Document B5Lrjaazoxna1O0vyBdKmz3NJ

cena ogan- 375 AR226- 09577 AIT-5 Draft Report Cnn TR ona 26-Week Capsule Toxicity Study with PerfluorooctaneSulfonic Acid Potassium Salt (PFOS; T-6295) in CynomolgusMonkeys TR& EPA-OTS 000811794u g z - SADFOR: or ro yee 8 E ro =J - | 002636 covaNCE Sponsor: St. Paul,3MMinnesota UNAUDIATprEilD 1D2R,2A0F0T0 FINAL REPORT Study Title: 26-WePeoktaCsaspisuumleSaTlotx(icPiFtOySS;tTu-d6y2w9i5t)hiPneCryflnuoomroolocgtuasneMoSunlkfeoynisc Acid Author: Peter J. Thomford, PhD Study Completion Date: To be determined Testing Facility: C3o3v0a1nKcienLsambaornaBtooruileesvaInrcd. Madison, Wisconsin 53704-2595 Laboratory Study Identification: Covance 6329-223 Sponsor Study Identification: 3M Study No. T-6295.7 Volume Iof IT Page 1 of 821 : 002637 -_-- CovanIcMeTT6e6322w99.s5272 COMPLIANCE STATEMENT 26-WePeoktaCsaspisuumleSaTlotx(iPciFtOySS;tTu-d6y29wi5t)hiPneCrfylnuoomroolocgtuasneMoSunlkfeoynisc Acid AGloloadspLeacbtosroafttorhyssPrtaucdtyicweeSrteanidnaarcdcs,or4d0aCncFeRw7i9t2h.the Environmental Protection Agency PeterJ. Thomford, PhD Study Director Covance Laboratories Inc. SAtnuddryeMwoMni.tSoeracat, PAD 3M Date Date 2 002638 -_ CovawnIcrMeT6e63229s0-25t273 STUDY IDENTIFICATION 26-WePeoktaCsaspisuumleSaTlotx(icPiFtOySS;tTu-d6y2w9i5t)hiPneCrfylnuoomroolocgtuasneMoSnulkfeoynisc Acid Test Material PSealrtfl(uPoFrOoSoc;tTan-e62S9u5l)fonic Acid Potassium Sponsor Study Monitor 3M `BTuoixlidcionlgo2gy20S-e2rEv-i0c2e,s 3M Center St. Paul, Minnesota 55144-1000 Andrew M. Seacat, PhD 365M1.575.3161 Alternate Study Monitor . Study Location Study Director Study Timetable Study Initiation Date IInn--LLiiffee T(eErxpmeirniamteinotnalD)atSetart Date Experimental Termination Date M3aMrvTionxiTc.oClaosgey,SDerVvMic,esPhD 651.733.5180 C33o0v1anKcienLsambaonraBtoourileesvaIrndc. Madison, Wisconsin 53704-2595 Peter J. Thomford, PhD Covance Laboratories Inc. PO Box 7545 Madison, Wisconsin 53707-7545 608.241.7207 August 20, 1998 MAaugrucsht72,62,0109098 To be determined : 3 002629 --_-- CowvamnMcTTe-6e362w299-s252773 KEY PERSONNEL 26-WePeoktaCsaspisuumleSaTlotx(icPiFtOySS;tTu-d6y29wi5t)hiPneCryflnuoomroologctuasneMoSunlkfeoynisc Acid Study Director Peter J. Thomford, PhD Study Toxicologist Dale Aldridge, BS Study Coordinator Elizabeth A. Disch, BA Manager, Large Animal Toxicology ~~ Sharon Dunn, LATG, AT Supervisor, Dose Formulation Dixie Bushee, BS, LATG AsMseodciicaitneeDirector, Laboratory Animal DDioplnonmaJa.teC,leAmConLsA,MDVM, MS Clinical Pathologist Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) Supervisor, Clinical Pathology Ronald Markevitch, BS,MT (ASCP) Anatomic Pathologist Robert A. Leedle, DVM, PhD Diplomate, ACVP Supervisor, Anatomic Pathology Laurie J. Schuller, BA, LAT Supervisor, Anatomic Pathology Kimberly W. Durland, BS, HT Consultant Stephen L. Bistner, DVM DVeitpelroimnaatrey,OAphCtVhaOlmologist 4 002640 -_-- ComvanMTcTe -6e3622s99-25y273 VOLUME I OF II CONTENTS Page ABSTRACT cssssssssssssssssssssssmseeensd REGULATORY COMPLIANCE... 12. TEST MATERIAL, VEHICLE, AND SOLVENT.........ccoocemrmmmrrmmsmmrnnnn 12. GEIBHDCAPSULES... 13 DISPOSIION corse 13 TEST ANIMALS AND HUSBANDRY... 13 i P GrouR p DesiO gnatioC ns andE DOSAGD E LEVU lS.R ...E ...oS mro. mer. men. rem 1155 DOSING PrOCEAUIES...rrresremomsssnsnemeoseen 16 CBl1i0n0idcaHlOPIAMtNhOONIEOBDYEv AETMNANIONs . rs s s 1189 SAdedriutmioPnaFlOSSErLeUvmelCODUECCtHEONmo ittion...r ....rrrmss sssseeenn 2109 AUdriinteiOannadlFFeeceCslPSFAMOPSLELSevv el DErMno atON..r ......e .....os cero22s00 ITeDrmEiTnalLiLVIeVrEBrIBOiPoSpYsySASMPAIESMv PIES...c .....eee sseenn2201 AAnnaaLtOoMmIiCc PPaatthhoOlIoOgg--yY RTeErCmOiVnaElYSSAACCTTHHIICCEE...v .r.rrrro rrrrs r rs es s2241 RSEHCBOSTHACAR] AEBEBCINSOSENS....s.e.scesssesmssmsssseemoe oon2255 5 002641 _-- TCoveanMcTew-326s92.9257273 VOLUMET OF II CONTENTS (Continued) Page RESULTS AND DISCUSSION... svn 26 PCalilnmiiCtaolylPtCHOOIAOORXYic dase DEv IETMs ALION errrsens 28 28 BA1R0L0Od THCOTPOANNOEIODREYLr CTTUNr AON.rv.11 28 29 CONCLUSION ccm 30 OPHTHALMOLOGYREPORTcnn 33 PATHOLOGYREPORT.ccc 34 COMMENTS ONTHE DATA cocvcvvsvmsrmssssstsssssssmnoss4n1 COGDeEnSer,alABCBOREESVnIdATAIONBS,BANIDUNEITSV...IBHONS.vrsrs4434 B Abbreviations and Units for ClinicaL l HOMAtOIORY..............oooo 48 AAbbbbrreevviiaattiioonnss and and URts URLs for for Clinical CliCal CURIHBAIEYSIMS .I.S.T.Y.......rrrsonS5O2 Codes for ANBOMICPALNOIORY verse53 FIGURES 1 2 Mean Mean Body Body Weight Weight Data Data - TRReCBCMROLV.E.T.cYc.c.v.srisvrvsssemessmrrmesensn n5565 TABLES 1 2 SSuummmmaarryyooffCClliinniiccaall OObbsSeErrvvaattiioonnss - RTreecaotvmeernyt(.Da.y.s.1.86.t.h.ro.u.gho3w65e)r..r..s..n..r.n..n.56s1 3 4 SSumumarmyoomffOCpalihntihrcaallmyOibcseOrvBatSioEnsI- RVe=cAToHvEeEOrAyMN(EDSNays 36 through cv. S61)............ 62 64 5 6 SSuummmmaarryyooffOBpohdtyhaWlemiigchtODDSaCtaVR(EkeO)NS- T=IRCEACMOEVEcTvY...rnvrrerrors6676 7 8 SSuummmarmyooaffBCrolidyniycaWleiHgehmtatDoaltoag(yKgD)at-aR-EDCaOyV-T2Y7..wwnrerrrnnreesrnsT802 9 10 Summary Summary of of Clinical Clinical Hematology Hematology Data- Day Da=tDaa 37... 62... 84 88 11 Summary of Clinical Hematology Data=D&Y 91 crn 92 6 -- 002642 -_ CovanIcMeT6Te362w29.9s25273 VOLUME I OF IT CONTENTS (Continued) Page TABLES 12 13 Summary of Clinical SummaryofClinical Hematology Data HematologyData - Day Day 153 182 ..........................oo.o.oro.rs19060 14 15 SummaryofClinical Summary of Clinical Hematology Hematology Data - Day Data-Day 217 245 o ..... ...o ...o ..o. oo. 104 108 16 17 SummaryofClinical SummaryofClinical Hematology Hematology Da-tDaay Data - Day 327242....o ....o ....o oooo. oom. 112 116 18 19. SSumumamrymooffCaClliirnniiccyaall HHEeMmAa(tOoIlOoggYyDDaattaa -+DDaayy 43645..6.............ooooo. 120 124 20 21 SSuummmmaarryyooffCClliinniiccaall CHheemmaitsotlroygDyaDtaat--aDaDya-y 25476 ..............o.o.u.r..rooonon. 128 132 22 23 SSumumamrymooffaCCllirinniicycaallCCHhEemMiSsttrrYy DDaattaa - Day - Day 3 627........ ...oooo 138 144 24 25 Summary of Clinical SummaryofClinical CHEMStry ChMiStry Data Data - Day Day 91... 153... 150 156 26 27 SummaryofClinical SummaryofClinical Chemistry Chemistry Data Data - Day Day 182... 217 vo... 162 168 28 29 SSuummmmooffaaCClliirrnniiccyyaallCChheemmiissttrryy DDaattaa - Day Day 245 274 ...o .....or wnrerro omrene 174 180 30 31 SSumumamrymooffaCCllirinniicycaall Chemistry Data Chemistry Data = Day Day 3 364 ..2 ...2 ..... .o.o. oore. rvr 186 192 32 33 Summary of Clinical SummaryofClinical CREMiStry ChEMISrY Data-Day Data -DAY 456 S46... oo. 198 204 34 35 SummofaClirnicyal Summary of Clinical Urinalysis Urinalysis Data Data - DRY Day 3-277o cu w rn e s 221102 36 37 SummofaClirnicyal SummaryofClinical Urinalysis Urinalysis Data Data = - Day Day 6 91 ..2 ..... ... oo2n21164 38 39 SummaryofClinical SummaryofClinical Urinalysis Data = UrinalysisData - DaY Da 153 182 .v..e..r..nuoonn r222m108 40 41 SSuummm ooffaCClm liirnniiccya aall UUrr riinnaally yyssiiss Data Data - Day Day 2 245 1 ...7....... ......oownrs 222 224 42 43 Summary of Clinical SummofaClirnicyal Urinalysis Urinalysis Data Data = Day DaY 274 322 m oor o 222286 44 45 SSumumamrymooffCaClliirnniiccyaall UUrriinnaallyyssiiss DDaattaa - DY Day 34 64 o 36 r. e .o . 223320 46 47 SSuummmmaarryyooffPCallimniictaolylUrCinoaAlyOsXisidDaastea D-EDtaEyrT5UNA4LION6S........... ....ooonrnoor 234 236 48 SUMMAY Of Organ Weight Data....veverssesmonsosr 238 7 002643 _-- CovanIcMeT3Te269w2.2s5273 VOLUME 1 OF IT CONTENTS (Continued) Page TABLES 49 50 51 IIInnnccciiidddeeennnccceeeooofffMMMiaacccrrrooossscccooopppiiicccOOObbbssseeerrrvvvaaatttiiiooonnnsss---TTReeerrcmmoiivnneaarllySSSAaaCccItriiifffiicicec.ee...o ..........o .......... ......... ...... 227681 52 Incidence of Microscopic Observations Recovery SaCTIfice ou... ..... .228722 PIOLOCOL DVIBHONS sss 284 PPrroOt(oOcCoOl! AAMMEENIAAMMEENNtt NNOO.. 2L. c 306 315 IIInnndddiiivvviiiddduuuaaalll AOClpDihntEihc]aallFmOaitcBeODSBRESEr.IVVNAILIOONNSS.....v.v.v.r..rvosete3s3n3407n342 vsrnoee APPENDIX3... Individual Body WEight Data (KE)... 489 490 VOLUME II OF It APPENDIX Individual d Clinical HEi MAIOIOBYc Daao ..t . s, S04 S05 IInnddiivviidduuaall CClliinniiccaall UCrHiEnMaIlSyIsEisYD Da alt aa si ... S66357. IInnddiivviidduuaall PAaNlBmIitOoMyC] CPAOtAhOOIxOiBdYaDsAeLDEev terMilations.s ......u.....s .... , 668960 APSPuEmNmDaIryX a6 nd Individual Blos od HOMMONDetermt ination....... ............71.76o09 APCPelElNPDrIOUXf7Er.ation REs POTt c s c s o sso 880089 8 002644 ABSTRACT CovanMceT6632299-52273 `Thepurposeofthisstudywas toassesstheeffectofthetestmaterial, Perfluorooctane Sulfonic Acid Potassium Salt [PFOS; T-6295 (hereafter referredtoasPFOS)] on critical enzyme levels, hormones, and other selected biochemical parameters when administered daily by oral capsule to cynomolgus monkeys for atleast 26 weeks. The treatment period was followedbyan approximate 52-week recovery period. Male and female cynomolgus monkeys were assigned to four groups (six animals/sex in `Groups 1, 3,and4; four animals/sex in Group 2). Each group received dose preparations containing the vehicle, lactose, or 0.03, 0.15, or 0.75 mg of PFOS/kg of body weight/day (mg/kg/day). Two animals/groupinGroups 1, 3, and 4 werein arecoveryperiodand werenottreatedforat least52weeksfollowingthe26weektreatmentperiod. Food was provided ad libitum, except when animals were fasted. Water was provided ad libitum. The animals were observed twice daily (a.m. and p.m.)formortality and moribundity. At least once daily, animals were examined for abnormalities and signs of toxicity, and food consumption was assessed qualitatively. Ophthalmic examinations `were done before initiationoftreatmentand during Wecks26and 52. Bodyweightdata were recorded weekly before initiationoftreatment, on Days -1 and 1, and weekly thereafter. Blood and urine samples were collected for clinical hematology, clinical chemistry, and urinalysis tests before initiationoftreatment andatspecified intervals duringtreatmentand recovery. Blood was also collected for blood hormone and PFOS level determinations before, during, and after treatmentatspecified intervals. Feces and liver samples were also collected at specified intervals. On Day 155 (Week 23), `one male given 0.75 mg/kg/day died, and on Day 179 (Week 26), one male given 0.75 mg/kg/day was sacrificed due to poor health. On Days184and 185 (Week 27), four animals/sex/group (Groups 1 through 3) and four females and two males (Group 4) were anesthetized, weighed, exsanguinated, and necropsied. At necropsy at the scheduled and unscheduled sacrifices, serum sample was collected, macroscopic observations were recorded, selected organs were weighed, and selected tissues were collected and preserved. Microscopic examinations were done on tissues from each animalinthe control and high-dose groups and selected tissuesfromanimalsinthe low`and mid-dose groups. Tissues were also collected for palmitoyl CoAoxidase determination, cell proliferation evaluation, PFOS determination, and electron `microscopy. Additionally, the bile was collected from the gallbladder, and the gallbladder was preserved. At the recovery sacrifice on Day 549, the remaining Group 4 9 002645 -_-- CovaunMcTeT6e2320w9-5s227y3 oabnsiemravlastiwoenrsewaenreestrheetciozredde,dwaenidghsepdec,iefxisedanigsusiuneasteadn,dasnedrnuemcrwoeprseiecdo.lleMcatcerd.osRceompiacining animals in Groups 1 Covance 6329-268. and 3 were donatedortransferred to a follow-up study, oAxtiadlalsoesdeetelrevmeilnsa,tciloinnsicdaol onbostearpvpaetaiorntso, obpehatfhfaelcmtiedc boybsterrevaattimoennst,wiatnhd PpaFlOmSi.toyl CoA s`oTwmoemaadlveesrsgeivcelinni0c.a7l5ombgse/rkvga/tdiaoynsdi(ecodndsutrriincgtetdrepauptimle,npta.lTehgeusmesd,eaatbhnsorwmearlefperceesc,eded by reexcceusmsbievnet,salliovwatfiooond, lcaobnosruemdprteisopni)raatinodn,apdpeehayrderdatteobdeaprpeelaartaendcteo, thhyepoaadcmtiinvies,traattaixoinc,of bPoFdOyS.weiWghhetsn(cComApMa)rfeodrmwaitlhesangiimvaelns 0g.i7v5enmgt/hekgc/odntaryolwemrateersilailgh,tcloyvlaorwiaetrebaedgjiunsntiendgmaetan s`iWgeneikfi2c1a,ntalnydlofworerfebmeagliensnignigveant W0.e7e5kmg11/.kSgi/mdialyaCrdAeMcrebaosdeyswweeirgehtnsotwesreee,niinntgheneeortahle,r etfrfeeacttesdogrnobuposd;ytwheeirgefhotrse,wethries nfoitndaipnpgairsenlitkdeluyritnesgtrmeactoevreirayl.-reLlaotwed.fooTdesctomnastuemrpitali-ornelwaatsed 0n.o0t3edmgsp/okrga/ddiacayltlhyrfoourgahnoiumtatlrseaitnmtehnet.grTohupesignicvideenntcheeocfolnotwrolfomoatdecroianlsuamnpdtion was ggienveernatlhley choingthreorlimnattheerigarloaunpds agpipveeanr0e.d15toobre0.te7s5tmmatgeri/alkregalast/cedo.dmpDaaurreyidngtoreacnoivmearlys, effects on food consumption were reversed. 0Es.t7r5admigo/lkgv/adlauye,s waelrtheoguegnherbaelclayusleowoefrtohenvDaariyasti6o2n, i9n1,thaenddat1a8o2nlinymtahleeDsagyiv1e8n2 value was Dsiagynisfi3c7a,nt6.2,Easntdro9n1e, vaalltuheosuwgehrbeecgaeunseeroalfltyhheighvear riniaaltilniotfohtnehedatrteaanteodnefoemfatlheessoenvalues were significantly different, and this difference was not apparent on Day 182. 0`T.r7i5iomdgo/tkhgy/rodnaiyneonvaDlaueyssw9e1raenndot1a8b2l.y Wloiwtehrtihne bsoitnghlemaelxecsepatnidonofefmamlaelsegsigvievne0n.15 and `0o.c1c5asmigo/naklg/idnsatyaoncnesDainyw9h1i,cahllthvealhuoersmwoenree vsailgnuiefsiciannttrleyalteodwegrr.ouDpusridinfgferreecdovsleirgyhtwleyrferom wtheorseenooftccolnetarrollys,dobsuet-rtehloasteedd,ifafnedrednicdesnowteraeppneoatrctoonsbiestcelnetarolvyerreltaitmeedotrobtehetween sexes, maadlmeinsiasntrdatfieomnaolfetshuesetdestinmattheirsisatl.udyApcpoamrpelnictadtiefsfetrheenicnetseripnrtehteatsieoxnuoaftl hmaetuhroirtmyoonfebodattha. 10 002646 -_-- CovanMcreT6e2320w9-5s22773 `The only clinical pathology parameters considered related to the test material were lower c`thootlalesctheorloelstfeorrolanfiomraalnsigmiavlesng0i.v1e5n 0.75 mg or 0.75 /kg/day and mg/kg/day. lower These ehfifgehctdsewnesirteylriepvoeprrsoetdeiwnit hi n the first 5 and 9 weeksofrecovery, respectively. Altivterh,ehetpeartmoicnealllsualcarrifhiycpee,ritnrcorpehays,edanldivheerpwaetiogchetlsl,ulmaarcvraocsucoolpaitcioonbsienravnaitmiaolnssogfivmeonttled 0.75 mg/kg/day were considered related to PFOS treatment. However, the microscopic examination findings and nliovneerobfitohpseiemstaackroesncdoupriicnogbrseecrvoavteiroyndsimd andoet ianttdihcearteeacnoyvetresytsamcartiefriicaelw-reerleated acnoinmsaidlesriendthteesthmiagthe-rdioasl-erreleactoevde.ryTghreoruep.weTrheinsoin`dmiiccartoesscotphiacttfhiendhienpgastiicn the test liver from the `material-related effects were reversible. ``mTarleeatamnedntfewmiatlhePcFyOnSomboylgoruasl mcaopnskuelyesfaotardosleeasstup26tow0e.e1k5smsg/gkegn/edraayll.y Cwleilnli-ctaollearnadted in pathological findings considered to be associated with the treatment ofPFOS after at least 26 period. weeksoftreatmen Based on the data t were presen found ted in to be reversible during a 52-week recovery this report, the no-observable-effect level is r0e.s0u3ltmsg(/pkrgo/vdiadye.dDboysPeAaIn)alayresefsor(tphrcoovmiidnegd. by the Sponsor) and electron microscopy : n 002647 -_-- mCovTaIneMeT6-w3622s09.52773 PURPOSE lTehveelsp, huormoronfetsph,iasosntduosdtyhweearsselteoactsesdesbsitohcheemefifceacltopfatrhameetteesrtsmawthereinalaodmnincriisttiecarledednazilyymbey capsule to cynomolgus monkeysforat least 26 weeks. followed by an approximate 52-week recovery period. The treatment period was REGULATORY COMPLIANCE AAlgleanscpyecGtosoofdtLhaisbsotruadtyorywePrraectdiocneeiStnaancdcaorrdsd,an4c0eCwiFtRht7h92e.Environmental Protection `TEST MATERIAL, VEHICLE, AND SOLVENT Test Material `NToh.e t2e1s7t,miastearwiahli,tePetrofolfufo-rwohoictteanpeowSduelrf.oniIct wAacisdrePcoetiavsesdiautmCSoalvtan(cPeFOoSn; T-6295), Lot September 4, 1997. The test material was stored at room temperature, Ithnaftordemfaitnieonthoentessytnmtahteesrisiamletishoodns,filsetawbiitlihtyt,hepuSrpitoyn,socro.mposition, or other characteristics Vehicle `(TehxepivreahtiicolnedwataesFleabcrtousaery(S1p3e,ct1r9u99m),.NIet wwaBsrurnescweiivcekd,aNteCwovJaenrsceey)o,nLMoatrNcoh. 3N0N,01199982. `The vehicle was stored at room temperature. Information on synthesis that define the vehicle is methods, purity, stability, composition, on file with the manufacturer. or other characteristics 1" 002648 -_-- CovaMnIcMeTT6-302s20-925?273 Solvent dTahteesJoulnveen2t00w0a)s. aIctewtaonser(eScpeeicvterduamt,CGoavradnecneao,nCaJliufnoern2i3a,),1L99o7t.NoT.heLs2ol5v3en,tw(aexspsitroartieodnat room temperature. sIonlfvoernmtatiisoonnofnilesywnitthhestihsemmeatnhuofdacst,ucroemrp.osition,orother characteristics that define the Gelatin Capsules NGeol.at5i4n40c4ap3s,uleexsp,irSaitzieonNdoast.e2Au(gLoutstNo1., 21020229)32w,erexepimraantuifoancdtautreedJubnyeT1o2r,p2a0c03I)nca.n4d (Lot L(FoatirNfoie.ld5,4N40e4w3Jwearsseyr)e.ceLiovetdNoon. S1e2p2t9e3m2bewras1arnecdeNivoevdeamtbCeorva1n1c,e1o99n8.JunTehe12c,ap1s9u9l8e,sand `wmearneufsatcotruerdeartisromoaimntteamipneerdatiunrteh.e dAatca,opyofthe CertificateofAnalysis provided by the Reserve (Archive) Samples mAarteesreiarlv/elascatmospeledi(l1utgi)oonfewaaschtakloetnofatndhesttoerstedmaatterrioaolm, vteehmipcelrea,tuarned.eaTchhesteesstamples were transferredto Deviations). the Sponsor afier completionofthe treatment phase (see Protocol Remaining test material will be retained at Covance for use in possible future studies. `TEST ANIMALS AND HUSBANDRY Animals Young adult Products Inc. to adult cynomolgus monkeys were obtained from Covance Research (Denver, Pennsylvania) on June 30, 1998. The animals weighed 2.410 4.4 kg at initiationoftreatment. 13 002649 -_-- CovanMcTeTe623w299s-52273 Identification Each animal was assigned a permanent number upon arrival and identified with a collar tag before initiationoftreatment. All dataforan animal are recordedunderthis number. Justification PnoFnOhSiumsaankpnriomwantehsep(astuicchpaesrtohxeiscoymneopmroollgifuesramtoonr(kPePy)) rinetshpeornadts.imWihlaernlyetxophousmeadntso.a PP, (spiecc.ielso.w to no hepatic response) and therefore are an appropriate human surrogate Husbandry Animal Rooms 251 and 259 were usedforthis study. Environmental controlsforthe animal rooms were set to maintain 18 to 29C, arelative humidityof 30 to 70%,and a 12-hour light/12-hour dark cycle. Variations from these conditions are documented in thedataand are considered to have had no effect on the outcome of the study. `The animals were housed individually in suspended, stainless-steel cages. Coetrhteirfwiiesdepsrpiemciaftieedd.ietT(h#e87lo2t6nCu,mHbaerrlsaanrTeekrleacdo)rdweadsinprtohveiddaetda.onTcheeodriettwiics eroduatiilnye,luynless. analyzed by the manufacturer for nutritional components and environmental contaminants. Resultsof specified nutrient and contaminant analyses are on file with Covance-Madison. Fruits or additional supplements were provided, but did not require analysis. `Water was providedad libitum. Samplesofthe waarteanealyzred for specified `microorganisms and environmental contaminants. Theresultsareon fle with Covance-Madison. `There were no known contaminants in the diet or water at levels that would have. interfered with this study. " 002650 CovanMceT6232095.272 Acclimation `ATnwiemnatly-Rfoouormma2l5e1sfaonrd5274dafyesmableefsorweerineitrieacteiionvoefdtorenaJtumneent3.0,I1n9g9e8n,eraanld, aacncilmiamlastiendthinis shipment appeared healthy. During acclimation, the animals were examined for `abnormalities indicativeofhealth problems. Inaddition, three tuberculosis tests, a physical examination, and a fecal flotation for parasites were performedoneach animal. PROCEDURES This study was conducted in accordance with the Protocol dated August 20, 1998, and Protocol Amendment Nos. deviationsare in Appendix 1, 1. 2, and 3. The protocol, protocol amendments, and protocol Group Designations and Dosage Levels Selection ofanimals for the study was based on data collected during acclimation. Animals were assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment group. Group Dose Level /day)* Total Material Dose Level G /day) NumofbAneimarls Males Females 1 o 2 003 30 1s [3 6 4 4 3 0.15 6 4 075 30 6 6 6 6 a The control group (Group 1) gelatin capsules as the total received the equivalent material administered to amount of lactose Group 4. in b The low-dose (1:499, wow). (Group 2) received the test material diluted with lactose T`mhaetermiiadl-ddiolsuete(dGwriotuhpl3a)ctaonsed (h1i:g3h9-,dwoiswe)(.Group 4) groups receivedthetest d Ttrweaoteadnifomralats lienasGtro2u6pwsee1k,s3,, atnhedn4tdreesaitgmneanttewdaassdriesccoovnetrinyuaendi,maanlds were the `animals oftoxic were observed for effects for at least 5r2evewresiebkislitpyo,stpreresaitsmteenntc,e, or delayed occurrence: 15 002651 -_-- CovanIcMTeT6e6322w90.s252773 Dosing Procedures dVeohsiecplree.paDraotsieonlse,vetlhse wsepreceifbiaesdedamoonutnhte ovfehliacclteosaes wsausppwleiiegdhfeodr,Gtrroanuspfe1r.rFedoirnGtorgoeulpati1 n capsules, and the top and bottom halvesofeachcapsule prepared at least once weekly. were joined. Capsules were dTielsutteMdatoenrciealb.efoTrheeintietsitamtaiotneorfiatlrleaacttmoesnet;prceappasrualteisonwserfoerpGrreopuapresd2atthlreoasutghon4cewewreeekly. `Aasndpetchiefaipepdroapmroiuantetovfotleusmtemoaftaecrieatlownaeswawesiagdhdeedd,.plAafcteedr isntitrorianglambaenleudalmliyxiunntgilctohnetatiensetr, c`moantteariinaelr.waTshdeiscsoomlvpeodn,enthtesrweequrieremdixaemdoutnhtoroofuglhalcytousseiwnagsawsepiatguhlea.d aTnhdeptrraenpsafrererdetdetsotthe tmhaetearcieatlodnieluhtaidonevwaapsorsattirerde.d pPerreipoadriactailolnyswwheirlee daillluotweeddtotofasctialnidtaetexpcoapsseudlteotprheepaariartuinotni.l Samplesofthe finished mixture for dose analyses were taken directlyfromthe container. TThhee adpopsreopprrieaptaeraatmioonusntweorfepsrteopraerdeadttresotommatteermipaelrwatausrewebiegthweedenancdaptsrualnesfperrerpeadraitnitoonSsi.ze 2 (Days 1 through 8)or 4 (Day9s through 184) gelatin capsules and thetopand bottom fhaaclivleitsaotfeedaoscehacdampisnuilsetrwaetiroenj.oiInneddi.viSdiuzael 4dacialpysduolseesswweerreeubsaesdeidnsotneatdhoefmoSsitz2reecteontbleytter recorded body weight, with the exception of doses given on days when body weight `measurements were performed; on those days, the previous body weight was used. All capsule preparations were stored at room temperature until used for dosing. Method of Administration. Gelatin capsules were used to facilitate comparison with ldiaktealyfrrooumtpeorefveixopuosstuorxeicionlohguymasntsu.diePsatrhtaiatluosredintthaectorcaalpsruoluetse.weArleson,ootreadl iins tthhee vmoomsittus ofseveral animals on occasion; the resultsofthe study. however, this is not considered to have adversely affected `The dose preparations were administered orally in gelatin capsules once daily 7 days/weekforat least 26 weeks (see Protocol Deviations for exceptions). 16 002652 -- -_-- CwovTanMeeeT66w32299s-522773 Dose Analyses Homogeneity and stability analyses were the responsibiltyofthe Sponsor. `Smaamtpelrieasll(aacptporsoexpirmaetpealrya1tigooenanschD)aywe-r1e5tfaokrenhofmroogmetnheeittoyp,anmaildydsilse., aSnadmpbloetstocmoolfltechteedtest Afrsoemt tohfesmaimdpdlleeso(fatphperopxriempaatrealtyio1nsgweearceha)lswoerdeestiagkneantferdofmotrhperelsotwu-dyansdtahbiilgiht-ydaonsaelytseisst. `materiallactose preparations at the endofthe treatment phase for test material content analysis. ASlplonssaomrpfloersanwaelryesisst.oreRdesautlrtsooofmdtoesmepearnaatluyrseeusntwiilllsebnetpurnodveirdeadmbfiorenitncclounsdiiotniinontshteo ftihneal report. Observationof Animals Clinical Observations. The animals were observed twice daily (a.m. and p.m.) for `mortality and moribundity. Animalswerealso observed at least once daily (a.m) for signsofpoor health or abnormal behavior, and food consumption was assessed qsucahleidtuatlievdelsya;croinfilcye,abenaocrhmaanlimfianldwinagssowbesreervreedc;oradbendo.rmOanlcefiwndeienkglsoyraandnoinndtihceatdiaoyn ooff onbosremravlatwiaosnsrweecorredreedc(osredeedPrdoutroicnogl tDreevaitamteinotnsapfporroexxicmeaptteilonys3).0 tAodd9i0timoinnaultleys, apfotsetrdtohsee last dose administration; only abnormal findings were recorded. oOfphtrtehaatlmmenotl,obgeyf.orOepthhtehsaclhmeidculeexdamtienramtiinaolnssawcerrifeicdeodnuerionngeWaceheakn2i6m,alanbdefdourreiningitiation `Week 52 (see Protocol Deviations). The pupils were dilated with 1% Mydriacyl and the anterior portionofthe eye, optic media, and ocular fundus were examined with an indirect ophthalmoscope by a board-certified ophthalmologist. tBroedatymeWneti,gohntsD.ayI-n1di,viodnuatlhebfoidrsyt wdeaiygohfttdraetaatmweenrte, raencdowredeedklwyeethkelryeabfteefro.reBionidtyiawtieoingohtfs were also recorded for animals sacrificed at unscheduled intervals. : 17 002653 -_-- CovanMceT68e329w9-5s227?3 Clinical Pathology Blood and urine samples were collected fromeachanimal once before initiation of wreatment (Day -27); on Days 37, 62, 91, 153, and 182 oftreatment; and on Days 245, 271,274, 322, 364, 456, and 546 during recovery (see Protocol Deviations). Animals were fasted overnight, and urine was collected overnight on wet ice before blood sampling; water was provaid ldibietudm. Blood was collectedfromthe femoral vein. Potassium EDTA was the anticoagulant used for hematology tests; no anticoagulant was usedforthe chemistry tests. Blood samples were collectedfromthe animalthatwas. sacrificedatan unscheduled interval. Animals were bled in sequentialorder on Days 37, 62, and 91 and in random orderatall other scheduled collections; this is not expected to have an impact on the clinical pathology results. The following were evaluated (see Protocol Deviations for exceptions). Hematology red blood cell (erythrocyte) count hemoglobin differential blood cell count segmented neutrophil count hematocrit `mean corpuscular volume Iymphocyte count `monocyte count `mean corpuscular hemoglobin mean corpuscular hemoglobin eosinophil count basophil count concentration platelet count rbeltoiocdulcoeclyltmeocropuhnotlogy `white blood cell (leukocyte) count glucose urea nitrogen creatinine: altobtualmpirnotein globulin total bilirubin cholesterol triglycerides alanine aminotransferase aaslpkaarltinaetepahmoisnpohtartaansseferase `gamma glutamyltransferase: Clinical Chemistry sorbitol dehydrogenase creatine kinase: calcium sinoodriguamnic phosphorus. potassium chloride bile acids amylase lipase: hpaingchredaetnisci-tsypelciipfoipcroatmeyinla(sHeDL) (effective with collection on Day 153) . 18 002654 _-- svpoelciufmiec (garapvpirtoyximately 16 hours) pPrHotein kgeltuocnoesse CovamnIcMreT6a6322w09-s25273 Urinalysis bilirubin blood `umriocbriolsicnoopgiecnexaminationofsediment appearance Blood Hormone Determination iBnliotioadtisoanmopflterse(atampepnrto(xDiamyaste-5l50y,m-4L0), wanedre-2c7o)l;leocnteDdafyrso3m7,ea6c2h, a9n1i,maanldth1r8e2eofttirmeesatbmeefnotr;e faansdteodnoDvearynsig2h1t7., B24l5o,od27w4a,s3c2o2l,le3c6t4e,d4f5r8o,manadfe5m4o9radluvreinignrweictohvoeurtyu.siAnngiamanls were caonltlieccotaigounl;anste.ruSmawmpalsehsarwveerseteadl.loTwheed stoerculomtwaansd dcievnitdriefduignetdowtiwtohaipnp1rohxoiumraatfetleryequal sahliiqpupoetds taondAnsitoLryetdicins Ianfc.refezoerra,nsaeltystiosmoafincotratiinso-l6,0tetsot-o8s0teCr,onuen,tielstpraacdikoeld,oenstdrronye,iceestarniodl, tthheyrcooildlescttiimounlaotninDgahyor3m2o2nteh,e tsoatmalpltersiiwodeortehyarlosnoinaen,alaynzdedtoftaolrtfhryereotxriini.odBoetghiyrnonniinnge waintdh free thyroxin. BSleoroudmsPamFpOlSesL(eavpeplroDxeitmeartmeilyna2timoLn) were collectedfromeachanimal once before ainnidti2a6t,ionanodf2t7re(aDtmaeynt18(3D)aoyf-t2r7e);atdmuernitn;ganWdeedukrsin1g(WDeaeyk7s),227,4(,Da6,ys8,11824,, 1168,52,0a,n2d4,187), 6258,(6D9,ay731,970)7,, 2a9nd(D7a9y(s1c9e8)P,ro3t0oc(oDlaDye2v0i4at)i,o3n1s)(.DaAyni2m1a1l),s 3w5e,r3e9,fa4s3t,ed47o,ve5r1n,ig5h3,t 5a7n,d61, awnataenrtiwcaoasguplraonvti.deSdaamdplleibsitwuemr.eBcleonotrdifwuagsedcowliltehcitned1 fhrooumraaffeermocroalllevcetiinonwaintdhosuetruusminwgas phaarcvkeesdteodn. dSreyriucemasnadmpslheisppweedretostthoereSdpoinnasofrrefeozrera,nasleytsitso.maRienstualitns -wi6l0ltroe-p8o0rtCe,dunil separately. : 19 002655 CovanScMe 6T3622925273 Additional Serum Collection At the scheduled terminal necropsy and the necropsy ofAnimal No. 105506 (Group 4 male), blood samples (approximately 20 mL) were collected from the vena cava at the. timeof exsanguination. Samples were collected without using an anticoagulant and centrifuged within 1 hourofcollection. Serum was harvested andstoredin a freezer, set to maintain -60to -80C,untilpacked ondryiceand shippedtothe Spofnorpsososibrle future analysis. An aliquot (0.8 ml)ofthe additional serum collection samples collected fromallanimals fromGroups1, 2, 3,and 4sacrifiatctehdeterminal necropsywere senton dry icebythe Sponsor to AniLyticsfortotal triiodothyronine, total thyroxin, free triiodothyronine, and free thyroxin determinations. Urine and Feces PFOS Level Determination Urine [at least 2mL (see Protocol Deviations)] and feces (at least 5 g)werecollected overnight on'the first dayof recovery (Day 184) and on Days 189, 216, 275, 321, and 366 during recovery. Inaddition, a 24-hour sample of urine and feces was collected before the completionof 52 weeksofrecovery. Except for the first dayofrecovery, animals were not fasted. Samples were stored ina freezer set tomaintain-10 to -30C, until they `were packedondry ice and shipped tothe Sponsor. The samples will be analyzed for PFOS. Results will be reported separately. Additional Fecal Samples During Week 23, a fresh fecal sample (up to 5 g) was collected from all animals in the `control and high-dose groups. Samples were collected in white polypropylene containers after pans were cleaned in the morning to ensure that the fecal samples were not more than 6 hours old (see Protocol Deviations). Samples were packedondry ice andshipped to the Mayo Clinic for analysis. Interim Liver Biopsy Samples Asample of liver (approximately 1 to 2 g) was collected by biopsy from animals in Group 4only during recovery [Week 57 (Day 393), on the same day as the serum PFOS. blood collection]. This sample was divided into four portions as follows. 002656 20 -_-- CowvaTnIcMee 6Tw322s09257273 Osencetisounebds,amstpalienewdawsiptrhehseemravteodxiynli1n0%anndeuetorsailn-b(duufpfleirceadtfeosrlmiadleisn,weermebeprdedpeardedin),paarnadf.fin, examined microscopically. `Tmhaeinstaeicnon-d60stuobs-a8m0plCe, wunatsilfslhaishp-pferdozteontihne lSiqupidoniftnorrogsaennaolaynsrdisst(osreeedPirnoatforceoelzer, setto, Deviations). Results will be reported separately. `TThhee ttihsisrudesbulboscakmspalnedwaahsepmraotcoexsysleidntaonbdloecoksisnt-asgteaifnoerdeslleicdterofnormliicgrhotsmciocpricosecvoapluyawtieorne. transferred to PAL PAL Areport will Tissues willbeprocessed and evaluated by be providedby PAI for inclusion in thefinal electron report. microscopy by `The fourth subsample was flash-frozen in liquid nitrogen and storedin a freezer,setto `maintain -60 to -80C, until transferredto the Sponsorfor possible future analysis. `Terminal Liver Biopsy Samples AGrsoaumppl3edoufrliinvgerre(caopvperroyx[imWaetcekly801 g()Dawyas55c4o)l,leocnteedwbeyekbaifotpesrythferosmearlulmanPiFmOalSsbilnood caonldlescttoiroendi(sneea Pfrreoetzoecro,lsDeetvtioamtaiionntsa)i.n T-h6i0stosa-m8p0leC,wuanstifllsahsihp-fpreodzetnoitnheliSqpuiodnsnoirtrfoogren `analysis. Results will be reported separately. Anatomic Pathology - Terminal Sacrifice NDeacyro1p5s5y(.WeAenke2c3r)opasnydwAansimdaolneNoo.n A10n5i5m0a6l(NGor.ou10p545m0a9l(eG)rothuapt 4wmaaslsea)ctrhifaitceddieidnaon `moribund condition on Day animals/sex/group (Groups 179 (Weck 26). 1 through 3) and During Week 27 four females and (Days 184 two males and 185) four (Group 4) were tfeassttse,deoxvsearnngiugihnta,teadn,esatnhedtniezcerdowpsiitehdk.etAanmiimnaelasnwdexryelanzeicnreo,pswieeidghiendr,abnldeodfmoorrrdeeqr.uired ohrieficneesc;rtohpesycriannicalludceadviatym;atchreosexctoeprincalexsaurmfiancaetioofntohfetbhreaine;xttehrenanlassaulrfcaacveiotfytahnedbody;all paranasal sinuses; cervical cavities and viscera. tissues and organs; and the thoracic, abdominal, and pelvic 21 002657 -_-- CovwanMrcTe 66e32299s-522t73 pOrregseannt)Wweeigrhetwse.igAhtesdc;hpeadiurleeddoarngdansunwsecrheedwueliegdhseadcrsiefpiacreas,tetlhye.followingorgans (when adrenal (2) brepaiidindymis (2) lkiivdenrey (2) ovary 2) pteasntcisre(a2)s thyroid (2) with parathyroid Organ-to-body weight percentages and `organ-to-brain weight ratios were calculated. Palmitoyl CoA Oxidase Determinations. Representative samplesofthe right lateral lobeof liver flash-frozen iwnerlieguciodllneicttreodgefnr,oamnedascthoraendimianlaatftreheezers,chseetdutloemdasianctraiifinc-e,60wetiog-h8e0d,C, until analyzed for palmitoyl CoA oxidaseactivity. Cell Proliferation liver, left and right tEevsatlesu,atainodn.panRcerperaessewnetraeticvoellseacmtpeldeasnodfptrheeselrevfeldatienrazlinlcobfeoorfmatlhien. A. i`sgehctonedstseesto,fatnidsspuaenscr(eraesp)repsreenstaetrivveed sianmfpolremsaloiftn hweitlheoftutlaztienraclwleorbeeoaflstoheprleipvaerr,edl.eftAafntder Ifnitxaetrinoant,iosnaamlp(lPeAsIw)erfoer permobleifdedreadtioinncpealrlafnfuicnleaanrdasnhtiigpepned(PtoCPNaAt)hoelvoaglyuaAtsisoon,ciiantcelsuding the examinationofslides stained with hematoxylin and eosin (see ProtocolDeviations). Results were provided by PAI for inclusion in the final report (Appendix 7). Liver PFOS Determination. A section of liver (approximately 20 g) was collected from each animal at the scheduled sacrifice, weighed, flash-frozen in liquid nitrogen, and stored in a freezer,set to maintain -60 to -80C, until shipped with plasma samples to the Sponsor. Results will be reported separately. Gallbladder and Bile Collection. At the scheduled terminal sacrifice for each`animal, bile was collected from the gallbladder, measured, transferred into acryovial, and flash-frozen in liquid nitrogen. The gallbladder, once emptied, was weighed, and a section (approximately 4 to 5 mm) from the mid-portion was collected. The remaining gallbladder was placed in a cryovial and flash-frozen in liquid nitrogen. The bile and gallbladder samples were storedondry ice until transferred to a freezer set to maintain ) 2 002658 _-- CovawnMcreT6e232w89-5s227y3 60to-80C.Sampleswerepacked future analysis. ondry ice andshipped tothe Sponsorforpossible w`eTrisescuoelPlreecsteerdvaatnidopnr.esTehrevefdolinlo1w0in%g nteiustsruaesl-(bwufhfeenrepdrefsoernmta)lionr,ruenplreesssenottahteirvewissaemples specified (see Protocol Deviations). aoardrteanal (2) brcaeicnum cervix colon duodenum eseopipdhiadgyumsis (2) eyfeisxa[t(i2v)epfroersaelrlvseadcriinfDiacevdidasnoimna'lss] fesmuurrfawcietohftbhoenedimsatarlreonwd)(articular gallbladder heart ileum jejunum kidney (2) lesions liver lung `memsaemnmtaerriycgllyanmdph node ovary 2) pancreas pituitary prostate rectum salivary gland sciatic nerve [mandibular (2)] sskeemlientaall mvuessiccllee ((t2h)igh) skin spinal cord lumbar) (cervical, thoracic, and spleen sternum stomach with bone marrow tessotlsut[(i2o)n pfroersaelrlvseadcriinfiBcoeudiann'ismals] thymus tthryarcohieda (2) with parathyroid urinary uterus bladder vagina Hwiitshtohpeamtahtoolxoyglyi.n Tainsdsueeossi(na,saanpdpreoxparmiaitnee)dwmeirceroesmcboepidcdaeldlyinfrpaormafefaicnh, saencitmiaolneidn,tshteained caonndtrtohlyamnuds hfiogrha-lldoasneimgarlosupisn t(hseeelPorwo-toacnodlmDiedv-idaotsieongsrofourpsexacnepdtsipoinnsa)l.cIonrdadgdriatyiomna,tltievrer sftraoimnefdewmiatlhesheinmatthoexlyolwi-naannddmciosdi-nd,oasendgreoxuapmsinweedremiecmrboescdodpeidcailnlyp.arOatffhienr, tsiescstuieosn,eda,s appropriate, will be retained for possible future examination. - 002659 -_-- CovaMnMcTeT6e2329w9-5s22773 Bone marrow smearsfrom the steronfeuacmh animal at scheduled and unscheduled sacrificeswere prepared, stained with Wright's stain, and retained for possible examination. Electron Microscopy. A sample ofthe liver was collected from cach animal at the: scheduled terminal sacrifice. Tissues were processed into blocks and, along with a hematoxylin and eosin-stained slide, were shipped to PAI for analysis. Results will be: provided for inclusion in the final report. Anatomic Pathology - Recovery Sacrifice Termination. Remaining animals in Group 1 were donated on Day 549 and remaining animals in Group 3 were transferred to Covance 6329-268 on Day 560. On Day 549, remaining animals in Group 4 were fasted overnight, anesthetized withketamineand xylazine, weighed, exsanguinated, and necropsied. `The necropsyofthe animals in Grou4p includead macroscopic examinatofitohne extemal surface of the body; all orifices; thecranialcavity; the external surfaceofthe brain; the nasal cavity and paranasal sinuses; cervical tissues and organs; and the thoracic, abdominal, and pelvic cavities and viscera. Liver Samples. Samples of liver were collected from animals in Group 4 as follows. One sample was preserved in 10% neutral-buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (duplicate slides were prepared), and `examined microscopically. `The second sample was flash-frozen in liquid nitrogenandstored in a freezer,setto `maintain -60 to -80C, until shipped to the Sponsor for analysis. Results will be reported separately. `The third sample was processed to block stage for electron microscopic evaluation. The tissue blocks and a hematoxylaindn eosin-stained slide for light microscopy were transferred to Pathology Associates International (PAI). Tissues will be processed and evaluated by electron microscopy by PAL Areport will be provided by PAI for inclusion in the final report. 2 002660 _-- CovawnMcrTe-e6632w299-s2527r3 bArdadiin,tiaobndaolmTiinsaslufeata,nhdearSte,r(uamppSraomxpilmaetse.lyS3amgpelaecsh)o,falnudngb,ilkeidannedy,sesprluemen(,eatchhyraosidm,uch asstoproesdsiibnale)frweeerzeer,cosleltetcotemda.inTthaeisne-s6a0mtpole-s80weCr,eunftlialshs-hfirpopzeedntiontlhiequSipdnointsroorgefnorapnodssible. future analysis. Statistical Analyses Lheetveernoeg'esnetietsty (oLfevvaerniea,nc1e96a0t)pw<as0.0d5o,ntertaonstfesotrmfaotrivoanrsiwanecreehuosmeodgetno esittabyi.lizIen tthhee vcaarsieanocfe. Comparison tests took variance heterogeneity into consideration. `Oanneal-ywzae yinaitniaallybsiosdyofwevairgihatnsc,eo[rgAaNn OweViAght(sW,inpearl,mi1t9o7y1la)C]owAasoxuisdeads(eifacatpipvliitecsa,blceo)nttoinuous scliignniicfailcapnat,thDoulnongeyttv'aslute-st,esatn(dDubnlnoeotdt,ho1r96m4o)newadsetuesremdinfaotriocnosn.tro1lf tvheersAusNtOreVaAtedwgarsoup `comparisons. Owenieg-htwsa,ywaintahlyisniistoalfcboovdayriweaingchets[AasNtCheOVcoAvar(iWaitnee.r,If1t9h7e1A6N] CwaOsVuAsewdatsosainganliyfizceanbto,dy covariate-adjusted means were used for control versus treated group comparisons. `5G.r0o%u,ptcwoom-ptaairliesdopnrsob(aGbrioluiptys l2evtehlr.ouOgnhly4 dvaetrasucsolGlreoctuepdo1n) owerraefeervtalhueatfeidrstatdtahyeof treatment were analyzed statistically. collected during recovery. Statistical analyses were not performed on data Record Retention Athlilsrsatwuddyatwai,lldboceuamrecnhtiavteidoni,n trheecosrtdosr,agperoftaocciollit,ieasnodfsCpoevcainmceen-sMgaedniersaotnefdoarsaapreersiuoldt ooff f1inyaelard.ispOonseityieoanroaffttheer mtahteesruiablmsi.ssAilolnorfatwhdeatfainsatlorreepdorotn, mtahgenSeptoincsmoerdwiialladnedtethrem.ine the protocol, study correspondence, by Covance-Madison. and an original copyof the final report will be retained 2 002661 _-- Covawn3crMeT6e632n299-s25273 f`rWoimthtihne1 Sypeoanrsaofrt'esr dseusbimginseseison(oAfntihLeytfiicnsalIrnec.p,or3t,MalEl.oTft.h&e Sa,foMraeymoenCtliionniec,d amantderials 3PaMt)hoblyogtyheASspsooncsioatre'ss Idnetseirgnnaeteiso.nal) will be sent to the Sponsor (Andrew Seacat, PHD, RESULTS AND DISCUSSION Observationof Animals iCnldiinviicdaulaOldbastearavrateioprness.enCtleidniicnalApopbesenrdviaxti2o.nsInadrievsiduumamlaarniizmeadlifnatTeabdlaetsaa1r,e2,alasnod 3; presented in Appendix 2. tAnhiemaslcNhoesd.ul1e0d5t5e0r6miannadl1s0ac5r5i0fi9ceg.ivAelnlo0t.7h5ermagn/ikmga/ldsaysu(rGvriovuedp t4omtahleessc)hdeiddulneodt sstuurvdiyve termination. No clinical observations sacrifice or fecovery were attributable notedinthe animals that survived to the administrationof PFOS. to the terminal DAaniym1a5l4N(oW.e1e0k552029) o(bGsreoruvpat4iomnasleo)fdcioendstarfitcetreddopsuipnigl oinnbDoatyh e1y5e5s(aWnedepkal2e3g).umOsnwere bnoltaecdk.-coOlbosreerdvafteicoesnsannodtleodwonfoDoadyco1n5s5upmrpitoirotno. dAopspinrgoxiinmcaltuedledy f1e5wm,imnuuctoeisda,ftleirqduoids,inagn,d athneimaanlimaallsowaapspeoabrseedrvdeedhaysdrhaytpeodacatnidvweawsitcholldabtootrhedetroeuspcihr.atiTohneasnedobpasleervgautmiso.ns Tpheirssisted Suhntoirltalypptrhoexreiamfattere,lyth3e0amniinmuatledsiepdosdtudroisneg wahneenxatmhienaantiimoanlbwyaaslaalbsoorantootreydaansirmeaclumbent. dVeetteerrinmairnieadn.toAbne peunllamrogneadrlyivneercrwoassisdewtietchtseedvbeyrepaalcpuatteioinn.flTamhmeatciaouns.eofdeath was cOonndDitaiyon1.79Lo(Wwefcokod26c)o,nAsnuimmpatlioNno.wa1s05n5o0t6ed(oGnrDoauy4p1m7a8le()Weweaks s2a6c)riafnidceadt itnhea am.omr.ibund observation interval on Day 179. Approximatel5y to 10 minutes postdose on Day the animal had excessive salivation, labored respiration, and hypoactive and ataxic 179, behavior. With the exceptionof excessive salivation, these findings continued to be observed approximately 3 hours postdose. determined. The cause of the moribund condition was not 26 002662 -_-- ComvanTIcMeTe6-362s28.9s25273 Tthweograodudiptgioinvaeln atnhiemcaolnstrhoaldmnaotetreiwaol,rtAhnyiombaslerNvoa.ti1o0n5s5d2u9r,iwnagstreexaatmmeinnte.d bOynea fleambaolreationry aapnpiemaarlanvcetee,rcilneaarrioarnaolnaDnadyna5sa(lWedieskch1a) drgue,etexocoebssseirvveastailoinvsaotifond,ehaynddraatuidoinbl,etrheisnpiration. `aTnhidsaanntiibmiaolticwsa.s`dTihiasgnaonsiemdalwihtahdprneecouvmeorneidabaynDdatyre1at4ed(WweitehkL2a)c.taAtneidmRailngNeor.'s10so5l5u3t4ion t(rGeartoedu4wpitfhemparlaez)iqwuaanstedli.agNneoistehderwiintfaehcttiaopnewwaosrmtesitnfmeactteiroinald-uerliantgedW.eek 23 and was Clinical observations during recovery were typical of laboratory primates. Oarpehtprheaslemnotledogiyn.ApOppehntdhiaxl2m.ic observations are summarized in Table 4; individual data `wTehreeretewsetrmeatneorioapbhrtehlaaltemdi.c oAbnsiemravaltNioon.s1a0t5t5h2e9W(eGerkou2p61orfeWmealeek) 5w2asexnaomtiendaatsiohnasvithnagt `ienxcarmeiansaetdiomnyse.lBiencataiuosneoftthhiseirsiaghpteorpmtaincennetr,vceoantgetnhietbaalsceolnidnietaionnd,Witeweaks5n2otoepdhtahtatlhmeic baseline and recovery examinations only and is not related to treatment with PFO. Body Weights. `Table5s and 6; Body weightdataare illustrated in Figures individual data are presentiend Appendix 3. 1 and 2 and summarized in C0o.v7a5rmigat/ek-ga/ddjauystwehdemneaconm(pCarAeMd)wbiotdhymawleeisghgtisvewnerteheslciognhttrloyllmoawteerriianlmbaelgeisnngiinvgenat `Week21; the difference was significantat Weeks 23 and 27. In females given 0.75 mg/kg/day, CAM body weights were significantly 19 through 23, and 25 through 27 when compared with lower at Weeks 11 through females giventhe control 16, material. These decreases were likely test material-related. Differences in body weights were not apparent during the recovery period. F(SouomdmCaornysoufmpCltiinoinca.l OFbosoedrvcaotnisonusm)p;tiiondnidvaitduaaalrdeastuamamraeriinzceldudiendTianbtlheesi1n,d2i,viadnudal3 clinical observations in Appendix 2. 2 002663 _-- CovanmIcMeT6Te322w90.s5227y3 Lcoontwrofloomadtceroinaslumapntdi0o.n03wamsg/nkogt/eddays.porTahdeicailnlcyidfeonrcaenoifmalloswifnotohdecgornosuupmspgtiivoennwtahes ggienveernaltlhye choingthreorlimnattheerigarloaunpds agipvpeenar0e.d15toabned t0e.s7t5mamtge/rkiga/ldrealyaatsedc.omDpuarirnegdtroecaonviemray,ls instancesoflow food consumption `control and treated groups. were sporadic and were similarfor animals in the. Clinical Pathology Hthermoautgohlo4g5y;,incdliivniicdaulalchdeamtiasatrrey,praensdenutreindailnysAipsdpaetnadairxe4.summarized in Table7s AdministrationofPFOS was associated with moderately to markedly lower total cchhoolleesstteerroollffoorrmmaalleess aanndd ffeemmaalleess ggiivveenn 00..7155 omrg/0k.g75/dmagy/kagn/ddahyi.ghDduernsiintgytlhiepotprreoattemiennt poenricohdo,letshteereoflfewctasonretvoetarlsecdhowlietshtienro5l wbeeeckasmoefprtohgereensdsiovfetlryeawtomresnet,ovaenrdttihmeee.ffTehcetoenffehcitgh dunecnesrittayilnirpeolpartoitoenisnhcihpotloeasdtemrionliswtarsatrieovnerosfePd FwOitShiwna9s wleoewkersotofttahl ebielnirduboifn tcroenactemnetnrt.atiOofn 0f.or75mamlge/skgg/idvaeyn. 0.T7h5esmeg/pkotge/ndtaiyalaenfdfehcitgsohefrthseertuemstbmialetearciiadl cwoenrceenvterraytimoinlfdo,ramnadlenseitghievren was considered adverse. Palmitoyl CoA Oxidase Determination Palmitoy] CoA oxidase determinations are summarized in Table 46; individual data arc presented in Appendix 5. Resultsofpalmitoyl CoA oxidase determinations were not considered to be related to the test material. Blood Hormone Determination Summary and Individual Blood Hormone Data are presented in Appendix 6. . 2% 002664 -_ 0 CovaSnMceiT6.3e6299-w52273 Estradiol values were generally lower on Days 62, 91, and 182 in malesgiven 0.75 mg/kg/day, although becauseofthe variation in the dataonly the significant. Estrone values were generally higher in allofthe treated Day 182 value femaleosn was Days 37, 62, and 91, although because ofthe variatiionn thedatanoneofthesevalues `were significantly different, and this difference was notapparent on Day 182. `Triiodothyronine values were notably lower onDays91 and 182 in bothmalesand females given 0.15 or 0.75 mg/kg/day. With the single exceptiononDay91ofmales given 0.15 mg/kg/day, all values were significantly lower. There were several other. instances in which the hormone values in treated groups differed from thoseofcontrols, `but these differences were not consistent over time or `between sexes, were notclearly dose-related, and did not. appear to be related to the administrationofthe testmaterial. `During recovery were occasional instances in which the hormone `values intreated groups differed slightly from those ofcontrols,but those differences were not consistent over time or between sexes, were not clearly dose-related, and did notappear to beclearly related to the administrationofthe test material. Apparent differences in the sexual `maturityofbothmalesand females used in thisstudy complicates the interpretation ofthe hormone data. Anatomic Pathology Terminal body weights, absolute organ weights, organ-to-body weight percentages, and `organ-to-brain weight ratios are summarized in Table 47; incidences of `macroscopic and `microscopic observations are summarized in Tables 48 through 51. Individual data are presented in Appendix 5. `Two of four males receiving 0.75 `mg/kg/day (high dose) did not survive to the scheduled terminal sacrifice at Week 27. At the terminal sacrifice, females in the group receiving 0.75 mg/kg/day had increased absolute liver weight, liver-to-body weight percentages, and liver-to-brain weight ratios. In males, liver-to-body weight percentages were increased in the high-dose group compared to the control group. Absolute and relative liver weight increases were. regarded as test material-related. `Among the macroscopic observations, only "mottled" liver was considered test `material-related. "Mottled" livers were observed in the two high-dose males andinone high-dose female. Of the two `males not surviving until the scheduled terminal sacrifice, one had "mottled"and"large" liver. 002665 _-- CovawnMcreTe6232w99-5s227y3 sMaiccrriofsicceodpiatcaenxaumnisncahteidounleodf ianntiemravlasl ysiaecrlidfeidcetdesattmaWteeerikal2r7ealantdeodftohbseertvwaotimoanlseisn the tlihveerr. cIennttrhirleoeobuflflaoruyrohriwgiht-hdoouste rmaelgeasrttdoherleocwataisonhewpiatthoicnelhleuplaatrichylpoebrutlerso.phHy,ypoecrcturrorpihnyg, o2bssdeersvcartiiboendsoifn htehpeamtaolceesl,luwlaasr ohybpseerrtvreodpihnyawllerheigrhe-fdloescetefdeimnalheisg.heTrhleivmericwreoisgchotpsiicn the. high-dose animals. females. Hypertrophy was regarded as test material-dependentinhigh-dose. hCiegnthr-idloosbeulaanrimoarlsd,ifofcucseurhreipnagtoicnetllwuoloafrfvoacuurofleamtailoenswaansdatlswootoesftfmoautremrailaels-.deMpuelntdiefnotcianl ohenpeatoifcfvoaucruloolwe-sdwoesreemanloetsr,eagnadridendaascotnetsrtomlaatnerdiaall-odwe-pdeonsdeenftebmaelcea.use they were found in Microscopic did not have examination of any testmaterial liver taken atbiopsy related findings. on Week 57fromthe high-dose animal `Amtactrhoesrceocpoivceorbysesarcvraitfiiocnes,.onTehoeftobwsoermvaalteiosnasndin otnheeoofthtewroafneimmaallseswedriedonfotadhhaevseions on cthoensliudnegrsedorlteisvtemraatnerdianlo-dreullaetsedi.n tOhneltyholriavcerisc wcaevrietye.xaNmoinneeodfmtihcersoescoobpsiecravlaltyi,oansndwnaos test s`maactreirfiiacle-arenldatuendsocbhseedruvlaetdiodnesatwheraenimmaadles.waTshenohteppraetsiecnvta,ciunodliactaitoinngtohbsaetrtvheedhienptaetrimcitneaslt material related effects were reversible. CONCLUSION `TmraelaetamnedntfewmiatlhePcFyOnSomboylgoruaslmcoapnskuelyefsoartadtosleeassutp26towe0.e1k5smigs/gkegn/edraayll.y Cwleilnli-ctaollearnadted in lpeaatshtol2o6giwceaelkfsinodfitnrgseactomnesnitdweererde tfooubnedas1s0obceiarteevderwsiitbhlethdeurtirnegatame5n2t-woefePkFrOeScaofvieerryat ipser0i.o1d5. mBg/aksge/ddaoyn.thDeodsaetaanparleysseenste(dprinovtihdiesdrebpyortth,ethSeponnos-oorb)searnvdabellee-catdrvoenrsmei-cerfofsecctoplyevel results (provided by PAI) are forthcoming. 30 002666 0YCove an3eMs 6T362s 92.52273 SIGNATURES ElSitzuadybeCtohoArdiDniastcohr,BA Covance Laboratories Inc. PeStteurd]y.DiTrheocmtofrord,PD Covance Laboratories Inc. Pe rr ' 31 002667 _-- `CovamnMcTTe-6e362w299.s252773 REFERENCES Dunnett, C. W., "New Tables for Multiple Comparisons witah Control," `Biometrics, 20:482-491 (1964). LSteavteisntei,csH,.(,e"dRs)ob1u. sOtlkTienstestfaolr,ECqhu.al2i5t,ypopf.V2a7r8i-a2n9c2e,s,S"taCnfoornd UtnirveristiobtyPurPorbteasbsii:lioSttynaanfnsordd, California (1960). In`WEinxepre,rBi.me.n,t"aDleDseisgingna,ndSeAncaolnydsiEsd.ofCShi.ng3l,ep-pF.ac1t4o9r-2E6x0p,erMicmGernatws-,H"iStlalt:istNiecawlPYrionrcki,ples New York (19712). `SWeicnoern,dBE.d.J,.,C"hA.na1l0y,spips.o7fC5|o2v-a81r2i,anMcceG,r"aSwt-aHtiisltli:calNPreiwncYipolreks,iNneEwxpYeorirmke(n1t9a7l10D)e.sign, 2 002668 -_-- CovamnMcTeT6e322w05.s272y3 OPHTHALMOLOGY REPORT aOnpdht5h2a.lmTihcereexawmeirneatnioonosphwtehraelmdiocnoebbseefrovraetiionnitsiaattitohnoefWtereekat2m6enotraWnedcdkur5i2ng Weeks 26 examinations that were test material-related. noted as having increased myelination of the Animal No. 105529 (Group 1 female) right optic nerve at the bascline and was ``wWaesenko5t2edaopthtthhealbmaisceleixnaemainnadtrieocnosv.erByeecxaaumsientahtiisoinssaopnelrymaannedinstn,octonrgeelnaitteadltcootnrdeiatitomne,ntit with PFOS. There were no test material-related observations at anyofthe examinations. DSitpelpohmeantIe.,BiAsCtnVerO, DVM Date Veterinary Ophthalmologist } 002669 -_ 0000 CoSnncie 3T2e699.25273 PATHOLOGY REPORT SUMMARY `The purposeofthestudywasto assess the effectofthe test `material, Perfluorooctane Sulfonic selected Acid Potassium Salt (PFOS), biochemical parameters when on critical enzyme administered daily levels, hormones, and other by capsule to cynomolgus `monkeysfor at least 26 weeks. 0.15, and 0.75 mg/kg/day. One The test material was. administered at dose levels of 0.03, male given 0.75 mg/kg/day died on Day 155afterdose paodomrihneialstthr. atainodn one male given 0.75 mg/kg/day was sacrificed on Day 179 due to Administration of PFOS was associated with `moderately to markedly lower total cholesterol for males and females given 0.75 mg/kg/day and high density lipoprotein cholesterol formalesand females given 0.15 or 0.75 mg/kg/day. During the treatment period, the effect ontotal cholesterolbecame progressively worse over time. The effect oncholesterol was reversed withi5n weeksoftheendoftreatment, andtheeffect onhigh density lipoprotein cholesterol was reversed within 9 weeksofthe endoftreatment. Of `uncertain relationship to administration of PFOS waslower total bilirubin concentration for males given 0.75mg/kg/dayand `higher serum bile acid concentration for malesgiven 0.75 mg/kg/day. These potential effects of the test material were very mild, and neither `was considered adverse. Two of four males receiving 0.75 mg/kg/day did not survive to the scheduled terminal sacrifice at Week 27. Atthe terminal sacrifice, females in the group receiving 0.75 mg/kg/day hadincreased absolute liver weight, liver-to-body weight percentages, and liver-to-brain weight ratios. In males, liver-to-body weight percentages were increased in the high-dose group compared to controls. These absolute and relative liver `weight increases were regarded as test material-related. Among the macroscopic observations, only "mottled" liver was considered test material-related. "Mottled" livers `were observed in the two high-dose males and in one `high-dose female. Of the two `males not surviving until the scheduled terminal sacrifice, one had "mottled" and "large" liver. Microscopic examination of animals sacrificed at Week 27 and the two unscheduled sacrifice males yielded test material-related observations in the liver. In three of four high-dose males there was hepatocellular hypertrophy, occurring either centrilobullary or hl 002670 _-- CovanMceTT6a3e2m9s.w273 without regard to location within hepatic lobules. Hypertrophy,asdescribed in the males, was also observed in four of four high-dose females. The microscopic observationsof hepatocellular hypertrophy were reflected in higher liver weights in the `high-dose females. Hypertrophy was regarded as test `material-dependeinnt high-dose `animals. Centrilobular or diffuse hepatocellular vacuolation was also test `material-dependent in `high-dose animals, occurring in twooffour females and twooffour males.Multifocal hepatic vacuoles were not regarded as test material-dependent because they were found in `oneof four low-dose males, andin acontrol and alow-dose female. dMiidcrnoostchoapviec aenxyamtiensattmiaotneroifall-irveelrattaedkefninadtibngiso.psy on Week 57 fromthehigh-doseanimal Forthe recovery sacrifice at Week 79, only high-dose animals were necropsied. Oneof twomalesand one of two females did not have macroscopic observations. The observations in theotheranimals wereofadhesions on the lungs or liver and nodules in the thoracic cavity. Noneofthese observations was considered test material-related. Only livers were examined microscopically. No test material-relatedobservationswere made. The hepatic vacuolation observed in terminal sacrifice and unscheduled death `animals was not present, indicating that the hepatic test material-related effects were reversible. METHODS Four groupsofmale and female cynomolgus monkeys were administered the test `material daily by capsule at a dose level of0 (control group; received capsules containing lactose), 0.03, 0.15, or 0.75 mg/kg of body weight/day (mg/kg/day). There were six `animals/sex in the control group and the groups given 0.15 or 0.75 mg/kg/day; two animals/sex in these groups were designated as recovery animals to be observed for approximately 26 weeks posttreatment. The recovery period was laterextendedan `additional 26 weeks. There were four animals/sex in the group given 0.03mg/kg/day. One male given 0.75 mg/kg/day died on Day 155 following dose administration, and one `male given 0.75 mg/kg/day was sacrificed on Day 179 due to poor health. 002671 Cone 3T2a9m2s2?3 Blood and urine were collected for hematology, clinical chemistry, andurinalysistests `once before initiation of treatment (Week -4), on Days 37, 62, 91, 153, and 182 and on Days 217, 245, 364, 456, and 546 (recovery animals). High density lipoprotein cholesterol was not measuredatthe clinical pathology intervals before Day 153. The terminal sacrifice and necropsy occurred on Days 184/185;`macroscopic observations were recorded, organ weights were obtained, and tissues were placed in fixativeas specified by the protocol. Samplesoftheright lateral lobeofthe liver from each animal were frozen for analysisofpalmitoyl CoA oxidase activity. Sections of liver fromeachanimal wereprocessed to block for electron`microscopyandshippedto the Sponsor's designee for ultrastructural evaluation. Samples of liver from each animal `were frozen and shipped to the Sponsor for PFOS determination. The gallbladders and bile from each animal were collected, frozen, and shipped to the Sponsor. Sampleofs pancreas, left and right testes, and the left lateral lobeofthe liver were preservedforcell proliferation evaluatiboyn the Sponsor's designee using proliferation cell nuclear antigen. Light microscopic examinations were done on collected tissues from control animals and animals given 0.75 mg/kg/day. In addition, liver and thymus from all animals given 0.03 or 0.15 mg/kg/day and spinal cordgraymatter from females given 0.03 or 0.15 mg/kg/day were examined by light microscopy. During Week 57, liver biopsieswereperformed on therecoveryanimals thathadbeen given 0.75 mg/kg/day. The tissue was divided into four samples. Throfetheesamples `were used for light microscopic examination, ultrastructural examination by the `Sponsor's designee, and PFOS analysisbythe Sponsor, respectively. The fourth sample `wasfrozen and shippedtothe Sponsor for possible analysis. At theend of the recovery period (Week 79), the control animals were returned to the Covance stock colony, the `animals that had been given 0.15 mg/kg/day were transferred to a subsequent study for the Sponsor (Covance 6329-268) following liver biopsy for PFOS analysis by the `Sponsor, and the animals that had been given 0.75 mg/kg/day were sacrificed and necropsied. At this recovery necropsy, macroscopic observations were recorded, and three samples ofliver were collected for light microscopic examination, ultrastructural `examinationby the Sponsor's designee, and PFOS analysis by the Sponsor, respectively. Statistically significant differences cited in the Results and Discussion are based on comparisons between the control and treated groups. * 002672 RESULTS AND DISCUSSION Connee 6a3r29s.2)3 Mortality Two of four males receiving 0.75 mg/kg/day (high dose)ofthe test material did not survivetothe scheduled terminal sacrifice at Week 27. One was founddeadon Day 155. `The other was sacrificed on Day 179duetopoor health. The causeofdeath in the animal found dead was judged to be pulmonary necrosis with severe acuteinflammation. Microscopically, the pulmonary lesion appeared to be an acute exacerbation ofa chronic lesion. The cause of death in the moribund animal was not determined. All other animals survived to their scheduled sacrifices. Clinical Pathology Week 4. Resultsofclinical pathology tests indicated no obvious. group or individual health abnormalities. Several animals, however, had mildly elevated alanine aminotransferase activities at Week -4. These animals were primarily females and were dispersed among allofthe groups. By Day 37, their alanine aminotransferase activities `were no longer elevated. The transient increase in this liver enzyme was considered `lickyenloymdoulegutso msoubnckleiynsic,alanidnfdeicdtinoontwnietghatHievpealtyiitmA ipsacvtireuvsa,luaactioomnomfotnhienfteescttimoanteirnial. Days 37, 62, 91, 153, and 182. Although there were several statistically significant or otherwise notable differences for clinical pathology test results between the control and treated animals, most of the differences were inconsistent over time or similar to differences present before initiation oftreatment (i.e., Week ~4). The only differences considered to represent effectsofthe test material were lower total cholesterol for`males and females given 0.75 mg/kg/day and lower high density lipoprotein cholesterol for `males and females given 0.15 or 0.75 mg/kg/day. The effect on total cholesterol became progressively worse over time. At Day 182, the mean total cholesterol concentrations for the males and females given 0.75 mg/kg/day were approximately 68% and 49% lower, respectively, than those for the control males and females. The effect on high density lipoprotein cholesterol, measured only at Days 153 and 182, was proportionately greater than that for total cholesterol. At Day 182, the mean high density lipoprotein cholesterol `concentrations for the males and females given 0.75 mg/kg/day were approximately 79% and 62% lower, respectively, than those for the control males and females. 7 002673 _-- TrCovaenMceTs62329s05.273 `cOofncuenncterrattaiionnrefloartmiaonlsehsipgitvoeandm0i.n7i5stmrga/tkigo/ndoafyP(FstOaStiswtiacsalllyowleorwetortaolnbDilaiyrsub9i1n, 153, and (1s8a2t)isatncdalhliyghheirghseerrounmDbialye a1c8i2d ocnolnyc)e.ntTrhaetisoenpfootrenmtailalesefgfievctesno0f.t7h5emtge/sktgm/adtearyial were `very mild, andneitherwas considered adverse. Statistically significant differences for other test results were consideredincidentaland `unrelated to administration ofthe test material. These differences were inconsistent over time or were similar to differences that existed before the initiationoftreatment. Unscheduled sacrifice Animal No. 105506, a male given 0.75 mg/kg/day, was sacrificed on Day 179. The most notable findings for this animalwere a moderateneutrophilic leukocytosis, mildly increased serum bile acid and globulin concentrations, and c`hmooldeesrtaetreolly. to markedly decreased total cholesterol and high density lipoprotein Days 217, 245, 364, 456,and546 (Recovery animals). The effect on cholesterol was reversed by Day 217, and the effect on high density lipoprotein cholesterol was reversed bocycDuraryen2c4e5.ofTthoexriec were no effects other findings considered to represent persistent or delayed Anatomic Pathology Organ Weights. At the terminal sacrifice (Week 27) femalesinthegroup receiving 0.75 mg/kg/day (high-dose) had increased absolute liver `weight, liver-to-body weight percentages, and liver-to-brain weight ratios. This increase was regarded as test materialrelated. In females the absolute pancreas weights were higher inanimalsreceiving 0.03 mg/kg/day (low-dose) than in those receiving 0 mg/kg/day (control). Increased pancreatic weights were not regarded as test material-related. Tn males, liver-to-body weight percentages were increased in the high-dose group compared to controls. The increase was regarded as test material-related. Left adrenalto-body weight percentages were also increased in high dose males compared to controls. `The significance of the increase left adrenal weight percentage was not known. Organ weights were not taken at the recovery sacrifice at Week 79. 38 002674 CovanMceT6e32299-52273 Macroscopic Observations. Twenty one of 30 animalsatthe terminal sacrifice had at least one macroscopic observation. One of the observations was obesity; it was not regarded as pathologic. Thus, 20 animals had macroscopic observations.Ofallthe `macroscopic observations, only "mottled" liver was considered test material-related. "Mottled" liver was observedinthe two high-dose males and in one high-dose female. `Theother observations included adhesions and darkorred foci in any of several organs. Among high-dose females, eachhadat least one observatoifoandarkorred focus. In `addition, adrenal cortices of the two high-dose males and two of four high dose females `were recorded as dark. In one high-dose female (Animal No. 105540), the red focus in the lung correlated microscopically with hemorrhage. There were no microscopic correlatesforthe other observations. Hence, dark orred foci were consideredtoononspecific tobeconsidered test material-related. Additionally, dark and red foci were. `observed in animals in other dose groups. Dark adrenal cortices did not have a `microscopic correlate; they, too, were not considered testmaterialrelated. Ofthe two males not surviving until the scheduled terminal sacrifice, one did not have. any macroscopic observations. The other (Animal No. 105509) had several observations. Ofthese observations, those in theliverwere considered test material related. The liver observations were "mottled" and "large". For the recovery sacrifice at Week 79, only high dose animals were necropsied. One of2 `males and oneoftwo females did not have macroscopic observations. The observations inthe other animals wereofadhesions on the lungs orliverand nodules inthethoracic cavity. None of these observations was considered test material-related. Microscopic Observations.Atthe terminal sacrifice, all tissuesfromthe control and high dose groups were examined. All tissues were examined on the two high-dose males not surviving until the terminal sacrifice; the microscopic data were grouped withthatof the terminal sacrifice animals. Liver, thymus, and spinal cord were examined from `animals in the low and intermediate dose groups. In three of four high-dose males there: `was hepatocellular hypertrophy, occurring either centrilobullary or without regard to location within hepatic lobules. Hypertrophy, as described in the males, was observed in four of four high-dose females. The microscopic observations of hepatocellular hypertrophy were reflected in higher liver weights in the high-dose females. Hypertrophy was regarded as test material-dependent in high dose animals. 002675 - 0 CovnNceiT3e2699.25273 Centrilobular or diffuse hepatocellular vacuolation was also test material-dependent in `high-dose animals, occurring in two of four females and two offour `males. Multifocal `hepatic vacuoles were not regarded as test material-dependent. Multifocalhepatic vacuoles were found in one of four low dose males, andin a control and alow-dose female. `Two other observations were suggestiveofpossible test material effects upon `examination ofonly control and high-dose animals. When tissues were examinedfrom the low and intermediate dose groups as well, test material-dependency was not supported. In females, thymic atrophy was more pronounced in dosed animals than in controls. However, there is no clear dose-dependentpatternand thus, thymic atrophy cannot be considered test material-dependent. Intraneuronal pigment was observed in the spinal cord gray matter of two of four high dose, four of four mid-dose, three of four low-dose and one of four control females. And thus, spinal cord pigment was considered unrelated to the test material. Microscopic examination of liver taken at biopsy on Week 57from the high-dose animal did not have any test material related findings. At the recovery sacrifice, livers were examined from `high-dose animals. No test `material-related observations were made. The hepatic vacuolation observed in terminal sacrifice and unscheduled death animals was not present, indicating that the hepatic test `material-related effects were reversible. In those animals from which all tissues were examined, the width ofthe femoralgrowth plates were assessed as a general indication of the maturityofthe animals. Basedonthis indicator, males were less mature than females. Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) Robert A. Leedle, DVM, PhD Diplomate, ACVP Date Date " 002676 -_-- CovmnMcrTT66e322s9025s273 (COMMENTS ON THE DATA Vdaatraioiunsthmiosdsetludsyo.fcBaelccualuasteordsi,ffceormepntutmeordse,lasnrdocuonmdopfuftoerr ptrroungcraatmesnwuemrbeerusseddiftfoeraennatllyy,ze svlailguhetslyinfrsoommtehotsabeleisn (ot.hge.,r mtaebalness,,fsrtoanmdianrddidveivdiuaatliloyncsa,locruliantdeidviddautaa,l ovralfureos)m mstaatyisdtiifcfaler adinfafleyrseinscedsa.ta. Neither the integritynorthe interpretationofthe data was affected by these `Some tabular data were compiled using Excel Version 7.0 software. `mTgh/ekgun/idtasyf.orthe dose levelsonthe data collection system (PTS) summary tables are `oTbhseernvuamtbioenrsofreaflneicmtasltshelinsutemdbienrotfhae nhiemadailnsg aosfsithgenesdutmomeaarcyh tgarboluefpoartctlhiensitcaarltofthe study. `cTohnedistuimonmawrays otabbsleefroverdclwiintihcaolutobrseegravradttioontsheisnpdeicciaftiecs ntahteunreu,msbeevreroiftay,niremvaelrssibfiolritwyh,ich a numberofincidences/animal, or the length of time the condition persisted. Oobnsleyrvoabtsieornvsattiabolnes.other than normal are indicated on the summaryofclinical Tinhdeicsapteecdifwiictdhetaai"lCs"fcorancobmemfeonutnsd aitn tthhee einnddiovfideuaaclhclgirniocuapl ofobrseeravcahtisoenx.s tablesthatare: `tThheestdaarytooffinaisttiuadtyiowneocfktr(ce.agt.m,eantboisdy"Dwaeyig1h,tWreeecokrd1e.d" oBnoDdayywe1iigshctodnastiadearreedenatWereeedakt1 body weight, a body weight recorded on Day 8 is considered a Week 2 body weight). Dpiaftfheorleongcyesarienetxhpelapoipnueldaotniotnhesiiznedi(vNi)douanltdhaetsautmabmleasr.y tablesforclinicalandanatomic Rinetseurlvtasloafpcpleianricoanl pthaethionldoivgiydsuaalmpclliensiccaolllpeacttheodlofogryatnaibmleaslfsosratchriefinceexdtastcahneduunlsecdhiendtuelrveadl. a`nTahleyseserse.sultsare not on the summary tables and are not included in the statistical Statistical analyses were not done when there were two or fewer values for a parameter. : a 002677 -_-- CovanMcTeT-6e632w299s25273 COMMENTS ON THE DATA (continued) "Baiblesovloulteumoergiasnlowceaitgehdto(ngrtahmesi)n"d.ivTihduealvaalnuaetolimsitcedpfaotrhobilloegiystnaobtleauwnediegrhtthaenhdeiasding of designated witha unit of mL. VSeotmeericnlairnyicSaelrovbisceersvfatoiromnasnddiswciulslsbeed airncthhieveredpwoirtthwtehreeradowcudamtean,tbeudtotnhetyhedoRenqouteasptpefoarr on the individual clinical observations tables. 2 002678 _-- CovanSucMe r6T362e29252s73 `CODES, ABBREVIATIONS, AND UNITS General Codes and Abbreviations Codes for Clinical Pathology Abbreviations and Units for Clinical Hematology Abbreviations and Units for ClinicaCl hemistry Abbreviations and Units for ClinicalUrinalysis Codes for Anatomic Pathology Note: The following liofscotdess, abbreviations, andunitsare used by Covance. Some, but not necessarily all,ofthis information may be needed for this report. 3 002679 -_-- WK N Mean; MEAN CAM SD; S.D.; STAND DEV; STANDARD DEV; sd * NA P c UDINSSPCAHTECDH TBW # VETEXAM AMOBS 30-9008 General Codes and Abbreviations CovanmIcMeT6Te3622w9-9s252737 Week Number ofmeasurements Arithmetic mean ina group CSotvaanrdiaartde-daedvjiautsitoend mean t`hGermouepamneoafnthise sciognntirfoilcganrtoluy pdi(ffGerroenutp f1r)oamt p<005 PNroesveanltue; not applicable; not present `Comment found for each sex at the endofeach group Unscheduled `Ombosdeurlveaotfiotnhsetdraatnasfceorlrleedctfiroonmstyhseteimnltiofethe n`neeccrrooppssyy.mOobdsuelrevaftoirornesfearreendcuepdluircaitnegs of tTheermliasntailn-bloifdeyowbeseirgvhattions. Number Veterinary examination Ombosrenrivnagtioobnsserrveactoirodneidntdeurrvianl.g the daily, ``minOutebposstdreoesceroorbdsveedrvdaautriitonngitinhteeor3va0ln.tos90 " 002680 _-- NS QS/QNS NR FS sc SH H SL L st 1 NF u DDTB/DOT be] TE RE EE SE PC PD Pr PL PA co HB PLASMO NO AGG FR UTD NO COAG Codes for Clinical Pathology CovanMcrTe-e663w2299s252773 GENERAL CODES No sample NQuoanrteipteyatno(tsasmupflfieciveonltume not sufficient for repeat analysis) Fibrin strands Sample clotted HSleimgohtllyyzbeedmolyzed LSliipgehmtilcy lipemic Slightly icteric Ieteric AUnnsicmhaeldnuoltedf/asmtoerdibund bleed ADineidmadlurdiinegd bolneetdesitng ``TTeecchhnniiccailanerjruodrg(miennsttrtuomernetpeoarttteecsthnician error that results in supnialclecde,pteanbtlreyodfatian,vael.gi.d, duantaac)ceptable instrument output, sample Rspeeclolridnginegrreorr,roirnc(orrerceocrtdeddatien)correct data, .g., wrong number, SEantmrpylienrrgoerr(rionrcorrect keyboard entry) Platelets clumped PPllaatteelleettss dineccrreeaasseedd Platelets large CPloaltoerleitnstaerpfpeeraers waidtehqutaetste HPelianszmobdoidiuems observed FNroacatgigoruesgation NUnoabcolaegutoladteitoenrmine . fis 002681 -_-- CovamnMcTeT62e3299w-5227y3 Codes for Clinical Pathology (Continued) RESULTS NOT INCLUDED IN STATISTICAL ANALYSES SHaemmpolleyszefdrocmlianniciamlalcsheamtiusntsrcyhoerducolaegduilnatteirovnalssamples APcrtoitvhartoemdb ipanrttiiamletsh(rPoTm)bogprleaastteirntthiamne5s0(sPeTcTo)n ds gre at er than 110 seconds Bleed times (BLETIME) greater than 30 minutes CODES FOR BLOOD CELL MORPHOLOGY p`Tohiekifloolclyotwosiinsg (sPcaOlIeK)w,aspoulsyecdhtroommaesaisaur(ePOtLhYe)d,eghryepeoocfharnoimsaosciytao(sHisYP(OA)N,ISaOn)d,toxic neutrophils (TOXNEUT): Scale f 1 2 3 -- 4 Degree Normal for the species. Sight Moderate Marked Not appplliacabtlee: Presence Not present Rare Few Moderate MMaanyy A Pale B Straw C Yellow --yD Do arkyew low Color E Amber F Bown G Red HHOGrween URINE APPEARANCE 1 Black P Blue/green Q Blue RROOwragngee Clarity 7 Clear K Hazy L Cloudy Miscellaneous M Debris O Feces 0 4% 002682 -_-- CovanmIcMeTT6e362s99-2s5273 Codes for Clinical Pathology (Continued) URINE CHEMISTRY MULTISTIX STRIP Urine Glucos_e = Negative + 100mg/dL +++++ 255000 mmgg//ddLL +h 1,000mg/dL HH 22000mgdl __ UrincRetone ~ Negative + Smg/dL +++++ 4105 mmgg//ddLL +++ 80mg/dL Here 160mgldL Urine Blood = Negative + Small +++++ MLaordgeerate Urine Urobilinogen = 02mgdL ++ 21mmgg//ddLL ++++++ m4mgg//ddLL (I' mg = approximately 1 Ehrlich unit) Urine Bilirubin = Negative +++ MSmoadlelrate +++ Large `URINE SEDIMENT Cells, Crystals, Casts, andCoB mmea nts cteria A Amorphousurates B Amorphousphosphates C Uricacid DTriple phosphates E Calcium oxalate F Calciumcarbonate Q Sperm R Fecalcontamination S$ Pinw ovoafr oum nd T Pinwormlarvae found U Parasite ova found 0 Notpresent. 1 Few 2 Moderate 3 Many G Granular casts HHyaline casts I Cellularcasts J Waxy casts K Unknowncrystal ----P_ oMuecorusetharedads 0 1 Notpresent 2 1-5perfield 3 6-10perfield 4 11p-er2fie0ld >W>2p0eperrffieeldd . 47 002683 -_-- CovanMcTe 66a322n00-s522y73 Abbreviations and Units for Clinical Hematology Test HReemdogbllooobdicnell count Hematocrit Mean corpuscular volume MMeeaann ccoorrppuussccuullaarr hheemmoogglloobbiinn concentration MPleaatenleptlcatoeulnett volume Reticulocyte count HAbesionlzubteodryetciocuunlotcyte count PErryotthhrroocmybtiensteidmiementation rate. `AcTthirvoamtbeidn ptairmteial thromboplastin time FiAbcrtiivnaotgeedncoagulation time PFliabtreilne/tfaibgrgirneoggaetniodnegradation products Collagen AlApdhean2o-sainnteipdliapshmoisnphate Bleeding time PMleatshmeamohgelmoobgilnobin EMysetliomiadt/eedrmyytehlrooiidd/reartyitohroid ratio D`iWfhfietreenbtlioaoldbcleololdcoceulnltcount Nucleated red blood cell count Corrected white blood cell count Segmented neutrophil count Band neutrophil count Lymphocyte count Monocyte count Eosinophil count ABnaissoapchyitlocsiosunt PPooilkyilcohcryotmoassiisa Abbreviation (Units) HRBGCB ((EG6//DLU)L or X10%uL) HCT (%) MCV (FL) MCH (PG) ~~ MCHC (%) MPLPTV((EF3L/)UL or X10%L) RETIC (%) RHEETIINCZ ((E%3)/UL or X10%/uL) PETSR(S(EMCM)/HR) PTT (SEC) TT (SEC) AFBCRT ((MSEGC/)DL) FDP (UG/ML) PPAAGGGG//ACDOPL ((%%)) ANTIPLAS (%) BLE TIME (SEC) PMLEATHHGGBB ((%M)G/DL) M/E RATIO EST ME RATIO WBC (E3/ULor X10%/uL) NRBC (/100 WBC) COR WBC (E3/UL or X10%/uL) N-SEG (E3/UL or X10%/uL) and % N-BAND (E3/UL or X10%4L) and % LYMPH (E3/ULor X10%4L) and % MONO (E3/UL or X10*/uL) and % EOSIN (E3/UL or X10/uL) and % BANAISSOO((E131/,U2L3)or X10"/4L) and % POLY (12,3) POIK (+123) " 002684 -_-- CowvarnMceeT6w39295s22773 Abbreviations and Units for Clinical Hematology (Continued) Test HHoywpeolclh-rJoolmlaysibaodies `BTaosxoipchineluitcrsotpihpipllsing Atypical lymphocytes AAqquueeoouuss wwhhiittee bblloooodd cceellll ccoouunntt ((lreifgthetyeey)e) Abbreviation (Units) HHIYBPOOD(Y12(-.13,)2.3,4) BTAOSXTNIEPU(T1.(2-.13,)23,4) ATYPLYM (-1.2.3.4) LREEYYEE ((WWBBCC//UULL)) . wo 002685 -_-- CovaMnIcTMeTe6032w299-s252T73 Abbreviations and Units for Clinical Chemistry Test Glucose UUrreeaa nitrogen CrTeoattalinpironteein Albumin GAllobbuumliinn/globulin ratio DTiorteacltbbiililriurbuibnin Indirect bilirubin Cholesterol Triglyceride TUorteaalnliitpriodgsen/creatinine ratio Phospholipids LHiogwh--ddeennssiittyy lliippoopprrootteeiinncchhoolleesstteerrooll Uric acid AAslpaanritnaetaemainmoitnroatnrsafnesrfaesrea:se Alkaline phosphatase S`oGrabmitmoal gdleuhtyadmryo]getnraansseferase Lactate dehydrogenase AmCryelataisneekinase Lipase Palmitoyl CoA oxidase: Calcium Tonized calcium Inorganic phosphorus PoStoadsisuimum Chloride Magnesium Zinc Strontium Tron Abbreviation (Units) GULNU(M(GM/GD/LD)L) UREA (MG/DL) CREAT (MG/DL) TPRO (G/DL) ALB (G/DL) GLOB (G/DL) A/G RATIO TBILI (MG/DL) D BILI (MG/DL) IBILI (MG/DL) CHOL (MG/DL) UTRNI/GCR(EMGA/TD(LR)ATIO) TLIPIDS (MG/DL) P LIPIDS (MG/DL) HDL (MG/DL) LDL (MG/DL) UASAT(IMSGG/ODTL()IU/L) ALT/SGPT (IU/L) ALK PHOS (IU/L) GSDGHT ((IUU/LL)) LDH (IU/L) CK (UL) ALIMPYALSAES(IEU(LT)UL) CPCAO(AMOG/(DILU)/G) ION CA (MG/DL) TPHOS (MG/DL) NA (MMOL/L) K (MMOL/L) CMLG((MMMEOQLI/LLo)r MG/DL) ZSRN ((MMGGI/LL oorrPPPPMM)) FE (UG/DL) 50 002656 -_-- CovanMceTT66e322w99-s52273 Abbreviations and Units for Clinical Chemistry (Continued) Test TEoxtcaelssiriornonbinding capacity P`eUrncbeonutndiriornosnatbuirnadtiinogncapacity Plasma cholinesterase RBerdaibnlochoodlicneelsltcehraosleinesterase HiCpaupdoactaemppuutsamen CFerroenbtealllcuomrtex BSiecarrubmohneamtoeglobin FSeecraulmbiblieleacaiciddss. Average fecal weight Fecal bile acids (calculation) Osmolality Electrophoresis Abbreviation (Units) TEIXBFCE((UUGG//DDLL)) UIBC (UG/DL) FE %SAT (%) CHEP (MUML) CCHHEEBR ((MMUU//MMLL)) HCIAPUPDOPCUATM ((UUMMOOLL//GG)) FCECROERBTEELXL((UUMMOOLL//GG)) SBEIRCAHRGBB((MMMGO/LD/LL)) FSBBAA ((UUGM/OMLL/)L or MG/DL) FCC WGT (G) FBA (MG/Day) (OSMO (MOSM/KG) Albumin AlAplphhaa--21--gglloobbuulliinn Beta globulin H`iGgahm-mdeangsliotbyulliipnoprotein Low-density lipoprotein InsuVleirny-low-density lipoprotein CoArdtriensooclorticotropic hormone Glucagon TTrhiyiroodxoitnheyronine Creatine kinase isoenzymes BB MMMB E ALB (G/DL) EEAA-21((GGD/DLL)) E BETA (G/DL) EE-GHADLMM(%A) (G/DL) E-LDL (%) IEN-SVULLDILN((%)UUML) ACTH (PG/ML) GCOLRUTCIASGOOLN(U(GP/GM/LM)L) T3 (NG/DL) T4 (UG/DL) CK-BB (UL) CCKK--MMBM ((UULLL)) 51 00657 -_-- : Abbreviations and Units for Clinical Urinalysis CwovainMcreTe3229w9.52s273y Test 8Urhionuervuorliunemevolume USpreicniefiocsmgorlaavliittyy Quafnltuiidtaptriovteeiunrinary/cerebrospinal Urine protein excretion UriUnreincehepmHistry Multistix strip UUrriinnee gprloutceoisne UUrriinnee bkieltiornuebsin Urine blood Urine urobilinogen Urine reducing substances Microscopic-examination ofurine sediment `RWehditbelboloodocdelclesllpseprehrihgihg-hp-opwoewrefrieflideld Epithelial cells per high-power field Bacteria per high-power field Casts per low-power field Crystals per low-power field Abbreviation (Units) 8UHVROLVO(LML()ML) SPGR U OSMO (MOSM/KG) QUAN PRO (MG/DL) PROEXC (MG) UPH UUGPLRYO (MG/DL) UUBKIELTI U BLOOD UROBILI URE SUB RWBBCC((PPEERRHHPPFF)) EPITH (PER HPF) BACT (PER HPF) CASTS (PER LPF) CRYSTALS (PER LPFI or CUormimneenaptpsearance PER LPF2) CUROIMNMEEANPTPSI or URINE APP2 Miscellaneous Codes and Abbreviations for ClinicalPathology Fecal occult blood Fecal parasite detection Not applicable Not applicable `Hemolytic potential Not applicable 52 002688 -_-- Code T u D M PIO 6 EX NOT TAKEN MISSING UNSUITABLE AEUXTCOLLUYDTEIC Codes for Anatomic Pathology M`TCovaenMceTw62320s9-527273 Definition ANIMAL DEATH CODES Terminal sacrifice FRoeuconvdedreyasdacrifice Moribund Donated `Transferred to Covance 6329-268 MACROSCOPIC CODES I`nOdrigcaantweestihgahttonrogtatnakweeni;gehxtpilsaenxatciloundegdivfernoimncnaleccurloaptsiyonnsotes Organ missing or lost Organ technically unsuitable for weighing `OWregiagnhtauwtaoslytzaekedna,nbdutcowualsd not be weighed excludedfromall calculations MICROSCOPIC CODES BC-odes Prefacing NeopPlraismtaircyF,ibnedniinggnsneoplasm MN- MPertiamsatrayt,icmanleiogpnlaanstmneoplasm I X- OLotchaelrlnyeionpvlaasisvme neoplasm Distribution of Findings Focal Diffuse Multifocal b = 002659 -_-- CovaMnIcTMeT6e632w299-s25273 Codes for Anatomic Pathology (Continued) Code Definition Grades 1 for Severity or Amount Minimal -theleast amountof changethatcan be observed withthe 2 light microscope: Sligh-t lessthanaverage amountof change, but readily discernible as abnormal 3 Moderate lesion - the average amount of change thatis expected fora 4 pMoosdseirbalteelloyssseovfefruen(ctmiaornkeodf)th-eaamfaferckteedd caemllosuonrtoofrgcahnasnge with 5 Stheevearfefe-categdrceealtl aomroourngtaonfscahnadnfgreeqwuientthlpyrionbvaobllveeslolsasrgoefafruenacstioofn of the organ TOLther Microscopic CoTdoetasl P Finding present - Finding not present Abbreviation LF IRNT GL STOMACH, GL STOMACH, NONGL SALIV GL, MANDIB LN, ANT MES/PANC AUDITORY SEB GL HLAECMRAITMOANLEGOLPALNADS,IAEX LACRIMAL GL, INT CAVITY, ABDOM SALIV GL,PAROTID LN, TRACHEOBRON THYROID/PARA TISSUE ABBREVIATIONS Definition Left LRiygmhpth node Gland Glandular stomach Nonglandular Mandibular salivary gland AAnutdeirtioorrymseesbeancteeroiucs/gplaanncrdeatic lymph node Exorbital lacrimal gland Hematopoietic neoplasia Internal lacrimal gland Abdominal cavity Parotid salivary gland `Tracheobronchial lymph node `Thyroid with parathyroid 54 002690 . _-- . Tt ee ST T " T aA TaWANN, m fe ell . X rel IIrl Re Ear pa ---- % ares Y 8 omSy Wg i ens NA NA Tr Ty JaTri T olpy Ee } go . & | me i in cos 2 g == : prada 58 . ; 8 & 8 3 &  % 2 8 2 @ 8 8 " 3? g 3 8 P & maz a 8 "Boh m0 oB ob oK BOB oho a oh dh i ish 2 pil E EEE EE 3 2B oH OH Hd 0B OB OB OH OB .g 33 a 8 8 n 8 3 8 mas ts in sc 1 88 3z TM 3 ob ob koh dn ku 5nN 2 38 , g | . Bod bob db ds 3 n n So g 32 ; Fw we BOT toy ORR OR a we ame g i5 Foon na Ta SW SL LUT me ee wen we < 23 5% u Hho oa BT EE Ed 2 3% Tho ofl WA Ra BEL GME en em mm we g ph Tew we BOT OR ORT BL TC Im 2 1 8 " Fa of 5 A Whe BSL ABE TM ee oo mm me 38B ws Thaw ff BOT OR ORR WILTON 388 ws Soe o lnm worn a Foon nf 5 TL Wa BL MRC nm me wen we 8 8 8 0 | I Fa alfa BTR ORO WML TOE g83 2 N She off ET AL EW. BFL uBR 0 0M mo sem we 88 a Sha ole mary o Cen [RN mnon cs TAL Td 0 RAE Sage acto BOTW ORR Lm we ame i2 3 EY Fa of A A Wha BBL GB CU em oo mem we 3g8 83 a Shoe ui WTR ORR ML TO LAE 82 23 2 Ba of BL AL Wh. BEL GB CU ee oo mm ome 883 She off Bo VOW ROT WM TC LE 38 3 EY Fw of 5 ST Wha BL GSC me me mee ome 2 ss Fwy wf GT AT We ESL GB CT ee oo men wer g8 88 ) [---- Sheer wif BOTW ROR WM TC Lame 88 3&5 9% Hua of ET SL Wh. BOL GB CU ee me wee we 8 &38 Tow 2 E EL T SR i BFW OR mm owe me g 388 Sh wile BTW ROT aM TC oA 3: 102 owe oT EL ATL Wa BL WL me oe oem we a&3d 0 mary of Chasen wmsclons tas Shue ole WTR WMT LA 32 a3 104 Fhe cf BT SFR Ra EFL BR. vee eo men me g 333 105 he wie BOTW ORR aM TOL 833 106 Shue oe NFL SL RP BEL MN. Cen eo men we 338 107 Thaw of BOT OW ORT aM TCE 8g338 08 or A Foon of 5 ST Wa BL MR. 0 em ee ome g a33 0 2 3 i" Shuey oe NFL ST Wha BEL MG UR ee oo mem we 2 33 a m Thay of BOT OR ROT all Tam 38g 3 mn Hen of Ea AW EV. BR MR. CTU tem mo mem me & 3 Thaw wf BOT OR ORT aM TLE 38g3 8 ne Thi oF BT EL ER EEL MR. CU ee oo mm we ga3 ns Baa ola BTR WML TE LSE S<$ 4a3 1s Bue na Wa SV Wa EL HP. men eo mm se 8H & Shen alfa BOTW OR ON af TO aN a3 a us Br y-- Ee olf EF SUL RP. BL BE. CT tee oo mie woe a 119 : Sh of am a ne g 3 8 . She wa reer ony 200 aE Sn OR TR SR frou aero MOT OW Rw pm we nol 3a " He Cr oC er om pues sro crs | a A ER , 32 a3 -] m mary of chiens wemscoons ca ; The olf FBTR ROR adh TC al 8 a3 @ 124 oy Huey of BSL ST SME WBE CU wee mo man we 833g us gg 2 Hua oT BL VL Se JL MR UU men wen me ome g8 2 " Shae alfa BROT OR ORT fh TC WME 28 33 4 8 Woon of S00 JD SJL HR mn ew wer g 332 ns De wife MOT OR OW ON af TC LEC 130 Shaw oe AFR AVL ED. JUL oBR ve vt eer em omer Bl g8 8g 35 8 a38 1  3 g i Seyt cto aire a 8 83 5 1" Shee SE Tn SEWER OSL WR OE SR. 2% DR ] 138 Wwe TET MTET MRS Wh Bh Ba VEE URE CUD 3 139 Dae Sw WR Bn edn "a 140 8 3 - j 0 mie oa She Sh oS EROWUR OSL WL EE am. 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Sh oDE 28 &B 156 BLTR SR Soa UT EEE OW BLS WE WE ou eg 1 | sass ars sorrymn on armen sc ero Sheen oS fw BROWER ONL WR OEE OM. 3h BL e2 &8& 3 1 : 3g " 2 5 8 i 228 28@ 193 Dhar Se WEE he eh a 22 194 2: g Sue NET NUED OEE EE Sa WE Uma 228 2&8 196 1: & " Meee SR Wa ERLOUR NR OWR CAE JR. ROP Sh 2 i 198 ies ow 136 aiaey TET NTET GR Sn Bh Wa SWE URE tar g8 &2 199 3g32 a 0 g82gS 8 om | g i = he Se WE Ba whe Su g 8 8 = 2g 8 8 or Shay MET UEC MIE WL BL Sa VEN mE cue 2 28 23 20s Sha Se WEL Me wb Sn 8g< 2a2 26 Swe oR fw EEOUE MA WR OEE SE. 2% BL Sn 5< 8&z 207 228 za& 2 g3e a=2 2 Be TE we um <&8 2&a 20 BE, Vm ma om 288 23 m Be te a om 8&< z2 an ; rey cp re on <2 3 " wero3 Ee, twa em 822 [A] 2 Th ETH om g5zg ns [Re -- 2 26 | ha tm me om 343 nm Ba tre em a 2 Bt we em 22a 2 | 4 " g38g m Tha TE Tm g8 g 2 3 g 3 2 zg  & 23 = 2z3 g3g 2a 20 | i 3 2 :: - : i 3 33 3i 33 - | eg 3% 239 2 z a 240 mary of cpa veion uta a aa wee Cin gem a :8 En ums of orp weighs Bas 225 33 20 } i g8 %3 w . sem omesomereysme yo smu, Se mor. 228 2 us meot org in sa g23 %g 6 me fr its <g3 Z3 0 23 2&z us may of ore tn sn epST HET 5REES om e5gc Z 249 J g 82 & x0 << 8 = 388g 2< 2 i 2 288 z 2 254 238 2@ = 25 g 32 = 256 3g 8 257 oo EECaO YTT IRST ars " wa EF OG CWE WF ea BF WTRIBS 23 258 pr ER ain WE mage em a 822 22 259 ame nonmt 8 5 28 ) 1SE PH ARRR nen g2 3 & x0 Sey of cr wt wa g 8 & 21 mcg of orp wats saa 328 22 ~ 2 eon avi 3s = osST PT ER nen 30 8g 263 26mm useopesm nyrE, SIC mor. 2 22 i 204 33 g 265 ame of on i a avncum oCSPN ENESET S Ee a g3g 88 26 ama of ce i ts 88 g 267 Ena 38 bd 268 Sciam of cross hsertions 5g< 54g] 2% <g33 3 0 TEBTEemy, FeORSETrALL. s atc of eres Grin ox us roc anor: a= A= 5c 8 g m g S EEEEEen 3 gg tripe m e cm eT 3 & m sm ---- seg xfeee ee SE a 252 g Ea | Em Sh 8 88s PriirEid ns : eo pms STTT ISSSR a g 8 RB 276 35 8 m | g3 88 m sms cars sr sm e we rte sronc 83e ]B m so REE ER gg3 & 280 | ;g . etme o once neato 26-vgas capssony eT wiThFELONS BAIT LEEHR a J a eg38 5 w ee APPENDIX 1 Protocol Protocol Deviations Protocol Amendment No. 1 Protocol Amendment No. 2 `Protocol Amendment No. 3 Come 029223 ew . 283 002318 `CovanIcMe T66202.925273 Protocol Deviations Protocol. Reserve (Archive) Samples. "These samples will be transferredtothe `Sponsor after completion of the in-life phase." Actual Procedure. These samples were shipped to the Sponsorafterthe dosing phase wascompeted, which was priotro the endofthe in-life phase. Protocol. Dosing Procedures. Method of Administration. "Orally by gelatin capsules, daily (7 days/week)forat least26weeks." Afcemtaulael)Pdriodcneodturreec.eiOvenaDdaoyse3,aAdnmiimnailsNtorsa.td1i0uo5en52t0o (blGereoduipng1amnadleir)raintadti1on0i5n5t2h9e(tGhrrooautp. 1 Dosing was discontinued for Animal No. 105530 (Group 1 female)fromDays 10 through 14 due toa sore inside its mouth. Pwriloltobceolo.bseOrbvseedr;vaatbinoonromfalAnfiimnadilnsg.s ColrianniicnaldiOcbasteirovnattihoantst.he"aOnnicmealwweeakslyn,ocramcahlawniillmabel: recorded." Awceteukally Porbosecrevdautrieo.n oDfurnionrgmatlhoefacobunrosreomfalthreecstourddye,dsoenvevraarliaonuismsaclhsedduidlendotdahyasv.e a Ptrroetaotcmoeln.t aOnbdsebrevfoarteioenaocfh Asncihmeadlusl.edOspachrtihfaiclem;itcheExaanmtierniaotriopnorst.ioBneofforteheineiytei,atoipotnicof meds, and ocular fundus will be examined using an indirect ophthalmoscopeby aboardcertified ophthalmologist. Actual Procedure. An ophthalmic examination was completed during Week 52 instead ofprior to the recovery sacrifice during Week 79. 002319 284 Covance6329-223 IMT-62957 Protocol Deviations (Continued) P{irnoittoiactoilon.oCfltirneiactamlePnatt;haofltoegya.t lFerasetqu3e0n,c6y0., 9S0c,he1d5u0,leadndCo1ll8e0ctdiaoynsso.ft"rOenactemebnetfo(rbeefore the daily dose); and afear least 30, 60, 90, 135, 180, 274, and 365 daysofrecovery" AhcotwuevaelrP,rtohceesdeusraem.plOens wDearye1n8o4t, ruerqiuniersedambpyltehsewperroetoccoollleacntdedweforresdeivsecraarldaendi.mals; Protocol. Clinical Pathology. Tests. Hematology. Eleven hematological parameters weretobetestateldl intervals. iAnctteurvaall,Prmoecaenduprleat.elIentavdodliutmieo,nrteodtbhleoroedqucierlleddihsetmriabtuotlioongywitdetshts,aptltahteeleptreditsrteraitbmuetniton width, and plateletcrit were also inadvertently tested for. Protocol. Serum PFOS fasted overnight; blood Level Determination. (approximately 2 mL) MethodofCollection. will be collectedfrom a "Animals will femoral vein be `without an anticoagulant." ADcatyua1l97P;rohocweedvuerre,. aAnniimamlaslswewreerefansottedfatshteedfoolvleorwniingghtifgohrttahendsccohleldeuclteidoncowlalsecdtoionneoonn Day 198. Pwirloltoncootl,be Ufraisnteeda(nedxcFeepctesfoPrFthOeSfiLrestvedlayDoefterremcionvaetriyo)n;.urMienteh(oatdloefasCto2llmecLt)ioan.nd"fAenciesma(lats least 5 g) will be collected overnight." Actual Procedure. The amountofurine collected was not documented at thetime of each collection and therefore cannot be verified. : 285 002920 CovanMceT-6362299-25273 Protocol Deviations (Continued) Parfotteorncoooln.afAtdedriptaionnsaalrFeecclaelanSeadmiplnetsh.em"oTrhneisnagmtpoleesnwsiulrlebtheatcotlhleeycaterdeninotmheoreaertlhyan hours old." Actual Procedure. On Day 160, some fecal samples were collected in the late morning rather than the early aftemoon. Protocol. Interim Liver Biopsy Samples. "Kris J. Hansen or her altemate will be notified regarding the shipmentofthe samples." ActualProcedure. Thephone call these samples was not documented madeto notify Kris J. Hansen prior and therefore cannot be verified. to shipment of bPreoctoolcloelc,teTdebrymibniaolpsLyivferroBmiaolplsayniSmaamlpsleins.Gr"oAupssam3pdluerionfglriveecro(vaeprpyr(oxWiemeakte7l9y, 1wi8t)hwiinll 1 dayofthe serum PFOS blood collection)." Actual Procedure. These samples were collected during Weck 80, and were not collected within one day of the serum PFOS blood collection. Psraomtpolceosl.forPoprsotlmioferrtaetimoPnrcoecleldnuurcelse.arCaenlltiPgreonli(fPerCaNtiAo)n eEvvaalluuaattiioonn.wi"lAfbeerefmibxeatdidoen.din paraffin and shipped t0:" Actual Procedure. PCNA analysis was not doneforAnimal No. 105509 (Grou4p male). Ptrheottoecromli,naTlenremcirnoaptsiyo,n.a sPaomsptlmeoorftelmunPgro(caepdpurroexsi.maAtdedliyt5iogn)alwiLlulnbgeTcioslsleuceteSdafmrploemsa.n"At ntirmogaelni,natnhde hsiogrhe-ddoisneagfrroeuepz.er,Thsits s0ammapilnetawiilnl6b0e wteoi8g0hedC,,fulanslh-sfhriopzpeendi.n lTihqiusidfrozen 286 002921 CovanMceT32299-25273 Protocol Deviations (Continued) Nsaom.pl1e0,35a0n9d(aGsraomuppl4eomfalleu,ngdi(eadpJparnoxuiamrayt2e7l,y 119g9.9)stwoirleldbiensfeonrtmasleipna)rafterloymuAnndiemral appropriate conditions (ambient or packed on dry ice) to: Kris J. Hansen, PAD 3BlMdEg,.2T3.E&-S0.9 9St3.5PaBuuls,hMAivnneensuoeta 55106 `Telephone:651.778.6018 Facsimile: 651.778.6176 Kris J. Hansen or her alternate will be. notified regarding the shipment. of the samples." Actual Procedure. This procedurewasinadvertently not done at the terminal sacrifice; thus no samples were available for shipment toKris J. Hansen, PhD. Protocol. Termination. Postmortem Procedures. Tissue Preservation. "The following tfiosrsmuaelsin(,whunelnespsreostehnetr)wfirseomspeeaccihf.iaendi,mfaolr wpiolslsibbeleprfeusteurreve`dmiicnro1s0c%op`inceuetxraamli-nbauftfieorne.d" Actual Procedure. Femoral bone marrow from Animal Nos. 105517, 105519, 105527 (Group 1 males), 105507, 105509, 105512 (Group (Group 4 4 males), 105531, 105535 (Group 1 females); mammary gland from Animal females), 105536, 105540, and Nos. 105508, 105519 (Group 1 105551 males), and 105512 (Group 4 male); and parathyroid from AnontimeaxlamNion.e1d0m5i5c4r4os(cGorpiocuapll1y.femAaddliet)iwonearlelyi,nsounffeicoiveanrt.y These tissues, therefore, were ofa possible two wesexamined fcroommmeAnntismailn tNhoe.p1a0t5h5o3l6og(yGdraotuaps4hefeetmsalfeor).inMdiivsisdiunagl tainsismuaelssa.reSluimstmedarwiytthaabplpersodproiantoet. include them as having been examined. These deviations are not expected to have affected the results of the study. 287 005522 CcoOmaVAseNtCcoEut. `Sponsor: 3M St. Paul, Minnesota "PROTOCOL Study Title: 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus`Monkeys Date: | August 20, 1998 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard `Madison, Wisconsin 53704-2595 Laboratory Study Identification: Proposal No. 90545B Covance 6329-223 Sponsor Project.Identification: 3M Study No. T-6295.7 288 UU2923 CovanIcMeT6-36229-925273 Page? S2t6u-dWyeekCapsuleToxicity Stady with Perluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys PTseuolarespcsoteessdesbtihoecehfefmeicctaolfptahreatmeetsetrmsatwehrieanl oadnmcirniitsitcealreednzdayimleylbeyveclasp,suhleotorcmynoomanonldeogtushse,r `monkeys for at least 26 weeks Sponsor 3ToMxicology Services `Building 2202-02, 3M Center St. Paul, Mivnesota 55144-1000 SAtnuddryeMwoMn.itSoearcat, PhD 3M `FTaeclseipmhiolneeNNoo..:: 665511..753735..13717631 Alternate Study Monitor `3MMarTvoinxiTc.oClaosgey,SDerVvMic,esPhD Telephone No.: 651.733.5180 Facsimile No.: 651.733.1773 Study Location Covance Laboratories Inc. `3M3a0d1isKoinn,sWmiasncoBnosuilnev5a3r7d04-2595 `Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 . 289 002924 ee Stady Director `CPoevtearnJc.eThLoabmofroartdo,riPehs DInc. Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 CovanMceT6632299-52273 Pawed SDatluedAylTdorxiidcgoel,oBgiSst Covance Laboratories Ine. ``EPxrpoeproismeedntSatlaSdtyarTtiDmaettea:blAeugust 26, 1998 TIanvlliiffeeSTtearrmtiDnaattei:onADuagtues:tT2o6,b1e9d9e8termined AEuxdpiertiemdeDnrtaafltTReerpmoirntatDiaotne:DaTtoe:beTodebteerdmeitneerdmined G"RToehogiusdlsLatatubodoyrrywaiCtloolbrmyepPlcrioanactdniuccceeteRdeignulcaotmipolnisaanscseetwiftohrtthhienEnTivtilreo4n0moefnttahlePUroSteCcotidoenoAfgFeendceyral Regulations, Part 792, issued November 29, 1983 (effective December 29, 1983), and with any applicable amendmeas. "AARlengliupmlraaotlcieoCdnausrr,ee9siaCnntFdhRiUss1pe4r.oStItoanctotelhmaeernoeptiinnicoonmpolfitahneceSpwointshotrheanAdnistmuadlyWdeilrefcatrore,Acthte study does `not unnecessarily duplicate any previous work. TQAushaseluirprtaoyntcoAecsosUlnu,irstat(nucdQyeAUco)n.duTcht,e aprnodliffienralatrieopnorcetlwlimlulcbleeaarudaintteidgebnyevtaheluCaotvioanncdaetQauaanlditryeport sielrlumbeanauddliitveedrbPyEtOhSeaQnaAlUysoifsPaantdhroelpoogrytAwsislolbcieaatuedsiItnetderbnyattihonealQ.ATUhoefd3osMe analysis and `Environmental Laboratories. The blood hormone determinations andreportwillbe audbyithteQeAdUof Ani LyticsInc. : 290 002325 CovanIcMe6T3629225273 J Test Material E------ IPdeernftliufoircoaotcitoanne Sulfonic Acid Potassium Salt (PFOS; T-6295) TLhoetlNotumnbumebrerwill be maintainedintherawdata. PRuesrpiotnysibilityofthe Sponsor SRteasbpiolnistiybilityofthe Sponsor SAttorroaogem Ctoenmdpeirtaitounrse CIhnafroarcmtaetriiosntoincssynthesis methods, composition,o othercharacteristics that define the test material is on file with the Sponsor. Vehicle ILdaecnttoisfeication TLhoet Nloutmnbumebrerwillbemaintainedintherawdata. POunriftilye with the manufacturer SOtnabfiillietwyith the manufacturer S"Attorroagoem Ctoenmpdeirtaitounres: 002926 CovanIceM6T32299.5273 wes `CGbaeeplssauutplipenlsCia(dSpisbzuyeltNehose.m2a)nuofbatcatiunreedrf.roGemlTaotripnacca,psIuncl.es(wFialirlfbieelds,tNoreewdatJerrsoeoy)m; tleomtpmeurmatbuerrew.iAll CopyoftheCertificateofAnalysisprovidedbythe manufacturerwilbe maintainedinthe daa. `ARelrsueesterorvnve(e1(sAagrmcephalicevhoe))wfiSelaalmcbpheltleoatskoefnteasntdmsattoerreidaal,tvrehoiocmle,taemnpdeeraatcuhrte.esTthmeatseerisaal/mlpalcetosswei.llbe transferred to the Sponsoraftercompletion of the in-life phase. `DAinsyproesmitaiionnionfgtTeessttmaMtaetreiarlivalil beretainedat Covanceforuseinpossiblefuturestudies. Animals SCpyencoimeoslgus monkey SCoouvracnece Research Products Inc. YAgoeunagt aIdnuiltti/aatdiuolntofTreatment AWpepirgohxtimaattIenliyti2atti0on5 okfgTreatment N22ummableers aanndd2Sexfemales Identification Collar tag Husbandry HInoduisviidnugal; animals will be housed in suspended, stainless steel cages. 2 002927 CovanIcMe6T3e229922537 -_-- PPiagpess Diet rCoeurttiinfeileyd parniamlaytzeeddibeytt(h#e87m2an6uCf,aHcatrulraenr Tfoerklnautdr)itoinocnealocrotmwpiocneednatisly.anTdheendviiertoinsmental ``cCoonvtaanmcien-anMtasd.isRoens.ulFtrsuoifts,pveecgieftaibeldesnu,torrieontthaerndscuopnptlaemmiennatsntmaanaylbyseepsroarveiodnedfibluetawtill not require analysis. Water Ad libitum. Samplesofthe waterareroutinely analyzed for specified microorganisms and environmental contaminants.Theresults are on file at Covance-Madison. TChoenrteamarien5a0ntksnown this study. contaminants in the di et or water a t levels t ha t might interfer with EnrEvenlivartioirvnoemnhemunmetinadtlictoyntorfo3l0sftoo 7t0h%e,anainmdaalr1o2-ohmowuirlllibgehts/e1t2t-hoomuaridnatrakincyc1l8te.oT2h9edCaar,.k cycle `may be interrupted to accommodate in-life procedures. AMcicnliimmautmioonf4 weeks AnRianmdaolmsiwzialtliboenweighed,stratifiedbybody weight, andallototcheanutmbeerodf b`lbolocckkswielqluatlhetnotbheean ssiu gnem dotfb oagnre iomuapr lsstusoibneg csoemlpeuctteedfr-ogreenaecrahtegdroruap.ndAonminmuamlbsiernse.ach JPuFsOtSifiiscaatkionnownhepatic peroxisome proliferator (PP) in the rat. When exposed to PP, `Gnioe.n,hluomwantporniomahteepsat(iscurcehsapsontshee)caynndomtohelrgeufsormeoanrkeeayn)arpepsrpoopnrdiastiemihluamrlayntosuhrurmogaantse species. 293 002928 CovanMcTe -63028925773 a -- A `GroupDesignations and Dosage Levels err------ DosTeTLevel ----TTouTl MTaterial Dose NumbeeropfAnr imal-- s Group (mg/kg/day)* Level (mg/kg/day) Males Females 1 o 30 & & 2 0.03 15 4 4 3 4 0.15 0.75 6 6 6 30 6 6 aDeqousivealleevneltsavmiolulnbteopfrolavcitdoesde.Tinhgeelactoinntrcoalpsgurloeuspa(sGtrhoeutpot1a)l wmialtlerrieacleiavdemainnisterteod Group 4. b T(1h:e49l9o,ww-dwo)s.e (Group 2) will rec thee tesi t mav terieal diluted with lactose T`mhaetemriiadl-ddiolsuete(dGwriotuhpla3c)taosned (h1i:g3h9-,dwoisve)(.Group 4) groupswillreceivethe test d Tawotal2ne6 awieseimknstaG,rtlohuepsnst1,3r, aendwa4idlelstbiegdnmiastceeodnatsnirneutcedo,vaenrdy tanhiemaalnsiwiml wbaielltlrbseeatedfor leoabssetr1v3ewdefeokrsrepvoesrtsiubriclaittmy,enpte.rsiBsatseendceo,noprldaeslmaayetdesotcmcautrerreinacleleovfetlosxaincdefcfleicntiscaflor at pathology findings, the recovery may be reduced or extended, at the discretion of `the study director and after consultation with the Sponsor. Dosing Procedures MOreatlhlyobdoyfgeAldatmiinnciasptsrulaetsi,odnaily (7 days/week) for at least 26 weeks Reason for Dosing Route `To compare with data from previous toxicology studies using the oral route, which is the most likely routeofexposure Dose Preparation Caopnstuhleepshwysiilclablecphraerapcatreerdisattilcseaosfttwheeektelsyt.mTatheersiai,zetahenddonsuembleevrelo,fcanadpstuhleewsewiiglhltdoefptehne:d m`woenikghetys., wIintdhivtihdeueaxlcdeapitliyodnoosfebsowdilyl wbeeibgahstecdoolnlectthieomnodsatysrewcheentnintdhievpirdeuvailobuosdbyody `weightwill be used. Dose levels willbe based onthevehiclaes suppfolriGreoudp 1. 294 002929 _-- CovanIcMe 6T3a22.925273 Ths3 FacoerttohneeGarnodupdislu2tetdhrwoiutghhl4acdtoossee(p1r:e4p9a9r,at1i:o3n9s,,atnhde t1e:s3t9m,awtierwi,alrewsiplelctbievdeilys)soolnvceedbienfore `ipnrietpiaatriaotnioonf. tArlelatdmoenste.preDpialurtaitoinonwsiwthilllacbteossetiosrendecaetsrsoaorymttoefmapceirliattautreecaupntsiulledosed. Dose Analyses Doseanalyseswillbe donebytheSponsor. oH`fSoamtmhopelgetesenst(eaimpattpyerroixali/mlaatcetol1sye gperaepcahr)awtiiolnsbefcoorltlheecltoedwf-raonmdthhiegtho-dpo,smeidgdrloeu,pasnadnbdottom a untilnanalafyozredtl.estmyaterzial ceontednt.Al samples willbestoredatroom temperature: Stability Homogeneity samples collected from the middleofthe preparations will be used for titanhkveleipnfrfeerpsohtamudstyehseatnladboiwlain-tayalaynnzdaelhdyisfgiohsr.-dteoOssnteetmeastseettromifaatlsecaroimnaptlle/enlsta.c(taopsperporxeipmaartaetliyon1sagtetahcehe)nwdilolftbeh. e Sample Shipping Samples will be shipped under ambient conditions to: Kris J. Hansen, PhD 3ME.T.&S ' Bldg. 236-09 935 Bush Avenue TSetlePapuhlo,neM:in6n5e1s.o7t7a8.565011086 Facsimile: 651.778.6176 KArniaslJy.siHsoafnfsoenrotershtemraatletreimaalctoenwtielnltbweilnlotbiefideodnreeoganrtdhinegstahmeplses.hReisuoplftstmwhielselabmenp:lets. provided for inclusioninthe final report. Observationof Animals Clinical Observations Efiancdhinagnsiwmialllbwielrlebceorodbesdearvsetdhetywiacreedoabisleyr(vaend.. and p.m.) for mortalityandmoribundity; : s 002930 CovanMceT63s295.2723 _-- Fa Eaabcuhoramnailmafilnwdiilnlgbsewoilblsebervreedcdoardieldy.anOdnfcoeodweceoknlsy,umepatcihoannwiimlallbweialslsbeessoebdsqeuravleidt;atively; tarbenaotrmmenatl,feiandcihnagnsi,moarlawnilinldbiecaotbisonertvheatd tfhoerasniigmnsalofispnooormhaealwtihllobreabrnecoorrmdaeld,beDhuarviinorg approximately 30 to 90 minutes afer the last animal on tes has been dosed; abnormal findings will be recorded. Additional findings will be recorded as they are observed. Body Weights calculations. `Weekly before initiationoftreatment, on the first dayoftreatment, and weekly thereafter. An additional body weightwill be recorded on Day -1forthe Day 1 dose Ophthalmic Examinations Before initiationoftreatment and before each scheduled sacrifice; theanteriorportion ofthe eye, optic media, andocular fundus willbeexaminedusing an indirect ophthalmoscope by a board-certified ophthalmologist. Clinical Pathology Frequency ``UWnhsecahpeodsuslibelde,Cobllloeocdtiwoilnlsbecollectedforclinicalpathologytessfrom animals sacrificed at unscheduled intervals Scheduled Collections O90n,caenbdef1o8r0edianyitsioafttironeoaftmtreenatt;maenndt;afaenrda(tbeefaosret t3h0e, d6a0i,lyanddos9e0) dafatyesroaft rleeacsotv3e0r,y60, AMneitmhaoldswoifllCoblelefcatstieodnovernight; blood willbecollectedfrom afemoralvein. Anticoagulant will be potassium EDTA for hematology tests. No anticoagulant is required for clinical chemistry tests. Urinewill becollected overnightonwetice: 296 002931 CovanIcMeT6-36229925273 -_-- hewn Tests Hematology red bloodcell (erythrocyte) count plateletcount hemoglobin `hematocrit `diwfhfietreebntlioaoldbcleolold(lceelulkcocoyutnet) count `mean corpuscular volume `mean corpuscular hemoglobin bloodcellmorphology reticulocyte count `mean corpuscular hemoglobin concentration Clinical Chemistry glucose ureanitrogen aspartate aminotransferase gamma. glutamyltransferase creatinine. total protein sorbitol dehydrogenase: creatine kinase, albumin globulin calcium inorganic phosphorus total bilirubin sodium directbilir(ufbtoitanl bilirubin is potassium greater than 2.0 mg/dL) cholesterol chloride bile acids triglycerides alanine aminotransferase: amylase lipase alkaline phosphatase: pancreatic-specific amylase: Urinalysis appearance volume (approximately 16 hours) glucose ketones specific gravity oH bilirubin blood protein urobilinogen `microscopic examinationofsediment Blood Hormone Determination Frequency `Three times before initiation of treatment; and afattleearst 30, treatment; 60, and 90 after at daysof least 30, 60, recovery 90, and 180 days of 297 002932 -_-- Number of Animals An CovanIcMe T632299-52273 men1 AnMiemtahlosdowfilCoblelefcatsiteodnovernight;bloodwilbecollectedfromafemoral vei. cAlpoptrfooxrismeartuemlysa5mmplLeos.fblood will be collected without anticoagulanndt allowed to `SSaammpplleesfHoarnsdelriunmgwillbecentrifwuitghiend1 hourafter collecatndisoernu,m will bsethoraerdviensatedf.rSeeresuzemetwitrollmbaeidntiaviinde-d6i0nttoo-t8w0oaCupnptriolxipmaactkeeldyoenqduarlyaliicequaontdsasnhdipped to: Dr. Saroj Das Ani Lytics Inc. G2a0i0thGeirrsabrudrgS,trMeeatr,ySluaintde 22000877 TFaeclseipmhiolneeNNoo..:: 330011..99772.10.4031368 Ani Lytics Inc. will be notified regarding shipmentofsamples. Tests eSsatmrponlee,sewsitrlilolb,etahnyarloyizdesdtibmyulAantiinLgythiocrsmoInnce,,ftorriicoordtoitshayl,rotneisnteos(tTe3r)o,nea,nedstradiol, thyroxin (T4). Serum PFOS Level Determination Frequency Before initaition of treatment, 26,28,30,32, 34, and 39. and during Weeks 1 (Day 7), 2, 4, 6, 8, 12, 16, 20, 24, Numberof Animals Al AnMiemtahlosdwoiflCbolelfeacsttieodnovernight; blood (approximately 2mL) will be collected from a femoral vein without an anticoagulant. 298 0002Z9s7te -_-- CovanIcMeT6632299-25273 wPage en12 Sample Handling `hSaarmvpelseteswd.iSlelbreucmesanmtprleisfwiwuilgtlhbeiednst1orheodurianftaefrrceoelzleercsteiotnt,oanmdaisnetraiunm-w6i0ltlob-e80C. Sample Shipping Serum sampleswilbe packedondry iceand shippedto: Kis J. Hansen, PhD SME. T.&S Bldg. 2-3E-09 935 Bush Avenue St.Paul, Minnesota 55106 F`aTcesliempihloen:e: 665511..777788..66107168 AKnrailsyJs.iHsoanfsseneor rfhoerruPalFtmOemSawteilwilblebdeonnoetoifnitedherseagmaprdliensg.thRe sehipsmweinlutlboeflptrheotvsiadmspeldes. for inclusion in the final report. AAdtdsicthieodnualledSaenrdumunCsoclhleedcutlieodnnecropsics, blood (as much as possible, up to 20 mL) will ebxescaonlglueicntaetdiofnr.omSathmeplveesnawiclalvbaewcietnhtoruitfuagnedanwtiitchoiangu1lahnoturfraofimerancoilmlaelcstiaotn,thaentdismeeroufm will 2bendhasrtvoersetdedin. aSfreereuzmerfrsoetmteoamcahianntaiimnal-6w0iltlob-e8t0raCnsufnetrirledsihnitpopeadntaopptrhoepSrpiaotnesocorntainer (Ksis J. Hansen) for possible future analysis. Urine and Feces PFOS Level Determination Frequency On thefirstdayofrecovearndy afteratleast 6, 30,and90 daysofrecovery Number of Animals Au Method of Collection A(napipmraolxsimwaitllelnyot$ bgerafmasst)edw;ilulribneec(oalplpercotexdimoavteerlnyig2htm.L) and feces 002934 299 BE `SSaammpplleeswHialnldbleisntgoredin a freezerset to maintain-1010 30C. CovanMceT638295.2723 313 `SSaammpplleeswSihlilpbpeipnagckedon dry ice andshippedto: Kris J. Hansen, PhD BSlMdEg..2T3.6-&0S9 935 Bush Aveane TSetlePpahulo,neM:inn65e1s.o7t7a8.565011086 Facsimile: 651.778.6176 KAnrailsyJs.iHsaonfsueraionerahnedrafletceersnfaoterwPiFlOlSbewnioltlibfieeddorengeaornditnhgetshaempslheisp.mReenstoulfttshweislalmbpeles. provided for inclusion in the final report. `Termination `Unscheduled Sacrifices and Deaths aNned xcylarzionwep i,lwslebiieghdeeodn,se.blA ed fon r reti qouibrem edsatcea srtisf,il acenddws eixlslabneguainneasttehde.tizedwithketamine Scheduled Sacrifices tAhfetnearnaetstlehaestti2z6edwweietkhskeotfatmrienaetmaenntd, xfyoluarziannei,mawlesi/gsheexd/,greoxuspanwgiulilnbaetefda,satenddnoevecrrnoipgshite,d. `rA xeymlf aaziiannt tienl,gee waaesntiir g2mh6aeldws,eweeixklslsaonbfgeturfiaensaattteedmdeo,nvaetnrandingndahetct,rlotephasesinetda1.n3eswteheetkisz`ewditwhiotuhtkterteaatmmiennet,andt.he PostmortemProcedures Necropsy Thenecropsy will include examination of: all orifices `cerxatneiranlalcasviutyrfoafcthee brain;theextemalsurfaceof the spinal cord andcutsurfaces of thebrain andspinal cordwillbeexaminedwhentissuetrimmingis performed. cervical tissues and organs 200 002935 `tehxotrearcniacl,saurbfdaocmeionfatlh,eanbdopdeylvic cavities and viscera `nasal cavity and paranasal sinuses CovanIcMeT6-32692292537 Page ld Organ Weights pFrreosemntc)acwhilalnbiemawleiagthsecdh;epdauilreeddaonrdgaunnsscwhieldlubleedwesaicgrhiefidcesse,ptahreatfeloyl:lowing organs (when bradariennal (2) keipdindiedyy(m2i)s (2) liver poavnacrryea(2s) ttehsytriosi(d2)(2) with parathyroid Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. PReaplrmeisteonytlatCivoeAsaOmxpildeassoeftDheeterrimgihntaltatieornalslobe of liver will be collected from each aanfirmeaelzearttshete tsocmhaeidnutlaeidns-ac6r0iftioce-,8w0eiCghuendti,l falnaaslhyz-efdrofozirenpnallimqiutiodynliCtrooAgeonx,iadnadssetoarcteidviitny. RCeeplrePsreonltiafteirvaetisoanmpElveasluoafttihoenleft lateral lobe of the liver, eft and right testes, and `pancreas willbecollectedandpreservedinzincformalin. Afetmeberfdidxeadtiionn,pasraamfpfilnesanfdorsphrioplpiefedrtaot:ioncell nuclear antigen (PCNA) evaluation will be Sandra R. Eldridge, PhD P1a5tWhoorlomgayn'AsssMoiclilatCeosuIrnttemational Suite I Frederick, Maryland 21701 Telephone: 301.663.1644ext.2036 Facsimile: 301.663.8994 PPaCtNhoAloegvyalAusastoicoinatweilslIbnetedmoanteioonnalthwielslabmeplneost.ifiReedsurletgsarwdiilnlgbsehpirpomveindteodffsoarmipnlcelsu.sion in the final report. 201 002936 CovanMceT623299-52273 Pages Liver PFOS Determination sAscheecdtuiloend (saacprpirfoixcei,mawteeilgyhe2d0,gf)loafslhi-vferrozweinllinbleiqcuoildlneicttreodgfern,omaneadcshtoarneidmainlaatfrteheezerset tanoamlaysiinst.aiRnes-u6l0ttsowi-l8l0beCupntril oshifvpopreiidnwcdiltuhseipolndaisnmtahsefaimnplaelsrtepoor3t.M (Kris Hansen) for Tissue Preservation T1h0e%fnoelultorwailn-gbutfifsesrueeds f(owrhmeanlipnr,esuennlte)ssfrotohmeerawicsheasnpiemcailfiweidl,lfboer pporsessiebrlveefduitnu.re, `microscopic examination: a0admreanal (2) brain cecum cervix colon deupoiddiednyumims (2) esophagus e3fei(x(at2i)vetofobresparcerisfeircveeddainnimDaalvsidosnolny']s femur with bone marrow (articular `surfaceofthe gallbladder distal end) ihleeaurmt jKeijdunneuym2) lesions liver l`umnagmmary gland ovary (2) mpeasnecnrteearsic lymph node pituitary prostate rectum salivary gland [mandibular (2)] ssceimaitniaclnevresviecle (2) sskkeilnetal muscle (thigh) spinal cord (cervical,thoracic,and Tumbar) sstpleereanum with bone marrow tsetsotimsa[c(h2) tobe preseinrBovuiensd' thsyomluutsion for sacrificed animals only] thyroid (2) with parathyroid trachea. uurtienraursy bladder vagina BoneMarrow Smear Fherlodmtfohreposstseirbnluemofuftueraecehxaamniinmaatliaotn)u.nscheduled and scheduled sacrifices (made and 302 002937 CovanIcMeT6632299-52.273 Page 16 `HTiissstuoepsaftrhoomloegaychanimalinthe control and high-dose groups willbeembeddeidn. `pTairsasfufeisnf,rsoecmtailolnoetd,hesrtaainniemdawlistwhilhlebmearettoaxinyeladnifdnorpeoossisni,balnedfuetxuarmeiexnaemdinmiactrioosnc.opically. Reports ALleetttteerrRreeppoorrttwill be prepared following the terminal sacrifice. Final Report A draft report that includes the following information will be prepared and submitted: Experimental Design and Methods Results `clmionritcaallitoybservations body weights `foopohdthcaolnmsicumepxtaimoinnations clinical pathology results phaolrmmiotonye]aCnaolAysoexsi(dparsoevaicdteidvibtiyesAni Lytics) sceelrlupmr,oluirfienrea,tifeocneesv,aalnuadtliiovnesr(PpFrOovSidleedveblys (PpartohvoildoegdybAyss3oMci)ates International) dose analyses (provided by 3M) organ weights organ-to-body weight percentages `organ-to-brain weight ratios `macroscopic observations `microscopic observations LSteavteisntei'catlesEtvwaillulabteiodnone to test for variance homogeneity. In the case of heterogeneityofvariance at 5 0.05, transformations will be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration. . 303 002299338 CovanIcMe T636259-52273 _-- ew `Obnoed-ywwaeiygahntasl,ypsaislomfivtaory]iaCnocAe (oxAiNdaOsVeAa)ctiwviiltliebse, uasneddc(oinftaipnpuloiucsabcllei)nitcoalanpaaltyhzoeliongiytial vtraelauteesd. gIrfotuhpecAoNmpOarViAsoinsss.ignificant, Dunnett's t-test wil be used for contral versus `Ownieth-iwnaityiaalnbaloydsyiswoeficgohvtsaraisatnhceec(ovAaNriCaOteV.IAf)twhielAbNe uCseOdVtioAsansailgynzifeibcaondt,ycwoeviagrhitast,eadjustedmeanswil be used for control versus treated group comparisons. Group comparisons (Groups 2 through 4 versus Group 1) wil be evaluated at the 5.0% two-tailed probability level. Only data collected on or after thefirstday of treatment will be analyzed statistically. Attheendof 1yearafterissuanceoftheauditeddraft report,ifnorequestedrevisionsor instructions to finalize have been communicated by the Sponsor, then the audited draft reportwillbeconsidered final'andissuedasthe and submitted to the Sponsor. final report, signedbythestudydirector, Any modificationsorchangestotheauditeddraftreportrequested 1yearafte issuance will be performed at additional cost to the Sponsor. Record Retention All raw data, documentation, records, protocol, specimens, and finalreportgenerated as a resultofthisstudy generatedby Covancewillbearchivedinthestoragefacilities of Covancefor aperiod of 1 year following submissionofthe final reportto te Sponsor. Oneyearaftersubmissionofthe final report, allofthe aforementioned materials will be: sentio the Sponsor, and areturn fee willbecharged. `The Sponsor may elect to have the `materialsretainedinthe Covancearchivesforanadditionalperiodoftime,andCovance will charge a storage fee. Ifthe Sponsor chooses to have Covance dispose of the `materials, a disposal fee wil be charged. All raw data stored on magnetic media will be. retained by Covance. `Within 1yearaftersubmissionofthefinalreport, all oftheaforementionedmaterialsfrom the Sponsor's designees (Ani Lytics Inc., SME. T. & S, and Pathology Associates International)willbe sentto the Sponsor (Andrew Seacat, PAD, 3M) by the Sponsor's designees. : 304 002939 PROTOCOL APPROVAL CovanMcTe.6632259.5272 men13 CoAnddreweM. sSeacat,0PHSD st 3StMudy Monitor DPe ays 1. fa) CStouvdaynceDLirabcoiorratories Inf? gdofop : 305 002930 COVANCE PROTOCOL AMENDMENT NO. 1 Covance 6329-223 3MT-6295.7 26-Week Capsule `Toxicity Study with Perfluorooctane SulfoniAc cid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys `Sponsor: 3M, St. Paul, Minnesota. Study Monitor: ~~ Andrew M. Seacat, PhD SudyDincior. Testing Facility: Study Director: Peer Thombodd ~ Covance Laboratories Inc., Peter J. `Thomford, PhD Madison, Wisconsin "Thisamendmentmodifiesthefollowingportionsof theprotocol. Effective August 20, 1998 1 Page 4, Vehicle, Storage Conditions. `Toinclude thesolventusedtodissolvethe testmaterialbeforemixing withthelactose,addthe following after this section. Solvent Acetone Identification Lot Numbers The lotnumberswillbemaintainedin therawdata. Purity On file with the manufacturer Stability On file with the manufacturer Storage Conditions At room temperature 002941 306 CovanIcMe 6T3-2692-925273 -_-- Protocol AmeadimPenut Neo.221 ICnhfaorramcatteiroinstoincssynthesis methods, composition, or other characteristics that detfhesiolvnenteisonfilewiththemanufacturer. 2 Pcoargreec7t,anGreroruorp, Ddeelseitgenathtiisosnesnatenndce.Dosage Levels, Footnote a, Sentenc1e. To Effective September 2, 1998 3. cPaapsguele,s,GdeellaetteinthCisapsseualteesn,ceSaenndtreenpcleac1.ewTiotchhtahnegfeotlhloewisnigz.eofthe gelatin `Cuapmsbuelreswi(lSlibzeesNuopsp.li2edorb4yt)hobetmaainnuefdacftruormerT.orpac, Inc. (Fairfield, New Jersey); lot Effective December 15, 1998 4. PPaagrealg4,raPop3sh,tSmeonrtteemnPc2re.ocTedoiunrcelsu,dCeeelvlaPlruoaltiiofneroaftsiloindEesvasltuaaitneidonw,ith whietmhattohxeyfloilnloawnidnge. osinithne PCNA evaluation,deletethis sentence and replace. ePoCsiNn)Aweivlallbueatdioonne(oinnctlhudeinsgamepxlaemsi.nationofsides stainedwithhematoxylin and Effective January 21, 1999 5. aPdadgieti1o0n,al CclliinniiccaallchPeamtihsotlroygtye,stT,eastdsd,thCleifnoilclaolwCihnge.mistry. To include an High deasity lipoprotein (HDL) `Effective January 21 and 28 and February 2, 1999 6. Pthaegree1q2u,estUorfitnheeaSnpdoFnescoerstoPcFoOllSecLteavdedlitDieotnealrmfiecnaaltsiaomnp,leSsam(epflfeectSihviepping. At JJaannuuaarryy2218,a1n9d9F9)eabnrdutaor2cly,ar1i9f9y9t)h,easdadmtphlee cfoolllloewcitnigonarfteeqruithriesmseencttsio(ne.ffective. 307 002942 ProtocolCAovmaenIcnMedT6m322e99Nn-o52t.2731 -_-- hPaugee3s f`ArDdoudrmiitn6igmoWanelaeleksFa2enc3ad,la6Sffarememspahllfeeessceaalcshaimnptlheeocfoanlteraoslatn5dgh,iigfhp-odssoisbelger,owuiplsb,seucrovlilveaclted `pcsearmmpliltetsieinwngi.tlahlTebehmnceosorlanlemiepnclgtdetesdoiweninlwshubiretecetophloallteytcphtreeoydpaiynlrteehn1ee0ecamoronltryaeaitfntheearrsnnao6nohdnosauefrnist opolnadd.nrsyTaihroeeeto: JPoorepMhycrCionnsnaenldl Nutritional Chemistry Group Mayo Clinic R2o0c0heFsitrsetr,StMrieentneSsWota 55905 LTaebleTpehloenpeh:on5e0:7.520874..228145.03232 Dr.McConnellwillbecontactedbeforeshippiagthe samples. `Effective January 21and February 12 and 16, 1999 7. Pdeacigseio,noCfltihniecSalpoPnastohrolaongdy,thFerSetquudeynDciyr,ecStcohretdoulaedddCaolcloelclteictoinosn.intTeorvraelfalfectterthe 1r5e0codvaeyryso(fetfrfeecattivmeeFnetb(reufafrecyti1v2e,Ja1n99u9a)r,ya2n1d,to1c9l9a9r)i,fyttoheextceonldltehcteilonernegqutoifhrement (feoflfleocwtiinvge.February 12,1999),deletethetextinthissectionandreplacewiththe oOfncterebaetfmoerneti(nbietfioarteiotnheofdtarielaytmdeonset);;aannddaafftteerraattlleeaast3s300t,,6600,,9900,,115305,,aaandd180days 180 days of recovery Effective February10and 16, 1999 8 `PTaogmeo1d2i,fytUhrienaemaonudntFeocfessamPpFlOeStLoebveeclolDleetcteerdm(ienfaftecitoinv,e FMeebtrhuaordyof10C,o1l9le9c9t)iaonn.d t(eofpfeercmtiivtecFeobrlualryeoc16f,tsa1i9m9po9l)en,sdeflreotmetfhaestteedxtaniinmtahlisssoenctthieonfiarnstddraeyploafcreewciotvhetrhye following. `2AnmiLm)alasnwdiflelcneostb(eatlfaesatsed5t(ge)xwcielpltbfoerctohlleefcitresdt doavyeronfirgehct.overy);urine (atleast 308 002943 CovanIcMe6T3-2692.92537 -_-- Protocol AmendmePataNgoee,sd1 Effective February 12, 1999 9. dPeacgiesi7o,nofGtrhoeupSpDoenssiognraatnidontsheaSntduDdoysDaigreeLcetvtoeolrse,xFtoeontdtnhoteeledn.gtThoofrerfelceoctvetrhye, deletethetextinthisfootnoteandreplacewiththe following. d trTewatoedafonraitilmenaGasrtol2u6psswe1e,ks3,,atnhden4trdeesaitgmneanttewdialslbreecdoivsecronytainniumeadl,sawnditlhbee. t`oaxniimcaelfsfweicltlsfboeroabtsleeravsetdf2o6rwreeevekrssipbiolsitttyr,epaetrmseinstt.ence,or delayedoccurrence of Effective February 12and 15, 1999 10. Pdaegciesi1o1no,fStehreuSmpPonFsOoSrLaendvetlheDSetutdyeDrirmecitonr atForteexqitueoenndncty,h.eTloenrgetfholefcrttehcoevery du(refifnecgtriveecFoevberruya(rye1f2,f19eF99ec)brtaunaidrytv1o 5er,evi1s9e99t)h,edreelqeutiertehmeetnetxstfionrtthhiessseecctoilloencatinodns rewpithltheafollcowieng. 2`B6e,faornedi2ni7ti(aDtaioyn o1f83t)redautrmienngt;trdeuartimnegntW;eaenkdsdu1 r(iDnagyW7e)e,k2s,42,76,(D8a,y1s2,18146,, 12805,,2a4,nd 187), 28 (Day 51,a0d 53. 190), 29 (Day 197), 30 (Day 204), 31 (Day 211), 35, 39, 43, 47, 11. `PSapognes1o3r,anTdertmhienSattuidoyn,DiSrcehcteodrultoedexStaecnrditfihceelse.ngTtohoreffrleecctotvheerdye(ceifsfieocntoifvtehe (FeefbfrecutairvyeF1e2b,r1u9a9r9y) 1a5n,d1t9o99pr)i,odreilteizteetthheetneuxmtbienrtohifsasneicmtailosndaensdigrneaptleadcefwoirtrhetchoev.ery following. Afteratleast 26weeksoftreatment, up tofouranimals/sewxil/lbgerfaosutepd overnight, then and necropsied. anesthetizedwith ketamine and xylazine, weighed, exsanguinated, Atfwtoeraantilmeaalsyt/s2e6x/wgereokuspowfiltlrbeeatfmasetnetdanovdeartnilgehats,tt2h6ewneaenkessthweittihzoeudtwtirtehaktemteantm,ine. andxylazine, weighed, exsanguinated,andnecropsied. : 309 002944 ProtocoCl oAvmaennIcdMemT6e-3n622t9-9N25o2..371 -_-- has5 Effective February 12 and 16, 1999 12. oPfatghee1S0p,onBsloorodanHdotrhmeoSnteudDyeDtierremcitnoarttiooenx,teFnrdeqtuheenlceyn.gthToofrerfelceocvtetrhye(defefceicstiiovne dFeelbertuetarhyet1e2x,t1i9n9t9h)isasnedcttioocnlaarnidfyretphlearceeqwuiitrehmtehnetfso(leofwfeicntgiv.e February 16, 1999), `tTrheraetemteinmte;asnbdefaofreeriantilteiaatsito3no0f,tr6e0,at9m0e,nt1;3a5,fearndat1le8a0sdtay30so,6f0r,e9c0o,vearnyd180days of 13. Page 12, Urine and Feces PFOS Level Determination, Frequency. To reflect rtehceodveecrisyi(oefnofefcttihveeFSepbornusaorryan1d2,th1e99S9t)udaynDditroeccltaorirftytoehxeterneqdutihreemleenntgsth(oefffective February 16,1999),deletethetextinthissectionandreplace withthe following. Ornectohveerfyirstdayofrecoveryandafteratleast 6,30,90,135,and 180daysof Effective February 15, 17, and 18, 1999 14. Page 13,PostmortemProcedures. Tofacilitate arequestbythe Sponsorto tgraalnlsbfleardsdaemrpalneds biolfel(uenfgfetcitsisvueeF(eebfrfuecatriyve1F7,eb1r9u9a9r)y, a15ddatnhde f18o,ll1o9w9i9n)g.and A`AtddtihteitoenramlinLaulnnegcrToipsssyu,e aSasmapmplleesoflung (approximate5lgy) will be collected ifnrloiqmauindannimialinttahnredshtioogrhe-gddiosneeagfrrnoeuepz.,er,Tsheisttsoammpalienwtialilnb-ew6et0io g-h8e0d,C,fluansthi-lfrozen fshoirpmpaeldi.n)TfhriosmfArnoizemnalsaNmop.le10a5n5d09a s(aGmrpoluepo4flmuanleg,(daipepdroJxainmuaatreyl2y71,g1,9s9t9o)rewdililnbe seat separately under appropriate conditions (ambient or packed on dry ice) to: 3KrMiEs .J.TH.an&sSe.n, PhD 9Bl3d5g.Bu2s-h3EA-v0e9nue TSte.lePpahulo,neM:in6n5e1s.o7t7a8.565011086 Facsimile: 651.778.6176 310 002299445 Covan3cMe 6T3-2692292537 Protocal Amendment No 1 --_--_-- Pe sKarmpilJse. sH.ansenorheralternate willbenotifiedregardingtheshipmentofthe Additional Gallbladder and Bile Samples Aatctchoersdchietndogulaespdenceicarlopcsoilelsec,ttihoenagnaldlfbilxaadtdieornapnrdobceidluerwei.llbFercoozlelnecgtaeldalnbdlsatdodarneedrd bile sampleswillbepacked ondryice andtransferred to: Kris J. Hansen, PhD 3BlMdgE..2T-.36&-0S9 935 Bush Avenue STte.lePpahulo,neM:in6n5e1s.o7t7a8.565011086 Facsimile: 651.778.6176 samples. Kris J. Hansenor heralternatewillbenotifiedregardingtheshipmentofthe Effective February 24 and March 9, 1999 15. Page 13, Postmortem Procedures. To facilitate arequest bythe Sponsor to `psraemppalrees (oefffleicvterivfeoFreeblreucatrroyn2m4i,c1r9o9s9c)opayndantdratnosfperre(peafrfeecatnivdetMraanrscfher9h,em1a9t9o9x)ylin and eosin-stained slides for light microscopy (effective March 9, 1999), add the. following. Electron Microscopy At the terminal necropsy, samplesofliverwill be collected and processed to block stage for electron microscopic evaluation. Thetissueblocks and ahematoxylin andeosin-stained slide for light microscopy will be transferred to: DSihvairsoinonADmibrreoctsoer `4P9a1t5hoDl.ogPyrAossspoeccitautsesDrIinvtee.rnational Durham, North Carolina 27713 `FaTcesliempihloen:e: 991199..554444..35221587 002946 311 CovanIcMe 6T36299.5273 -_-- Protocol Amendment No, 1 Paee77 `ATsissoscuiesawtielslIbnetpermoatcieosnsaeld(aPnAdI)e.vaAlrueaptoedrbtywielllebcetprronovmiidcerdobsycopPyAbIyfoPraitnhocllougsyiion t`hmeaifnitnealnraenpcoerotf.anPyALriaswredsaptoansoirbslpeefcoirmtehnes pquraoldiutycaesdsubryaPncAeLauditingand S`hsaamrpolensA. mbroseorheralternate wil be notifiedregarding theshipmentofthe Effective April 22,1999 16. srPteaaptglieasct1iec6wa,liatnRhaelptyohsreitsfsoo,lfloSotrwaigtnaigs.ntwiceailgEhvtaalnudathioornm,oPnaervaaglrueasp,hde2l,eSteetnhtisesnec1ate.enTcoe aanddd. `inOintiea-lwbaoydaynawleyisgihstosf,vpaarlimaintcoye!(CAoNAOVoxAi)dawsiellabcteiuvsiteeds,(cioanptpilniucoaubslec)litnoicaanlalyze: `pathology values, hormone values, and organweight data. Effective April 26, 1999 17. PPaargaeg1r4a,phPo1s.tTmooratdedmaPsreoccoenddurseesto,fCteilslsuPersofloirfecrealtlipornolEivfaerlautaitoinoenv,aluation, add the following to this paragraph. lAefstaencdornidghstetteosftetsi,ssaunedsp(arnecprreesaesn)tpartievseersvaemdpilensfoorfmtahleilnewfitlhatoeurtalzIionbcewoifltlhalesolibveer, prepared. Effective April 28, 1999 18. rePtaugrenso,ftDhoesesaAmnpalleyss,deesl,eStteatbhiilsistye,ntSeenncteeanncder2e.plTaocecwhiatnhgtehtehfeolslcohweidunlge.forthe hOingeh-sdeotoseftseastmpmlaetser(iaaplplraocxtiomseatperleypa1rgateiaocnhsa)wtitlhlebeentdoakfetnfherotmrteahtemleonwt-phaansde and wll be analyzedfortestmaterialconteat. i I. 0029944'7s CovanIcMeT6.362299.5272 -_-- Protocnt AmadmePntagNeos51 Effective May 6, 1999 19. rePpaogret,16d,eleRteeptohrettse,xLteitnttehriRsseepcotriton.aTnodrcehpalnagceetwhietlhettheerfroelploorwtintgo.aninterim pAIrnnetpieanrrtieemrdiRfmeroeplplooorrwtti,ngintchleutdeirnmgianlaltseacmrsiffirceo.m the finalreportasappropriwaitleb,e: Effective May 17, 1999 20. trPaearpggleeatc1eo6rw,giatPnhostshfteomrfoaornltilomewamilnsPgr.ionctehdeuIroews-, aHnidstmoipda-tdhoosleoggyr,ouSpesn,tdeenlceete2t.hiTsoseinntcelnucdeesnd Is`pniaendadmlictbioornedi,gndrpiaavrdeyarmfeaafnitnddt,teshrefycrmtouimosnffeeodmr,aaslltealisanineindmtawhliestjJhonhwt-heaelmnodawm-tiado-nddaxomnsyidedegl-ordsioiounns,pesgawrniodleulxpbaseamnidned `microscopically. examination. Other tissues, as appropriate, will be retained for possible future Effective May 18, 1999 21. tPeargmien3a,tioPnr,otphoesuendauSdtiutdeyd Tdriamfettianbtleer.imTroepoirntc,luadnedptrhoepaousdeidteddatdersafftorreipno-rltif,edelete. thetextin thissectionandreplace withthefollowing. IEnxcpiefreimSetanrttalDaSttea:rtADuagtue:stA2u6g,u1s9t9286, 1998 UInn-aluifdeiTteerdmDirnaafttiIonnteDraitmeR:epAuogrutstDa2t7e,: 1999 May 11, 1999 EAxupdeirtiemdenDrtaafltTReerpmoirntatDiaotne:DatDee:ceTmobebre 2d8e,te1r9m9i9ned : 313 002948 -- --w--n . - Pg AMENDMENTAPPROVAL Lr) PN.Sail Andrew M. Seacat, PhD Study Monitor 3M 1. a FhD Study. `Covance Laboratories Inc. DCatelef :3 : 314 002919 COVANCE> PROTOCOL AMENDMENT NO. 2 Covance 6329-223 3M T-6295.7 26-Week Capsule Toxicity Study with Perfluorooctane SulfonicAcid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Sponsor: 3M, St. Paul, Minnesota Study Monitor: ~~ Andrew M. Seacat, PhD `Testing Facility: Covance Laboratories Inc., Madison, Wisconsin Study Director: Peter J. Thomford, PhD `This amendment modifies the following portions of the protocol. Effective February 19, 1999 L Page 9, ObservationofAnimals, Clinical Observations, Paragraph 2, Sentence 2. To addan observation onthe day ofscheduled sacrifice, delete this sentence and replace with the following. Once weekly and on the dayofscheduled sacrifice, each animal will beobserved; `abnormalfindingsor an indication that the animal is normal will be recorded. Effective June 11, 1999 2 Page 11, Blood triiodothyronine Hormone Determination, Tests. (T3) and free thyroxin (T4) tests, To add free delete the textinthis section andreplace with the following. Samples willbeanalyzed by Ani Lytics Inc. for cortisol, testosterone,estradiol, `estrone, estriol, thyroid stimulating hormone, total and free triiodothyronine (T3), and total and free thyroxin (T4). : 315 002950 Covan3cMe 6T32299-52273 -_---s Protocol AmendmePntwNeo.222 3. mPeaagseur16e,sRteatpiostritcsal, aFnianlaylsiRsoefpocrhto,leSsttaetrisotlicaanldEtvoatlaluaatnidonfr.eeTtoriaidoddortehpyeraotneidne (T3), add the following to the end of the section. Chmoelaessutreesroalnaanlydstiostoaflcaonvdarfiraenecteri(iAodNotChOyrVoAni)nepr(oTc3e)dwuirlel wbiethanaavleyrzaegdebpyrerterpeeaattmeednt omreaAsNurCeOmeVnAtspfroorctehdeurpeasrawmileltebresaevsalcuovaatreidataetsp.=Tr0e.a05tmleenvetl.effAelcltspousntd-ehrocANOVA. rceocnotvroelr-yv)ewrisluls-bterecaotneddu-cgtroeudpumseinagnDcuonmnpeatrti'ssomnasn(yi-nocnl-uodinneg tvparlouceesdduurrei.ng Analyses both. will be carried out with SAS procedure PROC MIXED or BMDP, or Effective August 24, 1999 4. dPeacigseio,noGfrtohuepSDpeosnisgonratainodntshaenSdtuDdoysDaigreecLteovreltso,exFtoeontdnothteelde.ngTtohorferfleeccotvtehrey, delete the text in thisfootnoteand replace with the following. d Ttwreoataendifmoarlast lienasGtro26upwsee1k,s3,, athnedn4trdeeastimgennattewdilalsbreecdoivsecornytainnuiemda,lsanwidllthbee oafnitmoaxlisc ewfiflelcbtsefoobrsaetrvleeadstfo5r2rewveeerskisbiploistty,irpeeartsmiesntte.nce, or delayed occurrence: 5. dPeacgiessio,noCfltinhiecaSlpPoantshoorloagnyd,tFhereSqtuuednycDyi,reSccthoerdtuoleexdteConldltehcetiloennsg.tThoofrerfecloevcetrtyh,e delete the text in this section and replace with the following. t`rOenactembeenfto(rbeeifnoirteiatthieonodafitlyredaotsmee);nta;nadftaefrteartaltealsetas3t0,306,0,609,09,01,501,35a,nd1810,8027d4a,ysanodf 365 daysofrecovery 6. PthaegeSp1o0n,sBorloaonddHthoerSmtoundyeDDietreercmtiontorateixotne,ndFrtehqeuleenncgtyh.ofTroerceofvleercyt,tdheeledteecitshieon of textin this section and replace with the following. o`Tfhtrreeeattimmeents;baenfodraeftienirtaitatlieoansotf3t0r,ea6t0,me9n0t,;a1f35e,ra1t80,le2a7st4,30a,nd603,6950,daaynsdof1r8e0cdoavyesry 316 002951 Covanc3eMT6-36292-925237 -_-- Protocol AmendmenetPawgNeoe. 2 7. dPeacgiesi1o1no, fSethreumSpPoFnsOoSraLnedvetlheDeStteurdmyiDniarteicotno,r tForeexqtueenndctyh.e Tleongrtefhloefcrtetchoevery, delete the text in this section and replace with the following. 2B6e,foarnedi27n(iDatyoi1f8at3r)tedauitrmioenngnt;trdeuartimnegntW;eaenkdsdu1r(iDngaWye7)e,k2s,247, 6(,D8a,y1s2,18146,, 1208,5,24a,nd 51387,)5,7,2861(,D6a5y, 16990,)7,32,977(,Daaynd17979), 30 (Day 204), 31 (Day 211), 35,39, 43, 47, 51, Effective August 24, 1999, and January 12 and21,2000 8. PSapognes1o3r,aTnedrtmhienaSttiuodny,DiSrcehcetdourlteodexSatcernidfitchees.lenTgothroefflrecetcotvheerdyec(iesffieocntoivfet.he Aneucgruosptsy24(,ef1f9e9c9t)i,vetoJarnefulaercyt t1h2e, d2e0c0i0s)i,oanonfdtthoesuSbpsoenqsuoernttloyccalnacreilfythteherreecqovueirryement (effective January following. 21, 2000), deletethe text in thissection and replace with the oAvfetrenriagthtl,eatshte2n6anweesetkhseotfizterdeawtimtehnkte,tuapmtinoefaonurd axnyilamzailnse/,sewxe/iggrhoeudp,weixlsabnegufiansatteded, and necropsied. rAfetmearinaitnlgeaasnti2m6alwseweiklslobfetrreeamtomveendt farnodmatthleeassttu5d2y awseefoklslowwist.hout treatment, the `Two animals/sex in Group 1 will be returned to the Covance stock. Two animaly/sex in Group (Covance 6329-268). 3 wilbetransferred to a subsequent study k`eTtwaomainniemaalnsd/sxeyxlaizninGer,owuepig4hweidl,l ebxesafnasgtueidnaotveedr,niaghntd,ntehcernopasnieesdt.hetized with Effective August 24 andSeptember22, 1999 9. lPiavgeres1a2m,pAldedsibtiyobniaolpsFyec(aelffSeactmipvleeAsu.guAsttt2h4e,re1q9u9e9s)taonfdtthoe mSodpifoy tnthoescporloolcerecdtures (effective September 22, 1999), add the followingafte this section. 002952 317 Covan3cMe 6T3229025273 _--mm--m-- Protocol AmendmePntaNeo.s32 IAntsearmipmleLoifvleirveBrio(paspyprSoaxmimpalteesly 1 to 2 g) willbecollected by biopsy from aPnFiOmaSlsblionoGdrcoolulpec4tioonnl).y dTuhriisngsarmepcloveewriyll(bWeedeikvi5d7e,dwiitnhtoinfo1udrapyorotfitohnes saserum follows. `paOrnaeffsiunb,ssaemcptlioenewdil,lsbteaipnreedsweirtvhedheinma1t0ox%ynleiuntraanld-beuofsfienre(ddufpolrimcaaltiens,leidmebsewdidlledbein prepared), and examined microscopically. `frTeheezsere,cosnetdtsoumbasianmtpalien will -60 tboe-f8l0asCh,-furnotzielnsihnilpipqeudidfonritarnoagleyns,iasntod:stored ina 3KiMsE.J.TH.a&nsSe.n, PAD B9l3d5g.Bu2s-h3EA-v0e9nue TSte.lPeapuhlo,nMeiNnon.e:so6t5a1.5757180.66018 Facsimile No.: 651.778.6176 sKarmipslJe.sH.anRseseunlotrshweirlalbteerpnraotveidweidlfboer niontcilfuiseidonreignatrhdeinfgitnahlersehpoirptm.entofthe `eTvhaelutahtiirodn.subTshaemptilsesuweilbllobcekspraoncdesasehdemtaotbolxoyclkinstaangde cfoosrienl-escttarionnedmisclridoescfooprilcight microscopy will be transferred to: DSihvairsoinonAmDbirroescteo:r P4a9t1h5olDo.gPyrAossspoeccitautseDsrIinvteemational TDeulrehpahmo,neNoNrot.h: C9a1r9ol.i5n4a4.257257713 Facsimile No.: 919.544.3218 TAissssouceisatweisllInbteempartoicoensasled(PaAnYd).evAaluraeptoerdtbwyilellebcetprornovmiidcerdosbcyoPpAyIbyfoPraitnhcolluosgiyon in sthheipfmineanltroefptorhte. sSahmaplreosn.Ambrose or her alternate will be notified regarding the 318 002953 Protocol CAomveannIdcmMeeT3n62t92N-9o25.2723 -_-- Pages5 f`rTeheezfeor,usrethttsoubmsaaimnptlaeinwi-l6l0bteo f-l8a0shC-,faronzdetnriannslfiqeurirdednittorotgheenSapnodnssotrorfeodripnosasible future analysis. Effective January 12 and 21,2000 10. lPiavgeres1a2m,pIlnetsebryimbiLoipvseyr,tBhieonptseyrmSianmaptleesth.e AsttutdhyearnedqutestrofatnthheesSapnfoinmesaolrrsttoocaollect (seufbfseecqtiuveentJasntuuadryy (2e1f,fe2c0t0iv0e),Jaandudartyhe1f2o,l2l0o0w0i)ngaanfdttrotmhoisdisefcytitohne.requirement TAesrammipnlaelofLliivveerrB(iaopppsryoxSiammaptleleys1 g) will be collectedbybiopsyfromall animals cionlGlercotuiopns).3Tdhuirsinsgarmepcloevewriyl(bWeefelkas7h9-,frwoiztehninin1ldiqauyiodfntihterosgeenruamndPFstOoSredblioonad freezer, set to maintain -60 to -80C, until shipped for analysis to: Kris J. Hansen, PhD 3BlMdgE..2T3.E&-S0.9 935 Bush Avenue TSte.lPeapuhlo,nMeiNnon.e:so6t5a1.5757180.66018 Facsimile No.: 651.778.6176 sKarmipslJe.sH.anRseseunltosrwhierlableteprrnaotveidweidlfboer inontcifliuesdirioengatrhdeinfigntalheresphoirtp.mentofthe Effective January 21, 2000 11. aPnaagleyz1e2,adAddidtiitoniaolnaslerSuemrsuammpCloellsefcotriosnp.eciTfoierdeftlheycrtotihdedheocrimsoinoenso,fadthdefSoplloonwsiongr,to to the endofthis section. Aannimaallisqufotro(m0.G8rmou)opsft1,he2,a3ddaintdio4naslacsreirfuicmedcaotltlehcetitoenrsmainmapllenseccroolplescytewidlflrboemsaelnlt toontadlrTy3i,ceanbdyttohtealSTp4ondseotrertmoiDnra.tiSoanrso.j Das, Ani Lytis,forfree T3, free T4, 319 002954 CovanMceT623289-52273 -_---s Protocol AmendmentWNmo. s52 12. tPhaegdeec1i2s,iUornoifntehaendSpFoencseosrPtoFcOoSlleLcetvae2l4D-ehtoeurrmiunrainteioann,dFfreceaqluseanmcpyl.eaTfoterreoflneect Year ofrecovery, delete the text n this sectionandreplace with the following. rOencotvheeryf.irstIndaadydoiftiroenc,oave2r4y-ahnoudrafstaemraplteloefausrti6n,e30a,nd90f,ec1e3s5,wialnldbe18c0odlalyesctoefd before the completion of 52 weeksof recovery. 13. Ppraogceed1u6r,ePsofsotrrmeocrotveemryParnoicmeadlusreisn,thHeishtiogpha-tdhoosleoggryo.upT,oadddeftihnee ftohlelpoowisntgmoarfiteerm this section. PAotsntemcorrotpesmyoPfrtohceedruerceosvfeorryRaenciomvaelsryinAnthiemahilgshi-ndotshee gHrioguhp-,DporsoeceGdruorueps e(ixnacmliundaitnigoonr)gdaenscwreiibgehdtspraenvdiocuoslllyecatrieonnootfrteiqsusiureesd.forThhiestfooplaltohwoilnoggipcraolcedures will be done. NTehcernoepcsryopsy will include examination of: all orifices cranial cavity extseurrnfaalcessuorffatcheoefbtrhaienbarnadins;ptihnealexctoerrdnawlilslubrefaecexaomfitnheedspwihnealnctoirsdsuaentdricumtming is performed cervical tissues and organs `tehxortaecmiacl,saubrdfoamceionafl,thaendbopdeylvic cavities and viscera nasal cavity and paranasal sinuses Liver Samples dAivsiadmepdlienotfolithvreere (paoprtpiroonxsimaastfeollylo1w1s0. 2 g) will be collected. This sample will be pOanreaffsiunb,ssaemcptlieonweidl,lsbteaipnreedsweirtvhedheinma1t0ox%ylnieuntaranld-beuofsfienre(ddufpolrimcaaltien,sleidmebsewdildledbein prepared), and examined microscopically. `frTeheezsere,cosentdtsoumbasianmtpalien w-i6l0l tboe-f8l0asCh,-furnotzielnsihniplipqeudidfonr ainatlyrsiasnodtogs:toerend in a 320 002955 -_-- Kris J. IME TH.an&sSe.n, PhD Bldg. 2-36-09 935 Bush Avenue `StT.elPeapuhlo,nMeiNnon.e:so6t5a1.5757180.66018 Facsimile No.: 651.778.6176 Covan3cMe 6T32289.5272 Protocol AmendmentyNoe.r27 sKarmipslJe.sH.anRseseunltosr whielrlableteprrnoatveidweildlfboer inontcilfuiseidonreignatrhdeinfigntalheresphoirtp,ment ofthe `eTvhaelutahtiirodn.suTbshaemtpilsesuweilbllobcekpsraoncdesasehdetmoabtloocxkyslatiangnde efoosrienl-escttarionnedmisclriodsecfoopricl:ight `microscopy wil be transferred to: Sharon Ambrose: Division Director P4a9t1h5olDo.gPyrAossspoeccitautseDsriIvnet.ernational DTeulrehpahmo,neNoNrot.h: Ca9r1o9l.i5n4a4.252757713 Facsimile No.: 919.544.3218 TAissssouceisatweisllInbteerpnraotcieonsasled(PaAnId).evAaluraetpoerdtbwyilellebcetprornovmiidcerdosbcyoPpAyIbyfoPraitnhcolluosgiyon in the final report. shipmentofthe sSahmaprleosn.Ambrose or her alternate will be notified regarding the ASdadmiptlieosnoaflluTnigss,ukeiadnnedy,Sseprlueemn,Sathmyprloieds, brain, abdominal fat, heart, bile, (`Tahpepsreosxaimmaptleelsyw3ilgl ebaechf)l,asahn-dfrsoezernumin(laisqmuiudcnhitarsogpeosnsainblde)stwoirleldbien caolflreecetzeedr.,set to `maintain -60 to -80C, until shipped for possible future analysis to: Kris J. Hansen, PhD 3ME T.&S. Bldg. 2-3E-09 935 Bush Avenue TSte.lPeapuhlo,nMeiNnon.e:so6t5a1.5757180.66018 Facsimile No.: 651.778.6176 321 002956 -_---- m- Connce 6329.223 Protocol Ame3MnTdP-6u2N9o5.3:7 KrsaimspJl.esH.ansenorheralterate willbenotifiedregardingthe shipmentof the Effective February 11 and 15,2000 14. Page 3, Proposed Study Timetable. To change proposed dates for in-life termination (effective February 11,2000) andthe auditedraftreport (effective February 15, 2000), delete thetextin thissectionand replace with the following. Experimental Start Date: August 26, 1998 In-life Start Date: August 26, 1998 In-life Termination Date: March 7, 2000 Unaudited Draft Interim ReportDate:May11, 1999 Audited Draft Report Date: May 29, 2000 Experimental Termination Date: To be determined AMENDMENT APPROVAL Cone TN. Andrew M. Seacat, PhD Study Monitor 3M Saneat~ 1 ford; Study r Covance Laboratories Inc. 322 3 [30/2000 Date Date 002957 COVANCE PROTOCOL AMENDMENT NO. 3 Covance 6329-223 3M T-6295.7 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys `Sponsor: 3M, St. Paul, Minnesota Study Monitor: ~~ Andrew M. Seacat, PhD "Testing Facility: Covance Laboratories Inc., Madison, Wisconsin Study Director: _ Peter J. Thomford, PhD "This amendment modifies the following portions of theprotocol. Effective August 20, 1998 1. Page 16, Reports,Final Report, Statistical Evaluation, Paragraph 4. To correct an omission, add the followingtothe endofthe section. Dsaamtpalceolsliezec.ted during recovery will not be analyzed statistically due to the small Effective January 21, 2000 2. Protocol Amendment No. 2, Item 13. To correct a typographical error, change the font to bold on the following heading. Postmortem Procedures for Recovery Animals in the High-Dose Group Effective February 18, 2000 3. Page 12, Serum PFOS Level Determinations, Sample Shipping, Paragraph 3, Sentence 3. To indicate that the resultsofthe samplesforPFOS will be reported separately, Sponsor's delete this analysis of serum sentence and replace `with the following. Results will be reported separately. 2 323 0022958 Covan3cMe T663229925273 _-- Protocol AmeadmenetPaNgweoe.3? 4. tPhaagteth1e2,reIsunlttesroifmtLhievSeproBnisoorp'ssyaSnaamlpysliess,ofPtahreaignrtaerpihm3l,ivSeernbtieonpcsey s2.amTpoleisndwiiclalte be reported separately, delete this sentence and replace with the following. Results will be reported separately. 5. Pinadgieca1t2e,tThaetrtmhienraelsuLlitsvoefrtBhieopSpsoynSsoarm'psleasna,lyPsairsaogfrtahpehte2,rmSiennatlelnicveer2b.ioTposy samples will following. be reported separately, delete this sentence and replace with the. Results will be reported separately. 6. `PTaogiend1i5c,atPeotshtatmothreteremsuPlrtsocoefdtuhreeSsp,oLnisvoerr'sPFanOalSysiDseotfertmhienalitvieornssa,mpSleenstewniclle b2.e reported separately, delete this sentence and replace with the following. Results will be reported separately. 7. Preamgoev1e6,reRpeepaotretdsm,eFaisnuarleRsetpatoirstti,caSltaatniasltyisciasloEfvcahloulaetsitoenro,lPaanrdagtortaalphan4d. fTreoe triiodothyronine (T3) at the request of the Sponsor, delete the following. `Cmheoalseusrteesroalnaalnydsitsootaflcaonvdarfiraeencteri(ioAdNotChOyrVoAni)nepr(oTc3e)dwuirlel wbietahnaavleyrzaegdebpyrerterpeeaattmeednt moreaAsNurCeOmeVnAtspfroorcethdeurpeasrawmileltebreseavsalcuovaatreidataetsp. =Tr0e.a05tmleenvetl.effAelcltspousntd-ehrocANOVA r`ceocnotvreorly-)vewrislulsb-terceoatneddu-cgtreodupumseinagnDcuonmnpeatrti'ssomnasn(yi-nocnl-udoinneg tvparlouceesdduurrei.ng. bAontahl.yses will be carried out with SAS procedure PROCMIXEDor BMDP, or Effective February 18 and 22, 2000 8. P`Sapgoens1o6r,'sRaenpaolrytssi,s oFifntahleRseeprourmt,anRdesluilvters.saTmopliensdi(ceaftfeectthiavtetFheebrreusaurltyso1f8,th20e00) s`aenpdaruartienley,anddelfeetceetshesafmopllleoswi(negffective February 22, 2000) will be reported serum, urine, feces, and liver PFOS levels (provided by 3M) 32 002959 Protocol CAomveannIdcmMee6Tn3t22N99o.5.2723 -_-- Page33 9. tPGharegoleui1pv6e,r,LsiPavomesprlteSmsaomrwpitllelemsb.ePrrToecopeoidrntuderidecassteefpoatrrhaaRtteetlchyoev(reeefrsfuyelctAtsinvoiefmFatehlbesrSuipanorntysho1e8r,H'si2g0ah0n-a0lD)yosasinesdotfo dmeoldeitfeythteheteaxmtoiunntthiosfselicvteironcoalnldecrteepdlaatcenewcirtohptshye(feoflfleoctwiivneg.February 22, 2000), `Samples of liver will be collected as follows. ppOarnreaepfafsrieandm,)p,sleeacntwdiioelnexdba,emisptnraeeisdneemrdivcwerdiotishncoh1pei0mca%atlonlexyuy,tlrianl-abnudffceorseidnf(odrumpalliicna,teemsblieddeds ewdillinbe s`eTthetosemcaoinndtasianm-p6l0etwoil-l80beC,fluansthi-lfsrhoziepnpeidn floiqruaindalnyistirsogteon: and stored in a freezer, 3KrMiEs.J.TH.a&nsSe.n, PhD 9Bl3d5g.Bu2s-h38A-v0e9nue TSte.lePapuhlo,nMeiNnon.e:so6t5a1.5757180.66018 Facsimile No.: 651.778.6176 sKarmipsl1e.sH.anRseseunlotrshweilrlabletermenaptoertweidllsebpearnaotteilfyi.ed regarding the shipmentofthe me`Tvihacelruotahstiicroodnp.syaTwmiphlleletbwiesistlureabnbeslfpoercrokrcseedastnsode:dahtoembalotcokxysltiagneafnodr ceolseicnt-rsotnamiincerdosslciodpeifcor light DSihvairsoinonAmDbirroecsteo:r 4P9at1h5oDlo.gPyrAossspoeccitautseDsrIinvteemational TDeulrehpahmo,neNoNrot.h: C9a1r9ol.i5n4a4.257257713 Facsimile No.: 919.544.3218 tAThsiessosfcuiienaasltweriselplIonrbttee.rpnSraohtcaieorsnoasnledA(mPaAbnDrd)o.esveAalrouerapthoeerdrtbawlyitleellrnebacetterporwnoilvmliibdceerdnoobstcyiofPpiAyeIdbyrfeoPrgaaitrnhdciolnlugosgitoyhne in shipmentofthe samples. 325 002960 Covan3cMe T663229925273 -_-- Protocol Amendmh entPNaa og.es3 Effective February 22, 2000 10. PPaargaeg1r3a,pUhri2,neSeanntdenFceece3s. PTFoOiSndDiceatteertmhiantatthieorne,suSlatmopftlheeShSippopnisnogr,'s analysis ofthe urine and replace with the feces samples following. will be reported separately, delete this sentence and Results will be reported separately. 11. PGraoguep1,6,AdPdoisttiomnoarltTeimssPureocaendudrSeesrfuormRSeacmopvleesr,y APanriamgraalipnsh t1.heTHoigmohd-iDfoysethe `foalmloouwnitngo.f tissue collected at necropsy, delete this paragraph and replace with the S(aapmpprloexsimoaftelulnyg3, kgidenacehy,,isfpploesesni,blteh)yr,oaidn,dbbrialien,anadbdsoemriunmal(efaact,hhaesarmtu,ch as paonsdsisbtloer)edwiilnlabefrceeozlelre,ctseedt.toThmeasientsaaimnp-l6e0stwoil-l80beC,fluansthi-lfsrhoizepnpeidn lfoirqupiodssniibtlre.ogen future analysis to: Kris J. Hansen, PhD 3BlMdEg..2T-.3&8-S0.9 935 Bush Avenue St. Paul, Minnesota 55106 TFaeclseipmhiolneeNNoo..:: 665511..777788..66107168 KriJs. Hansen or her alternate will be notified regarding the shipment ofthe samples. Effective April 6, 2000 12. Pfeacgael3a,naRlyesgiusldaotnoeryatCtohmeplMiaaynoceC.liTnioc,idneclleutdeetahecotmepxltiianntcheisesxeccetpitoinoannfdorretphleace `with the following. `ATgheisncstyudGyoowidllLbaebocroantdourcytPerdacitniccoemRpelgiualnacteiownisthsthseetEfnovritrhoinnmTeinttleal40Porofttehcteion DUeSceCmobdeeorf2F9,ed1e9r8a3l),Reagnudlawtiitohnsa,nyPaarptpl7i9c2a,bilsesauemdenNdomveenmtbsewri2t9h,th1e98f3ol(leofwfienctgive exception. be done in Analysis of fecal samples compliance with GLPs. for urobilinogen atthe Mayo Clinic will not : Eo 002961 CovanIcMeT6-36229925273 _-- Protocol AmendmehntPeNaog.ess3 13. aPtatghee3M,aQyuoalCiltiyniAcs,saudrdantchee.foTloloiwnicnlgudteoatnheexecnedptofiotnhefsoercttihoen.fecal analysis done A`cnoamlpylsiiasncoef fweictahlGsLamPpsleasndfowrilulronobtilbeinaougdaietntedthbeyMaaQyuoalCiltiyniAcsswiulrlannocte bUenidt.one in 14. rPeaqgueir1e2m,eAndtdsiftoirofneaclalFaencaallysSiasm,paldeds.theTofoclllaorwiifnygantaoltyhseisenadnodftrehpiosrtsiencgtion. `cTohleorsmaemtprliecsaswsialyl.beTahnearleyszueldtsfoofrtuhreosbeilainnaolgyesnesuswiinllg aberopurtoivniedsetdafnodraridniczleudsion in the final report. 15. Preaqgueir1e7m,eRntescofrordfReecatlenatnialoyns,isP,adrealgetreapthhis2.paTraoignrcalpuhdaentdhereprleaccoerdwirtehtetnhteion following. `mWaittehriinal1sfyreoarmatfhteerSspuobnmsiosrs'siodnesoifgtnheeesfi(naAlnireLpyortti,csalIlnocf.,th3eMaEf.orTe.m&entSi,oMnaedyo C(lAinndicr,ewanSdeaPcaatth,olPohgDy,A3sMso)cibaytetsheInStpeornnsaotrio'nsald)eswiiglnleebse.sentto the Sponsor Effective April 7, 2000 16. Pseacgtei2on,aPnudrproepslea.ceTwoitmhodthiefyfotlhleowpiunrgp.oseofthe study, delete the text in this. `oTtohearssseeslsetchteedefbfieocctheomfitchaeltpesatrammateetreiraslownhcernitaidcmailneisntzeyrmeed dlaeviellys,byhocramposnueles,toand. cfoylnloomwoeldgbuys amnonakpperyosxfiomraattele5a2s-twe2e6kwreeeckosv.erTyhpeertiroeda.tment period will be : 327 002952 CovanIcMeT6-36292-925273 _-- Protocol AmendmePntPgNaog.ess3 AMENDMENT APPROVAL AnStdurdyewMoMn.iSteoarcat, PRD 3M PeSttuedryJD.Tirheocmtforord, PRD Covance Laboratories Inc. Dae Dae 328 002953 --- Covance 6329-223 MT3MeT6w2s957. APPENDIX 2 Individual Animal Fate Data Individual Clinical Observations Individual Ophthalmic Observations 329 ------------ 002954 o BoB 323 a2 EY) 282 &82 EY) 223 82 wm seamen hy Fe. 2& cares oncommon a oi 22& m -- EC er TA ene og PE ne 3g3S #5oro0: 1 oc: 32 PA 8 |wr o: I: 833 Ss as 5g< B5:oro1 1: R 56 3< ##7000: 1i wm 83g3 I E PooE rt & = | BEEIEEEm aman Tw | SERRE des o meer g$ 5 $2- BPir:o 1 @2 0 BERET EE Tei oe BERTmeros nsever < go 3s53 sn 223 #1BE} aor zo1 o:o::: 3 wn 223 gb10: 1i @ 3 ws 22S @bh] & 4 sens Om oggg& EE : : = us s83 2g 6 .g Bio zZ w 288 <3&8 8 - I 282 2= 9 32g mor : : 2 82& 350 33g8S & 1 323 22 2 ---- 5 Om e28 |:I 22& ES 323 | SE 22 354 3Z 228 822 356 g23 rooii 33g = 32g3g #0 3 5 g3 gi oo: oc: 2g 3 . g oJ s &32 ||I EEEE - 32 cg ----" g 8 Bio0: ::i 7 o o=: o o g 3 0 a Om <g 3g 2FE SE g sa 2233 |# : E0 E &3 0 l wt SET 23 223? 04 wr 5 3 222 88S 365 . Tone 2 gg wor: Bio 06 323g g gi r o or p: g3 a ns eon 2 #1 0: 1 8g 8 g2 B go i o oo Fb 2g 0 22 wI or i 2823 BEi:EE: } & m 233S mgobio : : oc:i: g3 m 222 rows Feces 223 #Bo 000: : : m 33322 Bi io: m 22 [gI fSE0: E1 i 882 ws 225 22B 16 g3332 E I$10T f 1A: E : i 8 m RE TTSR eresue a g EEREEm 33g HS - SL m  J-- se 3gg Fr 3 EA - Senn $2 fF 0: &52 0 2 2325 & m0 -23 PF 23 w 2 z332 & wm  wor z332s 2 . w ost s rm. 8g o as 283 #00 83 6 sevin 8 eng OEY 8 BE : @ : 33382 [oI d : 3 : w 2232 g0 } 10 0 ! i 8& 558  323 8a EE 2 oro: 8i 3 on - EE gi: 28 m 3sS B Fdor :0: 1: : 85 n | sm, pms sco otrcs arene ee 23 2PorI : il 238 moro: 04 eit Stent hares 223 |BoPSE 3gg 95 RT TS me 3SSs gBiE : ! i 82 6 2 F rr 33 82 m | tis en comoricn - g 1 2 882 08 283 332 EE) fatTCS eed I lg3e iF 0: i &32 ao = 8 coos 32333 "0 < Ed ff @ - gor 2 323 ra  REE wr - 388 a0  FI z32z - :g a 2 gr 0 5 2 8 a oo TE SE SE ---" o83 Bo 3& ooo. -  i 223 2a an 2 E3=At @ a0 3 85 0b 2 2 an 2 # :; i 2 an o wor ob 332@2 a 323 Robb 2s ae -- 5 LH FoF 238 g as > 2 Boro bd 2333 a6 25 #0: 1 2g& air 222 aor : : 3 3F& as Sepanain 2 Wm a Bg: oF 5323 ao 22 HoE F : : 2: g25 a a 225 Ee aa$ w #foh,!oo: p F: oiC a 2 Bobo g22 3 - . 5 L353 - Fo 25 2 Ey 2 gid 23g a 2 g3 FJ SE g& as 2 ge 2 83 soe oe a5 = & SE *a3 ar 22 i 1 fF os 22 g #00 f 00 0 b 8 a 2g223 B moroo3 bP : a 0 o-- Ra &3&232 ##100:: 3oF om 3 -- hee 1 3 g 3 a | &23 m wp: 2g Wor : : : 3 as 3g3 BPD: Fsi os fe hell 2 Bro e3 8 a 5s2 gio Po S 5) EES Sa 323 2 - - EE Sy . moe 2g3 @ "0 222 &gS a 2g3 Bop: 2 3 w ES Sy RN 22 gi : 2 & "w o22 |IEEE: 2S 3 "  2 328 as Si SE a 2 3 #i 1 i : e I ws te 3 = 205 23 Bi : i@ Pod 28 wr | asSine comers 29 2&2 ws war . #1 0: 23222 I w is EEE 323 Ei o: 1i:i 3@ 0 3233 8 5 ps eben [ERI 888 &3 a g88gg |I ELE oi | i & 83gg 8 uss  JE 8 g 6 g g 8 ER Sy n+ < #1 0: 1 g & ws oo ES Sm os Bio: E 2 - TSSy oe ;3 "w 460 s8gg 8 " 3 : i a ya SeSm _ -8gg gi: : 88 s Bioioro g 465 soni 3 TRE gg & " 2 $fi 858 8 s somes 8 8 gio: : . : 288 2 " 58& 2a an :z gio: 8 g8 ~ an i ha 888 8m sonop sven ) <g 8g 2g g - i ivi ppm coercion 1g<4 8BBg a6 i. 205 88828 m . Sp - gg & B888 2 PA sonsop cvting ' g2g8& w minsapt orton 8Bgg8 " 8B88 3 aw . ina escomin ) 88gB5 8 8 5 a 888 88 as 2288 8i a6 88g 88 wo ts 28:g --- CoMvanTIcMeeT6-3s6229s9.52772 APPENDIX 3 Individual Body Weight Data (kg) 003124 489 os esun om ggg wd 8 a 0 | S8a8 mT 8 * wo ink ey seicns th) 889 g 3 = " riogs 1 het) 888 & Fa atsio gooens 00 . i : oonon 8 888 8@ a0 a aSs o8 a5 [---- 88 8 os tvial 200 wigs saa th 8a 8 8 a TR 88gg H - } in Ss ou 288 25 o 8 8 a sw gg " H+138eCAonevmAicNaCs E e"v4no0s1e423500888 aSsaiassen a"aurs2tsaeammu 2000 ema Vit heCovaenbcsitee couance asonsronies M5a6s8s341 145us5227) Vseenr5s4A1x8UsSk 221) oartr4cs,142U2Cscons oMritn4e3r2G9ar78r3y001 THE DEVELOPMENT SERVICES coupaNY Shaping Solutions