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0 13 r) IN VITRO MICROBIOLOGICAL MUTAGENICITY OF 3H COMPANY'S COMPOUND T-3609 ASSAYS Final Report September 1984 By: Debra E. Verbaere, Microbiologist Microbial Genetics Department and lz4e@ Edward S. Riecio, Assistant Director 14icrobial 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 Jon B. Reid, Director Toxicology Laboratory 0 PJ47.@, W. A. Skinner, Vice President Life Sciences Division lntemational tm; (!)& 333 Ravenswood Ave. o Menlo Park,CA94025 (4151326-6206 o TWX: 910-373-20469 Telex:334-486 SUMHARY 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 TAIOO of the bacterium Salmonella typhimurium. Compound T-3609 was also screened for recombinogenic activity in the yeast Saccharomyces carevisiae D3 assay. Both assays were performed in the presence and absence of a rat-liver metabolic activation system. Compound T-3609 was reproducibly nonmut agenic and nonrecombinogenic when tested according to these procedures* CONTENTS SUMMARY.* ...............4..................... INTRODUCTION ......................... 1 MATERIALS .........................o ...... 3 )MTHODS.O .......o.o.o ...... *.o.........o...o. 5 RESULTS AND DISCUSSION .............*.*........o... 11 TABLES Table 1 ...........0...........0...0 ........... 12 Table 2 .......v.............e....*.......... 13 Table 3 .........0...0 .......................1.4 Table 4 .........4*..............*............. 15 INTRODUCTION SRI International examined 3M Company's Compound T-3609 for mutagenicity 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 tested for recombinogenic activity in the yeast Saccharomyces cerevisiae 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 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 60Z 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. 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 MATERIALS 9 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 1. typhimurium; D3 for S. cerevisiae - Source: Dr. Bruce Ames, Universi@y of California, Berkeley, for the Salmonella; Dr. P. X. Zimmermann, W. Germany, for the yeast Metabolic Activaton Aroclor 1254-induced,,rat 3.iverS-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 o 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 3 2-Nitrofluorene, c&s No. 607-57-8 Manufacturer: Aldrich Chemical Co., Milwaukee, WI Sodium Azide, CAS No. 26628-22-8 Manufacturer: Difco Laboratories, Detroit, MI 1,2,3,4-Diepoxybutane, CAS No. 1464-53-5 Manufacturer: Pfaltz and Bauer, Stamford, CT Sterigmatocystin, CAS No. 10048-13-2 Manufacturer: Calbiochem,' La Jolla, CA Counters Used - New Brunswick Scientific biotran 110 Automated Colony Counter, Model Clll, SRI No. 0030 0151 00 - New Brunswick Scientific BactronicO Colony Counter, Model C110, SRI No. 0013 0788 00 4 METHODS Salmonella typhimurium Strains T-Al535, TA1537, TA1538, TA98, and TA100 The Salmonella tvohimurium 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. tvvhiinuriumstrains 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 genes involved in the synthesis of the vitamin biotin (ii-0) and in the repair of ultraviolet (uv)-induced DNA damage @@rB). 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 matagens that cause base-pair substitutions. Strain TA100 is derived from TA1535 by the introduction of the resistance transfer factor, plasmid pKH101. This plasmid is believed to cause an increase in error-prone DNA repair that leads to many more mutations for a 5 given dose of most mutagens. In addition, plasmid pKK101 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 Bl, and 7,12-dimethylbenz(a)anthracene]. Strains TA1537 and T&1538 are reverted by many frameshift autagens. Strain TA98 is derived from TA1538 by the addition of the plasmid pKM101, 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 -80*C in 1-ul aliquoto 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 (his. rfa. uvrb, bio) and for the presence of the plasmid. For each experiment, the 1--mlfrozen 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 37'C, 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 (eog*, 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. tvphimurium 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 fir-thday, the rats are killed and the liver homogenate is prepared as follows. 6 The livers are removed aseptically and placed in a preveighed sterile glass beaker. The organ veight is determined, and all subsequent operations are conducted in an ice bath. The livers are vaihed 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 vet 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 -80*C. The metabolic activation mixture for each experiment consists of, for 50 ml: 0 5.0 al of S-9 fraction 0 1.0 al of Mgcl2 (0.4 M) and KC1 (1.65 M) 0 0.25 ml of g@ucose-6-phosphate (1 M) 0 2.0 al of NADP (0.1 M) 0 25.0 ml of sodium phosphate buffer (0.2 M, pE 7.4) 0 16.75 al of sterile E20. The amount of S-9 fraction delivered to each plate is 50 iLl. Plate Incorporation Assay Prior to testing, the test article is seriallydiluted 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 lover 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 aliquote are used. All assays are repeated at least once on a separate day. The plate incorporation assay is performed in the folloving way. To a sterile 13 x 100-mm test tube placed in a 43*C heating block we add: (1) 2.00 ml of 0.6% agar containing 0.6% NaCl, 0.05 uM biotin, and 0.05 uM histidine 8 (2) 0.05 ml of indicator organisms (about 10 bacteria) 7 (3) 0.05 al of a solutionof the test article (4) 0.50 ml of metabolic activationmixture (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 37*C. The number of hist revertant colonies is counted using a SioTran Il 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 nutagens using steps (1), (2), (3),and (4): 0 Sodium azide for the base-pair substitution mutants TA1535 and TA100. 0 9-Aminoacridine for the frameshifr mutant TA1537. 0 2-Nitrofluorene for the frameshift mutants TA1538 and TA98. a 2-Anthramine for all tester strains, in the presence of metabolic activation. 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 organism 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 0.05% MgS041o 0.15% KH2po4t 0.45% (NH4)2so4t 0.35% peptone, 8 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 108 cells/al in 67 =14phosphate buffer (pH 7.4). To a sterile test tube are added: 0 1.00 ml of the resuspended culture 0 0.50 ml of either the metabolic activation mixture or buffer 0 0.20 ml of the test chemical 0 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 for 4 hours on a roller drum. The sample is then diluted serially in sterile physiologic saline, and 0.2 ml of the 10-5 and 10-3 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 lr5 dilution. The plates are incubated for 3 days at 300C, followed by 1 day 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. 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--independeutcolonies per plate. The results of the S. carevisiae D3 assay are tabulated by calculating the number of mitotic recombinants per 105 survivors. A positive response in 9 this assayis 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 105 survivors. 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 carcinogens. Proc. Nat. Acad. Sci. USA 69, 3128-3132 (1972). Ames, B. N., W. E. Darston, E. Yamasaki, and P. D. Lee. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. USA 709 2281-2285 (1973). Ames, B. N., P. D. Lee, and W. E. Durston. An improved bacterial test system for the detection and classification of uttitagenasnd carcinogens. -Proe. Rat. Acad. Sci. USA 70, 782-786 (1973). Ames, B. N.9 J. McCann, and E. Yamasaki. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian,--microsomemutagenicity test. Mutat. Res. 3 347-364 (1975). Brusick, D. J., and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Pe'rspect. 6 83-86 (1973). Kier, L. D., E. Yamazaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat. Acad. Sci. USA 71j 4159-4163 (1974). McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA.L2, 97-983 (1975). McCann, J., and B. M. 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. X.,,and R. Schwaier. Induction of mitotic gene conversion with nitrous acid, 1-methyl-3-nitro-l-nitrosoguanidineand other alkylating agents in,Saccharomyces cerevisiae. Mol. Gen. Genet. 100, 63-76 (1967). 10 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, .andTA100. 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 gg/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 105 survivors were observed in either assay. In conclusion, Compound T-3609 was reproducibly nonmutagenic and nonrecombinogerlic when tested according to these procedures. Table I IN VITRO ASSAYS WITH SALMONELLA TYPHIMJRIUH COMPOUND T-3609 Experiment Date: 26 July 1984 Compound Metabolic Activation Compound Added per Plate Negative Control Acetone + 50 pl 50 Positive Controls Sodium Azide - 9-Aminoacridine - 2-Nitrofluorene - 2-Anthramine - + + 1 pg 50 5 1 1 2.5 2.5 TA1535 Histidine Revertants per Plate TA1537 -TA1538 TA98 TAl 22 19 5 4 11 13 26 19 140 9 12 10 6 20 23 25 41 123 497 491 21 25 ill 115 429 256 275 1005 14 111 905 567 533 16 19 28 152 119 96 85 339 77 50 59 Compound T-3609 - 10 pg 24 28 6 14 17 19 27 19 151 - 50 40 27 9 7 12 27 37 151 - 100 25 26 67 13 9 31 @9 149 - 500 37 26 10 3 8 14 18 27 144 - 1000 27 21 6 9 13 7 21 19 150 - 5000 27 22 76 5 12 31 24 126 + 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 27 21 34 31 132 25 19 35 34 169 21 21 33 42 139 18 25 34 37 148 27 21 36 38 135 Table 2 IN VITRO ASSAYS WITH SALMONELIA TYPHIMURIUM COMPOUND T-@609 Experiment Date: 3 August 1984 Compound Metabolic Activation Compound Added per Plate Negative Control Acetone + 50 pl 50 Positive Controls Sodium Azide - 9-Aminoacridine - 2-Nitrofluorene - 2-AnthrAmine - + + Compound T-3609 - - - - - - 1 tLg 50 5 1 1 2.5 2.5 10 pg 50 100 500 1000 5000 + 10 + 50 + 100 + 500 + 1000 + 5000 TA1535 Histidine Revertants per Plate TA1537 TA1538 TA98 TA10 25 30 8 10 8 7 12 11 8 17 24 27 130 19 20 40 34 142 502 472 18 19 117 124 36 32 39 32 19 3@ 25 19 28 31 38 31 13 13 15 9 16 15 8 13 12 12 96 503 254 281 1079 1074 696 587 15 15 119 116 20 25 141 97 102 401 .11 13 52 60 88 9 11 11 8 6 3 75 7 11 16 16 27 29 167 13 12 30 36 150 7 20 33 35 162 15 17 31 36 155 12 16 36 31 169 9 12 20 29 167 9 13 11 9 14 12 77 66 86 28 21 39 42 162 20 24 39 42 160 24 19 47 44 167 26 19 37 37 166 19 24 42 43 137 22 27 36 41 147 Table 3 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-3609 Experiment Date: 27 July 1984 Compound Mitotic Percent Surviving Recombinants Re Metabolic Concentration Cells pe ml Survivors per Activation (w/v) (x 10-Y (x 10-Mi s Negative Control Acetone + 4.2 100 5 4.o 100 5 Positive Controls 1,2,3,4-Diepoxybutane Sterigmatocystin + 0.025 0.005 0.005 4.7 100 4.8 100 4.8 100 881 1 4 158 Compound T-3609 - 0.05 4.4 100 7 - 0.1 4.4 100 6 - 0.5 4.6 100 7 - 1 4.6 100 4 - 5 4.6 100 6 + 0.05 4.4 100 4 + 0.1 4.1 100 6 + 0.5 4.6 100 5 + 1 4.8 100 6 + 5 4.1 100 3 Calculations are expressed using two significant figures. Table 4 IN VITRO ASSAYS WITH SACCRAROMYCES CEREVISIAE D3 COMPOUND T-3609 Experiment Date: 10 August 1984 Compound Metabolic Activation Percent Surviving Concentration cells pe; al (w/v) (x lo-,) Survivors Mitotic Recombinants per (x 10 mi Reco p Sur Negative Controls Acetone + Positive Controls 1,2,3,4-Diepoxybutane Sterigmatocystin Ln + Compound T-3609 - - - - - 0.025 0.005 0.005 0.05 0.1 0.5 1 5 3.9 100 3.7 100 4.7 100 4.0 100 4.1 100 4.5 100 4.3 100 4.6 100 4.3 100 4.5 100 6 1 5 1 933 200 4 1 150 37 8 1 4 8 1 6 1 7 1 + 0.05 + 0.1 0.5 + 1 + 5 4.1 100 4.3 100 4.6 100 4.6 100 4.7 100 5 1 5 1 5 1 5 1 4 Calculations are expressed using two significant figures.