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I 'I IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY COMPOUND T-2816CoC Final Report, Revised March 1980 (ooi ] By: Kristien E. Mortelmans, Ph.D. Director, Microbial Genetics Department and Anne Pomeroy, Microbiological Technician ] Prepared for: ] 3M COMPANY Medical Department General Offices, 3M Center St. Paul, Minnesota 55101 Attention: W. C. McCormick Toxicology Services SRI Project LSC-8958 [ Approved: David C. L. Jones, Director Toxicology Laboratory W. A. Skinner, Executive Director Life Sciences Division 333 Ravenswood Ave. Menlo Park, California 94025 1415) 326-6200 Cable SRI INTL MPK TWX: 910-373-1246 SUMMARY SRI International examined 3M Company's Compound T-2816CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TAJLOO of Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3. Each assay was performed in the presence and in the absence of a rat liver metabolic activation system. Compound T-2816CoC was not mutagenic or recombinogenic in any assay performed. 1 INTRODUCTION SRI International examined 3M Company's Compound T-2816CoC 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 proce dures 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 jj. 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 s. cerevisiae is about 60% reliable in detecting 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 hazardous or nonhazardous to man. 2 METHODS Salmonella typhimurium Strains TA1535, TA1537, TA1538, TA98, and TA100 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 containing a trace of histidine, only those cells that revert to histi dine 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 S_. 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 aromatic 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. TA100 is derived from TA1535 by the introduction of the resistance transfer factor, plasmid pKMIOl. This plasmid is believed to cause an increase in errorprone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasmid pKMIOl confers resistance to the 3 antibiotic ampicillin, which is a convenient marker to detect the presence of the plasmid in the cell. The presence of this plasmid also makes strain TA100 sensitive to some frameshift mutagens [e.g., ICR-191, bento(a)pyrene, aflatoxin B t, 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 supplemented with an excess of biotin and histidine. The plates with the plasmid-carrying strains also contain ampicillin (25 yg/ml) to ensure stable maintenance of the plasmid pKMIOl. New stock culture plates are made every 4 to 6 weeks from single colony isolates that have been 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 37#C in nutrient broth (Oxoid, 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 S_. typhimurium 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. 4 The livers are removed aseptically and placed in a preveighed sterile glass beaker. The organ weight is determined, and all sub sequent operations are conducted in an ice bath. The livers are washed 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 asthe S-9 fraction, is quickly frozen in dry ice and stored at -80C. The metabolic activation mixture for each experiments consists of, for 10 ml: 1.00 ml of S-9 fraction 0.20 ml of MgCl2 (0.4 M) and KC1(1.65 M) 0.05 ml of glucose-6-phosphate (1 M) 0.40 ml of NADP (0.1 M) 5.00 ml of sodium phosphatebuffer (0.2 M, pH 7.4) 3.35 ml of H 20. 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 3 days. The number of his+ revertant colonies is counted and recorded. * The 0.6% agar contains 0.05 mM histidine, 0.05 mM biotin, and 0.6% NaCl. tMinimal agar plates consist of, per liter, 15 g of agar, 10 g of glucose, 0.2 g of MgS0fc*7H20, 2 g of citric acid monohydrate, 10 g of K 2P0fa, and 3.5 g of NaHNH^PO^^HjO. 5 For negative controls, we use steps (1), (2), and (3) and 0.05 ml of the solvent used for the test chemical. For positive controls, we test each culture by specific mutagens known to revert each strain, using steps (1), (2), (3) and (4). Saccharomyces 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 recombinogenic activity of a compound or its metabolite is determined from the number of red-pigmented colonies appearing on test plates. A stock culture of J3. cerevisiae is stored at 4C. For each experiment, broth containing 0.052 MgSO*, 0.15% KHaP0,., 0.45% (NH^aSO*,, 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 in 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 4 hours on a roller drum. The sample is then diluted serially in sterile physiologic 6 saline, and 0.2 ml of che 10~9 and 10" 3 dlluclons Is spread on places containing che same Ingredients as che broth plus 2.0Z agar; five places are spread wlch che 10" 3 dilution and three places are spread vlch che 10" 3 dilution. The places are Incubated for 2 days ac 30C, followed by 2 days at 4C to enhance che development of che red pigment Indicative of adenine-deficient homozygosity. Plates containing the 10"* dilution are scanned wlch a dissecting microscope at 10 X 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" 3 dilution. The number of mitotic recombinants is calculated per 10s 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 recombinants per milliliter as well as in the relative number of mitotic recombinants per 103 survivors. 7 RESULTS AND DISCUSSION Compound T-2816CoC was tested for mutagenicity in the Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3 in the presence and in the absence of a metabolic activation system. The compound was tested at least twice on separate days in both assays. The results are presented in Tables 1 through 5. In the Ames Salmonella/microsome assay, T-2816CoC was initially tested in a preliminary assay with strain TA100 over a wide range of concentrations, from 10 to 5,000 pg/plate. Toxicity was observed at a dose of 5,000 pg/plate (Table lj. Ethanol was used as the solvent in all assays. The results of our tests of T-2816CoC with five strains of _S. typhimurium in the Ames Salmonella/microsome assay are presented in Tables . 2 and 3. No toxicity or dose-related increase in the number of revertants was observed in these assays. The results of the microbiological assays with S_. cerevisiae D3 on T-2816CoC are presented in Tables A and 5. The compound was tested at concentrations from 0.05 to 5.02. No toxicity or significant doserelated increase in the number of mitotic recombinants above background was observed. We therefore conclude that Compound T-2816CoC was not mutagenic with S_. typhimurium or recombinogenic with _S. cerevisiae D3. 8 Table 1 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUH COMPOUND T-2816Coc Experiment Date: 13 February 1980 Compound Negative Control Ethano1 Pos itive Controls 2-Anthrnmlne Sodium Azide Compound T-2816Coe Me tabo1ic Amount of Compound Art iva tion Added per Plate Histidine Revertants per Plate TA100 - 50 pi 114 + 50 111 - 1.0 pg 102 + 1.0 427 - 0.5 pg 367 - 10.0 pg 127 - 50.0 123 - 100.0 93 - 500.0 102 - 1,000.0 97 - 5,000.0 toxic -* 10.0 pg 146 + 50 .0 116 + 100 .0 110 t 500 .0 108 + 1,000.0 108 + 5,000 .0 toxic Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUH COMPOUND T-2816CoC Experiment Date: 20 February 1980 Compound Negative Control Ethanol Positive Controls 2-Anthramlne Sodium Azide 9-Aminoacrldine 2-Nitrofluorene Compound T-2816oC Metabolic Amount of Compound ____________ Histidine Revertants per Plate Activation Added per Plate TA1535 TA1537 1^1538 TA98 TAIo O - 50.0 pi 19 14 15 7 18 18 30 29 100 117 - 50.0 26 21 5 14 98 31 27 120 91 + 50.0 31 27 20 13 24 24 44 55 92 108 + 50.0 25 32 21 20 55 26 67 56 140 114 - 1.0 pg 12 13 38 31 125 115 + 1.0 320 330 299 319 542 550 - 2.5 21 26 8 15 + 2.5 201 197 110 109 - 0.5 pg - 1.0 293 370 260 245 - 50.0 pg 180 193 - 5.0 pg 530 490 402 399 - 5.0 pg - 10.0 - 50.0 - 100.0 - 500.0 - 1,000.0 + 5.0 + 10.0 + 50.0 + 100.0 + 500.0 + 1,000.0 17 18 32 20 28 31 28 18 26 19 29 28 18 30 26 24 19 30 26 38 39 31 36 30 9 11 79 15 16 16 9 69 68 17 22 12 13 8 12 16 12 18 23 69 8 18 8 15 17 15 14 15 15 16 18 12 27 37 36 27 30 27 19 28 35 37 19 42 39 37 38 28 36 38 33 34 27 40 37 32 42 99 61 55 39 44 49 53 42 61 51 63 111 93 105 110 104 97 98 122 140 126 113 113 114 89 111 99 108 103 114 120 99 101 117 115 Table 3 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-2816CoC Experiment Date: 25 February 1980 Metabolic Amount oT Compound _________ Compound______ Activation Added per Plate TA1535 Negative Control Ethanol + 50.0 pi 50.0 20 16 36 31 Positive Control 2-Anthramlne Sodium Azide 9-Amlnoacridine 2-N1trofluorene + + 1.0 Pg 2.5 1.0 2.5 0.5 p g 1.0 50.0 pg 5.0 pg 21 26 219 176 304 331 Compound T-2816Coc + + + + + + 5 .0 p g 10 .0 50 .0 100 .0 500 .0 1,000 .0 5 .Opg 10 .0 50 .0 1 0 0 .0 500 .0 1,000 .0 25 24 19 26 38 34 32 35 33 33 22 26 38 37 23 35 30 39 27 34 34 28 25 33 Histidine Revertants per Plate TA1537 * TA1538 T98 79 19 3 16 13 24 19 45 48 77 69 99 137 138 13 19 163 134 43 40 250 230 357 506 87 78 43 69 12 6 67 17 9 8 20 9 15 21 13 16 14 9 13 934 813 17 15 97 21 13 10 11 7 10 14 18 36 33 21 31 20 21 27 34 28 23 22 28 517 461 33 44 35 54 54 53 56 41 46 51 41 48 70 62 46 68 55 53 47 53 50 57 59 52 k Retested on 6 March; control values of 25 February were invalid. XAIOO* 90 96 137 139 126 96 662 706 403 369 111 87 137 114 111 97 122 87 98 104 97 123 126 116 124 117 140 124 93 89 110 97 116 110 Table A I VITR0 ASSAYS WITH SACCHAROMYCES CEREVISIAE COMPOUND T-2816Coc Experiment Date: 13 February 1980 Compound Negative Control Ethanol Positive Control 1,2,3,A-Dlepoxybutane Compound T-2816Coc Metabollc Activation Percent Concentration (w/v or v/v) Survivo rs Cells per ml (x 10-7) Percent Mitotic Recombinants Per ml Per 10s (x 10_s) Survivors - 6.3 100 6.0 9.0 + 5.6 100 7.0 12 - 0.025 6.A 102 980 1,500 + 0.025 7.3 116 1,000 1,A00 - 0.05 3.3 50 7.0 21 " 0.1 6.6 105 5.0 7.6 0.5 5.7 90 15 26 -- 1.0 5.0 79 7.0 1A 5.0 2.9 A6 5.0 17 + 0.05 3.A 61 3.0 8.8 + 0.1 6.A 11A 7.0 11 + 0.5 5.3 95 11 21 + 1.0 5.7 102 A .0 7.0 + 5.0 5.2 93 11 21 Compound Negative Control Ethanol Positive Control 1,2,3,4-Diepoxybutane Compound T-2816Coc Table 5 I VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE COMPOUND T-2816Coc Experiment Date: 21 February 1980 Metabol lc Activation Percent Survivor s Concentrt Ion Cells per ml (w/v or v/v) (x 10"7) Percent Mitotic Recombinants Per ml Per 105 + 5.4 100 5.5 100 4.5 8.5 6.0 11 + 0.025 0.025 5.8 107 1,100 5.7 104 1,100 1,900 1,900 - 0.05 9.4 174 10 11 " "* 0.1 9.4 174 16 17 0.5 8.0 148 12 15 1.0 8.1 150 9.0 11 5.0 9.7 180 8.0 8.2 + 0.05 8.0 160 9.0 11 + 0.1 8.1 147 9.0 11 + + + 0.5 8.6 156 5.0 5.8 1.0 8.4 153 10 12 5.0 8.4 153 4 .0 4.8