Document zzojrxx3Oj78kEyeQ1wqrGN8B

6 _ II^B FINAL REPORT IN VIVO RAT MICRONUCLEUS ASSAY COVANCE STUDY: 22900-0-4540ECD STUDY SCHEDULE: Study Initiation Date: 18-Oct-2001 Initial Dose Date: Study Termination Date: Final Report Date: 23-Oct-2001 19-Nov-2001 25-Jan-2002 rc<Us3s2sy3 G>?3 ae T J fT l "GO cr* --z t<rn 3-r3s* Opn Ho o o- 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: Telomer Research Program Consortium administered by RAND Corporation 1700 Main Street 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 of the Telomer Research Program Consortium. No part of it may be transmitted, reproduced, published, or used by other persons without the permission of the Telomer Research Program Consortium. Covance 22900-0-4540ECD 000068 8-2 Alcohol ii 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 INTRODUCTION........................................................................................ 8 MATERIALS.................................................................................................9 Test Article.....................................................................................................9 C ontrols.......................................................................................................... 9 Vehicle Control Article................................................................................... 9 Positive Control Article................................................................................ 10 Animals and Husbandry........................................................................... 10 Selection, Randomization, and Identification of Rats............................11 DOSE RANGEFINDING STUDY............................................................11 Study Design................................................................................................11 Rationale for Dose Selection........................................................................ 11 Test Article Formulation, Administration, and Duration of Dosing........................................................................................................... 12 Clinical Observations and Mortality........................................................13 Results.......................................................................................................... 13 MICRONUCLEUS STUDY...................................................................... 14 Study Design.................................................... 14 Rationale for Dose Selection........................................................................ 14 Test Article Formulation, Administration, and Duration of Dosing........................................................................................................... 14 Clinical Observations and Mortality........................................................15 Extraction of Bone Marrow...................................................................... 15 Preparation of Slides.................................................................................. 16 Slide Analysis...............................................................................................16 Data Presentation....................................................................................... 17 EVALUATION OF TEST RESULTS......................................................17 Assay Acceptance Criteria......................................................................... 17 Covance 22900-0-4540ECD 000069 8-2 Alcohol iii 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 Criteria......................................................................... 18 RECORDS TO BE MAINTAINED.........................................................18 STUDY RESPONSIBILITIES.................................................................. 19 RESULTS..................................................................................................... 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 oom m 8-2 Alcohol iv Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. LIST OF TABLES PAGE Dose Rangefinding Study Design.............................................................. 11 Micronucleus Study Design..........................................................................14 Micronucleus Summary Data....................................................................... 23 Micronucleus Test - 24-Hour Harvest Individual Male Data.................... 24 Micronucleus Test - 48-Hour Harvest Individual Male Data.....................25 Rat Micronucleus Historical Control Data - 1/2000 through 12/2000........................................................................................................ 27 Covance 22900-0-4540ECD 000071 8-2 Alcohol v LIST OF APPENDICES Appendix A - Experimental Data Tables...........................................................................22 Appendix B - Historical Control Data................................................................................26 Appendix C - Quality Assurance and Compliance Statements.........................................28 Covance 22900-0-4540ECD 000072 8-2 Alcohol 6 SUMMARY Objective: The objective of this study was to evaluate the test article, 8-2 Alcohol, for 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. 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/lcg and a dose volume of 20 mL/kg. The test article produced no lethality or signs of clinical toxicity. Micronucleus Study Design and Parameters: Based on the results of 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 of 20 mL/kg. Five of 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 0000*73 8-2 Alcohol 7 Results: The test article, 8-2 Alcohol, produced no lethality or signs of clinical toxicity. The test article, 8-2 Alcohol, was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio) at up to 2000 mg/kg. The test article, 8-2 Alcohol, did not induce a statistically significant increase in micronucleated PCEs at any of the dose levels tested. Conclusion: The test article, 8-2 Alcohol, produced no signs of clinical toxicity in male rats exposed up to 2000 mg/kg, the regulatory limit dose for this assay. In addition, 8-2 Alcohol was not cytotoxic to the bone marrow at up to 2000 mg/kg. Note, if observed, a statistically significant decrease in the PCE:NCE ratio would be evidence of bone marrow cytotoxicity induced by the test article. In the micronucleus assay, 8-2 Alcohol did not induce a statistically significant increase in micronucleated PCEs at any of the doses tested. Therefore, 8-2 Alcohol is considered negative in the rat bone marrow micronucleus assay under the conditions of this assay. Covance 22900-0-4540ECD 000074 8-2 Alcohol 8 1. INTRODUCTION The objective of this study was to evaluate the test article, 8-2 Alcohol, for 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; Heddle et al., 1983; Heddle et al, 1991). Micronuclei are small chromatin bodies, consisting of 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 of these non-nucleated cells. Detection of 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 000075 8-2 Alcohol 9 2. MATERIALS 2.1 Test Artide Test Artide: Haskell Number: CAS Number: 8-2 Alcohol (identified as Purified 8-2 Alcohol at receipt) 24691 678-39-7 Date Received: 08-Aug-2001 The test article, 8-2 Alcohol, described as a white solid, was stored at ambient temperature. The Sponsor is responsible for the determination and documentation of analytical purity, composition, and stability of 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 00007 8-2 Alcohol 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 of 10 mL/lcg. 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-IACUC. 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 000077 8-2 Alcohol 11 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/lcg, the accepted limit dose. Covance 22900-0-4540ECD 000078 8-2 Alcohol 12 3.2 Test Article Formulation, Administration, and Duration of Dosing The test article, 8-2 Alcohol, 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 of the vehicle, 0.5% aqueous methylcellulose, to a pre-weighed quantity of 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 of 20 mL/kg. The animals were dosed on 23-Oct-2001. A total of six animals, approximately 8 weeks old at the time of dosing, with a weight range of 262 to 276 g and 188 to 195 g for the males and females, respectively, were used for this study. The weight variation of the animals did not exceed 20% of 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 000079 8-2 Alcohol 13 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 of this study for toxic signs and/or mortality. 3.4 Results All animals appeared normal immediately after dosing and no signs of toxicity were noted until the end of 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 090080 8-2 Alcohol 4. MICRONUCLEUS STUDY 14 4.1 Study Design The treatment regimen for the micronucleus study is shown in Table 2. Table 2. Micronucleus Study Design Target Treatment (nig 8-2 Alcohol/kg) Dosing Volume mL/kg No. of Animals/TIarvest Timepointa 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 - The 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 of the dose rangefinding study, dose levels of 0, 500, 1000, and 2000 mg/kg at a dose volume of 20 mL/lcg 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 of Dosing The test article, 8-2 Alcohol, 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 100 mg/mL dosing concentration of 8-2 Alcohol was prepared by adding the appropriate volume of the vehicle, 0.5% aqueous methylcellulose, to a pre weighed quantity of test article and mixing to form a homogeneous suspension. Lower Covance 22900-0-4540ECD om m i 8-2 Alcohol 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 of 0, 500, 1000, or 2000 mg/kg at a dosing volume of 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 of 10 mL/kg. The animals used were dosed on 05-Nov-2001. A total of 54 male animals, approximately 8 weeks old at the time of dosing, with a weight range of 222 to 276 g were used for this study. The weight variation of the animals did not exceed 20% of 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 of 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 of the diaphragm. The hind limb bones (tibias) were removed for Covance 22900-0-4540ECD 000082 8-2 Alcohol 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 of the study. 4.5 Preparation of Slides Following centrifugation to pellet the cells, the supernatant was removed by aspiration and portions of 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 of 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 of 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 of micronuclei were those of Schmid {Schmid, 1976). Micronuclei were darkly stained and generally round, although almond- and ring-shaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCEs. 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. Covance 22900-0-4540ECD 000083 8-2 Alcohol 17 The staining procedure permitted the differentiation by color of PCEs and NCEs (brightorange and ghost-like, dark-green, respectively). The historical background frequency of micronucleated cells was expressed as percent micronucleated cells based on the number of PCEs analyzed. The historical background frequency of 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 000084 8-2 Alcohol 18 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 of variance (Winer, 1971) on untransformed proportions of 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 of 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 of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of 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 of Covance-Vienna for at least 1 year following submission of 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 000085 8-2 Alcohol 19 facilities of Covance-Vienna for an additional period of 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 Responsible Personisi Gregory L. Erexson, PhD, DABT Rebecca L. Axilbund, BS Carol S. Spicer, BS 8. RESULTS 8.1 Mortality and Clinical Observations The test article, 8-2 Alcohol, produced no lethality or signs of clinical toxicity until the appropriate harvest timepoints. 8.2 Micronucleus Evaluation (Appendix A -Table 3 Summary Data, Tables 4-5 Individual Data) The test article, 8-2 Alcohol, was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). 8-2 Alcohol did not induce a statistically significant increase in micronucleated PCEs at any of 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 of 3.10% 0.29%. Covance 22900-0-4540ECD 00008o 8-2 Alcohol 20 9. CONCLUSIONS The test article, 8-2 Alcohol, produced no signs of clinical toxicity in male rats exposed up to 2000 mg/kg, the regulatory limit dose for this assay. In addition, 8-2 Alcohol was not cytotoxic to the bone marrow at up to 2000 mg/kg. Note, if observed, a statistically significant decrease in the PCEtNCE ratio would be evidence of bone marrow cytotoxicity induced by the test article. In the micronucleus assay, 8-2 Alcohol did not induce a statistically significant increase in micronucleated PCEs at any of the doses tested. Therefore, 8-2 Alcohol is considered negative in the rat bone marrow micronucleus assay under the conditions of 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. Heddleeta l., 1983. Heddle JA, Hite M, Kirkhart B et al. The induction of micronuclei as a measure of genotoxicity, Mutation Res (1983); 123:61-118. Heddle et al, 1991. Heddle JA, Cimino MC, Hayashi M et al. Micronuclei as an index of cytogenetic damage: past, present, and future, Env and Mol Mutagen (1991); 18:277-291. Covance 22900-0-4540ECD 8-2 Alcohol 21 OECD, 1998. Organisation for Economic Cooperation and Development (OECD). Genetic toxicology: mammalian erythrocyte micronucleus test. OECD Guidelines for the Testing of 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 000088 8-2 Alcohol Appendix A. Experimental Data Tables 22 Covance 22900-0-4540ECD 000089 8-2 Alcohol 23 Table 3. Micronucleus Test in Bone Marrow Cells Summary Data - Male Rats Test Article: 8-2 Alcohol Study No.: 22900 Treatment Dose Harvest Time (HR) % Micronucleated PCEs Mean of 2000 per Animal S.E. Males Ratio PCE:NCE Mean S.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, psO.Ol 0.5% MC=0.5% aqueous methylcellulose; CP=Cyclophosphamide; PCE=Polychromatic erythrocyte; NCE=Normochromatic erythrocyte Covance 22900-0-4540ECD 00009V 8-2 Alcohol 24 Table 4. Micronucleus Test in Bone Marrow Cells - 24-Hour Harvest Individual Male Data Test Article: 8-2 Alcohol 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=Polychromatic erythrocyte; # MN PCEs=Micronucleated PCEs; NCE=Normochromatic erythrocyte Covance 22900-0-4540ECD 000091 8-2 Alcohol 25 Table 5. Micronucleus Test in Bone Marrow Cells - 48-Hour Harvest Individual Male Data Test Article: 8-2 Alcohol Study No.: 22900 Treatment Animal Number # MN PCBs/2000 PCEs Ratio PCE:NCE Vehicle Control 0.5% MC Test Article 500 mg/kg 1000 mg/kg 2000 mg/kg 6232 6241 6245 6255 6257 6225 6233 6251 6273 6316 6231 6250 6252 6260 6262 6230 6237 6249 6261 6269 1 1 2 2 1 1 2 4a i 3 0 0 1 4 4 1 3 1 2 2 1.10 0.97 1.11 1.17 1.21 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; # MN PCEs=Micronucleated PCEs; NCE=Normochromatic erythrocyte Covance 22900-0-4540ECD 000092 8-2 Alcohol Appendix B. Historical Control Data 26 Covance 22900-0-4540ECD 000093 8-2 Alcohol 27 Table 6. Rat Micronucleus Historical Control Data - 1/2000 through 12/2000 POOLED VEHICLE CONTROLS 24 hour harvest Minimum Maximum Average 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 Minimum Maximum Average N 0.00 0.35 0.080 V 0.002 50 0.54 1.55 0.866 V 0.004 50 POSITIVE CONTROLS Cyclophosphamide, 60,0 mg/kg 24 hour harvest Minimum 0.75 0.21 Maximum 5.60 1.39 Average 2.260 V 0.021 0.781 V0.003 N 80 80 PCE - Polychromatic erythrocyte; NCE = Normochromatic erythrocyte; N = Number of animals Covance 22900-0-4540ECD 000094 8-2 Alcohol Appendix C. Quality Assurance and Compliance Statements 28 Covance 22900-0-4540ECD 000095 8-2 Alcohol 29 QUALITY ASSURANCE STATEMENT 8-2 Alcohol: In Vivo Rat Micronucleus Assay The report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Good Laboratory Practice (GLP) regulations as set forth in the Food and Drug Administration (FDA) Title 21 of the U.S. Code of Federal Regulations Part 58; the Organisation for Economic Co-operation and Development 2(EFO9enE,dvCei1rr9oDa8n)l3mPRre[eirnnegctvuaiipsllaliPeotisnoronoetsfefGfcPetoicaootrnidtve7LA9agS2beeo,nrpiacstteysoumr(yeEbdPPerArNac-1oTt8ivcS,eeCm1, 9AEb8N)e,9rV]T)2/;iMt9lae,nC1d4/C90a8Hno3yEfaM(tehpfpef(le9iUcc8a)tSibv1leCe7;oaDtdmheeeecneodmfmbeenrts The following inspections were conducted and the findings reported to the Study Director and study director management. Written status reports of 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 06-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 000096 8-2 Alcohol 30 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 of the U.S. Code of Federal Regulations Part 58; the Organisation for Economic Co-operation a(C9no8dd)De1e7ov;fethlFoeepEdmenervnaitrlo(ROnemEgCeuDnlat)atiPlorPninsrcoiPtpealcertstio7on9f 2GA,ogioesdsnucLeyadb(oENrPaotAovr-eyTmSPbCraeAcrt)i2,c9eT,,itE1leN984V30/M(oeCffft/ehCceHtiEvUeMS 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 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. Although the test substance characterization (ISO 9001) was not performed under GLP standards, the accuracy of the data is considered sufficient for the purposes of 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). Covance 22900-0-4540ECD 000097 8-2 Alcohol 31 Study Director: O*?'- Gregory L.jgf'^son, PhD, DABT Covance, Genetic and Molecular Toxicology 9200 Leesburg Pike, Vienna, VA 22182 Study Completion Date Covance 22900-0-4540ECD Testing Facility Management: UL Timothy E.uLawlor, MA Associate Director Genetic and Molecular Toxicology 0( ' Date L 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 000098