Document JQKaLNjOymRqjLZOoL3y16yO

fifi. & & -- 06 If PFOS Genotoxicity - o O U00048 IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY'S COMPOUND T-2997CoC Final Report January 1981 Prepared for: 3M COMPANY Medical Department General Offices, 3M Center St. Paul, Minnesota 55101 Attention: Mr. William C. McCormick Toxicology Services, 220-2E-02 SRI Project LSC-8958 Approved by: David C. L. Jonee, Director Toxicology Laboratory Iaj Ql. X ^ W. A. Skinner, Executive Director Life Sciences Division 333 Ravenswood Ave. Menlo Park, California 94025 (415) 326-6200 Cable: SRI INTL MPK TWX: 910-373-1246 000049 SUMMARY SRI International examined 3M Company's Compound T-2997CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TA100 of Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3 using FDA-approved GLP procedures. Each assay was performed in the presence and in the absence of a rat liver metabolic activation system. Compound T-2997CoC was not mutagenic ot recombinogenic in any assay performed. ii 000050 CONTENTS S U M M A R Y ............................................. QUALITY ASSURANCE STATEMENT ........................ INTRODUCTION ......................................... MATERIALS ........................................... METHODS ............................................. RESULTS AND DISCUSSION ............................... TABLES Table 1 ......................................... Table 2 ......................................... Table 3 ......................................... Table 4 ......................................... ii iv 1 2 3 9 10 11 12 13 iii 000051 QUALITY ASSURANCE UNIT Final Report Statement SRI International assures the quality and integrity of this study, the Ames Salmonella Microsome assay and the Saccharomyces cerevisiae D3 recombinogenic assay of the 3M Company's compounds T-2997 CoC. The study was inspected on December 12 and 22, 1980 during the dilution, plating, and plate counting of the two assays. The findings of the Quality Assurance Unit Inspector were reported at the time of each inspection to the Study Director. SRI management was also informed of the inspection results on December 12 and 22, 1980, respectively. This final report accurately describes the methods and standard operating procedures and reflects the raw data of the study. Any deviations from the approved protocol and standard operating procedures were made with proper authorization and documentation. / iv m m sz INTRODUCTION SRI International examined 3M Company's Compound T-2997CoC for mutagenicity by in vitro microbiological assays with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3. An Aroclor 1254-stimulated, rat liver homogenate metabolic activation system was included in the assay procedures to provide metabolic steps that the bacteria either are incapable of conducting or do not carry out under the assay conditions. The assay procedure with JS. typhimurium has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has about the same reliability in identifying chemicals that are not carcinogenic. The assay procedure with Si. cerevisiae is about 60% reliable in detect ing carcinogens as agents that increase mitotic recombination. However, because the assay systems do not always provide 100% correlation with carcinogenicity investigations in animals, neither a positive nor a negative response conclusively proves that a chemical is carcinogenic or noncarcinogenic to man. The assays with Compound T-2997CoC were begun on 12 December 1980 and completed on 7 January 1981. Copies of the final report will be kept in our files (Building 28, Room 213) and in SRI's Records Center. The laboratory notebook will be retained in Building 28A, Room 10 for one year after it is filled, and then will be stored in SRI's Record Center. All that is left of Compound T-2997CoC will be kept for six months in our chemical storage room (Building 28, Room 217), then returned to the 3M Company. 1 000053 MATERIALS Test Compounds - Name: T-2997CoC. - Date Received: 8 December 1980. - Description: Waxy amber solid. - Stability: Stability assured by client. - Storage Condition: Stored at room temperature. - Special Testing Conditions: None. Indicator Organisms - Species: Salmonella typhimurium LT2 and Saccharomyces cerevisiae. - Strains: TA1535, TA1537, TA1538, TA98, and TA100 of S. typhimurium; D3 of j^. cerevisiae. Metabolic Activation Aroclor 1254-induced rat liver S-9; SRI Batch D 7 , D8, and NIEHS RLI003, ^ 30 mg/ml protein. Solvent Used Dimethylsulfoxide (DMSO). 000054 METHODS Salmonella typhimurium Strains TA1535, TA1537, TA1538, TA98, and TAlOO The Salmonella typhimurium strains used at SRI are all histidine auxotrophs by virtue of mutations in the histidine operon. When these histidine-dependent cells are grown on minimal medium agar plates con taining a trace of histidine, only those cells that revert to histidine independence (his'*') are able to form colonies. The small amount of histidine allows all the plated bacteria to undergo a few divisions; in many cases, this growth is essential for mutagenesis to occur. The his+ revertants are easily visible as colonies against the slight background growth. The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar, the mutation frequency is increased, usually in a dose-related manner. We obtained our jS. typhimurium strains from Dr. Bruce Ames of the University of California at Berkeley. In addition to having mutations in the histidine operon, all the indicator strains have a mutation (rfa) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved in the synthesis of the vitamin biotin (bio) and in the repair of ultraviolet (uv)-induced DNA damage (uvrB) . The rfa mutation makes the strains more permeable to many large molecules, thereby increasing the mutagenic effect of these molecules. The uvrB mutation causes decreased repair of some types of chemically or physically damaged DNA and thereby enhances the strains' sensitivity to some mutagenic agents. Strain TA1535 is reverted to his~*~ by many mutagens that cause base-pair substitutions. TAlOO is derived from TA1535 by Uie introduction of the resistance transfer factor, plasmid pKMIOl. This plasmid is believed to cause an increase in error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasmid pKMIOl confers resistance to the anti biotic ampicillin, which is a convenient marker to detect the presence 3 000055 of the plasmid in the cell. The presence of this plasmid also makes strain TA100 sensitive to some frameshift mutagens [e.g., ICR-191, benzo(a)pyrene, aflatoxin B j , and 7 ,12-dimethylbenz(a)anthracene]. Strains TA1537 and TA1538 are reverted by many frameshift mutagens. Strain TA98 is derived from TA1538 by the addition of the plasmid pKMIOl, which makes it more sensitive to some mutagenic agents. All indicator strains are kept at 4C on minimal agar plates supple mented with an excess of biotin and histidine. The plates with the plasmid-carrying strains also contain ampicillin (25 pg/ml) to ensure stable maintenance of the plasmid pKMIOl. New stock culture plates are made every i to 6 week* from sing1 colony isolates that have beer checked for their genotypic characteristics (his, rfa, uvrB, bio) and for the presence of the plasmid. For each experiment, an inoculum from the stock culture plates is grown overnight at 37C in nutrient broth (0X0ID, CM67). Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic chemicals (e.g., of the aromatic amino type or the polycyclic hydrocarbon type) are inactive unless they are metabolized to active forms. In animals and man, an enzyme system in the liver or other organs (e.g., lung or kidney) is capable of metabolizing a large number of these chemicals to carcinogens. Some of these intermediate metabolites are very potent mutagens in the JS. typhlmurium test. Ames has described the liver metabolic activation system that we use. In brief, adult male rats (250 to 300 g) are given a single 500 mg/kg intraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated biphenyls). This treatment enhances the synthesis of enzymes involved in the metabolic conversion of chemicals. Four days after the injection, the animals' food is removed but drinking water is provided ad libitum. On the fifth day, the rats are killed and the liver homogenate is prepared as follows. The livers are removed aseptically and placed in a preweighed sterile glass beaker. The organ weight is determined, and all sub sequent operations are conducted in an ice bath. The livers are washed 4 000056 with an equal volume of cold, sterile 0.15 M KC1 (1 ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 M KC1, and homogenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x , and the supernatant, referred to as the S-9 fraction, is quickly frozen in dry ice and stored at -80C. The metabolic activation mixture for each experiments consists o f , for 10 ml: 1.00 ml of S-9 fraction 0.20 ml of MgCla (0.4 M) and KC1 (1.65 M) 0.05 ml of glucose-6-phosohate (1 M) 0.40 ml of NADP (0.1 M) 5.00 ml of sodium phosphate buffer (0.2 M, pH 7.4) 3.35 ml of HaO. Assays in Agar To a sterile 13 x 100 mm test tube placed in a 43C heating block, we add in the following order: (1) 2.00 ml of 0.6% agar (2) 0.05 ml of indicator organisms (3) 0.50 ml of metabolic activation mixture (if appropriate) (4) 0.05 ml of a solution of the test chemical. This mixture is stirred gently and then poured onto minimal agar plates.^ After the top agar has set, the plates are incubated at 37C for 2 days. The number of his'1' revertant colonies is counted and recorded. For negative controls, we use steps (1), (2), and (3) and 0.05 ml of the solvent used for the test chemical. Dimethylsulfoxide (DMSO) was used as the solvent for T-2476ChR. For positive controls, we test each * The 0.6% agar contains 0.05 mM histidine, 0.05 mM biotin, and 0.6% NaCl. j* Minimal agar plates consist of, per liter, 15 g of agar, 10 g of glucose, 0.2 g of MgS0fc7Ha0, 2 g of citric acid monohydrate, 10 g of KaHPO*, and 3.5 g of NaNHj.HPO**4H30. 5 000057 culture by specific mutagens known to revert each strain, using steps (1), (2), (3), and (4). Saccharomvces cerevisiae D3 The yeast S_. cerevisiae D3 is a diploid microorganism heterozygous for a mutation leading to a defective enzyme in the adenine-metabolizing pathway. When grown on medium containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated from the heterozygotes by mitotic recombination. The frequency of this recombinational event may be increased by incubating the organisms with various carcinogenic or recombinogenic agents. The recuuiblnogeuic activity of a compound or its metabolite is determined from the number of red-pigmented colonies appearing on test plates. A stock culture of j>. cerevisiae is stored at 4C. For each experiment, broth containing 0.05% MgSO*, 0.15% KHaPO*, 0.45% (NHOaSO*, 0.35% peptone, 0.5% yeast extract, and 2% dextrose is inoculated with a loopful of the stock culture and incubated overnight at 30C with shaking. The iri vitro yeast mitotic recombination assay in suspension is conducted as follows. The overnight culture is centrifuged and the cells are resuspended at a concentration of 10* cells/ml in 67 mM phosphate buffer (pH 7.4). To a sterile test tube are added: 1.00 ml of the resuspended culture 0.50 ml of either the metabolic activation mixture or buffer 0.20 ml of the test chemical 0.30 ml of buffer. Several doses of the test chemical are tested in each experiment, and appropriate controls are included. The suspension mixture is incubated at 30C for A hours on a roller drum. The sample is then diluted serially in sterile physiologic saline, and 0.2 ml of the 10~s and 10" s dilutions is spread on plates containing the same ingredients as the broth plus 2.0% agar; five plates 6 000058 are spread with the 10"* dilution and three plates are spread with the 10"* dilution. The plates are incubated for 2 days at 30 C, followed by 2 days at 4C to enhance the development of the red pigment indicative of adenine-deficient homozygosity. Plates containing the 10" * dilution are scanned with a dissecting microscope at 10* magnification, and the number of mitotic recombinants (red colonies or red sectors) is recorded. The surviving fraction of organisms is determined from the total number of colonies appearing on the plates of the 10" * dilution. The number of mitotic recombinants is calculated per 10* survivors. A positive response in this assay is indicated by a dose-related increase of more than 3-fold in the absolute number of mitotic recombJ^ant **? milliliter as well as in the relative number of mitotic recombinants per 10* survivors. Statistical Method No statistical method was needed. Results are a tabulation of the number of colonies appearing on the plates after incubation. References Ames, B. N., E. G. Gurney, J. A. Miller, and H. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc. Nat. Acad. Sci. USA 6 9 , 3128-3132 (1972). Ames, B. N., U. E. Durston, E. Yamasaki, and F. D. Lee. Carcinogens are mutagens: A simple test system combining liver homogenates for activa tion and bacteria for detection. Proc. Nat. Acad. Sci. USA 70, 22812285 (1973). Ames, B. N., F. D. Lee, and W. E. Durston. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat Acad. Sci. USA 70, 782-786 (1973). Ames, B. N ) *J MeCaitn, and E. Yamasaki. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364 (1975). Brusick, D. J., and V. W. Mayer. screening techniques with yeast. (1973). New developments in mutagenicity Environ. Health Perspectives 83-86 7 000059 Kier, L. D . , E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat. Acad. Sci. USA 71, 4159-4163 (1974). McCann, J . , E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcino gens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA 72, 5135-5139 (1975). McCann, J., N. E. Spingarn, J. Kobori, and B. N. Ames. Detection of carcinogens as mutagens: Bacterial tester strains with R Factor plasmids. Proc. Nat. Acad. Sci. USA 72, 979-983 (1975). Mortelmans, K. E . , and B.A.D. Stocker. Segregation of the mutator property of plasmid R46 from its ultraviolet-protecting property. Mol. Gen. Genet. 167, 317-327 (1979). Ziiumermann, T. K. , and R. Schwaier. lodu^tiun of mitotic gene conversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alky lating agents in Saccharomyces cerevisiae. Mol. Gen. Genet. 100, 63-69 (1967). 8 OOQOQ RESULTS AND DISCUSSION Compound T-2997CoC was tested for mutagenicity with the Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3 assay in the presence and in the absence of a metabolic activation system. The compound was tested twice on separate days in both assays. Dimethylsulfoxide (DMSO) was used as the solvent in all assays. In the Ames Salmonella/microsome assay, Compound T-2997CoC was tested over a wide range of concentrations, from 10 to 5,000 yg/plate (Table 1). Because no toxicity occurred at 5,000 pg/plate, the second test was run using a higher dose range, from 50 to 10,000 pg/plate (Table 2). Still no toxicity or dose-related increase in the number of revertants per plate was observed in either assay. In the microbiological assays with S_. cerevisiae D 3 , T-2997CoC was tested over a wide range of concentrations, from 0.05 to 5.0% w/v (Tables 3 and 4). No toxicity or dose-related increase in the number of mitotic recombinants was observed in either assay. We conclude that Compound T-2997CoC is not mutagenic with murium or recombinogenic in S_. cerevisiae. typhi- 9 000061 Table 1 IN VITRO ASSAYS WITH SALMONELLA TYPH1MUR1UM COMPOUND T-2997CoC Experiment Date: 12 December 1980 Compound Metabolic Activation Compound Added (h r /plate) Negative Control DMSO Positive Controls Sodium Azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine Compound T-2997CoC + - - + + -- - + + + + + + 1.0 50.0 5.0 1.0 1.0 2.5 2.5 10.0 50.0 100.0 500.0 1,000.0 5,000.0 10.0 50.0 100.0 500.0 1,000.0 5,000.0 ____________ Histidine Revertants per Plate___________ TA1535 TA1537 TAJ 538 TA98 TA100 15 16 16 13 57 73 12 7 18 9 20 16 38 31 115 130 104 110 336 324 9 20 252 275 24 27 19 17 19 16 28 15 30 15 20 19 76 15 9 3 14 9 13 16 8 9 16 508 527 77 77 101 35 34 56 55 68 73 12 12 85 77 59 9 16 13 8 726 738 17 7 271 294 57 2 13 12 8 59 58 87 13 12 18 14 12 7 16 19 21 26 7 16 467 442 372 381 38 37 158 147 126 140 519 518 26 29 24 31 21 32 18 28 25 24 18 30 36 43 38 28 26 49 37 40 34 33 31 24 99 149 124 116 115 112 99 129 111 113 101 97 122 135 125 125 108 112 126 134 129 127 105 92 290000 Table 2 VITRO ASSAYS WITH SALMONELLA TYPHIMUR1UM COMPOUND T-2997CoC Experiment Date: 15 December 1980 Compound Metabolic Activation Compound Added (pg/plate) Negative Control DMS0 Positive Controls Sodium Azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine - + - - - - + + ] .0 50.0 5.0 1.0 1.0 2.5 2.5 Compound T-2997CoC - - - + + + + + + 50.0 100.0 500.0 1,000.0 5,000.0 10,000.0 50.0 100.0 500.0 1,000.0 5,000.0 10,000.0 TA1535 24 15 68 224 276 13 14 257 209 16 9 17 8 15 9 16 17 13 12 9 15 69 77 6 12 5 15 45 67 Histidine Revertants per Plate TAL537 TA1538 TA98 14 8 78 8 13 25 8 25 24 18 27 721 604 16 8 64 51 5 13 8 15 73 22 68 16 59 88 76 65 44 56 691 863 9 15 154 149 13 12 9 12 94 88 46 47 5 19 14 14 17 9 15 13 15 9 12 14 337 420 15 18 153 161 12 25 17 19 20 16 17 16 7 12 14 20 24 37 18 32 19 19 16 22 20 21 17 16 110 108 347 72 357 125 108 112 113 97 57 114 123 103 105 104 97 000064 Compound Negative Control DMSO Positive Control 1,2,3,4-Diepoxybutane Compound T-2997CoC Table 3 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-2997CoC Experiment Date: 17 December 1980 Metabolic Activation Percent Survivors Concentration Cells per ml (w/v or v/v) (x 10"7) Percent Mitotic Recombinants Per m l Per 10s (x 10 3) Survivors - 7.4 100 2.0 2.8 + 6.5 100 2.0 3.0 - 0.025 + 0.025 - .05 - .1 - .5 - 1.0 5.0 + .05 + .1 + .5 + 1.0 + 5.0 6.6 89 1100.0 5.2 80 530.0 6.5 88 7.0 95 6.2 84 6.4 86 6.5 88 7.0 108 5.9 91 6.4 98 6.2 95 6.5 100 1.0 1.0 1.0 1.0 3.0 1.0 1.0 1.0 1.0 2.0 1700.0 1000.0 1.5 1.4 1.6 1.6 4.6 1.4 1.7 1.6 1.6 3.1 00006S Compound Negative Control DMSO Positive Control 1,2,3,4-Diepoxybutane Compound T-2997CoC Table 4 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-2997CoC Experiment Date: 7 January 1981 Metabolic Activation Percent Survivors Concentration Cells per ml (w/v or v/v) (x 10 7) Percent Mitotic Recombinants Per ml Per 105 (x 10~3)1 Survivors 6.8 100 5.0 7.5 + 6.5 100 3.5 5.4 - 0.025 5.1 75 900.0 1800.0 + 0.025 5.6 86 1000.0 1800.0 - .05 - .1 - .5 - 1.0 - 5.0 + .05 + .1 + .5 + 1.0 + 5.0 6.4 9a 2.0 3.1 6.4 9-* 4.0 6.3 6.9 101 3.0 4.3 6.7 99 1.0 1.5 5.0 74 6.0 12.0 6.1 94 2.0 3.3 6.7 103 8.0 12.0 6.5 100 5.0 7.7 5.4 8 3 3.0 5.6 6.3 97 7.0 11.0