Document 5Ddgk4ZooXDopKrjoNGGRwb1R

IN VITRO MICROBIOLOGICAL NUTAGENICITY ASSAYS OF 3M COMPALNY COMPOUNDS T-2540 CoC and T-2541 CoC Final Report August 1979 By: Kristien E. @lortelmans,Ph.D. Director, Microbial Genetics Department and Nancy Marx, Microbiologist Prepared for: 3M COMPANY Medical Department General Offices 3M Center St. Paul, Minnesota 55101 Attention: W. C. McCormick, Ph.D., Manager Toxicology Services SRI Project LSC 4442-16 Approved: o David C. L. Jonel , Director n Toxicology Laboratory W. A. Skinner, Executive Director Life Sciences Division lnternatorud w 333 Ravenswood Ave. - Menlo Park,Californi9a4025 (415)326-6200- Cable:SRI INTL MPK - TWX: 910-373-1246 SUMMARY SRI International examined 3M Company compounds T-2540 CoC and T-2541 CoC for mutagenic activity with strains TA1535, TA1537, TA1538, TA98.,and TA100 of Salmonella typhimurium in the standard Ames Salmonella/ microsome assay, in an assay conducted in desiccators, 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. Neither T-2540 CoC nor T-2541 Coc was mutagenic or recombinogenic in any of the assays performed. INTRODUCTION SRI International examined 3M Company compounds T-2540 CoC and T-2541 CoC 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., in an assay conducted in desiccators, and with the yeast Saccharomyces 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. 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 .2 3 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 METHODS Salmonella typhimurium Strains TA1535, TA1537, TA1538,__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 containing a trace of histidine, only 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. 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 100-fold, usually in a dose-related manner. We obtained our S. typhimurium strains from Dr. Bruce Ames of the University of California at Berkeley. 1,4.-7 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 aromatic 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 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 pKK101. This plasmid is believed to cause an increase in errorpro.ne DNA repair that leads to many more mutations for a given dose of most mutagens 7 In addition, plasmid pKM101 confers resistance to the 3 antibiotic ampicillin, which is a convenient marker to detect the presence of the plasmid in the cell.8 The-..presencoef this plasmid also makes strain TA100 sensitive to some frameshift mutagens [e.g., ICI-191, benzo(a)pyrene, aflatoxin Bi, and 7,12-dimethylbenz(a)anthracenel. Strains TA1537 and TA1538 are reverted by many frameshift mutagens. Strain TA98 is derived from TA1538 by the addition of the plasmid pKK101, 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 jig/ml)to ensure stable maintenance of the plasmid pKK101. New stock culture plates are made every four to six weeks from single colony isolates that have been checked for their genotypic characteristics (his, rfa, uvrb, I?io) 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. 6,9-" 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.9 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. 4 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 (1 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 &, and the supernatant, referred to as the S-9 fraction, is quickly frozen in dry ice and stored at -80*C.' The metabolic activation mixture for each experiment consists of, for 10 ml: 0 1.00 ml of S-9 fraction 0 0.20 ml Of MgCl2 (0.4 M) and KCI (1.65 M) 0 0.05 ml of glucose-6-phosphate (1 M) 0 0.40 ml of NADF (0.1 M) 0 5.00 ml of sodium phosphate buffer (0.2 M, pH 7.4) 3.35 ml of H20- Assays in Agar To a sterile 13 x 100 mm test tube placed in a 43*C we add in the following order: heating block, (1) 2.00 ml of 0.6% agar (2) 0.05 ml of indicator organisms (3) 0.50 ml of metabolic activation mixture (optional) (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% NaCl. 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 K2HPO,, and 3.5 g of NaHNH4PO4o4H20- 5 For negative controls, we use steps (1), (2), and (3) (optional), and 0.05 ml of the solvent is used for the test chemical. Dimethylsulfoxide (DMSO) was used as the solvent for T-2540 CoC and T-2541 CoC. For positive controls, we test each culture by specific mutagens known to revert each strain, using steps (1), (2), (3), (optional), and (4). Assays in Desiccators for Volatile Compounds The standard Ames plate test is not entirely suitable for testing 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, with lids removed, are placed side by side on a perforated shelf in a 9-liter desiccator (Figure 1). A known 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 an additional 64 hours. The number of his revertants is counted and recorded. 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 . 2 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.3 6 DESICCATOR ASSAY PORCELAIN SHELF FA C;-::= Figure 1 7 PETRI PLATES WITH SALMONELLA GLASS PETRI PLATE FOR TEST CHEMICAL MAGNETIC STIRRER A stock culture of S. cerevisiae is stored at 4*C. For each experiment, broth containing 0.05% MgSO4, 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 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 ml of the resuspended culture 0.50 ml of either the metabolic activation mixture or buffer 0.20 ml of the test chemical 0.3 ml of buffer. DMSO was used as the solvent for T-2540 CoC and T-2541 CoC. Several dose levels of the test 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 then diluted serially in sterile physiologic saline, and a volume of 0.2 ml of the 10-5 and 10-' dilutions is spread on plates containing the same ingredients as the broth plus 2.0% agar; five plates are spread with the 10-3 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 40 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 10-5 dilution. The number of mitotic recombinants is calculated per 105 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. 8 'RESULTS AND DISCUSSION Tables 1 and 2 present the results of our tests of T-2540 CoC and T-2541 CoC in the Ames Salmonella/microsome assay. The data in each table show the results of a duplicate assay performed on separate days. Both compounds were tested over a wide range of dose levels, from 10 to 5,000 ug/plate, both with and without metabolic activation. Compound T-2540 CoC was toxic to the bacteria at 5,000 ug/plate. No dose-related increase in the number of histidine revertants over the background count was observed in either assay. Therefore, we conclude that compounds T-2540 CoC and T-2541 CoC were not mutagenic in the standard Salmonella plate incorporation assay. Table 3 presents the result s of an assay of T-2540 CoC and T-2541 CoC conducted in desiccators with strains TA98 and TA100. The compounds were tested over a range of dose levels from 0.1 to 5.0 ml per desiccator. Toxicity was observed at 1.0 ml per desiccator with T-2540 CoC and at 5.0 ml per desiccator with T-2541 CoC. Because no mutagenic response was observed with or without metabolic activation, no further testing was performed in desiccators. The results of microbiological assays with S. cerevisiae D3 on T-2540 CoC are presented in Tables 4 through 6. In a preliminary experiment conducted over a range of concentrations from 0.1 to 5.0% (Table 4), organisms exposed to T-2540 CoC showed less than 50% survival at concentrations of 0.5% and higher. Therefore, the compound was retested over a range of concentrations from 0.025 to 0.25% (Table 5). There was a slight increase in the number of mitotic recombinants both with and without metabolic activation at concentrations from 0.075 to 0.25%, and this increase was not dose-related. Compound T-2540 CoC was tested once more within a narrower range, from 0.07 to 0.2% without activation and from 0.09 to 0.4% with activation (Table 6). In this assay, there was an increase in the number of mitotic recombinants only 9 at 0.3% with activation. Because the increases in mitotic recombinants observed in both assays were neither dose-related nor reproducible, we do not believe that T-2540 CoC was recombinogenic with S. cerevisiae D3. Tables 7 through 9 present the results of assays with T-2541 CoC with S. cerevisiae D3. A preliminary experiment determined that T-2541 CoC was toxic to the yeast at 0.5% without activation and 5.0% with activation (Table 7). The compound was tested twice more at concentrations from 0.025 to 0.25% without activation and 0.25 to 2.5% with activation (Table 8), and at concentrations from 0.07 to 0.2% without activation and 0.2 to 1.0% with activation (Table 9). T-2541 CoC showed several increases in the number of mitotic recombinants per 103 survivors. However, as with T-2540 CoC, these increases were neither dose-related nor repeatable; therefore, we conclude that T-2541 CoC was not recombinogenic in this assay. In stimmary,we conclude that compound T-2540 CoC and T-2541 CoC were - not mutagenic with S. typhimurium, or recombinogenic with S. cerevisiae D3. 10 Table I IN VITRO ASSAYS WITH SALMONELLA TYPH114URIUM T-2540 CoC and T-2541 CoC Compound Metabolic Activation Negative control (DMSO) + Positive control Sodium azide - 9-Aminoacridine - 2-Nitrofluorene - 2-Anthramine - + + T-2540 CoC F4 + + + + + + Micrograms of Compound Added per Plate 1.0 50.0 5.0 1.0 1.0 2.5 2.5 10 50 100 500 1,000 5,000 10 50 100 500 1,000 5,000 TA1535 27 24 28 28 Histidi.neRevertants pe TA1537 TA1538 9 12 43 48 28 18 50 41 569 531 21 26 182 194 26 19 19 37 19 24 16 27 32 18 19T* 19T 33 25 31 37 24 29 30 31 21 39 26 28 576 261 18 7 81 67 12 8 7 13 87 48 19 9 6T 5T 36 29 33 39 40 26 18 30 30 27 18 -19 727 537 17 20 251 306 18 20 16 15 14 14 16 19 18 17 15T 12T 28 28 24 29 26 32 26 33 36 30 38T 25T Compound T-2541 CoC Table 1 (Concluded) IN VITRO ASSAYS WITII SALMONEI.I.A TYPIIIMURIUM T-2540 CoC and T-2541 CoC Metabolic Activation - + + + + + + Micrograms of Compound Addedj!Rr Plate 10 50 100 500 1,000t 5,000t 10 50 100 500 i,ooot 5,ooot TA1535 27 31 20 28 33 29 19 28 32 25 29 25 25 18 29 17 28 27 20 28 38 29 30 46 Histidine Revertants per TA1537 TA1538 12 7 26 17 45 15 15 1 66 12 6 17 6 12 9 17 20 12 21 13 13 15 17 21 30 29 17 19 36 32 25 1 28 26 19 29 1 .30 30 15 21 1 32 26 37 30 27 24 26 30 1 T, toxic. t The compound formed a precipitate at this concentration. Compound Metabolic Activation Negative Control DMSO + Positive Control Sodium azide - 9-Aminoacridine - 2-Nitrofluorene - 2-Anthramine - + + T-2540 CoC - - - - - - + + + + + + Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM T-2540 CoC and T-2541 CoC Micrograms of Compound Added 1)erPlate TA1535 21 20 7 21 Histidine Revertants pe TA1537 TA1538 5 10 28 9 16 15 1 13 28 3 1.0 50.0 5.0 1.0 1.0 2.5 2.5 10 50 100 500 1,000 5,000 10 50 100 500 1,000 5,000 505 380 19 24 221 256 20 38 25 28 33 17 29 20 21 16 T* 6T 17 13 19 16 10 19 16 4 18 5 14 15 442 390 56 131 158 68 7 14 7 14 12 3 12 5 T 1T 21 13 22 14 25 15 14 12 19 15 14 14 708 678 38 21 20 2 420 258 12 9 17 2 19 14 2 16 20 2 89 1 13 7 2 9T 5T I 15 17 3 14 15 2 17 18 2 7 20 2 14 23 2 18 16 1 Compound T-2541 CoC Table 2 (Concluded) IN VITRO ASSAYS WITH SALMONELTA TYPIIIMURIUM T-2540 CoC and T-2541 CoC Metabolic Activation - + + + + + + Micrograms of Compound Added 1)erPlate 10 50 100 500 i,.ooot 5,0001- 10 50 100 : 500 1 000 t 5,000t TA1535 29 21 26 28 34 24 21 28 20 26 15 19 7 15 12 15 18 15 9 8 14 14 13 14 llistidineRevertants p_g TA1537 TA1538 88 47 13 7 89 13 13 9 14 13 9 11 7 86 14 8 18 27 12 18 9 13 68 13 9 88 13 7 12 7 26 17 17 15 25 21 12 24 24 17 19 24 T, toxic tThe compound formed a precipitate at this concentration. IEOT tzl 8@T TTT IT!; T#IT' 9ZT 091 .L 19T 89T g[T I is OTT ZST 9L@T SZLT T9T TIET .166 @ZT 6@T gET J,9T 60T L6 SZT IL ILT 6ET EZT I IL 66 OET ET5;T 9L@T Z@T 09T 6Z 6Z TE 9f7 ICT 6Z 9z tz I .16 zi; 9c I lgz @T 6Z too L" Tg oc 6Z oz Ti 9E 6T TZ @z LE 1 19T zs oz 1 *IZT oz 9z LTS LS;L EE 9T OOTVI R6VJ, 93UTcl jed S3UVl39A9H 8UTPTISTH 0*9 OIT s;lo T*O 019 O'T 5;*o T'O 0*9 O'T S*O T*O 0*9 O'T S*O T*O O*T O'T jo3uDaTsaa uT punodmoo jo 819ITTTTTTH + + + + + + + + + + UOTIPATIOV z)TToqleiaN SHOIVDDISga DOD Tf7SZ-1 PUV DOD OVgZ-1 HHflSOcIXZ 11flOH-B--- NI KnIHflWlHd)U VIIZNOHQVS HIIM SKVSSV O'DJLIANI gtqel Table 4 IN VITRO ASSAYS WITH SACCRAROMYCES T-2540 CoC CEREVISIAE D3 Compound Negative Control DMSO Positive Control 1,2,3,4 Diepoxybutane T-2540 CoC Metabolic Activation + + + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (X 10 7) Percent 6.5 100 7.2 100 0.025 0.025 0.1 0.5 1.0 5.0 0.1 o.5 1.0 5.0 2.5 38 5.8 81 5.6 86 2.3 35 1.5 23 T T 7.7 107 3.5 49 1.1 15 T T -Mitotic Reco Per ml (X lo@-3) 3.5 3.5 608 928 5.0 2.0 3.0 T 6.0 4.o 3.0 T T, toxic. Table 5 IN VITRO ASSAYS WITH SACCHAROMYCES T-2540 CoC CEREVISIAE D3 Compound Negative Control DMSO Positive Control 1,2,3,4 Diepoxybutane T-2540 CoC 4 Metabolic Activation + + + + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (X 10 7) Percent 7.2 100 7.6 100 0.025 0.025 3.6 50 7.0 92 0.025 0.05 0.075 0.1 0.25 0.025 0.05 0.075 0.1 0.25 6.8 94 6.5 90 5.8 81 5.7 79 2.7 38 7.4 97 7.0 92 5.2 68 5.1 67 5.3 70 -Mitotic Reco Per ml (X 10-3) 2.0 2.5 923 1040 4.0 7.0 11.0 6.0 10.0 8.0 3.0 8.0 6.0 14.0 Table 6 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 T-2540 CoC Compound Negative Control DMSO Positive Control 1,2,3,4 Diepoxybutane T-2540 CoC co Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells per ml (x 10 Percent -Mitotic Reco Per ml (x io@-3) + 0.025 + 0.025 - 0.07 - 0.08 - 0.09 - 0.1 - 0.2 + 0.09 + 0.1 + 0.2 + 0.3 + 0.4 8.3 100 3.0 7.6 100 4.0 5.2 63 880 6.3 83 680 7.8 94 6.o 8.2 99 3.0 6.0 72 5.0 5.7 69 3.0 4.7 57 7.0 9.3 122 5.0 6.1 80 5.0 5.8 76 4.0 3.2 42 10 3.0 39 3.0 Table 7 IN VITRO ASSAYS WITH SACCHAROMYCES T-2541 Coc CEREVISIAE D3 Compound Negative Control DMSO Positive Control 1,2,3,4 Diepoxybutane T-2541 CoC Metabolic Activation + + - + + + + Percent Survivors Concentration Cells per ml (w/v or v/v) (x 10-7) Percent 0.025 0.025 6.5 100 7.2 100 2.5 38 5.8 81 0.1 0.5 i.o 5.0 0.1 0.5 1.0 5.0 5.0 77 2.3 35 T*. T T T 7.0 97 5.6 78 5.1 71 2.1 29 Mitotic Recc Per ml (x io-3) 3.5 3.5 608 928 1.0 4.0 T T 4.0 2.0 4.0 6.0 T, toxic. Table 8 IN VITRO ASSAYS WITH SACCHAROMYCES T-2541 CoC CEREVISIAE D3 Compound Negative Control DMSO Positive Control 1,2,3,4 Diepoxybutane T-2541 CoC 0 Metabolic Activation + + - + + + + + Percent Concentration (w/v or v/v) Survivors Cells per ml (X 10 7) Percent 0.025 0.025 7.2 100 7.6 100 3.6 50 7.0 92 0.025 0.05 0.075 0.1 0.25 0.25 0.5 0.75 1.0 2.5 5.5 76 5.5 76 5.1 71 5.7 79 2.7 38 5.5 72 5.1 67 3.6 47 3.1 41 T* T -Mitotic Recom Per ml (x 10-3) 2.0 2.5 923 1040 6.0 3.0 3.0 5.0 7.0 8.0 7.0 2.0 7.0 T T, toxic-. Table 9 IN VITRO ASSA-YS WITH SACCHAROMYCES CEREVISIAE D3 T-2541 CoC Compound Negative Control DMSO Positive Control 1,2,3,4-Diepoxybutane T-2541 CoC Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells per ml (x 10 7) Percent + 0.025 + 0.025 8.3 100 7.6 100 5.2 63 6.3 83 0.07 6.08 0.09 0.1 0.2 + 0.2 + o.4 + 0.6 + 0.8 + 1.0 8.2 99 7.7 93 6.0 72 6.4 77 5.2 63 6.5 86 8.7 114 6.2 82 5.4 71 4.4 58 Mitotic Rec Per ml (x 10 3.0 4.0 880 680 5.0 3.0 6.0 8.0 6.0 4.0 5.0 6.0 11 5.0 REFERENCES 1. 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