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PiR11-OM30 CO RN ING Hazleton MUTAGENICITY TEST ON T-6564 IN AN IN VIVO 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.: 17750-0-455 SUBMITTED TO 3M Corporation Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE November 1, 1996 CHV Study No.: 17750-0-455 1 of 26 f 000176 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT Project Title: In Vivo Mouse Micronucleus Assay Project No.: 20996 Assay No.: 17750 Protocol No.: 455 Edition No.: 17, Modified for 3M Corporation Quality Assurance inspections of the study and review of the fined 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/07/30/1996 07/30/1996 C. Orantes Harvest/08/1/1996 08/1/1996 C. Smith Draft Report Review/09/17,18/1996 09/18/1996 C. Orantes/C. Smith Final Report Review/11/01/1996 11/01/1996 C. Smith Ci --_________*// 1/ y > Qtality Assurance Unit Date Released CHV Study No.: 17750-0-455 2 000177 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: V-QAMJ I j J f c i . LLK.k Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology jLli\S h _____ Study Completion Date CHV Study No.: 17750-0-455 3 000178 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 I .......................................................................................... 9 10.1 Dose Selection 10.2 Dosing Information 10.3 Results and Interpretation 10.4 Conclusion CHV Study No.: 17750-0-455 4 < 000179 CORNING Hazleton 11.0 . DOSE SELECTION STUDY II .................................................................................... 11 11.1 Dose Selection 11.2 Dosing Information 11.3 Results and Interpretation 11.4 Conclusion 12.0 MICRONUCLEUS ST U D Y ............................................................................................13 12.1 Dose Selection 12.2 Micronucleus Assay Dosing Information 13.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS................14 14.0 EVALUATION CRITERIA ............................................................................................ 15 14.1 General 14.2 Data Presentation and Interpretation 15.0 RESULTS AND INTERPRETATION............................................................................ 16 16.0 CONCLUSION ................................................................................................................16 17.0 REFERENCES ................................................................................................................17 18.0 DEVIATION FROM THE SIGNED PROTOCOL...........................................................17 19.0 EXPERIMENT DATA TABLES .....................................................................................18 CHV Study No.: 17750-0-455 5 GGOISO C O R N IN G Hazleton SUMMARY Mutagenicity Test on T-6564 in an In Vivo Mouse Micronucleus Assay The objective of this in vivo assay was to evaluate the ability of the test article, T-6564, 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 1000,1510, 2010, 2540 and 3010 mg/kg in a first trial and then dosed at 1010, 1520, 2020, and 2530 mg/kg in a second trial. Six animals (three males and three females) were assigned to each dose group. Animals were observed for four 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 about 2000 mg/kg. In the micronucleus assay, the test article was solubilized in deionized water and dosed by oral gavage at 498, 995, and 1990 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, euthanized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24,48 and 72 hours after dosing for extraction of the bone marrow. The test material, T-6564, 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.: 17750-0-455 6 000181 C O R N IN G Hazleton Mutagenicity Test on T-6564 in an In Vivo Mouse Micronucleus Assay 1.0 SPONSOR: 3M Corporation 2.0 MATERIAL (Test Article) 2.1 Client's Identification: T-6564 L-13167 FC1015-X 2.2 Date Received: May 30, 1996 2.3 Physical Description: Clear colorless liquid 2.4 Genetics Assay No.: 17750 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: June 4, 1996 5.2 Experimental Start Date: July 11,1996 5.3 Experimental Termination Date: August 22, 1996 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-6564, 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.: 17750-0-455 7 0001S2 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-IACUC prior to the initiation of dosing. Animals were housed five per cage during quarantine, and housed five per cage at randomization. The temperature and relative humidity were maintained at 72 6 F and 55 15%, respectively, except on July 31,1996, for the definitive study, when the humidity was recorded as 75.1%. 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 days 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 euthanized by C 02/0 2 inhalation, followed by C 02 inhalation and penetration of the thorax. Any extra animals not used for the study were saved for training. 9.0 SOLUBILITY AND STABILITY The test article, T-6564, was supplied as a clear colorless liquid. The specific gravity of the test article was calculated in the testing laboratory as 1.22 g/ml. This specific gravity was used to prepare dosing stocks for the assay. Deionized water was used as the vehicle in this assay. The determination of the test article stability under the dosing conditions of this assay and the test article characteristics as defined in the GLP regulations of FDA (21 CFR 58.105), EPA-TSCA (40 CFR 792.105), and EPA-FIFRA (40 CFR 160.105) is the responsibility of the sponsor. CHV Study No.: 17750-0-455 8 G001S3 C O R N IN G Hazleton 10.0 DOSE SELECTION STUDY I 10.1 Dose Selection Dose levels of 1000,1510,2010, 2540 and 3010 mg/kg were administered by oral gavage for the first dose selection study. 10.2 Dosing Information The animals used in the first dose selection assay were dosed on July 11,1996. The weight range of the animals used in the dose range finding assay was 30.9-35.1 and 22.2-27.6 grams, for the males and females, respectively. Dosing solutions were prepared just prior to dosing and were prepared by making a 301 mg/ml stock for the high dose (3010 mg/kg ). This was prepared by adding deionized water (CHV, Lot # 19) up to a volume of 15.0 ml to 3.7 ml of T-6564, resulting in a clear colorless solution. Dilutions of this stock were prepared for the 2540, 2010, 1510 and 1000 mg/kg dose levels. Dosing was achieved using a 10 ml/kg dosing volume. All animals were eight weeks and three days old at the time of dosing. An outline of the dosing scheme is found in the following table. Dosing Scheme for Dose Selection Assay I Treatment__ T-6564 1000 mg/kg 1510 mg/kg 2010 mg/kg 2540 mg/kg 3010 mg/kg Male Female 33 33 33 33 33 A total of 30 animals was used in this assay. 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 (four days) for toxic effects and/or mortalities. The animal observations for this assay are summarized in the following table: CHV Study No.: 17750-0-455 9 000184 CO R N IN G Hazleton Animal Observations for Toxicity for Dose Rangefinding Assay I Time After Dose Dosing Level (hours) (mg/kg) Observations =0.2 all Normal 1000 Normal =16.8 Male # 9761 and Female #s 9776 & 9788 found dead. 1510 Remaining males appeared hunched and hypoactive. Remaining female appeared normal. 2010, 2540, Normal 3010 =40.2 1000, 1510, 2010, 2540 Normal 3010 Male #s 9765 & 9767 found dead. Remaining male and all females appear normal. 1000 Normal =67.0 1510 2010, 2540, 3010 Male # 9766 found dead. Remaining male and female appeared normal. Normal 1000 Normal 1510 Remaining male # 9774 found dead. Remaining female appeared normal. =88.5 2010, 2540 Normal 3010 Remaining male #9764 appeared hypoactive and hunched with a rough hair coat. All females appeared normal. The mortality data for this assay are summarized in the following table: CHV Study No.: 17750-0-455 10 000135 C O R N IN G Hazleton Summary of Mortalities for Dose Rangefinding I Treatment______________ Male_______ Female T-6564 1000 mg/kg 0/3 0/3 1510 mg/kg 3/3 2/3 2010 mg/kg 0/3 0/3 2540 mg/kg 0/3 0/3 3010 mg/kg 2/3 0/3 10.4 Conclusion Based on these results, the maximum tolerated dose could not be determined. 11.0 DOSE SELECTION STUDY II 11.1 Dose Selection Dose levels of 1010, 1520,2020 and 2530 mg/kg were administered by oral gavage for the second dose selection study. 11.2 Dosing Information The animals used in the second dose selection assay were dosed on July 24, 1996. The weight range of the animals used in the second dose range finding assay was 30.1-35.8 and 23.6-28.4 grams, for the males and females, respectively. Dosing solutions were prepared just prior to dosing and were prepared by making a 253 mg/ml stock for the high dose (2530 mg/kg ). This was prepared by adding deionized water (CHV, Lot # 20) up to a volume of 14.0 ml to 2.9 ml of T-6564, resulting in a clear colorless solution. Dilutions of this stock were prepared for the 2020, 1520 and 1010 mg/kg dose levels. Dosing was achieved using a 10 ml/kg dosing volume. All animals were eight weeks and two days old at the time of dosing. An outline of the dosing scheme is found in the following table: CHV Study No.: 17750-0-455 11 000186 C O R N IN G Hazleton Dosing Scheme for Dose Selection Assay II Treatment__ T-6564 1010 mg/kg 1520 mg/kg 2020 mg/kg 2530 mg/kg Male Female 33 33 33 33 A total of 24 animals was used in this assay. All doses given were on an acute (one-time only) basis. 11.3 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. The animal observations for this assay are summarized in the following table: Animal Observations for Toxicity for Dose Rangefinding Assay II Time After Dose Dosing Level (hours) (mg/kg) Observations =0.1, 1.1 & 15.6 All Normal =17.9 1520 Female #1417 found dead. =21.6 1520 Male # 1405 found dead. =39.5 All Normal =66.6 AH Normal =90.4 All Normal The mortality data for this assay are summarized in the following table: CHV Study No.: 17750-0-455 12 0001S7 C O R N IN G Hazleton Summary of Mortalities for Dose Rangefinding II Treatment_______________Male_______ Female T-6564 1010 mg/kg 0/3 0/3 1520 mg/kg 1/3 1/3 2020 mg/kg 0/3 0/3 2530 mg/kg 0/3 0/3 11.4 Conclusion Based on these results, the maximum tolerated dose was estimated as about 2000 mg/kg. 12.0 MICRONUCLEUS STUDY 12.1 Dose Selection Based on results from the dose selection study, dose levels of 498, 995, and 1990 mg/kg were tested in this study. 12.2 Micronucleus Assay Dosing Information The animals used in the micronucleus assay were dosed on July 30,1996. Cyclophosphamide (CP), (CAS # 6055-19-2; Sigma, Lot # 26H0473), the positive control, was solubilized in sterile deionized water (Lot # 19, prepared at CHV) and was administered by oral gavage at 80.0 mg/kg. The vehicle control, deionized water (CHV, Lot # 20), 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 31.0-37.7 and 22.4-28.3 grams for the males and females, respectively. The dosing solutions for the assay were prepared by making a 199 mg/ml stock for the high dose (1990 mg/kg ). This was prepared by adding deionized water (CHV, Lot # 20) up to a volume of 30.0 ml to 4.9 ml of T-6564, resulting in a clear colorless solution. Dilutions of this stock were prepared for the 995 and 498 mg/kg dose levels. A second group of animals (designated Secondary Dose Group) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group. CHV Study No.: 17750-0-455 13 ( G001S8 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, euthanized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanized 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 T-6564 Treatment 498 mg/kg Number of Animals Assigned Primary Dose Groups Secondary Dose 24 Hr 48 Hr 72 Hr Groupa MF MF MF MF 55 55 55 -- 995 mg/kg 55 55 55 -- 1990 mg/kg 55 55 55 10 10 Vehicle Control, deionized water, 10 ml/kg 55 - - - - -- Positive Control, Cyclophosphamide, 80.0 mg/kg 5 5 - - - - -- a The animals assigned to the secondary dose groups were dosed and were only used to replace animals which died in the primary dose group at the high dose level. All extra animals not used as replacements were euthanized at the completion of the trial. A total of 130 animals was used in this assay. The age of the animals at the time of dosing was eight weeks and one day. Volumes dosed were 10 ml/kg and were based upon individual animal weights. 13.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At the appropriate harvest time, the animals were euthanized by C 02/0 2 inhalation, followed by C 02 inhalation and penetration of the thorax. 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. CHV Study No.: 17750-0-455 14 0001S9 C O R N IN G Hazleton The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were 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. 14.0 EVALUATION CRITERIA 14.1 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). 14.2 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 judgment. CHV Study No.: 17750-0-455 15 C00190 CO RN ING Hazleton 15.0 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 test article dosed groups appeared normal immediately after dosing. Approximately 22 hours after dosing, one male (#1605) from the secondary dose group was found dead. All other animals appeared normal at this time and remained healthy until the appropriate harvest times. The test article, T-6564, 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. The positive control, Cyclophosphamide, induced significant increases in micronucleated PCEs in both sexes as compared to the vehicle controls, with means and standard errors of 3.86% 0.55% and 3.58% 0.79% 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. 16.0 CONCLUSION The test material, T-6564, 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.: 17750-0-455 16 I 000191 C O R N IN G Hazleton 17.0 REFERENCES Dunnett, C.W.: A multiple comparisons procedure for comparing several treatments with acontrol. J. Am. Statist. A s s o c . , 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., 1:9-15, 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. 18.0 DEVIATIONS FROM THE SIGNED PROTOCOL 1. In both trials of the dose rangefinding studies, animals were observed for four days and not three days after dosing. This had no impact on study integrity. 2. Due to a technical oversight, more than 1000 erythrocytes were analyzed for animal #'s 1576 and 1628. This had no impact on the integrity of the study. 3. On July 31,1996, for the definitive study, the humidity was recorded as 75.1%. This had no impact on the integrity of the study. CHV Study No.: 17750-0-455 17 000192 C O R N IN G Hazleton 19.0 EXPERIMENT DATA TABLES CHV Study No.: 17750-0-455 000193 C O R N IN G Hazleton TABLE 1 SPONSOR: 3M Corporation MICRONUCLEUS DATA SUMMARY TABLE TEST ARTICLE: T-6564 ASSAY: 17750 TREATMENT DOSE HARVEST % MICRONUCLEATED PCEs TIME MEAN OF 1000 PER ANIMAL S.E. (HR) MALES FEMALES TOTAL CONTROLS VEHICLE POSITIVE Water CP 80.0 mg/kg 24 hr 24 hr 0.08 0.06 3.86 * 0.55* 0.08 0.04 3.58 0.79* 0.08 0.03 3.72 0.46* RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.53 0.08 0.61 0.05 0.73 0.07 0.96 0.17 TEST ARTICLE 498 mg/kg 24 hr 48 hr 72 hr 995 mg/kg 24 hr 48 hr 72 hr 1990 mg/kg 24 hr 48 hr 72 hr 0.20 0.06 0.02 0.02 0.08 0.06 0.12 0.06 0.12 0.04 0.10 * 0.08 0.30 0.13 0.00 0.00 0.08 0.06 0.06 0.06 0.08 0.04 0.12 0.07 0.18 0.16 0.06 0.04 0.10 0.06 0.08 0.06 0.04 0.02 0.12 0.04 0.13 0.05 0.05 0.02 0.10 0.04 0.15 0.08 0.09 0.03 0.10 0.05 0.19 0.08 0.02 0.01 0.10 0.03 0.71 0.08 0.59 0.05 0.52 0.08 0.69 0.12 0.63 0.03 0.45 0.08 0.69 0.07 0.60 0.06 0.49 0.11 0.92 0.05 0.53 0.04 1.12 0.43 1.03 0.13 0.54 0.06 0.85 0.21 0.75 0.07 0.56 0.07 0.78 0.21 * Significantly greater than the corresponding vehicle control, p<0.05. CP = Cyclophosphamide PCE = Polychromatic erythrocyte NCE = Normochromatic erythrocyte CHV Study No.: 17750-0-455 19 { 000194 CORNING Hazleton TABLE 2 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-6564 ASSAY NO.: 17750 TREATMENT 24 HOUR HARVEST MALE a MN ANIMAL p l r RATIO NUMBER 1000 PCEs PCE:NCE VEHICLE CONTROL Water 1557 1 0.52 1574 0 0.79' 1576 0 0.62 15*5 3 0.39 1614 0 0.35 1 POSITIVE CONTROL CP *0.0 mg/kg 1564 46 0.62 'i 1566 32 0.49 1569 25 0.77 1607 56 0.64 1612 34 0.53 TEST ARTICLE 498 mg/kg 1562 3 0.73 1570 1 0.73 1581 3 0.97 15*6 3 0.61 1587 0 0.4* 995 mg/kg 156* 1 0.69 ; 1571 0 0.64 1 1591 3 1.04 J 159* 2 0.80 i 1602 0 0.29 1990 mg/kg 155* 2 1567 0 1580 8 1594 3 1611 2 0.72 0.61 0.96 0.55 0.63 CP = Cyclophosphamide PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte * 1059 erythrocytes scored CHV Study No.: 17750-0-455 1 20 00195 C O R N IN G Hazleton TABLE 3 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-6564 ASSAY NO.: 17750 TREATMENT 24 HOUR HARVEST FEMALE a MN ANIMAL RATIO NUMBER 1000 PCEs PCE:NCE VEHICLE CONTROL POSITIVE CONTROL TEST ARTICLE Water CP 80.0 mg/kg 498 mg/kg 995 mg/kg 1990 mg/kg 1669 1 1671 2 1678 0 1679 l 1683 0 1641 51 1653 33 1663 56 1664 25 1667 14 1627 0 1630 0 1635 0 1644 3 1652 0 1633 0 1637 0 1660 0 1666 8 1672 1 1643 0 1650 0 1668 l 1681 3 1682 0 0.65 0.81 0.82 0.88 0.50 0.72 1.06 0.57 0.89 1.56 0.97 1.00 0.74 0.90 1.01 0.78 0.77 0.96 1.26 1.39 0.89 0.67 0.88 0.53 0.78 CP = Cyclophosphamide PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17750-0-455 21 000196 C O R N IN G Hazleton TABLE 4 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-6564 ASSAY NO.: 17750 TREATMENT 48 HOUR HARVEST MALE ANIMAL NUMBER # MN PCEs/ 1000 PCEs RATIO PCE:NCE TEST ARTICLE 498 mg/kg 995 mg/kg 1990 mg/kg 1563 0 0.60 1582 0 0.75 1593 1 0.43 1603 0 0.54 1618 0 0.62 1572 1 0.67 1589 1 0.66 1596 2 0.52 1604 2 0.66 1615 0 0.62 1577 0 1579 0 1583 0 1595 0 1606 0 0.75 0.68 0.60 0.41 0.53 PCE = Polychromatic erythrocyte # MN PCEs = Micronucieated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17750-0-455 22 000197 C O R N IN G Hazleton TABLE 5 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-6564 ASSAY NO.: 17750 TREATMENT 48 HOUR HARVEST FEMALE ANIMAL NUMBER #MN PCEs' 1000 PCEs RATIO PCE:NCE TEST ARTICLE 498 mg/kg 1628 2 0.58* 1636 I 0.66 1638 0 0.39 1654 1 0.48 1662 0 0.54 995 mg/kg 1621 1 0.36 1649 0 0.44 1651 0 0.57 1657 0 0.71 1665 2 0.59 1990 mg/kg 1626 0 0.40 1632 1 0.64 1634 0 0.48 1639 1 0.77 1677 0 0.51 PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte * 1005 erythrocytes scored CHV Study No.: 17750-0-455 ! 23 0001S8 C O R N IN G Hazleton TABLE 6 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-6564 ASSAY NO.: 17750 TREATMENT 72 HOUR HARVEST MALE M MN ANIMAL RATIO 1000 PCEs PCE:NCE TEST ARTICLE 498 mg/kg 995 mg/kg 1990 mg/kg 1561 0 0.65 1565 0 0.74 1599 0 0.44 1600 3 0.34 1609 1 0.42 1555 1 0.48 1556 0 0.17 1559 0 0.8 1590 4 0.45 1597 0 0.57 1554 3 0.21 1560 1 0.78 1573 0 0.63 1613 0 0.24 1617 0 0.58 PCE * Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE Normochromatic erythrocyte CHV Study No.: 17750-0-455 24 000199 C O R N IN G Hazleton TABLE 7 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-6564 ASSAY NO.: 17750 TREATMENT 72 HOUR HARVEST FEMALE # MN ANIMAL RATIO NUMBER 1000 PCEs PCE:NCE TEST ARTICLE 498 mg/kg 995 mg/kg 1990 mg/kg 1619 0 0.60 1620 1 0.73 1624 0 0.45 1656 4 2.82 1680 1 0.99 1645 2 1648 3 1658 0 1674 0 1675 0 1.60 0.79 0.66 0.33 0.88 1622 I 0.67 1625 2 0.73 1629 0 0.34 1640 2 1.58 1642 I 0.56 PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17750-0-455 25 000200 CO RN ING Hazleton TABLE 8 MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95 POOLED VEHICLE CONTROLS MIN MAX AVG N % MICRONUCLEATED PCEs PER 1000 PCE MEAN OF 1000 PER ANIMAL S.E. MALES FEMALES TOTAL 0.00 0.22 0.087 0.007 47 0.00 0.24 0.081 0.008 47 0.01 0.17 0.084 0.005 47 RATIO PCE:NCE MEAN S.E. MALES FEMALES 0.31 0.85 0.550 0.021 47 0.24 1.03 0.587 0.025 47 POSITIVE CONTROLS Cyclophosphamide, 80.0 mg/kg MIN MAX AVG N 2.00 5.68 3.682 0.240 19 PCE = Polychromatic erythrocyte NCE = Normochromatic erythrocyte N = Number of harvests 1.50 6.36 3.170 0.245 19 2.41 538 3.426 0.184 19 0.41 0.72 0.577 0.020 19 0.40 0.79 0.588 0.026 19 CHV Study No.: 17750-0-455 26 0002