Document 93O6bL78qE9Ga4ndeXbom3yje

C 0 V AV -C@-E.@ THE DEVELOPMENT SERVICES CO#APANY Sponsors: APME Ad-Hoc APFO ToxicologyWorking Group PROTOCOL Study Mde: 26-Week CapsuleToxicityStudywithAmmonium Perfluorooctanoa(tAePFO) inCynomolgus Monkeys Date: September 23, 1998 PerforniingLaboratory: Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704-2595 Laboratory Study Identification: ProposalNo. w6806a Covance 6329-231 Covance 6329-231 Page 2 Study 26-Week Capsule ToxicityStudy with Ammonium Cynomolgus Monkeys Perfluorooctanoate(APFO) in Purpose To assessthe effectof the testmaterialon criticaelnzyme levels,hormones, and other selectedbiochemical parameterswhen administereddailyby capsuleto cynomolgus monkeys for atleast26 weeks Sponsors APME Ad-Hoc APFO Toxicology Working Group Study Monitor Paul Lieder,PhD, DABT 3M Toxicology Services Building 220-2E-02,3M Center St.Paul, Minnesota, 55144-1000 Telephone No.: 612.737.2678 FacsimileNo.: 612.733.1773 AlternateStudy Monitor John Butenhoff,PhD, DABT 3M Telephone No.: 612.733.1962 FacsimileNo.: 612.733.1733 Sponsor's Representative David Farrar,PhD ICI Chemicals & Polymers Limited Occupational Health The Heath, Runcom, PO Box 13 Cheshire,WA7 4QF Telephone No.: 01928.513953 FacsimileNo.: 01928.515146 Study Location Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Covance 6329-231 Page 3 Mailing Address: PO Box 7545-2595 Madison, Wisconsin 53707-7545 Study Director Peter J.Thomford, PhD Covance LaboratoriesInc. Telephone No.: 608-241-7207 Facsimile No.: 608-242.2736 Toideologist Dale Aldridge,BS Covance LaboratoriesInc. Proposed Study Timetable Experimental StartDate: September 29, 1998 In-LifeStartDate: September 29, 1998 In-LifeEnd Date: July 2, 1999 Audited DraftReport Date: October 22, 1999 Experimental Termination Date: To be determined Regulatory Compliance 'Mis study willbe conducted in compliance with theEnvironmental ProtectionAgency Good Laboratory PracticeRegulations as setforthin Title40 of theUS Code of Federal Regulations,Part792, issuedNovember 29, 1983 (effectivDeecember 29, 1983),and with any applicableamendments exceptthatbileaciddeterminationsdone by Universityof Dundee willnot be done in compliance with GLPS. Animal Care and Use Statement All procedures in thisprotocolare incompliance with the Animal Welfare Act Regulations,9 CFR 1-4. In theopinion of the Sponsor and study directort,he study does not unnecessarilyduplicateany previouswork. Quality Assurance The protocol,study conduc@ and finalreportwillbe auditedby the Covance Quality Assurance Unit (QAU). The proliferatiocnellnuclearantigenevaluationdata and report willbe auditedby the QAU of Pathology AssociatesInternationalT.he serum, urine, feces,and liverA.PFO analysisand reportwillbe auditedby the QAU of 3M Environmental Laboratories.The blood hormone determinationsand reportwillbe audited by theQAU of Ani LyticsIncand DuPont, as appropriate.The receptorlevel determinationsand reportwillbe auditedby theQAU of DuPont. Bileacid determinationsdone by the Universityof Dundee wiE not be audited. Test Material Covance 6329-231 Page 4 Identification Ammonium Perfluorooctanoate (APFO) Lot Number ne lot number will be maintained in the raw data. Purity Responsibility of the Sponsor Stability Responsibilityof the Sponsor Storage Conditions At room temperature Characteristics Informationon synthesismethods, composition,or othercharacteristitchsatdefinethe testmaterialison filewith theSponsor. Gelatin Capsules Capsules (SizeNo. 2) obtainedfrom Torpac,Inc.(FairfielNde,w Jersey);lotnumber will be suppliedby themanufacturer. Gelatincapsuleswillbe storedatroom temperature.A copy of theCertificatoef Analysis provided by themanufacturerwillbe maintained in the data- Reserve (Archive) Samples A reserve sample of each lotof testmaterial (I g each) will be taken and stored at room temperature. These samples willbe transferredto the Sponsor aftercompletion of the in-lifpehase. Disposition of Test Material Any remaining testmaterialwillbe returnedafterauthorizatiofnrom the Sponsor. Animals Species Cynomolgus monkey Covance 6329-231 Page 5 Source Covance Research Products Inc. Age at Initiationof Treatment Young adultladult Weight at Initiationof Treatment 3 to 5 kg Number and Sex 22 males Identification Collar tag Husbandry Housing Individual;animals willbe housed in suspended,stainlessteelcages. Diet Certifiedprimatediet(#8726C, Harlan Teklad) once or twicedaily.The dietis routinelyanalyzedby the manufacturerfornutritionaclomponents and environmental contaminants. Resultsof specifiednutrientand contaminant analysesare on fileat Covance-Madison. Fruit,vegetables,or othersupplements may be provided but will not requireanalysis. Water Ad libitum.Samples of the water areroutinelyanalyzed for specifiedmicroorganisms and environmental contaminants. The resultsareon fileatCovance-Madison. Contaminants There are no known thisstudy. contaminants in the dietor water atlevelsthatmight interferewith Environment Environmental controlsforthe animal room willbe setto maintain 18 to 29*C, a relativehumidity of 30 to 70%, and a 12-hourlight/12-houdrark cycle. Acdimation Minimum of 4 weeks Covance 6329-231 Page 6 Randoniizadon Animals willbe weighed,stratifibeydbody weight and allocatetdothenumber of blocksequaltothenumber ofanimalstobe selectefdoreach group. Animals ineach blockwillthenbe assignedtogroupsusingcomputer-generaterdandom numbers. Justification APFO isaknown hepaticperoxisomeprohferato(rPP)in therat When exposedto PP, nonhuman primates(suchasthecynomolgus monkey) respondsimilarltyohumans (i.e.l,ow to no hepaticresponse)and thereforaerean appropriatheuman surrogate species. Group Designationsand Dosage Levels Group I (control) 2 (low-dose) 3 (mid-dose) 4 (high-dose) Dose Izvel (mg/kg/day) oa 3 10 30 Number ofMales 6b 4 6b 6b a The controlgroup(Group 1)willreceivempty gelaticnapsules. b Two animalsinGroups 1,3,and 4 designatedasrecoveryanimalswillbe treatefdor26 weeks,thentreatmentwillbe discontinueda,nd theanimals willbe observedforreversibilipteyr,sistencoer,delayedoccurrenceof toxiceffectfsoratleast13 weeks posttreatmentB.ased on plasma test materialevelsand clinicaplathologyfindingst,herecoverymay be reducedorextendedatthediscretioonf thestudydirectorafter consultatiowniththe Sponsors. Dosing Procedures Method ofAdniinistration Orallyby gelaticnapsulesd,aily(7days/week)foratleast26 weeks Reason forDosing Route To compare withpreviouslyconductedtoxicologystudiesusingtheoralroute Covance 6329-231 Page7 Dose Preparation Capsules will be prepared at leastweekly. The size and number of capsules will depend on the physicalcharacteristicosf thetestmaterial,thedose level,and the weight of the monkey. Dose levelswillbe based on thetestmaterialas supplied.Individualdaily doses willbe based on the most recentindividualbody weights,with the exceptionof body weight collectiodnays when thepreviousbody weight willbe used. All dose preparationswillbe storedatroom temperature untildosed. Dose Analysis Because the testmaterialisnot being mixed with a vehicle,dose analyses are not required. Observation of Animals ClinicalObservations Each animal willbe observed twicedaily(am. and p.m.)formortalityand moribundity; findingswillbe recorded as theyare observed. Each animal willbe observed dailyand food consumption willbe assessedqualitatively; abnormal findingswillbe recorded.Once weekly, each animal willbe observed; abnormal findings,or an indicationthatthe animal isnormal willbe recorded. Additionalfindingswillbe recordedas they are observed. Body Weights Weekly before initiatioonf treatment,Day -I (forthe Day I dose calculations),otnhe firstday of treatment,and weekly thereafter OphthaliWe Exandnations Before initiatioonf treatmentand beforeeach scheduledsacrificeT.he anteriorportion of the eye, opticmedia, and ocularfundus willbe examined using an indirect ophthalmoscope. ClinicalPathology Frequency Unscheduled Collections When possible,blood willbe collectefdorclinicaplathologytestsfrom animals sacrificedatunscheduled intervals Covance 6329-231 Page 8 Scheduled Collections Before initiatioonf treatment;and (beforethedailydose) afteratleast30, 60, 90, and 180 days of treatment;and afteratleast30, 60, and 90 days of recovery Number of Animals All Method of Collection Animals willbe fastedovernight;blood willbe collectedfrom a femoral vein. Anticoagulantswillbe sodium citratfeorcoagulationtestsand potassium EDTA for hematology tests.No anticoagulanitsrequiredforclinicaclhemistrytests.Urine willbe collectedon wet iceovernight(approximately16 hours). Tests Hematology red blood cell(erythrocytec)ount plateletcount hemoglobin white blood cellOeukocyte) count hematocrit mean corpuscularvolume differentibalood cellcount blood cellmorphology mean corpuscularhemoglobin reticulocytceount mean corpuscularhemoglobin concentration Coagulation activatedpartialthromboplastintime prothrombin time fibrinogen ClinicalChemistry glucose urea nitrogen creafinine totalprotein albumin globulin totalbilirubin directbilirubi(niftotalbilirubiins greaterthan 2.0mg/dL) cholesterol triglycerides alanineaminotransferase a&Aine phosphatase aspartateaminotransferase gamma glutamyltransferase sorbitoldehydrogenase creatinekinase calcium inorganicphosphorus sodium potassium chloride bileacids amylase bpase pancreatic-specifiacmylase Covance 6329-231 Page 9 Urinalysis appearance volume (approximately16 hours) specificgravity pH protein glucose ketones bihrubin blood microscopicexamination of sediment urobilinogen Blood Hormone Determination Frequency Tbrw times beforeinitiatioonf treatment;afteratleast30,60, 90,and 180 days of treatment;and afteratleast30, 60, and 90 days of recovery Number ofAnimals All Method of Collection Animals willnot be fasted;blood willbe collectedfrom a femoral vein (an alternative vein may be used ifnecessary).Approximately 6 mL of blood forplasma samples willbe collectedintotubes with potassium EDTA as theanticoagulant. Approximately 6 mL of blood forserum samples willbe collectedwithout anticoagulantand allowed to clot.Blood samples willbe maintainedat room temperatureuntilsenim isharvested. Blood samples willbe maintained chilleduntil plasma isharvested. Serum Sample Handling Samples for serum willbe centrifugedwithin I hour aftercollectiona,nd serum will be harvested.Serum willbe divided intotwo approximatelyequal aliquotsand storedin a freezersetto maintain -60 to -80'C untilpacked on dry iceand shipped to: Dr. SarojDas Ani LyticsInc. 200 Girard Street,Suite200 Gaithersburg,Maryland 20877 Telephone No.: 301.921.0168 FacsimileNo.: 301.977.0433 Ani LyticsInc.willbe notifiedregardingshipment of samples. Covance 6329-231 Page 10 Serum Tests Samples willbe analyzedby Ani LyticsInc.,forestradiole,stronee,striolt,hyroid stimulatinhgormone, totaalnd freetriiodothyroniMn)e, and totaland freethyroxin (T4).Resultswillbe providedforinclusioinnthefinalreport. Plamna Sample Handling Samplesforplasmawillbe centrihgedwithinIhour aftecrollectionP.lasma willbe harvestedand storedina freezersettomaintain-60to-80'C untilpacked on dryice and shippedto: Jon C. Cook, PhD, DABT E. I.du Pont de Nemours & Company, Inc. HaskellLaboratoryforToxicologyand IndustriaMledicine BuildingIIRoom 1325 1090 ElktonRoad Newark, Delaware 19711-0500 TelephoneNo.: 302.366.5495 FacsimileNo.: 302.366.5003 DuPont willbe notifiedregardingshipmentof samples. Plasma Analysis Samples willbe analyzedby DuPont HaskellCompany forcholecystokininR.esults willbe providedforinclusioinn thefinalreport. Serum APFO Level Determination Frequency After7 daysoftreatmentand every2 weeks thereaftdeurringtreatmentand recovery Number ofAnimals All Method of Collection Animals willnotbe fastedb;lood (approximatel2ymL) willbe couectedfrom a femoralvein(analternativeinmay be used ifnecessaryw)ithoutan anticoagulant. Sample Handling Samples willbe centrifugewdithinI hour aftercollectioann,dserum willbe harvested.Serum sampleswillbe storedina freezesrettomaintain-10to -30*C. Covance 6329-231 Page II Sample Shipping Senun samples willbe packed on dry iceand shipped to: Kris J.Hansen, PhD 3M E.T.& S Bldg. 2-3E-09 935 Bush Avenue St.Paul,l@Ennesota 55106 KrisJ.Hansen, PbD orher alternatweillbe notifiedregardingtheshipment ofthe samples. Analysisofserum forAPFO willbe done on thesamples. Resultswillbe providedforinclusionin thefinalreporl AdditionalBlood Collection At scheduledand unscheduledsacrificebsl,ood (asmuch aspossibleu,p to20 mL) win be collectedfrom animalsatthetime ofexsanguination.Approximately equallysized samples of serum (collectewdithoutanticoagulanta)nd whole blood and plasma (both collectedusing EDTA asan anticoagulantf)rom each animal willbe transferreidnto containers(approximately5 mL each).Whole blood,serum, and plasma samples willbe storedina freezersettomaintain-60 to -80*C untilshippedto3M (KrisHansen) for possiblefutureanalysis. Urine and Feces APFO Level Determination Frequency After7 days of treatmentand every 2 weeks thereaftedruring treatmentand recovery (concurrenwtithserum APFO sample collection) Number of Animals All Method of Collection Animals willnotbe fastedu;rine(collecteodn wet ice)and feceswillbe collected overnight. Sample Handling Samples of urine(atleast2 mL) and feces(atleast5 grams) willbe storedina freezer setto maintain-10 to -30*C. Covance 6329-231 Page 12 Sample Shipping Samples willbe packed on dry iceand shipped to: Kris J.Hansen, PhD 3M E.T. & S Bldg. 2-3E-09 935 Bush Avenue St.Paul,Nhnnesota 55106 Kris J.Hansen, PhD or her alternatweillbe notifiedregardingthe shipment of the samples. Analysisof urineand fecesforAPFO willbe done on the samples. Results willbe provided forinclusioninthefinalreport. Terxrdnation Unscheduled Sacrificesand Deaths Necropsieswillbe done. Animals tobe sacrificewdillbe anesthetizewdith ketamine and xylazine,weighed, and exsanguinated. Scheduled Sacrifices After atleast26 weeks of treatment,fouranimals/sex/groupwillbe fastedovernight, then anesthetizedwith ketamine and xylazine,weighed, exsanguinated,and necropsied. After atleast26 weeks of treatmentand atleast13 weeks without treatment,the remaining animals willbe fastedovernight,then anesthetizedwith ketamine and xylazine,weighed, exsanguinated,and necropsied. Postmortem Procedures Necropsy The necropsy willincludeexaminationof: allorifices cranialcavity externalsurfaceof the brain;the externalsurfaceof the spinalcord and cut surfacesof the brain and spinalcord willbe examined whenever tissueuimming isperformed cervicaltissuesand organs thoracic,abdominal, and pelviccavitiesand viscera externalsurfaceof the body nasal cavity and paranasalsinuses Covance 6329-231 Page 13 Organ Weights From each animal atscheduled and unscheduled sacrifices,the following organs (when present)willbe weighed; pairedorgans willbe weighed separately: adrenal(2) brain epididymis(2) kidney(2) liver pancreas testi(s2) thyroid(2)with parathyroid Organ-to-body weight percentagesand organ-to-brainweight ratioswillbe calculated. PalniitoylCoA Oiddase Deterniinations The rightlateralobeof liverwillbe collectedfrom each animal at thescheduled and unscheduled sacrificesw,eighed, flash-frozenin hquid nitrogen,and storedin a freezer setto maintain -60 to -80*C untildispositionof the samples isdetermined by the Sponsor. CellProliferatioEnvaluation Representativesamples of leftlateralobe of theliver,leftand righttestesa,nd pancreas willbe collectedfrom each animal atthe scheduled and unscheduled sacrificesa,nd preservedin zincformalin. Afterfixations,amples forproliferatiocnellnuclearantigen(PCNA) evaluationwillbe embedded in paraffm maintained atambient temperatureuntildispositionof the samples isdetermined by the Sponsor. BileAcid Deterniination All availablebile(upto 5 mL) willbe collectedfrom each animal atthescheduled and unscheduled sacrificesf,lash-frozeinn liquidnitrogen,and storedin a freezersetto maintain -60 to -80*C. Bile samples willbe packed on dry iceand shipped to: Covance 6329-231 Page 14 Biomedical Research Centre Level 5 NinewellsHospitaland Medical School Universityof Dundee Dundee DDI 9SY UK Dr. CliffElcombe Biomedical Research Centre NinewelisHospitaland Medical School Universityof Dundee Dundee DD I 9SY, UK Telephone: +44 (0)1392-632641 Fax: +44 (0)1392-425596 Resultsof bileaciddeterminationsanalyseswillbe provided forinclusionin the final report. Receptor Level Deterndnation Samples (approximately2 g) of liverand pancreaswillbe collectedfrom each animal at thescheduled and unscheduled sacrificesf,lash-frozeinn liquidnitrogen,and storedin a freezersetto maintain -60 to -90*C. Liver and pancreassamples willbe packed on dry iceand shipped to: Jon C. Cook, PhD, DABT E. I.du Pont de Nemours & Company, Inc. HaskellLaboratory forToxicology and IndustriaMledicine Building 11 Room 1325 1090 Elkton Road Newark, Delaware 19711-0500 Telephone No.: 302.366.5495 FacsimileNo.: 302.366.5003 DuPont willbe notifiedregardingshipment of samples. Resultsof receptorlevel determinationswillbe provided forinclusionin thefmal reporl Tissue Preservation Three samples (approximately5 g each) of theliverand allremainingpancreas and left and righttestestissuei,fany, (dividedintothreeapproximatelyequal samples) willbe collectedfrom each animal at the scheduled and unscheduled sacrificesw,eighed, flash-frozeinn liquidnitrogen,and storedin a freezersetto maintain -60 to -800C for possiblefutureanalysis. Covance 6329-231 Page 15 The followingtissues(when present)from each animal willbe preservedin 10% neutral-bufferefdormalin,unlessotherwisespecifiedf,orpossiblefuture microscopic examination: adrenal(2) aorta brain cecum colon duodenum epididymis(2) esophagus eye [(2)to be preservedinDavidson's fixativferom sacrificeadnimalsonly] femur with bone marrow (articular surfaceof the distalend) gallbladder heart ileum jejunum Iddney(2) lesions liver lung mammary gland mesentericlymph node pancreas pituitary prostate rectum salivargyland [mandibular(2)] sciaticnerve seminalvesicle(2) skeletamluscle (thigh) sldn spinalcord (cervicalt,horacic,and lumbar) spleen stemum withbone marrow stomach testis[(2)tobe preserved in Bouins' solutionfrom sacrificedanimals only] thymus thyroid(2)with parathyroid trachea urinarybladder Bone Marrow Smear From the sternum of each animal atunscheduled and scheduled sacrifices(made and held forpossiblefutureexamination) Liver APFO Determination A sample of liver(any non-formahn treatedliverremaining aftersampling for histopathology)willbe collectedfrom each animal atthescheduled and unscheduled sacrificesw,eighed, flash-frozenin liquidnitrogen,and storedin a freezersetto maintain-60 to -80*C untilshipped with serum samples to 3M (KrisHansen) for analysis.Analysis of liverforAPFO willbe done on thesamples. Resultswin be provided forinclusionin thefinalreport. Covance 6329-231 Page 16 Histopathology Tissues from each animal in the control and high-dose groups and each animal that dies or issacrificedat unscheduledintervalwillbe embedded in paraffm,sectioned,stained with hematoxylin and eosin,and examined microscopically. The adrenals,pancreas,liver,spleen,and testesfrom each animal in the low- and mid-dose groups willbe embedded in paraff-m,sectioned,stainedwith hematoxyhn and eosin,and examined microscopically. Suspected targetorgans noted atthe high-dose willbe embedded in paraff-ins,ectioned, stainedwith hematoxylin and eosin,and examined microscopicallyfrom allanimals (atthe Sponsor's requestand added by amendment). Reports A draftreportwhich includesthe followinginformationwillbe prepared and submitted: Experimental Design and Methods Results mortality clinicalobservations body weights body weight gains food consumption ophthalmic examination results clinicalpathology results hormone analyses(providedby Ani Lytics,Inc.and theDuPont Haskell Company) serum, urine,feces,and liverAPFO levels(providedby 3M) organ weights organ-to-body weight percentages organ-to-brainweight ratios macroscopic observations microscopic observations palmitoylCoA oxidaseactivitie(sprovidedby theSponsoes designee) ceH proliferatioenvaluations(providedby PathologyAssociatesInternational) bileaciddeterminations(providedby UniversityofDundee) receptordetermination(providedby DuPont) Covance 6329-231 PaLe 17 Statistical Evaluation Levene's testwill be done to testfor variance homogeneity. In the case of heterogeneityof varianceatp :50.05,transformationswillbe used to stabiliztehe variance. Comparison testswilltakevarianceheterogeneityintoconsideration. One-way analysisof variance(ANOVA) willbe used (ifapplicablet)o analyzeinitial body weights,body weight gains,continuousclinicaplathologyvalues,palmitoylCoA oxidase activitieasn,d organ weight data. IftheANOVA issignificantD,unnett'st-test willbe used forcontrolversustreatedgroup comparisons. One-way analysisof covariance(ANCOVA) willbe used to analyzebody weights,with initiablody weights as thecovariate.IftheANCOVA issignificantc,ovariate-adjusted means willbe used for controlversus treatedgroup comparisons. Group comparisons (Groups 2 through 4 versusGroup 1)willbe evaluatedat the 5.0% two-tailedprobabilitylevel.Only datacollectedon or afterthefirstday of treatmentwillbe analyzed statisticallDya.ta collectedbeforethefirstday of treatment or during recoverywillnot be analyzedstatistically. At the end of 1 year afterissuanceof the auditeddraftreport,ifno requestedrevisionsor instructionsto finalizehave been communi by the Sponsor, then the auditeddraft reportwillbe considered 'finala'nd issuedas thefinalreport,signed by the study director, and submitted to the Sponsor. Any modifications or changes to the auditeddraftreportrequestedI year afterissuance willbe performed at additionaclostto theSponsor. Record Retention All raw data,documentation,records,protocol,specimens,and finalreportgeneratedas a resultof thisstudy willbe archivedin thestoragefacilitieosf Covance-Madison fora period of I year followingsubmissionof the finalreportto theSponsor. One year after submission of the finalreport,allofthe aforementioned materialswillbe sentto the Sponsor, and a returnfee willbe charged. The Sponsor may electto have the materials retainedin the Covance archivesfor an additionalperiod of time,and Covance willcharge a storagefee. Ifthe Sponsor chooses to have Covance dispose of the materials,a disposal fee willbe charged. All raw datastoredon magnetic media willbe retainedby Covance. Within one year aftersubmission of thefinalreport,allofthe aforementioned materials from theSponsces designees(Ani LyticsInc.,E. 1.du Pont de Nemours & Company, Inc.,3M E.T. & S, Pathology AssociatesInternationala,nd Universityof Dundee) win be sentto the Sponsor (PaulLieder,PhD, DABT, 3M) to be maintained. PROTOCOL APPROVAL Covance 6329-231 Page 18 Paul Lieder,PhD Diplomate, ABT Study Monitor 3M David Farrar,PhD ICI Chemicals & Polymers Limited P@(e@-J-.Thot"rd, Phi) Study Director Department of Toxicology Covance LaboratoriesInc. Date Date Dat@