Food and Chemical Toxicology 65 (2014) 1–11

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The safety of b-carotene from Yarrowia lipolytica Daniel Grenfell-Lee a,⇑, Samuel Zeller a,1, Renato Cardoso b, Kresimir Pucaj b a b

Microbia, Inc., 60 Westview Street, Lexington, MA 02421, USA Nucro-Technics, 2000 Ellesmere Rd #16, Toronto, ON M1H 2W4, Canada

a r t i c l e

i n f o

Article history: Received 8 August 2013 Accepted 8 December 2013 Available online 16 December 2013 Keywords: b-Carotene Yarrowia lipolytica Subchronic Toxicity Genotoxicity

a b s t r a c t Crystalline b-carotene from genetically modified Yarrowia lipolytica is an alternative source of b-carotene for use as a nutritional supplement. To support the use of b-carotene from Y. lipolytica as a food ingredient, the genotoxic and subchronic toxicity potential of this compound was determined. Genotoxicity was examined using Salmonella typhimurium and Escherichia coli (Ames test), a chromosomal aberration assay in Chinese Hamster Ovary WBL cells, and the micronucleus test in CD-1 mice. All three assays showed no significant results due to b-carotene from Y. lipolytica. In a subchronic toxicity study in SD rats, b-carotene from Y. lipolytica was administered by oral gavage for 13 weeks at 0, 125, 250 or 500 mg/kg per day. Adverse effects were not observed following clinical, clinical pathology and gross- and histopathological evaluations of dosed rats; thus, the no-observed-adverse effect level (NOAEL) for b-carotene from Y. lipolytica was 500 mg/kg, the highest dose used in the study. In conclusion, b-carotene derived from Y. lipolytica was shown in genotoxicity models and a standard rat subchronic rat study to have a safety profile similar to that of the current commercial products (synthetic and natural) with no unexpected finding attributable to the alternative source. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Crystalline b-carotene produced using the genetically modified microorganism, Yarrowia lipolytica, is an alternative source of bcarotene for use as a nutritional supplement (as an important source of vitamin A) and, consistent with existing Generally Recognized As Safe (GRAS) affirmed food categories, as an exempt color additive (21 CFR §73.95). The main dietary sources of b-carotene are carrots, oranges, tomatoes and dark green leafy vegetables. bCarotene from synthetic sources, from the fungal organism Blakeslea trispora, as well as mixed carotenes from plant and algal sources are widely used to impart yellow to orange color in foods, as a nutritional ingredient in foods and as ingredients for use in dietary supplements in order to help individuals meet the recomAbbreviations: ANS, Panel on Food Additives and Nutrient Sources added to Food; DMSO, dimethyl sulfoxide; EFSA, European Food Safety Authority; FDA, Food and Drug Administration; GLP, Good Laboratory Practice; GRAS, Generally Recognized As Safe; HOSO, high oleic sunflower oil; IOM, Institute of Medicine; NCE, normochromatic erythrocytes; NOAEL, no observed adverse effect level; OECD, Organization for Economic Cooperation and Development; PCE, polychromatic erythrocytes; SCF, Scientific Committee on Food; SD, standard deviation. ⇑ Corresponding author. Current address: DSM Nutritional Products, 60 Westview Street, Lexington, MA 02421, USA. Tel.: +1 718 259 7614; fax: +1 781 862 0615. E-mail addresses: [email protected] (D. Grenfell-Lee), samuel.zeller@ unilever.com (S. Zeller), [email protected] (R. Cardoso), pucaj@ nucro-technics.com (K. Pucaj). 1 Current address: Unilever, 800 Sylvan Ave, Englewood Cliffs, NJ 07632, USA. 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.12.010

mended dietary allowances for vitamin A as determined by the Institute of Medicine (IOM, 2001). A number of reviews, monographs and comments on the safety of b-carotene have been published (e.g. Bauernfeind et al., 1981; Heywood et al., 1985; Rock, 1997; IARC, 1998; Omenn, 1998; Palozza, 1998; SCF, 1998; Woutersen et al., 1999). The Scientific Committee on Food (SCF) assembled the scientific data relevant to the safety of use of b-carotene from all dietary sources but limited its conclusions only to food additive uses (SCF, 2000a). As summarized in the 2000 SCF opinion, no adverse effects of highdose oral b-carotene supplementation were observed in several standard toxicological studies in various experimental animals (rat, mice, rabbits) (IARC, 1998; Woutersen et al., 1999). These studies included acute toxicity, up to 5000 mg/kg bw/day in Sprague Dawley rats (Woutersen et al., 1999) and up to 2000 mg/kg bw/day in Wistar rats (Buser, 1992; Strobel, 1994), sub-chronic/chronic toxicity/carcinogenicity up to 1000 mg/ kg bw/day for life in rats (Hummler and Buser, 1983; Heywood et al., 1985) or mice (Buser and Hummler, 1983a; Heywood et al., 1985), and teratogenicity and reproductive toxicity (up to 1000 mg/kg bw/day for 3 generations, or during days 7 to 16 of gestation, in rats; up to 400 mg/kg bw/day during days 7 to 19 of gestation in rabbits) (Komatsu, 1971, cited in Kistler, 1981; Buser and Hummler, 1982; Heywood et al., 1985; Woutersen et al., 1999). In beagle dogs (Buser and Hummler, 1983b; Heywood et al., 1985) no toxic effects were observed (up to 250 mg/kg bw/ day for 2 years).

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Y. lipolytica is an avirulent yeast species historically used for the production of citric acid, c-decalactone and long-chain poly-unsaturated fatty acids (Groenewald et al., 2013). It is approved by the United States’ Food and Drug Administration (FDA) as a secondary direct food additive in citric acid production. Y. lipolytica is also routinely found associated with cheeses and meats. Although in rare cases the organism may lead to opportunistic infections in severely immunocompromised or otherwise seriously ill people, these infections either resolve spontaneously or can be effectively treated with standard antifungals. Therefore, Y. lipolytica is deemed ‘‘safe-to-use’’. Y. lipolytica has been developed as an alternative source of biobased b-carotene that is composed predominantly of all-trans bcarotene with minor amounts of 9-cis b-carotene, 13-cis b-carotene, 15-cis b-carotene and other naturally-occurring carotenoids. To support any intended use of b-carotene from Y. lipolytica as a food ingredient, the safety of this product was evaluated. Toxicological studies have been conducted to assess the genotoxic potential of b-carotene from Y. lipolytica, and to assess its subchronic toxicity. The following standard genotoxicity assays were performed: the Ames test for assaying bacterial reverse mutation, the chromosome aberration assay in Chinese Hamster Ovary WBL cells, and the in vivo mouse micronucleus assay. To investigate the subchronic toxicity of b-carotene from Y. lipolytica, a 13-weekoral toxicity study in rats was performed.

2. Materials and methods 2.1. Test article preparation Crystalline b-carotene (CAS No. 7235-40-7;molecular weight 536.88; molecular formula: C40H56) produced by fermentation using genetically modified yeast Y. lipolytica, was obtained from Microbia, Inc. (Lexington, MA; currently DSM Nutritional Products). Briefly, the process involved biomass isolation from the fermentation media, washing, and physical rupturing of the cells. The b-carotene was isolated by solvent extraction, crystallized from the mother liquor, washed, dried, and packaged. The production process was controlled by Good Manufacturing Practice procedures, with appropriate hygiene controls and appropriate control of raw materials. The final crystalline b-carotene from Y. lipolytica met the specifications as outlined in the Food Chemicals Codex (8th ed.) (i.e., purity not less than 96%; Table 1). The specifications and test methods for b-carotene from Y. lipolytica were the same as those incorporated in 21 CFR §184.1245, direct food substances affirmed as

Table 1 Technical and chemical description of b-carotene from Yarrowia lipolytica. Chemical name: b,b-carotene; 1,18-(3,7,12,16-tetramethyl1,3,5,7,9,11,13,15,17-octadecanonaen-1,18-diyl)-bis-(2,6,6trimethylcyclohexene) Common and usual name: b-carotene obtained by a fermentation process using the genetically modified yeast Yarrowia lipolytica Chemical structure:

CAS No.: 7235-40-7 Molecular formula: C40H56 Molecular weight: 536.88 Physical state: Solid Melting range: 176–182 °C, with decomposition Color: Red to purple-violet in color Solubility: Insoluble in water; practically insoluble in ethanol, slightly soluble in vegetable oil Purity: Not less than 96% A455/484: 1.14–1.18 A455/340: Not less than 1.5 1% solution in chloroform: clear Loss of weight on drying: Not more than 0.2% Lead (as Pb): Not more than 2 ppm

Generally Recognized As Safe. b-Carotene from Y. lipolytica is predominantly alltrans b-carotene with minor amounts of 9-cis b-carotene, 13-cis b-carotene, 15-cis b-carotene and other carotenes. The compositional attributes are similar to those of commercial counterparts, especially synthetic b-carotene and b-carotene from the fungal organism B. trispora (data not shown). For the in vivo studies, the b-carotene was provided as a 31% wt/wt suspension in high oleic sunflower oil (HOSO) and was a red, viscous liquid. The control compound used for the in vivo studies was HOSO, CAS No.: 8001-21-6, also provided by Microbia, Inc. The in vivo and in vitro genotoxicity studies and the 90-day subchronic toxicity study were performed by Nucro-Technics (Scarborough, Ontario, Canada). Experiments were performed in compliance with Good Laboratory Practice (GLP) requirements as described in the ‘‘Good Laboratory Practice for Nonclinical Laboratory Studies’’ of the US FDA (FDA, 2006) and ‘‘OECD Principles of Good Laboratory Practice and Compliance Monitoring’’ (OECD, 1998b). The use and the number of animals utilized in the in vivo studies were approved by the institutional Animal Care Committee of Nucro-Technics (AUP211905). 2.2. Bacterial reverse mutation assay (Ames test) The mutagenic potential of b-carotene from Y. lipolytica was evaluated using the Escherichia coli strain WP2 uvrA and Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, with and without metabolic activation. The tester strains were exposed to b-carotene according to the direct plate incorporation and preincubation methods. The experimental design followed the ‘‘OECD Guideline for Testing of Chemicals – 471, Bacterial Reverse Mutation Test’’ (OECD, 1997a). Bottom agar plates for S. typhimurium were made of minimal glucose agar that was based on a standard formula: 2% glucose, Vogel-Bonner medium E and 1.5% Bacto™ agar (Becton Dickinson Co., Sparke, USA). Bottom agar plates for E. coli contained 1.44% agar, 0.38% glucose, 0.24% casamino acids, 0.23 lg per mL tryptophan and 23.9% v/v Davis Mingioli Salt Solution. Top agar for the selection of S. typhimurium revertants was 0.6% BactoTM agar, containing 0.5% NaCl and supplemented with histidine and biotin to 50 lM each. Top agar for the selection of E. coli revertants contained 0.7% agar only. Liver microsomal 9000g fraction from liver homogenate of male Sprague–Dawley rats treated with Aroclor 1254 was used (Moltox Inc., Boone, USA). The solvent used to dissolve the test article was DMSO (CAS No. 67-68-5); DMSO alone was therefore used as the negative control for this assay. Positive controls for experiments without S9 were aqueous solutions of sodium azide (CAS No. 26628-22-8); and DMSO solutions of 2-nitrofluorene (CAS No. 607-57-8), methyl methanesulfonate (CAS No. 66-27-3) and 9-aminoacridine (CAS No. 52417-22-8). For experiments with S9, benzo[a]pyrene (CAS No. 50-32-8) and 2-aminoanthracene (CAS No. 613-13-8) were dissolved in DMSO; and cyclophosphamide monohydrate (CAS No. 6055-19-2) in water. All positive controls were purchased from Sigma–Aldrich Canada Ltd. (Oakville, Canada). Experiments were performed as described by Maron and Ames (1983). b-Carotene was dissolved in DMSO and tested at concentrations of 0, 0.062, 0.19, 0.56, 1.7 and 5.0 mg per plate. The selection of doses was based on the results of a previously conducted range-finding study (data not shown). Assays were performed in two independent experiments, using identical procedures, both with and without metabolic activation. Each concentration, including the controls, was tested in triplicate. The colonies were manually counted. For a test substance to be considered positive it had to generate at least a twofold increase in the number of reversions and present a dose-dependent increase in the number of revertants. 2.3. Chromosome aberration assay in Chinese Hamster Ovary cells WBL The potential for b-carotene from Y. lipolytica to induce structural chromosome aberrations in Chinese Hamster Ovary cells WBL was evaluated in vitro. The experimental design followed the ‘‘OECD Guideline for the Testing of Chemicals – 473, In Vitro Mammalian Chromosome Aberration Test’’ (OECD, 1997b). b-Carotene was dissolved in DMSO, and WBL cells were exposed to b-carotene, both with and without metabolic activation. The liver microsomal fractions were obtained from rats treated with phenobarbital and 5,6-benzoflavone (Moltox Inc. Boone, USA). Cultures were treated with b-carotene for 3 h with metabolic activation and for 3 or18 h without metabolic activation. The concentrations of b-carotene investigated were 0, 0.139, 0.417 and 1.25 mg per mL. Several solvents compatible with the chromosome aberration test were tested (data not shown), but none produced solutions at the highest concentration (5 mg/mL) indicated by OECD guidelines. Therefore, the highest used dose (1.25 mg/mL) was selected based on the maximum solubility of the test article in accordance with such guidelines (OECD, 1997b). Duplicate flasks were used for each dose level. Cells were cultured in McCoy’s 5A Modified Medium with 2 mM L-glutamine and 25 mM HEPES supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Co., Burlington, Canada). Incubation was performed in a humidified tissue culture incubator at 37 ± 2 °C and 5 ± 2% CO2. On the day before the experiment, 1  105 cells were seeded into each T-25 cm2 Falcon flask. The cultures were incubated overnight. Fifty lL of a dosing solution was added to each culture for all exposure conditions. DMSO was used as negative control. Mitomycin C (CAS No. 50-077) was used as a positive control (for cultures not treated with S9) at 1.0 lg/mL for

D. Grenfell-Lee et al. / Food and Chemical Toxicology 65 (2014) 1–11 the 3-h exposure period and at 0.2 lg/mL for the 18-h exposure period. Cyclophosphamide monohydrate was used at 12.5 lg/mL as a positive control for treatment with S9. All the controls were purchased from Sigma Chemical Co. (St. Louis, USA). For exposure without S9, the treatment medium was serum supplemented. For exposure with S9, the treatment medium was serum-free and contained 0.5 mL S9 mix. After the 3-h treatment, cultures were washed with Dulbecco’s Phosphate-Buffered Saline, re-fed and incubation continued for another 15 h in fresh culture medium until the time of harvest. For the 18 h exposure, the treatment proceeded until the harvest time. Cells were harvested according to standard protocols (Evans, 1976). One hundred metaphases were scored for chromosomal damage per slide. For the test substance to be considered mutagenic a significant dose-related increase in the number of structural chromosomal aberrations was required. However, both biological and statistical significance was considered. Test substance significance was established where P < 0.05.

2.4. Micronucleus assay in CD-1 mice The potential of b-carotene from Y. lipolytica to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of CD-1 male mice was evaluated in vivo. The experimental design followed the ‘‘OECD Guideline for the Testing of Chemicals – 474, Mammalian Erythrocyte Micronucleas Test’’ (OECD, 1997c). Animals (8 weeks old, Charles River Canada) were group housed, in ‘‘shoe box’’ cages, 7 per cage. Cages were in a room designated to maintain adequate environment conditions (18–26 °C, relative humidity 30–70%, 12-h photocycle and a minimum of 10 air-exchanges per hour). Tekland rodent diet and water were offered adlibitum. The animals were housed, cared for and used according to the Association for Assessment and Accreditation of Laboratory Animal Care International and the Canadian Council of Animal Care. Twenty-one mice each were administered with either a single oral dose of the vehicle (high oleic sunflower oil, HOSO) at 22 mL/kg (negative control), b-carotene in HOSO at 2000 mg/kg (22 mL/kg) or cyclophosphamide monohydrate at 70 mg/kg (positive control). Seven animals from each group were euthanized at 24, 36 and 48 h after dosing and bone marrow was removed from both femora. Femora were prepared according to the slightly modified procedure from Schmid (1975). The marrow from both femora was flushed out with 0.5 mL fetal bovine serum into a centrifuge tube containing 2.5 mL fetal bovine serum. After centrifugation at 1000 rpm for 5 min, the supernatant was decanted and cells were resuspended in 1 mL fetal bovine serum. A small drop of resuspended cells was spread on a slide, air-dried and fixed in 95% ethanol for 10 min. Slides were stained first with Jenner’s Giemsa followed by May-Grunwald-Giemsa, cleared in xylene and mounted with cover glass. At least 3 slides were prepared from each animal. At high magnification, 2000 PCE per animal (14,000 PCE per test or control compound per time point) were scored for the presence of micronuclei. The scored elements were the micronucleated cells, and not the number of micronuclei. The number of micronucleated normochromatic erythrocytes (NCE) was also scored. The ratio of PCE to NCE among 200 cells was determined for each animal. Any intergroup difference in mean number of PCEs with micronuclei was analyzed using ANOVA on ranks (Kruskal–Wallis at P < 0.05). For the test substance to be classified as positive in this study, it required that a reproducible and statistically significant positive response be detected for at least one of the experimental time points.

2.5. The 90-day subchronic toxicity study in rats The 90-day toxicity study was performed in accordance with the US regulations on GLP for nonclinical laboratory studies and with OECD Principles of GLP. The study design was based on the principles of the current test guidelines for repeated-dose toxicity studies as issued by the US FDA (2000), OECD (1998a), and the International Conference on Harmonization (ICH, 2003).

2.5.1. Preparation of dosing formulation The dosing formulation was prepared from the 31% (wt/wt) b-carotene stock dispersion every 4–8 days by diluting an appropriately weighed portion of the stock in the appropriate weight of HOSO, at designed levels of 2.5%, 5% and 10% (wt/wt) bcarotene. Preparation of dosing formulation was facilitated by the use of a PolytronÒ mechanical/ultrasonic mixer. Concentration, stability and homogeneity analyses of each concentration of dosing formulation were performed prior to the first day of dosing and then at the end of the 1st, 2nd, and 3rd month of study using a validated UV/VIS method (Nucro-Technics, 2009). The results indicated no more than ±12% difference from the targeted concentrations. The results also indicated that the stock suspension of b-carotene (31%) remained stable over the3-month study period, that dosing formulations were stable for at least 10 days after preparation, and that formulations were homogeneous.

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2.5.2. Animals and treatments A total of 80 rats were used. Four groups of 20 rats (10 males, 10 females) were administered the test substance formulated to supply targeted dosages of 0, 125, 250 and 500 mg/kg body weight per day for 90 days (groups 1, 2, 3, and 4, respectively). Animals in Group 1 received HOSO, which was used as a vehicle for dosing formulation in Groups 2 through 4. All animals were dosed orally at a dose volume of 5.5 mL/kg, using a blunt-tip G16 gavage needle. The upper limit usable in this study (500 mg/kg/day) was twofold lower than that used in other comparable studies (1000 mg/kg/day) and was due to a combination of the concentration of b-carotene in the stock (31%) dispersion in HOSO and the chosen delivery route of oral gavage (as opposed to beadlet forms of b-carotene delivered via feed as in previous studies). Animals used in this study were acclimatized for 3 weeks and treated and cared for in accordance with the guidelines recommended by the Canadian Council on Animal Care and the Association for Assessment and Accreditation of Laboratory Animal Care International. The experimental protocol for treating the animals was approved by the Institutional Animal Care Committee of Nucro-Technics. Rats used in this study were Sprague–Dawley (Rattus norvegicus), aged 7–8 weeks and were obtained from Charles River Canada Inc., Montreal, PQ. Each animal was identified with a unique tail tattoo. At the start of dosing, males weighed between 259 and 307 g and females weighed between 190 and 214 g. The rats were housed individually in NalgeneÒ rat cages with stainless steel cage covers under controlled conditions: temperature of 18–26 °C, relative humidity of 30–70%, a minimum of 10 air changes per hour, and a 12-h light and 12-h dark cycle. The animals were provided Teklad Certified Rodent diet (#8728C) and municipal water (using water bottles) ad libitum. 2.5.3. In-life data Animals were monitored closely after dosing, and were observed for clinical signs and mortality twice daily throughout the study. Detailed clinical examinations were performed once a week. A functional observation battery assessment for motor activity, grip strength, and sensory activity to visual, audio and proprioceptive stimuli was conducted on all animals during the last week of dosing. Body weights were recorded during the acclimatization period (days-13 and 6), on the first day of administration (day 1) prior to dosing, and then weekly thereafter. End of treatment body weights (unfasted) were recorded on day 90. Terminal body weights of main study rats were recorded prior to necropsy. Food consumption was recorded during the acclimatization period and weekly until scheduled necropsy. Funduscopic (indirect ophthalmoscopy) and biomicroscopic (slip lamp) examinations were performed on all animals prior to the start of treatment and on all study rats on day 86. 2.5.4. Clinical pathology Blood samples were collected on days 44 and 45 from five male and five female rats (fasted) by orbital sinus or jugular bleeds and limited clinical pathology (hematology and clinical chemistry) was performed. Blood samples were also collected on the day of necropsy from all study animals following an overnight fast. Blood was obtained from the abdominal aorta following anesthesia induced by Isoflurane, and routine clinical pathology investigations were performed as summarized in Tables 5–8. Hematology was performed on Ethylenediaminetetraacetic acid-treated samples, coagulation was determined from citrate-treated samples, and clinical chemistry was performed on serum. Hematology and clinical chemistry data were compared with the laboratory’s baseline historical data for Sprague–Dawley rats, and Charles River Technical Bulletin (CRTB, 1984). Urine was collected overnight (approximately a 12-h period) by placing rats in metabolic cages during the last weeks of dosing and recovery. Animals were fasted during urine collection. The urine samples were analyzed for color, appearance, volume, pH-specific gravity, bilirubin, urobilinogen, blood glucose, ketones, leukocytes, nitrite, and sediment microscopy. Hematology parameters were analyzed using an Advia-120 instrument (Bayer), serum chemistry parameters were analyzed using Vitros-350 (Ortho-Clinical Diagnostics), coagulation parameters were analyzed using STA-compact (STA60) and urinalysis using Multistix-10 S.G. (Bayer) and Clinitek-Status reader (Bayer). 2.5.5. Pathology On day 91 (males) or 92 (females), following an overnight fast (12 h), all animals were anesthetized by exposure to Isoflurane, exsanguinated, and subjected to necropsy. Necropsies consisted of an external examination. Organs that were collected and weighed included the adrenals, ovaries, brain, pituitary gland, epididymis, prostate, heart, spleen, kidneys, thymus, liver, testes, lungs, and uterus (horns, cervix and body). Paired organs were weighed together, and absolute and relative weights (relative to terminal body weight and brain weight) were calculated. The following organs and tissues were also collected: aorta (thoracic), cecum, colon, duodenum, esophagus, ileum, jejunum, liver (sample of central and left lobes), lymph nodes (mandibular and mesenteric), mammary glands (inguinal), salivary glands (submandibular), seminal vesicles, sciatic nerve, skeletal muscle (quadriceps), skin (inguinal) and subcutis, spinal cord, sternum and marrow, stomach, thyroid/parathyroids, and urinary bladder. Organs and tissues were fixed in

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10% neutral buffered formalin, except for the eyes, optic nerves, and testes, which were fixed in alcoholic formalin, paraffin-embedded, sectioned and stained with hematoxylin and eosin. Full histo-pathological examination was performed on tissues from control and high-dose animals, as well as on tissues with abnormal findings from all dose groups. 2.5.6. Statistical analysis Numerical data collected during the course of the study were subjected to calculation of group means and standard deviations. The data for males and females were separately analyzed for homogeneity of variance and for normality. Homogeneous data were analyzed using the Analysis of Variance (at P < 0.05) and the significance of intergroup differences were analyzed using Duncan’s or other appropriate tests. Heterogeneous data were analyzed using the Kruskal–Wallis test and the significance of intergroup differences between the control and treated groups were assessed using Dunn’s or other appropriate tests.

not show any cytotoxicity. All dose levels for all testing conditions produced P77% relative mitotic indices. For all three experimental conditions, a few chromosome aberrations were observed in all treated cultures. As demonstrated in Table 3, no relevant increases in the number of cells with structural aberrations after treatment with any concentration of b-carotene, either with or without metabolic activation, were observed. The differences in percentages of cells with chromosome abnormalities were not statistically significant by Chi-square test (P < 0.05) and did not result in a dose-related response. In contrast, positive control samples incubated with mitomycin C showed a significant increase in cells with aberrations. Therefore, b-carotene from Y. lipolytica did not meet the criteria for a mutagenic result.

3. Results

3.3. Micronucleus assay in bone marrow cells of the mouse

3.1. Bacterial reverse mutation assay (Ames test)

To determine the potential for b-carotene from Yarrowia to induce micronuclei, an in vivo mouse micronucleus assay was performed. As detailed in Table 4, there was no increase in the percentage of micronucleated PCE, or in the PCE/NCE ratio at any time point after treatment when compared to the corresponding negative control. Based on the above results, the test compound did not induce micronuclei in the mouse micronucleus test at the limit dose level of 2000 mg/kg administered orally by gavage. b-Carotene from Y. lipolytica was not clastogenic and did not interact with the mitotic spindle.

A standard bacterial reverse mutation assay was used to determine the mutagenic potential of b-carotene from Yarrowia. The numbers of revertant colonies on b-carotene-treated plates were at levels similar to the corresponding negative controls. There was a clear overlap between the colony counts (mean ± SD) per plate from b-carotene and the counts from the corresponding negative control plates (mean ± 2 SD), for all test conditions. The only exception observed was the strain WP2 uvrA treated with 0.19 mg b-carotene per plate in the absence of S9 mix in the preincubation test. However, since there was no dose–response and the same dose (0.19 mg per plate) in the plate incorporation test did not reproduce this increase in the number of colonies, this observation was not considered a mutagenic response. In contrast, the included positive controls induced a significant increase in the number of revertant colonies. See Table 2 for details. 3.2. Chromosome aberration assay in WBL Chinese Hamster Ovary cells The potential for b-carotene from Yarrowia to induce structural chromosomal aberrations was assessed. No cytotoxic effects were observed at any concentration when exposed to b-carotene for 3 h, both with and without incubation with S9. When exposed to b-carotene for 18 h in the absence of S9, there was a slight decrease in relative cell growth to 77 and 78% at 0.417 and 1.25 mg/mL bcarotene, respectively. The lower concentration, 0.139 mg/mL, did

3.4. The 90-day subchronic toxicity study in rats 3.4.1. In-life data A 90-day subchronic toxicity study was performed in Sprague– Dawley rats using b-carotene from Yarrowia. Daily cage-side observations showed no test compound-related systemic toxicity in groups dosed from 125 to 500 mg/kg/day for 90 days. Detailed weekly physical examinations (respiration, heart, abdominal palpations) also did not show any untoward signs of toxicity in any group or any individual animal. Neurological evaluations conducted during the last week of dosing did not show any apparent differences between the control animals and animals in the test groups. Red discoloration of feces was noted in all test rats. This finding was attributed to the red color of the test compound (b-carotene). All groups displayed satisfactory body weight gains, and no significant differences in body weights or body weight

Table 2 Summary of bacterial reverse mutation test results.a b-Carotene (mg/plate)

TA98 S9

S9

TA1535 +S9

S9

TA1537 +S9

S9

WP2 uvrA +S9

S9

+S9

Plate incorporation 0 0.062 0.19 0.56 1.7 5.0

20 21 16 22 20 19

30 27 23 25 23 29

109 100 112 119 117 108

119 111 118 122 113 109

17 20 16 16 16 18

16 13 11 17 15 14

15 12 12 13 12 9

14 13 13 10 10 13

23 26 21 22 28 23

33 39 32 34 28 35

Preincubation 0 0.062 0.19 0.56 1.7 5.0

16 18 16 17 17 14

30 32 32 29 25 29

117 111 108 109 114 113

137 125 103 127 118 127

16 17 12 16 13 12

11 12 14 14 11 12

13 9 13 12 15 12

17 16 13 13 6 13

19 22 27 21 21 21

32 26 28 25 25 33

1094

452

1786

1207

1549

147

1779

202

491

167

Positive control a

TA100 +S9

Note. Data represent the mean of the revertant colonies of 3 plates per experiment.

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D. Grenfell-Lee et al. / Food and Chemical Toxicology 65 (2014) 1–11 Table 3 Summary of chromosome aberration test results.a Cells

b-Carotene (mg/mL)

Not computedb

Chromatid type

Chromosome type

Simple tg 3 h Exposure 0 0.139 0.417 1.25 MMC 1.0 lg/mL

S9 mix 200 200 200 200

0.5 0.5 0.5 0.5

100 mix 200 200 200 200 100

18 h Exposure S9 0 0.139 0.417 1.25 MMC 0.2 lg/mL

3 h Exposure + S9 mix 0 200 0.139 200 0.417 200 1.25 200 MMC 0.2 lg/mL 100

sg

e

pp

tb

Complex isb

1.0 3.0 2.5 3.0

1.0 2.0 0.5 2.0

1.0

4.0

13.0

4.0

0.5 1.5 1.5 1.5 2.0

1.5 2.0 2.5 3.5 2.0

3.0 3.0 1.5 2.5 11.0

0.5

1.5 2.5 4.0 5.0 4.0

1.5 2.0 1.0 0.5 9.0

0.5

0.05 1.0 0.5 2.0

1.0 1.0 0.5

tr

qr

cr

id

ci

No. of aberrations per cell

Simple

Complex

sb

r

d

dm 0.01 0.025 0.005 0.02

0.5

11.0

1.0

2.0

3.0

1.0

2.0

1.0 1.0 1.0

4.0

0.5 1.0

1.0

3.0

1.0 0.5 0.5

0.5 0.5 2.0

11.0

1.0

1.0

1.5 0.5 6.0

0.5 1.0

1.0

% of cells with aberrations

0.5

1.0 2.5 0.5 2.0

0.37

32.0

0.035 0.04 0.025 0.03 0.22

3.5 4.0 2.5 3.0 20.0

0.02 0.03 0.025 0.025 0.32

2.0 3.0 2.5 2.0 28.0

Note. Tg, chromatid gap; sg, chromosome gap; e, endoreduplicated cells; pp, polyploid cells; tb, chromatid break; isb, isochromatid break; tr, triradial; qr, quadriradial; cr, complex aberration; id, interstitial deletion; ci, chromatid intrachange; sb, chromosome break; d, dicentric; r, ring; dm, double minutes; MMC, mitomycin C. a Pooled data from two cultures demonstrates incidence of aberrations per 100 cells. b Common practice is not to include gaps and numerical aberrations in total number of aberrations. Table 4 Micronucleus assay. Treatment HOSO

b-Carotene

CP

Dose (mg/kg)

Harvest time (h)

% of micronucleus PCEs (mean of 2000 per animal)

Ratio PCE/NCE (Mean)

0

24 36 48

0.06 0.065 0.06

0.89 0.89 0.80

2000

24 36 48

0.06 0.01 0.06

0.86 0.89 0.91

70

24 36 48

2.8 3.3 3.1

0.93 0.32 0.13

Statistical difference (p < 0.05) was noted when the cyclosphosphamide monohydrate (CP) group was compared to HOSO and b-carotene groups, at all time points. No significant difference between HOSO and b-carotene groups.

gains were observed between the control and b-carotene test groups (Fig. 1). Food consumption was also not significantly different between the control and b-carotene test groups. There were no ophthalmological findings that were considered treatment-related. 3.4.2. Clinical pathology and pathology Hematology profiles showed that red blood cell counts, reticulocytes, hemoglobin, hematocrit and red blood cell indices (MCV, MCH and MCHC; see Tables 5 and 6) were all within the normal physiological limits for the three test groups. Platelet counts, white blood cell counts and differential counts did not appear to be affected by the b-carotene treatments. There were also no coagulation parameter findings (APTT and PT; Tables 5 and 6) that were considered treatment-related, although APTT values were at the upper end of the normal ranges, sometimes exceeding these ranges. There was no dose response relationship between the APTT increases and doses, and in the control females the mean APTT values also slightly exceeded the upper limits of normal ranges. Some of these changes in APTT were considered to be partially or wholly related to bleeding techniques (Lewis, 1996). Summaries of the hematology and coagulation parameters at the end of treatment are presented in Table 5 (males) and Table 6 (females). Clinical chemistry profiles demonstrated that electrolytes (Na+, Cl and K+), calcium and phosphorus values were within the nor-

mal ranges for all groups and both genders (see Tables 7 and 8). Blood urea nitrogen and creatinine values were normal for all groups and both genders, indicating normal renal function. Glucose, triglycerides and cholesterol were all well within the normal physiological ranges. Hepatocellular/hepatobiliary panel showed that all examined parameters (ALP, total bilirubin, AST and GGT; Tables 7 and 8) were within the normal ranges in rats of both genders in the control and test groups of animals. Total protein, albumin, globulin and albumin/globulin ratios were not affected by the treatments. The lactate dehydrogenase (LDH) and creatine kinase parameters were also not affected by the treatments. Urinalysis parameters were unremarkable. Summaries of the serum chemistry parameters at the end of treatment are presented in Table 7 (males) and Table 8 (females). It should be noted, however, that for some parameters occasionally statistically significant differences were noted, and/or they were outside of the normal ranges. This especially applied to LDH, creatine kinase and P+, for which notable inter-group and intragroup variabilities were observed. Fluctuations in these parameters are known to be related to bleeding techniques (Friedel and Mattenheimer, 1970; Friedel et al., 1974), handling of rats, dosing, etc. (Yerroum et al., 1999). In the case of P+, the mean results for P+ for the control rats were either at the low end of normal range (control males) or were below the low end of the normal range

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D. Grenfell-Lee et al. / Food and Chemical Toxicology 65 (2014) 1–11

Fig. 1. Body weights of male and female rats during the 90-day subchronic toxicity study. b-Carotene was administered daily for 90 days at the doses indicated in the figure legend. Weight gain was similar and satisfactory between the control and all b-carotene test groups.

Table 5 Hematology profile for male rats at the end of treatment.

RBC (1012/L) Hb (g/L) Hct (%) MCV (fL) MCH (pg) MCHC (g/L) Platelets (109/L) WBC (109/L) Neutrophils (109/L) Lymphocytes (109/L) Monocytes (109/L) Eosinophils (109/L) Basophils (109/L) LUC (109/L) Reticulocytes (109/L) PT (s) APTT (s)

Group 1 HOSO (0 mg/kg)

Group 2 Low dose (125 mg/kg)

Group 3 Mid dose (250 mg/kg)

Group 4 High dose (500 mg/kg)

Normal historical ranges

8.88 ± 0.43 157 ± 7 45.3 ± 1.9 51.0 ± 1.4 17.7 ± 0.5 347 ± 3 1007 ± 140 8.01 ± 1.80 1.08 ± 0.37 6.57 ± 1.83 0.18 ± 0.05 0.12 ± 0.03 0.03 ± 0.01 0.04 ± 0.01 122.4 ± 21.1 22.5 ± 6.9 32.5 ± 15.1

8.60 ± 0.34 154 ± 6 44.4 ± 1.9 51.6 ± 1.7 17.9 ± 0.5 346 ± 3 1063 ± 143 6.82 ± 2.22 1.32 ± 0.48 5.18 ± 2.02 0.19 ± 0.05 0.08 ± 0.03* 0.02 ± 0.01 0.04 ± 0.03 122.5 ± 26.0 20.5 ± 3.8 31.3 ± 15.0

9.05 ± 0.28 158 ± 4 46 ± 1 50.9 ± 1.6 17.5 ± 0.5 344 ± 3 1010 ± 67 8.71 ± 1.82 1.10 ± 0.59 7.24 ± 1.35 0.21 ± 0.08 0.09 ± 0.03 0.03 ± 0.01 0.04 ± 0.01 115.7 ± 23.3 25.9 ± 7.0 45.2 ± 11.6

8.75 ± 0.48 154 ± 8 44.3 ± 2.4 50.6 ± 0.9 17.6 ± 0.3 347 ± 4 1081 ± 131 9.77 ± 2.33 1.59 ± 1.09 7.68 ± 1.87 0.26 ± 0.12 0.12 ± 0.04 0.03 ± 0.01 0.09 ± 0.10 135.2 ± 29.9 24.2 ± 4.5 37.6 ± 8.2

6.06–9.46 120–181 37.3–50.2 47.5–66.1 15.8–23.1 287–401 579–1641 5.00–15.28 0.05–2.37 1.67–14.00 0–0.46 0–0.21 0–0.06 0–0.14 100–400 11.6–23.3 4.7–37.4

Data are means ± S.D.  Statistically significant difference from control group (p < 0.05). n = 10. RBC, red blood cells; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cells; LUC, large unstained cells; PT, prothrombin time; APTT, activated partial thromboplastin time.

(control females), thus artificially ‘‘increasing’’ the mean P+ values in the test groups. Although it cannot be completely excluded that some of these slight differences were not treatment related, the magnitude of these differences was low, there was no dose–response relationship and thus they were not considered toxicologically significant or clinically relevant. The gross pathological findings (Table 9) and organ weights (Table 10) indicated that the treatments with b-carotene did not induce any treatment-related changes, and histopathological evaluation (Table 11) indicated that there were no conditions of possible or uncertain toxicological significance.

4. Discussion b-Carotene from Yarrowia is an alternative source of bio-based b-carotene that meets accepted specifications and is similar in composition to current commercial counterparts (including syn-

thetically- and other naturally-derived forms). This material was assessed using an accepted panel of studies to examine the potential as a mutagen, for in vitro induction of chromosomal aberration, for in vivo potential to induce micronuclei formation, and for subchronic toxicity. In the Ames test for mutagenic potential, the numbers of revertant colonies on b-carotene-treated plates were at levels similar to the corresponding negative controls. There was a clear overlap between the colony counts per plate from bcarotene and the counts from the corresponding negative control plates. The only exception noted was the strain WP2 uvrA treated with 0.19 mg b-carotene per plate in the absence of S9 mix in the preincubation test. Nevertheless, because of a lack of (i) a dose–response relationship and (ii) reproducibility of the result (same dose in the plate incorporation test did not induce an increase in the number of colonies), this observation was not considered a mutagenic response. Thus, it was concluded that b-carotene from Y. lipolytica was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA.

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D. Grenfell-Lee et al. / Food and Chemical Toxicology 65 (2014) 1–11 Table 6 Hematology profile for female rats at the end of treatment.

RBC (1012/L) Hb (g/L) Hct (%) MCV (fL) MCH (pg) MCHC (g/L) Platelets (109/L) WBC (109/L) Neutrophils (109/L) Lymphocytes (109/L) Monocytes (109/L) Eosinophils (109/L) Basophils (109/L) LUC (109/L) Reticulocytes (109/L) PT (s) APTT (s)

Group 1 HOSO (0 mg/kg)

Group 2 Low dose (125 mg/kg)

Group 3 Mid dose (250 mg/kg)

Group 4 High dose (500 mg/kg)

Normal historical ranges

7.82 ± 0.44 145 ± 7 41.2 ± 1.8 52.8 ± 1.5 18.6 ± 0.6 353 ± 3 931 ± 333 4.48 ± 1.26 0.63 ± 0.21 3.63 ± 1.25 0.11 ± 0.04 0.07 ± 0.04 0.01 ± 0.01 0.03 ± 0.03 117.5 ± 35.1 20.9 ± 4.3 38.2 ± 17.3

7.83 ± 0.51 146 ± 4 41.5 ± 1.1 53.1 ± 2.4 18.6 ± 0.9 351 ± 3 999 ± 274 4.45 ± 1.29 0.67 ± 0.20 3.60 ± 1.22 0.11 ± 0.04 0.05 ± 0.02 0.01 ± 0.01 0.02 ± 0.03 127.7 ± 35.2 18.8 ± 3.3 35.1 ± 15.8

8.00 ± 0.34 147 ± 6 41.7 ± 1.5 52.2 ± 1.5 18.3 ± 0.5 351 ± 4 993 ± 234 4.63 ± 1.28 0.66 ± 0.27 3.74 ± 1.13 0.11 ± 0.03 0.08 ± 0.03 0.01 ± 0.01 0.03 ± 0.02 132.9 ± 25.7 24.5 ± 6.9 55.6 ± 18.0

7.87 ± 0.25 147 ± 4 41.8 ± 1.2 53.2 ± 1.5 18.7 ± 0.5 353 ± 3 966 ± 303 5.13 ± 1.48 0.56 ± 0.20 4.33 ± 1.28 0.12 ± 0.05 0.08 ± 0.03 0.01 ± 0.01 0.03 ± 0.02 128.6 ± 17.8 23.1 ± 8.0 45.7 ± 23.9

6.16–9.09 127–172 35.3–47.5 47.5–64.0 17.9–21.6 325–385 526–1648 4.30–13.00 0.10–2.67 0.33–11.60 0–0.30 0–0.20 0–0.04 0–0.11 100–400 11.6–23.3 4.7–37.4

Data are means ± S.D. Statistically significant differences from control group (p < 0.05) were not observed. n = 10. RBC, red blood cells; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean cell hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cells; LUC, large unstained cells; PT, prothrombin time; APTT, activated partial thromboplastin time.

Table 7 Clinical chemistry profile for male rats at the end of treatment.

A/G ratio Albumin (g/L) Globulin (g/L) ALP (U/L) Total Bilirubin (lmol/L) BUN (mmol/L) Calcium (mmol/L) Chloride (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) LDH (U/L) Phosphorus (mmol/L) Potassium (mmol/L) Total protein (g/L) AST (U/L) ALT (U/L) Sodium (mmol/L) Triglycerides (mmol/L) Creatine kinase (U/L) Cholesterol (mmol/L) GGT (U/L)

Group 1 HOSO (0 mg/kg)

Group 2 Low dose (125 mg/kg)

Group 3 Mid dose (250 mg/kg)

Group 4 High dose (500 mg/kg)

Normal historical ranges

1.2 ± 0.1 31 ± 2 26 ± 2 133 ± 45 1.7 ± 0.1 4.6 ± 0.3 2.47 ± 0.07 103 ± 2 50 ± 3 8.3 ± 2 5674 ± 2413 2.04 ± 0.17 4.7 ± 0.3 57 ± 3 107 ± 21 40 ± 4 141 ± 1 0.78 ± 0.16 566 ± 227 1.81 ± 0.19