Document ypvnpJypgxVjjwo3OJN9YXxpn

. 25 3M Environmental Laboratory OB Final Report- Analytical Study Single-dose Intravenous Pharmacokinetic Study of T-6049 in Rabbits In-Vivo Study Reference Number: HWI#6329-129 Study Number: AMDT-112294.1 Test Substance: FC-95 (T-6049) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology & Services 935 Bush Avenue St. Paul, MN 55106 Method Numbers and Revisions: AMDT-M-1-0, Thermal Extraction of Fluoride by Means ofa Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, TFlounoArindaelyMzeearsurement by Means of an Orion EA940 Expandable AMDT-M-4-0, AMDT-M-5-0, Extraction of Fluorochemicals from Rabbit Liver AElneacltyrsoissporafyRMababsist SLpievcetrrEoxmtertarcyt for Fluorochemicals Using AMDT-M-14-0, Thermal Extraction of Fluoride by Means ofa Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Initiation Date: See attached protocol Author: James D. Johnson Approved By: Snr tmuedsy DDi.reJdoihdfirfon Has) 7s Completion Date 000447 Page 000001 _ Fy losyMmMARY 000 Rabbit rabbits serum and liver were analyzed were dosed intravenously with for fluorine content at various times after a single dose of FC-95 (T-6049). The fluorine 5in00limvegr/wkags(d3e0t0ecutge/dkgafFtCe-ar95s)i.ngTlehedolsieveorffl1u0o0rimdge/ikogn (60 ug/kg FC-95) or concentration in the after Group 5 animal (300 60 ug/kg the ug/kg), at 48 hours concentration was post dose about 0.4 was ppm. 1.T0hpeppmreasnendcfeorofthe rabbit dosed with perfluorooctanesulfonate was confirmed with electrospray mass spectrometry. 6T0heugm/ektg.hodAnwaoluyslidsporfobfalbuolryinbee aatbl2e8 tdoadyesteicntlaivderosaendsosmeerwuhmewriellbelitkweelyenpr1o0viadneda marker for dermal absorption for dermally applied fluorochemicals that are pbihoatrrmaancsofkoirnmeetdictostpuedrfylouforFoCo-c9ta5newshuilcfhonwaates acnairorni.ed Tohute tdoat2a8adgaryese wwiitthh maosreecoanndimals (HWI#6329-159). 2.0 INTRODUCTION - A-- This study was designed to octanesulfonate anion does gporotvoidtheeilnifvoerrmaantdioonthaesrttoiwsshueetshwehretnhetpheermfaltueorrioa-l is administered in an intravenous dose, and to ascertain the change in concentration with time after dose in serum and liver. Tt is known from studies octanesulfonate anion is done quite previously with rats long (>1 month). It that was the half-life of perfluoroexpected that the half-life in rabbits would also be long. Perfluorooctanesulfonate anion is a likely biotrans- formation productofseveral fluorochemicals that are to be tested for dermal absorption in anion is very roetlheevransttutdoietsh.esTehdeerpmhaalrmaabscoorkpitnieotnicsstoudfitesh.e perfluorooctanesulfonate 3 EST MATERIALS 3.1 Test, Control, and Reference Substances and Matrices 3.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent, 33..11..32 AAnnaallyyttiiccaall CReofnetrreonlcSeuMbasttrainxc:e: BoNvoinnee liver and bovine serum 3.1.4 Analytical Control Matrix: Bovine liver and bovine serum 000448 000002 3.2 Source of grocery store, bMoavtienreiaslesr:u3m MfrIoCmPS/iPgCmPaDCihviesmiiocnalfoCr oFmCp-9a5n,ybovine liver from 3.3. Purity and Strength of Reference Substance: Responsibility of Sponsor. 3.4 StabilityofReference Substance: To be determined by Sponsor. 3.5 For SbtioolroaggiecaClosnadmiptlieosnsthfeosrtToerasgteMiast-e2r0i+al1s0: Room C. temperature for FC-95. 3.6 Disposition GLP Regulation of Specimens: Biological tissues and fluids will be for the time period required for studies longer than retained 28 days. per 40 EXPERIMENTAL-Qvervi fSoerraunmalaynsdistifsosrufelsuforrionme acnoimmpaolusnddoss.edSianscedepsecrrfilbueodro(oHcWtIa#ne6s3u2l9f-o1n2at9e),anwieorneisavnaoitlable abniaoltyrsainssffoorrmfeldu,ortihnee.anaTlhyesifslwuoarsinaecdcaotmaplairsehreedlawtietdhdciroemcbtulysttioopnerafnlduosruoboscetqauneen-t isnutlefrovnaaltseacfotnecrednotsriatnigopnr.oTvhideesflduaotrainwehaincahlycsains obfe sientreurmprceotleldecptheadrmaatcdoikfifneerteinctatlilmye. 5.0 EXPERIMENTA-L Methods 5.1 AMDT-M-1-0, Dohrmann DX2000 Thermal Organic Extractionof Fluoride Halide Analyzer-Liver by Means ofa Modified 5.2 AMDT-M-2-0, Fluoride Expandable Ion Analyzer Measurement by Meansof an Orion EA940 5.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver E5.l4ecAtrMoDsTpr-aMy-M5a-s0s,SApneacltyrsoimsetorfyRabbit Liver Extract for FluorochemicalsUsing 5D.o5hArmMaDnTn-MD-X1240-000,OTrhgearnimcalHaElxitdraecAtnioanlyozfeFrl-uSoerriudem by Means ofa Modified 000449 000003 6.0 DATAANALYSIS ~~ The material T-6049 was a solution of FC-95 diluted to of 5, 10, 100, and 500 mg/kg are thus 3, 6, 60, and 300 0.06% ug/kg. in water. The doses These are very low doses. . Tpohset ldiovseer fwlausori1d.e0 piopnmcoanncdenftorratthieonraibnbitthedoGsreoduwpit5ha6n0imuagl/k(3g0t0heugc/okngc)e,ntarta4t8iohnowurass about 0.4 ppm. The control rabbit, the 5 mg/kg rabbit, and the 10 mg/kg rabbit concentrations were not above the practical limit of quantitation. Fthoerpleirvfelrsuoofrotohcetatnwesouhlifgohneastte dwoassesdeutseicntgabalnealaynsdistbheyeeslteicmtartoesdpraamyomuanstss sfpoerctthreom1e0t0ry, and 500 mg/kg rabbits were 0.3 and 0.6 ppm, respectively. TdehremsaeldaatbasosrhptoiwonthtaetstpseroffFlCuo-r9oo5ctaanndesfuolrfoannayteflwuiolrlobceheamivcearlysstehnastitairvee marker for biotransformed to perfluorooctanesulfonate. The fact that the material can be observed in liver after a 60 ug/kg dose is suggestive that a very large portion of the dose will be in liverifthe material is present systemically. O`mtehaenrsdoatfaa wmaosdicfoileldecDtoedhronmasnenruomrgsaanmipclheasliudseinagnatlhyezremralanedxtfrlaucotriiodneomfefalsuuorriedmeebnyt by means ofa supportive, in Orion expandable the opinion of the ion analyzer (see Study Director is appendices). not required This data, although to reach the conclusion stated here and therefore is not discussed in detail. c6o.n1ceCnitrrcautimosntaarnecefsroTmhacotmMbuasytiAofnfeacntaltyhseesQuoafltihteybioofltoghiecaDlatmaa:teTrihaels.e values The for recoveries are based FC-95. The fluorine on is spiking the biological measured by selective material with ion electrode known and the amounts of Orion meter is ccaolnicbernattreadtioovnesroaf5FCp-oi9n5t ortahnegre.thHaonwtehveepro,iintf utsheerdeaiss aa cdailfifberraetnitonrecshpeocnks,etahterdeifcfoeruelndt be a bias at those concentrations even measured accurately. This can come though the from other amount of variables in fluorine is being. the method such as the eafbfsiocliuetnecyvaolfuecso.mbTuhsetivoanl.ueTshaurse,ctehretavianllyuessufrfeipcoiretnetdtofomrackoenctehnetrpaotiinotntaharte tnhoet perfluorooctanesulfonate anion is present at 48 hours in liver. 000450 000003 4 l0 econcrusion000 6`T0heugm/ektgh.odAnwaoluyslidsporfobfalbuolryinbee aatb2le8 tdoadyesteicntlaivderosaendsosmeerwuhmewriellbleitkweelyenpr1o0viadneda marker for dermal biotransformed to absorption for dermally perfluorooctanesulfonate applied anion. fluorochemicals that The data agree with are a second pharmacokinetic studyof (HWI#6329-159). FC-95 which was carried out to 28 days with more animals 8.0 MAINTENANCEOFRAWDATAANDRECORDS~~ 8.1 Raw Data and Data: appropriate specimens, and Raw data, electronic approved protocol, approved data will be maintained in the final report, AMDT Archives. 2.0 APPENDICES ~~~ 000 9.1 Protocol and Amendments P9.h1a.r1maPcrootkoicnoeltiacndStFuidnayloRfeTp-or6t0:49HWiIn #R6a3bb2i9t-s1"2(9Pr"oStioncgolletDyopeseTPIn8t0r8a4v.enPoKusfor dosing of animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-112294.1 including methods. 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: Sec methods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-112294.1 9.10 Instrument Settings: See methods 000451 000005 5s 9.11 Data: See attached: 9.11.1 Summary and raw data; extraction followed by analysis uusginFg" iOnriwohnolieonlainvearlyazserd.etermined by thermal 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 9.11.3 Summary and raw data; ppm F* in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. 000452 000006 4 9.1.1 Protocol and Final Report: HWI#6329-129 "Single Dose Intravenous Pharmacokinetic Study of T-6049 in Rabbits" (Protocol type TP8084.PK for dosing of animals, tissue collection, etc.) 000453 000007 ? WPOSIT SOFCFIOCE NBOSK I75N45 MADISON. Wi 53/47 7548 Sponsor: El) St. Paul, Minnesota 3 CORNINGCorny FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6049 in Rabbits Author: Steven M. Glaza Study Completion Date: February 1, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-129 Phone 03.241-147 EXPRESS.MAIL DELIVERY Page 1 of 24 Fax 3301 XINSMAN BLYO MADISON. 000454 608-20410-7020270S Wi 53704 Page 2 of 24 QUALITY ASSURANCE STATEMENT HHT 6329-129 WTihsicsonrseipno,rtTnhca.s, beiennacrceovriedwaendcebwyitthhethQeualFiotoyd AasnsdurDarnucgeAdUmniitnisotfraHtaizolneton T(hFeOR)foGloloodwinLgaboirnastpoercytioPnrsactwiecree Rceognudluacttieodnsa,nd21finCdFRing5s8.3r5epo(rb)ted(6)to (t7)h.e SantdudyfinDdiirnegcstorareandismsauneadgetmoenHta.zletWornittmeannagsetmaetnuts mroenptorhtlsy oafccoirndsipnegcttioons standard operating procedures. Inspection Dates From To 1111//1066//9944 1111//1066//9944 0011//1300//9955 0011//1300//9955 Phase APnriomtaolcolObsReervviaetwion DReaptoar/tRepRoerrtevRieewview RepoDratteed to StudyDirector 1117/1068//9984 0011//3100//9955 Date to Management 1122//1100//9944 0022//1100//9955 Kl Repr{e1s1eant#a.tiDvaen,nerQuality Assurance Unit Date 000455 000000:Y Page 3 of 24 STUDY IDENTIFICATION Single-SDtousdey IonftrTa-v6e0n4o9usinPhRaarbmbaictoskinetic HHT 6329-129 Test Material Sponsor Sponsor's Representative Study Director Study Location StuEdxyperTiinmeenttaablleStart Date Experimental Termination Date T-6049 MTmoxicology Services 2St2.0-2PEau-l0,2 3MNM Ce55n1t4e4r JEodhn L. Butenhoff, PhD T22o0x-i2c6o-l0o2gy3MSerCveincteesr St. Paul, MN 55144 (612) 733-1962 HSatzelveetnonM.WiGslcaoznasin, Inc. MPa.0d.isoBno,x W7I54553707-7545 (608) 241-7292 HBauzilledtionng WNoi.sco3nsin, Inc. 3M8a0d2isoPna,ckWeIrs A5v3e70n4ue NNoovveemnbbeerr 1157,, 11999944 000456 000010 Acute Toxicoloay SSttuedvyenDMi.reGcltaozra Manager Francis (Bud) W. McDonald Study Coordinator PIant-r1iicfieaSuPpaedrgvhiasnor RReopsoertH.. SBurpiedrgveisor QualityAssurance MSahneargreyrR. W. Petsel Page 4 of 24 KEY PERSONNEL HHI 6329-129 aboratory Animal Medicine CDiipnldoymaJt.e,CarAyC,LAMDWM Supervisor AnatonicalPathology JDaecbkoraSherfL.ortP/irkel SNuepcerrovpissyors ASnunpeerMvoisshoerr Pathology Data 00000.457 000011 Page 5 of 24 CONTENTS QStuuadlyityTdAesnstuirfainccaetioSntatement SKeuymmaPreyrsonnel ObRejgeucltaitvoeryComp] iance Test and Control Materials Test System RPreoscueldtusres. DSiisgcnuastsuiroen Reference Table 21 IInnddiivviidduuaall CBloidnyicWaelighStisgns(g) AppPernotdoicxolA TP8084.PK HHT 6329-129 Page 23 16 77 7 8 19 1111 1 1123 1u5 000458 000012 Page 6 of 24) HHT 6329-129 SUMMARY Tahdimsinissttuedryedwasbydoinnetratvoenaosussessinjtheectiloenveltoofrabsbyisttse.mic exposure of T-6049 when F(eomnael/egHroruap:).(NZOWn)SPDFayra0,bbitthse waenriemalasssirgenceediveadt raansdionmgletoinfitvreavegnroouusps injection of Tt-h6e04v9e/hkigcleof(sbotdeyriwleeigwhatter(Gfroourpsinj1 ectthiroonu)gho5r, 5,res1p0,ect1i0v0e,lyo)r. 500Themgdoosfe volume was 0.5 ml/kg for all groups. 4C8linhiocuarls aofbtseerrvaitnitornasvenwoeurse cionnjdecutcitoend. atBoadpyprwoexiigmhattselwyer0e.5,det2,erm4,ine2d4,juasntd 4benfLo)rewatsestcomlalteecrtieadl fraodmninanistaruartiicounlar(Daayrt0e)r.y oAr mbalrogoidnaslameplaer v(eaipnproofxitmhaetely tahneimatlisme atof2-e,xpe4r-,ime6n-t,al8,ter1m2-i,natainodn 24(-4h8o-uhrosurspospto-sitn-jiencjteicotni.on),In apadpdriotxiiomna,telayt 2s0epanLratoefdblionotdo wsaesrunobtaanidnecdellfurloamreafcrhactainoinmsa,l. andAllsenstamptloesthewerSeponcseonrt.rifuged, Aspopdriouxmimpaetnetlobyar4b8ithaolu,rsblpeodst-viinajetchteiopno,stetrhieoranviemnaalscawvear,e aanndestehxestainzgeudinawtietdh. An caoblblreecvtieadt.ed Tghreosswhonleecrolpisveyr,exabimlien,atiaonnd wbaosthnoktidndeonyes, fhroowmeveearc,h taniismsauleswewreere collected and sent frozen to the Sponsor after termination of the in-1ife phase. T1h0emganTi-m6al0s49/tkrgeataepdpeawrietdh snotremrailletwhraotuegrhofuotr tihnejecsttuidoyn., 5AtmgthTe-6004.59,/kg2,, aanndd 4appheouarredposhty-pdooascetivoebsearnvdattihoen,anitmhael atnirmeaaltedtrewaitthed5w0i0thmg 1T0-06m0g49/1k-g604e9x/hkigbited a dsetcargegaesreedignaifto.od cIonnsaudmdipttiioonn, atthetheani4m8a-lhoutrreaptoesdt-wdiotshe o5b0s0ermvga/tkigons.howed a 000459 0000135 Page 7 of 24 HAI 6329-129 OBJECTIVE The objective of this study was to assess the level of systemic exposure to the test material, T-6049, when administered as a single intravenous injection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test mixtures for concentration, homogeneity/solubility, and stability was not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS dentification The test material was identified as T-6049 and described as a clear, colorless liquid. The control material was Sterile Water for Injection, USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. March 1, 1996), and was described as a clear, colorless liquid. Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). A sample of the test material/vehicle mixtures for concentration, solubility, homogeneity, and stability analyses was not taken before administration as this was not requested by the Sponsor. The purity and stability of the USP grade control material were considered to be adequate for the purposes of this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Any unused test material will be returned to the Sponsor after completion of Operating Procedure all testing (SOP). Any according remaining to Hazleton vehicle may Wisconsin (HWI) Standard be used for other testing and will not be discarded after issuance of the final report. 000460 000011 Page 8 of 24 Safety Precautions HWI 6329-129 The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were received from HRP, Inc., Kalamazoo, Michigan on October 19, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing . After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmental controls for the animal room were set to maintain a temperature of 19 to 23C, a relative humidity of 50% +20%, and a 12-hour 1ight/12-hour dark lighting cycle. In cases where variations from the required temperature and humidity conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal husbandry and housing at HNI complied with standards outlined in the "Guide for the Care and Use of Laboratory Animals". Animal Diet The animals were provided access to water ad 1ibitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodically analyzed by HWI. There were no known contaminants in the feed or water at levels that would have interfered with or affected the results of the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were selected at random based on health and body weight requirements. 000461 000015 Page 9 of 24 Study Design pFleamcaelde ainnitmoaltshewefioglhlionwgingfrosmtud2y,58g7routpos:2,857 g at initiation Group Treatment (mDaos1e-60L4e3v/ekla) _Dos(eni/Vkoal)une 1 (Con2trol) 71-6*049 05 0.5 05 43 T1--66004499 11000 0055 5 T-6049 500 05 * Sterile Water for Injection, USP. HHI 6329-129 of treatment were Number of Animals 1 11 11 Justification for Species Selection Hcihsotiocreicbaelclayu,sethoef Ntehew lZaeragleandamoWuhnitteofalbbiancokgrroaubnbditinhafsormbaeteniontheonantihmiasl spoefcies. PROCEDURES Dose Preparation and Administration Tshpeectiefsitc mcaotnecreinatlratwiaosndiflourteedachwitdhoseStelervielle WianteGrroufposr 2Intjhercotuigohn 4t.o aTchheievteesta bmautlekridaelnsiwatsy oafdn0i.n9i8stegr/emLd utondidleutteerdminaet tthhee d5o0s0emgv/oklugmed.oseAnlevienld,iviudsuianlg dtohsee of eonachitsrebsopdeyctiwveeightteston stohleutdiaoyn oorf ctornetartomlentw.as Tchaelcurleastpeedctifvoer teeascth sanoilmuatlionbawsaesd gaidvmeinnisasteraedslobwy piunsthrav(eanpopursoxiimnajteecltyion30 itnoto60a msaercgoinndasl ineardurvaetini.on).TheThdeose was Apfrteeprareaddmitneissttrsaotliuotni,onsanywerreemasitnoirnegd attestroosmolutteimopnesratwuerree udnitsiclardaeddm.inistered. Reason for Route of Adninistration Intravenous injection is an acceptable route to assess systemic exposure. Observations of Animals Clinical 48 hours observations were after intravenous cionnjdecutcitoend. at approximately 0.5, 2, 4, 24, and Body weights were determined just before test material administration (Day 0). 000462 000016 > Page 10 of 24 HUI 6329-129 sample Collection cAatbhleotoedriszaamtpiloen (oafpptrhoexiamuartiecluyla4rnla)rtwearsy corollfercotmedthefrmoamrgeiintahlereaerarveviina otfheall naencirmaolpssyat(a2p,pr4o,xi6m,ate8,ly 124,8-haonudrs24 pohosutr-sinjpeocstti-oinn)j,ectaipopnr.oximAtatetlhye time of 20 mL of blood wsatsoreodbtaaitnerdoomfrotmemptehreatpuorseteruintoirl vceneantrciafvuageodf eanadchseanpiamraalt.ed Ailnltosasmeprluensanwdere mcaeilnltualianr faratcetmipoenrsa.tureTheofbl-o2o0dCsa1m0plCesuwnteirle tshheinppesdtorteodthien aSpofnrseoerz.er set to Pathology Aptostt-eirnmjiencattiioonn),ofanthiemalesxpewreirmeenatnaelstphehtaiszeed(awpiptrhoxismoadtieulmype4n8t-ohboaurrbsital, bled via etxhaemipnoasttieorniorwasvennaotcacvoan,ducatnedd,exshaownegvueirn,atetdi.ssueAsn waebrbreevcioaltleedctegdr.ossThneecwrhooplsey pTliavecre,d boniled,ryanidce,botthhenkifdrnoezysenfrboynpleaacchinganiimnala wferreeezcerollseetctetdo maanidntiamimnediaately dtiesmcpaerrdaetdu.re of -20C +10C. After tissue/bile collection, the animals were sh 18 Akfitdneeryscomwpelreetisoenntoffrotzheen in(o-nlifderyphiacse)e ttohetbhelooSdponssaomprle(sJ,amelsiveD.rs,Johbnisloen,, an3dM ES.pEo.ns&orP.iCs.,reBslpdogn.sib2l-e3-f0o9r, th9e35reButsehntiAovnenuaen,d dSti.spoPsaiult,ionHN,of55t1h0e6)s.amplTehse. HcoIlledcoetsednoitn athcicseptstuadnyy rneosrpoanrseibtihleisteyrefsourlttshepraensaelnytseids oifn tthheissaremppolrets. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Report Trheetairnaewddaitna,therecaorrcdhsi,vesandofanHWIoriigninaaclcorsdiagnnceed cwiotphy oHHfI thSeOP.final report will be 000463 000017 Page 11 of 24 BodyWeights RESULTS Individual body weights at initiation are in Table 1. HMI 6329-129 linical Observations Individual clinical signs are in Table 2. The animals treated with sterile water for injection, 5 mg T-6049/kg, and 10 mg T-6049/kg appeared normal throughout the study. At the 0.5, 2, and 4 hour post-dose observation, the animal treated with 100 mg T-6049/kg appeared hypoactive and the animal treated with 500 mg T-6049/kg exhibited a staggered gait. In addition, the animal treated with 500 mg/kg showed a decrease in food consumption at the 48-hour post-dose observation. Pathology Aelxlamianneidmalgsrosssulryvivwehden tosactreirfmiicneadt.ion of the experimental phase and were not DISCUSSION The level of systemic exposure of T-6049 was evaluated in female albino rabbits when administered as a single intravenous injection at levels of 0, 5, 10, 100, and 500 mg/kg. Administration of this material resulted in hypoactivity at the 100 mg/kg dose level, and staggered gait and a decrease in food consumption at the 500 mg/kg dose level. SIGNATURE SSttuedvyen DMi.recGtlaozra Date Acute Toxicology REFERENCE 1. NIH Publication No. 86-23 (revised 1985). 2\4y 000464 000015 Page 12 of 24 Grow 1 2 3 4 5 Individual ody Veights (9) Dose. (Lmeavfeila) 0 Sex Female NAnuimmbaelr F52652 5 Female F52653 10 Female F52654 100 Female F52665 500 Female F52666 Dayo 2,634 2,587 2,620 2,857 2,79 HWI 6329-129 000465 000019 Dose Grow L(emvo/ekal) 1 0 2 5 3 10 4 100 5 500 Page 13 of 24 Table 2 Individual Clinical Signs HWI 6329-129 Sex ANnuimmbaelr _ Observation Female F52652 Appeared normal Female F52653 Appeared normal Female F52654 Appeared normal Female F52665 Appeared normal Hypoactivity Female F52666 Appeared normal Staggered gait Deccornesausmepdtifoonod 05 2 4 Hour ZA J77 v 7 v 7 J Vv 7 7 7 v Vv v - - - Vv J Vv 77 - - - - - Vv - vvJ - - Seay 7 Indicates condition exists. - Not applicable. 000466 00000 Page 14 of 24 APPENDIX A Protocol TP8084.PK HHI 6329-129 000467 000021 @HAzLETEN TSR Wit 0 at Page 15 of 24 + CORNING (arin sean: M St. Paul, Minnesota COpPyY F PROTOCOL TP8084.PK stage Tete of T-6049 in Rabbits Date: November 9, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard ratory sect vn iction HWI 6329-129 000468 000022 Page 16 of 24 : STUDY IDENTIFICATION TPB084. PK. Page 2 Single-Dose Intravenous PharmacokineticStudy of T-6049 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimenta] Start Date Experimental Termination Date Draft Report Date 6329-129 T-6049 3M 2T0ox-i5c6o-l0o2gyanSerCvaincteesr St. Paul, MN 55144 Jon L. Butenhoff, PhD Toxicology Services 220-2E-02 3M Center - St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Week of November 14, 1994 Week of November 14, 1994 Week of December 19, 1994 000469 00002.3 Page 17 of 24 PTaPge083 4P.K 1. SSitnugyle-Dose Intravenous Pharmacokinetic Study in Rabbits 2. papduimrsipsnoisasestseretdheasleavelsinogflesyisnttermaivceneoxupsosuirnejewchtieonn thteo treaspthmiaterial fs 3. TRheiasulsattuodryy Laboratory Compliance PwirlalctibceecoRnedugcuteldaitniaocncosr/daSntceanwidtahrtdhes following Good Ceoxncceepnttiroantitohna,t conducted: saonlaulbyisliistyo,f htohmeogteensetitmya,terainGadulisdtmeai]xbitinulerisetsynwipitolerl ih not be [(X1] 2C1onCdFuRct58as(aFOAN)onregulated Study [[']] 4400 CCFFRR 716902 ((EEPPAA--TFSICFAR)A) [[[J115N9o(t8i1Nf)oi3hc0saatn(iFoNinon.alN)o3.85(30013"C(DJ)a(pJaanpeasneeseMAHFOF)HN) 2 | dH{i0er}lefcahtrroeorc,AecdtutrheeRsegsutilunadtytihodinosse.sprontoIotncoutlmheecaorepesisniainroincloyomfplitahenceSpownietphr tnheedsAtniumdayl work. duplicate. any mosrrc) 4. QtTuhheealiJputrzoy]toAicstoyslu,ArsasnsuctreuadnyceconUdnuictt,in aandccotrhedanFciena)witrhepfoarztlewtiolln . be auditedby (HW) Standard Operating Procedures (SOPs) and poleinsHisar ie 5. Test Material A. TI-d6e0n4t3ification B. (PhTyosbiecaldocDuemsecnrtiepdtioinn the raw data) C. JdPenteeeursnhioar nnnastodiriSotnaasst bsiu(lmiy ientscylurdeisnpgonsuinbdierlittyestfocronpduirtiitoynsa)n.d stability SLhhaaookrmmeaocnngq))ennsaettiretaryti,iaolna/nvdeihfisrctelaqebuielmsiittxeytdurabeny(asl)ytsheefsoSrpwoicnlolsnocreb.netrtaTathkieeonsne,besSfaouommprplelsaecs]'o(tfiryf, terminatwiiolnl bfeorspeontssitboletheanaSlpyosnisso.r after exper inenta) 000470 0(000203 Page 18 of 24 \ 0. SRtooormagteemperature PPa8ge0844P.K E. ReResseerrvveeSasammpplleess will not be required for this study. F. RAentyenutnuisoend test material the final report, unless wdiilrlectbeed diostchaerrdweidse abfytertheisSspuoannscoer.of 6. SAsaferteyquiPrreedcabuytiWoWnIs SOPs and policies 6. ControlMaterial A. SItdeernitliefiwcaatteironfor injection 8. PClheyasri,calcolDoerslcersispti1oinquid - , C. P cTohensu ipduerar rinetddySiattnoadbt biesltiy aatdbyeiqluiattyeofforthithseUpSPurgproasdees omaftetrhiiasl stiusdy. 0. SRteofrriaggeerated E. ReSseeerSveectSiaomnp,le5.s E. Reserve Samples F. ARentyenrteimaoinning control material may be used for other testing and will not be discarded after issuance of the final report. 6. SAsaferteyquiPrreedcaubytiKoNnIs SOPs and policies 7. ExperimentalDesign A. Animals (1) SRapbebciites (2) HiSrtar:ai(nN/ZSHo)uSPrFc/eHRP, Inc. , (3) Age at Initiation AduTt 000471 0000x275 Page 19 of 24 P8084P.K Page (4) Meai tIg nith iatt ion 4 2.5t03.5kg (5) NumabndeSerx 5 females (6) Identification Individual numbered ear tag (7) Husbandry (2) Housing I(hnedaivvyidguaaulgley), in screen-bottom stainless steel cages (b) Food A measured amount of Laboratory (PMI Feeds, Inc.). The food is. Rabbit Diet HF #5326 routinely analyzed by (0) ater etnhveirmoannmuefnatcatlurecrontfaomrinnaunttrsi.tional `components and * ` Ad Tibitum from an water are analyzed automatic system. Samples by HWI for total dissolved of the solids, hardness, and specified microbiological for selected elements, heavy metals, content and organophosphates, and chlorinated `hydrocarbons. 4) Contaminants ` There are no known contaminants in the food or water () Enviroment that would interfere with this study. (F) Acclimation Environmental controls for the animal set to maintain a temperature of 19C room will be to 23C, a rTeilgahtti/v1e2-hhouumriddiatrykocfyc5l0e%. +20%, and a 12-hour At Teast 7 days (8) Selection of Test Animals Based on adequate health number and body of extra weight according to HWI SOPs. An animals will be purchased so that no animal in obviously poor health is placed on test. (9) Justification for Species Selection Historically, background information on this species the animal of the New Zealand White choice because of the albino rabbit has large amount of been 000472 00006 Page 20 of 24 , B. DoAsdeministration (1) Test Groups TPPa8g0e846.PK Group Dnoaske aL)evel NEuemnbzelresof 1 0 (Control) 1 2 5 1 3 10 1 4 100 1 5 500 1 C. Dosing Procedures a The dose volume will be 0.5 ml/kg for Groups weight 1-4 and approximately 0.5 mL/kg of body (depending on the bulk density of the test material) for Group 5. (1) Dosing Route Intravenous injection into a marginal ear vein over approximately 30 to 60 seconds. ) (2) R Intre avfenoa oruDsossinijneo gctRioonunteis an acceptable route to assess systemic exposure. (3) DoSisnignlegDduorsaetion (9) DoPsreeparation The test material will injection to achieve a be diluted with sterile water for specific concentration for each dadomsienilsetveelredinunGdrioluuptsed1-4a.t thTehe50t0estmgm/aktgerdioasle wlielvlel,beusing dtohseesbuwliklldebnesitcyalctuoladteetdermbianseed tohne dtohseeanviomluamle'.s bIonddyivwiediugalht taken just before test prepared test mixtures material administration. The will be stored at room temperature until administration. 0. ObservoafAtniimoalns (1) Clinical Observations aTthfoetxeircaintitmyraelaasttmeawniptlp.lroxbiemaotbesleyrve0d.5,for2.0c,lin4i.0c,al 24s,ignassdo4f8 hours 000473 000027 Page 21 of 24 ) PPa8ge087 4P.K (2) BoJudsytWbeeifgohrtestest material administration. (3) Sample Collections (a) F2r,e4q,ue6n,cy8, 12, 24, and 48 hours post-injection (b) ANl umofbAne imarls (c) M Bflrooome deisot tafhmCeplroh elsclaeo r(catvpid ipoarnoxthiematcealtyhet4ermLi)zatwiiloln boef cthoellected a4,ur6i,cu8l,ar12a,rteanrdy 2o4r hforuormstphoestm-airngjiencatlioena.r vein at 2, dpAoopscpturemorexiniotmreadtveelinyna2tc0haevmaLraowoffdabetalaco)hodawni(lialmcatlubaelatobvtotalheuimneetdinteofrbooefm the n20ecmrLoposfy b(l4o8odhowuirlsl pobset-cionljleeccttieodn)f.ronAmpoprriobxuinmdately , scaeannmitpmrlaielfssugdweiudlr,lingbaendtshetthoersetdusdeypa,atraartloesoom,setreimufpmepraoanstdsuibrcleeel.laundlaTrhtehen cfterelamlcputeliroaantrsurfersatocoftriedo-n2s0inCwial+l1f0rbeCee.zesreTnthseetfsreotzpoeanrmaatiteondtatihsneeruSamponasnodr after experimental ternination. Samples will be shipped to: J3MameEsE0. &JPo.hCn.son B9l3d5g.Bus2h-36A-v0e9n)ue St. Paul, MN 55106 J(a6n1e2)s 07.78J-5o2h3n4sonprwiiolrl tobethneotisfhiiepdmenbty otfeltehpehonsaempaltes. E. Termination (1) UAmnonsrycihbaeundniudmlalecodndSdyaiictnrigiofnd,iucrweiinslgalntdbhDeeessatutubhdjsyectoerdsatocriafnicaebdbreivniaated grercoosrsdedn.ecroApnsiymaelxsamiinnataiomnoriabnudndallconadbintoiromnaliwitlilesbweill be acanveas,theatnidzeedxsawnigtuhinsaotdeidu.m pentobarbital, bled via the vena 000474 00002sS Page 22 of 24 PPa8ge088 4P.K (2) SAspcetuhnretvadoipubvplairernodbgxiSitamtaoacltr,etilefybrilmce4ide8natvhiioaounrstwhieplolvsetnb-aeincjaaenvceats,itohnea,tndizaeendxismawanilgtsuhinsaotdeidu.m Adonnea,bbhroewveivaetre,d gtriossssuesnewcirlolpsybeecxoalmliencatteido.n will not ba (2) SdTuahremipnlwgeholtCehoelliesvcteutrdiyo,nandsacbirlieficferdonineacahmoarniibnaulnddycionngdition, okirdnseuyrsvivfirnogn tissues will beteaochptleaarcnmeiidnnaaltoniowdinlrlywilialclesobeibmecmoelcdloielacltteeecldty.eda,ftBeortThhe mcaoilnlteacitniona taendmpetrhaetnurpelacoefd-2i0naC 1fr0eeCz.er set to tTfhereromzeitnniastosinuoensd.ry(1Tiihvceeer,stoambptillheee,sSwpkioilndlsneoybrse)asfhwtiielprlpeedxbpeetorsiemntethnetal srpaeemrspsplooennss.ilbilsetedforinthSeecrteitonent7.i0o.n(3a)n.d (cd)i.sposTihteioSnpoonfsortheis ' F. SWtoatsitsattiicsatilcAanlalaynsaelysses are required. 8. ARepfoinratl report including those items 11sted below will be submitted. DDeessccrriippttiioonn ooff tthhee tteesstt asnydstceomntrol materials DPartoecseduorfesexperimental initiation and termination GDreosscsripptaitohnoloogfyanfyintdoixnigcs/gerfofsesctspathology report (if applicable) 000475 000029 Page 23 of 24 TPaPgae098eP.K | 9. LofOarcciaigt]ii{notalantoedfatRaaua,dwiDtoirantgcao.ptihReeescsottruhddesyr.eoadfun,rdinwFigilnliat]sbeRperapovogarrietlsasbleandatbeHifloreto raalecltcaeiponrteiagdnicneianloftphaetpheaerrcFhdiianvatleas, roeipfnocrHltMu.IdinagcWchoetrnhdoistnehge itftoienmaAlNTIirsteSoeppdo.rtbelioswcwomiplletbeed, DPorsoetocporlepaarnadtipornotorceoclordasmendments InB-oTdiyfeweriegchotrsds ObDsoeservaadtaifonnisstration PSaatmhpolleogcyollreeccotridosn records SFitnuadly creoprorretspo(nodreingicneal signed copy) Tnhoet bfeolalrocwhiinvgedsuwpiptohrtitnhge rsetcuodrydsdatwai.ll be retained at HWI but will AWnaitmearl anraelcyesiipst'/arceccloirmdastion records ) ARneifmrailgerraotoomrtaenmdpefrraeteuzreerantdemhpuemriadtiutrye rreeccoorrddss Instrument calibration and maintenance records ) 000476 0000350 Page 24 of 24 PPa8ge08140P.K PROTOCOL. APPROVAL John LTG!plGuotwernh2.ofLf,lPhoDbet) Sponsors Representative Nee NN . SStteuvdeynDiN.recGtloazra ` AHcauztleetoTnoxWiicsocloongsyin, Inc. resenLtaitive . QHauzalleittoynAsWsisucroannscien,UniItnc. (6329-129.protdskl) ne 1-18-9 Date aa Date w/a) ) 000477 0000751 9.1.2 Analytical protocol AMDT-112294.1 000478 000032 3M Environmental Laboratory Protocol - Analytical Study Single-dose Intravenous Pharmacokinetic Study of T-6049 in Rabbits In-Vivo Study Reference Number: HWI#6329-129 Study Number: AMDT-112294.1 Test Substance: FC-95 (T-6049) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Initiation Date: July 25, 1995 Proposed Completion Date: August 25,1995 Method Numbers and Revisions: AMDT-M-1-0, Thermal ExtractionofFluoride by Means ofaModified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ton Analyzer AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, AnalysisofRabbit Liver ExtractforFluorochemicals Using Electrospray Mass Spectrometry AMDT-M-14-0, Thermal ExtractionofFluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Author: James D. Johnson Approved By: `James BY/Johnson Study(Pirector hes Date Pl 2 Gli fifi JBohnutenPAhD offDat,e Sponsor Representative 000479 000033 lopurpOSE This study is designed to provide information as to whether the perfluorooctanesulfonate anion does go to the rabbit liver and other tissues when the `material is administered in an intravenous dose, and to ascertain the change in cinotnecrepnrtertadteironmawlitahbstoirmpetiaofntesrtduodisees iwnhseerreupmerafnlduolrivoeorc.taTnheessulefdoantaatewiislldibreecutsleyd to applied or is present as a biotransformation product. The persistence of perfluorooctanesulfonate as a marker in liver and serum or possibly other tissues is a critical part for the interpretation of these dermal studies. 2T 0 ESTMATERIALS 2.1 Test, Control, and Reference Substances and Matrices e2.q1u.i1vaAlnenatl.ytical Reference Substance: FC-95, lot 161 or 171. They are 2.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 2.1.3 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2li.v2erS)o, uSricgemaofCMhaetmeirciaallsC:o3mMpaInCyP/(2P.C1.P2,Di2v.i1s.4iosner(2u.m1).1), grocery store (2.1.2, 2.1.4 2.3 Number of Test and Control Samples: Liver and serum from 4 test animals and 1 control animal. Other biological tissues (kidney, bile, cellular fraction) will be available for analysis if deemed appropriate by the Study Director. 2.4 Identification of Test and Control Samples: The samples are identified using tnhuembHeWr,I palnuismtahleitdiesnstuiefiicdaetnitointynuamndbedraywhiidecnhtictoyns(issetrsumo).fa letter and five digit 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), -20 + 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 000480 00000035% 2 2.8 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. This study is in parallel with a 28 day dermal absorption study so all tissues will be retained. 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 3.0 EXPERIMENTAL - Overview The tissues from animals dosed as described (HWI#6329-129), are available for analysis for fluorine compounds. At the discretion of the Study Director, a series of analytical tests can be performed. The screening for fluoride in liver via combustion (See Methods--next Section) is the appropriate analysis to present definitive data for fluorine in the liver. For these analyses, bovine liver appears to be a good approximation to rabbit liver for matrix spikes and recovery determinations. Since perfluorooctanesulfonate anion is not biotransformed, fluorine content will be an accurate estimate of the concentrationof the compound. 4.0 EXPERIMENTAL - Methods 4.1 Liver and Serum screening methods: (attached) 4.1.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Meansof a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 4.1.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 4.1.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 4M.o1d.i5fiAeMdDDTo-hMr-m1a4n-n0,DXT2h0er0m0aOlrEgxatnriacctHiaolnidoef AFlnuaolryizdeerb-ySeMreuamns of a 000481 000035 SO0DATAANA ~~~LYS00I 000S 5.1 Data Reporting: Data will be reported as a concentration (weight/weight) of fluoride per tissue or fluid, or as FC-95 per tissue or fluid. Half-life of disappearance from serum will be determined from the linear regression of the averages of the concentrations at different time points for the post distribution part of the serum concentration versus time curve. 6D .0 MAA INTT ENAA NCEA OFRN AW DRECOR ~~ D ~S 6.1 Raw Data and Records: Raw data, approved protocol, appropriate specimens, approved final report, and electronic data will be maintained in the AMDT Archives. 21.0 REFERENCES ~~~ 0 7.1 AMDT-S-10-0, Sample Tracking System 80ATTACHMENTS 8.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 8.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M-4-0, Extractionof Fluorochemicals from Rabbit Liver 8.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 8.5 AMDT-M-14-0, Thermal Extraction of Fluoride by Means ofaModified Dohrmann DX2000 Organic Halide Analyzer-Serum 000482 000036 4 3M Environmental Laboratory Method Thermal ExtractionofFluoride by Means of a Modified Organic Halide Analyzer - Liver Dohrmann DX2000 Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: /o- #-1 Revision Date: None Author: Rich Youngblom Approved by: = foup Lead: ;{ 23 Quality Assurance ---- /fo/3/8< Date 2-7 9 Date Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Meansofan Orion EA940 Expandable fon Analyzer AMDT-EP-3 Routine Maintenanceof a Modified Dohrmann DX2000 Organic Halide Analyzer 000483 1 000037 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES f1l.1uoSrcidoepef:roTmhivsarmieotushomdatriiscfeosr. thTehoepefrlautoiroidneoisfatyDpiocharllmyacnonllDeXct2e0d0in0TwIhSeAnBitsiosluutsieodn tfoorexatnraalcytsis w1.i2thApapnliiocnasbelleecCtiovmepeoleucntrdosde:.Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological tissues, particularly liver. 2.0 KEYWORDS 2D.X12F0l0u0or,idfel,uofrlouocrhienmei,ceaxltsr.action, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, 3.0 PRECAUTIONS 3.1 Glassware and exhaust gases can be extremely hot 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 3.4 SPorlesvseunrtibzeadsegdasseasm,pplreospemracyofmlparshe,smseadygnaesehdantdolailnlgowprtachteimcetso required. dry down before starting run. 3.5 Potential equipment. biohazards due to the biological matrices. Use appropriate personal protective 4.0 SUPPLIES AND MATERIALS 4.1 4.2 Compressed Compressed Oxygen, Helium, Hydrocarbon free, High Purity Grade, regulated regulated to to 30 45 PSL. PSL. 44.34 QQuuaarrttzz ggllaassss csoammbpulsetbiooanttwuibte,h TReelfilaonnceTMGbliasnsg,G-D9o4h0r5m-a0n1n2 8o9r0e-q0ui9v7aloernetquivalent, 4.5 4.6 OSraimopnle94c0ol9l9e9ctTiootnavliaIlosn,icHDSPtEre.ngth Adjustment Buffer (TISAB II) or equivalent. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 44..190AcHtaimvialtteodnCShyarrcionagle,oEr.eMqueirvaclken2t0.05 or equivalent. 4.11 Miscellaneous laboratory glassware 50 0EQUIPMENT 000 5.1 5.2 RIoBsMemcoomupnattiDbolher3m8a6nnorD4X8260c0o0mpOurtgera.nic Halide Analyzer, modified for fluoride extraction. 5.3 5.4 DX2000 software, Excel Spreadsheet, vveerrssiioonn 15..000,ormgordeiaftieerd for fluoride extraction. 6.0 INTERFERENCES 6m.a1ySvaamryplferosimzemaitsrliixmittoedmattoriaxp.proximately 150 mg, depending on sample moisture content. This 000484 0002 038 2.0 SAMPLE HANDLING s7.a1mpSlaem.plUesseafroerncoetpstoorbeprhoabnedlteodtrwaintshfebrartiesshuaensd.s. Fluoride may leach from the skin to the c7l.e2aSnasmcpallpeeslofanldivceurttairnegcbuotarfdr.omTfhreozceunttliinvgerbaoanrddpalnacdesdcailnpaeltasrheoduladndbelacbleelaendewdewiigthh bwoaatte.r,Use a `methanol, or methanol-water solution after each liver is cut ALIBRAT! TANDAI Te 8.1 PreparationofCalibration Standards R8.e1f.e1rTthoeprsottaoncdoalrdfsorrtehqautirpreodjefcotr.cach project will need to be appropriate for that individual project. 88..11..32 FTyoprircaablbliyt 5li0v-e5r0s0tupdipems,FuCs-eb9e5eifnlimveetrhaansotlhestmaatnrdiaxr.dsCaurteauspeid.ece of frozenbeefliver (100 150 mg) and weigh it in a labeled and tared weigh boat. 82 Calibration - Overview c`Tuhrevenoirsmraelquciarleidbrtahteiponroicsetdhuerfeluliosrtieddebceulrovwe (isAuMsDeTd.-M-2). However,ifan optional spiked liver a8.n2d.1coAmbcuasltiibrnagtitohnecmuursvienfgorthtehesaDmXe20me0t0hoisdsgeannedramtaetdrbixytsyppiekiasngthseamspalmepslewsittho kbenotewsnteds.tandards 8.2.2 Typically, spiked. three replicatesof cach standard and five concentrations of standards will be 8R.e2c.o3veSrteadndFar(dugc)uornvethwielyl-abxeisp.lotGteenderaastMeaassreSgpreiskseidoFn c(uurgv)eoanndthcealxc-ualxaitseatnhde eSqtuaantdiaorndfMoarstshe line 8a.n2d.4thMea1s2svaSlpuiek.ed F (ug) = (Amount spiked in mL) x ( Conc. ofstandard in ppm) x (0.6004) 8.2.5 Standard*MFaCs-s95Reisco6v0e.r0e4d%FF(tuhge)re=fo(rTeI0S.A6B00v4oilutmhee finacmtoLr)usxe(dOtroiocnonrveeardtinFgC-i9n5pptron)F 8.3 Calibration - Procedure 8.3.1 Start Up 8.3.1.1 be used Run 2 or more if the previous sCalmepalneCsyccolnetsaiwnheednhisgtahrtcionngceinntsrtartuimoennst oefacflhuodraiyd.e. More clean cycles may 83.2 Blanks 83.2.1 8.3.2.2 PFroerpraarbebistasmtpuldeiesu,siunsgetbheeesfalmiveermaesthtohedsmaatnridx.tPryoefpapmraeteratixleaasstt3hesatemsptlseasmpolfe.beef liver (1008.3.2.3 150 mg) for Put sample blanks. in Dohrmann boat. Combust each sample as described in section 9.0 and analyze sample according to method AMDT-M-2 for the fon selective electrode analysis 000485 3 000039 8.3.2.4 For rabbit studies, the meter reading foar blank sample should be 0.03 ppm or lower jbuedfogreemepnrtoocfeetdhiengopweirtahttohrethcaelriberaadtiinogn.is Bstuamblesawmiptlhersesupneticlt tthoishilsitmoirticisa.lrreeaachdeidn,gsor(purnetvilioiunst4h8e hours). 8b.e3f.o2r.e5prFoocreneodni-nrgabwbiitth stthuediceasli,brtahteiobnl.ank readings should reach a predetermined ion concentration 8.3.2.6 It may be necessary to mix approximately 50 mg of charcoal with the sample to aid `combustion. 8.3.3 Standard Curve 8.3.3.1 Weigh out at least 15 matrix samples (5 standards with 3 replicates each) in tared and labeled weigh boats. study data. Store the For rabbit studies, matrix samples on weigh dry ice 1o0r0i-c1e5p0amckgsbetoefkelievperthseammpflreos.zenRuenctiolrdusweed.ights in 8.33.2 Place weighedbeef liver sample in Dohrmann sample boat. 8.3.3.3 Start with the lowest standard concentration. Using a Hamilton syringe, eject a fixed qourainntitthye obefetfhelisvetra.ndard on or in the matrix. For rabbit studies, use 4 uLofstandard and eject iton m8.a3y.3.b4e uAsteldeaasttth3erdeipsliccraetteisonsohfotuhled banealuysset.d for the lowest standard concentration; more replicates 8.3.3.5 8.3.3.6 Combust the sample Run all 15 standards. aIsfodensecrriebpeldicianteseisctsiiognni9f.i3caanntldyadniaflfyerzeentacfcroormditnhge to AMDT-M-2. other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and 8.3.3.7 When all that the new replicate standards have been will run, be used to calculate calculate the 12. 12 the regression curve. must be at least 0.95. Ifit is not at l8e.a3s.t3.08.95A,nceonwsuslttanwdiatrhdscuuperrvveissohro.uld be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion ofthe analyst. 8.4 Storage Conditions for Standards. 8.4.1 Storage requirements for standards are dependent on the individual standards used. `8T.y4p.i2caNlley,wsFtaCn-d9a5rdsstaarnedasrtdosresdhoautlrdoboemptreemppaerreadtuarteleianstploanstciec ascmroenwtht,op bottles. 9.0 PROCEDURES 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 Start Up Procedure: 9.2.1 [fthe program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the fumace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 000486 4 000050 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOATprogram found in the CELL CHECK menu. 9.2.6 See AMDT-EP-3 for details of the Dohrmann software. 9.3 Sample Extraction Procedure: 9m.i3x.1apOppreonxitmhaeteSlAyM5P0 LimEgoHfAcThaCrHcoaalndwiptlhactehethseamspalmeplteo aiindtchoemBbOuAstTi.on.It may be [fthis is ndeocnees,sacrhayrtcooal should also be mixed in while establishing the baseline and when generating the standard curve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriate volumeof TISAB solution or 1:1 TISAB: Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0mL of 1:1 TISAB:Milli-QTM water mixture. 9in.c3h.e4s.PlGaacseetsherevliaelasseod tdhuartitnhge ptyirpoolfystihse mCuOstMBbuUbSblTeItOhNrouTgUhBEthe iTsIiSnAtBh.e TISAB at least 0.25 93.5 Run the EOX-SOLIDS program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 Ifundiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. r9.e3m.o8vReinasneytThIe SeAndBofretmhaeincionmgbuosnttihoenttubueb.e with Milli-QTM water and wipe with a KIMWIPE to 9w.i3t.h9a Ocpotetnonthteipspaemdpalpephlaitccahtaoonrd vraecmuouvmeeadnoyutr.emIatimnainygbaeshnefcreosmstahteroyboastc.rapAspahrtciaclnesbeofrfetmheo.ved bottom boatto with aid in at hsepaCtluelaano rCyoctlhee.r similar device. A drop of Milli-QTM water may be added to the 9.3.10 Close the hatch. 99..33..1121 SRaumnpltheeisCrLeEadAyNfoBr OanAalTyspirsobgyraimo.n selective electrode (AMDT-M-2). 9.4 Sample Calculations 99..44..12 USasmeptlhee sMtaasndsaRrdeccouvrevreetdo cFal(cuugl)at=e (tTheISsAamBplvoel value. in mL) x (Orion reading inppm - intercept) (Slope) 10.0 VALIDATION 1100..11.1QuDaailliytyStCaornttrUoplCheck Samples: Once the standard curve is established, each day of analysis is started by concentration spiked analyzing QC samples. The samples used to generate the QC samples are siandard curve. to be Each the same as the lowest concentration must be dreopnleiciantetriispnlioctatneecuenslseasrsy.the first two replicates are within 20% of the standard curve, then a third 102 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samplesofdifferent matrices and different reference compounds. 10.3 Other Validation Parameters: NA 000487 s 000041 110 DATA ANA 1L1 Calculations ILLL 11.1.2 TFoorctahlecusltaatnedtahred fcluurovrei,deusceonrtergarcetsisoinoninantahleyssaimspilne,Exsceeel,mevterhsoidonAM5.D0To-rMg.re2a.ter. 112 Analyzing the Data a1r1e.2w.i1thri2n m2u0s%tobfeeaatclheaostth0e.r9a5nodr tghreeaotuert.lie"rOuitslgireerast"ermtahyanb2e0e0x%colfutdehdeifavtewroaogfetohfetthhorseeetrweoploicrates nleostsedthiannt5he0F%ionfalthReepaovretraalgoenogftwhiotshethtewor.easAonnyitswuachs ocuotnlsiierdsersehdoualndoubtelipero.inted out in the data and 1~ 20 ATTACHMENTS ~~ 000 None 13.0 REFERENCES 31439.1, RreovsiesimoonuBn,t Dohrmann December DX2000 1993) Organic Halide Analyzer Operator's Manual (Manual 915- 13.2 AMDT-M-2 Analyzer Fluoride Measurement by Meansofan Orion EA940 Expandable Ion 13.3 AMDT-EP-3 Analyzer Routine Maintenance ofa Modified Dohrmann DX2000 Organic Halide 14.0 REVISIONS Revision Number Reasonfor Change Revision Dae 000488 6. 000042 3M Environmental Laboratory Method Fluoride Measurement by Means of an Analyzer Orion EA940 Expandable Ion Method Identification Number: AMDT-M-2 Revision Number: 0 -_-- Author: Rich Youngblom Approved By: Gro] Leader Adoption Date: [0-445 Revision Date: None 2L3/2s Date Ze ALby BAL Quality ASsurance fo- #75 Date Software: MS Word 5.1a DAfofhercmtaednnDoDcXu2m0e0n0tsO:rgAanMiDcTH-aMli-d1e AnTahleyrzmearl ExtractionofFluoride by Means ofa Modified 000489 1. 000013 L0 SCOPE APPLICABLE COMPQUNDS, AND MATRICES I1o.1n SAnCaOlyPzEer:. This method is for the calibration and operation of an Orion EA940Expandable 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3 PH oAPfP6L.0. ICABLE MATRICES: Liquid samples in an appropriate buffer solution, Preferred 2.0 KEYWORDS 2.1 Fluoride, fluorine, ion selective electrode 3.1 No hazards identified with this method. 4.0 SUPPLIES AND MATERIALS 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer II (TISABII) orequivalent. 4.2 4.3 Orion Orion 9M4o0d9e0l790100000p1pemlefclturoordiedefisltlainngdasrodluotrioenqu(iAvgaCllen)to.r equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magnetic stir bars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100 mL volumetric flasks. 4.9 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. S0 EQUIPMENT 0000000 5.1 Orion Model EA940 Expandable lon Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode orequivalent. 5.3 5.4 MIaBgMnectoimcpaSttiirbPlleat3e8.6 or 486 computer (only neededifusing Orion 3 software). 5.5 Orion RS232 interface cable (only neededifusing Orion 3E software). 5.6 Microsoft Excel 5.0 (only neededif using Orion 3E software). 6.0 INTERFERENCES 6.1 It is recommended that the PH be ator near 6.0. A 1:1 mixture of TISAB and sample/Milli- Q6.2TMSwaamtpelrewtilelmpgeernaetruarlelymbaryinge.ffseacmtpflleuotroidpeHmeoafsur6.e0.ment. It is recommended that the sample be at room temperature as the standards were when the meter was calibrated. 6.3 The rate the samples are stirred at should be consistent with the rate the `standards were stirred. 000490 002 0033 6un4deArirelbeuctbrboldees. rapped under electrode can give erroneous readings. Make sure no air i trapped 2.0 SAMPLE HANDLING 7.1 No special handling necessary. 88..11.1PrMeepaasruarteio5n0 of Calibration mL of TISAB Standards into 5 100 mL plastic volumetric flasks. 8.1.2 8.1.3 LPiapbeetlteth0e.0f5l,as0k.s1,as0.05.,051.,0,0.a1n,d0.15,5m1.L0,oafnd110.05 ppm ppm fFl-u,oarildoengstwaintdhartdheindtaotethaenadppyroouprriinaitteilalys. labeled flasks. 88..11..45 SAhdadkeaptphreofxliamsaktsetloym3i0xmtLheosfolMuitilolnis-QTM water to cach flask. t8h.e1f6laEslkismoivneartethaeirabiurbbbulbebslefs.rom the flasks by tipping the flasks on their sides and rolling theairin 8.1.7 8.1.8 Bring Invert the volume in and shake the the flasks flasks for up the to the 100mL final mixing. mark with Milli-QTM water. 8.1.9 Record standards in Standards Log Book. 88..22.1CIaflniebcreastsiaorny, remove tape from electrode filling hole. 8.2.2 8.2.3 IEnjveecrttapfreobwedrtoopwseotf top seal. filling solution from bottomofelectrode to wet lower seal. 8.2.4 8.2.5 Fill The the electrode with filling solution. meter and the F- electrode are typically calibrated by direct measurement with no blank cthoerrmeacntuiofna,ctuusrienrg'ssiinndstarrudctsiownist.h concentrationsof 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, following 8.2.6 8.2.7 RCleecaonrdthteheelselcotpreodien tbhyeraipnpsrionpgrwiiatthe log book. Milli-QTM water and wiping the sides down with lab tissues. 88..33.1SCtaolriabgreatiCoonndsittanidoanrsdsfoarreSsttaonrdedaratdsroom temperature. 9.0 PROCEDURES 99..11.1CaTlhiebrsaatmipolneatnodbeMemaesausrureemdennte,edSsttaonbdearmdixmeedthwoitdh:TISAB using the proportions 9r.e1c.2omPmlaecnedaedsibrybatrheinTtIhSeAsBammpalneufaancdtuprlearc.e the sample on the stir plate. 9.13 Allow electrode is itnhseerstaedm,plmeaktoe msiuxreftohrearefaerwe sneocoainrdsbubbebfloerse tirnaspepretdinugntdheeretlheecterloedcet.rodWe.hen the 9.1.4 same The rate assamtphelecaslhiobrualtdiobne the same standards. 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. 000491 3 000045 9.2 Calibration And Measurement, Using Orion 3E Software: 99..22..11.1CaFloilblroawtisotnep:s 8.2.1 to 8.2.4 99..22..11..32 TPhreesscoFmupnuctteiornKscereye#n8wil(lFSa)s.k you to confirm the numberof standards to be used, Mcoankceentarnaytinoencoefstshareyscthaanndagredss,toanthdewihneftohremratoironnoptraesbelnatnekdiasntdo cbleicikncolnudCeOdNinTIthNeUcEal.ibration. 99..22..11..54 OPlbasceervtehetchelerceuaoddiengisnotnhethfiersgtrsatpahnidcadridsoplnatyhoensttihreplcaotmepuatnedr.cliWckhoennCtOheNTreIaNdiUnEgs.have 9st.a2b.i1l.i6zeRde,peparetsssteApC9C.2E.1P.T4 aRnEdA9D.2I.1N.5G.for the remaining standards. i9.n2t.e1r.c7epAtfatnedr ctohrerefliantailons.tanRdaercdo,rdthtehceosmlpopuet,eirntweirlcledpti,spalnady ctohrersellaotpieoonfinthtehecuarpvper,oparsiwaetlelloags bthoeok and click on CONTINUE. The calibration data is automatically copied to C:\Orion\Data\Calib.txt. 9.2.2 Data Spreadsheet: 190.2s.t2o.r1e Sdealteacitn.either NEW or OPEN from the FILE menu to open a new or existing spreadsheet 9.22.2 Record the name of the spreadsheet used in the appropriate log book. 9.2.3 Fluoride 9.2.3.1 Follow sMteeapssu9r.2e.m1etnhtr:ough 9.2.4 99..22..33..23 ECnltiecrk tohne tnhaemNeoEfWthSeAsMamPpLleEibnutthteonappropriate place on the screen a9.p2p.r3o.p4riWahteenlogthbeoorke.adings have stabilized, click on the RECORD button and write the result in the 10.0 VALIDATION 10.1 Quality Control: 102 Precision and Accuracy p1p03m fOltuohreirdeVuapli1d0ataisoantuPraatreadmseotleurtsionAocfcfolruodriindge.to Reference 13.2, the range ofdetection is 0.02 11.0 DATAANALYSIS 1L1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHMENTS None ~~~ 0000000 13.0 REFERENCES 000492 4 000036 13.1 Orion Model EA940 Expandable fon Analyzer Instruction Manual, Orion Research Incorporated, 1991. 13.2 Orion Model 960900 Solid State Combination Fluoride Electrode Instruction Manual, Orion Research Incorporated, 1991. 14.0 REVISIONS RNeuvimsbieorn ReafosrChoannge DRevuisieon 000493 000037 Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 - Adoption Date: /o-7/-55 Revision Date: None Author: Dave Christenson/Cynthia Weber Approved By: jroup Leader Le Buhl Quality Assurance Jo=3/-25 Date. las" Date ASfoffetcwtaerde:DoMcuSmeWnotrsd:, M6.-05, Analysisof Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. 000494 000045 1 1.1 SEtchoyple:acTethaitsemies tuhsoedd tios efxotrrtahcet fexlturoarcotcihoenmoicfafllsuforroocmhtehmeiclaivlesrsfrfoormarnaablbyistislibveyrs. 1.2 eAlpepcltircoaspbrlaey Cmoamssposupencdtsro:scFolpyu.orochemicals or other fluorinated compounds. 1.3 Matrices: Rabbit Livers. 2.1 Fcloumoprooucnhdemsi,ceaxltsr,acrtaibobni.t livers, electrospray mass spectrometer, fluorinated 3.1 Use gloves when handling the rabbit livers, they may contain pathogens. 4.0 SU4.P1PLISuEpSplAieNs DMATERIALS ~~~ 44..11..21 ESpyrpienngdeo,rcfaptaybpleoerofdimsepaossuarbilnegp1ip0e0tspL 4.1.3 4.1.4 PGllaosvtiecsgrinding tubes 44..11..56 PLlaabsetlisc centrifuge tubes, 15 mL 44.:1187 TNiitmreorgen 44..11..910 AFnilatleyrtsi,cTailtpainpentys:lognlassysrivnogleumfeitltreircs,p0ip.e2tpsm. 44..11..1112CrDiismpposacbalpeapultaosvtiiacls3. cc syringes. 4.2 4R.e2a.g1enAtqsueous Ammonium Acetate (Aldrich), approx. 250 ppm: Prepare a 2500 apcpetmataequ10eoau1s0s0olmuLtiovnoloufmaetmrmioc nfliauskm aancdetdaitleutbeytaodvdoilnugrn2e5w0imthgMaimllmio-nQ ium 4.2.2 `Swoadteiru.mDiclaurtbeotnhaitses/oSloutdiiounm1B:i1c0afrobronaa2t5e0Bpufpfmers(oJl.uTt.ioBna.ker), 2(nNd22,1C.O0/gNaofHsCoOd,i)um0.b2i5caMr:boWneatiegh(N2a6.H5CgOo;fisnotodiaum1 Lcavroblounmaetteri(cNaf,laCsOk,a)nd 4.2.3 DbirliuntgetoacveotolnuimteilweistohluMtiiloln,i-dQilwuatteearc.etonitrile 1:1 with Milli-Q water. 44..22..35 MEtehtyhlanAoceltate 44..22..76 M1iHl,l1i-HQ.2wHat,e2rH - perflugrooctanesulfonic acid (Aldrich) 4.2.8 FCI95 (3M Specialty Chemical Division) 000495 00003y 2 5.1 Ultra-Turrax T25 Grinder for grinding liver samples. 5.2 Vortex mixer 5.3 Centrifuge 5.4 Shaker 5.5 Analytical Evaporator 6.1 There are no known interferences at this time. 7.1 The rabbit livers are received frozen, and mustbe kept frozen until the extraction is performed. 8.1 8P.r1e.pa1rationareoafnIinntteerrnnaallstSatnadnadrdarodfsapproximately 12 ppm1H,1H,2H,2Hluorooctanesulphonic acid to be addedto each liver sample, 8.1.2 Weigh at least 0.1 g of1H,1H,2H,2H-perfluorooctanesulphonic acid into a 100 mL volumetric flask. Record the actual weight. 8.1.3 Bring it up to volume with methanol, this is the stock standard. 8.1.4 To a 250 mL volumetric flask, add 3 mLsofthe stock standard and bring to volume with Milli-Q water. Calculate the actual concentration of the standard. OPER Xml = actual mg luoroctane0.1L 250 mL. scualconcentadon, ppm 8.2 8P.r2e.p1arPereFpCar-e95FC-A9n5iosntanSdtaardnsdaforrdtshe standard curve. 8.2.2 Weigh approximately 100 mg of Record the actual weight. FC-95into a 100 mL volumetric flask. 8.2.3 Bring up to volume with dilute acetonitrile. 8.2.4 Dilute the solution with dilute acetonitrile 1:10 foar solution of aapsporlouxtiiomnatoeflyap1p0r0oxp.pm1.0 Dpiplmu.te this solution 1:10 with dilute acetonitrile for 8.2.5 Use the 10 ppm solution to make working standards with values close to 8.3 Prepare5.0Bpepemf,Li1.v0erppHmoamnodge5n00atpepb.to Use for Standards 8.3.1 Weigh 40 g of Bovine liver into a 250 mL Nalgene bottle containing 8.3.2 200 Add m1LmsLoMifltlhi-eQswoaltuetri.onGrtoinad1t5omaLhcoemnotgriefnuoguestusboel.utPiroen.pare a totalof eight s1 umspLenadlisqoulouttsioonf tbhye sshoaluktiinogn iitnbe15twmeLencaelnitqruioftusg.e tubes. Be sure to re- 000496 000050 3 8.3.3 sStpainkdearsdesveinn ostfetphe9.112mLo.fatlhiequportoscewdiutrhe.thOe nfeoll1 omwiLngalaimqouuotntsserovfe`swoarsktihen.g blank. =TT Bak Working Standard (Approximate Conc.) "Approximate final concentration of FC-95 in liver 8.4 Calculate the actual value of the standards: scofosntancdared_xntrati(inopmn) = final concentration. (ppm) 171 mg liver' / 1 ml homogenate of FC -95 in liver *Average weight of bovine liver in solution as determined by weighing 1mL homogenates of40 `mgliverin 200mL of Milli-Q water. The amount of [FC-95 is reported as equivalents of FC-95 potassium salt. 8.5 Calibration specrometer. 8.5.1 Extract the spiked beef liver homogenate following 9.13 to 9.23 of this method. Use these standards to establish your curve on the mass 8.5.2 Aliematively, a standard curve may be generated using ratiosofresponses coofntcheentpreartfilounoroofoctthaenpseurlfflounoartoeocatnainoensaulnfdontahteeinatneironn.al standard anion versus 8.6 Storage Conditions for Standards 8.6.1 cNoevweresdtapnldaasrtdisc careentprriefpugaeretdubweisthunetialchthaenaanlaylsyiss.isSotanntdhaerdmsasasresspteocrterdomineter is performed. 8.7 8S.t7o.r1ageBeeCfonlidvietrihoonmsogfeonrateSstamnadyarbdesfrozen after preparation. 9.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been 9:2 9.3 Upascekaagdeisdsewcittihngotshcearlpteilssuaensd. cut Weigh the sample directlyinto a off approximately tared plastic grind 1 in g g of liv tube. er. 9.4 9-5 Record the Putalabel liver weight in on the vial with tthheessttuuddyynnoutmebbeoro,k,weight, rabbit ID, date andanalyst initials. 000497 000051 4 99..76 G`Amrdiindnudt2e.ts5h,emunsLtaismlpwtlahete.esrP.aumtptlheeigsrainhdoermopgreobneeoiunsthseolsuatmiponlewiatnhdngorilnadrgfeorchaubnokust,2 99:98 ATRaiMnkDseTe-tEthhePe-grp2ir2no.dbeeroafpfaritntaontdheclseaamnpiltewiwtihthme2t.h5amnoLlsawfatterercaucshinsgamaplpei.petF.ollow 99--1110 iPCinapfpeotrtmh1aetmisLoanmipanslteotahaen1dg5rvimonLrdtiencxgentftuorbrief.1u5g(eSseetecuboAen.dMsLD.aTb-elM-th4e cWeonrtkrisfhuegeettufboer dwoicthumtehnetiidnengtitchael 9:12 Srpeimkaeintihnegbesetetplsi.)ver homogenates as described in 8.3. with the appropriate amount of FC-95 standard 99--1143 aAScpdeitdakte1em.thLeosfampthleessoadnidubmeceafrlbiovneratheo/msoogdeinuamtbeiscawribtohna1t0e0buufLfoefrinatnedrn1 amlLstaanmdmaordn,ium 99.-1165 UCsaipntghaensaanmapllyetiacanldpviopertt,exad2d05tmo L30estehcyolnadcse.tate. 99-.1187 CPeuntttrhifeumgeinftohre2s0htaok2e5rfomrin2u0temsi,nu,nl on the centrifuge to 25, the layers are well separated. Set the power 9-19 9:20 BmRleLomwgorvtahedeu4astamemdLplgeloafdssothwpeinpteootp.noTtrrhgaeannasinfacelrylratiyhceearlltaoebveaalpfotroreasththoe1r5fmroesLnhcetaeurnbtder.riyfnuegses twuibteh wniittrhoage5n, 93.:2221 Barpipnrgoxtihmearteemlayi3ni0ntgo Vortex 15 scconds. s4a0mpmilneutuepsi.n 1 mL dilute acetonitrilewithan analytical piper. 9-23 stTthreuadnsysafnmepurlmtebheeirnstaaonmadpflraeentsoiahmacel3ntnmruiLmfbusgeyerrtinbgee.oArtataacuhtovai0a.l2. Luambenlylthoentmuebseohrfviiltaelr,waintdh ftihleter 99.-2254 CCoamppalnedtehoAlMdDfTor-aMn-al4yswiosrbkysheeleetctarnodspartatyacmhastospsapgeectorfossctoupdyy.notebook. 10.0 VALIDATION 1100..21 PQrueacliistiyonCoanntdroAlc-cunrotacayp-plnioctabalpeplicable 10.3 Other Validation Parameters. not applicable 1L0 D1A1.1TANAonNeALYSIS ~~~ 00000 12.1 Worksheet AMDT-M-4 130R1E3.F1 ERAMEDTN-CEPE-2S2 Routine Maintenance of Ultra Turrax 1-2~5 ~ 14.0 REVISIONS Revision Number ReasonforChange. Revision Dae 000498 0000552 C FeE EE Worksheet AMDT-M-4 F rF m rr F F r F rt t EE E t P -- E----R-- rr t tr r fm ee rere -------- 000499 000053 3M Environmental Laboratory Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Number: 0 _-- Adoption Date: - 4 Revision Date: None Author: Dave Christenson/Cynthia Weber Approved By: roup Leader les Date QuaVlit3y Assurance < ouDae (Fe `SAoffftewcatrede:DoMcuSmeWnotrs:d,M6-.40, ExtractionofFluorochemicals fromRabbitLivers 000500 000054 1 LoscopE 0000000 1.1 Scope: This method is for the analysis of extracts of rabbit liver or other tissues or fluids for fluorochemicals using the electrospray mass spectrometer. Theanalysis sistapnedrafrodrmMe/dZb=y 4s2i7ng,leanidonotmhoenriatpoprrionpgrioaftFeCm-a9ss5esa.nion, M/Z= 499, the internal 1.2 Applicable Compounds: Fluorochemicals or other fluorinated`compounds. 1.3 Matrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 20KEYWORDS ~~~ 2.1 Fluorochemicals, fluorinated compounds, electrospray mass Spectroscopy, mass spectrometer, rabbit livers. 3.0 PRECAUTIONS ~~~ 3.1 Use caution with the voliage cable for the probe. When the voltage cable is plugged 3.2 iDnotontohterpurnobtheeDpOumNpOaTboTvOe UitC'sHcaTpaHcEitPy RoOf B4E00,0tphseir.eIifpsrreissksoufreelegcotersicoavlesrh4oc0k0.0 psi stop find and release pressure. the plug and replace tTheheplpuegagkedtutbuibninggm. aSyeebeAMplDuTg-geEdP.-T1r5oubleshoot back to 3.3 Do not run the pump to dryness. PP) AN] T 4.1 Supplies 4.1.1 4.1.2 NFliutorfoigxencoglausmrnegourlaeqtueidvtaole1n4t.0 psi. 4.1.3 100 uL or 250 uL flat tip syringe for sample injection. 4.2 Reagents 4.2.1 Dilute acetonitrile 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. 5.0 EQUIPMENT 5.1 VG Trio 2000 Electrospray Mass Spectrometer or equivalent. 5.2 ISCO Syringe Pump 5.3 Spectraphysics AS300 Autosampler 5.4 100 uL Assembly 5.5 Autovials or capped centrifuge tubes. 6I 0 NTERFERENCES 6.1 There are no known interferences at this time. 7.0 SAMPLE HANDLING 7.1 uKnetielprtehaedyextforracatneadlyssiasm.ples in capped 15 mL centrifuge tubes or in capped autovials 000501 000055 2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of Calibration Standards 8.1.1 Seven beef liver standards and one blank beefliverare prepared during the extraction procedure. (See AMDT-M-4, section 8.0) 8.2 Calibration 8.2.1 Run the seven beef liver standards twice, starting with the lowest standard 8.2.2 tToypoibctaalilnytohneestsatnadnadradrdcuirsvreu.n after each 5 to 7 samples. Choose a standard in the same rangeof concentration as the samples. 8.3 Storage Conditions for Standards 8.3.1 Fcroevsehresdtaplnadsatridcs caernetipfreupgaerteudbewsi tuhnieilacthheanaanlaylsyissi.s Standa on the rmdassasrespsetcotroemdeitner is performed. Samples and standards are NOTrefrigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8.4.1 Beef liver homogenates may be frozen after preparation, S20 PROCEDURE 9.1 Initial Set-up ~~~ or 9.1.1 9.1.2 RSeetcosrodftbwaacrkeitnog"pOrpeesrsautree on", lon Mode ES in the instrument log. 9.1.3 Fill the solvent cylinder with mobile phase. 9.1.4 Set the pumpto "Run". Set the flow to 1000 uL/min. Observesdroplets coming out of the tip of the probe. The pressure should be at 1700 to 1800 out the tp. 9.1.5 Beck the fused silica at the chips. The tip should be flat end of the probe. Use with no jagged edges. an If eye any piece chips to check for are found cutoff the tip of the silica with a column cutter and pull the silica through to the appropriate length. 9.1.6 Cihoegceknyoluerakniintgroagreonusnudptphley . Turn on tip of the ptrhoebeni.tArogfeinn.e Ther mist e should be no should be comin g 9.1.7 Carefully guide the further. Connect the probe into the voltage cable opening. Insert to the probe. it until it won't go any 9.1.8 aGpporotportihaete"Emdaistsoer"s ptaog4e2,7anadndse4t9I9o.nization Mode to ES', and the 9.1.9Ifitis not in single ion mode go to "Option" and set SIR. 9.1.10Stan Acquisition. Assigna file name, MO-DAY-YR + letter. Record it in the log book. 9.1. 11Run the beef liver samples first, running each standard twice at the beginning of the 5107 samples. run.. Run a QCcheck by running one standard afterevery 9.2 Manual Injection 9.2.1 Draw 150 uL injection port. of sample into Inject slowly. a syringe. Inject the sample into Record the sample ID in the log the rheodyne book. 99..22..32 WTauimt tthweomvailnveuttoe"aOsnn,d".inject the next sample. 9.2.4 Record the scan number for cach sample in the logbook. 000502 00005>63 9.3 9U.s3i.n1g the Autosampler Setup sample tray A, B, or C. 9.3.2 vRieaclosrmdatyhebesaimnpelaecsharnudn.their positioinns the instrumelnogt book. Up to 17 9.3.3 9S.et3-.u3p.t1he sampler: Push the sample bution 99..33..33.32 SeSettsianmjpelcetslaomoppsliez=e2 = 100ul 991.3333.54 SNeatmCeyctlhee ftiilem:eL=iv0ers 99..33.133.67 AIddedntitfhye tshaempwlaeysutsoeQdueue by pressing "Enter" 9.3.3.8 Press "Run to start 10.1 Q10u.a1l.it1yRuCnonatsrtoalndard every 5 to 7 samples. If a significant change(t 50%) in psteaankdahredigwhitllocbceurussesd.ioTphteherreumna.iOnnilnyg stahempslaemsplweilslbbeeforreeantahleylzaesdt.acceptable 10.2 1Pr0e.c2i.s1iSoeneaMnedthoAdccVualriadcatyion Report number AMDT-M-5.0.V1 1100..43 ROetfheerrtoVMaeltihdoatdiVoanliPdaatriaomneRteeprosrt Number AMDT-M-5.0.V1 11.0 DATA ANALYSIS ~~~ 0000 1111..21 PClaoltcutlhaetisotnasndard curve, using the mean of the two values obtained for each s1t1a.n2d.ar1d.Read peak heights or areas for the samples from the printout. Use linear 11.2.2Creaglrceuslsaitoen to the dmetgeromfiFnCe-t9h5e asnaimopnl,eorcoontcheenrtrfaltuioonrso,chemical in the total rabbit liver: `mg FC-95 anion in the total rabbi liver = mggmFsCo:f9l5iavneriounsfedrofomrsatnda_lycsuirsve x Total mass of liver, gms 1111..34 MPraikntea acrhersoulmtasttoagbrleamanfdoreentaecrhist aimnptlhee,swtiutdhy tbhoeopkeaks labeled with the sample or ssttuadnydafrodldIeDr.. WSrtiatpleetahlel csthurdoymantuomgbrearmosnttohgeetphreirntaonudt,niunimtbiael,rdpaatgee,sa.nd put it in the 000503 000057 4 1A 20 TTACHMENTS None 130REFERENCES 13.1 AMDT-EP-17 40REVISIONS 00 Revision Number Reasonfor change Revision Dae 000504 000055 5 3M Environmental Laboratory Method Thermal Extraction ofOrFglaunoriicdHeablyidMeeAannaslyozfear Modified - Serum Dohrmann DX2000 Method Identification Number: AMDT-M-14 Revision Number: 0 Adoption Date: jo-3-9 Revision Date: None -_-- Author: Rich Youngblom Approved by: Gomer) roup Leader /0/5/ 2s Date le Me BA Quality Assurance Fez Date Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Meansofan Orion EA940 Expandable Ion Analyzer AMDT-EP-3 Organic RHoaultidieneAnMaaliynzteernanceof a Modified Dohrmann . DX2000 000505 000059 1 . Pl LICA MPOUND: TRI f1l.1uoSricdoepef:roTmhivsarmieoutshomdatrisicfeosr.thTehoepefrlautoiroindeoisfatyDpoicharlmlyanconllDecXt2e0d0i0n TwIhSeAnBitsiosluutsieodn tfoorexatnraalcytsis w1.i2thApapnloincasbelleecCtiovmepeoleucntrdosd:e.Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological fluids, particularly serum. 2.0 KEYWORDS D2.X12F0l0u0or,idfel,uofrlouocrhienmei,ceaxltsr.action, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, 3.0 PRECAUTIONS 3.1 3.2 Glassware Glassware and exhaust gases can is fragile, broken glass be extremely hot. may cause injuries. 3.3 3.4 PSroelsvseunrtizbeadsegdasseasm,pplreospemracyofmlparseh,ssmeadygnaesehdantdolailnlgowprtachteimcetso required. dry down before starting run. 3.5 Potential equipment biohazards due to the biological matrices. Use appropriate personal protective 4.0 [ES AND 44..12 CCoommpprreesssseedd OHexlyiguemn,,HHiygdhroPcuarirtbyonGrfardeee,,rreegguullaatteeddttoo3405PPSSLI.. 4.3 4.4 Quartz Quartz glass glass sample boat combustion with tube, TeflonTM Reliance tubing, Dohrmann Glass G-9405-012 890-097 or equivalent, or equivalent 4.5 4.6 SOarimopnle94c0ol9l9e9ctTiootnavliallosn,icHDSPtEre.ngth Adjustment Buffer (TISAB II) or equivalent 4.7 Milli-QP water 4.8 Polystyrene pipettes. 44..910AcHtaimvialtteodnCShyarrcionagle,oEr.eqMueirvcakle2nt005 or equivalent. 4.11 Miscellaneous laboratory glassware: 5.0_EQUIPMENT 5.1 5.2 IRoBsMemcoomupnattiDbolher3m8a6nnorD4X8260c0o0mpOurtgera.nic Halide Analyzer, modified for fluoride extraction. 5.3 5.4 DX2000 software, Excel Spreadsheet, version version 1.00, modified 5.0 or greater for fluoride extraction. 6.0 INTERFERENCES 6.1 Sample size is limited to approximately 100 ul. This may vary from matrix to matrix. 000506 00000 2 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 Preparation of Calibration Standards 8.1.1 The standards required for cach 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, usebeef serum as the matrix. 8.2 Calibration - Overview The nor curve is mal calibration required the pr is the fluoride ocedure listed curve below (AMDT-M-2). is used. However, if an optional spiked serum 8.2.1 A calibration curve for the DX2000 is generated by and combusting them using the same methods and matrix spiking samples with type as the samples to known standards be tested. 8.2.2 Typically, three replicatesofeach standard and five concentrationsofstandards will be spiked. 8.2.3 Standard curve will be plotted as Mass SpikedF Recovered F (ug) on the y-axis. Generate a regression (ug) on the x-axis and curve and calculate the Standard Mass equationforthe 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 *FC-95 is 60.04%F therefore 0.6004 is the Standard Mass Recovered F (ug) = (TISAB volume factor used to convert FC-95 to F in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 Start Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used ifthe previous samples contained high concentrations of fluoride. 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 sPuatmpsleeruamccbolradnikngintoDmoehtrhmoadnnAMbDoaTt.-MC-o2mbfuosr tthseaimopnlesealsecdteisvecreilbeecdtriondeseacntailoynsi9s.0 and. 8.3.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 ppm or lower `before proceeding with the calibration. Bum samples until this limit is reached, or until in the judgementofthe operator the reading is stable with respect to historical readings. (previous 48 hours). 000061 8.3.2.5 For non-rabbit before proceeding with studies, the blank the calibration. readings should reach a predetermined ion concentration 83.2.6 It may be necessary to mix approximately 50 mg of charcoal with the sample to aid `combustion. 000507 3 8.3.3 Standard Curve d8.a3y.3(.=1 3I0fbmeLe)f.serum is frozen, thaw at least enough to complete the standard curve analysis for the 8.3.3.2 Pipette 100uLof beef serum into Dohrmann sample boat. q8.u3a.n3t.i3tyoSftatrht ewistthantdhaerldoowensotrsitnantdhaermdatcroinxc.entFroartiroanb.bitUsstiundgieas,Hausmeil4tounLosfyrisntgaen,daejredctaandfeijxeecdt it on or in thebeefserum. 8.33.4 `may be Atleas3t replicates should be used used at the discretionofthe analyst. for the lowest standard concentration; more replicates 88.33..33..65 RCuonmbaullst15thsetasnadmarpdlse. aIsfdoensecrriebpeldicianteseisctsiiognni9f.i3caanntdlyadniaflfyerzeentacfcroormditnhge toothAeMrDtTw-oM-2. replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and 83.3.7 When all that the new replicate standards have been will run, be cal used culat to calculate e the 12. r2 the regression must be at lea curve. st 0.95. Ifit isnotat 8l.ea3s.t3.08.95A, ncoenwsuslttanwdiatrhdscupuerrvveissohro.uld be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretionofthe analyst. 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for `Typically, standards are stored standards are dependent on the individual standards at room temperature in plastic screw top bottles. used. 8.4.2 New FC-95 standards should be prepared at least once a month. 90 PROCEDURES ~~~ 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 99..22.1StIafrttheUpprPorgorcaemdiusrneo:t stated, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the fumace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOAT program found in the CELL CHECK menu. 9.2.6 See AMDT-EP-3 for details ofthe Dohrmann software. 9.3 Sample Extraction Procedure: 9.3.1 Open the SAMPLE HATCH necessary to mix approximately 50 and pipette 100uLofsample into mgofcharcoal with the sample to the aid BOAT. It may be combustion. If this is done, charcoal should also be mixed in while establishing the baseline and when generating the standard curve. 9.3.2 Close SAMPLE HATCH. 000508 00096: 9l.a3b.e3ledAdsadmapplperocporlileactteiovnolviualm.e oTyfpTiIcaSlAlBy 0s.o6lumtiLontoor1S1:m1 LTIaSrAeBu:seMdi.llFio-rQrTMabwbaittesrtumdiixest,urueseto1a.0 or 29..30.4mLPloafce1:t1heTIviSaAlBs:oMtihlatlith-eQTMtipwoaftetrhemiCxtOuMreB.USTION TUBE is in the TISAB at least 0.25 inches. Gases 9.3.5 Run the released during EOX-WATER pyrolysis must program found bubble through the in the RUN menu. TISAB. 9.3.6 9.3.7 IWfhuendniltuhteeEd OTXISpArBogwraasmuissefdintioshceodl,lerctemtohevesatmhpelceo,llaedcdtiaonn eviqaulalfrvoomluthmeecoofmMbiulstliio-nQTMtuwbae.ter 10 the TISAB 10 make 1:1 TISAB:Milli-Q. 9.3.8 Rinse remove any tThIeSeAndB orfemtaheincionmgbuosnttihoenttuubbee. with Milli-QTM water and wipe with a KIMWIPEto 9w.i3t.h9aOcpotetnonthteipspaemdpalpeplhiactacthoraanndd/roermovvaecuaunmyerdeomuat.iniIntgmaasyhbfernoemcetshseabroyat.to sAcsrhapcpaanrtbieclreesomfofvtehde bbooatttotomawiidthinathsepaCtluelaanorCyoctlhee.r similar device. A drop of Milli-QTM water may be added to the 93.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample is ready for analysis by ion selective electrode (AMDT-M.-2). 9.4 Sample Calculations 9.4.1 Use the standard curve to calculate the sample value 9.42 Sample Mass Recovered F (ug) = (TISAB vol in mL) x(Orionreadinginppm-intercept) (Slope) 10.0 VALIDATION 10.1 Quality Control a1n0a.l1y.s1isDaiislsytaSrttaerdtbUypanCalhyezcikngSaQmCplseasm:plOesn.ceTthhee QstCansdaamrdplceusrvaereistoesbteabtlhieshseadm,eeaacshthdeayloowfest dcoonnceenitnrtartiipolincastpeikuenldesssamtphleefsirustsetdwotorgepelniecraattees tahree swtiatnhdianr2d0cu%rovfe.thEeascthancdoanrcdencturravtei,otnhmeunsattbhierd replicate is not necessary. F1l0u2oriPdreecNiostieobnooaknsd2A,c3,cuarnadc5y.: PSreeceismieotnhaondddaecvceulroapcmyevnatriaensalwyhsiesnaanndalsyazmipnlgesaanmapllyessiosfidnifferent matrices and different reference compounds. 10.3 Other Validation Parameters: NA 11.0 DATA ANALYSIS 1L1 Calculations ILL 11.1.2 FToorctahlecusltaatnedathred fcluurovrei,deusceonrtergarcetsisoinoninatnhaelyssaimspilne,Exsceeel,mevterhsoidonA5M.D0To-rM:gr-e2a.ter. 000509 000063 5 11.2 Analyzing the Data a1r1e.2w.i1th1i2nm2u0s%t boef aetaclheasotth0e.r95anodr gtrheeatoeurt.lie"rOuitslgireerast"ermtahyanb2e0e0x%colfutdehdeifavtewroaogfeothfethtohsreeetrweoploircates less than noted in t5he0%FinoafltRheepaovretraagleonogfwtihtohsethtewor.easAonnyitswucahs ocuotnlsiierdsersehdoualndobutelipro.inted out in the data and 1~ 2.0 ATTACHMENTS ~~ None 1~ 3.0 REFERENCES ~~ 13.1 349, Rosemount revision B, Dohrmann December DX2000 1993) Organic Halide Analyzer Operator's Manual (Manual 915- 13.2 AMDT-M-2 Analyzer Fluoride Measurement by Meansofan Orion EA940 Expandable Ion 13.3 AMDT-EP-3 Analyzer Routine Maintenance ofaModified Dohrmann DX2000 Organic Halide 10 40REVISI0 ONS 0000 Revision Number ReasonforChange Revision Date 000510 000064 6 9.3 Quality Assurance Unit Statement 000511 000065 Anachment D GLP Study Quality Assurance Statement Study Title: Single-dose Intravenous Pharmacokinetic Study of T-6049 in Rabbits Study Number: AMDT-112294.1 Name of Auditor: Kari Rambo `TThhiesfsitnuddiynghsaswebreeenreinpsoprecttteeodd bthye tshteudQyuadliirteyctAosrsaunrdanmcaenUangietmaesnti.ndicated in the following table. Inspection Dates Eom To Phase 10-11-95 11-03-95 Final Report MDaatneaIgnespmeecntioSntRuedpyorDtierdecttoor 11-03-95 11-03-95 pr Cu `QAU Auditor 47.95 Date 000512 000066 9.4 Key Personnel Involved in the Study 000513 000067 3M Environmental Laboratory Key Personnel . Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysisofliver extracts using electrospray mass spectrometry: Jim Johnson Dave Christenson Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo 000514 000065 9.11.1 Summary and raw data; ug Fin whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 000515 000069 Summary of Combustion Data - Liver AMDT-112294.1, EWI 6329-129 As Referenced in Final Report section 6.0 DATA ANALYSIS Total ug Fluoride in Whole Liver Mean per Dose Group ug Control group~~ 23.37 5.0 mg/kg dose (T6049) 23.28 10.0 mg/kg dose (T6049) 16.01 100 mg/kg dose (T6049) 35.87 500 mg/kg dose (T6049) 92.03 000516 000070 FO35 PK Actual Average Whole Total F-in RPTIZOLXLS BLNK LiInVER1 % pinplmivFe-r pinplmiveFr- bliuvmeerd wleiivgehrt ry (WW) (WW) (grams) (gams) `lwihvoelre (ug) Dosage (make) 1.052 0.1216 BBULNNKKLLIIVVEERR 23 0074528 00.11153551 BBUUNNKK LLIIVVEERR 45 00340783 00.11457032 UUVVEERRSSPPKKEE3312 8767%% 01.809254 00.11320704 ULVVEERRSSPPKK613263.1 8843%% 21109425 00..11125095 LUVVEERRSSPPKK 112266.23 8863%% 21365136 00.11151504 UUVVEERRSSPPKK225533-12 8802%% 33.956880 0.1309 0.1228 LLIVVEERRSSPPKK255036.-13 6896%% 64..419935 0.1242 0.1292 LLIVVEERRSSPPKK550066:.23 6638%% 76.31030 00.11127057 LUVVEERRSSPPKK 1100112212 7653%% 1142..284844 00.11217878 BLILVAENRKSLPIKVE1R0-112.3 64% 103..463325 0011114349 BBLLAANNKK LLIIVVEER2R3 00231093 0.1223 0.1402 FF5s226e5522-12 0032322 0273 00.1123229 562 2037 00 FFss22665532..13 00321495 00..11308153 FF5s22665533..32 00221276 024 0011115158 oes 2328 50 FF5e22665544.21 00210784 0204 0.1484 01173 7885 1601 100 FeF522666554.31 00.243810 00..11411818 FF5s22666665..12 00433874 0434 00.1135225 8272 387 1000 F5F2s626660.612 10094733 0988 00.11144165 9311 9203 5000 LFsV2E6R66S3PKA 66% 00799419 0.1161 0.1269 LLVVEERRSSPPKKCB 7637%% 0895 01128 0.1290 UUVVEERRSSPPKKED 7764%% 00..11435977 LLIVVEERRSBLPNKKF-1 74% 00..11325312 OSL? Page 1 01000000771 9.11.2 Summary and raw data; analysisofliver extracts using electrospray mass spectrometry. 000518 000072 HW 6329-129 sPtruodtyo:col Number: MaTtorsitxM:aterial: RReSsqpuoanrseedFaVcaltuoer: Amount: ADnaatley:st: IMnostthroudm:ent: LABBASE File: PSi8n0gl8o4-DPoKse Inravanous Pharmacakinatic TLi6v0or49 in Rabbits (FC-95) 019.0771.705 o32l8e195 0Fias2o8n0s5VCG 2000 Electrospray MS A-1 CovdlainP s Y Al Shai 4-7 Chuanbimon-- 4-1-4 rou Dose [loGmroaukpe77 ls[Gmraoupk2g: [1G0rmougpi3g7 [1G0r0omupaa:g [5G0r0omupg/5:kg ple # Ton Count Extracted wi Dlflon Area factor Fs6s2 ND. 0%o79 1 Fs2653 ND. 1.061 1 Fass Goss 129 1 Fsoses ames 1.0209 1 Fs2ses 7177 11125 1 Concentration Total mass hog offer ND. 85619 ND. s1.683 ootss 78656 027s s2718 0.5536 sain Tomar amount oF FC.95 por iver ND. ND. 0004 ocr 0052 +TfhaectcoornIcsetnhtrearteisounltwoafsacmailsccuallactueldatbiyounsIinnagptphleyisntganfdoarrmdulcaur8v.4eIannMdemtuhlotidplAyMiDnTg-tMh4e-r0es.ul1t3b7y m45g.ofThieve4r/5was used In tshhiosucladlcbuel.aBtiyonmurlattihpelryitnhgatnh7e1camlgc.ulTatheedccoonncceennttrraattiioonnsIInntthheessttaannddaarrddccuurrvvaebayre41t5h,etrheefocroerr5e4ctlraersguelrttIhsanobtthaeiyned. 000519 000073 WES moa Bol g \ LAR FCS x SFSaiRlmep:o0:a2095C from-sbheLoArDp-tDioAnSE - ThstuediesMS Dmat/a eSystesme 20/63/1095 10:55 8 g roo ---- 5ws 20 8 aon3 eo om Tan i gna goa as70 S396 F810 100. "15987ane Sieg 102 [Fs 2579 Bys0q E0 %15 se "Jada "150 60 .Z :VT 145W 20 eWaYRlsup "530 aden 5000 ha Ta ee g g FSialhmopncie:oazsssc 100 - a0 LAeBs]-DSE - The 1S eDataa Snystem ope S2C0m319%5 10:55 28of ar . . a ogg] we Cf ope pe, 100 '. Fis) PR. 1% 2 ls" 5% % vim wmpoege a( e0i " a qoey ee 5 2 * ~ g g wthooetems: om-shoL rpbton 0semtonhe15 5pu"tsS5 SIymEten eZamiss amis 33 ae soe zg - . he. EE H(Emr |S rel . pens gr (A fn we +b : g SFialmop:leeazmgsc erfro LAB-DAiSE - The HS Deaatzai-Siu2saten welts20/6a319%5 10:55 g 8 HY ia% sanEE ge hi p14 am 8 esq b asyr Awa ai hoo bers It 0 Lo seon22o00 h 7400 me yf |2600 2008 3900 3200 we A 3406 3608 To Ji SB ug 368 00a p0__4400 +X SFaimlpel0e3:2095 gt LAB-BAS> E ~The MS Da3ta29Sy7st0on foi2s0031o9c9t5 tess 2 1BI2895Cagen Tard. a -i- # aa1Go 2373 . opmi.e G2Lpf aooa 383 > ase bs: ars loo. WE - (22g auf HE bers lse Y a 54 ug St A A 525% I A a er 5 Ea ga a 4595 31  2 < SaFmipllesea;zasse LiOkBiE The 15 Du6t3a2S0un7t0an hofztvoysitws ws 8 hoo as we we Ho" E os YE i Ls if es (Ln EW ere, .i hoops 8 7wafuzen ad brs | lsc' 7 oe Soe i : EY Foes WH OWE Sing wun x= ose JLTil WR eT o ge goo 9.11.3 Summary and raw data; ppm F- in serum as determined by thermal Orion ion analyzer. extraction followed by analysis using This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 000526 000050 Summary of Combustion Data - Liver AMDT-112294.1, HWI 6329-129 Concentration of Fluoride in Serum Mean per Dose Group per Sampling Interval* Average ipn psmerFu-m 500 mg/kg dosed rabbit Ww) 48HR 0.633 24HR 0.707 12HR 0.789 SHR 0.620 . 6HR 0595 4HR 0614 2HR 0.705 100 mg/kg dosed rabbit 48 HR 0.341 24HR 0289 12ZHR 0395 SHR 0293 6HR 0.246 4HR 0265 2HR 0255 *2-24 hour calculated as the meanofduplicate samples, 48 hour analyzed in triplicate. 00527 00005e1 RPT120XLS FC95 PK iD Serum bik 1 Serum bik 2 Serum blk 3 Serum blk 4 Serum SPK 1 Serum SPK 2 Serum SPK 3 Serum SPK 4 Serum SPK 5 Serum SPK 6 SERUM BLANK-1 SERUM BLANK-2 FF5522666666.-7F55--4488 HR HR - 1 2 FF5522666666.-FF55.-4284 HR HR - 3 1 FF5522666666--FF55--1224 HR HR - 2 1 FF5522666666--FF55--812HHRR- -1 2 FF5522666666-.FF55--G8HHRR - 2 1 FF5522666666.-FF55--46HHRR - 2 1 FF5522666666--FF55--42HHRR - 2 1 SF5E2R6U6M6-BFL5A-2NHKR-1 2 SERUM BLANK 2 FF5522666655.-FF44..4488 HR-1 HR-2 F52665.F4-48 HR-3 F5F256266655-.FF44-.2244HHRR-.12 FF5522666655--FF44--1122HHRR.-12 FF5522666655--FF44..88HHRR.-12 F5F256266655--FF44--66HHRR--12 FF5522666655.-FF44..44HHRR--12 FF5522666655-.FF44-.22HHRR--12 Actual Average % pinpsmerFu-m ppm Finseum serum bumed rowry (0.W9W31) WW) (0m1l) 0.590 0.391 0.1 01 69% 0.321 2.10 0.1 01 184% 5.58 79% 2.40 01 0.1 78% 2.36 103% 313 0.1 01 98% 2.99 01 0511 0375 0.1 01 0.689 01 0582 0633 0.628 01 01 0.735 01 0680 0.835 0707 01 01 0743 0.636 0789 on 01 0605 0620 0.617 01 01 0573 0595 0.580 01 01 0649 014 0.755 01 01 0655 0705 0575 0.1 0.1 0.314 01 0.333 0350 0341 01 01 0.339 01 0.303 0276 0289 01 01 0.464 01 0326 0.288 03% 01 0.1 0209 0203 0.242 01 0.1 0249 026 0272 01 01 0257 0265 0.1 0.254 0286 0285 01 01 Dosage (mgikg) 500 500 500 500 500 500 500 100 100 100 100 100 100 100 Paget 000528 foe