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L->77?^ IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY'S COMPOUND T-3290CoC Final Report --- ---% November 1982 Oi.i 1 L i- f j- t. f t h <LH ^eJsrrL*ocus-->____ By: Kristien E. Mortelmans, Ph.D., Director, Microbial Genetics Department and -ydyu*^- U & ^hcuuU L s Debra E. Verbaere, Microbiologist Prepared for: 3M Corporation Medical Department General Offices, 3M Center St. Paul, MN 55144 Attention: Bill McCormick Toxicology Specialist SRI Project 3145 Approved by: Ip-*-1-- David C. L. Jones, Director Toxicology Laboratory 'h*/ic-- '-- W. A. Skinner, Vice /President Life Sciences Division 333 Ravenswood Ave. Menlo Park, CA 94025 (415)859-6200 . TWX: 910-373-2046 . Telex: 334 486 000222 SUMMARY SRI International examined 3M Company's Compound T-3290CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TA100 of bacterium Salmonella typhimurium in the standard Ames Salmonella/mlcrosome in vitro mutagenicity assay. Compound T-3290CoC was also screened for recombinogenic activity with the yeast Saccharomyces cerevlslae D3 assay. Both assays were performed in the presence and absence of a ratliver metabolic activation system. Compound T-3290CoC was found to be neither mutagenic nor recombinogenic when tested using these procedures. ii 000223 CONTENTS SUMMARY.......................................... INTRODUCTION..................................... MATERIALS ....................................... METHODS ....................................... RESULTS AND DISCUSSION........................... TABLES Table 1.................................... Table 2.................................... Table 3..................... Table 4 .................................... ii 1 2 3 9 10 11 12 13 iii 000224 INTRODUCTION SRI International examined 3M Company's Compound T-3290CoC 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 ceievisiae D3. An Aroclor 1254-stimulated, rat-liver homogenate metabolic activation system was included in the assay procedures to provide metabolic steps that the microorganisms 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 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 carcinogenic or noncarcinogenic to man. 000225 1 MATERIALS Test Compound - Name: T-3290CoC - Date Received: 8 October 1982 - Description: Yellow-amber liquid - Storage Conditions: Room temperature - Special Testing Conditions: None Indicator Organisms - Species: Salmonella typhimurium LT2 Saccharomyces cerevisiae - Strains: TA1535, TA1537, TA1538, TA98, and TA100 S. typhimurium; D3 for S. cerevisiae Metabolic Activation Aroclor 1254-induced rat-liver S-9; SRI Batch E-8 ; ~ 31 mg/ml protein. Solvent Used Sterile water 000226 METHODS Salmonella typhimurlum Strains TA1535, TA1537, TA1538, TA98, and TA100 The Salmonella typhimurlum 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 histidine independence (his'1') 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 hls+ 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 molecules, thereby increasing the mutagenic effect of these molecules. The uvrB mutation renders the bacteria unable to use the accurate excision repair mechanism to remove certain chemically or physically induced DNA lesions 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 3 000227 increase in error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasmid pKMlOl confers resistance to the 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, benzo(a)pyrene, aflatoxin B1, 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 pKMlOl, which makes it more sensitive to some mutagenic agents. All indicator strains are kept frozen in nutrient broth supplemented 9 with 10% sterile glycerol at -80C in 1-ml samples containing about 10 cells. New frozen stock cultures are made every 3 months 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, the frozen 1-ml samples are allowed to thaw at room temperature before inoculation in 50 ml of glucose minimal liquid medium supplemented with an excess of biotin and histidine. The cultures are grown at 37C, unshaken for 4 hours, then gently shaken (100 rpm) for 11 to 14 hours. All strains are genetically analyzed whenever experiments are performed. Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic chemicals (e.g., of the aromatic amine 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 Sprague-Dawley rats (200 to 250 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 4 000228 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 subsequent operations are conducted in an ice bath. The livers are washed with an equal volume of cold, sterile 0.15 M KC1 minced with sterile surgical scissors in three volumes of 0.15 M KC1, (3 ml/g of wet organ), and homogenized with a PotterElvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g_, and the supernatant, referred to as the S-9 fraction, is quickly frozen on dry ice and stored at -80C. The metabolic activation mixture for each experiment 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 phosphate buffer (0.2 M, pH 7.4) 3.35 ml of HjO. Plate Incorporation Assay Prior to testing, the test article is serially diluted from an initial stock. The dose levels are based on the results of a preliminary range finding experiment. The article is usually tested over a minimum of six dose levels, the highest nontoxic dose level being 10 mg/plate unless solubility, mutagenicity, or toxicity dictates a lower upper limit. All assays are repeated at least once on a separate day. 5 000229 The plate incorporation assay is performed in the following way. To a sterile 13 x 100 mm test tube placed in a 43C heating block we add: (1) 2.00 ml of 0.6% agar containing 0.6% NaCI, 0.05 mM biotin and 0.05 mM histidine 8 (2) 0.05 ml of indicator organisms (about 10 bacteria) (3) 0.05 ml of a solution of the test article (4) 0.50 ml of metabolic activation mixture (if appropriate). This mixture is stirred gently and then poured on plates containing about 25 ml of minimal glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37C. The number of his+ revertant colonies is counted using a BioTran II automated colony counter when possible. When accurate counts cannot be obtained (e.g., because of precipitate), the plates are counted manually using an electric probe colony counter. Concurrent sterility, negative (solvent), and positive controls are run with every experiment. Sterility controls include plating out separately steps (3) and (4). For negative controls, we use steps (1), (2), (4), and 0.05 ml of the solvent used for the test article. For positive controls, we test each bacterial culture with the following mutagens using steps (1 ), (2 ), (3), and (4): Sodium azide for the base-pair substitution mutants TA1535 and TA100. 9-Aminoacridine for the frameshift mutant TA1537. 2-Nitrofluorene for the frameshift mutants TA1538 and TA98. 2-Anthramine for all tester strains, in the presence of metabolic activation. 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 6 000230 or recotnbinogenic 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 _S_. cerevisiae is stored at 4C. For each experiment, broth containing 0.05% MgSO^, 0.15% KH2PO4 , 0.45% (NH^^SO^, 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 O resuspended at a concentration of 10 cells/ml in 67 mM phosphate buffer (pH 7.4). To a sterile test tube are added: 1 .0 0 ml of the resuspended culture 0.50 ml of either the metabolic activation mixture or buffer 0 .2 0 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 saline, and 0 .2 ml of the 10 and 10 dilutions is spread on plates containing the same ingredients as the broth plus 2 .0% agar; five plates are spread with the 10 dilution and three plates are spread with the 10"^ dilution. The plates are incubated for 3 days at 30C, followed by 1 day at 4C to enhance the development of the red pigment indicative of adenine-deficient homozygosity. _o Plates containing the 10 J dilution are scanned with 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"-* dilution. 7 000231 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 recombinants per milliliter as well as in the relative number of mitotic recombinants per 10 ^ survivors. 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 69, 3128-3132 (1972). Ames, B. N., W. E. Durston, E. Yamasaki, and F. D. Lee. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. USA 70, 2281-2285 (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. McCann, 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. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives j>_, 83-86 (1973). 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 carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA _72_> 979-983 (1975). McCann, J., B. and N. Ames. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion. Proc. Nat. Acad. Sci. USA 73, 950-954 (1976). 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). Zimmerman, F. K., and R. Schwaier. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alkylating agents in Saccharomyces cerevislae. Mol. Gen. Genet. 100, 63-69 (1976). 8 000232 RESULTS AND DISCUSSION 3M Company's Compound T-3290CoC was screened for mutagenic activity with the standard Ames Salmonella/microsome in vitro mutagenicity assay using the five standard Ames strains of Salmonella typhlmurium: TA1535, TA1537, TA1538, TA98, and TA100. This compound was assayed on two separate days,11 October and 18 October 1982, each time over a dose range of 10 to 5,000 ug/plate, with two plates per dose level, using sterile water as the solvent. All assays were performed both in the presence and in the absence of a rat-liver metabolic activation system. This compound foamed when vortexed; however, this did not appear to interfere with the testing. No dose-related increase in the number of histidine-independent revertants was observed in either of the two assays. Therefore, we conclude that Compound T-3290CoC is nonmutagenic when tested by these procedures. Data from these assays are presented in Tables 1 and 2. Compound T-3290CoC was also assayed for recombinogenic activity using the yeast Saccharomyces cerevisiae D3 assay for mitotic recombination. This assay was performed on two separate days, 11 October and 18 October 1982. This compound was tested twice over the dose range of 0.05% to 5.0%, both with and without a rat-liver metabolic activation system. Compound T-3290CoC was found to be reproducibly nonrecombinogenic when tested by these procedures. No dose-related increase in the number of mitotic recombinants per 10^ survivors was observed. Data from these assays are presented in Tables 3 and 4. 9 000233 Table 1 IN VITRO ASSAYS WITH SALMONELLA TYP11IMURIUM Compound T-3290CoC Experiment Date: 11 October 1982 Compound Metabolic Activation Compound Added per plate Negative Controls Sterile water -- + Positive Controls Sodium Azide - 1 yg 9-Am ino ac rid ine - 50 2 -Nitrofluorene - 5 2-Anthramine O o foO Compound T-3290CoC Co H + _ + -- _ , - 1 1 2.5 2.5 10 50 100 500 1000 5000 + 10 + 50 + 100 + 500 + 1000 + 5000 TA1535 21 20 7 13 528 509 21 18 105 115 25 24 17 11 24 15 25 14 17 21 18 20 15 9 89 87 79 79 76 Histidine Revertants per Plate TA1537 TA1538 TA98 4 11 79 10 12 16 17 19 24 25 33 173 166 89 69 65 7 13 96 66 78 85 65 86 96 97 57 53 2 10 605 595 17 20 111 116 285 283 19 19 129 101 16 14 17 10 18 20 11 13 10 11 11 12 18 29 19 26 18 19 17 14 18 21 16 18 18 19 18 19 21 24 17 23 18 18 14 21 28 27 31 28 36 21 39 39 33 26 24 21 TA100 176 154 179 139 507 464 173 184 392 441 164 155 170 165 164 161 159 177 171 164 153 139 162 160 144 171 177 167 131 170 176 164 166 151 Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM Compound T-3290CoC Experiment Date: 18 October 1982 ____ Compound____ Negative Controls Sterile water Positive Controls Sodium Azide 9-Arainoacridine 2 -Nitrofluorene 2-Anthramine Metabolic Activation _ + - + _ + Compound Added _per plate 1 ug 50 5 1 1 2.5 2.5 Compound T-3290CoC _ _ _ _ - + + + + + + 10 50 100 500 1000 5000 10 50 100 500 1000 5000 ________________Histidine Revertants per Plate TA1535 TA1537 TA1538 TA98 21 16 65 46 56 12 6 13 18 17 13 24 20 377 445 94 103 16 15 100 127 20 19 29 18 18 16 11 17 16 24 15 15 10 13 79 87 86 46 5 14 46 61 47 64 53 48 86 65 12 7 11 9 66 68 74 75 12 7 450 462 10 12 158 171 292 277 22 14 187 170 13 16 98 8 17 9 12 10 10 11 12 24 20 20 26 20 16 27 29 18 15 20 14 24 13 19 19 14 18 11 19 20 17 21 19 31 21 20 19 29 30 25 28 31 20 14 19 122 137 411 116 445 125 101 137 125 160 126 124 145 134 116 117 131 o o o w CO H <75 M Compound Negative Controls Sterile water Positive Controls 1,2,3,4-Diepoxybutane Sterigmatocyst in Compound T-3290CoC Table 3 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 Compound T-3290CoC Experiment Date: 11 October 1982 Metabolic Activation Percent Survivors Concentration Cells per ml weight/volume (x 10"7) Percent 4.8 100 + 5.3 100 - 0.025 5.2 100 - 0.0005 4.9 100 + 0.0005 5.7 100 0.05 4.6 96 - 0.1 4.4 92 - 0.5 5.1 100 -1 4.9 100 .- 5 4.5 94 + 0.05 4.8 91 + 0 .1 4.7 89 + 0.5 4.7 89 +1 5.0 94 +5 4.0 75 Mitotic Recombinants Per ml Per 105 (x 10"3)1 Survivors 4 8.3 6 11.3 877 1686 5 10.2 399 700 2 4.3 2 4.5 4 7.8 4 8.2 1 2.2 3 6.3 4 8.5 2 4.3 36 6 15 000237 Compound Negative Controls Sterile water Positive Controls 1,2,3,4-Diepoxybutane Sterigmatocystin Compound T-3290CoC Table 4 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 Compound T-3290CoC Experiment Date: 18 October 1982 Metabolic Activation Percent Survivors Concentration Cells per ml weight/volume (x 10 7) Percent 4.0 100 4- 4.1 100 - 0.025 4.6 100 - 0.0005 4.3 100 + 0.0005 4.3 100 __ 0.05 3.8 95 -- 0 .1 3.6 90 -- 0.5 3.6 90 --1 3.5 88 -5 3.8 95 + 0.05 4.4 100 + 0.1 4.3 100 + 0.5 4.2 100 +1 4.7 100 +5 3.6 88 Mitotic Recombinants Per ml Per 105 Cx 10'3]1 Survivors 3 7.5 6 14.6 946 2057 3 7.0 414 963 4 10.5 6 16.7 5 13.9 5 14.3 2 5.3 4 9.1 4 9.3 2 4.8 4 8.5 3 8.3