). A total of 20 patients (48.7%) were hospitalized and treated with antielapid antivenom, with a mean time to initiate antivenom therapy of 3 hours. Only 11 cases were asymptomatic and all patients progressed to cure with no sequelae outcome. Discussion: The higher frequency of elapid accidents in the pediatric age group compared to the general population is consistent with the tendency of previous studies, and it can be explained by the eye-catching color pattern of coral snakes, which attracts the curiosity of children. The clinical evolution of pediatric patients victims of Micrurus snakebites is usually favorable; however, it can lead to serious neurological manifestations and respiratory failure, especially in children younger than 1 year old or who do not receive adequate medical care. Therefore, preventive guidance for children to avoid walking barefoot, prompt health assistance in case of accident, and correct identification of the snake are decisive measures to avoid potentially fatal complications. Acknowledgments: We thank CIATox/SC for providing the data. The authors have no conflict of interest to declare.

Clinical-epidemiological profile of elapid accidents in pediatric population

registered at CIATox/SC

Silva, STephanie SoareS1; MeSSiaS, nayara caSagranDe1,2; BreSolin, nilzeTe liBeraTo1,3; Silva, DeniSe BouSFielD1,3; SanToS, clauDia regina1,2,4

1 Federal University of Santa Catarina (UFSC), Brazil; 2 Poison Control Center of Santa Catarina, Brazil; 3 Children’s Hospital Joana de Gusmão; 4 Department of

Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

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Background/Introduction: The widespread use of pesticides has caused health problems and fatalities worldwide, often due to occupational exposure and accidental or intentional poisoning. According to the World Health Organization (WHO), pesticide poisoning is one of the main means used in suicide attempts in development countries, especially those with a high proportion of rural residents who work in small-scale agriculture. In Brazil, between 2007 and 2015, rodenticides had the highest percentage of intoxication among the pesticides, with more than 40% of cases, followed by pesticides for agricultural use with 36.5%. In this context, cases of suicide attempts stand out, representing 53.6% of all cases of intoxication, which 5% evolved to death as a result of exposure. The combination of choices of sample preparation technique and detection method is of paramount importance in order to obtain an adequate methodology for an analysis of pesticides. In this sense, this work analyzed the suitability of using the combination between micro-QuEChERS sample preparation technique and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine pesticides in blood samples of patients after suicide attempt. Objective: Evaluate the suitability of micro-QuEchERS extraction method combined with LC-MS/MS detection of the following pesticides in blood plasma of patients after suicide attempt: aldicarb-sulfone, mevinphos, aldicarb, atrazine, carbofuran, fenthion, chlorpyrifos, 2,4-D and fipronil. Methods: micro-QuEChERS technique was employed for sample preparation, which 250 μL of blood were used, added to 25 μL of internal standard (diazepam-d5, 200 ng/

mL in methanol) and 500 μL of ice-cold acetonitrile, in addition to 100 mg of QuEChERS salt (Q-sepTM, Restek®). The tubes were closed, shaken for 10 min and centrifuged at 14,000 rpm for 10 min. Lastly, 125 μL of the organic phase was transferred to another tube containing 375 μL of the aqueous mobile phase. Two microliters of the dilution were injected into a LC-MS/MS system, model LCMS8060 (Shimadzu, Kyoto, Japan). Three blood samples collected from individuals over 18 years old who attempted suicide and were treated at the emergency unit of Hospital de Clínicas (UNICAMP) were analyzed with the developed method. The samples were refrigerated (2-8 ºC) until the moment of analysis. Results: The method was able to quantify nine different pesticides in blood, presenting linearity greater than or equal to 0.99 for all the pesticides. The recovery for the analytes was higher than 13.9% and the matrix effect observed was less than 41.2%, except for the analytes fenthion and chlorpyrifos. The detection limit for analytes was defined as 5 ng/mL, except for 2,4-D, which was five times higher (25 ng/mL). The quantification limit presented a concentration value equal to the detection limit. Discussion/Conclusion: micro-QuEChERS extraction combined with LC-MS/MS allowed the detection of different pesticides with different chemical properties in a single methodology. Thus, the analytical method developed can be used for analysis of blood samples for detection and quantification of the evaluated compounds. Acknowledgments: The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (process number 2020-10809-5).

Determination of pesticides in blood samples from patients after suicide attempt

by micro-QuEChERS and LC-MS/MS

goDoi, alexanDre Barcia1,2; Silva, Mariana criSTina1,2; coSTa, joSé luiz1,2

1 Faculty of Medical Sciences, University of Campinas, Campinas, SP, 13083887, Brazil; 2 Campinas Poison Control Center, Faculty of Medical

Sciences, University of Campinas, Campinas, SP, 13083859, Brazil.

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Introduction: Psychoactive drugs are the main agents involved in cases of intoxication according to the Toxicological Information Center of Rio Grande do Sul (CIT-RS) and the National Toxic-Pharmacological Information System (Sinitox). Within the classes of drugs involved, the class of tricyclic antidepressants (ADTs) stands out, generating severe effects in cases of intoxication, with cardiotoxic effects, which can be fatal. Objective: The objective is to develop an analytical method by dispersive liquid-liquid microextraction (DLLME) to determine tricyclic antidepressants and their respective biotransformation products in whole blood using liquid chromatography with a diode array detector (LC-DAD). Methods: The extractive technique was based on dispersive liquid-liquid microextraction (DLLME) using hexane as low density extracting solvent and methanol as dispersing solvent. Whole blood was the biological matrix of choice for the determination of the

tricyclic antidepressants amitriptyline, imipramine and doxepin and their respective biotransformation products nortriptyline and desipramine. The samples were analyzed by liquid chromatography with a diode array detector, the wavelength for the analysis was doxepin, amitriptyline and nortiptyline at 239 nm, imipramine and desipramine at 249 nm and the internal standard medazepam 255 nm. Results: The limit of quantification was 10 ng/ml for amitriptyline and doxepin, 20 ng/ml for nortriptyline and 30 ng/ml for imipramine and desipramine. The mobile phase was constituted by KH2PO4 buffer (pH 2.5) and methanol (60:40) with a flow rate of 1.2 ml/min. Conclusion: The DLLME developed in whole blood for the determination of ADTs developed proved to be a simple, reliable, robust and reproducible method that can be used in toxicology laboratories. Acknowledgements: We wish to thank CAPES for providing financial support.

Determination of tricyclic antidepressants in whole blood by liquid chromatography

with diode diarray detector employing dispersive liquid-liquid microextraction

BerlaTo, Dener goMeS1; roSa, vicTória goMeS2; pacheco, anDré lucaS Bezerra2; SanToS, rachel2; ugalDe, guSTavo anDraDe1; carDoSo, leonarDo corrêa1; reginaTo, FernanDa ziegler1; chiMenDeS, nayoMi

anDraDe2; SanToS, lara celeSTina2; naSciMenTo, Marcelo henrique SanTana2; BairroS, anDré valle1

1 Nucleus Applied to Toxicology, Postgraduate Program in Pharmaceutical Sciences, UFSM; 2 Nucleus Applied to Toxicology, Course of Pharmacy, UFSM.

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Introduction: In the early 1980s, some patients presented symptoms of a pathology with an unknown cause. Identified in 1983, about 37 million people live with HIV, and it’s estimated that 27 million are on antiretroviral treatment (UNAIDS, 2020). Once the HIV treatment introduction in the 80s, it’s known that adequately treatment infected individuals can present undetectable viral load (SIEDNER; TRIANT, 2019). However, there’s no way to perform therapeutic monitoring directly, and currently the therapy is monitored based on viral load, pill counts, and patient self-reporting (GARBIN; GATTO; GARBIN, 2017). Tenofovir is one of the most used drugs in combination therapy regimens and the adherence to the treatment is fundamental to reach undetectable viral load. A valid strategy to evaluate adherence to tenofovir treatment is through urine concentrations of the drug. Methodology: Urine samples were prepared by simple dilution in purified water (1:10, v/v). After centrifugation, a 2 µL aliquot of the diluted sample was injected into an Acquity I-Class liquid chromatography system associated with a Xevo TQD triple quadrupole mass spectrometer, both from Waters (Milford, USA). Chromatographic separation was performed on an Acquity HSS T3 column (100 x 2.1 mm, d.p. 1.7 µm), also purchased from Waters. Mobile phase A was composed of purified water with 0.1% formic acid and mobile phase B was composed of acetonitrile with 0.1% formic acid. Elution was performed in gradient mode, with an initial mobile phase composed of 98% mobile phase A, maintained for 2 minutes, followed by a linear gradient to 70% A in 3.5 minutes, followed by a new linear gradient to 30% of A in 4 minutes. This condition was maintained until 4.5 min, with the return to initial conditions in 4.6 min. The mobile phase flow rate was 0.4 mL/min, and the column was maintained at 30°C. Tenofovir was detected by electrospray ionization in positive mode. The operating conditions of the mass spectrometer were capillary voltage of 5 kV, cone voltage of 50 V, source temperature of 250 °C, desolvation gas flow of 1000 L/h and curtain gas flow of 40 L/ H. The monitored mass transitions were 288/136 (quantification) and 288/176 (qualification), with a collision energy of 25 V. The method was validade

according to FDA bioanalytical guidelines and applied to urine samples from 21 patients treated with tenofovir. Results: Tenofovir retention time was 1.75 minutes. Chromatographic run time was 6 min. The method was linear between 100 to 50,000 ng/mL. The precision and accuracy of the assay were tested at concentrations of 100, 300, 5000 and 30000 ng/mL. The intra-assay precision was from 2.64 to 4.56%, the inter-assay precision was from 2.33 to 5.15% and the accuracy presented values between 94.97 and 103.27%. The matrix effect, tested in urine from 10 volunteers, was -11.9 to 13% at a concentration of 300 ng/mL and from -4 to 13.7% at a concentration of 30000 ng/mL. Tenofovir was quantified in 18 samples, at concentrations between 8011 and >50000 ng/mL. Patients with undetectable concentrations had poor adherence according to the Morisky-Green questionnaire. Discussion and Conclusion: In previous studies, Koenig et al. (2017)but current adherence measurements are inadequate for real-time adherence monitoring. We developed and validated a urine assay to measure tenofovir (TFV demonstrate that through urinary concentrations of tenofovir it is possible to indirectly evaluate how long ago the patient used the last dose of the drug, being a marker of therapeutic adherence. Tenofovir urine testing is a non-invasive strategy to evaluate adherence, with high patient acceptance, and does not require prior preparation, with the sample collection performed at any time of the day. It’s an accessible way to monitor the treatment, allowing pharmaceutical interventions to increase adherence to therapy and control of viral spread.

REFERENCESGARBIN, C. A. S.; GATTO, R. C. J.; GARBIN, A. J. I. Adesão à terapia antirretroviral em pacientes HIV soropositivos no Brasil: uma revisão da literatura. Archives of Health Investigation, v. 6, n. 2, p. 65–70, 2017. KOENIG, H. C. et al. Urine assay for tenofovir to monitor adherence in real time to tenofovir disoproxil fumarate/emtricitabine as pre-exposure prophylaxis. HIV Medicine, v. 18, n. 6, p. 412–418, 2017. SIEDNER, M. J.; TRIANT, V. Undetectable = Untransmittable and Your Health: The Personal Benefits of Early and Continuous Therapy for HIV Infection. Journal of Infectious Diseases, v. 219, n. 2, p. 173–176, 2019. UNAIDS. Estatísticas mundiais sobre o HIV. Aidsinfo.Unaids.Org, p. 1–6, 2020.

Development and validation of a method to measure Tenofovir concentrations

in urine of HIV-positive patients

SanToS, raFaela knak; linDen, raFael

Feevale University/RS.

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Introduction: Antivenom serum therapy consists of the administration of concentrated heterologous serums of immunoglobulins, usually from equine hyperimmune plasma, being the main treatment in poisoning by venomous animals. It is a procedure intrinsically related to the possibility of occurrence of systemic hypersensitivity reactions. Early reactions can occur during the infusion of antivenom and within two hours and are classified as common and severe. Severe early reactions are uncommon but are of great importance as they carry a risk of complications and occasionally lead to anaphylaxis. Objective: To analyze the frequency of administration of different types of antivenom serum and the association between type of antivenom serum and early reactions. Method: This is a cross-sectional and descriptive study with cases of poisoning by venomous animals treated at the Information and Toxicological Assistance Center of Londrina (CIATox-Londrina) of the University Hospital of the State University of Londrina. Data were collected from the Brazilian System of Intoxication Records of Toxicological Information and Assistance Centers (DATATOX). The study period comprised the visits performed from January/2017 to December/2021, for which the administration of antivenom serum therapy was indicated. The CIATox-Londrina advises that the serums be diluted and administered after infusion of pre-serotherapy medication, which includes antihistamines and corticosteroids. The frequency of administration of antiarachnidic, antibothropic-crotalus, antibothropic, anticrotalic, antielapidic, antiscorpionic, antilonomic and antiloxoscelic serums was quantified and the prevalence of early reactions to each type of serum was verified. Furthermore, it was observed that the antivenom serum was diluted and the use of pre-serum therapy medication was used. For data analysis, the Epi Info® program, version 7.2.5.0 was used. Results: 808 cases of antivenom

administration were analyzed, observing the following frequency: 491 (60.77%) antibothropic, 197 (24.38%) anticrotalic, 51 (6.31%) antiscorpionic, 33 (4.08% ) antiarachnidic, 26 (3.22%) antiloxoscelic, 18 (2.23%) antibothropic-crotalus, 6 (0.74%) antilonomic and 3 (0.37%) antielapid. Dilution occurred in 805 (99.63%) antivenom serum infusions and pre-serum medication was administered to 780 (96.53%) patients. As for early reactions, it was observed that there were 72 (8.91%) cases. Of these, there were early reactions in 23.53% of the anti-bothropic-crotalic serum infusions, 14.29% of the anti-crotalic, 6.15% of the anti-bothropic, 3.85% of the antiloxoscelic and 2.04% of the antiscorpionic serum. In the analysis of the relationship between antivenom serum therapy versus early reactions, a statistically significant association was identified in cases that received anticrotalic serum (p=0.0002). Discussion/Conclusion: It was observed that, among the accidents with venomous animals that required antivenom serum therapy, snakebites were the most prevalent. With regard to early reactions, some factors favor their occurrence, which are related to the antivenom serum (dose, type of serum, concentration of proteins and immunoglobulins and infusion rate), the form of administration (with or without dilution; with or without pre-serotherapy medication), and to the patient (atopy and prior sensitization to the horse or equine products). In the literature, an association could be found between administration of anticrotalic serum and early reactions. In this case, early reactions were more frequent and severe in children who received the anticrotalic serum, when compared to children who received the antibothropic. Therefore, studies and analyzes are needed to investigate and deepen evidence of this relationship. Acknowledgments: We thank CIATox-Londrina, as an institution that promotes care and knowledge sharing in clinical toxicology for the population.

Early adverse reactions to different types of anti-poison serum in the period from

2017 to 2021: a descriptive analysis

prieDolS, guSTavo aBuD1; alveS, jonaS alher Meira1; oliveira, jorDana MeirelleS1; giroTTo, eDMarlon2; guiDoni, caMilo Molino2

1 Medical student at the State University of Londrina - UEL - Londrina (PR), Brazil and CIATox trainees Londrina (PR); Brazil; 2 Professor at the Department of Pharmaceutical Sciences at the Health Sciences Center at the State University of Londrina - UEL - Londrina (PR), Brazil.

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Introduction: Antivenom serum therapy is the main treatment for poisoning with venomous animals. On the other hand, there are risks related to systemic hypersensitivity reactions, highlighting early reactions during serum infusion, which occur within two hours after infusion of antivenom serum therapy. Objective: To analyze the frequency of administration of antivenom sreum therapy, prevalence of early reactions and factors associated with early reactions. Method: This is a cross-sectional and descriptive study with cases of poisoning by venomous animals treated at the Information and Toxicological Assistance Center of Londrina (CIATox-Londrina) of the University Hospital of the State University of Londrina. Data were collected from the Brazilian System of Intoxication Records of Toxicological Information and Assistance Centers (DATATOX). The study period comprised the visits for which the antivenom serum therapy administration was guided from January/2017 to December/2021. A descriptive analysis was performed, with calculations of frequencies, and association, using early reactions as the dependent variable and the independent variables sex, age, pregnant woman (in the case of females), skin color and need for antivenom serum therapy complementation. For data analysis, the Epi Info® program, version 7.2.5.0 was used. Results: Of the 808 cases of antivenom serum therapy administration, there were more indications for men (n=602; 74.50%), adults (n=669; 82.80%)

and whites (n=598; 74.01%). Only two (0.24%) pregnant women received antivenom serum therapy. The most common outcome for cases that required antivenom serum therapy was cure (n=787; 97.40%) and antivenum serum therapy complementation occurred in the minority of times (n=108; 13.37%). As for the early reactions, the occurrence was recorded in 64 (7.99%) initial administrations and 8 (0.99%) additions of the antivenom serum therapy. No statistically significant factors associated with early reactions were found during serum administration. Discussion/Conclusion: As for sex and age, the data analysis refers to residents of rural areas, where males are more abundant in the state of Paraná, and who perform agricultural work, being more exposed to venomous animals. As for the most prevalent color, the data coincide with the ethnic distribution of the state of Paraná. Cure, in turn, as the most common outcome, demonstrated the effectiveness of antivenom serum therapy in the management of accidents with venomous animals and in reversing the toxicity of their venoms. Finally, regarding the administration of the antivenom serum therapy or the complementation, although not significantly related to the variables analyzed, it was shown that the early reactions are infrequent. Acknowledgments: We thank CIATox-Londrina, as an institution that promotes care and knowledge sharing in clinical toxicology for the population.

Epidemiological variables and anti-poison serotherapy: a descriptive

analysis of early adverse reactions

alveS, jonaS alher Meira1; prieDolS, guSTavo aBuD1; oliveira, jorDana MeirelleS1; giroTTo, eDMarlon2; guiDoni, caMilo Molino2

1 Medical Student at the State University of Londrina - UEL - Londrina ( PR), Brazil and trainee CIATox Londrina (PR), Brazil; 2 Professor at the Department of

Pharmaceutical Sciences at the Health Sciences Center of the State University of Londrina - UEL - Londrina (PR), Brazil and on-duty CIATox Londrina (PR), Brazil.

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Background: Dried blood spots (DBS) are minimally invasive samples increasingly used for therapeutic drug monitoring. Despite the advantages, there are still some challenges related to this technique, such as the hematocrit (HCT) measurement. The variability of patients HCT and the impact of it on blood viscosity and assay accuracy, as well as the differences on blood to plasma ratios, are obstacles for the implementation of this technique in clinical toxicology and therapeutic drug monitoring. In this study, potassium (K+) was used as a marker of HCT, considering that 98% of the total body K+ is located within the intracellular compartment. Objective: To evaluate the applicability of an electrode-based method for the quantification of K+ and estimation of HCT in capillary DBS samples. Method: Quality controls and calibrators were prepared by adding or removing plasma from a blood sample with known HCT. Calibration curve was performed with HCT 20, 25, 30, 35, 40, 45, 50, 55 and 60% and controls HCT 25, 40 and 55 %. Precision and accuracy were tested in triplicate within 3 days. Capillary and venous blood samples were collected from 32 healthy volunteers. Capillary blood was collected from finger prick and applied to Whatman 903® paper card, dried for at least 3 hours until analysis. The venous blood was collected with venipuncture and stored in EDTA

containing tubes. The K+ was measured in DBS from volunteers and calibrators after extraction of a 6 mm punch with 300 uL of ultrapure water incubated for 40 minutes at room temperature and 1000 rpm. The aqueous extract was analyzed into the K+ LAQUAtwin (HORIBA) compact meter for ion quantification. The HCT of venous blood samples was quantified using the Sysmex Kx-21n hematology analyzer. Results: Precision (CV%) ranged from 2.4 to 2.7%, while accuracy ranged from 94.6 to 111.8%. The HCT measured in venous blood ranged from 34 to 43.5%, with a mean standard deviation (SD) of 40.11 ± 2.8%. While capillary HCT estimated from K+ measurement in DBS resulted in K+ ranging from 22.36 to 35.9%, with a mean SD of 28.2 ± 3.35%, representing 72 ± 7% of the values from venous blood. Passing-Bablok linear regression performed with a confidence level of 95% demonstrated the existence of a systematic error, thus the equation DBS HCT = -24.837358 + 1.389067 venous HCT was applied to estimate HCT values in DBS. The estimated values of HCT from capillary blood ranged from 33.97 to 43.71% with a mean SD of 38.18 ± 2.4%, representing 98 ± 6% of the venous HCT, with no proportional errors. Discussion/Conclusion: The quantification of K+ in DBS samples can be used to estimate the HCT as long as a correction factor is applied.

Estimation of hematocrit values by potassium quantification in capillary

dried blood spots (DBS)

Silva, laura cé; alveS, peDro aDolFo pereira; linDen, raFael; anTuneS, Marina vezon

Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo - RS, Brazil.

124

It is estimated that, in 2020, cancer was associated with approximately 10 million deaths worldwide.1 Among children, the most common types of cancer include leukemias and lymphomas, central nervous system tumors, kidney cancer and malignant bone tumors.2 The most common childhood bone cancer, osteosarcoma (OS), is treated with a sequence of neoadjuvant therapy, tumor surgical resection and postoperative adjuvant therapy. However, since therapy for recurrent OS is limited by poor survival rates, novel therapeutic agents are urgently required.3 In recent years, chromone-related compounds have been described to inhibit several processes related to cancer and to OS in particular.4 The aim of the present study was to evaluate the potential toxicity of a group of polyhydroxylated phenylchromones to OS cells, using in vitro models. For this, human OS cell lines - MG-63, Saos-2, HOS, and 143B - were incubated for 48 h with 10, 20, 40, 80 and 160 µM phenylchromones with diverse hydroxy substituents at positions C-3, C-5, C-6, C-7, C-8, C-3’, C-4’, and C-5’. Subsequently, sulforhodamine B (SRB) assay was used to determine cell growth inhibition, followed by spectrophotometric measurement at 510 nm. Moreover, cell viability was investigated upon incubation with WST-8 reagent, followed by spectrophotometric measurement at 450 nm. The obtained results suggest that the presence

of hydroxy substituents simultaneously at C-3 and C-8 of C-ring improve the in vitro cytotoxic activity of the tested compounds in OS. A higher cytotoxicity was also observed in compounds with hydroxy substituents simultaneous at positions C-3’, C-4’ and C-5’ of the B-ring combined with hydroxylation at C-3 and/or C-7 of the C-ring. These preliminary results enable the study of the potential application of these compounds in OS therapy. Acknowledgments: This work received financial support from the European Union (FEDER funds through COMPETE POCI-01-0145-FEDER-029243) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project PTDC/MED-QUI/29243/2017 and from PT national funds (FCT/MCTES) through grant UIDB/50006/2020. ATR and CP thank to FCT for the funding through the project PTDC/MED-QUI/29243/2017. JMPFO thanks FCT for funding through program DL 57/2016 – Norma transitória (ref. SFRH/BPD/74868/2010). MF acknowledges her contract under the CEEC Individual (2020.04126.CEECIND/CP1596/CT0006).

REFERENCES1. H. Sung et al., CA Cancer J. Clin. 2021, 71(3), 209-249.2. R.L. Siegel et al., CA Cancer J. Clin. 2021, 71(1), 7-33.3. M.E. Anderson, Orthop. Clin. North Am. 2016, 47, 283-292.4. J. M. P. Ferreira de Oliveira et al., Pharmaceuticals (Basel). 2021, 14(7), 640.

Evaluation of cytotoxic potential of polyhydroxylated phenylchromones in

human osteosarcoma in vitro models

oliveira, joSé Miguel p. Ferreira1; proença, carina1; SanToS, raquel1,2; Moreira, BeaTriz1,3; ruFino, ana T.1; FreiTaS, MariSa1; riBeiro, Daniela1,4; FernanDeS, eDuarDa1

1 LAQV, REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; 2 Department of Biology, Faculty of

Sciences, University of Porto, 4169-007 Porto, Portugal; 3 Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal; 4 Faculty of Agrarian Sciences

and Environment, University of the Azores, 9700-042 Angra do Heroísmo, Açores, Portugal.

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Introduction: Metabolites are biomolecules of low molecular weight with different organic functions, which are formed as intermediates in biochemical reactions. The metabolites profile and concentrations may be related to the physiological state of an organism. Gas chromatography coupled with mass spectrometry (GC-MS) has been extensively used in metabolome analysis due to its high separation efficiency, which can resolve very complex biological mixtures. However, the limitation of GC-MS is that the analytes must be volatile to be separated on a gas chromatography column. Therefore, analysis of biological samples by GC-MS requires derivatization so that non-volatile or thermally unstable species can be detected. Objective: In this study, the optimization of sample preparation was performed by variation of some factors in order to analyze how the experimental variables interact and which condition generates the largest number of molecular features. Results and Discussion: A pool of plasma from 10 healthy individuals was prepared and stored at -80°C. The sample preparation was based on existing protocols in the literature and the experimental variables were: 1) different solvents used in the deproteinization step; 2) methoximation reaction time and 3) silylation reaction time. Proteins were precipitated before derivatization of the sample, using 100% acetonitrile or a ternary mixture of isopropanol:acetonitrile:water (3:3:2) followed by a clean-up step, adding acetonitrile:water (50:50). The supernatants collected were evaporated to dryness using a SpeedVac Concentrator System. Methoxymation was performed by adding 10 µL of O-methoxyamine hydrochloride in pyridine to each sample and incubated at room temperature for 16 h or 18 h or at 70°C for 90 min. For silylation reaction, 10 µL of BSTFA with 1% TMCS was added and left for

1 h at 70°C with or without agitation of the samples. Then 100 µL of C18:0 methyl ester (10 ppm) in-heptane was added as an instrumental internal standard and analyzed by GC-MS. The analysis was performed with a GC instrument 7890A coupled to an inert mass spectrometer with triple-Axis detector 5975C from Agilent Technologies. Chromatography and MS parameters followed the protocol proposed by Fiehn et al. (2016). Samples were injected (2 µL) with the injector temperature set at 250°C in a split mode (1:10), using helium as a carrier gas at 1.1 mL/min flow. The capillary column used was a DB5-MS 30 m length, 0.25mm i.d. and 0.25 m (Agilent Technologies®) with temperature programming set at 60°C for 1 min, then raised by 10°C/min until it reached 325°C and maintained for 10 min before cooling down. MS parameters were: transfer line a 280°C, filament source at 230°C, quadrupole at 150°C, electron ionization energy of 70eV, mass range of 50-600 m/z. The data were acquired using the Agilent MSD ChemStation Software. The analysis was performed in triplicate, resulting in 21 samples, plus five quality controls and two blanks. The data processing was performed using the R software to obtain the matrix of results (number of ion peaks). Conclusion: statistical calculations showed that the sample preparation condition that provided the identification of a greater number of molecular features was achieved when the samples were submitted to two extraction steps (deproteinization followed by sample clean-up), a methoximation reaction time of 16 hours at room temperature and the silylation reaction at 70°C with agitation for 1 hour. Keywords: Metabolomics profile, GC-MS, untargeted metabolomics. Acknowledgement: To CNPq.

Evaluation of experimental conditions for sample preparation to identify the untargeted

metabolomic profile of plasma by GC-MS.

nolaSco, Daniela M.; pireS, SuMaia araújo; Souza, Tainá BruMaTe; Souza, Mirna Maciel D'auriol; paiva, Maria joSé nuneS; anDré, leiliane coelho

Toxicological Analysis Laboratory, Department of Clinical and Toxicological Analysis, College of Pharmacy - Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

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Introduction: Accidental ingestion and consequent methyl ethyl ketone peroxide (MEKP) poisoning during occupational activity as well as due management of the affected patient were reported. From the perspective of the MEKP, which it is applied in the industrial field, this colorless liquid causes oxidative stress and is therefore considered a highly toxic compound. Objectives: Delineate a rare accidental poisoning during the period of occupational activity by methyl ethyl ketone peroxide (MEKP), in addition to treatment and toxicological analysis. Methods: Oral administration of N-acetylcysteine (NAC) with a loading dose of 20 sachets (600 mg/sachet) with subsequent doses (10 sachets/4h) for three consecutive days. Application of Dipyrone ampoules (2 mL to 500 mg/mL) causing emesis. Biochemical, endoscopic, hematological and toxicological tests were performed. Results: Changes were observed in creatinine (1.27 mg/dL), creatinine phosphokinase (223 U/L), hematocrit (58.5%), hemoglobin (20 g/dL), leukocytes (31420/µ L), rod leukocytes (22%), urea (59 mg/dL). Ulcerative lesions (≥ 22 cm) in the gastrointestinal tract with necrotic stains exhibited by digestive endoscopy. Diagnosis of Zargar grade IIB acid erosive esophagitis, as well as the presence of Zargar grade IIIA acid gastric ulcers. The use of NAC is unquestionably appropriate due to its high efficacy in the elimination of free radicals, resulting from the metabolization of MEKP. Regarding the reaction between NAC and MEKP, (2S)-3-[(2-carboxy-2-acetamidoethyl)dissulfanyl]-2-acetamidopropanoic acid and 2-hydroperoxybutan-2-ol are then formed, which will be subsequently converted into MEK and

thus excreted in the urine. The aspects of this case were favorable to the exclusive oral administration of NAC, even with the patient presenting emesis, in which, in situations like this, the NAC is contraindicated. It was collected, 24 hours after the exposure, ethylene diamine tetra acetic acid (EDTA)-blood samples (stored at 23ºC/48h), which allowed the identification of MEKP (monomer/dimer) with LC-QTOF/MS, thus demonstrating something unprecedented. GC-FID (32.85 µg/mL) quantified methyl ethyl ketone above the occupational limit (0.1-10 µg/mL). During hospitalization, hepatic markers were elevated and occupational exposure (10 years without the use of PPEs) should be considered, since Butanox® has dimethyl phthalate in its composition, therefore, is an hepatoxic agent absorbed by the respiratory and dermal pathways. In addition, with a small working area (4m2) and no ventilation, in which the individual was, this caused anosmia, which culminated in the accidental ingestion of the solvent in a potentially lethal volume (10-100 mL). Conclusion: Determination, in the blood collected, of MEKP and MEK. Under the perspective of oral administration of NAC, success was achieved in the poisoning by MEKP. However, using N-acetylcysteine as the exclusive therapy is considerably critical, which requires proper supervision and assistance to the patient during hospitalization. Acknowledgments: We acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES) for the Master’s scholarship provided. Keywords: Methyl ethyl ketone peroxide; N-acetylcysteine; Occupational aspects; Toxicological analysis.

Exclusive therapy of N-acetylcysteine in accidental butanox intake: toxicological

analysis of methyl ethyl ketone peroxide

SanToS, rachel1; BairroS, anDré v.1; SalDanha, geovane a.1; BerlaTo, Dener g.1; MoraeS, liliana S.1; günDel, auguSTo r.2; carvalho, joSé a.M.2; haBiB, iSaBela a.3;

De carli, Diego M.3; oliveira, Tiago F.4; oliveira, Sarah c.W.S.e.F.4

1 Nucleus Applied to Toxicology, Center of Health Sciences, Federal University of Santa Maria, Santa Maria, Brazil; 2 Laboratory of Clinical Analysis, University Hospital of Santa

Maria, Federal University of Santa Maria, Santa Maria, Brazil; 3 Gastroenterology Unit, University Hospital of Santa Maria, Santa Maria, Brazil; 4 Mass Spectrometry Research

Group, Federal University of Health Sciences of Porto Alegre, Porto Alegre, Brazil.

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Background: Vitamin A performs several functions in the body. It is involved with the formation of rhodopsin in the retina, acts as a bone growth factor and is a cofactor in the synthesis of steroid hormones. From animal foods, vitamin A is absorbed as retinol. Vitamin A deficiency may be associated not only with insufficient intake, but also with chronic lipid malabsorption, liver disease, intestinal parasites, and alcoholism. It also causes night blindness, skin dryness, hair loss and in cases of severe deficiency, irreversible blindness. Toxicity can occur due to administration of high doses of vitamin A for long periods, causing a hypervitaminosis. Acute symptoms are tiredness, headache, nausea and vomiting, which result in chronic disorders such as bone fractures, intracranial hypertension, skin lesions, hepatotoxicity and teratogenicity. The aim of the study was to validate an easy and fast preparation method for the analysis of retinol. Methods: Validation was performed using a Xevo TQS micro tandem mass spectrometry sensitive liquid chromatography (LC-MS/MS). Analytical specificity is ensured using multiple reaction monitoring with fragmented ions that are exclusive to retinol, quantifier ion 269.15 > 93.00 and qualifier ion 269.15 > 83.00. The procedure involves a small amount of 50 µL of serum, followed by liquid-liquid extraction. The samples are then submitted to reverse phase separation on a Zorbax Eclipse Plus C8® analytical column (50.0 x 2.1 mm, 1.8

μm). Mobile phase was A: 2.0 mM ammonium acetate/ 0.1% formic acid (aq) B: 2.0 mM ammonium acetate/ 0.1% formic acid/ MeOH, using a gradient with an initial ratio of 40:60 (v/v) with a flow rate of 0.3 ml/min. Quantitation is achieved by the comparison of the responses from a given sample with the responses of calibrators with known concentrations. Linearity was observed in the expected concentration range from 2.0 to 64.0 mg/L and serum samples were evaluated on six different concentrations and six times each at the same time point of the study. Albumin was used as a biological matrix for the study, retinol standard (Sigma – Aldrich). Results. The linearity coefficient of determination (R2) was 0.998746. The method showed 100% of selectivity and interference of residual effect of less than 5%. In order to determine the average inter-assay CV%, three different concentrations were analyzed over three days and the results for each low, medium, and high concentration level were 4.60, 5.72 and 4.24%, respectively. The accuracy of the method was cheeked by analyzing samples of known concentration and expressed as a percentage. A retention time (RT) of 1.6 min was obtained and the total analysis time was 3.5 min. Conclusion: The method was fast and efficient for the determination of serum retinol. The efficiency and selectivity combined with the technical robustness can be used in the diagnosis of nutritional and absorption disorders and also in cases of toxicity.

Fast method of quantitative analysis of serum vitamin A (retinol) using LC-MS/MS

SoareS, Marcela De oliveira; SugaWara, eDuarDo kinio; MazeTe, FernanDa pine S.; raMaDan, DeBora r.; TuFik, Sergio

Associação Fundo de Incentivo à Pesquisa - AFIP.

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Background: Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor, considered first-line therapy for the treatment of chronic myeloid leukemia (CML). A significant portion of patients using imatinib experience adverse events, up to 45% of patients discontinue therapy after 8 years, 6% due to adverse effects and 16% due to unsatisfactory therapeutic effect. The relation between IM though plasma concentrations and therapeutic response has been demonstrated. Therapeutic drug monitoring (TDM) of imatinib has been performed for improving treatment outcomes and as a tool to evaluate adherence. Also, norimatinib, a CYP mediated metabolite of has similar pharmacological activity to imatinib and represents approximately 20–25% of the steady-state concentration of the original drug. Up to date there is no report of the use of volumetric absorptive microsampling (VAMS) for capillary blood measurements of imatinib. It could help increasing the access to TDM, with less invasive collection at pharmacokinetically appropriate time, with high stability and logistic advantages, once samples do not need to be refrigerated. Objective: to develop and validate a method for imatinib and norimatinib quantification in capillary blood collected with VAMS by LC-MS/MS. Methods: The 20 µl VAMS tip were prepared by liquid extraction with 100 µL of water and formic acid 0,1%, incubated for 20 min at 45 °C and 1000 rpm. After the proteins were precipitated with 300 µL acetonitrile 0,1% formic acid containing IS (Imatinib-D8 80 ng/mL). After 5 min mixture, samples were cooled at -20°C for 10 min and centrifugated for 10 min. The supernatant was transferred to vial and

5 µL injected into the LC-MS/MS with electrospray ionization in positive mode. Source temperature was 350°C, capillary at 200 °C, auxiliar gas nitrogen at 15 arb and sheath gas at 50 arb. Chromatographic separation occurred in an Acquity® UPLC BEH C18 (150 x 2.1 mm x 1.7 μm) at 40 °C. Mobile phase was acid formic in water 0,1% (eluent A) acid formic in acetonitrile 0,1% (eluent B) in initial gradient 15% to 60% (A:B, v/v) eluted at 0.25 mL/min -1. The following transition were monitored as quantifier: imatinib m/z 494 - 378, norimatinib m/z 480 - 394, and IS m/z 502 - 378. The validation tests are following the recommendations of FDA and EMA. Results: Total analytical run time was 7 min, with retention time of 3.5 for norimatinib and 4 min for imatinib and IS. The method was linear from 100 to 2500 ng/ml for both analytes (r2 ≥ 0.99), accurate 89-103% for imatinib and 92-112% norimatinib, precise with CV% 4.3-11.3% for imatinib and CV% 5.9-13.5% for norimatinib. Matrix effect was compensated by the use of the deutered internal standard, from -14 to 3% for imatinib and -12 to 10% for norimatinib. No significant effect of hematocrit (Hct) was observed (85-115%), with extraction yield ranging from 98% for Hct 25% to 85% for Hct 55% for imatinib and 95% for Hct 25% and 87% for Hct 55% for norimatinib. Discussion/Conclusion: The method presented adequate performance for imatinib and its metabolite norimatinib determination in blood VAMS. The method is going to be applied in samples from patients with CML for clinical validation. Acknowledgments: Financial support FAPERGS and CAPES.

Finger-prick volumetric absorptive microsampling (VAMS) for imatinib

therapeutic drug monitoring: method development and validation

guTerreS, FernanDa S.1; kruTzMann, Maria eDuarDa2; kohlrauSch, raMona1; linDen, raFael1,2; anTuneS, Marina vezon1,2

1 Institute of Health Sciences, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

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Introduction: Garlic is widely used in cooking as a seasoning for food. In popular culture it is recommended in the treatment of skin conditions. However, topical use may cause chemical burns on the skin, which may have varying degrees, including the appearance of blisters and vesicles at the injury site. These burns are due to sulfuric substances present in garlic. The Toxicological Information and Assistance Center of Santa Catarina (CIATox/SC) has received calls referring to patients exposed to garlic, mainly when using this product for the “treatment” of injuries caused by venomous or non-venomous animals. Methods: This is an observational, cross-sectional, descriptive and retrospective study of a series of cases of patients registered in the DATATOX system (DATATOX BI Pentaho / Saiku version 2.6) due to CIATox/SC instructions. Data was collected from 2014 to 2021 and was analyzed in the Microsoft Excel®. The variables were: age range, gender, circumstance, injury site, time from exposure to attendance, symptoms, severity, and outcome. Results: There were 47 cases of garlic burns ranging from 01 to 79 years old, predominantly between 60 to 69 years old (19,14%), and with a female profile (51.06%). Most patients (57.44%) applied garlic on the skin due to sting/bite/contact with venomous animals or when doubting if

the animal was venomous or not, in order to relieve the pain. Regarding the injury site, 42.55% were on the lower limb, and 40.42% were on the upper extremities. Also, 40.42% of patients searched for attendance 12 to 24 hours after exposure. The symptoms were: pain, paresthesia, hyperemia, erythema, edema, itching, burns, vesicles, and eruptions on the skin. Headache, fever, nausea, and vomiting were also registered. All of the cases were classified as mild and had good outcomes. Conclusion: The use of crushed garlic as a remedy for pain relief is especially frequent in the eastern cultures. Its curative properties are widely used for over 4000 years for a variety of conditions such as headache, tumors and intestinal worms. Therefore, its use in the skin is responsible for varying degrees of irritant reactions, of which may be affected by its concentration, freshness, the total duration of exposure on the skin, the anatomical area of the body applied, and individual’s skin sensitivity. The concept of “natural remedy”, when applied incorrectly, might be responsible for many injuries. These results show that there is still part of the population that is not aware of the irritant effects of garlic when applied on the skin. Educational measures in schools, workplaces, social media, and television programmes would be very useful to make that clear to various groups of people.

Garlic burn injuries: a clinical-epidemiological profile of cases registered at CIATox/SC, Brazil

corDeiro, gaBriela BaTiSTa cavalcanTi1,2; arruDa, FernanDa WolFF Da Silva1; peTry, anDrea2; reSener, MariSeTe canello3; SanToS, clauDia regina1,2,3

1 Federal University of Santa Catarina (UFSC), Brazil; 2 Poison Control Center of Santa Catarina, Brazil; 3 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

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Introduction: Acetaminophen intoxication has been considered the most common cause of liver transplant indication for liver failure in the world and the main one in the United States. Its toxicity comes from increased production of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Used as an antipyretic, it is easily acquired and considered by many to be harmless. Despite a good safety profile and antidote N-acetylcysteine (NAC), intoxication can lead to severe liver damage when ingested in large quantities as in suicide attempts (SA). Treatment with NAC is indicated in cases that reach a toxic dose (TD) or with serum levels of acetaminophen with potential or high risk of hepatotoxicity. In the United Kingdom, the stablished TD is above 7.5g, while the USA and some other countries adopted a higher dose, with prediction of lower toxicity below 200 mg/kg or 10g. Object: Evaluate the risk of hepatotoxicity in intoxication with acetaminophen by means of SA with doses between 7.5g and 10g that were treated at the Assistance and Toxicological Information Center of Santa Catarina (CIATox/SC), Brazil, between 2014 and 2021. Methods: Retrospective analysis of SA with acetaminophen between 2014 and 2021 recorded at the service’s information data source (DATATOX BI Pentaho / Saiku version 2.6). Were included patients with SA with acute ingestion of acetaminophen between 7.5g and 10g who had records of laboratory tests 24 hours after ingestion and/or serum acetaminophen dosage available. Were excluded those who, after initial care,

had confirmed a dose outside the predicted range or who lost follow-up. Results: Of the total of 224 records of SA with acetaminophen at doses between 7.5g and 10g in the years from 2014 to 2021, 173 met the inclusion criteria. Of these, 101 (58%) had laboratory tests 24h after the ingestion and 72 (42%) had serum dosage of acetaminophen. 70% were women and 30% were men. The main age group was between 15-39 years old (80%). 137 cases (79%) ingested doses between 7.5g and 10g, while 36 cases (21%) ingested a dose of 10g. Laboratory alterations 24h after ingestion and/or the need to repeat the NAC regimen were observed in 8.6% of those with an ingestion of 7.5g, 8.8% of those with intakes between 7.5g and 10g and 19.4% of ingestion of 10g. 50% of the cases with serum levels compatible with potential or high risk of hepatotoxicity ingested 10g. Discussion/Conclusion: In this retrospective analysis, we observed that less than 10% of the cases evolved with laboratory alterations or with potential/high of toxicity. Besides that, we observed that ingestion of a dose of 10g have a twofold increased risk of toxicity when compared to those between 7.5g and 10g. More studies at a national level are needed, as well as serum dosages made available, in order to stablish the safest toxic dose of acetaminophen to the Brazilian population and to standardization of treatment protocols. Acknowledgements: CIATox/SC for the partnership and provision of data.

Hepatotoxicity risk assessment in suicide attempts by acetaminophen with doses

between 7.5g and 10g at a reference center in Santa Catarina, Brazil

FonSeca, karoline kuhnen1; coSTa, ana carolina conchon1; corDeiro, gaBriela BaTiSTa cavalcanTi1; arruDa, FernanDa WolFF Da Silva2; reSener, MariSeTe canello1

1 Assistance and Toxicological Information Center in Santa Catarina; 2 Medical Student in Federal University of Santa Catarina.

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Introduction: Pesticides exposure is considered one of the main risk factors for human health. Such substances can produce toxic effects in the several body systems. In this context, the liver and kidneys are target of these chemicals causing a disbalance in the elimination process of xenobiotics. In addition, due the relevance of these organs, the changes in their functions increase the susceptibility. Objective: The objective of the study was to verify changes in liver and kidney functions as a possible result of the exposure to pesticides of rural workers from Casimiro de Abreu (RJ). Experimental: This is a cross-sectional study with individuals residing in the municipality of Casimiro de Abreu, Rio de Janeiro, Brazil, whose study inclusion criteria were: both sexes, residing in the rural area and aged above 18 years. Between March and July (2021), biological samples were collected from 95 workers/residents of Casimiro de Abreu, being 75 rural workers and 20 urban workers (comparison group). The samples were transported from the collection site to the Clinical Pathology Service of the HC1/INCA, in accordance with sanitary regulations. The biochemical parameters evaluated were creatinine, alkaline phosphatase, transaminases (alanine aminotransferase - ALT and aspartate aminotransferase – AST). The analysis of parameters evaluated and their respective cut-off points took into account the limit values of the Clinical Pathology Service of the HC1/INCA. From them, the subjects were classified as having or not having alterations in the parameters. The non-parametric Mann-Whitney test was used for statistical analyses. This project was approved by the Research Ethics Committee of INCA

(CAAE 64799217.3.0000.5274). Results/Discussion: Among the 75 individuals evaluated in the rural area most were man (83.2%), aged between 36 and 60 years (58.0%). Regarding education, 67.9% had elementary school, followed by 18.5% who reported not knowing how to write. In additional 83.9% lived in the municipality for more than 10 years and 9.9% have lived for less than 5 years. The mean (SD) of the biochemical parameters of each of the studied groups was performed. The mean values (±standard deviation) of ALT (21.90 (±11.10) U/L) and AST (21.72 (±7.84) U/L) for rural workers were higher than in comparison group (21.00 (±4.58) U/L and 20.22 (±7.44) U/L, respectively), except to phosphatase (30.05 (±17.53) U/L) that were almost 3 times lower in rural workers than in urban workers (86.06 (±32.84)U/L) . All observed results were in agreement with the reference limits (ALT ≤ 40 UL; AST ≤ 41 UL, phosphatase 40 – 129 UL and creatinine 0,3 – 1,3 mg/dL), despite the groups showing different mean values. Of the parameters analyzed, only creatinine showed a statistically significant difference (p value =0.024). Conclusion: The results obtained allowed to conclude that no relevant differences were observed when the results of the two groups were compared and suggest that there is a possible environmental exposure unable to change the parameters of liver and kidney functions. Therefore, the present study contributes to the risk assessment to pesticides exposure and to the health surveillance of rural workers in Casimiro de Abreu, an agricultural region in the Rio de Janeiro state.

Liver and kidney function at the pesticide’s exposure of rural workers

from Casimiro de Abreu - RJ

nuneS, raFaella Ferreira naSciMenTo1,2,3; poça, káTia SoareS2,3; caBral, yngriD DoS SanToS2,3; aguiar, gilBerTo SanToS; Siqueira, janaS; geralDino, BarBara roDrigueS;

oTero, uBirani BarroS2; MarTinS, iSariTa1; Sarpa, Márcia Sarpa De caMpoS2,3

1 Laboratório de Análises de Toxicantes e Fármacos (LATF)/ Universidade Federal de Alfenas (UNIFAL-MG), Programa de Pós-Graduação em Ciências Farmacêuticas/Doutorado em Ciências

Farmacêuticas;. 2 Área Técnica Ambiente, Trabalho e Câncer- Instituto Nacional de Câncer José Alencar Gomes da Silva – INCA; 3 Laboratório de Mutagênese Ambiental, Departamento de

Bioquímica, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro (UNIRIO); 4 Programa de Saúde do Trabalhador, Secretaria Municipal de Saúde de Casimiro de Abreu.

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Introduction: Senecio spp. is one of the most frequent plant-related poisoning in cattle and its ingestion generates the disease seneciosis, characterized by serious hepatic damages. Liver biopsies and serum liver markers dosage are tools used in seneciosis diagnosis; however, a large number of cattle breeding is undiagnosed. The microRNAs are small non-coding RNA molecules, stable in biological fluids, and the difference in expression levels may indicate the presence, absence and/or stage of the poisoning. Objective: Identification of a miRNA profile in cattle serum intoxicated to Senecio brasiliensis. Methods: Liver biopsy was performed in all the cows and the histopathological analysis were performed in order to confirm intoxication. Blood samples were collected to evaluate miRNAs and liver biochemical parameters. Expression of miR-21-5, miR-885-5p, miR-122-5p, miR-181b-5p, miR-30a-5p, miR-378-5p, and let-7f-5p were evaluated in serum of naturally exposed cattle, by RT-qPCR. The normality of the variable distribution was evaluated by the Kolmogorov-Smirnov test. Continuous variables were compared using Mann-Whitney test and the correlation was evaluated using Spearman’s correlation. A ROC curve analysis was performed and the AUC was calculated to evaluate the diagnostic value. The optimal cut-off value, sensitivity (SE), and specificity (SP) were determined by calculating the Youden index.A p<0.05 was considered statistically significant. Results: Twenty animals were considered as intoxicated because presented one or more histological change as fibrosis, megalocytosis, ducts proliferation and binucleate cells in liver. Control group (eight animals) did not presented any alteration in histology of liver. In poisoned animals, lower quantity of albumin (**p<0.01) and high activity of aspartate aminotransferase (AST) and alkaline

phosphatase (ALP) (*p<0.05) were also detected. No alteration was observed in γ-glutamyl transferase (GGT) and alanine aminotransferase (ALT) activities. MiRNAs miR-30a-5p (*p=0,0478), miR-378-5p (*p=0,0358), miR-21-5p (****p<0.0001), miR-885-5p (*p=0,0154), and miR-122-5p (**p=0,0026) were significantly more expressed in intoxicated than in healthy animals. Furthermore, miR-122-5p (SE and SP 100%), miR-885-5p (SE 73.4% and SP 100%) and mainly miR-21-5p (SE 71.4% and SP 100%) signatures demonstrated high sensitivity and specificity based in ROC curve, indicating a potential application for detecting cattle poisoning by Senecio brasiliensis. The miRNA concentrations were also correlated with worse clinical parameters such as albumin and ALP. Results demonstrated a predominance of degenerative lesions, associated with mild proliferation of the ductal epithelium and fibrous connective tissue, according to hepatotoxicity induced by Senecio spp. Discussion: Despite miR-21-5p was highly expressed in cattle liver, no study demonstrated its role in liver function. MiR-21-5p was described as identical in humans being found to be upregulated in liver diseases as hepatocellular carcinoma, hepatitis C virus and autoimmune hepatitis. MiR-21-5p is upregulated in many biological processes, including inflammation and fibrosis and we hypothesized that high miR-21-5p levels could occur to induce liver tissue regeneration. Conclusion: Although, the present study has some limitations, including the small sample size, it is unprecedented and brings some insights related to farm animals poisoning with plants containing pyrrolizidine alkaloids as well as showing the necessity of additional studies. Acknowledgments: FAPESC

microRNAs as serum biomarker for Senecio brasiliensis poisoning in cattle

WinTer, evelyn1; ciSiloTTo, julia2; goeTTen, anDré l.F.1; veiga, ângela1; raMoS, aDriano T.1; ziMerMann, Francielli c.1; reck, carolina4; creczynSki-paSa,Tânia B.3

1 Department of Agriculture, Biodiversity and Forest, PPGMVCI, UFSC, Curitibanos; 2 PGFAR, UFSC, Florianópolis; 3 Department of Pharmaceutical Sciences, UFSC,

Florianópolis; 4 VERTÁ, Laboratory of Veterinary Diagnostic, Curitibanos.

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Introduction: Poisonings are events that produce harmful effects on the physiological state of the organism and drugs are identified as the main agents involved in these events. Drug-associated toxicological events (ETMs) in women of childbearing age have been shown to be even more frequent, which are usually associated with two or more drugs, especially those that act on the central nervous system. Objective: To characterize drug-associated toxicological events in women of reproductive age. Methods: Cross-sectional study with data from consultations at a Toxicological Information and Assistance Center (CIATox) of all cases of drug-associated toxicological events in women of childbearing age (10 to 49 years). Data were extracted from the Brazilian System of Intoxication Registration of Information Centers, referring to the period from 2017 to 2020. Variables related to the patient, exposure and clinical conditions were explored. The final outcome (negative - moderate or severe manifestations, or death - or not) was the dependent variable analyzed. Logistic Regression was used for association analysis with calculation of the Odds Ratio (OR), using the Statistical Package for the Social Sciences (SPSS), version 19.0. Results: 3,304 cases of drug toxicological events were evaluated in women of childbearing age, with a mean age of 26.0 (±10.2) years. Of the drug toxicological events, about 90.1% involved drugs exclusively, and it is noteworthy that 5.5% of the events were associated with ethyl alcohol. The suicide attempt occupied 89.0% of the circumstances that led to these ETMs, followed by self-medication (5.6%) and medication error (2.1%). On average, 2.02 were used (±1.44) drugs per event and 20.2% of these drugs are antidepressants, being the most recurrent therapeutic class, followed by

the class of hypnotics and sedatives (17.1%). The most frequent drugs were benzodiazepines (16%), followed by serotonin reuptake inhibitors (10.4%). In the association analysis, a higher prevalence of negative final outcome was identified among cases that involved suicide attempts (OR1.55; 95% CI 1.15-2.09), four or more medications (OR 1.94; 95% CI 1.52-2.48), with time between exposure and care of more than 4 hours (OR 1.60; 95% CI 1.30-1.96) and that occur during the night (00:00h – 05:59h) (OR 1.39; 95% CI 1.05-1.82). Discussion/Conclusion: In view of the data presented, it is concluded that the drugs most commonly used in drug-associated toxicological events act on the central nervous system and, considering that the main circumstance involved in the events were suicide attempts, it is expected that psychiatric disorders and/or or mental factors were important motivations for the events identified. In addition, considering that antidepressants are among the main agents in toxicological events, it is believed that depressive and anxiety disorders are predominant in the population served. Finally, the circumstance of the event, number of agents involved, exposure time and occurrence shift were factors associated with the negative final outcome, which demonstrates the importance of urgency and emergency services to give more attention to patients who are involved in ETMs from suicide attempts and with multiple medications. Also, that the management of this patient is as fast as possible to minimize the negative impact, especially when these events occur during the night. Acknowledgments: We would like to thank the entire team at the CIATox responsible for the care.

Profile of drug-associated toxicological events in women of reproductive age

coSTa, quezia DoS SanToS1; prieDouS, guSTavo aBuD2; Brunello, giovanna criSTina Spagnuolo2; alFieri, Daniela Frizon3; guiDoni, caMilo Molino3; alveS, jonaS alher Meira2; giroTTo, eDMarlon3

1 Pharmacy student at the State University of Londrina– Londrina/PR, Brazil; 2 Medical Student at the State University of Londrina– Londrina/PR, Brazil; 3 Professor at the Department of Pharmaceutical

Sciences at the Health Sciences Center at the State University of Londrina– Londrina/PR, Brazil.

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Background/introduction: Globally, suicide is responsible for more than 800,000 deaths every year, being the second leading cause of death among the young population. Suicide, suicide attempt or suicidal ideation are multifactorial, mainly associated with previous psychiatric disorders and demographic characteristics. Consequently, it becomes important to evaluate the relationship of the variables among themselves and their influence on individuals, to characterize the profiles of people who attempt suicide and thus to contribute to the improvement of public healthcare services. Objective: This work aims to identify a profile of poisoning by suicide attempt in Rio Grande do Sul, as well as evaluating the impact of the COVID-19 pandemic on these cases. Methods: Data were collected from cases of poisoning attended by Toxicological Information Center of Rio Grande do Sul (CIT/RS), registered between 2010 and 2020. Only cases where the circumstance was suicide attempt were selected. Statistical analysis was performed using the IBM SPSS 28.0 software. Association between variables and groups of toxic agents was performed using Pearson’s Chi-Square test. For temporal evaluation, AutoRegressive Integrated Moving Average Model (ARIMA) was performed. Results: The total number of suicide attempt cases was 55,131. The most incident groups of toxic agents were medicines (47,295 cases; 85.8%), chemicals (3,034 cases; 5.5%), rodenticides (2,895 cases; 5.3%) and pesticides (2092 cases; 3.8%). About 73% of patients were women and 50% of the cases involved patients between 20 and 39 years old. The gender variable was associated with several groups, mainly, female patients associated with the use of medication (p<0.001), while male patients were associated with drugs of abuse, pesticides, and chemical products (p<0.001). As for the age group, the use of medication was associated with adolescents and young adults (p<0.001). The use of drugs of abuse was associated

more strongly in the age group of 20 to 39 years (p<0.001), while the use of pesticides is associated with the age group of 40+ years (p<0.001) and rodenticides with 60+ years (p<0.001). The evolution variable was associated with the medicines group for cure (p<0.001) and pesticides and chemicals for death (p<0.001). The association of these two groups with death is mainly due to the lethality rate, being 3.6% for pesticides and 1.2% for chemicals. In the temporal analysis, the best model was ARIMA (0,1,4), with a good correlation between time and the number of cases up to the first quarter of 2020 (R2=0.87). Although it was expected an increase in the cases, from the beginning of the pandemic in Brazil (end of 2020/1) an intense decrease in relation to predicted cases was observed in the following three quarters of 2020, with drops respectively of -37.1%, -38.7% and -28.8%, with all exceeding the confidence interval (CI95%). Discussion/conclusion: The main profile of suicide attempt is characterized by women, with 15 to 29 years-old, using medicines as a toxic agent, as representing the majority of cases. An important association between the variables can be seen, especially from the type of the agent involved in the intoxication with the sex and age of the patient, thus being able to identify different patterns of use among the groups. Considering the reduction in cases in 2020 with the pandemic, there are numerous hypotheses for explain this data, such as a real decrease in attempts, effectiveness of suicide, change of suicidal method and/or underreporting of poisonings. From this, other factors involved in suicide (attempt) must be studied to synthesize a global panorama and, thus, evaluate the real impact of the COVID-19 pandemic. The results of this study provide a basis for toxicovigilance system, as well as the development of mechanisms to prevent suicide attempts. Acknowledgments: CAPES; FAPERGS.

Profile of suicide attempts by self-poisoning in the Rio Grande Sul, Brazil, between 2010-2020

and the influence of the COVID-19 pandemic

SanToS, Bruno pereira1,2; eller, Sarah1; gouveia, giovanna criSTiano1; BorgeS, gaBriela raMoS1; SeBBen, viviane criSTina3; arBo, Marcelo DuTra2; oliveira, Tiago Franco1

1 Graduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil; 2 Faculty of Pharmacy, Federal University of Rio Grande do Sul

(UFRGS), Brazil; 3 Toxicological Information Center of Rio Grande do Sul (CIT/RS), Brazil.

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Introduction: The Nucleus Applied to Toxicology (NAT) is a service that helps the University Hospital of Santa Maria (HUSM) performing toxicological analysis of patients with suspected or proven intoxication, in order to contribute to the proper diagnosis, monitoring and treatment. Objective: To present the toxicological occurrences in Santa Maria and region during the period of 2019-2021 assisted by NAT. Methods: NAT advises health professionals through telephone contact. The analysis of biological matrices were performed by means of immunoassays, spectrophotometry, HPLC-UV/Vis or DAD and/or GC-MS, depending on the case. Results: During the last three years, until October 2021, the NAT carried out 39 toxicological analysis, in addition to 17 toxicological supports to health professionals. Among the most detected substances in the analysis are cocaine (found in 15 patients), benzodiazepines (9) and cannabinoids (5). In 2019, four immunochromatographic assays for drugs of abuse and medications and a quantification of paracetamol by spectrophotometry were performed. In 2020, in addition to six immunoassays for drugs of abuse and medications and two quantitative determinations of paracetamol, two analysis related to pesticides were performed: one case of suspected paraquat intoxication, in which the colorimetric method was performed with sodium dithionite, and in another case, quantification of serum butyrylcholinesterase was performed in order to estimate the degree of intoxication by malathion and fusilade. In 2021, 23 immunochromatographic assays for drugs of abuse and medications, two quantifications of paracetamol and one of butyrylcholinesterase were performed, the latter aiming to elucidate a suspicion of carbamate intoxication. In this year, analysis in gaseous (GC) and liquid (LC) chromatography began, aiming at the

detection and quantification of substances, in view of the growing complexity of the cases. Since the NAT does not have CG equipment and the LC equipment has limited performance, the techniques were developed in another laboratory, which has a high demand for work, a fact that restricts the execution of the NAT analyses. The CG-MS equipment was used in the research of adulterants (levamizole) in cocaine in a biological matrix, and determination of valproic acid, quetiapine, ketamine and amitriptyline. LC-UV/Vis was used in the analysis of trans, trans-muconic acid, quetiapine, nortriptyline and amitriptyline in biological matrices. Discussion/Conclusion: Intoxication cases usually represent a defiance for the health professionals, as they are difficult to define the diagnosis, treatment, often there are several agents involved and potential risk of fatal complications. Although the NAT is not a Toxicological Information Center and has a limited technological apparatus, it has significantly contributed to the advice of professionals and elucidation of toxicological occurrences attended at the HUSM. In addition to improving the patient’s health status, NAT contributes to the economy of public spending. With the correct identification of the agent causing the damage and thus the proper treatment and monitoring, there is a faster hospital discharge and there is often no need to use an ICU bed, generating savings of R$ 1,600.00 / patient / day, given that this is the amount that a SUS patient at the HUSM costs the public coffers per day, according to the AuditaSUS expenditure table in 2020. Thus, the important role that NAT plays for Santa Maria and the region is highlighted, both for health and for the economy through its toxicological support and analyses. Acknowledgments: UFSM.

Services provided by the Nucleus Applied to Toxicology (NAT) during the period 2019-2021

reginaTo, FernanDa ziegler; BairroS, anDré valle; SanToS, lara celeSTina; roSa, vicTória goMeS; chiMenDeS, nayoMi De anDraDe; SanToS, rachel; pacheco, anDré

lucaS Bezerra; carDoSo, leonarDo corrêa; ugalDe, guSTavo anDraDe

Núcleo Aplicado a Toxicologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria.

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Introduction: Suicide is an important public health problem, with impacts on society as a whole. According to the World Health Organization (WHO), it is the fourth leading cause of death for young people aged 15 to 29 years. A survey revealed that between 2010 and 2019, the Southern region of Brazil had the highest suicide rate among all regions, with Santa Catarina being the second state with the highest rate in the country. The data also show that poisoning corresponded to 60% of the means of aggression recorded in this period, followed by sharp objects (16.8%). The COVID-19 pandemic has exacerbated risk factors associated with suicidal behaviors such as job or economic loss, trauma or abuse, mental disorders, and barriers to accessing healthcare. Thus, understanding the characteristics of these individuals can contribute to the planning of public policies to reduce the impact of suicide attempts. Objective: Evaluate the epidemiological profile of suicide attempts in the state of Santa Catarina before and during the Covid-19 pandemic, assessed from 2014 to 2020, and to evaluate whether the pandemic had any influence on suicide attempts. Methods: Data regarding attempts of suicide of adult patients older than 15 years, both man and woman, with suicide attempts by poisoning, were analyzed for this work. The data regarding suicide attempts were extracted from the open source DATATOX BI Pentaho / Saiku version 2.6, used in the service routine of the Assistance and Toxicological Information Center of Santa Catarina (CIATox/SC), while data regarding deaths by suicide by means of poisoning were extracted from Datasus,

following the same criteria, from the years 2014 to 2020. Statistical analysis was performed at GraphPad Prism (version 9.0). Results: Based on the data from CIATox/SC, there was an increase in suicide attempts from 2014 to 2019, with a decrease in 2020. This decrease, although not significant when compared to 2019 (p=0.88), is significant when compared to the growth observed throughout these years (p=0.01). The majority of patients who attempted suicide belong to the age groups of 20-29 (29.3%) and 30-39 (23.3%). The toxicant group with the highest attempts was medicaments (77%) and women had a higher rate of suicide attempts than men (69.2% vs. 30.8%). As for the number of deaths by suicide in Santa Catarina, extracted from Datasus, no statistical significance was observed between 2020 and 2019 (p=) or between 2020 and the previous years (p=0,99). Discussion/Conclusion: A decrease in attempts of suicide in the State of Santa Catarina among adult patients in the year of 2020 was observed, when compared to the growth in these cases during the past years. Besides that, we observed that there was no increase or decrease in deaths by means of poisoning during the year of 2020 when compared to the previous years. Since the number of deaths in 2020 remains within the normal rates from the past years, we can infer that the decrease observed in the suicide attempts in 2020 in the state of Santa Catarina does not reflect the deaths by poisoning, which could be explained by the fear of Covid-19, which made patients not seek health care services in suicide attempts. Acknowledgements: CIATox/SC for the partnership and provision of data.

Suicide attempts in Santa Catarina, Brazil, before and during Covid-19 pandemic: epidemiological profile

coSTa, ana carolina conchon1; Taruhn, lillian FreiTaS2; reSener, MariSeTe canello1

1 Assistance and Toxicological Information Center in Santa Catarina; 2 Medical Student in Federal University of Santa Catarina.

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Introduction: Suicide is a critical public health issue and one of the leading causes of death in adolescence, a period marked by significant physiological, psychological, and social transformations. Because this phenomena is multifaceted, additional research and regional assessments are needed to intervene in suicide prevention and enable more suitable treatments in those at risk. Objective: To investigate the clinical and epidemiological aspects of adolescent suicide attempts related to toxicological events assisted by a Poisoning Information Center. Methods: From 2017 and 2020, a cross-sectional and descriptive study was conducted utilizing data from the Londrina Poisoning Information Center (CIATox-Londrina), involving adolescent patients aged 10 to 19 who were treated for attempted suicide. Variables relating to the patient, their exposure, and their clinical conditions were investigated. The dependent variable was the ultimate outcome (negative - mild, severe manifestations, or death - or not), which was analyzed using Logistic Regression and the computation of the Odds Ratio (OR) with the Statistical Package for the Social Sciences (SPSS), version 19.0. Results: We analyzed 1,462 cases of toxicological events due to suicide attempt in adolescents, whose mean age was 16.0±2.1 years. Suicide attempts were more common in females (79.8%), as well as white and yellow people (76.0%). The events happened most frequently in October and November (10.2% and 10.4%, respectively), particularly on Mondays (17.8%), with the periods between 6:00pm and 11:59pm being the most common (37.9%). More than half of the suicide

attempts (52.9%) were carried out with a single agent, and the most common strategy was ingestion of medication (87.9%), followed by drugs of abuse (4.8%), domestic cleaning/chemical products (4.8%), and rodenticides (4.1%). Furthermore, depression was the main reason of suicide attempts (14.1%), followed by problems with romantic relationships (10.5%) and family issues (10.4%). Almost all of the cases (99.8%) were treated or remained asymptomatic, with the remaining cases resulting in death (three deaths). In the association analysis, a higher prevalence of negative final outcome was identified among cases involving males (OR 1.45; 95% CI 1.08-1.95), with time between exposure and care equal to or greater than 300 minutes (OR 1.80; 95% CI 1.24-2.62), which took place in January and December (OR 1.89; 95% CI 1.61-2.56), and charcoal administration activated (OR 1.54; 95% CI 1.19-1.96). Discussion/Conclusion: Although adolescent females try more suicides, young males have a more serious outcome, possibly because they employ more hazardous drugs and more severe self-harming methods. The findings show that the presence of depression symptoms is the most common reason for suicide attempts. Additionally, in recent years, these incidents have resulted in a more serious outcome, with the shorter time between exposure and treatment serving as a key prognostic factor. As a result, emergency services must be prepared to identify risk factors and manage toxicological incidents appropriately in order to ensure more humane health procedures and contribute to the reversal of this extreme public health scenario.

Suicide attempts in adolescents assisted by a toxicological information and assistance

center due to toxicological events

Brunello, giovanna criSTina Spagnuolo1; alveS, jonaS alher Meira1; coSTa, quezia DoS SanToS2; prieDouS, guSTavo aBuD1; alFieri, Daniela Frizon3; guiDoni, caMilo Molino3; giroTTo, eDMarlon3

1 Student of Medicine at the State University of Londrina – Londrina/PR, Brazil; 2 Pharmacy Student at the State University of Londrina – Londrina/PR, Brazil; 3 Professor, Department of

Pharmaceutical Sciences, Health Sciences Center, State University of Londrina – Londrina/PR, Brazil.

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Introduction: Rodenticide and pesticides are chemical products mainly used on agriculture and may trigger severe even lethal intoxication in patients who come into intentional or accidental contact, topically or orally. In 2020, Assistance and Toxicological Information Center of Santa Catarina (CIATox/SC) recorded 559 cases of poisoning involving pesticides. Among these records, an expressive number of suspected toxic agent was unknown. In cases of cholinesterase inhibitor poisoning, when it involves “chumbinho”, as it is sold as an illegal rodenticide, the composition may variate and contain carbamates or organophosphates. Although both cause (reversible or irreversible) inhibition of cholinesterases, the enzymes responsible for degrading acetylcholine. In clinical practice the evolution of the two cases may be different, in terms of management and severity. Thus, although not always available, recognize the toxic agent helps to confirm the diagnostic suspicion, justify prolonged antidotal treatment and identify the composition commercialized. This strategy, as it involves chromatographic analyses, is more restricted, being most common the determination of the activity of cholinesterases, usually butyrylcholinesterase. Objective: Report a clinical case of acute intoxication by a cholinesterase inhibitor, along with the medical management, evolution of the patient’s clinical condition and toxicological analyzes performed during hospitalization. Case Report: Male patient, 49 years old, taken unconscious by a family member to the Health System, after swallowing “chumbinho”. On admission, he presented Glasgow 7 on the coma scale, normotensive, with sweating and tremors only, with no other symptoms of the cholinergic syndrome being described. The patient quickly received doses of atropine, an antidote used to relieve and control

the patient’s condition. One day after ingestion, erythrocyte (AChE) and plasma (BChE) cholinesterase activity was determined, with 19.9 U/mmol Hb and 1000 U/L, respectively, far below the reference values (352-779 U/mmol Hb and 7000 - 19000 U/L, respectively). The patient continued to be treated with atropine, sometimes requiring a new bolus dose. The patient was followed up for 14 days with recurrent analyzes of both cholinesterases and it was observed that the greatest inhibition was recorded one day after ingestion for BChE and 2 days for AChE. There was a slow and progressive reduction in inhibition, with AChE at 47.77 U/mmol Hb and BChE at 6000 U/L in 14 days. The patient continued receiving atropine infusion until the 15th day and was discharged from the hospital 21 days after ingestion. During hospitalization, analysis was performed to identify cholinesterase inhibitors in serum, by Gas Chromatography with Mass Spectrometry (GC-MS), using a validated method. The result made it possible to detect the presence of Terbuphos and Chlorpyrifos organophosphates, proving and emphasizing the severe clinical condition with slow evolution. Conclusion: The intoxications caused by “chumbinho” always deserve attention, since it depends on the involved agent, the evolution is slow and demands a sufficient atropine for the adequate treatment of the patient. Both AChE and BChE are significantly inhibited in acute intoxication, being the most rapid reversal of BChE. The identification of the Terbuphos and Chlorpyrifos in this case was extremely important for understanding the evolution, the necessary management and the appropriate recording of intoxication. Keywords: “chumbinho”; acute intoxication; acetylcholinesterase, butyrylcholinesterase, GC-MS, Terbuphos, Chlorpyrifos.

Variation of cholinesterases activity and the importance of the involved toxic agent

identification on the acute intoxication evolution by an acetylcholinesterase

inhibitor – Case Report

BauerMann, lauren1; França, liana conraDo2; nogueira, janer alveS2; paula, eliza Bianchini1; Marchioni, caMila3; SanToS, clauDia regina3

1 Federal University of Santa Catarina (UFSC), Brazil; 2 Laboratory of Toxicological Research - Unit II - Clinical Analysis Unit - HU-UFSC-EBSERH; 3 Department of

Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

04 DRUGS OF ABUSE

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Background/Introduction: Synthetic cathinones (SC), one of the most popular groups of New Psychoactive Substances (NPS), are closely related to amphetamines and marketed as legal alternatives to such conventional illicit stimulants. These new chemicals pose a threat to public health, as little is known of their toxicological properties, such as potency, adverse effects, and metabolism; moreover, they are usually not detected by conventional analytical methods, thus the development of new ones applicable to SC is of high importance in forensic toxicology. Furthermore, the concept of Green Analytical Chemistry (GAC) is gaining attention in toxicological sciences with the search for environmentally benign approaches during method development. Objective: The present work aimed at developing and validating a quantitative method for the analysis of 15 SC in urine and postmortem blood while applying the principles of GAC. Methods: A dispersive liquid-liquid microextraction (DLLME) was developed and the Design of Experiment (DOE) statistical tool was used to find optimal conditions for extraction of SC from both matrices. Full factorial and Central Composite designs were chosen to analyze the solvent for precipitation, pH of extraction, ratio and volume of extraction and dispersive solvents. Analysis of Variance (ANOVA) was used to find the solvent yielding the best analyte recovery. The ANSI/ASB Standard 036, 1st edition 2019 international guidelines were used for method validation. All analyses were performed using a UPLC-MS/MS with the multiple ion monitoring (MRM) approach. Ethical Committee approval nº 46404121.8.3001.0067. Results: Hazardous solvents, such as chloroform, are commonly employed in DLLME, which are not fit when sustainable applications are intended. Thus, ethyl acetate (EA) was proposed as a greener alternative to chlorinated solvents in DLLME. First, a comparison

between EA and chloroform was performed and equivalent, when not higher, analyte recovery for SC was achieved using EA (p < 0.05). In the following optimization step, it was observed that 200 µL of a EA:ACN mixture (1:2.5) yielded higher SC recovery, while the system was maintained at pH 8.0. Once optimized, the method was validated according to international guidelines. For blood: limit of detection (LOD) and quantitation (LOQ) of 0.25 and 1 ng/mL, respectively; linearity 1-100 ng/mL; whereas for urine: LOD and LOQ of 1 and 10 ng/mL, respectively; linearity 10-1000 ng/mL. For both matrices: no carryover was observed within linearity range; bias and precision (> 85%); analyte recovery varied from 27.4-60%; and selectivity studies have confirmed the absence of interfering peaks due to endogenous and exogenous substances at the retention times of all analytes. In addition, non-labeled internal standards were not identified as an impurity. Curiously, the matrix effect (ME) was higher than 100% for all analytes. Finally, the method was applied to the analysis of two authentic samples. Discussion/Conclusion: We demonstrated for the first time that EA is suited to replace chloroform in DLLME, at least for SC, as higher – when not equivalent – analyte recovery was observed. Moreover, although the overall analyte recovery was not high, the sensitivity sufficed for the intended applications. In this regard, even with a high ME, acceptable precision and bias using only 200 µL of sample was achieved. Furthermore, this is the first method including N-ethyl Heptedrone. We successfully developed and validated an analytical method for SC, while incorporating multiple GAC concepts, e.g. low sample and solvent volumes (i.e. miniaturization of extraction techniques), use of less hazardous organic solvents, reduced generated residues, and simple sample treatment. Acknowledgements: CNPq – 142056/2020-0; CAPES/INSPEQT Nº16/2020.

A green dispersive liquid-liquid microextraction for quantitative analysis of synthetic

cathinones in urine and postmortem blood

FaBriS, anDré luiS; yonaMine, Mauricio

Department of Clinical & Toxicological Analyses, School of Pharmaceutical Sciences, University of Sao Paulo.

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Background/Introduction: Analysis of oral fluid generally requires some type of sample preparation to concentrate the analyte and reduce matrix effects for sensitive and reproducible analyses. The use of disposable pipette extraction (DPX) tips has been previously shown to offer advantages over other solid-phase extraction (SPE) in terms of speed and feasibility with automation. Most SPE methods for analyzing drugs of abuse in oral fluid incorporate a strong acid in order to achieve high recoveries of basic drugs using strong cation exchange sorbent. Moreover, there have been reports that this acid may lead to conversion of cannabidiol (CBD) to tetrahydrocannabinol (THC). In this research, the use of mixed mode DPX-XTR tips without the addition of a strong acid to avoid potential conversion of CBD to THC was evaluated. The study focused on analyzing 41 drugs of abuse commonly found in driving-under-the-influence (DUI) cases. The analysis used a single sample extraction method and liquid chromatography - tandem mass spectrometry (LC-MS/MS). An additional methodology is also presented for differentiation of delta-8 and delta-9 THC (for positive THC cases). Objectives: The objective of this study was to develop a DPX based methodology for analyzing comprehensive drugs of abuse in oral fluid for driving-under-the-influence casework. The use of mixed-mode sorbent is evaluated to provide reproducible and high recoveries of acidic, neutral and basic drugs commonly found in DUI case work. A separate LC method from the comprehensive method is used in order to differentiate delta-8 THC from delta-9 THC. Methods: Synthetic negative saliva buffer solutions (500 µL) were fortified with 50 µL of a comprehensive drug standard (45 drugs) that includes opiates, opioids, benzodiazepines, barbiturates, THC (delta-8 and delta-9, as well as CBD), antidepressants, amphetamines, and cocaine. Concentrations ranged, depending on the analytes, as low as 0.1 ng/mL to as high as 500 ng/mL. An automated liquid handler

was used to perform all extractions of oral fluid. The extraction involved the steps of conditioning with 800 µL 50% methanol in water, aspirating and dispensing the solution 5 times, washing with 800 µL of water, and elution with 500 µL of 48/48/4% acetonitrile/methanol/ammonium hydroxide. The solutions were subsequently evaporated and reconstituted in 125 µL 10% methanol in water. All analyses were performed using LC-MS/MS using mobile phases of pH 3.6 ammonium formate in water and methanol. For comprehensive analysis, a biphenyl column (50 x 3.0 mm, 2.6um) was used. For selective analysis of CBD, delta-8 and delta-9 THC, a fluorophenyl column (2.7 µm, 100 x 2.1 mm) was used. Results: The extraction studies included the analysis of 24 samples extracted weekly for 2 months, for a total of 192 samples. The extractions using the DPX-XTR tips in average took less than 10 minutes to complete (i.e., for 96 samples simultaneously). Almost all of the drugs had recoveries greater than 80%. Drugs fortified at suggested cutoffs (from the literature) were readily detected at less than 8% C.V. The comprehensive LC-MS/MS method was used to detect THC and CBD, but a separate methodology was used to quantify delta-8 and delta-9 THC at levels as low as 0.5 ng/mL. Conclusion/Discussion: A novel sample analysis workflow using DPX-XTR technologies was developed for the analysis of drugs of abuse oral fluid samples often collected DUI cases. 45 drugs and metabolites were efficiently extracted (>%80 recovery for all drugs) using mixed-mode DPX-XTR tips as an alternative to traditional SPE columns, which can help laboratories to enhance throughput while maintaining drug quantification requirements. Acknowledgments: The author would like to acknowledge William E. Brewer, Kaylee Mastrianni from DPX Technologies and Karl Scheidweiler from Immunalysis for the technical development and method implementation of these assays.

A novel workflow for the analysis of drugs of abuse in oral fluid samples using disposable

pipette extraction technologies (DPX-XTR)

caBriceS, oScar g.

G-Flo Scientific Laboratory Testing & Consultants (USA).

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Background: Despite its well-known harmful effects to the society, drinking and driving remains a serious public health concern worldwide. Another issue that has recently raised concern is the risk posed by associating alcohol intoxication and cell phone use while driving. Objective: To examine the driving impairment effects of alcohol and of alcohol combined with texting. Methods: Fifteen licensed drivers with similar drinking habits completed a lap in a closed-course section in six different situations: (I) sober; (II) sober and while replying to a cell phone message via WhatsApp; (III) 30 minutes after ingesting a moderate dose of ethanol (0.50 g/kg); (IV) 30 minutes after drinking and while texting; (V) 60 minutes after drinking, (VI) 60 minutes after drinking and while texting. Driving performance was analyzed by means of mean speed (measured as time to complete one lap in the course, LT) and maximum speed (Smax); braking time (BT) and braking distance (BD); and off-course excursions (i.e., when the drivers hit a traffic cone or exceeded the boundaries of the course). For the evaluation of LT, Smax, BT, BD a non-parametric Friedman test was performed. Pairwise comparisons using the Bonferroni-Dunn post hoc test were performed when significant main effects were present (p < 0.05). Off-course excursions were evaluated by means of a Cochran Q test. The ethanol levels were measured at 30 and 60 minutes after drinking by using a breathalyzer. Results: Paired sample t-test showed no statistically significant differences between the blood alcohol levels (BAC) in 30 and 60 minutes after drinking. All participants presented BAC levels above 0.020 g/dL in both periods. Therefore, according to the Brazilian legislation, they would have their drivers’ license retained for one year. Additionally, 60% of the participants (N=9) presented BAC levels above 0.06 g/dL in at least one period of time. In a real situation, they would have been arrested. Regarding

LT, the Friedman test result showed statistically significant difference between the treatments. The post hoc comparisons revealed that Situation III (30 minutes after drinking) and Situation V (60 minutes after drinking) were statistically different from every other conditions, except when comparing one to another. Comparing with the baseline (Situation I, LT median value of 48 seconds), the Situations III and V presented a decrease of 8 and 11 seconds on the median, respectively. Concerning Smax, the pre-alcohol treatments were statistically different from the post-alcohol conditions. The median Smax values for Situations 1 and 2 were 30 and 31 km/h, respectively; whereas for Situations 3, 4 and 5, the median Smax increased to 40 km/h, and for Situation 6 was 39 km/h. Neither alcohol intake nor texting had a significant effect on the evaluated braking parameters. None of the participants exceeded the boundaries of the course. However, one third of the participants (N=5) knocked down cones at least in one condition and, interestingly, this happened only in the driving and texting experiments. Discussion/Conclusion: Our findings strengthen the hypothesis that even moderate alcohol doses significantly impair the driving performance by increasing the probability of risky driving decisions, which highlights the importance of lowering the BAC legal limit to reduce the risk of traffic crashes. On the basis of the parameters evaluated, alcohol is the main driving impairment factor in speed behaviours, whereas texting directly influenced the ability to control the car. Therefore, alcohol and texting have complementary effects on driving impairment, and their combination represents a significant risk factor for accidents. Acknowledgments: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Alcohol and alcohol combined with texting: effects on speed and braking

behaviours in a closed-course section.

coSTa, Bruno ruiz BranDão1; FreiTaS, Bruno ToleDo2; Bigão, víTor luiz caleFFo piva1; perDoná, gleici Da Silva caSTro3; De MarTiniS, Bruno SpinoSa2

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo. Avenida do Café, s/nº Ribeirão Preto - SP 14040-903, Brazil. 2 Faculdade de Filosofia, Ciências

e Letras de Ribeirão Preto - Universidade de São Paulo. Av. Bandeirantes, 3900, Ribeirão Preto - SP, 14040-900, Brazil. 3 Faculdade de Medicina de Ribeirão Preto – Universidade

de São Paulo. Av. Bandeirantes, 3900, Ribeirão Preto - SP, 14049-900, Brazil.

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Background/Introduction: In recent years, Brazil has foreseen the possibility of including drug screening devices as a traffic enforcement tool for the detection of impaired drivers. However, these devices have a high level of heterogeneity in terms of accuracy, and must be evaluated according to local needs before implementation. Looking for the development of an evidence-based policy, a large-scale study was conducted to evaluate different point-of-collection testing devices for drug detection aiming their application on Brazilian traffic enforcement scenarios. Objective: To evaluate the reliability of point-of-collection testing devices for the detection of psychoactive substances in oral fluid. Methods: Samples of drivers stopped at roadblocks in 10 different Brazilian locations were screened by trained highway police officers using one of the four different devices available. Paired oral fluid samples were collected in all positive screened samples and in around 15% of the negative samples through Quantisal®, and confirmatory analyses were performed by a validated HPLC-MS/MS method for the detection of cocaine, cannabinoids, amphetamine, and methamphetamine. Sensitivity, specificity and accuracy of each device for each drug class were calculated according to each device’s cutoffs. Results: Of the 8.990 samples screened by the devices, 5.9% were positive for cannabis, 3.1% for amphetamine, 2.7% for cocaine, and 0.9% for methamphetamine. Sensitivity for cocaine among the four devices ranged between 76.9 - 97.0%; specificity between 96.5 - 98.8%; and accuracy between 96.5 - 98.2%. For

cannabinoids, devices presented sensitivity ranging between 0 - 96.8%, specificity between 90.2 - 98.1%; and accuracy between 87.2 - 97.0%. For amphetamine, devices presented sensitivity between 72.0 - 100.0%, specificity between 93.2 - 97.8%; and accuracy between 93.4 - 97.7%. For methamphetamine, devices presented sensitivity ranging 72.0 - 100.0%, specificity between 97.4 - 99.6%; and accuracy 96.9 - 99.7%. Discussion/Conclusion: The D2 was the only device that presented the minimum of 80% for all reliability parameters for the detection of all classes of drugs. The D4 device showed sensitivity of 72% for amphetamines and methamphetamines; the parameters for cocaine and cannabinoids were greater than 80%. D3 and D1 devices showed very low sensitivity for cannabinoids (0% and 12.1%, respectively). The point-of-care testing devices analyzed in our study presented acceptable reliability for detecting most substances, showing the applicability of the devices as point-of-care testing for screening proposes. As seen in other studies, the sensitivity for cannabinoid detection is the critical parameter of most devices, and therefore this analytical limitation must be considered and evaluated within public policy contexts. The low values of positive predictive value highlights the importance of performing mandatory confirmatory analysis considering the legal scenario of traffic enforcement. Acknowledgments: We thank the Secretaria Nacional de Políticas sobre Drogas for their institutional and financial support, and the Brazilian Federal Highway Police for their institutional support. We also thank all the volunteers who participated in the study.

Analytical evaluation of four screening devices for drug detection

in Brazilian traffic enforcement

Scherer, juliana n.1,2; BorgeS, gaBriela raMoS1,3; SanToS, Bruno pereira1,3; Dalanhol, carolina Silveira1; gouveia, giovanna1,3; viola, paTrícia pacheco1; carlSon, renaTo roMera1;

govoni, Bruna1; Mello, raiSSa1; vaSconceloS, MailTon1; pechanSky, Flavio1

1 Center for Drug and Alcohol Research, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 2 Post-Graduate Program in Collective Health, Universidade do Vale do Rio dos Sinos, São Leopoldo, Brazil;

3 Pharmacosciences Department, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil.

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Opioids are analgesic drugs, used to control moderate and severe pain, including cancer pain. This class of drugs is derivate from opium, which is extracted from poppy (Papaver somniferum L.). Opioids act by regulating pain in the descending pathway that can be associated with the release of serotonin, through their binding to G protein-coupled receptors, mu, delta, and kappa in the central and peripheral nervous systems. Its action can be presynaptic, inhibiting the release of neurotransmitters from primary afferent endings in posterior horn neurons, or postsynaptic, reducing the excitability of dorsal horn neurons. Opioids cause changes in pyramidal physiology, attenuating the excitatory response, so that the longer the exposure to the drug, more difficult will be to control over their ingestion. There is a reduction in metabolic activity in the frontal cortical regions, associated with changes in the expression and function of G protein-coupled channels, harmful to neuronal activation, however, when activated, it is more difficult for them to return to basal levels, these are also associated with changes, increased of impulsivity, cognitive inflexibility, memory and concentration impairment, in addition, can lead to euphoria and generate hallucinations, reasons which lead to its illicit use. These drugs can cause addiction and are illegally used for recreational purposes. In addition to being associated with changes in sleep parameters, leading to apnea and consequently hypoxia. Oxycodone is a potent opioid drug, classified as semisynthetic because it is derived from thebaine, it is a mu receptor agonist, which also has activity at the kappa receptor, used for the treatment of acute and chronic pain. Therefore, the objective of

this work was to evaluate the toxicity of oxycodone, through in vitro studies using neuroblastoma (SH-SY5Y) cell lineage. Cells were exposed to oxycodone at concentrations of 1, 10, and 100 µM for 24 hours. The drug effect was evaluated by the following tests: cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test; DNA damage was analyzed using the alkaline comet assay; cell death by apoptosis and/or necrosis detection method with the combination of the compounds Annexin V (ANX) and 7-amino-actinomycin D (7-AAD). For the tests, at least three independent experiments were performed. Data were statistically analyzed using Prisma Graphpad software, version 6.0, using the Kruskal-Wallis test followed by Dunn’s test and presented as median ± interquartile range. Effects were considered significant when p<0.05. Kruskal-Wallis test showed no effect for oxycodone in the cell viability test at any concentrations tested. For the comet assay, no statistically significant difference was observed when the concentrations tested were compared to the control, however, the damage caused to the DNA was ~7 times greater in the cells exposed to 100 µM when compared to the control group (non-exposed cells), being possible to observe an alteration generated to the cellular DNA from exposure to oxycodone. For the death and apoptosis test, the Kruskal-Wallis test was not reveal statistically significant difference, in any of the concentrations tested, when compared to the control group. At the concentrations and time tested, no toxic effects were observed. In the future, models that mimic an acute exposure to dopaminergic neuron-like will be tested.

Assessment of possible neurotoxic effects of oxycodone in SH-SY5Y cell lineage

liMa, luiza Siqueira1,2; coSTa, nayara De Souza1,2; pereira, Meire ellen1,2; alMeiDa, WilliaM3,4; ceSTari, MarTa MargareTe3,4; irioDa, ana carolina1,2; oliveira, cláuDia Sirlene1,2

1 Programa de Pós-Graduação Strictu Sensu em Biotecnologia Aplicada à Saúde da Criança e do adolescente, Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, PR,

Brazil; 2 Faculdades Pequeno Príncipe, Curitiba, PR, Brazil; 3 Programa de pós-graduação em Ecologia e Conservação; 4 Universidade Federal do Paraná, Curitiba, PR, Brazil.

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The consumption of alcoholic beverages can be considered one of the main factors responsible for the high incidence of traffic accidents with fatal victims, becoming a public health problem. Low doses of ethanol in the body can cause changes such as euphoria and relaxation, which can lead to imprudent driving. The objective was to evaluate the relationship between the toxicological examination of alcohol and road traffic accidents (ATT) with fatal victims in Chapecó/SC and region. This is a descriptive observational epidemiological study, developed at the Médico-Legal Institute (IML) of Chapecó, with the General Institute of Expertises of Santa Catarina (IML-IGP/SC). Data were obtained from criminal reports, autopsies and records of fatal victims from traffic accidents in the city of Chapecó and region, in the period between 2014 and June 2019. For data analysis, descriptive statistics, rate, standard deviation and frequency distribution (%) were used. The association between the variables was analyzed using Pearson’s Chi-square test or Fisher’s exact test. The significance level adopted was 5% (p < 0.05). Considering the 437 criminal reports of fatal victims of road traffic accidents evaluated, 76.0% were men, 25.4% were aged between 21 and 30 years, 87.9% were white and 47.1% had fundamental school completed. The

afternoon was the period with highest occurrence of traffic accidents with fatal victims (32.3%). The months with the highest frequency were June and February, with 10.8% and 10.5% respectively, being 2017 the highest number of cases. Most victims were motorcyclists (18.8%), followed by drivers of motor vehicles with four wheels or more (18.3%). As for the results of the toxicological test, 27.2% had positive blood alcohol levels and 5.0% of the cases were positive in the detection of narcotics. Among the fatal victims of ATT with positive alcohol toxicology, 41.0% were men, 46.4% were between 21 and 30 years old, 36.3% were white and 42.3% were drivers of motor vehicles with four wheels or more. Road accidents in individuals who tested positive for alcohol were associated with being male (p = 0.000) and aged between 21 and 30 (p = 0.005). From the results, it is concluded that there was a high percentage of ATT with fatal victims presenting positive blood alcohol levels. In this sense, greater inspection is suggested through existing laws, in order to prevent accidents and raise awareness among the population. Keywords: ethanol, road traffic accidents, fatal victims. Acknowledgments: We thank all participants and collaborators of this research, as they contributed to a scientific and social well-being.

Blood alcohol levels in fatal victims of traffic accidents

everTon BoFF1; Daniel, caroline2; Sá, cloDoalDo anTônio2; Brugnera, DéBora SchMiTz2; Silva, Maria iSaBel gonçalveS2; Marcon, Scheila2; corralo, vaneSSa Da Silva2

1 Universidade do Oeste de Santa Catarina - UNOESC; 2 Universidade Comunitária da Região de Chapecó - UNOCHAPECÓ.

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Introduction: Human urine and feces contain chemical markers of consumed products, and the concentration of these markers in residual waters found in wastewater treatment plants (WWTP) can be used for the estimation of the consumption of specific products by a given population, a strategy named wastewater-based epidemiology (WBE). However, obtaining representative samples for analysis is challenging. The most used sampling strategy in WBE studies is the use of active sampling, with specialized devices resulting in composite samples. Usually, active sampling studies are limited to short sampling campaigns and characterized by low temporal representativeness and high variability. Alternately, the use of passive samplers can provide integrated temporal estimates with a smaller cost. Among the passive samplers, the polar organic chemical integrative sampler (POCIS) is used for the measurement of concentrations of hydrophilic compounds such as drugs of abuse. Sampling with POCIS is usually performed over several days and result in a time-weighted average concentration (CTWA). The capability of the POCIS to accumulate compounds present in the wastewater is characterized by its sampling rate (Rs). Rs values are not consensual and must be defined by calibration experiments. The target compounds of this study were biomarkers of tobacco, cocaine, amphetamines, cannabis, and caffeine consumption. Objective: Develop an analytical strategy integrating passive sampling with the quantification of drug of abuse consumption biomarkers by ultra-high performance liquid chromatography associated with mass spectrometry (UHPLC-MS/MS). Methods: POCIS samplers were constructed as sandwiched structures containing Oasis® HLB sorbent between two polyethersulfone membranes in triplicate one day before the exposure. POCIS are replaced every two weeks at the inlet of the WWTP that was monitored for 1 year. The sorbent was removed from the POCIS and extracted with

methanol and the methanolic extract was submitted to double solid-phase extraction (SPE). The extracts were injected in UHPLC-MS/MS, with separation in reverse phase and with electrospray ionization in positive and negative modes. Matrix effects were evaluated by the post-extraction addition method. The efficiency of the POCIS extraction procedure was evaluated in quintuplicate. Rs values for each target compound were calculated by the water concentration decrease method. The CTWA in wastewater of the target compounds were calculated using Rs values determined in the experiments. Results and Discussion: In this study, an analytical method integrating POCIS sampling, selective SPE of POCIS extracts, and analysis by UHPLC-MS/MS was developed and validated for 12 biomarkers of substance use. Chromatographic separation was achieved in 11 min. The selective SPE procedure, combining both cation and anion exchange cartridges, resulted in low matrix effects (-19.3 to 22.8% for compounds with a deuterated analog internal standard) and high extraction yields (93.4 to 106.4%). The lab made POCIS sampler was adequately calibrated for 9 compounds (4 drugs of abuse, 4 drug metabolites, and 1 human non-drug lifestyle markers). The method was applied in a WWTP in Brazil, with continuous monitoring for 392 days in 2020-2021. The highest calculated wastewater concentration among drugs of abuse and metabolites was found for benzoylecgonine (431.5±190.6 ng L-1), followed by 11-nor-9-carboxy-∆9-THC (58.3±21.1 ng L-1), biomarkers of cocaine and cannabis consumption, respectively. Conclusion: Passive sampling of wastewater is an interesting and cost-effective strategy for the quantification of biomarkers of drugs consumption in epidemiological studies. Particularly when combined with a sensitive analytical method as UHPLC-MS/MS, POCIS sampling allows the long-term monitoring of a wide range of analytes. Acknowledments: Feevale University, CAPES

Determination of a comprehensive set of drugs of abuse, metabolites and human

biomarkers in wastewater using passive sampling followed by UHPLC-MS/MS analysis

hahn, roBerTa zilleS; BaSTiani, MarcoS Frank; lizoT, lilian De liMa FelTraco; linDen, raFael

Universidade Feevale, Brazil.

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Introduction: Cocaine adulterated with levamisole as a cause of purpura rectiformis progressing to full-thickness skin necrosis was first documented in 2003 and currently comprises over 200 reported cases. Agranulocytosis in cocaine users is a newly recognized worldwide condition, this novel cutaneous vasculitis syndrome can be recognized by its characteristic rash and skin pathology, along with leukopenia and autoantibody production. Certain clinical features can be attributed to the adulterant levamisole, although cocaine may also play a role in its pathogenesis. Objective: To characterize the seized samples for the presence of cocaine and levamisole through the association of thin layer chromatography (TLC) and Mass Spectrometry (GC-MS) tests in the samples seized in the south of Santa Catarina in the period of March, April and May of 2020. Methods: SAMPLES – The samples used were provided by the Division of Forensic Analysis of Criciúma of the scientific police of Santa Catarina, which covers the entire southern region of Santa Catarina, classified into mesoregions. The Criciúma mesoregion includes Balneário Rincão, Cocal do Sul, Criciúma, Forquilhinha, Içara, Lauro Muller, Morro da Fumaça, Nova Veneza, Orleans, Siderópolis, Treviso and Urussanga; Araranguá mesoregion includes Araranguá, Balneário Arroio do Silva, Ermo, Maracajá, Meleiro, Morro Grande, Timbé do Sul and Turvo; Sombrio mesoregion includes Balneário Gaivota, Jacinto Machado, Passo de Torres, Praia Grande, Santa Rosa do Sul, São João do Sul and Sombrio; Tubarão mesoregion includes Armazém, Braço do Norte, Capivari de Baixo, Grão Pará, Gravatal, Jaguaruna, Pedras Grandes, Rio Fortuna, Sangão, Santa Rosa de Lima, São Ludgero, São Martinho, Treze de Maio and Tubarão. THIN LAYER CHROMATOGRAPHY (TLC) – In the months of March, April and May, an N of 239 samples of white substance in the form of powder was obtained, which were admitted to the laboratory of the scientific police. For the analysis of the selected samples, 1 mL of methanol was added for each 1mg of white matter in powder form. These were shaken and submitted to the physicochemical TLC method. A drop of the mixture was applied to the plate under the following operating conditions: stationary phase on silica gel G60F254; mobile phase methanol 100 mL, plus 1.5 mL of ammonium hydroxide; main comparison standards: cocaine and levamisole. Afterwards, they were allowed to dry naturally, the acidified iodoplatinate reagent was sprayed on the plate and the color and retention factor (Rf) of the stains were observed, comparing them with the samples and reference standards. GAS CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY (GC-MS) – From the N of 239 samples, 8 samples were positive for cocaine and levamisole with the TLC method. Sample preparation consisted of weighing 1 mg of

cocaine, adding 1 mL of ethanol, both in a microtube. They were shaken for 5 seconds and the microtubes were transferred to a microcentrifuge, centrifuged for 3 minutes at 10000rpm. After centrifugation, 500µL of the sample supernatant was transferred to vials, coupled to a support, which were accommodated in the chromatographic equipment and analyzed according to the standards of the scientific police. Agilent Technologies® 7890A GC coupled to the Agilent Technologies® 5975C mass selective detector, with electron impact ionization source (70 eV), and Agilent Technologies® CTC GC sampler 80 autosampler. HP-5MS column (30m x 0.25mm x 0.25µm) was used. The software used for data acquisition was Agilent MassHunter Data Acquisition. Injections were performed in split mode with a ratio of 1:10. The injection volume was 1 µL. The injector temperature was set at 280 °C. The oven was programmed with an initial temperature of 200°C for 0.5 minutes, followed by linear heating from 40°C/min to 230°C and then linear heating from 30°C/min to 290°C, being maintained at this temperature for 1.5 minutes. Helium was used as the mobile phase with a flow rate of 1 mL/min. The transfer line was set at 280°C. Under these conditions, the total running time was 6.0 minutes. The substances were collected by the equipment and identified in the SWGDRUGS version 3.8 library. Results: From the analysis of the samples seized from March, April and May 2020, when added up, an N of 239 samples was observed, these went through the TLC method and when comparing the bands, it was identified that 3.35% contained not only cocaine, as well as the substance of interest in the work, levamisole. These were sent to the GC-MS method, which confirmed the result already seen by TLC and, in addition, the technique observed other peaks of other substances, caffeine, lidocaine and tetracaine and the alkaloids benzoylecgonine and the cis and trans isomers of cinnamoilocaine. Discussion/Conclusion: From the analysis of the samples seized between March and May 2020, it was observed that out of an N of 239 samples, 3.35% contained levamisole adulteration. Based on the results obtained in this research, we confirm that the substance is present in the region and that it will serve as a basis for future studies, so that the incidence of cases related to adulteration by levamisole can be investigated. In conclusion, the methods used in this work proved to be effective in determining the presence of levamisole in samples of cocaine seized in the south of Santa Catarina in the period of March, April and May 2020. In addition, with the CG-EM it was possible to detect other adulterants such as caffeine, tetracaine and lidocaine and the benzoylecgonine alkaloids and the cis and trans isomers of cinnamoilocaine, from the extraction process.

Determination of levamisole in cocaine samples seized in the south of Santa Catarina

BoMBazar, aManDa1; MarTinS, anDré BiTTencourT2; STeiner, BeThina TreviSol1; ávila, ricarDo anDrez MachaDo1

1 University of the Extreme South of Santa Catarina; 2 Santa Catarina Scientific Police.

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Background: Variations in the illegal drug market increase overdose risk. Drug checking is a public health intervention that allows people who use drugs to submit substances for content analysis, receive information and counseling. It is also useful for obtaining information like patterns of use, drug effects and adverse reactions, that would not be obtained from other sources such as seizures. Drug checking has been used as a harm reduction strategy for over 50 years in other countries and has proven to be an effective tool in monitoring changes in the drug market, including the emergence of NPS. However, in Brazil, it is only emerging and there are no scientific studies in this field. Objective: The main objective was to start to set the basis for the development of a community-based drug checking service in Brazil and study the impacts of this intervention. Methods: Data collection was carried out in an electronic music party held in December 2021. Prior authorization and the access to the party was based on a collaboration with a harm reduction organization that has extensive experience in health education in party contexts. The area reserved for harm reduction offered various services such as psychological support, rapid hepatitis test, distribution of informational material and there was also a specific space for drug checking. This service used field techniques such as colorimetric reagents (Marquis, Mecke, Simon, Mandelin, Liebermann, Scott, Erlich, Hoffman) and Thin Layer Chromatography (TLC). Participants also answered a self-administered questionnaire on sociodemographic data, pattern of substance use and reactions to the result of the analysis. Results: A total of 22 samples were

analyzed. Among tablets, 5 were positive for MDMA, 3 were negative and 1 contained MDMA with an unidentified mixture. Among crystal samples, 4 were positive and 3 negatives for MDMA. Negative samples were suggestive of containing MDA. One blotter paper was positive for LSD. Among powdered samples, 1 was positive for cocaine, 1 contained cocaine mixed with ketamine, 2 contained cocaine with an unidentified mixture and 1 was positive for ketamine. In summary, approximately 27% of the samples did not contain the expected substance and 18% contained some type of mixture. The questionnaire analysis showed that 13% of participants gave up using the substance after knowing the test result, another 36% reported that they would use less amount of the substance. 91% of respondents stated that they always or almost always plan consumption in advance. 92% of respondents rated the service as very useful. Discussion/Conclusion: This preliminary data suggests that electronic music party attendees positively engage with harm reduction services when offered. The high rate of adulteration represents a great risk to drug users and shows the potential of drug checking services to prevent the inadvertent use of unknown substances, to reduce the amounts ingested and thereby decreasing the risk of intoxication. The fact that the vast majority of respondents plan consumption in advance demonstrates that there is an opportunity for interventions. As future directions, we intend to compare the results obtained by colorimetric reagents and TLC with analysis in the laboratory. Acknowledgments: The authors thanks CAPES by the financial support

Developing a drug checking service in Brazil and the potential of harm reduction

strategies to reduce intoxication risks

MaluF, ana criSThina SaMpaio1,2,4; coSTa, joSé luiz1,2,3

1 Campinas Poison Control Center, University of Campinas (UNICAMP), Campinas, SP, Brazil; 2 Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil;

3 Faculty of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil; 4 ResPire Harm Reduction Project, Centro de Convivência É de Lei, São Paulo, SP, Brazil.

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Background: Synthetic cannabinoids, originally developed for research of CB1 and CB2 cannabinoid receptors, were first identified in herbal incense in 2008. These herbal incenses, usually called ‘spice’, have since gained global popularity because they are sold on the Internet and in many head shops under the disguise of normal herbal products. JWH-018 was shown to have psychoactive effects similar to those of strong agonists of cannabinoid type 1 (CB1) receptor, for example, Δ9-tetrahydrocannabinol (THC). AB-CHMINACA, a recently introduced synthetic cannabinoid, was first reported in Japan in 2013 and has spread rapidly worldwide. It is a highly potent agonist of the CB1 receptor and can cause serious adverse health effects. Hair analysis is useful for monitoring exposure to drugs, and is a unique material for the retrospective detection of drug consumption, due to its large detection window, and it is easy to collect, store and transport. Objective:This study aimed to develop and validate a sensitive LC-MS/MS method for the quantification of JWH-018 and AB-CHMINACA and their principal metabolites in hair samples. Methods: In this method, hair samples were cut with scissors to fragments smaller than 3mm, and then 15 mg was weighted in 2.0 mL polypropylene microtubes. The samples were decontaminated sequentially with dichloromethane and methanol and were dried at ambient temperature. Then, 0.75 mL of methanol and a labeled internal standards solution were added. The microtubes were capped and incubated overnight at 40°C. The supernatant was transferred to a 2.0 mL glass vial and dried at 40°C with a nitrogen sample concentrator. The samples were resuspended with

0.15 mL of methanol and 10.0 uL were injected into an LC-MS/MS system. Chromatographic separation was performed on an Agilent 1290 Infinity II coupled with a Sciex 6500+ QTrap mass spectrometer. The system was equipped with a HALO Biphenyl column and used a mobile phase constituted by water and methanol with 0.1% of formic acid. Results: The present method was successfully validated for carryover, linearity, precision, recovery, detection and quantification limits. The chromatographic method is suitable to analyze all of the analytes with a baseline separation, principally the isomers JWH 018 N-(4-hydroxypentyl), JWH 018 N-(5-hydroxypentyl) metabolites. The analytes JWH-018 and metabolites (JWH 018 N-(4-hydroxypentyl), JWH 018 N-(5-hydroxypentyl), and JWH 018 N-pentanoic acid) achieved the linearity of 1.25 – 50.00 pg/mg, the detection limit of 0.09 - 0.19 pg/mg, and 1.25 pg/mg of quantification limit. The precision was less than 6%, and recovery was between 91 – 113% for all analytes. The analytes AB-CHMINACA and metabolite AB-CHMINACA M2 achieved the linearity of 2.50 – 100.00 pg/mg, the detection limit of 0.44 and 0.34 pg/mg, and 2.50 pg/mg of quantification limit. The precision was less than 5%, and recovery was between 93 – 101% for all analytes. Conclusion: We develop and validate a sensitive and precise method to detect and quantification synthetic cannabinoids in hair samples. The method does not need additional steps for clean-up the samples to achieve sufficient sensibility to detect these drugs at very low concentrations and can be used at routine analysis.

Development and validation of a highly sensitive method for detection of Synthetic

Cannabinoid JWH-018, AB-CHMINACA, and their metabolites in hair samples by LC-MS/MS

paulo, Breno F. pereira; zauli, Danielle alveS goMeS Breno

Group Hermes Pardini - Vespasiano, Brazil.

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Introduction: Suicide is a serious public health problem that affects people of almost all ages and has several social implications. However, its causes are preventable, where a comprehensive multisectoral strategy is necessary in order to revert to improvements in the health of the population. Objectives: To analyze suicide in the State of São Paulo using a new data source, the Public Security Secretariat (SSP) and the Legal Medical Institute (IML), and to compare it with the Mortality Information System of the Ministry of Health (SIM/MS). Method: a database related to suicide cases that occurred in 2015 was assembled from two databases: one from the SSP and the other from the IML, both in the state of São Paulo. The variables related to the characteristics of the victim, the event, injuries, presence of alcohol and/or drugs at the time of death and possible motivation for suicide were studied. Data were compared to SIM/MS data. Results and Discussions: 2469 cases were obtained, 7.5% higher than the SIM/MS data (2297 cases) showing an under-enumeration of suicide cases from the official data. Males predominated over females (ratio: 3.8) and, in terms of age, the highest frequency occurred in the group between 20 and 39 years old. Regarding suicide methods, hanging represented 60.2% of the total, followed by self-poisoning (16.1%), fall from heights (9.3%) and gun shooting (7.2%), there was a 92.5% decrease among unspecified suicides in relation to SIM/MS database, resulting in a data quality enhancement. Regarding intoxications, the highest frequency was due to the ingestion of medications, in which the cases resulting from the use of antidepressants stand out. Regarding ingestion of other substances, there was a greater number of poisonings by pesticides. The use of chemical substances for the

practice of suicide is common and can be seen by the significant gain in relation to official data from SIM/MS (from 100 to 172 in drug intoxications and from 155 to 225, when intoxication by other substances is analyzed). Toxicological tests were positive in 51% of the cases performed, with 902 substances found. The highest occurrence was ethanol (31%), highlighting blood alcohol concentration ≥ 0.8 g/L (82.1%), leading to the conclusion that the majority of suicides with a toxicological test for ethyl alcohol were drunk at the time of the act; the second group of substances corresponded to drugs (37%), where positivity in women was almost three times that found in men. Cocaine was detected in 75 cases, being more frequent in adult men. It was found in 55.2% of positive exams in cases of hanging, in 50% of suicides by sharp instruments and firearms, and in 42.9% of suicides by precipitation from a high place. Pesticides were detected in 93 cases, with carbamate being the most frequent followed by organophosphates. Carbon monoxide and liquefied petroleum gas were found in 20 cases, being higher among adolescents. Conclusion: Data from SIM/MS, regarding suicides, are still incomplete, since, for various reasons, deaths are under-enumerated, sometimes not specifying the correct cause of the event. The methodology of this research allowed quantitative and qualitative gains in relation to suicide cases. These results point to the importance of working together, data from death certificates (SIM/MS) and from police information and autopsy reports. It is possible to estimate the appreciable gain that public safety data would generate in health data and, consequently, in prevention measures. Keywords: suicide, epidemiology, mortality, health information systems. Acknowledgments: FAPESP, CNPq.

Epidemiological aspects of suicide in the State of São Paulo

Souza, karla apareciDa De oliveira1; gianvecchio, vicTor alexanDre percinio2; gianvecchio, Daniele Muñoz2; jorge, Maria helena praDo De Mello3; coSTa, joSé luiz1,4

1 Faculty of Medical Sciences, University of Campinas, (UNICAMP), Campinas, SP, Brazil; 2 Superintendence of the Technical-Scientific Police, Institute of Legal Medicine, SãoPaulo, SP, Brazil; 3 Faculty of Public Health, University of São Paulo (USP), São Paulo, SP, Brazil; 4 Faculty

of Pharmaceutical Sciences, University of Campinas (UNICAMP), Campinas, SP, Brazil.

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Introduction: The high number of intoxications by drugs of abuse treated by the Sistema Único de Saúde (SUS) contributes to the overload of the system. Measures established to restrict movement during the Corona Virus Disease pandemic (COVID-19) have altered social dynamics, with possible impacts on mental health. In this context, understanding the profile of cases reported before and during the pandemic can help in planning prevention strategies. Objective: To analyze epidemiological data on intoxications by drugs of abuse reported in Sistema de Informação de Agravos de Notificação (SINAN), in the previous period and during the COVID-19. Methods: Exploratory, descriptive, and retrospective study with analysis of secondary data obtained from SINAN, available on the DATASUS website, referring to the previous period and during the COVID-19 pandemic (2018-2019 and 2020-2021). Results: In Brazil, in the analyzed period, 66,889 intoxications by drugs of abuse were registered, 44,191 (66.07%) cases in the first period (2018 and 2019). The circumstance “abusive use” and male gender were similar in the analyzed periods, with a mean occurrence of 78.5% and 73.3%, respectively. Suicide attempt (SA) was reported in 3.6% of cases in 2018-2019 and 4.9% in 2020-2021. The age group with increased records corresponded to 20-39 years, first and second period, 55.1% and 56.3%, respectively. Regarding education, the data were similar in both periods, with the most prevalent being ignored data 54.0% (2018-2019) and 49.8% (2020-2021). In both periods, a low percentage of cases underwent clinical and laboratory diagnosis, 2.7 and 2.5%, respectively. Single-acute exposure was the most recorded in both periods (33.7% and 31.06%). Most notifications in the first period came from the Southeast region (62.2%), the city of São Paulo with the highest percentage of notifications (31.2%), followed by the Northeast region (14.7%). In the second period, most of the notifications were also registered in the Southeast (59.5%), followed by the

South region (15.8%, São Paulo maintained its position (35.2%). The highest percentage of evolution was cure without sequelae, 68.8% (2018-2019) and 71.8% (2020-2021); 20.5% (2018-2019) and 20.8% (2020-2021) with interrupted or unreported follow-up. The number of deaths was 589 (2018-2019) and 320 (2020-2021). Discussion/Conclusion: The pandemic did not change the profile of reports of intoxication by drugs of abuse, despite the 32.1% reduction, possibly resulting from underreporting, explained by fear of contamination and overcrowding of services. Between 2020 and 2021, there was an increase (1.2%) in cases of SA, which may indicate a correlation with the pandemic. In both periods, the majority of patients were male, with an increase (1.5%) in the most recent period. The prevalent age group was young adults, which indicates the male adult stage as the main target for prevention actions. A high percentage of cases with no schooling data was observed, making discussion unfeasible and demonstrating the need to improve the training of professionals responsible for collecting and recording data. Data show that clinical laboratory diagnosis could be more applied in the future as a confirmation criterion for drug use. The type of acute-repeated exposure was more prevalent, with an increase of 3.4% between the analyzed periods. The Southeast region, which ranked first in both periods, needs better prevention strategies, specifically the city of São Paulo, which showed an increase of 3.9% in notifications. Although most of the reported cases evolved to a cure without sequelae, there was a significant number of deaths. Thus, the relevance of knowledge of the epidemiology of cases of exogenous intoxication is evidenced so that prevention and intervention actions can be thought and carried out to reduce cases. Keywords: Intoxication, Epidemiology, Drugs. Acknowledgments: The State University of Maringá, Postgraduate Program in Biosciences and Pathophysiology (PBF/UEM), CAPES e CNPq.

Exogenous poisoning by drug abuse in Brazil: before and during the COVID-19 pandemic

Baccule, nicole SanToS1; ScanFerla, DeBorah ThaiS palMa2; lini, renaTa Sano2; Marchioni, caMila3; MoSSini, SiMone apareciDa galerani1,2

1 Laboratory of Toxicology, Department of Basic Health Sciences, State University of Maringá, Paraná, Brazil; 2 Postgraduate Program in Bioscience and Physiopathology, State University of Maringá,

Paraná, Brazil; 3 Department of Pathology, Federal University of Santa Catarina, Florianopolis, Brazil.

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The city of Balneário Camboriú is internationally known for its tourist attractions and electronic parties where there is great consumption of illicit drugs. Tourism is the main economic activity in the city, which is known, not only for its natural beauty, but also for its real estate market, which attracts large financial investors to the region. New psychoactive substances (NPS) or designer drugs comprise a group of modified drugs developed by changing molecular structures of known illegal substances such as cannabis, cocaine, lysergic acid diethylamide (LSD), and phenethylamine derivatives (MDA, MDMA, MDE, etc). NPS are illegally produced in a variety of preparations (powder, tablets, or capsules), and they may be injected, ingested orally, snorted, or smoked. Infrared spectroscopy is widely employed as a qualitative and quantitative method for the determination and structural analysis of drugs. Fourier Transform Middle Infrared Spectroscopy (FTIR) analysis can be a particularly useful tool for the rapid screening of seized drugs tablets and powders. Attenuated Total Reflectance (ATR) accessory for FTIR allows direct sample measurement with minimal preparation and with the potential to recover the sample if necessary. This study aimed to analyze and identify the content of synthetic drugs seized

in the region of Balneário Camboriú using ATR-FTIR. The seized material was first subjected to colorimetric analysis using the reagents Marquis, Simon and Mecke, and then analyzed using (FTIR) with Attenuated Total Reflectance (ATR), featured by the following elements: detector with element in Deuterated Triglycine Sulfate (DTGS), Zinc Selenide (ZnSe) Beam Splitter and spectral range from 500 to 4000 cm-1. The sampling interface used was Monolithic Diamond and thirty-two scans were collected at a resolution of 4cm-1. Most of the synthetic drug seizures in the region were marked in the form of tablets in various shapes and colors. The most common substances found in its composition were 3,4 methylenedioxymethamphetamine (MDMA) and alpha-methyl-3,4-methylenedioxy-phenethylamine or tenamphetamine (MDA) with prevalence of MDMA in the form of crystallized powder and of MDA in the form of tablets. In 2020, however, the first seizures of methamphetamines began to appear in the region. Keywords: New Psychoactive Substances; Balneário Camboriú; ATR-FTIR. Acknowledgments: Chemical Laboratory team of the Forensic Analysis Institute – Scientific Police Superintendence in Balneário Camboriú.

Identification of synthetic drugs seized in the region of Balneário Camboriú using ATR-FTIR

coSTa, karina oliveira1; Bagio, jéSSica2

1 Biochemical Criminal Expert - Scientific Police Superintendence in Balneário Camboriú; 2 Pharmacy graduate students- Vale do Itajaí University - UNIVALI.

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The emergence of New Psychoactive Substances (NPS) is observed worldwide. These compounds correspond to changes in the chemical structures of the original prohibited substances to create alternatives to consumption and legislative prohibition. The rise of these new drugs represents a challenge for identifying and depicting their main attributes. This study aimed to evaluate NPS by using silico methods to assess the principal characteristics by predicting their biological, therapeutical, and toxicological properties. We used the MetadrugTM software for calculating seventy-five variables among these properties for five different groups of compounds collected from the literature: amphetamines (15 molecules), benzodiazepines (13 molecules), cathinones (16 molecules), synthetic opioids (14 molecules), and synthetic cannabinoids (14 molecules). We calculated structural data, CYP450 QSAR Models, Protein binding QSAR Models, ADME QSAR Models, the predicted therapeutic activity, and predicted toxic effects. This tool is part of the MetaBaseTM API to access the knowledge base behind all systems biology products of Clarivate Analytics (https://www.cortellislabs.com/page/?api=api-MB). Multivariate analysis was applied to verify the behavior of these groups of substances. Unsupervised approaches PCA (Principal Component Analysis) and HCA (Hierarchical Cluster Analysis) was used to verify which variables better describe each group. Observed clusters were submitted to supervised learning through SIMCA (Soft Independent Modeling of Class Analogy). We observed that the five different groups were classified through PCA and HCA. SIMCA confirmed these classes, and no misclassification was found. Different characteristics were found for each category, as follows: Class 1. Amphetamines. We found four variables with high modeling power among those with the prediction of therapeutic activity: potential antiallergic, antianginal, and antihypertensive activities besides action against skin diseases. However, among the prediction

of toxic effects, we found potential for inducing carcinogenicity in rats and mice, kidney necrosis, and pulmonary toxicity. Class 2. Benzodiazepines. This class was modeled for variables related to the prediction of therapeutic activity, showing potential for antifungal, analgesic, antiviral and antiallergic activities. Besides, Protein binding QSAR Models showed potential to bind the androgen receptor and human hERG (human ether a-go-go-related gene) channel inhibition. The potential for inducing kidney necrosis was also found as a predicted toxic effect. Class 3. Cathinones. The main discriminant variables for this group were: human serum protein binding % (ADME QSAR Models); potential to be metabolized by CYP1A2 (CYP450 QSAR Models); potential for inducing hepatotoxicity (Prediction of toxic effects); potential anticancer activity (Prediction of therapeutic action). Class 4. Synthetic opioids. For Protein binding QSAR Models, these molecules presented the potential to bind to Estrogen receptors at 100 uM or less. Among the possibility of toxic effects, we found as variables: growth inhibition of MCF7 cell line (human Caucasian breast adenocarcinoma), pGI50; potential for inducing carcinogenicity in male mice, and cardiotoxicity. Finally, this group presented potential therapeutic activity against skin diseases and antifungal action. Class 5. Synthetic cannabinoids. Besides antifungal action, this group presented potential therapeutic activity against skin diseases and heart failure. Regarding the toxic effects, the potential for inducing carcinogenicity in male rats was an essential variable for modeling this class. This study was adequate to show different properties regarding different groups. All groups presented both potential for therapeutic and toxic effects. We thank the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 465450/2014-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) for financial support.

In silico evaluation of biological, therapeutical, and toxicological properties

for five groups of NPS: amphetamines, cathinones, benzodiazepines, synthetic

opioids, and synthetic cannabinoids.

SanToS, chriSTiano; Bruni, aline ThaiS

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

154

NBOMe is an abbreviation for compounds that contain an N-methoxybenzyl group. They are considered NPS (New Psychoactive Substances), used as alternatives to LSD (Lysergic Acid Diethylamide). ATS is the abbreviation for amphetamine-type stimulants, synthetic NPS used to mimic the effects of classic controlled substances such as MDMA. Both groups of NPS are compounds that present similar structures once NBOMes can give an amphetamine partial structure (https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/dta.1889). These compounds’ pharmacologic and biological properties are unknown and might be elucidated. However, the growth of NPS can cause many challenges in understanding the properties of these drugs. The main goal of this study is to use in silico methods for investigating the biological and toxicological behavior of these substances. For this study, we applied the MetadrugTM tool, part of the MetaBaseTM API, to access the knowledge base behind all systems biology products of Clarivate Analytics (https://www.cortellislabs.com/page/?api=api-MB). We used MetadrugTM software for calculating seventy-eight variables for 21 amphetamines, 21 cathinones, and 24 NBOMes. SMILES were used to simulate structural properties, CYP450 QSAR Models, Protein binding QSAR Models, and ADME QSAR Models, predicting therapeutic activity and toxic effects. We evaluated

the influence of the variables on the samples by multivariate tools. We used PCA (Principal Component Analysis) as an unsupervised approach and SIMCA (Soft Independent Modeling of Class Analogy) to observe classification effectiveness. PCA results showed there are mainly two classes regarding NBOMes and ATS. However, it is possible to observe some differentiation between ATS amphetamines and cathinones structures. SIMCA evaluation confirmed three different classes, but significant similarities between amphetamines and cathinones. Mouse carcinogenicity showed a higher modeling power for NBOMEs. The retention time for the affinity to human serum albumin was the most crucial variable in modeling the ATS class. Our findings showed a great potential of the in silico tools to foresee the properties and provide valuable information about unknown substances. We found that even NBOMes with amphetamine core structures differ in behavior from ATS. The computational approach helped to promote an understanding of the biological and toxicological characteristics of these NPS. It can support decision-making in public policies and law enforcement. We thank the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 465450/2014-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) for financial support.

In silico parameters and chemometrics applied to the study of NBOMes and

amphetamine-type stimulants

MarioTTo, lívia Salviano; roDrigueS, caio henrique pinke; Bruni, aline ThaiS

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

155

Context: New psychoactive substances (NPS) continue to proliferate in recreational drug markets worldwide. Synthetic cathinones is one of them. After administration of high doses or repeated use, they can produce serious medical complications, including tachycardia, hypertension, agitation, psychosis, aggressive behavior, seizures and, in some cases, death. N-ethylpentylone is a newly-emerging synthetic cathinone, but little information is available about its toxicology and pharmacology. Objective: Characterize the analytical quantification and describe the clinical presentation of analytically-confirmed cases of N-ethylpentylone intoxication. Materials and Methods: The quantification of N-ethylpentylone in blood obtained from human cases was carried out using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the clinical symptoms exhibited by the intoxicated individuals who were taken to the hospital emergency room for medical care and laboratory are described. Results: Our LC-MS/MS method assayed N-ethylpentylone concentrations with limits of detection and quantification of 1 and 5 ng/mL, respectively. Quantitation was linear from 5 to 500 ng/mL, and the method displayed excellent specificity and reproducibility. Five intoxication cases were monitored by Campinas Poison Control

Center, men and women aged 18 to 35 years reported circulating concentrations of N-ethylpentylone ranging from 7 to 170 ng/mL and symptoms included palpitations, tachycardia, agitation, hallucinations, coma and death. Discussion/Conclusion: Until the present study, only two published articles reported the amount of N-ethylpentylone. We present a validated method to quantify N-ethylpentylone in human case work. All cases accompanied by laboratory results showed high levels of creatine kinase, a symptom often associated with synthetic cathinone intoxication. The two highest concentrations of N-ethylpentylone that we measured were found in a fatal overdose case (ie, 170 ng/mL) and a severe intoxication with long-lasting cerebrovascular complications (ie, 149 ng/mL). Importantly, N-ethylpentylone was the only psychoactive compound detected in our fatal case, confirming that the drug can have lethal consequences, as well as a variety of symptoms, including palpitations, tachycardia, agitation, aggression, hallucinations, and coma. Acknowledgments: The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (process number 2015/10650-8 and 2018/00432-1) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (process number 830525/1999-8).

Intoxication case series by N-ethylpentylone (N-ethylnorpentylone or ephylone)

Denkena, iSaDora locilenTo1,2; cunha, kelly FranciSco2; lanaro, raFael2; cunha, ricarDo leal3,4; coSTa, joSe luiz1,2

1 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo 13083-859, Brazil; 2 Campinas Poison Control Center, Faculty of Medical Sciences, University of Campinas, Campinas, São Paulo 13083-859, Brazil; 3 Institute of Chemistry, Federal University of Bahia, Salvador, Bahia

40170-115, Brazil; 4 Institute of Analysis and Forensic Research, Aracaju, Sergipe 49100-000, Brazil.

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Introduction: 3,4-methylenedioxyamphetamine (MDA) is an entactogen often used in music festivals, mostly sold as MDMA, that showcases similar effects as the last, including euphoria, self-awareness, compassion and acceptance for self and others. Nevertheless, little is known about its pharmacokinetics in humans and data about its hepatotoxicity is scarce. Following the resurgence of MDA in the streets and the prevalence of ecstasy tablets containing both a mixture of MDA and MDMA or just MDMA - according to police reports-, insights on its pharmacology and dose response for a safer use under the perspective of harm reduction is imperative. Similarly, considering its entactogen properties and the advances in psychedelic research aiming to find alternatives to treat neurological disorders, pre-clinical information about MDA could enable further investigation of the drug as a potential therapeutic candidate. Objective: The aim of this study was to assess the toxicity of MDA in silico to provide more information about its pharmacology and sustain further pre-clinical studies. Methods: Toxicity assessment was run through statistical and machine learning-based IS-TOXTM platform (Altox Ltda, funded by FAPESP process number 2016/08322-5) under the following softwares: iS-LiverTM, to predict in vitro and in vivo hepatotoxicity; Acute-ToxTM, for acute toxicity; DevTox-IsTM, for developmental toxicity prediction; Pred-CYP2DTM, for metabolites prediction; Pred-OralTM, for bioavailability and permeability prediction; and lastly Genotox-IsTM, to predict genotoxicity; Results: Assessment of the molecule has returned structural alerts regarding potential DILI and action as agonist of the Aryl Hydrocarbon Receptor (AhR). The cytotoxicity assay using HepG2 cells predicted a weak-moderate toxicity (above 4,8 mg/L). MDA was classified as hepatotoxic, producing DILI in rodent but not humans. Its main metabolite, alpha-methyldopamine however,

was not predicted as hepatotoxic. As of metabolism, MDA is a substrate of the following CYP (cytochrome P450) isoforms: 2B6, 1A2, 3A4, 2C19, 2E1, 2A6, 2C9 and 2C8; acting as an inhibitor of the isoforms 2B6, 2D6, 1A2 and 3A4. The assessment of developmental toxicity predicted MDA as toxic in vivo, but not toxic in terms of reproductive toxicity. High permeability was predicted using Caco-2 cells (23,3 x 10-6 cm/s) and Paralell Artificial Membrane Permeability (PAMP) was moderate (1,2 x 10-6 cm/s). Rat oral acute toxicity was estimated in 125,8 mg/kg (OECD Category 3). MDA was not considered genotoxic. Discussion/Conclusion: Based on the predictions obtained MDA produces acute toxic effects in lower doses compared to its analogue and parent molecule MDMA. The toxic concentration predicted does not seem to pose serious risks to most users in controlled environments, once most tablets contain less than 120 mg of MDA, which would not be enough to produce hepatic damage. Although the data regarding its toxicity both in vitro and in vivo are scarce, set and setting seem to play an important role in MDA-induced toxicity, as for MDMA, once its use can cause hyperthermia, increased blood pressure and heartbeat rate. Considering the consumption pattern features of MDA it is likely that the adverse effects reported are due to secondary events concerning recreational use and not due to its intrinsic pharmacological properties. Sales of ecstasy tablets allegedly containing MDMA but being in reality MDA or a mixture of both might also contribute to missdosage among inexperienced users which may lead to scenarios of increased anxiety and panic attacks. Therefore, novel pre-clinical studies with MDA under therapeutic/recreational doses are encouraged not only to support medical assistance in cases of intoxication but to potentially bridge its use in assisted psychotherapy. Acknowledgments: None.

MDA in silico toxicity assessment suggests low hepatotoxicity and calls for further pre-clinical investigations

oliveira, arThur liMa1; MiranDa, raul ghiralDelli2; DorTa, Daniel junqueira1

1 Universidade de São Paulo (USP), Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Química, Ribeirão Preto, SP, Brazil; 2 University of São Paulo (USP),

Faculty of Pharmaceutical Science of Ribeirão Preto, Ribeirão Preto, SP, Brazil.

157

Introduction: 3,4-methylenedioxymethamphetamine (MDMA) is an entactogen widely used in music fes-tivals owing to its effects of increased compassion and acceptance for oneself and others, interpersonal closeness and changes in emotions recognition. How-ever, MDMA is thought to cause drug-induced liver injury (DILI) and numerous reports of intoxications and fatalities related to ecstasy tablets containing MDMA have arisen in the past couple of decades. In contrast, no clinically relevant adverse events were found in controlled administration of the drug, which raises the hypothesis of toxicity induced by second-ary events such as hyperthemia, lack of attention to somatic cues, hyponatremia, pre-existent cardiac and metabolic disorders. Set and setting are also funda-mental to assess whether the consumption scenario may offer risks of adverse effects, since most of them include uncontrolled environments associated with vigorous exercise, poly-drug use and lack of hydra-tion. Nevertheless, many studies have linked the ad-verse effects of MDMA alone to a net of events that do not necessarily owe to its pharmacological prop-erties. Objective: The aim of this study was to assess the toxicity of MDMA in silico as a mean of predicting its toxicity in vivo. Methods: Toxicity assessment was run through statistical and machine learning-based IS-TOXTM platform (Altox Ltda, funded by FAPESP pro-cess number 2016/08322-5) under the following soft-wares: iS-LiverTM, to predict in vitro and in vivo hepa-totoxicity; Acute-ToxTM for acute toxicity; DevTox-IsTM, for developmental toxicity prediction; Pred-CYP2DTM, for metabolites prediction; Pred-OralTM, for bioavail-ability and permeability prediction; and lastly Geno-tox-IsTM, to predict genotoxicity. Results: Assessment of the molecule has returned structural alerts regard-ing potential DILI and action as agonist of the Aryl Hydrocarbon Receptor (AhR). The cytotoxicity assay using HepG2 cells predicted a weak-moderate toxic-

ity (above 9,4 mg/L). MDMA was classified as hepa-totoxic, producing DILI in both rodent and human. Its main metabolite, MDA, was also considered hepato-toxic and weighed in the final toxicity result. In terms of metabolism, MDMA is a substrate of the following CYP (cytochrome P450) isoforms 2B6, 1A2, 3A4, 2C19, 2E1, 2A6, 2C9 and 2C8; acting as an inhibitor of the iso-forms 2B6, 2C19, 2D6, 1A2 and 3A4. The assessment of developmental and reproductive toxicity predicted MDMA as toxic in vivo. High permeability was predict-ed using Caco-2 cells (11,2 x 10-6 cm/s) and Paralell Artificial Membrane Permeability (PAMP) was also high (11,9 x 10-6 cm/s). Rat oral acute toxicity was esti-mated in 179 mg/kg (OECD Category 3). MDMA was not considered genotoxic, which is consistent with exper-imental findings. Discussion and Conclusion: Based on the toxic concentration prediction MDMA does not seem to represent serious health risks under the common recreational dose (100-125 mg) which would result in a peak plasmatic concentration of around 225 ng/mL, unlikely to produce liver injury. This find-ing, nevertheless, contradicts clinical reports of DILI after MDMA intake and studies using animal models, which might be explained by difficulties in translation of the results obtained in pre-clinical studies owing to its non-linear pharmacokinetics. Additionally, as sup-ported by the predictions obtained there seem to be factors influencing intoxications and fatalities – that were not considered in silico –, other than MDMA’s in-trinsic pharmacology which could be better assessed mimicking the real consumption pattern, to raise awareness on how environmental and secondary so-matic features might affect adverse outcomes. In this context, designing experiments to better mimic rec-reational use with models that recreate human phys-iology settings such as 3D cell culture might present itself as an alternative to increase translational relia-bility. Acknowledgments: None.

MDMA in silico toxicity analysis reinforces need of mimicking consumption patterns

towards translational reliability

oliveira, arThur liMa1; MiranDa, raul ghiralDelli2; DorTa, Daniel junqueira1

1 Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo; 2 University of São Paulo (USP), Faculty of

Pharmaceutical Science of Ribeirão Preto, Ribeirão Preto, SP, Brazil.

158

Introduction: New psychoactive substances (NPS) have similar effects to “classic” drugs, such as cocaine, “ecstasy”, THC and lysergic acid diethylamide (LSD), and they have become a global phenomenon over the last decade. Up to August 2020, over 1000 individual NPS have been reported to the United Nations Office for Drugs and Crime (UNODC) Early Warning Advisory. Brazil reported the identification of 116 NPS, being stimulants and classic hallucinogens the main groups. In Rio de Janeiro State (RJ/Brazil), NPS sold as blotter papers were mostly related to phenethylamine (NBOMe and NBOH families) between 2006 and 2019. The lack of knowledge about the side effects and toxicity of NPS, makes its use a risk to society, representing a public health problem. Objective: The goal of this work is to conduct a retrospective study of seized materials containing NPS, analyzed by the Police Civil of the Rio de Janeiro state (Brazil) during the COVID-19 pandemic, between March 2020 and February 2022. Methods: 38 drug samples representing a total of 3164 blotter or infused papers obtained from different regions of the Rio de Janeiro state were analyzed by gas chromatography coupled with mass spectrometry (GC-MS), high-resolution mass spectrometry (Orbitrap-HRMS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Results: From the data obtained, synthetic cannabinoids (SC) were the predominant group in this study with 5 different substances identified in the samples. Among these, ADB-BUTINACA compound was the main constituent, discovered in 37.1 % of the infused papers seized in the Rio de Janeiro state. Two of the evaluated samples presented both MDMB-4en-PINACA and ADB-BUTINACA. In 2021, the ADB-4en-PINACA and 4F-ABUTINACA were identified for the first time in 0.3% and 1.3% of the materials seized, respectively. These substances are already

controlled in the country, through Ordinance SVS/MS no 344, of May 12, 1998, being framed in Structure B10 of the structural classes of SC. During the COVID-19 pandemic, phenethylamines were the second group with the highest number of seizures (n=13). The NBOHs (25I-NBOH, 25C-NBOH, 25B-NBOH and 25E-NBOH) were discovered in 373 blotter papers (12.9% of the materials seized). The 25E-NBOH was the most frequent derivative in these periods (9.8%). A fentanyl analog (furanylfentanyl or an isomer thereof) was observed in a single seized with 35 blotter papers in the Green Coast region in 2020. In this study, synthetic cathinones were not identified. Discussion/Conclusion: The market for illicit synthetic drugs remains complex and very dynamic, although it goes through periods of innovation and stagnation. During the COVID-19 pandemic, new substances continued to emerge; especially substances from the SC group seized in prisons in the metropolitan region of the state of RJ. Prior to the pandemic period, there were only one report of the seizure of SC. Quite remarkably, the second most frequently encountered compounds belonged to the class of phenethylamines. The ongoing COVID-19 pandemic and related response measures have affected existing synthetic drug markets by promoting changes in the types of synthetic drugs seized. Mapping trafficked drugs is essential because the information obtained from monitoring drug seizures is important to alert the judicial authorities and to help the legislative effort to ban NPS and promote the updating of analytical methods by forensic laboratories. Acknowledgments: Carlos Chagas Filho Foundation for Supporting Research in the State of Rio de Janeiro (FAPERJ) and National Council for Scientific and Technological Development (CNPq).

Profile of new psychoactive substances (NPS) in seized materials analyzed in the Rio de

Janeiro State during the COVID-19 pandemic

oliveira, aDriana SouSa1; alMeiDa, FernanDo g.1; Bhering, cecília De a.2; anTonio, ananDa Da S.2; Wurzler, gleicielle T.2; coSTa, gaBriela vanini2

1 ICCE - Instituto de Criminalística Carlos Éboli/PCERJ, Rio de Janeiro, RJ, Brasil, 20060-050; 2 UFRJ - Universidade Federal do Rio de Janeiro, Instituto de Química,

NAF – LADETEC / IQ – UFRJ, Rio de Janeiro, RJ, Brasil, 21941-909.

159

Introduction: The use of drugs of abuse is a global risk factor for disability and premature deaths, accounting for over 585,000 lives lost prematurely each year. Previous studies estimate that the national prevalence of illicit drug use is approximately 10%, and alcohol is, directly or indirectly, the drug most associated with the risk of death. Although the health harms of drug of abuse are well researched, studies are mostly concerned with the effects of specific substances, with limited attention to the concomitant use of drugs such as alcohol and cocaine. Estimating the prevalence and morbimortality associated with the use of multiple drugs of abuse is important to quantify the extent and severity of drug use and to refocus public health interventions. Objective: Describe the epidemiological profile of users intoxicated by multiple drugs of abuse associated with death registered at the Assistance and Toxicological Information Center of Santa Catarina (CIATox/SC) and identify the specific drugs most frequently involved in the deaths. Method: Observational, cross-sectional, descriptive and retrospective study of a series of cases registered between 2014 to 2021 at CIATox/SC. The data were obtained from the attendance records of cases of intoxication by drugs of abuse with death outcome related to the event. The following variables were evaluated: sex, age group, agent, route, type of exposure, and initial classification. Results: During the period of the study, 96 cases of death associated with drug abuse were registered, 44 deaths (45.8%) occurred by the use of a single type of drug of abuse, 18 deaths (18.75%) were caused by the use of multiple drugs of abuse, 20 deaths (20.8%) occurred by the association of drugs of abuse and medicines, 12 deaths (12.5%) by the combination of drugs of abuse and pesticides and 2 deaths (2.08%) by the concomitant

use of drugs of abuse, medicines and pesticides. From the majority of the deaths related with a single type of drug, cocaine/crack was the main drug of abuse consumed (86.36%), followed by alcohol (7.89%) and N-Methyl-3,4-methylenedioxyamphetamine (MDMA) (7.89%). Among the deaths involving intoxication by multiple drugs of abuse, the use of cocaine was present in 16 cases (89%), and the combined use of cocaine and alcohol was the main association found, being present in 7 cases (38.8%). The profile of deaths from multiple drug abuse was predominantly male (66.6%) in the age between 20-29 years old (55.6%). As for the circumstance, all 18 cases were classified as substance abuse and were admitted to the health service with a severe condition due to acute exposure. The main route of exposure was nasal and oral, which occurred in 12 cases (66.6%). Conclusion: In the current study, cocaine/crack was the drug responsible for most deaths when consumed isolated or in combination with other substances. Poly-drug use occurs mainly by association of cocaine, alcohol, and/or marijuana, being related with the intention of enhancing the desired effects and reducing undesirable effects, such as withdrawal syndrome. Simultaneous use of cocaine with alcohol produces a new compound, cocaethylene, which is pharmacologically active and related to potentially unfavorable outcomes. In previous studies, the use of multiple drugs of abuse predominated among younger (<30 years old) male users. The pattern of drug usage in this study showed the same tendency. Keywords: illicit drugs; alcohol intoxication; drug overdose; epidemiology, drug abuse. Acknowledgments/conflict of interest: We thank CIATox/SC for providing data. The authors have no conflict of interest to declare.

Risk factors associated with deaths by multiple drugs of abuse intoxications

MeSSiaS, nayara caSagranDe1,2; Silva, STephanie SoareS1; reSener, MariSeTe canello1,2; alBino, Danielle BiBaS legaT1,2; BaroTTo, aDriana Mello1,2; SanToS, clauDia regina1,2,3; Marchioni, caMila1,2,3

1 Federal University of Santa Catarina (UFSC), Brazil; 2 Poison Control Center of Santa Catarina, Brazil; 3 Department of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

160

NBOMes, also known as “N-Bombs” or “N-Bomb” consist of substituted class 2C phenethylamine derivatives. These molecules are already cataloged by the Convention on Psychotropic Substances by UNODC, present in Schedule I of 1971. As well as LSD, it has potent hallucinogenic effects; its consumption has increased over the years, despite being a recently discovered substance and having adverse effects linked to its toxicity. This work targeted the use of in silico methods for investigating the biological and toxicological behavior of these substances. We employed the MetadrugTM software for calculating seventy-eight variables for 23 NBOMes. We simulated structural properties, CYP450 QSAR Models, Protein binding QSAR Models, ADME QSAR Models, the predicted therapeutic activity, and predicted toxic effects. This tool is part of the MetaBaseTM API to access the knowledge base behind all systems biology products of Clarivate Analytics (https://www.cortellislabs.com/page/?api=api-MB). We used multivariate analysis to evaluate the behavior of these substances. PCA (Principal Component Analysis) and HCA (Hierarchical Cluster Analysis) were used as unsupervised approaches. We performed Supervised learning through SIMCA (Soft Independent Modeling of Class Analogy). PCA and HCA results showed that NBOMes were divided into four main classes, according to the similarity in structures. SIMCA confirmed these classes. Different properties were found for each category, as follows: Class 1. Molecules: 2C-B, 2C-B-BZP, 2C-C, 2C-E, 2C-T-2, 2C-T-7, 2C-TFM. We found three variables with

higher modeling power are related to the prediction of therapeutic activity: potential activities against heart failure and Parkinson’s disease. They also showed antithrombotic activity. Class 2. Molecules: 4-MA-NBOMe, 4-MMA-NBOMe, 25-APB NBOMe. This class was modeled mainly for variables related to the prediction of therapeutic activity, showing potential for antidiabetic, anti-osteoporosis, and antithrombotic activities. Besides, CYP450 QSAR Models showed the potential to inhibit CYP1A2 at 10 uM or less. The potential for inducing epididymis toxicity was also found as high modeling power. Class 3. Molecules: 25B-NBOMe, 25C-NBOMe, 25H-NBOMe, 25N-NBOMe, 25T-NBOMe, 30C-NBOMe. This class showed high modeling power for potential activity against arthritis and heart failure. However, they have the potential for inducing carcinogenicity in rats and mice. Class 4. Molecules: 25D-NBOMe, 25E-NBOMe, 25G-NBOMe, 25P-NBOMe, 25T2-NBOMe, 25T4-NBOMe, 25T7-NBOMe. This group presented a potential for inducing cardiotoxicity. liver lipid accumulation, nephrotoxicity, epididymis toxicity. In conclusion, we have observed that even considering the same type of NPS; these substances presented different activities according to the structure. Particular attention should be given to those molecules which showed predicted therapeutic properties. We thank the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 465450/2014-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001) for financial support.

Study of NBOMes using MetadrugTM and Chemometrics

MarioTTo, lívia Salviano; Bruni, aline ThaiS

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

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New Psychoactive Substances (NPS) consist of structurally modified chemical compounds that intend to mimic the effects of the controlled classic substances besides circumventing prohibition. The growth of NPS can cause many challenges in understanding their behavior regarding detection, identification, and biological properties1. This work studies synthetic cannabimimetic (SCB) substances that aim to mimic the THC (Tetrahydrocannabinol) chemical structure regarding their metabolites. THC metabolism is mainly at the hepatic level. It involves phase I reactions: aliphatic hydroxylation, oxidation of alcohols to ketones and acids, β-oxidation, degradation of the pentyl side chain, epoxidation, decarboxylation, and conjugations. Given the large variety of reported structures, in silico tools can be an alternative to get information about the metabolism of SCBs2. SMILES structures were used to simulate the metabolites of 44 synthetic cannabinoids, using the iS-Tox® platform (In Silico Toxicology Platform) developed by the company Altox®3. The computational tool Pred-CYP2D was used. It combines artificial intelligence and is based on knowledge of models to predict the molecular metabolism network (MMN). We analyzed the metabolic reactions, as well as the formation rate and structures of the metabolites predicted. The three most likely metabolites for 44 cannabinoids were selected. We calculated information on molecular, distribution and toxicological properties using the Cambridge University Pkcsm platform, which results in 14 (fourteen) variables4. The molecules were divided into six different classes according to the structural similarities among the SCB. We evaluated the influence of the variables on the samples by multivariate tools. We used Principal Component Analysis – PCA as an unsupervised approach and

Soft Independent Modeling of Class Analogy – SIMCA to observe classification effectiveness5. PCA results showed there is some pattern among the structures. SIMCA evaluation confirmed this pattern but also showed that metabolites for different substances can present similar properties. The Blood-brain Barrier Permeability showed to be the most discriminant variable for discriminating the differences in structures. According to the results, we concluded that in silico tools could generate the metabolites for synthetic cannabinoids. It can help indicate experimental determinations. For the studied variables, our findings showed that they have a kind of pattern depending on the structures. Still, there is a need for an in-depth investigation of new variables using different software. WethankAltox®for the partnership and access to the platform, theBrazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 465450/2014-8) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, FinanceCode 001) for financial support.

REFERENCES1. United Nations. UNODC World Drug Report 2019. in.2. United Nations Office on Drugs and Crime. Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products. (2013). doi:10.18356/1e8e4f16-en.3. ALTOX. IS-TOX PLATFORM An artificial intelligence platform developed by the Brazilian company Altox will avoid using animals in tests. https://is-tox.com/publications-and-news/84-an-artificial-intelligence-platform-developed-by-the-brazilian-company-altox-will-avoid-using-animals-in-tests.4. Pires, D. E. V., Blundell, T. L. & Ascher, D. B. pkCSM: Predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. J. Med. Chem.58, 4066–4072 (2015).5. Ferreira, M. QUIMIOMETRIA. Conceitos, métodos e aplicações. (2015).

Study of synthetic cannabinoids regarding their metabolites: an in silico approach

caSTro, jaDe SiMõeS; roDrigueS, caio henrique pinke; Bruni, aline ThaiS

Departamento de Química, FFCLRP/USP-RP; INCT Forense/FFCLRP/USP-RP.

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Introduction: Methamphetamine is a stimulant substance widely used as a recreational drug, but also used medically for the treatment of attention deficit hyperactivity disorder and obesity. It is highly addictive and has established itself in mighty consumer markets like the United States. Surveillance of methamphetamine trade presented a real challenge due to easy to make synthetic routes with readily available over the counter (OTC) reagents which turns control very difficult. Most recently, UNODC reported continued growth in the supply of methamphetamine on East and Southeast Asia. Meanwhile, Afghanistan which has been a fertile ground for heroin production appears to have shifted to the methamphetamine business. This scenario of emerging consumer markets and new producers shine light onto Brazil, an avid market for drugs of abuse which has been notoriously distant from the methamphetamine commerce. The Brazilian Federal Police reported in 2020 that there have been few cases of methamphetamine since 2017 but showed worry due to two recent meth labs that were found that year inside Brazilian territory. The report also suggests other evidence that points to domestic production aiming the national market. One is that states were seizing small quantities of the drug in plastic bags which indicates it was packed for use. Also, the Federal Police seized methyl-2-phenyl acetoacetate, a precursor for phenyl-2-propanone (methamphetamine precursor). The Federal Police would not be able to indicate whether methamphetamine is being sold and used in the national drug scene, since the Federation states are responsible for handling the “street cases”. Therefore, it is relevant that the states study and publish their data to show new trends in Brazilian drug markets. Objective: Evaluate methamphetamine seizures in the Joinville region (Santa Catarina), from 2014 to 2021. Methods: Descriptive and retrospective study of a series of seizures between 2014-2021 at Polícia Científica de Santa Catarina (Joinville region). Data

was obtained from Sirsaelp (Sistema de Registro de Solicitações, atendimentos e emissão de Laudos Periciais) and processed on an Excel® spreadsheet. Results: During the observed period, there had been few cases of methamphetamine seizures. There were 4 cases in 2017, including a large seizure of 1108 pills, and 2 cases in 2018. Before (since 2014) or after these years, methamphetamine had been completely absent from police seizures in Joinville region. Then, since june 2021, methamphetamine seizures have been progressively increasing, totalizing 18 in 2021, an 800% increase in cases. While in 2017 and 2018 pills contained only methamphetamine, mixtures became very common in 2021. Substances detected with methamphetamine included caffeine, 3,4-Methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and even Alprazolam in one case. Discussion/Conclusion: The 2021 surge of methamphetamine cases in Joinville region points to a new trend in the regional drug market. Although there had been cases in the past, in 2017 and 2018, these were few; all seizures involved pills and no other substance was detected. In 2021, however, there were a sudden rise of cases involving methamphetamine, most being seizures of small amounts which possibly indicate use. Another difference in the recent scenery is that methamphetamine is being found in different forms, such as crystal and powders. It is also a novelty that it is being mixed with other drugs and adulterants. Particularly, the mixture of methamphetamine with classical ecstasy drugs like MDMA and MDA has become common. That is worrying as it exposes occasional users of ecstasy to a highly addictive drug. The variation in forms of presentation and mixtures corroborate with the evidence pointed in the federal police 2020 report that indicate a market being established in Brazil. Keywords: Methamphetamine; recreational drugs; illicit drugs seizures

Tracking methamphetamine surges in the Joinville region (Santa

Catarina) from 2014 to 2021

joSé, guSTavo pinheiro coelho1; pericolo, Suellen1; paraBocz, giSele chiBinSki1; coSTa, karina oliveira1; Marchioni, caMila2; raMoS, Silvia apareciDa3

1 Polícia Científica de Santa Catarina; 2 Universidade Federal de Santa Catarina; 3 Universidade da Região de Joinville.

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Introduction: The World Health Organization currently estimates that about 300 million people suffer from depression and about a third of the population does not respond appropriately to at least three different antidepressants. The available antidepressants currently possess profile of effectiveness and similar mechanisms of action, act in the modulation of monoamino the cerebral ones and lead up to 2 weeks to start to be efficient. The psychedelic medicines as psilocibina, dietilamida of acid LSD (LSD) and ayahuasca, induce modified states of conscience acting in the receivers 5-HT2A of the brain, beyond the 3,4-metilenodioximetanfetamina (MDMA) that it presents effect of “alteration in the mind” by means of different neurochemicals ways, given the number of works reporting on the effects of psychedelics as an aid in the treatment of anxiety and depression, we saw the need to produce this article. Objectives: To revise the advances of clinical studies on the effectiveness of the psicodélicos in the treatment of the depression and anxiety. Methodology: Descriptive narrative-type study of the literature, selected articles published in the last five years (2017-2022), the database used was PUBMED, with the following descriptors: LSD, anxiety, data depression, ayahuasca, psilocybin, MDMA. The inclusion criteria for this study were clinical trials and

controlled clinical trials, in English and Portuguese. A total of 42 articles were found, 4 of which were used for this review and those that did not meet the inclusion criteria were excluded. Results: According to the study by Bershad et al (2020), the effects of micro-dose LSD increases amygdala connectivity with other brain areas correlated with positive mood after the drug. However, psilocybin, another studied psychedelic, has positive, lasting and faster effects compared to existing pharmacological therapies and with fewer side effects. Palhano-Fontes et al., states that ayuhasca, when compared to placebo, has a greater antidepressant effect and a high response to treatment with only a single dose of the psychedelic and low adverse effects. MDMA, on the other hand, was well tolerated in all sessions and brought significant improvement compared to participants who ingested the placebo, but when comparing MDMA to other psychedelic therapies, it has greater side effects and can last up to 7 days. after use. Conclusion: It is concluded that psychedelic therapy (PD) can bring benefits to patients with anxiety disorder and depression refractory to other unsuccessful pharmacological therapies. More clinical trials with larger samples and a diverse population should be carried out to better assess the effects of PD.

Use of psychedelics in the treatment of anxiety and depression disorder - literature review

Silva, luíza MaDureira1; holanDa, Wiron piMenTel2; honório júnior, joSé eDuarDo riBeiro3

1 Academic of Nursing in the University Center Christus, Laboratory of Neurociência Translacional-Neurocit; 2 Academic of Biomedicina in the University Center Christus, Laboratory of Neurociência

Translacional-Neurocit; 3 Doctor in Biotechnology for the RENORBIO/UFC. Professor in the University Center Christus, Coordinator of the Laboratory of Neurociência Translacional-Neurocit.

05 ENVIRONMENTAL

SAFETY AND HEALTH

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Introduction: Over the past decades, the zebrafish (Danio rerio) has emerged as a cost-efficient model to be used in toxicological assays which may support risk assessment of environmental contaminants. Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is an herbicide commonly used in Brazil, particularly in sugar cane cultivation. Following application, diuron is degraded in the environment to some metabolites, including DCA (3,4-dicloroaniline) and DCPMU (3-(3,4-dichlorophenyl-1-methylurea). There are studies with rodents reporting the hazardous potential of these substances, but few studies have shown their toxicity to aquatic organisms. However, changes induced by pollutants in fish are among the most sensitive indicators of environmental disturbances. Objective: To assess the potential for inducing harmful alterations, embryos, larvae and adult male zebrafish were exposed to a doses’ range of Diuron, DCA or DCPMU from 0.5 to 100 µM, which included Brazilian environmentally relevant concentrations. Methodology: This study was approved by the local Ethics Committee on the Use of Animals (CEUA, Certificate No. 1304/2019). Embryos and larvae were generated at this laboratory; adult zebrafish were supplied by an external breeder (Powerfish pisciculture, Itaguaí/RJ, Brasil). Embryo’s evaluation followed the OECD 236 protocol “Fish Embryo Acute

Toxicity (FET) Test” slightly modified: instead of a 96-hour post fertilization (hpf) evaluation period, the study was extended up to 144 hpf. For evaluation of adult animals, the OECD 203 “Fish Acute Toxicity (FET) Test” protocol was used. Results: At concentrations of 0.5 – 10 µM Diuron, DCA and DCPMU induced teratogenic effects in the zebrafish embryos. Exposures above 10µM were letal to embryos, larvae and adult animals. There were no conspicuous histological alterations in the brain, heart, spleen, and liver of adult zebrafish exposed to the different test chemicals for 96 hours. However, groups exposed to 10 µM of Diuron, DCA or DCPMU presented mild cytoplasmic vacuolization of hepatocytes. Discussion/Conclusion: Identification of adverse outcomes induced by any chemical agent is a first step for risk evaluation and regulatory decision. Our results indicate that during the early phases of their development (embryo and larvae) the zebrafish is susceptible to the toxic effects of Diuron, DCA or DCPMU. In the adult zebrafish tissues alterations were considered not relevant, but the meaning of hepatocyte vacuolation is under evaluation. The current data also indicate an age-dependent zebrafish susceptibility to diuron and its main metabolites. Keywords: Diuron, DCA, DCPMU, zebrafish, development, histological parameters. Acknowledgement: Financial support: FAPESP – Proc. No. 2017/2542-5; TOXICAM.

Acute toxicity of Diuron, DCA and DCPMU in the early life and adultohood

of the zebrafish (Danio rerio)

SaleS, Bianca caMargo penTeaDo1,2; anDraDe, íTalo BerToni lopeS1,2; peixoTo, paloMa viTória liMa1,2; caMargo, joão lauro viana1,2; pereira, lilian criSTina2,3

1 São Paulo State University (Unesp) Medical School, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM); 3 São Paulo

State University (Unesp) School of Agriculture, Botucatu, SP, Brazil.

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Background: Environmental exposure to mercury in preschool children can impact their health by increasing their blood levels, which can cause negative health outcomes. However, we do not have reference values for Brazilian children. Objective: This study aimed to set the reference value for blood mercury in preschool children from a megacity in Brazil, and investigating associated risk factors. Methods: Blood samples were collected of 2,463 children and the present study reports preliminary results from 1,020 1-4-year-old children attending 29 daycare centers (DCC) located in São Paulo, Brazil, 2013. Guardians answered sociodemographic and potential risk factors questionnaires. DCC and metal contaminated sites (MCS) were georeferenced (QGIS™). Blood total mercury levels (BTM) were determined by Cold Vapor Atomic Absorption Spectrophotometry (CV-AAS). BTM was dichotomized (low/high levels) in a cut-off point of 1.06 μg.L⁻1, which represents the 95th percentile reference value for total mercury established by the U.S. Centers for Disease Control and Prevention (CDC). Summary data and multiple logistic regression were performed (p<0.05). Results: The geometric mean (n=1,020) for BTM was 1.71 μg.L⁻1 (95%CI: 1.64-1.78 μg.L⁻1) and the 95th percentile was 5.43 μg.L⁻1 (95%CI: 4.89-6.26 μg.L⁻1). DCCs located in the Northwest

geographic zone were associated with high BTM when the model was adjusted for fish intake, age, sex, geographic zone, mother schooling, and amalgam (p=0.018). No associations were found between high BTM and fish consumption, amalgam teeth fillings, and the georeferenced vicinity of the DCC to MCS. We also concluded BTM analyses for 2,438 children (total sample) and we will present the complete statistical analyses for the whole sample at the XXII CBTOX Congress. For the total sample, the geometric mean for BTM was 1.87 μg.L⁻1 (95%CI: 1.82-1.92 μg.L⁻1) and the 95th percentile was 5.50 μg.L⁻1 (95%CI: 5.18-5.92 μg.L⁻1), results very similar to the partial analyses, which included multiple logistic regression. Conclusions: BTM in Brazilian preschoolers (95th percentile) was more than five times higher than U.S. children’s levels. Even the Brazilian geometric mean was higher than the U.S. BTM 95th percentile. More studies are necessary to identify potential mercury exposure sources for preschool children. Nevertheless, these results showed the need to formulate public health policies, intending to better understand and eliminate mercury sources. Acknowledgments: Funded by Fapesp (2011/13076-0, 2011/23272-0, 2012/21840-4, 2018/18391-0) and Capes.

Blood total mercury levels of preschool children from Sao Paulo, Brazil,

and associated risk factors

olyMpio, kelly poliDo kaneShiro1; SalleS, FernanDa junqueira1; pereira, elizeu chioDi1; oliveira, allan SanToS1; coSTa, eric auguSTo caravaggio2; coSTa, BrenDa naTaSha Souza3;

pereira, joão paulo goeS3; jeSuS, iracina Maura3; queiroz, ThaiS karolina liSBoa3; liMa, Marcelo De oliveira3; Silva, agneS SoareS4; carDoSo, Maria regina alveS5

1 Departamento de Saúde Ambiental, Faculdade de Saúde Pública, Universidade de São Paulo, FSP/USP; 2 Centro de Engenharia, Modelagem e Ciências Sociais Aplicadas, Universidade Federal do ABC, SECS/

UFABC; 3 Seção de Meio Ambiente, Instituto Evandro Chagas, SEAMB/IEC; 4 Communicable Diseases and Environmental Determinants of Health (CDE), Pan American Health Organization, PAHO/WHO;

5 Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, FSP/USP.

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Background: Pesticides are active substances potentially toxic to humans and their consumption and production have increased significantly. Therefore, monitoring exposure to these pesticides is extremely important for public health. As an alternative way to assess exposure to pyrethroid pesticides, wastewater-based epidemiology (WBE) is a tool capable of providing epidemiological information through the analysis of wastewater, through the quantification of endogenous metabolism products. Commonly the sampling performed for WBE is single point collection, but to minimize the limitations of this type of sampling, the passive sampling, with Polar Organic Chemical Integrative Sampler (POCIS) is an attractive tool, with better cost-benefit, used to estimate exposure to the polar compounds of interest. Objective: Develop and validate analytical strategies to determine the concentration of biomarkers of pyrethroid pesticides exposure in wastewater using POCIS. Methods: The biomarkers of pyrethroid exposure monitored were 3-PBA, cis, and trans-DCCA. The POCIS constructed in-house was assembled in a sandwich format, with 200 mg of Oasis® HLB sorbent between polyester sulfone membranes. The sorbent was extracted with methanol, and after the methanol extract, it underwent solid phase extraction (EFS) using an ion-exchange cartridge (Oasis® MAX). The extracts were analyzed in liquid chromatography coupled to a triple quadrupole mass detector (LC-MS/MS), as an electrospray ionization source in negative mode. Collections took place every fortnight and in triplicate, from FEB/2020 to MAR/2021, at

a wastewater treatment plant in Novo Hamburgo, which serves about 5 thousand inhabitants. Results/Discussion: A method for determining biomarkers of exposure to pyrethroid pesticides has been developed and is sensitive for detecting the compounds of interest. In total there were 24 collection cycles. POCIS proved to be efficient for collecting the compounds of interest, being possible to detect them in all collections. The amount of residual sorbent ranged from 151-195 mg, which represents a maximum of 24.5% loss. The EFS protocol was efficient, allowing high recoveries, ranging from 95.7-100.4% with variation between replicates of 1.9-9.6%. The results obtained in ng g-1 of POCIS for 3-PBA, cis-DCCA, and trans-DCCA, were in the range of 208.6-2456.9, < Limit of quantification-331.3 and 57-494,4, respectively. To define the sampling rate, an experiment that assesses the decay of the substance was used, and only for 3-PBA was it possible to determine it. The sampling rate for 3-PBA was 0.096 L day-1, so the values found in sewage in ng L-1 ranged from 24.3 to 335.2. Conclusion: In all cycles, it was possible to detect the presence of the analytes of interest, thus showing the sensitivity of the method developed, as well as proving the efficiency of the POCIS sampling device, assembled in-house. The fact that we were only able to define the sample rate for 3-PBA does not represent a limitation, as this marker represents at least 20 different pyrethroids. To our knowledge, there is no other study that determines biomarkers of exposure to pyrethroids using POCIS sampling. Acknowledments: Feevale University, CAPES

Determination of biomarkers of exposure to pythroid pesticides through

wastewater-based epidemiology

lizoT, lilian De liMa FelTraco; BaSTiani, MarcoS Frank; hahn, roBerTa zilleS; linDen, raFael

Feevale University.

168

Background/Introduction: Genistein is a phytoestrogen, which is structurally similar to 17β-estradiol. It is present in plants, food, and as a contaminant in effluents. Danio rerio fish, known as zebrafish, are small vertebrate, teleost fish of freshwater. They were chosen as a model organism in this work due to their rapid development, ease of maintenance, availability of acquisition and applicability. Objective: to demonstrate the effects of embryonic exposure to three different concentrations of genistein (10 μg/L, 40 μg/L, and 80 μg/L) using the zebrafish model. Methods: Zebrafish eggs were exposed to genistein (10 μg/L, 40 μg/L, and 80 μg/L) during the first 72 h post fertilization (hpf). Heart rate was evaluated at 48 hpf and mortality rate was assessed during the first 72 hpf. The Light/Dark (LDT) and Open Field (OFT) behavioral tests were applied to the larvae (6 dpf), and the Novel Tank (NTT), Social Preference (SPT), Light/dark (LDT), and sexing tests were performed on adult fish (90 dpf). This study was approved by the Animal Use Ethics Committee

(CEUA) of the University of Passo Fundo, UPF, Passo Fundo, RS, Brazil (protocol n°: 041/2019). Results: Embryonic exposure to genistein causes anxiolytic-like behavior in larvae. These effects are persistent, since were also observed when fish reach adulthood. Adult fish also presented hypermotility and antisocial behavior in the concentration of 40 μg/L. There was an increase in the mortality rate in all concentrations when compared to the control and an increase in heart rate at the concentration of 80 μg/L. Exposure to 10 μg/L generated a higher frequency of females when compared to the control group. Discussion/Conclusion: Our results show that exposure to genistein during the embryonic phase provoke damage in the short and long term as it increases the mortality rate and leads to behavioral disorders both in the larval stage, with persistence until adult stage. Acknowledgements: This study was supported by Postgraduate Support Program for Community Higher Education Institutions - PROSUC, attached to Ordinance No. 149, of August 1, 2017.

Embryonic exposure to genistein induces persistent anxiolytic and antisocial behaviors in zebrafish

FreDDo, naTália1; MenegaSSo, aloMa SanTin1; SoareS, Suelen MenDonça2; ForTuna, Milena2; MaFFi, vicToria coSTa3; MozzaTo, MaTeuS TiMBola3; BarcelloS, leonarDo joSé gil1,2,3; roSSaTo-granDo, luciana grazzioTin1

1 Programa de Pós-graduação em Bioexperimentação, Universidade de Passo Fundo, Passo Fundo, Rio Grande do Sul, Brazil; 2 Programa de Pós-Graduação em Farmacologia,

Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil; 3 Curso de Medicina Veterinária, Universidade de Passo Fundo, Passo Fundo, Rio Grande do Sul, Brazil.

169

Population growth demands food production increase, which has resulted in an exponential increase in the world’s consumption of pesticides. Among the most used pesticides worldwide are glyphosate and imidacloprid. Assessments of the sublethal effects resulting from exposure to these pesticides are generally carried out in experiments with animal models, and the results are often used in extrapolations relating to human health. Poecilia reticulata is an easy-to-handle fish, whose characteristics favor research on such themes. Thus, the aim of this work was to evaluate changes in energetic metabolism (EM) and antioxidant system (AS) of P. reticulata after chronic exposure to these pesticides. Twelve 10 L aquariums containing 10 fish each were divided into groups: Control (dechlorinated water) and three groups, each treated with Glyphosate (G), Imidacloprid (I), and Glyphosate+Imidacloprid (GI). The animals were kept at a temperature of 25±2°C, constant aeration, and a photoperiod of 12h/12h. We exposed these animals for 6 months (September 2020 to February 2021). The muscle from 8 animals in each group was collected and prepared for enzymatic analyses (Hexokinase-HK, Malate Dehydrogenase-MDH, Aspartate Transaminase- AST, Creatine kinase-CK.MB, Pyruvate Kinase-PK, Lactate Dehydrogenase-LDH, Citrate Synthase-CS) and AS (Glutathione peroxidase-GPx, Glutathione reductase-GR, Glutathione transferase-GST, Thiols). The variable matrices were analyzed using principal component analysis (PCA), and the factor loadings of the first dimension of both matrices were related through linear models for each experimental group. In all statistical tests, the significance level used was 0.05. In relation to the control group, homeostasis of the antioxidant system and balance of energy metabolism and ATP synthesis were observed. The group exposed to imidacloprid showed a greater imbalance in

antioxidant system, and greater consumption of ATP. The group exposed to glyphosate also showed a greater imbalance in its antioxidant system but resulting in a lower production of ATP. In the GI group we observed a marked production of ATP, indicating a metabolic alteration with increased activity of LDH, CS, and MDH. Some studies have reported neurological and behavioral effects in fish after exposure to pesticides. Imidacloprid interacts with nicotinic ACh receptors, preventing ACh binding, causing their constant stimulation, and constant consumption of ATP (Almeida et al., 2021). In addition to the high demand for ATP, studies have shown that imidacloprid can accumulate in muscle tissues (Frew et al., 2018). In natural conditions, the change in cholinesterase activity and constant muscle contraction lead the fish to fatigue, making them vulnerable to predation and, therefore, with less behavioral ability. As for the group exposed to glyphosate, this xenobiotic binds to membrane receptors, which activate messengers that promote the release of Ca++ by the endoplasmic reticulum, thus altering mitochondrial activity (Cavalli et al., 2013). Carrée et al. (2021) showed that rainbow trout exposed to glyphosate had significant increase in the LDH, and this result may indicate a need to produce more energy via anabolism rather than aerobic metabolism. Finally, the analysis of the energy metabolism and antioxidant system of fish exposed to sublethal doses of pesticides alone or in synergism showed a reduction in antioxidant capacity and alteration in ATP synthesis, thus promoting greater vulnerability of the animal to predation events and decrease of survival potential. In this way, we emphasize the importance of pesticide risk analysis, environmental impact assessment, and, mainly, an inspection of the misuse of such products. Keywords: guppy; xenobiotics; iseticide; herbicide. Acknowledgments: CNPq and FAPEMIG.

Energetic metabolism and antioxidant system modulation after fish chronic

exposure to glyphosate and imidacloprid

SoBjak, ThaíS Maylin1; zazula, MaTheuS Felipe2; Macarini, leanna caMila3; rizzo, elizeTe1; guiMarãeS, ana Tereza BiTTencourT⁴

1 Departmento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 2 Departamento de Biologia Celular,

Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Brazil; 3 Programa de Pós Graduação em Conservação e Manejo de Recursos Naturais, Universidade Estadual

do Oeste do Paraná, Cascavel, Paraná, Brazil; ⁴ Programa de Pós Graduação em Biociências e Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, Paraná, Brazil .

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Introduction: Racism is a structuring system capable of promoting avoidable and unjust disparities between social groups, based on ethnicity or race. In Brazil, ethnic-racial inequalities exist in virtually all fields, obstructing access to goods, services and opportunities, including health services. Studies in other countries report ethnic-racial disparity in drug poisoning and drugs of abuse, but studies evaluating other toxic agents (such as pesticides) are lacking. Objective: The present study aimed to examine the relationship between race/ethnicity and general exogenous intoxications, intoxication by medicaments, by pesticides and by drugs of abuse in different Brazilian states. Methodology: An ecological study was carried out based on the data available in the Notifiable Diseases Information System (SINAN) and population data from the IBGE, for the year 2017. Data on the notification of exogenous intoxications in general and by toxic agents were collected: medicaments, pesticides (for agricultural use, domestic use, public health, rodenticides and veterinary products) and drugs of abuse, in all Brazilian states. Data were divided into: white and non-white people (black, brown and indigenous), in addition to ignored skin color. The percentages of notifications with unknown skin color were recorded and the ratio between the percentage of intoxications of whites and non-whites and the population percentage of each of the two groups was calculated. Values above 1 for the intoxication/population ratio among non-whites indicated racial-ethnic disparity. Results: A high percentage of unknown race/skin color was found in Amazonas, Federal District and in states in the Northeast and Southeast regions. Regarding the ethnic-racial disparity measured by the

ratio between the percentage of general intoxications and population data among non-whites, there was greater disparity in the states of Pernambuco (ratio equal to 1.5), followed by Ceará, Rio Grande do Norte and Paraíba (all with ratio equal to 1.4). In addition, the type of intoxication that presented the highest data was related to drugs of abuse, with a disparity of 1.6 in the Federal District and Paraíba. In general, in Brazil, racial disparities due to medications and pesticides are almost non-existent. Conclusion and Discussion: Racial inequality is a consequence of a political and historical process, varying according to the construction of society. This whole process made racism persist and structure itself at different levels. This condition affects blacks, brown people and indigenous in a critical way and makes them more likely to suffer from health problems, including exogenous intoxications. This inequality imposes worse living conditions, lower wages, low education and less access to knowledge, increasing vulnerability to intoxication scenarios. Among the toxic agents investigated, drugs of abuse have the most expressive rates of ethnic-racial disparity. In the country, non-white families are known to be more vulnerable and with people more subject to drug abuse. Regarding the spatial distribution of ethnic-racial disparity, the states of the North and Northeast regions stood out with the highest rates. In these regions, there is a higher percentage of non-white population when compared to the states of the South and Southeast. Finally, it should be noted that this type of study lacks completely accurate data due to underreporting and poor completion of notification forms. This scenario is partially revealed in the study by the high percentage of unreported skin color in several states.

Ethnic-racial disparity in exogenous intoxications in Brazil

MoraeS, niely galeão Da roSa; Silva júnior, Flavio Manoel roDrigueS

Universidade Federal do Rio Grande - FURG.

171

Background/Introduction: Mining is one of the most traditional economic activities carried out in Brazil. The state of Minas Gerais (MG) extracts more than 160 million tons of iron ore per year. Vale is the world’s largest company producer of iron minerals, with 133 dams in the country (90 of tailings) whereby 80% are in Minas Gerais. On January 25, 2019, a rupture in one of the ore tailings dams (B1), belonging to Córrego do Feijão Mine in Brumadinho - MG, resulted in 270 deaths. The mud reached the company’s areas, neighboring communities, and a drainage channel of the Ferro-Carvão stream, affecting the Paraopeba River, responsible for water supply to several Minas State municipalities. Objective: This study aimed to evaluate the concentration of toxic elements in several water samples collected from the Paraopeba River in Brumadinho – MG a few days after the Dam failure. Moreover, a toxicity test was performed on root cells of Allium cepa in order to evaluate water quality. Methods: Altogether, 136 samples from seventeen areas were collected in the present

study. pH, temperature, electrical conductivity, and dissolved oxygen were measured in loco using a meter containing multiparameter probes. The determination of chemical elements in the samples was carried out using an inductively coupled plasma mass spectrometer (ICP-MS). Results: All parameters evaluated in loco showed a significant variation according to the location of selected collection points. Concentrations up to 1,000 times the maximum value allowed by the CONAMA Resolution nº 357 were found in some collection points for the toxic elements: Iron (Fe), arsenic (As), nickel (Ni), aluminum (Al), and lead (Pb). Discussion/Conclusion: The present study provides evidence that it is essential to monitor the water quality indices of the Paraopeba River to avoid human and animal exposure to toxic elements and impacts on the environment. It is also crucial to promote conditions to guarantee contamination reduction and the environmental recovery of the affected area. Acknowledgments: CAPES, FAPESP, CNPq.

Evaluating the levels of toxic elements and toxicity testing in water samples of Paraopeba

River after the Brumadinho dam rupture

Devóz, paula pícoli; rocha, cecília criSTina De Souza; BarBoSa jr, FernanDo

University of Sao Paulo – Faculty of Pharmaceutical Science of Ribeirao Preto, Ribeirão Preto - SP, Brazil.

172

Background/Introduction: Dye compounds have been used for many years in various industrial segments, mainly in the field of textile products. Their excessive use has been causing more and more damage to the environment, such as preventing the passage of solar radiation affecting living beings that inhabit aquatic ecosystems. One option to reduce the impact of these pollutants is phytoremediation, which consists of a process that uses plants to purify contaminated environments, preventing the pollutant from dispersing further. Some phytoremediation plants are already known, the species Salvinia Auriculata is one of them. In agriculture, it is common to use nutrient solutions to help the environment reproduction. Objective: The objective of this study is to prove the effect of a nutrient solution on the phytoremediation potential of the macrophyte Salvinia Auriculata. Methods: For this, a calibration curve of the dye was performed in the Spectra Max M5 equipment, at the wavelength of the blue dye, 570 nm, the calibration curve was made with 6 different concentrations, all in mg/l: 50, 100, 150, 250, 350 and 400. With the absorbance reading at these concentrations, we obtained the equation of the straight line y = 0.0005x + 0.0031 with r2 of 0.998. Experiment treatments were performed with a solution of water and dye at a concentration of 350 mg/l. A nutrient solution adapted from Hoagland-Arnon was prepared for 100 mL, in the composition (weighed on an analytical

balance): calcium nitrate (4.609 g), potassium nitrate (1.998 g), potassium phosphate (1.324 g), magnesium sulfate (2.719 g), sodium chloride (0.788 g), boric acid (0.116 g), manganese chloride (0.136 g), molybdic acid (0.130 g) and Iron EDTA (1.850 g). The experiment was divided into two groups and placed in a transparent plastic container: group A, containing 100 ml of water-dye solution and plant and group B, containing 100 ml of water-dye solution, plant and 1 ml of nutrient solution. Each group was done in quadruplicate. Collections were performed at 0, 2, 4, 8, 16, 24, 48, 72, 96 and 120 hours and the samples were stored in the refrigerator until the absorbance reading was taken. Results: At hour 4, the dye concentration in the solution had dropped to approximately 79% in both groups; from then on, it was noted that the concentration of group B began to decline faster than that of group A, reaching 26% at hour 48, while group A, at this same hour, had a dye concentration rate of 41%. In the last collection of the experiment, group A presented 35% of the initial concentration of dye, while group B, which contained the nutrient solution, presented only 19% of initial concentration. Discussion/Conclusion: It is therefore concluded that the best remediation strategy for dye is to combine phytoremediation techniques by the macrophyte Salvinia Auriculata with the use of nutrient solutions, such as Hoagland-Arnon. Acknowledgments: Capes, CNPq, Federal Universitty of Pampa.

Evaluation of the effect of a nutrient solution on the phytoremediation potential of a blue dye

by the aquatic macrophyte Salvinia auriculata

SchMiTT, Maria laura viDeiro1; carriço, Murilo ricarDo Sigal1; nogueira, caroline lacerDa1; roDrigueS, Marina Diaz2; Molina, higor Severo2; DenarDin, elTon luiS gaSparoTTo2; roehrS, raFael3

1 Student of the Postgraduate Program in Biochemistry, Federal University of Pampa, Campus Uruguaiana; 2 Pharmacy Student, Federal University of Pampa,

Campus Uruguaiana; 3 Professor, Federal University of Pampa.

173

Introduction: The contamination of aquatic ecosystems by mining activities can cause numerous human and ecosystem health. Periphytic diatoms have been used to monitor environmental disturbances associated with anthropogenic activities. Currently, environmental monitoring tools require complex and laborious metrics for ecological assessment and management. Diatoms are increasingly being evaluated using new non-traditional metrics of low-cost labor and primary taxonomic expertise. These new non-taxonomic approaches complement traditional metrics, making environmental assessments more sensitive, especially in cases of metal contamination. Objective: The present study assesses the response and resilience of periphytic diatoms’ cell size (cellular biovolume) to exposure to iron ore tailings. Methodology: The bioassay was conducted in isolated recirculating systems with replicates of control and treatments. The experiment was conducted in experimental rivers (ERs) colonized by periphyton from the lower Doce Doce River Valley (ES, Brazil). The periphytic community of the ERs was exposed to iron ore tailings from the Fundão dam, with initial turbidity of 1,200 NTU and a progressive reduction to less than 10 NTU. Changes in diatom cell

size were evaluated after 3 and 7 days of exposure to mining tailings, corresponding to short and long-term exposure. Results: There was a reduction in the average size of algae in both experiments. Compared to the control experiment, the initial phase of the treatment showed an increase in cell size variability and a sharp reduction after a short period of time. Initially, exposure to tailings caused, on average, an increase in the proportion of thicker cells and later, a more accentuated reduction in the proportion of narrower algae. Discussion and Conclusion: The subsequent reduction in variability in cell size modifies the composition of the diatom community, leading to the decline in species richness. These short-term responses to the disturbance can be related to the increasing mortality rates of sensitive algae to environmental stress. Combining these metrics reveals a high potential for evaluating management options in fluvial ecosystems. Combining the main elements of sensitivity of periphytic diatoms to environmental disturbances with the capacity to control key environmental variables in ERs bioassays allows for simulating ecological impacts and providing an early warning system for environmental management.

New approaches to periphytic diatoms as bioindicators of contamination by

iron ore tailings from the collapse of the Fundão dam in the Doce River (Brazil)

Souza, luiz clauDio cinDra1; reiS, luciane ayreS caSTro2; rangel, lara luiza piMenTa3; BarroSo, gilBerTo FonSeca4

1 Federal University of Espírito Santo; 2 Federal University of Espírito Santo; 3 Federal University of Espírito Santo; 4 Federal University of Espírito Santo.

174

Introduction: The health of aquatic ecosystems is one of the main concerns of this century. However, developing and refining tools to assess contamination and changes in aquatic ecosystems is a challenge. For cause-effect assessments of contaminants and biological communities, especially in highly dynamic ecosystems, such as a river, enclosed mesocosms can become the only reliable way to conduct investigations. Experimental approaches are also valuable for assessing environmental disasters associated with tailings spills, providing essential guidelines to prevent or, at least, mitigate environmental impacts. As a result, the use of bioassays has advanced rapidly since 90’s, with many well-documented applications. For running waters, Experimental Rivers (ERs) stand out for providing a better understanding of the factors of interest through controlling key variables, offering certain realism, and integrating ecological responses of different biological groups. The community level has been considered the most effective for ecotoxicological responses. In this sense, the use of periphyton stands out as a micro-ecosystem with critical ecological functions, among which it is the primary production in fluvial ecosystems. Combined with the short life cycle, easy development of bioassays, sensitivity response for environmental contaminants, and requiring little space, the approach of the Periphytic Bioindication in ERs (PBERs) is promising. Nevertheless, the main obstacle is the lack of procedural review and methodological protocols that promote its effectiveness. Objective: To systematize knowledge of ecotoxicological responses of periphyton in ERs and to propose key guidelines for PBERs. Methodology: The study was carried out in two stages: 1) a systematic literature review using an indexed database, Clarivate Web of ScienceTM, with pre-selected keywords associated with bioassays of PBERs, such as types of resources used (i.e., water, periphyton, sediment, light), and the community, population, and ecophysiological parameters analyzed; 2) organize a database and select key guidelines for the use of PBERs. Results and

Discussion: The survey yielded 116 scientific papers addressing ecotoxicological bioassays with PBERs. Although various experimental designs and metrics analyzed the capacity to simulate a certain degree of realism, even in indoor experiments, the main advantage is the realism simulation and control. Realism is the experiment setup mimicking the structure and dynamics of fluvial ecosystems. On the other hand, control refers to the ability to relate cause and effect, to manipulate, replicate, and repeat experiments with reliability. In general, ERs are primarily developed outdoors, limiting control variables. However, in PBERs, most (58%) of the bioassays are indoors, which despite the complexity of the periphytic community, the dimensions of ERs required for its development are small, and the simulation of the main variables limiting the periphyton development are relatively simple. As for the experimental design, 74% of the PBREs case studies were designed with water recirculation, with sufficient renewal to maintain the nutrients in the water (22%), or adding culture media (30%). In general, 110 anthropogenic stressors were investigated in the ecotoxicological assessment, with pesticides and fungicides being the most common (33%), followed by metals (18%). Most experiments (84%) were planned with treatment replicates with different contaminant concentrations as independent systems, although multiple stressors approach has also been investigated. The exposure of periphytic communities (over 7 days of colonization) ranged from 16 to 30 days. Very few studies (6%) investigated the resilience after the disturbance. In this review, 117 different periphyton metrics were found, grouped in terms of functional, structural, molecular, and physiological processes of the entire community structure of its biological groups, algae, protozoa, fungi, bacteria. Microalgae was the most studied group (85%). Conclusions: The PBERs bioassays review indicated a very diverse approach for ecotoxicological assessments. Guidelines and protocols for bioassays are needed to provide more concise and standardized results for environmental regulation.

Periphytic community in experimental rivers as a tool for the assessment of anthropogenic

stressors in fluvial ecosystems

reiS, luciane ayreS caSTro; alMeiDa, STeFano zorzal; BarroSo, gilBerTo FonSeca

Universidade Federal do Espírito Santo - UFES.

175

Background: Mining areas can result in a series of damages to the environment and also to human health. People who work in these areas, as well as the population that lives close to these places, are subject to pollution from these activities. Coal mining and processing can trigger pollution by different potentially harmful elements to the exposed population. Among these elements, lead (Pb) stands out due to its high toxicity, especially for children. Objective: Therefore, the aim of this study was to evaluate urinary Pb levels in schoolchildren living near a coal mining region. Methods: A cross-sectional study was conducted with 92 school-age children residing in 7 municipalities in southern Brazil. The municipalities evaluated in this study were Aceguá, Bagé, Herval, Hulha Negra, Pedras Altas, Pinheiro Machado and Candiota, with two coal mines and two coal-fired thermoelectric plants located in the latter. Demographic and socioeconomic data were collected using a semi-structured questionnaire. For the determination of urinary Pb levels, atomic absorption spectrophotometry in a graphite furnace (AAS-GF) was used and they were normalized from the creatinine concentration. A spatialization map was prepared to identify the sites that contained the samples with the highest urinary Pb levels. To assess the factors associated with urinary Pb levels was used Poisson regression with a robust estimator and based on the model theoretical. Results: Urinary Pb levels in the study population ranged between ~0 and 21.6 µg.g-1-creatinine and presented 50% and 95% percentiles of 3.50 and 11.13 µg.g-1-creatinine, respectively. The highest urinary Pb levels were found in samples from children living in Candiota and in areas closer to potentially polluting sources. In the Poisson regression, it was observed that some conditions such as mothers with less schooling, non-

white, living in Candiota, fathers with occupational exposure and underweight children were associated with high urinary Pb levels (p < 0.01). Discussion: Urinary Pb levels identified in Candiota are above the reference values available from the Centers for Disease Control and Prevention for the unexposed population. While in our study the 95% percentile was 11.13 µg.g-1-creatinine, the CDC value for the population in the same age group was 1.14 µg.g-1-creatinine. Our spatialization map showed that the samples with the highest urinary Pb levels are located in Candiota, as can be seen in other studies that evaluate the exposure of metals in mining areas. In addition, studies carried out in this region have already shown that Pb levels in soil samples are low/moderate, but increase as the samples approach the pollution sources and it was also seen that the Pb concentration in surface water samples was above limits recommended by Brazilian legislation. In the literature, it has already been seen that the concentration of Pb in the urine of children can reach 3.95 µg.g-1-creatinine in areas with recognized pollution from the activities of coal-fired thermoelectric plants. In addition, as in our study, it has already been observed that socioeconomic factors may be associated with high levels of Pb, such as age, parents who smoke, parents’ education, drinking water, family income and housing conditions. Conclusion: Therefore, we conclude that coal mining areas can have a great impact on high urinary Pb levels in children living close to these areas and that these levels can also be associated with mother’s educational level and skin color, father’s occupational exposure, and child’s weight. Acknowledgments: We thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) for funding our research.

Urinary lead levels and factors associated with their increase in schoolchildren living in a coal

mining area in the extreme south of Brazil

BruM, roDrigo De liMa1,2; BaiSch, ana luíza Muccillo1,2; Silva júnior, Flavio Manoel roDrigueS1,2

1 Laboratório de Ensaios Farmacológicos e Toxicológicos, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Brazil. 2 Programa de Pós-graduação em Ciências da

Saúde, Faculdade de Medicina, Universidade Federal do Rio Grande, Rio Grande, Brazil.

176

Introduction: Manganese (Mn) is an essential metal used for metallurgical industries purpose. Despite its importance, Mn is considered a potential source of contamination, which includes damage to human health, and compromise of aquatic ecosystem. In this context, Luzerna is the capital of metalworking in middle west of Santa Catarina State - Brazil in which 35.3% of workers have employment in the metalworking sector. Although, there is a lack of information of occupational or environmental metal exposure in the region. Objectives: Then, the purpose of this work was to evaluate the environmental impacts of metals and water quality from the main river of the city, named Rio do Peixe, which is used for water supply to several cities around. Methodology: To determine water quality physical-chemical and bacteriological parameters, water samples were analysed in duplicate from two sites of the river (non-contaminated site and contaminated site) using in situ instrumentals. Results: Water parameters in reference site showed as fallows: pH = 7.67 ± 0.04; temperature (ºC) = 27.0 ± 0,14; dissolved oxygen (mg/l) = 7.85 ± 0.07; electrical conductivity (μS/cm) = 106.5 ± 10,61 and refractive index = 0.0 ± 0.0. In exposure site the values were: pH = 7.36 ± 0.02;

temperature (ºC) = 26.9 ± 0.01; dissolved oxygen (mg/l) = 7.90 ± 0.28; electrical conductivity (μS/cm) = 103 ± 4.24 and refractive index=0.0 ± 0.0. Total and fecal coliform bacteria were found in both sites. Metal analysis are being conducted by atomic absorption spectrometry. Conclusion: Although our results are still preliminary and only include duplicate samples collected, it highlight the importance of monitoring the environmental, since the results found were so far similar between two sites of the river. In addition, in order to analyse the environmental impact of metals and occupational exposure, we are evaluating the concentration of manganese metal as a biomarker of occupational metalworking in the same region, using nails and hair from 60 participants (30 controls and 30 exposure group) as sample. These future results will make it possible to implement appropriate prevention and control measures to avoid the harmful consequences arising from the bioaccumulation of metals, mainly, the compromise of the respiratory and neurological system resulting from exposure to manganese. Acknowledgments: This research was made possible in part by a grant from Federal Catarinense Institute– IFC/Luzerna, FURB, UNOESC and in part by CAPES scholarship.

Water quality assessment of the Rio do Peixe: a preliminary study

geMelli, Bianca apareciDa MarTinS1; SuMMy, Maria julia1; ceSaro, huMBerTo luiS1; alveS, rôMulo couTo1; Saraiva, illyuShin zaak1; reMor, aline perTille2; Baú, Morgana2;

liMa, Daina3; alMeiDa, eDuarDo alveS4; Müller, gaBrielle Do aMaral e Silva1

1 Instituto Federal Catarinense - IFC; 2 Universidade do Oeste de Santa Catarina-UNOESC; 3 Universidade Federal de Santa Catarina – UFSC; 4 Universidade Regional de Blumenau- FURB.

06  EXPERIMENTAL

TOXICOLOGY

178

Introduction: The 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the components of the herbicide 2,4-D (DMA® 806) considered one of the most used herbicides in the world due to its low cost and good performance. This pesticide is an eye irritant and has been classified as extremely toxic to humans. In 2018, the International Agency for Research on Cancer (IARC) classified this herbicide as possibly carcinogenic to humans. Also, it has been classified as dangerous to the environment and slightly toxic to fish and aquatic invertebrates. However, in 2019, 2,4-D was classified as a low toxic product (class IV) by Global Harmonized System (GHS). Objective: In this context, the objective of this study was to elucidate the potential teratogenic and understand the toxic effects induced by the active ingredient, 2,4-dichlorophenoxyacetic acid, using embryonic stage of zebrafish (Danio rerio) as a model organism. Methods: Embryo’s evaluation followed Fish Embryo Toxicity (FET) Test n. 236 (OECD, 2013). After the fish reproduction, 140 fertilized eggs in the blastula stage were collected and placed in 24-well culture plates. Assays were performed in triplicate, and each replicate consisted of 24 embryos for the negative control group (ISO-7346 standard water), 20 embryos for the positive control group (4 mg/L 3,4-dichloroaniline) and 20 embryos for each concentration of 2,4-dichlorophenoxyacetic acid: 0.1; 0.4; 1; 10; 100 and 200 µM. These concentrations are realistic since that have been already found in the

environment. The culture plates were incubated at 26 ± 1 °C, in cycles of 14 hours light and 10 hours dark and the development of zebrafish embryos and larvae were evaluated at 8, 24, 48, 72, 96, 120 and 144 hpf (hours post fertilization) through of a stereomicroscope (Leica M205FA). Mortality, yolk sac edema, swim bladder inflation defects, pericardial edema and caudal fin deformation were analyzed. The values were submitted to the analysis of variance test (One-way ANOVA) and a posteriori Dunnet’s test. Results: After 144 hpf of exposure, the negative and positive controls resulted in compliance with the test (mortality ≤ 10% and mortality ≥ 30%, respectively). Regarding to mortality, there were significant (p ≤ 0.05) only in the concentrations 100 and 200 µM (144 hpf), with 100% embryo mortality. There were not differences about Yolk Sac Edema, Pericardial Edema, Non-Inflated Swim Bladder and Caudal Fin Deformity, between the treatments, resulting in a maximum value of 20% affected embryos in each deformity classification. Discussion and Conclusion: We can conclude that the active ingredient 2,4-dichlorophenoxyacetic acid, one of the components of the herbicide 2,4-D (DMA® 806) can cause more toxic effects than teratogenic effects in zebrafish embryos. Acknowledgments: Acknowledgments to Coordination for the Improvement of Higher Education Personnel - (CAPES) and TOXICAM.

Acute toxicity of 2,4-dichlorophenoxyacetic acid in the early life of the zebrafish (Danio rerio)

viriaTo, criSTina1,2; anDraDe, íTalo B.l.2,3; peixoTo, paloMa v.l.2,3; pereira, lílian c.2,4

1 São Paulo State University (Unesp), Institute of Biosciences, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM); 3 São Paulo State University (Unesp),

Medical School, Botucatu; 4 São Paulo State University (Unesp), School of Agriculture, Botucatu.

179

Introduction: Lead (Pb) poisoning is a public health problem, as it presents high toxicity to the body, acting on several biochemical targets, and the central nervous system is especially sensitive to damage caused by this metal. Some of the most important sources of environmental lead contamination include mining, smelting and, in some countries, the use of leaded paint, and leaded fuels. Oxidative stress (EO) is considered a possible molecular mechanism involved in Pb neurotoxicity. Objectives: Considering the vulnerability of brain structures to Pb, this study evaluate the effects of chronic Pb administration (16, 64 and 128 mg/kg) on the activity of antioxidant enzymes and on Na+K+-ATPase activity (128 mg/kg) in rat cerebral cortex and hippocampus. Methods: 60-day-old male Wistar rats received the following treatment via gavage for 35 days, separated in four groups: Group I: Saline Solution; group II: Pb 16 mg/Kg; group III: 64 mg/Kg and group IV: 128 mg/Kg. Rats were sacrificed by decapitation, without anesthesia, the brain was removed and the hippocampus and cerebral cortex were homogenized in 10 volumes of appropriate buffer. The following antioxidant enzymes were measured: catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and the activity of Na+K+ATPase, data were analyzed by one-way ANOVA followed by Duncan’s test when the F test was significant (p<0.05). Results: Chronic administration of lead acetate (128mg/kg) decreased SOD activity in the cerebral cortex [p<0.05]; and, at doses of 64mg/Kg and 128mg/Kg, decreased SOD activity in the hippocampus [p<0.001] of rats. With

regard to CAT activity, chronic administration of lead acetate (128mg/Kg), in the cerebral cortex [p<0.01] increased this enzyme’s activity, as compared to the control group. Regarding GSH-Px activity, chronic administration of lead acetate decreased this enzyme’s activity at a dose of 128mg/kg in the cerebral cortex [p<0.01]. However, CAT and GSH-Px activities in the hippocampus were not altered by chronic administration of Pb. With regard to Na+K+-ATPase activity, chronic administration of lead acetate (128mg/Kg) decreased its activity in the hippocampus. Discussion: The brain structures chosen for this study derive from the importance of their functions. Hippocampus is fundamental to process emotions and information associated with memory. The cerebral cortex has an overlap of functions related to several processes such as motor behavior, receiving information, coordinating complex movements, multisensory information processing and language comprehension. Results indicated that Pb causes significant alterations in the antioxidant enzymes, thus demonstrating an oxidative stress. Alterations were different in the analyzed cerebral regions, which suggest that the oxidative potential is different in these regions in relation to the susceptibility to Pb. Conclusion: Pb alters the activity of antioxidant enzymes, causing oxidative stress, and compromises the activity of Na+K+-ATPase, important for brain homeostasis, contributing to brain dysfunction caused by chronic exposure to Pb. Keywords: Lead exposure, oxidative stress, cerebrum, antioxidant enzymes

Alterations in the activity of antioxidant enzymes and NA+K+-ATPase in lead poisoning

in the cerebral cortex and hippocampus of rats

Ferrazza-heiTich, MagDa1; liMa, Daniela DelWing1,2; BorgMann, gaBriela1; plauTz, kaTherine1

1 Programa de Pós Graduação em Saúde e Meio Ambiente; 2 Departamento de Medicina - UNIVILLE, Joinville, SC, Brasil.

180

Introduction: Carvacrol is a phenolic monoterpenoid found in essential oils of oregano, thyme, pepper, wild bergamot and other plants, possessing a wide range of useful bioactivities for clinical applications such as antimicrobial, antioxidant, anti-inflammatory and anticancer activities. Due to its anti-inflammatory action, it is possible to make use of its functions in acute lung injury (ALI) induced by cigarette smoke, which causes a condition characterized by tissue damage and an increase in inflammatory markers. Objective: Investigate the pharmacological activity of carvacrol (CAR) in the acute lung injury induced by cigarette smoke in mice. Methods: Mice C57BL/6, male, (25-28g) were divided into two groups: control and cigarette smoke (CS). The CS group were exposed to 12 cigarettes per day for 5 days. The control group was exposed to sham smoking. The CS group was treated with CAR (1, 3 or 10 mg/mL) or vehicle by inhalation (15 min/daily) for 5 days. After 5 days bronchoalveolar lavage (BAL), trachea and lungs were collected for inflammatory profile, functional and morphological analysis. Significant difference when p<0.05. Results and discussion: The number of inflammatory cells increased in the BAL of the CS group when compared to control one (0.02 ± 0.01 vs 0.34 ±

0.03; p<0.001). The treatment with CAR 1, 3 and 10 mg/mL reduced this number (0.23 ± 0.02, p=0.02; 0.18 ± 0.01, p=0.002 and 0.010 ± 0.01, p=0.001, respectively). The treatment with CAR 10 mg/mL reduced the airway hyperresponsiveness in tracheal segments in the electro and pharmachomecanical coupling (p<0.05). The pulmonary parenchyma analysis showed an increase of the epithelium damage (76.3 ± 2.26 vs 112 ± 3.62 control group; p<0.001), lung inflammation (3.5 ± 0.17 vs 1.9 ± 0.21 control group; p<0.001) and bronchoconstriction index (4.66 ± 0.2 vs 2.99 ± 0.18 control group; p<0.05). CAR recovery epithelium damage with 1 mg/mL (91.9 ± 2.12 p<0.01) and 3 mg/mL (102.3 ± 2.51 p<0.0001). The lung inflammation was reduced by all the doses (1 mg/mL 2.25 ± 0.09; 3 mg/mL 2.40 ± 0.11 e 10 mg/mL 2.59 ± 0.08; p<0.001). The bronchoconstriction index was reduced in 1 mg/mL (3.79 ± 0.17; p<0.05) and 10 mg/mL (3.85 ± 0.16; p<0.05). Alveolar septal volume density (VvSept) and mean linear intercept (Lm) did not show any difference. Conclusion: Carvacrol has anti-inflammatory and bronchodilator activity without change in the lung parenchyma in the lung injury induced by cigarette smoke. Acknowledgments: UFERSA and UERN

Carvacrol and its anti-inflammatory effect under cigarette smoke-induced acute lung injury

Moura, Maria joana nogueira1,2; Silva, aline gaBrielle goMeS1,2, SanToS, caio ceSar araújo1; pereira, arTeMia kelly holanDa1; Felix, renaTa gleySiane De SouSa1; liMa, crySTiane calaDo3, golçalveS, a.p.3;

Melo, paolo oliveira4; Silva, gerlane MoDeSTo1; FeiToSa, eManuel keneDy1; BorgeS, ciBele DoS SanToS1

1 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-

Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM), State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande

do Norte, Brazil; 3 Higher Institute of Biomedical Sciences, State University of Ceará (UECE), Fortaleza, Brazil; 4 Heart Institute, University of São Paulo (USP), São Paulo, Brazil.

181

Introduction: Methylphenidate is the main treatment for Attention Deficit and Hyperactivity Disorder (ADHD) in children. It is believed that there is an overdiagnosis and consequently over treatment of this problem and many children end up using methylphenidate without real need. The consequences of this exposure are still unknown, thus, in this context, the zebrafish is a suitable animal model for studying neurobehavioral changes related to exposure to drugs in a translational perspective. Objective: we aimed to evaluate whether the chronic exposure to methylphenidate during juvenile period leads to behavioral changes regarding the response to a new environment in zebrafish during adulthood. Methods: we exposed juvenile zebrafish (n= 12) to methylphenidate 2 mg/L from the post-natal day (PND) 30 to PND 60. The treatment was added in the tank water twice a week. A non-exposed group (control, n=12) was kept

in the same maintenance conditions for comparisons. The Novel Tank test (NTT) was performed on during adulthood, on PND 120. Results: animals exposed to methylphenidate presented behavioral changes regarding the exploration of the novel environment, spending less time on the bottom of the tank and more time on the top zones compared to the controls. Discussion/Conclusion: The behavioral pattern observed here might indicate that animals presented decreased anxiety-like behavior. On a translational perspective, we can suggest that the decreased anxiety due to exposure to methylphenidate during childhood and adolescence might affect normal behavior during adulthood. Acknowledgements: Universidade de Passo Fundo for the financial support and Laboratório de Fisiologia de Peixes for providing the structure, materials and help during the study development.

Chronic exposure to methylphenidate during juvenile period alters

zebrafish behavior in adulthood

narDi, jeSSica1; FreDDo, naTália1; BiazuS, inara carBonera2; oliveira, ana paula2; SoareS, Suelen MenDonça3; ForTuna, Milena3; poMperMeier, aline1; Siqueira, liSiane1; cole, aManDa carolina3;

TaMagno, Wagner3; aMaral, Francieli uBirajara inDia1; coSTa, viToria caDore4; MozzaTo, MaTeuS TiMBola4; BarcelloS, leonarDo joSé gil1,3; granDo, luciana grazzioTin roSSaTo1

1 Programa de Pós-graduação em Bioexperimentação, Universidade de Passo Fundo, Passo Fundo, Rio Grande do Sul, Brazil; 2 Curso de Farmácia, Universidade de Passo Fundo,

Passo Fundo, Rio Grande do Sul, Brazil; 3 Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil; 4 Curso de

Medicina Veterinária, Universidade de Passo Fundo, Passo Fundo, Rio Grande do Sul, Brazil.

182

Introduction: Excessive ethanol consumption is a risk factor for cardiovascular disease (CVD), one of the leading causes of death and disability worldwide. However, the mechanisms underlying ethanol effects on the cardiovascular system are not fully understood. Considering that alterations of gut microbiota composition or gut microbiota-dependent metabolites are key to the development and progression of diseases, including CVD. The hypothesis of the present study is that excessive ethanol consumption alters the gut microbiota composition/function, leading to gut dysbiosis. In addition, whether ethanol-induced gut dysbiosis plays role in ethanol-induced vascular dysfunction is not comprehended. Objective: The goal of this study is to determine whether the gut microbiota plays a role on vascular dysfunction induced by excessive ethanol consumption. Methods: Male C57BL/6 mice were treated according to the modified National Institute on Alcohol Abuse and Alcoholism (NIAAA) model. Animals were randomized into control or ethanol groups. All mice from the ethanol groups were submitted to one-week-adaptation period with 5% (v/v) ethanol. Then, mice either received 1) 10% (v/v) ethanol in their drinking water for 10 day and binge feeding on day 11 with 5 g/kg body ethanol solution (T1) or 2) 10% (v/v) ethanol for 20 days combined with feeding on days 11 and 21 with higher doses of alcohol. Animals from the control groups received water. Ethanol levels were measured by gas chromatography. Abdominal aortic tissues were used to perform functional experiments.

Analysis of the gut microbiota was performed by high-throughput sequencing of the 16S rRNA gene in fecal pellets. Results: Similar ethanol levels were observed after the short (T1) and long (T2) treatments (T1 = 298.3±15.4 mg/dL, n=8; T2= 303.9±13.1, n=5). Conversely, changes in abdominal aorta reactivity were observed only after chronic ethanol feeding for 10 days plus a single binge dose of ethanol. In this treatment (T1), changes in reactivity were observed in abdominal aortic rings with intact endothelium and without perivascular adipose tissue (PVAT). Reduced maximal contraction to phenylephrine was observed in ethanol-treated mice versus the control group (Emax: Control= 181.1±4.9, n=7; Ethanol= 128.8±6.3, n=8). Chronic plus binge ethanol feeding led to the loss of the anticontractile effect of PVAT (Emax – PVAT (-): Control= 181.1±4.9, n=7; Ethanol= 128.8±6.3, n=8; Emax – PVAT (+): Control= 148.5±4.4, n=8; Ethanol= 116.2±6.2, n=10). No alterations were observed in acetylcholine- or sodium nitroprusside-induced relaxation. These results were accompanied by increased abundances of Firmicutes and Proteobacteria phyla. Conclusion: Ethanol-induced alterations in vascular reactivity was not related to ethanol levels. Negative effects of ethanol in the PVAT contribute to vascular dysfunction. Gut dysbiosis may be associated with vascular dysfunction induced by chronic plus binge ethanol feeding, but this remains to be tested. Keywords: Ethanol; Vascular dysfunction; Abdominal aorta; Gut dysbiosis. Financial support: FAPESP.

Chronic plus binge ethanol feeding synergistically induces abdominal aorta

dysfunction and gut dysbiosis in mice

Silva, carla Brigagão pacheco1; roDrigueS, vaneSSa FernanDeS1; coSTa, Bruno ruiz BranDão2; SarTori, Daniela carloS1; De MarTiniS, Bruno SpinoSa3; ToSTeS, riTa c.1

1 Ribeirão Preto Medical School; 2 School of Pharmaceutical Sciences of Ribeirão Preto; 3 Faculty of Philosophy, Sciences and Letters of Ribeirão

Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

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Background: The use of pesticides has increased, due to their significant role in the economic development of Brazilian agriculture. Pesticides are used on a large scale, as they guarantee crop productivity while preserving the species of interest. However, its use is also related to negative impacts on the environment and on human health, leading to ecological and toxicological issues when incorrectly used. Analyses on different organisms and exposure conditions are conducted to assess the hazard and risk that these compounds may induce. Some of the mechanisms by which xenobiotics can exert their cytotoxicity are mitochondrial pathways, as it is an organelle vital to the maintenance of several biological processes, such as energy balance and cell death. The study of isolated mitochondria can provide information about toxicity mechanisms induced by chemical compounds. Thus, this study aims to investigate the toxicity mechanisms by which 2,4-D, one of the most widely used herbicides in Brazilian agriculture, may be harmful to the liver mitochondria. For this purpose, the redox state of mitochondria isolated from the liver of young Wistar rats, was evaluated after in vitro exposure to 2,4-D and its isolated components, 2,4-dichlorophenoxyacetic acid (C8H6Cl2O3) and 2,4-D dimethylamine (C10H13Cl2NO3). Even though 2,4-D is one of the most studied chemicals, little is known about the toxicity mechanism of this herbicide, and its cytotoxic action remains unclear. Objective: To identify the toxic mechanism of 2,4-D and its isolated components in hepatic mitochondria by in vitro exposure. Methods: The exposure of mitochondria isolated from the liver of Wistar rats to 2,4-D

was carried out for ten minutes, in four different conditions: [1] the commercial formula (DMA 806 BR®); [2] acid component (2,4-dichlorophenoxyacetic acid); [3] amine salt component (2,4-D dimethylamine); and [4] mixture of the acid and salt components, emulating their commercial concentrations, thus free from excipients and other components found in DMA 806 BR. Concentrations ranging from 0.1 to 200 µM were used, as these comprises the concentrations found in the environment. So far, the production and accumulation of Reactive Oxygen and Nitrogen Species (RONS) using the H2DCFDA probe and the redox state of pyridine nucleotides based on their auto-fluorescence has been assessed. The results were evaluated by analysis of variance (ANOVA) followed by the Dunnett test, assuming a significant difference when p ≤ 0.05. Results: The assays showed that, at the concentrations evaluated, the generation of RONS was not detected in any of the four different conditions. The highest concentrations (200 µM) of 2,4-D dimethylamine and 2,4-dichlorophenoxyacetic acid evaluated were able to oxidize the mitochondrial NAD(P)H, as well as the 10 µM concentration from the latter and the 100 µM concentration from the proportional mixture. Discussion: The oxidation of NAD(P)H may suggest a possible imbalanced redox state-related mechanism of action, which will be further investigated through other analyses such as the evaluation of antioxidant enzymes, since 2,4-D and its isolated compounds do not induce RONS production or accumulation. Financial Support: TOXICAM

Commercial 2,4-D and its isolated compounds, 2,4-dichlorophenoxyacetic acid and 2,4-

D dimethylamine, induces changes in mitochondrial function parameters

poliDo, lucaS roBerTo Ferreira1,2; rizzi, joyce SanTana1,2; pereira, lílian criSTina2,3

1 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM), Botucatu;

3 São Paulo State University (Unesp), School of Agriculture, Botucatu.

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Introduction: Cigarette smoke can induce the lung acute injury with inflammatory cells and lung parenchyma damage. Eugenol (EUG) is a component of clove oil, with anti-inflammatory and antioxidant activities. Objective: To evaluate the pharmacological activity of the EUG in cigarette smoke-induced acute lung injury. Methods: C57BL/6 mice, male were exposed to 12 cigarettes per day for 5 days (CS group). The control group was exposed to sham smoking. The CS group was treated with EUG (100 mg/mL) or vehicle by inhalation (15 min/daily) for 5 days. The anti-inflammatory markers and lung morphology were evaluated. Results and Discussion: The leucocytes number from mice BAL increased 3x on the CS group compared to control and the treatment

with EUG (100 mg/mL) reduced 46% when compared to the CS group (p<0.05). The number of macrophages (142%) and neutrophils (3x) increased in the CS group. EUG (100mg/mL) reduced in 60% (p<0.001) and 63% (p<0.001), respectively. The lung inflammation score showed the increase of 100% of inflammation in the CS group (p<0.001). The treatment with EUG 100 mg/mL reduced this score by 30% (p<0.01). The bronchoconstriction index was increased in the CS group (65%; p<0.01) and reduced in 29% (p<0.05) by treatment with EUG 100 mg/mL. Conclusion: Eugenol has an anti-inflammatory effect to attenuates the inflammatory response induced by cigarette smoke. Acknowledgments: UFERSA and UERN

Could Eugenol promote the reduction of the inflammatory process in acute injuries to the

lung of rats caused by cigarette smoke?

Silva, aline gaBrielle goMeS1,2; Moura, Maria joana nogueira1,2; SanToS, caio ceSar araújo1; pereira, arTeMia kelly holanDa1; Felix, renaTa gleySiane De SouSa1; araujo, Bruno vinicioS Silva1; BarBoSa, Maria

clara De oliveira1; Melo, paolo oliveira3; FeiToSa, eManuel keneDy1; BorgeS, ciBele DoS SanToS1

1 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-

Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM), State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande

do Norte, Brazil. Heart Institute; 3 University of São Paulo (USP), São Paulo, Brazil.

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Introduction: lung cancer is one of the biggest causes of death in the world, accounting for approximately 2.1 million new cases per year. In Brazil, it is the fourth most common type of cancer among men and the fifth among women. The use of platinum derivatives associated with other chemotherapeutics is still the main choice for the treatment of lung cancer, however the use of these drugs causes serious adverse effects and can present several resistance mechanisms. Objective: To identify new antitumor prototypes by virtual screening of coumarins from the LaSOM library and the in vitro assays. Methods: five coumarins were selected by the rank-by-rank method and their physicochemical and toxicological properties in silico were analyzed. From these results, three coumarins (LaSOM 235, LaSOM 229 and LaSOM 234) were selected to continue the in vitro studies. In vitro cell viability, oxidative stress and mitochondrial membrane potential assays were performed using the human lung cancer strain A549. Results: the results showed that LaSOM 235 presented a decrease in cell

viability at concentrations of 30, 40 and 50 µM when compared to the control group (100% of cell viability), revealing to be the most promising substance among the three evaluated in vitro. Additionally, in order to investigate the action mechanism of coumarins the inhibition of the CD73 enzyme (ecto-5’-nucleotidase) was investigated. Ecto-5’-NT has been associated with some pathological conditions, such as myocardial ischemia and cancer. The enzymatic activity was tested at concentrations of 50, 75, 100 and 200 µM and no enzymatic inhibition was evidenced at these concentrations. Discussion/Conclusion: the in vitro evaluation of coumarins LaSOM 229, LaSOM 234 and LaSOM 235 against the cell line derived from human lung cancer A549 showed that with the results obtained so far, LaSOM 235 is the most promising hit, with chances of becoming a potential candidate for an antitumor drug prototype. More tests should be performed to corroborate the results obtained. Acknowledgments: PNPD/CAPES

Cytotoxicity assessment of potential antitumor 4-methyl coumarins

göeThel, gaBriela1; Souza, joão peDro Silveira1; neveS, guSTavo MachaDo1; kagaMi, luciano porTo1; peruzzi, caroline porTela2; caTTani, ShanDa2; garcia, Solange criSTina2;

BaTTaSTini, ana Maria oliveira3; Figueiró, FaBrício3; eiFler-liMa, vera lucia1

1 Laboratório de Síntese Orgânica Medicinal (LaSOM), Pharmaceutical Sciences Graduate Program, College of Pharmacy, Federal University of Rio Grande do Sul, Avenida Ipiranga,

2752, Porto Alegre – RS, Brazil. 2 Laboratório de Toxicologia (LATOX), Pharmaceutical Sciences Graduate Program, College of Pharmacy, Federal University of Rio Grande do Sul, Rua São

Luís, 150, Porto Alegre – RS, Brazil. 3 Laboratório de Bioquímica (Laboratório 22), Graduate Program in Biological Sciences: Biochemistry, Institute of Basic Health Sciences, Federal

University of Rio Grande do Sul, Rua Ramiro Barcelos, 2600, Porto Alegre – RS, Brazil.

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Background/Introduction: Humans are exposed to a multitude chemicals: pesticides, pharmaceuticals and industrial chemicals, among other substances whether through production, consumption, presence in the environment or incorrect disposal. The bioaccumulation and biomagnification of their residues may increase to inadequate exposures and lead to high incidence of human diseases. Experimental studies have the capacity to predict the toxicity of these emerging contaminants and are valuable tools for understanding biological mechanisms potentially involved in the hazard definition and consequently, help in risk assessment to human health. Currently, the use of so-called alternative methods to animal use for assessing the toxicity of chemicals is an international trend in response to the 3R’s (Reduce, Refine and Replace) policy that restricts the use of animals in research. Among them are included cell cultures applied to toxicology. Objective: This study characterizes the in vitro toxicity to herbicide Triclopyr and the personal care product (PCPs) used in cosmetics, Decamethylcyclopentasiloxane (D5), in an attempt to elucidate their mechanism based on mitochondrial dysfunction and cellular injury using HepG2 cells. Methods: As a first step HepG2 cell lines were cultivated and plated to 96 or 24-well plates, incubated for 24 hours, then exposed to Triclopyr and D5 at different concentrations between 0.1 to 50 µM for 24 and 48 hours for MTT and SRB, respectively. Cell viability analysis was performed using the using MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide] test. The Sulforhodamine B (SRB) assay was used for cell

density determination, based on the measurement of cellular protein content. Furthermore, the Agilent Seahorse Cell Mito Stress Test kit was performed to assessing mitochondrial function, using concentrations of 0.5 to500 µM. This test provided multiple mitochondrial parameters like basal respiration, ATP-linked respiration, maximal and reserve capacities. Results: There were no significant results for MTT assay. However, outcomes of SRB demonstrated that Triclopyr and D5 affect cell proliferation, with statistical difference between the negative control and the tested concentrations of 0.1, to 50 µM. Effects induced by mitochondrial multiparameter using a high-capacity respirometer after exposure of HepG2 cells showed that both compounds have the ability to induce mitochondrial damage at intermediate concentrations of 10 and 100 µM, inhibiting basal and spare respiratory capacity. Discussion/Conclusion: The present results indicate that both emergenting contaminants are mitotoxic. A decreasing at mitochondrial respiration and ATP production was observed. In the SRB assay there was a decrease in cell proliferation. Although no effect on the ability to metabolize the MTT dye was observed, there was an alteration on cell density. Nonetheless, mitochondrial damage can have different consequences for the cell that will be investigated in more detail. Acknowledgments: This work was financially supported by grants from the Coordination for the Improvement of Higher Education Personnel (CAPES). Process number: 88887.600147/2021-00 and Fapesp (2018/00229-1 e 2020/11128-1).

Cytotoxicity evaluation in human hepatic cells HepG2 induced by emerging contaminants

Decametilcyclopentasiloxane and Triclopyr

kohori, naTália akeMi1,2; TeoDoro, joão Soeiro3; palMeira, carloS Manuel MarqueS3; pereira,, lilian criSTina1,4

1 Botucatu Medical School, Department of Pathology, São Paulo State University (UNESP); 2 Center for Environmental Assessment on Human Health (TOXICAM), Botucatu, São Paulo,

Brazil; 3 Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal; 4 Department of Bioprocesses and Biotechnology, Faculty

of Agronomic Sciences of Botucatu, São Paulo State University, Botucatu, SP.

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Introduction: Obesity is considered a global public health problem and diets rich in fats and sugars are increasingly being consumed. Associated with the intake of high fat-sugar foods is a sedentary lifestyle, the number 1 aggravating factor of weight gain problems. In addition to the problems intrinsic to obesity, others are also directly related, such as reduced fertility. It is also known that obesity directly impairs reproduction, altering hormone levels and gamete production. Objective: The present study aimed to evaluate and compare the possible beneficial or deleterious effects of physical training associated with diets rich in cashew nut fat or lard in reproductive systhem of female adult rats. Methods: Adult female Wistar rats were divided (n=8/group) into standard chow group (control, 10% lipid and 20% protein content) without physical exercise (sedentary) - SDCNT; standard chow with physical exercise group - EXCNT; cashew nut group (diet enriched with vegetable fat from cashew nut, 40% lipid and 20% protein content) sedentary - SDCSC; cashew nut group with exercise - EXCSC; lard group (diet enriched with fat of animal origin based on lard, 40% lipid and 20% protein content) sedentary - SDBHP and lard group with physical exercise - EXBHP. During 90 days, the food was offered ad libitum, and consumption was monitored weekly. The physical exercise groups were submitted to 30 minutes of daily physical exercise, following the running protocol on the treadmill (8m/min for 5 minutes, 12m/min for 20 minutes and 8m/min for the last 5 minutes, during the experimental protocol). In the last 15 days, the estrous cycle of the rats was monitored and after this period, in the first estrus, the rats were euthanized for collection, weighing and fixation of the ovaries and uterus, followed by further histopathological analysis. Ovaries and uterus were fixed in formaldehyde, processed and stained with eosin-hematoxylin for histopathological (qualitative) analysis. The study was

approved by the Ethics Committee of UERN 007/19 and Ethics Committee of UFERSA (PIA10006-2021). Results and Discussion: Feed consumption showed a significant difference (p<0.05) between groups SDCSC (14.23+0.83), EXCSC (16.54+0.60), SDBHP (13.69+0.47), EXBHP (16.56+0.58) when compared to both EXCNT (22.15+0.41) and SDCNT (19.19+0.68). The final body weight of the rats revealed a significant increase (P<0.5) in the weight of the exercise groups when compared to the sedentary groups, except the SDCSC group compared to the EXCNT group. The uterine weight of the SDCSC group was also significantly lower compared to the EXBHP group. On the other hand, the weight of the ovaries did not change (p>0.05). There was no alterations on ovary histology (p>0.05). All groups presented well-defined gonads, characterized by a cortex with the presence of ovarian follicles in various degrees of development, mainly corpora lutea (70%), since the rats were euthanized in estrus. Uterine histology revealed in the SDCSC an increased presence of leukocyte infiltrates compared to the SDCNT. In the exercise group, both EXCNT and EXCSC showed an increase in the presence of leukocyte infiltrates in the uterine endometrium (100% of the animals evaluated). In the group EXBHP, the presence of leukocytes was observed, but at a lower frequency when compared to the other groups. As for cyclicity, it was observed that there was a lower percentage (p<0.05) of proestrus in the EXCSC group (7.80+2.07) in relation to the SDCNT group (19.94+4.03), but not directly impacted the size and number of estrous cycles when compared between groups (p>0.05). Conclusion: Therefore, it can be concluded, based on this experimental model, that both diets, rich in vegetable or animal fat, do not have a direct impact on reproductive systhem of female adult rats, at least not on gonadal morphology. Acknowledgments: PIBIC (CNPq) and PIVIC UFERSA Scholarship.

Different diets and physical exercises: assessment of the impact on the female

reproductive system after 90 days

Moura, Maria joana nogueira1,2; Silva, aline gaBrielle goMeS1,2; SanToS, caio ceSar araújo1; goMeS, FranciSca Tayná Da Silva3; pereira, arTeMia kelly holanDa1; Felix, renaTa

gleySiane De SouSa1; FonSeca, ivana alice Teixeira3; BorgeS, ciBele DoS SanToS1

1 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-

Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM); 3 Multicenter Graduate Program in Physiological Sciences (PPGMCF),

State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

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Introduction: Numerous antidepressants and antipsychotics drugs have been continuously approved and released for sale that have not yet had all their effects evaluated, both in the short and long term. These drugs are aimed at different types of treatment of mental disorders. Among these drugs is Aripiprazole. Aripiprazole has high affinity for serotonin, dopamine and noradrenaline receptors for the treatment of mental disorders, mainly schizophrenia and depression. It is known that the 5-HT system can directly influence the reproductive function of vertebrates, due to its communication with the sex steroid system. This interaction between the systems occurs through estrogen and progesterone receptors located in central serotonergic neurons, demonstrating an important signaling pathway, in which it can modulate several neural processes, including thyroid secretion and sexual behavior. However, there are few reports of the effects of second-generation antidepressants and antipsychotics on the reproductive system and sexual behavior of male rats, mainly with regard to aripiprazole. Objective: The present study aimed to evaluate the possible effects of exposure to different doses of aripiprazole on the male reproductive system, with emphasis on sperm quality and sexual behavior. Methods: Adult male Wistar rats were divided (n=12/group) into control group (CTRL, treated with vehicle solution - 66% saline and 33% DMSO); group treated with 3.0mg/kg of aripiprazole diluted in vehicle (EXP1) and group treated with 6.0mg/kg of aripiprazole diluted in vehicle (EXP2). The animals were treated during 28 days. After the end of the treatment, a set of animals (n=7/group) were weighted and euthanized by overdose of anesthetic xylazine and ketamine. Blood samples were collected to hormonal levels. Vital organs (kidney, adrenal, heart, liver, brain, pituitary and thyroid) were collected and

weighed. The reproductive organs (testis, epididymis, ventral prostate, full and empty seminal vesicle) were also collected and weighed. The right testis and epididymis were fixed in modified Davidson’s solution (MDF) for further histopathological processing. The left epididymis was removed and the sperm sample was used to determine the sperm motility and count. The remained animals (n=5/group) were used to perform sexual behavior. The study was approved by the Ethics Committee of UFERSA 02/2020 (Protocol number 23091.014948/2019-20). Statistical analysis: ANOVA and Tukey test for parametric data and Kruskal Wallis and Dunn’s test for non-parametric data (P<0.05). Results and Discussion: No alterations were observed in the final body weight of the rats exposed to different doses of aripiprazole, as well as no alterations in the absolute weights vital organs, when compared to the CTRL group. However, when compared the reproductive organ weights there was a significant increased in the full seminal vesicle from EXP2 group when compared to CTRL group (p<0.05). The evaluation of the sperm motility showed a significant reduction on mobile sperm with progressive moviment from EXP1 compared to CTRL group (57.7% x 37.7%; p<0.05). The sexual behavior showed the same results, once the number of mounts before the ejaculation was significantly decreased in EXP1 group when compared to the CTRL group (16.0 + 3.2 x 6.0 + 1.5; p<0.05). Conclusion: Therefore, it can be concluded at this moment, based on this experimental model, that the exposure to aripiprazole during adulthood does not cause systemic intoxication effects. However, although the differences were occured only in some variable, it can cause impairment on male sperm quality and/or libido. Acknowledgments: PIBIC-CNPq UFERSA Scholarship and UFERSA/PROPPG (grant number 23091.014593/2019-02).

Discreet alterations on sperm quality promoted by Aripiprazole

Moura, Maria joana nogueira1,2; Felix, renaTa gleySiane De SouSa1; pereira, arTeMia kelly holanDa1; angelo, ana BeaTriz Silva1; SanToS, caio ceSar araújo1; Silva, aline

gaBrielle goMeS1,2; Silva, ana lucelha DoS SanToS1; Freire, livia horrana ForTe1; DanTaS, joao arTur DiogeneS1; Silva, MaTeuS liMerio carloS1; BorgeS, ciBele DoS SanToS1

1 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-Arid Region -

UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM), State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

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Background: Studies have indicated that the diuron herbicide induces toxicity in different tissues, especially in the urinary bladder of Wistar rats, where at high doses it may lead to the development of tumors. However, events involved in its intimate toxic mechanism remain unclear. In this context, Caenorhabditis elegans (C. elegans) has been widely used as an experimental model to understand adverse events related to chemical exposures. Objective: This study aimed to identify the toxic mechanism of diuron and its metabolites, 3,4-dichloroaniline (DCA) and 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) in C. elegans. Methods: Wild-type N2 worms in L1 larval stage were acutely exposed (1 hour) to the test chemicals in a six-concentration range of 0.5 to 500 μM and analyzed after 24 and 48 hours of incubation for % lethality. Then, exposures were carried out using the highest concentration (500 μM) to evaluate % survival, reactive oxygen and nitrogen species (RONS) , glutathione (GSH) and ATP levels, as well as behavior. The QU1:izEx1 (Pllg-1::GFP) and BY200 (Pdat-1::GFP) strains were used to determine autophagy and dopaminergic neurodegeneration, respectively. Results were evaluated by analysis of variance (ANOVA) followed by Dunnet’s or Tukey’s post hoc test, and significant differences were set at p < 0.05. Results: Increased % lethality was found for all chemicals at high concentrations, with statistically significant difference for 500 μM DCA. Significant difference was also observed in the % survival for DCA. No changes in RONS production were observed for diuron and its metabolites. Nevertheless, GSH levels were significantly increased upon DCA

treatment. Moreover, ATP levels were decreased for all test chemicals. There were no alterations in the autophagy process. Under the conditions of the present study, dopaminergic neurotoxicity was observed for all tested chemicals, but only diuron was induced alterations in the moving average and smoothed speed (μm/s) of the worms. Discussion/Conclusion: In this study, increased GSH levels acted as a compensatory mechanism against the generation of RONS by diuron and its metabolites. In turn, exposure to diuron and metabolites was sufficient to impair ATP levels, indicative of an alteration in oxidative phosphorylation that takes place in mitochondria. Such conditions may trigger autophagy as an adaptive survival mechanism, but this was not observed in C. elegans; in fact, necrosis and apoptosis have been shown as the main types of cell death associated with the test chemicals in previous studies. Although dopaminergic neurodegeneration typically triggers motor impairment, only diuron elicited this outcome; alteration in the specific parameters evaluated may occur at different time point for the metabolites and was not accessed in the present study. Altogether, the results suggest that the diuron metabolites are more toxic than the parent compound, with DCA in particular appearing to play an important role in the toxicity observed on C. elegans. Acknowledgments: Breno Pannia Espósito (Institute of Chemistry, University of São Paulo) for technical assistance. São Paulo Research Foundation (FAPESP) [Grant No. 2017/25402-5] and Coordination for the Improvement of Higher Education Personnel (CAPES) [CAPES-PrInt Grant No. 88887.467311/2019-00].

Diuron herbicide-induced toxicity on Caenorhabditis elegans

liMa, Thania rioS roSSi1,2; MarTinS jr., airTon cunha3; pereira, lílian criSTina2,4; aSchner, Michael3

1 Botucatu Medical School, Department of Pathology, São Paulo State University (UNESP); 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM),

Botucatu, SP, Brazil; 3 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA; 4 Faculty of Agronomy Sciences, Department of Bioprocesses

and Biotechnology, São Paulo State University (UNESP), Botucatu, SP, Brazil.

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Breast cancer causes a great number of deaths worldwide, mainly in women. One of the most aggressive breast cancer subtypes is triple-negative breast cancer (TNBC), which represents about 20% of the diagnosticated breast cancers. TNBC does not express estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2). In this way, hormone therapy is not an option, and TNBC usually is treated with chemotherapy. Unfortunately, chemotherapy treatment causes several side effects to patients. Selenium (Se) is an essential micronutrient, which has antioxidant and anti-inflammatory effects. On the other hand, some studies have been demonstrating that some Se-containing compounds have pro-oxidant effects. Thus, the aim of this work was to evaluate the antiproliferative effects of sodium selenite in breast cell lines. Two TNBC cell lines [BT-549 (primary tumor) and MDAMB-231 (metastatic tumor)] and a non-tumoral breast cell line (MCF-10A) were exposed to 1, 10, 50, and 100 µM sodium selenite for 48 hours. The following assays were carried out (1) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test; (2) identification of apoptotic and necrotic cells test; (3) colony-forming unit test; and (4) cell migration. For all tests, at least three independent experiments were performed. Data were statistically analyzed using Prisma Graphpad software, version 5.0, using the Kruskal-Wallis test followed by Dunn’s test and presented as median ± interquartile range. Effects were considered significant when p<0.05. Kruskal-Wallis test revealed an effect of exposure to sodium

selenite on the cell viability on the three cell lines evaluated. In fact, MCF10-A and BT-549 cell lines exposed to 50 and 100 µM sodium selenite had a statistically significant decrease in cell viability, as well as MDAMB-231 cells exposed to 100 µM sodium selenite also had a statistically significant decrease in cell viability. Kruskal-Wallis test revealed an effect of sodium selenite on the percentage of apoptotic and necrotic cells. In fact, the exposure to 50 and 100 µM sodium selenite caused a statistically significant increase in the percentage of late apoptotic and/or necrotic MCF-10A cells. However, only the exposure to 100 µM sodium selenite caused a statistically significant increase in the percentage of late apoptotic and/or necrotic MDAMB-231 and BT-549 cell lines. Sodium selenite exposure totally inhibits colony growth at concentrations of 10, 50, and 100 µM in the three cell lines evaluated. Exposure to sodium selenite (1 µM) caused a statistically significant increase in the migration of MCF-10A and MDAMB-231 cells when compared to control (unexposed cells). On the other hand, caused a statistically significant decrease in cell migration of the BT-549 cell line. Sodium selenite showed promising antiproliferative results in different breast cell lines. In conclusion, it was observed that sodium selenite has the potential to be tested in pre-clinical studies of animal breast cancer models. Keywords: Selenium, selenium inorganic, breast cancer, triple-negative breast cancer. Acknowledgments: We greatly appreciate the Pequeno Príncipe Complex by scholarship.

Effect of inorganic selenium on triple-negative breast cancer cell lines

coSTa, nayara De Souza1,2; liMa, luiza Siqueira1,2; oliveira, Franciele apareciDa MenDeS1,2; galiciolli, Maria eDuarDa De anDraDe1,2; oliveira, cláuDia Sirlene1,2; irioDa, ana carolina1,2

1 Programa de Pós-graduação Strictu Sensu em Biotecnologia Aplicada à Saúde da Criança e do adolescente, Instituto de Pesquisa Pelé Pequeno Príncipe,

Curitiba, PR, Brazil; 2 Faculdades Pequeno Príncipe, Curitiba, PR, Brazil.

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The HET-CAM method can be used to determine the irritation potential of certain substances on corioalantoic membrane in chicken eggs, and in addition, it can determine anti-inflammatory activity of this substances. The test is based on the exposure of membrane to irritating agents with posterior observation about vascular events triggered. HET-CAM is considered a test in vivo that replaces the use of animals, being a test considered useful and well established to determine the potential of pharmaceutical products. It should be emphasized that ocular toxicity assessment of cosmetic products is done alternatively to use of animals, since it is prohibited. The species Myrcia pubipetala, commonly known as Goiabão, is a tree registered on states of Santa Catarina and Rio Grande do Sul, occurring in the Atlantic Forest and Dense Anthropophilous Forest. The present study aimed to evaluate the irritability of Myrcia pubipetala extract by the HET-CAM methodology. The hydroalcoholic extract (with 70% of ethanol) from the leaves of Myrcia pubipetala was kindly ceded by Professor PhD Micheli A. Debiasi. The fertilized chicken embryos were pre-incubated to 38º Celsius with 60% of humidity for 8 days. After a hole highlighted with pencil was sanitized and drilled gently over the overhead bag with tweezers to not damage the eggshell, and the vascular zone was easily identified in corioalantoic membrane (CAM). Two drops of Saline solution 0,9% have been added to dampen the inner membrane of the casing adjacent to the CAM. After clamping and raised by ophthalmic

tweezers, the membrane and CAM were separated effortlessly, and in sequence, a space with 1x1cm on membrane will be seccionated to expose vascular zone. It was applied 300 µL of NaCl 0.9 % (negative control), NaOH 1M (positive control) and extract of Myrcia pubipetala (3, 30 and 300 µg/mL) directly on vascular zone. After the compounds application, the eggs have been observed for 300 seconds. During this period, bleeding, coagulation, and vascular lysis it was observed, and the experiment was performed in triplicate. For the positive control group for irritation was applied 300 µL of NaOH at 1 M, inducing a positive response to irritation with the appearance of vascular lysis, bleeding and coagulation, with an average score of 20, categorized as severe irritation. In the negative control group, the eggs were treated with NaCl 0.9 %, with a negative response to irritation, with a score of 0. Myrcia pubipetala test, 300 µL of plant extract were applied at 300, 30 and 3 µg/mL and no irritative effects were observed at all doses (p<0,001 vs negative control). Thus, the extract was categorized as non-irritating, with viability for dermal topical use. Although the HET-CAM method is an established and reliable, this test remains the most viable alternative method to the use of animals, and inexpensive for the purpose of controlling topical and ophthalmic irritation of substances. We can suggest that the extract of Myrcia pubipetala met the hypotheses raised by the research group, feasible for topical dermal use, because it does not cause irritation.

Evaluation of irritability of Myrcia pubipetala miq. by HET-CAM methodology

perini, caMila Maria; kohler, criSToFer joSé Weege; Wagner, eDuarDo joSé; MachaDo, iSaBel DauFenBack

Universidade Regional de Blumenau – FURB.

192

Introduction: Plastics are used wordwild due to their excellent mechanical properties, versatility and low cost. After their release into the environment, plastics are degraded and fragmented into smaller particles, at micro (<5 mm) and nano (<100 nm) scale, which increases their toxic potential. Micro (MPs) and nanoplastics (NPs) can be found in the air, sediment, water and terrestrial and aquatic organisms. Bisphenol A (BPA) is a industrial product used in the plastic production and is known as an endocrine disruptor. Bisphenol S (BPS) is an analogue BPA and it is used as an alternative to this contaminant. BPS has metabolism and mechanisms of action similar to BPA, which represents a potencial human health risk. Food and water contaminated with bisphenol adsorbed in MPs and NPs are the major sources of human exposure to these particles and contaminants. The consequences for human health are still unknown and there is no internationally accepted definition for ingestion and exposure to these MPs and NPs. In addition, few studies have shown the toxicity of MPs and NPs in liver cells, in vitro and in vivo. Objective: To evaluate whether micro and nanoplastics associated to BPA or BPS can alter the metabolic activity of liver cells. Methods: Commercial spherical micro and nanoplastics (Thermo Fisher) were used. Liver cells (HepG2) were co-exposed to BPA or BPS (100 µM) and to two different sizes of MP (0.2 or 0.5 µm) at three different concentrations (10, 100 and 300 µg.mL-1) for 24 hours. For the 72 hours experiments, HepG2 cells were co-exposed to BPA or BPS (100 µM) and to two different sizes of NP (0.02 or 0.1 µm) or one size of MP (0.5 µm) at two different concentrations (5 and 10 µg.mL-1). Methylmethanesulfonate (600 µM) was

used as a positive control. HepG2 metabolic activity was measured by resazurin (spectrofluorimeter – 540/590 nm). Results: At the 24 hours experiments, co-exposure to BPA or BPS and 0.2 µm MP (10 µg.mL-1) decreased the cellular metabolic activity compared to the group treated with MP only. The same results occurred in the group BPA + MP (100 and 300 µg.mL-1). No change in the cellular metabolic activity was observed when the cells were exposed to BPA or BPS only. Cellular metabolic activity decreased when the HepG2 cells was co-exposed to BPA or BPS and 0,5 µm MP (10 µg.mL-1) when compared to the control group and the MP group. In addition, BPA + MP group decreased the HepG2 metabolic activity compared to the BPA group. At the 72 hours experiments, co-exposure to BPA or BPS and 0.02 µm NP (5 and 10 µg.mL-1) decreased the cellular metabolic activity compared to the control and NP groups. Futhermore, BPS + NP group decresead the HepG2 metabolic activity in comparison to the BPS group. When co-exposed to the 0,1 µm NP or 0.5 µm MP at both concentrations (5 and 10 µg.mL-1) and BPA or BPS, the cellular metabolic activity decreased in comparison to the control group and the group treated with plastic only, but not in comparison to the bisphenol group. Discussion/Conclusion: At 24 hours, the 0.5 µm MP co-exposed to the BPA is able to intensify the reduction of HepG2 viability, demonstrating that the decrease in cellular metabolic activity of HepG2 depends on the concentration and size of the plastic. After 72 hours exposure, the plastic size influences the co-exposure response to MPs or NPs and bisphenol, but not the concentration. Acknowledgments: This work was supported by CAPES, FAPESP and CNPq.

Evaluation of metabolic activity of HepG2 cells co-exposed to micro or

nanoplastics and bisphenol A or S

rocha, cecília criSTina De Souza; Devoz, paula pícoli; anTuneS, luSania Maria greggi; BarBoSa jr, FernanDo

University of Sao Paulo – School of Pharmaceutical Sciences of Ribeirao Preto, Ribeirão Preto - SP, Brazil.

193

Introduction: Incorrect nutrition with the absence of physical activity associated with other factors can contribute to the development of obesity. In rats, specifically, the weight gain induced by high-fat diets, represents an experimental model that has been widely used to study the physiological and molecular mechanisms of obesity development, as these animals share similar gene expression patterns with humans. In addition, it is known that the accumulation of fat can directly harm reproduction, promoting hormonal problems as well as fertility. Objective: To investigate the possible impacts of different ketogenic diets with or without physical activity during a period of 180 days on reproductive morphological parameters of adult wistar rats. Methods: Adult female Wistar rats were divided (n=8/group) into standard chow group (control, 10% lipid and 20% protein content) without physical exercise (sedentary) - SDCNT; standard chow with physical exercise group - EXCNT; cashew nut group (diet enriched with vegetable fat from cashew nut, 40% lipid and 20% protein content) sedentary - SDCSC; cashew nut group with exercise - EXCSC; lard group (diet enriched with fat of animal origin based on lard, 40% lipid and 20% protein content) sedentary - SDBHP and lard group with physical exercise - EXBHP. The physical exercise groups were submitted to 30 minutes of daily physical exercise, following the running protocol on the treadmill (8m/min for 5 minutes, 12m/min for 20 minutes and 8m/min for the last 5 minutes, during the experimental protocol). The treatment occured during 180days and in the last 15 days, the estrous cycle of the rats was monitored. After that, in the first estrus, the rats were euthanized for collection, weighing and fixation of the ovaries and

uterus, followed by further histopathological analysis. Ovaries and uterus were fixed in formaldehyde, processed and stained with eosin-hematoxylin for histopathological (qualitative) analysis. Results and Discussion: There were no statistical differences observed in the body weight of the animals and also in the uterine and ovarian absolute and relative weights (p>0.05). In addition, no alterations were observed in the number of estrous cycles and in the duration of these cycles between the groups (p>0.05). However, when each phase was analyzed individually, a higher frequency of the diestrus phase was seen in the EXCSC group (54.81+6.41) when compared to the SDBHP group (25.96 + 8.09). In the histopathological evaluation of the ovaries, no significant changes were identified. All groups had well-defined and characteristic gonads. Histopathological analyzes of the uterus showed that both, in the sedentary and in the groups submitted to exercises, the presence of leukocyte infiltrates in at least half of the animals of all groups, including the group treated with standard chow. On the other hand, only in the group exposed to lard, both in the sedentary group and in the group submitted to exercise, alterations were observed in the glandular epithelium in at least 20% of the animals. Conclusion: Therefore, based on the present experimental model, there was no deleterious effects caused by the different types of high-fat diets and physical activity in the on the female reproductive system. Which, on the one hand, opens new perspectives on the intake of different types of fat on health and fertility studies. Acknowledgments: PIBIC (CNPq) and PIVIC UFERSA Scholarship.

Evaluation of the female wistar rats reproductive system submitted to different diet formulations

and activity patterns during 180 days

DanTaS, joao arTur DiogeneS1; Silva, aline gaBrielle goMeS1,2; Moura, Maria joana nogueira1,2; SanToS, caio ceSar araújo1; goMeS, FranciSca Tayná Da Silva3; pereira, arTeMia kelly holanDa1;

Felix, renaTa gleySiane De SouSa1; FonSeca, ivana alice Teixeira3; BorgeS, ciBele DoS SanToS1

1 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-

Arid Region - UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM); 3 Multicenter Graduate Program in Physiological Sciences (PPGMCF),

State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

194

Background/Introduction: Worldwide, the use of natural products of plant origin grows both as nutraceuticals and in drug therapy. Not only in developing countries, but also in developed ones, there is a greater acceptance of medicinal plants in health treatment. However, many of these products are used without studies that determine safe doses and absence of harmful effects. Objective: We intend to evaluate the safety associated with the use of tucumã (Astrocaryum vulgare Mart.) fruit oil, based on its effects on renal and hepatic parameters of rats. Methods: Virgin tucumã pulp oil (TO), purchased commercially, was applied daily, cutaneously, for 28 days, to three groups of five Wistar rats each (n=5), in increasing doses of 250, 500 and 1000 mg/kg . The control group (n=5) received saline solution. The test followed the OECD 410 protocol and was approved by CEUA-UFSM (8956120620). At the end of the 28 days of treatment, the rats were euthanized. For euthanasia, anesthesia was induced by ketamine in combination with xylazine hydrochloride at a dose of 100 mg/kg ketamine + 10 mg/kg xylazine (intraperitoneally). When the absence of caudal and limb reflexes was detected, blood was collected by cardiac puncture. The blood was centrifuged and the serum used to measure AST, ALT, blood urea nitrogen (BUN) and creatinine using a semi-automatic biochemical analyzer (Bioplus: Bio -2000). In addition, liver and kidney were weighed and their relative weights were

determined. To analyze if the data presented a normal distribution, the Kolmogorov–Smirnov test was used. Results that showed a Gaussian distribution were analyzed by parametric test one-way ANOVA followed by Tukey’s test. Kruskal-Wallis and Dunn’s were used as statistic tests when the requirements to perform a parametric test were not satisfied. Difference was considered significant when p < 0.05. Results: The measurement of the absolute and relative weights of liver and kidney showed no significant difference between the different treatment groups. Likewise, the dosage of renal markers BUN and creatinine remained similar between the groups and within the values considered normal for the species. The determination of the activity of liver enzymes ALT and AST was also within the reference values, however there was a reduction in the activity of AST in the group that received the highest dose of TO in relation to the control group. Discussion/Conclusion: End-of-study organ weights are relevant markers in medicinal plant toxicity studies. Furthermore, liver and kidney are organs very sensitive to xenobiotics and their markers reflect the effect of the compound on the health of the animal. Since our results were all within the normal range and there was no difference between the different groups, it is suggested that TO was not toxic at the doses and conditions used. Keywords: Tucumã, Astrocaryum vulgare, medicinal plants, topic, skin toxicity. Acknowledgments: UFSM.

Evaluation of the oil toxicity of Astrocaryum vulgare Mart. through renal

and hepatic indicators of Wistar rats

reginaTo, FernanDa ziegler; guex, caMille gauBe; FiguereDo, káSSia caroline; graiczik, jaMeS raMireS penTeaDo; caSSanego, gaBriela BuzaTTi; pappiS,

lauren; heck, aManDa SzyManSky; BauerMann, liliane De FreiTaS

Departamento de Fisiologia e Farmacologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria.

195

The species Myrcia tijucensis is a tree of Myrtaceae family, widely distribute and abundant in areas of anthropophilic forest and sandbank of Atlantic Forest. Some trees of Myrtaceae family, like M. tijucensis, are commonly know as “Guamirim” or “Ingabaú”. Chemical and toxicological studies about this species is scarce. The objetive of this study was assess the toxicity of crude hydroalcoholic (EBH), dichloromethane (DCM) and ethyl acetate (EBAE) extracts of M. tijucensis using Artemia salina toxicity model according Meyer et al. (1982). M. tijucensis leaves was collected in Blumenau (SC), on August 2018. Leaves were separated from the branches and ground in knife mill. The dry and grounded material was extracted by maceration during 7 days in solvents of different polarities. The extracts were filtered and evaporated until dryness in vacuum rotary evaporator. Artemia Salina larvae grown in saline solution 38 g L-1 during 72 hours with light and constant aeration at room temperature. The extracts were diluted in saline solution (38 g L-1) with

Tween 2% at concentrations 1000, 500, 250 and 120 ppm. About 10 Artemia salina larvae were tranferred to each well of a 24 wells microdilution plate containing extracts and saline solution in the concentration cited. The count of live and died animals was performed after 24 hours of incubation. Tests were conducted in triplicate using K2Cr2O7 solutions (10 to 50 ppm) as positive control. After counting of larvae, the results were analised by probitos statistical method. Results show that in all concentrations tested, the extracts of M. tijucensis presented a toxic profile (LD50 < 1000 ppm). In the lowest concentration tested (120ppm), mortality results it were 72,50 ± 9,01, 78,10 ± 19,18 and 98,99 ± 1,74% for the extracts EBH, EBAE and DCM, respectively. The results suggest toxicity of tested species, opening up the possibility of further studies in the area. So, future trials will be carried out using other methodologies and in minor concentrations in order to check the LD50 of these extracts.

evaluation of toxicity of extracts from Myrcia tijucensis in Artemia salina leach model

kohler, criSToFer joSé Weege; perini, caMila Maria; alBerTon, Michele DeBiaSi

Universidade Regional de Blumenau, Blumenau – SC, Brazil.

196

Introduction: Numerous antidepressants and antipsychotics drugs have been continuously approved and released for sale that have not yet had all their effects evaluated, both in the short and long term. These drugs are aimed at different types of treatment of mental disorders. Among these drugs is Aripiprazole. Aripiprazole has high affinity for serotonin, dopamine and noradrenaline receptors for the treatment of mental disorders, mainly schizophrenia and depression. It is known that the 5-HT system can directly influence the reproductive function of vertebrates, due to its communication with the sex steroid system. This interaction between the systems occurs through estrogen and progesterone receptors located in central serotonergic neurons, demonstrating an important signaling pathway, in which it can modulate several neural processes, including thyroid secretion and sexual behavior. However, there are few reports of the effects of second-generation antidepressants and antipsychotics on the reproductive system and fertility of females, mainly with regard to aripiprazole. Objective: The present study aimed to evaluate the possible impacts of exposure to different doses of aripiprazole on the female reproductive system, with emphasis on estrous cyclicity. Methods: Adult female Wistar rats were divided (n=11/group) into control group (CTRL, treated with vehicle solution - 66% saline and 33% DMSO); group treated with 0.3mg/kg of aripiprazole diluted in vehicle (EXP1); group treated with 3.0mg/kg of aripiprazole diluted in vehicle (EXP2) and group treated with 6.0mg/kg of aripiprazole diluted in vehicle (EXP3). The animals were treated during 15 days and the estrous cycle of the rats was monitored. After the end of the treatment, in the first estrus, six female rats per group were weighted and

euthanized by saturation of anesthetic xylazine and ketamine. Blood samples were collected to hormonal levels. Vital organs (kidney, adrenal, heart, liver, brain, pituitary and thyroid) were collected and weighed. The ovaries and uterus were collected, weighed and fixed in modified Davidson’s solution (MDF) for further histopathological processing. The study was approved by the Ethics Committee of UFERSA 02/2020 (Protocol number 23091.014949/2019-90). Statistical analysis: ANOVA and Tukey test for parametric data and Kruskal Wallis and Dunn’s test for non-parametric data (P<0.05). Results and Discussion: No alterations were observed in the final body weight of the rats exposed to different doses of aripiprazole, as well as no alterations in the absolute and relative weights of reproductive and vital organs, when compared to the CTRL group. However, when compared between the experimental groups, the relative weight of the pituitary was significantly increased (p<0.05) in the EXP1 group (0.07+0.01mg/g) in relation to the EXP2 group (0.04+0.01mg/g). The evaluation of the estrous cycle showed a significant decrease in the number of estrus in the EXP3 group (2.4+0.5) when compared to the CTRL group (5.2+0.7). On the other hand, when performing sexual behavior, the only group that showed a significant reduction in relation to the CTRL group was the EXP1 group (62% x 34%; p<0.05). Conclusion: Therefore, it can be concluded at this moment, based on this experimental model, that the exposure to aripiprazole during adulthood does not cause systemic intoxication effects. However, although subtle, it can cause impairment on female cyclicity and/or libido. Acknowledgments: PIVIC UFERSA Scholarship and UFERSA/PROPPG (grant number 23091.014593/2019-02).

Exposure of adult female rats to different doses of the antipsychotic Aripiprazole:

a look at the reproductive system

Silva, aline gaBrielle goMeS1,2; Moura, Maria joana nogueira1,2; pereira, arTeMia kelly holanDa1; Felix, renaTa gleySiane De SouSa1; angelo, ana BeaTriz Silva1; SanToS, caio

ceSar araújo1; Silva, ana lucelha DoS SanToS1; Freire, livia horrana ForTe1; DanTaS, joao arTur DiogeneS1; Silva, MaTeuS liMerio carloS1; BorgeS, ciBele DoS SanToS1

1 Laboratory of Tissue Biology and Developmental Toxicology – BioTox, Department of Bioscience, Biological and Health Sciences Center - CCBS, Federal University of the Semi-Arid Region -

UFERSA; 2 Multicenter Graduate Program in Biochemistry and Molecular Biology (PMBqBM), State University of Rio Grande do Norte - UERN, Mossoró, Rio Grande do Norte, Brazil.

197

Introduction: Imidacloprid (IMI) is a neonicotinoid insecticide employed worldwide for crop protection. IMI’s mode of action occurs through the agonism of postsynaptic nicotinic acetylcholine receptors (nAChRs), with high specificity for insect nAChRs although there are reports of mammals’ toxicity. Objective: Studies on IMI’s neurotoxicity are not conclusive; therefore, the aim of this study was to evaluate the behavioral and biochemical effects of an IMI based commercial pesticide on rats. Methods: Adult male Wistar rats received 1.5, 5, and 15 mg/kg of an IMI suspension via the oral route for 45 consecutive days. Behavioral toxicity was evaluated using rota-rod for motor coordination, spontaneous locomotor activity, and novel object recognition and OX maze tests for memory and learning. At the end of the treatment, the animals were euthanized, and blood was collected for biochemical measures (urea, creatinine, transaminases, γ-glutamil-transpeptidase, lactate dehydrogenase, alkaline phosphatase, creatine kinase

MB fraction, butyrylcholinesterase, total proteins, C reactive protein, and glucose). The experiments were approved by the University Ethics Committee (number 37572). Results: No alterations were observed in motor coordination and short- and long-term memory tests. However, IMI-based insecticide caused an increase in rearing and time spent at the periphery in the locomotor activity test and a decrease in time spent to finish the OX maze task (p<0.05; ANOVA/Bonferroni). In blood, it was observed only a significant increase in serum butyrylcholinesterase activity (p<0.001; ANOVA/Bonferroni). Conclusion: Subchronic administration of an IMI-based-pesticide caused behavioral and systemic impairments in rats. The behavioral altrations could point to an anxiogenic effect. The increase in serum BuChE activity is possibly a way to metabolize the acetylcholine excess. Keywords: imidacloprid, acetylcholine, locomotor activity, OX maze, butyrylcholinesterase.

Imidacloprid-based commercial pesticide causes behavioral and biochemical

impairments in Wistar rats

TonieTTo, Bruna DucaTTi1,2; laurenTino, ana olívia MarTinS3; coSTa-valle, Marina Tuerlinckx4; ceSTonaro, lariSSa vivan1,2; anTuneS, BiBiana pereira1; ManFio, cleoFaS SaTeS3; SanToS, nícolaS guiMarãeS1;

Dallegrave, eliane4; garcia, Solange criSTina1,2; leal, Mirna Bainy3; arBo, Marcelo DuTra1,2

1 Laboratório de Toxicologia (LATOX), Departamento de Análises, Faculdade de Farmácia - Anexo I, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre – RS. 2 Programa de Pós-Graduação

em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre - RS. 3 Laboratório de Farmacologia e Toxicologia Neurocomportamental, Departamento de Farmacologia,

Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre – RS. 4 Laboratório de Pesquisa em Toxicologia (LAPETOX), Departamento de Farmacociências,

Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre – RS.

198

Background: Attention deficit hyperactivity disorder is one of the most diagnosed neuropsychiatric disorders in school-age children and adolescents. The pharmacological treatment is carried out with psychostimulants, being Lisdexamfetamine Dimesylate (LDX), a prodrug of dextroamphetamine (D-AMF), one of the most used. The safety profile of this drug is similar to other psychostimulants, But it is still conflicting and needs to be better studied, mainly when the target population belongs to a critical period of development, such as peripuberty. Considering this fact and the benefits of its use, studies regarding aspects of toxicological evaluation and drug safety in this period are still scarce. Objective: the aim of this study was to evaluate the toxicological impacts immediately after exposure to LDX during the juvenile period to peripuberty in male rats. Methods: Male Wistar rats (23 days old) were divided into 4 groups: Control (deionized water); and three different doses of LDX: 5.2; 8.6 and 12.1 mg/kg/day. They were treated for 31 consecutive days, orally (gavage). During the treatment, clinical signs of toxicity, body weight, water and food consumption and play behavior were evaluated. On postnatal day (PND) 54, the animals were killed for organ weight, hematological and biochemical analysis. Results: We observed a reduction in the daily food consumption in all experimental groups compared to the control, as

well as in the daily water consumption in the group exposed to 8.6 and 12.1 mg/kg of LDX. Also, during treatment, the animals in the highest dose group had a reduction in body weight since the 15th day of treatment (PND 37) until the end. On play behavior evaluation, the animals of the highest dose group presented a reduction in the percentage of social behaviors. We observed an increase in the relative weight of the liver (12.1 mg/kg), the spleen (12.1 mg/kg) and the seminal gland (8.6 and 12.1 mg/kg), as well as a reduction in the erythrocyte count and total protein levels in these groups and reduction of albumin level in the lowest dose group. Discussion/Conclusion: The data regarding body weight and food intake are supported by previous finds in the literature, being able to corroborates with the information regarding this growth delay in children. The altered organ weight, hematological and biochemical parameters point to systemic toxicity of this substance. Thus, we can conclude that the continuous use of LDX during this period can lead to significant toxicological changes in the parameters and organs evaluated, negatively impacting the health of the individual during its exposure. Histopathological analyses of liver and kidney are underway to support our hypothesis of LDX toxicity. Acknowledgments: FAPESP (2021/04119-9) and Capes (88887.602916/2021-00). Ethics committee: 3148130421

Immediate toxicological impairment of Lisdexamfetamine Dimesylate in male rats

treated during juvenile period and periuberty

STein, julia1; jorge, BárBara caMpoS1; reiS, ana carolina caSali1; nagaoka, lívia Trippe1; Manoel, BeaTriz De MaToS1; valenTe, leTícia carDoSo1; arena, arielle criSTina1,2

1 Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil.

2 Toxicological Assistance Center (CEATOX), Institute of Biosciences of Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil.

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Introduction: Atherosclerosis is the main cause of morbidity and mortality, making it worrying for humans and causing an incessant search for experimental models to better characterize it and for therapeutic alternatives to prevent or reduce its evolution and severity. Because of this, studies with supplements and medicinal plants have become very promising. Among the plants that have potential for the prevention and treatment of atherosclerosis, we have Plinia cauliflora, which according to literature data is effective as a vasodilator and vascular oxidative stress reduction agent. Objective: Thus, the objective of the present work was to evaluate the lipid-lowering and antiatherogenic effect of P. cauliflora bark extract in an animal model. Materials and Methods: With the approval of the UNIPAR ethics committee, male New Zealand rabbits with approximately 2 kg of live weight were used, which were divided into 6 groups with 6 animals per group, being the negative control, control positive, doses of 10, 30 and 100 mg/kg of the extract and simvastatin 2.5 mg/kg group, thus totaling 36 animals. The negative control group was fed a normal commercial diet and the other groups were fed a high-cholesterol diet (CKD) for 60 days. Thirty days after the beginning of the diet with CKD, treatment was started orally with P. cauliflora extract at doses of 10, 30 and 100 mg/kg and with simvastatin 2.5 mg/kg, once a day, for 30 days. At the end of 60 days, the animals were euthanized and blood was

collected for analysis of biochemical parameters and indices of cholesterol and serum lipids, along with histopathological analysis of the metabolizing and cardiovascular organs (arteries, heart, liver, spleen and kidneys). Results and Discussion: The results showed that oral administration of P. cauliflora extract significantly reduced cholesterol and triglyceride levels in animals fed CKD when compared to the positive control group and simvastatin. In addition, when we looked at the lesions in the aorta, abdominal and iliac arteries, the reduction occurred in all treatments. Regarding the relative weights of the organs, no significant differences were observed in relation to the treated groups. The animals treated with CKD did not show increases in liver enzymes (ALT and AST) when compared to the negative control group. However, an increase in these enzymes (ALT and AST) was observed in the group treated with a dose of 100 mg/kg of the extract, which may indicate the beginning of liver injury, however, these can also be found in other metabolizing organs. Conclusion: In view of the results found, it can be concluded that all doses of Plinia cauliflora bark extract analyzed in the study were effective in reducing cholesterol and triglycerides, thus, it has the potential to be a new supplement to be used directly in the treatment and prevention of atherosclerotic disease. Keywords: Atherosclerosis, Cholesterol-rich diet, Animal Model, Plinia Cauliflora

Investigation of the lipid-lowering and antiatherogenic effects of Plinia

cauliflora bark extract in an experimental model of atherosclerosis

DalMagro, Mariana1; DonaDel, guilherMe2; pinc, Mariana MoraeS3; BerTa, joão anTonio4; BorBa, Maria eDuarDa4; gaSparoTTo junior, arquiMeDeS5; jacoMaSSi, eliza6; zarDeTo, giuliana6; hoScheiD,

jaqueline6; ceranTo, Daniela De caSSia Faglioni BoleTa6; lourenço, eMerSon luiz BoTelho7

1 Student Master in biotechnology applied to agriculture UNIPAR; 2 PhD student in animal science at UNIPAR; 3 PhD student in biotechnology at UNIPAR; 4 Undergraduate

Student Veterinary Medicine UNIPAR; 5 Professor at the University of Grande Dourados –UFGD; 6 Professor at UNIPAR; 7 Unipar Research and Graduate Coordinator.

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Introduction: Levamisole (LVS) is an anthelmintic that was withdrawn from the USA market due to its clinical complications, but it still can be found in Brazilian market. It is a cheap white powder that acts as a nicotinic agonist in the worms. Studies shown that LVS participates in the release of some neurotransmitters and it is metabolized in some active metabolites, such as aminorex, which has amphetamine-like effects. However, since 2002, the use of LVS as a cocaine adulterant increased worldwide and many case reports associate diseases, such as agranulocytosis, systemic vasculitis, neutropenia, and renal injury, with LVS-adulterated cocaine use. Nevertheless, there are no consistent data about toxicity of LVS. Objective: The aim of this study was to evaluate acute and subchronic toxicity of LVS in Wistar rats. Methods: This study was approved by CEUA/UFRGS (protocol nº: 34357). Male adult Wistar received saline (control group) or LVS at the doses of 12 mg/kg, 24 mg/kg and 36 mg/kg in the acute toxicity assay (n=5/group) (adapted from protocol OECD 420); and at 3 mg/kg, 6 mg/kg and 12 mg/kg in the subchronic toxicity assay (n=10/group) (adapted from protocol OECD 407). All the administrations occurred by intraperitoneal route. Doses were calculated through the relative doses usually ingested by users considering recent studies and then, it was extrapolated for the treatments. Toxicity was investigated through behavioral (rotarod, spontaneous locomotor activity and novel object

recognition test), renal, hematological, biochemical, and histopathological parameters. Results: In the acute toxicity assay we observed behavioral alterations in all LVS groups (p < 0.05), decreased GSH levels and urinary alterations in LVS 24 mg/kg group (p < 0.05), and death (p < 0.05) and macroscopic alterations in LVS 36 mg/kg group. Moreover, in the subchronic toxicity assay we observed behavioral alterations in the rotarod test in LVS 6 and 12 mg/kg groups (p ≤ 0.001 and p < 0.0001), decreased recognition index in short and long-term memory tests in LVS 6 mg/kg group (p < 0.05), increased liver relative weight in LVS 6 and 12 mg/kg groups (p < 0.0001), increased ALP activity in LVS 6 mg/kg group (p < 0.05), increased erythrocytes GSH levels in LVS 12 mg/kg group (p < 0.01), and decreased total thiols levels in the liver of all LVS groups (p ≤ 0.0001). Besides, it was also observed some hematological, urinary, and macroscopic alterations in all LVS groups (p < 0.05). Conclusion: LVS promoted toxic effects and its use as a cocaine adulterant is unsafe. We hope that our results contribute to the clinical practice by helping to correctly diagnose the unusual clinical manifestations found in cocaine users and avoiding more critical complications. Acknowledgments: CREAL-UFRGS, CNPq, CAPES, FAPERGS (Process nº 33416.465.34568.26032018), and PROPG/UFRGS for the support.

Levamisole, a cocaine adulterant, promotes acute and subchronic toxic effects in wistar rats

laurenTino, ana olívia MarTinS1,2; SaloMón, janaína1; SeBBen, viviane3; TonieTTo, Bruna DucaTTi4; ceSTonaro, lariSSa vivan4; Dallegrave, eliane5; garcia, Solange4; arBo, Marcelo DuTra4; leal, Mirna Bainy1

1 Programa de Pós-graduação em Ciências Biológicas: Farmacologia e Terapêutica da Universidade Federal do Rio Grande do Sul; 2 Curso de Psicologia da Universidade do Sul de

Santa Catarina; 3 Centro de Informação Toxicológica do RS, 4 Programa de Pós-graduação em Ciências Farmacêuticas da Universidade Federal do Rio Grande do Sul; 5 Programa de Pós-

Graduação em Patologia da Universidade Federal de Ciências da Saúde de Porto Alegre.

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Phthalates represent a class of molecules detected in different concentrations in breast milk, in the urine of pregnant women, and in the maternal-fetal tissue. Previous studies have shown that perinatal exposure to isolated phthalates increased susceptibility to prostatic perturbations. Thus, the study seeks to investigate proteomic profile and identify the most important pathway altered on ventral prostate (VP) of rats exposed to a mixture of phthalates in the perinatal period (F1), as well as their descendants (F2). For this, Sprague-Dawley pregnant rats were exposed to different concentrations of phthalates mixture (C: control; vehicle; T1:20µg/kg/day and T2:200mg/kg/day). The pregnant females received treatment by oral route from gestational day (GD) 10 to postnatal day (PND) 21. Exposed and unexposed animals were mated to obtain the F2 generation. Male rats were euthanized on PND22 and PND120 (F1) and PND22 (F2). At the end of the treatment, VPs from all groups were collected for proteomic analysis. The data were analyzed by string platform, to create a PPI network and enrichment analysis; PiNet platform (to show interaction with kinases) and WebGestalk platform

were performed to evaluate the pathway enrichment. The results showed a great number of downregulated proteins among treatments and generations. Among them, Rabs, Histone and, Chaperones clusters were altered in both treated groups and generations. Fourteen downregulated proteins interacted with one or more Kinase (39), that are involved in VEGF, Wnt and GnRG signaling, and Aldosterone synthesis and secretion. Furthermore, there were five proteins (Ywhae; Ywhag; Ywhaz; Hsp90ab1 and Hsp90b1) which have enriched for the Pi3k-Akt signaling pathway, the most deregulated pathway in prostate cancer and other prostatic disorders. In conclusion, the exposure of fetuses and newborns to this mixture, containing the most abundant phthalates found in the urine of pregnant women, alter proteins and cell pathways related to prostate homeostasis, increasing the susceptibility for developing prostatic diseases in F1 and F2 generations. Ethical approval: 1040/CEUA. Funding support and Acknowledgments: FAPESP: (2017/08306-2; 2018/50002-3; 2018/09510-5 and 2019/13823-1) and CAPES.

Maternal exposure to a phthalate combination associated with prostate

lesions and cancer in F1 and F2 offspring

aquino, ariana MuSa1; coSTa, luiz guilherMe alonSo1; SanToS, Sérgio alexanDre alcanTara1; MagoSSo, naTália1; Souza, paTrick vieira1; rocha, vaneSSa aguiar1; oliveira, MarcoS anTônio FernanDeS1; BarBiSan,

luiS FernanDo1; juSTulin junior, luíS anTonio1; FlaWS, joDi a.2; Scarano, WellerSon roDrigo1

1 Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil; 2 Department of Comparative

Biosciences; University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

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Organisms exposed to methylmercury (MeHg) usually present morphological, metabolic and behavioral damage, and the central nervous system is an important target of this metal. However, the effect of MeHg on heart development remains poorly explored. The heart is the first functional organ in vertebrate embryos, modeling itself from a single-chamber tubular structure to a four-cavity organ. Thus, the aim of this study was to investigate the effects of a single dose of MeHg on heart morphology and on cardiomyocytes compartments in cardiac ventricles, using Gallus domesticus embryos as a model. Fertilized eggs were incubated at 37.5ºC (± 0.5) and 65% humidity. At 33 hours of incubation (i.e., E1.5, corresponding to the developing heart with a single chamber), embryos were exposed in ovo to 0.1 µg MeHg/50 µL saline solution, being analyzed at 10th embryonic day (E10, corresponding to the developing heart with four chambers) (n = 45 embryos). Control embryos were exposed to 50 µL of saline solution at E1.5 and analyzed at E10 (n = 45 embryos) (Ethics Committee of the Federal University of Santa Catarina, CEUA - protocol 5843231018). For morphological analysis, the heart was submitted to light microscopy and transmission electron microscopy techniques. Initially, a significant reduction in heartbeats was observed after exposure to MeHg (76 ± 6 heartbeats/min) compared to the control (104 ± 8 heartbeats/min; p < 0.01). About 60% of embryos exposed to MeHg showed changes in the tissue organization of the heart. Therefore, changes in the morphology of most embryos exposed to MeHg were observed, such as differences in the

organization of the walls of the cardiac ventricles and in the appearance and consistency of the trabecular muscle fibers. Moreover, after exposure to MeHg, a reduction in the thickness of the left ventricular wall was observed (61.78 μm; ± 17.86) compared to the control (154.83 μm ± 25.90; p < 0.05), while for the right ventricle no differences were observed. Additionally, MeHg induced in 100% of exposed embryos, the appearance of ultrastructural changes in the ventricular trabeculae, as well as subcellular changes in mitochondria (dilatations in mitochondria ridges), increase in perinuclear space and increase in the number of vesicles (electrontransparent, electrondense and autophagic). These results demonstrate that exposure to MeHg was able to promote important cellular changes in the walls of the heart chambers. Morphological changes, such as the observed reduction in trabecular thickness, have been shown to compromise the structure of trabecular cardiomyocytes. These alliterations may be closely related to the reduction in heart rate. Furthermore, the interference of MeHg in the heartbeat of embryos is a determining factor for the development of the embryo. The morphological evaluation performed in this study can be used as an indication of patterns for visualization such as biomarkers. We would like to thank the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Laboratório Multiusuário de Estudos em Biologia (LAMEB) and the Laboratório Central de Microscopia Eletrônica (LCME).

Methylmercury toxicity induces structural and cellular damage in cardiac ventricles

krüger, naThália ronconi zilli; pinheiro, jacqueline; SiMioni, carMen; nazari, eveliSe Maria

Laboratório de Reprodução e Desenvolvimento Animal / Universidade Federal de Santa Catarina (LRDA/UFSC).

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Background: The use of pesticides is of concern because of the possible toxic effects on human health. Among herbicides, Diuron, a compound derived from urea, is the twelfth best-selling herbicide in Brazil, despite its classification by the North American Environmental Protection Agency (USEPA) as “probable carcinogen to the human species”. Successive studies on the potential carcinogenic mode of action (MoA) of Diuron in the urothelium of rats have been published by our research group TOXICAM in journals with selective editorial policy abroad. However, it remains unknown what is the mechanism of cytotoxic action of this herbicide, and which of its forms is responsible for cytotoxicity, whether the parental Diuron or any of its metabolites. Objective: Considering that the initial biotransformation of Diuron occurs in the liver, the present study aims to assess the in vitro damage induced in mitochondria isolated from the liver after exposure to Diuron and its metabolites, DCA and DCPMU. Methods and Results: Hepatic mitochondrial isolation was performed by differential centrifugation and the mitochondrial membrane potential was analyzed using safranine-ο as a probe; a significant decrease in the membrane potential was observed at highest concentration. Mitochondrial Respiration was monitored polarographically in

an oxygraph equipped with a Clark electrode using succinate and glutamate and malate as oxidizable substrates. Mitochondrial swelling was assessed by the decrease in the apparent absorbance of the suspension of mitochondria resulting from a decrease in the turbidity of the suspension. Since mitochondrial swelling was observed after exposure to DCA 500 µM, the effect of substances against mitochondrial swelling modulators was evaluated: Cyclosporin A (3 μM), Ruthenium Red (0.5 μM and 3 μM) , N-Ethyl-Maleimide (NEM) (15 μM) and N-acetyl-cysteine (NAC) (12 μM). Discussion/Conclusion: Our findings suggest that Diuron and its metabolites can be classified as mitotoxicants, inducing uncoupling of oxidative phosphorylation, which was evidenced by the dissipation of the mitochondrial membrane potential and the consumption of basal oxygen. In addition, DCA appears to play an important role in the chemical toxicity, since the metabolite caused mitochondrial swelling at 500 µM, a morphofunctional indicator of severe organelle damage. Acknowledgments: Supported by São Paulo Research Foundation (FAPESP) [Grant No. 2017/25402-5] and National Council for Scientific and Technological Development (CNPq) [Grant No. 133611/2019-1].

Mitochondrial injury induced by Diuron and its metabolites in the rat liver

SeloTo Danielle gaBriel1,2; liMa Thania roSSi rioS roSSi1,2; caMargo joão lauro viana1,2; pereira lilian criSTina2,3.

1 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM), Botucatu, São Paulo, Brazil;

3 São Paulo State University (Unesp), School of Agriculture, Botucatu.

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Background: Benzo(a)pyrene (BaP) is a ubiquitous chemical and has recently been classified as an endocrine disruptor. In our research lab it has already been shown to have harmful effects on the reproduction of males exposed to BaP, but whether these changes persisted until F2 generation in their female offspring was still unknown. Objective: to evaluate the reproductive repercussions of F2 generation in female offspring rats after exposure to BaP via paternal germ cells. Methods: Pubertal males were allocated to two experimental groups: control (vehicle) and BaP-exposed (0.1 μg/kg) during post-natal day (PND) 23 to 53 by gavage; and in adulthood they were mated with unexposed females to generate F1. The males of the F1 generation, in adulthood, were mated with unexposed females to generate F2. In this study, we evaluated the sexual development of the female rats, as well as estrous cyclicity, fertility potential, sexual behavior, hormone dosage, and the histological structure of the ovaries and uterus. All data obtained in this analysis were submitted to statistical tests. Results: The anogenital distance (AGD) were unchanged in all measurements, but the females of the BaP-group showed a decrease in weight on PND1 and 13. There was a precocious

vaginal opening and first estrus in the BaP-group. The frequency of lordosis in female sexual behavior was not significantly altered, but the score of lordosis was decreased in the BaP-group. In addition, the fertility of the F2 female offspring decreased when compared to control, as well as an increase in placental weight and pre-implantation loss rate. The percentage fetuses large for gestational age was increased in the BaP-group. Estradiol, progesterone, FSH and LH levels remained similar between the experimental groups. In the follicular composition of the ovarian on PND 90, the percentage of atrophic follicles increased and the corpora lutea had an abrupt decrease in the BaP-group. The morphometric measurements of the uterus showed an increase in myometrial thickness and luminal area in the BaP-group. Discussion/Conclusion: We can conclude that F2 generation showed impairment reproductive parameters, with negative transgenerational impacts in this experimental models, probably due to changes in the germ cells of the generation directly exposed to benzo(a)pyrene by epigenetic mechanisms (next analyses). Ethics committee, nº 1148/2019. Acknowledgment: CNPq – 140826/2019-0; FAPESP - 2019/03264-5; 2019/03284-6.

Paternal Origins of Health and Disease: BaP exposure via paternal germ cells causes

negative reproductive consequences in female offspring (F2) in rats

jorge, BárBara caMpoS1; STein, julia1; reiS, ana carolina caSali1; nogueira, jéSSica Bueno1; paSchoalini, BeaTriz rizzo1; Moreira, Suyane Da Silva1; Manoel, BeaTriz De

MaToS1; kaSSuya, cânDiDa apareciDa leiTe2; arena, arielle criSTina1,3

1 Department of Structural and Functional Biology. PPG Pharmacology and Biotechnology, Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo State, Brazil; 2 College of Health Science, Federal University of Grande Dourados, Dourados,

Mato Grosso do Sul, Brazil; 3 Center of Toxicological Assistance (CEATOX), Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo, Brazil.

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Introduction: Dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) are decomposition products of dichlorodiphenyltrichloroethane (DDT). These compounds are equivalent in the aspects of environmental contamination and toxic effects. Despite extensive knowledge about these substances, investigations are needed on the synergism of DDD and DDE at trace concentrations, simulating environmental conditions of exposure to multiple generations. Objectives: Describe the pathological findings of multiple generations of Wistar rats exposed to trace concentrations of organochlorine residues of DDT. Methodology: The study (CEUA/Unioeste protocol n. 21-20) was carried out with two generations (F1 and F2) of Wistar rats, exposed to trace concentrations of organochlorine DDT residues (DDD and DDE), since the period intrauterine until the end of life. These offspring were obtained from parental generations (10th week), not consanguineous. The groups were defined from day 0 of pregnancy as: Control, DDD (0.0147 µM of DDD), DDE (0.0056 µM of DDE) and DDD and DDE (association of the two substances). The placement of substances was carried out via water ad libitum, continuously, throughout the life of the multigenerations. At the end of the 5th and 15th week of life of the F1 and F2 generations, males (n=6-8 per group) and females (n=6-8 per group) were euthanized with specific anesthetics, followed by exsanguination and submitted to autopsy for identification of morphological changes in internal organs (heart, kidneys, liver, pancreas, brain, adipose tissue and organs of the male and female reproductive system). Cases of urinary bladder stones and their frequencies were compared between groups, in each generation, using the Chi-Square test for k proportions, followed by Marascuilo test. Histopathological studies were performed according to the identification of macroscopic findings and

described qualitatively. Results: We identified the presence of pathological morphological changes in the renal and hepatic systems. In a male from the DDD group (5th week, F1) the presence of changes in appearance, loss of turgidity and asymmetry in size and weight of the right kidney (1.76x1.2 cm; 0.842 g) in relation to the left kidney (1.48 x0.89 cm; 0.530 g). In the 15th week of both generations, urinary bladder stones were found, which more frequent in the DDE group (F1 = 57%; F2 = 33%) when compared to the other groups, with significant differences between this group and Control, especially in F1 generation (p=0.048). Regarding the hepatic system, in the 15th week of F2 generation, the presence of liver nodules was identified in a male from the DDE group and a female from the DDD/DDE group. The nodules were similar, both sessile, measuring approximately 343 mm3, with a translucent surface and serous-looking liquid content. Through histopathological analysis, a cavity surrounded by dense fibrous connective tissue was observed, associated with intense angiogenesis and chronic inflammation, predominantly plasmacytic, with Russell bodies and eosinophils. Additionally, the presence of xanthomatous macrophages was observed bordering the cavity lumen, which was filled with mucoid material, suggesting a possible evolving hepatitis. The other analyzed organs showed no morphological alterations. Discussion/Conclusion: Studies show the residues of the organochlorine DDT, mainly with regard to DDE, as inducers of nephrotoxic and hepatotoxic effects. However, the mechanisms and toxic effects generated by long-term exposure to low concentrations still generate gaps, especially with regard to the chronicity of the effects in multiple generations. Thus, although these findings are preliminary, these described pathologies suggest possible relationships with exposure to trace concentrations of DDT residues. Acknowledgments: Cardiff University, GCRF, CNPq.

Pathological findings arising from continuous exposure to trace concentrations

of organochlorine DDT residues in multigeneration of wistar rats

guiMarãeS, ana Tereza BiTTencourT1; Silva, FernanDa colerauS1; quiozini, naThaly De MaToS1; SiMon, jaqueline1; pavlak, jaíne luana1; azeveDo, caMilla De Marchi SancheS1; rangel, ana lúcia carrinho ayroza2

1 Department of Biosciences and Health, Universidade Estadual do Oeste do Paraná, UNIOESTE, Brazil. 2 Department of Oral Pathology, Universidade Estadual do Oeste do Paraná, UNIOESTE, Brazil.

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Background: Massive amounts of synthetic dyes and pigments are used around the world. In textile dyeing, the consumption of fresh water contributes to shortages and contamination of freshwater bodies. Also, the dye synthesis is problematic when it requires the use of toxic chemicals such as heavy metals. Additionally, certain workers can become sensitized to these compounds, and the incidence of some cancers is higher in highly exposed workers. Due to these problems, a new take on the natural colorants is emerging. The goal is to develop biodegradable, renewable, and sustainable alternatives to synthetic dyes. There is a long tradition of harvesting colorants from natural sources. Some of these include fungal anthraquinones and bisorbicillinols, and indigo from plants. More recently, some of the color-coding genes have been identified. They could be inserted to a host, enabling colorant production in large-scale tanks, reducing the need for farming land. In addition, waste material could be deployed following circular economy principles. Several colorants can also be used in waterless dyeing media, supercritical CO2, reducing the need for freshwater resources. One dye source of interest is Cortinarius sanguineus fungus, a producer of several Sc-CO2 soluble anthraquinone dyes. Objective: Despite their commercial potential, almost nothing is known about the toxic properties of the anthraquinones produced by C. sanguineus. Our aim was to study their toxicity in vitro to assess their acceptability for widescale use. Methods: First, dermocybin and dermorubin were extracted from bloodred webcap by multiple liquid-liquid partition and their purity was verified by HPLC-DAD-MS and NMR. Their genotoxic potential was studied in five strains of Salmonella typhimurium in a miniaturized Ames test protocol with and without S9 fraction. Thereafter, the dyes were studied in human HepG2 hepatocytes and human THP-1 monocytes. The cells were exposed to six dermocybin and dermorubin

concentrations of 0.035-7 and 0.03-10 µg/ml, respectively. Following the exposure, cell viability was studied with MTT and lactate dehydrogenase (LDH) assays, cytotoxicity with propidium iodide–digitonin (PI-DI) assay, and oxidative stress endpoints with H2DCFDA (DCF), dihydroethidium (DHE), and MitoSOX assays. In addition, their allergic potential was tested with KeratinoSens, a reporter gene assay kit which follows OECD in vitro skin sensitization guidelines. Results: High-purity fungal dyes were obtained. Neither caused genotoxic effects. Dermocybin caused slight cytotoxicity in HepG2 cells in the LDH assay at the two highest concentrations, and reduced the HepG2 cell viability in MTT test to 76.7% at the highest concentration of 7 µg/ml. Dermocybin also caused mitochondrial superoxide production in both cell lines at the two highest concentrations, 3.5 and 7 µg/ml. Dermorubin results were similar: some cytotoxicity was detected in LDH assay with both HepG2 and THP-1 cells at the concentrations of 1-10 µg/ml. No effects were seen in other viability tests. Dermorubin also caused mitochondrial superoxide production in THP-1 cells at the 10 µg/ml concentration. In KeratinoSens assay, neither dye caused the sensitization pathway activation. Conclusions: As the interest in the natural dyes increases, the need for toxicity studies arises. Natural origin is not a safety guarantee. Our results offer new information on the potential toxicity of natural anthraquinone dyes. However, more studies are needed on natural anthraquinone mixtures, which is normally the form of the dyes when first isolated from plants. Their ecotoxicological potential should also be evaluated. Acknowledgments: This research is a part of the BioColour (Bio-based Dyes and Pigments for Colour Palette) Consortium funded by the Strategic Research Council at the Academy of Finland. In addition, this study was funded by FAPESP and CAPES. A travel grant was awarded by the UEF Water research community.

Production and in vitro toxicological studies of natural dyes

yli-öyrä, johanna1; herrala, Mikko1; alBuquerque, anjaina FernanDeS2; FariaS, naTália oliveira2; MoraleS, Daniel alexanDre2; räiSänen, riikka3; FreeMan, harolD S.4; uMBuzeiro, giSela De aragão2,4; rySä, jaana1

1 School of Pharmacy, University of Eastern Finland, Kuopio, Finland; 2 School of Technology, University of Campinas, Limeira, Brazil; 3 Helsinki Institute of Sustainability Science, Craft Studies, University of Helsinki, Helsinki, Finland; 4 Wilson College of Textiles, North Carolina State University, Raleigh, USA.

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Background: Lisdexamfetamine Dimesylate (LDX), is the newest psychostimulant drug with prolonged action and the first and unique prodrug of dextroamphetamine (D-AMF) used in the treatment of attention deficit hyperactivity disorder, a condition most associated with children and adolescents, with a higher incidence in male sex. This treatment aims to increased neurotransmission levels, like dopamine and noradrenaline, improving the symptoms of this disorder and bringing a lot of benefits. On the other hand, this can compromise hormonal pathways, affecting the release of hormones, including those related to reproductive function, which can negatively impact the reproductive system of this individuals. Besides that, the gonads are susceptible to direct influence of neurotransmitters derived from local synaptic innervation or brought by the circulation, so, altered levels may impact its normal function. Objective: evaluated the impacts of LDX in reproductive and health parameters in adulthood of male rats exposed to this drug on peripuberty. Methods: Male Wistar rats (23 days old) were divided into control group (deionized water) and three groups exposed to LDX in therapeutical doses (correct by body surface of the rat): 5.2; 8.6 and 12.1 mg/kg/day. The treatment occurred from post-natal day 23 (PND) to 53, by gavage. During the treatment, puberty onset, an important reproductive parameter, were evaluated. In adult life (PND 90), the animals were evaluated in relation to male sexual behavior and fertility parameters. On PND 120, the animals were killed to blood and organ collection, to evaluation of organ weight, sperm motility and hematological

parameters. Results: No experimental group had the day of preputial separation altered compared to control, but the weight of the animals in the highest dose group were reduced in this day. The male sexual behavior was not altered between the groups. In the fertility test, also in the 12,1 mg/kg animals, we found a reduced placental weight and increased percentage of fetuses small for gestational age, as well as an increased number of corpora lutea, implantations and live fetuses. The experimental groups did not differ among themselves in the organs weight. Besides that, there were an increased in type C sperm (immobile) in the animals exposed to 8,6 mg/kg of LDX and hematological alterations (reduced neutrophiles and increased lymphocyte percentage) in the lowest dose group. Discussion/Conclusion: The reduced placenta can be related to a higher incidence of fetuses small for gestational weigh, and it can be related to paternal influence since placental development depend on both from maternal and paternal influence. However, the others fertility alterations may be related to maternal facts, independent of this treatment. The altered motility may represent an important alteration in sperm function which could compromise the reproductive system, but further analyses are being performed to confirm and support its finds. Thus, so far, LDX was able to impact negatively some important parameters related to reproductive function of the animals, but other analyses should be conducted to enrich these statements. Acknowledgments: FAPESP (2021/04119-9) and Capes (88887.602916/2021-00). Ethics committee: 3148130421

Reproductive repercussions in adulthood of male rats exposed to Lisdexamfetamine

Dimesylate on peripuberty

Stein, Julia1; Jorge, Bárbara Campos1; Reis, Ana Carolina Casali1; Nagaoka, Lívia Trippe1; Manoel, Beatriz de Matos1; Valente, Letícia Cardoso1; Pupo, André Sampaio2; Arena, Arielle Cristina1,3

1 Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil. 2 Department of Biophysics

and Pharmacology, Institute of Biosciences of Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil. 3 Toxicological Assistance Center (CEATOX), Institute of

Biosciences of Botucatu, UNESP – Univ. Estadual Paulista - Botucatu, São Paulo State, Brazil.

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Leucena leucocephala is an invasive plant that has attractive properties for use in folk medicine to treat different conditions, such as diabetes, antioxidant, gastric diseases and abortifacient. Its active ingredient is L-mimosine, a metabolite that, although much investigated for its antitumor properties, is poorly studied during fetal development. Thus, we aimed to evaluate the possible maternal-fetal toxicity in Wistar rats treated with increasing oral doses of L-mimosine during pregnancy. Thirty-four pregnant rats were divided into 4 groups: control (n=8); and 3 experimental groups treated with 60 (n=9), 100 (n=8) and 140 (n=9) mg/kg of L-mimosine. which was administered orally from the 6th to the 19th gestational day (GD). The control group only received the vehicle through the same route and period. Feed and water were provided ad libitum. Every 3 days the rats’ weight and feed consumption were measured. In the 20th GD, an exploratory cesarean section was performed for maternal-fetal assessment and blood and lymphoid organs (thymus and spleen) were also collected. During the experimental period, no statistically significant changes were observed between groups in feed and water consumption (P>0.05). Likewise, the hematological and biochemical parameters were within the reference range and without statistical differences between the groups, with the exception

of the ALT enzyme, which was increased in the groups that received 60 and 100 mg/kg of L-mimosine (P<0.05). No statistical differences were observed in relation to the relative weight of lymphoid organs and spleen cellularity of these animals, showing no evidence of immunotoxicity. In relation to reproductive parameters, no changes were observed between the groups. There was a decrease (P<0.05) in weight gain (WG) of the 100 L-mimosine group between GD15 and GD18, but without impacting the total WG of these animals. Skeletal analysis showed an increase in the number of affected fetuses only in the higher dose group (140 mg/kg) and an increase in delayed ossification of the skull and in sternal and vertebral bodies variations in all treated groups, in relation to control. Thus, with the results obtained in this study of the administration of L-mimosine during pregnancy, it can be seen that the doses used were sufficient to cause skeletal variations, although they were not sufficient to change the other parameters evaluated here in a dose-dependent manner. These results suggest a negative effect of L-mimosine on the skeletal development of the animals, which may be an indication of toxicity on the offspring. Complementary visceral analysis studies will help to understand these results. Keywords: immunotoxicity, skeletal variations, phytotoxin

Skeletal abnormalities caused by L-mimosine: development toxicity study in rats

alMeiDa, elaine renaTa MoTTa1; pereira, eDiMar criSTiano2; hueza, iSiS MachaDo1,2

1 Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, S.P., Brasil; 2 Instituto de Ciências Ambientais, Químicas e

Farmacêuticas, Universidade Federal de São Paulo (ICAQF-UNIFESP), Diadema, S.P., Brasil.

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Vaccination is considered one of the greatest techniques in terms of disease control and eradication. However, with the decrease in cases of vaccine-preventable diseases, the population’s attention has turned to the potential adverse effects caused by vaccines, especially concerning preservatives and metal-based adjuvants used in their composition (thimerosal and aluminum hydroxide, respectively). Based on this, this study evaluated the effects during 24 h, 96 h and 21 days after the exposure to thimerosal and aluminum hydroxide (isolated or in mixture) in kidney of Danio rerio (zebrafish) through biochemical biomarkers. The zebrafish were divided into four groups (n=30 per group) and exposed to the compounds intraperitoneally. The groups were: control (saline), thimerosal (TMS - 7.5 mg/kg), aluminum hydroxide (Al - 175.0 mg/kg) and mixture (TMS+Al - 7.5 mg/kg of TMS + 175.0 mg/kg of Al). Concentrations were chosen after conducting a pilot study, and the highest concentration that did not cause animal mortality within 96h was selected for this study. The bioassay was divided into three periods: 24 h, 96 h, and 21 days. After this, the animals were anesthetized with 0.001% benzocaine and euthanized by spinal section. The kidney was removed for analysis of the enzymes superoxide dismutase (SOD) and glutathione S-transferase (GST); and, for the levels of reduced glutathione (GSH) and metallothioneins (MT). The results were statistically analyzed using the Graph Pad Prism 5. For homogeneous data, one-way ANOVA was performed, followed by Dunnett’s test. The Grubbs test was performed to remove the outliers. The results were considered statistically significant when p<0.05. After 24 h of exposure, fish exposed only to Al showed a significant increase in renal SOD activity [F (3, 23) = 6.334; p = 0.0027] and

GST [F(3, 22) = 6.336; p = 0.0029] when compared to the control group; and animals exposed only to TMS showed a significant decrease in renal GSH levels [F(3, 22) = 4.954; p = 0.0089]. There was no change in MT levels. Ninety six hours after the exposure, animals exposed intraperitoneally to Al (Al and TMS+Al groups) showed a significant decrease in SOD activity [F(3, 35) = 14.15; p < 0.0001] and MT levels [F(3, 8) = 4.724; p = 0.0351] when compared to the control group. The GST and GSH biomarkers were not altered. Finally, after 21 days of exposure, animals exposed to Al have a statistically significant decrease in renal SOD activity [F (3, 33) = 15.51; p<0.0001] and renal GSH levels [F (3 35) = 3114; p<0.0385] when compared to the control group. Animals exposed to TMS + Al had a statistically significant decrease of renal SOD activity [F (3, 33) = 15.51; p<0.0001]. There was also a statistically significant increase in GST renal activity [F (3, 34) = 3.702; p = 0.0209] in animals exposed to TMS. Renal MT levels were not altered. Exposure to metals present in the composition of vaccines, thimerosal and aluminum hydroxide, caused biochemical changes in Danio rerio kidney when evaluated alone or in a mixture, and these alterations remain up even 21 days after the exposure. The increase observed in the first hours may indicate an adaptive response of the organism and the inhibition observed in the 21 days probably occurs because the compounds are bioaccumulated in the kidney, due to the excretion process. We emphasize the lack of scientific information on the subject and the importance of further studies to assess possible long-term effects since a better understanding of these compounds will result in a better understanding of the possible adverse effects after vaccination, contributing to better solutions to the problem.

Study of the toxicological effects of thimerosal and aluminum hydroxide on Danio rerio’s kidney

galiciolli, Maria eDuarDa a.; Silva, juliana F.; guiloSki, izoneTe c.; oliveira, cláuDia S.

Programa de Pós-graduação Strictu sensu Aplicada à Saúde da Criança e do Adolescente. Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, PR, Brazil; Faculdades Pequeno Príncipe, Curitiba, PR, Brazil.

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Background: Among the substances with the potential for endocrine disruption, benzo(a)pyrene (BaP) is a persistent organic pollutant diffused in the environment in a ubiquitous way. This substance has some well-established mechanisms at high doses; however, our group has demonstrated that BaP can directly impact the sperm quality of male rats at low doses. Our study showed the potential endocrine disruptor of BaP in a highly androgen dependent phase: juvenile period and peripuberty. Objective: Thus, this study aimed to evaluate if BaP in low-dose causes immediate toxicity and testicular disorders. Methods: For this, juvenile male Wistar rats (23 days, n = 8 animals/group) were allocated in control group (corn oil + DMSO - vehicle) and BaP (vehicle + 0.1 µg/kg); and the exposure to BaP occurred for 31 consecutive days (gavage), until post-natal day (PND) 53. The clinical signs of toxicity, the body weight progression and water and food of consumption of these animals were evaluated. On PND 54, the rats were killed for immediate toxicological and testicular histological evaluation. The organs of these animals were weighted and blood collection to hematological, hormone and biochemical tests. All data obtained in this analysis were submitted to statistical tests. Results: During the treatment, the animals showed no clinical signs of toxicity; however, the body

weight gain of BaP-treated group was reduced when compared to control group although water and food intake is not altered. On PND 54, the relative weight of kidney and liver were decreased. The leukocyte counts also decreased in treated group and the cholesterol have biological increased. The tubules seminiferous diameter and height was altered in treated group, as well as Leydig cells atrophy. Testosterone was decreased in BaP-group and FSH was increased. Discussion/Conclusion: The data on immediate systemic toxicity at low doses of BaP were similar when compared to high doses already established in the literature. Although indirect involvement due to systemic toxicity cannot be ruled out, the decrease in testosterone in the present study is key to support the hypothesis that BaP can occupy the steroidogenic acute regulatory protein (StAR) receptor, preventing cholesterol from entering the mitochondria at the proper physiological rate. This hormonal decrease negatively affects the reproductive system and testicular structure of the animal. Expression of steroidogenic enzymes and StAR are being performed to support our hypothesis. Approved by ethics committee, nº 1148/2019. Acknowledgment: CNPq – 140826/2019-0; FAPESP - 2019/03264-5; 2019/03284-6.

Systemic toxicity and testicular damage produced by exposure to benzo(a)pyrene in low-

dose from juvenile to peripuberty in male rats

jorge, BárBara caMpoS1; reiS, ana carolina caSali1; STein, julia1; paSchoalini, BeaTriz rizzo1; nogueira, jéSSica Bueno1; arena, arielle criSTina1,2

1 Department of Structural and Functional Biology. PPG Pharmacology and Biotechnology, Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São

Paulo State, Brazil; 2 Center of Toxicological Assistance (CEATOX), Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo, Brazil.

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2,4-D is one of the most used herbicides in agriculture, despite its classification as “possibly carcinogenic to humans”. Some of its components in commercial formulations have high lipophilicity, resistance to degradation and bioaccumulative potential, being evaluated as persistent organic pollutant (POP), probably at high levels in the environment (air, water and sediments), biota and humans. In this context, the objective of this study was to elucidate the potential teratogenic of the herbicide 2,4-D (DMA® 806), using embryonic stage of zebrafish (Danio rerio) as a model organism. Embryo’s evaluation followed Fish Embryo Toxicity (FET) Test n. 236 (OECD, 2013). After the fish reproduction, 140 fertilized eggs in the blastula stage were collected and placed in 24-well culture plates. Assays were performed in triplicate, and each replicate consisted of 24 embryos for the negative control group (ISO-7346 standard water), 20 embryos for the positive control group (4 mg/L 3,4-dichloroaniline) and 20 embryos for each concentrations of 2,4-D (DMA® 806): 0.1; 0.4; 1; 10; 100 and 200 µM. The culture plates were incubated at 26 ± 1°C, in cycles of 14 hours light and 10 hours dark and the development of zebrafish embryos and larvae were evaluated at 8, 24, 48, 72, 96, 120 and 144 hpf (hours post fertilization) through of a stereomicroscope. Mortality, yolk sac edema, swim bladder inflation defects, pericardial edema and caudal fin deformation were identified. The values were submitted to the analysis of variance test

(One-way ANOVA) and a posteriori Dunnet’s test. After 144 h of exposure, the negative and positive controls resulted in compliance with the test (mortality≤10% and mortality≥30%, respectively). There was 15% embryo mortality in the maximum concentration treatment (200 µM) 144h, with no difference, as in all other concentrations. Regarding to the deformities Non-Inflated Swim Bladder, Caudal Fin Deformity, Pericardial Edema and Yolk Sac Edema, there was not differences in the lowest concentrations tested (0.1, 0.4 and 1 µM). Despite of this results, these treatments had adverse effects. In all type of deformities observed, there were significant differences (p<0.05) in the treatments of concentrations 100 and 200 µM in 100% of the embryos in these two concentrations. The yolk sac edema deformation, in addition to the differences obtained at concentrations of 100 and 200 µM, there was also significant (p<0.05) in the treatment of 10 µM concentration in 50% of the embryos. This last result evidences the susceptibility of zebrafish to this pesticide, as it is a concentration of the commercial formula 2,4-D (DMA® 806) that approaches values found in the environment, being a substance with teratogenic potential, requiring further studies that will be done in our laboratory (TOXICAM). Acknowledgments to Coordination for the Improvement of Higher Education Personnel - (CAPES) and TOXICAM.

Teratogenic and toxic effects induced by the herbicide 2,4-D (DMA® 806) in

zebrafish (Danio rerio) embryos

viriaTo, criSTina1,2; anDraDe, íTalo B.l.2,3; peixoTo, paloMa v.l.2,3; pereira, lílian c.2,4

1 São Paulo State University (Unesp), Institute of Biosciences, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM); 3 São Paulo State University (Unesp),

Medical School, Botucatu; 4 São Paulo State University (Unesp), School of Agriculture, Botucatu.

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Introduction: Given the modest sperm antioxidant defenses and knowing that excessive damage in male reproductive organs can lead to infertility. All aspects of sexual reproduction can be affected if the balance of free radicals and antioxidant defense goes awry. That way, seeing the organochlorine dichlorodiphenyltrichloroethane (DDT) and its residues, Dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), are associated to metabolic alterations, causing free radical productions and oxidative stress. The present study becomes relevant to evaluate if the spermatozoa motility was affected by oxidative system changes induced by continuously exposed to DDT residues. Objective: Evaluate the association of oxidative system to sperm motility in Wistar rats continuously exposed to DDT residues. Methodology: The study (CEUA/Unioeste protocol n. 21-20) was performed with one generation of Wistar rats, exposed to trace concentrations of organochlorine DDT residues (DDD and DDE), since the intrauterine period until the end of life. The offspring was obtained from parental generation (10th week), not consanguineous. The groups were defined from day 0 of pregnancy as: Control, DDD (0.0147 µM of DDD), DDE (0.0056 µM of DDE) and DDD/DDE (association of the two substances). The placement of substances was carried out via water ad libitum, continuously, throughout the life. At the end of 5 and 15 weeks of age, males (n=6-8 per group) were euthanized with anesthetics, followed by collected of right testicle and sperm samples through the deferens ducts. The oxidative parameters, peroxidase glutathione (GPx), glutathione-S-transferase (GST), reductase glutathione (GR), superoxide dismutase (SOD), catalase (CAT) and lipoperoxidation (LPX), were measured in the testicle samples. The spermatozoa motility was evaluated at the 15 weeks of age, differentiating into

motile and immotile spermatozoa. Data collected at 5 and 15 weeks of age were compared between groups using one-way ANOVA followed by Tukey-HSD test. Matrices of oxidative system data from 5th and 15th weeks were evaluated using Principal Component Analysis (PCA). The factor loadings resulting from the first two dimensions of each data matrix were later related to the number of immotile spermatozoa, using linear models evaluated through Analysis of Variance (α=0.05). Results: There were no significant differences in the variables means among the groups at 5 and 15 weeks of age (p>0.05). However, when performing the integrative assessment (PCA) at the 5th week of age, opposite linear relationships between the number of immotile spermatozoa and GST and GR activities were observed, and direct linear relationships between the number of immotile spermatozoa and SOD activity. The results indicate that the changes in antioxidant function at 5 weeks of age in animals exposed to DDD/DDE led to the increased number of immotile spermatozoa observed at 15 weeks of age. The integrative assessment at the 15th week of age showed the direct linear relationship among the number of immotile spermatozoa and SOD, CAT and GR activities. The results indicate that an induction of antioxidant system at 15 weeks of age, especially in the DDE group, led to the increased number of immotile spermatozoa. Conclusion: The testis is a site of intense cell division and relies heavily of NADPH, hence it is susceptible to oxidative stress and depends of antioxidant enzymes to repair the damages that can affect the spermatozoa. In this way, knowing that DDT can contribute to oxidative damage in different tissues, the results showed like small changes in oxidative system can affect the quality of sperm, contributing to possible male infertility in experimental animals exposed to DDT residues. Acknowledgments: Cardiff University, GCRF, CNPq.

The relationship between oxidative system and sperm motility in wistar rats continuously

exposed to organochlorine DDT residues

Silva, FernanDa colerauS1; quiozini, naThaly De MaToS2; SiMon, jaqueline2; SchWengBer, heloySa Talia3; leSnieWSki, iSaBela raMoS3; azeveDo, caMilla De Marchi SancheS1; iTinoSe,

ana Maria4; Marek, carla Brugin4; guiMarãeS, ana Tereza BiTTencourT1

1 Department of Biosciences and Health, Universidade Estadual do Oeste do Paraná, UNIOESTE, PR, Brazil; 2 Pharmacy, Universidade Estadual do Oeste do Paraná, UNIOESTE,

PR, Brazil; 3 Odontology, Universidade Paranaense, UNIPAR, PR, Brazil; 4 Department of Toxicology, Universidade Estadual do Oeste do Paraná, UNIOESTE, PR, Brazil.

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Introduction: The organochlorine dichlorodiphe-nyltrichloroethane (DDT) and its residues, Dichlo-rodiphenyldichloroethylene (DDE) and dichlorodi-phenyldichloroethane (DDD), are persistent organic pollutants (POPs) that are resistant to degradation and can accumulate at high levels in the environment and human body. Besides that, DDT and its residues have been associated with several pathological con-ditions including endocrine disruption and immuno-logic dysfunction. Studies reveal that DDT has es-trogen-like properties and can affect some immune cells functions. That way, the present study becomes relevant to evaluate the white blood cells behavior in multigenerations of female Wistar rats exposed to DDT organochlorine residues. Objectives: To evaluate possible changes in the white blood cells of Wistar rats exposed to trace concentrations of DDT residues. Methodology: The study (CEUA/Unioeste protocol n. 21-20) was carried out with two generations (F1 and F2) of female Wistar rats, exposed to trace concen-trations of organochlorine DDT residues (DDD and DDE), since the intrauterine period until the end of life. These offspring were obtained from parental gener-ations (10th week), not consanguineous. The groups were defined from day 0 of pregnancy as: Control, DDD (0.0147 µM of DDD), DDE (0.0056 µM of DDE) and DDD/DDE (association of the two substances). The placement of substances was carried out via water ad libitum, continuously, throughout the life of the multigenerations. At the end of the 5th and 15th week of life of the F1 and F2 generations, females (n=6-8 per group) were euthanized with specific anesthetics, followed by cardiac puncture to blood collect. White blood cells, mononuclear cells and polymorphonu-clear cells, were estimated using the hematological analyzer Mindray bc 2800 vet. White blood cell fre-quency data were evaluated using linear models, con-sidering experimental groups and time of experimen-tation as predictors. The time of experimantation was representaded of ages (5th and 15th weeks of life) in

the respective generations (F1 and F2). The models were evaluated for their significance through Anal-ysis of Variance (α=0.05). Results: When evaluating the data, it was possible to verify that during the de-velopment of two generations of Wistar rats exposed to the trace concentrations of DDD and DDE there was a white blood cells productions modulation. The ani-mals of the DDE and DDD/DDE group of the F1 gener-ation, with 5 weeks of life, showed a polymorphonu-clear cells reduction, especially neutrophils. However, in the following ages and respective generations (F1-15th week, F2-5th week and F2-15th week) there was an increase in the polymorphonuclear cells frequen-cy, indicating permanent primary defense activation (r2=0.25; F3.105=4 .50; p=0.005). In relation to mon-onuclear cells, the opposite behavior was observed, especially in the DDE and DDD-DDE groups, with an increase in the frequency of these cell types, especial-ly by lymphocytes, among the animals with 5 weeks of life of the F1 generation and subsequent decrease in the following periods, indicating a possible loss of antibody production cells capacity (r2=0.22; F3,105=2.24; p=0.088). Discussion/Conclusion: Knowing that DDT is classified as a xenoestrogen, these results are in agreement with studies which suggest that polymor-phonuclear cells alterations is partially caused by the impact of estrogen-like effect on differentiation of those cells, especially for the neutrophilic way. The main way of biological effects induction of xenoestro-gens in neutrophils is by their interaction with estro-gen receptors, being able affect neutrophilus abilities, like chemotaxis, adhesion and phagocytosis. As this way, the results suggest an important modulation of white blood cells caused by DDT residues, especial-ly for the DDE. Being important to conduct an exten-sive research and make a comprehensive assessment of DDT and its residues effect on immunocompetent cells. Acknowledgments: Cardiff University, GCRF, CNPq.

The white blood cells behavior in multigenerations of wistar rats exposed

to DDT organochlorine residues

Silva, FernanDa colerauS1; quiozini, naThaly De MaToS2; SiMon, jaqueline2; azeveDo, caMilla De Marchi SancheS1; vieira, Bruna ToDeSchini3; Marek, carla Brugin4; guiMarãeS, ana Tereza BiTTencourT1

1 Department of Biosciences and Health, Universidade Estadual do Oeste do Paraná, UNIOESTE, PR, Brazil; 2 Pharmacy, Universidade Estadual do Oeste do Paraná,

UNIOESTE, PR, Brazil; 3 Veterinary laboratory, VitaLab, PR, Brazil; 4 Department of toxicology, Universidade Estadual do Oeste do Paraná, UNIOESTE, PR, Brazil.

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Reproductive health has received special attention as the incidence rate of human infertility has increased. Exposure to environmental xenobiotics constitutes a serious risk, particularly for male infertility. Despite some evidence, however, the molecular processes underlying lower fertility remain unknown. The aim of this study was to investigate toxicological effects of Color Index Disperse Red 1 (DR1) in mice F0 and F1 sperms (sperm count, mitochondrial activity, vitality and acrosome integrity). DR1 is a textile azo dye commonly discharged into water bodies that can be consumed by humans. Herein, mice were distributed in 6 groups, including: 1 - negative control: treated with filtrated water; 2 - positive control: intraperitonealy treated with 3 doses of n-etil-n-nitrosourea (ENU; 100mg/kg body weight); 3 - vehicule control: orally

treated with 0.5% DMSO; 4, 5 and 6 - orally exposed to DR1 at doses of 5, 50 and 500 µg/kg/b.w., respectively. Treatments lasted 14 days, and mating took place 35 days later. Animals were euthanized two days after mating (F0) or at the age of eight weeks (F1). The highest DR1 dose (500 g/kg/b.w.) reduced sperm count in both F0 and F1 mice; mitochondrial activity damage was observed in all DR1-treated groups of animals. Changes in sperm vitality and acrosome integrity were also observed in F1 mice. In conclusion, DR1 was able to cause transgenerational damage, implying that water contamination with these substances could be hazardous to human health. Acknowledgments: CNPq (141322/2020-9) and CNPq-INCT DATREM (465571/2014-0).

Toxicological effects of Color Index Disperse Red 1 textile azo dye in sperm of mice orally exposed

TanaMachi, aManDa roDrigueS1; FernanDeS, FáBio henrique1; liMa, geovana criSTina riBeiro1; Mariani, noeMia apareciDa parTelli2; Silva, alan anDreW DoS SanToS2; Silva, erick joSé raMo2; SalvaDori, DaiSy Maria Fávero1

1 Medical School, UNESP - São Paulo State University, Botucatu, SP, Brazil; 2 Institute of Biosciences of Botucatu, UNESP - São Paulo State University, Botucatu, SP, Brazil.

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Introduction: The use of medicinal plants has been a therapeutic alternative for thousands of years, mainly in Middle Eastern countries and Asia in the treatment of health disorders, prevention of disease epidemics and microbial control, with more than 235 species of plants medicinal plants do not have a detailed study of their beneficial health effects. Crude extracts of medicinal plants are the most used form in research both in vitro and in vivo studies, in addition to the use of isolated bioactive compounds in order to evaluate possible mechanisms of action. Among the medicinal plants, Eugenia uniflora L. stands out, belonging to the Myrtaceae family, whose origin is the Brazilian Atlantic Forest. It is characterized by having great economic exploitation potential for the Northeast region, in which it is effective for the treatment of various diseases, having anti-inflammatory, antioxidant, analgesic, antihypertensive, antibacterial and antifungal pharmacological activities, and its action It takes place through the consumption of its leaves and fruits, as these are where there is a source of nutrients. Objective: The objective of this work is to perform the administration and evaluation from a vehicle (vaginal gel) of E. uniflora at concentrations of 5%, 10% and 15%, in an animal model (female Wistar rats). Materials and Methods: With the approval of the UNIPAR ethics committee by protocol 38108/2020, 56 female animals were used, divided into 8 groups of 7 animals per group, where they were

evaluated for the estrous cycle, On-site Tolerance test of Administration, clinical signs such as pain, edema, pruritus, irritation, secretion and sensitivity of the vaginal gel and aqueous extract of E. uniflora, being carried out macroscopic analyzes of the metabolizing organs (liver, kidneys and spleen), histological diagnoses of the vaginal cavity, uterus and ovaries, blood count and biochemical analyses, estradiol and progesterone dosage. Results and Discussion: The results obtained after the statistical tests of the PRISMA program using the one-way ANOVA test followed by the post-bonferroni test, against the parameters evaluated for estrous cycle, macroscopic analysis and relative weight of liver, kidneys and spleen, together with histology of the reproductive system (ovaries, uterus, vaginal cavity) and local tolerance test were not significant and there were no changes in blood count, biochemical and hormonal tests. Conclusion: Due to the arguments presented in the study, it is concluded that, after carrying out the tests in the vaginal region with the gel and extract at concentrations of 5, 10, 15%, they did not show significant differences in terms of hematological, biochemical, hormones, estrous cycle and sensitivity in the region of administration. However, the use of Eugenia uniflora gel or extract for the vaginal region does not pose a risk of toxicity in the application site and may be suggested as a dermocosmetic. Keywords: Medicinal Plants; Pitanga; Vaginal Gel; Toxicity.

Toxicological safety studies of plant extract of Eugenia uniflora L. at different safety doses

DonaDel, guilherMe1; DalMagro, Mariana2; pinc, Mariana MoraeS3; BerTa, joão anTonio4; BorBa, Maria eDuarDa4; gaSparoTTo junior, arquiMeDeS5; zarDeTo,

giuliana6; hoScheiD, jaqueline6; lourenço, eMerSon luiz BoTelho7

1 PhD student in animal science at UNIPAR; 2 student Master in biotechnology applied to agriculture UNIPAR; 3 PhD student in biotechnology at UNIPAR; 4 Undergraduate

Student Veterinary Medicine UNIPAR; 5 Professor at the University of Grande Dourados –UFGD; 6 Professor at UNIPAR; 7 Unipar Research and Graduate Coordinator.

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Background: Benzo(a)pyrene (BaP) is a chemical substance formed by the union of a molten benzene into a pyrene produced by the incomplete burning of organic compounds. Because it is ubiquitous, it can cause adverse events and behave as an endocrine disruptor at low doses; also targeting the reproductive system. Puberty, a critical period of development, is vulnerable to the effects of substances, leading to effects throughout life or in next generations. Transgenerational repercussions involving low-dose BaP exposure and reproductive aspects is still unexplored; and furthermore, we seek to fill in gaps about the importance of paternal health in offspring. Objective: to evaluate the transgenerational (F2) reproductive repercussions in male rats offspring, first generation non-exposed, which the paternal generation (F1) was exposed to benzo(a)pyrene by germ cells. Methods: For this, juvenile male Wistar rats (F0, 23 days, n = 8 animals/group) were allocated in control group (received corn oil + DMSO - vehicle) and BaP-group (exposed to vehicle + 0.1 µg/kg/day) for 31 consecutive days (gavage). In adult life, they were mated with untreated females to obtain male offspring (F1) and, later, the F2 generation, which was evaluated in relation to their initial development and reproductive parameters in male offspring (n = 8 litters/group). All data obtained in this analysis were submitted to statistical tests. Results: There was a reduction in absolute and relative anogenital distance (AGD) in post-natal days (PND) 1, 13 and 22, and

decreased on the body weight (PND 1) in F2 generation male offspring. The preputial separation was advanced in BaP-group. In adulthood (PND 90), there were no changes in male sexual behavior and total ejaculations number; however, fertility potential was reduced in BaP-group. There was a reduction in body weight and an increase of reproductive organs weight. Type A sperm percentage (progressive motile) was decreased, and Type B/C were increased (immobile) as well as in the epididymal transit time in BaP-group. In the stereological analysis of the epididymal caput, we found that the height of the epithelium was decreased while the luminal area was dilated in the BaP-group. Testosterone levels was reduced in BaP-group, as well as Leydig cells number and volume. Discussion/Conclusion: The results obtained so far on the reproductive repercussions of the male F2 generation indicate that these animals showed losses in both sexual development and fertility potential when compared to the control group. This damage was generated in the sperm of the F1 generation by indirect exposure to BaP, probably due to changes in its epigenetic pattern (next analyses) that modified the functioning of the reproductive system of the F2 generation. These data also reinforce the importance of paternal health in offspring development. Molecular analyses will be performed to confirm our mechanistic hypothesis. Ethics committee, nº 1148/2019. Acknowledgment: CNPq – 140826/2019-0; FAPESP - 2019/03264-5; 2019/03284-6.

Transgenerational reproductive effects (F2) in male offspring exposed to benzo(a)

pyrene by paternal germ cells in rats

jorge, BárBara caMpoS1; reiS, ana carolina caSali1; STein, julia1; paSchoalini, BeaTriz rizzo1; nogueira, jéSSica Bueno1; Moreira, Suyane Da Silva1; Manoel, BeaTriz De MaToS1; arena, arielle criSTina1,2

1 Department of Structural and Functional Biology. PPG Pharmacology and Biotechnology, Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São

Paulo State, Brazil; 2 Center of Toxicological Assistance (CEATOX), Institute of Biosciences of Botucatu, Univ. Estadual Paulista – Botucatu (UNESP), São Paulo, Brazil.

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Background: The herbicide triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid, CAS 55335-06-3), is already considered an environmental problem due to the high concentrations found in the environment. This fact occurred, probably, due to incorrect disposal, leaching and dispersion aerial. This type of contamination can pose risk to both environment and human health. Studies have evaluated the metabolism, absorption, excretion and active transport of triclopyr but there is no clear information about its potential mode of action (MoA), and its cytotoxic action remains unknown. In this context, mitochondria have been used to assess the toxicity of xenobiotics because it is a complex and highly regulated organelle. It is essential for the maintenance of various biological processes such as cell survival and metabolic homeostasis and, therefore, for the interaction of potentially toxic substances in various points in this system can result in mitochondrial dysfunction and consequently cytotoxicity. Objective: Identify the toxic mechanism of triclopyr in Rat liver mitochondria isolated from young Wistar rats. Methods: The exposure of isolated mitochondria with triclopyr was carried out in vitro using different concentrations between 0.5 to 500 µM as it comprises concentrations found in the environment, in biological samples of animals and humans. The formation of reactive oxygen species and nitrogen (RONS), redox state of pyridine nucleotides, dissipation of membrane potential, membrane lipoperoxidation, mitochondrial levels of membrane protein sulfhydryl (P-SH) groups, reduced glutathione (GSH) and oxidized glutathione (GSSG)

levels and mitochondrial respiration was assessed. The results were evaluated by analysis of variance (ANOVA) followed by the Dunnett test. Results: The results showed that was no statistical significance in ADP/O rate, RCR value and Oxygen consumption rate in state 3 (V3) when compared to the negative control. However, the Oxygen consumption rate in state 4 (V4) was increased at highest concentration (500 µM) demonstrating that triclopyr can has an uncoupling characteristic. However, ERONs was not detected. In addition, neither inducing the formation of malondialdehyde (MDA), nor alterations in the levels of P-SH in the mitochondrial membrane were observed. Therefore, at 500 µM was able to oxidize NAD(P)H but did not alter the redox homeostasis from GSH/GSSG ratio. Discussion/Conclusion: All results taken together suggests that it can act as an uncoupler of oxidative phosphorylation, which associated with dissipation of the membrane potential demonstrates that triclopyr is able to induce in vitro alterations on parameters of mitochondria, not involving damage related to the redox state due to the normality of the GSH/GSSG ratio, suggesting that the oxidation of NAD(P)H may be related to other contexts besides those analyzed so far. Triclopyr also does not have the properties of formation of RONS, membrane lipoperoxidation and preserves the thiol groups of the mitochondrial membrane. Acknowledgments: Coordination for the Improvement of Higher Education Personnel (CAPES) [Grant No. 88887.481968/2020-00] and São Paulo Research Foundation (FAPESP) [Grant No. 18/00229-1].

Triclopyr induces changes in mitochondrial parameters

rizzi, joyce SanTana1,2; SeloTo, Danielle gaBriel1,2; pereira, lílian criSTina2,3

1 São Paulo State University (Unesp), Medical School, Botucatu; 2 Center for Evaluation of Environmental Impact on Human Health (TOXICAM), Botucatu;

3 São Paulo State University (Unesp), School of Agriculture, Botucatu.

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Introduction: Studies involving zebrafish have highlighted genetic, anatomical, physiological and cellular similarities as well as mammals, allowing the science to expand to tests on human research. Some toxicological research, which elucidate disease prevention, has recently adopted the use of zebrafish, after proving that this model would be adequate. The small teleost stands as an alternative and innovative proposal for use in toxicological experiments and tests, and may offer several benefits, such as rapid development of the nervous system, low costs, easy handling, high level of neural homology with humans and a complex immune system with mammalian characteristics. Objectives: To identify in the literature the use of Zebrafish as an animal model for toxicological tests, highlighting its advantages and benefits. Materials and Methods: Descriptive study of the narrative type of literature, with selected articles published in the last five years. Data were obtained through a search in the PUBMED and MEDLINE databases, using the descriptors: Zebrafish, Toxicology, Animal Model. The inclusion criteria for this study were clinical trials and comparative studies, in Portuguese and English. Those who did not meet the inclusion criterion were excluded. Results: The comparative study between mice and zebrafish was carried out with the aim of evaluating the latter as a high-throughput model for a first screening of New Psychoactive Substances. Each model was exposed

to different concentrations of Methiopropamine and APINAC, and to equivalent amounts for each species, and resulted in similar behaviors. In another study, PfrHEPA also had its toxicity evaluated in different models; and their results corroborate each other, confirming that drug screening in zebrafish can become effective in toxicology, which may benefit several fields of study in the future. Another study done to observe neurotoxicity from acute exposure to acrylamine, an alkene widely used in water treatment, mineral processing and cosmetics, which showed very satisfactory results, and how it was observed that the nervous system and neurotransmitter systems are similar to humans. The zebrafish is a widely used model used in neuropharmacological research. It is observed in another clinical study, in which a library with 87 chemicals were used, as generally toxic or neurotoxic for development, or unknown, and were exposed in zebrafish and planarians; resulting in similar behavioral trends, but the zebrafish presented itself as a more sensitive indicator of bioactivity. Conclusion: It is possible to conclude that the animal model presented gives a positive response in the experiments used, revealing its scope in the types of neurotoxicological compounds that can be studied from zebrafish. Its potential is evidenced for further studies to be carried out, enabling a better assessment of the capacity of this organism.

Use of zebrafish as animal model for research for neurotoxicological

substances: a literature review

Silva, luíza MaDureira1; pinheiro, Dávylla rennia SalDanha2; Braga, áDrya lariela liMa2; holanDa, Wiron piMenTel2; honório júnior, joSé eDuarDo riBeiro3

1 Academic of Nursing in the University Center Christus, Laboratory of Neurociência Translacional-Neurocit; 2 Academic of Biomedicina in the University Center Christus, Laboratory of Neurociência

Translacional-Neurocit; 3 Doctor in Biotechnology for the RENORBIO/UFC. Professor in the University Center Christus, Coordinator of the Laboratory of Neurociência Translacional-Neurocit.

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Introduction/Objective: Exposure to chemical substances during pregnancy can result in abnormal development and congenital malformations of the fetus. OECD 414 is a guideline for developmental toxicity testing designed to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism; this may include assessment of maternal effects as well as death, structural abnormalities, or altered growth in fetus. This OECD mandates that all fetuses should be screened for skeletal and soft tissue changes, whether the test animal is a rodent or a non-rodent. Specifically for the skeletal assessment, although required by the guideline, there is no description of the methodology for its performance. A methodology was developed by Bioagri toxicology laboratory for the skeletal assessment of rabbit fetuses. Techniques described in the literature were adapted for the analysis of whole-mount skeletal preparations. This technique allowed detecting changes in skeletal patterns and evaluating the skeletal maturation rhythm through specific bone and cartilage staining. Methods: After evisceration of rabbit fetuses, proceed with the removal of the skin, eyes, adipose tissue and bubbles from the body cavity. Fix the embryos in 95 % ethanol alcohol (EtOH) overnight at room temperature. Place the samples in acetone for three days at room temperature. Stain for cartilage by submerging the embryo in a vial containing at least enough Alcian blue solution to cover it. Incubate the sample for 01 to 08 hours at room temperature. Wash fetuses in 70% EtOH twice in a row. Incubate fetuses in 95% EtOH, at room temperature, for 24 hours. Wash the fetuses in deionized water. Submerge them in 1% potassium hydroxide (KOH) solution, at room temperature for 01

to 04 hours. If soft tissue clearing has not occurred, change the 1% KOH solution for another one and leave the fetuses submerged for the same period. Remove 1% KOH solution by washing fetuses in deionized water. Submerge the fetuses in a solution of Alizarin Red S, at room temperature, for at least 04 hours. A period longer than 24 hours should be avoided due to over-coloring effects. Replace the Alizarin Red S solution with a 50% liquid Glycerin:50% KOH 1% solution, with enough quantity to cover all fetuses. The fetuses should remain incubated in this solution, at room temperature, until the excess of the reddish-purple color is removed. When the fetuses reach a gelatinous and transparent appearance, transfer them to a container with 100% liquid glycerin. Results/Discussion: Alcian blue and Alizarin red stain cartilage and bone, respectively. As a cationic dye, Alcian blue binds strongly to sulfated glycosaminoglycans and glycoproteins, while Alizarin red, an anionic dye, binds to cationic metals such as calcium. Fetuses can have different size, which influenced the time of immersion in each of the solutions of the double staining process. Care must be taken to not have an excess of blue or reddish coloration, as well as for the time of immersion in the KOH solution; which can irreversibly damage the fetus, especially the smallest ones. As expected, it was possible to evaluate the shapes and sizes of the rabbits’ fetuses skeletal elements in their appropriate locations. Conclusion: In view of the results, the use of whole-mount skeletal staining is valid to the teratogenicity evaluation of the rabbit’s fetuses, as long as each step is carefully followed. Acknowledgments: Bioagri Laboratórios Ltda (Merieux NutriSciences), Piracicaba-SP, Brazil; Faculdade de Ciências Agrárias e Veterinárias/UNESP Jaboticabal-SP, Brazil

Whole-mount skeletal staining in rabbits fetuses for teratogenicity evaluation (OECD 414)

MaTSui, anDreSa; alveS, paula Daniela SaBino De FreiTaS; SanTana, ThaTiane nuneS; vecina, juliana FalcaTo; BechTolD, Bruna aSSunção; Fava, luiS paulo

Mérieux NutriSciences / Bioagri Laboratórios Ltda, Piracicaba-SP, Brazil.

07 FOOD SAFETY

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Introduction: Aluminum occurs naturally in the environment and may be in drinking water; unprocessed foods such as fruits and vegetables; food of animal origin and processed foods. Due to its technological properties, it is widely used, e.g. in food packaging, kitchen utensils and food additives. At high levels aluminum can be toxic to human health. It is a neurotoxic metal, especially in patients with renal failure on dialysis, and it is related to several diseases, such as osteomalacia and microcytic anemia. Joint FAO/WHO Expert Committee on Food Additives (JECFA) established provisional tolerable weekly intake (PTWI) for aluminum of 2 mg/kg bw. The Brazilian Health Surveillance Agency (ANVISA) specified as PTWI for aluminum in food additives 1 to 7 mg/kg bw, but it did not establish any maximal level for aluminum in food. Taking this scenario into consideration, it is necessary to study aluminum occurrence in foods, especially in fruit and vegetables that are directed impacted by water and soil contamination and also the influence of materials in contact to food in aluminum occurrence in processed food. Objective: The main objective of this study was to evaluate the world situation of aluminum contamination in fruits and fruit/vegetable juices. Methods: The worldwide occurrence of aluminum was evaluated in fruits and fruit/vegetable juices from data available in GEMS/Food database. The inclusion criteria were: i) all regions; ii) results from edible parts. The exclusion criteria were i) dried fruits; ii) concentrated juices; iii) results in dry basis; and iv) samples with limit of detection (LOD) and limit of quantification (LOQ) higher than the average levels observed. The results of aluminum in foods

were expressed as mean, median, standard deviation and 95th percentile. When results were not detected, they were replaced by zero in lower bound treatment (LB) and by LOD value in upper bound (UB). Results: It was evaluated 443 results of aluminum in fruits with 53.5% of positive samples. LOD and LOQ ranged from 0.02-0.236 mg/kg and 0.1-0.5 mg/kg, respectively. The mean level of aluminum at LB was 0.57 ± 0.85 mg/kg and at UB was 0.64 ± 0.81 mg/kg. Median and 95th percentile were respectively 0.24 mg/kg and 2.2 mg/kg. Fruit and vegetable juices had 48.08% of positive samples (n=52) with mean and median levels at LB of 0.51 ± 0.86 mg/kg and zero, respectively. At UB mean and median levels were 0.58 ± 0.81 mg/kg and 0.22 mg/kg, respectively. The 95th percentile in LB and UB was 2.33 mg/kg. LOD and LOQ for fruit and vegetable juices ranged from 0.05-0.236 mg/kg and 0.218-0.5 mg/kg, respectively. Discussion/Conclusion: The mean levels of aluminum found both in fruits and their derived products and in fruit juices were very similar, suggesting that the processing used by food industry does not interfere in aluminum concentration in final product. This is probably due to the most industrial equipment is made of stainless steel and not aluminum. However, in Brazil, which is one of the largest aluminum producers, most homes have aluminum utensils in their kitchens, which can favor migration of this metal to the foods, increasing aluminum exposure. Further studies evaluating migration of aluminum from kitchen utensils to food are necessary to understand its impact in the accumulation of this metal in human body has on public health. Acknowledges: CAPES

Aluminum levels in fruits and in fruit and vegetable juices

pinelli, juliana junqueira1; cuSTóDio, Flávia BeaTriz1,2

1 Curso de Pós-Graduação (lato sensu) em Tecnologia e Qualidade na Produção de Alimentos da UNIFAL; 2 BioTox - Laboratório de Bioquímica e Toxicologia de

Alimentos, Departamento de Alimentos, Faculdade de Farmácia da UFMG.

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Introduction: In the North Region, cassava flour (Manihot esculenta) stands out for being a versatile food and its production plays an important role in the economy of the involved sectors, mainly for the producing municipalities in the west of Pará. Traditionally, flour is produced from cassava known as “brava”, but a new flour allegedly produced from cassava, a subspecies of cassava, has been sold in natural food stores. The difference between the two products is that the flour produced from cassava can be consumed immediately after a short cooking period, while cassava is indicated for consumption only after prolonged preparation to reduce cyanogenic compounds. Cassava is being a preferred product in the market, as sellers point out to consumers that cassava has a high content of hydrocyanic acid (HCN) compared to cassava flour. Considering that there are no quality standards for the manufacture of products derived from cassava, only Normative Instruction nº 52 for table flour, it was proposed to verify the content of cyanogenic compounds in commercialized cassava flour. Objective: To evaluate the physical-chemical parameters and free cyanogen residues of cassava flour. Material and Methods: The study was carried out with 3 different samples from each supplier from the municipality of Bragança-PA. Analyzes were performed in triplicate for moisture, pH, soluble solids (ºBrix), foreign bodies, titratable acidity, based on the methodology of the Instituto Adolfo Lutz, 1985. For the determination of free cyanide it was based on the study by Essers et al (1993), adapted. Results and Discussion: In the verification of foreign bodies in all the samples were found: ants, ash and insect wings, which indicates that the manufacturing process

directly influences this contamination of foreign matter, it is believed than by the lack of structure, being the flour produced in open places, with dirt floors, presence of domestic animals and wooden utensils. In the analysis of soluble solids, the following mean values were obtained: 2.1; 1.6 and 4.3, where only the A3 sample presented a discrepant value. In the pH verification, the samples obtained an average of 5.15 meq NaOH (0.1N) /100g, thus, according to the regulation n° 52 of BRAZIL, 2011 is considered high acidity for values above 3.0 meq NaOH (0.1N) /100g. In the analysis of titratable acidity, they presented values of 5.84%; 4.85%; 8.58%. The moisture levels of the samples were between 4.5 and 9.0%, according to the standards of normative nº 52 of BRAZIL 2011. As for the analysis of free cyanide, the average found was respectively 0.75; 0.96; 12.78 mg/HCN/kg. Second, the WHO (World Health Organization) and FAD (Organization of Food and Agriculture United Nations) have set a limit of 10 mg HCN/kg. According to the Pinto study (2020), in their physicochemical analyzes of cassava flour, such as pH, titratable acidity and free cyanide, they presented respectively: 4.95; x ≥ 3%; 1.42 mg/HCN/kg. Thus, most samples of cassava flour had a low content compared to cassava flour. Conclusion: In view of the results obtained, cassava flour has a low free cyanide content compared to cassava flour. It is worth mentioning the presence of foreign bodies in all samples, so it is pertinent to evaluate good production practices. Therefore, quality control is necessary in the production of flour, to provide food security to consumers. Acknowledgments: I thank the collaborators who participated in this study.

Analysis of physical chemical parameters and cyanogen residues in macaxeira flour

(Manihost esculenta Crantz), produced in a municipality of Paraense

araújo, Maria eDuarDa liMa; eSTuMano, Saulo Braga; oliveira, SiDney julio vieira; SanTiago, Mayla anDra De anDraDe; coSTa, eliene DoS SanToS Da Silva; oliveira, cláuDia SiMone BalTazar

Laboratório de Toxicologia, Centro Universitário Fibra, Belém-PA, Brasil.

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Cocoa and its products are foods widely consumed by the population in all age groups. For cocoa to be transformed into the final product to be marketed, it must go through several processing steps that can have a great influence on the contaminants present in the final products. Among the possible contaminants are polycyclic aromatic hydrocarbons (PAHs), a group of organic compounds consisting of two or more fused aromatic rings, which are related to cellular mutation processes and carcinogenicity. European legislation sets the maximum limit of Benzo[a]pyrene (BaP) at 5.0 ng.g-1 fat, and 30.0 ng.g-1 fat for the sum of Benzo[a]pyrene (BaP), Benzo[a]anthracene (BaA), Chrysene (Chr), Benzo[b]fluoranthene (BbF) in cocoa beans and derived products. This work aimed to determine the incidence and concentrations of BaP and ΣPAH4 in commercial chocolate samples consumed in Brazil. Eighty-eight samples (n=88) of various chocolates were purchased from local markets in the city of Ribeirão Preto, Brazil. PAH concentrations were determined in all collected samples using a liquid chromatograph system equipped with a fluorescence detector and a Supelcosil LC-PAH column, 250 x 4,6 mm, 5 μm particle size. Gradient elution with mobile phase composed of water and acetonitrile was used at 1.0 mL/min. Around 0.7 gram of lyophilized sample was extracted with n-hexane on ultrasound for 1 hour. The extract was centrifuged, and the solvent evaporated to near-dryness. The residue was dissolved in hexane and eluted through a Strata SI-1 silica cartridge (5g, 20 mL). PAHs were eluted from the column with n-hexane/dichloromethane (70:30, v/v). The eluate was dried, dissolved in 200 μL of acetonitrile, and injected into the liquid chromatography system. The

method evaluated presented quantification limits of 0.45 ng.g-1 BaA, 0.45 ng.g-1 Chr and BbF, and 0.45 ng.g-

1 BaP. Analytical curves were evaluated in terms of linearity, following the necessary criteria for linear regression and quantification of analytes using the external calibration method. Recovery rates ranged from 62 to 88%, and accuracy (n = 3) varied between 3 and 18%. The results of BaP obtained are described as follows (n; mean ± standard deviation; range): milk chocolate (n=18; 2.8 ± 2.2 ng.g-1; <LOQ – 7.1 ng.g-1 ), bittersweet (n=22; 3.2 ± 1.7 ng.g-1; 0.40 – 7.0 ng.g-1), bitter (n=10; 2.9 ± 1.9 ng.g-1; 1.0 – 5.0 ng.g-1), white (n=12; 3.8 ± 1.9 ng.g-1; <LOQ – 7.0 ng.g-1), milk chocolate diet (n=13; 2.0 ± 2.4 ng.g-1; <LOQ – 8.4 ng.g-1); bittersweet diet (n=4; 1.4 ± 0.7 ng.g-1; 0.79 – 5.5 ng.g-1), bitter diet (n=4; 3.5 ± 4.6 ng.g-1; <LOQ – 10.0 ng.g-1), white diet (n=5; 2.8 ± 1.0 ng.g-1; 1.5 – 3.8 ng.g-1). ΣPAH4 results are described as follows (n; mean ± standard deviation; range): milk chocolate (n=18; 36 ± 29 ng.g-1; 2.0 – 89 ng.g-1 ), bittersweet (n=22; 44 ± 23 ng.g-1; 7.0 – 85 ng.g-

1), bitter (n=10; 28 ± 25 ng.g-1; 6.8 – 90 ng.g-1), white (n=12; 43 ± 31 ng.g-1; 2.0 – 115 ng.g-1), milk chocolate diet (n=13; 17 ± 17 ng.g-1; < LOQ – 60 ng.g-1); bittersweet diet (n=4; 15 ± 9 ng.g-1; 5.5 – 26 ng.g-1), bitter diet (n=4; 27 ± 37 ng.g-1; <LOQ – 80 ng.g-1), white diet (n=5; 26 ± 11 ng.g-1; 17 – 42 ng.g-1). Twelve out of 88 samples (13%) had a BaP concentration greater than 5.0 ng.g-1

fat, while 44 out 88 samples (50%) presented ΣPAH4 values greater than 30 ng.g-1 fat. These preliminary results show a high incidence of BaA, Chr, BbF, and BaP in cocoa-derived products manufactured in Brazil. The absence of PAH legislation in several food products in Brazil can be revised by regulatory agencies in the future.

Assessment of the incidence and concentration of polycyclic aromatic

hydrocarbons in cocoa-derived products

Silva júnior, cloviS reiS; alMeiDa, gaBriela De oliveira; MoreTTi, gaBriela; jager, aleSSanDra vincenzi

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil.

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Background: The stimulating and performance‐enhancing properties of caffeine are often explored in pre‐workout supplements (PWS) and energy drinks (ED). However, despite the great popularity of these products, previous studies have reported incompatibilities between what is described in their labels and their actual caffeine content. Objective: This study aimed to apply simple and fast methods to quantify caffeine in PWS and ED, and to analyze commercial samples marketed in Brazil to estimate the caffeine daily intake. Methods: The PWS sample preparation consisted of weighting 50 mg of the supplement and adding 5 mL of a lidocaine solution prepared in isopropanol (internal standard – IS – at 25 μg/mL). Then, the extraction was performed by ultrasonic bath (70°C for 10 minutes - conditions that were previously optimized by central composite rotatable design). After the extraction, the samples were centrifuged (10 minutes at 2800 rpm), and then 100 μL was transferred for a vial containing 900 μL of the IS. The ED sample preparation consisted of a simple dilute and shoot: 20 μL of the ED was transferred for a vial containing 980 μL of a 2,2′-Bipyridyl solution prepared in isopropanol (IS for ED analysis – at 40 μg/mL). Both PWS and ED samples were analyzed by gas-chromatography with nitrogen phosphorous detector (GC-NPD). In addition, both methods were previously validated following the ANVISA Guideline on Analytical Method Validation. Fifty-two PWS and 37 ED commercial samples marketed in Brazil were analyzed. Results: The presence of caffeine was declared in 40 of the 52 PWS samples (77%). However, the GC-NPD analysis revealed that actually 45 products contained caffeine (87%). The actual caffeine concentration ranged from 1.95 to 904 mg/g of supplement. From the 36 PWS labels that

in fact specified the caffeine amount, seven (19%) presented more than 120% of the declared quantity, whereas 15 (42%) contained less than 80% of the labeled caffeine. Additionally, six products presented undeclared caffeine. Considering the label stated doses, five supplements exceeded the safe caffeine daily intake (400 mg). Regarding the ED products, caffeine was declared and detected in all samples. The determined caffeine concentrations ranged from 11 to 68 mg/mL. Only one ED sample (2,7%) presented more than 120% of the declared quantity, whereas four (10.8%) contained less than 80% of the labeled caffeine. No ED products exceeded the safe caffeine daily intake. Discussion/Conclusion: More than 50% of the PWS products presented inconsistencies in caffeine content. These issues may affect consumers in two ways: either the product contains less caffeine than expected, so the consumer is paying for a supplement that may not provide the desired effect; or it contains more caffeine than expected, leading to higher caffeine intake than the amount that is considered safe, which may cause adverse effects. The quantified caffeine content in ED samples was much more consistent with the labels. Thus, the production and/or quality control of these products are more efficient comparing to PWS. Therefore, the present findings showed that a stricter quality control is still needed to ensure that PWS actually contain what is declared. This is an issue that deserves more attention from consumers, manufacturers, and regulatory agencies. Acknowledgments: This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPQ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Caffeine content in pre-workout supplements and energy drinks marketed

in Brazil: are the actual levels safe and in accordance with the labels?

coSTa, Bruno ruiz BranDão1; el haDDaD, lohanna pereira2; FreiTaS, Bruno ToleDo2; Marinho, paBlo alveS3; De MarTiniS, Bruno SpinoSa2

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Faculdade de Filosofia, Ciências e Letras de Ribeirão

Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 3 Instituto de Criminalística da Polícia Civil do Estado de Minas Gerais, Belo Horizonte, Brazil.

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Introduction: Banana is a world-renowned fruit, cultivated in several countries with predominantly tropical and subtropical climates. The fruit is known for its nutritional value, being an excellent source of vitamins and minerals. In addition to in natura consumption, part of its production is transformed into banana flour and biomass. This process takes place through the use of green bananas. The use of green bananas, a nutritional flour, minimizes future losses that occur in the production chain, due to factors such as distribution and storage. Green banana flour is a source of carbohydrates, phenolic compounds, vitamins and minerals. This food has been gaining prominence in the functional food market for having high concentrations of resistant starch that helps in the weight loss process due to its composition. Several tests are done to determine the composition and physicochemical properties of this type of food, however, tests that can determine its toxicity are necessary to determine its safe and effective use through a cytotoxic and mutagenic investigation due to the increasing consumption of natural products. The Allium cepa (also known as onion) test is a simple and widely used test as a cytotoxicity and genotoxicity of plants, aquatic systems and extracts. In addition, an efficient test for the analysis of several environmental substances. Objective: The goal of this study was to evaluate the cytotoxicity of the green banana flour (Musa sinensis) using Allium cepa test. Methods: Hydroalcoholic extract (80:20 EtOH:H2O)

of green banana flour was obtained by extraction in an orbital shaker (150 rpm, 24h, 35 ºC). After, the material was dried out in a rotary evaporator. The flask was weighed before and after the process to obtain the corresponding mass and then, the extract obtained was resuspended in a 50% ethanol solution (Conc.=5.0 mg.mL-1) for posterior analysis. For Allium cepa assay, roots were cultivated for 48h (25ºC) using a cylindrical reservoir containing water. After, they were divided in three groups: (a) negative control (NC) (distilled water); (b) banana flour extract (BFE) (0.5 mg.mL-1); (c) positive control (PC) (5% glyphosate solution). Three Allium cepa bulbs size were measured at the initial treatment and every 24h during 120h (total exposure). Root growth rates were analyzed statistically using one-way ANOVA /Tukey test (p≤0.05). Results: Our results demonstrated that both positive control (glyphosate solution) and extract (BFE) reduced root growth significantly when compared to NC. In relation to negative control (NC) the growth of 18.5% and 3,5% for BFE and PC, respectively. Discussion/Conclusion: According to results obtained, Banana Flour Extract exhibits, for 5 mg.mL-1, an apparent toxicity, however, lower than the positive control. Mitotic index and genotoxicity assay will still be carried out, aiming at greater knowledge about the toxicological effects of this food, as well as tests using animal models. Acknowledgments: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Cytotoxic activity of green banana (Musa sinensis) flour by Allium cepa test

TaDeu, viTória coSTa1; roehrS, raFael2; FariaS, FaBiane Moreira1; kieling, keTelin Monique cavalheiro2; nogueira, caroline lacerDa1; DenarDin, elTon luiS gaSparoTTo1

1 Laboratório de Estudos Físico-Químicos e Produtos Naturais (LEFQPN); 2 Laboratório de Análises Químicas Ambientais e Toxicológicas (LAQAT), Campus Uruguaiana,

Universidade Federal do Pampa (Unipampa), Uruguaiana – RS, Brasil.

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BACKGROUND: Counterfeiting alcoholic beverages can lead to harmful effects on consumers’ health due to the lack of quality control, which can originate a formulation that contains alcohol congeners (i.e., substances which are responsible for the sensorial properties of the beverage) in improper concentrations or even in hazardous levels. In this sense, the monitoring of alcohol congeners can serve as counterfeiting markers, aiding the elucidation of possible frauds. Objective: To develop and optimize a HS-SPME-GC-MS method for the qualitative analysis of volatile alcohol congeners in seized whiskeys in cases of fraud suspect. Methods: A pool of six authentic whiskeys samples was used for the optimization of the method. For the SPME fiber selection, two types of stationary phase coating material were evaluated (Carboxen/PDMS and PDMS). The pool of whiskeys was analyzed in triplicate on the following conditions: extraction temperature (40ºC), extraction time (40 min), and equilibration time (12.5 min). The responses evaluated were the number of peaks and the total chromatographic area. A Student’s t-test was applied to assess any statistical significance between the mean of the responses for the two treatment groups. Next, a two-level-three-factor Full Factorial Design (FFD 23) was conducted in triplicate to examine the impact of the factors pH (pH 3-5) and salting-out (0-1g of NaCl) on the responses. The data were evaluated by ANOVA to verify if any of the four possible combinations resulted in any statistically significant differences in the responses. Finally, a Box-Benhken Design (BBD) with 16 experiments was conducted to model the response in function of the following factors: extraction temperature (30-50ºC), extraction time (20-60min.), and equilibration time (5-20min.). The geometric mean of the total chromatographic area was adopted as the response. The desirability

function of Derringer & Suich was conducted after BBD optimization to find the theoretical optimum condition for the HS-SPME extraction. For validation of the model, experiments in triplicate on the predicted optimum conditions were conducted and the relative error was measured. Results: The fiber type evaluation results revealed that Carboxen/PDMS presented better responses than PDMS. The Student’s t-test revealed a statistically significant difference between the means of the total chromatographic area for each type of fiber (p=0.041). Since Carboxen/PDMS presented a higher total chromatographic area, we choose it for further experiments. Regarding the pH and salting-out effects, the ANOVA results indicated no statistical difference between the means of each of the four treatment combinations (p>0.05). Thus, we decided to choose the treatment with no pH and salt adjustment. The predicted optimal settings for the SPME factors obtained through the BBD 23 and the desirability function were: 60 minutes for extraction time, 5 minutes of equilibration time, and 50ºC for extraction temperature. The experimental validation of the model presented a relative error of -1.59%, which demonstrates the adequacy of the model. Discussion/Conclusion: The HS-SPME-GC-MS method was developed and successfully optimized through the aid of the design of experiments, which propitiated a reduction in the number of experiments and standard experimental work required. The method will be applied to 39 seized whiskeys provided by the Superintendência da Polícia Civil Técnico-Científica de Minas Gerais to verify materiality in possible causes of fraud. Acknowledgments: This research was supported by CAPES – Brazil (Financing Code 001). We also thank the Superintendência de Polícia Técnico-Científica de Minas Gerais.

Design of Experiments as a tool for HS-SPME-GC-MS method development

applied to qualitative analysis of volatile alcohol congeners in seized whiskeys

Bigão, víTor luiz caleFFo piva1; coSTa, Bruno ruiz BranDão1; goMeS, nayna cânDiDa1; Marinho, paBlo alveS2; Silva, jonaS joaquiM MangaBeira3; De MarTiniS, Bruno SpinoSa3

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP. 2 Superintendência de Polícia Técncio-Científica da Polícia Civil de Minas Gerais –SPTC/PCMG. 3 Faculdade de

Filosofia, Ciências e Letras de Ribeirão Preto – Departamento de Química – USP.

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Background/Introduction: Scombrotoxin fish poisoning or also known as histamine fish poisoning, occurs after the consumption of dark-muscled fish that has been not properly handled. Histamine is produced from putrefied fish after the inadequate chilling of raw fish enables allows bacterial conversion of free L-Histidine. Histamine intoxications have been reported to occur at concentrations lower than 1000 mg/kg, the United States Food and Drug Administration uses a guidance level of 50 mg/kg for histamine in fish, whereas in the European Union Critical levels for histamine in fish and simple fish products are 100 mg/kg histamine. So far, the established analytical methodology to quantitatively measure histamine involves complex laboratory instrumentation such as high-pressure liquid chromatography. However, most fish processors do not have the expertise or the infrastructure to deploy these technologies on the field to make rapid decisions. In this research, the validation of a simplified quantitative workflow for the field testing of histamine in fish samples using a new lateral flow immunochromatography assay is discussed. Objectives: The objective of this study was to develop a new quantitative and portable methodology that enables fish processors to analyze histamine in fish samples in 3 minutes. The use of an aqueous extraction in combination with lateral flow immunochromatography was evaluated and developed to provide reproducible and high recoveries of histamine in spiked fish samples and quality control materials at critical levels, as well as the comparison of these results with those derived by other analytical methods. Methods: The following fresh and thawed frozen raw Histamine-free fish samples were used for the analysis: anchovy, sardine, tuna, cod, mackerel, fish meal. These matrices were spiked at both 50 mg/kg and 100 mg/kg. The extraction involved a

homogenization step with distilled water. The extracts were filtered subsequently diluted for analysis. 100 µL of the extracts were loaded into testing wells and were analyzed using Symmetric Histamine Lateral Flow immunochromatography kits (3-minute incubation time), subsequent quantitative analysis using the portable Lateral Logic S-Flow reader. Both technologies were produced by Prognosis Biotech. Results: The overall lateral flow method’s recovery for histamine was above 90% and coefficient of variation of all the fish matrices spiked and the quality control materials were within acceptable range (%CV < 15%). The extraction process using distilled water in average took less than 5 minutes to complete, in comparison to other methodologies that use long acylation procedures (i.e., more than 20 min) and require expertise to avoid errors in accurate histamine measurements. Comparison to other analytical approaches showed good correlation between methods (R2> 0.9), and no significant difference in the results between fresh and thawed frozen raw fish samples was found. Conclusion/Discussion: A new quantitative histamine assay was developed using lateral flow immunochromatography technology. Histamine was efficiently extracted (>%90 recovery) using a simplistic aqueous extraction protocol. The 3-minute analysis time as an alternative to other analytical techniques can help fish processors to maintain product integrity on the field while maintaining histamine quantification requirements by regulatory agencies. This approach provides portability and reduces the complexity presented by other methodologies. Acknowledgments: The author would like to acknowledge Research and Development team at Prognosis Biotech for the technical development and method implementation of this assay.

Development of a new quantitative field-testing assay for the analysis

of histamine in seafood samples

caBriceS, oScar g.

G-Flo Scientific Laboratory Testing & Consultants (USA).

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Introduction: In the Food Safety context, packaging and materials that come in food contact must not: endanger human health; change the composition of the food and/or cause organoleptic deterioration. Through migration, packaging can transfer components into the food. Migrants are low molecular weight substances, with mobility and ability to be extracted and/or absorbed by food, which may represent a health risk to the consumer. Food packaging are classified as: Cellulosic; Elastomeric; Metallic; Plastic; Regenerated Cellulose; and Glass. Objective: Approach the legislative and toxicological aspects of migrants in food packaging, emphasizing Anvisa’s role in Food Safety. Methods: Bibliographic review of articles, legislation and national regulations that address the regulatory and toxicological aspects of food packaging. Results and Discussion: In Brazil, the National Health Surveillance Agency (Anvisa) is responsible for regulating the use of materials intended to come into contact with food. Thus, it establishes the maximum tolerated limits of contaminants relevant to human health through the Collegiate Directorate Resolutions (RDC), organized by type of packaging material. In this scope, the RDC’s describe that every substance used in the composition of a package must appear in the positive lists, which present the list of substances proven safe for the intended application, once the specifications, restrictions and limits established are met. Until the present moment, there is no negative list about this subject, that is, with substances prohibited to be used in food packaging materials. A substance is not listed as positive when: use is unsafe; safety has never been studied; there is not enough information to conclude on safety; inclusion has not been requested. When a component is not in the positive list, it cannot be used in the application to which this list refers. Some

RDC’s establish parameters for: total migration, specific migration, and the composition of the material. Therefore, because of packaging migrants, the human health risk assessment is related to the hazard of the component (toxicological aspects of that migrating material) and the potential exposure to this given component (amount that will migrate from the packaging to the food). In the cellulosic packaging issue, it is possible to highlight RDC’s No. 88/2016, No. 89/2016 (coction and hot filtration) and No. 90/2016 (coction or oven heating). The positive list present in RDC No. 88/2016, which approves the technical regulation about cellulosic materials, packaging and equipment intended to come into contact with food and makes other provisions, has numerous substances. Among these substances, with the function as additive for raw materials and authorized as accelerator for lignin and cellulose separation, Anthraquinone (CAS No. 84-65-1), is classified within the Carcinogenicity class - Category 1B (risk phrase: may cause cancer), by the European Chemicals Agency (ECHA). RDC No. 88/2016, in terms of risk assessment, establishes for Anthraquinone the maximum limit of 0.10% by weight of lignocellulosic material and the specific migration limit of 0.01mg of Anthraquinone/kg of food. Conclusion: According to what has been analyzed, it can be concluded that Anvisa, through the parameters described in the RDC’s, acts clearly stamping the toxicological concepts of risk assessment concerning the composition and possible migration of food packaging materials, ensuring the adequate protection of human health in Food Safety. As was also evident the importance of toxicological studies to establish the conditions under which foods can be ingested without causing harm. Acknowledgments: To Intertox Ltda. and everyone who participated in the development of this work.

Food Safety: regulatory and toxicological aspects of food packaging

alMeiDa, giulia Forni; pinheiro, FaBriciano

Intertox Ltda. - São Paulo, SP.

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The market for plant-based products has been expanding over the last few years due to consumer’s concerns about vegetarianism, healthier and/or more sustainable lifestyle. Then, vegetable products analogous to conventional animal-products has been developed, such as the plant-based “milks”. Peanut is appreciated both for its flavor and for its high nutritional value, despite its allergenic potential and mycotoxin contamination. Aflatoxins are carcinogenic and immunosuppressive mycotoxins whose presence in peanut grain and derivatives is commonly reported in the literature. If present in the peanut grain, aflatoxins can be carried into the extract during processing, reaching the peanut “milk” and compromising the product safety. In the same way, allergenic proteins found in peanut are resistance to process of milk production. Thus, we studied the “peanut milk” ozonation process, evaluating the kinetics of degradation of aflatoxins AFB1, AFB2, AFG1, and AFG2, considering their effect on lipid oxidation and the allergenicity of peanut proteins and oxidative parameters. Aflatoxins degradation was evaluated through ozonation of commercial peanut milk samples for 0, 5, 10, 20, 30, 60, 90, 120, 150, 180 and 210 min (initial concentrations: AFB1 = 43 μg/L, AFB2 = 38 μg/L, AFG1 = 59 μg/L, and AFG2 = 30 μg/L). Oxidative parameters evaluated comprised the fatty acid profile and the volatile compounds formation. Ozonation effect on the allergenic proteins from peanut milk was measured at times 0, 10, 30, 60, 120 min. Ozonation was efficient to degrade AFB1 and AFG1 within 30 min, despite AFB2 and AFG2 were more resistant even after 210 min. A higher

ozone consumption occur between the first 30 min, suggesting that the most accessible and reactive points on the molecules from peanut milk were exhausted after this time. The fatty acid profile shown a reduction of oleic acid and an increasing of volatile compounds whose signal the oxidative degradation. Nonanal and hexanal are markers of lipid oxidation and can be detected by the human nose at levels from 1-5 ppb. The oleic acid degradation after ozonation produced nonanal and hexanal levels several times above these threshold levels, even after only 10 min of process. Thus, it suggests that the evaluation of the improvement of the ozonation process should be done in this initial range of time. In addition, additional strategies to prevent lipid oxidation and improve the ozonation efficiency could be useful. Ozonation was suitable to degrade the most toxic aflatoxins, AFB1, and AFG1 from peanut milk.In addition, the ozonation of peanut milk has shown the potential to reduce the concentration of the allergenic peanut proteins. However, the process should be improved to find the best compromise to the reduction of the undesirable compounds and the quality maintenance of the ozonated product. Acknowledgments: This work was supported by the National Council for Scientific and Technological Development (CNPq, Brazil) (CNPq, Brazil) (funding the productivity grant of P.E.D. Augusto 306557/2017-7 and 310839/2020-3); Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) [funding the postdoctoral fellowship of A.C.Romero 88887.356778/2019-00]; São Paulo Research Foundation (FAPESP) [2019/05043-6].

Ozone processing of peanut “milk”: degradation of aflatoxins, reduction of allergenic

proteins and impact on lipid oxidation

roMero, aleSSanDra De cáSSia1; SarTori, alan giovanini De oliveira1; caeTano-Silva, Maria eliSa2; alencar, Severino MaTiaS1,3; calori-DoMingueS, Maria anTonia1; auguSTo, peDro eSTeveS DuarTe1

1 Department of Agri-food Industry, Food and Nutrition (LAN), “Luiz de Queiroz” College of Agriculture (ESALQ), University of São Paulo (USP), Piracicaba, Brazil. 2 College of Agricultural,

Consumer & Environmental Sciences, University of Illinois at Urbana-Champaign, IL, USA. 3 Food and Nutrition Research Center (NAPAN), University of São Paulo (USP), São Paulo, SP, Brazil.

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Background/Introduction: Lead (Pb) is the second most toxic metal after arsenic (As), comprising 0.002% of the earth’s crust. It is estimated that exposure to lead was responsible, in 2004, for 143 thousand deaths and 0.6% of the global disease burden, taking into account mild mental under development and cardiovascular outcomes. Beyond health surveillance actions and analytical monitoring and systematic evaluation of the data produced is required. Objective: Investigate the content of Lead in foods using the R language as a technique for filtering, analyzing and modeling distributions according to the varieties of food consumed, countries, analytical conditions and dates. Find out where and which foods are most contaminated and create possible hypotheses of cause. Create a reproducible approach that can be reviewed and easily shared among the scientific community. Methods: All calculations and graphs were made in Rstudio version 1.3.959 and R base 3.6.3 and the GEMS\FOOD database (WHO). Results: Among developed countries, European Union presents the highest levels of contamination with most outliers. Most foods with high lead values have a very limited intake: “Animal Feed”, “Stimulant beverages”, “Herbs, spices and condiments”. Only “Products for special nutritional use” are critical as they may result in a

higher intake for longer periods. They show mean values of There are no indications that imported samples (to the European Union) are quantified more frequently than domestic ones, indicating contamination at source. In the European Union, 91% of all samples are below the 0,1mg/kg and 99% are below 1.21mg/kg. Stimulant Beverages, especially tea, present a high continuous source of Lead in the diet. Just to address how much, 50% of tea infusion samples are higher than 0,2mg/kg while 95% of other samples fall on this range. Discussion/Conclusion: In the 10 years with most data from the European Union and the most studied sample (Tea Infusion), no difference was noted in the mean value of contamination. No difference in the number of reproved samples was found among those from accredited laboratories and unaccredited ones. During risk analysis, samples with a reported LOQ are disregarded. We found that there are no statistical differences (Tukey HSD, p<0.01) in quantified results from laboratories with a reported LOQ below 0.01mg/kg and those who didn’t report the LOQ. meaning that in practical terms the lack of LOQ doesn’t mean an inferior result. Acknowledgments: The authors acknowledge the help of Minas Gerais State government for supporting

Using R language to study worldwide Lead contamination distribution on food using the

GEMSFOODS(WHO) database during 1995-2020

Silva, FaBiano; couTo, nilTon; alMeiDa, Mariana

Fundação Ezequiel Dias - FUNED - Minas Gerais.

08 FORENSIC

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Introduction: The actual illicit market for synthetic drugs is characterized by a wide variety of psychoactive substances of different chemical and pharmacological classes, such as amphetamine-type stimulants and new psychoactive substances. Knowledge about its chemical composition, as well as the identity and quantity of the active substances present, is important for emergency care in cases of intoxication by these substances and to establish adequate chemical and toxicological analysis procedures in forensic laboratories. Aim: The objective of this work was to study the prevalence of amphetamine-type stimulants and new psychoactive substances (NPS) in the northeast region of Brazil (States of Bahia and Sergipe), involving seized samples by local police in the period from 2014 to 2019. Methods: In a total of 121 seized samples (between tablets, blotter papers, powders, and crystals) in which ecstasy tablets predominated (n=101), were used GC-MS and 1D-NMR techniques, in order to unequivocally identify the nature of classical synthetic drugs and NPS present in these materials. One-dimensional nuclear magnetic resonance spectra (1H-NMR and 13C-NMR) were acquired using two different instruments. Crystal samples were analyzed on a Bruker Avance III 600 NMR spectrometer, operated at 14.1 T (600 MHz for 1H, 150 MHz for 13C), while the other samples were analyzed on a Varian INOVA 500 NMR spectrometer

operating at 11.7 T (500 MHz for 1H, 125 MHz for 13C). On the other hand, the quantity of psychoactive and adulterant substances present in ecstasy tablets was determined using an optimized and validated analytical method by GC-MS (Agilent 7890A/5975 GC-MSD). Results: Twenty-one substances were identified, of which sixteen NPS of different chemical and pharmacological classes (25I-NBOH, 25B-NBOH, methylone, N-ethylpentylone, N-ethylpentedrone, diphenidine, fentanyl and others) and five classical synthetic drugs (MDMA, MDA, methamphetamine, amphetamine and clobenzorex). Analyzes of 101 ecstasy tablets showed that MDMA was the main component, being found in 57% of the samples, in amounts between 27.3 and 187.1 mg per tablet. In addition, mixtures of MDMA, MDA, caffeine and synthetic cathinones were observed in 34 samples analyzed. Conclusions: The results obtained with the identification of several new psychoactive substances (NPS) in the states of Bahia and Sergipe, in northeast region of Brazil, demonstrate that the population of this region is exposed to the illicit trade of these drugs. These results are somewhat similar to those found in previous studies carried out in other Brazilian regions.

REFERENCE1. BRASIL, Ministério da Justiça e Segurança Pública, MJSP (2020). RELATÓRIO 2019 – DROGAS SINTÉTICAS. SEPLAB/DPER/INC/DITEC Polícia Federal.

An overview of NPS in northeast Brazil: NMR-based identification and analysis

of ecstasy tablets by GC-MS

cunha, ricarDo leal1,3; oliveira, celinalva Da Silva liMa2; oliveira, aline liMa4; MalDaner, aDriano oTávio5; pereira, peDro aFonSo De paula3

1 Polícia Científica de Sergipe, Aracaju, Brazil; 2 Departamento de Polícia Técnica da Bahia, Salvador, Brazil; 3 INCT E&A, Universidade Federal da Bahia, Salvador, Brazil; 4 Instituto de Química, Universidade

de Brasília, Brasília, Brazil; 5 Instituto Nacional de Criminalística, Polícia Federal, Brasília, Brazil.

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Introduction: Diethylene glycol (DEG) is a toxic, colorless and odorless reagent, used for the production of antifreezing devices, among other applications. In Minas Gerais - Brazil, several people presented intoxication symptoms related to DEG ingestion, after the consumption of a brand of beer contaminated with DEG, possibly from the leakage of an antifreeze liquid. Clinical manifestations are gastrointestinal symptoms (nausea, abdominal pain, among others), inebriation, with altered mental status and metabolic acidosis, kidney injury and neurological symptoms. Several victims had a common history of use in previous days of a specific beer. In the chemical analysis of this beer, carried out by another laboratory, DEG was detected on it. The hepatic metabolism of DEG is carried out by the enzymes alcohol-dehydrogenase and aldehyde-dehydrogenase. Recent studies demonstrated that DEG shows its toxic action through diglycolic acid (DA). Studies indicated that DA is slowly excreted, being detected even when DEG is not. Objective: The aim of this study was to detect DA in biological samples (whole blood, vitreous humor, urine, cerebrospinal fluid (CSF), liver and kidney) collected from victims related to beer intoxication, using gas chromatography coupled to a mass spectrometer (GC-MS). Methods: Prior to extraction, solid organ samples (liver and kidney) were homogenized using a bead tissue disruptor at 20 Hz for 10 minutes. In a 1.5 mL polypropylene microtube, 20 μL of fluids (blood, vitreous humor, urine or CSF) or 10 mg of tissue homogenates (liver or kidney) were mixed with 45 μL

of acetonitrile and 50 μL of methanol. The tube was then vortexed for 15 seconds, sonicated for 5 minutes and centrifuged at 5000 rpm for 15 minutes. The supernatant was transferred to a 2 mL vial and dried at room temperature with air flow. The dried extracts were derivatized with 50 μL of BSTFA/1% TMCS for 20 minutes at 70°C. After cooling, the samples were transferred to inserts and injected into the GC-MS. Results: In the 9 autopsied victims, whole blood, urine, vitreous humor, CSF, kidney and liver samples were evaluated. For DA analysis in the samples collected from these cases, 2 victims presented positive results in all matrices. Another 4 presented negative results in all matrices, while 2 presented negative results only in the liver, and one negative result in blood and vitreous humor. Although DEG can be detected using the analytical method presented in the study, DEG was not found in the necropsied victims. Conclusion: DA was successfully used to assist in the investigation of poisoning cases after the consumption of beer contaminated with DEG. The existence of one case with a negative result for DA research in the blood, with the presence of a positive result for DA research in other matrices, is an indication that in cases of necropsy, it could be interesting to evaluate other matrices besides blood. Finally, it was noticed that, in samples in which DEG could not be observed, DA was found. Acknowledgments: This study was carried out with the support of the Civil Police of Minas Gerais and the Public Ministry of Minas Gerais.

Analysis of diglycolic acid in victims intoxicated by the consumption of beer containing diethylene glycol

goularT, criSTiano o.l.1,2; BorDoni, leonarDo S.1,3,4; naScenTeS, cléSia c.2; coSTa, leTícia M.2

1 Instituto Médico Legal André Roquette, R. Nícias Continentino, 1291, Gameleira, 30510-160, Belo Horizonte, Brasil; 2 LEAQUAA, Departamento de Química – ICEx, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, 31270-901, Belo Horizonte, Brasil; 3 Universidade Federal de Ouro

Preto, R. Dois, Campus Morro do Cruzeiro, 35400-000, Ouro Preto, Brasil; 4 Faculdade de Medicina de Barbacena, Praça Presidente Antônio Carlos, 8, São Sebastião, 36202-336, Barbacena, Brasil.

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Background: The harmful use of ethanol has become a global health problem, associated with increasing risk for many harmful health conditions as injuries, violence, risky sexual behaivors, as well as long-therm health risks as high blood pressure, heart disease, cancer, mental health problems, social problems and alcohol dependence. Phosphatidylethanol (PEth), a direct ethanol biomarker, formed in the membranes of the erythrocytes with a 4-6 days half-life. It has been used as a highly specific biomarker for alcohol abuse and classification of drinking patterns, with detection window of 2-4 weeks. The used dried blood microsampling would facilitate logistics for PEth measurements, with high stability, low invasiveness of collection and no need for refrigeration during transport and storage of the samples. Objective: To develop and validate a method for the determination of PEth in dried blood samples (DBS) by LC-MS/MS. Methods: DBS samples were prepared by liquid extraction from 8 mm punch with 500 µL of methanol and acetonitrile (80:20, v/v) containing the internal standard IS (phosphatidyl propranolol 17ng/mL). Samples were homogenized for 30 minutes at 25 °C and 1000 rpm. The organic layer was evaporated at 45 °C for 45 minutes. The extract was resuspended with 100 µL of water and methanol (50:50, v/v), 10 µL

was injected into the LC-MS/MS with electrospray ionization in negative mode. Chromatographic separation occurred in an phenyl column (100 x 2.1 mm x 3 μm) at 40 °C. Mobile phase was ammonium acetate 4mM in water and acetonitrile 28:72 (v/v) eluted at 0.4 mL/min -1. A electrospray source was used for ionization in negative MRM mode, with source temperature of 350 °C, capillary temperature of 200 °C, auxiliary gas nitrogen at 10 arb and sheath gas 50 arb. The quantification transition was m/z 781 – 281 for PEth and m/z 741 – 281 for IS. The validation tests are following the recommendations of the international forensic guidelines (SWGTOX, 2013), Results: Total analytical run time was 6 min, with elution of PEth in 2.1 min and IS in 2.5 min. The method was linear from 10 to 3,000 ng/ml (r2 = 0.99), accurate 87% to 109%, precise with CV% within 5.4 to 12.3% Mean extraction yield was 70%. There was no significant impact of the hematocrit, 25% to 55% in assay accuracy (85-115%) or recovery (p=0.23). The use of the IS was satisfactorily compensated the matrix effect (-2 to +17%). Discussion/Conclusion: The method presented adequate performance for measuring PEth and can be used for evaluating patterns of alcohol consumption in clinical studies. Acknowledgments: Financial support CNPq and CAPES.

Determination of phosphatidyl ethanol in dried blood spots by LC-MS/MS

guTerreS, FernanDa S.1; Tegner, Mariane2; oTT, iSaBela riTTer2; linDen, raFael1,2; anTuneS, Marina vezon1,2

1 Institute of Health Sciences, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

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Introduction: There are three different database in Brazil with toxicological information, the Information System of Grievances Notifications (SINAN) of Health Ministry, the National System of Toxic and Pharmacologic Information (SINITOX) of Oswaldo Cruz Foundation and the National Health Surveillance Agency and Brazilian System of Intoxication Data (DATATOX) of Brazilian Association of Information and Toxicological Assistance Centers. Unfortunately, there is no collection by any of them of criminal toxicological information because of the clinical approach to the toxicology of these systems. Such absence of criminal toxicological information and identified analyte data creates a gap of knowledge that is of vital importance for official laboratories to use in delineating criminal toxicological investigations. The ToxCrim system, as a pilot of a national database of criminal toxicological information, provides an important knowledge of the scope of intoxication cases in the Brazilian scenario, especially in the involvement with crimes in these cases. Objective: To present the development of a national database on toxicological information prepared by official forensic laboratories in the first semester of 2021. Methods: The records in the ToxCrim system (www.toxcrim.com.br) were based on data from forensic toxicologists reports issued/signed (regardless of the examination entry date) and collected by the participants designated by the official forensic laboratories. The information required were: a) Identification, b) State, c) Age, d) Sex, e) Cause of death, f) Middle of the Cause of death, g) Biological matrix , h) Analytical technique and i) Analyte. The data were extracted, filtered and consolidated that

have been identified and/or detected by the official forensic laboratories of the States of Bahia, Espírito Santo, Goiás, Sergipe, Paraíba, Rio Grande do Sul, São Paulo and Rondônia between January and June 2022. For this first report only data from post mortem cases of traffic accidents and homicides were exposed. Results: 2,975 post mortem cases were consolidated with 10,734 records of analytes detected/identified in post mortem blood samples. 825 cases were classified as traffic accidents. Of these 825 cases, 711 records of toxicological tests to quantify ethyl alcohol in blood (alcohol levels) and 206 records of other tests were recorded. In 92 cases both records were recorded. In this legal cause of traffic accidents, the most detected/identified analytes in post mortem blood samples were ethyl alcohol (711), benzoylecgonine (118) and methylecgonine (82). Homicide cases were all classified directly in this way, whether intentional or negligent or due to bodily injury or after robbery or robbery with resistance to arrest and consisting of the following means: firearm, melee weapon, other blunt agent, fire or other. There were 461 homicides containing 1117 records of analytes detected/identified in post mortem blood samples. The most detected/identified analytes were alcohol (280), benzoylecgonine (216) and methylecgonine (182). Several other data were obtained from the records. Acknowledgments: We would like to thank the participating Official Experts Ana Cecília Bandeira (BA), Fabrício Pelição (ES), Mariana Dadalto (ES), Sophia Wieczorek (GO), Ricardo Cunha (SE), Victor Gianvecchio (SP), Carolina Lupi (RS), Ana Júlia Frazão (RO) and Carolina Diniz (RO).

First Report of national database on toxicological criminal information (ToxCrim system)

coSTa, rony anDerSon rezenDe1; coSTa, joSe luiz2

1 Official Expert of Cientific Police Institute of Paraiba State and PhD student of Pharmacology Program at UNICAMP; 2 Executive Coordinator of Campinas Poison

Control Center and Professor of Pharmaceutical Science at UNICAMP.

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Introduction: Forensic Entomotoxicology is the application of the study of insects and other arthropods, associated with forensic procedures involving toxicological analysis. Among the species of flies of the Calliphoridae family, the blowfly (Lucilia cuprina) is commonly linked to post-mortem phenomena. The larvae, more precisely at the L3 stage, of this species are used as an alternative matrix in the analysis of toxicants, especially in bodies in an advanced state of decomposition and tissue unfeasibility. Objective: Due to its great importance and influence in the area of forensic entomotoxicology, the objective of this study was to monitor the development stages of these insects, from the egg, larva, pupa to adults. Methods: For a better understanding of the facts, a protocol for rearing Lucilia Cuprina flies was started, meeting all the requirements of their biological cycle, to be developed in the laboratory. The rearing room where the larva, pupa and adult stages were kept at 25ºC, relative air humidity of 70% (± 5%) with a 12-hour photo phase. Entomological cages (30 x 30 x 50 cm) were used for rearing. The adult’s food is made up of honey, placed on top of rice grains, placed in a Petri dish, in small glass jars, and water is kept freely on a filter paper strip so that they can hydrate themselves without to die by drowning. So that there is egg laying, a cut of beef liver was placed next to the same feeding cage as the adults. The postures should be carefully removed with a damp brush and transferred to another Petri dish. This should contain wet dog food (Pedigree® sachet for dogs), so that when the eggs advance to the larval state, they maintain their saprophagous and/or carnivorous habits. The plate

containing the substrate and eggs was placed on the sterilized sand in a plastic pot (350 mL). The pot was closed with organza tissue and rubber tie. The larvae’s food was changed every two days and, to do this, the plastic pot was filled with water, sifted the sand and the content containing the larvae was poured into a sieve. Then, larvae at stage L1, L2 and even L3 must be transferred to a new pot containing sand and food. Larvae in L3 that had already migrated to the sand and pupae, should be transferred to pots containing only sterilized sand. After leaving the pupae, the adults were changed cage to continue the cycle. Results: As its life cycle is relatively short, it was easy to monitor its development, allowing for a continuous collection of material when the creation of insects is established. L. cuprina completed its whole life cycle in 29 to 45 days where the eggs hatched within 1–2 days and the larvae went through three instars with an average duration of 6.5 days. The pupal stage lasted for 14 days and longevity of moths averaged 36.25 days. Conclusions: Therefore, through the aforementioned facts, the advantages of studying this matrix are understood, as it is considered to be easy to create, because it has a smaller amount of matrix interfering when compared to other more complex biological matrices and for maintaining the preservation of analytes, that can undergo biological and/or abiotic alteration in the decomposing tissue to be analyzed. Acknowledgements: We wish to thank the staff of the insect rearing center at Parasitic Disease Laboratory (LAPAVET/UFSM) for providing the space and share the knowledge about the insects rearing.

Forensic entomotoxicology: application of studies in the development and rearing of

Lucilia cuprina flies (Diptera: Calliphoridae)

chiMenDeS, nayoMi De anDraDe1; BairroS, anDré valle1; ugalDe, guSTavo anDraDe1; SanToS, lara celeSTina1; pacheco, anDré lucaS Bezerra1; reginaTo, FernanDa ziegler1; BerlaTo, Dener goMeS1; roSa, vicTória goMeS1;

naSciMenTo, Marcelo henrique SanTana1; pereira, aleSSanDra De oliveira2; MonTeiro, Silvia gonzalez2

1 Nucleus Applied to Toxicology (NAT); 2 Parasitic Disease Laboratory (LAPAVET).

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Introduction: Organophosphate pesticides are one of the main classes of pesticides related to intoxication. When the human body is in a state of decomposition, it presents few alternatives of biological matrices. Therefore, the flies larvae of the species Lucilia cuprina, a saprofable insect, is an alternative matrix in these situations. However, studies with organophosphates in larvae of flies Lucilia cuprina are scarce. Objective: The objective of this study was to evaluate the behavior of the larvae matrix using larvae at the third-stage of development (L3) in the determination of organophosphates and their respective biotransformation products using dispersive Solid Phase Extraction (dSPE). Methods: The analyzes were performed in GC-MS with RTX®-5MS column (30 m x 0.25 mm, film thickness 0.25 µm) using ultrapure helium gas at a constant flow of 1.20 mL/min. The chromatographic parameters used were: Injector temperature 175°C; 1 µL of injection volume in splitless mode; Column oven configuration: 50°C (maintained for 4 min) → 50°C (35°C/min) → 190°C (maintained for 7 min.) → 190°C (40°C/min) → 300°C (maintained for 1 min) and total chromatographic analysis time was 15.75 minutes. The dSPE method was used using 1g of the L3 homogenized and weighed in a Falcon tube of 15 mL, followed by the analytical standards (Azamethiphos, Carbofenotione, Chlorpyrifos ethyl, Chlorpyrifos methyl, Ethion, Fenitrothion, Formotion, Malathion, Malaoxon, Paraoxon ethyl, Paraoxon methyl, Pirimiphós ethyl, Pirimiphós methyl, Triazophos) and the internal standard Medazepam were added in duplicate at a concentration of 50 ng/g. Then, 2 mL of acetonitrile

were added and tubes were manually shaken for 1 minute. Then, 1 g of NaCl and 4 g of MgSO4 were added. After, it was centrifuged (4,000 RPM) for 10 minutes, 1 mL of the supernatant was transferred to another 15 mL Falcon tube, with 150 mg of NaCl and 25 mg of previously weighed primary secondary Amine (PSA). Finally, the tubes were manually shaken for a further 1 minute, centrifuged at 4,000 RPM and 500 µL of the supernatant was transferred to a 2 mL flask and dried in a sample concentrator. It was resuspended in 50 µL of ethyl acetate and 1 µL was injected in GC-MS. The analytes were analyzed in their respective ions: Azamethiphos (109, 125, 215) Carbophenothion (342, 157, 199), Chlorpyrifos ethyl (314, 199), 197), Chlorpyrifos methyl (79, 125, 286), Ethion (125, 153, 231), Fenitrothion (125, 260, 277), Formothion (93, 125, 198), Malathion (125, 127, 173), Malaoxon (127, 195, 268), Paraoxon ethyl (109, 149, 220), Paraoxon methyl (230, 109, 247), Pirimiphós ethyl (318, 304, 168), Pirimiphós methyl (180, 276, 290) and Triazophos (161, 162, 172). Results: For the analysis of the results, ions 125 and 109 were used for the quantification of Azamethiphos and Paraoxon ethyl, respectively. The retention time (RT) of the analytes were Azamethiphos RT:13.339 minutes and Paraoxon ethyl RT: 9.001 minutes, but in the blank sample, there are interference peaks of the matrix shown at the same retention time as Azamethiphos and Paraoxon ethyl, which makes it impossible to determine and quantify these analytes. Thus, the importance of the study of matrix behavior from larvae of flies Lucilia cuprina is relevant for toxicological analysis. Acknowledgments: Acknowledgments to CNPQ for financial incentives.

Interference of larvae matrix of flies Lucilia cuprina in the determination of

Azamethiphós and Paraoxon ethyl using dispersive solid phase extraction (dSPE)

pacheco, anDré l.B.1; ugalDe, guSTavo a.1; chiMenDeS, nayoMi a.1; SanToS, lara c.1; BerlaTo, Dener g.1; naSciMenTo, Marcelo h.S.1; reginaTo, FernanDa z.1; roSa, vicTória

goMeS1; SanToS, rachel1; carDoSo, leonarDo correa1; MonTeiro, Silvia gonzalez2; STainki, Daniel rouliM2; pereira, aleSSanDra De oliveira2; BairroS, anDré v.1

1 Nucleus Applied to Toxicology, Center of Health Sciences, Federal University of Santa Maria, Santa Maria, Brazil; 2 Parasitic Disease Laboratory, Federal University of Santa Maria, Santa Maria, Brazil.

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Introduction: The vitreous humor (VH) has in recent years become an important alternative biological fluid for post mortem toxicology. Due to its anatomical position, this important biological fluid is protected by the eyeball, being less affected by post mortem phenomena such as putrefaction and redistribution. A case of fatal nortriptyline overdose was investigated after the death of a 27-year-old woman who used this pharmaceutical to treat depression. Her body was embalmed and buried, but after a period of four days, the exhumation was performed by court order to clarify the real cause of death. Thus, considering the formaldehyde used in the preservation of the body, the VH was collected under appropriate conditions for carrying out the toxicological analysis. An analytical method using micro-QuEChERS and LC-MS/MS was applied to determine antidepressants in VH in order to evaluate a possible overdose case. Aim: The aim of this work was to determine nortriptyline in VH as an alternative biological sample and to correlate with the concentration in post mortem blood (PB) to confirm a case of fatal intoxication. Methods: For sample preparation, 300 μL of acetonitrile was transferred to a polypropylene tube containing 100 mg of QuEChERS salt mixture (MgSO4/NaAc 4:1), followed by 200 μL of ultrapure water, 100 μL of blank post mortem vitreous (negative samples previous analyzed at laboratory) and 10 μL of internal standard (citalopram-d6 5 μg/mL in methanol). The tube was homogenized in vortex for 5 minutes and centrifuged at 14000 rpm/10 min in a refrigerated centrifuge at 10°C. The upper layer (300 μL) was transferred to a microtube, dried at a gentle nitrogen flow and reconstituted with 40 μL of methanol in a small insert glass. After this step, 2 μL was injected on LC-MS/MS system model LCMS-

8040 (Shimadzu, Kyoto, Japan). Chromatographic separation was performed in a Restek Raptor Biphenyl Column (100mm x 2.1mm, 2.7μm) maintained at 40°C with mobile phase composed by ultrapure water (A) and methanol (B), both containing 0.1% formic acid and 2 mmol/L ammonium formate in a gradient elution starting at 5% MeOH/95% H2O to 100% MeOH. The flow rate was set to 0.4 mL/min with 6.0 minutes total run time. The mass spectrometer was equipped with an electrospray ionization (ESI) source operating in positive ionization mode. The source parameters were: heat block temperature of 400°C; capillary voltage of 4.5 kV, nebulizer gas (N2) flow of 3 L/min, desolvation line (DL) temperature of 250°C, drying gas (N2) flow of 15 L/min and collision induced dissociation gas (Ar) pressure of 230 kPa. The analyses were performed in multiple reaction monitoring mode (MRM), in which two MRM transitions were chosen, one for quantitation and one for confirmation. Results: The calibration curve for nortriptyline was linear from 10 - 500 ng/mL, achieved an r > 0.99, and the limit of detection 5 ng/mL. Nortriptyline was quantified in VH at a concentration 209 ng/mL and the ratio between VH and PB concentrations (VH/PB=0.13) proposed by Øiestad et al was used to determine the concentration in PB (1607 ng/mL). Conclusions: A sensitive method based on micro-QuEChERS and LC-MS/MS was applied to quantify nortriptyline in post mortem VH samples (209 ng/mL) and to correlate with the concentration in PB (1607 ng/mL). Therefore, the concentration obtained for PB was very high for nortriptyline considering the therapeutic (7-200 ng/mL) and lethal (1500-2000 ng/mL) range, where the cause of death was concluded as fatal nortriptyline intoxication.

Nortriptyline overdose and quantitative analysis in post mortem vitreous humor – a case study

cunha, ricarDo leal1,2; DalBoSco, juliana SanToS1; BriTo, Maria carolina SanToS riBeiro1; coSTa, joSé luiz2,3

1 Instituto de Análises e Pesquisas Forenses, Polícia Científica – Aracaju, SE - Brasil; 2 Faculdade de Ciências Farmacêuticas, UNICAMP – Campinas, SP – Brasil; 3 Centro

de Informação e Assistência Toxicológica, UNICAMP – Campinas, SP – Brasil.

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Background/Introduction: Considering the various alternative biological matrices used in forensic toxicology, the vitreous humor (VH) presents several attractive characteristics including the absence of metabolic activity; low levels of endogenous interferers; less susceptibility to postmortem redistribution; and location in an isolated compartment, reducing the putrefaction interference when compared to other matrices. Drugs and medicines have been already detected in VH, such as cocaine, opioids, alcohol, antidepressants, and benzodiazepines. However, a few studies have developed methods for the analysis of a wide range of compounds in this biological fluid. Therefore, there is a need of the implementation of effective, fast, and automated procedures develop in accordance with the principles of green chemistry. Objective: The aim of this study was to optimize a straightforward solid phase-based microextraction employing cork sheet stripes for LC-MS/MS determination of 18 pharmaceuticals in VH. Methods: The extraction methodology was performed of 3 steps, all conduced in a 96 deep well plate. First, a 1.5 cm x 0.5 cm cork sheet stripe is submerged in 1 mL of the conditioning solvent for two minutes at 300 rpm. Second, this stripe is transferred to 1 mL of a diluted sample solution, containing 200 μL of vitreous humor and 800 μL of ultrapure water and extraction occurred for 15 minutes at 300 rpm. Third, the cork stripe is transferred to 1 mL of the desorption solvent for two minutes at 300 rpm. Subsequently, this solvent was transferred to an Eppendorf tube, dried with assistance of an air flow, resuspended in 20 μL of methanol and analyzed. A

LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Japan) was used for the analysis. For the optimization studies, three experiments were planned to determine the optimal conditions for the extraction procedure. Two experiments were based in simplex-centroid design to evaluate three different solvents in the conditioning and desorption steps. For the conditioning solvent, acetonitrile, methanol, and acetone were tested, and for the desorption step, acetonitrile, ethyl acetate and methyl tert-butyl ether (MTBE) were used. In addition, a Doehlert design was performed for optimization of pH sample and extraction time. The time variable was analyzed in five levels, from 5 to 25 minutes, and the pH factor in three levels, acid, neutral, and basic (5, 7 and 9). Results: The optimal conditioning solvent determined was a 1:1 mixture of acetonitrile and methanol, and the desorption solvent ideal conditions were a 1:1:1 mixture of acetonitrile, ethyl acetate and MTBE. Considering sample extraction factors analyzed, the optimal conditions found were a neutral pH and 15 minutes of extraction time. Discussion/conclusion: In conclusion, this study was able to develop an efficient and fast microextraction method for 18 substances in vitreous humor samples, using small cork sheet stripes for extraction and a 96 deep well plate as recipient. Moreover, this approach system could be possible to automate. In addition, this analytical tool can be used to support the study of other substances in these alternative biological sample, supporting broad applications in forensic toxicology. Keywords: vitreous humor, benzodiazepines, antidepressants, cork. Acknowledgements: CAPES.

Optimization of extraction procedure for benzodiazepines and antidepressants

analysis in vitreous humor using cork sheet

oSSaneS, Daniela Souza; Birk, leTícia; eller, Sarah; oliveira, Tiago Franco

Graduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil.

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Background/Introduction: Volatile organic compounds (VOCs) is a group of substances with different structures presented in a wide variety of products of domestic and industrial use, therefore people are frequently in contact and have easy access. Inhalation or prolonged exposure could lead to poisoning, and its lipid solubility and volatility enhance their toxicity, having great importance for forensic analytical toxicology. In addition, these types of substances are also attractive as they are legal, cheap and the effect occurs and disappears quickly. Depending on the amount inhaled, they produce depressant effects on the nervous system, which can cause psychological dependence. Several countries report the abuse of inhalants among children and adolescents in order to produce effects, including pleasure, disinhibition, euphoria and hallucinations. The main harmful effects presented by the user occur in the heart, lungs, kidney, neurological system, liver and bone marrow. Studies show that more than 50% of the deaths after inhalants consumption were caused by direct toxic effects of the substance, such as cardiac toxicity and respiratory depression, other causes included plastic bag asphyxia, aspiration of stomach contents and trauma due to dangerous behavior. Objective: The aim of this work was to evaluate the prevalence of inhalants consumption in São Paulo state between January and June 2021. Methods: The authentic samples were analyzed by the Sao Paulo State Police, being VOCs identified by GC–MS, according to laboratories standard operational procedures and the SWGTOX recommendations. Reports are entered into the GDL system (Sistema de Gestor de Laudos, in Portuguese) and data from the first half of 2021 were evaluated.

Results: More than 11,000 cases were registered in the period, with 382 (3.5%) of the reports positive for VOCs used as inhalants. Five different substances were identified: trichlorethylene (63.5%), chloroform (30.6%), dichloromethane (5.2%), toluene (0.5%) and benzene (0.2%). In 46.1% of the cases, more than one compound was detected, being the most frequently combination trichlorethylene with chloroform (94.1%). Prevalence of abuse is most common in males (63%) and aged between 15 and 24 years old (60%). The toxicological analyzes positive for inhalants were requested mainly by the city of São Paulo (68.6%) and those belonging to the metropolitan region (22.5%). Discussion/Conclusion: The use of inhalants is not gender specific but is most common among males, being most prevalent in young population. Brazilian data from 2010 report this group as the most used by high school and the second by university students, just behind marijuana. Data also shown that consumption at least once in a lifetime is around 10% higher in the city of São Paulo than in the rest of the country. In 2015, the inhalants most identified in seized solvents were trichlorethylene (51%), dichloromethane (29%), ethanol (15%) and chloroform, ether or ethyl chloride (4%). As observed six years earlier, in toxicological analyzes, trichlorethylene still is the most prevalent, but chloroform appears as the second, being six times higher than dichloromethane. This data showing the consumption profile of this group of substances could help to alert judicial authorities and guide them to take measures aimed to reduce the access and consumption of these substances, especially among young population. To our knowledge, there are few studies that report the prevalence and profile of inhalants consumption in Brazil.

Prevalence of volatile organic compounds in forensic toxicology reports of São Paulo State

roDrigueS, TaíS B.1; MeDeiroS, elviS a.2; chinaglia, kauê o.1; gianvecchio, vicTor a.p.2; coSTa, joSe luiz1,3

1 Campinas Poison Control Center, University of Campinas, Campinas-SP, Brazil; 2 Superintendence of the Technical-Scientific Police, Institute of Criminalistics, São Paulo-SP,

Brazil; 3 Faculty of Pharmaceutical Sciences, University of Campinas, Campinas-SP, Brazil.

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Introduction: The widespread use of synthetic chemical substances for recreational purposes, allied to their ease of acquisition, has been considered a serious global public health problem. According to the UNODC (United Nations Office on Drugs and Crime) World Drug Report 2021, about 275 million people used drugs worldwide in the last year, while more than 36 million suffered from disorders associated with drug use. The market for these illicit synthetic substances is considered complex and very dynamic. According to data from the European Monitoring Center For Drugs and Drug Addiction in 2019, the production of synthetic drugs has become more sophisticated and diversified around the world, with a new trend having been observed with the use of unusual and more easily acquired precursors. Traditionally, the term “synthetic drugs” was used to refer to amphetamines, substituted amphetamines (MDMA and its analogues) and LSD, synthetic substances used for abuse. With the emergence of the so-called new psychoactive substances (NPS), this concept had to be expanded to include this new class of substances. Objective: The aim of this work was to carry out a survey on the synthetic drugs most commonly identified by the Forensic Toxicology Center of the Forensic Expertise in the State of Ceará on the samples seized between January 2019 and December 2021.Methods: The data were collected from the Software Catalog/Database used by the Forensics team – Galileu System, which is a platform that holds the record of all information related to forensics investigations, that also allows communication and data exchange with the Police Information System (SIP - Sistema de Informação Policial) of the Civil Police of the State of Ceará. The

data were extracted from reports issued by Galileu, referring to samples received by PEFOCE between January 2019 and December 2021, registered in the system as suspected synthetic drugs. After identifying the reports in which synthetic drugs were analyzed, the substances identified and reported in each report were also verified. Results: During the study period, PEFOCE received 199 suspected synthetic drug samples, of which 137 were analyzed, while the others are still being analyzed at the laboratory. The most identified substance was MDMA, with 75 appearances, followed by its analogue MDA, identified in 33 samples. Other synthetic drugs identified were: LSD, ketamine, eutylone, methamphetamine, ethylone, PMK-glycidate, isosafrole, N-ethyl-pentylone, 4-chloro-eth-cathinone and derivatives of phenethylamines 2C-I, 2C-E and 25H-NBOME. In 7 samples analyzed, the presence of any substance prohibited or controlled by 344/98 of ANVISA was not identified. Discussion/Conclusion: The three substances most identified in the analyzed samples of synthetic drugs belong to the group of “Classic Drugs” (MDMA, MDA and LSD). These findings do not reflect the reality only in the state of Ceará. Data from the latest Federal Police Synthetic Drug Reports reveal that, for four consecutive years (2017 to 2020), the synthetic drug most seized in Brazil was MDMA; and in the 2020 report, this substance was preceded by MDA and LSD in the ranking of seizures. Given the data presented, it is necessary to create policies to monitor the routes of origin of these substances marketed in Brazil, as well as to investigate the existence of clandestine laboratories in the country.

Profile of synthetic drugs seized between January 2019 and December 2021 analyzed by

the Forensic Toxicology Center of the Forensic Expertise in the State of Ceará (PEFOCE)

oliveira, juliana riBeiro iBiapina leiTão; MagalhãeS, Danielle De paula; MarTinS, Mayane eManuella Melo lopeS; alMeiDa, vivian roMero SanTiago; SaBoya, anDrea luiza rocha; holanDa júnior, WanDerley pinheiro

Forensic Expertise in the State of Ceará (PEFOCE).

242

Introduction: According to the World Health Organization (WHO) suicidal behavior comprises from the planning of the act, the attempts and even the success when reaching the purpose, being a serious public health problem. Around 800,000 people worldwide commit suicide annually, being the second leading cause of death among young people aged 15 to 29. In Brazil, Santa Catarina is in second place in relation to the rate of death by suicide, with a rate of 8.62 deaths per 100 thousand inhabitants, according to 2015. Suicide is considered a form of violent death, therefore, cases of death by suicide must undergo analysis by the Scientific Police of Santa Catarina (PCI/SC). Objective: Analyze the epidemiological profile of suicides committed between 2015 and 2020 registered by the PCI/SC, with emphasis on the results of toxicological analyses. Methods: Study using data obtained from the PCI/SC forensic toxicology department, based on an internal case registry worksheet. The analyzed data were: number of suicides, sex, region of the state, result of toxicological analysis and alcohol dosage. Screening of blood, urine or gastric content samples was performed by immunoassay (Randox®). The confirmation was made in a gas chromatograph coupled to mass spectrometry and alcohol dosage in whole blood by headspace and gas chromatography with flame ionization detection. Results: 11.44 deaths per 100,000 inhabitants were observed in the state of Santa Catarina. The percentage of male cases increased over the years analyzed, reaching 82.2%

of cases in 2020. Cocaine detection was present in 380 cases (10.7%), with a prevalence of more than 90% in men. Furthermore, an increase in the number of suicides was observed with increasing age, with a peak in the age group of 50 to 59 years, followed by a decrease. The mesoregion with the highest suicide rate was the west of Santa Catarina, with 15.71 deaths / 100 thousand inhabitants and with the lowest rate the north of Santa Catarina with 7.61 deaths / 100 thousand inhabitants. Toxicological analysis detected the presence of toxic substances in 43.18% of the cases and the presence of alcohol was detected in 27.49% of the suicides. Discussion/Conclusion: The data showed that special attention should be given to cases of suicide that are increasing in the state, especially among males. There is a high prevalence of suicide among men and also most cases involving cocaine are in this group. In relation to 2020, despite the consequences of the COVID-19 pandemic, there was no significant increase in suicide cases compared to previous years. Almost half of the reports showed some substance detected, the most frequent being: antidepressants, anxiolytics and cocaine. Although the data be limiting in terms of substance dosage and direct correlation with the cause of death, they show the need for discussions and preventive measures and/or public health actions. Keywords: Suicide. Toxicological Analysis. PCI/SC. Drugs. Acknowledgments: The Scientific Police of Santa Catarina for the partnership and the provision of data.

Profile of toxicological analysis of suicide cases reported by the Santa Catarina

Scientific Police in the last 5 years

Silva, Bruna eSpínDola1; BoFF, Bruna De Souza2; Silveira Filho, jair2; SchroeDer, SaMilla DrieSSen2; rezin, kéTTulin zoMer2; Marchioni, caMila3

1 Resident Pharmacist, Hospital University, Federal University of Santa Catarina, Florianópolis, Brazil; 2 Scientific Police of Santa Catarina, Florianópolis, Brazil; 3 Department

of Pathology, Federal University of Santa Catarina, Florianópolis, Brazil.

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Introduction: Several studies worldwide report that alcohol is major responsible for traffic accidents, worsening morbidity and consequently increasing mortality. Since 2008, the Brazilian legislation has established severe punishment for drivers caught driving after drinking alcohol or under the influence of any other psychoactive substance. At the end of 2012, the law was changed, becoming stricter, but even so, in the global scenario, Brazil accounts for almost half of all deaths from traffic accidents. Objective: Describe the demographic profile of traffic accident victims which had their blood alcohol content tested at the Forensic Toxicology Center (NUTOF) of PEFOCE from January 2020 to December 2021. Method: The data extracted from the Gallileu system, forensics management software developed and used by PEFOCE, and tabulated in Excel® 2019 and Stata/MP version 2016 programs. In the period from January 2020 to December 2021, from the total number of blood alcohol levels (BALs) requested to the NUTOF, the cases of traffic accidents by collision/trespassing and with ethanol detection test performed were considered for the description of the victims’ profile. The variables analyzed were sex, age, BALs verification, in vivo and post mortem. It was considered as detected, values equal or greater than 1,0 dg of ethanol per liter of blood. Results: PEFOCE received 7,015 requests for BALs tests in the years

2020 (51.0%) to 2021 (49.0%), of which 2,433 (34.7%) were related to traffic accidents. Of these, 847 (35%) samples were analyzed, with a higher prevalence of males (86%; n=729), and ethanol was detected in 47.8% (n=405) of the total analyzed. Most analysis (92%; n= 782) were performed on post-mortem samples. Of the samples with the presence of ethanol, 61.8% (n=250) had a value between 15,0 and 29,0 dg/L. The positivity of the test was higher in males (92%; n=372). The presence of ethanol was detected in 51% of males (H) and 28% of females (M). The median age was 37.2 years, with 38.7 years (M) and 37.1 (H). The age group 30 to 39 years had the highest prevalence among the cases analyzed (26%; n=218), as well as among the positive cases (15%; n=125). Conclusion: Ethanol was detected in a significant portion of the cases analyzed, which reflects the involvement of alcohol consumption in traffic accident cases. A high prevalence of male gender was observed in cases of traffic accidents, especially in the young adult age group, up to 40 years old. We also observed a higher number of positivity among males. Thus, we conclude that there is a need for investments in public policies of awareness and more efficient enforcement. Acknowledgments: The Forensic Experts of the state of Ceará and the colleagues from the Forensic Toxicology Center.

Profile of victims of traffic accidents with blood samples collected for alcohol tests and analyzed

by the Forensic Expertise of the State of Ceará (PEFOCE) from January 2020 to December 2021

MagalhaeS, Danielle De paula; holanDa júnior, WanDerley pinheiro; SaBoya, anDréa luiza rocha; oliveira, juliana riBeiro iBiapina leiTão; MarTinS, Mayane eManuela Melo lopeS; SanTiago, vivian roMero

Forensic Expertise of the state of Ceará (PEFOCE).

244

Introduction: Counterfeiting alcoholic beverages can lead to harmful effects on consumers’ health due to the lack of quality control, which can generate formulations that contain ethanol content in improper concentrations or even at hazardous levels. In this context, the monitoring of ethanolic content may be used as a possible counterfeiting marker, aiding the elucidation of possible frauds. Objectives: This study aimed to develop and validate a method for quantifying ethanol content in alcoholic beverages. Methods: Ethanolic solutions of 11, 21, 31, 41 ,51, 61% v/v (ethanol:water) were used for calibration and validation of the method. For the ethanol extraction, 50 µL of the sample, 0.25 g of NaCl, 15 µL of acetonitrile (internal standard, IS), and 935 µL of distilled water were incubated in a headspace (HS) vial for 5 minutes at 80 ºC with an agitation of 500 rpm. A Gas Chromatographic with Flame Ionization Detector (GC/FID) system with a CP Wax 52 CB capillary column was applied for the analysis. The oven temperature was set as follows: 50 ºC (hold for 2 min); then ramped to 70 ºC at 10 ºC.min-1. Next, the temperature was ramped at 30 ºC.min-1 to 200 ºC. The injection was performed in the split mode (120:1). Nitrogen 5.0 at 1 mL.min-1 was used as the carrier gas. The parameters limit of detection (LOD), the limit of quantification (LOQ), selectivity, linearity, weighting, precision, and accuracy were evaluated during the validation of the method following the recommendations of the UNODC Guidance for Validation of Analytical Methodology. Precision and accuracy were expressed as relative standard deviation (RSD%) and relative error (RE%), respectively. The heteroscedasticity at an α=5%,

carry-over, and stability were evaluated according to the RDC Nº27/ANVISA. The ethanol stability in the working solution was assessed during the analytical process by determining the precision and accuracy at two concentration levels (21 and 61% v/v) in triplicate. Freeze-thaw stability was evaluated for 2 cycles of thawing at room temperature followed by re-freezing at 4°C for 24 h. The short-term stability was determined from the ethanolic solutions kept at room temperature for 5 h before processing. Results: The method presented LOD of 2 µg.mL-1 and LOQ of 5 µg.mL-1. The analyzed interferents did not interfere in the retention time, identification, and quantification of the analyte. No residual effect was observed either. The F-test showed the model is heteroscedastic, so a 1.Y-2 weighted linear regression was employed and the method is linear (r>0.999) in the working range of 11 to 61% v/v. The method also showed good precision (RSD=4.08 to 7.97%) and accuracy (RE=-0.05 to -1.31%). For short-term stability, the accuracy ranged from 2.85 to 3.34% with a RSD from 3.35 to 4.41%. Concerning the Freeze-thaw stability, RE ranged from -0.66 to -3.05% and RSD ranged from 2.84 to 3.52%. Discussion/Conclusion: The method developed and validated met all the criteria required by the Guidance for Validation of Analytical Methodology (UNODC, 2009) and the RDC nº27 (ANVISA, 2012). The method will be applied to forty-four seized alcoholic beverages (whiskey, rum and tequila) kindly provided by the Superintendência da Polícia Civil Técnico-Científica de Minas Gerais. Acknowledgments: This research was supported by CAPES – Brazil (Financing Code 001).

Quantifying ethanol content in alcoholic beverages by HS-GC/FID

Bigão, víTor luiz caleFFo piva1; coSTa, Bruno ruiz BranDão1; SanToS júnior, WilSon joSé raMoS1; De MarTiniS, Bruno SpinoSa2

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP; 2 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – Departamento de Química – USP.

245

4-aminopyrine (4AP) is a palliative drug generally recommended for the febrile condition. This substance usage showed relations to the decreasing of white blood cells, bone marrow suppression, and carcinogenesis, reasons which the drug was banned from some countries. Furthermore, 4AP is usually found as an adulterant in cocaine samples, either to mimic its pharmacological properties or to increase the sample volume for higher trafficking profits.1 One way to monitor this substance is through the electrochemical method. Among other analytical techniques, it exhibits some advantages such as low cost, simple handling, cheap instrumentation, and real-time response. In addition, the selectivity and sensibility of the electrochemical method can be improved by modification of the working electrode surface.2a reduced graphene oxide sensor associated to the batch injection analysis system with amperometric detection was investigated for the simple, fast and sensitive monitoring of sulfanilamide in lake water and synthetic biological fluid (saliva, sweat and urine Prussian blue (PB) is a well-known electroactive inorganic compound, highly employed for electroanalytical purposes. It can be easily produced by an electrochemical approach directly over the working electrode surface. Although PB shows good stability in acid media, the presence of graphene (and derivatives) as support for PB species can not only enhance its pH stability range but the electron transfer signal too.3,4 In this work, we show an all-electrochemical preparation of a stable reduced graphene oxide/Prussian blue (rGO/PB) nanocomposite and some of its spectroscopic and microscopic characterizations. Moreover, the electroactivity of the material was evaluated towards the 4AP presence, which led to the application as a selective electroanalytical sensor of 4AP in seized cocaine samples. The nanocomposite was produced over a glassy carbon electrode in a typical three-electrode system by cyclic voltammetry (CV) immersed in a GO dispersion with Fe2+ ions. After dried, this precursor was submitted to CV in an acid ferricyanide solution, obtaining the rGO/PB nanocomposite. Several electrochemical tests of the material in the absence and the presence of 4AP were executed. The determination of the analyte in cocaine-seized samples was made by amperometry along with a batch injection analysis (BIA) system. Moreover, the material,

along with its isolated components, was characterized by spectroscopic and microscopic techniques. While the CV of the first step of preparation showed a typical signal of graphene electroreduction, the second step CV attested the formation of PB particles by the current growth of its characteristic redox pair through the cycles. Its composition was also certified by the presence of several standard assignments in Raman and FTIR spectra. SEM images showed well-distributed PB particles over and between rGO sheets, which also had its elemental constitution confirmed by EDX. The nanocomposite exhibited higher stability at pH= 4 (KCl 0,1 mol L-1), which was the media employed in the entire work. Also, it demonstrated a good response for the electrooxidation of 4AP by CV around 0.7 V. For amperometry/BIA determination of the analyte, some parameters were optimized for better results. The analytical curve of standard 4AP injections was obtained at 0.8 V showing good sensitivity (0,168 µA L mol-1) and linearity between 0.25–10.0 µmol L-1 (R2 = 0.999). The selectivity test showed no response (3-fold) of 8 different usually founded components in cocaine seized samples. Furthermore, the injection of the seized sample showed no significant signal difference with and without a known concentration of 4AP, suggesting that the sample obtained was not filled with the analgesic. In conclusion, the rGO/PB nanocomposite can be successfully prepared by an easy two-step electrochemical method, as attested by electrochemical, spectroscopic, and microscopic techniques. This material exhibits excellent response to 4AP electrooxidation, which provides its application as a highly sensitive and selective electroanalytical sensor for the analyte in complex cocaine seized samples. We would like to thank the support from CAPES, FAPEMIG, CNPq, IQ-UFU, GQMIN, NUPE, INCT Nanocarbon, Criminalistic Institute of São Paulo, and SBTox.

REFERENCES1. W. R. de Araujo, A. O. Maldaner, J. L. Costa and T. R. L. C. Paixão, Microchem. J., 2015, 121, 213–218.2. L. V. de Faria, T. P. Lisboa, T. A. Matias, R. A. de Sousa, M. A. C. Matos, R. A. A. Munoz and R. C. Matos, J. Electroanal. Chem., 2021, 892, 115298.3. S. C. Silva, R. M. Cardoso, E. M. Richter, R. A. A. Munoz and E. Nossol, Mater. Chem. Phys., 2020, 250, 123011.4. Y. Yang, Y. Cao, X. Wang, G. Fang and S. Wang, Biosens. Bioelectron., 2015, 64, 247–254.

Selective determination of 4-aminopyrine in seized cocaine samples by a reduced graphene

oxide/Prussian blue nanocomposite

reiS, karSonn B.; BorgeS, peDro h.S.; rocha, raquel g.; raMoS, DaviD l.o.; Muñoz, roDrigo a.a.; richTer, eDuarDo M.; noSSol, eDSon

Instituto de Química, Universidade Federal de Uberlândia, 38400-902, Uberlândia, MG, Brazil.

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Introduction: Betim is considered the fifth largest municipality in the state of Minas Gerais, with a population estimated by the Brazilian Institute of Geography and Statistics, in 2021, of 450.024 inhabitants. The rapid industrial growth provided the migration of a large number of people to the municipality in search of employment. Most of the time without professional qualification, these people started to live in situations of social vulnerability, opening doors for the growth of drug trafficking, making Betim one of the most violent cities in the State of Minas Gerais. Objective: This study aimed to analyze the sociodemographic profile of the authors involved in the arrest of illicit drugs by the Civil Police in the region of Betim-MG. Methods: This is a descriptive observational study designed according to the guidelines proposed by the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). A total of 568 reports were analyzed regarding arrest of illicit drugs (marijuana, cocaine and crack) issued by the Criminal Investigation from January 2017 to December 2018, in Betim-MG. The variables collected were: sex, age, marital status and education. The database was created using the Questionnaire Development System (QDS) version 2.6.1.1, and later, the data were exported to the Statistical Package for Social Sciences (SPSS) version 19, for statistical analysis. Results: According to the data obtained, the sociodemographic profile of the authors indicted in the reports of arrest of illicit drugs (marijuana, cocaine and crack) in Betim-MG is mainly

composed of male individuals (86.6%), with a median age of 20 years old (IQ25% = 17 ; IQ75% = 24), with single marital status (88.0%) (including divorced and separated), and with incomplete primary education (33.2%). As for the age of the authors, the youngest was arrest at 14 years old and the oldest at 61 years old, and 17 years old was the age with the highest number of individuals apprehended. Discussion/Conclusion: The sociodemographic profile described for the municipality of Betim is consistent with that presented, in general, for the profile of drug users. These are generally young, male, single and with a low level of education. These results demonstrate the need of the studied municipality, as well as the country, in general, to invest in education, to prevent more and more young people from entering the world of drugs. The knowledge of the profile of the perpetrators of drug arrest in this specific region (Betim-MG), allows preventive measures for trafficking and use to be created, thus reducing public health and safety problems caused by the use and abuse of these substances. Keywords: Illicit Drugs. Arrest. Prisoners. Acknowledgments: The present work has carried out with the support of the Coordination of Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001. We thank the Federal University of São João del-Rei (UFSJ), Dona Lindu Center-West Campus (CCO) for the support. To the management and other teams of the Betim Civil Police for the reception and support during the realization of the research.

Sociodemographic profile of authors involved in illicit drug arrest in the

city of Betim, Minas Gerais

coSTa, anna carolina De Moura; oliveira, lara luiza FreiTaS; SaleS, ThaiS lorenna Souza; SancheS, criSTina; chequer, Farah Maria DruMonD

Federal University of São João del-Rei (UFSJ), Dona Lindu Center-West Campus (CCO), Divinópolis-MG, Brazil.

247

Introduction: Cocaine (COC) is a drug that causes dependence and overdose deaths, which makes this drug a public health problem in Brazil and worldwide. To make greater profits from the sale of COC, dealers mix cocaine with adulterants such as talc, boric acid and sodium bicarbonate. Dilution decreases COC activity, therefore, to mask this dilution, various drugs are added to COC-containing mixtures such as lidocaine, caffeine, carfentanil (found recently in adulterated COC in other countries), levamisole and others. At the same time, the concomitant use of COC with other drugs such as ethanol increases the lethality of COC. In this way, it is important to know which drugs are found along with COC in individuals who consume it, this can help in the creation of public policies to contain damage, such as the purchase of antidotes such as naloxone, to prevent death and/or other side effects produced by adulterants. Objective: The objective of this study was to evaluate which drugs were found concomitantly with COC in the blood, urine, stomach contents and liver of victims of violent death in the state of Minas Gerais/Brazil, in the year 2021. Methods: Retrospective analysis of 283 positive COC cases analyzed in 2021, obtained by gas chromatography coupled to mass spectrometry and from liquid/liquid extractions (stomach contents and liver) and solid phase extraction (urine and whole blood). Results: Analytes were found in the analysis: caffeine 174(61%), cocaethylene 82(29%), lidocaine 51(18%), levamisole 23(8%), theobromine 16(6%), diazepam 13(5%), midazolam 11 (4%), chlorpheniramine 11(4%), tetracaine 10(4%), tramadol 6(2%), phenobarbital 6(2%), cyclobenzaprine 5(2%),

nortriptyline 5(2%), amitriptyline 5( 2%) (2 cases in common with nortriptyline), orphenadrine 5(2%), ketamine 4(1%), probarbital 4(1%), clomipramine 3(1%), codeine 3(1%), propofol 3 (1%), methadone 3(1%), promethazine 3(1%), carbamazepine 3(1%), methamphetamine 2, propylhexedrine 2, efavirenz 2, diphenhydramine 2, orphenadrine 2, ethosuximide 2, phenytoin 2, clopidogrel 2, biperiden 2, metoclopramide 1, hydroxyzine 1, propafenone 1, pheniramine 1, olanzapine 1, metoprolol 1, mirtazapine 1, fluoxetine 1, fentanyl 1, phenacetin 1, carisoprodol 1, ibuprofen 1, temazepam 1 (common case with diazepam), adrafinil 1, escitalopram 1, sertraline 1, trazodone 1, bupropion 1, doxepin 1, meprobamate 1, chlorpromazine 1, ticlopidine 1 , fluconazole 1, phenacetin 1, aminopyrine 1, bupivacaine 1 and lamotrigine 1. Conclusion: A high number of cases containing the analytes caffeine 174 (61%), lidocaine 51 (18%), levamisole 23 (8%), theobromine 16 (6%), chlorpheniramine 11 (4%) and tetracaine 10 (4%) were found. The drugs discussed are COC adulterants. Cocaethylene was found in 82 (29%) of the cases. This metabolite increases the risk of heart attack. Few opioids were found (tramadol 6(2%), codeine 3(1%), methadone 3(1%) and fentanyl (1). This is an indication that the COC adulteration by opioids had not yet reached Minas Gerais in the year 2021. Finally, many cases of benzodiazepines diazepam 13 (5%), midazolam 11 (4%) were found. This is an indication that COC users may be using benzodiazepines to treat crises of insomnia possibly caused by this drug. Acknowledgments: This study was carried out with the support of the Civil Police of Minas Gerais.

Survey of drugs/adulterants found concomitantly with cocaine, in victims

of violent death, in the state of Minas Gerais, in the year 2021

corrêa, Brunna F.1; BorDoni, leonarDo S.1,3,4; couTo, Tauer j.g.1; goularT , chriSTiane g.l.; goularT, criSTiano o.l.1

1 Instituto Médico Legal André Roquette, R. Nícias Continentino, 1291, Gameleira, 30510-160, Belo Horizonte, Brasil; 2 Universidade Federal de Ouro Preto, R. Dois, Campus Morro

do Cruzeiro, 35400-000, Ouro Preto, Brasil; 3 Faculdade de Medicina de Barbacena, Praça Presidente Antônio Carlos, 8, São Sebastião, 36202-336, Barbacena, Brasil.

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Introduction: Illicit drugs has been persistently a global problem. In this sense, the analysis of the seizures of these drugs can be used as an indicator to reflect the dimensions of its market. Its existence in society has the consequences on violence, corruption and countless health events. In the last decade, drug trafficking and the use of illicit drugs has shown a continuous incremental trend, remaining in the world as a challenging problem in the society. Objective: To verify the evolution of incarcerations for crimes related to illicit drugs and the amount of these substances seized. Methods: A descriptive documental study was carried out, observing secondary data from the reports issued by the System of the National Penitentiary Department (SISDEPEN) that is available on the website http://antigo.depen.gov.br/DEPEN, from January 2009 to December 2019, as well as the data issued by the Federal Police on the Statistics of Drugs Seized in Brazil, that is available on the website https://www.gov.br/pf/pt-br/acesso-a-informacao/estatisticas/diretoria-de-investigacao-e-combate-ao-crime-organizado-dicor/drogas_apreendidas_por_uf.pdf/view, in the same period above. Results: According to the data from SISDEPEN, in the first semester of 2009, the number of prisoners in Brazil was 469.546, and after ten years this number increased by 61%, reaching 755.274 people. In relation to the group of drug-related crimes, the percentage increased from 18,21% in 2009, with 85.506 individuals, to 26,56% in 2019, with 200.583 prisoners. Drug trafficking was the crime responsible for the most convictions, in which it represented, in 2009,

93,04% in relation to all drug crimes (laws 6,368/76 e 11,343/06); while in 2019, this percentage dropped to 84.3%. As described by the Federal Police Seized Drug Statistics, in 2009 about 131.4 tons of marijuana were seized, and in 2019 this number increased to 266 tons, totalizing a percentage increase of 102%; in relation to cocaine, 24 tons were seized in 2009, increasing to 104.6 tons in 2019 (an increase of 336%); 49,457 ecstasy were seized in 2009, growing to 561,951 pills in 2019, totalizing an increase of more than 1,000%; and over Lysergic acid diethylamide (LSD) were 48,424 stamps in 2009 dropping to 10,643 stamps in 2019 with a 78% drop. Conclusion: The use of illicit drugs varies over time, and it is extremely important to do correctly document data related to this problem in the community. In Brazil, through a database, it is possible to observe a growing number of individuals convicted of drug-related crimes, and an increase in the seizures of drugs by the Federal Police between 2009 and 2019. It is, therefore, necessary to work on solutions to the control of the illicit drug market to contain its advance in society. In this way, it would be possible to resolve much of the damage caused by them. Keywords: Illicit Drugs; Drug Trafficking; Marijuana; Cocaine; Crack; Prisoners; Lysergic acid diethylamide. Acknowledgments: The authors would like to thank the Federal University of São João del-Rei (UFSJ)- Dona Lindu Midwest Campus for its support and financial support as well. (PIBIC Scholarship). This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - financing code 001.

The increase in the number of incarcerations in Brazil involving drug crimes and the main

drugs seized over ten years (2009-2019)

BonFioli, MaySa guilherMe; coelho, júlia França FiguereDo; Melo, Saulo naSciMenTo; Belo, viníciuS Silva; chequer, Farah Maria DruMonD

Universidade Federal de São João del-Rei, Campus Centro-Oeste Dona Lindu (UFSJ-CCO), Divinópolis, Minas Gerais, Brasil

249

Introduction: First synthesized in 2008 by Diaz and colleagues as a selective agonist to cannabinoid receptor type 2 (CB2R), MDA-19 or its formal name BZO-HEXOXIZID (N′-[(3Z)-1-(1-Hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]ben-zohydrazide) was targeted as a potential treatment for neuropathic pain. Thereafter, several researches have been conducted and promising results were published evidencing the therapeutic potential of this compound in different clinical conditions, such as melanoma, osteosarcoma, neurodegenerative disorders, and hepatocellular carcinoma. Notwithstanding this potential as a medicine, MDA-19 was unexpectedly apprehended as drug of abuse by law enforcement in Spain and Germany a few years later, in 2016. Furthermore, this substance was prohibited in China in July 2021, while in the United States this compound was seized by police officers for the first time more recently. Objective: To report the first apprehension of BZO-HEXOXIZID in Brazil followed by its chemical characterization and to discuss pharmacologically the possible reasons why a CB2R agonist has been incorporated to the illicit market. Methods: Suspected seized samples were forwarded to the Laboratory of the Scientific Police of the State of Sao Paulo. After the screening, samples were confirmed for the presence of MDA-19 by using GC-MS, FTIR, and NMR techniques. Results: A total of 53 samples were analyzed; 25 (47,17%) contained only MDA-19, whereas 28 (52,83%) were associated with other illicit substances. ADB-BUTINACA, caffeine, and cocaine were found associated with MDA-19 in 8 (36.6%), 5 (22.7%), and 9 (30.9%) of these samples, respectively, representing 22 cases. Cases containing two or more substances were also found, there

represented 6 cases that are 2 (33.3%) were MDA-19, cocaine, and caffeine, and one (16.6%) of each for cocaine, sildenafil; ADB-BUTINACA caffeine and lidocaine; THC and ADB-BUTINACA; and THC, ADB-BUTINACA, and cocaine, ever with MDA-19. Most of the cases (41) were apprehended in Sao Paulo Metropolitan Area, while the remaining 12 cases were submitted to the laboratory in coastal cities of the state. Discussion/Conclusion: We herein have described the first apprehension followed by identification and characterization of BZO-HEXOXIZID in Brazil by the State Police of Sao Paulo. Of those, 47,1% had MDA-19 only, while 52,83 % it mixed with other compounds. Interestingly, ADB-BUTINACA, a CB1R agonist, was found in 11 of these samples (only with MDA-19 or with two other rugs), which could be an attempt to mimic the effects of cannabis consumption - mixing a CB1R agonist with a CB2R agonist. However, most of these seized materials had only MDA-19; hence, the reason behind its individual use remains unclear. Speculations go from its isolated use to induce analgesia states by activation of opioid receptors or achieve any reward sensation by stimulation of the mesolimbic pathway when associated with other synthetic cannabinoids, such as ADB-BUTINACA, that significantly stimulate the CB1R resulting in a cannabis-like effect. Acknowledgments: INSPEQT (Investigation of new psychoactive substances in Forensic Chemistry and Toxicology) project, funded by the Coordination for the improvement of Higher Education Personnel (CAPES), through Public Notice 16/2020- Academic Cooperation Program in Public Security and Forensic Sciences (PROCAD), process 88887.61228/221-00.

The mystery behind the apprehensions of the selective agonist to cannabinoid type 2 receptor

BZO-HEXOXIZID (MDA-19) as a drug of abuse

araujo, karen raFaela gonçalveS1; FaBriS, anDré luiS1; neveS júnior, luiz F.2; ponce, júlio De carvalho2; coSTa, joSé luiz3; yonaMine, Mauricio1

1 School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP 05508-000, Brazil; 2 Superintendence of the Technical-Scientific Police, Institute of Criminalistics, São Paulo, SP 05507-

060, Brazil; 3 Campinas Poison Control Center, University of Campinas, Campinas, SP 13083-859, Brazil.

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Background: The Division of Postmortem Inspection is a public institution of the National Public Health System. The main goal of this service is the elucidation of deaths that occurred of natural causes but under suspicious conditions, by request of the responsible physician and the family authorization. In 2020, there was an expansion of this service in the country with the building and improvement of the units, including one in Porto Alegre, Brazil. In this scenario, toxicological examination can provide valuable information for the investigation of these inconclusive deaths. However, the complexity associated with the analysis of biological matrices mainly in postmortem cases represents a challenge for toxicologists. Consequently, the use of sensitive and specific methodologies is essential in order to guarantee the correct determination and appreciation of the results. Objective: This study aimed to investigate the presence of psychoactive substances of toxicological interest in the cases attended by the Division of Postmortem Inspection of Porto Alegre in the year of 2021. Methods: Blood samples were prepared through a protein precipitation methodology according to the article published by Franco de Oliveira et al. (2019). An aliquot 800 µL of an acetonitrile:methanol (80:20, v/v) mixture was added to 100 µL, followed by vortexing for 30 seconds and centrifugation at 10,000 rpm for 5 minutes. An aliquot of 50 µL of the supernatant was transferred to a vial and 3 µL were injected into the analytical system. A Nexera UFLC system coupled to a LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Japan) was used for the analysis. The analytical method was developed for the detection of 67 substances, including drugs of abuse and pharmaceuticals such as anesthetics, antidepressants, sedatives, and anxiolytics. Results: During the year of 2021, a total of 75 blood samples were analyzed resulting in 13 positive samples for the presence of one or more

than one psychoactive substance. Sixteen substances were identified being as it follows: benzoylecgonine (12.9% of cases, N=4; 0.113 – 0.396 µg/mL), cocaine (9.7%, N=3; 0.016 – 0.166 µg/mL), amitriptyline (9.7%, N=3; 0.079 – 0.203 µg/mL), nortriptyline (9.7 %, N=3; 0.103 – 0.198 µg/mL), cocaethylene (6.5%, N=2; 0.024 – 0.062 µg/mL), diazepam (6,5%, N=2; 0.110 – 0.460 µg/mL), nordiazepam (6.5%, N=2; 0.250 – 0.270 µg/mL), lidocaine (6.5%, N=2; 0.165 µg/mL), promethazine (6.5%, N=2; 0.013 – 0.042 µg/mL), ecgonine methyl ester (6.5%, N=2; 0.027 – 0.078 µg/mL), norcocaine (3.2%, N=1; 0.001 µg/mL), fluoxetine (3.2%, N=1; 0.087 µg/mL), norfluoxetine (3.2%, N=1; 0.030 µg/mL), carbamazepine (3.2%, N=1; 3.410 µg/mL), alprazolam (3.2%, N=1; 0.138 µg/mL) and phenobarbital (3.2%, N=1; 27.410 µg/mL). One interesting analytical finding was the presence of promethazine along with cocaine derivatives in two samples, which can indicate the use of this pharmaceutical as a cutting agent in cocaine. Discussion/Conclusion: Considering these findings, there can be an influence of the use of drugs of abuse and pharmaceuticals in deaths that occur without traumatic events. Nevertheless, these data suggest that it can be an understatement the use of these substances in the population, since epidemiological data usually is based on information from the poison control centers and forensic medical institutes. The toxicological analysis will continue in the samples provided by the service, being able to compare data across the years and identify trends in substance intake. Acknowledgments: CAPES.

REFERENCESFranco de Oliveira, S. C. W. S. E., Zucoloto, A. D., de Oliveira, C. D. R., Hernandez, E. M. M., Fruchtengarten, L. V. G., de Oliveira, T. F., & Yonamine, M. (2019). A fast and simple approach for the quantification of 40 illicit drugs, medicines and pesticides in blood and urine samples by UHPLC-MS/MS. Journal of Mass Spectrometry. doi:10.1002/jms.4369

Toxicological findings in cases attended by Division of Postmortem

Inspection of Porto Alegre in 2021

Birk, leTícia1; BarBoSa, FáBio De Souza1; peTry, aDriana uBirajara Silva1,2; MenezeS, FranciSco paz2; gonzaga, alexSanDro pinTo2; eller, Sarah1; oliveira, Tiago Franco1

1 Graduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre (UFCSPA), Brazil; 2 Division of Postmortem Inspection, Associação Hospitalar Vila Nova - Porto Alegre, Brazil.

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Background/Introduction: Biological matrices can be used to assess human exposure to xenobiotics. Among these, blood stands out for having a better relationship with the effects on human performance and adverse effects. Microsamples bring many benefits for a minimally invasive quantification of drugs of abuse and allows stabilization of analytes in the dried samples. Transport and storage of specimens is also facilitated as dried blood is not considered biohazardous. Recently, new alternatives for volumetric microsampling of blood have emerged, in addition to the already known dried blood on paper, such as the hemaPEN device, which allows capillary collection of exact volumes of blood from a capillary puncture, regardless of blood hematocrit. Furthermore, as the sample is safely protected inside the device, its use is attractive in the context of forensic toxicology. Objectives: Develop and validate a method for quantification of cocaine, benzoylecgonine and cocaethylene using liquid chromatography associated to tandem mass spectrometry (LC-MS/MS) and capillary sampling with the hemaPEN device. Methods: The hemaPEN devices were opened with a special tool provided by the manufacturer. Afterwards, the 4 filter paper discs inserted into the device were separated and transferred to a 2 mL polypropylene tube. The tubes with the disks were added with 500 µL of a methanolic solution of internal standards and homogenized for 45 minutes at room temperature, at 1000 RPM. Afterwards, a 440 µL aliquot of the supernatant was transferred to another tube and evaporated at 45 °C. The dry extract was recovered with the LC mobile phase for subsequent injection into the LC-MS/MS. The analytes were quantified in an Acquity-Xevo

TQS-micro system, using a separation on an ACQUITY UPLC BEH C18 column (1.7 µm 2.1 x 50mm), kept at 40°C, with mobile phase A composed of 0.1% formic acid in water and mobile phase B, composed of 0.1% formic acid in acetonitrile. Calibrators were prepared at concentrations of 2, 6, 20, 100, 400 and 1000 ng/mL. The method has been validated according to international guidelines, including evaluation of matrix effect, hematocrit effect on accuracy, precision and accuracy, stability, and extraction yield. Results: The analytes were stable in the hemaPEN devices for seven days, both at room and elevated temperature (45 °C). The accuracy in evaluated in blood with hematocrits between 25 and 50% presented values between 89 and 108.8%, demonstrating a small influence of the blood hematocrit. The matrix effect was adequately compensated by the internal standards, presenting values between -0.42 and 9.75%. The intra-assay precision was between 2.57 and 18.15% and the inter-assay precision was between 2.70 and 10.82%. Accuracy ranged from 89.5 to 118.0%. The method was applied to samples obtained from patients admitted to a chemical dependency treatment unit. Discussion/Conclusion: A method for the determination of cocaine, benzoylecgonine and cocaethylene using LC-MS/MS in dried blood samples obtained with the hemaPEN microsampling device was developed and validated. The developed method presents adequate performance and is being applied in an ongoing study with patients. The hemaPEN device can be a safe and convenient alternative for assessing exposure to drugs of abuse in both clinical and forensic toxicology. Acknowledgments: CAPES, Universidade Feevale, and Trajan.

Validation of methods for the determination of cocaine, benzoylecgonine, and

cocaethylene in dried blood using the hemapen microsampling device

SMiDT, Mariana1,2; BaSTiani, MarcoS Frank1; hahn, roBerTa zilleS1; linDen, raFael1,2

1 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil; 2 Graduate Program on Toxicology and Analytical Toxicology, Feevale University, Novo Hamburgo-RS, Brazil.

09 GENOTOXICITY AND

CARCINOGENESIS

253

Introduction: Monolayer models, widely disseminated and used, are not able to reconstruct the complex and heterotypic in vitro cellular microenvironment, and animal models face increasingly reinforced ethical barriers. Meanwhile, organotypic culture promotes physiological parameters of organs and tumors, and allows to mimic the architecture of the parental tissue, being important to understand the role of the microenvironment, transcriptional plasticity and agents for targeted therapy in heterogeneity, selection of cells with intrinsic or acquired resistance and in the reprogramming of oxidative metabolism by tumor cells. The 3D cell culture model allows the study of changes in the behavior of cells and the performance of tests to elucidate the mechanism of drug action, being an interesting platform for toxicogenomic tests. Objective: To standardize the technique for obtaining and isolating the three-dimensional model of spheroids, based on tests with different proportions of melanoma and fibroblasts; to observe the behavior of the 3D cell aggregate in a reconstructed human skin model; and to investigate the tumor heterogeneity and expression of proteins of the melanoma proliferation and invasion pathways from the analysis of SKMEL-28 clones. Methods: Clonal subpopulations were stochastic isolated from the initial parental SKMEL-28 cell line, then 48-well culture plates were prepared using 1.5% soft-agar, and stemming from the hanging drop method, 2,5.104

cells were plated in 30 uL, in different proportions of melanoma and fibroblast. After the incubation period, 3D aggregates were obtained and subsequently fixed for histological processing. In a model of reconstructed human skin, protocolled and standardized by the group, spheroids were added to the dermis and histologically analyzed. Results: The spheroids obtained with a higher proportion of fibroblasts showed higher levels of compaction and density, highlighting the importance of non-cancerous stromal cells for the creation of the tumor microenvironment. The SKMEL-28 cell line, being metastatic, showed an invasive phenotype in all spheroid proportions when sedimented, and in the reconstructed human skin model. Discussion/Conclusion: The size of the spheroid is not only determined by the number of cells, but the interaction effects of compaction inducers, being important for the architecture of the three-dimensional cell aggregate. The use of organotypic culture ensures a more accurate understanding of the cellular interaction with the extracellular matrix and other cell types, further studies are needed to compare the results of 2D and 3D models, and to elucidate the mechanism of drug action and emergence of resistance, with the possibility of using this standardized platform for testing antimelanoma compounds. Acknowledgments: FAPESP (17/04926-6, 18/14936-1 and 21/05150-7), CNPq (#408769/2018 and #304339/2017-2) and CAPES.

Cellular spheroids as a platform for toxicogenomic assays in cancer

queijo, roDrigo gonçalveS; carvalho, lariSSa anaSTacio Da coSTa; Maria-engler, Silvya STuchi

Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, Avenida Professor Lineu Prestes, 580, São Paulo 05508-00, SP, Brazil.

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Background/introduction: The recent discovery of small-molecule nitrosamine impurities in marketed drugs, starting with nitrosodimethylamine (NDMA) in batches of Valsartan in 2018, has led to significant regulatory response, including drug recalls and regulatory guidance that requires the evaluation of all synthetic and formulation routes for the potential presence of nitrosamine impurities. Due to the wide range of potential routes of formation for nitrosamines1, many active pharmaceutical ingredient (API) structures are themselves liable to be nitrosated, either during the later stages of the synthetic process or as the formulated drug product. Objectives: This work describes the formation of a data-sharing initiative, coordinated by Lhasa Limited, which has the following aims: Firstly, since many APIs are generic drugs and are manufactured by multiple companies, the reduction of duplicate testing and the availability of an Ames test result conducted to the highest standards and in appropriate conditions should reduce potential uncertainties for regulatory submissions. Secondly, the consortium aims to investigate differences between these compounds and the small molecules which comprise the majority of publicly available nitrosamine data2 and elucidate any structure activity trends and significant differences. Methods: Pharmaceutical industry partners share Ames data on structurally-complex nitrosamines, which is input by Lhasa (in an anonymised form) into a shared database to enable read across and reduce duplicate testing. Results: The consortium currently consists of 6 respected members within the pharmaceutical industry,

with additional pharmaceutical companies in the process of joining. It is estimated that mutagenic data from 60 compounds will be donated during Q2 2022. Discussion/Conclusion: Extensive testing of complex nitrosamines – synthesized on purpose and fully characterized – is currently underway as part of the second stage of the regulatory response to the current crisis. The need for large numbers of Ames tests, and potential in vivo follow-up assays, leads to pressure on the few laboratories able to carry out these tests. As a result, the sharing of testing results amongst companies – both discovery pharmaceutical companies and generic manufacturers, leads to a reduction in the testing burden for each member. The majority of toxicity data that has been reported is for small-molecule nitrosamines, with nitrosodiethylamine and nitrosodimethylamine the most studied as well as among the most potent, while a far smaller amount of data is currently available for more complex structures. The proportion of complex nitrosamines which are Ames negative may be higher than the proportion of small molecule nitrosamines, for reasons such as differences in metabolic potential or the proposed adducts formed. Full testing of this hypothesis requires the curation of a large high-quality dataset, which can only be achieved via cross-industry collaborations such as this.

REFERENCES1. Lopez-Rodriguez et al (2020), Org. Process Res. Dev. 24, 1558-1585. 2. Thresher et al (2020), Regul. Toxicol. Pharmacol., 116, 104749. 3. Brambilla and Martelli (2007), Mutat. Res., 635, 17-52. 4. Schmitsdorff et al (2022), Arch. Pharm. e2100435.

Collaborative analysis of complex nitrosamines

avila, carolina MarTinS; ponTing, DaviD. j.; Werner, anne-laure D.; TennanT, rachael e.; oliveira, anTonio anax F.; hegheS, crina

Lhasa Limited, Leeds, United Kingdom.

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Background/Introduction: melanoma is the deadliest type of skin cancer due to its high metastatic capacity. Accumulation of genetic mutations in melanocytes confers phenotypic changes, especially in MAPK signaling pathway which might develop point mutations in BRAF protein (V600E, the most common) or NRAS protein (Q61K and Q61R). Targeted therapy has revolutionized the treatment of cancer. Melanoma is one of the biggest beneficiaries considering the emergence of BRAF inhibitors (iBRAF, e.g., vemurafenib) along with MEK inhibitors, in combination or as a single treatment option against NRAS+ mutation tumors which present poor prognosis. However, even when therapy does not initially fail, high heterogeneity of melanoma cells leads to a selection and emergence of treatment-resistant cells that culminates in disease relapse. This fact highlights the need to search for new therapeutic alternatives, whether isolated or combined, to overcome these resistance phenotypes. In this context, metallodrugs (e.g., cisplatin) appear as anticancer drugs candidates, in which we can highlight copper(II) complexes. Copper(II) complexes have already been studied for their ability to trigger programmed cell death, also claiming an activity with more tolerable adverse effects. Design, synthesis, and biological evaluation of dinuclear copper(II) complexes on metastatic melanoma cell lines were already reported, showing cytotoxic activity displaying apoptotic and autophagic profiles, yet their activity against BRAF inhibitor-resistant cells, as well as, with different mutations in NRAS have not

been investigated thus far. Objective: The present study evaluates the cytotoxic activity of the dinuclear copper(II) complex [Cu2(apyhist)2(dpam)]ClO4) against strains resistant to iBRAF (vemurafenib) and with different mutations in NRAS. Methods: MTT assay and trypan blue (TB) exclusion techniques were used to evaluate cell viability of naive and vemurafenib-resistant WM164 and SK-MEL-28 (BRAFV600E) and SK-MEL-173 (NRASQ61K) metastatic melanoma cell lines. Concentrations ranging from 0,03µM to 100 µM were tested in a 24-hour incubation period. Results: The copper(II) complex demonstrated a concentration-dependent cytotoxicity in a micromolar range potency, both assessed by MTT and TB exclusion. IC50 values were slightly higher for vemurafenib-resistant strains compared to the naive cell lines. Regarding the NRAS+ cell line (SK-MEL-173), the IC50 value was about double compared to the BRAF mutated cell lines. In addition, the copper(II) complex was also more potent than the anticancer metallodrug cisplatin and the first-line anti-melanoma drug dacarbazine. Interestingly, the copper(II) complex was 40x more potent against WM164 cells compared to cisplatin cytotoxicity. Discussion/Conclusion: These results support the hypothesis that the copper(II) complex can overcome the vemurafenib-resistance phenotype, in addition to the more aggressive cell line NRAS+. This scenario configures a viable alternative therapy for patients with BRAF and NRAS mutations with no longer responsive to existing treatments.

Copper (II) complex as a potential treatment to overcome BRAF inhibitor resistance

and aggressive NRAS point-mutation

MoraeS junior, Manoel oliveira1; carvalho, lariSSa anaSTácio Da coSTa1; nuneS, cleia juSTino2; Ferreira, ana Maria Da coSTa2; Maria-engler, Silvya STuchi1

1 Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil; 2 Department of Fundamental

Chemistry, Chemistry Institute, University of São Paulo, São Paulo, Brazil.

256

The worldwide increased incidence of mosquito-borne diseases has led the population to look for preventive measures, as the use of insect repellents. Although insect repellents are considered safe, there is an increase of their simultaneous use combined with UV filters due to high incidence of solar radiation in Brazil. Consequently, studies that evaluate the interaction of the combination of these substances are required to ensure the safety of the population exposed to these compounds. In the literature we can find many studies about the undesirable effects of UV filters and repellents as isolated raw materials. There are also studies that demonstrate interactions between UV filters and insect repellents. Thus, this study evaluated the photomutagenic potential of UV filters and IR3535 combination in an in-house skin model, an in vitro assay, based on Reconstructed Skin Micronucleus (RSMN) assay, recently accepted into the OECD’s test guideline development program, for mutagenic potential evaluation. The combination of UV filters avobenzone, ethylhexyl methoxycinnamate and octocrylene with IR3535 (UVF+IR3535) were diluted in sesame oil and applied onto in house reconstructed human skin models, so as the positive control 8-methoxypsoralaen (8MOP). After 24 hours of incubation, the skin models were irradiated and incubated with cytochalasin B until the isolation of the cells, that were dropped onto glass slides, stained, and analyzed by fluorescence microscopy. Regarding mutagenicity, the results of the frequency of micronuclei obtained in non-irradiated models

indicate that the compounds did not show mutagenic potential. Regarding photomutagenicity, it was observed that the treatment with positive control 8MOP (+UV) showed statistically higher micronuclei values than the values of UVF+IR3535 and sesame oil, considered non-photomutagenic. The percentage of binucleated cells between treatments submitted or not to UV radiation, did not show any significant variation. The cytokinesis-block proliferation index (CBPI) indicated that the irradiation dose, experimental conditions, and concentrations studied did not interfere in cell proliferation and were not cytotoxic, since no variation was observed among treatments and those pairs submitted or not to UV radiation. The insect repellent IR3535, according to TOXNET (Toxicology Data Network) and HSDB (Hazardous Substances Data Bank) platforms, has no evidence of genotoxicity or photogenotoxicity. According to literature, in vitro assays and in vivo micronucleus assay have also shown that the IR3535 was not mutagenic. In conclusion, the substances studied did not show any mutagenic potential. Regarding photomutagenicity, the combination of UV filters and IR3535 and the vehicle (sesame oil) also did not show any photomutagenic potential. This work was supported by “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP), “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq), and “Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior” (CAPES).

Evaluation of photomutagenic potential of UV filters and IR3535 combination

Fuzinaga, ThaíS y.T.1; gluzezak, ana j.p.1; TavareS, renaTa S.n.1; kaWakaMi, caMila M.1; aBe, Flávia r.1; oliveira, Danielle p.1; Maria-engler, Silvya S.2; gaSpar, lorena r.1

1 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo; 2 School of Pharmaceutical Sciences, University of São Paulo.

257

Background/Introduction: Genetic alterations affecting RAS proteins are commonly found in human cancers. Roughly a fourth of melanoma patients carry activating NRAS mutations, rendering this malignancy particularly challenging to treat. Although the development of targeted as well as immunotherapies led to a substantial improvement in the overall survival of non-NRASmut melanoma patients (e.g. BRAFmut), patients with NRASmut melanomas have an overall poorer prognosis due to the high aggressiveness of RASmut tumors, lack of efficient targeted therapies or rapidly emerging resistance to existing treatments. Understanding how NRAS-driven melanomas develop therapy resistance by maintaining cell cycle progression and survival is crucial to develop more effective and specific treatments for this group of melanoma patients. With the goal of improving the low effectiveness of MEK inhibitors in metastatic NRAS-mutated melanoma, it is necessary to research new therapies for these patients. In others studies, the gliotoxin (GT) exhibits cytotoxic effect and a therapeutic potential for prevention of naïve BRAFmut melanoma metastasis. GT is a fungal secondary metabolite that mimics the peroxiredoxin activity by reducing hydrogen peroxide and being reduced by the NADPH electrons from TrxR system. Objective: Our aim is to evaluate the cytotoxicity of GT in NRAS-mutated melanoma and its therapeutic potential. Methods: The cell lines used were NRAS-mutated

melanoma (SK-MEL-173 and SK-MEL-147), primary melanocytes, and primary fibroblasts as control. Cell viability assay using trypan blue was used to calculate IC50 (Inhibitory Concentration) of melanoma cells treated with GT. The clonogenic assay evaluates the ability of a single cell to grow into a colony and determine the effectiveness of cytotoxic agents. The wound healing assays creates a “wound” in the cell monolayer and the captured images during cell migration are followed during the closure or not of the wound. Results: The trypan blue viability assay has IC50 values of nM to melanocytes (50), SK-MEL-147 (57), SK-MEL-173 (58) and fibroblasts (73), respectively after 24-hour GT treatment. In clonogenic assay, all cell lines (Skmel-147, 173 and fibroblasts) had a significant decrease of colonies after GT treatment with concentrations IC25 and IC12,5. The wound healing assay showed that SK-MEL-147 had a significant decrease of migration after GT treatment (p<0.05) in 24-hour. Discussion/Conclusion: Our results show that gliotoxin proved to be efficient at low concentrations with higher cytotoxicity and selectivity to melanoma than fibroblasts and also impacted the migration process. Therefore, GT has attributes as a promising molecule that can be further explored for NRAS-mutated melanoma. Acknowlegments: We thank to financial support of CNPq (#408769/2018, #304339/2017-2 and 142058/2020-3), FAPESP (18/14936-1 and 17/04926-6) and CAPES.

Gliotoxin and its potential cytotoxicity against NRAS-mutated melanoma

noMa, iSaBella haruMi yonehara; carvalho, lariSSa anaSTacio Da coSTa; Maria-engler, Silvya STuchi

Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

258

Background/Introduction: Melanoma is a malignant neoplasm that originates from melanocytes and although it corresponds to only 3% of skin cancers, it is responsible for 80% of deaths; mostly due to its resistance to specific inhibitors of mutated proteins in the MAPK pathway, such as BRAF. Researchers has shown an interesting relationship between adenosine kinase (ADK) activity and cell growth and proliferation, but its importance has never been investigated in melanoma. ADK plays a key role in purine metabolism regulation and studies have shown that one of the ADK isoforms might be related to cancer biology. In silico screening studies of our group, using TCGA and GEO databases, identified ADK among a set of genes differentially expressed between nevus and invasive melanoma. Objective: In this study, we investigate how ADK may contribute to the mechanisms that lead to melanoma progression and resistance. Methods and Results: Analysis of a TCGA cohort of cutaneous melanoma samples revealed that 9% of the samples had genetic alterations in ADK, mainly characterized by mRNA increase. To validate this data, we checked the gene and protein expression levels in a broad panel of human melanoma cell

lines, with sensitive and resistant cells to a BRAF inhibitor by RT-qPCR and Western Blotting assays, in which we observed that metastatic melanoma showed increased ADK expression when compared to melanocyte. Furthermore, we observed a decrease in both genetic and protein ADK expression in cells resistant to the BRAF inhibitor. Cell proliferation assay showed that the inhibition of ADK, either by a specific inhibitor (ABT702) or by genetic knockdown using shRNA, in ADK-high melanoma cells resulted in an increase of proliferation compared to their controls. Discussion/Conclusion: These data suggest that ADK downregulation might have an impact in melanoma cell growth and proliferation. Therefore, exploring the impacts of ADK, both by pharmacological and genetic inhibition, may contribute to understanding the role and regulation of ADK regarding the molecular mechanisms that support the progression of melanoma and resistance to therapy. Acknowledgments: This work has been supported by the following Brazilian research agencies: FAPESP, CAPES, CNPq. The first author is funded by the grant # 2020/14878-1, São Paulo Research Foundation (FAPESP).

Strategies for overcoming toxicity and resistance in melanoma progression and in adaptative resistance to braf

inhibitors using ADK modulation

Silva, julia rezenDe1; oliveira, érica apareciDa2; carvalho, lariSSa anaSTacio Da coSTa1; Maria-engler, Silvya STuchi1

1 Clinical Chemistry and Toxicology Department, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil; 2 Centre for Evolution and

Cancer, The Institute of Cancer Research, London, SM2 5NG, UK.

259

Background/introduction: melanoma is the most aggressive type of skin cancer and more than 50% of the patients harbor a BRAF mutation. Although BRAF inhibitors (BRAFi) has revolutionized therapy, the minimum residual disease is still refractory. Microenvironment and transcriptional plasticity propitiate distinct subpopulations within the tumor and the use of BRAFi contributes to the selection of intrinsic resistant cells or acquired resistant phenotype. MITF is the key biomarker for phenotype switching (proliferative versus invasive). MITFhigh/AXLlow phenotype is linked to proliferation and BRAFi sensitivity and MITFlow/AXLhigh to invasion and resistance. MITF controls oxidative metabolism by regulating PGC-1α, responsible for mitochondrial biogenesis and antioxidant proteins expression. BRAF mutation suppresses MITF, PGC-1α, OXPHOS, antioxidant proteins and ROS, while BRAFi increases it. Naïve cells show multiple transcriptional states, but it is poorly explored if all cells respond to BRAFi similarly and what is the role of antioxidant defense at the single cell level. PRX1 and PRX2 are central players in redox signaling acting as sensors, antioxidants and biomarkers. Since the lack of PRX is advantageous for melanoma progression, we tried to overcome the resistance associated to the heterogeneity using gliotoxin, an antioxidant metabolite from fungi containing a disulfide bridge that mimics PRX. Objective: understand the role of PRX1 and PRX2 in heterogeneity and overcome intrinsic and acquired resistance to BRAFi targeting PRX by using gliotoxin. Methods: we produced a SK-MEL-28R with acquired

resistance to BRAFi and stochastically isolated clonal subpopulations (C1, C2 and C3) from naïve melanoma (SK-MEL-28P). Functional profiles, phenotypes and survival were analyzed using 2D models and 3D reconstructed skin. Results: C1 is less proliferative, more migratory and invasive, less sensitive to BRAFi, less dependent on OXPHOS and more sensitive to oxidative stress; C2 is more proliferative, less migratory and invasive, more sensitive to BRAFi and less sensitive to oxidative stress; and C3 is less proliferative, more migratory and invasive, less sensitive to BRAFi, more dependent on OXPHOS and more sensitive to oxidative stress. The intrinsically resistant clones C1 and C3 have lower MITF, PGC-1α, PRX1 and C1 has higher AXL, linking PRX1 to clonal heterogeneity, intrinsic and acquired BRAFi resistance. PRX2 expression is equal among the clones, depleted in SK-MEL-28R and act as a redox sensor in melanoma. Gliotoxin exhibited a potent cytotoxicity in 85-160nM, with different IC50 among the clones and SK-MEL-28R, inhibiting colony formation and showing to be a promising agent to overcome intrinsic and acquired BRAFi resistance. Discussion/Conclusion: our results illustrate new roles of PRX1 and PRX2 in melanoma as contributors of heterogeneity and resistance, demonstrating that redox sensitive reactions could be a new target in melanoma resistance once it is linked to tumor heterogeneity and could be overcome with a PRX mimetic. Acknowledgments: FAPESP (18/14936-1 and 17/04926-6), CNPq (#408769/2018 and #304339/2017-2) and CAPES.

The peroxiredoxin mimetic gliotoxin overcomes heterogeneity, intrinsic and acquired resistance

to BRAF inhibitor in metastatic melanoma

carvalho, lariSSa a.c.1; noMa, iSaBella h.y.1; uhera, aDriana h.1; Siena, áDaMo D.D.2; haruMi, luciana3; Mori, MaTheuS p.4; goDing, colin5; pinTo, naDja c. Souza4; FreiTaS,

vaneSSa M.3; Silva-jr, WilSon a.2; SMalley, keiran S.M.6; Maria-engler, Silvya S.1

1 Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, USP, SP, Brazil; 2 Department of Genetics, Ribeirão Preto Medical School, USP, SP, Brazil; 3 Department of

Cellular Biology and Development, Institute of Biomedical Sciences, USP, SP, Brazil; 4 Department of Biochemistry, Institute of Chemistry, USP, SP, Brazil; 5 Ludwig Institute for Cancer Research,

Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 6 Department of Molecular Oncology, Comprehensive Melanoma Research Center, Moffitt Cancer Center, FL, USA.

10 IMMUNOTOXICOLOGY

261

Eugenia caryophyllus is a tropical plant belonging to the Myrtaceae family and is popularly known as the clove. It is widely applied in culinary and cosmetic products as a flavoring agent. The essential oil obtained from buds of Eugenia Caryophyllus L. is generally used in traditional medicine. Previous studies have reported its biological activities such as antioxidant, antibacterial, antifungal, antiallergic, insecticidal, anticarcinogenic, and antimutagenic effects. There is a lack of toxicological information on the clove essential oil. Therefore, this study aimed to analyze the toxicity of clove essential oil in human lymphocytic cells, through in vitro and in silico methods. Clove essential oil was bought commercially from the IBD-certified firm, BioEssência®. A gas chromatograph linked to a mass spectrometer (GC/MS) was used to identify and quantify its main constituents. The identification process included interpreting chromatogram peaks and comparing retention times with those found in the National Institute of Standards and Technology (NIST) databases. Seven major components were identified and quantified: eugenol (86.7%), β-caryophyllene (10.4%), humulene (1.9%), eugenyl acetate (0.3%), caryophyllene oxide (0.3%), terpinen-4-ol (0.2%), γ-terpinene (0.1%) and others components (0.2%). To analyze the toxicity, the lymphocytes were cultivated and exposed at five different concentrations of clove essential oil: 1, 10, 100, 500, and 1000 µg/mL, in addition to negative and positive controls. The lymphocytes quantification was evaluated using the Neubauer chamber. For in silico study, the canonical forms of eugenol, β-caryophyllene, and humulene identified in clove essential oil were found in the PubChem database and submitted to computational

tests via module DIGEP-Pred from Way2Drug and GeneCards platforms. Only genes of the species Homo sapiens were investigated for potential interactions (upregulation and downregulation with probability greater than 70%) between the main components of the oil and genes associated with the cell cycle or genetic maintenance of the cell. The results in vitro analysis showed that the minor concentration did not affect the lymphocytes total count. On the other hand, the concentrations of 10, 100, 500, and 1000 µg/mL decreased lymphocytes count as the concentration increased. The computational analysis of Clove essential oil compounds demonstrated that the main component Eugenol induces alterations in the expression of approximately 21 genes, causing both upregulation and downregulation. Especially, our results showed that Eugenol causes downregulation of the H1FX gene, a protein-encoding that participates in the formation of histones, which are responsible for the nucleosome structure of the chromosomal fiber in eukaryotic cells. To form higher-order chromatin structures, the chromatin fiber is further compacted by the interaction of a binding histone, H1, with the DNA between the nucleosomes. As a consequence, the organization of DNA in the cell is affected and the cell cycle is interrupted. This mechanism may explain the lymphocytes decrease when exposed to higher concentrations of the clove essential oil. Therefore, according to our results, Eugenia caryophyllus essential oil is likely to interfere with gene expression and, consequently, cell toxicity. However, more studies are needed to certify the mechanism involved in the reported effects. Acknowledgments: CAPES, CNPQ, FAPERGS e UNIPAMPA.

Analyzes of clove essential oil immunotoxicity based on in vitro and in silico studies

eTcheverry, BiBiana FraSSon; goMeS, gaBriela criSTiane MenDeS; rioS, naThália vieira; chaveS, paMella eDuarDha eSpinDola; SoTelo, êMily clori; zuravSki, luíSa; MachaDo, Michel ManSur

Grupo de Pesquisa em Imunologia e Genética Aplicada (GIGA), Universidade Federal do Pampa, Uruguaiana, RS, Brasil.

262

Background: Exposure to combustible products of cigarette smoke (CS) aggravates rheumatoid arthritis (RA) symptoms and impairs immune response. Hence, non-combustible tobacco products, such as heat-not-burn tobacco (HNBT), which mainly release nicotine, glycerin and propylene glycol, could reduce those toxicities caused by CS. Nevertheless, this hypothesis needs to be proved. Regarding the immunotoxic effects of non-combustible tobacco products, further experimental data are required. Neutrophils are innate immune cells generated by a process named granulopoiesis in the bone marrow (BM). Neutrophil delivery into blood is a sustained process of homeostasis and host defense, also these cells play a pivotal role during RA course, and its production and functions can be directly affected by xenobiotics exposures, including those released by CS. However, the impact of HNBT in neutrophil generation and functions, remains unclear. Recently, our group reported that mice exposed to CS displayed a worsen in antigen-induced arthritis (AIA) symptoms, with higher neutrophil migration into synovial cavity; conversely HNBT did not cause these effects. Objective: We here investigated the impact of CS or HNBT exposures on BM and circulating leukocytes profile on AIA model and the impact of these exposures in neutrophil functions in vitro. Methods: AIA was induced in C57B6 mice by s.c administration of 500 μg of methylated bovine serum albumin on day 0; boosted on days 7 and 14 and challenged intra articular (i.a.). on day

21; 24h later, samples were collected. AIA-mice were exposed to airflow, CS or HNBT from day 14 to 20 after first immunization, for 1h/twice a day under the Health Canada Intense (HCI) smoking regime (55 mL of smoke for 2 s puffing, for every 30 s). The cellularity and profile of BM and circulation leukocytes were performed by manual counting and morphological analysis. Neutrophils from naive mice were exposed to airflow, CS or HNBT (30min, 2 s of smoke/vapor followed by 58 s of airflow) to evaluate fMLP-induced chemotaxis and NETs formation by flow cytometry. Results: AIA increased BM cellularity in airflow-exposed mice, which was higher in CS-exposed group, due to the enhanced number of immatures, banded and segmented neutrophils; conversely, HNBT exposure did not enhance BM cellularity as CS did, and reduced the number of immature and blast cells. Elevated number of circulating leukocytes was detected in airflow and CS exposed mice under AIA, which was lower in HNBT exposed animals. In vitro assays demonstrated that neutrophils exposed to HNBT or nicotine, but not to CS, showed impaired fMLP- induced chemotaxis and enhanced activation state and NETs formation in comparison to airflow exposure. Conclusion: Associated data obtained show that CS and HNBT exposures differently affect granulopoiesis during AIA, and nicotine in either CS or HNBT impairs pivotal neutrophil functions during the host defense. Acknowledgments: FAPESP and CNPq.

Cigarette smoke and heat-not-burn tobacco vapor exposures alter

granulopoiesis and neutrophil functions during experimental arthritis

ScharF, paBlo1; heluany, cínTia1; SanDri, Silvana1; SchneiDer, ayDa henriqueS2; BarBiM, paula DonaTe2; Fock, ricarDo1; cunha, FernanDo2; FarSky, SanDra1

1 Department of Clinical and Toxicological Analyses School of Pharmaceutical Sciences University of São Paulo – Brazil; 2 Department of Pharmacology, Ribeirão Preto Medical School, University of Sao Paulo.

263

Introduction: Neuroinflammation is a pathological factor involved in the development of neurodegenerative diseases like Parkinson’s and Alzheimer’s diseases. Lipopolysaccharide (LPS), in turn, is a membrane element of gram-negative bacteria, which induces neuroinflammation via toll-like receptor 4 (TLR-4) and NF-kB activation. Marinobufagenin is a cardiotonic steroid isolated from Bufo marinus toad venom. This molecule has the ability to connect Na+/K+ -ATPase and recent studies have demonstrated anti-inflammatory effects in vivo and in vitro based on modulation of cell migration and production of proinflammatory cytokines like IL-6 and IL-1β. Aim: The present work seeks to evaluate the cytotoxic and immunomodulatory effects of marinobufagenin on glial cells challenged with LPS. Methods: The initial step was to evaluate

the cytotoxicity effects of different concentrations of marinobufagenin (10,100,1000 and 10000 nM) in primary culture of glial cells. Thereafter, the glial cells were treated with concentrations of marinobufagenin mentioned above and challenged with LPS (1ug/ml) for 18 hours. Lastly, the culture medium was collected to evaluate the levels of cytokines TNF-α, IL-6 and IL-10 by ELISA. Results: Marinobufagenin did not show cytotoxic effects at any concentration tested on glial cells in the MTT assays. Also did not interfere with the production of cytokines by glial cells challenged or not with LPS. Conclusion: Preliminary data suggest that marinobufagenin did not show cytotoxic effects in glial cells challenged or not with LPS. However, further studies are needed to better understand the effects of marinobufagenin on neuroinflammation. Financial Support: CNPq.

Cytotoxicity of Marinobufagenin in Glial Cells Chalenged Whith LPS

FariaS, evelyn rayani araújo1; Silva, rivia regina lopeS1; Souza, naTacha MeDeiroS2; MarqueS, ana Maria2; Scavone, criSToForo2; quinTaS, luíS e.M.3; leiTe, jacqueline alveS1

1 Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Goiás, Goiânia, Brazil; 2 Departament of Pharmacology, Institute of Biomedical Sciences, University of

São Paulo; 3 Laboratory of Biochemical and Molecular Pharmacology, Institute of Biomedical Sciences, Health Sciences Center, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro.

264

Foeniculum vulgare, popularly known as fennel, is a perennial herb that grows in tropical and temperate regions and receives applications in culinary, food, and cosmetics industries, as well in traditional medicine for therapeutic purposes. Computational methods include a variety of tools that contribute to processes like molecules design, prediction of pharmacokinetics and toxicological parameters, and identification of potential biological targets, proposing pharmacological activities or mechanisms of action from the compounds under analysis. Due to the biological activities attributed to this species and the relevance to determine the security of his use, in addition to the advantages of the use of computational methods, we aimed to evaluate the potential cytotoxicity of Foeniculum vulgare essential oil in human lymphocytes and predict the mechanisms of action through in silico analysis. For this purpose, Foeniculum vulgare essential oil was purchased from the company BioEssência, with a certificate of organic and natural ingredients. The phytochemical characterization was performed by Gas Chromatography Coupled to Mass Spectrometry (GC/MS), the retention times obtained were compared to data previously described in literature and present in the databases of the National Institute of Standards and Technology (NIST). Lymphocytes cultures were prepared from venous blood obtained by venipuncture from a donor who did not ingest alcohol, medicines, or smoke in the last 72 hours. Human peripheral blood mononuclear cells (PBMC) were isolated using Histopaque® 1.077 g/mL and maintained in RPMI 1640 supplemented with fetal bovine serum and antibiotics for 24 hours at 37ºC in a 5% CO2 environment. In the sequence, lymphocytes were isolated and stimulated with phytohemagglutinin-M. Lymphocytes were

exposed to Foeniculum vulgare essential oil at a range of concentrations (1 µg/mL - 1000 µg/mL), while the negative control contained only supplemented medium and the positive control received colchicine (1 µg/mL). After 24h of exposure, was performed the cell count. For the in silico analysis, the canonical form of the majority compounds of Foeniculum vulgare essential oil was searched in the platform PubChem and posteriorly submitted to in silico tests in the platforms Way2Drug using the DIGEP-PRED module and GeneCards for evaluation of the gene interactions (upregulation and downregulation with probabilities above 80%) associated to the cell cycle or with genetic aspects of the cell. The statistical analysis was realized in specific software. The CG/MS analysis detected and quantified 99.6% of the total constituents, being trans-anethole the majority compound identified in the essential oil. The results in vitro showed that Foeniculum vulgare essential oil affects the total lymphocytes count with LC50 of 501.2 µg/mL, and it is possible to predict the mechanisms related to this effect through in silico simulation. According to these analyses, Foeniculum vulgare interferes in the cell cycle promoting a downregulation of H1FX, a gene that encodes the protein H1x and it is related to chromosome condensation and regulation of DNA replication. Therefore, our findings showed that Foeniculum vulgare essential oil is cytotoxic for human lymphocytes. Moreover, the majority compound trans-anethole interferes in gene expression of H1FX affecting the histones replication, as predicted through in silico platforms. Subsequent studies are necessary to elucidate the role of H1FX in cytotoxicity and also the potential activity against tumor cells. Acknowledgments: CAPES, CNPq, FAPERGS and UNIPAMPA.

In vitro and in silico analysis of the cytotoxicity of Foeniculum vulgare essential oil in human lymphocytes

goMeS, gaBriela criSTiane MenDeS; eTcheverry, BiBiana FraSSon; chaveS, paMella eDuarDha eSpinDola; rioS, naThalia vieira; caMpoS, Dyene naSciMenTo; zuravSki, luíSa; MachaDo, Michel ManSur

Grupo de Imunologia e Genética Aplicada (GIGA), Universidade Federal do Pampa, Uruguaiana, RS.

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Mentha arvensis, popularly known as Brazil mint, Japanese mint, or menthol mint is a herbaceous, perennial, and aromatic plant belonging to the Lamiaceae family. It is widely cultivated in tropical and subtropical regions. The essential oil obtained from the leaves of Brazil mint is extensively used in culinary, cosmetics, and traditional medicine as a therapeutic resource for fever, colds, asthma, digestive and cardiovascular disorders, among other diseases and conditions. Recent studies with this oil mainly aimed to prove the effectiveness of its biological properties in preventing diseases and/or maintaining health and not verify the safety of its use. In that regard, the present study had as objective analyze the lymphotoxicity of Mentha arvensis essential oil. Initially, the compounds of the oil were identified and quantified through the analytical method of Gas Chromatography Coupled to Mass Spectrometry (GC/MS) by retention times obtained which compared with those reported in the literature and present in the databases of the National Institute of Standards and Technology (NIST). In the sequence, for in vitro tests, the peripheral blood was collected by venipuncture of a healthy self-declared individual, the Peripheral Blood Mononuclear Cells (PBMC) were separated by density gradient using Histopaque-1077® (1:1) and incubated in RPMI 1640 supplemented with SFB, penicillin-streptomycin, and gentamicin at 37°C in 5% CO2 overnight. The lymphocytes that remained in suspension were separated from monocytes which were adhered to the contact surface. Human purified lymphocytes were exposed to a range of concentrations of Brazil mint (1, 10, 100, 500, and

1.000 μg/mL) and incubated at the same conditions for 24h, the protocol was performed in triplicate. After this period, the total lymphocytes were counted in Neubauer’s hemocytometer and the results were expressed as lymphocytes/mL. In addition, we submitted the majority of compounds identified in the oil to in silico platforms. For this, the canonical form of the main constituents found were submitted to the platforms Way2Drug using the DIGEP-PRED module and GeneCards. We analyzed the possible interactions (upregulation and downregulation with probabilities above 70%) between the majority compounds and the genes related to the cell cycle or with the genetic maintenance of the cell. The in vitro assay results were expressed as mean ± standard deviation and the LC50 value was calculated by nonlinear regression method. We used a one-way analysis of variance (ANOVA), followed by Tukey’s posthoc test. The results were considered statistically for p<0.05. The Menthol (49.6%), Menthone (24.1%), and Isomenthone (12.4%) represent the majority of compounds found in the oil analyzed, totalizing 86%. The LC50 value calculated was 9.752 µg/mL to human lymphocytes exposed to Brazil mint for 24h. The used platforms in our study predicted probabilities higher than 77% for downregulation of genes CDCA4, CENPJ, and HAUS8 which were related to cell multiplication and can be possibly involved in the observed effects. Therefore, we can conclude that Brazil mint has a high probability of interfering with gene expression inducing lymphotoxicity. CAPES, CNPq, FAPERGS, and UNIPAMPA supported the study.

Lymphotoxicity of Brazil mint essential oil: an in vitro and in silico study

chaveS, paMella eDuarDha eSpinDola; rioS, naThália vieira; goMeS, gaBriela criSTiane MenDeS; eTcheverry, BiBiana FraSSon; caMpoS, Dyene naSciMenTo; zuravSki, luíSa; MachaDo, Michel ManSur

Grupo de Pesquisa em Imunologia e Genética Aplicada (GIGA), Universidade Federal do Pampa, Uruguaiana, RS, Brasil.

266

Introduction: Imidacloprid is a neonicotinoid insecticide that appeared on the market due to the resistance related to other classes of insecticides and the low-risk potential for mammals due to the greater specificity for insects. Mitochondria are responsible for synthesis of almost all cellular ATP necessary to maintain cellular structure and function through oxidative phosphorylation. Oxidative phosphorylation occurs in the inner mitochondrial membrane. Electrons removed from the citric acid cycle by NADH and FADH2 are used to drive the pumping of protons from the matrix to the intermembrane space. These organelles represent a preferred and critical target for the action of drugs, toxins, or their reactive metabolites. Complex II consists of two types of prosthetic groups and four different proteins, and the enzyme succinate dehydrogenase (SDH) is one of them. In this complex, there is the reduction of ubiquinone to ubiquinol with FADH2 electrons. Objectives: This study aimed to investigate the potential involvement of mitochondria in imidacloprid-related toxicity in RAW 264.7 cells. Materials and Methods: Cells were seeded in 6-well

plates at a density of 1x106 cells/ml (final volume 0.2 ml/~260,000 cells/cm2). The effects of complex II was evaluated after 24 and 96-hours incubation-time of 150, 500, and 1000 mg/L of imidacloprid (MUCH 600 FS) at 37 °C. Cell suspensions were analyzed in a FACS Calibur flow cytometer to determine the mitochondrial mass and membrane potential. Single dye controls were used to set compensation. Ten thousand events corresponding to intact cells were analyzed. Results: It was observed a significant increase in complex II activity as well as in SDH activity after 24h of incubation time with 1000 mg/L imidacloprid (ANOVA/Bonferroni p < 0.001). Interestingly, this increase was more significant after 24 h compared to 96 h in SDH (two-way ANOVA/Bonferroni p < 0.001) and complex II activity (two-way ANOVA/Bonferroni p < 0.05). Discussion/Conclusion: Mitochondrial complex II represents one of the intracellular targets of imidacloprid. Overall, the results pointed to a potential immunotoxic effect of imidacloprid in RAW 264.7 cells. Acknowledgements: Financial support from CNPq/Brazil and FAPERGS.

Mitochondria as a target for imidacloprid toxicity on RAW 264.7 cells

ceSTonaro, lariSSa v.1; SchMiTz, Felipe2; Ferreira, FernanDa S.2; conTe, FernanDa M.1; piTon, yaSMin v.1; WySe, angela T.S.2; garcia, Solange c.1; arBo, Marcelo D.1

1 Laboratory of Toxicology (LATOX), Department of Analysis, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil;

2 Laboratory of Neuroprotection and Neurometabolic Disease, Department of Biochemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

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Background/Introduction: Inflammation is a form of defense against tissue damage or pathogens entry. Although being essential for the homeostasis, this process must be stopped, avoiding this way its exacerbation. Non-steroidal anti-inflammatory drugs play an important role stopping the inflammation, although they have described many adverse effects that can range from nausea to gastric ulceration and renal hypertension. In this context, it is necessary to keep continuously searching for new bioactive compounds. Pyrazolines are molecules characterized by having a five-membered heterocycle with high structural versatility, presenting many biological activities such as antioxidant, anti-inflammatory, anti-carcinogenic, among others. Objective: this study aims to evaluate the toxicological parameters using in silico methods and anti-inflammatory potential of new pyrazoline compounds. Methods: Initially, a predictive pharmacological and toxicological evaluation of the compounds was performed in silico, using the SwissADME and Qsar Toolbox platforms. To evaluate its anti-inflammatory potential, different methodologies were performed, such as dosage of nitric oxide (NO), pro-inflammatory cytokines levels such as IL-1β and TNF, using the ELISA method, and a chemotaxis assay was performed as previously described using an agarose gel and fMLP as chemotactic agent, all of them using macrophages and neutrophils, the main cells involved in the acute inflammatory response. Results/Discussion: The results obtained in the MTT assay showed that none of the four compounds (PH0, PH3, PH4 and PH7), at all concentrations evaluated, showed cytotoxicity. In the in silico analysis of compounds PHO, PH3, PH4 and PH7, it was possible to observe using SwissADME, an online platform, that all compounds have good

pharmacological characteristics such as molecular weight lower than 500 g/mol, only one of the four compounds, PH0, demonstrates to be able to pass the blood brain barrier, also PH0 and PH7 has shown a good probability to be absorbed by GS, different from PH3 and PH4, that seems to have a lower probability to be easily absorbed by GS due to the presence of an chloride its stucture. Using the Qsar Toolbox software, parameters related to the prediction of toxicological potential of the compounds were observed, such as genotoxicity, carcinogenicity, hepatotoxicity, effects on the endocrine system, among others. Analyzing the results, it was possible to conclude that both PH3 and PH4 compound has alerts to hepatotoxicity and carcinogenicity related to the presence of one halogenated benzene in the molecule. The others compounds showed no predictive potential to have negative or toxic effects. The results of nitric oxide measurement, both in macrophages and neutrophils, showed that all of the four compounds were able to significantly decrease nitric oxide levels in the supernatant of cell culture stimulated with Escherichia coli lipopolysaccharide (LPS), with PH4 showing the highest inhibition rates. As in the nitric oxide dosage, the compound that also showed a satisfactory effect in decreasing TNF levels was PH4, with a higher average inhibition. The chemotactic assay was performed using only the compound PH4, considering that it presented the best initial results, and also showed satisfactory results regarding in vitro chemotaxis of neutrophils. Conclusion: The results obtained in the study so far has shown the promising effects of the compound PH4, but further analyzes will be performed to determine if the compound has potential to go to in vivo rodent tests.

New pyrazoline compounds: effects on neutrophils and macrophages functions

golDoni, FernanDa c.1; BenvenuTTi, lariSSa1; vaz, Milena Menegazzo1; carloS raFael1; Buzzi, FáTiMa c.2; quinTão, nara l.M.1; SanTin, joSé roBerTo1

1 Postgraduate Program in Pharmaceutical Science, Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brazil; 2 Pharmacy, Courses, Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brazil; Postgraduate

Program in Pharmaceutical Science, Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brazil.

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The practice of health care and prevention through natural products such as aromatherapy is something that has been drawing attention over the years. Aromatherapy is a practice that consists of the use of pure essential oils to prevent and treat pathologies. One of the most sought-after essential oils in this practice is Pogostemon cablin essential oils. Pogostemon cablin is a branched, perennial, aromatic herb of the Lamiaceae family, native to Southeast Asia and widely cultivated in many countries. It is commonly known as Patchouli and has recently gained prominence in academia due to its antidepressant, anti-inflammatory, antiseptic, immunomodulatory, aphrodisiac, and astringent properties. Despite having numerous biological effects and being used in ethnopharmacology for many years, little is known about its cellular toxicity. Thus, this work aimed to evaluate the cytotoxicity potential of the major compounds from the essential oil of Pogostemon cablin. For this, human peripheral blood was collected by venipuncture (UNIPAMPA Ethics Committee, nº 27045614.0.0000.5323), and peripheral blood mononuclear cells (PBMC) were isolated by density gradient with Histopaque® 1.077 g/mL and maintained overnight in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in an atmosphere of 37ºC and 5% CO2. After this period, the lymphocytes were separated from the monocytes that adhered to the contact surface. Lymphocytes were treated with concentrations of Patchouli ranging from 1 to 1000 µg/mL and stimulated by the addition of PHA-M (1 mg/mL) for 24 hours and then the total lymphocytes

were counted in Neubauer hemocytometer. The determination of the LC50 was realized using non-linear regression analysis. For the in silico tests, a previous analysis was performed by GC-MS (gas chromatography coupled to mass spectrometry) and the major compounds in the oil were determined. The canonical form of the major compounds Pogostol and α-gurjunene was searched in the PubChem database and submitted to computational tests on the Way2Drug platform using the DIGEP-PRED Module and later the GeneCards website was used to correlate the genes found. These tools investigated gene expressions, where up and downregulation were considered with probabilities above 70%. In this study, the calculated LC50 value was 66.1 µg/mL for human lymphocytes exposed to Pogostemon cablin essential oil. This toxicity can be explained in part by in silico forecasts, which showed a reduction in the CENPJ gene caused by the major compounds of Patchouli (Pogostol and α-gurjunene). CENPJ is a gene capable of encoding a protein that belongs to the centromere family of proteins. During cell division, this protein plays a structural role in maintaining centrosome integrity and normal spindle morphology and is involved in microtubule disassembly in the centrosome. Alterations, in quantity or quality, in this protein interfere with the cell’s ability to proliferate, as well as remain metabolic active. It is concluded that from the results presented, new studies related to this essential oil are of paramount importance for the construction of its toxicological profile and determination of its safety. Agradecimentos: CNPQ, CAPES, FAPERGS e UNIPAMPA.

Patchouli (Pogostemon cablin) essential oil: safe or a hidden risk?

rioS, naThália vieira; chaveS, paMella eDuarDha eSpinDola; goMeS, gaBriela criSTiane MenDeS; eTcheverry, BiBiana FraSSon; SoTelo, êMily clori; zuravSki, luíSa; MachaDo, Michel ManSur

Grupo de Imunologia e Genética Aplicada (GIGA), Universidade Federal do Pampa, Uruguaiana, RS, Brasil.

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Introduction: Lung cancer is the world’s leading cause of cancer due to its death aggressive manifestation, high ability to promote metastasis, and late diagnosis. Despite the development of several antitumor drugs, the prognosis of lung cancer is relatively low. The search for new compounds with antiproliferative potential that respond selectively to tumor cells with less toxicity to non-tumor cells have been the focus of many studies. Trilobolide-6-O-isobutyrate (TBOI) is a natural eudesmanolide lactone isolated from Sphagneticola trilobata Pruski (Wedelia paludosa D.C.) and W. prostrata which have a wide range of pharmacological activities reported as microbicidal, anti-inflammatory, and antitumor, but the study of this natural drug against lung carcinoma is still limited. Objective: The present study aims to evaluate the TBOI in silico pharmacological and toxicological characteristics and the in vitro action of this compound on normal cell lines and against A549 (lung adenocarcinoma) and NCI-H460 (large cell carcinoma) lung cancer cells. Methods: The TBOI structure in silico study was carried out to evaluate theoretical drug-likeness and toxicological parameters using the SwissADME and Qsar Toolbox platforms. MTT assay was performed to determine the cytotoxic effects against lung cancer cells A549 and NCI-H460, and LLC-MK2, VERO, and AMJ2-C11 normal cell lines. The cells were treated for 24-72 h with different TBOI concentrations (12 -100 µM) and the cytotoxic concentration of 50% (CC50) and the tumor selectivity index (TSI) was determined. The TBOI hemolytic

activity was also evaluated (3-200 µM). Results: In silico predictions showed good drug-likeness potential for TBOI with high oral bioavailability and intestinal absorption, no blood-brain barrier penetration, and no interaction with the main five CYP isoforms. The presence of any pan assay interference compounds (PAINS) was also not identified. Also, the TBOI was predicted as non-genotoxic, non-carcinogenic, non-mutagenic, and non-hepatotoxic. TBOI structure also predicts the in vivo mutagenicity (Micronucleus) alerts due to the oxalane (tetrahydrofuran) moiety, which acts as nucleoside analogues, a common chemical characteristic present in cancer chemotherapeutic agents, by inhibiting the function of DNA polymerase and/or being incorporated into DNA as nucleosides fraudulent. The in vitro analyses showed that TBOI treatment of lung cancer cell lines was able to reduce cell viability at all concentrations tested in a dose- and time-dependent manner defining the CC50 after 24h for A549 (73µM ±0.06) and NCI-H460 (54µM ±0.03). The CC50 for LLC-MK2, VERO, and AMJ2-C11 was also determined as 335 µM (±0.01), 69 µM (±0.02), and 83 µM (±0.01), respectively. It was determined in vitro TSI A549 of 4.6, 1, and 1.1 for LLC-MK2, VERO, and AMJ2-C11 respectively, and TSI NCI-H460 of 6.2, 1.2, and 1.5 for LLC-MK2, VERO, and AMJ2-C11 respectively. The TBOI tested concentrations also showed no hemolytic activity in vitro. Conclusion: This study revealed that TBOI may be a promising candidate for the development of antitumor drugs, targeting lung carcinoma.

Trilobolide-6-O-isobutyrate shows in silico and in vitro potential for antitumor activity of A549 and NCI-H460 lung cancer cell lines

MiranDa-Sapla, Milena Menegazzo1; concaTo, virginia Marcia2; gonçalveS, Manoela Daiele3; MaToS, ricarDo luiz naSciMenTo3; conchon-coSTa, iveTe2; arakaWa, nilTon Syogo3; pavanelli, WanDer rogério2

1 Laboratory of Toxicology, University of Vale do Itajai, Itajaí, SC, Brazil; 2 Laboratory of Immunoparasitology of Neglected Diseases and Cancer, State University of Londrina, PR, Brazil;

3 Laboratory of Biotransformation and Phytochemical, State University of Londrina, PR, Brazil.

11 NANOTOXICOLOGY

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Background/introduction: Nanotechnology is the principle of engineering nanomaterial with different compound and structures allowed to be used in several areas of science, including in pharmaceutical. The interaction of nanoparticles (NPs) with the immune system is a key element both in the assessment of nanotoxicity and in the development of nanomedicines. Annexin A1 (AnxA1) is a protein with many anti-inflammatory properties and is capable to act on macrophages, promoting anti-inflammatory macrophage phenotype in in vitro models polarization, also improving the efferocytosis. Multi-wall lipid core nanocapsules (MLNC) and surface-functionalized metal-complex multi-wall lipid core nanocapsule (MLNC functionalized) are able to carry efficiently lipid and protein molecules in the core and in the polymeric wall, according to the characteristic of the substance. The metal complex with Zn2+ provides better binding of the protein to the MLNC structure by interacting with histidine residues present in the C-terminal region in the proteins. This supramolecular structure optimized and enhancing bioaviability and driving medicines to the pharmacological targets. Herein, we aimed to functionalize recombinant AnxA1 on MLNC to investigate the cytotoxicity and effectiveness on macrophage polarization. MLNC was prepared by covering lipid core nanocapsule (LNC) with chitosan, which coordinates metals to the specific protein chemisorption site. Therefore, MLNC was linked to Zn2+ and rAnxA1 was added to form the MLNC-AnxA1. The cytotoxicity of four different concentrations of recombinant AnxA1 (rAnxA1), MLNC and MLNC-AnxA1 was evaluated in Raw 264.7 cells and

(3-(4,5-Dimethylthiazol-2-yl) assay (MTT assay) was assess as a screening test. Only the concentrations that showed mitochondrial activity less than 70% in the MTT assay were tested by the LDH activity, necrosis, apoptosis and late apoptosis assay by flow cytometer in 24 and 48 hours. The uptake of nanoparticles by Raw 264.7 cells was performed in a dark field hyperspectral microscopy (CytoViva®). The polarization of macrophages was evaluated using primaries cells obtained from the bone marrow of C57Bl/6 mice. Primaries macrophages were then differentiated into inflammatory (M1) by the addition of LPS (100 ng/mL) and treated with AnxA1, MLNC or MLNC-AnxA1. After 48 hours the cell phenotype was evaluated by flow cytometry (F480, CD80 and CD206) and the cytokines of the supernatant were quantified (TGF-β and IL-10). LNC, MLNC and MLNC-AnxA1 presented 129,5, 152,47 and 162,87 nm, respectively, and equivalent polydispersity index; incorporation of chitosan inverted the negative potential zeta; the encapsulation efficiency of AnxA1 was 92.22%, and transmission electron microscope photomicrograph showed MLNC-AnxA1 spherical shape. MLNC-AnxA1 was able to be taken up by Raw 264.7 cells at a concentration that did not generate cytotoxicity and both MLNC and MLNC-AnxA1 was able to differentiate M1 macrophages in anti-inflammatory macrophages (M2). Altogether the results improve the knowledge about the cytotoxic capacity of MLNCs and showing MLNCs actively act on macrophages, an important cell type present in the innate immune response. Acknowledgments. USP, CNPq and FAPESP.

Cytotoxicity and effectiveness in macrophage polarization of Annexin A1-

surface-functionalized metal-complex multi-wall lipid core nanocapsule

Broering, Milena F.1; leão, MaTheuS c.2; ScharF, paBlo1; SanDri, Silvana1; uchiyaMa, Mayara k.3; araki, koiTi3; guTerreS, Silvia S.4; pohlMann, aDriana r.5; FarSky, SanDra h.p.1

1 Department of Clinical & Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of Sao Paulo, SP, Brazil. 2 Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences,

University of Sao Paulo, SP, Brazil. 3 Department of Fundamental Chemistry, Institute of Chemistry, University of Sao Paulo, SP, Brazil. 4 Department of Production and Control of Medicines, Faculty of

Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. 5 Department of Organic Chemistry, Chemistry Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.

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Introduction: Nanotechnology comprises the science that works with materials at molecular levels, whether for manipulation and/or creation of new materials and products. The properties of materials at the nanoscale can be very different from those at a larger scale. Metal nanoparticles have been widely explored for biomedical purposes, especially gold nanoparticles, due to their low cytotoxicity and with this, they are used clinically to improve the potential of drugs, altering the pharmacokinetics, biodistribution and cellular absorption. Although nanoparticles modify the physical properties of several compounds and result in several benefits, the evaluation of the toxicity of these materials is poorly understood. Objectives: To evaluate the degree of acute renal (urea and creatinine) and hepatic (AST and ALT) toxicity of gold nanoparticles in 60-day-old male mice. Methods: Adult male mice (20 - 30 g) of the Mus musculus species were divided into groups and treated once a day, for four days, with nanoparticles (doses of 0.025; 0.07 and 0.22 mg/kg) or saline solution (0.1 mL/10g) intraperitoneally. One day after the last administration, a blood sample was obtained for plasma separation and spectrophotometric measurement of the level of hepatic and renal biomarkers was conducted. Data are presented as mean ± standard error. After the synthesis, the

AuNPs solution was characterized by the authors using the ultraviolet-visible (UV-Vis) spectroscopy technique. The values obtained were 10 nm, as expected, and this was confirmed by the Biological and Analytical Transmission Electron Microscopes (TEM) image, which showed predominantly spherical gold nanoparticles (GNP). Results: Gold nanoparticles doses of 0.025; 0.07 and 0.22 mg/kg and saline resulted in a blood level of oxaloacetic transaminase of 79.8 ± 4.7; 108.0 ± 9.9; 100.1 ± 6.3 and 99.3 ± 9.0 U/L; glutamic transaminase of 20.3 ± 1.6; 17.2 ± 1.2; 15.8 ± 1.4 and 16.9 ± 2.1 U/L; of urea, 52.1 ± 6.3; 56.2 ± 1.2; 52.8 ± 1.2 and 55.6 ± 2.0 mg/dL and creatinine of 0.27 ± 0.01; 0.28 ± 0.01; 0.30 ± 0.01 and 0.29 ± 0.01 mg/dL, respectively. Discussion/Conclusion: The data found allow us to infer that nanoparticles, in short-term treatment, are not significant promoters of hepatotoxicity or nephrotoxicity, being well tolerated in this sense regarding the safety of their use in this period. It is possible that longer periods of administration imply a different profile of toxicity, which may motivate other studies, as well as investigations of toxicity linked to mutagenesis, carcinogenesis and teratogenesis, which are essential in the screening of adverse effects of new compounds with biological activity. Keywords: Toxicity; Metallic Nanoparticles; Acute Toxicity; Gold Compounds.

Preclinical evaluation of short-term gold nanoparticles toxicity

plauTz, kaTherine1; gaSTalDi, aleSSanDra BeTina1; Maia, Thayná paTachini1; pereira, eDuarDo Manoel3; Ferreira, gaBriela kozuchovSki4; BorgMann, gaBriela1; caBral,

heloiSi1; DelWing-Dal Magro, DéBora5; DelWing-De liMa, Daniela1,2

1 Programa de Pós-graduação em Saúde e Meio Ambiente - UNIVILLE, Joinville, SC, Brasil; 2 Departamento de Medicina - UNIVILLE, Joinville, SC, Brasil; 3 Departamento de Farmácia

– Universidade da Região de Joinville – UNIVILLE – Joinville, SC; 4 Departamento de Saúde - UniSociesc – Joinville, SC; 5 Departamento de Ciências Naturais - FURB, Blumenau, SC

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Introduction: nanotechnology products are being increasingly used in the consumer industry; such as pharmaceuticals, food, medicine, cosmetics, pesticides. Zinc oxide (ZnO) and cerium dioxide (CeO2) nanoparticles (NPs) can be highlighted, due to their high consumption in these industries. ZnO NPs are used due to their antimicrobial action and it’s the raw material for sunscreens and cosmetics. CeO2 NPs are used in sunscreens, cosmetics, in polishing agent (mainly for the production of ophthalmic lenses), in diesel additives and cigarettes. The NPs can be atmospheric pollution agents, which can cause adverse effects on human health (causing lung diseases and eye irritation or corrosion). NPs toxicity studies still use animals, however, animal welfare, financial and legal issues encourage the consolidation of alternative predictives methods to cytotoxicity is necessary to promote the reduction and replacement the animal use. Objective: the present study evaluated the cytotoxicity of ZnO and CeO2 NPs using SIRC cells (to assess eye irritation and A549 human alveolar epithelial cells to assess lung toxicity. Methods: the average size of NPs were evaluated by dynamic light scattering (DLS); the surface charge by zeta potential; morphology by transmission electron microscopy (TEM); cytotoxicity was evaluated by electrical impedance and MTT assay for 72h. Results: ZnO and CeO2 NPs have an average size (in water) of 241.20 ± 11.49 nm and 212.29 ± 15.38, and with a polydispersion index (PDI) of 0.20 ± 0. 03 and 0.35 ± 0.07 nm, respectively. After 24h of stabilization, the ZnO NPs had an average size in water of 188.90 ± 42.68 nm and a PDI of 0.13 ± 0.03, whereas the CeO2 NPs was 140.02 ± 4.26 nm and 0.18 ± 0.05 nm. The average size of the NPs after contact with the culture medium (Dulbecco’s Modified Eagle’s Medium low glucose (DMEM)

supplemented with 10% fetal bovine serum (FBS) and stabilized with bovine serum albumin (BSA) specific for the cells of this study was evaluated. The ZnO and CeO2 NPs had an average size of 192.8 ± 13.62 nm and 167.49 ± 22.62 nm, with PDI of 0.18 ± 0.09 and 0.12 ± 0.01 nm, respectively. In relation to Zeta potential, the NPs demonstrate stability in water, presenting a surface charge of 22.6 ± 0.63 for the ZnO NPs and 37.71 ± 7.63 mV for the CeO2 NPs; there was a significant difference in the surface charge of NPs after contact with the culture medium, with a surface charge of -8.23 ± 0.69 mV for ZnO NPs and 3.81 mV for CeO2 NPs, probably due to protein corona formation. NPs have characteristic spheroidal morphology. The potential for irritation and cytotoxicity will be analyzed after exposure of NPs in cells at concentrations of 2.5; 25; 50; 75; 100 µg/mL, for 72h. Discussion: electrical impedance is a non-colorimetric technique, applied to cell viability analysis, provides kinetic information in a non-invasive way and with high temporal resolution through growth curves recorded in real time. The performance of this technique can be efficient to evaluate the cytotoxicity of NPs, being able to be used as an alternative test system for the toxicity of NPs in vitro, interference-free, allowing the consolidation of an alternative test to the use of animals. Conclusion: cell viability tests without colorimetric interference appear as a promising and important alternative for the nanotoxicology field. Mainly to assess the safety of ZnO and CeO2 NPs, reducing the potential irritation risk and eye and lung corrosion. Acknowledgments: thanks to the Ministry of Science and Technology and Innovations of Brazil, Foundation Carlos Chagas Filho Research Support of the State of Rio de Janeiro (FAPERJ), the National Institute of Metrology, Quality and Technology. The Unigranrio and UEZO universities.

Predictive analysis of ocular and lung damage in cells exposed by zinc oxide or

cerium dioxide nanoparticles using electrical impedance compared to MTT test

alexanDre, angela De oliveira1,2; Souza, WanDerSon2,3; Dal-cheri, BeaTriz kopke De aSSiS1,2; granjeiro, joSe Mauro1,2,3; pereira, leonarDo Da cunha BolDrini1,2,3

1 Postgraduate Program in Translational Biomedicine, University Grande Rio, Duque de Caxias, Brazil; 2 Directory of Life Sciences Applied Metrology, National Institute of Metrology

Quality and Technology, Rio de Janeiro, Brazil; 3 Postgraduate Program in Biotechnology, National Institute of Metrology Quality and Technology, Rio de Janeiro, Brazil.

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Introduction: Nanotechnology, the science of creating devices and materials, from the control of matter on the nanometer scale is currently considered an innovative area of scientific and economic growth; arouses great interest from various sectors and its use, due to its characteristics, especially its size (0-100 nm) is already widespread in many areas, including the pharmaceutical, electronics, paint industry and manufacturing, among others. However, toxicological studies are insufficient to accurately identify the effects to health and to the environment resulting from the use of nanocomposites. Among the extensive use of nanoparticles, the Chromium (III) Oxide nanoparticle (Cr2O3 NP) have a wide variety of applications. Objectives: the aim of this study was to evaluate the toxicity of Cr2O3 NP in reconstituted seawater (RSW) through acute and chronic toxicological tests using microcrustacean marine Mysidopsis juniae. Methods: the characterization of Cr2O3 NP was carried out by different physicochemical techniques, in different means, through toxicological testing toxicity of Cr2O3 NP, acute exposure to defining the LC50(96h) and chronic and transgenerational test evaluating: birth, mortality and biometrics during the period of 63 days. Finally, it was conducted the analyzing of the concentration of Cr (III), Cr (VI) ions and total chromium to the concentrations of acute test (25, 50, 75, 100 and 125mg/L-1) and chronic (5mg/L-1). Results: The results of the characterization of Cr2O3 NP by Transmission Electron Microscopy (TEM), Scanning with Field Emission (SEM-FEG) and X-ray Diffraction showed that the particle diameter is in nanometer scale (21,8 nm), showing further that there

is formation of large agglomerates of suspension NP, especially in saline, which was confirmed by analysis of the hydrodynamic diameter. For acute toxicological evaluation, it was observed that concentration below 5 mg L-1 have no mortality and from the concentrations of 25mg/L-1, always occurred mortality, being the concentration of 125 mg/L-1

the responsible for overall mortality of organisms in 96 h of exposure. The LC50(96h) found for the test organism exposed to Cr2O3 NP was 58.86 mg/L-1. Chronic and transgenerational test showed that only the parameter mortality of 2nd generation exposed to concentration of 5mg/L-1 of Cr2O3 NP was affected by the studied substance (p<0.05). Finally, it was possible verify on analysis of chromium ions for both concentrations of acute test as to the chronic test, the presence of Cr (VI), and already in the concentration of 5 mg L-1 of Cr2O3 NP the concentration of Cr (VI) was 0.25mg /L-1, a result above the standard limit for discharge of effluents defined by Resolution CONAMA 430/2011, exponentially increasing for the other evaluated concentrations. Discussion/Conclusion: this study shows that Cr2O3 NP when dispersed in saline and when found in very small diameters tend to have low stability and can contribute to the agglomeration of NP, being a possible cause of decrease toxicity which would hinder the absorption by the organism, however, this same instability also contributes to the release of ions and in this study, it was found the presence of Cr (VI), which can be, in this case, the most responsible for the toxicity found Keywords: Nanoparticles; Chromium oxide; Acute toxicity; Chronic toxicity; Mysidopsis juniae.

Study of the acute and chronic toxicity of chrome III oxide nanoparticles on the marine

microcrustacean Mysidopsis juniae (Silva, 1979)

plauTz, kaTherine1; Fugazza, jonaS1; gaSTalDi, aleSSanDra BeTina1; kleine, TaMila1; MaTiaS, WilliaM gerSon2; oliveira, Therezinha Maria novaiS1

1 Programa de Pós Graduação em Saúde e Meio Ambiente – UNIVILLE, Joinville, SC, Brasil; 2 Departamento de Engenharia Sanitária e Ambiental - UFSC, Florianópolis, SC, Brasil.

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Nano-enabled pesticides have been developed with the potential to promote more sustainable agriculture since they improve efficiency and reduce the number of pesticides in the field. However, little is known about the interaction of these nanomaterials (also called nanopesticides) with plants. In this sense, nanostructured lipid carriers (already used as nanopesticides) associated with hydrophobic gold nanoparticles were synthesized using the solvent emulsion/evaporation method, to study the interaction of these nanocarriers in aquatic macrophytes of the Lemna valdiviana species. The labeled nanocarrier (ca. 250 nm) presented a polydispersion index below 0.2, zeta potential of -17.3 ± 1.5 mV, and excellent encapsulation efficiency (99.9%) of hydrophobic gold nanoparticles with the lipid core of the nanocarriers. Moreover, the interaction of nanoparticles with aquatic plants was evaluated using Atomic Absorption Spectroscopy (AAS), and the detection of labeled nanocarriers was concentration- and time-dependent in macrophytes. Also, X-ray diffraction (XRD) and

infrared spectroscopy (FTIR) analysis showed changes in the spectrum of plants when exposed to water (as standard) and labeled nanocarriers, which indicates the presence of nano-enabled materials associated with the aquatic macrophytes. Furthermore, histological sections of the plants allowed detecting aggregates of nanocarriers by optical microscopy analysis, corroborating with the X-ray fluorescence analysis (FRX) that showed the presence of 0.37% of gold ions in the plant. Therefore, this study reveals a new labeled nanopesticide able to comprehend the interaction of nanopesticides with plants as well as can help to better understand their mechanisms of action and toxicological effects in the environment. Acknowledgements: The authors acknowledge the FAPESP (#2020/12769-0, #2019/20124-2, #2017/21004-5), CNPq (#161360/2021-1, #427498/2018-0), CAPES for the financial support. Also, the Environmental Nanochemistry Lab, UNESP-Sorocaba for the support.

Study of the interaction between a potential labeled nano-enabled pesticide and aquatic plant

Forini, Mariana M.l.1; anTuneS, DéBora r.1; cavalcanTe, luiz a.F.1; ponTeS, MonTcharleS S.2; SanTiago, eTenalDo F.2; SancheS, alex o.1; MarTinS, aline r.1; grillo, renaTo1

1 São Paulo State University (UNESP), Faculty of Engineering, Ilha Solteira, SP, Brazil; 2 Center for Natural Resources Study (CERNA), Mato Grosso do Sul State University (UEMS), Dourados, MS, Brazil.

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Introduction: Flavonoids are bioactive compounds that have protective effects on the neuronal circuit due to their antioxidant properties, free radical scavenging and their possible impact on intracellular redox impact. Studies have reported anti-inflammatory, antioxidant and inhibitory action against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The encapsulation of bioactive compounds through nanotechnology techniques has helped in the stability, improvement of the physical property and in the absorption of these bioactive compounds such as naringenin. Although naringenin in its free form does not present a high level of toxicity, studies are needed to promote the investigation of the toxicity of its encapsulated form. Objective: To investigate the toxicity of naringenin loaded with nanoparticles using the survival rate of Drosophila melanogaster. In addition, the activity of naringenin against the cholinergic enzymes AChE and BChE was evaluated by enzymatic kinetics assays. Methodology: Poloxamer 407 (0.9g, Sigma-Aldrich, encapsulant), ethanol (37.5mL, Dynamic), Tween80 (0.009g, Dynamic) and naringenin (0.09g, 95% purity, Sigma-Aldrich) were used to obtain the nanoparticles. Poloxamer 407 and Tween80 were solubilized in ethanol for 5 min under gentle agitation at 40°C, naringenin was added and solubilized for 1 min, then the solution was sonicated (Fisher Scientific 120, 120W, 1/8” tip) for 3 min. in a pulsed regime (30s on, 10s off) in an ice bath. The obtained dispersion was evaporated at 40°C for 3h. The nanoparticles were characterized by Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR). The survival test was performed as follows: 100 flies were exposed for

five days to nanoparticle-loaded naringenin (160, 210, and 250 µg naringenin.mL-1) of nanoparticles and also to free naringenin (n=5 for each treatment). The enzymatic kinetics of naringenin against AChE and BChE enzymes was determined by the method of Ellman et al (1961) with modifications. Results: The same thermal transition temperatures were found in free naringenin and nanoparticles, while the FTIR showed absorption peaks related to the chemical groups of naringenin. The survival rate curve did not show significant changes in relation to controls (water or ethanol) at the concentrations evaluated. No significant changes were detected in the mortality of flies when compared to the control group (water or ethanol) at all concentrations. The mechanism of AChE and BChE enzyme inhibition was determined using the Lineweaver-Burk plot and the type of inhibition was determined by the position at which the lines in the plot intersected. Discussion/Conclusion: DSC and FTIR analyses detected the interaction between Poloxamer and naringenin, suggesting that encapsulation occurred satisfactorily. No toxicity was detected in the Drosophila melanogaster survival rate test for pure naringenin (as expected) and also for the nanoparticles, pointing towards the safety of the nanoparticles. Naringenin acted as an efficient inhibitor following a reversible inhibition of the non-competitive type behavior. In fact, naringenin is identified as a neuroprotective agent, acting as an inhibitor of cholinesterase enzymes by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Acknowledgments: This work was in part financed by CAPES/Brazil - Financial Code 001. Authors also thank CPNq for the support.

Survival rate of Drosophila melanogaster exposed to nanoparticles containing Naringenin

and in vitro determination of inhibition kinetics against cholinergic enzymes

cunha, viviane auguSTa De MeDeiroS garcia1; roSSi, Bruna Franzon2; leiMann, FernanDa viTória1,3; ineu, raFael porTo1; FoleiS, vaneSSa kapluM1; gonçalveS, oDinei heSS1,3

1 Post-Graduation Program of Food Technology (PPGTA), Federal University of Technology – Paraná – UTFPR Campus Campo Mourão, Campo Mourão, PR, Brazil; 2 Food and Chemical

Engineering Department (DAAEQ), Federal University of Technology – Paraná – UTFPR Campus Campo Mourão, Campo Mourão, PR, Brazil; 3 Centro de Investigação de Montanha (CIMO),

Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal.

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Introduction: The increased use of nanoparticles in pharmaceuticals and also in the food industry is in part explained by the growing demand for bio-based additives and nutraceuticals by consumers. However, most naturally occurring bioactive substances present low water solubility and thus low bioavailability and nanotechnology may overcome this challenge. This is the case of silymarin, which is extracted from Silybum marianum, consisting of a mixture of flavonolignans with several biological activities. Although silymarin presents low toxicity, it is important to shed light on the possible toxicity of silymarin-loaded nanoparticles and also on how they could affect key metabolic systems. Objective: To determine the survival rate of Drosophila melanogaster (DM) exposed to silymarin-loaded nanoparticles and to evaluate the activity of silymarin-loaded nanoparticles against the cholinergic enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Methods: Poloxamer 407 (9 g, Sigma-Aldrich) was used as an encapsulant, ethanol (375mL, Dinânica) as the solvent, and Tween80 (0,09g, Dinânica) as surfactant. Silymarin (0,9g, 95% purity) was acquired from Sigma-Aldrich. Encapsulant and Tween80 were solubilized in ethanol for 5 min under gentle stirring at 40°C. Then, silymarin was added and solubilized for 1 min, then the solution was sonicated (Fisher Scientific 120, 120W, 1/8” tip) for 3 min in a pulsed regime (30sec on, 10sec off). The obtained dispersion was evaporated at 40°C for 24h. The nanoencapsulated compound was characterized by Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy Fourier Transform (FTIR). The effect of silymarin and silymarin-loaded nanoparticles on DM was evaluated by a survival rate test (five days, 100 flies each treatment, n=5). AChE and BChE activities

were determined ex vivo (Ellman et al., 1961 with modifications). Results: The DSC of silymarin showed peaks at 218.0°C and 281.8°C. In the physical mixture of silymarin and Poloxamer, the silymarin melting peak appeared at 290.9°C, while in the nanoparticles this peak was shifted to 311.6°C. The FTIR signal referring to C=O (1640 cm-1) present in silymarin was attenuated in the physical mixture and in the silymarin-loaded nanoparticles. No significant changes in the mortality of flies were detected when compared to the control group (water or ethanol) in all concentrations. The AChE and BChE activity was significantly inhibited at 160 and 250 µg.mL-1 for silymarin in water and ethanol. Nanoparticles did not significantly influence the activity of the enzymes. Discussion/Conclusion: DSC and FTIR indicated that interaction occurred between the Poloxamer and silymarin, suggesting encapsulation took place as already demonstrated by our group for curcumin. Microscopy and UV-Vis analyses (not shown here) confirmed the formation of the nanoparticles containing silymarin. No toxicity was detected in the survival rate test in Drosophila melanogaster for silymarin (as expected) and also for the nanoparticles, suggesting that encapsulation is a safe strategy to improve the technological properties of silymarin. Silymarin is a neuroprotective agent, acting as an inhibitor of cholinesterase enzymes by catalyzing the hydrolysis of the neurotransmitter acetylcholine. It is worth noting that encapsulation does not affect silymarin activity meaning that these particles deserve to be further investigated as a pharmaceutical tool as a reversible cholinergic inhibitor. Acknowledgments: CAPES/Brasil-Finance Code 001; and CPNq.

Survival rate of Drosophila melanogaster exposed to Silymarin-loaded nanoparticles and

the ex vivo inhibition of cholinergic enzymes

Souza, Daniela criSTina1; roSSi, Bruna Franzon2; leiMann, FernanDa viTória1,3; gonçalveS, oDinei heSS1,3; appelT, paTrícia1; ineu, raFael porTo1

1 Post-Graduation Program of Food Technology, Federal University of Technology – Paraná – UTFPR/Campo Mourão, PR, Brazil; 2 Food and Chemical Engineering

Department, Federal University of Technology – Paraná – UTFPR/Campo Mourão, Campo Mourão, PR, Brazil; 3 Centro de Investigação de Montanha (CIMO), Instituto

Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal.

12 RISK ASSESSMENT

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Introduction: Great strides have been made in the ability to use data from non-animal assessment methods to establish safety of cosmetics and cosmetic ingredients. However, a lack of familiarity with these data and processes inhibits uptake, both for product developers and for those who must assess the safety of those products. Objective: To increase confidence and enable regional capacity to perform non-animal safety assessment of cosmetics and cosmetic ingredients while addressing the needs of the regulated and regulatory communities and other stakeholders. Methods: We facilitated the creation of the AFSA collaboration, a group of like-minded industry add non-profit organizations with expertise in non-animal safety assessment to create an education and training program based on scientific knowledge and experience. Results: The Animal-Free Safety Assessment (AFSA) Collaboration has created an Education and Training Program that covers the entire risk assessment process of cosmetics and cosmetic ingredients in eight modules: Problem Formulation, Consumer Exposure, In silico Tools, Exposure-based waiving, Internal Exposure, In vitro

Data Synthesis, and the Overall Risk Assessment. A ninth module covers the regulatory landscape of chemicals and consumer products. The program is built on established principles and processes and illustrated throughout with case examples from our members’ experience. Discussion: The first step in this approach for risk assessment is to understand the product, its use, and the safety question that needs to be addressed. The assessment is exposure-led, so that consumer exposure is the first consideration. If exposure suggests there is a possibility of risk, hazard assessment is carried out, followed by refinement of the exposure estimate if necessary. Finally, all this information is combined in the risk assessment which includes estimates of uncertainty. The AFSA Cosmetics Education and Training course covers this process and will be freely accessible to all. Conclusion: Broad dissemination of this program is intended to familiarize global stakeholders in using modern, non-animal predictive tools to assess the safety of cosmetics and cosmetic ingredients, aiming to better protect people and our planet and hasten the end of animal testing.

Animal-Free Safety Assessment of Cosmetics: a global education and training program

Marigliani, Bianca1; WilleTT, caTherine2

1 Research & Toxicology Department, Humane Society International, Brazil. 2 Research & Toxicology Department, Humane Society International, USA.

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Introduction: The use of pesticides to support agricultural productivity varies between different crops, in terms of frequency of application, dose and proximity to harvest time. Fungicides triazoles are potent inhibitors of the CYP51. In humans, a product derived demethylation mechanism catalyzed by CYP51 is cholesterol, which is required for the synthesis of bile acids, mineralocorticoids, glucocorticoids and sex steroids. Biological monitoring plays an important role in estimating human exposure, allowing the health of workers to be assessed. Objective: Verify the dependence between the presence of triazoles fungicides in the urine of rural workers in southern Minas Gerais and the decrease in the hormone androstenedione. Methods: Cypronazole, epoxiconazole, metconazole, propiconazole and triadimenol were quantified in urine samples with a vortex-assisted liquid-liquid microextraction analyzed in gas chromatography-mass spectrometry. The dosage of androstenedione in serum samples was performed by chemiluminescence. Statistical analysis of test G were performed with a level of 5% for significance, using the BioEstat version 5.0. This study was approved by the Ethics Committee of the Federal University of Alfenas-MG-Brazil (CAAE: 34644620.2.0000.5142). Results: The dependence between the presence of triazoles in the urine and

the androstenedione dosage equal to or below the reference value (0.6 ng mL-1) for adult men was tested (n=30). Then, with only the samples in which triazoles were detected, another test was performed to assess whether the relationship could be dose-dependent. Samples with concentration of triazoles in urine above the limit of quantification were compared with samples that presented concentrations between the detection and quantification limit of the method and the androstenedione dosage equal to or below the reference value (n=16). For both cases, an association was found, p=0.0174 and p=0.0086, respectively. Conclusion: Therefore, the two variables are dependent, so there is a concentration-dependent relationship between the presence of triazoles in the urine and a trend towards lower doses of androstenedione in the serum of workers exposed to fungicides. The next step of the study is to analyze a greater number of samples from workers exposed to fungicides in southern Minas Gerais to assess occupational exposure and check verified if the association between exposure to triazoles and decrease in the hormone androstenedione is increasingly stronger. Acknowledgments: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Biomonitoring of occupational exposure to triazoles and the disruptive effects

of steroidogenesis on androstenedione biosynthesis in humans

Marciano, luiz paulo De aguiar1; coSTa, luiz Filipe1; Freire, joSiane oliveira1; FelTriM, FernanDo1; MachaDo, SiMone caeTani2; Silvério, aleSSanDra criSTina pupin2; MarTinS, iSariTa1

1 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences, Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-

000, Alfenas, MG, Brazil; 2 University José do Rosário Vellano - UNIFENAS.

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Introduction: The use of triazole fungicides is widespread in rural areas, due to the favorable conditions for the proliferation off fungi in different cultures. These pesticides, despise being efficient, do not present specificity in relation to the target organisms, and may cause toxic carcinogenicity and also endocrine disruption. Objective: The present study, in a pioneering way, aims to assess the risk of exposure of rural workers to triazole fungicides, using the micronucleus test, as a biological indicator of genotoxic effect, in a sample of the oral mucosa, in order to assess whether there is correlation between the results of this test and exposure, through the investigation of urinary triazoles, subsidizing the risk assessment and implementation of public polices to monitor workers exposed this class of pesticides. Methods: 87 farmers (men) occupationally exposed to triazole fungicides and 55 women living in rural areas in the Southern region of Minas Gerais are being evaluated. In this population, a questionnaire was applied to collect epidemiological and clinical data, and the collection of cells from the oral epithelium and urine are also performed for the relevant analyses. For the micronucleus test, the slides are being analyzed under a fluorescence microscope, with 0,001% acridine orange, staining, observing the presence of cellular alterations such as the presence of micronucleous, karyolitic cells, karyorhexis, binucleated and pyknotic. The investigation of urinary triazoles is being carried out using gas chromatography- mass spectrometry with previous miniaturized liquid-liquid extraction. Statistical analysis was performed with a level of 5% for significance, using the BioEstat version 5.0.

This study was approved by the Ethics Committee of the Federal University of Alfenas-MG-Brazil (CAAE: 34644620.2.0000.5142). Results: Based on the recommendations of national and international validation guides, the method used showed linearity in the range of 10 to 200 µg L-1 for cyproconazole, metconazole and triadimenol and 30 to 200 µg L-1

for epoxiconazole and propiconazole. Of the 86 urine samples analyzed, 2 had more than one pesticide, 10 samples had concentrations between 10 µg L-1 and 70 µg L-1 and 27 samples were between the detection limit (2 µg L-1 for cyproconazole, metconazole and triadimenol; 5 µg L-1 for epoxiconazole and propiconazole) and quantification (10 µg L-1 for cyproconazole, metconazole and triadmenol; 30 µg L-1 for epoxiconazole and propiconazole). Partial results showed statistical differences for kariolytic, binucleated, pyknotic and karyorrhexis cells (p < 0,05) more frequently in men (n= 43) when compared to women (n = 19), both living in rural areas. In addition, a higher frequency of karyorrhexis cells was found in men with urinary triazoles above the LQ (n = 8), when compared to men with urinary triazoles below the LQ (n= 11). Conclusion: At the end of the study, it will be confirmed, or not, the hypothesis that exposure to triazole fungicides increases the risk of genetic mutations, which can trigger serious diseases, thus contributing to the area of occupational health, in the prevention of intoxications from the occupational exposure. Acknowledgments: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Buccal micronucleus cytome assay as a biomarker of genotoxic effect rural

workers exposed to triazole fungicides

coSTa, luiz Filipe1; Marciano, luiz paulo De aguiar1; Freire, joSiane oliveira1; FelTriM, FernanDo1; Silvério, aleSSanDra criSTina pupin2; MarTinS, iSariTa1

1 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences, Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-

000, Alfenas, MG, Brazil; 2 University José do Rosário Vellano - UNIFENAS.

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Background/Introduction: Cigarette smoking is indicated as one of the risk factors for a number of serious diseases, including lung cancer and cardiovascular disease. It is generally accepted that smoking-related diseases are associated to exposure to the thousands of chemical constituents created during the process of burning tobacco. There has been significant growth in the number of smokers using next generation tobacco and nicotine products including e-cigarettes and Tobacco Heating Products (THPs). As THPs heat tobacco at temperatures of 200-300°C instead of burning (900°C), the toxicants within the aerosol are greatly reduced (~95%) compared to cigarettes and hold promise as reduced risk products. Using various guidelines for assessing the modified risk status of a novel tobacco products, BAT has recently published a 9-step framework proposing the use of pre-clinical, clinical and population studies to assess the reduced risk potential of THPs at the individual and population level. Objective: To investigate changes over a six-month period in biomarkers of exposure and potential harm in smokers switching to the THP gloTM or quitting. Methodology: An ambulatory four-arm switching study enrolling 500 participants was carried out at 4 UK clinical sites conducted with the ethical principles of the Declaration of Helsinki, Good Clinical Practice. Regular smokers were randomised to either continue to smoke their own brand cigarettes, or to switch to using gloTM for the period of the study. A further group, (cessation) consisted of regular smokers who had intentions to quit and were provided with assisted smoking cessation . A final control group was made up of participants who have never smoked and were followed during the study period. Participants attended the clinic approximately every 30 days for one year, though the

never smokers attended less frequently. Breath, blood and urine were collected and analysed for a range of exposure and potential harm biomarkers, in addition, physiological, questionnaire and safety measures were also performed. Clinical Trial Registration: https://www.isrctn.com/ISRCTN81075760. Results: In smokers who switched to using gloTM, exposure to harmful cigarette smoke toxicants were significantly reduced, in many cases to the same levels observed in smokers who quit smoking (cessation), or in never-smokers. The reduction in these exposure biomarkers were rapid and sustained for the 6-month period. In cigarette smokers who switched to using gloTM, a number of biomarkers of potential harm were significantly changed in a favourable direction including biomarkers associated with cardiovascular disease and lung cancer. Discussion/Conclusions: These data demonstrate gloTM has much lower toxicant levels, resulting in significantly lower levels of biomarkers of exposure to toxicants and a favourable change in a number of biomarkers of potential harm compared to cigarettes when used by smokers over a six-month period. These data suggest that the negative health impacts of cigarette smoking may be reduced in smokers who completely switch to using gloTM. This conclusion is consistent with the UK Committee on Toxicology which found that “it is likely that there is a reduction in overall risk to health for conventional smokers who switch to heat-not-burn tobacco products.” Acknowledgments: The authors wish to acknowledge Covance (Leeds, UK), Celerion (Belfast, UK), Richmond Pharmacology (London, UK) and Simbec Orion (Merthyr Tydfil, UK) for their management and professional conduct of the clinical study.

Changes in biomarkers of exposure and potential harms in adult smokers following

180 days of gloTM tobacco heating product use

eSTeveS, iuri1; zeBele, paTricia1; gale, naThan2; harDie, george2; MceWan, Michael2; gaca, Marianna2; gooDall, Sharon2

1 BAT Brasil, a member of British American Tobacco, Republica do Chile 330, Rio de Janeiro, Brazil; 2 British American Tobacco (Investments) Ltd, R&D, Southampton, SO15 8TL, UK.

283

Introduction: COVID-19, caused by the SARS-CoV-2 virus, has been impacting a large number of people due to high mortality and variable susceptibility among individuals. It is crucial to assess chemical-exposed groups that may be more predisposed to severe COVID-19. Firefighters are often exposed to a range of occupational hazards due to the broad spectrum of activities and in the COVID-19 pandemic, these professionals are considered essential. As a result, firefighters were more exposed than the general population. One of the most frequent activities in the routine of firefighters is fighting fires, whether urban or forest. In these situations, workers are exposed to high levels of toxic substances and low oxygen supply. Objective: In this context, the objective of this review is to explore the current literature on a possible relationship between susceptibility (severe signs and symptoms) to COVID-19 and the occupational exposure of firefighters to smoke. Methods: The scoping review protocol used followed the methodology proposed by the Joanna Briggs Institute (JBI). Published and unpublished studies were included. The databases used were MEDLINE (via Pubmed), CINAHL, Scopus, LILACS, Embase and Web of Science. Regarding gray literature, they were consulted via Google Scholar or relevant research organizations/groups. Studies in English, Spanish or Portuguese were included. Two independent reviewers performed the screening, data

extraction and selection of articles. A third reviewer was used in cases where there was disagreement regarding the inclusion of a study. Results: Articles focusing on firefighters or smoke exposure in the context of susceptibility to COVID-19 were included in this review. As it is a current topic, studies are still limited, so studies that evaluated respiratory infections in the general public from exposure to smoke were also reviewed. It is understood that the general population may present different health risks and lower exposures when compared to firefighters, who generally have physical fitness and training, but there is no availability of a similar occupational group. It has been observed in some studies that increased concentrations of PM2.5 in atmospheric air due to fires, especially wildfires, have correlated with increased COVID-19 cases and deaths in some locations. Conclusions: The results obtained allowed us to conclude that exposure to smoke from forest fires can cause lung irritation, inflammation and alteration of immune function, which can increase susceptibility to respiratory infections such as COVID-19. In addition, some studies already show a possible relationship between exposure to smoke and cases of COVID-19. This information is important for the development of policies to protect firefighters who often work in conditions of high smoke exposure.

COVID-19 and susceptibility of firefighters exposed to smoke: A scope review

Silva, raFael araújo; Marciano, luiz paulo De aguiar; coSTa, luiz Filipe; nuneS, raFaella Ferreira naSciMenTo; SakakiBara, iSariTa MarTinS

Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences, Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-000, Alfenas, MG, Brazil

284

Introduction: The harmful effects of pesticides on human health have been increasingly studied. The lack of action selectivity represents a risk for man and other forms of life present in the environment. Brazil is one of the biggest consumers of pesticides in the world and more than 90% of rural workers depend on these substances for pest management. Triazole fungicides represent the second largest group of pesticides used in the southern region of Minas Gerais. These fungicides are potent inhibitors of CYP51, its main mechanism of action, thus actively interfering with the production of steroid hormones in humans and other health hazards. Currently, occupational exposure monitoring is performed through biological parameters, called bioindicators, which can be measured and evaluated expressing a correlation with exposure. Objective: Determination of triazoles in the urine of rural workers for application in biomonitoring of occupational exposure. Methods: Cypronazole, epoxiconazole, metconazole, propiconazole and triadimenol were chosen utilizing as criteria the most used in coffee growing in the southern region of Minas Gerais, Brazil. This study was approved by the Ethics Committee of the Federal University of Alfenas-MG-Brazil (CAAE: 34644620.2.0000.5142). Sample preparation was optimized with 1 mL of urine and 100 μL β-glucuronidase enzyme (diluted 1: 28 in 0.5 mol L-1 acetate buffer, pH 5.0). The sample was incubated at 38 ºC for 12 h. At room temperature, 20 µL tebuconazole-(tert-butyl-d9) internal standard was added, followed by the addition of 1 mL of acetonitrile, 2 mL of phosphate buffer pH 7.0 and 200 μL of toluene, vortexed for 1 minute, then centrifuged at 1134 g for 5 minutes. Next, 200 μL of the toluene fraction was

collected, dried at 30 ºC and resuspended in 100 μL of toluene and analyzed by gas chromatography–mass spectrometry. Statistical tests were performed with a level of 5% for significance, using the Computational System R. Results: Confidence parameters were evaluated and linearity (coefficients of determination higher than 0.98), precision (below 15% for quality controls and 20% for lower limit of quantification), accuracy and residual effect were satisfactory according to the validation guidelines. The calibration curve ranged from 10 to 200 µg L-1 for cyproconazole, metconazole and triadimenol analytes and from 30 to 200 µg L-1 for epoxiconazole and propiconazole. Linear regression model was significant (p <0.0001) with normality (Shapiro-Wilk), independence (Box-Pearce) and homoscedasticity (Breusch-Pagan) were verified and non-significant. To show the applicability of the method, samples of rural workers who reported exposure to triazole fungicides were analyzed (n=10). Epoxiconazole was found in five samples (70.0; 49.99; 47.74; 43.67 and 40.18 µg L-1); Triadimenol in one sample (45.46 µg L-1); and four samples with cyproconazole, a sample was quantified (14.98 µg L-1) and three between the limit of detection and the lower limit of quantitation (2 µg L-1 - 10 µg L-1). Conclusion: With these data it is possible to conclude that the validated method permitted to determine urinary triazoles in farmers exposed to these fungicides and the bioindicator of the internal dose is a reliable tool to assess the occupational exposure. Acknowledgments: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Determination of urinary triazoles as reliable biological indicator for the

application in coffee growers

Marciano, luiz paulo De aguiar1; coSTa, luiz Filipe1; Freire, joSiane oliveira1; FelTriM, FernanDo1; MachaDo, SiMone caeTani2; Silvério, aleSSanDra criSTina pupin2; MarTinS, iSariTa1

1 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences, Federal University of Alfenas - UNIFAL-MG, Gabriel Monteiro da Silva St. 700, 37130-

000, Alfenas, MG, Brazil; 2 University José do Rosário Vellano - UNIFENAS.

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Background: The p-hydroxybenzoic acid esters, or parabens, are preservatives in foods, medicines and personal care products, cosmetics and perfumes (PCPCP), belonging to the class of emerging contaminants that have the ability to promote xenoestrogenic and oncogenic effects. Studies in the literature have shown that they act as endocrine disruptors, as they mimic the functions of estradiol, and have been associated with the incidence and increase of breast cancer in animals and humans. Thus, the ability to interfere with the endocrine system is the main question to be clarified regarding the safety of parabens in human exposure. Objective: In this context, the objective of this study is to develop an analytical method, applying Dispersive Liquid-Liquid Microextraction (DLLME) technique for the sample preparation of body cream and analyze performed by liquid chromatography, with UV detection. Methods: To development method, the best conditions of extraction and detection/ quantification were evaluated. Blank sample was diluted in Milli-Q water: methanol (4:1, v/v) and centrifuged. Subsequently, the supernatant was extracted with acetone (dispersing solvent): chloroform (extracting solvent) (2:1, v/v). The extract was analyzed by liquid chromatography using a chromatographic NST 08 100 A (250 mm x 4.6

mm) column, in isocratic mode, at 35ºC, with water: acetonitrile (70:30, v/v) as mobile phase in a flow of 1 mL min-1, and detection at 254 nm. The range evaluated was between 60 to 480 µg mL-1, covering the Brazilian limit (0.4%, m/m, to isolated paraben). To assess the applicability of the method, four samples of body cream from different brands were analyzed. Results: The method was linear and showed precision and accuracy. Average concentration of parabens in the four samples analyzed was 0.011% m/m for MeP, and below LoQ for EtP and PrP (LoQ: 0.0058% and 0.0051% m/m, respectively). Discussion/Conclusion: The results demonstrated that the method is very efficient in recovering and improving the detectability of analytes studied. DLLME was efficient to prepare the samples and pre-concentrate the analytes, as well as being a practical and fast sample preparation method, respecting the Green Chemistry principles, as the decrease the spend of toxic solvents. The method demonstrated satisfactory analytical performance to be applied as a potential tool to analyze parabens from body cream. Acknowledgments: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001.

Dispersive liquid-liquid microextraction to determinate parabens in body cream by liquid chromatography

raMoS, ThaliTa Da Silva1; BarBoSa, alyne Maria Da coSTa1; raTh, SuSanne2; MarTinS, iSariTa1

1 Laboratory of Toxicants and Drugs Analysis – LATF, Faculty of Pharmaceutical Sciences, Federal University of Alfenas - Unifal-MG, Gabriel Monteiro da Silva St. 700, 37130-

000, Alfenas, MG, Brazil; 2 Institute of Chemistry, Department of Analytical Chemistry, University of Campinas, P.O. Box 6154, 13084-971 Campinas, SP, Brazil.

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Background/introduction: Quantum dots (QDs) - fluorescent types of semiconductor nanoparticles with unique electrical and optical properties – have clinical, electronic and energy applications. However, QDs uses raise health concerns due to toxic effects previously reported, of which some are related to surface chemistry of this nanoparticle. Objective: The objective of this study was evaluate the cytotoxicity of a cadmium-selenium containing QD (ITK™ – carboxyl functional group) on keratinocyte-based systems because the skin is a potential route of exposure especially in the occupational setting. Methods: Immortalized human keratinocytes (HaCaT cell line) and neonatal primary keratinocytes (HEKn) were exposed for 24 h to different concentrations of QDs (2.5, 5, 10, 20 and 40 nM), and the cytotoxicity was measured by the MTT assay. Intracellular reactive oxygen species (ROS) were also quantified (by labeling with H2DCFDA and quantifying fluorescence by flow cytometry) to verify whether the generation of ROS is related to cytotoxicity. Results: The tested QD caused cytotoxicity in both keratinocyte-based systems; however, the level of cytotoxicity differed

between the two systems. While HaCaT showed an IC50 (half-maximal inhibitory concentration) of 32.75 nM, the HEKn showed a lower value of IC50 (23.14 nM). A significant increase in ROS generation was not verified under the conditions tested. Discussion/Conclusion: The values of IC50 of HaCaT and HEKn, demonstrate that the primary cells are more prone to the effects of QDs than the cell line. The cytotoxicity observed for this nanoparticle is probably not mediated by oxidative stress, which is an important mechanism of QD-induced toxicity; however, additional investigation is needed to elucidate this hypothesis. In conclusion, our data showed that ITK™ QD is cytotoxic for skin keratinocytes. This result is in concordance with the cytotoxicity previously verified to human neural progenitor cells. Additionally, these findings draw attention to the correct choice of the keratinocyte-based test system because cell line- and primary cell-based systems have different sensibilities, and it can affect the determination of safety levels and protection of human health. Acknowledgments: CAPES, CNPq.

Evaluating the cytotoxicity of quantum dots using two different keratonocyte-cell based systems

Souza, iSiSDoriS roDrigueS1; cruz, juliana varella1; Thá, eManoela lunDgren1; gagoSian, viviana STephanie coSTa1; araujo-Souza, paTrícia Savio1; ceSTari,

MarTa MargareTe1; FauSTMan, elaine M.2,3; leMe, Daniela MoraiS1,4

1 Graduate Program in Genetics, Department of Genetics, Federal University of Paraná (UFPR), Curitiba, PR, Brazil; 2 Department of Environmental and Occupational Health

Sciences – University of Washington, Seattle, WA, USA; 3 Institute for Risk Analysis and Risk Communication – University of Washington, Seattle, WA, USA; 4 National Institute for

Alternative Technologies of Detection, Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM), Institute of Chemistry, Araraquara, SP, Brazil.

287

Human health risk assessment (HHRA) involves characterization of both exposure and hazard. The traditional approach defines the hazard from mammalian toxicity studies, many of which do not contribute data to the HHRAs, only then evaluating exposure to determine risk. To address this challenge, we have explored ways of predicting exposure based primarily on the use scenario and comparing the exposure to reference dose values derived by international regulatory agencies to identify mammalian toxicity studies that are relevant to HHRA. The premise is that exposure can be predicted based on the use scenario, regardless of the chemistry or pesticidal mode of action. The use scenarios were defined based on registered agricultural uses for fungicides, insecticides, and herbicides as they are applied in USA and Europe. For human dietary exposure, values were based on existing residue data for substances with comparable use on the same or similar crops. To provide a point of comparison for the exposure predictions, data were collated for acute, chronic and occupational reference dose values derived by US EPA, JMPR and EU Commission that are publicly available. The exposure predictions and range of hazard endpoints were compared using

the ILSI HESI Risk 21 risk matrix plots (https://risk21.org/) in order to visualise and contextualise the level of potential concern for the exposure prediction. In addition, an approach is proposed to categorise the likelihood of acceptability of risk based on where the exposure sits relative to the distribution of reference dose values. The approaches proposed in this study allow for exposure prediction based on the Good Agricultural Practice (GAP) in conjunction with the use of existing hazard data for crop protection products in order to make an initial determination on acceptability of risk and to identify key studies that are required for HHRA and also opportunities for study waivers. This work is sponsored by Crop Life International Exposure Based Assessment Team (CLI EBT) to Exponent and the manuscript is accepted as Parsons et al., 2021 in Critical Review in Toxicology. Acknowledgments: Wolf D (Syngenta Crop Protection, USA), Aggarwal M (Corteva Agriscience, USA), Rupprecht JK (Bayer, USA), Mehta J (ADAMA, UK), McEuen S (FMC, USA), Lautenschalaeger D (Bayer, Brazil), Ramanarayanan TS, Weidling R (Exponent US), Gill P (Exponent International), Byron N (Exponent International), Williams G (Exponent International).

Exposure driven data generation strategies for dietary and non-dietary risk evaluation

of crop protection products to inform safety and minimise unnecessary animal testing

caTalano, ShaDia M.i.1; paiS, Mariana caSTello novo2; laTorre, anDreia oliveira3; Faria, paTricia MiranDa3; SoareS, Daniel4; FreeMan, elaine5

1 Corteva Agriscience, Barueri, SP, Brazil; 2 Syngenta Crop Protection, Sao Paulo, SP, Brazil; 3 BASF SA, Sao Paulo, SP, Brazil; 4 Bayer SA, Sao Paulo, SP,

Brazil; 5 Exponent International Limited, Washington, DC, USA.

288

Chemical Safety is achieved when all activities involving chemical products are carried out in a way that guarantees the safety of human health and environment. It is a science composed of many scientific and technical components, including toxicology, ecotoxicology and the chemical risk assessment process. Chemicals are part of our day-to-day and are present in any analysis laboratory. The UN, aware of it, in order to harmonize the criteria for classifying and communicating hazards worldwide, has developed the GHS (Globally Harmonized System of Classification and Labeling of Chemicals), which has been adopted and become mandatory in Brazil for any chemical product used in the workplace. Within this context, the objective of this work is identifying the main chemical products used in laboratories, discussing the GHS classification of these compounds and evaluating the knowledge of professionals who work in laboratories regarding the dangers and potential risks these products present. For this survey, there carried out an exploratory research using the BABBIE methodology, in a group of 28 laboratory workers, applied in an anonymous online format. The group answered three open questions: (1) What type of laboratory do you work in?; (2) What is(are) the main chemical product(s) used in your daily work?; (3) Do you know if the mentioned product is classified as dangerous according to the GHS?. According to the answers of the first question, more than 50% of the interviewees worked at a physical-chemical laboratory, while the other interviewees diversify at school laboratory, research and development, organic and inorganic chemistry, toxicology and microbiology and parasitology. The second question, which allowed an answer with up to 3 components, provided the identification of the main chemical products used in these laboratories, the most present

ones were Ethanol (29%), Methanol (18%), Hexane and Hydrochloric Acid (14%). The last question resulted in an index of 46.4% of then answered that the chemicals used were classified according to the GHS, another 28.6% ones replied yes was classified, but they could not say if it was according to the GHS , while almost 10% of them informed they had never observed the issue of hazard classification and almost 15% reported that the chemicals handled were not classified as hazardous. Based on that, it was possible to observe that chemicals classified as hazardous by the GHS are present in a large proportion in laboratory environments and many of them can even pose significant risks to human health, such as skin corrosion or damage to the central nervous system and optic nerves, if handled without proper protection. The survey also showed a concern about knowledge and training about GHS in Brazil, given that some of the participants were not able to identify the danger communications that should be easily visible and adhered to the bottles of handled chemical products. The results of this research raise the discussion that such information may not has been transmitted to professionals during their academic training or even through training provided for in legislation. Regardless of the reason, this knowledge gap increases the risk of accidents in laboratories, since safety precautions may not be carefully followed due to the lack of danger knowledge. Thus, it is necessary to narrow the approach to the safe management of chemical products, the dangers and potential risks that professionals from Brazilian laboratories may be involved, encouraging the discussion of the topic in educational institutions, in order to contribute to the strengthening of the chemical safety culture in public and/or private institutions. Keywords: GHS. Chemical. Hazards and Risks

GHS classification of chemicals frequently used in laboratories: A risk assessment

Silvério, kérolyn apareciDa; pinheiro, FaBriciano

Intertox.

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Introduction: Nitrosamines are a class of potentially mutagenic impurities which were found to be present above the permitted limits in several batches of drug products in the past few years, leading to global recalls of several drugs. As a result, regulatory agencies have issued guidance for industry to ensure all drugs on the market will be assessed for the risk of nitrosamines, however this is a complex evaluation needing a range of different aspects to be considered. Objective: Here we aim to evaluate how in silico systems and data sharing initiatives can be applied to a nitrosamine risk assessment. Methods: The assessment was done by using in silico models and proprietary data shared within a consortium. Results/Discussion: A nitrosamines risk assessment should start by considering all the potential sources of nitrosamines within the manufacturing and storage of a drug product, including the drug substance, excipients, and their interaction. Nitrites can be found within excipients, and under specific conditions may form nitrosating agents that can react with vulnerable amines in drug formulations, potentially forming nitrosamines. A data sharing initiative in which companies share nitrite levels with each other can help in this stage. Such a database containing typical results could reduce duplicate testing by the industry and increase scientific knowledge. If there is a theoretical risk of forming a nitrosamine, then a risk assessment should follow as outlined by ICH M7, the guideline for the assessment and control of mutagenic impurities in drug products. This guideline describes that in silico systems can be used to predict

the mutagenicity potential of impurities, in lieu of performing the Ames test, if two complementary systems are used – such as Derek and Sarah Nexus. However, in silico systems may not always be conclusive and performing the Ames test may be necessary. Such a test is time-consuming, requires resources, and can be challenging for low level impurities which are not easily obtained in their pure form. An alternative to overcome these challenges are data sharing initiatives where different companies can share Ames test results for their nitrosamines and have access to results shared by other companies. This then avoids rework and allows for the science to advance quicker. Lhasa Limited, as a not-for-profit organisation, acts as an honest broker to allow the industry to benefit from such initiatives. Finally, if a nitrosamine is found to be mutagenic, the acceptable limit must be defined, followed by assessing the levels of the impurity. Purge calculations are a way of assessing this through in silico systems, which avoid the need for analytical procedures to be developed and validated. Purge assessments are described by ICH M7 and can also be used for nitrosamines. Conclusion: If the risk is mitigated with either approach – finding that nitrite levels are not of concern, that the nitrosamine is not mutagenic, or that it is sufficiently purged during the synthetic process – the need for an analytical test is avoided. Therefore, by applying the use of in silico systems and data sharing initiatives, unnecessary testing can be avoided using a science-based and risk-driven approach.

How can nitrosamine risk assessments be supported by in silico systems

and data sharing initiatives?

WaechTer, FernanDa; kockS, grace; avila, carolina MarTinS; BurnS, Michael j.; ponTing, DaviD j.; TennanT, rachael e.; oliveira, anTonio anax F.; hegheS, crina

Lhasa Limited.

290

Field work and literature data show that the tailings disposed on the soil after the failure of Dam I (VALE S/A), in Brumadinho, are rich on iron, silica, aluminum and manganese; they are also composed by fine particles (from 10 to 100 µm), ultrafine particles (from 10 at 1 µm) and colloidal (less than 1 µm) ones. Inhalation of particles and their consequent deposition in the airways is an important risk parameter for human health. This work presents the conceptual model of human environmental exposure to iron ore mining tailings after dam failure and the preliminary estimate results of particle deposition in the lung by inhalation exposure using MPPD Multiple-Path Particle Dosimetry Model. After the selection

of values for the input parameters based on primary data and literature, the preliminary results indicated that the concentration, the density and size of the particles are important factors for the differential deposition of the particles in the respiratory system. It is important to emphasize that the decrease in uncertainties in the results obtained in this work, from a preliminary approach, will be possible from the availability of air quality data in the monitored areas in Brumadinho. This information will be forwarded to local institutions and other stakeholders as a subsidy for discussions on the environmental impacts of the iron mining dam I failure.

Human health risks assessment by iron mining dam failure (VALE S.A) in Brumadinho-MG

DoMingoS, líllian Maria BorgeS; caSTilhoS, zuleica carMen

CETEM- Centre for Mineral Technology.

291

Background: Histamine is a biogenic amine naturally present in different foods or produced due to the activity of histidine decarboxylase from added or contaminating microorganisms. At low concentrations histamine is a vasoactive and neuroactive substance, which is involved in three primary areas of the body, e.g., immune system function, digestion and brain/nervous system as a neurotransmitter. However, at high concentrations, histamine can cause intolerance, with symptoms like itching, hives, sneezing, watery eyes, asthma, headaches, abdominal pain, diarrhea, tachycardia, and hypotension. The European Food Safety Authorities (EFSA), in 2011, established no observed adverse effect levels (NOAEL) for histamine per meal for normal (50 mg) and for histamine intolerant (0 mg) individuals. Objective: To investigate the dietary exposure to histamine by the Brazilian population. Methods: The levels of histamine in different Brazilian foods were compiled from literature and used to calculate dietary exposure. The food consumption profile of the Brazilian population was obtained from the Family Budget Survey (POF) 2017-2018 (IBGE). The daily portion of each food with detectable levels of histamine was considered. The risk characterization was evaluated by the relationship between Brazilian daily acute exposure to histamine from the consumption of the main histamine containing foods and the acute reference dose per meal (50 mg). Results: The Brazilian foods containing higher histamine levels were in mg/100 g:

grated cheese (115); Parmesan cheese (65.5); soy sauce (39.5); Gouda cheese (19.5); Italian sausage (18.5); eggplant (12.5); bean sprout (8.8); tuna with tomato sauce (8.4); canned grated tuna (5.6); sausage (4.7). The total intake of histamine by Brazilian population can reach acute toxic levels. Cheese was the main contributor to total histamine intake. Considering the consumption of grated cheese and the other foods, the total histamine intake was 94.9 mg. If it the consumption of Parmesan cheese was contemplated instead of grated cheese, the total histamine intake (67.3 mg) would still be above the acute reference dose. Discussion/Conclusion: Histamine legislation is only available for Scombroid fish (tuna, bonito) and other histamine formers. However, fermented products including cheese, sausages and soy sauce, as well as germinated products were the food items which showed higher levels of histamine. Cheese, mainly grated and Parmesan, accounted for a high intake of histamine. Histamine intolerant individuals must be careful regarding histamine free food products. The NOAEL can be easily reached in a meal, especially when fermented products are present. One must also consider that alcoholic beverages (wine and beer), which were not included in the survey, also contribute with significant amounts of histamine and can potentiate its toxic effects. Acknowledge: We thank Capes, CNPq and Fapemig for the financial support.

Occurrence and dietary exposure to histamine by the Brazilian population

Diniz, FaBiana BarBoSa1; Braga, DouglaS evangeliSTa2; cuSTóDio, Flávia BeaTriz1; gloria, Maria BeaTriz aBreu1,2

1 Faculty of Pharmacy; 2 Veterinary School, UFMG.

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Background: The prevalence of obesity and other diet-related chronic noncommunicable diseases is increasing worldwide. In this scenario many consumers started to choose foods containing sweeteners instead of sugars, one of the largest contributors to the high prevalence of these diseases. Non-nutritive sweeteners are used in small amounts to reduce the caloric and sugar contents of foods and beverages. One of the main sweeteners in the market is aspartame, a synthetic one 200 to 300 times sweeter than sucrose and with a low caloric value. Aspartame has been incorporated into over 6,000 products available in the market, mainly beverages. It is therefore consumed by millions of people across the world. Objective: With the increasing availability of products containing aspartame in its formulation in the market, the objective of this study was to investigate the exposure of the Brazilian population to this sweetener through consumption of soft drinks. Methods: Soft drinks sold in wholesale and retail stores in the city of Belo Horizonte, Minas Gerais, Brazil were included in this study. The labels of these products were analyzed to obtain information regarding the brand, presence or absence of aspartame, and the amounts declared (when available). Exposure was estimated using the mean levels of aspartame declared in labels and the mean consumption of beverages divided by the mean body weight of Brazilian population. The food consumption profile and the mean body weight of the Brazilian population (≥10 years old) was obtained from the Family Budget Survey (POF) 2017-2018 (IBGE). The risk characterization was evaluated by the relationship between Brazilian daily exposure to aspartame from the consumption of soft drinks and the chronic

toxicological reference value, described for aspartame as the Acceptable Daily Intake (ADI) of 40 mg/kg body weight for neurological damage related to the generated metabolites: phenylalanine, aspartic acid and methanol. Results: Overall, 15 different types of soft drinks from eight different brands were available in the market. Among them, only six (40%) had aspartame. The declared aspartame contents varied from 0 to 0.35 mg/mL, with an average of 0.1 mg/mL. All the samples were within the limit established in the legislation for beverages with total replacement of sugars, which corresponds to 0.75 mg/mL. All risk assessment scenarios for aspartame showed that the daily consumption of aspartame through soft drinks is not greater than 5% of the ADI for this sweetener, even in the most pessimist consumption scenario. The subpopulations with the highest aspartame intake were adolescents (by age group), male (by gender) and residents in the South of Brazil. Discussion/Conclusion: Soft drinks are the non-alcoholic beverages widely consumed in the modern diet and aspartame is an important sweetener used in them. Low-sugar or sugar-free beverages account for about 30% of the total non-alcoholic beverage market. Consequently, this has led to an increased use of sweeteners in product development. This whole scenario requires the protection of consumers’ health by carrying out a risk assessment. The estimated daily intake of aspartame by the Brazilian population showed levels below the ADI indicating low risk. The results of the present study are in agreement to other works in the scientific literature. Acknowledgement: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil – Finance Code 001.

Occurrence and exposure to aspartame from soft drinks by the Brazilian population

SouSa, roBerTo ceSar SanToS; cuSTóDio, Flávia BeaTriz; gloria, Maria BeaTriz a.

Graduate Program in Food Science, Faculty of Pharmacy, UFMG, Belo Horizonte, MG.

293

In Brazil, the publication of RDC 301/2019 and the 14 Normative Instructions became the regulatory framework for Good Manufacturing Practices. This regulation establishes general guidelines for good drug manufacturing practices, in which ANVISA requires companies to have a Quality Risk Management that should include precautions to mitigate cross-contamination, in addition to a toxicological evaluation of all active ingredients present in multipurpose manufacturing areas. Within this context, the determination of the Permitted Daily Exposure (PDE) emerged, defined as the specific dose of a substance that is unlikely to cause adverse effects to an individual exposed daily during lifetime based on data from preclinical and clinical studies. Thus, this study aimed to determine the PDE of the dry extract of Ginkgo biloba leaves, utilized as an input in a pharmaceutical solid dosage form administered orally in preclinical studies in mice. Ginkgo biloba was chosen because this plant is the fourth greatest in reported adverse effects, which include gastrointestinal disorders, allergic skin reactions, hypotension, palpitations, hemorrhages, among others. For that reason, acute oral toxicity tests were performed based on the OECD 423 guideline, exposing three male and female mice to a single dose of 2000 mg/Kg body weight of Ginkgo biloba dry extract and monitoring them for 14 days. In addition, the repeated dose toxicity test was performed based on the OEDC 407 guideline, with the administration of Ginkgo biloba

dry extract at doses of 120, 240 and 480 mg/Kg to 10 mice in each dosing group and evaluated for 28 days. Finally, a genotoxicity assessment was carried out based on comet and micronucleus assays. The former evaluated the DNA damage index in lymphocytes, and the latter determined the polychromatic immature lymphocytes of the bone marrow and treatment differentiated polychromatic and normochromic erythrocytes. The results indicated that the dry extract of Ginkgo biloba leaves causes low acute toxicity, with no liver toxicity or alteration of blood glucose levels in males and females. Changes in weight gain were not demonstrated in the groups tested, but in males, the food intake was lower during the first week of treatment at the highest dose of extract (480 mg/Kg). The hematological parameters were not altered in males, whereas in females receiving 480 mg/Kg there was a reduction of leukocytes and lymphocytes and an increase of neutrophils. The lipid profile of males was not altered by the doses administered, whereas females treated with 480 mg/kg presented higher total cholesterol levels. In the genotoxicity tests, no significant difference was observed in the micronucleus and comet assays. The results found in this work permitted calculation of the PDE, which was estimated at 0.1 mg/day; when correlating to the maximum residual concentration of 250 µg/mL, it is concluded that the current cleaning process is safe and does not need to be re-evaluated.

Permitted Daily Exposure (PDE) from preclinical studies of ginkgo biloba dry extract

laMB, liliane WeBer BolFe1,3; roDrigueS, gaBriela ziMMerMann praDo2; Saraiva, Thalia eMManoella SeBulSqui1; Souza, DouglaS1; Berna, gaBriel Da coSTa1; garcia, ana leTícia hilário2; BerTolDi,

FernanDo4; kaySer, juliana MachaDo1,2,3; Ferreira, julia gaBriele De jeSuS1,3, veiverBerg, anDriele1; Marco, Mariana1; gehlen, günTer2,4; BeTTi, anDreSa heeMann1,3; MaTToS, criSTiane BaSToS1,3

1 Bioanalysis Laboratory, Health Sciences Institute, Universidade FEEVALE, Novo Hamburgo-RS, Brazil; 2 Compartive Histoly Laboratory, Health Sciences Institute, Universidade

FEEVALE, Novo Hamburgo-RS, Brazil; 3 Postgraduate Program on Toxicology and Analytical Toxicology, Universidade FEEVALE, Novo Hamburgo, Brazil; 4 Postgraduate

Program on Environmental Quality, Universidade FEEVALE, Novo Hamburgo, Brazil.

294

Thyroid function during gestational age is a determinant factor for fetal neurologic development since studies have been demonstrating that prenatal thyroid dysfunction may result in an impairment of cognitive and behavior functions and also other outcomes such as birth weight, fetal stress, malformations and prematurity. Pesticides are of an increasing concern about their effects on human and environmental health. Mancozeb (MZ), an ethylene bisdithiocarbamate manganese (Mn)-based, is the most used fungicide in Brazil, Europe Union and United States of America and considered as an important endocrine disruptor chemical. The aim of this study is to assess the Mn exposure and thyroid and sex hormones concentrations in pregnant women of Recôncavo Baiano - Brazil, and to evaluate their correlation. Blood, occipital hair and toenails were collected from volunteers in the second trimester. Mn in hair (MnH) and Mn in toenails (MnTn) were determined by graphite furnace atomic absorption spectrometry (GF-AAS). Thyroid hormones such as thyroid stimulant hormone (TSH), thyroxin (T4), triiodothyronine (T3) and the sex hormones estradiol (E2) and testosterone (T) were measured by chemiluminescence. Sociodemographic and pesticide-related questioners were applied. A total of 146 pregnant participated, with mean of 26.5 years-old and 18.1 gestation weeks at enrollment. Median (minimum and maximum) of hormones were: TSH 1.11 µIU/mL (0.14-3.41), FT4 0.91 ng/dL (0.71-1.24), T3 132.15

ng/dL (84.11-207.46), E2 5263.82 pg/mL (450 – 9030) and T 0.56 ng/dL (0.26-2.52); and for Mn biomarkers of exposure: MnH 0.20 µg/g (0.05-3.04) and MnTn 0.68 µg/g (0.05-22.35). A total of 16 (11.3%) women reported that have already worked with pesticide before and 28 (20.1%) husbands/partners reported that are now working with them. MnTn levels were significantly different in those pregnant that work with pesticide (p=0.026), and also T concentrations were different in this group (p=0.015). Significant difference of MnH levels in women that have already worked with pesticides before (p=0.03) and in those who still work with those (p=0.011) were observed. However, these results are controversy, showing higher levels of these biomarkers and hormones in groups with no pesticide contact. No correlation was observed between biomarkers of Mn exposure and thyroid or sex steroid hormones. It could be a result of our number of volunteers that is still not enough. This study is still in progress as well as a panel of pesticides analysis and the recruitment of more pregnant women. The present work is one of the fields of investigation of the Brazilian Birth Cohort called Socio-Environmental Determinants of Neurodevelopment at 12 months-old (DSAN-12m). The authors would like to thank the participation of all pregnant and nursers in the project, and CNPq Universal Edital 421550/2018-0 and FAPESB/PPSUS EFP_00019898 for funding. Keywords: pesticide exposure, pregnancy, neurodevelopment, neuroendocrine.

Thyroid and sex hormonal profile and manganese exposure in pregnant women from

a Brazilian birth cohort: preliminary results

SanToS, naThália riBeiro1,2; Bah, hoMegnon anTonin Ferréol1,3; goMeS-júnior, erival aMoriM1,4; MarTinez, vicTor oTero1,2; coSTa, DaiSy oliveira1,2; MenezeS-Filho, joSé anTonio1,2,3,4

1 Laboratory of Toxicology, College of Pharmacy, Federal University of Bahia; 2 Postgraduate Program of Pharmacy, College of Pharmacy, Federal University of Bahia; 3 Postgraduate Program of Collective Health, Institute of Collective Health, Federal University of Bahia;

4 Postgraduate Program of Food Science, College of Pharmacy, Federal University of Bahia.

295

Background: Arsenic (As) is a widely distributed element in nature and its exposure can cause carcinogenic effects to humans. One of the sources of arsenic dietary exposure is fish consumption. The main compound found in fish is arsenebetaine, a non-toxic and non-carcinogenic organic arsenic compound. Current speciation analyses are still expensive and not widely available. However, to evaluate the real risks of fish consumption, it is necessary arsenic speciation or to distinguish non-toxic and toxic arsenic. Objective: The objective of the work was to identify the occurrence of total and inorganic arsenic in fish and crustaceans and to evaluate inorganic arsenic exposure of Brazilian population by fish consumption. Methods: All data of arsenic in fish available in GEMS/Food database (Global Environment Monitoring System - Food Contamination Monitoring and Assessment Programme from World Health Organization) up to 2021 was downloaded for the first analysis. Global data from fish and crustaceans that had clearly defined name or species were selected. Fish were categorized by species, family or commercial name and crustaceans were categorized in four groups: shrimps and prawns, lobster, crabs and crayfish. Descriptive analyses were done for each category and for fish and crustaceans. The intake of inorganic arsenic was estimated by fish consumption and global inorganic arsenic occurrence estimated from GEMS/Food. Fish and crustaceans consumption data were obtained from the Family Budget Survey 2017-2018 (POF) of Instituto Brasileiro de Geografia e Estatística (IBGE). Risk characterization was evaluated by calculation of margin of exposure (MOE), i.e., the ratio of the toxicological reference dose (BMDL0.5 = 3 µg/kg bw day for lung cancer) and estimated intake.

Results: It was evaluated 12941 results of total arsenic (92% of total data) and inorganic arsenic (8%) in fish (89%) and crustaceans (11%). Tuna (1,339 results), cod (3,608 results), salmon (1,455 results) and shrimps (1,018 results) were the most representative groups in database. For fish, the global range of total As level was 0-110.0 mg/kg (mean= 2.92 mg/kg) and the global inorganic As levels varied from 0-0.16 mg/kg (mean=0.0028 mg/kg). For crustaceans, the global range of total As level was 0-100.4 mg/kg (mean=3.08 mg/kg) and global inorganic As levels were 0-0.90 mg/kg (mean= 0.05 mg/kg). The proportion of total As and inorganic As obtained for fish ranged for 0.02 to 16.4% (mean=0.10%) and for crustaceans, 0.14% to 2.2% (mean=1.50%). Brazilian fish and crustacean consumption varied from 8.8 to 49.5 g/day and 0.2 to 1.2 g/day respectively. Brazilian intake of inorganic As from fish and crustaceans consumption was estimated in 0.0004-0.0022 µg/kg bw and 0.0001-0.0009 µg/kg bw respectively. MOE values ranged from 83,370 to 468,959 and 14,031 to 83,901 respectively. Discussion/Conclusion: Total As levels were at most above Brazilian legislation of 1 mg/kg for fish and crustaceans, although the inorganic species, the main toxic forms, in all samples were largely below this value. This reinforces the need for As speciation and/or revision of Brazilian legislation. By the MOE calculation it was possible to conclude that, at first sight, the ingestion of inorganic As from fish consumption does not present substantial risk to the Brazilian’s health. The obtained results are important to understand and evaluate risks of arsenic intake by fish consumption in Brazil, being a base to risk analysis and decision making in food safety area. Acknowledgments: CAPES

Total and inorganic arsenic in fish and exposure to inorganic arsenic by fish consumption in Brazil

raMoS, viTor Serrão1; vaSconceloS neTo, MilTon caBral2; cuSTóDio, Flávia BeaTriz1

1 BioTox - Laboratório de Bioquímica e Toxicologia de Alimentos, Departamento de Alimentos, Faculdade de Farmácia da UFMG; 2 Instituto Octávio Magalhães - IOM, Fundação Ezequiel Dias - Funed.

13 TOXICOLOGIC HAZARD

IN THE WORKPLACE

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Introduction: Dichlorodiphenyltrichloroethane (DDT) was the most used chemical substance in the 20th century, due to characteristics such as high environmental persistence, toxicity and low degradability. In Brazil, it was widely used to control malaria in the period 1945-1997, with workers in health campaigns exposed to DDT for years. In the Amazon, full coverage was reached at the end of the 1960s. In the 80s and 90s, the incidence of the disease grew alarmingly in Brazil, with 99% of cases located in the Amazon region. During public health campaigns using DDT, workers involved in home spraying were vulnerable to contamination due to high exposure due to factors such as lack of training, lack of use of PPE or resistance to its use by workers. Objective: Evaluate the distribution of serum levels of pp’-DDT and pp’-DDE in serum samples from health agents who worked in public health campaigns to combat malaria until 1997, attended at Instituto Evandro Chagas from 2009 to 2015. Methods: This study is epidemiological, longitudinal and retrospective , where 460 health workers were treated, exposed to activities such as transport, preparation, handling, storage, disposal and vaporization of DDT in the internal and external areas of residences in endemic areas by the minimum period of up to 5 years. Serum samples were extracted with n-hexane and analyzed by Gas Chromatography electron capture detection (GC-ECD) model CP 3800 Varian with extraction Data were analyzed using the following applications: StatisticalPackage for the Social Science (SPSS), version 20.0. This work was submitted to the Research Ethics Committee of the Instituto Evandro Chagas under the Certificate of Presentation of Ethical Appreciation nº 52618121.4.0000.0019 and approved through the

opinion nº 5.178.481. Results: The detection frequency of p,p’-DDT and pp’-DDE corresponded to 40% and 87.82% of the samples, respectively. The average levels of pp’-DDT were 1.70 ± 3.92 μg/L, while pp’-DDE presented 9.78 ± 10.99 μg/L. The DDE/DDT ratio found was 5.75 μg /L. The predominant age group in the research was from 41 to 50 years old (38.9%). The variables significantly associated with p,p’-DDT levels were: > 20 years of work, fish intake and symptoms of cramps and low sexual performance (p<0.05). The variables significantly associated with p,p’-DDE levels were: > 20 years of work, working in mining areas and eating fish (p<0.05). Of these, < 20 years of work was the variable that most influenced the concentration of p,p’-DDE (β=5.68; p<0.001). Discussion/Conclusion: The high means of p,p’-DDE in relation to p,p’-DDT indicate that the concentrations in the study are the result of old exposure, probably until 1997 when spraying officially ended in Brazil. The high index of statistical significance of p,p’-DDE concentrations achieved in the multivariate analysis for working time (p<0.001) is a strong indication that the concentrations in the study are the result of a high occupational exposure. Despite the indication of old exposure, the ratio between p,p’-DDT and p,p’-DDE was relatively low when compared to other studies, indicating a natural decline in DDT levels, considering that the last spraying occurred in 1997 and the Participants only carried out the collections from 2009 onwards. Despite the absence of more specific clinical data, the results lead to the creation of a health care program for agents, due to the persistence of DDT in the body. Acknowledgments: CAPES and FUNASA for their logistical and financial support.

Assessment of serum levels of DDT and metabolites in workers in malaria control

campaigns in the state of Pará, Amazon, Brazil

rocha, Thiago l.1; rocha, cáSSia c.S.2; MiranDa, anTônio M.M.2; MenDeS, roSivalDo De a.2

1 Postgraduate Program in Epidemiology and Health Surveillance/Instituto Evandro Chagas; 2 Environment Section/Instituto Evandro Chagas.

298

Background: The hazard classification of the active substances (AS) used in pesticides is a fundamental step on the reevaluation process. Recently, the Brazilian Health Regulatory Agency (Anvisa) approved a legislation setting new parameters for the classification of pesticides for a better alignment between the Brazilian assessment and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Objective: To detail Anvisa’s challenges and approach on the hazard classification of pesticides during the reevaluation of AS, in order to comply with both GHS criteria and the legislation adopted by the normative RDC n⁰ 294/2019. Methods: All toxicological data about the AS from regulatory and non-regulatory (scientific literature) studies were considered in a weight of evidence approach (WoE) to select the endpoints with toxicological relevance for humans and their corresponding lowest observed adverse effect level (LOAEL). Then, the AS were classified according to the hazard categories of the GHS, by comparing the available data with the criteria from GHS and RDC n⁰ 294/2019. When gaps on the classification criteria were identified, scientific judgment was used to decide whether a category should or not be attributed to the AS. Results: Case study #1: substance A was classified in Category 1 for Specific target organ toxicity – single exposure (STOT-SE) due to testicular toxicity in rats after single oral exposure at doses ≥ 50 mg/kg body weight. In addition, this test substance was also placed in Category 1B for reproductive toxicity – as a presumed human reproductive toxicant – due to its adverse effects on reproductive physiology and on embryofetal and neonatal development. Although the GHS defines that the same endpoint should not be

used for the classification in two distinct categories, in this case, it was considered necessary to inform specifically the occurrence of testicular effects after a single dose exposure. Moreover, the chronic effects used to the reproductive classification included but were not limited to the testicular effects. Case study #2: substance B was not classified for Specific target organ toxicity – single exposure (STOT-SE) since the thyroid effects (altered hormone levels) were not considered relevant in this kind of exposure. On the other hand, sub chronic and chronic studies showed disruption of thyroid homeostasis with relevant histological damages and changes on the hormone levels, which led to a classification in the Category 2 for Specific target organ toxicity – repeated exposure (STOT-RE). This AS was also classified in Category 1B for reproductive toxicity due to the likelihood of the changes on the thyroid hormone levels to cause neurodevelopmental impairments of the offspring exposed during critical windows of development. Although both classifications were supported by the damage observed in the same organ, the outcomes used for the classifications were considered distinct and important to be communicated. Discussion/Conclusion: To properly communicate the hazard to the user in the AS reevaluation, a careful analysis of all available scientific data is critical to select the toxicological endpoints that will be used to classify the substances within the different hazard categories of GHS and RDC n⁰ 294. However, for some AS the classification criteria cannot promptly be used, because of a potential overlap on the endpoints assigned to each Category. When facing these situations, Anvisa is relying on a case-by-case scientific judgment.

Case Studies: challenges on implementing the GHS classification of pesticides in the reevaluation of active substances

aguiar, lariSSa MuraTori; Braz, juliana MachaDo; Moreira, caMila queiroz; SanToS, Thiago SanTana; FreiTaS, Daniel roBerTo coraDi

Brazilian Health Regulatory Agency (Anvisa).

299

Brazil is one of the world leaders in pesticide use. The adverse effects caused to the health of workers in contact with these substances depend on the toxicological profile of the product and mainly on the intensity and time of exposure. Eventual damage due to individual exposure can be minimized with the correct use of personal protective equipment (PPE). The study aimed to analyze the adverse effects due to exposure to pesticides used in tomato and onion crops without the correct use of PPE. Forty workers who plant both crops on their rural properties and commonly do not use PPE completely and correctly were clinically and laboratory analyzed. Were measured plasma acetylcholinesterase levels, the mutagenicity parameter evaluated through the micronucleus test and investigation of oxidative stress through measurements of the parameters of superoxide dismutase (Sod), myeloperoxidase (MPO), glutathione-S-transferase (GST), vitamin C (VIT C), thiobarbituric acid reactive substances (TBARS), protein carbonyl (PC) and DNA damage. All results were compared to the control group consisting of 20 professional accountants from offices in the urban area of the city of Caçador (SC). The clinical evaluation was performed by an occupational doctor registered at the Regional Council of Medicine of Santa Catarina (CRM/SC). The ethical opinion approved has protocol

No. 4,255,852. All the laboratory parameters of the farmers showed significant differences in relation to the control group. In the clinical evaluation, no apparent alterations were observed in any of the exposed and non-exposed individuals. Referring to the significantly lower plasma levels of acetylcholinesterase in exposed workers, demonstrates that incorrect, incomplete and depending, even non-use, are compromising the exposed bodies. The micronucleus test with significantly higher results in rural producers demonstrates a greater possibility of mutagenicity development. Finally, all parameters evaluated for oxidative stress were significantly different between both groups, being unfavorable results for the group of farmers exposed to pesticides, which have a greater tendency to diseases and/or injuries due to the excess of Radicals Oxygen Species (ROS). It was evident that the incorrect, incomplete or even non-use of PPE can bring numerous damages to the exposed worker’s body, especially in the case of chemical substances with biocidal powers, which is the case of pesticides. used in tomato and onion crops. Keywords: personal protective equipment, pesticides, health of workers. We thank all participants and collaborators of this research, as they contributed to a scientific and social well-being.

Clinical and laboratorial evidenced organic damage due to the incorrect use of PPE in

rural producers of tomato and onion crops in the contestado region - Santa Catarina

BoFF, everTon; kaMpMann, Micheli gaBarDo; BenvenuTTi, régiS carloS

Universidade do Oeste de Santa Catarina - UNOESC.

300

Introduction: The great challenge of occupational toxicology is the prevention of chronic non-communicable diseases, since these diseases are often disabling, reducing workers time and quality of life. Studies to evaluate the use of biomarkers, especially those with an early effect, are essential for technical-scientific advancement and the prevention of irreversible chronic effects in exposed workers. Occupational exposure to crystalline silica (CS), which is classified as a group 1 carcinogen by the International Agency for Research on Cancer, it can trigger several pathological processes, such as silicosis, pulmonary fibrosis, chronic obstructive pulmonary disease and cancer. In the vast majority of cases, the diagnosis of these diseases is late, and ceasing exposure to the causative agents does not guarantee that they will stop developing, neither that the damage caused to stabilize, as it is a progressive disease. In this line, biomarkers, including genes and proteins, can be detected and/or quantified in biological fluids as a response to pathological changes in the face of chronic exposure to different xenobiotics, thus contributing to early diagnosis and/or indicating the severity of diseases. Objective: This study aimed to investigate potential early peripheral biomarkers of inflammation through protein and gene expression in workers occupationally exposed to CS. Methods: The non-occupationally exposed workers at CS (NEW) group (n=22) and CS exposed workers (CSEW) were divided into two groups: workers diagnosed with silicosis (CSEW I, n=26) and workers without silicosis (CSEW II, n=28). Serum reactive C-protein (RCP) was measured by immunoturbidimetry. Lymphocytes and monocytes protein expression of L-selectin, CD18 and CD54 by flow cytometry and gene expression

of CXCL2 and IL-8 by RT-PCR were performed. CEP: 1.868.122. Statistical analyses: IBM SPSS (version 22). Significance was accepted at p≤0.05. Results: Lymphocytes protein expression of L-selectin was significantly decreased in the exposed groups (CSEW I: 25.73 ± 1.51 %; CSEW II: 40.56 ± 1.73 %) compared to the NEW group (48.51 ± 2.18 %) (p<0.05) and was significantly different between CSEW I and CSEW II groups (p<0.001). No significant difference was observed between the CD18 and CD54 groups. Significant higher values of RCP in CSEW I (16.36 ± 5.08) than in CSEW II (1.91 ± 0.54) and NEW (2.40 ± 0.97) (p <0.05), indicating an increase of inflammatory activity. Otherwise, CESW groups presented significant lower gene expression of CXCL2 and IL-8 (CSEW I: 0.15 ± 0.03 and 0.53 ± 0.07, respectively; CSEW II: 0.27 ± 0.06 and 0.52 ± 0.07, respectively) when compared to the NEW group (1.88 ± 0.33 and 1.03 ± 0.03, respectively) (p<0.001). Also, were observed that the more T cells were activated, the lower were the gene expression of these inflammatory markers (L-selectin vs. IL-8 r= -0.293 p<0.05; L-selectin vs. CXCL2 r= -0.420 p<0.001). Discussion/Conclusions: As the exposure to CS causes an excessive inflammatory response, increasing adhesion molecules levels in plasma, we can suggest that the downregulation of IL-8 and CXCL2 chemokines is a negative feedback mechanism for organism protection against inflammation. Our findings help us to increase our understanding of the complex mechanism of silicosis pathogenesis and to identify new early biomarkers of precocious diagnosis. These results may be considered for designing new studies with more details in the future. Funding: This work receives financial support by FAPERGS (PqG 02/2017) and CNPq. Acknowledgments: BANRISUL.

Evaluation of proteins and genes expression in workers exposed to

crystalline silica in Southern Brazil

peruzzi, caroline porTela1,2; göeThel, gaBriela1,2; FleSch, ingriD1,2; naSciMenTo, SaBrina1,2; narDi, jeSSica1,2; arBo, Marcelo DuTra1; garcia, Solange1

1 Laboratory of Toxicology (LATOX), Department of Analyzes, Faculty of Pharmacy, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; 2 Postgraduate

Program in Pharmaceutical Sciences, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; 3 FUNDACENTRO - Jorge Duprat e Figueiredo Foundation, Porto

Alegre, RS, Brazil; 4 Regional Worker Health Unit, Ametista do Sul, RS, Brazil.

301

Background: The manipulation of anticancer drugs must follow strict safety rules in order to minimize the risks of occupational exposure to professionals. Contamination of the external surface of medication bottles is recognized as a health risk. The identification of potential sources of contamination becomes important for the adequacy of protection measures and effective monitoring. Objective: To determine if there is residual contamination on the surface of vials and packages of the anticancer drugs doxorubicin (DOX) and cyclophosphamide (CP) using ultra high performance liquid chromatography coupled to a mass spectrometer (UHPLC-MS/MS). Methods: Samples were collected using a Whatman® Qualitative Filter Paper Grade 2 moistened with 1 mL of methanol and kept in a test tube until analysis. Samples were prepared by solid-liquid extraction. 5 mL of methanol and ethyl acetate (1:1, v/v) were added to the tube containing the filter paper and homogenized by vortex for 30 minutes at room temperature. After centrifugation, 2 mL of supernatant was filtered to a new tube and evaporated at 60 ºC. The dried extract was recovered with 200 µL of water with 0.1% formic acid and methanol (1:1, v/v). After homogenization, an aliquot of 10 µL was injected on UHPLC-MS/MS system. Chromatographic separation occurred in an Acquity UPLC BEH C18 (50 x 2,1 mm; 1.7 μm) at 40 °C. Mobile phase was water with 0.1% formic acid (eluent

A) and acetonitrile with 0.1% formic acid (eluent B) eluted in gradient mode from 90:10 to 50:50 (A:B, v/v) at flow rate of 0.3 mL min -1. Monitored transitions for quantitation were 261.9/141 for CP and 544/360.8 for DOX. Retention times were 2.7 and 2.8 minutes for CP and DOX, respectively. Calibration and control samples were analyzed in all batches, with linear intervals ranging from 1 to 1000 ng/patch. Results: A total of 198 samples were analyzed, 66 of CP and 132 of DOX. All CP samples belong to the same manufacturer, while DOX samples are from two different manufacturers. For each manufacturer, 33 samples of vials and 33 samples of packages were collected. CP levels were detected in 9 samples (13.63%), 3 were below the lower limit of quantification (LLQ) and the other 6 showed contamination levels ranging from 1.24 to 28.04 ng/patch. DOX levels were detected in 25 samples (18.93%), 2 were below the LLQ and the others showed levels between 1.32 and 664.84 ng/patch. 85.29% of samples with residual contamination were from vials. Conclusion: This study demonstrated that cytotoxic drug vials and packaging may contain residual contamination. These results highlight the need to implement strategies to prevent and monitor the effectiveness of safety measures, in order to minimize the risk of exposure of professionals involved in the preparation of medicines. Acknowledgments: FIPE/HCPA.

Evaluation of residual contamination in anticancer drug packaging using UHPLC-MS/MS

Silva, luciana STein1; Silva, laura cé2; MachaDo, ciBele Da Silva BarBoSa1; capp, eDiSon1; linDen, raFael2; neSS, SanDro luiS riBeiro1; anTuneS, Marina vezon2

1 Department of Gynecology and Obstetrics, Federal University of Rio Grande do Sul, Porto Alegre-RS, Brazil; 2 Analytical Toxicology Laboratory, Feevale University, Novo Hamburgo-RS, Brazil.

302

Brazil is one of the largest tobacco producers in the world, the production is concentrate at country south, where 97% of the crops come from and employ more than 600.000 people. In tobacco production, the tobacco workers expose themselves to substances such as nicotine, present in tobacco leaf and pesticides. The exposure to high nicotine concentration can occur during several stages of tobacco chain production, mainly at harvesting the leaves, arranging the leaves for drying and also while sorting leaves for quality selection. Handling tobacco leaves cause nicotine poisoning, an acute intoxication also known as Green Tobacco Sickness (GTS). The mains symptoms are nausea, vomiting, weakness, dizziness, abdominal pain and sometimes fluctuations in blood pressure or heart rate. Risk of nicotine poisoning increases when the leaves or skin is wet, allowing nicotine to get onto the skin and pass into the bloodstream more easily. The objective of this study was to observe the symptoms associated to GTS occurrence and to evaluate the life quality of tobacco workers. This is a cross-sectional descriptive study with a quantitative approach, using two questionnaires as instruments: (i) sociodemographic and health conditions data of tobacco workers, mainly those associated to GTS; and (ii) life quality information (WHOQOL-bref). The majority of the participants were men, under the age of 40 (39.9 ±11.92 years old) and incomplete elementary school education. Few workers reported chronic disease as depression, obesity and asthma. Most of them (93.55%) were non-tobacco smokers. In regarding of GTS symptoms, most of the participants reported dizziness (41.93%), headache (38.71%), weakness (38.71%), abdominal pain (35.48%), vomiting (22.58%), nausea (32.26%), drooling (12.9%) and diarrhoea (9.68%) after the harvest phase, which reinforces the evidence of green tobacco sickness occurrence. Those results were in accordance with

studies conducted in Rio Grande do Sul at 2014 and 2016. It is important to emphasize that symptoms related to GTS and pesticide poisoning are similar, as well the tobacco workers are exposed to both, nonetheless tracked symptoms were reported only after harvest phase. In addition, 77.42% of the questionnaire respondents reported using personal protective equipment (PPE), however, participants did not specify whether the PPE used were regular cotton clothing or the full standard garment consisting of waterproof pants or long sleeve shirt and gloves. Some home measures are adopted by 41.94% of tobacco farmers in an attempt to relieve these symptoms, such as the use of teas and cola drinks. The descriptive results of the WHOQOL-bref by domains, facets and total quality of life, revealed that tobacco growers presented good averages in all domains: physical (68.45%), psychological (66.85%), social relations (73.12%) and environmental (59.30%). Environmental domain showed the lowest satisfaction index, highlighting greater interference mainly from the facets “Health care”, “Recreation and leisure” and “Financial resources”. The “Health Care” demonstrate a strong influence of the performed activity: the production of tobacco on the health of the producer, which is directly affected by DFVT, also to pesticide exposure and the physical effort required. Similar was observed in other studies regarding countryside populations, especially exhaustive workload and pesticide poisoning. The present study demonstrated evidence of GTS among tobacco farmers in south Brazil. The tobacco workers activity directly interferes with their life quality. Many are unaware that the symptoms experienced after harvesting are GTS or nicotine intoxication, so the need for access to information is emphasized, as well as the importance of using PPE correctly.

Green tobacco sickness occurrence and evaluation of life quality among tobacco

workers in Paraná countryside

BorToli, STella; SchaMne, TaTiane; kalv, Danielle crySTiane; peDroSo, Bruno; velloSa, joSe carloS reBuglio

Universidade Estadual de Ponta Grossa - UEPG.

303

Introduction: In Brazil, health professionals face difficulties in diagnosing, registering and referring patients with pesticide poisoning1. Across the national territory, countless varieties of products from agriculture are marketed. The southern region is considered one of the country’s agricultural granaries - in Santa Catarina (SC), mainly in the micro-region of Corupá, agriculture is family-owned - and the number of cases of poisoning in the region is proportionally high compared to the rest of the country2. In 2017, 1.196 cases of poisoning and 25 deaths were reported in the southern region. This points to the importance of protection through personal protective equipment (PPE)3,4. Objectives: Determine the main pesticides used in banana cultivation in Corupá - SC and raise awareness among banana growers about the importance of taking care with the use of pesticides and the proper use of PPE. Methods: This study is a literature review with subsequent extension activity through the lecture “Poisoning by Pesticides in Banana Farming” at the event “Prevention and Care in the Use of Agricultural Chemicals and Water Quality, promoted by the Association of Banana Growers of Corupá (ASBANCO). Results and Discussion: According to acquired information from ASBANCO at the event, the compounds most used in banana plantations in Corupá are thiazides and strobilurin. Thiazides are herbicides and are used to control weeds and broadleaf. Strobilurin, on the other hand, are fungicides derived from a secondary metabolite produced by the fungus Strobilurus tenacellus and, therefore, they are so called, and are often used with thiazides5. The event highlighted the importance of extending scientific knowledge to banana communities, demonstrating the need for the correct use of PPE: water repellent cotton jumpsuit with long sleeves, waterproof wide-brimmed hat, waterproof apron, face shields, disposable mask, covering the nose and mouth, gloves and rubber boots. In addition, according to VSPEA-SC data from 2020, 251 cases of poisoning were reported in Santa Catarina, however only one of them happened in Corupá6. In 2021, also according to VSPEA-SC data, there weren’t any

notifications of poisoning in Corupá7. Considering that Corupá is a large banana producer, the low amount of poisoning events in this city could be justified by the use of natural based pesticides - such as strobilurin - and the correct use of PPE by the banana growers. Conclusion: There is a large amount of annual poisoning in Santa Catarina and it is necessary to intensify prevention and control actions. However, specifically in Corupá, the pesticides used are of light classification, which is a positive point for the region and contributes to minimize poisoning cases. Even so there are risks in their use, if the necessary precautions are not taken. Keywords: Pesticides; Poisoning; Banana growers; Personal protective equipment.

REFERENCES1. LONDRES, F. Agrotóxicos no Brasil: um guia para ação em defesa da vida. Rio de Janeiro: AS-PTA - Assessoria e Serviços a Projetos em Agricultura Alternativa, 2011.2. Instituto Brasileiro de Geografia e Estatística. Área plantada, área colhida, quantidade produzida, rendimento médio e valor da produção dos produtos das lavouras temporárias, segundo a Unidade da Federação, suas Mesorregiões, Microrregiões e Municípios. Acesso em: 10 out. 2021. Disponível em: https://www.ibge.gov.br/estatisticas/economicas/agricultura-e-pecuaria/9117-producao-agricola-municipal-culturas-temporarias-e-permanentes.html?=&t=resultados3. Ministério da Saúde: Sistema Nacional de Informações Tóxico-Farmacológicas. Casos Registrados de Intoxicação Humana por Agente Tóxico e Centro. Região Sul, 2017. Acesso em: 10 out. 2021. Disponível em: https://sinitox.icict.fiocruz.br/4. Ministério da Saúde: Sistema Nacional de Informações Tóxico-Farmacológicas. Óbitos Registrados de Intoxicação Humana por Agente Tóxico e Centro. Região Sul, 2017. Acesso em: 10 out. 2021. Disponível em: https://sinitox.icict.fiocruz.br/5. RODRIGUES, MAT. Classificação de fungicidas de acordo com o mecanismo de ação proposto pelo FRAC. 2006. 249 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, 2006. Disponível em: http://hdl.handle.net/11449/972246. Governo de Santa Catarina: VSPEA-SC. Informativo VSPEA-SC Setembro/2020. Acesso em: 15 fev. 2022. Disponível em: http://www.vigilanciasanitaria.sc.gov.br/index.php/comunicacao/noticias/149-noticias/noticias-2020/1212-2-informativo-vspea-2020.7. Governo de Santa Catarina: VSPEA-SC. Informativo VSPEA-SC Dezembro/2021. Acesso em: 15 fev. 2022. Disponível em: http://www.vigilanciasanitaria.sc.gov.br/index.php/150-noticias/noticias-2021/1313-1-informativo-vspea-sc-2021.

Pesticide poisoning in banana cultivation in Corupá city, Santa Catarina

BorgMann, gaBriela1; plauTz, kaTherine1; ziMMaTh, Michel2, TenFen, aDrielli2

1 UNIVILLE, Programa de Pós-Graduação em Saúde e Meio Ambiente; 2 UNISOCIESC Jaraguá do Sul.

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Introduction: Ultraviolet (UV) light can inactivate coronaviruses, but the practicality of UV light as an engineering control in public spaces is limited by the hazardous nature of conventional UV lamps, which are mercury-based and emit a peak wavelength (254 nm) penetrating human skin and has carcinogenic potential. During the SARS-CoV-2 pandemic, the far-UVC light has emerged as a safe tool to sanitize populated environments, once it is preconized to be absorbed by the stratum corneum without reaching the genetic material of living cells. Recent advances in the development and production of KrCl excimer lamps hold promise in this regard, as this lamp emits a filtered shorter peak wavelength (222 nm). The studies performed so far have evaluated the capacity of the 222 nm light to produce cyclobutane pyrimidine dimers and 6-4 photoproducts on the DNA and inflammatory responses in the skin and eyes. Objective: None of the previous studies has evaluated molecular alterations such as ROS formation, alterations in the protein levels and photo-aging. Our goal is to study these phenomena on in vitro full-thickness reconstructed human skin (RHS). Method: We developed RHS using human primary keratinocytes and fibroblasts embedded in a collagen type I matrix. We added 50 µM DCF-DA on top of the RHS to assess ROS formation. Afterwards, we exposed the RHS to 0, 6, 25, 500 and 1500 mJ/cm2 of 222 nm light and collected immediately the fluorescence emission using EnSpire 2300 equipment (Perkin Elmer). RHS were snap-frozen and the cryo-slices analyzed for the formation of pyrimidine dimers using immunofluorescence, immunohistochemistry and an in-house developed Fiji script software. The RHS was

also irradiated with 500 or 1500 mJ/cm2 of 222 nm and collected after 0 h, 24 h or 48 h of exposure. The proteins of epidermis were extracted and digested for MS evaluation. The MS data were analyzed in MaxQuant and Perseus and the list of quantified proteins were submitted to pathway enrichment. In all experiments a traditional germicidal UVC light at 254nm was used as a control. Results: The RHS cultivated over eight days, as expected, developed a fully differentiated epidermis on top of a dermis, as evidenced by the presence of cytokeratin 14 in the basal layer and cytokeratin 10 in the upper layers. As already reported, even at the higher tested doses, 222 nm produced a less intense and more superficial pyrimidine dimers staining than 254 nm, which were more promptly removed form the skin following the regular process of skin desquamation. However, UV at 222 nm induced ROS formation in a dose-dependent manner, with the signal being detected with a dose as low as 25 mJ/cm2, the preconized occupational threshold limit value (TLV) of exposure to 222nm in 8 hours. Moreover, the preliminary data showed differential proteins associated with important molecular functions and biological processes of the epidermis. Conclusion: Our study showed for the first time that skin exposure to filtered KrCl excimer lamp, even within the regulatory TLV, can increase ROS production, alterations on cellular functions and photo-aging processes. Taken together, our results raised important read-flags for the use of this lamp, even within the TLV. Acknowledgments: We would like to thank Instituto Tecnológico da Vale, Embrapii and CNPEM for the project funding.

Skin exposure to KrCl excimer lamp emitting at 222 nm cause tissue and

cellular alterations of concern

TavareS, renaTa Spagolla napoleão; giraSSole, aleSSanDra; aDaMoSki, DouglaS; caznok, ana clara; DoMingueS, roMênia; leMe, aDriana paeS; carvalho, Murilo; DiaS, SanDra MarTha goMeS

Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Sao Paulo 13083-970, Brazil.

305

Introduction: Cyanide present in Manihot succulent crantz is a highly toxic and hypoxemiant substance, as it has properties capable of inhibiting the cytochrome oxidase enzyme and, therefore, the chemical state of iron. In this way, preventing the reception of oxygen in the red cells of exposed individuals, increasing the production of methemoglobin, for example those who work in the production of cassava flour. Objective: To evaluate the influence of occupational exposure to cyanide on methemoglobin levels in cassava flour producers. Material and Methods: Observational, cross-sectional, analytical study developed in a community located in the northeast region of the state of Pará in the year 2018 to 2019. Participants were 40 producers of both sexes between 13 and 55 years old with recent exposure and 20 residents of the same community non-producers. Both groups had lived in the communities for more than a year and had not had contact with other methemoglobinizing substances, such as tobacco, use of illicit drugs, contact with mercury, without infectious diseases, who had not practiced recent physical exertion and had in the production of flour its main source of income. By UV spectroscopy, the concentration of free cyanide and the percentage of methemoglobin were analyzed. Hematocrit and hemoglobin were also measured, however using a complete blood count device. The tests were analyzed at the Toxicology Laboratory of the NMT/UFPA and at the University center FIBRA. The protocol of this study was prepared in accordance with the rules of resolution 466/2012 and approved by the Research Ethics Committee of the Nucleus of Tropical Medicine in accordance with

ethical opinion number 2892820 of September 13, 2018. Results: The mean cyanide in blood samples from producers with recent contact was 3.45 ± 0.8 mg/HCN, minimum of 0.7 and maximum of 5.2. In those who declared non-producers, the average of cyanide was 1.3 ± 0.12, minimum of 0.5 and maximum of 2.5. A significant difference was observed according to the Z test for two independent samples. In the measurement of methemoglobin, the value obtained was 4.5 ± 0.8 among producers and 1.2 ± 0.9 among non-producers. A moderate positive correlation was observed between free cyanide concentrations and Methemoglobinemia r=0.45 p<0.05. In the hematocrit measurement, the mean value was 43.6 ± 4.5 and Hemoglobin was 13.8 ± 1.2. However, it was not observed between hematocrit and hemoglobin levels. Conclusion: Cyanide concentrations between groups were statistically different, indicating the influence of occupation on exposure to the chemical agent. The methemoglobin values show a probable participation of cyanide in the oxidation of iron present in the red blood cells of rural producers, although no influence was observed on the hematocrit and hemoglobin values. Therefore, this population needs to be monitored more, when considering the toxicity of the chemical compound and the risks it offers to the health of exposed individuals. In addition, we also admit that it is important to develop preventive measures regarding this form of occupational exposure. Graded: I thank my advisors and everyone who participated in the research and for the willingness in the process of obtaining data.

The influence of occupational exposure to cyanide on metahemoglobinemia

in cassava flour producers

eSTuMano, Saulo Braga1; oliveira, cláuDia SiMone BalTazar1,2; araújo, Maria eDuarDa liMa1; coSTa, eliene DoS SanToS Da Silva1; chiSTé, renan caMpoS2; laMeira, chriSTian

neri1; aMaro, BeaTriz oliveira3; pinheiro, Maria Da conceição naSciMenTo2

1 Centro Universitário FIBRA - FIBRA; 2 Universidade Federal do Pará - UFPA; 3 Instituto Evandro Chagas - IEC.

306

Background: This cross-sectional study was conducted at the Brazilian open dump named as Structural dump, also known as Jóquei dumpsite, which was considered the second largest active dump in the world until its closure in 2018. According to SLU, until 2018, most of the waste pickers in the Federal District were self-employed professionals working at the open dump. After the open dump closure, they were requested to organize themselves into associations (coops) to be reallocated to the new sorting plants. This transfer brought quality gains to work in terms of safety, however the impact on health related to the less ventilated environment at the sorting plants could contribute to a greater chemical exposure and should be monitored. Objective: the aim of this study was to determine the metal concentration in the hair of waste pickers who worked sorting mixed waste including e-waste, in Brasilia, Brazil providing metal background values to support further public policies. Methods: There were 2112 waste pickers registered at the SLU (urban cleaning service) in 2017. A total of 359 waste pickers hair samples were collected. 39 samples were excluded that had been dyed or straightened less than 3 months before collection. All the 320 remaining samples were washed to remove external contamination. Samples and Certified Reference Material ERM-DB001, underwent digestion using an ETHOS EASY and analyzed by ICP-MS. Regarding the statistical analysis, participants were grouped prior bivariate comparisons according to their workplace (sorting plants or open dump), gender, and time as waste picker using Chi-square tests.The comparisons between age and gender were analyzed using the non-parametric Mann-Whitney test. Results: The results showed that the arsenic, cadmium, barium, copper, lead, tin, especially cobalt, manganese and molybdenum were found in significantly higher concentrations than previously reported. Results: Most participants are women and

have less As, Be, Cd, Co, Mn, Sn, Pb, Ba, Mo, Zn, Fe, and Cu in their hair compared to men. Regarding working conditions, it was not yet possible to verify significant differences, since the migration of part of the waste pickers from dumpsite to the sorting plants was still in transition in 2017. Discussion/Conclusions: Waste pickers are a vulnerable population to be exposed to toxic metals. According to our study, most of them are women working in this job for many years and have less metal concentration in their hair compared to men. In relation to working conditions, it was not yet possible to verify significative differences, since the migration of part of the waste pickers from dumpsite to the sorting plants was still in transition. Many waste pickers from the sorting centers still were performing their occupation in the dumpsite as extra-income in 2018. This work brings initial data (background values) for monitoring the population of waste pickers in the Federal District and supports future studies already in progress. It is expected that another sample collection carried out between 3 to 5 years after the closure of the dump, the difference between the work environments can probably be noticed in the metal concentrations, mainly regarding to released particles which are better dispersed outdoors, resulting in lower concentrations in the workers’ breathing zones and the group 1 workers had cooperatives that operate in indoor facilities (sorting plants) without proper ventilation sorting electronic materials containing Cd, Cu, and Pb. It is very important to continue metal monitoring, as they can have toxic effects after many years of exposure and metal long-term poisoning might have an insidious beginning, difficult to diagnose, which might lead to irreversible sequelae being costly for public healt. Acknowledgments: This research received no external funding; however, all samples were analyzed at no cost by the non-profit Belgium Center for Occupational Hygiene (BeCOH).

Toxic metal backgrounds among waste pickers in hair sampling: a cross-sectional

study in Brazil (Federal District)

gonçalveS, Michelly roDrigueS1; verpaele, STeven2; MarqueS, carla pinTaS1; BaShaSh, MorTeza3,4; cruvinel, vaneSSa reSenDe nogueira5; SanToS, vivian Da Silva6

1 MSc, University of Brasília, Faculty of Ceilândia, Brasília (DF), Public Health, Brasilia, Brazil. 2 MSc, Belgian Center for Occupational Hygiene asbl, Zwijnaarde, Belgium. 3 University of Southern California, Department of Preventive Medicine at the Keck School of Medicine,

Los Angeles, CA, USA. 4 PhD, Ryerson University, School of Occupational and Public Health, Toronto, Canada. 5 PhD University of Brasília, Faculty of Ceilândia, Brasília (DF), Public

Health Department at Ceilândia Faculty, Brazil. 6 PhD, University of Brasília, Faculty of Ceilândia, Brasília (DF), Pharmacy Department at Ceilândia Faculty, Brazil.

14 OTHER

308

Introduction: Suicide is a behavior of self-injury with the intention of ending one’s self-life. It is a complex, multifaceted phenomenon which includes ideation, attempt, and consummation of the act. It is recognized as a serious public health concern worldwide and it is related to several personal and social factors. According to some studies, there was an increased risk of suicide attempt during the Covid-19 pandemic in Brazil. The use of substances is one of the well-established methods in suicide attempts and leads to exogenous intoxications. Research on the toxic agents used motivated by suicide can contribute to policies and strategies for case prevention and health promotion. Objective: To delineate the toxic agents and analyze the temporal trend of cases of intoxication by suicide attempt (IBSA) in the period 2015 to 2019 and compare with the year 2020 regarding the pandemic by covid-19 in Brazil. Method and Material: Descriptive study and time series of the cases notified in the Injury Notification System (Sinan) of the confirmed exogenous intoxications by suicide attempt, referring to the years 2015 to 2021 by tabnet/DATA/SUS. The following parameters were applied: notifications by year of 1st symptom(s), all types of toxic agents and evolution (except loss to follow-up), single and repeated acute exposures. The following softwares were used for data tabulation, statistical calculations: Pearson chi2 tests, prevalence ratio (PR), 95% confidence interval (95%CI) and verification of the temporal series: Microsoft Excel 2019®, Stata® 2016 and Joinpoint Regression Program (version 4.9.0.1). The analysis was performed according to stratification by sex: male (M), female (F) and toxic agent. The crude rate of intoxication by suicide attempt (CR-IBSA) per 100,000 inhabitants was calculated by the following equation: (number of notifications)/(estimated brazilian population-year)x100,000. Population data were sourced from

the Brazilian Institute of Geography and Statistics. Results: During the period between 2015 and 2020, 206,118 cases of IBSA were reported, the highest number of intoxications were in 2019 (25.0%) and 72.8% (N=205,816) belonging to the female gender.Regarding the annual temporal analysis there was growth in CR-IBSA until the year 2019, annual percent change (APC) = +26.4 (p<0.05) and then decrease, not significant, in 2020 (APC = - 43.9; p=0.054). Regarding the monthly average, in the period from 2015 to 2019 there was non-significant growth of CR-IBSA in the second half of the year, monthy percent change (MPC) = +4.5 (p=0.074) starting in June, in 2020 the highest coefficients were in the first half with a decrease until May: MPC = -15.5 (p<0.05). Medications were the most used substances by both genders, being from 2015 to 2019: 85.5% (F); 66.0% (M), PR=1.29* (95% CI: 1.28-1.30), in 2020: 88.3%(F); 71.5%(M), PR= 1.23*(1.21-1.25). In comparing the period from 2015 to 2019 with the year 2020, it was observed that among women there was an increase in drug of abuse poisonings RP=1.93* (1.58-2.36) and decrease in agricultural pesticide use (2.4%; 1.4%, p<0.01). In addition, among men, we observed an increase in drug use: (66.0%; 71.5%, p<0.01), lower use of agricultural pesticides (8.91%; 7.01%, p<0.01) and rodenticides (12.5%; 8.84%, p<0.01) in the corresponding periods studied. Conclusions: There was an expressive increase in the number of IBSA cases in the last five years before the Covid-19 pandemic (2015 to 2019) in Brazil and decrease in the pandemic year (2020) and with greater implications for women. Drug intoxications were predominant in suicide attempts in both genders. A change in the profile of the types of substances involved in suicide attempts in the pandemic period was evidenced. We emphasize the importance of knowing the toxic agents used in the attempts in order to implement specific preventive measures for vulnerable groups.

Attempted suicide: temporal series and agents involved before and during

the COVID-19 pandemic in Brazil

holanDa júnior, WanDerley pinheiro; MagalhãeS, Danielle De paula; oliveira, juliana riBeiro iBiapina leiTão

Forensic Expertise of the state of Ceará (PEFOCE), Brazil.

309

Introduction: Although several non-animal methods have been accepted in Brazil since 2014, due to lack of clarity animals are still routinely used for the safety assessment of school supplies such as paints and glues. According to Brazilian regulatory requirements, all school supplies with more than 3 grams of paints, glues, gouaches, watercolors, and powered material per unit use must have their safety confirmed regarding acute oral toxicity. Those susceptible to skin contact must also have their safety confirmed for skin irritation. Acute oral toxicity and skin irritation could be assessed by in vivo or in vitro methods, which means the laboratories were free to choose between testing on living animals or using alternative methods (cell cultures). In addition, in Brazil safety testing has traditionally been performed on finished products, which is expensive, time consuming and has significant limitations. Objective: To change the Brazilian regulatory requirements for school supplies to allow the toxicological safety assessment of the products based on existing information on its ingredients, prioritizing animal-free approaches and assuring the use of animals only as a last resort. Methods: A series of meetings was held with national stakeholders including the Brazilian Association of Technical Standards (ABNT), school supplies industry representatives and laboratories that perform safety assessment of school supplies to discuss the proposal

made by the Humane Society International (HSI) to update the ABNT norm on school supplies (ABNT Norm 15236). Results: The HSI proposal for an approach that prioritizes animal-free safety assessment was discussed by the national stakeholders with the support of the National Council for the Control of Animal Experimentation (CONCEA). The ABNT committee on school supplies have accepted HSI proposal and published the updated norm (ABNT NBR 15236:2021) in September 2021. This norm is referred to in the Inmetro ordinance number 423, published in October 2021, which makes ABNT 15236:2021 the mandatory requirement for school supplies compliance in Brazil. Discussion: Although in vivo methods are still allowed, the ABNT NBR 15236:2021 now prioritizes the safety assessment of school supplies based on existing information on its ingredients for both acute oral toxicity and skin irritation. Another important change is the requirement that animals are used only as a last resort and only when technical justifications are present. Conclusion: The changes made in the Brazilian regulatory requirements for the safety assessment of school supplies now prioritizes animal-free approaches and the use of animals only as a last resort. This is in accordance with the 3Rs Principle, closer to regulatory harmonization with other countries, and enables avoiding the unjustifiable use of many animals.

Brazil now prioritizes animal-free safety assessment of school supplies

Marigliani, Bianca

Research & Toxicology Department, Humane Society International, Brazil.

310

Background/Introduction: Early exposure to environmental pollutants like toxic metals has been identified as neurotoxic and interaction with social determinants may be a mechanism of that toxicity. The possibility of that interaction makes it necessary to innovate in the way of approaching and studying their effect on human population. Bronfenbrenner’s Bioecological Theory (BBT) is a framework that considers human development as a result of a joint effect of its biological characteristics, immediate setting, and the other settings of society. Objective: To develop a conceptual framework that allows encompassing the social and biological characteristics of children from the gestational period to childhood, considering exposure to toxic metals. Methods: a literature review was conducted and the BBT besides other concepts such as the social and structural determinants of health, allostatic load, embodiment, genetic and epigenetic concept were used to design our framework. Results: Our model presents how mechanisms based on structural determinants (like skin color or ethnicity, socio-historical context, power, and privilege) from the distal level of society

define the social status of a mother (and her family). In the immediate setting, life circumstances linked to social context define the exposure to (chronic) stress and toxic metals, affect parents’ mental health and their relation with the child. We argue that exposure to heavy toxic metals and stress due to social status may act together at a biological level to influence the neurodevelopment of the child depending on the quality of interaction between the growing child and its immediate setting. Discussion/Conclusion: Assessment of true neurotoxicity of pollutants cannot be done separately from the ecological environment in which they act. Although this model is based on a specific contaminant, it opens new horizons on how biological sciences, such as neurotoxicology, can articulate with the theoretical models from human sciences to have a broader approach to study human development. Acknowledgments: We are thankful to the National Council for Scientific and Technological Development (CNPq), the Bahia State Research Support Foundation (FAPESB) and Coordination of Superior Level Staff Improvement (CAPES).

Early exposure to toxic metals and child neurodevelopment: proposal

of a theoretical model based on Bronfenbrenner’s bioecological theory

Bah, hoMègnon anTonin Ferréol1,2; SanToS, naThália riBeiro2,3; coSTa, DaiSy oliveira2,3; carvalho, chriSSie Ferreira4; MarTínez, vicTor oTero; goMeS-júnior, erival aMoriM; MenezeS-Filho, joSé anTonio1,2,3

1 Institute of Collective Health, Federal University of Bahia, Brazil; 2 Laboratory of Toxicology, College of Pharmacy, Federal University of Bahia, Brazil; 3 Graduate Program

in Pharmacy, College of Pharmacy, Federal University of Bahia, Brazil; 4 Department of Psychology, Federal University of Santa Catarina, Santa Catarina, Brazil.

311

Toxicology is a science that studies the effects of chemical substances on organism in determinate exposure conditions and require of knowledge from several areas. This complexity demonstrates the level of demand for the student during the contact with toxicology. Alternative teaching proposal are a good tool for fixation and improvement of knowledge, one example is comic books. This didactic capacity of comic books has successfully applied in our society, from entertainment to state policy campaigns. We developed a fictional history of José da Silva, the Junkie, who lives in an urban center, having drug dependence. When José da Silva uses drugs consciously, objectively, predicting their future biological effects, these effects are improved in high levels. However, the protagonist still has side effects like any normal person. In your universe, we will approach the toxicology and adjacent themes like abuse drugs, pesticides, industrial solvents, metals, new psychoactive substances, medicinal plants, sociology, law, risk and toxicity evaluation, etc., important to science and for the society. In this sense, pharmacy students employed their toxicological knowledge to develop a story on comic book based in anti-hero journey, which it will be share with other health area students. The scripts for the comics will be written by NAT members as well pharmacy students

during clinical toxicology and forensic toxicology disciplines. For NAT members, the tales will be chosen by project coordinator while other students were free to choose the substances that will be used on story, explain our action mechanism, effects, and consequences. Posteriorly, the material will be sent for industrial drawing department for illustration and opinions about narrative. It is estimated 2 comics with 2 pages for each group, which 10 pharmacy students will be involved. In the first comic book we present José, the place where he lives, his wife and some friends. The main drugs involved in this story are cocaine and crack, ranging from abstinence to use. The mechanism of these drugs is explained, also abstinence, and illustrated to show us the effects on the body and brain. All events bring a context to build the story of José and his power when using drugs, giving many possibilities to explain and explore other drugs. José’s past is unknown, and time will tell the truth and how he received these powers. Ten forensic toxicology students participated in the development of this first story. We want a popularization of toxicology content and other pertinent subjects in this science, which can help the learning process for undergraduate students approaching the students involved on comic production and other students, including high school, via digital divulgation.

Junkie – Teaching project in comic book for the development of learning in toxicology

BairroS, anDré valle1; reginaTo, FernanDa ziegler2; ugalDe, guSTavo anDraDe2; BerlaTo, Dener goMeS2; pacheco, anDré lucaS Bezerra2; naSciMenTo, Marcelo henrique SanTana2;

oliveira, leTícia ciMó2; SanToS, lara celeSTina2; chiMenDeS, nayoMi De anDraDe2; roSa, vicTória goMeS2; DalMazzo, anDré kuSSer3; Maia, aFFonSo enriqueS MonTagner4

1 Nucleus Applied to Toxicology (NAT) Federal University of Santa Maria (UFSM); 2 NAT - UFSM; 3 Industrial Drawing Department (IDD) - UFSM; 4 IDD - UFSM.

312

Introduction: Paracetamol, also known as acetaminophen, is present in a number of over-the-counter (OTC) and prescription drugs for its antipyretic and analgesic properties. The effects of acetaminophen, especially in its analgesic properties, are primarily due to activation of the decreased serotonergic pathway. The main site of action of paracetamol may still be the inhibition of prostaglandins, a multifunctional substance derived from the synthesis of polyunsaturated fatty acids. Enzymatic reactions of molecular mechanisms may reduce the formation of glutathione, a coenzyme for enzymes such as prostaglandin synthase, which inhibits prostaglandin synthesis. Many health conditions can affect male fertility, underscoring the need for a thorough evaluation of the patient to identify treatable or reversible lifestyle factors or medical conditions. The causes of male infertility vary widely, but may be related to congenital, acquired, or idiopathic factors that impair spermatogenesis. Widespread use of acetaminophen at high doses has been found to cause various forms of toxicity, such as reproductive toxicity. The effects of paracetamol on semen quality and sperm parameters have been found in many published studies. Studies demonstrated increased percentage of abnormal sperm head, reduced sperm count, impaired motility and reduced anogenital distance and decreased testosterone production. Objective: To evaluate the literature studies of the use of Paracetamol and its relation to male infertility. Methodology: A bibliographic

search was carried out using electronic databases (Scielo, Pubmed, and Lilacs) for articles, published in English, Portuguese and Spanish, between 2000 and 2022. The descriptors used were infertility, male infertility, Paracetamol, Acetaminophen. Results and Discussion: The electronic searches identified 20 studies, of which 3 were selected that met the criteria for inclusion. A study done by Ratnasooriya et al., in 2000, showed that Long-term high-dose administration of paracetamol impairs reproductive performance in male rats. They propose that this effect is reversible, not due to general toxicity, but due to increased oligospermia, insufficient motility of normal and hyperactive sperm, and decreased sperm fertilization capacity. Another study done by Yano and Dolder, in 2002, found that Seminiferous tubules were altered and some degeneration, Sertoli cell fragmentation, and sperm cell morphology were altered in Wistar rats treated with a single dose of acetaminophen (4.4 mmol/kg). Banihani in his study concluded that the effects of acetaminophen on semen quality may occur by increasing reactive oxygen species production, inducing spermatocyte apoptosis, reducing nitric oxide production, inhibiting prostaglandin synthesis, and inhibit testosterone synthesis. Conclusion: We can conclude that the long term use of high doses of Paracetamol by men appears to modify testosterone levels, a decrease in the sperm parameters, specially sperm morphology, and consequently semen quality.

The use of paracetamol as cause for male infertility

Silva, luíza MaDureira1; holanDa, Wiron piMenTel2; capelo, MeliSSa FigueireDo2; honório júnior, joSé eDuarDo riBeiro3

1 Academic of Nursing in the University Center Christus, Laboratory of Neurociência Translacional-Neurocit; 2 Academic of Biomedicina in the University Center Christus, Laboratory of Neurociência

Translacional-Neurocit; 3 Doctor in Biotechnology for the RENORBIO/UFC. Professor in the University Center Christus, Coordinator of the Laboratory of Neurociência Translacional-Neurocit.

313

INDEX OF AUTHORS

AAbe, Flávia R. – 256Adamoski, Douglas – 304Aguera, Raul Gomes – 109Aguiar, Gilberto Santos – 131Aguiar, Larissa Muratori – 298Aguilera, Mariana – 62Alberton, Michele Debiasi – 195Albino, Danielle Bibas Legat – 159Albuquerque, Anjaina Fernandes – 206Albuquerque, Polianna Lemos Moura Moreira – 114, 115Alcântara, K.C. – 80Alencar, Severino Matias – 229Alexandre, Angela de Oliveira – 273Alfieri, Daniela Frizon – 133, 137Allen, David – 62Almeida, Eduardo Alves – 176Almeida, Elaine Renata Motta – 208Almeida, Fernando G. – 158Almeida, Gabriela de Oliveira – 223Almeida, Giulia Forni – 228Almeida, Jéssica Azarias – 42Almeida, Mariana – 230Almeida, Stefano Zorzal – 174Almeida, Vivian Romero Santiago – 241Almeida, William – 144Alves, Carolina Esmeraldo Lima – 68Alves, Gessé de Souza – 109Alves, Gutemberg – 64Alves, Jonas Alher Meira – 121, 122, 133, 137Alves, Paula Daniela Sabino de Freitas – 219Alves, Pedro Adolfo Pereira – 123Alves, Rômulo Couto – 176Amaral, Francieli Ubirajara India – 181Amaro, Beatriz Oliveira – 305

Andrade, Ana Rosa Brissant – 37, 56Andrade, Ítalo Bertoni Lopes – 41, 165Andrade, Ítalo B.L. – 178, 211Andrade, Wanessa Machado – 42André, Leiliane Coelho – 113, 125André-Miral, Corrine – 48Angelo, Ana Beatriz Silva – 188, 196Antonio, Ananda da S. – 158Antunes, Bibiana Pereira – 197Antunes, Débora R. – 275Antunes, Lusania Maria Greggi – 192Antunes, Marina Venzon – 84Antunes, Marina Vezon – 77, 102, 123, 128, 234, 301Appelt, Patrícia – 277Aquino, Ariana Musa – 201Arakawa, Nilton Syogo – 269Araki, Koiti – 271Arantes, Ana Carolina Furiozo – 101Araujo, Bruno Vinicios Silva – 184Araujo, Karen Rafaela Gonçalves – 249Araújo, Maria Eduarda Lima – 222, 305Araujo-Souza, Patrícia Savio – 286Arbo, Marcelo D. – 266Arbo, Marcelo Dutra – 112, 134, 197, 200, 300Arena, Arielle Cristina – 198, 204, 207, 210, 216Arroteia, Kelen Fabiola – 49Arruda, Fernanda Wolff da Silva – 129, 130Aschner, Michael – 189Assis, Silvia Romano – 32Augusto, Pedro Esteves Duarte – 229Avila, Carolina Martins – 254, 289Ávila, Ricardo Andrez Machado – 147Azevedo, Camilla de Marchi Sanches – 205, 212, 213

BBaccule, Nicole Santos – 151Bagatin, Julia de Toledo – 27, 32Bagio, Jéssica – 152

Bah, Homegnon Antonin Ferréol – 294Bah, Homègnon Antonin Ferréol – 310Bairros, André V. – 126, 237

Bairros, André Valle – 47, 73, 74, 75, 116, 119, 135, 236, 311Baisch, Ana Luíza Muccillo – 175Bando, Érika – 109Barbim, Paula Donate – 262Barbisan, Luis Fernando – 201Barbosa, Alyne Maria da Costa – 285Barbosa, Fábio de Souza – 250Barbosa, Ingrid Lopes – 83Barbosa Jr, Fernando – 85, 171, 192Barbosa Junior, Fernando – 90Barbosa, Maria Clara de Oliveira – 184Barcellos, Leonardo José Gil – 168, 181Barotto, Adriana Mello – 159Barroso, Gilberto Fonseca – 173, 174Barros, Silvia Berlanga de Moraes – 32Bashash, Morteza – 306Bastiani, Marcos Frank – 81, 146, 167, 251Batista, José Márcio Machado – 114, 115Battastini, Ana Maria Oliveira – 185Bauermann, Lauren – 138Bauermann, Liliane de Freitas – 194Baú, Morgana – 176Bechtold, Bruna Assunção – 40, 219Bedor, Danilo César Galindo – 56Belo, Vinícius Silva – 248Benvenutti, Danyela Francine – 103Benvenutti, Larissa – 38, 57, 59, 267Benvenutti, Régis Carlos – 299Berlato, Dener G. – 126, 237Berlato, Dener Gomes – 73, 74, 116, 119, 236, 311Berlinck, Débora Zorrón – 89, 104Berna, Gabriel da Costa – 293Berta, João Antonio – 199, 215Berti, Fernanda Vieira – 70Bertoldi, Fernando – 293Betti, Andresa Heemann – 293Bhering, Cecília de A. – 158Biazus, Inara Carbonera – 181

Bigão, Vitor Luiz Caleffo Piva – 94, 106Bigão, Vítor Luiz Caleffo Piva – 142, 226, 244Biondi, Ilka Borges – 68Birk, Letícia – 95, 239, 250Boff, Bruna de Souza – 242Boff, Everton – 299Bombazar, Amanda – 147Bondan, Amanda Pacheco – 81Bonfioli, Maysa Guilherme – 248Borba, Maria Eduarda – 199, 215Bordoni, Leonardo S. – 233, 247Borges, Amanda Cecília Guimarães – 53Borges, Cibele dos Santos – 180, 184, 187, 188, 193, 196Borges, Gabriela Ramos – 78, 112, 134, 143Borges, Pedro H.S. – 245Borgmann, Gabriela – 179, 272, 303Bortoli, Stella – 302Bosquetti, Bruna – 58Bouez, Charbel – 46Braga, Ádrya Lariela Lima – 218Braga, Douglas Evangelista – 291Braga, Jacqueline Ramos Machado – 68Braz, Juliana Machado – 298Bresolin, Nilzete Liberato – 117Bresolin, Tania Mari Bellé – 103Brito, Maria Carolina Santos Ribeiro – 238Broering, Milena F. – 271Brohem, Carla Abdo – 58Brugnera, Débora Schmitz – 145Brum, Rodrigo de Lima – 175Brunello, Giovanna Cristina Spagnuolo – 133, 137Bruni, Aline Thais – 61, 67, 153, 154, 160, 161Bruno, Vitor – 87Buchele, Maria Luiza Caneiro – 25Burns, Michael J. – 289Busato, Maria Amelia de Castilhos – 84Buzzi, Fátima C. – 267Buzzi, Fatima de Campos – 103

CCabral, Heloisi – 272Cabral, Yngrid dos Santos – 131Cabrices, Oscar G. – 141, 227Cademartori, Pedro Henrique Gonzalez – 55Caetano-Silva, Maria Elisa – 229Calori-Domingues, Maria Antonia – 229Camarena, Denisse Esther Mallaupoma – 27, 32Camargo João Lauro Viana – 203Camargo, João Lauro Viana – 165Campos, Dyene Nascimento – 264, 265Canavez, Andrezza Di Pietro Micali – 58Capelo, Melissa Figueiredo – 312Capp, Edison – 301Cardoso, Leonardo Correa – 237Cardoso, Leonardo Corrêa – 74, 116, 119, 135Cardoso, Maria Regina Alves – 166Cardoso, Marilia Santoro – 72Carlos Rafael – 267Carlson, Renato Romera – 143Carneiro, Gabriel Reis Alves – 76, 96, 100Carriço, Murilo Ricardo Sigal – 86, 92, 172Carvalho, Angélica Romão – 44Carvalho, Chrissie Ferreira – 310

Carvalho, Deoclécio Lustosa – 37, 56Carvalho Filho, Sérgio de Morais – 51, 63Carvalho, José A.M. – 126Carvalho, Larissa A.C. – 259Carvalho, Larissa Anastacio da Costa – 32, 253, 257, 258Carvalho, Larissa Anastácio da Costa – 255Carvalho, Murilo – 304Cassanego, Gabriela Buzatti – 194Castilhos, Maria Amélia – 81Castilhos, Zuleica Carmen – 290Castro, Jade Simões – 61, 161Castro, Juliana Cristina – 109Catalano, Shadia M.I. – 62, 287Catarino, Carolina Motter – 58Cattani, Shanda – 185Cavalcante, Luiz A.F. – 275Cavichion, Rafael Filipe Battisti – 28, 29Cazarin, Karen – 66Caznok, Ana Clara – 304Ceranto, Daniela de Cassia Faglioni Boleta – 199Cesaro, Humberto Luis – 176Cestari, Marta Margarete – 144, 286Cestonaro, Larissa V. – 266

Cestonaro, Larissa Vivan – 197, 200Chaves, Pamella Eduardha Espindola – 261, 264, 265, 268Chequer, Farah Maria Drumond – 246, 248Chimendes, Nayomi A. – 237Chimendes, Nayomi Andrade – 116, 119Chimendes, Nayomi de Andrade – 73, 74, 135, 236, 311Chinaglia, Kauê de Oliveira – 89, 101Chinaglia, Kauê O. – 240Chisté, Renan Campos – 305Choksi, Neepa – 62Cianci, Julio Cesar – 40Cisilotto, Julia – 132Coelho, Júlia França Figueredo – 248Coelho, Maria Paula Mancini – 40Coelho, Matheus Campelo da Costa – 76Cole, Amanda Carolina – 181Colla, Guilherme – 28, 29Concato, Virginia Marcia – 269Conchon-Costa, Ivete – 269Conte, Fernanda M. – 266Cordeiro, Gabriela Batista Cavalcanti – 129, 130Cordenuzzi, Dorys Angela – 57, 59Corralo, Vanessa da Silva – 145Corrêa, Brunna F. – 247Corrêa, Rogerio – 103Costa, Ana Carolina Conchon – 130, 136Costa, Anna Carolina de Moura – 246Costa, Brenda Natasha Souza – 166Costa, Bruno Ruiz Brandão – 94, 106, 142, 182, 224, 226,

244Costa, C.D.D. – 80

Costa, Daisy Oliveira – 294, 310Costa, Eliene dos Santos da Silva – 222, 305Costa, Eric Augusto Caravaggio – 166Costa, Gabriela Vanini – 158Costa, Jose Luiz – 89, 98, 155, 235, 240Costa, José Luiz – 72, 83, 101, 104, 118, 148, 150, 238, 249Costa, Karina Oliveira – 152, 162Costa, Larissa Gabrielli – 40Costa, Letícia M. – 233Costa, Luiz Filipe – 280, 281, 283, 284Costa, Luiz Guilherme Alonso – 201Costa, Meg Cristina da Castilho – 58Costa, Nayara de Souza – 144, 190Costa, Quezia dos Santos – 133, 137Costa, Rony Anderson Rezende – 235Costa-Valle, Marina Tuerlinckx – 197Costa, Vitoria Cadore – 181Couto, Angélica Garcia – 103Couto, Nilton – 230Couto, Tauer J.G. – 247Creczynski-Pasa,Tânia B. – 132Cruvinel, Vanessa Resende Nogueira – 306Cruz, Juliana Varella – 55, 286Cunha, Fernando – 262Cunha, Kelly F. – 98Cunha, Kelly Francisco – 104, 155Cunha, L.C. – 80, 105Cunha, Ricardo Leal – 155, 232, 238Cunha, Viviane Augusta de Medeiros Garcia – 276Custódio, Flávia Beatriz – 221, 291, 292, 295

DDakic, Vanja – 40, 46Dalanhol, Carolina Silveira – 78, 143Dalbosco, Juliana Santos – 238Dal-Cheri, Beatriz Kopke de Assis – 273Dallegrave, Eliane – 99, 197, 200Dalmagro, Mariana – 199, 215Dalmazzo, André Kusser – 311Daniel, Caroline – 145Dantas, Joao Artur Diogenes – 188, 193, 196Debiasi, Michele Alberton – 38De Carli, Diego M. – 126Delwing-Dal Magro, Débora – 272Delwing-De Lima, Daniela – 272De Martinis, Bruno Spinosa – 82, 94, 106, 108, 142, 182, 224,

226, 244

Demets, Mariana Barbieri Alvarez – 88Denardin, Elton Luis Gasparotto – 86, 92, 172, 225Denkena, Isadora Locilento – 155Deolindo, Carolina Turnes Pasini – 97De Vecchi, Rodrigo – 40, 46Devoz, Paula Pícoli – 192Devóz, Paula Pícoli – 171Dias, Sandra Martha Gomes – 304Dias, Wanessa Amorim – 34, 69Diniz, Fabiana Barbosa – 291Domingos, Líllian Maria Borges – 290Domingues, Romênia – 304Donadel, Guilherme – 199, 215Dorta, Daniel Junqueira – 156, 157

EEberlin, Samara – 50Echterhoff, Marcelo Rodrigo Franke – 38Eifler-Lima, Vera Lucia – 185El Haddad, Lohanna Pereira – 94, 224Eller, Sarah – 78, 95, 99, 112, 134, 239, 250

Esteves, Iuri – 282Estumano, Saulo Braga – 222, 305Etcheverry, Bibiana Frasson – 261, 264, 265, 268Everton Boff – 145

FFabichak, Ana – 97Fabris, Andre Luis – 87Fabris, André Luis – 140, 249

Facchini, Gustavo – 50Faria, Patricia Miranda – 66, 287Farias, Beatriz Valentim – 115

Farias, Evelyn Rayani Araújo – 30, 33, 263Farias, Fabiane Moreira – 225Farias, Natália Oliveira – 206Farsky, Sandra – 262Farsky, Sandra H.P. – 271Faustman, Elaine M. – 286Fava, Luis Paulo – 40, 219Favreto, Camila – 81Feitosa, Emanuel Kenedy – 180, 184Felix, Renata Gleysiane de Sousa – 180, 184, 187, 188, 193,

196Feltrim, Fernando – 280, 281, 284Fernandes, Eduarda – 124Fernandes, Fábio Henrique – 214Ferrazza-Heitich, Magda – 179Ferreira, Ana Maria da Costa – 255Ferreira e Silva, Hendyelle Rodrigues – 114, 115Ferreira, Fernanda S. – 266Ferreira, Gabriela Kozuchovski – 272Ferreira, Julia Gabriele de Jesus – 293Ferreira, Maria Augusta Drago – 114, 115Figueira, Ana Carolina Migliorini – 49Figueiró, Fabrício – 185Figueredo, Kássia Caroline – 194Fitarelli, Bruna – 97

Flaws, Jodi A. – 201Flesch, Ingrid – 300Fock, Ricardo – 262Foleis, Vanessa Kaplum – 276Fonseca, Ivana Alice Teixeira – 187, 193Fonseca, Karoline Kuhnen – 130Forini, Mariana M.L. – 275Fortuna, Milena – 168, 181França, Liana Conrado – 138Freddo, Natália – 168, 181Freeman, Elaine – 287Freeman, Harold S. – 206Freire, Josiane Oliveira – 280, 281, 284Freire, Livia Horrana Forte – 188, 196Freitas, Bruno Toledo – 142, 224Freitas, Daniel Roberto Coradi – 298Freitas, Mariana – 81Freitas, Marisa – 124Freitas, Vanessa M. – 27, 259Fugazza, Jonas – 274Furtuoso, Marcella Miranda Siqueira – 36, 39Fusco-Almeida, Ana Marisa – 44Fuzinaga, Thais – 50, 52Fuzinaga, Thaís Y.T. – 256

GGaca, Marianna – 282Gagosian, Viviana Costa – 55Gagosian, Viviana Stephanie Costa – 286Gaitani, Cristiane Masetto – 88Gale, Nathan – 282Galiciolli, Maria Eduarda A. – 209Galiciolli, Maria Eduarda de Andrade – 190Ganzerla, Melissa Dibbern – 49Garcia, Ana Letícia Hilário – 293Garcia, Cristina – 46Garcia, Solange – 200, 300Garcia, Solange C. – 266Garcia, Solange Cristina – 185, 197Gaspar, Lorena R. – 256Gaspar, Lorena Rigo – 50, 52Gasparotto Junior, Arquimedes – 199, 215Gastaldi, Alessandra Betina – 272, 274Gehlen, Günter – 293Gemelli, Bianca Aparecida Martins – 176Geraldino, Barbara Rodrigues – 131Giannini, Maria José Soares Mendes – 44Gianvecchio, Daniele Muñoz – 150Gianvecchio, Victor Alexandre Percinio – 150Gianvecchio, Victor A.P. – 240Gimenes, Izabela – 64Giovagnoli, Stefano – 103Girassole, Alessandra – 304Girotto, Edmarlon – 121, 122, 133, 137Gloria, Maria Beatriz A. – 292Gloria, Maria Beatriz Abreu – 291Gluzezak, Ana J.P. – 256Gluzezak, Ana Júlia Pasuch – 52Goding, Colin – 259Godoi, Alexandre Barcia – 118Godoi, Manuella Machado – 28

Göethel, Gabriela – 185, 300Goetten, André L.F. – 132Golçalves, A.P. – 180Goldoni, Fernanda C. – 267Gomes, Francisca Tayná da Silva – 187, 193Gomes, Gabriela Cristiane Mendes – 261, 264, 265, 268Gomes, Geovana Maria de Lima – 96, 100Gomes-Júnior, Erival Amorim – 294, 310Gomes, Nayna Cândida – 82, 106, 108, 226Gomes, S.A. – 105Gomes, Thaisângela Rodrigues Lopes e Silva – 30, 33Gonçalves, Manoela Daiele – 269Gonçalves, Michelly Rodrigues – 306Gonçalves, Odinei Hess – 276, 277Gonzaga, Alexsandro Pinto – 250Gonzalez, Marcelo – 64Goodall, Sharon – 282Goulart , Christiane G.L. – 247Goulart, Cristiano O.L. – 233, 247Gouveia, Giovanna – 143Gouveia, Giovanna Cristiano – 78, 112, 134Govoni, Bruna – 143Graiczik, James Ramires Penteado – 194Grando, Ana Paula – 77, 102Grando, Luciana Grazziotin Rossato – 181Granjeiro, Jose Mauro – 273Grillo, Renato – 275Guex, Camille Gaube – 194Guidoni, Camilo Molino – 121, 122, 133, 137Guiloski, Izonete C. – 209Guimarães, Ana Tereza Bittencourt – 169, 205, 212, 213Gündel, Augusto R. – 126Guterres, Fernanda S. – 128, 234Guterres, Silvia S. – 271

HHabe, Priscila – 62Habib, Isabela A.3 – 126Hahn, Roberta Zilles – 77, 81, 102, 146, 167, 251Hardie, George – 282Harumi, Luciana – 259Heck, Amanda Szymansky – 194Heghes, Crina – 254, 289Heluany, Cíntia – 262

Herrala, Mikko – 206Hoff, Rodrigo – 97Holanda Júnior, Wanderley Pinheiro – 241, 243, 308Holanda, Wiron Pimentel – 163, 218, 312Honório Júnior, José Eduardo Ribeiro – 163, 218, 312Hort, Mariana Appel – 48Hoscheid, Jaqueline – 199, 215Hueza, Isis Machado – 208

IIndolfo, Nathalia de Carvalho – 49Ineu, Rafael Porto – 276, 277

Irioda, Ana Carolina – 26, 43, 144, 190Itinose, Ana Maria – 212

JJacomassi, Eliza – 199Jager, Alessandra Vincenzi – 223Jesus, Iracina Maura – 166Jorge, Bárbara Campos – 198, 204, 207, 210, 216

Jorge, Maria Helena Prado de Mello – 150Jorge, Roberta Jeane Bezerra – 68José, Gustavo Pinheiro Coelho – 162Justulin Junior, Luís Antonio – 201

KKagami, Luciano Porto – 185Kahl, Júlia M.M. – 89Kakuda, Priscila – 79Kalv, Danielle Crystiane – 302Kampmann, Micheli Gabardo – 299Kassuya, Cândida Aparecida Leite – 204Kawakami, Camila M. – 256Kawakami, Camila Martins – 50, 52Kayser, Juliana Machado – 293Kieling, Ketelin Monique Cavalheiro – 225Kishishita, Juliana – 37, 56

Kleemann, Cristian – 97Kleine, Tamila – 274Kocks, Grace – 289Koepp, Janice – 28, 29, 70Kohler, Cristofer José Weege – 191, 195Kohlrausch, Ramona – 128Kohori, Natália Akemi – 186Krotulski, Alex J. – 98Krüger, Nathália Ronconi Zilli – 202Krutzmann, Maria Eduarda – 128

LLamb, Liliane Weber Bolfe – 293Lameira, Christian Neri – 305Lanaro, Rafael – 101, 155Latorre, Andreia Oliveira – 62, 66, 287Laurentino, Ana Olívia Martins – 197, 200Leal, Leila Bastos – 37, 56Leal, Mirna Bainy – 197, 200Leão, Matheus C. – 271Leimann, Fernanda Vitória – 276, 277Leite, Jacqueline Alves – 30, 33, 263Leme, Adriana Paes – 304Leme, Daniela Moraes – 55Leme, Daniela Morais – 65, 286Lesniewski, Isabela Ramos – 212Lima, Aliny Pereira – 30, 33Lima, Crystiane Calado – 180Lima, Daina – 176

Lima, Daniela Delwing – 179Lima, Geovana Cristina Ribeiro – 214Lima, Luiza Siqueira – 144, 190Lima, Marcelo de Oliveira – 166Lima, Natália Cavalcante Barbosa – 68Lima, Thania Rios Rossi – 189Lima Thania Rossi Rios Rossi – 203Linden, Rafael – 77, 81, 84, 102, 120, 123, 128, 146, 167, 234,

251, 301Lini, Renata Sano – 54, 109, 151Lizot, Lilian de Lima Feltraco – 81, 146, 167Logan, Barry K. – 98Lopes, Bruna Gonçalves – 38Lourenço, Emerson Luiz Botelho – 199, 215Lovatel, Ivy Bauer – 75Lucinda-Silva, Ruth Meri – 57, 59Luz, Heloisa Peres – 93

MMacarini, Leanna Camila – 169Macedo, Larissa Matuda – 30, 33Machado, Cibele da Silva Barbosa – 301

Machado, Isabel Daufenback – 38, 191Machado, Michel Mansur – 261, 264, 265, 268Machado, Sérgio de Paula – 76

Machado, Simone Caetani – 280, 284Machinski Junior, Miguel – 109Maffi, Victoria Costa – 168Magalhaes, Danielle de Paula – 243Magalhães, Danielle de Paula – 241, 308Magalhães, Karla do Nascimento – 115Magalhães, Washington – 55Magosso, Natália – 201Maia, Affonso Enriques Montagner – 311Maia, Thayná Patachini – 272Maldaner, Adriano Otávio – 232Maluf, Ana Cristhina Sampaio – 148Manfio, Cleofas Sates – 197Manoel, Beatriz de Matos – 198, 204, 207, 216Marchioni, Camila – 93, 107, 109, 138, 151, 159, 162, 242Marciano, Luiz Paulo de Aguiar – 280, 281, 283, 284Marco, Mariana – 293Marcon, Scheila – 145Marek, Carla Brugin – 212, 213Maria-Engler, Silvya S. – 256, 259Maria-Engler, Silvya Stuchi – 27, 32, 52, 253, 255, 257, 258Mariani, Noemia Aparecida Partelli – 214Marigliani, Bianca – 279, 309Marinho, Pablo Alves – 224, 226Mariotto, Lívia Salviano – 154, 160Marize Campos Valadares – 53Marques, Ana Maria – 263Marques, Carla Pintas – 306Martinez, Victor Otero – 294Martínez, Victor Otero – 310Martins, Aline Franco – 83Martins, Aline R. – 275Martins, André Bittencourt – 147Martins, Isarita – 131, 280, 281, 284, 285Martins Jr., Airton Cunha – 189Martins, Mayane Emanuela Melo Lopes – 243Martins, Mayane Emanuella Melo Lopes – 241Massucato, Lucas Eduardo – 54Matias, William Gerson – 274Matos, Ricardo Luiz Nascimento – 269Matsui, Andresa – 219Mattos, Cristiane Bastos – 293Mattos, Guilherme – 46Mazete, Fernanda Pine S. – 127McEwan, Michael – 282

Medeiros, Elvis A. – 240Meirelles, Gabriela de Paula – 91Mello, Raissa – 143Melo, Paolo Oliveira – 180, 184Melo, Saulo Nascimento – 248Mendanha, Sebastião Antônio – 60Mendes, Michele Polyana Rocha – 113Mendes, Rosivaldo de A. – 297Mendonça, Izadora Caroline Furtado – 31, 51, 60Menegasso, Aloma Santin – 168Menegatti, R. – 105Menezes-Filho, José Antonio – 294, 310Menezes, Francisco Paz – 250Messias, Nayara Casagrande – 117, 159Migliato, Ketylin Fernanda – 44Milanez, Guilherme Paier – 83Miranda, Antônio M.M. – 297Miranda, Raul Ghiraldelli – 156, 157Miranda-Sapla, Milena Menegazzo – 269Molina, Higor Severo – 86, 172Monteiro, Fabíola Branco Filippin – 25Monteiro, Silvia Gonzalez – 74, 236, 237Moraes Junior, Manoel Oliveira – 255Moraes, Liliana S. – 126Moraes, Natália Valadares – 88Moraes, Niely Galeão da Rosa – 170Morais, Déborah Araújo – 85, 90Morales, Daniel Alexandre – 206Mora, Tamara Dal – 25Moreira, Beatriz – 124Moreira, Camila Francisco – 113Moreira, Camila Queiroz – 298Moreira, Rhubens Levy Rodrigues – 114Moreira, Suyane da Silva – 204, 216Moretti, Gabriela – 223Mori, Matheus P. – 259Mossini, Simone Aparecida Galerani – 54, 109, 151Motomura, Larissa Tiemi Akamine – 54Moura, Kézia – 50Moura, Maria Joana Nogueira – 180, 184, 187, 188, 193, 196Mozzato, Mateus Timbola – 168, 181Muccillo-Baisch, Ana Luiza – 48Müller, Gabrielle do Amaral e Silva – 176Muñoz, Rodrigo A.A. – 245Murata, Rosana Zoriki Hosomi – 62

NNagaoka, Lívia Trippe – 198, 207Nardi, Jessica – 181, 300Nascentes, Clésia C. – 233Nascimento, Marcelo Henrique Santana – 47, 73, 74, 116,

119, 236, 311Nascimento, Marcelo H.S. – 237Nascimento, Sabrina – 300Nazari, Evelise Maria – 202Nerilo, Samuel Botião – 109Ness, Sandro Luis Ribeiro – 301Neves, Gustavo Machado – 185Neves Júnior, Luiz F. – 249Nogueira, Caroline Lacerda – 86, 172, 225

Nogueira, Janer Alves – 138Nogueira, Jéssica Bueno – 204, 210, 216Nolasco, Daniela – 113Nolasco, Daniela M. – 125Nold, Juliana C. Lago – 27Noma, Isabella Harumi Yonehara – 257Noma, Isabella H.Y. – 259Nonino, Elisa de Castro Wille – 65Nossol, Edson – 245Nunes, Cleia Justino – 255Nunes, Rafaella Ferreira Nascimento – 131, 283Nunes, Viviane Abreu – 70

OOgasawara, Maryanne – 62Oliveira, Adriana Sousa – 158Oliveira, Aline Lima – 232Oliveira, Allan Santos – 166Oliveira, Ana Paula – 181Oliveira, Antonio Anax F. – 254, 289Oliveira, Arthur Lima – 156, 157Oliveira, Celinalva da Silva Lima – 232Oliveira, Claudete C. – 72Oliveira, Cláudia S. – 209Oliveira, Cláudia Simone Baltazar – 222, 305Oliveira, Cláudia Sirlene – 26, 43, 144, 190Oliveira, Danielle P. – 256Oliveira, Danielle Palma – 55Oliveira, Érica Aparecida – 258Oliveira, Franciele Aparecida Mendes – 190Oliveira, Jordana Meirelles – 121, 122Oliveira, José Miguel P. Ferreira – 124Oliveira, Juliana Ribeiro Ibiapina Leitão – 241, 243, 308

Oliveira, Lara Luiza Freitas – 246Oliveira, Leandro Leal Rocha – 30, 33Oliveira, Letícia Cimó – 311Oliveira, Marcos Antônio Fernandes – 201Oliveira Neto, J.R. – 80Oliveira, N.R.L. – 80Oliveira, Sarah Carobini Werner de Souza Eller Franco – 116Oliveira, Sarah C.W.S.E.F. – 126Oliveira, Sidney Julio Vieira – 222Oliveira, Silvana Ruella – 85, 90Oliveira, Therezinha Maria Novais – 274Oliveira, Tiago F. – 126Oliveira, Tiago Franco – 78, 95, 99, 112, 116, 134, 239, 250Olympio, Kelly Polido Kaneshiro – 166Osaki, Luciana Harumi – 27Ossanes, Daniela Souza – 239Otero, Ubirani Barros – 131Ott, Isabela Ritter – 234

PPacassa, Pâmela – 38Pacheco, André L.B. – 237Pacheco, André Lucas Bezerra – 73, 74, 116, 119, 135, 236,

311Padua, Amanay Sousa – 40Pais, Mariana Castello Novo – 62, 287Paiva, Maria José Nunes – 113, 125Palmeira, Carlos Manuel Marques – 186Pappis, Lauren – 194Papsun, Donna M. – 98Parabocz, Gisele Chibinski – 162Paschoalini, Beatriz Rizzo – 204, 210, 216Paula, Eliza Bianchini – 107, 138Paula, Favero R. – 103Paula, Fávero Reisdorfer – 47Paulo, Breno F. Pereira – 149Paulucci, Leticia Trevisan – 111Pavanelli, Wander Rogério – 269Pavlak, Jaíne Luana – 205Paz, Maria Elizabeth Gomes – 86, 92Pechansky, Flavio – 143Pedralli, Bruna Cristiane Oliveira – 35, 36Pedroso, Bruno – 302Pego, Ana Miguel Fonseca – 99Peixoto, Paloma Vitória Lima – 41, 165Peixoto, Paloma V.L. – 178, 211Pepato, Ana Claudia de Andrade – 66Perdoná, Gleici da Silva Castro – 142Pereira, Alessandra de Oliveira – 74, 236, 237Pereira, Artemia Kelly Holanda – 180, 184, 187, 188, 193, 196Pereira, Edimar Cristiano – 208Pereira, Eduardo Manoel – 272Pereira, Elizeu Chiodi – 166Pereira e Silva, Jefferson – 91Pereira, Henrique Marcelo Gualberto – 76, 96, 100Pereira, I.B. – 105Pereira, João Paulo Goes – 166Pereira, Leonardo da Cunha Boldrini – 273Pereira, Lílian C. – 178, 211Pereira Lilian Cristina – 203

Pereira, Lilian Cristina – 165Pereira,, Lilian Cristina – 186Pereira, Lílian Cristina – 41, 183, 189, 217Pereira, Meire Ellen – 144Pereira, Pedro Afonso de Paula – 232Pericolo, Suellen – 162Perini, Camila Maria – 191, 195Perjessy, Gisele – 62Peruzzi, Caroline Portela – 185, 300Pestana, Cynthia Bomfim – 65Petry, Adriana Ubirajara Silva – 250Petry, Andrea – 129Pimenta, Camila de Almeida Perez – 37Pinc, Mariana Moraes – 199, 215Pinelli, Juliana Junqueira – 221Pinheiro, Adriano da Silva – 50Pinheiro, Ana L.T.A. – 50Pinheiro, Dávylla Rennia Saldanha – 218Pinheiro, Fabriciano – 228, 288Pinheiro, Jacqueline – 202Pinheiro, Maria da Conceição Nascimento – 305Pinto, Nadja C. Souza – 259Pires, Janaina Aparecida Cardoso – 62Pires, Sumaia Araújo – 125Piton, Yasmin V. – 266Plautz, Katherine – 179, 272, 274, 303Poça, Kátia Soares – 131Pohlmann, Adriana R. – 271Polido, Lucas Roberto Ferreira – 183Pompermeier, Aline – 181Ponce, Júlio de Carvalho – 249Pontes, Montcharles S. – 275Ponting, David J. – 289Ponting, David. J. – 254Presgrave, Octavio – 64Priedols, Gustavo Abud – 121, 122Priedous, Gustavo Abud – 133, 137Proença, Carina – 124Pupo, André Sampaio – 207

QQueijo, Rodrigo Gonçalves – 253Queiroz, Maria Eugênia Costa – 79Queiroz, Thais Karolina Lisboa – 166Quintão, Nara Lins Meira – 38

Quintão, Nara L.M. – 267Quintas, Luís E.M. – 263Quiozini, Nathaly de Matos – 205, 212, 213

RRäisänen, Riikka – 206Ramadan, Debora R. – 111, 127Ramborger, Bruna Piaia – 92Ramos, Adriano T.1 – 132Ramos, David L.O. – 245Ramos, Silvia Aparecida – 162Ramos, Thalita da Silva – 285Ramos, Vitor Serrão – 295Rangel, Ana Lúcia Carrinho Ayroza – 205Rangel, Lara Luiza Pimenta – 173Rath, Susanne – 285Reck, Carolina – 132Reginato, Fernanda Z. – 237Reginato, Fernanda Ziegler – 73, 74, 75, 116, 119, 135, 194,

236, 311Reis, Ana Carolina Casali – 198, 204, 207, 210, 216Reis, Emily Marques – 28, 29, 70Reis, Karsonn B. – 245Reis, Luciane Ayres Castro – 173, 174Remor, Aline Pertille – 176Resener, Marisete Canello – 129, 130, 136, 159Rezin, Kéttulin Zomer – 242Ribeiro, Daniela – 124Ribeiro, Milena Mariano – 26, 43Ricci, Maurizio – 103Richter, Eduardo M. – 245Rigaudeau, Anne-Sophie – 46Rios, Nathalia Vieira – 264

Rios, Nathália Vieira – 261, 265, 268Rizzi, Joyce Santana – 183, 217Rizzo, Elizete – 169Rocha, Anna Carolina Furaer – 59Rocha, Cássia C.S. – 297Rocha, Cecília Cristina de Souza – 171, 192Rocha, Daniela Barbosa – 42Rocha, Danilo Galvão – 68Rocha, Raquel G. – 245Rocha, Thiago L. – 297Rocha, Vanessa Aguiar – 201Rodrigues, Caio Henrique Pinke – 61, 67, 154, 161Rodrigues, Gabriela Zimmermann Prado – 293Rodrigues, Leonardo Costalonga – 89Rodrigues, Marina Diaz – 86, 92, 172Rodrigues, Taís B. – 240Rodrigues, Vanessa Fernandes – 182Roehrs, Miguel – 75Roehrs, Rafael – 86, 92, 172, 225Romero, Alessandra de Cássia – 229Romoli, Jéssica Cristina Zoratto – 109Rosa, Victória Gomes – 73, 74, 116, 119, 135, 236, 237, 311Rossato-Grando, Luciana Grazziotin – 168Rossi, Bruna Franzon – 276, 277Rudolf, Carline – 59Rufino, Ana T. – 124Rysä, Jaana – 206

SSaboya, Andrea Luiza Rocha – 241Saboya, Andréa Luiza Rocha – 243Sá, Clodoaldo Antônio – 145Sakakibara, Isarita Martins – 283Saldanha, Geovane A. – 126Saleh, Najla Adel – 25Sales, Bianca Camargo Penteado – 41, 165Sales, Thais Lorenna Souza – 246Salles, Fernanda Junqueira – 166Salles, Gabriela Pereira – 114, 115Salomón, Janaína – 200Salvadori, Daisy Maria Fávero – 214Sampaio, Amanda Mello Kasper Vaz – 75Sanches, Alex O. – 275Sanches, Cristina – 246Sandri, Silvana – 262, 271Santana, Davi Pereira – 37, 56Santana, Thatiane Nunes – 40, 219Santiago, Etenaldo F. – 275Santiago, Mayla Andra de Andrade – 222Santiago, Vivian Romero – 243Santin, José Roberto – 38, 57, 59, 103, 267Santos, Bruno Pereira – 78, 112, 134, 143

Santos, Caio Cesar Araújo – 180, 184, 187, 188, 193, 196Santos, Carlos Eduardo Matos – 103Santos, Christiano – 153Santos, Claudia Regina – 93, 107, 117, 129, 138, 159Santos, Fabiana Pereira – 87Santos, Jeniffer Farias – 70Santos, Jordana Andrade – 34, 53, 69Santos Junior, Wilson José Ramos – 106Santos Júnior, Wilson José Ramos – 244Santos, Lara C. – 237Santos, Lara Celestina – 73, 74, 116, 119, 135, 236, 311Santos, Nathália Ribeiro – 294, 310Santos, Nícolas Guimarães – 197Santos, Rachel – 74, 116, 119, 126, 135, 237Santos, Rafaela Knak – 120Santos, Raquel – 124Santos, Sérgio Alexandre Alcantara – 201Santos, Thaís Rosa Marques – 53Santos, Thiago Santana – 298Santos, Vanessa Farelo – 76, 96, 100Santos, Vivian da Silva – 306Saraiva, Illyushin Zaak – 176Saraiva, Thalia Emmanoella Sebulsqui – 293

Sarpa, Márcia Sarpa de Campos – 131Sartori, Alan Giovanini de Oliveira – 229Sartori, Daniela Carlos – 182Scanferla, Deborah Thais Palma – 109, 151Scarano, Wellerson Rodrigo – 201Scavone, Cristoforo – 263Schamne, Tatiane – 302Scharf, Pablo – 262, 271Scherer, Juliana – 78Scherer, Juliana N. – 143Schimith, Lucia Emanueli – 48Schmitt, Maria Laura Videiro – 172Schmitz, Felipe – 266Schneider, Ayda Henriques – 262Schroeder, Samilla Driessen – 242Schuck, Desiree Cigaran – 58Schwengber, Heloysa Talia – 212Sebben, Viviane – 200Sebben, Viviane Cristina – 112, 134Seloto Danielle Gabriel – 203Seloto, Danielle Gabriel – 217Sérgio de Morais – 63Siena, Ádamo D.D. – 259Silva, Adny Henrique – 25Silva, Agnes Soares – 166Silva, Alan Andrew dos Santos – 214Silva, Aline Gabrielle Gomes – 180, 184, 187, 188, 193, 196Silva, Ana Cléia Cardoso – 26, 43Silva, Ana Lucelha dos Santos – 188, 196Silva, Artur Christian Garcia – 31, 34, 45, 51, 53, 58, 60, 63Silva, Bruna Espíndola – 93, 242Silva, Carla Brigagão Pacheco – 182Silva, Claudia Larissa Viana – 32Silva, Denise Bousfield – 117Silva, Erick José Ramo – 214Silva, Fabiano – 230Silva, Fernanda Coleraus – 205, 212, 213Silva, Gerlane Modesto – 180Silva, Graciele Machado – 111Silva, Gustavo Henrique – 50Silva, Jonas Joaquim Mangabeira – 226Silva, José Wellithom Viturino – 56Silva-Jr, Wilson A. – 259Silva, Juliana F. – 209Silva, Julia Rezende – 258Silva Júnior, Clovis Reis – 223Silva Júnior, Flavio Manoel Rodrigues – 170, 175Silva, Laura Cé – 77, 102, 123, 301Silva, Luciana Stein – 301

Silva, Luíza Madureira – 163, 218, 312Silva, Maria Isabel Gonçalves – 145Silva, Mariana Cristina – 118Silva, Mateus Limerio Carlos – 188, 196Silva, Priscilla Muniz Ribeiro – 40Silva, Rafael Araújo – 283Silva, Rivia Regina Lopes – 263Silva, Samanta de Matos – 44Silva, Stephanie Soares – 117, 159Silva, Victória da Costa – 114, 115Silveira Filho, Jair – 242Silvério, Alessandra Cristina Pupin – 280, 281, 284Silvério, Kérolyn Aparecida – 288Simioni, Carmen – 202Simon, Jaqueline – 205, 212, 213Singulani, Junya de Lacorte – 44Siqueira, Gabriella Ferreira – 54Siqueira, Janas – 131Siqueira, Lisiane – 181Smalley, Keiran S.M. – 259Smidt, Mariana – 251Soares, Daniel – 287Soares, Marcela de Oliveira – 111, 127Soares, Suelen Mendonça – 168, 181Sobjak, Thaís Maylin – 169Sotelo, Êmily Clori – 261, 268Sousa Junior, Wellington Tavares – 90Sousa Júnior, Wellington Tavares – 85Sousa, Roberto Cesar Santos – 292Souza, Asley Thalia Medeiros – 37, 56Souza, Daniela Cristina – 277Souza, Douglas – 293Souza, Isisdoris Rodrigues – 286Souza, Israel Donizeti – 79Souza, João Pedro Silveira – 185Souza-Kaneshima, Alice Maria – 54Souza, Karla Aparecida de Oliveira – 150Souza, Luiz Claudio Cindra – 173Souza, Mirna Maciel D'Auriol – 125Souza, Natacha Medeiros – 263Souza, Patrick Vieira – 201Souza, Tainá Brumate – 113, 125Souza, Wanderson – 273Stainki, Daniel Roulim – 237Steiner, Bethina Trevisol – 147Stein, Julia – 198, 204, 207, 210, 216Stival, Ana Clara Silva – 45Sugawara, Eduardo Kinio – 111, 127Summy, Maria Julia – 176

TTadeu, Vitória Costa – 225Tamagno, Wagner – 181Tanamachi, Amanda Rodrigues – 214Taruhn, Lillian Freitas – 136Tavares, Kvetta Pinheiro Teixeira – 36, 39Tavares, Renata S.N. – 256Tavares, Renata Spagolla Napoleão – 52, 304Tegner, Mariane – 234Tenfen, Adrielli – 303

Tennant, Rachael E. – 254, 289Teodoro, João Soeiro – 186Thá, Emanoela Lundgren – 58, 286Tonietto, Bruna Ducatti – 197, 200Tostes, Rita C. – 182Trajano, Christian Farias – 96, 100Tufik, Sergio – 111, 127Tumas, Vitor – 79

UUchiyama, Mayara K. – 271Ugalde, Gustavo A. – 237Ugalde, Gustavo Andrade – 73, 74, 75, 116, 119, 135, 236, 311

Uhera, Adriana H. – 259Umbuzeiro, Gisela de Aragão – 206

VValadares, Marize Campos – 30, 31, 33, 34, 35, 36, 39, 45,

51, 58, 60, 63, 69Valente, Letícia Cardoso – 198, 207Vasconcelos, Mailton – 143Vasconcelos, Mayrla Emilia Dantas – 88Vasconcelos Neto, Milton Cabral – 295Vaz, Milena Menegazzo – 267Vecina, Juliana Falcato – 40, 219Veiga, Ângela – 132

Veiverberg, Andriele – 293Vellosa, Jose Carlos Rebuglio – 302Verpaele, Steven – 306Viana, Roberta Rodrigues – 99Vicente, Eduardo – 50Vieira, Bruna Todeschini – 213Viola, Patrícia Pacheco – 143Viriato, Cristina – 41, 178, 211Vivani, Riccardo – 103

WWaechter, Fernanda – 289Wagner, Eduardo José – 191Wagner, Theodoro – 103Walton, Sara E. – 98Werner, Anne-Laure D. – 254

Willett, Catherine – 279Winter, Evelyn – 132Wurzler, Gleicielle T. – 158Wyse, Angela T.S. – 266

YYli-Öyrä, Johanna – 206 Yonamine, Mauricio – 87, 91, 140, 249

ZZardeto, Giuliana – 199, 215Zauli, Danielle Alves Gomes Breno – 149Zazula, Matheus Felipe – 169Zebele, Patricia – 282Zimermann, Francielli C. – 132

Zimmath, Michel – 303Zoghaib, I.V.J. – 105Zuravski, Luísa – 261, 264, 265, 268

A L T E R N A T I V E T O X I C O L O G Y

PATROCINADOR OURO:

PATROCINADOR PRATA:

PATROCINADOR BRONZE:

PATROCINADORES:

EXPOSITORES:

APOIO INSTITUCIONAL:

ORGANIZAÇÃO: AGÊNCIA OFICIAL: