Document kmBvpgbYdkb0LbLwox5yN9nDb

/waa-oMsq C O R N IN G Hazleton MUTAGENICITY TEST ON T-6342 MEASURING CHROMOSOMAL ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS: WITH A CONFIRMATORY ASSAY WITH MULTIPLE HARVESTS FINAL REPORT AUTHOR Hemalatha Murli, Ph.D. PERFORMING LABORATORY Coming Hazleton Inc. (CHV) 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT IDENTIFICATION CHV Study No.: 17073-0-437CO SUBMITTED TO 3M Corporation Building 220-2E-02 3M Center St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE September 16, 1996 CHV Study No.: 17073-0-437CO 1 of 35 C00295 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT Project Title: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells: With a Confirmatory Assay With Multiple Harvests Project No.: 20990 Assay No.: 17073 Protocol No.: 437CO Edition No.: 4, Modified for 3M Corporation Quality Assurance inspections of the study and review of the final report of the above referenced project were conducted according to the Standard Operating Procedures of the Quality Assurance Unit and according to the general requirements of the appropriate Good Laboratory Practice regulations. Findings from the inspections and final report review were reported to management and to the study director on the following dates: Inspection/Date Findings Reported Auditor Dosing-09/06/1995 09/06/1995 M. Murphy Draft Report Review-11/06,07/1995 11/07/1995 C. Orantes, K. Newland Revised Draft Report Review-01/04/1996 01/04/1996 C. Orantes Final Report Review-09/16/1996 09/16/1996 C. Orantes Quality Assurance Unit CHV Study No.: 17073-0-437CO '/`/ f t Date Released 2 0002S6 C O R N IN G Hazleton STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Food and Drug Administration (FDA) Title 21 of the U.S. Code of Federal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) with any applicable amendments . There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation of the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria. All test and control results in this report are supported by an experimental data record and this record has been reviewed by the Study Director. All raw data, documentation, records, protocol and a copy of the final report generated as a result of this study will be archived in the storage facilities of Coming Hazleton Inc. for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Coming Hazleton Inc. for an additional period of time, or sent to a storage facility designated by the Sponsor. Submitted By: Study Director: Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology Date CHV Study No.: 17073-0-437CO 3 000297 C O R N IN G Hazleton TABLE o f f o m e n t s Page No. ABSTRACT.................................................................................................................................. 6 1.0 SPONSOR.................................................................................................................... 8 2.0 MATERIAL (TEST ARTICLE) .................................................................................. 8 2.1 Client's Identification 2.2 Date Received 2.3 Physical Description 2.4 Genetics Assay No. 3.0 TYPE OF A SSA Y ..........................................................................................................8 4.0 PROTOCOL NO..............................................................................................................8 5.0 STUDY DATES ........................................................................................................... 8 5.1 Initiation Date 5.2 Experimental Start Date 5.3 Experimental Termination Date 6.0 SUPERVISORY PERSONNEL ....................................................................................8 6.1 Study Director 6.2 Laboratory Supervisor 7.0 OBJECTIVE ..................................................................................................................8 8.0 RATIONALE................................................................................................................. 9 9.0 EXPERIMENTAL DESIGN......................................................................................... 9 10.0 MATERIALS AND METHODS..................................................................................10 10.1 Test Cells 10.2 Cell Culture Medium 10.3 Negative and Solvent Controls 10.4 Positive Control Agents 10.5 Rangefinding Assays 10.6 Aberrations Assay Without Metabolic Activation CHV Study No.: 17073-0-437CO 4 C00298 C O R N IN G Hazleton 10.7 10.8 10.9 10.10 Aberrations Assay With Metabolic Activation Harvest Procedure Slide Preparation and Staining Aberrations Analysis and Assay Evaluation 11.0 RESULTS .................................................................................................................... 15 11.1 Solubility and Dose Determination 11.2 Rangefinding Assay Without Metabolic Activation 11.3 Rangefinding Assay With Metabolic Activation 11.4 Chromosomal Aberrations Assay Without Metabolic Activation 11.5 Chromosomal Aberrations Assay With Metabolic Activation 12.0 CONCLUSION............................................................................................................20 13.0 REFERENCES ............................................................................................................21 14.0 EXPERIMENTAL DATA TABLES............................................................................22 15.0 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED C E L L S..........................................................................................................................33 CHV Study No.: 17073-0-437CO 5 000299 C O R N IN G Hazleton ABSTRACT The objective of this in vitro assay was to evaluate the ability of T-6342 to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. For the dose rangefinding assay with and without metabolic activation, the test article was dissolved in sterile deionized water at a concentration of 498 mg/ml. Concentrations of 0.166, 0.498, 1.66, 4.98, 16.6, 49.8, 166,498, 1660, and 4980 pg/ml were tested in the dose rangefinding assays. All dosing was achieved using a dosing volume of 1% (10 pl/ml). In the nonactivation assay, complete cytotoxicity was observed in the culture dosed with 4980 pg/ml, and reductions of 100% in the mitotic index as compared with the solvent control cultures were observed in the culture dosed with 1660 pg/ml. In the activation assay, reductions of 19%, 33%, 27%, and 63% as compared with the solvent control cultures were observed in the cultures dosed with 166, 498, 1660, and 4980 pg/ml, respectively. Based on the data from the dose rangefinding assay, replicate cultures of CHO cells were incubated with 125, 250, 500, 750, 1000, 1500, and 2000 pg/ml without metabolic activation and with 250, 500, 1250, 2500, 3750, and 5000 pg/ml with metabolic activation in 20.0 hour aberrations assays. Cultures treated with 750, 1000, 1500, and 2000 pg/ml without metabolic activation and with 1250, 2500, 3750, and 5000 pg/ml with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations was observed in the nonactivation assay. A significant increase in cells with chromosomal aberrations was observed in the cultures dosed with 3750 and 5000 pg/ml with metabolic activation. In the confirmatory trial, replicate cultures of CHO cells were incubated with 125, 249, 498, 746, 995, 1490, and 1990 pg/ml without metabolic activation and with 249,498, 1250,2490, 3730, and 4970 pg/ml with metabolic activation in 20.0 and 44.1 hour assays. Cultures treated with 746, 995, 1490, and 1990 pg/ml from the 20.0 hour assay and with 249,498, 746 and 995 pg/ml from the 44.1 hour assay without metabolic activation, and with 1250, 2490, 3730, and 4970 pg/ml from the 20.0 and 44.1 hour assays with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations was observed in the nonactivation assay. A significant increase in cells with chromosomal aberrations was observed in the cultures dosed with 4970 pg/ml in the 20.0 hour assay with metabolic activation. A significant increase in percent polyploidy was observed in the cultures dosed with 4970 pg/ml in the 44.1 hour assay with metabolic activation. This positive response was investigated under nonactivation conditions by treating cells for 3 hours under the same conditions as the assay with metabolic activation. CHV Study No.: 17073-0-437CO 6 000300 C O R N IN G Hazleton In this repeat trial, replicate cultures of CHO cells were incubated with 250, 500, 1250, 2490, 3740, and 4980 pg/ml without metabolic activation for 3 hours and harvested 20.0 and 44.0 hours after initiation of treatment. Cultures treated with 250,500,1250 and 2490 pg/ml from the 20.0 hour assay and with 500, 1250, 2490, and 3740 pg/ml from the 44.0 hour assay were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations was observed in the 20.0 hour assay. A weakly significant increase in cells with chromosomal aberrations was observed in the cultures dosed with 3740 pg/ml in the 44.0 hour assay. A significant increase in percent polyploidy was observed in the cultures dosed with 3740 pg/ml in the 44.0 hour assay. The test article, T-6342, was considered weakly positive for inducing chromosomal aberrations in CHO cells without metabolic activation with a three hour treatment. T-6342 was considered positive for inducing chromosomal aberrations in CHO cells with metabolic activation. T-6342 was considered positive for inducing polyploidy with and without metabolic activation. CHV Study No.: 17073-0-437CO 7 000301 C O R N IN G Hazleton Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells: With a Confirmatory Assay With Multiple Harvests With T-6342 1.0 SPONSOR: 3M Corporation 2.0 MATERIAL (TEST ARTICLE): 2.1 Client's Identification: T-6342 2.2 Date Received: July 21, 1995 2.3 Physical Description: Clear, colorless liquid 2.4 Genetics Assay No.: 17073 3.0 TYPE OF ASSAY: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells 4.0 PROTOCOL NO.: 437CO, Edition 4, Modified for 3M Corporation 5.0 STUDY DATES: 5.1 Initiation Date: August 15, 1995 5.2 Experimental Start Date: August 24, 1995 5.3 Experimental Termination Date: December 04, 1995 6.0 SUPERVISORY PERSONNEL: 6.1 Study Director: Hemalatha Murli, Ph.D. 6.2 Laboratory Supervisor: Carol S. Spicer, B.S. 7.0 OBJECTIVE: The objective of this in vitro assay was to evaluate the ability of the test article, T-6342, to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells, with and without metabolic activation. CHV Study No.: 17073-0-437CO 8 000302 C O R N IN G Hazleton 8.0 RATIONALE: The assay is designed to establish whether the test article or its metabolites can interact with cells to induce chromosome breaks. Chemically induced lesions may result in breaks in chromatin that are either repaired by the cell in such a way as to be undetectable or result in visible damage. Aberrations are a consequence of failure in repair processes such that breaks do not rejoin or rejoin in abnormal configurations (Evans, 1962). 9.0 EXPERIMENTAL DESIGN: Results from the rangefinding assay were used to determine the dose range to be used in the chromosomal aberrations assay. In the rangefinding assay, the cultures were harvested 20.0 hours after initiation of treatment. Mitotic index and visual observations of the cultures were evaluated for evidence of toxicity. A summary of the treatment schedule for the rangefinding assay is given below. Summary of Rangefinding Assay Treatment Schedule in Hours Test Test Article - S9 0 + S9 0 Wash 17.8 3 Colcemid Fixation 17.9 20 17.9 20 In the chromosomal aberrations assays, replicate cultures were used at each dose level, and negative and solvent controls. Single cultures were used for each of two doses of the positive control. The aberrations assays were conducted with a 20.0 hour harvest time in the initial trial and with 20.0 and 44.1 hour harvest times in the confirmatory trials. Chromosomal aberrations were analyzed from the cultures treated at four dose levels and from only one of the positive control doses. A summary of the treatment schedule for the chromosomal aberrations assays is on the following page. CHV Study No.: 17073-0-437CO 9 GG0303 C O R N IN G Hazleton Summary of Chromosomal Aberrations Assay Treatment Schedule in Hours Test Test Article Initial Trial - S9 0 + S9 0 Confirmatory Trial - S9 0 + S9 0 - S9 0 + S9 0 Repeat Trial - S9 0 - S9 0 Wash Colcemid Fixation 17.8 18 3 18 20 20 17.6 18 3 17.9 41.8 42.1 3 42.1 20 20 44.1 44.1 3 18 3 42 20 44 10.0 MATERIALS AND METHODS: 10.1 Test Cells: The Chinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent cell line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells have since been recloned to maintain karyotypic stability. This cell line has an average cycle time of 12 to 14 hours with a modal chromosome number of 21. 10.2 Cell Culture Medium: The CHO cells were grown in McCoy's 5a culture medium which was supplemented with 10 % fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin and streptomycin, at approximately 37 C, in an atmosphere of about 5% C 02 in air. CHV Study No.: 17073-0-437CO 10 00304 C O R N IN G Hazleton 10.3 Negative and Solvent Controls: In the nonactivation assays, negative controls were cultures which contained only cells and culture medium. Solvent controls were cultures containing the solvent for the test article at the highest concentration used in test cultures. In the activation assays, the negative and solvent controls were the same as described in the nonactivation assays but with the S9 activation mix included. 10.4 Positive Control Agents: The positive control agents which were used in the assays were mitomycin C (MMC) for the nonactivation series and cyclophosphamide (CP) in the metabolic activation series. Mitomycin C (CAS# 50-07-7, Sigma, Lot # 25H0619) is a clastogen that does not require metabolic activation. Cyclophosphamide (CAS # 6055-19-2, Sigma, Lot # 67F0155) does not act directly but must be converted to active intermediates by microsomal enzymes. In the chromosomal aberrations assays, two concentrations of MMC (0.08 and 0.10 pg/ml, initial and confirmatory trials; 0.50 and 1.0 pg/ml, third trial) and CP (5.0 and 10.0 pg/ml) were used to induce chromosomal aberrations in the CHO cells. One of the dose levels was analyzed in each of the aberration assays. Both MMC and CP were dissolved in water. 10.5 Rangefinding Assays: In these assays, the cells were cultured for approximately 24 hours prior to treatment by seeding approximately 0.3 x 106cells per 25 cm2flask into 5 ml of complete McCoy's 5a culture medium. 10.5.1 Assay Without Metabolic Activation: The cultures were incubated with the test article for 17.8 hours at =37C. Then, the test article was washed from the cells with phosphate buffered saline and fresh complete medium containing Colcemid (final concentration 0.1 pg/ml) was added. The cultures were then trypsinized and harvested 2.0 hours later. (See Sections on Harvest and Slide Preparation and Staining). 10.5.2 Assay With Metabolic Activation: In this assay, the CHO cells were exposed to the test article for three hours CHV Study No.: 17073-0-437CO 11 000305 C O R N IN G Hazleton at =37C in the presence of a rat liver S9 reaction mixture (S9 15 pl/ml, NADP 1.5 mg/ml, and isocitric acid 2.7 mg/ml). The S9 fraction (Molecular Toxicology, Inc., Lot #0583) was derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254 to induce the mixed function oxidase enzymes which are capable of metabolizing chemicals to more active forms. The three hour incubation time was used because prolonged exposure to the S9 mixture might be toxic to the cells and the enzyme activity of S9 is lost rapidly at about 37C. The medium did not have FBS during the exposure period to avoid possible inactivation of short lived and highly reactive intermediates produced by the S9 enzymes by binding to serum proteins. After the exposure period the cells were washed twice with buffered saline. Complete McCoy's 5a medium was added to the cultures which were then incubated for 16.8 hours with Colcemid (final concentration 0.1 |ig/ml) added for the last 2.0 hours to collect metaphase cells. The cultures were then trypsinized, harvested, fixed, and slides were prepared and stained as was described for the nonactivation rangefinding assay. 10.5.3 Assay Evaluation: Mitotic index was analyzed from the highest five surviving dose levels by analyzing the number of metaphases present in 1000 consecutive cells. 10.6 Aberrations Assay Without Metabolic Activation: Cultures were initiated by seeding approximately 1.2 x 106cells (20.0 hour assay) and 0.8 X 106cells (=44.0 hour assay) per 75 cm2flask into 10 ml of complete McCoy's 5a medium. One day after culture initiation, for the initial and confirmatory trials, the cells were incubated at =37C with the test article at predetermined doses for about 17.7 (20.0 hour assay) and 41.8 (44.1 hour assay) hours. The cultures were then washed with buffered saline and complete McCoy's 5a medium containing 0.1 pg/ml Colcemid was placed back onto the cells. Approximately two hours later, the cells were harvested and air dried slides were made. The slides were then stained in 5 % Giemsa solution for the analysis of chromosomal aberrations. For the third trial, one day after culture initiation, the cultures were incubated at =37C for three hours in the presence of the test article in McCoy's 5a medium without FBS. After the three hour exposure period the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. The cells were incubated for the rest of the CHV Study No.: 17073-0-437CO 12 000306 C O R N IN G Hazleton culture period up to the time of harvest with 0.1 pg/ml Colcemid present during the last =2.0 hours of incubation. The metaphase cells were then harvested and prepared for cytogenetic analysis. 10.7 Aberrations Assay With Metabolic Activation: Cultures were initiated by seeding approximately 1.2 x 106cells (20.0 hour assay) and 0.8 X 106cells (44.1 horn assay) per 75 cm2flask into 10 ml of complete McCoy's 5a medium. One day after culture initiation, the cultures that were treated under the conditions of metabolic activation were incubated at =37C for three hours in the presence of the test article and the S9 reaction mixture in McCoy's 5a medium without FBS. After the three hour exposure period the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. The cells were incubated for the rest of the culture period up to the time of harvest with 0.1 pg/ml Colcemid present during the last =2.0 hours of incubation. The metaphase cells were then harvested and prepared for cytogenetic analysis. 10.8 Harvest Procedure: Prior to the harvest of the cultures, visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium. The cultures from the dose rangefinding assay were trypsinized first to collect mitotic and interphase cells and were treated with 0.075 M KCL hypotonic solution. This treatment helps to swell the cells and thus disperse the chromosomes. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1, v:v) fixative and were washed several times before air-dried slides were prepared. 10.9 Slide Preparation and Staining: Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were then air-dried and cover slipped using Depex mounting medium. CHV Study No.: 17073-0-437CO 13 000307 C O R N IN G Hazleton 10.10 Aberrations Analysis and Assay Evaluation: Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 1 (range 20-22) were analyzed. One hundred cells, if possible, from each replicate culture at four dose levels of the test article and from the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962; See Section 15.0). At least 25 cells were analyzed for chromosomal aberrations from one of the positive control cultures. For control of bias, all slides except for the positive controls were coded prior to analysis. Cells with aberrations were recorded on the data sheets by the microscope stage location. Mitotic index was assessed by analyzing the number of mitotic cells in 1000 cells and the ratio was expressed as a percentage of mitotic cells. The following factors were taken into account in the evaluation of the chromosomal aberrations data: 1. The percentage of cells with any aberrations. 2. The percentage of cells with more than one aberration. 3. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks and may possibly be induced by toxicity. Percent polyploidy was analyzed from the 44.0 hour assay and results were tabulated. Historical control data are presented in Table 10. A cell classified as "GT" is considered to contain 10 aberrations for statistical purposes but a ">" is also included in the tables for this classification to indicate that it is a minimum number. Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the solvent controls. A linear trend test of increasing number of cells with aberrations with CHV Study No.: 17073-0-437CO 14 C O R N IN G Hazleton increasing dose (Armitage, 1971) was also performed. Test article significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test article was based upon scientific judgement. 11.0 RESULTS: 11.1 Solubility and Dose Determination T-6342 was described as being freely soluble in water, so its solubility for this study was evaluated in sterile deionized water (Lot #17, prepared 06/16/95 at CHV). A clear, colorless solution was obtained at a concentration of 498 mg/ml. This solution was diluted to 1% (10 pl/ml) in McCoy's 5a culture medium in the absence of any cells. At a concentration of 4980 pg/ml in culture medium, the test article remained completely soluble and a pH of 7.5 was determined. Sterile deionized water was therefore chosen as an appropriate vehicle for this assay. A stock concentration of 498 mg/ml and dilutions thereof were prepared for dosing the rangefinding assays. All dosing was achieved with a 1% (10 pl/ml) dosing of each stock solution, and the solvent control culture was dosed with 10 pl/ml of sterile deionized water. Concentrations of 0.166, 0.498, 1.66,4.98, 16.6,49.8, 166. 498, 1660, and 4980 pg/ml were tested in the rangefinding assays with and without the S9 metabolic activation system. The stability of the test article under the preparation and dosing conditions used in this assay is the responsibility of the Sponsor. 11.2 Rangefinding Assay Without Metabolic Activation In the culture treated with 4980 pg/ml, no visible mitotic cells were observed; the cell monolayer was <5% confluent, and the culture consisted only of debris and dead cells, floating or attached. A severe reduction in the number of visible mitotic cells was observed in the culture dosed with 1660 pg/ml. The cell monolayer looked unhealthy and the degree of confluency was reduced about 15%; floating dead cells and debris were noted. Floating debris also was observed in the culture dosed with 498 pg/ml, but the culture otherwise was similar to the negative control. Mitotic indices were analyzed from the cultures dosed with 16.6, 49.8, 166, 498, and 1660 pg/ml (Table 1) and compared to the solvent control. At 1660 pg/ml, the mitotic index was essentially zero. However, little or no reduction in mitotic CHV Study No.: 17073-0-437CO 15 000309 C O R N IN G Hazleton index was observed at the next lowerdose of 498 pg/ml. Based on these results, the first trial of the chromosomal aberrations assay without metabolic activation was conducted with a 20 hour harvest at test article concentrations of 125, 250, 500, 750, 1000, 1500, and 2000 pg/ml in order to ensure a wide toxicity range. 11.3 Rangefinding Assay With Metabolic Activation With S9 metabolic activation, an unhealthy cell monolayer was observed in the culture dosed with 4980 |ig/ml. The cell monolayer confluence was reduced by approximately 85%, and a severe reduction in the number of visible mitotic cells occurred (about 63%, Table 1). At the next lower dose of 1660 pg/ml, the monolayer confluence was the same as the solvent control, and the mitotic index was only somewhat reduced (about 27%). Based on these results, the initial aberrations assay with metabolic activation was conducted with a 20 hour harvest at test article concentrations of 250, 500, 1250, 2500, 3750 and 5000 pg/ml. 11.4 Chromosomal Aberrations Assay Without Metabolic Activation Initial Trial Chromosomal aberrations were analyzed from the cultures dosed with 750, 1000, 1500, and 2000 pg/ml (Table 2). No significant increases in cells with chromo somal aberrations were observed for the cultures analyzed. This assay covered a wide range of toxicity. Unhealthy cell monolayers, floating dead cells and debris, severe loss of visible mitotic cells, and about 25% reduction in cell monolayer confluence were observed in the cultures dosed with 1500 and 2000 pg/ml. Slightly unhealthy cell monolayers, floating dead cells and debris, a reduction in the number of visible mitotic cells, and about 15% reduction in cell monolayer confluence were observed in the cultures dosed with 1000 pg/ml. At the lowest analyzed dose of 750 pg/ml, the cell monolayers appeared to be almost normal, with only a slight reduction in the number of visible mitotic cells and about a 15% reduction in the degree of confluence. Severe reductions in mitotic index relative to the solvent control cultures occurred at all dose levels, eg., 95%, 86%, 67%, and 57% at 2000, 1500, 1000, and 750 pg/ml, respectively. Based on the toxicities obtained in this trial, the confirmatory trial of the nonactivation aberrations assay was conducted at test article concentrations of 125, 249, 498, 746, 995, 1490, and 1990 pg/ml, using two harvest times of 20 and 44.1 hours. CHV Study No.: 17073-0-437CO 16 000310 C O R N IN G Hazleton Canfirniat-sre Trial For the 20 hour harvest, chromosomal aberrations were analyzed from the cultures dosed with 746, 995,1490, and 1990 pg/ml (Table 3). No significant increases in cells with chromosomal aberrations were observed for any of the cultures analyzed. As obtained in the first trial, the toxicity range was wide, ranging from a severe 95% reduction in mitotic index at 1990 pg/ml to 25% reduction at 746 jig/ml. The cell monolayer confluence was reduced by about 25% at 1990 pg/ml, and only 83 metaphases could be scored from one of the replicate cultures. To compensate, 117 metaphases were scored from the replicate culture, in order to score a total of 200 metaphases for this dose. For the 44.1 hour harvest, chromosomal aberrations were analyzed from the cul tures dosed with 249, 498, 746, and 995 jig/ml (Table 4). Again, no significant increases in cells with chromosomal aberrations were observed for any of the cultures analyzed. The percent polyploidy is scored at this harvest time, and no significant increases were noted. The concentrations of test article were lower relative to the 20 hour harvest because the longer exposure time caused greater toxicity. The two highest applied doses of 1990 pg/ml and 1490 |ig/ml were lethal. Treatment with 995 pg/ml was severely toxic, resulting in about 40% reductions in monolayer confluence and 79% reduction in mitotic index. At 746 pg/ml, however, the toxicity was much less (about 15% reduction in confluency and 18% reduction in mitotic index compared to the solvent control cultures). The results obtained from the two assay trials in the absence of an S9 metabolic activation system appeared to show the lack of any clastogenic activity by the test article. However, in another assay having an exposure time of only 3 hours, the induction of chromosomal aberrations was demonstrated (see Section 11.6). 11.5 Chromosomal Aberrations Assay With Metabolic Activation Initial Trial Chromosomal aberrations were analyzed from the cultures dosed with 1250, 2500. 3750, and 5000 pg/ml in the presence of the rat liver S9 metabolic activation system (Table 5). Significant increases in cells with chromosomal aberrations were observed in the cultures dosed with 3750 and 5000 pg/ml. The treatment with 3750 pg/ml was only slightly toxic, but the 5000 pg/ml treatment was highly toxic, causing about 95% reduction in monolayer confluency for one culture and CHV Study No.: 17073-0-437CO 17 (/0311 C O R N IN G Hazleton about 75% reduction for the replicate culture. Floating dead cells and debris were abundant, and only 94 metaphases were analyzable from the culture with greater toxicity. However, the mitotic index was reduced by only 13% compared to the solvent control cultures. Based on these results, the confirmatory trial was conducted with essentially the same test article concentrations, using two harvest times of 20 and 44.1 hours. Confirmatory Trial For the 20 hour harvest, a significant increase in cells with chromosomal aberra tions was observed only for the cultures dosed with 4970 pg/ml (Table 6). This toxic treatment reduced the confluency by about 70% and caused a 43% reduction in the mitotic index. The treatment with 3730 pg/ml was much less toxic, causing about 30% reduction in confluency and 18% reduction in mitotic index. Even though the treatment with 3730 pg/ml appeared to be somewhat more toxic than the similar dose in the initial trial, there was no hint of any clastogenic activity. Therefore, only the highly toxic treatment at the dose limit of the assay (5000 pg/ml) induced a repeatable, but large, increase in percent cells with chromosomal aberrations. In both trials, both simple and complex aberrations were observed. For the 44.1 hour harvest, chromosomal aberrations were analyzed from the cul tures dosed with 498, 1250, 2490, and 3730 pg/ml (Table 7). No significant increases in cells with chromosomal aberrations were observed for any of the cultures analyzed. Although the 4970 pg/ml treatment was too toxic to yield sufficient metaphases for analysis, it was nevertheless noted that the percent polyploid cells increased significantly. At 3730 pg/ml, there was little or no toxicity evident. The successful activation by the metabolic system was illustrated by the large increase in percent cells with chromosomal aberrations (at 20 hours harvest) in the cultures exposed to cyclophosphamide, the positive control article, which requires metabolic activation to become clastogenic. The initial interpretation of these results was that the test article was converted to clastogenic substance(s) in the presence of the S9 metabolic activation system. However, according to information provided by the Sponsor, the test article is not metabolized. The appearance of clastogenic activity in the activation portion of the assay was not in doubt, but this activity should also have been observed under CHV Study No.: 17073-0-437CO 18 000312 C O R N IN G Hazleton nonactivation conditions if the S9 system had no effect on the test article. This situation raised the possibility that an unrecognized toxicity in the nonactivation assay was actually eliminating the cells with induced aberrations before the cultures were analyzed. The treatment periods under nonactivation conditions were only about 2 hours less than the harvest times, whereas the test article was removed after 3 hours exposure for the activation assays. If the continued presence of the test article was causing the demise of cells with induced aberrations, then the aberrations should be observable if the treatment time was made equivalent to the activation assay, ie, reduced to 3 hours. Accordingly, the nonactivation assay was repeated, using a 3 hour exposure period. 11.6 Chromosomal Aberrations Assay Without Metabolic Activation; 3-Hour Treatment The positive response observed in the assay with metabolic activation was investigated by treating cells under the same conditions without metabolic activation. Cells were treated for 3 hours at test article concentrations of 250, 500. 1250, 2490, 3740, and 4980 pg/ml, using harvest times of 20 and 44 hours after the initiation of treatment. For the 20 hour harvest, chromosomal aberrations were analyzed from the cultures dosed with 250, 500, 1250, and 2490 pg/ml (Table 8). No significant increases in cells with chromosomal aberrations were observed. Unfortunately, these treatments were not very toxic (about 30% reduction in confluency at 2490 pg/ml), and the next higher dose of 3740 (ig/ml was so toxic that only a small number of metaphases could be examined. As footnoted in the table, 7 of 38 metaphases obtained from the replicate cultures had chromosomal aberrations. Thus, approximately 18% of the cells were indicated as having aberrations. Due to the severe depression of mitotic index, the percent cells with aberrations likely would have been higher at a longer harvest time. Obviously, much closer dose steps would have been necessary to achieve toxicities comparable to the activation assays and to compare the observable clastogenic responses. It is interesting to note that the test article was clearly more toxic for the 3 hour treatment period than when the S9 activation system was present. Genotoxic events (mutation or chromosomal changes) are necessarily associated with toxicity, and the metabolism of test articles to clastogenic substances is therefore expected to be accompanied by increased toxicity, not less toxicity. Thus, the observation of more toxicity in the absence of S9 was very suggestive that the CHV Study No.: 17073-0-437CO 19 000313 C O R N IN G Hazleton controlling factor in observing clastogenic activity for this test article was the attainment of a certain high toxicity for a short time period, and not the metabolic conversion of the test article. For the 44 hour harvest, chromosomal aberrations were analyzed from the cultures dosed with 500, 1250, 2490, and 3740 pg/ml (Table 9). A small, but significant 7.5% of cells with chromosomal aberrations was observed in the cultures dosed with 3740 (ig/ml. A significant increase in percent polyploidy also was observed in these cultures. The 3740 pg/ml treatment resulted in about 55% reduction in monolayer confluency relative to the solvent controls, and the 2490 pg/ml treatment gave about 30% reduction. The mitotic index was also significantly reduced (57%) for the high dose. These results were consistent with the interpretation that a highly toxic treatment with this test article, applied for a short period of time, will give rise to cells containing chromosomal aberrations that can be observed in the 20 or 44 hour time period after initiation of treatment. The harvest time for the observation of maximum numbers of aberrant cells was not determined. The addition of S9 to the cultures simply reduced the test article toxicity, so that finer adjustments in the dose levels than achieved in this study are necessary to make comparisons between equi-toxic treatments under activation and nonactivation assay conditions. The initial observations of clastogenic activity only in the presence of the S9 activation system can be interpreted to be a consequence of a more limited toxic action over 3 hours exposure, compared to the long exposures for the standard nonactivation assays, rather than being due to a metabolic effect. The test article is therefore evaluated as clastogenic only for highly toxic treatments for short enough exposure periods (eg, 3 hours) such that the cells containing chromosome aberrations do not disintegrate before they can be observed at 20 to 44 hour harvest times. 12.0 CONCLUSION: The test article, T-6342, was evaluated as being clastogenic to cultured CHO cells for highly toxic treatments of short duration (eg., 3 hours) both in the presence and absence of an S9 metabolic activation system. Polyploidy was also induced for the highly toxic treatments. CHV Study No.: 17073-0-437CO 20 000314 C O R N IN G Hazleton 13.0 REFERENCES: Armitage, P. Statistical Methods in Medical Research. John Wiley & Sons, Inc., New York, NY, 1971. Evans, H.J.: Chromosomal aberrations produced by ionizing radiation. International Review of Cytology, 12:221-321, 1962. Sokal, R.R., and Rohlf, F.J.: Biometry. Ed. 2, W.H. Freeman and Company, New York, 1981. CHV Study No.: 17073-0-437CO 21 000315 C O R N IN G Hazleton 14.0 EXPERIMENTAL DATA TABLES CHV Study No.: 17073-0-437CO 22 000316 C O R N IN G Hazleton TABLE 1 RANGEFINDING ASSAY FOR ASSESSING TOXICITY Compound: T-6342 Assay No.: 17073 Trial No.: I Date: 08/24/95 Metabolic Activation: -S9 Lab No.: CY8225 Treatment NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE McCoy's 5a Water 10.0 gl/ml 16.6 Hg/tnl 49.8 Hg/ml 166 Mg/ml % Mitotic Index 9.8 2.0 5.4 9.7 9.1 Confluence* % Solvent Control 100 100 100 100 100 498 Hg/ml 1660** Hg/ml 9.2 0.0 100 85 Metabolic Activation: +S9 Lab No.: CY8225 Trial No.:I Treatment % Mitotic Index Confluence* % Solvent Control NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE McCoy's 5a Water 10.0 jil/ml 49.8 Ug/ml 14.8 17.1 17.3 100 100 100 166 498 1660 4980 Hg/ml Ug/ml Ug/ml gg/ml 13.9 11.5 12.5 6.3 100 100 100 14 *This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. Actual cell counts are not taken and any hypertrophy o f the attached cells cannot be evaluated. At the time o f the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells. ** Toxic dose level. CHV Study No.: 17073-0-437CO 23 000317 C O R N IN G Hazleton CHV Study No.: 17073-0-437CO TABLE 2 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 20.0 Hours After Treatment Assay No.: 17073 Trial #:1 Lab #: CY9055 Metabolic Activation:-S9 Compound:T-6342 Date:09/06/95 NUMBER AND TYPE OE ABERRATION Co n t r o l s NEGATIVE: McCoy's 5a SOLVENT: Water POSITIVE: MMC NOT COMPUTED SIMPLE CELLS TG SG ! UC TB ! SB SCORED # OF % . % COMPLEX OTHER ABERRA CELLS CELLS TIONS WITH WITH>1 % ID j TR I QR j CR ; D ! R ! Cl I DF GT PER ABERRA-ABERRA-MTIUnC CELL TIONS TIONS INDEX A 100 2 I B 100 3 A+B 200 5 1 I0.0fil/ml A 100 2 B too 2 1 A+B 200 4 1 0.08iig/ml A 25 9 275 1 1 1 1 3 0.01 1.0 0.00 0.0 0.01 0.5 0.01 10 0.00 0.0 0.01 0.5 0.60 28.0* 0.0 0.0 0.0 0.0 0.0 0.0 8.0** 4.4 5.6 5.0 8.6 7.2 7.9 4.5 TEST ARTICLE 750ng/ml A B 100 2 1 100 4 A+B 200 6 1 1000ug/ml A B 100 8 1 100 4 A+B 200 12 1 1500|ig/ml A B too 4 100 A+B 200 4 2000|ig/ml A B 100 100 4 A+B 200 * Significantly greater than the solvent controls, p<0.01 ** Significantly greater than the solvent controls, p<0.05 4 oo Co h* oo 1 0.01 1.0 0.0 3.5 0.00 0.0 0.0 3.3 1 0.01 0.5 0.0 3.4 0.00 0.0 0.0 2.5 0.00 0.0 0.0 2.6 0.00 0.0 0.0 2.6 0.00 0.0 0.0 1.4 2 0.02 2.0 0.0 0.7 2 0.01 1.0 0.0 1.1 0.00 0.0 0.0 0.3 0.00 0.0 0.0 0.4 0.00 0.0 0.0 0.4 CHV Study No.: 17073-0-437CO TABLE 3 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 20.0 Hours After Treatment Assay No.: 17073 Trial #:2 Lab #: CY9155 Metabolic Activation:-S9 Compound:T-6342 Date:09/l3/95 NUMBER AND TYPE OF ABERRATION CONTROLS NEGATIVE: McCoy's 5a SOLVENT: Water POSITIVE: MMC NOT COMPUTED SIMPLE CELLS TG I SG : DC TB j SB SCORED # OF % % COMPLEX OTHER ABERRA CELLS CELLS TIONS WITH WITH>1 % ID 1TR ! QR ! CR j D j R j Cl j DF o r PER ABERRA-ABERRA-MTIOnC CELL TIONS TIONS INDEX A 100 8 1 B too 8 1 1 0.01 1.0 0.0 6.1 1 0.02 2.0 0.0 6.4 A+B 200 16 1 1 1 1 0.02 1.5 0.0 6.3 I0.0nl/ml A 100 7 1 B too 1 2 1 0.01 1.0 0.0 7.4 0.00 0.0 0.0 8.3 A+B 200 8 3 1 001 0.5 0.0 7.9 0.1()0|ig/Iiil A 25 3 61 74 21 1 0.88 52.0* 24.0* 4.2 TEST ARTICLE 746(ig/ml A B 100 3 100 4 1 A+B 200 7 1 995pg/ml A B 100 9 100 1 1 1 1 A+B 200 10 1 2 1490ng/ml A B 100 3 1 100 4 1 1 A+B 200 7 1 1 1 I990(ig/ml A 83 4 1 1 It 117 12 1 1 1 A+B 200 16 2 2 * Significantly greater than the solvent controls, p<0.01 1 1 0.02 2.0 0.0 5.8 1 0.01 1.0 0.0 6.0 2 0.02 1.5 0.0 5.9 1 1 0.03 3.0 0.0 2.3 0.01 1.0 0.0 2.1 1 I 0.02 2.0 0.0 2.2 1 0.02 2.0 0.0 0.6 1 0.01 1.0 0.0 1.3 2 0.02 1.5 0.0 1.0 0.01 1.2 0.0 0.2 0.00 0.0 0.0 0.4 0.01 0.5 0.0 0.3 C O R N IN G Hazleton 25 GO033.9 C O R N IN G Hazleton a U a h2 CHV Study No.: 17073-0-437CO Assay No.:17073 Compound:T-6342 CONTROLS NEGATIVE: McCoy's 5a SOLVENT: Water TEST ARTICLE tCo\ oo o to oto TABLE 4 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 44.1 Hours After Treatment Trial #:2 Lab#: CY9I55 Metabolic Activation:-S9 10.0nl/m! Date:09/13/95 NUMBER AND TYPE OF ABERRATION NOT COMPUTED SIMPLE CELLS TG SG j UC TB 1 SB SCORED # OF % % COMPLEX OTHER ABERRA CELLS CELLS TIONS WITH WITH>1 % % R 1 Cl : DF GT PER ABERRA-ABERRA- POLY-MTIOnC CELL TIONS TIONS PUOIDYINDEX A 100 3 2 B 100 1 A+B 200 3 3 A 100 3 B 100 2 1 A+B 200 5 I 1 t 0.00 0.0 0.0 0.0 4.4 i 0.01 1.0 0.0 2.0 4.9 i 0.01 0.5 0.0 1.0 4.7 0.00 0.0 0.0 4.0 3.4 1 0.02 2.0 0.0 1.0 3.3 1 0.01 1.0 0.0 2.5 3.4 249)tg/ml 498)ig/ml 746)tg/ml 995)tg/ml A 100 4 2 B 100 2 2 A+B 200 6 4 A 100 4 2 B 100 2 1 A+B 200 6 3 A 100 3 1 B 100 5 A+B 200 8 1 A 100 2 1 B 100 3 AGI 200 5 1 2 2 11 11 31 21 5 11 1 1 12 11 23 1 1 2 0.04 3.0 1.0 0.0 5.1 0.03 3.0 0.0 2.0 4.8 0.04 3.0 0.5 1.0 5.0 0.01 1.0 0.0 0.0 4.2 0.00 0.0 0.0 0.0 2.8 0.01 0.5 0.0 0.0 3.5 0.05 4.0 1.0 2.0 2.5 0.02 2.0 0.0 2.0 3.0 0.04 3.0 0.5 2.0 2.8 0.03 3.0 0.0 5.0 0.9 0.01 1.0 0.0 4.0 0.5 0.02 2.0 0.0 4.5 0.7 CHV Study No.: 17073-0-437CO TABLE 5 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 20.0 Hours After Treatment Assay No.: 17073 Trial#: 3 Lab #:C Y 11275 Metabolic Activation:-S9 Compound: T-6342___________________________________________ Date: 11/28/95_______________________________________ NUMBER AND TYPE OF ABERRATION CONTROLS NEGATIVE: McCoy's 5a SOI.VENT: Water POSITIVE: MMC NOT COMPUTED SIMPLE CELLS TG 1 SG : UC TB I SB SCORED UOF % % COMPLEX OTHER ABERRA- CELLS CELLS TIONS WITH W1TH>1 % ID 1TR 1QR 1CR : D 1 R 1 Cl ; DF GT PER ABERRA-ABERRA-MTInC CELL TIONS TIONS INDEX 10.0[il/ml 0.500(ig/ml A 100 2 1 B too 2 A+B 200 4 1 A 100 2 B 100 1 A+B 200 2 1 A 25 5 3 1 1 1 1 6 11 2 64 1 1 3 l1 41 1 0.01 1.0 0.01 1.0 0.01 10 0.03 3.0 0.03 3.0 0.03 3.0 0.80 64.0* 0.0 0.0 0.0 0.0 0.0 0.0 16.0* 7.9 7.8 7.9 5.2 7.4 6.3 7.2 TEST ARTICLE 250(ig/nil A B 100 too 2 3 1 0.03 3.0 0.01 1.0 A+B 200 2 31 0.02 2.0 500jig/ml A B 100 2 1 100 1 13 3 0.04 4.0 0.04 4.0 A+B 200 2 1 1 16 0.04 4.0 1250(ig/ml A B 100 4 100 I 1 1 0.01 1.0 1 0.01 1.0 A+B 200 5 1 2 0.01 1.0 2490(ig/ml A B 100 2 1 100 10 1 2 1 0.02 2.0 2 1 0.05 5.0 N A+B 200 12 1 21 3 1 0.04 3.5 3740(ig/nil** A 0 B0 A+H 0 t^o4 * Significantly greater than the solvent controls, p<0.01. ** Toxic dose level. Very few metaphases on slides. 5/20 and 2/18 metaphases from cultures A and B, respectively, had chromosomal aberrations. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.9 7.6 8.3 10.2 10.7 10.5 16.9 12.3 14.6 6.3 3.1 4.7 0.3 0.5 0.4 C O R N IN G Hazleton 0003 C O R N IN G Hazleton CHV Study No.: 17073-0-437CO TABLE 6 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 44.0 Hours After Treatment Assay No.: 17073 Trial 3 Lab #:CYI 1275 Metabolic Activation:-S9 Compound: T-6342 CONTROLS NEGATIVE. McCoy's 5a SOLVENT: Water 10.0iil/ml Date: 11/28/95 NUMBER AND TYPE OF ABERRATION NOT COMPUTED SIMPLE CELLS TG 1SG 1DC TB j SB SCORED # OF % % COMPLEX OTHER ABERRA CELLS CELLS TIONS WITH WITH>1 % % ID ! TR 1QR j CR 1 D ! R i Cl ; DF GT PER ABERRA-ABERRA- POLY-MBI1C CELL TIONS TIONS PLOtDY INDEX A too 2 t B too 2 A+B 200 2 3 A too 3 1 B too l A+B 200 4 1 1 1 11 1 1 21 1 12 1 321 21 21 0.04 4.0 0.0 1.0 8.5 0.05 4.0 1.0 2.0 7.5 0.05 4.0 0.5 1.5 8.0 0.03 3.0 0.0 2.0 9.8 0.01 1.0 0.0 3.0 8 2 0.02 2.0 0.0 2.5 9 .0 TEST ARTICLE 500ng/ml A B too too A+B 200 I250ag/ml A B too too A+B 200 2490|tg/nil A B too too A+B 200 3740(ig/nil A B too 100 A+B 200 * Significantly greater than the solvent controls, p<0.0l. * * Significantly greater than the solvent controls, p<0.05. 23 3 53 51 2 71 31 2 51 6 6 12 1 11 21 1 12 22 21 21 1 1 1 1 2 2 51 71 0.00 0.03 0.02 0.00 0.01 0.01 0.02 0.03 0.03 1 >0.13 2 >0.29 3 >0.21 0.0 3.0 1.5 0.0 1.0 0.5 2.0 3.0 2.5 4.0 11.0 7.5** 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 2.0 1.5 2.0 1.0 1.5 1.0 3.0 2.0 3.0 2.0 2.5 37.0 12.0 24.5* 95 9.7 9.6 9.7 9.9 9.8 6.3 56 6.0 4.3 3.4 3.9 NOQ) ooo Co C O R N IN G Hazleton CHV Study No.: 17073-0-437CO TABLE 7 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 20.0 Hours After Treatment Assay No.:17073 Trial #:1 Lab #: CY9055 Metabolic Activation:+S9 Compound:T-6342 Date:09/06/95 NUMBER AND TYPE OF ABERRATION TRTRE5------- NEGATIVE: McCoy's Sa SOLVENT: Water POSITIVE: CP NOT COMPUTED SIMPLE COMPLEX CELLS TG : SG i UC TB I SB ID ; TR : QR \ CR ! D SCORED I0.0fil/ml S.OOng/ml A 100 1 4 B 100 2 1 A+B 200 3 5 A 100 2 1 B 100 2 1 A+B 200 4 2 A 25 9 11 1 1 11 2 1 11 1 21 1 12 4 1 2 7 1 # OF % % OTHEBjABERRA- CELLS CELLS TIONS WITH WITH>1 % Cl : DF GT PER ABERRA-ABERRA-KflOnC CELL TIONS TIONS INDEX 0.01 1.0 0.03 2.0 0.0 10.0 1.0 7.8 0.02 1.5 0.5 8.9 1 0.11 2.0 1.0 9.1 0.03 3.0 0.0 8.6 1 0.07 2.5 0.5 8.9 2 1.88 56.0* 48.0* 2.8 TEST ARTICLE I250ng/ml A 100 1 2 1 1 B 100 3 1 11 A+B 200 3 2 2 11 1 1 2500ng/ml A B 100 2 41 100 4 1 2 1 1 2 1 1 A+B 200 2 4 1 6 1 1 1 2 11 3750fig/nil A B 100 13 4 6 1 3 3 1 1 1 5 100 5 1 5 2 2 6 11 1 2 2 A+B 200 18 4 1 11 2 3 9 14 2 3 1 7 5000|ig/nil A 94 29 4 2 17 4 2 4 6 4 1 2 4 B 106 23 2 14 3 4 10 21 4 4 2 11 A+B 200 52 6 2 31 7 6 14 27 8 5 4 15 N'O> Significantly greater than the solvent controls, p<0.01 0.04 0.02 0.03 0.06 0.07 0.07 0.21 0.31 0.26 1 0.57 0.69 1 0.64 1.0 2.0 1.5 40 6.0 5.0 11.0 18.0 14.5* 25.5 36.8 31.5* 1.0 0.0 0.5 1.0 1.0 1.0 6.0 8.0 7.0* 12.8 17.0 15.0* 10.5 9.1 9.8 11.2 10.6 10.9 10.5 111 10.8 4.3 11.1 7.7 O o o 4 & CO CHV Study No.: 17073-0-437CO TABLE 8 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 20.0 Hours After Treatment Assay No.: 17073 Trial #:2 Lab#:CY9!55 Metabolic Activation:+S9 Compound:T-6342 Date:09/l3/95 NUMBER AND TYPE OF ABERRAHON 5NTRLS NEGATIVE: McCoy's 5a SOLVENT: Water POSITIVE: CP NOT COMPUTED SIMPLE CELLS TG SG i UC TB j SB ID ; TR SCORED o 73 n a COMPLEX # OF % % OTHER ABERRA CELLS CELLS TIONS WITH W1TH>1 % R j Cl I DF GT PER ABERRA-ABERRA-MBOIIC CELL TIONS TIONS INDEX lOOfil/ml 5.00ng/ml A 100 B 100 A+B 200 A 100 B 100 A+B 200 A 25 2 52 72 4 6 10 11 4 11 1 l 11 34 3 3 3 1 1 11 1 2 0.03 2.0 0.00 0 .0 0.02 1.0 0.03 3.0 0.00 0.0 0.02 1.5 0.48 36.0* 1.0 0 .0 0.5 0 .0 0 .0 0 .0 8.0** 17.0 11.2 14.1 13.3 13.3 13.3 4.7 TEST ARTICLE I250ftg/nil A B 100 6 1 100 2 2 2 21 A+B 200 8 1 2 2 2l 2490(.ig/ml A B 100 2 1 100 5 I1 1 A+B 200 7 1 12 3730ng/ml A B 100 9 11 100 9 1 1 1 A+B 200 18 1 1 1 1 1 4970ng/ntl A B 75 29 1 2 17 9 2 9 10 1 2 75 32 3 17 2 3 10 19 4 2 18 5 A+B 150 61 4 2 34 11 5 19 29 5 4 * Significantly greater than the solvent controls, p<0.01 ** Significantly greater than the solvent controls, p<0.05 1 13 0.03 2.0 0.02 2.0 1.0 12.8 00 9.0 0.03 2.0 0.5 10.9 002 2.0 0.01 1.0 0 .0 13.1 0 .0 15.6 0.02 1.5 0 .0 14.4 0.02 2.0 0.01 1.0 0 .0 13.6 0 .0 13 8 0.02 1.5 0 .0 13 7 0.79 41 3 0.83 44.0 28.0 24.0 8.3 6.9 0.81 42.7* 26.0* 7.6 C O R N IN G Hazleton CHV Study No.: 17073-0-437CO TABLE 9 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS Cells Fixed 44.1 Hours After Treatment Assay No.: 17073 Trial ^:2 Lab#:CY9155 Metabolic Activation:+S9 Compound:T-6342 " CONTROL'S NEGATIVE: McCoy's 5a SOLVENT. Water lO.Oal/ml Date:09/13/95 N U M B ER A N D TY PE O F A B ER R A TIO N NOT COMPUTED CELLS TG \ SG IIJC SCORED SIMPLE TB ! SB # OF % % COMPLEX OTHER ABERRA CELLS CELLS TIONS WITH WITH>1 % % ID ! TR ! QR I CR 1 D I R I Cl I DF GT PER ABERRA-ABERRA- POLY-MTIUnC CELL TIONS TIONS HOOEJYINDEX A too 3 1 1 B too 2 A+B 200 5 1 1 A too 2 2 B too 2 2 1 11 A+B 200 4 4 12 1 1 3 3 0.01 1.0 0.0 1.0 6.1 0.01 1.0 0.0 2.0 6.5 0.01 1.0 0.0 1.5 6.3 0.01 1.0 0.0 0.0 5.6 0.05 5.0 0.0 3.0 4.3 0.03 3.0 0.0 1.5 5.0 TEST ARTICLE 498(ig/ml A B too 6 12 1 too 4 1 A+B 200 10 1 1 2 1 1250ng/ml A B too 3 too 5 2 A+B 200 8 2 2490ng/mt A B too 1 2 too 6 2 1 1 A+B 200 7 4 11 3730ng/ml A B too 2 2 too 3 1 2 1 A+B 200 5 3 3 4970jig/ml** A B 0 0 A+B 0 1 * Significantly greater than the solvent controls, p<0.0l ** Toxic dose level. O C CO N 1 1 2 t 1 1 1 1 1 0.04 3.0 0.01 1.0 0.03 2.0 0.00 0.0 0.01 1.0 0.01 0.5 0.02 2.0 0.01 1.0 0.02 1.5 0.03 2.0 0.01 1.0 0.02 1.5 1.0 0.0 4.2 0.0 2.0 5.0 0.5 1.0 4.6 0.0 2.0 5.1 0.0 2.0 4.4 0.0 2.0 4.6 0.0 4.0 5.2 0.0 2.0 5.6 0.0 3.0 5.4 1.0 6.0 6.4 0.0 4.0 5.8 0.5 5.0 6.1 15.0 0.8 26.0 2 1 20.5* 1.5 C O R N IN G Hazleton TABLE 10 CONTROL DATA OF CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS 6/95 THROUGH 8/95 Negative Control Solvent Control Positive Control Mitomycin C Negative Control Solvent Control Positive Control Cyclophosphamide Activation Without Without Without With With With MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N #Of Aberrations Per Cell 0.00 0.06 0.015 35 0.00 0.03 0.010 35 0.14 2.88 0.553 26 0.00 0.03 0.014 34 0.00 0.12 0.020 34 0.36 4.28 1.299 25 % Of Cells With Aberrations 0.0 4.0 1.19 35 0.0 2.0 0.74 35 14.0 68.0 31.38 26 0.0 3.0 1.10 34 0.0 7.0 1.44 34 24.0 88.0 51.20 25 % O f Cells With >1 Aberrations 0.0 0.5 0.07 35 0.0 0.5 0.04 35 0.0 60.0 11.38 26 0.0 1.0 0.09 34 0.0 4.0 0.29 34 8.0 72.0 30.40 25 CONTROL DATA OF POLYPLOIDY IN CHINESE HAMSTER OVARY CELLS 4/95 THROUGH 9/95 Negative Control Solvent Control MIN MAX AVG N MIN MAX AVG N Without Activation 0.00 4.00 0.540 46 0.00 2.00 0.430 58 With Activation 0.0 4.0 0.63 46 0.0 3.0 0.50 58 CHV Study No.: 17073-0-437CO 32 000326 C O R N IN G Hazleton 15.0 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS NOT COMPUTED TG Chromatid gap: ("tid gap"). An achromatic (unstained) region in one chromatid, the size of which is equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals of aberrations as they may not all be true breaks. SG Chromosome gap: ("isochromatid gap, IG"). Same as chromatid gap but at the same locus in both sister chromatids. UC Uncoiled chromosome: Failure of chromatin packing. Probably not a true aberration. PP Polyploid cell: A cell containing multiple copies of the haploid number (n) of chromosomes. Not counted in the cells scored for aberrations. E Endoreduplication: 4n cell in which separation of chromosome pairs has failed. Not counted in the cells scored for aberrations. SIMPLE TB Chromatic break: An achromatic region in one chromatid, larger than the width of a chromatid. The associated fragment may be partially or completely displaced. SB Chromosome break: Chromosome has a clear break, forming an abnormal (deleted) chromosome with an acentric fragment that is dislocated. DM "Double Minute " fragment: These are small double dots, some of which are terminal deletions and some interstitial deletions and probably small rings. Their origins are not distinguishable. CHV Study No.: 17073-0-437CO 33 000327 COMPLEX ID Interstitial deletion: TR Triradial: QR Quadri radial: D Dicentric: DF TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring RC Ring Chromatid: RF CHV Study No.: 17073-0-437CO C O R N IN G Hazleton Length of chromatid "cut out" from midregion of a chromatid resulting in a small fragment of ring lying beside a shortened chromatid of a gap in the chromatid. An exchange between two chromosomes, or one chromosome and an acentric fragment, which results in a three-armed configuration. As triradial, but resulting in a four-armed configuration. An exchange between two chromosomes which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF. Dicentric with fragment. An exchange between three chromosomes which results in a chromosome with three centromeres. Often associated with two to three AF. Such exchanges can involve many chromosomes and are named as follows: four centromeres, up to four AF five centromeres, up to five AF six centromeres, up to six AF A chromosome which forms a circle containing a centromere. This is often associated with an acentric fragment in which case it is classed as RF. Single chromatid ring (acentric). Ring with associated acentric fragment. 34 000328 CI Chromosome Intrachange: T Translocation: AB Other GT/> C O R N IN G Hazleton Exchange within a chromosome; e.g., a ring that does not include the entire chromosome. Obvious transfer of material between two chromosomes resulting in two abnormal chromosomes. When identifiable, scored as "T" not "2Ab." Abnormal monocentric chromosome. This is a chromosome whose morphology is abnormal for the karyotype, and often the result of a translocation, pericentric inversion, etc. Classification used if abnormality cannot be ascribed to; e.g., a reciprocal translocation. A cell which contains more than 10 aberrations. A heavily damaged cell should be analyzed to identify the types of aberrations and may not actually have >10, e.g., multiple fragments such as those found associated with a tricentric. CHV Study No.: 17073-0-437CO 35 000329