Document 5GJxX2MYmxYrdrwwmn5KvYMR

IN VITRO MICROBIOLOGICAL MUTAGENICITY OF 3M COMPANY COMPOUND T-2816CoC ASSAYS Final Report, Revised March 1980 By: Kristien E. Mortelmans, Ph.D. Director, Microbial Genetics Department and Anne Pomeroy, Microbiological Technician Prepared for: 3M COMPANY Medical Department General Offices, 3M St. Paul, Minnesota Center 55101 Attention: W. C. McCormick Toxicology Services SRI Project LSC-8958 Approved: . David C. L Jon477Director Toxicology Laboratory W. A. Skinner, Executive Director Life Sciences Division SFII lntemational 333 RavenswoodAve.oMenloPark,Californ9i4a025 (415)326-6200- Cable SRI INTL MPK - T,AIX:910-373-1246 SUMMARY SRI International examined 3M Company's Compound T-2816CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TAIOO 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. 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 SacchaEREyces 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 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 ME"MODS Salmonella typhimurium Strains TAIS35, TA1537, TA.1538, TA98, and_TAI.00 The Salmonella typhimuri= strains used at SRI are all histidine auxotrophs by virtue of mutations in the histidine operon. When these histidine-dependenc 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 ",he University of Calilornia at Berkeley. In addition to having mutations in the hist4-4-ineoperon, all the indicator strains have a mutation (.r@'CaL) that leads to a defective lipopolysacchar4-de coat; they also have a deletion ".hatcovers genes involved in the synthesis of the vitamin bictin (bio) and in the repair of ultraviolet (uv)-induced DNA damage (uvrB). The rfa mutation makes the strains more permeable to =any large aromatic molecules, thereby increasing the mutagenic effect of these molecules. T.ie uvrb mutation causes decreased repair of some t-zpes o.@r chemically or phys4-cally damaged DNA and thereby enhances the strains' sensiti,7-;-=-r7:o some mutagenic agents. + Strain TA1535 is reverted to his by many mutagens tnat cause base-pair substitutions. TA.100 is derived from TA1535 by the introduction of the resistance transfer factor, plasmid piC4101. 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 pMlOl confers resistance to the 3 antibiotic ampicill:Ln,which is a convenient marker to detect the presence of the plasmld In the cell. The presence of this plasmid also makes strain TA.100sensitive to some frameshift =tagens [e.g., ICR-191, benzo(a)pyrene, aflatoxin Bi, and 7,12-dimethylbenz(a)anthracene]. Strains TA1537 and TAIS38 are reverted by -any fra-eshift mutagens. Strain TA98 is derived from TA.1338by the addition of the plasmid pKM101, 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 plasm4-d-carryingstrains also contain ampicillin (25 @ig/ml)to ensure stable maintenance of the plasmid PKMIOI. 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 370C in nutrient broth (Oxoid, CM67). Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic chemicals (e.g., of the aromatic amino type or the polycvclic 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 inter-mediate metabolites are very potent mutagens in the S. typhimurium test. Ames has descr4-bed the live.-metabolic activation system that we use. In brief, adult male rats (250 to 300 g) are given a single 500-mg/kg intravericoneal injection ot 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 animal-s'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 subsequent operations are conducted in an ice bath. The livers are washed with an equal volume of cold. sterile 0.15 M KCI (I ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 M KCI, and homogenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x .1,and the supernatant, referred to asthe 5-9 fraction, is quickly frozen in dry ice and stored at -80*C. The metabolic activation mixture for each experiments consists of, for 10 ml: 1.00 ml of S-9 fraction 0.20 ml Of MgC12 (0.4 M) and KCI (1.65M) 0.05 ml of glucose-6-phosphate (I M) 0.40 ml of NADP (0.1 M) 5.00 mi of sodium phosphate buffer (0.2 M, pH 7.4) 3.35 =1 of E20- Assays 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 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'-N"aCl. tl-iinimaalgar plates consist of, per liter, 15 g of agar, 10 g of glucose, 0.2 g of MgSO,-7H20, 2 & of citric acid manohydrate, 10 g of K2POI, and 3.5 g of NaH.Nli,PO.-4H20- 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 S. cerevisiae is stored at 4*C. For each experiment, broth containing 0.05t MgSO4.*,0.152 KH2PO,, 0.45% (NHI)2SOI, 0.35Z peptone, O.5Z yeast extract, and 2Z dextrose is inoculated with a loopful of the stock culture and incubated overnight at 30*C 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/mi in 67 mM phosphate buffer (pH 7.4). To a sterile test tube are added: 1.00 mi of the resuspended culture 0.50 ml of either the metabolic activation mixture or buffer 0.20 ml of the test chemical 0.30 =1 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 for 4 hours on a roller drum. The sample is then diluted serially in sterile physiologic 6 saline, and 0. 2 ml of the 10@-3and 10-3 dilutions is spread an plates containing the same ingredients as the broth plus 2.OZ agar; five plates are spread w:Lth the 10-3 dilution and three plates are spread with the 10-3 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 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 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 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 I 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 ;ig/plate. Toxicity was observed at a dose of 5,000 pg/plate (Table 1). Ethanol was used as the solvent in all assays. The results of our tests of T-2816CoC with five strains of S. lyyhimurium in the Ames SalmonellaLmicrosome 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 4 and 5. The compound was tested at concentrations from 0.05 to 5.0%. No toxicity or significant doserelated increase in the number of mitotic recombinants above background was observed. 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