Document X7r90yDvK1VdJE9ooe5Y97XBw

AR226-0 206 3E M nvir Labo oratn ory mental Final Report- Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits In-Vivo Study Reference Number:HWI#6329-138 Study Number: AMDT-122094.2 Test Substance: FC-129 (T-6054) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology & Services 935 Bush Avenue St. Paul, MN 55106 Method Numbers AMDT-M-1-0, and Revisions: Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940Expandable Ton Analyzer AMDT-M-4-0, AMDT-M-5-0, Extraction of Fluorochemicals from Rabbit Liver Analysis of Rabbit Liver Extract for Fluorochemicals Using AMDT-M-8-0, `AEnlaelcytsriossoprfayFMluaosrsidSepUesctirnogmtehterySkalar Segmented Flow Analyzer with Ton Selective Electrode Initiation Date: See attached protocol Author: James D. Johnson Approved By: / es D. Jghson tudy Dirdefor 000416 Wezf2s Completion Date . 10SUMMARY aLinvaelryzseadmpfolrestoattal4o8rghaonuircs fplousotriinnetraanvdenpoeursflduoosreooocftaFnCe-su1l2f9on(aTt-e6.054) in rabbits were oAfrtgearniacnfilnutorrianveenionulsidveorseato4f8FhCo-u1r2s9poinstradbobsites,atthdeorseesisraandgeitnegctfarbloemi0n.c1r2e8asteoo1f2t.o8tal pmegr/fklgu.oroAocstuabnsetsaunltfioanlaatem.ouTnhtisofphtahirsmatoctoaklionregtaincicstfulduyorsihnoewiss itnhatthtehferoermisoaf convenient marker (perfluorooctanesulfonate) for assessing the extent of dermal absorption. 2I 0 NTRODUCTION dTheirsmasltuadbysowrapstipoenrsftourdmyed(HiWnIo#r6d3e2r9t-o1p3r3o)v.ideKndoatwaifnogr the the assessment disposition a subsequent of an intravenous dose of FC-129, facilitates interpretation ofa dermal absorption study. Analysis For total of liver organic falnudorsideeru(mcosmabmupslteisonfraonamlyrsaibsb)itasnddosspeedciifnitcrcavoemnpoousulnydwsith FC-129 a(reeleicntrtohseprlaivyemraast s48spheocutrrsompeotsrty)dopsreovainddewdhaettahaesrtpoerwfhleutohreorocftlaunoersiunlatfeodnactoemipsounds present, previous wPeorrfkluhoarsooschtoanwensuilt ftoonbaetepeirssaisvteernyt good marker for a in liver and serum dinerrmaabblitsstu(dbyiosliongciecal half life >1 month). 3T .0 ESTMATERIALS 3.1 Test, Control, and 3.1.1 Analytical Reference Reference Substances Substance: and Matrices FC-95, lot 161 or 171. They are equivalent. 3.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 3.13 3.14 Analytical Analytical Control Control Substance: None Matrix: Bovine liver and bovine serum 3g.r2ocSeoruyrscteoreo,fbMoavtienreiaslesr:u3mMfrIoCmPS/iPgCmPaDCihviesmiiocnalfoCroFmCp-a9n5,y.bovine liver from 3.3. Purity and Strength of Reference Substance: Responsibility of Sponsor. 3.4 Stability of Reference Substance: To be determined by Sponsor. _ 00417 2 3bi.o5lSogtiocraalgseaCmoplnedsittihoensstofroargTeeisst-2M0at1er0iaCl.s: Room temperature for FC-95. For 3SS.tE6uPdDyiRsiespgoiusnliapttairioaonlnloefflorSwpittehheciiamm2e8ensdp:aeryBiiaoobdlsorogeriqpcutaiilroentdisstfsuoudreyss,tausndoideasflllutioidnssgseuwerisltlwhibalnel 2rb8eetadriaenyteasid.npeTedh.ris SToerraunmalaynsdistifsosruoestaflroormgaanniicmafllsuodroisneedaansddfelsucorriibneadte(dHWcIo#m6p3o2u9n-d1s3.8)T,hewesraempalvaeislable wT"xeotreoesnstaanorafyldytezorepmdrabolvyiacdboesmosbrupuftsfiitocinioenontfadFnadCt/-ao1rf2oe9rlet(chHteWroIisn#tp6er3ra2pyr9me-ta1as3tsi3o)sn.poecfatrsoemectornydtsotutdhye eoxntetnhte 5D.o1bArMmDanTn-MD-X12-000,0 TOhregramnailc HEaxltriadcetAinoanloyfzeFrlu-oLriivdeerby Means ofaModified 5E.x2paAnMdDaTb-leM-I2on-0An,alFyluzoerride Measurement by Meansof an Orion EA940 5.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 5.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 5.5 AMDT-M-8-0, Analysis of Fluoride Using the Sklar Segmented Flow Analyzer with Ton Selective Electrode 6D 0 ATAANALYSIS TQuhaentdiatataatihaorne)0,.at06t1.a,1ch21e,1d8,0,.61F46o,4r,1c.ao2nm8d,bu2as2nt3di9o1nu2g.a8/nwamhlgoy/slkiesglfihovaredtro1tra8els(pobercegtlaionvwiecltyhf.elulToerhvieenleb,ooftdhpyerwarceatibigbchialts 00418 = 3 of the 12.8 mg/kg rabbit (F52752) was 2.7 amount of total organic kg. Thus, the total dose given fluorine in this dose is 19 mg. this rabbit The total was 34.6 mg. The organic fluorine in liver after the intravenous dose in rabbit F52752 represents. 11.8% of the dose. Thecdtaatbalefpoerrafnlueolreocotcrtoaspnreasyulmfaosnastsepienctarllomterteartyedanraalbybsiitss.isTahtetachhiegdh. dTohseerreabibsit . If dete (F52752) had about dose is e3x3p5reusgsepderafsluFoCr-o9o5c,ta(ntehsautlifs,onaastseupmriensgen1t0i0n%l`ivbeirotarta4n8sfhoorumrastion) the 34.6 dose mg is 31.8 mg. The This r3e3p5reusgenptrses2e`nstubisntawnhtoiallealmiovuernitnotfhibsioatnriamnaslforrempraetsieonnotsf the F1C.-0152%9oftothpeerdfolsueo.rooctanesulfonate at 48 hours. eOltehcetrroddaet(aswecasapcpoelnldeiccteesd)u.sTihnigsSdkaatlaa,rasletghmoeungthesdupfploorwtiavnea,liynzetrhewiotphiniioonnsoelfetchteivse not Study Director is not required to reach the conclusion stated here and therefore i discussed in detail. t6h.i1s CainralcyusmisstiasntchaetsitnhaatphMaarmyacAofkfiencetttihcesQtuuadylirteypoorftetdheseDpaatraat:elTyh(eHWpIr#o6b3l2e9m-w1tih5at9th), there was a delay inperfluorooctanesulfonate unexplained distribution pattern. `concentration observed in serum The level dropped dramatically at 12 indicated an and 24 hours but returned to a maximum at 48 hours indicated a biological half-life of> with a subsequent decay 1 month. This delay in in serum levels that peprefrflluuoorrooooccttaannecssuullffoonnaattee lisevreelssulmtaiyngbferoshmif`tbeidottroanasfloartemrattiiomne, when There may actually be igher levelof ent perfluorooctanesulfonate in liver to 48 hours. This would indicate at a that later time period and this perfluorooctanesulfonate is an ah study only w even better marker for a dermal absorption study than would be indicated by this data. J0CONCLUSION After an intravenionulsidvoerseato4f8FCho-u1r2s9aitndroasbebsitsr,antghienreg is a detectable increase of total from 0.64 to 12.8 mg/kg. A orgsatnainctifalluoarmioneuntofthis sub nate. total This organic fluorine is in the formof pharmacokinetic study shows that there is a Ppeermfvleunoireonotctmaanreksuelrfofor assessing the extent of dermal absorption. 00419 4 8.0 8.1 MAINTENANCE OF Raw Data and Data: RAWDATAAND RECORDS Raw data, approved protocol, approved final report, appropriate specimens, and electronic data will be `maintained in the AMDT archives. 9-- 0APP-- ENDIC-- ES -------------- 9.1 Protocol and Amendments 9.1.1 Protocol and FuidnayloRfe"pTo-r6t0:5H4 WiIn#R6a3bb2i9t-s1"38(Pr"oStioncgollet-yDpoese`TIPn8t0ra8v4e.nPoKusfor `Pharmacokinetic St dosing of animals, tissue collection, etc.) 9.1.2 AnalyticalprotocolAMDT-122094.2 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 9.4 Key Personnel Involved in the Study: Seeattached 9.5 Materials and Equipment: See methods 9.6 Solutions, Reagents, and Standards: See methods 9.7 Sample Preparation: Seemethods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-122094.2 9.10 Instrument Settings: See`methods 000420 , 9.11 Data: See attached. e9x.t1r1a.c1tSiounmfmoalrloywaedndbyraawnadlaytsai;s ug F" using iOnriwohnolieonliavnearlyazserd.etermined by thermal 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. e9x.t1r1a.c3tiSounmfmoalrloyweadndbyraawnadlaytsai;s uusginFg" iSnkawlhaorlseelgimveenrtaesddfeltoewrmainnaeldyzbeyr twhietrhmiaoln selective electrode. 00421 6 9B.o1s.1e PIrnottroacvoelnoaunsdPFhianramlaRceopk0oirn8te4:t.iHcPWKIS1tf#uo6dry3d2oo9fs-iT1n-3g680of5a"4nSiiinmngalles-, Rebbits" (Protocol type TP tissue collection, etc.) 006422 = 2? WTPOBSITTsSQEOCFwFIiOC aN80S00I7M5Na4d Sponsor: st. Paul,"Minnesota FINAL REPORT + CORNING Cony RECEEIVE FEB 27 15 Gy,erm SH 6& 27 ieoLls Study Title: Single-SDtousdey IonftrTa-v6e0n5o4usinPhRaarbmbaictoskinetic Author: Steven H. Glaza Study Completion Date: February 24, 1995 PerformingLaboratory: MHaa3dz3i0ls1eotnoK,ninWsWimisasccnoonnBssoiiunnl,ev5aI3rn7dc0.4 LabPorojreca tIdetntio ficr atiy on: HHI 6329-138 Phone Ee 8a0i8-e241b-e44u7e1ar Page 1 of 28 3301 XINSMAL BLVD. MADFaEx 808.2415.37720247 000423 Page 2 of 24 HHT 6329-138 QUALITY ASSURANCE STATEMENT TThiSsrreprort hheas.beiennryarcecPvoriraedcwatenidcceebywRiettghhuelatQthiueoanlFsiostoyd2A1asnSdCuFrBRiruu5cg8e.A3dU5nnii(ntbi)sotf(r6aH)ttaoiz(olt7ne)h.teon O(hFReoRy)enGDoiiordencLtgaobraorirenaasntdpioesmcsatunieaodgnestmoevneHtra.ezlceWotnrodinutctmteaenndagsetamandteunstFinmvdeoipnnotgrhstlsyreoafpcocroitrnedsdipnegctitoons SStuandiarndgosperating procedures. Date Inspection Dates Phase Reported to StudyDirector Date to Management from To 1125//0199//9044 1122//0199//9344 APnriontaolcalObsReervviaetwion 1127//0199//9944 02/02/95 0011//1100//9955 03/10/95 07/02/95 02/02/95 Data/Report Review ffeiprtessentW.atiDvaen,nerQuali2ty Assurance Unit Date 000424 Page 3 of 24 HI 6329-138 STUDY IDENTIFICATION Single-SDtousdey IonftrTa-v6e0n5o4usinPhRaarbmbaictoskinetic Test Material Sponsor sponsors Representative Study Director Study Location T-6054 3MDTeopxairctomleongty Service Medical ?3.M0.CenBtoexr,332Bl2d0g. 220-2E-02 St. Paul, MN 55133-3220 J3oMhnToxL.icoBlutoegnyhoSfefr,viPcheDMedical 3MDeCpeanrttemre,nt.Bldg. 220-2E-02 sP.t0.. PBaoulx,33M2N2055133-3220 (612) 733-1962 HStaezvleentonM. WiGslcaoznasin, Inc. Mp.a0d.isoBno,x W7I5_4553707-7545 (608) 241-7292 HBauzilledtionng WNio.sco3nsin, Inc. M38a0d2isoPna,ckeWIrs A5v3e7n0u4e StuEdxypeTriimmeenttaablleStart Date Experimental Termination Date DDeecceenmbbeerr 17, 19, 11999944 000425 Acute Toxicology SSttuedvyenDHi.recGtlaozra Manager SFtruadnycisCoo(rBdudi)naWt.orMcDonald PIant-r1iicfieaSuPpaedrgvhiasnor RReopsoertM. SBurpiedrgveisor Quality Assurance MshaenrargyerR. W. Petsel Page 4 of 24 HHT 6329-138 KEY PERSONNEL LaboratoryAnima)Medicine CDiinpdlyomaJt.e,CarAyC,LAMDVM Supervisor Anatonical Pathology DJeabcokraSherfL.ortP/irkel NSuepcerrovpissyors ASnunpeervMiossohrer Pathology Data 00426 Page 5 of 24 CONTENTS QSutauldiytyIdAesnstuirfainccaetioSntatement KSuemymaPreyrsonnel ObRejgeucltaitvoery Compliance TTeesstt SaynsdteCmontrol Materials RPreoscueldtusres DSiisgcnuastsuiroen Reference Table 21 IInnddiivviidduuaall CBloidnyicWaelighStisgns(a) AppPerontdoicxolA TP8084.PK HUI 6329-138 Page 32 46 77 87 119 nu un 1123 115 cooar? Page 6 of 24 HWI 6329-138 SUMMARY This study was done to assess the level of systemic exposure of T-6054when administered by intravenous injection to rabbits. FemaleHra: (NIW)SPF rabbits were assigned at imals received random to five groups a single intravenous injectionof (one/group). On Day 0, the an cle (sterile water for injection) or 0.128, 0.64, 1.28, or 12.8 mg of respectively). The dose volume the vehi T-6054/kg of body weight (Groups 1 through 5, was 0.5 mL/kg for all groups. Clinical observations were after intravenous conducted injection. at approximately 0.5, 2, 4, 24, Body weights were determined and just 48 hours before test material administration (Day 0). an auricular artery A or blood sample(approximately marginal ear vein of the 4 nL) was collected from animals at 2-, 4-, 6-, 8, 12, and 24-hours post-injection. In addition, at (48-hours post-injection), approximately the time 20 nL of oblfooedxpewraismeonbttaalinetdermfirnoamtieoanch animal. All samples were centrifuged, sent to the Sponsor. sAeppparroaxtiemdateilnyto48serhuomursandposcte-lilnujleacrtiforna,ctitohnes,aniamndals were anesthetized with exsanguinated. An sodium pentobarbital, bled abbreviated gross necropsy via the posterior vena cava, and examination was not done, however, kidneys from each tissues were animal were collected. The whole liver, collected and sent frozen to bile, and both the Sponsor after termination of the in-life phase. AT five animals appeared normal throughout the study. 00428 oT Page 7 of 24 OBJECTIVE HAI 6329-138 tThhee toebsjtecmtaitveerioafl,thiTs-60s5t4u,dywhweans atdomiansisesstseretdheaslevaelsinogflesysitnetmriacveneoxupsosuirnejecttoion to rabbits. REGULATORY COMPLIANCE ATdhuiisnisstturdaytiwoans'sconGdooudcteLdaboirnataocrcyordParnaccteicweithRegtuhleatUi.So.ns Fofoodr and Drug Nonclinical mLiaxbtourraetsoryforStucdoinecse,ntr2a1tiCoFnR, 58h,omwoigtehneitthye/seoxlcuebpitliiotny,thaatndansatlaybsiilsityofwatshenottest AcnoinmdaulcteWde.lfaArlelAcptrocReegduulraetsiounsse.d inInthtihse osptiundiyonweroef tihnecSopmopnlsioarnceanwditshtudtyhe director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification TThhee tcoensttromlatemraitaelriawlaswaisdeSntteirfiileed WasateTr-60f5o4r aInndjedcetisocnr,ibeUdSPas(Abanbotatmber liquid. aLacbloeraart,oriceosl,orlLeostsNol.iqu8i6d-.748-DH-02; Exp. March 1, 1996), and was described as Purity and Stability TdheeterSmpionnastoironasssu(miensclurdeisnpgonsuinbdierlittyestforcontdeisttiomnast)e.rialA spaumrpiltey oafndthsetabteisltity smattaebriilailt/yveahniacllyesesmixwtasurensot ftoarkecnoncbeenftorraetiaodnm,inissotlrubaitliiotny,as hothmiosgenweaistyn,ot and mraetqeureisatledwebryethceonsSipodnesroerd. toThbee paudreiqtuyateandforstathbeiliptuyrpoosfestheofUStPhisgrasdtuedyc.ontrol StoanrdRa eteg ntieon Tshteoretdestrefmraitgereiraaltedw.as Asntyoreudnusaetdrotoesmttmeamtpeerriaatlurew.as Trheeturcnoendtrotlo mtahteerSipaolnswoars OapfetrearticnogmplPertoicoenduroef a(lSlOP)t.estAinnyg raecmcaoirndiinngg tvoehiHcalzeletmoany Wbeiscuosnedsinfor(HoWIt)herStandard testing and will not be discarded after issuance of the final report. 00429 Page 8 of 24 HI 6329-138 SafetyPrecautions Tahned tpeoslticiaensd. control material handling procedures were according to HWI SOPs TEST SYSTEM TestAnimal AKadlualmtazaolob,inMoicrhaibgbaintsonofNothveembHerra:(16N,ZW)1S9P9F4 santdramianiwntearienerdeceativetdhefHraozmleHtRPo,n Inc., Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing ADfutrienrgreaccecilpitm,atitohne aanndinatlhsrowueghroeutaccthleimastteuddy,forthea paenriimoadlsofweraet lienadsitvi7dudaalylsy. choonutsreodlliendsqcuraeretne-rbso.ttomEnvsitraoinnmleenstsalsteceolntrcoalgsesfoirn ttheempeanriamtaulre-rooamndwehruemidsietty-to m1a2i-nhtoauirn1aigthetm/p1e2r-ahtouurredoafrk191ightotin2g3Cc,yclae.relaItnivcaesehsumwihdeirtey ovfari5a0t%ion2s0%,froamndthae rceoqnusiirdeedredtemtoperhaatvuerehadandnohuamdivdeirtsye ecfofnedcittioonnstheexisstteudd,y otuhtecyomwee.re dAnoicmuamlented and husbandry and housing at HHI complied with standards outlined in the "Guide for the Care and Use of Laboratory Animals"." Animal Diet TLhaeboraantiomraylsRawberbeitprDoievtideHdF a#5c3c2e6s,s tPHoIwaFteeedrs,adInIci.bitTuhneanfdeeda miesasruoruetdinealnyount of aconnatlaymziendanbtys.the Smaamnpulfeasctoufretrhe fwoartenrutrairetiopnearliocdoimcpaolnleyntasnalayndzedenvbyiroHnHIm.entaTlhere were no known contaminants in the feed or water at Tevels that would have interfered with or affected the results of the study. Selection of Test Animals Twheereansiemlaelcstedweraet riadnednotmifibeadsedbyonanihmeaa]lthnumanbderboadnyd cweoirgrhetspornedqiunigremeeanrtst.ag and 00430 Page 9 of 24 HHI 6329-138 StudyDesign FPelnaacleed ainnitomaltshewefioglhlionwgingfrosmtud2y,70g2routpos:2,891 g at initiation of treatment were Dose Level Dose Volume Number Group Treatment (7m-g6054/kq) _(ml/ka) oAfnimals 1 (Co2ntrol) T-60*54 00.128 00.55 11 31 TT--66005544 10..2684 0055 11 5 T-6054 12:8 05 1 * Sterile Water for Injection, USP. for Selecti Hcihsotiocreicbaelclayu,sethoef Ntheew ZleaarlgeandamWohunitteofalbbiancokgrroaubnbditihnafsormbaeteniontheonantihmiasl spoefcies. PROCEDURES DP oserepar andaAdt mini isto ratn ion The test specific mcaotnecreinatlrawtaiosndiflourteedachwitdhoseStelervielle. WaAtnerifnodriviIdnujaelctdioosnetoofaceahciheve a rietsspebcotdiyveweitgehstt osnoluttheiondayorocfonttrreoaltmewnats.cajTchuelarteesdpecftoirveeacthestanismoallutiboansedwason administered by given as a slow piunsthrav(eanpopursoxiimnajteecltyion30 intoto60a msaercgoinndasl ienadrurvaetini.on).TheThdeose was pArfetpearreaddnitneissttrsaotliuotni,onsanwyerreemasitnoirnegd taetstroosmolutteimopnesrawteurree duinsticlardaeddm.inistered. Reason for Route of Administration Intravenous injection is an acceptable route to assess systemic exposure. ObservaotfAinoimnalss 4C8linhiocuarls aofbsteerrvaitnitornasvenwoeurse cionnjdeucctitoend. at approximately 0.5, 2, 4, 24, and Body weights were determined just before test material administration (Day 0). 00431 Page 10 of 24 HUI 6329-138 sampleCollections AAtbhleotoedriszaamtpiloen (oafpprtoheximaautreilcyula4rmLa)rtwearsy coorllfercotmedthefrmoamrgeiintahlereaerarveviina tohfeall aCatirOmoatplssayant(aat2p,prf4or,xoim6m,tahte8e,lyp1o24s,8t-eharoniudorrs24vpeohnasoutr-csianvjpaeocsottfi-oienna)jc,ehctaainpoipnmr.aolx.iAmtaAtletllhye sta2i0mmpelnlesofowferbelood SSraooiTnedttaainrat farratoceotmmipoetnresam.tpuerreaTthueorfebl-ou2on0dtiClsa1mc0pelnCetsriuwnfteuirgleed stahhneidnppessdetpoartreoadttehiden aintforeeszeerrum saetnd to Sponsor. Pathology APPthoestt-epironmsjitenecarttiiiooornn)v,ofnaanthicemavaale,sxpewarenirdmeeenxatsnaaelnsgtuhpiehntaaistezeedd(.awpiptArhnoxsaiombdabitrueenlvyiapte4en8dt-ohbogaurrrobssistanle,crobplseyd via rPilevarecneradtboiinolned,rwya"asincden,obtotthhceonnkdiudfcnrteoeyzdse,n fhrboyowmepveleaarcc,hintgainsiinsnauleas wfewrreeereezceorcolllsleeectcttetedod.maandinThtieamimnwehdoi2laetely Demperature of 20C $10C. After tissue/bile collection, the animals were discarded. Shipom f Teisn suets AKfitdenreyscowmeprleetisoenntoffrotzheenin(-o1nifderyphiacse)e ttohetbhleaoSdponssamoprle(sJ,amelsiveDr.s,Johbnisloe,n, an3dH oSepeonpsooersCisnsorteBsalpcdocgne.spitb2l-ae3nEy-f0or9re,spt9ho3en5sirbeBiutslehinttAyivoenfnouraen,dthedSti.sapnoPaasluiylts,iiosnMNo,off5t5th1he0e6s)as.ammppllTeehsse. Hollected in this study nor are these results presented in this report. StatisticalAnalyses No statistical analyses were required by the protocol. Location of Raw Data, Records. and Final Report Trheetairnaewddaitna,therecaorrcdhsi,vesandofanHHIoriigninaaclcorsdiagnnceed wciotphy HoWfIthSOeP.final report will be 00432 page 11 of 24 RESULTS HNT 6329-138 BodyWeights Individual body weights at initiation are in Table 1. Clinical Observations Individual clinical signs are in Table 2. All five animals appeared normal throughout the study. Pathology AexIamianneidmaglrsosssulryvivwehdentosatcerirfmiicneadt.ion of the experimental phase and were not DISCUSSION rThaebbiTtevselwhoefn saydsmtienmiisctereexdposausrea 00.128, 0.64, 1.28, and 12.8 osfinTg-l6e05i4ntwraasveneovuasluaitnejdectiinonfemaatlelevaellbsinoof ma/kg. All animals appeared normal throughout the study following administration of this material. SIGNATURE StevenNW. W Glaza WN ASctuutdey DToixrieccotloorgy AW o FETE Date REFERENCE 1. NIH Publication No. 86-23 (revised 1985). 00433 Page 12 of 24 Individual Body letghts (9) LDeovseel Animal Group (maka) Sex Number Da0v 1 0 Female F52792 2,784 2 0.128 Female F52793 2,805 3 0.64 Female F52750 2,891 4 1.28 Female FS2751 2,702 5 12.8 Female F52752 2,783 HT 6329-138 00434 -= LDeovseel Group (mask) 10 2 0.128 3 0.64 4 1.28 5 12.8 Page 13of 24 IndividuaTlablCeli2nical Signs HI 6329-138 Animal Mew Sex Number _Observation~~ 0.5 2 4 28 48 Female F52792 Appeared normal J J J J J Female F52793 Appeared normal J J J J J Female F52750 Appeared normal J J J VJ Female F52751 Appeared normal J J J J J Female F52752 Appeared normal J J J J J 7 Indicates condition exists. 00435 Page 14 of 24 APPENDIX A Protocol TP8084.PK HHI 6329-138 00436 @HnzLeTon OSE AR EH Page 15 of 24 + ComnaNG Cr Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits Date: ) December 13, 1994 PerformingLaboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LabPorojrectaIdtentioficratiyon: HWI 6329-138 00437 Page 16 of 24 . PPa8ge0824P.K STUDY IDENTIFICATION Single-Dose IonftrTa-v6e0n5o4usinPhRaarbmbaictoskinetic Study HW No. 6329-138 Test Material T-6054 Sponsor 3MDeTpoaxritcmoelnotgy Service Medical P3.H0.CenBtoexr,332B2l0dg. 220-2E-02 st. paul, WN 55133-3220 Sponsors Representative 3JHohnToxL.icoBluotgeynhoSfefr,vicPehDMedical 3MDeCepnatretrn,entBldg. 220-26-02 SPt..0. PaBuolx, 3M3N22055133-3220 ) (612) 733-1962 Study Director HSatzelveetnonH.WiGslcaoznasin, Inc. MPa.d0.isoBno,x W7I54553707-7545 (508) 241-7292 Study Location HBauzilledtionng NWoi.sco3nsin, Inc. M3a8d0i2soPna,ckWeIrs A5v3e7n04ue PrEoxppoesreidneSnttuadly STtiamrettabDaltee DErxapfertinReenptoarlt DTaetremination Date DDeecceemnbbeerr 1197,, 11999944 January 23,1995 ) = 00438 Page 17 of 24 PPa8ge083 4P.K 1. SStiundglye-Dose Intravenous Pharmacokinetic Study in Rabbits 2. PTauodrnpianosissesestseretdheaslevaelsiongflesysitnetmriacveneoxupsosuirnejewchteinonthteo freasbtbitmsaterial is 3. ReTLghauiblsoraastttoourrdyyyCwPoirmlaplcltiibacenecceRoengduulctaetdionisn/Satcacnodradradnsce/Guwiidtehlitnheesfwoiltlhowitnheg Good ecoxncceepnttiroantitohna,t conducted: asonlaulbyisliistyo,f htohmeogteensetitmya,terainadl smtiaxbtiulrietsy wfiolrl not be [.] Conduct as a Nonregulated Study [['X]] 2410 CCFFRR 51860(F(DEAP)A-FIFRA) I[]1 4C0(81C)F3R079(2Fin(aElP)A-T(SOCEAC)D) [[11 5N9otiNfoihscaantioNno.No3.850313(Ja(pJaanpeasneeseMAFMFO)N) ) dAWieNrlefcaptrroeorc,AecdtutrheeRsegsutilunadtytihoidnosse.sprontoIotncoutlhnenaercoeepsisniainroincloymopfdluitpahlneicceSaptwoeintshoarnytahpnerdAenvsiitmouaudlsy work. 4. QThuealiprtoytoAcsoslu,ranscteudy conduct, and the final report will be audited by t(hHeNI)QuSatlaitnydarAdssOuprearnacteinUgnitProinceaducrceosrda(nScOePs)witahndHapzolliectioens.Wisconsin 5. Test Material A. TI-d6e0n5t4ification B. P(ThoysibcealdoDcuensecnrtiepdtioinn the raw data) C. TPhueritSyponasnodrStaassbuinleistyresponsibility for purity and stability dteesttermmiantaetriioanls/ve(hiincclleudimnigxtuurned(esr) tefsotr ccoonndcietnitornast)i.on, Ssaomlpulbeisliotfy, haodmnoigneinsetirtayt,ionandifsrteaqbuielisttyedanbaylytsheesSwpiolnlsorb.e tTahkeensebesfaomrpeles (if ttaekrenni)natwiiloTn bfeorsepnotssitboletheanaSlpyosnisso.r after experimental 00439 Page 18 of 24 ) 0. SRtooormagteemperature PPa8ge084 4P.K E. ReRseseerrvveeSasammpplleess will not be required for this study. F. ARneytenutniusoend completion toefstthmeatienr-iiaflewiplhlasebeorfettuhrenesdtudtyo. the Sponsor after G. SaAsferteyquPirreecdaubtyioHnHIs SOPs and policies 6. ControlMaterial A. ISdteenritliefiwcaatteironfor injection . B. PhClyesairc,alcDoelosrclreispsti1oiqnuid ) C. P cThoensu ipduearr rinetddyi Statnoadt bbieslty iaatbdyielqiutayteofforthitsheUSpPurpgorsaedse omaftetrhiiasl stiusdy. D. SRteofrraiggeerated E. ReSseeerSveectSiaomnp,le5.s E. Reserve Samples F. ARanenytdenwritelimloaninnoitngbecondtirsoclardmaetderaifatlermaiyssbueancuesedoffotrheotfhinearlteresptoirntg. 6. SaAfserteyquPirreecdaubtyioHnHsI SOPs and policies 7. ExperimentalDesign A. Animals (1) SRpaebcbiites (2) FSirtar:ai(nN/ZSWo)uSrPFc/eHRP, Inc. ) 3) fIgaennaitttiation _. 00450 , ) . 3 Page 19 of 24 (4) M 2.5e 10ai 3t.5Ig nkigth iatt ion PPa8ge0854P.K (5) N5umfbeemraalensdSex (6) IIdnednitviidfuiaclatinuomnbered ear tag (7) Husbandry (a) HIonudsiivnigdually, (heavy gauge} in screen-bottom stainless steel cages (b) FA(oPHomIdeasFeuerdesd, amIonuc.n)t. ofThLeabofrooadtoriys RraobubtiitnelDyigetanHalFyz#e5d326by ethnevirmoannmuefnatcatlurceorntfaomrinnaunttrsi.tional components and (c) MAadteTribitun fron an water are analyzed bayutHoNmIatifcor styosttaelm. disSsaomlpvleeds of the solids, fhoarrdnseeslse,cteadndeSlpeemceintfsi,edhemaivcyrobmieotlalosg,ical content and organophosphates, and' chlorinated hydrocarbons. (d) CTthhoaentrteawmoiaurnleadnntoisntkenrofwenrecownittahmintahnitssstiundyt.he food or water (e) EsEenntvviirrtooonnmmmeaenintntatlaincoanttroelmsperfaotrurteheofani1m9alC rtooo2m3wCi,llabe rTeilgahtti/v1e2-hhouumriddiatrykocfyc5l0e%.+20%, and a 12-hour (f)D Acclimay tion (8) S Baadsee eqdualtoen enohuefmacTbleterhtstoai fnAdneiobxmotadrnlyaswaeniigmhatlsawciclolrdibnegputorcHhMaIsedSOPsso. thaAtn no animal in obviously poor health is placed on test. (9) HisJtusotriifciacaltliyo,n the animal of "ftohre NSepweciZeesaTaSnedleWchtiitoen choice because of the laalrbgienoamroaubnbtitofhas been background information on this species. ota41 Page 20 of 24 3 : B. DoAsdeministration PPa8ge0864P.K (1) Iest Groups Dose Level Nunber of Group Ama/ke) [Females 21 0 0(.C1on2t8rol) 11 13 10..2684 5 12:8 11 1 a wTehieghdto.se volume will be 0.5 ml/kg of body C. DosingProcedures (1) DaoIpnsptirrnoagxvieRnmooauuttseleyin3j0ecttoion60isnetcoonadsm.arginal ear vein over ) (2) R Intre avfenoa orusDos isnijneo gctRioon untefs systemic exposure. an acceptable route to assess (3) DSoisnignlgeDudorsaetion (4) DPTiohnrsejeeectpteaistornamatttoieraoicnahlievwiella bsepecdiifluitcedcownictehntrsatteiroinlefworateearchfor adthdoeasienainlsietvnreaallt,'isonbI.onddyi"vTwihedeuiagplhrtepdaotrsaeekdsenwtiejlsultstmbibexetfcuoarrleecsutlweaistlteldmabbteaesrseidtaolroend at room temperature until administration. D. ObservoafAtniimoalns (1) CltThoiexniicacinatilmyOalbastsewaripvlpalrtoixboienmsaotbesleyrve0.d5,for2.0c,lin4i.c0a,l 24s,igannsdo4f8 hours after treatment. (2) BJoudsytWebeifaohrtestest material administration. (3) sample Collections J) (a) E2r,e4q,ue6n,cy8, 12, 24, and 48 hours post-injection - 00442 Page 21 of 24 98084P.K . Page 7 (b) ANnumofbAniemarls (c) M fBlrooome deoist tafhmCeprolhelselaeo r(catvpid ipoarnotxhiematcealtyhet4ernLi)zatwiiloln obef tchoellected 4au,r6i,cul8,ar12a,rteanrdy 2o4r hforuomrsthpeosmta-rignijneacltioena.r vein at 2, Adpopcruomxeinmtaetdelnyth20ealraovfdbaltoso)dwi(lalctubaelobvtoaliumneedtofrabne the npeocsrtoeprsiyor(v48enahoucrasvapoosft-eaicnhjecatniiomnal). atApthperoxtiinmeateolfy a20nimnlalsofdubrlionogd twihlel sbteudyc,olallescot,ediffrpoomsmsoibrlieb.undThe cSeanmtprliefsugweidl,l abnedsttohreedseaptarratoeomsteermupneraantdurceellanudlarthen tFermapcetriaotnusrestoorfed-20inCa +1f0reCe.zerThseetsetopamraaitnedtaisneruam and . ctehleluSlpaornsofrracatfitoenrsewxiplelrimbeentsaelnttefrrmoizneantioonn.dry ice to Samples will be shipped to: ) JManEesED. 8JoPh.nCson Bldg, 2-3-09 S9t3.5 PBauuslh,AHvNenue55106 JtaenleesphoD.neJoahtns(o6n12)or7h7i8s-5a2l9t4erpnraitoer wtiollthebesnhoitpimefnitedofby the samples. E. Termination (1) UAnmnosyrcihbaeundinumdlalecdoSndadyiictnrigiofndi,ucreiwsnialglntdhbDeeeasstutubhdjsyectoerdsatocriafnicaebdbreinviaated TgZrenocesossrtdhenedet.cirzoeApdnsiywmiaetlhxsansioinndaitauinomnopreinabntudonbdaarllbciotnaadblintoir(omvanilaiwtiiilnlejsebcewtiiloln be in Tethexersmaminagnruagitininaoatnle,d.e(a3)rTiSvseasimunp)el,se, bCloaseldledcvetisiacornit,bheedwivleilnna sbeceacvctaoi,lolneacnEt.ded. ) 00443 Page 22 of 24 . PPa8ge0884P.K (2) SActheadpuplreodxiSmaactreilfyi4c8e hours post-injection, animals pseunrtvoibvairnbgittaol te(rvimainaitnijoenctwiiolnl inbethaenemsatrhgeitniazledewairthveisno)d,ium gbrloesds vinaecrtohpesyveneaxamcianvaa,tioanndwielxlsannogtuinbaeteddo.ne, Anhowaebvberre,viated tissues will be collected. (3) SaThmeplwehCoolellTeicvteiroannd bile fron each animal dying during the stteurdnyi,natsiaocnriwfiilcledbeincoallmeocrtiebdu.nd cBootnhditkiiodnn,eysorfrsoumrveiavcihng to oannindarly wiiclel iamlmseodibaetelcyollaefctteerd.collTehcetitoinssuaensd wtihleln pbelacpeldaceidn a freezer set to maintain a temperature of -20C 10C. dTrhye tiicsesuteosth(eivSepro,nsobrilea,ftekirdneexypse)rimweinlltalbetesremnitnaftrioozne.n on STheectisoanmp7l.e0s.(w3i)l.l(cb)e. shTihpepedSptoonsothreisperrseosnponlsiisbtleed ifnor the retention and disposition of the samples. : F. StNoatsitsattiicstailcAanlalaynsaelysses are required. 8. RAepfoirnatl report including those items 1isted below will be submitted. DDeessccrriippttiioonn ooff tthhee tteesstt sanydstceomntrol materials PDartoecseduorfesexperimental initiation and termination GDreosscsripptaitohnoloogfyanfyindtionxgisc e(fiffecatpsplicable) GrDoisrsecptaotrh)ology report (if applicable and requested by the Study ) 00444 Page 23 of 24 PPa8ge089 4P.K ! 9 . LoOrciagtiinoanl odfatRaa,wDoartcao,piReescotrhdesr,eoafn,dFwiilnlalbReeapvoariltable at WWI to afcacceipltiatnactee aofudithteingfintahle rsetpuodryt.duriWnhgenittsheprfiongarlessrepanodrtbeisfocreompleted, raleltaoirniegdinianl tphaepearrcdhaitvae,s oifncHlWuIdinagccotrhdoisengittoemHWlIistSeOPd. below will be PDroosteocporlepaarnadtipornotorceoclordasmendments InB-oTdifyeweriegchotrsds DOobsseervaadtmiionnisstration sSahmipplpeingcolrelceocrtdison records SPtautdhyolocgoyrreRsecpoornddsence Final report (original signed copy) Tnhoet bfeollaorwcihnigvedsuwpiptohrtitnhge sretcuodrydsdatwail.l be retained at HHI but will AWnaitmearl anraelcyesiipst/raecccolridmsation records ) ARneifmrailgerraotoonrteanmdperfarteuerzeeratndempheurmaitduirtey rreeccoorrddss Instrunent calfbration and maintenance records ) 00445 Page 24 of 24 ) Fae to TPB084. PK. PROTOCOL APPROVAL Ql 2. Bulla] John L. Butenhoff, PhD 3SpMonTsooxri'cso.lRoegpyreSseernvtiacteivMeedical Department Jams Date NeW Date ven - Steven MN. Glaza Study Director ) Acute Toxicology Hazleton Wisconsin, Inc. Representatiye Quality Assurance Unit Hazleton Wisconsin, Inc. 12.2.2 Date (6329-138. protdsk2) 600446 9.1.2 Analyticalprotocol AMDT-122094.2 600447 - 3M Environmental Laboratory Protocol - Analytical Study Single-Dose Intravenous Pharmacokinetic Studyof T-6054 in Rabbits In-Vivo Study Reference Number: HWI#6329-138 Study Number: AMDT-122094.2 Test Substance: FC-129 (T-6054) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facilit3yM: Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Initiation Date: July 25,1995 Proposed Completion Date: August 25, 1995 Method Numbers and Revisions: AMDT-M-1-0, Thermal Extraction of Fluoride by Means ofa Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, AElneacltyrsoissproafyRMababsist SLpievcetrrEoxmtertarcyt for Fluorochemicals Using AMDT-M-8-0, Analysis with Ton SoeflFelcutiovreidEeleUcstirnogdethe Skalar Segmented Flow Analyzer Author: James D. Johnson Approved By: [ames D. Johnson Study ctor 1S Date lon 1. Bodlfff vof5ef5 John Butenhoff, PhD Date Sponsor Representative 00448 - 1.0 PURPOSE This study is performed in order to provide information necessary to assess the extent of dermal absorption of FC-129 (T-6054) in a subsequent dermal absorption study, HWI#6329-133. The study is designed to provide information as to whether FC-129 and its metabolites are detectable in liver and other tissues, either as total organic fluorine or atos sapsecceirftiacincwohmeptohuenrdpserwfhleunortohoectFaCna-t1e29wilisl apdrmoivniidsetearmedaraksearnfiorntdrearvmeanoluasbdsoosrept;iaonn.d 2T 0 ESTMATERIALS 2.1 Tes2t.,1.C1onAtnraolly,tiacnadl RReeffeerreennccee SSuubbssttaannccee:s aFnCd-9M5a,trloitce1s61 or 171. They are e2.q1u.i2vaAlneantl.ytical Reference Matrix: Bovine liver and bovine serum 2.13 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source of Materials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4liver), Sigma Chemical Company (2.1.2, 2.1.4-serum) 2.3 Number ofTest and Control Samples: Liver and serum fro4m test animals and 1 control animal, other biological tissues (kidney, bile, cellular fraction) will be available for analysis if deemed appropriate by the Study Director. 2.4 IdentificationofTest and Control Samples: The samples are identified using the HWI animal identification number which consists oaf letter and five digit number, plus the tissue identity and day identity (serum). 2.5 Purity and StrengthofReference Substance: To be determined by Sponsor. 2.6 Stability of Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room 20+ 10C (2.1.2, 2.1.4). Test and Control samples tweimlplebreatruerceei(v2e.1d.1a)c,cording to AMDT-S-10-0. 00449 . 2L.8PDiRsepgousliattiioonnofforSipttehheciatmi2em8nesdp:aeyrBiidooedlrormgeaiqcluaialrbetsidosrsfpuoterissotanunsdditeufsdlyuliodsnsogwaelirlltthibasesnur2ees8tawdiianlylesdb.peeTrhis Srtetuadiyneids.in parallel w i2.r9eS.afUetsyePcraeuctaiuotniwohnse:nRheafnedrlitnogakpnpirovpersifaotrecMutStDiSng,thWeesaarmpalpepsr.opriate laboratory 3.0 EXPERIMENTAL - Overview Toheeltaintsesuefsoranadnasleysriusamnfafolrryotfimlcuaaolnriitmneeastlcssocmdaopnsoeubdnedapsse.rdfeoAsrtcmrteihdbe.eddiT(shHcerWeIstci6ro3en2eon9fi-ntg1h3eSf8ot)r,ufdaltyueoride in Oy De prosent Mnetemrabiuesstioofn (See definitive data for Mfeltuohroidnes--innetxhte serum, Sleivcetri.onF)oir ctohnefaiprpmraotpiroinatoefatnhaelypsriessetnoce electrospray mass spectrometry canbe of Pesifc compounds n tissues and used. 4.0 EXPERIM ENTAL -Methods 4.1 Liver and Serum screening methods: (attached) 4D.o1h.1rmAaMnDnTD-XM2-010.00,OrTghaenrimcalHaElxitdreacAtniaolnyzoefrF-lLuiovreirde by Means of a Modified 41.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable lon Analyzer 4.1.3 AMIDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver U4.s1i.n4gAEMlDecTt-roMs-p5ra-y0,MaAsnsalSypseicstroofmReatbrbyit Liver Extract for Fluorocheicals 4".A1n.a5lyAzMerDwTi-tMh:l8o-n0S,elAenctailvyesiEsleocftrFoldueoride Using the Sklar Segmented Flow 00450 5 5.0 DATA ANALYSIS f5.l1uoDriadteapRereptoirsstuiengo:r flDuaitd,a owrilalsbFeCr-e9p5or(teeldecatsroaspcroanycemnatsrastisopnec(twreoimgehttr/yw)eipgehrtu)niotfof tissue or averages fluid. Statistics used, at the discretion and standard deviations from different of the Study dose groups. Director, may include If necessary, simple standard statistical tests such as the Students t test may be applied to determine statistical difference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw approved Data final and Records: Raw report, and electronic data, data approved protocol, will be maintained appropriate specimens, in the AMDT archives. 7.0 REFERENCES 7.1 AMDT-S-10-0, Sample Tracking System 8A 0 TTACHMENTS D8.o1hArMmDanTn-MD-X12-000,0 TOhregramnailc HExatlriadcetiAonnaloyfzFelru-oLriivdeer by Meansof a Modified 8.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver E8.l4ecAtrMoDsTpr-aMy-M5a-s0s,SApneacltyrsoimsetorfyRabbit Liver Extract for Fluorochemicals Using 8.5 AMDT-M-8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 00451 4 3M Environmental Laboratory Method Thermal Extraction of Fluoride by Means ofa Modified Dohrmann DX2000 Organic Halide Analyzer - Liver Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: /o- #-1< Revision Date: None Author: Rich Youngblom Approved by: i p Lea of /7< Dale Z 2RA Quality Assurance [02--1-15-1 Date Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-EP-3 Routine Maintenance ofa Modified Dohrmann DX2000 Organic Halide Analyzer 00452 1 1.0 SCOPE,APPLICABLE COMPOUNDS, AND MATRICES P 1.1 Scom pe: Thivsarmieotushomdatirsicfeosr.thTehoepefrlautoiroindeoisfatyDpoicharlmlyanconllDeXct2e0d0i0n TwIhSeArBitsiosluutsieodntfoorexatnraalcytsis w11i32thMAaaptnprlfiioccneassbe:lleeBciCtoiolvomegpieolceauclnttrdiossdse:u.eFs,lupoarrtoicchuelmarilcyallisveorr.other fluorinated compounds. 22.01 FKluoEriyde,wflouorRinDe, Sextraction, pyrolysis, ionization, fon selective electrode, Dohrman, filide, DX2000, fluorochemicals. 3.1 3.2 33 PGGrlleaassssssuwwraairrzeeedaisngadfsreaegsxi,hlaep,ursobtpreograksecenosmgpclarasnessbsmeeadeyxgtcaraseumhseaelnyidnljhiuonrtig.esp.ractices required.n before starting run. 3344 SPootrevnetnitalbbaiseodhaszaamrpdledsuemtaoytfhleasbhi,olmoagiycanlemeadtr1i0ceasl.loUwsteheapmptroopdrriyatdeopwersonal protective equipment. 4.0 SUPPLIES AND MATERIALS 4.1 4432 CCoommpprreesssseedd Quart glass sOHeaxlmyipgulemen,,boHHaiytgdhwrioPtcuharirTtbeyofnGlrofarndeTMee,,trurebegigunulgl,aattDeedodhttroom34a05nPPnSSI8L.90-097 or equivalent. +444 Qruiaorntz5g4l0a9ss95coTmobtuasltTioonnictuSbter,enRgetlhiaAndcjeuGsltamsesntGB-u9f4f0e5r-(0T1I2SoArBeq11u)iovarlenetq.uivalent. 4.6 Sample collection vials, HDPE. 444...987 PMAociltlyilsvita-ytQreeTMdnCewhapatirepcerotatles,.E. Merck 2005 or equivalent. 44.1110 HMiasmcielltloanneSoyursinlgaeboorrateoqruyivgallaensts.ware 5-- 0EQU-- IPMEN-- T ------------ 55.:12 RIoBsMemcooumpnsatotfiDtbwolahrere3,m8av6nernosriD4oXn82610.c00o00m,pmOuortdgeiarfn.iiecdHfaolridfeluAonraidleyzeexrt,ramcotidoinf.ied for fluoride extraction. 55.:43 EDxXc2e0l0S0preadsheet, version 5.0 or greater 6.0 INTERFERENCES 6.1 Sample size is limited to approximately 150 mg, depending on sample moisture content. This may vary from matrix to matrix. 00453 2 10 SAMPLEHANDLING s7.a1mpSlae.mplUesseafroerncoetpstoorbeprhoabnedlteodtrwaintshfebrartiesshuaensd.s. Fluoride may leach from the skin to the c7l.e2aSnasmcpallpeeslofanldivceurtatriengcbuotafrdr.omTfhreozceunttliinvgerbaoanrddpalancdesdcailnpaeltasrheoduladndbelacbleelaendewdewiigthh bwoaatte.r,Use a `methanol, or methanol-water solution after cach liver is cut. 8.1 Preparationof Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 88..11..32 FToyprircaablbliyt i50v-e5r0s0tupdipems,FuCs-eb9e5eifnlmievetrhaansotlhestmaatnrdiaxr.dsCaurte uaspeide.ceoffrozen beefliver (100+ 150 mg) and weigh it ina labeled and tared weigh boat. 8.2 Calibration - Overview "The normal calibration is the fluoride curve (AMDT-M-2). However, ifan optional spiked liver curve is required the procedure listedbelowis used. a88..n22d..12coTAmypbicucasaltliilbynra,gttithohrneecemurruesvpielnifgcoartttheesheosfaeDmaXec2h0me0st0thaoinsddagsreadnneadrnadmtaeftdirvbiexyctosynppcieeknaitsnrgatthsieaomnspsalmoepfslsewtsiatnthodakbrendsotewwsintlelds.tbaendards spiked. 8.2.3 Standard curve will be plotted as Mass SpikedF Recovered F(ug)on the y-axis. Generatae regression (ug) on the x-axis curve and calculate and the Standard equation Mass for the line. and the r2 value. 8.2.4 Mass Spik*eFdC-F9(5uigs)6=0.(0A4m%ouFntthesrpeifkoerde i0n.m60L0)4 xis(tCheonfca.ctoorfussteadntdaordcoinnvperptm)FCx-9(50.t6o00F4)* 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 83.1 Start Up 83.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may `be usedif the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8312.1 Prepare sample using the same methods and type ofmatrix as the test sample. 83.2.2 For rabbit studies, use beef liver as the matrix. Prepare at least 3 samplesof beef liver (100 - 150 mg) for blanks. 8.3.2.3 analyze Put sample in Dohrmann boat. Combust sample according to method AMDT-M-2 each sample as described in section 9.0 for the ion selective electrode analysis. and 000454 3 8.3.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 pprm or lower jbuedfogreemepnrtocoefedtihnegopweirtahttohretchaelirberaadtiionng. isBsuabmlesawmiptlhersesupnteiclt tthoishilsitmoirticiaslrreeaacdhiend,gsor(purnetvilioiunst4h8e h8o.u3r.s2)..5 For non-rabbit studies, the blank readings should reach a predetermined ion concentration before proceeding with the calibration. 8.3.2.6 It may be necessarytomix approximately 50 mg of charcoal with thesample to aid `combustion. 8.3.3 Standard Curve 8.3.3.1 Weigh out at least 15 matrix samples (5 standards with 3 replicates each) intaredand lsatbuedlyeddatwae,igShtobroeattsh.e mFaotrriraxbbsiatmpstluedsioesn,dwreyigiche 1o0r0i-c1e5p0acmkgsbteoefkleievperthseammpflreosz.enRuenctiolrdusweed.ights in 833.2 Place weighed beef liver sample in Dohrmann sample boat. 8333 Start with the lowest standard concentration. Using a Hamilton syringe, ejecat fixed qourainnttihteyobfetehfelisvetra.ndard on or in the matrix. For rabbit studies, use 4 uL ofstandard and eject it on 83.3.4 Atleast3 replicates should be used for the lowest standard concentration; more replicates `may be used at the discretion ofthe analyst. 8.3.3.5 8.3.3.6 RCuonmbaullst15thsetasnadamrpdlse. aIsfdoensecrriebpeldicianteseisctsiiognni9f.i3caanntldyadniaflfyerzeentafcrcoormdtihneg to AMDT-M-2. other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that thenewreplicate will be used to calculate the regression curve. 833.7 When all standards have been run, calculate the 12. r2 mustbe at least 0.95. If itis not at least 0.95, consult with supervisor. c8.h3a.n3g.e8d.ANneeww ssttaannddaarrdd ccuurrvvee mshaoyuladlsboebreurnuwnhaetnthtehdeicsocrmebtuisotnoifotnhteubaenaolryssta.mple matrix is 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for standards are dependent on the individual standards used. Typically, standards are stored at room temperature in plastic screw top bottles. 8.4.2 New FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.13 Vaporization/Drying tim=e 240 seconds. 9.1.4 Bake time = 300 seconds. 99..22.1StIafrttheUpprPorgorcaemduisrneo:t started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the fumace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. . 4 00455 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOAT program found 9.2.6 in the CELL `CHECK menu. See AMDT-EP-3 for details of the Dohrmannsoftware. 9.3 SampletEhxetSraAcMtiPoLn EPrHocAeTduCrHe:and place the sample in the BOAT. It may be necessatryo If this is done,charcoal 9.3.1 Open `mix approximately 50mgofcharcoallis`hwiintgh ttheh.e"sbaasmepllinee taonadidwhcoemnbugesnteiroant.ing. the standard curve. s99.h33o..u32ldCAaldlosdsomeapbpleSpermAociMpoxrlPeileadLctEtienivoHwnohAlviiuTlamelC.eHTesyo.tpfaiTbcIaSlAlBy 0s.o6lumtiLontoor151:m1 LTIaSrAeBu:sMedi.ll`iFo-rQrTMabwbaittesrtumdiixets,urueseto1a.0 or l29.a.3b0.e4lmeLdPlsoaafce1:t1heTIviSaAlBs:oMtihlaltit-heQTMtip`owfattehremiCxtOuMreB.USTION TgUhBtEheiTsIiSnAtBhe. TISAB at least 0.25 inches. Gnatshese rEeOleXa-sSedOdLuIrDinSg p`rpyorgorlyasmisfmouusntd `ibnutbhbleeRtUhrNoumenu. rom the combustion tube. 9.3.5 Ru When the EOX program is finished, remove the collection to collect the sample, add an `vial f equal volumeof Milli-QTM water 9910..33t..h67e TIfIunSdAiBluttoedma"kTIeS1A:B1TwIaSsABus:eMdilli-QTMw.ith `Milli-QTM water and wipe: with a KIMWIPE to 9r9..e33m..o89vReOinpasenenytTthhIeeSesAnaBdmorpfeltmeha`eihnactiotcnhomgrbaunoosrdntvrtiaheocemnuottuuvubmebeee.adnoyutr.emIatimnainygbaesRhfercoemstlhrye tboosactr.apAspahrtciacnlebsoefrfetmhoeved a amtiitohnatisppapteudlaapoprlioctaher similar device. A drop.`of Milli-QTM water may be added to the `bottom boat to w aidin the CleanCycle. 999...333...111012 CRSlauomnspetlhteehei`shCraeLtacEhdA.yNfoBrOanAaTlyspirsobgyrajmo.n selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 9.42 Use Sam the ple standard Mass Re curve to: covered calcula F (vg) te = (tTheISsaAmBplveolvianlume.L) x(Orion readin(gSilnoppep) m - intercept) 10.0 VAL] DATION 1100..11.1QuDaailislisyttyaSrtCtaoerdnttbrUyopalnCahlyezcikngSaQmCplaemsp:leOsn.ceTthhee <QtCansdaamrdplcceuursrvvea.ereisEtoaescbtheabctlohienschseeandtm,reeaat2ic5ohtnhdmeauylsootwfebset aodnnoacnleeynsitinrsatrtiipolincastpeikuneldessasmtphlefeisrsutstedwotorgeeplniecraattees tahree s`wtiatnhdianr2d0% of the. standard curve, then athird replicate is not necessary. 1F0l.u2oriPdreecNiostieobnoaonkds Accuracy: 2,3, and 5. PSreeceimsieotnhoadnddaecvecluorpamceyn"tv.arainesalwyhsiesnaanndalsyazmipnlgesaanmapllyesisosfidnifferent `matrices and different reference compounds. 10.3 Other Validation Parameters: NA 00456 s 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1 11.1.2 For the standard To calculate the curve, use. regression analysis in Excel, version 5.0 or greater. fluoride contraction in the sample, see method AMDT-M-2. 11.2 Analyzing the Data H11e.2w.hr1aitni2n5m20u0%so%tfbotfeehaaetcalhevaeosrttah0ge.er9o5afnodtrhtgohrseeeaottuewtrlo,i.e"rOAuintslygireseruastc"ehrmotauhtyalinebr2es0es0xh%coluoulftddehbdieefpatoviewnrotaoegfdetoohufettithnhorsteeheetrwdeoapltoiarcaatneds noted in the Final Report along with the reason it was considered an outlier. None 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915- 349, 13.2 revision B, December 1993) AMDT-M-2 Fluoride Measurement by Means. of an Orion EA940 Expandable lon Analyzer 13.3 AMDT-EP-3 Routine Maintenance ofa Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS Revision Number `ReasonforChange Revision Dae 00457 6 Environmental Laborator Method Fluoride Measurement by MeaAnnsaloyfzaenr Orion EA940 Expandable Jon Method Identification Number: AMDT-M-2 Revision Number: 0 Adoption Date: /0-445 Revision Date: None I Author: Rich Youngblom Approved By: Gr Leader 13/2s Date Ar 4 BAX Quality Assurance fo- 4705 Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal `Extraction of Fluoride by Means ofa Modified Dohrmann DX2000 Organic Halide Analyzer 1 00458 - PE, AP BI 1.1 SCOPE: This method is for the calibration and operationofan Orion EA940 Expandable fon Analyzer. 1.2 APPLICABLE COMPOUNDS: Fluoride. P1H3 OAPfP6L.0. ICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred 2.0 KEYWORDS 2.1 Fluoride, fluorine, ion selective electrode 30PRECAUTIONS 00000000 3.1 No hazards identified with this method. 4.0_SUPPLIES AND MATERIALS 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer IT (TISABI) or equivalent. 4.2 Orion Model 900001 electrode filling solution (AgCI) or equivalent 43 Orion 940907 100 ppm fluoride standard or equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magneticstirbars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100mL volumetric flasks. 49 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. 50EQUIPMENT 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 336 or 486 computer (only neededifusing Orion 3E software). 5.5 Orion RS232 interface cable (only needed if using Orion 3E software). 5.6 Microsoft Excel 5.0 (only needed if using Orion 3E software). 6.0 INTERFERENCES 6.1 It is recommended that the pH be at or near 6.0. A 1:1 mixture ofTISAB and sample/MilliQTM water will generally bring sample to pHof 6.0. 6at.2roSoammptleempteermapteurraetuarsetmheasytaenfdfeacrtdsflwueorriedewhmeeanstuhreemmeentte.r wIatsiscraleicboramtmede.nded that the sample be: 63 The rate the samples are stirred at should be consistent with the rate the standards were stirred. 00459 2 6.4 Air bubbles under electrode. trapped under electrode can give erroneous readings. Make sure no air is trapped 1~ 0SAMPLEHANDLIN~ G ~ 7.1 No special handling necessary. 8S .0 CT ALIBA RATN ION AD NDARDIZAT~I ~~ON 8.1 Preparation of Calibration Standards 8.1.1 Measure 50 mLof TISAB IT into 5 100 mL plastic volumetric flasks. 8.1.2 Label the flasks as 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, along with the date and your initials. 8.1.3 Pipette 0.05, 0.1, 0.5, 1.0, and 1.5 mL of 100 ppm fluoride standard into the appropriately labeled flasks. 88..11..54 SAhdadkeaptphreofxliamsaktsetloym3i0xmtLheosfoMluitliolnis-.QTM water to each flask. t8.h1e.f6laEslkismoivneartethaeirabiurbbbulbebslefsr.om the flasks by tipping the flasks on their sides and rolling the air in 8.1.7 Bring the volume in the flasks uptothe 100 mL mark with Milli-QTM water. 8.1.8 Invert and shake the flasks for the final mixing. 8.1.9 Record standards in Standards Log Book. 8.2 Calibration 8.2.1Ifnecessary, remove tape from electrode filling hole. 8.2.2 Invert probe to wet top seal. 8.2.3 Ejecta few drops of filling solution from bottomof electrode to wet lower seal. 8.2.4 Fill the electrode with filling solution. 8.2.5 Themeterand the F- electrode are typically calibrated by direct measurement with no blank correction, using standards with concentrations of 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, following the manufacturers instructions. 8.2.6 Record the slope in the appropriate log book. 8.2.7 Clean the electrode by rinsing with Milli-Q water and wiping thesidesdown with lab tissues. 8.3 Storage Conditions for Standards 83.1 Calibration standards are stored at room temperature. 9~ 20 PROCEDURES ~~ 9.1 Calibration and Measurement, Standard method: 9.1.1 The sample to be measured needs to be mixed with TISAB using the proportions recommended by the TISAB manufacturer. 9.1.2 Place a str bar in the sample and place the sample on the stir plate. e9l.e1.c3trAoldeloiswitnhseersteadm,pmleaktoemsiuxreftohaerrefaerwesneocoainrdbsubbebfloersetirnaspepretdinugntdheeretlheecterloedcet.rodWe.hen the 9.1.4 Thesample should be the same temperature as the calibration standards and stirred at the same rate as the calibration standards. 9.1.5 When the readings have stabilized, record the reading in the appropriate log book. 3. 00460 9.2 Calibration And Measurement, Using Orion 3 Software: 9.2.1 Calibration: 9.2.1.1 Follow steps 8.2.1 to 8.24. 99c..o22n..c11e..n23trTParhteeisoscnooFfmutpnuhctteeiosrtnsancKdreaeyredn#s,8wial(nlFdSa)sw.kheytohuertoocfonnoftiarmbltahnekniusmtboebreoifncsltuadneddaridnstthoe bcealiubsreadt,ion. M99..a22k..11e..54aPOnlbyasnceeercvtehesestaehrleyerccethardoaidnenggeissnottnhoettfhhieersgitrnsaftpoahrnimdcaatrdidiosnopnlpartyehsoeensntttiehrdeplacaontmdepcaulntiedcrkc.loinWckhCoeOnnNCTtOhIeNNTrUeIEaNd.iUnEg.s have s99t..a22b..i11l..i67zeRAdfe,tpeeprartetshssetefAipnCa9Cl.2Es.tP1a.nT4daaRrndEd,A9tD.h2Ie.Nc1.oG5m.pfourtethrewriellmadiinsiplnagystthaendsalrodpse.of the curve, as well as the ianntderccleipctkaonndCcOorNreTlIatNiUonE..ReTchoercdatlhiebrsaltoipoen,dianttaericsepatu,toamnadticcoarlrleylactoiopnieidn ttoheC:a\pOprrioopnr\iDaatteal\oCaglbiob.otkxt. 99..22..22.1DaStelaecStprceitahdesrhNeeEt:W or OPEN from the FILE menutoopen a new or existing spreadsheet t9o.2s.t2o.r2eRdeactaoridn.the nameofthe spreadsheet used in the appropriate log book. 99.22..33.1FlFuoolrliodwe sMteeapssu9r.2e.m1etnhtr:ough 9.2.4 9.23.2 9.233 9.23.4 Enter Click When tohne tnhaemNeoEfWthSeAsMamPpLleEibnutthteonappropriate the readings have stabilized, click on the place on the screen. RECORD button and write the result in the `appropriate log book. 100VALIDATION 10.1 Quality Control: 10.2 Precision and Accuracy 103 ppm fOltuohreirdeVuaplitdoaatisoantuPraatreadmseotleurtsionAocfcfolurodriidnte.og Reference 13.2, the rangeofdetection is 0.02 11.0 DATAANALYSIS ~~ 11.1 Calculations None necessary. ---------- 112 Analyzing the Data None necessary. 12.0 ATTACHMENTS None 13.0 REFERENCES ec ------ 00461 + 13.1 Orion Model EA940 Expandable lon Analyzer Instruction Manual, Orion Research Incorporated, 1991. 13.2 Orion Model 960900 Solid State Combination Fluoride Electrode Instruction Manual, Orion Research Incorporated, 1991. 14.0 REVISIONS m--S--------------eeeee Revision Number Reason forChange. Revision Date 5 00462 Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 rrr Author: Dave Christenson/Cynthia Weber Approved By: ip Leader Adoption Date: /o-2/-55 Revision Date: None ------------ Jo=3/-25 Date Glbf Bk Quality Assurance lie1s Date Software: MS Word, `Affected Documents: 6.0 M-5, AnalysisofRabbit Extractfor Fluorochemicals Using.Electrospray `Mass Spectroscopy. 00463 ' losc1o.1rEScope: This method is for the extraction of fluorochemicals from rabbit livers. eEltehcytlroasceptraatyemias susseSdpteocterxotsrcaocptyf.luorochemicals from the livers foranalysisby 11..32 AMaptprliicceasb:leRaCbobmiptoLuivnedrss.: Fluorochemicals or other fluorinated compounds. 20KEYWORDS 2.1 `Fcloumorpoocuhnedmsi,caexltsr,acrtaibobni.t livers, electrospray mass spectrometer, fluorinated 30PR3.E1CAUUseTgIloOveNs wShen handling the rabbit livers, they may contain pathogens.= 4.0 SUPPLIES ANDMATERIALS ~~~ 4.1 4S.u1p.p1lieSsyringe, capofamebasulrieng 100 yuL. 41:2 Eppendontypeor disposable pipes 44..11..43 PlaGsltoivcesgrinding tubes 44..11.65 PLlaabsetlisc centrifuge tubes, 15mL 44.:11.87 TNiitmreorgen 44.11:.190 AFnialtleyrtsi,cTailtpainpnesy;lognassyrvionlguemfeitltreircs,p0i.p2esj.m. 44..11..1112 DCirsipmopsacbalpeapultaosvtiiacls3.ccsyringes. 4.2 4R.e2a.g1enAtqsueous Ammonium Acetate (Aldrich), approx.250ppm: Preparae 2500 `apcpemtaqa1u0tee2ou1s0s0omluLtiovnoloufmeatmrmicofnliasukmanacdedtialtuetbetyoavddoilnugm2e5w0imthgMailmlmio-Qnium 4.2.2 `Swoatdeir.umDiclaurtbeotnhaitses/oSloutdiiounm1B:i1c0arfobronaa2t5e0Bupfpfmers(oJl.uTt.ioBna.ker), a(nNdC2O1./0 NgAoHfCsoOd,i)um02bi5caMr:boWneatiegh(N2a6.H5CgOo;fisnotodiau1mLcavroblounmaetteri(cNaf,laCsOk,a)nd 4.2.3 DbirliuntgetoacveotolnuimteilweistohluMtiilolni,-dQilwuatteerac.etonitrile 1:1 with Milli-Q water. 44..22..45 EMtehtyhlanAoceltate 441.22..67 M1iHl,li1-HQ2wHa,te2rH - perfluorooctanesulfonic acid (Aldrich) 4.2.8 FC'95 (3M Specialty Chemical Division) 00464 2 5.1 Ultra-Turrax T25 Grinder for grinding liver samples. 5.2 Vortex mixer 5.3 Centrifuge 55..54 SAnhaalkyetrical Evaporator 7.1 Tpheerfroarbmbeidt.liav re e recr eivsed frozen, and`mustbekept frozen untiltheextractioins 8.1 Preparation of Internal Standards 8.1.1 Pperretplauroeraoonciinatneersnuallpshtoannidcaarcdoidftoapbperoaxdidmeadtteolyea1c2hplpivmer1sHa,ta1pHl,e,2H,2H- 8.1.2 W10e0igmhLatvolleuasmtet0r.i1cgfloafsk1.H,Re1Hc,o2rHd,t2hHe.-apcetruafll`uwoeriogohctt.anesulphonic acid into a 8.1.3 Bring it up to volume with methanol, this is the stockstandard. 8.1.4 Toa 250mL `volume with volumetric flask, add 3 mLsof the Milli-Q water. Calculate the actual stock standard and bring concentration of the to standard. --acstupalhmognpeirfclauocroictdane- Xml = 0.1L 250 mL. acwal concentation, ppm 8.2.1 Prepare FC.95 standards for the standard curve, 8.2 Prepare FC-95 Anion Standards 8.2.2 RWeeciogrhdapptrhe oacxtiumaaltweeliygh1t0.0 mgof FC-95 into a 100 mL volumetric flask. 8.2.3 Bring up to volume with dilute acetonitrile. 8.2.4 aDiplpurtoexitmhaetseollyut1i0on0 wpiptmh.dDiliultueteactehtiosnistorliulteio1n:110f:1o0rwasitohludtiliuotne aocfetonitrile for 8.2.5 Uasseoltuhteio1n0opfpamppsroolxu.ti1o0n ptopmm.ake working standards with values close to 5.0 ppm, 1.0 ppm and 500 ppb. 8.3 Prepare Beef Liver Homogenate to Use for Standards 8.3.1 Weigh 40 gof Bovineliver into a250mL Nalgenebottlecontaining 200 mLs Milli-Q water. Grind to ahomogenous solution. 8.3.2 Add 1 mLof the solution to a 15mLcentrifuge tube.Prepare atotalofeight s1umspLenadlisqoulouttsioonf tbhye sshoalkutiinogniitnbe15tmweLenceanltirquioftusg.e tubes.Be sure to re- 00465 3 8.3.3 Spike seven of the | mL aliquots with the following amountsof working sbltaannkd.ards in step 9.12 of the procedure. One 1 mL. aliquot serves as the Working Standard (Approximate Conc.) Approximate Tid `concentration of FC-95 in liver 8.4 Calculate the actual valofuthee standards: soxf_sctanodarnd cenvat(iinoppnm) = final concentration. (ppm) 171 mg liver'/ 1 ml homogenate of FC -95 in liver *Average weight of bovine liver in solution as determined by weighing F1Cm-L95hiosmroegpeonrattedesaosfe4qu0ivmaglelnitvseorfinF2C0-095mpLootfaMsisilulmi-sQaltw.ater. The amount of 8.5 Calibration spectrometer. 8.5.1 Extract the spiked beef liver `homogenate following 9.13 to 9.23 of this `method. Use these standards to establish your curve on the mass. 8.5.2 Altematively, a standard curve may be generated using ratiosofresponses ocfotncheentpreartfilounoorfootchteanpseurlfflounoartoeocatnainoensualnfdontaheteinatneironna,l standard anionversus 8.6 Storage Conditions for Standards 8.6.1 Ncoevwersetdapnldaasrtdiscacreentprriefpuageretdubweisthunetialchthaenaalnyasliyss.isSotanntdhaerdmsasasresspteocrterdoimneter is performed. 8.7 Storage Conditions for Standards 8.7.1 Beef liver homogenates may be frozen afterpreparation. 9.1 pOabctakiangefdrowzietnhloitvherersatmispslueess.. In spent tissue, note that the liver has not been 9.2 9.3 UWesieaghditshesescatminpglescdailrpeecltlaynidnctuatootfafraepdpprloaxstiimcagtreilnydi1nggotfublei.ver. 9.4 Record the liver `weight in the study note book. 9.5 iPnuittiaalsl.abel on the vial with the. study number, weight, rabbit ID, date and analyst 00466 4 99..76 Add 2.5mLs water. Grind the sample. Put the grinder probe in the sample and grind for about 2 9-8 R`miinnsuetetsh,eupnrtiolbetohfe fsianmtpolethies a homogeneous sample with 2.5 smoLlustiwoantewrituhsinnog large chunks. a pipet. 9.9 Takethegrinder apart and clean it with methanolafter each sample. AMDT-EP-22. Follow 99..1110 PCiappetth1me sLamipnltoeaan1d5mvoLrtecxenftorrif1u5gesetcuoben.dsL.abel the centrifuge tube with the identical 9.12. Sripenimfkoaerimntahitnigboensstatesplsti.hv)eergrhionmdoignegntautbee.s(wSieteh AthMDaTpp-rMop-r4iaWtoerakmsohuenett offorFdCo.c9u5mesnttainndgartdhe as described in 8.3. 99..1143 aAScpdeitdakte1e.tmheLosafmtphleessoadnidubmeecfarlbiovneart`ehoomuogennatbeesarwbitohna1i0e0ufLroefiintsemaalLstaanmdmarodn.ium 99..1156 CUsaipntghaensaanmapllyetiacanldpvioprett,exad2d0 5tom3L0 estehcyolndacse.tate. 99..1187 CPeuntttrhifeumgeinfotrhe2s0h1a0k.e2r5fmoirn2u0temsi,nu,nilthe layers are well separated. Set the power 9.19 Ronemthoe vce4entmrLifsuogeftthoe2t5.op organic layer to fresh 15mLcentrifuge tube with a mL graduated glass pipet. Transfer the label to the fresh tube. 9.20 Blow the sample down approximately 30 to 40 on the analytical minutes. evaporatotornear dryness withnitrogen, 9:21 Bring the remaining sampleupin 1mLdilute acetonitrile with an analytical pipet, 9.22 Vortex 15 seconds. 9.23 Transfer the sample to the sample into a fresh a 3 mL. syringe. Attach a 0.i2m nylon centrifuge tubeor a autovial. Label the mesh filter, tube or vial and with filter the 9-24 sCtaupdyannd yeholdafonrdaananliymsaislpbuy etleectrospray mass spectroscopy. 9.25 CompleteAMDTM-4worksheet and attachto page of study notebook. 1100..12 QPrueacliistiyonCoanntdroAlc-cunortacayp-plnioctabalpeplicable 10.3 Other Validation Parameters notapplicable 11.1 None 12.1 Worksheet AMDT-M-4 13.1 AMDT-EP-22 Routine Maintenance of Ultra-Turrax T-25 RevisioNnumber ReafosrChoangne. RevisionDac 00467 5 Worksheet AMDT-M-4 Sway 7 NSuammbpelre | awaplCp50r55opppxpmm || aacptpuraElo0x L3pppomm || aacpalpCr9$o5ppxop.mm | InBiutiefo3arnSldds. set hw w aw T r -- -- o -- TT u r --------r ---- F r r rar rr r e g r 1 rr p rrr t r r t t t ] r t t t Ft t r r t t t 11 r tt t fff [memberyh hl be oe peleame 00468 6 3E M nvir Labo oratn orymental Method Analysis of RaEblbeicttrLoisvperrayExMtarsasct SfpoercFtlruoosrcoocphyemicals using SOP Identification Number: AMDT-M- -_--Revision Number: 0 Adoption Date: (- 35 Revision Date: None Author: DaveChristenson/CynWtehbiear Approved By: roup Leader ds Z QualityAssurance A, Date C4 (7 Date Software: MS Word, 6.0 Affected Documents: M-4, Extraction ofFluorochemicfarlomsRabbitLivers 00469 1 L0SC1O.1PESS_lciodpse:fTorhifslumoertohcohedmiiscaflosr tuhseinagnatlhyesiesleocfterxotsrparcatysmoafsrsabsbpietctlrivoemreotrero.thTehretainsasluycssiosr 1.2 iSAstpappneldriafcroadrbmMlee/dZCbo=ym4sp2io7ng,ulneadnisdo:notmFholenurioatrpooprcrihonpegrmioiafctaFelCsm-a9osr5seoastn.hioenr,fMlu/oZri=na4t9e9d,ctohempiontuenrdnasl. 1.3 Matrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 20KE2.Y1 WOFlRuoDrocShemicals, fluorinated compounds, electrospray mass Spectroscopy, mass spectrometer, rabbit livers. 3.0 PR3.E1CAUUseTcIauOtiNonSwith the voltage cable for the probe. When the voltage cableis plugged 3.2 DinotontohterpurnobtheeDpOamNpOaTboTvOeUitC'sHcaTpaHcEitPyRofOB40E0,0tphseir.eIifsprriessksoufreelgeoctersicoavlesrh4oc0k0.0 psi stop and release pressure. The peak tubing may be plugged. Troubleshoot back to 3.3 Dfiondntohterpulnugthaenpdurmepptlaocedrtyhneepslsu.gged tubing. Sec AMDT-EP-15 4.0 SUPPLIES AND MATERIALS 4.1 S4.u1p.p1liNeistrogen gas regulated to 140 psi. 44.11..32 F1l0u0oufiLxocro2l5u0mnuoLrfelqatuitviaplseynrti.nge for sample injection. 4.2 R44..e22a..g12enDMtiisllultie-Qacweattoenirt,rialllewmaotbeirleuspehdasien,tdhiilsumteetahceotdonsithroiulled1b:1e wMiitlhliM-iQllwia-tQerw.ater. 50EQ5.U1IPVMGETrNioT2000 Electrospray Mass Spectrometer or equivalent. 55..23 ISpSeCcOtaSpyhryisnigcesPAuSm3p00 Autosampler 53 5.5 A2u0t0ovuiLalAssosrecmabplpyed centrifuge tubes. 6.0 INTERFERENCES se-- cre -- 6.1 There are no known interferences at this time. 2.0SA7.M1 PLKEeeHp AtheNeDxtrLacIteNd sGamples in capped 15 mL centrifuge tbesor in capped autovials until ready for analysis. 00a70 2. 8S .0 CT A8.L1IA BPRrAepTN arIatOiNoD nAoNf A DCalibrR ationD StandI ards ZATION 8.1.1 eSxetvreanctbioenefprliovceerdusrtea.nd(aSredes aAnMdDoTn-eMb-l4a,nksbeceteifolniv8e.r0)are prepared during the 8.2 Calibration 8.2.1 tRouonbttahienstehveensbteanedfalridvecrursvtea.ndards twice, starting with the lowest standard 8.2.2 Tinyptihceaslalymeonreansgteanodfacrodnicsenrturnatafitoenr easacthhe5staomp7lseasm.ples. Choose a standard 8.3 8S.t3o.r1agceForveCesorhnesddtiaptlniadsoatnridscscaferonctrrpiSrfetupgaaenrdteuadbrewdsistuhnelacthheanaanlaylsyissi.sSotnantdhearmdasassrespsetcotreodmeitner is performed. Samples and standards are NOT refrigerated. 8.4 8S.t4o.r1agBeeeCfonlidvietrihoonmsogfeonrateBseemfayLibveefrroHzeonmaofgteernparetpeasration. 9.1 I9n.i1t.ia1l SSetets-ouftpwareto "Operate on", Ion Mode ES". 99..11..32 FRiellcotrhde sboalcvkeinntgcyplreisnsduerrewiinththmeoibnislterpuhmaesnet.log. 9.1.4 cSeotmithnegpouumtpotfoth"eRtuinpo"f.tSheet ptrheobfel.oTwhteo p1r0e0s0suurLe/msihonu.lOdbbseeravte1s7d0r0optloet1s800 9.1.5 Cpshieck the fused silica at the chips. The tip should be flat weintdohfntohjeagpgroebde.edUgesse.aInfaenyye cpiheicpestaorechfeocukndfor tchuet aopfpfrtohperitiapteofletnhgetshi.lica with a column cutter and pull the silica through to 9.1.6 nCihtercogkeynoluerakniintgroagreonusnudptphley.tTpoufmthoenptrhoeben.itArogfeinn.e Tmhisetreshsohuoludlbdebceonmoing 9.1.7 out of the tip. Carefully guide the probe into the opening. Insert it until it won't go any 9.1.8 fGurothteor.thCeo"nEndeicttort"hepavgoel,taagnedcasebtleItoonitzhaetipornobMeo.de to ES', and the 9.1.9 1ap3p1rosprnioattienmSaisnsgleesoton4m37oadndgo4t9"9.Optionn"d se SIR. 9.1.10StthaerltoAgcqbuoioski.tion. Assign a file name, MO-DAY-YR + letter. Record it in 9-1. L1bReugnintnhienbgeoefltihveerruns..a.mplReunsafQiCrscrhuencnikngbyearcuhnnsitnagndoanred sitcanedaartdtahfeter every. 5107 samples. 9.2 9M.a2n.u1alDrIanwje1c5t0iounL of sample into a syringe. Inject the sample into the rheodyne 9.2.2 Tinujemcttihoenvpaolrtv.e Ito"njOecnt"s.lowly. Record the sample ID in the log book. 99..22..43 RWeaciotrtdwtohemisncuatnesn,uamnbderinfjoerctetahcehnseaxmtpslaempilnet.he logbook. -- 00471 2 9.3 9U.s3i.n1g StehteupAustaomsplaemptlraeyrA, B, or C. 9.3.2 vRieaclosrmdatyhebesaimnpelaecsharnudn.their positions in the instrument log book. Up to 17 9:3.3 9S.e3c.u3p.t1he sampPluesrh the sample button 9911331133132 SSeettsianmjpelcetslaoompplsei=2z=e 100ul 913.34 SetCycletime=0 991.33..33..65 INadmeetthneheftilwe:aiyLiufvseeyrds 99.133..3387 PArdedsst"heRusanm"ptloessttaonQueue by pressing "Enter" 100V10A.L1 IQDuaAlTitIy OCoNntrol 10.1. 1`Rpueank ahesitgahntdoacrcduervsesrtyop tthoe7rusna.mpOlnelsy. tIhfeassaimgpnilfeiscabnetfcorheantghee(ltas5t 0ac%c)epitnable. standard will be used. The remaining samples will be reanalyzed. 10.2 P1r0e.c2i.s1iSocneaMnedthAodccVualriadcatyion Report number AMDT-M-5.0.V1 1100..43 ReOftehrertoVMaeltihdaotdiVoanliPdaatriaomneRteeprosrt Number AMDT-M-5.0.V1 11.0DATAANALYSIS 1111..21 PClaoltcutlhaetisotnasndard curve, using the mean of the two values obtained for each 1st1a.n2d.ar1dR,ead peak heigorharteass for the samples from the printout. Use linear 11.2. 2CreaglrceuslsaitoentthoedmetgeorfmiFnCe-t9h5e asnaimopnl,eorcoontcheenrtrfaltuioornosc.hemical in the total rabbit liver: `mg FC-95 anion in the total rabbit liver = mggmFsC-of95liavneriounsefdrfoomrsatnda.lcysuirsve x Total massof liver, gms 1111..43 PMraikntea2crhersoumlasttoagbrleamanfdoreentaecrhist aimnptlhee,swtiutdhy tbhoeokp.eaks labeled with the sample or ssttaunddyafrodldIeDr.. WSrtiatpleetahlel scthurdoymnatuomgbrearmosnttohgeetphreirntaonudt,niunimtbiael,rdpaatgee,sa.nd put it in the 00472 a, 1A 2.0 TTACHMENTS None 13.0REFERENCES 13.1 AMDT-EP-17 140REVISIONS Revision Number Reasonforchange Revision Dae 00473 . 3M Environmental Laboratory Method Analysis of Fluoride Using Ton tSheelecStkiavlearElSeecgtmreondeted Flow Analyzer With Method Identification Number: AMDT-M-8 Adoption Date: /o-s-g Revision Number: 0 Revision Date: None Author: DebWr/i Cyng thiahWebter Approved By: up Leader F4e BAL Quality Assurance 1/5/75 Dae 1-27.45 Date Software: IBM MS Word, 6.0 Affected Documents: AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer 00474 1 lescope 00 1.1 This method is for the analysis for fluoride, thermally extracted from samples. using Tthoen DSeolherctmiavnenElDeXc2tr0o0d0e ((IASME)D.TT-hMe-1a)na,lyasnids cisolpleercfteodrmiendTuIsSiAngBtfhoerSaknaallayrsis with an Segmented Flow Analyzer with SE. 1.2 Samples can be tissues, serum, biological material,orother materials extracted on the Dohrmann. 20KEYWORD 00S 000 2.1 Skalar, segmented flow, fluoride. 3.0 PRECAUTIONS 3.1 Follow standard laboratory safety practices. ~~~ 4.0SUPPLIESANDMATERIALS ~~~ 4.1 Supplies 44..11..21 SAuatmoppliepectusp,so,x4fomrdLorpleaqsutiicvacluepnstwwiitthhcpalasstic tips 4.1.3 Polypropylene volumetric flasks, 100 mL. 4.1.4 Cartridge components, refer to the Skalar Methods for components and part 4.1.5 Snaummbpelresp.refilters, Evergreen 4.2 Reagents 4.2.1 Brij 35, 30% S.F.A.S. Detergent 4.2.2 TTIISSAABB IIII abudfdfe2r.5somluLtioorn:1P0u0rpchpamseflTuIorSiAdeBsIoIluftrioomn Oarnidon1.mTLo1Brliijt.er of 4.2.3 Sampler rinsing solution: Dilute TISAB II 1:1 with Milli-Q water. 4.2.4 `NmitLrsiccoancciednstorlautteidonnfitorricdeaccoidnt(aHmNinOa,t)ioton,2510NmL(lsabogfraMdiel)l:i-SQlwoawtleyr.adBdri6n4g the volume up to 1 L with Milli-Q water. 4.3 S4.t3a.n1daSrtdosck solution, 100 ppm F: purchased from Orion. 4.3.2 IwnittehrMmieldilait-eQ swtaatnedra.rUd,se10poplppmr:opDiylluetnee1v0olmuLmseotfrisctoflcakskss.olution to 100 mLs 4.3.3 Wvoolrukmiensgofstainndtaerrdm:edMiaatkeeourpstocthekfsotlalnodwairndg iwnodrikcaitnegd sotnantdhaertdasblbey, uasdidnig.ng the `oxford or pumpmate pipets, to 50 mLs of TISAB and diluting to 100 mLs with Milli-Q water. [00 op pm m TT-- o0i3s | --Omosmpepmn---- -- 1 ----1 ~---- 7 15--T|| [--ospem 03 | 0047B 5 ? 5.1 Skalar Segmented Flow Auto Analyzer SansTM System equipped with ISE 6.1 High concentrations of alkalinity, chloride, phosphate, sulfate or iron can cause interferences. `within 30 days. 8.1 Preparation of Calibration Standards 8.1.1 Prepare calibration standards as in section 4.3. 8.2 Calibration 8.2.1 The standards are analyzed at the beginnoifnthge run. 8.3 Storage Conditions for Standards 8.3.1 Standards are stored in capped polypropylene volumetric flasks. New standards are prepared at a minimum of `every six months, or as necessary. 9.1 9St.a1r.t1 UCplaPmrpodcoewdnurtehe pumpdecs,airbars and sampler-pump tubing. 9.1.2 Put the fluoride electrodes in the electrode chamber. 9.1.3 Tum on the powerofthe sampler, pumps, offset potentiometer andheating 9.1.4 Pbauttht.he reagent-lines in the appropriate botles. 9.1.5 Tum on the interface, computer, display and printer. Make sure you 9.1.6 Lteutrntheonsysttheem isntatbeirlfiazeceforbeafpoprreoxitmhaetecloym3p0utmeinru.tes. 9.2 9St.a2r.t1inCgreaatReuansample table by selecting FILES, TABLE, and CREATE, type in 9.2.2 Ptrhientnatmheseaomfptlheeftilaeb,lea,nidnsperretsesdEiNn TthEeRs.ystem table by pushing ESC, 9.2.3 PDiRaIlNtTh,e GsaRmpOlUerPse1.ttTihnigss twoiltlhepraipnptrotphreieantteirneurmubn.er of samples, number of 9.2.4 seconds for sample Fill the sample tray wash, and number with the standards, osfasmpelceosn,dswafsorhetsheasnadmpdrlief.is. IW and FW/RUNOUT cups on the sampler do not needt be filled. 9.2.5 Set the baseline. 00476 3 9.2.5. 1Select GRAPHICS, REAL TIME. If you cannot get real-time, you may be in the Data Handling Panel. Switch to the Analysis Panel by selecting 9.2.5.2Use the CONTROL PANEL and small screwdriver for the pushing F7. offset potentiometer to set the. base line. Adjust the baseline until it is approximately 3/4 inch from the bottom of the screen. 9.2.5. 3Check the highest standard and adjust the gain,if necessary, with the interface screw #3. Y 9.2.6 G`wioltl onoCtObeNTruRn.O(LSePleAcNtEoLr,deasnedltecotaannaallyyssiiss pbayneplr.eDsessinegleEcNtTtEheR.an)alPyrseissstThaatb to retum to the Analysis Panel. 9.2.7 Press the spacebar to bring up the local menu. 9.2.8 Select START to start the analysis. 9.2.9 Type your ID (initials), the sample table which you created under 9.2.1 (or press ENTER for choices), choose running with or without the system table and select START ANALYSIS. 9.2.10After starting the software, start the sampler. Make sure that the sampler is Fd the right number of samples and that the sample/wash/air times are 9.2.118elect GRAPHICS, REAL TIME to view the progress of the analysis. 9.3 Loading and Printing the Data-File 9.3.1 Go to CONTROL PANEL, press the spacebar to bring up the local menu ahingdhlsieglhectttLheOAfiDle.toSebleecptriAnUteTdOaCndApLrCesUsLEANTTEIRO)N. and enter the filename (or 9.3.2 To view the calibration curve, go to GRAPHICS, CALIBRATION CURVE. 9.3.3 To printthehigh level curve, push PRINT SCREEN. 9.3.4 To print the low level screen, push ESC to get out of graphics. Select SETTINGS. Change the maxy value to approximately 900. Go to CAL CURVE and press ESC, and Enver, Press PRINT SCREEN. 9.3.5 Returnto SET press ENTER TanIdN GPSRIaNnTd change the max value bac SCREEN to print sample k to 4095, peaks. go to EDIT, 9.3.6 To print the results go to CONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. Select PRINTER for the Epson or PRN for the Laser. 9.4 Shutdown 9.4.1 Putall the reagent-lines in Milli-Q water. 9.4.2 Let the system rinse for approximately 30 minutes. 9.4.3 After the system has rinsed completely, tumoffthe sampler, pump and offset potentiometer. Tum off the heating bath on weekends. Leave liquid in the lines. 9.4.4 Take the electrode out and soak in 100 ppm F ovemight. 9.4.5 Release thpe omp decks, air bars and sampler pump-tubing. 9.4.6 Select FILES, press ALT F and select QUIT to exit the program. 9.4.7 On Friday, turn off the computer, display and interface for the weekend. 1 VA ATION 10.1 Quality Control 10.1. Run a standard (mid to high concentration) every 10 samples. Ifa significantchange in peak height occurs, only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 00477 2 10.2 1P0r.e2c.is1iSoeneaMnedthoAdccVualriadcatyion Report number AMDT-M-8.0.V1 10.3 10.4 Other Refer to VMaeltihdoatdiVoanliPdaatriaomneRteeprorst Number AMDT-M-8.0.V1 11.1 1C1a.l1c.ul1aTthieonstsandard curve is plotted by the Skalar softwage. 11. 1.2bAeltlterc.alculationsare done by the Skalar software. r* should be 0.995 or 1111..32 PwWiretehpeaarltlehpesapscrtkeuaaddgysehndeueomtcbsuemtroeonsntusmtmahaenrdpirpzilneatcodeuattisan,.tiIhnneictislatulud,dedyastfaeomlptdhelere.pvrMoianltkuomeueta,, cawonepidygihotnfsdtuhtseoegdetehtce.r osnutmomsatruydyshdeiestkaannddtbaapcekiunptodtihsekss.tudy notebook. Back up all data and spreadsheets 11.4 E1l1e.c4t.ro1niGcLDPatsatudies: Electronic data study, and also data is copied iosnctoopaiefdloopnptyo dtihesStthuadty fslooprpeyddiinstkh forIbe.ach 11.4.20therstudies: Alldatais copied onto a floppy disk that is stored in the lab. None. I~ 30R1E3.F1EARMEDTN-CM-E1,SThermal Extractionof~ Fluorideby Means ofaModified Dohrmann 1133..32 SDkXa2la0r0M0etOrhgoadnsi,c#H3a3l5i,deSkAanlaalryMzeert-hLoidvserManual AMDT-EP-26, Operation and Maintenanceofthe Skaler Segmented Flow Analyzer Number Reasonfor change: Dae 600478 5 9.3 Quality Assurance Unit Statement 00479 - Attachment D GLP Study Quality Assurance Statement Study Tide: Single-dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits Study Number: AMDT-1220942 Name of Auditor: Kari Rambo T"ThheisfsitnudidnyghsawserbeeernepionrstpeedctteodtbhyestthuedQyudailrietcytAosrsaunrdanmcaenaUngietmeansti.ndicated in the following table. Pom Inspection DaTot.es Pe 10/1495 10/19/95 Final Report MDaatneaIgnespmeecntionSRmedpvoDrtiemdetloor 10/1995 101995 A 7~ Li QA Audiior 44D9ae5S 00450 .1 - 9.4 Key Personnel Involved in the Study 600481 . ' 3M Environmental Laboratory Key Personnel - `Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson RDiecbh WYroiugnhgtblom Deann Plummer Analysis of liver extracts using electrospray mass spectrometry: Jim Johnson Dave Christenson Thermal extraction followed by analysis using Skalar segmented flow analyzer with fon selective electrode: Jim Johnson Deb Wright - Rich Youngblom Deann Plummer Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo cocasz . 9.11 Data 00483 N; 9.11.1 Summary and raw data; ug F" in whole liver as Odertieornmiionnedanbaylytzheer.rmal extraction followed by analysis using 600484 . Summary of Combustion Data - Liver AMDT-122094.2, EWI 6329-138 As Referenced in Final Report section 6.0 DATA ANALYSIS `Total ug Fluoride in Whole Liver Mean per Dose Group Control Group 1u8.g0 0.128 mg/kg dose (T6054) 60.5 0.64 mg/kg dose (T6054) 118 1.28 mg/kg dose (T6054) 164 12.8 mg/kg dose (T6054) 2239 00485 RPT138XLS Fo129 PK Liver BliaDnk-1 pAcptmuFal- % revy iniiver (WW) 1.11 LiLivveerr BBllaannkk-32 00237513 LiLvievrerSSppikiek-e12 ~~ o95%% 01..80640 F5F2a725705-012 11573 FF5s22779530.13 0185349 FFs522779933..23 0098500 FF5s227r9022-12 00.22543 FeFs22775012.31 02.61487 FsFr5s27t5.123 223347 Fs752.1 233 FF5s22775522-32 223405 LLiivveerr BBllaannkk-21 00.237624 LUivveerrssppiikkee 6633-21 6976%% 01.09885 LUivveerrssppiikkee 112266-21 8925%% 21.3786 Liverspike 126-3 83% 231 ApvpemraFg-e inliver (WW) 159 0879 025 245 238 bLuimveerd (g0r.a14m5s) 00..113278 00..114618 00.112118 00..114310 00112235 00.11421 00..114088 00113333 0.143 00..114327 00..113049 00.113478 00112412 0.109 Wlhiovelre TotalFinwhole weight liver (grams) (ug) 739 118 688 605 82 180 ees 164 848 2239 Dosage (maka) oss 0.128 00 128 128 Paget 00486 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 00487 . HWiK6325-138 PrTsoowtaadoyMc:aotlerNautm:bers RMeasSrpqeuo:anrseedVFaaclleo:rAmount: AaDiuytaa!t: LMIAnesBttBrhuAm:SeEntF:re: TfSig5ileD0enoRsa5tIhni4vav(aFrCu-s1P29h)armacokinc a1L6to2re6s00 eools oFpuoroneiossVshG)2000GeckospMeSy A-l Contsins poe A-{ Hoos, p. 1-2. 95 Se ww es T[on=CounExvacied wi DlBuoloorn ~~] rane ome ime 1 [E ms ome ow 1 =~ amo ome aw 1 = om ots ums 1 [1o2r8omupos:n rome mas ues ' Couregen Touoltmaes omw sms orm wae ome nem ows enn sss oasesz FTCoSSRpTeremevSer sex oom oom oom 03s * atoof M499fonCounlhonCeouznt "{{TShahhicoetuccoolnrdacsbeeltn.htcerBaYeutmailoaunaltawhotaaftirstcnomhligancscnihasel1tc7ce1uadlmlagbtcy.iauoTsnehiidnneagccpootpnnhlcceyeeisnnntttgraranafttdioioaronrnmdsIucuihr0ev.he4eaIsanntnMdadenmatdurhadlorpddcluAcryuWvriDevnTeoyt-a4h-r%4r,et0sh-uheler1tce3boo7yrrmsre543c.tf rTrergsee4r5e twaeosnmunsr ceeyd.In 00488 A-2 HWI# 6329-138 Cone. 0.4 0.8 1.2 1.6 4 8 12 499 lonCount 427lonCount M499L.C/M427LC. 9091 74303 0.1224 63720 112333 0.5672 117051 146156 0.8009 183223 145956 1.2553 342495 135711 2.5237 654597 157098 4.1668 774077 128461 6.0258 Sampl#e 499lonCount 427lonCount M499 LC/M427IC. F52792 F52793 F52750 F52751 F52752 2399 12826 41969 41908 256267 97837 54664 84226 83555 94129 0.0245 0.2346 0.4983 0.5016 2.7225 00489 A-3 Sample Method C:DLCLIVE1 Run date 07-26-19O5p5era1t6o:r12:19 version: 35 StraiPgrhitnteLdineonFi0t7-2f6o-r1c9e9d5 tAhTrou1g6h:12Or:i3g2in. Component #:1 6.8258 5 a R E a 4.1668 2.537 + 1.2553 + .8889 + .5672 1224 41.2 4 81.6 8 LEVEL avouwT EXTAERRENCAAoLmpSoTneAnNtDARD1 =AMOUNT CALIB`RATION 21 0.0.84000000 1 Yel. s:3 4112.602000000000 3T 1 o he Bom 76 128..00000000 64 Y= SPE + X + INTERCEPT AmRousAnrqteuaared= ==16..991588.2426E1.2060E-0+1 + AArmeoaunt ++ 00..00000000EE+:0000 12 nf we o Olas oe 00490 ` * Tos kpnlas , ht me aa g(reeams) ' Filio:adcseos8a fe case LAB-BASE ~ Tho MSelDyattaas Symsetem 06/84/1995 00:14 2 a a _ oo. ES be] a r2e303408 Hig RieadBEE | Le sen 0 reULL en | e Zeee soee ages sae | e 9B [lag (rena \ FFirreemein roo) HE Li .7 le ol wm LOB-ASE - The HsS ipata System ww wa ws 86/04/1995 00:14 .3 we dE 2 a |\ am g= Fur CITTER SH aij of of 3 SeEenleivaLnse. : 1 0 ' 1 } oi33 Sanciotiors iimLAB-BASE = The MS tee 86/84/1995 08:14 ee "ip HE appre gray 2 ors. gage gos 3 -. T wth aT afl Eomml a? n i258 X - Jyo} ibzokdie ti Log | site a Fogang pa AA lsof Bo Tage eget gag vm :~ f " LOO C333 re AEEi 1 bs vn : sna 0 00 gre EE ag a WR 4s 2H 2 hrs] WB E I\ \ ma e Lun EN \ 2h ILS 3 go ae FF = &Eam E \ sap , A \ Lame < i Filo:04ac05n LOB-BASE - The MS Data System Samplaty " mi rol [5 De oop HEHE age Ee rs. wpe Eb 493g 433 o 100 = 833 am Eo ob LN sen PT 53A90 5306 \ coos \ + cree 06/04/1995 80:14 us a Ju g2 3 ae lu @ @ | 00| 9.11.3 Summary and raw data; ug F" in whole liveras determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selectiveelectrode. This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated in dFeitnaaill.Report Section 6.0, and therefore is not discussed in 00496 * ~ RE: 6329-138 LIVER SAMPLES AMDT 122894.2 Dateof Analysis: 4-28-95 Analyst: DOW [-- The samples are burned in the Dohrman at 958 C using between 8.1 and 8.2 grams of the liver. The gas is collected in 1.8 mL of 1:1 TISAB/Milli-Q water then an additional 1 mL of 1:1 TISAB/Milli-Q is added to allow for sufficient volume for Skalar analysis. The samples are then analyzed on a Skalar Segmented Flow Analyzer using the lon Specific Electrode (SE)Method. TISAB buffer is added to each sample as it proceeds through the system. The sample then goes through a heated mixing coil before the potential between the ion selective electrode and the reference electrode is measured. The signal is amplified and related to the fluoride concentration. The instrument was calibrated in the ranges of 8.815 - 8.15 ppm and 8.15 - 1.50 ppm fluoride. The standard curve for the high range was plotted using the inverse logarithm option. The standard curve for the low range is linear. All standards and samples were then calculated by the Skalar software using these curves. All results below 8.8601 ppm appear on the raw data as #.####. A quality control standard was analyzed every 18 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection volume and any subsequent dilutions. Ques 00497 S`ULReoMrrMs1Oa2Fu2A0gp0sL4Rze5s1Y38 tGaotwe mEBimonoSBm aoBwo nuooww aWn o mPEY 2oE [I T ows c o mnieeBkzoO omawoeBR J a anoSI SN 2&i 8 [ocwso TO m-moB oonuR 10 on uuImemm JO -- .. ox 8 eowe s mFomaa G ox 3muomm 1a8m @gmmom biows m mGoRmA WoSw ENzono wuLemm aosme J mFrom i nemo wr 3 O1e9r5u0o7r061557 <0DW Ou of 9302081 DA aasFs lAenNsaymes:1C9/0S4K20A0L7A1R2DATAVWIDATALI50V4E20RAS1 TauvER ALS :Lombew 5 Na 1i0n 1wo0n owam 3 sswwddtz o0nms o0nms omwm 7 SSmmaadds 006 oomm ldoow s5o sswwawadms o01ns o0n5 omwm BEST COPY AVAILABLE oSi10 Sswwaanns 000 0oam woem SBib sswwaamn a1m0 iLne wowm SEui 6 TSomm mk ib da ow aio 20 ows 1m wvoo sommxaa [I] aa0 ow 2200 2% aanued own o1mx im ooeo SGoo ooss oonm ouwm n noaw zw oaonnn 0200 0 mo6s ann 1i5i5s In hhoossee wDeeeas nose Dim c5u oomma 50m o0m0s 0% 2B000 0oDs ormo wawmn oasks 0 om 0 ssw on 2n%o Dwina 0 wwe ww = om1a [w3 020 0omumw o0n mmiimm um ot IEe weny3 EoIma aw 00 oowm sWmw wmwiws m0m 385583 [i Foot : az RP sens 5 Nnoom mma ooonna 0B Io s oohmm aasmn wswmmma wmveiwem 5 BFR am ma oo0nw IBoo oooounmn bbonoa wmmaa lweann N5oooobkNe im we om 0 wm ou &bboo sSnmo ooann 4a on B BBo oom oumn oien Bo ow in oGooumw fBoammm mi mi PFaA Ba" me otneow FE Gm me ooonsw ooomnn uuwnmm oonn oamn oomm gg S$ BESTCOPY AVAILABLE ENco Poge2 1995-04-20 09:36 output of : 950420A1 Software : version 6.1 c1990,93 operator + bow Date of the Analysis : 1995-04-20 07:12 Analysis File Name C:\SKALAR\DATA\HWIDATA\LIVERS\$50420AL QAOMDTTi1l2e2s094.2 HoT 829-138 des mpi FClauloirbirdaetio1.n5order = Inverse Logarithm stope is = pansn Result = 100 [x=-5e=t] xci == ccoorrrreecctteedd vvaalluuee ooff tthhee scaomnpcleentration 1 5 = Slope of the electrode aazi == -00..0000000605 20 = -124984 FClauloirbirdaetioLn order = 2 Correlation : r = 0.99991 Res=ua2l*xt? + al * x + a0 2a21=- 00.0000002000 20 = o0loooto sampler Nyupmeber +ii5A1000 WSaasmhple TTiimmee :: 15200 sseecc.. MTarke upTime :1Singlseec. nspeeecdilael Height :: N7o0nemm. Diluter dneieldulteionHoiFgahcttor :: 8100 mm Redsilaumtpiloen Volume i: i2.5 nl. Dilution runs: 1 uRseeprrocfeisle :: No Lr = 006501 1995-04-20 09:36 Output of : Fluoride 1.5 sPiagtnhalnumtbyepre :: De3bubbled DseycsotleomrNumber :: 0Yes Rdeislaumtpele `i NNoo idiGl Touhtrpeusthold :: 04095 Window event : Off 5s21 ssTTaannddaarrdd :: IIggnnoorree s543 ssTTaannddaarrdd :: IIggnnoorree ss65 ssTTaannddaarrdd :: Igno0r.e150 5s87 ssTTaannddaarrdd :: 00..360000 55910 ssTTaannddaradr:d: 11..250000 DOirmdeenrsi:onInv: erPsReM Logarithm tstrairgtgerVaLliumeit :: 1580000 SDeUc PSetaAkrtshiagpneore :: 6P0oin=teSdec MeeNadsureigwnionredow :: 71520 S% ec RFielgteenreration :: NNoo ofourtmuplaut:: #4.404 Fluoride L PSaitgnhalnumtybpeer :: De0bubbled DseycsotleomrNumber :i 0No RdeisLaumtpele i: NNoo idiGl oTuhtrpeusthold :: 04095 Window event : Off 950420A1 _ 00502 . ~ 1995-04-20 09:36 OutPut of : 950420A1 s512 sstTaannddaarrdd :: 00..001350 ss3 ssTTaannddaarrdd :: 00..006900 ss56 ssTTaannddaarrdd :: 00..112500 s578 ssTTaannddaarrdd :: IIggnnoorree 55910 ssTTaannddaarrdd :: IIggnnoorree oDirmdeenrsi:on2 : PPM sttrairgtgerValLuiemit :: 1508000 DU Sec PSotaAkrtsihgapneore :: P60ointeSedc MeeNadsureigwnionrdeow :: 17250 Sec FRielgteenreration :: NNoo ofourtmuplaut:: c8.:4z8c838 . a 00503 - 1995-04-20 09:36 Output of : 950420A1 Fluoride 1.5 Pluoride L eM 12 Pos Typ Ident Ch Result F Time Ch Result PF Time 1 We iw InitialTraWcaesrh 33 10..405576 21605 4400..70904021 00 32 aow DWraifsth 33 01..045760 632864 4400..80000061 00 54 ss21 SSttaannddaarr21dd 33 00..007603 S70373 4400..00310427 00 76 s5d3 SSttaannddaarr43dd 33 00..100867 11206815 4400..00360043 00 98 5s65 SSttaannddaradr6d5 33 00..115258 11641316 440.0.11510950 00 1110 ss87 SSttaannddaarr87dd 33 00..621842 11975895 440.0.42462092 00 1132 5s910 stSatnadnarddar19d0 33 11..426372 22311315 440.0.67996858 00 514 oaw DWraisfht 33 01..045863 22742875 44 00..08000616 0 0 167 uu BBLLKK--12 33 00..017112 32081316 4400..00392866 0 0 189 uu SSPPRK--12 33 00.110035 33316837 4400..00888597 0 0 2210 uu 5500--21 33 00/.112224 33751348 44 00..11111456 0 0 2232 uu 9530--13 33 00..019233 43086806 44 00..01710320 o0 2254 unu 9933--32 33 00.008888 44421348 44 00..00662213 00 2276 wa DWraisfht 33 01..045667 44754808 4400..07090818 00 2298 uu 9922--21 33 00..006635 54131316 44 00..00119413 00 3310 ouu 5912--13 33 001.106631 55426835 44 00..10518167 0 0 3332 uu 5511--32 33 00..117742 55861337 44 00..11760856 0 0 3354 au 5522--21 33 00..745272 65196839 44 00..35406583 00 3376 ouu BL5K2--13 33 00..077245 66531359 440.0.04396188 0 o 3398 wa DWriafsth 33 01..055067 66963809 4400..08010813 00 401 ouu SPK-B6L3K--12 33 00..100667 77201344 4400..00283988 00 4432 onuu SPSKPK-16236--12 33 00.115016 77336960 440.0.10495062 00 4454 uu SSPPKK 112266--32 33 00..114433 77971440 4400..11337715 00 4476 oaw DWraisfht 33 01..045965 88302340 4400..8010203% 00 Wt rw RunOut Wash 3 0.056 8365 40.0001 Page 1 of 1 00504 cottration curve of sos- price BESTCOPY AVAILABLE | I wel [bo| | | 1 | | A A / In i 1| | i1 i :ii | C| S I -- * rz J / tests 7/// IIll l|Il| I |ll || von TM| 00505 -- ! !| i| u| | 5 CdCo i IY | | REST COPY AVAILABLE =; 71i J ( I | So 1 / ll | / i | Jose | L{io iwOOre rddeerr:rInves rse Le ogaritl hn os 00s 0 or ss::eiw62s05te| 00506 PC-- i11 WH1nl! Fil J A a V HI s} H oAL H L eT ---- = oe, TOMAR ar CL i ol TLL al r 1 I | I | in I WI ony co lo Lom [i [ ml | J | qllo TTI | Daas | f TL fe | j /{ lenna] WU | Al 000509 0 oP a AIRE au data of S504 Fors I ll i | |PoTdl ol F| ol| |4 iI fl Hi | iI Lol mil | | iH || 34] IH || / [ / N 1 1 us | I] | Io $5 d]1 ] ) Ll H [+] I | Pi--------------------| oe00510 B