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A R 3 0 6 - 0 130 -3 C O R N IN G Hazleton MUTAGENICITY TEST ON T-6295 IN AN IN VIVO MOUSE MICRONUCLEUS A S S A Y 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.: 17403-0-455 SUBMITTED TO 3M 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE May 23,1996 CHV Study No.: 17403-0-455 1 of 26 C013Q7 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT Project Title: In Vivo Mouse Micronucleus Assay Project No.: 20996 Assay No.: 17403 Protocol No.: 455 Edition No.: 17 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 Harvest/03/21/1996 03/21/1996 C. Smith Draft Report Review/05/15,16/1996 05/17/1996 C.Orantes Final Report Review/05/23/1996 05/23/1996 C.Orantes CHV Study No.: 17403-0-455 2 001508 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 axe 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.: 17403-0-455 3 C01S09 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 RANGEFINDING STUDY I ................................................................................... 9 10.1 Dose Selection 10.2 Dosing Information 10.3 Results and Interpretation 10.4 Conclusion CHV Study No.: 17403-0-455 4 001310 C O R N IN G Hazleton 11.0 DOSE RANGEFINDING 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............................................................................ 15 16.0 CONCLUSION.................................................................................................................17 17.0 REFERENCES .................................................................................................................17 18.0 DEVIATIONS FROM THE SIGNED PROTOCOL ...................................................... 17 19.0 EXPERIMENT DATA TABLES .................................................................................... 18 CHV Study No.: 17403-0-455 5 001311 C O R N IN G Hazleton SUMMARY Mutagenicity Test on T-6295 in an In Vivo Mouse Micronucleus Assay The objective of this in vivo assay was to evaluate the ability of the test article, T-6295, to induce micronuclei in bone marrow polychromatic erythrocytes of Crl:CD-l(ICR) BR mice. In the dose selection study, the test article was suspended in deionized water and dosed by oral gavage at 500, 1630,2750, 3880 and 5000 mg/kg, and the dose selection study was repeated testing dose levels of 700,1000, and 1300 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 rangefinding study, the maximum tolerated dose was estimated as 950 mg/kg. In the micronucleus assay, the test article was suspended in deionized water and dosed by oral gavage at 237.5,475, and 950 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-6295, 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.: 17403-0-455 6 O01312 C O R N IN G Hazleton Mutagenicity Test on T-6295 in an In Vivo Mouse Micronucleus Assay 1.0 SPONSOR: 3M 2.0 MATERIAL (Test Article) 2.1 Client's Identification: T-6295 2.2 Date Received: January 22, 1996 2.3 Physical Description: Off-white mixture of powder and flakes 2.4 Genetics Assay No.: 17403 3.0 TYPE OF ASSAY: In Vivo Mouse Micronucleus Assay 4.0 PROTOCOL NO.: 455, Edition 17 5.0 STUDY DATES 5.1 Initiation Date: January 22, 1996 5.2 Experimental Start Date: March 13,1996 5.3 Experimental Termination Date: April 18,1996 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-6295, 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 Heddleetal. (1983). CHV Study No.: 17403-0-455 7 001313 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 seven 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 March 10 and 16,1996, for the second dose rangefinding study, when the relative humidity was recorded as 36.1% and 39.0%, respectively. A 12-hour light/12-hour dark cycle was maintained. A commercial diet (Purina Certified Laboratory Pellets # 5002) and water were available M 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 inhalation, followed by penetration of the thorax. Any extra animals not used for the study were euthanized by C 02inhalation, followed by penetration of the thorax. 9.0 SOLUBILITY AND STABILITY The test article, T-6295, was supplied as a off-white mixture of powder and flakes. The solubility of the test article was evaluated in com oil, 0.5% high viscosity carboxymethyl cellulose, and acetone: com oil, 20%:80%, v:v. Suspensions suitable for dosing was not obtained with these vehicles. The test article was crushed in a mortar and pestle and a translucent, off-white suspension was obtained in deionized water, that passed easily through an 18G needle after mixing in a Tissumizer. Deionized water was the vehicle of choice for this assay. The stability of the test material under the dosing conditions of this assay is the responsibility of the sponsor. CHV Study No.: 17403-0-455 8 001S14 C O R N IN G Hazleton 10.0 DOSE SELECTION STUDY I 10.1 Dose Selection Dose levels of 500,1630, 2750, 3880 and 5000 mg/kg were administered by oral gavage for the first dose rangefinding study. 10.2 Dosing Information The animals used in the first dose rangefinding assay were dosed on March 13, 1996. The weight range of the animals used in the first dose range finding assay was 30.8 - 40.3 and 24.4 - 30.8 grams, for the males and females, respectively. Dosing solutions were prepared just prior to dosing and were prepared by making a 250 mg/ml stock for the high dose (5000 m g/kg). This was prepared by adding deionized water (Lot # 19, prepared at CHV) to 3.5025 g of T-6295 up to a volume of 14.0 ml, resulting in a cloudy cream colored suspension. This was mixed for ~2 minutes with a Tissumizer. Dilutions of this stock were prepared for the 3880, 2750,1630 and 500 mg/kg dose levels. All dosing stocks were placed on magnetic stir plates during the dilution and the dosing procedure. Dosing was achieved using a 20.0 ml/kg dosing volume. All animals were nine weeks and two days old at the time of dosing. An outline of the dosing scheme is found in the following table. Dosing Scheme for Dose Rangefinding Assay I ___ T reatment____ T-6295 500 mg/kg 1630 mg/kg 2750 mg/kg 3880 mg/kg ______ 5000 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 (three days) for toxic effects and/or mortalities. All animals appeared CHV Study No.: 17403-0-455 9 C0135 C O R N IN G Hazleton normal immediately after dosing. The toxicity observations are shown in the following table: Animal Observations for Toxicity for Dose Rangefinding Assay I Time After Dose Dosing Level (hours) (mg/kg) Observations 500 Normal 1630 Slightly hypoactive 1 2750 Hypoactive with dyspnea 3880 Hypoactive with dyspnea 5000 Hypoactive with dyspnea, lacrimation and tremors 500 Normal 1630 Hypoactive 18 2750 All animals found dead, except female # 6978, which was hypoactive, cold to the touch, pale and had tremors 3880 All animals found dead 5000 All animals found dead 500 Normal 42 1630 Males # 6963 and 6967, and female # 6984 found dead, all other animals appeared normal 2750 Female # 6978 found dead 500 Normal 72 1630 All remaining animals appeared normal The mortality data for this assay are summarized in the following table: Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T-6295 for Dose Rangefinding Assay I Treatment______________ Male_______ Female 500 mg/kg 0/3 0/3 1630 mg/kg 2/3 1/3 2750 mg/kg 3/3 3/3 3880 mg/kg 3/3 3/3 5000 mg/kg 3/3 3/3 CHV Study No.: 17403-0-455 10 001816 C O R N IN G Hazleton 10.4 Conclusion Based on these results, the maximum tolerated dose could not be determined. 11.0 DOSE RANGEFINDING STUDY II 11.1 Dose Selection Dose levels of 700,1000 and 1300 mg/kg were administered by oral gavage for the second dose rangefinding study. 11.2 Dosing Information The animals used in the second dose rangefinding assay were dosed on March 16, 1996. The weight range of the animals used in the second dose range finding assay was 32.7 - 39.8 and 25.0 - 31.3 grams, for the males and females, respec tively. Dosing solutions were prepared just prior to dosing and were prepared by making a 65.0 mg/ml stock for the high dose (1300 mg/kg). This was prepared by adding deionized water (Lot #19, prepared at CHV) to 780.0 mg of T-6295 up to a volume of 12.0 ml, resulting in a cloudy cream colored suspension. Dilutions of this stock were prepared for the 1000, and 700 mg/kg dose levels. All dosing stocks were placed on magnetic stir plates during the dilution and the dosing procedure. Dosing was achieved using a 20.0 ml/kg dosing volume. All animals were nine weeks and five days old at the time of dosing. An outline of the dosing scheme is found in the following table. Dosing Scheme for Dose Rangefinding Assay II Treatment T-6295 700 mg/kg 1000 mg/kg 1300 mg/kg Male Female 33 33 33 A total of 18 animals was used in this assay. All doses given were on an acute (one-time only) basis. CHV Study No.: 17403-0-455 11 00181.7 C O R N IN G Hazleton 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. All animals appeared normal immediately after dosing. The toxicity observations are shown in the following table: Animal Observations for Toxicity for Dose Rangefinding Assay II Time After Dose Dosing Level (hours) (mg/kg) Observations 700 Normal =1 1000 Slightly hypoactive 1300 Hypoactive and hunched 700 Normal =24 1000 Normal 1300 Male #7259 found dead, all remaining appeared hypoactive and hunched 700 Normal =43 1000 Female # 7255 found dead, all others appeared normal 1300 All remaining males and females #'s 7263 and 7261 found dead, one surviving female appeared normal 700 Normal =70 1000 All remaining appeared normal 1300 All remaining appeared normal The mortality data for this assay are summarized in the following table: Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T-6295 for Dose Rangefinding Assay II Treatment_______________Male_______ Female 700 mg/kg 0/3 0/3 1000 mg/kg 0/3 1/3 1300 mg/kg 3/3 2/3 CHV Study No.: 17403-0-455 12 001818 C O R N IN G Hazleton 11.4 Conclusion Based on these results, the maximum tolerated dose was determined to be 950 mg/kg. 12.0 MICRONUCLEUS STUDY 12.1 Dose Selection Based on results from the dose rangefmding study, dose levels of 237.5,475, and 950 mg/kg were selected for testing in this study. 12.2 Micronucleus Assay Dosing Information The animals used in the micronucleus assay were dosed on March 19, 1996. Cyclophosphamide (CAS # 6055-19-2; Sigma, Lot # 44H0486), 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 (Lot # 19, prepared at CHY), was administered concurrently with the test article at a volume of 20.0 ml/kg. The weight range of the animals used in the micronucleus assay was 29.9-37.0 and 23.1-29.2 grams for the males and females, respectively. The dosing solutions for the assay were prepared by making a 47.5 mg/ml stock for the high dose (950 mg/kg). This was prepared by adding the vehicle to 2.3751 g of ground up T-6295 up to a volume of 50.0 ml, stirring with a spatula and a cloudy cream colored suspension was obtained. Dilutions of this stock were prepared for the remaining dose levels. All dosing stocks were placed on magnetic stir plates during the dilution and the dosing procedure. 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. 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: CHV Study No.: 17403-0-455 13 001319 C O R N IN G Hazleton Dosing Scheme for Micronucleus Assay T-6295 Treatment 237.5 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 - 475.0 mg/kg 55 55 55 - 950.0 mg/kg 55 55 55 55 Vehicle Control, deionized water, 20.0 ml/kg 5 5 - - - - - 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 120 animals was used in this assay. The age of the animals at the time of dosing was nine weeks and one day. Volumes dosed were 20.0 ml/kg (except positive control) and were based upon individual animal weights. Volumes dosed to positive control were 10.0 ml/kg. 13.0 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At the appropriate harvest time, the animals were euthanized by C 02inhalation followed by 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 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 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%. CHV Study No.: 17403-0-455 14 C01320 C O R N IN G Hazleton 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. 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 and CHV Study No.: 17403-0-455 15 C O R N IN G Hazleton about one hour after dosing. The toxicity and mortality observations of the test article dosed animals are shown in the following table: Animal Observations for Toxicity/Mortality Time After Dose Dosing Level (hours) (mg/kg) Observations 237.5 Normal 475.0 Normal 950.0 Males # 7304 (24 hour harvest), 7314 (48 hour harvest), and 7305 =22 (secondary); and females # 7369 (48 hour harvest), all 72 hour harvest, and 7332 (secondary) were found dead. Two males went into convulsions upon opening of their cages but recovered after a few minutes. All others remaining appeared normal. 237.5 Normal 475.0 Female # 7343 (48 hour harvest) found dead, all remaining =46 appeared normal 950.0 Males # 7300 (48 hour harvest) and 7286 (secondary), and females # 7368 (48 hour harvest) and 7379 (secondary) were found dead. All remaining appeared normal. 237.5 Normal =71 475.0 All remaining appeared normal 950.0 Male # 7282 (secondary) found dead, all remaining appeared normal The test article, T-6295, 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. Bone marrow toxicity was manifested by the significant reduction in the PCE/NCE ratios of the 48 and 72 hour males from the 237.5 and 950 mg/kg dose groups, 72 hour males from the 475 mg/kg dose group, 48 hour females from the 475 mg/kg dose group, 72 hour females from the 950 mg/kg dose group, and the positive control females. 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 3.36% 0.97% and 4.52% 0.72% 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. CHV Study No.: 17403-0-455 16 C O R N IN G Hazleton 16.0 CONCLUSION The test material, T-6295, 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. 17.0 REFERENCES Dunnett, C.W.: A multiple comparisons procedure for comparing several treatments with a control. J. Am. Statist. Assoc., 5Q: 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. 18.0 DEVIATIONS FROM THE SIGNED PROTOCOL 1. On March 10 and 16,1996, for the second dose rangefinding study, the relative humidity was recorded as 36.1% and 39.0%, respectively. This did not affect the animals or the integrity of the study. 2. The actual dose levels achieved for the dose rangefinding study were 3880 and 1630 mg/kg and not 3875 and 1625 mg/kg, respectively. These dose levels are very close to the dose levels (<0.05% difference) mentioned in the protocol amendment and hence there was no impact on the integrity of the study. CHV Study No.: 17403-0-455 17 C01323 C O R N IN G Hazleton 19.0 EXPERIMENT DATA TABLES CHV Study No. : 17403-0-455 18 001324 C O R N IN G Hazleton SPONSOR: 3M TABLE 1 MICRONUCLEUS DATA SUMMARY TABLE TEST ARTICLE: T-6295 ASSAY: 17403 TREATMENT DOSE HARVEST % MICRONUCLEATED PCEs T IM E MEAN OF 1000 PER ANIM AL S.E. (HR) MALES FEMALES TOTAL CONTROLS VEHICLE Water 24 hr 0 .0 2 0 .0 2 0.14 0.06 0.08 0.04 POSITIVE CP 80.0 mg/kg 24 hr 3.36 0.97* 4.52 0.72* 3.94 0.60* RATIO PCErNCE MEAN S.E. MALES FEMALES 0.79 0.08 0.58 0.05 0.77 0.08 0.55 0.03** TEST ARTICLE 237.5 mg/kg 24 hr 48 hr 72 hr 475.0 mg/kg 24 hr 48 hr 72 hr 950.0 mg/kg 24 hr 48 hr 72 hr 0.06 0.04 0.16 0.07 0.04 0.02 0.14 0.05 0.04 0.02 0.14 0.05 0 .0 2 0 .0 2 0.07 0.05' 0.08 0.04 0.10 0.03 0 .0 2 0 .0 2 0.12 0.05 0.04 0.04 0.05 0.03' 0.08 0.04 0 .0 2 0 .0 2 0.03 0.032 0 .0 2 0 .0 2 0.08 0 . 0 2 0.09 0.04 0.08 0.03 0.09 0.03 0.04 0.02 0.11 0.03 0 .0 2 0 .0 1 0.06 0.03 0.05 0.02 0.57 0.11 0.52 0.10 0.48 0.04** 0.80 0 . 1 0 0.39 0.11** 0.42 0.14 0.76 0.11 0.59 0.08 0.71 0.05 0.37 0.07'** 0.29 0.06** 0.40 0.12 0.56 0.13 0.59 0.08 0.54 0.08'** 0.44 0.112 0.17 0.05** 0.17 0.05** * Significantly greater than the corresponding vehicle control, p<0.05. ** Significantly less than the corresponding vehicle control, p<0.05 1Out of four animals 2Out of three animals CP = Cyclophosphamide CHV Study No.: 17403-0-455 19 001325 C O R N IN G Hazleton TABLE 2 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 24 HOUR HARVEST MALE # MN A N IM A L RATIO PCEs/ NUMBER PCE-.NCE 1000 PCEs VEHICLE CONTROL POSITIVE CONTROL TEST ARTICLE Water CP 80.0 mg/kg 237.5 mg/kg 475.0 mg/kg 950.0 mg/kg 7283 7285 7292 7297 7303 7266 7274 7277 7294 7313 7265 7281 7291 7296 7321 7272 7284 7293 7307 7320 7267* 7273 7287 7295 7318 0 1 0 0 0 34 3 38 29 64 0 2 0 1 0 1 2 0 3 I 0 0 0 1 0 0 .6 6 0.64 0 .6 8 1 .0 0 0.99 0.58 0.64 0.42 0.53 0.72 0.71 0.58 0.91 0.34 0.30 1.16 0.56 0.84 0.73 0.53 0.53 0 .6 8 1 .0 2 0.33 0.26 * Secondary dose group animal CP = Cyclophosphamide MN = Micronucleus PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic eiythrocyte CHV Study No.: 17403-0-455 20 C01S26 C O R N IN G Hazleton TABLE 3 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT # MN ANIMAL RATIO NUMBER 10^ Es PCE:NCE 24 HOUR HARVEST FEMALE VEHICLE CONTROL POSITIVE CONTROL TEST ARTICLE Water CP 80.0 mg/kg 237.5 mg/kg 475.0 mg/kg 950.0 mg/kg 7324 7327 7336 7350 7360 7328 7330 7334 7359 7366 7349 7355 7363 7370 7376 7337 7347 7362 7364 7377 7325 7335 7340 7361 7365 0 2 2 3 0 61 26 63 36 40 2 1 0 I 1 0 0 2 0 0 1 0 0 0 0 0.56 0.73 0 .8 6 0 .6 6 1.04 0.54 0.53 0 .6 6 0.52 0.49 0.67 0.17 0.71 0.43 0.63 0.89 0.59 0.58 0.43 0.46 0.63 0 .8 6 0.49 0.41 0.55 CP = Cyclophosphamide MN = Micronucleus PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17403-0-455 21 001327 C O R N IN G Hazleton TABLE 4 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 48 HOUR HARVEST MALE UUN ANIMAL I TM RATIO NUMBER 1000 PCEs PCE:NCE TEST ARTICLE 237.5 mg/kg 475.0 mg/kg 950.0 mg/kg 7279 7288 7308 7312 7322 7270 7306 7317 7319 7323 7269 7271 7280** 7301 7314* 1 4 2 1 0 0 1 0 1 0 0 0 2 1 0.35 0.45 0.60 0.47 0.55 0.65 0.66 0.69 0.62 0.92 0.57 0.32 0.71 0.56 * Animal found dead ** Secondary dose group animal MN = Micronucleus PCE = Polychromatic erythrocyte # M N PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17403-0-455 22 CQ1S28 C O R N IN G Hazleton TABLE 5 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT 48 HOUR HARVEST FEMALE U MISI ANIMAL I L T , RATIO NUMBER IQOOPCEs PCE:NCE TEST ARTICLE 237.5 mg/kg 475.0 mg/kg 950.0 mg/kg 7326 7352 7353 7372 7383 7329 7341 7343* 7356 7374 7338 7358 7368* 7369* 7373 1 0 0 0 0 0 1 1 0 1 0 0 0.79 0.96 0.77 0.43 1.04 0.58 0.31 0.29 0.30 0.41 0.64 0.28 * Animal found dead MN = Micronucleus PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17403-0-455 23 C01329 C O R N IN G Hazleton TABLE 6 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAYNO.: 17403 TREATMENT 72 HOUR HARVEST MALE U MN ANIMAL I TM . RATIO NUMBER 1000 PCEs PCE:NCE TEST ARTICLE 237.5 mg/kg 475.0 mg/kg 950.0 mg/kg 7264 7289 7298 7310 7315 7275 7278 7290 7302 7316 7268 7276 7299 7309 7311 1 1 0 0 0 1 2 1 3 0 0 2 0 1 1 0.32 0.33 0.72 0.52 0.07 0.25 0.18 0.15 0.40 0.47 0.26 0 .1 1 0.31 0.08 0 .1 0 MN = Micronucleus PCE = Polychromatic erythrocyte # MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No. : 17403-0-455 24 001330 C O R N IN G Hazleton TABLE 7 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT t MN ANIMAL p 'p , RATIO NUMEER 10^ Es PCE:NCE 72 HOUR HARVEST FEMALE TEST ARTICLE 237.5 mg/kg 475.0 mg/kg 950.0 mg/kg 7331 7348 7351 7378 7380 7339 7346 7357 7371 7375 7333. 7345 7367 7381* 7382 1 3 1 1 0 0 1 2 0 1 0 0 1 0 0 0.28 0.07 0.92 0.40 0.46 0.87 0.30 0.38 0.25 0.19 0 .1 0 0 .1 1 0.34 0 .1 1 0.18 * Secondary dose group animal MN = Micronucleus PCE = Polychromatic erythrocyte MMN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte CHV Study No.: 17403-0-455 25 C01S31 C O R N IN G Hazleton TABLE 8 M OUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95 POOLED VEHICLE CONTROLS MIN MAX AVG N % MICRONUCLEATED PCEs PER 1000 PCE M EAN OF 1000 PER ANIM AL S.E. MALES FEMALES TOTAL 0 .0 0 0 .2 2 0.087 0.007 47 0 .0 0 0.24 0.081 0.008 47 0 .0 1 0.17 0.084 0.005 47 RATIO PCE:NCE M EAN 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 .0 0 5.68 3.682 0.240 19 1.50 6.36 3.170 0.245 19 2.41 5.38 3.426 0.184 19 0.41 0.72 0.577 0.020 19 0.40 0.79 0.588 0.026 19 PCE = Polychromatic erythrocyte NCE = Normochromatic erythrocyte CHV Study No.: 17403-0-455 26 C01332 CHV STUDY NO. ______________ PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton IN VIVO MOUSE MICRONUCLEUS ASSAY Corning Hazleton Inc. (CHV) will conduct this study in compliance with Good Laboratory Practice (GLP) Regulations. This protocol, critical phase(s) of the work in progress and the final report will be subject to audit by Quality Assurance in accordance with SOPs at Corining Hazleton Inc. The study will be conducted by CHV at 9200 Leesburg Pike, Vienna, Virginia 22182. PART 1. SPONSOR INFORMATION AND APPROVALS I. SPONSOR IDENTIFICATION Company Name: Address: ___ 3 / K ______________ a-, ,mH II. TEST ARTICLE IDENTIFICATION: T ' - H I . TEST ARTICLE ANALYSIS Determination of the test article stability and the test article characteristics as defined in the GLP regulations is the responsibility of the Sponsor. IV. NOTIFICATION OF REGULATORY SUBMISSION In order to comply with the GLP regulations, consulting laboratories must be notified if all or part of a study is intended for regulatory submission. CHV maintains a master schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the results of this study: if===^rf 11--- 'I ii ii D-- !l ii ..n Undetermined 1'-- ^ MAFF if- ii It-- a MOHW Ii ""'il FDA II-- 0 EPA-TSCA Ii------- ii Ir---~il = = y OECD IL==a OTHER ii ii It-- a EPA-FIFRA ____________________ 4/95 1 of 10 001833 PROTOCOL NO. 455, EDITION 17 V. STUDY DATES Proposed Experimental Start Date: Proposed Experimental Termination Date: VI. APPROVAL OF STUDY PROTOCOL Study Director: Hemalatha Murli, Ph.D. Sponsor's Authorized Representative: C O R N IN G Hazleton Date: Date : y / z z ' / f ' T ' 4/95 2 of 10 001834 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton PART 2 - STUDY PROTOCOL IN VIVO MOUSE MICRONUCLEUS ASSAY I. OBJECTIVE The objective of this study is to evaluate a test article for clastogenic activity and disruption of the mitotic apparatus in polychromatic erythrocyte stem cells in mouse bone marrow in vivo. II. DEFINITIONS Micronucleus: a small chromatin body, consisting of entire ' chromosome(s) and/or of acentric chromosome fragment(s), which lags behind at mitotic anaphase. After telophase, these chromosome(s) and fragmentes) may not be included in the daughter nuclei, and may form single or multiple micronuclei in the cytoplasm. III. RATIONALE The micronucleus test can serve as a rapid screen for clastogenic agents and test articles which interfere with normal mitotic cell division (Schmid, 1975; Heddle et. al., 1983). Micronuclei are formed from chromosomes or chromosome fragments left behind during anaphase and can be scored during interphase because they persist (Schmid, 1975). In this assay, polychromatic erythrocytes (PCEs) in the bone marrow are scored for the presence of micronuclei. During maturation from erythroblast to erythrocyte the nucleus is extruded, while micronuclei, if present, remain in the cytoplasm. Detection of micronuclei in non-nucleated cells is thus facilitated, and time involved in searching for metaphase spreads in treated cell populations is eliminated. Test articles affecting spindle-fiber function or formation as well as clastogenic agents can be detected through micronucleus induction (Schmid, 1975). IV. MATERIALS A. Animals Young adult male and female mice of the ICR strain, 810 weeks old at the time of dosing, will be purchased from Charles River Laboratories, Inc., or Harlan Sprague-Dawley, Inc. This strain has been selected to i/95 3 of 10 C01S35 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton maximize genetic heterogeneity and at the same time ensure access to a common source. B. Control Articles Cyclophosphamide (CP, 80 mg/kg; dosing volume of 10 ml/kg) will be used as the positive control article and will be administered by oral gavage. The vehicle control article will consist of the solvent or vehicle used for the test article and will be administered by the same route as, and concurrently with, the test article and in amounts equal to the maximum volumes administered to the experimental animals. The dosing volume will not exceed 20 ml/kg for oral gavage and IP administrations. The vehicles generally used in the assay are water, 0.5Z aqueous carboxymethylcellulose solution, or corn oil. V. EXPERIMENTAL DESIGN A. Animal Husbandry All applicable CHV SOPs will be followed. Animals will be isolated by sex. Animals will be housed up to seven per cage during quarantine, and will be housed up to five prior to experiment initiation. Animals are housed under the following climatic conditions: temperature, 72F 6F; humidity, 55Z 15Z; light cycle, 12 hours light/dark. A commercial diet (Purina Certified Laboratory Chow #5002) and tap water will be available ad libitum. The feed is analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water is analyzed biannually on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. Animals will be quarantined for at least 7 days before being placed on study. Animals will be assigned to study groups at random according to Coning Hazleton Standard Operating Procedures. Animals will be weighed prior to dosing. They will be dosed based upon the individual animal weights. Animals will be uniquely identified by ear tag. Treatment groups will be identified by cage label/card. 4/95 4 of 10 001S36 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton Sanitary cages will be used. Personnel handling animals or working within the animal facilities will be required to wear suitable protective garments and equipment. B . Dose Selection The high dose generally will be selected as 80Z of the maximum tolerated dose. The high dose should produce some indication of toxicity (e.g., death, depression of ratio of PCEs to normochromatic erythrocytes (NCEs). One-half and one-quarter of this high dose will normally be used as the intermediate and low dose levels, respectively. Use of a high dose increases the likelihood that a weak clastogen will be detected, and is therefore recommended. If no appropriate range finding data are available, a range finding study can be performed. The top dose tested in the dose rangefinding study will be 5000 mg/kg. The dose levels tested will be issued as an amendment. DOSE RANGEFINDING STUDY The dose rangefinding study will be conducted using five treatment groups. Each of the five groups will consist of 3 male and 3 female mice. Group Designation and Treatment Regimens GrouD No. Number of Mice Male Female Route Duration (Days) 1 3 3 P0 3 2 3 3 PO 3 3 3 3 PO 3 4 33 PO 3 5 3 3 PO 3 4/95 5 of 10 001337 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton The route of administration will be oral gavage. In the event that test article characteristics preclude oral gavage, IP injection will be employed. These routes of administration have been selected because they are the most common routes of administration for this test procedure. The dosing volume will not exceed 20 ml/kg for oral gavage and IP administrations. Other routes of administration that may be used are intravenous, intramuscular, sub-cutaneous administrations or by feed. The test material will generally be solubilized in one of the following solvents: water, 0.9Z saline, 0.5Z aqueous carboxymethylcellulose solution, or corn oil. All animals will be dosed based upon individual body weights. Dose levels will be assigned by a protocol amendment. Body weights will be taken prior to dosing. Dosing formulation will be prepared just prior to dosing. Dosing solutions will be prepared and held at ambient temperatures until dosing (0-2 hours). All animals will be euthanized 3 days after receiving a single dose. The animals will be observed daily for toxic signs and mortality for the duration of the study. Animals will be euthanized by C02 inhalation followed by penetration of the thorax. The daily observations of toxic symptoms and/or mortalities data will be used to estimate the Maximum Tolerated Dose (MTD). Doses will then be assigned for the subsequent cytogenetics assay. MICRONUCLEUS STUDY C . Dosing Schedule and Route of Administration Normally an acute dosing regimen (single administration) will be used (see Table below). Harvest will be approximately 24, 48, 72 hours after administration of the test article, and at approximately 24 hours after administration of the control articles. A total of 110 animals will be used. Equal numbers of males and females will be used at each treatment group. An additional group of animals consisting of 3-10 males and 3-10 females may be dosed as a secondary dose group with the high dose of the test material. This group will be dosed if toxicity is expected at the high dose and the animals in this group will only be used as replacements for any which die prior to euthanasia. The use of the secondary dose group will be determined by the study director. Freshly prepared solutions will be 4/95 6 of 10 G01S3S PROTOCOL'NO. 455, EDITION 17 C O R N IN G Hazleton employed. The animals will be observed daily for toxic signs and mortality. NUMBER OF ANIMALS USED FOR MICRONUCLEUS ASSAY Group No. Treatment Harvest Times After Treatment (Males and Females) 24 Hours 48 Hours 72 Hours 1 Positive Control 5 + 5 -- --- 2 Vehicle Control 5+5 -- -- 3 Low Dose 5+5 5+ 5 5+5 4 Medium Dose 5+5 5+5 5+5 5 High Dose 5+5 5+ 5 5+5 Total 5+ 5 5 +' 5 15 + 15 15 + 15 15 + 15 TOTAL 25 + 25 15 + 15 15 + 15 55 + 55 The route of administration will be oral gavage. In the event that test article characteristics preclude oral gavage, IP injection will be employed. The dosing volume will not exceed 20 ml/kg. These routes of administration have been selected because they are the most common routes of administration for this test procedure. Other routes of administration that may be used are intravenous, intramuscular, sub-cutaneous administrations or by feed. D. Extraction of Bone Marrow Euthanasia will be with C02, followed by penetration of the thorax, and hind limb bones will be removed for marrow extraction. The marrow will be flushed from the bone and transferred to centrifuge tubes containing 3-5 ml bovine serum (one tube for each animal). E. Preparation of Slides Following centrifugation to pellet the tissue, the supernatant will be removed by aspiration and portions of the pellet will be spread on slides and air-dried. The slides will then be fixed in methanol, stained in May-Grunwald Solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides are coded for analysis. 4/95 7 of 10 001839 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton F. Scoring the Slides An attempt will be made to score one-thousand PCEs per animal. The frequency of micronucleated cells will be expressed as percent micronucleated cells based on the number of PCEs analyzed. The normal background frequency of micronuclei in the ICR mouse strain is around 0.0-0.4Z. The frequency of PCEs versus mature erythrocytes (NCEs) will be determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes on the slide. vi. DATA The criteria for the identification of micronuclei are those of Schmid (1976). Micronuclei are darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and are generally between 1/20 and 1/5 the size of The PCE. The unit of scoring is the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus is counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bluish-grey and red, respectively). Data Presentation The data reported will include the number of PCEs scored, the number of micronucleated PCEs, the percentage of micronucleated PCEs, and the ratio of polychromatic to normochromatic erythrocytes for each experimental animal. Evaluation Criteria The criteria for a positive response is 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 induces neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level is considered negative. In either case, the final decision is based upon scientific judgement. 4/95 8 of 10 001340 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton VII. TEST INTERPRETATION The analysis of this data will be performed using an analysis of variance (Winer, 1971) on either untransformed (when variances are homogeneous) or rank transformed (when variances are heterogeneous) proportions of cells with micronuclei per animal. If the analysis of variance is significant (p<0.05), a Dunnett*s t-test (Dunnett, 1955; 1964) will be used to determine which dose groups, if any, are significantly different from the negative control. Analyses will be performed separately for each harvest time and sex combination. VIII. 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.:482-491, 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., jl.:9-15, 1975. Schmid, W. : The micronucleus test for cytogenetic analysis. In. 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. IX. REPORT FORMAT CHV employs a standard report format for each assay design. The final report will provide the following information. Sponsor identification. Quality Assurance statement. Statement of GLP Compliance. Signature of study director. Test article identification and CHV Study Number. A physical description of the test article and date of receipt will be included in this section. Type of assay and protocol number. Dates of study initiation and completion. Study director and senior technician. Methods. 001S41 4/95 9 of 10 PROTOCOL NO. 455, EDITION 17 C O R N IN G Hazleton Evaluation criteria. Interpretation of results. Conclusions. References. Test results presented in tabular form. X. CHANGES OR REVISIONS Any changes or revisions of this approved protocol will be documented, signed by the Study Director, dated, and maintained with this protocol. X I * ANIMAL CARE AND USE STATEMENT In the opinion of the Study Director, no alternative testing methods are appropriate, the study does not duplicate any previous work with this material, and the number and species selected are appropriate. This protocol will he reviewed by the CHV-IACUC for compliance with regulatory guidelines concerning the care and use of animals. If not in compliance, a modification will be required. Any changes or revisions of this approved protocol will be sent to the CHV-IACUC for their review. XII. RECORDS TO BE MAINTAINED All raw data, documentation, records, protocols, and the final report generated as a result of this study will be archived in the storage facilities of Coning 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 Corning Hazleton Inc. for an additional period of time or sent to a storage facility designated by the sponsor. 4/95 001842 10 of 10 AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO.: 17403-0-455 Page 1 of 1 Amendment #1 Section 2, Part V.B. Based on the solubility test, the test article will be suspended in water for the study. In the dose selection study, dose levels of 500, 1625, 2750, 3875, and 5000 mg/kg will be tested. STUDY DIRECTOR Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology Date C01S43 AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO.: 17403-0-455 Page 1 of 1 Amendment #2 Section 2, Part V.B. The dose selection study will be repeated testing dose levels of 700, 1000, and 1300 mg/kg. STUDY DIRECTOR Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology 3 !is/U Date 001.844 AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO. : 17403-0-455 Page 1 of 1 Amendment #3 Section 2, Part V.B. Based on the results of the dose selection study, the mouse micronucleus assay will be conducted testing dose levels of 237.5,475, and 950 mg/kg. A secondary dose group will be used. STUDY DIRECTOR iMc.L '_______ Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology 3jisj% Date CQ1S45