Document mpqg1VnjZZpvkedo0n04nMM9J

REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5869 IN AN IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH L5178Y MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT) NOTOX Project 115954 NOTOX Substance 38196 - page 1 of 22 - 005631 RECEIVED m \! - 9 7934 TOXiCOLOGY 1-5869 STATEMENT OF GLP COMPLIANCE NOTOX Project 115954 NOTOX B.V., 's-Hertogenbosch, The Netherlands The study described in this report was conducted in compliance with the most recent edition of: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. With the exception that the stability of the test substance in the vehicle was unknown. Study Director Ing. E.3. van de Waart Da - page 2 - 0SG3Z T -5869 QUALITY ASSURANCE STATEMENT NOTOX Project 115954 NOTOX B.V., 's-Hertogenbosch, The Netherlands. Study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals. This report was audited by the NOTOX Quality Assurance Unit and the methods and results accurately reflect the raw data. DATES OF QAU INSPECTIONS/ AUDITS January 24, 1994 February 10, 1994 March 28, 1994 REPORTING DATES January 24, 1994 February 14, 1994 March 28, 1994 Quality Assurance Manager C.J. Mitchell B.Sc. Date: J2o ' I - page 3 - 005633 T-5869 REPORT APPROVAL STUDY DIRECTOR: NOTOX Project 115954 Ing. E.O. van de Waart MANAGEMENT: Dr. I.C. Enninga Technical Director Date 2 - page 4 - 005634 T -5869 NOTOX Project 115954 PREFACE Sponsor Study Monitor Testing Facility Study Director Technical Coordinator Study Plan 3M Belgium - Chemical EBC Canadastraat 11 B-2070 ZWIJNDRECHT Belgium Mr. R.H. Cox NOTOX B.V. Hambakenwetering 3 5231 DD 's-Hertogenbosch The Netherlands Ing. E.O. van de Waart C.M. Verspeek Start : February 01, 1994 Completed : February 22, 1994 TEST SUBSTANCE Identification Description Batch Purity Specific Gravity Instructions for test substance storage Stability under storage conditions Expiry date Stable for at least 4 hours in vehicle T -5869 Cream solid 2408 95% 1.5 At room temperature in the dark Stable January 01, 1996 Dimethylsulphoxide: not indicated VEHICLE The test substance was suspended or dissolved in dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use. The final concentration of the solvent in the culture medium amounted to 0.8% (v/v). - page 5 - 005635 T -5869 NOTOX Project 115954 GUIDELINES The study procedures described in this report were based on the following guidelines: - Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 476: "Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Tests", (adopted April 4, 1984). - European Economic Community (EEC), Directive 87/302/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; "Other Effects-Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test". EEC Publication no. L133 (adopted May 30, 1988). ARCHIVING NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test article reference sample, all specimens and raw data. OBJECTIVE Purpose of the study The objective of this study was to evaluate the test substance for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. The assay was conducted in the absence and presence of a metabolic system (S9-mix). The TK mutational system detected base pair mutations, frame shift mutations and small deletions. Justification for selection of the test system L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK'/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells can not incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which are lethal to the cells. Thus, cells which will survive in culture medium containing TFT are mutated, either spontaneously or by the action of the test substance, giving rise to a TK deficient phenotype. A test article which induces a positive response in this assay is presumed to be a potential mammalian cell mutagen. - page 6 - 005636 T -5869 NOTOX Project 115954 MATERIALS AND METHODS TEST SYSTEM Test System L5178Y mouse lymphoma cells Rationale Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC). Source Dr. A.G.A.C. Knaap, Department of Radiation Genetics and Chemical Mutagenesis of the State University of Leiden, The Netherlands (1981). This mouse lymphoma cell line was originally derived from the Fischer L5178Y line, isolated by Clive (1975)* *) Clive, D. and Spector, J.F.S., 1975, Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells, Mutation Res., 31, 17-29. Stock cultures of these cells were stored in liquid nitrogen (-196C) The cultures were checked for mycoplasma contamination. CELL CULTURE Cell culture conditions L5178Y mouse lymphoma cells were cultured in F10 complete culture medium. Cultures were incubated in a humid atmosphere containing 5% CO2 in air at 37C. Cell density was preferably kept below 7 x 105 cells/ml. Cells were exposed to the test substance in FIO complete culture medium without serum, buffered with 20 mM HEPES. F10 complete culture medium FIO complete culture medium consisted of Ham's FIO medium without thymidine and hypoxanthine (Gibco), supplemented with 10% horse serum, Lglutamine (2 mM) and penicillin/streptomycin (50 U/ml and 50 yg/ml respectively). Selective medium Selective medium consisted of FIO complete culture medium which contained, in addition 0.33% Agar Noble (Difco) and 5 yg/ml TFT (Sigma). Cloning medium Cloning medium consisted of FIO complete culture medium, supplemented with 10% horse serum, which was transformed into a gel by addition of 0.33% Agar Noble. - page 7 - &05637 T-5869 NOTOX Project 115954 Environmental conditions All incubations were carried out in a humid atmosphere (80-95%) containing 5% CO2 in air in the dark at 37C. The temperature and CO2 percentage were monitored during the experiment. REFERENCE SUBSTANCES Negative control: The vehicle of the test article. Positive controls: Without metabolic activation (-S9-mix): Ethylmethanesulphonate (EMS; CAS no. 62-50-0; purity 98%; Janssen Chimica) (2 mM) was used. EMS causes direct alkylation of DNA. With metabolic activation (+S9-roix): Dimethylnitrosamine (DMN; CAS-no. 62-75-9, purity 99%, Janssen Chimica) (0.5 mM) was used. DMN had to be activated by microsomal enzymes present in the S9-mix, resulting in a methyldiazonium ion which could react with cellular DNA. Solvents for Reference Substances Hank's balanced salt solution (HBSS) without calcium and magnesium. Solutions of reference substances were prepared immediately before use. METABOLIC ACTIVATION SYSTEM Preparation of S9-homogenate* Rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from BRL, Switzerland. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2 -EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196C). - page 8 - 0SG3s T -5869 NOTOX Project 115954 *) Ames, B.N., Me Cann, J. and Yamasaki, E.t 1975, Methods for detecting carcinogens and mutagens with the Sal monella/mammalian microsome mutagenicity test. Mutation Res., 31, 347-364. Preparation of S9-mix S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per ml: 1.02 mg MgC^.fi^O; 2.46 mg KC1; 1.7 mg glucose-ephosphate; 3.4 mg NADP; 4 pmol HEPES and 0.5 ml S9. The above solutions were mixed and filter (0.22 ym)-ster1l1zed (apart from the S9-fraction, which was added after filter-sterilization of the S9-mix components). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to each ml of cell suspension. EXPERIMENTAL PROCEDURE Selection of Dose Levels/Cytotoxlcity Test Prior to the actual mutagenicity test cytotoxicity data were obtained by treating 6 x 106 cells, suspended in 6 ml of F-10 medium buffered with 20 mM HEPES, in the absence of serum in a sterile 30 ml centrifuge tube with a range of test substance concentrations in approximately half log steps, both in the absence and presence of S9-mix. The centrifuge tubes were rotated for 3 h on a roller mixer at 37C. After the exposure, the cells were separated from the treatment solutions by 3 centrifugation steps (115 g, 8 min), each followed by removal of the supernatant and resuspension of the cells, twice in Hank's balanced salt solution and finally in F-10 medium. The cells in the final suspension were counted in an "Improved Neubauer" haemocytometer. Relative cytotoxicity, expressed as the reduction after approximately 24 h and 48 h growth compared to nontreated control cells, was used to determine a suitable concentration range (4 doses) of the test substance to be applied to cultures prepared for mutagenicity testing. Because the test substance was not toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. Cleansing Prior to mutagenicity (and cytotoxicitv) testing, the cells were grown for 1 day in culture medium containing 10`* M hypoxanthine, 2 x 10'7 M aminopterin and 1.6 x 10*5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to normal medium at least for 1 day before starting the experiment. - page 9 - 005639 T -5869 NOTOX Project 115954 Mutagenicity test* The test substance was tested both with and without S9-mix in two independent experiments. 6 x 106 cells (106/ml), or 12 x 106 cells (106 /ml) for test substance concentrations expected to be strongly toxic, for each selected dose cells were exposed for 3 h to the test substance in HEPESbuffered Ham's F-10 medium without serum. For this purpose the cell suspensions were placed in 30 ml centrifuge tubes on a roller mixer at 37C. Solvent and positive controls were included. After exposure, cells were washed twice with HBSS, counted and seeded in F-10 culture medium for expression of the mutant phenotype. The cultures were subcultured at least every other day in order to maintain log phase growth. The expression period for TFT-resistant mutants was 3 days. Immediately after exposure to the test substance 3 x 200 cells of each dose were plated into P90 petri dishes containing 15 ml cloning medium to determine cell survival (cloning efficiency). The cell survival was counted after 10-14 days with the Artek colony counter or the naked eye. *) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M., 1979, Validation and characterization of the L5178Y/TK Mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. *) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. and Salsburg, D.S., 1980, Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation, Mutation Res., 72, 447-474. *) Jotz, M. and Mitchell, A.D., 1981, Effects of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells. In: Evaluation of short-term tests of carcinogens. F.J. de Serres and J. Ashby (Eds.), Elsevier-North Holland. *) Van der Hoeven, J.C.M., Bruggeman, I.M. and Debets, F.M.H., 1984, Genotoxicity of quercetin in cultured mammalian cells, Mutation Res., 136, 9-21. *) Clive, D., Caspary, W., Kirby, P.E., Krehl, R., Moore, M., Mayo, J. and Oberly, T.J., 1987, Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity, Mutation Res., 189, 143-156. Mutant selection After the expression period a total number of 1.5 x 10^ cells.were plated in ten P90 petri dishes, each containing 15 ml selective medium (TFTselection). TK-deficient mutants formed microcolonies in 10-14 days, which were counted with the naked eye. Cloning efficiencies at the time of mutant selection were determined as described above. The mutant frequency was expressed as the number of mutants per 105 surviving cells. - page 10 - 005640 T-5869 NOTOX Project 115954 ACCEPTABILITY OF ASSAY A mutation assay was considered acceptable if it met the following criteria: a) The absolute cloning efficiency of the solvent controls was > 5096. b) At least three of the four doses of the test substance had an acceptable number of surviving cells (10^) analysed for expression of the TK mutation. c) The spontaneous mutant frequency in the untreated or solvent control was < 5 per 105 clonable cells. d) The positive controls (ethylmethanesulfonate and dimethylnitrosamine) induced significant (at least 2-fold) increases in the mutant frequencies. DATA EVALUATION AND STATISTICAL PROCEDURES No formal hypothesis testing was done. A test substance was considered positive (mutagenic) in the mutation assay if: a) It induced at least a 2-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and b) The results were reproducible in an independently repeated test. A test substance was considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations showed a mutant frequency at least twice that of the solvent control. b) The results were confirmed in an independently repeated test. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. - page 11 - 005641 T -5869 NOTOX Project 115954 RESULTS CYTOTOXICITY TEST/OOSAGE SELECTION Preliminary solubility tests indicated that T-5869 precipitated in the exposition medium at concentrations of 100 ug/ml and upwards. Table 1 shows the results of the preliminary cytotoxicity test with T-5869 in the presence and absence of a metabolic activation system (S9-mix). In the absence of S9-mix T-5869 inhibited the growth of the lymphoma cells in suspension at a test substance concentration of 33 yg/ml by 22% and at 100 pg/ml by 72%. In the presence of S9-mix the test substance did not inhibite the growth of the lymphoma cells in suspension. MUTAGENICITY TEST Based on the results of the cytotoxicity test (Table 1), the following dose range was selected for mutagenicity testing: Without S9-mix: 3 to 100 ug/ml culture medium With S9-mix : 3 to 100 yg/ml culture medium EMS (2 mM) was used as a positive control in the test without metabolic activation, whereas DMN, (0.5 mM) served as a positive control for the assay with metabolic activation. Tables 2 and 3 show the percentages of cell survival and the mutant frequencies for various concentrations of T-5869. Individual colony counts of cloning and selective plates, and cell counts during subculturing are listed in Tables 4-9 of the appendix. Both in the presence and absence of S9-mix the test substance induced no significant increase in the mutant frequency in both independent experiments. The spontaneous mutant frequencies in the solvent-treated control cultures were within our historical control data range (1.9 + 1.2 in the absence of S9-mix and 1.4 + 0.7 in the presence of S9-mix; indicated are means + S.D. for n=44 and 45 respectively). Mutant frequencies induced by positive control chemicals were increased by 7- to 9-fold for EMS and by 21- to 23-fold for DMN. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly. CONCLUSION In conclusion, T-5869 was found to be not mutagenic in the TK mutation test system under the experimental conditions described in this report. - page 12 - 00SG42 7-5869 NOTQX Project 115954 TABLE 1 PRELIMINARY CYTOTOXICITY DETERMINATION IN L5178Y MOUSE LYMPHOMA CELLS Dose (ug/ml) Cells/ml (X 105 ) After After After 0 h 24 h 48 h Suspension growth Total1 X of control Solvent control 1 3 10 33 100* 6.1 6.4 7.4 5.7 4.9 5.0 Without metabolic activation ( -S9-mix) 4.9 6.1 5.1 6.5 4.5 5.9 5.3 5.6 4.7 6.2 2.5 4.1 71.2 82.9 76.7 66.1 55.8 20.0 100 116 108 93 78 28 Solvent control 1 3 10 33 100* 7.9 6.6 7.0 6.4 6.3 6.6 With metabolic activation (+S9 mix) 5.7 6.8 119.6 6.3 5.7 92.6 6.4 6.0 105.0 7.7 5.8 111.7 6.9 6.2 105.3 6.8 6.4 112.2 100 77 88 93 88 94 Solvent control = DMSO Subculture 1.6 x IO5 cells/tnl 1 Total growth = Cell count after O h x (24 h cells/ml) x (48 h cells/ml) cells subcultured cells subcultured directly after after 24 h treatment * Test substance precipitated slightly in the exposition medium - page 13 - 00S643 T -5869 NOTOX Project 115954 TABLE 2 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-5869 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM EXPERIMENT 1 DOSE (pg/ml) C.E. AT DAY 0 (56 OF CONTROL) C.E. AT DAY 3 (ABSOLUTE %) MEAN NO. OF MUTANTS PER PLATE MUTATION FREQUENCY X 105 Solvent control 3 10 33 1001 2 mM EMS Without metabolic activation (-S9-mix) 100 84 98 77 101 80 82 89 89 90 104 79 2.0 1.0 1.2 1.8 1.5 14.1 1.6 0.9 1.0 1.3 1.1 11.9 Solvent control 3 10 33 1001 0.5 mM DMN With metabolic activation (+S9-mix) 100 88 86 82 76 84 96 87 80 88 44 49 1.0 1.0 0.8 1.1 1.9 13.6 0.8 0.8 0.6 0.8 1.4 18.5 C.E. Cloning Efficiency Solvent control = DMSO EMS = Ethylmethanesulphonate DMN Dimethylnitrosamine 1 Test substance precipitated slightly in the exposition medium - page 14 - 005644 T-5869 NOTOX Project 115954 TABLE 3 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-5869 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM EXPERIMENT 2 DOSE (wg/ml) C.E. AT DAY 0 (* OF CONTROL) C.E. AT DAY 3 (ABSOLUTE %) MEAN NO. OF MUTANTS PER PLATE MUTATION FREQUENCY x 105 Solvent control 3 10 33 1001 2 mM EMS Without metabolic activation (-S9-mix) 100 86 111 88 102 79 58 88 44 95 83 81 1.2 1.1 0.6 2.4 2.9 9.3 0.9 0.8 0.5 1.8 2.0 7.7 Solvent control 3 10 33 1001 0.5 mM DMN With metaboli c activation (+S9- mix) 100 87 95 81 75 88 91 81 82 75 33 49 0.7 0.6 0.9 0.6 0.7 7.9 0.5 0.5 0.7 0.5 0.6 10.7 C.E. * Cloning Efficiency Solvent control * DMSO EMS = Ethylmethanesulphonate DMN - Oimethylnitrosamine 1 Test substance precipitated slightly in the exposition medium - page 15 - 005645 T-5869 NOTOX Project 115954 APPENDIX Individual Colony Counts and Cell Counts during Expression Period FORMULAS AND CALCULATIONS FOR L5178Y MOUSE LYMPHOMA TEST SYSTEM. Total number of mutant Mutant frequency = ______ 100__________ X colonies on selective plates per 105 survivors cloning efficiency number of seeded cells Cloning efficiency = Average No. of colonies on cloning plates X 100% 200 L5178Y MOUSE LYMPHOMA TEST SYSTEM - Cell counts during expression period - Cloning efficiency immediately after exposure - Mutation experiments, individual colony counts Tables 4-6 Tables 7-9 Experiment 1 Experiment 2 Abbreviations used: DMN, dimethylnitrosamine EMS, ethylmethanesulphonate Solvent control: Dimethylsulphoxide - page 16 - 005646 T -5869 NOTOX Project 115954 TABLE 4 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CELL COUNTS AND SUBCULTURE DATA Experiment l 1 DAY 0 1 DAY 2 | DAY 3 1 dose |Total amount of|Total amount of| 1 Subculture cells before 1cells after _| .. 1 x 106 1 1 1 Subculture ICell count! 1 x 106 11 ICell count 1 (ug/ml)treatment x ,10" jtreatment x IO6 !*1/ |Total amount'5/1c/ml x IQ5 !**/1Total amount4/ 1c/ml x 10* X2> Without Metabolic Activation (S9-mix) DMSO 3 10 33 100 EMS 6 6 6 6 12 6 5.2 87 3.0 6.6 100 4.0 3.9 65 3.0 6.5 98 4.0 5.5 92 3.0 5.9 89 4.0 4.5 . 75 3.0 5.2 79 4.0 6.6 55 6.0 5.2 79 4.0 4.1 68 3.0 6.3 95 4.0 5.8 100 6.5 112 6.0 103 5.9 102 6.0 103 6.2 107 With Metabolic Activation (+S9-mix) DMSO 3 10 33 100 DMN 6 6 6 6 6 6 4.8 80 3.0 6.6 100 4.0 5.9 100 4.7 78 3.0 7.1 108 4.0 5.9 100 4.7 78 3.0 5.7 86 4.0 6.4 108 3.6 60 3.0 6.9 105 4.0 5.5 93 4.5 75 3.0 6.1 92 4.0 5.4 92 5.4 90 3.0 2.4 36 4.0 4.8 81 (1) cells after treatment x 100* cells before treatnent (2) cell count__________ x 100% cell count of control (3) cell density 0.4 x 10* c/ml (4) cell density 1.6 x 10s c/ml - page 17 - 005647 T -5869 NOTOX Project 115954 TABLE 5 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CLONING EFFICIENCY DAY 0 EXPERIMENT 1 Dose (yg/ml) No. of colonies/ cloning plate 12 3 Mean No. of colonies/plate Cloning efficiency absolute relative (% of control) Solvent control 154 151 169 3 161 149 INF 10 143 174 159 33 122 135 INF 100 167 129 126 EMS 163 165 INF Without S9-mix 158 79 155 78 159 80 129 65 141 71 164 82 100 98 101 82 89 104 Solvent control 187 188 169 3 145 167 INF 10 125 140 145 33 163 167 188 100 148 147 138 OMN 80 76 84 With S9-mix 181 156 137 173 144 80 91 78 69 87 72 40 100 86 76 96 80 44 INF Plate Infected with fungi - page 18 - 005648 T-5869 NOTOX Project 115954 TABLE 6 L5178Y MOUSE LYMPHOMA TEST SYSTEM - SELECTION DATA AND CLONING EFFICIENCY EXPERIMENT 1 Dose yg/ml |Number of colonies/Selection piate|Total|No. of colonies|Mean|MF |.................................. | No. |/cloning plate | | i 1 2 3 4 5 6 7 8 9 10| 12 3 Without Metabolic Activation (-S9-mix) Solvent control 3 10 33 100 EMS 1 1o 2 03 12 0 12 1 11 0 | 22 14 42 00 12 04 02 21 16 22 11 01 21 21 13 14 23 11 31 04 13 9 14 21 21 02 13 32 8 10 20 10 12 18 15 141 161 173 171 162 153 148 161 159 156 177 177 181 174 188 177 156 161 157 168 1.6 154 0.9 159 1.0 178 1.3 180 1.1 158 11.9 With Metabolic Activation (+S9-mix) Solvent control 3 10 33 100 DMN 11 0 10 1 10 1 13 0 11 1 1 6 13 01 00 02 12 23 15 15 30 20 01 20 20 16 14 21 21 02 00 42 18 13 11 22 11 03 22 12 14 10 10 8 11 19 136 177 181 170 162 157 170 163 171 INF 182 167 169 171 178 179 94 94 105 176 0.81 163 0.8 j 167 0.61 173 0.81 176 1.41 98 18.5| MF * Mutant frequency per 105 survivors INF = Plate infected with fungi - page 19 - 005649 T-5869 NOTOX Project 115954 TABLE 7 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CELL COUNTS AND SUBCULTURE DATA Experiment 2 1 DAY 0 1 DAY 2 1 DAY 3 1 1Total amount of|Total amount Of| 1 Subculture | 1 1 Subculture 1 1 dose cells before cells after (tig/ml) jtreatment x ,106 treatment x .1 .. I x 10 ICell lO^IX1/JTotal amount'9/1 c/ml count 1 1 x x 103 |%ZZ|Total 106 |Cell amount4 / jc/ml count x 10 1 ,,X |X2> Without Metabolic Activation (-S9-mlx) OMSO 3 10 33 100 EMS 6 6 6 6 12 6 4.3 72 3.0 6.3 100 4.0 5.9 100 3.9 65 3.0 6.4 102 4.0 5.3 90 3.9 65 3.0 5.2 83 4.0 5.7 97 3.1 52 2.9 3.4 54 4.0 5.5 93 3.6 30 3.5 2.0 32 4.0 4.8 81 3.8 63 3.0 5.9 94 4.0 4.7 80 With Metabolic Activation (+S9-mix) DMSO 3 10 33 100 DMN 6 6 6 6 6 6 3.8 63 3.0 8.4 100 4.0 4.3 100 3.9 65 3.0 8.7 104 4.0 5.2 121 4.2 70 3.0 7.5 89 4.0 5.0 116 3.8 63 3.0 6.1 73 4.0 5.6 130 3.7 62 3.0 7.4 8B 4.0 5.1 119 4.4 73 3.0 3.2 38 4.0 3.5 81 (1) cella after treatment x 100X cells before treatment (2) cell count__________ x 100X cell count of control (3) cell density 0.4 x 10s c/ml (4) cell density 1.6 x 105 c/ml - page 20 - 005650 T -5869 NOTOX Project 115954 TABLE 8 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CLONING EFFICIENCY DAY 0 EXPERIMENT 2 Dose (yg/ml) No. of colonies/ cloning plate 12 3 Mean No. of colonies/plate Cloning efficiency absolute relative (_% of control) Solvent control 3 10 33 100 EMS 130 140 ; 129 135 154 ; 154 140 .121 ; 145 70 80 ; 82 51 60 ; 62 112 101 ; 116 Without S9-mix 133 67 148 74 135 68 77 39 58 29 110 55 100 111 102 58 44 83 Solvent control 204 180 ; 198 3 192 170 ; 191 10 152 138 ; 149 33 177 179 ; 174 100 159 162 ; 155 DMN 69 67 ; 56 With S9-mix 194 184 146 177 159 64 97 92 73 89 80 32 100 95 75 91 82 33 - page 21 - 005651 T -5869 NOTOX Project 115954 TABLE 9 L5178Y MOUSE LYMPHOMA TEST SYSTEM - SELECTION DATA AND CLONING EFFICIENCY EXPERIMENT 2 Dose yg/ml Number of colonies/Selection plate|Total|No. of colonies|Mean|MF j................................ I No. /cloning plate j 1 2 3 4 5 6 7 8 9 101 |1 2 3 | j| Without Metabolic Activation (-S9-mix) Solvent control 3 10 33 100 EMS 1 10 I0 I0 I0 I1 18 0 0 0 4 4 5 14 02 00 44 24 59 1 1 0 1 2 2 12 161 179 173 0 2 4 2 1 0 11 183 170 172 0 0 2 0 2 2 6 159 149 165 3 1 1 4 2 1 24 170 185 171 6 2 3 3 2 2 29 192 199 178 8 6 10 13 18 11 93 157 156 173 171 0.9 175 0.8 158 0.5 175 1.8 190 2.0 162 7.7 With Metabolic Activation (+S9*mix) Solvent control 3 10 33 100 DMN 1 13 10 I2 10 I2 16 0 0 0 0 0 8 00 31 02 00 10 89 20 00 02 10 11 66 10 01 02 00 21 00 20 12 0 1 10 8 9 11 8 7 6 9 6 7 79 175 181 162 161 159 167 180 171 175 164 163 160 152 136 160 96 91 106 173 0.5| 162 0.51 175 0.7 j 162 0.51 149 0.6 j 98 10.7j MF = Mutant frequency per 105 survivors - page 22 - 056S2