Document xdYV8JNk6Vv0XeMrz3yz9j3RQ

bSrRiPshT-6295.8, T-6316.4, T6868.2, T-7071.1, 7132.1 AR26-1. 8// 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Title: Comparative Molecular Biology of Perfluorooctanesulfonate (PFOS, `T-6295), N-ethyl perfluorooctanesulfonamido ethanol (N-E(FOSE, T-6316), N-Ethyl perfluorooctanesulfonamide (N-EtFOSA, T-6868), Perfluorooctanesulfonamido acetate (FOSAA, T-7071), and/or Perfluorooctanesulfonamide (FOSA, T-7132)of in Rats and Guinea Pigs following Oral dosing. Final Report Date: May 25,2004 Study Numbers. T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 Strategic Toxicology Study Number: DT-15-B. Sponsor: Study Location(s): Study Director: Study Toxicologist: In-Life Start Date In-Life End Date In-Life Start Date In-LifeEnd Date In-Life Start Date In-Life End Date 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 1. 3M Strategic Altemative Toxicology Laboratory 3M Center, Building 270-SB-181 Saint Paul, MN 55133-3220 2. University of Minnesota, Duluth Dept. of Biochemistry and Molecular Biology School of Medicine 10 University Drive Duluth, MN 55812-2496 Andrew M. Seacat Ph.D., DABT Toxicology Specialist 3M Medical Dept. Corporate Toxicology and Regulatory Services Deanna Luebker M.S Senior Toxicologist 3M Medical Dept. Protocol: Protocol: Amendment #1: Amendment #1: Amendment #2: Amendment #2: 11/16/1998 11201998 03/01/1999 03/05/1999 02/192001 02/23/2001 ' 650014 SDRIPTSTH-6205.8, T-6316.4, T-6868.2, T-1071.1, T7132.1 ``STuambmlaeoryf.Contents somm---- sormm-- 3 MetIhDoTdISs-BProtocol Procedr rcs..-- .....-- ..ov-- roormnrr -- A od7 `AAmemndmeentnNNuudmmbbeemrr2|PerPorcocneedduutrreess ...........oor... rm-- en 7 8 ResAunlatlsyticalmethods ree re--en--m-- st---- | LiBvioelrogFilcualorPoacrahmeemtiercsa.l..Concentrations... nmremmmemm--_]12 CPlinaicla mChCieonitAsotoxyi.yd.a]se (PCoAO)activity. rerre--n--m-------- mn----gly SiCgOnnatuuSrIeOPnasge....c... r--er--e--a--m------ Tstl SumTmabalreIy. TAavbelreasg.eCumulative SEDose.--.--. erm 1199 TTaabbll3ee2.. SSuummmmaarryyooffOBrogdaynwweeiigghhttssannddOBrWganto Body WcehiagnhgReast.i.o.... rna20 "TTaabblle4e.. LPievrecre-ntanInditKiaildBnoedyytoWeBiogdhyt,CWeoimgbhitnReadtiDoast,a.CombinedJEData. ...._______..... 223 "pTearblf6e.oLriamvteehrdePerUflBuorIosuVifOofEnRamSiOdeisCaynHd MEeta.bol.ite.Values fromRatssndGuinea Pigs. Arneanleeyses 24 "ATnaabllyes7e.spSeurmfmoarrmyedoaftLithveerIPVefElNuVoErOoNsMuElNfaolnLamai.ad.ne.ds Metabolite Values fomRatsand Guinea Pigsh.e} A"Tapblep8HIe.epDaetvniicatPdiaomnisitotyolxthCeoPArOoxtiodcoalseActivity, CombcinsedD--at --------o.-oo. 228 `ApApepndpi23xe.- CnAunamdluyltiaitcxiavleLDiovseer SIandmipvlieduLaalbanIddeSntuimfmicaartiyodna.ta...._................. 2 nn 3) A`pAppenpdei45xn..OBdrogidayxnWweeiigghhtts,InOdirvgiadnuatloaBndWSrautmiomsa. rIyndividualDaaan..d..S..u..m..m.a..r..y...data... a4s0 `AAp.pendi6Rxa.taLnidveGruFliunoPreoiacgheLmiivceraFlCDactoan.cIenndtiravtiidounasl(aAnlldunSiutsmamrearuyggd)a.taA.nalysesai the rUniveersityo4fS RB.OCESIREaRtandeGouinea Pig Liver FC PeorfDocseEevalunatiotns. Analyseasiethee Universiotfy 45 Roc.hestRera.tLiver PEOSX Analyses aA the 5M EnvironmentalLab....... Asd (D.A.ll uRanatvaeindgs/Ggu.iTnheaePIiMg LEinvveirrFoCnmPeenrtcaelnLtaobfoDnolsyeaEvanluaatironalst.lyiAvenrzaleyasdttehsepies). 3M environmental5 LabE.. Te$c5hnical Report: Liver Fluorocarbon Metabo~lUniivteresisty of ROChESCF.............. 59 `ApIpennddixi7:vCalniidnSdiucmuamlaaCrhlyemCilsirnyi.c.a.l.C.h.e.m.i.s.t.r.y. Data. sioon-------- 65 AB.. SStattiasttioocsnnsCiClliincniiccsaall CChhemeimstirsistffororiGrauetis,nmeaalPeigasn,dmfelmealanedvafleumeas.lcvaoluems bcoimbinneed,d... 17018 `AAppppenedi98nx..HdEefpfiaetcxitcofPacauteFlCCmoa-dmAii0nxisittdraasoteiaonycotn]caitavlaisetaynd.a.cy.lCoA oxidase exprsion.1313s9 ``AAppppeennddiixx 1110:: FFiinnaallRReeppoorrttffoorrFFOOSSAA(AT(1T3-2T1O)7L.1.).......r-.r.orerrrrrrrr 1i1e3 `Ap"Ap. eCnodriexla12t,ioEnnodfpoeinft fCboeyrdriecflfaettrienostn.totLreiavtemerntPgFrOouSp.s 10 ivertPFtOSri --a--is... 118822 CBo.mbCionmede.l.a.tion ofeffbeydcifftersen treatmem nt group-- s o liv-- er PROS-- in Guinea Pigs, maleB s andT female 2 Le01S SoRTPITST-B6295.8, T-6316.4, T68682, T-7071.1, T-7132.1 Summary Adult male and female Sprague-Dawley rats and Harlan guinea pigs received oral gavage (doPsFeOsSo,fTv-e6h2i9c5l)e,c4o0ntmrokld(o2r%1T6w0emeknd0N)-,Et4h0ym!gp/ekrgf/lduaoyro(omcktda)nepseurlfflounoarmoiodcotaentehsaunloflon(aNt-e 6E8F6O8S)Ef,orTf-o6u3r16d)a,ysoran4d0 wmegr/ekg/hduamyanNe-lEythsya!crpiefricfelduoornootchteafniefsthuldfayo.namMiadlee(SNp-rEa(gFuOcS-A, T- Dpearwflleuyorroaotcstraenceesiuvlefdonoarmalidgoaavcaetgaetdeo(sFesOSofAAve,hiMc5l5e6c,onTt-r7ol07(1p)r,oopryl4e0nemgkldycol), 160 mkd perfluorooctansulfonamide on the fifth day. (FOSA, T-7132) for four days and were humanely sacrificed All ats animals survived. by 40 mkd PFOS The and percent initial body weights were 160 mkd N-E(FOSE significantly significantly decreased reduced body weight in `compared to controls to 85% and 87% inital body weight, at average liver PFOS concentrationsof > 600 ug/g. 40mkd FOSA and 160 mkd FOSAA significantly reduced body weight to 93% and 95% of initial body weight, respectively, at average ElitvFerOPSFEoOfS 4co0nmcekndtrNat-iEo(nsFOofSA14h0adga/sgigannidfi2ca0n0tgefgfe,ctroenspbecotdiyvewley.iNgehittihnerrat4s.0 mIkndthNe-rat, liver to body weight ratios were significantly increased by 40 mkd PFOS at average liver PraFtsOfSrcoomncalelnottrhaetriodnossoe fg>ro6u0p0s,gb/ugt.thIenscercehaasendgleisvewretroe bnootdysiwgeniifgihctanrtatcioomspwaerreedftoounthdein control. `aTnhde4g0uimnkeadpPiFgOpeSr1c0en9t1bo%dyanwdei9g3h%t winaistiasligbnoidfyicawnetilgyhd,ecarteaasveedrabgye 1l6iv0ermPkFdONS-E(FOSE craotnicoesnwtrearteiounnscohfan4g1e9d.ugG/ugianneda 1p7i1g kgi/dgn,eyre1s0pebcotidvyelrya.tioGsuwienreeaspiiggnilfiivcearnttolybiondcyrewaesiegdhbty treatment in all the dose groups measured, i.. 40 mkd N-EtFOSE, N-EWFOSA, or PFOS. Ianspaatrst,atcehaolmeisntoetrroalns(fCeHrOaLse),(AtSriTg)lywceerriedessig(nTiRfIicGa)n,tlaylklaolwienreepdhoinsprhaatstatsreea(teAdLwKiPt)h,4a0ndmkd iPnFrOatSs atrnedat1e6d0wmitkhd4N0-mEk(dFOPSOE.S,Si4g0nimfikcdanNt-deEcFreOaSsEesainndse4r0ummkpdotNa-sEsIiFuOmS(AK.")Ioncrcautrs,re4d0 macktidviPtyF,ObSutca1u6s0eNd-aEsiiFgOniSfEicdanitdinnoctr.eaEsxepionsuherpeaotifcrpaaslmtoit1o6y0l CmkodANo-xiEdLa-sFeO(SPECcoaAuOs)ed a tdhoeubcloinncgeonfttrahteiosnpoecfimfiRc NacAtiveintcyoodfinlagurfyolrCPoCAoAoxOidiansreat(iLvCero,AbOu)t acnatdalaas2e-faocltdiviintcyraeansde in catalase mRNA were unchanged. Exposure of rats to 40 mkd PFOS caused a 2-fold riantsc.reTarseeaitnmLenCtOwAiOtha4ct0ivmiktydanFdOSaA3 otro 61-6f0olmdkidncFreOasSeAiAn PcaCuoseAdOamsRigNniAficeaxnptreinscsrieoanseinin hepatic cytochrome P40 content and Acyl CoA oxidase activity in male ras. PsiFgnOiSficwaanstlaypipanrdeuncteldyPaCmooAreOpaoctteivnittypewrhoexriesaosmeN-prEoFliOfeSrEatoirnctrheaanseNd-,EbIuFtOSdiEd. noPtFOS significantly induce PCoAO activity, even though both treatments achieved similarly high liver PFOS concentrationsofgreater than 600 pg/Ke. PCOAO activity was not 3 60016 SDRTPITST-H6295.8, 6316.4, T-6868.2, T-1071.1, T-7132.1 determined for the 40 mkd FOSA and 160 mkd FOSAA treated male rats, buat 2-fold induction of acyl CoA oxidase activity occurred at relatively lower average liver PFOS concentrations of 193 ug/g and 140 pig/g,respectively, with an equivalent or greater fractionofthe total liver fluorochemical was contributed by the parent compound or the metabolite FOSA from the FOSAA treated rats. These data suggest that FOSA may be an equally potent peroxisome proliferator to PFOS, but not the N-acetyl metabolites of N-Et FOSE. In the guinea pig, potassium (K") values were significantly reduced by 40 mkd PFOS and N-EFOSA. Neither40mkd PFOS or N-EFOSA, nor 40 mkd or 160 mkd N-E(FOSE caused peroxisome proliferation in either gender ofguinea pigs. `These results showed that PFOS, N-EWFOSE, FOSA and FOSAA al caused indications ofperoxisome proliferation in rats, but not in guinea pigs. These results are in concordance with effectsof classical peroxisome proliferators, in tha the response is specific to certain species. 4 600017 DSRTPITSTB-62958, 6316.4, T8682, T0711, T-71321 Introduction: p`Tehreooxbijseocmteivpeoroflitfheirsatsitoundyinwraatsstaondingveusitniegaatpeigasn.dTchormepeacreomtpheoumnodlescduelrairvemdecfhraonmisms of perfluorooctane sulfonate and known to cause peroxisome proliferation in the rat were: (inNit-iEaltlFyOtSesAt)eda,npderNf-lEutohryo!ocptearnfelsuuolrfoonoacttean(ePsFuOlSf)o,naNm-iEdtoheytlhapneorlfl(uNo-rEo(oFcOtaSnEe)s.ulBfoynamide apemrefnldumoernotoc,ttanweosoutlhfeornacmoimdpeo(uFndOsS,A)pewrefrleuoardodoecdtatnoesthuelfsotnudaym.idoThaeceutlatteim(atFeOSmeAtAa)bolaintde of N-EtFOSA, N-EFOSE, FOSAA and FOSA was presumed to be PFOS, however there is some debate about that The hypothesis tht these fluorochemicals would induce peroxisome proliferation in the rat, but not the guinea pig, was based on several lines of evidence indicating that guinea pigs and primates are resistant to peroxisome prreoslpiofenrsaetioofn.theHsoewsepveecri,estthoe pmeorloexcuilsaormeanpdrobliifoecrhaetmoriscaflormtehcehpaenrifslmusortohsautldfiofnfaemriendteisatweatshe unclear. The specific aimsofthis study were to: 1. To elucidate the molecular response in both rats and guinea pigs by measuring the induction of MRNAofgenes that are associated with peroxisome proliferation. 2. To measuring the hepatic activity of peroxisomal enzyme systems and fatty acid binding proteins. 3. To perform standard toxicity testsofserum clinical chemistry and to examine the liver for histological changes which may indicate an explanation for the species differences seeninresponse to these compounds. 4. To correlate any observed alterations of the above functions to liver and serum levels ofperfluorosulfonamides and their metabolites. "This studywaspart ofa series of investigations designed to understand the `vimvool.ecu`lTahirsasntdudbyiowcahsemciacrarliemd-eocuhtainnicsomlslafboorrattheioenffweictths ootfhtehresienvcesotmipgaotuornsd.sDorb.sKerevned in- Wallace, UniversityofMinnesota Duluth, who has been engaged in studies designed to understand the effectsofthese perfluorosulfonamides on mitochondrial bioenergetics, performed molecular and biocheical analyses ofthe inductionofgenes associated with opfetrhoxei3soMmaElnvpirorloinfmeeranttiaoln Taencd/honrolcoeglyreapnldicaStaifoent.y SDerr.viKcreiss lHaabnpseernfaonrdmeLdisqauaAn.tiSttateivveenson serum and liver perfluorosulfonamide metabolite analyses. Dr. Lin Xu performed quantitative liver perfluorosulfonamide Marion Anders lab at the University of Rmoecthaebsotleirt.e analyses on some samples in Dr. Study Timelines: `This study (DT1wa5sco-ndBuct)ed in three parts, the protocol and protocol amendment numbers 1 and 2. The protocol had an in-life start date of 11/16/98andan in-life end date of 11/20/98. Amendment number 1 had an in-Jife start date of 3/1/99 and an in-life end date of 3/5/99. Amendment number 2 had an in-life start dateof2/19/01 and an inlife end dateof 2/23/01. Amendment #3 was procedural only. s 600018 DSRTPITST-H6295.8, T-6316.4, 768682, T7071.1, T-7132.1 Regulatory Compliance: 0`T9h5i0s,wSatsraatnegeixcplToorxaitcoorlyosgtyudLyabanGdLtPhuPsrcolgasrsaimfiPerdoacsednuoren.-GLP as explained in TOX SOP Test Material: dTohceumsepnotnasotriopnroofvitdheedsstaarmtpilnegsmoaftearlilalflwuaorsotchheemriecsaplosnstiobtihletyinovfteshtiegastpoornss.orA.naAlyctihceamlical composition temperature. specification Test material sheet was kept on was stored tightly file. Compounds vere stable sealed at room temperature. at room `sTthreucTt-urneusmboefresa,chcohfemtihcealconmapmoesu,ndasbbtrheavtiwateiroensteussteedd ifnorthtihsessetusdaympalreesg,iavenndbtehleocwh.emTihceal currently accepted abbreviations for each compound are in bold 1. Vehicle control: 2% Tween 80, or propylene glycol. 2. T-6295 Perfluorooctane sulfonic acid, potassium salt (perfluorooctanesulfonate), PFOS, FC-95, Formula: CSOs K*, MW = 538.1 g/mole). 3. T6316: N-ethylperfluorooctane sulfonamido ethanol, (narrow Range NEthyl Perfluorooctanesulfonamido ethy! alcohol), N-EAFOSE. EtFOSE, FC-10, Formula: CyF:SON(CHICH,CH,OH, MW = 571.06). 4. T-6868: N-ethyl perfluorooctane sulfonamide, (perfluorooctane sulfonyl ethylamide), PFOSA (as sated in the protocol, but not used for this compound in this report), lab), FX-12, NF-orEmFuOlSa AC,uFPyF:OSSOE;ANH(CaybMbrWevi=a5t2i7o.n2u)sed by the 3M Environmental 5. TC-I7F0S71O:NHCH.COP0er,flMuoWroo~ct5a3n6esgu/lmfoolnea)mido acetate, FOSAA, M536, (Formula 6. T-7132 by the 3M EnvirPoenrmfelnutoarlooLcatba)n,esFuolrfomnualmaidCeaF, 1F7OSS0A:,NPH,FOFSOASA(,abMbreWvi=at4i9on9.u0s6ed mole). 7. Wyeth14643 (WY, MW = 323.79 g/mol) was obtained from Chemsyn Science Laboratories, Lexena, KS. Wyeth-14643 (WY), was added to DTISA as positive control dose group for hepatic peroxisome proliferation. 6 n0019 SDRIPTSTH-6295.8, T-6316.4, T8682, T7071, T-7132.1 Methods mTehtehmoedtshaordesgainvdendoinsethgeropurpostofcoorleaancdh apamretnodfmtehnitsss.tuDdeyviaarteiosnusmtmoartihezepdrobteolcoowl.arDeetliasitleedd in Appendix 1 DTIS-B Protocol Procedures "UTnhdeeprrotthoecoplrohtaodcoaln, iing-hlitfemtaalretadnadteeiogfht1f1e/m16a/l9e8SapnrdagauneiDn-alwifleeeyndRadtast,e1o0-f1211w/e20e/k98s.old weighing approximately 250-300 grams at the time of initiation were obtained from oHladrlaanndLwaebiograhtionrgieasp,prInocx.imEaitgehltym6a0l0e1a0n7d5e0igghrtafmesmaalethHeatritmleeyoGfuiininteiaatiPoingswe1r0e-1o2btwaeienkesd from Harlan Laboratories, Inc. Each dose group contained 2 animals/sex/species. The dose groups were vehicle control (2% Tween 80), 40 mg/kg/day N-Et FOSE, 40 `cmogm/pkog/udnady wNa-sEpFrOeSpaAr,edanind240%mTgw/ekegn/d8a0y iPnFagOlSa.sAs 2ti0ssmuge /grmiLndesru.spTehnesiaonnimoaflesacrhecteeisvted four consecutive daily doses. The dayofthe frst dose was designated day zero, thus the doses were administered on days 0,1,2 and 3ofthe study. Rats received the test compound suspended in 2% Tween 80 or the vehicle control by oral gavage at a volume of2 mi/kg body weight. The guinea pigs receivedtheirdaily oral dose volumeof 2 ml/kg by droplet in the backofthe mouth. day4 The animals were humanely sacrificed on Amendment Number 1 Procedures Amendment number | had an in-life start date of 3/1/99 and an in-life end date of 3/5/99. "The purposeofamendment 1 was to add groupsofboth male and femal rats and guinea pigs treated with vehicle control, N-EFOSE (T-6316) or PFOS (T-6295). The histological and clinical chemistry resultsof the treatment groups under the protocol were. not remarkably different than in the controls, and the tissues for northern blot analysis 1w0erreepdlaecleaytehde dsupreicnigmeshnisptphiantgwaenrdewleosrteadnedgrealdeveadt.e Tthheerdeofsoeroe,faNm-eEn(dFmOeSnEt a1dwmainsisdteesriegdnetdo achieve a more effective level, pTrwoetlovcoelraatmseannddmtewnetlv#e1.guEianceha pdiogssetgortaolup(6cmoanltea,in6edfe2maalnei/msaples/csieexs/)spweecrieesu.sed under the `The dose groups were vehicle control (2% Tween 80), 160 mg/kg/day N-Et FOSE and 40 mg/kg/day PFOS. For N-E(FOSE, a suspension of 80 mg/m] N-EFOSE in 2% Tween 80 was prepared and a volume of2 ml/kg was administered by oral gavage to the ats and by droplet in the backofthe mouth o the guinea pigs on days 0 through day 3ofthe study. `PTChiOs AdoOsaewctaivsitcyominpathreabralteitnoat4hewceuemkulfaeteidviengdossteuodyfwNi-thEt3F0O0SpEptmhaNt -iEndFuOceSdEp(eRreofx.is3oMmal Medical Dept. T-6316.1), and was less thanhalfof the LDS0. 7 n0020 SBRrIiTsTs.2988, T3164, T8682, T2071, T1521 For PFOS, a suspensionof 20 mg/ml of PFOS in 2% Tween 80 was prepared, and a volume of2 ml/kg was administered by oral gavage to the rats and by droplet in the back of the mouth to the guinea pigs on day zero through day 3 of the study. The cumulative dose of PFOS delivered under protocol amendment #1 was ~160 mg/kg, as was used in the protocol. The cumulative dose was below the LDS0 for PFOS and well above the threshold for inducing peroxisome proliferation in the rat. `Amendment Number 2 Procedures `The purpose of amendment 2 was to add groups male rats treated with vehicle control, dpeorsfelsueoqruoiovcatlaennetsutloftohneadmiodsoeascoeftaNt-eE(XFFOOSSAEAa)ndorPpFeOrSf,luroersopoeccttainveelsyu,lfionnparmoitdoeco(lFOSA) at amendment #1 in order to compare the effects and metabolite profiles ofthese `compounds in rats at equivalent doses. Amendment number 2 had an in-life start date of 2/19/01 and an in-life end dateof2/23/01. Nine male rats were used under the protocol amendment #2. Each dose group contained 3 male Sprague Dawley Rats, 10-12 weeks old weighing approximately 250-300 grams at the timeofinitiation were obtained from Harlan Laboratories, Inc. The dose groups were vehicle control (propylene glycol), 160 `mgga/vkagg/edaatyaFvOoSluAmAe,oafnd m4l0/kmgg/bkogd/ydawyeiFgOhStAo.n Tdahyesvzeehriocltehcroonutgrholdawyas3odefltihveersetdudbyy. oAral wlaerrgeerdovsoeldumbey oorf5al mgLav/aKgge,wwahserueseads tthheanguininperaotpoicgoslcaamnenontdmbeendtos#e1dbbeycaoursale goanvlayger.ats For FOSAAa, suspension of 32 mg/ml FOSAA in propylene glycol was prepared and a ivcohliuomveedoaf5cummlu/lkagtiwvaesdaodsmeinoifs6t4e0remd ibky eoraFlOgSaAvAa.ge to the rats on days 0-3. This dose For FOSA, a doseof 40 mg/kg body weight was administered via gavage to rats on day wzearso ptrherpoaurgehd,daaynd3aofvtohleumsetudoy.f5 Aml/skusgpwenassiaodnmoinf8istmegr/emdlboyfoFraOlSgAaviangeprtoopytlheenreatgs.lycTohlis dose achieved a cumulative dose of 160 mg/kg after four daysofdosing. The dose of FOSA was the same protocol. The LDSO as for tFheOSdoAseios fnoPtFkOnSowand,mibnuitsttheerecdumuunldaetrivaemedonsdemeonftF#O1SoAf this administered under this protocol was below the LDS0 for PFOSof251 mg/kg for PFOS in corn oil, as a pointofreference. Specimen Handling: Liver and sera were collected and frozen rapidly after euthanasia according to the details described in the protocol and amendments. A one to two gram aliquotofthe liver samples shipped in dry ice to the analytical laboratories listed in the protocol and amendments. The identificationof each liver sample sent to each lab is listed in Appendix 2. cAooz1 SRbTPITSET6205, T-6316.4, 68632, T1071, T-7132.1 Analytical methods cCoenrtteanitn,lLiavuerrosya]mpCloeAs woxeirdeasseenatctfiovritayna(lPysoiosscbhyapnrdevYiaomuaslzyakpiub1l9i8s6he)danmdetphroodtesinfocronPt4e5n0t (Bradford Biology at 1976) in the laboratory of Ken Wallace Deptof the UniversityofMN by the following methods: Biochemistry and Molecular Enzyme Sample supernatant ofa Preparation 10% (wtvol) ~ The enzyme fraction consisted of homogenate of 0.5-1.0 g frozen liver the 6,000 g issue in 300 emstMimmaatnenditaoclc-o1r0dimngM10HEthPeESm-et1homdMofEBGrTaAdfo(rpdH 7.2). using Protein concentration was commercial bovine serum albumin as standard. sLu-sCpoeAndOexdidinas6e0AmssMayKH~;TPh0e:-e0q.u0i2val%enTtriotfonca.X,1500ug(/pmHl 7t.i4s)suceonthaoimnoigngen|atmeMwaps- hydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 uM FAD, and 60 uM lauryl-CoA (LCoA). The reactions were allowed to incubate at 37C for 30 min in a 100 shaking water bath and mM sodium carbonate terminated (pH 10.5). by adding 3 volumes The concentration of of 2 mM KCN in H;0; generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength fluorescence was calibrated with of 317 nm commercial and H;0; emission at 405 and the results are nm. The expressed as nmol peroxide generated/min/mg mitochondrial protein. Protein was quantitated by the Bradford method. Catalase Assay original method - The activity of catalase was estimated published by Claiborne and Fridovich (J. by a modification Biol. Chem. 254, of the 4245- 52,1979), which is based on the direct measurement of H;O; disappearance as wquaasntdiifliuetdedspienc5tr0opmhMotopmoettaiscsailulmy pahto2s4p0hantme. (pIHn h7i.0s).proTcheedumree,dituhemtiwsassuewsaarmmpelde 10 27C and the reaction initiated by adding 10.3 mM Hz0;. The progress of the reaction was monitored at 240 nm for 5 min, Catalase activity was estimated fprrootmeinth(eTaibnilteial1).liOnneaer urnaitteof(acEt*i=vi4t3y.i6s dmefMi'necdmaTMs)thaatndameoxupnrteosfseednzasymueniwthsi/cmhg catalyzes the decomposition of | umole of Hy; per min. Northern Blot Analyses - Quantitation of mRNA for both acylCoA oxidase (ACOA) and catalase were performed by Northern blot analysis of quick frozen liver was samples from treated rats and guinea pigs. powderized in liquid nitrogen using a Approximately mortar/pestle. 1 g offrozen Total RNA liver was recovered using the PERFECT RNATM isolation kit and the concentration quantified spectrophotometrically at 260nm. The RNA was electrophoresed on a 1% agarose gel, blot transferred t0 a cellulose membrane and hybridized to the fcorrormespproinmdeirnsgtoracna.d,om3l5y0 [baPs]e sleabqeuleendceoloigfotnhueclreeostpiedcetsivtehartatwelirveerPgCenRe.ampmliRfNieAd 9 cneoRR DSRrPITST-H6295.8, 6316.4, T-68682, T-7071.1, T-7132.1 band density software. was quantified autoradiographically using phospho-imaging Certain Liver samples were sent for analysis for the parent compound (s) and the. metabolites by the 3M methods (Hansen et al. Environmental Technology and Safety Services 2001). The details of the methods used and the using published resultsofthe analyses that were completed were issued in an analytical report from the 3M LEnIvMiSroNnom.eEnOt1a-l01L2a9b).onTMhaeyre8s,ul2t0so03f (an3aMlySsteusdtyhaNto.weFreACcTom-pTlOetXed~1a0r7e,in3cMluLdaedboirnatthoirsy report and integrated with the body weight and liver weight data. The abbreviations that athbeov3eM, aEnndviarroenfmoeunntdalaslsaobciuasteeddtwoitihdetnhtiefpyrciommarpyoudantdastwabelreesdfirfofemretnhte tlahba.nTthheeolnaebs listed analyzed fora metabolite, perfluorooctanesulfonamidofethyDacetate (PFOSAA), that awnadsanneavleyrzdeodsefdo.r inThlievearbbsraemvpilaetsioanrseugsievdenfobreelaocwh, afnoalllyotweedthbatytthheelcahbemuisceadalsfosrtmaunldaaradnsd the abbreviation used in this report. PFOS= PFOSA= PFOSAA = EFOSE= MsS6= PFOSEA= Perfluorooctanesulfonamide (Formula G,F,S0y-, PFOS) PPeerrfflluuoorrooooccttaanneessuullffoonnaammiiddoe(et(hFyolramcueltaatCey.F1(T,hSiOs:cNHoym,poFuOnSdA)was also FreAfeCrTredT1O0Xas1P07e)r.fl(uFoorromoucltaanCessuFl1f5oSnOam:iNd(oCaaceHtsa)tCeH2inCOthOe'r,aNw-dEaWtFaOtSaAblAe)s.of narrow Range N-Ethyl Perfluorooctanesulfonamido ethyl alcohol (FPeorrfmluuloarCooicStOa:neNs(uCl:fHon:aImCiHd,oaCcHe:tOaHte, N-(FEoWrFmOuSlEa)CiF,:SO:NHCH,COO FOSAA) Perfuorooctane sulfonyl ethylamide EFOSA) (Formula CyFy:SONHC:Hy, N- Certain other liver samples were analyzed for parent compounds and metabolitesof the fluorocarbons by LC-MS/MS by Dr. Lin Xu in the laboratory of M. W. Anders DpuebplairsthmeednmtoetfhPohdasrm(aHcaonlsoegnyetanald. 2Ph0y0s1i)o.loTghy,eUrneisvuletrssiatryeorfeRpoorctheedsitnerthuissirnegpoprrtevainodusly integrated with the body weight and liver weight data. Certain other liver samples were palmitoy] Co-A Oxidase activity sent to Covance in Madison WI and were analyzed as an indicator of peroxisome proliferation using a for validated method based on published methods (Lazarow 1981). The principleof the aisnsvaoylviesstthhaet irnedtuhcetipornesoefncNeAoDf ptaolNmiAtDoHyl,-Cwoh-iAc,htcheanthbierdmsetaespoufrtehdespbe-cotxriodpahtoitoonmestpririaclally at 340 nm. The results were reported the 3M on October 22, 2002 ina letter report from `Covance and the data is included in this report. No formal report was written by Covance. 1 G20923 oSRrPiTsT.a6295.8, 316.4, T68682, T0711, T71321 Results Biological Parameters. All animals survived to the end ofthe study. No gross observations were noted during the in-life phase or at necropsy. The cumulative doses ranged from approximately 33 to 53 mg fluorochemical inrats,and fTrhoem1a6p0prmogx/iKmga/tdealyy 6d7osteog1r2o3upmganfilmuaolrsocrheecemiivceadl pirnogpuoirntieoanaplilgys rheicgehievricngum4u0lamtgi/vkeg/ddoasye.s (Table 1). The body weightsof the guinea pigs that were dosed at different times were greatly different, which accounted for the wide range in cumulative dose in the guinea pigs. Individual and summary cumulative dose data are shown in Appendix 3. Average body weights decreased significantly over the dosing period for female rats given 40 mkd PFOS and 160 mkd N-EtFOSE (Table 2). Average body weights decreased significantly over the dosing period for male rats given 40 mkd PFOS or FOSA and 160 mkd N-EtFOSE or FOSAA (M556). Individual and summary body weight data are shown in Appendix 4. `The male rats given N-E(FOSE at 40 mkd had significantly increased liver weights (Table 3). Male rats given PFOS at 40 mkd haad significant increase in liver to body wsiegingihfticraanttilosy. loMwaelreedraktisdtnreeyatweediwgihtths PaFnOdS,kiNd-nEe(yFtOo SbEoadnydweNi-gEhtFraOtiSoAs. atF4e0mamlkedkialdlnehyad weight and kidney to body weight ratios were not significantly different from control values. Kidney weight data was not obtained for the other dose groups. Individual and summary body weight dataare shown in Appendix 5. dMoasleegarnodupfeamnadleanpaelryczeendtotfoigneitthiearl. Tbohdeycwoemibghitnsedonradapyerfcoeunrtwbeordeycwoemibgihntedwafsor each significanly decreased by 40 mkd PFOS, FOSA, and FOSAA, and by 160 mkd N- E(FOSE (Table 4). The combined guinea pig percent body weight was significantly decreased by PFOS, N-EtFOSA and N-EtFOSA at 40 mkd, and by N-EtFOSE at 160 mkd. Organ weight ratios (liver to body weight and kidney to bodyweight where available) `were combined for males and females independently from cach dose group in all parts. ofthe study increase the and analyzed together. N for each dose group Combining for analysis the and relative organ weights was done is justified because the organ to `weights have been normalized by body weight. PFOS at 40 mkd significantly increased liver weight to body weight ratios in the rat combined data (Table 5). The combined male and female guinea pig liver to body weight ratios were different from the control group values for any of the treatments given. not significantly Significantly increased kidney to body weight ratios were found in guinea pigs treated with N- EtFOSE, N-EtFOSA, or PFOS at 40 mkd. n n0o24 SDTRITSHT-6295.8, T-6316.4, 768682, T7071.1, T7132.1 Liver Fluorochemical Concentrations `The liver fluorochemical concentrations in ats and guinea pig livers are summarized Efnrvoimrtohnemeannatlaylselsabp(eTrafbolreme7)d.aTthteheinUdniivviedrusailtyanodfsRoucmhmeasrtyerl(ivTearblfelu6o)r,oacnhdemaitcathle 3M concentrationsdataare liver samples from the presented in Appendix 6. The 3M PFOS 40 mkd and the N-EFOSE Environmental lab analyzed 160 mkd rat dose groups and did not measure fluorochemical levels in the guinea pig livers. Aliquotsofthe livers that wtheereUnainvaelrsyizteyd obfyRtohcehe3sMterE.nvTihreonimnednitviadluallabrawtelrievearlsdoataanaslhyozweeddbtyhaDtroangeAlnotdorfetshleab at samples analyzed at Rochester had a high background ofFOSA in the control group. The Rochester analyses tended to have higher PFOS and FOSA determinations than the 3M Environmental lab, however the values for PFOS are lstedas + 35% accuracy, for FliOstSedAaAs qua5l0it%ataicvceuornalcyy,inantdheffoirnaFlOrSeApo,rtFO(SFAAAC,T TEXOFXOS17E0)a.ndThNe-reEfoFrOe,StAhethdeifvfaelrueensceasre in the measurement of PFOS and FOSA fall within the experimental error inherent with the methods used. The percentofthe dose that was in the liver was calculated for each dose group from the amountofthe total liver PFOS containing species, derived from the sumofthe TcoLnPceFnOtSraXt,ioannsdoffaorlthfeluaomrooucnhteiocfaPlFsOpSeciitesseldfeitnecttheedliivnert.heThlievearmaonudntde(smigg)noafteadll fluorochemical species detected in the liver was derived from the TLPFOSX times the liver weight. The percent of fluorochemical dosed present in the liver was derived for oboftRhocthheesTtLerPlFabOaSnXdatnhde 3foMr PEFnvOiSrointsmeelnftfaolr tlhaeb (vaAlpupeesnddeirxiv6,edBafrnodm Db)o.thInthreatUsndiovesresdity with PFOS, the percentofPFOS dosed present in the liver as PFOS ranged from about 17 p10i.g2s3dpoesrecdenwtitihn bPoFtOhSm,atlheeapnedrcfeenmtaalgee roaftst,haentdotwalasamcoounnsitsotfentPFbOeStwedeonsleabtsh.atIwnagsuinca pwreerseenotnliyn tahneallyivzeerds awtatshefaUrnlievsesrstihtayn oinf tRhoechraets,tbeertswoeeanco3m%paarnidso5n%.betGwueiennealapbisgfloirvetrhse fbelusoereonchfermoimcatlhecodnatteanptriensetnhteeldivienrsToafblgeusinBeaanpdigDs cinanAnpoptebnedidxet6ertmhiatnetdh.e HToLwePvFeOrS,Xit can values derived at the University of Rochester and at the 3M Environmental for rats treated with 160 mg/kg/day N-EWFOSE are about the same at each lab. `wTihtehUeniitvheerrs4i0tymokfdRoPcFhOeSs,teNr-aEn(aFlyOzSeEd obrotNh-EraWtFaOnSdAg,uionre1a6p0igmklidveNrs-EftrFoOmSaEn.imTahles treated TpaLrtPiFcuOlaSrXlyiinngtuhienemaalpeisgsgutrienaetaedpiwgsi.thT1h6e0 mpekrdceNn-tEo(fFtOheSEdowsaesthlaotwwearsthparnesientnhteinrtaht,e PliFveOrSasitPseFlfO,SiXnwbaotshapgpuirnoexaimpaigtselaynd2-ifnolmdosttheaptesrcternetatoefd wthiethdoNs-eEp(rFesOeSnEt ainndthFeOiSvAe.r as However, the female rats treated with 160 mkd N-EFOSE had greater than 70% of the pTrLesPeFntOSasXPaFsOPSFOinSithnethlievierr loifvearst.s Ctorenavteerdsweiltyh, t1h6e0pmerkcdenFtOagSeoAfAthhaeddhoisgehtlheavtewlsasof FOSAA itselfand FOSA, with low levelsof PFOS. 2 000025 SDRTPITST-H6295.8, T6316, T-6868.2, T-1071.1, 7132.1 Clinical Chemistry Serum clinical chemistries for rats and guinea pigs are presented in Appendix 7. Clinical chemistry values were performed. were combined from males and females in ach species and statistics In ats, cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALKP), and aspartate aminotransferase (AST) were significantly lowered in rats treated with 40 mkd PFOS and 160 mkd N-E(FOSE. Significant decreases in serum potassium (K) occurred minosattssitgrneiaftiecdanwtitdhec4r0eamskesd oPcFcOurSr,in4g0inmkthde N4-0Em(kFdOSPEFOanSd d4o0semgkrdouNp.-EA(lFbOuSmAi,n w(iAtlhb)thaend total protein (TP) were significantly increased by 40 mkd PFOS, and creatinine. g(rCoRupEsA.TT)hewsaescshigannigfeicsanitnlcyliinniccraleacsheedmiinsttrhye a4r0emckondsiPstFeOnSt wiatnhd p4r0evmikodusNs-tEudIiFesOSwiAthdose PFOS and N-EtFOSE. Noneoftheotherclinical chemistry parameters were significantly. different from control values in rats. In the guinea pigs combined male and female clinical chemistry analysis, there were no significant changes in cholesterol, triglycerides, alkaline phosphatase, or aspartate aminotransferase. Albumin, total protein and creatinine were not significantly changed. rPeodtuacsesdiubmy(4K0')mvkadluPesFOfoSr amnadle40anmdkfdemNa-lEeAtFhOeSgAu,inseiamipliagrstcootmhbeirnaet.d Awlearneinsiegnificantly aminotransferase (ALT) was significantly increased by treatment of guinea pigs with 40 mkd POS. . None of the other clinical chemistry parameters were significantly different from control values in ats. Palmitoyl CoA oxidase (PCoAO) activity Hepatic palmitoy] CoA oxidase (PCOAO) activity data for males and females from cach dose group was combined and analyzed together. PFOS at 40 mg/kg/day for four days. caused a significant increase in hepatic PCOAO activity in rats (Table 8). N-E(FOSE at c1h6a0nmgekdocfcuorr4reddayinstdhiedgnuoitnseiagnpiigf.icIanndtilyviidnucarleaasnedhseupamtmiacrPyCPOCAoQAiOn rvaatlsu.esNoforsimganlifeiscaanntd females separately are shown in Appendix 8. Peroxisomal enzyme activity and gene expression The effects of PFOS for four four days of oral dosing on catalase and acylCoA of 160 oxidase mg/kg/day N-EWFOSE or 40 gene expression and enzyme mg/kg/day activity in liver tissue from exposed rats and guinea pigs are presented in Appendix 9. These data were presented as a poster at the 2001 Society of Toxicology meeting (Wallace er al. 2001). activity Acute exposure of LCoAO and of a rats 10 2+fold 160 mkd increase N-Et-FOSE caused a in the concentration doubling of the specific of mRNA encoding for 5 n0o26 SDRTPITSTB-6295.8, 6316.4, T-6868.2, T-1071.1, 7132.1 cPactaolAaOse imnRlNivAerwferroemubnocthhanmgaeldebayndexpfoesmuarlee troat1s6(0AmpkpednNdi-xEt9-).FOSCEatianlalsieverasctfirvoitmybaontdh bseoxtehs.sexEexsp,oasnudrepoosfsriabtlsy0a 4sl0igmhtkdinPcrFeOasSecianucsaetdalaas2e-faocltdiviintcyreianselivienrLfCroomAOmalaect,ivbiutty nfootr fPeCmOalAeO, rmatRs.NAAceuxptreesesxipoonsutrhaetowfarsatmsotroe4p0romnkodunPcFedOSincfaeumsaelde aco3m-ptaor6e-dfotlodmianlcereraatsse. in Exposure of guinea catalase activity in pigs 10 either N-E-FOSE sex. The and PFOS guinea pig did not mRNA stimulate LCoAO activity or encoding for PCOAO was undetectable, even following exposure to N-Et-FOSE or PFOS (Appendix 9). "Treatment of rats with 40 mkd FOSA caused significant increases in hepatic cytochrome P450 content and Acyl CoA oxidase activity (Appendix 10). Treatment of rats with 160 mCkodA FoxOiSdaAsAe accatuivsietdys(iAgnpipfeincdanitxi1n1c)r.eaTsehseiinnhdeupcaetdicthceyetxopcrhersosmieonoPf4t5h0ecsoentoefntthaensde Acyl proteins in liverofrats indicated that FOSA and FOSAA were peroxisome proliferators in rats. For the 40 mkd FOSA and 160 mkd FOSAA treated male rats, a 2-fold induction ofacyl CoA oxidase activity occurred at average liver PFOS concentrations of 193 pig/g caonndce1n4t0ragtieo,nsriensptehcetiPveFlOy.S TthreeastemelnitvegrrPouFpO.S concentrations were lower than the liver In FOSA treated rats, the parent compound, FOSA, contributed an equivalent or greater tfrhaecsteifonloufortohcehetmoitaclallisvpeercfileusorroecprheesmeinctaeldaaspdpirdoxtihmeaFteOlSyA0.me3t%aboofltihtee dPoFsOe Sinatnhde elaivcehr.oIfn contrast, the FOSA-glucuronide present in FOSA treated rats contributed only a small fraction of the TLPFOSX (Appendix 10). In FOSAA contributed treated an 2 10 rats, the 3 times, mreetsapbeocltiitveelyF,OtShAe,amanodunttheopfatrheenttoctaolmlpiovuerndf,luForOoScAheAm,ical than did the the metabolite PFOS, and represented liver, respectively (Appendix 11). approximately 0.14% and 0.24%ofthe dose in Taken together, these data suggest that FOSA may be an equally potent peroxisome pEr(oFliOfSerAaAt,oratnodPFFOOSSAiAn)ratasr,eawnedatkheart ptheeroNx-iasceotmye!pmreotliafbeorlaittoerss othfaNn-eEitthFerOSPEFO(SN-or PFOSA. The compounds FOSA and FOSAA, were not tested in guinea pigs. Discussion `The analytical data showed that FOSA was consistently identified as a metabolite of oPfFtOhSe,awmhientohegrroPuFpOiSs nwoatsreaaddmiilnyiasptpearreedntd.ireFcOtlSyAorwafosrpmreedseanstainmestoamboeloiftte,hebuctonttheroslource ssuabmmpiltetsesduabtmdiitftfeedrefnotrtainmaesl.ysTehseatfaorgmiavteinontoimfe,PbFuOtSnAotfirnoomthPeFrOgSroturpesaotfecdoanntirmoallsswaampsles determined in allof the liversofall PFOS treated animals that were analyzed at the University of Rochester, and at the 3M environmental lab for the same samples, which I] cnaoz7 SDRTPITSTH-6295.8, 6316.4, T-68682, T-1071.1, 7132.1 had no background Clarificationofthe PFOSof PFOSA formation of the in the concurrent conirol FOSA following treatment from with cither PFOS lab. should be obtained in further studies. FOSA N-glucuronide. Whatever its route of formation, FOSA was metabolized to cNo-uElWdFaOrSsEe aflrcoomhotlhegiNv-edseertihseyltaotaiornaonfgeNo-fEmWaFjOoSrEanaldcomhionlo,ramnedtaNb-olEiIteFsO.SAFAOScoEuladlcaorhiosle EbyAFthOeSEoxiadlactoihoonlowfoutlhedaglicvoehotlhe10obtsheercvaerdboNx-yElitcFaOcSidE. alGclouhcoulrognliucduartoinoinodefs.thFe OpaSrAenAt Naclocuolhdolb,eofrobromtehd. bFyOtSheANc-odueledthbyelaftoironmoedfbNy-tEhFe NO-SdeAeAthoyrlabtyiotnhoefoxNi-daEtXiFonOoSfAFoOrSbEy.the mreamyovbaelofofrtmheedcbayrbtohexygmleutchuryolngidraotuiponofofFOFOSSAAA. asLoglsysooxyfltahtee.carFbOoSxyAmeNt-hgyllucgurroounipdferom FOSAA would give FOSA or lossofthe glycine moiety would give PFOS directly c"Tohnececnotrrraeltaitoinosnwoefrae faenwaolyfzetdhebmyodsotsseiggnriofuipcainnt attoxsicandengdupionienatspitog.s (liAvpeprePnFdOixS 12). P"TFheOSdeactre4a0semkindtahendpeNr-cEenttFiOnSitEialatbo16d0ymwekightto 8wa5s%maonsdt8s7tr%onignliytiaelffbeocdteydwienigrhatts,bayt aalvseoradgeecrleiavseredPFinOrSatcsobnyceFntOrSatAioants4o0 fm> k6d00anpdig/Fg.OSTAhAe p(eMr3ce6n)t iantit1a6l0bmokddy wte0i9gh3t%waansd 95 %ofinitial body weight, respectively, at average liver PFOS concentrations of oafptphreoxFimOaSteAlAy t1r4e0atte0d2a0n0imga/lgs.waAsgprreeasteenrtfarsactthieonpoarfetnhtectoomtaplofulnudoorroacshemthieclmeitnabtohleiltiever FOSA. Similarly, the FOSA treated animals had a high liver concentrationofthe. supnemceiteasbolilkiezleydcopnatrreinbtucteodmptooutnhed.boTdhyuwsetihgehsteepfefrecftlutooraosgurlefaotenramdiedgerefelutohraonchtheemical metabolite PFOS in the FOSAA and FOSA treatment groups. b"TohdeyrawnekigohrtdeirnofrattshewaesffPecFtOoSfatthe40dimffkerden>t FreOaStAmeantt4g0romukpds >onNi-nEc(reFaOsSesElaitve1r6w0emigkhdt>to FOSAA at 160 mkd occurring at average liver PFOS concentrations of> 600 jig for tPrFeaOtSedaanndimNa-lEsX,FrOesSpEe,ctaivpeplyr.xiAmlaltdeo1ls9ey3ggr/ougpsfohraPdFiOnScAre,asaenddli1v4e0r tuog/bgodfyorwFeOiSghAtA.ratios, ibuntcroenalsyedthceomlipvaerre1d0 tboodcyonwtreoilghvtalruaetsiobsyofDtuhnenePtFt'OsSt-ttreestatedCorartrselwaetrieonosifgtnihfeicaclnitnliycal ccohnecmeinsttrraytieonndspoiinnrtastosfaproetnaostsisuhmo,wnchdolueestteortohlealnidmittreidglnyucmerbiedreosftodalitvaerpoPiFnOtsSfor each determination. b`TyheNd-eEc(rFeaOsSeEinatth1e60pemrkcedntanidniPtaFlObSodayt 4w0eimgkhtdwaansd m0os9t1s%taronndgl9y3e%ffeicnitteidalibnogduyinweeaigphitg,s aftraacvteironaogfetlhievertoPtaFlOfSlucoornoccehnetmriactailonisnotfhe41l9ivearondf1t7h1e Ng/-gE,(FrOesSpEecttirveealtye.d guAingeraeaptiegrs was. present as the metabolites FOSAA and N-EIFOSAA than occurred in rats. Thus, these. is 600028 pSRiTtyT2955, T3164, 48602, T2071, 71321 perfluorooctanesulfonamide fluorochemical species likely contributed to the body weight `eqfufieccktlyinthgauningeuainpeiagsp.igTsh,espeerdhaatpas sduugegetsottthheatinrdautcsibmieltiatbyoolficzyetNo-cEhtrFomOeSEP4t3o 0PsFiOnSramtso,raes. were shown in this report for rats, but was not determined for guinea pigs. `The percent weight by N initial body wei -Et FOSA at 40 gmhkt dwaasnadlgsorodwetchrewaassedheilndgtuoin9e9a%piingistitaol 94% initial b body weight ody by 40 `mkd N-EtFOSE, at N-E(FOSA treatme average nt group liver PFOS c and 66 g/g on in centrations of approximately 100 ug/g in the the N-EWFOSE treatment group, respectively. Only PFOS and FOSA were analyzed for in the liverofthe N-EtFOSA treated guinea pigs. However, the 40 mkd N-EtFOSE treatment group guinea pig livers had a large fractionofthe total fluorochemical in the liver present as the metabolites N-EtFOSAA and FOSAA which when combined, were equal to or greater than the concentration of PFOS in the liver and thus effects on body weight. may have made a significant contribution to the observed `The liver weight to body weight ratios in guinea pigs, in contrast to rats, were all decreased, although non of these changes were significantly different than control. The rank orderofthe decreased liver to body weight effect in guinea pigs was N-EtFOSA at 40 mkd > is roughly N-EtFOSE the inverse at 40 mkd > orderofthe PFOS decrea at 40 sed b mkd > N-E ody weight {efFfOecStEiantgu1i60nemakpdi.gsTfhoirseoarcdhe r of thesedosegroups, Given th ack ofhe Ives esponscs in guinea pigs af eer hepatomegaly or induction ofperoxisome proliferating enzymes coupled with the fact that an Intraperitoneal injection of 100 mg/Kg PFOS caused death in guinea pigs bu not mechaniom. in rats (See DT15 A), suggests that the peroxisome proliferation in the rat is a protective Correlation of of the effectof the clinical chemistry endpoints in guinea pigs showed that the rank the different treatment groups on decreased serum potassium levels order was PFOS at 40 mkd > N-EtFOSaAt 40 mkd, occurring at average liver PFOS concentrations of 148 ug/g and 100pg/g, respectively. In contrast, treatmenot fguinea pigs with 40 `mkd N-EtFOSE had no effect on the serum potassium levels at average liver PFOS concentrationsof approximately 45 ug/g in female and 88 g/g in male guinea pigs. Cholesterol and triglycerides different than control values, concentrations and showed no in guinca pigs were not significantly particular trend between dose groups or strong correlation to liver PFOS concentrations. not guinea pigs, caused by these acute exposures. These dats provid strong evidence Conclusions All treatments caused increased liver to body weight ratios in rats with the increase caused by PFOS being the greatest among all but the not guinea pigs, fluorochemicals tested. The evidence revealed the classical signs of peroxisome proliferation in rats, but that: 1) N-Et-FOSE and PFOS stimulate both the transcriptional and translational expression of acylCoA oxidase in rats in vivo, and 2) there is a marked difference in the 'tnoon9 SDRrPITST-S6295.8, T-6316.4, T6868, T7071, T1321 rreessuplotnssaereofvreartys caonndsigsutiennetawpiitghst1h0eisnuvgigveosteixopnostuhartetthoetsheefselutowroocfhleumoircoaclhecmoimcaplosu.ndTsheasree "clpaesrsoicxailso"mpeerporxoilsifoemreatporrosl"ifienrartaotrs"acnhde,mimcuaclsh, lgiukienewahaptighsasarebereensisdteamnotnostrtahtisedefffoercttohef fluorochemical exposures. " 610030 Prepare ty dia) WW Sel Andrew M. Scacat, Ph.D, DABT. Study Director sfesley Date . Gn0031 SIRPSTTB-6295.8, T-6316.4, T68682, T-10711, T-7132.1 Summary Tables VarieeT=e `Table 1. Average Cumulative Dose comilative bose (ng) sGeeecies Dc0onStE'_GRO N-EtFOSAMOmKd N-EtFosEleOmkd N-stroseiOmka erosdonkd Mos6-160mkd cont FOSMOmKg N-EcROsAOmKd N-EtFOSEIGOmKd N-EtFosEdGmkd Pros4Omkd sex dNeana0.0 sNo 02 0 Msoean 838..86 Nean 2163.4 sNWo 24.3 ean 01.062.2 N eana66.7 NsWD M3i2s.4sing MseDan MMiissssiinngg dneana0.0 sNo i0.s0sing Mseoan MMiissssiinngg N danZ366 KN 21.5 ean 21.284.3 N eanZ39.1 sNo d14 esoan 3333.2 ua 00..00 2122.5 02.0 1i6s3.8 2117.6 13 .0 7a5l.e0 3166.1 71.5 00..00 340.0 12.3 2 0. | 2173.5 62.3 502..6 41 6.6 3. 10 00032 SRDPITSHT6295, T-6316.4, T6868, T-1071.1, T7132. Ta2b.Sulmmaery ofBody weights and BW chang SPECIES 005E_GRO BT DO BDI(g) EMOD o cont vf9) ` ` wSea7n 241 423s107 uE-erosatonkd Fa wean 67 2 51 z 95 - 5woz1s 22 1z ELFOSE160rd wean 253 233 9% uw x 26 32 32 Etrosesonkd dean 635 623 99 prosionkd sNod 2i o3 dSea2n 14325 3 19 %2 SSE-160nka NMean MMiissssiinngg MMiissssiinngg MMiissssiinngg con 5v0 4Missing Miissing Miissing M=ean 2304 z3 n 32 ROSMOmkg Mesn MMiissssiinngg MMiissesiinngg MMiissssiinngg u- w50 zMissing M2issing M2issing EtFOsRdOnkd wean 21 208 9 5 Fxa 2 i2 22 EtFosE160mkd wean 200 pst gt ue wozs 21 23 Er0sEdomkd Men 220 229 10 rosiomkd Node 5i ii dsenu205 gip! sid! Signihcanly diffrent rom controvaluesby Dumnet's ex WISWDD BDI(g) BHDO 2 @ ` d2e6s 520030 1610 22 2 m1 o s : w1 222 25 5 1203 ow1 222 m Fetm 0100 awo ias `& 223558 s23umg 83sg 78 f7o s7 2B 7 s297 1207 2353 s3 as s3al 2 216 23 0i 3 m1 0 11 02 222 26 ast gt 25 225 s2 s1: z 331 6 01`01 12290 Igtm! g3 f 2 70033 SbRrPiTsaT6205, T-6316.4, T6868, T0711, T71321 "Table 4. Percent Initial Body Weight, Combined Data = Sov -- MSoane108 wetrosaoma NYear at wetrossicomid Nso 1 wetrossioma N YMeoanafnag2p"" erostonka vYooun 3 551 3 conn wYFoonon106 weptrossaomd YNFelan io1 weetrosatomka NVEeSan 4100 wse-teoma VNnean 3og! Fosatonkg vVo esn3a wetrossicomd Nd erosonka eEvoNan got Msooan at 3 `Significantlydifferentfromcontrolvalues byDunnett's t-test 2 0034 SBRrPiTsTs.2958,T3164, T0482,TLL 121 Table 5. Liver- and Kidney- to Body Weight Ratios, Combined Data. ow [Ee --TERenFfon :bone : weston YSeneB4s03 oo; nie! fen Bno3ok8: Mwissning S Bean e Bost gooluernt . [er [FA PPE S BRen C 000 Mvisseing oso BBKBeeannSoSot0t4o6 :MS0iosn%ssing Pe Bosh fnenodboes wdoomeme RS fHonosBoats :Mmisusinng BBonososes S0one [7 SiGe oie `Significantly different from control values by Dunnett's t-test n0935 SRPT T-6295.8, T-6316.4, T-68682, T-1071.1, T-7132.1 DTISB `Table 6. Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the UniversityofRochester. (SAPlEuC nSaiErXetgs/DgOSE FOS TOSA FOSAR ETFOSAR FOSE ELFOSE NETFOSE FOSA 18S. Gre _Roc _Roc _R ROC ALC ROC _gluc _gluc Tw F com NO 75T TROCO T o esoan NwaA NNAA NNAA NNAA NNAANNAA nNAA nAA NB-erosa no 20o o0 o 2 10mka Mean 104.6 0.6 NA NA wa NA nA 0.2 N- sno z30.1 02 8 Nz A N2A N2A N2A N2A [2 2 E1L6F0OmSkEa Mseoan 335309..23 71.60.2 211073.09 3%8.81.2 208..87 01..79 63.51 00.13 uE-BLFOSE noo2 22z 22 2 2 40mkd Mean 44.7 7.1 30.4 105.0 3.2 0.9 1.2 0.3 PROS snNo 435.3 3319 60.2 20 .5 1o4 o01 0o.2 o0ko 4omka Mean 140.8 24.3 NA NA won na na Mw ocoar soNO 11 10.5 301.5 N0A W0A NoAoNA nA [3 NA o Msepan W0A.1 NNAA NNAA WWAA NNAANNAA uwAa nnAA NE=LFOsA no2 200 c 0 o 2 toma Mean 96.3 0.5 NA NA NANA na 0.1 N- Nsoo 223.0 02 2 W2A W2a 2A NA 2 nA 2 0.0 2 B16r0omskea MseOan 4598.88.6 64..26 5162.04.4 216113..68 18.63.0 20..23 21..10 00.05 NB-LFose Nz z 2 2 20 2 2 2 4omkd Mean 88.4 4.9 20.2 54.0 3.5 1.3 0.4 0.4 pros nsND 84 4.0 0` 3 8.6 o1.6 1[I 6 0.5 00 1 00l0 40m Mean 134.5 41.7 NA NA won na na RF cow NSO 113118 23311 oMA 0NA N[A ANA NA n0A sMeoan N0A.0 04.44 NNAA NNAA NBAANNAA NA NA na nA ns-cross N22 222 22 2 2 T60mkd Mean 891.9 18.1 67.5 201.9 47.2 6.4 0.2 0.2 eros sNo z3.6 21.4 05.5 0334 2o406o1.5 0o.1 0oX0 2 n0936 SDTIRSE PTT-T631-6.4,6T-68268.29, T-57071..1, 8T-71,32.1 Tomes Mean 866.4 18.3 NA NA mow wm na uo owsse so 1343.0 43.2 3WA W0A No MA0 N0a Ao T60mkt Mean 140.3 316.5 555.2 NA mow om A cor sxo 825.8 1514.1 1013.8 N0A NoA oNM W0A Ao Mseoan 00..00 110193..26 NNAA NWAA [WLE owSYom NAA 0fmoksg N03 30 0 oo 3 Mseoan 12963..54 1167.72.9 NNAA WWAA mNoNw oNmA 00.14 N EtrosN e 2 2 2 2 22 2 2 T60mkt Mean 1124.3 18.5 84.3 284.6 12.1 3.8 0.3 0.1 eros SnNo 1266.0 927.5 203.7 1015.8 2o.5 o1.2 0.0 0o.0 soma Mean 969.9 17.8 NA WA mow om NA SD 8.2 1909 Na wa Nw wa NA 2 n0o37 SRPTT-6205.8, T-6316.4, T-68682, T-1071.1. T-7132.1 DTISB `Table 7. Summary of Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the 3M Environmental Lab. SEPsEC SEX oDOrSeE _BR3OMS 3FOMSR EWF- OSAR TMETFOSE 3FOMSRR NE-tFOSA_3M LE T E2 N J am T T sMeoan W0A.151 00.00 00.02 nnaA N0.1ANAmA n- No 2 22 2 21 E1c6r0omsked Mseoan 65.0645.7000 11019..30 2244.47.5 3610.22.5 11138.4.5 N0A.9 L 40mkaE] 22 o 2o Mseoan 870.34.75500 00.01 00.01 nnAa 00.01 mWAA Mw cor Mxean 02 .298 02.0 02.2 noA 01 .1 oma n- nsoo 20.13 20.0 02 1 2nA W2 ANA2 E1t6r0omskEd MSeOan 900.85.10000 89.95.0 3117.77.5 4124.74.0 11609.6.5 00..14 p4r0osm nN 2 22 o 2o MSeban 8160.05.10100 00.:10 00.:30 ANA 0010 NwAa % ~0038 SDRIPTSHT6295, T-6316.4, T-6868.2, T-1071.1, T-7132.1 `Table 8 Hepatic Palmitoyl CoA Oxidase Activity, Combined Data 53 conT T Means2 N-ELFOSAAOMKE so 04.8 esoan 21.0 N-ECFOSEIGORKE ean 33 N-ELFOSEAORKE so 0` .6 wsoean 20.6 PrOS40mkd Neo an 3 cour sxo a1.6 vsoean 3.2 N-BtFOSELGOmKA N eand13 PrOSCOmka sNo s1.6 Mean 1g sp 5. Significantly different from control values by Dunnett's ies e 7 670939 SBRhPTs1.62958, T3164, T60682, TTL T1321 Appendix 1. Deviations to the Protocol `pTehrefl3uMoroMoecdtiacnaclsuDlefpoanratmimdeen,tTi-d6en8t6i8f,icwataisonnontumlibseterd fionrtNh-eEtphroytlocol. The protocol used abbreviations for N-Ethyl perfluorooctanesulfonamide (T-6868) of PFOSA and FX-12. Protocol amendment numbe2r lists the abbreviations for perfluorooctanesulfonamide (T-7132) as FOSA, PFOSA and FOSAmide. The aanbbdrefvoriapteirofnlsuuosreodocitnatnheissulrefpoonratmifdore Nw-aEsthFyOlSAp.erfluorooctanesulfonamide was N-EtFOSA `The protocol stated that the animals would be dosed on days `zero through 4of the study". Thatwas a typographical error, as evidenced by the fact that even the example calculation in the protocol was for a 4-day dosing period, not a 5-day dosing period. The actual dosing period was for days zero through three ofthe study, and the animals were sacrificed n day fourofthe study. Antal wir ot weighed in mos cases an dey one of the study, and insome cases on day three of the study. In those instances, the previous days' body weights were used for determinationof dosing volume. Protocol amendment #2 stated the Dr. M. Wempe at the Universityof Rochester would perform themetabolite analysisofthe liver samples from animals treated with T-7071.1, T-7132.1. performed tDhueesetmoeptearbsoolninteelacnhaalnygseess,inDsrt.eaXd.in Lu at the Universityof Rochester KCliidnniecaylwcehiegmhitstwrayswnaost noobttapienrefdodrumreidngonnealclrospesryumunsdaemprlaesm.endment number 2 on 3/5/99. pHiesrtfolluoogriocsaullfevoanlaumaitdieosnaonfdlmiveetra,bkoildinteeyvaalnudestewsteirsewenorteonbottaipneerdf.ormed. Serum aInnddluocrticoenllorfemplRicNaAtionfowretrhee following genes not analyzed for associated with peroxisomal proliferation in liver: Peroxisome Proliferation Activating Receptor (PPAR), Liver fatty acid binding protein (L-FABP) and Proliferating Cell `Nuclear Antigen (PCNA). 5rooa0 DSTPITSBT-6295.8, T-6316.4, T-6868.2, T-1071.1, T-7132.1 Ta3b.Sulmmaery ofOrgan weights and Organ to Body Weight Ratios. SsPECIE ap R DOSE FLW LWB GRP Wrawtio CONT ON Men 4 196 4 0083 NEFOSA SD N 727 2 oes 2 40mkd SMDen 11488 000002i N16E0FmkOdSE N 2 2 SMeDan 0953 0000003 oN-mEkFdOSE N 2 2 D Mean 120850 003 oPRmOkSd ON 4 4 SMDen 61938 0000335 MSSe160mkd N Mising Missing SMeDan MMiissssiinngg MMiissssiinngg CONT OMNean 41 40038 FOSA SND M0i9ssing 0M0i0s3ing omkg Mean Missing Missing NEFOSA SND 2Missing 2Missing oma Mean 80 0038 NEFOSE NSD 024 2oom 160mkd SMeDan O7l4 00000412 oNmEkFdOSE N 2 2 Mean 99 0043 PROS OsND 4or 4000 oma Men 82 oon' SD__07 000 Significantly different fromcontrolvaluesby KW 2 4ol3 2 o4ss 0 MiMsissiinngg 2 0502 2 0411 Missing MMiissssiinngg ~~ 219 M0i1sing MMiissssiinngg 2 18 00 2 MMiissiinngg 2 20 202 17 oi Dunnett's Lest KWBW mio 2 00000016 2 00000018 0 MiMsissiinngg 2 00000008 2 0000007 Missing MMiissssiinngg 20000 0M0i0s0ig MMiissssiinngg 2 0008 00001 MMiissiinngg 2 0009 20001 0009 000 M LW LWB KW @ Wo 4 197 40032 9 6 101 2 0003 2 09 2 22667 00000337 0S1S 220 O8061 00000317 MMiisssiinngg 22 2 21684 0000031% 0s1s 42 91564 00000348 0s1o 330 01.008 00..000434 MMiissssiinngg 7 121 700a 22 9 1d 3 0003 3 00 0 01.07 00..000436 MMiissssiinngg 222 141 0046 25 0: 3 02001 001 11230 00.004080 MMiissssiinngg 22 2 iso' 0047 27 12 0a004 201 132 12 0o0o0n4' 2020 KWB Ww 02006 000i 2 00000080 0 MMiisssiinngg 2 00000007 2 00000018 0 MMiissssiinngg 02009 0000 0 MMiissssiinngg 2 goos 00000 MMiissssiinngg 2 gost 02000 o0o000s' 2 620041 SriRsEeTT.623196.54,.T688,68, T0711, T7132.1 Appendi2x - Analytical Liver Sample Lab Identification. [[RSetcuodry7d [[Tnuubmeber n[1e11e6528| ploeRrooaigcaosz 11116106|__3(8R0A04TIF IPnFaO'S,| SOir W Fe [rer ee | omJee] IFOsE, 8 [gGroosusp [[DNaetecropey[STpypeecimenl[Asmenttto [Afomtsant [[sAemntttol[sAeTnTtto | (CovanceR|orcih4e9s0t1e [[3EMnv [MUNniv la19b-01()1- [[Dualiuteh [[cCoonm || TTiura2o0smleliivveerr || _0034281=17sg-- I[--1T9a[[WNAA || [Cont |Tira0/seliver oon rides |O4ddl-tq |-Tg [NA] arenIspe] El l Oaal l WiA RNAclwwll lo is or [pepe] Iacal lI Tir20|a lo Ce ee [ooi e Fe[[FFoossee ri[ee |e | ob | re E|oo n[ep] i errosa| ioS TIg N CH Sr peeuTe rreee] = ' Talo co|sF T720G8 er [besed TC [|33/a1r/s9e9-40(70[sRo0ABalN [Com| eel1iver iP1m6Rk0OdmS:k [[o2212Ro[5oRR00000a44G6ESM7IF 0[CC[ooFNenmtt|||__S3fs5ei9b09lliivveerr 0 7s 075 = Fre | | 070a771--11a_|[-=1ag [ |=1a a| | 0088186-14 [1g[1a 4fence3 [| ROCHE R[ ote Se |omr e TTeg | [w[ecoentnrol80 158 [8R00471| INF-eote 3/5/99) [LIE bSRrPITsTa-62058, T3164, T68682, T0711, T7132.1 pe ee[ee ep [27 [oRooaerii [pfFroosse| wemalver | 083=Tg |-7g |-7g| [[2289 [[oorRooo0orasllFm [pprross|| vyssmmoollivveerr || 007s0tl3~11a_|J--TTqo ||--1Tgg|| [30 JoR00i7alF [pros| aiseBliver | 0776=1g_|-Ta |-Ta | UdSCR CRRsA 5 i AA Fl ll es I I f[ e eae] 1 ee ee pe 2 oi |e [re fe] Ir Fose Pr [pe A Fose Tere, | [we pe 3 FOSE | ge | | or[ep] a Fose [oiere me [|rm e pee | o [e 1 5Re pros[seme [! omeTe | fe | | | Dro | [los oraZW[Cot|BESOflii er [NA [Te JA[To | [liFoSmuAa||[--Z[3T7/R1uR01c0774a143lMMR[[CCooOnntt| |022Tzp02r0a2030a10ililili8ivvveeerrrIMINNNAAA_[|I|=~-F111ga9OISNNNAAAA|||-A--TT7agg|||| oe Ven" |sl8[1iR0074R91M o[FO0SA |72222as30/t0Tl0 lilvveel rr--NMIANA--_|F -[=11Oag SIJNNAAA [|A-T1qT a] contol [["S[ieARlTotRR00o07o77a4asS7|]MMMII[FFFOOOSSSAAA|||2222rasi1 b0itllii2 vveerr_y_IIN0 NNAAA_1 _J|J---l 1TTaga iII[NNNvAAA e |J=--111r aqg || 0 neoa3 SoRTPITsTs.62958, T6316, T8682, T7071, T1521 Appendix 3. Cumulative Dose Individual and Summary data. SEE Fa wmcrosnomks NecroseiGomka NercrosEiomks rrosioma Wetrosionkd Nescrostigonka Westroseiomk SSSeoornreses .Bsenono:80 EH ci N|u oseo5i36n 3s:6h00e05n3 113686 YERea fos :8i01e407 h110%3 Ywoon do22 osSeooooystns 9 3 .bseeainoose 3:ber wow coSooiunimi 0 YsH oonurs 000 woo ,fsoeise) }1323 [wrI :6h0o0o01r7 }116617 Mw ean gfess 6SS0o1r4i7s9s 111550 noone wr To ed Bfpm El fe Fa fae 1 N-EtFOSE4Omkd BR04042 40 e pet - 192045 eSsRPTT3T164., 6686282,T907151,8T13,21 uVSeRnST 900 5 oe sosndonka sitRooooiriss 4309 Y5en 50h,e0 w-prosadonkd H5sRoEi03e 51 : b3ean Eosi \estrossisoaka Sxsooookees 11:8745 b3ean 1019s wertrosEionka ueSRoo10i34 55:3 Yon oEi26 srostonka sSSrhroootosotosessrs 453s0 uYSoonres &{is i 55 55 n0046 DSTPITSBT-6295.8, T-6316.4, T-6868.2, T-0071.1, T-7132.1 34 Cn0ooa7 id 9% pg Beg oa Se 9 sum Blenas ds T F.73 Tod 9 rat FTIFeLN lof g Ig me Se e EF BRESR.E8 3 3.5 3.8% 8 8 8 223% 3 -i ZE | Blo Ecgeg geEo 2 Pr 5s%oo gcainfe g 3 :3 3 2 I J & g 2z z3 flA &: zl| 2 gf 58 < [G5 n0o18 bh oagg5dz gy FE En Edge ffE8 e. 39, lo "Bop om mg on 8a a. Zo Boegs.38 FAS 8 8.83 shAE mme.gd 22 Ld of gg SEERA EEN ERLE EFT c seer Er oz Torr or or oor o gol gr peer EE dOREEILEE PREP GIR ELE MHLE FoRERE.8Y BRET BEN 7 BAAS REISER Tp EBER.Ea B.Ie R.Es Beds BESELEe & = 25 i n00A9 & 4d : i5s L 3 83 3 2 3 z 8 8 I n2950 0 zSCE8EEY c|e TE s . ag %F - 208 o "ne 2 . 2a To ) a hE goBgesasas. ii 823.80 s 8.8 g 8.80 8 z i LiseiEENR0e dE Bis BRR A 8 = z z 3 i i < Hd 3 g go os g BR u5 - no05% 2 gpiigs HERE Elis 85550 g i 2:z a 3 5 n2052 ED PBlasii 88 2 T = 88 F 7 er % 8F . J a 88 = uf FoF dais i f.8f & dudS 3 34 i rr 2 roer Elkeidigr oc ERIE 7 oc 3 OLE 3 i3 EEE 22 SBT EE z ff E Plzzzo 38 2 PEesc.d 2 BE: sass 8 = 88 2 oo 88 no xd SEE EF 4. SE z= :E f [|fePssE F2s85 =58 E 2sgB8ABD1= E5 A2 d2 I. EF 8 3 RaEs82d% 3agayd EOE RRiRREe i: i2 IkB Sch i El BE f BBE RLF BEBE Hgr .Ii8 L2i BLii BN 0053 z gz 388 5288 853 [Bade SoEEnce 6 88% once poz 33s FoF & g zB 88g ofacds pails 18: 18 2g H SE 2 : Bt +3 RH i 2: . g Is g n0054 GEESE RRAEER OR ORRRG BABY RENE 3 153 188 fi i: . 25 CNa955 11 7 gr zefepr 22 regs 3S8H%E8S8 3E:Roi9E3E%N5F5E5S.c25E8Enc8e8 F3 H31EoHE8 5& 3oof g%% 85 8% % Fiisci: 38 1 8% $f 3 AEEadd HHoFFEFFENcEEacoc F EEoEE & aa g SpE ISmEmRE EEE i z dS.ddsdsdind4d) 88.95 5 44 g 855.85 3 52. ip0EEiEssd.dp 2 2E.ip & g: 3 lo 4EG $3F de 2 88 = 3% 3 zg i. 153 fgai g 8 & n0956 z Elis i I 2 &L5H | er roe me 8 GEE E.0s f EEi.is E2D 8g i 2 188 18% Ee n0os7? i z Bolssss.ss 883 z [iy E [Besssoss 5 sos 5 fecssss 5 sues 2 8 4 3 EXE585c55 S058 Fs2: Ff [esssoss 5 sss ZfF5 ls I 2 [Blssssoss 5 sess 3 esr E 5iEBE E| esssoss 822g.3f2g E oFE 3 L pHEEsfeE g E 2 eo B883.0s Bia 5i o5 }3 |. 25 s 8 8 78 ERAT Csr son oan 2 230 3038 4 F030 E REE 588 & 2083 doco w 28r8mg 8 8 2.86 4 5.50 5s e28s38583 5329, 5.RE 3.88 5 4 iin, 5 8 E808888d2pue0e?5 5 2fa:z siaE woaf 8 B.8s B.Es y2 g8e 3 L iPe i r 5 0% Bas C0058 ssoss ssssoss 5 SEE 3 IEE EIR. of ssoss ssssoss 3 sess Gad 0 Gu 5 sg 505s s3ssoss 5 sess Gail eid 5 38 sfoss ssssoss 5 sos Saino cand os gs ssoss ssssoss ssoss ssssoss oo PR Pz F8%E52 g lo2s8 22 HH2 sess seodeods J 83 - f= goss 3 RMSE 6 dad) goss 5 sess 2.3% fF dads 5 ss DIET CIINSREIIAE F8 if. i E3 i8% ai% 2 5d8r3 eag8i% gnt o3a $s3e tess gasEe i gs gz B gy w1 10059 oss ssssoss 5 sess S50 sess 5 sss. sos% 5550s 8 sess 4 Suid 5 goss 3 sss. 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