Document 93X6JrrayKVR52JL0RvRVM7LR

FYS, I -0500- 1378 ---- pans-ous3fort'd) ATtotxaiccohlmoegnytsSttuodLieetstearndtoOCt.heArueInrfdoartmeadtiMoanyon25P,F20O0A0 (85000000/ 01 4) 1) 6 Final RepionrRta,bbAintasl,yt3icMalReSft.udNyo,.SLin1g3l4e9-2Do(s4e5I%ntpreaarlvfcelonuhooourlso)o,PcShtatarunmadaiyccNoakociAindMetDicTS-tudy of ooeudtyl1a.mNmoovenmibSuetmruds2ya8l,otfi1Tn9-9w65a;0t6err7epaionnrdtRaiibnsbcoilptursdo,epsyHlaFzienalltoRnepWoirstc,oSnisnignl,en-Dco,seSttuade yNo.h i HWn632e9-15t1, February 1, 1995 2) F5i0n6a8l,Raenpdor1t-,6A0na6lyitnicRaalbbSittusd,y3, MSiEnngvlier-DoonsmeeAnbtsalorTpetcihonnoFlFaooglxyiRc&elpyoSreSrttv,uiSdciyenosg,flSeTt-Bu6do0ys6eN7oT.- Abortion1T0Io9nx5ci..c1,iSttNyuodSvyteuNmdoby.eoHrf2WT8-,66013692799,5-;T1r-5e62p0,o6rM8t,aiyannc2ld6u,Tde-1s690965;9 in Rabbits, Hazelton Wisconsin, 16067 is 13492, 45% perfiuorooctanoate terabutyl ammonium salt in wale? and isopropyl alcohol; 1tt6e0ab6u8tyils afambmrointireuamtesdalwtitinhwhaitgehrlaevnedliosfopLr1o3p4y9l2a,lc4o5ho%l:perfluorooctanoate 1te-t6ra0b6utyils faambmriocntireuamtesdalwtitinhwlaotwerleavneldoifsoLp1r3o4p9yl2,al4c5oh%olperfluorooctanosie 3) ADME studies with FC-143-C 2) FLianbaolraRteoproirets,, AInbcs.o,rJpatniuoanryof1F4,C-1194830-4C in Rats After a Single Oral Dose, Riker ) CFihneamliRceaprosrtD,ivSiysnitohnesainsdaRnidkeCrhaLraabcotreartiozraiteiso,nIonf,FCJ-a1n4u3a-r'y*C23,,C1o9m8m0ercial ) CFianrabloRne-p1o4rti,n EMxatleentanadndFeRomuatleeoRfaEtxscArfetteiroan SainndglTeisIsVueDoDissetroifbuFtCi-o1on43f-1T%oCt,alRaiker Laboratories, Inc., February 7, 1980 4 "FAifntaelraReSpionrgtl,eEOxrtaelntDaonsdeRoofuAtmemoofnExicurmeti'oCn-oPferTfoltuaolroCoacrtbaonroa-t1e4,iRiFkreergnantRats Laboratories, Inc, July 1, 1983 ) FCihnaalleRsetpyorratm,inEenhTarenactemdenEtl,imRiinkaetrioLnaboofraFtCo-r9i5e-s,1%nCca.ndMaFrCc-h14134-,'/1C95i0n Rats with 4 CFionmalinRegpoHratz,elStionnglIen-cD,oSsteuIdnytNroa.venCoHusWPh6a3r2m9a-c1o8k6i,neAtpiriclSt18u,dy1o9f97T-6564 in Rabbits, 5) FCionmalinRegpHoarzte,l5t-oDna,ilIyncD.oSsteudDyerNmo.alCAHbsWor6p3t2i9o-n1T8o5x,icAiptryilSt28u,dyo19f97T-6564 in Rabbits, 6 c0z55 Su ~ 3M Environmental Laboratory Final Report- Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Tn-Vivo Study Reference Number: HWI#6329-151 TSetsutdySuNbusmtabnecre:: ALM-D13T4-91220(6T-9640.617) Name and AddressofSponsor: 336M7 SGrCoDveDiSvtirseieotn St. Paul, MN 55106 Name and Address of Testing Facility: 3M 935 Environmental Bush Avenue Technology & Services St. Paul, MN 55106 Method Numbers AMDT-M-1-0, and Revisions: Thermal Extraction of Fluoride by Meansof a Modified AMDT-M-2-0, FDlouhorrimdaenMneDasXu2r0e0m0enOtrgbayniMceaHanlsiodfe Analyzer-Liver an Orion EA940 Expandable AMDT-M-4-0, Ton Analyzer Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, EAlneacltyrsoissproafyRMababsist SLpievcetrroExmtertarcyt for Fluorochemicals Using AMDT-M-8-0, Analysis with Ion SoefleFcltuiovreidEeleUcstirnogdethe Skalar Segmented Flow Analyzer AMDT-M-14-0, DTohehrrmmaalnnExtDrXa2ct0i0o0n oOfrgFalnuiorciHdaelbiydeMAenaanlsyzoefra-SMeorduimfied Initiation Date: See attached protocol Author: James D. Johnson Approved By: "JSatumdeys DDi.gggforJ6hhson 25/55 Completion Date | iF gp "leks - 3D 1 9253' 8 LJ Tissue and serum samples were analyzed for total organic fluorine aftersingle dientrrmaavlenaobussordpotsieosn tsotuidnyd.ividual rabbits in order to collect information in support ofa ahSaerlrfa-pulimidfesdaeimscproleneasstehweienorrsedeearrnuaomlyfl4zeevhedoluarotsf2.,to4t,al6,or8g,a1n2i,c24f,luaornidne48wihtohurtsimpeo.stTdhoeseb,iolTohgeircaelis iTnhterraeveisnovuesrydolsiettloeftoLt-al13o4r9g2an(iTc6f0l6u7o)ri.neTphreespehnatrimnacliovkeirnoertiscerstuumdayts4u8ggheosutrsstphoatst interpretationof dermal absorption of L-13492 (to be done and reported in a f4sle8upoahrroiauntreeswsahtnaudtdyet)vheewrillitlvheebreesxmatomeprnlteeodsfifadftiec2ru8mltad.layaItsbsiwsoirulplntliciooknne.tlayitnhaatnythdeetseecrtuambleleovrelgsanaitc This study was designed to provide information as to whether L-13492 when wadhmoilneisltieverredatas48a hsoinugrlsepionsttradvoesneouosr dinosseerreusmulatts 2i,n 4t,ot8a,l o12r,ga2n4i,cofrlu4o8rihdoeurisn,creTahsiesin information is to be acquired in order to interpret a subsequent dermal absorption asbtsuodrypwtiiotnh stthuedsyawmiellcboempreopuonrtded(HsWepIa#r6at3e2l9y-.152, AMDT-011 095.1). The dermal 3.1 Test, Control, and Reference Substances and Matrices 3.1.1 Analytical Reference Substance: equivalent. FC-95, lot 161 or 171. They are 3.1.2 33..11.43 Analytical AAnnaallyyttiiccaall Reference Matrix: Bovine liver and bovine serum CCoonnttrrooll MSautbrsitxa:ncBeo:viNnoeneliver and bovine serum g3.r2ocSeoruyrscteoreo,fbMoavtienreiaslesr:u3mMfrIoCmPS/iPgCmPaDCihveismiiocnalfoCroFmCp-9a5n,ybovine liver from 3.3. Purity and Strength of Reference Substance: Responsibility of Sponsor. 3.4 Stabilityof Reference Substance: To be determined by Sponsor. 0253% . Lo ST 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples the storage is -20+10 C. b3.e6rDeitsaipnoesditpieornGoLfPTeRsetgualnadtiCoonntforrolthSeutbismteanpceersio:d Brieoqluoigriecdalfotrisstsuuedsieasnldonfglueirdsthwainll 28 be days. This retained. study is in parallel with a 28 day absorption study, so all tissues will 4.0 EXPERIMENTAL - Overview Serum and tissues from animals for analysis by combustion with sduobsseedqausendtesacnrailbyesdis(bHyWIs#el6e3c2ti9v-e1i5o1n)ewleecrteroadveaiolrabblye electrospray total organic mass spectrometry. fluorine to collect The tissue information and serum in order to samples support were analyzed for a dermal absorption sstaumdpyl.esTahreiseextfernotmoaf parneavliysoiuss wsitluldybe(HatWIth#e63di2s9c-r1et5i1o)noifntwhheicShtusdiyngDlierercatbobirt.s Twheree dosed intravenously protocol for dosing, with L-13492 at doses tissue collection, etc.) ranging 4 to 24 mg/ke. In the final report from (See attached HW on this s1t6u0dymgt/hkegd.osAinrgabibsiftrdoomse4dtoat2440mmgg/k/gk.g In the died 5 original minutes protocol, the dosing is to after injection. No tissues were saved. 50EXPERIMENTAL-METHODS ~~~ 5.1 AMDT-M-1-0, Dohrmann DX2000 Thermal Organic Extraction of Fluoride Halide Analyzer-Liver by Means oaf Modified E5.x2paAnMdDaTbl-eM-Io2n-0An,alFylzueorride Measurement by Means of an Orion EA940 5.3 AMDT-M-4-0, ExtractionofFluorochemicals from Rabbit Liver E5.l4ecAtrMoDsTpr-aMy-M5a-s0s,SpAneacltyrsoimsetorfyRabbit Liver Extract for Fluorochemicals Using w5.i5thAIMoDnTS-eMle-c8t-iv0e,ElAencatlryosdies of Fluoride Using the Skalar Segmented Flow Analyzer D5.o6hArMmaDnTn-MD-X1240-000,OTrhgeanrimcalHaElxitdreacAtnioanlyozfeFrl-uSoerriudem by Meansof a Modified cezs34 0) WN 60DATAANALYSIS ~~~ 0000000 `dTohsee.datTahearteotaatltaocrhgeadnifcorftloutoarlinoergfaonriwchfollueorliinveeranfaolryscionstorfolliavneirmaatls4,8 4homugr/skgpo,st 16 66 mg/kg, ug, and and 24 54 ug. mEga/ckhgdaitnatrpaovienntoursepdroesseesntos ofane45an%imsaoll.utAionn are 20 animal ug, 43 dosed ug, with 40 mg/kg died 5 minutes after injection. T12h,e2d4a,taanadre48athtoaucrhesdpofosrt serum dose. cWohnactenitsraitmimoendsioaftteoltyalapopragraennitcifsltuhoartinteheat 2, 4, 6, 8, cbioonlcoegnitcraaltihoanlfs-ldiefceraepapseearraspitdolbyewaibtohutti4mehaonudrsa(reseneopna-dgeetsec8t4a-b8l6)e.at 48 hours. The fItemapapleearrasbbliitkse.lyItthiastntohtisksnalotwonfipfertfhleureoriosoactsaenxoadtieffiesrernacpeidilny tehleimeilniamtiendatfiornoomfthe perfluorooctanoate in rabbits as is observed with rats. Other data segmented was flow collected analyzer wuistihngioenlescetlreocstpirvaeyemleacstsrosdpeec(stereomaeptpreyndaincdesS)k.alTahris data, acolntchlouusgihosnupstpaotretdivhee,reinatnhdetohpeirnefioorneoifs the not SdtisucduysDsierdeicntodretaisiln.ot required to reach the 6.1 this aCniarlycsuimssitsatnhcaetsthtehraetiMs anoyt Affect a good tbihoeloQguiaclailtmyaorfketrheanDdattah:us The the problem with pdhearrmmaalcaobksionrepttiiconstsutduydydoes not provide a good avenue for the interpretation of a 1.0 CONCLUSION `dTohseereofisLv-e1r3y49li2t.tle Tthotealphoarrgmaaniccokfilnueotriincesptruedsyenstugigneslitvserthaatt4i8ntheoruprrestaptoisotn ionftrdaevremnaolus adibfsfoircpultti.on of L-13492 (to be done and reported in a separate study) will be very 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8a.p1proRpraiwatDeastpaecainmdenDs,ataan:d Raw data, electronic daaptparwoivleldbperomtaoicnotla,inaepdprionvtehde final report, AMDT archives. 0253 . Cr 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-151 "Single-Dose Intravenous dPohsairnmgacoofkainniemtailcs,SttuisdsyueofcoTl-le6c0t6io7n,inetRca.)bbits" (Protocol type TP8084.PK for 9.1.2 Analytical protocol AMDT-120694.1 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 9.4 Key Personnel Involved in the Study: See attached 9.5 Materials and Equipment: See methods 9.6 Solutions, Reagents, and Standards: See methods 9.7 Sample Preparation: See methods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-120694.1 9.10 Instrument Settings: See methods 9.11 Data: See attached. 9.11.1 Summary and raw data; extraction followed by analysis ug F using in whole liver as determined Orion ion analyzer. by thermal e9x.t1r1a.c2tiSounmfmoalrloyweadndbyraawnadlaytsai;s pupsimngFOirniosneriuonm aansaldyezteerr.mined by thermal 9.11.3 Summary and raw data; ppm F" in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 9.11.4 = Summary and raw data; analysis of liver extracts using electrospray -_ 02536 9".S1i.n1glPer-oDtoocsoelIanntrdaFviennaolusRePphoarrtm:aHcoWkIi#n6e3t2ic9-S1t5u1dy of Tdo-s6i0n6g7ofinanRiambablists,"ti(sPsruoetcooclollecttyipoen,TePtSc.0)S4.PK for 02538 Woveny  FWISSCTONaSnIgN comme. Sponsor: St. pau,3MMinnesota FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Author: Steven M. Glaza SCtuodympletiDoatne: February 1, 1995 Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 aboratory Project Identification: HI 6329-151 shone 80s 241.447 Page 1 of 28 02538 Fax sop 241r22s Page 2 of 28 QUALITY ASSURANCE STATEMENT HUI 6329-151 This report has been reviewed by the Quality Wisconsin, Inc., in accordance with the Food Assurance Unit of Hazleton and Drug Administration (FDA) Good Laboratory Practice The following inspections were Regulations, 21 CFR 58.35 (b) (6) (7). conducted and findings reported to the Study Director and management. Written status Standard and findings are issued to Hazleton management reports monthly of inspections according to operating procedures. nate Inspection Dates From To Phase Reported to Study Director Date to Management 11/27/94 12/09/94 12/28/94 01/17/95 01/30/95 11/27/94 12/09/94 12/28/94 01/19/95 01/30/95 Protocol Review Sample Collection Protoco] Amendment Data/Report Review Report Rereview 11/27/94 12/09/94 12/28/94 01/19/95 01/30/95 12/10/94 01/10/95 01/10/95 02/10/95 02/10/95 i172 M. Danner Representative, Quality Assurance Unit 3), Jos Date coz539 Page 3 of 28 STUDY IDENTIFICATION HWI 6329-151 Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Test Material T6067 Sponsor Sponsor's Representative El Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Study Director SHatzelveetnonM. WiGslcaoznasin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location 3802 Packers Avenue Hazleton Wisconsin, Inc. Building No. 3 Madison, WI 53704 Study Timetable Experimental Start Date Experimental Termination Date December 9, 1994 December 11, 1994 2540 i. Acute Toxicology Steven M. Glaza Study Director Manager SFtruadnycisCoo(rBdudi)natW.orMcDonald Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Quality Assurance MaShneargreyrR. W. Petsel Page 4 of 28 KEY PERSONNEL HWI 6329-151 Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Patholoay Thomas E. Palmer, PhD Anatomical Pathologist Supervisors Jack Serfort/ Deborah L. Pirkel Necropsy Anne Mosher SPautpheorlvoigsyorData co=542 Page 5 of 28 contents Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Pathology Report Table 1 Individual Body Weights (g) 2 Individual Clinical Signs Appendix A Protocol Deviation Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-151 Page 2 3 4 6 7 7 7 8 9 11 11 1n 1 12 13 14 15 16 17 27 CORS4R, Page 6 of 28 HAI 6329-151 SUMHARY aThdimsinissttuedryedwasbydoinnetratvoenaosussessinjtheectiloenveltoofrabsbyisttse.mic exposure of T-6067 when tF(heoemnae/lvgeerhHoirucapl:)e.((NsZOtWne)rSiDPlaFey rw0a,abtbeittrhse fwoaernriemianljasescstirigeoncnee)diveoadrt arasnidnognletoifnitvreavgernoouusps injection of 4, 16, 24, or 40 mg of T-6067/kg of body weight at a dose volume of 0.5 mL/kg. C4b8leifnohiorcuearlstesaotfbtsmeearrtveairtniitaorlnasveanwdoenuriseniscitonrnjaedtcuitciotonend.(DaatyBoa0dp)yp.rwoexAiigmhbatltsoeoldwyers0e.a5m,pdleet2,er(m4ai,pnper2d4o,xjiumasanttdely 4sumrlv)iviwnags addition, acnoilmlaelcstedat f2r-o,n at the time of 4ae-nx,pear6u-ir,miecn8ut-la,alr12ta-er,rtmeir2ny4a-thoiorounrmsar(gp4io8ns-ahtlo-uirnesjaercptoviseotin-n.inojfeIcntthieon), ssaeapnmptprloetxsoimwatethreeelySpcoe2n0nstornriL,fuogfe"Tdbh,elooosndeepwaaarsnaitmeoadbltaii(nn4t0eodnsgef/rrkuogmm)aenfadochuncdesludlreuvalidavrindgufrriaancngtiimoatlnh.se, sAtalunlddy bwtlahesed4nve8ic-arhooputsrhfeepdpoosstbt-ueitnrjiieoscrstuiveoesnnapweecrravieao,dnotwanedrseaevexadsn.aensgtuhAienntiaimtzaeeldds. wsiuAtrnhvivasibonbdgrieuvmtioaptetenhdetobgearnrodbsisotfal, cnToielvcelrreo,cptsebyidleexa,andmiannsdaetntiboontfhrowazkseindnnetoytosdtohfnereo,nSpeohanocswhoervearna,ifmtaetlristsseuuerrsmviinvwaietnrigeontcooolflteectrthmeeidn.aitni-o1Tnihefewewrheole phase. Tsthuedya.nimaTlhse dosing. At tannericenraaotlpesdyt,raetatth0,eed 4l,uantg1s6t,heina4n0dthim2sg4/kmagngi/mkdagolseawpelpreeevaerleddifdfineuodsrmealwlyitdhtiahnrrkou5grmehdio.nuuttetTshhiesof change was attributed to acute death and postmortem change. 02548 sir Page 7 of 28 HN 6329-151 OBJECTIVE to rabbits. The objective of this study was the test material, T-6067, when to assess the level of systemic exposure to administered as a single intravenous injection REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Studies, 21 Laboratory Practice Regulations CFR 58, with the exception that for Nonclinical analysis of the test mixtures for concentration, conducted. All procedures homogeneity/solubility, used in this study were and stability was in compliance with not the Adniirmeac]torW,elftahreesAtcutdy Rdeigdulantoitonusn.neceIsnsatrhielyopdiunpilonicaotfe tahneySpproenvsioorusandworskt.udy Identification TEST AND CONTROL MATERIALS The test liquid. material was identified The control material was as T-6067 Sterile and Water described as a for Injection, clear, colorless USP (Abbott Laboratories, Lot No. 86-748-DM-02; a clear, colorless liquid. Exp. March 1, 1996), and was described as Purity and Stability The Sponsor assumes responsibility for test material determinations (including under test conditions). A purity sample and stability of the test msattaebriilailt/yveahnailcylseesmiwxatsurensot fotrakceonncbeenftorraetiaodnm,inissotlruabitliiotny,as hotmhiosgenweaistyn,ot and requested by the Sponsor. The material were considered to be purity and stability of the USP grade control adequate for the purposes of this study. Storage and Retention The test material was stored refrigerated. stored at room temperature. Any unused test material was The control returned to material was the Sponsor after completion of all testing according to Hazleton Wisconsin (HNI) Standard Operating Procedure testing and will not (SOP). Any remaining vehicle be discarded after issuance may be of the used for other final report. cozsig Page 8 of 28 HUI 6329-151 SafetyPrecautions Tahned tpoelsitciaensd. control material handling procedures were according to HHI SOPs TEST SYSTEM Test Animal AKWadilusalcmtoanzsaoilonb,infMoaiccirhlaiibgtbayintsaotno3f8N0ot2vheemPbaHecrkrae:r(1s6N,ZAWv)1eS9nP9uF4e,asntMdraadmiinasionwnte,arienWiersdeccoeantisvietndh.e fHraozmleHtRoP,n Inc., Housing DAufrtienrgraeccecilpitm,atitohne aanndimatlhsrouwgehroeutaccthleimasttueddy,forthea housed in screen-bottom stainless steel cages in apneirmiaolds owferaet temperature- ilenadsitvid7uadlalyys and hunidity- m1ca2oi-nnhttorauoirlnle1dia gqthuetam/rp1te2er-rahsto.uurre dEaonrfvkir1ol9nimgehtntotian2lg3Ccc,yocnltaer.orleslaIntfiovcreastehhseumwiahndeiirmtaeyl vofarroi5oam0t%iwoe+n2rs0e%,fsertaonmdttohae croenqsuiidreerdedtemtpoerhaatveurehadandnohuamdivdeirtsye cefofnedcittioonnstehexisstteudd,y touhtecyomwee.re Adnoicmuamlented and hfuosrbatnhderyCaraendanhdousUisengofatLHaWbIoractoomrpylieAdnimwailtsh".standards outlined in the "Guide Animal Diet TLhaeboraantiomraylsRawbebriet pDrioevtideHdF access #5326, PtHoI waFeteedrs,ad In1ci.bitTuhmeanfdeeda miesasruoruetdinealmyount of caonnatlaymziendanbtys.theSmaamnpulefsactoufretrhe fwoartenrutrairteiopnearliodciocmaplolnyentasnalanydzedenvbiyroHnWIm.entaTlhere wienrteerfnoerekdnowwnithcoonrtamaifnfaencttsed inthetheresfueletdsorofwattheerstautdyl.evels that would have Selection of Test Animals Twehreeansiemlaelcstedwerate riadnednotmifibeadsedbyonanihmeaallthnumabnedrboadnyd wceoirgrhetsporenqduiinrgemeeanrts.tag and Coz54 +O > Study Design Page 9 of 28 Wl 6325151 Female placed bose Level animals weighing from 2,511 to 2,781 g into the following study groups: Grop Treatment (mq 1-6067/ka) 1 (Control) *- 0 70 rae : 3 T-6067 16 HE: bi 4 T-6067 40 at initiation Dose Volume mL/kg) 00.35 030.5 0.5 of treatmentwere Number of Animals 11 i1 1 * Sterile Water for Injection, USP. ification for Spectes Selcts Historically, the choice because of New the Zealand White albino rabbit has been the animal of large amount of background information on thisspecies. broparati pithealinnPROCEDURES The test specific material was diluted with Sterile Water for Injection to achieve concentration for each dose level. An individual dose of each a respective test its body weight solution or on the day control was of treatment. calculated for each animal based on The respective test solution was administered by given as a slow intravenous injection into a push (approximately 30 to 60 marginal ear vein. The dose seconds in duration). The was prepared test solutions were stored After administration, any remaining at room temperature test solutions were until administered. discarded. Reason for Route of Adninistration Intravenous injection is an acceptable route to assess systemicexposure. Observations of animals 1C8linhiocuarls oafbtseerrvantiivoansvewneerse cSoansdauccttieodn at approximately 0.5, 2, 4, 24 and Body weights were deternined just before test material administration (Day 0). c02546 Page 10 of 28 Sample Collection HWI 6329-151 Blood samples (approximately 4 mL) were collected from either ear via the catheterization of the auricular surviving animals at 2, 4, 6, 8, artery or from the marginal ear vein 12, and 24 hours post-injection. At of all the time of necropsy (approximately 48-hours post-injection) approximately 20 mL of blood was obtained from the posterior vena cava of each surviving animal. All samples were stored at room temperature until centrifuged and separated into serum and cellular fractions. The blood samples were then stored in a freezer set to maintain a temperature of -20C 10*C until shipped to the Sponsor. pathol abbreviated gross necropsy examination and any SOnoreaNsin ers rerendey The animal found dead immediately after dosing (40 mg/kg) was subjected to an No tissues were collected from this animal. At termination of the experimental phase, surviving animals were anesthetized with sodium cpoelnlteocbtaerdb.ita)TheanwdhoelxesanTgivueirn,atebdilew,ithaonudt bontehcrokpisdnyeyhsowefvreorm tissues were each animal surviving to termination were collected and immediately placed on dry ice, then frozen by placing in a freezer set to maintain a temperature of -20C 10C. After tissue/bile collection, the animals were discarded. Shipnent of Tissues After completion of the in-1ife phase the blood samples, livers, bile, and kidneys were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3-09, 935 Bush Avenue, St. Paul, MN, 55106). The Sponsor is responsible for the retention and disposition of the samples. HMI does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. ocation of Raw Data. Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of HWI in accordance with HHI SOP. CoR54% Page 11 of 28 Body Weights RESULTS Individual body weights are in Table 1. HHI 6329-151 Clinical Observations Individual clinical signs are in Table 2. The animals treated and 24 mg/kg appeared normal throughout the study. The animal at 0, 4, 16, treated at 40 mg/kg died within 5 minutes of dosing. Pathology The animals treated at experimental phase and 0, 4, 16, and 24 mg/kg survived to termination of the were not examined grossly when sacrificed. The animal treated at 40 mg/kg died on the day of treatment and was necropsied. The lungs in this anima] were diffusely dark red. acute death and postmortem change. This change was attributed to DISCUSSION The level of systemic exposure of T-6067 was evaluated in female albino rabbits when administered as a single intravenous injection at levels of 0, 4, n16o,rma2l4, tahnrdou4g0houmtg/ktgh.e sTtuhdey.aniAmadlmsinitsrteraatetdionat of0, th4,is 1m6,atearnidal24atmg/4k0gmga/pkpgeared resulted in the death of the animal. SIGNATURE NWR WN Steven M. Glaza ASctuutdey TDoixrieccotolrogy WN. = 2s Date REFERENCE 1. NIH Publication No. 86-23 (revised 1985). coz548 Page 12 of 28 HL 6329-151 PATHOLOGY REPORT wTahserenecwarsopsoineed.Group-4 female rabbit treated at 40 mg/kg that died on Day 0 and AbwatosthneaKctritodrpnisebyyu,tsedtfhreotnoTutanhcigussteaidnnienatathlhiswaenardneimpaoilsntanwdeovrreetretendnitcflhfyaunsgneeol.ty daTrhke |reidv.er, Thbiilse.chaanndge collected as required by Protocol. After necropsy, the animal was discarded. PTaontse/he.ologhistoFfrner,dPAa 1(262322994-151.51h) ate RFS cozsqq Page 13 of 28 Group 1 2 3 4 $ Individual TaBboldey W1eights (g) Lever (mg/kg) 0 Sex= Female Animal Number F52762 4 Female F52763 16 Female F52768 0 Female F52769 2% Female F52770 DaQy 2,781 2,770 2,735 2,511 2,718 HWI 6329-151 C2550 Page 14 of 28 HWI 6329-151 Table 2 Individual Clinical Signs Group 1 Dose Level (ma/kg) J Animal Hour Sex Number _Observation~~ 0.5 2 4 24 48 Female F52762 Appeared normal J ST 0 2 4 Female F52763 Appeared normal 77777 3 16 Female F52768 Appeared normal J 7 Jv 7 4 40 Female F52769 Deapdost(-=i5njmeicntuitoens) + 5 4 Female F52770 Appeared normal J77J / Indicates condition exists. - Not applicable. Co2552 Page 15 of 28 APPENDIX A Protocol] Deviation Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-151 CORSSR Page 16 of 28 Protocol Deviation HHI 6329-151 -- Protocol Page 8, 7. Experimental Design, E. Termination, (2) Scheduled Sacrifice, (a) Sample Collection. The whole liver and bile from each animal dying during the study, sacrifice in a moribund condition, or surviving to termination will be collected. Both kidneys from each animal will also be collected. PrAo ctucaledure The liver, bile, and kidneys from the animal that died on test (Group 4, No. F52769) were inadvertently not collected at necropsy. This deviation is not considered to have had an adverse effect on the outcome of the study. COR55F @H STONE N [OI AR HH Page 17 of 28 +CORNING rior Sponsor: St. Paul,3MMinnesota PROTOCOL Toa0a4p.K Study Title: Single-Dose IonftrTa-v6e0n6o7usinPhRaarbmbaictoskinetic Study Date: | December 6, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. Ma3d3i0s1onK,inWsimsacnonBsoiunleva3r7d04 aboratory Project Identifications WW 6329-151 COR55 Page 18 of 28 STUDY IDENTIFICATION PPa8ge082 4P.K Single-Dose IonftrTa-v6e0n6o7usinPhRaarbmbaictoskinetic Study HN No. 6329-151 Test Material T-6067 Sponsor Mm 2T2o0x-i2co-l0o7gy3HSeCrevincteesr St. Paul, HN 55144 Sponsors Representative JNohn L. Butenhotf, pho 2T2o0x-i2co-l0o2gy3HSeCrevnitceers S(t6.i2)Pau7l3,3-1M9H52 55144 ! Study Director HSatzelveetnonN.WiGslcaoznasin, Inc. MPa.0d.isoBno,x W7I54553707-7545 (608) 241-7292 Study Location HBauzilledtionng WMoi.sco3nsin, Inc. M3a8d0i2soPna,ckWeIrs 5A3v7e0n4ue PrEoxppoesreidmeSnttuadly STtiamrettabDlaete Experinental Termination Date DDeecceemnbbeerr 19,1, 19193434 Draft Report Date January 13,1995 02556 Page 19 of 28 TPrasg0en'gs. ox 1. S$tiu5d6yte-tose Intravenous Pharmacokinetic Study in Rabbits 2. Purpose TSointanssiesstseratdhe3l'esvelSinogfle'sysntiermviecneoxnpeosunrgeenwhteinm thheo LET test material is 3. gemlatory comlianee bSCUToxhsncibcesiaeprntastittkroouanrdtyyitohPwnair,tlalctSaiombc1seeutbyRcTseo1tng1sdutulyoca,tfteidhhooenmisongteeSanstcetocinotdmryaad.rkadansrcosienadGswariSoctEnhLoArtrThceesEsSfSoFolHtOleano)wiinnogdt Good be [Conduct a5 a Nonregulated Study {[X7] 5210 CEFtR 51680! ((FDEA0)k-Frrm) [ ] 40 CFR 792 (EPA-TSCA) [ 1]5(581N)03n0san(FNion.al)365(0.OEC(DS)apanese MAFF) , WAd[eIil1YreacpNrtrooeotrci,eAfdcihutcrhaeeRtseigoSuntilunadNtyoti.hodinas3ss1.s3prno(otIJkanacpouta]mneeeascoreeepsiMsnOfaiHnroNin)claynopfdlfutahpnecoeSmpownritehonrytehenpAeniiimma pi 4. QualityAssurance The the protocol, study conduct, and the final report will be audited Quality Assurance Unit in accordance with HazletonWisconsin by 5. ATestsLsdetsnteifrication (HWI) Standard Operating Procedures (SOPs) and policies. B. PhTyhsiScealoDteusmcerriiapttioSYn the raw data) C. puarndiStatbilyity The Sponsor assumes responsibility for purity and stability RodSEtaseaekstnoeteignrnm)mtmnisaewnttaretTtaryiti:oiabnolesn/a'nvg(ee(FhimisnrcicellaqeSubaudi-eimTsntiitghxEeetyduurSmneBopd(ysoeshnr)rsTsohteerefnsotSSrwPiprcceoaotnnmdciemestnniitporTenahsnot)ekei.sooncne,fSlSotsaelomrlpdoulrbeeisliiotfey, Heraination far sessile snatyrie J co2556 Page 20 of 28 D. SRotoomragteemperature ThPa0gaed4.px E. RReesseerrvvee sSaammpplleess will not be required for this study. F. Retention Acnoympluentuisoend otfestthmeatienr-iTailfewiplhlasebeofretthuernesdtudtyo. the Sponsor after G. SaAsferteyquPirreecdaubtyioWnRsI SOPs and polictes 6. ControlMaterial A. Identification Sterile water for injection B. Physical Description Clear, colorless liquid C. Puar ndi Stat bily ity ) cTohnesipduerrietdy atondbestaadbeiqluiattyeofforthitsheUpSuPrpgorsaedse mofatetrhiiasl stiusdy. D. RSetforriaggeerated E. ReserveSamples See Section, 5. E. Reserve Samples F. ARneyternetmiaoinning contro) material may be used for other testing and will not be discarded after issuance of the final report. G. SafetyPrecautions As required by HNI SOPs and policies 7. ExperimentalDesign A aims (1) sRpaebbciites (2) Strain/Source Hra: (NZW)SPF/HRP, Inc. B (3) AAIgaenraitttiation OR558 Page 21 of 28 (4) MeattInaitihatiton 2.5t03.5kg TPPaBgOeB4. PK (5) N5umfbeemralaensgsex (6) Identification Individual numbered ear tag (7) Husbandry (3) Housing (Ihnedaivvyidugaalulgye,] in screen-bottom stainless steelcages. (b) FAomoedasured amount of Laboratory Rabbit Diet HF #5326 (PMI the Feeds, Inc.). manufacturer for The food is nutritional routinely analyzed components and by environmental contaminants. (c) Mater 1 Ad Tibitum from an water are analyzed automatic system. Samples of the by HWI for total dissolved solids, hofarorgrdannseoesplshe,ocstpeahdnadteelssep,meecniatnfsdi,cedhhlemoairvcrynmoaebtiteoadllosgh,iycdarlocacrobnotnesn.t and (@) CTtohhneartteawmoiaunrleadnntosinktneorwfnerecointtahmintahnitssstiundyt.he food or water (e) EEsnentvviirrtooonnmmmeaenintntatlainconatrtoelmsperfaotrurteheofani1n9alC rtoon33wCi,ll abe rTeilgahtti/v1e2-hhouumriddiatrykofcyc5l0e%. +20%, and a 12-hour (F) AAteclTiemaasttio7ndays (8) SaBadeseleqeducattioeonnneuoanflbteThrestoafndneixbntoardlayswaeniigmhatlsawciclolrdibnegputrochHHaIsedSOPvsy. thaArn no animal in obviously poor health is placed on test. (9) HistoJrusiticfiacaltilony, f"tohre lewSpecieZs ealanSdeleWchtiitoen albino rabbit has been. ) btaheckgarniomuanld oifnfcohromiatcieonbeocnaustehisofsptehceielsarge amount of O=558 Page 22 of 28 B. DAosdeministration PTargaeoas. pk (1) TestGroups Group DoseLevel --(ma/kg)* Number of Females 321 3 0 163(Control) 1 111 1 5 160 1 2 Twheeigdhotse volume will be 0.5 ml/kg of body C. DosingProcedures (1) DosingRoute aIpnptrroaxviemnoautselyin3j0ecttioon60 insteoconadsm.arginal ear vein over (2) Refoa rDos sino gRon ute ) sIynsttreamviecnouesxpoisnujreec.tion is an acceptable route to assess (3) DSionsgilneg pdausreation (4) DTPIohnrsejeeecTpteaisotrnamatttoieraoicnahlievweilla bsepedcii|fliuctedcownictehntrsatteiroinlefowrateearchfor ad5tdohnseiernoaionTsmietmvreatallet.'mispoenrbI.aontdduyir"vTewihdeeuiuanpglthritelpdatoraasdekemedsinnwtijieslustltsrtambtiebixeotcfnuao,rrleecsultwaeitslteldmabbteaesresidtaolroend D. ObservoafAtniimoalns (1) CTtalhfoietxneiircacinatitlymraelaOasttbosewseniprtlpv.lraotxibieonnasatbesleyrve0d.5,for2.0c,lin4i.0c,al24s,igsnnsdo4f8 hours J Co2589 Page 23 of 28 @) i TPaag0e847. PK (3) sample Collections (a) F2r,e4qu,e6n,c8y, 12, 24, and 48 hours post-injection (6) Number of Animals AN (c) Meot f Ch ollo ectd ion Blood samples (approximately 4 mL) will be collected from either ear via 476.8, 12, auricular artery or the catheterization of the from the marginal ear vein at 2, pAopsptreorxiiomratelya'n2d0 a2l4 ohfourbslapbost(-sintjees)ctivoaet,ine to be docunented in the raw data) will be obtained from the vena cava of e3ch animal a the sine op necropsy (48 hours post-injection). Approximately 20 nL of blood will be collected from moribund animals during the study, also, if possible. The samples will centrifuged, be stored at room temperature and then and the separate serum and cellular tfermapcetrioantsurestoofred-20inC3$1f0reCe.zerThseetsetopamraaitnetdaisnerua.m and cellular fractions after experimental will be sent termination. frozen to the Sponsor samplesSJW5t3aw.E5mielEsbplau,us0blh.e,LAJsvHhohNehi,nnpuspeo5en5d10t6o: Bldg. 2-3-09 the samples. James D. Johnson or his alternate telephone at (612) 778-5294 prior will be notified by to the shipment of E. Termination (1) UnARgronsorycsdhsbaeundninumdelacelrcdooSpdnsaydyiictnrigeioxfnadi,nucirenwisanitg]ainotndbheDeaenSSadtutubhdj31sye1ctoesrdonstaeocvraaispfiisciseeadrraimuniitaa)tedce i raenceosrtdheedt.izedAnwiimtahlssoidniuammoprenitboubnadrbciotnadliti(ovnia wiilnljecbetion in etxhseanmqaurigniantaeld:ear vein), bled via the vena cava, and co256 Page 24 of 28 FTioaaenssspx. (2) ScheduledSacrifice At approximately 48 hours post-injection, animals BentabarbiLal surviving to termination will be anesthetized with sodium (via jection in the sapeiacs en eos! bled via the vena cava, and exsanguinated. An abbreviated Grass necropsy examination wii] nok be aone, norco. tissues will be collected. (2) Sample Collection iKodTihrsudersnisuonwueyghrssovlitWevhfIeirTnoTTgnsitvbueteaedorcyh,pf.aienradssmacnibceanirdalilotefniiWocfndeIrrdo]ywmiiAneJlleaesacobhemmbosaercneiiodambliabuialtnetsddcaocytneoieysnqnaddgsi,ittbieoorPnnin,e RCaoitnltascitni'osn stnedmptehreanturpelaocefdoTbnoe3 HFortoem"e sot fo Trhoezetnisasunesdry(liIvceer,fo. bithlee', Spkoidnnseoyrs)aFwsielrl exbpeersiepnetntal , SFaenstpaosn,sibe for the retention and liporyiiss ni the termination." The samples will be shipped to the person listed in Section 7.0.(3).(c). The Sponsor is F. StRaotiSsHtEiScaelAanalTyassetysses are required. 8. Report A final report including those items listed below will be submitted. Description of the test and control materials PDreosccardiupeteison of the feet paten Dates of experimental initiation and termination Description of ay toric sFhects Gross pathology findings (if applicable) GrDoisrsecptaotrh)ology report (if applicable and requested by the Study ) i cozbed Page 25 of 28 PPa8g0e849.PK 9. LoOfrcaicagitiliniaotlnatoedfafaRaua,dwiDtoirantgcso,ptRiheeescsottrhudedsry.eodafun,rdinwFgiilnliatslbeRpearpvoogarrieltsasbleandatbHeUfIoreto acceptance of the final report. When the final report is completed, arleltaionreidginianl thpeapearrcdhaitvae,s oifncHlMuIdinagccotrhdoisneg ittoemHlIistSeopd. below will be Protocol and protocol amendments Dose preparation records InB-oldiyfeweriegchotrdss DOobsseervaadtmiionnisstration SSPhaaimtpphploielnoggcyolrlReeeccocortrdidsosn records SFitnuadly rceoprorretspo(nodraingicneal signed copy) Tnohte bfeolalrocwihnigvedsuwpiptohrtitnhge rsteucdoyrdsdatwai.ll be retained at HNI but will AVnaitmearl arnaelcyesiipst/arceccolridmsation records ) ARInenifsmrtairlugmeerrnoattoomrcatlaeinmbdprearftariteouenrzeeranadntdemmahpiuenmrtiaedtniuatrnyacerreeccrooerrcdodsrsds ) COR56R, Page 26 of 28 ToPaognes1.0pK PROTOCOL APPROVAL Fo 2. Cuts lus] 3HJohn L. Butenhoff, PhD Sponsor's Representative Steven Gaza Study Director Acute Toxicology ) Hazleton Wisconsin, Inc. LA QHuaizlreettsyoennAtsWasitusicrvoaennscien,UniItnc. (6329-151. protdsk1) 12-1z-6y Date' Tite 2 me 5/54 J ----- 2564 Page 27 of 28 WISETO NSN ) RB SR I NIRSS 0 1348 CORNING Cam Single-Dose PROTOCOL TP8084P.K IonftrTa6v0en8o7usinPhRaarmpaecsokinetic Study HW1 6329-151 Sponsor. 3M Toxicology Services 220-2-02 3M Center St. Paul, MN 55144 Contractor Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Study Director remeee John L. Butenhoff, PhD Steven M. ee Glaza Aaendsent No. 1 , This amendment modifies the following portion of the protocol: Effective December 9, 1994 1. Page,7.ExperimentalDesian;B. DoseAdministration:(1)TestGroups, fDoure tthoe tGhreoudpeat5hanoifmatlheasGroiunpdic4ataendimably t(h4e0 fmgo/lklgo)w,ingmoudnifdyerltihneeddoscehanlgeev:el Group Domsae/kLaev)el NFuemmbaelresof 1 0 (Control) i 2 ! i 3 16 1 i io 1 5 24 1 a The dose volume will be 0.5 ml/kg of body weight coz564 ) Amendment No. 1 Page 28 of 28 PROTOCOL APPROVAL HPIaIge63225-151 Gln 7 Cutty JSophonnsaLr.'sButReenphroefsfe,ntaPthiDve 3M Steven W. Glaza - Study Director AHaczulteetoTnoxWiicsocloongsyin, Inc. ) aFreseLnititiHve Ci2) ) QHauzalleittoynAWsissucroannscien,UniItnc. (6329-151.Am1.dsk2) elge BE Date' -- A Tate NAS te Jy JasJac 0256 9.12 Analytical protocol AMDT-120694.1 Corset it 3M Environmental Laboratory Protocol - Analytical Study Single-Dose Intravenous Pharmacokinetic Studyof T-6067 in Rabbits In-Vivo Study Reference Number: HWI#6329-151 TSetsutdySuNbusmtabnecre:: ALM-D13T4-91220(6T-9640.617) Name and Address of Sponsor: 3M SCD Division 3St6.7PGaurlo,vMe NStre5e5t106 Name and Address of Testing Facility: 933M5 EBnuvsihroAnvmeennuteal Technology and Services St. Paul, MN 55106 Proposed Initiation Date: July 25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers and Revisions: AMDT-M-1-0, TDhoehrrmmaalnnExtDrXa2ct0i0o0n oOfrFglaunoircidHealbiydeMAenaanlsyzofera-LMiovderified AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ton Analyzer AMDT-M-4-0, AMDT-M-5-0, Extraction of Fluorochemicals from Rabbit Liver Analysis of Rabbit Liver Extract for Fluorochmicals Using Electrospray Mass Spectrometry AMDT-M-8-0, wAniatlhylsoins oSfeFllecutoirviedEeleUcstirnogdethe Skalar Segmented FlowAnalyzer AMDT-M-14-0, DTohherrmmaannnExDtXra2c0t0io0n OorfgFalnuiocriHdaelibdyeMAenaanlsyzoerfa-SMeorduimfied Author: James D. Johnson Approved By: WAN) 45 "James D Johnson // Study Director Date Pl 2 BiteEf ifpnfiy -- John Butenhoff, PhD Sponsor Representative Date "ss Tadomiansissetssertehde alsevaelsionfglseysitnetmriacveenxopuossiunrjeecwthioenntothreabtbeisttsm.aterial L-13492 (T-6067) is 2.1 Test, Control, and Reference Substances and Matrices 2.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 2.1.2 Analytical 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), liver), Sigma Chemical Company (2.1.2, 2.1.4-serum) grocery store (2.1.2, 2.1.4- 2.3 Number of Test and Control Samples: Liver and serum from 3 test animals and 1 control animal. One animal died after injection ofa 40 mg/kg dose, and was not replaced. available for anOatlhyesrisbiiofldoegiecmaeldtiaspspureosp(rkiiadtneebyy, btihlee,ScteuldlyulDairrfercatcotri.on) will be 2.4 Identification of Test and Control Samples: The samples are identified using the HWI animal identification number which consists oaf `number, plus the tissue identity and day identity (serum). letter and fivedigit 2.5 Purity and Strength of Reference 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 temperature (2.1.1), 204 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 2.8 Disposition of Specimens: Biological tissues and fluids will be retainedper GLP study Regulation for the is in parallel with time a 28 period required for studies longer day dermal absorption study so all than 28 tissues days. will be This retained. 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. oo 02569 3.0 EXPERIMENTAL - Overview ~~~ The tissues analysis for from animals dosed as fluorine compounds. described (HWI#6329-151), At the discretionof the Study are available for Director, a series of (anSaeleytMiectahlotdess-ts-nceaxntbSeecpteirofno)rmisedt.he Tahpperospcrrieaetneinagnafloyrsifslutooripdreesiennltivdeerfivniiaticvoemdbautsatifoorn fluorine in the liver. 4E 0 XPE -MR ethodI s M ~ E ~ N~ TAL 4.1 Liver and Serum screening methods: (attached) 4.1.1 AMDT-M-1-0, Thermal Extraction of Fluoride Dohrmann DX2000 Organic Halide Analyzer-Liver by Means of a Modified 4.1.2 AMDT-M-2-0, Fluoride Expandable Ion Analyzer Measurement by Means of an Orion EA940 4.1.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 4.1.4 AMDT-M-5-0, AnalysisofRabbit Using Electrospray Mass Spectrometry Liver Extract for Fluorochmicals 4.1.5 AMDT-M-8-0, Analysis of Fluoride Analyzer with Ion Selective Electrode Using the Skalar Segmented Flow 4.1.6 AMDT-M-14-0, Thermal Extraction of Modified Dohrmann DX2000 Organic Halide Fluoride by Meansof Analyzer-Serum a SODATAANALYSIS~~~ 000 5.1 Data Reporting: fluoride per tissue or Data fluid, will or as bFeC-re1p4o3rt(eedleacstraocsopnrcaeynmtarastsiosnpe(cwteriogmhett/rwye)igphetr)unoift of tissue or fluid. 6R .0 MA AINW TEND ANA CE OT F AANDRECORDS 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. 3 rr 7.1 AMDT-$-10-0, Sample Tracking System 8.1 AMDT-M-1-0, Dohrmann DX2000 Thermal Organic HEaxtlriadcetiAonnaloyfzFelru-oLriivdeer by Means ofa Modified 8.2 AMDT-M-2-0, Fluoride Expandable Ion Analyzer Measurement by Means of an Orion EA940 8.3 AMDT-M~4-0, Extraction of Fluorochemicals from Rabbit Liver E8l.4ecAtrMoDsTpr-aMy-M5a-s0s,SApneacltyrsoimsetorfyRabbit Liver Extract for Fluorochmicals Using w8i.5thAIMoDnTS-eMle-c8t-iv0e,ElAencatlryosdies of Fluoride Using the Skalar Segmented Flow Analyzer D8.o6hArMmaDnTn-MD-X1240-000,OTrhgearnmicalHaElxitdraecAtinoanlyozfeFrl-uSoerriudem by Means ofa Modified -_-- 4 COR57@ 3M Environmental Laboratory Method Thermal Extraction ofOFrlguaonriicdeHablyidMeeaAnnasloyfzearM-oLdiivfeired Dohrmann DX2000 Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: /o- #-1 Revision Date: None _--_-- Author: Rich Youngblom Approved by: i Zoup Lead, ---- . /of3/7< Date QuaZlityeAsS surVanocR e Fk Lo-0 1.f7~ Date Software: MS Word 5.1a Affected Documents: AMDTE-xMp-an2daFbllueorliodneAMneaalsyuzerrement by Meansof an Orion EA940 AMDT-OErgPa-n3ic RHoaulitdieneAnMaaliynzteernance ofa Modified Dohrmann DX2000 1 0_SCOPE, APPLICABLE COMPOUNDS, AND MATRICES wA1i.u1tohrSiacdnoepiefo:rnosTmehlivesacrtmiieoveutshelomeadcttirrsiocdfeeos.r.thTehoepefrlautoiroindeoifatyDpoicharlmlyacnonllDeXct2e0d0in0TwIhSeAnBitsoisluutsieodn tfoorexatnraalcytsis 11..32 MAaptprliicceasb:leBiCoolomgpiocaulntdisss:ueFs,lupoarrtoicchuelmarilcyallisveorr. other fluorinated compounds 2.0 KEYWORDS 2D.X120Fl0u0or,idfel,uofrloucohrienmei,caelxst.raction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, 3.0 PRECAUTIONS 33..21 GGllaasssswwaarree iasnfdraegxihlea,usbtrgoakseensgclaasnsbmeaeyxtcraeumseeliynjhuort.ies. 333...543 PPSorotelesvnsetunirtailbzbeaidsoeghdaaszseaasrm,dpsplredosupeemratocytohmfelparbshie,oslmsoegadiycganalesmeahdtarntiodcleaisl.nlgoUwprstaechtaeipmcpterosodprrreqiyuaitdreeodpw.enrsboneaflorperosttearcttiinvge run. equipment. 4.0 SUPPLIES AND MATERIALS 44..21 CCoommpprreesssseedd HOexlyiguemn,, HHiygdhroPcuarirtbyonGrfardeee,, rreegguullaatteedd to to 30 45 PSL PSI 44..43 QQuuaarrtzz ggllaassss scoammbpulsetbiooanttwuibteh, TReelfilaoncneTMGtluabsisng,G-D9o4h0r5m-a0n1n2 8or90e-q0u9iv7aolrenetquivalent. 44..65 SOarmiopnle94c0ol9l9e9ctTiootnavliaIlosn,icHDSPtrEe.ngth Adjustment Buffer (TISAB II )orequivalent, 44..87 PMoillylsity-rQeTMnewaptipeerttes. 44..910AcHtaimvialtteodnCShyarrcionagle,oEr. eMqueirvcakle2nt005 or equivalent 4.11 Miscellaneous laboratory glassware: 5.0_EQUIPMENT 5.1 5.2 RIoBsMemcoomupnattiDbolher3m8a6nnorD4X8260c0o0mpOurtegra.nic Halide Analyzer, modified for fluoride extraction. 55..43 EDxXc2e0l0S0prseoafdtswhaereet,, vveerrssiioonn 15..000,ormgordeiaftieerd for fluoride extraction. 6.0 INTERFERENCES 6m.a1ySvaamrpylferosimzemaitsrliixmitotedmattoriaxp.proximately 150 mg, depending on sample moisture content. This 2 C0257 7.0 SAMPLE HANDLING s7-a1mpSlaem.plUesseafroerncoetpstorbeprhoabnedlteodtrwaintshfebratriesshuaensd,s. Fluoride may leach from the skin to the cm7le.e2tahSnaansmocpla,llopeesrlmoaefntldhivacenurtoatlri-enwcgaubttoeafrrrdso.omluTtfhireoonzceaunftttlirinvgeearbcaohanrdldipvalernacdiessdccuaitlnpaeltasrheoduladndbelacbleelaendewdewiigthh bwoaatte.r,Use a 80 CALIBRATION AND STANDARDIZATION 8.1 Preparation of Calibration Standards R8.e1f.e1rTthoeprsottaoncdoalrdfsorrtehqautiprreodjefcotr each project will need to be appropriate for that individual project. 88..11..32 TFyoprircaablbliyt f5i0v-e5r0s0tupdipems,FuCs-eb9e5eifnlimveetrhaanotlhestmaatnrdiaxr.dsCaurte auspeidcce of frozenbeefliver (100150 me) and weigh it in a labeled and tared weigh boat. 82 Calibration- Overview `cTuhrevenoirsmraelquciarleidbrtahteiponroicsetdhuereflluiosrtieddebceulrovwe i(sAuMsDedT.-M-2). However,ifan optional spiked liver 8a8.n22d12coTAympbicucasaltliilbynr,agttithohrneeecmurruesvpielinfcgaotrtehtsehoesfaDmcXae2cmh0e0st0thaonisddasgredanneadrnadmtaeftdirvibexycotsnypcpieeknaitsnrgatthsieaomsnpsaolmefpslsetwsaint0dharkbdensotewwsintlesd,tbea:ndards R8spe2ic.ko3evde.SrteadndFar(dugc)uornvethweily-abxeisp.loGtteenderaastMeaassreSgpreiskseidonFc(uurgv)eoanndthcealxc-ualxaitseatnhed eSqtuaantdiaorndfoMastshe line 8a.nd24thMears2svaSlpuiek*.eFdC-F95(uigs)6=0.(0A4m%ouFntthesrpeifkoerde i0n.6m0L0)4 xis(tChoenfca.ctoofrstusaenddatrodcoinnvperptm)FCx-9(50.t6o00F4)" 82.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in pprm) 83 Calibration - Procedure b88e33..u11s.e1dSitRfaturhtneU2poprrevmiooruse sCalmepalneCsyccolnetsaiwnheednhisgtharcionngceinntsrtartuimoennstoefacflhuodraiyd.e. More clean cycles may 888333..222..12BlFParonerpkrasarbebistasmtpuldeiesu,siunsge tbheeesfalmiveermaesththoedsmatarnidx.typPeroefpamraetarixleaasstt3hesatemsptlseasmpolfeb.eefliver a8(n31a.0l20y.z3e1Ps5ua0tmmpsgla)empaflcoercobirlndaiDnnokgshtrommaentnhobodatA, MCDoTm-bMu-st2efaochthseaimopnlese3lsecdteisvcereilbeecdtriondseeacntailoynsi9s.0 and 3 02578 b8.e3f.o2r.e4prFoocrereadbibnigt wsitutdhietsh,e tchaleibmreatteiorn.reaBduirnmg fsoarmpalbelsanunktislamthpilselismhiotusldrebaech0e.d0,3 oprprunntoilr ilnotwheer jhuoudrgse)m.ent of the operator the reading is stable with respect to historical readings (previous 45 b8e3f.o2r.e5prFoocreneodni-nrgabwbiitthstthuediceasli,brtahteiobnl.ank readings should reach a predetermined ion concentration c8.o3m.b2u.s6tiItonm.ay be necessary to mix approximately 50 mgof charcoal with the sample to aid 88..33..33.1StWaenidgahrdouCtuartlveeast 15 matrix samples (5 standards with 3 replicates each) in tared and sltaubedlyeddatwae.igShtobroeattsh.e mFaotrrirxabsbiatmpsltuedsieosn, dwreyigihce1o0r0i-c1e5p0acmkgsbetocfkleievpetrhseammpflreosz.enRuenctiolrdusweed.ights in 8833.33.32 SPtlaratcewiwtehitghheedlboeweesftlisvtaenrdsaardmpcloencienntDroathironm.anUnssianmgpaleHabmoiatl.ton syringe, ejecta fixed oqruainnttihteyboefetfhelivsetra.ndard onor in the matrix. For rabbit studies, use 4 uLof standard and eject it on m83a3y.b4e uAsteldeaasttt3herdeipslcirceatteisosnhoofutlhdebaenaluyssetd.for the lowest standard concentration; more replicates 883333..65 RCuonmbaullst15thsetasnadmarpdlse.Iasfdoensecrriebpeldicianteseictsiiognni9f.i3caanntldyadniaflfyerzeentacfcroormditnhge toothAeMrDtTw-oM-2. orlepdlirceaptleisc,atreuannadnotthhaterthseanmpelwerfeoprlitchaatteswtialnldabred.useIndtdioccaatleciunladtateathtehartetghreesnseiwonrceuprlvieca.te replaces the l8e.a3s.t3.07.95W,hceonnsaullltswtiatnhdasrudpserhvaivseorb.een run, calculate the r2. r2 must be atleast 0.95. Ifit is not at c8h3a3n.ge8d.AnNeeww ssttaannddaarrdd ccuurrvvee mshaoyuladlsboebreurnuwnhaetnthtehedicsocrmebtuisotnoifotnhtebaenaolrysst.ample matrix is 8.4 Storage Conditions for Standards T8.y4p.i1caSltloyr,asgteanrdeaqrudisreamreensttsorfeodr ast rtooam tanermpededreaapteunrrdeedinntposlnasttihcesicnrdeiwvitdoupalbostttlaensd.ards used. 8.4.2 New FC-95 standards should be prepared at least once a month, 9.0 PROCEDURES 99..11.1TyCpoimcbaulsOtpieornattuibnegtCeomnpdeirtaitonusr=:e 950C. 99..11..32 VOaxpyogreinzaatniodnH/eDrlyiiunmgftliomwe ==52040ces/emcionnudtse.. 9.1.4 Bake time = 300 seconds. 99..22.1StIafrttheUpprPorgorcaemduisrneo:t started, start the EOX program on the PC. 99..22..32 POuptetnhethfeumSaYcSemToEdMulSeEaTnUdPthweicnedllowi.n the READY mode. 9.2.4 Close the SYSTEM SETUP window. 4 a O257% 9:2.5 found When in the tCheEoLvLenChHaEs CrKeacmheenduthe READY temperature, run the CLEAN BOAT program 9.2.6 See AMDT-EP-3 for details of the Dohrmann software. `99m..i33x.1SaapOmpppreolnxeitmEhaxetteSrlaAycMt5iP0oLnmEgPorHofcAceThdaCurrcHeo:aalndwiptlhactehethseamspalmeplteo aiindtcheomBbOusAtTi.on.It Imfatyhisbeisndeocnees,scahrayrtcooal 9s.h3o.u2ldCallossoebeSAmiMxPedLEin HwhAiTlCeHe.stablishing the baseline and when generating the standard curve. 2l9.0.am3b.e3lLedAdsoadfmpapl1:perTocIpoSlrAlieBact:teMioivnollvliuialm-.eQTMToyfwpTaitIceaSrlAlmByix0st.ou6lrumet.iLontoor151:m1 LTIaSrAeBu:sMedi.llFio-rQrTMabwbaittesrtumdiiexst,urueseto1.0aor i9n3c.he4s.PlGaacseetsherevliealasseodtdhuartitnhgeptyiproolfystihse mCuOstMBbuUbSblTeItOhNrouTgUhBtEheiTsIiSnAtBh.e TISAB at least 0.25 99.33..65 WRuhnenthteheEEOXO-XSpOrLoIgDraSmiprofgirniasmhefd,ournedmionvethtehRe UcoNllemcetniuo.n vial from the combustion tube. 910.3t.h7eITfIuSndAiBluttoedmaTkIeSA1:B1 wTaIsSAuBs:eMditlolico-lQlTMec.t the sample, add an equal volume of Milli-QTTM water r9.e3m.o8vReinasneytThIeSeAndBorfemthaeincionmgbuosnttihoenttuubeb.e with Milli-QTM water and wipe with a KIMWIPE to w9i3t.h9a Ocpotetnonthteipspaemdpalpephlaitccahtoaonrdvraecmuouvmeeadnoyutr.emIatimnaiyngbeasnhecfersosmarthyetoboastc.rapApsahrtciaclnesboeffrtehmeoved bbooatttotom.awiid tinh2thsepaCtluelaanorCyoctlhee.r similar device. A drop of Milli-Q water may be added to the 99.33..1110 CRluonsethteheChLatEchA.N BOAT program. 93.12 Sample is ready for analysis by ion selective electrode (AMDT-M-2). 9.4 Sample Calculations 99..44..12 SUasmeptlhee sMtaasnsdarRdeccouvrevreteodF(caulcgu)lat=e(tTheISsAamBplvoelvianlume.L) x(Orionreadinginppm -intercept) (Slope) 10.0 VALIDATION a11n00a..l11y.s1QisDuaaiilslisyttyaSrtCtaeordnttbryUopalnCahlyezcikngSaQmCplseasmp:leOsn.ceTthhee QstCansdaamrpdlceusrvaereistoesbteabtlhieshseadm,eeaacshthdeayloowfest rdceoopnnlceiecniatntretaiitspilnoionctastnpeeicukenesldseassrsaym.tphleefsirustsetdwotoregpelniecraattees atrhee wsiitahnidnar2d0cu%rovfet.heEascthancdoanrcdencturravtei,otnhemnusatthbierd mF1al0ut2ocrciPedrseeacNinosdtiedobinoffoaeknrsden2tA,c3r,cefuaernradecn5yc.:e PcSroeemcepisomiueotnnhdaosndddeavcecluroapcmyenvatriaensalwyhsiesnaanndalsyazmipnlgesaanmapllyessisoifndifferent 10.3 Other Validation Parameters: NA 5 O2576 11.0 DATAANALYSIS~~~ 1L1 Calculations 0000000 111L.L1.12 TFoorctahlecusltaatnedtahred fcluurovrei,deusceonrtergarcetsisoinoninatnhaelyssaimspilne,Exsceeel,mevterhsoidonA5M.D0Tor-Mgr.ea2ter. 11.2 Analyzing the Data aI1rs1e.s2w.ti1htahn2in5m20u0%so%tfobtfeheaeatcalhveaeosrttahg0e.ero9a5fntodhrotgshreeeaottuewtrol..i"eOrAuintslygireseruasct"ehrmotauhtyalnieb2res0es0xh%cooluulfddtehbdeiefapt ovienrtaw oegfdeto oohfutethitonhsreteeh.etrdewaptloaircaanteds noted in the Final Report along with the reason it was considered an outlier. 12.0 ATTACHMENTS None 1~ 3.0 REFERENCES ~~ 00000 3143.91, rReovsiesimoonuBn,tDDeochermmbaenrn1D9X932)000 Organic Halide Analyzer Operator's Manual (Manual 915A1n3.a2lyzAeMrDT-M-2 Fluoride Measurement by Meansofan Orion EA940 Expandable Ion A1n3a.3lyAzMerDT-EP-3 Routine Maintenance ofa Modified Dohrmann DX2000 Organic Halide 14.0 REVISIONS Revision Number ReasonforChange Revision Dae 6 c02576 3M Environmental Laboratory Method Fluoride Measurement by Means of an Orion EA940 Expandable Ton Analyzer Method Identification Number: AMDT-M-2 Revision Number: 0 -_-- Author: Rich Youngblom Approved By: | God] Pome Leader Adoption Date: /0-4-45 Revision Date: None 13 /0s Date Le LRA Quality Assurance Lot Date Software: MS Word 5.1a ADfofhercmtaednnDoDcXu2m0e0n0tsO:rgaAnMiDcTH-aMli-d1e ATnahleyrzmearl Extraction of Fluoride by Meansof a Modified 1 CoRS Ty 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES 1.1 SCOPE: Ton Analyzer. This method is for the calibration and operationofan Orion EA940 Expandable 1.2 APPLICABLE COMPOUNDS: Fluoride. P1.H3 oAfP6P.0L. ICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred 2.0 KEYWORDS 2.1 Fluoride, fluorine, on selective electrode 3.0 PRECAUTIONS 3.1 No hazards identified with this method. 4.0 SUPPLIES AND MATERIALS 44..12 OOrriioonn M9o4d0e9l9990To0t0a0l1IeolneicctSrtordeenfgitlhlinAgdsjoulsuttmieonnt(ABugfCf)eorrIeTq(uTiIvSalAeBnItI.) or equivalent. 44..43 MOriilolni-9Q4TM09w0a7ter10o0rpeqpumivaflleunotr.ide standard or equivalent, 44..56 MLaagbnteitsiscuess.tr bars. 44..87 PSlaamstpilce1c0ol0lmecLtiovnolvuiamles.tric flasks. 44..910PoMliysscteylrleanneeopiupsetltaebs.oratory glassware. 50 EQUIPMENT 00 5.1 5.2 Orion Orion Model Model EA940 960900 ESxolpiadndStaabtleeCloomnbiAnnaatliyoznerFolruoerqiudievaelleenctt.rode or equivalent. 55.43 MIaBgMnectoimcpaSttiirbPlleat3e8.6 or 486 computer (only needed if using Orion 3E software) 55..65 MOirciroonsoRfSt23Ex2ceilnte5r.f0a(coenclayblneee(doenldyifneuesdiengd Oifriuosinn3gEOrsiooftnwa3rEe)s,oftware). 6.0 INTERFERENCES Q6.1MItwiasterrecwiolmlmgeenndereadlltyhabtrtihneg psaHmpbeleattoorpnHeaorf6.06..0. A 1:1 mixtureof TISAB and sample/Millia6t.2roSaommptleempteermapteurraetuarsethmeasytaenfdfeacrtdsflwueorriedewhmeeansuthreemmeentte.r wIatis carleicbroamtemde,nded that the sample be 63 The rate the samples are tired at should be consistent with the rate the standards were stirred. 2 A------ 925 78 u6n.4deArirelbeuctbrboldees. trapped under electrode can give erroneous readings. Make sure no air is trapped 1.0 SAMPLE HANDLING 7.1 No special handling necessary. 88..11.1PrMeepaasruarteioSnOomfLCaolfibTrIaStAioBnIStinatnodar1d0s0 mL plastic volumetric flasks. 88..11..23 PLaibpeelttthee0f.0l5,as0k.s1,a05.50,.051.,0,0.a1n,d0.15.,5 1m.0L, aonfd110.05ppppmmfFl-u,oarlidoengstwaintdhartdheindtaotethaenadpyproouprriinaitteiallys, 8l.a1b.e4leAddfdlasakps.proximately 30mLofMilli-Q" water to each flask. 8.15 8.1.6 Shake the Eliminate flasks to mix the air bubbles from solutions the flasks by tipping theflaskson their sides and rolling theairin 8t.h1e.f7lBasrkisnogvtehretvhoelauirmebuibnbltehse.flasks up to the 100mL mark with Milli-QTM water. 88..11..89 IRnevceorrtdansdtasnhdaakrdesthine SftlaasnkdsarfodrstLhoegfiBnaolomkixing, 82 Calibration 88..22..12 IInfvneerctespsraorbye, troewmeotvetotpapseealf.rom electrode filling hole. 88..22..34 EFijlelcttae efleewctdrroodpesowiftfhilfliillnigngsosloultutiioonn.from bottomofelectrode to wet lower seal. 8c.o2r.r5ecTtihoen,meutseirnganstdatnhdearFd-sewlietchtrcoodnecaernetrtaytpiiocnaslloyfc0al.i0b5r,a0te1d,0b.y5,dir1e.c0t, maenads1u.r5epmpenmtFw-i,tfholnloowbilnagn.k 8t.h2e.m6aRneucfaocrtdurtheer'ssloipnestirnuctthieonas.ppropriate log book. 8.2.7 Clean tissues the electrode by rinsing with Mill-Q"TM water and wiping the sides down with lab 88..33.1SCtaolriabgreatCioonndsittanidoanrsdsfoarreSstoarneddaratdsroom temperature. 9.0 PROCEDURES 99..11.1CaTlhiebrsaatmipolneatnodbeMemaesausrueremdennte,edSsttaonbdearmdixmeedthwiotdh:TISAB using the proportions r9.e1c.2omPmlaecnedaedstibrybatrheinTIthSeAsBammpalneufaancdtuplraecre the sampleon the sir plate. 9el1e.c3trAodleloiswitnhseerstaedm,plmeaktoemsiuxreftohaerrefaerwesneocoainrdsbubbebfloersetirnaspepretdinugntdheeretlheecterloedcet.rodWe.hen the s9a-m1e4 rTahtee assamtphelecaslhiobrualtdiobnestthaendsaardmse. temperature as the calibration standards and stirred at the 9.1.5 When the readings have stabilized, record the reading in the appropriate log book 3 coz58g 9.2 Calibration And Measurement, Using Orion 3E Software: 99..22..111CaFloilblroawtisotne:ps 8.2.1 08.2.4. 99..22..11..32 TPhreesscoFmupnuctteiornscKreeyen#w8il(lF8a)s.k you to confirm the numberof standards to be used, cMoankceentarnaytinoencoefstsahreyscthaandnagredss,toanthdewihneftohremratoironnoptraesbelnatnekdiasntdo cbleicikncolnudCeOdNinTIthNeUcEal.ibration. 99..22..11..54 POlbasceervtehetehleercetardoidnegisnotnhetfheisgtrsatpahnidcadridsoplnatyhoensttihreplcaotmepauntedrc.licWkhoennCtOheNTreIaNdiUnEg.s have. s9t.a2b.i1l.i6zeRde,peparetssseApC9C.2E.P1.T4 aRnEdA9D.2I.N1.G5.for the remaining standards. i9n.t2e.r1c.e7pAtfatnedr ctohrerefilnaatliosnt.anRdearcdo,rdthtehceosmlpopuet,eirntweirlcledpti,spalnady ctohreresllaotpieoonfitnhtehecuarpvper,oparsiwaetellloags bthoeok and click on CONTINUE. The calibration data is automatically copied to C:\Orion\Data\Calib.txt. 99..22..22.1DaStealecStpreeitahdesrhNeeEt:W or OPEN from the FILE menu to open a neworexisting spreadsheet 190.2s.t2o.r2eRdeactoaridn.the name of the spreadsheet used in the appropriate logbook. 99..22..33.1FlFuoolrliodwe sMteeapssu9.r2e.m1etnhtr:ough 9.2.4 9.2.3.2 Enter the name of the sample in the appropriate place on the screen. 99..22.33..43 WClhiecnk othnetrheeadNiEngWs hSaAveMsPtLabEilibzuetdt,ocnlick on the RECORD button and write the result in the appropriate log book. 10.0 VALIDATION 10.1 Quality Control: 10.2 Precision and Accuracy p1p0m3 fOltuohreirdeVuaplitdoaatisoantuPraatreadmseotleurtisonAocfcflourodriindge.to Reference 13.2, the rangeofdetection is 0.02 1.0_DATA ANALYSIS 1L1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 2.0 ATTACHMENTS None 13.0 REFERENCES a Co=s8g 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. Revision Number ReasonforChange Revision Dae 5+ c02582 3E M nvir Labo oratn orymental Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AMDT-M-4 Adoption Date: /o0-7/-55 Revision Number: 0 Revision Date: None ----r---------------------- Author: Dave Christenson/Cynthia Weber Approved By: roup Leader Jo=Da3e/-25 Le kd Quality Assurance M3015 Date Software: MS Word, 6.0 Affected Documents: M:-S, Analysisof Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. sss 1.1 eESltcehocyptler:aocseTpthraiatsyeimmaestushseosddpeticostferxootrsrcatochpteyel.xutorraoccthioenmiocfalflsuforroomchtehmeiclaivlesrsfrfoormarnaablbyistislibvyers. 11..32 MAaptprliicceasb:leRaCbobmitpoLuivnedrss.: Fluorochemicals or other fluorinated compounds. 2.1 Fcloumoprooucnhdemsi,caexltsr,acrtaibobni.t livers, electrospray mass spectrometer, fluorinated 3.1 Use gloves when handling the rabbit livers, they may contain pathogens 4.1 4S.u1p.p1"lieSsyringe, capableofmeasuring 100 uL 44..111.32 EGlpopveensdorf type or disposable pipets 44..11.154 PPllaassttiicc cgernintdiifnuggetbteubses, 15 mL 44.111.176 NLaibterlosgen 44..11.89 FTiilmteerrs, Titan nylon syringe filters, 002.2 um. 44..11..1110 DAinsaploysdacballepiplpaesrtsi:cg3lacscs vsyorliunmgeetsr.ic pipet. 4.112 Crimp cap autovials. 4.2 4R.e2a.g1enAPtqspueoaquuseAoumsmsoonluituiomnoAfcaetmamteon(Aildurmicahc)e,taatpeprboyx.ad2d5i0ngpp2m5:0 Pmrgepaarmemoan2i5u00m awacteetra.teD1i0:l2 ut1e0t0himsLsolvuotliuomnet1r:i1c0ffolarskaa2n5d0dpilpumtestooluvtoiolnu.me with Mill-Q 4.2.2 a(SnoNddai2,1uC.mO0JcgNaorAfbHsoConOad,tie)u/mS0o.b2di5icuaMmr:bBoiWnceaatiregbho(nN2aa6t.eH3CBuOfo,ffeisrno(td1oiaT.u1mBLackaverorbl)ou,nmaetteri(cfalcasoka)nd 44..22..33 Dbirliuntge1s0cveotlounimielwesiotlhutMiiolnl,i-dQilwuatteera.cetonitrile Ethyl Acetate 1:1 with Milli-Q water. 44..22..65MiMleltiha-nQowlater 44..22..87 FTCH'9.5I(H32MHHSpe-cipaelrtfyluCohreomoictcaanlesDuilvfiosnioinc)acid (Aldrich) 0W 25-- 53-- 5$51.21 VUolrttrae-xTmuirrxaexrT25 Grinder for grinding liver samples. 55.34 SCheankveirfuge 5.5 Analytical Evaporator 6.1 There are no known interferences at this me. 7.1 pTehreforrambebidt.livers are received frozen, and must be kept frozen until the extraction is 8.1 8P.r1e.p1arapPterreifpolanuroeorafocncItinnattneecrrsnnuaalllpshuSoantniadcnaadrcdaoirdfdtsaopbperoaxdidmeadtteolyca1c2h pLipvmer1sHa,mp1lHe,,2H,2H- 8.1.2 W1e00igmhLatvloelausmtet0r.1icgfolafsk1.HR,e1cHo,r2dH,t2hHe-apcetrufalluwoeriogohctt.anesulphonic acid into a 88..11..34 vTBoorail2nugm5ei0twumiptLhtoMvvioolllluium-emQterwiwacitftelhra.smCke,atlhacadundloalt3,emtthhLiesoaifcstttuhhaelecssottnoocccekknststrtaaantndidaoarnrdd.ofanthde.bring to standard. : --acsruallpmghopenrifleuoarcocit~dan~e- X 3mL = OIL 250ml actual concentration, ppm 8.2 8P.r2e.p1arePreFpCar-e9F5C-A9n5iosntanSdtaardnsdfaorrdtshe standard curve. 8.2.2 RWeeciogrhdatphperaocxtiumaaltweeliygh1t0.0mg of FC-95 into a 100 mL volumefric flask. 88..22..43 BDirliuntgeutphetosvoloultuiomnewwiitthhdidliultueteacaecteotnointirtriille1e.:10for a solution of aapsporlouxtiimonatoeflyap1p0r0oxp.pm1.0 pDiplmu.te this solution 1:10 withdiluteacetonitrile for 8.2.5 5U.s0eptphme.101.p0ppmpsmolauntdio0n t0o mppabk.e working standards with values close to 8.3 8P.r3e.p1arWeeiBgehef40LigvoefrBoHvoimnoegleivneartinetotao25U0semLfoNralSgteannedabrodrse containing 8.3.2 2A1 0dm0dLm1LamlsiLqMuiooltfsltioh-efQstwhoaeltusetoril.ounGttirooinnadin1t5o1m5aLmhLcoemcneotngrteirfniuofguuegstesubotelu.ubetsPi.orne.Bpaeresuaretottoalreo-feight suspend solution by shaking it between aliquots ------ CORSE Sank. 8.3.3 Spike seven standards in osftetphe9.11m2Lofaltihqeuportoscewdiutrhe.thOenfeoll1 omwLinagliaqmuooutntsserovfe`swaosrktihen.g Working Sardar Approve Tal (Approximate Conc.) I concentration of 1 FC-95 in liver Bak | 8.4 Calculate the actual valuoe f thestandards: ux1L7o1_fmcsgtaloinvedrna'/rdTcmel nhomtogrenaate.t(iinpopmn) = final concoefnFtrCat-i9o5nin(plipvme)r Averageweightof bovineliverin solutionasdetermined by weighing F1Cm-L95hios rmeopogrteend aaostfeeq4us0imvaglleinvtesorifnFC2-0905mpLotoafsMsiilulmi-saQltw.ater. The amount of 8.5 Calibration method 8.5.1 Extract the spiked beef liver `homogenate following 9.13to9.23 of this Spectro.meUtesre.these Standards 10 Sa0lish your cure on he ras 8.5.2 Altematively, a standard curve may be generated using ratios. ofresponses of the perfluorooctansulfonate anion and the internal `concentrationofthe perfluorooctanesulfonate anion. standard anion versus 8.6 Storage Conditions for Standards 8.6.1 icNsoevpewerrefstodarpnmldeaadsr.tdiasc rceenptrriefpuagreetdubweisthunetialchthaenaalnyasliyss.isSotanntdhaerdmsasasresspteocrterdomineter 8.7 8S.t7o.r1agBeeeCfonldvirthioomnosgefnoarteSstamndabraedfsroyzen afer preparation 9-1 pOabctakiangefdrowzietnhloitvherersaemnpluees. In spenttissue, note that the liver has not been, 9:2 9-3 WUseeiagdhistsheecstaimnpglescdailrpeecltlaynidntcoutaoftafraepdpprloaxsitimcagtreilnydi1ngg toufbel.iver. 99:.54 iPRuernacoaarbdetlheolnivtehrewieihghtwiitnhtthehsstuaddy nmoutembboeork.welgh, rabbi ID, dat and anys \ . COR53E 99..67 GArdidnd2.t5hemsLasmpwlaet.erP.ut the grinder probe in the sample and grind for about 2 9-8 minutes, until the sample is Rinse the probe off into the a homogeneous sample with 2.5 solution with no mLs water using large chunks. apipe. 519 TAaVkDe TheESgr3i5nd:er span and clean x with rcinancl shes ach non 9.10 Cap the sample and vortex for 15 seconds. remaining sep 9.11 Piinpfeotrm1atmiLoinnatsothae1g5rminLdcinegnttruibef.ug(eSeteubAe.MLDaTbe-lMt-h4e cWeonrtkrisfhuegeettufboer dwoitchumtehnetiidnengtitchael s 9.12 9.13 fe 9.14 aSspidkeesctrhiebbeedfinli8v.e3r.homogenates with the appropriateamountof FC-95 standard Spike the samples andbeefliver homogenates with 100uLofintemal standard. Add 1 mL of the sodium carbonate/sodium bicarbonate buffer and 1 mL ammonium 9.15 Using an analytical pipet, add 5mLethyl acetate. 9.16 Cap the sample and vortex 20 to 30 seconds. 99..1178 PCuetnutihfeumgeinfotrhe2s0hatkoe2r5fomirn2u0temsi,n.until the layers are well separated. Set the power 9-19 Ronemtohevceen4tmrLifsuogefttohe2t5.oporganic layetro a fresh 15mL centrifuge tube with a 5 appr 9:20 BmlLowgrtahdeuastaemdplgeladssowpinpeot.n Ttrhaenasnfaelryttihceallaebvealptooratthoerfrteosnhetaurbed.ryness with nitrogen, oxima 3010 4 mies. 99:.2212 BVroirntgexth1e5rseemcaoinndisn.g sample up in 1mLdilute acetonitrile with an analytical pipet. 9.23 tThreanssafmeprlteheinstaomapflreestho ace3ntmrLif.usgyertiunbgee.orAtat ach a 0.2 autovial, um nylon Label the mesh tube o filter, r vial and with filter the 9.24 9.25 Cstaupdyanndumhboledrfaonrdaananliymsails nbuymebleerc.trospray mass spectroscopy. Complete AMDT.M-4 worksheetand attach to `poafsgtuedy notebook. 10.1 Quality Control - notapplicable 10.2 10.3 OPtrehceirsiVoanliadnadtiAocncPuarraacmye-tenrots.apnpolticaapbplleicable 11.1 None 12.1 Worksheet AMDT-M-4 A reTT 2 4.0 REVISIONS Revision Number Reason forChange Revision Dae ---- COR556 WorksheetAMDT-M:4 ee 1Ft ayT p wapt a E r F Et tE s y F A --E---- r FF r E tt E S SRE -- ------ F F tF ---- EE eeF Lee F pe--a -- s --] erTe -------- -- a EE ---------- Pee mlof20oo Amaia eee--------gg e E Er e T ---- ee-- Peete | E emSa --_-- e -- a = ----] ---- pE eE -- | O---- cozssg 3M Environmental Laboratory Method Analysis of Rabbit Liver Electrospray Extract Mass for Fluorochemicals Spectroscopy using SOP Identification Number: AMDT-M-5 Revision Number: 0 Adoption Date: (- -95 Revision Date: None Approved By: Author: Dave Christenson/Cynthia Weber iroup Leader 4 Z Quality Assurance sl/es Date 1d Date Software: MS Word, 6.0 Affected Documents: M-4, ExtractionofFluorochemicals from Rabbit Livers . cm 1 02559 1.1 Sflcuoipdse:fTohrifslumoertohchoedmiiscaflosr uthseinagnatlhyesiesloefcterxotsrparcatysmoafsrsabsbpietctirvoemretoerro.tThehretainsasluyessiosr isstapnedrafrodrmMe/dZb=y 4s2i7ng,laenidonotmhoenritapoprrionpgrioaftFeCm-a9s5seasn.ion, M/Z= 499, the intemal 11..23 AMaptprliicceasb:leRaCbobmitpoLuivnedrss:(sFalmpuloerso)c,heBmeiecfLalisveorr (ostthaenrdafrlduso)r,inoattheedrctoismspuoeusnadnsd. fluids. 2.1 sFpleucotrroocmheetmeirc,alrasb,biftluloirvienrsa.ted compounds, electrospray mass spectroscopy, mass 3.1 Uinsteo cthaeutpiroonbweiDthOthNeOvToltTaOgeUcCabHleTfHorEtPheRpOrBoEbe,.tWhehreenistrhieskvoolftaelgeecrcraibclael isshpolcku.gged 3.2 lDoopnoatnrdurneltehaeseppurmespsuarbeo.vTehiet'specaapkaucibtiynogfm4a0y0b0eppsil,ugIgfepdr.essTurroeubgloeesshoovoetrb4c00k0topsi 3.3 fDionndottherpulnugthaenpdurmepplatcoedrtyhenepslsu.gged tubing. See AMDT-EP-15 4.0 SUPPLIES AND MATERIALS 4.1 S4.u1p.p1liNeistrogen gas regulated to 140 psi. 44.11123Fl1u0o0fuiLxocr 2o50 ulolrefulqautimivpalsneynrtinge for sample injection 4.2 4R.e2.a1genDtislute acetoniuile mobile phase, dilute acetonitrile 1:1 with Milli-Q water. 4.2.2 Milli-Q water, all water used in this method should be Milli-Q water. SOEQ5.U1 IPVMGETrNioT2000 Electrospray Mass Spectromet~er~or~equivalent 55.32 ISpSoCcOmsSpyhryisnigcesPAuSm3p00 Auosampler 55..45 A10utovuiLalAsssoremcbaplpyed centrifuge twbes. 000000 6I .0 6N .1 TT here arE e no knR own intF erferencE es at thR is ime. E ~~~NCES 1.0 SAMPLE HANDLING 7.1 Keep the extracted samples in capped 15 m_L cS entrifuE ge tubes or in-- capped autovials unil ready for analysis. -----coz599 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of Calibration Standards 8.1.1 Seven beefliver standards and one blank beefliverare prepared during the `extraction procedure. (See AMDT-M-4, section 8.0) 8.2 Calibration 8.2.1 1R0uonbttahienstehveensbaenedfalridvecrursvteandards twice, starting with the lowest standard 8.2.2 Typically one standard is run after each 5to 7 samples. Choose a standard in the same rangeof concentration as the samples. 8.3 Storage Conditions for Standards 8.3.1 cForveesrhesdtapnldasatridcsaCrenecApTruegpearteudbewsituhneiachheaAnanlaylsyissi.s Sotnanidnarmdassasrespsetocrieodmientr is performed. Samples and standards are NOT refrigerated. 8.4 S8.t4o.r1ageBeeCfonidviertihoonmsogefonrateBseemf LbiaevefrryozHeonmiotgesepnraeptaersation. 9.1 Initial Set-up po 9.1.1 Set software to "Operate on", Ion Mode ES". 9.1.2 Record backing pressure in the instrument log. 9.1.3 Fill the solvent cylinder with mobile phase. 9.1.4 Set the pump to "Run". Set the flow to 1000 uL/min. Observes droplets coming out of the tip of the probe. The pressure should be at 1700 to 1800 9.1.5 Check the fused silica at the end of the probe. Use an eye piece to check for chips. The cutoffthe ttiipposfhothueldsibliecaflwatitwhitahcnoolujmanggceudtteedrgaensd. If any chips are found pull the silica through to the appropriate length. 9.1.6 nCihtercokgeynoluerakniintgroagreonusnudptphley.tiTpoufntohen the nitrogen. probe. A fine There should be no mist shouldbe coming. out of the tip. 9.1.7 Carefully guide the probe into the opening. Insert it uniil it won't go any further. Connect the voltage cable 1 the probe. 9.1.8 Go to the "Editor" appropriste masses ptaog4e2,7anadndse4t9I9o.nization Mode to ES', and the 9.1.9 If itis not in single ion mode go to "Option" and set SIR. 9.1.10Start Acquisition. Assign a file name, the log book. MO-DAY-YR + letter. Record it in 9.1.11 an the beef liver samples iss, running each sandard twice at the `beginning of the run.. Run a QC check by running one standard after every 9.2 ManualSIenjescatimopnes 9.2.1 Draw 150 ul of sampleinto a syringe. Inject the sample into the rheodyne injection port. Inject slowly. Record the sample ID in the log book. 99..22..23 TWauimt the valve 10 "On". wo minutes, and inject the next sample. 9.2.4 Record the scan number for each sample in the logbook. ow OR590 9.3 Using the Autosampler 99..33..12 vRSieeatclosurpmdastyahmebpeslaeimnptcrlaaeycshAa,rnudnB.,thoerirCp.ositions in the instrument log book. Up to 17 9.3.3 S9e.t3-.u3p.t1he sampl"ePrus:h the sample buon 99..33..33.32 SSeetts inja eclyom soapms=pip lz2ee=l 100e ul. 99..33..33.54 9.3.3.6 SN et Cyacthleeftiim lme:eLi=e v0ers Identifythe tray used 9.3.3.7 9.3.3.8 `Add the samples to Queue by pressing "Enter" Press "Rutonsta"rt 100 VALIDATION 10.1 Q1u0a.1l.it1yRuCnoantsrtoalndard every 5 107 samples. If a significant change( 50%) in `Spaenadkahredigwhitlolcbceurusseds.toTphteherreumn.aiOnnilngy stahempslaemsplweislbbeforreeantahleylzaesdt.acceptable. 10.2 Precision and Accuracy 10.2. 1See Method Validation Report number AMDT-M-5.0.V1 10.3 Other Validation Parameters 10.4 Refer to Method Validation Report Number AMDT-M-5.0.V1 11.1 Calculations 11.2 Plot the standard curve, using the mean of the two values obtained for each s1t1a.n2d.ar1dR.ead peak heights or areas for the samples from the printout. Use linear regression to determine the sample concentrations. 11.2. 2lCiavlecrulate the mg of FC-95 anion, or other fluorochemical in the total rabbit mg FC-95 anion in the total rabbit liver = mgmsE ofliF vaenrioC unsfedr. ofomrsat9 ndalcyus5 irsve x Total mass of liver, gms 1111..34 PMraiknteaacrhersoulmtasttoagbrleamanfdoernetaerchitsaimnptlhee,swtiutdhy book. the peaks labeled with the sample or standard ID. study folder. Write the Staple all study number on the printout, initial, date, and chromatograms together and number pages. put it in the 3 - E502 1A 2.0 TTACHME ~~N ~ TS None 13.0 REFERENCES ~~~ 13.1 AMDT-EP17 14.0 REVISIONS ReNvuimsbieorn Reafosrchoannge DRevuiseion Lo--n--n--ed- a. C 02592 3M EnvironmentalLaboratory Method Analysis of Fluoride Using Ton tSheeleSctkiavlearElSeecgtmreondteed Flow Analyzer With Method Identification Number: AMDT-M.-8 Adoption Date: Jp-s.q Revision Number: 0 _-- Revision Date: None Author: Deb Wri/gCynhthtia Weber Approved By: up Leader LBL g a on Quality Assurance 1/5/75 Date 1-27-95 Date Analyzer Software: IBM MS Word, 6.0 Affected Documents: AMDT-EP-26, `Operation and Maintenance of the Skalar. Segmented Flow pa 0259% 1.1 This methodi the Dohrmann for the analysisfor fluoride, DX2000 (AMDT-M-1), thermally extracted from samples using Lon Selective Electrode (ISE). The analyasnids cisolpleercftoedrmiendTuIsSiAngBtfhoerSaknaallyasis with an Segmented Flow Analyzer with ISE. 1.2 Samples can be the Dohrmann. tissues, serum, biological material, or other materials. extracted on 2.1 Skalar, segmented flow, fluoride. 3.1 Follow standard laboratory safety practices. 4.1 S4u.p1p1liSeusmple cups, 4 mL plastic cupswith caps 4.1.2 4.1.3 PAoultoyppirpoeptysl,eonxefvoorldoumreetqruiicvfallaesnkst,w1i0t0h pmlLa.stic tips 4.1.4 4.1.5 Canridge components, refer to Snuammbpelres,preflters, Evergreen the Skalar Methods for components andpart 4.2 Reagents 4.2.1 Brij 35, 30% S.F.A.S.Detergent 4.2.2 TISABI buffer solution: Purchase TISAB II rom Orion. To | lier of 4.2.3 TISAB IT add 2.5 mL or Sampler rinsing solution: 100 ppm fluoride Dilute TISAB II solution 1:1 with and | mL Brij. Milli-Q water. 4.2.4 tmNhiLetsrvioccloanuccmiedenstuorplauttteoido1nnLitfrowirict`ahdceiMcdioln(tlaHim-NiQnOaw)tatiteoorn.,2510NmL(lsabogfraMdiel)l:i-SQlowawtleyr.adBdrin6g4. 4.3 Standards 4.3.1 Stock solution, 100 ppm F: purchased from Orion. 4.3.2 IwinttherMmieldliia-tQe swattaenrd.ardU,s1e0poplpymp:roDpiylluetnee1v0olmuLmsetorficstfolcaksksso,lution to 100mLs with MllEQ war 4.3.3 OvWxooflroukrmidensoogrfsptuaimnndtpaemrradm:tedeMiaaptikpeeerosur,pstttoohce5k0fSomtllaLonswdioanrfgdTiwInodSriAckBaitneagdnsdotndaintldhuaetritdnasbglbe6y, adding the u1s0i0ngLs S Ceno ---- mmee y7e--S--an--d om em Sem-- Ty | 2 02594 S.1 Skalar Segmented Flow Auto Analyzer SansTM System equipped with ISE cn 7-1 wSiatmhpilne3s0shdaoyusl.dbe stored in polyethylene botles. Samples should be analyzed 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 `beginningofthe run. 8.3 Storage Conditions for Standards 8.3.1 sSttaannddaarrddssaarree psrteopraerdeidn actaappmeidnpiolmyupmroofpyelveenrey vsoixlummoenttrhisc,floarskass.nNeceewssary. S13 RTE pte bt 9.1 Start 9-11 UCplaPmrpodcoewdnurtehe pumpdecs, air bars and sampler-pump tubing. spt 9-1.3 Tum on the power of the sampler, pumps, offset potentiometer and heating 99.11..54 Pbuattht.he r Tum on eage the nitn-tlerifnaecsei,n ctohmepauptperro,p rdiiastpelabyoraensd. printer. Make sure you 9.1.6 LD oe turn on the interface before the computer. 52 girig aptiuno otats htor1 sthe computer 92.1 Create a sample able by selecting FILES, TABLE, and CREATE, type in 9.2.2 PPrRiInNt Tt,heGsRamOpUlePta1.blTeh,iisnsweirltledpriinntthteheseynsttierme traunb,le by pushing ESC, on BIS Bea unk 9.2.3 Dial the sampler settings to the appropriate number of samples, number of of spe 9.2.4 Fill the sample tray 0.2.5 SOUT FW/RUNOUTcups with the standards, samples, washes and on the sampler do not need (0 be filled, drifts. IW and -- 6025983 9-2.5. mIaScylebcet iGnRtAhePHDaItCaSH,anRdEliAnLg TPaInMeEl..SIwfiytocuh ctaontnhoetAgneatlyrseails-Ptiamnec,yl obyu selecting CONTROL PANEL and pushing F7. 9.2.5.2Use the base line. sAmdajlulstsctrheewbdarsievleirnefournttihle iotfifsseatppprootxeinmtaitoemleyte3r/4toisnecth the from the bottom of the screen. 9.2.5. 3Check the highest standard the interface screw #3. and adjust the gain, if necessary, with 9.2.6 Gwioltl onoCtObeNTrunR.O(LSePleAcNtEoLr,deasneldetcotanaanlalyyssiisspabnyeplr.esDseisnegleEcNtTtEheR.an)alPyrseissstThaatb 9.2.7 tProersesttuhme tsoptaheceAnbatalorysbirsinPganuepl.the local menu. 9.2.8 9.2.9 STeylpeectySouTrAIRDT(itnoitsitaalnst),thtehaenaslaymsipsl.e table whigh you created under 9.2.1(or press ENTER for choices), choose running with or without the system table 9.2. 10AafntdesrelsetcatrtSinTgAtRheTsAofNtAwaLrYe,SIstSar,t the sampler. Make sure that the. sampler is sOeKt.to the right numbeorf samples and that the `sample/wash/air times are 9.2. 11Select 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 and select LOAD. Select AUTOCALCULATION and highlight the fileto be printed and press ENTER). enter the filename (or 9.3.2 To view the calibration curve, 0 10 GRAPHICS, CALIBRATION 9.3.3 CURVE. To print the high level curve, push PRINT SCREEN. 9.3.4 To print the low level screen, push ESCtoget out of graphics Select SETTINGS. `CURVE and Change the press ESC, maxy value t2o pproximare ly 900. Go and Ler Press PRINT SCREEN. to CAL 9.3.5 pRreetsusrnENtoTSEERTTanIdNGPSRIaNnTd cShCaRngEeEtNhetomparxinvtaslauempblaeckpteaoks4.095, go EDIT, 9.3.6 Toprint 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 rinsefor approximately30minutes. 9.4.3 Aoffftseert tphoetesnytsitomeemthears.rTinusmedofcfomtphleehteealtyi,ngtubmatohfofnthweeseakmepnldesr., pump Leave and liquid in 9.4.4 Tthaekelinthes.clctrode out and sok in 100 pp F overigh, 9.4.5 Release the pump-decks, air bars and sampler pump-tubing, 9.4.6 Select FILES, press ALT Fand select QUIT to exit theprogram. 9.4.7 On Friday, tum off the computer, display and interface for the weekend. loovapamion 0000 10.1 Q1u0a.l1i:t1yRuCnonatsrtoalndard (mid to high concentration) every 10 samples, Ifa ascigcneipftiacbalnet csthaanndgaerdinwpielakbehuesiegch.t Tochceurrse,maoinnlyintghesosmapmipelseswbief5orertehneeulnasseted. 4 oz594 10.2 10.3 10.4 Precision and Accuracy 10.2. See Method Validation Report number AMDT-M-8.0.V1 Other Validation Parameters Refer to Method Validation Report Number AMDT-M-8.0.V1 11.1 1C1a.l1c.1ulTahtieonsstandard curve is plotied by the Skalar software. 1111..32 11.1.2All calculations are done by the Skalar software. better. * shouldbe 0.995 or WPrreiptaertehespsrteuaddyshneuemtbsetroosnumtmhaerpirzientdoautsa,.inIinicallu,dedastaempthleepvroinltuomuet,, awnedigbhitnsdutsoegdeicniec,r siutmhmaallrypsahcekeatgeanddocpumeenitnstoatnhde psltaucdeyinnottheebosotk.yBfaocldkeru.p Malalkdeat4a caonpdyospfrtehaedsheets onto study disk and backup disks. 11.4 Electronic Data 11.4. 1GLP studies: Electronic data is copied onto the Study floppy disk for each 11.4. study, 20ther and also data is copied onto a floppy disk that is stored in studies: All data is copied onto a floppy disk that is stored the lab. in the lab. EE -- DX2000 Organic Halide AnalyzerLiver 13.2 Skalar Methods, #335, Skalar Methods Manual 13.3 AMDT-EP-26, Operation and Maintenance of the Skalar `Segmented Flow Analyzer 40REVISIONS Revision Number Reason forchange 0000000000000 Revision Dae ; GRAeey coz;bog 3M Environmental Laboratory Method `Thermal Extraction ofOrFglaunoriicdHeablyidMeeAannaslyozfaer Modified - Serum Dohrmann DX2000 Method Identification Number: AMDT-M-14 Revision Number: 0 Adoption Date: /0-3-7 Revision Date: None -_-- Author: Rich Youngblom Approved by: For) roup Leader. /0/5/ 7s Date gE A Quality Assurance Fr2z Date Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Expandable Ion AMneaalsyuzrerement by Means ofan Orion EA940 AMDT-EP-3 Organic RHoaultiidneeAnMaaliynzteernance ofa Modified Doi hrmann DX2000 TE. 562 0259g 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES wf1.il1tuohSraicndoepiefo:rnosTmehlivesacrtmiievoetushelomeadcttririsocdfeeos.r. thTehoepefrlautoiriodneoisfatyDpiocharllmyacnonllDecXt2e0d0in0TwIhSeAnBitsiosluutsieodntfoorexatnraalcytsis 11.23 MAaptprliicceasb:leBiCoolomgpiocaulndflsui:dsF,lpuaorrtoiccuhleamrilycaslesruomr.other fluorinated compounds. 2.0 KEYWORDS D2.X12F0l0u0or,idfel,uofrloucorhienmei,caelxst.raction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, 3.0 PRECAUTIONS 33..12 GGllaasssswwaarree iasnfdraegxihlea,usbtrogkaesensgclaasnsbmeaeyxtcraeumseeliynjhoutr.ies. 333...534 PPSorotelesvnsetuniratilzbbeaidsoeghdaaszseaasrm,dppslredosupeemrtaocyothmfelparbsehi,oslsmoegadiycganalesmeahdtarntidocleaisl.nlgoUwpsrtaehctaeipmcpertsooprdreriqyuaitdreeodpw.enrsboenfalorperostteacrttiinvgerun. equipment. 4.0 SUPPLIES AND MATERIALS 44..21 CCoommpprreesssseedd OHexlyiguemn,,HHiygdhroPcuarirtbyonGrfardeee,,rreegguullaatteeddttoo 3405PPSSLI. 44..43 QQuuaarrttzz ggllaassss scoammbpulsetbiooanttwuibteh, TReelfilaoncneTMGtluabsisng,G-D9o4h0r5m-a0n1n2 o8r90e-q0u9iv7aolrenetquivalent, 44..65 SOraimopnle94co0l9l9e9ctTiootnavliaIlosn,iHc DStPrEength Adjustment Buffer (TISAB 11) or equivalent 44..87 MPoillylsity-rQeTMnewaptipeerttes. 44..910AcHtaimvialtteodnCShyarrcionagle,oEr.eqMueirvcaklen2t005or equivalent 4.11 Miscellaneous laboratory glassware 5.0 EQUIPMENT 5.1 5.2 IRoBsMemcoomupnattiDbolher3m8a6nnorD4X8260c0o0mpOurtgear.nic Halide Analyzer, modified for fluoride extraction. 55.34 EDxXc2e0l0S0presoafdtswhaereet,, vveerrssiioonn 15.0.0o0,rmgordeiaftieerd for fluoride extraction. 6.0 INTERFERENCES 6.1 Sample size is limited to approximately 100 2. This may vary from matrix to matrix. 2 c--oz--2c5u99 7.0 SAMPLE HANDLING 7.1 Samples are to be handled with plastic pipettes. A new pipette is to be used for each sample. 8.0 CALIBRATION AND STANDARDIZATION 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. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit serum studies, usebeefserum as the matrix. 8.2 Calibration - Overview `The normal calibration is the fluoride curve (AMDT-M-2). However, if an `optional spiked serum curve is required the procedure listed below is used. 8.2.1A calibration curve for the DX2000 is generated by spiking samples with known standards a8.n2d.2coTympbiucsatlilyn,g tthhreeemruespilincgattehseosfaemaecmhestthaonddasrdanadndmaftirviexctoynpceeantsratthei.onssamopflsetsantdoabredstewsitleld.be spiked. R8.e2c.o3veSrteadndFar(dugc)uorvnethweilyl-abxeisp.lotGteenderaastMeaasrsegSrpeiskseidonFcu(ruvg)e on the x-axis and calculate and the Standard equation Mass for the line and the r2 value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Conc. ofstandard in ppm) x (0.6004)* 8.2.5 Standard*MFaCs-s95Reics o6v0e.r0e4dF% F(tuhge)re=fo(rTeI0S.A6B00v4oilsutmhee ifnacmtLor)uxse(dOtroiocnonrveeardtinFgC-in95pptom)F 8.3 Calibration - Procedure 83.1 Start Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be usedif the previous samples contained high concentrationsoffluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and typeofmatrix as the test sample. 8.3.2.2 For rabbit studies, use beef serum as the matrix. 8.3.2.3 analyze sPautmpsleeruacmcobrldainkngintoDmoehtrhmoadnAn MboDaTt.-MC-o2mbfuosr tthseaimopnlesealsecdteisvecreilbeecdtriondeseacntailoynsi9s..0 and j8bu.e3df.og2re.em4epnrFtoocroefreadtbihbneigtopwseitrtuadhiteotshr,ettchhaeleirbmereaatdtiieonrng.riesaBsduianbmglesfawomiarptlhbelrsaesnupkneticsltatm0hpilsheilsistmohirotiucilasdlrrebeaeacd0hie.dn0,g3sop(rppurmnetvoiilroilunoswt4eh8er hours). 8.3.2.5 For non-rabbit studies, the blank readings should reach a predetermined ion concentration before 8.3.2.6 proceeding It may be with the calibration. necessary to mix approximately 50 mgof charcoal with the sample to aid `combustion. , CORS00 883333.1SItFabnedefasrcdrCuumrvi efrozen, thaw at least enough to complete the standard curve analysis for the d8a3y3.(2=30PimpLe)t.te 100uLofbeef serum into Dohrmann sample boat. `8q3ua3ni3tySotafrtthweistthantdhaerldoowneostrsitnantdhaermdatcroinxc.entFroartiroanb.bitUsstiundgieas,Haumseil4tounLosfyrsitnagen,daeredcaatndfeijxeecdt it on o8r33in.t4heAbteleefassetr3umr.eplicates should may be used at the discretionofthe banealuyssetd. for the lowest standard concentration; more replicates 833.5 83.3.6 RCuonmbaulst1Sthsetasndaamrpdlse. Iasfdoensecrriebpeldicianteseictsiiognni9f.3icaanntldyadniaflfyerzeentacfcroormditnhge to AMDT-M-2. other two roelpdlirceaptleisc,atreuannadnothtahterthseanmpelwerfeoprlitchaatteswtialnldabred.usIendditocactaelciunldaatteathtehartetghreesnseiownrceuprlviec.ate replaces the. 8le3as3t.07.95W,hceonnsaullltswtiatndhasrudpserhvaivseorb.een run, calculate the r2. r2 must be at least 0.95. Ifit is not at 8c.h3a.n3g.e8d.ANneeww ssttaannddaarrdd ccuurrvvee mshaoyuladlsboebreurnuwnhaenthtehdeicsocrmebtuisotnoifonthteubaenaolrysst.ample matrix is 8.4 Storage Conditions for Standards 8T.y4p.i1caSltloyr,asgteanrdeaqrudisreamreensttsorfeodr astanrdoaormdsteamrpeedraetpureenidneponlnatsttihcesicnrdeiwvitdoupalbostlaensdards used. 8.42 New FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES 99..11.T1yCpoimcbaulsOtpieornattuibnegtCeomnpedriattiuornes:= 950C. 99..11.32VOaxpoyrgieznatainodn/HDerlyiiunmg ftliomwe == 52040ccs/emcionnudtse.. 9.1.4 Bake time = 300 seconds, 99..22.1StIafrttheUpprPorgorcaemduisrneo:t started, start the EOX program on the PC. 99..22.32 POuptetnhethfeumSaYcSeTmoEdMulSeEaTnUdPthweicnedllowi.n the READY mode. 99..22..54 WClhoesne found in the tthhee oSvYenSThaEsMreSaEchTeUdPthweiRndEoAw.DY CELL CHECK menu temperature, run the CLEAN BOAT program 9.2.6 See AMDT-EP-3 for detailsofthe Dohrmann software. 99..33.1SaOmppelnetEhxetSraAcMtiPoLnEPrHocAeTdCurHe:and pipette 100uL of sample into the BOAT. It may be sdntoeancneed,sascrahdraycrtucroovameli.sxhoauplpdroaxlisomabteelmyix5e0dmignowfhiclheaersctoaablliwsihtihngthtehesabamspelleinteoaanidd cwohmebunsgteinoenr.atiIfngthtihseis 9.3.2 Close SAMPLE HATCH cGu026--0 l9.a3b.e3ledAdsadmpaplperocporllieactteiovnolviualm.e oTyfpTiIcaSlAlBy 0s.o6lumtiLontoor151:m1 LTIaSrAeBu:seMdi.llFio-rQrTMabwbiattesrtumdiixest,urueseto1a.0 or 92..3.04mLPloacfe t1:heTIviSaAlBs:oMtihlatlith-eQTMtipwoaftetrhemiCxtOuMreB.USTION TUBE is in the TISAB at least 0.25 9in.c3h.e5s.RuGnastehse EreOleXa-seWdAduTrEinRgppryrooglryasmisfmouusntd binubtbhleeRtUhrNoumgehnuth.e TISAB. 99..33..67 IWFhunedniltuhteeEd OTIXSpArBogwraasmuissefdintioshceodl,lercetmtohevesatmhpelceo,llaedcdtiaonn evqiaulalfrvoomlutmheecoofmMbiulstliio-nQTMtuwbae.ter 91.03t.h8eRTiInsSeAtBhetoenmdakoef 1:1 the TISAB:Milli-QTMTM combustion tube with Milli-Q water and wipe with a KIMWIPE to 9r.e3m.o9vOepaenny tThIeSsAaBmprleemhaaitncihnganodn rtehemotuvbee.any remaining ash from the boat. Ash can be removed bwiotthtoamcwotittoanhtsippapteudlaapoprlioctahteorrsiamnidl/aorrdveaviccueu.meAd odurt.opIotfmMaiyllbie-nQeTMcewsastaerry tmoasycrbaep apadrdteidcletso othfef. the. 9b.o3a.t1t0 oCaldosine tthhee Chlaetcahn. Cycle. 99.33..1112 SRaumnpltheeisCrLeEadAyNfoBr OanAalTyspirsobgyraimo.n selective electrode (AMDT-M-2). 9.4 Sample Calculations 99..44.21 USsaemptlhee sMtaasnsdarRdeccouvreveretdo cFal(cuugl)at=e(tTheISsaAmBplvoelvianlume.L) x Orion reading inppm - intercept) (Slope) 10.0 VALIDATION 10.1 Quality Control a1n0a.l1y.s1isDaiislsytaSrttaerdtbUypanCahlyezcikngSQamCplseasm:pleOsn.ceTthhee QstCansdaamrdplceusrvaereistoesbteabtlhieshseadm,ecaaschthdeayloowfest dcoonnceenitnrtartiiploincastpeiukneldesssamtphleefsirustsetdwotoregpelniecraattees tahree swittahnidnar2d0cu%rovfet.heEascthancdoanrcdencturravtei,otnhmenusattbhierd replicate is not necessary. F1l0u2oriPdreecNiostieobnooaknsd 2A,c3c,uarnadc3y.: PSreeceismieotnhaondddaecvceulroapcmyevnatriaensalwyhseisnaanndalsyazmipnlgesaanmapllyessisoifndifferent matrices and different reference compounds. 10.3 Other Validation Parameters: NA 11.0 DATA ANALYSIS 111 Calculations 1111..11.21 TFoorctahlecusltaatnedtahred fcluurovrei,deusceonrtergarcetsisoinoninatnhaelyssaimspilne,Exscceel,mevterhsoidonAM5.D0To-rMg.re2at.er. CORREO 11.2 Analyzing the Data a1r1e.2w.i1th2inm2u0s%tobfe eaatlcehasotth0e.r95anodr tghreeaotuetrl.ie"rOuitslgireerast"ermtahyanb2e0e0x%colfudtehdeifavtewroaogfetohfetthhorseeetrweoploircates less than noted in t5he0%FionfaltRheepaovretraagleonogfwtihtohsethtewor.easAonnyitswuachs ocuotnlsiiedrsersehdoualndoubtelipero.inted out in the data and None 13.1 349, Rosemount revision B, Dohrmann December DX2000 1993) Organic Halide Analyzer Operator's Manual (Manual 915- A1n3.a2lyzAeMrDT-M-2 Fluoride Measurement by Meansofan Orion EA940 Expandable fon A1n3.a3lyAzMerDT-EP-3 Routine Maintenance ofaModified Dohrmann DX2000 Organic Halide 140 REVISIONS Revision Number ReasonforChange 000 Revision Date 6 GE 02603 9.3 Quality Assurance Unit Statement ---- oz604 AtachmentD GLP Study Quality Assurance Statement Study Title: Single-dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Study Number: AMDT-120694.1 NaofAmuditeor: Kari Rambo `TThhiesfstuidynhawdsebrieeernnepiongrstpseecdtetdo tbhyetshteuQduyadliirteyctAosrsaunrdanmcaenUangietmeansti.ndicated in the following table. Inspection Dates Eom Te -- Phase 1109.95 11-1395 Final Report Date Inspection Reported to Management StudyDirector 1-13.95 11-1395 4 Ll sess AU Auditor Date 1 C 02608 9.4 Key Personnel Involved in the Study a c02606 Environmental Laborato Key Personnel `Thermal extraction Jim Johnson followed by analysis using Orion fon analyzer: Deb Wright Rich Youngblom Deann Plummer Analysisof liver extracts using electrospray mass spectrometry: Jim Johnson Dave Christenson `Thermal analyzer weixtthraicotniosnelfeocltlivoeweedlebcytraondael:ysis using Skalar segmented flow 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 | CIA CoRe0g 9.11 Data cozsog d9.e1t1e.r1miSnuemdmbayrtyhearnmdalraewxtdraatcat;ionugfoFl"lionwewdhoblyealniavleyrsiass using Orion ion analyzer. C02n 689 Summaryof Combustion Data - Liver AMDT-120694.1, HWI 6329-151 As Referenced in Final Report section 6.0 DATA ANALYSIS `TotalMuegaFnlupoerridDeosineWGhrooluepLiver ug Control Group 19.7 4.0 mg/kg dose (T6067) 42.9 16 me/kg dose (T6067) 66.4 24 mg/kg dose (T6067) S44 o261@ RPTISILXLS Lrassz pk % PAcMtEua.l inlver Apveormag.e inliver buivmeerd Wvheolre weight TinawholFl-e ler Dosage ivveerrbbiik2k1 verbk 3 revry o(s02Wo8W16) (WW) (g00r11a45m50s) (grams) (rg) 0252 0138 (mg/kg) iivveerrssppikkee21 9o1%% 11.0080 iverspke3 84% 0985 a01a2a7 0129 FFs2a7r2ez.-21 Fsarez.3 00328s4 020 0o%i e67777 02 oz 77 197 op FsF22rr6633--12 Fs2763-3 o07m32 oso ooiuis T7sp os2 0 72s a9 40 FFss22rree88-21 Fszress 007792%7 ams o01i2s s85s7 oso 011s 857 ees FFss22r77r00.12 Fs21703 00870s8 ome o01t0 6s0z2 0718 oz es2 see iivveerrssppikkeeds 9867%% 11.0179 0oa1t3 Liver bank ozs 012s co261 Pat w------ d9.e1t1e.r2miSnuemdmbayrtyhearnmdalraewxtdraatcat;iopnpfmolFl*oiwnedsebryuamnaalsysis using Orion ion analyzer. COR61R HWI 6329-151 AADnoMahlDrysmTiasn1n2Da0St6ee9s4r:.u10m7/A2n1a/l9y5si-s07/31/95 All serum samples were thermally extracted by a modified Dohrmann TeDxIXpS2aA0nBd0a0bsolOlerugtiiaoonnni.acnTaHlahyleziedsrea.mApnTlaehlseyzwDeeorrhearnmmdeaacnosnlulrweeacdtseodcnailnainbarOa1rt:e1idomnuilsEliiAng9Q43w04aptemr,and 40ppm, 62pm, 100ppm, 124ppm, 250ppm, and S00ppm FC-95 standards. sTulshoiepnegO,r0ii.no0tne5rpwcpeampst,,c0aa.ln1idbprpcamotr,erde0l.ba5ytpidpoinmr,ewcet1r.me0eparpesmcuorarendmdeedn1ti.n5wptihptemhaFnp*oprsbtolapanrdniakartdceso,rlroegcTbthoioeokn. A2).su`mTmhearvyaltuaebloefi"sNiDnc"luhdaesd,besehnoweinntgertehdefporpmanFy"sianmepalcehwsiatmhpalne O(rsieeonpage reading of below 0.05. An initial calibration curve with standard deviation, %RSD, R? value and equation of the line is on pages 3 and 4. wsPiaatmghpels"e5NsDaw"nedrie6nsbseerhitonewgd atwnhheaelreyexzceetdhl.esOpPrraeigaoednsshr7eeeaatdnitdnhga8tisswhabosewlgotehwnee0rs.a0at5me.ed wsphreenadtshheeet ` aot r ammo. Coos on fuego 5,445 wwoddad Yo Juport on J2onhaj.udtPadgoengwsilTJTen2upmioSnd Page Lof8l Te. "sles ~ coz618 ------- Linz PK Dosage: Omgikg Dosage: 4mgig Dosage: 16 mokg Dosage: 4286mahg PTY STUD#Y 6326-151 SERUM Fluoride concentrHaWtiIon63in2r9a4b5bi1t serum (ppm F-) Sample 20Mhour 4.0hour 60hour 8.0hour 12.0 hour 24.0 hour 48.0 hour FS2762 125 58 ND 35 298 122 ND Fs2763 400 325 227 189 201 141 ND Fs2788 149 805 43 287 272 ND ND FS2770 410 194 127 808 440 NO ND C 02614 . `2 Ts1cHART 0SChr 12 Wnwas among o _ _o 3 --- oe o . a v3 3 " - ne hpes ndnl Page 1 ~ p can Ow 1 Ay wns 16mang w e _ _-- -- e -- e -- ee 0 e FR e = -- e a --_-- rrrr ee , BE a 2 . me spootdone! . " es 8 aot a2 1S1CHART.XLS Chart 10 MW has 24mg/kg. pe_ ------ _---- 100 _ -- e _-- _ --_-- ---------- -- 2 & tpg n s 2 2 2DO ot ~~ NOMA SERUM CURVE 1 7 Aki sae TISAB FC Cone ES CR 0%1 Ack Mads Mas B UTu(mpeinmfrdas || reoGammdnesgaO0ogt1iyfaa v300o spbokmseepwde edeso%oon n terecelncoouevexmcryy. Pi1boehmmsFmme- GeowopgkoeyeFdsTraceewcopriiveelimred we ( Dffowmmmmmsa Gomi oooommmms 000111 oom 01 2ZZ0oo ooommmiu do oom awa ielmew uiwsiess occeoomww oooreresms wseev ousF db be lhe oom Ge GGGToaommmmsiai oaoDoimwmess o000i111 22ZZ00oo Gooommmmee tBmm eoewlJeemk ha lass lik oGommm ocorwreemnm aseev obs ia sev omwsu0s9 aos DBBeiiwmmmsai Genz BOOhRmm 000111 Dum 01 Z3Z0oo ooSmmdee 30 bos diimmm wswkee mm uuumdb dom ooommmm om 5bb5ea8en wee soe ame ame msTmU HBGoowmwmmnias Bomms SSihswah o0Gl11 own 01 23Z00o obooommsa 0dmmm do om 3 wlmowmw io Gniamae ere booemmmm oom aboeunans wseev onm amor( SSSiimmmnmsls cSiaummme 000111 ZxZooo oObmomis sssmm akrewe weomeme jiSahamems dobomewess swewee oweUsN SERUMCURVE 1 NORMAN (0722556) | os yrVoroasomms . : || l|| gZwe : |[ | He | | | = . : | or | I I) om ow Er) Too ao on| MASSSPIKED (ug) | -- o2618 TEW-ld3s serum curve1 nomen ras ffu : : for R Nz . yd1 [I B= = ~ En r= = Too w+ STUDY# 6326-151 SERUM Sam0ple sBaONRkKz BBLAANNKKA sBAnNkKsS ec2PPPPMM1z c@22pPPPMMa4 @c2PpPMss Z20oPPPPMMT2 F2s0eP22P0MM3R FFszriees2a0MkR FFeTaoa200HHRR FFSeZeIE4LoAOdHRR FeoeAMotRR 2@0PPPPwMzA 2@0PPPPMs2 SSSEEECRRRUUUMMMBBBLLLAAANNNKKK SSEERRUUMMBBLLAANNKK SSPPKKEEGe221z SSPPKKEE2S0231 SSPPKKEEZZSR0032 SSEERRUUMMBBLLAANNKK FFaoerzeesR FFFoarTreOeSsaGeRR FFoeresse rAeacduinlg SaGmlpyle TfnIaSiAvoBl G0o0m8F0)7 -- (m6ior1g 7(0ml) 0000ms 0011 2200 c0o0es 0011 2200 o00os 0011 2200 000105 0011 oom 01 222000 0o0o7Mse 0011 2200 00211930 0011 2200 0002658 0011 2200 o02m0e5 0011 2200 0220052 0011 2200 00410623 0011 2200 ootae 0011 2200 00211s 0011 026 01 2200 20 0001408 0011 2200 000033502 0011 2200 0000335s0 0011 2200 oooos 0o1f 2200 0o1o75i5s 0011 2200 0o2z7 0o1i 2200 0000323868 0011 2200 00011242 o0i1 2200 o0e2s0 0011 2200 oooiM 0o1n 2200 ois 01 20 mSLpFkCeSdS Comsocl.dFloCnOS oom) oooooo @@ o00o0o @ o00o0ok @ oooonn 2200 004 20 oooooo G@2 000044 22500 ooo @ oooooo @ oooooks 26 oooom 220% recov%ery pAocmuPsl(ogi) _inTsoarmple aarsos 00780177 sw o7e1r 1w2e%e 2ime 1t0o0m% 11%4 awvwe 1134 m71k% 44271 410205 sios 3S2ose i2o0ss aissen 2am Msw% 40a22 eh o2s60 os0s7008 a0r70s0 se%n 11mm se2%n 13i40 7s%m 4s5z4 oTsor Sooders i127 T3a%s 287 (MsapshFesd oroecMeassEesyed oGoTo 0a00e61s7 ots coo0s0e7t om ooitss ootzeo ooitss ooiie 0ot8s 0or0e 006a0 osesy o01d2s8 4T1a0 oosss 0T8o0ks ootms 0o2s1m0 006600 00disss 015 ooozstse 0o0o6r800s 0o0a7t0s0 ooiiss oorms 001ss 0o3t1s 0o8s0 o0dss oootsa777 0002247s 0i247s1 aa3tsse 0287 _-- < C0620 yg 2 STUD#Y 6326-151 SERUM Simple rAecatduianlg Salmyple GomF) ior TfnIaSlAvoSl mi) msLpFeCOdS CosnocllFoCn S oom) regcio%gve)ry ipmApcaumsg.le sM(oa7rse)sd repMmsaisssnd FFoiEeziOzR o01d4o8r FBR 0101 0o1T 01 222000 2T0%8 201 S0520s8 0201 SSSPPKKKEEESZZ200112 o01o6d7 0225 0011 01 2200 20 ooooo os 23 2om% 230 7% a1ms 4m o01msy omy eoarea eam FFeZeTiOzARR 0012%0 0011 Freeze oom 01 222000 2id%o 0024702 122 0122 FFeTsLz2RR 0O0o0M4z 0011 Foz 00 oi 2200 20 oTsans 0J0a88rs 07s oom FFeemvuerr 0000%4 0011 Feder 000 01 222000 o0s5a874 0520 000058247 00820 FaBTLNOKMIHR BLANK 0O0000 0004 0011 01 222000 0T7e1ss oer o0o16n8s 0027 SsSPKPKkKEEEZSG2Oz11z O00088M 0157 0o01i1 2200 20 o00o0o6 odd @G2 ssww 20 sw i1%z ais ooiis os oorrm oo SSPPKKEEz20023 00220 0011 2200 o0o0d4 2200 s8m% 44370a bbsso oouuss COR622 CJ a STUD#Y e326-151 SERUM Sam>ple wAacduisnlg 7) SaQmpyle @iorg fTaItmSvAoB mtLpFkCeUdS Consco.wiFoCnBS reco%vvo)ery imAncsmtaursne eMaesydsey r"eMamanisessss -- BB-- AANNKK-- SZ 00S003r8s5 0011 -- 7300 Goo p) NnNemooete wr) GNNooof) BBLLAANNKKS4 BUNKe 0000338098 000 0011 01 223000 PPM oo oi 20 ooo N>o No NNoo \o @ use 1m ois orn pPePwMs2 Pema 0O0180s ome o0f1 01 2300 20 ooooooooo @@ u1s%w @ 10m 2t:e 1% 0o1lss 01s oorsem 01 cc2rPeemMes ZOPPMI ooaormss 010 0011 01 2200 20 ZPPM2 0213 01 20 aoooso o0o0k4 1w0w0% 20 a 1144 sss oottss oe oirees sees a0 nw 43 oe osm FPameMe2S0nR O02o2 Feo 0205 0011 01 3200 20 ood 20 en 1a2a8 i oe o0o1ti2sd8 FFFaerreeoa2aoooWlRRR o2r05e 022 000111 232000 "Sitaso o4Ts5e0s FFTeei4OoHR ootus FaToaoR osm 0011 01 2200 30 2302ss ht 00830ss Tot ecppePMMa2 ZoPPMA 0o1t%e 0221 0011 01 2200 20 ooooon coos G2 aies 20 7% 2a0 44 ootss oss ooze ose Jp0PPMPMs2 o0t2o6 SERUMBLANK 00488 o0i1 01 2200 30 o0o0o4 20 mgamn 240s oowa oeseme o No SSSCEERRRUUUMMMBBBLLLAAANNNKKK 0000330% 003% 000111 2300 20 NNoo NNoo No No SESRPUIMKBELSA!NK G000m3e% SkEszz ooess o0f1 01 2200 20 0o0o0od @ wwws 1Nom ism ootss oNios orm SSSPPPIKkKEEEZe2020s12 o00o2170i51s 0o1i 01 2200 20 ooooo oo 230 2:0 swmw s1a4o os; ootss os o0s1s osm SSSEERRPUUKMMEBB2LL0AA3NNKK 0000022087%8 000111 723000 oan 2:0 7m eTNeoxr om abN1uo7n FFFaarrmeesiseeeRR o0ooxu2zt0e 0011 01 7200 20 2No2 5 0N22o7 ois FFFoarrosseaRnR oomses ood 0011 01 2200 20 S2e7 Ta o0Tar3e%s Feesin owe 01 20 28 027 COR -- ---- STUY8 6328.151 SERUM ESame ple orAecamudis)nlg mCSaiQmoprlge fTnISaAlB mSLFeCdSS Cosnooc.uiFamCn9sgmo)%o wsAecmtaubsn _eM(eaat9 r"oMwanrsstg FFSgKwaaEiRam1re oo01lms 060111 SKEZOY ow 01 23000 Zo aamoes wm x Zz0oor Ge as ofozeso zo gn 3h oe oo FFSePakTreomsioiRz o0Omr0se 000111 FezeR oom 61 232000 20 aos zo te ipoHnb em oooiumsn i oi FFFaaammeosaaawteRRR oOoovoues 00a111 223000 Fein 00m 41 0 iNwaoo orwiso No FFFaaamLmsamanRRR 0oOo0oms.s 600111 222000 awe amo 01 Zo NNoo \00 i% oes SPBRaeEwzaG O0oo0wmsss 000111 swemor ot ol 222000 20 aGGoamo aoobwm wsww 11\%mo 33 oooens oowrgom ons SsPPKKEeZzO03z 0o2z0 ooii 2200 boamm xx aamw iiss ooes ooeums cozez = ---i, F. 9.11.3 Summary and raw data; ppm F' in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. ---- coreg RE: 6329-151 SERUM SAMPLES AMDT 120694.1 Date of Analysis: 8/2/95 Analyst: DOW LOW "219s The samples are burned in the Dohrman at 958 C using 8.18 mL of the serum. The gas is collected in 2.8 mL of 1:1TISAB/Milli-Q water. The samples are then analyzed on a Skalar Segmented Flow Analyzer using the lon Specific Electrode (ISE) 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. Ril results below 8.8881 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. (Qu Qt C-- 2 by s/o 1/T 02626 SU6M 329M-1A51ROYF SERUM SAMPLES AMDT120694.1 20 "hits Dose Leve0 Bre 1s as MW 3% sw te oss DoseLevel 4mph Bes a0 as 26 2m 2s 10 os DoseLevel: 16mpg Be 16 sm 4m 3% 208 os os DoseLevelC2S 3663s Bm ms ma us em . se om om Fore le1nx a Prddrocsst wna hangedAononysis Mic esaststs.sM 02626 Paget a SERUM CURVE 1 N7O3R1M.9A5N saw luis Stata Que Smple RSeutl Gem SSkKMI2 00m SkM3 oo SSSkEkieOa3 00o0w0 SSskaK@sE2 ao0mmm SSkKiI0N2 O01I2 SKIBS 0 SSSkEkiEes 0oo6nm SSKkIsOeL2 003% Sku03 0% SSKkNiO0La skied 0047 0% | | i lr = DGIrTaINvD MSLoFoCSnSFCOC3oSnle oh Ske Gem SMpiuksed sR) RtoMmserd Re%c aE 2200 20 0oMo oom MMe0o Meo o00m8 om oOn1y7 ole i2lemw STANDARRDDSEDVATION: 102505 222000 0ooo0xmM ww0em0 0oo1ll0 o0oni1se6 lliememe STANDARoDrDsEV.ATION: oS3s0s7 222000 0oo0oo @aGmmm oooliisss ooommi llaaiw STANDpARrDDEVIATION: 2o6ums 2200 20 o00mM ook l0o0o leo o0oxMu o02x ox liomw how STANDA"RaDsDoE,VATION: foiirie 222000 0ooomoe 00hee 00o0%m o0oxn2 Idageenc STANDARRDSDDE.VATION: a0077s 2200 20 o0oo0omMm 2z0o0o mao o0w@ om 0o8m7 oe IwUm% lam STANDAKRDDDE.VATION: 1o5n1s% 2200 20 0o0oM oom XW00013120 S00 1m 0ol3mw4 7a%m STANDARDrDsEpV.IATION: 0S0auemz SERACURVE |DORM TS Jomianoom || 1 I = T = 1 | _-- }== err || i| | 1 | | | = a - = =| ------ eee) seRcRviN sum cozszyPaget 3 fErE,Ioe-- rento-- PCOmE eei Pomme oun PBPPoooommEEEe oWOowEm ooWoooommm Pod. olowiz om gFO gopbFbE ommoomaEmAmEem:Re ooE m m mon no@noomwoowmomooawu LE Pom so = sas PK250-1 7 om Bool am ee am oon ow one : PET ae 28% 020 20 ot 3% 00M 25000 060 040 "% Eg + SE consons POE oe fPoOmdEay o ww n ouoB Fol iE " wb EB osoman Gomme ow PoE oe 8 PPoEE 8oh g ETH eB PE oe poo EBEnR ow oa ow wow -- msn ot of; on Softwar:e version 6.1 c1930,9) Operator w Bt of th dies: sae 03 i il CSA BO nailtaein1.5sce = avs Lope so casrm oa) come lo of se wal 51 TERRA nul hd--im--m nGloirnttieo se +1 Canation 1 = 0.10 [IPI uWusl avVoaemm wie moSele tin om 0 ae utnhednTm aehe wrerarl Caa Ne ipdlnri gtyoa: ww 15 a Satiete 1 sonntite to n arwen*ihlnas' HW 32945) vo 4 026GE saan 0 oth of : gs Hrite 15 sintghuslatbreer:: s3 el brosltarBabe+r01a asltelesia d1 atrtosald:0{035 distor wnat : ott 0 ssttuaddi:: IInonn s0tasntduadrit: IIppeo 65 astatdaud Isqis aa swtait:d 00k oattaald:: U1i0 oDicndssi:onT:ame7r0s Lagrithe rstairtgnaLsi:nit : 5108007Sc Ptaik gen oi60steSde Haoareivgisndor + 7I5 s4 e gn eunae tion i3s afotils Muriel smitgsul tbree: :se0ksi bsotloenrTaber: 0do daiagtee kIe dAooBgeeets 1(0035 visto ovat: att 7 COR63 5-041 00 oath of : ssn 0sestteeaams 0 sadtdesd 0d0u0 5 otm e 010 ssttaatdut: digornee S810 ssttatsa:tIIaygenordrse oSredsesri+on2 : p10 rmiagotriinnit: 1s80w50 aalktsi re60nteSde Masongveander I$75 s1 e pnitnt 1ds faotndtly: cLheae 1 o263g a na oun of: sm Hols wris m m Pomme Oe frie 0 etre oDToenetsmaiot0iLLmoEs m6 ooommoo ow oom iCyo smawlh 3d0osw Sod sted) om a mom on modems oTo O ssutuldd)) Sms o0mm mowas moo (doowwn ow SWoad Lod ssmmha]lmmKn swans om sa (demmo come DWoans swans Wan LU6momDi0 drm uw (Goewm em 0 D5Wooomomnmrwihd3oeewm 0ome (om owl oa WDuaoSWAmKIuC Wass ToUmS dom Doaw neeamw mode o HBBoaauoorommeEaKsEridooemms ounw oa (ceoawm dom BHWaaaooawmdwaidddeoLmmm oooummn n ccoommeo BnByaaosoweaswsidw oomu oaw sno ooomm 1o WWoomm Baw deommowusoddeemmm em sn (ume o DWaooonnan Boom deamm ans aosen ccoummm oom 0 WMaenoaw)) oUme BE mi rm aews (emesno gm emo nWGay oomimmedeenmomnu odwmn dems 0 dGWaaaoommoeawra)ideetmmm omnee ne odoomm dew 0GGuooomswamaodenoeom mmwi ome d(emeemo Gow WGWooiNnoenE))DOfonm Wwin ou (deewn (emo oS Sya omAmHCwa CaTln eosm owmn ns oooem dla note -- so Ccoz639 WANE wen meta tai 1s nis "m Gu 0 wHSo1UolmmMmomEwimLeamn nmse aaiamew GB BB rlOMm EEIL) E mudamme 0 B oB B1y0 i lm GE eRoEmmonam BBBH lom L1D Em L) mmmam mqmeh BBBLRLiUOoOr MmMeRd moi mmn m nHBHeUOiMaEmdRRhE)LE B BWm m Gm 4 WBBniOeoOoMOEmRan om oAm F B8BLO1OmMme aaaee BsBB elooIOlMREn LL L mBlL nma BBBHiLOLOOMMMME LL Amuee nm) jnaee E FrE ) R " coz63 rn cre of st rion I CTTW WRI0T 000 ZMH | ly | | | ) Ig |a.; | Po LI o CS I I L[i 0 oco]me1s A ' Fa 7 / 4/ // /J A yd |[| I IIHl 1 | ||| L|| o 0 eOrdewr: 2e' TT Measured e0.m39908 900 |i S-- c02636 n | Co I sss |[1| | rte cr of st rin 5 || 4 SoHl / Hl |i s |J //A / H | | i] |P |Y g // |i || /J/ ||: ||| 1{|| J/JVoa. i|!| lo pd y p| + oms"I LLoL" oarrkte : mtoesme oomiri actaowwe | LY 3 0263 pi I| Ti |-7 HPllll aol C| }l aIll | uIlLl all COU al. Te]al | ' ' Val| il ran J | .-- cozeag"' T oo Hl Ww | |] LT] lL']1 tall|a]]ll | i ||to l | aigo | Vasil | w || coz639 ' Co | NB | || | iIl , | |]|; |] I | ert]| /h|n| | willlll TM| lit |Mm J---- co2s10" (A -- | | ||1 woe [1|[odI | 1I Ll I Il| aI. lo] 1 Lo] r|o i | me as | UT re || AlTl.TeaTtteahll ! H|l HL AAS Ls 2 Ci EP -- --_---- cozsat 9.11.4 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated in Final Report detail. Section 6.0, and therefore is not discussed in c0284 I corntacit e :x WET vo oeko aimseARTTORYE CB on n$$ SoapYienquwaenkeiiesthaadp,hy sereened for UH on oily and) 2 es we seem File:Loon:oH1UT9e5r6329-151: 11L5A4D9-BACSPEX)- The MS Data Susten [= an oe 8 off gf shogl og 8 or BILE 81 I rs. i. ue ea lse Pr a cds % 01/88/1995 14:50 a. gy 8 g ag 103 das = o:: I -- nr [oe C(Dox) 3 ogo ook sen EST SFaimlmoicea?goiose LAD-DASE - The MS Data System BLOBS 14:22 A a "K &e8 SFialmoesro?setoss fe LA- S ho HSS Data ston = 0101995 14:22 or] iF losePlaces 471.00 26.00 [47r2.i00f 453.00|aH 7a.005E , 5330S0ansa 570P.00H io i can.o0 Z+: eS tf FiSBlaOmoB1p:9l50Eeg:0192652E ou (6-987) LAB-BASE ~The MS Data System 427_C.O0R0BINET (267 Fo 2667=CCCIB0 Eo o1ea/1995 14:22 T5775 Ea 3007) 1-55T8onal 407.00 106.00 A230 lr: fo Ree ee gee o=n 0< 83e FiSalmop:leeo:n195E 1B0OaR) 1535633.2010 5.648) LAB-BASE - The MS Data System 81/00/1995 14:22 COMBINE (321 6 3267= (C204 Eo 0273635947) 1-668222] a1.00 fers. 10.00 (418,00. 40 tiie Td gt gy - seaousszon Ls - TN 2 ? Ma ams Laue (Pk) g b Fitecommiose CM 17 < bers] 2 IH3Ys &8 os = LAD-BASE - The NS Data Syston . TR. 172 666 1166 A277 1398 lect i = 01/80/95 15:07 TI TrLan $ Se g =e +85ep i t iy as ae h! Zusasor as w