Document JNYQDoZ9bNYx5Q42B2qQ6b7yX

B=E=f= ~~ Pathology Associates International 4ComofScpiencaeApplnicatyions Intemational Coporaton =r -pape-- AnEmplOwoedyComepae ny SECOND DRAFT CELL PROLIFERATION REPORT 104-WEEK DIETARY CHRONIC TOXICITY AND CARCINOGENICITY STUDY WITH PERFLUOROOCTANE SULFONIC ACID POTASSIUM SALT (PFOS;T-6295) IN RATS COVANCE STUDY NUMBER 6329-183 PREPARED FOR: 3M TOXICOLOGY SERVICES BUILDING 220-2E-02, 3M CENTER ST. PAUL, MN 55144-1000 PREPARED BY: PATHOLOGY ASSOCIATES INTERNATIONAL * 15 WORMAN'S MILL COURT, SUITE I FREDERICK, MD 21701 AUGUST 24, 1999 Worman's Mill Court, Suite 1 + Frederick, Maryland 21701 + (01) 663-1644 + (01) 663.8994 FAX 002375 - TABLE OF CONTENTS Contents Narrative Tables Figures Signature Page Quality Assurance Statement Appendix Seco`nCdovDarnacfet SCetluldyPrNoluimfebrearti6on32R9e-p1o8r3t Section I on m v v vi Page 12 002376 "SeconCdovDarnacke CStluldyPrNoluimfebreartio6n32R9e-p1o8r3t CELL PROLIFERATION REPORT 1W0I4-TWHEEPKERDFILEUTOARRYOOCCHTRAONNEICSUTLOFXOINCIICTYACAINDDPCOATRACSISNIOUGMESNAILCTIT(YPFSOTS;UTD-Y COVANCE S62T9U5)DYINNRUAMTBSER 6329-183 PURPOSE Twhheenpurpaodsmieniosfttehredsitnutdhyewdiaest ttooraastssefsosrthae cleharsotni1c04owxeiceiktsy.and carcinogenicity of the test materia `reTnehtpiirstelseredenpto"sr1t0t,h4es-uWcbeelmeliktptreoDldiiefbteayrraytPiaoCtnhhrfooilnnodigicyngATsosxsaionccdiitaiytnetasernpIdrnettCeaamtraictoiinnoofngoarelnCi(ocPivAtaIyn)cSettouSdtthyuedwysittNuhduymPbeSrpelorunos6ro3or2o,0c-i31aM8n,3e SPAuAglTef'noscnyipcoGrAotcioioddn PLooaftbatohsriasstiousrmtyuSdPayrlatcwt(eiPrcFeeO(ScG;oLnTdP-u)6c2tRe9ed5g)uliinanticRooanmtspsl"a.isansAceltelfawosripttehhctitnshTeoiftEltnehve4i0rtoaosnfkmsetnhatesasUlocSiParCtoeotdedcewtiitoohnf FaepdpleircaalblReegaumleatnidomnesn,tPsarwti7t9h2,theisfsouleldoNwoinvgemexbceerpt2i9o,n.198A3n(eexfpfiecrteidvereDaegceentmbwears 2u9s,ed19i8n3)t,haenBdradnUy iixmmppm.aucn4to/h9t/ih9se9t)so.cciheneTtmhiifeiscisrvtayaliinsditinatgiynwionafgstphjeruosdctgeueddduy.rteo (bmeosuasteisfIagcGt,oryV;ectthoerr,efloorte,#thPiKs-G40L0P2,dePviAaItioNno.doKes35n1o,t MATERIALS AND METHODS Praliferating C igen (PCNA) Sta Tissue Collection for Cell Proliferation Fhgeirpvoaeutpoacn(eilgmlrauolluasprspper1ro-l5is)feexwraaptsieornf.igxreAodupsineciztngiinorcnouoffoprstmhael1ilnte,hfrploarutogecrheals5sleowdbe,eraeonfsdtahceremiflbiievcederddefadrtowtmoeeepakacsrhaf4offiann5bdlroa1cs4k pfbeoyrr Csdteoatvieancintnicgoe.n poFefrrporpomrloietfaeocrcahotlibnlsgopccekce,ilfaincuasctlliiedoeansrw.aanstTipigrseenspu(aePreCbdNloAfc)okr,sHawn&eerEnedeosvghaelinupoaputeisdomntaoarnkPdeArIiomfmfucoernlolshpeircsottliioofcnehirenagtmiioacnan.ld Immunohistochemistry for Cell Proliferation S(Seucpteirofnrsoostf pPalursa,ffFiins-heemrbeSdcdieedntitfiiscs,uePsitwtesrbeurcguht, aPtA5)ytmo aenndsurpelacaeddheosnionposdiutrivienlgy pcrhoacregsesdinsglifdeosr PStCaNndAa.rd SOptearnadtairndgiPmrmoucneodhuirsetofcohremIimcmaulnomheitshtoocdhsemwiecraeluSsteadintiongst(aiSnOPtis#s7u0e7s)]f.or PBrCieNfAly,[tPiAsIs'use saKencd3t2ir7oe)nasgmewenettrhseordeiqnfucoiurrbteahdteefddoerwtiettchhetiaaovnmiodonifont-chbleiooantnaitnlipgaeennrt-oiaxbniotddiaybsoteody(PAcCoBNmCpAlKe(ixtD.;AVKPeOcCt,NorlA,otel#xo0tp1r#6ePs;KsPi-oA6n1I0iN0no;c.ePllAAsI1i8nN9o9a,l) hp(ehDmaAasBte;osxoySfliitgnh.meacelClhceymcilcea(lG,,CoS.,,G,loatnd#M18)H8w2a0s1)l.ocalizTeidssbuye thSeeccthironosmawgeerne3,c3o"udnitaemrsitnaoibneendz.idwiinteh. Page 1:3 002377 = SecoCnodvDarnafcteCSetuldPyroNluifmebreatrio6n32R9e-p1o8r3t Cell Proliferation Measurements T2h0e00pehrecpeanttoacgyeteosfinpratlfleeaasti6ngfiecledlslospfrolliivefre.raItnimnogsitndceaxs,es,PIa)t wlaeasstd3e0t0e0rmhienpeadtobcyytsecsorwienrgeastcoeraesdt ians 1m0anfiyelcdesl,lsb.utSilnidfeosurwearneimfailrssttpheerruesweadsatinlsouwffimcaigennitftiicsastuieopnr(es1e0n0tXo)nttohjeusdlgiedeqtuoalbietyabolfesttoaisncionrge, epxrtorcaehsespiantgocealnldulasrecptrioolniifnerga,tioann,dsupcahttaesm Koufpffceelrl cellalbse,libnigle (deu.gc.t, epcietnhterliiluomb,ulaerndootrheplainulmo)b,ulaanrd, ehaicshtomaonripmhaolloegviaclucahtaendgefso.r Tcehlel lpartotleirfweraastifounr.theCreallsseprsosleidfebryatieovnalwuaatsingthtehne sqeurainatlifHi&edEatslihdieghfeorr `magnification (200X) as described above. A#Cn9e3g0a0ti8vethcaotnwtraoslnsoltidienwcaubsaitnecdlwuidtehdtihnetpherismtaarinyiangntriubnodayn.d consisted of study tissue from animal Statistical Analysis TbhetewSeteundecnotn'tsrotleasntd(ttrweoa-tsmiednetd,gruonuepqsuaulsivnagriMainccreo)swoafts Euxsceedltvoetressitofnor5.s0t.atisAticPalvsailguneifoifca0n.c0e5inwaPsI judged tobe statistically significant. dine (BrdU) Staini d Evaluati Tissue Collection for Cell Proliferation Ssuervgeicnaldlayysibmepfloarnetetdheiwneievke52aniinmtaelrsimpsearcrisfeixce,peorsmgortoiucppuinmpgsrocownstai|nianngd205 mfgo/rmhlepBartodcUellwuelraer oprnollyi)ferwataison.taAkseenctaitonnoefctrhopeslyivearnadndedmuboeddedneudmtforopmareaafcfhinofb$lorcaktsbpyer Cgorvoaupnc(egrpoeurpsp1roatnodcoSl sslpiedceifiwcaatsionpsr.epTairsesduefobrlocHk&s Ewereevaslhuiaptpieodn toaPndAIifmomrusneochtiisotnioncgheamnidcsatlaindientge.ctFiornomofcacBhrdblUo,ck,ana exogenous markerofcell proliferation. Immunohistochemistry for Cell Proliferation S(Seucpteirofnrsoosft pPalursa,ffFiins-heemrbeSdcdieendtitfiiscs,uePsitwiesrbeurcguht, aPtA5)utmo aennsdurpelacaeddheosnionposdiutriivnegly pcrhoacregsesdinsglifdoesr rStaandardSOapnedraactidngmiPmraonceodhuirsetofcohreIimcmaulnomheitstoodcshewmeircaeluSsteadinfiongs(aiSnOPti#s7su0e7s)].for BBriredflUy,(tPiAssLuSe sNeoc.tiAon2s0w8e9r)eainndcurbeaatgeedntwsitrheqauimroendocflorontahle aanvtiidbiond-byitootiBnrpdeUro(xBiedcatsoen(DAicBkCenKsiotn;,Veloctto#r3,16l0o4t;#PPKA-L 6in1c0o0r;porPaAtIionNoi.ntoK3ce5l1ls) imneSthopdhasfeorofthethedetceelclticoyncloef wtahes alnotciagliezne-dantbiybotdhye ccohmrpolmexa.gen B3r,3d'Ucdoiuanmtienrosbteaniznieddiwnieth(hDeAmBa;toxSyliignm.a Chemical Co., lot #108H8210). Tissue sections were Cell Proliferation Measurements Thehpeatpoecrycteenstaigne1o0ffBiredlUds-loafbelilveedr.celSllsid(elsabweleirneg ifinrdsetxp,eLrIu)sewdasatdleotewrmmaignneidfibcyatsicoonri(n1g0a0tX)leatsotj2u0d0g0e pquaanlliotbyuloafr,steaxitnrianhge,paptroocceelsluslianrg aprnodlifseercattiioonni,ng,Supcahtteasm of cell labeling Kupffer cells, (e.g., bile centrilobular or duct epithelium, endothelium), and histomorphologic changes. The latier was futher assessed by evaluating the Page 4 002378 "SeconCdovDarnacfet CSetluldyPrNoluimfebreartio6n32R9e-p1o8r3t "hiHgAhEerslmiadgenfiofriceaatcihonan(i2m0a0lXe)vaalsudaetsecdrifobredceallbopvreo.liferation. Cell proliferation was then quantified at #ACn9e2g5at3i4vethcaotnwtraoslnsoltidienwcaubsaitnecdlwuidtehd tihnetpherismtaarinyianngtriubnoadyn.d consisted of study tissue from animal Statistical Analysis `bTehtewSeteundecnotn'trsolteasntd (ttrweoa-tsmiednetd,gruonuepqsuaulsivnagriMainccreo)swofats Euxsceedltvoetressitofnor5.s0t.atisAticPal vsailguneifoifca0n.c0e5inwaLsI judgedtobe statistically significant. RESULTS PCNA Cell Proliferation I1n-d4)ivaindudaglraanpihimcaalllayndingrSoecutpimoneIaTnT(PFiCgNurAescel1l apnrodli2)f.eraTthieonndeagtaatiavree cpornetsreonltteidssiune SweactsiojnudIgIe(dTatbolbees fhreepeatforcoymtesba(cPIk)grinoutnredatsetdaianniinmga.lsAdtid4noatnddif1fe4rwseigenkisficoanntlsytufdryo,m ctohnetrpoelrcaennitmaagles foofr epriotlhiefresreaxt.ing BrdU Cell Proliferation aInnddivgirdaupahlicaanlilmyailnaSnedctgiroonuIpNm(eFainguBrred3U).celTlhperonleifgeartaitvieoncodnattraolarteipssrueesewnatesdjiundSgeecdtitoonbIeI f(rTeaeblfero5m) dbiafcfkergrsiogunnidfisctaanitnliyngf.roTmhecopnetrroclenatnaigmealosffloarbeeiltehderhespeaxtoacty5t2eswe(LeIk)sionntsretautdeyd. animal groups did not Histopathology SSteacitnieodnswfirtohm htehmeastaomxeyltiinssuaenbdloecokssiunse(dHf&oEr)prefpoarrathiiosntoopfatPhColNoAgi-canedvaBlrudatUi-osntaiton_efdacsilliidaestewetrhee pirnteesrepnrteetadtiionnAopfpetnhediixmmI.unostained slides. Individual animal findings and group summaries are `rTathse,rwehsiulcths wsohuolwdedalttehratthneoinstiegrnpirfeitcaatnitoncohfantgheesPwCeNreAoobrseBrrvdeUd sitnatinhiengliivnerthfirsosmtumday.le and female DISCUSSION lIinvtehreofprmeaselnetasntdudfye,maelle rpartosliffreoramticoonntwraols (mGeraosuupce1d),byloPwCdNosAe i(mGmruonuopbi2)s,tamcihdemdiosstey(wGirtoiunp 3t)h,e `mid-high dose (Group 4), and BrdU immunohistochemistry ihnigthhedloisveer(fGrroomupco3n)trgorlou(pGsroaufpte41r) aanndd 1h4igwheedkosseon(Gsrtouudpy, Sa)ndmablye caenldlsfeamta5l2e rwateseakfsterw5a2swebeaksesdononsttuhdey.iTnchreeadseecdissieonnsittoivuistye BofrdcUonltaibneuloinugs tloabdeeltiencgt pwriotlihfeBrartdinUg `caodmmpianirsetderteodPviCaNoAsmiontidectpecutmipngcaopnrtoilniufoeursatliyvfeor7erspdoanysse. in the liverofrats. In this case, BrdU was iAndsetaxti(sPtIi)caallty4 saignndif1i4cawneteiknsc,reoarsebyinthceelBlrpdrUolilfaebrealtiinogn,inadsexdeatte5r2miwneeedksb,y wtahes PnoCtNoAbsperrovleidferianttihneg plirvoelrifeorfatmiaveleresorponfseematloethreattse.st maAtletrhiaolu,gbhiosltoagtiicstailcasliganniafliycsainsce dwiads envoitdernetveianlinadivsiigdnuiaflicaannitmaclesl.l Biological significance for an individual animal is defined as a doubling in PI or LI over controls Pages 002379 TT SecoCnodvDanrcae SCetluldyPrNouimfbresrio6n32R9e-p1o8r3t ainncdregarseeatoevretrhacnontthreolhsi)ghaenstd cionnctirdoelncvealu(en.umbTehruso,ftahenidmaatlasasrehopwriensegntbeidoliongitcearlmssigonfifsiecvaenrciet)y.(foAlsd sheiegnh dinosTeexatftTerab4lewe1e,kbsioolnogisctauldlyy asingdniifnic1a/n5t cmeallleprroaltsifaetratthieonmiwda-shiegvhiddeonsteinan2d/5mmiadledorsaetsafatterth4e wweaesklsesosntshtaundyt.he cTohnitsroalppPaIreantt14anwdemeikns,imaalndprwolaisfernaottivesursetsaipnoendseasseeevnidinenmcaeldebryatsthaetl4acwkeoefksa r`weesepkosn.seThaetrewfeoerke,1t4h.e pFruorlitfheerramtiovree,rensopnonesoefs steheenaninimtahlesseeixnhdiibviitdeudaal mparolleifaenriatmiavlesrweasspoanspeosastib5l2e `mild response evident only at4 weeks. Text Table 1. Biologically Significant Cell Proliferation in Liver of Male Rats Group |SeverityV'eIekn&cidence|?Severity'WeekI1n4cidence|'Severi~tWy'eeki52n,cidence' 1| [Lowdose T <I T O5 T <rT 05 1 - T------ [Midhighdose[TO | 1/5 <r [05 [| --- | ""FNoulmdbienrcroefasaenoivmearlscownittrholas PI or LI double the mean control value and greater than the highest control animal `Inmicdo-nhtirgahstdotsoem(a2l/e5 rraattss,) bainodloagtitchaellhyigshigdniofsieca(n2t/5cerlaltsp)roalfiftee4rratiwoenekwsa,saenvdidatenttheinlofwemdaolseer(a2t/s5atratthse) aprnodlimfeirdatdivoeser(e1s/p5onrsates)oabfsteerrve1d4 wienekfesmaolnesstwudays, agrseasteeern tihnanTexthtatTasbeleen2.in Tthhee dmaelgerse.e ofTthhee prreoslpiofnesraetoivbeserrevspeodnaste5o2bsweerevkesd.atBawseeedko4nwtahse nsoetvesruisttyaianneddianfctierde1n4ceweoefktsh,enporrolwiafesraatipvreolriefserpaotnisvee sisejeundginedthteohiingdhudcoesaetfreamnaslieesnt, parnodlitfhereadtiovseerreessppoonnsseeirnetlhateiiovneshriopfsfeeemnaalte4rawtesaetkst,hethheigthesdtomsaet.erial Text Table 2. Biologically Significant Cell Proliferation in Liver of Female Rats | Weekd |__ Weekd |_ Group Severity! Incidence? | Severity! | Incidence' | Severity' | Incidence"| |Lowdose [ 10 [ 05 1 26 [ 25 [ ~- T - | [Middose[TT 1 05 1 12 | U5 |- T --~ } [Midhighdose|-- T8 | 25 | <r[05 | 1-1] [FHoildghindcreesasee o|ver 3 cont5rols 1 25 [| <r | O05 | <r 1 05 | c"oNnutrmoblearniomfalanimals with a PI or LI double the mean control value and greater than the highest Iwnhtiecrehstwinagsly,noatrasreeepnatitneranoyf PoCtNheAr-lgarboeulpisn,g nwoars aotbstheerveeadrliinerhimghedopsoeinfte.malTehreatsstaatiwnienegkW1a4s, cvehianrsaocftethreizlbievyder.ag"rTahneulsairgcnyiftiocpalnacsemoifc tshtiasintiynpgepoafttlearbnellioncgalpiaztetdertno itshenohtepkantoocwynt;eshoawreovuenrd,ceintthraasl bReeceenntslpye,cuilahtaesd bteheatn ist hmoawyn rtehpartePsenNtAmitcoacnhornodsrsiarleaDtNwAitshynptehreosxiisso(muanlpumbulilsihfendciobosnearvaptriootne)i.n h(iMgahyedro,seetanali.m,al1s99m8)a.y bTehuass,sociitaistepdowsisitbhlpeerthoaxtisthoemecse.ntrilobularstaining pattem observed in the. Page 1-6 002380 "Secon`dCovDarnacfet CSteuldyPrNoluimfberearti6on32R9e-p1o8r3t Trahtse. celIlnprtohleiffeermaatiloen rraattse,s icneltlheprwoleiefkera1t4iomnalraetersatastwweereekall14hiwgeherre thhiagnhetrhotsheainnattheweweekek44inmatlhee Cfoonritrhoel,colnoswisdtoenstelyanhdigmhierdcdeollseprgorloiufeprsa,tibountrnaottesiinntthheemwiede-khi1g4h aonrihmiaglhs disosneotgkronuopwsn.. THhoewreevaesro,n ableltswleiednesrufnrsomcatnhbee4rualnedd o1u4t.wOeneke ptoismseibiploiitnytsis twheartethsettaiisnseudestwogeertehehra,ndsloedvadriifafteiroenntliyn isntatienrimnsg oCfomfpiaxraitisoonnsacnadn pbreocmeasdsiengb,etwwheeincheamcahytrheaatvmeenatffgercotuepd atnhed tshteaicnoinntgr.ol gDreosupiptfeortahisspedciifffiecreincmee, point. SUMMARY Tsihnevetechreeilthlyip(grhhoilgdihofsedreoastgeiroownuaspse,elnaesnisdnttwhhaaenslitvtwerora-nofsfoilemndatlgeirneraanttaesatruttrhe4awn(ei.ceco.kn,tsrwowlaass)s,nnooocttcdsuourssrete-adrienilenadtteawdt,o 1wo4afswfiemveieknsai)nm.iamlalIisnn sfaeenvmieamrlaieltsyraciinos,mbpcoeatlhreptdrhoetloihmfieagrlhaetaironandtsimn(itgdhr-eehalitigevrhertdhwoaasnsetdwgoors-oefu-oprlsed.laateBtdahasetehdwiegohenkdot4sheeb)su,etafnniodntdoiacntcguwsr,ereeckedlli1n4p,trowlgoirfeoearftaeftriivoienn wefafsecntsoseeveindean4tcewoefehkespiantotcheellrualtasrwperroleifceorantisoniidnemtroableeedorrefleatmeadlteortaetsstamtattheerihailg.hAdtos5e.2 weeks, there LITERATURE CITED Maye"prr,oliDf.e,ratFionrgscteellr,nucKl.e,arBeainetri,geKn.c,rosasn-dreaVcotlkwli,thtAh.epe(r1o9x98i)s.omaMlomnuolctliofunnacltiaonntailbpordoiteesin.agaAinnaslt Biochem 256: 135-137. Page 17 002381 - = SecoCnodvDarnacfet CSetluldyPrNoluimfbereartio6n32R9e-p1o8r3t II. TABLES Legend BrdU, bromodeoxyuridine LI, labeling index, percentageofcells in S phaofsthee cell cycle as determined by BrdU staining ND, not determined PCNA, proliferating cell nuclear antigen PGeLiEprmomliifneerdatbinygPCinNdeAx,sptenricnegntage of cells in S, G1, G2 anMd phasesofthecell cycle as SEM, standard error of the mean 002382 COVANGE STUDY NO. 6329-183 TABLE 1. PCNA CELL PROLIFERATION IN LIVER OF MALE RATS AFTER 4 WEEKS ON STUDY Group So [1-0ppmPFOS(Control) |M [1-0ppmPFOS(Control) |M || Animal || ProPlCifNerAating | number | index | Co2524 | 0.000% | | Co2526 | 0.130% | [T TT MeT an |o.os2% | [TsT ew |o.o2a% | [2-0S5[pWpMmP|FCoO a57S4 |(o 0.0w 00% )| [2-0SppmPFOS(Low) |" WM | co2575 | 0.027% | [2-05ppmPFOS(Low) | WM _ | Co2610 | 0.054% | [2-0S5| ppm M P|FcoO 261S8 |(o 0.0w 27%)| [TMeT a | 0.038% | [s-20p| omP WM_F|OCoS 265(0 |Mi 0.02d7% )| [C TTMeT an | 0.089% | [TsT w | oo028% | [45.0 ppm PFOS(Mid-High) |__M |Co2718| 0.053% | TT Mean | 0.043% | [TsT m | 0.025% | [T I T Mean | 0.085% | "ProlferatinE g cellE nuclear antigen; e percee ntage of cells inEG1, e5. Ge 2 ane d M e phases . Page l-1 002383 `COVANGE STUDY NO. 6325-163 TABLE 2. PCNA CELL PROLIFERATION IN LIVER OF FEMALE RATS AFTER 4 WEEKS ON STUDY | | | PoNa Group | Sex | NAunmimbaelr || Proliinfdeerxa*ting [1-OppmPFOS(Contre) |F |Co2s1s[m0 | [2-0SppmPFOS(Low) |F [sam T0.032%| |co2045 | 0.086% | [2-0Sp | pmFP| FoO cozS e82 | (o 0.0w 53%)| TT Men [ 0.055% | [1 sm [ oo015% | [s-20p | pmFPF | CO o2S s% [ (M 0.0d 84% )| [s-20p | pmFPF | cO o30S 06( [ M 0.0d s3%)| [TMeT an [ 0.088% | [7 TT sem | 0.013% | [TMeT an [0.097% | [TsT m o.036% | [5-200ppmPFOS(Hig | F | C93114 | 0.056%_| 5-m20m0 papm PFOS (High) f | __F ten sa349% Costas 0.430% 1 |__Mean 0.183% [Tre |m o.oss% | C1 1 1 PageIl2 002384 COVANCE STUDY NO. 6326-183 `TABLE 3. PCNA CELL PROLIFERATION IN LIVER OF MALE RATS AFTER 14 WEEKS ON STUDY Group sex [L~g0 epapm rP0FOsS(c(Coonntrcolh[wm 1-0 ppm PFOS (Control M__ || NAunmimbaelr prolIinfdeerxa*ting [cessn | osa1% | | cozsei 0.588% [7 men | ossa% | [2-oSpe | mMP _ F |cO o6S 16|(o 0.2w 40%)| [2-oSp| pmMP| FcO oseS 21 ( | o 0.2w 96%)| | TT Mean | o.7s% | [TsT m | o.os% | [3-20pomPFOS(Mid) |M |cozee2 | 0.242% | [TmeT an [| o242% | [TMeT an[0.335% | mean |o210% | 1-------- 1 1 Page Il3 002385 TABLE 4. PCNA CELL PROLIFERGAOTVIOANNGINELSIVTEURDOYFNFO.EM6A3L2E6-R1A8T3S AFTER 14 WEEKS ON STUDY || || Animal I| Pona proliferating Group ! sex | Number | index [1 sm | o.o23% | 20.5 ppm PFO(LoSw) [TMeT an | 0.257% | [1 T sm |o1as% | 0.054% [s-20pp|m F PT FOC0S 302(3|M0i .35d 6% )| [s-20pp|m F PT FOC9S 303(5|M0i .05d 3% )| [1 T Mea | 0.120% | [TsT m | o.0se% | [| Mea | 0.055% | [TsaT m | 0.000% | 520.0 pom PFOS (High 5 - 20.0 ppm PFOS (Hig! F coats 0.000% [o.02a%| rT Page l-4 002386 `COVANCE STUDY NO. 6326-183 TABLE 5. BRDU CELL PROLIFERATION IN RAT LIVER AF5T2WEEERKS ON STUDY Treatment Group Animal Gender | Number Brau Labeling Index" [+ 1 wae | ocos12 | 010% | [11 mae | cos3s| 000% | [CT wae cozsez |026%| [ [ 1 Male | cMo7 e2sa70| |0.02 26%4%|| [s |m oor% | [55 | MMaaloe | cCoo2r7r9e5 | 000.831%% | [5 | male [ coves| o0o% | [5 | Male | ocosetz | ose% | [me1 an| 022% | [s [m ooe% | 1 Female Co2676 [1 | Female | copes | 123% | [11 || FeFemmaallee || oCc9o2p9s2o3e| 164% | [1| Female | C92930 0.11% [ 1 T mseamn T| T T 212.620%% | [15 Female | coatiz | 0.26% [|5 Female | co3120 [15 Female | coai5 [5 | Female | costes| 027k | [5 | Femsle | ocoatr+| oa3% | [Me | om ie% | [Te TT om os% | 1 "Bromodeoxyuridine; percen1 tageof cel1 s in S phase 1 Pagal 002387 "7 SecondDraftCel Proliferation Report `Covance Study Number 6329-183 IIL. FIGURES 002388 0s 04s 04 io3s 2 03 E2 os go. ggos ot 00s 5 88 FIGURE 1. PCNACELL PROLIFERATION IN LIVER OF MALE RATS - [wesc meek 14] . opp Group 1 osm Group 2 2Gr0oumpm3 Fain SGroouppm4 2G0r0ouppp5m FIGURE2. PCNA CELL PROLIFINELIRVEAROTFIFEOMANLE RATS | OS 04s 04 fo 0.35 gEgz 03 ozs mwwweoeko41k|4] 3 oe ; Eos 01 00s 8 0 Gowt Gowz Gow Gawd Grows & 0 ppm 0.5 ppm 2.0 ppm 5.0 ppm 20.0 ppm g Faiz | 2 $. 2 i. os gg 88g FIGURE 3. BRDU CELL PROLIFERATION IN LIVER OF RATS AFTER 52 WEEKS a j iSiaki2 i | i| i| i| a 8|| ||| eh Wea 25emi E a: m orn : "Secon`dCovDaranfcte CStluldyPrNoluimfebreartio6n32R9e-p1o8r3t IV. SIGNATURE PAGE `Submitted by: PAI Project Manager: SaE ndra R. ET ldridge, PhD. SDaR e ---- PAI Project Pathologist: CarolynE Moyer, DT .V.M., DiT plomate, TA.ECVP. DJ ae 002392 "7 SecoCnodvDarncseSCteulldPyrNoulmibefrer63aR2te5pi-o1or8nt3 V. QUALITY ASSURANCE STATEMENT 002393 Pathology Associates International sh ACom ofScp ienca e Appn licaty ionsIntemationalCorporation Cm mato e ee r ------ es Cell Proliferation Report 104-Week Dietary Chronic Toxicity and Carcinogenicity Study With Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Rats Covance Study No. 6329-183 `QUALITY ASSURANCE STATEMENT This cell `Assurance pUrnoiltife(rQaAtiUo)n project has as required been by the iGnosopedctLeadboarnadtorayudPirtaecdtibcey (tGhLeP)PArIeguQluaatliiotnys pprroolmiuflergaattieodn rbeyporttheisUa.n aSc.cuErnavtierroenfmleencttiaolnoPfrtotheectrieocnorAdgeedndactya.(TEhPAe-fToSlCloAw)i.ng tTahbelecieslla Tecord ofthe inspections/audits performed and reported by the QAU. Date of Inspection `PhaseInspected. Date Findings Report to PAI Management/ `ProjectManager/Pathologist 08/18/98 10/07/98 10/07/98 0058/71200/2949199 SLatbuedlyiDnagt(a1a0n0d% slide/block match) DSruapfptorCteilnlgPrDoolciufmereanttiaontiRoenport SSteacionnidngDraanfdtCCoevlelrPsrloilpipfienrgation Report 08/18/98 10/08/98 10/08/98 05/10/99 08/24/99 2LE QualitKyaArsesnurE.anBcuellSeprecialist slik Date " 002394 J 75 Worman's Mil our, Sult | Frederick, Maryland 21701 + (801) 663-1644 + (301) 663-6994 FAX "Second `CovDarnacfetSCteuldlyPNruomlbiefrer63aR2te9-ip1oo8rn3t VI. APPENDIX 002395 Ce Cone SudyNumber 339.183 Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 4 Weeks on Study Tissue: Liver Ami ome | Number || ~]Sex| || DoseGroup Tome | | HistoloLgiicvFeirndings | O CE c|I TomRosac)m [ I oNmL | Tomes Ccoan|WFLoSang| [Or[Mentor | rene | [[[[ OOmmoomm]W|||| TTT0IeOsrrpmmmseSoSnhToyn| | NMoosS FFaadnse|| [omW| opm ros ty_|FMooS ne] [om| Tom ese oom | WM | 320pm PROS MG) No Significant Findings om || anes I 52 [1 M Co 9IT3E T927a8 |TM] CA a Il omy TOTS okay (Mid-high) Phi 75.o0vidpepnmigPhFyO! 373.0 ppm PFO! TRE 775.0 ppm PFO. (Mid-high) samo Em 3720.0ppm PFOS (High) i 16 Significant Findings To Significant Findings To Significant Findings No Significant Findings | | Co29 | 5 | S-200ppPmFOS (High) |ComSE [MC| S00pom PROS High NoSignificant Findings | G Significant Findings Page VI-1 002396 = Covance Sudy Numb632e9-1r83 Individual Animal Findings P1er0f4l-uWoereokoctDaineetarSyulfCohnriocniAcciTdoxPiocittayssainudm SCaalrtci(noPgFeOnSi;cTi-t3y29St5u)dyin wRiatths Histopathologic Findings after 4 Weeks on Study Tissue: Liver [omar Tse] DoeCow- ][tog Fading: |1 [[[oC omoTym|eF FFr|rm T0pmpm PROrsosS CaCmonmanl omn| | TopmeOSCoy| [oommeTrF] TTaassmpmmeerses Ttoonn OC lc No Significant Findings | MoS Fang:| We " wim Ohi NAC ic I 53038 I I 730 ppmSPFO l No Significant Findings | SIF] Taicd-higah) [vee|| [7 53070 [71 35."0opiptmhePyFO omTT Ta N.o Sigaificant oe Findings | | |[ [T30o O 84 m F [F s[h73.0vsS pmpmmmosPFeOES R|NoSfo pSiwgnieficanntrFinadimngsn|g| [|[ o omo o e | |m t |F || sSS7T0O0mpppommsPROS gy|SNo tF:| High) "No Significant Findings Page VI-2 002397 = Covance Study Number 6329-18 Individual Animal Findings Pe1r0f4l-uWoereokoctDaineetarSyulfCohnriocniAcciTdoxPiocittayssainudm CSaalrtci(noPgFeOnSi;cTi-t3y2S95t)udyinwiRtahts Histopathologic Findings after 14 Weeks on Study Tissue: Liver [AnCimalSSVex| ToDmosPRoOS mCoer--a| ||S NHoistolt ogEic eFFioen: dings --|||| [oom m|aW| TToommess CCoonry IWW Ci 9255 |M_| 10pm PFOS (Contoh Significant Findings [Om| Tose sen I NL Nd WAC [om] | id-high) O5"7T 728 TT M C33.C0E papdm iPFO: CSTs[WM | S200ppm PFOS (High) "NoSignificant Findings 7 Significant Findings [om moms|osm | Com M_| 5-200ppm PFOS (High) I I a GC) "NoSignificant Findings | foSignificant Findings | PageVI-3 002398 - - "CoStuv dyNa umbn er6c 329e -133 . Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 14 Weeks on Study Tissue: Liver [ Animal | Sex| Dose Group Histologic Findings | CTP |Ta Fs [Coos |F| 0 ppm PFOS (Control) [C omeC |L|A rCoEsLmmpLroEsDTo)n [o|m F [resem Aros No Significant Findings. [ [ToIm TFF| SS0mpmmOPRSOS O)| [ [CoT|v F F|| ssoowwnn rroossoeIny RoSigmanFindings | |O33T 0 TT] Rm 3-50 ppm PFOS I I I 777 No Significant Findings. [OW TT am (Mid-high) |ees|1 [I CO3S31I09 [IF50045v.70mp7pm5PrOS PFWOShy NoSignificant Findings [om T2506 POS (Righ) [o|F mown FOS S|eFo ne Franm s | : Pagsve 002399 o Gurdon Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 52 Weeks on Study Tissue: Liver EEAe [ Doecow THistologle IFTindings -- 1 I OORT [ToemoCCon| NoseFr] [Com ome | Tose) res [Con | TopmrosECm |smears| [ oF so uommsowsy||o F Ses samm e] -- [[oOmT{frS 5 m oT r ||--O TexoSvp--t--R F--oi--|] : Tgp vis 002400