Document kD5O2j5pa3prYXrrLeoj48aob

AR226-3226 FINAL REPORT IN VIVO RAT MICRONUCLEUS ASSAY 'COVANCE STUDY: 22900-0-454OECD STUDY SCHEDULE: Sudy nisionDate: 18-0012001 Initial Dose Date: 23-01-2001 StudyTerminatDiaoten: 19Nov-2001 Fina Report Date: 25.3202 STUDY DIRECTOR, TESTING FACILITY. AND SPONSOR [er -- Gregory L. Erexson, PD, DABT - Testing Facility: C5V2oi0ve0annL,ceeveLisarbgooinrnaiPtaiokr3ei2e1s8I2nc. (Covance) 1700 Main Street `Santa Monica, California 90407 Trhiis nsosypewasosrosEncioodnonemiscnGCoosoopderdaabtrinosntoainrtdyherCvodvvoasnpcmee(nSGtaLn)(dORrEedCgDOa)piePrnetsin,nig2l1PeoCasfFGe,ouForadst h5e; Fhoeod DE brie ENVNCCHEMO adopt November 26, 1997, ad he Eho y Environmental. vers 35.358 (cece. Dever Protection Agency (EPA-TSCA), Title 40 o29f,th1e98U3SeCvodieoonf Tse Sepember Federal Regulations RThis deoacumaemnatsytahercoanfnideent.ialrperporpoedrutcyedo,fpoUUIeda-- wid he persons wiht the permison of Covance 22900-0-4540ECD Company Santirar, rane. A ~ or? `TABLE OF CONTENTS 0 PAGE 22 221 222 23 24 3. 3.11 32 33 4. 4.1 4.1.1 4.2 HO43 - 44 46 Ss4.7 51 COBIIOIS cosrrrssrssrssrsssesnssmssessssssssssesssssssssssssssssssssssssssssesssssss Vehicle Control Article... ------------------ rat asin Positive Control Article............coooeieieirene nsnsnimmmiinsimmmiiion 10 Animals and HUsHandry.....mmsssssssmssssssssssmssssssmmssssssssssasssss 10 Selection, Randomization, and Identification ofRatS...........cusecsnes 11 DOSE RANGEFINDING STUDY..ccouvmmmmmmmmmmmsssssssssssssssssssssssssssssssssssssss 11 Rationale for DOSE SEleCtiON.......cooouurresuvrerrssmsensrsssssssssssssssssssssessnns 11 Test Article Formulation, Administration, and Duration of Clinical Observations and MOFAlity .....iusmmessmsnn 13 MICRONUCLEUS STUDYcevessssmmssmsmssssssssssssssssssssassssssssssssssssssassses 14 Study Desigh cms gs. 14 Rationalefor DOSE SEIECtiON........cvvvresevverrssesrerrrsssnarsessssssssrssssssneesss 14 Test Article Formulation, Administration, and Duration of DOSE cerns 14 hrm Clinical Observations and Mortality ........cmmmmmmmms 15 Extraction of Bone Marrow o.oo 15 SHAE ADBIYSIS conn 16 Data Presentation mms 17 EVALUATION OFTESTRESULTSmi] ASSAY ACCEPLANICE CTHET um Cove e 290s 34540811 Compr senSos rca TSAGB - Donnan) ii 5U1D1 52 6. 1 AN CCEPLaDIECOe NITOIS ce rv nah -- Assay EVAIURLON CHIteria wovvuevsessmmmssssssssssssssssssssssssssssssssssssssssss 18 RECORDS TO BE MAINTAINED ccovvvumsmnsssssssssssssssssssssssssssssssssses 18. E01 STOR TOIT 1:31 3 OL-------------- 81 FE ---- 82 Micronucleus Evaluation (Appendix A -Table 3 Summary Data, Tables 4-5 Individual Data)........smsssssmssssmssssssssssessssesssss 19 9. CONCLUSIONS cccnsswcrssrsemsversessmemsmmsssmsmsssssmnssmssesmrssessmsssssssssss 30 10. LIST OF REFERENCES ..ccoovsrrmsmsssssmssssssssssssssssssssssssssssssssssses 20 _ Covance 22900-0-4540ECD (Company Sanitized. Does not contain TSCA CBI - [ri] iv Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. LIST OF TABLES PAGE Dose Rangefinding Study Design .............cceuusveserssssussssssmssssessssssssssens 11 Micronucleus Study Design rereveveveveee nmmsssesanmsssaaremmrsssoreness 1 Micronucleus SUMmMAIY Data.........vsussssssssessssssssssssssssssissssssns 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 Covance 22900-0-4540ECD Company Saniized.Doesnotcontain TSCA CB! - [om] v LIST OF APPENDICES Appendix A -- Experimental Data Tables .........ew "3 Appendix B --Historical CONtrol Data ............ccouvureesrsusnerssssssnsscsss Appendix C -- Quality Assurance and Compliance Statements... -- 28. Covance 22900-0-4540ECD Company Seized.Dass ncomanTSCACBI - [ean] . SUMMARY Objective: The objective of this study was to evaluate the test article, 8-2 Alcohol, for in vivo clastogenic activity and/or disruptionof 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 adose volume of 20 mL/kg. The test article produced no lethaliotry signsofclinical toxicity. Micronucleus Study Design and Parameters: Based on the resultsofthe 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 levelharvest timepoint at 0, 500, 1000, or 2000 mg/kg ata dosing volume of 20 mL/kg. Fiveofthe 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 Santized. Dossnot contain TSCA CEI --~ (emu) 7 Results: The test arti oroduced no lethality or signs ofclinical toxicity. "The test articl 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 Ej) (on: not induce a statistically significant increase in micronucleated PCEs at any ofthe dose levels ested. Conclusion: The test ic Rc no signsofclinical toxicity in male is exposed up 10 2000 my, the regulatory limit dose for his assay. In additional] --.. not cytotoxic to the bone marrow at up to 2000 mg/kg. Note,ifobserved, a statistically significant decrease in the PCE:NCE ratio would be evidenceofbone marrow cytotoxicity induced by the test article. - In the micronucleus ws not induce a statistically significant increase in micronucleated PCEs at anyofthe doses ested. Thereof considered negative in the rat bone marrow micronucleus assay under the conditions of this assay. Covance 22900-0-4540ECD Company Santized. Does not conttaainin TSCA CB - [emmei] : 1 INTRODUCTION The objectiveofthis study was to evaluate the teswt ilemer vive clastogenic activity and/or disruption ofthe 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. agents that cause breaks in chromosomes) and agents which interfere with normal mitotic cell division (Schmid, 1975; Heddle et al. 1983; Heddle etal, 1991). Micronuclei are small chromatin bodics, consistingofentire 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 cither daughter nucleus, thus forming single or multiple micronuclei in the cytoplasm. The nucleus is extruded during maturation of hematopoietic cells from erythroblasts to erythrocytes Micronucleiifpresent, persist in the cytoplasm of these non-nucleated cells. Detection ofmicronuclei in non-nueleated cels eliminates the need to search for metaphase spreads in treated cll 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 presenceofmicronuclei. } _ Covance 22900-0-4540ECD ormpany Seized. Dogsnotconan TSC cal - [ f 2) ' 2 MATERIALS 21 Test Article Test Article: TomS s -- Haskell Number: 9924691 receip) CAS Number: --] Date Received: 08-Aug-2001 The test a as a white solid, was stored at ambient temperature. The Sponsor i responsible forthe determination and documentation of analytical purty, composition, and stabilityofthe test article 22 Controls 221 Vehicle Control Aricle "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, th test article, os single dose at 20 mLkg. _ Covance 22900-0-4540ECD Company Saniized. Dass ot contain FSCACB ~ [re 1 222 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 levelof 60 mg/kg and a dose volume of 10 mL/kg. FE `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-ACUC. _ `The animals were acclimatedforat 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 concentrationsofspecified 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 JR ---- - (| u Personnel handling the animals or working within the animal facilities were required to wear suitable protective garments and equipment. 24 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 31 Study Design ----- The treatment regimen for the dose rangefinding study is shown in Table 1. aoEomhrTable 1. eemraoie Dose Rangefinding Study Design gsco e, a 311 Rationale for Dose Selection Since no appropriate toxicity data was available (.8., the same species, strain, same route, eic?),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 ConnySets, bosses 6A! - [mil] 2 32 Test Article Formulation, Administration, and Durationof Dosing `The test wc was a solid at room temperature. In order to ensure homogeneity, the test aricle was placed in a heated water bath (approximately 60 ~ 80C) uniil liquefied. The dosing solution was prepared by adding the appropriate volume of the vehicle, 0.5% aqueous methylcellulose, to a pre-weighed quantityoftest article and mixing to form a homogeneous suspension. The dosing solution was stored at ambient temperature uniil 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 volumeof20 mLkg. "The animals were dosed on 23-Oct-2001. A totalofsix animals, approximately weeks old a the timeofdosing, witah 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 variationofthe animals did not exceed 20% of the mean weight. The animals were weighed prior to dosingand dosed based upon the individual animal weights. All animals were dosed on an acute (one-time only) basis. _ Covance 22900-0-4540ECD Company Santtized. Does not coniaitn aiaTSCACE! - [ims] 13 33 Clinical Observations and Mortality During the 7-day acclimation period, the rats were observed at last once daily for abnormalities in appearance or behavior. During the dosing period, all animals were examined immediately after cach dose, approximately 1 hour after each dose, and atleast daily for the duration ofthis study for toxic signs and/or mortality. 34 Results All animals appeared normal immediately after dosing and no signsoftoxicity were noted until the end ofthe 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 Santtized. Doatscontsain TSCA CBI - [--] " 4 MICRONUCLEUS STUDY a1 Study Design "The treatment regimen for the micronucleus study is shown in Table 2. Table2. Micronucleus Study Design oToggi DosingnVeolume No.oif tiAonnnalsHarve Tintogugri i02o00n00 .`` ``` FVPeoetstineeonCCboeoonftnen,, eC0o5ld%opsapqspupeiaouresidmoetu,hsycelonesmlupseeeto AyF__S10oF al`` o miles`: Sa 411 Rationale for Dose Selection Based on resultsofthe dose rangefinding study, dose levels of0, 500, 1000, and 2000 mig at a dose volumeof 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. 42 Test Article Formulation, Administration, and Duration of Dosing The est aJJrcas solid at room temperature, In onder to ensure homogeneity, the test article was placed in a heated water bath (approximately 60 ~ 80C) until liquefied. The 100 mg/mL dosing concentration Pn. prepared by adding the appropriate volume of the vehicle, 0.5% aqueous methylCellulose 1 apreweighed quantity of test article and mixing to form a homogeneous suspension. Lower _ Covance 22900-0-4540ECD Company Sanfized. Damotscorisain TSCACES - [amma 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 levelsof0, 500, 1000, or 2000 mg/kg ata dosing volume of20 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 timeofdosing, with a weight rangeof 222 t0 276 g -_ `were used for this study. The weight variationofthe animals did not exceed 20%ofthe mean weight. The animals were weighed prior todosingand dosed based upon the individual animal weights. All animals were dosed on an acute (one-time only) basis. 43 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 cach dose, approximately 1 hour after each dose, and at least daily for the durationofthis study for toxic signs and/or mortality. _ } 44 Extraction of Bone Marrow At the appropriate harvest timepoints, the animals were euthanized by CO; inhalation followed by incision of the diaphragm. The hind limb bones (tibias) were removed for . Covance 22900-0-4540ECD CompanySantzed Doss acon TSCAGBI - | 1 `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 (onetubeperanimal). Animals not needed for bone marrow collection were euthanized at the completionof the study. 45 Preparationof 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 controlofbias, all slides were coded prior to analysis. - 4 Side Anlyss 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 ofmicronucleated PCE from at least 2000 PCE per animal. "The PCE:NCE ratio was determined by scoring the numberof PCEs and NCEs observed in the optic fields while scoring at least the first 500 erythrocytes on the slide. The criteria for the identification ofmicronuclei 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 ofthe PCEs. The unit ofscoring 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 companySaid. BosotonaTSCACEI - --) " `The staining procedure permitted the differentiation by color ofPCEs and NCEs (brightorange and ghostlike, dark-green, respectively). The historical background frequency ofmicronucleated cells was expressed as percent micronucleated cells based on the numobfPeCrE analyzed. The historical background frequencyof micronuclei in the Crl:CD(SD)IGS BR strain in this laboratory is about 0.0-0.4%. 47 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 51 Assay Acceptance Criteria 511 Acceptable Controls : "The vehicle control group was within the historical control range, having less than approximately0.4% 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 - Pom) " 512 Acceptable High Dose `The high dose reached the regulatory dose limit dose for this assay (2000 mg/kg). Assay data analysis was performed using an analysisofvariance (Winer, 1971) on `untransformed proportionsofcells with micronuclei per animal and on untransformed. PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. Ifthe analysisofvariance was statistically significant (p< 0.05), a Dunnett's t-test (Dunnett, 1955; Dunnett 1964) was used to determine which dose groups,ifany, 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 ofa statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose- related response. A test article that did not induce bothofthese responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevanceofthe results in the final evaluation. 6. RECORDS TO BE MAINTAINED All raw data, documentation, records, the protocol, and the final report generated as a resultofthis study will be archived in the storage facilitiesofCovance-Vienna for at least 1 year following submissionofthe 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 ComprSos. stan TSCA - [ron'} 1 facilitiesofCovance-Vienna for an additional periodof 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 Person(s) 8. RESULTS 81 Mortality and Clinical Observations - The test i oc no lethality or signsofclinical toxicity until the appropriate harvest timepoints, 82 Micronucleus Evaluation (Appendix A ~Table 3 Summary Data, `Tables 4-5 Individual Data) The test ricefg vs not cytotoxic to the bone marrow (i.., no statistically significant decrease in the PCE:NCEwai)fc not induce a statistically significant increase in micronucleated PCEs at anyofthe 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 `Company San Gossi wotz one anTd SCA. CS} - oi] 0 9. CONCLUSIONS "The test artcl produced no signsof clinical toxicity in male rats exposed up to 2000 the regulatory limit dose for this assay. In sadiionJMR not cytotoxic (o the bone marrow at up to 2000 mg/kg. Note, ifobserved, a statistically significant decrease in the PCE:NCE ratio would be evidence of bone marrow cytotoxicity induced by the test article. In the micronucleus we [a not induce a statistically significant increase in micronclested PCE at anyof th doses teste. Therefore NBR considered negative in the rat bone marrow micronucleus assay under the conditionsofthis assay. 10. LIST OF REFERENCES Dunnett, 1955. Dunnett CW. A multiple comparisons procedure for comparing several treatments with a contrJoAlm, 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 ef al. The inductionof micronuclei as a measure of `genotoxicity, Mutation Res (1983); 123:61-118. Heddleer al, 1991. : . Heddle JA, Cimino MC, Hayashi M et al. Micromuclei as an indexof cytogenetic damage: past, present, and future, Env and Mol Mutagen (1991); 18:277-291 Covance 22900-0-4540ECD Company Sanized. Doss rot cantain TSCA CBI _ Jen] x 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 BJ, Statistical Principles in Experimental Design; McGraw-Hill, New York, (1971) Second Edition. _ Covance 22900-0-4540ECD - a) 2 Appendix A. Experimental Data Tables _ `Covance 22900-0-4540ECD Company Seiad Us a! contin TSCATE! - [nas] zn Tabled. Mictonucleus Test in Bone Marrow Cells Summary Data -- Male Rats Tet f-- J StudyNo: 23900 HaTrivmees|t Mea%neoMfic2r0o0nupleetAendiPmaEs+S Tomer Doe GR) Mas RaMtieoaPnCsENCE Vales controls Vaid oswMC ah ooo 12600 ashe oon Laos Posie CPOOOmPRs he s100290 o7m0006 Tetanide SOmpkg 24h 008002 Linsogs whe 0092002 Lossont omg 2h ois007 Limos _ she 009005 0551008 wong 24h 010003 L100 ashe 00002 120.008 SSiinginciangleoswetreatnanthtehecocromaepsopnodnidnigngveehiiccleecocnotrno,,pp0011 N0.C5E4-MNCo-r0mo5c%hciaomuespmeotheylase; CP-Cydlaphasphanide; PCE=Polychromatic eybrocycs Covance 22900-0-4540ECD Desrotconsi TSCAcsi Company SwAlizod. ~ [--] Tabled. Micronucleus Test in Bone Marrow Cells ~ 24-Hour Harvest Individual Male Data Test Avice -- Study Nos 22900 Teamen NAunmimbaelr PCECI0OPCEs PRCaENiCE Vehicle Conte! sume aas2 21 i10n1 aa6n 0| 113306 an 0 ist PosdveConrol CP eDOmghe aai aa oons aais 85 ored aes @ 076 Tet Arle some aan 2 110s - aasss i 019184 ass 1 13 1000 mgt aass 7s 11306s aasss 02 11012s aot 1 128 2000 mats a9 +1 0130s7 aaun 2| 11306 as 2 Tos + #0.M5%NMPCC=E0s-.M5i%craoqnuueoeulseimeedtPhCyESl;aNsCeE;-NCoPr-mCyolcahprhoomstpihaennitdh;ocPyCtEe=Folychromaic nto _ Covance 22900-0-4540ECD Company Saniized. Doesot comain TSCACSI ~ [e--] = Table. Micronucleus Test in Bone Marrow Cells - 45-Hour Harvest Individual Male ip . re H y T = Data -- w--m " Bod i ~ wr .2w=J 3 2ni w-- 8 % r Tr EAS Covance 22900-0-4540ECD GompanySeniized. Doesnolcapisfa TSCACBI - [r---- . Appendix B. Historical Control Data -- Covance 22900-0-4540ECD company Santzed. Doss nt contain TSCA - Ic Taled, Ra Morons itor] Cnt Data 200 rh 122000 ee seppare o i ieey rr we po a we m-- memn. w rh i _ Covance 22900-0-4540ECD Company Sanilized. Does riot conta - [oi] Appendix C. Quality Assurance and Compliance Statements _ Covance 2900-04540ECD Company Saniized. Doesnolcontain TSCA CS! | - Tn] QUALITY ASSURANCE STATEMENT [me]. Vivo Rat Micronucleus Assay `The report has been reviewed by the Quality Assurance Unitof 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. Codeof Federal Regulations Part 58; the Organisation for Economic Co-operation and Development (OECD) Principles of 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, 19891); and any applicable amendments 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. Dates Reported to Study - Inspection Dates Phase Director and Study ~~ Auditor Director Management 22.0ct-2001 Protocol Review 22-0t:2001 J. Howard 06-Nov-2001 Slide Preparation 06-Nov-2001 S. Ballenger 03-Dec-2001 Draft Report Review 04-Dec-2001 P. Caceres 21-Dec-2001 Revised Draft Report Review 21-Dec-2001 P. Caceres 25-Jan-2002 Final Report Review 25-Jan-2002 P. Ciceres Ee heniting Representative, Quality Assurance Unit oi[28/02 Date _ Covance 22900-0-4540ECD CompanySanitizsd.Deoiecoms SSCACS! - [imi] 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 21ofthe U.S. Code of Federal Regulations Part 58; the Organisation for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice, ENV/MC/CHEM (98) 17; the Environmental Protection Agency (EPA-TSCAY), 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 of the study or the interpretationofthe 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 contextofthe 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 thedatais considered sufficient for the purposesof this study. ~ The dosing preparations were not analyzed for stability, homogeneity, or accuracy of concentration. The procedure used by trained staftof 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 + 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: - a nLLlolSenta aeeSA - ipolaa sloa CoA enes _ Covance 22900-0-4540ECD es notcontain TSCA CB! Company Sanitized. DOS [oy 3 Testing Facility Management: pp4 2 LL "ATsismooctihayteE\Directloorr, MA Genetic and Molecular Toxicology ol28 0% Date Report Reviewed and Accepted for E.I. du Pont de Nemours and Co.by: Gerald Kennedy Date `Sponsor Study Monitor = Maria Donner, PhD `Sponsor Technical Project Monitor Date Covance 22900-0-4540ECD Company Saniized. Doss notcontain TSCA CBI