Document zoOM6nYLrn6pV4zYVdrZnoj1z

-- CHARLAEBSORARTIORVIEERS Sponsor: 3M St. Paul, Minnesota Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-E(FOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EWFOSA; 3M T-7091.1) in Rats Study Number: TRC 1132-100 Performing Laboratory: Gene Logic, Inc. (formerly, Therlmmune Research Corporation) 15 Firstfield Road Gaithersburg, Maryland 20878 Prepared by: Pathology Associates DivisionofCharles River Laboratories, Inc. (formerl1y5,WPoartmhaolno'gsyMAislslocCioautrets, ISnutietrenaItional) Frederick, MD 21701 Date: Original Final Report: August 9, 2000 Amended Final Report: November , 2004 IS Worman's Mill Court, Suite 1+ Frederick, Maryland 21701 (R01) 663-1*6(34014) 663-8994 FAX TABLE OF CONTENTS Report Narrative Summary Tables Individual Animal Data Tables Appendix 1 ~ Inlife Report Appendix 2 ~ Electron Microscopy Report Appendix 3 -- Palmitoyl CoA Oxidase Report Appendi4x ~ Study Protocol and Amendments Signature Page `Study Number:TRSCpon1s1o3r2;-310M0 `Amended Final RPeapgoer2t Section 1 un m wv v vi vit vin Study Number: TR`SCpo1ns1o3r2:-130M0 `Amended Final RPeapgoert 1 REPORT NARRATIVE `Study Number: TRSCpon1s1o3r:2-310M0 `Amended Final RPeapgoerdt STUDY REPORT (C6e3l1l6P.r1o1l)i,fePreartfilounoSrtouodcytawnietShuNl-foEntihcylAcPiedrfPloutoarsosoicutmanSeaslutl(fPoFnaOmSi;do3EMthTa-n6o2l95(.N1-6)E,AFaOnSdEN;-E3tMhyTl- Perfluorooctancsulfonamide (N-EFOSA; 3M T-7091.1) in Rats Study Number: TRC 1132-100 SPONSOR: B3uMilCdoirnpgo2ra0t-e26T-ox0i2c,o3loMgyCenter St. Paul, MN 55144-1000 SPONSOR REPRESENTATIVE: Andrew Seacat, Ph.D, (previously, Marvin T. Case, D.V.M, Ph.D., who retired prior to final rSeppoonrts)or contact: M3eMliCnodrapoMriattceheTlolxicology PFahxonNeo.N:o.:65165713733-61-7074343 Email: mitchell @mmm com TEST FACILITY: Gene Logie (formerly, Therlmmune Research Corporation) G1a5iFtihresrtsibeulrdg,RoMaardyland 20878. STUDY DIRECTOR: GTahreyrlWm.muWnolefeR,esPeha.rDc.h CDoArBpo.rTat.ion PFahxo.neNoN:o.30310133303-03-7337823 Email: gwolfe@lab.row.com PRINCIPAL INVESTIGATOR: `PSaatnhdorlaoRg.y ElAdsrsiodcgiea,tPehs.DD.ivision of Charles River Laboratories, Inc., (formerly, Pathology Associates International) Study Number:TRSCpon1s1o3r2:-310M0 `Amended Final RPeepgoer'ts PFhaoxnNeo.No3.01306136-2849-924036 Email: seldridge@criver.com HISTOPATHOLOGIST: CPaartohlolyongMyoAysesro,ciDa.tVe.sMI.n,teDmiaptlioomnaatle, A.C.V.P. No longer with Pathology Associates atthe time report finalized ULTRASTRUCTURAL PATHOLOGIST: JPaatmheoslBo.gyNoAlsds,ocDi.aVte.sM.I,ntPehrn.aDt,ioDnailplomate, A.C.V.P. No longer with Pathology Associates at the time report finalized STUDY TIMELINE: Initiation: January 12, 1999 `Completion of inlife phase: April 6, 1999 FAimnealndReepdorFti:naAluRgeupsotrt9:, 2N0o0v0ember 8, 2004 REGULATORY COMPLIANCE "This study was conducted in the sprit ofGood Laboratory Practice (GLP) regulations. PURPOSE "The objective of this study was to assess cell proliferation and peroxisome proliferation in rats administered test material in the diet. INTRODUCTION TadhmiisnissttuedryedwaNs-EdtehsyilgnePdertfoluoasrsoeoscstacneelslulpfroonlaimfeirdaotioEnthaanndolpe(rNo-xiEsWoFmOeSEp:rol3ifMeratTi-o6n31i6n.1r1a)t,s Perfluorooctane Sulfonic Perfluorooctancsulfonamide A(cNi-dEFPOoStAas;si3umM TS-aTl0t91.(1P)F.OS;In 3adMditi_oTn-,62r9a5t.s16w)e,re and N-Eihyl administered Wenyd-p1o4i,n6t4s3ev1a5luaatepdosfiotrivteestcoanrttricolle effofrecctelalreprloilstiefderiantiToenxtanTdabpleero1.xisBoemceauspreoltihfiesrasttiuond.y The was a maunldtiidnidsicviipdluianlarsytudtieeasmaerfeforretp,orttheedreinsutlhtes are summarized Appendix. in Sections 1, If and III of this report, sorwa iT=Em ToTt. sperm Epossami Endpoint Examined eis Location of Report ot r m , Clinical chemists Sections I, IL, Il FINAL REPORT AMENDEMENTS `The study report was originally finalized August 9, 2000. The present study report (amended final report dated November 8, 2004) has been revised to reflect a correction in the test article abbreviation for N-ethyl perfluorooctanesulfonamide from "PFOSATM to N-EtFOSA, and in the i Rr SL CL huh en concentration of Wy-14,643 from "1000 ppm" to 100 ppm. These corrections have been made to all Sections of this report, except for Section IV (Appendix 1), which is the report from the `obtainable in pdf format and therefore, unable to be revised. Additionally, due to the inability to obtain signatures from all participating investigators involved in this study, this amended final report is signed only by the principal investigator, Dr. Sandra Eldridge, on behalf of original signatures from Dr. Carolyn Moyer (study pathologist, Pathology Associates; originally signed August 9, 2000), Dr. James Nold (EM pathologist, Pathology Associates; originally signed October 4, 1999), Dr. Gary Wolfe (study director, Therlmmune; originally signed May 2, 2000), and Dr. Ron Markevitch (Palmitoyl CoA Oxidase activity, Covance; originally signed December AB TERILt S SD METHODsSnd no J 9) LaE ni final study report unsigned. i pte Experimental De sirsign ws losin To Tbe Study Number:TRSCpon1s1o3r2:-130M0 `Amended Final RPeapgoer?t "Text Table 2. Group Designations, Dietary Levels and Scheduled Sacrifice Time Points Bh CT NumberoMafleRats Group Number| (Time Point) | Control | (0 ppm) | N-EWFOSE | 300 100 30 ppm | PFOS| 20 ppm| N-EWFOSA| Wy-14,643| Total No. 100 ppm| 100 ppm|of Animals @81hr) 0 [fof 0 | 10 10 5 65 2 0 |s|s|s 5 5 5 40 (7 days) (14 d3ays) 0 |s|s]| s 5 5 5 "0 Qwkrec4overy) 0 | ||s||s|s 5 a 5 5 "0 ~ (4 wk re5cover 0 |s|s|s 5 5 5 40 Total No.of anal So dnln0 l la Tn-life PortionofStudy `fTohretihne-iifnevlpifoertpioorntoifotnhiosf sthtiusdystwuadys,cionncdluucdtiendg attesTthaerrtilcmlemufnoermuRleasteiaornchanCodrpaodrmaitniisotnr.atMioent,hotedsst animals and husbandry, IV, Appendix 1 observations, termination and tissue collection are reported in Section Cell Proliferation Staining and Evaluation Rcoelplreecsteendtaatnidveprseasmeprlveesd oifn tfhoermlaleifn.latrAafltelrobfeixaotfiotnh,eelaicvhersaanmdplaenyofmalicvreorscwoapsicprloecseisosnesdwaenrde stained same tpirsosluieferbaltoicoknscelwlenrueclesatraiannetdigewnit(hPCNheAm)a.toxInylaidnditiaonnd, liver sections prepared from the cosin (H&E) and examined microscopically. cSheactrigoends solfidpeasra(fSfuipner-fermobsetddPeldust,isFsiusehsewrerSceiecnuttifaitc,apPpirtosxbiumragthe,lyPA5)umto aenndsuprleacaeddheosnioponsidtuirvienlgy processing for PCNA. Standard immunohistochemical methods for PCNA were used to stain tissues. Briefly, tissue sections were incubated with a monoclonal antibody to PCNA (DAKO, Study Number: TRSpCon1s1o3r2:-130M0 `Amended Fina RPeapgoerst CBaurrpliinntgeraimae,,CCAA)) amnedthroedagfeonrtsthreeqdueitreecdtiofnorotfhteheavainditni-gbeino-tainntipbeordoyxicdoamspele(x.ABCPCKiNt,AVeexcptraesstsaiionn, wMaOs).loTcailsiszueedsbecytitohneswcehrroemcaoguennte3r,s3ta'idnieadmiwniotbhenhezmiadtionxeyl(iDn.AB; Sigma Chemical Co, St. Louis, T3h0e00pehrecpeanttoacgyeteosfihnep1a0toficeyltdesosfinliSv-epr.hasAe (nleagbaetliivnegcionndterxo,lLsIl)idweawsadsetienrcmliudneedd ibnytshceosrtianignaitnglerausnt and consistedofstudy tissue that was not incubated with the primary antibody. Fquoarlictelyl opfrosltiafienriantgi,onpervoacleusastiinognsa,nsdlisdeesctwieonriengf,irsptotpeenrtuisaeldpaattlteomwsmaogfniceflilcualtaironpr(o1li0f0eXra)titoonj,udagned histomorphologic changes. (200X) as described above. Cell proliferation was then quantified at higher magnification Histomorphology was further assessed byevaluatingthe H&E slide prepared from the same tissue block for each animal evaluated for cell proliferation. Clinical Chemistry jAungiumlaalrsvweienreinftaostaedn oEveDrTnAi-gchtoabteefodretuabnei.malS'esrsucmheedunlzeydmeneclreovpelssy;obflaoloadniwnaes acmoilnloetcrtaendsffreroamsae (ALT), alkaline triglycerides were dpehtoesrpmhianteadseby(LAaLbPC)o,rp,asapnardtarteeporatmeidnsoetpraarnastfeelryatsoePa(tAhSoTl)o,gy cAhsosloecsitaetreosl. and `Tissue Collection for Electron Microscopic Evaluation Sections of liver from all animals were collected, minced to approximately one millimeter cubes, lpalbaocreadtoirnyMfcoDropwreoclels-sTinrgu,mpsecftiixaotniivneg, aanndd esvuablmuiattitoend. toEPlaetchtroolnogmyicArssoosccioaptyesw'sasNopretrhfoCramreodlionna select animals as described in Section V, Appendix 2. Palmitoyl-CoA Oxidase Tissue Collection and Analyses `Aansiamamlpslean(dappflraosxhi-mfartoezleyn 5i0n0lmiqeu)idofnitthreogreing.ht lTatheerallilvoebreotifstshuee wlaivserswtoarsedcoilnleactefdreferzeorm sseetlectto msaaimnptlaeisn a-n6a0lytzoe8d0incCluudnetidl aalnl asltyuzdeyd abnyimCaolsv,anecxecefpotr fpoarltmhietoWyyl--1C4o,A6O4x3idanaismeaalcstiavintdy. allThaenilmiavlesr from will the 4-weck be analyzed recovery groups. In for palmitoyl-CoA addition Oxidase to this study, activity and samples reported from a previous 3M study separately; these samples consistofliver samples from 35 rats and 35 guinea pigs. Remaining Liver Tissue `The remaining liver tissue was frozen and is being stored at -60 to -80 C for possible future analysis. Statistical Analysis Study Number: TRSCpon1s1o3r:2-130M0 `Amended Final RPeapgoerdt The Student's rtest (two-sided, unequal variance) was used to test for statistical significance in Land clinical chemistry between control and treatment groups using Microsoft Excel version 5.0. AP value of less than 0.05 was judged to be statistically significant. Analysis of variance using Dunnett's procedure was used to analyze body and organ weight data with a 7 value of less than 0.05 judged to be statistically significant. Record Retention Aolflthriaswsdtautday, wdiolclumbeenatracthiiovne,driencotrhdes,stporroatgoecolfa,cislpieticeismoefnsP,atahnodlofignyalAsrespoocritatgeesnefroartaedpearsiaodreosufl|t year following submissionofthe final report to the Sponsor. One year after submission of the final report, all ofthe aforementioned materials will be sent to the Sponsor and a return fee will becharged." All raw data stored on magnetic media will be retained by Pathology Associates. RESULTS Cel Proliferation and Histopathology Group mean PCNA labeling indices are presented in Section II, Table 1. The severity and incidence of cell proliferative responses are presented in Section II, Table 2. The severity is d`enfuimnbeedr oafsatnhiemaflolsdwiitnhcrienaasetrienatPmCenNtAgrLoIupcowimtpharaeLdItgorecaotnetrrotlhs.an tThheehiignhceisdtecnocnecurreprreenstecnotnstrtohle value. Individual animal cell proliferation data are presented in Section II, Table 1 Statistically significant increases in cell proliferation were revealed in only one treatment group: 100 ppm N-EIFOSA at the 7 day time point. Although statistically significant, the labeling indices for individual animals were within the range seen in the control group; therfore, this response was not biologically significant. Biologically significant increases in cell proliferation, as determined by a severity of at least 2-fold and an incidence of at least onc animal in the tthreeattrmeeanttmegnrtouppe,riwoedraeti4d8enhtiofuiresdaonndly7idnatyhse. pDosuirtiinvge ctohnetrreolcogvreoruyppe(r1i0o0d,pbpimolWogyi-c1a4l,ly64s3ig)nidfuircianngt. increases in cell proliferation were seen in 300 and 30 ppm N-EtFOSE, 20 ppm PFOS, and 100 ppm N-EWFOSA, but not in 100 ppm N-EWFOSE or 100 ppm Wy-14,643, at the 4 weck recovery period. Histologic findings are presented in Section III, Table 3. Examination of the H&E slides revealed no significant changes in the liver of rat sacrificed at 48 hours or 7 days. At 14 days, and 1 and 4 week recovery time points, lipid vacuolization was observed in animals from all groups, including controls. In addition, livers from Wy-14,643 treated animals exhibited mild Kupffer cell hypertrophy and multifocal, minimal hepatocellular necrosis at 14 days. At the 1 an4d week recovery time points, no significant treatment related findings were noted, except for Study Number: TRSpCon1s1or3:2130M0 `Amended FinalPRaegpeor1t0 the mild Kupffer cell hypertrophy seen in Wy-14,643 treated animals which was absent 4 weeks post-dosing. Clinical Chemistry Group mean clinical chemistry parameters are presented in Section II, Table 3. Individual animal clinical chemistry data are presented in Section I, Table 2 Statistical differences in ALT, ALP and AST were seen sporadically among treatment groups and time points, but were within the nomal range for cach parameter. Triglycerides were significantly decreased as well as outside the normal range in the following treatment groups: 300 ppm N-E(FOSE at 7 days and 1 week recovery, and 100 ppm N-EFOSA at the same time points. Cholesterol was significantly decreased as well as outside the normal range in all treatment groups at one or more time points during the dosing period. At the 1 week recovery period, but not the 4 week recovery period, cholesterol remained decreased in all groups except 20 ppm PFOS and 100 ppm Wy-14,643. Body and Liver Weight Group mean body weights, liver weights and liver to body weightratiosare presented in Section AIIp,peTanbdliex 41.. Individual animal body and organ weight data are presented in Section IV, Body weights were not affected during the dosing period. Mean body weight was reduced only at the 1 week recovery period in groups 300 ppm N-EFOSE, 100 ppm N-EFOSA and 100 ppm Wy-14,643. Liver weights were significantly clevated at ane or more time points during the dosing period in all treatment groups except 30 ppm N-EFOSE and 20 ppm PFOS. At the 48 hour time point, liver weights were elevated in groups 100 ppm N-EWFOSE and 100 ppm Wy14,643. At the 7 day time point, liver weights were elevated in groups 100ppm N-ETFOSA and 100 ppm Wy-14,643. At the 14 day time point, liver weightswereclevated in group 300 ppm NEFOSE. At both the 1 and 4 week recovery periods, liver weight was elevated in the 300 ppm N-EtFOSE group. Liver to body weight ratios were significantly increased at one or more time points during the dosing period in all treatment groups except 20 ppm PFOS. At both the 1 and 4 week recovery time points, liver to body weight ratios were significantly increased in treatment groups 300 ppm N-EWFOSE and 100 ppm N-EWFOSA, whereas Wy-14,643 was elevated at the 1 week recovery period, but not the 4 week. Peroxisome Proliferation Electron microscopy (EM) and analysis of palmitoyl CoA oxidase activity were used to assess peroxisome proliferation. Palmitoy] CoA oxidase group summary data are presented in Section Study Number: TRSpCon1s1o3r2:-310M0 `Amended FinalPRaegpeor11t IL, Table pamitoy] 5. The EM CoA oxidase draetpaoratreipsrperseesnetnetdeidn in Section Section VI, AV,ppAepnpdeinxd3i. x 2, and individual animal ANotn4e8 ohofutrhse, oWtyh-e1r4t,r6e4a3tmienndtugcreoduapsdoeuxbalmiinngeidn(t1he00nupmpbmerNo-fEpteFrOoSxEi,so2m0epspcmomPpFarOeSdotro c1o0n0trpoplsm N-EtFOSA) revealed an increase in mean numberofperoxisomes per hepatocyte. Palmitoyl CoA oxidase activity did not differ remarkably among controls and all treatment groups at all time points examined. DISCUSSION pInertohxeispormeesepnrtolisfteurdayt,ionmiunlttihpeleliveenrodpforianttss were given examined N-EFOSE, tPoFOaSs,seNss-Ec(eFllOSpAr,oloirfeWrayt-i1o4n,6a4n3d 2rsecaovpeorsyitpievreiocdonatrreolpretessetntmeatderiinaTl.extATabsluemsm3arayndo4f, the findings respectively. during the dosing period and Ainscreexapseecitnedh,eptahteocpeolsliutliavrepcroonltirfoelrattiesotn maantderliiavle,r w1e0i0ghptpwmitWhyo-u1t4a,6c4on3copmriotdaunctedintchreeaasnetiincilpiavteerdassociated serum enzymes. Cholesterol was lowered as expected. These changes were reversed upon cessationofdosing. Iwnhercoenatsr,astd,urnionngethoef the test recovery materials period, induced cell hepatocellular proliferation proliferation dwuarsingintchreeadseodsining period, the N- dEuAeFOtSo Et,hePlFoOwScoanntrdolN-gEro(uFpOmSeAagnrolaubpesl.ingThiinsdeaxppsaereenntat rtehsepwoenseek m4aryechoavveerybteiemne apnoinatb.errTahteisoen animals, which PCNA labeling were index 13 weeks of 0.17% of age at has been rtehpiosrtteidmef,orexchoinbtirtoeldaatscoofnttrhoel LI of same only 0.016%. age (Eldridge A and Greoclodvsewroyrtthiym,e 1996). point isTahpupsr,oxtihemactoenltyrol10l-afboledlibngelionwdetxhavtalwuheicsheehnasinbteheins study at the week 4 previously reported. Furthermore, the response was not dose-related in the N-EtFOSE treatment groups. cLaiukseeWay-d1e4c,re6a4s3e,itnhechtoelsetstmeartoelr.iaNls-EdiWdFOnoStEa(ff3e0c0tplpivme)r-aanssdocNi-atEeFd OsSerAum(1e0n0zpypmme)leavleslos,cabuusteddiad idnetcerreeassteinignttorniogltyectehraitdeWsyt-h1a4t,w6a4s3 roenvleyrslioblweerweidthtirnig4lywceereikdsesfaotl7lodwaiynsgocfedsossatiinogn,obfutdonsoitnga.fterIt 1i4s daysofdosing. mPaatlemriitaolys]evCaolAuatoexdidbaessiedeascttihveitpyosaintdivepecornotxrioslomWayl-1p4r,o6l4if3e.ration were not elevated in any test Taken together, proliferators in these rats. findingsdonot support N-EFOSE, PFOS Although these test materials produced or N-EWFOSA an apparent to be peroxisome cell proliferative Tesponse in the liver of rats, the proliferative response did not occur upon initial dosing as scen TESponsor: 3M recovery time point may have been an aberration due to the abnormally low control mean labeling index. Like Wy-14,643, these materials decreased cholesterol in rats, but the lowering effect was reversible up to 4 weeks post-dosing as it was with Wy-14,643. "Text Table 3. Summary a ae ofPrFoilnidfienragtsitoon Assess Hepatocellular Proliferation During the Dosing Period and Peroxisome Cellproliferation [~~[1- -[-1 Tincrease| [Bodyweigh|. T - [- T -[-[ - |-1 [[LLiivveerrwbeoidgyhwtt| . [Inc[riencaresasee |ncrease| - |_| |Tncrease|Increase| ~~ | Increase Increase | | Increase Increase | | Ae ee JeTr] far ce Te - T e yeeSe7mel-er fener o] em pee] =Fov C mrtl1 C rlrT ltreltrl [(Peroxisomes |~~ -|- | -1- | - 1] *An increase or decrease identified statistically and/or descriptively (judged to be biologically relevant) at any time during the 14 day dosing period. ND, not determined i"_i Text Table 4. Summary of Findings to Assess Hepatocellular Proliferation and Peroxisome Proliferation During the Recovery Period Cell proliferation| [Increase| - [Increase|Increase[Increase| | ER Ee LehLolm LL er [Liverbodywt. | Tnerease|[| |increase| far 1 [= ae === fast____[- 1-|-[-T-T-T- m F|o m fm] T m [== oxidase [Peroxisomes | ND | ND|ND|ND |ND|ND|Nb| "An increase or decrease identified statistically and/or descriptively (judged to be biologically relevant) at any time during the recovery period. SUMMARY N-EFOSE, PFOS and N-EFOSA are not peroxisome proliferators, do not have overt hepatocellular proliferative effects, but do exhibit a cholesterol lowering effect in rats that is reversible within 4 weeks following cessationofexposure. LITERATURE CITED of B6C3F1 mice and F344 rats: effects of age, gender,and choice of marker. Fund. Appl. IL SUMMARY TABLES Study Number: TRSpCon1s1o3r2:-310M0 `Amended Final[Re2port `Table II-1. Group Summary of Cell Proliferation Data TjrTosa--r Gop NNEefrroorotsseet wwomrm WVeirsne ammm LFaoabrnan LpaFbrroandor a Geurm oFohm Gdooomm oaoooonm GGaom aamoenn LaroTeoomnxpyies LabFtrrooginuodes pCS raims os oFoYm wSsooowmm odooooommm CVuemm odoomm Wek Bacon Wek cv 4WakRacor4WekRacovry . Lbnioane Lsoanghiex bmg Libwisgodex [e Nrerros-- t oorpm wSaamn ooooe mm CGomm odooomm jNFEiovseaemorm dpteammrecdd.oomm SSGoooomn ooomouemn ries comm wm oom Gm do "Soeconti, e005. LabFawnoogxnnen LabwFsooxwen. a oeme oFmeYn SGShoEooRnn oooooamn tSeer ooaonm Study Number: TR`SCpo1n1so3r2:-130M0 `Amended FinalPRaegpeor1t6 `Table 11-2. Severity and Incidence of Proliferative Responses in Rat Liver LibaFiaoongu.nnds Trotman Group Sever NNNEEErRroOosSSeEEsWwmomnnm 001762 $n7e0r8ospAomomn 0088 Weiss ion 67 LsbeaJlongyeiodex incidence" a2io1n0o n1o0 w" Lapbes7i0nngmaindex Sov o00r7 a2 i LsJbe0otmgmyindex nigance ooosss aa55 w LabFewotwnuognsinydex LabeotPnmogayindex Seve cdance o3s5 o%%s 0088 0os 0 os L4oWbossikoRgryIendecx 1oWLesvbeekoiRemnegcalroovdeeyrxy TrosterGroup Sev ncidence WLeaebbkaoRngniendecx o+LWaevbeakrieRgeycrIonvdeyerxy Sevrty_ntdance WNNEEEFFFOOOSSSEEE3OD0r0opmomm o1is8 ooswss a"io 2&%5 PnWeOerSFso2SsAoOommpen i13 08 o0iss 32 F=d To i oWtercfarcmvelcwoionhvoPCNA ein rot hnheistitvik il bre rs EE EERE IEEE Ried z ls 513% "5184 ZIRE]|2]S|=|BI)= A EE |Z 7 iEH 2 3 3FR-3 EERE= CSZ tg H2E = " =El g i 3E32 MEnEERgeenEE Rdaeg ER aEbE] 5 =<| 2 5 <2 :Z| i2 E213757| 73 =mE2Z|3EloEllE=|E||EZ=|RoZ[E5}F 2 A=|RolEe oEleE:s Z2s 5 3 z zE :xz2 e23e z=| cP [=| 218/755=(5] #22 98) o8l E[Bl2=i82gl2E] [(98 [Zs=ElsiE=lEEle] [(93 (EeBeaa8s] AFEEEERnRsnEyRE(FMREEREER IE(HEnFmnEanET O|z|z|z|Z|z|2] S|2|2|2|E] 22] O|z|zZ||2z)|2] zjeaek 2f i #78 =/2|5|cl=2 22 ES 725552 5 g ii3 El Hsl<|g|xlol Z 2 =| <=] = =| ZFi e iE LA BSEElE3NR EESso E. z E|E2N=EE] ggg 1 Z| 23 2 =E2Nl |E3FE 9Eoss G22l A ls .5=7 is SHEER il 4 EREEEPEE El -(8zlz8z8=Slo8f =F|ESE25E 25)2E 5|E2E |5Z] EEsEEdEEE ; il i Hee BEE IEREE PEE gl |E||a-lp[z 318B8Io58z 23 B2ES8EE5EE5EE5S2EE2ER5]H]2 2H83E2 fg . ASf10 [HeaEEaEssEEsE (EE[dEAdzRlReEiEsE) | REsAsEe ir25 3g "It & u 3 . ReEeEEkER i2 E5EE|R=|ELgEEleE|EEslHEz AEE25E 22R 558 :Bl IFEdERdE |5lelznglseclE|aE 25EE 8 2 EEEEERE REREREN 5 z g =2 g5 LI|.5 2 gmanor FEREIEE ERERREE pl0H elses REERHE EEEESREEHSEREER EEERE & B I1 Sz| 4 3 [2 : |slSo2sEEas2a2n &(8 | &[&[s[S gl 5 [558 | BE 2E] gl |zz(=(8=z| feEiines |fe22ziey fo g2Emns Lal 555E8zz5] [[Fa[lFEaRnERaE]E [=|] g 1H2 E[E5l2ls EEREEEE |E] |= Sol [|51SA] eEE5|8Z|5Z] e|sl|s2eREoRlElE ei] if | 5 3] 5 E: g |g gg]3 2S1388225|13852e = S=1203 (855E5l28)5=]:7 Table 11-5. Group Summary of Palimitoyl CoA Oxidase Activity ConTiroelsamentGroup NN--EEIIFFOOSSEE310000ppoomm PNR-OESFO2S0Ep3p0mpom N-EIFOSA 100pom TonTroesstmentGrowp NNC-EEIFFOOSSEE310000ppppmm PN-REOISF2OS0E3p0mpom N-EFOSA 100pom He COAG" smn PCOAO dsHe PCOAO SH Sample Me5a Ss D. sRoange Si0z0e 55 329 aedms w1o0 95 21 eennw10 7 vos 1 P1C4ODaAyO PC14ODAayO PC14ODAayO Sa1m4pDalye MeGan S[DER. REamRge Sire 33 01s 612s.4 5s 35 004s 4a6s 55 3 uoo2a P7CODAO Me5an s7 67 6 Week RePcCoOveArOy M7e 4s 66 5 PDCaOyAO PDCaOAyO SDamapyle S3D. 4Rsane Si1n0e 5wo 0 as s 4 1s ss os a ss RIecWoeveekry R1ecWoeveekry RWeceoveekry PCOAO SD. PCOAO Range Sample Size 2B33360010os 08 19 1a6 ss5 014s 3 68 7s s "Pamitol CoA Oxidase activity, 1U/g `Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPaRegpeo2r1t IIL INDIVIDUAL ANIMAL DATA TABLES Study Number: TRSCpo11n3s2o-13r0M:0 `AmeFinnaldReepodrt Page22 `Table 111-1. Individual Animal Cell Proliferation Data 48 Hour7, Day and 14 Day Sacrifices Zi Trstment Gros AnimalNumber|Labi nde | AnimalNum Labonndg|AnimaN>umber LobelFonindex [Cons RRHiioooawsrz SOrooiessre | | RRRoutwtss| | oooovo.e ||| RRmiooowwss || ooowwemex [RRooaswt s| ees SoSoworesen ||| Riunouiwsts |oCow 3om. || mm--iasrso || ooaniosse] re- RRRiooouuwsrs tuoiamernns| | | imiobsiusssss | oooosowsmx | || Rmiioooiiwsessa | ooommdeean Inerose opm Rooowwwtez `oaooons| || mimaiiiksres | ooooooomo | mfRooowwsss | | ocoomswemsx RRRiiooowsss SGooososm || ERmiiioeswes| | oomce || RmRooosswssso || accoooswesst fe J RRiicoawr oSSeovesseonn--] - = = cose Gam RFFiiickccessr oOSfeoitroonn || Riioieusss | | ooomoaowme || mRissonesr | osomsueemn RRiicoiasr uOmr | | mRiibeesr|| ocoowms || sRisoss || oommoewr Ficus orn Tiwi omen. -- =] [NeFosEsm pn | eRRiiow oss | o[oeeowxos| | | MRioomissr |ooouwst|| iReors| s oomron RRRoiiaroosss Goer | 7 i7ow o1o1m | 1 1 Mus--|-- oom RRRiooonwweer oaaocozeonen | fo-rosmpm R1 ios3 | oom Boson I Rio o7s0s3 CRoi-- os goss RRRiiioooitizssst SEoowaTers | Emeis oom ERRoorsss-- oooonr RRriioeous SSsoooiaanrr. Rios oom i --t e" ros Toop "RRoiowtss | Riou Sooomm || C mRosw |C coommee--||-- mieows-- oomus | RRRiiooowste Goooozoew | Rios | oCt1e7w | memo-- | oom Roar ose sass -- tcorpnToRRuiioosnts|[SoTmhoein | | iM moowwameWwoo | || wmeeess TRS Roser eaor[ mm ee ois Geierna || iRososszs oaomoeomnmt ootes| Study Number: TRSCpon1s1o3r2:-130M0 `AmeFinnaldReepodrt Pages `Table 111-1. Individual Animal Cell Proliferation Data 1and 4 Week Recovery I Twa ssoc k TeRacor SPiodn [CTom reGraoupt||AmneRRsioosNsnuazmsbter Laboringeindex = AnRRsoissueemrsber |LabelDai0ngoo0I%ndex RsFo0os5sa3s) oooeuor.. FRRuiocceecrsoo aaaccczeeooonnn. RRRiiooosssoesta|| oaatnoioann | RRRooeaanrrs oot"ooom. eroseiomn| -- RRoissesse | oosos-- omw | RRuioosrssm oosouuoor.. = | RRRooosssaasse oaGswosotono.n--TRiRoRisoosTsTreo ooSaeons [Roose oh Rios| ons erosE opm] Ra Rioossz oLomT.-- Ross aaccoon% RRiiooss | oomnnaen moRsoesss Sowen. IeFosEmpm| RRiRiooSssS" RRioossso oDoSzomoanr. RiRmoiosessss | Soacoromn oSuuna. Rmiososs|| aaooseeown proszgom |I RRossess ----oas0oo0s RomRoossssa ||ooooromietn hoFsoessse aocwas. RRoossass | oSoeonn | IeesosSaAIStngm| RaRirooossssee RRiiosotse oa ounse.n. Roossas-- s oom onus ooense RRiooestoa || 0o0aerks sasstooni Rios3 oo0oo0s0io%x | | RRmioiosooeosrr ||onSowoonom | Rios Ooorzme |Romwosss| Sooooo.w ictstom ctck tesiso spat inn Study NumTRbSpCoen1s1o3rr2:-1:30M0 `AmendedFinalPRaepgoerst `Table 111-2. Individual Animal Clinical Chemistry 48 Hour Interim Sacrifice foe Tmoemewr | mAw SSwRo e m SE "rRioowws |m -- mm --"e w -- e w = @ | DomRReooswws|| om % aomems mww rew ea w-= ET Ro3ss | --% ---- -- ww--e-- ----wo 3a= | mE Rooww || w% T omam oww--m--aon wo- mRmeoowwwse | || m mawmawwosw m = m ao Rowe | Riows | am 2 wai wr a| Ei i IeroseER om RRmoiewoes [--3 |--% wm a m me aw rw ww" mow | % wm a " RRooors | S %|E emoow e a no&r mmmoowwwos || 2m |B mawwmowr | oo%ww =o 1 E Rows | sS aw aa----" @" Sms Rom | %mamm --oem----w ow e 77 mpRiooewws || w % m awe a --we-- on oor = fproszion Co1mmorT e|w%w R eaw ---- wa -- s wow ---- @5 RmioomsE| m| % mm----ee-- m ae----wa -@S A Rmoewm | mnoaww w w--w a "b=i RRooww| | %2 | ames mmowh == ETE T ioe T aI w m------w--- E7s S-- Rmoowws|| x w am omm%a a mohRwowem|| sow m Tm omw m m wTw MeT aaHi Tag og immoowwow|| swom weawwww ow =57 Rmmooowwws ||| y ow mwoem m wwmw w "= mows | wm won = Std N"uAmmTebFRSEnpre1o1d3nRr3ees-p1oo:dr0rt0 Fas3es "Table 11.2. In7dDiaviyduIanlteArniimmSaalcrCilfiinciecal Chemistry Conils RRoouas Rous Sso t| am | ar a | ou | 1s a% 3 7 7 Rmoowss 32 || oamm || ene = 57 -- CRs 1) S st a |seow|ee s me et s - R[RIsIMsS 0| 208 a | ms | 26 is 5aa "s7os2r Ne=FoesE 0pm|CoRmieoss a 0 | aisnt ||m iz m = meRie ess 4e3r ||3z 3 || e tfears 8 qe a% r NErOSE 0pm| RR RIo04s6e0i e3tiwe 3 | os || mew su | i i1mm w afaor E3S | sw - Roses Bn a5 us 7 u erosesopm | RrR1io0vw4ie6er5s |3sw4t |ew2s0s1 |[i1d0es4 o aa o ar RmR10o46io8 os3w4r || 2as61se || 1id0oer3 4=a5 asxt. PFos20ppm | Rmieoamt |m33a|w6 |12tsw arw| o w s| Ris a0 | ais| qa 25 er NerosatOoFoTpm | RmR1oo04rw7se4 Rim | s22a a|| w1ma8rs8 || | 1ii3s8e am =8=Wow0=5 | -- RRR iimmosw o2r|| zw0 ee |||77d im a728 6 E2} |Wy-141050p4p3m Rmiioovwaeesr 3 wm ||| smomsst | | | 1s 1 21a 17a Rio0ds8ss 3 Ey | 22 Bi || 6 us Ey " st & "Table 1112. Individual Animal Clinical Chemistry 14 Day Interim Sacrifice Study Number TRSCpo1n13s2o-13r0M:0 `AmendedFinalPRaegpeo2rt6 [Contos RRii0via8?0 s 3owTe ||omor 5u 77" RRiiootss 2 || 2om || awwm >0 ES . RiR0iio%t0 u || zoem || ww t w= on5 ---- ~RRimi0oi#is0gs4e s a smiw ee|| m ws w7 I2Te --| NEFOSE 00pm RR110048%965 2sw || aoms || wrse 33s 5 E-- Ris R10408" 3 | waa |e 34 | | 1g 19a 1g "2 o RiRC10S400 NeFosE 00pm| Ricso1 JENA st | am | 1m 7|A -- " 7 -- RRii008s0032 @nm | is || oowm wat o wwm TT Riosos 3 148 | 169 a8 ss| erosesopm| RR10i505o. s 43 5 | 2a0e5 | 1u1st4 s34 e aswo | = oo--RiRo1s05o0s7 aSs| |t|2er6| | | 206 wm 341 50 5 pros=zoppm|CRimRcsOi1s0osiasu [2a || 2w6o | om ||u11e3 bw tiea s5ar || Rwiisstn Rivsts 5EO 0 70 [rer aiia a | wm [mo = 330 NC EFOSA 0T 0pm| rRousss || m200|| uuss 22 Rose a aw | ws ES se | [Wet.T5oap4p5m RiRR0i1d0ss52z10t9 3m s4 m[ao2a38 || 114 rw ii21s5 22n7 RRii00ss223z @ || ammis || aoew 35s w@or RRii0css224s wo | | wwes || iwwe 45 EoS `Table 111-2. Individual Animal Clinical Chemistry 1 Week Recovery Sacrifice Study Number: TRSCpon1s1o3r2:-130M0 `AmeFinnaldPReeegpeodr2t? Gonos RRiTc0s522r5 u5| aTme || 2m8e aa5 [ot3 RRii00s52280 Ri0s30 7s || wim || 1w0r6 wu | tes | 10s 32is 2or w ---- Rosy zr| ve | es wm CRissz 0 | on | ess #2 58 = wRRo ii00s5s 3354 TmIFI | 181M1] wo |ow O2M E55a0 NEFOSES0pomCeRI0oSH sw amsn || ow ter | | 1 tas 6 [st 7 [a CT RRimoss Twar m2i5I| mm2Taw 3 0% | EOSE 00pm|~RiRR01O0s5iS420 642 || 1w9ze3m || amae 359 ai37 JJ E-- Rossa ar [eo R10545 38 144. [ios | 2 | 28 | 107 36 Ei) neFosCEoO0pe|m RmRioicrss| 3swmo || 2azs0s || 1f|1srst | 2te| a wi RiR0I0s5s400 aBwo | owme || m1e0 73 2wo proszop| pm Rtosst | mes 48 ar | taewer|[wTs w|a s2t8T|sO wesw ss ws | ws 2 [8 oo IN-EFOSA 100ppm| RRR1110005S55546. 43w2o | 2we1se4 || 1u8ms 27%1) E63D2 -- CRs | 3 | me | te x | 2 oo | wmioss|s sz | wr RR1100555690 | 40 e186 | [re 1138 |7 1 32 45 w3n Wyet4.1060p4o3m RiR0i0sSoS2T R103 ngeo | m wn |ee 2 | ow | mw 55 PF) 1o05 0 RRi1008645 w0s || wets || o1at 7" 0&] `Table 111-2.4InWdeievikduRaelcAovneirmyalSaCclriinfiiccael Chemistry StudyNAummeTSbRpnFCoinn1ead1sl3oRr2ree-:p1o:3d0rM0t Page 28 [Contos RRii0s5e6s7 30 008 [| m0 52 a27 RiR100556680 as || w2ss || e1s 2w a5 meRm1o0s5s70 waoo |N| ower || wm | dw| 1N sa N EF) a W5 i fRI0SC74 -- 57 1w3ss | [iame 5%1 aao NEFoSEs00ppm| RRIi005S7T5S 3551 wwoo | |e 23 3& -- CT fmCes--a2 |mas0 || 1 161 % EC -- 3 = RR10i56s0 e aSr|| tieess|| wisets ae aotr NEFOSE 00pm| RiRisose82T sws | | a2 || oteml 503 339 RRisuems aS| eaes || tees 0 3 eosw INEFosEsoppm | RiRoisseess ast 2ot | aTo r 3ast 25 a RRii0osses87 501 ia s | 15 42 "a7 RiR100558600 3i5 206 || 225 5& =7 pCrooszopem - R Riosoit sewwe ||wwrr || Riss 4s |fae | ae s mx a tee ws| RiR01s0e5s64 B saw s | |e 3& i7s IN-EEFORSA 100ppm| RiR0oss6s7e w5 s| ms| | 21 F2a Ewrs) 5 RiR0i56o9sss 135| 1os7 | 22a0 E3Y% 5wm | [Wy-114004pp3m| RR1100660010 72 uuse| | 166s8 25 r6y RiR010660023 3moms0s || 1t6o8 1532 &5 RRii00660054 s o | mton | dot 2se oorr Tcs eiie `Table IT1-3. Individual Animal Histopathology Findings E E=EE iowr =fndege m= EEEEEEe=e= SEEEE=e [Rows Nosignicant ee =EFs E Rican hosarodogs = fe EgEE EEEE= =EE s EEEEES RT0i09 [Nosgndcantnanis -- fF rosso ci mRioiizz h[Nosopcstopd i| en Bi(EBE BEEB= eeEiEiE= = =Ee esEsEsESss [rn Rois Noteindno Rios Nosepteon Study Number, TRSpCon1s1o3r:3-130M0 `AmFienalRPneapodgr3e0t `Table 11:3. Indi7viDdauaylIAnntiemriamlSHaicsrtiofpicaethology Findings Treatment Group _Animal Number FiLnidvienrgs Controls RRiIoaTdd NNoo ssiggnniifiiccaannt nnddiinnggss | | RRI1M0S5 NNoosisnniiffccaanntt nnddiinnggss -- R1R0I0445S0T NNoo ssiggnniifficcaannttnnddiinnggss | -- - "RR110065 NNoossignficcaanntt nnddiinnggss N-EFOSE00TpmR|IRRO11040S545 NNNooossgisnngiinfficccaaannntttinnndddiinnngggsss RRI1004S87 NNoossiggniifccaannttfnnddiinnggss ~ _RR1I0046i059 NNoossiigginiffccaanntt nnddiinnggss INa-EFOSE 100pOprm| R1Ri0bi46621 NNoosisggnniifticcaannttfnidndiinnggss | RR11044663 NNoossiiggnniiffiiccaanntt nnddiinnggss N-EFOSE0pm | RR11040645 NNoosgsnniifccaannt finndinnggss RR11040657 NNoo ssiiggnniiffiiccaanntt fniinnggss | RRi100609 NNoosisniginiicfcaanntt nddiinnggss ros Topo RR11074712 NNoo ssiiggnniiffiiccaanntt ndiinnggss RRI1G7S4 NNoo ssiiggnniiffiiccaanntt ndiinnggss N-EFOSA00pm RRIIOTSS NNoosiignniiffccaanntt nndciinnggss RRIISGTST NNoo ssiaginiffcicaannt idinnggss RR11040705 NNoosisgnniiificcaannt nddiinnggss Wy-1410604pp3m RR11040821 NNoossiignniicfcannt nddiinnggss RR11004843 NNoo ssigganiiificcaannt nddiinnggss R10485 No sgniicant ndings StudyNumber: TRSCpon1s1o3r2:-130M0 Amended FinalPRaegpeortt `Table 111-3. Individual Animal Histopathology Findings 14 Day Interim Sacrifice rm [CTarsnsotment Group AnimRailouimthber Tp vacsmton Tp. minaLiler Rik10e48r8 RiokeD iNlppoasvgvonaccucaaknuatfloinnde,itnpadgds,t.mmoiinnnaa.ll = ios |aa0garogantsm,ice aptsmima:tocsy. mio, mira RIOHSH [|0NooSroggtet,micFeinidifnag_smmaoorty. mutoca, minifmali-- R10i52 lp vocuotzton, pd, inal | Rioisd iagpgrdgevteas.cmiubnsaapmmemtmaointa,olcrey, muito, minimsalin, neeiFoses00ppm | Ri0ASS ibis i|Iopd0ovrvooacgutocek.zmwaitcioenf:mfaotemdd.ommmiinadnaolroty. motos, minima. ~ RIOAST Ip vocation. i. mid iRdRRI100Gv5500S0 ooIppdbvvoaccuusokkzzaatttoonn..oppdd..nmmaisdipemid | INEFOSE 100ppm | R10S01 iadgorvocaatcs,umiaxnailtaommnmi,a.tcrey,moc, minimal 1 RRiioosasz [iIpp vvoeccuuooktzzaatoonn,, IIdd,. mmidd: aggregate,mixednfamatory ce, mtfccl, minal RRIi05s04 pNoostonaimcattnengiss ris ircato,misoaoc, tc, inl NEFOS0Epm| RRRi11o00s5500075s iIipppvvvoeaccucoukouaktazoantt.oopindn,.. mmmiiidad pros20pom | R11I00O55S01H90T (iIv[ppIpovvococcuuoudiozitilloooaznedd,..,ttmmmoiimnninnamaa,,l:l CRs 10513 NNagooosrspcganicuoasin,cmtaixnda inngdsngm s e co,mut tta, minal 1 RR1i0o5s1t5a__IlNopsvgencuiootnzastfoni,tnosd.minimal INEFOSA 00pm | RIRIOOSSTIe? i[pIspvvoocucouizsaatoan,tHioodn.. miisnal | RiR01s05t1o8 iipa pvvoeccwuoolizzaatonn,iipd..mminmimaal [Wy-14643 10050m| 1R100552201 ~~[pIoprvopckuyoi,zKautpnf.eirdc.l,moamda:r nopatocohsarnecrosis moc, minima, Rp Jeippatvceoliooor urtecelatpogg,mmtisrtoaocns, minima R10523 |tpiervvoocpwyo.Ktzuapteorino,,l,mminiidma:l ioppatvooccouhsiaznaectrootsnoi,s.mtoicta, minima. Ri0s24 | |lhmepiaptcvoeliriorKvurnepcforeorisppcsaa,h,mmmtaydomc:as, minima. 0525 peeprattocheyl.irKrifeecrtce,l,mumtildo:cs, mn. - |osdorveogcmeu,stzmsiiocne, bit.ammimniyma:co,motto,minimal Study Nu`mAbmere:nTdRSFEponn1ls1o3rRs3e-p1o3r0Mt0 [er "Table 113. Ind1iWviedeuaklRAenciomvaelryHiSsactroipfaitcheology Findings Trestment Group _ Animal Number HistoRathliLgiyver Frags Gonos R025 [NG Scanntos _R1100525827[plvaiipgadigdcrvguaaoctuleosil,aztmaiiotsni.oolIninppfi,,da, mmmiilmd:at oorly, ------ mutocal minal RR1I00552390 g[[iIoppardvgvaaeccusou,lzaatmtioionnd., kppdf,l.ammmmiiadt:orycol, mlfocl minimal | RRw11i0055s33n12 {[|iiipipdivvdaevccauucoouzloaaitbznoa,niHhtoppnp.. mmmiinmneraaall = INEFOSE 00pm| RR1I00553345~~|iippiddvvaacucokuzoatlona.HtHpip..ommnii.dnal 10538 iid vacolzaton. Ho. mi: = | RRI100S5ST3_8_ |ii0gddrovvgaaacevcso,uzaomtolnda.ltyipindf..olmamnmimd,datory col, muwlfaocsl minimal RRiI0OSS3H90 iippavvaaccuuooitzzaattoonn,, lppd,mmiiara I\EFOSE 1005pm| R1i005S4H12 |iappiddvcvaacovclurzoamitiooznneag.dspioedf.n.ltmmmiainetinmoaasnl:lo,l, muldocal minal RR1i0o5s R10545 pipidddvvaaccvuuooliaeztaitocnn,. tHlsppp,.gtmmmiianodimlanl NEFOSE 0pm | RR11005457 lioiddvveaccvuloazaattoonn.. ikppa.. mmiinderal R1R11000555544006_~_~[[iI[ppididvpavciaucdouvzoaaltcaount.oiholppyndzd...ammmiiiionnnnaaa:lll ros20pm 1R100555512 [|iippiiddvvaaccuuoollzaattiioonn.. HHoy.. mmiinnaall - ces INEFOSA 100ppm | - |Wy-14,643 100ppm _ RR1100565643 _~_~|iopi0vip0ddraveacgcavueosoa,aktmoien.zdkaInpift.da,immmmiioannliinmmoaa,rlly: c ol, cet-- mutocal mi na l 1100555585 i|iipddvvoaccvvllazaattoonn., ltpp..mmiidnal -- RRRi11000S55S5708 [iNNpoossvgignanifcfauicnoantitzfdianntdhiosnpng.s,. minimal oo R10550 lipidvacuolizatioind, minima_l _ ~ R10561 n|ineydppeavrttaorcooopllhiyz,aaKrtuponfnefc,reoirsdcie.sl,l,imumnitlfado;lcs, minima spon. ollcuar erase,moderate R10562 |khydpervtarcoophlyz,aKtuopnf.feirdce.ll,mimniilmd;al R10563 #10564 | iIpneytpetvroatrcpouhpohyly,a,KtKuiupopnff.feerirscccoe,lll,,mimmniiilcmd:;al lips vacolzaton. isc. imal R10565 |G ipoi vacn uzatKounp.fertcpel.l,mimniildm;al Study Number: TRSpCon1s1o3r:2-130M0 `Amended FinalPaRegpeo3r3t `Table 111-3, Individual Animal Histopathology Findings 4 Week Recovery Sacrifice Treatment Group _ Animal Number HistopathLoilvoegry Findings [Gonos 10566 ppidd vvaaccuuoolliaztaitoonn,, Tpidd., mmiildd: RR1100556687 aligpgidrevgaactueosl,zamtioxne,dpiindf,lammimnaitmoarly cel, multifocal, minimal R10569 lipid vacuolzaton, pid, mid CTTRRT00S5T701__ apgigvrdaecgautoelsa.tmiioxne,dpidnf,lammimnaitmoarly coll, multifocal, minimal | RR51075737 laigpvigadrceugoaltiesz.amtiixoend, iipen. mfinaimmalc_mea,tmlloforcayl, minimal -- R10574 ppiidd vvacauoclzuatooni,izpdia,,tmmioinninimma,al: N-EFOSE 300 ppm| RR1100557765 apgigdrevgaactueosl,atmiioxne,dliindf,lammimladtory ce, mtfocal, minimal RR1100S57778 pliipidd vvaaccuuoollzzaattoonn,, ppdi,, mmiindimal R10579 pagigdrevgaactueosl,izmaitxone,dpiinf,lammimda;tory cell, multifocal, minimal [N-EFOSE 100 pp| m| RR1100585180 iiiidd vvaaccuuoollaattiioonn,, lpidd,, mmiilndimal RR110058632 lNiopidsivganciufoilcianztatfoinn,dinpgisd,minimal -- R10584 Npiodsivganciufoilcainztatfoinn,dipngis, mid: R10585 _ apgigdrovgaactueosl,izmaitoxne,diipnf,lammimnaitmoar:y co, mutfocal, minimal N-eFosE 30pom | RR1100558867 aNgogrseiggnaitfeisc,anmtifxineddinignsflammatory col, multifocal, minimal RR1100558898 lliippiidd vvaaccuuoollzzaaitoonn, ipdd,, mmiinniimmaall Pros 20 ppm RR1100559%10 Nagogrsieggnaitfeisc,anmtiixnecdiinngfslammatorycel, mulfocal, minimal RR1100559823 lliippiidd vvaaccuuoollziaziaotno,n pidd,, mmiinniimmaall R10594 pagigdrevgaactoelsz,amtioxne,dpidnf,lammimnaitmoray:cel, multfocal minimal N-EFOSA 100 ppm| RR1100559965 aaggggrreeggaatteess,, mmiixxeedd iinnffllaammmmaattoorryy cceell,l mmuulltfiofoccaall,, mmiinniimmaall RR1100558978 lipid vacuolzaion pd, minimal No significant findings i Ri0599 Npoisidgvnaicfucoalnztatfoinnd,inigps, minal: [Wy-T41060p4p3m ~~ R10600 R10601 aggregates, mixed inflammatory cel, mulfocal, minimal Nosignificantfindings RR1100660023 NNoo ssiiggnniiffiiccaanntt ffiinnddiinnggss tpeisdtevsa,csupoelirzmatgorna,nuildo,msm;inima: RRI100660054 epNiodisidgynmiifsic,anstpfeirnmdigngrsanuloma Study Number: TRSpCon1s1o3r2:-1I0M0 `Amended FinalPaRegpeo3r4t IV. APPENDIX 1 - IN-LIFE REPORT Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalPaRegpeo3r5t V. APPENDIX 2 - ELECTRON MICROSCOPY REPORT Study Number: TRSpCon1s1o3r2:.31M0 `Amended FinalPaRgepeo3r6t ANCILLARY PATHOLOGY REPORT ELECTRON MICROSCOPIC EVALUATION OF LIVER IN CRL:CD@(SD)IGS BR RATS) CELL PROLIFERATION STUDY WITH N-ETHYL, PERFLUOROOCTANESULFONAMIDO ETHANOL (N-EtFOSE; 3M T-6316.11), PERFLUOROOCTANE SULFONIC ACID POTASSIUM SALT (POS; 3M T-6295.16), AND N-ETHYL PERFLUOROOCTANESULFONAMIDE (N-E(FOSA 3V1 T-7091.1) IN RATS TRC STUDY NUMBER 1132-100 PAI EM PROJECT NUMBER EM 99.62 SUMMMARY An increase in the mean numberofperoxisomes per hepatocyte was detected by electron microscopy in male Crl:CD(CD) IGS BR rats given 100 ppm Wy-14,643 by diet after 48 hoursof treatment. The mean number of pheepraotxoicsyotmeewsewraesnoatpprreomxairmkaatbellyy ddiofufbelreendtofvoerracnoinmtarlosl gviavleuens.10T0hpepmmeNa-nEnGuFmObSeEr,so2f0ppepromxPiEsOoSme,sorpe1r00 ppm NEFOSA for 48 hours when compared with control values. PROCEDURES The purpoofsthies study was to examine by electron microscopy the livers from selected rats administered the test materials to assess peroxisome proliferation in hepatocytes. Male Crl:CD(CD) IGS BR rats were given the test material in the dit according to Text Table 1. weoli Text Table 1. Group Designations, Dietary Levels and Scheduled Sacrifice Time Points. Numberof Male Rats (NTuimmebPeorin"t) | 1 (0 [30 10 ppm) | 0 0 EtFOSA 30 | 20 | 100ppm| 100 ppm pp pp No. of Animals PS T rra T I 4 s5[s 5 r ee Pr T 1] Animals ! in AGnriomuaplss4iannGdro5urpesce1ivtehdrotuhgehte3strdeiceetivfeodr the test 14 days dfioetllfoowre4d8bhyouars1 ,or7d4aywse,eaknrdec1o4vdearyysp,errieosdp,ecrteisvpeelcyt.ivAelnyi.mals After the prescribed dosing or recovery period, the animals were fasted overnight, bled for serum samples, anesthetized with CO, weighed, and exsanguinated. Postmortem procedures included weighing the liver and `analysis, and electron microscopy. Liver samples for cell proliferation and routine histopathology were collected in zinc formalin and submitted to Pathology Associates International (PAI) Maryland for evaluation. Liver samples for palmitoyl-CoA Oxidase activity analysis were flash frozen in liquid nitrogen and submitted to C`polvacaendcien LMacbDoroawteolrlie-sTrfourmapnafliyxsaetsi.ve L(iMvceDroswaemlplleEsMf,or1e9l7e6c)t,roanndmiscurbomsictotpeyd etvoatlhuiastliaobnorwaetroeryth(iPnA-IsliNcoerdtahnd Carolina) for electron microscopy processing and evaluation. Per sponsor request, only liver samples from selected ratswere processed and evaluated ultrastructurally (Text Table 2). Only liver samples from animals dosed for 48 hours were examined by electron microscopy. enoEiE Text Table 2. Animals Selected for Electron Microscopic Evaluation R10404 R10439 R10443 RET stained with 5% methanolic uranyl acetate and Reynold's lead citrate, and examinedon aZeiss 900 transmission electron microscope. preferentially examined. Five repr Centrilobular hepa esentative electron tocytes, photomi where clearly identifiable crographsofhepatocytes in li were ver section taken and s, wer e significant ultrastructural features were summarized for each photograph and animal on a designated transmission electron micrograph interpretation form. The numberofperoxisomes in hepatocytes was h`meapnautaolclyytecowuanstemdanfuoralelaychcaplhcoutloagtredapbhyedsuhmempaitnogcytthee annudmbreecroorfdpede.roTxhiesommeeasncn ountu edoim nffpieb vreohxe eipsaotr moecsytpeesrper Individual interpretationsofelectron micrographs for cach animal selected for evaluation follow this report narrative. The original signed/dated raw data sheets and photomicrographs are maintained in the archived study After 48 hours oftreatment, the numbers ofperoxisomes appeared significantly increased over control values only for animals given 100 ppmof Wy-14,643 (Text Table 3). The mean numberofperoxisomes was approximately double the control values. The mean numbersofperoxisomes per hepatocyte, however, were not remarkably different for animals given 100ppm N-EtFOSE, 20 ppm PFOS, or 100 ppm N-EtFOSA when compared with control values. Figures 1 throug4h show hepatocytesfromcontrol and non-affected treated an animal given 100 ppm Wy-14,642, illustrating the increased numbersofperoxisomes. - Control 48 hour R10384 11.4 R10388 13.6 R10404 R10430 38 pp R10443 294 No other ultrastructural abnormalities were identified in the samples evaluated. `CONCLUSION BAsRervaatlsugaitveednb1y0e0lpecptmronWym-i1cr4o,s6c4o3pyb,yhdeipeattaofcteelrl4ul8arhopuerrsooxfitsroemaetsmewnetr.eTihnecrmeeasaend niun mmbaelreoCfrple:rCoDxi(soCmDe)sIwGaSs `raepmparrokxaibmlatyedliyffdeoruebnltefdoroavneirmcaolnstrgoilvevanlu1e0s0. pTphme Nm-eEa(nFOnuSmEb,e2r0sopfppmerPoFxOiSs,omoers1p0e0rphpempaNto-cEy(tFe OwSerAefnorot48. McDowell, EM and Trump, BF: Histologic fixative for routine diagnostic light and electron microscopy. Arch. StyNu"mNbaendredTRSiCpnosn1s1oR3re2sp-o13r0Mt0 Tag4e0 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Swdy No: _1132:100(EM99.62) AnalNo: _R_ IO, Treatment Group: Contd Sex: Malte `Speciew/Strain:CLIGCSDBOKSBDaLt. Tse Live BlockNo(s): _99Z6i2t:4 Photo/Negative No(s). __ZI7350 SignificantLesions (checkonc): Yo _X MN a Patbwiogst (gnats Nerney G0 add Sept 20, 192 "Fv2ea3altduoarmeclseyZ.IsTnThdeGi iLgbihovireedre.srteadHibenypiaandgocjcaaydcoietenbtel1hie0ap3lact2eo0clKyltTceysHt0ophl(awseom isspideevasidaetcnndoto.nsisnMicuisst3ooyicdhstnonvideaerilir.ally 204roughcadoptasmicreeulum (RER)profssefrequentthroughthecyioplasnof the hepatocyte. The intervening cytosol appearsfinely granularandcontains ribosomes, lgelfytcaongdeunpapnedr rsimgohottmhaerngdionpsl.aPsemriocxriestoimceusluamre(iSnEfRr)e.qBueinlte;cfaonuarli(c)ulpiearroexpirseosmeenstcoonutnhtee.d. pZr1bo7f3o4el7s,hveLeifrvieergq.huteaHnentpatthooecueygfteth.,t1eM0i.ct3yo2co0hpXoh.niTduhrniiaossfhnetdhpearteooucpgyhteeeniedso.pbolraAdsemprirecodrmbeiytnieecnnudtlomtuihmce(lrRioaEvlRlcie)loluss. | `borderisevidentatthetop rightmarginofthiscell. Nine (9)mitochondriacounted. | ZI7348, Liver. Hepatocyte. 10.320X.Thishepatocyteis surrounded by two adjacent h{empaetroceytdeis atnhd maistioncuhsooniddrailaonagndtohebreirghotrgmaanreglilne.sT.SheevceerlallhmasiatbuoncdhaaRonEtRnredgurlicya- `2Ss1hi7anp3he4itd9a.pndcLgoicveohecntrto.aaninHtnedicponyatntootpcdeylatalesek.mfirtcs1io0on.emv3ae2g0liXan.rmaTmethblirlioaasnn.hdoEeuiepgabhtisnotccls(yu8pts)reipecoensor.enoAntxatniinsnetosshmeeenvseexcrtoadrulanlctioeepcilded.ldtrioppphlraeectsesee.,nt || T7hi1or7tp3e5ee0n,(a1sLe3i)vppereersreoHnxetipmsmooicmysetshec.pouuni10to.ec3dy2.0tK..ThSereiaretoabZu1n7dv3a4n9t,lmseiveartalomcedhiuaomn-dnsindzuemrderpioiuasd RERpoles evident. Twenty-two (22) protisomes ound Study NumTbRSCpo1en1s3or2r:-1:30M0 "Amended FinalPRaegpoerst TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_1132:100(EM99.62) Animal No: RUGEE ee TreatmentGroup:_Control Sex: Maol o e `Species/Strain:CCOMSIIGSBR Rat Tissve _L _ iver Block No(s) 99.628 ZI781 + Photo/Negative No(s):_Z17355 SignificantLesions (checkone): Yes __X__ No Interpreting Pathologist (signature and date): eB S200 TinecahraWtrEewRn:ppeoZclmiIiomTne.Lrt TmLhaicenkreorlai,eTaiTlgshooeocscsbytuieno,dgelaa1tsnIs0c 1s3felThagiepsadbmesigaoarmr:oacnpydeiicecbohaisrcskomseonm.maSmevreworimatlecbhmioliencdleetuas || Saamalbiecuploatnaecpyrtesee:ntavbeergsenohsisococlsntdheaccaycentpBepcoteoss.ies.cAihdeaighhteEcagoamn iosofkheik | SrAheemgtioeacghio.v.TrocHTleeemmwu(c1ce2o)sutpseoinsi1ps0o3o2lm0yeXso.loNauunsmtdeerdeomreasllmyis1a0c2h0o6n4d.iaAabndeRiEgRhptrmoafrigleisnaares p1r9e0scat 1n || oRrakenr depooteemcyptreosx himeaspap1efhysaevseesamscasamf:epnesclaertgeirn aand cwyopreiamsumm.eroFnistemeinto(c1h5o)ndia DerIaxiToniiLcisrveecerontvH.eepdmTchytpc.ot10.e3208a.w cOenntaheufteadthsincboadeasdoftheenCdysopkasienlcoinnsv wey inamebe srrlen seenta. nFIts e5)npneraiilseomaesicmoeeWda Rt. Som proceso | 1a73ns00iSnLeivv.eerr,aalltH1heofuurgaohncassltce4.guml e1s0a.t32o0pfXr.suentTmhoebdcuotriaoplrnesheepnetaisooencsytshioee eisatlbtboiuodtneedrryeibtdyhihohnecapytaest)oaecnydtaens evvaensnatte 4n19d) pdptsman pcreosemnet.. NPeEIrORoNGALdEaSrk, 1nOoSrUEtSe34SPOUTTGHOSHOASRIIEISEWETRS 7"Te0re7y1dh8i5ofnTfas.uert,oSdHinelpfacetronceyntbest.se 1r1e0athc.i1e2F0f.HTtChiToEsppbheapnadrebssutrencoonftaCionspfhoaomgs aaph10nt weed bdysl pater iin ceca cele Thicon (15) perosomss ust. Study Number: TRSpCon1s3o2r:.130M0 Amended FinaPlaRgeepo2rt TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No. _1132:100(EM99.6) Animal No. R10803 Trestment Group: _N-EIFOSE100ppm Sex Male `Speciev/SCOtBrMSaDiInGS:BCKrKlit Tissues Liver Block No(s): 99.622.1273356 Photo/Negative No(s):_Z17360__ Significant Lesions (check one): Yo X No Interpreting Pathologist (signature snd done): Seam fd o Septn 20,1, 999 Faepaptruorxeism:ate1l7y3t3e6n,smaLlivle-r0. Hmeepdaituonc-ystiez.ed10li,p3i2d0dXr.opTlhetiss.hTehpaetoccyyttoeplcaonstmaihnass numerous amdjiacteontchhoeannpddrRaEioRatnptrohoeflicelefstaypnredtstehnee.riBsgihlte.cEanalcicuyli atreiphnressiiencutsoboeitdcswaereyenprltehisseecnetlsol nsnd three margins. Some stain debri/anifoct is present on the photograph. Eight (8) p2e1r7o3x5i7s,omLeisvecro.unHteepda.tocyte. 10320X. This hepatocyte is bimacleated. Anendothelial ceolnltiasipnrsenseunmteartouthsetmoitpo,cbhuotnadlrliatahnedrmRarEgRinpsraofrieleasd.jaOcneentdhisetpiantcotclyitpeids.dTrohpelectytiopprleassemn, aTnwdetnhteyr-etahrreeenu(2m3e)rpoeursoxsimsaolmle0s cmoeudniteudn.-sized dark peroxisomesandor ysosormes. 217358, Liver. Hepatocyte. 10.320. A ucleus is not evident in this ploafsenctieon Toorgratnheilslheespaaptpoecaytrev.eFroyusrimmeildairu0m-Zs1i7z3e5d7.lipSidodmreopllyetssosaormeepsrceosnetna,isnndcltehaer votahceuroles within tZhIe7m3aS)n,d Lmiavnery.sHeeepiartroecgyutlea.ly1s0h,ap3e2d0..ThHierpiacteonc(y1t3ec)poenrtaoixnissoonmeeslcaorguentleidp.id droplet. Twenty-tiree (23) peroxisomes courted. Some stain debrivarifactpresen on the photograph. 17360, Liver. Hepatocyte. 10.320X. Multiple small to medive-sized lipid droplets re present. Sixteen (16) peroxisomes counted. Conclusions: Normal hepaocy 5.6 average peroxisomes per hepitocyic 1 Study Number TRSpCon1s1o3r2:-130M0 "Amendod FinaleRepyort TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100(EM99.62) Animal No. Ra. Treatment Group:N-EIFOSE, 100ppm Sex: __Male Species/Suain:CALCGDSAB(RSRDa)t Tissue: Liver -- Block Note): 99.6724274361 - Photo/Negative Nofs).:_Z17365 SignificantLesions (check ane): Yo xX Ne Imerpreting Pabologit (signature and date): QSyfemeen Gp1t4emb3e0,1r 999 mFiecartounrielsl:ousZIboTrIdEeLradTjvaecrentHe0paitomcytie. oTnUhTiEc.maTihnsscaonndtbaoBrdeeprwsowciyhehxeieiaci3cent hhepeacpytae.otTnhtoerheecpabtyoecrtymtaereghiansss.fouilncdceanalccalrecsuiaddelnqtuwieth3fweosomfahlel10mceednio sized liipeildydrgoprlaemtis.arTahnedrceaoennunmesrcoautsrmediiytsooncohmeosanaandddrRpiEraRoxpireosf.s.TSihxeirceem(a16) einriongncsyotomwoels chZiIo7su3p6dh2o.,toLgirvaeprh., Hweiptahtocytes.od10a.32h0eX.opTheiftbaenpdatbooystosma.pPpaeratrsofso3msewohadt daiamnonidg-cshlalpaenddiin aaunrcedlpaurbesusniedsta.cnvtiSdRieaEn(R6pa)rpohefeirloteonssonmdfetosmficttohoucnehipcoldno.dteoi.saAplho.,Thneuhmeerpoautsolysohsaosmesraonudndafceenwtpraelrmouxicsuosmes hZcooIsveTede,dscrLiivbeer.dshHoevpeat.oSceytvee.ral1s0m.3a2l0lX.pidTdhriophepastorcysthe apppreesseesnm.orpEhololgi(c1a1)vllpyersoeixmisiolnmateors oZihnIe3gr6pir4go,hficlLoeinovtefari.ansiHlseeepvcaeoarcanylatmleoi.cduu10lm.i.3p27r0eXe.sden4Mtnbdoeotnowsefeegttnwteohbitewgdoacdtrlookpcl.yetTi.rhseEeipcrgoehmstep(nl5te)itnpeehrheiopsxapitesooacmryests.eonA counted riZgLhTt3o65f,thLeipvhero.togHreappatho.cTytweo. c10r.320yX.iThahirse iprsraesoleanrtcgienhytehpaettsoicneyutseosniedx.tTtho eahseinpuasotidoucthytahtseeatop lIayrsgoesroomuensidaennuidcflpfeeiurcsouax.insdoSnmuvemseeinrnotuh(es16m)cytiopptelmaouosmo.rcnCehseacanooduRdnmiEesddRi.nprcrioofinilaetsw.eAebno,Sthienrepareermueltninplee and Conchutons: Normal hepatocyte. Mean of 114 peroxisomes per Wpaioey Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalPRaegpeo4r4t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_1132:100 (EM99.62) Animal No.: __R1042) "Treatment Group: POS 20ppm Sex: _ Male Species/Strain:C1iGCSBDRORSaDt Tissue: __ Liver Block Nos): __992.6127-43166Photo/Negative No(s).._ZI17370 Significant Lesions (check one) _____ Yes __X___ No Interpreting Pathologist (signature and date): Sept. 30.1999 pFoelaytguorneasl: nu7c1l7e3u6s6s.urLoivuenrd.edHebpyatcoyctytoep.las1m0r3i2h0Xi.n mTihtioschhonedrpiaaanthdaRosEacRcepynrtortaflsel.y lMouclattiepdle sgrmaanlullatroamneddicuomnt-asiinzsesdolmiepidlydrsoopsloemtessaraendalpseorporxeisseonmte.s.ThTewreenmtayi-nsienvgency(t2o7p)lpaesrmoaxpipseoamressfinely cZoIuTn3tTe7d,. Liver Hepatocyte. 10,320X. This hepatocyte has adjacent hepatocytes both dorsally ``aAnndevnednotrtahlellyi.l-Tliontehdeslienftuisoak ssinausooipdrfeisneendt bayloanngetnhdeotrihgehltiamlarcgeilln.anAdpcpornotxaiimniantgelayn neirnyethsrmoaclylt. ZlIipTi3d6dSr.oplLeitvsera.rHperpeasteonctyitne.the10cy,t3o2p0l.asmI.n tFhiifsteheenpa(t1o5c)ytpeertohxeimsiotmoecshoconudnrtieada.ppear smaller and more condensed. ProfilesofRER and SER are casily evident inthe cytoplasm. Five (5) pZ1e7r3o6x9i,somLeisvecro.unHteepda.tocyte. 10,320X. Several small and one large lipid droapreplreesetnt in `hwiisthhienpsaitnoucsyotied.s. AtThtehehteoppatroicgyhltea'nsdnbuctleoums ilsefntotmaprrgeisnenstontfthhisplhoatnoegroafepchtairoene.ryNthurmoecrytoeuss c`mointtoacihnosnSdrEiRa aannddRotEhRrporrogfainleesllrees viinscilblued.ingThseomientpeerrveonxiinsgomcyetsosaonldiyssfoisnoelmyesg.ranSuloamre.and ccroyusnttaeldl.oid structures appear very prominent in some peroisomes. Fifteen (15) peroxisomes ZisInTo3t70e.vidLeinvteri.nthHepaptloacnyeotfe.sec1t0i.o3n20pXh.otHoegpraatpohceydt.eSaepvpeeraarlssvmearlylsitmoilmaerditoumZ-1s73i6z9e.d liApindudcrloepulsets are present. Sixteen (16) peroxisomes counted. Conclusions: Normal hepatocyte. Mean of 15.2 peroxisomes per hepatocyic Sty Nu`mAbmeern:deTdRSnpEoln1s1o3Rr3es-p13o0rM0t Page 45 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No. _1132:100(EM99.62) Animal No: _RI0430 Treatment Group: _PEQS 20.0pm Sex: __Mals. SpecieCsd/COSMEtDr) IaGSiIRnK:at "Tissue: Liver Block Note): __99.62Z:5i0 m- Photo/Negative No(s). __Z17375 `SignificantLesions (check one): Yes X Ne Intcapeting Pabologs (signature an de): Sion 8(ah Sept. 30,1999 Features: ZI7371, Hepatocyte. 10.320X. This hepatocytehasaneccenirically located oval Nnuucmleeurso.usItimistboocrhdoenrderdiba,yaRnEsRinpruosfoiildesa,titnhectlogpaeanndlrairghctyatonsdol.anaontdhearfhoepatsovcayllteiopnthderolepfite.s re ppreersoexnitsoinmetsheocyutnopdlasm. Some smalllysosomes are scaticred in the cytoplasm Five (5) SSZioinItdm7he3ees7le2cx,ycyteopLspilvoeatrsh.sem.taHoo3en5ppmdawwtehfoeicesyclawthse.pipesa1rl0io.nexu3s2ic0skwXdo.r.meagTechuTseilhsaeprhrcyeelpsslaehtnoho.pcseFycotbuesurmisra(s4ln)utrpfroeormRuoeEnxddRiiesudpomrbm-oyesfisihlzceeeospduaatninotpdceiysddtiedosrocophnloeatnlslr:ia spZIrTofiilees. LSipoveumrte.olHiyepipdsmdorccoyoptnlteea,tin1ai0rne3gp20r.ecge.TshLueyssheopsnaatncoecseytaeaadpdppeenarrosnoisssovmcetslinleyarmanvei. aTaooi(dp2r)eKvEioRusly ZopUrfTot3bh7ae4bp,lreepvLieiorvuoesxrliysHoeemxpeaasmtioccnoyeutdnec.cde.l1l0.w3e2r0eX,.anTdhiis hoecpaatteodcybteeneiatshoacmeewrhoatbulilghaterrvsesinnianlgonhgahnemisgeht. PcSeroryomstxeailsclooolimdleasigreunnctbthoeerhsre.parEaeitgeohvctiyd(epanerteriboexhtieesroSdpmeaefcsiencoeofdvDheiadsn.einbmenoesatthotthheerenedlds.haenladlhacvle mparrogmiinnent SemZvaIreZgr3i0an5l..eTryLhtiebvrecroe,clytlHesesp.maitAcodrcjoyatveci.elonut1sh0e:hp3oa2rt0doXce.yitsTesnhsesrtcheo1p0natthoheceyetngdeohitthaeblnoirdadleerifentdoefdbytsaheocsoinsniorilwdheaipkcaohrnocgcayhnleei.noepA nluiclpeus imsintoocphronedsreitai3n dthiRsEpRlanpoefoiesse.ctiZon.eTh0e) cypleonpslsaomsefs aeeycliglryaniuslcoeamnidblceontom Conclusions: Nomeal bepatoeyt. Average 33 perovipoorempamioeeyse Study Number: STpRoCns1o3r2:.130M0 AmendedFinal Report Pages TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_1132:100 (EM99.62) Animal No: _RIOSI Treatment Group: N-E(FOSA 100ppm Sex: __ Male Specics/Strain:Cel CSSD) GS BR Tissue: Liver Block No(s): _99.62:5 Photo/Negative7No1(s7):-.3Z_IZ71T73638906 Significant Lesions (check one): _____ Yes _X__ No InterpretingPathologist (signatureanddate): Sept. 30,1999 pFoelaytguorneasl: nZuIcl7eTu6s,suLriovuen.deHdepbaytoccyyttoep.la1sm03c2o0nXta.iTnihnginuimgehrtosuasmimnitgochheopnadtrioac.yeRhEaRceanntdrSaElR prNoifniele(9s)arpeeproxrisoemeassndcteohuentnreda.tre grethaanatdeozren smalltomedium-sized lipid droplets. ZprIe7c3e7d.ingLeixvaemr,pleH.epTathoecyctyet.opl1a0s.m320isX.fiTnheliysghreapnaultaoreaynedstmaiintsoschoomnedwrihaaatpdpeaarrkedrentshearn.the Pofetreonxdisoimefs aftnodidisfcofmerueenlltiyatsteosfroommaercsacghenoethrearl.lysTmwaelnlteyr-atnwdod(2a2)rptekrhoaxenitshroememsictouonctehdondarndiar,e A2d1j7a3c7e8n,t tLoi:vesri.nuHseopida.toTcyhtee.nuc1l0e,us32i0sXn.oTheevtiopdmaergnhiitnsopfltahnieoscfesllescthioownfsoartmhiicrsohveiplaltooucsybteo.rdeTrhe ScyotmoepllasmycosntaoiannssdapbeournodmxainsteomSesEsR.bSeitxiwceennt(h1e6)mpiteorcohxoinsdormieasacnodunottedh.e organelle, including hZeIpTatAoTeytL'ivserc.ytoHpelpaastmo.cyAte.si1n0gl.e32m0eXd.iuMm-siize tlipidodancrdRohEipRpplroreoesfenitnltesdnaarrerptrheoimtionpeabntoirtdohifesthris cel. Asinusoid lindbyan endothelial cll is presentslongthe Ie margin. Seventeen (17) pZeIr7o3s8i0s,omOevsecroeuxnptoesde.d negative; notprinted. ZeI7p39a%.tLoivoser.ythetHewpeiatdsoecyatned. b1o03t2.0X,ATthteheceonptiesraesdihneupsaotoicdyftieneidbbyaonrenddobtyehealrdijaaelceednltl and cSounrtraoiunnidnegdsboymenugmrearnouluoscymtietsocahnodnderriytaharnocdyRteEs.RTphreofhilee. pSacattiheoraesdcdaayrcektnetraeplerpooxliysgoomneaslanruesleus present. Tweny-fve (25) peroxisomes counted. Conclusions: Normal hepatocyte Averags 17.8peroxpeshaepmaioeeyst. Study Number:TRSCpon1s1o3r2:-130M0 Amended FinalPRaegpeo4rt7 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No.: __R10430 "Treatment Group: N-EWFOSA 100ppm Sex: __ Male Specics/Strain:CrL1CGDSBERSRDa)t Tissue: _ Liver Block No(s). _99.6221:759381 Photo/Negative No(s): ZIT385 Significant Lesions (check one): Yes X__ No Interpreting Pathologist (signatureand date): October 1,199 pFreeasteunrtesi:n ZI7381, Liver. this hepatoeyte. ItHeispastuorcryotuen,de1d0b,y32n0uXm.erAoumsemdiiutmo-csihzoenddraroniudandRnEucRlepurosfiisles. iFnicvleusliiopinds;drtohpislemtasyabreeevsiodmeentstianitnhpereccyitpoitpaltaes.m.SeSvoermael mliytsoocshoomnedsriarachparveesednatr,kbut zero Z(0I)7p3e8r2o.xiLsiovemre,s aHreepactloeacryltye.dis1c0e,m3i2b0lXe.. This hepatocyte is somewhat lighter staining ptrhoafni2l1es7.38T1h.e cTyhteoprloausnmdcnounctlaeiunss issevseurrarlosumnadleld-btyo mneudmieurmo-ussimzietdoclihpoindddrriopaleatnsd. RER SZiIm7i3l8a3r.ly,Lsievveerr.alHelpyastooscoymtees. ar1e0,p3r2e0sXe.nt,Tbhuitszsrliogh(0t)ypdearrkoexri-ssoamiensinargehceleparalty odicschyeartsneaible. `SiDnuumseorioduslimnietdobcyhoanndreinadoatnhedliRaElRceplroaftilietss.upSpeevreriaglhltimpiadrgdrionp.leTthsearceyptroepsleants.m cNoinnteai(n9s) p71e7r3o8x4i.somLeisvecro.unHteepdatocyte. 10,320X. Erythrocyte containing sinusoids are evident on i{nhetheesciydetsopolftahsimsofhtehpiastohceyptaet.ocySteev.eraTlhmeerediaurem-ntuomelraorgues-smiizteodclhiopniddrdiraopalnedtsRaEreRpprreosfeinltes i7n1t7h3e85c.ytoLpivlears.m.HeFpiavteo(c3y)tep.er1o0x,i3s2o0mXe.s cTohuentceydt.oplasmofthis hepatocyte contains `bnoudmye,raoruespRreEseRntp.rofiSloems eansdcamtitteorcedholnydsroisao.meSsevaenrdalpelirpoixdidsroompleestsa,reonpreewseintth,aanladmaerlelar difficult to clearly differentiate from each other. Eleven (1) peroxisomes counted Study Number: TRSpCon1s1o3r2:-130M0 Amended FinalPRaegpeo8rt TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_1132:100 (EM99.62) Animal No.: __R10441 "Treatment Group: _Wy-14.643 100ppm Sex: __ Male Specics/Strain:Cl1CGDSBORSRDs)s Tissue:__ Liver Block No(s): __99.62:61 217386 Photo/Negative Nos):_Z17390 Significant Lesions (check one): _X__ Yes No Interpreting Pathologist (signatureand date): Oct. 01,1999 Fsienautsuoriedss:onZ1b7o3t8h6t.heLriivgehrt.anHedplaetfotcmyatreg.ins1.03T2h0eX.cenTthrials phelpaattoocfyttherheae shaepnatoovcayltneuschlaesus which is surrounded by numerous mitochondria and finely granular cytoplasm. Peroxisomes are the darker staining organelles. Twenty-two (22) peroxisomes counted Z17387. Liver. Hepatocyte. 10320X. Four medium-sized lipid droplets ae present in {his hepatocyte. The cytoplasm is finely granular and contains numerous mitochondria and some RER profiles. Three erythrocytes are present in the sinusoid atthe topofthe pZhIo7t3o8g8r,aphL.iveTrh.irHteyp-asteovceynte(3.7) p10e,r3o2x0iX.somTehsicsohuenptaetdo.cyte appears ultrastructurally similar {coontthaoisneidnegsncurmibeerdoaubsomviet.oIcthohnadsriaa,cefnitnreallyrgoruannudlnaurcclyetuossoslu,rRroEuRndperdofbilyesc,ytaonpdlsaosmme lipid derroyptlhertosc.ytAeddiniatiosnianlulsyoisdoimeprvaersieabnalty-tshiezeodpdarrikg-hsttmaairnignignopefrtohxeispohomteosgarraephp.resTewne.ntAy-n t1w7o38(292,) pLeirvoerx.isHoempeastoccoyutnet.ed.10,320X. Hepatocyte appears ultrasiructurally similar 717385. Forty-one (41)peroxisomes counted. 717390. Liver. Hepatocyte. 10,320. Only a small segmentof nucleus is present in his hepatocyte. This hepatocyte is bordered by adjacent hepatocyte on three sides and by an endothelialinedsinusoidon the lef. Is cytoplasm contains numerous mitochondria, RER profiles, scattered dark peroxisomes, and some lysosomes. Twenty-six (26) peroxisomes counted. Study Number:TRSCpon1s1o3r2:-130M0 `Amended FinaPRaegpeo4r9t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_1132-100 (EM99.62 Animal No: _RI043 Treatment Group: _Wy-14,643 100ppm Sex: __ Male Species/Strain:CrLCDO(BSDR1RGaSt Tissue: __ Liver Block No(s).: __99.62:63 217391 - Photo/Negative Nofs):._Z17395 Significant Lesions (check one): _X_ Yes No Interpreting Pathologist (signature and date): Oct. 01,1999 Fceenatteurreeds:inZtIhe73p9h1ot.ogLriavpehr.. HAetpatthoectyotpe.is a10s,i3n2u0sXo.id Aconptoaliyngionngala-nsehnadpoetdhehleipaaltcoeclyltaenids esriyntuhsrooicdyitsepornestehnet laettfthaendboattpeormoisfintuhseoipdhaoltloigpriadp-hst.orTinhgecehlelpaattotchyetreigchotn.taAinnostnhuemrerous cmointtoacihnosndsrcaitatearnedd RdaErRk plryosfoilseosm.esThaendipneterrovxeinsionmgesc.ytoTshoilrtisyf(i3n0e)lypegrroanxuilsaormaesndcoaulnstoed. 2s1ma7l3l9-2t.o mLievdeiru.m-Hseipzateodcylitpei.d dr1o0p,l3et2s0.X.NuThmiesrhoeupsatRoEcyRteprcoofnitlaeisnasngdremaitteorcthhoanndraidaoazreen pZr1e7s3e9n3t.inLtihveerc.ytHoepplaatsomc.yteT.wen1t0,y3-2s0iXx.(2T6h)ipsesrooxmieswohmaetslcioguhntteerd-.staining hepatocyte is bevoirddeenrtedinbtyhiasdjpalcaennetofhespeacttioocny.tesSeovneraalll mlaiprigdidnrsopilnetthsisarpehoptreosgernatp.h.FoArtnyu-colneeu(s4i1s)not pZe1r7o3x9i4s,omLeisvecro.unHteepda.tocyte. 10,320X. This hepatocyte appears morphologically similar 10217393, except it has a large central polygonal nucleus. Thirty-eight (38) peroxisomes c2o1u7n3t9e5d.. Liver. Hepatocyte. 10,320. `This round hepatocyte appears essentially s"TihmeilianrtteorvZe1n7i3ng91c.ytTohsoelhiespfaitnoeclyytgercaonnutlaairnasnnduamlesroocuosntmaiitnoscshcoantdtreireadadnadrRk ElyRsoprsoofmieless.and peroxisomes. Tw(1e2)pleroxvisoemes counted. Conclusions: Increased peroxisomes. Average 29.4 peroxisomes per hepatocyte. Study Number: TRSpCon1s1o3r2:-130M0 `Amended Final Report Page $0 VI APPENDIX 3 - PALMITOYL CoA OXIDASE REPORT REi .S.n o.m fon [rE ER ER "Table VI-1. Individual Animal Palmitoyl CoA Oxidase Activity -- Es ===== E BL E E r Em FEE FE m gm rr r8g B Er EE h hge =r E B E BE TE o y m=er r e E s r e - m o: m BECCLEE or CET om -- et [mem theeIf Emery Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalPRaegpeosr2t VIL APPENDIX 4 - STUDY PROTOCOL AND AMENDMENTS Study Number: TRSCpon1s1o3r:2-310M0 `Amended FinalPRaegpeo5r3t -- CHARLCEABSORRATIOVRIEERS Sponsor: 3M St. Paul, Minnesota PROTOCOL Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EFOSA 3M T-T091.1) in Rats Date: January 12, 1999 Performing Laboratory ROW. Sciences 15 Firstfeld Road Gaithersburg, Maryland 20878 Laboratory Study Identification: Study Number: (1132-100) PAI Project Number: (Histology number tobeassigned by PAI by protocol amendment) WoISrman'sil Cour, Sue 1+ Frederick, Maryland 21701 + (01) 663-1644 + (01) 663-8994 FAX Study Cell Proliferation Study with N-Ethy Perfluorooctanesulfonamido Ethanol (N-E(FOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16),and N-Ethyl Perfluoroctanesulfonamide (N-EFOSA 3M T-7091.1) in Rats Purpose "Toassesscell proliferation and peroxisome proliferation in rats administered test material in the diet. Sponsor 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 Study Representative MarviTn. Case, D.V.M, Ph.D. 3M Corporate Toxicology PhoneNo. 651 733-5180 Fax No.:651 733-1773 Email: micase@mcmom Alternative Study Representative Andrew M. Seacat, Ph.D. 3M Corporate Toxicology Phone No.: 651 575-3161 Fax No. 651 733-1773 Email: amseacat@mmcmom. Test Facility `Therlmmune Research Corporation (formerly, R.O.W. Sciences) 15 Firstfield Road Gaithersburg, Maryland 20878 Study Monitor Sandra R. Eldridge, Ph.D. Pathology Associates Intemational Phone No. 301 624-2036 Fax No. 301 663-8994 Email: srepaisaic@caoml Study Director Gary W. Wolfe, Ph.D, DAB.T. ROW. Sciences Phone No. 301 330-3723 Fax. No: 301 3303738 Email: gwolfe@lab.row.com Principal Investigator Sandra R. Eldridge, Ph.D. Pathology Associates Intemational Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@sol.com Study Pathologist Carolyn Moyer, D.V.M, Diplomate, A.C.V.P. Pathology Associates Intemational Phone No. 301 624-2928 Fax No. 301 663-8994 Study Number:TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeosrst Proposed Study Timetable In-life Start Date: To be added by protocol amendment; Day 0 In ie End Date: Tobeaddedby protocol amendment `Audited Draft Report Date: To be added by protocol amendment Study Number:TRSCpon1s1o3r2:-310M0 Amended FinalPRaegpeo6rt Regulatory Compliance "This study willbe conducted in the spiritofGood Laboratory Practice (GLP) regulations. Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 14. In the opinion ofthe Sponansdstoudry directhtesoturd,y does not unnecessarily duplicate any previous work. Quality Assurance Not applicable. Test Materials Test Material: |N-EFOSE (completedby 3M) ee. Identification: |N-EWFOSE LotNumber: [LoFtMs339002305, | 30037, 30039 Purity: 99.2% Subility: | >5years Storage room temp. Conditions|: Characteristics:|waxy solid | PFOS N-EFOSA | (completbedy | (tobe added by 3m) protocol amendment) PROS N-EFOSA [217 [Loser Wy-14,643 | (1be added by protocol amendment) wy | 9% >5 years >5 years oo room temp. womtemp. | | | white powder | amber waxy solid StudyNmber: TRSCpon1s1o3r2:-310M0 `Amended FinalPaRegpeo5r7t Reserve (Archive) Samples A reserve sample (approximately Sg)ofcach lot will be taken and stored at room temperature. "These samples will be transferred to the Sponsor after completionofthe in-Jife phase to be retained in accordance with 40 CFR 792.195. DispositionofTest Material `After authorization from the Sponsor, any remaining test material wil be returned to: Marvin Case, D.V.M., Ph.D. 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 PhoneNo: 651-733-5180 Fax No: 651-733-1773 Animals Species: Strain: [Ra | cacosp) Gs BR Source: Charles River Laboratories, Inc, Raleigh, NC `Age at Initiationof Treatment: | Preferable 6 weeks ofage, but not more tha8n weeks of age `Weight at Initiation of 15010300g oo Treatment: Number and Gender: males Identification: unique identificationbyindividual car tags and cage cards. wdo3s [Tousing: |SiTnegkllaedho70u1s2edCiernthiafniegdinRogsdewnitnlDeiests.teFerlewsihrfeocoadgweisllb_ epro_ vide_ d wee_ kly. | Water: a ---- `metals, aflatoxin, chlorinate hydrocarbons, organophosphates, and specified nutrients. Specified nutrients analysesare on file at R.O.W. Sciences. "Tap water, provided ad libitum via an automatic watering system or water `bottles. The water is analyzed at least two timesper year for contaminants and specific microbes. The resultsofthese analyses are on file at RO.W. substances potentially present in animal feed and water, including the test `material itself or possible structurally related materials as well as the items. listed in (2) and (3) above. Noneofthese contaminants are reasonably expected to be present in animal feed or water at levels sufficient to interfere Environment. | The targeted temperatures arc between 64 and 79F with a relative humidity `between 30% and 70%. Temperature and humidity are monitored continuously. A 12-hour light/12da-rkhcoyculre willbemaintained. Ten Acclimation: Randomization: as "Animals will be acclimated to the facility for a minimum of7 days prior to the startofdosing. Animals will be observed for general health and suitability for testing during this period. Animals that are diseased or | Using computer-gencrated random numbers wit assignment (0 groups, At the tshiomuelodf rnoatndeoxmiczeaetidon2,St.hDe.owfeitghhetmveaarinatwieoingohftt,haenadntihmeamlseoafneabcodhysweexiugshetds for each group ofeach sex will notbestatistically different. quirementsfor arodent species. Study Number:TRSCpon1s1o3r2:-310M0 `Amended FinaPlaRgepeo3r9t Group Designations, Dietary Levels and Scheduled Sacrifice Time Points ER CT NumberofMaleRas Group Number] (Time Point)| Control | (0 ppm)| N-EWFOSE | 300 100 30ppm| PFOS| 20 ppm| N-EWOSA| 100 ppm| Wy- | Toul No. 14,643 |of Animals -1 100 ppm| 1 (48h) 2 (dn 0 [10 0 | 10 10 5 65 a cameo 0 | s]s|s 5 5 5 "0 cece - spe cca 3 0 cle Oddy) | 1wk recovery) sso] |s|s|s 5 5 we 5 40 ecm] (4 wk r5ecovery) 10 "Total No. of Animals _| 50| 5 5 0 |3% 5 40 so | 2s | 25| Dosing Procedures Methodof Administration Dietary. Animals in Groups 1 throug3h will receive test diet for 48 hours, 7days, and 14 days, respectively. Animals in Group4s and 5 will receive test det for 14 days followed bya 1or 4 week recovery period, respectively. Reason for Dosing Route The potential human exposure is bytheoral route. Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalPaRgepeo6rt0 Dose Preparation Before initiationof treatment, dose preparationof each test material will be mixed. All dose preparations will be stored at room temperature. Dose preparation will be: documented and reported. See AttachmentI for test diet preparation procedures. Retention Sample `Samples (approximately 100 g) will be taken from the dose preparation and stored at room temperature. Unless used for analyses, these samples wil be discarded atleast 1 `month after completionofthe in-ife phase. Observation of Animals Clinical Observations Each animal will be observed twice daily (a.m. and p.m)for morality and moribundity; findings will be recorded as they are observed. Body Weights Prior to treatment (at randomization), weekly for Week 1 through 4 weeksofrecovery. Food Consumption `Weekly for Week 1 through 4 weeksofrecovery. Clinical Chemistry Animals will be fasted overnight before animal's scheduled necropsy; blood wilbe collected from ajugular vein into an EDTA-coated tube. Serum enzyme levels ofalanine aminotransferase (ALT), alkaline phosphatase, aspartate aminotransferase (AST), cholesterol and triglycerides will be determined. Study Number: TRSpCon1s1o3r2:1M00 `Amended FinalPaRegpeo6r1t Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with COs, weighed, and exsanguinated. Scheduled Sacrifices Interim Sacrifices At48 hrs, 7 days, and 14days, animals will be fasted overnight, bled for serum samples, anesthetized with CO, weighed, and exsanguinated. NOTE: Two serum samples will be needed, (10).5 mi sample for clinical chemistry and (2)a 1.5 ml sample for compound level analysis. "The abdominal cavityofeach animal will be opened, the liver will be removed and weighed, and liver sampleswillbe collected. Animals will be discarded after liver collection. Terminal Sacrifices After 1 and 4 weeksofrecovery, animals will be fasted overnight, bled for serum samples, anesthetized with COs, weighed, exsanguinated, and necropsied. NOTE: Two serum samples will be needed, (1) 0.5 ml sample for clinical chemistry and (2) a 1.5 mi sample for compound level analysis. Postmortem Procedures Study Nu`mAbmeern:deTdRSFCpionna1sl1o3Rr2e:-p31o0rM0t Pa? Neeropsy "The necropsy will include an examinationoftheextemal features of the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Cell Proliferation Tissue Collection and Immunohistochemical Representative samplesofthe left lateral lobe ofthe liver and any Evaluation macroscopic lesionsofthe iver will be collected and preserved in zinc formalin. After fixation, cach sample of iver wil be delivered to SPaatnhdorlaoRg.y EAlsdsroicdigaet,esPhI.nDt.emational F1r5eWdeorrimcka,n'MasrMyillalnCdou2r1t7,0S1uite Proliferation cell nuclear antigen (PCNA) evaluation wil be done on the samples. In addition, liver sections prepared from the same tissue block will be stained with hematoxylin and eosin and examined microscopically. Palmitoyl-CoA Oxidase A sample (approximately Tissue Collection and Analyses S00 me)ofth right lateral lobe ofthe live will also be collected from select animals and flash-frozen in liquid nitrogen. See Attachment II for procedure. "The liver tissue will be stored in a freezer set to maintain -60 to -80 C until analyzed by Covance for palmitoyl-CoA Oxidase activity. The liver samples to be analyzed wil include al study animals, EXCEPT for the Wy-14,643 animals and al animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study wil be analyzed for palmitoyl-CoA Oxidase activity; these samples consist of liver samples from 35 rats and 35 guinea pigs. "Tissue Collection for Electron Microscopic Evaluation Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeo6r3t Sections of liver from all animals wil be collected, minced to approximately one millimeter cubes and placed in a fixative appropriate for electron microscopy. The containers and fixative will be provided by PAIL Electron microscopy will be performed on one animal per treatment group exhibiting the highest cell proliferative response as well as one control animal at the discretion of the Sponsor, from one time point as well as the 4-week recovery. "Thus, EM will be performed on one animal from the control, N-E(FOSE (one dose only to be determined), PFOS, N-EFOSA, and Wy groups at one of the time points, as well as the 4 week recovery, foar totalof 10 animals Remaining Liver Tissue The remaining liver tissue will be frozen and stored at -60 to -80 C for possible future analysis. Organ Weights Atthe scheduled sacrifices, the liver willbe weighed. Histopathology Liver from cach animal that is examined for cell proliferation will be stained with hematoxylin and eosin, and examined microscopically for histopathologic changes. Reports One copyofthe draft report will be sent to the Sponsor. The report wil include the following information: Experimental Design and Methods Results dose analyses `mortality clinical observations body weights body weight changes fteosotdmactoenrsiuamlpctoinosnumption cplailnmiciatlopyalt-hCoolAogoyxirdeassueltasctivities macroscopic observations mulitcrraosstrcuocptiucraolbosbesrevravtaitoinosns cell proliferation assessments Study Number: TRSpCon1s1o3r2:-I10M0 `Amended Final Report Paget Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the storage facilites of PAI for a periodof 1 year following submissionofthe final report to the Sponsor. One year after submission of the final report, allofthe aforementioned materials will be sent to the Sponsor and a return fee willbe charged. All raw data stored on magnetic media willberetain by PAL Study Number: TSRpon1s1o3r2:-1M00 `Amended FinalPaRegpeo6r5t PROTOCOL APPROVAL Marvin Case, D.VM, PhD. Date Study Representative 3M Corporate Toxicology Gary W. Wolfe, PhD, DABT Date Study Director ROM. Sciences Sandra R_ Eldridge, PhD. Date PPraitnhcoilpolgeyIAnvsessotciigaatteosr International Attachment: 1 `Test Diet Preparation Procedures Study Number: TRSCpon1s1o3r2;-130M0 `Amended FinaPlaRgepeo6r6t 1). Determine the amount of test dit (feed) thati to be prepared and weigh out that amount of feed. 2) Calculate the amount of test article that is needed to prepare the test diet at the desired concentration. 3) Accurately weighoutthenecessary amountoftest article. 4) Transfer the weighed test article 10 a container and add a small volume of acetone to container. Manually mix to dissolve the test material adding acetone as necessary (typical natio of test material to acetone is 1 g 1520 ml acetone). Visually inspect test materialacetone for solubilityoftest materia. 5) Prepare a pre-mix by transferring the dissolved test material into 4 kg of feed in a Hobart mixing bowl. Mix for 10 minutes. Transfer the premix to a larger mixer, add remaining `amountofweighed diet, mix for 30 minutes. Study Number: TRSpCon1s3o2r.:130M0 `Amended FinPaaRgeepo6r7t Attachment: 11 CollectionofTissue Samples for Biochemical and Molecular Analysis Becauseofthe extreme instabilityofcertain enzymes and biomolecules, itis ssental that tissues be harvested as soon after death as possible and flash frozen immediately in liquid nitrogen. Failure to follow these procedures may lead to lossofthe entire sample and all the energies and resources that were invested into generating the samples. Therefore, make every effort to comply with the following: 1) Harvest the tissue samples as soon as possible afler death. Delays may allow for biodegradation andor inactivationofthe desired endpoint. 2) Immediately submerse the tissue sample directly into liquid nitrogen. Dry ice or other altematives will not suffice. It is important that the tissue be immersed directly in liquid nitrogen; transferring it o a dry vessel (or sample container) suspended in liquid nitrogen will not suffice. The tissue may freeze to the vessel wall and will then be impossible to remove without completly destroying the vessel (or sample container). 3)Beabsolutely sure to maintain the tissue frozen. It should be stored in a scaled container at 70C and shipped or transferred on dry ice. If needed, the frozen sample can be fractured (broken nto portions for different applications) by placing in a crucible which contains liquid nitrogen to keep the sample frozen while grinding/fracturing. PROTOCOL AMENDMENT #1 Study Number: TRSpCon1s1o3r:2-130M0 `Amended FinalPaRgepeo6r8t Date: February 24, 1999 Study Number: 1132-100 Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EWFOSE; 3M T-6316.11), Perflurooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Changelreplace the following: 4) Title page: PAI Project Number: 99-1233 b)Paged: In-lfe Start Date: 2/23/99 In-life End Date: 46/99 Draft Report Date: 5/18/99 ) Pag4e: Test Materials N-EFOSA JWy-14,643 (Cayman Chemical) | [d-- entificat-- ion: --INEFwyOSA [LotNumber [sat Theo | [Puity: Notprovided |Notprovided | [Swbili:_-- [>Syes TSiyer | [Characterisies: ___-- Tamberwaxysolid |whitepowder | Page 5: Animal identification is by car tag ) PageP8h:yOsbiscearlvEaxtaiomnionfatAinoinmsas; add, `Weekly at each weigh inerval. 9 Entire protocol: Change R.O.W. Sciences to Therlmmune Research Corporation ) Page 8: Clinical Chemistry blood will be collected from ajugular vein into a serum-separator tube. Study Nu"mAbmeern:deTdRSFpCionn1as1o3Rr2e:-p1oM0r0t Page 69 h) Page`9S:erSucmhesdaumlpeldesSafcorrifcilicneiscal chemistry will be performed by PAI at Chevy Chase, MD. `Serum samples for compound level analysis will be stored in a freezer set to maintain -60 10-80C and packed on dry ice and shipped to: 3DrM. EKrTis.J&. HSansen 9Bu3i5ldBiunsgh2A-v3e6-n0u9e TSte.lPeapuhlo,neM:N61525170768-6018 Fax: 612 778-6176 For both Interim and Terminal Sacrifices: cNhOeTmiEs:trTywaonds(e2r)utmhesarmepmlaeisniwniglsbaempnleeedfeodr,c(o1)mpaotufnedasltev0el.2a5namllysissa.mple for clinical ) Page L1i0v:ePraslammiptloeysl-fCoroApaOlxmiidtaosyel-TiCsosAuoexCiodlalseectaicotinviatnydwAinllalbyesisshipped on dry ice to: CDro.vaRnocneMLaarbkoervaittocrihes Inc. M33a0d1isKoin,ngWsIma5n37B0o4ulevard Telephone: 608 242-2712 ext. 2337 Fax: 608 241-7227 3) Page L1i0:veTrisssaumeplCeosllfeocrtiEoMn fsohroEulledbcterosnhMiipcpreodstcoo:pic Evaluation PShAaLroNnCAmbrose 4915 D Prospectus Drive Durham, NC 27713 K) Page 10: "The Remaining Liver Tissue remaining liver tissue will be frozen and stored at 60 to -80C in PAI's long-term 1) Page a1r0:chCiveelfPorroploisfseriabtlieonfuTtiusrseuaenaCloylslse.ction and Immunohistochemical Evaluation m) Page 1l0i:veTrwisislulebeColcloellcetcitoendfaonrdElpercetsreornveMdicirnofsocrompailcinE.valuation `Sections ofthe left lateral lobeofthe lver from all animals... Study Number: TRSpCon1s1o3r2:-1M00 `Amended FinalPRaegpeo1r0t 1) 0) Page Page 71:3,ChItaenmge#5:doPsreeopfarWeya-p1r4e,-6m4i3x fbryomtr1an0s0f0erprpinmg ttohe1d0i0sspoplmved test material into feed. Mix unil homogeneous. Reason for Amendment: 4b)) ADsastiegsndeedtearfmtirnperdotafotceorlparpoptrocoovledsigned; study is non-GLP, therefore report wil not be audited ) Information obtained after protocol signed a)) SPhpyeslilcianlg eerxraomrs should be conducted in addition to body weights 1)TNeostainntgicfaocaigluitlyancthaisnguesdednfaomreclinical chemistry 1) Provide shipping instructions and specify minimum amountofserum needed for clinical c)hePmriosvtirdye shipping instructions ) Provide shipping instructions 1m))PSrpoevciidfeysltoobreagoefilnisvterrutcotiboenstaken for EM 1w)ei1g0h0tpapndmiWnycr,ea1s4e,6in43peirsoaxdiesqoumaetse for inducing a cell proliferative response, increase in liver ) Accurate descriptionoftest diet preparation procedure Approvals: Marvin CaseT,ED.RVE-M., PhD. S3tMudCyorRpeporreasteenTtoaxtiivceology ---- Gary W.-- Wolfe,-- Ph.D, DABT STthuedrylmDimruencetoRresearch Corporation Date isoneeeeesee, Date --S-a--ndra R_ Eldridge, Ph.D. Date PPraitnhcoilpolgeyIAnvsessotciigaatteosr Intemational Study Number: TRSpCon1s1o3r:2-130M0 `Amended FinalPRaegpeo7r1t PROTOCOL AMENDMENT #2 Date: April 16,1999 Study Number: 1132-100 Title: C3eMlTP-r6o3l1i6f.er1a1t)i,onPeSrtfulduyrowoictthanNe-EStuhlfyolnPicerAfcliudorPoootcatsasnieusmulSfaolnta(mPiFdoOSE;th3anMolT-(6N2-95E.F1O6S),E; `and N-Ethyl Perfluorooctanesulfonamide (N-EXFOSA 3M T-7091.1) in Rats Add the following: a) Page 9: Necropsy Gross lesions will be collected, processed and stained with H&E for microscopic evaluation Reason for Amendment: 4) For histopathologic examinationofgross lesions. Approvals: -Marvin Case, DVM, PhD. Date S3tMudCyorRpeoprraesteenTtoaxtiivceology -Gary W. Wolfe, Ph.D, DABT Date Study Director "Therlmmune Research Corporation _Sandra R_ Eldridge, Pho. Date Principle Investigator Pathology Associates Intemational Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalPaRegpeo7r2t Date: August, 1999 PROTOCOL AMENDMENT #3 3MStudyNumber: TRC 131-100 Tite: "3CMellT-P6r3o1l6i.f1er1a)t,ioPnerSftluudryoowcittahneN-SEutlhfoyn!icPeArcfilduoProotoacstsainucmsuSlaflotn(aPmFidOoS:Et3haMnoTl-6(2N9-5E.1(6F)O,SE; `and N-Ethyl Perfluoroocianesulfonamide (N-EWFOSA 3M T-7091.1) in Rats" Original: Page 10: Tissue Collection for Electron Microscopic Evaluation Sections of liver cubes and placed finroamfiaxlaltaivneimaaplpsropwrililatbeefocrolelleecctterdo,n mmiinccreosdcotpoya.ppTrhoexicmoanttealiyneornseanmdilfliixmaettievre gwirlolubpeepxhriobviitdiendg btyhePhAiIgheEslteccetlrlonprmoilcifreorsactoivpey rweisllpobnesepearsfowrelmledaosnonoenecaonntirmoall apneirmtarleaattmetnhte dpiesrcfroertmioendoofnthoeneSpaonnismoarl, ffrroomm tohneectoinmteroplo,iNnt-aEsFwOelSlEa (otnhee d4o-sweecoknlryectoovebrey.detTehrumsi,neEd)M,wPiFllOSbe,: N-E(FOSA, and Wy groups at oneofthe time points, as well as the4 week recovery, for a total of 10 animals. ChangelPraegpelac10e:tThiesfsouleloCwoilnlg:ection for Electron Microscopic Evaluation Sections of liver cubes and placed finroamfiaxllatiavneimaaplpsropwriilatbeefocrolelleecctterdo,n mmiincrcoesdcotpoy.appTrhoexicmoanttealiyneornseanmdilfliixmaettievre will be provided group from the by PAI 48-hour tEilmeectprooinntm.icTrhoussc,opEy Mwilwlilble performed on be performed two animals per on two animals treatment from the control, N-EtFOSE (100 ppm dose group only), PFOS, N-EIFOSA, and Wy-14,643 groups at the 48-hour time point for a total of 10 animals. ReasonAfnorimAamlesnsdemleenctte:d on the basisof ell proliferation results. Approvals: T Marvin Case,I DVM, PhD. e eee Date e S3tMudCyorRpeoprreasteenTtaotxiivceology Gary W. Wolfe, PhD, DABTeee Date e ee eee Study Director ROW. Sciences Sandra R. Eldridge, Ph.D. Date Principle Investigator Pathology Associates Intemational Date: May 30,2000 PROTOCOL AMENDMENT #4 Study Number TRSpCon1s3o2r-:130M0 `Amended FinalPRaegpoerst Study Number: TRC 1132-100 Title: C3eMllTP-r6o3l1i6f.er1a1t)i,onPeSrtfulduyrowoictthanNe-EStulhfyolniPcerAfcliudorPoootcatsasnieusmulSfaolnta(mPidFoOSE;th3anMolT-(6N2-95E.F1O6S),E; and N-Ethyl Perfluorooctanesulfonamide (N-EFOSA; 3M T-T091.1) in Rats Change/replace the following: 4P)erGfllouboarloloycrteapnleascuelfPoFnaOmSiAdewtihtaht Nis-cEuWrrFeOntSlAy abecccaeupsteedthiseNa-crEoFnOySmAf,orrNa-thFetrhtyhlan PFOSA. b) Page 4: Revise table because 3M provided the lot numbers, and purity informationis changing with ongoing analysis at 3M. Test Material: |N(p-rEovFiOdeSdEby 3M)|P(RpOroSvided by 3M)|P(FpOroSviAded by 3M)| (WyC-a1y4m,a6n43 or ---- | Chemical_) Identification: |N-EWFOSE PFOS PFOSA Wy |Lot Number: |3030003395,c3o0m0b3i7n,ed |[217 541 1 119904 Purity: Responsibility of| Responsibility of | Responsibility of| 98% oo Stability: aM aM SSyers |>Syers Mm Ssyars [>2yes Storage: room temp. room temp. room temp. Toom temp. Conditions: "waxy soolido |whitepowder | amber waxy solNidL| white powd Approvals: S`MtaurdvyinReCparsees,enDt.aVt.iMv,e PhD. Date 3M Corporate Toxicology Gary W. Wolfe, PhD, DABT Date `StTuhdeyrlDmimruencteoRresearch Corporation PSrainndcriaplRe. IEnlvdersitdiggeat,oPrh.D. Dae Pathology Associates Intemational satya TE-- rRoEeSrO VIIL SIGNATURE PAGE Sut Tie: C6e3l1l6P.r1o1l)i,fePreartfilounoSrtouodcytawnietShuNl-foEntihcylAcPiedrfPloutoarsosoicutmanSeaslutl(fPoFnaOmSi;do3MEthTa-n6o2l95(.N1-6)E,{FaOnSdEN;-E3tMhyTl- Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Sud Namber: TRC 1131-100 Ld CH BPS