Document KGGMGg6VRGo368dRJo0rrZZb0

2 5 / J UN, 25. 1 9 9 9 0 1 2 : 4 1 P M p c f K E D I C A L 2 2 0 2E 0 2 n , u o u t e r s 57331773 NO, 0773 n P. 30 001 A W 2&-0253 REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF T-6906 IN AN i n v i t r o m a m m a l i a n c e l l g e n e m u t a t i o n t e s t WITH L517BY MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT) NOTOX Project 223458 NOTOX Substance 7B741 page 1 of 31 9 TELEFAX oaTUu /OAT^: *aM/TO/H:M waB/enow/ce-,__ eET,/9UiJ./eO-0 N> * 3 3 i 5 4 AANTALOAa^NlM4CC*; -i/- SUtiSfcLisaEJB. ^i3ax. ? 9 I 001362 T - 6906 STATEMENT OF GLP COMPLIANCE NO, 0773 NP. 33 502 NpTOX Project 223456 NOTOXB.V., 'S'Nertogenbosch, The Netherlands The study described in this report has been correctly reported and 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 (T5CA), Title 40 Code of Federal Regulations Part 792. Study Director: C.M. Verspeek-Rip Management; Ing. E.J. van de Waart Head of Genetic & Ecotoxieology page 2 001363 25/JUN. 25. 1999012:41PM pcfmeDICAL 220 2E 02<e n . uouters * 07331773 NO. 0773 nP. 34 P 0 3 T-69Q6 QUALITY ASSURANCE STATEMENT NOTOX Project 223458 NOTOX B.V,, `S'Hert0 9 enbo8Ch, The Netherlands This report was audited by the NOTOX Quality Assurance Unit to ensure that the methods and results accurately reflect the raw data. The dates of Quality Assurance inspections and audits are given below. During the on-site Inspections procedures applicable tothls type of Study were inspected. DATES OF QAU INSPECTIONS/ AUDITS on-site inspections 16-01-199B 27-01-1990 19-02-1998 10-03-1998 19-03-1998 protocol inspection 31-12-1997 report audit 22-04-1998 REPORTING OATES 16-01-1998 27-01-1998 20-02-1998 12-03-1998 19-03-1998 31-12-1997 22-04-1998 Head of Quality Assurance C.J. Mitchell 0 .Sc. Date: 1 aj - 9 * - page 3 001364 ' 25/.JUN. 25. 19993112:41PM p c f m e D K A L 220 2E 02<e n ; u o u t e r s * 87331773 NO. 0773 N|P. 35 BEST COPY AVAILABLE T-6906 NOTOX Project 223458 SUMMARY This report describes the effects of T-6906 on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in 15178Y mouse lymphoma cells in the presence end absence of S9-mix. T-6906 was tested up to concentrations of 42 and 45 pg/ml in the absence of S9-mix 1n the first and second experiment respectively. T-6906 was tested up to concentrations of 1000 and 750 pg/ml m the presence of 8% (v/v) 59fraction, in the first and second experiment respectively. At these dose levels appropriate cytotoxicity was observed. In the presence of S9-m1x, in both mutation assays the'toxicity of T-6906 in the highest, precipitating concentration 1s less severe than the toxicity in lower, non-precipitating concentrations. The toxicity observed in the cell count durinQ the expression period 1s not observed in the cloning efficiency of the initially surviving cells. These observations were confirmed in other experiments. Therefore this rare finding 1n toxicity is probably a test substance related effect and not an accidental finding. In the absence of S9 metabolic activation in the first experiment T-6906 did not induce a significant increase in the mutant frequency at the TK-locus. In the second experiment, T-6906 showed a 1.7-fold increase 1n the mutant frequency at the TK-locus. However, this Increase was observed only at one toxic concentration. The relative cloning efficiency of the remaining cells was 22% compared to the solvent control, whereas 44% of the cells Initially survived the treatment. This means that the actual survival of the cells after treatment was only 10% (22% of 44%). Since, no dose response relationship was observed, the effect was observed only in one experiment and the increase was less than two-fold compared to the solvent control, this increase was considered not biologically relevant and T-6906 1s considered to be not mutagenic 1 n the absence of S9-mix. In the presence of 8% (v/v) S9 metabolic activation. T-)5?Q6 showed 22- and 3.2-fold increases in the first and second experiment respectively in the mutant frequency at the TK-locus. In the first experiment, the Increase was observed only at ona very toxic concentration. A severe delay 1n the cell growth at the concentration of 1000 pg/ml was observed after 48 and 72 hours of subculture compared to the cell growth of the solvent control and a reduction of 61% was observed in the cells plated for the determination of the cloning efficiency of day 3. In the second experiment, the increase in the mutant frequency was observed at test substance concentrations of 250 and 750 P9 /ml, however no dose response relationship was observed. Since the increases were observed 1n two experiments and the increases m the second experiment were both in toxic and non toxic concentrations, these Increases are considered to be biologically relevant. Therefore, T-6906 1s considered mutagenic in the presence of 69-m1x. page 4 001365 25/JUN 25 199901L;:4^FM p c f MKDICAL ''U 11 U ^ e n .u q u t e r s e?33i?73 Nl). U'/7J rtf. jb D05 best csn r in n u u n E T-6906 NOTOX Project 223450 In the first experiment, both the number of small and large mutant colonies were increased compared to the solvent control values. In the second experiment, mainly the large mutant colonies were increased compared to the solvent control values. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. Mutant frequencies induced by positive control chemicals were Increased by 8,6 and 15.7*fold for EMS 1n the first and second experiment respectively, and by 10.5- and 11.6-fold for OMN, In the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9*mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-m1x) functioned properly. It is concluded that T-6906 is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. - page 5 I 001366 d^jUK. 3. T-6906 ^ M t U l l A L \) I t U^EN.WDUTERS -* 87331773 UT U K Ji t?06 A iM fe NOTOX Project 22345B PREFACE Sponsor Study Monitor Testing Facility Study Director Technical Coordinator Study Plan 3M USA St. Paul, M1NES0TA 55133-3220 U.S.A. Mr. R.H. Cox 3M Belgium NV - Chemical EBC Cenadastraat 11 B-2070 ZWIJNDRECHT Belgium NOTOX B.V. Hambakenweterlng 3 5231 DD *s-Hertogenbosch The Netherlands C.M. VerspeeK-Rip 8. van Oort Start : 12 January 1998 Completed : 14 April 199B ; TEST SUBSTANCE Identification Description Batch Purity Test gubstance storage Stability under storage conditions Expiry date Stability in vehicle T-6906 Creme coloured solid 040016 97% At room temperature 1n the dark Stable 31 December 1998 Dimethylsulphoxide: at least 96 h The sponsor 1a responsible for the completeness and GLP Compliance of all test substance data. ARCHIVING l NOTOX B.V. will archive the protocol, report, test article reference sample and raw data for at least 10 years. page 6 001367 J'JN. 25. ;999 1 2 :35PM MEDICAL 220 2E 02 NO. 3723 ?. 7 T-6906 NTOX Project 223458 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), Annex V of the EEC Directive 67/54B/EEC, Part B: Methods for the Determination of Toxicity; 'Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test'. Directive 87/302/EEC, EEC Publication no. L133 (adopted May 30, 1988) and Directive 96/54/EEC, EEC Publication no. 1248 (adopted September 30, 1996). OBJECTIVE Objective The objective of this study was to evaluate the mutagenic potency of T-6906 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. Background 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 1s able to detect base pair alterations, frame shift mutations and small deletions. Cells deficient 1n thymidine kinase (TK), due to the forward mutation (TK+ /to T K'/-) 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. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between the two different classes of TFT-resistant mutants (small and large colonies) which are believed to represent the different types of lesions induced in the DNA by the test substance. A test article which Induces a positive response in this assay 1s presumed to be a potential mammalian cell mutagen. page 7 001368 JUN. 25. 2399 12:35PM MEDICAL 220 2E 02 NO. 3773 ?. 8 T-6906 NOTOX Project 223458 MATERIALS AND METHODS TEST SYSTEM Test System L5178Y mouse lymphoma cells Rationale Recommended test system in international guidelines (e.g. EPA, OECD, EEC). Source Dr. A.Q.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). Stock cultures of these cells were stored in liquid nitrogen (-196'C) The cultures were checked for mycoplasma contamination. CELL CULTURE F10 complete culture medium FIO complete culture medium consisted of Ham's FIO medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2 mM) and peniclllin/streptomycin (50 U/ml and 50 yg/ml respectively). Cell culture conditions L5178Y mouse lymphoma cells were cultured in FIO complete culture medium. Cell density was preferably kept below 7 x 105 cells/ml. Exposition medium Cells were exposed to T-6906 for 3 hours in FIO culture medium, buffered with 20 mM HEPES or for 24 hours in FIO complete culture medium. Selective medium Selective medium consisted of FIO complete culture medium, supplemented with 10% (v/v) horse serum and 5 yg/ml TFT (Sigma). Non-selective medium Non-selective medium consisted of FIO complete culture medium, supplemented with 10% horse serum. Environmental conditions All incubations were carried out in a humid atmosphere (80*95%) containing 5% C02 in air in the dark at 37*C, The temperature and C02 percentage were monitored during the experiment. ` Paa` * " 001369 MEDICAL 220 2E 02 NO. 3773 ?. 9 T-6906 NQTOX Project 223458 TREATMENT OF THE TEST SUBSTANCE The test substance was dissolved in dlmethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use. REFERENCE SUBSTANCES Negative control: The vehicle of the test article, being dimethylsulphoxide. Positive controls: Solvents for Reference Substances Hank's balanced salt solution without calcium and magnesium. Solutions of reference substances were prepared immediately before use. Without metabolic activation (59-mix): Ethylmethanesulphonate (EMS; CAS no. 62-50-0; purity 98%; Janssen Chimica) (1 mM) was used. EMS causes direct alkylation of ONA. With metabolic activation US9-m1x): Dimethylnltrosamine (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 1n a methyldiazonium Ion which could react with cellular DNA. METABOLIC ACTIVATION SYSTEM Preparation of S9-fract1on Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. 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). - page 9 - 001370 KO, 0773 ?. IO T *6906 NOTOX Project 223458 The livers of the rats were removed asepticaHy, and washed in cold (O'C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2 -EDTA. Subsequently the livers were minced 1 n a blender and homogenized 1 n 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (59-fraction) was transferred into sterile ampules, which were stored in liquid nitrogen (196 *C ). Preparation of S9-mlx S9-mix was prepared Immediately before use and kept on ice. S9 *mix contained per ml: 1.63 mg MgCl2 -6H2 O ; 2.46 mg KC1; 1.7 mg glucose-ephosphate; 3.4 mg NADP; 4 pmol HEPES. The above solution was filter (0.22 pm)-steril1zed. To 0.5 ml S9-mix components 0.5 ml S9-fraction (batch 97-6 or 98-1) was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 ml S9*mix to each ml cell suspension. The concentration of the 59-fraction in the exposition medium was 8% (v/v). EXPERIMENTAL PROCEDURE Cleansing Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in F10 complete culture medium containing 10' 4 M hypoxanthine, 2 x 10*' M aminopterin and 1,6 x 10* 5 M thymidine (HATmedium) 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. Dose range finding test In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 6 x 1 0 cells, suspended 1 n 6 ml exposition medium, 1n a sterile 30 ml centrifuge tube, with a number of test substance concentrations increasing with approximately half log steps. T-6906 was tested in the absence and in the presence S9-fraction. The centrifuge tubes were rotated for 3 hours on a roller mixer at 37*C. The highest tested concentration was determined by the solubility of the test substance in the culture medium. After 3 hours 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 complete culture medium. The cells 1 n the final suspension were counted with a microscope with an 'Improved Neubauer' haemocytometer. page 10 - 001371 JUN. 25. :399 12-.36PK MEDICAL 220 2E 02 T-6906 HO. 3723 ?. 11 NOTOX Project 223458 The cells were seeded for determination of the cloning efficiency (CE). For that, the cel! suspensions were diluted and seeded 1n wells of a 96well dish. 1 cell was added per well (2 x 96-well microtiterplates per concentration) in non selective medium. After 9 days the plates were scored for empty wells, i.e. wells 1n which no visible colonies were present. The microtiterplates were scored with the naked eye or with a microscope at a magnification of 4x. The calculation of the cloning efficiency was determined by deviding the number of empty wells by the total number of wells. This value was called P {0), the zero term of the Poisson distribution. P(O) = number of empty wells/total number of wells. CE = -In P(0)/number of cells plated per well. The P(O) and the CE are expressed as percentage, Mutagenicity test Treatment of the cells T-6906 was tested both in the absence and in the presence of S9-m1x in two Independent experiments. Per culture 8 x 10 cells (10/ml) were used. If test substance concentrations were expected to be toxic, 16 x 10 cells (lO/m1) were used per culture. The concentration of the S9*fraction in the exposition medium was 8% (v/v ) Cell cultures in the absence of S9-mix were exposed for 24 hours to the test substance in exposition medium (FIO complete culture medium) and the cell cultures 1n the presence of S9-mix were exposed for 3 hours to the test substance in exposition medium (FlO culture medium buffered with 20 mM HEPES). The cell cultures were placed in 30 ml centrifuge tubes on a roller mixer at 37*C. Solvent and positive controls were included. After exposure, cells were separated from the exposition medium 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 complete culture medium. The cells in the final suspension were counted with a microscope with an 'Improved Neubauer* haemocytometer. Cloning efficiency directly after treatment Immediately after exposure to the test substance 2 x 96-well microtiter plates with 1 cell/well were plated for each dose to determine the survival of day 0. The plates were scored after 9 days incubation for determination of the CE. Expression period and determination of the mutant frequency For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the 24 hours exposure time for the cell cultures without S9*mix. The cells of the cell cultures with S9-mix were cultured for 3 days after the 3 hours exposure time. During this culture period at least 4 x 10 cells (1f possible) were subcultured. Three days after the start of the treatment with the test substance the cells were plated for determination of the CE (CE3 ) and the mutant frequency (MF). page 11 001372 JN. b. . m U : J t W . tit U I CAL HW U n u . j y ; 3 ?. 12 T-6906 N0T0X Project 223458 At least four doses of the test substance were selected for the mutation assay, both in the absence and presence of S9-mix. For determination of the CE3 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) 1n non selective medium. For determination of the MF a total number of 5.76 x 105 cells/ concentration were plated in three 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection). The microtiter plates for CE3 and MF were incubated for 12 days at 37*C in humified air with 5% CO2 . After this incubation period the plates for the TFT-selection were stained for 2 h, by adding 0.5 mg/ml MTT (3-[4,5-Dimethy1thiazol-2-yl]-2,5diphenyltetrazolium bromide, Sigma) to each well, The plates for the CE3 and MF were scored with the naked eye or with the microscope. The calculation of the mutation frequency was determined, as follows: MF = {-In P(0)/2000)/CE3 . The mutant frequency was expressed as the number of mutants per 105 surviving cells. DETERMINATION OF THE MUTANT COLONIES The colonies were divided in: Small colonies Large colonies Morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. Morphological less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony was classified as one small colony. A well containing more than one large colony was classified as one large colony. A well containing one small and one large colony was classified as one large colony. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies can be associated with the induction of chromosomal mutations. - page 12 - 001373 JN. 5. .aay vcJ'/Ft' ML U U A L '\i UL (It). UY/3 ?. 13 T-6906 NOTOX Project 223458 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 > 50%. b) At least at three of the four doses of the test substance, an acceptable number of surviving cells (10) could be analysed for expression of the TK mutation. c) The spontaneous mutant frequency in the untreated or solvent control was < 10 per 105 clonable cells. d) The positive controls (ethylmethanesulfonate and dimethylnitrosamine) induced significant (at least three-fold) increases 1n the mutant frequencies. e) In the absence of S9-m1x the selected dose range had to include a clearly toxic concentration. 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 3-fold increase in the mutant frequency compared to the solvent control 1n 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 of at least three-fold compared to 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 13 - 001374 j m 25. : 399 12:37PM MEDICAL 220 2E 02 KO. 0773 14 T-6906 NOTOX Project 223458 RESULTS DOSE RANGE FINDING TEST T-6906 precipitated 1n the exposition medium at a concentration of 1000 pg/ml. Therefore, this concentration was used as the highest test substance concentration, In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 1000 jig/ml 1n the absence and in the presence of S9*mix (Table 1). In Table 1 the first column, contains the relative cell counts (expressed as the percentage of the control culture) of cultures directly after 3 hours exposure to the various concentrations of the test substance, whereas the second column expresses the relative cell survival directly after exposure as the percentage cloning efficiency of the remaining cells (individual plate counts are listed 1n Appendix 1, Table 4). In the absence of S9-mix, the number of cells counted after 3 hours treatment was reduced at a test substance concentration of 333 pg/ml by 67%, whereas the number of cells counted at the concentration of 1000 pg/ml was reduced by 52% compared to the solvent control. The initially surviving cells at all concentrations tested showed no clear reduction in the cloning efficiency compared to the cloning efficiency of the solvent control. In the presence of S9-mix, no reduction was observed in the cell count of the remaining cells directly after treatment. The initially surviving cells showed no clear reduction in the cloning efficiency compared to the solvent control. In the absence of S9-m1x the selected dose range did not include a concentration with toxicity up to 90% compared to the solvent control. / Therefore, cell cultures 1n the mutagenicity test, in the absence of S9-mix, were exposed for 24 hours to the test substance in exposition medium (FIO complete culture medium). MUTAGENICITY TEST Tables 2 and 3 show the percentages of cell survival and the mutant frequencies for various concentrations of T-6906. Individual colony counts of cloning and selective plates, and cell counts during subculturing are listed in Tables 5-12 of Appendix 1. Experiment 1 Based on the results of the dose range finding test and the solubility test, the following dose range was selected for mutagenicity testing, in the absence of S9 metabolic activation: 10, 33, 100, 175, 333, 560 and 1000 pg/ml exposition medium. However, the dose levels 100 pg/ml and upwards were too toxic for further testing; since not enough dose groups were left for mutagenicity testing, the experiment was repeated. - page 14 - 001375 NO. 3773 ?. lb T-6906 NOTOX Project 223458 The following dose range was selected for mutagenicity testing: Without S9-mix: 10, 18, 33, 42, 56, 75 and 100 j^g/ml exposition medium With S9*mix : 33, 100, 333 and 1000 pg/ml exposition medium Evaluation of the toxicity In the absence of S9 metabolic activation, the dose levels of 56 pg T-6906/ml and upwards were too toxic for further testing. In the presence of S9 metabolic activation, no severe toxicity was observed and all dose levels were evaluated. The dose levels selected to measure mutant frequencies at the TK-locus were: Without S9-mix: 10, 18, 33 and 42 pg/ml exposition medium With S9*mix : 33, 100, 333 and 1000 pg/ml exposition medium In the absence of S9-mix (Table 2), the cell count of all concentrations tested showed no reduction compared to the cell count of the solvent control after 24 hours treatment. The cloning efficiency of the remaining cells was comparable to the solvent control even at the highest tested dose. . In the presence of S9-mix, after 3 hours treatment, the cell count of the test substance concentration of 1000 pg/plate showed no reduction compared to the cell count of the solvent control, whereas the cells counted at the concentration of 333 pg/ml showed a reduction of 44% compared to the solvent control. The cell count after 48 hours of subculture (Table 6) at test substance concentrations of 333 and 1000 pg/ml showed reductions of 61% compared to the cel! count of the solvent control. The cloning efficiency of the remaining cells (Table 2), at the test substance concentration of 333 pg/plate was reduced by 44%, the other dose levels showed no reduction 1n the cloning efficiency. Further investigation showed that at a concentration -6906 already precipitated 1n the exposition medium. Theref was selected as highest dose in the absence and presence of S9'mix, all other concentrations did not precipitate 1n the exposition medium. Experiment 2 Based on the results of experiment 1 and the solubility test, the following dose levels were selected for mutagenicity testing. Without S9-mix: 10, 20, 30, 40, 45, 50 and 55 pg/ml exposition medium With S9-m1x: 10, 33, 100, 175, 250, 375, 500 and 750 pg/ml exposition medium Evaluation of the toxicity In the absence of S9 metabolic activation, the dose level of 10 ug/ml was not selected for mutant frequency measurement, since this concentration was considered non toxic and not relevant for mutant frequency measurement. The dose levels of 50 and 55 pg/ml were too toxic. In the presence of S9 metabolic activation, the dose level of 10 pg T-6906/ml was not selected for mutant frequency measurement, since this concentration was not relevant for mutant frequency measurement. The dose levels of 375 and 500 pg/ml were not selected for mutant frequency measurement, since these concentrations were too toxic. - page 15 001376 NO. 3773 ?. 16 T-6906 NOTOX Project 223458 The dose levels selected to measure mutant frequencies at the TK-locus were: Without S9-mix: 20, 30, 40 and 45 pg/ml exposition medium With S9-mix : 33, 100, 175, 250 and 750 ng/ml exposition medium In the absence of S9-m1x (Table 3), after 24 hours treatment the cell count of the test substance concentration of 45 pg/ml was reduced by 56% compared to the cell count of the solvent control. The cloning efficiency of the remaining cells directly after treatment of 45 ug/ml showed a reduction of 78%. In the presence of S9-mix, the cell count of the test substance concentration of 750 ig/plate showed no reduction compared to the cell count of the solvent control, whereas the cells counted at concentrations of 100, 175 and 250 pg/ml showed reductions of 62, 49 and 40% respectively. In the cloning efficiency of the remaining cells of the test substance concentration of 750 ^ig/plate no reduction was observed compared to the cloning efficiency of the solvent control, whereas the cloning efficiency of the concentration of 175 pg/ml showed a reduction of 66%. In the presence of S9-mix, in both mutation assays the toxicity of T-6906 in the highest, precipitating concentration 1s less severe than the toxicity in lower, non-precipitating concentrations. The toxicity observed in the cell count during the expression period is not observed in the cloning efficiency of the initially surviving cells. These observations were confirmed in other experiments (Tables 13-16). However, the results of the mutagenicity tests were not acceptable and the experiments were not used to evaluate the mutagenic activity of T-6906, the individual colony count of day 0 and the cell count during the expression period are reported in Appendix 2. Therefore this rare finding in toxicity is probably a test substance related effect and not an accidental finding. Evaluation of mutaoenicitv The spontaneous mutant frequency/in/the solvent-treated control cultures in the absence of S9-mix (first and second experiment) and in the presence of S9-mix (first experiment) wereiii^t-outside the maximum value of the historical control data r a n (HO.7-) and 5.8 x 105 (mean 3.0 x IQ*) in the absence of S9-mix) and /{i7o^>and x 10^ (mean 2.9 x 105 ) in the presence of S9-mix>; for n66 ano-i>3r respectively). But these values were below the 10 mutants per 105 clonable cells, which was defined in the "Acceptability of assay* as an acceptable background for this assay. Therefore, this test is adequate and considered to be acceptable. The spontaneous mutant frequency in the solvent-treated control culture, in the second experiment in the presence of S9-mix, was between the minimum and maximum value of the historical control data range ({1.0- and 6.6 x 10^ (mean 2.9 x 105 ) in the presence of S9-mix); for n=63). Mutant frequencies induced by positive control chemicals were increased by 8.6 and 15.7-fold for EMS in the first and second experiment respectively, and by 10.5- and 11.6-fold for DMN, in the first and second experiment respectively (Table 2 and 3). It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. - page 16 - 001377 Dii. 25. ! 399 1 2 :38PK MEDICAL 220 2E 02 T -6906 KO. 2773 NOTOX Project 223458 \i * op-,.**\*V' Vl In the absence of S9 metabolic activation in the first experiment (Table 2) T-6906 bid not induce a significant increase in the mutant frequency at the TK-locus. In the second experiment (Table 3), T-6906 showed a 1.7-fold increase in the mutant frequency at the TK-locus. However, this Increase was observed only at one toxic concentration. The relative cloning efficiency of the remaining cells was 22% compared to the solvent control, whereas 44% of the cells initially survived the treatment. This means that the actual survival of the cells after treatment was only 10% (22% of 44%). Since, no dose response relationship was observed, the effect was observed only in one experiment and the increase was less than two-fold compared to the solvent control, this increase was considered not biologically relevant and T-6906 is considered to be not mutagenic in the absence of S9-mix. In the presence of 8% (v/v) S9 metabolic activation, T-6906 showed 22- and 3.2-fold increases in the first and second experiment respectively in the mutant frequency at t^JQC^lfl4is-r-4fl-the-flrst experiment, the increase was observed only at,,one"very toxic concentratioru>A severe delay in the cell growth at the conc^irtrratiTiTroTDLMQ^ observed after 48 and 72 hours of subculture compared to the cell growth of the solvent control and a reduction of 81% was observed in the cells plated-for the determination of the cloning efficiency of day 3. In the second experiment, the increase in the mutant frequency was observed at test substance concentrations o_250-- - and 750 pg/ml, however no dose response relationship was observed<r"ince the increases were observed in two experiments and the increases in the second experiment were both 1n toxic and non toxic concentrations, these increases are considered to be biologically relevant. Therefore, T-6906 is considered mutagenic in the presence of S9-mix, , In the first experiment, both the number of small and large mutant colonies were increased compared to the solvent control values. In the second experiment, mainly the large mutant colonies were increased compared to the solvent control values. The difference between the numbers of small and large mutant colonies 1n the first and second experiment is a rare finding, usually the amount of small and large colonies the Independent experiments are comparable. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. CONCLUSION In conclusion, T-6906 1s mutagenic in the TK mutation test system under the experimental conditions described in this report. 2 * page 17 001378 j m 25. . 999 12:38PM MEDICAL 220 2E 02 KO. 3773 ?. 18 T-6906 NOTOX Project 223453 REFERENCES 1 Amacher, O.E., Paillet, S.C., Ray, V. "Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicology testing,' Mutation Research, 64:391-406 (1979). 2 Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. and Salsburg, D.S., 1960, Point mutations at the thymidine kinase locus 1n L517BY mouse lymphoma cells. II. Test validation and interpretation, Mutation Res.. 72, 447-474. 3 Ames, B.N., Mc Cann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Saimonella/mammalian microsome mutagenicity test. Mutation Res., 31, 347-364.. 4 Bradley, M.O., Bhuyan &., Francis, M.C., Langenback, R., Peterson, A., Huberman, E. 'Mutagenesis by chemical agents in V-79 Chinese hamster cells: a review and analysis of the literature: a report of the GeneTox Program,* Mutation Research, :81-142 (1981). 5 CUve, 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. 6 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. 7 Clive, D., Spector J.F.S. 'Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,' Mutation Research, 31:17-29 (1975). 8 Cole, J., Arlett, C.F., Green, M.H.L., Lowe, J. and Muriel, W., 1983, A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells, Mutation res., Ill,, 371-386. 9 Cole, J. and Arlett, C.F., 1984, The detection of gene mutations in cultured mammalian cells. In: Mutagenicity testing, a practical approach Ed.. Venlt, S. and Parry, O.M., IRL press limited. 10 Jotz, M. and Mitchell, A.O., 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. 11 Van der Hoeven, J.C.M., Bruggeman, I.M. and Debets, F.M.H., 1984, Genotoxlcity of quercetin 1n cultured mammalian cells, Mutation Res., 136. 9-21. - page 18 - 001379 J'JR 25. ; 399 1 2 ;39PM MEDICAL 220 2E 02 T-6906 NO, 0773 ?. 19 NQTOX Project 223458 TABLE 1 DOSE RANGE FINDING TEST WITH L5178Y MOUSE LYMPHOMA CELLS Relative survival, % of control DOSE (pg/ml) Solvent control 3 10 33 100 333 10001 CELL COUNT AFTER EXPOSURE CLONING EFFICIENCY AFTER EXPOSURE KCE Without metabolic activation 100 100 101 84 120 70 85 86 97 63 33 71 48 113 # c. Ref* 8cc (oo "i5 <1-3 UJL,,3 _ oV. Solvent control 3 10 33 100 333 10001 With metabolic activation 100 96 110 99 107 114 90 100 132 141 120 117 90 136 i. (fc,e> \i.'l ITS V-5 Vo3 V*Z- Solvent control = dimethylsulphoxide ' T-6906 precipitated in the exposition medium v 5*\ page 19 001380 JUN. 25. 2399 12 : 39PM M EDICAL nr 2E T-6906 NO. 3773 ?. 2 0 " ' BEST COPY AVAH A LE NOTOX Project 223458 TABLE 2 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-6906 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM EXPERIMENT 1 iAjJlb tuj<7 DOSE (Pfl/m1) CELL COUNT C.E. AFTER AFTER TREATMENT TREATMENT % OF CONTROL % OF CONTROL ACTUAL SURVI VAL OF THE CELLS % OF CONTROL* C.E. AT TOTAL NO. DAY 3 OF WELLS ABSOLUTE % WITH MUTANTS MUTATION FREQUENCY X 10= Solvent control IQ 18 33 42 yu EMS Without metabolic activation 100 102 93 90 80 4-*%.\c 107 100 BB 147 69 97 77 N( 100 90 137 62 78 62 133 46 (19s 271) 6.5 125 39 (18s 211) 5.8 135 36 (21s 151) 4.9 152 32 (13s 191) 3.9 111 26 (12s 141) 4.3 79 169 (47s 1221). > 55.9 Solvent control 33 100 333 10001 DMN With metabolic activation 100 100 92 95 131 B9 56 56 121 105 102 65 100 87 117 air, rL 127 J ' 66 93 95 111 94 (?? 31 36 (23s 21 (15$ 20 (17s 32 (IBs 131 (72s 131) 61) 31) 141) 591) 108 (53s __551>; 7.2 4.0 3.2 6.3 160.0 t r 75.8 C.E. s Cloning Efficiency Solvent control = dimethylsulphoxlde EMS s Ethylmethanesulphonate OMN * Dimethylnitrosamlne ' T-6906 precipitated in the exposition medium * The actual survival of cells (% of control)=the cell count after treatment (% of control) x the C.E. after treatment (% of control) s = small colonies 1 = large colonies - page 20 001381 J'jN. 25. 1999 1 2 : 39PM ktui UL ilm ci uc T-6906 BEST COPY AVARISI! NOTOX Project 223458 TABLE 3 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-6906 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM EXPERIMENT 2 DOSE {(ig/ml ) CELL COUNT C.E. AFTER AFTER TREATMENT TREATMENT % OF CONTROL % OF CONTROL ACTUAL SURVI VAL OF THE CELLS % OF CONTROL* C.E. AT TOTAL NO. DAY 3 OF WELLS ABSOLUTE % WITH MUTANTS MUTATION FREQUENCY X 10s n V Without metabolic activation Solvent control 100 20 111 SO 64 40 78 45 44 5o W cc EMS 7B 100 67 84 71 22 10 100 74 54 55 10 8 95 36 (10s 261) 7.0 74 31 ( 7s 241) 7.7 B8 21 ( 6s 151) 4.3 61 25 ( 6s 191) 7.4 49 32 ( 2s 301) 12.1 ___ 18 94 (12s 821)} 109 8 V W=c' S"o Solvent control 33 100 175 250 '750' DMN With metabolic activation 100 100 B7 113 30 85 51 34 60 95 65 U B BO 41 100 9B 32 17 i 57 2. 100 1 33 147 37 ( 2s 351} 4.7 182 77 ( 6s 711) 8.5 162 38 ( 4s 341) 4.4 165 39 ( 4s 351) 4.4 - iv' 147 102 ( 5s 971) 14.9 157 85 (10$ 731} 11.1 32 85 (12s 73 l)x - 54.6 C.E. a Cloning Efficiency Solvent control = dlmethylsulphoxide EMS = Ethylmethanesulphcnate DMN a Dimethylnitrosamine 1 T-6906 precipitated 1n the exposition medium * The actual survival of cells (% of control)*the cell count after treatment (14 of control) x the C.E. after treatment (14 of control) s = small colonies 1 = large colonies page 21 - 001382 0 . 2 5 . :999 12 : 39PM MEDICAL 220 2E 02 NO. 3773 ?. 22 T-6906 NOTOX Project 223458 APPENDIX 1 INDIVIDUAL COLONY COUNTS AND CELL COUNTS DURING EXPRESSION PERIOD - Cloning efficiency of the dose range finding test Cell counts during expression period - Cloning efficiency immediately after exposure - Mutation experiments, individual colony counts Table 4 Tables 5*8 Tables 9-12 Dose range finding test Experiment 1 Experiment 2 Abbreviations used: DMN, dimethylnitrosamine EMS, ethylmethanesulphonate Solvent control: dimethylsulphoxide TABLE 4 DOSE RANGE FINDING TEST Dose Empty wells/ (ug/ml) cloning plate 12 Total no. of empty wells Cloning efficiency absolute relative (% of control) Without metabolic activation Solvent control 20 41 69 102 3 33 40 01 66 10 51 43 94 71 33 41 39 SO 80 100 52 49 101 64 393 45 40 93 72 1000 < 25 36 61 115 100 04 70 06 63 71 113 With metabolic activation Solvent control 49 47 96 69 3 39 30 77 91 10 37 36 73 97 33 39 45 04 03 100 44 41 05 01 333 47 50 103 62 1000' 36 39 75 94 100 132 141 120 117 90 136 ' T*6906 precipitated in the exposition medium page 22 001383 T -6906 NOTOX Project 223458 APPENDIX 1 continued EXPERIMENT 1 TABLE 5 CELL COUNTS AND SUBCULTURE DATA Without metabolic activation 1 DAY Q 1 DAY 1 1 DAY 3 1 ITotal amount ofITotal amount of 1 Subculture ! 1 Dose Icells before Icells after .1 x 10s ICell count ' (pfl/ml) 1treatment x 106 ltreatment x 10s % l'ITotal amount3 Me/nn x 10s 1 %2 / Solvent ! control 1 10 1 is - I 33 1 42 e 1 56 1 75 f I 100 5 ) 1 EMS 1 e 8 8 8 8 16 16 16 8 1 4.1 1 4.2 1 3.8 1 3.7 1 3.3 1 4) 1 4) 1 4) 1 4.4 51 3.7 1 1.9 100 53 3.8 1 3.3 174 48 3.4 1 4.7 247 46 3.3 1 3.0 158 41 3.0 1 4.2 221 1 1 1 55 4.0 1 3.0 158 (1) ceils after treatment x 100% cells before treatment (2) cell count x 100% cell count of control (3) cell density0.4 x 10s c/ml (A) eell death (5) not used for mutation assay TABLE 6 CELL COUNTS AND SUBCULTURE DATA With metabolic activation DAY 0 1 DAY 2 1 DAY 3 '1 1Total amount ofITotal amount ofl 1 Subculture 1 1 1 Subculture 1 1 Dose !cells before Icells after .1 -.1 x 106 .ICell count 1 .1 x 10 ICell count %2)(pg/ml)1treatment x 106Itreatment x 106 l%*/ITotal amount3/le/ml x 10s l%2/ITotal amount4 )lc/rnl x 10>! Solvent! control! 33 1 100 1 333 1 1000 5)1 DMN 1 8a 8 a8 8 1 5.2 1 4.8 1 6.8 1 2.9 1 6.3 1 5.3 65 4.0 1 4.6 100 4.0 1 6.1 100 60 4.0 1 4.9 107 4.0 1 6.0 98 85 4.0 1 4.3 93 4.0 1 4.8 79 36 2.8 1 1.8 39 4.0 1 4.4 72 79 4.0 .1 1.8 39 4.0 1 0.7 11 66 4.0 1 1.9 41 4.0 1 3.9 64 (1) cells after treatment x 100% cells before treatment (2) cell count ______ x loo% cell count of control (3) cell density 0.4 x lof e/ml (4) cell density 1.6 x 105 c/ml (5) T -6906 precipitated in the exposition medium - page 23 - 001384 T -6906 NOTOX Project 223458 APPENDIX 1 continued EXPERIMENT 1 TABLE 7 CLONING EFFICIENCY DAY 0 Dose (pg/ml) Empty wells/ cloning plate 12 Total no. of empty wells Cloning efficiency absolute relative (14 of control) Solvent control 10 IB 33 42 56 75 100 EMS 45 ; 44 ; 20 ; 55 ; 461 ; * z 54 ; Without metabolic activation 35 BO 45 B9 33 53 49 104 36 62 43 97 68 77 129 61 85 68 100 88 147 69 97 77 Solvent control 33 100 333 1000' DMN 50 ; 4 ; 51 : 65 ; 49 ; 62 ; 49 53 55 67 47 63 With metabolic activation 99 66 102 63 106 59 132 37 96 69 125 43 1 T-6906 precipitated in the exposition medium * Cell death directly after treatment 100 95 89 56 105 65 page 24 001385 T-6906 NOTOX Project 223458 APPENDIX 1 continued EXPERIMENT 1 TABLE B SELECTION DATA AND CLONING EFFICIENCY Mutant colonies 1Cloning efficiency (at day 3) Dose Mfl/ml [Number of wells with mutants ! par select.i.o.n.p.l.a.t.e... . il 2 3 ITotal INo. of 1 INo. of empty we lisi Total 1 1 cloning piste 1 No. ofl CE empty 1 11 2 1 wells 1 1 1 MF 1 Without metabolic activation Solvent control 10 16 33 42 EMS 1s 1 1 15 9 ia B 16 7 12 6 15 S 113 43 s1 7 10 S6 82 65 45 11 43 51 7a S7 76 5e 34 23 36 11 11 1 46 1 29 1 39 1 31 1 36 1 24 1 32 1 24 1 26 1 30 1 1G9 1 41 11 11 22 1 51 1133 24 1 55 1125 26 1 50 1135 IS 1 42 1152 33 1 63 1111 46 1 87 1 79 1 1 1 6.5 1 5.8 1 49 i a9 1 4.3 1 55.9 With metabolic activation Solvent control 33 100 333 1000' OMN 1 15 5 12 5 16 1 16 5 (22 14 117 17 62 41 01 77 21 23 24 15 12 6 90 11 1 52 29 22 12 23 1 1 36 1 21 1 20 1 32 1 131 1 108 1 I 1 1 1 1 1 41 39 32 41 75 74 1 35 1 76 35 1 74 31 1 63 34 1 75 83 1158 67 1141 1 1 93 1 95 1111 1 94 1 19 1 31 1 1 7.2 1 4.0 1 3.2 1 6.3 1160.0 I 75.8 MF a Mutant frequency par 10s survivors ' T-6906 precipitated in the exposition medium page 25 - 001386 J'JK, 25. 1999 12:40PM MEDICAL 220 2E 02 NO. 2773 ?. 26 T-6906 NOTOX Project 223458 APPENDIX 1 continued EXPERIMENT 2 TABLE 9 CELL COUNTS ANO SUBCULTURE DATA Without metabolic activation 1 DAY 0 I 1 DAY 1 1 DAY 3 ITotal amount oflTotal amount ofl 1 Subculture 1 Dose 1celIs before fcells after .1 .. 1 x 106 (Cell count (jig/fnl) 1treatment x 106 ltreatment x 10l%1MTotal amount3 !1c/ml x 10s %2> Solvent 1 " otrSi 1 1 20 i 30 1 40 i 4550 fat I 1 55 5) 1 EMS i B 8 a 6 a a 16 16 a 1 4.5 1 3.9 1 5.0 1 2.9 1 3.5 1 2.0 1 1.7 1 1.7 1 3.5 56 4.0 1 4.2 100 49 3.7 1 2.6 67 63 4.0 t 3.0 71 36 2.7 1 3.1 74 44 3.3 1 2 9 69 25 1.9 1 1.7 40 11 1.7 1 4) 11 1.7 1 4) 44 3.3 1 1.1 26 (1) ' cells after treatment x 100% cell* before treatment (2) cell count ______ x 100% cell count of control345 (3) cell density 0.4 x 10s c/ml (4) cell death (5) not used for mutation assay - page 26 001387 J'J#. 25. 2399 12.40PK MEDICAI 220 2E 02 NO, 3773 3 37 T-6906 NOTOX Project 223458 APPENDIX 1 continued EXPERIMENT 2 TABLE 10 CELL COUNTS AND SUBCULTURE DATA With metabolic activation 1 DAY 0 1 DAY 2 1 DAY 3 1 (Total amount of 1Total amount ofl 1 Subculture 1 1 1 Subculture 1 1 Dosa Scella before 1cells after J ..1 x 10 ..ICell count 1 .1 x 10 iCel1 count 1 (pg/ml)1treatment x 101 treatment x 106 l%1'ITotal amount3!Ic/ml x 10s (%2)lTotal amount4)Ic/ml x 10J 1*3) Solvent! control 1 10 >i 33 ! 100 1 175 1 250 ,.l 375 )| 500 1 750 7 >! DMM 1 B 8 8 6 8 8 16 16 16 S 1 5.5 1 4.5 1 4.B 1 2.1 1 2.8 ! 3.4 1 4.9 . 1 4.9 1 9.3 1 4.4 69 4.0 ! 4.8 100 4.0 1 4.4 100 56 4.0 1 3.8 79 4.0 1 4.6 105 60 4.0 I 3.8 79 4.0 1 3.7 64 26 1.9 1 2.8 56 4.0 1 4.4 1Q0 35 2.5 1 1.6 33 5) 1 3.8 86 43 3.0 1 2.7 56 4.0 1 2.5 57 31 4.7 1 0.2 4 5} ! 0.2 5 31 4.7 1 0.2 4 5) ' 1 0.2 5 56 8.0 1 3.5 73 4.0 1 4.1 93 55 4.0 1 1.4 29 5) 1 1.9 43 (1) cell after treatmant x 100% calls bafora treatment (2) cell count_________ x 100% cell count of control34567 (3) call density 0.4 x 10 c/iol (4) call danslty 1.6 x 10s c/ml (5) no subculture (6) not used 1n the mutation assay (7) T-6906 precipitated 1n the exposition medium page 27 - 001388 T -6906 N0T0X Project 223458 APPENDIX 1 continued EXPERIMENT 2 TABLE 11 CLONING EFFICIENCY DAY 0 Dosa (pg/mi) Empty wells/ cloning plate 12 Total no. of empty wells Cloning efficiency absolute relative (% of control) Solvent control 10 ao 30 40 4S 50 55 EMS 36 ; 56 ; 54 ; 52 ; 55 ; 62 ; 93 ; 96 ; 88 ; Without metabolic activation 41 77 41 97 50 104 38 90 45 100 75 157 96 189 95 191 87 175 91 68 61 76 65 20 2 1 9 100 75 67 84 71 22 2 1 10 Solvent control 10 33 100 175 250 375 500 750' DMN 42 ; 40 ; 37 ; 41 ; 66 ; 40 ; 95 ; 56 ; 35 ; 66 ; 34 40 30 46 73 40 93 64 29 63 With metabolic activation 76 93 80 88 67 105 87 79 139 32 80 88 IBS 2 120 47 64 110 131 38 100 95 113 85 34 95 2 51 118 41 ' T>6906 precipitatati in tha exposition medium page 28 001389 JUN. 25. '999 12:40 PM MEDICAL 220 2E 02 NO. 3723 ?. 29 T -6906 NOTOX Project 223458 APPENDIX 1 continued EXPERIMENT 2 TABLE 12 SELECTION DATA AND CLONING EFFICIENCY 1 Mutant colonies iCloning efficiency (at day 3) Dose jig/mi 1Number of wells with mutants 1 per selection plate |..................... . i1 2 9 ITotal INo. of empty wells 1 Total 1 INo. of 1 cloning plate 1 No. ofl CE limitants empty 1 1 11 2 1 wells 1 1 l 1 1 MF 1 1 Without metabolic activt ion Solvent control 20 30 40 43 EMS 1s 1 1 1 5 10 14 8 12 7 1 1 10 1 1 10 1 5 28 s1 28 28 14 24 17 4 27 S1 38 18 34 35 0 13 3 27 11 1 1 36 1 36 1 31 1 49 1 21 1 43 1 25 1 49 1 32 1 60 1 94 1 84 II 38 1 74 1 95 42 1 91 1 74 37 1 80 1 88 5S 1104 l 61 58 1118 1 49 77 1161 1 18 1 7.0 1 7.7 1 4.3 1 7.4 1 12.1 1109.8 Solvent control 33 100 175 250 750 DMN I I 1 0 10 1 0 26 1 2 10 11 8 1 1 40 1 5 20 1 3 25 1 14 3 24 2 13 2 19 3 29 2 31 5 22 With metabolic activation 330111 4 222211186411e 11111!11 1033B3789258757 11 1 1 11 11 72221120066822 18 15 20 15 22 20 70 1 44 1 31 1 38 1 37 1 44 1 40 1140 1147 1182 1162 1165 1147 1157 1 32 1 4.7 1 8.5 1 4.4 1 4.4 1 14.9 1 11.1 1 54.6 MF Mutant frequency per 10s survivors page 29 001390 J'JH. 25. 1399 12:40PM T -6906 MEDICAL 220 2E 02 NO. 0723 ?. 30 NOTQX Project 223458 APPENDIX 2 INDIVIDUAL COLONY COUNTS AND CELL COUNTS OF NOT REPORTED EXPERIMENTS EXPERIMENT 3 TABLE 13 CELL COUNTS ANO SUBCULTURE DATA With metabolic activation 1 DAY 0 1 DAY 2 1 DAY 3 1 ITotal amount ofITotal amount of 1 1 Subculture 1 1 1 Subculture 1 1 Dose 1cel s bifore Jcells after J ..1 x 10 ICall coun X 106 ICell count) (ig/m1)l treatment x IO6 1treatment x 10l%JlITotal amount3)1c/ml x 10^!%*)!lrotai amount4 te/ml x 105| %2> Solvent 1 control ! 100 1 250 1 500 1 750 =) 1 DMN 1 8 8 e 8 8 B 1 4.4 1 2.6 1 3.0 1 3-4 1 4.1 1 4.1 55 4.0 1 3.3 100 4.0 1 6.5 100 33 2.4 1 3.4 103 4.0 1 5.5 85 38 2.8 1 2.5 76 4>Q 1 5.2 80 43 3.1 1 1.3 39 6) 1 3.a 58 51 3. B 1 Q.a 24 6) 1 1.5 23 51 3.a l 2.1 64 4.0 1 3.1 48 (1) cells after treatment x 100% cells before treatment (2) cell count_________ x 100% cell count or control (3) cell density 0.4 x lof c/ml (4) cell density 1.6 x 10s c/ml (5) T-6906 precipitated in the exposition medium (6) no subculture TABLE 14 CLONING EFFICIENCY DAY 0 Dose (ng/mi) Empty wells/ cloning plate 12 Total no. of empty wells Cloning efficiency absolute relative (% of control) Solvent control 100 250 500 750 OWN 46 ; 52 ; 47 ; 41 ; 57 : 50 ; 43 48 45 41 45 52 With metabolic activation 89 77 100 65 92 74 82 85 102 63 102 63 100 84 96 110 82 82 (1) T-6906 precipitated in the exposition medium page 30 001391 JUKI 25. 2399 12:4 Pii T-6906 MEDICAL 220 2E 02 HO. 3 7 13 ?. 31 NOTOX Project 223456 APPENDIX 2 continued EXPERIMENT 4 TABLE 15 CELL COUNTS AND SUBCULTURE DATA With metabolic activation 1 DAY 0 DAY 2 1 DAY 3 1 1Total amount of(Total amount of! 1 Subculture 1 1 1 Subculture 1 1 Dose (cells before Icells after J .. 1 x 10 _ (Cell count ) ,,1 x 10 ^ ICetl count! (ug/ml)ltreatment x 10ltreatment x 101 >(Total amount3'1c/ml x 10s!%2'JTotal amount4'le/ml x 105) %2> Solvent! control 1 100 1 260 1 375 1 600 1 750 aii DMN 1 8 8 8 8 16 16 8 1 5.6 1 5.2 1 3.3 1 3.1 1 9.3 1 11.1 1 4.7 70 4.0 65 2.4 41 9.0 39 2.9 58 3.1 69 3.8 59 3.8 3.5 100 3.1 89 1.6 46 1.8 51 1.4 40 1.1 31 1.1 31 4.0 40 40 | ) ) 1 6.4 ! 6.6 1 3.4 ! 4.B 1 4.0 1 0.6 1 1.4 100 103 53 75 69 9 22 (1) calls after treatment * 100% cells before treatment (2) cell count x 100% cell count of control lof(3) cell density 0.4 x c/ml (4) cel! density 1.6 x 10s c/ml (5) T-6906 precipitated 1n the exposition medium (6) no subculture TABLE 16 CLONING EFFICIENCY DAY 0 Dose (pg/ml) Empty wells/ cloning plate 12 Total no. of empty wells Cloning efficiency absolute relative (% Of control) Solvent control 100 250 375 500 750 1) DMN 51 ; 48 ; 53 ; 62 ; 65 ; 55 ; 74 ; 58 50 61 57 57 49 76 With metabolic activation 109 57 98 67 114 52 119 48 122 45 104 61 150 25 100 118 91 84 79 107 44 (1) T-6906 precipitated 1n the exposition medium - page 31 001392