Document 10qmZ4ypzQXVEejerL25YvY5m

c 0 V A-N--C--E@ THE DEVELOPMENT SERVICES COMPANY Covance LaboratoriesInc. P.O. Box 7545 Madison, Wisconsin 53707-7545 Packages: 3301 Kinsman Bouleva Madison. Wisconsin S3704 Tel:608/241-4471 Fax: 608/241-7227 THE AMERICAS Sponsor: St.Paul.Minnesota FINAL REPORT Study Title: 5-Dallv Dose Dermal Absorption/ToxicityStudv of T-6684 in Rabbits Author: F. Bud W. McDonald Studv Completion Date: December 1,1997 Testing Facility: Covance LaboratoriesInc. -')30K1insman Boulevard Madison, Wisconsin 53704 Testing FacilityProjectIdentification: Covance 6)29-200 EUROPE Page I of 52 ASIA/PACIFIC AFRICA QUALITY ASSURANCE STATEMENT Covance 6329-200 Thisreporthasbeenreviewedby theQualityAssuranceUnitofCovance Laboratories Inc.i,naccordancewiththeFood and Drug Administratio(nFDA) Good Laboratory Practiceegu ations, e o owing inspectiownesrecon ucte an in ini-!s reportedtotheStudy Directorand management. Writtenstatusreportsof inspectionasnd findingsareissuedtoCovance management accordingtostandardoperatin2procedures, Inspection Dates From To 04/15/97 05/05/97 05/21/97 07/14/97 04/15/97 05/05/97 05/21/97 07/15/97 Phase ProtocolReview Dose Administration ProtocolAmendment Data/ReportReview Date Reportedto Study Directorand Management 04/'5l/97 05/05/97 05/21/97 07/15/97 RepreserdatQiu@ael'i,tAyssuranUcneit /Z 7 7 '5ate 2 STUDY IDENTIFICATION 5-DailyDose Dermal Absorption/ToxicitSytudy of T-6684 inRabbits Covance 6329-200 Test Material Sponsor Sponsor's Representative Study Director TestingFacility Study Timetable Study InitiatioDnate Experimental(In-lifSet)artDate In-lifEend Date ExperimentalTerminationDate Study Completion Date T-6684 1)i@vi Toxicology Services Medical Department ')M Center,Bldg.2'-10-2E-02 P.O.Box ')-')220 St.Paul,NIN 551')3-)220 Rozer G. Perkins.PhD, DABT 3M Toxicology Services Medical Department ')M Center.Blda. "-@'-)0-2E-02 P.O. Box ')3220 St.Paul.MN 55 1 (61'-7'3)3-3222 F.Bud W. McDonald Co%,ance LaboratoriesInc. P.O. Box 7545 Madison, Wl 53707-7545 608.242.7901 Co%-ance LaboratoriesInc. ')')OKlinsman Boulevard Madison,Wl 5)704 April 18,1997 May 5, 1997 June 2, 1997 June 2, 1997 December 31, 1997 3 Acute Studies StevenM. Glaza Manager F.Bud W. McDonald Studv Director JeffreyB. Hicks In-lifSeupervisor Rose M. Bn'dge AdministrativeSupervisor Toxicology Support Kathy N/lyers Mana(yer CalvinL. Horton Supervisor KEY PERSONNEL Covance 6329-200 Laboratory Animal Medicine Donna J.Clemons, DVM Diplomate,ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Deborah L. Pirkel LaurieSchuller Supervisors Necropsy Qualitv Assurance Nancy M. Centanni Manaizer 4 CONTENTS Covance6329-200 QUALITY ASSURANCE STATEMENT ........................................2................... STUDY IDENTIFICATION ...................................................3................... KEY PERSONNEL ...........................................................4.................. SUMMARY .................................................................7................... OBJECTIVE .................................................................9.................. REGULATORY COMPLIANCE ...............................................9................... TEST AND CONTROL MATERIALS ...........................................9.................. Identificati.o.n.............................................................9.................. Purityand Stabilit.v.......................................................1.0................... Storageand Retention......................................................1.0................... SafetvPrecautions.........................................................1.0................... TEST SYSTEM .............................................................1.0................... TestAnimal ...............................................................1.0................... Housing ..................................................................1.0................... Animal Diet...............................................................I.I................... SelectionofTestAnimals ...................................................I.I................... Study Design ..............................................................I.I................... JustificatiofnorSpeciesSelection............................................I.I................... PROCEDURES .............................................................1.2................... Preparationof Exposure Area ................................................1.2................... Dose Administration.......................................................1.2................... Reason forRoute ofAdministratio.n.........................................1.3................... ObservationsofAnimals ....................................................13................... Blood Sample Collections/Shipmen.t.........................................I.................... Pathology ................................................................1.4................... Shipment of Bile and Tissues ........................................................1.4...................... StatisticAanlalyses ........................................................1.4................... Locationof Raw Data,Records,and FinalReport...............................1.4................... 5 ----C-o-vance@6329-200 PESULTS ...................................................................................... 15 Body Weights ................................................................................ 15 ClinicalObservations........................................................................... DerrnalIrritation 15 Pathology ................................................................1.5................... 16 DISCUSSION ................................................................................... 16 SIGNATURE ...............................................................1.7.................... R-EFEP,ENCE .................................................................................... 17 TABLE I Individuaalnd Mean Body Weights (a).....................................1.8.................... IndividualClinicaSligns .................................................1.9.................... IndividualDermal IrritatiSocnores........................................).0.................... Group I (Control-)DimethylSulfoxide(0.0mg/kg/day).....................)..o................ 4 IndividualDermal IrritatiSocnores........................... )I Group 2 -T-6684 (').m0g/kg/day)...............*.".*.*.'*.*.'.*.*.*.'.*.*.**.*.*.".*........).I.................... APPENDIX A III IndividualAnimal PathologyData...............................................2,..)................... APPENDIX B .................................................................................... 35 ProtocolDeviation........................................................3.6...................... ProtocolTP6798 ..........................................................3.7..................... Amendment No. I To The Protocol...........................................5.1..................... 6 SUMMARY Covance 6329-200 This study was done toassessthesystemicabsorption/toxiciatnyd relativsekinlititancv ofT-6684 when appliedtotheskinof rabbitsforfiveconsecutivedays. The study was conducted usingthreemale and threefemale acclimatedrabbitsof the Hra:(NZW)SPF strainforeach treatmentgroup. Group I (Control) Control/ Dose Level TestMaterial (mg/kg/day) Dimethyl O.Oa Sulfoxide T-6684 .Oa Number of Animals Mates Females 3 3 3 a Administered at a dose volume of 0. 1 mL/kg of body weight. The back of each rabbit was clipped free of hair and a single dose of the respective controlor testmaterialwas administeredtotheskinof therabbitsfor5 consecutivedays. The treatmentsitesrema'ned intact.The areaof applicatiownas covered witha -,auze patchsecured with papertapearound alledi,,easnd overwrapped with Saran Wrape and Elastoplast,t@a'pe to providean occlusivedressingforeach approximate 2-)'-hourexposure period. Clinicalobservationswere conductedpredoseon Day I onlyand atapproximately1.2.5, and 4 hours aftereach controlortestmaterialadministratioonn Days I through5. Additionalclinicaolbservationswere conducteddallythereaftetrhroughDay 29. Mortalitycheckswere conductedtwicedailyon Days 2 through29 (a.m.mortalitycheck onlyon Day 29). Body weightswere determinedon Day -5 forrandomizationpurposes, beforethe firsctontrolor testmaterialadministratio(nDay 1),and atthescheduled sacrificienterva(lDay 29).The initiadlermal irritatiroenadingwas made beforethe first controlor testmaterialadministratio(nrecordedas theDay I reading).Subsequent readingsof dermal irritatiwoenre made aftereach patchremoval on Days 2 through6 and on Day 13. A blood sample (approximately4 mL) was collectedfrom eachanimal on 7 Covance6329-200 Days 8, 15,and 23. Inadditiona,tthetimeof necropsv(Dav 29).approximately20 to 40 mL of blood was obtainedfrom each controlanimaland approximately20 mL of blood was obtainedfrom each Group 2 animal. All samples were centrifugedand separatesamples of serum and cellularfractionwsere obtainedand sentfrozenon dry ice tothe Sponsor. On Day 29,theanimalswere anesthetizewdith sodium pentobarbital. bled viatheposteriorvena cava,exsanguinatedand necropsied.The whole liverb,ile,an approximate I-cm x I-cm sectionof thedermal applicationsite,and bothkidneys (collecteads one sample)were collectedfrom allanimals.weighed (volume oniv determined forbile)a,nd sentfrozentothe Sponsor. Applicationof T-6684 did notresultinany testmaterial-relatcehdanizesinbody weight ,ainor macroscopic findingsatnecropsy.All animalsappeared normal throughoutthe studywith theexceptionof one Group I animaland two Group 2 animalswhich had decreasedfeedconsumption forone tothreedays withinthe firsntinedavs of thestudv. Slightdermal irritatiwoans observedinone controlanimal while sil2htomoderate dermal irritatiwoans observedintheotherfivecontrolanimalstreatedwithdimethyl sulfoxide(DMSO). In theanimalstreatedwithT-6684,slightdermalirritatiwoans observed inone animal while slightomoderate dermal irritatiwoans observed inthe otherfiveanimals. No macroscopiclesionswere seen inany oftheanimalsatnecropsy. nr 8 OBJECTIVE Covance 6329-200 The objectiveof thisstudywas toassessthesystemicabsorption/toxiciatnyd relative skinirritancoyf testmaterialswhen appliedtotheskinof rabbitsforfiveconsecutive days. REGULATORY COMPLIANCE This study was conducted inaccordancewiththeUnited StatesFood and Drug AdministrationGood LaboratorvPracticeRegulationsforNonclinicalLaborator-v, Studies,21 CFR 58.withtheexceptionthatanalysisof thetestmaterialmixturesfor concentrationh.omogeneity/solubilitayn.d stabilitwyas not conducted.All procedures used inthisstudywere incompliancewith theAnimal Welfare Act ReLulations.Inthe opinionofthe Sponsor and studydirectort.hestudvdid notunnecessarilyduplicateany previouswork. All proceduraltimespresentedinthisreportfallwithintheacceptable rangesas specifiedintheCovance LaboratoriesInc.(Covance) StandardOperating Procedures(SOPs). TEST AND CONTROL MATERIALS Identification The materialswere identifieadnd describedasfollows: Identification PhysicalDescription T-6684 (test) off-whiteliquid DMSO (control) Clearcolorleslsiquid Dimethyl sulfoxidewas manufacturedby MallinckrodtChemical. 9 Covance 6329-200 Purity and Stability The Sponsor assumes responsibility for test material purity and stabilitydeterminations (includingunder testconditions).Analysis of the testmaterialmixtures for concentration.homogeneity/solubility,and stabilitwvas not conducted. The purity and stabilityof the controlmaterialwere considered to be adequate for the purposes of this study. Storage and Retention The control and testmaterialswere storedat room temperature. A reserve sample of each the control and testmaterialswere taken and stored in a freezerset to maintain a temperature of -20'C IO'C. The controlmaterialreservesample will be retainedat Covance for one year. The testmaterialreservesample was sent to the Sponsor. Any unused testmaterial will be returnedto the Sponsor. Any remaining controlmaterialma,., be used for other testingand will not be discarded afterissuance of the finalreport. Safet-,P,recautions The controland testmaterialhandling procedures were according to Covance SOPs and policies. iF-- TEST SYSTEM Test Animal Adult albino rabbitsof the Hra:(NZW)SPF strainwere received from Covance Research Products Inc.,Kalamazoo. Michigan on April 16, 1997. Housing After receipt.the animals were acclimated fora period of at least7 days. During acclimation and throughout the study,the animals were individuallvhoused in suspended stainlesssteelcages. Environmental controlsfor the animal room were setto maintain a temperature of 16 to 22'C, a relativehumidity of 50% t20%, and a 12-hour light/12-hour 10 Covance 6329-200 dark lightingcvcle.In caseswhere variationfsrom therequiredtemperatureand humidity conditionsexistedt,heywere documented and consideredtohave had no adverse effecton thestudyoutcome. Animal Diet The animalswere providedaccesstowaterad libituamnd a measuredamount of LaboratorvRabbitDietHF #5')26,PMI Feeds,Inc.The feedisroutinelyanalyzedby the manufacturerfornutritionaclomponents and environmentalcontaminants.Samples of the water arepen'odicaliavnalyzed.There were no known contaminantsinthefeedor water atlevelsthatwould be expectedtointerferweithoraffecttheresultsof thestudy. Selectionof Test Animals The animals were identifiebdy animal number and correspondingeartagand were placed intostudy groups usinga stratifibeoddy weightrandomizationprogram. The randomizationbody weightswere obtainedon Day -5. Study Design Animals weighing from 2,067 to2,500 izand approximately15 weeks of aizeatinitiation oftreatmentwere placedintothefollowingstudygroups: Group Control/ Dose Level Dose Volume Number ofAnimals Test Maten*ai (mg/kg/day) (mL/kg/day) Males Females I (Control) Dimethyl 0.0 0.1 Sulfoxide T-6684 1.0 0.1 3 13 Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of the largeamount of background informationon thisspecies. of choice because PROCEDURES Covance6329-200 PreparatioonfExposureArea On thedaybeforethefirsctontroolrtesmtateriaalpplicatitohne,backand,ifnecessary (toobtainunblemished skin),the flanksof each rabbitwas clippedfreeof hairwithan electriclipper.The clippedareamade up approximately20% ofthetotalbody surface area.The testsites(intacstkin)were inspectedforinterferinlgesionsi,rritatioonr. defectsthatwould precludetheuse ofany oftheanimals.The animalswere clippedas needed throughoutthe study(Days 3,6.13 and 29). Dose Administration All animalsreceivedfiveconsecutivedays ofadministratioonf therespectivecontrolor testmaterial.The firsdtay oftreatmentwas desianatedas Day 1. Group 1. An individualdose was calculatedand measured based on each animal's body weight on the firsdtay of treatment.The controlmaterial(dimethylsulfoxide) was appliedtothetestsiteata dose volume of0.1 mL/kg ina thinand uniform laver. Group 2. An individuadlose ofthetestmaterialmixture(T-6684 and dimethyl sulfoxide)ata concentrationof ')0mg/mL was calculatedand measured based on each animal'sbody weighton thefirsdtay oftreatment.The testmaterialmixture was appliedtothetestsiteata dose volume of0.1 mL/kg of bodv weight ina thin and uniform layer. Each areaofapplicationinGroups I and 2 was coveredwith a 4-piv5-cm x 5-cm -,auze patch. Each gauze patchwas securedwith papertapearound alledges and overwrapped with Saran WrapD and Elastoplasttla&petoprovidean occlusivedressing.Collarswere used torestraitnheanimalsduring eachapproximate23-hour exposure period.One Group 2 male was found withoutitscollaron atthea.m.mortalitycheck on Day ').The collarwas placedback on theanimalatthistime(seeprotocoldeviationspage). Approximately 23 hours aftereach controlortestmaterialapplicationt,herestraining collarsand patcheswere removed and any residualmaterialremoved from theapplication sitesusingtap water and disposablepapertowels. 12 ReasonforRouteofAdministration Theden-narlouteisa potentiraolutoefexposurienhumans. Covance6329-200 ObservationosfAnimals ClinicaolbservatiownesreconductepdredosoenDay Ionlyandatapproximate1l.y2.5, and 4 hours aftereach controlor testmaterialapplicatioonn Days I through5. Additionalclinicaolbservationswere conducteddailythereaftetrhrough Dav 29. Mortalitychecks were conductedtwicedailyon Days 2 through29 (a.m.mortalitycheck only on Day 29). Body weightswere determinedon Day -5forrandomizationpurposes.beforethefirst controlortestmaterialadministratio(nDay I).and atthescheduledsacrificienterval (Day 29). The initiadlermal irritatiroenadingwas made beforethe firsctontrolortestmaterial applicatioanccordingto I theDralze technique(recordedas theDay I reading). Subsequent readingsofdermal irritatiwoenre made within')0to37 minutesaftereach patchremoval (Days 2 through6) and on Day I Blood Sample Collections/Shipment A blood sample (approximately4 mL) was collectefdrom a marginalearveinofeach animal on Days -3.8, 15.and 2'). Inadditiona.tthe timeofnecropsy(Day 29), approximately20 to40 mL ofblood was obtainedfrom each controlanimal and approximately20 mL of blood was obtainedfrom each Group 2 animal viathe posterior vena cava. All samples were storedatroom temperatureuntilcentrifuged.After centrifugations.eparatesamples of serum and cellularfractionwsere obtainedand stored in a freezersetto maintaina temperatureof -20'C -IO'C untiIshipped tothe Sponsor. The serum and cellularfractionsamples obtainedon Days -3.8,and 15 were shipped frozen(on dry ice)totheSponsor (JamesD. Johnson.')M E.T.& S,Bldg.2-3E-09,9)5 Bush Avenue. St.Paul,NlN, 55106) on Day 16. The serum and cellulafrractionsamples obtainedon Days 23 and 29 were shippedto theSponsor theday aftertheexpen'mental (in-lifed)ateofterminationinthesame manner asthesamplesobtainedpriorto Day 2-'1. The Sponsor isresponsiblefortheretentionand dispositioonfthesamples. Covance 13 Covance 6329-200 does not acceptany responsibilitfyortheanalysisof thesamples collectedinthisstudy nor aretheseresultspresentedin thisreport. Pathology At terminationofthe in-lifeexperimentalphase (Day 29),animalswere anesthetizewdith sodium pentobarbitalb,led viatheposteriorvena cava.exsanguinated.and necropsiedin random order.The sitesofcontrolortestmaterialapplicatiownere washed with warm tapwater beforethenecropsyprocedure.Allanimalswere subjectedtoan abbreviated (-,ronsescropsy examinationand any abnormalitiewsere recorded.The whole liverb,ile. an approximate I-cm x I-cm sectionof thedermal applicatiosnite,and bothkidneys (collecteads one sample)were collectedfrom allanimals,weighed (volume only deter7ninedforbile)a,nd placed ina freezersettomaintaina temperatureof-20'C t 10 Afternecropsy.theanimalswere discarded. Shipment of Bileand Tissues One week afterin-lifeexperimentalterminationt.hetissues(whole liversd,er-Mal applicatiosnitesa.nd kidneys)and bilewere sentfrozen(on dry ice)tothe Sponsor (James D. Johnson.')M E.T.& S.Bldg.2--')E-0993,5 Bush Avenue. St.Paul,NIN. 55106).along with documentationof theircorrespondingweightsor volumes. The Sponsor isresponsiblefortheretentionand dispositioonfthesamples. Covance does not acceptany responsibilitfyortheanalysisof thesamplescollectedinthisstudynor are theseresultspresentedin thisreport. StatisticaAlnalyses No statisticaanlalyseswere requiredby theprotocol. Location of Raw Data, Records, and FinalReport The raw data,records,and an originalsignedcopy of thefinalreportwillbe retainedin thearchivesof Covance in accordancewith Covance SOP. 14 RESULTS Covance 6329-200 Body Weights Individualand mean body weightsare n Table I- All animals,with theexceptionof one controlanimal.exhibiteda weightlossfrom randomizationtoinitiati(oinn-lifweh)ich can be attributetdotheacclimationoftheanimalstorestrainincgollarsconducted for 3 days (approximateiv21-2'3hours/day)beforetheinitiaclontroland testmaterial administrationA.ll animalsgainedweight from Day I toDay 29. ClinicalObservations IndividualclinicaslignsareinTable2. All animalsappearednormal throughoutthe studywith theexceptionof one Group I animal and two Group 2 animalswhich had decreasedfeedconsumption forone tothreedays withinthefirsntinedays of thestudy. Dermal Irritation Individualdermal irritatisocnoresareinTables ')and 4. Slighterythema.edema and fissuringreactionswere observed inone controlanimal while slightomoderate ervthema and edema and slightdesquamation reactionswere observed in theotherfive controlanimalstreatedwithdimethylsulfoxide.Of thesefivecontrolanimals,two animals alsoexhibitedslightcoriaceousnessreactions.In the animals treatedwith T-6684. one exhibitedslighterythema.edema and fissurinrgeactionso.ne exhibited slighterythema and slightomoderate edema reactionso,ne exhibitedslighto moderate erythema and slightedema and fissurinrgeactionso,ne exhibitedslighttomoderate ervthema and slightfissurinrgeactionso.ne exhibitedslighttomoderate erythema and edema and slightdesquamation and coriaceousnessreactionsa.nd one exhibitedslighto moderate erythema and edema and slightdesquamation.coriaceousnessa.nd fissuring reactions. 15 Covance 6329-200 Pathology Individualanimal pathology data (tissueweights, bilevolumes, and gross pathology observations)are presented in Appendix A. No macroscopic lesionswere observed in the animals at necropsv. DISCUSSION The acute systemic absorption/toxicityand relativeskin irritancyof T-6684 was evaluated in male and female albino rabbitswhen administered dermally for five consecutive days. Application of thismaterialdid not resultin any testmaterial-related changes in body weight gain or macroscopic findingsat necropsy. All animals appeared normal throughout the study with the exception of one Group I animal and two Group 2 animals which had decreased feed consumption for one to threedavs within the firstnine days of the study. Slightdermal irritatiownas observed inone controlanimal while slightto moderate dermal irritatiownas observed in the other five controlanimals treated with dimethvi sulfoxide.In the animals treatedwith T-6684. slightdentialirritatiownas observed in one animal while slightto moderate dermal irritatiownas observed in the other five animals. 16 F. Bud W. McDonald Studv Director Acute Studies SIGNATURE Covance 6329-200 _13 11-7 Date REFERENCE 1. Dralze.J.H.. "Acute Dermal Toxicity(SingleExposure),"In: Appraisal of the Sqfel@, of Chemicals in Foodv, Drugv and Cosmetics - Dermal Toxicitv,Association of Food and Drug Officialsof the U.S., pp. 54-56 (1959). 17 Table I Individual and Mean Body Weights (g) Covance 6329-200 Animal Number Males Random -Ization Day 1 '?9 Animal Number Females Random -ization Day 1 29 Group I (Control)- Dimethyl Sulfoxide (0.0mg/kg/day) F6')Ol 1 F63012 F6')O 1 2,-'1)5 2,430 2.447 Mean 2,)97 2.277 2,500 2.)88 2,529 '-)7,6 6 2,708 2.)88 2.668 F6-'1017 F6')018 F63019 2.424 2.')80 2.2 6 -') 2,-'3)-') 2.755 2,277 2,78') 2.244 2.619 Mean 2.)56 2.285 2,719 Group 2 - T-6684 (3.0 mg/kg/day) F63014 F63015 F6')016 2.404 '-?4,8 5 2,447 Mean 2,445 2.402 2,444 2.)90 2,722 '-',754 2,805 F63020 F63021 F 6 -'0)2'-' 2.262 2.4')8 95 '@).412 2.760 Mean 2.365 2,067 2,429 1 2.506 2.804 7 ')5 2.272 2.682 18 T2ble 2 IndividualClinicalSigns Covance 6329-200 Animal Number Sex Observation 'Day I Hour 1 2.5 4 Day 2 Day 3 Day 4 Day Hour Hour Hour Hour 1 2.5 4 1 2.5 4 1 2.5 4 1 2.5 4 6 7 Day(s) 8 9 10-15 16-29 Group I (Control)- Dimethvi Sulfoxide(0.0mg/kg/dav) F63011 @M Appeared normal F63012 M Decreased food consumption Appeared normal F63013 M Appeared normal /V --- --- --- --- --- o' V F63017 F Appeared normal F63018 F Appeared normal 1:63019 F Appeared normal eV vv Group 2 -'1'-668(43.0mWkg/day) 1:63014 @M Appeared normal F63015 M Appeared normal Decreased tood consumption f:63016 Ni Appeared normal V - - - - - - - - - -/ V VV F63020 F Appeared normal F63021 Decreased t*ood consumption F Appeared normal F63022 F Appeared normal --- --- -- - a Each animal appeared normal priorto controlor testmaterialadministration. Condition existed. Condition not evident. 19 Covance 6329-200 Table 3 IndividualDermal IrritationScores Group 1 (Control)- Dimethyl Sulfoxide(0.0mg/kg/day) Dermal Reaction Ervthema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Er-,,thema Edema Atonia Desquamation Coriaceousness Fissuring Males Study Dav 1 2 34561 Animal No. F63011 0001 222 000 1 0000000 000000 1 0000000 0000000 Animal No. F6)012 01 11 1 11 00000 1 1 0000000 0000000 0000000 000000 1 Animal No. F6')0 1 0 1 1 1 12 1 000 12 1 0000000 00000 0 1 00000 00 00 000 00 Females Study Day 1 2 3 4 5 6 13 Animal No. F6)017 00 1 000 1 22 1 0 00 000 0 000000 1 00001 00 000 000 0 Animal No. F6')018 01 1 1 1 01 1 1 00 0000 0 000000 1 00000 1 0 00 000 00 Animal No. F63019 01 1 - -- 011112 00 000 00 00 000 0 1 00 00000 00 00000 qr 20 Table 4 IndividualDermal IrritatioSncores Group 2 - T-6684 (3.0mgAkg/day) Covance 6329-200 Dermal Reaction Males Studv Day 1 2 3 4 5 61 Animal No. F6'10 14 Erythema Edema Atonia Desquamation Coriaceousness F i' ssuring 001 1 1 1 1 00 1 1 220 0000000 0 000000 00 00000 0000 00 0 Animal No. F63015 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 01 11 1 1 1 001 1 11 1 0 000000 00 0 00 00 0000000 0 00000 1 Animal No. F6')0 16 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 01 1 1 121 0000 1 1 1 0000000 0 00000 1 00 00000 0000 00 0 Females Stud,-Day 1234 61 Animal No. F6-')020 00 1 2 22 1 000 1 2 2 1 000 00 00 0 00000 1 00 000 1 0 00000 1 0 Animal No. F6')021 01 22222 0 1 1 1 I- 1 00 00 000 00 000 0 1 00000 1 0 0 000 00 0 Animal No. F63022 00 1 1 22 1 0 0 1 1 1 '-, I 000 00 00 0 000 00 0 0000 000 000 000 1 21 APPENDIX A IndividualAnimal Pathology Data Covance 6329-200 I I t !i t I v 22 zzzz W LaW (4 C4 F .I z @4 0 E. cz - coo EW4x, 00 z- '141 . . 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U 0 @44 -o La cn 0u cn rA0 w .4 cn Ul z Cf) Cl)> 0 C4 0 WOEW 0 1=4 W (D -, C-3 M Wz a.@ INF@ w E. 3 iz > .3 Z IM X 10, xr4wH: E- F- (n 5440 26 14 0 w m C4 zzzz 14 W W W @4 x Y. .14 4444 uz C4cnC) 0 0M o:c E-Z 0 m W 0 . L, J p LIZ3 @C 0 Z En Z -- > :4 0w L44 I:=1,=4 0U ID WW -0 w z m:t f- E- Zz L4 X o@ 0 C4 L4 uw LQl 14 w m w E-4 E- 0 C4 W uCXl lu cn CL EL @. 0 =)cn Ecn. 0 00 C4 U .3 0, w 0 n 1 I0n CL 11, U0 ce.E0 0 C) 0 0 14 w 0 ol r@ m Lf) W . LI) - Z , @; : - C) 00 cn r4 W. 0 'WI 0 ul 0 U) cn 0 w ..i M 00@ W) mw >ca lm En 0 r. 0 t3)_: c: 5 0 ux Cn > 0 ix F" u0 C)Owz. low w 0 ru w 0w w C.D z c3x r@ @ . . .10 ru w r.3 H F- cn 440 0 C3 0. IM EW- In w 4 -0 > Z Law m w 0> 0 @4.3im : z 14 r4 0 z w IDD@ZW 00 27 > 0 L) > r. 0 0 4)U tn r.0 .r. .U) L0) m x zzzz wwww c@ Z @4 Y. @4 W. Lo 4444 w cil0 f- .Hzo w loaco. I wo C) z -.WI C) w U3 CzO 40 CA z zw >Z :r r4 co w c@ .0 @ L'ZW z 03 In .. .. .. E14i o C4 CX 3 F- Zz 0 C4 W3 C-0 0 > L.3 > < ci@r4 r4 L) L,3 Emx L4 10, U) ID 14 LCat , 0 :r tn x 1, C4 0 . . ... .. 14 E. -C 0 1 . I :0 0 ED 0 C) 0 0 00 U0) @ W 0 0 U) = : 0 k- r4 0 0 @3 < cn cn r,j3 IDCo 04 0 H U) : IL CL 0-.o co w M 0 Cl w l0y 1>4 th cn IX 0u U3 L,) 0U UZ Ix xI 0 im > a, 4 w @ ioxF@ @ r4 u 0 00 z ca 0 r4 . x W E@ In r4 a. Y. C6 1< 0 Z > r4 W C, Z U3 W Cl > -3 0 C3 f- E- 'A 440 IZ a. x .,o L) O,z mW 0 28 ID u r_ re > CP 0 CL ul E4 C4 E- uz zzzz w 94 C4 L4 z Coco 0 WWWW tz z L.]> Z 01, :@ 0 c:l .4 M L.) - 4- z z C) 0 L) - CD w C4 L4 z w.:r Ly. 1 U) cn k- oc 0m 0-- a) u In tn r. 0 .@4 01 u0 x 4z > L,3 a@ C) L4d LD w c C4 (L C'/'E)O ZI C) E=.- O:c cn 00 E 0 ca L4 17 0 o (D U3 L'i0 0 Ecy 9 IL cl. L.1 0 L. r.3 Di 0 r- OD 0 L4 cn U. m >@ @ 4 c:i 17, XDL4 E- cq W CQ 09 CF,0 (h 0 0 En !2 : 0n Cl z -o. rA C4 u0 0 Ua , (A uz cr.' L.1 > co 14 0 ou > ,t CIO @UO o C4 Z L. C',w ow w E- x C4 0 z C3 X @lW C) co : C4 C.3 0> @ w %,a zw > a @-4n EUl E0 UJ :U Z 5 :w 0 ,Z C) lm 29 r4 1 u > 0 u 0 CL ai a > u C: 0' ,3 (D u 0m ux a r4 C4E@ zzzz C4 w w @4 @4 @4 44 4 -C H E- k- k- c@@ 0 U Z .4 < @ -- - - - - 0 L%lE.3LO C4 U) Z r4 > :r L4 ct >. @4 0 co r4 Ld C L,3 [4 w ce.:r L4 go 0 f- :,: 2 C4 cx 0 Ecn La x w E-. @C U Wc C4 < @. 14 L. E- C) 141). (4 ce X 0 U 3: C, 0 cn E:3. :K C) 0 2 > C) H CD co ()0 0 u C) E- w L) En E4 0 0 0 Cl) ,:1 0 1- w CL CL 14 cn cn ua 0 zz C) r"@ L4 >x x W. 0 L) x Ll 0 C4 0 0 z 0M In 0 Ix 0w L) iL L4 cnU)!@ .@ UZ, > co IC40 IZ) r- Cf)> u0 00 Z, w r4 u co z MX 10,@ @@ 1 W 14 E@ F0 4m iL U IE4- U, C, 4 O@ E Z > x Ed 0 z CQ : 0 :Z W C4 00 30 rm > 0 L) m 0 a I x ,a (L) m CL > w a 0 tn r_ 0 wu ,.m 0m ux z ZZZZ UZ a@ 0 Z 0 uz 0 F'n 'D U3 W :r X :C 3: 0 Z ul z 1 11 on om co w 1-114 :D 4@ LO)I-) 0 w C4 w 0 W 0 I-.@. :: ::: X .. . C.)W Z:t 0 . z 0 Z 0 C, F-IJ. > Li LLJ L4 L'Z Z C. 9 ID 0, L4 XH H > WX x Mo u W Ix- C, IL44 U3 =) 0 L-34 E- :wr 3" (L 0 im (Do 0 0 L7] cn , 0 0 0 (D 0 UJ r4 0 0 E- 0 U3 iL W0 Z W0 "Z 4 D >4 r- 0 W-. E- 0 C4 C4 Ln En 0 (d ..I M 0@x Im, 0 3 CD >W y >'EX4 E4 cn UO @W CL Z 0w U 0u od w UZ 0 a) ri) > lox E- u0 oowz ul %D W IL Y. w 0 C. w : 1. ow , 4 0 1 .. .. = Z W. 0 wM4-w: D] a w ulC4 ilaa > OX, w 0 0-0@e4m caUl E- E. E- cn 440 0 a ial L) z C4 0 u Z E4 z14 0 31 0 cn u 0= 0 u m c 0 u x LiZ r,3 ri)C) zzzz cl) W Ed W C4 E. U Z .I < m co 0z m W> Z C) L4 n F. CQ -.1< , , 0r2) m9 0"(4w,o u C4 'A 0 0 C-4 E4 u2 c4 , ,u : : : : 0 1:1 E- cl) -ctZ C4 > w C) L4 0 w4 141 3: C 0 Ix, 00 IL I (D = W@ : CD 0 E@ 0 H C4 u -I x cn C4 0 'A x 0 F. w cz 4 : 3. a c)m o H u) 142 r.30 m 0 ol X :D- cn 0 ux 0 0 C4 u(A IL 0u E4 c/) > z nx 10, 440 in co o zww 0> n C4 @IU Z ED 32 (D 14 E14 U) C) 00 <UZM :40 0 c@@ an w m U) E- Ln ZZZZ C4 W W E&3 L.3Ul W :c 3: :r .14 0 1:1 E. t) c In 0 wu CD r_ 0 cn, 0 ct x . . . . . -4@ : @. 2 a- 14 0 [4 w: - Lu Z) 0 14 4 @. CL .3 C:l.3 0 cl 0 'a 00 :0 C) 0 ou L.3 U n0 1(4n C4 w 14 0 @ L'i , E- 0 M ..Dm X =)@ W E- rn ril0 w -.4 0 LC@ CK 0 >. La W EY 1) .0 E4 cf) ou -XW: uz .4 m cn cn > a. 0 E. uw C) owz C. W 0 U=l ca co ca w E- 3 a z Cl X ,01, @ -w F- H ot 4 0 C/I z .4 ww :4 C4 0 > ct , 0 - 0 @4..3 03 La 10 u z 0 Zw 0a 33 ucu Z Z > I o:c 4 4 4 Z 0 (Z W.0 - W ;7 4 .44 M.T 3:r o cL E E :rE4 cx 0 Z co Ed o@ -0 ci o;C 4z UO -0 W E4 L) L,3 Ln E. LI) w 3: 0 L) LL, in x Zml , IE--@,<1 0 ul a L,4U1= L4 m L% H 14 c> 2 c@ 0 Ul 0 H0 CD 0 0 E- mo 1c4n C=, c@ 0 k- L4 E- m oce 0 u) u) u) c W 0 c@ 11 4z w0 o)x L4 L4 Ln 0 cn Oll 0 CD Y. 14 w. 0u ELI c)io6 C) L) D>l oz clio U0 cow= -z ww .. I w o r& L% 0w r4 w F. 2: m 4 " c4 EWE-M, mox: z .4 LO, 14 H F- cn 440 ol r4 CL* 04 Z wz 0, 0>.2: w:w.:I- 0 IZ x o3 E4 0 Z: w Z 0 APPENDIX B ProtocolDeviation ProtocolTP6798 Amendment No. I To The Protocol Covance 6329-200 35 Protocol Deviation Covance 6329-200 Protocol Actual Procedure Page 7, 7. Experimental Design, B. Dose Administration, (3) Dose Administration, Ninth Sentence: The rabbitswill be collaredduring each approximate 2)-hour applicationperiod. Animal No. F6'30 16 (Group 2 Male) was found without itscollar on at the a.m. mortalitycheck on Day'). The collar was placed back on the animal at this time. The patch appeared to have remained in place on the applicationsite This deviation is not considered to have had an adverse effecton the outcome of the study. 36 Covance 6329-200 c 0 V A44-C@E@ Covence Laboratories ln@ P.0, Bo. 7545 Mad.son. Vsconsi- 53707-7545 P..kagOS: 3301 Kinsman Boulevard madison. Wisconsin 53704 Tel: 608/241-4471 Fax: 608/241-7227 Sponsor: 3im St.Paul,Minnesota PROTOCOL TP6798 Study Titic: 5-Dail\D.ose Dermal Absorption/ToxicitSvtud@of T-6684 inRabbits Datc: April18.1997 TestingFacility: Covance LaboratoriesInc. 3301 Kinsman Boulevard i@vladisoWni.sconsin 53704 TestinaFacilirPvrojectldentirication: Covance 6329-200 TWE AIAERICAS EUITOPE ASIA/PACIFIC AFRICA 37 Covance 6329-200 Covance 6329-200 TP6798 Page 2 STUDY IDENTIFICATIOLN 5-Daiiv Dose Dermal Absorption/ToxiciSttuydy ofT-6684 inRabbits Covance No. Testiviaterial 6329-200 T-6684 Sponsor 3 i%@l Toxicology Services Medical Department 3,1@Clenter, Bldg. 220-2E-02 P.O. Box 33220 St.Paul,iviin55133-3220 Sponsor's Represcntati%-e Roger G. Perkins,PhD, DABT 3 i'vl Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St.Paul.MN 55 133-3220 (612) 733-3222 Studv Director Testing Facility F. Bud W. '@-IcDonald Covance Laboratories Inc. P.O. Box 7545 Madison, Wl 53707-7545 608.242.7901 Covance LaboratoriesInc. ')')OKlinsman Boulevard Madison, Wi 53704 Proposed Study Timetable Experimental StartDate Experimental Termination Date Draft Report Date Week Week Week of May 5, 1997 ofjune 2. 1997 ofjuly 14, 1997 38 Covance6329-200 Covance6329-200 TP6798 Pa@o3e 1. Studv 5-Daily Dose Dermal Absorption/ToxicityStudy in Rabbits 2. Purpose To assess the svstemic absorption and toxicityand relativeskin irritancyof a test material when appliedto the skin of rabbitsforfiveconsecutive days 3. Regulatory Compliance This studn.%,@ilble conducted inaccordance with the following Good Laboratory Practice Regulations/Standards/Guidelines%viththeexception thatanalysisof the testmaterial mixtures forconcentration.solubilityh,omogeneity, and stabilitywill not be conducted: Conduct as a Nonregulated Stud,, [X] 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30 (Final()OECD) 59 NohSan No. 3850 (Japanese MAFF) NotificationNos. 313 and 870 (Japanese IMOHW) All procedures in thisprotocolare incompliance %%,itthhe Animal kk'clfarAect Regulations. In theopinion of the Sponsor and study director,the studn.does not unnecessarily duplicateany previous work. 4. Quality Assurance The protocol,study conduct,and the finalreportwillbe auditcd by the Quality Assurance Unit in accordance with Covance LaboratoriesInc.(Covance) Standard Operating Procedures (SOPS) and policies. 5. Test iNl2terial A. Identific2tion T-6684 B. Physical Description Off-white liquid 39 Covance 6329-200 Covance 6329-200 TP6798 Page 4 C. Purit.v and Stability The Sponsorassumes responsibiliftoyrpurityand stabilitdyeterminations (includinugndertestconditions)S.amplesof testmaterial/vehicmiiexture(s) forconcentration,homogencitv/solubilitya,nd stabilitavnaivseswill not be taken. D. Storage Room temperature E. Reser-%-eSamples Resen,e sample(s) of each batch/lotof testmaterialwillbe taken forthisstudy. The testmaterialreservesample(s) %%,ilble storcdat Covance in a freezersetto maintain a temperature of -20'C : IO'C untilretumed to the Sponsor after completion of the in-lifephase of thestudy. F. Retention An%- unused testmaterialwill be returned to the Sponsor. G. S2fCt-%. Precautions As requiredby Covance SOPs and policies 6. Control Nlaterial A. Identification DimethN.1Sulfoxide(DMSO) B. Ph%-sicalDescription Clear. colorlessliquid C. Purit-%a.nd Stability To be documented by Covance (informationfrom the supplier) D. Storage Conditions Room temperature 40 Covance 6329-200 Covance 6329-200 TP6798 Page 5 E. Reser%,e Samples Reserve sample(s)ofeach batch/lotofcontrol materialwill be taken. T'hecontrolmaterialreservesample(s) willbe storedat Covance in a freezerset to maintain a temperatureof-20'C i lOoC. F. Retention Any remaininq controlmaterialmay be used forother testingand willnot be discarded afterissuance of the finalrepori. G. Safety Precautions As required by Covance SOPs and policies 7. Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(\'Z%@@SPF/Covance Research Products Inc. (3) Age at Initiation Adult (4) Weight at Initiation 2.0 to 3.0 kg (5) Number and Sex 6 males and 6 females (6) Identification Individualnumbered ear tag (7) Husbandry (a) Housing Individually,in suspended stainlesssteelcages 41 Covance 6329-200 Covance 6329-200 TP6798 Page 6 (b) Food A measured amount of Laboratory Rabbit Diet HF -,"5326(Pi'l-II Feeds, Inc.).The food isroutinelyanalyzed by the manufacturer fornutritionalcomponents and environmental contaminants. (c) Water Adlibittinftrom an automatic system. Samples of the water are periodicallyanal@-zedfor specifiedmicroorganisms and environmental contaminants. (d) Contaminants There are no known contaminants in the food or .%-atetrhat would interferewith thisstudy. (e) En%-ironment Environmental controlsfor the animal room will be setto maintain a temperatureof 19'C to 23'C, a relativehumidity of 50% -20%. and a 12-liourlight1/2-hour dark c,.-cleT.he dark cycle may be interrupteddue to in-lifperocedures. (f) Acclimation At least7 days Selectionoftest Animals Based on health and body weight according to Co,.-anceSOPS. An adequate number ofextra animals willbe purchased so that no animal inobviously poor healthisplaced on test.The animals will be placed intostudy groups using a stratifiebdody weight randomization program %vithinnine days of study initiation. (9) JustificatfioorSnpecieSselection HistoricatlhleNye,w ZealanWdhitealbinroabbihtasbeentheanimalof choice because of the largeamount of background information on this species. 42 ;r7 f- Covanc6e329-200 Covanc6e329-200 TP6798 Page 7 B. Dose Administration (1) Test Groups Qr= I (Control) 2 Control/ Test 'vlaterial Dose Level (m-/k-c/dav DNISO T-6684 0.0* 3.0* NumberofAnimal LiaLc.@ Females 3 3 3 3 To be administered at a dose volume of 0.1 mL/kg of body .Neight (2) Preparation of Exposure Area On the day beforethe initiatlestand controlmaterial application.the back and, ifnecessary (toobtainunblemished skin),the flanksof each rabbit%villbe clippedfreeofhair with an electriclipper. The shaved area %villconstituteapproximately 20% of the totalbody surface area. The treatmentsites(intactskin)will be inspectedfor interferinglesions, irritationo,r defectsthat %-ouldpreclude the use ofany ofthe animals. The animals %%-ilble clipped as needed throuchout the studv. (3) Dose Administration All animals %%-ilrleceive ft%-consecutive days of administration ofthe respecti%-ceontrolor testmaterialapplied to the same respective applicationsiteeach day. The testmaterial/vehiclmeixture willbe prepared fresheach day of administration.The firstday oftreatment Aill be designated as Day 1. The controlmaterial willbe applied undiluted at a dose volume of 0.1 mL/k-g. The Group 2 testmaterialwill be diluted with DMSO and appliedata dose volume of 0.1 mL/kg. The doses for the animals in Groups I and 2 willbe based on theanimal's body weight taken on Day I beforethe firstadministrationand appliedto the area of exposure in a thinand uniform layer.The area of application(Groups I and 2) %villbe completely covered with a 4-ply 5.0-cm x 5.0-cm gauze patch. All patcheswill be secured with paper tape around alledges and overN,Tapped with Samn Wrap* and Elastoplast'tape to provide an occlusive dressing. The rabbitswillbe collaredduring each approximate 23-hour applicationperiod. The prepared testmixtures willbe storedat room temperature untiladministration. 43 4A Covance6329-200 Covance6329-200 TP6798 Page 8 (4) Reason for Route of Administration The dermal route isa potentialrouteof exposure in humans. (5) Removal oftest LNIaterial Approximately 23 hours aftereach testor controlmaterialapplication, thepatches and collarswillbe removed and the residualmaterial will be removed using water or an appropriatesubstance,ifnecessary. The sites of testand controlmaterialapplicationwillbe washed with warm tap water on Day 29 priorto thetissuecollectionprocedure. C. Obscr-%'2tionof Animals (1) ClinicalObservations Before controlor testmaterialadministrationa,t approximately 1,2.5, and 4 hours aftereach administrationof controland testmaterial on Days 1-5 forclinicalsigns,dailythereafterforclinicalsigns.and twice daily (a.m.and p.m.) for mortalitystartingon Day 2 and continuing to Day 29 (a.m. mortalityonly on Day 29). Observations may be extended %vhen directed by the study director. (2) ReadingofDerM21 Irritation Before the initiaclontrolor testmaterialadministration,the der-Mal irritatiorneading willbe made and recorded as the Day I reading (Attachment 1). Additionaldermal irritatiorneadings willbe made within 30 to 60 minutes aftereach patch removal (Days 2-6) and on Study Day 13. Individualdermal irritatiornecords %villbe maintained for each animal. (3) Body Weights For randomization, before the initiatlestor controlmaterial application (Day 1),on Day 29, or at death (when survivalexceeds I day) (4) BloodSampleCollections (a) Frequency Predos(eanytimferomuptofourdaysbeforteheinitidaolse administrationto Day 1),on Days 8, 15,22, and atexperimental termination(Day 29) 44 AMR Covance 6329-200 Covancc 6329-200 TP6798 Page 9 (b) Method of Collectioni/Number ofAniM21S Blood samples (approximately 4 mL each) willbe collectedfrom a marginalcarveinofallanimalsattheintervalpsredosethrough Day 22. From the posteriorvena cava,approximately 20 mL of blood will beobtainedfrom eachanimalsacrificeidna moribundcondition (ifpossible).approximately 20 to 40 mL of blood %villbe obtained from each controlanimal sacrificedon Day 29 (themaximum volume possible %%-ilble obtained),and approximateiv 20 mL of blood willbe obtained from each Group 2 animal sacrificedon Day 29. The blood samples willbe stored atroom temperature and then centrifuged,and the separateserum and cellularfractionsstored in a freezersetto maintain-20*C t IO'C. The serum and cellular fractionsobtained through Day 15 %villbe sent frozen on dry ice to the Sponsor one to t%vo%veeks priorto in-lifetermination. The serum and cellularfractionsobtained afterDay 15 @.vilble sent frozen on dry iceto the Sponsor .vithionne %veckafterin-life termination. The Sponsor isresponsibleforthe retentionand dispositionof the samples. The serum and cellularfractionsamples %%illbe shipped to: James D. Johnson 3i@vEi.T. & S Bldg. 2-3E-09 935 Bush Avenue St. Paul, '@fiN55106 James D. Johnson or hisalternatewillbe notifiedregarding the shipment of the samples. D. Pathology (1) Unscheduled S2cririceSand Deaths Any animaldying duringthestudyor sacrificeidna moribund condition will be subjected to an abbreviatedgross necropsy examination and all abnormalitieswillbe recorded. Animals ina moribund condition willbe 45 Covance 6329-200 Covance 6329-200 TP6799 Page 10 anesthetize%dvithsodium pentobarbit(avliainjectioinnthemarginalear vein),bled via the vena cava, and exsanguinated. The whole liver,bile (allavailable)a,n approximate 1-cm x 1-cm sectionof the dermal applicationsiteand both kidneys (collectedas one sample) willbe collectedfrom allanimals dying during the study or sacrificedin a moribund condition,weighed (volume only determined for bile),and placed intoa freezerset to maintain a temperature of -20'C t IO'C. After necropsy, the animals %villbe discarded. (2) Scheduled Sacrifice On Day 29, the animals %%-ilble anesthetized .%-itshodium pentobarbital (viainjectionin the mareinal ear vein),bled via the%-cnacava, exsanguinated, and subjected to an abbreviated gross necropsy examination. The animals willbe necropsied inrandom order (via computer-generated random numbers) and allabnormalities%villbe recorded. The whole liver,bile(allavailable)a,n approximate I-cm x 1-cm sectionof the dermal applicationsite.and both kidneys (collected as one sample) %%-ilble collected,%vcit!hed(volume only determined for bile),and placed intoa freezersetto maintain a temperature of-20*C =10'C. After necrops%,,theanimals will be discarded. (3) Tissue Sample Shipment The samples (liver,bile,dermal applicationsite.and kidne,,.sc)ollected at the scheduled sacrifice%villbe sent frozen on dry iceto the Sponsor %vithinone week- aftercollection.Samples collectedat unscheduled sacrificesand deaths (ifapplicable)willbe sent with and in the same manner as the samples from thescheduled sacrifice.The samples and theircorresponding weights or volumes willbe shipped to the person listedin Section 7.C.(4).(b).The Sponsor isresponsibleforthe retention and dispositionof the samples. E. StatisticalAnalyses No statisticaalnalyses are required. 46 Covance6329-200 Covance6329-200 TP6798 Page II 8. Report A finalreportincludingthose items listedbelow willbe submitted. Description of the testand controlmaterials Description of the testsystem Procedures Dates of experimental initiatioannd termination Tabulation of mortalitydata by sex and dose level Descriptionof any toxiceffects/dermalirritation Tabulation ofmean body %veightsby sex and dose level Gross pathology findings Gross pathology repori(ifrequestedby the Study Director) Individualanimal tissue %-eiehtasnd bilevolumes 9. Location of Ra%v Data, Records, Rescr%-cSample(s), and Final Report Original data,or copies thereof,%%-ilble availableat Covance to facilitataeuditing the study during itsprogress and before acceptance of the finalreport. When the Finalreportiscompleted. controlmaterialrescne samplc(s),alloriginalpaper data, including those items listedbclo%vwillbe retainedin the archives ofcovance for a period ofone ),carfollo%vinesigningofthe finalreport.One year aftersigning of the 1-inarleport,allof the aforementioned materialswillbe sentto the Sponsor and a return fee %%-ilble charged. The Sponsor may electto ha%c the materials retained in the Covance Archives foran additionalperiod of time and Co,.-ance%%-Iilcharc!ea storal,,fcee. Ifthe Sponsor chooses to have Co,6,ancedispose of the materials,a disposal fee %%,ilble charged. Protocol and protocolamendments Dose preparation records In-liferecords Body weights Randomization data Dose administration Observations Anatomical pathology records Sample collectionrecords Shipping records Study correspondence Final report(originasligned copy) 47 Covance 6329-200 Covance 6329-2DO TP6798 Page 12 The foilo%kinsgupportingrecordswillberetaineadtCovance butwillnotbe archived%%iththestudy data. Animal receipt/acclimationrecords %%latearnalysisrecords Animal room temperature and humidity records Refrigeratorand freezertemperature records Instrument calibratainodnmaintenanrceecords 48 wI PROTOCOL APPROVAL Covance 6329-200 Covance 6329-200 TP6798 Page 13 Roc'erV. Perkins,PhD, DABT Sponsor'sRepresentative 3m i@,@ 4)1@ @ F. Bud W. NlcDonald StudyDirector Acute Studies Covance LaboratorieIsnc. 5", Representative Quality Assurance Unit Covance LaboratoriesInc. Date @-/R-q7 Date Ll-If @"7 Date 49 -.0 Covance6329-200 Attachment I Scoring Scale for Acute Dermal Reactions Covance6329-200 TP6798 Pa@-e 14 Er%,thema 0 - None I - Slight 2 - I@toderate 3 - Se,,-ere Edcma 0 - None I Sti@iit(bareivperceptibleto %%elldefined by definiteraising) @%toderate(raisedapproximateln- I mm) 3 - Sc%-ere(raisedmore than I mm) Atonia 0 - None I - SliLht(slightimpairment ofelasticity) 2 - %loderate(slow returnto normal) I@larked(no elasticity) Desquamation 0 - None I - Slight(slii!hstcaling) 2 - ',.toderat(cscalesand flakes) 3 - @viarked(pronounced flakine,%-itdhenuded areas) Coriaccousness 0 - None I - SliQht(decreasein pliability) 2 - Moderate (leatherytexture) 3 - Marked (tough and brittle) Fissurina, 0 - None I - Slicht(definitceracks inepidermis) 2 - Moderate (cracksin dermis) 3 - Marked (crackswith bleeding) 50 Covance6329-200 C 0 V A-9-CE. t.1OlvatoomEmr Sta@cas Co..." AINIEND,NIENNTO. ITO THE PROTOCOL Covance LaboratoriesInc. P@0.BOX 7545 Madison. Wimonsin 537O7.7S45 P&Ck:::S: 330t Kinsman Mad, , Wi"ons.n T.I: 609/241-4471 Fa.:604/241-7227 53704 PROTOCOL TP6798 5-DailyDose Dermal AbsorptionrroxiciSttyudy of T-6684 inRabbits Covancc 6329-200 Sponsor Testing Facilit% 3,,M ToxicologyServices NtedicalDepartment 3M Center,Bldg.220-2E-02 P.O. Box 33220 St.Paul,NfiN55133-3220 Sponsor's Representative Co%,ancc LaboratoriesInc. 3301 Kinsman Boulc%,ard Madison,Wi 53704 Stud%,Director Roger G. PerkitisP.liD,DABT F.Bud %k'.',IcDonLild This amcndnient niodifictshefollowingportionsoftheprotocol: Effecti-.N-leav 20, 1997 'rhcobscr%-ailocfc Memorial DaN coincideswithDa),22 of thestud\.T(ia-o.idiia%-itnog conduct the Day 22 bleedson a holida@,m,odifv thefolio%vintgwo sectionsot'thc protocoltoniove theDay 22 bleedstoDay 23. I. I'ame8, 7.ExperimentalDesign,C. Obscr%-ationof Animals,(4)Blood Sample Colicctions(,a)FrequcncN,.Modifv thissectionwiththefollo%vinuenderlined change: Predose(anytimefrom up tofourdaysbeforethe initiadlose administratiotno Day 1).on Days 8, 15,21,and at experimentii terminatio(nDay 29) TNE AMERICAS EUROPE ASIA/PACIFIC AFRICA 51 Covance 6329-200 Amendment No. I Covance 6329-200 TP6798 Page 2 2. Page 9, 7. Experimcnt2i Desion, C. Observation of Animals, (4) Blood Sample Collections, (b) Method of ColicctiontNumber of AniM215. Modify the first paragraphinthissectionwith thefollowingunderlinedchange: Blood samples(approximatel4ymL each)willbe collectefdrom a marginalear veinof allanimalsatthe intervalpsredosethroughDay 21. PROTOCOL ANIEND.NIE.N'TAPPROVAL IT.-g@@-(FcrkinsP,liD,DABT Date Sponsor'sReprescntatiN-c Bud W. %IcDonal(P' Stud%,Director Acute Studies Co,.,ancLcaboratoriesInc. /,- L'@ - prcsentati%,c QualityAssuranceUnit Covancc LaboratorieIsnc. Date q7 Date 52 1,11i1rlicliendpcunbtl.ichie.lvdcompann, iii)%,cr15 coLintr@CSdtido%,er30 ot-t-ices @vorld%v]LI%Cv.ithfic@id(luirtcirnsPrinceton,Neiv istheiiiarkctlnn,i,me forCo%,anceInc. ,IIIiLtI@ lroutidthe %%,orldt,he principil' trti:i,tedon riiilsi@igeT.he USe L)t- iiitiiir,cll()rrett'crt@oone or more @)t- tlic,icti@i)thcrLlhll(il,lrlcs. THE AMERICAS P,,n(@eton 1 800.773,0011 EUROPE Brusseis ,32.2773 29 10 ASIA/PACIFIC S nqaccre -65 774-1233 COVANCE LABORATORIES Maci,sorINI USA 888 5,ilLABS 5227 v,-r,naV.A. USA 800-742-8378 Harroqate. UK 01 1 44 1423 50001i Munster Germany 01 1 49 251 97960 COVA-MCE- THE DEVELOPMENTSERVICESCOMPANY Si,,IpillSgolutions