Document N227Voe1O6dEwzgLYLkM1wNNQ

3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 9M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 LIMS E00-1668 StudyTitle ARR26_ 1103 Extended Recovery Study Following a 26-Week Capsule Todcly Study (FACT-TOX-030) with Perfluorooctane Sulfonate Acid Potassium Salt (PFOS; T-6295.22) in Cynomolgus Monkeys Analytical Laboratory Report Title Determinaotfitohne Presence aonfdGyCnonocmeonltgrautsioMnonofkePyFsOS in Serum and Liver Samples DatNaotRAepqpuliicraeblmeent . 3M EnvironAmuetnthaolrLaboratory Study Completion Date May 3, 2002 Performing Laboratories Sera andLiverAnalyses Sera and Lvor Extractions aM Environmental Laboratory Pace Analytical Senices, c.--Tier Faciy Buiding2S:t3P5a.u0l9,,M93N55B5u1s0h6 Avenue 17M0i0nnEeampoSliirse,atM,NS5u5e411400 Projectidentification 3M M`eCdoivcaanlceDeIpna-rLtfemeSnttudSyt:ud6y3:29T--2662985.22 Analytical Report: FACT TOX-160 "3M LIMS No. E00-1668 Total NumberofPages 13 = e 22 tE=Z] =2 38 =3 3M EnvironmaenwtEanlviLraobnomreanttoalryLaboratory 000055 roirry Page CAIN NO CBI Page 1 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: LFIAMCSTET0O0X--1166608 `This page has been reseforr svpeecifdic country requirements. 3MEnvironmentalLaboratory 3M EnvironmentalLaboratory 000056 Page 2 Page2 3M Medical Department Study: T-6295.22 aM Medical Dopartmnt Sucy:T-6205.22 GLP Compliance Statement Analytical Report: FACT-TOX-160 LIMS E00-1668 Anayical Report [FiAvCeTTO0X1-616680 Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS in Serum and Liver Samples of Cynomolgus Monkeys `Study Identification Numbers: T-6295.22, FACT TOX-160, LIMS-E00-1668 `This study was conducted in compliance with United States Environmental Protection Agency (EPA) Good Laboratory Practice (GLP) Standards 40 CFR Part 792, with the `exceptions in the bulleted list below. Exceptions to GLP compliance: There were two study directors in this study. This study was designed as four `separate studies. The in-life study phase was considered to end at the generation and shipment of specimens. The analytical study phase was considered to start at the receipt of these specimens for analysis. This resulted in having two separate `study directors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception. There were two in-life studies and two analytical studies that utilized the same test system. These studies include in-life studies Covance 6329-223 and Covance 6329-268 and analytical studies FACT-TOX-030 and FACT-TOX-160. Thepurity and stabilofitthye reference standards are not included in this report, they are not known at this time. Chndites T)_Saeal `Andrew Seacat, Ph.D., Study Director Fh 2. Riplaf John Butenhoff, Ph.D., Sponsor Representative May 32nd pon May 3, 2002 Date #13 & Clemen, Principal Analytical Investigator Wilip amFoager n, PhD. Anal-- yticalLaboratoryManager 4Eninmariat Laborsiors 3MEnvironmental Laboratory 0s, Date oBasts ty ho 000057 Paces Page 3 3M Medical oo---- Department Study: T-6295.22 arm Analytical Report: FACT-TOX-160 nr LIMS E00-1668 rgie fraud GLP Study--Quality Assurance Statement PFOS in Serum and Liver Sampleosf Cynomolgus Monkeys. Eat `Study Identification Numbers: T-6295.22, FACT TOX-160, LIMS-E00-1668 This study has been inspectedby the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the`study | noone [ee Date Reported to ee] ocBnEanrErRe ||eorn |[wowne ||oowwne| 02/01/02,02/06/02-02/08/02, Ta QAURe tative S/1, ate ira ro000058 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 `Analytical Report: FLIAMCSTET0O0X--1166608 Table of Contents GLP COMPIENGE SIEM... d (GLPStudy - Quality ASSUTANCE SIBEMN srs LAEVIS assmmmmmmimibommmmmmg------------_ Stuy PerSOnNel 81d COMIBULOTS vcs. LL `Specimen Receipt and MaINENANC.......c.uwwmmmmmssmssssiond Chemical Characterization of the Reference SUBSIBNCE ummm:10 ET ----| A. A| L AVIE EAI wmsonmmeorsusmsmmmmmesmomsom-- m----g Le L `Summary of Quality CONG! ANL BYSES RESUS...v.vrronrvr-- nrrenrmrsmrenrnn1e4 . SSUIMEIOMMEOTNYEOOFfADMaPt SQURAIEHSYUIc S...v.v.v vrrcornrss rvsvsvsrssssessinsosososmonen11e68n Statistical Methods and CalGUIBHIONS sven:AT SHEOfECONCNIISI!ON rns AT APE A COP MRE anges AS Appendix B: Protocol, Amendments, and DEVBHON(S) vives19 AppONix C: EXracion and ANalical MOtNOGS..........rsrsnnnne4s0 EHPTLSC--8E4I.e2c,t1"E0xStprraacyti/oMnasofsFSlpueocrtorcohmeemtriyc,a"l (C1o5mPpaoguendss)f.r.om.S.errwurmmfosrmAnnarlymsmismUissinmgrrs 41 ETS-8.6.0, "Extraction of Potassium Perfluorooctane-sulfonate or other Fluorocherical `GCAoPmIpGouEnNdsrsfrtogm eLitvermfrorsAnmalmymsims UmsminrgiHPmLCm-mEleecrtrsomspgraoy/mMsasssaSpmecTtrmommet--ry--,--" ----" 0 ESTeSr-u8m-5E.x2t,ra"cAtnsalUyssiinsgofHPPLoCta-sEslieucmtrPoesrpflruaoyrMoaoscstaSnpceecsturlofmoentartye.o"r(O1t1hPeraFiugoreochsem)ica.ls.in.70 ELiTvSe-r8-E7x.tr0a,c"tAsnUasliysnigsHoPfLPCo-tEalsesciturmosPperrafylu/oMraososctSapneec-tsruolmfeotnrayt,e"o(f1o0tpheraFlguoerosche)mi.cal.s .in 81 ARPT DONS SURREY TH mms 3m Environmental Laboratory 3M Environmental Laboratory 000059 Page 5 Pages 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 `3M Medical Department Study: T-6295.22 Analytical Report: FLAMCSTET0O0X--1166608 APPONGiX F: EXMPIE CalGUBNONS...rsnssssnninenmismmsnosnss 107 Appendix G: Iai Corticate(s) of ANBIYSIS ......ovevcvsrsensrasersrnren 108 Appendix H: Report SIGNatUre Page... 113 List of Tables Table 1. SHY THONG... Table 2. Cynomoigus Monkey Specimen Receipt for Study (6329-268) rnd Table 3.PCAhaCrTaActCeKriAzRatDi.onoftmhmemAanarlyntsicsalaRmesfemrmennctemSmubnsitnanpcmeminmSitmudmymsonmionon 0 Table4. Target 100s Monitored in OM Laboratory ANBIYSeS.........c.rurmmmnne1n3e Table5. Determinationsofthe LOQ For the Extracted Curve In the AnaloyfSseerusm Table 6. LIVEr Matrix Spike RECOVENIES....uruvronsssnannnsnsnsesnsnnn1s8 J ---- `Table 8.SCUhAarYacFtAeCriTz-aTtiOoXn-oAfBt0hec Controlo Matricr es Useo d for Sen rum ane d Livern Analyse es in 18 Table 9. Data Summary for PFOS in Serum FACT-TOX-16-0R/ML vrs 91 Table 10. Data Summary for PFOS in Liver FACT-TOX-160 - Kg/Gvuvvvurcrararrnsn OT 3M EnvironmeSnMtaElnvLiaobnomraetnotrayLavoratory 000060 Pages Page6 3M Medical Department Study: T-6295.22 3M Medical Department Stuy: T-6296.22 _St--udy Personnel and Contributors Analytical Report: FACT-TOX-160 LIMS E00-1668 `Analytical Report: FACT TOX-160 uwsEwess `Study Director Andrew Seacat, Ph.D. 3M Corporate Toxicology Building 220-2E-02 St. PaMuN5l 51, 44 Analytical Chemistry Laboratories Sorum and LiverAnalyses 3M Environmental Laboratory (3M Lab) Lisa Clemen,PrincipalAnalytical Investigator Sponsor 3M Corporate Toxicology 3M Medical Department `Building 220-2E-02 St. Paul, MN 55144 John Butenhoff, Ph.D., Sponsor Representative Sorum andLiverExtractions Pace Analytical Services, Inc.--Tier2 Facility 3M Lab Contributing Personnel Rhonda S. Dick* Kelly Dorweiler* Kristen J. Hansen Marlene M. Heying* "HCaroonlrdaicOat. pJroehsnsisoonns sav apiopees Ognjenka Krupljanin Kelly J. Kuehiwein Sally A. Linda* Bob W. Wynne" Location of Archives 2 according to 40 GFR Pant 792 requirements. All original raw data, protocol, and analytical report have been archived atthe3M Environmental Laboratory and will be retained according to 40 CFR Part 792 requirements. The test substance and analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study are archived at the 3M Environmental Laboratory and will be retained 5M Envirnmants Laboratory 3M Environmental Laboratory 000061 Pace? Page 7 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 _------ Introduction and Purpose Analytical Report: FACT-TOX-160 LIMS E00-168 Analytical Report: FLIAMCSTET0O0X--1166680 `"Tmhoenkpeuyrpsoesreaoafntdhelisvetrusdyamips ltoesdattaekremnifnreotmheCopvraesnecnecsetaunddy#c6o3nc2e9n-t2r6a8t,io"nExotfePnFdeOdSRienccoyvneormyoSltguudsy 6Fo2l9l5o.w2i2n)gian C2y6n-oWmeoelkgCuaspMsounlkeeySst.udyTwhitehaPneirmfallusorforoocmtasnteuSduyif6o3n2ic9-A2c6id8 wPoetraespsrieuvmioSuasllty(aPsFsOiSg;neTdtPoeraficuoomrpoloectteadneinS-ullffeoCniocvAacnicdePsottuadsys#i6u3m2S9a-l2t23(,FF"O2S6;-WTe-6e2k95C.a7p)suinleCyTonxoimcoitlygSutsuMdoynwkitehys" and a C`caopmspulleeteTdoxaincailtyytSictauldsytwuidtyh#PFeArCfTlu-oTrOoXo-ct0a3n0e,su"lAfnoanliyctiAccaildLPaobtoraastsoiruymRSeaplotr(tTf-6r2o9m5t.h7e) 2in6C-yWneoemkolgus. (MPoFnOkSe)ysionnLtivheer aDnedteSremrinuamtiSoanmopfltehse".PrDeusreinncgesatnuddyCo6n3c2e9-n2t2r3a,ttihoefnanPiemrfelsuorreocoeciivaendes0u.f1o5nate rmegc/okvge/rdy.ayAotftPhFeOenSdaosfathseinignilteiadlairleycoCvaeprsyultehedaonsiemfaolrsawt elerasttr2an6sfweererkedstfoolalofwoeldlobwy-uap 5s2tu-dwyesk t(hCeovfoalnlcoew-6u3p2s9t-u2d6y8., T-6295-22) for evaluation of extended recovery. Animals were not treated in ``TChoevasnecrea aAnnadlyltiivcearlsRaemspelaerscfhorLtahbiosrsattuodryieasreuntdheerpsrotduudcyt 6of32t9he-2i6n8-if(eT-r6e2c9o5v.e2r2y).stAundyalcyosmepsleoftesderbay ``aTnOdXi-v1e6r0s(aEm0p0l-1e6s6w8)e,reancdomtphleetreedsibsy otfheth3eMseEanvniarloynsmeesnatraelpLraebsoernatteodrynutnhdiesrresptourdty. nTuhmebeanralFyAtiCcTalportion of this study was initiated on 27 August, 2001. Table 1.StudyTimeline es Recovery Extended Recovery 3M EnvirSoMnmEennvtiarlonLmaebnotarlatLoarboyratory 000062 Paces Pages 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 `Analytical Report: LFIAMCSTET0O0X-.1166808 Specimen Receipt and Maintenance "tThhee3inM-IfeEnsvtiurdoyn#m6e3n2ta0l-2L6a8bofrraotmo0ry9rMeacye,iv2e0d0c0yntoom1o5lMgaurscmh,on2k0e01y.spAlelcsipmeecnismceonlslewcteerd artetcheeiveendd of `frSopzeecniminengsotohdatcownedrietieoxntorancdtreyd iactePaancdewTeirere2iwmemredeisahtieplpyetdrafnrsofzeernreodntdorsytiocrea.ge at -50C = 20C. [Roce | Tnepor | specimen | amberrecs | `Table 2. Cynomolgus Monkey SpecimenReceiptfor Study (#6329-223) [oso |Weeks| Som | 4 | [o7oa0|Weoki7 |"Senmirmeeces| amis | [om000|Weok2s| Sowm | 4 | [102400 |"Weok33|Senmiur|nerFwmeacos| [Zero|"Weskat| Sewn | 4 | | ariaot |"Weeks3|Serumin|elFaiesce| s [Lherung [4%] [Koney|Spiwaeen| [Abdom| inai4Fat| [ Hean | B 5 addiwtl oanal ve als| from sample 105552 f`Croonmtrcolommmaetrrcicieaslussoeudrcienssaernadaanred plirveesreanntaeldysinesApppeernfdoirxmeAd.dSuraimnpglFeAsCaTn-aTlyOzXe-d1a6t0twheer3eMobtained 7En9v2irreoqnumiernetmaelntLsa.boratory will be stored and maintained at the laboratory according to 40 CFR Part 3MEnviro3nMmeEnnvtiarlonLmaebnotraaltLoarbyoratory 000063 Pace 9 Page 3M Medical Department Study: T-6295.22 . 3M Medical Department Study: T-6295.22 Analytical Report: FLAICMTS-TEO00X--1166608 Analytical Report: LFIAMCSTET0O0X--1166680 Chemical Characterization of the Reference Substance Potassium`pGeArSflNuuomrboeocrt:an2e7s9u5l-f3o6n-a3te (KPFOS) Chemical Formula: CaF 805K" Molecular Weight: 537.9 Cprheesmeinctaeldcihnatraabcutlearrizfaotrimonbienlfoowr.mation on the reference substances KPFOS used inthisstudy is Table 3. Characterization of the AnalyticalReferenceSubstances In Study FACT-TOX-160 PrOS | (Surogate Standard) Substance | noe | TcRoooross [own | a0| mows | [[ ConEcE tones p[ ||rocnco am usroreso e| ||tnaet n mcwcouem io o e| | [Eoe)n[ro| wweee r r| 10 ara low 1 5 ow| w EEE pwntomar tr Aer. 3M Envir3oMnmEennvtiarlonLmaebnotraaltLoarboyratory 000064 Page 10 Page 10 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FLIAMCSTET0O0X--1166608 r eee ye ----eeee Sample Preparation and Analysis Sora Analyses As per the study protocol, all serum samples were analyzed In this phase study. me`Stehrya-staemrptlbeustywleerteheerxt(rIaBcEt)ed.beSgaimnnpilnegeoxntr3a1ctAsuwgeurste,a2na0l0y1zeudsiunsgiangn hiionghp-apierirnfgorrmeaangceentliaqnuidd cmhordoemavteorgsruasphayn-eexltercatcrtoesdprraabbyittasnedream cmuarsves.spPeFcOtrSomleetvrelys(wHePrLeCq-uaEnStMitSaMteSd)biyn etxhteermntailpclaleibrreaatcitoino.n Liver Analyses. As per the study protocol, al liver samples were anelyzed in this phase study. Lmievtehryls-aemrptlbeustywleerteheexrtr(aVcItBeEd).begSianmnpilnegoexntr0a5ctsSewpetreembanearl,y2z0e0d1usuisnignghiagnih-opneprafiroirnmganrceealgieqnuitdand cmhordoemavteorgsruasphayn-eexltercatcrtoesdprraabybittalnidveermcmuarvses. sPpeFcOtrSomleevterlys(wHePreLCq-uaEnStiMtSaMteSd)biynetxhteermnuallticpalleibrreaatciotni.on Method Summaries FEonlvliorwoinnmgenistaalbLraiebfordaetsocrriyp.tDieotnaoifletdhedemsectrhipotdisonussoefdtdhuerminegththoidssanuaslyetdicianlthsitsudstyubdyytahree 3lMocated in Appendix C. g 3M Environmental Laboratory PREPARATORYMETHODS EElToSc-t8r4o.s2p,ra"yEMxatsrsacSpteiocotfnroFmieutorryo"chemical Compounds from SerumforAnalysis Using HPLC- Arneaalgyetnitcawlassamapdldeesdwteoraeleaxbtorraacttoerdy ussaimnpglaenanfodn-tphaeirainnaglyetxteriaocntipoanirprwoacsedpuerrei:iAonneidoni-nptaoimreitnhgylutnoirltbdurtyy.l-Esatchehr(exMtBrEac)t.edThlaeboMrIatBoEreyxstaramcptlweawsasthreencornesmtiotvuetdedainnd1.p0utmnLtoofamneitthraongoenl eavnadpopraastsoerd through a 0.2 um nylon fiter using a 3 mL. disposable plastic syringe into glass autovials. 3M Envir3oMnmEennvtiarolnLmaebnotarlatLoarboyrators 000065 paca 11 Page 11 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 `3M Medical Department Study: T-6295.22 Analytical Report: LFAICSTET0OoX--1166680 ETS-`8C-o6m.p0o,u"nEdxtsrafcrtoimonLiovfeProftoarsAsnialuymsiPserufsliunogroHoPcLtGa-nEel-escutlrfoonstperoafyo/tMhasesr SFpleucotrroocmheetmriyc"al `LiTvHePrFsOaSmpalnedsewxetrrsachtoedmougseinngizaendioinn-wpaatierri.ngAenxtarlaicqtuiootnopfreoaccedhuhreo.moAgnenioant-epawiraisngspriekaegdenwtitwhas t`raandstdfoeetrhdteroesadamcpelenatndrtithuefbaeunaagnlyedtepiuotnpoanitrowansitrpoagretnieovnaepdorinattoorMIuBntEi.l dTrhy.eEeaxctrhaecxttwraacstwas dreicsopnosstaibtluetepdlaisnt1ic.0symrLinogfemientohgalnaoslsaanudtopsaasmspelderthvriaolusg.h a 0.2 um nylon fter, using a 3 mL. ANALYTICAL METHODS `SEeTrSu-m86E.x2t,ra"cAtnsalUyssiinsgofHPPLoCt-aEslseicutmrPoesrpfrlauyo/rMoaocstsaSnpeescutirfoonmaetteryo"r Other Fluorochemicals in * EExTtSr-a8c:t7s.0U,si"nAgnaHlPyLsiCs-EolfePcottraossspiruamy/PMearsflsuSoproeoccttraonmee-tsruyl"fonateorother Fiuorochemicals in Liver `pTrhiemaarnyaliyosnecshawrearcteerpiesrtfiocromfead pbayrtmiocnuilatrorfilnugoroonceheorricmaolruespirnogdHucPtLiCo/nEs SseMlSeIcMteSd.fFroormeaxasmipnlglee, tmoolpercoudluacreiioonn49999,(sFeSleOc)t.edTahsetchhearparcitmearirsytiiocniofnor9P9FwOaSs (mCoonFi:t,oSrOeyd-)foarnqaulaynstiist,atwiavse afnraalgymsiesn.ted ANALYTICAL EQUIPMENT `The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-PackardSeries 1100 Liquid Chromatograph system CAnoallyutmincatlecmopleuramtnu:rKe:ey3s0toCne BetasilTM C15 2x50 mm (5 um) Mobile phase components: C`Coommppoonneenntt BA:: m2emtMhaanomlmonium acetate FInljoewctriaotne:vo3l0u0meL:/m10inyl Solvent Gradient: 5.0 minutes Time (m0in0utes) 1%08% 5150 9150%% 8705 9150%% 3M Envir3onMmEennvtiarlonLmaebnotraaltLoarbyoratory 000066 Pace 12 Page 12 3M Medical Department Study: T-6295.22 `3M Medical Department Study: T-6296.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: LFIACMTTE0O0X--1166608 MSoafstwsaSrpee:cMtarsosmeLtyenxr"; M3i.c4romass"APIMass Spectrometer Quattro II" Triple Quadrupole system CCoollniesiVoonltGaagse:E3ne0r-g7y0:V20-50 eV M`Sooduer:ceEBlleocctkroTsepmrpaeyrNaetguartei:ve150C 10C AEnlaecltyrsoidseT:yZp-es:prMualytiple Reaction Monitoring (MRM) [ Tabl4e. Ta arget lo|nsm MeroinmiaytiooIo nrnteSaMdnL)a|bo_rr aptroorcywAcntale oynsaemsy| [TwreossT | wre| Tw mmeo|| Deviations/Amendmonts `dTehveiraetiwoenrseforonmetahmeeonridgmineanltpraontdocsoelvaennddmeveitahtoidonssafrreoimntchleudoerdigiinnatlheprAoptpoceonld.ixAmB.endments and Data Quality Objectives and Data Integrity `The following data quality objectives (DQOs) were indicated in the protocol for this study: Li`naenadreiqtuyal:tTohoercgoreefaftiecrietnhtaonf0d.e9t9e0rmfionrasteiroan a(1n%a)leyqsueaslutsoionrgg1r/exawteeirghtthianng.0.985 for iver analyses + cLailmiibtrastoiofnQcuuarnvtei,tdaetfiionned(aLsOG)th:eTlhoeweLstOsQtasndeaqrduthtaottsheboltohwe2sttiamcecseptthaebmlaelsrtiaxnbdlaarndkianntdheis calculated within + 30% of the expected concentration. Ac`caneaplytsaesb,lperoPvriedceidsitohne:y aQruaelqiutaynctointtartoeldswaimthpilnetshearseelreecqtueidrecdaltiobrmatieon+er2an5tg%e.precision for AocfcelipvetraabnldesSepraikseamRpelceosvesrhioeusl:d sMhatorwixssppiikkeerseacnovdermiaetsriwxitshpiink7e0d~u1p3li0c%a.tes requfoiranraleysdis Confirmatory be witen. Method: If a confirmatory method is used, an amendment to this protocol will + Dreetmeontnisotnrtaitmieo(napopfrSopxeicmiaftiecliyty8:.0P0FmOiSneidse)n,tifbiycatthieoncwhialrlabcteersiusbtsitcanptriiamtaerdyblyonc(hr4o9m)ataongdraphic `characteristic product ion (99). 3M EnvirSonMmEennvtiarlonLmaebnotraaltLaobroyratory 000067 Pace 13 Page 13 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACT TOX-160 e Ee e ese rst LIMS E0e o-1668 in Data Summary, Analyses, and Results DLaabtoarqautaolriytyproobtjoeccotlivfeosrfFoArCtThe-TanOaXl-yt1i6ca0l(psheaesAepopfetnhdiisxstB)udwyeoruetlmineetdwiinththteh3eMexcEenpvtiiroonnsmennottaeld in this report. `Summaryof Quality Control Analyses Results + aLinnaelayrsietsy:anTdhe20c.oe9f9f0icfioernsteorfadaentaelrymsiensa.tion (19) of the standard curve was 20.985 for iver `Caanlalbyrsiast,i1o/nxSwteaingdhatreddso:f oQunaentoirtattwioonexotfratchteetdarmgaettriaxnacluyrtveesswbarsacbkaestiendgoenaclhinegarroruepgorfesssaimopnles, edexaccelpitvaatsendottoedprionvtihdee daevbieatteironlisnueamrmiatroyv.erHtihgehcaunrdvleorralnogwepomionstts oapnptrhoeprciuartveetmo athyehdaatvae.bLeeonw tcourdvisequpaoliinftysawidtahtpaeraakngaeretahsatlmesasythhaanvetwboeetinmseisgntihfaitcaonfttlyheafefxetcrtaecdtiboyn bblaacnkkgsrowuenrde ldeevaecltsiovfattehde `baenealnytdee.acOtcicvaastieodn.alQluya,natistiantgiloenomfied-arcahngaenacluyrtveewpaosibntatsheadtownastahen roebsvpioounsseoouftliaenremsapyechifaivce p`ArnoadluytcitcaflonM(est)huosdisn)g.the mitple response-monitoring mode ofthe instrument (see Appendix C, LicmaliibtrsatioofnQcuuarnvteittahattioisnw(iLtOhiQn):3T0h%eofLOthQetisheeoqruetailctaotlhvaeluleo,weasntdaicscaetptleaabsltetswtoantdiamreds nthethe analyte peak area detected in the extraction blanks. I TT `oTfabSleer5.umDeatnedrmLiinvaetriEoxntsroacfttshe LO_- QFortheExtractedCurve intheAnalyses. [rosie[momo | Blsaimnpklisfy:aAnlal lbylsaensksthwaet rweerbeelcoowmptlhieclaotweedrblyimeint dofogqueannotuitsalteivoenlfsoorfthfelucoormopchoeumnidcsalosfinintuenreesxtp. osTeod smuointakbeleyssuerrraowgahtiechmawterriex aabnodvaelltrhaebbliotwseerrlaibtlaonfkqsuawnetriteatwiiotnh,inrcarbibtietrias.era was selected as a Prweicthiisni2o5n%:,pprreecciissiioonnwwaassddeetteerrmmiinneeddbybyanaanlalyyssiissooffCCCGVsVinn sievrearaannddwwaassrreepprroodduucciibblleettoo within 30%, Ma`tanrdisxerSapsiakmesp:lesMaatnridxasnpailkyezseadnadt tmhaetr3ixMsEpnivkierdounpmleinctaatlesLwaeborreaetxotrrya(ctseede wtiatbhleesa5chansd 6oe)f. Tvtweor rsapbibkietswlieverremnaottrixwistphiikne+s,30ex%torfacttheedt0h9e/o0r5e/t0i1c,alwceornecewnittrhaintiCornitwerhieananadnatlwyozemdon08k/e1y7/l0i1ve.r mTahterix emnodnokgeeynoliuvser lmeavterlisxpsrpeiskeenst wien rtehensoatmpprleepsa.reAdddaitttihoenaalppmroonprkieaytelicvoenrcmeanttrriaxtsipoinkbeassaenddoonne monkey liver sample wore re-extracted on 10/02/01 at lovels appropriate to the endogenous 3M EnvirSoMnEmnevnitraolnmLeanbtoarlatLoabroyratory 000068 Pace 14 Page 14 3M Medical Department Study: T-6295.22 SM Medical Department Stuy: -6296.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analcal Report: FLAiCuTTE0O0X--1166808 ilenvceolnss.isTtehnet rsea-mepxtlreacctoendcesnptirkaetsiodni,d innoctomnesiesttecnrtitmeartiraiaxnsdptihkee rexetcroavcetriioens,waansdshuisgphecsturdruoegattoean values - refer to deviations attached to this report for more information. Additional monkey liver s`mpaitkreixssapnidkessamapnldeowneermeoinnkaegyreleivmeernstamwiptlhetwheerienitrieal-eexxttrraacctteidonoonn1019//0075/0/10.1.TThheeexrtabrbaictteldiver `matrix spike averages extracted with the samples on 09/05/01 and the monkey liver matrix spike averages extracted on 11/07/01 were within + 35% of the theoretical concentration. versus an unextracied (solvent) curve. All sera matrix spike recoveries, evaluated versus extracted and unextracted curves, were `within + 25% of the theoretical concentration. Extraction efficiency and absolute recovery were >100%inthesera matrix, based on average recoveries of 101%, 111%, 111%, and 116% Table 6. Liver Matrix Spike Recoveries Date Extraction Tver Type |% Recovery| Average T Com [oE e 2] OT | 100401 | ankey |__55|% SEES consist Monkey |_60%| = ESN een" [IGT | wonkey surogate | Reanabss| zug |_| [rm| doTvi0a2io4n1for MHonkey |6%| analysis T0201 | Wonkey|[_a 17w 0||% ton| 2990 [ot%| mex Sampo | M1o0nugklegy|200%| m7 ASOT |[M2on6key|[10o0m%|| ex WToonukesy [ei| 3M Envir3oMnmEennvtiarolnLmaebntoarlatLoarboyratory 000063 Pace 15 Page 15 3M Medical Department Study: T-6295.22 at eps uc Tass28 Analytical Report: FACT-TOX-160 srr a pie ri 'LIMS E00-1668 a a i STSeroa,| [rmyr||]| "Table7. Sera Matrix Spike Recoveries m2m |E|e foEienEe,r|20n5%ey [[1o0%n|] oH EE |py | | fSinoctiiee || eoenyr Evaluated Monkey Jen UnVeexrtsruascatned| 20 ug/ml. nw Surrogates: The surrogate (THPFOS) was added to alsl amples.and standards. THPFOS was not usedfor quantitation, but was used to monitor for gross instrument failure. The surrogate foesvtiA i 50% oprLansLof dE rs on 02e 40, Ths sami dreetsepromnisneestwheatraitwdiitdhinnot+v5a0ry%meoxrceeptthafnor+an5a0ly%sifsroofmltihveermdeilauntiownisthoinn e1a0/c2h4/a0n1al.ytTihcaelsreuns.amApllles sitomant of ata custy `extracts met surrogate criteria requirements. tis not possible to verify true recovery of endogenous analyte from tissues without radio-labeled rinedfiecraetnecsetmhaattetrhiaels.eTdhaetaonalrye mqueaanstiutraetimveenttoo2f6a%ccourrgarceyaatveariilnabsleeraatatnhdis3t5im%eo,rmagtrreiaxtesrpiiknelisvteurd.ies, ti `Summaroyf Sample Results PPFOS results (those obtained using lot # 217)havebeen correfocrtpueritdyof the analytical Samples from Control Animals: No control animalswere included in this study. + Be CS ie free mlSo Samples from Dosed Animals: In general, PFOS levels found in sera of the test animals. `decreased over time. PFOS levels in male liverswere approximately half that determined in 3MEnvironmental Laboratory 3MEnvironmental Laboratory 000070 Page 16 Page 16 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 `3M Medical Department Study: T-6295.22 Analytical Report: FLIAMCSTET0O0X--1166608 eeeeeesoeeesmmseepbeo etreebopoeeemememeernseteer Statistical Methods and Calculations SFftaotrisetixcaalmpmleethcoaldcsulwaetrioenlsimuisteedd ttootgheenecraalctuelatthieoinvoefrmaenadnssearnudm sstaamnpdlaerddadteaviiantiFoAnCs.T-STeOeXA-p1p6e0n.dix Statement of Conclusion aUnnddelrivterheofcoalnldirteicoonvseorfy tshteudpyrcesyennotmostlugduises,motnhekefylusoroorcighienranlilcyaldoP sed wF wiathstO ohbesteesrS tvseudbsintatnhecesdeurruimng the in-lfe phase of the study #6329-223 (TOX-030). References None 3M EnvirSoMnmEenvnitraolnLmaebnotraalLtaobroyratory 000071 Pace 17 Page 17 3M Medical Department Study: T-6295.22 SM Medical DepaSry.m7e62n95t22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Avaya RorFeACTeTOx.e160 Appendix A: Control Matrices [Ia ESo-- mme |T Woorwmoysoram| | FweonwST asrum| prwomm ] TaSbtuldey8.FACChTa-raTcOtXe-ri1z6a0tionoftheControlMatrices Used forSerum andLiverAnalysesin [n[Perssatteoscrvon| | neoyassoiusm| | oremwrseonn| | rowu ie]| AM Frvirnmantal Laboratory 3M Environmental Laboratory 000072 Pana 18 Page 18 3M Medical Department Study: T-6295.22 `3M Modical Departmont Study: T-6206.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: LFIAMCSTET0O0X--1166608 Appendix B: Protocol, Amendments, and Deviations 3M Envir3onMmEennvtiarlonLmaebnotraaltLoarbyoratory 000073 Page 19 Page 19 3M Medical Department Study: T-6295.22 nand SSerT v R S1t PoPnatW,MMNN 55s1s333331 Analytical Report: FACT-TOX-160 Protocol#FACT-TOXE -160 a Study Title : Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys ANALYTICAL PHASE PROTOCOL Author Lisa Clemen Date: August 27, 2001 Performing Laboratories 3M EnvirSoenmreantaalndTecLhinvoelrogAyn&alSayfseetysSarvices Sera aPnadcoLTiiveerr2 FEaxctilrlayctions 3M Env9i3r5oBnmuesnhtaAlveLnabuoeratory 17M0i0nnEeampolSitsre,etM,NS5u5e4124.00 St.Paul, MN 55106 Laboratory Project Identification FACT TOX-160 Covance Inife Study Number: 6329-268 3M Medical Department Study: T-6295.22 ET&SS LIMS: E00-1668 3MEnvironmental Laboratory 3MEnvironmental Laboratory 000074 Page 10f9 Page 20 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol #FACT-TOX-L1I6M0S E00-1668 Study Identification `Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Sponsor 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Sponsor Representative John Butenhof?, Ph.D. 3M Corporate Toxicology 3M Medical Department `Building 220-2E-02 St. Paul, MN 55144 Telephone: 651-733-1962 Study Director Andrew Seacat, PhD. 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Telephone: 651-575-3161 Principal Analytical Investigator (PAI) Lisa Clemen Phase Locations In vivo Testing Facilty Covance Laboratories, Inc. M3a3d0i1sKoni,nsWmiasncoBnosuilnev5a3r7d04 Analytical Testing Laboratories (sera andliveranalyses) 3M Environmental Laboratory Building 2-38-09 935 Bush Avenue St. Paul, MN 55106 (sera and liver extractions) Pace Tier? Facility 1700 Elm Street, Suite 200 Minneapolis, MN 55414 ProposEexpdeSritmuednytaTlimSteatratbDlaete Experimental Completion Date 27 August 2001 29 April 2002 3M EnvironmentalLaboratory 3M Environmental Laboratory 000075 Page 2019 Page 21 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol HFACT-TOXL-I1M6S0 E00-1668 1. Stuoy Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) `with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys. 2. Purpose `This study is designed to continue the assessmentofperfluorooctanesulfonate (PFOS) levels in sera and liver during an extended recovery timeofapproximately one year following the daily administrationofthe test material by capsule to cynomolgus monkeys for at least 26 weeks and at least 52 weeksofrecovery. `The animals used in this study were previously assigned to a recovery phase ofa completed studyforat least 52 weeks. At the endofthis initial recovery phase study Covance #6329223,a partial hepatectomy was conducted on the animals, they were allowed to recover from the surgical procedure, then transferred to this follow-up study for evaluation ofrecovery for an additional 52 weeks. `The serum andliverofcynomolgus monkeys will be analyzed for PFOS. Additional tissues or fluids may be analyzed at the discretionofthe PAI or study director. The in-life portion of this extended recovery study was conducted at Covance Laboratories, study #6329-268. 3. REGULATORY COMPLIANCE "This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. 4. QUALITY ASSURANCE `The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. 5. TESTMATERIAL Refer to Covance Laboratory protocol for study #6329-268. Animals were not treated during this study. The FACT TOX-160 study is an extended recovery studyof cynomolgus monkeys from Covance study 6329-223 (analytical work performed under 3M Environmental Study # FACT-TOX-030), where animals received 0.15 mg/kg/day of PFOS lot #217 in a daily single capsule fora least 26 weeks, followed by a S2-week recovery period. 6. ConTROL MATRICES `Typesofcontrol matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. 7. REFERENCEMATERIAL FOR FACT TOX-160 Potassium perfluorooctanesulfonate (KPFOS), CF,,SO;K', CAS 2795-39-3 3M Environmental Laboratory 3M Environmental Laboratory 000076 Page 30f9 Page 22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol #FACT-TOXL-I1M60S E00-1668 `The target analyte is perfluorooctanesulfonate (PFOS), CF, SO; 8. TestSystew Cdyensocrmioblegd uinsmthoenokreigyisnawleCroevuasnecdeasprtohteotceoslt#s6y3s2t9e-m2,2a3nd(TwOeXr-e0m3a0i)n.taTiwneod amanldedoasndedtawos. efxetmeanldeemdornekceoyvserwyhsitcuhdywearned adlolsweedrdeuarlilnogcastteuddyto#G63r2o9u-p221.3T(hTeOsXe-a0n3i0m)alwserreecienicvleuddnedo in this further treatment during the recovery study. 9. SPECIMENANDSaMPLERECEIPT f`lTuhied3s MfrEonmvtihreoninmdeinctaatledLpaobionrtastoirnythreecsetiuvdeydfsrpoemciCmoevnasnocftehLaebforoaltloorwiiensg abtotdhyeteinsdsouefstahnedinlife phaseofthe study. All specimens were frozen and packed on dry ice for shipping. Body tissue/fluid Collected Expected # of specimens Fd] 2f,ol4l,o6w,i8n,g 1i0n,itainadtio1n2omfosnttuhdsy At scheduled sacrifices B2sa8mplsesen Total numberoftest animals: 4 tMhoediPfAiTcaatnidontshetostthuedynudimrbecetroor.fsamples analyzed may be implemented at the discretion of tShpeeSciammepnlse sTernatctkoin3gMSyEsnvtiermoSntmaenndtaarld LOapbeorraattionrgiePsrwoceerdeurreec.eDievteadilasnodftsrpaecckeidmeacncionrsdpiencgtitoon pfroresdeanmtaegdei,nrtehceeipphta,ssetorreapgoer,t.identification, chain ofcustody, protocols and data will be 10. PREPARATORY METHODS 10.1 CEToSm-p8o-u6n,dEsxtfrracotmiLoniovferPofotraAsnsailuymsiPserUfsliunorgoHocPtLaCn-eEsluelcftonraotseprorayO/tMhaesrsFSlpueocrtorcohmeentircyal 10.2 ETS-8-4, ExtractionofFluorochemical Compounds HPLC-Electrospray/Mass Spectrometry from Serum for Analysis Using 10.3Ipfroptroecpoalrawtiolrlybmeewtrhitotdesn.otAhenrythdeavnitahtoisoenslifsrtoedmatbhoevsee maerethuosdeds,wainllabmeenddocmuemnetnttoetdhaisnd included with the study data. IMEnvironmental Laboratory 3MEnvironmental Laboratory 000077 Page 4of9 Page 23 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol#FACT-TOXL-I1M60S E00-1668 11. ANALYTICAL METHODS 11.1 ETS-8-7, AnalysisofPotassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry 11.2 ETS-8-5, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in `Serum Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 12.DATAQUALITY OBJECTIVES `The numberofspikes/duplicates, useof surrogates, and information on other data quality mietn: dicaraeitncolurdesd in the analytical methods.Inaddition, the following criteria will be 12.1 Linearity `The coefficient of determination () ofthe extracted liver standard curve must be equal to or greater that 0.985 using linear regression or quadratic fit. `The coefficient of determination (r)ofthe extracted serum standard curve must be equal to or greater that 0.990 using linear regression or quadratic fit. 12.2 Limits ofQuantitation (LOG) "The LOQ will be equal to the lowest acceptable standard in the calibration curve, defined as the lowest standard that is both 2 times the matrix blank and is calculated `within + 30%ofthe expected concentration. 12.3 Acceptable Precision Quality control samples are required to meet + 25% precision, provided they are quantitated within the selected calibration range. 12.4 Spike Acceptable Recoveries Matrix spikes and matrix spike duplicates required for analysisofliver samples should show spike recoveries within 70%~130%. After liver samples have been analyzed, sample concentrations will be evaluated. Ifa `measured sample (s) concentration exceeded the calibration range of the method and `was diluted with methanol into the validated calibration range, then additional matrix spikes will be prepared at the approximate measured concentrationsofthe samples then subsequently diluted with the same volumeofmethanol as the sample into the validated rangeofthe calibration curve. `aTnhdesaenluinveerxtmraatcrtiexdscpailkiebsrawtiilolnbceurevvea.luIafttehdevdeirlsuutsedalnievxetrrmaacttreidxmsaptirkiexscdaolimbortatsiohnowcurve recoveries within 70-130% versus the extracted matrix calibration curve, then the 3MEnvironmental Laboratory 3M Environmental Laboratory 000078 Page 5of9 Page 24 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol HFACT-TOXL-I1M60S E00-1668 `sample concentrations, for the spike recovery. at the same dilution factor as the matrix spikes, will be corrected `For serum matrix spikes, a comparisonofextracted (matrix) versus unextracted (solvent) calibration standards will be performed at two concentrations spanning the validated calibration rangeof themethod. This comparison will demonstrate the absolute recovery (recovery + serum matrix affect)ofanalyte from the matrix for determining the extraction efficiencyof serum versus an unextracted calibration curve. After serum samples have been analyzed, sample concentrations will be evaluated. Ifa `measured sample (s) concentration exceeded the calibration rangeofthe method and was diluted with methanol into the validated calibration range, then additional matrix spikes will be prepared at the approximate measured concentrationsofthe samples then subsequently diluted with the same volumeofmethanol as sample into the validated rangeofthe calibration curve. `These serum matrix spikes will be evaluated versus an extracted matrix calibration curve and an unextracted calibration curve. Ifthe diluted serum matrix spikes do not show recoveries within 70-130% versus the extracted matrix calibration curve, then the sample concentrations, at the same dilution factor as the matrix spikes, willbe corrected for the spike recovery. 12.5 Use of Confirmatory Methods Ifa confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration of Specificity 'PFOS identification will be substantiated by chromatographic retention time (approximately 8 minutes), by the characteristic primary fon (499) and the characteristic product ion (9) Minor modifications to the Data Quality Objectives may be implemented at the discroeftthiePoAnL.Thesewillbedocumented intherawdataandtheanalytical report 13.SUB-CONTRACTED ANALYSIS 13.1 Al sera and liver extractioans detailedin this protocolwillbe performed at Pace. `Tier, 1700ElmStreet, Suite 200, Minneapolis, MN 55414. 13.2 All seca and liver analyses as detailed in this protocol will be performed at 3M `Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 13.3 An amendment to this protocol will be writtenifextractions and analyses are performed at laboratories other than the 3M Environmental Laboratory or Pace Tiet2. 14. STATISTICAL ANALYSIS Statistical methods will be limited to the calculationof means and standard deviations. Examplesofthe calculations used in the analyses will be included in the analytical phase: report. 3MEnvironmentalLaboratory 3MEnvironmentalLaboratory 000079 Page 60f9 Page 25 3M Medical Department Study: T-6295.22 AnalyticalReport: FACT-TOX-160 Protocol #FACT-TOXL-I1M6S0 E00-1668 15.RepoRT A reportofthe resultsofthe study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: 15.1 Name and addressofthe facility performing the phase 15.2 Dates upon which the phase was initiated and completed 15.3 A statementofcompliance by the PAI addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved phase protocol, including any `amendments to the original phase protocol 15.5 Identity, purity, stability and the solubilityofthe reference standard under conditions of use 15.6 A descriptionofthe methods used to conduct the test(s) 15.7 A descriptionofthe specimens 15.8 A descriptionofany circumstances that may have affected the quality or the integrity of the data 15.9 The nameofthe PAT and the namesofother scientists, professionals, and supervisory personnel involved in the phase: 15.10A descriptionofthe transformations, calculations, or operations performed on the data, a summary and analysisofthe analytical chemistry data, and a statementof the conclusions drawn from the analyses 15.11Statistical methods used to evaluate the data,ifapplicable 15.12 The signed and dated reportsof each ofthe individual scientists or other professionals involved in the phase, ifapplicable 15.13Thelocation whererawdataandthe final reportare obe stored 15.14 A statement prepared by the QualityAssuranceUnit listing the dates that study inspections and audits were made, and the dates ofany findings reported to the Study Director and Management Ifitis necessary to make corrections or additions to a final report afer it has been accepted, the changes will be made in the form ofan amendment issued by the Study Director. The amendment will clearly identify the part ofthe final report that is being amended, the reasons for the amendment, and wibeslignled by the Study Director. 16.LOGATIONOFRaW DATA, RECORDS, AND FINAL REPORT Original data, or copies thereof, will be available at 3M Environmental Laboratory to facilitate auditsofthe study during is progress and before acceptanceofthe phase report. `When the phase report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory. All corresponding 3MEnvironmental Laboratory 3M Environmental Laboratory 000080 Page 7019 Page 26 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol HFACT-TOXL-I1M60S E00-1668 training records, calibration records, instrument maintenance logs, standard operating `procedures, equipment procedures, and methods will be retained at the 3M Environmental Laboratory. 16.17 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.25tudy correspondence. 16.1.3Shipping records 16.1.4 Raw data 16.1.5Approved final report (original signed copy) 16.1.6 Electronic copiesofdata 16.2 The following supporting records will be retained separately from the study folder according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17.SPECIMEN RETENTION 5 `Specimens remaining after the analytical phase is completed will be sent to and maintained by: Lisa Clemen 3M Environmental Laboratory Building 2-38-09 935 Bush Avenue St. Paul, MN 55106 Telephone: (651) 778-6176 18. PROTOCOL AMENDMENTSAND DEVIATIONS Planned changes to the protocol will be in the formofwritten amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and willbeattachedtothe final protocol. All changes totheprotocol will be indicated in the final report. Any other changes wil be in the formofwritten deviations, signed by the Study Director and Sponsor Representative and filed with the raw data. 3M EnvironmentalLaboratory 3MEnvironmental Laboratory 000081 Page 8of9 Page 27 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Protocol #FACT-TOXL-I16M0S E00-1668 19. ATTACHMENTS 19.1 Attachment A: Material Safety Data Sheets (MSDS) forReferenceStandard 20. SIGNATURES And eacat, Ph.D., Study Director Olen 7. BillBoA John Butenhoff, Ph.D., Sponsor Representative fuA Chip Lisa Clemen, Principal Analytical Investigator Date 27 August 200) Date ola Date aMEnvironmental Laboratory 3MEnvironmental Laboratory 000082 Page9ofs Page 28 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study Title `Extended Recovery Study Following a 26-WeekCapsuleToxicity Study (PFOS; (FACT-TOX-030) witTh-P6e2r9f5l)uoirnoCoyctnaonmeoSluglufsonMiocnAkceiydsPotassium Salt PROTOCOL AMENDMENT NO. 1 Amendment Date: "April 4,2002 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT TOX-160 Covance In-life Study Number: 6329-268 3M Medical Department Study: T-6295.22 ET&SS LIMS: E00-1668 aM Environmantal Laboratory 3M EnvironmentalLaboratory 00083 of Page29 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT-TOX-160 Amendment No. 1 `This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: Section 10 states that method ETS-8-6.0 will be used for extracting liver samples and section 12 of method ETS-8-6.0 references the useofequipment procedure AMDT-EP-22 for cleaning the tissu grinder. AMEND TO READ: Section 12 of method ETS-8-6.0 changed to reference equipment procedure ETS-9-52.0 as of 11/0701. REASON: Equipment procedure AMDT-EP-22 was replaced by equipment procedure ETS-9-52.0 on 11/07/01. Both cover the tissue grinder's cleaning procedure. 2. SPeRcOtiToOnC1O1LstRaEtAesDSth:at method ETS-8-7.0 will be used for analyzing liver extracts using HPLCElectrospray/Mass Spectrometry AMEND TO READ: Method Modification Method: ETS-8-7.0 "AnalysisofPotassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: 10.3.2, 14.5.1, add sections 143.2-143.6 Effective dateofmodifications: July 22, 1999 Section 103.2 Method reads: Analyze a mid-range calibration standard after every tenth sample, with aminimofuonmeperbatch. Modify method to read: Analyze a mid-range calibration standard at least after every ten samples, with a minimumofone per batch. Section 14.5.1 Method reads: Continuing calibration verification percent recoveries `mustbewithin +30% ofthespikedconcentration. Modify method to read: One continuing calibration verification per ten samples must show a percentrecovery within +-30%ofthe spiked concentration. 3M Enviro3nMmEennvtiarlonLmaebnotraaltLoarboyratory. 000084 Page 30 3M Medical Department Study: T-6295.22 Section 1432 Method reads: NA Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT-TOX-160 Amendment No. 1 Modify method to read: The second (bracketing) calibration curve may be deactivated if instrumental drift affects the data. The first curve and acceptable calibration checks shall bracket usable data. Section 1433 Method reads: NA Modifymethodtoread: Calibrationstandardswithpeakarcaslessthan 2 timesthecurve matrix blank shouldbe deactivatedtodisqualify adatarangethatmaybeaffected by background levelsofthe analyte. Section 1434 Method reads: NA Modify methodtoread: Loworhighcurvepoints maybedeactivatedtooptimize alinear rangeappropritoatthe data. Section 143.5 Method reads: NA Modifymethodto read: Acurvepointmaybedeactivatedifitdeviatesmorethan30%from thetheoreticalvalue whenthecurve isevaluatedover alinearrangeappropriatetothedata. Section 143.6 Methodreads: NA" Modify methodtoread: A validcalibration curvemustcontainat least5activepoints. `RMeeatshoodnm: odiarfeimiprocvemeantst/clairifiocatnionsstothecurrentmethod. 3MEnvironmental Laboratory 3M Environmental Laboratory 000085 Page31 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT-TOX-160 Amendment No. 1 Amendment Approval fn Az Sere C/V `Andrew Seficat, Ph.D., Sponsor Representative/Study Director Date Fe A One Lisa A. Clemen., Principal Analytical Investigator otfotfra Date SrA-- 04/65/20 WilliamK. Reagen, Ph.D., Performing Laboratory Management Date 3m Environmental Laboratory 3MEnvironmentalLaboratory 000086 Page 32 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS 00-1668 Record of Deviation SFuAdCT-TTOPXj-1e6c0N,to.E00-1668 _ Deviation type sop D(ocCumienttonu)mber XProtocol FACT-TOX-160, E00-1665 I__identification Method Equipment Procedure Other. Baiofocccusren)ce oonsiol RePrqoutoicroleFdApCroTce-dTurOe1Xl6p0-r,o0c0e-s1s:638 sais: Mate spikes and atx spike duplicates reqive for analysis of ver samples shouldshow piksecoverwiithein70%-130%. "Afr iver amples avbeen alyzed, saceplconcentraionswillbe evaliied. Ifameasuredsamp () Gonceniationexceeded hecalibrationranofgheemethod 3d was diluted with methanol inoi validated Csaalmipblreatsitonhernnsgub,sehqeunendtlyidoiluntedmwaitthitshpeiskaemewivlolbuemperoefpmareethatatnheo3aslptpreoxsiammaptleemientaosutrhedvacloindcaetnetdrraatnigonesofotfhtehe `nCealxbirraaticoindccralvib,raTthioenssculrvvee. mFhaeristpiekedswiivlelrbmeaeivralxisaptiekdesvedrosu0sa0 shexotwrarcetceodvemraietsiwictahliinbr7at0i-o1n3c0u%rvveerasnudsatnhe extacidmatrixclfriaciv,ohnen thsampleconcentrations, willbecomecedforthe sperecovery. ai - te same dilution factoa the mate spikes, "ATchteuMaKlLpOrSo0c9e0d1or-el2p5r0opcpebs-s-:0330_3 _a_s_sp_ke/matxspike_du-plicate (MS/MSts) averaigEerecoverywas 359%. Thesesampleswere nt spiked a he appropriate eve in relation to endogenous levels presentin he. sample (suchasamaTinlc.imAecnttisounesd,TSaOkPernovision, tc Newmispewereprepared at 20g & 10 ug/g them extracted on 10/0201 Recorded by . Ob A ine Kithorized By sen 3. pte [72 SsehnaioyrORneiprcehsren:tatiAvued: John5 Buknho Date 12lioloy Date 12/15/01 = 12/13/01 (ssidbySty D`iDsetvrioartPiroonjetNLoe.st Hm nToF dy rR) Avachrent A 3M Environmental Laboratory DocumeEntTatSio4n8of2Deviations Pigeons 000087 Page 33 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation dy Precio FACLTOX-160, EOO.IG68 Deviationtype. SOP T_ identification `Method Equipment Procedure _(Checkone) _ XProtocol Other: DFoAcCuTm-eTnOtXn-u1m6b0e,rE00-1668 `100D40a1ofeocc(ursren)ce a RePrqoutoicroleFdpAroCceTdu-reT/prOoEcXCeGs-s1:16386s_0e_s_,:_ Mati__sp_ik_es_an_d_a_i_r spi_k_e_ duplicates required for analysis of liversamples should showspkrecoveries withinT0%-130%. "fer versinpls avebeeanalyzed,snp Goncniztons illBs cvluited. Ifamessuredsample 6) Ccoanlicbernaiiaonironnegx,ceehdeenddihteicoanlbarlamtaitornixanspgiekeosf hweimlbelethpordepaanrdeawdshie eppdroxwiimtahtmeemtehasou!retdocotnceenvtarlaitdiaotnesdof the sCaalmipblraetsiotnhceunrsvueb,seTqhueesnstllyidvielrumteadtwiitshptkheesswaimlevboeleuvamleuoatfemdevtehrasnuoslaanetxtersaactmepdlmeatniothcealviabrlaitdiaotnedcurravnegaenodftahe `uneiaracceiemdatcaellrcsatlibornatsiaonvec.arIvfet,htedeinehde sTavemrpmacroincesnptirkaetsiodnso,naottshhsowareecdoivefrtioesnwfiatchtoirna70h-e13m0%avexrssupsiktehs,e willbeconecedforthe spikerecovery. - . A"cTthueaulgprmoacetduereslppkreo/cmeastsr:n spi_ke_Gu_pi_ca_ls_(V_MS_IVISD) a_ve_ra_ge recovery was 35%and te 10ug/S g MSE AD. was oodlcusing a 1500dukon acer. "Toe TOs VES vs 5 utuedcvhiansgomel1lrl5c.0imAedcinlttuitisioosnunsosnd,dTSaaOnkaPlreyenzveidsoionn,10/1601 The 150 iaiof n 2 5sMSMSDswasalsoFeaslyzedon 101601toconfiem theorginal lowrecovery. Recordedby - On A Doman Authorizbeyd pg = 1) WI Seen" SporverSReaentotifeoc:bTsohSoakbike? (sip Date 120] fy 12(13/01 StyDDiesvtiraotPiorneNLoo.k HR 2Sr) tachment A 3M EnvironmentalLaboratory DocumentaEtTioSn4o8fD2eviations v Page Lof1 000088 Page 34 3M Medical Department Study: T-6295.22 3M Confidential Record of Deviation Analytical Report: FACT-TOX-160 LIMS E00-1668 FSAeCdTTOPX-r10o0,. BOO688 Deviation type. SOP `Method (Checkone) _ XProtocol __Other: Equipment Procedure FADCoTcLumTeOnXt-n1u6m0b,er00-1668 D1a0t1e(6s0)1ofoccurrence 1I._Description Required procedure/process: a . Protocol FACT-TOX-160, E00-1668 states: Matrixspikes and matrix spike duplicates requiredforanalysis of liver samples should show spikerecoverieswithin 70%-130%. "Afterliversamplhaevsebeen analyzed, sample concentrationswillbe evaluated. If a measured sariple (5) `concentration exceededctahelibrationraon fthg emetehod andwas diluted with methanol into the validated" acmalpilbreastiohnerasngueb,stqhueenandtdyitdiioluntaelmdwaitrtihxhspeiskaeswivlolbluemperoefpamreetdhaatntohlea1ptprhosxipmalte mnetaostuhreevd coancentlrraatnigdoenosofefthtehe `calibration curve. Theselivermatrixspikes willbeevaluavetresuds an extracted matrix calibrationcurve and an |unextractedcalibration curve. Ifthediluted livermatrix spikes do not show recoveries within 70-130% versus the A NERA |extracted matrix calibration curve, then the sample concentrations, at the same dilution factor as the matrix spikes, ATctuh2aulgep/rgoacneddu1r0eu/gpromcaetsse:siksepiko emduplaio catte(MeSMSnD) sverige recoverieswee9%} and 52% Toc ITV 3 9SSU3NT ple(suwcheatsalmleIiln.atmAeecndttisroenods,hTSeaOokPgerenivi]sixoni,acetcs F505 syed on 16/24/01. EE - OfA |Recordebyd Conan ------ A Date Abo2% aon. punt Dus ir 00 Duper I. Saea" 72/001 Spontor Repreaenlatine> Tohn Loos Shady Dicclor' Andrews Stack (sind SyDDeivriatPioontNLoo.n_B_E _38 ___) __ tachment A 3M Environmental Laboratory DocumeEntatsionsofDevisions Pagetoft . 000089 Page 35 3M Medical Department Study: T-6295.22 3M Confidential Record of Deviation Analytical Report: FACT-TOX-160 LIMS E00-1668 - 1._Identification `FSAtuCdTy T/POrXoj1e6ct0N.o.Bo0-668 ie Deviation type (Checkone) _ SOP Method XProtocol Other: Equipment Procedure DoFcACuTm-eTnOtXn-u1m6b0e,r 00-1668 D102a410to1feocc(urrsenc)e. |PRreoqtuoicroledFpAroCceTdu-reTipOroEXc0e0s--s1:61686i0te,:Mati spikesiad ati spike duplicatesrequicdforanalysisof iAvfe savmeprlseasmsphloeuslhdasvheobweespniaknearleyczoevedr,iseasmwplieci7o0%n-1o30a% zwilalbetvoanles.If msured ipl () `ocanlciebrnattiaoinornanegxec,etehdnedadhdeiiaobnallatiionsasnpgiekeosfwihlelmbeethproedpainrddwaatstedilauptperdowxiitmhatetmheasnurnedocotnhcenviazlaitdiaotnesd ofthe samplesthensubsedqiluuedewinthtthlsaymevolumeofmetshthasamnpleiotoltevalidated angeofthe caleixbtrartaicon curv,These vesrarspikes calibration curve, ieiid wil be valuedversa an extracted ati callbrationcurveand ver max spikes donotshow recoverieswithin 70-130% versus the extractedmati calibration curve,ten hesample oncentatons,at hesamedilution cor 3 thematispikes, willbe correctedforthespikerecovery. Co . A"Tcht2uuagl/pgronceddu1r0uegl/pgrmocaetsis:spel spikeduplicate(VISMSD) averige iecoveies vere 119% and 242%. "Thesamplerecoverywas otconsistentwithth previousanalyses, "Toesamplesewv-cxiaiad(osnu1c1h0a7s1a1m,Hotlrh.ceimAeacntttieisdosnu1es:d5,0TSaoOkdPeannalyzreviseon, d ec) Recorrdedby oT or o On A, Olena i} Abs gs 2ft Cont og z SSphonasyorDrReegcYrhesee:ntaAkinved.e* sJoShcnaBtukenhot ) erro Date liloor Diere, 12/3 for DEceoaPr ieNLoa.d Tr L Atachment A 3M Environmental Laboratory PocomEeTSn4ot8faD2etviiatoionns Page tort 000090 Page 36 3M Medical Department Study: T-6295.22 3M Confidential Record of Deviation Analytical Report: FACT-TOX-160 LIMS E00-1668 Ferg a |FACTTOX160, EOO-888 ete] (DCelviaactkioonnet)ype. _XPSrOowPocol MeOtthheord: [Equipment Procedure FDoAcCuTm-eTnOtX.n1u6m0b,erE00-1668 ----T 1601 Date(s)ofoccurrence PRreoqtuociorleFdApCrTo-cTeOdXu-r1e6/0p,roc0e0s-s1:668 ics Matspiaknd eatsi sike duplicate eidfoeailysd of ve AR Tsavmeprlessaosphloeuslhdasvhebowesnpikaenlryaeed,csaomwpvlieheco7r0n%ci-e1ep30zs%awiiolns valid. If measured spe ) cloinbcreanteiaontiroanenxgc,etahdeedadhdeitciaolniablramtaioinaispgikeosfwtihellmbeetphroedpaoreddawtaheisippdrowxiitmhatme meessturnehdoCtahoenvcnaelniodtartlaetdoifonhes saalmlpblraetsitohnecnursvueb,seTqhueesntelyvdeieadowistphltheswamlelvoelvualnioaifemdevtehrasnolaesxsttrhaescatmepdlme tnoxtchalivbraatioln ciurradvnegatenoedftadh0e euxtnreaxcatecdemdacauliibcraaltiibornactuiornvec,urTvFe,hteedniheedsamveprlmecatornicxespnitkersatdoioaonftshs,hsoawmreecdoivkeirtioens cwitthoinar7t0h-1e m0a%trivxesrpsiuksetsh,e willbecorecied for tespike rsovery. ATchteua1l0 uprpocmeadtuirseplipkrlomcaetsis:pikeduplats(MSIVSD)average recowvise6r7%y. o "T5% 10s VISwaAsmSot e(suschn05 talomceIolrlnyc.fiimAremzcnttriieescosounvedesdr,yTSsaOakPeertnvhies2ieln gfcMSD erg reser, b`anjalyezedcdwuierirnvgwteihtehssianmcerrun,iwfoarts i9e5sa%nraanlyidtwiicatahlirnuth.eTshtaeteadcceruirtecryiao.rAltshoe,lavlel CCdaVtsaainndtoitshesrtuddaytwa quiableictyhl.angled from.- 30%to+/-35% inthefinalreport. - oo -} OlA Clinae Recorded by - To Authorized by_y fon >. SR A lk Date Datiersbzr 2 , Suan" L2t3/0) Sponsor Representoie'. ohn BueahoSt Study Drechr Andrews Stacok ioeby SyDDiercvtiraotriPornocNLoe.w _2__of5E_T__D Atachment A 3MEnvironmental Laboratory DocumenEtaTtSio4n 8of2Deviations Page tort 000091 Page 37 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation SadProjecNot. [DeIviIatDion typerat usguomt D(oCcheucmkeonnet) nuber ~~ Ei37.0 T._Identification insSOgurPis dosssionXg Metshimoradn - `Equipment Procedure - ProtoCcoTl T Other: | Dae)ofocimenee 11-1601 i Description |RAeccqouridrinegdtopsreoccteidonure11/.p1rTohceeasvse:rageoFwosandr curveswill b~ploiedbylinear rpegression. ~ FAocrttuhaeldpariocseedtumr1ef1p1r1o6c5eshse: inxircied curvewasplofed wing o3 nquadratic fi. A fiear urve ~ th mehe t2 eri andxiended t250pp (5% eve oftheCCV)couldntbsgered. - - (uch as amTeiln.dmAecnttisosunesd,TSaOkPenvision, oc LECTETIEEIER, oo cqpresnes - Recordebd y" TY, " mr "Date LTC (J a 1-260, [r Bo= Mire vopock GoAnl(cmuaact,a.gn St/uPrdojeyct SE Phir hay Shdy comfls we exolunded, versus. Exhneled "abix Cues. Ue "nifarl Authorized by ob [2 itor - Date. moro SSteuddyerDuReacpkr:antAonedr.ews TSoeahcedBOune r eZanneonL rrrL E-- r-- ) Atachment A 3M Environmental Laboratory DocumETeS4n5o.tf1aDetviiatoionns Page 1071 000092 Page 38 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FLAICMTS-TEO00X--1166608 Record of Deviation T__identification F`ACST-/tTPOroXuj-e1cd6t0Ny,o.E00-1668 ; } Deviation type SOP `Method EquipmentProcedure (DCohceucmkenontne)umb_er _ XProtocol Other: Bofoy ccurence I FACT-TOX-160, E00-1665.--711601 P`rRoetqouciorledFApCrTo-cTeOdXu-re1/6p0r,oEc0es0s:68sses:Maissp~essndmatrixspike duplicatesrequiredforanalysisof fiver samplesshoukd showsik recoveries wiiT0%-130%. cAofntceerntlrivaetsisoanmpelxecseebdaevdetbheeecnaabnbaelayizoend,rsaangmepolfectohnecmeenttrhaotdiaonnsdwwilalsbdeeivealduawtietdh.1meftaamneoalsunrfeodsthaemvpallied(a5t)ed libration ange,thenadditonalatispikeswillbeprepared a heapproximatemeasuredconcentration ofte caslaimbprlaetsitonhcnursvueb,seTquheenstelylidvelrmeadtrwiix tsptikhessvaimlelvboeleuvmaleuoaftmedevtehrasnuoslaa tehxetrsaacmtpedlmeaftortioxtcaelibvraaltiidoantecdurravenasnodfahne umextacedcal curve. 1 hedled vermari spikesdopotshowrecoveries within 70-130% versus the extraciedmaicalibration ve,thenth sampleconcentrations,atthesame ifuonfacorastemati spikes, willbecomet fo thespikerecovery. |A1c)Tthuael1p0ruogc/egdmurae/iprsoickeesmsa:sixspike dup~licate(MS/MS)averI ags recoveywas 67%. N b2e)cNauose1t/h500wlriovnergdliiveossnamwpalserweapsourtseedddfuoripnrgtephaercionugrtsheaotfMtSiMsSsiDodyl.evTehl.e1/500 iverdiutionwastoodilute 2 a5amiein.ciAnecnttionssued, TSaOkPen ovsin, oc) To Tvervali werComeciad{oc hi owrecoverysince On 5 ofMS/MSDsWerewinGers 0d55 averagetecaveryfortis stofMS/MSD werewithinthexinded!65-135% ereriaasatedina cartier devinion. 2)Dilutionwasnotrexirictedrdilicdusin thecore sump since the1/50dilutionwaswithincreaand nis1/50 lutionwasse to sho hanofectwasobservedwhealvedion werepesded. `Recordedby Date LiA.Cslemaen (Jf, A. (loan 04/09/02 04 Jpg). Authorizedby Date Foo 2. Ralf] yin me 0 Sued Srrfor Snr RepenatatSoh e] Srccor 5 Mndies> aca](sip bySyDeDviioartiPocnsNLoo.d odof3 ty Pp 3M Environmental Laboratory o 93 Page 39 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS 00-1668 Analytical Report: LFIAMCSTET0O0X--1616680. `Appendix C: Extraction and Analytical Methods `This appendix includes the following methods: EETlSe-c8t-r4o.s2p,r"aEyxMtarsasctSipoencotfroFmleutorryo,"ch(e1i6cpaalgeCso)mpounds from SerumforAnalysis Using HPLC`ECTSo-m6p-o6u.n0d,sExftrroamctLiivoenrofforPoAtnaalsyssiiusmuPseirnfgiuHoPrLoCo-cEtalneec-tsruoifsopnraatyeMoatssheSprecFtlruoomreotcrhye,m"i(c1a4l pages) ETS-8:5.2, "Analysis of Potassium Perfluorooctanesulfonaotre Other Fluorochemicals In `SerumExtracts Using HPLC-Electrospray/Mass Spectrometry," (11 pages) EExTtSr-a8c.t7s.0U,si"nAgnaHlPysLiCs-oEflePocttarsossipurmayPMearslusoSrpoeoccttraonmee-tsruyi,f"on(a1t0epoargeosth)er Fluorochermicals in Liver SM Environmental Laboratory 3M EnvironmentalLaboratory. 000094 Page 20 Page 40 3M Medical Department Study: T-6295.22 3M ENVIRONMENTAL LABORATORY. Analytical Report: FACT-TOX-160 B+, estCopLyIoMfSOEg0i0-)1668 Keero polHo E : METHOD EXTRACTION OF FLUOROCHEMICAL COMPOUNDS FROM SERUM FOR ANALYSIS `USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-4.2 : Adoption Date: 03/01/99 Effective Date: 2/2101 Approved By: [a LaBratory Manager GroA up Leader . 02feat Date Date 1.0 SCOPEANDAPPLICATION 11 Scope: Thismethod isforthe extraction offlsorochemical compounds from serum. 12 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 13 Mtahetrviacliedsa:tiRoanbrbeipto,rtrat, bovine, monkey, and human serum or other fluids as designated in 2S 0 uMvARYoFMEmROR 21 Tsuhlifsonpaetrefo(rPmFaOnSc)e-obrasoetdhemreftuhoordocdheesrcriicbaelsstuhrefapcrtoacnetdsufrreofmorseerxutmr,acotrinogtpheerrfflluuoidrso,ocutsainneg-an tfhoen psaaimrpilneg raenadgethnet aannadlmyetet-hiyoln.tpearitr-ibsutpyalrteitthieorne(dMiBnEto)M.BAEn.ioTnhpeaiMriBngEreeaxgternatctisisadded to r1e.0momvLedofanmedtphuatnooln,tothaenniftirltoegreend etvharpoourgahtaor0.un2tuilmdrnyy.lonEacfhierxtursaicntgsar3e-cmoLn.stpilatsuttiec:d in Word 695 3MEnvironmental Laboratory Frain ofFEeTrSe:h8.a4n2ess frm Soro 000095 Page 10115Page 41 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 4 22 syringe into glass antovials. (Application of this method to seven fluorochemicals was sdteamnodnasrtdra(tseede:3P.F0ODSef,iPniFtOiSoAns,).PFOSAA, EtFOSE-OH, PFOSEA, M556, and a.surrogate These sample extracts are analyzed following method ETS-8-5.2 or other appropriate method. dp31eemPRoOSn: pserfluorooctanesulfonate (anionofpotassium sat) CyFisSO;" 0000000 32 PFOSA: perflvorooctane sulfonylamide CyFirSO:NHy 33 PFOSAA: perflucrooctane sulfonylamido (ethyDacetate CyFirSON(CH:CH;)COHy 3.4 CEitFFOSiE-rOHS:O2(NN-(etChCyHHlp;;erCCfHlHuOor,Hooctane sulfonamido)-ethyl alcohol 36 M556: CFnSONGH)(CH,COOH) 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CyF17SO;N(CH,CHy)H . 3.7 Surrogate standard THPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid 40 Warns AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when `bandling animal tissue, which may contain pathogens. 5.0 INTERFERENCES . 5.1 Therearenointerfkneowrneatnthicsteimse. S0 OFoow0 weer00000000000 61 Tachceepftoalblloew.ing equipmiseunsetd whileperformingthis method. Equivalent equipment is 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.12 Centrifuge, Mistral 1000 or [EC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance (0.100 g) : 10 SuaoMarenars 71 Gloves 7.2 Eppendorf or disposable pipettes, plastic or glass (or equivalent) 73 Polypropylene bottles, capable of holding 250 mL and 1 L (Nalgene or equivalent) 3MEnvironmental Laboratory ETS842 Page20015 000096 Page 42 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 74 Volumetric flasks, glass, type A 7.5 , 7.6 $77 78 7.9 710 7.11 7.02 40 mL glass vials (I-CHEM or equivalent) Centrifuge tubes, polypropylene, 15 mL Labels Bottle-top Dispenser - 3.0 to 100 mL or a 5-10 mL graduated pipet, glass Syringes,capabloof measuring 5 pLto 50 uL Graduated pipettes. Syringes, disposable plastic, 3 mL (B&D or equivalent) Syringe filters, nylon, 0.2 um, 25 mm 713 Timer 7.14 Crimpcapglass autovanidacalpss 7.15 Crimpers Note: Prior to using glassware andbottles, rinse 3 times withmethanoland 3 times with water. Rinse syringes aminimoufm9 timeswithmethanol, 3rinsesfrom 3 separate vials. 8.0 REAGENTS ANDSTANDARDS 81 Reagent grade water, Mill-QTM, NanopureII or equivalent 82 Sodium hydroxide (NAOH), JT Baker or equivalent 83 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent - 84 Sodium carbonate (Na:COy), LT. Baker or equivalent 85 Sodium bicarbonate (NaHCO), J.T. Baker or equivalent 86 Methyl-T-Butyl Ether, Omnisoly, glass distilledorHPLC grade 88.87 MSeetrhuamnoorl,blOomondi,soflrvo,zegnlafsrsodmisstuipllpeldioerr HPLC grade 89 Fluorochemical standards 89.1 KPFOS (PFOS) (3M Specialty Chemical Division), molecular weigh=t 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 89.3 Hosa (PFOSAA) (3M Specialty Chemical Division), molecular weight = 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 89.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H 89.8 OCtyhFe1r3SfOl3uHo,roc[hTeHmPiFcOaSlYs,))a, molecular weight sppropriste = 428 ETsa42 Page 30f15 3M Environmental Laboratory - - 000097 Page 43 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 810 Reagent preparation NOTE: Whe preparing different volumes preparation, adjust accordingly. than listed in reagent, standard, or surrogate * 8.10.1 110N000msoLdbieuamkheyrdrcooxntiadien(inNgaO5H0)0:mWLeiwagthera,pmpirxouxntiimlaaltle2sl0oly0idgs NaraeOdHi.ssoPlovuerd.inSttooare ina 1 L polypropylene (or equivalent) bottle. 8102 110NNsoNdaiOumHhsyoldurtoixoindei(nNtaaoO1H0)0:mDLilvuotleum1et0NricNfalaOsHka1n:d10b.riMnegastouvroelu1m0meuLsionfg water. Store ina 125mLpolypropylene (or equivalent) bottle. . 8.10.3o0f.5TMBAteitnrtoabaut|yLlavmomlounmieutmrihcycdornotagienninsgul5fa0t0em(TLBAw)a:teWre.iAgdhjuasptptrooxpiHmat10eluysi1n6g9 `asaptphreoxpiHmactehlayng4e4staob5r4upmtlLy)oDfil1u0tNetNoaOvHol.u(mAedwditthhewaltasetr.1.S0tmorLeiofnaNa1OL.H slowly, `polypropylene (or equivalent) bottle. . 8.10.3.1TnBeeAdreedquusiirnegs 1aNchNecakOprHiosrotluoteioanc.husetoensure pH = 10. Adjustas 8.10.4 0ap.p2r5oMximsaotdeiluym2c6a.r5bgoonfatse/osdoiduimumcabribcoanrabtoena(tNe&b;uCfOfser) (aNnady2C1O.y0/gNGofHCsOoyd)i:umWeigh Sbitcoarrebionnaate1 L(NpaolHyCprOo)pyilnetonea(1orL evqouliuvmaeletnrti)cfblotatslke.and bring to volume with water. 811 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2Pfrleupoarroecohetmhiecralflsutoarnodcahredmsiacraelasctcaenpdtaarbdlse, a(fsorapepxraomprpilaet,e.onMeuwlotrikcionmgposnteanndtard + PsoFlOutSiEonA,coMnt5a5in6inagndapEpCroFxOiSmEa-tOelHy).1.00 pg/L.of PFOS, PFOSA, PFOSAA, 8.11.3 Wtheeiagchtuaaplpwreoixgihmta.teFloyr1s0t0anmdagrodfs wPiOthSK-inotrooath1e0r0msalLtsv,omluulmteitplryicbfylaaskcoarnrdecrteicoonrd `fawcetiogrh.toFforPeFxOaSmp=le4,99th,eCmaollceuclautlearthweeicgorhrteocftiKoPnFfaOcStor=b5y38u,sianngdthtehefomlolloewciunlgar equation: molecular w. PROS (499) _ ives . "molwte.KcPFaOSi(a53r8) 09713 (Correctionfactor) 8.11.4 Bring to volume with methanol for a stock standardofapproximately 1000 pg/mL. 8.11.5 Dilute the stock solution with methanolfor aworking standard 1 solution of approximately 50 pg/mL. (100t0pegmrLxiS m)L _ pm 8.116 Dilute working standard 1 with methanol for a working standard 2 solution of appro. 5.0 pg/mL. 3MEnvironmental Laboratory ETS842 000098 Pagdeof15 Page dd 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 (Sompmgimxtt)omt) _sons (Batatzion) arn i 8.117 Dilute working standard 1 with methanol for a working standard 3 solution of `approx. 0.50 pg/mL. Sougimixiont) 8.12 Surrogate THPFOS stock standard preparation 8.12.1 Weigh approximately 50-60 mgof surrogate standard 1-H,1-H, 2-H, 2-H, CsF13SO;H (THPFOS) into a 50 mLvolumetric flaskandrecord theactual weight. 8.12.2 Bring to vowl ithmuethm anoleforasursrtockoofgapparoxitmateely 1000- : 1200 pg/mL. 8.12.3 Prepare a surrogate working standard. `Transfer approximately 1mLofsurrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a `working standard of 100 pg/mL. Recordthe actual volume transferred. 9.0 SAMPLE HANDLING 91 All samples are received frano dmuz stbe eken pt frozen until the extractionisperformed. 92 Allow samples to thaw at room temperature or in lukewarm water prior to extraction. 100QuanrvConrmor, 10.1 Solvent Blanks, Method Blanksand Matrix Blanks 10.1.1 Solvent Blanks: An aliquot of 1.0mLmethanol is used as a solvent blank. 10.1.2 Method Blanks: Following this procedure, extract two 1.0 mL aliquots of water and use as method blanks. 10.1.3Matrix Blanks:Matrixblanksare prepfarromeondeofthree sources: 1) a study control matrixfrom a study control animal received with each sample set; 2) a commercially obtained sampleofthe same species as the study animals; or 3) a surrogate matrix, also obtained commercially,butof adifferent species than the `study animal (e.g.ifrabbitisused to generate standard curves and CCVs foar `monkeyorratsera study).The matrix tousedependson whatmatrixisused for the curve. 10.1.3.1Studycontrol matrix curve--TIf the study control matrix is used for the curve, prepare two (2) matrix blanks using the study control matrix. 10.1.3.2 Commercially obtained (same species) matrix curve--If the curve is prepared using commercially obtained matrix in the same species as the study animal, prepare two matrix blanks using this same commercially available matrix. 3M Environmental Laboratory ~ _ETSSa2 000099 Pagesof 15 Page ds 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 10.1.3.3 Surrogate continuing mcaatlriibrxactiuornvveer--ifIiafcastiuornrosgaamtpelemsa:trix is used for the curve and. 1 2) prepare two (2) matrix blanks usingthe surrogate matrix. b) A`lmsator,ipxrseppiakre/emaatmraitxrsipxibkleandkupwliitcahteeaacthosneetolefvemla)truisxinsgpaikceos(mmaesrceiiastlaly available matrixof the same species asthestudyanimals. 102 Matrix spikes 10.2.1 Study Control Matrix Curve No matrix spikes are prepared. 10.2.2 Commercially obtained matrix curve (same species) No matrix spikes are prepared. 1023 Surrogate matrix curve, `Matrixspikesarenecessaryifmatrixisnotavailableinthe samespeciesasthe rsatbubdiyt asneirmaamlaayndbea suusrerdotgoatgeemnaertartiexsistuansdeadrfdocrutrhveecsufrovrea amnodnkCeCyVssearmapslteusdy(,c.dgu.e, atonamleyazseumraatbrliexlspiekeoavfnednmedaotgrleixnossupsiakneadluyptleiicnatethseammpolneksetyosveerrai)f.y tPhreepaacrceuraancdy of the extraction for target analytes. 10.2.4 Prepare each MS and MSDattwo (2) levels (usually a lowandmid-range gcornocuepntarnaitmiaoln)reucseiinvgeda wsiatmhpleeacchhossaemnplbeystehte. analyst, typically sera from a control 1024.1 mfatthreirxesipsikneostussufifnigciceonmtmseerrcaiaavlaliylaabvlaeilfarbolemmtahtercioxnftrroolmgtrhoeups,amperepsapreec:ies as the study animal. 10.2.5 pPerre2pa0rseaomnpelemsa,twriitxhspaimkie nainmdummaotrfix2mspaitkreidxupspliikceastpeeartleeavcehl pleevreblalticshte.d Iinfa1b0a.2i.4c.h includes more than 20 samples,additionalspikes may be prepared at the same, Tow, or high range levels. 10.26Ifmore than 25%ofthe samples are SLOQ, two matrix spikes should be prepared at approximately 2-5 times the expected LOQ. 10.2.7 Iafdtdhiteiomnaaljmaotrroiifxttshpyeikseasmsphloeusldarbeeatproerpaabroevdettohaephpirgohxriamnagteeosfatmphleeccuornvcee,ntrations. 10.3 Continuing calibration verifications (CCVs) 10.3.1 vPerreipfaircaeticoonntdiunruiinnggacnaalliybsriast.ioPnrevpearirfeicaantdioannsaalmypzeleCsCfoVr ifnosrtreuvmeernytasstsaabiylirtuyn, regardless of the matrix type used to prepare the standard curve. 10.3.2 pPrreeppaarree tehaechinctoianlticnuurivneg(cia.l.i,beriatthieorn tvehreisfitcuadtyiocnonftrrooml tmhaetrsiax,mecommamterrixciuasledmattorix of same species, or surrogate matrix). 3MEnvironmental Laboratory ETS 842 0 00100 Page 6015 Page 46 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 10.3.3 Tcahleiberxatpieocntceudrcvoen;cteynptircaaltliyo,nstoheftlhoew CtoCmVisd-wrialnlgfeallofwitthheincutrhvee.range of the initial 1 10.3.4 cPornecpeanrter,aattioan,mipneirmgurmo,uptwoof c1o0nstaimnpuliensg.caFloibrreatxiaomnplveer,iifficaatsioanmsp,leeasceht ata different = 34, eight c(o8)nccehnetcraktsiaorne. prepared; four (4) at alow range and four (4) at a mid-range 1035 A`mdidditoirohniaglhc-ornatnigneucionngcceanltirbartaitoino.n verificationsmaybe included at the same, low, 110 CALIBRATION AND STANDARDIZATION 111 Prepare matrix calibration standards 11.1.1 Transfer available 1smerLai.oufseapdp,ropprreipaatreesetrwoummattroiaxb1l5amnkLsc,enutsriinfgutgheetuvboel.uImfeocfosmemrear.cially available for most study animals. 11.1.1.1 aDveapielanbdlie,ngauspurornogsattuedymagtorailxsmoariyfbaenaulystee-ffodrreegceonemrmaetricoinoafltsherea cisalniobtration . rsattansdtaurddi.esiFofrqueaxnatmiptlaet,iornoabfbietxtserreammealyylboewulseevedlassofatshureroangaaltyetemaistrriexqufiorred. 11.1.2IVfomlousmtessaemqplueaolvotlhuemessamaprleelevsoslutmheasn1..0DmoLn,otexetxrtarcatcsttlaensdsatrhdasnw0i.t5h0mmaLtrioxf matrix. Record each sample volume on the extraction sheet. 11.1.3 sWehrialebeptrweepeanrianlgiqauottost.alofthirteen aliquots in 15-mL centrifuge tubes, mix or shake 11.1.4 TTywpioca1l-lymLusaelitqhueosttsa,nodraortdhceorncaepnptrropartiiaotnesvaonldumsepi,ksienrgvaemaosucnutrsvleismtaetdriinxTbalbalnkes.1 at tthweoenmadtorfitxhbilsasnekcst,iaonndttowspoikmeeotnheodstbalnadnaksr.d curve,for atotalofninestandards, 11.15 RreafnegretsoanvdatlhiedaLtiinonearerpCoarltiEbrTaSt-i8o-n4R.a0n&geE(TLSC-R8)-5f.o0r-cVa-li1b,rawthiiocnhculrisvtesst.he working 11.1.6 sSteaendSaercdtsi.on 13.0 to calculate actual concentrationsofPFOS in calibration 112 sTuorreoagcahtestwaonrdkaridn,gbsltaannkd,acrodnttoinaucihnigevceheackc,onasntdanstacmopnlceenatdrdatainonaptphraotpfrailalstewiatmhoiunnttheof cacloinbsrtaatnitocnocnucrevnetrraatnigoenoifn 2al.l5sanmgp/lmeLs~a1t0a0p0prnogx/immLa.teUlsyua5l0l0y,nsga/mmpLleosraortehesrpickoendcfeonrtraation as determined by the analyst. 113 tEoxtersatcatblsipsihkeeadchmaitnriitxalsctuanrdvaerodsnftohlelmowaisnsgs1p2e.c6t-r1o2m.e1t6eorf.this method, Use these standards 3MEnvironmental Laboratory ETS842 Page 7015 000101 Page 47 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 , Approximate spiking amToaubnltes1for standards and spikes i Using 1.0 mL of matrix Working standard approx. conc.) uw `Approx. final conc. of ein matrix 11 Bak | [S0 170 5 1 pp 0025pom m | [0170 7790 1 p 0500m pom | [ or__50 20 0 "|p10p pmm| R10 O2P1eoOcbetapion wfreozen sam0 ples and allow to thaw a0 t room temperatureorin0 lukewarm water, 122 aLnaableylstai1n5itmiaLl.poSleyepraotptyalcehendecweonrtkrsihfeuegte (tAutbteawcihtmhenttheAstourdsyimmuimlabrewro,rskasmhpeleet)IDf,ordateand documenting the remaining steps. 12.3 V1o5rmteLxpmoilxypfroorpy1l5esneecocnedntsr,itfhugeentturbaen.sfer 1.0mLor other appropriate volume to the 124 Return unused samples tofreezeafter extraction amounts have been romoved. 125 Rwoerckosrhdeetth)e.inital volume on the extraction worksheet (Attachment A or similar 126 sStpainkdeaarldl sasamdpelsecsr,ibbeldanikns1a1.n2d.standards, that ae ready for extraction with surrogate 127 dSepsickreibceadchinca1l1i.b1r,atoironTasbtalnedaLridnmtahtartisxecwtiitohn,thfeoraptphercoaplriibartaetiaomnocuunrtvoefssttaannddaardrsd.asAlso `prepare matrix spikesifnecessary and continuing calibration standards. 128 Vsaomrptleexsmfioxr t1h5essetcaonnddasr.d curve samples, matrix spike samples, and continuing calibration 129 Check to ensure the 0.5 MTBA reagent is at pH 10. Ifnot, adjust accordingly. 1210 bTiocaerabcohnastaempblueff,era.dd 1 mL 0.5 MTBA and 2mLof0.25M sodium carbonate/sodium 1211 Using an bottle butyl ether. top dispenser or 5-10 mL graduated ghss pipette, add 5 mLmethyl ert- 12.12 Cap cach sample and put on theshakeratasettingof300 rpm for 20 minutes. 3M Environmental Laboratory ETS842 000102 Pages ris Page 48 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.13 Centrifuge for 20 to 25 minates atasetting of 3500 pm or until layers are well separated. 12.14 Labelafresh 15mL centrifuge tube with the same information as in 12.5. ; 12.15 Remove 4.0mLof the organic (top) layer to this clean 1m 5 Lcentrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until ry, approximately 1 to 2 hours. 12.17 Add 1.0 ml ofmethanoltoeach centrifuge tube using agraduated pipette. 12.18 Vortexmixfor 30 secondsor, lonifg neeeder d. 12.19 Labela 1.5mLglass autovial (or low-volume autovial when necessary) with the study `number, animal num anb dgee nder r, sample timepoint, matrix,finalsolvent, extraction date, and analyst(s) performing the extraction. 12.20 Attach a25mm,0.2pm nylon mesh filter to a 3mLsyringeandtransferthesamplefrom step 12.18 to this syringe. Filter intothelabeled autovial. 12.21 Cap andstoreextractsat room temperature or at approximately 4 C untilanalysis. 12.22 Complete the extraction worksheet (AttacAhomrseimniltar worksheet) and include in the study binder. 13.0 DATA ANALYSIS AND CALCULATIONS 131 Calculations 13.1.1 Calculate actual concentrationsofPFOS, or other applicable fluorochemical, in calibration standards using the following equation: mLofstdxconcetratonofsd (si mL) = Fotomate mLof std +m ofsurrogatesid +initial matrixvolume (mL) Fag AL -- lof 2108 om 1M 40 ethopPERFORMA 00N 000C 00E 0 141 The method detection limit (MDLi)s analyte and matrix specific.Referto MDL report for specific MDLandlimit ofquantitation (LOQ)values(see AttachmentBs and C). At thediscretionofthe PAI, MDL may not be defined for some studies. 142 The following quality control samples are extracted with cach batch ofsamples to evaluate. the qualityofthe extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2Ifsurrogatematrix isusedforcurve and QC,matrixspikeandmatrix spike duplicate samples to verify extraction efficiency. 14.2.3 Cionnctaincuailnigactailoinbrcautriovne.check samples to determine the continued accuracy of the 14.3 Refer to section 14of ETS-8-5.2 for method performance criteria. 34Environmental Laboratory Erss42 TTT Fagen 000103 Page 49 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-168 15.0 POLLUTION PREVENTION AND WASTEMANAGEMENT , 15:1 Dispose of sample waste, flammable solvent wasteandused glass pipette waste using i proper methods and by placing each in their designated waste container. 16.0 Recorps 16.1 Complete include in tthhee esxtturdaycbtiinodnewro.rksheet attached to this method (or a similar worksheet), and 17.0 ATTACHMENTS 17.1 `AAttttaacchhmmeenntt A, A) Extraction worksheet (a similar worksheet may be substituted for 17.2 AttachmenBt, MDLval/ uesaLndsOummQ ary 17.3 sAutbtsatcihtumteendtfCo,r AItotnaPcahimreSnttanCd)ard Curves worksheet (a similar worksheet may be B0 oRerg0 menc0 es 000000000 18.1 Thevalidation report associated with this methoidsETS-8-4.0 & 5.0-V-1. 182 FHPALCCT--EMl-e3c.t1r,os"pArnaaylyMsaissosfSSpeecrturmomoertrOyt"her Fluid Extracts for Fluorochemicals using 183 ESTeSr-u8m-5E.x2t,ra"cAtnsaUlyssiinsgofHPPoLtCa-EslseicutmroPserpfrlauyo/rMoaocstsaSnepseucltfroonmaetteroyr"Other Fluorochemicals in 19.0 AFFECTED DOCUMENTS 19.1 ESTeSr-u8m-5E.x2t,rac"tAsnaUlsyisnigsoHfPLPCo-tBalsesciturmoPseprrfalyu/orMoaoscstaSnpeescutlrfoomneatteryo"r Other Fluorochemicals in 20.0 REVISIONS r---------------- Revision Revision Num1ber Secon1221 Changedto inclsdeRseaampsloe nsFtosraRtervoiomsiteomnperature. [D=atre Section 12.13Added hesheespc. 2 SSeeccttoniLoln, 12.127.F1inBailmivnoaltuem`epitsa1s.i0vmmL'nortoam upetflorfooorcnneisauvfoilausmtees es than 10m. 89.1 AK 0PFOS 109231A02H4, 104P.F22OSAAddRinformation sboutwingsrogae mati,clcifycontrol mates nd sp1i0k3iCnlgatrihtycupruvrepo,scehaonfgceontpiinkueisngtocavleirbryat2i0onsacmhpelcekss 11.1 Addedwerdingaboutcontol aml srs, using and po sing commercially available eIqnugievnaelreanlt,bmoaxkelmeessybseevece.for epipmendsuppies. Ste watefo Mili Nlgene ae 3MEnvironmental Laboratory ETS 842 or "600104 Page 100115 Page 50 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 SMd ar Extraction Worksheet ETS-8-4.2 [|sScuomppgal|| spRpOoMx.s05|| aFppOrMoxxS| | aFprMon. | Comme hain [Voroi WBDoe . |o | . jm |. pgnl " wml DaAtvesS:piked W_ rr rT -- ww [TTT -- wo|rT --Tr rT rr rT rr rT ---- rr TTT rr 17 rrr rr TT rr rT 7 -- T -- r T r T r r C rr r r rrr rrrrrr rr Tr TT rT rr rr11 rr r r T r r T ] r rrrrTT E r m r p Tr Fini Maia lewilslsreaBM Vom [Foc nlaosMTAA A= sav [FeToLoo CoV HCO ((a CpomemteiopsteeSelormebiiaier T 1 ------ [m w m wpm e a] (E Furniss m e r T ] | Cont Cul Venicatons uscd same mati 3fo i rv. Aosendix A: Extraction Wockshest 3MEnvironmental Laboratory ETsa42 000105 Buse 110115 Page 51 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 MDIL/LOQ values for rabbit serum Compound|(uMgD/mLl)| (nLgO/mQL)||LAipnperaorxCiamlaitberactoinocnenRtarnatgieon(sLCtoRb)e used for preparing i the Standard Calibration Curve [PFOS |174 | 555 [5 ng/mL-1000 ng/mL. [ EtFOP SE-F OH|3O46S[A 20A 5 [5 ng/mL-1000 5ng/mL~1000 ng/mL ng/mL. M536 [6|0193 2 [5ng/mL1000 ng/mL [PF|O 571S |E 182A[5ng/mlL--1000 ng/mL MDL/LOQvalues inrat, bovine,monkey, andhuman serum,andmonkeyplasmawere notstatistically determined. Twocurvesineachofthesematriceswereextractedandanalyzedwiththerabbitserom curvestodetermine equivalence.Responsesintherat,bovine,monkey,andhumanwere equivalenttothe rabbit responses, therefore, their MDL andLOQwillbethe same valuesasdeterminedinrabbitserum. PleaseseeLOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-1 for further information. AgpendiBs:MDULOQ Values snd Summary ETS842 3M Environmental Laboratory 0001.06 Page 120115 Page 52 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 'LIMS E00-1668 ans i| yTT | Prepared range PELCR from % [WERecovery RSD 4[[rinicee|| wo(nmg |[a(ngn/ml)|| woom [|swoeoo|| [[rpeee[oaonn[|sooamn|| on||sowwosn|| `Compound: PFOSA Prepared range | LCR from | % Recovery Rabbit Serum of standards curve. Range RSD Range [[p[[ rp iievmiee-- |||T swso=rnn s[|[[|o awwsmo=nsom ||||m wwaawnn[[||o sssomomw wo|||| Preparcdrange| LCRfrom| % Recovery RSD Rabbit Serum| of standards curve Range Range [[fP[ iparremese[[aoomr amoannn[[|orwsmomao somn||||wweownrw [[||oownnanasnnr|||| (ng/mL) ng/mL) JArpt DUrOesQ Visor ETsaaz | 0R0"2QY pis_- 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Compound: or EtFOSE-OH : Rabbit Serum of standards * (ng/m curve (ng/mL) Range Range ama | smes was [xTwwmae |Tommaos |wn[waansas| `Compound: PFOSEA Preparedrange Rabbit Serum| ofstandards LCR from curve % Recovery Range RSD Range te] TE | RE ompound:M556 Preparedrange |LCRfrom| RE%Recovery (ng/mL) ng/ml) [mee[omon[mone | ew | EDRSD a] oom | ses sos 3MEnvironmental Laboratory Page 54. 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analyst(s) 4 Prep date(s): Analyte(s): - EXAMPLE Ton Pair Standard Curves ~ Fluids Study number: Final solventandTN: Blank fluid/identifier: (Recon! matcix used o prepare StudyAnimal Matrix : FERED Method/revision: `Targetanalyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: scien ated ' `Surrsotdgapaprtoxe. 100 teppm: NIe Er jg r enn comE et. [0500 T~0507T0552T0501 |o0sal |0501| 0010 ]1015| - [0500Toor1055 T0s0r|osat | osot | 0020 | noes | [500[507 53% [soi| sat I sot|os | tow| [500WTM5%7 | 532 |soi_| sai soi |ooo| tos| [5.00L_soTS(r 567m "gsorm lgomiK oso)|0020 | Loa | [00 so. KsWH sii ff @s@& 301|ocos | too| [500B_safi7[8524hA.0.0 1bool8 sod | 000| ros| [[5s0o0oTT77550010TT775s5322T[ssoor[sa52r1 [ | 500s | | ooooo ios | |1r t0o25w| | Calculated concentorfsatatndiarodnsisnthesera matrix: Rabbit | 493 "T~s00 T7524 TT494 Tso | 513 T 100 [oos5 | [276 1 os 1"toa "97 | 9s | t02 | [aosTH 500lo2 r fg d | 501MShad[Finalconc [ors = Wyo 18 8THous @IWoo m Faod| ogni [s | mma w 77 owe 76] Co 1 oss To on | 0n 5 | oir | [E > r =Te `Validated ranges ~ approximate concentrations [Sem |"PFOS__PFOSA __PFOSAA BIFOSE-OH_PFOSEA _ Msse | [Bovine |Estimatesonly.Usevalvesforrabbit | [FionkeRya&tPlas |EEssttiimmaatteessocnollyy..UUsseevvaalluueessffoorrrraabbbbiitn, || [Human Testimatesonly.Usevaluesformabbun | 3MEnvironmentalLaboratory 000109 Page 55 3M Medical Department Study: T-6295.22 . - 3M ENVIRONMENTAL LABORATORY Analytical Report: FACT-TOX-160 LIMS E00-1668 loCdo"pyofJroingl) ChE re METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICAL COMPOUNDS FROMLIVERFOR ANALYSIS USING HPLC- ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wymne * Approved By: 4 To -- Laboratory Manager Dit fo Group Leader "Techlniachal RQevioewser Adoption Date: pH2Llag Revision Date: - NK Date on fog F499 Date oDah te m 1.0 SCOPE AND APPLICATION 1.1 Sotchoepre:fluTohrioschmeemtihcoald icsofmoprotuhnedesxtfrracotmiolinovefr.potassium perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants or other fluofinated compounds. 1.3 Mvaaltirdiacteiso:n rReapobrbti.t, rat, bovine, and monkey livers or other tissues es designated in the Word 6.095 3MEnvironmental Laboratory ETS 860 - 000110 Page lof1s Page 56 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 SuMMARY oF METHOD 4 21 (TBhiFsOmSe)tohroodthdeerscfrliuboersocthheeapmircoacledsuurrfeacftoarnetxstfrarcotminlgivpeor,taossoituhmepetrisfsluueosr,ouoscitnagneasnulifoonnaptaeiring erxetargaecntteda:nPdFmOetSh,ylP-FfeOrStA-b,utPyF!OetShAeAr ,(MEIXBFEO).SEI-nOtHhi,s mPeFtOhSoEd,A,seMv5e5n6f,luaonrdocshuermriocgaaltsecan be. stpaarntdiatrido.neAdnintioonMpaBiEri.ngTrheaegMeIntBiEsaedxdtreadcttoithtreanssafmeprlreedantodathceonatnrailfyutgee tiuonbepaainrdisput onto a : fniilttreorgeednthevraopuogrhaato3rcucntpilladsrtyi.c sEyaricnhgeexattrtaaccthiesdrteocons0t.i2tputmedniynlo1.nf0imltLerimnettohagnloalsstahuetnovials. . 2.2 T`hmeetsheodssa.mple extractsareanalyzed following method ETSo-rot8he-rap7prop.ria0te 3.1 PFOS: perfluorooctanesulfonate (anionof potassium salt) C,F,SO, 32 PFOSA: perfluorooctane sulfonylamideCF, SO,NH, 33 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetateCyF,SO;N(CH,CH,)CH,CO, 34 EFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol CiF,SO,N(CH,CH,)CH,CH,0H 35 PFOSEA: perfluorooctane sulfonyl ethylamide C,F,,SO;N(CH,CHi, 3.6 MSS6: CF,SO.N(H)(CH,COOH) 37 Surrogate standard: 1H-1H-2H-2Hperflucrooctane sulfonic acid 4.0. WARNINGS AND CAUTIONS _-- 41 Health and Safety Warnings: 41.1 hUasnedluininvgearnsialmaplretciasusutei,onwsh,iecshpemcaiayllcyonltaaboirnaptaotrhyocgoeantss., goggles, and gloves when - 5.0 INTERFERENCES presse ---- 5.1 There are no interferences known at this time. 660 0.1 EoTuh0 eefmoelln0 orwing eq0 uipmen0 t is use0 d while0 perfor0 ming thi0 s method0 . Equi0 valent e0 quipme0 nt is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.12 Vortex mixer, VWR, Vortex Geni2e. 613 Centrifuge, Mistral 1000orIEC 6.1.4 Shaker, Eberbach or VWR 3MEnvironmental Laboratory ETS860 TTT 000111 Page2of14 Page 57 3M Medical Department Study: T-629522. _ Analytical Report: FACT-TO1X6-0 LIMS E00-1668 6.15 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity 1.0.10 g) 7.0 SUPPLIES AND MATERIALS 71 Gloves 72 Dissecting scalpels 73 Eppendoorrf disposable pipettes 7.4 Nalgene bottles, capableofholding 250 mL end 1 L. 7.5 Volumetric flasks, glass, type A 7.6 1-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 79 Labels 7.10 Oxford Dispensor ~3.0 to 10.0 ml 7.11Syringes,capableof measurin5gkLto 50 pL 7.12 Graduatedpipettes < . 7.13 Syringes, disposableplastic,3 cc 7.14 Syringe filters, nylon, 0.2 um, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers > Note:PrQitorwtaotuesri.ngRgilsaessswyarrienagnedbaomttilens,irimnosuefm3timmeesswwiitthhmmeetthhaannooll,an3dsa3tsismfesowmit3hMsielplris-te s. B8R810 ETyA pelG reageEnt grN ade wT aterS , MilA li-QN TM orD equivSalenT t allA walerNuseD d in tA his meRthodDshoS uld `beMilli-QTM waterandbeprovidedby a Milli-QTOC PlusTMsystem 82 Sodium hydroxide (NaOH), J.T Baker or equivalent . 83 Tetrbutylammonium hydrogen sulfate(TBA), Kodak orequivalent . 84 Sodium carbonate (N&;COy), IT. Bakeror equivalent . 85 Sodium bicarbonate (NaHCO), J.T. Baker or equivaltat 86 Methyl tert-butyl ether, Omnisoly, glass distilled or HPLC grade 87 Methanol, Omnisoly, glass distilled or HPLC grade 88 Liver,frozenfrom supplier 89 Dryice from supplier 810 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight= 538 29EvironmentLaboratory remETSi860ss 000112 Page30f14 Pages 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight= 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 i 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight= 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight =527 . 8.10.6 MSS6 (3M Specialty Chemical Division), molecular weight = 557. 8.10.7 SCu,rFr,o,gSaOt,eHs)tamnodlaercdu:l4a-rHw,epiegrhftlu=o4r2o8octane sulfonic acid(1-H,1-H, 2-H, 2-H 8.10.8 Other fluorochemicals, as appropriate 811 Reagent preparation `NpOreTpaEra:tiWohn,eandjpursetpaacrcionrgdilnagrlgye.rvolumesthanlistedinreagent, standard, orsurrogate. 811.111000N0smoLdibuemakheyrdcroonxtiadieni(nNga5O0H0):mLWeMiiglhlia-pQpTMrwoatxeirm,amti2ex0luy0ntilNaalOsHo.liPdosaurriento a dissolved. Store ina 1 L Nalgene bottle. 8.~ 11.2110N NsNodaiOuHmhsyoldurtoixoindient(oNaaO1H0)0:mDLilvuotleum1e0tNriNcfalaOsHk a1n:1d0d.ilMuetaetsuorveol1u0mmeLuosifog . Milli-QTM water. Store ina 125mLNalgenebottle. 8113 0.5TMBAteitntaobuat1yLlavmomlounmieutmrihcycdornotgaeinnisnuglf5a0t0e (mTLBAM)i:llWie-iQghwaaptperr.oxAidmjautsetlyto169 g POfHNa10OuHs,inagdadppsrlooxwilmyabteeclayu4se4 ttohe5p4HmcLhaonfge1s0 aNb'rNupatOlyH)(.WDhiilluteeadtodivnogltuhmeelawistthmL Milli-QTM water. Store ina 1 L Nalgene bottle. 8113.1TnBeeAdreedquusiirnegs1&cNheNcakpOrHiosroltuoteioanc.huseto ensurepH = 10.Adjustas 8.11.4 0a.p2p5roMximsaotdeiluym2c6a.r5bgoonfastoeldsioudimumcabribcoanrabtoena(tNeb,uCfOf;e)r(aNn2d,2C1O.,0/gNoafHsCoOd,)i:umWeigh bQiMcarwbaotnera.teS(tNoArHeCiOn,a)1iLntNoa1lgeLvnoelbuomttelte.ric flask and bring to volume with Milli- 812 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. - 8.12.2 Pfrleupoarorcehoetmhiecrafllsutoarnodcahredmsiacraelasctcaenpdatradbsl,e a(sfoarpeprxoapmrpilaet,e.onMeulwtoirckoimnpgosnteanntdard s1o.l1u0tpiopnmcoEnttFaOinSiEn-gO1H..0)0 ppm PFO, 1.02 ppm PFOSA, 0.987ppmPFOSAA, and 8.12.3 Weigh approximately 100 mg ofPFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standardofapproximately 1000 ppm (kg/mL). 8.12.5 Daiplpurtoexitmhaetsetloyck50soplputmi.on with methanol for a working standard 1 solution of 3M Environmental Laboratory ETS860 Tm 0QoM13 Page dof 14 Page 59 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIS E00-1668 8.12.6 Daiplpurtoex.th5e.0stpopcmk.solution with methanol for & working standard 2 solution of 1 8.12.7 Daiplpurtoex.th0e.5s0topcpkms.olution with methanol for a working standard 3 solution of 813 Surrogate stock standard preparation 8.13.1 We`iC.gFh,a;pSpOr,oHxiinmtaotael5y05m0l-6v0olmugmeotfrsiucrfrloagkataensdtarnedcaorrdd1t-hHe,a1c-tuHa,l2w-eHi,gh2t.,H, 8.13.2 Brpipnmg.to volumewith methanol for a surrogate stockofapproximately 1000-1200 8.13.3 Psrtepoacrketaosaur1r0omgaltveowolrukminegtsrtfaliansdckaradn.dbTrriannsgfteor avpprooxiwlmiatteuhlmyemt1.h0amenolloffosuarrrogate `working standard of 10-20 ppm. Record the actual volume transferred. 2.0 SAMPLE HANDLING 9.1 Allsamplesarereceivedfrozenandmustbekept frozenuntilteextractionisperformed. 10.0 QuavTy ConTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used as asolventblank. 10.1.2 aEsxtmreatchtotdwobl1a.n0kms.L aliquotsof Milli-TM water following tis procedure and use 10.1.3 E2sxtmraatcrtitxwbola1n.k0s.mLReafleirqtuoots11of1i6v.er homogenate folowingthisprocedure and use: 102 Matrix spikes + 102.1Prtehepaarcceuarancdyaonfatlhyezeexmtartarcitxiosnp.ike and matrix spike duplicatesamplesto determine 10.22 Prerceepiavreedcwaicthhsepaickhe ussaimnpgleassta.mple chosen by the analyst, usually acontrol liver - 102.3 AEdxdpietcitoendalcsopniceknetsrmaatiyonbsewiincllaudledinanthdemmaiyd-fraalngienotfhtehlowi-nirtaianlgceaolifbtrhaetiionnitciuarlve. calibration curve. 10.2.4 Pmrienpiarmeuomnoe fm2atmraitxrisxpiskpeikaensdpmeartbraitxhs.pike duplicate per 40 samples, with a 10.3 Continuing calibration verifications 10.3.1 iPnrietpaalrceacloibnrtaitniuoinncgucravlei,bration verification samples to ensure the accuracy ofthe 10.3.2 oPfre1p0arsea,maptleasm.inFiormuemx,amopnlee,coinftaisnauminpglecasleitbr=at3i4o,nfvoeurrifviecraitfiiocnatsiaomnpslaerepeprregpraoruepd and extracted. 3MEnvironmental Laboratory ETS 860 TTT 000114 Page Sof 14 Page 60 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 103.3 Pprreeppaarreetehaechincitoinatlicnuurivneg. calibration verification from the same matrix used to i 103.4 cTahleiberxaptieocntecdurcvoen.ceAndtdriattiioonnaslwsilplikfeasllmwaityhbine tihneclmuidde-drathnagteofaflthien initial the low-range of tohnelyintithieallocawleibnrdaotfitonhceurcvael.ibrTahtiisoniscunrevcees(sfaorryeixftamhpelea,na5lypsptbmu--s1t0q0uapnptbi,tartaethuesring than 5 ppb -- 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION _ 111 Prepare matrix calibration standards ir 11.1.1 `WemisgMhialplpir-oQxTMimwaatteerl.y 4Gr0ignodftloiavehrionmtoogean2e5o0usmsLolNuatilogne.ne bottle containing 200 11.1.2 14gi0snotavailable,use appropriateamountsofliverand water ratio. to ensurae 1:5, 11.13 Refer to 13.0 to calculate the actual densityofliver homogenate and the concentrationofsolid liver tissue dispersed in 1.0mLofhomogenate solution. 11.1.5 sAhdadki1ngmbLeotfweheonmoalgieqnuaottsewthoiale1p5rmepLarcienngtraitfoutgaelotufbeei.gRhet-eseuns1pemnLdsoalluiqtuiootnsboyf homogeneous solution in 15mLcentrifuge tubes. 11.16 Two 1 mL aliquots,orother appropriate volume, serve as matrix blanks. 11.1.7 tThyepiecnadlolfytshiestsheectsitoann,datordspcioknec,enitnrdautpiloincsataen,dtswpoiksitnagndaamroducnutrsvelsi,stfeodriantoTtaablleof1, at eighteen samples,twomatrix blanks, andtwomethodblanks. 11.1.8 `RwehfiecrhtloisvtasltihdeatwioornkrienpgorrtasnEgTeSs-a8n-d6.th0eaLnidneEaTrSC-a8l-i7b.r0at-iVo-n1RoarnAgtet(aLcCRh)mfeonrBt, calibration curves. 11.19 UstsaendAatrdtsa.chRmeefenrttCo 1as3.a0ntaoicdailncuclaaltceulaacttiunagl ctohnecceonntcreanttiroantsioonfsoPfFOtSheiwnocrakliibnrgation standards. - 11.2 sTuorreoagcahtewowrorkkiinnggssttaannddaradr,dbfolrat,heocronccoennttirnautiinognvteoriffailcawtiiotnh,inatdhdeacpaplriobprraitaitoencaumrovuenrtaonfge 5 `ppb 1000ppb. 3M Environmental Laboratory ETS860 000115 Page6orls Page 61 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 11.3 Extract spiked liver homogenates following 12.14-12.25ofthis method. Use these 3 standardsto establish eachinitial curveonthe massspectrometer. `Table 1 Approximate Spiking Amounts for Calibration Standards TT Bak | `Working Standard (Approx. Conc) ul Approx. final conc. of PFO in liver 00500pppmm [ | 2010 | om00ss0ppm m|| 0S0pm | #0 0152500 gmm m| ComSompm 1[4 50 10070pmmm | 120 ProcEpuRe. --_-- 12.1 Obtain frozen liver samples. - 122 Cu`tpearpfoprrmoexdiqmuaitckell1yy, gnooft lailvleorwiunsgintgheadliivsesretcotitnhgaws.calpel. "Thispartoftheprocedisubreset 123 Weigh the sample directly into ataredplasticsampulevial. 12.4 Record the liver weight in the study notebook. 12.5 Returnunusedliver portionstofreezer. . 12.6 Add 2.5mLsof watetro sampule vial. 12.7 Gruintnidltthhee ssaammppllee. iPsuhtotmhoeggernienoduers.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 a: pipette. 129 Take the grinder apart and cleanitwith methanol after each sample. Refer toAMDT-EP22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory mss 000116 paged Page 62 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.11 cPeinptertitfeug1e.0tumbLe,, Loarboetlhetrheapcperntorpirfiuagtee tvuobleumwei,thotfhheoimdoegnteincaalteinifnotrom2at1i5onmaLs ptohelyspsrroappyulleene `vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquotsofMilli-QTM water to centrifuge tubes. These will serve as `method blanks. 12.13 Spike all samples, including blanks and standards ready forextractionwith surrogate. standard as describedin section 11.2. 12.14 Spike each matirx with thie appropriate amountofstanasddeascr ribded in 11.1,or Table 1 ocfotnhtaitnusiencgticoanl,ibfroarttihoen scatlainbdraartdiso.n curve standards. Also prepare matrix spikes and 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16Checktoensure 0.5 MTBAreagentisatpH 10. Ifnot,adjustaccordingly. 12.17 Toeachsamplaed,d 1 mL-0.5 M TBA and 2mLofthe 0.25 M sodium `carbonate/sodium `bicarbonate buffer. . 12.18 Using an Oxford Dispenser, add 5 mL methyl-ert-butyl ether. 12.19 Capeach samanpd plutoentheshakeart asettingof 300rpm,for20 minutes. 12.20 Centrifugefor 20 to 25 minutesat a setoft35i00nrpg m, or until layersarewell separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL ofthe organiclayer to the fresh 15 mL centrifuge tube. 12.23Puteach samopnltheeanalyticalhitrogenevaporunatitldoryr, approximately 1 to 2 `hours. 12.24 Add 1.0mLtoeachcentrifugetubeusing agraduatedpipette. ~ 12.25 Vortex mixfor30seconds. 12.26 Attach 2.0.2 umnylonmesh filter to a3cosyringeandtransferthesampletothissyringe. `Filterinto a 1.5mLglass autoorlvow-ivoalulme autwohenvneicesasarly. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, * `matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at. approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attachetdo this document,andtape in study notebook: or include in study binder, as appropriate. 3M Environmental Laboratory ETS:860 000117 ESM Page 63 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: q 13.1.1 `Ctaelncsuelpaatreattehe1.a0vemrLagaeldieqnuostistoyoffthhomeoglievneartheo.mogenate by recording each mass of Average density (mg/mL) =Averagemass(me)ofthealiquots 1.0m aliquot, 13.12 Cdailscpuelrasteed stohleiadmtoisusnuteopfelrimveLro(fmhgo)mpoegre1n.a0tmeLsuhsopmeongsieonna)tues(ionrgctohnecfenotlrlaotwiionngof equation: gofLiverxAv(egroafLgeivdeenrs+itgyo*fofWahtoemro)genate(mg/mL) *referto 13.1.1fordetails. | 13.13 sCtaalncdualradtse uacstiunagltchoencfeonltlroawtiinognseqoufatPioFnO:Sandofhes fluorochemicals incalibration. LCLoofSnt`amncgdaLriedvexrn/ 1tmLrhoamotge(niuatgoe/*mnL) = FoifnaPlFCOoSncienntLriavteiron (ug/g or m/ke) *refer to 13.1.2for details. "114410MTSEphTeecHimfOeicDthMPoDEdRLdFetaOenRcdtMilAoinmNiCltioEmfitqu(anMtDitLait)sioann(aLlyOtQea)nvdalmuaetsri(rxefsepretciofAict.taRchemetfnotsMeBDrLanrdepCo)r.t for 142 Tthheeqfuoalllitoywoinfgthqueaelxittryaccotnitornoalnsdamanpalleyssiasr.e extracted with cach batchofsamples to cvalusts 14.2.1 Method blanks and matrix blanks. - 14.22 M`parterciisxiosnpoifketahnedemxattrarcitxisonp.ike duplicatesamplestodetermine accuracy and 14.23 Coofntthieniuniintgalcaclailbirbartaitoinonvecruirfviec.ation samples to determine the continued acouracy 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 1515..01 PhSOiaLgmLhpUlBTeTIwUOaNsctPoenRtEiasiVndEeirNsspT,oIsaOendNdAiunNsbeDidoWghAlaazSsasTrdpEicMpoeAntttNaeAiwnGaeEsrstM,eEfiNlsTadmimsapbosleedsionlvbernotkweansgtleasiss dciosntpaoisneedrsin located in the laboratory. 3M Environmental Laboratory E5860 000118 Page9ofid Page 64 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS 00-168 16 Recoros , 16.1 oCromipnclleutdeetihnetehxet3r-arctiinognswtoudryksbhienedetr,atatsacahpepdrotporithaitse.method, and tape in the study notebook 4 17.0 TABLES. DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet `180 R Lo 18.1 The validation reportassociatedwiththismethod is ETS-8-6.0 & 7.0-V-1. 182 AMDT-EP-22, "Routine MaintenanceofUltra-Turrax T-25TM 183 FLiAvCeTr-fMo-r1A.n1a,ly"sEixstrUascitnigonHPoLfCB-FEOlSecotrrOotshprearyA/nMiaosnsicSpFelcutorroomcehtermyi"cal Surfactants from 1A 919..01 v ETS-8p -7.0,e "Analc ysisor fLivee r Extp ractsD for Flo uorocc hemicu als usm ing HPe LC-Eln ectrot sprays Mass Spectrometry" 20.0 Revisions Revision Number, pree----m ---- ie Revision `Reason ForRevision Date. 3M EnvironmentalLaboratory ETS860 000119 Peles Page 65 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 SudyF_____ Matrix |SumogweSd | FCMixS@ | approx. ppm| appr0.o5pxp.m | aFpCpMrixSoSpxo@m.| | apFprCoxM. x50Sp@pm| Comments Box actual ppm| actual ppm| actal ppm| actual ppm WeDay______(4 |e |e |a" Date Spiked/Anal eo Trrr7r-- FT tr ws rr rr 1 Ele ---------------------- Fer---- A rr r T feeeee e--]-- ------------) err "grea foesemt fr---- rT Be eee e---- -- er ----e -- ee r -- ea er et---- -- r rr rT ------------ rr 071 eT e r ee A rar -- rrlla eeem eelra ee r To] r ec-. ------------------------ ----Se-- ri-- e ] r rr rr r rr rT 71 ==ssrrrT"T=--------///--//------'ee ~~ -------- eee rr]re e-------- e r e a m r T ea E ErEr rrTrer ter mw--ar---- E weE ns TS `Cont. Cal. Verifications used the samematix38fore sandard curve. Atachament Bt MDLILOQ Vales 3M EnvironmentalLaboratory ETS8.60 000120 Page 110f16 Page66 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 MDL/LOQ values for rabbit liver Compound| MDL | LOQ| Linear Calibration Range (LCR) (ppb) | (ppb)| Approximate concentrations to be used for preparing the 1 Standard Calibration Curve [PES 845 | 269 |30ppb- 1200 ppb PEOSA |3.|5T10 .1 [12ppb--1200 ppb [ FTFP OSE-F OH||O 1 24S60|A |378 48A 53|6|030ppppbb~-9102000pppop*b [M[53P6 F|O3832S93 |E|1206A52|| 6300ppopbb--11220000pobp,b 'MDL/LOQvalues inrat,bovine,andmonkey liverwere not statisticallydetermined. Two curvesineachofthesematriceswereextractedandanalyzedwith therabbitliver curvesto determineequivalence. Responsesintherat, bovine,and monkey livercurves wereequivalent to `therabbitresponses,therefore, theirMDLandLOQwillbeassumedtobeequivalenttothose `valuesas deteforrtmheriabn bite livd er. . Referto LOQ SuraamndaMrDyLstudyinETS-8&-7.60-.V-01forfurtherinformation * EtFOSE-OHestimatesonlyforMDLand LOQ. Didnotmeetcriteriaforvalidation. [om Jeon om | em `Compound:PrPepFaOreSd| Ramgeof CROW] Ramgeol mLaivneir ||osrnammdgaediosf ||| ob aevoaevngee_oaiveiwiuiven|| | ob) lcoawvswed ont | mn LORE Tamgeof Glmoewnsd]l - heigehs 3Gp a) pnt + IOREGR] oFmrmeadds] 5b Gt [Jn[oon `Compound:PrPepFaOreSd| A Ramgeof oLiveer || mmogesof|| aSveenge | ee |wn po | S CEmGeRkEvSeSr,|| | Rlaoswgseodf sLeCIGRVnf3oQ]mLTS|| RhaSinggeheeofd b{CeLhOiREgOhmZa] [im [Cm|eomow [wen| . Compound:FrPoFpaOedS|AA Wamgeof UCKfiom| Wamgeof mLiavecr || psoryngdeunotfsy||| 62v0c,eavgee weave| lawnsed oo pm | 39) LOlRofwosdm'|| Rhagmhgeeodf a)mpenst)||Go)amGoerm gLOhRGdam | ament Atachment B: MDLILOQ Values 3M Environmental Laboratory ETS560 000121 Page 12616 Page 67 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Compound: EtFOSE-OH SE ror eT i| ver | mogeof | aemge weeurve | lows lowsd | highed highs [om |[rSmEs So[o=n [vS a TE Gh Compound: PFOSEA we| EToEr | r Te T T er T To mavix | sands| cave piiieii| curve curve| curve Ceurve| [nor s ST m]oETnTE]E THE| ompound:M556 - Lier | mogeol| avenge ave lowsd war | highs + high| mais| osangduyds || oanmte) E8S)SaEt os cavee s o50wied] ] oscaapevs e Htiy eGgr AttachmeCnt: Standard Calculations 3MEnvironmentalLaboratory ETS$60 i. 000122 Page 130f 14 Page68 3M Medical Department Study: T-6295.22 `Analytical Report: FACT-TOX-160 'LIMS E00-1668 Ton Pair Standard Curves -- Tissue 4 PArnealpydtae(tse:: ESqtuanidpamredntnunmubmebre:r: Sample matrix: BFliananlksloilvveern/tidaenndtiTfeNr:: TaMretgheotdasneavliysitoen):: . FFCC mmiixxssttdd aappppr05r..50o00o0ppspp.ms.:: FC mix std approx. 50.0 pp: Surrogatestd approx. 100ppm: R ir [or [oE r[Er [EE T aeT [aa [2 ActualconceonfsttanrdaardstinitheoFCnmisx Sticonc|Stdcone| Stdeons| Sudcons| Sulcone| Stdcons| Sulconc | Austspiced| Density [[0C500000TT00550000[[0055000[|00550000 || 00550000 [C0500|0500| 0500|os | 0500 ||G050000[ [ | evoo| ooir[[waiiee]] | 0500| Goro ater] - [[0C5050000T[00550000|[00350000[|0500500 || 00550000||wass0000|[| wGaoaso ||ooeire]] [[C55000[550m0T[5s00o 5s000||5s0000[[550000| | | otoaro[toreerr|] ([o SoT[530000[|550000 || 5000[|550000 ||s50000||| oasso0r JJoorree]] CaFFlOicSulla||tedFcFFoiOnSmceAn|tr|aPFtFliOcoSonmsAoeA|f|stEaIFnNoduOalSrEd||s iPnFFtOhaSelEsA|a|mplMFeSimlsat|rixSc_- one|SSorarcosgsaice|| AAmTt [59wvse9[|swoe [5%[50we0|Tsww | 5w0e0[ | | oTows| F [29a 5 [2e 09 [a 29|a9e[2a9e9 |a20e9 [1 | Suse [C2m9[Tmm9T [2m 0 [TaeoeTm o[9e | 1IowEsemR| I[CCo | m0 9 | s0 0 |sw[ 00|OswY [7] IE 0 0 00 0 CE | Validatedranges- approximate concentrations sro [R= et Tss im els ------------] -- tachmenCt:Standard Cllations 3M Environmental Laboratory ETS60 000123 Page t4ot1e Page 69 3M Medical Department Study: T-6295.22 Analytical Report: FLAICTS-TE0O0X--116660 bCetGzcgy of Oring) 3E3MMNEVNVIIRRONOMENNTMALELANBOTRAATOLRYLABO~R~ AT~ORY METHOD ANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATE OR OTHER 'FLUOROCHEMICALS IN SERUM EXTRACTS USING 'HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-52 Author: Lisa Clemen, Kris Hansen `Adoption Date: 03/01/99 RevisionDate: ,2) ./01 Approved By: Wer Le Laboratory Manager lite fe | Group Leader Aa LOG (os Technical Reviewer 02DaLeory oz/Deaitefe] 22Da-toe l:ol 10 Scope AND APPLICATION 1.1 uSscionpge:HPLTCh-islemcettrhoosdprdaesyc/rmiabsess sthpeecatnraolmyestisroy.fserum extracts for fluorochemical surfactants 12 oAtphpelricioanbilzeabCleomcpoompuonudnsd:s.Flaorochemical surfactants or other fluorinated compounds, or 13 vMaaltirdiacteiosn:reRpaobrbti.t, rat, bovine, monkey, and human serum,orother fluids as designated in the Word 695 3MEnvironmental Laboratory Ate ntSEorTSB5o2r immense 000124 Page lof Page 70 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 SUMMARY OF METHOD. i 21 pAelrtfhoorumgahncseu-pbpaosreteddmbeythaovda.liCdaarteifounlfaotrtmenotsitoncsomhmoonblueypulasidedd matrices, this is a to method QC as there is great evaxrtiraabcitleitdyfirnosmersa.erTuhmisormeotthheordfdlueisdcs,ruibseisntgheHPaLnCal-yesliescotfrofslpuorraoyc/hmeamsiscaslpescutrrfoacmteatnrtys, or cshiamrialcatresryissttiecmaofsapaaprptriocpurilaatre.fluTohreocahneamliycsails, issupcehrfaosrtmheedpberyfmlounoirtooorcitnagneassuilnfgolneatieon(POS) taonfiounr,thme/rvze=ri4f9y9.thAeddidietnitointaylolfy,ascampolesmmpayboybedeuatneancltyidznegdduasugihntegr aitoanndseomftmhaesspasrpeencttrfoomn.eter 3.0 DEFmvrTIONS 31 `quAatdmroupsoplheesryisctePmrsesaslulorwe Lfoornivzaartiiouosn m(eAPtTh)o:dsTohfeioMniiczraotmiaosnbsyQuuatitltirzoinIglvaanrdioUulstsiomuarctreisp,le Aprtomboessp,haernidcinPtreersfsaucerse.chTehmeisceailnlcolnuidzeatbiuotna(rAePnCo)t,liTmhietremdotsop:rEalye,ctertoc.sprTahyeIoinoniizzaattiioonn(pErSo)c,ess in these techniques occurs at atmospheric pressure (i. not under a vacuum). 32 presEslureec,trwohseprreabyyIioonnisizantsioolnut(iEoSn, aErSeDt:raansmfeetrrheoddtoofitohenigzaastpihoanspeevrifaotrinmyedcahtaratgmeodsdprhoeprlietcs. These charged droplets are produced bytheapplicationofastrong electrical field. 33 (MSM/aMsSs):SpTechterAoPmeTtQruya,ttMraosIsTSapnedcUtlriommeatterrip(leVSq)ua,dTruapnodleemsyMsatesmssSapreecetqruoimpepetderwith rqautaidor(ump/ozl)eamnadsssusbesleeqcuteivnetldyetdeectteocrtse.d.ToAnssianrgeleseMleSctmivaeylybdeisecmripmlionyaetdedfboyr imoansdsettoeccthiaornoger a Series (MS/MS) for more specific fragmentation information. 34 `quaCdrounpovleenstysitoenmvsasl(.poZs-tsp1r9a9y8)purtoibliezeinat*eZr-fsacper:ay"Thceonlfaotresmtatmioodne.lsTohfeMsipcrraoymeamsisttterdipflreom a dpirroebcetliys aotrtthheogcoonnael atpoertthuerce,onafetaeprepratsusrien.g Itnhrtoheugchonavteonrttiuoonuasl pcaotnfhowramyaitniotnheitciosuantiemred e`lmeacitnrtoednea.ncTehaoreugthhetsheamceo.nfHigouwreavteiorn,iZs-dsipffrearycacto,mtphoenmeentthsodasnodfcoopnevreanttiioonn,alclceoamnpinogn,enatnsd are `ncootmcpaotmipbalteibwiltehwistohmeonoethaenroZth-esrp,rabuystyosntleymwsi,tehtcs.i)milar systems (ic., Z-spray components are 35 Mass Lynx Software: System software designedforthe specific operation of these. 4Q.u0atvterrsoioInIst.ripAllelvqeurasdirounpsoalreesyssitmeilmasr.. CFuorrremnotrlyedMeatsaslLssyenex htahes mWainnudaolwsspe9c5ifiacndtoWtihnedowsNT Uisnesrt'rsumGeunitd(e)M.icromass Quattro II or Ultima triple quadrupole MassLynx or MassLynx NT 4W 0 amvncsaMoCaumons 41 Health and Safety Warnings: 4.1.1 eUmspelcoayustiaovnowlittahgetohefvaoplpiraogxeimcaabtleelsyf5o0r0t0heVoplrtosb.e. When engaged, the probe Word 695 3M Environmental Laboratory _EmSS2 0goqR5 Fus2olt Page 71 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 'LIMS E00-1668 4.12 When handling and clothing. samples or solvents wear appropriate protective gloves, eyewear, 42 Cautions: : 4.2.1 IDfothneobtaocpkerparteesssuorleveenxtcepeudmsp4s 0abobavre, ctahpeaHciPtLy Cofw4i0l0ibniatria(t5e3a0u0topmsai)tibcacskhuptrdeosswunr.e. 4.22 "Do not run solvent pumps to dryness. 50 Iremepmevces 0000000 5.1 sTtoormaigneimoirazneyipntaerrtfeorfenicnesstrwuhmeenntaantailoynztihnagtscaommpeless,inTceofnltoanctshwoiutlhdtnhe bsoaemupstleedorfoerxtsraacmtp.le SOFouewesr 000 6.1 Equipment listed belowmaybemodifieidn order to optimizethesystem. Document any `modificationsintheraw data as method deviations. 6.1.1 wMiitchroamnaeslsecQturaotstprraoyIiToonrizUalttioinmasoturirpclee quadrupole Mass Spectrometer equipped 6.12 HP1100 or Agilent low pulse solvent pumping system, solvent degasser, column `compartment, and autosampler 77S .01 u Suppe liespuesANDMaTERIALS 7.1.1 nHiitgrhogpeunristyysgtreamd)enitrogengas regulated to approximately 100 psi (Houaisroer 7.2 iHnPtLheCraanwaldyattiac.al column, specifics to be determinedbythe analyst and documented 7.13 Capped autovialsandcapped 15 mL centrifuge tubes 8.0 REAGENTS AND STANDARDS 81 Reagents 8.1.1 Methanol, HPLCgradeor equivalent 8.1.2 eMqiulilvail-eQnTMt,waatnedr,maalyl wbaetperrouvsieddedinbythaisMmielt-hoQdTsOhoCulPdlubse sMyisltleim-oQrTMowtahteerrvoerndor 8.13 Ammonium acetate, reagent grade or equivalent 82 Standards : 8.2.1 pTyrpeipcaarleldydtuwroinmgetthheoedxtbrlaacntkiso,ntpwroocmeadturriex.blSaeneksE,TaSn-d8-e4i.g2h.teen matrix standards are 3MEnvironmental Laboratory _Emses2 000126 Pessofll Page 72 3M Medical Department Study: T-629522 Analytical Report: FACT-TOX-160 LIMS E00-1668 9.0 SAMPLE HANDLING 91 Fresh matrix standardsare prepared with cach analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 92 Ifanalysis will be delayed, extracted standards and samplescanbe refrigerated at `approximately 4 C, or at room temperature, until analysis can be performed. 10.0 QuaLITY CONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 `Soonlcveednutrbilnagntkhse, mcoeutrhsoedobfltahnekssatnuddymattordiextebrlmainnkesifarceopnrteapmairneadtiaonndaoncacluyrzreeddadturilnega,st `sample prep. 10.1.2 Analyze at least onesolventblank prior to each calibration curve. 10.1.3 eMxattrraictxsb.lanks should be analyzed with each sample listthatincludes undiluted 10.2 Matrix Spikes 102.11curanvdmeethsod QCareprepared in asurrogate matrix (e.g. curves in rabbit sera,sam arep monl keye sers a),matrixspikesandmatrixspikeduplicates are prepared in blank samplematrix (e.g., monkey sera) and analyzed to verify extraction efficiency. 10.2.2I`fmcautrrivxesspiakneds m are re equQirt Ceda.rh epreo pared dinthesamematrix as samples, no additional 10.3 Continuing Calibration Verifications (CCVs) 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 103.2 A`snaamlpylezse,twwiothcaalimbirnatiimoun msotfatndwaordspe(ronbeatactheaancdh oalf2waylesvfeilnsi)sahfitnegraenveirnjyeoctnieonto ten sequence withatleast two calibration standards. 11.0 CALIBRATIONAND STANDARDIZATION 11.1 Awinlallbyezpelottheteexdtbryalcitneedamrartergirxescsailoinb,rawteiionghstteadnd1a/rxd,snportiofrortcoeedatchhrsoeutgohfzeexrtor,acutssi.ngTMhaescsuLryvnex or other suitable software. 11.2 If thecurve does not meet requirements, perform routine maintenance, reextract samples, or reanalyze the standard curve. 113 Fraonrgep,uript mosaeysboef anceccuesrsaacryywthoeunsequeaintthietratthineglloewveelnsodfoarnatlheytheiagthtehnedolifmtihtseocfatlihberactuirovnecurve rather than the approximately full rangeof the standard 10 ppb of analyte, it may curve. Example: when be beneficial to generate attempting to quantitate 2 calibration curve cpopnbsitsoti1n0g0o0ftphpbe).staTnhdiasrdwisllfrroemdu5ceppibnatcocu1r0a0cyppatbtrriabtuhteerdtthoanlitnheearfurlelgrraensgsei.onofwetiheghctuirnvge o(f5 ETsesz Page dof 11 3M Environmental Laboratory ren 000127 Page 73 3M Medical Department Study: T-6295.22. Analytical Report: FACT-TOX-160 LIMS 00-1668 haingdhachoingchencturravtei.onIfstthainsdaisrddso.neI, insoalmsooraecctehpatnabolneetpooibnrtesahkotuhledlbieneaursreadnigneciontmomaolnobwectuwreveen . pthpebcaunrdvesth.eFhoirghexcaumrpvlee,mathyienlcolwudceutrhvee mpoaiyntisn:c2l5u0d,e t5h0e0,fo7l5l0o,wi1n0g00po,in1t2s5:01,pp5b,.25, 100, 250 12.0 PROCEDURES 12.1 Acquisition Set up 12.1.1 dOensitghneatMoarslseLttyenr,xmlaasit n2 page, set up asample lst name. digitsoftest year-mo-day, and a Save the list as instrument letter that will increase throughthe alphabetwitheachadditional is for that day. Example Sample List:IYYMMDDa or D010712a Ininstrument name (D for "Davey" YMM==ymeoanrtohfotfesttes(t01()07) a=DfDir=sdtasyoafmptleesltis(t1(2r)un)ofthe day(thenext sample lst will end with `b," the next `c" and s0 on.) 12.1.2 dAasys,iagnnda fai3l-ednigaimte suequsenttihieailnfnisltgeruummebnetrdtehsaigtnsattaorrtslweitttehr, |tahnedliastn2dcigritesboyafoysneeaerf-osmroeach filename. `Example File Name:TYYMMDD## orD010712001 IY==iynsetarurmeonfttnesatm(e01()D for "Davey") MDDM==dmaoynotfhtoefstte(s1t2)(07) ##=3-dligit sequential file numberstartingwith 1 through 999 (001) Ablotst,leanspumabcetro,ftanheisnajemcptiloenlvsot,lausmseiganndasmaemtphloedd(eMscSr)ipftoironasc.quiring,aninletfile, a 12.1.3 TseoleccrteSatIeRa(SmientghloedI,ocnlRicekcoorndMinegt)hoordMEdRitMor(bMuutlttoinplienRtehaecMtiSonStMaotnuistoPrainneg)a.ndSet TseotnitzhaetiaocnquMiosidteionasstaaprptaronpdrsitatoepatnidmemsa.sSsatvoe4a9c9quiosritoitohnermeatpphroodp.rIiaftMe Sma/sMseSs. Also instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.4 Teyxptircaacltleydtmhaetrainxalsyttaincdalarbdast.ch run sequenceBi begins with solvent blanks and aset of 12.1.5 Sampleextractsare analyzed with two CCVs injected every one to ten samples. aSnoldvaernetnboltanckosnssihdoeurleddbseaamnpalleyzexetdrpaecrtisobduitcamllayytboemoinnciltuodrepdoasssisbulceh.analyte carryover 3M Environmental Laboratory ETSS852 Th ---- Page Sof 11 000128 Pages 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-168 12.2 Using the HPLC 12.2.1 Set up sample tray according to the sample lst prepared in Section 12.1.1, : 12.2.2 Set-up the HPLC to the following conditions or at conditions the analyst considers * `Taopgpbrooopkri:ate for optimal response. Record actual conditions in the instrument 1222.1 Sample size = 10 pL injection 1222.2 Injectsample = 1 122.2.3 Cycletime = 10.0 minutes. 122.2.4 Flow rate = 300 pLimin = 122.2.5 Mobile Phase (program) acetate(in HO) [00min | To%I o0% | [100m | 10%| 00% | [50min | 05% | 5% | [7.50mn| 05% | 56 | (80min | 10% | 90% | 123 Ynstrament Set-up 123.1 RefertoETS-9-24formore details. 123.2 Checkthesolventlevelinreservoirsandrefillifnecessary. 12.3.3tChethipt.hTeehsetctaiinplkesshsosutledeblceafplialtlwairtyahtntohejeaggenodefdtdgheesp.rIofbteh.etUispeiasnfeoyuenp1di0ebtce:oe check upnrsoabteissfhaoctuolrdy,bedicshaescskeemdblweetehkelyp.robe and replace the stainless steel capillary. The 12.3.4 SetHPLC pumpto"On".Settheflowto10- 500 L/minorasappropriate. Observe droplets coming outofthe tipofthe probe. 12.3.5Turnonthenitrogen. Afiremistshouldbeexpelled with nonitrogenleaking around thetipof the probe. Readjust the tip oftheproibfneo mist is observed. 12.3.6 Tchhaenignestinruomrednetrutsoesoptthiemsiezeptahrearmeetseprosnsaet:the following settings. These settings may 12.3.6.1 Drying gas 250-400 lters/our 12.3.62 EST nebulizing gas 10-15 liters/hour 123.63 HPLC constant flow mode, flow rate 10 500 wLimin 12.3.6.4 Pressure <400 bar (This parameterisnot set, it is a guide to ensure the HPLC is operating correctly.) 1237 Carefully guide the probe into the opening. Insert probe unil it will not go any further. Connect the voltage cables to the probe. 3M EnvironmentalLaboratory ETS852 Page6af 11 000129 Page 75 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.3.8 Psruimntmtahreytupnaegepaagnsd,sMtoSrefiinlet,hHePsLtuCdypabrianmdeetrewrsi,thsaamcpolpeyltisatpaendditnhteo MtihecrionssotfrtumWenotrd log. 1 12.3.9 vCelriscikoonsn,ssteaertabpuptrtoopnriiantethMeaMsassLsyLnyxnUxSmEaRin'SpaGgUeI(DtEhi)s.maEynsvuarrey among MassLynx startandend sample `number includes allsamplesto be analyzed. 1D30aTAANALYSISANDCALCULATIONS 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: } % Recovery = ObservedResula t~MatrixBlank Result x 100 13.1.2 Calculate percent difference using the following equation: % Difference _= ExpectedCone.CCaellcuileated Cone. x 100 (1n3g./1m4L):_Calulate actual concentrationof PFOS, or other fluorochemical, in matrix {Con(IcniotifalPVFoOlSuCmealof MfartormiSxudn.lC)urv+e_(mngL/omfL)SurrxoDgialteutSitaonndFaarcdt)or) T010p05 "FinalVolume (mL) 14.0 METHOD PERFORMANCE 14.1 Method Detection Limit (MDL) and LimitofQuantitation (LOQ) are method, analyte, and `MmaDtrLixasnpdecLifOicQ. vPalleuaesse. see ETS-8-4.2, Attachment B, fora listing ofcurrent validated 1422 SolventBlanks,Method Blanks, and Matrix Blanks 14.2.1 `Saocltviveentstbalnadnakrsd, imnetthheocdalbilbarnaktsi,onancudrmvaet.rix blanks values must be below the lowest 14.3 Calibration Curves 14.3.1 The coefficientofdetermination value for the calibration curve must be 0.990 or better. 3M Environmental Laboratory ETS8.52 TS ---- Page Tor 11 000130 Page 76 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS 00-1668 14.3.2 Atlhle aecxtcievpeticaolniborfatthioenLcOurQvepopionitn,tswhmiucshtmbaeywidtehviinat2e5u%potfot3h0e%t.heoretical value with 4 14.3.3 Cmaulsitbrbaetidoenacsttiavnadtaerddstowidtihsqpuealaikfyaraedaastlaersantghaenthtawtomtaiymebsetahfefceuctrevdebmyatbeaicxkbglraonuknd levelsofthe analyte. 143.4 Ltootwheordahtiag.h curve pointsmaybe deactivated to optimize a linear range appropriate 14.35 N`moatyibneclduedaicntgivlaotweodirfhtihgehypdoeivnitastedrmooprpeedthtaono2p5ti%mifzreotmhethleintehaerorreatnigcea,l cvaulruveewpohiennts thecurveisevaluated over alinear rangeappropriate tothedata. 14.3.6 OhnasepaopienatkbaerleoawlteshsethLaOnQtwmoaytirmeemstahienamcattirveixebvleannkif;ihtodweevviaetre,stmhoerLeOthQawnil3lb0e%or definedat the lowest pointwithacceptable deviation. 14.37 AthveaLliOdQca.librationcurve must containatleast 5 active points above and including 144Matrix Spikes 14.4.1 Tcohneceanvterraatgioenm.aRtercioxvsepriikeespoeurtcseindteroeftcohviesrireasnsgheosuhlodublde wbietdhiisnc+us3se0d%inofthteheresppoirkte.d 145 Continuing Calibration Verifications (CCVs) 14:5.1 Cpeorncteinntuirnegcocvaelirbyrwatiitohninsa2m5pl%esofwtithheinstphiekeldinceoanrcreanntgreaotifotnh.eIfraunCmCuYstisshouotwsiade of tbhriascrkeectoevderbyy,tshuebcsuerqvueenatnddaptaasssihnogulCdCnVot.be accepted. Acceptable data must be. 14.6 pIefrcfroitremreiadtonetdheinstyhisstmeematnhoddsapmeprlfeosrrmeaanncaelsyezcetdioonroitsnh'etrmaectt,iomnasiantsedneatnecremimnaeydbbeythe analyst. Document al actions in the appropriate logbook. . 14.7 fIofodtantoataerdeotnobteabrleepsoarntdeddiswchuesnspeedrifnotrhmeaencxetcorfittehriraehpaovret.notbeenmet,thedatamustbe 15.0 POLLUTION PREVENTIONAND WASTE MANAGEMENT. 15.1 `SpaipmeptlteeweaxstrtaectiswdaisstpeosaenddifnlbarmomkaebnleglsaoslsvceonnttiasidneirsspolsoecdatiendhiingthhBe TlaUbocroanttoariyn.ers, and glass 16.0 Recoros 161 Tinhfeorfmiasttipoangiencolfuedaecdheidtahtearpianctkheethgeeandeerr,atiendtfhoerafoostteurdoyrmhuasntdhawrvietttehne ofonltlhoewipnagge: study or project number, instrument, sample matrix and time point, date, and analyst. 162 `Acodmatpaoupnadckseutmimnaclruyderseptohretffoolrloewaicnhg:tardgaettaarneavliyteew,squumanmtairfyycfaolrimb,raMtiaosnsrLeyponrxtqfuoarnteiafcyh target analyte (curve), method report, Word document isting set-up parameters, tune. 3M Environmental Laboratory ETS852 or PageSor 11 000131 Page 77 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 method quantity rseapmorptl,eMraesposrLty(ncxhrsocmaantnionggramme)t.hod report, HPLC method report, sample list, and : 163 `Fpoarraemaectherasn,alMyassiss,Laytnexrspcrainntniinnggtmheetthuonde mreeptohrto,darnedposrat,mpWloerldistd, occoupmyeanntdltiastpiengi,nsteott-huep instrument runlog. The original is maintainiend the data packet. 16.4 Oqunanetaicfhy spaamgpeoleftrehpeorqtua(ncthirfoymcaotmogproaumn)d, rtehpeofrto,llqouwainntgifiynfcoalrimbartaitoinonmruesptorbte(icnucrlvued)e,daenidther `inmetthheodh,eacdaelri,btrahteifoono(ttehre,moerthhaonddawnrditctaelniobrnattihoen paraegeu:suasltluydaysosrigpnreodjetchtensuammbeern,amiens)t,rument, analyst, and date. 16.5 Tarheeelaencatlryosnticmaulsltyidnactleuadneddinointieaaltchhepfaigres.tIpfaignietiinalsaapnacddkaettaesalroenngoatsetlheecitrrionniitciaalllsyainndcdluadteed, they must date and initial each page. 16.6 AStutmamacrhimzeentdaAtafuosrianngesxuiatmapblleesooffatwsaurmem(aer.gy.sEpxrceeald)shfeoert.inclusion in the final report. See 167 bBaacckkuuppeelleeccttrroonincicddaattaiantioanpsptrorpurmiaeltnoegtmebdoioukm.. Recordthefilenameandlocationof 17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATIONDATA 17.1 Attachment A: Datasummaryspreadsheet. OReverevees 18.1 cFoAmCpTo-uMn-d4s.1f,ro"mExSterarcutmiofnoorfAPnoaltyassissiUusmiPnegrfHlPuLoCr-oEolcetacntersouslpfroanayt/eMaosrsOStpheecrtFrloumoertorcyhemical 18.2 TEoTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornomaentderMaQiunattetnraonIceotrfiptlheequMaidcrruopmoalsesSyAsttmeomssp"heric Pressure: 18.3 "The validation report associated with this method is ETS-8-4.0 & 5.0-v-1. 1A 90 rcgcrepDocumewts 19.1 ETS-8-4.2, "ExtractionofPotassium Perfluorooctanesulfonate or Other Fluorochermical `Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory ETS852 Te Page9of 11 0001.32 Page 78 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 20.0 REVISIONS Revision 4 Number, 1 ReasonForRevision Section 6.1.2 ClarificationofHP1100 system components. Spleoctttiionngli1n1.e1arAvreegrraegsseoiofntawnodacdudrevedst,hneot1/sxtawnediagrhdtivanlguoefst,haerecuursveed.for Section 12.2.2.4 Clarificationofsolvent ramp. Section 17.1 Changed from attachmenBt to A. 2 eats 10.1Clawr heniblf anki sare erudn. sos Ronen hewion cae 10.3Specifyrequirements for CCV. ber he pes 111.52 CRlaoriwfywthatte odoeifcs curvedoesnotmeetrequirements. 121.21..113CClalrairfyy staypmipclaellriusnt.ID. 12P .32C5hVannges tdomiobivleephcasleetgrnaedoiensnstatane domspetcifccyofhlmoepwenrraseten.kalploet. iv 114333ti Wehesn dvceiocnsasancdnldaefnsmCsCiaVnd0ed wile her rn bA rekewhwia ipftonntsson decom. Revision Date 04/02/99 Aman Soy Sst 3MEnvironmental Laborato" Essa Page 011 000133 mo e 79 3M Medical Department Study: T-6295.22 Analytical Report: FLAICMTS-TEO00X--1166608 Laboratory Study # ESouey-- MMeatihxoFdiRneavlisSoolnv:ent: AInnasltyrtmiecnatESqoufitpwmaerneVteSryssitone:m Number: FRielSeqnuaamree:d Value: SiIgen:ept DDaatteeooffExArnaalcytsoin//AAnnaallyysts:t: [mr omer cme Tore Toe| "oie| SGtroopueor/DTaaskee:n fTaokmenlnfcoarmrtohmrsetsuidoynfcoaevasi,on. SCoanmcpelnetrsaTtaioknen(farotmmt:hesTtoukdeynffooldmerthe MassLyn nearaton summary. IDinltatlioVnoFlaucmtoer(:mTLa):keTnafkreonmfthoemsttuhdeysftodlydf.older. Final Cone.(sghnL:.Calculated by dividingthe ntlvolumefrom thconcentration Auschment A: Summary Spreadshet ETS852 3MEnvironmental Laboratory HS te Page tof 11 000134 Page 80 3M Medical Department Study: T-6295.22 - AL LABORAT Analytical Report: FACT-TOX-160 LIMS E00-1668 ExtCopy of Or Lee a) Ts J MeTHOD ANALYSIS FOFLUPOORTOACSHSIEUMMICPAELRSFLIUNOLIRVOEORCETXATNREASCULTFSOUNSAITNEGOR OTHER 'BPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-87.0 : Author:Lisa Clemen, Glenn Langenburg Adoption Date: 03/2211% Revision Date: NK T `Approved By: 1 Laboratory Boe ito thr Group Leader (nb Cleanse `Technical Reviewer flay Date D2a/te14(39 olla Date 1.0. SCOPE AND AppICATION 1.1 Scope: This method is forthe analysisofliver extracts for fluorochemical surfactants using 1 .2 HPLC-electrospray/mass spectrometry. Applicable Compounds: Fluorochemical surfactants or o ber fluorina te d compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation Wordre5posrt Ersen0 Page taf10 3M Environmental Laboratory 000135 Page 81 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 SUMMARY OF METHOD. i 21 HThPiLsC-meeltehcotrdodsepsrcaryi/bemsastshespaneacltyrsoimseotfryf,luoorrsoicmhielmaircsaylstsuermfaacstaapnptrsoepxrtiraatcet.edThfreoamnalliyvesrisusising `performed by monitoring a single ion characteristic ofa particular fluorochemical, such as athnealpyezreflduuosrionogctaanteasnudlefomnmataess(PsFpOecSt)roamneiotne,r tmo/zfu=rt4h9e9r.verAidfdyittihoenaildleyn,tistyamoplfaescommapyobuend by detecting daughter ionsofthe selected parent ion. 3.0 DeFnTIONs 3.1 AStysmtoesmpshaelrliocwPfroersvsaurrieouIsomnieztahtoidosno(fiAoPnIi):zaTthieonMbiycruotimlaiszisngQuvaatrtiroouIs storuirpcleesq,upardorbuepso,laend `iPnrteesrsfuacreesc.heTmhiecsaeliInocnliuzdaetibounta(rAePCno)t, Tlihmeirtmeodstpor:aEyl,ecettrc.osTprhaey iIoonniizzaattiioonnp(rEoScIe)s,sAitnmothsepshe:eric techniques occurs at atmospheric pressure (i.e. not under a vacuum). 32 pErleescsturroe,spwrhaeyrIeobnyiziaotnsioinn(sEolSu,tEioSnDa:reatmreantshfoedrroefditoonitzhaetgiaosnpphearsfeorvmiaedtiantyacthmaorsgpehderdircoplets. Thesechargeddropletsareprodubcyethde applicatoifoan strongelectricalfield. 3.3 `MTahsesAPSIpeQcutartotmreotIryt,riMplaesqsuaSdpreucptorloemmeatsesr s(pMeSc)t,roTmaetnedreims eMqausipspSepdewcittrhotmweoteqrua(dMrSup/oMlSe.): `cmhaasrsgeserlaetcitoi(vem/dze)teacntdorssuabnsdeqauecnoltlliysidoenteccetlle.d.ToAnssianrgelseeMleSctimvaeylybdeisecmrpilmionyaetdedfboyrmioanss to adentdectthieosneofrraagnmefonntsmmaayybebesealneacltyezdeidninthtehfeirsstecquoanddrquupaodlreu,pforlea.gmented in the collision cell, 3.4 tCroinplveeqnutaidornuaplolvse.(Zp-osstpr1a9y98p)ruotbielizientae"rZfa-csep:raTyh"econlaftoesrtmmaotidoenl.s oTfhMeiscprroamyamsistQtueadtfrroomIla dpirroebcetliys aotrtthheogcoonnael atpoetrhtuerceo, naefearpepratsursei.ngItnhtrhoeugchonavetnotritouonuasl pcaontfhowramyaitniotnheitcoisunatiemred : `elemctaroide.ntThaeoreuntghahetnshaecmceeo.nfHiogwureavteiorn,iZs-dsipfrfearyencto,mtphoenmeentthsoadnsdofcoopnevreanttiioonn,alclceoamnpinogn,enatnsdare ncootmpcaotmipbalteibwliethwoitthheornZe-sapnrotahyers,ysbtuetmso,nleylcw.i)thsimilarsystems (i.c. Z-spray components are 3.5 MatsrisplLeyqnuxadSroufptowlaersey:stSeymss.teCmursroefnttwlayreMdaesssiLgynnedxfhoarstWheisnpdeociwfsic9o5pearnadtiWoinonfdtohwessNeTQu4a.t0tro iVenrsstirounmse.nAtl(lMivcerormassasirQeouanttsrsoimiIlaTr.tFrioprlme oquraeddreutpaoillesMreafesrstLoytnhxeomraMnausalssLpyencxiNfiTctUostehre's Guide). 4W 0 ARNINGSANDCAuTIONS 41 Health and Safety Warnings: 411 Uesmpelcoayustiaonvowlittahgetohfe vaoplptraogxeicmaabtleelsyf5o0r0t0heVporltosb.e. When engaged, the probe 3M Environmental Laboratory FS870 000436 ruezori0 Page 82 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 42 Cautions: 4.2.1 Operate pressure the solvent pumps below a back pr exceeds 400 bar, the HP1100 will einsistuiraeteoafu4t0o0mabtairc (s5h8u0t0dopwsin).. Ifthe back 422 Donotrun solvent pumpsto dryness. 50IntemeemeNces 51 To minimize interferences when analyzing samples, Teflon shall not be used for sample storageor anypart ofinstrumentthaattcoimoens in cownithttheasamcpletor extract. SoFoueweNr 6.1 Equipmentlistedbelowmaybemodifiedinordertooptimizethesystem.Documentany `modificatioinns therawdataasmethoddeviations. 61.1 Micromass QuattroI riple quadrupole Mass Spectrometerequippedwith an electrospray ionization source. 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column. compartment, and autosampler 7.0_ SUPPLIES AND MATERIALS 7.1 Supplies - 7.11 Highpuritygradeairregulatteod approximately 100psi (hoairusyssteem) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented : in the raw data. 71.3 Cappedautovialsor capped 15 mlcentrifuge tubes 8.R 0 EAGENTSANDSTANDARDS 81 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this methodshould be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other `vendor 8.13 Ammonium acetate, reagentgrade or equivalent 8.13.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 3M Environmental Laboratory temperature. 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g `ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room ETS870 000137 Page 3010Page 3 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 82 Standards J 82.1 T`pyrpeipcaarleldydtuwrionmgetthheoedxtbrlaacntkiso,ntpwroocmeadturrei.x bRleafnekrs,taonEdTeSi-g8h-t6e.e0n.matrix standards are 2.0 SampLE HANDLING rm -- _ 9.1 Farreesshtmoraetdriixnsctaapnpdeadrdasuatroveiaplrsepoarrceadpwpietdh1c5amchlacneanltyrsiifsu.geExtturbaecstuendtisltaannadlayrsdiss.and samples. 9.2 Itefmapnearlaytsuirsew,iolrlrbeefrdieglearyaetde,deaxttarpacptreodxismtaantdealryd4saCn,dusnatmipllaensalmysaiys bceanstboerepderaftorromoedm. 10.0 QuALITY CoxTrOL peer ------ ecm 101 Method Blanks and Matrix Blanks 10.1.1Soealcvhenbattbclhantoksd,emteertmhiondebcloanntkasm,iannatdimoantorricxabrlraynokvsera.re preparedand analyzed with 10.1.2 Ana amelthoydblaznkaend amatrix blankpriortoeachcalibrationcurve. 102 Matrix Spikes 102.1 Mreactorviexsrypiekfefiscaireencpyr.eparedandanalyzedto determinethematrixeffectonthe 10.22 rMeactorviexrsyfpiokreeaducphliacnaatleystea.re prepared and analyzed to measure the precision and the 10.2.3 A`nmailnyizmeumaomaf2trisxpiskpeiskpeearnbdamtcaht.rix spike duplicate per forty samples. With a 10.2.4 Matrixspikeand matrix spike duplicate concentrawtiillofnalsl in the mid-range of trhaengienoitfiatlhcealiinbirtiaatliocnalciubrrvaet.ionAdcduirtvieo.nal spike concentrations may fall in the low- +103 Continuing Calibration Checks 103.1oCfotnhteincuailnibgrcaatliiobnractuirovnev.erifications are analyzed toverifythecontinuedaccuracy 10.3.2 Aonnaelpyezrebaatmcihd.-range calibrationstandardeverytenth sample, with a minimumof 11.0 CALIBRATION AND STANDARDIZATION 11.1 ATnhaelayvzeertahgeeoefxttrwacotesdtamnadtarridxcsutravnedsarwdislplrbieorptloottaenddbfyolllionewairngreegarcehsssieoton f(ysa=mmpxle+ebx)t,racts. weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 11.2 Istfatnhdeacrudrcvuerdvoee(sifnnoetcmeesseatryr)eqaunidrermeeanntaslypzeer.form routine maintenance of reextract the 3MEnvironmental Laboratory S370 000: 901233 Pagedof 10 Page 84 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 11.3 uFsoertphuerploosweseonfdoafctchueraccayliwbhraetnioqnuacnutrivteatriantghelrotwhalnevtehles ofufllanraalnygteeo,fitthmeaystbaendnaercdescsuarrvey.to 4 caElxiabmrpaltieo:nwchurevnecaotntseimspttiinnggotfotqhueasnttiatnadtaeradpsprfoxirmSaptoeplbymt1o0 1p0p0bpopfbanarlyatet,thgaenhnetrheaetfeurall rreagnrgeesosfitonheweciugrhvtein(5gopfpbhitgoh1c0o0n0cepnptbr)a.tiTohnisstawnidlalrrdesd.uce inaccuracy attributed to linear 2OProceouRes 12.1 Acquisition Set up 00000000000 12.11 Setup the sample list. 12.1.1.1 lAeststiergnofatshaemapllpehalbisettsftialretnianmgewuistihnag MO-DAY-last digitofyear-increasing. 12.112 Assign amethod(MSfile) for acquiring 12.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 TspoecctrreoatmeetaemrehtehaodidncglsicaknodnsemleetcthoSdIRin(StihnegAlceqIuoinsiRteicoonrcdoinntgr)o]opraMneRlMthe(nMumlatsipsle ReaappcrtoiproinatMeonmiatsosreisn.gA). fSueltlsTcoanniziastuisonuaMlolydecoalsleacptperdoparlioantgewaintdhtmhaessSItRos.499Soarveother afcrqaugimseinttiaotnimoentihnofdo.rmIaftMioSn/mMaSyibnesctorlulmeecnttesd.arReeefmeprltooyMeidc,taodmdaitsisonMaalspsrLodyumcxt ion `GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3T`ympaitcraixllsyttanhdearadnsa.lyticalbatchrunsequencebeginsandendswith asetofextracted \ 12.1.4 aSfatmeprleevsearryetaennathlyszaempdlw e. Si aolcvoentnttinbuh lianngkscsalhioburladtiboen vaenrailfyizceadtipoenriinojdeicctaeldlysttaondard `mionncilutdoerdpoassssiubclhe.analyte carryover and are not considered samples but may be 122 Using the Autosampler 122.1 Setup satmraypacclordieng tothesamplelstpreparedinSection 12.1.1. - 12.2.2 Saental-yusptthcoenHsiPd1e1rs0a0p/praouprtioatseafmoarptoltpehtreifmoalllroewsiponngsce. oRecnorddoacritautatlcocnoidnidtioitoinonsntsshien the instrument logbook: 12.2.2.1 Sample size = 10 uL. injection. 12.222 Inject/sample= 1 1222.3 Cycle time = 9 minutes 3M Environmental Laboratory ESE Page Sof10 000139 Page 85 3M Medical Department Study: T-6295.22 _ Analytical Report: FACT-TOX-160 LIMS E00-1663 12.2.2.4 Solvent ramp conditions Time MeOH 20mM 4 : [ooomm [a | am m | Ammonium acetate [Tomin. [4|0 em%| [[EES5mminn1|95o%s%[5% sn || [Town |a [Go| (Omi1a0% | 6%] 12.2.2.5Pressthe "Start" button. 12.3 Instrument Set-up : . 12.3.1 Referto ETS-9-24.0, "Operation and MaintenaonftcheeMicromass Quattro II `Triple Quadrupole Mass SpectrometerFitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refillif necessary. 12.3.3 Chthe e stc ainlk ess steel capialtlthaeernydoftheprobe. Use an eyeptioechcecek tuhnesattiips.fTachteortyi,psdhisoauslsdebmeblfelatthweiptrhobneo ajnagdgreedpeldagceest.heIfsttahienlteispsisstfeelocauptinlolbadrey. 12.3.4 Tum on the nitrogen. 12.3.5 Open the tune page.Clicks on operate to initiate source block and desolvation `heaters. 12.3.6 Open the Inlet Editor. . 12.3.6.1 Set HPLC pump to "On" 12.3.62Setthe flotwo 10 - 500wLrminorassppropriste 12.3.6.3Oetbxhspeeetlrplvoeedfdwrtiohtpehlpenrtoosnbic etrifnoo goenom muilseti atokifisntn gohbeasrg teoirpuvonedfdtthheetpirpoboef.theAfprionbeem.isRtesahdojuulstd be ~ 12.3.6.4 Allow to equilibrate forapproximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. Thess settings may changeinorder to optimize the response: 12:37. Drying gas 250-400 lters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/our 1122..33..77.3.HPPreLsCsucreo<4n00sbfat lro(a Twhminosdtpeafrlaomwertearteis 1n0ot--s5t00tuiLs/amgiunide to ensure the . HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 123.76 Desolvation temperature 250 3M Environmental Laboratory T5870 000140 Page 6010 Page 86 3M Medical Department Study: T-6295.22 _ Analytical Report: FACT-TOX-160 LIMS E00-1668 12.3.8 + tPraipnetdtihnetotutnhee piangset,ruwmietnhttlsogp.arameters, and sore tin the study binder with a copy .i 12.3.9 `MCelanidscsskLaoymnpnlxsetavrnetrubsmiubotentsro,nirniencfleturhdeteosAcaaqplulpirssoaipmtrpiilaoetnesCMotaontsbrseoLlaynPanalxnyezUelsde.(rth'issGmuaiydev)a.ryEanmsuornegstart and 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Caleulations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: %Recovery= OBbsearvecdREexkspuelctgt-edrResuolt uRensuldtx 100 13.15 Calculate percent difference using the following equation: %Differenc=e ExpectedCo`nEex,pe-cCtaeldcCuolnact.edCone, x 100 . 13.1.6 Calacctuaulcolnceantrtatieonsinmatrix (ug/g): | {ngofPFOS ca(lnci.tfirao]msWtedi.ghCtuorvfeLixvDeirlu(tg)ionFactor) x 1l00u0g0g Final Volume (mL) 14.1 `MmeattrhioxdspDeectieficct.iRoenfLeirmitto(EMTSD-L8)-6a.n0d,LAitmtiatochfmQeunanttBitfaotrioanl(isLtOinQg)ofacruermreetnhtovda,liadnaatleydteM, DanLd and LOQ values. 142 Solvent Blanks, Method Blanks and Matrix Blanks . 14221Ssotlavnednartdbilnantkhse,cmaeltibhroadtiboinnckursv,ea.ndmatrix blanksmustbebelowthelowest 143 Calibration Curves | 143.1 The value for the calibration must be 0.980 orbetter. 144 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within + 30%ofthe spiked concentration. 145 Continuing Calibration Verification ~ 14.5.1 Cspoinkteidnucionngcecnatlriabtriaotni.on verification percent recoveries must be within 30%ofthe 14.6 Ipfecrrfitoerrmieadliosntetdhienstyhsetmeemtahnoddspaemrpfloersmraenacnealsyeczteidonoraortehneortamcetti,onmsaaisntdeentaernmcienmeadybybe.the analyst. Document all actionsinthe appropriate logbook. 3MEnvironmentalLaboratory E5870 Tr ---- 000141 Page 7f10 Page 87 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 147 Iffodoattmoataerdeotno btaebrleespaorntdeddiwshceusnspeedrifnotrhmeantecxetocrfitthereiarhepaovret.not been met, the data must be * 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 pSiapmepttleeweaxsttreacitswdaisstpeosaenddifnlbarmomkaebnlgelsaoslsvceonnttaisindeirsspolsoecdatiendhiingthhBe lTaUborcaotnotrayi,ners, and glass 60Recors 00000 16.1. Ehachepaogarehgadenndewreraittertdefnoronatshteupdaygmeu:sstthuadvyeotrhperfoojlelcotwniunmgbienrfo,racmqautiisointiinocnlmuedtehdoedi,therinthe. integration method, sample name, extraction date, dilution factor (fapplicable), and analyst. : 162 P`riantpthpertuonseptpuradgyief,aosltdaemrep.lCeolpisyt,tahnedsaecqpuaigseistainodntmaeptehiondtfotrhoemMinasstsruLmyennxttrouinnlocgl.udeinthe 163 Psltootrtehienctahleibsrtautdiyonfocludrerv.eby linearregression,weighted 1/x,then printthesegraphsand 164 Parnidntsdtaotreaiinnttehgerasttiuodnysfuomldmearr. y, intmeethgod,rana d cht romiatoogranmsfromMassLynx ~ 165 AStutmamcahrmieznetdaAtafoursianngesxuaitmapblleeosfofatwsaurmem(aErxcyeslp5r.e0ad4s)haeentd.store in the study folder, refer to 166 Banadckloucpateiloencotfrobnaicckduatpaetloecatprpornoipcrdiaattae.medium. Record in study notebook the file name 17.0 TABLES. DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 AttachmenAt: ETS-8-7.0 Data summary spreadsheet IO0 1R8.e1 wFeA0 RCTe-NM-c0 2e.1s, "E0 xtracti0 onofPot0 assium0 Perflu0 orooctan0 esilfon0 ateorO0 therFl0 uoroche0 mical `Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" - 182 IEoTnSi-z9a-t2i4o.n0/,Ma"sOspeSrpaetcitornoamentdeMraQiunattetnraoIncetorfiptlheequMaidcrruopmolaessSyAsttmeomssp"heric Pressure 183 The validation report associated with this method is ETS-8-6.0 & 7.0-V-1 + 1A 20 rgcrecDocowews 19.1 ETS-8-6.0, "ExtractionofPotassium Perfluorooctanesulfonate or Other Fluorochemical `Compounds fromLiver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3MEnvironmental Laboratory ETS870 PageBot10 000142 Page 88 3M Medical Department Study: T-6295.22 _ DARGVGIONS Revision ; Number ReasonForRevision Analytical Report: FACT-TOX-160 'LIMS E00-1668 Revision Date 3M Environmental Laboratory ETS8.70 Page9of10 000143 Page 89 3M Medical Department Study: T-6295.22 Analytical Report: FLAICMTS-TEO00X--1166608 Laboratory Study # TSteusdtyM:ateria: MMeatuhFodiRneavilsSioolnv:ent: IAnnasltyrtuimceanltESqoufivpamreenVterSsyison:m Number: RFielSeqnuaamree:d Vale: YSlnotpeer:eept ` DDuatetoefoEfxAiuaycstiosnA/nAamlyyss:t: - GSrpouEp/DoseH: NTa=kenfomtehesoud nfolderS.a `SCaomnpcelnet:ratTiaoknen(fago)m:thTeasketnayfroodmehre MassLy nigraion suis. InDiiltuitailWoneF.a(c9t)o:rT:aTkaeknenrfoommthtehesstyudfyolfdoelrd.er. Final Con. ug) Clete by divitdh intnig!volumefromtheconcentration - -- 3M EnvironmteancthalmLeanbAot:raStuomrmyary Spreadshet St af u5rTi SB:i8r70rc Ting BAS Page 10010 000144 Page 90 3M Medical Department Study: T-6295.22 vv -- EE Appendix D: Data Summary Tables Analytical Report: FACT-TOX-160 non reere0xe LIMS E00-1668 Table 9. Data Summary for PFOS In Serum FACT-TOX-160--pg/mL. [ome[| = mmoF | e se | nrows | [o|e me=m e aee Cm T = | F m eE e [e mee [e] e [me mn] SETIEne, op EEA peat = [Cm=m] =e] RE TE ISe J 3MEnvironmentalLaboratory ~ 000145 Page 91 3M Medical Department Study: T-6295.22 `3M Medical Department Study: T-6295.22 -_-- Appendix E: Data Spreadsheets Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: LFIAMCST0T0O-X1-816680 SMEnvironmental Laboratory 3M Environmental Laboratory 000146 Pago 22 Page 92 PE p tA erk -- a. 3MMedical artmentSt : T-6295.22 =F crraoniaE i rAanalryE tiecal To t: FAvCsT-TsOoXo-t160 E i e rEEa E=EEY EEcmEEm S ERree BSEn oe [son= sEaviass[ie -- T= = ea=s cimoiatns| | com oem] en [= H TEs T TTaAra EE [os Te a a hlCe Tiaee WE T Ba ar Tw eta [= rr =r r[r=r TT ew Bovey ml he oulasloa 3MEnvironmental Laboratory 000147 Page 93 3M Medical Department Study: T-6295.22 sor Coser A 3308 -- LIMS E00-1668 AnalyticalReport:FACT-TOX-160 E jo T ats Prd Nemesssc. TBooaEwssnn ssswsnaamotsstlienrem bSE= = iemi iESisyem LEn BZuEi -- P soE emTr EEE TE Ea] [mete[1% E|[E ee]E meeE[r Slewey | Pee] Te I A 1+3J TIee| ee le |= TeEeE le EeilseA mToarTaor] |= [om ow s Twe T her Tae l wu ] |= [ Te To we eres sree ms To 5 oT1 al] ow] [=[e= e T re eTT e ree lere eeT s wu TLoee la| [--oeme [aTTEel ee eegerea ] ms TCao lew]| |= EEE] TER EE Ue ge 3M EnvironmentalLaboratory 000148 Page54 3M Medical Department Study: T-6295.22 rtm tetNumb Sb SE=EEEerlonS r Vebminain seria `Covance#6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 rote eto TeyPS: TE)CranspHeers E= = E2Em5EE rity RA =] 5] Emm Il<5 I =PV vy | T FEEe Tm Tala - [ammenrn [ow Tol wo | L0Q=10 nn bedsir 2. em. mim neUC ouster 3MEnvironmental Laboratory 000149 Page 95 3M Medical Department Study: T-6295.22 EATEN Analytical Report: FACT-TOX-160 a REE rare ome 629.268 LIMS E00-1668 Mg=EE eeaTn: m fEE o RIrE, sites E=BeyoeEr-- ESerEueEn e ET =T EmE [eE E n =E a [r a]] E B I rtE E e t e Eefea eam mn em ea ] w UC odlaston 3M Environmental Laboratory 000150 Page 96 3MMedicalDepartment Study: T-6295.22 st ACTOR bins me Se foam Bf BumoEo mata. : Supe an voce seu PArCcTrTOrXo.xIie AnalyticalReport: FACT-TOX-160 Covancel 6329268 LAM gea1a0s REeideRSTypWae CiTs 6 Pens Tat m ne fe pSrhe Toinio)tonot ES See h [oe TEm T t or T iT se wo | TLLoo0a05 Ri E I I l I eteiit5taeoe 0-01 tm DDeuVaenned eumnica We ilar 3MEnvironmentalLaboratory 000151 Page97 3M Medical Department Study: T-6295.22. Some, Analytical Report: FACT-TOX-160 LIMS E00-1668 -- Pts: frei ri faot. Sf [one Br eoteit:r mesy ns CeTotyee il OTA Ge Tavem masts fry hseeheen Toau waontn Sumi = sony5)seam = ETE E5 E = Femme[TT To FT emma = a=] II OS A =RJ ot pony | Eee TERE eal LLo m E i mte 15 6 rs ne011 i EooEpE BaiRn S ue ge 3M Environmental Laboratory 000152 Page 98 3M Medical Department Study: T-6295.22 mmm [E-- BE ox: Commer Analytical Report: FACT-TOX-160 LIMS E00-1668 a i Es mst lL Bees TER BEE ashe em] 3M Environmental Laboratory 000153 Page 99 3M Medical Department Study: T-6295.22 I Comme 5345 -- pro eTmi ESeT mcom E beans Ee rd EEE, Be Sem Analytical Report: FACT-TOX-160 LIMS E00-1668 mse | mmER TT TwTres er To | TY mm WC palyston 3MEnvironmental Laboratory 000154 Page 100 3M Medical Department Study: T-6295.22 rte TT Analytical Report: FACT-TOX-160 Covasces 6325-168 Is onmytns LIMS E00-1668 pr [Eeem 8iTm T m| Hl EEEe rar Fa m= Ue oufasha 3MEnvironmentalLaboratory 000155 Page 101 3M Medical Department Study: T-6295.22 [rm Covances 329-268 Tio -- EE emrEe... EERIE rot EET asan a `Analytical Report: FACT-TOX-160 LIMS E00-1668 mem ---- [Emerlls &[aTBT ree | EEE EEE FREE EEE ge aslo ea a 3M Environmental Laboratory 000156 Page 102 3M Medical Department Study: T-6295.22 -- iis Analytical Report: FACT-TOX-160 LIMS E00-1668 iia me Fadl Logi & Jot ule]] ow] SEE. Ea oon UCaslon 3M Environmental Laboratory 000157 Page 103 3M Medical Department Study: T-6295.22 ---- Conc 6529268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Ee. mn E- EF LS eoE or EET ivan En B- EF i EE a Ba E RT E yTey REE BEL la 3M EnvironmentalLaboratory 000158 Page 104 3M Medical Department Study: T-6295.22 RR Covanced6329-268 EEE ps OBE OEE Analytical Report: FACT-TOX-160 'LIMS E00-1668 HEEL BE = 5 CEEE EEE a EL = Teoalaslor 3MEnvironmental Laboratory 000159 Page 105 3M Medical Department Study: T-6295.22 BRI Covances 329.268 Analytical Report: FACT-TOX-160 LIMS E00-1668 ---- RR Si EE Ei Sree CE = Ter EEE re (Ea Re Ei ia Ce EE=E TEerErEe [EEETEE[EEEE]a,| om Ss 3MEnvironmental Laboratory 000160 Page 106 3M Medical Department Study: T-6295.22 `3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: LFIAMCSTET0OoX--1166608 Appendix F: Example Calculations Formula UsedforSera Analyses InStudy FACT TOX-160 AR (ng/mL) x DF x_FV (mL) x_10pg = ReportedConcentration(ug/mL) EV (ml) ~10000g Calculation Used for Group 1, Week 17, Animal ID 105505M 4575ng/mLx200x 0106m3Lm x 110O00pngg = 14.5 pg/mL AR-- Analytical result from MassLynx summary FDVF----FiDnialulteixotnrafcatctvoorlume (1.0 mL unless otherwise noted) EV--Volume ofsera extracted Formula UsedforLiverAnalysesinStudy FACTTOX-160 AR (ng/g) x 3dcsuamrpvlee x DF x 1_01000pngg = Reported Concentration (ug/g) 3curveisassumtoed be;_1gliver SmLH0 Calculation Used for Group1, Week 53, Animal ID 105505M 6607ng/g x1011g60/gS/mSLmL x50 x 110O00ungg = 325pgg AR-- Analytical 3 curve--Density result from MassLynx summary of theliverstandard curve,assumedtobe 1gliver/Smiwater 3 sample--Density of the liver sample (g sample/ 5 mL H;0) DF-- Dilution factor 3M Environmental Laboratory 3M Environmental Laboratory 000161 Page 23 Page 107 3M Medical Department Study: T-6295.22 `3M Medical Department Study: T-6205.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: LFIAMCSTET0O0X--1166508 Appendix G: Interim Certificate(s) of Analysis `3M Environmental Laboratory 3MEnvironmental Laboratory 000162 Pace 24 Page 108 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 --_----------------HIMSTor1e88 CentreAnalytiactaalCollLage,aPbA oT6r80a7 torwiiesca,nirIanbcc.om 'e Phone:(814) 231-6052 Fax:(814)231o-r(1614)22315-16380 INTERIM CERTIFICATE OFANALYSIS Centere ntre AnyaabLaobroarattoriieess:COA Referencee:#: 023-00231-01 84 3MPrRoedfuecrte:nc#Pe: FSD-OL0o1St82,17 Parity: 869% hCc v - fabs Eo eimrm EEEATrinrge g] | =] CTaee ntiication 1 1 ewe | al1.s (CaClPcMium 1 0.008wesw 23.. MSaogdnieusmium 25. 0L040310wwiinm 54.. NPioctkaeslsium 54. <608.00901wwit/nokt% 67. kMaonnganese 67.._<000.00501wwiiniust% [Toul Impurity MR)|7] TSTwih% | e Related Compounds -- w [ResidualSolvents (TGA)|| NoneDetesied | TnorgIa.niCcAhlnoiroindse 25.. FBlruoomriiddee 45. NNiittee 67.. PSuhlofsaptheate Organs1. ATciFdAs TM10) 2. 3. HPFFPBAA Elem4entaNlFPAAnaly" 21.. HCayrdbroongen 45. Nsuiltfroorgen 5._Fiuorine 21. ThTeohreetiocarlVVeaalltuueei==c107a%.8l% 43. ThTeohreetiocarlVVeaalltuueei==c05%.a95l% 5. Theoretical Value =60% 21 <000s1S9wwnm%s 45. <<0000d009wwism 65. <<00.000067wweiiwwit%% 7. B36wiimt% 21. <<0011wwietnss 4300280wwitiiwi%h 21. 0122448wwesa% 43 s17a4aww/inn%d) 5. satwilwit coansansa Page tor 3MEnvironmental Laboratory 000163 Page 109 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 - ------------------I-- MSE0-- 0-1668 Ce30n78tRerseearAchnDaivlaytiSctealColeLg,aPAboT ratorieCsa,n AIRnBcE.T Phone: (814)231-8032 Fax: (814)231o- r(81 14)2 2315 -15380 INTERIM CERTIFICATE OFANALYSIS Centre Analytical LaboratRoerviiessioCnO3A Reference #: 023-0184 DateofLast Analysis: 08/31/00 `Expiration Date: 08/31/06 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/06 "Purity = 100% - (sum ofmetal impurities, 1.45% +LC/MS impurities,8.41% Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) `Total impurity fromalltest=s 13.07% Purity = 100-% 13.07=% 86.9% Potassium is expected in this salt form and is therefore not considered an impurity. oPbusreirtvyebdyfDorStChisissgaemnpelrea.lly not applicable to materials of low purity. No endotherm was "Sulfurinthesampleappearstobe converted to SO andhencedetectedusing the inorganicanionmethod conditions. Theanionresult agrees well with the sulfur deteir nthm eeli emenn tala anat lysii s,lo endn ing conftoi thisdinteerpn retc atie on. Based `on the results, the SOx is not considered an impurity. "tA [-- HFBA Heptafluorobutyric acid NFPA 'Nonafluoropentanoic acid PFPA Pentafluoropropanoic acid "Theoretical value calculations based on the empirical formula,CiF17SO;K"(MW=538) This work was conductedunderEPA Good Laboratory Practice Standards (40 CFR 160). ECE 3M EnvironmentalLaboratory 000164 Page2o3 Page 110 3M Medical Department Study: T-6295.22 --_-- Analytical Report: FACT-TOX-160 JIMSF00-1668 C3e04n8tRerseearchADnivaolytiStcalalColLage,aPAboT6r30a1 torwiWeicsa,niiIanbce.on A Phone: 616)2918022 Fax. (914) 291-1or2(55143) 231.1580 INTERIM CERTIFICATE OFANALYSIS Centre Analytical LaboratRoerviiessioCnO3A Reference #: 023-018A LOMS Pury Profile: C I erm 1% SO =rw we -- wm ----& ua CC mom 7 sar Note: The C4 andC6valueswere calcusiungl theaC4tanedCd6 standardcalibration acunrdveCs8, rsetsapnedcatridvecluyr.veTs.heLCikSewviasleu,e twhaesCc7alvcaulluaetwedasusicnaglctuhleataevdeursaignegtrehseultavferroamgethreesCu4lt from the C6 and C8 standard curves. PreparedBy: le fo Charles Si Scientist, Centre Analytical Laboratories softe_r Date Reviewed By: ply Flaherty Date `Laboratory Manager, Centre Analytical Laboratories cono018a 3M Environmental Laboratory 000165 Page 30r3 Page 111 3M Medical Department Study: T-6295.22 _--m Analytical Report: FACT-TOX-160 LMSE00-1668 3M ENVIRONMENTAL LABORATORY Note to File Project or Study Number: FACT-TCR-001 Associated Study Number: LIMS #E00-1682 ToecapdtsoarPiOSnor 71 217 (50091 S008) beced 5yum (R310) 1sabilywasdemWISo TSCln i,WAi TTS hc oc)wdBOLOMe | (biodegradation). -- ~ We oli RLeiisa csoarCdlCleeedmeBny: 3MEnvironmental Laboratory Date (3) 08/03/01 orloslo, FomeTs 10 . 000166 actmyoto0leidgo intel Date Page 112 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.22 Appendix H: Report Signature Page Analytical Report: FACT-TOX-160 LIMS E00-1668 Analytical Report: F[A8CTT0O0X-1166008 Soul Andrew Seacat, Ph.D., Study Director May 3, ror Gloon 7. Bulloinlfefr fy 3, 2002. John Butenhoff, Ph.D., SponsorRepresentative Date Cli A Clin, . Lisa Clemen,PrincipalAnalytical Investigator osfoifoa Date Pr. aa William Reagen, Ph.D., Analytical Laboratory Manager oTora Date am EnvironmentalLaboratory 3MEnvironmental Laboratory 000167 Pace 25 Page 113