Document BoOGyvqqkQQZ5dOVQ9ZpvyvJ

AR224- 1905 Sponsor: St.Paul,aMMinnesota <n OCnHcARstLnLEdABSpOyRnRATIrOVsREeInERiS Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA; 3M T-7091.1) in Rats Study Number: TRC 1132-100 Performing Laboratory: Gene Logie, Ine. (formerly, Therlmmune Research Corporation) Gaither1s5buFrirgs,tfMiaerldylRaonadd20878 Prepared by: Pathology Associates DivisionofCharles (formerly, Pathology Associates River Laboratories, International) Inc. 15 Worman's Mill Court, Suite I Frederick, MD 21701 Date: Original Final Report: August 9, 2000 `Amended Final Report: November 8, 2004 TS WormalnlCo'urst, Sute 1+Frederic,Marstand 21701 + GO1) 663-1604 + GOT) 63-994 FAX CONTAIN NO CB: 2/ TABLE OF CONTENTS Report Narmative Summary Tables Individual Animal Data Tables `Appendix 1 ~ In-ifeReport `Appendix 2 ~ Electron Microscopy Report Appendix 3 ~ Palmitoyl CoA Oxidase Report Appendix 4 ~ StudyProtocoland Amendments Signature Page Study Number: TRSCpon1s1o3r2;-130M0 `Amended Final RPeapgoer2t Section 1 n m wv v vi vit vin 292 `Study Number: TRSpCon1s1o3r2:-1I0M0 `Amended Final Report Page3 L REPORT NARRATIVE Sud NoimbeerdTRSFEpooTorSp:1r03t01 Fort STUDY REPORT Cell Proliferation Study with N-Ethyl `Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA; 3M "T-7091.1) in Rats Study Number: TRC 1 132-100 SPONSOR: 3M Corporat Toxicology `Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 SPONSOR REPRESENTATIVE: Andrew Seacat, Ph.D, (previously, Marvin T. Case, D.V.M, Ph.D, who retired prior tofinal rSMepipotonirtsn)odra cMointtcahecltl Fao: 651 T1773 3M Corporate Toxicology Phone No.: 651 736-0443 Email: mmitchell@mmm.com `TEST FACILITY: Gene Logic (formerly, Therlmmune. Research Corporation) 15 Firstfield Road Gaithersburg, Maryland 20878 STUDY DIRECTOR: Gary W. Wolfe, Ph.D., DAB.T. "TherImmune Research Corporation Phone No.: 301 330-3723 Fax. No.: 301 330-3738 Email: gwolfe@lab.row.com PRINCIPAL INVESTIGATOR: Sandra R. Eldridge, Ph.D. Pathology Associates Division of Charles River Laboratories, Inc., (formerly, Pathology Associates International) LY `Study Nu`mAbmeern:deTdRSFpCionna1sl1o3Rre2:p-o31r0tM0. Pages FPahxonNeoN.o3.013061636-2849-924036 Email: seldridge@criver.com HISTOPATHOLOGIST: CPaatrhoollyongMyoAysesro,ciDa.tVe.sMI,n,teDrinpaltioomnaatle, A.C.V.P. No longer with Pathology Associates at the time report finalized ULTRASTRUCTURAL PATHOLOGIST: PJaatmheosloB.gyNoAlsds,ocDi.aVte.sM.I,ntPehm.aDt.ionDailplomate, AC.V.P. No longer with Pathology Associates at the time report finalized STUDY TIMELINE: `InCiotmipatlieotni:oJnaonfuianr-ylif12e,p1ha9s9e9: April 6, 1999 FAimnealndReepdorFti:naAluRgeupsotrt9:, N2o00v0ember 8, 2004 REGULATORY COMPLIANCE This study was conducted in the spirit ofGood Laboratory Practice (GLP) regulations. PURPOSE Tadhmeinoibsjteecrteidveteosft mthaitsersitauldiyntwhaesditoeta.ssess cell proliferation and peroxisome proliferation in rats INTRODUCTION PaTdehmriifsnliussottrueodroyecdtwanaNes-EdtSeuhslyfilognniePcdertfAolcuiaodsrsoeoPscsoitaacneselssliulupfmroonlaiSfmaelirtdaoti(oPEnFthOaaSnn;dolp3e(MrNox-iETs-Fo6Om2Se95E.;p1r6o)l3i,fMeraantTdi-o6n3N1i-6nE.t1rh1ay)t,ls WPeyr-f1l4u,o6ro4o3ctaasncasulpfoosintaimviedeco(nNtr-oElFOfoSrA;cel3l MproTl-i7f0e9r1a.t1i)o.n In and apdedirtoixoins,ormaets pwroelrieferaadtmiionn.isteTrheed emnudlptodiinstcsipelvianlauraytetdeafmoreftfeosrtt,artthieclreeesfufletcstaarreesluimsmteadriinzeTdexitn TSaebaltieon1s. Because 1, I and this study III of this rwepaosrta, and individual studies are reported in the Appendix. 245 Study Number:TRSCpon1s1o3r2:-130M0. `Amended Final RPeapogrte. `Text Table 1. Experimental Endpoints Examined Performing Laboratory LocaHteiroenwoifthRienport [Bodyand liverweights| Therlmmune | Sections I,I,IV -Appendix 1| FINAL REPORT AMENDEMENTS `fiTnhaelsrteupdoyrtrdepaotretdwNaosveomribgeinrall,y2f0i0n4a)lihzeads Abuegeunsrtev9i,s2e0d00t.o reTflheectparecsoernrtecsttiuodnyirneptohrett(esatmaerntdicelde acbobnrceevnitartaitoinonofofrWNy--et1h4y,l64pe3rfflruoomro"o1c0t0an0cspuplmf"ontaomi1d0e0fprpomm."PTFheOsSeAc"ortroecNt-iEontsFOhaSvAe,baenedn imnatdhee tteostalilngSefcatciiolnitsyo(fTthhiesrlrempmourtn,eexRceespetarfcohr SCeocrtpioornatIiVon(;Apppreensdenitxly1,),GwehniechLiosgitche) rtehpaotrtwfarsomontlhye obtainable in pdf format and therefore, unable to be revised. Additionally, due to the inability to orebptoaritn issigsniagtnuerdesonflryombyallthpearptriicnicpiaptailngiinvnevsetsitgiagtaotro,rsDir.nvSoalnvderdainEltdhrisidsgteu,dyo,ntbhieshaalmfeonfdeordigfiinnaall signatures from Dr. Carolyn Moyer (study pathologist, Pathology Associates; originally signed August October 9, 4, 2000), 1999), Dr. James Nold (EM Dr. Gary Wolfe (study pathologist, Pathology Associates; originally director, Therlmmune; originally signed May 2, signed 2000), `1a,nd19D9r9.).RonThMearskteuvdiytcphro(tPoaclomlit(ooyr]igCinoalAdOaxtieddasJeanaucatirvyity1,2,Co1v9a99n)cea;nodriaglilnaplrloytoscioglneadmDeendcmeemnbtesr have final also been revised to reflect study report unsigned. the corrections noted above, and are included in this amended MATERIALS AND METHODS Experimental Design `The experimental design was as follows in Text Table 2. oo RK Study Nu`mAbmeern:deTdRSFpCionna1sl1o3Rr2e:-p1o30rM0t Page? `Text Table 2. Group Designations, Dietary Levels and Scheduled Sacrifice Time Points I `NumberofMale Rats - (GTrioumpeNPuomibenr)||(C0onptpmr)ol |[300N-1E0t0F3O0SEppm PFOS | N-EfFOSA| Wy-14,643| Total No. |20pp | 100ppm| 100ppm _| ofAnimals| 1 10 (8h)| 2 10 (days| ) 10| 10 10 10 oe woes 5s 5 5 eo este 10 5 ll rte 65 sess 5 5 40 pers rc] cn 3 (dda) 4 | (1 wkrecover 10 5s 5 | Lf) 10 5s 5 5 5 5 sr a 5 5 5 40 ese 40 (4TwotkalreNcoo.veorfy) |_ Animals Ble ollwe| : 30 25 | In-life Portion of Study `The in-life portion ofthis study was conducted at Therlmmune Research Corporation. Methods for the animals ian-nldifheupsobratnidorny,ofobtsheirsvasttuidoyn,s,intcelrumdiinnagtitoenstanardtitcilsesufeorcmoulllaetctiioonn and are administration, test reported in Section IV, Appendix 1. Cell Proliferation Staining and Evaluation Representative samples of the left lateral lobeofthe liver and any macroscopic lesions were collected and preserved in formalin. After fixation, each sample of liver was processed and stained proliferation cell nuclear antigen (PCNA). In addition, liver sections prepared from the same tissue blocks were stained with hematoxylin and eosin (H&E) and examined `microscopically. Sections of paraffin-embedded tissues were cut at approximately 5 pm and placed on positively charged slides processing for (Superfrost Plus, PCNA. Standard FiimsmhuenrohSciisetnoticfhiecm,icPaitltsmbeutrghho,dsPAf)or to ensure adhesionduring. PCNA were used to stain tissues. Briefly, tissue sections were incubated with a monoclonal antibody to PCNA. (DAKO, 2L2Y7 Study Nummebnerd,eTdRSFEpoo1sns1Ro5er3p1o0r0t Page Carpinteria, CA) and reagents required for the avidin-biotin peroxidase (ABC Kit, Vectastain, Burlingame, CA) method for the detectionofthe antigen-antibody complex. PCNA expression `was localized by the chromagen 3,3'-diaminobenzidine (DAB; Sigma Chemical Co., St. Louis, MO). Tissue sections were counterstained with hematoxylin. `The percentageof hepatocytiens S-phase (labeling index, LI) wasdetermined by scoring at least 3000 hepatocytes in 10 fields of liver. A negative control slide was included in the staining run and consistedofstudy tissue that was not incubated with the primary antibody. For cell proliferation evaluations, slides were first perused at low magnification (100X)to judge quality of staining, processing and sectioning, potential pattems of cellular proliferation, and histomorphologic changes. Cell proliferation was then quantified at higher magnification (200X) as described above. Histomorphology was further assessed by evaluating the H&E slide prepared from the same tissue block for each animal evaluated for cell proliferation. Clinical Chemistry `Animals were fasted overnight before animal's scheduled necropsy; blood was collected from a jugular vein into an EDTA-coated tube. Serum enzyme levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), cholesterol and wriglycerides were determined by LabCorp, and reported separately to Pathology Associates. `Tissue Collection for Electron Microscopic Evaluation Sectionsofliver from all animals were collected, minced to approximately one millimeter cubes, placed in McDowell-Trump fixative, and submitted to Pathology Associates's North Carolina laboratory for processing, sectioning and evaluation. Electron microscopy was performed on select animals as described in Section V, Appendix 2. Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately 500 mg)ofthe right lateral lobeofthe liver was collected from select animals and flash-frozen in liquid nitrogen. The liver tissue was stored in a freezer set to `maintain -60 to -80 C until analyzed by Covance for palmitoyl-CoA Oxidase activity. The liver samples analyzed included all study animals, except for the Wy-14,643 animals and all animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study will be analyzed for palmitoyl-CoA Oxidase activity and reported separately; these samples consistofliver samples from 35 rats and 35 guinea pigs. Remaining Liver Tissue aTnhaelyrsiesmaining liver tissue was frozen and is being stored at -60 to -80 C for possible future YF Statistical Analysis Study Number: TRSCpon1s1o3r2:-130M0 `Amended Fina RPeapgoerdt TLIheanSdtucdleinnti'csalrctehsetmi(sttwroy-sbideetdw,eeunnecqounatlrovlarainadncter)eawtamsenutsegdrotuopstesutsifnorg sMtiactirsotsicoafltsEixgcnieflicvaenrcseioinn u5.s0i.ngADuPnnveatltu'esopfrolecsesdtuhraenw0a.s05uwsaeds tjoudagneadlytzoebbeosdtyatiasntdicaolrlgyasnignwiefiigcahntt.datAanawliytshisaoPfvvaarliuaencoef less than 0.05 judged to be statistically significant, Record Retention Aolflthriaswsdtautday, wdiolclubmeenatracthiiovne,d riencotrhdes,stporroatgoecofla,cislpietceisomefnsP,atahnodlofignyalAsrespoocritatgeesnefroartaedpearsiaodreosful|t year final following report, all submissionofthe final report to of the aforementioned materials the will Sponsor. be sent o One year afier submissionofthe the Sponsor and a return fee will be charged. All aw data stored on magnetic media will be retained by Pathology Associates. RESULTS Cell Proliferation and Histopathology Ginrcoiudpencmeeaonf cPelClNpArolliafbeeraltiinvge irnedsipcoenssearse aprerepsreensteendteidn Section II, in Section Table 1. I, Table The severity and 2. The severity is `denfuimnbeedr oafs atnhiemaflolsdwiitnhcrienaasetrienatPmCenNtAgrLoIupcowimtpharaeLdItgorecaotnetrrotlhsa.n tThheehiignhceisdtecnocnecurreprreenstecnotnstrtohle value. Individual animal cell proliferation data are presented in Section II, Table 1. S1t0a0tisptpicmallNy-sEiWgnFiOfiScAantatintchreea7sesdaiyn cteilmleprpooliinfte.ratAilonthwoeurgehrsetvaetaislteidcailnlyonsliygnoifniecatnrte,attmheentlagbreoluipn:g riendsipcoenssefowraisndniovtibdiuoallogainciamlallyssiwgenirfeicawnitt.hinBitohleogriacnaglelysseiegnniifnictahnet icnocnrteraoslesgirnoucpe;llthperroeliffoerrea,titohni,s atrseadtemteenrtmignreodupb,yweareseivdeernittiyfioefd oatnllyeaisntth2e-pfoolsditainvde aconntirnoclidgernocuep o(f10a0t plepamstWoyn-e14a,n6i4m3a)l diunritnhge thinecrteraesaetsmeinntceplelriproodliafte4r8atihoonurwseraendse7ednayisn.30D0urainndg3t0heprpemcoNv-eErytFpOerSiEo,d,2b0ioplpogmicPalFlOySs,igannidfic1a0n0t ppm N-EFOSA, period. but notin 100 ppm N-EtFOSE or 100 ppm Wy-14,643,atthe 4 week recovery Histologic findings are presented revealed no significant changes in in the Section liver of IIT, rats Table 3. sacrificed Examination at 48 hours or of the 7 days. H&E At 14 slides days, garnodup1s,anidnc4ludwienegkcornetcroovles.ry Itnimaeddpiotiinotns,,lliivpeirds vfarcoumolWizya-t1i4o,n6w4a3stroebasteerdvaendimianlasniexmhailbsitferdommialldl aKnudpf4fewreceekllrehcyopveerrtyrotpihmye apnoidntmsu,ltniofosciagln,ifmiicannitmatrleahtempeanttocreelllautleadrfniencdrionsgisswaetre14nodtaeyds,.exActeptthefo|r Pel Study Number: TRSCpon1s1o3r2:-3100 `Amended FinaPaRgepeor1t0 the mild Kupffer post-dosing. cell hypertrophy seen in Wy-14,643 treated animals which was absen4t weeks Clinical Chemistry `clGirnoiucpalmcehaenmicsltirnyicdalatcahaermeisptrreysepnatreadmeinteSresctairoenpIrIeIs,eTnatbeldei2n Section II, Table 3. Individual animal Statistical and time differences points, but in ALT, ALP and AST were within the normal were scen range for sporadically among treatment groups each parameter. Triglycerides were s3i0g0nipfipcmantNl-yEd(eFcOreSaEseadt 7asdwaeylslaansdo|utwseideektrheeconvoerrmya,lanrdang1e00inppthme Nf-olEl(oFwiOnSgAtrateatthmeenstamgerotupism:e ptorienattsm.entChgorloeusptseraotlonweasorsmigonriefitcainmtelypodienctrsedausreidngasthweedlolsiansg outside period. the At normal range in all the 1 week recovery 2`p0erpiopd,mbPuFtOnSotatnhde 140w0epepkmrWeyc-o1ve4r,y64p3e.riod, cholesterol remained decreased in all groups except Body and Liver Weight Group mean IL, Table 4. body weights, liver weights Individual animal body and and liver to body weight organ weight data ratios are presented in Section are presented in Section IV, Appendix 1 atBotdhye w1 ewieghetks rweecroevenroyt paefrfieocdteidn during groups t3h0e0dopspimngNp-eErWioFdO.SEM,ea10n0bpopdmy weight was N-EtFOSA reduced and 100 only ppm Wy-14,643. dosing period Liver in all weights were significantly elevated at one or more treatment groups except 30 ppm N-EWFOSE and 20 fime ppm points PFOS. during At the the 48 hour time point, liver weights were elevated in groups 100 ppm N-EtFOSE and 100 ppm Wy- 1140,06p43p.mAWty-th1e4,76d4a3y. tAitmetphoein1t4,dlaiyvetriwmeeipgohitnst,wleirveerewleeivagthetdsiwnegrreoeulpesva1t0e0dpinpgmrNou-pE3T0F0OpSpAmaNn-d EAFOSE. At both N-EFOSE group. the 1 and 4 week recovery periods, liver weight was elevated in the 300 ppm Ldiovseirngtopebroidody iwneialglhttreraattimoesntwegrreouspisgneixfciecpanttl2y0ipnpcrmeaPsFedOSa.t oAnteboorthmotrhee 1tiamnedp4oiwnetsekdurreicnogvetrhye tNi-mEe(pFoOinStEs,alnidve10t0o bpopdmy Nwe-iEgFhtOrSaAti,oswwheerreeassigWnyif-i1c4an,t6l4y3inwcarseaseeldeivnatterdeaattmtehnet1grwoeuepks r3e0c0ovpeprmy period, but not the 4 week. Peroxisome Proliferation `Epleercotxroinsommiecprroolsicfoerpayti(onE.M)PaalnmditaonyallyCsiosoAfopxaildmaisteogyr]oCupoAsuomxmiadraysedaacttaivairtey pwreerseenutesdedintoSeacstsieosns 2S Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeo1r1t IL, Table palmitoy] 5. The EM CoA oxidase draetpaoratreipsrpesreensteendteidn SinectSieocntiVoIn, VA,ppAepnpdeinxd3i.x 2, and individual animal ANo1n4e8ohfotuhrse, oWtyh-e1r4t,r6e4a3tmienntdugcreoduapsdoeuxbalmiinngeidn (t1h0e0nupmpbmerNo-fEpFeOrSoxEi,so2m0epspcmomPpFarOeSdotro 1co0n0trpolpsm. N-EFOSA) revealed an increase in mean numberofperoxisomes per hepatocyte. Palmitoyl CoA oxidase activity did not differ remarkably among controls and all treatment groups at all ime points examined. DISCUSSION pInerothxeisoprmeeseprnotlisfteurdayt,ionmuinlttihpeleliveenrodpforianttss were given examined N-E(FOSE, to assess cell proliferation and PFOS, N-EFOSA, or Wy-14,643 rasecaovpeorsytpiveecoarnrterpoirletsoeesnttdmeadtienriaTle.xtATasbulmems3arayndo4f,trheespefcitnidvienlgys. during the dosing period and iAnscreexapseecitnedh,eptahteocpeolsliutliavrepcroonltirfoelrattiesotn maantderliiavle,r w1e0i0ghptpwmitWhyo-u1t4a,6co4n3copmriotdaunctedintchreeaasnetiincilpiavteerdassociated serum enzymes. Cholesterol was lowered as expected. These changes were reversed upon cessationofdosing. In contrast, none of whereas, during the the test recovery materials period, induced cell hepatocellular proliferation proliferation during the dosing was increased in period, the N- dEuAeFOtSoEt,hePlFoOwScoanntdrolN-gEro(uFpOmSeAangrloaubpesl.ingThiinsdeaxpspeaernenatt rtehsepwoenseek m4aryechoavveerybteiemne apnoiantb.errTahteisoen aPnCimNaAls,lawbehliicnhg iwnedreexo1f3 0w.e1e7k%s hoafsabgeeenatrtehpiosrtteidmef,orexchoinbtirtoledraatscoonfttrhoel sLaImoefoangely(E0l.dr0i1d6g%e.anAd Goldsworthy, recovery time 1996). point is Tahpupsr,oxthiemactoenltyrol10-lfaobledlibngelionwdetxhavtalwuheicsheehnasinbteheins study at the week 4 previously reported. Furthermore, the response was not dose-related in the N-EXFOSE treatment groups. cLaiukseeWay-d1e4c,re6a4s3e, itnhechtoelsetstmeartoelr.iaNls-EditdFOnoStEa(f3fe0c0t plpivme)r-aanssdocNi-aEteWdFsOeSrAum(1e0n0zpypmme)leavleslos,cabuusteddida dientcerreeassteinigntotrniogtlyectehraitdeWsyt-h1a4t,w6a4s3 roenvleyrslioblweerweidthtirnig4lywceereikdsesfaotl7lodwaiynsgocfesdsoastiinogn,obfudtonsoitngaf.terIt 1i4s. daofdyosisng. Palmitoyl CoA oxidase activity and peroxisomal proliferation were not elevated in any fest materials evaluated besides the positive control Wy-14,643. Taken together, proliferators in these rats. findings do Although nthoetsseuptpesotrtmNat-eErtiFalOsSEp,roPdFuOceSdoranN-aEpIpaFrOeSntA to be peroxisome cell proliferative response in the liver of rats, the proliferative response did not occur upon initial dosing as seen o 2S/ ml RR with Wy-14,643, rather it appeared during the recovery period following 14 days of dosing and was not associated with overt cytotoxicity. The cell proliferative response seen at the week 4 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 Text Table 3. [ee | = [ee SummaryofFindings to Proliferation ADsusreisnsgHtehpeatDoocseilnlgulPaerrPiroodliferation and Peroxisome [Bodyweight | - T - [ -- [7-777 7T-T.| [Liverweight| |Tncrease| Increase | - | - |Inorease | Increase| [Liverbodywt.| - |increase[Increase |Tncrease| | Increase | Increase| f1 ac -1 face-- TT fo m : R Te fast | - [ -[1111 -1] oxidase [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. ~ 252 orof n `Text Table 4. Summaryof Findings to Assess Hepatocellular Proliferation and Peroxisome Proliferation During the Recovery Period" ae| [a Cell proliferation|__|Increase| - |Tncrease |Increase |Increase|_- | RR RR [Liverweight| [Livedbody wt.|= Tnerease increase [=[|.= | | |=|= ||Tnorcase|Tncrease| fT ar T To face 1 [ast fI oomme T mm m Te T oxidase [Peroxisomes |ND |ND | ND|Nb|Nb| Wb | Wb] "An increase or decrease identified statistically and/or descriptively (judged to be biologically relevant) at any time during the recovery period. sn ND, not determined `Nh-eEpattFoOceSlEl,ulaPr FprOoSlifearnadtivNe-eEffteFctOsS, Abutardeoneoxthibpietroaxichsoolmeesteprroollifleorwaetrorisn,g do not effect in have rats overt that is reversible within 4 weeks following cessation ofexposure. LITERATURE CITED EldroifdgBeC, 3S.FR1.,maincdeGaonlddsFw3o4r4thryat,s:S.efMf.ectCsoelflapgreo,ligfeenrdateiro,narnadtecshionicceoomfmmoarnkcearn.ceFrutnadr.geAtptpils.sues `Toxicol. 32: 159-167, 1996. oo 253 - II. SUMMARY TABLES 259 Study Number: TRSCpon1s1o3r2:-130M0 `Amended Final Report Page 15 Table I1-1. Group SummaryofCell Proliferation Data TresmentGro NNEENrFEFoOOsSSeEE 000pmmm Vf Nairtosy aA T1oo0p0pmm LibFalooughineodes ase aaoaooesn acoSeeiesnt L[iba=lnngndex stu aSScoooounns,. aaaozosns. LaFb7son0ug0nrndes LabpelToooguxodex d oemt omsSotous ooSuoennn avaaccioensr Sahulo aooimzes. WLoiobakpioRegnoadesc1oLaWbeveelkioenRguacnronrayry WLeaabakbtRongiandecx 4oLWaovvoPokoRonenIcrnodveeyxry NS TErFoosmseent g0rpm asieso n asceeur o ooemsa aSsoiun NPErNEoOFsOsSpEe0or0p7mm Sooosoeem saocoeuzn. aaaccoeonn ofSnroeesad. NVEirtoaSeA1To0o0smpmm Sfrew. aooonze oonmiox. SSoerean "St ressvincocndo,P25. LabePlwoowmw de Laba4Ften0ngmndex e asassoen oSroie aaocneoonsn, ooSooerrse oprrioont. SSooiinn StudyNumber: TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeor1t6 Table 1-2. Severity and IncidenceofProliferative Responses in Rat Liver LabaFoneu.lner baFioaougnnndex TrsimentGrosy Sova? Incoeca Lair7moiougindex Libru:ogiwndes Sey anne Labtwvgooioudex_ Labwroonndes Sey iaonta NNNEeeFrrOooSssEtcRioOomRmmmm 00176 ahno Niearcoyasekyomemn ao6n7 aiiwoo oaar oaas fra2] ooWssn o5s5 o=s aais @@& WLaobotkiRuginnedescAoWLeaevkboRiuarcndoevyrxy Trstmeniosy Sony liints 4LWaebekaRonunades c4WLooboakt`nRrrgeoicnonvdyeerxy Seven icine NNN7Ee7eIr0rFo5Oos2SstEpe3osmo0mr9nnm a11s83 i ooisw ws aii" 2&% 3 = Wersosi0omnm 0183 @" pyi =Wn romdibnacseamiorverisn POA aba dsra:0 ht lab acy : Lleol loll Hlslslzlelelelal EEE EN et ft Tf i213lslslslslzlls ed Pl SSE slelalaslel Ae z 3 22 =|5|2]4 EI 78 HFa E 2 i 3gt&l3alss :3 55 g #513) 5 kd El 4 slesEnelelela EEE |g SEE ae PE 28 EllelzizlalelIzeEl Alslslzlalze 2 8[RI5(N8=E| [2122= 2 EE & is : : : i2 dg nBEE |HZE]Er2 eRl =| RolEe] oRo[5gO]rlEelE=|RoEllEoEle 3 =| 8 2eluleleElelEelRolEelES EolEelRolEZ] EleRlelEaleElaElals | 1glgsl23l (S glslslalEE] Bi gg8g2e8g15508 (8(2 228888las282| AeEEEg AEs [222] =| |Z] =|Z|2] |g [Egle ((E55528 AEs SEER zs iH sp1If] EEEEEE =]: rE 1 GRAIRET |eREE 4 defense g 4 Adnan gE z :3H z2 3g i: DEEESFEERERREDEeeErERE g2 g8 2|15af/=5](23]/e5]z3] 8 (2d (28 dl 358 loa 3 Bll Effie $ ZB |5|15=/|5(|2]|5 | =| : |g (2d (gE2 | SEE eas [3 2 EE een. ISEEEEES 252 jack . 3 i3:3 N=|k) 1Sl3s [ER5I2SE8=E] 2 252 353 55 5 : g dPlaasteed] 2 i Bl % I" z c[eHcaeibdeaoaf l(lElleat Ex (frie ol E+] = | zIal EE8R3R8ER2a8a5lRelsElaelnelaazEleRl els AEeBRdSEeEl i 2 g g g 7 i5H 2d dodHefEaofeezea fd dgnd[B5 EEEaaiag : 8 B 32 de [del dd dBzZ]) lel 2 SSeIlzRRlRz[laElgR] 2[{2=zefl2all=Ell S|EE=R 3]8 :ME Ss 25 1E525=81|852i5s LLEEEREERLL 2 S58 2 S ((2 38e 3(S2 85s8 E8 ES5E5 EE EER `Table ILS. Group Summary of Palimitoyl CoA Oxidase Activity ConTtrroelasmentGrop NN-EEIFSOSEE 310000pppom PNF-OESFOSpEp3m0 pom N-EFOSA100 ppm ToTmreoamentGrop NNC-EEIIFFOOSSEE 310000pppomm PNRCOESIFO2S0Ep3m0 pom N-EIFOSA 100ppm PwOhAC PaCuOA.O P@CEOAnO SMameple MeT m SD.s Range Sinze 85s o2m9 s amawow 5s aw1 oeenw1 7 sos wo P1CODAO P1C4ODaAyO PC1ODaAyO Sa1m4Dpalye MeJ an TSD. Range Sie 3 5 sos asuss S 50ss aasess 3 uoo24 os 7PCDOyAO PDCOaAyO PDCaOAyO STaDmaplye Me5 n SD. Raansge Smi0ne 7 s s2s 3w0ss asas asomss on ass Week Recovery RIecWoveekry ReIcoWveesrky R1ecWoeveekry MPeCOaAnO PCSOD.AO PRCaOngAeO Sasmipele sTnBoos3e0hos saass aassss aux easrss "palmitoy CoAOxidseactivi Ug 260 Study Number: TRSCpan1s1o3r2:-130M0 `Amended FinalPRaegpeo2r1t IIL INDIVIDUAL ANIMAL DATA TABLES db/ naann Ee-- EneSe `Table III-1. Individual Animal Cell Proliferation Data 48Hour, 7Dayand 14 DaySacrifices =E i E f=E e=E n Epee pEs E etESeT EgE ea Eh Swis Be=y= e g i= g hE eE me e i e E teRe Eae EE oo omnes Saar. E: ee EttShull [ET Rose oom | me] ------ ee -- m E e heE E e E a E SE Fe ie E e E me e e e 1 ------ Ea E P EO S -- e E = S-- e E SE t SioEe m w --E E |Eoo omR E--|ER t wa Eor R-- osOuE--| mE e eeeE EEe Er Ee -- e re - e E E s mooea e e e Ee he E me E ar = E =E ee =E =R==R = -- r a f _m--r] ca E wn o mee Riis Some Ee e -- e eS SHERios 1 suppareE--sT_iE--] Page23. `Table III-1. Individual Animal Cell Proliferation Data 1.and 4 WeekRecovery I EE -- -- -- Ee e -- E h-- e-- e -- e -- -- E -- e -- -- e1 -- Ee eeHee11 pe ea trr 1 e a F=e 11 Fa eH E r --ee eee r r HE-- E1 E r e rrE B= = =e r E =t =E=Ewe=e r T rr T r uw a BoE 243 `Study Tpawem NumTb RC1e13r2-1:00 `Table 11-2. Individual Animal Clinical Chemistry 48 Hour Interim Sacrifice t t S==== S=r sss ssti =s1ss pei E E S e E d E E e e E g Ee i E a E pSP E a r =m=={ E=e e s=E f sREi e EE sss pe r E E r l e EEE S sSSse s= y e S=S=EE S sEsSeS=es le Ea E SC Ee EE EREg Rse mEe RS a E i a rEtei i e F-- moa TwTaw Tw a0 es | pm { oss fE 4 p ber RIOM0 [722 ams v0| a Ta | t Er t mee w w wwe| pE E eteB r tE L eo rsE p F b EE g EoE `Table INI-2. In7dDiavyiduIanlteArniimmSaaclrCilfiinciecal Chemistry Sty N"uAmmTeb`RFSCpna1oeln1ds3Ror5ere-:p1o:3d0rMt0 Page 25 [Coos RRiioouTs Rims |a ae [ostr 6 2tta6r a [iw | ew w r Tn wi| RRiiesss mam | ar |w t1ise = e 55 | [ -- R RR10i451ei sa 3s1 [|me a3s013 |ot1wa78ss |w s83e s w 6815 || [I WEFoSER0pom 1 RRios ssw3a 0J |s tet ||e feJ t |__e | 3 ||| [m Jm J i w1 [ J s rs s | | Rioase[67 | 228 | 168 18 | 41 | [RimRRe 1i04d60 w |3S %a | | s A0746| || w113e27a2i7r o m 3S9 mmoowms w owww ow ws r sw s 5 [[EoFoosEsom |[ mRRioioowsieeerssm 33a m 1 [200 1 [||w t10ea |a a1 | ] Tu [3 i [P[ ros20pm |Rim ver [|asso |i stess[|et2rs8 [|sa aa2r | ss3mi3 || wwRirieemne|m 2eas00 [oas mmee e ie remr w ms e s r w ] ]| [Ri as |a | wies Si -- a 2 ssp5 r| Rwomamwerw [we[n ai me| (WTAE Tonpom| Roast | 85 | pst| foe 21 a791 | RiRR0ioodds8os3ss 3m EE 63w1 |s 146 4 w s&i 26S `Table ITI-2. IndividualAnimalClinical Chemistry 14 Day Interim Sacrifice Study`NAomTSmepRFoiCbnnn1as1eldo3Rr2ere:-p1o3:d0rMt0 Page26 [Cowos mRoioess3e % [see||ftese[|8ss n Tr w| 159 050> a} [ [ w w i m | %ae |e i oi || tae116s4 w e 12 w 75 I"-eF3o0asEom RiRDivaigsse. 2 ease twe wEzs 2st mRiioisosr we 3 eww w mwre 0a 31 heI eroseoom[R1 iRsicaooJt--|| 0 s5e1 || agsess0|A AS -- [ 1 wis [se w 201 [i] ---- I FJ rom [meew-- w -- hRimse |e ee m[ate f[ ea ra e s| [P[ ROS 20pm Rm ioJ s Tesos SO o as 60| wtJ eiee s|A a S si||| Iesoskam iRsiotstss --|[ar{o[ma0|| t15 a10o 7=| a50o [Romoss | ee at|asew | wa s s 2| 3 || RRii0ss20ew29 ma0|awwe 6 a3r] [ [ mm m [s oeems o asis we e[sr s s z3 e o7r Riosad ww 264 [Controls wi roel Table 111-2. Individual Animal Clinical Chemistry 1 WeekRecoverySacrifice | Risze RI0527 | or [ iss | | 34 243 114 | 45 81 8 61 RI0528 | 59 87 52 g 62 [ [m m [ [s2 i i o | |tanen | |s s m es |r 5e s | 8 80| ] RI0534_ R10535 E:) TesTaT= | 35 0 |e as da 50 | [ [m m | | 2i o a0r | 2t7es5i | |s s i7az 81 | a 2 3 e s 0s aars]| | Ri08a2 2 [wz[ie] 3 R10543 so | 150[ed| [= moss | 3 [1 mioss| | | eo | os | 3 tes | dor 3% a| ds] [NEFGsESupp | Riosds | 34 | 2s | 1s | 20 TL4} a R10649. ([ prosz0gem |Rmiioosssto 43 || aass || 22 toeat || 18 ie0s3 | ||" ar | 25[d3 e | || [m mises | | _e ar | |1aa59 | |s mwas aatis 8 50 | | miss mises a2 wm | ote eo | |e am w[6 ss| ) I[ NEFoSAToopomm | Rios o sao7 || s w0s || 1w2e5s [|x204 [ 1 y 3392 || -- [ miess [sz | miosss | 40 | aor | wz | me | 8| ar [45 zz | st | | ete iE 70 [et| ts [3s as | 247 StudyNmumebTRFSEneop11o5rR5ieo-p1,or00 Page28 "Table 11-2. IndividualAnimalClinicalChemistry 4 Week Recovery Sacrifice res eg Tig ep Te SHO R10367 38 | ios[171 32 62 Riese ma @ RT m R10568 a88 | 125 [181539 309 55) Rost i) [R sa |i m7 | is s ar |4 | [m mei m |ow3%o ||o1w36 s ||m 186 |i H ds |s 4a39 || Riots 3 | qr mm [N-EFOSE300ppm | R10576 | 61 | 133 | 187 36 R077. EE) 136 32 63 38 [7 miosre |_a7 | 10 | wet | 33 58 | [m | a7 |o vss| iss| as 4 ar | [| miosso | eo | 163 |"isa | 48 61 | press Re eefe aa | Rios&2 46 | ss [22| [mo a7 |s 126 | teee | doa s 64 | b [ ro m mo |e sr |w s 152 |m teae |w 31 s e 4 | N-EFOSE3Oppm | R10586 | 54 | 120 | 200 | 21 84 | [m| o si |s tsa | dare | 49Ty as | [TT r_m miosss r | s 40 |e 8s | T 206 T | s 28 Tae 7 | [PFOS20pp Riosot | 46 | 147 | 235 | [mo |s der | 183e | % s 8 | eros Toon | idaes -- R10584 R10595. R10597 R10596. ST los {iss | 4 38 | 115 | 148 | 36 43 108 | 173 |286| 28 125 | tar | oar | a8 i45 | 49 38 | [Ri 7s | 6 |s Mes 25o 48 | Rios0z 3 T6832| EH} -- 124. 52 67 [7"wioeos 38 | doi | 101 | 64. 67 28 Sua NumTbRSEep11o3rn2s-1:o0r0 "AmeFinnaldrRieepsoedrnt `Table I1I-3. In4di8vHidouuarl AInnitmerailmSHaicsrtiofpiactehology Findings [Gonos Roieort hNooseigneiacnmrriioso|s -- fis fosicmiescs| FfFiton--hfoiiisssms fihoocsssimicicmmiiionnssenns|||s E-- -- E his-- -- hosnioane| m cero ot-- ho hsseqnmcetir oans | f i fioo feessmiiassns|| ao io-- r or rice| [Rf EFOSEi i gam| e Rioatt leorsnc+ia=cnmttreamines || fT1oiifun is hhooosssssioacmimciiomesinnassn|||s ffiuiiinn rnoosomssieinsee|| Fase -- fiisn hoess-- ioiceatreieasen||s Tio osiicaiosns mi me ffinm s toseoe ss| f f for o so it sss e s otir ct t os ng | Er eae: hie ee r] 209 StyNuAmbmereSTpFRnioEnnad1ls1Ra3eer3p-o13dr0mt0 Tage 30 `Table 11-3. Individual Animal Histopathology Findings 7DayInterimSacrifice iT "TreatmentGroup |Animal Number EFTindings Liver I VEF-- OS50S3E-- pm| R1i0o4d8s5s ( NNoossiggnnY iffcicaanntt inO dnigngs L-- ESo ---- Raker No gnicantfinangs [ RioiI o Nosgnifcentinangs | EE -- [INv-sErtFoOsSEE3s0ppop n RRi1o04t6s6 otinteminany [Nosignificantfinding [ RidisI s Nosonifcontfangs| RR110047732 NNoossgglifcfacntanntdingndings RR10047745 NNoossggniiffccaannttfinnddiinngg I---- sRr Rui1oi8n75aicINNoar sgnnfctan nddiinnggss | W543 100 on| RIGBY Nosignfcant ndings RRiid0i4s843 NNoossggniifcfanctainnndiintngg I RiDiss Nosqnifcan fing 270 oo `Study Number:TRSCpo1n32s.o1r30M:0 `AmeFinnadlPRaeegpeo3dr1t `Table I1I-3. Individual Animal Histopathology Findings 14 Day Interim Sacrifice pe ger | RR1I04De9 i[Nposvgrifcaamtcincgs tabao,tn, | T0750 poodgrreeggvaactsso,, cumomaootddoInnih,a.mmamtiomroarytccll.,mmuotocca,l,mmiinniamall EE -- R10455 T ip vooT wizston: i minima-- | | RwiDdmes ogregacs,mcd nfamatay cel,mutocs,minimal pdvacsolaatooni. mma 10155 ipoarsgae, vacmuodlaptdi.omni.aoryco,mutta,minal I O( S-- -- -- TT T N [EFOSE T0upmm| RRiI0O5S0010 [kvpiiooaddregvacesa, ocutomiiiszaeadltitidoi,donmmni,k: cry cal,mf,minima. 10803 icporegavtso, csolgmatoond:Inda.tmioc:r cel,mutta,minimal Er S --ea= Rm 10305 |Cs iTipvacoomtsepsptdomn, cot.stom,t [PROSZoom 3 T CTT T -- ---- | RRII0CSST1T0 Iippcovrvsaec,uoolcatuimoionx:eldiaiia.ftmmimionnitinmam:lacof,muti, misal. CEFOTSG Agom| RRiioosst1eT. RRIiGoSs1T0E Ip vocvolaaton. pd minimal [Frees 100m | Ri0s21~~ Ipooirevvoapphcy:uaKnunpellfcreizorospaoi,sai,oimoiunfnecemi,:ctul,mainirma T|| RIOS ephopercvephay Konpeercroc,dlmutfmiod:cl,inst 4vacatkid,ominima,t. TGRES~~ I[fupeericoky,pKuspnfeecrtrosciosa,.mmucifco:cea,mainral T0828 | 0IperrvvoaeochpyuKaouneplcteraorsoctiosli,o,mmmoiainclnidmf:a,ohlc,umaart pkvocokzatonip, minima || RiGEES pIarnropt.aKnulepcreoorsiceso,mmueifco:lcalu,mainarl; (5Corvegaaes,cumoinledaIidtfa.immmoianntimo,ay:ci, mtfocal, minimal oo 27/ - Study Number TRSEpon1s5o3r-130M0 "AmeFinnaldPRueegpeodsrzt `Table III-3. Ind1iWveideukalReAcnoivmearlyHiSsactroipfaitcheology Findings [[rCosTntrmreoaeltsnmetntcGroup _|AannimiRatilvSuzmm5bter]No Sear odin Li+veir | 1Ri00552287 iipplvvaaccuuooltatziaiotin.o,mni.dmid: apgregates,mixed nfammatryco,mutiocal, minimal RRI00S5370 [iippavvaaccwoootlzzIaiaptt..oommnnik, ---- grote, mise naman col mutfoca,minimal ---- [[ T RT io RR1i005Ss 3353[iiplipipadvvvaacacuoclvizouatlioosnl,lallpeitopiiddao..,mmmntiiinn.odiimmnaall EFOSS00pEom| Ri0s26 [pd vacuniIzaitmoind,: E[ E -- : RRi10s53%9F lT aiipgpgvrveeagT anctoebsl,aomslitxzoedann.itdni.foplmanmmiim.dagtT ory cell,-- multifocal, minimal 1| [R105i 40 f[iappo itdvvaaeccuus noilfI1zzopda.ammmttiaoronalnn:l R105 ipgvraceuogmtiazseadtitpifedlo.ammsnmia.t,aory cl,malfoclminimal [Rli dveouolaaton,pis minal t| TT -- |_| RiR#11000555454098 liiipppidvavaaccucuolioozaolionts,pzaipdipdat...mmtomiininoniinmmna-aalll | [R Rios lioi nddvvaeccuuoollaaattioonn,.ipis c.mmiinnimmeall s|| R10554 laigpgirdevgacautoelsi,zamtiixone,dlinpfida,mmmineitmoarly:cl, mutocal,minal pidvacuoliizpaidt,miioninma,l RRR11100055555587_ NpNoiodsviasgnfgcat cuucoalnitfzianntddiiinonpggnis,d,mild L | [523 4 100m| RR1Ri100o585s6190T ll|iFipypipdevvraatccouupoonlilyze.aaKttiuoopnn,.:ipiddc:,olmmiinniimmmial:all | [hiplan. aevoaculpollizaaatnrieotcn,roppsaoiirdass,, ommiuntilommacolad;ule,rmaaitneirma: TRIGB2 a lpidavraic,uolKuplr aict,lii,ommina,l -- Cr | lipid vacuolization, lipid, minimal R10564 i[iPydpervtarocpahyt.iKounpf,ferlpc,ellm,imniildm;al R10565 p|Piyspevratcroupohly,ationKupfer.tpce.llm, mmilda; Sas oo EE Study NumTbRSCp1eo1n3sr2or-1:30M0 `Amended FinaPaRgeepo5r3t Table ITI-3. Individual Animal Histopathology Findings 4 Week Recovery Sacrifice TreatmentGroup |TAnimal NumberT FitopathoLliovgeyr Findings [Controls 1T Rios_lipGivdavcaicuoollzzasttioonn.pidi, meid: | |TRi0Ri8os6e78__ iagdorvogaactuoosl.zmaitxoe,dpnef,mmmaitnoarlycll, muliocal, minimal Rioss pd vacuolzaton. pd mi |iagdrevgaactueosl.zmaioxne:dpiindfammimnetmoayFcel, mulifoca, minimal INEFOSE300pom| R10576 ind vacuolation, pi mid I T -- N 1 R10577 lipidvacuolizatiloipnid,,mild [1|rRRo1i00s5s7880 ||lilspipiiodd vvgaaccuurooliiezzmaatitgiovonena,,dpltiin,pfiadme,mimmsnailtido,m;raylcl, mutfocal,minimal 1 neirose 3050 Pros pom NEFOSA T0055 | YL -- R10584 R10565__ TNioasgdiggrvnoigaofticecsau,ntmofiitxnedzid1nagnpstf. aomnHmE,aElocerly,mufoca, minimala Ri0585__appodrevgaacluoosl,atmiiosne,dIinpf,lammmiantoirmyalcel,moles, minimal. 1R100858889 Tpiiddvvaaccuuoollazattiioonn idid..mmiinimlal Ri10085810 Noogosreggaiens,fcanmtbineddingfslammatory cal, muRfocalminial 1100550623 ppididvavcaucouollaattiioonn,. iid.. mmiinniimmaall R10504 aTigpgidrevgaactuoelsi,zamtiixoen,d liipnidf,ammimntimoarly; col, mltfocal, minimal _ RRR11i000555690756 sliggodrevgaaotceus,orlizemamtbiiipxoeen,ddtIi fplea,n mmsmaf ito.nrl ayla cceom l.,mmmuulfta oifcoat call,,ommiir nniinmyaayl | R10596 Nosgniantfindings T2545 1005p ||__RR1100660010 R10602 Naogsggnirfceanmgtinxadeidtningefsasmm,atory No sgniantfncinga ce, mttocal, minimal -- 10808 TNvioasconufocalnztandlionIgnps, RTE -- R10504__{eastpos ispiersdmpgoyrrmamigorimansis:om,a. Study Number: TRSpEon1s1o3r2:-130M0 "Amended FinalPRaegpeo4rt IV. APPENDIX 1 - IN-LIFE REPORT 27 Study Number:TRSCpon1s1o3r2:-130M0 `Amended FinalPaRegpeo3r5t V. APPENDIX 2 - ELECTRON MICROSCOPY REPORT `Study Number: TRSpCon1s1o3r2:-310M0 `Amended FinaPRaegpeo3r6t ANCILLARY PATHOLOGY REPORT ELECTRON MICROSCOPIC EVALUATION OF LIVER IN CRL:CD(SD)IGS BR RATS) ECTEHLALNOPRLO(LNI-FEEFROSAET;IO3NMSTT-6U31D6Y.1W1)I,TPHENR-FELTUHOYRLOPOECRTFALNUEOSRUOLOFCOTNAINCEASCUILDFPOONTAAMSISDIUOM SALT (PFOS; 3M T-6295.16), AND(NN--EEFTOHSYAL3PMETR-F70L9U1.O1R) OINOCRTAATSNESULFONAMIDE TRC STUDY NUMBER 1132-100 PAI EM PROJECT NUMBER EM 99.62 SUMMMARY ACnr:iCnDcre(asCeDi)n tIhGeSmBeaRnrantusmgbievreonf1p0e0rpoxpimsWoym-es14p,e6r4h3epbaytodciyctteafwtaers4d8etheocutresdobfytreelaetcmternotn.miTchreomsceoapnynuinmmbaelreof hpeepraotxoicsyotmeewsewraesnaoptprreomxairmkaatbellyy ddiofufbelreendtofvoerracnoinmtarloslgviavleuens.10T0hpepmmeNa-nEnFuOmSbeEr,so2f0ppeprmoxPiFsoOm,esorpe1r00 ppm NEFOSA for 48 hours when compared with control values. PROCEDURES t`eTshtempautreproisaelosftothaisssesstsudpyerwoaxsitsoomeexpraomliifbenryeatieolnecitnrhoenpmaitoccryotsecso.py the livers from selected ratsadministeredthe Male Crl:CD(CD) IGS BR rats were giventhetest material in the diet accordingto Text Table 1. Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinaPaRgepeo3r7t Text Table 1. Group Designations, Dietary Levels and Scheduled Sacrifice Time Points `NumberofMale Rats Group |Contro| N-EFOSE | PFOS| N- |Wy-14643| Toul (TNiummebPeorint) | 1 [30 10 30 | 20 E[1t0F0OppSmA| 100ppm| Noof. ppm) | 00 ppm | ppm Animals mL PPT ero] (48hrs) IPT INOI (7 days) PS a A a 5 55 5 rec(o1vwerk LEeTLP]=] Animals i1 n AGnriomuaplss4iannGdro5urpesce1ivtehdrotuhgehte3strediceetivfeod t1h4edtaeystsdfioeltlfoowre4d8bhyoaurs1,or7 d4awyse,ekanrdec1o4vedrayysp,errieosdp,ecrteisvpeelcyt.iveAlnyi.mals AfanteesrtthheetizperdeswcirtihbeCdOd,oswienigoghrerde,coavnedreyxspaenrgiuoid,nattheed.aniPmoasltsmowretreemfapsrtoecdeodvueremsigihntc,lubdleedd wfeorigsheirnugmtshaemlpilveesr,and acnoalllyescitsi,ngasnadmeplleecstroofnivmiecrrofsocropceyl.l pLroilviefrersaatmiponlesstufdoiresc,elrlouptroilniefehriasttioopnatahnodlorgoyu,tipnaelhmiisttooypla-tChoolAoOgxyiwdearse.e activity collected in zinc formalin and submitted to Pathology Associates Intemational (PAI) Maryland for evaluation. CLiovvearnscaempLalbeosraftoorrpiaelsmfiotroaynla-lCyosAesO.xiLdiavesre sacatmipvlietysafnoarleylseicstwroenremifclrasohscforpozyeenvianlulaitqiuoidnnwietrreogtehnina-nsdlicseudbmaintdted to pCalraocleidnian) MfocrDeolwecetlrolnTmriucmrposfcioxpatyivpero(cMecsDsionwgelanldEeMv,alu1a9t7i6o)n,.anPderssupbomnitstoerdretqoutehsits,loanbloyraltiovreyr s(aPmApIlNeosrftrhom sdeolseecdtefdorra4t8s wheourrespwreocreesesxeadmainndeedvbalyuealteecdtruolntrmaistcrruocstcuroaplyl.y (Text Table 2). Only liver samples from animals 277 cTEeE Text Table 2. Animals Selected for Electron Microscopic Evaluation =[Trc| amAnie malNn umbet r G [--rTio mepoiw nt p| R10388 we R10404 R10439 R10443 `The selected livers were then processed into Spurr's resin for ultrastructural examination. Thick (1) sections `were cut, stained with toluidine blue, and examined to `microscopy processing. Thin sections (approximately select representative areas 90 nm) were cut, mounted for on further electron 200-mesh copper grids, stained with 5% methanolic uranyl acetate and Reynold's lead citrate, and examined on a Zeiss 900 transmission electron microscope. Centrilobular hepatocytes, where clearly identifiable in liver sections, were preferentially examined. Five representative electron photomicrographs ofhepatocytes were taken and tsirgannisfmiicsasnitounlterlaescttrruocntumriaclrfoegartuarpehs iwnetrereprseutmamtiaornifzoerdm.forTheaecnhupmhboetroogfrappehroaxnidsaonmiemsalinohneapadteosciygtneastewdas `manually counted for each photographed hepatocyte and recorded. The mean numberofperoxisomes per `hepatocyte was manually calculated by summing the numberofperoxisomes counted in five hepatocytes per animal and dividing by five. RESULTS AND DISCUSSION Individual interpretationsofelectron micrographs for each animal selected for evaluation follow this report narrative. The original signed/dated raw data sheets and photomicrographs are maintained in the archived study file at Pathology Associates' North Carolina facility. After 48 onlyfor ahnoiumraslosftgrievaetnme1n0t0,ptphmeonfumWbye-r1s4o,f6p4e3ro(xTiexstomTeabslaep3p)e. arTehdesimgenaifnicnauntmlbyeirnocfrpeeasreodxiovseormecsonwtraosl values `approximately double the control values. The mean numbersofperoxisomes per hepatocyte, however, were not remarkably different for animals given 100 ppm N-EFOSE, 20 ppm PFOS, or 100 ppm N-E{FOSA when compared with control values. Figures 1 through 4 show hepatocytes from control and non-affected treated animals. Some normal ultrastructural features are illustrated in these figures. Figure 5 shows a hepatocyte from an animal given 100 ppm Wy-14,642, illustrattihneg increased numbersofperoxisomes. 275 = Text Table 3. QuantitationofHepatocellular Peroxisomes `Treatment Group `Timepoint Animal Number `Mean Number of Peroxisomes Control [se] RR1100338884 1131.46 R10404 114 TEE ppm R10439 50 pp RI0443 24 No other ultrastructural abnormalities were identified in the samples evaluated. ABsRervaatlsugaitveednb1y0e0lpecptmronWym-i1c4ro,s6c4o3pyb,yhdeipeattaofcteelrl4ul8arhopuerrsooxfitsroemaetsmewnetr.eTihnecrmeeasaend ninummbaelreoCfrple:rCoDxis(oCmDe)sIwGaSs approximately doubled over control values. The mean numbersofperoxisomes per hepatocyte were not remarkably different for animals given 100 ppm N-EtFOSE, 20 ppm. PFOS, or 100 ppm N-EtFOSA for 48 `hours when compared with control values. REFERENCES `McDowell, EM and Trump, BE: Histologic fixative for routine diagnostic light and electron microscopy. Arch. Pathol. Lab. Med., 100:405-414, 1976. 279 Study Number:TRSEpon1s1o3r2:-130M0 `AmendedFinalPReapgo4re0t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No: _LUIZ-IQEMBE Animal No:_ RIOISone Treament Groups Coml Sex: _ Mile oo SpecievSininiC.UCSO1B8DBIat Te liver BlockNo(s): 96ZI2 msae-4 Photo/Negative No(s).:__ZIT350 SignificantLesions (chockone): Yo _X Ne tri Fag gn vd: No8.0n add 21 1220 Feaaltureets:eZsITd bLoirvdee.redHbepyeataodeejyacieen,t hTe0p3at2o0cy%tTeshosnhwepoastiodceysteacnodnstianiunssoi#dcscvoerntarlally "2r0d4droorusgalhleyn.dTophlealsimgihiceretstiaciunliunmg (eRnEdoRt)heplrioafliclelaceyftropelqausemntitervoiudgehntth.eMciyttoocphloansdesioaf the hepatocyte.Theinterveniag cytosol ppearsfinelygranularandconta ribosomes, glelfytcaongdeunpapnedrsrmigohottmhaerngdionpsl.aPsemriocxriestoicmuelsuamre(iSnEfRr)e.qu`eBnitl;efcaonuarli(c5u)lpiearroexpirseosmeeastcoonutnhteed. Zr17o3fS4lo7et,tLshieevrefireg.qhutseHnenptaUtthnoecoyJategteht..eM1i0t.3oy2c0hXo.nodTrhiioas`afhntedpeaothuogceyhteeeniydsob.polradAsepmrriecdorbmeyitenincedunolmtuihmce(rloRivaEilRlcl)eolulss `eb7op1ra7edt3teo4ric8iya,sieecsLvswiiaivdneeta,hdtmaaHtesiiptnahuteistooociopydrtsic.lgohan1htng0tm.doah3ore2bg0rnXeii.ngrdohTotrfhmgritaahsrinbgiseecilpenlala.lte.To.cShNyeieivnceeirlas(9ls)bmamsiuitaotmcbhoouonncdbrudyihaaIRanovcrnEoeotRunadrndetdjgeeaudcr.leadnirtlay: hapedsdcontaincyoplasiciavagiaaion. Eight (8)peroxisomescounted Z1iwh7eh34it9co,phorLniiegvhectro,.natnHaedipna0stotdchyeatleke.tro1su0on3md2em0lXea.mme"bTlrhlaiansrhodeuepsabtnioccilysaptsreieocnso.ennttAaiinnnste.hseenveedrxtarloalciepcblieldluledlarirsolsppplraeiectssee,l.nt ||| ThZiUoTrp1ci5e0sn,sr(Le1i3pv)rpeeersreoHnextpiamstoohmcieyhtseec.poaunt1to0ec.dy3.t2e0.X.TShiemrielarretoabZuInTd4an9t,mseivetralomcehdionudnmnmdusmrierzioepuiadsd RERprofi evident. Twenty-two (22) eronisomescounted. Camco ei hep dni SE weep WP WTS Study Nu"mbAerm: TeRSFpEinonna1sld1oR3re2:ep-o13dr0Mt0 Page st `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No: _LL32-100(EMO9.62) Animal No: RIQIEE en Treatment Group: _Conwrol Soni Mie SpecieyStrain:CILCOMSDB)RIKGaS Tissue Liver BPlhootcoklNNoefgast)iv_e _N9o9(62:28_7_Z_3I_1_T_3_5_S SignificantLesions(checkone: Yes X__ No InterpretingPathosilgnaoganiddstct):SwenB084 SepL20,18 ||eeamtbemeseZtiiIntoTe: TahCeiree.a TaiTgsaaeoebcsateno,degTl0nyc.asIcccsTuehgipsadrhsseioagcmann.aponidiAceooLnhnaBstvonmszesshe.oSnmreayvreriiranamle1asWkwieeieeen | e S reiteo rae ghhm,vT.aeoslHemepwa1ibno2dae]rypmeeb ,arnoxhOiSsyo2me 0oeNls.nodNuuamt dedroiuse mBiocn honda nd VER to proafreirsecst in m r r eypeerotmhes eapp]t o1escam4 bRSsSoe135d y rorstuserFsilmeiroc(h1o3n)ciia petAoneisontteiisev,cetRoEnvHEeeedptTrohisesyetese..poeHe0sIee2vUeKir.al3pOcanegrhaaenxesdtphsidncbdaarsokp3sa.f(eeSonm5elcspprlemaslsicsalaoniiess1zndwe|y | koemeoeerltoHioerpenvhoenass.tsne.aFsigv1 eSaet250o0pf .pmiratiTnhsebdocnuo1 rsertessoheewopeaedoonnctytathlee lsiistdBcBoniOuthdNcriEsyibIlhoEecpygaiu)toRacnyydteasn thaore a(pl1a3d)hpdercarpees amennpcrresieend.. NPesTomOdah[sAoArSi5amdSPOAEOIDAB PI1OEh eSrsTdeEinfckTuvilero,SdTiuefpmemormcehintctt.e 10h.i12i0.Tshihs bepeeasts contains eect op td Ltn Tc pfouhr soitl n0eddiOykalees im endeinclelslls Thetecn 13) peragsemss sounted. Comision Noval paise, MeanOf 136 persue pe hepatocyte counted: 2/ Study Number,TRSCpon1s1o3r2:-130M0 `Amended FinsPRaegpeo4r2t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No: 1132-100(EM99.62) Animal No. _RI003 Treatment Group: _N-EA\FOSE100ppm SEX: ee e MA ee Specien/StrainCL.GSCDBRSKSaDt Tose lver Block No(s): ~ 99.622:1273356- Photo/Negative No(s):__Z17360 Significant Lesions (check one): _____ Yes _X No Iterprting Pathologist (signature ad de): Se\am bd e Sepn ,29,1, 999 Fapepartouxriemsa:te7l1y73t3en6,sasLailv-ert.o Hmeepdaitocystiez.ed10li,p3i2d0dXr.opTlheti.s hTehpealoccyyttoeplcaonstmaihnass numerous amdijtaoccehnotnhdriea apndRaEoRtnptrohofeillceefstaypnredsttehnete.figBshitl.e cEanalcicuyli atreiphnrseirsneutsoobiedctswaeryeenprttehisseecnetlsolannd three margins. Some stain debris/antifoct is presocnathte photograph. EIght (8) peroxisomes counted. Z17357, Liver. Hepatocyte. 10.320X. This hepatoeyte is bimuclcated. An endothelial cceolnltiaipnrsensuenmtesrtouthsetmoipto,cbhuotnadlrliaotahnedrmRarEgRinprsoafrilees.adjOanceentdhiestpiantcotclyitpieds.dTrohpleectyitsopprleassemn. aTnwdeatthye-retharreesn(u2m3e)preoruosxismsaolmlestocomuendtieudm.-sized dark peroxisomesandor lysosomes. ZUT358, Liver. Hepatocyte. 10.320X. A mucleus is not evident in this ploafsenctieon foorrgtahnielslheespaaptpoecyatrev.erFyousrimmieladriu10m-Zs1i7z3e5d7.lipSidomdreopllyestsosaormeeps cronteaiasnndcetlehnaerotvtahc.euroles within. 2t1h7e3m5a7n,dLmiavenry.aHreepiartroecgyultaer.ly1s0h.a3p2e0dX..ThHierptaeteoncy(1t3e)capnetraoixnissoonmeeslcaorugentleidp.id droplet. Twenty-three (23) peroxisomes counted. Some stain debrivlartifact present on the photograph. $1236, Liver. Hepatocyte. 10320X. Multiple smal to medium-sized lipid droplets ar present. Sixteen (16) peroxisomes counted. [| Cometsioms: Normal Ippe10. 5.6 1 e7age ; Prone per iINpioye. 2 StudyNumber: TRSCpon1s1o3r2:-130M0. `Amended FinalPRuegpeosr3t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.. 1132-100(EM99.62) Animal No. __RI0404 Treatment Group: N-ESFOSE100p0om Sex _Male Species/Strain:CA.ICGSOBMRSRDa)t TRU, crnBEccascsvasosasnie Block Nots): 9.246732612- 4 Pholo/Negative Nofs).:_Z17365 Significant Lesions (check one): Yes. X _ No Interpreting Pathologist (signature and date)S: innBiaaldSeptember 30.1090 mFiecartouvrielso:usZbIoTrdWeLradLjiavecre,ntFe0psuiiocnyue,soo1o0i-h1di2cs0e.marTghisnscoannrdablorbdeeprastwoictyhetehexeadhjacent lhhieppaetdorcpoyptlea est.osT.t nhTthehorehereeec posattry hoeecrpytmutaemrNeegrsionsuss.omBuiitnlodecccheaonnnatdlariilcaunsluaicldareeuRseEvRaipndrdeoqnfutiilwteiset.h3Ttfhweoewosrefamtalhietnaidojnamgcceeydn(itoosnoliized cTionuenlyedg.ranular andcontains catered lysonomen and peroxisomes. Siziecn (16) perorisomes muZicIks7c3pu6ho2i,iosgLcrivvaieprdh..ewnHiaeltphatthoesciy0ntpues.oei1tda0ot32t0hheKe.ptohpToholiegsfrtbaaepnphdu.tbToochyoiecmh.spPpeeasteosofsoh3smaetswiharontovdueicacsmieonnniyotdnr-gaicsltheualdpaceenadldiislns aarnedparbeusnedna.ntSiRxE(R6)pperroofxeissaonmdemscitooucnhiodn.dca, Also, numerouslysnomesanda few peroxisomes ZUTS63, Liver, Hepocyte. 10.2UX. This hepatocyte appearsmorphologicallysimilarto ZctohUuo7ns3te6ed4de,.scLriivbeerd. above. Several small lipid droplets are abso present. Hepmocyte. 10.320X. Mostoftwo hepatocytes ae Eleven present (11) in i peroxisome s picture, s A Ttohngrpirghotficloenotfainbsiskevecraalnmaedliciuupmrl-esucensentdbonee ltargthwe litepwidoecdrlonspl.etT.hEeicgohtmp(l8)etpeerhoenpiastoomeeys on counted riZg1h7t3o6f5,thLeipvheort.ogHreappatho.cTytweo. e1r0.y3t2h0rXo.cyThatiresesispa lrargeie nhtesphaetesoicnnyutseotinde.xTtthoeashienpuastooicydtsethtahse top. a yTarsprooundasnnudcopleeumrsoasenodmsneusmienrtohues cmyittoopclhaomo.diCioleaanrddRiEiRncpiroofnileBs,eAebno,mheerepaerre cmxulotmieplse 30d is diffcuk. Sixteen (16) peroxisomes counted. Conclusions: Normal hepatosyte. Mean of 1173 perosisomes per epaioeyt. 83 Study Number: TRSCpon1s1o3r2:-130M0 Amended Fin)PRaegpeodrat TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No: _R10421 Treatment Group: _PFOS20ppm_ Sex: _ Male Species/SCDtO(rSaD)i0nS CBRrRlat Tissue: L __iver Block No(s):_ 99.624 Photo/Negative No(s):217_Z31676370 Significant Lesions (check one): Yes _X__ No Interpreting Pathologist (signature and date): Sept. 30,1999 pFoelaytguorneasl: nu7c1l7e3u6s6s,urLriovuenr,dedHebpyatcoyctyotpel.as1m0r3i2c0hXi.nTmhitioschhonedripa aantdhaRosEcRcepynrtofrtialleels.y lMouclattiepdle Sgmraalnlu-latroamneddicuonmt-asiinzssedomliepidldryoplsetsoaanesdaploesroopmxriesseeonmtes.s.ThTerweemnatiyn-isnegvecnyt(o2p7)lapsemroaxpipseoamressfinely cZoIuTnAteTd.. Liver, Hepatocyte. 10.320. This hepathaoscadyjatceent hepatocytes both dorsally `aAnndevnednoitrahlellyi.aT-ointehdeslienfusiosiadisianlussoopirdelsienendtblyanoetnhdnotrhigeglhitamlarcgliln.anAdpcprooxinmatteaalnyienriynntehirsomcnayltlge. liZpIi7d3d68r. oLivpearr.lepHereeptsaetsnoctyite.the1c0y.t3o2p0la.sIm.ntFhiifstheeepnat(o1c5y)tpeertohexmisiomets coourctedha.ppoeanrsdmalrleiraand `pmeorroexicsoondmeenssceodu.ntPerdo.files of RERand SaErccaRsily evidenitn the cytoplasm. Five (5) 17369. Liver {his hepatocyte. HAettphaetotcoypter.igh1t0a,n3d2b0o.ttSoemvelreafltmsamrgailnsanodfotnheeplahgeoliptid doarroepgleertrytahraeroppcryetsheesnt in wmiitthoicnhosnidnrusioaidasn.dRTEheRphreopfaitloecsytaer'esvmiuscilbleeu.s Tishneoipnrteesrveenntiinngtchyitsopsloalnieoffisneelcytigorna.nulNaurmaenrdous ccroynsttaailnlsoiSdEsRiarcntduoretshearppoeragranveelrlyesp,rionmcilnuednitngisn osmoemepepreorxoixsiosmoemseasnFdilfyiseonso(m1e5s).peSroomxei.somes cZoIu7n3t7e0d,. Liver. Hepatocyte. 10320X. Hepatocyte appears very similatro Z17369. A nucleus a{5renoptreevsiednetntSiinxtteheenpla(1n6)eofpersoecrtiisoonmepshoctoougnrtaepdh.ed. Several small- to medium-sized lipid droplets "Conclusions: Normal hepatocyte. Meanof15.2 peroxpiersheopamtoceytse. a%/ Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpoerst TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No: 1132-100(EM99.62) AnimalNo: _RIO0 Treatment Group: _P2F0pOomS Sexi Mie SpecieCsl./CDSMtSr)IaGSiURnK:at Tiswe: Liver Block No(s): 99.62.50____ Photo/Negative No(s)Z.i.7_3Z71173-75 Significant Lesions (check one): Yes X No Interpreting Pathologist (signature and date): SpomBets seo 01000 Features: ZIZ371, Hepatocyte. 10330X. This heptiocye has an cecemiricaly ocaied oval nucleus. It isborbdyae nsirnuseoiddat thetopand rightand anotherhepatocyteon the left. pNruemseenrtoiunsthmeitcoycthoopnldarsima., RSEoRmeprsomfaillels,lfiynelsygroaansulaosrcacmyttcoreseodlsi,natnhdeacfyetwopslmaaslmlFliivpied (dr5o)plets are: peroxisomes counted. SZidIeTsT2e,xLcivteehre,poHtepp,awthoicchyitse. a1s0i.n3u2s0oXi.d.TThihsehceelpahtuoscsybitusendsaunrtrRouEnRdepdrboyfhieplaaetnosdcymtietsoocnhaolnldria `nStohme clytoyplassm,aansodwaelfslewaspoemucloixmpilseoeimrersgsaurleatpyr-essehno.pFeodsumra(l4)p0emreodxiiusmo-mseiszceodulniipeidd.droplets. ZUT3T3, Liver. Hepatocyte. 10,320K. Thehepatocyteappearsexentally similar to previously described hepato ytes, containing 4 lage wud wecleus aud numerous miwchunddis an RER pprroofbialebsl.e pSroomteisloimpiedsdrcoopulnettesd.are present. Lysosomes andperoxisomesare rare. Two (2) o2f1t7h3e7p3r,evLiiovuesrl.yeHxeapamtionceytdec.ell1s0w3e2r0eX..anTdhiiss lhoecpaatteodcbyetneeiastshoamecwehattlriglhtoerbvsteoaiilanilanognrtghatnhemiagsht. P`SeoomxeicsoolmleagseninftihbeerhseapraetoecvyitdeenatreinbethteesSdpeafcienoefdtDhiasnseinbemnoesattohtthehrecnedlolt.healnidalhcaevlel mparrogmiinn,ent c`ZrmIyaTsrHtgIailn5l..oiTdLhisevtercrue.cltlu'Hrseepmsai.tcorEcoiyvgtihelt.l(o8u1)s0pb3eo2rr0odXxe.irsioTsmhneiessxtchoeouptnathteoedc.yetnedoitshebloiradleriendedbysi3oussioniuds,oiwdhkicohncgotnhteaitnosp seavuecreaulseriystnhortocpyrteesse.nAadjathciesnptlhaenpeaotfoceyctteiosna.r Tohnetchytroipglhatsamndisefifneolfytghreacnoontlraaanhdepcaotnotcayintse, A nuliple mitochondrainad RER profiles. Zoro (0) perosisomes aeclearly discernible Conclusions: Normal hepatocyte. Average 3.8 peroxisomesper hepatocyte. 2YS Study Number: TRSCpon1s1o3r2;-130M0 `Amended FinalPRaegpeodr6t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No: __R10431 `Treatment Group: _N-EFOS, m Sex: __ Male SpecieCsi C/DOS(StD)aGSiBnRR:at Tissue: __ Liver. Block No(s): _ 99.6251____ 717376 - Z17380 Photo/Negative Nos).._Z17396 Significant Lesions (check one): _____ Yes _X__ No Interpreting Pathologist (signature and date): Sept. 30,1999 Fpoelaytguorneasl: nZu1c7l3eu7s6,suLriovuern.deHdepbaytoccyyttoep,las1m0,c3o2n0tXa.iniTnhgisnulimgehrto-usasimniintgochheopnadtroicay.teRhEasRaacnedntSrEalR Nprionfeil(e9s)apreerporxeisseontmeasndcotuhnetreeda.re greater than a dozen small- to medium-sized ipid droplets. pZrIeTcAeTd]i,ngLeivxearm.pleH.epaTthoecyctyet.op1l0a,s3m20isX.finTehliysghreapnautloacryatnedsmtaiitnoscshoonmderwihaaatppdaerakredretnhsaenrthe Pofetreonxidisfofmiceusltatnoddsifofmereenltyisaotseofmreosmaceacghenoetrhael.lyTsmwaelnlteyr-atnwdo (d2a2r)kepretrhoaxnistohememsitcooucnhtoendd.ria, and are `ZdUjTa3c7e5n,t tLoiavesri.nuHseopida.tocTyhtee.nuc1l0e.u3s20iX.notTheevitdoepntmiargtihinsofpltahniesofceslelcsthioonwsfoarmtihcirsohveiplaltooucsytbeo.rdTehre cSyotmoepllayssmocsoonmteasinasndabpuernodxainstomSeEsR. bSeitxwteeeenn (t1h6e)mpietorcohxoinsdormieasacnodunottehder organelles, including Z`h1e7p3a7t9o,cytLeisvecry.toHpelpaastmo.cytAe.si1n0gl,e32m0eXd.iuMmi-tsoiczheonlidpriidadraonpdleRtEiRpprreosfeinltesneaarre ptrhomtionpebnotrdnerthoifsthis cell. A sinusoid lined by an endothelial cel is present long the let margin. Seventeen (17) pZeIr7o3x8i0s.omOevsecroeuxnptoesde.d negative; not printed. 2h1ep7a3t9o6c,yteLisvoern.thHeepwaitdoecyatned. b1o0f.i3o2m0.X.AtTthhee cteonpitera edsihneupsaotiodcyitneeidsbbyoradnereenddobtyhealdijaalcceenltl and cSounrtraoiunnidnegdsboymenugmrearnouluoscymtietsocahnodnderriyatharnocdytReEs.RTprhoefihleesp.atSoccayttteehreadsdaarckeentrrpaelrpooxliysgoomneasl naurccleus present. Twenty-five (25) peroxisomes courted. Conclusions: Normal hepatocyie. Average 17.8 peroxisomes per Repatocyte Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalpRaegpeodr?t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No: 1132-100(EM99.62) Animal No.: __RI0439 Treatment Group: _N-EFOSA100ppm Sex: __ Male `Species/Strain:CriGCDSB(RSRDa)t Tissue: ___ Liver Block No(s): __99.6721:759381 Photo/Negative No(s).: Z17385 Significant Lesions (check one): Yes X__No Interpreting Pathologist (signature and date): October 1,1999 Fpreeasteunrtesi:n thZiIsTh3e8pLa,tocLyitvee.r. ItHeispastuorcryotuen.de1d0b,y3n2u0mXe.r_oAumsemdiituomc-hsoinzderdiaroaunnddRnEucRlepurosfiilses. Fiincvleusliiopinds;drtohipslemtasyarbeeesviodmeentstianitnhpereccyitpoiptlaatse.m.SeSvoermael mliytsoocshoomnedsriaarehparveesdeanr,kbut 20 2h(01a)7n3p8e22r1.o7x38iL1si.ovemre,TshaHereerpaoctuloenacdrylntyuec.dlise1cu0es,rn3iis2b0slXue.r.roTuhnidsehdebpaytnocuymteeroisussommietwohcahotnldirgihatearnsdtaRinEiRng Spriomfiillaersl.y,Tsheevecryatlolpylsaossmocmoenstaairnespsreesveenrta,l bsumtazlerlo-0(m0)edpieurmo-xsiiszoemdesliapricdcdlreoaprlleytsd.iscernible. 7S1in7u3s8o3i.d lLiinveedrb.yHaenpaetnodcoytthee.lia1l0c,l3l20aXt.itTshuipspelrigrhitglhtymdaarrkgeirn-.stTaihneincgythoepplataoscmyctoenhtaaisnas Tumerous mitochondria andRERprofiles. Several lipid droplis are present. Nine (9) p1e7r3o8x4is,omLeisvecro,unHteepda.tocyte. {hrce sidesofthis hepatocte. 1S0e,v3e2r0aXl.meEdryituhmr-octyotleacrogen-tsaiizneidnglpsiidnudsrooipdlsetasreareevipdreenstenotn iinntthhee ccyyttooppllaassmm.ofFtihvies(h5e)ppaetorcoyxties.omTehsercoeuanrteedn.umerous mitochondria and RER profiles ZT1u7m3e8r5o,usLiRvEerR. pHroefpialteoscaynted.mi1t0oc,h3o2n0d.riaT.heSecvyetroaplllaispmiodfdtrhopilsethse,paotnoecwyilethcoantlaaimnesl.lar bdiofdfyi,culatretoprcelseeanrtl.y diSfofemreenstciaattetefrredomlyesaocshoomtehsera.ndElpeevrenox(1i1s)opmeareroesxipsroesmeenst,coaunndteadr.e "Conclusions: Normal hepaiocyte. Average 5.0 peroxisomes per hepatocyte. Study Number:TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeo4r8t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No: __R10441 Treatment Group: _Wy-14,643 100ppm Sex: _ Male Species/Strain:CrlGCDSB(RSRDa)t Tissue: __ Liver Block No(s): _99.62:6 217386 Photo/Negative No(s).._Z17390__ Significant Lesions (check one): __X__ Yes ___ No Interpreting Pathologist (signature and date): Oct. 01,1999 Fsienautsuoriedss:onZbIo7t38h6t.herLiivgehrt.anHedlpeafttocmyatreg.in1s0.,3T2h0eX.cenTthriasl hpleaptaetoofcytthereheahsepaantoovcayltneuschlaesus `Pwehriocxhiissosmuersraoruendtehed dbayrnkeurmestraoiunsinmgiotrogcahneolnldersi.a Tanwdenftiyne-ltywgor(a2n2u)laprecryotxoipsloamsems.counted. ZIT3817. Liver. this hepatocyte. HTehpeatcoyctyotpel.as1m0i,s3f2i0nXe.ly Fgoruanrumleadriaunmd-csoinzteadinlsipniud mderorpoluestsmiarteocphroensdenrtiain pahnodts ograpo hR.ERTm hpirrotfiyel-esse.veTnh(r3e7e)epreyrtohxrioscoytmeessacroeupnrteesde.ntinthesinusoidatthetop ofthe Z17388, Liver. Hepatocyte. 10,320X. This hepatocyte appears ultrastructurally similar {tothose described above. It has a central round nucleus surroundbeyd cytoplasm containing numerous mitochondria, finely granular cytosol, RER profiles, and some lipid droplets. Additionally some variably-sized dark-staining peroxisomes are present. An erythrocyte ina sinusoid is present at the top right margin ofthe photograph. Twenty- Zt1w7o3(8292.) pLeirveorx.isHoempeastoccoyutnete.d.10,320X.Hepatocyteappears ultrastructurallysimilarto ZZ1I77339808,. LFiovretry.-oHneepa(t4o1c)yptee.rox1i0s,o3m2e0sX.couOntneldy.a small segmentofnucleus is present in this hepatocyte, This hepatocyte is bordered by adjacent hepatocyte on three sides and by an endotheliallined sinusoid on the let. Its cytoplasm contains numerous mitochondria, RER profiles, scattered dark peroxisomes, and some lysosomes. Twenty-six (26) peroxisomes counted. Conclusions: Increased peroxisomes. Average 29.6 peroxisomes per hepatocyte. `Study Number: TRSCpon1s1o3r2:-310M0 `Amended FinalPRaegpeo4r9t TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100(EM99.62) Animal No: __R10443 Treatment Group: _Wy-14,643100ppm Sex: __ Male, `Species/Strain:C1.GCSDOB(RSRDa)t Tissue: __ Liver. Block No(s): _99.62:63 217391 Photo/Negative No(s).._Z17395 Significant Lesions (check one): __X__ Yes _____ No Interpreting Pathologist (signature and date): 0ct 01,1999 Features: centered in ZI7391. Liver. the photograph. Hepatocyte. At the top is 10,320X. a sinusoid A polygonal-shaped hepatoyte is containing an endothelial cell and esriyntuhsrooicdyitsepornestehnetalefthaendboattpeormiosfitnuhseoipdhaoltloigpriadp-hs.torTihngechelelpaattotchyetreicghotn.taAinnostnhuemrerous `cmointtoacihnosnsdcraitatearnedd RdaErRk plryosfoilseosm.esThaendipnteerrovxeinsionmgecsy.toTshoilrtisyf(i3n0e)lypegrroanxuilsaormeasndcoaulnsoted. 217302, Liver. Hepatocyte. 10,320X. This hepatocyte contains greater than a dozen `pSrmeasleln-ttoinmtehdeicuymt-ospilzaesdm.lipTidwednrotpyl-etssi.x (N2u6m)epreorouxsiRsoEmRespcroofuinlteesda.nd mitochondria are 217303, Liver. Hepatocyte. 10,320X. This somewhat lighter-staining hepatocyte is bevoirddeenrtedinbtyhiasdpjalcaenneot fhespeacttioocny.tesSeovneraalll mlaiprigdidnrsopilnetthsisarpehoptroesgernatp.h.FoArtnyu-colneeu(s41is) not p71e7r3o0x4i,somLeisverc.ounHteepda.tocyte. 10,320X. This hepatocyte appears morphologically similar 10217393, except it has a large central polygonal nucleus. Thirty-eight (38) peroxisomes c7o1u7n3t0e5d,. Liver. Hepatocyte. 10,320X. This round hepatocyte appears essentially Similar to Z17391. The hepatocyte contains numerous mitochondria and RER profiles. `pTehreoxiintseormveesn.inTgwceyltvoseol(1i2s)fpienerloyxigrsaonmuelsarcoaunndteadl.so contains scattered dark lysosomes and Conclusions: Increased peroxisomes. Average 29.4 peroxisomes per hepatocyte. Study NumTbR`CSe1p1o3nr2s-o13,r0M:0 `AmendedFinPalaRgepeorStI Table VI-1. Individual Animal Palmitoyl CoA Oxidase Activity Com ETE a he hae ae fhe heae ne stae m--t----| hfe | a Pe ame | ei Et tee er 1 erSe on | ro e T= ye wa Er ss e + [EEE fe he hem F R h ee n a | a -- reme eee e m r ------ e -------- ro otes ten i--e ----w-- ar --m--e-- --r-- ] E E e a hae e rheenhe me e ] Et ert mm em r mt 1menet ere | ee etnt en e t | srrr P ----ee ee] 297 `Study Number:TRSCp1on1s3o2r:-130M0 `Amended Final Report Pages2 VIL APPENDIX 4 - STUDY PROTOCOL AND AMENDMENTS Study Number,TRSCpon1s1o3r2:-130M0 `Amended FinaPaRgepeo5r3t -- eCHyARLb LEABSOg RRATIOVt RIEERS Sponsor: St. Paul,3MMinnesota PROTOCOL Study Title: Cell ProlifePreartfilounorSotoucdtyanweitShulNf-oEntihcylAcPiedrfPloutoarsosoicutmanSeasltul(fPoFnOaSm;id3oMEtTh-a6n2o9l5(.1N6-)E,WFaOndSEN;-E3tMhyTl-6316.11), Perfluorooctanesulfonamide (N-EFOSA 3M T-7091.1) in Rats Date: January 12,1999 Performing Laboratory ROW. Sciences Gaither1s5buFirrgs,tfMiaelrdylRaonadd20878 Laboratory Study Identification: StudyNumber: (1132-100) PAI Project Number: (Histology number o be assigned by PAI by protocol amendment) TS Worman's Mill Cour, Suite 1+ Frederick, Maryland21701 + (30D) 663-1644+ GOD 3-F9 AX 94 2793 Study Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamide (N-EIFOSA 3M T-7091.1) in Rats Purpose To assess cell proliferation and peroxisome proliferation in rats administered test material in the diet. Sponsor 3M Corporate Toxicology Building 220-26-02, 3M Center St. Paul, MN 55144-1000 Study Representative Marvin T. Case, D.V.M, Ph.D. 3M Corporate Toxicology Phone No.: 651 733-5180 Fax No: 651 733-1773 Email: micase@mmm.com Alternative Study Representative Andrew M. Seacat, Ph.D. 3M Corporate Toxicology Phone No. 651 575-3161 Fax No: 651 733-1773 Email: amseacat@mmim.com Test Facility `1T5hFeirrsltmfmiuelndeRReosaedarch Corporation(formerly, R.O.W. Sciences) 5 Gaithersburg, Maryland 20878 Study Monitor Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: stepaisaic@aol.com Study Director Gary W. Wolfe, Ph.D, DAB.T. ROW. Sciences Phone No.: 301 330-3723 Fax. No. 301 330-3738 Email: gwolfe@lab.ow.com Principal Investigator Sandra R. Eldridge, Ph.D. Pathology Associates Intemational Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@aol.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: TRSpCon1s1o3r2:-130M0 `Amended Fina Report Page 55 295 Proposed Study Timetable In-life Start Date: Tobeaddedbyprotocol amendment; Day 0 In life End Date: To be added by protocol amendment Audited Draft Report Date: To be added by protocol amendment `Study Number;TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeo3r6t Regulatory Compliance "This study will be conducted in the spiritofGood Laboratory Practice (GLP) regulations. Animal Care and Use Statement Al procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 14. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance Not applicable. Test Materials TestMaterial: |(Nc-oEmpFlOetSeEd by ||P(FcoOmSpleted by 3M) 3m Identification: |N-EFOSE FOS || N(tEo bFeOadSdAed by | Wy-ldeds | (to be added by protocol protocol amendment) amendment) N-EFOSA Wy (LotNumber: |FM3929 [217 |Letsal | Lots 30035, Purrimtpye:reeeeneeee S99O2% IIG | 99%pemmsessssn frmissenensesmsssmepmmemsmt Stability: >Syears >Syears >Syeans Storage room temp. | room temp. Conditions: Characteristics: `white powder room temp. `amber waxy solid 296 Study Number: TRSCpon1s1o3r2:130M0 `Amended FinalPaRgepeo5r7t Reserve (Archive) Samples A reserve sample (approximately g)ofeach lot wil be taken and stored at room temperature. `These samples will be transferred to the Sponsor ater completionofthe in-ife phase to be retained in accordance with 40 CFR 792.195. Dispositionof Test Material After authorization from the Sponsor, any remaining test material will be retumed to: M3aMrvCionrpCoarsaet,eDT.oVx.iMc,o,loPghyD. BStu.ilPdauiln,gM2i2n0n-e2sEo-t0a2,535M14C4e-n1t0e0r0 PFahxonNeo.N:o.65615.17-3733.31-7571380 Animals [Species: Rat - Strain: Crl:CD%(SD) IGS BR Source: | Charles River Laboratories, Inc,Raleigh,NC | `Age at Initation of Treatment:_| Preferable 6 weeksofage,butnot more than 8 weeksof age Weight at Initiation of Treatment: 50103008 Numberand Gender: |males `unique identification by individual car tags and cage cards 297 Study Number: TRSEpon1s1o3r2:-130M0 `Amended FinalPaRgeep'osrst Husbandry [DHioeuts:ing | S"iTnegklleadho7u0s12edCiernthiafniegdiRnogsdteanitnlDeisests.teFerlewsihrefocoadgweisll _ be pr_ ovide_ d wee_ kly.| F`meeetadlsi,s aafnlaaltyozxiend,bcyhltohreinmaatneuhfyadcrtoucraerrbfoonrs,coonrcgeanntorapthioosnpshoaftsepse,cainfidesdpehceiafviyed Water "nTuatpiweanttesr,Sppreocviifideedd naudtrliiebnittsumanalvysiesa an aaurteoomnatiicl waatteRr.iOn.gWs.ysStcieemncoersw.ater baontdtlessp.eciTfhicemwiactreorbeiss.anTalhyezeredsualttlseoafstthtewsoetainmaelsypseesr yareearonfofrilceonattaRmOinWan.ts Contaminants: | STchieenscteusd.y director and/or the Sponsor have considered possible interfering S`muabtsetraianlceitsseploftoerntipaolslsyibplreessetnrtucitnuraanlilymarlelfaeteedd amantderwiaatlesr,asinwcelluldiangtthheetteesmts leixspteecdtiend(t2o) baendpr(e3s)eanbtoivne.anNimoanleofefetdhoersewactoenrtaatmilneavenltsssaurfefirceiaesntontaobilnyterfere Environment | wTihtehttahrigsestteuddyt.emperaartbuetrweeesn 64 and 79F wih arelative humidity bevween 30% continuously. aAnd1720-h%o.urTleimgphetr/a12t-uhroeuranddarhkucmyicdlietywialrlebmeonmaiitnotraeidned. Ten Acchimation: | oArnigmraealtserawiilrlcbheaancgcleismoauterdwtiolltbheemfaaciinlittayifnoerda. minimumof 7 days prior to shueitasbtialrtitoyfdfoosritnegst.ingAndiumrianlgstwhiilslpbeerioobds.eArnviemdaflosrtgheanteraarle dheiaslctahseadndor Randomization: | unsuitablefortesting will be removedfromthe study. Using computer-gencrated random numbers wit assignment [0 groups, Al the tsihmoeuoldfnroatndeoxmciezeadtion2,St.hD.e woeftihghetmvearainatwieoinoghft,thaenadntihmeamlseoafnebaocdhysweexiugshetds for ach groupofeach sex will not be statistically different. quirementsfor a rodent species. 298 _ `Amended Final Report Page 59 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points TT umob fMale eRatsr "Group Number| Control N-EtFOSE PFOS| N-EtFOSA| (Time Point) | (0 ppm)| 300 100 30 ppm| 20 ppm| 100ppm ~ Wy- Total No. cle 14,643 |ofAnimals 100 ppm (7 daiy wien& } : BE : --- 1 [| |: " lf ee] Ae al el Animals 30] 30 30 25 I ssedta Dietary. Animals in Groups 1 through 3 will receivetest diet for 48 hours, 7days,and Animals in Group4s and 5 will receive test diet for 14 days followed by a 1 or 4 week recovery period, respectively. Reason for Dosing Route The potentialhumanexposure is by the oral route. Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPaRgepeo6r0t Dose Preparation Before initiation oftreatment, dose preparationofeach test material will be mixed. All dose preparations willbestoreadt room temperature. Dose preparation wil be. `documented and reported. See Attachment I for test dit preparation procedures. Retention Sample Samples (approximately 100 g) will be taken from the dosepreparationand stored at room temperature. Unless used for analyses, these samples will be discarded at least 1 `month after completionofthe in-ife phase. Observationof 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 WeeklyforWeek 1 through 4 weeksofrecovery. Clinical Chemistry Animals will be fasted overnight before animal's scheduled necropsy; blood will be collected from a jugular vein into an EDTA-coated tube. Serum enzyme levelsofalanine aminotransferase (ALT), alkaline phosphatase, aspartate aminotransferase (AST), cholesterol and triglycerides will be determined JB Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeo6rlt Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with COy, weighed, and exsanguinated. Scheduled Sacrifices Interim Sacrifices At48 hrs,7 days, and 14 days, animalswillbe fasted overnight, bled for serum samples, anesthetized with COs, weighed, and exsanguinated. NOTE: Two serum samples will be needed, (1) 0.5 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 samples will be collected. Animals will be discarded aftr 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 wil be needed, (1)a0.5 mi sample for clinical chemistry and (2) 2 1.5 ml samplefor compound level analysis. Postmortem Procedures Boy Study Number:TRSCpon1s1o3r2:-130M0 Amended FinalPaRegpeo6r2t Necropsy "The necropsy will include an examinationofthe extemal featuresofthe carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Cell Proliferation Tissue Collection and Immunobistochemical Evaluation Representative samples of the left lateral lobe ofthe liver and any macroscopic lesions ofthe liver will be collected and preserved in zine formalin. After fixation, each sampleofliver will be delivered to: `PSaatnhdorlaoRg.y EAlsdsroicdigaet,ePshI.nDt.emational 15 Worman's Mill Court, Suite I Frederick, Maryland 21701 Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition, liver sections prepared from the same tissue block will be stained with hematoxylin and cosin and examined microscopically. Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately 500 mg) of the right lateral Iobeofthe liver 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 will include all study animals, EXCEPT for the Wy-14,643 animals and all animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study will be analyzed for palmitoyl-CoA Oxidase activity; these samples consistofliver samples from 35 rats and 35 guinea pigs. Tissue Collection for Electron Microscopic Evaluation 302 Study Number: TRSCpon1s3o2r:.130M0 `Amended FinalPaRegpeo6r3t Sectionsofliver from all animals will 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 PAL 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 discretionofthe Sponsor, from one time point as well as the 4-week recovery. "Thus, EM will be performed on one animal from the control, N-EGFOSE (one dose only to be determined), PFOS, N-EFOSA, and Wy groups at oneofthe time points, as well as the 4 week recovery, for a total of 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 At the scheduled sacrifices, the liver will be weighed. Histopathology Liver from each 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 will include the following information: Experimental Design and Methods Results dose analyses `mortality clinical observations body weights SutyNunter TEa NSE 0 `Sponsor: 3M Page 64 `body weight changes food consumption test material consumption el patholo rls palmitoyl-CoA oxidase activities. Fcosopieonervatons `microscopic observations om promon sestents ultrastructural observations 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 facilities of PAI for a period of 1 year following submission ofthe 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 will be charged. All raw data stored on magnetic media will be retain by PAIL 1 Study Number: TRSpCon1s1o3r2:-130M0 `Amended FinalPRaegpeo6r5t PROTOCOL APPROVAL Marvin Case, DVM, PhD. Date Study Representative. 3M Corporate Toxicology GaryW. Wolfe, Ph.D, DABT Date Study Director RO.W. Sciences. Sandra R. Eldridge, Ph.D. Date Principle Investigator Pathology Associates Intemational 308 Attachment: 1 `Test Diet Preparation Procedures Study Number:TRSCpon1s1o3r2:-130M0 `Amended FinaPaRgepeo6r6t 1) Determine the amountoftest diet (feed) that is to be prepared and weigh out that amount of feed. 2) Calculate the amount of test article that is needed to prepare the fest dict at the desired concentration. 3) Accurately weigh out the necessary amountoftest article. 4) Transfer the weighed test article to a container and add a small volume of acetone to container. Manually mix to dissolve the test material adding acetone as necessary (typical ratio of test material to acetone is 1 g: 15-20 ml acetone). Visually inspect test `material/acetone for solubilityoftest material. 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 amoofuweinghted diet, mixfor 30 minutes. 206 Study Number. TRSCpon1s1o3r2:-310M0 `Amended FinalPRaegpeo6r7t Attachment: 11 CollectionofTissue Samples for Biochemical and Molecular Analysis Becauseofthe extreme instability of certain enzymesandbiomolecules, it i essential 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 after death. Delays may allow for biodegradation and/or inactivation ofthe desired endpoint. 2) Immediately submerse the tissue sample directly into liquid nitrogen. Dry ice or other alternatives will not suffice. It is important that the tissue be immersed directly in liquid nittogen; transferring it toa 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 completely destroying the vessel (or sample container). 3) Be absolutely sure tomaintainthe tissu frozen. Itshouldbe stored in asealed container at = 700C and shipped or transferred on dry ice. If needed, the frozen sample can be fractured (broken into portions for different applications) by placing in a crucible which contains liquid nitrogen to keep the sample frozen whilegrinding fracturing. PROTOCOL AMENDMENT #1 `Sudy Number: TRSpCon1s3or2:.130M0 `Amended FinalPaRgepeo6r8t Date: February 24, 1999 Study Number: 1132-100 Title: C3eMllTP-r6o3l1i6f.er1a1t)i,onPeSrtfulduyrowoictthanNe-ESutlhfyo]niPcerAfcliudorPoootcatsasnieusmulSfaolnta(mPiFdoOSE;th3anMolT-(6N2-9E5.t1F6O)S,E; and N-Ethy! Perfluorooctanesulfonamide (N-EFOSA 3M T-7091.1) in Rats Changelreplace the following: 2) Title page: PAT Project Number: 99-1233 b)Page3: Indife Start Date: 2/23/99 In-ife End Date: 4/6/99 Draft Report Date: 5/18/99 ) Page 4: Test Materials [LotNumber N-E(FOSA. N-EFOSA [5a 'Wy-14,643 (Cayman wy_-- = Tuo [Swbilt:_____-- [Storage Conditions: |Characteristics: [>Syeas [roomtemp____ Tamberwaxysolid Dlyar [woomtemp. |whitepowder ) Page 5: Animal identification isbyear tag. ) Page 8: Observationof Animals; add, Physical Examinations Weekly at each weigh interval. 1 Entire protocol: Change RO.W. Sciences to Therlmmune Research Corporation 8) Page 8: Clinical Chemistry blood will be collected from ajugular vin nto a serum-separator tube. Chemical) 1 | | | | `Study Number;TRSCpon1s1o3r2:-130M0 `Amended FinalPaRegpeor6t0. h) Page`9S:erSucmhesdaumlpeldesSfaocrricfilciensi.cal 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: Dr. Kris J. Hansen 3MET.&S Building 2-38-09 935 Bush Avenue St. Paul, MN 55106 Telephone: 612 778-6018 Fax: 612 778-6176 For both Interim and Terminal Sacrifices: cNhOeTmiEs:trTywaonds(e2r)utmhesarmepmlaeisniwnilglsbaempnleeedfeodr,c(o1m)paotulneadstlev0el.2a5namliyssisa.mple for clinical ) Page L1i0v:ePraslammiptloeysl-fCoroApaOlxmiidtaosyel-TiCsosAuoexCiodlalseectaictoinvaitnydwAinllalbyesisshipped on dry ice to: Dr. Ron Markevitch 3C3o0v1anKcienLgasbmoarantBooriuelsevIancr.d Madison, WI 53704 Telephone: 608 242-2712 ext. 2337 Fax: 608 241-7221 3) Page L1i0v:eTrisssaumeplCeoslfloerctEiMon fsohroEulledcbtreosnhMiipcpreodstcoo:pic Evaluation Sharon Ambrose: 4P9A1L5NDCProspectus Drive. Durham, NC 27713 X) 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 a1r0c:hCievlelfPorroploisfseirabtlieonfuTtiusresuaenaCloylsliesc.tion and Immunohistochemical Evaluation m) Page l1i0v:eTriwsislulebCeolclolelcetcitoendfaonrdElpercetsreornveMdicirnofsocrompailcinE.valuation Sectionsofthe left lateral lobeoftheliver from all animals.. Study Number: TRSpCon1s1o3r2:-310M0 `Amended FinalPRaegpeoTrt0 1) 0) Page Page 7: Change doseofWy-14,643 from 1000 ppm 13, Item #5: Prepare pre-mix by transferring to 100 ppm the dissolved test material into feed. Mix until homogeneous... Reason for Amendment: ba)) ADsastiegsndeedtearfmeirnperdotaoftceorlparpoptrocoovledsigned; study is non-GLP, therefore report will not be audited ) Information obtained after protocol signed ) ) Spelling Physical error exams shouldbe conducted in addition tobody weights 1)) TNeostainntgicfaocaigluitlyancthaisnugseeddnfaomreclinical chemistry h) Provide shipping instructions and specify minimum amount of serum needed for clinical ci)hePmriosvtirdye shipping instructions 3) Provide shipping instructions Km))PSrpoevciidfeysltoobreaogfeliinvsetrrutcotiboenstaken for EM 1) 100 ppm Wy, 14,643 is adequate for inducing a cell proliferative response, increase in liver w0)eiAgchcturaantdeidnecsrceraispetiinonpoefrtoexsitsdoimeetspreparation procedure: Approvals: `Marvin Case, D.V.M,, PhD. Date S3tMudCyorRpeoprreasteenTtoaxtiivceology Gary W. Wolfe, Ph.D, DABT Date Study Director `Therlmmune Research Corporation Sandra R. Eldridge, Ph.D. Date PPraitnhcoilpolgeyInAvsessotciigaatteosr Intemational Co 3/6 `Study Number: TRSCpon1s1o3r2:-130M0 `Amended FinalPRaegpeo7r1t PROTOCOL AMENDMENT #2 Date: April 16,1999 Study Number: 1132-100 Title: C3eMllTP-r6o3l1i6f.e1ra1t)i,onPeSrtfulduyrowoictthanNe-EStulhfyolniPcerAfcliudorPoootcatsasnieusmulSfaolnta(mPidFoOSE;th3anMolT-(6N2-9E5.t1F6O)S,E; and N-Ethyl Perfluorooctanesulfonamide (N-EfFOSA 3M T-7091.1) in Rats Add the following: a) PageG9r:oNsesclreospisonys will be collected, processed and stained with H&E for microscopic evaluation. Reason for Amendment: a) For histopathologic examinationofgross lesions. Approvals: `Marvin Case, DVM, PhD. Date S3tMudCyorRpeoprreasteenTtoaxtiivceology Gary W. Wolfe, Ph.D, DAB.T Date Study Director Therlmmune Research Corporation Sandra R. Eldridge, Ph.D. Date Principle Investigator Pathology Associates Intemational -- oo 3// `Study NumTbRSCpo1en1s3or2r-:1:30M0 `Amended FinalPaRgepeo7r2t Date: August, 1999 PROTOCOL AMENDMENT #3 3M Study Number: TRC 1131-100 Title: "3CMellT-P6r3o1l6i.f1er1a)t,ioPnerSftluudryoowcittahneN-SEutlfhoynlicPeArcfilduoProotoacstsainuemsuSlaflotn(aPmFidOoS;Et3hManoTl-6(2N9-5E.1t6F)O,SE; `and N-Ethyl Perfluorooctanesulfonamide (N-EFOSA 3M T-7091.1) in Rats" Original: Page 10: Tissue Collection for Electron Microscopic Evaluation Sections of liver cubes and placed ifnroamfiaxlaltiavneimaaplprsopwirlilatbeefocrolelleecctterdo,n mmiincrcoesdcotpoy.appTrhoexicmoanttealiyneorsneanmdilfliixmaettievre gwirlolubpeepxhriobviitdiendg btyhePhAiLgheEsltecctelrlonprmoilcifreorsactoivpey rweisllpobnesepearsfowremlledasonoonneecoanntirmoall apneirmtarleaattmetnhte d`ipsecrrfeotrimoendooftnhoeneSpaonnismoarl, ffrroomm othneectoinmteroplo,iNnt-EasXFweOlSlEas(otnhee 4do-sweeeoknlryectoovebrey.detTehrumsi,neEd)M, wPiFllOSb,e NCEWFOSA, and Wy groups at one ofthe time points, as well as the 4 week recovery, for 2 otal of 10 animals. ChangefPraepgleac10e:tThiesfsouleloCwoilnlge:ction for Electron Microscopic Evaluation Sections of liver cubes and placed ifnroamfiaxllataivneimaaplprsopwriilatbeefocrolelleecctterdo,n mmiinccroesdcotpoya.ppTrhoexicmoanttealiyneornseanmdilfliixmaettiever wgirlolubpefprroomvitdheed4b8y-hPoAuLr tEilmeectprooinntm.icTrhoussc,opEyMwilwlilblebpeerpfeorrfmoerdmeodn townotawnoimaanlismaplesr tfrreoamtmetnhte 4co8n-throoul,rNti-mEeFpOoiSntEo(1ra00tpotpamlodfos10e garniomuaplso.nly), PFOS, N-E(FOSA, and Wy-14,643 groups a the Reason`fAonriAmamlesndsemleenctte:d on the basisof cell proliferation results. Approvals: `MarvinCase, D.VM,PhD. Date S3tMudCyorRpeoprraetseenTtoaxtiivceology Gary W. Wolfe, PhD, DABT Date Study Director RO.W. Sciences Sandra R. Eldridge, Ph.D. Date Principle Investigator Pathology Associates Intemational 2 Date: May 30, 2000 PROTOCOL AMENDMENT #4 Study Number: TRC 1132-100 Study Number: TRSCpon1s3o2r.:130M0 `Amended Final Report Pages Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perflurooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-E(FOSA; 3M T-7091.1) in Rats Change/replace the following: a) Globally replace PFOSA with N-EtFOSA because the acronym for N-Ethyl Perfluorooctanesulfonamide that is currently accepted is N-EtFOSA, rather than PFOSA. wb)itPhaogneg4o:iRnegvainsaelytsaibsleatbe3cMa.use 3M provided the lot numbers, and purity information is changing TestMaterial. |N-EGOSE | PROS [PFOSA | Wyi46s3 | TT (providedby 3M)| (provided by 3M)| (provided by 3M) | (CChaeymmiacna) Identification: | N-EtFOSE PFOS PFOSA Wy _ Lot Number: Purity: SSttaobrilaigtey: | Conditions: |30s0o305c,o3m0b03i7n,ed |217 |"541 11990d MmResponsibility of| 3MResponsibility of| 3MResponsibility of| 98% >`r5oyoematremsp. _|T>ooSmyteempa. rs >ooymeatresmp. [`rZoom2tevmpe. ms Approvals: Marvin Case, D.V.M., Ph.D. Date 3StMudCyorRpeoprraetseenTtoaxtiivceology Gary W. Wolfe, Ph.D, DABT Date Study Director Therlmmune Research Corporation SandraR. Eldridge, Ph.D. Date Principle Investigator Pathology Associates International _ 3/3 VIIL SIGNATURE PAGE StudyNummbeernsTiRSFCponn1s1o3Rr3es:p13o0r0t Past Study Title: C6e3l1l6P.r1o1l)i,fePreartfilounoSrtouocdtyawnietShuNl-foEntihcylAcPiedrfPloutoarsosoicutmanSeaslutl(fPoFnaOmSi;do3MEthTa-n6o2l95(.N1-6)E,tFaOnSdEN;-E3tMhyTlPerfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Study Number: TRC 1131-100 AvLa GY,h SHR TeLJ PPLLS TPooncoe soenrosr Divisio of Chie Rives Libortrs ye