Document jMbb4ZGDw6Kp9nK7QMaxQvLy

REPORT EVALUATION OF THE ABILITY OF T-5869 TO INDUCE CHROMOSOME ABERRATIONS IN CULTURED PERIPHERAL HUNAN LYMPHOCYTES (WITH INDEPENDENT REPEAT) NOTOX Project 115943 NOTOX Substance 38196 page 1 of 26 - 9 T-5869 STATEMENT OF GLP COMPLIANCE NOTOX Project 115943 NOTOX B.V., Is-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 ... ................... Date: 5-/o5/m, 9 page 2 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: 11-5-cit4_ page 3 - T-5869 REPORT APPROVAL STUDY DIRECTOR: MANAGEMENT: NOTOX Project 115943L..@ Ing. E.J. van de Waart Date: 05104@&+ Dr. I.C. Enninga Technical Director ......................... Date: c>6106 linch page 4 T-5869 NOTOX Project 11594@- 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 Is-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 concentrationswere prepared directly prior to use. The final concentration of the solvent in the culture medium amounted to 0.9 % (V/V). page 5 - 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: NGenetic Toxicology: In Vitro Mammalian Cytogenetic TestO, (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.10: mother Effects-Mutagenicity:In Vitro Mammalian Cytogenetic Testm. EEC Publication no. L383 (adofte-dDecember, 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 T-5869 NOTOX Project 115949 - Justificationand rationale of the test system Stimulated cultured human lymphocytes were used because they are sensitive indicatorsof 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 clastogeniceffects 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-treatmentmitosis could be considerablydelayed, 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 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.194) experiment 1: age 34 AGT = 16.6 h (Dec.193) experiment 2: age 31 AGT n 15.6 h (Dec.'93) AGT 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 250C. Within 4 h after withdrawal lymphocyte cultures were started. F10 complete culture medium F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% heatinactivated (56OC; 30 min) foetal calf serum (Gibco), L-glutamine(2 mM), penicillin/streptomycin(50 U/ml and 50 ug/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 F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added. Environmental conditions All incubationswere carried out in a humid atmosphere (80-95%) containing 5% C02 in air. in the dark at 370C. The temperature, humidity and C02-percentage were monitored during the experiment. - page 8 T-5869 NOTOX Project 115943L: REFERENCE SUBSTANCES Negative control: The vehicleof the test article. Positive controls: Without metabolic activation (-S9-mix): MitomyeTn(-CMMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.2 ug/ml (solvent: HBSS) for a 24 h treatment period and 0.1 ug/ml (solvent: HBSS) for a 48 h treatment period. With metabolicactivation (+ 9-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 concentrationof 15 ug/ml (solvent: HBSS) for a 3 h treatment period (24 h fixationperiod). 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 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 intraperitoneallywith 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 3.2hours preceding sacrifice).The livers of the rats were removed aseptically, and washed in cold (OOC) 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 (-1960C). Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/Mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. page 9 T-5869 NOTOX Project 115943-- 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 MgC12.6H20; 2.46 mg KCI; 1.7 mg glucose-6phosphate; 3.4 mg NADP; 4 umol HEPES and 0.5 ml S9. The above solutions were mixed and filter (0.22 um)-sterilized(apart from the S9-fraction, which was added after filter-sterilizationof 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 SR-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 -rinsedonce with 5 ml of HBSS and incubated in 5 ml growth medium for another 20-22 h (first fixationperiod) 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 inhibitorcolchicine (0.5 ug/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 0.56% potassium chloride solution at 370C. 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 T-5869 NOTOX Project 11543@: Preparation of slides Fixed cells were dropped onto previously cleaned (24 hours immersed in a 1:1 mixture of 96% ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and group number) slides. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water. 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 coverslip. 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 'Forcontrol 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 reasonablyfall within the laboratory historical control data range. b) The positive control substances should produce a statistically significant (Chi-squaretest, P < 0.05) increase in the number of cells with chromosome aberrations. page 11 T-5869 NOTOX Project 115943 DATA EVALUATION AND STATISTICAL PROCEDURES A test substance was considered positive (clastogenic) in the chromosome aberrationtest if: a) It induced a dose-rele@tedstatisticallysignificant(Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations. b) A statisticallysignificant 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 concentrationsinduced a statisticallysignificant (Chi-square test, P < 0.05) increasein 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-1) x___Cad-bc)2 (a+b) (C+d) (a+c) (b+dy where b = the total number of aberrant cells in the control cultures. d - the total number of nonaberrantcells in the control cultures. no = 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 no and n, if p X2 > (N-1) x (ad-bc',2 (a+b)(C+d)(a+c)(b+d) two-tailed) is small (P< 0.05) the hypothesis that the incidenceof 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 significantlydifferent from the control group at the 95% confidence level. page 12 T-5869 NOTOX Project 115943-: RESULTS DOSE SELECTION In a preliminary study blood cultures were treated with 1, 3, 10, 33 and 100 ug 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 T-5869 PILOT STUDY OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS Test substance concentration (ug/ml) 24 h fixation period Controla) 1 3 10 33 loob) Number of metaphases per 1000 cells Absolute Percentage of control Without metabolic activation (-S9-mix) 616 100 73 ill 77 117 56 85 52 79 42 64 48 h fixation period Controla) 38 100 1 35 92 3 37 97 10 53 140 33 loob) 47 124 27 71 ----------------------------------------------------------------------- With metabolic activation (+S9-mix) 24 h fixation period Controla) 79 100 1 85 108 3 67 85 10 82 104 33 ()Ob) 71 90 82 104 a) DMSO b) Slight precipitation of the test substance in culture medium. page 13 - 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 With S9-mix : 3, 10, 33 and 100 ug/ml culture medium : 3, 10, 33 and 100 ug/ml culture medium Tables 2 and 3 show the mitotic index of cultures (from blood of a healthy male donor) treated with various test @ubstance concentrations or with the positive or negative control substances. Based on these observations the following doses were selected for scoring of chromosome aberrations: Experiment I Without S9-mix With S9-mix : 10, 33 and 100 ug/ml culture medium (24 h fixation period) 100 ug/ml (48 h fixationperiod) : 10, 33 and 100 ug/ml culture medium (24 h fixationperiod) 100 ug/ml (48 h fixationperiod). Experiment 2 Without S9-mix With S9-mix : 10, 33 and 100 ug/ml culture medium. (24 h fixation period) : 10, 33 and 100 ug/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 (inclusiveand 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 statisticallyand biologicallysignificantincreasein the number of cells with chromosome aberrations. page 14 T-5869 NOTOX Project 115943@@ The number of cells with chromosome aberrations found in the solvent control cultureswere within the laboratoryhistoricalcontrol data range (i.e. 1.0 + 1.1 (mean + standard deviation) aberrant cells per 100 metaphases (without S9mix; gaps excluded) and 0.6 + 0.7 aberrant cells per 100 metaphases (with S9mix; 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 experimentalconditions of this test. page 15 T-5869 NOTOX Project 115943 TABLE 2 MITOTIC INDEX OF DONOR OF T-5869 Experiment I CULTURES TREATED WITH VARIOUS CONCENTRATIONS Test substance concentration (ug/ml) 24 h fixationperiod Contrala) 3 10 33 iooc) MMC-C; 0.2 ug/ml 48 h fixation period Controla). 3 10 33 iooc) MMC-C; 0.1 ug/ml Number of metaphases per 1000 cells b) Absolute Percentage of control Without metabolic activation (-S9-mix) 29 - 50 43 - 45 47 - 45 47 - 45 41 - 31 21 - 23 100 ill 116 116 91 56 35 - 32 100 41 - 60 151 55 - 44 148 40 - 36 113 -45- 38 124 23 - 24 70 ----------------------------------------------------------------------With metabolic activation (+S9-mix) 24 h fixation period Controla) 3 10 33 iooc) CP; 15 pglml 76 - 87 100 108 - 85 118 75 - 63 85 79 - 68 90 76 - 75 93 34 - 34 42 48 h fixation period Controla) 3 10 33 iooc) 87 - 64 97 - 74 68 - 99 89 - d) 87 - 101 100 113 ill 118 125 a) DMSO b) duplicate cultures C) Slight precipitationof the test substance in culture medium. d) Culture infected with fungi - page 16 T-5869 NOTOX Project 115943 TABLE 3 MITOTIC INDEX OF DONOR CULTURES OF T-5869 Experiment 2 TREATED WITH VARIOUS CONCENTRATIONS Test substance concentration (ug/ml) Number of metaphases per 1000 cells b) Absolute Percentage of control 24 h fixation period Controla) 3 10 33 looc) MMC-C; 0.2 ug/ml Without metabolic activation (-S9-mix) 42 - 39 44 - 46 23 - 46 60 - 26 39 - 43 41 - 43 100 ill 85 106 101 104 -------------------------------------------------------------------- With metabolic activation (+S9-mix) 24 h fixation period Controla) 3 10 33 iooc) CP; 15 ug/ml 70 - 66 77 - 64 65 - 56 73 - 56 79 - 72 54 - 34 100 104 89 95 ill 65 a) DMSO b) duplicate cultures c) Slight precipitationof the test substance in culture medium. page 17 T-5869 NOTOX Project 115943L.: TABLE 4 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED CONCENTRATIONS OF T-5869 (Without Sg-mix)a) 24 h fixation period Experiment 1 WITH VARIOUS CUSo,n,c ml Culture omso 10 (0.9% viv) uglel A 8 A+B A 8 33 ug/ml A+B A B 100 tigiml mmc_c 0.2 ug/ml A+B A B A+B A B A+B No. of' calls *cured 100 100 200 100 100 200 100 100 200 100 100 200 50 50 100 No. of calls with aberrations 2 3 5 1 4 5 2 1 3 4 6 10 38 32 70 (+ gap$) No. of calls with aberrations gaps) g. go 1 2 3 0 3 3 1 1 2 4 3 7 36 31 67 11 11 1 3 32 3 bt 12 bw 3 11 '3 3 25 30 33 2 mo exch. 1 13 B die d' misc. total aborr (+ gaps) 23 24 21 46 49 43 total aborr gaps) a3 11 43 43 Abbreviations used for various types of aberrations are listed in appendix 1. The numerical variations ondareduplication (ando) and polyploidy (poly) were not counted as an aberration. misc. - (miscellaneous) aberrations not belonging to the ones mentioned Significantly different from control group (Chi-square test), P < 0.05, ** P ( 0.01 or *** P < 0.001. above. page 18 T-5869 NOTOX Project 115943 TABLE 5 Conc ug/ml Culture CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH CONCENTRATIONS OF T-5869 (With Sg_mix)a) 24 h fixation period Experiment 1 DMSO 10 (0.9% viv) tsgIml A B A+B A B 33 tig/al 100 ugial A+B A B A+B A B CP 15 vg/ml A+B A B A+B VARIOUS No. of cells scared 100 100 200 100 100 200 100 100 200 100 100 200 50 100 150 No. of cells with aberrations (+ gaps) 4 2 6 A 0 4 3 3 6 2 2 4 25 44 69 No. of cells with aberrations gaps) gI go b- bu 3 1 4o 00 11 4 11 1 3 1 4 2 1 3 25 44 69 12 31 1 '2 1 2 2 35 52 38 mo exch. 37 dic dt misc. total aberr (+ gaps) 42 40 43 22 41 71 total aberr (- gaps) 32 o-o 31 22 41 67 a) Abbreviations used"for various types of aberrations are listed in appendix 1. The numerical variations andoreduplication (endo) and polyploidy (poly) were not counted as an aberration. misc.- (miscellaneous) aberrations not belonging to the ones mentioned Significantly different from control group (Chi-square test), P (.0.05, P ( 0.01 or *** P ( 0.001. above. page 19 T-5869 NOTOX Project 115943- TABLE 6 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS.DF T-5869 (Without Sg-mix)a) 48 h fixation period Experiment 1 cttoln;c /ml Culture No. of sc:tlr:d DMSO 100 (0.9% V/V) mg/ml A B A+B A B mmc-c 0.1 ugiml A+B A B A+B 100 100 200 loo 100 200 50 50 100 No. of cells with aberrations 5 4 9 3 6 9 -28 30 58 (+ gap$) No. of cells with aberrations 3 4 7 2 5 7 27 30 57 gaps) I g. 2 11 35 gN b- 24 21 26 30 bft 2 3 11 10 exch. 1 56 die ds misc. total aberr (+ gaps) 54 36 45 53 total aborr (- gaps) 34 25 42 48 a) Abbreviations used for various types of aberrations are listed in appendix 1. The numerical variations endoreduplication (enda) 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 T-5869 NOTOX Project 115943.: TABLE 7 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (With S9-mix)a) 48 h fixation period Experiment 1 C:nc u /ml DHSD loo (D.9% vIV) ugial Culture A B A+B A B A+B No. of cells scored 100 100 200 100 100 200 No. of cellswith aberrations 0 2 2 3 2 5 (+ gaps) No. of cellswith aberrations 0 1 1 1 1 2 gaps) g. 1 21 go bl 1 1 bv exch. dic d' misc. totalaberr (+ gaps) 0 2 32 totalaborr (-gaps) 0 1 11 a) Abbreviationsused for varioustypesof aberrationasre listedin appendix1. The numericalvariationsendareduplicatio(nenda)and polyplaidy(poly)were not countedas an aberration. misc.- (miscellaneousa)berrationsnot belongingto the ones mentioned above. Significantldyifferentfromcontrolgroup(Chi-squarteest), P < 0.05,-- P ( 0.01or ***P ( 0.001. page 21 T-5869 NOTOX Project l.L5943@- TABLE 8 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS OF T-5869 (Without Sg-mix)a) 24 h fixation period Experiment 2 Conc ug/ml DKSO 10 (0.9% VIV) lagiml 33 lAgIml 100 uglal mmc-c 0.2 ug/ml Culture A B A+B A B A+B A B A+B A 8 A+B A B A+B No. of calls seared 100 100 200 100 100 200 IOD 100 200 100 100 200 100 100 200 No. of cells with aberrations 1 1 2 1 1 2@ 1 1 2 1 1 2 29 28 *5*7* (+ gaps) No. of Ce113 with aberrations 1 0 1 0. 0 0 0 0 0 0 0 0 25 24 49 gaps) g. 1 45 go bl 1 b- 10 a 14 10 exch. 28 dic d' mi3c., total aberr (+ gaps) 11 11 11 11 30 31 total aberr gaps) 10 00 a0 00 26 26 a) Abbreviations Used for various types of aberrations are listed in appendix 1. The numerical variations andoreduplication (endo) and polyplaidy (poly) were not counted as an aberration. Misc. - (M13cellaneaus) aberrations not belonging to the ones mentioned above. Significantly different from control group (Chi-square test), P 0.05, P o.ol or P < 0.001. page 22 T-5869 NOTOX Project 115943 TABLE 9 CHROMOSOME ABERRATIONS IN DONOR CULTURES TREATED CONCENTRATIONS OF T-5B69 (With Sg-mix)a) 24 h fixation period Experiment 2 WITH VARIOUS Conc uglal Culture of4so la (0.9% V/V) -AVial 33@ ug/ml A 8 A+13 A 6 A+B A 8 100 ugial A+B A B CP 15 mg/ml A+B A 9 A+B No. of calls scared 100 100 200 100 100 200 IGO 100 200 100 100 200 100 100 200 No. of calls with aberrations 1 1 2 (+ gaps) No. of cells with aberrations a 0 0 0 1 gaps) 1 2 0 1 1 21 24 45 1 2 0 D 0 17 17 34 49 1 b- 1 9 13 bl 1 97 'A exch. 4 die d' misc. endo total aberr (+ gap3) II 1 01 26 30 total abarr (-gaps) 1 0 0 01 11 00 i 22 207 a) Abbreviations used for various types of aberrations are listed in appendix 1. The numerical variations endareduplication (ando) and polyplaidy (poly) were not counted as an aberration. mist.w (miscellaneous) aberrations not belonging to the ones mentioned Significantly different from control group (Chi-square test), P ( 0.05, -- P ( 0.01 or *** P 0.001. above. page 23 - T-5869 NOTOX Project 115943 APPENDIX 1 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE PORTRAITS Aberration Abbreviation Description Chromatid gap 9 Chromosome gap go Chromatid break b" Chromosome break bm Chromatid deletion dy Minute mp Double minutes MN Dicentric chromosome dic Tricentric chromosome tric 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 "brokenn 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 T-5869 NOTOX Project 115943L: APPENDIX 1 Continued Aberration Abbreviation Description Ring chromosome Exchangefigure r exch. Chromosome intrachange Pulverized chromosomes Multiple aberrations intra p ma Polyploidy poly Endoreduplication endo A ringstructurewitha distinctlumen. An exchange(s)between two or more chromosomesresultingin the formation of a tri-or more-armedconfiguration. A chromosomeintrachangeis scored afterrejoiningof a lesionwithinone chromosome. A fragmentedor pulverizedchromosome A metaphasespread containingten or more of the above mentionedaberrations (chromatidand chromosomegaps not i-ncluded). A chromosomenumber that is a multiple of the normaldiploidnumber. A form of polyploidyin which each centromereconnectstwo or four pairs of chromatidsinsteadof the normalone pair. page 25 T-5869 NOTOX Project 115943- APPENDIX 2 STATISTICAL EVALUATION OF TEST RESULTS Chi-square Test TOTAL NUMBER OF CELLS WITH ABERRATIONS; TREATMENT/CONTROL (INCLUSIVE/EXCLUSIVE GAPS). COMPARISON, Experiment 1 TREATMENT DOSE (ug/ml) S9-mix GAPS P-VALUE two-sided DECISION AT 95% CONFIDENCE LEVEL 24 h fixation period MMC-C (0.2) + CP (15) + + 48 h fixation period mmc-c (0.1) + <0.0004 "@0.0004 <0.0004 @0.0004 <0.0004 ZO. C)004 significant significant significant significant significant significant Experiment 2 TREATMENT DOSE (ug/ml) S9-mix GAPS 24 h fixation period MMC-C (0.2) + CP (15) + + P-VALUE two-sided <0.0004 'ZO.0004 <0.0004 'ZO.0004 DECISION AT 95% CONFIDENCE LEVEL significant significant significant significant page 26