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IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY COMPOUNDS T-2247 CoC AND T-2248 CoC Final Report 5 September 1978 By.: Vincent F. Simmon, Ph.D., Manager Microbial Genetics -Program Nancy Marx, Microbiologist Prepared for: 3M COMPANY Medical Departmerit General Offices 3M Center St. Paul, Minnesota 55101 Attention: J. E. Long, Sc.D., Manager Toxicology Department SRI Project LSC-4442-016 Approved by: David C. ]@. o es, Director Toxicology Laboratory W. A. Skinner, Executive Director Life Sciences Division lntemabonmal 333 Ravenswood Ave. Menlo Park,Californi9a4025 (415)326-6200 o Cable:STANRES, Menlo Park - TWX: 910-373-1246 SUMMARY SRI International examined 3M Company Compound T-2247 CoC and T-2248 CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Salmonella typhimurium in the Ames Salmonella/microsome assay and with the yeast Saccharomyqes cerevisiae D3. T-2247 CoC was also testedin desiceators. Each assay was performed in the presence and in the absence of a metabolic activation system. T-2247 CoC and T-2248 CoC were not mutagenic or ricombinogenic in any of the assays performed. INTROD UCTION SRI International examined 3M Company compounds T-2247 CoC and T-2248 CoC for mutagenicity by in vitro microbiological assays with five strains of the bacterium Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, and TA100) in the standard Ames Salmonella/microsome assay, in desiceators, and with the yeast Saccharomyces cerevisiae D3. An Aroclor 1254-stimulated, rat liver ho=genate 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. 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. The combination of the two assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. However, because the 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 T-kl535,T-U537, TA 538, 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 petri plates containinga.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 nany cases, this growth is essential for mutagenesis to occur. The his revertants are easily scored 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 2- to 'LOO-fold. We obtained cur S. tvphimurium strains from Dr. Bruce Ames of the University of California at.Berkeley.1-5 In addition to having mu,-@a- tions in the histidine operon, all the indicator strains have a muta- tion (rfa that leads to a defective lipopolysaccharidecoat; they also have a deletion that covers genes involved in the synthesis of vitaminbiotin (bio-)and in the repair of ultraviolet(uv)-induced DNA dqnage (u@vrB-). The rfa mutation makes the strains more permeable to many large aromatic molecules, thereby increasing the mutagenic effect of tlhesemolecules. The uvrb- mutation decreases repair of some types of chemically or physically damaged DNA and thereby enhances the stra4&ns'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 pK@LOl. This plasmid '6s believed to cause an .Lncreasein error-prone DINArepair that leads to many more mutations 3 for a given dose of most mutagens.5 in addition, plasmid pKM101 confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of the plasmid in the cells. The presence of this plasmid also makes strain TA100 sensitive to some Lrameshift mutagens (e.g., ICR-191, benzo(a)pyrene, aflatoxin Bi, and 7,12dimethylbenz(a)anthracene). Strains TA1537 and TA1538 are reverted by many fratn,-shifmtutagens. TA1537 is more sensitive than TA1338 to mutation by some acridines and benzanthracenes, but the difference is quantitative rather than qualitative. Strain TA98 is derived from TA1538 by the addition of the plasmid pIM01, which makes it more sensitive to some mutagenic agents. All indicator strains are kept at 4* C on minimal medium plates, supplemented with a trace of biotin, and an excess of histidine. The plates with the plasmid-carrying strains contain in addition ampir-illin ('&1j5ig/ml), to ensure stable maintenance of the plasmid pDjl0l. New stock culture plates are made every two months*from single colony reisolates that were checked for their genotypic characteristics (his, rfa, uvrb, @io) and --Forthe presence of the plasmid. For each experiment, an inoculum from the stock culture plates is grown overnig'ntat 37* C in nutrient broth (O.-.oidC,M67). After stationary overnight growth, the cultures are shaken for 3 to 4 hours to ensure optimal growth. Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic chemicals, either of the aromatic.amino type or 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 . 4,6-6 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-6 In brief, adult male rats (250 to 300 g) are given a single 500-mg/kg intraperitoneal injection of a polychlorinated biphenyl, Aroclor 1254. This treatment enhances the synthesis of enzymes involved in the metabolic 4 conversion OILchemicals. 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 in 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 X KC1, and homogenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x.&, and the supernatant, referred to as the S-9 fraction, is quickly frozen in dry ice and stored at -800 C. 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 (1.65M) * 0.05 ml of glucose-6-phosphate (1 M) * 0.40 ml of NADP (0.1 M) * 5.00 ml of sodium phosphate (0.2 M, pH 7.4) * 3.35,ml Of B20- 5 Assays in Agar To a sterile 13 x 100 mm 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) 0.05 ml of a solution of the test chemical (4) 0.50 ml of metabolic activation mixture (optional). For negative controls, we use steps (1), (2). and (4) (optional) and 0.05 ml of the solvent used for the test chemical. Because the majority of organic compounds are not sufficiently water soluble-particularly at the higher concentrations-we routinely use dimethylsulfoxide (DY.SO). Other solvents that are occasionally used are water, ethanol, or benzene. For positive controls, we test each culture by specific mutagens known to revert each strain using steps (1), (2), (3), and (4) (optional). This mixture is stirred gently and then poured onto minimal agar t 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. 0. 6 % agar contains 0. 05 ml-Ihistidine , 0,05 mM blotin, and 0. 1 M NaCl Minimal agar plates consist of, per liter, 15 g of agar, 10 g of glucose, 0.2 g of MgSO4-7H2O, 2 g of citric acid monohydrate, 10 g of K2HP04, and 3.5 g of NaHNH4PO4o4H2O- 6 Assays in Desiccators for Volatile Compounds 'fhestandard Ames plate test is not entirely suit*ablefor the testing of highly-volatile chemicals, so we have modified the procedure .to conduct such testing. The Salmonella plates are prepared as described for the assays in agar, but no test chemical is added. The plates, the lids having been removed, are placed side by side on a perforated shelf in a 9-liter desiccator. A Imova volume of the test chemical is added to a glass-petri plate that is placed in the center of and attached to the bottom of the shelf. A control chemical is tested similarly in each experiment. The desiccator is sealed and placed on a magnetic stir plate in a room maintained at 37* C. A magnetic stirrer with vanes, placed in the base of each desiccator, ensures adequate dispersion of the chemical. After incubation for 8 hours, the plates are removed from the desiccators, their lids are replaced , and they are incubated at + 37* C for ariadditional 42 hours. The number is his revertants is counted and recorded. DESICCATOR ASSAY PORCELAIN SHELF FAN C;:= 11 11 11 11 IL PETRI PLATES WITH SALMONELLA GLASS PETRI PLATE FOR TEST CHEMICAL MAGNETIC STIRRER 8 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.9 When grown on medium containing adenine, cells homozygous for this mutation produce a red pigment. These homozyaous mutants can be generated from the heterozygotes by mitotic recombination. The frequency of this recombinational event may be increasedby incubating the organisms with various mutagens. The degree of mutagenicity of a compound or of its metabolite is determined from the number of redpigmented colonies appearing on the plates.10 The S. cerevisiae tester strain is stored at -80* C. For each experiment, the tester strain is inoculated in 1% tryptone and 0.5% yeast extract and grown overnight at 37* C with aeration. 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 104 cells ml/in a 67 mM phosphate buffer (pH 7.4). To a sterile test tube are added: 0 1.30 ml of the organisms 0 0.50 ml of either the metabolic activation mixture or buffer 0 0.20 ml of the test chemical. Because many organic chemicals are not appreciably water soluble, dimethylsulfoxide (DMSO) is used routinely as the solvent for the test chemical. Other solvents that are used occasionally are ethanol, benzene, or water. Several doses of the chemical (up to 5%, w/v or v/v) 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 diluted serially in sterile physiological saiine, and a volume of 0.2 ml of the 10-3 and 10-3 dilutions is spread on tryptone-yeast agar plates; five plates are used for the 10-3 dilution and three plates are used for 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- 9 deficient homozygosity. Plates of the 10-3 dilution are scanned with a dissecting microscope at 10 X magnification, and the number of red colonies or red sectors (mitotic recombinants) is recorded. The surviving fraction of organisms is determined'from the number of colonies appearing on the plates of the 10-3 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 mote than threefold in the absolute number of mitotic recombinants per milliliter as well as in the relative number of mitotic recombinants per 10'ssurvivors. 10 RESULTS AND DISCUSSION Table 1 presents the results of testing T-2247 CoC in the Ames Salmonella microsome assay. The data are an average of two assays performed on separate days. The compound was tested over a wide range of concentrations, from 10 to 5000 pg/plate, both with and without metabolic activation. Because no.dose-related increase in the number of mutants over the background count was.observed, we conclude that T-2247 CoC was.not mutagenic in S. typhimutium. As Table 2 shows, T-2248 was tested at dose levels of 10 to 5000 Pg/plate. Toxicity was observed irxstrain TA1538 at 1000 Pg/ plate and in strains TA1535, TA1537, TA1538, TA98, and TA100 at 5000 ug/plate without metabolic activation. T-2248 CoC was toxic at 1000 pg/plate in strain TA1537 and at 5000 ug/plate in all strains when tested with activation. No mutagenic activity was observed with T-2248 CoC in the Ames Salmonella/microsome assay. Table 3 presents the averaged results of testing T-2247 CoC in the desiccator assay on strairxs.TA98 and TA100. The assay was conducted in duplicate (two plates per strain per dose) with an exposure of 8 hours. The sample was exposed to a wide range of doses of from 0.1 to 5.0 ml per desiccator. No mutagenicity or toxicity was observed. Tables 4 and 5 present the results of assays on T-2247 CoC with S. cerevisiae D3. T-2247 CoC was tested at concentrations of from 0.1 to 5.0% (Table 4) and at concentrations of from 1.0 to 5.0% (table 5). The compound was not toxic in these assays and did not cause a doserelated increase in the number of mitotic recombinants. Therefore, we conclude that T-2247 CoC was not recombinogenic in S. cerevisiae D3. Tables 6 and 7 present the results of testing T-2248 CoC with S. cerevisiae D3. A slight toxic and apparent mutagenic response was seen at 5.0% without metabolic activation (Table 6). T-2248 CoC was retested at concentrations from 1.0 to 5.0% (Table 7). No dose- related increase in the number of mitotic recombinants was observed; therefore, we conclude that T-2248 CoC was not recombinognic to S..cerevisiae D3. We conclude that T-2247 CoC is not toxic or mutagenic in either S. typhimurium or S. cerevisiae D3. T-2248 CoC is toxic at higher doses in the Salmonella/microsome assay and is not mutagenic in either S. typhimurium or S. cerevisiae D3. 12 REFERENCES 1. J. McCann, E. Choi, E. YaTm-gsaki,and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA, 72, 51355139 (1975). 2. B. N. Ames, E. G. Gurney, J. A. Xiller, and E. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc. Nat. Acad. Sci. USA, 69, 31283132 (1972). 3. B.-N. Ames, 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-r786(1973). 4.- B. N. Ames, W. E. Durston, E. Ya-asaki, and F. D. Lee. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. '&'.TaAtc.ad. Sci. USA, 70, 2281-2285 (1973). 5. J. McCann, N. E.Spingarn, J. Kobori, and B. N. Ames. Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids., Proc. Nat. Acad. Sci. USA, 72, 979-983 (1975). 6. L. A. Poirier and V. F. Simmon. Mutagenic-carcinogenic relationships and the role of mutagenic screening tests for carcinogenicity. Clin. Toxicol.,9 (5), 761-771 (1976). 7. L. D. Kier, E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat. Acad. Sci. USA, 71, 4159-4163 (1974). 8. B. N. Ames, J. McCann, and E. Yamsaki. Methods for detecting carcinogens and mutagens with the Sal=nella/mAmmalian-microsome mutagenicity test. Mutation Res., 31, 347-364 (1975). 9. F. K. Zimmerma= and R. Schwaier. Induction of mitotic gene conversion with nitrous acid, 1-methyl-3-nitro-lnitrosoguanidine and other alkylating agents in Saccharomyces cerevisiae. Mol Gen. Genet., 100, 63-69 (1967). 13 10. D. J.'Brusick and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives, 83-96 (1973). 14 IN VITRO ASSAYS Table 1 WITH SALMONELLA T-2247 CoC TYPHIMURIUM Compound Negative control (H2,O) Positive controls Sodium azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine T-2247 CoC Metabolic Activation Micrograms of Compound Added per Plate + - 0.5 - 50.0 - 5.0 - 1.0 - 2.5 + 1.0 + 2.5 10 50 - 100 - 500 - 1000 - 5000 + 10 + 50 + 100 + 500 + 1000 + 5000 Average_Histiding R TA1535 TA1537 TA 11 8 9 5 141 63 11 19 186 138 19 8 14 10 18 11 16 13 18 12 17 12 7 5 10 7 9 6 8 11 8 5 13 5 Compound Negative control (H20) Positive controls Sodium azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine T-2248 CoC T, toxic. IN VITRO ASSAYS Table 2 WITH SALMONELLA T-2248 CoC TYPHIMURIUH Metabolic Activation Mici7ograms of Compound Added per Plate + - 0.5 - 50.0 - 5.0 - 1.0 - 2.5 + 1.0 + 2.5 - 10 - 50 - 100 - 500 - 1000 - 5000 + 10 + 50 + 100 + 500 + 1000 + 5000 Average Histidine Re TA1535 TA1537 TAI 11 8 9 5 141 63 8 11 19 2 186 138 19 13 12 12 24 13 15 10 10 7 9T T 8 12 11 6 8 9 12 9 8 7T T T Table 3 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM T-2247 CoC; 8-HOUR EXPOSURE IN DESICCATORS Compound Negative control (H20) Positive control 1,1-Dichloroethylene T-2247 CoC FJ 4 Metabolic Activation + + + + + + Amount of Compound in Desiccator_Spll 1.0 1.0 0.1 0.5 1.0 5.0 0.1 0.5 1.0 5.0 Revert TA98 13 27 31 35 12 11 15 7 33 18 27 19 Table 4 IN VITRO ASSAYS WITH SACCHAROMYCES T-2247 CoC CEREVISIAE Compound Negative control (H,O) Positive control 1,2,3,4-Diepoxybutane T-2247 CoC co Metabolic Activation + + - + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (X.10-7) Percent 0.025 0.025 6.2 100 6.1 100 6.8 ill 6.1 98 0.1 5.8 94 0.5 6.9 ill 1.0 5.6 90 5.0 7.5 121 0.1 6.5 107 0.5 6.9 113 1.0 6.4 105 5.0 5.1 84 Mitotic Re Per ml (x io@-3) 3.5 3.5 460 350 1.0 3.0 5.0 -0- 3.0 3.0 2.0 2.0 Table 5 IN VITRO ASSAYS WITH SACCHAROMYCES T-2247 CoC CEREVISIAE Compound Negative control (H20) Positive control 1,2,3,4-Diepoxybutane T-2247 CoC %0 Metabolic Activation + + - + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (X 10-7) Percent 0.025 0.025 6.8 100 8.3 100 6.9 101 7.0 84 1.0 6.9 701 2.0 5.7 84 4.0 4.9 72 5.0 7.6 112 1.0 8.4 101 2.0 5.3 64 4.0 5.7 69 5.0 7.4 89 'Mitotic RE Per ml (x io@-3) 3.5 2.5 610 570 2.0 1.0 3.0 3.o 2.0 2.0 3.0 2.0 Table 6 IN VITRO ASSAYS WITH SACCHAROMYCES T-2248 CoC CEREVISIAE Compound Negative control (H20) Positive control 1,2,3,4-Diepoxybutane T-2248 CoC Metabolic Activation + + + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (x 10-') Percent - 0.025 0.025 6.2 100 6.1 100 6.8 ill 6.1 98 0.1 5.6 90 0.5 6.5 105 1.0 5.0 81 5.0 3.1 50 0.1 7.0 115 0.5 7.3 120 1.0 6.5 107 5.0 6.1 100 Mitotic Rec Per ml (x 10@-.3) 3.5 3.5 460 350 3.0 3.8 6.0 10.0 1.3 -01.0 3.0 Table 7 IN VITRO ASSAYS WITH SACCHAROMYCES T-2248 CoC CEREVISIAE Compound Negative control (H20) Positive control 1,2,3,4-Diepoxybutane T-2248 CoC FA Metabolic Activation + + - - + + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (x 10-7) Percent -Mitotic Rec Per ml (x lors) 0.025 0.025 1.0 2.0 3.0 4.0 5.0 1.0 @2.0 3.0 4.0 5.0 6.6 8.6 4.6 5.6 6.4 7.1 5.6 5.8 7.0 5.2 6.5 6.4 7.5 5.9 100 2.5 100 1.0 70 520 65 560 97 1.0 108 1.0 85 5.0 88 7.0 106 3.0 60 6.0 76 1.0 74 5.0 87 1.0 69 2.0