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IN VITRO MICROBIOLOGICAL MUTAGENICITY OF 3M COMPANY'S COMPOUND T-2997CoC ASSAYS --71 IF]y@ Final Report January 1981 By: L,,@ Kristien E. Mortelmans, Ph.D. Director, Microbiai Genetics Department and Anne L. Pomeroy, Microbio Prepared for: 3M COMPANY Medical Department General Offices, 3M St. Paul, Minnesota Center 55101 Attention: Mr. William C. McCormick Toxicology Services, 220-2E-02 SRI Project LSC-8958 Approved by: David C. L. Jonei7lirector Toxicology Laboratory W. A. Skinner, Executive Director Life Sciences Division or-ism lntemational) 333RavenswoodAve.-MenloPark,Calif@rn9i4a025 (4153)26-620-0Cable:SRIINTLMPK - TWX: 910-373-1246 SUMMARY SRI International examined 3M Company's Compound T-2997CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TA100 of Salmonella tnphimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomvces 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 -,iotnuLagenic 6, reco:nbinogen4-c4.r,any absay perfcrmed. CONTENTS SUMMARY .. ... . .. .. . .. ... .. ... . . QUALITY ASSURANCE STATEMENT . .. ... .. ... . . iv INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 MATERIALS .. .. .. .. . .. .. .. .. ... . . 2 METHODS . .. ... . .. . .. .. ... . ... . . 3 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 9 TABLES Table 1 . . . . . . . . . . . . . . . . . . . . . 10 Table 2 . . . . . . . . . . . . . . . . . . . . . 11 Table 3 . . . . . . . . . . . . . . . . . . . . . 12 Table 4 . . . . . . . . . . . . . . . . . . . . . 13 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. Quality A ut@Ace Unit/Title &/Date iv INTRODUCTION SRI International examined 3M Company's Compound T-2997CoC for mutagenicity by in vitro microbiological assavs with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomvces 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 S. tvphimurium has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has about the same reliabilitv 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 carcinogenicitv 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 Januarv 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. MATERIALS Test Compounds - Name: T-2997CoC. - Date Received: 8 December 1980. - Description: Waxy amber solid. - Stabilitv: Stability assured by client. - Storage Condition: Stored at room temperature. - Special Testing Conditions: None. Indicator Organisms - Species: - Strains: Salmonella tvphimurium LT2 and Saccharomvces cerevisiae. TA1535, TA1537, TA1538, TA98, and TA100 of S. tvphimurium; D3 of S. cerevisiae. Metabolic Activation Aroclor 1254-induced rat liver S-9; SRI Batch D7, D8, and NIEHS RLI003, @,30 mg/mi protein. Soo@"@vent 'U'sed Dimethyisuifoxide (DMSO). 2 METHODS Salmonella typhimurium Strains TA1535, TA1537, TA-1538, TA98, and TA100 The Salmonella tvphimurium 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 S. tvphimurium 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 -e,.iesi-nvol,@,e4d--.the E@-itbesis 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 strainst sensitivity to + some mutagenic agents. Strain TA1535 is reverted to his by many mutagens that cause base-pair substitutions. TA100 is derived from T pKM101. y '6I'le i,itroduction uf Liie resistance transfer This plasmid is believed to cause an increase faCLor, plasmid in error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasmid pKM101 confers resistance to the anti- biotic ampicillin, which is a convenient marker to detect the presence 3 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 Bi, 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 pKMI01, which makes it more sensitive to some mutagenic agents. All indicator strains are kept at 4*C on minimal agar plates supplemented 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 pKM101. New stock culture plates are made every 4 to 6 wpekq from sitial- color3, iqola-r-oct---$,ahtave 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 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 verv potent mutagens in the S. typhimurium test. has described the liver metabolic activation system that we use. Ames 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 subsequent operations are conducted in an ice bath. The livers are washed 4 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 y,, and the supernatant, referred to as the S-9 fraction, is quickly frozen in dry ice and stored at -800C. The metabolic activation mixture for each experiments consists of, for 10 ml: 0 1.00 ml of S-9 fraction 0 0.20 ml Of MgCl2 (0.4 M) and KC1 (1.65 M) 0 0.05 ml of glucose-6-phosDhate (1 M) 0 0.40 ml of NADP (0.1 M) 5.00 ml of sodium phosphate buffer (0.2 M, pE 7.4) 3.35 ml of H20- Assavs in Agar To a sterile 13 x 100 = test tube placed in a 43*C 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. t This mixture is stirred gently and then poured onto minimal agar plates. After the top agar has set, the plates are incubated at 37'C for 2 days. + The number of his 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. t Minimal agar plates consist of, per liter, 15 g of agar, 10 g of glucose, 0.2 g of MgSO,-7H20, 2 g of citric acid monohydrate, 10 g of K2HFO,, and 3.5 g of NaNH,HPO,-4H20- 5 culture by specific mutagens known to revert each strain, using steps (1), (2), (3),and (4). Saccharomvces cerevisiae D3 The veast 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 va@.lous carcinogeniz or recom"-@-'@nogenica6encs. "A."he L .L C 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 4*C. For each experiment, broth containing 0.05% MGSO,, 0.15% KH2POI, 0.45% (NH,)2SOI, 0.35% peptone, 0.5% yeast extract, and 2% dextrose is inoculated with a loopful of the stock culture and incubated overnight at 300C 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 /'.4). To z ster@@le test tube are adu'ad; 0 1.00 ml of the resuspended culture 0 0.50 ml of either the metabolic activation mixture or buffer 0.20 mi 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 30*C ior 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 6 are spread with the 10-3 dilution and three plates are spread with the 10-5 dilution. The plates are incubated for 2 days at 30*C, followed by 2 days at 4'C to enhance the development of the red pigment indicative of adenine-deficient homozygosity. Plates containing the 10-3 dilution are scanned with a dissecting microscope at lOx 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 10-5 dilution. The number of mitotic recombinants is calculated per 103 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 recornbi-art- --- milliliter as well as in the relative number of mitotic recombinants per 105 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 carc4--iogens. Proc. "'at. Acal@. 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, 22812285 (1973). Ames, B. N. , F. D. Lee, and W. E. Durston. An improved system for the detection and classification of mutagens Proc. Nat Acad. Sci. USA 70, 782-786 (1973). bacterial test and carcinogens. P,INES,B. N. , j. aiid E. Yamasak.-L. Methods for detecting carcinogens and mutagens with the Salmcnella/ma-malian-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 6, 83-86 7 Kier, L. D., E. Yamasaki, and B. N. Ames. activitv in cigarette smoke condensates. 4159-4163 (1974). Detection Proc. Nat. of mutagenic Acad. Sci. USA. 71, McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. gens as mutagens in the Salmonella/microsome test: Proc. Nat. Acad. Sci. USA 72, 5135-5139 (1975). Detection Assay of of carcino300 chemicals. McCann, J., N. E. Spingarn, J. Kobori, and B. N. Ames. carcinogens as mutagens: Bacterial tester strains with Proc. Nat. Acad. Sci. USA 72, 979-983 (1975). Detection of R Factor plasmids. Mortelmans, property of Gen. Genet. K. E., and B.A.D. Stocker. Segregation of plasmid R46 from its ultraviolet-protecting 167, 317-327 (1979). the mutator property. Mol. K. , a,-,-:,. '.,7,,iu-L-;-u,, M4-totic gei-.c with nitrous acid, 1-methyl-3-nitro-l-nitrosoguanidine and other alky- lating agents in Saccharomvces cerevisiae. Mol. Gen. Genet. 100, 63-69 (1967). RESULTS AND DISCUSSION Compound T-2997CoC was tested for mutagenicitv with the Ames Salmonella/microsome assav and with the veast Saccharomvces cerevisiae D3 assav in the presence and in the absence of a metabolic activation svstem. The compound was tested twice on separate days in both assays. Dimethvlsulfoxide (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 jig/plate (Table 1). Because no toxicity occurred at 5,000 @ig/plate,the second test was run using a higher dose range, from 50 to 10,000 jig/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 D3, 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 assav. 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