Document 4ak7yqq3oKRBRLB9QRz7Np19e

AR M6-0135 C O R N IN G Hazleton MUTAGENICITY TEST ON T-6342 IN AN /TVF7FD MOUSE MICRONUCLEUS ASSAY / 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.: 17073-0-455 SUBMITTED TO 3M Corporation Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE December 14,1995 CHV Study No.: 17073-0-455 1 of 23 000330 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT Project Title: In Vivo Mouse Micronucleus Assay Project No.: 20996 Assay No.: 17073 Protocol No.: 455 Edition No.: 17, 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 Randomization of Animals/09/18/1995 09/18/1995 S. Ballenger Draft report Review/10/31/1995 10/31/1995 C. Orantes Final Report Review/12/14/1995 12/14/1995 C. Orantes CHV Study No.: 17073-0-455 2 000331 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 Study Completion Date CHV Study No.: 17073-0-455 3 000332 C O R N IN G Hazleton TABLE OF CONTENTS Page No. SUMMARY.................................................................................................................................. 6 1.0 SPONSOR..................................................................................................................... 7 2.0 MATERIAL (Test Article) ............................................................................................7 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 ..........................................................................................................7 4.0 PROTOCOL NO..............................................................................................................7 5.0 STUDY DATES ........................................................................................................... 7 5.1 Initiation Date 5.2 Experimental Start Date 5.3 Experimental Termination Date 6.0 SUPERVISORY PERSONNEL ....................................................................................7 6.1 Study Director 6.2 Laboratory Supervisor 7.0 OBJECTIVE ..................................................................................................................7 8.0 MATERIALS ................................................................................................................8 9.0 SOLUBILITY AND STABILITY: ................................................................................8 10.0 DOSE SELECTION STUDY ........................................................................................8 10.1 Dose Selection 10.2 Dosing Information 10.3 Results and Interpretation 10.4 Conclusion CHV Study No.: 17073-0-455 4 000333 C O R N IN G Hazleton 11.0 MICRONUCLEUS STU D Y ..................... 11.1 Dose Selection 11.2 Micronucleus Assay Dosing Information 10 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS ........... 11 13.0 EVALUATION CRITERIA ........................................................................................ 12 13.1 General 13.2 Data Presentation and Interpretation 14.0 RESULTS AND INTERPRETATION........................................................................ 13 15.0 CONCLUSION............................................................................................................ 13 16.0 REFERENCES ............................................................................................................14 17.0 EXPERIMENT DATA TABLES ................................................................................ 15 CHV Study No.: 17073-0-455 5 000334 SUMMARY C O R N IN G Hazleton Mutagenicity Test on T-6342 in an In Vivo Mouse Micronucleus Assay The objective of this in vivo assay was to evaluate the ability of the test article, T-6342, to induce micronuclei in bone marrow polychromatic erythrocytes of Crl:CD-l(ICR) BR mice. In the dose selection study, the test article was solubilized in deionized water and dosed by oral gavage at 500,1625,2750, 3875, and 5000 mg/kg. Six animals (three males and three females) were assigned to each dose group. Animals were observed for three days after dosing for toxic signs and/or mortality. Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg. In the micronucleus assay, the test article was solubilized in deionized water and dosed by oral gavage at 1250, 2500, and 5000 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized approximately 24 hours after dosing were included in the assay. The animals dosed with the test article were euthanatized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material, T-6342, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test. CHV Study No.: 17073-0-455 6 000335 C O R N IN G Hazleton Mutagenicity Test on T-6342 in an in vivo Mouse Micronucleus Assay 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: In Vivo Mouse Micronucleus Assay 4.0 PROTOCOL NO.: 455, Edition 17, Modified for 3M Corporation 5.0 STUDY DATES 5.1 Initiation Date: August 15, 1995 5.2 Experimental Start Date: August 29, 1995 5.3 Experimental Termination Date: October 16, 1995 6.0 SUPERVISORY PERSONNEL 6.1 Study Director: Hemalatha Murli, Ph.D. 6.2 Laboratory Supervisor: Monica Vegarra, B.S. 7.0 OBJECTIVE The objective of this in vivo assay was to evaluate the ability of the test article, T-6342, to induce micronuclei in bone marrow polychromatic erythrocytes of Crl:CD-l(ICR) BR mice. This study was conducted using modifications of the procedures suggested by Heddle et al. (1983). CHV Study No.: 17073-0-455 7 000336 C O R N IN G Hazleton 8.0 MATERIALS Adult male and female mice, strain Crl:CD-l(ICR) BR, were purchased from Charles River Laboratories, Portage MI.. This healthy, random bred strain was selected to maximize genetic heterogeneity and at the same time assure access to a common source. The protocol for this study was approved by the CHV-ACUC prior to the initiation of dosing. Animals were housed up to seven per cage during quarantine, and housed up to five per cage at randomization. The temperature and relative humidity were maintained at 726F and 5515%, respectively. A 12-hour light/12-hour dark cycle was maintained. A commercial diet (Purina Certified Laboratory Pellets # 5002) and water were available ad libitum for the duration of the study. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, alkalinity, heavy metals, and halogens. Sanitized caging was used for housing the animals. Personnel handling animals or working within the animal facilities were required to wear suitable protective garments and equipment. Animals were quarantined for seven before being placed on study. Animals were randomly assigned to study groups and were individually weighed prior to dosing. All animals were dosed based upon the individual body weights. Animals were uniquely identified by ear tag. Dose or treatment groups were identified by cage card/label. At the termination of the study all surviving animals were euthanatized by C 02 inhalation, followed by penetration of the thorax. Any extra animals not used for the study were euthanatized by C02inhalation, followed by penetration of the thorax. 9.0 SOLUBILITY AND STABILITY: The test article, T-6342, was supplied as a clear colorless liquid. The vehicle selected for this assay was deionized water. The stability of the test material under the dosing conditions of this assay is the responsibility of the sponsor. 10.0 DOSE SELECTION STUDY 10.1 Dose Selection Dose levels of 500, 1625, 2750, 3875, and 5000 mg/kg were administered by oral gavage for the dose selection study. CHV Study No.: 17073-0-455 8 000337 C O R N IN G Hazleton 10.2 Dosing Information The animals used in the dose selection assay were dosed on August 29,1995. The weight range of the animals used in the dose range finding assay was 26.5 - 33.5 and 21.1 - 25.1 grams, for the males and females, respectively. Dosing solutions were prepared just prior to dosing and were prepared by making a 500 mg/ml stock for the high dose (5000 mg/kg). This was prepared by adding 6.6 ml of deionized water (Lot # 17, prepared at CHV) to 6.0007 g of T-6342, resulting in a clear, colorless solution . Dilutions of this stock were prepared for the other dose levels. Dosing was achieved using a 10 ml/kg dosing volume. All animals were eight weeks and one day old at the time of dosing. An outline of the dosing scheme is found in the following table. A total of 30 animals was used in this assay. DOSE GROUPS TREATMENT M F T-6342 500 mg/kg 1625 mg/kg 2750 mg/kg 3875 mg/kg 5000 mg/kg 33 33 33 33 33 All doses given were on an acute (one-time only) basis. 10.3 Results and Interpretation All animals were examined after dosing and daily throughout the duration of the study (three days) for toxic effects and/or mortalities. All animals appeared normal immediately after dosing and remained healthy until the end of the observation period. The mortality data for this assay are summarized in the following table: CHV Study No.: 17073-0-455 9 000338 C O R N IN G Hazleton Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T-6342 Observations Jl&atfflgai______________ Male______________Female 500 mg/kg 0/3 0/3 1625 mg/kg 0/3 0/3 2750 mg/kg 0/3 0/3 3875 mg/kg 0/3 0/3 5000 mg/kg 0/3 0/3 10.4 Conclusion Based on these results, the maximum tolerated dose was estimated to be >5000 mg/kg. 11.0 MICRONUCLEUS STUDY 11.1 Dose Selection Based on results from the dose selection study, dose levels of 1250, 2500, and 5000 mg/kg were selected for this study. 11.2 Micronucleus Assay Dosing Information The animals used in the micronucleus assay were dosed on September 19,1995. Cyclophosphamide (Sigma, Lot # 43H0269, CAS # 6055-19-2), the positive control, was solubilized in sterile deionized water (Lot #17, prepared at CHV) and was administered by oral gavage at 80 mg/kg. The vehicle control, deionized water (Lot # 17, prepared at CHV), was administered concurrently with the test article at a volume of 10 ml/kg. The weight range of the animals used in the micronucleus assay was 28.7 - 37.6 g and 21.1 - 25.6 g grams for the males and females, respectively. The dosing solutions for the assay were prepared by making a 500 mg/ml stock for the high dose (5000 mg/kg). This was prepared by adding deionized water to 11.0008 g of T-6342 up to a volume of 22.0 ml. A clear, colorless solution was obtained. Dilutions of this stock were prepared for the remaining dose levels. CHV Study No.: 17073-0-455 10 000339 C O R N IN G Hazleton Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, euthanatized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanatized approximately 24,48 and 72 hours after dosing for extraction of the bone marrow. An outline of the dosing scheme is found in the following table: Dosing Scheme for Micronucleus Assay A total of 110 animals was used in this assay Number of Animals Assigned Treatment 24 Hr 48 Hr 72 Hr MF MF MF T-6342 1250mg/kg 55 5 5 55 2500 mg/kg 55 5 5 55 5000 mg/kg 55 5 5 55 Vehicle Control, deionized water 10 ml/kg 55 Positive Control, Cyclophosphamide, 80 mg/kg 5 5 The age of the animals at the time of dosing was eight weeks and one day. Volume dosed was 10 ml/kg and was based upon individual animal weight. 12.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At the appropriate harvest time, the animals were euthanatized with C 02followed by penetration of the thorax and the adhering soft tissue and epiphyses of both femora were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 - 5 ml bovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped using Depex mounting medium. The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were CHV Study No.: 17073-0-455 11 000340 C O R N IN G Hazleton used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this Crl:CD-l(ICR) BR strain is about 0.0-0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes. 13.0 EVALUATION CRITERIA: 13.1 General The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively). 13.2 Data Presentation and Interpretation Data are summarized by sex and dose groups for the different time points. Individual animal data are also presented. The analysis of these data was performed using an analysis of variance (Winer, 1971) on either untransformed (when variances are homogeneous) and rank transformed (when variances are heterogeneous) proportions of cells with micronuclei per animal. If the analysis of variance was significant (p<0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination. The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific j udgment. CHV Study No.: 17073-0-455 12 000341 C O R N IN G Hazleton 14.0 RESULTS AND INTERPRETATION: All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All animals from the 1250 and 2500 mg/kg dose groups appeared normal immediately after dosing and remained healthy until the appropriate harvest times. Immediately after dosing, all animals in the 5000 mg/kg dose group appeared normal. Approximately 24 hours after dosing, 2 males (#'s2712, 48 hour harvest group; 2714, 72 hour harvest group) from the 5000 mg/kg dose group were found dead. One male (#2718, 72 hour harvest group) appeared hunched and weak with squinted eyes. All other animals appeared normal at this time. Approximately 48 hours after dosing, 1 male (#2718, 72 hour harvest group) and 1 female (#2780, 72 hour harvest group) from the 5000 mg/kg dose group were found dead. All other animals appeared normal at this time and remained healthy until the appropriate harvest times. The test article, T-6342, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. Due to toxicity, the PCE/NCE ratios of the males and females from the 5000 mg/kg dose group at the 72 hour harvest group were significantly lower than the vehicle control animals. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes as compared to the vehicle controls, with means and standard errors of 5.44% 0.37% and 2.50% 0.33% for the males and females, respectively. The data summarized by dose group are presented in Table 1 and individual animal data are found in Tables 2 through 7. Historical control data are presented in Table 8. 15.0 CONCLUSION: The test material, T-6342, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay. CHV Study No.: 17073-0-455 13 000342 C O R N IN G Hazleton 16.0 REFERENCES: Dunnett, C.W.: A multiple comparisons procedure for comparing several treatments with a control. J. Am. Statist. Assoc., 50:1096-1121. 1955. Dunnett, C.W.: New tables for multiple comparisons with a control. Biometrics, 20:482491,1964. Heddle, J.A., Hite, M., Kirkhart, B., Larsen, K., MacGregor, J.T., Newell, G.W. and Salamone, M.F.: The induction of micronuclei as a measure of genotoxicity. Mutation Res., 123:61-118. 1983. Schmid, W.: The micronucleus test. Mutation Res.. 31:9-15. 1975. Schmid, W.: The micronucleus test for cytogenetic analysis. Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4 (A. Hollaender, ed.). Plenum, pp. 31-53, 1976. Winer, B.J.: Statistical Principles in Experimental Design, McGraw-Hill, New York, Second Edition, 1971. CHV Study No.: 17073-0-455 14 000343 C O R N IN G Hazleton 17.0 EXPERIMENT DATA TABLES CHV Study No.: 17073-0-455 15 000344 C O R N IN G Hazleton SPONSOR: 3M Corporation TABLE 1 MICRONUCLEUS DATA SUMMARY TABLE TEST ARTICLE:T-6342 ASSAY:17073 TREATMENT DOSE CONTROLS VEHICLE POSITIVE-CP Watar 80.0 m g / k g HARVEST TIME (HR) 24 hr 24 hr % MICRONUCLEATED PCEs MEAN OF 1000 PER ANIMAL S.E. MALES FEMALES TOTAL 0.14 0.02 5.44 0.37* 0.02 0.02 2.50 0.33* 0.08 0.02 3.97 0.54* RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.51 0.06 0.64 0.07 0.69 0.11 0.63 0.05 TEST ARTICLE 1250 D g / k g 2500 m g / k g 5000 m g / k g 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr 0.28 0.05 0.00 0.00 0.06 0.02 0.12 0.04 0.02 0.02 0.20 0.07 0.06 0.04 0.15 0.09 0.17 0.03 0.00 0.00 0.02 0.02 0.02 0.02 0.04 0.02 0.02 0.02 0.06 0.04 0.10 0.04 0.06 0.02 0.03 0.03 0.14 0.05 0.01 0.01 0.04 0.02 0.08 0.02 0.02 0.01 0.13 0.04 0.08 0.03 0.10 0.04 0.09 0.03 0.69 0.09 0.68 0.08 0.36 0.08 0.69 0.07 0.79 0.07 0.47 0.07 0.80 0.08 0.56 0.10 0.17 0.04* 0.75 0.17 0.74 0.06 0.59 0.06 0.64 0.05 0.65 0.01 0.64 0.07 0.79 0.09 0.63 0.06 0.24 0.05* "Significantly different from the corresponding vehicle control. p<0.05. CHV Study No.: 17073-0-455 16 000345 C O R N IN G Hazleton TABLE 2 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY N O . :17073 TREATMENT 24 HOUR HARVEST MALE ANIMAL NO.MN NUMBER PCEs (1000) RATIO PCE :NCE VEHICLE CONTROL Water POSITIVE CONTROL CP 80.0 rag/kg TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2682 2689 2694 2720 2721 2696 2698 2715 2723 2728 2683 2691 2697 2703 2717 2679 2685 2693 2702 2724 2676 2684 2709 2713 2727 1 2 1 2 1 46 61 52 65 48 4 1 3 3 3 2 1 0 2 1 0 0 1 0 2 MN = Micronucleus PCE = Polychromatic erythrocyte No.MN PCEs = Micronucleated PCEs NCE=Normochromatic erythrocyte 0.32 0.54 0.48 0.66 0.52 0.79 0.80 0.53 0.58 0.49 0.67 0.78 0.98 0.45 0.59 0.74 0.47 0.90 0.72 0.60 0.51 0.90 0.81 0.98 0.78 CHV Study No.: 17073-0-455 000346 17 C O R N IN G Hazleton TABLE 3 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY N O . :17073 TREATMENT ANIMAL NUMBER NOTMN PCEs RATIO PCE :NCE (1000) 24 HOUR HARVEST FEMALE VEHICLE CONTROL Water POSITIVE CONTROL CP 80.0 mg/kg TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2741 2744 2773 2776 2779 2734 2746 2749 2760 2783 2735 2742 2750 2771 2781 2732 2733 2738 2755 2772 2743 2757 2761 2763 2768 0 1 0 0 0 31 17 29 17 31 0 0 0 0 0 0 0 0 1 1 0 0 2 2 1 MN = Micronucleus PCE =* Polychromatic erythrocyte No.MN PCEs = Micronucleated PCEs NCE=Normochromatic erythrocyte 0.76 0.76 0.70 0.27 0.94 0.78 0.71 0.62 0.54 0.48 1.03 1.25 0.69 0.39 0.39 0.56 0.64 0.81 0.54 0.67 0.92 0.56 0.83 1.04 0.61 CHV Study No.: 17073-0-455 000347 18 C O R N IN G Hazleton TABLE 4 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT ANIMAL NUMBER "NO". MN PCEs ' RATIO P C E :NCE (1000) 48 HOUR HARVEST MALE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2680 2681 2695 2705 2710 2675 2688 2707 2726 2729 2677 2699 2700 2708 2712* 0 0 0 0 0 1 0 0 0 0 1 1 4 0 *Animals found dead MN = Micronucleus PCE = Polychromatic erythrocyte No.MN PCEs * Micronucleated PCEs NCE=Normochromatic erythrocyte 0.44 0.54 0.77 0.77 0.89 0.52 0.81 0.96 0.82 0.83 0.32 0.64 0.47 0.79 CHV Study No.: 17073-0-455 000348 19 C O R N IN G Hazleton TABLE 5 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY N O . :17073 TREATMENT ANIMAL NUMBER TOT PCEs (1000) RATIO PCE :NCE 48 HOUR HARVEST FEMALE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2730 2736 2747 2754 2775 2737 2745 2758 2759 2762 2739 2753 2756 2767 2770 0 0 1 0 0 0 1 0 0 0 1 0 1 1 0 MN = Micronucleus PCE = Polychromatic erythrocyte No.MN PCEs = Micronucleated PCEs NCE=Normochromatic erythrocyte 0.94 0.62 0.74 0.75 0.64 0.67 0.69 0.62 0.65 0.63 0.43 0.78 0.68 0.67 0.61 CHV Study No.: 17073-0-455 000349 20 C O R N IN G Hazleton TABLE 6 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT ANIMAL NUMBER TOT PCEs (1000) RATIO P C E :NCE 72 HOUR HARVEST MALE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2678 2687 2716 2719 2722 2692 2701 2704 2711 2725 2686 2690 2706 2714* 2718* 0 1 1 1 0 0 3 4 2 1 1 2 2 Animals found dead MN = Micronucleus PCE = Polychromatic erythrocyte No.MN PCEs = Micronucleated PCEs NCE=Normochromatic erythrocyte 0.26 0.30 0.17 0.51 0.56 0.28 0.37 0.52 0.49 0.70 0.11 0.16 0.26 CHV Study No.: 17073-0-455 000350 21 C O R N IN G Hazleton TABLE 7 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR:3M Corporation TEST ARTICLE:T-6342 ASSAY NO.:17073 TREATMENT ANIMAL NUMBER TJ7HN"' PCEs (1000) RATIO P C E :NCE 72 HOUR HARVEST FEMALE TEST ARTICLE 1250 mg/kg 2500 mg/kg 5000 mg/kg 2740 2751 2766 2769 2778 2748 2752 2764 2774 2777 2731 2765 2780* 2782 2784 0 0 1 0 0 1 0 0 0 2 0 0 1 0 `Animals found dead MN = Micronucleus PCE " Polychromatic erythrocyte No.MN PCEs = Micronucleated PCEs NCE=Normochromatic erythrocyte 0.68 0.48 0.68 0.40 0.71 0.68 0.45 0.54 0.85 0.66 0.24 0.38 0.18 0.18 CHV Study No.: 17073-0-455 000351 22 C O R N IN G Hazleton TABLE 8 MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 2/95 THROUGH 9/95 POOLED VEHICLE CONTROL MIN MAX AVC N % MICRONUCLEATED PCEs per 1000 PCE MEAN OF 1000 PER ANIMAL S.E. MALES FEMALES TOTAL 0.00 0.26 0.08 0.06 49 0.00 0.16 0.06 0.04 49 0.01 0.18 0.07 0.04 49 RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.32 0.82 0.56 0.13 49 0.34 1.03 0.61 0.14 49 POSITIVE CONTROL Cyclophosphamide, 80 mg/kg MIN MAX AVG N 1.72 5.44 3.25 1 0.94 22 1.50 6.36 3.00 1.05 22 1.81 5.38 3.13 0.81 22 0.44 0.72 0.58 0.09 22 0.44 0.81 0.62 0.11 22 CHV Study No.: 17073-0-455 G00352 23