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Safety Assessment of DHA-Rich Microalgae from Schizochytrium sp.
Regulatory Toxicology and Pharmacology 35, 255–265 (2002) doi:10.1006/rtph.2002.1535, available online at http://www.idealibrary.com on
Safety Assessment of DHA-Rich Microalgae from Schizochytrium sp. IV. Mutagenicity Studies Bruce G. Hammond,∗ Dale A. Mayhew,† Larry D. Kier,∗ Richard W. Mast,‡ and Wayne J. Sander§ ∗ Monsanto Company, St. Louis, Missouri 63198; †NutraSweet Company, Mt. Prospect, Illinois 60056; ‡TPSRC, Inc., Severance, New York 12872; and §OmegaTech Inc., Boulder, Colorado 80301 Received August 2, 2001
The purpose of this series of studies was to assess the genotoxic potential of docosahexaenoic acid-rich microalgae from Schizochytrium sp. (DRM). DRM contains oil rich in highly unsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA n-3) is the most abundant PUFA component of the oil (∼29% w/w of total fatty acid content). DHA-rich extracted oil from Schizochytrium sp. is intended for use as a nutritional ingredient in foods. All in vitro assays were conducted with and without mammalian metabolic activation. DRM was not mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains. Mouse lymphoma suspension assay methodology was found to be inappropriate for this test material because precipitating test material could not be removed by washing after the intended exposure period and the precipitate interfered with cell counting. The AS52/XPRT assay methodology was not subject to these problems and DRM was tested and found not to be mutagenic in the CHO AS52/XPRT gene mutation assay. DRM was not clastogenic to human peripheral blood lymphocytes in culture. Additionally, DRM did not induce micronucleus formation in mouse bone marrow in vivo further supporting its lack of any chromosomal effects. Overall, the results of this series of mutagenicity assays support the conclusion that DRM does not have any genotoxic potential. C 2002 Elsevier Science (USA)
INTRODUCTION Docosahexaenoic acid (DHA n-3)-rich dried microalgae is produced via fermentation using Schizochytrium sp. (Barclay, 1992; Bajpai et al., 1991a,b). There are no reports of this organism producing toxic chemicals nor is it pathogenic and chemical analysis confirms the
absence of the common algal toxins, domoic acid, and Prymnesium toxin. Schizochytrium sp. dried microalgae (DRM) has been utilized in aquaculture applications, including enrichment of DHA in Artemia and rotifers used to feed larval fish and shrimp (Barclay and Zeller, 1996). OmegaTech (Boulder, CO) commercialized a product for aquaculture applications (HUFA 2000, a spray-dried form of Schizochytrium sp. dried microalgae), which has been successfully utilized for over 7 years as an excellent, stable dietary source of DHA in shrimp larvaculture and finfish (red seabream, Japanese flounder) culture with no adverse effects. Dried Schizochytrium sp. microalgae has also been determined to be generally recognized as safe (GRAS) for use as a DHA-rich ingredient in broiler chicken and laying hen feed at levels up to 2.8 and 4.3%, respectively. DRM contains oil rich in highly unsaturated fatty acids (PUFAs). DHA is the most abundant PUFA component of the oil [∼29% of total fatty acid (TFA) content]. Docosapentaenoic acid (DPA n-6) is present in DRM but as a minor component (∼10% of TFA). DHA-rich extracted oil from DRM is intended for use as a nutritional ingredient in foods. Fatty acid and sterol components of oil extracted from DRM have been characterized and identified as normal constituents of common human foods. Direct consumption by man of thraustochytrids, especially those of the genus Schizochytrium, is primarily through consumption of mussels and clams. Indirect consumption, through the marine food chain, is more widespread. Exposure to components contained in the extracted oil from its use as a nutritional ingredient in foods is within the normal range of exposures from consumption of foods with these components and long chain PUFA intake is within the limits determined by FDA to be affirmed as GRAS for Menhaden oil (FDA, 1987). In this paper we report the results of a comprehensive series of genotoxicity studies on DRM.
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0273-2300/02 $35.00 C 2002 Elsevier Science (USA)
All rights reserved.
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MATERIALS AND METHODS1 Chemicals DRM (Lot No. 35003, 35004, and 40026) was obtained from Monsanto Company, NutraSweet Kelco Unit (San Diego, CA). In the Ames assay and the CHO/AS52/XPRT assay Schizochytrium sp. cells were fully lysed by homogenization at pressures greater than 9000 psi (Zeller and Peik, 1996). Intact Schizochytrium sp. was used in the cytogenetic and mouse micronucleus studies. In all studies, the concentration, stability, and homogeneity of DRM suspensions/solutions were verified prior to and upon completion of the study. All positive control chemicals were at least reagent grade and obtained from Sigma (St. Louis, MO). Ames/Salmonella Reverse Mutation Assay2 Test strains. The Salmonella typhimurium test strains (TA98, TA100, TA102, TA1535, and TA1537) were obtained from the laboratory of Dr. B. N. Ames (Berkeley, CA). The proper phenotype of each culture was verified. S9 preparation and mix. The S9 preparations from livers of Aroclor 1254-induced male Sprague–Dawley rats (Hilltop Laboratories, Scottsdale, PA,) were purchased from Molecular Toxicology, Inc. (Annapolis, MD; Lot No. 0340, 41.4 mg protein/ml). The procedures used in preparation of the S9 supernatant solutions and the preparation of the S9 metabolic activation system (S9 mix) were those described by Ames et al. (1975). Plate incorporation and preincubation tests. The general procedures used were basically those described by Ames et al. (1975). Plate incorporation tests were performed by mixing DRM or control solutions, 0.1 ml of bacterial culture and, if appropriate, 0.5 ml of S9 mix (as described above) with 2 ml of histidine–biotin top agar. The mixture was poured onto minimal glucose agar plates. Preincubation tests were performed by mixing DRM or control solutions, 0.1 ml of bacterial culture, and 0.5 ml of S9 mix or S9 mix buffer. The mixture was incubated at 37 ± 1◦ C in a shaking incubator for 20 min. After incubation, 2 ml of histidine–biotin top agar, maintained at 44–48◦ C, was added and the mixture was poured onto minimal glucose agar plates. Initially, a toxicity test was conducted using plate and preincubation methods with test strain TA100 with and 1 All studies were conducted in accordance with U.S. Food and Drug Administration (FDA) Good Laboratory Practices (GLP) Regulations [(21 CFR Part 58) March 21, 1994] and the GLP principles of the OECD (1981). 2 This study was performed in general accordance with: OECD Guideline 471 (OECD, 1983a,b,c, 1995a,b), U.S. Food and Drug Administration Redbook II (FDA, 1993), and ICH (1994). The study was conducted by Monsanto Company, Environmental Health Laboratory, St. Louis, MO, and completed in June 1996.
without S9 to define dose levels for mutagenicity testing. Once dose levels were established, mutagenicity testing was conducted using plate incorporation and preincubation assay methods in five Salmonella test strains (TA98, TA100, TA102, TA1535, and TA1537) with and without metabolic activation. In the mutagenicity test, three replicate plates were prepared for each strain/S9/DRM dose level combination. Concurrent positive and negative controls (solvent and nonsolvent) were conducted for all mutagenicity tests. Solvent control plates used the same volume of water per plate as that selected for DRM. Plates were examined and revertant colonies were counted after at least 48 h at 37 ± 1◦ C. Revertant colonies on other plates, except as noted, were counted with an Artek Model 880 automatic colony counter or counted by visual examination (≤10 revertants/plate). Revertant colonies for plates with more than 500 revertant colonies/plate were estimated by counting colonies in several fields under a stereomicroscope and multiplying the counted colonies by a factor relating the total plate area to the area of the counted fields. Statistical analysis. Statistical analysis was performed on plate incorporation assay results after transforming revertant/plate values as log10 (revertants/plate). Analysis included Bartlett’s test for homogeneity of variance (Bartlett, 1947) and comparison of treatments with controls using within-levels pooled variance and a one-sided t test (Dixon and Massey, 1969; Ling, 1974; Munroe, 1951). Grubbs’ test was performed to determine if outliers were present (Grubbs and Beck, 1972). Statistical significance of dose response was evaluated by regression analysis for log10 transformed doses and revertants/plate (Draper and Smith, 1966). Evaluation criteria. A critical level of P ≤ 0.01 was used in determining statistical significance. As a general guide, results were considered clearly positive for a strain/microsome combination if revertants/plate values were significantly elevated over control values (P ≤ 0.01) at three or more treatment levels, and there was a statistically significant dose response (P ≤ 0.01). CHO AS52/XPRT Gene Mutation Assay3 The mutagenic potential of DRM was tested in cultured Chinese hamster ovary/ xanthine–guanine phosphoribosyl transferase (CHO AS52/ XPRT) gene locus assay. 3 This study was performed in general accordance with: OECD Guideline 476 (OECD, 1983a,b,c, 1995a,b), GLP principles of the OECD (1981), U.S. Food and Drug Administration Redbook II (FDA, 1993), and ICH (1994). The study was conducted by Monsanto Company, Environmental Health Laboratory, St. Louis, MO, and completed in September 1996.
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
Cell line. The AS52 cell line was obtained from Dr. L. F. Stankowski, Jr. (Pharmakon International Laboratory, Waverly, PA). The AS52 cells were maintained as logarithmic phase growing cultures, periodically examined for mycoplasma contamination, and cloned to prevent a high spontaneous mutant frequency. Chemicals. Solutions of the DRM (Lot No. 35003) were prepared on the day of use in Ham’s F12 treatment medium as the solvent. DRM was suspended in medium at the desired concentrations before being added to the appropriate treatment flasks. The positive controls were benzo[a]pyrene (B[a]P) with S9 mix and actinomycin D (AcD) without S9 mix. The positive controls were prepared in DMSO and added to medium to yield final concentrations of 2 µg/ml B[a]P and 0.005–0.05 µg/ml AcD. The S9 preparations (Ames et al., 1975) were purchased from Molecular Toxicology, Inc. (Lot No. 0340, 41.4 mg protein/ml). The different concentrations of S9 in this study represent the percentage of S9 (v/v) in the S9/cofactor mixtures. Two milliliters of the S9/cofactor mixture was added to 8 ml of medium for cytotoxicity and mutagenicity testing. Cytotoxicity determination. AS52 cells were seeded in 75-cm2 plastic culture flasks at 0.5 × 106 cells/flask in Ham’s F12 medium, supplemented with 5% heatinactivated fetal bovine serum plus 200 U/ml penicillin, and 200 µg/ml streptomycin (F12FCM5 ), 18–24 h before treatment. On the day of treatment, medium was changed to Ham’s F12 medium without serum in the presence and absence of S9. Suspensions of DRM were prepared in treatment medium at the desired concentrations and then added to the appropriate treatment flask. After an incubation of 5 h at 37 ± 1◦ C, the treatment medium was discarded. The cells were washed with 5 ml Hanks’ balanced salt solution and removed from the flasks by trypsinization, resuspended, and counted. Three aliquots of approximately 200 cells were plated per sample for determination of cloning efficiency. The plates were returned to incubation for 6–9 days. The colonies that developed were fixed with methanol, stained with 10% Giemsa, and manually counted. Cytotoxicity was expressed as relative survival (relative survival = cloning efficiency treated/cloning efficiency control). The cytotoxicity results (data not shown) were used for dose-selection purposes for the mutagenicity experiments. Mutagenesis. For mutagenesis experiments the total number of cells surviving treatment and used at each stage of processing were at least 1 × 106 cells.4 4
As recommended in OECD Guideline 476 (OECD, 1984, 1995a,b), this number of cells is approximately 10 times the inverse of the spontaneous AS52/XPRT locus mutant frequency of 1 × 10−5 . For cell survival, anticipated being ≥50%, this was accomplished by treating at least duplicate flasks with 1 × 106 cells/flask as described above for cytotoxicity.
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AS52 cells were plated on the day before treatment as described for cytotoxicity determination. The next day, they were treated for 5 h with the DRM (five dose levels in triplicate with and without S9), positive controls, or solvent control. The cells were then processed as described above for cytotoxicity determination, except that in addition to plating 200 cells for cloning efficiency, the treated cells from each flask (approximately 106 to 107 treated cells) were plated in 10 ml of subculture medium (hypoxanthine-free FI2FCM5 ). The cells were subcultured every 2–3 days as attached cultures for 7–9 days for the expression of the mutant phenotype. Mutant selection was performed as previously described (Li, 1982) using selective medium consisting of hypoxanthine-free Ham’s F12 medium supplemented with 10 µM 6-thioguanine (6TG) and 5% heat-inactivated dialyzed fetal calf serum plus 200 U/ml penicillin and 200 µg/ml streptomycin. Approximately 106 cells/sample were plated in 100-mm plates (five plates, 2 × 105 cells/plate) in 8 ml of selective medium per plate for mutant selection. Three aliquots of approximately 200 cells/sample were plated in 3 ml of selective medium without 6TG and incubated for 6–10 days for the determination of cloning efficiency (C.E.). Colonies developed were fixed, stained, and counted by hand. Results were expressed as mean mutation frequency (MF) where MF = (number of mutant colonies/106 cells plated) × (1/cloning efficiency) = number of mutant colonies per 106 viable cells. Statistical analysis. Mutagenicity data were analyzed according to the statistical method of Snee and Irr (1981) designed originally for the CHO/HGPRT mutation assay.5 Student’s t test was then used to compare treatment data to solvent data. Evaluation criteria. A chemical was considered negative if, when tested to approximately 10–20% relative survival, to the limit of solubility, or to a maximum dose of 5 mg/ml, there was no increasing dose response (P ≤ 0.05) and no significantly elevated (P ≤ 0.05) mutation frequencies over control values. A chemical would be considered positive if a reproducible dosedependent increase in mean mutant frequency with a positive slope statistically different (P ≤ 0.05) from zero was observed. In addition, at least one treatment level should have had a mean mutant frequency statistically greater (P ≤ 0.05) than control values, and at least twofold greater than the solvent control values. 5 In this analysis, mutant frequency values were transformed according to the equation y = (X + 1)0.15 , where y = transformed mutant frequency and X = observed mutant frequency. The Snee and Irr analysis also allowed the evaluation of dose–response relationships as linear, quadratic, or higher order. A computer program obtained from Dr. Irr (Dupont) was used to calculate mutant frequency transformation.
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In Vitro Mammalian Cytogenetic Test6 The clastogenic potential of DRM was assessed based on its ability to induce chromosome aberrations in human peripheral blood lymphocytes (HPBL). Test materials. DRM (Lot No. 40026) was suspended in sterile distilled water. Mitomycin C (MMC) was dissolved and diluted in sterile distilled water for use as the positive control in the non-S9-activated test system at final concentrations of 0.13 and 0.25 µg/ml. Cyclophosphamide (CP) was dissolved and diluted in sterile distilled water for use as the positive control in the S9 activated test system at final concentrations of 25 and 50 µg/ml. For each positive control one dose with sufficient scorable metaphase cells was selected for analysis. Water was used as the solvent control at the same concentration as that found in the DRM-treated groups. RPMI 1640 complete medium (containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, and 100 µg streptomycin/ml) supplemented with 1% PHA or S9 reaction mixture (20 µl S9/ml of medium) was used in the untreated control. Chromosome aberration assays. The chromosome aberration assay was performed using standard procedures (Evans, 1976). The initial assay was performed by exposing replicate cultures of HPBL to nine concentrations of DRM as well as positive and negative controls. In the initial assay, cells were harvested at approximately 20 h from the initiation of treatment. Due to precipitation of DRM, the highest dose level demonstrating sufficient unobstructed metaphase cells and three lower doses were selected for microscopic evaluation of structural chromosome aberrations. In the repeat assay, replicate cultures of HPBL were exposed to DRM at concentrations of 125, 250, 500, and 750 µg/ml. The concentrations tested were selected based on the findings of the initial assay. For the repeat assay, cells were harvested at three time points in the nonactivated study, 20 and 44 h after the initiation of treatment and at 20 h after the initiation of a 4-h pulse treatment, and in the S9 activated study at two time points, approximately 20 and 44 h after the initiation of treatment. For the chromosome aberration assays, 0.6 ml heparinized blood was inoculated into centrifuge tubes containing 9.4 ml complete medium supplemented with 1% PHA. The tubes were incubated at 37 ± 1◦ C in a humidified atmosphere of 5 ± 1% CO2 in air for approximately 44–48 h. Treatment was carried out by refeeding with approximately 9 ml fresh complete medium or S9 reaction mixture to which was added 1 ml of dosing solution of DRM or control in solvent or solvent alone. An untreated control consisting of cells in complete medium or S9 reaction mixture was also included. 6 Conducted in accordance with OECD Guideline 473, May 1983; and EPA Guideline 40 CFR Subpart 798.5375.
In the nonactivated studies, the cells were exposed for 4, 20, or 44 h at 37 ± 1◦ C in a humidified atmosphere of 5 ± 1% CO2 in air. In the S9-activated studies, the cells were exposed for 4 h at 37 ± 1◦ C in a humidified atmosphere of 5 ± 1% CO2 in air. In the S9 activated treatment groups and the nonactivated 4-h pulse treatment group, after the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium free-phosphate-buffered saline (CMFPBS), refed with complete medium containing 1% PHA, and returned to the incubator for an additional 16 h for the first harvests and 40 h for the delayed harvest. For all treatment and control groups colcemid was added to the cultures at a final concentration of 0.1 µg/ml 2 h prior to the scheduled cell harvests at 20 or 44 h after treatment initiation. Collection of metaphase cells. Two hours after the addition of colcemid metaphase cells were harvested from both the activated and the nonactivated studies by centrifugation. The cell pellet was resuspended in 5 ml 0.075 M KCl and incubated at 37 ± 1◦ C for 20 min. At the end of the KCl treatment and immediately prior to centrifuging, the cells were gently mixed and approximately 0.5 ml of fixative (methanol : glacial acetic acid, 3 : 1 v/v) was added to each tube. The cells were collected by centrifugation, the supernatant was aspirated, and the cells were fixed with two washes with approximately 3–5 ml fixative and stored in fixative overnight or longer at approximately 2–6◦ C. Slide preparation. To prepare slides, the fixed cells were centrifuged, the supernatant fluid was aspirated, and the cells were resuspended in 1 ml cold fresh fixative. The cells were then collected by centrifugation and the supernatant was aspirated, leaving 0.1 to 0.3 ml fixative above the cell pellet. An aliquot of cell suspension was dropped onto a glass slide and allowed to airdry overnight. The slides were then stained with 5% Giemsa and permanently mounted. Evaluation of metaphase cells. Metaphase cells with 46 centromeres were examined under oil immersion without knowledge of treatment groups. A minimum of 200 metaphase spreads (100/duplicate treatment condition) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The mitotic index (% cells in mitosis/500 cells counted) was recorded. In the delayed harvests, the percentage of polyploid cells was recorded per 100 metaphase cells. Evaluation of results. Statistical analysis of the percent aberrant cells was performed using the Fisher’s exact test (Fisher, 1946). In the event of a positive (P ≤ 0.05) Fisher’s test at any dose level, the Cochran– Armitage test was used to determine dose responsiveness (Snedecor and Cochran, 1980). The assay was considered valid if the frequency of cells with structural chromosome aberrations in either the untreated or the
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
solvent control was no greater than 6% and was significantly increased (P ≤ 0.05) in the positive control. Mouse Bone Marrow Micronucleus Assay7 Animals and test materials. The animals used in this study were 8- to 10-week-old male CD-1 mice (Charles River Laboratories Inc., Raleigh, NC). Upon receipt, the animals were quarantined for a minimum of 10 days. The animals were housed one or two per cage prior to dosing and one per cage after dosing in suspended stainless-steel cages with stainless-steel mesh bottoms. Animals were assigned to experimental groups by a computer-generated randomization scheme. Certified Rodent Diet No. 5002 (PMI, St. Louis, MO) and tap water (municipal water system, St. Louis, MO) were provided ad libitum. The animals were housed in environmentally controlled rooms (12-h light:dark cycle, temperature range of 64–79◦ F, and relative humidity of 40–70%). Animal housing and husbandry were in accordance with the provisions of the “Guide for the Care and Use of Laboratory Animals,” USPHSNIH Publication No. 86-23. Experimental design. At least 10 male mice per group were administered a single oral dose of nonlysed DRM (Lot No. 35004) suspended in distilled water (dose volume of 10 ml/kg) at dose levels of 500, 1000, and 2000 mg/kg. Vehicle controls received 10 ml/kg distilled water and the positive controls were dosed with 40 mg/kg CP. All animals were observed for signs of toxicity and mortality on the day of treatment and daily thereafter until sacrifice (24 and 48 h after treatment). Body weights were obtained just prior to dosing and again at the time of sacrifice. All animals were sacrificed by CO2 asphyxiation. Their femora were removed, and their bone marrow was flushed and pooled for slide preparation. Two slides were prepared from each sample and the remaining cell suspension was stored refrigerated to prepare additional slides, if necessary. Following preparation of the smears the slides were allowed to air-dry overnight, fixed in methanol for 5 min, and air-dried. The two slides from each animal were stained using acridine orange (Hayashi et al., 1983) and read the same day using a fluorescence microscope. Scoring of slides. For each animal, two scorers each evaluated: (a) 500 total erythrocytes for polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) and (b) 1000 PCEs for micronucleated polychromatic erythrocytes (MN PCEs). All slides were coded and scored blindly. Scoring data from each animal were
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used to calculate the ratio of PCEs per total erythrocytes (PCEs plus NCEs) and the number of MN PCEs per 1000 PCEs. Statistical analysis. The individual animal was the unit for analysis of MN PCE frequency, PCE/total erythrocyte ratio, and body weight change. MN PCE frequencies for each animal were transformed as the square root prior to analysis (Snedecor and Cochran, 1967; MacGregor et al., 1987). PCE/total erythrocyte ratios were not transformed. Dunnett’s test (one-sided) was used for comparison of treatment group and positive control values with vehicle control values (Dunnett, 1955, 1964). RESULTS Ames/Salmonella Reverse Mutation Assay The potential of DRM to cause point mutations (both base pair and frameshift) in a bacterial system was assessed in the Ames test. Results of the toxicity screens (plate incorporation and preincubation, not shown) using TA100 showed precipitation of DRM at the 500 µg/plate treatment level and higher. DRM precipitation interfered with the counting of revertant colonies at treatment levels of 1500 and 5000 µg/plate. No toxicity was observed at DRM levels of 5000 µg/plate with or without metabolic activation. The maximum level chosen for mutagenicity testing, 500 µg/plate, was based on precipitation and the ability to count revertant colonies. This maximum level is in accord with international guidance for this assay (Gatehouse et al., 1994). Additional lower DRM treatment levels selected for mutagenicity testing were 5, 15, 50, and 150 µg/plate. The results of the plate incorporation and liquid preincubation studies are presented in Tables 1 and 2, respectively. Positive controls specific to each of the five tester strains resulted in the expected increases in the number of histidine revertants. Precipitation of DRM was observed at the 500 µg/plate treatment level for all experiments. DRM precipitation was also observed at the 150 µg/plate level in some experiments. Dose–response evaluation and overall statistical analyses of the plate and preincubation assays demonstrated that DRM was not mutagenic towards any of the S. typhimurium test strains used (TA98, TA100, TA102, TA1535, and TA1537) either in the presence or in the absence of an Aroclor 1254induced rat liver homogenate metabolic activation system. CHO AS52/XPRT Gene Mutation Assay
7 Conducted in accordance with OECD Guideline 474, “Micronucleus Test” (OECD, 1983c, 1995a,b) and the GLP principles of the OECD (1981).
Experiments were initially conducted using the mouse lymphoma suspension assay methodology (not shown). However, the DRM test material precipitated
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TABLE 1 Bacterial Mutagenicity of DRM in the Ames Plate Incorporation Assay Revertants per platea Material
Amount/plate (µg)
S9
TA98
TA100
TA102
TA1535
TA1537
Solvent control Solvent control Positive controlb Positive controlc DRM
− − − − 5 15 50 150 500
− + − + − − − − −
21 ± 6 33 ± 6 122 1420 32 ± 6 20 ± 5 20 ± 4 25 ± 8 21 ± 4(p)
109 ± 19 133 ± 14 938 1980 114 ± 10 101 ± 14 106 ± 13 111 ± 14 111 ± 24(p)
264 ± 23 306 ± 33 1330 1680 275 ± 20 271 ± 18 293 ± 30 272 ± 4 231 ± 35(p)
14 ± 5 15 ± 5 1930 296(t) 13 ± 6 11 ± 4 15 ± 11 13 ± 3(p) 12 ± 1(p)
11 ± 2 12 ± 6 10,300 867 13 ± 11 16 ± 3 20 ± 4 9 ± 4(p) 9 ± 5(p)
DRM
5 15 50 150 500
+ + + + +
36 ± 2 28 ± 5 43 ± 4 36 ± 5 31 ± 6(p)
134 ± 15 137 ± 5 128 ± 9 125 ± 18 132 ± 12(p)
324 ± 3 343 ± 30 342 ± 25 345 ± 30 242 ± 14(p)
13 ± 1 15 ± 2 19 ± 4 10 ± 1(p) 14 ± 5(p)
18 ± 1 11 ± 2 16 ± 2 16 ± 3(p) 13 ± 5(p)
Note. (t), toxicity observed; (p), precipitated DRM, which did not prevent the counting of revertants. Revertants/plate were determined by visual counting. a Average of three plates. b Positive controls used without S9: 4-nitroquinoline-N-oxide (0.2 µg, TA98 and Ta100), cumene hydroperoxide (100 µg, TA102), sodium nitrite (5000 µg, TA1535), and 9-aminoacridine (100 µg, TA1537). c Positive controls used with S9: 2-acetylaminofluorene (30 µg, TA98;), benzo[a]pyrene (2 µg, TA100), danthron (50 µg, TA102), and 2-aminoanthracene (10 µg, TA1535 and TA1537).
in medium at all levels tested and the precipitate could not be removed by washing. The precipitate was found to interfere with automated cell counting. For these reasons the mouse lymphoma suspension assay methodol-
ogy was considered to be inappropriate and DRM was tested in an alternate in vitro mammalian cell gene mutation assay used attached cells. The ability of DRM to induce forward mutation at the XPRT gene locus, as
TABLE 2 Bacterial Mutagenicity of DRM in the Ames Liquid Preincubation Assay Revertants per platea Material
Amount/plate (µg)
S9
TA98
TA100
TA102
TA1535
TA1537
Solvent control Solvent control Positive controlb Positive controlc DRM
− − − − 5 15 50 150 500
− + − + − − − − −
20 ± 6 29 ± 5 108 1720 23 ± 3 20 ± 4 20 ± 7 19 ± 3(p) 19 ± 5(p)
84 ± 9 117 ± 33 1040 1500 94 ± 8 81 ± 3 90 ± 10 103 ± 6∗ 88 ± 11(p)
219 ± 33 295 ± 31 1380 1590 226 ± 38 221 ± 11 222 ± 25 261 ± 38 223 ± 10(p)
16 ± 5 14 ± 5 1660 168(t) 15 ± 5 17 ± 3 16 ± 1 14 ± 2 13 ± 5(p)
12 ± 4 14 ± 3 3380 1450 12 ± 4 14 ± 2 14 ± 2 14 ± 2(p) 10 ± 4(p)
DRM
5 15 50 150 500
+ + + + +
27 ± 4 21 ± 1 22 ± 4 24 ± 4(p) 19 ± 3(p)
84 ± 8 105 ± 4 108 ± 16 124 ± 3 120 ± 11(p)
285 ± 41 303 ± 12 283 ± 13 263 ± 31 274 ± 32(p)
18 ± 1 13 ± 0 15 ± 2 11 ± 2 11 ± 2(p)
13 ± 0 16 ± 4 18 ± 2 15 ± 7(p) 14 ± 4(p)
Note. (t), toxicity observed; (p), precipitated DRM, which did not prevent the counting of revertants. Revertants/plate were determined by visual counting. a Average of three plates. b Positive controls used without S9: 4-nitroquinoline-N-oxide (0.2 µg, TA98 and TA100), curnene hydroperoxide (100 µg, TA102), sodium nitrite (5000 µg, TA1535), and 9-aminoacridine (100 µg, TA1537). c Positive controls used with S9: 2-acetylaminofluorene (30 µg, TA98;), benzo[a]pyrene (2 µg, TA100), danthron (50 µg, TA102), and 2-aminoanthracene (10 µg, TA1535 and TA1537). ∗ P < 0.05.
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
indicated by the induction of 6TG-resistant mutants, was measured in AS52 cells. The solubility of DRM and cytotoxicity to AS52 cells were evaluated in two range-finding experiments (data not shown). AS52 cells were treated with DRM, suspended in Ham’s F12 treatment medium, using a range of test concentrations (10–5000 µg/ml) and different S9 concentrations (0, 1, 5, and 10%). All dosing solutions were a suspension. No other obvious precipitation other than DRM, assessed by visual inspection at the beginning and end of treatment, was observed in the absence or presence of 1, 5, and 10% S9 mix. No significant cytotoxicity (≤50% relative survival) was observed for any treatment levels without S9. Significant cytotoxicity was observed at 1400 µg/ml and higher with 1% S9, at 800 µg/ml and higher with 5% S9 and at 1000 µg/ml and higher with 10% S9 mix. In this initial experiment (Fig. 1), no statistically significant increases in mean mutant frequency or dose responses were observed in any of the DRM-treated cultures without S9 and with 5% S9. In the presence of 1% S9, a statistically significant dose response and a statistically significant increase in mean mutant frequency at the 1250 and 1300 µg/ml DRM treatment levels were observed. A statistically significant higher order dose response was observed in the cultures treated in the presence of 10% S9 mix. In order to determine the reproducibility of the statistical increases observed, the 1% S9 experiment was repeated and the highest treatment level of DRM increased to 1600 µg/ml. In the repeat experiment (Fig. 2), there was no significant cytotoxicity observed in the presence or absence of S9 even though the highest DRM treatment level was increased from 1400 to 1600 µg/ml. Further, there were no statistically significant increases in mean mutant frequency and no statistically significant dose responses in the absence or presence of 1% S9.
FIG. 2. Repeat of the initial AS52 mutagenicity assay of DRM in the presence and absence of 1% S9 mix.
Based on the two initial screening studies, DRM was tested at 200, 500, 1000, 2000, and 5000 µg/ml without S9 mix and at 200, 700, 850, 900, and 1000 µg/ml with 5% S9 mix. The results are presented in Table 3. The positive controls (B[a]P and AcD) yielded the expected positive responses in mutagenicity demonstrating the adequacy of the experimental conditions. No statistically significant increases in mean mutant frequency were observed for cultures either in the presence or
TABLE 3 CHO AS52/XPRT Gene Mutation Assay with DRM
Treatment Medium control AcD −S9 DRM
Medium control B[a]P +5% S9 DRM
FIG. 1. Initial mutagenicity determination of DRM in the AS52 assay at different S9 concentrations.
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Dose (µg/ml)a
Relative survival after treatmentb
Mean mutant frequency/106 viable cells
— 0.05 200 500 1000 2000 5000
1.00 0.53 0.80 0.94 0.93 0.98 0.87
56.2 ± 10.9 150 ± 25 54.9 ± 15.0 43.2 ± 3.0 58.4 ± 6.8 51.5 ± 7.0 85.1 ± 31.5
Dose responsec 1.00 2 0.08 200 0.81 700 0.39 850 0.29 900 0.34 1000 0.07 Dose responsed
46.4 ± 6.3 853 ± 564 64.6 ± 21.2 72.5 ± 57.2 75.5 ± 31.0 75.1 ± 20.0 93.6 ± 23.1
—
a Dose solution of DRM was a suspension at all levels; no other obvious precipitation was observed. b Average results from duplicate cultures. c,d Dose–response relationship as determined by the Snee and Irr (1981) statistical method. In the absence of S9 the linear model was significant (P < 0.05). The quadratic and higher order models were both nonsignificant. In the presence of S9 all models were nonsignificant.
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TABLE 4 Cytogenetic Analysis of Human Peripheral Blood Lymphocytes Treated with DRM in the Absence of S9 % Cells with aberrationsc
Treatment (µg/ml)
Treatment/harvest time (h)
Mitotic index
Aberrations/cella,b
None Water DRM 125 250 500 750 MMC 0.25
4/20 4/20
3.3 4.0
0.000 ± 0.000 0.010 ± 0.141
0.0 0.5
4/20 4/20 4/20 4/20 4/20
3.5 3.6 3.7 2.5 3.7
0.000 ± 0.000 0.000 ± 0.000 0.005 ± 0.071 0.010 ± 0.141 0.215 ± 0.548
0.0 0.0 0.5 0.5 16.0∗∗
20/20 20/20
3.7 2.4
0.000 ± 0.000 0.000 ± 0.000
0.0 0.0
20/20 20/20 20/20 20/20 20/20
3.4 3.9 2.4 2.0 1.4
0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.105 ± 0.353
0.0 0.0 0.0 0.0 9.0∗∗
44/44 44/44
7.9 6.0
0.005 ± 0.071 0.020 ± 0.172
0.5 0.0
0.5 1.5
44/44 44/44 44/44 44/44 44/44
6.4 5.4 3.8 2.8 1.6
0.030 ± 0.198 0.010 ± 0.141 0.015 ± 0.122 0.015 ± 0.122 1.105 ± 1.705
0.0 0.0 0.0 0.0 0.0
2.5 0.5 1.5 1.5 42.0∗∗
None Water DRM 125 250 500 750 MMC 0.25 None Water DRM 125 250 500 750 MMC 0.25
Numericald
Structural
a 200
cells were scored for each treatment. damaged cells were counted as 10 aberrations. c Fisher’s exact test; ∗∗ P ≤ 0.01. d Data not collected for 20-h harvest time. b Severely
absence of 5% S9. No significant cytotoxicity was observed at any of the DRM treatment levels without metabolic activation. With 5% S9 mix, significant cytotoxicity was observed at the 700 µg/ml treatment level and higher of DRM. Although a statistical dose– response trend in mean mutant frequency was observed in the absence of S9 mix, this was due to a small increase in mutant frequency at the highest treatment level tested. Because none of the treatment levels, including the highest treatment level, produced a statistically significant increase in mutant frequency in the absence of S9 mix the result for this experiment was judged to be nonmutagenic. In Vitro Mammalian Cytogenetic Test The clastogenic potential of DRM was studied for its ability to induce chromosome aberrations in HPBL in vitro. In the initial assays (data not shown), HPBL were exposed to nine concentrations of DRM suspended in water ranging from 0.5 to 5000 µg/ml. Exposure of HPBL to DRM was for 20 h in nonactivated cultures and for 4 h (harvest at 20 h) in the presence of S9. While DRM precipitation was observed on the slides
at all concentrations tested, excessive DRM precipitation precluded microscopic analysis of cells at concentrations greater than 500 µg/ml. Therefore, the highest concentration of DRM evaluated microscopically for chromosome aberrations was 500 µg/ml. At this concentration, the mitotic index was the same as control in the absence of S9 and was reduced 8% relative to control in the presence of S9. The percentage of cells with structural aberrations was not significantly different from control in either the presence or the absence of S9. In the repeat assay, cytogenetic analysis of groups treated in the absence or presence of S9 is presented in Tables 4 and 5, respectively. Precipitation of DRM was observed on slides prepared from all dose levels in the presence or absence of S9; however, the precipitation did not obscure the cells to the extent that they were unscorable. In the absence of S9 and at the highest DRM concentration evaluated (750 µg/ml), for chromosomal aberrations, the mitotic indices in the 4-, 20-, and 44-h groups were reduced relative to control 38, 17, and 53%, respectively (Table 4). At the same DRM concentration in the presence of S9, the mitotic indices relative to control in the 20- and 44-h groups were reduced 31 and 6%, respectively (Table 5). The percentage of cells with numerical or structural
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TABLE 5 Cytogenetic Analysis of Human Peripheral Blood Lymphocytes Treated with DRM in the Presence of S9 Treatment (µg/ml)
% Cells with aberrationsc
Treatment/harvest time (h)
Mitotic index
Aberrations/cella,b
4/20 4/20
2.9 3.9
0.020 ± 0.140 0.010 ± 0.100
2.0 1.0
4/20 4/20 4/20 4/20 4/20
2.5 3.5 3.8 2.7 0.5
0.000 ± 0.000 0.005 ± 0.071 0.015 ± 0.122 0.000 ± 0.000 0.330 ± 0.673
0.0 0.5 1.5 0.0 25.0∗∗
4/44 4/44
3.7 3.3
0.000 ± 0.000 0.000 ± 0.000
0.0 0.5
0.0 0.0
4/44 4/44 4/44 4/44 4/44
2.6 3.8 4.1 3.1 0.8
0.000 ± 0.000 0.010 ± 0.100 0.015 ± 0.122 0.000 ± 0.000 0.725 ± 1.748
0.0 0.0 0.0 0.0 0.0
0.0 1.0 1.5 0.0 23.5∗∗
None Water DRM 125 250 500 750 CP 0.25 None Water DRM 125 250 500 750 CP 0.25
Numericald
Structural
a 200
cells were scored for each treatment. damaged cells were counted as 10 aberrations. c Fisher’s exact test; ∗∗ P ≤ 0.01. d Data not collected for 20-h harvest time. b Severely
aberrations following DRM treatment was not significantly different from control (P ≤ 0.05) either in the absence or in the presence of S9. In Vivo Mouse Bone Marrow Micronucleus Assay The ability of DRM, administered by oral gavage, to induce chromosome effects was assessed in mouse bone marrow cells. This assay detects in vivo damage to chromosomes or mitotic apparatus by determining the presence of micronuclei in the PCE in the bone marrow of mice. Induction of micronucleus formation in this assay is indicative of either clastogenic effects or
malsegregation of chromosomes. An advantage of this assay is that it evaluates effects on somatic cells of mice that are treated in vivo and thus is relevant to prediction of potential in vivo mammalian effects (MacGregor et al., 1987). In the preliminary range-finding study (data not shown), DRM was found to be nontoxic to male or female mice at oral dose levels of up to 2000 mg/kg. Therefore, oral DRM doses of 500, 1000, and 2000 mg/kg were chosen for the micronucleus study. There were no deaths, no clinical signs of toxicity, and no significant effects on body weight observed in control, DRM, or positive
TABLE 6 Summary of Micronucleus Results in Male Cd-1 Mice Treated with DRM by Oral Gavage Mean PCE/total erythrocyte ratio Time (h)
Number
Vehicle controla
24 48
5 5
0.56 ± 0.03 0.56 ± 0.07
500 mg/kg
1000 mg/kg
2000 mg/kg
Positive controlb
0.55 ± 0.04 0.60 ± 0.06
0.58 ± 0.06 0.53 ± 0.05
0.57 ± 0.07 0.57 ± 0.05
0.55 ± 0.11
Mean micronucleated PCE/1000 PCE Time (h)
Number
Vehicle controla
500 mg/kg
1000 mg/kg
2000 mg/kg
Positive controlb
24 48
5 5
1.0 ± 0.6 0.8 ± 0.7
0.9 ± 1.0 1.5 ± 0.8
1.1 ± 1.1 0.9 ± 0.9
0.9 ± 0.5 1.3 ± 0.8
16.6 ± 4.3∗∗
a Vehicle
control, distilled water (10 ml/kg body wt). control, cyclophosphamide (40 mg/kg). P ≤ 0.01 (Dunnett’s test).
b Positive ∗∗
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control groups. There were no statistically significant decreases in mean PCE/total erythrocyte ratio and no statistically significant increases in mean micronucleated PCE frequencies compared to control in any DRM treatment group (Table 6). DISCUSSION As part of a comprehensive safety evaluation program, the genotoxicity of DRM was examined in a wide range of in vitro and in vivo assays. All in vitro assays were conducted in replicate cultures using multiple doses in the absence and presence of exogenous metabolic activation. Positive and negative controls were included to assure the validity of each assay. Overall, the data fully support the conclusion that DRM is neither mutagenic nor clastogenic. In the AS52 assay, initial mutagenicity testing was conducted using a range of S9 concentrations (0, 1, 5, and 10%) followed by a confirmatory experiment with 0 and 5% S9. The range of test concentrations, up to clearly cytotoxic levels, varied depending on the concentration of S9 utilized. Overall, greater cytotoxicity was observed with increasing concentrations of S9. In the initial study, a statistically significant increase in mean mutant frequency and a statistically significant dose response was observed with DRM only in the 1% S9 treatment group. However, the increases were very small (<1.7-fold over control values) and were not reproduced in the subsequent repeat study. In the confirmatory study, a statistically significant linear dose response was observed without S9 but none of the treatment levels were statistically increased compared to the control values. Additionally, no treatment-related trends were observed without S9 in two other experiments (the initial mutagenicity assay and subsequent repeat study). This trend in a single experiment was not judged to be DRM treatment related. The authors conclude that DRM was not mutagenic in the AS52/XPRT assay in the absence or presence of 1, 5, or 10% S9. The results of the Ames assay and the in vitro mammalian cytogenetic assay demonstrate that DRM was not mutagenic in any of the five S. typhimurium test strains used and was not clastogenic to human peripheral blood lymphocytes. Finally, in vivo DRM did not induce micronucleus formation in mice further supporting its lack of any chromosomal effects. This series of genotoxicity studies demonstrates that DRM does not have any genotoxic potential. REFERENCES Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for Detecting carcinogens and mutagens with the Salmonella mammalian-microsome test. Mutat. Res. 31, 347–364. Bajpai, P., Bajpai, P. K., and Ward, O. P. (1991a). Production of docosahexaenoic acid by Thraustochytrium aureum. Appl. Microbiol. Biotech. 35, 706–710.
Bajpai, P., Bajpai, P. K., and Ward, O. P. (1991b). Optimization of production of docosahexaenoic acid (DHA) by Thraustochytrium aureum ATCC 34304. J. Am. Oil Chem. Soc. 68, 509–514. Barclay, W. (1992). Process for the Heterotrophic Production of Microbial Products with High Concentrations of Omega-3 Highly Unsaturated Fatty Acids, U.S. patent 5,130,242. U.S. Patent Office, Washington, DC. Barclay, W. R., and Zeller, S. (1996). Nutritional enhancement of n3 and n-6 fatty acids in rotifers and Artemia nauplii by feeding spray-dried Schizochytrium sp. J. World Aquacult. Soc. 27(3), 314– 322. Bartlett, M. S. (1947). Multivariate analysis. J. R. Stat. Soc. Ser. R 9, 176–197. Dixon, W. J., and Massey, F. J. (1969). Introduction to Statistical Analysis, 3rd ed., McGraw–Hill, New York. Draper, N. R., and Smith, H. (1966). Applied Regression Analysis. Wiley, New York. Dunnett, C. W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121. Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482–491. Evans, H. J. (1976). Cytological methods for detecting chemical mutagens. In Chemical Mutagens: Principles and Methods for Their Detection (A. Hollaender, Ed.), Vol. 4. Plenum, New York. Fisher, R. A. (1946). In Statistical Methods for Research Workers, para. 21.01. Oliver and Boyd, Edinburgh, UK. Gatehouse, D., Haaworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Ohta, T., Venitt, S., and Zeiger, E. (1994). Recommendation for the performance of bacterial mutation assays. V. 12, 217–233. Grubbs, F. E., and Beck, G. (1972). Extension of sample sizes and percentage points for significance tests of outlying observations. Technometrics 14, 847–854. Hayashi, M., Sofuni, T., and Ishidate, M. (1983). An application of Acridine Orange fluorescent staining to the micronucleus test. Mutat. Res. 120, 241–247. International Conference on Harmonization (ICH) (1994). Draft Guideline on Specific Aspects of Regulatory Genotoxicity Tests, Fed. Regist., September 22, 1994, Part VII. Department of Health and Human Services, Food and Drug Administration, Washington, DC. Li, A. P. (1982). Quantification of mutation at the Na+ -K+ -ATPase and hypoxanthine guanine phosphoribosyl transferase gene loci in Chinese hamster ovary cells. J. Tissue Culture Methods 7, 27–32. Ling, R. F. (1974). Comparison of several algorithms for computing sample means and variances. J. Am. Stat. Assoc. 69, 859–866. MacGregor, J. T., Heddle, J. A., Hite, M., Margolin, B. H., Ramel, C., Salaake, M. F., Tice, R. R., and Wild, D. (1987). Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutat. Res. 189, 103–112. Munroe, M. E. (1951). Theory of Probability. McGraw–Hill, New York. OECD (1983a). OECD Guideline for Testing of Chemicals—Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline No. 471, adopted May 26, 1983. OECD, Rome, Italy. OECD (1983b). OECD Guideline for Testing of Chemicals—Genetic Toxicology: In Vitro Mammalian Chromosomal Aberration Test, Guideline No. 473, adopted May 1983. OECD, Rome, Italy. OECD (1983c). OECD Guideline for Testing of Chemicals—Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline No. 474, adopted May 1983. OECD, Rome, Italy. OECD (1984). OECD Guideline for Testing of Chemicals—Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test, Guideline No. 476, adopted April 4, 1984. OECD, Rome, Italy.
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE OECD (1995a). OECD Guideline for Testing of Chemicals— Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline No. 471, proposed revision September 1995. OECD, Rome, Italy. OECD (1995b). OECD Guideline for Testing of Chemicals—Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test, Guideline No. 476, proposed revision September 1995. OECD, Rome, Italy. Scott, D., Danford, N. D., Dean, B. J., and Kirkland, D. J. (1990). Metaphase chromosome aberration assays in vitro. In Basic Mutagenicity Tests: UKEMS Recommended Procedures (D. J. Kirkland, Ed.). Cambridge Univ. Press, New York. Snedecor, G. W., and Cochran, W. G. (1980). In Statistical Methods, 7th ed., Iowa State Univ. Press, Ames, IA.
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Snee, R. D., and Irr, J. D. (1981). Design of a statistical method for the analysis of mutagenesis at the hypoxanthine guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells. Mutat. Res. 85, 77–93. U.S. Food and Drug Administration (FDA) (1987). Substances affirmed as generally recognized as safe: Menhaden oil. Fed. Regist. 62, 30751–30757. U.S. Food and Drug Administration (FDA) (1993). Draft Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, “Redbook II.” U.S. FDA, Washington, DC. Zeller, S., and Peik, J. (1996). “Lysed” EX 7566 Test Material for In Vitro Genetic Toxicity Studies, March 6, 1996. NutraSweet Kelco Company, a unit of Monsanto Company, San Diego, CA.
Safety Assessment of DHA-Rich Microalgae from Schizochytrium sp. IV. Mutagenicity Studies Bruce G. Hammond,∗ Dale A. Mayhew,† Larry D. Kier,∗ Richard W. Mast,‡ and Wayne J. Sander§ ∗ Monsanto Company, St. Louis, Missouri 63198; †NutraSweet Company, Mt. Prospect, Illinois 60056; ‡TPSRC, Inc., Severance, New York 12872; and §OmegaTech Inc., Boulder, Colorado 80301 Received August 2, 2001
The purpose of this series of studies was to assess the genotoxic potential of docosahexaenoic acid-rich microalgae from Schizochytrium sp. (DRM). DRM contains oil rich in highly unsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA n-3) is the most abundant PUFA component of the oil (∼29% w/w of total fatty acid content). DHA-rich extracted oil from Schizochytrium sp. is intended for use as a nutritional ingredient in foods. All in vitro assays were conducted with and without mammalian metabolic activation. DRM was not mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains. Mouse lymphoma suspension assay methodology was found to be inappropriate for this test material because precipitating test material could not be removed by washing after the intended exposure period and the precipitate interfered with cell counting. The AS52/XPRT assay methodology was not subject to these problems and DRM was tested and found not to be mutagenic in the CHO AS52/XPRT gene mutation assay. DRM was not clastogenic to human peripheral blood lymphocytes in culture. Additionally, DRM did not induce micronucleus formation in mouse bone marrow in vivo further supporting its lack of any chromosomal effects. Overall, the results of this series of mutagenicity assays support the conclusion that DRM does not have any genotoxic potential. C 2002 Elsevier Science (USA)
INTRODUCTION Docosahexaenoic acid (DHA n-3)-rich dried microalgae is produced via fermentation using Schizochytrium sp. (Barclay, 1992; Bajpai et al., 1991a,b). There are no reports of this organism producing toxic chemicals nor is it pathogenic and chemical analysis confirms the
absence of the common algal toxins, domoic acid, and Prymnesium toxin. Schizochytrium sp. dried microalgae (DRM) has been utilized in aquaculture applications, including enrichment of DHA in Artemia and rotifers used to feed larval fish and shrimp (Barclay and Zeller, 1996). OmegaTech (Boulder, CO) commercialized a product for aquaculture applications (HUFA 2000, a spray-dried form of Schizochytrium sp. dried microalgae), which has been successfully utilized for over 7 years as an excellent, stable dietary source of DHA in shrimp larvaculture and finfish (red seabream, Japanese flounder) culture with no adverse effects. Dried Schizochytrium sp. microalgae has also been determined to be generally recognized as safe (GRAS) for use as a DHA-rich ingredient in broiler chicken and laying hen feed at levels up to 2.8 and 4.3%, respectively. DRM contains oil rich in highly unsaturated fatty acids (PUFAs). DHA is the most abundant PUFA component of the oil [∼29% of total fatty acid (TFA) content]. Docosapentaenoic acid (DPA n-6) is present in DRM but as a minor component (∼10% of TFA). DHA-rich extracted oil from DRM is intended for use as a nutritional ingredient in foods. Fatty acid and sterol components of oil extracted from DRM have been characterized and identified as normal constituents of common human foods. Direct consumption by man of thraustochytrids, especially those of the genus Schizochytrium, is primarily through consumption of mussels and clams. Indirect consumption, through the marine food chain, is more widespread. Exposure to components contained in the extracted oil from its use as a nutritional ingredient in foods is within the normal range of exposures from consumption of foods with these components and long chain PUFA intake is within the limits determined by FDA to be affirmed as GRAS for Menhaden oil (FDA, 1987). In this paper we report the results of a comprehensive series of genotoxicity studies on DRM.
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0273-2300/02 $35.00 C 2002 Elsevier Science (USA)
All rights reserved.
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MATERIALS AND METHODS1 Chemicals DRM (Lot No. 35003, 35004, and 40026) was obtained from Monsanto Company, NutraSweet Kelco Unit (San Diego, CA). In the Ames assay and the CHO/AS52/XPRT assay Schizochytrium sp. cells were fully lysed by homogenization at pressures greater than 9000 psi (Zeller and Peik, 1996). Intact Schizochytrium sp. was used in the cytogenetic and mouse micronucleus studies. In all studies, the concentration, stability, and homogeneity of DRM suspensions/solutions were verified prior to and upon completion of the study. All positive control chemicals were at least reagent grade and obtained from Sigma (St. Louis, MO). Ames/Salmonella Reverse Mutation Assay2 Test strains. The Salmonella typhimurium test strains (TA98, TA100, TA102, TA1535, and TA1537) were obtained from the laboratory of Dr. B. N. Ames (Berkeley, CA). The proper phenotype of each culture was verified. S9 preparation and mix. The S9 preparations from livers of Aroclor 1254-induced male Sprague–Dawley rats (Hilltop Laboratories, Scottsdale, PA,) were purchased from Molecular Toxicology, Inc. (Annapolis, MD; Lot No. 0340, 41.4 mg protein/ml). The procedures used in preparation of the S9 supernatant solutions and the preparation of the S9 metabolic activation system (S9 mix) were those described by Ames et al. (1975). Plate incorporation and preincubation tests. The general procedures used were basically those described by Ames et al. (1975). Plate incorporation tests were performed by mixing DRM or control solutions, 0.1 ml of bacterial culture and, if appropriate, 0.5 ml of S9 mix (as described above) with 2 ml of histidine–biotin top agar. The mixture was poured onto minimal glucose agar plates. Preincubation tests were performed by mixing DRM or control solutions, 0.1 ml of bacterial culture, and 0.5 ml of S9 mix or S9 mix buffer. The mixture was incubated at 37 ± 1◦ C in a shaking incubator for 20 min. After incubation, 2 ml of histidine–biotin top agar, maintained at 44–48◦ C, was added and the mixture was poured onto minimal glucose agar plates. Initially, a toxicity test was conducted using plate and preincubation methods with test strain TA100 with and 1 All studies were conducted in accordance with U.S. Food and Drug Administration (FDA) Good Laboratory Practices (GLP) Regulations [(21 CFR Part 58) March 21, 1994] and the GLP principles of the OECD (1981). 2 This study was performed in general accordance with: OECD Guideline 471 (OECD, 1983a,b,c, 1995a,b), U.S. Food and Drug Administration Redbook II (FDA, 1993), and ICH (1994). The study was conducted by Monsanto Company, Environmental Health Laboratory, St. Louis, MO, and completed in June 1996.
without S9 to define dose levels for mutagenicity testing. Once dose levels were established, mutagenicity testing was conducted using plate incorporation and preincubation assay methods in five Salmonella test strains (TA98, TA100, TA102, TA1535, and TA1537) with and without metabolic activation. In the mutagenicity test, three replicate plates were prepared for each strain/S9/DRM dose level combination. Concurrent positive and negative controls (solvent and nonsolvent) were conducted for all mutagenicity tests. Solvent control plates used the same volume of water per plate as that selected for DRM. Plates were examined and revertant colonies were counted after at least 48 h at 37 ± 1◦ C. Revertant colonies on other plates, except as noted, were counted with an Artek Model 880 automatic colony counter or counted by visual examination (≤10 revertants/plate). Revertant colonies for plates with more than 500 revertant colonies/plate were estimated by counting colonies in several fields under a stereomicroscope and multiplying the counted colonies by a factor relating the total plate area to the area of the counted fields. Statistical analysis. Statistical analysis was performed on plate incorporation assay results after transforming revertant/plate values as log10 (revertants/plate). Analysis included Bartlett’s test for homogeneity of variance (Bartlett, 1947) and comparison of treatments with controls using within-levels pooled variance and a one-sided t test (Dixon and Massey, 1969; Ling, 1974; Munroe, 1951). Grubbs’ test was performed to determine if outliers were present (Grubbs and Beck, 1972). Statistical significance of dose response was evaluated by regression analysis for log10 transformed doses and revertants/plate (Draper and Smith, 1966). Evaluation criteria. A critical level of P ≤ 0.01 was used in determining statistical significance. As a general guide, results were considered clearly positive for a strain/microsome combination if revertants/plate values were significantly elevated over control values (P ≤ 0.01) at three or more treatment levels, and there was a statistically significant dose response (P ≤ 0.01). CHO AS52/XPRT Gene Mutation Assay3 The mutagenic potential of DRM was tested in cultured Chinese hamster ovary/ xanthine–guanine phosphoribosyl transferase (CHO AS52/ XPRT) gene locus assay. 3 This study was performed in general accordance with: OECD Guideline 476 (OECD, 1983a,b,c, 1995a,b), GLP principles of the OECD (1981), U.S. Food and Drug Administration Redbook II (FDA, 1993), and ICH (1994). The study was conducted by Monsanto Company, Environmental Health Laboratory, St. Louis, MO, and completed in September 1996.
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
Cell line. The AS52 cell line was obtained from Dr. L. F. Stankowski, Jr. (Pharmakon International Laboratory, Waverly, PA). The AS52 cells were maintained as logarithmic phase growing cultures, periodically examined for mycoplasma contamination, and cloned to prevent a high spontaneous mutant frequency. Chemicals. Solutions of the DRM (Lot No. 35003) were prepared on the day of use in Ham’s F12 treatment medium as the solvent. DRM was suspended in medium at the desired concentrations before being added to the appropriate treatment flasks. The positive controls were benzo[a]pyrene (B[a]P) with S9 mix and actinomycin D (AcD) without S9 mix. The positive controls were prepared in DMSO and added to medium to yield final concentrations of 2 µg/ml B[a]P and 0.005–0.05 µg/ml AcD. The S9 preparations (Ames et al., 1975) were purchased from Molecular Toxicology, Inc. (Lot No. 0340, 41.4 mg protein/ml). The different concentrations of S9 in this study represent the percentage of S9 (v/v) in the S9/cofactor mixtures. Two milliliters of the S9/cofactor mixture was added to 8 ml of medium for cytotoxicity and mutagenicity testing. Cytotoxicity determination. AS52 cells were seeded in 75-cm2 plastic culture flasks at 0.5 × 106 cells/flask in Ham’s F12 medium, supplemented with 5% heatinactivated fetal bovine serum plus 200 U/ml penicillin, and 200 µg/ml streptomycin (F12FCM5 ), 18–24 h before treatment. On the day of treatment, medium was changed to Ham’s F12 medium without serum in the presence and absence of S9. Suspensions of DRM were prepared in treatment medium at the desired concentrations and then added to the appropriate treatment flask. After an incubation of 5 h at 37 ± 1◦ C, the treatment medium was discarded. The cells were washed with 5 ml Hanks’ balanced salt solution and removed from the flasks by trypsinization, resuspended, and counted. Three aliquots of approximately 200 cells were plated per sample for determination of cloning efficiency. The plates were returned to incubation for 6–9 days. The colonies that developed were fixed with methanol, stained with 10% Giemsa, and manually counted. Cytotoxicity was expressed as relative survival (relative survival = cloning efficiency treated/cloning efficiency control). The cytotoxicity results (data not shown) were used for dose-selection purposes for the mutagenicity experiments. Mutagenesis. For mutagenesis experiments the total number of cells surviving treatment and used at each stage of processing were at least 1 × 106 cells.4 4
As recommended in OECD Guideline 476 (OECD, 1984, 1995a,b), this number of cells is approximately 10 times the inverse of the spontaneous AS52/XPRT locus mutant frequency of 1 × 10−5 . For cell survival, anticipated being ≥50%, this was accomplished by treating at least duplicate flasks with 1 × 106 cells/flask as described above for cytotoxicity.
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AS52 cells were plated on the day before treatment as described for cytotoxicity determination. The next day, they were treated for 5 h with the DRM (five dose levels in triplicate with and without S9), positive controls, or solvent control. The cells were then processed as described above for cytotoxicity determination, except that in addition to plating 200 cells for cloning efficiency, the treated cells from each flask (approximately 106 to 107 treated cells) were plated in 10 ml of subculture medium (hypoxanthine-free FI2FCM5 ). The cells were subcultured every 2–3 days as attached cultures for 7–9 days for the expression of the mutant phenotype. Mutant selection was performed as previously described (Li, 1982) using selective medium consisting of hypoxanthine-free Ham’s F12 medium supplemented with 10 µM 6-thioguanine (6TG) and 5% heat-inactivated dialyzed fetal calf serum plus 200 U/ml penicillin and 200 µg/ml streptomycin. Approximately 106 cells/sample were plated in 100-mm plates (five plates, 2 × 105 cells/plate) in 8 ml of selective medium per plate for mutant selection. Three aliquots of approximately 200 cells/sample were plated in 3 ml of selective medium without 6TG and incubated for 6–10 days for the determination of cloning efficiency (C.E.). Colonies developed were fixed, stained, and counted by hand. Results were expressed as mean mutation frequency (MF) where MF = (number of mutant colonies/106 cells plated) × (1/cloning efficiency) = number of mutant colonies per 106 viable cells. Statistical analysis. Mutagenicity data were analyzed according to the statistical method of Snee and Irr (1981) designed originally for the CHO/HGPRT mutation assay.5 Student’s t test was then used to compare treatment data to solvent data. Evaluation criteria. A chemical was considered negative if, when tested to approximately 10–20% relative survival, to the limit of solubility, or to a maximum dose of 5 mg/ml, there was no increasing dose response (P ≤ 0.05) and no significantly elevated (P ≤ 0.05) mutation frequencies over control values. A chemical would be considered positive if a reproducible dosedependent increase in mean mutant frequency with a positive slope statistically different (P ≤ 0.05) from zero was observed. In addition, at least one treatment level should have had a mean mutant frequency statistically greater (P ≤ 0.05) than control values, and at least twofold greater than the solvent control values. 5 In this analysis, mutant frequency values were transformed according to the equation y = (X + 1)0.15 , where y = transformed mutant frequency and X = observed mutant frequency. The Snee and Irr analysis also allowed the evaluation of dose–response relationships as linear, quadratic, or higher order. A computer program obtained from Dr. Irr (Dupont) was used to calculate mutant frequency transformation.
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In Vitro Mammalian Cytogenetic Test6 The clastogenic potential of DRM was assessed based on its ability to induce chromosome aberrations in human peripheral blood lymphocytes (HPBL). Test materials. DRM (Lot No. 40026) was suspended in sterile distilled water. Mitomycin C (MMC) was dissolved and diluted in sterile distilled water for use as the positive control in the non-S9-activated test system at final concentrations of 0.13 and 0.25 µg/ml. Cyclophosphamide (CP) was dissolved and diluted in sterile distilled water for use as the positive control in the S9 activated test system at final concentrations of 25 and 50 µg/ml. For each positive control one dose with sufficient scorable metaphase cells was selected for analysis. Water was used as the solvent control at the same concentration as that found in the DRM-treated groups. RPMI 1640 complete medium (containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, and 100 µg streptomycin/ml) supplemented with 1% PHA or S9 reaction mixture (20 µl S9/ml of medium) was used in the untreated control. Chromosome aberration assays. The chromosome aberration assay was performed using standard procedures (Evans, 1976). The initial assay was performed by exposing replicate cultures of HPBL to nine concentrations of DRM as well as positive and negative controls. In the initial assay, cells were harvested at approximately 20 h from the initiation of treatment. Due to precipitation of DRM, the highest dose level demonstrating sufficient unobstructed metaphase cells and three lower doses were selected for microscopic evaluation of structural chromosome aberrations. In the repeat assay, replicate cultures of HPBL were exposed to DRM at concentrations of 125, 250, 500, and 750 µg/ml. The concentrations tested were selected based on the findings of the initial assay. For the repeat assay, cells were harvested at three time points in the nonactivated study, 20 and 44 h after the initiation of treatment and at 20 h after the initiation of a 4-h pulse treatment, and in the S9 activated study at two time points, approximately 20 and 44 h after the initiation of treatment. For the chromosome aberration assays, 0.6 ml heparinized blood was inoculated into centrifuge tubes containing 9.4 ml complete medium supplemented with 1% PHA. The tubes were incubated at 37 ± 1◦ C in a humidified atmosphere of 5 ± 1% CO2 in air for approximately 44–48 h. Treatment was carried out by refeeding with approximately 9 ml fresh complete medium or S9 reaction mixture to which was added 1 ml of dosing solution of DRM or control in solvent or solvent alone. An untreated control consisting of cells in complete medium or S9 reaction mixture was also included. 6 Conducted in accordance with OECD Guideline 473, May 1983; and EPA Guideline 40 CFR Subpart 798.5375.
In the nonactivated studies, the cells were exposed for 4, 20, or 44 h at 37 ± 1◦ C in a humidified atmosphere of 5 ± 1% CO2 in air. In the S9-activated studies, the cells were exposed for 4 h at 37 ± 1◦ C in a humidified atmosphere of 5 ± 1% CO2 in air. In the S9 activated treatment groups and the nonactivated 4-h pulse treatment group, after the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium free-phosphate-buffered saline (CMFPBS), refed with complete medium containing 1% PHA, and returned to the incubator for an additional 16 h for the first harvests and 40 h for the delayed harvest. For all treatment and control groups colcemid was added to the cultures at a final concentration of 0.1 µg/ml 2 h prior to the scheduled cell harvests at 20 or 44 h after treatment initiation. Collection of metaphase cells. Two hours after the addition of colcemid metaphase cells were harvested from both the activated and the nonactivated studies by centrifugation. The cell pellet was resuspended in 5 ml 0.075 M KCl and incubated at 37 ± 1◦ C for 20 min. At the end of the KCl treatment and immediately prior to centrifuging, the cells were gently mixed and approximately 0.5 ml of fixative (methanol : glacial acetic acid, 3 : 1 v/v) was added to each tube. The cells were collected by centrifugation, the supernatant was aspirated, and the cells were fixed with two washes with approximately 3–5 ml fixative and stored in fixative overnight or longer at approximately 2–6◦ C. Slide preparation. To prepare slides, the fixed cells were centrifuged, the supernatant fluid was aspirated, and the cells were resuspended in 1 ml cold fresh fixative. The cells were then collected by centrifugation and the supernatant was aspirated, leaving 0.1 to 0.3 ml fixative above the cell pellet. An aliquot of cell suspension was dropped onto a glass slide and allowed to airdry overnight. The slides were then stained with 5% Giemsa and permanently mounted. Evaluation of metaphase cells. Metaphase cells with 46 centromeres were examined under oil immersion without knowledge of treatment groups. A minimum of 200 metaphase spreads (100/duplicate treatment condition) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The mitotic index (% cells in mitosis/500 cells counted) was recorded. In the delayed harvests, the percentage of polyploid cells was recorded per 100 metaphase cells. Evaluation of results. Statistical analysis of the percent aberrant cells was performed using the Fisher’s exact test (Fisher, 1946). In the event of a positive (P ≤ 0.05) Fisher’s test at any dose level, the Cochran– Armitage test was used to determine dose responsiveness (Snedecor and Cochran, 1980). The assay was considered valid if the frequency of cells with structural chromosome aberrations in either the untreated or the
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
solvent control was no greater than 6% and was significantly increased (P ≤ 0.05) in the positive control. Mouse Bone Marrow Micronucleus Assay7 Animals and test materials. The animals used in this study were 8- to 10-week-old male CD-1 mice (Charles River Laboratories Inc., Raleigh, NC). Upon receipt, the animals were quarantined for a minimum of 10 days. The animals were housed one or two per cage prior to dosing and one per cage after dosing in suspended stainless-steel cages with stainless-steel mesh bottoms. Animals were assigned to experimental groups by a computer-generated randomization scheme. Certified Rodent Diet No. 5002 (PMI, St. Louis, MO) and tap water (municipal water system, St. Louis, MO) were provided ad libitum. The animals were housed in environmentally controlled rooms (12-h light:dark cycle, temperature range of 64–79◦ F, and relative humidity of 40–70%). Animal housing and husbandry were in accordance with the provisions of the “Guide for the Care and Use of Laboratory Animals,” USPHSNIH Publication No. 86-23. Experimental design. At least 10 male mice per group were administered a single oral dose of nonlysed DRM (Lot No. 35004) suspended in distilled water (dose volume of 10 ml/kg) at dose levels of 500, 1000, and 2000 mg/kg. Vehicle controls received 10 ml/kg distilled water and the positive controls were dosed with 40 mg/kg CP. All animals were observed for signs of toxicity and mortality on the day of treatment and daily thereafter until sacrifice (24 and 48 h after treatment). Body weights were obtained just prior to dosing and again at the time of sacrifice. All animals were sacrificed by CO2 asphyxiation. Their femora were removed, and their bone marrow was flushed and pooled for slide preparation. Two slides were prepared from each sample and the remaining cell suspension was stored refrigerated to prepare additional slides, if necessary. Following preparation of the smears the slides were allowed to air-dry overnight, fixed in methanol for 5 min, and air-dried. The two slides from each animal were stained using acridine orange (Hayashi et al., 1983) and read the same day using a fluorescence microscope. Scoring of slides. For each animal, two scorers each evaluated: (a) 500 total erythrocytes for polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) and (b) 1000 PCEs for micronucleated polychromatic erythrocytes (MN PCEs). All slides were coded and scored blindly. Scoring data from each animal were
259
used to calculate the ratio of PCEs per total erythrocytes (PCEs plus NCEs) and the number of MN PCEs per 1000 PCEs. Statistical analysis. The individual animal was the unit for analysis of MN PCE frequency, PCE/total erythrocyte ratio, and body weight change. MN PCE frequencies for each animal were transformed as the square root prior to analysis (Snedecor and Cochran, 1967; MacGregor et al., 1987). PCE/total erythrocyte ratios were not transformed. Dunnett’s test (one-sided) was used for comparison of treatment group and positive control values with vehicle control values (Dunnett, 1955, 1964). RESULTS Ames/Salmonella Reverse Mutation Assay The potential of DRM to cause point mutations (both base pair and frameshift) in a bacterial system was assessed in the Ames test. Results of the toxicity screens (plate incorporation and preincubation, not shown) using TA100 showed precipitation of DRM at the 500 µg/plate treatment level and higher. DRM precipitation interfered with the counting of revertant colonies at treatment levels of 1500 and 5000 µg/plate. No toxicity was observed at DRM levels of 5000 µg/plate with or without metabolic activation. The maximum level chosen for mutagenicity testing, 500 µg/plate, was based on precipitation and the ability to count revertant colonies. This maximum level is in accord with international guidance for this assay (Gatehouse et al., 1994). Additional lower DRM treatment levels selected for mutagenicity testing were 5, 15, 50, and 150 µg/plate. The results of the plate incorporation and liquid preincubation studies are presented in Tables 1 and 2, respectively. Positive controls specific to each of the five tester strains resulted in the expected increases in the number of histidine revertants. Precipitation of DRM was observed at the 500 µg/plate treatment level for all experiments. DRM precipitation was also observed at the 150 µg/plate level in some experiments. Dose–response evaluation and overall statistical analyses of the plate and preincubation assays demonstrated that DRM was not mutagenic towards any of the S. typhimurium test strains used (TA98, TA100, TA102, TA1535, and TA1537) either in the presence or in the absence of an Aroclor 1254induced rat liver homogenate metabolic activation system. CHO AS52/XPRT Gene Mutation Assay
7 Conducted in accordance with OECD Guideline 474, “Micronucleus Test” (OECD, 1983c, 1995a,b) and the GLP principles of the OECD (1981).
Experiments were initially conducted using the mouse lymphoma suspension assay methodology (not shown). However, the DRM test material precipitated
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TABLE 1 Bacterial Mutagenicity of DRM in the Ames Plate Incorporation Assay Revertants per platea Material
Amount/plate (µg)
S9
TA98
TA100
TA102
TA1535
TA1537
Solvent control Solvent control Positive controlb Positive controlc DRM
− − − − 5 15 50 150 500
− + − + − − − − −
21 ± 6 33 ± 6 122 1420 32 ± 6 20 ± 5 20 ± 4 25 ± 8 21 ± 4(p)
109 ± 19 133 ± 14 938 1980 114 ± 10 101 ± 14 106 ± 13 111 ± 14 111 ± 24(p)
264 ± 23 306 ± 33 1330 1680 275 ± 20 271 ± 18 293 ± 30 272 ± 4 231 ± 35(p)
14 ± 5 15 ± 5 1930 296(t) 13 ± 6 11 ± 4 15 ± 11 13 ± 3(p) 12 ± 1(p)
11 ± 2 12 ± 6 10,300 867 13 ± 11 16 ± 3 20 ± 4 9 ± 4(p) 9 ± 5(p)
DRM
5 15 50 150 500
+ + + + +
36 ± 2 28 ± 5 43 ± 4 36 ± 5 31 ± 6(p)
134 ± 15 137 ± 5 128 ± 9 125 ± 18 132 ± 12(p)
324 ± 3 343 ± 30 342 ± 25 345 ± 30 242 ± 14(p)
13 ± 1 15 ± 2 19 ± 4 10 ± 1(p) 14 ± 5(p)
18 ± 1 11 ± 2 16 ± 2 16 ± 3(p) 13 ± 5(p)
Note. (t), toxicity observed; (p), precipitated DRM, which did not prevent the counting of revertants. Revertants/plate were determined by visual counting. a Average of three plates. b Positive controls used without S9: 4-nitroquinoline-N-oxide (0.2 µg, TA98 and Ta100), cumene hydroperoxide (100 µg, TA102), sodium nitrite (5000 µg, TA1535), and 9-aminoacridine (100 µg, TA1537). c Positive controls used with S9: 2-acetylaminofluorene (30 µg, TA98;), benzo[a]pyrene (2 µg, TA100), danthron (50 µg, TA102), and 2-aminoanthracene (10 µg, TA1535 and TA1537).
in medium at all levels tested and the precipitate could not be removed by washing. The precipitate was found to interfere with automated cell counting. For these reasons the mouse lymphoma suspension assay methodol-
ogy was considered to be inappropriate and DRM was tested in an alternate in vitro mammalian cell gene mutation assay used attached cells. The ability of DRM to induce forward mutation at the XPRT gene locus, as
TABLE 2 Bacterial Mutagenicity of DRM in the Ames Liquid Preincubation Assay Revertants per platea Material
Amount/plate (µg)
S9
TA98
TA100
TA102
TA1535
TA1537
Solvent control Solvent control Positive controlb Positive controlc DRM
− − − − 5 15 50 150 500
− + − + − − − − −
20 ± 6 29 ± 5 108 1720 23 ± 3 20 ± 4 20 ± 7 19 ± 3(p) 19 ± 5(p)
84 ± 9 117 ± 33 1040 1500 94 ± 8 81 ± 3 90 ± 10 103 ± 6∗ 88 ± 11(p)
219 ± 33 295 ± 31 1380 1590 226 ± 38 221 ± 11 222 ± 25 261 ± 38 223 ± 10(p)
16 ± 5 14 ± 5 1660 168(t) 15 ± 5 17 ± 3 16 ± 1 14 ± 2 13 ± 5(p)
12 ± 4 14 ± 3 3380 1450 12 ± 4 14 ± 2 14 ± 2 14 ± 2(p) 10 ± 4(p)
DRM
5 15 50 150 500
+ + + + +
27 ± 4 21 ± 1 22 ± 4 24 ± 4(p) 19 ± 3(p)
84 ± 8 105 ± 4 108 ± 16 124 ± 3 120 ± 11(p)
285 ± 41 303 ± 12 283 ± 13 263 ± 31 274 ± 32(p)
18 ± 1 13 ± 0 15 ± 2 11 ± 2 11 ± 2(p)
13 ± 0 16 ± 4 18 ± 2 15 ± 7(p) 14 ± 4(p)
Note. (t), toxicity observed; (p), precipitated DRM, which did not prevent the counting of revertants. Revertants/plate were determined by visual counting. a Average of three plates. b Positive controls used without S9: 4-nitroquinoline-N-oxide (0.2 µg, TA98 and TA100), curnene hydroperoxide (100 µg, TA102), sodium nitrite (5000 µg, TA1535), and 9-aminoacridine (100 µg, TA1537). c Positive controls used with S9: 2-acetylaminofluorene (30 µg, TA98;), benzo[a]pyrene (2 µg, TA100), danthron (50 µg, TA102), and 2-aminoanthracene (10 µg, TA1535 and TA1537). ∗ P < 0.05.
MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
indicated by the induction of 6TG-resistant mutants, was measured in AS52 cells. The solubility of DRM and cytotoxicity to AS52 cells were evaluated in two range-finding experiments (data not shown). AS52 cells were treated with DRM, suspended in Ham’s F12 treatment medium, using a range of test concentrations (10–5000 µg/ml) and different S9 concentrations (0, 1, 5, and 10%). All dosing solutions were a suspension. No other obvious precipitation other than DRM, assessed by visual inspection at the beginning and end of treatment, was observed in the absence or presence of 1, 5, and 10% S9 mix. No significant cytotoxicity (≤50% relative survival) was observed for any treatment levels without S9. Significant cytotoxicity was observed at 1400 µg/ml and higher with 1% S9, at 800 µg/ml and higher with 5% S9 and at 1000 µg/ml and higher with 10% S9 mix. In this initial experiment (Fig. 1), no statistically significant increases in mean mutant frequency or dose responses were observed in any of the DRM-treated cultures without S9 and with 5% S9. In the presence of 1% S9, a statistically significant dose response and a statistically significant increase in mean mutant frequency at the 1250 and 1300 µg/ml DRM treatment levels were observed. A statistically significant higher order dose response was observed in the cultures treated in the presence of 10% S9 mix. In order to determine the reproducibility of the statistical increases observed, the 1% S9 experiment was repeated and the highest treatment level of DRM increased to 1600 µg/ml. In the repeat experiment (Fig. 2), there was no significant cytotoxicity observed in the presence or absence of S9 even though the highest DRM treatment level was increased from 1400 to 1600 µg/ml. Further, there were no statistically significant increases in mean mutant frequency and no statistically significant dose responses in the absence or presence of 1% S9.
FIG. 2. Repeat of the initial AS52 mutagenicity assay of DRM in the presence and absence of 1% S9 mix.
Based on the two initial screening studies, DRM was tested at 200, 500, 1000, 2000, and 5000 µg/ml without S9 mix and at 200, 700, 850, 900, and 1000 µg/ml with 5% S9 mix. The results are presented in Table 3. The positive controls (B[a]P and AcD) yielded the expected positive responses in mutagenicity demonstrating the adequacy of the experimental conditions. No statistically significant increases in mean mutant frequency were observed for cultures either in the presence or
TABLE 3 CHO AS52/XPRT Gene Mutation Assay with DRM
Treatment Medium control AcD −S9 DRM
Medium control B[a]P +5% S9 DRM
FIG. 1. Initial mutagenicity determination of DRM in the AS52 assay at different S9 concentrations.
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Dose (µg/ml)a
Relative survival after treatmentb
Mean mutant frequency/106 viable cells
— 0.05 200 500 1000 2000 5000
1.00 0.53 0.80 0.94 0.93 0.98 0.87
56.2 ± 10.9 150 ± 25 54.9 ± 15.0 43.2 ± 3.0 58.4 ± 6.8 51.5 ± 7.0 85.1 ± 31.5
Dose responsec 1.00 2 0.08 200 0.81 700 0.39 850 0.29 900 0.34 1000 0.07 Dose responsed
46.4 ± 6.3 853 ± 564 64.6 ± 21.2 72.5 ± 57.2 75.5 ± 31.0 75.1 ± 20.0 93.6 ± 23.1
—
a Dose solution of DRM was a suspension at all levels; no other obvious precipitation was observed. b Average results from duplicate cultures. c,d Dose–response relationship as determined by the Snee and Irr (1981) statistical method. In the absence of S9 the linear model was significant (P < 0.05). The quadratic and higher order models were both nonsignificant. In the presence of S9 all models were nonsignificant.
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TABLE 4 Cytogenetic Analysis of Human Peripheral Blood Lymphocytes Treated with DRM in the Absence of S9 % Cells with aberrationsc
Treatment (µg/ml)
Treatment/harvest time (h)
Mitotic index
Aberrations/cella,b
None Water DRM 125 250 500 750 MMC 0.25
4/20 4/20
3.3 4.0
0.000 ± 0.000 0.010 ± 0.141
0.0 0.5
4/20 4/20 4/20 4/20 4/20
3.5 3.6 3.7 2.5 3.7
0.000 ± 0.000 0.000 ± 0.000 0.005 ± 0.071 0.010 ± 0.141 0.215 ± 0.548
0.0 0.0 0.5 0.5 16.0∗∗
20/20 20/20
3.7 2.4
0.000 ± 0.000 0.000 ± 0.000
0.0 0.0
20/20 20/20 20/20 20/20 20/20
3.4 3.9 2.4 2.0 1.4
0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.105 ± 0.353
0.0 0.0 0.0 0.0 9.0∗∗
44/44 44/44
7.9 6.0
0.005 ± 0.071 0.020 ± 0.172
0.5 0.0
0.5 1.5
44/44 44/44 44/44 44/44 44/44
6.4 5.4 3.8 2.8 1.6
0.030 ± 0.198 0.010 ± 0.141 0.015 ± 0.122 0.015 ± 0.122 1.105 ± 1.705
0.0 0.0 0.0 0.0 0.0
2.5 0.5 1.5 1.5 42.0∗∗
None Water DRM 125 250 500 750 MMC 0.25 None Water DRM 125 250 500 750 MMC 0.25
Numericald
Structural
a 200
cells were scored for each treatment. damaged cells were counted as 10 aberrations. c Fisher’s exact test; ∗∗ P ≤ 0.01. d Data not collected for 20-h harvest time. b Severely
absence of 5% S9. No significant cytotoxicity was observed at any of the DRM treatment levels without metabolic activation. With 5% S9 mix, significant cytotoxicity was observed at the 700 µg/ml treatment level and higher of DRM. Although a statistical dose– response trend in mean mutant frequency was observed in the absence of S9 mix, this was due to a small increase in mutant frequency at the highest treatment level tested. Because none of the treatment levels, including the highest treatment level, produced a statistically significant increase in mutant frequency in the absence of S9 mix the result for this experiment was judged to be nonmutagenic. In Vitro Mammalian Cytogenetic Test The clastogenic potential of DRM was studied for its ability to induce chromosome aberrations in HPBL in vitro. In the initial assays (data not shown), HPBL were exposed to nine concentrations of DRM suspended in water ranging from 0.5 to 5000 µg/ml. Exposure of HPBL to DRM was for 20 h in nonactivated cultures and for 4 h (harvest at 20 h) in the presence of S9. While DRM precipitation was observed on the slides
at all concentrations tested, excessive DRM precipitation precluded microscopic analysis of cells at concentrations greater than 500 µg/ml. Therefore, the highest concentration of DRM evaluated microscopically for chromosome aberrations was 500 µg/ml. At this concentration, the mitotic index was the same as control in the absence of S9 and was reduced 8% relative to control in the presence of S9. The percentage of cells with structural aberrations was not significantly different from control in either the presence or the absence of S9. In the repeat assay, cytogenetic analysis of groups treated in the absence or presence of S9 is presented in Tables 4 and 5, respectively. Precipitation of DRM was observed on slides prepared from all dose levels in the presence or absence of S9; however, the precipitation did not obscure the cells to the extent that they were unscorable. In the absence of S9 and at the highest DRM concentration evaluated (750 µg/ml), for chromosomal aberrations, the mitotic indices in the 4-, 20-, and 44-h groups were reduced relative to control 38, 17, and 53%, respectively (Table 4). At the same DRM concentration in the presence of S9, the mitotic indices relative to control in the 20- and 44-h groups were reduced 31 and 6%, respectively (Table 5). The percentage of cells with numerical or structural
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MUTAGENICITY STUDY OF DHA-RICH SCHIZOCHYTRIUM SP. MICROALGAE
TABLE 5 Cytogenetic Analysis of Human Peripheral Blood Lymphocytes Treated with DRM in the Presence of S9 Treatment (µg/ml)
% Cells with aberrationsc
Treatment/harvest time (h)
Mitotic index
Aberrations/cella,b
4/20 4/20
2.9 3.9
0.020 ± 0.140 0.010 ± 0.100
2.0 1.0
4/20 4/20 4/20 4/20 4/20
2.5 3.5 3.8 2.7 0.5
0.000 ± 0.000 0.005 ± 0.071 0.015 ± 0.122 0.000 ± 0.000 0.330 ± 0.673
0.0 0.5 1.5 0.0 25.0∗∗
4/44 4/44
3.7 3.3
0.000 ± 0.000 0.000 ± 0.000
0.0 0.5
0.0 0.0
4/44 4/44 4/44 4/44 4/44
2.6 3.8 4.1 3.1 0.8
0.000 ± 0.000 0.010 ± 0.100 0.015 ± 0.122 0.000 ± 0.000 0.725 ± 1.748
0.0 0.0 0.0 0.0 0.0
0.0 1.0 1.5 0.0 23.5∗∗
None Water DRM 125 250 500 750 CP 0.25 None Water DRM 125 250 500 750 CP 0.25
Numericald
Structural
a 200
cells were scored for each treatment. damaged cells were counted as 10 aberrations. c Fisher’s exact test; ∗∗ P ≤ 0.01. d Data not collected for 20-h harvest time. b Severely
aberrations following DRM treatment was not significantly different from control (P ≤ 0.05) either in the absence or in the presence of S9. In Vivo Mouse Bone Marrow Micronucleus Assay The ability of DRM, administered by oral gavage, to induce chromosome effects was assessed in mouse bone marrow cells. This assay detects in vivo damage to chromosomes or mitotic apparatus by determining the presence of micronuclei in the PCE in the bone marrow of mice. Induction of micronucleus formation in this assay is indicative of either clastogenic effects or
malsegregation of chromosomes. An advantage of this assay is that it evaluates effects on somatic cells of mice that are treated in vivo and thus is relevant to prediction of potential in vivo mammalian effects (MacGregor et al., 1987). In the preliminary range-finding study (data not shown), DRM was found to be nontoxic to male or female mice at oral dose levels of up to 2000 mg/kg. Therefore, oral DRM doses of 500, 1000, and 2000 mg/kg were chosen for the micronucleus study. There were no deaths, no clinical signs of toxicity, and no significant effects on body weight observed in control, DRM, or positive
TABLE 6 Summary of Micronucleus Results in Male Cd-1 Mice Treated with DRM by Oral Gavage Mean PCE/total erythrocyte ratio Time (h)
Number
Vehicle controla
24 48
5 5
0.56 ± 0.03 0.56 ± 0.07
500 mg/kg
1000 mg/kg
2000 mg/kg
Positive controlb
0.55 ± 0.04 0.60 ± 0.06
0.58 ± 0.06 0.53 ± 0.05
0.57 ± 0.07 0.57 ± 0.05
0.55 ± 0.11
Mean micronucleated PCE/1000 PCE Time (h)
Number
Vehicle controla
500 mg/kg
1000 mg/kg
2000 mg/kg
Positive controlb
24 48
5 5
1.0 ± 0.6 0.8 ± 0.7
0.9 ± 1.0 1.5 ± 0.8
1.1 ± 1.1 0.9 ± 0.9
0.9 ± 0.5 1.3 ± 0.8
16.6 ± 4.3∗∗
a Vehicle
control, distilled water (10 ml/kg body wt). control, cyclophosphamide (40 mg/kg). P ≤ 0.01 (Dunnett’s test).
b Positive ∗∗
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control groups. There were no statistically significant decreases in mean PCE/total erythrocyte ratio and no statistically significant increases in mean micronucleated PCE frequencies compared to control in any DRM treatment group (Table 6). DISCUSSION As part of a comprehensive safety evaluation program, the genotoxicity of DRM was examined in a wide range of in vitro and in vivo assays. All in vitro assays were conducted in replicate cultures using multiple doses in the absence and presence of exogenous metabolic activation. Positive and negative controls were included to assure the validity of each assay. Overall, the data fully support the conclusion that DRM is neither mutagenic nor clastogenic. In the AS52 assay, initial mutagenicity testing was conducted using a range of S9 concentrations (0, 1, 5, and 10%) followed by a confirmatory experiment with 0 and 5% S9. The range of test concentrations, up to clearly cytotoxic levels, varied depending on the concentration of S9 utilized. Overall, greater cytotoxicity was observed with increasing concentrations of S9. In the initial study, a statistically significant increase in mean mutant frequency and a statistically significant dose response was observed with DRM only in the 1% S9 treatment group. However, the increases were very small (<1.7-fold over control values) and were not reproduced in the subsequent repeat study. In the confirmatory study, a statistically significant linear dose response was observed without S9 but none of the treatment levels were statistically increased compared to the control values. Additionally, no treatment-related trends were observed without S9 in two other experiments (the initial mutagenicity assay and subsequent repeat study). This trend in a single experiment was not judged to be DRM treatment related. The authors conclude that DRM was not mutagenic in the AS52/XPRT assay in the absence or presence of 1, 5, or 10% S9. The results of the Ames assay and the in vitro mammalian cytogenetic assay demonstrate that DRM was not mutagenic in any of the five S. typhimurium test strains used and was not clastogenic to human peripheral blood lymphocytes. Finally, in vivo DRM did not induce micronucleus formation in mice further supporting its lack of any chromosomal effects. This series of genotoxicity studies demonstrates that DRM does not have any genotoxic potential. REFERENCES Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for Detecting carcinogens and mutagens with the Salmonella mammalian-microsome test. Mutat. Res. 31, 347–364. Bajpai, P., Bajpai, P. K., and Ward, O. P. (1991a). Production of docosahexaenoic acid by Thraustochytrium aureum. Appl. Microbiol. Biotech. 35, 706–710.
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