Document 2JxEz2Z5ep9OJ3eY77vX3Vavg

3M General Othices 96 ff S3M FCemer sas om 612733 1110 . AR22-6 [036 HR pia BEHR 090 - 37% sepempers. 200 Document Processing ener (407) UOfSf.icEeonvfiTrooxnmiecnStuablsPtraontceecstion Agency 401 M Street, SW. `Washington, DC 20460 Attn: TSCA Section 8(e) Coordinator BEKQ-O-373 ooo 24a Dear Section 8(e) Docket Coordinator: . Re: TSCA 8(e) Supplemental Notice on Sulfonate-based Fluorochemicals `With this letter, 3M is providing final reports and other supplemental information related to previous TSCA Section 8(e) notifications. Manyof the enclosed items are analytical reports providing bloodserum and liver levelsoftest materials for which the in-life report referring to administered doses has already been submitted to the 8(e) docket.In other cases `where the 8(e) notification consisted ofpreliminary data, we are submitting a final study report. All ofthe enclosed items are already in EPA'spossessionand available in TSCA Docket AR-226. We believe, however, that placing these items in the 8(e) docket may allow for more convenient access to information directly related to previous 8(e) notifications by 3M. `The table below lists the enclosed items and references the stourd day ta which already has been the subjectofan. 8(e) notification by 3M: Attached Submission RelateoddSSttuuddyaDaattaaAAllready |. ADlmefetonnedanemtiddiAeonyanlyothfiatcnhadel!SPitrnueTdsywe,noc0eNGa-ennEedtrhCayotlnicpoeennftRlravatotrRieoopncrtooafdnuPecFtOiSon., ||| CDoevmeblionpemdenOtraall a(nGdaPveargien)atFalerPoist.natal Reproduction Toxicity Study of N- | MMCi5os3lS6-6,,CDPPBFFROOVSSAAAFAA/,.PlaPunFsdORPSaFtAsOaSEnxAdpoiEnsIteFdhO0LSiEEv.eFrHOaSniEnd-tPOeRHOS,Se,r3aMof || 6E3S1F6O.5S,EJiunneRa3t0s,, 319M99R,effuelrlernecpeorNto. | submited February 15, 2000 0 T- Reference No. T-6316., Anaptcal Report TOX013. | supplement adie filing o 1 LRN-U2095, June 11, 2001. =| 55 3S= 00000/ ContaitinNo cgESEF 3 . 1 TSCA Section 8(e) Docket Coordinator : Page 2 AttaA ched Submission RelateodtSStudypDataa Already | 2. Analytical Laboratory Report, Determination ofthe | Combined Oral (Gavage) Fertility, V7| SFPPrReueOsameSnociaaenadmGnLadoinvvCeoeern,cnoefnaLtSarrpabrataoigor(uneCrDoAyfSRPaNeoputomavrsbtseirNu,Roms2U72E50x00pe.o,3se5d.) nh. | || TiRDneenvRpeoalSdsopuposmAnetsrnooetrnas'lsTRSaoensredetayPrsecrNhiSonLaatn2baol9r/oaP5otfso5PtrniFuaeOtsaeS,l || 3. Requenr rj No. Sw TON 3345. Osta 37, 1968, Report Amendment |, Combined OralGavage) Fert, || cF1ea0b1rr9ur0a0ri,y1nf5g,l2p00r0tsubppilenmendting || Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats, Argus Research Laboratories, Inc., Protocol 418-008, Sponsor's Study No. 35Av5a5t,icAlpReepo15r.t,2D00t0rminaon ofthe Prsetce and | 13-Week Dietary StuyofNth! Concentrationof Perfluorooctanesulfonate, Perfluorooctanesulfonylamide, M556, and M570 in the Ties onsen Samples Evironment Libortry | 314.1, Covina Study No, 525.25, Ref. No. U2636, TOX-028, February 23, 2001 Perfluorooctanesulfonamido Ethanol | (N-MeFOSE) in Rats, 3M Ref. No. T- dated June 30, 2000, Section 8(e) filing. 5. Amaitial Laboratory Repo, Determination ofthe | lFiynal2R6e.p30o0,0Oral(Gavage) Concentration of PFOS, PFOSA, PFOSAA, and EtFOSE- | Developmental Toxicity Studyof2(N- rhe Sor and ivr ofCALCOBR VATPs Rats |Emylperurocsianesiforamido: Exposed to Report No. TN-OEXt-F0O9S8E,,La3bMorEantvoirryoRnemqeunetsatlNLoa.boUr2a4t0o2r,y 3M | ethanol 6316.7, in Rats, 3M Reference No. T- December 17, 1998, submitted Ref. No. T-6316.7, February 6, 2001. to Section 8(e) docket per letter of August 21, 2000 & PAnaiteicaalnsLdaCboornacctaotrayteRieopnortofoPn tahsDeserminaionof he. |DeFvienopRemprrt,alOraTloSxtiocsmaScrhyTuboef) Perfluorooctanesulfonate (PFOS) or another metabolite of |EtFOSE in Rabbits, 3M Reference No. | ToEervromsmentoal LoibramtoyaRfoorramNoye.aTbOsXe-0l970 | TR-6316.a331,Tp3a0m0a0r 11,1999, brite EtFOSE) in Liver and Serum Specimens, 3M toSection 8(e)docketper letter of Laboratory Request No. U2452, 3M Ref. No. T-6316.8, 7 February 8, 2001 FE il Reporte Ale3xanDdere,e Br.FMoarayli,ty USnuveesrosfoWforkers || r$l0mdionckeeytidnete a subomfiDtee ecde0mbSeeri15n, Minnesota, April 26, 2001. 2000 000002 . 2 TSCA Section 8() Docket Coordinator Page3 Acesubsion | rtm Related Study/Data Already | 8. Final Report, Acute Oral Toxicity Screen with T-3290CoC | Acute Oral Toxicity Screen with T- | binoAlrbainnoeR,atsn, eSafPertoyjeEcvtalNuoatiGoBnBL2aAbRorDa2tGo2r,y,MRikReerference| 3E2v9a0sCiooCninbAolbriononR,atsR,iSkaefrety aNnod. 4T-03%290H,(0,40L-%67K7'8,PFF-O6S8A73A, iLno3t %SOIE)t,ONHo,ve1m7b%erIP5A,. || L0a8b8o2raAtRo0r3ie6s2,,I3ncM., RPerofjeercetncNoe.No. T- w1a9s82to[BbieblmioovgerdapthoyTeSnCiAr i8n(eD)odcokcektetA]R-226.final report | 3127990%(P40A%anKd'4P0F%OSHA,0A,iLn637%78,EIFO-H, || s6u8b7m3i,tLtoetd tSo01S)e,ctNioovne8m(be)rdo5c,k1e9t82in, `(Awuhgiucsht e2r1r,o2n0eo0u0ssleylrfe-faeurdsittoetraebrbits rather than rats) 9. GPieersfyl,uoLrPo.octaanndesKu.lfKoannanteana,ndAcRceulmautleadtFilounoroofchemicals in | 8P(ree)lidmoicnkaertyMdaatya 2s6u,bm1i9t9t9ed to Section Fish Tissue, Michigan State University, June 20, 2001 10. GPeirefslyuo,roIPo. ctaanndesKu.lfKoannanteana,ndAcRceulmautleadtFilounoroofchemicals in Mink and River Otters, Michigan State University, June 20, 2001. 11. GRieelsayt,eJd.PF.l,uaonrdocKh.eiKcaanlnsani,n OPyesrtfelru,orCoroacstsaonsetsruelafoVniragtienaicnad, From the GuoflMexifco and Chesapeake Bay, Michigan State University, June 20, 2001 12. GRieelsayt,edJPF.luaonrdocKh.emKiacnanlasn,inPFeirsfhl-uEoartoioncgtaWnaetsearlfBoinradtse,and Michigan State University, June 20, 2001. 13. GPieersfyl,uoLrPo.ocainandeKsu.lKfaonnantaena,nAdccRuemlualtaedtiFolnuoorfochemicals in Marine Mammals, Michigan State University, June 20, 2001 If you have any questions about ths submission, please contact me at (651)737-4795. Sincerely, Je Milne Georigh Adams Manager, 3M Corporate Product Responsibility Enclosures 000003 - 3 CPAoSsNsumbPere2r75r8o3c83anesoua rWM Eevornmeneal LToroy MR 516% ------ ee ------ ANALYTICAL LABORATORY REPORT onTHE Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (CAS Number: 2759-393) in the Serum and Liver of Sprague-Dawley Rats Exposed to PFOS via Gavage Laboratory Report No. <U2006> Requester Project No. <3M TOX 6295.9> -_-- StudyDates SL rea Study Initiation: 26 May 1998 | Study Completion: At Signature 8Epa-oTs ARR 0008118260 00000 z i 2 = % EQ ~~ PR HW PCoAtSasNsuummbPeert2u7o5r8so3c5a3nssutonss TABLE OF CONTENTS Laboratory RaMcueEsmtvuRomenpbmoeerrrti(sNLoRLabTo0or2act0oo0r:6y StudyPErSANOdCONMTBNULDETS ! cvs 3 SIOfCR OMEPINENC....rsesssssen d QUAHYASSUTNGS SIEM... B (CECECHe ATGCIEZBHON...... ssn 8 PAOD!DEVIBIONS...cesremmmssooersssee ee 13 DataSSSuUUmMmTMaRrTUyYO,ToffASEQBBUYTaNlPStIYSyEC,oRnEBtNSrUoRRlSEATSaUl.yS.S.ecs..R.rES.UcSe.vm.s.mi.ss.s.eo.s.so....orose1213 1 D812QUaltyOBIEANCADHAEVINMESGHSY cos 18 SHE Of CN ONCS! ION sss 18 REBIENGES cn mmm 1 Attachment A: Report Signature Page Attachment 8`:STparbalgeuoef-DRaewsiuelytsRaotfsAnAadmliynsissotfeLrievdePrSFaOmSplDeaslfyrboymGFaOvaMgaleeand Female AttachmentC:STpabrleoafgReuseuRsa-oifsDAAnaadlmywisniiissoteefrSeyedrPaSFaOmSplDeaslfyrboymGFaOvaMgaeleandFerale AttachmentD:SpTraabglueeo-fDaRveiseuylRsaoifsAAnadlmyisniissotfeLrievdePrSFaOmSplDeaslyfboymGFa1vMaagleeandFemale: `Attachment In-feProtocolandAnalyticalStudyProtocol AtiachmenFt: PrepaandrAnalaytitcaloMetrhoyds AttachmenGt:Dose Analyses, 000005 Pagezatts Pots Parsocaneuttos (CASNumber2756383. STUDY PERSONNEL AND CONTRIBUTORS Analytical Chemistry Laboratories 3M Environmental Technology and Safety Services Fl3uMorEinnveiArnoanlmyetnitcaall LCahbeomriatsotryyTeam (FACT) 293358B0u0sh Avenue St Paul MN 551068 Kris Hansen, Ph.D., AnalyticalStudyDirector Lisa Clemen, Analytical Chemist(s) Indife Testing Laboratory Argus Research Laboratories, nc. 9Ho0r5sShhaeme,hPyADri1v9e0,4B4uilding A Raymond G. York, Ph.D.,StudyDirector Sponsor 33MM TCoexnitceorlogy Services BStuiPianulg, 2M2N0-525610424 Marvin Case, SnonsorRepresentative SM Eneonmenal abort Laboratory RacuetNumRebpeorrt(NFoN.)T-e0x2001028 000006 6 Pon3ctte CPAotSasNsorirmoPeorr2t7u5o6r.e3d8a3ncsutorsts WM EnvorRmeepnotratlNoLaTboorxaotorr?y LabReocuerstNuarote (oLRN)rU20y08 STATEMENT OF COMPLIANCE Study Te: oAcntaalnyetiscuafloLanbaotreaitnorSyeRreupmoarntdoLinPverreosfeSnpcreaagnude-CDoancweinetyraRtaitosnoEfxPpootsaesdstiuomPPFeOrfSlvuoiraoGavage Study Identicaton Number. FACT Tox012 "T(hGiLsPs)tuRdegyuwiaatsicoonnsfdourcNtoendciiniccoamlplLiaabnocraewtoirtyhSFtoudoideasn[dDaDtrauRgeAqdumiirneimsetnrta(tsi)o:n G2o1oCdFLaRbo(Praatrotr5y8)P,rawcittihce bthyeaenxicenpdteipoennsdienntthqeubaulltlyeatsesduisrtabnecleowu.niI.naAduddiittiopnr,otchedeuprreessaenntdsftiunddyihngasfsobreaenuasudpietrefdorretmreodsaptetcthieve3lMy pEenrvtiinreonntmteontthailsLsatbuodryaitnoarrycahnidveastaptatrthicei3paMtilnagbcoornattroarcyatlnadbowriatlobreireesatarienheodfuosreadtwlietahsdto1c0uymeeanrtsa.tTihoen `aEnnavliyrtoicnamlepnotratlioTneccohmnpolleotgeydaantdtShaef3etMyESnevrivriocnemseSnttaanldLaarbdwOapesrpaetrifnogrPmreodcienduarcecso.rdancewith 3M Exceptions to GLP compliance: Storage batch nucmobnetra,ineexrpsifroatritohne draetfe,erosetncoersaubgsetaconncdei,tiPoFnsO.S,werenotlabeledwithname,CAS number, + Destuabisltsanocfet,hPepFrOeSp,araarteiounn,kmnaoxwinm.umstoragetime,andstabiltypropertesofthereference Tathtehiedteinmieyo,fsttrheensgttuhd,yp;urhtoyw,eavenrd,caonmaployssietsitonoddeetfailnitnhgetchheatreacstteorrizcaotiiroonlwaerrtiecloewngaosinngoattdettheertmiimneeodf this analytealstudy. + Two separatestudydirectorswereassignedtothe invivo andtheanalyticalportions ofthisstudy. From during1t5haStepmteepmebreirod1n99o8tunnctom1pOlcitaonbcee.r 1998,theprotocolwasnot sponsor approved andwas Deviatons were notconsistently appr.vedbythestudydirectorasrequiredbyGLPreguiatins. Dat correctionswerenotaways recordedasrequiredbyGLPreguiations. Dat werenot awaysattributed totheindividualresponsibleforrecordingthedata. Records n electronic ormdonotmeetthecriteriasetforth under 21 CFR(11). dAofcinuamleinzetdssdaimdpelxeiltoag-nidnwerarcekuisngesdysotleomgwiansanndotriancpkascamepaltesth.e meofthestudy;however,draft 000007 7 Pagedal1e [Baenona --T-- y * Notallrawdatawereverified bythe studydirector. + Noin-phaseaudits wereconducted duringthe presentstudy. Ate andmettihmoeodtfrtaihneipnrge;hsoewnetsvteurd,yp,epresrosnonnenlefileseshvidenboeteanluwpadyastceodn,taainnddotchiusmneonntcaotmipolniaofnceequhiapsment beenresolved. Signature ofStudyDirector: fh ho 27 0c 95 SnareofSeySponsM or eiTO 2041, 000008 FR PCotAaSsNsioumoPra2a7c55a3n5e5sioras Laboratoy ReWcEuensv:NkuoRmnebpmeoerrnt(tNRloNLUaTo2bxo0as0rys8 GLP Stupy QUALITY ASSURANCE STATEMENT Study Title: APnearlfyutoircoalocLtaabnoersautlofroynaRteepoirntSeornuPmraesnednLcievearondfSCponrcaegnuter-aDtiaovnieofyPoRtaatsssEixumposed to PFOS via Gavage Study ID Number: FACT Tox012 "iThisnstuddiynihathscebfeoaleonwitinnsgpeteacbtdleed.Tbyhethfei3ndMinEgnsvwierroenrmeepnotratleLdabtootrhaetsotryuQduyadlirteycAtsosraunradnmcaenUangietm(eQnAUt)as INSPECTION DATES Piase DATE RepORTETDO [N] 13 spotron | rome ss |r| JR somtimesme leo Representative 10-15Tate 000009 Pagesal16 PoCtAaSsNsuummbPeerd2u7o6r5o3c03anesonste RTTeter INTRODUCTION Laboratory ReWquEensrtoNnuRmmebeeonrnt(lLLRaNb)Toro2art0oor0sy8 ---------------- Potassium Perfluorooctanesulfonate CAS Number: 2795-30-3 `Chemical Formula:CyF SOs Molecular Weight 538 V`IenThwitechelenepcr8oe0n.st(erTnothleFsstOuerdday,tossgweresorucpesadomfoiFnOisrrtaettsrorewcdeoornneceleayndtTmrsiwanetiiesopntnes8ro0eof.dM00.a.01nl,2e,0F04.Od,0a81,n.6i0,.mo3ar2,3w.ae2nrdmeg0t.rP6e4FatmOegSd4/km2o.ld)daayGysrionpur0pio.sr5o%f wpteroirmoeratetoxinpmgoatsaiennddgt,tohttrhhoruoeguchghhethgmeeiscmtaaattliiionnngu,tpaeonrridoodau;npdftoedmu2ar0liendgaFlyOaasctnfaiotmliloaonlw.siFnwogelirltoeewrianddegmlWiienveiarsnyt.ienrFge1adtmPa2Ol1edSaanyddsioafyafra4mg2alede,arytss asenldecbtioeldogFi1caalnpiamraalmsewteerrestwreeraetemdodnuirtionrgeddeivneFlOo,pFm1e,ntanadndF2praondiumcatlison of F2 animals, Various physical IPdnoFtsOheSed.ana(anlAiynmtaaillcysasle(ssgtweunederyrerateippoeonrrftFoeOrd)hmaeendrdett,ohediveretreoafmninsidpnsreientrghae(sgpaernmeepsrlaeetniscoecnooFlf1cE)tIweFedOrfSerEo,amnPatlFhyOziSetAda,floPrpOhopAauAlpa<rteiPsoFneOnoScfeEAof, wPeFrOeSnAot,reapnodrttehde.)moncester, however, these data were colected for informational purposes ony, and Lc`iehvxretorrmascatatimoogpnrlpareposhcyew/deeurlreee.chtorTmohosegpereraxytirztaeacdtn,sdweaemnrdmeeaqsursansatipntedacttsireveorlamyestaarnmyapl(lyHezLesPdwCueIsrEieSngeMxShtMirgSahc)t-.epdraebnsyduarPneFloOnuS-pialiefveilnsg were aevvaailuaabtleediangtahienssttuedxytrbaicntdeedrsmtaainndtaaridns.ed bAnyaltyhteic3aMlFdlueotraAnaarleytiinccalludCehdeimnisitsyrTepeoarmt f(uFtAhCeTr)details are ``AwmneeartlehyocsdoesnsdaaunscdsteessdtsaiinnndcgaortmdhpelopiteoaxrniaccteoiwlnoiggptirhcoalGceoedfoeudcrteLsasobwfoerrPaeFtOoSrfyolPlirnoawtchetdicdveuerRriensgguatlnhadetispoernerspaoa(rf2a1tSiGporFnaRagnu5de8)-a.DnVaaawllylisediyasotrefadttshe samples associated with this study. ------- SAMPLE RECEIPT tthhTriesosu3ugMehEsnJaavnmiupralorenysm,ew1ne9tr9ae9l.LreaScbaeomirpvaeltdoersfyrroeamcneAdritgvrueasndswRfeeerrsreeepadtraocchkaaLfgarbeeoedrziaentrdofrroiyresscteso.proaSrgpaeed.ciSciaamlme,pnisrewoermreceAeruiepgtgu.isstdt,een1rt9ei9dc8wa,ittohn, sitsolroacgaet,edanindcthheai3nMoafrccuhsitveosd.yprotocols anddataarelocatedinthestudyfolderforthisreport hefolder a`SnpdewciilmebnessatnoarleydazetdtahtetihabeorIaMtEonrvyairton2m0enCta+lL1a0boCr.atorywilbemaintainedfora periodof 10years 000010 : Poce7at16 STARTS cmarEtEs 'MATERALSAND METHODS `Chemical Characterization oe SEit "Tabl1e presents information regarding characterization of the test, control, and reference substances Com romoonaimam | [orm Twexposed to light [Con owen eT | [miomm eSets Tou wommmm] [|Sr omi EeoEna e r ea ame] [Cooemm Toowwenn E Oa ena r e Cor-- Fesod ett 0000| Page8of 16. reesmeats es) reprrien Laboratory Request Number (LRNJ-J2006 Tee SBoengouoaineerSmSaapPsoEor SEoees eamme a OACTTRo H30 wr Seotrm eSetmen [o[AsttatFchmenrt F) amazre oFCnrreymDarrousnesryAsTr m-- ww rr ] ] Cf ToomC mSatT erandT To amasICeoCneal Err] ad Perfluorooctanesulfonate In Serum andLiver ofSprague-Dawley Rats. Exposed to PFOS via Gavage (continued) m Felina,n reoiignt rcrrsrttant rol arvee rgerent Method Summaries Da dre es esas ne epi Following isabriefdescroiftphetmeithoodnsusedduringt:hisanalyticalstudy on bythe3M +PrepE i MmHaeraaetognryAFoMAE eTstChToodAi1s:18TEEm oeentin otf MosBae EnrsPtervvseoerrCs ens Midt.otemerOuch ooirmnkas en rere Eat env tai Orlov mot PCoAtaSsNsioommaPror2u7o5r6c3oc5a3nesutrats LaborateyReWcMueEsvtrNouRrremmpeaoerrrtt(NaLLoRabNToYroUax2ta0orr0ys6 Me`tCohmopdouFnAdCsTf-rVo-m3.S0e:ruExmtorracottihoenrofFlPuoidtafosrsAinuamlyPseirsfuUosrionogctHaPnLeCs-uEfloencattreosoprrOatyh/eMrasFlsuSopreocctherronmiectarly. re`Saegreanstawmpalsesadwdeerdeioextthreactseadmpulseiangnadnthoen-apnaailfyitnegieoxntpraacitiwoanspropceadurre.tIinnistuomtmmeaitrhyyo, tanenrteo-bnudtpyaliring etdihy.erEa(cMhBeEx)t.raFcotuwrmals.reocfotnhsetiMtIuSteEdexnt1r.a0cmtwi.aosfrmeemtohvaendoalannddpfuetronetdotharnoiutgrhogean3e-vcacpoplraasttiocrsuyrnitnge: `tachedto a0.2 ymnylonfterinto gas autovials. Analytical Methods Method FACT-M-2.0: `Spectrometry AnalysisofFluorochemicals in Liver Extract Using HPLC-Electrospray/Mass Tshuecahnaaslytshiespwearsfupoerrofoocrtmaendesbuylomonnaittsor(iPnFgOaSs)ianngiloeni,omncz =h49a8.rAdadiotfcoanpatalr,teiscaumlraprlifeusowsreorctheerimicca, d`eatneacltyiznegdduasuignghtaetraonndseomfmtahsesselsecptedepacretnttroofnou.rtmheervetrdeytrhe Kdoefacnomtpouynd by MeStehruomdorFoAtCheTrMF4lu0id:UAsnianlgyHsiPsLCof-EPloetcatsrsoisupmrPaeyrMfalsusorSopoecctiarnoemseulornyateorother Fluorochemical in s`Tuhcehaansaltyhseispwerafslupderrofocotramneedsublyfomnoantieto(rPiFngOSa)sianngiloen,iozn c=har4a9c9t.eriAsdtdiictoofnaaplayr,tiscaumlpalrfelsuowreorcehemical, adneatleycztiendgudsaiungghtaetraonndsemomf atshspsarepnteon.ctrtoofutmheervetreytrh ideofnatcoimpotunyd by CaonnafliyrsmeastoarryedporseseeanntaeldyisneAstwaecrhemceonntduG.cted folowingtheendof thestudy,andthe resultsofthese. 000013 Page 100016 PoCtAaSsNsuimmbPearr2o7r5s9o3c5an3esutonats LabRooWcMuEersnvoRaeenmpmboeternrtt(oNlRoLN.-arT0bo3o0rry0dr8iy Analytical Equipment . seFtotlionwgisnvgararyestoympiecwalhaantadluytriicnaglaecqtuuiaplmdeanttacsoeltliencgtsifoonr.Etxhaecptrsoecteidnugrsedusruisngedalinptrheaspreessoefnctasttaudcyo.lTehcetsoen: arerecorded andpresentedinthe analysissectionofthestudybinderfor FACTTox-12. HPLC System: Hewlett-Packard Series 1100 Liquid Chromatograph Column: Keystone Betasil C18 Column 2X100 mm, Sum particle size Flow rate: 300 pLmin SoventA: 20mMammonium acetate Sovent 8: Methanol SowventGradient: 40% 090% Bn 85minutes RHoeltduamtt9o 40%0%BfBori3n.10.0mimniuntuets.e Hold at 40% B for 1.0 minute Injection Volume: 100 Run Time: 135 minutes Electrospray Tandem Mass Spectrometer: Micromass Quattro ll API Mass Spectrometer Mass Lynx 3.1 Software `Cone Votage: 30--60v Colision Gas Energy: wev Mode: Electrospray Negative Source Block Temperature: 115C Desolvaton Temperature: 250C Electrode: Zspray Primary lon/Daughterlons: 499/98,1030,, 180, 230amu Page t1at16 PCoAtaSsNsaiummbePrau2o7r55o3c8a3nesuonsts WMEmrorRmaegntoanlLeaTbaoxrorny LaboratoryRecuest Number(LRNHU2008 Protocol Deviations. nTuhmebreewra2s17o;nheodweevvieatri,onottontuhmebperrot1o93cowkaTshuesperodtdoucroilncgaltlheedanfaolrtythiecuaslpeoortfiroenfoefrtehnecsesttuadnyd.ardlot . DATA SUMMARY, ANALYSES, AND RESULTS Summary of Quality ControlAnalyses Results + leCasatelverbyC1rh0esacakmtSptlaiensdtoaormdnosn:itAomridi-nlsetvreulm,eenxtarcitt.eCdamlattartiixocnaclthreactikosntcahndeacrkdwsawseraencaolmypzleidaantt, andalcalibrationcriteriaweremet(within 30%). + blBlaannkkss:weFrouarpbrelpaanrkesdwweirtehewxattrearcatesdcaonscuuorrgeanttelymwaittrhxe.aTcwhboaatdcdhitoofsnaamlpslaemsp.lTeswoofebxatnrakction `cumrvaexs.wAelrebtaynpikcsawlerpreenopnadreetdwectt forratbbhietcIovmeproousnedr(as)aonfdweterreeussted forextractedcalibration + Dounpelciocnattreomlaatnimaslp.iWkietahnaalfyesweesxicnecpltudiionngsa,lrletcaorvgeertiaensalwyetreeswweirtienptrheepaarcceedpatnadblaenarlayngzeeodffo7r0130%. ATnhaelsyesirseocfovtewroiesvaerrembaetrlioxwstphiekelsoawmeprlbeosurnedsatriyteofditnhe5a1c%ceapntdab6l1e%rraencgoev;ehroiewsevoefr,PtFeOSs.econd scethofamartiastchpekstesmeamarptliesspwziekeerse.withintheacceptablerange. NoactionwastakenIofurther Alniamiltoyfstehseoafbcoctehspettsaorfbasnlegera.mAlattousgph1kmesLreosfusletreadwinasPFuOsSerdefcoovtehreieansgalryesaitsoyfabPoFvOeSthleevheilgsh isnptihkeepsreerpaaorafttieosnt;atnhiimsallism,iotend yquaanstmtyalolfammaoturnitxomfaysherauvmeb(5e0e0nuLa)swoausrcaevoaifaebalre.foAr sthtehmeatrix paerxttrcacatrilonyisssusccaelpetdi-bdloewonotossaocfcosomlmvoednattdeutehteoaevvaaploarbalteimoant.Trh,etsheereevsualptionrgaetxetralocstsiess, which ``cowuoluddrensoutletfifnecatghreePatFeOr-Shacnon-ceexnptercatteidonPF(OPSFcOoSncheanstraaltoiwovnaipntohreperxetsrsaucrt.e)iWnhethneaenxatrlaycste,sof `seramatrix spkesareperformedwih afull1mLofsera,high PFOSrecoveriesare unusual + MWaittrhiamx Spiikesn:Moaiftwiompspeiurkbeastmacnhdmduartintgasnapliykteidcuapllaincaaltyessewse.rTeheanraelsyuzlesdoefverheys4e0ansaalmypsleessa,re locatedinthearchstiudvybiendder. 000015 Page 120116 (PCoAtSasNsuimumbePerr2t7u5o9r3o6oc.i3anesutonste LaboratoryReWqMueEsmtvNeRuoermpmboeerrret(aNlLoRL.Nab)ToUor2xat0oo0rr62y `SummaryofSample Results + aPnFimOaSls.wasPFdeOtSecwtaesd idnettheectievdrisnotfhaellsecroantsraolmpalnedsecxoplolescetdedgfrrooumpsaolfofFOthaenciomnatlroslaannddFd1osed tFhOe msaalmepsleasndfirnomsecronatrsoalmapnliemsacloslwleecrteedqufirtosmkaollw--dgoesneedraFlOlfyem1a0l0e-sl.sTshtehaPnFlOeSvellsedveeltsecftoeudndinin `tghreoulpo,wg-ednoesrealglryouipn.aPdoFsOe-Srecloantceedntmraantnieorn.in exposed animals increased with increasing dose. tPhFanOSlevleelvesldseitnebcottehd isnersaamapnldelsivceorlslaecmtpeldefsrcoomlltehcetceodmfersopmofnedmianlgegFrOouapnoimfaFlOs wmealrees.much less. FP1FOanSimcaolnscesnhtorrattyioanfsermebiarstuh rweedreinrpoouoglhleyd eliqvuearlstaomoprlleoswceorltlheactnedcofnrcoemnttrhaeteixopnsosmeedasgruoruepdsinof 3t.h2emcogrrPeFspOoSnpdeinrgkggrioduaypswoefrFeOsufbemmailtese.dfoNroasnaalmypsliess.Tcaobllecete1dsfruommFOmfaemrraelsueilsttzshoaefttrhseeceived `quantitative analyses performed on the FOfiverand sera samples examined. Grow 0.0 mgkglday [-- CONCEDNoTsRATION (ant) 0.1 mokgiday 0.4 mgkgiday 0.08 16 mgkgiday 32mohgay [32 Sampleswer nt recoved CONCEN- TRATONOF Ere [= CONCAEvNeTrRaAcTeON OF [E(2r2)p Female: 00307 | Female: 0.171 Male: 0.0244 Male: 0.685 Female: 528 Female: 14.8 Male: 105 Male: 84.9 Female: 18.9 Female: 58.0 Male: 45.4 Male: 176 Female: 82.0 Female: 184 Male: 152 Male: 323 Female: NR" Female: NR* Male: 273 Male: 1360 Table 2.DaPwFlOeSyCRoantcsen.trations In Serum and Liver Samples from FO Male and Femal0e0Sp0ra0gue- Page 13alts PCoAtSaNsusmbePreo2r7s5o03c8a5nesutonss Laboratory RSaqMueEsntvacmrRbmepeeonrrtt(lNL1oab2Toro0act00o1r8y2 gPrFoOupSscoofncFe1ntarnaitmiaolnss smheoarstyuraefdtrinbiprotolweedreverrousgahlmypltehes ecqolulaelcttoedorfolomwetrhethcaonntcroonlceanntdraetixopnossed tmheaatsruerceeidveind t3h.e2 cmogrkrges/pdoanydiwnegrgersouubpmsiotfeFdOffremaanlaelsy.sis.NoPsFaOmSplweasscodlelteecctteeddfiontmheF1vemraloefsaloroftehmearles. tehxepoFs1esdergarosuapmspolfeFs1exaanmimianlesd.. Table 2 summarizes resus ofthe quanttative analyses performed on Grown Dost corto Joo owtoey Jor foarggsn Joo root [io seroroer__Jo2 T Saverrsrptecsaved (accion) L -- EPp-- EecsT foo Jown | fem few | Jew me | Jew me | owe fw | `Table 3. PFOS Concentrations in Liver Samples from F1 Male and Female Sprague-Dawley Rats Pakeppetfroaantdleealsecttaronpiecricoodpoifestoef raaswedsattaablainsdhesdtubdyyrreegpuolrattsiogneanenrdabteydthdeur3inMgStthaenpdraersdenOtpesrtautdiiensgwill be EPnrvocierdounrmeesn.taPlaTpeecrhannodloeglyecatnodniScafcetyoSpeorfviirceeepsorlsta,boprraottoorcyolwsi,l abnedremteatihneoddswgientehreastteuddybfyoltdheerf3oMr at least a period of time as established by reauation and by the 3M Standard Operating Procedures. ---- STATISTICAL METHODS cFaolrcudlaattaedgeunseirnagteMidcbroystofhte E3xMcEen,viaronnmreenlattailveLsabtoarnadtaorrdyd,evmieaatinosnaswnedrsetacnadlacrudladteevdiamtainounasllwye.re aSntaalnydsaesr.d dReevliaattiivoensitsaandmaerdasdeuvriaetmioennoptfreasnealnyttsicaalmepraescuisrieono;fhthse miaigsniutsueddetoofgtahuegsettahnedaprrdecdiesviiaotnioonf.the nex? - oy? (n=) iMnediavnidsuaalreenctaitliceust.aStteadnbdyaraddddienvgiaitnidoinvsidwuaelreentciatliceuslaanteddvuisdinigngthteheeqrueastuolnta:nt sum by the number of sRuabsteqhuesnatnmdueldtidcaetaonnosf twheerqeuoctieanttbyt1y00 his ft sandarcU o b on tn Pago thts PCotAaSsMsaiummbePre2o7m5a5c3a5n3esutonss LaboratoryRoScMueEsmtiNoruRmeperorrtt(sNLLoRNaT)Uab2c0oo0s8r DATA QUALITY OBJECTIVES AND DATA INTEGRITY tNhoecicartcau.mTshteafnocleosweixnigsdtaetdaduqruianlgttyhoebpjrecetsievnetss(tDudQyOtsh)atwewroeuflodlhlaovweeadfdfuercitngedththeesqtuuadlyf:yor integrityof Ntohecdiartac.umTshteanfcoelsoewxiinsgtdeadtdaurqiunagltthyeopbrjeecsteinvetsst(uDdQyOtSh)atwweorueldfholaovweeadfdfuercitnegdtthheesqwuayl:i orintegrityof + uLsiing n1xewTeiahgehrtcionrtgrelyati--oncoefficient(R?) ofthestandardcurvewasequ toorgreaterthan 0.58 Limits of detecton--PFOS in serum=1.75 ppb Limts ofquantiaton--Equal to thelowestacceptablestandardinthe cabrationcurve: + Duplcate requencylacceptable precision--<30% Spike frequencylacoeptable recoveries--70% to 130% + Useofconfirmatorymethods--noconfimatorymethodswereused Demonstraion `mass spectralodafugsphetceirfiicointyc--hsapreaccftiecrtzyawtaonsdemonstratedbychromatographicretention tmeand EE STATEMENT OF CONCLUSION , `Statement of Conclusion: Ulnivdeersratnhdesceornadiotfioalnlsogfrtouhpesporfersaetnstesxtpudoiseesd, ptoottahesscihuemmipcearlfdluuroinrgoothcetiannveisoufpoownratatosneoofbstheervsetuddiienst.he m ---- o-- eS-------------------------- REFERENCES None 000018 Paget5ct16 PCoAtSaNsuhmbePret2u7o5r0o3ca5n3esutonsts Laboratory RaWcMuEemsvNruuRmnabmpeerna(t4aL.aT0bo2ara0tdo0rd8y --_-- ATTACHMENTS AttaA:Rcepohrtsmigneaturnepatge + DAatwtlaechymreatnstaB:TdabmleiofrnePsiFuOssSodfatainyaelbyysrigsaovefaigvdee.rsamplesfromFOmaleandferale Sprague. ADtatvaecyhrmaetnsCat:TdabmleiofnrePsiFulOssSodfatlalnayelbyysrigsaoevfasgdeer.asamplesfromFOmale andferale SpragueAtDatwaiceyhrmaesnaDt:TdabmleiofnrePsiFutOsSsocftaanyaelbyysrisgoaefviadvgee.rsamples romF1maleandfemale SpragueAttachmenEt: In-feproantdsotudcyprootolcol Attachment: Prepaandarnalaytitcalometrhoyds AtiaG:cDohsemAnealnystes 000019 Paget60t1s CPoe msms IET ATTACHMENAT REPORT SIGNATURE PAGE 1 1 far J KristeJn. HaPn h.Ds .,Ste udyDn irec, tor W.. alr Marvin T. Case, D.V.M., Ph.D., Study Sponsor L/lj -- Back,Labora Director aocsoyRecon NomiTs T0 C2008 lo)z7/99 Date. 200 fell tooy Date (ol22/57 Tate 000020 Page 7at18 i mses ATTACHMENBT EmE=eEmr eEaela====n] Cl LE kis; Le PF e sz z=P la rn = rom =EEEE EFBE. : Z EZEE EEEE{|E= EEEEEE EiE| = Ll. EEE Ea E EaH . To SE ULTE Tr Ees Se Propranid Ceontfiadenrtiyal 000021 PSeaesmePnesos avy ReEcumtoNinRreosre(tL.C5aTo0su0oor8os ATTACHMENTB (CONTINUED) FT Now | Woon Cuca AE w oo)E Swed Rena Dewanon aamorouay | esr Ie e153 es) eis[Ce] smo) |prsest prTeosa assses woss asose re po ros sz ps sis sso ne on wsnaan Tsoour TTenEse =by ssan sroosts Jutso n stom sor warms oTmommoamyr || sSsef TToosre oron =mn wasre 11a0a2t2e mpoenn =we soar I) wos ao wwoiw saEaQn EZ er 3us aamn 10a tors mun se m 0s nz Szrorgen |aWaR s0ome o=rs on = 1 Coamomty | nwe ww w=" "I= w= =w= au=n frTeoesm Ereast ane sisoonu 1to0t20r2 zoemeartaea woor ass ass sess no + Samo 8001F vatewexh fm clans. Tabl4e. AannadlCyosnecsenoftrLaitvieornSaofmpPloetsasfsrioummFP0erSfplruaogruoeoc-tDaanwelseuylfRoantasteinitnhSeeSrtuumdyaonfdtLheiPvroeefsence `Sprague-Dawley Rats Exposed to PFOS via Gavage (continued) 000022 Prostar and Coc PoCtAsSsMsairmPeeet2u7r5o5c3a8n3esutons LaboraReWquMeEsvtroNruFmmeeenprtuaiLlhRLoaNbHToUro2aot0or0r?8y ATTACHMENT C EElem ol le le w| Err Noo Cupcruossnp AToowraor Concenmanon PFOS [= Goi vancagars | wosurkz | avo pr Marc Bak | R(a=t)ver wot MankBank | REatnbeaiver ao Comm [seoortrruuss0 || 6702 aFna somes | 100 I somo | 5 Comaratay | ss01FM 5 ox pase cent 51s sz casts oos2 arm [527 aosz7 assem | 460 oo Omonorey | seen | 552 ae Oomomy) p[rscr | s0uo 7a31 asm [os 67 ssroent EM 7 COammeongi)e [[ssoooermm || seizruesso 6i7 wos [ame ar osrsmn || ssusn BsYs Emre | sstoEm a Ta mmm) | sens sass 9 wwarmm [ois [eso 20 eso worm | sro a5 + Same80017va ctinchdd thcals Mo(wiPgoF)OS as SRteaomo Ee =) wou TM ss oozes o2te o"wnr 2u6s `Table 5. AannadlCyosnecseonftrLaitvieronSoafmpPloetsasfsrioummFP1erSfplruaogruoeoc-tDaanwelseuylfRoantasteinitnhSeeSrtuumdyanofdtLhievePrroefsence Sprague-Dawley Rats Exposed to PFOS via Gavage ProsritayandCotter 000023 PoCtAaSsNsoummbPeeru2c7r5o6c3i8a3nessonsts WMEmionRmeepnoarltL.abToroautodr2y LabortryRcuestNumber(LRNHUZ006 ATTACHMENT D lS e [mm 138 f Jae fae | Dose Grow J Ee Mar gare Mat Ba ac-- 10pm Omak Trane uzmm) Oamonger 08mom) 0 pv Rast ver@ank2) | wot awoirais sassooimFaassD ssomaiso aaancorre 0u1z27 aasaceeirr oazsn aous sr oozes som 0006 ssttoomm ocuosrtm sstoonm oan ome aaer aor anezr ssoanaer ssaaamn os8 sstaesam ses 1o enue 104 anor 0s saenosssrr 110s4 oar 2 ssose 101 ssrirranm esiirrme pr pan [oy 05" ogre oassor 6 oos0 oou 248 000805 or os ososze Table 6. AS2npnardlaCygosuneecsDeonaftwrlSaeetryiaoRSnoaaftmpsPEloextspaosfssrieoumdmtFoPOrSPfpFirOoaSrgouvoei-cDtGaaawnvleaesgyueRfaotnsaienitnhSeeSrtuumdyoanfadthre Pcrehse2nc4e Proper and Content PCoRsSNsaummbaPrao27c5o5s3a6n3esukras absty RoScMueEsntoNnRmteepnoaLlr0tL9-aTUbo2oo0rt0as8 Dose Grou EE TEE EE fn pr J 16 mopiey ior 2 mm) oassazsirF m"2 soar a 2a se ee ws zo a siaomw 1a ezzm 15s s0n1 S2moroter w= = @stmom) Jwom I"o " "w= "= ne waaunn om massmm m + Samia8001 va otcdsn hi calculi Tables. APnraelsyesnecse oafndSeCroanSceanmtprlaetsiofnroofmPFoOtSapsrsaiguumeP-eDrafwllueoyroRoacttsanienstuhlefoSntautdeyionfStehreum and Liver of Sprague-Dawley Rats Exposed to PFOS via Gavage (continued) PrpatndCtonadotyel 000025 pCoAtSaNsambePorr2h7o5o6c3a6n3esuforste Laboratory RoSuMeEsntvNruRmrebmpeeornt(aNRLoNaHbToUor2ac0to0o?6 ATTACHMENT E IN-LIFE PROTOCOL AND ANALYTICAL STUDY PROTOCOL Provetay and Content 000026 [nesT eanL cy Argus50R5esSehaerachhyLDarbiovrea,tBouriilesd,inigncA. Horsham, Pennsylvania 19044 -------------------------------------------------- STUDYTITLE: PURPOSE: TESTINGFACILITY: STUDY DIRECTOR: SPONSOR: } PROTOCOL 418-008 SPONSOR'S STUDY NUMBER: 6295.9 PCeormibnaitnaeld/POorstanla(taGlavRaegper)odFuecrttiiiotny,TDoxeivceiltoypSmteundtyalofaPnFdOS in Rats. The purposeofthis studyis disturbances resulting from to test for toxic effects/ PFOS treatment of Cr:CDBR VAF/Plus male and female rats before cohabitation tehvraoluuagthemsaItiCnHg,HagresmtoantiisoendanTrdiplaarcttiatteioGnu.idTehliisnestsutdayges A through Fofthe reproductive process and should detect egfefsetcattsioonn, tpahretuersittrioonu,slcaycctlaet,iotnubaanldtrmaantspeomrat,l ibmephlaavnitoatriionn, ftreemaatleed rmaatsl,eoanntdhefedmeavleelroaptsm,enatndofptehremiotffdseptreicntgioofn tohfe functional effects (e.g., effects on libido or epididymal sperm emxataumriantaitoino)ntshaotf mmaalyenroatt breepdreodtueccttievdeboyrghainstso.logBieccalause manifestations of effects induced during this period may be tdherloauygehd pirnotdhuectoiffosnproifngF,2ogbesneerrvaattiioonnsliwtielrls.be continued A9r0g5uSshReeesheyarDcrihveL,abBourialtdoirnigesA, Inc. Horsham, Pennsylvania 19044-1267 Telephone: (215) 443-8710 Telefax: (215) 443-8567 Raymond G. York, Ph.D., DABT Associate Director of Research 33MM TCoexnitecro,loBguyilSdeirnvgi2c2es0-2E-02 St. Paul, Minnesota 55144-1000 000027 " Protocol 418-008 Page2 STUDYMONITOR: TMealrevpihnoTn.e:Cas(e6,12D).V7.3M3.-,5P1h8.0D. Telefax: (612) 733-1773 SATLUTDEYRMNOANTIETOR: TAenlderpehwonMe.:Sea(6c1a2t), 5P7h5.-D3.161 Telefax: (612) 733-1773 REGULATORYCITATIONS: rISnettpuerdroyndauDtcietosinioagnlnfCoaorsnmfMeeodrdieicfniicnceaaltoinpornoHdaofur:cmtosUn..iSs.FateFidooeonrd;alaGuRneidgdieDslrtiuenrge,AoSdnemipdnetitesemtcbrtaeitorinoo2n2f,(t11o9x99i94c4i),t.yVtoo, 59, No. 183. 2U1.SC. FFRooPdaratn5d8.Drug Administration. Good Laboratory Practice Regulations; Final Rule, fJoarpSaanfeestey SMtinuidsitersyoofn HDeraulgtsh, aMnHd WWelOfradriena(n1c9e87N).umGoboedrLa21,bMoarracthor2y6P,ra1c6t9i7c.e Standard maEecuncrdeoapptteiaanoncneEocbnoycntoohmmepilEciuaCrnoocmpemeuawnintihEtcpyori(nn1oc9mi8pi9l)ce.sCooCfmogumnouconidiltldyaebcooifrsaaitnoonrOyoEnprC2aDc8tidJcueelc.yis1Oif9of8nic/9iraoelsnJootmuh.renal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17 REGULATORY COMPLIANCE: rTehgiuslsattiuodnyswcililtebdeacboonvdeu.cted in compliance with the Good Laboratory Practice (GLP) ADlilrecchtaonrgaensdotrhereSvpiosinosnosr,ofdtahtisedpraontdocmolaisnhtalalinbeeddwoicthumtehnetperdot,ocsoilg.ned by the Study PaTrnhodecweQiudlaulriientssypoAefcstsAurcrrgiautinsccaRleepsUhneaiastre(csQhAoLfUa)tbhowerialslttoaurudidyeisti,nthIanecc.cporortdoacnolc,e twhiethrtahwedSattaanadnadrdthOeperreapotritn,g 2aTchceauprfpailntiaecllayrbelrpeeofrlGteLcwtPislrtiehngecurlluaadtwieodanastsatwaeotrbeetmaefionnlteldsoiwdgeudnreiindngbttyhhetehcpeoenSrdtfuuocdrtymoaDfnitcrheeecotsfotrtuhdthyea.sttStuhhdeoyuralendpdortthat tSoiggneitfhiecrawntitdehvhiaotwiotnhsefdreovmiaGtiLoPn mreigguhltataifofencstotchceurq,uaelaitcyhowrilinltbeegrdietyscorfitbheedsitnuddeyt.ail, 7000028 Protocol 41P8a-g0e038 `STUDYSCHEDULE: See ATTACHMENT 1 to the protocol. TA ESTRTIANC D VEL HICE LE: Identification: TestArticle: Name: PFOS. LPhoyts/iBcaatlchDeNsucmrbipetri:on: ~~ 2L1i7g.ht-colored powder. Specific Gravity: ~06. Purity: 98.9%. Expiration Date: May, 2000. Information on the with the Sponsor. identity, composition, strength and purity of the test article is on file Vehicle: 0.5% Tween 80 in Deionized Water). RSeuvpeprliseerdaOnsdmloost iisdeMnteifmibcartaionneofPrTowceeesnse8d0DteoiobneidzoedcuWmaetnetre(dR.inO.the raw data. tNoeibteheprrtehseenStpionntshoervneohirctlheetShattudwyouDlidreicnttoerrfiseraewwairthe tohfearneysuplottsenotfitahliscosnttuadym.inTahnetrsefliokreel,y no analyses other than those mentioned in this protocol will be conducted. SafetyPrecautions: fGolromvuelsa,timoanskpr,epaaprpartoiporniaatnedeydeospargoteecatdimoinniasntrdataiounn.ifoTrhme/iMaabtecroiaatlaSraefteotybeDawtaomShdeuerting (MSDS) is attached to the protocol (ATTACHMENT 2). Storage: VBeuhlikcTleesCtoAmrtpiocnlee:nts: Prepared Prepared Vehicle: Formulations: Room temperature. RRoooomm tteemmppeerraattuurree.. Frozen (-20C). Altlieasn\tarGuilbciensskhii,pmeManntasgertootfhFeoTremsultaitnigoFnasc,ilaittytshehouplrdeviboueslay ddrciessed to the telephone number. P Y poTYEY attention and of Protocol 41P8a-g0e084 `Shipments should cartons should be include labeled information conceming storage conditions and appropriately. The recipient should be notified shipping in advance of shipment. [EQRMULATION: Ereqou fPreepanrac tioyn: Formulations (suspensions) will be prepared daily at the Testing Facility. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). AdjustfomrPeurnitty: `The test article will be considered 100% pure for the purpose of dosage calculations. Testing FacilityReserve Samples: `The Sponsor wil reserve a sample (1g) of each lot of the bulk test article used during the course of this study. The Testing Facilty will reserve a sample (5 mL) of each lot of the vehicle components used during the courseofthis study. Samples will be stored under the previously cited conditions. ANALYSES: Samples additional to those described below may be taken if deemed necessary during the courseof the study. Bulk TestArticleSampling: NInofoarnmaaltyisonesoonftthheesbtuabliklitteysotfatrthieclbeuwliklltbesetcaortnidcluectisedondufirlienwgitthhethceouSrpsoensoofrt.his study. AnalysesofPrepared Formulations: Statiity Stability data for prepared formulations bracketing the rangeofconcentrations and tchoendciotniodnusctofotfhtihsisstsutduydya.reSounspfeilneswiiotnhsthwiellSbpeonpsroerpaarnedd wdiallilnyoattbtehedeTteesrtimnigneFdacdiulrtiyn.g 000030 | Protocol 41P8a-g0e0s8 HomogeneityAnalyses: . Homogeneity of the test article in prepared suspensions will be verified during the tcoopu,rmsieoddfltehiasndstubdoyt.toAmosfytrhinegehiwgihlesbtecuosnecdenttorawtiitohndroanwtshaemfpirlset sda(y5 mofLpreeapcahr)atfiroon.m the Each sample (5 mL) will be divided into two aliquots, one of 2 mL and oneof 3 mL. OatntehealTieqsutoitn(g2 FmaLci)liwtiyllasbeasbhaicpkpeudp fsoarmapnlael.ysiBsa;ctkhuepostahemrplaleisquwoiltl (b3emsLt)orweildl ubnedreerttahineed pSrpeovnisoours.ly cited conditions and discarded at the Testing Facility upon the request of the ConcentrationAnalyses: Concentrationofthe prepared test article suspensions will be verified during the course of this study. A syringe will be usedtowithdraw samples (5 mL each) from each concentration during the first and sixth week of dosage administration. Each sample (5 (2 mL each) will be divided intotwo mL) will be shipped for analysis; aliquots, the other one of aliquot 2 mL and one (3 mL) will be of3 mL. retained One at the aliquot Testing Facilty previously cited as a backup sample. Backup samples will be stored under the. conditions and discarded at the Testing Facility upon the request of the Sponsor. `Shipping Instructions: `Samples to be analyzed will be shipped (frozen on dry ice) to: Kris J. Hansen, Ph.D. 3M Environmental Technology and Safety Services 9B8u3i5ldBiungsh2-A3v6e-n0u9e St. Paul, Minnesota 55133-3331 Telephone: (612) 778-6018 Telefax: (612) 778-6176 Both the recipient and the Study Monitor will be notified in advance of sample shipment. DISPOSITION: Prepared article wil formulations be returned will be discarded at the to the Study Monitor at Testing Facility. All the previously cited remaining address. bulk test 000031 Protocol 418-008 Pages JEST SYSTEM: SpeciaendsRe/asSont forrSea leci tionn: The Cr:CDBR VAF/Plus (Sprague-Dawley) rat was selected as the Test System because: 1) this strain of rat has been demonstrated to be sensitive to reproductive and developmental toxins and has been widely used throughout industry for reproductive and developmental toxicity evaluations; 2) historical data and experience exist at the Testing Facility; and 3) the test article is phamacologically active in the species and strain. Number: Initial population acclimated: 195 virgin male and 205 virgin female rats. Population selected for study: 175 male rats (35 per dosage group) and 175 female rats (35 per dosage group). Ten mated female rats wil be assigned to Caesarean-sectioning on day 10 of presumed gestation; the remaining female rats will be permitted to deliver litters. 250 F1 generation pups (25 per sex per dosage group) will be selected at weaning on day 21 postpartum for continued postnatal observation. BW ode y iganh dAgte: Male rats will be ordered to weigh from 300 g to 325 g each at receipt, at which time they will be weigh from expected to be 200 g to 225 g at least each at 60 days of age. receipt, at which Female rats will time they will be be ordered to expected to be at least 60 days of age. Actual body weights will be recorded the day after receipt and will be documented in the raw data. The weight ranges will be included in the final report. Sex: Both Fo and F1 generation male and female rats will be evaluated. male and female rats will be given the test article. Only Fo generation Source: Charles River Laboratories, Inc., Raleigh, North Carolina. The rats will be shipped Laboratories, Inc., to the in filtered cartons Testing Facility. by air freight andor truck from Charles River 000032 Protocol 4P18a.g0e0?8 Identification: FGoeneration: RCoa.t,s Ianrc.e, pNeor.maMnSePntTly20i1d0en1t)i.fieMdaulseinagndMofnemeallesreaftfs-paireercaisngsieganredtatgesm(pGoeryarByannudmabnedrsTaatg rbeecfeoirpet aadnmdingiisvternatuionniqoufethpeerfimrastndeonstaigdeeontfiftihceatitoenstnaurmtibcleer. s when assigned to the study E1/F2Generations: iPnutpesrmwislol fntothebelititnedr.ivAidtuawlelayniidnegnt,ifeiaecd hdurraitnsgelleaccttaetdiofno;r aclol nptairnaumeedtoebrssewrivlal tbieonevwialllubaeted identified with a Monel self-piercing ear tag. ANIMAL HUSBANDRY: All cage sizes and housing conditions are in compliance with the Guide for the Care and Use of Laboratory Animals. Housing: [G Eo eneRatr s/Fa 1Gent erai tiono Littn ers: Fo generation rats will be individually housed in stainless steel wire-botiomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of rats will be housed in the male rat's cage. Beginning no later than day 20 of presumed gestation, Fo generation female rats assigned to natural delivery will be individually housed in nesting boxes. Each dam and delivered litter will be housed in a common nesting box during the postpartum period. EG 1 eneRatr s/Fa 2 Gent erai tiono Littn ers: After weaning, the F1 generation rats will be individually housed before cohabitation, housed in pairs (one male rat per female rat) during cohabitation, and individually housed after cohabitation. The same type of caging will be used as described for the Fo generation rats. Beginning no later than day 20 of presumed gestation, F1 generation female rats will be individually housed in nesting boxes. Each dam and delivered litter will be housed in a common nesting box during the postpartum period. 000033 : Protocol 418-008 Pages Nesting Material: Bedding material (bed-0'cobs) will be supplied to female rats assigned to natural delivery. ABneadldyisnegswiflolrbpeoscshibalnegecodnatasmoifntaetnioans anreececsosnadruyctteodkeaennputahlelyaannimdadlsocdurmyeanntdecdleinant.he raw data. RT ooe mAim r,peraat ndu Humr idie ty: The animal room is independently supplied with at least ten changes per hour of 100% freshairthat has been passed through 99.97% HEPA filters (Airo Clean room). Room temperature will be maintained at 64F (18C) to 79F (26C) and monitored constantly. Room humidity wil also be monitored constantly and maintained at 30% to 70%. Light: An automatically controlled 12-hour light:12-hour dark fluorescent light cycle wil be maintained. Each dark period will begin at 1900 hours EST. Diet: Rats will be given Certified Rodent Diet #5002 (PMI Nutrition Intemational) available ad libitum from individual feeders. Water: Water will be available ad libitum from individual bottles attached to the cages or from an automatic watering access system. All water wil be from a local source and passed through a reverse osmosis membrane before use. Chlorine will be added to the processed water asa bacteriostat; processed water is expected to contain no more than 1.2 ppm chlorine at the timeof analysis. Water is analyzed monthly for possible bacterial contamination and twice annually for possible chemical contamination. Contaminants: Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the certified diet, the drinking water of the nesting material at levels that would interfere with the resultsofthis study. Therefore, no analyses other than those routinely performed conducted. by the feed supplier or those mentioned in this protocol will be 000034 | Protocol 41P8a-g0e0s8 RANDOMAINDZCOAHATBIITATOIONN: EGoeneration: Upon arrival, rats will be assigned to individual housing on the basis of computerSgteundeyraotnedthreabnadsoism oufnipths.ysiAcfatleraapcpcelaimraatnicoen,amnadlbeoadnydwfeeimghatlse rraetcsorwidleldbedusreilnegcted for acclimation. The rats will be assigned to dosage groups based on computer-generated (weight-ordered) randomization procedures. Wciothahbiintaetaicohn,doonseagmealgeroruapt,pceornfseemcaultievreato.rdTehrewiclolhbaebiutasteidontopearsisoidgnwirllatcsontosist ofa vmaagxiniamlucmonotfe1nt4sdaanyds./orFeamcaolpeulraattosrwyipthlusgpoebrsmeartvoezdoainosbistuerwivlel dbeincaonssmiedaerreodf the to be at day 0 of presumed gestation and assigned to individual housing. Female rats not mated within the first 7 days of cohabitation will be assigned altemate male rats that have mated (same dosage group) and will remain in cohabitation for a maximum of seven additional days. The first ten female rats per dosage group with a confined date of mating will be. assigned to Caesarean-sectioning on day 10 of presumed gestation. The remaining female rats will be permitted to naturally deliver litters. A table of random units will be used to assign five rats per group to a pharmacokinetic sample collection at scheduled sacrifice after completion of the cohabitation period (male rats siring litters with dams allowed to naturally deliver a litter) or on day 21 postpartum (female rats allowed to naturally deliver liters). EA/F2Generation Pups: Day 1 of lactation (postpartum) is defined as the day of birth and is also the first day on which all pups in a litter are individually weighed (pup body weights will be recorded after all pups in a litter are defivered and groomed by the dam). On day 4 postpartum, a table of random units will be used to select pups to be culled, and litters wil be reduced to eight pups each. Whenever possible, the same number of male and female pups perlitterwill be continued on study. Atweaning of the F1 generation pups on day 21 postpartum, a table of random units will be used to select 25 male and 25 female pups per group, resulting in a total of 250 F1 generation rats (125 per sex) chosen for continued evaluation. At least one male pup and one female pup per liter, when possible, will be selected. 00035 + Protocol P41a8g-e00180 ADMINISTRATION: RR outee andasfoo rChon ice: ~Thdieetaorryarlo(ugtea,vatghee)erxoaucttedwoassagseelceacntebdefoarccuusreatbeelcyauadsmei:ni1s)teirnecdo;mapnadri2s)ointiwsitohnetohef the possible routesof human exposure. Meat ndFh reqo uend cy: aDpopsraogxeismawtielllybethaedsjuasmteedtfiomretehaecmhodsaty.recently recorded body weight and given at FGo eneraMatlieRoatns: (Mamlaexiramtusmwi1ll4 bdeaygsi)veanndthecotnetsitnuaritnigcltehornocugehdtaihleydbaeygibnenfionrge 2s8acrdiafyicse.beMfoarlee croahtasbwiitlal tbieon sacrificed after completion of the cohabitation period. EoGeneration FemaleRats: Female rats cohabitation will be given the (maximum of 14 test article days) and once daily continuing beginning 28 days before through day 9 of presumed agsesstiagtnieodn t(orantastaursasligdneelidvteoryCtaheastardeoann-ostedcetliiovneirnag)l,ittdera)yor24daoyf p2r0epsousmtepdargteusmta(triaotsn t(hraatts delivera liter). EG1eneration: eFx1pgoesneedrattoitohneptuepstsawritlilcnleotdubreindgirmecattleyrgniavlegnetshteattieosnt (airntiuctlee,robuetxpmoasyurbee) poorsvsiiablmyaternal `milk during the lactation period. RatfoirDo osan geSa elel ctie on: Dosages will with the test be selected article. by the Sponsor on the basis of previous studies conducted 000036 Ja : Protocol P41a8g-e00118 DoCsaogen Levcele s, ntratanidVooln umess: EET [osloI ow ToTousossomuomven] [wlsloo[oo Fv[el we Tow | 5[oumomconmmnvon] Ts [ormcorommmmnven] LvTeel sa Toe TsTeemamconmomven] Thetotacewtb comadared 100%pureforthepurposeofdosagecaatons. TM ESTSE .ANAA LYSS ESAU ND REME -FoN GENET RATIS ON: Via- MbaleiandlFemialteRayts: All Periods: At least twice daily. Clinical ObservationsandlorGeneral Appearance -MaleandFemale Rats: Acclimation Period: Atleast once. Dosage Period: Twice daily. Prior approximately one to dosage administration hour postdosage. and once. Matemal Behavior: aDbanyosrm1,al4,b7e,ha1v4iaorndwil2l1bpeosrtepcaorrtduemd.daAilnyy. observed aCplipnriocparlioabtseebrvyatthieonSstumdayyDbireecrteocroarndde/domroSrteudfyreMqouneinttolry.than cited above, if deemed BW odyeig-h Malt eRas ts: Acclimation Period: Dosage Period: Sacrifice: At least once. Weekly. Terminal weight. 000037 ! Protocol 4P1a8g-e00182 Body Weights -F Rats: Acclimation Period: Dosage Period: Atleast once. Weekly to cohabitation. Daily during presumed gestation and on Days 1, 4, 7 and 14 postpartum (rats assigned to natural delivery). Sacrifice: Terminal weight. FCeedonsumVapluets-MialoeRants (recorded and tabulated): Dosage Period: Weekly. EC eedonsuVmalupes-tFei malo eRan ts (recorded and tabulated): Dosage Period: Weekly to cohabitation. Daily during presumed gestation. Days 1, 4, 7 and 14 postpartum (rats assigned to natural delivery). Feed consumption not tabulated after day 14 postpartum, when it is expected that pups will begin to consume maternal feed. FC eed onsu Valum es -Mp alet andFi emao le Rn ats: Feed consumption values may be recorded more frequently than cited above if it is necessatroy replenish the feed. During cohabitation, when two rats occupy the same cage with one feed jar, replenishmentofthe feed jars will be documented. Individual values will not be recorded or tabulated. EstrousCyclingandMating: Atable estrous of random cycling by units will be examination used to select 15 female rats per group for evaluation of vaginal cytology for 14 days before the startof the of cohabitation period. During cohabitation, all female rats wil be evaluated daily until spermatozoa are observed in situ. in a smear of the vaginal contents and/or a copulatory plug is observed DuraoftGesitaotionn: The duration of gestation is calculated from day 0 of presumed 000038 gestation to the day the first pup is observed. : Protocol 4P1a8g-e00183 EertilityParameters: Fertiity Index (percentage of matings that result in pregnancies). Gestation Index (percentage of pregnancies that result in birth of live litters). Number of offspring per litter (ive and dead pups). Number of implantation sites. General condition of dam and liter during the postpartum period. Viability Indices (percentage of pups bom that survive 4 and 7 days). Lactation Index (percentage of pups bom that survive 21 days). Caesarean-Sectioning Observations: Rats will be Caesarean-sectioned on day 10 of presumed gestation. Placentae that appear abnormal (size, color or shape) will be noted in the raw data. The rats wil be `examined for number and distribution of: Corpora Lutea. Implantation Sites. Viable and Nonviable Embryos. (A viable embryo is oval or crescent shaped, pink, fim and enclosed in an `amniotic sac filed with clear fluid. A nonviable embryo is amorphous, small, pale pink to tan or deep red to black, soft and enclosed in an amniotic sac filed with clear, cloudy, or opaque fluid.) NaturalDelivery: Female rats will be evaluated for: Clinical Observations During Parturition. Duration of Gestation (day 0of presumed gestation to the time the first pup is observed). Length of Parturition (timeofdelivery first pup divided by N-1 pups in each of last litter). pup minus the timeofdeliveryof the Litter Size (defined as all pups delivered). . 000039 : Protocol 418-008 Page 14 Pup Viability at Birth. MS ETHA ODOC F RIF -FoI GENEC RATIE ON: Rats will be sacrificed by carbon dioxide asphyxiation. Embryos will be discarded after examination. NEC-RFoOGENPERASTIOYN: Gross lesions will be `evaluation (a table of retained random in neutral buffered 10% formalin for possible units will be used to select one control group future at of each `sceoxntfrorlotmiwsshuiecshfaolrl atinsysupeossseixbalemihniestdopaattnhoelcorgoipcsayl weivlallbueatrieotnasinoefd,grionsosrldeesriotnosp).rovUindleess specifically cited below, allother tissues will be discarded. MaP leah ndFa emalr eRam tsAsa signc edtookine Sampt leCoi llectc ion: At scheduled sacrifice after completion of the cohabitation period (male rats siring liters with dams allowed to allowed to naturally deliver a naturally deliver a litter), five litter) and on day rats per group will 21 be postpartum (female assigned to a rats bpehlaorwm,acbolkoiondetsiacmpslaemspl(eapcporlloexcitmiaotn.elyIn4amddLitpieonr rtaot)thweillapbperocporlilaetceteedvfalruoamttihoensindfeesrciorribed vena cava into (approximately serum 2 mL) separator tubes and centrifuged. wil be immediately frozen on dry The resulting serum ice and maintained frozen a(-n7d0aCs)aumntpilleshsiecptmieonnt(tloattehraelSlpoboen)sowirlfobreafnraolzyseins.anTdhreetlaiivenredwilalt b-e70exCciusnetdi,l swheiipgmheendt, to the Sponsor for analysis. Abfetesrhicpopmepdle(tfiroonzeonf osnamdprlyeicceo)llteoctKiroins,Js.eHraunmseann,d Pihv.eDr.,seacttitohne (plraetevriaoluslloybec)itseadmapdldersewsisl for analysis. Both sample shipment. the recipient and the Study Monitor will be notified in advance of ScheduledSacrificeofMaleRats: Anfetcerrocposmypolfetthioenothfotrahceicc,ohaabbdiotmaitnioanl paenrdiopde,lvmiaclveisrcatesrawiwlilllbebesapcerriffiocremdeda.ndTahegrfooslslowing organs testes, ewpilildbideyemxicdiesse,dparonsdtawteeiagnheddseamnidnarletvaeisniecdlfeosr(pwoesisgihbelde histologic evaluation: with and without fluid). nTehuetrtaelstbeusffweirlledbe10fi%xefdorinmaBloiuninf'ors psooslsuitibolne fhoirs4to8pattoh9ol6oghiocuarlseavanlduatthieonn.reTtahienerdeminaining organs will be retained in neutral buffered 10% formalin. 000040 Protocol 4P1a8g.e00185 ScheduledSacrifice-FemaleRatsAssignedto Caesarean-Sectioning: On day 10 of presumed gestation, female rats will be sacrificed, Caesarean-sectioned, aUtnedraiofgraopspsarneenctrloypnsoyonfprtehgenathnotrarcaitcs,wialbl dboemisntaailnaedndwiptehlv1i0c %viascmemraonwiillubmesuplefrifdoertmoed. wciolnlfbiermretthaeianebdseinncneeuotfrailmpbluafnftearteidon10si%tefsor.malUtienrifoorfpnoosnsipbrleegnfuatnutreraetvsalaunadtiaolnl.ovaries. Scheduled Sacrifice-FemaleRatsAssignedto NaturalDelivery: Rats that do not deliver a litter will be sacrificed on day 25 of presumed gestation and examined for gross lesions. Uteri will be stained with 10% ammonium sulfide to confirm the absence of implantation sites. After completion of the 21-day postpartum period, female rats will be sacrificed, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The number and distribution of implantation sites will be recorded. DS amu swirthv Noivin Pupgs: Dams with no surviving pups will be sacrificed after the last pup is found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Postpartum data for these dams will be excluded from summary tables. Rats FoundDeadorMoribund: Rats that die or are sacrificed because of moribund condition or abortion will be `meaxdaem.ineTdhfeorrtathsewiclal ubseeeoxfadmeiantehdofrormogrriobsusnldescioonndsi.tiToenstoens,theepdidaiydtyhmeidoebss,erprvoasttiaotneisand seminal vesiclesofmale rats will be recorded (seminal vesicles weighed excised and individual with and without fluid). organ weights will be The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. `The remaining organs will be retained in neutralbuffered 10% formalin. Pregnancy sdtealtiuvseraenddpuutpesriwnielcboenteexnatmsionfefdemtoalteheraetxstewinltl pboessriebcloer.dedO.varAibeosrtwieldl bfeetruesteasinaendd/ionr neutral buffered 10% ammonium 10% formalin. Uteri of apparently sulfide to confirm the absence of nonpregnant rats wil implantation sites. be stained with 0004! Protocol 4P1a8g-e00186 TM ESTSE ,ANAA LYSS ESANUD REME -F1N GENET RATS ION: Viability: Preweaning Period: Litters will be observed for dead pups at least twice daily. The pups in each litter will be counted once daily. Postweaning Period: Twice daily. Clinical Observations and/or GeneralAppearance: Preweaning Period: Once daily. Postweaning Period: Once weekly. Maternal Behavior: Dabanyosrm1,a4l,b7e,ha1v4iaorndwil2l1bpeosrtepcaorrtduemd.daAlny.y observed CalpipnriocparlioabtseebrvyatthieonSstumdayyDbireecrteocroradnedd/omrotrhee fSrteuqduyenMtolnyittohra.n cited above, if deemed BodyWeights: Preweaning Period: Postweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. Weekly. Presumed Gestation Period: Lactation Period: Sacrifice: Days 0, 7, 10, 14, 17 and 20 (female rats only). Days 1,4, 7, 10 and 14 (female rats only). Terminal weight, EeedConsumptionValues (recorded and tabulated): Preweaning Period: Not recorded. Postweaning Period: Presumed Gestation Period: Lactation Period: Weekly except during cohabitation. Days 0,7, 10, 14, 17 and 20 (female rats only). Days 1.4.7, 10 and 14 (femaleratggry 1, 2 Protocol 4P1a8g.e00187 Freepeldenciosnhstuhmepfteieodn. vDaulruiensgmcaohyabbietarteicoon,rdwehdemnortweofrreatqsueonctclyupifyittihsenseacmesescaraygetowi_th one food jar, values will be documented when feed jars are filled. These intervals wil not be tabuiated. PreweaningDevelopmentalObservations: Tcohnetinnuumebseurntiolftphuepdsamyeethteincgritthereiocnriitseraitotnaiisnerdecboyradleldpuopnseiancthhdealyittoerf. testing. Testing Surface Righting Reflex (abilty to right in 5 seconds): From day 1 postpartum. Pinna Unfolding: From day 2 postpartum. Eye Opening: From day 12 postpartum. Acoustic Startle Response: From day 13 postpartum. Air Righting Reflex: From day 14 postpartum. Pupil constriction is evaluated once, on day 21 postpartum. PostweaningDevelopmentalObservations: `Sexual Maturation: Fpoesmtaplaertruamt.s wiMlallbeereavtsalwuilaltbede feovratlhueataegdefoorf tvhaegiangale poaftpernecpyu,tibaelgsienpnairnagtioonn,dbaeyg2in8ning on day 39 postpartum. Beginning at 24 where possible, 1 will day postpartum, one male be evaluated in a passive rat and one female rat from each litter, avoidancetestfor leaming, short-term retention and long-term retention. `PTlheexipgalsassiveliadsv.oiOdnaencceoampppaarrtamteunstciosnfsiitsttesd woiftah tawbor-icgohtmpliagrhttmaenndtPclheaxmigblearswiftlohorh.inTgheed ocutrhreerncto(m1pmaAr)tmceannt bisefdiettleidvewrietdh. aTgrhied tflwooorctoompwahircthmeantbrsieafr(e1sseepca)raptueldseobyfmaislldideilnegctric door. On each test trial, the rat is placed into the "bright" compartment, the sliding door is opened and the light is fumed on. The rat is allowed to explore the apparatus until it `enters the "dark" compartment. The sliding door is then immediately closed, the light is tumed off and the brief pulse of current is delivered to the grid floor. The rat is then removed from the apparatus and placed into a holding cage for 30 seconds before the startofthe next tial. Trials are repeated until the rat remains in the "bright" 000043 Protocol 4P1a8g-e00188 compartment for 60 seconds on two consecutive trials (the criterion for learning) or until ~ m15axtriiamlus mha6v0e-sbeeceonndcoimnptleervtaeld.is The latency recorded for to enter the each trial. dark compartment or the Each rat is tested twice. The test sessions are separated by a one-week interval, and the criterion is the same for both days of testing. Dosage groups are compared for the following dependent measures: The number of trials to to compare groups for the criterion in overall leaming tpheerffiorsrtmsaenscsei.on~this measure will be used `cTohmeplaarttemnecynt(inonsetrcioalnd1si)nttoheenftiresrt ttehset"sdeasrski"ocno-tmhpiasrtmmeeansturferowmilthbee"ubrsiegdhtt"o compare groups environment. for activity levels and exploratory tendencies in a novel The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on compare groups tfroiralsh2oirnt-ttheerfmirrsettteensttiosne.ssion~this measure wil be used to `The numberof trials be used to compare to the criterion in the groups for long-term rseetcenotnidont.est session~this measure will The latency (in seconds) compartment on tral 1 in to enter the the second "dark" compartment from the session--this value is another "bright" indication of fong-term retention. Watermaze Testing: eBaegcihnnliittnegr waitllabppereovxailmuaatteeldy 7in0adwaaytser-pfoisltlpeadrMt-umm,azoneefomraolveerrtatcaoonrddionnaetifoen,maslweimramtifnrgom ability, learning and memory. Each rat is tested is filled with water in to a a wdaetpetrhtoigfhtap1p6r-ogxaiumgaetesltyainnilneessinsctheeels,moadnidfitehde M-maze. The maze water is monitored for temperature (range of 21C + 1C). sOtnemeafcahrttheesstt tfrriaol,mtthheertawtowialrmbse)palnadcerdeiqnutioretdhetostsawrtiimngtopoosniteioonf (thbeasteowofgtohaelsMo-fmtahzee eMn-tmearzbeo,thinaorrmdseroftothbeemraezmeovbeedfofrreombetihnegwraetmero.veOdnfrtohme ftihrsetwtraitael,r.theThraetiinsitriaelquairrmed to cfahilotsoenmaoknetraialco1rriescdtesgoiaglnactheodictehewiitnhcionrr6e0ctsgeocaolnddusriinngantyhegirveemnaitnriianlgartreiaglsu.idReadtstotthhaet csoerpraercattgeoealacahndtriaalr.e then Each removed from rat is required the water. to reach a A 15-second intertrial interval criterion of five consecutive will eroriess trials to terminate the test session. The maximum Eh in any test 4 Protocol 4P1a8g-e01098 mseasxsiiomnumis 6105.-sLeactoenndciynt(emrevaalsuisrerdecionrdseedcofnodrse)actohctrhioalo,saestihsethcoernreucmtbgeoraloforertrhoers (incorrect tums in the maze) during each trial. Each rat is tested twice. The test sessions are separated by a one-week interval, and the correct goal and the criterion are the same for both test sessions. Dosage groups are compared for the following dependent measures: The number of trials to criterion on used to compare groups for overall the first day of testing~this leaming performance. measure will be The first daavyeorfatgeestniunmgb--ethriosfmeerarosrusr(einwiclolrraelcstotbuemsusiendthteo maze) for each trial compare groups for on the overall learning performance. The latency testing~this (in seconds) measure will to reach be used the correct goal on to compare groups trial 2 of the first day of for short-term retention. "bTeheusneudmbtoercoomfptrairales tgorocuriptserfioorn loonngt-hteersmecroetnedntdiaonyo. ftesting--this measure will The average number of errors for each trial on the second day of testing--this measure will aiso be used to compare groups for long-term retention. The latency testing---this (in seconds) to reach the correct goal on trial is another indicator of long-term retention. 1 of day 2 of ReproductiveCapacity: Atapproximately 90 days of age, the F1 generation rats within each dosage group will breanadsosmigunneidtstoofcrohaanbditoamtiuonni,t otanbelems,alweitrhatthpeerexfcelmuaslieonraot,f sbiabsliendg omantcinogmsp.uteTrh-egenerated cohabitation period will consist of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Female rats that do not mate within the first 7 days of cohabitation will be assigned alternate male rats from the same dosage group that have mated. Female rats will be allowed to naturally deliver and maintain litters through a 21-day postpartum period. MatingPerformance: As cited above for Fo generation rats. 000045 : Protocol 4P1a8g-e00280 DuraoftGesitaotionn: As cited above for Fo generation rats. Eertility Parameters: As cited above for Fo generation rats. ; Data: Viability, clinical observations and body weights for F2 generation pups will be recorded as cited above for F1 generation litters. METHOD OF SACRIFICE -F1 GENERATION RATS/F2 GENERATION PUPS: As previously cited for Fo generation rats. NEC-FR 1 GEO NERAPTIOS NRAY TS: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation (a table of random units will be used to select one control group rat of each sex from which all tissues examined at necropsy will be retained, in order to provide control tissues for any possible histopathological evaluationsof gross lesions). Unless specifically cited below, all other tissues will be discarded. `Scheduled Sacrifice - F1 GenerationMale Rats: Rats will be sacrificed after completionof the 14-day cohabitation period. A gross necropsyofthe thoracic, abdominal and pelvic visceral will be performed. Testes and epididymides of male rats wil be excised and individual organ weights will be recorded. `The epididymides will be retained in neutral buffered 10% formalin. The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. ScheduledSacrifice-F1GenerationFemale Rats: Female rats will be sacrificed after completionof the 21-day postpartum period. The number and distribution of implantation sites wil be recorded. Rats that do not deliver a liter will be sacrificed on day 25 of presumed gestation and uteri wil be stained with 10% ammonium sulfide to confirm the absence of implantation sites. A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Female rats without a confirmed mating date that do not delivera litter will be sacrificed on an estimated day 25of presumed gestation. 000046 : Protocol 418-008 Page 21 EG 14 ene Ratsr Foua ndDt eadoi rMoo ribn und: Rats that die or are sacrificed because of moribund condition or abortion will be examiried for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Testes, epididymides, prostate and `seminal vesicles of male rats will be excised and individual organ weights will be recorded (seminal vesicles weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. The remaining organs will be retained in neutral buffered 10% formalin. Pregnancy status and uterine contents of female rats will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible. Ovaries will be retained in neutral buffered 10% formalin. Uteriof apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites. [G 1 ene Damr swia thNot Suri vivio ngPun ps: Dams with no surviving pups will be sacrificed after the last pup is found dead, missing voirspcrereaswuimlledbecapnenrifboarlmiezde.d. PAosgtrpoasrstunmecdraotpasyfoorftthheesethdoaramcsicw,ilalbbdeomeixncalludaenddfpreolmvic summary tables. [E1/F2GenerationPupsFoundDeadonDay1Postpartum: sPtuaptsustahtatbidriteh.beTfhoereleuxnagmsiwnialltiboenroefmtohveeldittaenr dforimpmueprvsieabdiliintywwaitlerb.e Peuvaplsuwaittehd lfuonrgvsittalhat asinndk twoillhabveeiddeinetdifsiheodrtalsy satfitlelrbobrinr;thp.upPsupwisthwiltuhnggrsotshsatleflsoiaotnswiwlilllbebeidpernteisfeiredveads ilnivBeobuoirnn',s solution for possible future evaluations, it will Le noted evaluation. Should postmortem in the necropsy data. autolysis preclude these neration PupsFound Deaod r Morib: 2 ostpartum: Pups found lesions and dead or sacrificed due to moribund condition will for the cause of the moribund condition or death. be examined for gross Pups with gross lesions efvoaulnudatoinond;agyrso2sstole4sipoonsstopfarptuupms wfiollunbde opnredsaeyrsve5dtion 2B1oupions'tspsaorltuutimonwilflorbpeopssriebsleervfeudturine `neevuatlruaaltbiounfsfeirt ewdil1b0e%nfootremdaliinn.theShnoeuclrdoppsoystdamtoar.tem autolysis preclude these 000047 Protocol 418-008 Page 22 FAIE2GenerationPupsNotSelectedforContinuedObservation: F1 and F2 generation pups culled on day 4 postpartum will be sacrificed and examined for gross lesions; pups with gross lesions will be preserved in Bouin's solution. Necropsy will include a single cross-sectionof the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. All F1 generation pups culled on day 21 postpartum will be sacrificed and examined for gross lesions; gross lesions will be preserved in neutral buffered 10% formalin. Necropsy will include a single cross-sectionofthe head at the level of the frontal-parietal suture and examinationofthe cross-sectioned brain for apparent hydrocephaly. Scheduled Sacrifice-F2GeneratPiuposn: NOencdroapys2y1wiplolsitnpcalrutduema, pups will be sacrificed and single cross-sectionofthe examinedforgross head at the level of lesions. the frontal-parietal suture and examinationof the cross-sectioned brain for apparent hydrocephaly. 000048 Protocol 4P1a8g.e00283 PROPOSEDSTATISTICALMETHODS: aApvperroaprgieastea.ndAdpdeirtcieonnatlagpersocweildlubreescaalncdu/loarteadn.alLyistteesr vmaalyuebsewiplelrbfeorumseedd, wifhaeprpreopriate. TyofpTee st* I. Parametric" A. Bartlett's Test! Il. Nonparametric A. Kruskal-Wallis Test (75% ties) Significant at ps0.05 Not Significant Significant at p<0.05 Not Significant A wo of Variance J Test Significant at ps0.05 Dunnett's Test Not Significant B. Fisher's Exact Test (>75% ties) Hil. TPesrtfoorportiDoatna Variance Test for Homogeneity ofthe Binomial Distribution a. b. UStsaetidstoincallylytosiagnnaifliyczaentdaprtoabwaibtihlfhioesmoagreenreeiptoyrotfedvaarsieaitnhceer. ps0.05 or p.toon 9 c. d. TPreosptofrotriohnomdoatgaenaerietnyootfvinacrliuadnecde.in this category. Protocol 4P1a8g-e02048 DATA ACQUISITION, VERIFICATIONANDSTORAGE: DDiarteactwoirllabned/hoarnadp-praonpdr/ioartceommapuntaegre-mreecnotrdpeedr.sonRneeclorwditshiwinll21bedaryevsiaefwteedr gbeynetrhaetiSotnu.dyAl original records will be stored in the archives of the Testing Facilty. Al original data wil ybreeeqaubreoasuttn.edrPmaraneidsleiirnngvdeeodxfettdih.sesdAureascfotwipflyilnabolferasellptoorrrate,wd adafatttetarhewwihlTilecsbhteitnsigumpeFpaltcihieeldtSytpoaottnhsneoorScpwhoialnrlsgboeerfcouorpnootnnaected to determine the disposition of these materials. REC TO O BE MR AIND TAINS ED: TPersottoAcrotlicalen,dVAehmiecnldemaenndt/so.r Reagent Receipt, Preparation and Use. ARnainmdaolmiAzcaqutiisointiSocn.hedules. TMraetiantgmeHnitst(oiryp.rescribed by Staff Veterinarian). GCleinneicraallOCbosemrmveanttiso.ns and/or General Appearance. BBloodoydWSeaimghptlse.Collection, Processing and Shipment, CFaeeesdarCeoanns-uSmepcttiioonniVnagluOebss.ervations. LNiatttuerraOlbsDeerlviavetriyonOsb.servations. Reflex Gross NaencdroPhpyssyicOablseDrevvateiloonpsm.ent and Behavioral Observations - F1 Generation Pups. POhrogtaongWreaipghhst(sif (riefqrueiqrueidr)e.d). SFeteudd,y WMaatinetreannadncBeed(drionogm Aannadlyesnevsi.ronmental records). Packing and/or Shipment Lists. KPEEYRSONNEL: DExeicurtivoeefRDciersteecatoorrcrho:f RAelsaenarMc.h:HoMbielrdmraedn,S.PhC.hDri.s,tiDaAn,BTPh.D., ATS ADsirseocctioartoefDLiarbeocrtoartoorfyROepseeraartciohnas:ndJSothundyF. DiBraercnteotrt:, BR.aSy.mond G. York, Ph.D., DABT MMaCaonnmaamggieetrrteooeff:ASntDiuemdanylaCOoCop.redrLiaentbaioto,inoVsn.:aMn.VdDa.lMeerimebAe=r,ShIanrsptietru,tiMon.aSl.Animal Care anid 050 CMoannsaulgtearnto,f VReetgeurliantaorryyPCaotmhpolloigayn:ceW:.KRaathylBeernowAn.,MDo.rVa.nM,.,M.PSh..D. AGVP Protocol 4P1a8g-e02058 FINALREPORT: AbecfoimnpalriezheednfsoillvoewidnrgafctofnisnualltraetpioorntwwiiltlhbteheprSeppoanrseord.onThcoemrpelpeotritowniollftinhcelusdteudthyeand will following: Summary and Conclusion. EEvxapleuraitmieonntaolf TDeesstigRnesaunltds.Method. ADpaptae,ndPircoetso:colFiagnudreAss,soScuimamteadryAmaennddImnedinvtidsuaalnTdaDbelveisatSiuomnms,arSitzuidnygDtihreecAtobro'sve GLP QAU CSotmaptelmieanntc.e Statement, Reports of Supporting Data (if appropriate) and INSTITUTIONALANIMALCARE ANDUSECOMMITTEE STAEMN: `ITnshteitpurtoiocneadluArneismdaelsCcrairbeedanidn tUhsisepCrootmomciotltehea.veAblleeprnorceevdiuerweesddebsyctrhiebeTdesitnitnhgisFaprcoitloyc'osl dthiastcoimnfvoorltv,edsitsutdryesasnoirmaplasinwitlol tbheecaonnidmualcst.ed in a manner to avoid or minimize TnehceesSspiotnysoforr'csosnidguncattiunrge tbheisloswtuddoycaunmdentthse tfahcetftahcatttthhaits iinsfnoortmaatniounnnceocnecsesranriinlgythe pdurpolciecdatuirveesswteurdey mavaayilbabeleobftoarimneeedtfirnogmtthheesStaptoendsopru.rpoNsoeaslotfetmhaetisvteu(diyn.vitro) 000051 : Protocol 4P1a8g-e02068 REFERENCES: 1. CThersitsst.ianE,nvMi.rSo.nmaenndtaVloyPtreokt,ecPt.iEo.n (A1g9e8n2c).y,IWnaVsihviongRteopnr,odDu.cCt.ivNeatainondaMluTteacghenniiccailty Information Service, U.S. Department of Commerce, Springfield, VA 22161. 2. nCharlitsrteixaonn,eM(.PSr.o(c1e9e8d4i)n.gsReofprNoadlutcrteixvoenetoSxiycmitpyosanidumt,erNateowloYgoyrekvaAlcuaatdieomnsy ooff Sciences, Novembe7r, 1983), J. Clin. Psychiat. 45(9):7-10. 3. LCoanntgr,olP.DLa.t(a19i8n8t)h.e EChmabrrlyeos RainvdeFreCtralt:DCevDelBoRpRmaetn.taClhaTroxliecsitRyiv(eTerrLaatboolroagtyo)ries, LInacb.o,rWaitlormiiensg,toInn,c.)MA 01887-0630. (Data base provided by Argus Research 4. LInasbtoirtuatteoorfyLAanbiomraaltso.ryNAatniiomnaall RAecsaoduermcyesPr(e1s9s96,)W.asGhuiindgetofno,r tDh.eC.Care and Use of 5. SImaplleawnstkait,ioEn.ss(t1e9l6l4e)n. aFmarUbteemreusthdoedreRaztutem.mAarkcrho.skPoatphiosl.chEexnp.NaPchhawremiaksovlo.n 247:367. 6. Sthneedbeicnoorm,iaGl Wdi.strainbudtiCoon.chrStaant,isWti.cGal.M(e1t9h6o7d).s,V6atrhiaEndicteiotne,stlfoowrahSotmaotgeeUnneiivteyrsoifty Press, Ames, pp. 240-241. 7. SBoikoamle,trRy,R.W.aHn.dFRroeheHm,aFn.Ja.n(d19C6o9.),.SBaanrtFlertatn'csitsecsot,opfp.ho3m7o0g.e3n7e1i.ty of variances. 8. MSneetdheocdosr,,6tGh.WEd.itainodn,CloocwharaSnt,atWe.UGn.iv(e1r9s6i7t)y.PrAensasl,ysAimseosf,Vaprp.ia2n5c8e-.27S5t.atistical S. tDruenantemtetn,tCs.wWi.th(a19c5o5n)t.rolA. muJ.ltAimpleer.coSmtpata.riAsssooncp,r5o0c:e1d0u9r6e-f1o1r2c0o.mparing several 10. WSoHka.l,FrReRe.maanndanRdohCo,.,F.SJ.an(1F9r6a9n)c.isKcrou,skpapl.-W3a8l8l-i3s89T.est. Biometry, 11. 6D(u3n)n2,41O..J2.52(.1964). Multiple comparisons using rank sums. Technometrics 12. Siegel, S. (1956). McGraw-Hill, New Nonparametric Statistics York, pp. 96-104. for the Behavioral Sciences, 00 0052 : PROTOCOLAPPROVAL: FOR THE TESTING FACILITY OeNoy George E. Deariove, Ph.D., DABT Associate Director of Research Ze Az Ra) d G. York, Ph, Associate Director of Study Director ABT rch arbara J. Pat on, B.A. `Chairperson, Institutional Animal Care and Use Committee* Protocol 4P1a8g-e0z0r8 2mmmg ofS Date 20-05 Date = Ma, 55 Date FOR THE SPONSOR M 4 i Ta s Marvin T. Case, D.V.M., Ph.D. Study Monitor 22h ery Date 000053 rPRIMED!ICA _--mm Rl Ee Argus Research Laboratories, Inc TelTeeplheofnaex:: ((221155)) 444433--88378170 PROTOCOL 418-008 PERINACTOAMLB/IPONSETDNOARTAALL R(EGPAVRAOGDEU)CFTEIROTNILTIOTYX,ICDIETVYESLTOUPDMYENOTFAPLFAOSNDIN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 8 - 18 May 1999 -_-- 1. Concentration Analyses (page 5 of the protocol): cTohneducocntceedntartatthieondissacrmeptlieonsowfetrheesSepnotntsootrheanSdponnosorre.porStawmiplllbeeasneanltysteosthwiell be Testing Facility. ReaforsChoangne: This change was made at the request of the Sponsor to clarify the protocol. 2. MoaflteheapnrdotFoecomla)l: eRats Assianed to Pharmacokinetic Sample Collection (page 14 The liver analyzed aantdthseedriusmcrsetaimopnloefstwheerSeposnesnotrt.o the Sponsor Samples will be ReaforsCho angne: This change was made at the request of the Sponsor to clarify theprotocol. 3. AEmA/eEn2dmGeennetra5tioofnthPeupprsotNoocotlS):elected for Continued Observation (page 22 and The stomach contentsof these pups were sent to the Sponsor for analysis. Stomach contents will be analyzed at the discretion of theSponsor. 000054 . Pro"toAcmolen4d18m.e0n0t8 Page? Rea forsCho angn e: This change was made at the request of the Sponsor to clarify the protocol. <J pC 04 Assoorcgiea(tEe.DDieraercltoorveo,f 1879 PRhe.sDe.a,rDcAhBT Date forme AsRsaoychioantde X SS NOAN GD.irYecotor(ofPtRDe.seDaArcBhT sg -59 Date Study Director a hse... Ytdpmbed Lugny11 CDheaniarpCe.rsLoenb,o,InVst.iMt.uDt.ional Animal CaDraete Klason Jae. [tg 52 SMtaundiynMTo.niCtaosre, DV.M, PhD. Date and Use Committee | 000055 2T 30M1 EN NVIV RONMI ENTR ALLLAAO BBOORRN AATTOOV RRYY ENT ~~A ~ L PROTOCOL - ANALYTICAL STUDY Potassium Perfluorooctanesulfonate in Two Generation Rat Reproduction In-vivo study reference number: Argus 418-008 Study number: FACT 052798.1 Test substance: Potassium perfluorooctanesulfonate (PFOS) Name and addressof Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Experimental start date: Expected termination date: December 31, 1998 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic SSpurefcatcrtoamnettsrfyrom Liver for Analysis Using HPLC-Electrospray/Mass FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic. SSuprefcatcrtoamnettsrfyrom Serum for Analysis Using HPLC-Electrospray/Mass FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author; Lisa Clemen Vihortlr--_ KSrtiusdyHaDnirseecntor 2/5/79 Date Pe oT Gas [)F sr SpaornvsionrCRaesperesentative Date 000056 1 L0 0Poeos0 e 0000000 `The analytical portionofthis dosing study is designed to evaluate levels of potassium perfluorooctanesulfonate (PFOS), or a metabolite of PFOS designated by the study director, in the liver of the parent and subsequent generations of the test system, or in the serum as necessary. `The in life portion of this study was conducted at Argus Research Laboratories. 2R 0 eGULATORYCOMPLIANCE `This study is conducted in compliance withtheFood and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. 3.0 TESTMATERIALS 31 Test, control, and reference substances and matrices 3.11 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 312 Analytical reference substance matrix: Rat liver and serum 3.13 Analytical control substance: None 3.14 Analytical control substance matrix: Rat liver and serum 32 Source of materials 321 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 322 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report 32.3 Analy ical control matrix: 323.1 Ratlive-r Argus Research Laboratories; traceability information will be included in the final report 3232 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 33 Number of test and control samples. Liver samples for testing were received from 40 test animals and10 control animals. Serum samples will be tested at the discretion of the Study Director. 34 Identificationoftest and control samples: The samples are identified using the Argus Rescarch Laboratories identifiers, which consist of a letter followed by the Argus project `number, the animal number, the group designation, and the draw date; 35 Purity and strength of materials: Characterization of the purity and identity of the reference material i the responsibiolfitthye Sponsor. 000057 2 3.6 Stabilityoftest material: Characterizationofthe stability of the test material is the responsibility of the Sponsor. 37 Storage conditionsfor test materials: Test materials are stored at room temperature. Samples are stoart-e20d+ 10 C. 38 Dispositionoftest and/or control substances: Biological tissues and fluids are retained per GLP regulation. 39 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological `materials and preparing samples for analysis. 4E 0 XPERIVENTAL-O ~~v ~ ervie0 w `Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-008 are arneacleyitviecdalfotresatnsawliylslisbeofpfelrufoorrimneedcoomnpsoeulnecdtst.isAsutest.he discretion of the Study Director, a series of Initially, all liver samples (ES/MS). On the basis of will be analyzed for findings from these PFOS by analyses, electrospray/mass additional sample spectrometry matrices may be evaluated or other metabolites amendment wil be written. may be targeted.Ifadditional analysis is performed, a protocol 5.0 EXPERIMENTAL - Analvtical Methods 51 SuFrAfCaTc-taMn-t1s.f0r,omExLtirvaecrtifoonr oAfnaPloytsaisssUisuimnPgerHfPlLuCo-rEoloectcatnreossuplrfaonya/tMeasosr OSptehcetrrAonmieotnriyc 52 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry 53 FSuArCfTac-tMa-nt3s.0fr,omExSterarcutmiofnorofAnPaoltyassissiUusmiPnegrfHlPuLoCr-oEolcteacntersouslpfronaayt/eMaosrsOtShpeerctArnoimoentircy 54 FElAeCcTt-roMs-p4r.a0y/,MaAsnsalSypseicstroofmFelturoyrochemicals in Serum Extracts Using HPLC- 6.0 DATA ANALYSIS 6.1 Data transformations and analysis: Data will be reported as the concentration (weight/weight) of PFOS per tissue or sample, or of PFOS per unit of tissue or fluid. 62 Statistical analysis: Statistics used may include regression analysis of the serum `concentrations over time, and standard deviations calculated for the concentrations within each dose applied to egrvoaulpu.atIefsnteactiesstsiacarly,disfifmerpelneces.tatistical tests, such as Student's t test, may be 000058 3 1.0 MAINTENANCEOFRAWDATAANDRECORDS ~~~ 71 The following raw data and records willberetained in the study folder in the archives according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.12 Study correspondence 7.1.3 Shipping records 7.14 Raw daa 7.15 Electronic copiesof data 72 aScucpoprodritnigngtoreAcMoDrdTs-tSo-b8e rwieltlaiinnecdlsuedpeaartatleelaystftrhoemftohleloswtiundgy: folder in the archives 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.24 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens. 8.0 REFERENCES -- 81 3M Environmental Laboratory Quality System Chapters 1, 5 and 6 82 Other applicable 3M Environmental Laboratory Quality System StandardOperating Procedures 9.0 ATTACHMENTS 9.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfouate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 92 ElFeAcCtTr-oMs-p2ra.y0/,MaAsnsalSypseicstorfomFelturoyrochemicals in Liver Extracts Using HPLC- 93 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 94 FACT-M-4.0, Analysisof Fluorochemicals in Serum Extracts Using HPLC- `Electrospray/Mass Spectrometry 000059 4 PoCtAasSsMiaommPbaat2u1s5r5cc3i6n5ssutorat ATTACHMENT F: PREPARATORY AND ANALYTICAL METHODS Labora RoWcMueEsntrNoinRmmeapenonsrttLlNRLoNabToo1rc2a0o0r8z [r-- 000060 B3N M ENVAIRRO ONMe ENTAALL LAABBOORRAATTOORYY eee METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER ANIONIC 'FLUOROCHHEPMLICC-AELLSEUCRTFRAOCSTPANRTASY/FMRAOSMSLSIPVEECRTFROORMAENTARLYYSIS USING Method Number: FACT-M-1.0 Author: Lisa Clemen Adoption Date: 5/24/57 Revision Date: 0/4 Approved By: -- 2711 So Taboratory Manager Yuh to Group Leader OeAO Technical Reviewer S/ec/ 7% Date Shu l4 Date ley Date 1S 110..01 SScc CoOpPeE:o ATNhDisr AmPePtLhe IoCdATisIfa OoNrthn e extp ractioA nofPor tasse ium P0u erf0 lui oro0occ ta0ne0sa u0lfo0nam 0te0(0Po F0OS0)n 0or0 other fluorochemical surfactants from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated inthe validation report. 00006 | Microsoft 7.0.1/95 FACT-M-1.0 Extractionof PFOS from Liver Page 1 of 8 2.0 SUMMARY OF METHOD 2.1 Tflhuiosrmocehtehmoidcadlesscurrifbaecstahntoswftrooemxtlirvaecrt upsoitnagssfiounmpapierrifnlguorreoaogcetnatneasnudlf5o.n0amteLs(oPFfOeSt)hyolr other acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Funotuilr dmrLy.s oEfaecxhtreaxcttraicstries mroecvoendsttiotuatcedenitnri1f.u0gme tLubmeetahnadnpoulttohnetnofailntierterdogtehnroeuvgahpoara3tcoer plastic syringe attached to a 0.2 pm filter into glass autovials. 3.0 DEFINITIONS 3.1 None. 4.0 WARNINGS AND CAUTIONS 41 Health and Safety Warnings: 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. 5.0 INTERFERENCES 5.1 There are no known interferences at this time. 6.0 EQUIPMENT _ 6.1 aTchceepftoalblloew.ing equipment is used while carrying out this method. Equivalent equipment is 6.11 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.13 Centrifuge, Mistral 1000 or [EC 6.1.4 Shaker, Fberbach or VWR 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 73 Eppendoorrf disposable pipettes 7.4 Nalgene bottles, capable ofholding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL. 7.9 Labels 000062 Extrac tioFnAoCfTP-FMO-S1 .0 from Liver Page208f 7.10 Syringes, capable ofmeasuring 10 uL to 50 hL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendororf equivalent 7.14 Timer 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 um, 25 mm 7.17 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTM water. Rinse syringes a minimum of9 times with methanol, 3 rinses from 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagents - 8.11 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 10N: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-Q' `water, mix until all solids are dissolved. Store ina | L nalgene bottle. 8.12 MSeoadsiuurmeH1y0drmoLxiofdteh(e1.T1B0aNkNeraoOrHeqsuoilvuatlieonnt)i,nt(oNaa1O0H0)mILN.voDliulmuetteri1c0Nfla1s:k10a.nd dilute to volume using Milli-QTM water. Storein a 125 mL nalgene bottle. _ 813 Tapeptrroaxbiumtaytlealmym1o6n9iugmrahmysdroofgTeBnAsulifnattoea(|KoLdvaoklourmeetqruiicvacloenntta),in(iTngBA5)000.L5MMi:lWlie-iQgh water. Adjust to pH 10 using approximately 64 mL 10N NaOH and dilute to volume with Milli-QTM water. Add NaOH slowly while adding the last 1 mL of NaOH because the pH changes abruptly. Store ina 1 L nalgene bottle. 8.1.3.1 TneBeAderd eusqinug aiIcNrhNeecaskOHprisoorluttoioena.chuseto ensure pH = 10. Adjust as 8.1.4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T.Bakeror equivalent), (Na,CO,/NaHCO,) 0.25M: Weigh approximately 26.5 g of sodium carbonate (Na,CO,) and 21.0 gofsodium bicarbonate (NaHCO) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store ina | L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.19 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 82 Standards 8.2.1 Prepare PFOS standardsforthe standard curve. 00006 ExtractioFnAoCfT-FMO-S1.f0rom Liver Pag3e of8 8.2.2 tWheeiagchtuaaplpwreoixgihmta.tely 100 mg of PFOS into a 100 mL volumetric flask and record 8:23 Bring to volume with methanol for a stock standardof approximately 1000 ppm. (ng/mL). 8.2.4 aDpilpurtoexitmhaetsetloyck50soplputmi.on with methanol foar working standard 1 solution of 8.25 Daiplpurtoex.th5e.0stpopcmk.solution with methanol for2 working standard 2 solution of 8.26 aDpiplrutoex.th0e.5s0topcpkms.olution with methanol for a working standard 3 solution of 9.0 SampLE HANDLING : 9-1 All livers are received frozenand must be kept frozen until the extraction is performed. 10.0 QuALTy ConTROL 10.1 Matrix Spikes 10.1.1 tPhreepaacrceuraancdyoafntalhyezeexmtartarctiixons.pike and matrix spike duplicate samples to determine 10.1.2 Prepare cach spike using liver chosen by the analyst, usually control liver. 102 C1o0n.t1i3nuEixnpgecCtaeldibcroantceinotnraCthieocnksswill fallin the mid-rangeofthe initial calibration curve. 10.2.1 Pcroenptairneueadnldinaenaarliytyzoefctohnetiinnuiitinagl ccaalliibbrraattiioonncchurevcek.samples to determine the 1022 fOonuer cchheecckksisarpereppraerpeadrepderangdroeuxtporafctteedn. samples. For example, if2 sample set = 34, 102.3 Pprreeppatrheeeianicthiaclocnutrivneu.ing calibration check from the same liver homogenate used to 102.4 Tcuhrevee.xpected concentration will fall within the mid-rangeofthe inital calibration 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare Liver Homogenate to Use for Standards 111.1. Weigh approximately mLs Milli-QTM water. 40 gof Grind liver into a 250 mL Nalgen to a homogeneous solution, e bole co nt ain in g 200 1112 2117:450ra8tiiso.not available, use appropriate amounts ofiver and water in0ke0ep0i0ng6wi4th 11.13 See section 13.0 to calculate the actual density of liver. ExtractioFnoAfCTP-FMO-S1.f0rom Liver Page 4 of 8 11.1.4 shAiodxmtdoeeg|nemnILemoofLuhsaolsmiooqluugotetisononefboyuhossmhsaookglieuntngieoboneuttsowaseoel1nu5tamiloiLnquiconetns1t5wrhimfiLulgeecpetrnuetbpreai.rfRiungege-stauutsboeptsea.lndof 11.1.5 Tspwiokin1gmaLmoaulnitqusotlisssteerdvienatsabmleat1ritxo totaloffourteen samples. bslpainkke,s.inUdsueplithceatset,antdwaordstcaonndcaerndtrcautrivoenssfaonrda Approximate Spiking AmoTuanbtles 1for Calibration Standards Working Standard (Approx. Cone.) i "Approx. final conc. of PFOS in liver 00.500pppmm[ [2a 0{T 0.0o5o0iopppmm | [00pm[a0 | 50m TT 1m 0 | Gioop 0250ppm 5.0 ppm [20 30mm 50 0.500 ppm [70pm CL SOeemT&T Too0ppm 11.2 ILL See section 13.0 to calculate actual concentrationsof PEGSin calibration standards. Extract spiked liver homogenates following 12.14-12.24ofthis method. Use these standards to establish each initial curve on the mass spectrometer, 12.0 ProcEDURES 12.1 oOtbhtearitnisfsruoezse.n liver samples. In spent issue, note that the liver has not been packaged with 12.2. Cut approximately 1 gof liver using adissecting scalpel, 12.3 Weigh the sample directly intoatared plastic sampule vial. 12.4. Record the liver weight in the study notebook. 1122.56 ALadbdel2.t5hemsLasmopfulweatveiarltowistahmtphuelsetvuidayl number, weight, liver ID, date and analyst initials 12.7 uGnrtiilndthtehesasmamppllee.isPhuotmothgeengerionudse.r probe in the sample and grind for about 2 minutes, or 12.8 Rinse the probe into the sample with 2.5 mLs water using apipette. 129 Take the grinder apart and clean it with methanol afte each sample. Follow AMDT-EP.22. 12.10Capthe sampalnedvortexfor 15seconds. 000065 ExtractioFnoAfCTP-MF-S1.f0rom Liver Page Sof8 12.11 tPuibpeettweit|hmthLe hidoemnotigceanlaitnefionrtomaat1iSaosmnL.thpeoslaymppruolpeylveinale.c(eSntereiWfuogrekstuhbeee.tLfaobredlotchuemecnenttirnigf.uge the remaining steps.) 12.12 sSepcitkieonli1v1e.r1hoormoTgaeblneat1e.s with the appropriate amountofPFOS standard as described in 12.13iPnipsettrtuemetnwtob|lamnkLs.aliquots ofMilli-QTMwaterto centrifuge tubes. These will serve as 12.14 Add 1 buffer. mL 0.5 M TBA and 2 mLof the 0.25 M sodium carbonate/sodium bicarbonate 12.15 Using a volumetric pipette, add mLs ethyl acetate. 12.16 Cap each sample and put on the shakerfor 20 minutes. 12.17Centrifuge for 20 to approximately 3t5o 0205 rmpimn.utes, until layers are well separated. Set power on the centrifuge 12.18 Remove 4 centrifuge tmuLbes.oLfaobreglanthiics flaryeesrh,tuusbienwgiath5 tmhLe sgarmaeduiantfeodrmgalatsisonpiapsetitne,12t.o5.a clean 15 mL 12.19hPouutrsc.ach sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 12.20 Add 1.0 mL ofmethanol to each centrifuge tube using a graduated pipette. 12:21 Vortex mix for 30 seconds. 12.22 Attach2 0.2 Filter into a 1y.5mmnLylgolnasmsesauhtfoivlitaelr.to a 3 cc syringe andtransferthe sample to this syringe. 1223 mLaatbreilx,thfeinaaultsoovlivaelnwt,itehxttrhaectsitoundydantue,mbaenrd,aannaliymsatl(sn)uwmhboerpearnfdogremneddert,hesaexmtprlaectiiomne.point, 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to pageofstudy notebook. 1D11333..010ADCaATlTcAuAlAaNtiAAoLnYsSN:ISAANDLCAYLCUSLATIIOSNS ANDC0A0 tc0 ut0a00moNs 13.1.1 Calculate equation: the densityofliver (mg) in1.0 mL homogenate using the following ofLiver x Average weight Gof Live+r gooffWteant|meLaeliqouotss ()m; FACT-M-1.0 Extraction of PFC om Liver 000066 Page 6 of 8 13.1.2 fCaollcluolwaitneg aecqtuuaatliocno:ncentrationsof PFOS in calibration standards using the bL of Standard x Concentration (ug /mL) = Final Concentration (ug/g or mg/kg) mg Liver / 1 mL homogenate of PFOS in Liver *Average weightofliver in solutionas determined in 13.1.1, by weighing ten | `mL homogenatesof approximately 40 mg liver in 200 mLofMilli-Q water. 14.0 METHOD PERFORMANCE 14.1 The method detection limitisequal tohalfthe lowest standard in the calibration curve. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in l`hoicgahteBdTiUn tchoentlaaibnoerrast,orayn.d used glass pipette waste is disposed in broken glass containers J166O0ReReccoomreos 16.1 Complete the extraction worksheet and tape into the =0000000 study notebook. 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Thevalidation report associated with this method is FACT-M-1.0 & 2.0-V-1 10 188.00 RReE0 rFeEmReEnN0 CcEeSs 0000000000 18.1 AMDT-EP-22, "Routine Maintenance ofUltra-Turrax T-25" 19.0 AFFECTED DOCUMENTS 19.1 FMaAsCsT-SMp-ec2t,ro"mAentarlyy"sis ofLiver Extracts for Fluorochemicals usingHPLC-Electrospray R2e0.v0isRiEVoInSIONS RNeuvmibseiro,n Reason For Revision RevDiastieon 000067 FACT-M-10 Extractionof PFOS from Liver Page 70f8 Extraction Worksheet for FACT-M-1 Study # Sample Number PFOS approx. 0.5 ppm| PFOS approx. 5 ppm | PFOS approx. SO ppm| Date and Initials sett actual HW. ppm| actual Ww ppm| actual ppm| for Std wW___ [ [ Tmo 1 smkves1 ] Tr 7 rT r r 7rT1 [ 7 ----d--f 1 ---- 1 ---- r ------TT T1 feeee}meetrbr-- r t rr r TT r rr rr r r r r T 7 r1 T r r r r 77 1 ES SS --r r r T rr rT r r= r rrr T r1 7 ---trrr1 rl rr rr TT rT 1 ------ 1 1 1 Pomme Memes ET 1 Phegomn Soe = Tempe Cpee'rMeS/vMvyiSnDge/3_3_cCBonDt.sChveceks:ifS--piak0e--.d2_mSR--uL--ieorfiao 13mplpsmwsoimdmplevl 5Tora final concentration of Pom. MS/MSD used sample `Cont. Checks used same homogenaatse for std curve. ExtractioFnAc*TT>-7M0-S1f0rom Liver Page8of 00068 3SMS ENVITROOVNAMENNTTAALLJAABBOORRAATTOORRYY 0 METHOD ANALYSHIPSLOCF-FELLUEOCRTORCOHSEPMRIACYA/LMSAISNSLSIPVEECRTERXOTMREATCRTYS Using Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: D- / J 2 Laboratory Manager Yoko fbr Group Leader lx A Choma Technical Reviewer Adoption Date: 5/3/33 Revision Date: Na s/s2c fy Date s/esay Date 5/23/28 Date 10 SCOPEANDAPPLICATION 1.1 sSucrofpacet:antTshiussimnegtHhPodLCis-folretchteroasnparlayys/imsaosfsesxptercatcrtosomfetlriyv.er or other tissues for fluorochemical 1.2 sAuprpflaictcaanbtls,e oCroomtphoeruniodnsi:zabPloetcaosmspiuomunpdesr.fluorooctanesulfonate, anionic fluorochemical 13 vMaaltirdiacteiso:n reRpaobrbti.t, rat, bovine, and monkey livers or other livers as designatedinthe 00006 Word 70.1195 ACTM20 Analysisof__. Extract Using ES/MS Page 10f8 22S 0.0 SUU MMARM Y OF MM ETHOA D Ro VO o RMESROD 2.1 'THhPiLsCm-eetlehcotdrodsespcrraiyb/emsastshespaneacltyrsoimseotfryf.luTohreocahnaelmyisciaslissuprefrafctoarnmtesdexbtyramcotneidtforrionmglaivseirnugslieng `ipoenrfchlaurcarcotoecrtiasnteiscuolffoapnaarteti(cPuFlOaSr)flaunoiroonc,hMem/iZca=l,49s9u.chSaasmptlheespomtaayssailusmo be screened to Verify compound identification. dp0 3.e0 rDemr0 mmToiosn0 ss 0000000000 34 None. 44.90 WWaArRnNImNoGsSaANNpDCCvAUmToIOnNsS 41 Health and Safety Warnings: 0000 4.1.1 inUtsoetchaeutpiroonbweiDthOthNeOvTolTtaOgUe CcaHblTefHoErtPheROprBoEb,e.tWhehreenistrhieskvoofltealegcetcraibcalle is plugged shock. 42 Cautions: 42.1 `Dooverno4t00rubnars,oltvheenHtPp1u1m0p0s waiblolvienictaiaptaecaiutytoomfa4t0ic0sbhaurt(d5o8wn0.0 psi). Ifpressure goes 42.2 Do not run solvent pumps to dryness. 550 .00IIvNvTeEmRe0 FeEmReENnCcEeSs0000000 5.1 cToenftlaocntswhiotuhldthneostabmeplueseodrfeoxrtrsaacmt.ple storage or any part of instrumentation that comes in 6.0 EQUIPMENT 6.1 Equipment listed below may be changed inorderto optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.12 HP1100 low pulse solvent pumping system and autosampler. 17S 7..010 Su USPuPpLpIlr iEeSsANp D MAL TERIi ALS esa0n 000p0M0a00r 00e 000m 00L 0 0s 7.11 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.13 Capped autovials or capped 15 mL centrifuge tubes. 830 .0RREEAAGGEENTSSAND0 SSTANNDDAARRDDSS 000 81 Reagents 000070 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-2.20 Analysis ofLiver E:-= Using ESMS Page2of8 812 bMeiplrloiv-iQdTMewdatbeyr,a aMlillwlait-eQruTsOeCdPilnutshissysmteetmh.od should be Milli-QTM water and may 8.13 Ammonium acetate, HPLC grade or equivalent. 82 Standards 82.1 dTyupriicnagltlhyeoenxetrHac,t0iobnlparko,ceodnuerel.iveSrebelFanAkC,Ta-nMd-1se.ven liver standards are prepared 9.0 SAMPLE HANDLING 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are. stored in capped autovials or capped 15 mLcentrifugetubes until analysis. 92 Ifanalysis will be delayed, extracted standards and samples may be refrigerated until analysis can be performed. 10.0 QUALITY ConTROL 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blaandmnatrkix blankprierto cach calibration curve. 10.2 Matrix Spikes 102.1 Analyze a matrix spike and matrix spike duplicate with cach analysis. 10.2.2 cAEudxrdpvieet,citoendalcosnpcieknetrcaotnicoennstrwaitlilofnasllmianythfeallmiidn-trhaenlgoeowf-trahnegeionifttiahlecainliitbiraaltciaolnicburravteio.n 102.3 See section 13 to calculate percent recovery. 103 Continuing Calibration Cheeks 103.1 Analyze amid-range calibration standard after every tenth sample. Ifa significant rcuhna.nOgnel(y+ t3h0os%e)isnamppelaeksaarneaalyoczceudrsb,efroerleattihvee ltaostthaeccineipttiaalblsetacnadlairbrdactuironves,tasntdoaprdt.he will be used. The remaining samples must be reanalyzed. 103.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 Sasyssetsesmedsufiotrabeialcihtyr(uen..g. peak area, retention timeandpeak shape, etc.) will be 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted liver standards prior to and following each setofextracts. The mean offortwthoe sctaalinbdraartdiovnalcuuersv,eautseiancghMsatsansdLayrndxcoonrceontthreartsiuoint,abwliellsobfetpwlaortet.ed by linear`regression FACT-M-20 AnalysisofLive: =xtract Using ES/MS 30f8 000 oF 112 Tdihsecrre*tviaolnoufetfohrethaneadlaytsta.should be 0.98orgreater. Lowervalues may be acceptable at the 11.3Ifthe curve does not meet requirements, perform routine maintenance or reextract the standard curve(if necessarayn)d reanalyze. D1o2.P0ePoRcOCoEoDvUReEsSs 12.1 Acquisition Set up 0000000 12.1.1 Clickonstart button in the Acquisition Control Panel. Set up a sample list. Assign (aMfSi)lefnoarmeacuqsuiinrignlg,eatntedrt-yMpOe-DiAnYs-almapstleddiegsictroifptyieoanrs-.sample number, assign a method 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriate and mass to 499 or otherappropriate masses. A scan is usually collected along with the SIRs. Save method. 12.1.3 tThyepisceaclolnydtsheetosafmsptlaendlairstdsb.eginswiththe firstset ofliver `standards and ends with 12.1.4 Samples are analyzed with a continuing calibration check injected afier every tenth sample. Solvent blanks should be analyzed periodically to monitor possibleanalyte carryover and are not considered samples but may be included as such. 122 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1, 12.2.2 aSneatl-yuspttchoensHiPd1er1s0a0p/paruotporsiaamtpelfeorraotptthiemaflolrleoswpionngsec.onRdeictoirondsaocrtuaatlccoonnddiittiioonnsstihnethe instrument logbook: 12.2.2.1 Sample size = 10 uL injection with a sample wash 12.2.2.2 Inject/sample = | 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time MeOH 20mM `Ammonium acetate ([[oESCmmiminon|||_#995700%%%0[ 5 NO f 9m ] Note: In this instrument configuration, the run must be set up on the electrospray psroefstsweadreownitthheaH"PWaWiotriknsgtaftoiroinn.let start" `message before the "Start" button is 12.2.2.5 Press the "Start" button. 000072 FACT-M-2.0 AnalysisofLiver Ex: Using ESMS Page 4 of 8 123 Instrument Sep-up 12.3.1Referto AMDT-EP-31-for more details. 123.2 Check the solvent level in reservoirs and refill ifnecessary. 12.3.3 Check the stainless steel capillary at the end ofthe probe. Use an eye piece to check tuhnesattipi.sfaTchtoeryti,pdsihsoauslsdembbelefltathewiptrhonboe ajnadggreedpleadcgeest.hIeftsthaeinlteispsisstfeoeluncdaptiollbaery. 12.3.4 SOebtseHrPvLeCdrpopulmetpstcoo"mOinn"g. oSuettofthtehfeltoiwpotfot1h0e-p5r0o0beu.LA/lmlionwotroaseqaupiplriobprraitaetef.or approximately 10 minutes. 12.3.5 aTruorunnodnthtehetinpiotfrotgheen.pArobfei.ne mist should be expelled with no nitrogen leaking 12.3.6 Tchhaenignesitnruomrednetrutsoeosptthiemsiezeptahrearmeesteprosnsaet:the following settings. These settings may 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12:3.63 LC constant flow mode flow rate 10- 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter instrument is operating correctly.) is not set, it is a guide to ensure the 12.3.7 Cfuarrtehfeurl.lCyognuniedcettthheepvroolbteagientcoabtlheesotpoentihnegp.roIbnes.ert probe until it will not go any 123.8 Record tune parameters in the instrument log. 12.3.9 Uthseinangatlhyesicsroofsbsi-oflloogwiccoaulnmtaetrriecleesc.trode in the ES/MS source is recommended for 123.10toCpliocfksaomnpslteartlisbtu.ttEonnsuinrethsetaArctqaunidsietnidonsCaomnptlreolnuPmanbeelr. iPnrcelsusdetshealsltsaratmbpultetsontoabte analyzed. 1D133.00aDArTAaANAALYSnIS AaNDLCALyCULsATIiONSsanoCa00r0cu00a00m00o0n00s 131 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Re`sEuxlptec-teBdacRkesgurlotund Result x 100 13.12 Caleulate percent difference using the following equation: %Difference = Expected Conc. - Calculated Cone. x 100 `Expected Conc. 000073 Analy si s FACTM-2.0 ofLiver = -~=t Using ESMS Page Sof 8 13.13 Calculate actual concentrationof PFOS anionintotal liver (mg): (= PFOS anion calc. from stdze) Loflverwedforamlysis J poe ofiver 1000 ug/1 mg) 14.0 METHOD PERFORMANCE 14.1 The method detection limit is equal to at least three times the baseline noise inthe matrix `blank. 14.2 The practical quantitationlimitis equalto the lowest standard in the calibration curve. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Samplewaste is disposed in biohazard containers, flammable solvent waste is disposed in `high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. l01R6.e0 cREoCmORnDsS ~~ 000 16.1 iSntfoorermcahtrioonm atograms included in the either study in the folder. header oErahcahncdhwrroimttaetnoognr am s the hcohurlodmahtaovgertahme:folstluodwying `number, sample name, extraction date, and dilution factor(if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 163 Print sample list from MassLynx and tape into the instrument runlog. 16.4. Print data integration summary from MassLynx and tape into the instrument runlog, 16.5 Copy instrument runlog pages, including tape into appropriate study notebook. instrument parameters and sample results, and 16.6 Summarize data using suitable software and store in the study folder. 16.7 lBoaccaktiuonp oeflebcatrcoknuipc edlaetcatrtoonaipcpdraotpar.iate media. Record in study notebook the file name and 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V-1. 0 M18B.O0RReErFeEmReEnNcCeESs 00 18.1 AMDT-EP-31, "Operationof VG Platform Electrospray Mass Spectrometer" 000074 FACT-M-2.0 Analysis ofLive: =xtract Using ES/MS Page 6of 8 DA 1B9.O0 Ar FFECe TEDDs OCUMc ENTr S epPocume0s 000e 00s 0 19.1 UFsAiCnTg-MH-P1LC.-0E,l"eEcxttrroascptiroanyo/fMPaostsaSspseicutmroPmeertfrlyu"orooctanesulfonate from Liver for Analysis 20.0 Revisions Revision Number. Reason For Revision ReDvaitseion FACT-M-20 AnalysisofLive- Zxtract Using ESMS 000075 Page 7of8 Laboratory Study # Sud: Test Material: MatrixFinal Solvent: AMneatlhytoidcRaelvEisqiuoinp:ment System Number: IFinlsetnraummee:nt Softvare/Version: ReSquared Value: Slope: Intercept: DateofExtraction/Analyst: Dateof Analysis/Analyst: Group Concentration Final Conc. GSlroopuep:/DToaskee:n rTaokmenlifnreaormrteghresstsuidoynfeoqludaert.ion ``CSoanmcpelnetsr:atTiaokne(nufgr/ommL)th: study folder. Taken from the MassLynx integration summary. DIniiltuitailoVnoFlaucmtoer:(mLT)a:kenTfarkoemntfhromsttuhdeystfoulddyerf.older. Final Cone. (ug/L): Calculated by dividing th inital volume from the concentration 000076 FACT-M-2.0 Analysis ofLiver Extrac: Using ES/MS Page 8 of METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER ANIONIC FLUOROCHEMICAL SURFACTANTS FROM SERUM FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M-3.0 Author: Lisa Clemen Adoption Date: 1243 Revision Date: Ng Approved By: ) Laboratory Manager Lesto Hh Group Leader Shan ad Date ilr)ay Date Technical Reviewer Date 10 SCOPE AND APPLICATION 1.1 Sotchoepref:luTohrioscmheemtihcoadl sisurffoarcttahnetesxftrraocmtisoenorufmp.otassium perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants of other fluorinated compounds. 1.3 Matrices:Rabbit,rat,andbovineserumor other sera as designatedinthevalidationreport. Microsoft 7.0.1/95 FACT-M-3.0 Extraction of PFOS fom Serum Page 1 of8 000077 22S 0.1 Tu his mem thod dm escriba es howr toextrv actpoto assiur m perfM luorooce tanesum lf0on0ath 0e (0P0FOo 0S)0o0r0p ot0he0r eatnhiyolniacceftlauteo.roAchnemiiocnalpasiurrifnagcrteanatgsenftroismadsdeerdumtuosithnegasanmpilonepaanidritnhgeraenaagleyatte ainondp5a.0imisL of te`pvhaarrpotouirtgiahotnaoerd3uicnnctlopledatrshyty.ilcEasacyecrtihantgee.extarF tatcatcio hmseLrdeotcu oofn2se0tx.itr 2truajtcemtdainrnyel1ro.en0mmfoLivlteeodrfiamnntedothpgaulnatososln,atutothoaevnniiaftlisrl.otegreend 30pewmows 3.1 None. 00000 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.11 Uhasnedluinnigvearnsailmaplrescearuutmio,nist,measpyeccioanltlayilnabpoartahtoogernysc.oats, goggles, and gloves when 5.0 INTERFERENCES 5.1 There are no known interferences at this time. 6.0 6.1 EQUIPMENT Tachceepftoalblloe.wing equipment is used while carrying -- out this ---- method. Equivalent equipment is 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.12 Centrifuge, Mistral 1000 or IEC 6.13 6.1.4 NSihtarkoerg,enEbevearpboarcahtoorr, VOrWgRanom.tion 6.1.5 Balance, ( 0.100 gm) .0_SUPPLIES ANDMATERIALS 7.1 7.2 Gloves Eppendororf disposable pipettes 7.3 Nalgene bottles, capableofholding 250 mL and 1 L 7.4 Glass, type A, volumetric flasks 75 40mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 78 Syringes, capableofmeasuring 10 kL to 50 uL 7.9 Glass, type A, volumetric pipettes 7.10 Graduated pipettes 18 0000 ExtractiFoAnCofT-M>-S3.f0rom Serum Page2 of8 7.11 Electronic pipettor, Eppendororf equivalent 7.12 Timer 7.13 Disposable plastic 3ccsyringes 7.14 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.15 Crimp cap autovials Note:PQrMiowrattoeru.siRnigngsleasssyrwianrgeeasn2d mbiotntliems,umrionsfe3ttiimmeesswwiitthhmmeetthhanaonlo,l 3anrdin3setsifmresomwi3tshepMairlaltie-: vials. 80 REAGENTSANDSTANDARDS ~~ 81 Reagents 8.11 S20o0digurmamhsydNraoOxiHd.eP(o1urBiantkoera o1r00eq0uimvLableenatk),er(cNoanOtHa)ini1n0gN:50w0eilgitherasp(pLr)oMxiilmlai-tQe'ly `water, mix until all solids are dissolved. Store ina 1 L Nalgene bottle. 8.12 MSeoadsiuurmeh1y0drmoLxidoef (1L0TNBNaakOerHosroelquutiivoanleinntto),a(1N0a0OmHL). IvoNl.umDeitlruitce f1l0asNk 1a:n1d0.dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 81.3 Teatprparbouxtiymaatemlmyo1n6i9ugmrahmysdroofgTeBnAsulifnattoea(1KoLdvaoklourmeetqruiicvacloenntt)a,in(iTngBA5)000.L5MM:ilWlie-iQgh wvaotleurm.eAdwjiutshtMtiolpliH-1Q0TM uwsaitenrg.apApdrdoxNimaaOteHlysl6o4wlmyLwohfil1e0aNddNiangOtHheanladstdimlLuteofto NaOH because the pH changes abruptly. Store ina 1 LNalgene bottle. 8.13.1 TBA requires needed using Ia NchNeackOHprisoorluttoioena.ch use to ensure pH = 10. Adjust as 8.1.4 S(oNdai,uCmOycNarabHoCnOat,e)/s0o.d2i5uMm:bWiceairgbhonaaptperbouxfifmeart(e.lTy.2B6.a5kegroforsoeqduiiuvamlecnatr)b,onate (anNda,bCrOi,n)g atondvo2l1u.0mgeowfitshodMiilulmib-iQcTMarwbaotnera.teS(tNoareHCinOa.V1inLtnoaalg1eLnevbooltutlmee.tric flask 8.15 PFOS (3M Specialty Chemical Division), molecular weigh=t 538. 8.1.6 Otherfluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.8 Methanol, Omnisolv, glass distilled or HPLC grade. 8.19 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with cach sample set. 8.1.11 Mbeilplrio-vQidTMewdatbeyr,a aMlillwlait-eQrTuOseCdPilntuhsissysmteetmh.od should be Milli-QTM water and may 000079 FACT-M-3.0 Extractioofn 7 fromSerum Page 3 of8 82 Standards 82.1 Prepare PFOS standards for the standard curve. 822 Prepare other fluorochemical standards, as appropriate. 823 Weigh approximately 100 mg of PFOS into a 100mL volumetric flask and record the actual weight. 8.2.4 B(urgi/nmglt)o.volume with methanol for a stock standardofapproximately 1000 ppm 8.2.5 D`aiplpurtoextihmeatsetloyck50soplputmi.on with methanol for a working standard 1 solution of 8.2.6 Dilute the stock solution with methanol for aworking standard 2 solution of approx. 5.0 ppm. 82.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. W0 savpe0 Haouv0 e 000000 9.1 Allseraare received frozen and must be kept frozen until the extraction is performed. 10.0 QUALITY CONTRO} 10.1 Matrix Blanks and Method Blanks 10.1.1 Two 1.0 mL aliquotsofthe serum are extracted following this procedure and used as matrix blanks. See section 11.1.2. 10.12 Two used a1s.0mmetLhoadlibqluaontksos.fMilli-QTM water are extracted following this procedure and 10.2 Matrix Spikes 102.1 Pthreepaacrceuraancdyoanfatlhyezeexmtartarcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 Prepare each spike using serum chosen by the analyst, usually control serum received with each sample set. 10.2.3 AEdxdpietcitoendalcosnpcieknetsramtaiyonbsewiilnlclfualdleidnatnhde m`maiyd-fraallngieonftthehleowin-irtaianlgecoaflitbhraetiionnitciaulrve. calibration curve. 10.3 Continuing Calibration Checks 10.3.1 cProenptairneueadnldinaenaarliytzyeofcotnhteiinnuiitinagl ccaalliibbrraattiioonncchurevcek.samples to determine the 103.2 Ofonuer cchheecckksisarpereppraerpeadrepderangdroeuxtproafctteedn. samples. For example, HERE FACT-M-3.0 Extraction of 70S from Serum Page 4of8 1033 Prepare eachcontinuing calibrationcheckfromthesame serum used to prep the initial curve. 10.3.4 curve The expected concentration will fall withinthemid-rangeofthe initial calibration 10 CummmonawStoamzenoy 111 Prepare Serum Standards. 11.1.1 Transfer 1 mLofserum to a 15 mL centrifuge tube. 11.1.2 I`futsihneg msaejrourmitvyooflusmeersuimn tshaempsltaenvdaorldusmeeqsuaarlteolteshsethsaenru1m.0vmoLl,umeexstrianctsasmtpalnedsa.rdsDo `not extract below 0.50 mLofserum. Record the serum volume on the extraction sheet. 11.1.3 Minix15omr Lshaceknetrbieftuwgeeetnubaelsi.quots while preparing a totalofsixteen aliquotsofserum 11.1.4 Two 1 mL or appropriate aliquots serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves foar total of fourteen samples. 11.1.5 Refer to the validation report FACT-M-3.0-V-1 and FACT-M-4.0-V-1 which lists the working ranges for calibration curves. Approximate Spiking Table 1 Amounts for Standards and Spikes Using 1.0 mLofSerum Working Standard iL "Approx. final cone. of T CC(Approx. Conc.) T PbFOSmin serum | [5[ 0pm5 [0 510 | | p o0w0m0m sppmm || [50T0 oTm 0557050mm pmom | 11.1.4 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 000081 11.2 Extract spiked serum standards following 12.6-12.16 ofthis method. Use these standards to establish each initial curve on the mass spectrometer. FACT-M-3.0 Extracti=- -f PFOS from Serum Page Sof8 12.1 Obtain frozen serum samples andallowto thaw. 12.2. `Vpoorltyepxrmoipyxlefnoerc1e5ntsreicfuognedstutbhee.n remove 1.0 mL or appropriate volume toa 15 mL 123 Retum serum samples to freezerafierextractionamounthas been removed. 124 Reqeucaolrtdhtehienisteiarlusmevroulmuvmoeluomnet.he extraction worksheet. The final methanol volume will 125 L`waobreklshteheettfuobredwoictuhmtehnetsitnugdythneurmebmeari,nisnegrustmepIsD., date and analyst initials. See attached 126 STpaibklee Isfeorrutmhewictahlitbhreataipopnrocpurrivaetestaamnodaurndtso.fAPlsFoOsSpiskteanmdaatrrdaixssdpeiskcersiabnedd icnonsteicntuiionng11.1 or calibration standards. 127 Vsaomrptleexsmfioxr t1h5essetcaonnddasr.d curve samples, matrix spike samples, and continuing calibration 128 Add 1 mL 0.5M TBA an2d mLofthe 0.25 M sodium carbonate/sodium bicarbonate buffer. 129 Using a volumetric pipette, add mL ethyl acetate. 12.10Cap each sample and put on the shaker for 20 minutes. 12.11 C10enatprpirfouxgiemafotrel2y0 3t5o0205 rmpimn.utes, until layersarewell `separated. Setpoweron the centrifuge 12.12 cTernatnrisffueg4re tmubLeo.fLoarbgealnithcislafyreers,hutsuibnegwait5hmthLe gsraamdeuaitnedfogrlamsastpiiaopsenttien, 1t2o.5a. clean 15 mL 12.13Put cach hours. sample on the analytical nitrogen evaporator nil dry, approximately 2 to 3 12.14Apidpdet1..0 (mTLhiosrvaoplpuromperieaqtuealvsotlhuemienoitfiamlevtohlaunmoelotfoseearcuhmceunsterdiffuogretthuebeexutsriancgtiaong)raduated 12.15 Vortex mix for 30 seconds. 12.16Attach 2 0.2 um nylon mesh filter 0a 3c syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.17Lmaabtreilx,thfeinaaultsoovlivaelnwti,tehxttrhaectsitoundydantue,mbaenrd,aannailymsatl(sn)uwmhboerpaenrdfogremneddert,hesaemxtprlaecttiiomne.point, 12.18Cap and hold for HPLC-lectrospray/mass spectrometry analysis. Extracts may be stored at 4 C until analysis. 12.19 Complete `notebook. the extraction `Worksheet, attached to this document, and tape to pageof study 000082 ExtractionFAoCfT-7MI-S3.f0rom Serum Page 6 of 8 Extraction Worksheet for FACT-M-3 Sd Sample Number | approFx.O0S.5 ppm| approFx.OSppm| approFx.O5S0 ppm|| InDiatiealsanfdor actual ppm| acual ppm | actual ppm | Std. or [mommsetrx 1 #--W ------pHW-------- Ww Comments rr ee r --rT r ----] r r 1 r r r r --r r r r -- r rr rr r r r r r r 1 E rS r ByRS Sn: Be rr rr rT -- B r rr S r SrSnS--| rrr 71 --7--] Ee-- 1 -- r _---- [------ -- 1---- -------- 1--] Smee E aTY--o ---------------------------- B [Pp p oe emeri omn leico a l we o ] : Femaan Covet ey [Removesdol digvolofommicaver PfeceE d T owe --ml weei -- [romeee -------- ey MIMDP!o.CoMntS.ICMhSeDeu:idSpsaemple A aT pCoent.nChdecks wed sme seTrourmaaTsefaolrscodnccuernvi.aon oF ExvucionFcA:CT:0-M83.0fromserum 000083 Page 8of8 METHOD ANALYSIS OF FLUOROCHEMICALS IN SERUM EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: FACT-M4.0 Author: Lisa Clemen 2 Approved By: nN Laboratory Manager Adoption Date: 47,43 Revision Date: [4 g[22/o" Date Groupweb TecHhinsiecal ReCvliiemwaer Date Y/14(98 Date 1.1 Ssucrofpaec:tanTthsiussmiengtHhoPdLiCsf-eolretchteroasnparlayys/ismoasfsexstpreaccttrsomoeftsrye.rum or tissue for fluorochemical 12 Asuprpflaictcaanbtlse, oCroomtphoeruniodnsi:zabPloetcaosmspiouumnpdesr.fluorooctanesulfonate, anionic fluorochemical 1.3 Matrices: Rabbit, rat, and bovine serum or other seraas designated in the validation report. Word 7.0195 F: AnalysisofSz - T-M40 Extract Using ES/MS 000084 page1ors 2S 20.1 TU his meM thod dM escribA es the R analysiY sofflO uorochF emicalM surfacE tants eT xtractH e0d f0roO m0se0ruD 0m 0 suisnignlgeHiPoLnCc-halreacctterroisstpircayo/famapsasrtsipceucltarrofmleutorryo.cThehmeicaanla,lyssuicshisapsetrhfeopromteadssbiyummonitoring a `Vpeerriffylucooromopcotaunnedsuildfeonntaitfeic(aPtiFoOnS.) anion, M/Z= 499. Samples may also be screened to 3.0 DEFINITIONS _-- 3.1 None. 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.11 Uinsteo cthaeutpiroonbweiDthOthNeOvoTltTaOgeUcCaHbleTfHoEr tPheROprBoEb,e.tWhehreenistrhieskvoofletlaegcetrciacballe isshopclku.gged 42 Cautions: 42.1 Dovoerno4t0r0ubnars,oltvheenHtPp1u1m0p0swaiblolvienictaiaptaeciatuytoomfa4t0i0cbsahrut(d5o8w0n0.psi).If pressure goes 422 Donotrun solvent pumpsto dryness. 5.0 INTERFERENCES 5.1 Tcoenftlaocntswhiotuhldtnhoe tsbameplueseodr feoxrtrsaactm.ple storage or any partofinstrumentation that comes in 6.0 EQUIPMENT 6.1 Equipment listed below may be changed inorderto optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler. 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.11 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.12 HPLC column, specifics to be determined by the analyst. 7.13 Capped autovials or capped 15 mL centrifuge tubes. 8.0 REAGENTS AND STANDARDS 81 Reagents 8.11 Meethanol,HHPPLLCC grade or equiivalent3 000085 FACT-M4.0 Analysis of Seru=- Extract Using ES/MS Page 20f8 8.12 Mbeilprloiv-iQdewdatbeyra, lMillwlait-eQ TuOseCdiPlnutshissysmteetmh.od should be Milli-QTM water and may 8.13 Ammonium acetate, HPLC gradeorequivalent. 82 Standards 82.1 Typically one HO blank, one serum during the extraction procedure, See blank, and seve FACT-M-3. serum standards are prepared S90:S1 ovFarrmeegsshtMosareevrduoimnuscrtaavpnpedeadrdasuatoeviparlespoarrecdapwpietdh 1e5acmh0Lan0caelny0tsirsi0.fugE0extfru0abcets0eudn0stt]a0nadnaa0rldyss0ea.n0d s0amples 92 Ifanalysis wil be untilanalysiscan delayed, extracted be performed. standards and samples may be refrigerated at 4 C 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and amatrix blankpriorto each calibration curve, 102 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with eachanalysis. curve. 10.2.2 Expected concentrations will fall in the `mid-rangeofthe initial calibration curve. Additional spike concentrations `may fallin the low-rangeofthe initialcalibration 103 10.2.3 See section 13 tocalculatepercentrecovery. Continuing Calibration Checks 103.1 rcAuhnnaa.nlOgynezle(y=at3mh0iod%s-e)rsaiannpmgepelacekaslaiarbneraaaltyoizcocneudsrbsae,nfrdoerrleadttiahveetletaosetthvaecrciyneitptetinaaltbhlsetsaacnamdlpialbrerd.atcIiuforanvess.itgasnntidofipiactdahnet will be used. The remaining samples must be reanalysed. 103.2 See section 13 to calculate percent difference. 104 SystemSuitability 10.41 System suitability e.g, peak are, retention time, peak shape, etc) vill be assessed for each run. 110 CALIBRATION AND STANDARDIZATION meanoftwo standard values,ateach standard concentration, will be ploted by inca 11.1 Analyze theextractedserum standards prior to and following each set of extracts. The regression for the calibration curve `using MassLynx or other suitable software. Analysis of SeFrAuCmTE-xMtr4a.ct0Using ESMS000086 p50 11.2 Tdhisecretviaolnuoefftohrethaenaldyastta.should be 0.98 or greater. Lower values maybeacceptaabttlhee 11.3 sItfatnhdeacrudrcvuerdvoe(eisfnnoetcemseseatryr)eqaunidrermeeanntasl,yzpee.rform routine maintenance of reextract the 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample lst. Assign (aMfSi)lefnoarmeacuqsuiinrginlg,etatnedr-tMyOpe-DiAnYs-almapstledidgeistocrfipyteiaorns-.sample number, assign a method 12.1.2 oTStIohRecrr(eSaaiptnpegrloepmrlieoatntheRoemdcaoscrlsdieicskn.go)An. ssScceaatnnlbiousntiutzsoauntaiilnoltnyhMceoolAdlceeqcutaiessdiaatpliporononpgcrwoiniatttrheoltahnpedaSnmIeaRlssa.sntdSoas4ev9lee9ctor method. 12.13 tThyepisceaclolnydtsheetosafmsptlaendlairstdsb.egins with the first setofserum standards and ends with 12.14 sSaammppllee.s aSroelvaennatlybzleadnkwsisthhoaulcdonbteinaunianlgyzcealdipberraitoidoniccahlelycktoinmjoencitteodarfptoesrseivbelreyantaelnytthe carryover and are not considered samples but `may be includedassuch. 122 Using the Autosampler 12.2.1 Setup sample tray according to the sample list prepared in section 12.1.1. 12.2.2 iSanneastlt-yrusuptmtechnoetnsHliPodg1eb1ros0o0ak/p:paruotporisaatmefpolreaortpttihemaflolrleoswpionnsgec.onRdeictoirondsoacrtauatlccoonnddiittiioonnsstihnethe 1222.1 Sample size = 10 pL injection with a sample wash 1222.2 Injectsample = 1 1222.3 Cycle time = 15 minutes 12.224 Solvent ramp = Time Ammo2n.i0ummMacetate [[0(70Smmiinn1 TT 04 % | |5 stso%%%|| (Lomin OfSmin | TT 90% | a5% | 10% sm | | osNnootftthew:eaIrHenPwtWihotirshkasint"saWttariuiomtnei.nntg cfoornfinilgeturstaatni"tohmnee,srsuangemubsetfobree stehte0u"pS0toa0nrt0t"hbe8uet7lteocntrisospprreasysed 12.2.2.5 Press the "Start" button. FACT-M4.0 Analysis of Serum Extract Using ES/MS Page 4 of8 123 Instrument Set-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2Checkthe solvent level in reservoirs and refilifnecessary. 12.3.3chCehceckkththeetispt.ainTlheesstsitpeeslhocualpidlblaeryflaatttwhiethenndoojfagtgheedpredogbees.. UIsfetahne teiypeispifeocuentdoto be unsatisfactory, disassemble the probe and replace the stainless steelcapillary. 12.3.4 OSebtseHrPvLe Cdrpopulmetpstcoo"mOinn"g.Soeuttotfhtehefltoiwp tooft1h0e-p5r0ob0eu.L/Amlilnowortoaseaqpupirloipbrriataetef.or approximately 10 minutes. 12.3.5 Taruorunnodnthtehetinpiotfrtogheen.prAobefi.ne mist should be expelled with no nitrogen leaking 12.3.6 cThhaenignestirnuomrednetrutsoeospttihmeiszeeptahraemreetseprosanstet:he following settings. These settings may 12.3.6.1 Drying gas 250-400 litersour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 1236.3 HPLC constant flow mode flow rate 10-- 500 L/min 1236.4 HPrPeLssCuries <op4e0r0atbianrg(cTohrirsecptalyr.a)meterisnot set, it is a guide to ensure the 12.3.7 Cfuarrtehferu.llCyognuniedcettthheepvroolbteaignetcoatbhleesotpoetnhiengp.roIbnes.ert probe until it will not go any 12.3.8 Record tune parametersinthe instrument log. 12.3.9 tUhseinangaltyhseicsorofsbsi-oflloogwiccoaulnmtaetrreilceesc.trode in the ES/MS source is recommended for 12.3.10oCflsiacmkpolnestlasrtt.buEtntsounreintsthaertAacnqduiesnidtisoanmCpolnetrnoulmbPaenreli.nclPurdeesss atlhlessatmaprlbeustttoonbeat top analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.14 Calculate matrix spike percent recoveries using the following equation: %Recover=y OB bseravedcReEskxupletcg-tedrReso ult uRensuldtx 100 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Co`nEex.pe-ctCeadlcCuolnact.ed Cone, x 100 000088 FACT-M-4.0 Analysoifs Serum Extract Using ES/MS Page Sof8 13.1.6 Calculate actual concentration ofPFOS, or other fluorochemical, anion in serum (pg/mL): uIgniotifalPVFOolcualmce.offrsoemrsutmd.(CmuLr)ve x Dilution Factor x Final Volume (mL) 1M 144.01 e The mT ethodH detecO tion lD imit isP equalE tohaR lftheF lo~ wesO t~sta~ nR dard M in theA cal0 ibrN at0 ionC c0 urve.0 E 142 The practical quantitation limit is equal to the lowest standard in the calibration curve. 1P 155.01OSamL pleL extrU act wT asteIandO flaN mmWaAbP SleTsEoR MlvAeNnE tAiGsEV diMsEpE oNsTedN in hT igh BITUO contN ainerA s, aN nd glD ass. pipette waste is disposedin broken glass containers located in the laboratory. l0 60Recom0 s 00000 16.1 iStnofroermcahtrioomnaitnocglurdaemdseiinthtehreisntuthdey hfoeladdeerr. oErahcahncdhwrroimttaetnoognratmhemcuhsrtohmaavteogtrhaemf:olsltouwdiyng number, sample name, extraction date, and dilution factor(ifapplicable). 1622 Plot calibration curve by linear regression and store in the study folder. 163 Print sample list from MassLynandtape into the instrument runlog. 16.4 Printdataintegration summary from MassLynx and tape into the instrument runlog. 165 Ctaoppeyinitnosatprpurmoepnrtirautenlsotgudpyagneost,eibnocolku.ding instrument parameters and sample results, and 16.6 Summarize data using suitable software and store in the study folder. 16.7 `Baandckloucpateiloencotrfobnaicckduatpaetloecatprpornoipcrdiaattae.medium. Record in study notebook the file name 17.0 TABLES, DIAGRAMS, FLOWCHARTS. AND VALIDATION DATA 17.1 Anachment A: FACT-M4 Data reporting spreadsheet 172 The validation report associated with this method is FACT-M-3.0 & 4.0-V-1. 18.1 AMDT-EP-31, "OperationofVG Platform Electrospray Mass Spectrometer" 190 ArpecrepDocumenrs 00000000 19.1 FHPALCCT--EMl-e3c.t0r,os"pErxatyr/acMtaisosnoSfpFelcutorroomcethreym"ical Anions from Serum for Analysis Using 000089 FACT-M4.0 Analysis ofSerum Extract Using ESMS Page 6 of 8 WORevisows Revision Number, BN ReasonForRevision 0000000000 Revision Date FACTV4.0 Analysisof Serum . -_t Using ESMS 000090 Page 70f8 Laboratory Study # STMetasurtdyiM:xeFriiaalSolvent: MAneatlhyottiRcaelviEsqiuoinp:ment Syste Number IFnistnraummeent Sorenson: ReSquared Value: SDlaoiptneo:afrEexpr:cton/Anays: DateofAnalysis/Analyst: DGrooww Concuemnilr.aton| maI=V)al | DFaoctnor | FmwwilC.one SGlroopueprDToaskee:s FTraokmenTifnerraoem tgherdeysfcoqludaeiron SCounrcmepnlter:atTiaokne(nsf/rmoLm)t:heTdakyenfforlodmerthe Massy negation summary: TDialiutlioVnoFlaucmteo:(mTLa)k:enTafkreonmrthoemsthye sfoyldefrolder Final Conc. (ug/L): Calculated bydividingthe nia volun rom he concenrion 00009! FACT-M-4.0 Analysis ofSerum E>-~:ct Using ESMS Page 8of8 PCoAtSsNumbePror2h7a5r5o3oc5a5nesuionss Laboratory RecueEsntvNouT mnbmeern(tRNe LHaUbZo0re0r6 ATTACHMENT G: RESULTS OF CONFIRMATORY DOSE ANALYSES Prprtay and Condotal 000092 Sec C E ET E =] E E ETE ElTT Ea a Z Elton 000093 ATT | EBrrm 3B= Ee = || gp - || E EB e Elm 000094 . 3M SPECIALTY ADHESIVES & CHEMICALS ANALYTICAL LABORATORY To LeoGehl-h(3o09f727f) -3MChemi-2c36a-2lA-s01 Request #'s 53030 From: `TomKes-t(3n-5e63r3) SAZC AnalLyabt-2i36c-2a81l1 Subject: Date: -_-- FluorocIshomeermDisitrcibuatilon by "*F-NMR Spectroscopy December 1, 1997 SAFMCP-L9E5,DEloStC#R2I1P7T(ITO-6N2S9:5); Nominal product =C,F-SOs(-) K(+) INTRODUCTION: p`coTohsniscsiebsnltaerm.aptlieonwsaosfstuhbejefcltueodrotcohaem'icFa-lNiMsRosmpeersctarnadlaasnmalaynsyis omtehtehrioddenttoifdieabtleeirmmpuitrnhieetyidceontmiptoienseanntds raeslative EXPERIMENTAL: pA(rFe5pp3oa0rrt3ei0do.tn4ho0fe1ts)hawmeapsslaeamfcpqolureiarsneoaldliyudssiwisanasgnatdosVtaehlreliayzldnsU oiacsquN siiprolenudDlasI Mn4vSd0pO0elT -oFddtT;t-eY aNdtnMdhRtehNespnMecRat3ros7mp6eetMcetHrr.uzm.A"i*dFa-RNoMbRbsipnesctrum RESULTS: BTquhuoaernto"icFtha-etmiNviMecRaclosmpipesocostmiretriusomanwanaldsrteuhsrsuleetedsottwohhdeierctihemrwpmueririneteytdehcreoimviepddoeanfterinotetmsstaihnnetdshirinesgllsaetaimtvrpeilcaelo.n"cTFenh-teNrqaMtuRiaoslnpsietcotafrttalhaienavnndeaolmyisinsalare summarized in TABLE-1 on the following page. ycAoocumophplaeyvtoeifangtnhtyheqiNuseMwsotRriko.snpsaebctoruutmtahnedstehNesMpRecretsrualltass,sipgnmlenlteestdmaateakspnaogewe.arIeaatptacohedlffooorrytgohueirdreelzfaeyrieennce. If Tom Kestner c: Jim Johnso-n EEZ-2P3E-C09 FitReimer: LG$3030.0CH3 000095 ~ December 1, 1997 SA&C Analytical Lab Request # 53030 TABLE-1 _-- Te `Sample: T-6295 (FC-95, Lot 217) F-NMR Compositional Results CFy(CF2)-SO(-) K(+) (Normal chain, where x is mainly 7) CF5(CF2)-CF(CF;HCF2),- SO) K(+) (internal monomethyl branch, `wherex+yismainly 5, and x =0, y 0) (CF3),CF{CFa)- SOs(-) K(+) (Isopropyl branch, where x is mainly 5) 70.0% ee (Alpha branch, where x is mainly 6) (oul raach, where iy -- TE EEO (nent gCeF3n~(nCFia)-rCa(nCcFh3)w{eCrF:e),ke- SO5s(a-)itKh(+4),0 x20) 0.15% 000096