Chemosphere 80 (2010) 137–143

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Toxicity of Bromkal 70-5DE, a technical mixture of polybrominated diphenylethers, following 28 d of oral exposure in rats and impact of analysed impurities

Mattias Öberg a,*, Emma Westerholm a, Elena Fattore b, Natalia Stern a, Annika Hanberg a, Peter Haglund c,Karin Wiberg c, Anders Bergendorff a, Helen Håkansson a

a Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 210, SE-171 77 Stockholm, Swedenb Department of Environmental Health Sciences, ‘‘Mario Negri” Institute for Pharmacological Research, Via La Masa 19, 20156 Milano, Italyc Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 November 2009Received in revised form 26 February 2010Accepted 2 April 2010

Keywords:Flame retardantPolybrominated diphenyl etherPenta-BDESprague–Dawley ratPolybrominated dibenzofuran

0045-6535/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2010.04.006

* Corresponding author. Tel.: +46 8 52487517; fax:E-mail address: mattias.oberg@ki.se (M. Öberg).

The subacute toxicity of a commercial polybrominated diphenyl ether (PBDE) preparation, Bromkal70-5DE, was investigated. In addition to a vehicle control, the mixture was given orally to male andfemale Sprague–Dawley rats for 28 d at three dose levels; 2.5, 25 and 250 mg kg�1 b.w. d�1. The observedeffects include increased hepatic EROD activity (from 2.5 mg kg�1 d�1); increased liver weight (males),increased PROD activity and depletion of hepatic retinoids (from 25 mg kg�1 d�1); and increased liverweight (females), marked histological changes in the liver and lungs, as well as increased serum param-eters such as total protein, cholesterol and albumin (from 250 mg kg�1 d�1). Chemical analysis of thePBDE mixture with gas chromatography/mass spectrometry (GS/MS) showed impurities of polybromi-nated dibenzofurans and to a lesser extent dibenzodioxins, in total levels of about 7.0 lg g�1 of Bromkaltechnical mixture. The animals were thereby exposed to an estimated dose of dioxin-like equivalents cor-responding to 1.3–131 ng TEQ kg�1 b.w. d�1. It cannot be ruled out that this level of impurities canexplain the hepatic EROD induction and hepatic retinoid depletion, which are considered typical markersof toxicity mediated via the aryl hydrocarbon receptor (AhR).

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Technical mixtures of polybrominated diphenyl ethers (PBDEs),including Penta-BDE products such as Bromkal 70-5DE, have beenwidely used as flame retarding additives in various commercialand household products. The concern for human and environmen-tal health due to increased use of PBDEs combined with their ten-dency to bioaccumulate in humans has prompted the ban of penta-PBDE flame retardants both in the EU and in various states in theUSA (EU, 2003a,b; BSEF, 2009). However, some major applicationsrelate to long-lived consumer products such as domestic furnitureand cars, and thus, continued release to environment and humansmay be anticipated for many years to come.

Each BDE-product is not exclusively one pure congener, but con-tains several BDE congeners with different amount of bromination.Bromkal 70-5DE, the commercial penta-BDE mixture used in thepresent study, is known to contain 37% of 2,20,4,40-tetra-BDE(BDE-47), 35% of 2,20,4,40,5-penta-BDE (BDE-99) (Sundström andHutzinger, 1976) and 6.8% of 2,20,4,40,6-penta-BDE (BDE-100),3.9% of 2,20,4,40,5,50-hexa-BDE (BDE-153) as well as one additional

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tri-BDE, one tetra-BDE, one penta-BDE, three hexa-BDEs and onehepta-BDE (Sjödin et al., 1998). Similar congener composition hasbeen confirmed by more recent studies (Korytar et al., 2005; LaGuardia et al., 2006). In total 18 congeners have been reported fromthe commercial product in these studies. The exact congener pat-tern is likely to vary slightly between manufacturers and batches.

PBDEs are ubiquitous environmental pollutants; they are foundin rural and urban air, sewage sludge, sediments, as well as in ter-restrial, freshwater, and marine wildlife (de Wit, 2002; Murvollet al., 2005). When food items were analysed, the highest levelswere found in fish, meat and dairy products (Schecter et al.,2004). The dietary intake of PBDEs has been estimated to 27 ng d�1

for Swedish primiparous women (Lind et al., 2003). For the generalpopulation the dietary intake has been estimated to 50.9 ng d�1 inSweden (Darnerud et al., 2006) and 90.5 ng d�1 Great Britain(Harrad et al., 2004). PBDE congeners have also been found inhuman breast milk, serum and adipose tissue (Schecter et al.,2005; Johnson-Restrepo et al., 2007; Fängström et al., 2008; Lignellet al., 2009). The dominating congener pattern in human samples issimilar to the pattern in commercial penta-BDE products, namelyBDE-47, BDE-99, BDE-100 and BDE-153.

PBDE congeners have a low acute toxicity and long-term toxic-ity studies have shown that the thyroid and liver are major target

138 M. Öberg et al. / Chemosphere 80 (2010) 137–143

organs for BDE toxicity (reviewed by WHO-IPCS (1994), Gill et al.(2004), Talsness (2008)). The structural similarities between PBDEsand classes of dioxin-like chemicals (e.g. PCBs), suggests thatPBDEs might activate the aryl hydrocarbon receptor (AhR), whichis a critical mechanism of toxicity for dioxin-like compounds. Un-like the dioxin-like AhR ligands, PBDEs are usually not coplanarand the AhR binding affinities of PBDEs is reported to be 10�2–10�5 times that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)(Chen et al., 2001). The activation of AhR leads to induction ofCYP1A1/2 which can be assayed as ethoxyresorufin-O-deethylase(EROD) activity. Some of the previously observed toxicological ef-fects of Bromkal 70-5DE are also similar to effects related to diox-in-like chemicals, i.e. induction of CYP1A (Chen et al., 2001) anddepletion of hepatic retinoids (Hallgren et al., 2001; Fernie et al.,2005). Bromkal 70-5DE has been shown to cause an AhR depen-dent dioxin-like CYP1A1 activity in vitro (Hanberg et al., 1991)and enzyme induction has also been observed in vivo (Von Meyer-inck et al., 1990).

It has been suggested that the observed dioxin-like effects ofcommercial PBDE products might be due to contaminants. In astudy on pure individual congeners BDE-47, �99, �100, �153, and�154 in rodent hepatoma cell lines, no CYP1A1 (EROD) activitywas observed. When combined with TCDD, an inhibitory effect onthe TCDD induced EROD activity was present, indicating that theBDEs bind the AhR but do not cause any increased activity (Peterset al., 2006). The results by Peters et al. are only partly confirmedwhen the same congeners were tested in vitro by others (Chenet al., 2001; Hamers et al., 2006). In these studies the relative po-tency for individual BDE-congeners are found to be in the order of10�5 and 10�7, as compared with TCDD. Other experimental strate-gies to elucidate the contaminant hypothesis include separate test-ing of individual or grouped constituents of the technical mixture.An approximation of such design is given by the studies of Hallgrenet al. (2001) with BDE 47, and of Kuiper et al. (2006) and Van der Venet al. (2008), with purified and fractionated commercial mixtures.Taken together, the results of these studies are not fully conclusive,since dioxin-like effects observed in vivo have not been fully con-firmed invitro. Thus, the overall composition of the commercial mix-tures seem to produce dioxin-like activity and a characterization ofthe profile of impurities is necessary to interpret the results.

The aim of the present study was to investigate the toxicity ofBromkal 70-5DE, following 28 d of oral exposure in the rat and toevaluate to which extent dioxin-like contaminants are present inthe technical product and may account for the observed toxicity.To this end, the OECD TG407 was enhanced with markers of diox-in-like activity, i.e. retinoid levels and CYP1A1 (EROD) inductionand the content of polyhalogenated dibenzo-p-dioxins and dib-enzofurans were analysed in the commercial PBDE-product.

Table 1Weekly feed consumption, body weight gain, and relative and absolute organ weights in

Treatment(mg kg�1 d�1)

Feed cons.(g week�1)

Body weightgain (g)

Absoluteliverweight (g)

Relative liverweight(mg g�1 b.w.)

MaleVehicle 173 163 ± 20 15.8 ± 1.3 44 ± 22.5 169 167 ± 20 16.8 ± 1.5 46 ± 225 178 169 ± 11 18.3 ± 2.9 51 ± 6b

250 160 148 ± 24 26.7 ± 4.1 b 77 ± 9b

FemaleVehicle 140 67 ± 14 8.5 ± 4.9 45 ± 32.5 150 73 ± 9 9.9 ± 1.1 41 ± 325 137 76 ± 13 11.2 ± 0.9 47 ± 3250 141 61 ± 11 19.9 ± 3.3 b 88 ± 11b

a Values represent the mean ± sd of five animals.b Significantly different (Mann–Whitney, p 6 0.05) from the vehicle control.

2. Materials and methods

A full and detailed description of materials and methods are gi-ven in Supplements. The study was performed according to OECDGuidelines for Testing of Chemicals 407 and Standard OperatingProcedures (SOP). Shortly, the Bromkal 70-5DE mixture was dis-solved in peanut oil (Kemila AB, Sweden) (10 g/200 mL) to give ahigh-dose solution and diluted 10 and 100 times to give solutionsfor medium and low-dose administration respectively. The exper-imental protocol, including animal housing and care during thestudy was approved by the Stockholm Northern Animal Ethic Com-mittee (Stockholm, Sweden). Groups of five male and five femaleSprague–Dawley rats were given daily oral doses via gavage of 0,2.5, 25 and 250 mg kg�1 b.w. d�1 of Bromkal 70-5DE at a volumeof 5 mL kg�1 b.w. per animal and day for 4 weeks. The controlgroups received the vehicle (peanut oil) only. The rats were sacri-ficed after 28 d of exposure. Organs and specimens for histologicalexaminations were collected. Portions of liver, lungs and kidneyswere frozen and stored at �70 �C for biochemical determinations.

Chemical impurities were analysed by gas chromatographyinterfaced to a double focusing mass spectrometer. Quantificationwas made according to the isotope dilution technique and by usingall the 17 2,3,7,8-substitued PCDFs and PCDDs and ten of the2,3,7,8-substituted PBDFs and PBDDs (CIL) as reference com-pounds. The maximum proportion of 2,3,7,8-substituted congenerswas estimated from the chromatograms by using the retentiontimes of ten of the 2,3,7,8-brominated congeners and, in addition,the congener distribution between 2,3,7,8-congeners andnon-2,3,7,8-congeners within each homologue group. The weightadjusted potency relative to TCDD was adapted from Behnischand colleagues, who investigated the AhR activity of several2,3,7,8-substituted PBDFs by using the micro-EROD and DR-CALUXbioassays (Behnisch et al., 2003) and the TCDD equivalent dose(TEQ) (Van den Berg et al., 2006) was calculated.

O-Dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyres-orufin (PROD) were determined in the hepatic S9-fraction (Lubetet al., 1985). Frozen samples of liver, kidneys and lungs wereanalysed for retinoids (sum of retinol, retinyl acetate and retinylpalmitate) by high pressure liquid chromatography (HPLC)(Håkansson et al., 1987; Chu et al., 1995).

3. Results

3.1. Clinical observations, body and organ weights

No clinical signs of toxicity were observed and no animals diedduring the study. Animals in all groups gained weight (Table 1).

male and female rats treated orally with Bromkal 70-5DE for 28 d.a

Absolutekidneyweight (g)

Relative kidneyweight(mg g�1 b.w.)

Absolute thymusweight(mg g�1 b.w.)

Relative thymusweight(mg g�1 b.w.)

2.4 ± 0.1 6.6 ± 0.6 0.54 ± 0.1 1.5 ± 0.32.4 ± 0.2 6.5 ± 0.5 0.46 ± 0.3 1.6 ± 0.22.5 ± 0.2 6.9 ± 0.4 0.55 ± 0.1 1.5 ± 0.42.1 ± 1.2 7.6 ± 0.2b 0.51 ± 0.1 1.6 ± 0.4

1.7 ± 0.2 7.1 ± 0.6 0.43 ± 0.04 1.8 ± 0.21.6 ± 0.2 6.6 ± 0.5 0.49 ± 0.10 2.0 ± 0.31.7 ± 0.1 7.3 ± 0.2 0.41 ± 0.08 1.7 ± 0.21.5 ± 0.9 8.2 ± 0.8 0.44 ± 0.09 1.8 ± 0.5

M. Öberg et al. / Chemosphere 80 (2010) 137–143 139

However, the body weight gain in both male and female rats inhigh-dose groups tended to be lower (�9%) compared to the corre-sponding vehicle control groups. In male rats, this may partly berelated to a slight decrease in feed consumption as compared tocorresponding vehicle controls (Table 1). The relative and absoluteliver weights were increased with increasing dose in male and fe-male rats (Table 1). Significant group differences were observed inthe medium and high-dose groups of male rats and in the high-dose group of female rats. The relative kidney weight (but notabsolute weight) was increased in male rats of the high-dose group(Table 1). There were neither significant changes for absolute kid-ney or thymus weight (Table 1), nor in spleen, lungs, heart, adrenalglands, brain, testis, uterus and ovaries weights (data not shown).

3.2. Histopathological examinations

Few macroscopical changes were noticed at necropsy. Accentu-ated lobular pattern was observed in livers of two female rats in themedium-dose group, and in three male and two female rats in thehigh-dose groups (data not shown). Numerous rats belonging toboth experimental and control groups, showed histological hepaticchanges (Table 2). The lesions observed consisted of centrilobularhepatocellular hypertrophy of different grade and patchy fattychanges (lipidosis). In general, the prevalence and grade of hepaticchanges was found to be more frequent and severe at higher doses.Moderate centrilobular hepatocellular hypertrophy appeared in allanimals from the high-dose groups. The hypertrophy was slight tomoderate in medium-dose groups and did not appear in the controlgroups. The average grade of hypertrophy varied from minimal inlow-dose groups to slight and moderate in medium- and high-dosegroups, respectively. Minimal to slight fatty changes were as wellmost prevalent in rats from the high-dose groups. A variety of his-tological changes in the lungs were observed in both rats exposed tovehicle and Bromkal 70-5DE (Table 2). Interstitial pneumonia ofmoderate grade was observed in rats from vehicle-, medium- andhigh-dose groups. Lymphoid cuffing, alveolar oedema and atelecta-sis were occasional lung findings in rats from all dose groups. Pelvicdilatation was the only finding in kidneys in one of the male rats inmedium-dose group. Increased erythropoiesis in spleen was occa-sionally observed in a few female rats. No histopathologicalchanges were observed in any of the other examined tissues.

Examination of the haematological parameters showed no sig-nificant alterations in haemoglobin level, haematocrit, erythrocytecount, platelet count, and total and differential leukocyte count,MCV, MCH or MCHC (data not shown). Count of erythrocytes wasslightly increased in female rats of the control and high-dosegroups.

Table 2Incidence and average gradea of histological changes in lungs, liver, kidneys and spleen in

Organ Finding Vehicle 2.5 m

Female Male Fema

Liver Fatty change/periportalFatty change/patchyCentrilob. hypertrophyMicrogranuloma 1(+) 1+

Lungs Alveolar macrophages 1+

AtelectasisInterstit. pneumonia 3++ 1++ 3+

Lymphoid cuffing 4+ 3+ 4++

Alveolar oedema

Kidney Pelvic dilatation

Spleen Ingr. erythropoesis 1+ 1+

a Average grade of histological findings: (+) = minimal/very small, + = slight/small, ++b Five animals in each group.

3.3. Clinical chemistry and biochemistry

Total serum protein was increased in both male and female ratsof the high-dose groups (Table 3). Serum cholesterol levels wereincreased in male and female rats of the high-dose groups. In-creased serum albumin level and decreased albumin/globulin ratiowere observed in female rats of the high-dose group. The activity ofALAT was increased in male rats of the high-dose group, but with-out a clear dose-dependency. Serum concentration of magnesiumwas increased in male rats of the high-dose groups. Serum phos-phorus concentrations were increased in female and male rats ofthe medium and high-dose groups respectively and calcium con-centration increased in male rats of the medium and high-dosegroups. Sodium was slightly increased and potassium decreasedin female rats of the high-dose group. The concentration of ureawas increased in male rats of the medium and high-dose groups.There were no effects upon ALP and creatinine when comparedto vehicle groups (data not shown).

Hepatic EROD activity was increasingly elevated in low, med-ium and high-dose groups both in male and female rats (Table4). The high-dose groups showed an elevation of 78 and 137 timesas compared to the vehicle dose group, for male and female rats,respectively. The PROD-activity was also markedly elevated in asimilar manner (Table 4). Significant changes were observed inthe medium and high-dose groups of both male and female rats.Hepatic retinoid content was decreased as a response to increaseddoses in both male and female rats (Table 4). The high-dose groupsshowed a decrease in hepatic retinoid content of 36% and 47%, ascompared to the vehicle dose groups, for male and female ratsrespectively. Male rats in the high-dose group showed a tendencyof increased retinoid content in their kidneys and decreased con-centrations in the lungs, however, these values were not signifi-cantly different from the vehicle dose group.

3.4. Urine analysis

There were no effects upon solute particles, pH, protein, glu-cose, methylketone, haemoglobin, bilirubin, leucocytes and epithe-lial cells when compared to vehicle groups (data not shown).

3.5. Chemical characterization of Bromkal 70-5DE

The Bromkal 70-5DE dosing solution was analysed for polyhalo-genated dibenzo-p-dioxins and dibenzofurans (PXDD/F). Analysisof the chromatograms suggested that a multitude of congenerswere present. It was not possible to perform congener specificanalysis due to the lack of analytical standards for some of the

male and female rats treated orally with Bromkal 70-5DE for 28 d.b

g kg�1 d�1 25 mg kg�1 d�1 250 mg kg�1 d�1

le Male Female Male Female Male

2++

1(+) 3+ 1+ 1+ 1+

2+ 5+ 4++ 5++ 5++

3+ 1+ 3+

1+

2++ 1++

1++ 1++ 5+++ 3++

2+ 5++ 2++

2++

1++

1++

= moderate/moderate size, +++ = marked/large.

Table 4Hepatic enzyme activities and total amount of retinoids in liver, kidneys and lungs, in male and female rats treated orally with Bromkal 70-5DE for 28 d.a

Treatment ERODb (nmol mg�1 min�1) PRODc (nmol mg�1 min�1) Hepatic retinoidsd (lg) Renal retinoidsd (lg) Pulmonary retinoidsd (lg)

MaleVehicle 0.05 ± 0.02 0.2 ± 0.06 2133 ± 192 6.4 ± 1.4 15.2 ± 7.12.5 mg kg�1 d�1 0.09 ± 0.02e 0.1 ± 0.02 2146 ± 257 5.8 ± 1.6 14.7 ± 10.725 mg kg�1 d�1 0.6 ± 0.1e 0.4 ± 0.1e 1809 ± 219 8.0 ± 3.0 14.6 ± 6.9250 mg kg�1 d�1 3.9 ± 0.4e 6.1 ± 1.3e 1373 ± 182e 13.7 ± 7.3 9.1 ± 3.7

FemaleVehicle 0.03 ± 0.01 0.01 ± 0.002 2401 ± 309 2.0 ± 1.0 9.2 ± 7.22.5 mg kg�1 d�1 0.09 ± 0.02e 0.02 ± 0.01 2196 ± 130 1.8 ± 0.7 5.6 ± 2.825 mg kg�1 d�1 1.3 ± 0.6e 0.2 ± 0.1e 1907 ± 226e 2.0 ± 0.8 9.7 ± 8.3250 mg kg�1 d�1 4.1 ± 1.2e 1.4 ± 0.5e 1261 ± 141e 2.3 ± 1.6 9.9 ± 3.3

a Values represent the mean ± sd of five animals.b O-Dealkylation of 7-ethoxyresorufin.c O-Dealkylation of 7-pentoxyresorufin.d Sum of retinol, retinyl acetate, and retinyl palmitate.e Significantly different (Mann–Whitney, p 6 0.05) from the vehicle control.

Table 3Clinical chemistry parameters in serum from male and female rats treated orally with Bromkal 70-5 DE for 28 d.a

Treatment Totalprotein(g L�1)

Cholesterol(mmol L�1)

Albumin(g L�1)

A/G-ratio

ALAT(lkat L�1)

Mg(mmol L�1)

P(mmol L�1)

Ca(mmol L�1)

Na(mmol L�1)

K(mmol L�1)

Urea(mmol L�1)

MaleVehicle 67 ± 3 1.2 32 ± 1 0.9 ± 0.1 0.57 ± 0.09 0.97 ± 0.05 2.9 ± 0.2 2.6 ± 0.1 140 ± 1 4.5 ± 0.2 4.9 ± 0.22.5 mg kg�1 d�1 67 ± 2 1.1 ± 0.1 32 ± 1 0.9 ± 0.0 0.89 ± 0.67 0.98 ± 0.02 3.1 ± 0.1 2.7 ± 0.1 139 ± 1 4.5 ± 0.1 4.3 ± 0.525 mg kg�1 d�1 68 ± 2 1.2 ± 0.3 33 ± 1 0.9 ± 0.0 0.53 ± 0.07 0.98 ± 0.04 2.4 ± 0.2 2.8 ± 0.1b 140 ± 1 4.2 ± 0.4 7.2 ± 0.6b

250 mg kg�1 d�1 75 ± 3b 2.2 ± 0.3b 34 ± 2 0.9 ± 0.0 0.86 ± 0.09b 1.05 ± 0.04b 3.4 ± 0.1b 2.9 ± 0.2b 140 ± 1 4.5 ± 0.3 6.2 ± 0.6b

FemaleVehicle 67 ± 2 1.2 ± 0.1 33 ± 1 1.0 ± 0.1 0.50 ± 0.05 0.97 ± 0.10 2.2 ± 0.3 2.5 ± 0.1 137 ± 1 4.2 ± 0.3 7.6 ± 0.72.5 mg kg�1 d�1 69 ± 2 1.2 ± 0.3 33 ± 1 0.9 ± 0.0 0.53 ± 0.07 0.98 ± 0.04 2.4 ± 0.2 2.6 ± 0.1 137 ± 1 3.9 ± 0.2 7.2 ± 0.625 mg kg�1 d�1 69 ± 2 1.2 ± 0.1 33 ± 1 0.9 ± 0.1 0.52 ± 0.05 1.02 ± 0.06 2.6 ± 0.2b 2.6 ± 0.1 138 ± 1 3.8 ± 0.3 6.4 ± 0.4250 mg kg�1 d�1 85 ± 3b 3.0 ± 0.4b 39 ± 2b 0.8 ± 0.1b 0.43 ± 0.08 1.05 ± 0.07 2.5 ± 0.2 2.7 ± 0.1 139 ± 1b 3.8 ± 0.2b 6.5 ± 1.2

a Values represent the mean ± sd of five animals.b Significantly different (Mann–Whitney, p 6 0.05) from the vehicle control.

Table 5Concentrations (ng g�1 oil) in the dosing solution (5 mL oil = 4.575 g) and corresponding daily doses per body weight of individual polybrominated dibenzo-p-dioxins (PBDD),dibenzofurans (PBDF), and TCDD equivalent dose, in male and female rats treated orally with Bromkal 70-5DE for 28 d.a

Congener Concentration(ng g�1 oil)

Daily intake (ng kg�1 b.w. d�1) REPb Proportion of 2,3,7,8-substituted congeners

PBDF-TEQc (ng kg�1 b.w. d�1)

Low dose Medium dose High dose High dose

di-BDFs 6.0 0.27 2.8 27 – – –tri-BDFs 13 0.58 5.76 58 0.000077 – –tetra-BDFs 91 4.18 41.84 418 1.12 0.1 46.9penta-BDFs 260 11.9 119 1190 0.28 0.25 83.3hexa-BDFs 4.1 0.19 1.9 19 0.031 0.8 0.5hepta-BDFs 7.7 0.35 3.51 35 0.0057 1.0 0.2di-BDDs 0.039 0.00 0.02 0.18 – – –tri-BDDs 0.023 0.00 0.01 0.1 0.0065 – –tetra-BDDs 0.03 0.00 0.02 0.16 1.06 0.1 0.02penta-BDDs <0.20 <0.01 <0.09 <0.92 0.45 0.25 <0.1hexa-BDDs <0.90 <0.04 <0.41 <4.12 0.04 0.8 <0.2hepta-BDDs <4 <0.2 <1.8 <18.3 – 1.0 –RBDD/Fsb 384 18 176 1759 131.1

a Low dose = 2.5 mg kg�1 b.w. d�1, medium dose = 25 mg kg�1 b.w. d�1, high dose = 250 mg kg�1 b.w. d�1.b Relative potency values estimated from in vitro assays (Behnisch et al., 2003).c Estimated TEQ-dose assumes that only 2,3,7,8-substituted congeners contribute to the potency.

140 M. Öberg et al. / Chemosphere 80 (2010) 137–143

2,3,7,8-congeners and lack of the co-elution pattern of the non-2,3,7,8-congeners. Penta- and tetra-BDFs were the major constitu-ents among analysed PBDD/Fs, 260 and 91 ng g�1 oil respectively,while PBDD concentrations were low for all congeners (Table 5).Di- and tri-BDF were found in concentrations of 6 and 13 ng g�1

oil, respectively, while hexa- and hepta-BDFs were found at 4.1and 7.7 ng g�1 oil, respectively. These concentrations give rise to

a summarised PBDD/F intake of 1759 ng kg�1 b.w. d�1 in thehigh-dose groups. Calculated as concentration in the commercialBromkal 70-5DE, the level of PBDD/Fs was about 7 lg g�1. Themaximum proportion of 2,3,7,8-substituted congeners was esti-mated to 10 % of the detected amounts of tetra-, 25% of penta-,80% of hexa-, and 100% of hepta-BDFs. The TCDD equivalent dosein the present study was estimated to be about 131 ng TEQ kg�1

M. Öberg et al. / Chemosphere 80 (2010) 137–143 141

b.w. d�1 at the high dose level (Table 5). No detectable amounts ofPCDD/Fs were found in the Bromkal 70-5DE dosing solution.

4. Discussion

This subacute toxicity study in rats was initiated to improve riskassessment of commercial penta-BDE and to enable a comprehen-sive interpretation of the repeatedly observed dioxin-like patternof effects. The standard study design was therefore enhanced withmarkers for dioxin-like exposure and the test compound analysedfor halogenated dibenzo-p-dioxins and dibenzofurans.

The current study confirmed previous observations indicatingthat the liver is a major target organ for BDE toxicity (WHO-IPCS,1994; Gill et al., 2004; Talsness, 2008). Liver enlargement accom-panied by a broad spectrum of functional and structural changesare often responses appearing after acute as well as continuousexposure to hepatotoxic substances. In this study the relative liverweight was markedly increased, in high-dose groups (250 mg kg�1

b.w. d�1) relative liver weights were 75% and 96% higher in malesand females respectively as compared to vehicle controls. Relativeliver weight was also increased in medium dose. Hepatotoxic ef-fects after exposure to commercial penta-BDE products have beenrepeatedly found by industrial investigators from doses above 1–10 mg kg�1 b.w. d�1 (WHO-IPCS, 1994). In addition, Bromkal 70-5DE was previously investigated using male and female Wistar ratsand a repeated dose of 50 mg kg�1 b.w. for 28 d, which resulted inan increase of relative liver weight by 40% (Von Meyerinck et al.,1990). When another commercial available pentabrominated mix-ture (DE-71) was tested in female C75BL/6 mice, the relative liverweight was increased at all doses tested (>250 mg kg�1 b.w.) (Fow-les et al., 1994). Liver was also found to be the primary target organafter repeated exposure to DE-71 for 13 weeks in both rats andmice (Dunnick and Nyska, 2009). The same batch of Bromkal 70-5DE as tested in the present study was previously shown to causeincreased relative liver weight in both rats and mice when given18 mg kg�1 b.w. by gavage for 14 d. A similar effect was evidentin mice (not tested in rats) exposed to 18 mg kg�1 b.w. of BDE-47(Hallgren et al., 2001).

The most pronounced dose-dependent histopathological find-ings in liver were fatty changes (lipidosis) and centrilobular hyper-trophy of hepatocytes, leading to liver enlargement. The liver has acentral role in normal lipid metabolism and toxicants can cause anaccumulation of triglyceride in the liver resulting from an imbal-ance between uptake of fatty acids and their secretion. Fattydegeneration is a sign of ongoing degenerative processes and is of-ten followed by fibrosis during long-term exposure. The increasedactivity of ALAT, a marker of hepatocyte destruction, was observedin male rats of the high-dose group. Hepatocytes are the mostimportant sources of synthesis of plasma proteins, and an increaseof albumin and total serum protein can appear as a result of hyper-trophy of liver cells. In this study the serum concentration of totalprotein was increased in both male and female high-dose groupsand albumin concentration was increased in the female high-dosegroup. The levels of several serum electrolytes were slightly in-creased in the high-dose groups (Table 3). These changes exceedreference values and probably appeared as a result of increasedserum protein levels.

In the present study, hepatic enzyme activity was increasedfrom 2.5 and 25 mg kg�1 b.w. d�1 for EROD and PROD respectively,indicating that Bromkal 70-5DE is a mixed type inducer similar tocommercial PCB products. The observed effects are in the same or-der of magnitude as in a previous study using the same batch ofBromkal 70-5DE at doses of 18 and 36 mg kg�1 b.w. d�1 for ratsand mice, respectively (Hallgren et al., 2001). Similar effects havealso been observed in rats exposed to 50 mg Bromkal 70-5DE kg�1

b.w. d�1 for 28 d (Von Meyerinck et al., 1990). Zhou et al. (2001)examined hepatic enzyme activity in weanling rats administeredDE-71 (penta-BDE) at doses from 0.3 to 300 mg kg�1 b.w. d�1 andfound significantly increased EROD and PROD at P10 mg kg�1

b.w. d�1 (Zhou et al., 2001).Induction of hepatic EROD activity is known to be one of the most

sensitive markers of dioxin exposure with a reported Lowest Ob-served Effect Level (LOEL) of 0.3 ng TCDD kg�1 b.w. d�1 in mice (Vo-gel et al., 1997) and 1 ng TCDD kg�1 b.w. d�1 in rats (lowest dosetested) (Viluksela et al., 2000). Kohn and colleagues observed induc-tion of CYP1A1 mRNA at 0.1 ng TCDD kg�1 d�1 in rats (Kohn et al.,1993). In the present study we observed EROD activation in thelow-dose group (2.5 mg kg�1 b.w. d�1), which received an esti-mated daily dose of 1.3 ng PBDD/F-TEQ kg�1 b.w. Given a LOEL forEROD induction in rats of 0.1–1 ng kg�1 b.w., it may be reasonableto explain the increased EROD activity by the found impurities. Thisconclusion partly contradicts the previous study by Hallgren et al.(2001), using the same batch of Bromkal 70-5DE. They reported thatexposure to Bromkal 70-5DE (18 mg kg�1 b.w. d�1) could increaseEROD activity in rats and mice. In addition, they also observed a sim-ilar effect in mice (not tested in rats) given the same dose of the ma-jor PBDE constituent congener, BDE-47 (comprising 37% of theBromkal isomeric content). From these two experiments they con-clude that BDE-47 is the most likely candidate of causing dioxin-likeeffects. The stated purity of BDE-47 in the Hallgren study is >98%and it is not possible to fully elucidate if the observed effects iscaused by the compound or possible impurities. As mentionedabove, when tested in vitro, several research groups have failed toinduce EROD activity by BDE-47 (Hamers et al., 2006; Chen et al.,2001). In addition, Richardson and co-workers found an increasedEROD activity while Cyp1a1 mRNA expression was not significantlyincreased in mice after short-term exposure to 100 mg BDE-47 kg�1 d�1, indicating that BDE-47 is not an AhR agonist and in-stead constitutive androstane receptor (CAR) activity was suggestedto be involved in the response to BDE-47 since both PROD (from3 mg kg�1 d�1) and Cyp2b10 expression (from 10 mg kg�1 d�1)were increased (Richardson et al., 2008). When a purified fractionof DE-71 was tested in a 28 d study by Van der Ven et al. (2008) in-creased CYP1A2 mRNA and protein levels was observed along withan increased EROD activity. However, the original sample testednegative for AhR agonist activity in vitro (CALUX).

In addition to EROD induction and liver/thymus weights, hepa-tic retinoid levels are also often used as markers of dioxin-like tox-icity. We observed dose-related effects for hepatic retinoiddepletion, suggesting a dioxin-like toxicity of Bromkal 70-5DE,with a Lowest Observed Effect Level (LOEL) of 25 mg kg�1

b.w. d�1. A LOEL in rats of 1 ng TCDD kg�1 b.w. d�1 (lowest dosetested) is reported for depletion of hepatic retinoids (Fletcheret al., 2005). Looking at the significant decrease in hepatic retinoidsin the medium-dose groups, an impact of the estimated dailyPBDD/F-TEQ intake of 13 ng kg�1 b.w. is likely.

A relative potency of 10�6 as compared to TCDD has been estab-lished for dioxin-like effects of Bromkal 70-5DE in vitro (Hanberget al., 1991). Similar order of magnitude potency was found in vivo(Hallgren et al., 2001). However, single BDE-congeners present inBromkal 70-5DE, such as BDE-47 and -99, have failed to bind theAhR and increase EROD activity in vitro in several test systems (Chenet al., 2001; Peters et al., 2006; Hamers et al., 2006). Using the REPvalues (BDE-100 = 10�5, BDE-153 = 10�6) from Chen et al. (2001)combined with the congener specific quantification of Bromkal70-5DE (BDE-100 = 6.8%, and BDE-153 = 3.9 %) by Sjödin et al.(1998), a theoretical daily intake for the low-dose groups in the pres-ent study would be about 1.8 ng PBDE-TEQ kg�1 b.w. This is in thesame potency range as the estimated LOEL for EROD and hepatic ret-inoid depletion. Still, while Chen and co-workers classified BDE-100as a weak AhR inducer with a potency of about 10�5, the observed

142 M. Öberg et al. / Chemosphere 80 (2010) 137–143

inhibitory effect during cotreatment with TCDD by Peters and co-workers indicates binding to AhR without causing activation. Thesedata suggest the possible impact of dioxin-like impurities, e.g. the2,3,7,8-substituted PBDD/Fs, in the toxicity of the technical PBDEproduct. PBDEs have also been shown to be precursors to PBDFs,both through thermal and photolytic degradation (Eriksson et al.,2004; Buser, 1986). The impurity hypothesis is supported by Kuiperand co-workers who tested the commercial penta–BDE mixture DE-71 for AhR activation both in vitro and in vivo. The DE-71 was frac-tioned into clean DE-71, containing only PBDEs, and planar DE-71,containing PBDD/DF contaminants. The results indicate that theoverall composition of the commercial mixtures may produce diox-in-like activity but this effect is most likely not due to the PBDEs.However, a purified preparation of DE-71 was also tested for subacute toxicity in Wistar rats (Van der Ven et al., 2008). Although sta-ted to be free from 2,3,7,8-substituted BDD/Fs, as confirmed by DR-CALUX in vitro system (Hamers et al., 2006), the study showed nota-bly increased EROD activity and decreased hepatic levels of reti-noids at doses from 0.7 mg kg�1 b.w. and 0.5 mg kg�1 b.w.,respectively. These inconsistent results may indicate a mechanismindependent of direct binding and activation of the AhR.

In the present study, we performed a semi-quantitative analy-sis, showing a total PBDD/F concentration of 384 ng g�1 oil in theBromkal 70-5DE mixture. The most abundant compounds weretetra- and penta-BDFs, accounting for 91% of the total amount. Itis not likely that all formed PBDFs are laterally substituted.2,3,7,8-Substituted PBDD/Fs are known to induce CYP1A1 in vivoand in vitro with a similar structure activity relationship asPCDD/Fs (Safe, 1990; Behnisch et al., 2003). Several congeners ofPBDD/Fs have been shown to cause classical dioxin-like effects(thymic atrophy and weight loss in rats) with an excellent correla-tion for structure–activity relationship as compared to CYP1A1activity in vitro (Mason et al., 1987). In addition, PBDD/Fs have alsobeen shown to cause developmental toxicity (cleft palate in mice)(Birnbaum et al., 1991), and relative potency factors have beendetermined. Health effects of PBDD/Fs, including relative potencycalculations are also reviewed in (Birnbaum et al., 2003). In generalthese potency values show a similar structure-activity relationshipas the WHO-TEFs for chlorinated analogues (Van den Berg et al.,2006). The TCDD equivalent dose in the present study was esti-mated to be about 131 ng TEQ kg�1 b.w. d�1, but it has to be keptin mind that this is only a rough estimate of the TEQ concentrationfrom PBDD/Fs in the technical Bromkal 70-5DE product.

Considering the kinetics of different components of Bromkal 70-5DE, including impurities, the bioavailability and accumulation ofthe components can influence the body burden and therefore therelative contribution to toxic effects. Rats given Bromkal 70-5DEvia gavage for 2 weeks, showed a different PBDE congener composi-tion in plasma as compared with the administered mixture (Darne-rud et al., 2007). In addition, the PBDD/F impurities are believed tohave similar kinetic properties as their chlorinated analogues ande.g. the 2,3,7,8-TBDF is even more resistant to metabolism thanthe corresponding persistent chlorinated analogue (Birnbaumet al., 2003). This may further emphasize the contribution of impu-rities and the importance of tissue concentration data for riskassessment of technical PBDE mixtures, especially after repeatedexposures.

To establish to what extent the dioxin-like effects are caused bythe PBDE congeners or PBDF impurities, the analytical difficultiesneed to be solved. This also calls for synthesis of pure individualPBDFs and the need to report individual relative potency valuesin comparison to TCDD both in vitro and in vivo. There is also a needto test BDE-99 (the major constituent together with BDE-47) for ef-fects like hepatic EROD activity and depletion of hepatic retinoids.

In conclusion, this study describes the patterns of toxicity inmale and female rats for the commercial penta-BDE mixture Brom-

kal 70-5DE following 28 d of oral exposure. The effects include in-creased hepatic EROD activity (from 2.5 mg kg�1 b.w. d�1);increased liver weight (males), increased hepatic PROD activityand depletion of hepatic retinoids (from 25 mg kg�1 b.w. d�1);and increased liver weight (females), marked histological changesin the liver and lungs, as well as increased serum parameters suchas total protein, cholesterol and albumin (from 250 mg kg�1

b.w. d�1). It cannot be ruled out that the analysed levels of PBDFimpurities can explain most of the observed dioxin-like effects,such as hepatic EROD induction and hepatic retinoid depletion.

Acknowledgements

We thank Ellu Manzoor for retinoid and enzyme analyses. Thisstudy has been carried out with financial support from the SwedishFoundation for Strategic Environmental Research (MISTRA), theCommission of the European Communities, specific RTD Project,BoneTox (EU-QLK-CT-02-02528), and the Swedish ChemicalsAgency (KemI).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.chemosphere.2010.04.006.

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