Document G5R7j9x4dnE71MMx5wkg60Mvm

SDrRiYS.TA-629%8, T6849. T2164 HA 74322 3 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY `Title: Inter-Species Comparison of Mechanisms Fluorochemical Toxicity following Intraperitoneal dosingofperfluorooctanesulfonate (PFOS, T-6295) or ammonium perflyorooctanoate (APFO, T-6889) in Rats and Guinea Pigs and oral dosing of N-ethyl - , perfluorooctanesulfonamido ethanol (N-EtFOSE, T-6316) in Rats. onFianalnReporotes LGo7r7n7. 30. Tom EBI lo BE Sponsor: Study Location: Study Director: Study Toxicologist: Study Numbers. T-6295.8, T-6889.1, 6316.4. a Strategic Toxicology Study Number: DT15-A = 33MM CSepnetceira,ltByuCihledimnigca2l3s6Division `Saint Paul MN 55133-3220 5a3 EI == 33MM SCternatteerg,icBuAilltdeirnngat2i7v0e-TSoBx-i1c8o1logy Laboratory = Saint Paul, MN 55133-3220 a TAonxdirceowloMg.y SSpeeacciaatliPsht.D., DABT XY 3M Medical Dept. Corporate Toxicology and Regulatory Services 3M Center Building 220-2E-02 Deanna Lucker M.S Senior Toxicologist 3M Medical Dept. Corporate Toxicology and Regulatory Services In-Life Start Dates In-Life End Dates In-Life Start Dates IInn-LLiiffeeSEtnadrtDDaattees In-Life End Date PartA: Part A: Part B: PPaarrttCB:: Part C: 10/23/1997 and 11/17/1997 11/04/1997 and 12/1/1997 02/10/1998 and 03/11/1998. 0093//1160//11999988 and 03/13/1997 09/18/1998 a t oR Sl ~ oib s w 600% DSRTPITSTA.6295., T6889.1 T6316.4 `Tableof Contents Me"tTheosdsNisei. m------r-- en--e ProPcaerdturAe:s..P.roceduresforsingle nesperioneal dosingo"fPFOS (1.6295) and APFO (1p -6859i)n a lerts and E PartBOProrceedurmeesrfeoraTimea.CouC rse oA fPFOSE(1-6L29) ovicity inmaleand fermasaandgluineea pigs r fPoallroCtwinPgraocegdluer1fepo.srJheopatnicpOfePrFOoS axlgi0e4ns0BeiOonSdmEu.ca.ti.lon lowingoral dosingofN-EIFOiSrtEs. ...6 ResPaurltsAaRnedsDuilstcussosniotnh.e Tosichyof PFOSand APFO inratsand guineapig. stanm y RERASSPUALAS rr A FO { LiCovnecrlsuasmiponlsesfrsoumbPmaitteAd:for LLFABPanalysis rom rats and gine pgs neaedin PartA. nano PaRrB:ERsSuRlAUsSofPSTAiL:me.-Course ofPFOS tonicity a twodoses in aleans dfemar le rse and e vine pigs. 85 RPeasruClts::RGesuluts ipOirganslPdoaesrinagofBi.s..w.it.h._20._m_g_/.K.g.N.-oE.I.FOSE .. -- Conclusions a> rsh TablGeusi:neapig. np SII 3 "TTaabbllee 21. PPaarrttAA.- SSuummmmaarryy ooffTToxkoityxooffiPPFFOcOSSaiannddtAAPPFyFO iinn mmaalleergautis.nea pigs --in "TaTbalbel34e. PPaarrttB.. S Sumumooam ffBBrooddymyyWWeeiagihgthstr iisnnRGauy tinscMaPiegs,aaMnendaSnstsdaDnedvSitatdioDnesviations n6s Table . Part B Liver toBody Weigh ratios in rats... 7 FFiigguurr1ee2:: PPaatrBtB:: FMeamlaelRetRBtodBoydWy WeeiigVhsgVTsihmTe.te.. mn IIei FFiiggoor43ee:: PPaarnBB:: MFeamlaelGeuGiunienaePaigPgBoBdoydWy WeeiighVtgsVhaTtimeTme_....___._______________________.___.................oorrrr2200 ApFpiegnudri4en:1.PDaeviB:toinssleoGtuhinee PrPoitgocBaody Weght Vs Timem.m.o.---------- 2] `APpapretnAd:iIRx.atPdaartAta ToxicofiPFtOyS and APFO IndivanidSdumumaarly Dot LmI------ ApPparetndA:iIxG.uinPaeratPBi:gTdiameacourse with :PFOS. Individual Data... --2 PrtPaB.rBtI.ndLiivviedruaWl ReatidattgaTBhiodtmyecWoeuirgsehrwaitthoPiFnOmSa.leandeveras comc binedo doa SrAS ns ayso ... 228] PaPratrBtB.. FMemaalesrtalLtWLi/evBeWrrwaetiioghsusmmaurymmary... B--12 PPaartrtB. KFiedmnaelye WraetiLghWttBo WBroadtyioWSeiughmtmaatroyi.n _ma.l.e _n_d_fe_m_al__a_s_c_o_mb_i_n_ed_d_a_ta_,_SA_S_an_a_ly_sis. 336 ParPtarBt.BG.uGinueainigPe:iaIgndLiviiduvtaolBeDoadrtyaWeighRatio Summary Saisie: re e--38 AppenPadritxBI.V.KiPdanteyCWOeriaglhdtosBiodwniytghWeNig-htErIatFoiiOnnS2maEl5e aInNdVIfAeUmaSl]e0g4u8.n.a.igs. SAS analysis... 4a3 References SII a 2 620107 SDRTPITSTA-62058, T6889., T3164 "ITnhteroredsuecatricohnpresented in this report was designed to elucidate the mechanism(s) that initiate Tpleuroorxoicshoemmailcaplrso.liTfhereatrieosneairncrhatosb,jbeucttinvoetsnweecresestaoridleyteinrmgiunienicfatphiegsluoorrporcihmeamtiecsa,lsbydissormuept3tMhe fwuhnacttieoxntenotf.liTvherefiunttyeramceiddiabtiendoibnjgecptriovteesinwser(eL-0FAiBnPveSs)tiigatbeotthherahtyspaotnhdegsuiisntehaatpiFgCs acnodmipf5ou0n,dtso alelaedriLng-FtoAhBiPghleervellesvealnsdo/forunfuanscstoicoinaatleidtylaonngdcthhatinitfiastttyheacdiidssruipntitohneocfyftaotptlyasamc,idanpdrotcheastsaintgs and guinea pigs will respond differently. Further objectives were 0 investigate and compare the mfouloercouclhaermimceaclhsa;npiesrmflsuoofropoecrtoaxniessoumlefopnraotleif(ePrFatOiSo,n iTn-6r2a9s5)a,ndN-geutihnyela pigs. Three perfluorooctanesulfonamido ethanol (N-E(FOSE, T-6316) and perfluorooctanoate (APFO, TS6o8h8l9e)nwieusreeftaels.te1d99in3)thoirs sstuusdpyecttheadt owerceauesiethpeerrporxeivsioomueslpyroklniofewrnat(iSonohilnetnhiusrate.f Talh.e19h9y4p;othesis tested was that these fluorochemicals would induce peroxisome proliferation in the rat, but not tpheerogxuiinseoamepipgr,olainfdertahteiodnati a tshueppaotr,tbeudttnhoatt htyhpeotghueisniesa. pTigheweefrfeepcrteosfenttheedseincoanmpaobsutnrdasctoannd pionsvtesetripgrateosresntuesdinagt tthheesSeocfileutoyroofcheTmoixciacloslohgayvemeaelstoinsgubisneq2u0e0n1tl(yWaslhloawcen epfear.ox2i0s0o1)m.e Other p1r9o9l9i;feYraatnigonefwailth2s0o02m;eoBfertthheisaeucmoemapnodunWadlslaincevi2v0o02i)n.atsoror in-vitro (Maloney and Waxman Lin-vFesAtBiPgasteistohleateefdfefctroofm ctehrotsaeinflruaotsroacnhdemgiucianlesaopnigtshedobsienddiinngtohefacufrlrueonrtesscteundtylywelrabeeulesdedprtoobe to pLu-bLlFiAsBhePd.elTsheewhdeerteai(lNedabmbeetfholdds,19r9e8s;ulLtsueabnkdecrofnclalu.si2o0n0s2o)f,tahned aLr-eFoAnBlyPbarniaelfylsyemsewnetiroened in the results and discussion sectionofthis report. rGoudiennetas pbiegcsaumsae,ylsiekrevheuamsabnest,tetrhseuyrarroegarteesimstoadnetlos fpoerrhouxmisaonmeripsrkoalisfseersastmioenn.t Hthoawnedvoero,thtehre `pmeorloexciuslaormeanpdrobliiofcehaetomriscailn gmeencehraanliasnmdsftohratthdeifpfeerrfenltuioartoestuhleforneasmpiodnesse ionfptahretsiecuslpaerciisesuntcolear. Therefore, the specific aimsof this study were (0 1. tChheasreacctoemripzoeutnhdesperoxisomal enzyme and fatty acid binding proteins response in the liver to 2. Pinedrifcoartematnoxeicxiptlyantaesttisonsufcohr aasnysetrhuemspcelciiniecsaldicfhfeemriesnctersyianndretsopoinvseerohistthoelsoegcyotmhpatoumnadys. 3. Comelate any observed alterations of the above functions to liver and serum levels of perfluorosulfonamides and their metabolites Methods "wTrhiistesntudpyr,ot(oDcTol-1d5aAt)edw9a/s18c/o1n9d9u7cttehdatinhatdhebeeesnegampepnrtosv,edparbtystAh,e B3ManidnsCtituuntidoenaarl abnrioamdalyuse: 3 010205 DSTRITSAT6295, T-6889., T6316. committee (3M IACUC Animal use application # 2088). Part A consistedofastudy designed to compare the toxicity and peroxisomal proliferation in male rats and male guinea pigs dosed via intraperitoneal (i.p.)injection with PFOS or APFO. The resultsofPart A led to further testing under part B, which consistedof a dose-response and time-course for the observed effects of PFOS in both male and female rats and guinea pigs dosed via intraperitoneal (ip. injection. The resultsofparts A andB ledtothe conclusion that i.p. dosing was not the best route of administration, therefore an exploratory oral dosing procedure in rats was performed with NE(FOSE in Part C Test Materials . "The T-numbers, chemical names, abbreviations used for these samples, and the chemical structuresofeach of the compounds that were tested in this study are given below. The currently accepted abbreviations and the ones generally used in this report for each compound are in bold: 1. Vehicle control (2% Tween 80). 2. T6295: Perfluorooctane sulfonic acid, potassium salt (perfluorooctanesulfonate), PFOS, FC-95, Formula: CiFSOr- K', MW = 538.1 gmole). 3. T-6889: Ammonium perfluorooctanoate (PFOA, APFO, FC-143, Formula: CHFisCOONH.', MW = 431.1 g/mole). 4. T6316: N-ethylperfluorooctane sulfonamido ethanol (narrow Range N-Ethyl Perfluorooctanesulfonamido ethyl alcohol), N-EAFOSE, EFOSE, Formula: CUFSON(CHICHCHOH, MW = 571.06). 5. Wyeth-14643 (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. `The protocol stated that Formula CyF,:SO:NHC:H, T-6868, ) would Nb-eettehsytledp,ehrfolwueovreoro,ctTa-n6e8s6u8lfwoansamniotdete(sFtXe-d1i2n,DSTuIlSf-urAa.mide TM), Procedures Part A: Procedures foar single intraperitoneal dosing of POS (T-6295) and APFO (T-6889) in `mal rats and guinea pigs. For part A, a total of 15 male rats and 13 male guinea pigs, 6-8 weeks in age and weighing between 150 and 250 grams were purchased from Charles River. Threteo four male rats or `guinea pigs per treatment group were used. Following an adaptation periodof not less than one: week afer arrival at 3M, animals were administered treatments by intraperitoneal (.p.)injection, 4. 610209 DSRIPTSTA-6295.8, T-6889.1, T6316.4 at equimolar doses that were less than 40%of the rat ral LD 50 for PFOS or the rat i.p. 24 hour LD 50 for APFO. `There were solubility problems in com oil for the compounds. Therefore, dosesofeach test material at 32 mM in 2% Tween-80 and usinagdose volume of5mL/Kg were used. The doses were calculated to be to the highest dose that could be delivered at less than 40% of the LD 50 for each test material. The selectionofthese dose levels is discussed further in the protocol. Fresh solutionsoftest substances were prepared for each dosing. The dose groups and final number of animals in each dose group were as follows: 1. Control groups. The vehicle control group consistedof 2 male rats and three male. `guinea pigs. Each animal in the vehicle control group received a single i.p. injection 2% Tween-80. In addition, a no treatment control group consistedoftwo male rats that receivednotreatment were included in the study to check for any pronounced effectsofthe vehicle control on the subtle endpointsofgene induction planned for this study. The biological and clinical chemistry data from the vehicle control group and the no treatment control rats were combined. 2. PFOS. A single ip. injection of32 mM PFOS in 2% Tween-80 (final concentration =17.4 mg PFOS/mL) was given at a volume of5 mL/Kg (final dose ~ 87.15 mg/Kg) 10.a total three male rats and four male guinea pigs. 3. APFO. A single ip. injectionof 32 mM APFO in 2% Tween-80 (final concentration =13.8 mg APFO/mL) was given at a volume of5 mL/Kg (final dose ~ 4. 7W0yemtgh-/1K46g14)03 a(WtoYt)a.l fAousrimngallee r.ap.tsinajnedcttihorneeofmWalYe giunicnoeampoiigls.(final concentration = 52.45 mg/mL WY ) was given at a volume of | mL/Kg (final dose ~ 52.3 mg/Kg) to a total four maleratsand three male guinea pigs. Example dose calculation for 32 mM PFOS i.p. at S ml/Kg, assuming an average weight of 350for the guinea pig this came to: (0.35 kg)(0.005 Likg)(538.1 gmol)(0.032 mol/L) = 30.1 mg/guinea pig, or approximately 86 mg/Kg PFOS. Al animals were observed for mortality and signsoftoxicity during the first four hours after dosing, at 24 hours and daily thereafier for the durationof the study. Body weights were recorded immediately prior to dosing, daily thereafier, and at necropsy. Following the first exposure and up to four times throughout the in-lie phaseof the study, the animals were housed individually in metabolic cages overnight, and urine and feces were collected. Animals were sacrificed by CO; and gross necropsy performed on either day 12 or day 14 post-dose. Organ tissues (liver, kidney, testes and pancreas) and urine and feces were stored frozen at -80C for biochemical analysis. Sectionsofliver kidney, testes and pancreas were placed in 10% buffered formaldehyde and submitted for histological analyses by light microscopy. Some livers were perfused with gluteraldehyde and submitted for histological analysis by electron microscopy. `Bmleotoabdolsiatmepfloersmwaetiroencaonlldecttoemdoantinteocrrfooprscyhbayngheesaritnpbulnocotdurcheetmoisatnrayl.yze for test compound, s 6n0RL0 SDRTPITSTA6295, T-6889., T6316. Part B: Procedures foar Time:Course of PEOS (T-6295) toxicity in male and female rats and guinea pigs following a single i. injectionofPFOS a two doses. Based on the resultsof Part A (discussed below) the doses, dose volumes, and the time points chosen for sample analyses were modified. A time courseof3, 6, 12 and 28 days was conducted to measure peroxisome proliferation induction in the liverof male and female rats and guinea pigs treated with either 16 mg/Ke or 100 mg/Kg PFOS, A totalof22 rats and 20 guinea pigs, 12 male and 10female rats, and 10 male and 10 female: guinea pigs, 6-8 weeks in age and weighing between 150 and 250 grams were purchased from aChnairmlaelss Rwievreer.adFmoilnlisotweirnegdatnreaadtampetnattsiobnypienrtiroadpoefrintootnealles(si.tph.)aninojnecetiwoen.ek after arrival at 3M, Eight rats and eight guinea pigs (4/sex) were treated in each fluorochemical dose group. The idonsjeecgtrioounap,s1w0e0rmeg1/6mmLg/suKsgpePnFsiOoSn aonfdPF10O0Smwga/sKgmaPdFeOSb.y dTisosollimviitngth2e0v0omlgumPeFoOfSthieni0.p..5 mL. `com oil then adding 1.5 ml 2% Tween 80 and creating an emulsion in a glass tissue grinder. A tshiengl1e00i.pm.gi/nKjegctdioosneofgtrhoeup1a0n0immagl/s,maLnPdFaOt S& vsoulspuemnesoifo0n.1w6asmgLi/vKeneattoathveoalnuimmealosf1inmtLhe/K1g6 to mg/Kg dose group. One animalpersex per species per fluorochemical dose group were sacrificed on days 3, 6, 12 and 28 post-dose. ``oTnweovaenhiicmlaelscopnetrroslexanpiemraslpeeacicehsarteaceviovledumtehoefvvehoilculemecoontfr1olm(L2/K5g%,cocronrroeislpionnd2i%ngTtwoetehne 8100)0, mg/Kg dose group animals, and at 0.16 mL/Ke, corresponding to the 16 mg/Kg dose group. One anima per sexperspecies per dose group were sacrificed on days 3, 6, 12 and 28 post-dose. `Two male rats received WY as a positive control group. Oneof the positive control rats received a single i.p. injection ofa 100 mg/mL. suspension ofWY at a volume of 1 mL/Kg to foar total dose or mg/mL 100 mg/Kg suspension WY. The of WY ata other positive volume of control rat 0.16 mL/Kg received a single ip. for a total dose of 16 injection ofa 100 mg/Kg dose group WY. The positive control group rats were sacrificed on day 3 post-dose. Part C in rats Procedures for hepatic peroxisomal gene induction following oral dosing of N-EFOSE "The resultsofparts A and B led to the conclusion that ip. dosing was not the best route: oEfAaFdOmSinEisitnrPaatritonC,.tThehreefpourrepaonseexwpalsor(a0tgoernyeorraatledsoasmipnlgepsrfoocredpuerreoxiisroamtaslwgaesnpeeirnfdourcmteidonwaistshaNys- in liver, and for N-EWFOSE metabolite analysis in liver and serum. A doseof20 mg/Kg/day N- sEtAuFdOySwEitfhor30t0wopdpamysN-wEatsFcOhSosEetnhaatscaanusinetdiallisvecrreeefnfeicntgsdaonsed.inTdhiusceddospealwmaitsoyblasCeod oAnoaxidideatsaery activity dietary sitnudliyvwear socvaelrcaul4a-tweedektopbeeriaopdpirnoxwihmiactheltyhe23avmegr/agKegN/d-aEyWfFoOrStEhecsoencsounmdptwieoenkoinfttheh.e study (fRoerft.w3oMdayMse.diAca4lmDgep/tm.LT-s6u3s1p6e.n1s)i.onTohfreNe-EmGalFeOSraEtsinwe2r%e dTowseeednwi8t0hw2a0smpgr/eKpgar/eddayinNa-gElXasFsOSE 6 0n0%1 DSRTPITST--A6295.8, T-6889.1, T6316.4 tissue grinder and delivered to the rats by oral gavage at a dose volume of 5 mL/Kg for two consecutive days. Two ras were dosed with the vehicle control, 2% Tween 80aat dose volume of5mL/Kg for two consecutive days. The animals were euthanized and necropsied on day 3 post-dose. Livers were weighed and chunks of liver were frozen in liquid nitrogen then put in polypropylene vials, placed on dry ice and then stored at -80. Serum was obiained by centrifugation from blood collected by heart puncture at necropsy and stored at -80 The ~ 2g liver samples that were flash frozen in liquid nitrogen were sent on dry ice for analysisofgene: induction to Kendall B. Wallace, Ph.D. DABT. Professor, Deptof Biochemistry and Molecular Biology School of Medicine: 10 University Drive Duluth, MN 55812-2496 Results and Discussion Part A_ Results on the Toxicity of POS and APFQ in rats and guinea pigs. IRn erats, utRhaeltrsetwPeasrrte:Ano statistically significant changes in body weight. Liver weight and liver-tobody weight ratios were increased following treatment with al three compounds, particularly APFO; however, these changes were not statistically significant (Table 1; Appendix 1). `The serum clinical chemistry analysis revealed that cholesterol was lowered to below the limit of detection of45 mg/dL in rats treated with PFOS. Glucose was significantly lowered in rats treated with POS and WY. No saistically significant changes occurred in the clinical chemistry parametersof ats treated with APFO. `The histological results for 3 outoffour rats treated i.p. with 52 mg/Kg WY (Animal numbers TR4845 7R4847 and TR4837) showed macroscopic massesof white spots on the liver surface covered in thin scar tissue and evidence of mononuclear infiltration. Multifocal microscopic. subchronic granulomas were noted. "The histological results for rats treated i.p. with APFO (animal numbers 7R04851-53) showed enlarged livers and kidneys. Macroscopic lesionsofmotled black spots on oneliverwere noted (TRO4851). Microscopically, this animal had a padofscar tissue with an imbedded granuloma on the liver surface. "The histological results for one rat treated with PFOS (TR04838) showed signs of liver damage repair. All other rats, including the control group rat, had no significant change present in liver, Kidney, or pancreas. 7 Croz12 DSRIPTST-A6295.8, 6889.1, T6316.4 Results: Guinea pigs Part A One guinea pig (Animal number 7G0253) that was treated with 100 mg/Kg APFO died overnight following treatment. No tissues were collected from that animal and the inital body weight (358 g)ofthat animal is not included inthe tables or statistical analyses. The cause of death is unknown, but may have been compound related. The guinea pigs treated with PFOS had significantly lower weight gains compared to control. The guinea pigs treated with APFO had significantly greater liver to body weight ratios compared to control. One of four guinea pigs treated with 17.4 mg/Kg i.p. PFOS (7G02254) showed signsofliver damage repair. No significant changes were present in the liver, kidney, testes, or pancreas of any of the other guinea pigs examined microscopically (7G2004 through 7G2013). Tissues from two out of three control guinea pigs (7G2255 & 7G2256) were never analyzed and were disposed of. Liver samples submittedfor L-FABP analysis from rats and guinea pigs treated in Part A Certain liver samplesfrom control and PFOS treated rats and guinea pigs were used for the 7isGo0l2a0ti0o4n-coofnltirvoerlfgatutiyneaacipdigb,in7dGi0n2g0p0ro5t-ePinFOatStghueiUnneiavpeirgs,itTyRo0f4S84a4n-cFornatnrcoilscroat(aAnndim7aRlOs4848ePvFaOluSatreatd).byTthhee agboialliotyfotfhteheLFfAluBorPesscteundtyfwatatsy atociadssaensasltohgeueeff11ect- (o5f-FCs on L-FABP function as dimethylaminonapthalenesulphonyl) - undecanoic acid (DAUDA) to bind to L-FABP isolated from rats and guinea pigs treated and not reated with PFOS in vivo. Results showed a decreased maximum binding capacity of L-FABP from PFOS treated rats without an increase in Kd. The `complete methods and the resultsof thoseanalysesare reported elsewhere (Luebker ef al. 2002; Nabbefeld 1998). Conclusions from Part A: "The histological resultsofliver damage with evidenceofscar tissue on the surfaceofthe livers ofsome animals 12 or 14 days after treatment with WY, APFO, and/or PFOS led us to consider that the doses, dose volumes, routeof administration and the time points chosen for part A were. not necessarily the best mode for the objectives of the study. Further analyses of tissues for total organic fluorine, electron microscopy, or peroxisome proliferation from part A were aborted and PartB was planned. P`agrutinBe:aRpeisgsu.ltsof Time-CourseofPFOStoxicityattwodosesinmaleand femaleratsand Results: Rats Part B All ats reated with either 16 mg/Kg or 100 mg/Kg PFOS gained weight following dosing and `maintained body weights that were slightly less than control values (Table 3, Figures 1 and 2). Statistical analysesofbody weight suggested that there were no significant differences between treated and control, but had very little power becauseofthe low N (statistics not shown). Liver weights were increased in both male and female rats treated with 100 mg/Kg dose groups (Appendix 3). The liver to body weight ratios in rats were significantly increased in combined 8 000223 DSRTPITST.A6295.8, T-6889., T63164 male and female liver to body weight ratio values from the 100 mg/Kg dose group versus control values (Table 5, Appendix 3). The female rats had more ofan increase in liver to body weight ratios than did the male rats. Hypertrophy and white pigmentation were noted by gross observation at necropsy insomeofthe high dose group rats (Appendix 3). Kidney weights and kidney to BW ratios were not significantly affected in rats (Appendix 3). Results: Guinea pigs Part B PFOS was more toxic to guinea pigs than rats, and apparently more toxic to female guinea pigs than male guinea pigs. Threeofthe four female guinea pigs, and one male guinea pig treated with 100 mg/Kg PFOS i.p. ied on day 2ofthe study. The remaining female guinea pig lost 12%ofits initial body weight by day 6 andwas humanely euthanized on day 6. All other guinea pigs gained weight and maintained body weights that were slightly lower than control values throughout the time course (Table 4, Figure 4). The guinea pig kidney to body weight ratios from all animals, males and females combined, were significantly increased by treatment with 100 mg/Kg PFOS i.p. (Appendix II), Female guinea pigs in particular were found tobevery sensitive to PFOS toxicity, evidenced by the fact that three out of four female guinea pigs died within the first 24 hours aftr dosing with a 100 mg/Kg dose of PFOS in 25% com oil and 75% Tween 80 and the remaining female guinea pig lost weight up to day 6 post-dose. In contrast, one out of four male guinea pig died aftear ip.doseof 100 mg/Kg dose of PFOS in 25% com oil and 75% Tween 80 at that dose, and the remaining 3 male guinea pigs survived and gained weight following treatment. `The administrationofthe 100 mg/Kg PFOS dose mixed with 25% corn oil may have contributed to the observed toxicity of PFOS to guinea pigs dosed in part B. Noneof the male guinea pigs died following ip. administration of a 100 mg/Kg PFOS dose in 2% Tween 80 at a volume of5 `mL/Kg, whereas one male and three female guinea pigs died when the same dose was delivered in 25% corn oil ata volume of ImL/ Kg. There is a precedent for deliveryofPFOS in com oil by oral gavage to be more toxic than when delivered in an aqueous solution. The useofcorn oil in the dose preparation has previously led 10. clear difference in the oral LD 50of PFOS in rats "The rat 24 hour oral LD-50 for PFOS in a 20:80 acetone: corn oil suspension was determined to be 251 mg/kg (Dean ef al. 1978). In contrast, the rat oral LD-S0 for PFOS in an aqueous suspension was determined to be 1.25 - 2.5 g/kg (Gabriel 1976). No deaths or gross lesions were observed 48 hours after dosing male and female rats with a single oral gavage doseof 250 mg/kg aqueous suspensions of PFOS (Goldenthal ef al. 1979). Thus, the use ofa mixtureof 25% com oil in an aqueous suspension of 2% Tween 80 as the vehicle to deliver PFOS may have contributed to the observed increased toxicityof PFOS to guinea pigs when compared to an 2% `Tween 80 vehicle alone. ConclufsoriPoarntBs: Intraperitoneal administration of PFOS led to the formationofscar tissue on the liver. The findings in this study led to the decision that i.p. dosing was not the appropriate route of administration, and that subsequent studies should be conducted following oral administration of these compounds. 9 Cn0tt DSRIPTSTA-62958, T-6889.1, T6316.4 `There was no apparent effectof treatment on body weight or liver weight (Appendix IV). No signsoftoxicity or liver effects were apparent by gross observations at necropsy. The rats used `wereofdifferent ages and initial body weighs, therefore, statistics were not performed on these. data. The liver specimens were analyzed for inductionof mRNA for peroxisome proliferation specific genes. There was no apparent indicationofgene induction by N-E(FOSE by Norther Blot analysis, however the total mRNA was partially degraded in all samples, therefore, a quantitative analysisofthe results was not performed. Further analysis ofthese samples was aborted, and the tissues were disposed of. Conclusions Standard toxicity tessofserum clinical chemistry and microscopic examinationofthe liver obtained in Parts Aofthis study indicated that male guinea pigs weremore sensitive tothetoxic effects ofPFOS and APFO than were male rats. In Part B, three out offour female guinea pigs asancdriofnieceoduotnodfafyou6r dmuaeletoguexicneesaspiivgeswdeiiegdhotvleorsnsi,gfhto,llaonwdintgheipretmraeiantimnegnt fweimtahle10g0uimnge/aKpgigPwFOaSs. In contrast, noneofthe male or female. rats died following a100 mg/Kg i.p. doseofPFOS. `These data showed that the guinea pigs, females in particular, were very sensitive to PFOS toxicity relative to rats. The findings in this study led to the conclusion that intraperitoneal dosing was not the appropriate routeofadministration, and that subsequent studies should be conducted following oral administrationof these compoundsto est the stated hypotheses. An initial oral dose study with 20 mg/Kg N-EFOSE in male rats showed no apparent effects by gross observations therefore the doses were considered to be low as there were no apparent liver effects, and the gene induction studies were aborted. Therefore, Oral dosing and. higher doses of N-EIFOSE were deemed necessary for future experiments. 10 R072 DSRIPTSTA-02053, 6889.1, T6316. Signatures Prepared by: Cmdnas IV). Seat Andrew M. Seacat, Ph.D, DAB. Toxicology Specialist & Study Director SS 25/acod Date . " r0v35 Fa SDRIPTSTA-6295.8, T6880. T-6316.4 Part A. Histopathology Report. Elden Lamprecht 3M Lab Animal Research Services: Rats 7TR4845 Macroscopic mass imbedded in liver surfacecovered in thin scar issue. Its substance hasbeendissolved leavinag foamy matrix of protein and mononuclear cells. The interface of the mass and liver consists of foamy mononuclear cells. 7TR4847 Macrospopic mass protruding from liver surface covered by thin layerofscar tissue. Its substance has been dissolved leaving a foamy matrixofprotein and mononuclear cells. `The interface of the mass and liver consistsoffoamy mononuclear cells. TR48S1 A padofscar tissue with an imbedded granuloma on the liver surface. TR4837 Multifocal microscopic subchronic granulomas are present in liver. TR4838 Parietal pleura ofliver is markedly thickened with repair tissue. A generalized `mild periportal vacuolation ofhepatocytes is present. Some degredationofhepatocytes may be pArlelsoetnth.er rats had no significant change present in iver, kidney or pancreas. Guinea pigs 702254 -A macroscopic. padofgranulomatous inflammation present on the surface. It has a foamy appearance with pore contents being dissolved leaving.The matrix which remains is composedofmononuclear cells, some ofwhich are engorged or vacuolated. The pad is covered by an increasingly thick layerofscar tissue. Scar tissue interfaces with the liver parenchyma. No change in iver parenchyma. A second smaller pad is present on another locationofthe liver 72255 & 762256 - never analyzed and disposed of. 762004 - 7G2013 - No significant changes were present in the liver, kidney. testes or pancreas. [12 l0ia SRPTT-6295.8, 6889.1, T-6316.4 IISA fe ef To | Tables: Table 1. Part A. Summary of Toxicity of PFOS and APFO in male rats [Parameter vg 150 lovg 150 Ticown fovg150 Tocowm fovgTso Joc [ 1 TTTT TT 1 Bodywtigdayt|sul val zeal asl soa sulsess evel ool er wo) |selofsol ol wed wa] ose]vel seesed orl rr rr ~ -- rT1 [verwii | wl sal wes] vol 111]see ea] reed rose sal rau] fiverrbodywt |oolool ood ous 112]ooeo| wore] vere oos| oo ver ( r-- -- 1 -- 11 lClnicalChemiswy |[| | | TT[TT 1 fhotmgidt |elowes | oo | elwelwo] elss ind camgist 1 we ord ol oa sed vedoul ood ria oso sed] Phosmgiat | ze osd vol owl exe vss rel wed 17 oul wif Jbmgat |ssl oa sel ool ead srloss iedseowl wd unmg T a vss r sd wel vol ood wes woe esi] vars ze se] lorum 1goedt od aol se sod sed wee ser] slsol cod Jakp 1u ws r ed sul el sod] wal eel vid sel ef ef stu | wn l od ol eal sed el eo sed al wl sdf AT run o oddevl ol od er] esel wor] ef 12s ed oT ro so ve wae walesdssod i esd su owwd Pasmmort | veal vil wed si od wel ai vod a os ef [eel osd oof ol md esoltoosled va owl l mg ermmoit T tofo wl vo ord we]osm ee] ess soo wd |ool oul ow oof wdowl ool isd oulog ug [RGmga1 t vn[ wool ml sd esd wel si eed] vel wl wed 21.. TNhAeNloimtitapofpDleictaebclteion for Cholesterol (CHOL) was 45 mg/dL. *Significantly diferentfromcontrol values by Student's T-test 13 00x23 SBRrTiaT2988, Tap, TasIed Table parameter 2. ParJt[iAg.cSuo1mm5aw0ry| ofroofgTox[ic5ipty0roofscPoFmeO|rSfoavng_d A1aP5Fr0Ooibnimcamlw|es fguriungea pig 50 comer| --I II fJuBooiogynwtgisti)n za| tzoece]] rseodfwseralsroadl ssoon]demruacsues]] ssoodd mneollw1esd] ewedd [or ra wrol our lvr aser l wel vr olvr or dwor oed fvCerrbooywiar 1214| r ood oor oesr soul_rr zse]our s oad rir e] ows or u 12d ffoCniomiemagraGtromm[y|veoTouT l oval[ val evilalT cw ool] wT ela[ ul | wi F[Prhaotsmmagtoa 1o0u]osodd osews ovuall a51dd eorw oouwl] ro1edl volooouull 121]d fro mol ossoo soudd aenaloonol] swudd seunsloousl] asodl ssuolosre] umdd fJaokremuyner T1sssec uordevemsosseollwwad eonoo]] soonw]sseeess]] osdodd soovseo]mrae]] eeeedd bhretuor Twoon wuolssosao]nese]l easdd swisae]lszoon]] osoddsaallmsaell eendd Jioomemmol Tovnsoou osdel ensssoe]svo6a]] oorndlsresos0soo]T2c5oe]] 0s0odfsmoawos][srowe]domsee| JJoemmmmooirtt Tosoeso csoeddmsosso]srsensdod c1as]o]oowull smaellssoommoos ssooww]soowwe|| fJoooRerAumnga Tooeu]oedlpoewloool eoadd woenlosnall wndd osulloouoll wudd 2 TNhAe NTotrpapDlecet for Chole! (COL) was mpl + Sianfcaly ifn fom control ales by Sdn' st oxo SDRTPITTS-A6295.8, T-6889.1, T-6316.4 `Table 3. Part B. Summary of Body Weights in Rats, Means andstd Deviations CET [GossPFOS] Day [Mean A [ECTTT va T70[7} [F 5Te "TR m ATo] [F 6To 201n 712] [TF 72 I2C TWa[7} [c 25 20m WNATi} [FE T+1s T+Td [F 5Tww Twi r 1sTe ar 17[5] [FT T 7 26 T7717] Fe | 25a wali] [Fe Tse Ta 14) [FleTT Twi] [F 1 ee 16[5] [TF vTz w 127] [F 2 | 28w TW[7] en12] : 1s Jo] [T 75 T o WT o w Jo] [[W 6T 200C Tz] r Tv1 e 7TRA T7] [2 o T 4mn TTT Pw] ww T71 20 Td] [eTw am Ts WIT] [RT %T& = TT 7wIs fl wT=1 = 212] ewe 14T% w [7] [P T+T 7 =T 0 9Ta [7 5 270 TNA Ti} e TeTw am 17[3] [TF 7 5o Ta[7] [o | 26 t 386 o [WAT] [c T+To 2a Tm 9572} [MTCnt T5TW TW [0] [con T6120 iT[7] [C 72 o 73% T n W[7] CW]Cm|2 | 45 Twi] Is 0220 DSRTPITSTA-62958, T-6889.1, T-6316.4 ``GTeanbdleer 4D.osPeaPrFtOBS.DaSyuPomsmiaDroyseofMeBaondByW(W9e)iSgihdtDsevi.n NGuinea Pigs, MeansandStdDeviations (mga) Fo cont 1 s a 2 FFoo CCoonntt 63 xRNA0 20 FFo oocCoomm 2 0703 MNa 1 FFote oe 13 s Mz N 41 FFoootee 26 www oos2 . Foe = wo NA FFoooo 31 wNsR N2AO4 F100 6 wo oN 1 FFo 100 2 NM A NNA 0 Mo Cont 1 mw 7 2 MMoo CCoonntt 36 NR N0AO2 Mo Com 2 si Na 1 MMo osCom 21 ammNa a1 M6 3 mom 1 M Mo6s 25 w as u232 Mos 2 e021 MM o 00 31 wde 7NA 41 M M100 26 sm s wwa 21 M10 2 MRNA 0 16 R0Z2% Table 5. Part B Liver to Body Weight ratios in rats [DoseGroup [Cot [16mgKgPFOS-- [1omgKgPFOS [Control [temgxg [Tome ---- [Species [Gender |N [Mean | SldDev | TRat [M&F [4 To05s --Joor | |Rat [M&F [8 loos Toor | [Rat [MSF 8 loos" [oor rr 1 1 T1 GP 1 To04 4 T0003 | T" GP 1 Toosa % ooo | TGP aTo04 T0001 | --7 77 7 1 . 0020R SDRTPITST--A62958, T-6889.1, T-6316.4 Figures Figure 1: Part B: Female Rat Body Weight Vs Time. Time-Course ofPFOS toxicity at two doses, 16 mg/Kg and 100mp/K. Female Rat BW Vs Time 30 20 ET 5 2 ENE ge 100 50 01 3 6 2 Days Postdose ----Cont -- 100 0h5a0z2.3 SbRTTISTA-62958,T-6889., T63164 Figure 2: Part B: Male Rat Body Weight Vs Time. Time-Courseof PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Ke. Male Rat BWVs Time 0 a0 Sy 530 ie Gan 50 a asTo 10 o 1 3 6 2 Days Postdose Bo POZA BSrRiTn. 988, Tat), T0164 Figure 3: Part B: Female Guinea Pig Body Weight Vs Time. Time-Courseof PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Kg. Female Guinea Pig BW Vs Time 20wo == ie i 000 wo] o Ts os nom Days Posdom R025 DSTRITSTA-6295.8, T-6889.1, T6316. Figure 4: Part B: Male Guinea Pig Body Weight Vs Time. Time-Course of PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Kg. Male Guinea Pig BW Vs Time 0 0 5 50 SN i Ne 20 100 01 3 6 2 oz Days Postdose -- Gon J |. 2 C076 DSRTPITST-A6295.8, T-6889.1, T6316.4 Appendix 1. Deviations to the Protocol: 1. The protocol states that there would be Five male and five female rats and guinea pigs per treatment group, with 5 treatment groups fora total of 50 rats and 50 guinea pigs. In Part A, only male animals were used. Three to four animals per treatment group were used and a total of 15 male rats and 13 male guinea pigs were used in Part A. In Part B,atotal of 20 rats and 20 guinea pigs, 10 males and 10 femalesperspecies were used `Thus, in both partAs and B, a totalof35 rats and 33 guinea pigs were used. 2. The protocol stated that N-ethyl perfluorooctane sulfonamide (FX-12) and Ne`Tthheyslepetrwfoludoorsoeocgtraonuepssuwlefronearmeimdooveetdhafnorlom(FtCh-e1p0r,otNo-cEolW,FaOnSdE,a Tpo-s6i3t1i6ve)cwoonutrlodlb, eWydeostehd-. 14643 (WY), 2 model persoxisome proliferators, was added as a dose group. 3. The protocol stated that all treatments wouldbe administered in com oil. PFOS (T6295) and APFO (T-6889) were administered in 2% Tween 80. Wyeth-14643 (WY) was administered in 25% corn oil. 4. The protocol stated that Dunnett's t-test would be performed on all data Student's t-test were performed instead where noted. 5. The protocol stated that all treatments would be given by i.p. dosing. Dosing by oral gavage was performed in Part C. 2 n02.77 I [W wh nm h [TT TTT | T= 2 5[~a[Ag[so| 5[ 2 | 3[Aw]so| El lw al lf Lo 122 272 EbS e arr e ee e a osee =o pean TT | | 747] 60]72 4of 72] os] sa 830 iid a i ---- tI {1 TI I TI | [over | i251 159] [232] 17] 59) |__toi o e7sl9] 74 festsi) |33 34 20]30]3a oa3333 3] 323 oil ---- + rrrrr 1 [C| imcaiChem|iI stI n[|[TT| [Ch|oi52mg63ial 47MM S40] 82045 Jca5 a5|NA| NA| Camgidl 15 117] iTGMMM116]off ios fiz iil iio 02) eiLmgat[oi oi oilorl 03 od oi pubmgol13 57 s SIM ss ol 35] [rPmoal | 62] 64 iM 62]02] 63 JpuNmgioL |" 13 15 13] 20]153 33 ial |oL|Um algai 6]dol7Ji 263205 Tiel JakPun 127i] 326] 451]200 33a] 80.9 276] [isTon | oe se ial719] 255] sof sa] pron er 77) e2l oa] 7of of 70] jronun |332] 286] 556} [arr | taal iol ras] [emmon|2" el 7oll eslod s3 |c-mmoit 0100] o3fEll Toi] 1S es] JREAmgial [05] oS oe oi od joorur Ts 8 ej eo oo oi oi oi oq 36]32]34 02 62 se ei 02 wl is]i531 Tee] 6a]140.3] 26.3) 43] 200]337.0] e635 oo] se] eto] ef eof ei so id serl sao]saa 552) aa aa] taal 3a) es ss ee 17] oo ool gsol if od o4[ oa oof 8 8 so od Crnz 3 SRPTT-6295.8, T-6889.1, T-6316.4 DTISA PRaatridaa:ta. (con anmal I=T=2 TsT~783Tag|sol 11T2a || 2" 84 3%Avg |sD]} TRO000 8 s7 [PaBordaymwetter|TT (o= ra | a aso I as] as Pst Irns Iaa |Ezs |Ecto ParYo0PonAs [PsYr diz 340 a13 a 27 376 3083 Jutssn |ss]so] sof sr0] soa]"7 soo]v0] 113] oo]ss ens] [gain% |70% |38% |37%|11%|38%|02|39%|43%|43%|17%|35% 0.1} [iver | 2801166236]344[266]64)196]186]185]256]205]34] F me oon ose 00r|oan 000] ool]oos[00s ooo] [Kidney (@) | 30 35134]a8]3oloe)37]32134] 43]37]05] [Tests@)|36130]34]36 3alos)37]34]32137] 34]03] 11 1 1 1 1 1 [Cinic--a_t|cr| em| is| ty |[[| [" T] JChoimoral | 530]56.01520]7601600] 12}650]630]600] 530]603153] |CamgdL 1 1151114]ia] 123117]04) 726 1111.2] 108]1141os] [Phosmol| 11.22.0121]7as 126116) 9211720 1.5]1.51 11.8103] [TeiLmgal oT011ot 02]o1loi oToi]of oil oil00} [Abmgal134134]30]a2]37104)341 351 33]35]34101] |TPmya 161161]661 7.1]65]05]61 63]61] 66]63102] BUN mg/dl|140161 0117 60]13 45]0 13]170] 120] 730] 170] 48]26} [GLUmgiaL| 206[175|145]ag1|254[760|210 142|139]206 175] 40} JAPULTats] 284|370] 4151 37a]63] 3321 401] 284| 245]316] 67] [Aston eal a0]o80[76.0]880 00)760 80 760] 304 135]173} [ATULTes] os] se] vol e7| ef sal 7a 62] sol er] 12 JrNoatnun1340254]306 451|334 Go| 262 233]167]2205] 717]ve3] ER PP PP V0 SYPY PYIP PY [KemmortT7071 80] 91] ool8s Tol 73174165173 71104] |CEmmoin 701 100 tot[ 102 701] 100] e6] v6 102 eo 3} CREA mg/dL [0a 04] 05]04]00) 05] 04] 0a] 06] 05]01] |eToRITGun T 80] so] solsol soloof 80] sol 80] sol soloof [me | wor]violwo smo sor]sofsn]sof rs]sn] rs| eo = Histoiog "A=sacrificedonday 14ratherthan 12 Limitof detection for Cholesterol = 45mgidl -- -- 2 aen0z 9 SDRTPITSTA-62958, 6889.1, T-6316.4 Part A: Guinea Pig data |im_al|# " T ~2T~3TaA[svbgJ5 T2 T%3 T [orf[m=] epeeO] l [Parameter -- 11TT1 1 Er lw)vw =]ww) at TT ~aTAvg [so | TT Go |]dl ld Wd [moan Toi 86] oi so] 3] 7a 42] | sol sol 18) [ver I i72 i671 177] 172] os] daili64] 207] desl 175] 34} [{iverbodywi|0.04| 0.04|0041004|000] 003]004] 0061005] 005]001} [ioney @)| 307 341aT38]oal 351 so] 44] aa] aol od} [Tests@ |12] 16]15] 14]o2| 10] o08] oe] i6[ ii]o4} r -- r --+ rr rT 1 [ClinicalChemistry || | 1 1TT [Choimgial| 6ST e651 451 ss] dol oI as 48] e2| si] 7) | el wl wil wel Ap wil oi] wil wl wil of fProsmgat to 4a nT v2 2p] 71 8] ni] sl 1} |oa] 721oi] os] oe} of oi]oilof] oi] oo] [Abmgal 221 28124125]o3p 21] 22 22] 25" 231oz} |a6] ssl ao so] os) 45] 46] 47] 71] 47] 03] BUNmgalL |" al 21 200" tel 4)" wal da] 200 7] 6] 3] | piel e6sl217 267] ro) 17] 30] Tas] 222] ao] 4s) | a251273a3 g| i1 or| 276| sa0l 264 252] zea 30] |er] sos es si] isa) se 47] ao] eo sal el |se 71] esl es] sf 49 So] ss] 4s] ss 5) | 2771204] 203] oi q050| 783] tas] 173] 265] 763] 50) |ies i38 dao0 ] 4) daz] 30] Gaol a3] Gar 2] xe mmoin._| | 17] 99 25) so a3] aa 7s 53] 7s) [CCmRmEAoit10a] 1031 doa] doa] C1O10s] Foi] doo] os] dos] 4} ld Wd | 200 1 dol tel io] sal 31 +i] 33] 7} [Romo]157]37s] 275] 26s] dos] e6] 94] 0] Teo] is] 55) oih230 SDRTPITSTA-6295.8, T-6889.1, T-6316.4 GPauritneA:a Pig data (cont) + 1 1 TT1 7 "TT T | Ee,[= [= =e | | [= ee | [Parameter_----_---- Gorm[0]SR]ST ow] S Se]ee ] mv a | em]] Eee] Ee]ww) 12 [wigan--{46] oi] 46] 61] 26] [iverI 7681 feel vol 76] 12 |iverowdty| 005] 004] 005]005]000) [Ron(@e)[ y a2 301 38]aol02) | ai] os ol doloi) F Clinicat l Chemis1 t 1TT1 [45 T<a5 1 52] soINAI [Camga--l{-- 471 i] 2] ail if [PhosmgalI oT 8 ol of of |oT oi oiloa]oof | 237 22 26 24 oof | asl 46 si] a8] 03) I wsT ve sT ws + | ol wai] isl 30] ef ALKPUL |3231 260 367] 327] dof | es] a9] 60] 66] 200 [si ar[sss af | 342] is] 237] 264| eel | faa] a0] | 771 oe} | e202) | oz of fCREAmgiL] 031 03]04170331006] os wo]s ss 2p ELIE| 27] iii] ies] dao] def | si] ie2| 004] ail" oe] 7 78] so] eo] 2 jor| tea ieil is] 005] 005] 005] 000} 48] a6 45] od] is] os] 12] 06} 77 7 1 53] 45]as] as] | aliow o of 8[ sl sl 1] oi] oiloa oil ool 24 23724 24 01] a7] a5]48] a7] 02] so] 20] ss 18] 3 vai] 22]22] isa] sal ace] 2se[317] ses| 75] ee] ea] S53 zi vol ar] 20 a7 38 16] 2501 252]2i2] 238] _23 NA TWA] INTA NA] [Na Tha | 04 03] 03] 033] 006) wil nl sof w[ 1 27] a3] die] dee] 4] ["=sacrifcedon day 14ratherthontz | 2 023% b| o fi | 1 |ine| 1 4 kw e SH iaa p || ii LhdIGoT dad dL] Lfit 128 feellLatTtTtEs bbl DEi ids lo5 ed eiinels] ss Ea UFee SGlREmY mmnmae C0232 swe BPE Part B. Liver Weight to Body Weight ratio in male and female rats combined data. SASanalysis f=t <= 3a| i = >=4 OO) 22 [OO w= - ww [--i CO sooens st Duonenestrs. fra S J--m-- Spasyes jroraersan Sooinea: f sore p0TF J VoSEmmoaEmsk omCwseoanmsoaemwsee orCntay Tha 5 lw Swe ce L==otJ-- 3Tooolwe yooogmn En Doane a r=o 30--Erro--riYhessopkoeidhearmotwofancre vreresomm E=n $ Domm laoswms uTomm =zDtcHean eanpatoToo,usmsey oouotms6 o00uoSm & oom So ind pJ nCanboraJ nac'hoirw rooogtSrae desr cm OTIS 11698sud Gi0o Cdooooooorrsssn ootooooutmmes occ0oe0ann0ss CPoompsanssovnalwueishsahocwontaoifmneaonswtnhtsarWeet ican diferent r5o2siohsl core0s0 o3n Sdoooanr PGoasioenvals showpasofmeans ht re sicanty 26a0234 -- Part B. Male rat LW/BW ratio summary LW/BWByDosePFOS(mg/Kg) oo a NYA JAY q ow CIN Eos Lo SN oso N\&L / T q / oo 0 i0 com mwachPar fav eGommme BFeiee te, ss=aem e mCr o EUEpEa EE i wes oona BBONo--I -- RiE odgEmm siomae pBONo t MEfREIerRlv oEmRgIemts RyRoosoeCUES 2= var coma ens ogooll saomd ermeaeEounr a Eo ond . Cnnzas DTIS 11/16/98 study cont 00000088%8 00002120s3 o00t156621 CoPmpoasrtiesovnaslwuietshshaocwonpraoilrsousfimngeDauntnnheasttaMeetshiogndificant diferent. AbsO27L0oS6l0D2 Cont C1o0n0t "000012167234 16 "001848 aPoesirtiav.e values showpaisof means that are significant 31 000236 Part B. Female rat Liver weight summary Le By DesePFOSmB) SIN q 1 AA DTS 1698 sy "a NE N ESE omrotmene oo ! Rsaure SSeoarroyt oss RaRenkaoeranRnegSsiuorees ror afraehrn Coemeions tosumvie) % HsIoac oFT:o AnaailomfyoVhlahsrSiomiganmrsceeess MemedUSeSahmmeey nFeernrae 5- I 1 +4 LiotMeoSraanmeveTnayArescusamn S4Em xEon LPoohaun om Love SdtEe rmr uosnsdgs cSedoavnrsainmatSoufpeorrviScE om EiIn T DPR onhJ ees owe Tam ode DIienteM)eansCampoarconTsd otweh ircim Exn SI008RoMm sulow ApaCompforsach\rpcius0r0ssScoentns sboascoisD 20004 2a ole6 acRom tooza? DTIS 11/16/98 study. c1o6nt 072m0I7028 2248174528 23845147720 CoPmopsariiesovnasluweissthhaocwopnraolsuosfinmgeDaunnsntehtataMeetshiogndificant diferent 270o6l02 A1b0s0O-LSD 184c4o1n5t co16nt ""33z5s0176658 ePorseitnitv.e values show pais of means tha are significant BeRn0n2:.38 PartB. FeWmalEe rBatyLGoWs/ePBFoWSirnagtho Summary OTIS 111698 study oes. aN\ goo NV 2 LA)HED. 9. | aos < >) fl oon ~o~ { \l \ source ous os Rsqare SOonemvaayndororvhe osuteis RRNoeataumaartaeFnAesdasorrsos Err SSeoStsoesaniets Osavatons (or SumWits) (] oSdorac OF2ArGuom"ftoVlaoSrsaaonmcmons Me"oGnoSoaewye Fsnraakte CEraom 7 GOoooaosmmziso ocoooeoosr modaets wiooveMeansfomOooeway aAorvoenm s0a0r0e0 Gion I oGoloam aoooeo Low StNEeroramorresunsaensd8poagDMlaoeavsntsotninstSe6ofDeeryr variSancEe Maan CiIont I o0Ooodio0tomm ooooccooosemsneesso accooourses OSfo ean eanM]eansCampaorsoonTosoo oooezi6o 0015c6a0m0 SEoym 0"00G0os 0`0o0o0o0 o00o03m80 ApparComporaeacrh\paius0g0osSunts AToosiLSD 20o84o10n0 000077 oocrm P 620239 DITIS 11/1698 study C1o8nt 0000001027317 00001008022 000010481027 `PoCsieovalwmueisshphaocowanptarrdorllsuosfiinmgeDasnustohatt naMreetsshiogdifcanty ferent. Ab1s0(0Oi27}0L8S0D2 0.00C0o5n4t C1o6nt ""0o0o0e02718 aPfofseirievnetvalues show pairs of means that are significant 3 010240 DTIS 11/1698 study Pans. Kidney Weight to Body Weight rato in male and female rats combined data. SAS analysis. KWBIWBy DosePFOS(mgg el REA oe AA al N Al Y Pp \ NE Sy | -= em Ee ~Co~ r a) Shaoas r Ouavsers FS Roz NL DIS 111638 sudy Roe OSnomvaasrrotva 007634 HRReooanorfeaARnegSsoareosEre d"Cotooaoos0strss Otseneton orSum te = tosEsraermc oF2"AnaiisimavoanSriaaounceess MemSTahmse oFRoaoto OE sar peer Coa fo odmwess sarer owes LiondMeansfomr OoneevTeyoAnmcrasmnn sSoowrs Coint LoOmuras oooums Lov idHiEororrsveasndaSgaoDcllaednvsiimnatDoefrrorvorsnaceirhean iiCon TDoOOumrEesr oaOowownes oOooowmmes iDIwiceant earH]ears onp00r00c00n00s0%00 oGooooo0lo6dm oomrooeoizsms Con Boos oor oooomme AppaCompforeas fpri airsusoisonsScnentss Ai5xons 210000000008 So o0o%m6 0Siocceoorn Co Sos 50% aoe PC osi o vawsim shhoowpnitp soafnmgeDaa unnntorn eosrisnc arnt Abh xi231s7d 2 oaConu Ciot aSoooen PoGisrioenvalvesshowacsofmeansht se scanty 5 R02AT i aap [||a[T]TT as| ||| dL LE] uLo|TeE pot | | | assem sacs sopLLLbeastfsn, fon.| [1 [kl11] 1a] fi | [1][ed[LN[letl]] Joss | || Liesl.Last sss. |[LWIAHIL]T seca5 1ps aaanananuens| lLIa LaLLiIE Fp0kLLLHIIE So GR0ZA3 Part B. Guinea Pig Liver to Body Weight Ratio Summary Statistics: LWBWBy DosePFOS(mgig oat Ls AR C -om 0) T 0) EI Car a) aos Ca aos 640% a DTIS 111698 sy Rsaure Sruemvmaaryy inotva oosezse RReooaqtnuHaereRoeasSpaounnsee rer Qr"toicisoessira Ghaanatons (or SumWot) ti ESroosruece OF2 AvSauomt"foolvsoaSoraioaounomscseeoss Mem""STaooukrores FoRnaeos Ooms 00% probr Clow 18 Gomes does ow tioMveans oRruOmenr""evoacvysloan 1G0ooEsree Cion ooown Gaooos Liol 1NHrumobreeTsressndaSaopioolMurDeasodavsniestsoanstoSeiwogfDnrevr variaSnuceioroeamn Coin 4o oDoowmuo aoomass cGooneess O0fMeant ear]ears Compa0r0is0o0nos0o oocr6s oorceodn Ciom "coontss 0`0o0c0or0z0 0O0o00n0a0 ApnaCompfoaeacrh|piiussi0n0gosSunnts Aoo040 2160o00u0s oom oocomn Gion o"uGosizts Ooomzee coooes CPosoie vmewsiphs6hocawopmaaonssoifnimgeDaroosmthtnsaWsasshiegdicanty dirt. Ao0siN24L3S5D0 ai00 CIont Spormees PGorseteenvaluesshowpaisofmeans at re sigan 002" OTIS 11698 sy a DoseprOSimpg Part B. Kidney Weight to Body Weight ratio in male and female guinea pigs. SAS. analysis -) acne AL ZN L220 como <3 1 <b @) ( ) 00 [-- " Cont. Sonr. Niners a GO 6 DTIS 11/1698 study Rsquare OSruevmamyaanorvFyai osessr RRMoeqoatunroHfaaRnesSpquoanrsee Ero foQarscezede0sda? Obsenatons or Sum ts) % esoawnce 02 AnSaoifTmVoiaorooiakssnocw:ee MeOomoeo FeRsaso ESnerT Bie 0O0o0mmse ioomeeers poeoro LIo)vMeans omr OanervTayAonovaaian 1o0oErnr Coint 7i GOooaurks o00o00n1 Low S14Nre oombrersa easndaSgir aeDlaovns ietstoinmsatSe@ofDeeyre varSianEce bean CoIoont Ti7 0Go0ao0sEm ooOdooweoow 0do0oo0owo0isss O0fetoaiMaan[a --00000T0o0 0001c0a0m0 00011017 Co%nt 0000101 G0o0oo0oo1o 00oo0u0o0t0o0r AasCompforaecrh|paicusn0g0osSennss AoosiLSD 2r7eez O06 ooocnorm oooosos CoIm oomoan: oDsoss oDsooeesss FGE-- omwipn Saonit)ineDuoetsnestd AosoO2.4d15lS0505 00001070 So5m dQooo0c0aenros PoGseiveenvaluesshowprsofmeans ht aesigrifcany 2 n0z? DTIS 111698 study Appendix IV. Part C Oral dosing with N-EtFOSE in rats. Individual data. Animal# GendeDoseN-EIFOSE Specie BW BW LW(LWB Saciice [Necropsy r (damysg. /Kg/fdoray) twos. a @ 9 Ww ddaoyspeost [Date 5eRO217 M Control sR026 M Control 7R026 M Control Ral 38463687173 0.045 Ral 41634204201 0.048 Ral 45374612219 0047 3 anaes 3 enems 3 omess 5aR0217 M 7aRo217 1 8aRo217 WM 20Rat 5237 59302 0058 Rat 48564884238 0049 Rat 4973 503238 0.047 3 oname ERT ER Note, 20 mg/Kg is approximat3e0ly0 ppm ith diet. ERaWtFsOSwEe,rethdeonsesdacoinitcweodcoonns9e1c1u8t/i1v9e96days, 9/16/1998, and9/17/1998with 20 mg/Kgiday N- " --_ cen? 5g DIS 11/16/98 study References BSuelrftohniaatuem,ea,nJd. Na-nedthWyall-lpaecref,luKo.roB.oc(t2a0n0e2s)u.lfPoenrafmliuodrooeotchtaannoola;te,PePreorxfilsuoormoeocPtroalnieferation aDnedanM,itWo.chP.o, ndJreisasulpB,iDog.eCn.eneTshiosm.psTooxni,coGl.,ogRyoLmeitgt,erGsi, 129, 23 - 32. and D, P. (1978). Fluorad FRleusoeraorcchheamnidcaDlevSuerlfoapctmaennttFCCo-r9po5raactuitoen,orMaalttioaxwicaint,y M(IL.DS0) study in rats. International GPhaiblraideell,pKh.ia,L. P(A1.976). Acute oral toxicity in rats of T-13891. Biosearch, Inc., sGtoulddyenwtihtahl,FCE.-915. Jiensrsautps,. DI.nteCr.,naGteiioln,alR.ReGs.caarncdh JaenfdfeDresovne,lNo.pmDe.n(t19C7o9r)p.orMaetitoanb,olism MLuaetbtkaewra,n,D.MJI,. Hansen, K.J.,Bass, N. M., Butenhoff, J. L., and Seacat, A. M. (2002). I1n7t5e-r8a5c.tionsoffluorochemicals with rat liver fatty acid-binding protein. Toxicology 176, PMaPlAoRngeya,mEm. aK.b,yasntdruWctauxramlalny ,diDv.erJs.e(e1n9v9i9)r.ontmreanntsa-Alccthievamtiicoanlso.fTPoPxAicRoallApphpalaPnhdarmacol 161, 200-18. NBaibnbdeifnegldPr,otDe.in(.19I9n8)E.nvIinrvoenstmiegnattailonHoefatlhthe,Epf.fe5c0t.sUonfivFelrusoirtoycoafrbMoinnsneosnotLai,veMrinFantetaypoAlciisd.- (S1o9h9l4e)n.iuEsf,feAc.tsKo.f,pAenrdfelrusosroono,ctKa.n,oBiecrgasctidr-a-nad,poAt.e,ntSppyedreovxoilsdo,mOe.,praonlidfeDreatPoirerirner,aJt..-Wo.n MSoohrlreinsiuhse,pAa.toKm.,aE7ri8k0s0sCo1n,ceAl.ls,M.a,raHtocgesltlrloinme,. CB.i,oKcihmilmaBnido,pMh.,ysaAncdtaDe1P2i1e3r,re6,3-J7.4W,. o(x1i9d93a)t.ioPnearfnlduoortoheorctaacnteivsituilefsonkincoawcnidtiosabe paoftfeenctteidndbuycepreorfopxeirsooxmeispormoalilfefraatttoyrasciind mboetuas-e Wliavlelr.acPeh,aKr.maBc.,olLuTeobxkiecro,lD7.2,J9,0B-u3tenhoff, J. L., and Seacat, A. M. (2001). Ppeerrfolxuiosroomoectparnoelisfuelrfaotonrasteinanrdas2,-b(uNt-entohtylgpueirnfelauopirgoso.ctaaxniecsoullofgoinsatmi6d0o,)-3e4t8hyA!bsatlrcaochtolIDa:re 1657. IYnavnogl,vQem,enXtieo,ftY.h,eApleerxosxoin,soSm.eE.p,roNleilfesroant,orB-.acDt.i,vaatnedd DreePcieeprtroer,alJ.phWa.in(2t0h0e2) immunomodulation 1893-900. caused by peroxisome proliferators in mice. Biochem Pharmacol 63, ---------------------- " Cenzn9 oo 31 Cs assisaafsE | z2 H zeie ElEz ut #g2i8e2s: || 3 3 4Ei gErE2zel || =[2z oImE 2z3z8m 3 : iFR sz gz TOR30