Document vBn5XQwo1VNQeLjxoYVLbpzQb

ARa%-03l8 REPORT EVALUATION OF THE ABILITY OF T-5869 TO INDUCE CHROMOSOME ABERRATIONS IN CULTURED PERIPHERAL HUMAN LYMPHOCYTES (WITH INDEPENDENT REPEAT) NOTOX Project 115943 NOTOX Substance 38196 - page l of 26 - Rfc-C&VED NOV - 9 1994 T"C/a O t--'O Oi \ 005605 T -5869 STATEMENT OF GLP COMPLIANCE NOTOX Project 115943 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 following exception: Stability of the test substance in the vehicle was unknown. Study Director Ing. E.J. van de Waart - page 2 - 005606 T -5869 QUALITY ASSURANCE STATEMENT NOTOX B.V., 's-Hertogenbosch, The Netherlands. NOTOX Project 115943- 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 February 23, 1994 March 23, 1994 April 19, 1994 REPORTING DATES January 24, 1994 February 14, 1994 February 23, 1994 March 24, 1994 April 19, 1994 Quality Assurance Manager C.J. Mitchell B.Sc. Date: U - S ' % . - page 3 - 005607 T -5869 REPORT APPROVAL STUDY DIRECTOR: NOTOX Project 115943- Ing. E.J. van de Waart 71/1-- Date: MANAGEMENT: Dr. I.C. Enninga Technical Director - page 4 - 005608 T-5869 NOTOX Project 115943_ 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.J. van de Waart A.M.C. Bertens Start : January 26, 1994 Completed : March 30, 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 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 0.9 % (v/v). - page 5 - 005609 T -5869 NOTOX Project 115943- 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. 473: "Genetic Toxicology: In Vitro Mammalian Cytogenetic Test", (adopted May 26. 1983). - European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.lO: "Other Effects-Mutagenicity: In Vitro Mammalian Cytogenetic Test". EEC Publication no. L383 (adopted December, 1992). 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 structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic system (S9-mix). - page 6 - G05610 T-5869 Sf NOTOX Project 115943 - Justification and rationale of the test system Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes. In combination with a mammalian metabolizing system (S9-mix) also indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, could be tested for clastogenic effects in vitro. Following treatment, cell division was arrested in the metaphase stage of the cell cycle by addition of the spindle poison colchicine. Structural chromosome changes such as breaks, gaps, minutes, dicentrics and exchange figures were examined microscopically in cultures treated with the test substance and the results were compared with those of the control (vehicle-treated) cultures. Chromosome aberrations were generally evaluated in the first post-treatment mitosis. The appearance of the first post-treatment mitosis could be considerably delayed, due to toxic insult of the cells. Therefore, cells were harvested at 24 h and 48 h after beginning of treatment to cover the interval in which maximum aberration frequency was expected. A test article which Induced a positive response in this assay was presumed to be a potential mammalian cell clastogenic agent. - page 7 - 005611 T-5869 NOTOX Project 115943^ MATERIALS AND METHODS TEST SYSTEM Test System Rationale Source CELL CULTURE Cultured peripheral human lymphocytes Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC). Healthy adult male volunteers: pilot study : age 28 AGT = 15.4 h (Mar.'94) experiment 1: age 34 AGT = 16.6 h (Dec.'93) experiment 2: age 31 AGT = 15.6 h (Dec.'93) AGT s Average Generation Time Blood samples Blood samples were taken from a healthy adult male volunteer by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25C. Within 4 h after withdrawal lymphocyte cultures were started. FIO complete culture medium FIO complete culture medium consisted of Ham's FIO medium without thymidine and hypoxanthine (Gibco), supplemented with 20* heatinactivated (56C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 iig/ml respectively), sodium bicarbonate (2 g/1) and 30 U/ml heparin. Cell culture conditions Whole blood was cultured in FIO complete culture medium with Phytohaemagglutinin (Murex). Per culture (5 ml FIO complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added. 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, humidity and C02*percentage were monitored during the experiment. - page 8 - 005612 T -5869 NOTOX Project 115943C REFERENCE SUBSTANCES Negative control: The vehicle of the test article. Positive controls: Without metabolic activation (-S9-mix): Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.2 yg/ml (solvent: HBSS) for a 24 h treatment period and 0.1 yg/ml (solvent: HBSS) for a 48 h treatment period. With metabolic activation (+S9-mix): Cyclophosphamide (CP; CAS no. 50-18-0. Endoxan-Asta, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 15 yg/ml (solvent: HBSS) for a 3 h treatment period (24 h fixation period). Solvent for reference substances HBSS = Hank's Balanced Salt Solution without calcium and magnesium. METABOLIC ACTIVATION SYSTEM Preparation of S9-homogenate*: Rat liver microsomal enzymes were routinely prepared from adult male Wlstar or Sprague Oawley 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). *) Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Sal monella/Mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. - page 9 - 005613 T -5869 NOTOX Project 115943,1 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 MgCl2-6H20; 2.46 mg KC1; 1.7 mg glucose-ephosphate; 3.4 mg NADP; 4 ymol HEPES and 0.5 ml S9. The above solutions were mixed and filter (0.22 ym)-sterilized (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 cell suspension. EXPERIMENTAL PROCEDURE Cytogenetic test The test was carried out with minor modifications as described by Evans (1984)*. The test substance was tested both with and without S9-mix in duplicate in two independent experiments. Lymphocyte cultures (0.4 ml blood of a healthy male donor in 5 ml medium) were cultured for 48 h and thereafter exposed in duplicate to selected doses of the test substance for 24 h and 48 h without S9-mix or for 3 h with S9-mix. An appropriate range of dose levels was chosen to determine the concentrations which caused inhibition of the mitotic index. In case the test compound was difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. Concentrations exceeding 5 mg/ml were not tested. After 3 h treatment, the cells exposed to the test substance in the presence of S9mix were rinsed once with 5 ml of HBSS and incubated in 5 ml growth medium for another 20-22 h (first fixation period) or for 44-46 h (second fixation period). The cells which were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after treatment and were fixed immediately after 24 h and 48 h. During the last 3 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 yg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56SS potassium chloride solution at 37C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v). For the independent repeat the 24 h fixation period was needed only. *) Evans, H.J., 1984, Human Peripheral Blood Lymphocytes for the Analysis of Chromosome Aberrations in Mutagen Tests. In: Handbook of Mutagenicity Test Procedures, B.J. Kilbey, M. Legator, W. Nichols and C. Ramel eds, 405-427, Elsevier Science Publishers B.V., Amsterdam. - page 10 - 005614 T-5869 NOTOX Project 115943^ Preparation of slides Fixed cells were dropped onto previously cleaned (24 hours immersed in a 1:1 mixture of 9696 ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and group number) slides. Two slides were stained for p1r0e-p3a0remdinpewrithcul5t%ur(ev./v)SliGdieesmsawerseolaultiloonwedintotapdrywataenrd. thereafter Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in DePeX and mounted with a covers!ip. Mitotic index/dose selection The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. For the first fixation time (24 h harvest) chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50-20% (if present) whereas the mitotic index of the lowest dose level was approximately the same as the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. For the second fixation time (48 h harvest) one appropriate dose level was selected for scoring of chromosome aberrations. In order to obtain the appropriate concentration range for the chromosome aberration test a pilot experiment was performed. Experimental conditions were identical to those in the chromosome aberration test, except that one culture per concentration was used and with the omission of the 48 h fixation period in the presence of S9-mix. Analysis of slides for chromosome aberrations For control of bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. Only metaphases containing 46 chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated. ACCEPTABILITY OF ASSAY A chromosome aberration test was considered acceptable if it met the following criteria: a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably fall within the laboratory historical control data range. b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations. - page 11 - 00SG15 T -5869 NOTOX Project 115943 DATA EVALUATION AND STATISTICAL PROCEDURES A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant increase in the frequency of aberrations was observed in the absence of a clear dose-response relationship, but the results were reproducible in an independently repeated experiment. A test substance was considered negative (not clastogenic) in the chromosome aberration test if: a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics: X2 = (N-l) x (ad-bc)2 (a+b) (c+d) (a+c) (b+d) where b = the total number of aberrant cells in the control cultures. d = the total number of nonaberrant cells in the control cultures, ng = the total number of cells scored in the control cultures, a = the total number of aberrant cells in treated cultures to be compared with the control. c * the total number of nonaberrant cells in treated cultures to be compared with the control. n^ = the total number of cells scored in the treated cultures. N = sum of ng and n^ If P I" | X2 > |_ , (N-l) x (ad-bc)2 "I | (a+b) (c+d) (a+c) (b+d) _ | (two-tailed) is small (P< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 9535 confidence level. - page 12 - 005G16 T-5869 NOTOX Project 115943C RESULTS DOSE SELECTION In a preliminary study blood cultures were treated with 1, 3, 10, 33 and 100 yg T-5869 per ml culture medium with and without S9-mix. Higher concentrations could not be tested, because of the low solubility of the test substance in the culture medium. TABLE 1 MITOTIC INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 PILOT STUDY Test substance concentration (yg/ml) 24 h fixation period Control) 1 3 10 33 100b ) 48 h fixation period Control) 1 3 10 33 100b ) Number of metaphases per 1000 cells Absolute Percentage of control Without metabolic activation (-S9-mix) 66 100 73 111 77 117 56 85 52 79 42 64 38 100 35 92 37 97 53 140 47 124 27 71 24 h fixation period Control) 1 3 10 33 100b) With metabolic activation (+S9-mix) 79 100 85 108 67 85 82 104 71 90 82 104 a) DMSO b) Slight precipitation of the test substance in culture medium. - page 13 - G05G17 T-5869 NOTOX Project 115943. Based on the results of this pilot study the following dose levels were selected for the chromosome aberration test: Experiment 1 + 2 Without S9-mix : 3, 10, 33 and 100 yg/ml culture medium With S9-mix : 3, 10, 33 and 100 yg/ml culture medium Tables 2 and 3 show the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances. Based on these observations the following doses were selected for scoring of chromosome aberrations: Experiment 1 Without S9-mix With S9-mix 10, 33 and 100 yg/ml culture medium (24 h fixation period) 100 yg/ml (48 h fixation period) 10, 33 and 100 yg/ml culture medium (24 h fixation period) 100 yg/ml (48 h fixation period). Experiment 2 Without S9-mix With S9-mix 10, 33 and 100 yg/ml culture medium (24 h fixation period) 10, 33 and 100 yg/ml culture medium (24 h fixation period) CYTOGENETIC TEST The ability of T-5869 to induce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in two independent experiments. The results of duplicate cultures are indicated by A and B. The scores for the numbers of aberrant cells (inclusive and exclusive gaps) and the numbers of the various types of chromosome aberrations at the various concentrations of the test substance are presented in Tables 4-9. The criteria according to which the aberrations were classified are outlined in Appendix 1. Both in the presence and absence of S9-mix the test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. - page 14 - C5G18 T-5869 NOTOX Project 115943. The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {i.e. 1.0 + 1.1 (mean + standard deviation) aberrant cells per 100 metaphases (without 59mix; gaps excluded) and 0.6 0.7 aberrant cells per 100 metaphases (with 59mix; gaps excluded)}. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly. Finally, it is concluded that this test should be considered valid and that T-5869 is not clastogenic under the experimental conditions of this test. - page 15 - 005619 T -5869 NOTOX Project 115943^ TABLE 2 MITOTIC INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T -5869 Experiment 1 Test substance concentration (ug/ml) 24 h fixation period Control3 ) 3 10 33 100c ) MMC-C; 0.2 yg/ml 48 h fixation period Control3 ) 3 10 33 100c) MMC-C; 0.1 yg/ml Number of metaphases per 1000 cells b) Absolute Percentage of control Without metabolic activation (-S9-mix) 29 43 47 47 41 21 - 50 45 45 45 31 23 100 111 116 116 91 56 35 41 55 40 -45 23 - 32 60 44 36 38 24 100 151 148 113 124 70 24 h fixation period Control3) 3 10 33 100) CP; 15 yg/ml 48 h fixation period Control3 ) 3 10 33 100c) With metabolic activation (+S9-mix) 76 108 - 75 79 76 34 - 87 85 63 68 75 34 100 118 85 90 93 42 87 - 64 97 - 74 68 - 99 89 - d) 87 - 101 100 113 111 118 125 a) DMSO b) duplicate cultures c) Slight precipitation of the test substance in culture medium. d) Culture infected with fungi - page 16 - 005620 T-5869 NOTOX Project 115943 TABLE 3 MITOTIC INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 Experiment 2 Test substance , concentration (yg/ml) 24 h fixation period Control3 ) 3 10 33 100c ) MMC-C; 0.2 yg/ml Number of metaphases per 1000 cells b) Absolute Percentage of control Without metabolic activation (-S9-mix) 42 - 39 44-46 23 - 46 60-26 39 - 43 41 - 43 100 111 85 106 101 104 24 h fixation period Control3 ) 3 10 33 100c ) CP; 15 yg/ml With metabolic activation (+S9-mix) 70 - 66 77 - 64 65 - 56 73-56 79 - 72 54 - 34 100 104 89 95 111 65 a) DMSO b) duplicate cultures c) Slight precipitation of the test substance in culture medium. : page 17 - 005621 T -5869 NOTOX Project 115943-1 TABLE 4 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (Without S9-mix)a) 24 h fixation period Experiment 1 Cone ug/ml DMSO 10 (0.9% V/V) ug/ml 33 ug/ml 100 ug/ml MMC-C 0.2 ug/ml Culture A B A+B A B A+B A B A+B A B A+B A B A+B No. of cells scored 100 100 200 100 100 200 100 100 200 100 100 200 50 50 100 No. of cells with aberrations 2 3 5 1 4 ( gaps) 5 2 13 *** 4 6 10 38 32 70 No. of cells with aberrations (- gaps) 12 3 0 3 3 112 ** 4 3 7 36 31 67 9' 11 11 3 32 9" 1 1 3 b' 1 2 3 11 53 25 30 b" 3 3 n' 2 m" exch. die d' 1 13 8 misc. total aberr (+ gaps) 23 24 21 46 49 43 total aberr (- gap) 12 03 11 43 43 41 a) Abbreviations used for various types of aberrations are listed in appendix 1. The nunerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration. misc. - (Miscellaneous) aberrations not belonging to the ones mentioned above. *) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or ** P < 0.001. - page 18 - & 05622 T -5869 NOTOX Project 115943 TABLE 5 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (With S9-mix)a) 24 h fixation period Experiment 1 Cone ug/ml DMSO 10 (0.9% V / V ) ug/ml 33 ug/ml 100 ug/ml CP 15 ug/ml Culture A B A+B A B A+B A B A+B A B A+B A B A+B No. of cells scored 100 100 200 100 100 200 100 100 200 100 100 200 50 100 150 No. of cells with aberrations (+ gap) 42 64043 362 ** 2 4 25 44 69 No. of cells with aberrations 3 1 4 (- gaps) g' 1 1 g" b* 1 1 0 0 0 3 14 4 12 31 2 13 *** 25 44 69 1 21 2 2 35 52 b" 1 38 m* 1 m" exch. 37 die d* misc. total aberr (+ gaps) 42 40 43 22 41 71 total aberr (- gaps) 31 00 31 21 41 67 a) Abbreviations used for various types of aberrations are listed in appendix 1. The nunerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration. misc.- (miscellaneous) aberrations not belonging to the ones mentioned above. *) Significantly different from control group (Chi-square test), * P <, 0.05, ** P < 0.01 or *** P < 0.001. - page 19 - *05623 T-5869 NOTOX Project 115943 TABLE 6 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (Without S9-mix)a ) 48 h fixation period Experiment 1 Cone ug/ml DMSQ 100 (0.9% v/v) ug/ml MMC-C 0.1 ug/ml Culture A B A+B A B A+B A B A+B No. of cells scored 100 100 200 100 100 200 50 50 100 No. of cells with # aberrations 5 4 9 3 6 9 28 30 58 (+ Bps) No. of cells with #** aberrations 3 4 7 2 5 7 27 30 57 (- gaps) 9' 2 11 35 g" b' 24 21 26 30 b" 1 3 11 10 m* 1 m" 1 exch. 1 56 die d' misc. total aberr (+ gaps) 54 36 45 53 total aberr (- gaps) 34 25 c<o CM a) Abbreviations used for various types of aberrations are listed in appendix 1. The numerical variations endoredupllcation (endo) and polyploidy (poly) were not counted as an aberration, misc.- (miscellaneous) aberrations not belonging to the ones mentioned above. *) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001. - page 20 - 005624 T-5869 NOTOX Project 115943C TABLE 7 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (With S9-mix)a) 48 h fixation period Experiment 1 Cone ug/ml MSO 100 (0.9% V/V) ug/rnl Culture A B A+B A B A+B No. of cells scored 100 100 200 100 100 200 No. of cells with aberrations 0 2 2 3 2 5 (+ gaps) No. of cells with aberrations (- Saps) Oil 112 9' 1 2 1 9" b' 1 1 1 b" m* m" exch. die d' misc. total aberr (+ gaps) 02 32 total aberr (- gaps) 01 11 a) Abbreviations used for various types of aberrations are listed in appendix 1. The nunerical variations endoredupllcatlon (endo) and polyploidy (poly) Mere not counted as an aberration. misc.B (miscellaneous) aberrations not belonging to the ones mentioned above. *) Significantly different from control group (Chi>square test), P < 0.05, ** P < 0.01 or *** P < 0.001. - page 21 - 005625 T -5869 NOTOX Project 115943 - TABLE 8 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (Without S9-mix)a) 24 h fixation period Experiment 2 Cone ug/ml Culture No. of cells scored OMSO 10 (0.9% v/v) lig/ml A B A+B A B 33 pg/ml A+B A B 100 ug/ml A+B A B MMC-C 0.2 pg/ml A+B A B A+B 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200 No. of cells with aberrations (+ g a p * ) 112 112 112 CM *4 *** 29 2B 57 No. of cells with aberrations (- gaps) 10 *** 1 0 0 0 0 0 0 0 0 0 23 24 49 g' 1 11 11 1 45 g* 1 b' 1 10 8 b" 14 10 m" exch. 28 die d' misc. total aberr (+ gaps) 11 11 11 11 30 31 total aberr (- gaps) 10 00 00 00 26 26 a) Abbreviations used for various types of aberrations are listed in appendix 1. The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration. misc. (miscellaneous) aberrations not belonging to the ones mentioned above. *) Significantly different from control group (Chi-square test), # P < 0.05, ** P < 0.01 or *** P < 0.001. - page 22 - 05626 T -5869 NOTOX Project 115943 TABLE 9 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (With S9-mix)a) 24 h fixation period Experiment 2 Cone ug/ml Culture No. of cells scored DMSO 10 (0.9* V / V ) ug/ml A B A+B A B 33 ug/ml A+B A B 100 pg/ml A+B A B CP 15 yg/ml A+B A B A+B 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200 No. of cells with aberrations (+ gaps) No. of cells with aberrations (- gaps) g* g" b' b" 112 00 0 11 Oil Oil 1 112 112 1 1 Oil * 21 24 45 *** 0 0 0 17 17 34 1 49 1 9 13 97 n* " exch. die 4 d' misc. endo total aberr (+ gaps) 11 01 11 01 26 30 total aberr (- gaps) 00 01 11 00 22 20 a) Abbreviations used for various types of aberrations are listed in appendix 1. The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration. misc. (miscellaneous) aberrations not belonging to the ones mentioned above. *) Significantly different from control group (Chi-square test), * P < 0 .05, * P < 0.01 or *** P < 0 .001. - page 23 - 005627 T-5869 NOTOX Project 115943 APPENDIX 1 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE PORTRAITS Aberration Abbreviation Description Chromatid gap 9' Chromosome gap 9" Chromatid break b' Chromosome break b" Chromatid deletion d' Minute m' Double minutes m" Dicentric chromosome die Tricentric chromosome trie An achromatic lesion which appears as an unstained region in the chromatid arm, the size of which is equal to or smaller than the width of the chromatid and the apparently "broken" segments of the chromatid arm are in alignment. An achromatic lesion which appears as an unstained region in both chromatids at the same position, the size of which is equal to or smaller than the width of the chromatid and the apparently "broken" segments of the chromatids are in alignment. An achromatic lesion in a chromatid arm, the size of which is larger than the width of the chromatid. The broken segments of the chromatid arm are aligned or unaligned. An achromatic lesion in both chromatids at the same position, the size of which is larger than the width of the chromatid. The broken segments of the chromatids are aligned or unaligned. Deleted material at the end of a chromatid arm. A single, usually circular, part of a chromatid lacking a centromere. Two, usually circular, parts of a chromatid lacking a centromere. A chromosome containing two centromeres. A chromosome containing three centromeres. - page 24 - 005628 T-5869 APPENDIX 1 Continued NOTOX Project 1X59431 Aberration Ring chromosome Exchange figure Chromosome intrachange Pulverized chromosomes Multiple aberrations Polyploidy Endoreduplication Abbreviation Description r exch. intra P A ring structure with a distinct lumen. An exchange(s) between two or more chromosomes resulting in the formation of a tri- or more-armed configuration. A chromosome intrachange is scored after rejoining of a lesion within one chromosome. A fragmented or pulverized chromosome ma poly endo A metaphase spread containing ten or more of the above mentioned aberrations (chromatid and chromosome gaps not included). A chromosome number that is a multiple of the normal diploid number. A form of polyploidy in which each centromere connects two or four pairs of chromatids instead of the normal one pair. - page 25 - 005629 T -5869 NOTOX Project 115943^ APPENDIX 2 STATISTICAL EVALUATION OF TEST RESULTS Chi-square Test TOTAL NUMBER OF CELLS WITH ABERRATIONS; TREATMENT/CONTROL COMPARISON, (INCLUSIVE/EXCLUSIVE GAPS). Experiment 1 TREATMENT DOSE (yg/ml) S9-MIX GAPS P-VALUE two-sided DECISION AT 95515 CONFIDENCE LEVEL 24 h fixation period MMC-C (0.2) - <0.0004 <0.0004 significant significant CP (15) + + <0.0004 significant <0.0004 significant 48 h fixation period MMC-C (0.1) + <0.0004 significant <0.0004 significant Experiment 2 TREATMENT DOSE (yg/ml) S9-MIX GAPS 24 h fixation period MMC-C (0.2) - + CP (15) ++ P-VALUE two-sided <0.0004 <0.0004 <0.0004 <0.0004 DECISION AT 95% CONFIDENCE LEVEL significant significant significant significant - page 26 - 005630