Document jBvKV0oXGyeJddYX8o51RV5LO

AR226 - 1198 FINALREPORT. IN VIVORATMICRONUCLAESSUASY COVANCE STUDY: 22900-04540ECD STUDY SCHEDULE: StudyInitiationDate: 18-0012001 Initial DoseDate: Study TerminatDiaoten: Fina Report Date: Tu TSteutdnygDiFraecctiolr: 23-Oct-2001 19-Nov-2001 252002 IN AND SPONSOR GCroevgaonrcyeLL.abEorreaxtsoorni,esPhTDn., D(CAoBvaTnce) 9V2i0a0nL,esVbirugrignPiaik2e152 2S om E 33 o=m= EZF 3O3m s2 Sponsor: a`Tedlommer iResnebayricRhAsPNrDtogCreoarmproCroaentsiodorntium 1700 Main Steer Santa Monica, California 90407 TaOnnhdginDsirtsuagayoAwndamsfionrcsoEnrcdaoutcnotonemd'iseinGCaoocoocpoderrdLaaatnbicooernatwaoinrtdyhDPeCrvoavcctalinaccpeem(eSGntLtaPnO)dEaRreCdgDOupl)aetrPiarotininscn,igpP2l1reosCcoFefRdG,uorPeorsd;tt5h;e Ftohoed ECFoaavtbno7or9no3m,reymnaPerldacPiNrcoeov,teEmoXbnVeIrAMg3Ce0InCc1Hy9E8(8ME((PScAe8Ce)TtS1iC7vAea)dD,oepTctieemedbNe40ro3o9v,th1ee9U32S,[Creo1v9di9es7io;ofnanFeefdetetherivReegSuelpatiiboensr 15. 1989), TNhoisprdtocoumfenmtaysbtshetcroanmfsimdeintteida,l rperporpoedrutcyedo,fptheuTbelloimeasrRhueseseeddabr,ycohthPerogperrasmonCsonwsiotrhtoiuutm.the permissionofthe TelomerResearchProgramConsort, Covance 22900:0-4540ECD 000063 8-2 Alcohol it TABLE OF CONTENTS PAGE SUMMARY sss6. I INTRODUCTION cnn 2. MATERIALS consmmmmssmsmssmssssssssssssssssssmsnsd 22.1 Vehicle Control Article. mmmensimisees) 222 Positive Control Article....... ws IB 23 J ------. 24 Selection, Randomization, and Identification of RAtS....vews 11 3 DOSE RANGEFINDING STUDYcerca 11 31 311 SEY DESIZN corre11 Rationale for Dose Selection............ 1 32 Test Article Formulation, Administration, and Duration of DOIG mrss 33 33 Clinical Observations and MOFAIY users 13 34 A -- a MICRONUCLEUS STUDY corse 14 411 RationaleforDose Selection... -- 42 Test Article Formulation, Administration, and Duration of EE 43 Clinical ObSrvations and MOFAIIyesr: 15 44 A ------.T 45 a --T 46 SHAQ ABBIYSIS rrr 16 47 Data POSEN corse: 11 s EVALUATION OF TEST RESULTS cose 11 51 A$50Y ACCEPIANCE CHE 1 Covance 22900-0-4340ECD 000069 82 Alcohol I" 551112 AAcCcCeEpPtGabbIlEe CHiOgRhIDOOSLE.S.. rrr os7 52 J NT 6 RECORDS TO BE MAINTAINED cvs: 18 7. STUDY RESPONSIBILITIES .cnmms 19 88.1 RMoErStaUlLiTtySac nd Clinicalo OBSErVATOo NS..cvervs vrvrvrner: svsnrone 1199 82 MDaitcar,onTuacblleeuss4-E5vaIlnuiaVtiidoUna(lADpApeAn)d.ix A-Table 3 Summary 19 5. CONCLUSIONS susmmmmmmmmmnsnsnssnsssmssons 2 10. LIST OF REFERENCES ocovssssssssmsnssnsssssssssssssssnsnins 20 Covance 22900-0-4540ECD 000070 82 Alcohol iv Table1. Table. Table. Tabled. TableS. Table6. LIST OF TABLES PAGE Dose Rangefinding Study DESIgN rover 11 Micronucleus Study DESIgN oro -. Micronucleus Summary Data. ............. osm MicronTuesct~l24e-Huousr Harvest Individual Male Data.............. 24 MicroTneustc-4l8e-Huousr Harvest Individual Male Data. ............25 Rat Micronucleus Historical ControlData -- 1/2000 through 122000......... - commis Covance 22900-0-4540ECD 000071 82 Aleohol v LIST OF APPENDICES AppAe~nExpderiimenxtalDataTables... AppeB-nHidstoiricxal Control Data..... Appendix C - Quality Assurance and Compliance Statements will wasn smn 25 Covance 22900-0-4540ECD 000072 82 Alcohol 6 SUMMARY Objective: The objectiveofthis study was to evaluate the test article, 8-2 Aleohol, for in vivo clastogenic activity and/or disruptionofthe 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 ance by oral gavage to three males and three females at 2000 mg/kg and a dose volumeof 20 mUkg. The test article produced no lethalityorsignsofclinical 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/doselevelharvest timepoint at 0, 500,1000,or 2000 mg/kg at a dosing volumeof20 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 000073 82 Alcohol 7 Results: The test article, 8-2 Alcohol, produced no lethality or signsof clinical toxicity. "The test article, 8-2 Alcohol, was not cytotoxic to the bone marrow (i.., no statistically significant decrease in the PCE:NCE ratio) at up t0 2000 mg/kg. The test article, 8-2 Alcohol, did not induce a statistically significant increase in micronucleated PCE at any ofthe dose levels tested. Conclusion: The test article, 8-2 Alcohol, produced no signsofclinical 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 evidenceofbone marrow. cytotoxicity induced by the test article. Inthe micronucleus assay, 8-2 Alcohol did not induce a statistically significant increase in mictonucleated PCE at anyof 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 82 Alcohol 8 I INTRODUCTION "The objectiveofthis 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:CDY(SD)IGS BR rats for micronuclei. The assay design was based on OECD Guideline 474, updated and adopted July 21, 1997 (OECD, 199). `The micronucleus test can detect clastogenic agents (i.., agents that cause breaks in chromosomes) and agents which interfere with normal mitofic cell division (Schmid, 1975; Heddle et al., 1953; Heddle et al. 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 andor fragments are not segregated to cither daughter nucleus, thus forming single or multiple micronuclei in the cytoplasm. The nucleus is extruded during maturationofhematopoietic cells from erythroblasts to erythrocytes. Micronuclei,if present, persist in the cytoplasmofthese non-nucleated cell. 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 presenceofmicronuclei Covance 22900-0-4540ECD 000075 82 Alcohol 9 2 MATERIALS 21 Test Article Test Article: Haskell Number: CAS Number: Date Received: 8-2 Alcohol 24691 (identified as Purified 8-2 Alcohol at receipt) 678397 08-Aug-2001 `The test article, 8-2 Alcohol, described as a white solid, was stored at ambient temperature. The Sponsoirs responsible for the determination and documentation of analytical purity, composition, and stabilityofthe test article. 22 Controls 221 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 090075 82 Alcohol 10 222 Positive Control Article `The positive control article used in this study was Cyclophosphamide (Sigma Lot No. 108H0S68, CAS No. 6035-19-2) ata dose levelof 60 mg/kg and a dose volume of 10 mL/kg. 23 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 10air changes per hour. A commercial diet, PMI Feeds, Inc. Certified RodentDiet #5002 (Pellets) and tap water were available ad libitum. The feed was analyzed by the manufacturer for concentrationsof 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 n 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. Table 1. T(armgget)Treament 2000 Dose Rangefinding Study Design DosimngiVgolume 2 MaNol.oef AnimFaelmsale 3 3 301 Rationale for Dose Selection Since no appropriate toxicitydatawas available (c.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 000078 $2 Alcohol 2 32 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 quantityof tes 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 volumeof20 mL/kg `The animals were dosed on 23-Oct-2001. A total of six animals, approximately weeks old at the timeofdosing, witha weight rangeof262 to 276 g and 188 to 195 g for the males and females, respectively, were used for this study. The weight variationofthe animalsdid not exceed 20%ofthe mean weight. Theanimals were weighed prior to dosing and dosed based pon the individual animal weights. All animals were dosed on an acute (one-time only) basis. Covance 22900-0-4540ECD 000079 82 Alcohol 13 33 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 durationof this study for toxic signs and/or mortality. 34 Results All animals appeared normal immediately after dosing and no signsoftoxicity were noted until the endofthe 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 000080 82 Alcohol 1 4 MICRONUCLEUS STUDY a Study Design The treatment regimen for the micronucleus study is shown in Table 2. e Table2. Micronucleuse Study Design e (Tanrgg:s2TNrsamehnati DosmingiVeolume No.oEfY ArmlsHarvestTimeopouirnes sim000 2000 20 0 ` 6 6 ` PV"eoTthsiecilneeumCCbooentrtooef,,rC0y.c5ei%phanoqisueaepohluasmd imrdoeeut,y6el0cvlegosiseshe SST210oF To555rlsFo rom`: Tae aL Rationale for Dose Selection Based on results of the dose rangefinding study, dose levelsof 0, 500, 1000, and 2000 mg/kg at a dose volumeof20 mL/kg were selected. Only males were used in the micronucleus assay, since both sexes appeared to respond similarly in the dose rangefinding study. a2 `Test Article Formulation, Administration, and Duration of Dosing The test article, 8-2 Alcohol, waasosld at room temperature. In order to ensure homogeneity, the test article was placed ina heated water bath (approximately 60 - 80C) until liquefied. The 100 mg/mL dosing concentrationof8-2 Alcohol was prepared by adding the appropriate volumeof the vehicle, 0.5% aqueous methylcellulose, to a preweighed quantityof test article and mixing to form a homogeneous suspension. Lower Covance 22900-0-4540ECD 000081 82 Alcohol 15 concentrations were achieved by diluting the 100 mg/mL dosing solution with vehicle control article. The formulations were stored at ambient temperature util dosing and stirred during the dosing procedure. The test article was administered once to six males/dose levelharvest timepoint by oral gavage at dose levelsof 0, 500, 1000, or 2000 mg/ke ata dosing volumeof20 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 totalof54 male animals, approximately weeks old at the timeofdosing, with a weight range of 222 t0 276 were used for this study. The weight variationofthe 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 dosedon an acute (one-time only) basis. 43 Clinical Observations and Mortality During the acclimation period, the ras were observed at east once daily for abnormalities in appearance or behavior, All animals were examined immediately after each dose, approximately 1 hour afr cach dose, and at least daly for the duration ofhis study for toxic signs andor mortality. 44 Extraction of Bone Marrow At the appropriate harvest timepoints, the animals were euthanized by CO; inhalation followed by incisionofthe diaphragm. The hind limb bones (ibias) were removed for Covance 22900-0-4540ECD 000082 ee ---------------- e -- 82 Aleohol 16 marrow extraction from the frst five selected surviving animals. For each anima, 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 collectionwere euthanized at the completionofthe study. 4s Preparation of Slides Following centrifugation to pellet the cels, the supematant was removed by aspiration and portionsofthe pellet were spread on slides and ar dried. The slides were fixed in methanol, stained in acridine orange, protected by mounting with coverslips, and analyzed under uorescent microscopy. For control ofbias, al slides were coded prior to analysis. 46 Slide Analysis Slides prepared from the bone marrow collected from the five animals per group a 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 numberofmicronucleated PCE 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 eriteria for the identification ofmicronuclei were thoseofSchmid (Schmid, 1976). Micronucle were darkly stained and generally round, although almond- and ring-shaped `micronuclei occasionally occurred. Micronucle had sharp borders and were generally between 1/20 and 1/5 the sizeofthe PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cel with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. Covance 22900-0-440ECD 000083 82 Alcohol 1" "The staining procedure permitted th differentiation by color of PCE and NCES (bright orange and ghost-like, dark-green, respectively). "The historical background frequencyofmicronucleated cells was expressed as percent micronucleated cells based on the numberofPCE analyzed. The historical background frequencyofmicronuclei in the Crl:CD*(SD)IGS BR strain in this laboratory is about 00-04% a Data Presentation "The mean percent micronucleated PCE and PCE:NCE ratio and thei standard errors are summarized by dose group forthe different timepoints. Individual animal data are also shown. Study data are presented in AppendiAx. Historical control data are presented in Appendix B. s. EVALUATION OF TEST RESULTS s1 Assay Acceptance Criteria S11 Acceptable Controls "The vehicle control group was within the historical control range, having les 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 ---------------- 82 Alcohol 18 512 Acceptable High Dose `The high dose reached the regulatory dose limit dose for this assay (2000 mg/kg). 52 Assay Evaluation Criteria Assay data analysis was performed using an analysis ofvariance (Winer, 1971) on untransformed proportionsofcls with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. Ifthe analysisof variance 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 foar positive response was the detection ofa 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 bothofthese responses was considered negative. Statistical significance was not the only determinant ofa 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 result of his study will be archived in the storage facilitiesof Covance-Vienna for a least 1 year following submission ofthe final report to the Sponsor. After the I-year period, the Sponsor may elect to have the aforementioned materials retained in the storage Covance 22900-0-4540ECD -- 000085 8-2 Aleohal 19 facilitesof Covance-Vienna for an additional periodoftime or sent to a storage facility designated by the Sponsor. 7 STUDY RESPONSIBILITIES Function Responsible Person(s) Study Director Gregory L. Erexson, PhD, DABT Tnlife Laboratory Supervisor Rebecca L. Axilbund, BS Postlife Laboratory Supervisor~~ Carol S. Spicer, BS 8 RESULTS 81 Mortality and Clinical Observations The test article, 8-2 Alcohol, produced no lethalityorsignsofclinical toxicity until the appropriate harvest imepoinis. 82 `MTiacbrloensu4c-l5eIunsdiEvviadluuaaltiDoanta()Appendix A ~Table 3 Summary Data, The test article, 8-2 Alcohol, was not cytotoxic to the bone marrow (i.c., no statistically significant decrease in the PCE:NCE ratio). 8-2 Alcohol did not induce a statistically significant increase in micronucleated PCES at anyof the dose levels tested. The positive control article, cyclophosphamide, induced statistically significant increases in micronucleated PCE, as compared to the vehicle control, with a mean and standard error Of 3.10% 029%. Covance 22900-0-4540ECD --test-- eseee ------------ 000088 8:2 Aleohal 20 9 CONCLUSIONS "The test article, 8-2 Alcohol, produced no signs ofclinical toxicity in male rats exposed up 0 2000 mg/ke, 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 evidenceofbone marrow cytotoxicity induced by the tes article. In the micronucleus assay, 8-2 Alcohol did not inducae statistically significant increase in micronucleated PCEs at anyofthe doses ested. Therefore, 8-2 Alcohol is considered negative in the rat bone marrow micronucleus assay under the conditionsofthis assay. 10. LIST OF REFERENCES Dunnett, 1955. cDounntnreolt,tJCAW.m SAtatmiuslttAispsloec.co(m1p9a5r5i)s;o5n0s:1p0r9o6c-e1d1u2r1e.for comparing several treatments with a Dunnett, 1964. Dunnett CW. 20482491. New tables for multiple comparisonswitha control, Biometrics (1964); Heddle et al, 1983. `HgeednodtloexiJcAi,tyH,iMtuetMat,iKoinrkRheasrt(1B98e3r);al.12T3:h6e1-i1n1d5u.ction of micronucleias a measure of Heddle et al, 1991. dHaemdadglee:JpAa,stC,ipmriensoentM,Ca,ndHafyuatsurhei, MEnvefaan.d MMioclroMnuutcalgeeinas(1a9n91i)n;de1xo8:f27c7y-t2o9g1e.netic Covance 22900-0-4540ECD o-------------------- 000087 82 Alcohol 2 `OOErgCaDni,sa1ti9o9n8.for Economic Cooperation and Development (OECD). Genetic toxicology: Cmhaemmmiaclailasn1e9r9y8t;hrNoo.cyt4e74m.icronucleus test. OECD Guidelines for the Testing of Schmid, 1975. `Schmid W. The micronucleus test, Mutation Res (1975); 31:9-15. Schmid, 1976. S(Ecdh)m, iCdheWm.ic(a1l97M6u)tTahgeenmsi:crPorniunccilpeluess taenstd fMoertchyotodgsenfeotriTchaeniarlyDseitse,ctini:onH,ol1l9a7e6ndVeorl,,A4., Plenum, pp. 31-53. Winer, 1971. Winer (1971) BSJe,cSotnadtisEtdiictailonP.rinciples in Experimental Design; McGraw-Hill, New York, eet ------------ Covance 22900-0-4540ECD 000088 8-2 Alcohol Appendix A. Experimental Data Tables -_-- -- Covance 22900-0-4540ECD. 000089 82 Alcohol 2 sTettyvi:l:255.020Ate Table 3. Micronucleus Test in Bone Marrow Cells Summary Data -- Male Rats eC)HTaie|| eoifMciooMnmiseerlmoIcctaSsSE. ReNsoeSrisceesnce contro Veide oseMe ake oso pee anon 126009 Lito roe craoomts ath | S102 asamp Tene omg 24h aoson a oman Lisi00s jr omg 240 an | orsi007 0900s Linio0s assim ovr 2naem oo1o00n03 Lis 1a0s008 N0sSCi6EaNa-non0rtm5a%gomesesercatuhantteoieccosomrelpnoin:dnignCgP-siCiiysllocognohtnuog,hAa0m2i00:101PCE-Polhromati cosy: Covance 22900-0-4540ECD 000099 8-2 Alcohol 2 Tabled. --_-- MDaitcaronucleus Test in Bone Marrow Cells ~ 24-Hour Harvest Individual Male Tes Avice: 8:2 Alcohol S-tu_dy No: 2900 e eTereatment --NAunm mimbeaelr eePCCESO2a000EPOCES rere PCCREaNtSiCE Venice Cont sume peaes 2' i 0 t10o1 136 @an6 1 i3s0 Positive Conrol CP 600mg a@0 aa 0os3t6 aiis 55 26s a 0076 076 Test Arce somes awn 20 as 0 rLoiss ss aasss. ii 111340 1000 mere s3ss 2s esi 2 1130s6 1s Ears 0' 1in2 2000mykg aals 4i an 2 019057 10 Quue 2i i13o6 -_-- -- #0.M5N% MPCC=E0s-.M5i%craoqnuueoluesatmeedthPyCleEl;ulNoCsEe-;NCoPr-mCoycclhorpohmossipehamitdheer;oycC,yEi-cPolyshromatie crthocyes -------------------- Covance 22900-0-4540ECD. a 000091 82 Alcohol = r-- Taeblre.e MDiact-- raonurcmlaenus Test i-- n Bone Marrow Cea lls ~ 4-Hour Har-- vest Individual Male es Avice: 52 Aleohel S_tud--yNo: 22900 emer NAvuimmbaelr PCBAo0n0PCEs PRCaENiCE -- ossne Gaanr i1 o11s0 aatsss 22 ast i i1n i Test Avice Some aans 2! asi a 01030 03 a1n6 3i 015500 1000 mers aa0 an 001 00s % oss aa00 s: 110ss 20mgie aaaxw 5: i 019480 125 Gaast 22 i1o5s a059 MNMPCC=0E5%40s00qsPoCuEs # NINPCEs-Micon methPyOiEashlNoCsEs-;NCoPr=mCayscohpohmaasnphaenirde;iPCcE Plyhvomatic array Covance 22900-04540ECD 090092 ---------------------------------------- 8-2 Alcohol 26 Appendix B. Historical Control Data --_---- Covance 22900-0-4540ECD 000093 ---------------------------------- 82 Aleohol 27 Table 6. Rat Micronucleus Historical Control Data ~ 1/2000 through 12/2000 FOOHLbEDowVhEHaInCeLECONTMRiOLnSin ANovssim N F%ROMMIC20R0O0NPUCCELSEPAETREADNPICMBAsL MEAMNAYLSESE, oass aos vBom PMCEEANCNERSAET.IO MALES o1sse asv7om hohe NMeisioma 003s0 o15ss Avxe oss0"vace ossvases CFoopsTnIaVpE oComNiTnRO0LS0mg bouts MMianxionn oS6s0 o5n) PCE = Fol homie eyhoxseyee; NCE ~ Nom2a26r000r0myote = Number olasmv0on Covance 22900-0-4540ECD 000094 A 8-2 Alcohol 2 Appendix C. Quality Assurance and Compliance Statements -_-- Covance 22900-0-4540ECD. eeeEE------------ 000095 82 Alcohol 29 QUALITY ASSURANCE STATEMENT 8:2 Alcohol: In Vivo Rat Micronucleus Assay The report has been reviewed by the Quality Assurance UnitofCovance Laboratories tRIhneceg.u,FloianotdiaocancnsodrPdaDarrntuc5ge8;AwditmthihentOihrsegtarGnaoitosiaodtniL(oanFboDfrAoar)tEoTcrioytlnPeor2ma1ciotcifcCteoh-(eoGpLUeP.rS)a.triCeogonudleaantoidfoDnFseevdaeesrlsaoeltpmfeorntth in (OECD) PrinciplesofGood Laboratory Practice, ENV/MC/CHEM (98) 17; the Environmental Protection Agency (EPA-TSCA), Title 40 of the US Code of 2F9ed,e1r9a8l3R[ergeuvliastiiononesffPeacrttiv7e9S2,epitsesmubederNo18v,em19b8e5r])2;9a,n1d9a8n3y (aepfpfleicctaibvlee aDmeecnedmmbeentrs `The and following inspections were study director management. conducted and Written status the findings reported to the Study Director reportsof inspections andfindingsare issued to Covance management according to standard operating procedures. T Inspection Dates T Phase DasDRireectoorwadndeStsudwy~y~ Auditor Director Management 22:0ct:2001 Protocol Review 22002001 J Howard 06-Nov-2001 Slide Preparation 06-Nov-2001 S. Ballenger 03-Dec-2001 Draft Report Review 04-Dec-2001 P. Ciceres 21-Dec-2001 Revised Draft Report Review 21-Dec-2001 P. Ciceres I2d5F -oJnan2i -02000n 22 al FinalRRepoe rt Rep viewo~~r2t525J-JnRa2n-02e 00022 vi P.P.Ce Caicceerrw eess ; Representative, Quality Assurance Unit orf28j02 Dae Covance 22900-0-4540ECD 000096 ee 82 Alcohol 30 STUDY COMPLIANCE AND CERTIFICATION (TGhLePd)esrcergiubleadtisotnusdayswsaets fcoornthduicnttehde iFnocoodmpalnidaDncreugwiAtdhmitnhiesGtroaotdioLnab(oFrDaAt)orTyitPlreac2t1iocefthe aU.nSd. DCeovdeeloofpmFeendter(aOlERCeDgu)laPtriinocnisplPeasrotf58G;otohde OLragbaonriastaotriyonPrfaocrtiEcce,onEoNmVi/cACMo-Co/pCerHaEtMion C(9o8d) e17o;ftFheedEenrvailroRnemgeunltaatlioPnrsotPeacrtti7o9n2A,giesnscuyed(ENPoAv-eTmSbCeAr),29T,it1le98430(oeffftehcetiUvSe aDpepcliecmabbleeram2e9n,dm1e9n8t3s.[reTvhiesiroenweefrfeecntoivdeevSieatpitoenmsbferrom1t8h,e 1a9f8o9r]e)m;enatnidonweidthreagnyu.lations othrettheestsirgesnueldtsp.roTthoceorlatwhadtawtaouhladveafbfeeectn trheeviinetweegdribtyyofthethSetsutduydDyiorrectthoer,inwtheroprceetrattiifoinesof `tchoatnctlhuesieovnalwuiatthiionnothfetchoentteesxttoafrttihclee asstupdryesdeenstiegdn haenrdeienvarleuparteisoenntcsriatneriaap.prAolplritaetsteand hcoanstrboelenrerseulvtisewinedhibsyrtehpeorSttuardey sDuirpepcotrotr.ed by an experimental data record and this record Astlatnhdoaurdgsh,tthheetaesctcusruabcstyaonftcehechadraatcatiesriczoantsiiodner(eIdSOsu9f0fi0c1i)enwtafsornottheppeurrfpoorsmeesdofuntdheirs GstLudPy. c`Tohnecednotsraitnigonp.repTahraetipornoscewdeurree nuostedanbaylytzreadinfeodsrtsatfafbiltiotyp,rehpoamroegetnheeidtoys,inogr saoclcuutriaocnys of ensured: Tahnaelyatciccaulrabcayloafncceonaccecnutrraatetitoontbhreeceaudseecitmhaeltepsltacseusbsatnadnctehewaveshiwceliegihnedwhoincahnit iwnacsresmuesntpse.nded was aceurately measured in a flask graduated in 1-mL Hopmorotgieonnsewietrye breecmaouvseedthfeormdioxsteuraedsmiwneirsetrsattiirorne,d aprnidor to dosing and while Stakebpitltiotyambiecnaiumseumth(eatpipmreoxbiemtawteeelny d1ohsoeurp)r.eparation and administration was Covance 22900-0-4540ECD 000097 3585 82 Alcohal a -- e E elalree Slesdon.rmSetc eTcoonry SoakediBe Covance 25000451080 Testing Facility Management Tomo EgatzerLM3b GAesnseotciiactaenDdiMreoclteocrular Toxicology ol 28 0n be Report Reviewed and Accepted for EL du Pon de Nemours and Co. by: Gerad Kennredyeeset Sponsor Study Monitor -- Bae VEiaeDonneer, Pr Sponsor Technical Project Monitor BPae resi Covance 22900.0-4540ECD 000098 ee