Document V34NX4j7x4z1w2EmOzrQddz4w

AfUL26 - 06?lf- . IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY'S COMPOUND T-365I Final Report February 1985 By: Kathleen Okamoto, Microbiologist Microbial Genetics Department and Edvard S. Riccio, Assistant Director Microbial Genetics Department Prepared for: 3M Company Medical Department General O ffices, 3M Center S t . Paul, MN 55144 Attention: William C. McCormick Toxicology Specialist SRI Project LSC-3145 Approved by: Kristien E. Mortelmans, Director Microbial Genetics Department -Z7T. J , .Q u J ffr ) Jon B. Reid, Director Toxicology Laboratory (j X ..Ck- , W. A. Skinner, Vice President Life Sciences Division 333 Ravenswood Ave. M enlo Park, CA 94025 415 326-6200 TWX: 910-373-2046 Telex: 334-486 000073 SUMMARY SRI International examined 3M Company's Compound T-3651 for mutagenic activity in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Sal monella typhimurlum. Compound T-3651 was also screened for recombinogenic activity in the yeast Saccharomyces cerevlsiae D3 assay. All assays were performed in the presence and absence of a rat-liver metabolic activation system. Compound T-3651 wa6 reproducibly nonmutagenic and nonrecombinogenlc when tested according to these procedures. ii 000074 CONTENTS SUMMARY.................................................................................. 11 INTRODUCTION........................................................................ 1 MATERIALS............................................................................... 3 METHODS.................................................................................. 5 RESULTS AND DISCUSSION..................................................... 12 TABLES Table 1......................................................................... 13 Table 2 ........................................................................ 14 Table 3 ......................................................................... 15 Table 4 ........................................................................ 16 111 000075 INTRODUCTION SRI International examined 3M Company's Compound T-3651 for mutagenicity in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98, and TA.100 of the bacterium Salmonella typhlmurlum. Compound T-3651 was also tested for recombinogenic activity in the yeast Saccharomyces cerevislae D3 assay. An Aroclor 1254stlmulated, 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_. typhlmurlum has proven to be 80 to 902 reliable in detecting carcinogens as mutagens, and it has about the same r e l ia b ilit y in identifying chemicals that are not carcinogenic. The assay procedure with S_. cerevislae is about 602 reliable in detecting carcino gens 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 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 c ell divisions in the presence of the test chemical, the test is semiquantltatlve 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. 1 000076 The test chemicals are assayed at several dose levels within a non toxic dose range--with the exception of the highest dose le v e l, which sometimes exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial lawn growth, formation of pinpoint colonies consisting of surviving c e lls, and a decrease in the number of revertant colonies below the spontaneous background. A chemical is considered a mutagen in the Salmonella assay i f it e lic its 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, result ing 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 Z k illin g . Any concentration that induces 90Z killing is considered toxic. When the number of genetically altered colonies per m illiliter (y ie ld ) and the ratio of altered colonies to survivors (frequency) from the treated cells are unequivocally larger than those of the solvent-treated controls, we con clude that the exposure of the cells to the compound Induces mitotic recom bination. If this event is dose-related, the observation is termed a positive response. 2 000077 MATERIALS Test Articles - Names: T-3651 - Date Received: 28 November 1984 - Description: Amber liquid (foams at high concentrations) - Storage Conditions: Room temperature - Special Testing Conditions: None - Stability: Assured by Sponsor Indicator Organisms - Species: - Strains: - Source: Salmonella typhirnurium LT2; Saccharomyces cerevislae TA1535, TA1537, TA1538, TA98, and TA100 for S_. typhirnurium; D3 for S_. cerevisiae Dr. Bruce Ames, University of California, Berkeley, for the Salmonella; D r. F. K. Zimmermann, V . Germany, for the yeast Metabolic Activation Aroclor 1254-induced, rat liver S-9; SRI Batch F-4; ~ 2 6 .5 mg/ml protein Negative (Diluent) Control Material Sterile water Positive Control Chemicals 9-Aminoacridine, CAS No. 90-45-9 Manufacturer: Pfaltz and Bauer, Stamford, CT 3 000078 2-Anthr amine, CAS No. 613-13-8 Manufacturer: Sigma Chemical C o., St. Louis, MO 2-Nitrofluorene, CAS No. 607-57-8 Manufacturer: Aldrich Chemical C o., Milwaukee, WI Sodium A zide, 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 Sterlgmatocystln, CAS No. 10048-13-2 Manufacturer: Aldrich Chemical C o., Milwaukee, WI Counters Used - New Brunswick Scientific BioTran II Automated Colony Counter, Model C lll, SRI Nos. 0030 6126 00 and 0012 3318 00 - New Brunswick Scientific Bactronic Colony Counter, Model C110, SRI Nos. 0013 0788 00 and 0030 1471 00 4 000079 METHODS Salmonella typhimurlun Strains TA1535, TA.1537, TA1538. 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 agar plates con taining a trace of histidine, only those cells that revert to histidine independence (his~*~) are able to form colonies. The small amount of his tidine allows all the plated bacteria to undergo a few divisio ns; 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 >_. typhimurium strains from Dr. Bruce Ames of the University o f 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 (bio) and in the repair of ultraviolet (uv)-induced DNA damage ( uvrB) . 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 mutagens that cause base-pair substitutions. Strain TA100 is derived from TA1535 by the introduction of the resistance transfer factor, plasmid pKMIOl. 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 pKMIOl confers resis- 5 0000S0 tance to the antibiotic am picillln, which is a convenient marker to detect the presence of the plasmid in the c e ll. The presence of this plasmid also makes strain TA100 sensitive to some frameshift mutagens [ e .g . , ICR-191, benzo(a)pyrene, a flat ox in B^, and 7 , 12-dimethylbenz( a) anthracene]. Strains TA1537 and TA1538 are reverted by many frameshift mutagens. Strain TA98 is derived from TA.1538 by the addition of the plasmid pKMIOl, which makes it more sensitive to some mutagenic agents. A ll indicator strains are kept frozen in nutrient broth supplemented 9 with 10Z sterile glycerol at -80C in 1-ml aliquots containing about 10 c e l l s . New frozen stock cultures are made every three months from single colony Isolates that have been checked for their genotypic characteristics ( h i s , r f a , uvrB, b io ) and for the presence of the plasmid. For each experiment, the frozen 1-ml 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 37C, 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 ( e . g . , lung or kidney) is capable of metabolizing a large number o f these chemicals to carcinogens. Some of these intermediate metabolites are very potent mutagens in the S_. typhlmurlvm test. Ames has described the liver metabolic activation system that we use. In b r ie f , adult male Sprague-Dawley rats (200 to 250 g) are given a single 500-mg/kg intraperi toneal 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 0000S1 The livers are removed aseptically and placed in a preweighed, ster ile glass beaker. The organ weight is determined, and all subsequent operations are conducted in an ice bath. The livers are ashed with an equal volume of cold, sterile 0 .1 5 M KCl, minced with sterile surgical scissors in three volumes of 0 .1 5 M KCl (3 ml/g of wet o rgan ), and homo genized 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 on dry ice and stored at -80C. The metabolic activation mixture for each experiment consists o f, for 50 m l: 5 .0 ml of S-9 fraction 1.0 ml of MgCl2 (0 .4 M) and KCl (1 .6 5 M) 0 .2 5 ml of glucose-6-phosphate (1 M) 2.0 ml of NADP (0 .1 M) 2 5 .0 ml of sodium phosphate buffer ( 0 .2 M, pH 7 .4 ) 16.75 ml of sterile H jO . The amount of S-9 fraction delivered to each plate is 50 pi. Plate Incorporation Assay Prior to testing, the test article is serially diluted from an ini tial stock. In some cases, a preliminary experiment is conducted to find a suitable dose range for testing. The article is usually tested over a mlnlmun of six dose levels, the highest nontoxic dose level being 10 mg/ plate unless so lubility, mutagenicity, or toxicity dictates a lower upper lim it. When extracts are made, various undiluted aliquots are tested, usually over a doBe range of 5 to 100 or 200 p i/p late. 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--mm test tube placed in a 43 C heating block ve add: 7 oooos (1 ) 2 .0 0 ml of 0 .6 Z agar containing 0 .6 Z NaCl, 0.05 mM b io tin , and 0.05 mM histidine ( 2 ) 0 .0 5 ml of indicator organisms (about 10 bacteria) (3 ) 0.05 ml of a solution of the test article ( 4 ) 0 .5 0 ml of metabolic activation mixture ( i f 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 II automated colony counter when possible. When accurate counts cannot be obtained ( e . g . , because of p recip itate), the plates are counted manually 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 .0 5 ml of the solvent/diluent used for the test a rtic le , i f appropriate. For positive controls, we test each bacterial culture with the following mutagens using steps ( 1 ) , ( 2 ) , ( 3 ) , and ( 4 ) : Sodiun azide for the base-pair substitution mutants TA153 5 and TA100 9-Amino acridine for the frameshift mutant TA153 7 2-Nitrofluorene for the frameshift mutants TA1538 and TA98 2-Anthramine for all tester strains, in the presence of metabolic activation. Criteria for Interpretation P o s it iv e . 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 must occur for at least three dose le v e ls. Negative. A test article is considered a nonmutagen when no doserelated increase in the number of revertants is observed in at least two Independent experiments. The maximim dose level tested for nontoxic compounds is 10 mg/plate (unless dictated otherwise by solubility prob lems). For toxic compounds, only the highest dose level tested should show evidence of toxicity. 8 000083 Inconclusive. When a test article cannot be identified clearly as a mutagen or nonmutagen in the standard plate assay, the results are classified as inconclusive. Saccharomyces cerevlsiae D3 The yeast J_. cerevlsiae D3 is a diploid microorganism heterozygous for a mutation leading to a defective enyzme 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_. cerevlsiae is stored at 4 C. For each experi ment, broth containing 0.0 5 Z MgSO^, 0 . 15Z KH2P0^, 0.45Z (N H ^ ^ S O ^ , 0.35Z peptone, 0.5Z yeast extract, and 2Z dextrose is inoculated with a loopful of the stock culture and incubated overnight at 30C, with shaking. The in vitro yeast mitotic recombination assay in suspension is con ducted as follows. The overnight culture is centrifuged and the cells are resuspended at a concentration of 10 cells/ml in 67 mM phosphate buffer (pH 7 . 4 ) . To a sterile test tube are added: 1.0 ml of the resuspended culture 0 .5 ml of either the metabolic activation mixture or buffer 0.2 ml of the test chemical 0 .3 ml of buffer. Several doses of the test chemical are tested (up to 5Z w/v or v /v ) in each experiment, and appropriate solvent/diluent controls are Included. 1, 2: 3 , 4-Diepoxybutane without metabolic activation and sterigmatocystin with activation are used as positive controls. The suspension mixture is Incubated at 30C 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 9 cc '"84 the same Ingredients as the broth plus 2 .0 Z agar; five plates are spread with the 10" 3 dilution and three plates are spread with the 10~5 dilu tio n . The plates are incubated for 3 days at 30*C, followed by at least 1 day at A *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 10x 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. Criteria for Interpretation P o s it iv e . 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 m illiliter and in the relative number of mitotic recombinants per 1 0 s survivors. Negative. 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 was performed for any of the assays. The results of the plate incorporation assay are a tabulation of the number o f colonies appearing on the plates. The results of the S_. cerevisiae D3 assay are tabulated after calculating the number of mitotic recombinants per 1 0 5 survivors. All calculations are expressed with two significant digits. 10 00008 References Ames, B. N ., E. G. Gurney, J. A. M ille r, and H. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc. Natl. Acad. Sci. USA 6 9 , 3128 -3132 (1 9 7 2 ). Ames, B. N ., W. E. Durston, E. Yamasaki, and F. D. Lee. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. USA 7 0 , 2281-2285 (1973) . 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. S c i. USA J70_, 782-786 (1 9 7 3 ). Ames, B. N ., J. McCann, and E. Yamasaki. Methods for detecting carcino gens and mutagens with the Salmonella/mammal lan-micro some mutagenicity test. Mutt. Res. 31, 347-364 (1 9 7 5 ). Brusick, D. J . , and V. W. Mayer. New developments in mutagenicity screen ing techniques with yeast. Environ. Health Perspect. 6, 83 -86 (1 9 7 3 ). K ier, L. D . , E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Natl. Acad. Sci. USA 71, 4159-4163 (1974) . McCann, J . , E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcino gens as mutagens in the Salmonella/microsome test: Assay of 300 chem ic a ls . Proc. Natl. Acad. Sci. USA _72_. 5135-5139 (1 9 7 5 ). McCann, J . , and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion. Proc. Natl. Acad. Sci. USA 73, 950 -954 (1 9 7 6 ). Mortelmans, K. E . , and B .A .D . Stocker. Segregation of the mutator property of plasmid R46 from its ultraviolet-pro tec ting property. Mol. Gen. Genet. 167, 317-327 (1 9 7 9 ). Zimmermann, F. K ., and R. Schwa 1er. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alkylat ing agents in Saccharomyces cerevisiae. Mol. Gen. Genet. 100, 63-76 (1967). 11 000086 RESULTS AND DISCUSSION 3M Company's Compound T-3651 was screened for mutagenic activity in the Ames Salmonella/mlcrosome in vitro mutagenicity assay using the five standard strains of Salmonella typhimurium; TA1535, TA1537, TA1538, TA98, and TA100. The assays were performed in duplicate, both in the presence and absence o f a rat-liver metabolic activation system. Sterile water as used as the dilu en t. The microbial mutagenicity testing of this sample was performed on 12 and 18 December 1984. Dose levels ranging from 10 to 5000 pg/plate were used for all Ames assays (Tables 1 and 2 ) . Compound T-3651 foamed \Aien it was diluted with water to form the stock solution of 100 mg/ml; further dilutions were performed once the foam settled. No dose-related Increases in the number of histidine-Independent revertants were observed with the compound in either assay. Compound T-3651 was also screened for recombinogenlc activity in the yeast Saccharomyces cerevislae D3 assay for mitotic recombination. The assays were performed on 10 and 29 January 1985, both with and without metabolic activation (Tables 3 and 4 ) . Sterile water was used as the diluent. At the high doses, T-3651 foamed and appeared to react with water. No dose-related increases in the number of mitotic recombinants per 10^ survivors were observed with the test compound in either assay. In conclusion, Compound T-3651 was reproducibly nonmutagenic and nonrecomblnogenic when tested according to these procedures. 12 000087 Table 1 IN VITRO ASSAYS WITH SALMONELLA TVPHIMURIUM COMPOUND T-3651 Experiment Date: 12 December 1984 Compound Metabolic Activation Compound Added per Plate _____________Histidine Revertants per Plate__________ TA1535 TA1537 TA1538 TA98 TA100 Negative Control Water + 50 pi 50 19 24 10 10 14 11 25 39 159 146 19 7 10 6 17 21 42 38 155 165 Positive Controls Sodium Azide 9-Amino acr id ine 2-Nitrofluorene 2 -Anthramine - - - + + 1 H8 50 5 1 1 2.5 2.5 460 376 615 621 223 670 1554 1441 102 7 983 28 19 47 31 180 149 336 321 394 360 622 693 17 16 10 17 249 226 128 92 Compound T-3651 - 10 pg 23 24 9 9 21 15 21 20 143 139 - 50 13 18 1 7 21 20 21 19 151 149 - 100 13 11 4 4 18 12 15 17 139 115 - 500 21 20 9 15 17 24 37 20 124 141 - 1000 14 24 7 10 15 23 32 19 136 146 -- 5000 26 17 5 8 10 14 26 25 115 120 + 10 14 12 6 8 20 11 32 26 178 182 -f 50 9 9 12 6 15 15 31 37 147 161 + 100 15 14 13 8 17 9 28 32 137 152 + 500 17 8 6 12 10 14 34 37 177 168 + 1000 8 16 4 6 10 12 26 43 162 154 + 5000 7 11 12 13 11 21 31 30 170 156 0000S8 Table 2 Compound______ Negative Control Water Positive Controls Sodium Azide 9-Aminoacridine 2-Nltrofluorene 2 -Anthramine Compound T-3651 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3651 Experiment Date: 18 December 1984 Metabolic Activation Compound Added per Plate _____________Histidine Revertants per Plate_______ TA1535 TA1537 TA1538 TA98 TAlOO + 50 nl 21 26 7 8 10 10 24 25 128 150 50 8 16 8 8 19 17 28 32 116 123 * 1 U8 557 641 50 331 305 652 636 5 1115 1105 708 653 1 +1 22 12 137 122 19 27 105 171 151 319 105 343 2.5 18 20 7 16 + 2.5 257 252 30 36 - 10 pg 23 23 9 10 12 12 33 20 122 122 50 29 24 5 7 19 12 21 15 108 101 ** 100 21 27 4 5 16 15 14 15 111 128 500 25 17 7 10 20 8 20 29 103 94 1000 18 16 7 8 17 7 13 18 93 123 5000 23 24 9 8 18 11 17 20 106 115 + + + + + + 10 50 100 500 1000 5000 9 10 7 7 25 20 20 19 101 104 6 10 4 8 15 17 36 28 111 108 8 6 11 12 28 12 28 26 142 98 15 7 12 5 28 26 32 36 130 106 9 10 7 6 23 18 28 25 102 99 14 10 6 12 19 13 23 30 103 94 000089 Table 3 IN VITRO ASSAYS OF SACCHAROMVCES CEREVISIAE D3 COMPOUND T-3651 Experiment Date: 10 January 1985 Compound Metabol lc Activation Negative Control Ha ter + Positive Controls 1,2: 3 ,4 -Diepoxybutane Sterigmatocystln + Compound T-3651 - - -- + + + + + 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 (x 10" 7) 6.7 5.8 6.2 6.5 7.1 7.1 7.8 6.9 7.1 6.7 5.8 6.9 7.0 7.5 7.3 Survivors <*) 100 100 93 97 100 100 100 100 100 100 100 100 100 100 100 Mitotic Recombinants per ml (x 10-3) 4 2 971 4 210 10 6 12 2 3 4 8 6 5 5 Mitotic Recombinants per 1 0 5 ^ Survivors 6.0 3.4 1600 6.2 300 14 7.7 17 2.8 4.5 6.9 12 8.6 6.7 6.8 000090 *A11 calculations are expressed to two significant figures Table 4 IN VITRO ASSAYS OF SACCHAROMYCES CERE VIS IAE D3 COMPOUND T-3651 Experiment Date: 29 January 1985 Compound Metabolic Activation Negative Control Water + Positive Controls 1 ,2 :3 ,4 -Die poxybutane Sterigmatocystin + Compound T-3651 - -- + + + + + Percent Concentration (v/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 (x 1 0 " 7) 6.2 6.4 6.3 6.1 6.3 6.5 6.9 6.4 7.1 6.5 6.2 6.4 6.6 6.8 6.5 Survivors (X) 100 100 100 98 98 100 100 100 100 100 97 100 100 100 100 Mi to tic Recombinants per ml (x 1 0 " 3) 5 8 933 8 235 10 7 8 2 13 10 4 13 12 5 Mitotic Recombinants per 10 Survivors 8.1 13 1500 13 370 15 10 13 2.8 20 16 6.3 20 18 7.7 *A11 calculations are expressed to two significant figures. 000091