Document 4abG4K01EYk5NKL04eQdegJMe

PathologAyssociateisnfernational A Company ofScienceApplicationisntemationCaolrporation An &rp"v*.O~ CopwV PATHOLOGY REPORT (ANCILLARY STUDY) ELECTRON MICROSCOPIC EVALUA17ION OF LIVER IN CYNOMOLGUS MONKEYS 26-WEEK CAPSULE TOMCrrY STUDY WITH PERFLUOROOCTANE SUIXONIC ACED POTASSIUM SALT (PFOS,-,T-6295I)N CYNOMOLGUS MONKEYS CLIENT STUDY'NUMBER 6329-223 PAI STUDY NUM33ER EM 99.76 Prepared by: James B. Nold,D.V.NL,Ph.D.,Diplomate,A.C.V.P. PathologyAssociatesInternational 4915-D ProspectusDrive Durham, NC 27713 July 13,1999 PREPARED FOR 3M CORPORATE TO]ACOLOGYI 3M CENTER, ST. PAUL, MN 55144-1000 4915 D ProspectusDriveo Durham, NorthCarolina27713o (919)544-5257 (919)544-3218FAX PathologyNarrative Ught Mcroscopy and TransmissionElectron Mcrograph InterpretatiFonrms TransmissionElectronNficrographs QualityAssuranceStatement Section m IV L PathologyNarrative PATHOLOGY (ANCILLARY REPORT STUDY) AncillaPraythologRyeport CovanceStudyNo.6329-223 ELECTRON MICROSCOPIC OF LIVER IN CYNOMOLGUS EVALUATION MONKEYS 26-WEEK SULFONIC CAPSULE TOMCITY STUDY WITH PERFLUOROOCTANE ACIID POTASSILTM SALT (PFOS;T-6295) IN CYNOMOLGUS MONKEYS CLIENT STUDY NUMBER 6329-223 PAI STUDY NUMBER EM ".76 SLTUMIMARY Treatment of cynomolgus monkeys with 0.75 mg PFOS/kg/day for26 weeks resultedin hepatocellulavracuolationand hypertrophyby lightmicroscopy. HepatoceRular vacuolationand hypertrophy.were primarilycentrilobulairn distributioannd did vary in incidenceand severitybetween individualanimals. LTItrastructuraltlhye,rewere increasedlipiddropletswithinbepatocytes.Increasedlipiddropletswere present in both thehigh-dose males examined and intwo of fourhigh-dosefemales examined. Increased hepatocellularglycogen was presentin theremaining two high-dosefemales. These ultrastructuraclhanges correlatedwith hepatocellulahrypertrophy and vacuolationnoted by lightmicroscopy and with increasedliverweight parametersin thehigh-dose animals. The no-effectlevelforhepatocellularvacuolation(lipiddropletaccumulation) was 0.15 mg/kglday of PFOS (mid-dose group). Quantitationof peroxisomes withinhepatocytesby electronmicroscopy indicatedno remarkable differencesbetween controlliversand liverstreatedwith 0.03,0.15, or 0.75 mg PFOS/kgtday. Ultrastructuradlifferencesin cellularmembranes or mitochondria were not discerniblebetween controland treatedanimals in the samples examined. PROCEDURES The objectiveof thisstudy was to examine by electronmicroscopy theliversfrom selectedanimals in a 26-week capsuletoxicitystudy with PFOS in cynomolgus monkeys. In additionto a general qualitativevaluationfordifferencesbetween controland treated animals,the ultrastructureanldpointsincludedquantitatioonf peroxisomes within hepatocytesand membrane and mitochondrialchanges in hepatocytes.Light microscopy of paraffin-embedded liversectionswas alsoperformed. For the toxicitystudy,22 male and 22 female cynomolgus monkeys were given thetest articleor thevehicleonce dailyforatleast26 weeks accordingto Text Table 1. AncillaryPathologyReport Covance Study No. 6329-223 Text Table 1.Group Designationsand Dosage Levels Dosage Group Dose Level (mg/k2/day)' TotalMaterialDose Level(mg/kg/day)b Number ofMales Number of Females oa 302 6d 60 0.03 i5b 4 4 m 0.15 6c 6d 6d IV 0.75 30c 6d 6d Dose levelswere provided.The controlgroup(Group 1)receivedan equivalenatmount of lactosein gelatincapsulesas thetotalmaterialadministeredtoGroup 4. b The low-dosegroup (Group 2) receivedthetestmaterialdilutedwithlactos(e1:499,w:w). The mid-dose (Group 3) and high-dose(Group4) groupsreceivedthetestmateriadlilutedwith lactose (1:39,w:w). dTwo animalsinGroups 1,3,and 4.designatedas recoveryanimals,were treatedforatleast26 weeks, thenmtinent was discontinuedand theanimalswere observedforreversibifipteyr,sistenceo,r delayed occurrenceoftoxiceffectsforatleast13 weeks posttreatment. Animals to be sacrificedatscheduled terminaland recoverysacrificetimes were anesthetizedwith ketamine and xylazine,weighed, exsanguinated,and necropsied. Multiple tissueswere collectedand preservedin 10% neutral-bufferefdormalin according to theprotocol.Additionally,piecesof liverfrom each animal were thin-slicedand placed in @6 strengthKamovsky's fixative(2.0% parafonnaldehyde/2.5% glutaraldehyde in 0.IM phosphate buffer)forsubsequent ultrastructureaxlamination. These liverswere processed intoEpon blocks and submittedto thislaboratoryforsubsequent electron microscopy processingand evaluation.Hematoxylin and eosin(H&E)-stained slide sectionsof formalin-fixed,paraffin-embeddedliverswere submittedforlightmicroscopic examination. Per sponsor request,tissuesamples from terminalsacrificceontroland high-dose males and females were initiallpyrocessedand evaluatedultrastructural(lTyext Table 2). Two high-dose males, Animals Nos. 105506 and 105509, died or were sacrificedin a moribund conditionpriorto scheduled terminalsacrifice.Ultrastructureaxlamination of liversfrom these two unscheduled death animals was not performed. Following evaluationof the electronmicrographs from thecontroland high-doseliverst,he sponsor requested that electronmicroscopy be performed on thelow- and mid-dose liversto evaluatewhether increasedlipiddroplet accumulation was evident inthesedose groups. Text Table 2. Animals SelectedforElectronMicroscopy 0 mg/kg/dayPFOS (Group IControl) 0.03 mgtkg/day PFOS (Group2 low-dose) 0.15 mgtkgfdayPFOS (Group3 mid-dose) Males 105508 105517 Females 105530 105531 Males 105514 105515 Females 105537 105541 Males 105510 105518 Females 105532 105538 105519 105535 105516 105547 105524 105545 105527 105544 , 105521 105550 . 105528 105548 0.75 mglkg/day PFOS (Group4 High-Dose) Males Females 105507 105534 105512 105536 105540 105551 Thin sections(approximately90 nm) from theEpon-embedded liverswere cut,mounted on 200-mesh copper grids,stainedwith 5% methanolic uranylacetateand Reynold's lead 1-2 AncillaPraythologRyeport CovanceStudyNo.6329-223 citratea,nd examined on a ZeissEMLOCICR, a ZeissEMIO, ora JEOL IOOS transmissionelectronmicroscope. 'Centrilobulahrepatocytes,where clearlyidentifiable in liversections,were preferentialleyxamined. Representativeelectron photomicrographs of liverwere taken and significantultmstructurafleatureswere summarized foreach photograph and animal on a designated=mmission electron micrograph interpretatiofnorm. The number of peroxisomes in hepatocyteswas manually counted foreach centerphotographed bepatocyteand recorded. For light microscopy, theH&E slidesfrom thesame animals selectedforelectronmicroscopy were evaluated and significanfteatureswere summarized on a lightmicroscopic evaluation form. RESULTS AND DLSCUSSION Individualinterpretationosf lightmicroscopy slidesand electronmicrographs foreach al areshown in Section M Representativeelectronphotomicrographs were selected as reportfigures,labeled,and are shown in SectionIEL AR re ng electron micrographs aremaintained in thearchived study file. Li2ht Microscopy A summary of the incidenceand severityof lightmicroscopicfindingsin thecontroland high-dose liversisshown in Text Table 3. There was an increasedincidenceof vacuolationof the cytoplasm of hepatocytesin'high-dosetreatedanimals compared with controlanimals. The distributioonf thisvacuolationwas predominantlycentrilobular, but forone high-dose female,Animal No. 105534, therewas diffuseslighthepatocellular vacuolation.Affected animals generallyalso had enlargement(hypertrophy)of centrilobularhepatocytes.This enlargement appeared to be relatedto the presence of the small to largevacuoles with thecytoplasm. Some sporadicvacuolationnoted in low- and mid-dose animals did not appear to be treatment-relateadnd was not clearlyassociated with hepatocellularhypertrophy. Text Table 3. Incidenceand SeverityofLightMicroscopicLiverFindings MicroscopiFcinding I0 mglkgtday (Contmi) Males Females (numberexamined) (4) Vacuolathieopna.tocallular, centrilobular minimal slight moderately-severe Vacuolathieopna,tocellular, diffuse slight Vacuolathieopna.tocellular, multifocal minimIal (4) 0.03mglkg (L4Dw-Dose) Males Females (4) (4) I I 0.ISmg/kg (Mid-Dose) Males Females (4) (4) 0.75mg(kgtday (High-Dose) Males Female s (2) (4) 2 I 1-3 AncillaryPathologyReport Covance Study No. 6329-223 Text Table 3 Continued. Incidenceand Severityof LightMicroscopicLiverFindings tviieroscopFiicnding 0 mg/kg/day (Control) Males Females 0.03 mglkg (Low-Dose) Males Females (numberexamined) Hypertrophyh,epatocellular, centrilobular minimal slight moderate Clearingc,ytoplasmic. centrflobular ff@ninw slistht Clearing,cytoplasuu'c. diffuse rfdrdnud slijzht Infiltraltyem,phohistiocytic fftininua slight Inflammatione,osinophificgmnulonmtous minimal slijtht Pigment,hepatocellular, centrilobular slight Ectasiab.iliary minimal Angiectasis nliwmal (4) 1 4 I (4) 2 4 (4) 3 4 (4) 2 1 4 I 0.15 mg&g (Mid-Dose) Males Females (4) (4) - 1 1 2 I I 3 3 I 0.75mg(kg/d2y (High-Dose) Males Feniale s -(2) (4) 1 2 - - 1 2 1 3 - Electron Microscopy Control (0 mg/kg(day). Ultrastructumfleaturesof controlhepatocytesaredemonstrated in Figures 1-- 3. Hepatocytes characteristicalcloyntainnumerous n-@tochondriaand abundant endoplasrnicreticulum. Peroxisomes arecommon organellescomposed of a finegranularmatrix surrounded by a membrane and normally vary in sizefrom 0.1 - 1.5 microns in diameter.Hepatocytes exhibita microvillousborder which protrudesintothe perisinusoidalspace. Tight junctionsborderbilecanaliculibetween hepatocytes. Glycogen iscommon in the cytoplasm and may be in theform of small granules or slightlylargerrosettes.Residual bodies aresecondary lysosomes (phagolysosomes) containingelectrondense degenerativecytoplasmicmaterial.Lipid metabolism isa normal liverfunction,and it'snormal to see random lipiddropletswithinhepatocytes. There were no discernibledifferencesbetween controlmales and females in thisstudy, and allwere considered normal. A summary of ultrastructurfailndingsisshown in Text Table 4. One male, Animal No. 105527, appeared to have increasednumbers of peroxisomes compared with theothercontrolanimals. However, because so few animals and individualhepatocytes were examined, thisvalue may not be significantliyncreased. 1-4 PathologyAssociate.l#ntemational ikncillaPraythology A Company ofScienceApplicatiolnnstemationCaolrpLlov'ancSetudyNo. TextTabl4e. Summary of UltrastructumFlindings Treatment Group (mgtkgtday)/Sex Animal No. (0)Male 105508 (0)Male (0)Male (0)Male_ 105517 105519 105527 (0.03)Male 105514 (0.03)Male (0.03)Male 105515 105516 (0.03)Male 105521 (0.15)Male (0.15)Male (0.15)Male (0.15)Male 105510 105518 105524105528 (0.75)Male 105507 (0.75)Male 105512 (0)Female (0)Female (0)Female (0)Female (0.03)FenWe (0.03)Female (0.03)Female (0.03)Female (0.15)Female (0.15)FenWe (D.15)Female (0.15)Female (0.75)Female (0.75)Female (0.75)Female (0.75)Female 105530 105531 105535 105544 105537 105541 105547 105550 105532 105538 105545 105548 105534 105536 105540 105551 Comments Normal Normal Normal Normal. Increasedperoxisomes Mid increaseinlipiddroplets Slighitncreasienlipiddroplets Normal Normal Normal Normal Nomud, slightincreasein peroxisomes Normal Lipid dropletaccumulation,hepatocellulu,moderately severe. Increasedglycogen Lipid dropletaccumulation,moderate,hepatocellularI.ncreased peroxisomes Normal Normal Normal Normal Normal Normal Normal Normal Normal Increasedglycogen Normal Normal Increasedglycogen Lipid dropletaccumulation,moderatelysevere Lipiddropleatccumulationn,ildto moderate Increasedglycogen Peroxisomes were identifiedandquantitatiendrepresentathievpeatocytesF.orthe controlliverst,heaveragenumber ofperoxisomescountedperhepatocytephotographed was 11.50 forcontrolmales and 10.94pemxisomes percellforcontrolfemales. (Text Table5). Depending upon fixatioannd stainingp,eroxisomesmay be difficultto differentiaitneroutineelectropnhotorrlicrograpfhrsom primarylysosomesor marginal cutsthroughtheends of mitochondria.Cytochemicaland immunocytochemical methods areavailablfeorspecificalliydentifyipnegroxisomesby lightand electronmicroscopy, and utilizationf thesemethods would providea more specifiacnd accuratemethod of peroxisomequantitatio(nBeierK. 1992;Reddy UN, 1988). T-.5 4915 D Prospectu Drive o Durham. North Carolina 27713 o (919)544-5257 0 (919) 544-3218 FAX AncillaryPatholo.,Roeyport Covance Study No. 6329-223 Text Table 5. Quantitationof HepatocellulaPreroxisomes Animal Number 105508 105517 105519 105527 Mean 0 mg PFOS/kglday(Control) Male Female Number' of Animal Peroxisomes Number Number of Peroxisomes 7.50 105530 12.75 12.25 105531 13.75 3.50 105535 7.00 22.75 105544 10.25 11.50 Mean 10.94 0.03mg PFOS/kgtday(Low-Dose) Male Female Animal Number of Animal Number of Number Peroxisomes Number Peroxisomes 105514 6.00 105537 8.80 105515 15.00 105541 12.00 105516 7.20 105547 10.00 105521 10.80 105550 17.60 Mean 9.75 Mean 12.10 0.15 mg PFOS/kg/day (Mid-Dose) 0.75mg PFOS/kglday(High-Dose) Male Female Male Female Animal Number' of Animal Number of Animal Number of Animal Number of Number Peroxisomes Number Peroxisomes Number Peroxisomes Number Peroxisomes 105510 10.60 105532 4.20 105507 11.0 105534 5.50 105518 18AO 105538 7.20 105512 23.25 105536 3.75 105524 105528 23.20 14.60 105545 105548 9.00 17.00 105540 105551 12.50 9.50 Mean = 16.70 Mean = 9.35 Mean 17.12 Mean = 7.81 Average number of peroxisomesperhepatocyteforfourhepatocytecsountedperanimal forcontroland high-dosegroupsand fivebepatocytecsountedper animalforlow-and Mid-dosegroups. lUgh-Dose (0.75 mgtkgtday). Lipid droplets were increased in animals given 0.75 mg PFOS/kg/day (Text Table 4). This increasewas presentin both high-dose males examined (Animal Nos. 105507 and 105512) and in two high-dosefemales (Animal Nos. 105536 and 105540). These are illustrateidn Figures4 -7. Lipid dropletswere randomly distributedin thecytosoland variedin sizefrom lessthan .I rnicronto greaterthan severalmicron'sin diameter. They generallywere clear,but occasionallycontained wispy lamellarmaterialor faintsmooth electrondensity.They were not membrane-bound, but appeared to form a defined margin with surrounding cytoplasm. These lipiddroplets correlatewith hepatocellularvacuolationdescribedin theseanimals by lightmicroscopy. Increasedcytoplasmic glycogen was evidentin two high-dosefemales (Animal Nos. 105534 and 10555 1),and often formed distinct"pockets"of glycogen,resembling vacuoles or cleardroplets,but containingglycogen particle(sFigure8). The accumulation of glycogen apparentlycorrelateswith thehepatocellulavracuolation described by lightmicroscopy in thesetwo animals. Lipid dropletsdid not appear increased in thesetwo animals. For high-dose animals,the average number of peroxisomes per hepatocyte was 17.12 for males and 7.81 forfemales (Text Table 5). One male (Animal No. 105512) averaged 23-25 peroxisomes per hepatocyte,however, thisisnot remarkably higherthan thatfound in the controlmale Animal No. 105527. Animal No. 105507 averaged 11.0 peroxisomes per hepatocyte,similarto the averagecontrolvalue formales. Thus, administrationof 0.75 mg/kg/day of PFOS to monkeys for26 weeks didnot appear to induce peroxisome prolifiratioinnthe liver. 1-6 AncillaryPathologyReport Covance Study No. 6329-223 Differencesin mitochondria and cellularmembranes were not discerniblebetween highdose and controllivers.NUtochondria from controland high-dose liversdid not exhibit remarkable differencesultrastructural(lFyigures3,9, 10). Low-Dose (0.03mglkglday). Electronmicroscopicexaminationof theliversfrom monkeys given 0.03 mg/kg/day of PFOS did not identifyany atypicalul=tructural featurescompared with controlsample. Lipid dropletswere identifiedin some hepatocytes,and two low-dose males appeared to have slightlyincreasedlipiddroplet accumulation (Animal Nos. 105514 and 105515). However, increasedlipiddroplet accumulation was not seen in the low-dose females or in the inid-dosemales and females, and thisobservationin the two low-dose males was believedto representnormal biologicalvarianceand sampling. As noted earlier,lipidmetabolism isa normal liver function,and it'snormal to see random lipiddropletswithinhepatocytes.Figure II illustrateashepatocytefrom thelow-dose group. For-low-dose animals,the average number of peroxisomes per hepatocytewas 9.75 for males and 12.10 forfemales (Text Table 5). NEd-Dose (0.15mgtkgtday). There was no consistentultrastructureavlidence of increasedlipiddropletaccumulation in the liversof themid-dose monkeys. given 0.15 mg/kg/day of PFOS. This mid-dose level,0.15 mg/kgtday, istheno-effectlevelforthe PFOS-related effectof increasedhepatocellulalripidaccumulation (vacuolation)in monkeys given PFOS. One female,Animal No. 105538, had increasedglycogen, but a marked increasein glycogen was not seen in any othermid-dose liversexamined. For niid-doseanimals,the average number of peroxisomes per hepatocytewas 16.70 for males and 9.35 forfemales (Text Table 5). Figure 12 illustrateashepatocyte from the mid-dose group. CONCLUSION Ultrastructuraelvaluationof liversfrom cynomolgus monkeys treatedwith 0.75 mg PFOS /kg/dayfor26 weeks identifieidncreasedlipiddropletswithinhepatocytes. Increasedlipiddropletswere present inboth treatedmales exarru*nedand in two of four treatedfemales examined. Increasedhepatocellularglycogen was present in the remainintgwohigh-dofsemalesT.heseultrastruccthuarnaglecsorrelawtietdh hepatocellularhypertrophy and vacuolationnoted by lightmicroscopy and with increased liverweight parameters in thehigh-doseanimals. The no-effectlevelforhepatocellular vacuolation(lipiddropletaccumulation)was 0.15 mg/kgtday of PFOS (rnid-dosegroup). Quantitationof peroxisomes withinhepatocytesby electronmicroscopy indicatedno. remarkable differencesbetween controlliversand liverstreatedwith 0.03, 0.15 or 0.75 mg/kg/day. Ultmstructuraldifferencesin cellularmembranes or mitochondria were not discerniblebetween controland treatedanimals in the samples examined. 1-7 SIGNATURE: OF AUTHOR Dmft AncillaryPathologyReport Covance Study No. 6329-223 ke s B.2N @,@@M -@ Ph.D.,@ Diplomate,A.C.V.P. BaTte REFERENCES BeierK: Lightmicroscopicmorphometricanalysisof peroxisomesby automatici-mage analysisa:dvantagesof immunostainingoverthealkalinDeAB method.J.Histochem Cytochem.,40(l):115-121,1992. Reddy, LTN et.al:Tissuespecificitaynd speciesdifferenceisnthedistributioonf urate oxidaseinperoxisomes.Lab. invest.5,8(1)100-111,1988. IL LightNficrOSCOPYand TransmissionElectron NficrographInterpretatiFoonrms Forms aretypographicallcyonecmd versionsof siped/datedraw datasheetsmaintained inarchivedstudyfile. LIGHT NUCROSC'OPIC EVALUATION' Stud@ No.: 6329-223 (EM 99.76) Species/Strain,Cvr@c)moiLus- Mc)rik'@.v.@ Animal No: see below Sex: seebelow InterpretinPgathologist(signaturaend date): Tissue: @@-ivei- Animal Dose/Group Number MicroscopicFindingsand Comments 0 mg/kg 105508 1, InfiltraItme, ohistiocytmiucl,tifocamli,nimal (Control) --- 1-0--5-5-1-7---------I-. Infiltraltyelp,n@ohistioc c,multifocalm,inimal Males 105519 1. Infiltraltyem,phohistiocytimcu,ltifocalm,inimal 2, Clearingc,ytoplasmicc,entrilobulmairn,imal 105527 1 , Infiltraltyem,phohistiocytmiucl,tifocamlinimal 2. Angiectasifso,calm.inimal 0 mg/kg ...1.0.5530 (Control) 105531 1. Infiltraltyep,lpa_qih!Lt@2Sytmiucl,tifocal@,@nimaf 1. Infiltraltyem,phohisfiocytmiucl,tifocamli,nimal 2. Clearingc,ytoplasmic,entrilobulmairn,imal Females 105535 1- Infiltratley,mphohistic>cytmiucl,tifocalm,inimal 105544 2....C.l.e.a..r.i.ngc,ytoplasmicc,entrilobulmairn,i.m.a.l........ 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 2. Inflammatione,osinophilic-granulomatous, 0.75mgfkg 105507 1. Infiltraltyem.phohistiocytfiocc,alm,inimal (High-dose) Males 105512 2, Vacuolationh,epatocellulcaern,l-ilobular, 3 S!!..u.l...a..rc..,e..n.t.r.i..l..o..b...u..l...a.m.r.o,dera.t..e..... 1. Clearingc,ytoplasmicc,entrilobulsalri,ght 2. Hypertrophyh,epatocellulcaern,trilobulmairn,imal ...... 0.75mg/kg 105534 1. Infiltraltyem,phohistiocytfiocc,alm,inirrial (High-dose) Females ........... 105536 105540 2. Vacuolationh,epatoccllulzdlirf,fuseS,lighE u..l.a.r.c,.e..n..t.r.i.l.o..b.u.l.ma.ir.n,..i.m.a.l.. 1. Vacuolation,hepatocellular,ccntrilobulaf,moderately-severr@, .,@@p@..t.o.c.e.l.l.u.l.ca.er.n,.l.r.i.l..o.b.u.l.a.mr.o,..d..e...r.a.t.e.... 1 Infiltra)tyem,phohistiocytmiucl,tifocalm,i,nimal Vacuolationh,epatocellulacre,ntrilobulamriniTT@al '3@ Clearingc,ytopwmic, centirlobulasrl,ight ------------105551 .........c..e...n...t.r.i.lobulamri-n,in,@al 1. Infiltratleym,phohistiocyticm,ultifocal,tninimal 2. Vacuolationh,epatocellulcaern,trilobulzmlirn,imal .......... 3. Cleari_ng,cytoplasmicc,entrilobular, Animal Dose/Group Number MicroscopicFindingsand Comments 0.03mg/kg 105514 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 2. Inflammationc,osinophilic-granulomatfooucsa,lm,inimal 3. Pigment,hepatocellulmaurl.tifocacle,ntrflobulmairn.imal c to lasmicc,entiilobulsalri.ht (Low-dose) 105515 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 2. Vacuolationh,ep!!Lcellulcaern,trilobulamri,nimal Males 105516 1 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal .. ..............2.....C.l..e.a.ringc,ytoplasmicc,entrilobulsalri,ght 105521 1. Infiltraltyem,phohistiocytmiucl,tifbcamlinimal 2. Pigment,hepatocelfulamru,ltifocacle,ntrilobulsalri,ght 3. Vacuolationh,epatocellulmaurl,tifocalm,inimal 4. Clearingc,ytoplasmicc,entdiobulasrl,ight 0.03mg&g 105537 i 1. Infiltraltyem,phohistiocytmiucl,tifww,minimal 2. Cleatin&M,oplasinic,centfflobulasrl,ight (Low-dose) 105541 1. Infiltraltyem,phohistiocytimcu,ltifocamLi4nimal 2. Cl in ,cytoplasmiccc,ntdlobulamri,nimal Females 105547 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal b 105550 2. Ectasiab,ili focalm,inimal 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 2. Clearingc,ytoplasmicc,entfflobulmairn,imal 3. Vacuolationh.epatocallulamru,ltifocamli,nimal 0.15 mg/kg 105510 1. Infiltraltyem,phohistiocytmiucl,tifocamli,ninud 2. Clearingc,ytoplasmicd,iffimem.inimal (Mid-dose) 105518 1 . Infiltraltyem,phohistiocytmiucl,tifocamli.nimal 2. ClearingE,yto lasniic,entrilobulmairn,imal Males 105524 1 Clcan .1,cytopl raicc,entdiobulasrl,ilht 105528 1 Infiltraltyem,phohisdocytimcu,ltifocamli,nimal 2. Clearingc,ytoplasmicd.iffuses,light 0.15m gfkg ..1.-.055-32------ 1. InfiltraItmep,hohistiocticm,ultifocamli,nimal (Mid-dose) 105538 1. Infiltraltyem,phohistiocytmiucl,tifocamli,nimal 2. Clearin,c to lasmicc,entrilobul ht Females 105545 1. InfiltraItme, ohisti -timcu,ltifocamli,nimal 105548 1. Infiltraltyem,phohistiocytmiucl,tifocasll,ight 2. Clearingc,ytoplasmicc.entrilobulsalri,ght InterpretinPgathologist(signaturaend date): TRANSNIISSION ELECTRON 1%,IICROGRAPHINTERPRETATION StudyNo.: 6329-22-3(EM 9976) Animal No.: 105508 TreatmentGroup: 0 mL,/kg(Control) Sex: Male Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99-76-1 43401- Photo/NegativNeo(s).:43405 SignificaLnetsions(checkone):- Yes x No InterpretiPnagthologis(tsignaturaend date): Features: 4340 1: Liver.Hepatocyteexhibitinlgargecentranlucleus.The cytoplasm containsmany initochondriaand abundantendoplasmicreticulum.Bilecanaliculaire evidentatthelower rightand leftmarginsofthishepatocyte.A number (approximately 20-25)of smallto medium-sizeclearlipiddropletsarepresentp,rincipalliyn theupper portionof theceR. Mtochondria appearnormal;normal matrixgranulesareevidentin most. Severallysosomes and peroxisomesarepresent.Five(5)peroxisomescounted. 43402: Liver.Hepatocyteispossiblybi-nucleatbeu,t thismay be functionof cutthrough thenucleus.Some smallto medium-sizedclearlipiddropletspresents,imilarto 43401. Microvillousborderinspaceof Disseevidentattop marginofcell.Severallamellar myelin-likbeodiespresentwithinlysosomes.Five (5)peroxisomescounted 43403. Liver.Hepatocytewithbilecanaliculuastlowerright.Only a few s*mallto medium-sizeclearlipiddropletsareevident.Numerous mitochondriaand abundant endoplasmicreticulumareevident.Clearstructuraetthebottommay be dilatedGolgi, reticulumorartifactN.ine (9)peroxisomescounted. 43404: Liver.Hepatocytewithadjacentendotheliaclelland sinusoidatthebottom o. f the photograph. Hepatocytecontainseveralsmalltomedium-sizedclearlipiddroplets. One droplet contains some lamellar material. Eleven (11) peroxisomes counted. 43405: Liver.Hepatocytewithhighermagnificatioonfmitochondria.Seven riiitochondrpiraesentwith interveninrgough endoplasniirceticulumand cytosol.Outer membrane, cristaem,atrixand granulesappearnormal. Conclusions: Normal hepatocyte.Average7.5peroxisomescountedperhepatocyte. TRANSNIISSION ELECTRON NUCROGRAPH PMRPRETATION StudyNo.:-6329-223(EM 99-76) Animal No.: 105517 TreatmentGroup: 0 mg&g (control) Sex: Male Species/StraiQnn:omolgus Monka Tissue: Liver- BlockNo(s).: 99.76-2 43406- Photo/NegativNeo(s).: 43410 SignificLaenstions(checkone):- Yes X-- No InterpretiPnagthologis(tsignamm and date): Features: 43406: Liver.Hepatocytesurroundedby otherhepatocytewsithb.Ue canaliculiin thelower rightand upperleftmarginsoftheceR. There arenumerous mitochondriaand abundantendoplasmicreticulum.The ER and nuclearenvelope appear' slightldyilatedO.nly a couplesmallclearlipiddropletesvident.Severalsmau lysosomesorvacuoleswithmicrovesicleisnthem arepreSenLFive(5)peroxisomes counted. 43407: Liver.Hepatocytewithperisinusoidrarldcrovillobuosrderevidentinlower right and upperleftof photograph.A bilecanaliculuisspresenton therightmiddlemargin of thecell.Severalsmalltomedium-sizedclearlipiddropletsareevident.One largerone containssome lainellaprofilesF.our (4)peroxisomescounted. 43408: Liver.Hepatocytewith sinusoidcontaininegrythrocyteastthetop. A lipidcontainingperisinasoidaclell(Itocell)ispresentatthetopleftand a bilecanaliculuiss presentatthebottom. Random smallclearlipiddropletsarepresentinthehepatoceuular cytoplasmand some of theER appearsmildlydilated.Eleven(11)peroxisomescounted. 43409: Liver.Hepatocytecontainsnumerous dense lysosomesand/orperoxisomes intermixedwithmitochondriaand abundantER. Severalsmallclearlipiddropletsare evident.Twenty-nine(29)peroxisomescounted. 434 10: Liver.Enlargedphotographofmitochondria.Ten mitochondriareclustered togetherinthisphotograph.Althoughtheimage isa littloeutof focus,theseorganeues appearessendallnyorrnal.Theremay be mUd dilatatiofntheER inthecytoplasnl Conclusions: Normal hepatocyte.Average 12.25peroxisomesperhepatc)ryte. TRANSIVHSSION ELECTRON NUCROGRAPH UCMRPRETATION StudyNo.:-6329-223(EM 99.76) AnimalNo.:- 105519 TreatmentGroup:--0m /"k (control) Sex: Male Species/Straignm:omolgus Monkey Tissue: Liver BlockNo(s).-:99.76-3 43421 -43425, Photo/NegativNeo(s).: 43431 - - SignificaLnetsion(scheckone):_ Yes InterpretinPgathologis(tsignaturaend date): X No Features: 43421: Liver.Hepatocyte.At lowerleftisaprominentperisinusoidaflatstoringceH (Itocell)containingnumerous largeclearlipiddropletsT.he centered bepatocytecontainsone largeclearlipidropletand severalsmallerones. The nuclear envelopeand ER ismildlydilated.Moderate numbers ofglycogengranulespresent. Scatteredresidualbodiesnoted.Four (4)peroxisomescounted. 43422: Liver.@Etochondriaand surroundingcytosoland ER. Membranes of ER arenot very distinctG.lycogen incytosol.Mtochondria appearessentialnloyrmal. Larriinoaf membranes and cristaeappearparallelS.everalnormal granulesinthematrixof the rriitochondria. 43423: Liver.NfitochondriaS.ame view as43422 butslightllyessmagnificationS.ome dilatatioonf ER. l@Utochondriaappearnormal.A peroxisomeispresentinthelower left of thephotograph. 43224: Liver.Hepatocytewithseveralmedium-sizedand one largeclear'lipiddroplets. Small granuleswithincytosoland aroundER appeartobe glycogenparticleosr ribosomesand polydbosomes. Four (4)peroxisomescounted.Slightloyutof focus. 43425: Liver.Hepatocytesurroundedby otherbepatocytesand a sinusoidatlower right. Severalbilecanaliculeividentbetweenhepatocytes.A singlemedium-sizedclearlipid dropletispresentinthehepatocyteF.our (4) peroxisomescounted.A coupleoflarger dense lysosomes also identified. 43431:*Liver.The centerehdepatocytiesoblongdiagonallyin the photograph, and doesn'tcontainany distincUtpiddroplets.A few bpiddropletsarepresentin adjacent hepatocytes.There ismild dilatioonftheER and nuclearenvelope.Only a few Ilysosomalstructurensoted.Two (2)peroxisomescounted. Conclusions: Normal hepatocyte.Average 3.5peroxisomesper hepatocyte. TR,KNSNIISSIONELECTRON NUCROGRAPH INTERPRETATION StudyNo.:6329-223(EM 99.76) Animal No.: 105527 TreatmentGroup: -0 mg/kiz(control) Sex: Male Species/StraiCny:nomolzus Monkey Tissue: Liver BlockNo(s).: 99.76-4 43426- Photo/NegativNeo(s).:43420 SignificanLtesions(checkone): X ---Yes No InterpretiPnagthologis(tsignaturaend date): Features:43426: Liver.Hepatocytewithseveralsmalltomedium-sizedlipiddroplets (approximatel2y0). The adjacentlowerlefthepatocyteshows significadnitlatatioonfits ER. At topleftand rightmargin,theextracellulianrterstitihuamssome collagenfibers. Some residuablodiesnoted.Twenty-one (21)peroxisomescounted. 43427: Liver.NUtochondria.Membrane, cristaaend matrixappearnormal. Several normal granulespresentmatrix.Some rough ER presentadjacenttomitochondria. Lysosome presentatleftof centermitochondrion. 43428: Liver.Hepatocyte.There ismilddilatioonftheER and nuclearenvelope. Scatteredsmalltomedium-size clearlipid roplets(approximately18). Scattered residuablodiesnoted.Twenty-five(25)peroxisomescounted. 43429: Liver.Hepatocyte.Bilecanaliculaireevidentatthelowerrightand top ofthe cell.The ER ismildlydilated.Scatterecdlearlipidropletasreevident.Abundant numbers of dense bodies(peroxisomes)/Iysosomeasnd residualbodiesarepresent. Thirty(30)peroxisomescounted. 43430: Liver.Hepatocyteappearssimilartoabove describedcells.There aresome scatteresdmalltomedium-size clearhpid dropletsS.ome residualbodiesarepresent. Fifteen(15)peroxisomescounted. C nclusions:Normal hepatocyte.Appear tobe increasendumbers ofperoxisomes compared with othercontrolsamples.Average 22.75peroxisomesperhepatocyte. TRANSNEISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:6329-22-3(EM!9.76@- Animal No.:, ID5530 TreatmentGroup: 0 mz/kg (control) Sex, Female Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99-76-5 L4756 - Photo/NegativNeo(s).: L4 760 SignificaLnetsions(checkone):_ Yes X- No InterpretinPgathologis(tsignaturaend date): Features:L4756: Liver.HepatocytecontainsthreesmalllipidropletsT.here arebile canaliculaitthetop rightattoplefta,nd a perisinusoidna-liicrovwoubsorderatthe bottom.The nuclearenvelopeand ER shows some rnildartifactu(aflixationd)ilation. Numerous mitochandriaarepresent.Five(5)peroxisomescounted. L4757: Liver.Nfitochondrial.eghermagnificatioonffivemitochondriaM.atrixand dense granulesappearnormal. ER betweenmitochondriaisslightldyilated. LA758: Liver.Hepatocyte.A sinusoidispresentatthetopof thephotograph,linedby an endotheliaclell.Four (4)medium-sizedlipidropletasrepresentinthiscellT.he nuclearenvelopeand ER shows some n-dldartifactu(aflixationd)ilationN.umerous riutochondriaarepresent.Four (4)peroxisomescounted. L4759: Liver:Hepatocytecontaininsgcatteresdmalltomedium-sizedlipiddroplets (approximately14). Scatterepdrimarylysosomesand/orperoxisomespresentT.here is a perisinusoidamlicrovillousborderatthetopbeneathan endotheliaclelland sinusoid. Twenty-five(25)peroxisomescounted. L4760: Liver.Hepatocytecontaininagpproximatelnyine(9)smalltomedium-sized lipiddropletsS.eventeen(17)peroxisomescounted. Conclusions: Normal hepatocyte,Average 12.75peroxisomesperhepatocyte. TRANSIVUSSION ELECTRON NUCROGRAPH UIITRPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: -- 105531 TreatmentGroup:--0mg/kg (control) Sex: Female Species/-StraiCny:nomolgus -Monkey Tissue: Liver BlockNo(s).: 99.76-6 LA761 - Photo/NegativNeo(s).: LA765 SignificanLtesions(checkone): Yes InterpretinPgathologis(tsignaturaend date): X No Features: L4761: Liver.Hepatocytewithlargecentralnucleus.Two margins are straight-edgebdy thegridbar.A few smalltomedium-sizedcleartoslightlsyhaded lipid dropletsarepresent(approximatel3y2).ER profileasnd glycogengmules arescattered throughoutthecytosol.A moderatenumber ofprimarylysosomesand/orperoxisomes noted.Nine (9)peroxisomescounted. L4762: Liver.l@CtochondriaO.ne ovaland one club-shapedmitochondriapresent.Both appearnormal with cristaem,embranes, and matrix.The club-shapedone contains severalnormal densegranulesand some slightldyilatedcristaep,robablyfixationartifacl Some RER and smooth ER (SER) areevidentinthecytosol. LA763: Liver.Hepatocytesurroundedby 'Mterstiticuomntainingsome couagen fibers. Cellshas slightldyilatedER and nuclearenvelope.A singlesmalllipiddropletispresent inthiscellF.our (4)peroxisomescounted. LA764: Liver.This bepatocyteshows some dilatioonfER and severallamellaresidual bodiesinthecytoplasm.Four (4)smalllipiddropletasreevident.Numerous scattered glycogengranulesarepresent.Eight(8)peroxisomescounted. L4765: Liver.Hepatocytecontainin5g smalltomedium-sizedclearhpid droplets.Bile canaliculaireevidentatthelowerleftand toprightofthecellmargins. A clusteorf RER ispresentin thelower leftsideof thenucleus.Some largerdark lysosomes/peroxisomesarepresentamong some of theniitochondriTah.irty-fou(r34) peroxisomescounted. Conclusions: Normal hepatocyte.Average 13.75peroxisomesperbepatocyte. TRA,NSINUSSIONELECTRON ABCROGRAPH E@IMRPRETATION StudyNo.:6329-223(EM 99.76)- Animal No.: 105535 TreatmentGroup: 0 mg&iz (control) Sex: Female Species/StraiCny:nomol-eusMonkey Tissue: Liver BlockNo(s).: 99.76-7 14766- Photo/NegativNeo(s).:L4770 SignificLaenstions(checkone):- Yes X No InterpretiPnagthologis(tsignaturaend date): Features: LA766: Liver.Hepatocytecontainsome mUd dilatationfthenuclear envelopeand ER (fixatioanrtifactO)n. theleftand rightsidesarebilecanaliculia,nd ventralliysthesinusoidamlargin showingtheperisinusoidnailicrovmousborder.Several dense residuablodiesarepresentinthecytoplasm.Three smalllipiddropletso,ne containinga lamerarbody, areevident.One (1)peroxisomecounted. L4767: -Liver.NfitochondriaH.ighermagnificatioonffourmitochondriashow normal morphology. Membranes, cristaaend matrixappearnormal.Scatteredense granules arepresent.The ER inthecytosolismildlydilated. L4768: Liver.Hepatocyte.ThereismoderatedilatatiofntheER and nuclearenvelope. Severalscatteresdmalltomedium-sized clearlipiddropletsareevident,as aresome scattererdesiduablodies.Sinusoidsarepresentatthebottom rightand upper leftofthe photograph,and a bilecanaliculuisspresenton therightmargin of thiscentered hepatocytewithitsneighborbepatocyte.Two (2)peroxisomescounted. LA769: Liver.Hepatocytewithsinusoiidnupperrightcomer and canaliculpirominent alongthebottom border.The cejlexhibitdsilatedER and nuclearenvelopelikethe previouslydescribedcellsforthisanimal- indicatinsguboptimalfixationM.u.ltiple residualbodiesarepresent.Nineteen(19)peroxisomescounted. LA770: Liver.HepatocytewithsimilaorverallmorphologyasL4769. Six(6) peroxisomes counted. Conclusions: Normal hepatocyte.SuboptimalfixationA.verage 7.0peroxisomes per hepatocyte. TRANSrvHSSION ELECTRON ry]aCROGRAPH RMRPRETATION StudyNo.: 6329-223 (EM 99-76)- Animal No.: 105544 TreatmentGroup: 0 Ln ontrol) Sex: Female Species/StraiCny:noinolgHsMonkey Tissue: Liver BlockNo(s).: 99.76-8 L4771 - Photo/NegativNeo(s).:-IA775- SignificanLtesions(checkone)-. Yes X No InterpretiPnagthologis(tsignaturaend date): Featares:L477 1:Liver.Along leftmarginofhepatocytaeresome interstitiaunmd collagenfibersH.epatocytecontainaspproximately17 smalltomedium-sizedclearlipid dropletsA. few residuablodiesareevident.There issome minimaltomild dilatationf ER. Eight(8)peroxisomescounted. L4772: Liver.Enlargedphotographofthreemitochondria.No abnormalitiensotecl L4773: Liver.Hepatocytewithone largeand severalsmalltomedium-sizedlipid dropletisnthecytoplasm.Eight(8)peroxisomescounted. IA774: Liver.Hepatocytewithprominentmicrovwous borderalongbottom sinusoid. An endotheliaclellnucleusisevidentalongthisborder.'Inthehepatocyteareseveral medium-sizedclearlipiddropletsT.he ER does show some mad dilatatioannd thereare abundantglycogen granulesinthecytoplasm.Severaldark residuablodiesarcpresent. Eight(8)peroxisomescounted. LA775: Liver.Hepatocytewithsinusoidon therightand prominentcanaliculuosn top and leftmargins.No lipiddropletspresentincenteredhepatocytea,lthoughone mediumsizedlipiddropletispresentintopadjacenthepatocyte.Cytoplasm containssome RER butnotmuch SER, possiblya periportahlopatocyte.Random peroxisomesand/or primarylysosomes noted. Seventeen(17)peroxisomescounted. Conclusions:Normal hepatocyte.Average 10.25peroxisomesperhepatocyte. -A TP-A,NSNUSSIONELECTRON MICROGR APHINTERPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: 105514 TreatmentGroup: .03mg/.k-(glow-dos-el Sex: Male Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 9-9.76-15 Z16232 - Photo/NegativeNo(s)Z.1:6236 SignificanLtesions(checkone): x Yes No InterpretinPgathologis(tsignaturaend date): Features: Z16232. Liver.Hepatocyte.Hepatocytecontainsmultiplesmallto mediumsizedlipiddropletscatteretdhroughoutitscytoplasm.At thetopmargin isa microvilloubsorderand some collagenfibersinthespaceofDisse.Smooth endoplasmic reticulum(SER) and roughendoplasmicreticulum(RER) arepresenthroughoutthe cytoplasmwith many mitochondrialsothroughout.Ntochondria arewell-preserved and cristaeg,ranulesand matrixareclearlyevident.Severalirreguladrarkstaining residualbodiesarepresent.One (1)pemxisome counted. ZI 6233. Liver.Hepatocyte.A moderatenumber ofsmalltomedium-sizedlipiddroplets arepresentinthecytoplasm,as wellasseveralprominentresiduablodies.The upper left and lower leftbordersshow microvillaind collagenfibersadjacento a sinusoidor centravlein.The RER formsa distincptalisadea thebottomofthecell.Nine (9) peroxisomescounted. Z16234. Liver.Hepatocyte.Centeredhepatocyteshows minimalfixatioanrtifacwtith some slightdilatatioonf SER and thenuclearenvelope.There area few small to medium-sizedlipiddropletspresentF.our (4)peroxisomescounted. Z16235. Liver.Hepatocyte. Cellappearssimilartothosedescribedabove. There are random lipiddropletsand multipleresidualbodies.Five(5)peroxisomescounted. Z16236. Liver.Hepatocyte.Multiplesmalltomedium-sizedlipidropletpsresentin thecytoplasm.Only a smallnuclearprofiliesevidentinthiscell.Eleven(11)probable peroxisomescounted. Conclusions: Tissueappearswell-fixedforultrastructuerxaalmination.There appears tobe a mUd increasein numbers of lipidropletsA.verage 6.0peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:-6329-223(EM 99.76) Animal No.: 105515 TreatmentGroup: .03mglkg (low-dose) Sex: Male Species/StraiCny:nomolgus Monkey Tissue: - -Liver BlockNo(s).: 99.76-16 Z16237 - Photo/NegativNeo(s).:-Z16241- SignificanLtesions(checkone)- X Yes No InterpretinPgathologis(tsignaturaend date): Features: Z 16237. Liver.Hepatocyte.A centralveinispresentatthetopmargin and containsa largea.mount ofcollagenextraceuularlyA. bilecanaliculuisspresenton the leftbetween threehepatocytes.theendoplasinirceticulumand nuclearenvelopeare slightldyilate-d thisisa fixatioanrtifaclSeverallipidropletasrepresenlProminent glycogengranulesarepresentthroughoutthecytoplasm.Twenty-two (22)peroxisomes. Z16238. Liver.Hepatocyte.AbHe canaliculuisspresentatthetopborderwith another hepatocyte.One largeand severalsmallerlipiddropletasrepresent.Nfitochondria appearnormal and have finecristaem,atrix,and granules.Twenty-one (21)peroxisomes. Z16239. Liver.Hepatocyte.Largecentralnucleusisadjacentoa largelipiddroplet.A few smallerlipiddropletsarepresent.There isartifactudaillatationf theRER and the nuclearenvelope.Severalresiduablodiesarepresent.Fourteen(14)peroxisomes counted. Z 16240. Liver.Hepatocyte.The centeredhepatocytecontainsseveralmedium-sized lipiddroplets.There ismarked dilatationf theRER and thenuclearenvelope.A bile canaliculuisspresenton theleftmargin of thiscelland extracellulcaorllagenisevident intheupper right.Ten (10)peroxisomescounted. Z 16241. Liver.Hepatocyte.Hepatocytecontainstwo largerlipidropletsand several smallerones. The RER and nuclearenvelopearedilatedT.he ventralmargin shows a microvilloubsordernexttoan endotheliaclellatthebottom.A prominentbile canaliculuisspresenton thelefts,urroundedby darkdesmosomes. Eight(s) peroxisomescounted. Conclusions: Slight(equivocali)ncreasienlipidropletsA.verage 15.0peroxisomes per hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:6329-223(EM 99-76) Animal No.: 105516 TreatmentGroup: .03mp-/kg(low-dose) Sex: -Male Species/StraiCny:nomoigus Monkey Tissue: Liver BlockNo(s).: 97.76-17 Z16242 - Photo/NegativNeo(s).: Z16246 SignificaLnetsions(checkone):--,- Yes X No InterpretinPgathologis(tsignaturaend date): Features: Z16242. Liver.Hepatocyte. Hepatocyteisadjacentoa cellcontaininga largelipidropletp,robablya perisinusoidfaalt-storicnegll(Itocell).The hepatocyte containsnumerous nonnal-appearingmitochondriaand scatteredark irregularesidual bodies.Smooth and RER am presentthroughoutthecytoplasm.One (1)probable peroxisome counted. Z16243. Liver.Hepatocyte.Hepatocytehas a bilecanaliculupsresentalongitsright margin with an adjacenthepatocyte.The cytosolcontainsabundantsmooth and RER and numerous nlitochon&ia.A few residuablodiesam present.Two (2)peroxisomesseen. Z 16244. Liver.Hepatocyte. Hepatocyteexhibitvseryslighdtilatatiofnthe endoplasn-dcreticulumand nuclearenvelope.Glycogen granulesarepresentthroughout thecytoplasm,and scattererdesiduablodiesarepresent.Two (2)probableperoxisomes identified. Z 16245. Liver.Hepatocyte.A clearvacuolewitha centralrounddensityispresentto theleftofthenucleusof thehepatocyte.This appearstobe a cytoplasn-Liincvagination from theplasma membrane. Normal-appearingmitochondriaarepresentthroughoutthe cytoplasm.A few residuablodiesarepmsent. Fifteen(15)peroxisomescounted. Z 16246. Liver.Hepatocyte.The centeredhepatocyteappearsessentiallsyimilarto above-describecdells.There areglycogengranulesthroughoutthecytoplasm.Sixteen (16)peroxisomescounted. Conclusions:EssenfialnloyrmalhepatocytesA.verage7.2peroxisomepserhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:-6329-223(EM 99.76) Animal No.: 105521 TreatmentGroup: -0.03 mg&g (low-dose) Sex: Male Species/StraiCny:nomolgus monkey Tissue: . Liver BlockNo(s).: 99.76-18 Z16247 - Photo/NegativNeo(s).: Z 16251 SignificLaenstions(checkone):- Yes X No InterpretinPgathologis(tsignaturaend date): Features: Z 16247. Liver.Hepatocyte.Severallargeirregulaerlectron-densreesidual bodiesarepresentinthecenteredhepatocyteand thesubjacenthepatocyte.The SER and nuclearenvelopeare slightldyilatedA.lso,the mitochondria ppearsslightlsywollen, althoughthisappearstobe artifactuaallongwiththedilateSdER. A few smalllipid dropletsarepresent.Fourteen(14)peroxisomescounted. Z 16248. Liver.Hepatocyte.Clustersof finegranulesp,robablyglycogen,arepresentin thecytoplasm.The mitochondriaappearsslightldyilateda,s doestheSER inan adjacent hepatocyteto thetop left.The smooth ER ispoorlydefined.Severalsmalllipiddroplets arepresent.Two erythrocyteasrepresentatthelower rightof thephotograph. Eleven (11)peroxisomescounted. Z 16249. Liver.Hepatocyte.The RER, nuclearenvelom and SER inthehepatocyteare slightldyilatedc,onsistenwtithsome fixatioanrtifactS.everalsmalllipiddropletsand residualbodiesarepresent.A smallsegment ofa pedsinusoidaflat-storincgellispresent atthelower righta,djacentto an endotheualcell.Sixteen(16)peroxisomesidentified. Z16250. Liver.Hepatocyte.S*everalsmalllipidropletsarepresentinthehepatocyte. Multipledark,and irregularresiduablodiesarealsopresent.The ER isslightldyilated throughout.Three (3)probableperoxisomescounted. Z 16251. Liver.Hepatocyte.There isnulddilatationfthenuclearenvelopeand the endoplasmicreticulumthroughout.Mitochondriahave a palermatrixand may be slightly enlarged.On theleftisthehepatocyte'ns-Licrovillbooursder,withan adjacentendothelial cellanderythrocytienthesingsoidA. bilecanaliculiuspresenitnthelowerright.Ten 1(10) peroxisomes counted. Conclusions: EssentiallnyormalhepatocytesS.ome minimaltoslighftixationartifacts. Average 10.8peroxisomes perhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:-6329-223(EM 99.76) Animal No.: 105537 TreatmentGroup: -.03mg/kg.(Iowd-ose) Sex: Female Species/StraiCny:nomolgus h4onkey Tissue: Liver BlockNo(s).: 99.76-19 Z 16252 - Photo/NegativNeo(s).:Z16256 SignificaLnetsion(scheckone):- Yes X No InterpretinPgathologis(tsignaturaend date): Features: Z 16252. Liver.Hepatocyte. Fixationappearsgood. Dorsaltothe hepatocyteisa centralveinor sinusoidarea.Severalmedium-sizedlipiddropletsare presentinthe cytoplasm.Fine glycogengranulesarescatteretdhroughoutthecytoplasnl A drsmosome ispresentatthelowerleftmarginwithan adjacenthepatocyte.Ten (10) peroxisomes counted. Z16253. Liver.Hepatocyte.Hepatocyteshows veryslighdtilatationf endoplasmic reticulum.There isone largelipidropletand severalsmalldropletswithinthe cytoplasm.A bilecanaliculuisspresentatthetoprightmarginof thecell.Five (5) peroxisomescounted. Z16254. Liver.Hepatocyte.ThisceU appearsessentialsliymilartoZ16253, although therearelessernumbers of lipiddropletsT.wo irreguladrark-staininrgesidualbodiesare presenton theleftand rightsideofthenucleus.The one on therightissomewhat vacuolated.Eleven (11)peroxisomescounted. Z 16255. Liver.Hepatocyte.A medium-sizedlipidropletcontaininsgome lamellar materialispresenttotherightof thenucleus.Justa few smallerlipiddropletsarepresent elsewherein thecytoplasm.The endoplasmicreticulumisslightldyilated.At thebottom isthemicrovilloubsorderextendingintothespaceof Disse.A few smallresidualbodies arepresent.Six (6)probableperoxisomescounted. ZI 6256. Liver.Hepatocyte.In thishepatocytetwo mitochondriappearpalerand somewhat swollen.The restappearnormal.Twelve (12)probableperoxisomescounted. Conclusions: Essentiallnyor7nalhepatocytesQ.uantityof lipidappearswithinnormal expectationsA.verage 8.8peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99-76) Animal No.: 105541 TreatmentGroup: .03mg/kg (low-dose) Sex: Female Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-20 Z 16257 - Photo/NegativNeo(s).: Z16261 SignificaLnetsion(scheckone):- Yes x No InterpretinPgathologis(tsignaturaend date): Features: Z 16257. Liver.Hepatocyte.Mcrovillarbordersarepresentalongthelower leftand upperrightmarginsof thishepatocyte.A bilecanaliculuisspresentatthelower right.Lamellarmaterialwithina lipidropletispresenttothelowerrightofthecentral nucleus.There isabundantSER throughoutthecefla,nd theRER appearsslightly dilated.Seven (7)probableperoxisomescounted. Z16258. Liver.Hepatocyte.Severalmedium-sizelipidropletsarepresent,as wellas approximatelyeightresidualbodies,inthecytoplasm.The lowerleftmargin isa microvilloubsorder,and a bilecanaliculuisspresentintherriiadnd upper rightmargins of thishepatocyte.Eight(8)peroxisomescounted. Z 16259. Liver.Hepatocyte.ThisceH issurroundedby severalotherhepatocytesa,nd containsapproximatelytenmedium-sizedlipidropletsinitscytoplasm.Severalvery darkround toovalresidualbodiesarepresentatthetopof thecell.Severaldark mitochondrianoted;theseareoftendifficutlotdifferentiaftreom lysosomesor peroxisomes.Fourteen(14)peroxisomescounted. Z 16260. Liver.Hepatocyte.Hepatocyteappearsessentiallsyimilartothosedescribed above.Two lipiddropletnsoted.Thereisslightdilatatiofntheendoplasmicreticulum and nuclearenvelope.Twenty-three(23)pcroxisomescounted. Z 16261. Liver.Hepatocyte.Cellcontainsapproximately5 smalltomedium-sizedlipid droplets.At thebottom isa sinusoidcontaininga plateleotrsegment of a granulocytein thecirculationA.nothersinusoidamlarginand microvulousborderispresentatthetop. There isslightdilatatioonfthe.endoplasniircetiuclum. Eight(8)peroxisomes counted. Conclusions: Essentiallnyormal hepatocytes.Some lipiddropletsnoted,but within normal expectationfsorhepatocytes.Average 12.0peroxisomesperhepatocyte. TRANSiNlISSION ELECTRON rvIlCROGRAPH INTERPRETATION StudyNo.: 6329-223 (E@@,76@ Animal No.: 105547 TreatmentGroup: 0.03melkg (low-dosel Sex: -Female Species/StraiCnv:nomoteus Monkey Tissue: Liver BlockNo(s).: 99.76-'21 Z16262 - Photo/NegativeNo(s).Z:16266 SignificaLnetsion(scheckone):- Yes X No InterpretinPgathologis(tsignaturaend date): Features: Z 16262. Liver.Hepatocyte. Photographshows a hepatocytewithjusta smallnuclearprofileA. microvirousborderispresentatthetopright.Some artifactual knifemarks arepresent.Severaldarkresiduablodiesarepresenta,nd onlyabout six smalllipiddropletsareevidentinthecytoplasm.Abundant SER and RER arethroughout thecytoplasmintermixedwithnumerous mitochondria.Four (4)peroxisomesidentified. Z 16263. Liver.Hepatocyte.Numerous collagenfibersarepresentalong thedorsal extracellulmaarrgin. At thelowerleftthishepatocyte'rsriicrovillbooursderextendsin thespaceof Disse. There isslighdtilatatioonf thenuclearenvelopeand endoplasmic reticulum.Severalresidualbodiesand lipiddropletsarepresent.Fineglycogengranules arerandomly scatteretdhroughoutthecytoplasm.Six (6)peroxisomesclearlyidentified. Z16264. Liver.Hepatocyte.Thereisslighdtilatatiofntheendoplasmicreticulumand nuclearenvelope.A bilecanaliculuisspresentatthedorsalrightmargin of thecell,and anotherhepatocyteispresentalongtheventralmargin. The generalmorphology is similartotheabove describedcells.Only one lipiddropletisevidentin thiscell.Several residualbodiesareevident.Fourteen(14)probableperoxisomesidentified. Z 16265. Liver.Hepatocyte. A centralveinand sinusoidsurroundthishepatocyteon thelower and upper leftmargins.Erythrocyteasrepresentinthevascularlumens. Within thehepatocytethereisslightdilatationf theendoplasmicreticulumand nuclear envelope.Two smalllipiddropletasrepresent.Nine (9)peroxisomescounted. Z 16266. Liver.Hepatocyte.Hepatocyteappearsessentialsliymilartocellsdescribed above.Thereisslighdtilatatiofntheendoplasrririecficuluamnd nuclearenvelope.A largeirregularly-shapreedsiduablody ispresenttothelowerleftofthenucleus. Seventeen(17)probableperoxisomescounted. Conclusions: Essentiallnyormal hepatocytes.Average 10.0peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-2 3 (EM 99.76)- Animal No.: 105550 TreatmentGroup: .03mg/katLow-dosel Sex: Female Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-22 Z16267 - Photo/NegativNeo(s).: Z 16272 SignificaLnetsions(checkone):_ Yes X No InterpretiPnagthologis(tsignaturaend date): Features:Z16267-Liver.HepatocyteA.bundantnumbersofglycogegnranuleasrepresent throughoutthecytoplasmofthishepatocyteB.ilecanaliculaireevidento,n therightl@eftand lowerleftmargins.A largedarkresiduablody ispresentin an adjacenthepatocyteatthetop left.Severalsmallerresiduablodiesarepresentin thecytoplasmof thecenteredhepatocyte. Mitochondriappearnormal,havingcristasep,oradicgranulesanda finegrainymatrix.Five(5) probableperoxisomescounted. Z16268. Liver.Hepatocyte.Cytoplasmhasa somewhat lightappearancedue toslightdilatation oftheendoplasmicreticulum.The nuclearenvelopeisalsoslightldyilatedT.hese areartifactual fixation-relactheadnges.Prominentbilecanaliculairepresenton theleftand lowerrightcellular margins.No lipidropletesvident.Fiftee(n15)peroxisomescounted. Z16269. Liver.Hepatocyte.CellappearssimilatroZ16267 describeadbove.There appearsto be abundantglycogengranulesinthecytosol.A microvilloubsorderispresentattheupper rightextendinginto thespaceof Disse,whilebilecanaliculaireevidentalongthelowerleftand upperleftmargins.There aremany darkmitochondriwahicharedifficutlotdifferentiaftreom peroxisomes.Twenty-seven(27)probableperoxisomescounted. Z16270- Liver.Hepatocyte.Thishepatocytaeppearstobe binucleateA.n endotheliaclellis presentliningthesinusoidventrallaynd two erythrocyteasrepresentdorsaltothiscell.The endoplasmicreticulumand nuclearenvelopeisslightldyilatedT.wenty (20)peroxisomesseen. zi 272. Liver.Hepatocyte.Two largeandmultiplsemallerlipidropletasrepresentinthis hepatocyte.Dorsally,themicrovilloubsorderextendsintothespaceofDisseand isadjacentto an endotheliaclell.Ventrallyarea smallsegmentofa lipid-containcienlglp.robablya perisinusoidfaalt-storicneglland an adjacenhtepatocyteT.wenty-one(21)probable peroxisomescouinted. Conclusions:EssentiallnyormalhepatocytesA.lthough onecell(Zl6272)had a lotoflipid dropletst,heoverallimpressionisthatlipicdontentinthisliverwas withinexpectednormal limitsA.verage 17.6peroxisomesperhepatocyte. TRANS1%,IISSIOENLECTRON NUCROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: 105510 TreatmentGroup: .15mvjkiz(mid-dose) Sex: Male -- Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-23 Z16273 - Photo/NegativNeo(s).: Z16277 SignificaIn-tesion(scheckone):- Yes X - No InterpretiPnagthologis(tsignaturaend date): , Features: Z 16273. Liver.Hepatocyte.Numerous glycogengranulesarepresent randomly throughouttheSER and cytosol.Fixationappearsgood. One swollen"barbellshaped"n-Litochondriisapresent,butallothermitochondriaappearnormal. Scattered dark residuablodiesarepresenl Thirteen(13)peroxisomescounted. Z16274. Liver.Hepatocyte.Severalsmalltomedium-sizedlipidropletsarepresentin thecytoplasmof thehepatocyte.Along itsrightmarginitsmicrovilloubsorderfaces anotherhepatocyte.A poorly-definebdilecanaliculuisspresentatthelower left.The cytoplasmisfillewdithSER, RER, mitochondriand glycogengranulesthroughout. Some peroxisomesarenoted.ScatteredarkresiduablodiesarepresentT.en (10) peroxisomescounted. Z162.7.5L.iver.Hepatocyte.Centeredhepatocytehasonlya smallnuclearprofile.At thebottom isa centralveinsurroundedby an endotheliaclelland a moderatenumber of extracellulcaorllagenfibers.An endotheliaclellisthelightesrhadedcellatthetopright. Three lipidropletspresentinthecytoplasm.Thirtten(13)peroxisomescounted. Z16276. Liver.Hepatocyte.This cellappearssimilatroZ16273. Approximately10 smalltomedium-sized lipiddropletasrepresentinthecytoplasm.Numerous glycogen granulesareevidentinthecytosol.A lamellarbody ispresentatthebottom margin of thecellF.ourteen(14)peroxisomescounted. Z16277. Liver.Hepatocyte.Approximatelya dozen smalltomedium-sizedlipid dropletsarepresentinthecytoplasm.The clusteorf 5 dropletastthefarrightarein anothercell.The cytoplasmcontainasbundantfineglycogengranulesS.everalresidual bodiesarenoted.Three (3)probableperoxisomescounted. Conclusions: EssentiallnyormalhepatocytesM.oderate amount of glycogenin several cells.Number of lipiddropletsappearsnormal.Average 10.6peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.:--- 105519 TreatmentGroup:-.15 mg/kg (mid-dose Sex: L4ale Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-24 Z16278 - Photo/NegativNeo(s).:-ZI6282- SignificanLtesions(checkone): Yes InterpretinPgathologis(tsignaturaend date): X - No Features:Z 16278. Liver.Hepatocyte.At thetoprightthishepatocytiesborderedby a perisinusoidaflat-storicnegllcontainingseveralprominentlipidroplets.Four smah lipiddropletsarepresentinthehepatocyte.A bilecanaliculusa,djacento a large residualbody,ispresenton theleftmarginof thecell.Thereisslighdtilatatioonf the nuclearenvelopeand endoplasmicreticulum.Multipleirregularresiduablodies-are evident.Thirteen(13)peroxisomesclearliydentified. Z 16279. Liver.Hepatocyte.The centeredhepatocyteisborderedby a darkerhepatocyte on theright.On itsleftisa band of interstitial'coflTahgenn.uclearenvelopeand endoplasmicreticulumareslightldyilated.Severalendocytoticvesicleasrepresentalong therightcellulamrembrane. Sixteen(16)probableperoxisomescounted. Z16280. Liver.Hepatocyte.Hepatocytehas prominentbilecanaliculailongitsright and leftmarginswith theadjacenthepatocytes.One smalllipiddropletnoted. Mitochondriastainsomewhat more paleinthiscelland contraswtithdarkerperoxisomes. MultipleclusterosfRER evident.Twelve (12)peroxisomescounted. ZI 628 1. Liver.Hepatocyte.Thisbepatocyteappearstobe binucleateA.t.thetopleftis an erythrocytewithinthecentravleinor sinusoid.Severalsmalllipidropletsarepresent inthecyfoplasnlThere isslighto moderatedilatatioonf theendoplasmicreticulumand nuclearenvelope.Some shrunkenmitochondriaaredifficulttodifferentiaftreom lysosomesor peroxisomes.Thirty-two(32)probableperoxisomescounted. Z16282. Liver.Hepatocyte.Generalcellulamrorphologyissiniilarcteolldescribed above. There isslightdilatatioonf theendoplasrnirceticulumand nuclearenvelope. Nineteen(19)peroxisomescounted. I- Conclusions: EssentiallnyormalhepatocytesO.nly smallnumbers oflipiddroplets seen.Average 18.4peroxisomesper4epatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: 105524 TreatmentGroup: .15mvjkg (mid-dose) Sex: Male Species/StraiCny:nomoleus Monkey Tissue: Liver BlockNo(s).: 99.76-25 Z16283 - Photo/NegatiNvoe(s).: Z 16287- SignificanLtesions(checkone)- X Yes No InterpretinPgathologis(tsignaturaend date): Features:Z16283. Liver.Hepatocyte.Hepatocytecontainstwo medium-sized lipid dropletsT.he nuclearenvelopeand endoplasniirceticulumareslightldyilated.At the toprightisa bilecanaliculubsetween thiscelland an adjacenthepatocyte.Desmosomes arepresentaround thecanaliculusT.hirty-one(31)peroxisomescounted. Z16284. Liver.Hepatocyte.Thiscenteredhepatocyteissurroundedby other hppatocyteson allm-argins inthisphotograph.Bilecanaliculaireevidentatthe topleft and bottom leftmargins.No lipiddropletasreevidentinthiscell.Mitochondria,RER and SER appearnormal. Twenty-nine(29)probableperoxisomescounted. Z16285. Liver.Hepatocyte.Two erythrocyteasrepresentinthesinusoidadjacenttothe leftof thishepatocyte.Collagenfiberasrepresentinthespaceof Disse.Along theright margin of thiscellisa prominentbilecanaliculu.sA singlemedium-sizedlipiddroplet ispresentinthiscell.Twenty-seven (27)peroxisomescounted. Z16286. Liver.Hepatocyte. Cellappearsessentialsliymilartocellsdescribedabove. At topleftand bottom rightareerythrocytewsithinsinusoidsN.o distincltipiddroplets arepresentinthiscell.There isslighdtilatationf theendoplasmicreticulumand nuclearenvelope.Severaldarkresiduablodiesarepresent.Twenty-one (21) peroxisomescounted. Z16287. Liver.Hepatocyte.Cellhas two largeand severalsmallerlipiddropletspresent initscytoplasm.There isslighdtilatationftheendoplasmicreticulumand nuclear envelope.Severaldark residualbodiesarepresent.Glycogen granulesarepresent throughoutthecytoplasm.Eight(8)peroxisomecsounted. Conclusions:EssentiallnyormalhepatocytesS.lightincreaseinaveragenumber of peroxisomes.No increaseinlipidnoted.Average 23.2peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:6329-223(EM 99.76) Animal No.: 105528 TreatmentGroup: .15mglkg (mid-dose) Sex: Male Species/StraiCnv:homoigus Monkey Tissue: Liver BlockNo(s).: 99.76-26 Z16288 - Photo/NegativNeo(s).: Z16292 SignificanLtesions(checkone): Yes X No InterpretinPgathologis(tsignaturaend date): Features: Z16288. Liver.Hepatocyte.This hepatocytecontainsaboutfivemediumsizedand approximatelyfifteesnmalllipidropletisnitscytoplasm.Microvillous borderbeneathendotheliacleus areevidentattheupperrightand lower leftmargins. A bilecanaliculusurroundedby desmosomes ispresentatthelowerrightmargin. Scatteredark-staininrgesiduablodiesarepresentinthecytoplasm.There isslight dilatatioonf theendoplasmicreticulumand thenuclearenvelope.Five (5)peroxisomes clearlyidentified. Z 16289. Liver.Hepatocyte.Thishepatocyte'msicmvillousborderextendsintothe spaceof Disseattheupper leftmargin ofthephotograph.At thelower leftisa perisinusoidfaalt-storicnegll(Itocell)T.here isslighdtilatatioofntheendoplasmic reticulumand thenuclearenvelope.Two small.lipdirdopletasreevident.Sixteen(16) peroxisomescounted. Z 16290. Liver.Hepatocyte.Thiscellisa littldearkerstainintghanpreviouscells.At lowerlefta granulocyteispresentinthesinusoidor centravlein.At thetop righta perisinusoidaflat-storicnegll(Itocell)ispresent.Severalsmalllipidropletsarepresent inthehepatocyteand a few residuablodiesareevidentT.here isslightdilatatioonf the endoplasmicreticulumand thenuclearenvelope.Nineteen(19)peroxisomescounted. Z 1629 1. Liver.Hepatocyte.Much ofthecytoplasmhasa densegranularappearance, composed of SER, glycogengranules,and probablecytoskeletaclomponents. Approximatelyeighteensmalltomedium-sizedlipiddropletasm present.Nineteen(19) probableperoxisomescounted. Z 16292. Liver.Hepatc>cyte.HepatocyteappearssimilartoZ 16291. There areseveral lipidropletspresentinthecytoplasm.Fourteenprobable(14)peroxisomescounted. Conclusions: Essentiallnyormal hepatocytesL.ipiddropletnumbers withinnormal expectations.Average 14.6peroxisomesperhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:6329-223(EM99-76)- Animal No.: 105532 TreatmentGroup: .15mglg (mid-dose) Sex: Female Species/StrainC:ynomolgus Monkey Tissue: Liver BlockNo(s).:- 99.76-27 Z16293 - Photo/NegativNeo(s).: Z 16297 SignificanLtesions(checkone): Yes X No InterpretinPgathologis(tsignaturaend date): Features:Z 16293. Liver.Hepatocyte.Thisphotographhasa centeredhepatocyte.At thetop aremultiplelipiddropletswhich areinan adjacentperisinusoidfaalt-storincgell (Itocell)n,otinthishepatocyte.Therearenumerous glycogengranulesinthecytosol intermixedwith theendoplasmicreticulum.Two verysmalllipiddropletasrepresenl Mitochondriaappearnormal. They show finecristaes,ome granulesand finelygrainy matrix.Four (4)peroxisomescl6arliydentified. Z 16294. Liver.Hepatocyte.A singlelargelipiddropletispresentinthecytoplasm beneaththenucleus.SimilartoZ16293, therearemany finegranulesinthecytoplasm which appeartobe glycogengranules.Three (3)peroxisomesidentifieidnthis hepatocyte. Z 16295.Liver.Hepatocyte.Severalmedium-sizedlipidropletsarepresentinthis hepatocyte.At theupper rightisa "P"-shapedextracellulaarreacontainingcollagen fibers.Moderate amounts ofglycogenarepresentinthecytoplasmof thehepatocyte. Severalresidualbodiesarealsopresent.Five (5)probableperoxisomescounted. Z16296. Liver.Hepatocyte.Centeredhepatocyteappearssimilartothosedescribed above. Three lipiddropletsareevidentinitscytoplasm.At thebottomisa longitudinal profileof a bilecanaliculusand a cross-sectioonfa canaliculuastthetopmargin. Fine glycogengranulesarepresentthroughout.Six (6)peroxisomescounted. Z 16297. Liver.Hepatocyte.. Centeredhepatocyteappearssimilartothosedescribed above. Two medium-sized and severalsmalllipidropletpsresent.To therighti.san areaof intersfifiucmontainingsome collagenfibers.The nucleusprobablybelongstoan endotheliaclell.Three (3)peroxisomescounted. . -I ConclusionEss:sentianlolrymahlepatocytMeosd.emteamountosfglycogperneseinnt most cells.Lipiddropletnumbers appearnormal.Average4.2peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:-6329-223(EM 99.76) Animal No.: 105538 TreatmentGroup:-.15mg/kg (mid-dose) Sex: Female Species/StraiCny:nomolgus Monkey Tissue-:Liver BlockNo(s).-: -99.76-28 Z16298 - Photo/NegativNeo(s).: Z16302 SignificanLtesions(checkone): X Yes No InterpretinPgathologis(tsignaturaend date): Features: Z16298. Liver.Hepatocyte.Sectionhasa prominentsingleknifemark transectintghecellbeneaththenucleus.Numerous glycogengranulesarepresentin the cytoplasm.A singlesmalllipiddropletispresent.Mitochondriaand otherorganelles appearnormal. Five (5)peroxisomescounted. Z16299. Liver.Hepatocyte.Hepatocytehas a singlelargeand severalsmalllipid droplets.A sinusoidispresentattheupperleftmarginwhere themicrovilloubsorder extendsintothespace ofDisse.There areabundantglycogengranulesinthecytoplasm. Some mitochondriaarelightesrtainintghanthemajorityofdarkerone. These light mitochondriaaresomewhat difficutlotdifferentiaftreom lysosomesor peroxisomes. Eighteen(18)probableperoxisomescounted. Z16300. Liver.Hepatocyte.Cellcontainsabundantglycogenofteninirregulacrlear spacesor pocketsin thecytosol.A singlelipidropletispresent.Three (3)peroxisomes identified. Z16301. Liver.Hepatocyte.Thiscenteredhepatocytehasadjacenthepatocytedorsally and ventrallaynd a microviuous-sinusoidbaolrderatthetopright.Itscytoplasmcontains multipleirregulaprocketsof glycogengranules.Otherorganelleasppearsomewhat clumped between theglycogenpockets.Nine (9)peroxisomescounted. Z 16302. Liver.Hepatocyte.ThiscellappearssimilartoZ 16301,althoughithas more pocketsof glycogengranulesand hasan overallightappeamce. Other organelles appearclumpedbetweentheglycogengranulesO.ne (1)peroxisomeclearliydentified. Conclusions:EssentiallnyormalhepatocytesT.here isincreasegdlycogeninsome of thesehepatocytes.Average 7.2peroxisomesper hepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:6329-223(EM 99.76)- Animal No.: 105545 TreatmentGroup: .15mg&g (mid-dose) Sex: Female Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-29 Z16303 - Photo/NegativNeo(s).: Z16307- SignificanLtesions(checkone): Yes X No InterpretinPgathologis(tsignaturaend date): Features: Z 16303. Liver.Hepatocyte.Thiscellisborderedby hepatocyteson thetop rightand bottomrightand by an interstitmiaarlginon theleft.Prominentbilecanaliculi areevidentbetween adjacenthepatocytes.There isslighdtilatatioonf theendoplasn-dc reticulumand nuclearenvelope.No lipiddropletsareevident.Severaldarkirregular residuablodes arepresent.Six (6)probableperoxisomescounted. Z 16304. Liver.Hepatocyte.Centeredhepatocyteissurroundedby hepatocyteson all margins.Bilecanaliculaireevidentattherightand bottommargins.Itsendoplasmic reticulumand nuclearenvelopeareslightldyilated.Severalresiduablodiesarepresent. No lipiddropletsarepresent.Fourteen(14)probableperoxisomecounted. Z16305. Liver.Hepatocyte.HepatocyteappearssimilatroZ16303 above. At topleftis a centralvein,linedby an endotheliaclelland containinga segmentof an erythrocyte. No lipiddropletsareevidentinthishepatocyte.Seven (7)peroxisomesclearlyidentified. Z16306. Liver.Hepatocyte.Thiscellissomewhat lightesrtainintghanprevious hepatocytesa,lthoughthemitochondriastilalrestaininfgairldyarkly.*Cellon thefar rightmay be an endotheliaclell.Twelve (12)probableperoxisomescounted. Z 16307. Liver.Hepatocyte. AllmitochondriainthiscellaredarklystainingT.here is slighdtilatationftheendoplasmicreticulumS.ix (6)peroxisomesidentified. Conclusions:EssentiallnyormalhepatocytesN.o evidenceofincreasedlipiddroplets. Average 9.0peroxisomesperhepatocyte. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION StudyNo.:-6329-223 (EM 99-76) Animal No.:-105548 TreatmentGroup: .15mg&g (mid-dose) Sex: Female Species/StrainC:ynor@olgus Monkey Tissue: Liver BlockNo(s).: 99.76-30 Z16308- Z16309 Photo/NegativeNo(s).:-Z16311Z163'13 SignificanLtesions(check one):-Yes X No InterpretinPgathologis(tsignattiamnd date): FeaturesZ:16308,LiverH.epatocytTeh.ecenterehdepatocyitseborderebdyadjacenhtepatocytoens itsleftandrightmargins.On therighitsa prominenbtilecanaliculuAs.microvillobuosrderisevidentat thetopright.Fixatioanppearsgood andcytoplasmiocrganelleasppearnormal.No lipidropletasre presentinthecytoplasm.AbundantSER ispresenitnthecytoplasm.Therearea number ofshrunkendark, variably-sizmeidtochondriwah,ich aredifficutlotclearldyifferentifartoempossiblleysosomesor peroxisomes.One largelysosomeispresenattabout"4:40o'idlockf"romthenucleusa,nd containssome lineadrebrisT.wenty-nine(29)peroxisomeisdentified. Zi6309.Liver.Hepatocyte.Thishepatocyteexhibitsslighdtilatatioofntheendoplasmimcticulumand nucleaernvelope.Threesman tomedium-sizeldipidropletasrepresenlAn erythrocytiesevidentina sinusoiadtthebottomleftofthephotograph.Random darkand irregulraersiduablodiesarepresent. Seventeen(17)peroxisomesclearliydentified. Z 16310.Liver.Hepatocyte.Crystalloidnclusioinnssome mitochondrian. occasionaflindingin mitochondriaP.eroxisomesnotcountedinthishepatocyte. Z 16311.Liver.Hepatocyte.One largeandseveraslmallerlipidropletasrepresenitnthecytoplasm. Thereisslighdtilatatioofntheendoplasmicmticulumand nucleaernvelope.Prominentbilecanaliculaire presenotn thelefatnd rightmarginswithadjacenhtepatocytesS.ixtee(n16)probableperoxisomecounted. Z16312. Liver.Hepatocyte.Thishepatocytiesborderedby adjacenhtepatocytoen allmargins.Several smalllipidropletasrepresentinthecytoplasm.Tnereisslighdtilatatioofnthenucleaernvelopeand endoplasmicreticulumN.ine (9)probablpeeroxisomecsounted. Z 16313,Liver.Hepatocyte.Two medium-sizeadndseveraslmallelripidropletasreevidentinthe cytoplasm.At thebottomlefotfthephotographaresome interstitcioallagefnibersA. bilecanaliculuiss presenattthetoprightmarginofthecell.Thereisslighdtilatatioofntheendoplasmicreticulume,specially theRER, and thenuclearenvelope.A largelysosomeispresentincloseproximitytothenotedbile I canaliculuistc,ontainsome electron-denmsaeterialF.ourteen(14)peroxisomecsounted. Conclusions:Essentiallnyomud hepatocytesN.o substantiianlcreasienlipidropletnsoted.Average 17.0peroxisomesperhepatocyte. TRANSNUSSION ELECTRON NECROGRAPH rinRPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: 105507 TreatmentGroup:0.75 mg/kg (hi-dosel Sex: Male Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-943411- Photo/NegativNeo(s).: 43415 SignificanLtesions(checkone): X Yes No InterpretinPgathologis(tsignaturaend date): Features: 43411. Liver.Hepatocytecontaininga singleverylargeclearlipiddroplet and many smalltomedium-size clearlipiddropletsT.here appearstobe an increasein theamount of glycogena-rosettgeranules.Severalfociof ER appearirregularldyilated. A few scattereldysosomesand peroxisomesnoted. Nine (9)peroxisomescounted. 43412: Liver.Hepatocytecontainsalargenumber of smalltomedium-sizedclearlipid dropletsinthecytosol(approximatel1y25),most beingverysmall.Abundant glycogen as (x-rosettiesspresentinthecytoplasm.Twenty-six(26)peroxisomescounted. 43413: Liver.Hepatocytecontaininsgeveralverylargeclearlipidropletsand many smallerdroplets.Some interstittiiaslsuecontainingsome collagenfibersispresentatthe lowerleftof thephotograph. Some residuablodiespresent.Many glycogenrosettes presentincytoplasm.Three (3)peroxisomescounted. 43414: Liver.Severalmitochondriaareshown which appearessentiallnyormal. Some normal dense granulesarepresentinthematrixand thecdstaeand envelopingmembrane appearnormal.In thecytoplasmtherearemultiplesmalllipiddropletsu,sually surroundedby finedense granules(glycogenor ribosomes). 43415: Liver. Hepatopytewitha bilecanaliculuastthebottommargin and an interstitial fibroblasticcellattheupper right.The hepatocytecontainsabundantsmallto mediumsizedclearlipiddroplets(>170).Random residuablodiesarepresent.Six (6) peroxisomescounted. Conclusions: Lipidaccumulation,moderately-severhee,patocellularI.ncreased glycogen.Average 11.0peroxisomesperhepatocyte. TRANSINUSSION ELECTRON WCROGRAPH INTERPRETATION StudyNo.: 6329-223(EM 99.76) Animal No.: 105534 TreatmentGroup: .75mgA& (hi-dose) Sex: --Female Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-11 L4798 - Photo/NegativNeo(s).:L4802 SignificanLtesions(checkone): X Yes No InterpretiPnagthologis(tsignaturaend date): Features:IA798: Liver.Hepatocytesurroundedby adjacenhtepatocytes.Itcontains abundantnumbers of mitochondriaand ER. There israildilatationf thenuclear envelopeand of some ER. Scattererdesidualbodiesarepresent.Justone distincltipid dropletisevident.Four (4)peroxisomescounted. L4799: Liver.Enlargedview of fivemitochondria.Nfitochondriaappea@essentially normal. Severaldense granulesarepresentinthematrix.The cristaaend limiting membranes appearnormal. At therightsideisthemildlydilatednuclearenvelopeand the nucleus. LA800: Liver.Hepatocyte.A few smallclearlipiddropletasrcpresentinthecytoplasm There arealsoabundant glycogengranulesinthecytoplasm.A number of "clearareas" in thecytoplasmappeartocontainglycogengranuleswithouta distincltipiddroplet margin.Two (2)peroxisomescounted. LA801. Liver.Hepatocyte.Therearenumerous smalltomedium-sizedrarefied 44 pockets"inthecytoplasm(approximatel5y7).Some containsome wispy lameuar materiala,nd some glycogen granulesappeartobe inthesespaces.The margins ofthese dropletsarenotas sharplydefinedasclearround lipidropletussuallyare.Four (4) peroxisomescounted. L4802: Liver.HepatocyteappearssimilartoIA801 withsome smalltomedium-sized lipiddropletsand otherrarefieadreaswithglycogeninthecytoplasm(approximately29). Some containsome wispy laineuarmaterialT.here arealsoabundantglycogen granules throughoutthecytoplasm.Twelve (12)peroxisomecsounted. Conclusions:Increasedglycogeninthisanimal.Increasednumber of glycogengranules. Average 5.5peroxisomes perhepatocyte. TRANSrvilSSION ELECTRON ARCROGRAPH INTERPRETATION StudyNo.: 6329-223 (EM 99.76) Animal No.: 105540 TreatmentGroup: .75moai (M-dose) Sex: Female Species/StraiCny:nomolgus Monkey Tissue: Liver BlockNo(s).: 99.76-13 L4809 - Photo/NegativNeo(s).: LA813 SignificanLtesions(checkone): X Yes No InterpretinPgathologis(tsignaturaend date): Features: L4809: Liver.Hepatocyte.Only a smau segmentof nucleusispresentin this photograph.Abundant ER and glycogengranulesarepresent.There arescatteresdmall tolargelipidropletsb,ut nottoomany inthishepatocyte(approximatel1y5).A few -darkresidualbodiesareevident.Fifteen(15)pemxisomes counted. LA810: Liver.Enlargedphotographof severalmitochondria.No abnormalitiensoted. Cytoplasm containssome ER and scatteregdlycogen granules. L481 1: Liver.Hepatocytecontainsmultiplemedium tolargeclearlipiddroplets (approximatel4y1). There isabundantglycogeninthecytoplasm.Five(5)peroxisomes counted. L4812: Liver.Hepatocyte.SimilartoL481 1. Hepatocytecontainsmultiplesmallto largeclearlipiddropletst,oonumerous tocount.There isabundantglycogeninthe cytoplasm.Twenty-eight(28)peroxisomescounted. LA813: Liver.Hepatocytecontainsabundantmitochondriand ER. Only a few clear lipidropletsarepresent.An endotheliacleH ispresentatthetopof thephotograph. There isminimal to mild dilatationftheER and nuclearenvelope.Scatteredresidual bodiesarepresent.Two (2) peroxisomesclearliydentified. Conclusions:NEld tomoderateincreaseincytoplasmiclipidropletsI.ncreased .glycogeninmost hepatocytes.Average 12.5peroxisomesperhepatocyte. TRANSIVHSSION ELECTRON NUCROGRAPH INTERPRETATION StudyNo.:6329-223(EM'99.76) Animal No.: 105551 TreatmentGroup: .75m /"k (hi-dose) Sex: Female Species/StraiCny:nomolggs Monkey Tissue: Liver BlockNo(s).: 99.76-14 LA814 - Photo/NegativNeo(s).: 'LA818 SignificanLtesions(checkone): X Yes No InterpretinPgathologis(tsignaturaend date): Features: LA814. Liver.Hepatocyte.Thereisa dilatecdanaliculusatthetop rightof thehepatocyte.In thecytoplasmtherearemultiplerarefieadreasthatmostlyappear to containglycogengranules.However, a few have some lamellarmaterialin them. There isslightdilatatioonf thenuclearenvelopeand ER. Some residualbodiesarepresenl No distincltipiddropletsarepresentinthishepatocyte,however,thereareseverallipid dropletsintheperisinusoidfatllt-storcienUg(Itocell)atthetopleft.Nine (9) peroxisomescounted. IA815: Liver.Enlargementof a clusteorf niitochondriaT.hese organenesappear essentiallnyormal. LA816: Liver.Hepatocytecontainas singlemedium-sizedclearlipidroplet.The cytoplasm containsabundant glycogengranules.Severalresiduablodiespresent.Four (4)peroxisomesclearlyidentifiedS.everalsmalldarkbodiesappeartobe tipsof mitochondria. LA817: Liver.HepatocyteappearssimilartolA814. Thereisabundantglycogenin the cytoplasm,oftenmaking clearareascontaininjgustglycogengranules.Severalclusters of RER present.One smalllipiddropletpresentF.ourteen(14)peroxisomescounted. LA818: Liver.Hepatocytecontainsmultiplrearefie"dpockets"ofglycogengranules.No lipiddropletspresent.Eleven(11)peroxisomescounted. Conclusions: Increasedglycogen.Average9.5peroxisomesperhepatocyte. III.TransmissionEectron NcrograPhs Figure Legends DraftAncillaryPathologyReport Covance Study No. 6329-223 Magnification. Print ma-n0ification is indicated on the back of each photograph. The measurement bar on each photomicrograph equalsone rnicron(0.001mm). -Fi-gur1e. Liver. Hepatocyte showing normal cytoplasmicorganelles.Some peroxisomes are identifieadnd other normal organe.Ues.Ventrallythemicrovillousborder extends into theperisinusoidaslpace. Animal No. 105517,0 mg/kgtday (controlmale), 10,920X magnification. Figure 2. Liver. Hepatocyte. Severallipiddropletsarepresenl There isslight artifactuadlilatatioonf some rough endoplasmic reticulum.Some extracellulacrouagen ispresentin the interstitiuimn theupper rightcomer. An endothelw cellnucleus is presentliningthesinusoidin thelower rightcomer of thephotograph. Animal No. 105544, 0 mg/kglday (controlfemale),11,20OX magnification. Figure 3. Liver. Hepatocyte. Mtochondria showing outermembrane, cristaem,atrix and granules. These granuleswithin themitochondrialmatrixarenormal and are sitesof calcium accumulation. Animal No. 105519, 0 mg/kg/day (controlmale), 112,OOOX magnification. Fiszure4. Liver. Hepatocyte showing increasedlipiddropletaccumulation. Lipid dropletsvary from small to very large.Severalresidualbodies arepresent. Peroxisomes are scare. Animal No. 105507, 0.75 mg&g/day (high-dosemale), 10,920X magnification. Figure 5. Liver. Hepatocyte showing numerous lipiddropletsand peroxisomes. Several peroxisomes are labeled.Animal No.105512,0.75 mg/kg/day (high-dosemale), 10,920X magnification. Figure 6. Liver. Hepatocyte with many smallto largelipiddroplets.Representative peroxisomes arelabeled. Desmosomes and a bilecanaliculusisevidentat the righl Animal No.105536, 0.75 mg/kg/day (high-dosefemale),I1,20OX magnification. Figure 7. Liver. Hepatocyte with multiplelipiddropletsand abundant glycogen granules in thecytoplasm. Animal No. 105540,0.75 mg&g/day (high-dosefemale),11,20OX magnificafion. Figure 8. Liver. Hepatocyte containingnumerous "pockets"of glycogen. Animal No. 105551.0.75 mg/kglday (high-dosefemale),11,20OX magnificadon. DraftAncillarypathologyReport Covance Study No. 6329-223 Figure 9. Liver. Hepatocyte. NUtochondria showing outermembrane, cristaeand matrix. Small lipiddropletsare presentin thesurroundingcytosol.Animal No. 105512, 0.75 mg/kg/day (high-dosemale), 112,OOOX magnification. Figure 10. Liver. Hepatocyte. Mitochondria showing outermembrane, cristae,matrix, and granules.The nucleus ispresenton theright.Animal No. 105534, 0.75 mg/kg/day (high-dosefemale),95,20OX magnification. Figure 11. Liver. Hepatocyte. There ismoderate accumulationof small to mediumsizedlipiddropletsin thishepatocyte.Only a small nuclearprofileisevident in thiscell. The endoplasmic reticulumisslightldyilated.Animal No. 105514,0.03 mg/kg/clay(lowdose male), 11,20OX magnification. Figure 12. Liver. Hepatocyte. Mtochondria, endoplasmic reticulumand some residual bodies make up the majorityof thishepatocyte'scytoplasm. A few small-dark mitochondria arepresent,but no clearlyidentifiablpeemxisomes arepresent.No lipid dropletsarepresentin thiscell,although a singlelipiddropletispresentin the subjacent cellatthebottom of thephotograph. Animal No. 105545, 0.15 mgtkg/day (mid-dose female),11,20OX magnification DraftAncillaryPathologyReport Covance Study No. 6329-223 Abbreviation BC co CR D EC G GR LD m mv mx N om p RB RER SER Index of Labels for Ultrastructural Structures Structure Bilecanaliculus CoBagen Cristae Desmosome Endothelialcell Glycogen Granules Lipiddroplet Mtochondria Mcrovilli Matrix Nucleus Outer membrane Peroxisome Residual body Rough endoplasaiicreticulum Smooth endoplasmic reticulum 4A riin"rll If ik q% Id ir 7e. .0; LD rl 7p-- IeF 14" LD m CoSH: FANNER REEE ET cons ARREST PIERRE | . Ele ERS ER EEE FoEr Af R TOAr RR) AHkE Eoai VEE Na0 n NNV E neE d Ca ET RRR Ejo R Fe eC IgLE Lons FE er SR SL Foe It LZ PR 47 JHn BY cr BF oRePRs eah, ] FAS po? FESSETR i: g5a240d9 Co3s REE, 2 feng nic e pk e eR - = 58 Pao CE R Beeer aEgR e B TARR ho ee JAE (EHRR ANE b er eLIRA RS AaAR CT PR RL rre aT:y ee RCEHR Je 4 ed } H EE aR r LN AR Ja i AEE eE eORE E WRENRREGY ,. TEiRo he TUES BE BEY BDAE S a WGER RT BERET PE ay Sg St Con Dl FES, erp e ri akiia2E ST% BMoRk T "OERS Re {Raha Gr DARE" E ERNR, SW ERRE E ps i Ea SE PRR . " Qa a NET CER sg 2 a BE } RNRa3 il S PRRR MAEGR. 4e i fy d Seo aR ,o aEPNaRWELgE 3 2 AEE Aet . SE eR | = E 5 I EEE MReenR,UWE HCToSo T os TER I PyG RB Si || R BgElEdtT r. ore Bp ge ."46 LD LD LD 4t Y, i re 0 Al -,jp W. Vol c. UL LD ZP '4N IL LD PSI. It !LW., - LD .Flo Ile' AIW- 7 . tv. AL" Lt.. Ir z@e ir tp G 1% . "tit LD LD 4L A)A It -1 Ore. .4L t 4 JA"Z@ No,m 4r *Vold& 4L LD . b :6jL, 4k' 41 Ito OL Vb 46 IL LD ,Z7 jr: Tire Pop* LD ti -@a7, P"AV EC RPFE!.P'lP, .7f H:l LD IV. QualityAssumnce Statement pathologAyssocitaesinetmational A Company ofScienceApplicationsintemadonalCorpora6on An&rVoyft6o~ CAVMPV QUALITYASSURANCE STATEMENT ThiselectrmoincroscosptyudyhasbeeninspectaenddauditebdytheQuality AssurancUenitasrequirbeydtheGood LaboratoPrryacticReesgulatiopnrsomulgated bytheU.S.FoodandDrugAdministratPiAoInh.asa functionainndgresponsive QualitAyssurancUenitwhichreportdsirecttloymanagementT.hefollowiinsga record ofinspectonstauadnidttsheirresultirnegports: Date-of lns2ection Phaselns2ected DateFindingRsel2orted toManaaement andStudyDirector 4/1,2/99 4/2/99 7/13/99 7/13/99 DataAudit DrafRteporAtudit AudiotfDatafromAdditionGarlo.ups FinaRleporAtudit 4/2190 412/99 7/13/99 7/13199 @nedeward,B.S.R,OAP-GLP e uallAistsyurancSepecialist b7a(e Sponsor3:M CorporatTeoxicology TestArticlPeF:OS StudyNumber-6329-223P,AIStudyEM-99.76 4915D ProspectbursivoeDurhamN,ortCharolin2a7713(9195)44-5257(9195)44-321F8AX