Document X7Z1j2M3G0nLaeZ6ye1Er8zBg

AR226-3226 FINAL REPORT IN VIVO RAT MICRONUCLEUS ASSAY COVANCE STUDY: 22900-0-4540ECD STUDY SCHEDULE: Study Initiation Date : 18-Oct-2001 Initial Dose Date: 23-Oct-2001 Study Termination Date: 19-Nov-2001 Final Report Date: 25-Jan-2002 STUDY DIRECTOR. TESTING FACILITY. AND SPONSOR Study Director: Testing Facility: Gregory L. Erexson, PhD, DABT Covance Laboratories Inc. (Covance) 9200 Leesburg Pike Vienna, Virginia 22182 Sponsor: Santa Monica, California 90407 This study was conducted in accordance with Covance Standard Operating Procedures; the Food and Drug Administration's Good Laboratory Practice (GLP) Regulations, 21 CFR, Part 58; the Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice, ENV/MC/CHEM(98)17 adopted November 26, 1997; and the Environmental-Protection Agency (EPA-TSCA), Title 40 of the US Code of Federal Regulations Part 792, issued November 29, 1983 (effective December 29, 1983 [revision effective September 18, 1989]). This document is the confidential property o f the No part of it m aybe transmitted, reproduced, published, or permission of tha d by other persons without the Covance 22900-0-4540ECD Com pairys^-r^f. cm TABLE OF CONTENTS PAGE SUMMARY.................................................................................................................................. 6 1. 2. 2.1 2.2 2.2.1 2.2.2 2.3 2.4 3. 3.1 3.1.1 3.2 3.3 3.4 4. 4.1 4.1.1 4.2 4.3 4.4 4.5 4.6 4.7 5. 5.1 I N T R O D U C T IO N ..............................................................................................8 M A T E R IA L S ...................................................................................................... 9 Test Article.......................................................................................................... 9 C o n tr o ls ........................... 9 Vehicle Control A rticle.......................................................................................9 Positive Control Article.....................................................................................10 Animals and Husbandry.............................................................................. 10 Selection, Randomization, and Identification o f Rats............................. 11 DOSE RANGEFINDING STUDY............................................................... 11 Study D esign..................................................................................................... 11 Rationale for Dose Selection............................................................................ 11 Test Article Formulation, Administration, and Duration of D osing........................................................................... 12 Clinical Observations and M ortality.................... ....................................13 R esults.........^.................................................................................................... 13 MICRONUCLEUS STUDY..........................................................................14 Study D esign..................................................................................................... 14 Rationale for Dose Selection............................................................................ 14 Test Article Formulation, Administration, and Duration of D osing.................................................................................................................14 Clinical Observations and M ortality.......................................................... 15 Extraction of Bone M arrow ...:..................................................................... 15 Preparation of Slides....................................................................................... 16 Slide Analysis.................................................................................................... 16 Data Presentation................................................... 17 EVALUATION OF TEST RESULTS......L.................................................17 Assay Acceptance Criteria............................................................................. 17 Covance 22900-0-4540ECD Company SanffizedL D oes nrf contain TSCA CBf 5.1.1 5.1.2 5.2 6. 7. 8. 8.1 8.2 9. 10. Acceptable Controls...........................................................................................17 Acceptable High Dose....................................................................................... 18 Assay Evaluation C riteria........................................... 18 RECORDS TO BE M A IN TA IN ED ............................................................18 STUDY RESPONSIBILITIES......................................................................19 R E S U L T S ........................................................................................................... 19 Mortality and Clinical Observations...........................................................19 Micronucleus Evaluation (Appendix A -Table 3 Summary Data, Tables 4-5 Individual Data)......................................................... 19 CONCLUSIONS............................................................................................. 20 LIST OF REFERENCES............................................................................... 20 Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA C81 iv Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. LIST OF TABLES PAGE Dose Rangefmding Study D esign.................................................................. 11 Micronucleus Study D esign...............................................................................14 Micronucleus Summary D ata............................................................................23 Micronucleus Test - 24-Hour Harvest Individual M ale D ata...................... 24 Micronucleus Test - 48-Hour Harvest Individual Male D ata...................... 25 Rat Micronucleus Historical Control Data - 1/2000 through 12/2000.............................................................................................................. 27 Covance 22900-0-4540ECD tsca cbi Company eSannitiiK*d- Doe* s o n is i LIST OF APPENDICES Appendix A - Experimental Data T ables............................................................................... 22 Appendix B - Historical Control D ata.....................................................................................26 Appendix C - Quality Assurance and Compliance Statem ents........................................... 28 Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBS SUMMARY Objective: The objective of this study was to evaluate the test article, 8-2 Alcohol, for in vivo clastogenic activity and/or disruption o f the mitotic apparatus by examining bone marrow polychromatic erythrocytes in male Crl:CD(SD)IGS BR rats for micronuclei. Dose Rangefinding Study Design and Parameters: Since no appropriate toxicity data was available, a dose rangefinding study was performed. The test article was prepared in 0.5% aqueous methylcellulose and dosed once by oral gavage to three males and three females at 2000 mg/kg and a dose volume of 20 mL/kg. The test article produced no lethality or signs o f clinical toxicity. Micronucleus Study Design and Parameters: Based on the results o f the dose rangefinding study, the high-dose chosen was 2000 mg/kg. The test article was prepared in 0.5% aqueous methylcellulose and dosed once by oral gavage to six males/dose level/harvest timepoint at 0, 500, 1000, or 2000 mg/kg at a dosing volume o f 20 mL/kg. Five o f the 6 animals from the 0, 500, 1000 and 2000 mg/kg dose groups were euthanized for bone marrow extraction at approximately 24 and 48 hours postdose. Five animals dosed once with the positive control article, cyclophosphamide at 60 mg/kg, were euthanized for bone marrow extraction approximately 24 hours postdose. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBl Results: The test a r t i c l d H 0 ^ H i p r o d u c e d no lethality or signs o f clinical toxicity. The test a r t i c l H H H f l r f was not cytotoxic to the bone marrow (i.e., no statistically 4M)significant decrease in the PCE.NCE ratio) at up to 2000 mg/kg. The test articl c rdid not induce a statistically significant increase in micronucleated PCEs at any o f the dose levels tested. Conclusion: The test a rtic l^ B B B B B J jro d u c e d no signs o f clinical toxicity in male rats exposed up to 2000 mg/kg, the regulatory limit dose for this assay. In a d d i t i o r J j ^ j J I H m ^ K v a s not cytotoxic to the bone marrow at up to 2000 mg/kg. Note, if observed, a statistically significant decrease in the PCErNCE ratio would be evidence o f bone marrow cytotoxicity induced by the test article. In the micronucleus a s s a y j p m ^ i i d not induce a statistically significant increase in micronucleated PCEs at any o f the doses tested. T h e r e f o r ^ ^ m j j ^ s considered negative in the rat bone marrow micronucleus assay under the conditions o f this assay. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSC CB5 8 1. INTRODUCTION The objective o f this study was to evaluate the test a r t i c l e j p ^ ^ ^ ! |f o r in vivo clastogenic activity and/or disruption of the mitotic apparatus by examining bone marrow polychromatic erythrocytes in male Crl:CD(SD)IGS BR rats for micronuclei. The assay design was based on OECD Guideline 474, updated and adopted July 21, 1997 {OECD, 1997). The micronucleus test can detect clastogenic agents (i.e., agents that cause breaks in chromosomes) and agents which interfere with normal mitotic cell division {Schmid, 1975; Meddle et al, 1983; Meddle et al, 1991). Micronuclei are small chromatin bodies, consisting o f entire chromosomes and/or acentric chromosome fragments, which lag behind at mitotic anaphase. Micronuclei are not normally present in erythrocytes. At telophase, these chromosomes and/or fragments are not segregated to either daughter nucleus, thus forming single or multiple micronuclei in the cytoplasm. The nucleus is extruded during maturation of hematopoietic cells from erythroblasts to erythrocytes. Micronuclei, if present, persist in the cytoplasm o f these non-nucleated cells. Detection o f micronuclei in non-nucleated cells eliminates the need to search for metaphase spreads in treated cell populations. Clastogenic agents and test articles that affect spindle-fiber function or formation can be detected through micronucleus induction {Schmid, 1975). In this study, enucleated immature red blood cells (polychromatic erythrocytes, PCEs) were analyzed for the presence of micronuclei. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBl 2. MATERIALS 2.1 Test Article Test Article: Haskell Number: CAS Number: 24691 identified a: it receipt) Date Received: 08-Aug-2001 The test a r t i c l ^ B W H p |l e s c r i b e d as a white solid, was stored at ambient temperature. The Sponsor is responsible for the determination and documentation of analytical purity, composition, and stability o f the test article. 2.2 Controls 2.2.1 Vehicle Control Article The vehicle control article was 0.5 % aqueous methylcellulose (Covance Batch No. 08-25-01 and 09-14-01 for the dose rangefinding and micronucleus assay, respectively). The vehicle control animals were dosed with the vehicle control by the same route as, and in parallel with, the test article, as a single dose at 20 mL/kg. Covance 22900-0-4540ECD .Company Sanitise*!. D ees not oartara YSGA.CBI 10 2.2.2 Positive Control Article The positive control article used in this study was Cyclophosphamide (Sigma Lot No. 108H0568, CAS No. 6055-19-2) at a dose level of 60 mg/kg and a dose volume o f 10 mL/kg. 2.3 Animals and Husbandry Young adult male and female Crl:CD(SD)IGS BR rats were purchased from Charles River Laboratories, Raleigh, NC. This outbred rat maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles. The protocol for this study was approved by the Covance-LACUC. The animals were acclimated for at least 5 days before being placed on study. The animals were housed in sanitary, stainless-steel, hanging, wire cages. The animals were housed up to two animals per cage during acclimation and singly after randomization. The animals were housed under the following climatic conditions: temperature, 18C 26C (64F - 79F); humidity, 30% - 70%; light cycle, 12 hours light/dark; at least 10 air changes per hour. A commercial diet, PMI Feeds, Inc. Certified Rodent Diet #5002 (Pellets) and tap water were available ad libitum. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed biannually, on a retrospective basis, by Bioreliance and Montgomery Watson for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. Contaminants known or reasonably anticipated to in the diet or water were not at levels expected to interfere with fulfilling the study objective. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBf Personnel handling the animals or working within the animal facilities were required to wear suitable protective garments and equipment. 2.4 Selection, Randomization, and Identification of Rats The animals were randomly assigned to study groups, according to Covance's standard operating procedures, by a computer-based randomization program. Each animal was uniquely identified by ear tag. Treatment groups were identified by cage label. 3. DOSE RANGEFINDING STUDY 3.1 Study Design The treatment regimen for the dose rangefinding study is shown in Table 1. Table 1. Dose Rangefinding Study Design Target Treatment (mg/kg) 2000 Dosing Volume mL/kg 20 No. of Animals Male Female 33 3.1.1 Rationale for Dose Selection Since no appropriate toxicity data was available (e.g., the same species, strain, same route, etc'), a dose rangefinding study was performed using both sexes and the same treatment regimen used in the micronucleus assay. The dose level tested was 2000 mg/kg, the accepted limit dose. Covance 22900-0-4540ECD Company Sanitized. Does noi coniate TSCA GBi 3.2 Test Article Formulation, Administration, and Duration of Dosing The test artici was a solid at room temperature. In order to ensure homogeneity, the test article was placed in a heated water bath (approximately 60 - 80C) until liquefied. The dosing solution was prepared by adding the appropriate volume o f the vehicle, 0.5% aqueous methylcellulose, to a pre-weighed quantity o f test article and mixing to form a homogeneous suspension. The dosing solution was stored at ambient temperature until dosing and stirred during the dosing procedure. The dosing solution was administered once to three males and three females at 2000 mg/kg by oral gavage at a dosing volume o f 20 mL/kg. The animals were dosed on 23-Oct~2001. A total o f six animals, approximately 8 weeks old at the time o f dosing, with a weight range o f 262 to 276 g and 188 to 195 g for the males and females, respectively, were used for this study. The weight variation o f the animals did not exceed 20% o f the mean weight. The animals were weighed prior to dosing and dosed based upon the individual animal weights. All animals were dosed on an acute (one-time only) basis. Covance 22900-0-4540ECD Company Sanitized. Does not cordsiss TSC CB 3.3 Clinical Observations and Mortality During the 7-day acclimation period, the rats were observed at least once daily for abnormalities in appearance or behavior. During the dosing period, all animals were examined immediately after each dose, approximately 1 hour after each dose, and at least daily for the duration o f this study for toxic signs and/or mortality. 3.4 Results All animals appeared normal immediately after dosing and no signs o f toxicity were noted until the end o f the observation period. Therefore, 2000 mg/kg was selected as the high-dose for the micronucleus study. Only males (since both sexes appeared to respond similarly) were tested. Covance 22900-0-4540ECD Company Sanitized. D ees not oonteaa TSCA CBI 14 4. MICRONUCLEUS STUDY 4.1 Study Design The treatment regimen for the micronucleus study is shown in Table 2. Table 2. Micronucleus Study Design Tar^^TreatnraU Dosing Volume mL/kg No. of Animals/Harvest Timepoint0 24 Hour 48 Hour 500 20 6 6 1000 20 6 6 2000 20 6 6 Vehicle Control, 0.5% aqueous methylcellulose 20 6 6 Positive Control, Cyclophosphamide, ~60 mg/kg 10 6 - aThe number of treated animals/group ensures the availability of a minimum of five animals for micronucleus analysis. 4.1.1 Rationale for Dose Selection Based on results o f the dose rangefinding study, dose levels o f 0, 500,1000, and 2000 mg/kg at a dose volume of 20 mL/kg were selected. Only males were used in the micronucleus assay, since both sexes appeared to respond similarly in the dose rangefinding study. 4.2 Test Article Formulation, Administration, and Duration o f Dosing The test a r t i c l e , ^ H H ^ B v a s a solid at room temperature. In order to ensure homogeneity, the test article was placed in a heated water bath (approximately 60 - 80C) until liquefied. The 100 mg/mL dosing concentration o ^ j ^ m ^ w a s prepared by adding the appropriate volume o f the vehicle, 0.5% aqueous methylcellulose, to a pre weighed quantity o f test article and mixing to form a homogeneous suspension. Lower Covance 22900-0-4540ECD Company Sanitized. Does not ccnisfet TSCA CBi 15 concentrations were achieved by diluting the 100 mg/mL dosing solution with vehicle control article. The formulations were stored at ambient temperature until dosing and stirred during the dosing procedure. The test article was administered once to six males/dose level/harvest timepoint by oral gavage at dose levels o f 0, 500,1000, or 2000 mg/kg at a dosing volume o f 20 mL/kg. Cyclophosphamide, the positive control article, was dissolved in sterile deionized water (Covance, Batch No. 10-04-01) and administered by once by oral gavage at dose volume o f 10 mL/kg. The animals used were dosed on 05-Nov-2001. A total o f 54 male animals, approximately 8 weeks old at the time o f dosing, with a weight range o f 222 to 276 g were used for this study. The weight variation o f the animals did not exceed 20% o f the mean weight. The animals were weighed prior to dosing and dosed based upon the individual animal weights. All animals were dosed on an acute (one-time only) basis. 4.3 Clinical Observations and Mortality During the acclimation period, the rats were observed at least once daily for abnormalities in appearance or behavior. All animals were examined immediately after each dose, approximately 1 hour after each dose, and at least daily for the duration o f this study for toxic signs and/or mortality. 4.4 Extraction of Bone Marrow At the appropriate harvest timepoints, the animals were euthanized by CO2 inhalation followed by incision o f the diaphragm. The hind limb bones (tibias) were removed for Covance 22900-0-4540ECD Company Sanitized. Does no! contain TSCA CBi 16 marrow extraction from the first five selected surviving animals. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3-5 mL fetal bovine serum (one tube per animal). Animals not needed for bone marrow collection were euthanized at the completion o f the study. 4.5 Preparation o f Slides Following centrifugation to pellet the cells, the supernatant was removed by aspiration and portions o f the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in acridine orange, protected by mounting with coverslips, and analyzed under fluorescent microscopy. For control o f bias, all slides were coded prior to analysis. 4.6 Slide Analysis Slides prepared from the bone marrow collected from the five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number o f micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 500 erythrocytes on the slide. The criteria for the identification o f micronuclei were those o f Schmid {Schmid, 1976). Micronuclei were darkly stained and generally round, although ulmond- and ring-shaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size o f the PCEs. The unit o f 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. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBI 17 The staining procedure permitted the differentiation by color o f PCEs and NCEs (brightorange and ghost-like, dark-green, respectively). The historical background frequency o f micronucleated cells was expressed as percent micronucleated cells based on the number of PCEs analyzed. The historical background frequency o f micronuclei in the Crl:CD(SD)IGS BR strain in this laboratory is about 0.0-0.4%. 4.7 Data Presentation The mean percent micronucleated PCEs and PCE:NCE ratio and their standard errors are summarized by dose group for the different timepoints. Individual animal data are also shown. Study data are presented in Appendix A. Historical control data are presented in Appendix B. 5. EVALUATION OF TEST RESULTS 5.1 Assay Acceptance Criteria 5.1.1 Acceptable Controls The vehicle control group was within the historical control range, having less than approximately 0.4% micronucleated PCEs. The positive control group was significantly higher (p # 0.05) than the vehicle control group. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBI 5.1.2 Acceptable High Dose The high dose reached the regulatory dose limit dose for this assay (2000 mg/kg). 5.2 Assay Evaluation Criteria Assay data analysis was performed using an analysis o f variance (Winer, 1971) on untransformed proportions o f cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis o f variance was statistically significant (p< 0.05), a Dunnett's t-test (Dunnett, 1955; Dunnett 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time. The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant doserelated response. A test article that did not induce both o f these responses was considered negative. Statistical significance was not the only determinant o f a positive response; the Study Director also considered the biological relevance o f the results in the final evaluation. 6. RECORDS TO BE MAINTAINED All raw data, documentation, records, the protocol, and the final report generated as a result of this study will be archived in the storage facilities o f Covance-Vienna for at least 1 year following submission o f the final report to the Sponsor. After the 1-year period, the Sponsor may elect to have the aforementioned materials retained in the storage Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBf facilities o f Covance-Vienna for an additional period o f time or sent to a storage facility designated by the Sponsor. 7. STUDY RESPONSIBILITIES Function Study Director In-life Laboratory Supervisor Post-life Laboratory Supervisor 8. RESULTS 8.1 Mortality and Clinical Observations The test ^ i c l ^ p H H H |^ ) r o d u c e d no lethality or signs of clinical toxicity until the appropriate harvest timepoints. 8.2 Micronucleus Evaluation (Appendix A -T able 3 Summary Data, Tables 4-5 Individu al Data) The test a rtic le A H H ^ ^ H ^ w a s not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio) j^^ - lid not induce a statistically significant increase in micronucleated PCEs at any o f the dose levels tested. The positive control article, cyclophosphamide, induced statistically significant increases in micronucleated PCEs, as compared to the vehicle control, with a mean and standard error o f 3.10% 0.29%. Covance 22900-0-4540ECD CoiSiNiifF $ w i . TSESSf 20 9. CONCLUSIONS The test a rtic le ^ P B B B H jl produced no signs of clinical toxicity in male rats exposed up to 2000 mg/kg, the regulatory limit dose for this assay. In a d d i t i o n f i l B l B ^ ^ y a s not cytotoxic to the bone marrow at up to 2000 mg/kg. Note, if observed, a statistically significant decrease in the PCEiNCE ratio would be evidence of bone marrow cytotoxicity induced by the test article. In the micronucleus a s s a y , p p m i ^ l i d not induce a statistically significant increase in micronucleated PCEs at any o f the doses tested. T h e re fo re ^ P B M (J ^ is considered negative in the rat bone marrow micronucleus assay under the conditions o f this assay. 10. LIST OF REFERENCES Dunnett, 1955. Dunnett CW. A multiple comparisons procedure for comparing several treatments with a control, J Am Statist Assoc. (1955); 50:1096-1121. Dunnett, 1964. Dunnett CW. New tables for multiple comparisons with a control, Biometrics (1964); 20:482-491. Heddle et al., 1983. Heddle JA, Hite M, Kirkhart B et al. The induction o f micronuclei as a measure of genotoxicity, Mutation Res (1983); 123:61-118. Heddle etal, 1991. Heddle JA, Cimino MC, Hayashi M et al. Micronuclei as an index o f cytogenetic damage: past, present, and future, Env and Mol Mutagen (1991); 18:277-291. Covance 22900-0-4540ECD Company Sanitized. D oss not contain TSCA CBf OECD, 1998. Organisation for Economic Cooperation and Development (OECD). Genetic toxicology: mammalian erythrocyte micronucleus test. OECD Guidelines for the Testing o f Chemicals 1998; No. 474. Schmid, 1975. Schmid W. The micronucleus test, Mutation Res (1975); 31:9-15. Schmid, 1976. Schmid W. (1976) The micronucleus test for cytogenetic analysis, in: Hollaender, A. (Ed), Chemical Mutagens: Principles and Methods for Their Detection, 1976 Vol. 4, Plenum, pp. 31-53. Winer, 1971. Winer BJ, Statistical Principles in Experimental Design; McGraw-Hill, New York, (1971) Second Edition. Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBi Appendix A. Experimental Data Tables Covance 22900-0-4540ECD Table 3. Micronucleus Test in Bone Marrow Cells Summary Data - Male Rats Test Articim Study No.: 22900 *] Treatment Dose Harvest Time (HR) % Micronucleated PCEs Mean of 2000 per Animal S.E. Males Ratio PCE:NCE MeanS.E. Males CONTROLS Vehicle 0.5% MC 24 hr 48 hr 0.040.02 0.070.01 1.260.09 1.110.04 Positive CP 60.0 mg/kg 24 hr 3.100.29a 0.780.00* Test Article 500 mg/kg 1000 mg/kg 2000 mg/kg 24 hr 48 hr 24 hr 48 hr 24 hr 48 hr 0.040.02 0.090.02 0 . 150.07 0.090.05 0.100.03 0.090.02 1.130.05 1.060.11 1.170.06 0.950.08 1.100.07 1.200.08 Significantly greater than the corresponding vehicle control, psO.Ol ^Significantly lower than the corresponding vehicle control, piO.Ol 0.5% MC=0.5% aqueous methylcellulose; CP=Cyclophosphamide; PCE=Polychromatic erythrocyte; NCE=Normochromatic erythrocyte Covance 22900-0-4540ECD SM Kffia Do k C om p* TSC* CB1 Table 4. Micronucleus Test in Bone Marrow Cells - 24-Hour Harvest Individual Male Data ----------------r - .......... Test Article: Study No.: 22900 Treatment Animal Number # MN PCEs/2000 PCEs Ratio PCE:NCE Vehicle Control 0.5% MC Positive Control CP 60.0 mg/kg Test Article 500 mg/kg 1000 mg/kg 2000 mg/kg 6228 6238 6242 6267 6271 6239 6240 6246 6247 6265 6227 6234 6256 6259 6268 6235 6253 6254 6258 6264 6229 6236 6243 6244 6248 1 2 0 1 0 48 61 53 81 67 2 0 0 1 1 5 7 2 0 1 4 1 2 1 2 1.01 1.10 1.36 1.30 1.51 0.81 0.86 0.87 0.62 0.76 1.13 1.08 0.98 1.14 1.30 1.05 1.36 1.19 1.02 1.24 1.07 0.95 1.09 1.36 1.04 0.5% MC=0.5% aqueous methylcellulose; CP=Cyclophosphamide; PCE=PoIychromatic erythrocyte; # MN PCEs=MicronucIeated PCEs; NCE=Normochromatic erythrocyte Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CS1 Table 5. Micronucleus Test in Bone Marrow Cells - 48-Hour Harvest Individual Male Data Test Study No.: 22900 Treatment Animal Number # MN PCEs/2000 PCEs Ratio PCE:NCE Vehicle Control 0.5% MC 6232 6241 6245 6255 6257 1 1 2 2 1 1.10 0.97 1.11 1.17 1.21 Test Article 500 mg/kg 1000 mg/kg 2000 mg/kg 6225 6233 6251 6273 6316 6231 6250 6252 6260 6262 6230 6237 6249 6261 6269 1 2 4a 1 3 0 0 1 4 4 1 3 1 2 2 0.99 1.00 0.93 0.90 1.50 0.89 0.96 0.68 1.06 1.15 0.98 1.40 1.25 1.05 1.33 a# MN PCEs/4000 PCEs 0.5% MC=0.5% aqueous methylcellulose; CP=Cyclophosphamide; PCE=Polychromatic erythrocyte; U MN PCEs=Micronucleated PCEs; NCE=Normochromatic erythrocyte Covance 22900-0-4540ECD Company Sanitized. Does noi contain TSCA CBi Appendix B. Historical Control Data Covance 22900-0-4540ECD Company Sanitized. Does no! contain TSM CB1 27 Table 6. Rat Micronucleus Historical Control Data - 1/2000 through 12/2000 POOLED VEHICLE CONTROLS 24 hour harvest M inim um M axim um A verage N % MICRONUCLEATED PCEs FROM 2000 PCES PER ANIMAL MEAN V S.E. MALES 0.00 0.45 0.088 V 0.001 75 PCE:NCE RATIO MEAN V S.E. MALES 0.54 1.81 0.943 V 0.003 75 48 hour harvest M inim um M axim um A verage N 0.00 0.35 0.080 V 0.002 50 0.54 1.55 0.866 V 0.004 50 POSITIVE CON TRO LS C yclophospham ide, 60.0 m g/kg 24 hour harvest M inim um 0.75 0.21 M axim um 5.60 1.39 A verage 2.260 V 0.021 0.781 V 0.003 N 80 80 PCE = Polychromatic erythrocyte; NCE = Normochromatic erythrocyte; N = Number of animals Covance 22900-0-4540ECD Company Sanitized Does v",ot contain TSCA CBS Appendix C. Quality Assurance and Compliance Statements Covance 22900-0-4540ECD Company Sanitised. Does not content TSCA CBS 0 29 QUALITY ASSURANCE STATEMENT In Vivo Rat Micronucleus Assay The report has been reviewed by the Quality Assurance Unit o f Covance Laboratories Inc., in accordance with the Good Laboratory Practice (GLP) regulations as set forth in the Food and Drug Administration (FDA) Title 21 o f the U.S. Code o f Federal Regulations Part 58; the Organisation for Economic Co-operation and Development (OECD) Principles o f Good Laboratory Practice, ENV/MC/CHEM (98) 17; the Environmental Protection Agency (EPA-TSCA), Title 40 of the US Code of Federal Regulations Part 792, issued November 29,1983 (effective December 29, 1983 [revision effective September 18, 1989]); and any applicable amendments The following inspections were conducted and the findings reported to the Study Director and study director management. Written status reports o f inspections and findings are issued to Covance management according to standard operating procedures. Inspection Dates Phase Dates Reported to Study Director and Study Auditor Director Management___________ 22-Oct-2001 Protocol Review 22-Oct-2001 J.Howard O-Nov-2001 Slide Preparation 06-Nov-2001 S. Ballenger 03-Dec-2001 Draft Report Review 04-Dec-2001 P.Cceres 21-Dec-2001 Revised Draft Report Review 21-Dec-2001 P.Cceres 25-Jan-2002 Final Report Review 25-Jan-2002 P.Cceres Date Covance 22900-0-4540ECD Conipn y S u ttfeedLDees tmcmTBn-ISC * CBI STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice (GLP) regulations as set forth in the Food and Drug Administration (FDA) Title 21 o f the U.S. Code o f Federal Regulations Part 58; the Organisation for Economic Co-operation and Development (OECD) Principles o f Good Laboratory Practice, ENV/MC/CHEM (98) 17; the Environmental Protection Agency (EPA-TSCA), Title 40 of the US Code of Federal Regulations Part 792, issued November 29, 1983 (effective December 29, 1983 [revision effective September 18,1989]); and with any applicable amendments. There were no deviations from the aforementioned regulations or the signed protocol that would affect the integrity o f the study or the interpretation o f the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation o f the test article as presented herein represents an appropriate conclusion within the context o f 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. Although the test substance characterization (ISO 9001) was not performed under GLP standards, the accuracy o f the data is considered sufficient for the purposes o f this study. The dosing preparations were not analyzed for stability, homogeneity, or accuracy of concentration. The procedure used by trained staff to prepare the dosing solutions ensured: The accuracy of concentration because the test substance was weighed on an analytical balance accurate to three decimal places and the vehicle in which it was suspended was accurately measured in a flask graduated in 1-mL increments. Homogeneity because the mixtures were stirred prior to dosing and while portions were removed for dose administration, and Stability because the time between dose preparation and administration was kept to a minimum (approximately 1 hour). Study Director: Covance, Genetic and Molecular Toxicology 9200 Leesburg Pike, Vienna, VA 22182 Study Completion Date Covance 22900-0-4540ECD Covance 22900-0-4540ECD Sanitized. Does not contaiinnTSCA CBl Company 31 Testing Facility Management: Timothy E.\|L-awlor, MA Associate Director Genetic and Molecular Toxicology 0 ( 2-E - 0 ^ Date Report Reviewed and Accepted for E.I. du Pont de Nemours and Co. by: Gerald Kennedy Sponsor Study Monitor Date Maria Donner, PhD Sponsor Technical Project Monitor Date Covance 22900-0-4540ECD Company Sanitized. Does not contain TSCA CBS