Document NNkQ6QE10xQg36zdE1v6rO3z8

r] (ss) 2=3 R236-03/0 IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY'S COMPOUND T-3609 Final Report September 1984 By: C U t ) Debra E. Verbaere, Microbiologist Microbial Genetics Department and $ra p <:>. 'V1 < P Edward S. Riccio, Assistant Director Microbial Genetics Department Prepared for: 3M Company Medical Department General Offices, 3M Center St. Paul, MN 55144 Attention: Bill McCormick Toxicology Specialist SRI Project LSC-3145 Approved by: Kristien E. Mortelmans, Director Microbial Genetics Department /.f-rfacefo (f* Jon B. Reid, Director Toxicology Laboratory W. A. Skinner, Vice President Life Sciences Division 333 Ravenswood Ave. Menlo Park, CA 94025 (4151326-6200' TWX: 910-373-2046 Telex: 334-486 005275 SUMMARY SRI International examined 3M Company's Compound T-3609 for mutagenic activity in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Salmonella typhimurium. Compound T-3609 was also screened for recombinogenic activ ity in the yeast Saccharomyces cerevisiae D3 assay. Both assays were performed in the presence and absence of a rat-liver metabolic activation system. Compound T-3609 was reproducibly nonmutagenic and nonrecombinogenic when tested according to these procedures. 005276 ii CONTENTS SUMMARY............................................ INTRODUCTION....................................... MATERIALS.......................................... METHODS............................................ RESULTS AND DISCUSSION.................. TABLES Table 1 *...................................... Table 2 ....................................... Table 3 ....................................... Table 4 .......... ii I 3 5 11 12 13 14 15 005277 in INTRODUCTION SRI International examined 3M Company's Compound T-3609 for mutagenicity in the standard Ames Salmonella/mlcrosome assay with strains TA1535, TA1537, TAI538, TA98, and TA100 of the bacterium Salmonella typhimurium. Compound T-3609 was also tested for recombinogenic activity in the yeast Saccharomyces cerevislae D3 assay An Aroclor 1254stimulated, 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 J5. 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_. cerevislae 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. Evaluation of experimental results from the Salmonella assay consists of comparing the number of histidine-independent colonies on the treated agar plates with the number observed on the control plates. Because all the plated Salmonella indicator organisms undergo a few cell divisions in the presence of the test chemical, the test is semiquantitative in nature. The plate test procedure does not permit quantitative determination of the number of cells surviving the chemical treatment. It is the demonstration of a mutagenic dose-response relationship that is important in establishing mutagenicity. 1 005278 The test chemicals are assayed at several dose levels within a nontoxic dose range-- with the exception of the highest dose level, which sometimes exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial growth lawn, formation of pinpoint colonies consisting of surviving cells, and a decrease in the number of revertant colonies below the spontaneous background. A chemical is considered a mutagen in the Salmonella assay if it elicits a reproducible, dose-related increase in the number of histidine revertants per plate in one or more tester strains. The yeast Saccharomyces cerevisiae D3 is a eukaryotic microorganism capable of detecting mitotic recombination, as expressed through a mutation leading to a defective enzyme in the adenine-metabolizing pathway, resulting in a red-pigmented colony. In this assay, the yeast cells are exposed to several concentrations of the test chemical, usually ranging from a concentration that results in no killing to one that causes 50% killing. Any concentration that induces 90% killing is considered toxic. When the number of genetically altered colonies per milliliter (yield) and the ratio of altered colonies to survivors (frequency) from the treated cells are unequivocally larger than those of the solventtreated controls, we conclude that the exposure of the cells to the compound induces mitotic recombination. If this event is dose-related, the observation is termed a positive response. 2 OS279 V MATERIALS Test Article - Name; T-3609 - Date Received: 19 July 1984 - Description: Yellow, glue-like liquid - Storage Conditions: Room temperature - Special Testing Conditions: None - Stability: Assured by Sponsor Indicator Organisms - Species: Salmonella typhimurium LT2; Saccharomyces cerevisiae - Strains: TA1535, TA1537, TA1538, TA98, and TA100 for S_. typhimurium; D3 for S_. cerevisiae - Source: Dr. Bruce Ames, University of California, Berkeley, for the Salmonella; Dr. F. K. Zimmermann, W. Germany, for the yeast Metabolic Activaton Aroclor 1254-induced, rat liver S-9; SRI Batch F-3; ~ 22.0 mg/ml protein Negative (Diluent) Control Material Acetone Date Opened: 13 December 1983 Expiration Date: 13 December 1984 Manufacturer: American Scientific Products, McGraw Park, IL Positive Control Chemicals 9-Aminoacridine, CAS No. 90-45-9 Manufacturer: Pfaltz and Bauer, Stamford, CT 2-Anthramine, CAS No.. 613-13-8 Manufacturer: Sigma Chemical Co., St. Louis, MO 005280 3 2-Nitrofluorene, CAS No. 607-57-8 Manufacturer: Aldrich Chemical Co., Milwaukee, WI Sodium Azide, CAS No. 26628-22-8 Manufacturer: Difeo Laboratories, Detroit, MI 1,2,3,4-Diepoxybutane, CAS No. 1464-53-5 Manufacturer: Ffaltz and Bauer, Stamford, CT Sterigmatocystin, CAS No. 10048-13-2 Manufacturer: Calbiochem, La Jolla, CA Counters Used - New Brunswick Scientific bioTran II Automated Colony Counter, Model Clll, SRI No. 0030 0151 00 - New Brunswick Scientific Bactronic Colony Counter, Model C110, SRI No. 0013 0788 00 00S281 4 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+ ) 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_. typhimurlum 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 llpopolysaccharide 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. Strain TA100 is derived from TA1535 by the introduction of the resistance transfer factor, plasmid pKMlOl. This plasmid is believed to cause an increase in error-prone DNA repair that leads to many more mutations for a 5 005282 given dose of most mutagens. In addition, plasmid pKMIOl 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 B^, 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 frozen in nutrient broth supplemented 9 with 10% sterile glycerol at -80C in 1-ml aliquots 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 (hi s, rfa, uvrB, bio) and for the presence of the plasmid. For each experiment, the 1-ml frozen cell cultures 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_. typhimurlum 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 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. 6 005283 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 waished 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 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 50 ml: 5.0 ml of S-9 fraction 1.0 ml of MgCl2 (0.4 M) and KC1 (1.65 M) 0.25 ml of glucose-6-phosphate (1 M) 2.0 ml of NADP (0.1 M) 25.0 ml of sodium phosphate buffer (0.2 M, pH 7.4) 16.75 ml of sterile HgO. The amount of S-9 fraction delivered to each plate is 50 pi. Plate Incorporation Assay Prior to testing, the test article is serially diluted from an initial stock. 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. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 pl/plate. Occasionally, liquids are tested; the sample is not diluted, and various aliquots are used. All assays are repeated at least once on a separate day. 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% NaCl, 0.05 nM biotin, and 0.05 mM histidine 8 (2) 0.05 ml of Indicator organisms (about 10 bacteria) 7 005284 (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 j>. 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 recombinogenlc 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^, 0.15Z KH2PO4 , 0.45% (NH^^SO^, 0.35% peptone, ooszss 8 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 0 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 J 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 lO""* 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. Plates containing the 10 dilution are scanned with a dissecting microscope at 10x 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. Statistical Analysis No statistical analysis was performed for any of the assays. The results of the plate incorporation assay are a tabulation of the number of colonies appearing on the plates. A positive response is indicated by a reproducible, dose-related increase in the number of histidine-independent colonies per plate. The results of the S. cerevlsiae D3 assay are tabulated by calculating the number of mitotic recombinants per 10^ survivors. A positive response in 9 005286 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, 6 . 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. Scl. 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-mlcrosome mutagenicity test. Mutat. Res. 31, 347-364 (1975). Brusick, D. J., and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspect. 6, 83-86 (1973). Kier, L. D., E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat. Acad. Scl. USA 71, 4159-4163 (1974). McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella/mlcrosome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA _72_, 979-983 (1975). McCann, J., and B. 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). Zimmermann, F. K., and R. Schwaler. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alkylating agents in Saccharomyces cerevlsiae. Mol. Gen. Genet. 100, 63-76 (1967). 10 005287 RESULTS AND DISCUSSION 3M Company's Compound T-3609 was screened for mutagenic activity in the Ames Salmonella/microsome in vitro mutagenicity assay using the five standard strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA98, and TA100. The assays were performed in duplicate, both in the presence and absence of a rat-liver metabolic activation system. Acetone was used as the diluent. The microbial mutagenicity testing of this sample was performed on 26 July and 3 August 1984. Dose levels ranging from 10 to 5000 iig/plate were used for both assays (Tables 1 and 2). No dose-related increases in the number of histidine-independent revertants were observed in either assay. Compound T-3609 was also tested for recombinogenic activity in the yeast Saccharomyces cerevisiae D3 assay for mitotic recombination. This assay was performed on 27 July and 10 August 1984, both with and without metabolic activation. A dose range of 0.05 to 5% was used for both assays (Tables 3 and 4). No dose-related increases in the number of mitotic recombinants per 1 0 5 survivors were observed in either assay. In conclusion, Compound T-3609 was reproducibly nonmutagenic and nonrecombinogenic when tested according to these procedures. 005288 11 Table 1 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3609 Experiment Date: 26 July 1984 Compound Metabolic Activation Negative Control Acetone + Positive Controls Sodium Azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine - - - + - + Compound Added per Plate 50 pi 50 1 MS 50 5 1 1 2.5 2.5 TA1535 Histidine Revertants per Plate TA1537 TA1538 TA98 TA100 22 19 5 4 11 13 26 19 140 92 9 12 10 6 20 23 25 41 123 162 497 491 21 25 111 115 429 256 275 1005 905 567 533 14 16 19 28 152 111 119 96 85 339 77 50 59 428 165 349 Compound T-3609 - 10 pg 24 28 6 14 17 19 27 19 151 138 - 50 40 27 9 7 12 5 27 37 151 151 - 100 25 26 6 7 13 9 31 29 149 150 - 500 37 26 10 3 8 14 18 27 144 151 - 1000 27 21 6 9 13 7 21 19 150 146 -- 5000 27 22 76 5 12 31 24 126 151 + 10 + 50 + 100 + 500 + 1000 + 5000 14 14 7 14 10 9 7 13 12 12 14 14 88 9 12 10 9 14 9 88 12 12 24 19 43 32 130 142 27 21 34 31 132 141 25 19 35 34 169 159 21 21 33 42 139 150 18 25 34 37 148 140 27 21 36 38 135 151 00S289 Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3609 Experiment Date: 3 August 1984 Compound_____ Metabolic Activation Compound Added per Plate ___________ Histidine Revertants per Plate_______ TA1535 TA1537 TA1538 TA98 TA100 Negative Control Acetone - + 50 pi 50 25 30 8 10 87 12 11 8 17 24 27 130 150 19 20 40 34 142 154 osltive Controls Sodium Azide 9-Amlnoacridine 2-Nitrofluorene 2-Anthramine - - + + 1 pg 50 5 1 1 2.5 2.5 502 472 18 19 117 124 503 254 281 1079 1074 696 587 15 15 20 25 141 119 116 97 102 401 . 11 13 52 60 479 137 393 Compound T-3609 - 10 pg 36 32 8 8 16 16 27 29 167 163 - 50 39 32 9 11 13 12 30 36 150 152 - 100 19 33 11 8 7 20 33 35 162 170 - 500 25 19 6 3 15 17 31 36 155 164 - 1000 28 31 7 5 12 16 36 31 169 162 -- 5000 38 31 7 11 9 12 20 29 167 153 + 10 + 50 + 100 + 500 + 1000 + 5000 13 13 15 9 16 15 8 13 12 12 96 9 13 11 9 14 12 77 66 86 28 21 39 42 162 150 20 24 39 42 160 164 24 19 47 44 167 160 26 19 37 37 166 158 19 24 42 43 137 151 22 27 36 41 147 124 G05290 Table 3 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-3609 Experiment Date: 27 July 1984 Compound Metabolic Activation Percent Surviving Concentration Cells per ml Survivors (w/v) (x 10"7) (%) Mitotic Recombinants per mj (x 10 J ) Mitotic Recombinani per 10^ Survivors Negative Control Acetone + 4.2 100 4.0 100 5 12 5 13 Positive Controls 1,2,3,4-Diepoxybutane - Sterigmatocystin - + -p' Compound T-3609 o C/T ?) V - - " + + + + + 0.025 0.005 0.005 0.05 0.1 0.5 1 5 0.05 0.1 0.5 1 5 4.7 100 4.8 100 4.8 100 4.4 100 4.4 100 4.6 100 4.6 100 4.6 100 4.4 100 4.1 100 4.6 100 4.8 100 4.1 100 881 1900 4 8.3 158 330 7 16 6 14 7 15 4 8.7 6 13 4 9.1 6 15 5 11 6 13 3 7.3 Calculations are expressed using two significant figures. I Table 4 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-3609 Experiment Date: 10 August 1984 Compound Metabolic Activation Percent Surviving Concentration Cells pey ml Survivors (w/v) (x 10-7) <%) Mitotic Recombinants per ml (x 10"3) Mitotic Recombinants per 103^ Survivors Negative Controls Acetone - + 3.9 100 3.7 100 6 15 5 14 Positive Controls 1,2,3,4-Diepoxybutane Sterigmatocystin - - + 0.025 0.005 0.005 4.7 100 4.0 100 4.1 100 933 2000 4 10 150 370 Compound T-3609 -- 0.05 - 0.1 - 0.5 -1 -5 4.5 100 4.3 100 4.6 100 4.3 100 4.5 100 8 18 4 9.3 8 17 6 14 7 16 + 0.05 + 0.1 + 0.5 +1 +5 4.1 100 4.3 100 4.6 100 4.6 100 4.7 100 5 12 5 12 5 11 5 11 4 8.5 * Calculations are expressed using two significant figures. 00S292