Document 3NjX797Q60Mqn0OnDoze62rR6

PathologyAssociateslntemational A Company ofScienceApplicatioInnsternatioCnoarlporation May 30,2000 Dr. Andrew Seacat 3M Corporate Toxicolocv Buildinc- 220-2E-02 PO Box 33220 St.Paul,MN 55133-3220 o w E,@ W3 12D Wc Re: Draft Electron Microscopy Report, Study No. 6329-223 (Recovery Animals) Dear Dr. Seacat, Please find enclosed the draftelectronmicroscopy reportfor the recovery sacrifice animals on Covance Studv No. 6329-223. Li-2htand ultrastructuraflindinl-yisdentifiedin the liverof monkeys after26 weeks of treatment have disappeared in the tissuesfrom recoverv sacrificeanimals, indicatin(ryesolutionof the lesions. After your review and approval of thisdraftreport,I willprepare a signed finalreport. Please retainthe elossv reportfiguresand allglossy electronmicrographs for insertion into the finalreport.as these are originals. Ithas been a pleasureworkin2 with 3M and Covance on thisstudy. I hope thatyou will provide PAI with opportunitiesto work- with you again in the future. Sincerely. James B. Nold. DVM, PhD 'v'icPeresident,Southeast Operations Enclosure: Draftelectronmicroscopy report,6329-223 4915 D Prospectus Drive o Durham, North Carolina 27713 o (919)544-5257 (919)544-3218 FAX PathologyAssociatelsntemational A Company ofScienceApplicationIsntemationCaolrpora6on @mrim 0 AnEMP@VIO&O-W COTV&-rll DRAFT PATHOLOGY REPORT (ANCILLARY STUDY) ELECTRON MICROSCOPIC EVALUATION OF LIVER IN CYNOMOLGUS MONKEYS (RECOVERY ANIMALS) 26-WEEK SULFONIC CAPSULE TOXICITY STUDY WITH PERFLUOROOCTANE ACID POTASSIUM SALT (PFOS;T-6295) IN CYNOMOLGUS MONKEYS CLIENT STUDY NTuTi'vl[B6E3R29-1.23 PAI STUDY NUNMER EM 99.76 Prepared by: James B. Nold, D.V.M., Ph.D., Diplomate, A.C.V.P. Pathology AssociatesInternational 4915-D Prospectus Drive Durham, NC 27713 Mav 30,2000 PREPARED FOR 3M CORPORATE TOXICOLOGY, 3M CENTER, ST. PAUL, MN 55144-1000 4915 D ProspectusDriveo Durham, North Carolina27713 9 (919)544-5257 9 (919)544-3218 FAX PatholocyyNarrative LightMicroscopy and TransmissionElectron Micrograph InterpretatiFoonrms Transmission Electron iN@Ecrto@ cyraphs QualityAssurance Statement Section I n in IV 1. PatholoC)gyNarrative DraftElectronMicroscopy Report Covance Study No. 6329-223 Recovery SacrificAenimals DRAFT PATHOLOGY REPORT (ANCILLARY STUDY) ELECTRON MICROSCOPIC EVALUATION OF LIVER IN CYNOMOLGUS MONKEYS (RECOVERY ANIMALS) 26-WEEK SULFONIC CAPSULE TOXICITY STUDY WITH PERFLUOROOCTANE ACID POTASSIUM SALT (PFOS;T-6295) IN CYNOMOLGUS MONKEYS CLIENT STUDY NUM13ER 6329-223 PAI STUDY NLNI]BER EM 99.76 SUMMMARY Two male and two femalecvnomolaus monkevs treatedfor26 weeks with0.75 m-Lylk.PFOS were removed from treatmentforup to 53 weeks. Liversamples obtainedby biopsy atWeek 57 and necropsy atWeek 79 were examined forreversibilitpye,rsistence. or delavedoccurrenceof toxiceffects.Li(yhtand electronmicroscopicevaluationof liversidentifiendo patholo-icaclhan(2es.The treatment-relatcehdan-esof hepatocellular hypertrophvand -@.'acuolatinoont,ed at26 weeks and characterizeudltrastructuralblyy increasednumbers of lipiddropletswithinhepatocytes,had completeINd,isappeared durin2therecoveryperiod. PROCEDURES The objectiveofthisstudywas toexamine by electronmicroscopy theliversfrom selectedanimalsina 26-week capsuletoxicitsytudvwithPFOS incynomolgus monkeys. In additionto a Lyeneraqlualitativeevaluationfordifferencebsetween controland treated animals,theultrastructureanldpointsincludedquantitatioonfperoxisomes within hepatocvtesand membrane and mitochondrialchan2es inhepatocytes.Lightmicroscopy of paraffin-embeddedliversectionswas alsoperformed. This reportdetailsthe lightand electronmicroscopy findingsinthe fourhl-,h-dosmeale and female monkevs desianatedasrecoveryanimals. A previousreport(dated6-241999) indicatedthattreatmentof cynomol(t@yusmonkeys with0.75mgzn PFOS/kg/day for26 weeks resultedinhepatocellulavracuolationand hvpertrophvbv lichtmicroscopy. Hepatocellulavracuolationand hvpertrophvwere primarilvcentrilobulairndistribution and did vary inincidenceand severitybetween individualanimals.Ultrastructurally, therewere increasedlipiddropletswithinhepatocytes.Increasedlipidropletswere 1-1 DrafEtlectrMoincroscopRyeport CovanceStudyNo.6329-223 RecoverSyacfifiAcneimals presentinboththehi-Lyh-domsaelesexaminedandintwo offourhigh-dosefemales examined.increasehdepatocellulgalrycogenwas presentn theremain@i,nct,wo high-dose females.Theseultrastructucrhaalngescorrelatewdithhepatocellulhayrpertrophavnd vacuolationnotedbv li2htmicroscopyand withincreaseldiverweightparametersinthe hi-h-doseanimalsT.he no-effecltevelforhepatocellulvaarcuolatio(nlipiddroplet accumulationw)as 0.15mg/kg/dayofPFOS (m-id-dosceroup).Quantitationf peroxisomeswithinhepatocytebsy electromnicroscopyindicatendo remarkable differencebsetweencontroliverasnd livertsreatewdith0.03,0.15,or0.75mcy PFOS/kcy/davU.ltrastructudriaflferenceisncellulamrembranes orniitochondrwieare notdiscernibbleetweencontroland treateadnimalsinthesamplesexamined.The recoveryanimalswereexaminedtoassessforreversibiloirtpyersistencoefthese hepatocellulcahranges,orforthedelayedoccurrenceofadditionaPlFOS-inducedeffects. r*ortiiteoxicit@y,Ludv',-"n--Iialaend femalecviioinolcuisiiorikewyesreolventhetest articloerthevehicleoncedailyforatleast26 weeks accordicnct,oTextTable1. TextTable1.GroupDesiCc7natioannsdDosac-eLevels DosaL,e GroaL 111 IN, Dose Level (me/k-c-,/da@,)' ol 0.03 0.15 O@7@; TotalMaterialDose Level(m-FJkg/dav)b 30' 15b 6c 30@ Number of Males 60 4 6d 6d Number of Females 6' 4 6d 6d Dose levelswere pro@i.ded.The control2roup (Group I)receivedan equivalentamount of lactosein eelaticnapsulesas thetotalmaterialadministeredtoGroup 4. The low-doseLrouptGroup 2) receivedthetestmaterialdilutedwithlactose(1:499,w:w). The mid-dose(Group 1)and hi2h-dose(Group 4) s!rouprseceivedthetestmaterialdilutedwithlactose (1:39, w:",). Two animalsinGroup,,1.3.and 4.desi2natedas reco%,era,n,i,mals.were treatedforatleast26 weeks, thentreatmentwas discontinueadnd theanimals were observedforreversibilipteyr,sistencoer, delayed occurrenceoftoxiceffectfsoratleast52 weeks posttreatment. During Week- 57 (approximately 31 weeks into the recovery period), the 2 high-dose recovery males and 2 hilyh-dose recovery females were anesthetized and liverbiopsies were- suraicallv,obtained from each animal and chemicativ preserved and processed as described belo"-. DurinLy Week- 79. the 4 hl!zh-dose recovery animals were necropsied and liversamples obtained as described beloa-. The two control recovery animals were returned to the Covance stock animal colony, and the two Group M animals were transferred to a subsequent study (Covance 6329-268). Liver swnples were not obtained and examined 'in the control recovery or Group E[Irecovery animals. Animals to be sacrificed at recovery sacrificetimes were anesthetized with ketamine and xviazine, weilyhed. exsan-uinated, and necropsied. Multiple tissues were collected and preserved in lOc7cneutral-buffered formalin according to the protocol. Additionally, 1-2 DraftElectronMicroscopy Report Covance Study No. 6329-223 Recovery SacrificeAnimals pieces of liverfrom each animal were thin-slicedand placed in 1/2strengthKamovsky's fixative(2.0% paraformaldehyde/2.5% clutaraidehvdein0.1M phosphate buffer)for subsequent ultrastructuraelxamination. These liverswere processed intoEpon blocks and submitted to this laboratoryfor subsequent electronmicroscopy processing and evaluation.Hematoxylin and eosin (H&E)-stained slidesectionsof formalin-fixed, paraffin-embedded liverswere submittedfor licchtmicroscopic examination. The four hi-h-dose recovery animals are listedin Text Table 2. For each animal, there c was a 57-Week liverbiopsy sample and a 79-Week terminalnecropsy liversample, Text Table 2. Recovery Animals Examined by Light and ElectronMicroscopy Males 105511 105522 0.75 me-,Ika-/dPaFvO (Group 4 Hiah-Dose) Females 105533 105542 Thin sections(approximately 90 nm) from the Epon-embedded liverswere cut,mounted on 200-mesh copper zrids.stainedwith 5% methanolic uranyl acetateand Reynold's lead citratea,nd examined on a Zeiss 900 transmissionelectronnucroscope. Centrilobular hepatocytes,where clearlyidentifiablien liversections,were preferentialleyxamined. Representative electronphotomicrographs of liverwere taken and significant ultrastructurafleatureswere summarized for each photograph and animal on a designated transmission electronrrlicrozraphinterpretatiofnorm. The number of peroxisomes in hepatocvtes was manually counted for each center photographed hepatocyte and recorded. For licyhmticroscop@',the H&E slidesfrom the same animals selectedfor electron microscopy \x,,eer-e,.-aluataendd significantfeatureswere summarized on a light n-Licroscopice-v,aluatiofnorm. RESULTS AND DISCUSSION Individual interpretationsof llcyhmticroscopy slidesand electron micrographs for each animal are shown in Section 11.Representative electronphoto micro (cYraphswere selected as reportfi2ures,labeled,and are shown in Section M. All remaining electron n-licro2raphsare separatelyattached to thisreport. Li2ht Nticroscop-., A summary of the incidence and severityof IILhtmicroscopic findin2s in the control and hllyh-doseliversis shown in Text Table 3. There were no microscopic findingsthat appeared to be associatedwith testmaterialadministration.Hepatocellular hypertrophy and vacuolation,noted after26 weeks of treatment in some high-dose animals, were not identifiedin recovery samples at eitherthe 57-Week biopsy or 79-Week terminal necropsv samples. indicatincc,omplete reversalof the 112htmicroscopic liverchances. 1-3 DraftElectroMnicroscopyReport CovanceStudyNo. 6329-223 RecoverySacrifiAcneimals The livefrindicnansotedintherecoveraynimalwsereconsiderendormalbackground histopathologyof cynomoigus monkeys. Text Table 3. Incidenceand Severityof LightMicroscopicLiverFindings-- Recovery Animals 0.75mz/kp-/da(yHijah-Dose) Males Females (number examined) (2) (2) 57-Week Biopsy Infiltratley,mphohistiocytincu,nimal I Tem,u'nalNecropsy Infiltratley,mphohistiocytimci,nimal 1) 2 Hyperplasia,bileduct,slight I - lnflanunationl,ymphohistiocytiscl,ight I inflammation,eosinophilics,light 1 Cleari.n!czv.toplasmic,centrilobulamri,nimal - - Incidenceofzero. ElectronMicroscopv Electronmicroscopicevaluatioonfthe57-week biopsyand 79-week necropsyliver samples identifiendo abnormal featuresa,nd alltissuesamples were consideredto be ultrastructuranlolrymal (TextTable4). The hepatocellulahrypertrophvand vacuolation seen after26 weeks oftreatmenta,nd characterizeudltrastructurablylyincreasedlipid droplets,were not seen inthe recoveryanimal liversamples,indicatinrgecoveryofthese testmaterialrelatedeffects.The electronmicroscopicfeaturesdescribedinthese recover-vsamples were considerednormal ultrastructurfailndin-sinhepatocvtes.Ficures 1-4arerepresentativeelectronmicrographsfrom thesesamples and illustrantoermal liver and hepatocellulafreatures. Text Table 4. Summary ofUltrastructurFailndings- Recovery Animals Treatment Group (mF-Ikg/dav)/Sex Animal No. Comments (10.75M)ale, 57-week biopsy 105511 Normal ..(.0......7..5.).M..a..l.e..,..7..9..-..w..e.e...k.....n..e.c..r..o..p..s..v................1..0..5.5..1.1............................N..o..r.m..a..l................................................. ................... (0.75)Male, 57-week biopsy 105522 Normal ..(..0......7..5.).M..a..l.e..,..7..9..-.w...e..e..k..n..e...c...r..o.p..@..y.........................1...0...5...5..".."....................................N....o...r....m....a..l.................................. (0.75)Female, 57-week biopsy 105533 Normal ..(..0. ...7..5..)F..e..m..a.l..e...,...7..9..-...w.e..e...k...n...e.c.rp.p.s...v..................1.0..5.5..3.3...............................N..o.r..m..a.l....................................................................................... (0,75)Female,57-week biopsv 105542 Normal (0.75)Female,79-week necropsv 105541- Normal 1-4 DraftElectronMicroscopy Report Covance Study No. 6329-223 Recovery SacrificeAnimals Peroxisomeswere identifieadnd quantitateidnrepresentativhepatocvt,s(TextTable 5). Mean peroxisome numbers per hepatocytewere considerednormal. Text Table 5. QuantitatioonfHepatocellulaPreroxisomes 0.75m,- PFOS/k-Jday (Hi,h-Dose)Recoverv Animals Male Female 57-week biopsv Anim,-ii Number' of Number Peroxisomes 79-week necropsv Animal Number of Number Peroxisomes 57-week biopsv Animal Number of Number Peroxisomes 79-week necropsy Animal Number of Number Peroxisomes 105511 18.5 105511 19.2 105533 29.8 105533 20.8 105522 19.4 105522 25.8 105542 23.0 105542 15.2 Mean = 18.95 Mean = 22.5 Mean = 26.4 Mean = 18.0 Average number of peroxisomes per hepatocyteforfivehepatocytescounted per animal. For Animal No. 105511 (biopsy),peroxisomes were counted inonly fourhepatocytes CONCLUSION LlEhtand electronmicroscopicevaluationofliversfrom cynomolgus monkeys treated with0.75 mg PFOS[ka/day for26 weeks and allowed to"recover"untreatedfor31 and 53 weeks identifiendo pathologicaclhanges. The treatment-relatcehdanges of hepatocellulahr.vpertrophyand vacuolationc,haracterizeudltrastructurablylyIincreased numbers of lipiddropletswithinhepatocytesat26 weeks, had completelyresolvedduring therecoveryperiod. SIGNATURE OF AUTHOR James B. Nold, D.V.M., Ph.D., Date Diplomate,A.C.V.P. 1-5 U. Li2htMicroscopy and TransmissionElectron Micrograph InterpretatioFnorms I 1Forms areexact copiesof signed/datedraw data sheetsmaintained in archivedstudyfile. LIGHT MICROSCOPIC EVALUATION Studv No.: 6329-223(EM 99.76) Species/tSrain.C-vnomolzus Monkeys Animal No: seebelow Sex: see below InterpretinPca,t,holot@cli(sst-i-cnaturaend date): u Tissue: Liver --Ai)ri7l, 20W Animal Dose/Group Number 0.75mg[kc, 105511 (High-dose) Recovery 105522 Males 57 wk biopsv 0.75mgfk- 105533 (High-dose) Recovery 105542 Females 57 wk biopsv 0.75mg/k- 105511 (High-dose) Recovery Males 79 wk ................ 105522 necropsy 0.75mg[kg (High-dose) Recovery Females 798 week necropsy 105533 105542 MicroscopicFindingsand Comments 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 1. Infiltraltyem,phohistiocytfiocc,alm,inimal 1.I.n.f..i.l..t.r..a.l.t-y.-e.m-.,-p-h--o--h-i- stioc..y.t.m.i.u.c*l.,*.t"-i-f,--o*-c-a-ml--i,-n--i-m--a-l----------------- 1. Infiltraltvem,phohistiocytmiucl,tifocamli,nimal ........................................................................................... ............................. 1. Infiltraitvem,phohistiocytmiucl,tifocamli,nimal 2. Hyperplasiaf,ocals,lightb,ileduct 3. Inflammationl,ymphohistiocytmiucl.tifocasll,ight 4. Inflammatione,osinophilimcu,ltifocasll,ight 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 2. Clearingc,ytoplasmic,entrilobulamri,nimal TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223 (EM 99.76) Species/StrainC:ynomol2us Monkey Animal No.: 105511 57-week biopsy Treatment Group: .75 m2/k-@z(hi-dose) Sex: Male Tissue: Liver- centrilobular Block No(s).: 99.76-3 1- IA Z18823 - Photo/NegativeNo(s).: Z18827 SignificanLtesions (checkone): Yes x No lntlerpret-@-lPna(thologist(signatureand date),:Ora=-,, (i Y-)1-141, Nlay 26, 2WO FeaturZeIs8:823L.iveHre.patoc1y5t,e2.9TOhXi.hsepatohcayastsemarlolund centralnucleus. The surrounding cytoplasm containsnumerous round to oval rnitochondriaand severalsmall to largelipiddroplets.The darkerround organelles, mostly slightlsymallerthan themitochondria,areperoxisomes. Twenty-four (24) peroxisomes counted. The intervenincc,ytosolcontainsprofilesof endoplasmic reticulumand some finegranules,which may be glycogen. Z18824. Liver. 10,320X. Portionsof threecellsarepresent.The dorsaltwo hepatocytes exhibita mixture of normal appearingcytoplasmicelements,includingn-iitochondria, endoplasmic reticulumand scatteredperoxisomes. The largerventralcell'scytoplasm containsirregularclearspaces surrounded by membranous material.Only a couple of mitochondria are evident,and thiscellisprobably not a hepatocyte.A-round the periphery are severallargestructurecsontainingfinelygranularand reticulamraterial.This may representsmooth endoplasn-iirceticulumintermixedwith glycogen and/orribosomes. It appears to be membrane bound, but thismay be a pseudomembrane caused by compacting of the surrounding cytoplasm. Or, thesestructuresmay representcytoplasmic invaginationsof extracellulamraterialsuch as plasma. The nuclearprofilesin thesecells appear normal. Peroxisomes were not counted inthiselectronmicrograph because an individualhepatocyteshowing most of itscytoplasm isnot present. Z18825. Liver. Hepatocyte. 10,320X. This hepatocyteispolygonalwith a round central nucleus. Itssurroundingcytoplasm containsnumerous mitochondriaand a few small lipiddroplets.The interveningcytoplasm containsprofilesof endoplasm-icreticulum, scatteredfineglyco2en granules,and some dark round peroxisomes. Twenty-two (22) peroxisomes counted. Three adjacenthepatocytesappear essentiallnyormal. At the bottom rightof the photograph isa finelygranularstructurew,hich isoutsideof the centered hepatocyteand appears similartothatdescribedinZ18224. Z18826. Liver.Hepatocyte. 10,320X. This hepatocyteisborderedby an extracellular space alon(2itsrightmargin. The cellhas a small nuclearprofileand abundant cytoplasm containinc,numerous rru'tochondn*aand slightldyilatedendoplasmic reticulum.Several Ilipiddropletsare present.Seventeen (17)peroxisomes counted. 105511BiopsyContinued. Z18827.Liver.Hepatocyte1.0,320X.A largreoundnucleuisspresenitnthecentered hepatocyte.The surroundingcytoplasmcontainsnumerous mitochondriaand abundant endoplasmicreticulum.A few darkresidualbodiesarepresent.Eleven (11)peroxisomes counted. At theriahtdorsalmargin of thephotographarean endotheliaclelland a cyranulasrtructurethatappearsto be an inva-inationof plasma. Conclusions: Normal hepatocytes.Peroxisomes avera4g:)e 18.5perhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EMM-929-..76-)L Animal No.: 105511 79-week necropsy Treatment Group: .75mg/kg (hi-dose) Sex: Male SignificanLtesions (checkone): Yes Species/StrainC:ynomoigus Monkey Tissue:-Liver-- centrilobular Block No(s).: 99.76-32-2A Z18828 - Photo/NegativeNo(s).: Z18832 x No InterpretlnP,a-thologist(signatureand date): May 29, 2000 Features: Z18828. Liver. Hepatocyte. 10,320X. This hepatocyteissomewhat oval in shape with a smallround nucleus. Approximately 8-9 small to medium sizedlipid dropletsare presentinthecytoplasm. The majorityof the cytoplasm containsnumerous round to rod-shaped ni-itochondriian,terveningareasof smooth endoplasmic reticulum (SER) and "more clear"patchesof glycogen. Some profilesof rouch endoplasmic reticulum(RER) and round dark peroxisomes arealsoevident.Twenty-eight (28) peroxisomes counted. Bilecanalicullcan be seen between the hepatocytessituatedon the rightand leftwith the centralhepatocyte. Z18829. Liver. Hepatocyte. 10,320X. The centralvein and surroundingextracellular collagenare presentatthetop rightmargin of thiselectronmicrograph. A sinusoldis alsopresentatthe lower leftmargin. The interveninghepatocytehas a round nucleus and surroundingcytoplasm containingnumerous profilesof endoplasrnicreticulumand dark mitochondria.There ismild dilatatioonf theendoplasmic reticulumand nuclear envelope,which isa fixationartifactS.everaldark residualbodies and occasionalsmall lipiddropletsarealsoidentifiedD.ifferentiatiobnetween peroxisomes and n-iitochondria isdifficultE.leven (11)peroxisomes counted. Z 18830. Liver.Hepatocyte. 10,320X. The cytoplasm of thishepatocytecontains abundant SER, although some profilesof RER are alsoevident,especiallyaround some rnitochondria.A nuclearprofileisnot evident inthisplane of sectionof the centered hepatocyte.Approximately 20 lipiddropletsarepresent.Twenty (20)peroxisomes counted. This hepatocyte'sn-@crovilloubsorder isevidentboth dorsallyand ventrally along sinusolds.The nucleus of an endothelialcellispresentatthe lower right.Adjacent hepatocytesarepresentto the leftand rightof the centeredhepatocyte. Z 1883 1. Liver. Hepatocyte. 10,320X. The morphology of thishepatocyteisvery siniilar-Zt1o8828. The cytoplasm containsnumerous mitochondria,endoplasmic reticulumand "more clear"patches of glycogen. Severallipiddropletsarepresent. Nineteen (19)peroxisomes counted. The surroundinghepatocytesappear ultrastructuralsliymilar. 105511NecropsyContinued. Z18832. Liver.Hepatocyte.10,320X.ThishepatocytaeppearssimilatroZ18830. Its cytoplasm containsabundant SER. Other recognizableorganellesincludesome RER, lipiddroplets,mitochondria,peroxisomes and residualbodies. Eighteen(18) peroxisomes counted. At the lower rightmargin of thephotograph isa sinusold containingseveralerythrocytes.A prominent bilecanaliculusispresentat the right where threehepatocytesappear to meet. These hepatocytesare consideredto be ultrastructuralnloyrmal. Conclusions: Normal hepatocytes.Average 19.2peroxisomes per hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: 105522 57-week biopsy Treatment Group: .75 m@zPKs@(hi-dose) Sex: Male Sizg@ nificanLtesions(checkone): Yes InterpretinPgatholo-is(tsignaturaend date): Species/StrainC:vnomolaus Monkey Tissue: Liver - centrilobular Block No(s).: 99.76-33-IA Z18833 - Photo/NegativNeo(s).: Z18837 x No May 29, 2000 Features: Z18833. Liver.Hepatocyte. 10,320X. A sinusoldcontainingfinelygranular plasma isalongtherightmicrovillousmargin ofthishepatocyte.The hepatocytehas an oval nucleus. In thesurroundingcytoplasm areseveralvery electron-densreesidual bodies.There ismild dilatatioonfthe nuclearenvelopeand endoplasmicreticulum, which isa fixatioanrtifactE.ighteen(18)peroxisomescounted.Mitochondriaare numerous and arevariablyshaped. ZI 8834. Liver.Hepatocyte.10,320X. This hepatocyteappearsultrastructuraslilmyilar toZ 18833. There ismild dilatatioonftheendoplasn-lirceticulumand nuclearenvelope. The hepatocytelinesa sinusold,which containstwo erythrocyteasnd a granulated eosinophil.Eight(8)peroxisomescounted. Z18835. Liver.Hepatocyte.10,320X. Severalsmalllipidropletsarepresentinthe cvtoplasm of thishepatocyte.Ithas a slightloyvalnucleusand abundant cytoplasm which containsnumerous mitochondriaand profileosfendoplasmicreticulum.There is slightdilatatioonf theendoplasmicreticulumand nuclearenvelope.A distincmtyelin figureisevidentassociatedwith a lipidroplettotheleftof thenucleus.Twenty-two (22)peroxisomescounted. Z18836. Liver.Hepatocyte.10,320X. The hepatocytehas a largepolygonalnucleus. The cytoplasm containsabundant SER and RER, which exhibitslightdilatationS.everal small tolargelipiddropletsarepresent.Numerous round darkperoxisomesareevident. Thirty-fiv(e35)peroxisomescounted.Mitochondriaarenumerous and variablyshaped, but appear ultrastructuranlolrymal. Z18837. Liver.Hepatocyte.10,320X. A sinusoldispresentalonct,he leftand lower margins of thiselectronmicrograph. The lighter-stainiengdotheliaclellispresentalong the lower margin. The centeredhepatocytehas an ovalnucleusand numerous rnitochondriand endoplasmicreticuluminitssurroundingcytoplasm.There ismild dilatatioonf theendoplasn-iirceticulumand nuclearenvelope.Severallipiddropletsand residualbodiesarealsodiscernibleF.ourteen(14)peroxisomescounted. Conclusions: Normal hepatocytes.Peroxisomes avera e 19.4perhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Species/StrainC:ynomolizus Monkey Animal No.: 105522 79-week necropsy TreatmentGroup: .75me/kEz(hi-dose) sex-. Mate Tissue: Liver -centrilobular Block-No(s).: 99.76-34-2A Z18838 - Photo[NegativNeo(s).: Z18842 SignificaLnetsion(scheckone):_ Yes X No InterpretcinP-athologis(tsignaturaend date):D4@@, f?n,cH u Mav 29,2000 Fe--tureZs1:8838.LiverH.epatocyt1e0.,320XT.hishepatocyhtaesa !a-rr-oeund nucleus.At theverytop leftmargin oftheelectronmicrograph isa segment of sinusoid along itsmicrovillousborder.A bilecanaliculuissevidentatthelower leftmargin of the photograph,where thecenteredhepatocytemeets an adjacenthepatocyte.The centered hepatocvtehas abundant cytoplasm,which containsnumerous profilesof RER and SER. There issli2htdilatatioonftheendoplasmic reticulum.Mitochondriaarenumerous are round tooval inshape. Approximatelytwenty-three(23)peroxisomescounted. Z18839. Liver.Hepatocyte.10,320X. Two hepatocytesareclearlyevidentinthis electroniicrolraphT.hey appearultrastructuraslilmyilar.They exhibitmild dilatation of theirendoplasmic reticulumand nuclearenvelopes.They have numerous n-@tochondriand occasionallipiddropletsand residualbodies.Peroxisomes areusually round and more electron-denstehan therr@tochondriaS.eventeen (17)peroxisomes counted in theupper hepatocyte. Z18840. Liver.Hepatocyte.10,320X. This hepatocytehas a largeround central nucleus.The surroundingcytoplasm has numerous n-Litochondrainad many slightly dilatedprofileosf endoplasn-Lirceticulum.Both theSER and RER appearslightldyilated. Approximately 15-16smalltomedium sizedlipiddropletsarepresent,as area few very electrondense residualbodies.Twenty-one (21)peroxisomescounted. Z18841. Liver.Hepatocyte.10,320X. This hepatocyte'nsucleusisnotevidentinthis planeof section.A sinusoidcontainingan erythrocyteispresentalongtheverytop margin of thiselectronmicrograph.There isslightdilatatioonftheendoplasmic reticulumthrou(2houtthecytoplasm. Also,random smalltomedium sizedlipiddroplets arepresent.Numerous peroxisomesareevident.Forty(40)peroxisomescounted. Z 18842. Liver.Hepatocyte. 10,320X. This hepatocyteappearssimilartoZ 18838, and isultrastructuranlolrymal. Ithas an ovalnucleusand abundant surroundingcytoplasm containininzumerous mitochondriaand slightldyilatedprofileosfendoplasmicreticulum. Twenty-eight(28)peroxisomescounted.Sinusoidsareevidentatthe upperleftand lower rightmarizinsofthephotograph. Conclusions: Normal hepatocytes.Peroxisomes averac,2,5e.8per hepatocyte._ TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76-)-- Species/StrainC:ynomoigus Monkey Animal No.-. 105533 57-week biopsy TreatmentGroup: .75miz@iz(hi-dose) Sex: Female Tissue: Liver - centrilobular BlockNo(s).: 99.76-35-1A Z18843 - Photo[NegativNeo(s).: Z18847 SiCc7 ,nificalnetsions(checkone): Yes x No InterpretiPnagthologis(tsignaturaend date):k@!@R Mav 29,2000 Features: Z18843. Liver.Hepatocyte. 10,320X. This hepatocytehas a smallnuclear profile.A sinusoidlinedby an endotheliaclellispresentalongthe upper rightmargin of theelectronmicroszrapha,nd an adjacenthepatocyteispresentatthe lower leftmargin. A prominentbilecanaliculuisspresentbetween thetwo hepatocytes.The cvtoplasmofthe centeredhepatocyteisvoluminous and containsnumerous n-Litochondrainad endoplasmic reticulum.The endoplasn-Lirceticulum,nuclearenvelope,and mitochondriaappear to be sli-htldyilatedT.his dilatatioinsa fixatioanrtifactA. few smalltomedium sizedlipid dropletsarepresent.Forty(40)peroxisomescounted. Z 18844. Liver.Hepatocyte.10,320X. This somewhat triangular-shapehdepatocytehas a largeovalnucleus.Itscytoplasmcontainsnumerous mitochondriaand slightldyilated profileosf RF-R. Some glycogengranulesappear tobe intermixedwiththeSER. Eighteen(18)peroxisomescounted. Z18845. Liver.Hepatocyte.10,320X. This electronrriicrocrapahppearssimilarto Z18844, althourhtwo nuclearprofileasrepresent,which isprobablya functionofthe planeof sectionalthoughitmay be binucleated.Thirty-fou(r34)peroxisomescounted. Z18846. Liver.Hepatocyte.10,320X. The centeredhepatocytecontainsa smallround nucleus.The surroundingcytoplasmcontainsnumerous n-litochondrainad slightldyilated profileosf endoplasn-tirceticulum,bothSER and RER. One myelin figureand several lipiddropletsarepresent.Thirty-thre(e33)peroxisomescounted.Some extracellular coliagenand a sinusoldarepresentalongthe lower rightmargin ofthephotograph.The hepatocyteatthetop riahtismore lightlsytainingthanthecenteredhepatocyte,and its cytoplasm containabundant SER. Some mitochondriaand peroxisomes arealsoevident. Z18847. Liver.Hepatocyte.10,320X. This electronmicrographappearsverysimilarto Z18844. Althouah thereisslightdilatatioonftheendoplasmicreticulumt,hishepatocyte appearsultrastructuranlolrymal. Twenty-four(24)peroxisomescounted. Conclusions: Normal hepatocytes.Peroxisomesaverage29.8per hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Studv No.: 6329-223 (EM_29.76)_ Species/StraiCnv:nomolizusMonkey Animal No.: 105533 79-week necropsy Treatment Group: .75mlylk@(zhi-dosel Sex: Female Tissue: Liver - centrilobular BlockNo(s).: 99.76-36-2A Z18848 - Photo/NegativNeo(s).: Z18852 SignificanLtesions(checkone): Yes x No Interpretci@ nPgatholocgis(tsiillg.,@naturaend date): C) - - May 29, 2000 Feature-s: ZI 8848. Liver. Hepatocyte. 10,320X. This electron micrograph illustrates a hepatocytewithadjacenthepatocyteaslongeitherside.Sinusoidsarepresentalongthetop right and bottom leftmargins.Along thesinusoidst,hehepatocytesexhibitmicrovilloubsorders. Lightlystaininegndotheliaclellslinethesinusoids.The centeredhepatocytehasa largeoval nucleusand abundantsurroundingcytoplasm.The cytoplasmcontainslargeamounts of SER and numerous scatteremditochondria.A few lipidropletsand residuablodiesarepresent.RER is evidentas clustersu,suallyaroundmitochondria.Thirty-fiv(e35)peroxisomescounted. Z18849. Liver.Hepatocyte.10,320X. This pentagonaly-shapedhepatocytehas a somewhat "trapezoid"appearanceand isbounded by hepatocyteson fivesides.Ithas a round central nucleusand abundantsurroundingcytoplasm.The cytoplasmcontainsnumerous mitochondria and lotsof SER. Some profileosf RER arepresentaroundthemitochondria.Twenty-four(24) peroxisomescounted. Z18850. Liver.Hepatocyte.10,320X. Thishepatocyteisborderedby a sinusoidatitslower leftmargin.The sinusoidislinedby an endotheliaclelland containstwo prof'iioefs erythrocytesT.he hepatocytehas a smallcentralnucleus.Itscytoplasmcontainsnumerous areas of glycogen.These arelightlsytainedareaswithfinegranularmaterial.Mitochondria,SER and RER appearultrastructuranlolrymal.Eight(8)peroxisomescounted. Z18851. Liver.Hepatocyte.10,320X.Thiselectromnicrographappearsessentialsliymilatro Z18850. Near thetop centerthereisa sinusoidlinedby an endotheliaclelland containingan erythrocyteT.he hepatocyte'csytoplasmcontainsmultiplepatchesof glycogeninterspersed betweenSER and mitochondria.Eighteen(18)peroxisomescounted. Z18852. Liver.Hepatocyte.10,320X. Thishepatocyteisround,witha roundnucleus,and containsone medium sizedlipidropletinitscytoplasm.A sinusoidcontainina-n erythrocyties presentalon-thelowerfightmargin oftheelectronmicrograph.Abundant SER, mitochondria, and some glycogenarepresentinthecytoplasmofthehepatocyte.Approximatelynineteen(19) peroxisomescounted. Conclusions: Normal hepatocytes.Peroxisomesaverage20.8perhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Species/StraiCnv:nomoigus Monkev Animal No.: 105542 57-week biopsy TreatmentGroup: .75mL-/kiz(/hi-dose) Sex: Female Tissue: Liver- periportal BlockNo(s).: 99.76-37-1A Z18853 - Photo/NegativeNo(s).Z:18857 SignificaLnetsions(checkone):_ Yes x No InterpretiPnagthologis(tsignaturaend date):kic@p, L-) Mav 29, 2000 Features: Z18853. Liver.Hepatocyte.10,320X. Thishepatocyteappearssurrounded by adjacenthepatocyteson allsides.Ithas a largepolygonalnucleusand abundant surroundingcytoplasm.Justtothe lower leftof itsnucleusisa smallsegment of extracellulasrpacecontainingsome n-ticrovialnldi collagen,probablynextto a sinusold. Severalsmall lipiddropletsare presentin the cytoplasmof the centeredhepatocyte.The endoplasn-tirceticulumisslightldyilated,and severalresidualbodiesarepresent. Nineteen(19)peroxisomescounted. Z18854. Liver.Hepatocyte.10,320X. SimilartoZ18853, theendoplasmicreticulumof thishepatocyteisslightldyilatedw,hich alongwiththedilatednuclearenvelope,isan artifacotf fixationT.he mitochondriaappeartoalsoexhibitsome slightartifactual swellinc,M.ultiplebilecanaliculalreevidentalonc,themarginsof thiscell'scontactwith adjacenthepatocytes.Approximatelythirteen(13)peroxisomescounted. Z18855. Liver.Hepatocyte.10,320X. This hepatocytehas a larcyreound nucleus.It's surroundingcytoplasmcontainsnumerous profileosf SER, which areminimallydilated. Approximately 20 smalltomedium sizedlipiddropletsarepresentinthecytoplasm. Fifty-one(51)peroxisomescounted. Z18856. Liver.Hepatocyte.10,320X. Extracellulasrpacecontainingnumerous coliagenfibrilisspresentnext tothecenteredhepatocyte.The hepatocytehas a large ovalnucleusand abundant surroundingcytoplasmwithnumerous mitochondriaand endoplasnu'creticulum.A few smalllipidropletsarepresent.Thirteen(13) peroxisomes counted. Z18837. Liver.Hepatocyte.10,320X. Smooth endoplasmicreticulumand rfi,itochondrairaeabundant in thecytoplasmof thishepatocyte.The hepatocytehas a larc,reound nucleus and severalsmallto medium sizedlipiddropletsarepresent. Nineteen(19)peroxisomescounted. Conclusions: Normal hepatocytes.Peroxisomesaverage'-13.p0er hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:-6329-223(EM 99.76) Animal No.: 105542 79-week necropsy TreatmentGroup-. .75mg/kg (hi-dose) Sex: Female Siz@gnificaLnetsions(checkone):- Yes InterpretinPgathologis(tsignaturaend date): Species/StrainC:ynomoigus Monkey Tissue: Liver-centrilobular BlockNo(s).:99.76-38-2A Z18858 - Photo/NegativNeo(s).: Z18862 x No yi eg,,t Ma v 29.2000 Features: Z18858. Liver. Hepatocyte. 10,320X. This hepatocytehas a round central nucleus.To theleftof and beneaththenucleusareseveralmedium sizedlipiddroplets. The remaining cytoplasm containsabundantSER, patchesof glycogen,rru'tochondriaan,d some profileosf RER. Six (6)peroxisomescounted. Z18859. Liver.Hepatocyte.10,320X. The cytoplasmofthishepatocytecontains multipleareasofglycogen granuleaccumulation.These arelightlystainedareaswith numerous smallelectrondense granules.A few lipiddropletsarealsopresentinthe cytoplasm.The darkerand irregulacrytoplasmicmaterialisSER intermixedwith glycogen.Five(5)peroxisomescounted. Z18860. Liver.Hepatocvte.10,320X. Thirty-two(32)peroxisomesarecountedinthis hepatocvte'scytoplasm.The cellhas a largeround nucleus.Most of thecytoplasm is occupiedby SER, mitochondria,and occasionallipiddroplets.Some profilesofRER are evidentaround nu'tochondriaA. microvillousborderand adjacentsinusoidarepresent alongtherightmar-in of thishepatocvte. Z18861. Liver.Hepatocyte.10,320X. For thishepatocytei,tsnuclearprofileisnot presentinthisplaneofsection.A moderate number of medium sizedlipiddropletsare present.Severalof thesecontainsome membranous or myelin-likedebris. Numerous profileosf SER and RER arepresent.There isslightdilatatioonf theendoplasmic reticulum.Numerous variablyshaped n-@tochondriarepresent,but theyappear uItrastrucrtaullynormal. Eleven (11)peroxisomescounted. Z18862. Liver.Hepatocyte.10,320X. Thishepatocyteappearssomewhat triangular shaped in thisplaneof section.Ithas a largeround nucleusand abundant surrounding cytoplasm. Severalsmalltomedium sizedlipidropletsarepresent.Abundant SER, some profilesofRER, mitochondria,and peroxisomes occupy themajorityofthe cytoplasm.Approximatelytwenty-two(22)peroxisomescounted.A sinusoldwith an endothelialcelland erythrocytiespresentatthelower leftmargin of thephotograph. Conclusions: Normal hepatocytes.Peroxisomesaverage 15.2perhepatocyte. M. TransmissionElectronMicrographs Figure Legends DraftElectronMicroscopy Report Covance Study No. 6329-223 Recoven, SacrificeAnimals Magnirication. Printmagnificationisindicatedon theback of each photoe!raphT.he measurement bar on each photomicrographequalsone micron (O.OOIMM). .Fl-gu-1r.eLiver. Hepatocyteshowing normal cvtoplasmicorganelles.Some peroxisomes and othernormal organellesareidentifiedT.here isslightartifactual dilatatioonfthenuclearenvelope and endoplasm-icreticulum.Animal No. 105522,0.75 mg/k-g/day(high-dosemale).57-week biopsy,10,320X magnification. Figure2. Liver.Hepatocvte.Glycogen accumulationsareshown. Glycogen arethe smallgranuleswithintheclearareas.Animal No. 105511,0.75 ma/k-p-/da(vhigh-dose male),79-week necropsy,10,320X magnification. Figure3. Liver.Hepatocyteshowing normal cytoplasmicorganelles.There isslight artifactuadlilatatioonf theendoplasmicreticulumand nuclearenvelope.Animal No. 105542,0.75 mg/kg/dav (high-dosefemale),57-week blopsv,10,320X masznlfication. Figure4. Liver.Hepatocyteshowing normal cytoplasmicorganellesA.nimal No. 105533,0.75mgfk-2/day(high-dosefemale),79-week necropsy,10,320X magnification. Shidy: 6329-223 PAI EM No.: 99.76-33-1A Test ArtcleNo.: 105522 Biopsy Group/Sex: 4-M Negative No.: Z 18835 Magnification: 10,320x Date: 5/22/00 Tech: NW PathologyAssociatesInternational 4915-D ProspectusDr. Durham, NC 27713 (919)544-5257 FIGURE I FP Study-6329-223 PAI EM No.: 99.76-32-2A Test ArticleNo.: 105511 U -Sac Group/Sex: 4-M Negative No.: Zl 8831 LA Magnification: 10,320x Date: 5/22/00 Tech: NW PathologyAssociates International 4915-D Prospectus Dr. Durham, NC 27713 (919)544-5257 FIGURE 2 FiL. -OK 4w ER r . Study: 6329-223 PAI EM No.: 99.76-37-1A Test ArticlNeo.: 105542 Biopsy Group/Sex: 4-F NegativeNo.: Z 18854 Magnification: 10,320x Date: 5/22/00 Tech: NW PathologyAssociatesInternational 4915-D ProspectusDr. Durham, NC 27713 (919)5"-5257 Ll FIGURE 3 1, r-k oi, le R.;6 law- Study: 6329-223 PAI EM No.: 99.76-36-2A TestArficlNeo.: 105533 U-Sac Group/Sex: 4-F NegativeNo.: Zl 8849 Magnification: 10,320x Date: 5/22/00 Tech: NW PathologyAssociatesInternational 4915-D ProspectusDr. Durham, NC 27713 (919)544-5257 FIGURE 4 L/ s IK' -f7. .WR E 4W -Y*t t4 POP @3,SE -qp Abbreviation BC EV G L m N p RB RER SER DraftElectronMicroscopy Report Covance Study No. 6329-223 Recovery SacrificAenimals Index of Labels for UltrastructuralStructures Structure Bile canaliculus Nuclear envelope Glycogen Lipid droplet Mitochondria Nucleus Peroxisome Residual body Rough endoplasmic reticulum Smooth endoplasn-ticreticulum IV. QualityAssuranceStatement PathologAyssociatelsntemational A Company ofScienceApplicatiolnnstemationCaolrporation An FfrPoye-CA-d Comwr@v QUALITY ASSURANCE STATEMENT Thiselectromnicroscopsytudyhasbeen inspecteadndauditebdy theQuality Assurance Unitas requiredby the Good LaboratoryPracticesRegulationspromulgated by the U.S.Food and Drug AdministrationP.AI has a functioninagnd responsive QualityAssurance Unitwhich reportsdirectltyo management. The followingisa record ofinspections/audiatnsd theirresultinrgeports: Date of Inspection Phase Inspected Date FindingsReported to Management and Study Director 4/1,2/99 4/2/99 7/13/99 7/13/99 5/25,30/00 Data Audit DraftReport Audit AuditofData from AdditionalGroups FinalReport Audit Data and DraftReport Audit Recovery Animals 4/2/99 4/2/99 7/13/99 7/13/99 5/30/00 OKne deward, B.S.,RQAP-GLP t:rualiAtsysurance Specialist Dite Sponsor: 3M CorporateToxicology Test ArticleP:FOS Study Number: 6329-223, PAI Study EM-99.76 4915 D Prospectus Drive o Durham, North Carolina 27713 (919)544-5257 o (919)544-3218 FAX