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EI IN VITRO MICROBIOLOGICAL 6F- TM-COMPAW'S COMPOUND MUTAGENICITY T-3752 Final Report ASSAYS jUL 2 2 lv',i99g55 (@3 1-2r June 1985 By: Kathleen Okamoto, Microbiologist Microbial Genetics Department and dward S. Riccio, Assistant Director Microbial Genetics Department Prepared for: 3M Company Medical.Department General Offices, 3M St. Paul, MN 55144 Center- Attention: Janine R. Gleason Toxicology Specialist SRI Project LSC-3145 Approved by: Kristien E. Mortelman8, Study Director Microbial Gene" Department ()J noyn Zx.iBJ0cr.@iRdeidD D ,iercte'ctor oxicology L@aabbloorrarattoory W. A. Skinner, Vice President Life Sciences Division lnltemational 333 Ravenswood Ave. o Menlo Park,CA94025 l4l5j326-6200 o TWX: 910-373-2046 o Telex: 334-486 SUMMARY SRI International examined 3M Company's Compound T-3752 for mutagenic activity in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98, and U100 of the bacterium Salmonella typhimurium. Compound T-37@2 was also screened for recoubirogenic activity in the yeast Saccharomyces cerevisiae D3 assay. Both assays vere performed in the presence and absence of a rat-liver metabolic activation system. All tests were performed in compliance'with the United States Food and Drug Administration (FDA) Good Laboratory Practice Standards. Compound T-3752 was reproducibly nomutagenic when tested according to these procedures. and nonrecombinogenic CONTENTS QUALITY ASSURANCE STATEMENT ....................... iv INTRODUCTION.O..*..**.* ... MATERIALS*ooo*oooo**ooeooooooooooeeoo*oooooe...Oo. 3 5 RESULTS AND DISCUSSION ............ 12 TABLES Table 13 Table 14 Table 15 Table 16 QUALITY ASSURANCE UNIT Final Report Statement SRI Internationalassures the quality and integrityof this study, In Vitro Microbiological Mutagenecity Assays Of Compound T-3752, for the 3M Company. Inspections and audits were performed during different phases of the study, and the Study Dire@tor and SRI management were notifiedof the findingsof the Quality Assurance Unit. This report accurately describes the methods and standard operating procedures of the study. Any deviationsfrom the approved protocol were made with the proper authorizationand documentation. Any deviations from standard operating procedures were documented. nager of Regulatory Affairs ancr'QualityAssurance Date iv IliTRODUCTION SRI International examined 3M Company's Compound T-3752 for mutagenicity in the standard Ames Salmonella/microsome assay with strains TA1535, TA15379 TA15389 TA98, and TA100 of the bacterium Salmonella typbimuirium. Compound T-3752 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. 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 1002 correlation with carcinogenicity investigations in animals, neither a positive nor a negative response conclusively proves that a chemical is carcinogenic or noncarcinogenic to 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 organism 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 lawn growth, 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 solvent-treated controls, we conclude that the exposure.of the cells to the compound induces mitotic recoirbination. If this event is dose-related, the observation is termed a positive response. The assays with Compound T-3752 were begun on 24 May 1985 and testing was completed on 14 June 1985. Copies of the final report will be kept in our files (Building M, Room 213) and in SRI's Records Center. The raw data will be retained in Building 205, Room 13, for one year after the labora@tory notebook has been filled and then will be stored in SRI's Records Center. All that remains of Compound T-3752 will be kept for six months in our chemical storage room (Building M, Room 21-7)and then returned to 3H Company. 2 MATERIALS Test Article - Name: T-3752 - Date Received: 15 May 1985 - Description: Amber waxy solid - Storage Conditions: Stored at room temperature in a secondary container - Special Testing Conditions: None - Stability: Assured by Sponsor Indicator Organisms - Species Salmonella tvvhimurium LT2; Saccharomyces cerevisiae - Strains: TA1535, TA1537, TA1538i TA98, and TA100 for L- typhimurium; D3 for S. cerevisiae - Source: Dr. Bruce Ames, University of California, Berkeley, for the Salmonella. Dr. F. K. Zimmermann, W. Germany, for the Sacchar Metabolic Activation Aroclor 1254-induced, rat liver S-9; SRI Batch F-5; - 31.5 mg/ml protein Animal Supplier: Simonsen Laboratories, Gilroy, California Negative (Solvent/Diluent)Control Material Dimethyloulfoxide (DMSO), CAS No. 67-68-5 Date Opened: 3 and 8 May and 3 June 1985 Expiration Date: 3 and 8 May and 3 June 1986, respectively Manufacturer: American Scientific Products, McGraw Park, IL Purity: 0.12%-H 0 (for all) Lot No.: 4948 P@V& (for all) 3 t 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 2-Nitrofluorene, CAS 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 ND. 1464-53-5 Manufacturer: Pfaltz and Bauer, Stamford, CT Sterigmatocystin, CAS No. 10048-13-2 Manufacturer: Aldrich Chemical Co., Milwaukee, Wl Counters Used - New Brunswick Scientific BioTran 110 Automated Colony Counter, Model Clll, SRI Nos. 0030 6126 00, 0030 0151 00, and 0012 3318 00 - New Brunswick Scientific BactronicO Colony Counter, Model Clio, SRI Nos. 0030 1471 00, 0012 3108 00, and 0013 0788 00 4 METHODS Salmonella tyldhimurium Strains TA1535, TA1537, TA1538, TAVT$. and TALOU The Salmonella tvphimurium strains used at SRI are all histidine auxotropho by virtue of mutations in the histidine operon. When these histidine-dependent cells are grown on minimal medium agar plates corrtaining 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 autagenesis 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 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 (2!-1-0) and in the repair of ultraviolet (uv)-induced DNA damage (MrB). 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 autagens that cause base-pair substitutions. Strain TA100 is derived from TA1535 by the introduction of the resistance transfer factor, plagmid pM4101. 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 pKH101 confers resistance to the antibiotic ampicillin, which is a convenient marker to detect 5 the presence of the plasmid in the cell. The presence of this plasmid also makes strain TAIOO sensitive to some frameshift mutagens [e.g., ICR191, benzo(a)pyrene, aflatoxin Bl, and 7,12-dimethylbenz(a)anthracene]. Strains TA1537 and TA1538 are reverted by many frameshift mutagens. Strain T&98 is derived from T&1538 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-ml aliquots containing about 10 cells. New frozen stock cultures are made every three months from single colony isolates that have been checked for their genotypic characteristics (As, Lfa, uvrb. lio) and for the presence of the plasmid. For each experiment, the frozen 1--mlcell cultures are allowed to thaw at room temperature before inoculation in 50 ul 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-Stimul-atedMetabolic Activation System Some carcinogenic chemicals (e.g., of the aromatic amine type or the polycyr-lichydrocarbon type) are inactive unless they are metabolized to active forms. In animals and uum, 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 very potent mutagens in the S. typhimurium 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 fifth day, the rats are killed and the liver homogenate is prepared as follows. 6 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, minced with sterile surgical scissors in three volumes of 0.15 K KC1 (3 ml/g of wet organ), and homogenized with a Potter-Elvehjem apparatus. The homogenate is cetrtrifuged for 10 minutes at 9000 x.L, 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: 9 5.0 ml of S-9 fraction 0 1.0 ml of Ngcl2 (0.4 M) and KC1 (1.65 M) o 0.25 ul of glucose-6-phosphate (i M) o 2.0 ml of NADP (0.1 M) o 25.0 ml of sodium phosphate buffer (0.2 M, pH 7.4) o 16.75 ml of sterile R2 The amount of S-9 fraction delivered to each plate is 50 Plate Incorporation Assay Prior to testing,the test article is seriallydiluted from an ini- tial stock. Generally, a preliminary experiment is conducted to find a suitable dose range for testing. 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 lower upper limit. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 pl/plate. When liquids are tested, occasionally the sample is not diluted and various aliquots are used. All assays are repeated at least once on a separate day. The plate incorporation assay is performed in the following way. To a sterile 13 x 100-in test tube placed in a 43*C heating block we add: 7 (1) 2.00 ml of 0.6% agar containing 0.6% NaCl, 0.05 mM biotin, and 0.05 mM histidine (2) 0.05 ml of indicator organisms (about 108 bacteria) (3) 0.05 ml of a solution of the test article (4) 0.50 ml of metabolic activation mixture (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 his+ revertant colonies is counted using a BioTran Il automated colony counter when possible. When accurate counts cannot be obtained (e.g., because of precipitate), the plates are counted mmually using an electric probe colony counter. Concurrent sterility, negative (solvent/diluent), and positive con-trols 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/diluent used for the test article, if appropriate. For positive controls, we test each bacterial culture using steps (1)@,(2), (3), and (4)-vith the folloving mutagens: o Sodium azide for the base-pair substitution mutants TA1535 and TA100 o 9-Aminoacridine for the frameshift mutant TA1537 o 2-Nitrofluorene for the fr-eshift mutants TA1538 and TA98 9 2-Anthrenine for all tester strains, in the presence of metabolic activation. We rout inely check for true revertant (his+) colonies by replica plating from the parent to minimal glucose agar plates supplemented with biotin. Criteria for Interpretation Positive. A test article is considered a mutagen when it produces a reproducible, dose-related increase in the number-of revertants in one or more strains. This increase should occur for at least three dose levelso Negative? A test article is considered a noroutagen when no doserelated increase in the number of revertants is observed in at least two independent experiments. The maximum dose level tested for nontoxic 8 compounds is 10 mg/plate (unless dictated otherwise by the sponsor or by solubility problems). For toxic compounds, only the highest dose level tested should show evidence of toxicity. Inconclusive. When a test article cannot be identified clearly as a mutagen or nomutagen in the standard plate assay, the results are classified as inconclusive. Saccbaromyces cerevisiae D3 The yeast S. cerevisiae D3 is a diploid microorganism heterozygous for a mutation leading to a defective enyzme in the adenint--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 heteroxygotes by mitotic recombination. The frequency of this recombinational event may be increased by incubating the organisms with various carcinogenic or recombinogenic agents. The recombinogenic -activityof a.compound or its metabolite is determined frm the number of red-pigmented colonies appearing on test plates. A culture of S. cerevisiae is stored at 4*C. For each experiment, broth containing 0.05% MGSO 49 0-15% KH2po4' 0.45% M4)2SO41 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 108 cells/al in 67 mm phosphate buffer (pH 7.4). To a sterile test tube are added: o 1.0 ml of the resuspended culture 0 0.5 ml of either the metabolic activation mixture or buffer 9 0.2 ml of the test chemical 9 0.3 ml of buffer* Several doses of the test chemical are tested (up to 5% w/v or v/v) in each experiment, and appropriate solvent/diluent controls are included. 1,2:3.4-Diepoxybutane without metabolic activation and sterigmatoeystin with activation are used as positive controls. 9 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 10-5 dilirtion. The plates are incubated for 3 days at 30*C, followed by at least 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..theplates of the 10-5 dilution. Criteria for Interpretation Positivi.. A positive response In this assay is indicated by a dose- related increase of more than threefold in the absolute number of mitotic recombinants per'silliliter-andin the relative number of mitotic recombinants per 105 survivors. Neitative. When no reproducible recombinogenic activity is obtained in any of the assays performed, the test results are considered to be negative. Inconclusive. When a test article cannot be identified clearly as causing a positive or a negative response, the results are classified as inconclusive. Statistical Analysis No statistical analysis is performed for any of the assays. The results of the plate incorporation assay are a tabulation of the number of colonies appearing an the plates. The results of the S. cerevisiae D3 assay are tabulated after calculating the number of mitotic recombinants per 105 survivors. All calculations are expressed with two significant digits. 10 References Ames, E. G. Gurney, J. A. Miller, and B. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylamino- fluorene and other aromatic amine carcinogens. Proc. Natl. Acad. Sci. USA 69, 3128-3132 (1972). Ames, B. N., mutagens: A and bacteria (1973). W. E. Durston, E. Yamasaki, and F. D. I.,@-e.Carcinogens are simple test system combining liver hamogenates for activation for detection. Proc. Natl. Acad. Sci. USA.LO, 2281-2285 Ames, B. N., F. D. Lee, and W. E. Durston. An Improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. USA.LO, 782-786 (1973). Ames, B. N., J. McCann, and E. Yamasaki. Methods for detecting carcinogens and matagens with the Salmonella/ua=alian.--microsome autagenicity test. Mutat. Res. 31p 347-364 (1975). Brusick, D. J., and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Realth Perepect. 6. 83-86 (1973). Kier, L. D., E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Natl. Acad. Sci. US& 71 4159 -4163 (1974). McCann, J., E. Choi, E. Yan4saki, and B. N. Ames. Detection of carcinogens as mutagens In the Salmonella/microsome test: Assay of 300 cheiricals. Proc. Natl. Acad. Sci. USA 12, 5135-5139 (1975). McCarm, J., and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion. Proe. Natl. Acad. Sci. USA 73, 950-954 (1976). Mortelmens, 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. K., and R. Schvaier. Induction of mitotic gene conversion with nitrous acid, 1-aethyl-3-nitro-l-nitrosoguanidine and other alkylating agents in Saccharomyces cerevisiae. Mol. Gen. Genet. 100, 63-76 (1967). RESULTS AND DISCUSSION 3m Company's Compound T-3752 was screened for mutagenic activity in the Ames Salmonella/microsome in vitro mutagenicity assay using the five standard strains of Salmonella tvphimurium: TA1535, T&1537, TA1538, TA98, and TA100. The assays were performed in duplicate, using three plates per dose, both in the presence and absence of a rat-liver metabolic activation system. DNSO was used as the solvent. The microbial outagenicity testing of this sample was performed twice at dose levels ranging from 10 to 5000 pg/plate.(Tables 1 and 2). No increases in the number of revertant colonies over the spontaneous background were observed under any of the assay conditions used. Background lawn thinning was observed in the first assay only with strain TA1537 on all of the plates at 5000 pa/plate and on two of the three plates at 1000 pg/plate without activation. A light precipitate was noted at 5000 pg/plate; therefore, these plates were hand-counted. The results are presented in Tables 1 and 2. Compound T-3752 was also screened for recombinogenic activity in the yeast Saccharomycei cerevisiae D3 assay for mitotic recombination. The assays ver e performed twice on separate days, both in the presence@and absence of a rat-liver metabolic activation system. DMSO was used as the solvent. Since no toxicity was seen in the range-finding assay, the first experiment was performed at dose levels of 0.05, 0.1, 0.5, 1.0 and 5.0% (v/v). No increases in the number of mitotic recombinants were observed. The confirmatory assay was performed under conditions identical to those used in the initial assay. Again, no recombinogenic response was observed. The results of these two experiments are presented in Tables 3 and 4. In conclusion, Compound T-3752 was reproducibly noroutagenic and nourecombinogenic when tested according to the procedures outlined in this report. 12 Table 1 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3752 Experiment Date: 28 May 1985 Compound Metabolic Activation Compound Added per Plate TA1535 Histidine Revertants per Pl TA1537 TA1538 T Negative Control D14SO + 50 pi 19 22 27 6 7 3 22 29 15 19 .50 8 16 6 3 9 10 23 17 39 31 Positive Controls Sodium azide - 9-Aminoacridine - 2-Nitrofluorene - 2-Anthramine - + + Compound T-3752 - + + + + + + 1 pg 372 280 386 50 - 175 165 170 5 1686 1329 1284 818 1 18 33 28 19 1 230 214 808 97 2.5 28 18 17 .13 5 11 2.5 158 177 61 31 33 25 10 pg 17 16 24 10 3 8 24 18 17 20 50 10 9 15 5 5 6 9 20 28 14 100 15 14 22 5 7 6 14 15 20 13 500 12 11 21 4 6 7 23 17 27 17 1000 9 15 17 Bt 2t 13 14 15 21 19 5000* 14 11 18 5t 7t. 8t 15 24 15 21 10 50 100 500 1000 5000* 13 7 17 13 8 5 26 34 33 41 12 8 3 13 12 7 22 30 31 39 14 5 13 6 5 6 35 18 36 31 10 3 7 4 9 6 14 31 18 36 13 13 8 9 11 8 28 23 22 32 8 6 9 7t 5t 9t 28 35 27 43 *Experiment performed on 14 June 1985. tbackground lawn thinning. *Precipitated at this dose level; hand-counted. Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3752 Experiment Date: 6 June 1985 Compound Metabolic Activation Compound Added per Plate TA1535 Histidine Revertants per Plat TA1537 TA1538* TA9 Negative Control DMSO + 50 pl 50 31 37 27 18 14 13 3 10 3 998 16 15 18 20 17 30 24 23 38 24 Positive Controls Sodium azide 9-Aminoacridine 2-Nitrofluorene 2-Anthramine + + Compound T-3752 - - - - - - + + + + + + I pg 50 5 1 1 2.5 2.5 10 pg 50 100 500 1000 5000t 10 50 100 500 1000 5000t 562 586 621 456 307 390 1111 1017 842 675 452 24 18 20 23 17 202 160 256 109 108 33 30 38 13 13 12 214 182 177 51 59 49 42 24 34, 32 33 36 33 27 45 35 42 33 31 33 31 39 24 31 9 10 12 3 10 11 13 9 4 6 98 6 8 10 11 7 8 21 17 11 15 14 14 17 17 24 30 15 13 8 28 38 19 11 17 23 18 7 13 12 10 25 12 16 15 12 14 16 11 10 10 13 17 10 11 20 17 16 12 12 15 9 18 18 18 13 7 12 9 7 13 12 3 17 976 10 12 11 15 11 18 18 16 19 25 20 30 17 32 22 18 23 24 36 22 24 31 32 17 24 27 22 22 18 38 29 37 35 33 28 17 *Experiment performed on 14 June 1985. tprecipitated at this dose le*vel;hand-counted. Table 3 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 C014POUND T-3752 Experiment Date: 24 May 1985 Compound Metabolic Activation Negative Control DMSO + Positive Controls 1,2:3,4-@Diepoxybutane Sterigmatoeystin + Compound T-3752 - - - - + + + + + Percent Concentration (w/v) 0.025 0.005 0.005 0.05 0.1 0.5 1.0 5.0 0.05 0.1 0.5 1.0 5.0 Surviving Cells per ml 10-17)-- Survivors (z) 7.3 100 7.2 100 7.1 91 6.8 93 7.4 100 7.2 99 7*1 97 6.9 95 7.1 97 7.6 100 7.8 100 6.4 89 7.2 100 6.0 83 8.0 100 Mitotic Recombinants per ml (x io-3) 4.5 7.5 1331 2 303 9 6 7 11 5 7 9 8 6 7 *All calculations are expressed.to two significantfigures. Table 4 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-37.52 Experiment Date: 7 June 1985- Compound_ Negative Control DMSO Metabolic Activation Percent Concentration (W/V) Positive Controls 1,2:3,4-Diepoxybutane Sterigmatocystin + Compound T-3752 - + + + + + 0.025 0.005 0.005 0.05 0.1 0.5 1.0 5.0 0.05 0.1 0.5 1.0 5.0 Surviving Cells per ml (x 10-7) 7.3 6.9 7.3 7.3 7.3, 7.5 7.0 6.9 6.9 7.5 7.2 7.6 7.1 7.3 7.7 Survivors (Z) 100 100 100 100 100 100 96 95 95 100 100 100 100 100 100 mitotic Recombinants per al (x 10- 3) 8 10 1512 8 434 8 9 13 6 6. 6 7 9 11 11 *All calculations are expressed to two significant figures.