International Journal of Toxicology oculata Nannochloropsis Safety Assessment of EPA-Rich Polar...

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published online 9 October 2014International Journal of ToxicologyMichael L. Kagan, Dexter W. Sullivan, Jr, Shayne C. Gad and Corey M. Ballou

oculataNannochloropsisSafety Assessment of EPA-Rich Polar Lipid Oil Produced From the Microalgae

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Original Article

Safety Assessment of EPA-Rich PolarLipid Oil Produced From the MicroalgaeNannochloropsis oculata

Michael L. Kagan1, Dexter W. Sullivan Jr2, Shayne C. Gad2,and Corey M. Ballou2

AbstractAlmega PL is an eicosapentaenoic acid-rich o-3 oil that is isolated from Nannochloropsis oculata algae and developed as a dietarysupplement. The safety of the algal oil was evaluated in 14- and 90-day studies in Sprague-Dawley rats by oral gavage at dose levelsof 0, 250, 500, and 2500 mg/kg/d and 0, 200, 400, and 2000 mg/kg/d, respectively. No mortalities occurred and no signs of toxicitywere observed during the studies. No treatment-related effects were seen for body weight, food consumption, ophthalmology,neurological effects, urinalysis, clinical pathology, gross pathology, organ weights, or histopathology. Although statistically signif-icant effects were noted for some end points, none were considered to be of toxicological significance. The no observed adverseeffect level for Almega PL was 2000 mg/kg/d. Additionally, Almega PL was not mutagenic in Salmonella typhimurium or Escherichiacoli, did not induce chromosome aberrations in Chinese hamster ovary cells, and did not induce genotoxic effects in vivo in ratbone marrow erythrocytes.

KeywordsNannochloropsis oculata, algae, o-3, dietary supplement, eicosapentaenoic acid, EPA, docosahexaneoic acid, DHA, toxicity,genotoxicity

Introduction

Nannochloropsis is a small green microalga genus that is well

known due to its nutritional value and ability to produce valu-

able lipophilic and lipophobic materials.1 In the aquaculture

industry, Nannochloropsis is extensively used as feedstock in

growing small zooplanktons such as rotifers and in fish hatch-

eries2 and for producing green water.3 In addition, the bio-

chemical composition of Nannochloropsis makes it a

valuable food source for animals and humans.1

Nannochloropsis oculata is 1 of the 6 species contained in

the genus Nannochloropsis and was originally isolated off the

coast of Scotland. Species of the genus Nannochloropsis have

been shown to contain high concentrations of eicosapentaenoic

acid (EPA) while containing no docosahexaneoic acid

(DHA).4,5 Nannochloropsis oculata is a rich source of EPA,

and the oil extracted from the green microalgae is one of the

few sources of EPA that does not also contain DHA.4 Recent

studies comparing EPA and DHA have suggested that not only

is the EPA component primarily responsible for reducing

inflammation6 and depression7 but that any DHA component

acts to block the activity of EPA.8 Therefore, the oil extracted

from N oculata presents a means of maximizing the available

levels and desired beneficial aspects of EPA as evidenced in

humans.4

Almega PL is an EPA-rich o-3 oil isolated from N oculata.

It is being developed by Qualitas Health Ltd, (Jerusalem,

Israel) as a dietary supplement ingredient with a suggested

daily intake of 300 to 1250 mg (2.0-20.8 mg/kg/d for a 60-kg

person), thus providing a maximum of 312.5 mg/d o-3 fatty

acids.9 To evaluate the potential toxicity of Almega PL, a

battery of Good Laboratory Practice (GLP)-compliant in vivo

and in vitro assays were performed. In rat models, a 14-day

maximum-tolerated dose (MTD) study and a 90-day repeated

dose subchronic toxicity study were performed. Clinical obser-

vations, clinical chemistry, hematology, urinalysis, functional

observational batteries (FOBs), and gross and microscopic

pathology were conducted to determine whether or not

repeated oral exposure of Almega PL resulted in any signs of

toxicity. Additionally, to evaluate the genotoxic potential of

Almega PL, a bacterial reverse mutation assay, an in vitro

mammalian chromosome aberration test in Chinese Hamster

1 Qualitas Health Ltd, Jerusalem, Israel2 Gad Consulting Services, Cary, North Carolina, USA

Corresponding Author:

Michael L. Kagan, Qualitas Health Ltd, 19 Hartom St, Jerusalem 91450, Israel.

Email: [email protected]

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(Cricetulus griseus) ovary (CHO) cells, and an in vivo micro-

nucleus assay in rats were performed.

Materials and Methods

Test Item

Almega PL (Lots 130202a and 130610; Qualitas Health Ltd) is

a nonpurified, dark green oily paste obtained from N oculata.

The source biomass, N oculata, is cultured in raceway-type

pools and harvested daily followed by extraction of the oil from

the biomass, resulting in Almega PL. Almega PL consists of

approximately 55% total fatty acids, of which 25% is EPA.

Almega PL also contains chlorophyll (approximately 3%) and

15% polar lipids (phospholipids and glycolipids).

The test item was analyzed for the presence of organochlor-

ine and organophosphate pesticides. No pesticides were found.

An analysis for dioxin, dioxin-like polychlorinated biphenyls

(PCBs), and nondioxin-like PCBs indicated that the levels of

these substances are below the maximum levels for marine oils

as listed by the World Health Organization and when adjusted

for fat content. Toxin analyses conducted on Almega PL indi-

cated that no natural product toxins were detected above detec-

tion limits including microcystins/nodularin, anatoxin-a,

cylindrospermopsin, paralytic shellfish toxin/saxitoxins, oka-

daic acid, and brevetoxins.10

Chemicals and Materials

Extra virgin olive oil used as the vehicle for the in vivo studies

was supplied by Qualitas Health Ltd except for in the micro-

nucleus assay where it was supplied by Pharmaseed Ltd (Ness

Ziona, Israel). 2-Nitrofulorene and 2-aminoanthracene were

obtained from Sigma-Aldrich (Munich, Germany). 9-

Aminoacridine was obtained from Novel Organic Synthesis

(Andhra Pradesh, India). 4-Nitroquinoline-1-oxide, sodium

azide, ethyl methanesulfonate, cyclophosphamide monohy-

drate, and colchicine were obtained from Sigma-Aldrich (St

Louis, Missouri). Dimethyl sulfoxide (DMSO) was obtained

from RFCL Ltd (Faridabad, India). Oxoid nutrient broth No.

2 was obtained from Oxoid Ltd (Basingstoke, Hampshire,

England). Fetal bovine serum was obtained from Sera Labora-

tories (West Sussex, United Kingdom). Methanol was obtained

from Merck Specialties Private Limited (Mumbai, India).

Potassium phosphate monobasic, sodium phosphate dibasic,

and acridine orange hydrochloride hydrate were obtained

from Sigma-Aldrich (Buchs, Switzerland). DPX (Distyrene,

Plasticizer, Xylene) mountant was obtained from SD Fine

Chemicals Limited (Mumbai, India).

Animals and Organisms

For the 14-day MTD study performed at Pharmaseed Ltd, 24

(12 male and 12 female) 8-week-old Sprague-Dawley SD rats

obtained from Harlan (Israel) were housed in polyethylene

cages (3/cage). The acclimatization period was 3 days prior

to testing. Initial mean group body weights were 237 g for

males and 181 g for females. The minimum and maximum

initial weight in the groups were within a range of +20% of

the groups mean weight. Room temperature was maintained at

20�C to 24�C with a relative humidity of 30% to 70%, a min-

imum of 15 air changes/h and a 12-hour light and 12-hour dark

cycle. Autoclaved and acidified drinking water (pH between

2.5 and 3.5) and commercial rodent diet (Teklad Certified Glo-

bal 18% Protein Diet cat # 2018SC) were provided ad libitum.

For the 90-day repeated dose, subchronic toxicity assay per-

formed at Advinus Therapeutics, Ltd (Bangalore, India), male

and female Sprague-Dawley rats raised in house by Advinus

Therapeutics were used. The acclimatization period was 5 days

prior to testing. The rats were 6 to 7 weeks old at the beginning

of the assay. Initial mean group body weights ranged from 145

to 148 g for the male rats and 127 to 130 g for the female rats.

At the beginning of treatment, the weight variation of the test

animals did not exceed +20% of the mean body weight in each

group and sex. Rats were housed in groups of 2 per sex per cage

in solid floor standard polysulfone cages with stainless top

grills for providing pelleted food and drinking water in poly-

carbonate bottles with stainless steel sipper tubes in rooms with

12 hours light cycles. Steam sterilized clean corn cob was used

as bedding and changed along with the cage once during the

acclimatization period and twice a week during the treatment

period. Room temperature was maintained at 19�C to 24�Cwith a relative humidity of 58% to 68%, with a minimum of

12 air changes/h. Filtered deep-bore well water and Teklad

Global 14% protein rodent maintenance diet (Harlan Labora-

tories, An Venray, the Netherlands) were provided ad libitum.

The Salmonella typhimurium and Escherichia coli test

strains for the bacterial reverse mutation assays performed at

Advinus Therapeutics Ltd were obtained, respectively, from

Health Protection Agency National Collection of Type Cul-

tures (London, Great Britain) and The National Collection of

Industrial and Marine Bacteria Ltd (Scotland, United King-

dom). The CHO cell line CHO-51, (ATCC CCL-61, Lot

4765275) for the chromosome aberration assay performed at

Advinus Therapeutics Ltd were obtained from the American

Type Culture Collection (Manassas, Virginia).

For the in vivo erythrocyte micronucleus test performed at

Advinus Therapeutics Ltd, male and female Sprague-Dawley

rats (in-house random bred) were acclimated for 5 days before

dosing. The rats were at least 10 weeks old (range, 10-11 weeks)

at the beginning of the assay. Initial body weights ranged from

250 to 253 g for the male rats and 188 to 190 g for the female

rats. All rats were visually inspected prior to the start of treat-

ment. Rats were individually housed during acclimatization and

treatment in standard polysulfone cages with stainless steel top

grills in rooms with 12 hours light cycles. Steam sterilized corn

cob was used as bedding which along with cages was changed at

least once a week. Room temperature was maintained at 20�C to

23�C with a relative humidity of 65% to 67% and 12 to 15 high-

efficiency particulate air-filtered air changes/h. Deep bore-well

water (passed through activated charcoal filter and exposed to

ultraviolet [UV] rays) and Teklad Certified (2014C) Global 14%Protein Rodent Maintenance pellets were provided ad libitum.

2 International Journal of Toxicology



Guidelines

Because the 14-day rat study was a dose-range finding (DRF)

study and used to determine the MTD of Almega PL as well as

the doses for the subsequent subchronic (90 day) study, it was

not performed in full compliance with GLP guidelines. The

subchronic toxicity study in rats was performed in accordance

with the OECD Guideline for Testing of Chemicals, (408):

Repeated Dose 90-Day Oral Toxicity Study in Rodents11 and

under GLP in accordance with OECD12 Principles of GLP for

the Testing of Chemicals. The bacterial reverse mutation

(Ames) assays were performed in accordance with the OECD13

Guideline 471 for testing of chemicals: Bacterial Reverse

Mutation Test and under GLP in accordance with OECD14

Principles of GLP. The chromosome aberration assay was per-

formed in accordance with the OECD15 Guideline 473 for

testing of chemicals: In Vitro Mammalian Chromosome Aber-

ration Test and under GLP in accordance with OECD14 Prin-

ciples of GLP. The rat micronucleus assay was performed in

accordance with the OECD16 Guideline 474 for testing of che-

micals: Mammalian Erythrocyte Micronucleus Test and under

GLP in accordance with OECD12 Principles of GLP.

Experimental Design

Maximum-tolerated dose oral toxicity study. Groups of 3 male and

3 female SD rats each were administered a single dose of 0, 5%(250 mg/kg), 10% (500 mg/kg), or 50% (2500 mg/kg) Almega

PL by oral gavage on day 1. The administrations were per-

formed using 16-gauge gavage needles. The vehicle was warm

extra virgin olive oil, and the dose volume was 5 mL/kg. Fol-

lowing the treatment period, all rats were monitored for 14 days

and euthanized on day 15.

Rats were observed twice daily for signs of morbidity and

mortality and once daily for clinical signs until study termina-

tion. Body weights were recorded prior to the initial dosing,

twice weekly during the study, and at study necropsy. On day

15, rats were euthanized by carbon dioxide asphyxiation. After

killing the animals, the organs and tissues identified in Table 1

were removed and examined for gross pathological anomalies.

Subchronic oral toxicity study. Groups of 10 male and 10 female

Sprague-Dawley rats each were administered 0, 250, 500, or

2500 mg/kg Almega PL in extra virgin olive oil by oral gavage

once daily for 90 days. Doses were administered using dispo-

sable plastic syringes attached with a metal feeding cannula.

All doses were administered at a constant dose volume of

5 mL/kg. Following the treatment period, all animals were

euthanized on day 91.

Rats were observed twice daily for signs of morbidity and

mortality and once daily for clinical signs until study termina-

tion. Detailed clinical examinations were performed prior to

the item administration on day 1 and at weekly intervals (+2

days) thereafter. Body weights were recorded on day 1 prior to

the test item administration and weekly thereafter except for

week 13 when the body weights were recorded on day 5 of the

week. Fasting body weights were recorded prior to necropsy on

day 91. Food consumption was measured once per week on the

same days that body weight measurements were made. At the

end of the study, rats were fasted overnight (water allowed),

anesthetized with isoflurane, and exsanguinated.

Ophthalmological examination. Prior to the start of treatment (on

day 5 of acclimatization), ophthalmological examinations were

performed for all animals with an ophthalmoscope. Mydriasis

was induced before examination using a solution of 1% tropi-

camide. The eyes were examined in subdued light. Subdued

light was maintained in the animal room for the remainder of

the day. These procedures were repeated at the end of the

treatment period for all animals.

Functional observational battery tests. To assess the behavioral

and neurological status of each rat, FOB tests were performed

for all of the group rats on day 90 of the treatment period. The

FOB tests included home cage observations for posture and for

presence or absence of abnormal vocalizations and convulsion;

observations during removal from rat from home cage and

handling; open field observations; and functional tests includ-

ing sensory evaluation, landing hindlimbs foot splay, grip per-

formance, and motor activity (Table 2).

Urinalysis. Before killing, urine was collected from all rats in

urine collection tubes. Each rat was placed in specially fabri-

cated cages overnight (water allowed) and the next morning the

urine was collected for analysis. Urinalysis parameters exam-

ined in the collected samples included color, clarity, bilirubin,

erythrocytes, glucose, ketone bodies, leukocytes, nitrite, pro-

teins, pH, specific gravity, urobilinogen, and volume. Urine

was also subjected to microscopic examination for sediments

such as crystals, epithelial cells, and casts.

Clinical pathology. At the end of the treatment period, rats were

fasted overnight (water allowed) after which blood was col-

lected from the retro-orbital sinus plexus with fine capillary

tubes under isoflurane anesthesia. An aliquot of blood was

collected in tubes containing 3.2% sodium citrate solution for

determination of coagulation parameters, and the remaining

blood was collected into K2EDTA and lithium heparainized

Table 1. List of Evaluated Organs.

Organ/tissue Organ/tissue Organ/tissue

Adrenal glands Lungs Seminal vesiclesBrain Mandibular lymph node SkinCecum Mesenteric lymph node SpleenColon Mammary gland StomachDuodenum Esophagus TestesEpididymitis Ovaries ThymusEyes Pancreas TongueHeart Parathyroid glands TracheaIleum Thyroid Urinary bladderJejunum Pituitary UterusKidneys Prostate gland VaginaLarynx Rectum Sciatic nerveLiver Salivary glands

Kagan et al 3



tubes for hematology and clinical chemistry examinations

(Table 3).

Pathology. All animals were fasted overnight prior to terminal

killing on day 91. At the scheduled termination, all survivors

were euthanized by exsanguination under isoflurane anesthesia.

The organs identified in Table 4 were removed, weighed, and

examined for gross and microscopic pathological anomalies.

Bacterial reverse mutation assays. The mutagenic potential of

Almega PL was evaluated using the bacterial reverse mutation

assay (Ames test) using standard plate incorporation (experi-

ment I) and preincubation (experiment II) methods. The ability

of Almega PL to induce mutagenicity in S typhimurium tester

strains TA98, TA100, TA1535, and TA1537 and E coli strain

WP2uvrA was assessed in the presence and absence of rat

microsomal fraction S9 mix. Based on the observations of

preliminary experiments to evaluate toxicity, 5000 mg/plate

was selected as the highest dose for all test strains and condi-

tions. A stock solution of 50 000 mg/mL test item in DMSO was

prepared by mixing 500 mg Almega PL in DMSO and bringing

the volume to 10 mL in a volumetric flask with DMSO. For

experiment I, 4 lower dose levels (50, 158, 500, and 1581 mg/

plate) were prepared by serially diluting the stock solution with

DMSO. For experiment II, 4 lower dose levels (100, 266, 707,

and 1880 mg/plate) were prepared by serially diluting the stock

solution with DMSO. The positive controls in the absence of

S9 mix were 2-nitrofluorene for S typhimurium TA98, sodium

azide for S typhimurium TA100 and TA1535, 9-aminoacridine

for S typhimurium TA1537, and 4-nitroquinoline-1-oxide for

E coli WP2uvrA. The positive control for all bacterial strains in

the presence of S9 mix was 2-aminoanthracene. The vehicle

control for all strains in the presence or absence of S9 mix was

DMSO (100 mL).

Each of the tester strains from the master plates were inocu-

lated into tubes containing Oxoid Nutrient broth No. 2 and then

incubated at 37�C + 1�C for approximately 17 hours. For

experiment I, the bacterial suspensions were exposed to the test

item, vehicle, and the positive controls in the presence or

absence of the exogenous metabolic activation system. These

bacterial suspensions were then mixed with overlay agar and

plated immediately onto minimal medium, viz, his� for S

typhimurium and trp� for E coli, respectively. The plates were

then incubated at 37�C + 1�C for 67 hours. Revertant colonies

Table 3. Clinical Pathology Investigations.

Parameter Abbreviations

HematologyDLC, G/L and % HCT, L/LHGB, g/L MCH, pgMCHC, g/L MCV, fLMPV, fL Plat, G/LRBC, T/L Retic, T/L and %WBC, G/L

CoagulationPT, s APTT, s

Clinical chemistryA/G ALT, U/LAlb, g/L ALP, U/LAST, U/L BUN, mmol/LCa, mmol/L Cl, mEq/LCreat, mmol/L GGT, U/LGlob, g/L Glu, mmol/LPi, mmol/L K, mEq/LNa, mEq/L TBil, mmol/LTChol, mmol/L TPro, mmol/L

Trig, mmol/L Urea,a mmol/L

Abbreviations: DLC, differential leukocyte count; HCT, hematocrit; HGB,hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscularhemoglobin concentration; MCV, mean corpuscular volume; MPV, mean plate-let volume; Plat, platelets; RBC, red blood corpuscles; Retic, reticulocytes;WBC, white blood corpuscles; PT, prothrombin time; APTT, activated partialthromboplastin time; A/G, albumin/globulin ratio (calculated values); ALT, ala-nine aminotransferase; Alb, albumin; ALP, alkaline phosphatase; AST, aspartateaminotransferase; BUN, blood urea nitrogen; Ca, calcium; Cl, chloride; Creat,creatine; GGT, g-glutamyl transpeptidase; Glob, globulin (calculated values);Glu, glucose; Pi, inorganic phosphorous; K, potassium; Na, sodium; TBil, totalbilirubin; TChol, total cholesterol; TPro, total plasma protein; Trig, triglycer-ides; urea, urea (calculated values).aUrea values are not reported separately as both BUN and urea values are thesame when expressed in mmol/L.

Table 2. Functional Observational Battery (FOB) Tests.

Observations during removal of rat from home cage and handling

Ease of removal from homecage

Salivation

Handling reactivity Skin/fur examinationPalpebral closure Perineum wetnessEye examination RespirationPiloerection Muscle toneLacrimation Extensor thrust responseOpen field observations

Gait Bizarre behaviorPosture UrinationMobility score DefecationArousal level RearingClonic and tonic movements VocalizationsStereotypic behaviors

Functional test—sensory reactivity measurements

Approach response Tail-pinch responseTouch response Pupillary responseClick response Aerial righting reflexFunctional test—landing hindlimbs foot splay

The landing hind limb foot splay was performed by dropping an animalon a surface from a short height and measuring the distancebetween the feet upon landing.

Functional test—grip performance

Hindlimbs and forelimbs grip performance was tested over 3 trialsusing a computerized dual grip strength meter.

Functional test—motor activity

The motor activity of each rat was measured using an automatedanimal activity measuring system equipped with a computeranalyzer over a 30-minute duration.

Physiological observations and body weight measurements

Rectal body temperature, �C Rat weight at the end of functionaltests




were counted manually, and the plates were examined for bac-

terial background lawn. For experiment II, the test constituents

were mixed with the bacteria inside tubes, incubated in an

incubator shaker for 30 minutes at 37�C + 1�C, mixed with

overlay agar, and plated immediately onto minimal medium

his� for S typhimurium and trp� for E coli, respectively. The

plates were then incubated at 37�C + 1�C for 67 hours. Rever-

tant colonies were counted manually, and the plates were

examined for bacterial background lawn.

The tests were considered positive for mutagenicity if the

increase in mean revertants at the peak of the dose response

was equal to or greater than twice the mean vehicle control

value for TA98, TA100, or WP2uvrA or equal to or greater

than 3 times the mean vehicle control value for TA1535 or

TA1537. Data were not analyzed statistically.

Chromosome aberration assay. The potential of Almega PL to

induce structural chromosome aberrations in cultured mamma-

lian cells was evaluated using CHO cells. The maximum con-

centrations of Almega PL tested were selected based on

preliminary range finding test in which concentrations between

17.5 and 35 mg/mL with 4-hour exposure (presence of S9) and

35 and 70 mg/mL with 4-hour exposure (absence of S9) caused

cytotoxicity of at least 50% cell growth inhibition compared to

the vehicle control. Similarly, cytotoxicity was seen at concen-

trations between 4.4 and 8.45 mg/mL with 21-hour exposure in

the absence of metabolic activation. A stock solution of 10 000

mg/mL Almega PL was prepared by mixing 250 mg Almega PL

in DMSO and bringing the volume to 25 mL with DMSO in a

volumetric flask. Almega PL was exposed to CHO cells in

duplicate at concentrations of 0 (DMSO negative control),

1.9, 6, and 19 mg/mL and at 0 (DMSO negative control), 4,

13, and 40 mg/mL of the medium in the presence and absence of

metabolic activation, respectively, with 4-hour exposure.

Additionally, Almega PL was exposed to CHO cells in duplicate

at concentrations of 0 (DMSO negative control), 1.75, 3.5, and

7 mg/mL of the medium in the absence of metabolic activation,

with 21-hour exposure. Ethyl methanesulfonate (600 mg/mL)

was used as the positive control in the absence of S9 mix,

and cyclophosphamide monohydrate (55 mg/mL) was used as

the positive control in the presence of S9 mix. Following expo-

sure of the cell cultures to the test items for 4 or 21 hours, they

were treated with Colchicine to arrest the cells in a metaphase-

like stage of mitosis (c-metaphase). Cells were then harvested

and chromosome preparations were made. Preparations were

stained, and metaphases were analyzed under microscope for

chromosomal aberrations. Concurrent cytotoxicity for all treated

and control cultures was also assessed.

In vivo micronucleus assay. The micronucleus assay was per-

formed in order to determine the potential of Almega PL to

cause genotoxicity in the form of micronuclei containing lag-

ging chromosome fragments (clastogenicity) or whole chromo-

somes (aneugenicity). Prior to the main experiment, a DRF

study was performed to determine the MTD of Almega PL and

to select the doses for the main experiment. Due to a lack of

observed toxicity in the DRF study, the MTD was set at 2000

mg/kg/d, the highest dose tested. For the main study, groups of

5 male and 5 female Sprague-Dawley rats each were adminis-

tered 0, 500, 1000, or 2000 mg/kg/d Almega PL in extra virgin

olive oil by oral gavage, 2 times, at an interval of 24 hours. Five

male and five female rats served as positive controls and

received 15 mg/kg cyclophosphamide monohydrate in sterile

water (10 mL/kg) as a single intraperitoneal injection. The

vehicle control and Almega PL-treated rats were observed

twice daily for clinical signs and mortality on days 1 and 2 and

once on day 3. Positive control rats were observed twice for

clinical signs and mortality on day 1 and once on day 2. Body

Table 4. Tissue Collection and Organ Weighing.

Tissue/organ Tissue/organ Tissue/organ

Adrenalsa Kidneysa Sciatic nervesAll gross lesions Lacrimal glands Seminal vesicles and coagulating glandsa

Aorta Larynx SkinBone marrow smear Livera Spinal cord (cervical, mid thoracic, lumbar)Brain (cerebrum, cerebellum, and

medulla)aLungs (with bronchi and bronchioles) Spleena

Cecum Mandibular lymph nodes Sternum with marrowColon Mesenteric lymph nodes StomachDuodenum Esophagus Testesa

Epididymidesa Ovariesa Thymusa

Eyes with optic nerve Oviducts Thyroid with parathyroidsa

Mammary gland Pancreas TongueFemur bone with distal joint Pharynx TracheaFemoral muscle Pituitarya UretersHearta Prostatea Urinary bladderIleum with Peyer patches Rectum Uterus with cervixa

Jejunum Salivary glands (sublingual, submandibular, andparotid)

Vagina

aOrgans weighed.

Kagan et al 5



weights of rats were recorded on days 1, 2, and 3, except for the

body weights of positive control animals which were recorded

on days 1 and 2. All rats were anesthetized with isoflurane and

killed 23 to 24 hours following the second treatment of Almega

PL. After killing, 1 femur was removed from either side of the

body after clearing the musculature. Femur heads were

trimmed to expose the marrow canals, femur bone marrow was

flushed, collected in centrifuge tubes, and smears were pre-

pared and stained. Prepared slides were scored (minimum of

2000 polychromatic erythrocytes [PCE] from each rat) for the

incidence of micronuclei in immature erythrocytes or PCEs and

the proportion of immature erythrocytes among total erythro-

cytes (number of PCE divided by number of total red blood

corpuscle [RBC]) was determined for each rat by counting at

least 200 to 256 erythrocytes.

Statistical Analyses

Maximum-tolerated dose oral toxicity study. For the MTD oral

toxicity study, group means and standard deviations were cal-

culated for male and female body weights. Statistical analysis

was performed by 2-way analysis of variance (ANOVA) fol-

lowed by Bonferroni post hoc analysis.

Subchronic oral toxicity study. For all quantitative variables like

functional observation, parameters (neuromuscular observa-

tions, motor activity, body weight, and body temperature) were

tested for normality and homogeneity of variances within the

group before performing a 1-factor ANOVA modeling by treat-

ment groups. When the data were found to be heterogeneous, a

suitable transformation was performed. In the case of nonpara-

metric data, the Kruskal-Wallis test was used when the var-

iances were significantly different. Comparison of means

between treatment groups and control group was done using

Dunnett test when the overall treatment ‘‘F’’ test was found

significant. The data pertaining to male and female rats were

evaluated separately. All analyses and comparisons were eval-

uated at the 5% (P ¼ 0.05) level.

Chromosome aberration assay. Statistical analysis of the experi-

mental data was carried out using validated SYSTAT Statisti-

cal package ver. 12.0. Data were analyzed for proportions of

aberrant metaphases in each sample excluding gaps as aberra-

tions were analyzed. Pooled data from each test concentration

and the positive control were compared with the vehicle control

using Fisher exact test. All analysis and comparisons were

evaluated at 5% (P ¼ 0.05) level.

In vivo micronucleus assay. The statistical analysis of the experi-

mental data was carried out using validated copies of the

SYSTAT Statistical package ver. 12.0. All quantitative vari-

ables like change in net body weight were tested for normality

(Shapiro-Wilk test) and homogeneity (Levene test) of within

group variance before performing ANOVA. For percentages,

data were normalized using square root transformation before

ANOVA. Comparison of means between the control and the

treatment groups was done using Dunnett test where ‘‘F’’ test

was found to be significant in ANOVA. All analyses and com-

parisons were evaluated at 5% (P < 0.05) level.

Results

Maximum-Tolerated Dose Oral Toxicity Study

No mortalities occurred during the study. No abnormal clinical

signs were observed in any of the animals. Body weight

increases were observed in all groups over time as shown in

Figure 1. However, when compared to the control group, no

statistically significant differences between groups were identi-

fied. No gross pathological findings were observed in any ani-

mals at study termination. Under the conditions of this study, it is

concluded that Almega PL is safe and tolerated in rats at doses

up to 2500 mg/kg, the highest dose tested in this study.

Subchronic Oral Toxicity Study

No mortalities or treatment-related clinical signs were

observed at any of the doses tested. Dark-colored feces were

Figure 1. Mean body weights of male and female rats during the 14-day study.




observed in all rats of the high-dose group. The dark-

colored feces were attributed to the high chlorophyll content

of Almega PL and considered a treatment-related nonad-

verse effect. No statistically significant changes were

observed in food consumption for any treated group as com-

pared to the control group. No treatment-related changes

were observed in body weights at any of the tested doses

in either sex during the entire treatment period (Figure 2).

The results of ophthalmological examinations were normal

for all of the study animals.

Neurological examinations including functional tests

were conducted on both male and female rats and were gener-

ally negative. Statistically significant differences were identi-

fied for forelimbs grip strength in mid-dose males and low- and

high-dose females, hindlimbs foot splay in mid- and high-dose

females, body temperature in high-dose males and low- and

high-dose females, and motor activity in low- and mid-dose

females (Table 5). However, these changes were considered

to be incidental as either the changes were of minimal magni-

tude or not dose dependent.

Figure 2. Mean body weights of male and female rats during the 90-day study.

Table 5. Selected Results From the Functional Observation Battery.a

Sex Males Females

Dose, mg/kg/d 0 250 500 2500 0 250 500 2500

Forelimbs grip strength(GF)

1125.70 +80.22

1076.00 +46.54

1062.10 +19.46b

1160.60 +61.08

953.10 +17.39

996.10 +37.07b

961.80 +31.83

911.50 +32.71b

Hindlimbs foot splay,mm

84.50 +24.52

99.90 +12.37

80.00 +13.26

89.10 +12.96

52.10 +9.90

58.20 + 9.82 73.90 +12.61b

70.80 +9.65b

Body temperature, �C 36.61 36.67 37.09 37.36b 38.09 37.18b 37.76 37.21b

Motor activityAmbulatory time—interval 2, s

202.80 +37.95

222.10 +56.22

203.00 +44.12

219.90 +41.87

171.10 +45.09

209.80 +43.53

222.10 +26.81b

200.30 +37.43

Horizontal counts—interval 3

758.70 +213.88

864.10 +223.02

711.60 +227.76

815.20 +150.60

648.80 +325.66

1024.70 +202.89b

899.10 +246.39

914.40 +327.63

Ambulatorycounts—interval 3

508.20 +194.10

557.50 +165.95

472.20 +164.30

507.70 +133.73

404.70 +227.92

682.00 +161.77b

580.20 +197.71

581.90 +220.37

Ambulatorycounts—total

2955.90 +672.60

2940.80 +675.53

2676.40 +566.06

2471.10 +742.46

2515.90 +841.83

3128.50 +415.33

3482.70 +758.07b

3082.70 +898.64

aData for all end points except body temperature are presented as mean values+ standard deviations with N¼ 10; body temperature presented as mean with N¼ 10.bStatistically significant from the vehicle control group (P � 0.05).

Kagan et al 7



Data from the hematology, coagulation, and clinical chem-

istry analyses performed on day 91 are presented in Tables 6

and 7 for males and females, respectively. Statistically signif-

icant changes in hematology parameters were seen in high-dose

males including hematocrit (HCT), mean corpuscular

hemoglobin concentration (MCHC), and mean platelet volume

(MPV); MPV was also statistically different in mid-dose males.

In female rats, statistically significant hematological changes

including RBC, HCT, mean corpuscular hemoglobin, MCHC,

and MPV were seen in the mid- and high-dose groups. These

Table 6. Summary of Hematology, Coagulation, and Clinical Chemistry Values on Day 91—Males.a

Parameter 0 mg/kg/d 250 mg/kg/d 500 mg/kg/d 2500 mg/kg/d

HematologyWBC, G/L 9.72 + 2.64 9.28 + 2.21 9.14 + 1.85 9.04 + 1.39RBC, T/L 9.14 + 0.30 9.23 + 0.27 9.21 + 0.27 9.47 + 0.25HGB, g/L 168 + 5 166 + 4 166 + 4 170 + 5HCT, L/L 0.520 + 0.014 0.519 + 0.013 0.522 + 0.017 0.537 + 0.017b

MCV, fL 56.9 + 1.1 56.3 + 1.0 56.7 + 1.1 56.7 + 1.7MCH, pg 18.4 + 0.3 18.0 + 0.3 18.0 + 0.4 18.0 + 0.6MCHC, g/L 323 + 4 321 + 4 318 + 6 317 + 4Plat, G/L 859 + 73 898 + 114 902 + 114 837 + 98MPV, fL 8.3 + 0.7 8.4 + 0.6 8.8 + 0.6b 9.9 + 0.8b

Neut, % 19.2 + 4.6 19.2 + 5.9 16.5 + 4.9 16.7 + 4.7Lymp, % 76.6 + 4.7 76.7 + 6.3 79.4 + 5.2 79.3 + 5.4Mono, % 1.8 + 0.5 1.8 + 0.4 1.9 + 0.4 2.0 + 0.7Eosi, % 0.7 + 0.1 0.6 + 0.2 0.8 + 0.4 0.7 + 0.2Baso, % 0.4 + 0.1 0.3 + 0.1 0.3 + 0.1 0.3 + 0.1Neut A, G/L 1.83 + 0.57 1.72 + 0.53 1.47 + 0.39 1.50 + 0.43Lymp A, G/L 7.48 + 2.30 7.18 + 1.95 7.30 + 1.77 7.19 + 1.32Mono A, G/L 0.18 + 0.08 0.16 + 0.06 0.17 + 0.03 0.18 + 0.05Eosi A, G/L 0.07 + 0.02 0.06 + 0.01 0.07 + 0.02 0.06 + 0.01Baso A, G/L 0.04 + 0.02 0.03 + 0.02 0.03 + 0.01 0.03 + 0.01Retic, % 2.46 + 0.28 2.51 + 0.21 2.51 + 0.09 2.31 + 0.24Retic A, T/L 0.224 + 0.023 0.231 + 0.019 0.231 + 0.009 0.219 + 0.024

CoagulationPT, s 17.2 + 0.6 16.7 + 1.1 16.4 + 0.4 16.5 + 0.7APTT, s 16.2 + 1.7 15.5 + 2.9 12.9 + 2.1b 14.6 + 3.4

Clinical chemistryGlu, mmol/L 7.53 + 0.66 7.46 + 0.45 7.54 + 0.83 7.79 + 0.97BUN, mmol/L 4.43 + 0.51 4.79 + 0.39 4.88 + 0.41 5.41 + 0.43b

Creat, mmol/L 31 + 9 30 + 6 28 + 8 25 + 7AST, U/L 86 + 21 91 + 14 77 + 9 94 + 11ALT, U/L 56 + 7 56 + 8 60 + 8 78 + 13b

GGT, U/L 1 + 1 1 + 1 1 + 1 1 + 1ALP, U/L 118 + 23 108 + 16 113 + 15 120 + 20TBil, mmol/L 1.66 + 0.63 1.33 + 0.61 1.45 + 0.54 1.35 + 0.75TChol, mmol/L 2.65 + 0.17 2.66 + 0.23 2.63 + 0.29 3.20 + 0.43b

Trig, mmol/L 0.55 + 0.10 0.50 + 0.15 0.61 + 0.12 0.54 + 0.09TPro mmol/L 71.3 + 2.9 72.2 + 2.4 73.1 + 1.5 71.8 + 3.5Alb, g/L 42.2 + 1.8 42.3 + 1.6 42.7 + 0.6 42.8 + 1.9Pi, mmol/L 1.94 + 0.23 2.00 + 0.18 2.06 + 0.20 2.05 + 0.14Ca, mmol/L 2.65 + 0.07 2.63 + 0.06 2.61 + 0.07 2.51 + 0.09b

Na, mEq/L 147.2 + 2.2 148.6 + 0.5 148.7 + 0.5 148.8 + 1.0K, mEq/L 4.09 + 0.17 4.32 + 0.26 4.07 + 0.35 4.47 + 0.39b

Cl, mEq/L 111.5 + 1.1 112.2 + 0.7 111.9 + 0.6 112.1 + 1.2A/G, ratio 1.46 + 0.10 1.42 + 0.11 1.41 + 0.10 1.49 + 0.14Glob, g/L 29.0 + 1.9 29.9 + 2.0 30.4 + 1.8 29.0 + 2.6

Abbreviations: WBC, white blood corpuscles; RBC, red blood corpuscles; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, meancorpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Plat, platelets; MPV, mean platelet volume; Neut, neutrophils; Lymp, lymphocytes;Mono, monocytes; Eosi, eosinophils; Baso, basophils; Retic, reticulocyte counts; PT, prothrombin time; APTT, activated partial thromboplastin time; Glu, glucose;BUN, blood urea nitrogen; Creat, creatine; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, g-glutamyl transpeptidase; ALP, alkalinephosphatase; TBil, total bilirubin; TChol, total cholesterol; Trig, triglycerides; TPro, total plasma protein; Alb, albumin; Pi, inorganic phosphorous; Ca, calcium; Na,sodium; K, potassium; Cl, chloride; A/G, albumin/globulin ratio (calculated values).aAll data are presented as mean values + standard deviations with N ¼ 10.bStatistically significant from the vehicle control group (P � 0.05).




changes were considered incidental due to the lack of a dose–

response relationship and/or the changes being within the phy-

siological range. For coagulation parameters, a decrease in

activated partial thromboplastin time was seen in mid-dose

males, and an increase in prothrombin time was seen in high-

dose females. These values were considered incidental due to

the lack of a dose response and the minor magnitude of the

changes. Clinical chemistry values including blood urea nitro-

gen (BUN), alanine aminotransferase (ALT), total cholesterol

(TChol), calcium (Ca), and potassium (K) were statistically

Table 7. Summary of Hematology, Coagulation, and Clinical Chemistry Values on Day 91—Females.a

Parameter 0 mg/kg/d 250 mg/kg/d 500 mg/kg/d 2500 mg/kg/d

HematologyWBC, G/L 6.58 + 2.22 6.62 + 1.43 6.56 + 1.27 7.57 + 2.17RBC, T/L 8.21 + 0.28 8.45 + 0.22 8.68 + 0.32b 8.59 + 0.26b

HGB, g/L 156 + 4 156 + 3 159 + 5 159 + 4HCT, L/L 0.483 + 0.014 0.490 + 0.011 0.503 + 0.016b 0.500 + 0.014b

MCV, fL 58.9 + 1.2 58.0 + 1.3 58.0 + 1.5 58.3 + 0.8MCH, pg 19.0 + 0.5 18.5 + 0.3b 18.3 + 0.4b 18.5 + 0.3b

MCHC, g/L 322 + 4 319 + 4 316 + 5b 317 + 4b

Plat, G/L 868 + 122 943 + 120 949 + 140 982 + 105MPV, fL 9.1 + 1.1 8.6 + 0.6 9.7 + 0.9b 9.9 + 0.4b

Neut, % 15.3 + 4.2 14.5 + 5.6 16.6 + 5.7 12.0 + 2.2Lymp, % 80.6 + 4.6 81.7 + 5.8 79.5 + 6.2 84.1 + 2.3Mono, % 1.9 + 0.8 1.5 + 0.3 1.7 + 0.6 1.8 + 1.1Eosi, % 0.9 + 0.2 0.8 + 0.2 0.9 + 0.4 0.9 + 0.3Baso, % 0.3 + 0.1 0.3 + 0.1 0.3 + 0.1 0.3 + 0.1Neut A, G/L 1.00 + 0.37 0.98 + 0.56 1.10 + 0.51 0.89 + 0.24Lymp A, G/L 5.32 + 1.92 5.38 + 1.10 5.20 + 1.01 6.39 + 1.93Mono A, G/L 0.13 + 0.06 0.10 + 0.04 0.12 + 0.05 0.13 + 0.08Eosi A, G/L 0.06 + 0.01 0.05 + 0.02 0.06 + 0.02 0.07 + 0.03Baso A, G/L 0.02 + 0.01 0.02 + 0.01 0.02 + 0.01 0.02 + 0.01Retic, % 2.84 + 0.54 2.58 + 0.39 2.37 + 0.39 2.30 + 0.65Retic A, T/L 0.233 + 0.040 0.217 + 0.031 0.205 + 0.030 0.197 + 0.053

CoagulationPT, s 16.5 + 1.0 16.5 + 0.7 17.2 + 0.8 18.2 + 0.9b

APTT, s 14.6 + 3.1 14.8 + 2.2 13.2 + 3.6 13.4 + 2.5Clinical chemistry

Glu, mmol/L 7.36 + 0.52 7.76 + 0.51 8.13 + 1.52 7.36 + 0.71BUN, mmol/L 4.59 + 0.44 5.17 + 0.53 5.02 + 0.65 5.65 + 0.76b

Creat, mmol/L 30 + 8 27 + 9 27 + 9 33 + 6AST, U/L 118 + 22 87 + 14b 83 + 10b 92 + 14b

ALT, U/L 50 + 4 50 + 8 45 + 8 64 + 8b

GGT, U/L 0 + 1 1 + 1 0 + 1 2 + 1b

ALP, U/L 85 + 14 87 + 12 87 + 25 98 + 16TBil, mmol/L 2.04 + 1.17 1.78 + 0.76 2.31 + 1.19 1.68 + 0.65TChol, mmol/L 3.10 + 0.27 3.23 + 0.16 3.07 + 0.35 3.60 + 0.35b

Trig, mmol/L 0.47 + 0.09 0.45 + 0.05 0.44 + 0.10 0.50 + 0.09TPro, mmol/L 69.9 + 2.6 70.7 + 2.2 71.0 + 3.4 73.6 + 2.7b

Alb, g/L 42.4 + 0.7 44.2 + 3.1 42.6 + 1.8 42.75 + 2.2b

Pi, mmol/L 1.73 + 0.22 1.79 + 0.14 1.80 + 0.22 1.89 + 0.18Ca, mmol/L 2.59 + 0.07 2.59 + 0.05 2.53 + 0.18 2.62 + 0.09Na, mEq/L 147.9 + 1.5 147.6 + 1.0 147.9 + 1.4 148.7 + 0.9K, mEq/L 3.83 + 0.55 3.78 + 0.14 3.83 + 0.24 4.03 + 0.40Cl, mEq/L 112.6 + 1.0 112.2 + 1.0 112.6 + 0.9 112.9 + 1.0A/G, ratio 1.55 + 0.14 1.72 + 0.42 1.51 + 0.12 1.82 + 0.16b

Glob, g/L 27.5 + 2.5 26.5 + 3.6 28.4 + 2.3 26.2 + 1.8

Abbreviations: WBC, white blood corpuscles; RBC, red blood corpuscles; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, meancorpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Plat, platelets; MPV, mean platelet volume; Neut, neutrophils; Lymp, lymphocytes;Mono, monocytes; Eosi, eosinophils; Baso, basophils; Retic, reticulocyte counts; PT, prothrombin time; APTT, activated partial thromboplastin time; Glu, glucose;BUN, blood urea nitrogen; Creat, creatine; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, g-glutamyl transpeptidase; ALP, alkalinephosphatase; TBil, total bilirubin; TChol, total cholesterol; Trig, triglycerides; TPro, total plasma protein; Alb, albumin; Pi, inorganic phosphorous; Ca, calcium; Na,sodium; K, potassium; Cl, chloride; A/G, albumin/globulin ratio (calculated values).aAll data are presented as mean values + standard deviations with N ¼ 10.bStatistically significant from the vehicle control group (P � 0.05).

Kagan et al 9



different in the high-dose males when compared to controls.

The BUN, aspartate aminotransferase, ALT, g-glutamyl trans-

peptidase, TChol, total plasma protein, albumin, and albumin/

globulin ratio were statistically different in high-dose females

when compared to controls. Aspartate aminotransferase was

also statistically different in low- and mid-dose females. The

minimal increases in ALT activity (39% for males and 28% for

females) and TChol levels (21% for males and 16% for

females) seen at the highest dose (2500 mg/kg/d) were consid-

ered to be a test item-related, nonadverse effect due to the

severity of change being limited and below the threshold of

concern. In addition, the increased ALT activity was not

accompanied by correlating histologic changes in the liver. All

other statistically significant changes in clinical chemistry were

of a low magnitude and within the physiological range and

therefore considered toxicologically insignificant. No

treatment-related changes were seen in any of the urine para-

meters analyzed in terminally killed animals.

When compared to the control groups, no significant

changes attributed to treatment were seen in organ weights or

organ to body weight ratios for male or female animals at any

dose level evaluated (Tables 8 and 9). Statistically significant

changes were limited to increased thyroid weights (absolute

and relative) in low- and high-dose males, increased adrenals

weight in mid- (absolute) and high-dose (relative) females, and

increased relative liver weights in high-dose females. These

were considered incidental due to the absence of clinical signs

and histopathological changes.

Compared to their respective control groups, no test item-

related gross pathological lesions were evident in male or

female rats at terminal necropsy (day 91). No incidental find-

ings were seen in males. Incidental findings were found in

females as dilated uterus noted across the tested groups (2 per

test group including controls). The dilated uterus findings were

confirmed microscopically (mild to severe dilation across all

treatment groups including controls) and was not considered to

Table 8. Summary of Terminal Fasting Body Weights and Absolute Organ Weights on Day 91.a

Group

Males Females

0mg/kg/d

250mg/kg/d

500mg/kg/d

2500mg/kg/d

0mg/kg/d

250mg/kg/d

500mg/kg/d

2500mg/kg/d

Terminal fasting BW, g 420.72 +37.17

429.34 +37.14

421.29 +32.71

424.90 +32.04

252.36 +14.06

260.70 +14.49

259.48 +18.03

243.39 +12.44

Adrenals, g 0.0538 +0.0068

0.0544 +0.0055

0.0566 +0.0055

0.0572 +0.0048

0.0602 +0.0061

0.0643 +0.0050

0.0674 +0.0036b

0.0653 +0.0071

Brain, g 1.9636 +0.0804

2.0037 +0.0770

1.9731 +0.0938

2.0226 +0.1018

1.8508 +0.0689

1.8205 +0.0531

1.8805 +0.0527

1.8486 +0.0489

Epididymides, g 1.3437 +0.0321

1.3612 +0.0729

1.3159 +0.0566

1.3492 +0.1044

– – – –

Heart, g 1.3063 +0.1234

1.3493 +0.0890

1.3235 +0.1517

1.3993 +0.1269

0.8871 +0.0659

0.9263 +0.0507

0.9490 +0.0702

0.9084 +0.0849

Kidneys, g 2.2479 +0.1927

2.2841 +0.1547

2.1974 +0.2168

2.3741 +0.2300

1.4327 +0.0863

1.4397 +0.0871

1.4984 +0.1006

1.4403 +0.0869

Liver, g 11.2948 +1.5264

11.1451 +1.3716

11.3951 +1.1856

12.0852 +1.3774

6.4700 +0.3399

6.6165 +0.3849

6.5445 +0.5758

6.7227 +0.5350

Ovaries – – – – 0.0886 +0.0109

0.0794 +0.0138

0.0916 +0.0104

0.0899 +0.0143

Pituitary, gc 0.0128 +0.0021

0.0122 +0.0023

0.0122 +0.0028

0.0132 +0.0014

0.0141 +0.0014

0.0140 +0.0019

0.0142 +0.0035

0.0145 +0.0020

Prostate, g 1.1370 +0.1241

1.0702 +0.1731

1.1452 +0.1348

1.1797 +0.1418

– – – –

Seminal vesicles andcoagulating glands, g

1.8415 +0.2714

1.7696 +0.3712

1.7238 +0.1837

1.8494 +0.2565

– – – –

Spleen, g 0.7734 +0.0990

0.7919 +0.1067

0.07675 +0.0964

0.8095 +0.1058

0.6358 +0.0525

0.6008 +0.0362

0.6382 +0.1240

0.6201 +0.1160

Testes, g 3.8115 +0.1292

3.8862 +0.2085

3.8779 +0.2073

3.8856 +0.1647

– – – –

Thymus, g 0.4015 +0.0399

0.4209 +0.0750

0.4144 +0.0477

0.4041 +0.0814

0.3118 +0.0455

0.3084 +0.0466

0.3120 +0.0357

0.3741 +0.1857

Thyroid with parathyroids, gc 0.0282 +0.0041

0.0351 +0.0067b

0.0315 +0.0055

0.0394 +0.0049b

0.0259 +0.0040

0.0310 +0.0056

0.0299 +0.0087

0.0275 +0.0040

Uterus with cervix, g – – – – 0.7654 +0.3443

0.7138 +0.2771

0.8041 +0.5670

0.8099 +0.4375

aAll data are presented as mean values + standard deviations with N ¼ 10.bStatistically significant from the vehicle control group (P � 0.05).cData recorded on day 93.




be treatment related. All other microscopic findings (inflam-

matory foci in esophageal adnexal tissue, proteinaceous mate-

rial in kidney tubules, harderian gland alterations, hepatocyte

vacuolation, mesenteric lymph node erythrophagocytosis,

mandibular lymph node plasmacytosis and/or erythrophagocy-

tosis, unilateral ovarian cysts, prostate mononuclear cell infil-

tration, and rectum nematode) occurred at low frequencies in

the control and high-dose groups and were not considered treat-

ment related.

Under the conditions of this study, the ‘‘no observed adverse

effect level’’ (NOAEL) for Almega PL would be 2500 mg/kg/d

based on a lack of treatment-related effects at 2500 mg/kg/d,

the highest dose evaluated in both male and female rats. How-

ever, because dose formulation analyses revealed consistently

lower (approximately �20%) concentrations of the test item

from the corresponding theoretical doses, the highest intended

dose of 2500 mg/kg/d would result in an actual dose of 2000

mg/kg/d. Therefore, the NOAEL for the subchronic oral

gavage administration of Almega PL in rats is determined as

2000 mg/kg/d.

Bacterial Reverse Mutation Assays

Genotypic characterization of tester bacteria indicated that the

assays were valid because (1) all S typhimurium strains demon-

strated the requirement of histidine amino acid for their growth

and E coli strain WP2uvrA demonstrated the requirement of

tryptophan amino acid for its growth, (2) TA98, TA100, and

WP2uvrA demonstrated their typical resistance to ampicillin,

(3) the presence of characteristic mutations like the rfa muta-

tion was demonstrated by all S typhimurium strains by their

sensitivity to crystal violet, (4) The uvrA mutation in the E coli

strain and the uvrB mutation in the S typhimurium strains were

demonstrated by their sensitivity to UV light, (5) all of the

tester strains produced spontaneous revertant colonies which

were within the frequency ranges of historical control data, and

(6) the numbers of revertant colonies in the positive control

groups were greater than 3 times the number of revertant colo-

nies in the corresponding negative controls.

The summarized results for experiments I and II are pre-

sented in Tables 10 and 11, respectively. Slight precipitation of

Table 9. Summary of Organ to Body Weight Ratios on Day 91.a

Group

Males Females

0mg/kg/d

250mg/kg/d

500mg/kg/d

2500mg/kg/d

0mg/kg /d

250mg/kg/d

500mg/kg/d

2500mg/kg/d

Adrenals, % 0.0128 +0.0017

0.0128 +0.0016

0.0135 +0.0017

0.0135 +0.0015

0.0239 +0.0028

0.0247 +0.0021

0.0261 +0.0021

0.0268 +0.0024b

Brain, % 0.4690 +0.0319

0.4695 +0.0406

0.4697 +0.0246

0.4776 +0.0315

0.7354 +0.0474

0.7001 +0.0411

0.7276 +0.0498

0.7607 +0.0283

Epididymides, % 0.3216 +0.0291

0.3186 +0.0249

0.3136 +0.0221

0.3188 +0.0296

– – – –

Heart, % 0.3107 +0.0157

0.3150 +0.0156

0.3141 +0.0267

0.3292 +0.0135

0.3515 +0.0165

0.3556 +0.0148

0.3665 +0.0260

0.3728 +0.0225

Kidneys, % 0.5353 +0.0324

0.5337 +0.0338

0.5216 +0.0309

0.5585 +0.0265

0.5682 +0.0274

0.5538 +0.0447

0.5787 +0.0389

0.5918 +0.0207

Liver, % 2.6760 +0.1536

2.5917 +0.1568

2.7044 +0.1747

2.8375 +0.1362

2.5676 +0.1381

2.5415 +0.1492

2.5239 +0.1673

2.7601 +0.1251b

Ovaries – – – – 0.0351 +0.0038

0.305 +0.0054

0.0353 +0.0031

0.0369 +0.0056

Pituitary, % 0.0030 +0.0005

0.0028 +0.0005

0.0029 +0.0006

0.0031 +0.0002

0.0056 +0.0006

0.0054 +0.0007

0.0055 +0.0013

0.0059 +0.0007

Prostate, % 0.2733 +0.0475

0.2521 +0.0527

0.2730 +0.0363

0.2796 +0.0428

– – – –

Seminal vesicles andcoagulating glands, %

0.4386 +0.0609

0.4170 +0.1066

0.4097 +0.0385

0.4363 +0.0595

– – – –

Spleen, % 0.1835 +0.0117

0.1850 +0.0239

0.1825 +0.0216

0.1905 +0.0208

0.2524 +0.0227

0.2309 +0.0161

0.2462 +0.0434

0.2537 +0.0388

Testes, % 0.9117 +0.0790

0.9084 +0.0542

0.9252 +0.0840

0.9192 +0.0818

– – – –

Thymus, % 0.0958 +0.0100

0.0983 +0.0178

0.0988 +0.0133

0.0947 +0.0157

0.1234 +0.0158

0.1179 +0.0136

0.1203 +0.0121

0.1550 +0.0811

Thyroid with parathyroids, % 0.0067 +0.0007

0.0082 +0.0014b

0.0075 +0.0012

0.0093 +0.0011b

0.0103 +0.0015

0.0119 +0.0022

0.0115 +0.0034

0.0113 +0.0017

Uterus with cervix, % – – – – 0.3042 +0.1424

0.2732 +0.1023

0.3102 +0.2222

0.3324 +0.1753

aAll data are presented as mean values + standard deviations with N ¼ 10.bStatistically significant from the vehicle control group (P � 0.05).

Kagan et al 11



Almega PL was visually observable in all test strains both with

and without S9 mix at concentrations of 1581 mg/plate and

higher in experiment I and 1880 mg/plate and higher in experi-

ment II. However, this slight precipitation did not interfere with

the scoring of the bacterial background lawn intensity. No

toxicity was observed up to the highest concentration of 5000

mg/plate for experiment I or II, as the intensity of the bacterial

background lawn as well as the mean number of revertant

colonies were comparable to the DMSO control. For both

experiment I and II, no positive mutagenic responses were

observed up to the top dose of 5000 mg/plate in any of the tester

strains both in the presence and absence of metabolic activa-

tion. It is concluded that all criteria for the bacterial reverse

mutation assays are valid and that under the conditions of this

assay, Almega PL was not mutagenic at any of the doses tested.

Chromosome Aberration Assay

The results for the chromosome aberration assay are presented

in Table 12. After 4 hours of exposure and in the presence of

metabolic activation, cytotoxicity was seen at 19 mg/mL, the

highest concentration tested. Similarly, cytotoxicity was seen

at the highest doses tested in the absence of metabolic activa-

tion following 4 hours of exposure (40 mg/mL) and 21 hours of

exposure (7 mg/mL). For all doses and times, and in the absence

or presence of metabolic activation, no positive incidence of

chromosomal aberrations was observed following treatment

with Almega PL. The in vitro mammalian chromosome aberra-

tion assay is considered valid because (1) the incidence of

aberrations in the vehicle control cultures is in the range of

in-house historical control data and (2) the positive control

substances produced a significant increase in the incidence of

aberrations compared to the respective vehicle control.

In Vivo Micronucleus Assay

No mortality was seen in any of the tested animals, and body

weight gains were comparable to vehicle controls. No

treatment-related clinical signs were observed in any dose

groups except for dark-colored feces seen at 1000 and 2000

mg/kg/d on days 2 and 3. The dark-colored feces were attrib-

uted to the high chlorophyll content of Almega PL and consid-

ered a treatment-related nonadverse effect. Data for the total

RBC ratio and micronucleus incidence are summarized in

Table 13. No significant increases in micronucleated PCE were

observed between mice treated with Almega PL and the vehicle

control. A significant decrease in PCE:total RBC ratio was seen

in males at 500 and 2000 mg/kg/d and for combined sex at 500

Table 10. Summary of Results of Initial Mutation Assay.

Treatment, mg/plate

No. of revertants/platea

TA98 TA100 TA1535 TA1537 WP2uvrA (pKM101)

Vehicle control—DMSO (100 mL)With metabolic activation 21 + 3 112 + 4 18 + 1 10 + 2 137 + 4Without metabolic activation 24 + 3 106 + 8 19 + 1 11 + 2 132 + 4

50With metabolic activation 20 + 2 100 + 4 16 + 1 10 + 2 132 + 5Without metabolic activation 21 + 1 99 + 4 16 + 2 10 + 2 131 + 3





Positive controlWith metabolic activation 544 + 8b 787 + 9b 132 + 3b 123 + 6b 591 + 4c

Without metabolic activation 231 + 6d 578 + 6e 131 + 4e 125 + 4f 605 + 10g

Abbreviations: DMSO, dimethyl sulfoxide; SD, standard deviation.aValues are means + SDs of 3 replicates and are rounded off to the nearest whole number.bTA98, TA100, TA1535, and TA1537 with activation: 2-aminoanthracene (4 mg/plate).cWP2uvrA (pKM101) with activation: 2-aminoanthracene (30 mg/plate).dTA98 without activation: 2-nitrofluorene (2 mg/plate).eTA100 and TA1535 without activation: sodium azide (1 mg/plate).fTA1537 without activation: 9-aminoacridine (50 mg/plate).gP2uvrA (pKM101) without activation: 4-nitroquinoline-1-oxide (4 mg/plate).




and 1000 mg/kg/d. However, the values were comparable to

historical data ranges and therefore determined to be nonbio-

logically relevant. The test was considered valid because (1)

the incidence of micronucleated PCE in the vehicle control

group was within the historical control range, (2) the positive

control animals demonstrated a significant increase in micro-

nucleated PCE compared to controls, and (3) at the end of the

study, the weight variation in animals was minimal and did not

exceed +20% of the mean weight of each sex.

Discussion

Omega-3 fatty acids possess significant health benefits for

brain function and development as well as cardiovascular con-

ditions.17,18 Currently most of these fatty acids isolated for

human consumption are sourced from fish. However, due to

a limited amount of global fish stocks, alternative sources of

fatty acids are needed. Potential substitutes include oil extracts

from plants, krill, microalgae, and microalgae-like protists.

Of the potential substitutes, algae may be one of the more

promising alternatives. Due to their small size, algae are easy to

grow on a large scale. Algae do not require freshwater or arable

land and are capable of producing significant amounts of fatty

acids even under adverse conditions.18,19 In the past, the

potential for using microalgal oils on a larger scale has been

limited by cost, extraction, and purification methods.17 At pres-

ent, various strains of algae are being used for the production of

fatty acids for human consumption. For example, Schizochy-

trium sp and/or Crypthecodinium cohnii are included in infant

formulas, food additives, cosmetics, and pharmaceutical

products.17

Nannochloropsis oculata cultures have been used as a

source of live food for rotifers in marine fish hatcheries and

as crustacean feed for over 30 years.20 No data were located in

the published literature indicating a history of consumption of

N oculata-derived oil products by humans, either in conven-

tional food or dietary supplements. Currently, Almega PL is not

regulated by the US Food and Drug Administration and has not

been determined Generally Recognized As Safe (GRAS) as a

food ingredient. However, EPA has been evaluated and

approved in GRAS notifications and food additive petitions for

use as an o-3 fatty acid. In addition, the FDA has stated that

dietary supplements should not recommend or suggest in their

labeling a daily intake exceeding 2 g EPA and DHA.9

Previously, the acute and subchronic oral toxicities of

N oculata biomass were evaluated in male and female

Sprague-Dawley rats.1 The oral median lethal dose of N ocu-

lata was greater than 12 g/kg and the subchronic toxicity was

Table 11. Summary of Results of Confirmatory Mutation Assay.

Treatment, mg/plate

No. of revertants/platea

TA98 TA100 TA1535 TA1537 WP2uvrA (pKM101)

Vehicle control—DMSO (100 mL)With metabolic activation 23 + 2 106 + 12 18 + 1 11 + 2 134 + 4Without metabolic activation 26 + 4 103 + 10 19 + 2 11 + 2 130 + 3






Positive controlWith metabolic activation 556 + 4b 791 + 7b 128 + 2b 119 + 3b 596 + 6c

Without metabolic activation 235 + 5d 587 + 6e 130 + 2e 126 + 6f 610 + 5g

Abbreviations: DMSO, dimethyl sulfoxide; SD, standard deviation.aValues are means + SDs of 3 replicates and are rounded off to the nearest whole number.bTA98, TA100, TA1535, and TA1537 with activation: 2-aminoanthracene (4 mg/plate).cWP2uvrA (pKM101) with activation: 2-aminoanthracene (30 mg/plate).dTA98 without activation: 2-nitrofluorene (2 mg/plate).eTA100 and TA1535 without activation: sodium azide (1 mg/plate).fTA1537 without activation: 9-aminoacridine (50 mg/plate).gWP2uvrA(pKM101) without activation: 4-nitroquinoline-1-oxide (4 mg/plate).

Kagan et al 13



Tab

le12.

Sum

mar

yR

esults

ofC

hro

moso

me

Aber

ration

Ass

ayW

ith

and

Without

Met

abolic

Act

ivat

ion.a

Tre

atm

ent,mg

/mL

Exposu

re,h

No.ofm

etap

has

essc

ore

d

No.(%

)ofm

etap

has

esw

ith

aber

rationsb

Tota

lno.(%

)ofab

erra

nt

met

aphas

esc

Cel

lgr

ow

thin

hib

itio

n,%

Gap

sBre

aks

Exch

ange

s

Cs

Ct

Cs

Ct

Cs

Ct

RC

Incl

udin

gga

ps

excl

udin

gga

ps

With

met

abolic

activa

tion

DM

SO(1

50mL

)4

200

00

00

00

00

00

1.9

4200

1(0

.5)

1(0

.5)

00

00

02

(1.0

)0

26

64

200

02

(1.0

)0

00

00

2(1

.0)

042

19

4200

00

01

(0.5

)0

00

1(0

.5)

1(0

.5)

51

CPA

55

4200

42

(21.0

)9

(4.5

)1

(0.5

)70

(35.0

)42

(21.0

)31

(15.5

)0

158

(79.0

)127

(63.5

)d38

Without

met

abolic

activa

tion

DM

SO(1

50mL

)4

200

01

(0.5

)0

4(2

.0)

00

05

(2.5

)4

(2.0

)0

44

200

00

03

(1.5

)0

00

3(1

.5)

3(1

.5)

20

13

4200

2(1

.0)

3(1

.5)

1(0

.5)

4(2

.0)

00

010

(5.0

)5

(2.5

)25

40

4200

3(1

.5)

7(3

.5)

1(0

.5)

5(2

.5)

00

013

(6.5

)5

(2.5

)51

EM

S600

4200

15

(7.5

)19

(9.5

)51

(25.5

)91

(45.5

)0

33

(16.5

)4

(2.0

)144

(72.0

)132

(66.0

)d31

DM

SO(1

50mL

)21

200

1(0

.5)

1(0

.5)

00

00

02

(1.0

)0

01.7

521

200

01

(0.5

)0

1(0

.5)

00

02

(1.0

)1

(0.5

)26

3.5

21

200

00

01

(0.5

)0

00

1(0

.5)

1(0

.5)

39

721

200

1(0

.5)

2(1

.0)

02

(1.0

)0

00

5(2

.5)

2(1

.0)

52

EM

S600

21

200

9(4

.5)

33

(16.5

)6

(3.0

)91

(45.5

)9

(4.5

)45

(22.5

)0

121

(60.5

)107

(53.5

)d35

Abbre

viat

ions:

DM

SO,dim

ethyl

sulfo

xid

e;C

s,ch

rom

oso

me

type;

Ct,

chro

mat

idty

pe;

RC

,ri

ng

chro

moso

me;

EM

S,et

hyl

met

han

esulfo

nat

e.a A

ber

rant

met

aphas

esin

cludin

gga

ps

are

not

subje

ctto

stat

istica

lan

alys

is.

bV

alues

are

the

sum

oftw

ore

plic

ates

and

the

valu

esin

par

enth

esis

repre

sent

%.

c Met

aphas

epla

tes

with

one

or

more

aber

rations

are

consi

der

edas

one

met

aphas

epla

tew

ith

aber

rations.

dSt

atis

tica

llysi

gnifi

cant

from

the

vehic

leco

ntr

olgr

oup

(P�

0.0

5).

14 by guest on October 21, 2014ijt.sagepub.comDownloaded from


6 g/kg/d. However, this study focused only on the potential

hepatotoxicity and nephrotoxicity of N oculata. Furthermore,

the study was limited to the evaluation of N oculata biomass

and did not evaluate the potential toxicity of oil extracted from

the biomass. Therefore, the studies reported herein provide new

and relevant data on the safety of N oculata (as Almega PL) oil

extract.

The oral administration of Almega PL at concentrations of

up to 2000 mg/kg/d was well-tolerated by both male and female

rats in the 14-day MTD study and the GLP-compliant 90-day

subchronic toxicity study. Almega PL had no adverse effects

on health or growth as measured by survival, appearance, beha-

vior, food consumption, body weight, or body weight gain. No

treatment-related effects were seen for ophthalmology, neuro-

logical examinations, hematology, coagulation, clinical chem-

istry, gross pathology, absolute and relative organ weights, or

histopathology in animals of any dose group. Although statis-

tical significance was noted for several end points in the 90-day

subchronic toxicity study, these were not considered to be bio-

logically relevant or attributable to Almega PL because these

effects were (1) incidental or sporadic in nature, (2) within the

range of historical control data for rats of this age and strain,

and/or (3) occurred in the absence of corroborative clinical or

histopathological changes.

The battery of GLP-compliant in vitro and in vivo genotoxi-

city studies provide overall support that Almega PL is not

genotoxic. In 2 independent bacterial reverse mutation assays,

Almega PL was negative for mutagenicity at levels up to 5000

mg/plate using S typhimurium strains TA98, TA100, TA1535,

and TA1537 and E coli strain WP2uvrA, both with and without

metabolic activation. Almega PL was not clastogenic as eval-

uated by the in vitro chromosome aberration assay in the CHO

cell line at concentrations of 1.9, 6, or 19 mg/mL and 4, 13, or

40 mg/mL with or without metabolic activation, respectively,

for 4 hours exposure as well as concentrations of 1.75, 3.5, or 7

mg/mL without metabolic activation for 21 hours of exposure.

When evaluated in the in vivo erythrocyte micronucleus assay,

Almega PL was not clastogenic or aneugenic. No treatment-

related structural and/or numerical chromosomal damage was

seen in immature erythrocytes in rats administered Almega PL

at doses of 500, 1000, or 2000 mg/kg/d.

The available human toxicity data for Almega PL are lim-

ited to a randomized, crossover study conducted in 10 male

patients (aged 18-45 years) which compared the appearance

of fatty acids in the blood plasma of healthy humans after

consumption of a high fat meal followed by either Almega

PL or a commercially available krill oil supplement (NKO;

Neptune Technologies & Bioresources Inc, Quebec, Canada).4

Both oils were encapsulated in gelatin soft capsules manufac-

tured by Catalent (Eberbach, Germany). The capsules provided

approximately 1.5 g EPA and no DHA for Almega PL or 1.02 g

EPA and 0.54 g DHA for krill oil. Both oils were well tolerated.

Plasma samples collected before and 0.5 to 10 hours after

capsule administration indicated time-dependent increases in

EPA for both oils with the concentration of EPA being higher

with algal oil than with krill oil at multiple time points.

Described here is the nonclinical safety assessment (and

limited clinical assessment) of the algal oil Almega PL, which

is being developed as a dietary supplement in the United States,

providing a vegetarian and sustainable source of o-3 fatty acids

Table 13. Summary of PCE:Total RBC Ratio and Micronucleus Incidence.

Sex and dose,mg/kg/d

No. ofrats

PCE: Total RBC ratio

TotalPCEa

No. of PCEwith MN

PCE with MN

No. ofPCE/group

No. ofRBC/group

Ratio,mean + SD % Mean

Male—0 5 559 1134 0.49 + 0.01 10 265 4 0.04 0.04 + 0.04Male—500 5 531 1122 0.47 + 0.01b 10 337 5 0.05 0.05 + 0.03Male—1000 5 534 1116 0.48 + 0.01 10 643 7 0.07 0.07 + 0.04Male—2000 5 520 1096 0.47 + 0.01b 10 274 6 0.06 0.06 + 0.06Malec—15 5 455 1118 0.41 + 0.01b 10 317 129 1.25 1.25 + 0.26d

Female—0 5 549 1134 0.48 + 0.01 10 520 7 0.07 0.07 + 0.04Female—500 5 536 1117 0.48 + 0.01 10 359 6 0.06 0.06 + 0.04Female—1000 5 501 1066 0.47 + 0.01 10 296 6 0.06 0.06 + 0.04Female—2000 5 535 1111 0.48 + 0.01 10 881 3 0.03 0.03 + 0.02Femalec—15 5 446 1100 0.41 + 0.02b 10 535 125 1.19 1.18 + 0.43d

Combined—0 10 1108 2268 0.49 + 0.01 20 785 11 0.05 0.05 + 0.04Combined—500 10 1067 2239 0.48 + 0.01b 20 696 11 0.05 0.05 + 0.04Combined—1000 10 1035 2182 0.47 + 0.01b 20 939 13 0.06 0.06 + 0.04Combined—2000 10 1055 2207 0.48 + 0.01 21 155 9 0.04 0.04 + 0.05Combinedc—15 10 901 2218 0.41 + 0.01b 20 852 254 1.22 1.21 + 0.34d

Abbreviations: RBC, red blood corpuscle; SD, standard deviation.aA minimum of 2000 polychromatic erythrocytes (PCEs) were scored from each rat for the incidence of PCE with micronuclei (MN). Total PCE included PCEscounted to determine PCE: Total RBC ratio.bSignificantly lower than the control group by Dunnett test.cPositive control (cyclophosphamide) administered as a single intraperitoneal injection.dSignificantly higher than the control group by Dunnett test.

Kagan et al 15



in the diet, primarily EPA. Based on the results of the GLP-

compliant nonclinical studies, the NOAEL for the algal oil is

2000 mg/kg/d, which will provide a reasonable margin of

safety (96-fold) over the suggested maximum oral intake of

1250 mg/d Almega PL (20.8 mg/kg/d for a 70-kg individual).

Acknowledgments

This work was supported by Qualitas Health Ltd.

Author Contributions

M. Kagan contributed to conception and design, acquisition, critically

revised the article, gave final approval, and agrees to be accountable for

all aspects of work ensuring integrity and accuracy. D. Sullivan contrib-

uted to analysis and interpretation, drafted the article, critically revised

the article, gave final approval, and agree to be accountable for all aspects

of work ensuring integrity and accuracy. S. Gad contributed to analysis

and interpretation, critically revised the article, gave final approval, and

agrees to be accountable for all aspects of work ensuring integrity and

accuracy. C. Ballou contributed to interpretation, drafted the article,

critically revised the article, gave final approval, and agrees to be accoun-

table for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest

with respect to the research, authorship, and/or publication of this

article: Michael L. Kagan is an employee of Qualitas Health Ltd.

Funding

The author(s) received no financial support for the research, authorship,

and/or publication of this article.

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