Document QXXwbY70bNpeZ7aJNrL52MR2v

AR 826 _ 065D PFOS Repeat Dose - la 000066 PA athology Assoceiae ter s Inotersnatrieoornal Ea -:. =f May 30,2000 3DrM. CAonrdproerwatSeeTacoaxticology PBuOilBdoinxg3232202-02E-02 St. Paul, MN 55133-3220 NESET ws iam Re: Draft Electron Microscopy Report, Study No. 6329-223 (Recovery Amal) Dear Dr. Seacat, aPlneiamsaelsionnd Cenocvlaonsceed SthteddyraNto.el6e3c2t9ro-n22m3i.crLoisgchotpyanrdepuolrttrafostrutchteurraelcfoivnedriyngssacdtefnicteifed in rtheecolvievreryosfacmroifnikceeyasniamfatlsr,2i6ndwieceaktisngofretsroelauttmieonntohfatvheedliessaipopnseared in th tissues from PAflteearseyroeutrairnetvhiewglaonsdsyaprperpoorvtalfiogfurtehsisadnrdafatlregploorsts,y ewlielcltrpornepmairceroagsriagpnhesd ffoirnailnrseeprotrito.n into the final report, as these are originals. phraovsidbeePenAIa wpiltehasouprpeowroturnkiitnegswoithwo3rMk waintdhCyoovuanacgaeinonithtihsesftuutduyr.e.I hope that you will Sincerely. pen oo JVaicmeesPrBe.siNdoelndt,, SDoVutMh,eaPsthDOperations Enclosure: Draft electron microscopy report, 6329-223 4915 D Prospectus Drive Durham. North Carolina 27713 + (919) 544-5257 + (19) 544-3218 FAX 000067 Pathology Associates International A Companyof Science Applications Intemational Corporation =r Er curese Core, DRAF(TANPCAITLHLAORLYOGSTYURDEYP)ORT E`LOEF CLTIVREORN IMNICCYRNOSOCMOOPLIGCUESVMAOLNUAKTEIYOSN (RECOVERY ANIMALS) S2U6L-FWOENEIKCCAACPISDUPLOETTAOSXSIICUIMTYSASLTTUD(PYFOWSI;TT-H62P9E5)RFINLCUYORNOOOMCOTLAGNUES MONKEYS CLPIAEINSTTSUTDUYDNYUNMUBMEBREERM639299.-72623 Prepared by: James B. Nold, D.V.M. Ph.D., Diplomate, A.C.V.P. Patho4l9o1g5y.ADssPorcoisapteecstuIsntDerrinvaetional Durham, NC 27713 May 30,2000 PREPA3RMECDEFNTOERR3,MSTC.OPRAPUOLR,AMTNE5T51O4X4I-1C0O0L0OGY, 2815 0 Prospectus Dive + Dutham. Nori Carolina 27713 + (919) 544.5257 + (919) 544.3218 FAX 000068 Section Pathology Narrative 1 Light Microscopy and Transmission Electron Micrograph Interpretation Forms n `Transmission Electron Micrographs m Quality Assurance Statement ~v 000069 1. Pathology Narrative. 000070 DraftCoBveacntcoenSMtiucdyroNsoc.op3y29Re.p2o2r3t Recovery Sacrifice Animals DRAFT PATHOLOGY REPORT (ANCILLARY STUDY) ELECTRON MICROSCOPIC EVALUATION OF LIVER IN CYNOMOLGUS MONKEYS (RECOVERY ANIMALS) 26-WEEK CAPSULE TOXICITY STUDY WITH PERFLUOROOCTANE SULFONIC ACID POTASSIUM SALT (PFOS;T-6295) IN CYNOMOLGUS MONKEYS CLIENT STUDY NUMBER 6329-223 PAISTUDY NUMBER EM 99.76 SUMMMARY Two male and two female PFOS were removed from cynomolgus monkeys treatment for up to 53 treated weeks. for 26 Liver weeks with 0.75 mg/kg samples obtained by obriodpeslyayatedWoececkur5r7enacnedonfetcorxoipcseyffaetctWse. eLkig7h9t waenrdeeelexcatmrionnemdicfroorsrceovperiscibeivlailtyu,atpieornsiosftence, livers identified hypertrophy and no pathological changes. vacuolation. noted at 26 wTeheekstraenadtmcehnatr-arcetleartiezdecdhualntgreasstorufchtuerpaaltloyceblylular increased during the numbersof lipid recovery period. droplets within hepatocytes. had completelydisappeared PROCEDURES `sTehleecotbejdecatniivmealosf tihnisa s2t6u-dwyeweakscatopseuxlaemtionxeicibtyyeslteucdtyrownitmhicPrFoOscSopinyctyhenolmivoelrsgufsrommonkeys. Ianniamdadlist,iotnhetoulatrgaesnterruacltuqruaalleintadtpiovientesvailnucaltiuodnedfoqruadniififtearteinocnesofbpeetrwoexeinscoomnetsrowlitahnidn treated hepatocytes and membrane and mitochondrial changes in hepatocytes. of paraffin-embedded liver sections was also performed. Light microscopy `This report and female details the light and monkeys designated electron microscopy as recovery animals. findings in the four A previous report high-dose male (dated 6-24- w1e9e99k)sirnedsiuclatteedditnhhateptarteoactemlelnutlaorfvcaycnuoolmaotligounsamndonhkyepyerstrwoitphhy0.b7y5limgghtPmFiOcSr/oskcgo/pdya.y for 26 aHenpdatdoidcevlalruylairn vianccuiodleantcieonanadndsehveyrpietrytrboepthwyeewneriendpirviimdauraillyanciemnatlrsi.lobUulltarrasitnrudcitsutrrailbluyt,ion there were increased lipid droplets within hepatocytes. Increased lipid droplets were 1 000071 nz examined. Increased hepatocellular glycogen was present in the remaining two high-dose accumulation)was0.15 mg/kg/day of PFOS (mid-dose group). Quantitation of differences between control livers and livers treated with 0.03, 0.15, or 0.75 mg PFOS/kg/day. Ultrastructural differences in cellular membranes or mitochondria were recovery animals were examined to assess for reversibility or persistence of these `hepatocellularchanges, or for the delayed occurrence of additional PFOS-induced effects. For the toxicity study, 22 male and 22 female cynomolgus monkeys were given the test article or the vehicle once daily for at least 26 weeks according to Text Table 1. n 003 1st 4 4 m 0.15 & 6 v 075 30 6 Covance stock animal colony, and the two Group III animals were transferred to a subsequent study (Covance 6329-268). Liver samples were not obtained and examined in 000072 DraftCoEvleacnzcoenSMtiudcyroNsoc.op6y32R9e-p2o2r3t Recovery Sacrifice Animals pfiixeacteisvoefl(i2v.e0r% fpraoramfeoarcmhaladneihmyadlew/e2r.e5%thgilnu-tsalriacleddeahnyddeplinac0e.d1iMn %phsotsrpehnagttehbKuaffmeorv)sfkoyr's saunbdsseuqbumeintttueldtrtaostthriusctluarbaloreaxtaomriynfaotrisounb.seTqhueesnet leilveecrtsrwonermeicprrooscecsospeydpirnotcoesEspionng balnodcks epvaarlaufaftiino-n.embHeedmdaetdoxlyivleirns awenrdeeossuibnmi(tHt&eEd)f-osrtaliignhetdmsilcirdoessceocptiiconesxaomfifnoartmiaolni.n-fixed, w`Tahse afo5u7r-hWiegeh-kdolsiveerrebcioovpesryysaanmipmlaelsanadreali7s9te-dWieneTkextterTmainballen2.ecrFooprseyalcihvearnsiammalp,let.here Text Table 2. RecoveryAnimals Examined by Light and Electron Microscopy TM0a51e1 Tem 105533 e 105522 105542 `"oTnhi2n0s0e-cmteiosnhsc(oapppperrogxriidms.atste9ali0ynendmw)itfhro5m%tmheetEhpaononl-iecmbureadndyeldacleitveartse waenrdeRceuytn,omlod'usntleedad chietpraatteo,cyatneds,exwahmeirenecdleoanslayZiediesntsif9i0a0blteriannslmiviesrsisoecnteiloencs.trwoenrmeicprreofsecroepneti.alCleynetxrialmoibnuelda.r Ruletprraessternucttautriavlefeelaetcutrreosnwpehroetsoummicmraorgirzaepdhsfoofrleiavcehrpwheorteogtraakpehn aanndd asniginmiaflicoanntadesignated htreapnastmoicsystieosnwealsecmtarnonuamlilcyrocgoruanpthedinftoerreparecthatcieonntefrorpmh.otTohgeranpuhmedbehrepoaftopceyrtoexiasnodmerescionrded Fmiocrrloisghctopmyicwreorsecoepvya.lutahteeHd&anEd ssliigdneisfifcraonmt ftehaetusraemsewaenriemsaulsmmsaerleiczteedd foonraelleicgthrton microscopic evaluation form. RESULTS AND DISCUSSION aInndiimvailduaarle isnhtoerwpnreitnatSieocntsioofn lIIi.ghtRempircersoesntcaotpiyvesleildeesctarnodn eplheocttormoincmriocgrroagprhasphwserfeorseelaecchted as report figures, micrographs are labeled. and are shown in separately attached to ths Section report. IIL. Al remaining electron LightMicroscopy. A summary of the high-dose livers is incidence shown in and severity Text Table 3. of light There microscopic findings were no microscopic in the control findings that and. aapnpdevaarceudoltaotbieona,snsooctieadteadfewrit2h6twesetekmasteorfitarleaadtmmiennitstirnatsioonm.e hHiegpha-tdoocseellaunliamralhsy,pewretrreopnhoyt nideecnrtoipfiseydsianmprleecso.veirnydiscaamtpilngescoampeliethteer rtehveer5s7a-lWoefetkhebiliogphstymoircr7o9s-cWoepieckltievremricnhaalnges. 3 000073 Draft`CBolveacnrcoenSMtiudcyroNsoc.op6y32R0e-p2o2r3t Recovery Sacrifice Animals `hTihsetolpiavetrhofliongdyiongfscnyontoemdoilngtuhse rmeocnokveeyrsy.animals were considered normal background Text Table 3. Incidence and SReevceorvietryyofAnLiimghatlsMicroscopic Liver Findings 57-Week Biopsy (number exami[ ned) wTFw "Terminal Infiltrate, Necropsy lymphohistiocytic, minimal 1 2 Infilirate,Hylpyemrpphlohaissitai,obciylteicdu,cmt,inliimgahlt 21 -2 InflamImnaftliaomnm.atliyomnp,hoehoissitnioopchyitliicc., sslliigghhtt 1 1 - Clearing, cytoplasmic. centrilobular. minimal z 1 ~ Tncidence of zero. ElectronMicroscopy sEalemcptlreosnimdiecnrtiofsiceodpinco eavbanlouramtailonfoeafttuhrees5,7a-nwdeealkl tbiisospuseysaanmdpl7e9s-wweereek cnoencsriodpesryedlitvoerbe suleternaasfttreucrt2ur6alwleyenkosrmoafltr(eTaetxm:enTta,balned4)c.haTrhacetehreipzaetdocuelltlrualsatrruhcytuprearltlryobpyhyinacnrdeavsaecduolliapitdion tdersotplmeattse,riwaelrerenloattedseeefnfecitns.theThreecoevleecrtyroannimmiaclrolsivceorpsiacmfpelaetsu,reisnddiecsactriinbgedreicnovtheersyeof these. r1e-c4oavreeryresparmespelnetsatwievreeelceocntsriodnermeidcrnoogrrmaaplhsulftrroasmtrtuhcetsueraslafmipnldeisngasnidn ihlelupsattroactyetneos.rmaFligluirveesr and hepatocellular features. Text Table 4. Summary of Ultrastructural Finding--s Recovery Animals (T0r.e7a5)tmMeanlte,Gr5o7u-pwe(emkg/bkigo/pdsayyySex__ Animal No. 105511 (0.75)Male, 79- week necropsy 105511 (0.75) (0.75) Male, Male. 57-week 79-week biopsy necropsy 105522 105522 (0.75) Female, 57-week biopsy 105533 (0.75)Female, 79-week necropsy 105533 ((00..7755)) FFeemmaallee,. 5797.-wweeeekk bnieocrpospy 105542 105542 Comments `Normal Normal ___ Normal Normal Normal I. _ Normal Normal 5 000074 DraftCoElveacntcreonSMtuidcyroNsoc.op6y32R9e-p2o2r3t Recovery Sacrifice Animals Peroxisomes were Mean peroxisome niudemnbteifrisedpearndheqpuaatnotciyttaetewdeirnerceopnrsesiednetraetdivneorhmeapla.tocytes (Text Table 5). Text Table 5. Quantiationof Hepatocellular Perorisomes 0.75 mg PFOS/kg/day (High-Dose) Recovery Animals Mae heme NAunimmbaelr _NPuemrobsesromoefs|| NAunmimbaelr_ pNeuromxbiesromoefs|| NAummmbaelr PNeurmobxiesroomfe|| NAunimmbaelr _NPoermobiesromoefs lToossss Meam= 1198ss | j[1o0sSs2 1895 | Mesm=___ 215982 || Imoossss 225 | Mem= 229380 | l[joessss 264 | Mem= 210582 iso `1A0vSeSr1a1ge(nbiuompsbye)r. opfepreorxoixsiosmoemsewsepreercRoeupnatteodeiyncofnolryifvoeurhheeppaattooccyyeesscounted pe animal. For Animal No. CONCLUSION wLiigthht0a.n75d melgecPtrFoOnS/mkicgr/odsacyopfiorc2e6vawlueaetkisonanodf lailvleorswefdrotom"cryencoomvoerl"guusntmreoantkeedyfsortr3e1ataendd h5e3pawteoeckeslluidleanrthifyipeedrtnrooppahtyhoalnodgivcaacluoclhaatnigoens,.chTahraectterreiaztmeednutl-trrealsattreudctcuhraalnlgyebsyofincreased tnhuemrbeecrosveorfylippeirdioddr.oplets within hepatocytes at 26 weeks, had completely resolved during SIGNATURE OF AUTHOR James B. Nold. D.V.M.. PhD. Diplomate, AC.V.P. Dae Is 000075 IL Light Microscopy and Transmission Electron Micrograph Interpretation Forms " Finorarmcshiavreedexsatcutdycofiplie.es of signed/dated raw data sheets maintained 000076 LIGHT MICROSCOPIC EVALUATION Study No.: 6329-223 (EM 99.76) Species/Strain: Cynomolgus Monkeys Animal No: _scebelow Sex:seebelow Tissue: Liver Interpreting Pathologist (signature and date): OpeeB0MC puna DoselGroup| NAuvimmbaelr Microscopic Findings and Comments 0G7i5ghmdopseg)| 105511 || T_ Tflaie mphohismiuoscaymiincim.al Recovery 105532 Now _ 57 uMiaebsop | G0lR7eg5chmodvioeskreye)|||T1_0O55S4S_7 ||| T.1. fTieoain.lylmm pyonRsoo ceyye.. Fm mouslilfamoce eal mlsinae l me 57wFekmbailoepssy g07h5omsptg)| TOSSTT Toa. mph shocye, maleioel ral Rercoovery I necwrokpsy GSE 2I Hyfpeirpleasipa fochal, Sioghbt ilmeadluioeitocaelmis nal 43. Iinnfflfaammmmaattioonn., eboysimnpohpohitliisci.omcoyliicomcuallt.ifhoigchal,slight G0i7ghdmopstg)| 105533 1. nla. ymphohistoeyi. muifocal maT RFeecmoavleersy |__ _ 9a8towresek SSH || 21 IClenariinag, cyymopphloahsimsii.ocseyniiel,ombaullairo.camlinmimianlimal 000077 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No. 6329-223 (EM 99.76) Animal No.: _105511 Treatment Group: ST-week biopsy, _75 me/ke (hi-dose) Sex: Male Species/Strain: Cynomolgus Monkey Tissue: c__enLtivreir-lobular Block No(s). _99.7631-1A Photo/Negative No(s).Z:1_8Z8128382- 7 Significant Lesions (check one): _____ Yes ___X No Interpreting Pathologist (signature and date):Groeafnuel Miv262000 cFeenattruarlensu:cleZuIs8.82T3h.eLsiuvrerr.ounHdeipantgoccyytteo.pla1s5m2c9o0ntXa.inTshnisumheerpoautoscrytoeunhdastoaosvmaalll round mitochondria and several small to large lipid droplets. The darker round organelles, pmeorsotlxyissolmigehstlcyousnmtaeldl.er Tthheanitnhteermvietnoicnhgoncydtroisao,lacroenptearionxipsroomfeilse.s oTfweenntdyo-pfloausrmi(c24) 2re1t8i8c2u4l.umLainvedr.som1e03f2i0neX.graPnourlteiso,nwshoifcthhrmeaeyceblelsgalryecopgreens.ent. The dorsal two hepatocytes eexnhdiobpiltaasmmiicxtruerteicouflnuomramnadlsacpaptetaerreidngpecryotxoipsloamsemsi.c eTlheemelnatrsg,erinvcenlturdailngcemlil'tsocchyotnodprliaas,m mcointtoacihnosndirrrieagualrarecvliedaernts,paacneds tshuirsrcoeulndiesdprboybambelmybnroatnaouhespamtaoteeyrtieal.. AOrnoluynda cthoeuppleeriopfhery rareeprseesveenrtalsmloaorgtehsetnrudcotpulreassmciocntraeitniicnuglufmineilnytegrrmainxueldarwainthd greltyiccouglaernmaatnedroiarl.ribTohsiosmemsa.y c`aopmppeaarcstitongbeofmtehmebsruarnreoubnoduinndg,cybtuotptlhaissmm.ayOrb,ethaepsesesutrduocmtuermebsrmaanye rceapurseesdebnycytoplasmic ainpvpaegairnantoirmoanls.ofPeexrtroaxcieslolumleasrwmeatreerinaolt scuocuhntaesdpilnastmhai. eTlhecetrnounclmeiacrrporgorfaiplehsbienctahuesseeacnells 2in1d8i8v2i5d.ualLihveepra.toHceyptaetsochyotwei.ng1m0o,3s2t0 Xo.f i tTshciysthoepplaatsomcyits enoits preset. polygona l with a round centr al lniupcildeudsr.oplIettsss.urTrhoeunidnitnegrvceyntionpglcaystmocpolnatsamincsonntuaminesropruosfimlietsoocfhoennddroipalaasnmdica freetwicsumlaulml, scattered fine glycogen granules, and some dark round peroxisomes. Twenty-two (22) bpeortotxoimsroimgehts ocfoutnhteedp.hotTohgrreaephadijsaacefnitnehleypgartaoncuyltaerssatprupcetaurree,sswehnitciahlliys onuotrsmiadle. ofAtthtehe centered hepatocyte and appears similar to that described in Z18224. sZp1a8c8e26a.lonLgivietrs.riHgehptamtaorcgyitne.. T1h0e,3c2el0lX.haTshaissmhaelplatnouccylteaerisprboofridleearendd baybuanndeaxnttraccyetlloupllaarsm lciopnitdaidnroipnlgetnsumareeropursesmeni.tocSheovnednrtieaenand(1s7l)igphetrloyxdiisloamteedsecnodunotpelda.smic reticulum. Several 000078 103511 Biopsy Continued. 218827. Liver. Hepatocyte. 10,320X. A large round nucleus is present in the centered ehenpdaotpolcaytsem.icTrheetiscuulrurmo.unAdinfgewcydtaorpklraessmidcuoanltabiondsiensumareeropruessemnitt.ocEhloenvdernia(1a1n)dpaebruonxdiasnotmes counted. A the right dorsal margin of the photograph are an endothelial cell and a granular structure that appears to be an invagination of plasma. `Conclusions: Normal hepatocytes. Peroxisomes average 18.5 per hepatocyte 000079 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 6329(-EM2992.736) Species/Strain: CynomolgusMonkey Animal No: 105795 -wee1 k n1 ecropsy `Treatment Group: _75mg/kg(hi-dose) Sex: Male Tissue: __ Liver -centrilobular Block No(s).: _99.76:3224 Z18828 Photo/Negative No(s)..__Z18832 Significant Lesions (checkone): ______ Yes _X No Interpreting Pathologist (signature and date):DpemgOrd Mavzo2000 Fsheaapteurweist:haZsIm8a8l2l8.roLuinvdern.ucHleepusa.tocAyptep.rox1i0m,a3t2e0lX.y 8T-h9issmahlelpattoocmyetdeiiusmsosmizeewdhlaitpiodval in drrooupnldettso arroed-prsehsaepnetd imnitthoechcoyntdorpilaas,mi.nteTrhveenmianjgorairteyasoofftshemocoyttohpelnadsompcloanstmaiicnsrentuimceurloumus (SER) and "more clear" patches of glycogen. Some profiles of rough endoplasmic preetriocxuilsuomm(eRsEcRo)untaendd. roBuilnedcdaanraklipceurlioxciasnobmeesscaeren ablestoweeveindentth.e hTewpeatnotcyy-teeisghstit(u2a8t)ed on the sight and left with the central hepatocyte. Zc1ol8l8a2g9e.n aLrieveprr.eseHnetpaattoctyhtet.op 1r0ig,h3t20mXa.rgiTnheofcetnhtisraellevcetirnonanmdicsruorgrroaupnhd.ingAesxitnruascoelildulisar aslusroopurnedsienntg actytthoepllaoswmercolnefttaimnairngginn.umTehreouisntperrovfeinliensog fheepnadtoopclyatsemhiacsraertoicuunldunmuacnldeudsaraknd mitochondria. There is mild dilatation of the endoplasmic reticulum and nuclear envelope, which is a fixation artifact. Several dark residual bodies and occasional small lipid droplets are also identified. Differentiation between peroxisomes and mitochondria 2is18di8f3f0i.cultL.ivEerl.eveHnep(a1t1o)cypteer.oxi10s,o3m2e0sX.couTnhteedc.ytoplasm of this hepatocyte contains `abundant SER, although some profiles of RER are also evident, especially around some. h`meiptaotcohcoyntder.iaA.ppAronxuicmleaatrelpyrof2i0leliipsidnodtroepvliedtesntarien ptrheissenptl.anTewoefnsteyct(i2o0n)opfetrhoexciesnotmeersed caoluonntgeds.inuTsohiidss.hepTahteocnyutcel'esumsiocrfoavnilelnoduosthbeolridaelrceilslevisidpernetsebnottahtdtohresallolwyearnrdigvhet.ntrAadljlaycent hepatocytes are present 0 the left and right of the centered hepatocyte ZI8831. Liver. Hepatocyte. 10,320X. The morphology of this hepatocyte is very sriemtiilcaurlutom Za1n8d8"2m8o.reThcleeacry"toppaltachsemscoofntgaliyncsogneun.merSoeuvsermailtloicphiodnddrroipal,etesndaroeplparessmeinct. Nulirnaesttereunct(u1r9a)llpyesriomxiliasro.mes counted. The surrounding hepatocytes appear 000080 T0511 Necropsy Continued. ZI8832. Liver. Hepatocyte. 10,320X. This hepatocyte appears similar to Z18830. Its cliyptiodpdlraospmlectos,ntmaiitnoscahobnudnrdiaan,tpSeErRo.xisOotmheesr arencdogrnesiizdauballeboordgiaens.ellEeisgihntceleunde(1s8o)me RER, peroxisomes counted. At the lower right margin of the photographi a sinusoid containing several erythrocytes. A prominent bile canaliculus is presenta the right `where three hepatocytes appear to meet. These hepatocytesareconsidered to be ulrastructurally normal. `Conclusions: Normal hepatocytes. Average 19.2 peroxisomes per hepatocyte. 000081 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: _6329-223 (EM 99.76) `Species/Strain: CynomolgusMonkey Animal No: 105522 Treatment Group: 15m5g7/-kwge(ehki-bdioopssey) Sexi Male Tissue: Liver -centrilobular Block No(s). __99.76:33:1A Photo/Negative No(sZ)1.8_83Z318- 837 Significant Lesions (check one): Yes X No Interpreting Pathologist (signature andJ date): f vmzmao Features: ZI8833. Liver. Hepatocyte. 10320X. A sinusoid containing finely granular opvlaalsmnaucilseuasl.ongIntthheerisguhrtrmoiucnrdoivniglclyotuospmlaarsgminareofstehviesrahlepvaetroycyetle.ectTrhone-dheenpsaetorceysitdeuahlas an bodies. whichi Tahefirxeatiisomnialrdtifdaiclta.tatEiiognhotfeetnhe(1n3u)clpeearroexnivseolmoepsecaonundteedn.dopMliatsomcihconrdertiiacualruem, numerous and are variably shaped. 211082831488,33L.iveTrh.erHeepiastmoiclydted.ila1t0a,t3i2o0nXo.f tThheiesnhdeoppaltaoscmyitce arpepteiacrusluumltarnadstnruucctlueraarlleynvseilmoiplea.r e"oTshienhopehpialt.ocyEtieghltin(e8s)apseirnouxsiosido,mewshiccouhntceodn.tains two erythrocytes anda granulated ZI883S. Liver. Hepatocyte. cytoplasm of this hepatocyte. 1It0,h3a2s0aX.ligShetvleyraolvaslmanlulclleiuisdadnrdopalbeutsndaarnetpcreysteonptlainsmthe s`wihgihcthdciolnattaatiinosn noufmtehreoeunsdompiltaoschmoincdrreitaicaunldumpraofnidlensuocfleenadroepnlvealsompei.c Aretidciusltuimn.ct mTyheelrien is figure is evident associated with a lipid droplet o the left of the nucleus. Twenty-two (22) peroxisomes counted. Z18836. Liver. Hepatocyte. 10,320X. The hepatocyte has a large polygonal nucleus. "sTmhealclyttoopllaragsemlicpoindtdarionpslaebtsunadraenptreSsEenRt.anNdumReErRo,uwshriocuhnedxhdiabriktpselrigohxtidsiolmaetastairoen.evSiedveenrta.l Thirty-five (35) peroxisomes counted. but appear ultrastrucuurally normal, Mitochondria are numerous and variably shaped, m21a8r8g3i7n.s ofLitvheirs.eleHcetpratooncymtiec.rog1r0a,p3h2.0X.TheAlsiignhtuesro-isdtaiisnpirnegseenndtotahleolnigalthceelllefst apnrdesleontwearlong mthietolcohwoenrdrmiaargainnd. eTnhdeopcleanstmeircedrehteipcautlocuymtien hiatsssaunrroovaulndniuncglceyutsopalnadsmn.umeThreorues is mild dilatation of the endoplasmic reticulum and nuclear envelope. Several lipid droplets and residual bodies are also discernible. Fourteen (14) peroxisomes counted. 000082 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 6129(-EM2992.763) Species/Strain: CynomolgusMonkey Animal No: _7_9-w1ee0k n5ecr5op2sy 2 Treatment Group: _.75 mg/kg (hi-dose) Sex: Male Tissue: Liver - centrilobular Block No(s). __99.76-34-2A Z18838 Photo/Negative No(s)..__Z18842 Significant Lesions (check one): ______ Yes __X No Interpreting Pathologist (signature and date): pelt Mame Features: Z18838. Liver. Hepatocyte. 10,320X. This hepatocyte has a large round nucleus. At the very top left margin of the electron micrograph is a segment of sinusoid Palhoontgogirtaspmhi,crwohveirlelotuhsebcoerndteerr.edAhebpilaetoccayntaelimceuleutss iasnevaiddjeanctenatt htehpeatloocwyeter.lefTthmearcgeinnteorfedthe Thehpearteocisytselighhats daibluatnadtainotn ocfyttohpeleansdm,opwlhaiscmhiccornettaiicunlsumn.umeMriotuocshpornodfrilieasaorfe RnuEmReraonudsSaErRe, round to oval in shape. Approximately twenty-three (23) peroxisomes counted. ZI883, Liver. Hepatocyte. 10.320X. Two hepatocytes are clearly evident in this electron micrograph. They appear ultrastrucuurally similar. They exhibit mild dilatation oftheir endoplasmic reticulum and nuclear envelopes. They have numerous. `mitochondria and occasional lipid droplets and residual bodies. Peroxisomes are usually croouunntdeadnidn mtohereupepleercthreopna-tdoecnystee.than the mitochondria. Seventeen (17) peroxisomes 218840. Liver. Hepatocyte. 10,320X. This hepatocyte has a large round central dnuicllaetuesd.proTfhileessuorfreounnddoipnlgascmyitcoprleatsicmulhuams. nuBmoetrhotuhse mSiEtoRchaonnddRriEaRanadppmeaarnyslsilgihgthltyldyilated. Approximately 15-16 small to medium sized lipid droplets are present, as are a few very electron dense residual bodies. Twenty-one (21) peroxisomes counted. 2p1l8a8n4e1,of seLcitvieorn.. HAepastioncuystoei.d co1n0t,a3i2n0iXn.g aTnhiesryhtehpraotcoyctyeteis'sprneucsleenuasliosnngottheevviedreynttoinp this `rmeatrigciunluomf tthhirsoueglehcoturtonthmeiccyrtoogprlaapshm.. TAhlesroe, irsasnlidgohmt dsimlaaltlattioonmoefdtihuemensdizoepdlalsipmiidcdroplets are present. Numerous peroxisomesareevident. Forty (40) peroxisomes counted. 218842, Liver. Hepatocyte. is ultrastrucuurally norma. It h1a0s,3a2n0Xo.valThniusclheeupsataoncdytaebuanpdpaenatrssusrirmioluanrditongZIc8yt8o3p8l,aasnmd containing numerous mitochondria and slightly dilated profilesof endoplasmic reticulum. Twenty-eight (28) peroxisomes counted. Sinusoidsareevident at the upper left and lower right marginsofthe photograph. 000083 "TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 6320(-EM2900.763) Species/Strain: CynomolgusMonkey Animal No. _I05533 57-week biopsy Treatment Group: _75ma/ke(hi-dose) Sex: Female Tissue: _c_e_nLtivreri-lobular Block No(s): __99.7635-1A 718843 Photo/Negative No(s).:_Z18847 Significant Lesions (check one): Yes __X__ No Interpreting Pathologist (signature and date):SuoB0 May20.2000 Features: Z18843. Liver. Hepatocyte. 10,320X. This hepatocyte has a small nuclear tprhoefiellee.ctrAonsimniucsrooigdralipnhe,dabnydaanneanddjoatcheenltiahlecpealtloci ytpereissepnrteasleonntag thteheuplpoewrerrilgehftt mmaarrggiin.n ofA cpernotmeirneednhtebpialteoccayntaeliiscuvloulsuimsipnroeussenatnbdectowneteanintshenutmweorhoeupsatmoictyotcehs.ondTrhiea cayntdopelnadsomploafstmhiec rsleitgihctullyumd.ilatTehd.e eTnhdiospldialsamtiactiroentiiscaulfuimx,atniuocnlearatrifeacntv.eloApef,ewansdmamliltotcohmoenddriiuamapsipzeeadrltiopibde droplets are present. Forty (40) peroxisomes counted. 2al1a8r8g4e4.ovaLlivneru.cleuHse.patIoscyctye.top1l0a,s3m2c0oXn.taTihnissnsuommereowuhsatmittroiacnhgounldarri-ashaanpdedslhiegphattlyocdyitleatheads profiles of RER. Some glycogen granules appeartobe intermixed with the SER Eighteen (18) peroxisomes counted. 2Z1188884454,, aLlitvhero.ughHetpwaotoncuyctel.ear 1p0r,o3f2il0eXs. arTehpirseseelnetc,trwohnimcihcrisogprraopbhaabplpayeafrusncstiimoinlaorfttohe p2l1a8n8c46o.f sLeicvteiro.n aHletphaotuogchytiet.ma1y0,b3e2b0iXn.ucTlehaetecde.ntTehrierdtyh-efpoautroc(y3t4e) cpoenrtoaxiinssoamessmaclolunrtoeudn.d npruocflieluess.oTfheendsouprlraosumnidcinrgetciyctuolpuml,asbmotchonStEaiRnsannudmReErRo.usOmnietomcyheolnidnrifaigaunrde salnidghstelvyedriallated clioplildagdernopalentdsaarseinpursesoeindt.areThpirretsye-ntthraeleon(g33t)hepelroowxeirsroimgehts mcaourngtiend.of Sthoempehoetxotgrracaeplhl.ulaTrhe hepatocyte cytoplasm caontthaeintoapburnigdhatnits SmEoRr.e lSioghmtely msiationcihnogndtrhaina tahnedcpeenrtoerxeidsohmeepsataorceytael,soanevdiditesnt. 218847. 218844. LAilvterh.ougHheptahteorceyties.sli1gh0t,3d2i0lXa.tatiTohnisofeltehecternodnompilcarsomgircaprehtaicpupleuamr,s tvheirsyhseipmaitloacryttoe appears ultrastructurally normal. Twenty-four (24) peroxisomes counted. 000084 "TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Sex:___Female~~ 218848 - Photo/Negative No(s). _Z18852 Significant Lesions (check one) Yes X__ No Interpreting Pathologist (signature and date): Jpomforf Mazo Features: Z18848, Liver. Hepatocyte. 10,320X. This electron micrograph illustrateas 218849. Liver. Hepatocyte. 10,320X. This pentagonally-shaped hepatocyte haas somewhat ZI8850. Liver. Hepatocyte. 10,320X. This hepatocyte is bordered by a sinusoid at its lower Z18852. Liver. Hepatocyte. 10,320X. This hepatocyte is round, with a round nucleus, and 000085 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 6329-223 (EM 99.76) Species/Strain: Cynomolgus Monkey Animal No: _57I-w0eek5bio5psy42 `Treaument Group: _.75 me/kg(hi-dose) Sex Female Tissue: _L__iv-peeripor rtal Block Nos): __99.762:183875-31A__ Photo/Negative No(s). __Z18857 Significant Lesions (check one): Yes X_ No Interpreting Pathologist (signature and date):YrB 1 Muv202000 bFyeaatdujraecse:ntZhIe8p8a5to3c.ytLeisveor.n aHllepsaitdoesc.yteIt.has102.3l2a0rXg.e pTohliysgohneaplatnouccylteeusapapnedarasbusnudrarnotunded sexutrrraocuenldluilnagrcsyptaocpelacsomn.taiJnuisntgosothmeelmoiwcerroveifltlioafntds cnoulcllaeguens,ispraosbmaabllly sneegxtmeontaosfinusoid. `Seenvdeorpallassmmailclrelitpiicdudlruomplisetssliagrhetlpyredisleanttedi.n atnhedcsyetveorpallasrmesoidfutahlebcoedniteesreadrehperpeasteoncty.te. The Nineteen (19) peroxisomes counted. Zth1i8s85he4p.atoLicvyetre. isHselpiagthotclyytdei.lat1ed0,,3w2h0i.ch SailmoinlgawrittohZt1h8e8d5i3l,attehdeneuncdloeaprlaesnmvieclorpeet,icisulaunm of artifactoffixation. swelling. Multiple The bile mitochondria canaliculi are appear evident oalsoexhibit some slight artifactual along the margins of this cell's contact with aZd1j8a8c5e5nt. hLeipvaetro.cytHeesp.atoAcpyptreo.xi1m0a,t3e2l0yX.thiTrhtieesnh(e1p3a)topceyrtoexihsasomaeslacroguenrteodu.nd nucleus. I's sAuprprroouxnidmiantgelcyyt2o0plsamsamllcotnotmaiendsinuummesrizoeudslpirpoidfidlreospoleftSsEaRr.e pwrheiscehntarine tmhienciymtaolpllyasdiml.ated. Fifty-one (51) peroxisomes counted. 2co1l8l8a5g6e,n fLiibvreirl.s isHperpeasteonctytnee.xt1t0o,t3h2e0Xc.entEexrterdacheelplautloacrytsep.aceThceonhteapiantioncgyntuemhearsoauslarge `eonvdalopnluacslmeiusc raentdicaubluunmd.anAt fsuerwrsomuanldlinlgipciydtdorpopllaestsmawriethpnreusmeentr.ouTshimritteoecnho(n1d3r)ia and peroxisomes counted. Z18837, Liver. mitochondria are Hepatocyte. abundant in t1h0e3c2y0to.plaSsmmoooftthhiesnhdeoppaltaoscymtiec. reticulum and The hepatocyte has a lNairngeetreeonun(d19n)ucpleeruosxiasnodmseesvecroaulntsemda.ll to medium sized lipid droplets are present, 000086 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 6320(-EM2992.763) Animal No: [05542 79-week necropsy Treatment Group: _.75mak(hi-dose) Sex: Female Species/Strain: CynomolgusMonkey Tissue: c__e_nLtivreir-lobular Block No(s).: _99.76:38:2A. 218858 Photo/Negative Nofs).:_Z18862 Significant Lesions (check one): _____ Yes __X__ No Interpreting Pathologist (signature and date)Senn Marco Fnuecalteuurs.es:ToZIt8he83l8ef.t oLfivaenrd. bHeenpeaattohcytthee.nuc1l0e,u3s20aXr.e sTehviesrahlepmaetdoicyutmeshiazsedalriopuidnddrcoepnltertasl `sTohmeerepmroafiinliensgocfyRtEoRpl.asSmixco(n6t)apiensroaxbiusnodmaenst SER, patches counted. of glycogen, mitochondria, and 218859. multiple Liver. areas of Hepatocyte. 10,320X. The cytoplasm of this hepatocyte contains glycogen granule accumulation. These are lightly stained areas with ncyutmoeprlaosums.smTahle edlaerckterronanddenismeeggurlaanurlcesy.topAlafsemiwclimpaitderdiraolpliestsSEarRe ianltsoerpmriexseedntwiitnhthe glycogen. Five (5) peroxisomes counted. hZ1e8p8a6t0o.cytLeisvecr.ytoHpelpaastmo.cytTeh.e c1el0l,3h2a0sX.a lTarhgiertryo-utnwdo n(u3c2l)eupse.roMxoissotmoefstahreeccyotuonptleadsminitshis eovcicduepnitedarboyunSdERm.itmoichtooncdhroinad.riaA, amincdroovciclalsoiuosnablorlidpeird adrnodplaedtsj.aceSnotmsienupsroofiidleasreofprResEeRntare along the ZI8861. right Liver. margin of this Hepatocyte. hepatocyte. 10,320K. For this hepatocyte. its nuclear profile is not pprreesseenntt. inSetvhiesrapllaonfetohfesseecctoinont.ainAsomomdeermaetmebrnanuoumsoofrbmmeyeedliirun-mliskiezeddebrliisp.id drNoupmleetrsouarse rpreotfiicluelsumo.f SNEuRmearnoduRsEvaRriaarbelyprsehseanpte.d Tmhietrocehiosnsdlriigahtadrielaptraetsieonnt,obfutthetheenydoapppleaasrmic ZuIl1t8r8a6s2t.ructLuirvaelrl.y nHoerpmaatlo.cyteE.lev1e0n3(2101X).perTohxiissohmeepsatcoocuyntteeda.ppears somewhat triangular csyhtaoppeldasimn.thiSsevpelraanle somfaslelcttioonm.edItihuamss2lizaerdgelipriodudnrdopnluectlseuarseapnrdesaenbtu.ndaAnbtunsduarrnotuSndEiRn,g csyotmoeplaprsomf.ileAspopfroRxEiRm,atmeiltyocthwoenndtryi-at,waon(d22p)erpoexriosxoimseosmeoscccuoupnytetdh.e majority of A sinusoid the. with an endothelial cell and erythrocyte is present at the lower left margin of the photograph. 000087 IIL Transmission Electron Micrographs 000038 Figure Legends DraftCoElveacntcreonSMwidcyroNsoc.op6y32R9e.p2o2r3t Recovery Sacrifice Animals mMaegansiufriecmaetnitonb.ar Porninetacmahgpnhiofticoamtiicornogisrainpdhiceaqtueadlsonontehemibcarcokno(f0e.a0c0h1mpmh)o.tograph. The pFiegruorxei1s.omeLisvearn.d oHtehpeartoncoyrtmealshoorgwainnegllneosrmaraelicdyenttoipfliaesd.miTchoerrgeaneisllselsi.ghtSarotmifeactual dilatation of the nuclear envelope and mg/kg/day (high-dose male), S7-week ebnidopospyl,as1m0i,c32re0tXicmualgunmi.ficAantiiomna.l No. 105522, 0.75 sFmiaglulregr2.anuLilveesr.witHheipnattohceytcel.earGalreyacs.o"geAnniamccaulmNuola.t1i0o5n5s1a1r,e0s.h7o5wmng./kGgl/ydcaoyg(ehniagrhe-dtohsee male), 79-week necropsy, 10.320X magnification. aFritgiufraectu3.alLdiivleart.atiHoenpoaftotchyeteensdohpolwaisnmgicnorremtailcuclyutmopalnadsmniuccloeragraneenlvleels.ope.ThAerneimias lslNiogh.t 105542, 0.75 mg/kg/day (high-dose female), 57-week biopsy. 10.320X magnification. 1Fi0g5u5r3e3,4.0.L7i5vemrg./kHge/pdaatyoc(yhtieghs-hdoosweinfgemnaolre)m,al79c-ywtoepelkasnmeiccrooprsgya,ne1l0le.s3.20AXnmiamganlifNioc.ation. 000089 [PAEMpNo:-- 5075351 NTGeorgsutperrveseltniaoN:o. i#110n05853325 Bopey Nagniiator: 10850n Date: 522/00atnTlecoi hg:DyuAsNrsWaciaNti SesFi7n1ar5masonai ore sakgr FIGURE1 Sles3g 9| 2 u < Re LG IEE Sil ERR pag JInRcIS ERER RR CGoeOsR iN NRE AEra TT C El A Fe AehCo ny RRS TPEE NRo rA k EnR o a AN RANE ft TEa TERA RRR CCAoA sFto aharB RoloR A F STIENE A & : fi eg IOR e AEFE ASR SeWaERsE SWE Ls EC RN ER al LE a TRE AV SRA P S Efo RRR S RE LEYREe ESACRRAR REN (sO atrie eglR yea E spHiaaWg,d7] EE TANE SE oR PTby ra Gi H R iEpEe e Rl aEAag Fae TEaT cme BY a SS SSSe eEEg EaNpE i a t Hey LA a i FaNn aeS6aARneoe Eg BA BESET aol] iE SEW a a po La Pu Be SHOad EEESEC WE $38 "oon A MEA PRS EYOTASER . RFEAERE RUE asf S AR SFR SFeRrXEBa aa EtenhpF is E ST a t EoE i 4 ) bAe riSh EOAL R e RETE EagseHE CER I E AAR BEST COPY AvAILABLE 006091 sPAuIoEM2N0o2:23| 897653228 GNTeersgotauAtptiSiocoNlxoN.o. 2H110M85851511 USac DMaagen:if5ca2t2on0:0 Ta1c0h,:320NW Pathol4o9g1y5A.s0soPcrioatsepsencttrOuarst.ional Ou0t1t9a),s4N4G522757713 FIGUR2E z Vse3) ay., > Fu S 00009: Fe? EE BRE ARR eRe pe is Se BF aa Ao KaeREEP a Ahe SYRe FP EA T eo Phi" Vad oy haw ag ev oo SEN FS ad SE ASR baci i ne AL SG PNA te) br ep We ik SER ra Shao 0g De rimieel ga BONER22 CR Tepes eAgRS S a EE. KY, # .3 C: ; 5 Pxros SSE LC TEAR x NE a EE BER AE lel a, 2 Xda, RgLteSOU A EEN b--. oe ses.2 CO BBoREERn ) TAICECPAEAP CEA 0B dr7aLei8enRSeE e 3S I SAA HTIOROEN e Se Pe LR R[EEN aR: pet 0 ro Se? RRL TN Eras tr Be hey oCSA BEST COPYAVAILABLE 000093 SPnAoI:EMeNxzon:am $9.76:371A TcoostmAreticrle"No.: 14075542Biopsy MDaalgtonegi:afii5vce2at2io0nN:o:0 T1a02c.1h36:82504xNW Pathol1o5gy.As0soPcrioastpeesctIunstemational Du5r16a) ,suNsC-522757713 FIGUR3E Zlissso y vy\ 2 = Y5 000094 CR STS fe uBo asaliE a25 OS F eNe S TARSTCeevier ;ofaSr / ARSIE e S ny ig 0: Cl GT 0 HES "00 (EXE Ee & 4 orSe Ey ea l Se o a Se aSC2 ARE La SSTR he : la] Le : lieNS6L0Cd ELtge Sica aE Rit pB raa 5 HET RE PL BC Co A+ Sehees Se E SE yA RB NiSondSBE as Eo OEE a a OER EN:paASshREvSeh9. g y Sl a PN l El PEe EES y eR a eernor Ares gaa S eeR Syfn E E AD n JE or WE Cen Lr H b 3 erEe TaR3Lh 0 Seda og a ibP riee ih ne, een Bea SR and Sg vali E a h eel Br RE | OPY AVAILABLE 000095 TsPouAsaIt.AErM2lN0oo:sNo. 15005675530-662ac NeGgaotuivneaNo: ZA1Fas4o Magnification: ~~ 10,320x pat: ano AJo4sn5O1su5rcmaPimroeNspEesct7uIis7nt1oe5brrnational )se4-5267 FIGUR4E 2lssyq v Y'. y == =< Ys 000096 si El FEiye En pa Hi R2 A 5 N 3 2E , PE ahA hl Ka +g vSE iSeoie EYooBe sienc 38u.sNeesne iSR gr bem oo peA ae W fat rA SESEasL. Paiee s ENS Ce CEE bn & haw EP hy five. 0g Spec 2 ST er SC ESO2 REENERoi jeTEv ievy so g SE lRRMooRkEo B ed r oSn Ss a oge F feeseE n d 8iNRfKSroay,wx 0ia YC C o SrE Ae E Eor CHYEaN Fe Sel DEnPd Co S gilA t eS N Lpkie PPasatFe OEBSE ei ILaOnn GE H GEE NE sine ann Ia e ar al nL, IgE 0. ott La Pete LAS MBER ia Teen ge 5B BEST COPY AVAILABLE e0e00097ie Abbreviation BEVC G L M N RB RER SER Draft`CEolveacntcreonSMuidcyroNsoc.op6y29R.ep2o2r3t Recovery Sacrifice Animals Index of Labels for Ultrastructural Structures Structure Bile canaliculus NGulcylceoagrenenvelope LMiiptiodchdoronpdlreita NPuecrloexuissome RReosuigdhuaelnbdoopdlyasmic reticulum `Smooth endoplasmic reticulum 00009 IV. Quality Assurance Statement 000093 Pathology Associates International 4 Companyof Science Applications Intemational Corporation E~.154 aEe oreoCe oe QUALITY ASSURANCE STATEMENT AssuranTcreisUenlietcatsrornemqiuicrreodsbcyoptyhestGuodyodhaLsabboereantoirnyspPercatcetdicaensdReaguudliatteidobnys tphreomQuulalgiattyed QbuyaltihteyUA.sSs.uFroaondceanUnditDrwuhgicAhdmrienpiosrtirsatdiiorenc.tlyPAtIo mhaasnaagfeumnecntito.niTngheanfdolrleowsipnognsiisvaerecord of inspections/audts and thelr resulting reports: DaIntspeeoctfion PhaseInspected 1DM0aatneFaignedimnegsnRteported andStuyDirector an, 200 Data Audit ar2s99 ar2r90 Dreft Report Audit 42/00 nee Audit of Data from Additional Groups: mass nase Final Report Audit mse 525,3000 DRaetcaovaenrdyDArnaifmtaRlesport Audit 5/5000 almyo AdsasWuarradn,ceB.S5p.e,ciRaGliAsPt -GLP 5/3Di9e00 Sponsor: 3M Corporate Toxicology Test Aricle: PFOS Study Number: 6320-223, PAI Study EM-99.76 4915 D Prospectus Dive + Durham, North Caraina 27713 + (319) 544.5257 (19) 544-3218 FAX 000100