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3M Environmental Laboratory ( Final Report- Analytical Study Single-dose DermalAbsorption/Toxicity Study of T-6049 in Rabbits In-Vivo Study Reference Number: HWI#6329-130 Study Number: AMDT-020895.1 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 MAeMtDhTo-dM-N1u-m0b,ers TahnedrmRaevliEsxitornasc:tion of Fluoride by Means of a Modified AMDT-M-2-0, FDlouhorrimdaenMneDaXsu2r0e0m0enOtrgbayniMceaHanlsiodfeaAnnaOlryizoenr-ELAiv9e4r0 Expandable Ion Analyzer AMDT-M-4-0, AMDT-M-5-0, Extractionof Fluorochemicals from AnalysisofRabbit Liver Extract for Rabbit Liver Fluorochemicals Using Electrospray Mass Spectrometry Initiation Date: See attached protocol Author: James D. Johnson Approved By: ames D. JGfinson Study Difettor 1/20) 95 Completion Date 000352 Deqe 000001 of 55, i 1B 1.0 SUMMARY Samples of 0.30 mg/kg rabbit FC-95 liver at 28 (T-6049) days were post dermal analyzed by administration of 0, combustion for total 0.003, 0.06, and organic fluorine. There was no detectable levelof observed in control rabbit liver. total organic in anyofthe treated Since in a pharmacokinetic study groups above that (HWI1#6329-159), bitiowlaosgiecsatlabhallifs-hleidfethiantepxecrefslsuoorfooocnteanmeosnutlhfoanantdetihsatpepresrifsltuenotroionctraabnbeistullfiovenratweitwhilal serve as a good marker dermal absorption does for not aobcscourrptatiotnheosfFeCd-o9se5,leviteliss.cTohnecsleuddeodsethaltevdeeltseacrteabvleery low and possibly t00 low to make an assessmentof dermal absorption. 2I 0 NTRODU~C ~~ TI0O 000N 00 FC-95 is the potassium salt of perfluorooctanesulfonic acid. In a previous pharmacokinetic study (HWI#6329-159), it was shown that the biological half-life pisre>v1iomuosntshtu.dieIts iisnperartssi,sttehnetmionlseceurluem iasnndotlikvenroawfniertoabniionttrraanvsefnoorumsodrocseo.njuFgratoem. Tthheusa,moanuanltysoifs pbeyrfcloumobruooscttiaonneswuiltfhonsautbesepqrueesenntt.meaAstulraetemretnitmoefsf,ltuhoericodnecweinltlraetsitoinmaitne laisvseerssisdheirgmhaelr atbhsaonrptthieona,motuhentleivnelsoefrutmotaolnotrhgeanaivcerfalgueo.rinFeorinulsievearsias tmhaerrkeeforrteoa more sensitive test at 28 days post dose. ATERIAL 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 Analytical Reference Matrix: Bovine liver and bovine serum 3.1.3 Analytical Control Substance: None 3.1.4 Analytical Control Matrix: Bovine liver and bovine serum 3g.r2ocSeoruyrscteoroef,Mbaotveirniealsse:ru3mMfrIoCmPS/iPgCmPaDCihviesmiiocnalfoCroFmCp-a9n5,y.bovine liver from 3.3. Purity and Strength of Reference Substance: Responsibility of Sponsor 3.4 Stability ofReference Substance: To be determined by Sponsor 000353 000002, 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples, the storage is -20+10 C. 3.6 Dispositionof Specimens: Biological tissues and fluids will be GLP Regulation for the time period required for studies longer than 2r8etdaaiynse.d per 4.0 EXPERIMENTAL - Overview Samples of tissue and serum were available from HWI#6329-130 for analysis for total organic fluorine. The samples were combusted and analyzed for total organic fluorine. The data were assessed for extent of dermal absorption of FC-95. 5.0 EXPERIMENTAL - Methods 5.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Means ofaModified Dohrmann DX2000 Organic Halide Analyzer-Liver 5.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3 AMDT-M-4-0, ExtractionofFluorochemicals from Rabbit Liver 5.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 6.0 DATA ANALYSIS The data from combustion analysis at 28 days post dermal administration of FC-95 (T-6049) are attached. The dosing material T-6049 is a 0.06% solution of FC-95. Doses in this analysis are expressed as the FC-95 equivalents. The liver was combusted and subsequently analyzed for total organic fluoride. There is no evidenceofdermal absorption at any dose level for the groupsofrabbits in the control, 0.003 mg/kg, 0.06 mg/kg, and 0.30 mg/kg groups. In all cases the amount oforganic fluorine was below the practical quantitation levels and apparently below the detection limits. Electrospray mass spectrometry did detect perfluorooctanesulfonate anion, but again the results are nebulous and below the practical quantitation limits. There does not appear to be dermal absorption of FC-95 at these dose levels. 000354 000003 3 6.1 Circumstances That May problems with this analysis are Have Affected recognized that the Quality could affect of the the Data: No conclusion. aHboswoervbeerd, itthweoduolsdesbeaddmiifnfiicsutlrtattoedquaarnetistoatleo.wInthtahteehviegnihefraldlostehegdroouspe,h2adkgbereanbbits would receive only 600 ug total. For dermal dosing with analysis at 28 days and a half life on the order of a month, there would be less than 300 ug present in liver evenifall the absorbed perfluorooctanesulfonate had gone to the liver. 7.0 CONCLUSION FC-95 is not dermally absorbed at 0.30 mg/kg dose. This is a very low dose and possibly too low to make an assessmentofdermal absorption. 8D .0 MAA INTT ENAA NCE A OFRN AW DRECORD ~~S 8.1 Raw Data and Data: Raw data, approved protocol, approved final report, appropriate specimens, and electronic data will be maintained in the AMDT archives. 9.0 APPENDICES ---------- 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-130 "Single Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits (Protocol type TP-3016.AB for dosing of animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-020895.1 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: See methods 000355 000001 4 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-020895.1 9.10 Instrument Settings: See methods 9.11 Data: See attached: 9.11.1 Summary and raw data; ug Fin whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 000356 000005 5 9.1.1 Protocol and Final Report: HWI#6329-130 "Single Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits (Protocol type TP3016.AB for dosing ofanimals, tissue collection, etc.) 0003577 000006 HSAEzSLEaTneoaN Soansar: 3M Toxicology Service Medical Department St. Paul, Minnesota +oommorens FINAL REPORT EEE Ny oan of Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Author: Steven M. Glaza Study Compleion ate: June 27, 1995 berforning Laboratery: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 oratory prosect Identification: HWI 6329-130 Page 1 of 42 000358 000007 Page 2 of 42 QUALITY ASSURANCE STATEMENT HHI 6329-130 WT(ihFsiOsAc)onrseGiponoo,drt LIhanacb.so,rabteioenrnyacrcePovrriadecwatenidccee bwyRietgthuhleatthQieuoanlFsio,toyd 21AasnsdCuFRrDarun5gc8e.3A5Udnmii(tnb)isotf(r6)aHtaiz(ol7n)e.ton TSahntedudyffionDldilironwegicsntgorarienansdpiesmcsatuneiadognestmoewneHtra.ezlectWoornnidtutcmetanendagsetamnaedtnutsfimnrodenipntogrhstlsyraoecfpcoorirtndesidpnegcttoitootnhse standard operating procedures. Inspection Dates from _ To 0112//1201//3954 0112//1201//9954 0014//2043//9955 0014//2143//9955 0064//2073//9955 0046//2173//9955 __ Phase APnriomtaolcolObsReervviaetwion PDraottao/cRoelporAtnenRdenveinetw DRaetpaortReRveireewview RepoDratteed to Study Director 0112//1202//995 0041//1284//9955 0064//2174//9955 Date to Management 0021//1100//9955 0025//1100//9955 0057//1100//9955 Rfepirensednt,ativAe, oQuzality Assurance Unit D6at:e22.95 000359 000008 Page 3 of 42 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits HWI 6329-130 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Study Initiation Date Experimental (In-1ife) Start Date In-life End Date Experimental Termination Date Study Completion Date T-6049 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2-02 P.0. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD EL Toxicology Service Medical Department 3M Center, Bldg. 220-2-02 P.0. Box 33220 St. Paul, MN 55133-3220 (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 December 30, 1994 January 7, 1995 February 10, 1995 June 27, 1995 June 27, 1995 000360 000009 Page 4 of 42 Acute Toxicoloay SSttuedvyenDMi.recGtloarza Manager SFtruadnycisCoo(rBduid)natW.orMcDonald Patricia Padgham In-1ife Supervisor Rose M. Bridge Report Supervisor Quality Assurance Sherry R. W. Petsel Manager HHI 6329-130 KEY PERSONNEL LaboratoryAninal Medicine DCiipnldoymaJt.e,CarAyC,LAMDVM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Jack Serfort/ Necropsy Deborah L. Pirkel Supervisors Anne Mosher Supervisor Pathology Data 000361 000010 Page of 42 CONTENTS QSutauldiytyIdAesnstuirfainccaetioSntatement SuKemymaPreyrsonnel ObRejgeucltaitvoery Compliance TTeesstt SaynsdteConntrol Materials PRreoscueldtusres DSiisgcnuastsuiroen RePfatehroelnocgey Report Table 21 IInnddiivviidduuaall aClnidniMceaaln BSiogdnysWeights (g) 34 IInnddiivviidduuaall DPeartmhao]logIyrrCiotmamteinontsScores 5 Individual Animal Tissue Weights and Bile Volumes Appendix A PPrroottooccooll DTPe3v0i1a6t.iAoBns Protocol Amendment No. 1 HAI 6329-130 Page 32 46 88 98 1130 1124 1154 1167 2139 2 2278 a29 Of00362 000011 Page 6 of 42 HAT 6329-130 SUMMARY Tshkiisn sitrurdiytawnacsy dofoneT-6t0o49asswehsesn tahpepliseydstetomictheabsskoirnptoifon/rtabobxiitcsi.ty and relative TofhethsetudHyraw:a(sNZWc)oSnPdFuctsetdraiunsinfgortheraeceh mtarleeatmaenndttghrroeuep.female acclimated rabbits Group Test Material Dos_(ema/kLae)vel MNaulmebser of AFneimmaalless 1 (Con2trol) Distilled water T-6049 0 5 3 3 3 3* 31 TT--66004499 501000 33 33 * a * OAndeminainismtaelredsacatrifaicdeodseonvoDlauyne2ofdue2.0tomlp/oksgs.ible broken back and replaced with another female animal. Trheespebcatcikveofmaetaecrhiarlabbaitttwheas icnldiipcpaetdedfdroeseeoflevhealirwaasndadamisniinsgtleereddosetoofthetheskin woafs thceoverraebdbitwsi,th aThgeautzreeatbmaenndtagesitseescurreedmaiwnietdh pianptaecrt.tapeThearoaurneda of all application edges and dorveesrswirnagppefdorwiath24S-ahroaunr WexrpaopsuraendpeErliaosdt.oplast tape to provide an occlusive C4lihnoiucrasl aofbtseerrvtaesttionosr wceornteroclondmuatcetreidalpreaddomsienisatnrdataitona.pprAodxdiimtaitoenlayl 1,cli2n.i5c,aland ofobrser2v8atdaiyosn.s anTdhetwoincley eaxcdeapytmioorntsaltiotythicshecwkesreweonreDacyosndu4ctaendd 6dawihleyntthheereafter raefptlearcneomoenntmoratnaimlailtsy. cheBocdkywawseigihntasdvweerrteendteltyernmotinecdondonuctDeady f-4orfotrhertawnodomization piunr-pToisfees,terbmeifnoarteiotnest(Doary c2o9n)t.rol'Thematienriitailal addemrimnailstriartriiotnati(oDnayre1)a,dinagndwaast made breeafdoirneg)t.estSuobrsecqounternotl rmeaatdeirnigasl ofaddmeirnmiasltraitrirointat(iroencorwdeerde maasdetheapDparyoxi1mately w3e0remincuotlelsectaefdterfrobmandaagmeargrienmaolvalear(Dvaeyin2) ofandtheonanDiaymsals4 abnedfor8.e iBnl-o1oidfesamples iannditi23a.tionRep(lDaacyem1e),ntaapnpirmoaxlismawteerlye b2l4e-dhouornsDapyosstd1,ose2, (4D,ay8,2),15,onanDdays22."4, 9I,n 16, oabdtdiatiinoend, fratomtheeachtimaenimoafl.necArlolpsysamopnleDsaywe29r,e acpepnrtorxiifmuagteedlyand20smeLpaorfatebdiooidntowas tsheerumin-a1ndifecelplhuaslearanfdracnteicornosp.siedA.ll Tahneimawlhsolweerleiveeru,thabniliez,edanatatpeprrmoixniamtaitoenlo-fcn xkid1n-ecymssefcrtoinononeofmatlhee daenrdmaolneapfpelmiaclaetiionneascihtegrforuopm waellreacniomlallesc,tedandatbontehcropsy 000363 000012 Page 7 of 42 HHI 6329-130 csaeennldtluwlefairrgohzeefdnract(toviootlnhusem)e,Sponlolinyvseorrdse,taefrtbiemlrien,etderdemfriomnraaltbiiolanep)p.ioficaTtthhieeonbilno-soTiditefses,apmhpaalnsedes.ki(dsneeryusm waenrde Abopdpylicwaetiigohnt ogafinT.-60A4l9l diadnimnaoltsreaspupletareidn acnlyinitceastllymatneorrmiaall-rtehlraotuegdhoucthantgheesstinudy wsiatchriftihceedexconeptDiaoyn2odfueonetoGrionujpury2 a(npdososniebleGroburpoke3nfebamcaklse).aniTmhalessethaantimawlesrewere rtheeplasctueddy.in Ntohedesrtmuadly ainrdrittahetiornepwlaascemoebnstervaendinaaltsthaeppdeearrmeadl nosrcmoarlingthrionutgehrovuatls aTsevealsr.esulTtheroef wtehreeapnoplitceasttiomnateorfiadli-srteillalteeddwalteesrionosr To-b6s0er4v9edatatanynecorfoptshye.dose 000364 000013 Page 8 of 42 HWI 6329-130 OBJECTIVE Trheelatoibvjeectsikvine oifrrithtiasncystuodfyawatsesttomaatsesreisasl twhheensyasptpelmiiced totoxictihety/sakbinsoropftion and rabbits. REGULATORY COMPLIANCE material mixture prepared for the Group 2 animals for concentration, This study was conducted in accordance with the U.S. Food andDrug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test homogeneity/solubility, and stability was not conducted. All procedures used in this study are in compliance with the Animal Welfare Act Regulations. In tdhuepliocpaitneionanyofprtheevioSupsonwsoorrk.and study director, the study did notunnecessarily TEST AND CONTROL MATERIALS Identification The test material was identified as T-6049 and described as a clear, colorless Tiquid. The control material was distilled water 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). Analysis of the test material mixture prepared for the Group 2 animals forconcentration, homogeneity/solubility, and stability was not conducted or requested by the Sponsor. The purity and stability of the control material were considered to be adequate for the purposes of this study. Stoanrd Raetegntieon The test and control materials were stored at room temperature. A reserve sample of each test and control material was taken and will be retained in a freezer set to maintain a temperature of -20C 10 for 10 years in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any unused test material was returned to the Sponsor after completion of the in-1ife phase according to HWI SOP. Any remaining control material is retained for other testing and will not be discarded after issuance of the final report. 000365 000014 Page 9 of 42 safety Precautions HUI 6329-130 Tahned tpeoslticiaensd. control material handling procedures were according to HHI SOPs TEST SYSTEM Test Animal DAednuvletr,albPiennonsyrlavbabniitas oonf DtehecemHbrear:(N2Z8,W)S1P9F94starndainmawienrteainperdocuartedthefroHmazlHRePt,on Inc., Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing ADfutreirngreaccecilpitm,atitohne aanndimatlhsrowuegrheoutaccthleimasttueddy,forthea apneirmiaoldsowferaet lienadsitvid7uadlalysy. choonutsreodlliendsqcuraeretne-rbs.ottomEnvsitraoinnmleenstsalsteceolntrcoalgsesfoirn ttheempearniamtaulre-rooamndwehruemidsietty-to m1a2i-nhtoauirnTaigthetm/p1e2r-ahtouurredaofrk1T9ighttoin2g3Cc,yclae.relaIntivceasehsumwihdeirtey voafri5a0t%io2n0s%,froamnd a athdevseersecoenfdfietcitonsonextihsetesdt,udytheoyutcwoemree. documented and considered to have had no Animal Diet LThaeboraantiomraylsRawberbeitprDoievtideHdF a#5c3c2e6s,s PtHoIwaFteeedrs,adIn1ci.bitTuhneanfdeeda miesasruoruetdinealmyount of caonnaltyazmeidnanbtys.theSammapnluefsacotfuretrhe fwoartenrutrairteiopnearliocdoimcpaollnyentasnalaynzdedenvbyiroHnHmI.entaTlhere wienrteerfneorekdnowwnithconortamaifnfaencttsed inthetheresfueledtsorofwattheerstatudyl.evels that would have Selection of Test Animals Twheereapnliamaclesd wienrteo sitduednytifgiroeudpsbyusaniinmgala nsutnrbaetrifiaendd cboodryrewsepiognhdtingraenadromitzagatiaonnd wperioggrhatm.vari~Tahteiornandoofmitzhaetiaonnimablosdyfworeigehatcsh wgerroeupdoefteremacihnedsexonseDalyect-e4.d fTorhethe bsotduydywediigdhtnsotfoerxceeaecdh52grosutpandoafrdeacdhevisaetxiownesreofnotthestmaetainstiwceiaglhlty, diafndfertehnetmeaatn trheepla5c%edpraofbtaebrilitthyeylweveerle. treTawotedfemdaueletaonipmoaslssibl(eNosb.rokFe5n339b8ackasn.d F53355) were These animals wweerree trreepaltaceedd iwnitthhetwsoameothmeanrnefre.male animals (Nos. F52999 and F53351) which 000366 000015 Page 10 of 42 Study Design HUT 6329-130 Ainnitmoaltshewefioglhlionwgingfrosmtud2y,0g1r8outpos:2,625 g at initiation of treatment were placed Group Test Material Do_s(eng/kLqe)vel NMuamlbeesrof Animal females 1 (Con2trol) DistTi-6l0l4e9d water 05 33 33+ 34 TT--66004499 510000 33 33 * a* OAndeninainismtaelredsacatrifaicdeodseonvoDlauyme2ofdue2.0tomlp/oksgs.ible broken back and ratepliancietidatwiiotnh. another female animal. This animal weighed 2,625 g Justification for Species Selection Hcihsotiocreicbaelclayu,sethoef Ntehwe ZlaeragleandamWohuintteofalbbiancokgrroaubnbditihnafsormbaeteinontheon atnhimiasl spoefcies. PROCEDURES Preparation of Exposure Area Oonbtatihne udanyblebmeifsohreedtesskitn)m,atetrhiealflaanpkpslicoaftieoanc,h trhaebbibtackwasandc,lipipfednecfreesesaroyf h(atior The The cilnitpapcetd skairneaofmadtehe uptesatpprsiotxeismawtaesly in20s%pecotfedthefortotianlterbfoedyrinsgurflaecseionasr,ea. ainrirniatlastiowne,reorclidpepfeedctsasthnaetedewdoultdhropurgehcoluutdethtehestuusedyoftoanayidofinthveisuanailmiazlisn.g tThhee application sites. Dose Administration Amlalterainailm.alsThreecediayvedofatrseiantgmleentadwmaisnisdtersaitginoantedofastheDayres1.pective test or control Gornouepach1. aniAnmali'nsdivbiodduyalweidgohste o(n2.0thmel/dkagy)ofwastrecaatlmceunlta.tedThaendcomnetarsoulredmatbearsieadl a(pdpirsotxiilmlaetdelwyate0r.)05wamsl/acpnp.lied evenly to the test site at a rate of 000367 000016 Page 11 of 42 HHT 6329-130 G(rTo-u6p0s43)2, wa3,s mainxded4.witFhorditshteilGlreodup-w2atearnimtaolsa c(o5ncmegn/tkgr)a,tiotnheoftes5t00mamtge/rmilaland atpepmlpieerdatuarteaudntoisle vadomliunniestoefre0d..01 mTLh/ekgt.estTmhaetemriixatlurewaswasadmsitonriesdteraetdroom 5u0n0dilmug/tkegd, torestpheecttievsetlys)iteussinogf tthhee aGrvoeurpasge3bualnkd d4enasniitmaylsof(01.0909org/l to dreetsepremcitniev'eltyh)e. dosTehevoalrueameofforexpeaocshuredosfeorletvheel 5,(0.10100, aanndd0.55010 mmLg//kkgg, dose Tlheevelasppwraosxim4,ate25ra(t1e6 offoraprpelpilcaacteimoennt raanngiemdal)f,romand0.010060 ctmo?,0.r0e1s4pemcit/icnv'e.ly. An wiansdivciadlucaullatdeodsefoorf tehaechreasnpimeacltivbeasetdestonmaittesribaoldyowreitgehstt moantetrhiealdamyixotfure treatment. Esaecchureadreawitohf apapppelricattaipoenawraosundcovaellrededgweisthanad 1o0v-cemrwxrap1p0e-dcnwigtahuzeSarbaanndaWgreap and rEelsatsrtaoipnlasthte taanpiemaltso dpurroivnigdethaen o24c-chlouusriveexpdorsesusriengp.erioCdo.llars were used to Arpepsrtorxaiinmiantgelycol2l4arhsourasndabfatnedragteesstweorrecornetmroovledmaatnedriaanly raepspildiucaaltiotne,sttmhaeterial was removed with tap water and disposable paper towels. Reason for Route of Administration The dermal route is a potential route of exposure in humans. Observations of Aninals 4Clihnoiucrasl afotbesrervtaesttionosr wcoenrteroclonmdautcetreidalpreaddomsienisatnrdataitona.pprAodxdiimtaitoenlayl 1,cli2n.i5,caland ocbosnedruvcatteidondsailayndthtewirceeaftaerdayfomror2t8aldiaytsy. cheTchekson(lmyorenxicnegptainodnsaftteorntohoins)wewreereon cDaoynsduc4teadndfo6rwhtehne ttwhoe arfetpelrancoeomnentmoratnaimlailtsy. check was inadvertently not mBaotdeyriwaelighatdsminwiesrteradteitoenrmi(nDeady f1)o,r ranadndoatmiziant-iloinfeputrepromsiensatioonnDa(yDay-4,29)b.efore test aThdeminiinsittiraaltiodnernaaclcoridrirnigtattoionthereDardaiinzge'yatsecmhandiequbeef(orreecotredsetd oars ctohnetroDlay ma1 terial mrienaudtiensg').afteSrubsbeaqnudeangte rreemaodvianlgs (oDfayde2r)maalnd iornriDtaaytsion4 waenrde8.made approximately 30 000368 000017 Page 12 of 42 sample Collections HHI 6329-130 Ballloodanismaamlpslesbef(oarpepreoxxpiemraitmeelnyta4l mLi)niwteirateiocnoll(eDcayted1).fromSubasemqaurgeinntalcoelalrectvieoinn ooff b16l,oodanwdas23.condRuecptleadcemaepnptroxainimmaatleslywe2r4e-hobulresd opnostDdaoysse 1,(Da2,y 24),,8,and15,onanDdays22.4, 9I,n oadbdtiatiinoend, fratomthtehetipmoestoefrionrecrvoepnsaycaovnaDoafy e29a,chapapnriomxailm.ateAllyl' 2s0a'mnpLlesofwebrleood was wceernetritfhuegnedstoarndedseipnaraatferdeezienrtosesterutno maandintcaeilnlulaartemfrpaecrtaitounrse. ofTh-e2s0eCsa1m0plCes until shipped to the Sponsor. Pathology Awtithtesrmoidniautmiopnentoofbatrhbeiteaxlp,eribmleendtavliapthhaesepo(sDtayeri2o9)r, veannaimaclavsa,weerxesaanngeusitnhaetteidz,ed aanpdplinceactrioopnsiewdereinwarsahneddomwiotrhderl.ukewTahrensittapeswaotfertesbtefoarned ctohnetrnoelcrmoaptseyriparlocedure. Aanly aanbinmoarlmsaliwteirees swuebrjeectreedcortdoeda.n abThberevwihaotleed lgirveors,s necropsy bile, an examination approximate and 1-cm x f1r-ocmn tsheectifoinrstofmatlhee adenrdmaflemaalpepliincateiaocnh gsriotuepfwreormealclollaencitmeadl.s, Tahned tbiostshuekidneys nseacmrpolpessiefdoronallDayani2m,alwse,rewiwtehightehde e(xvcoelputmeiononloyf dteheteramniimnaeld fsoarcribifliec)edanadnd tiemmmpeedriaattuerley poflac-e2d0Con1d0ryC.ice,Afttehrennepclraocpesdy,inthaefarneiemzaelrssweetretodimsacianrtdaeidn. a Shipment of Blood, Bile, and Tissues Affrtaecrtioenxsp)e,rinleinvtearls, tbeirlmei,natdieormna,l thaepplbilcooadtiosnampslietses,(saenrdumkianddneycsellwuelraersent f2-r3oz-e0n9,(on935dryBusihce)Avetnouet,heStS.ponPasuolr, (MJNa,mes551D.06)J,ohnasloonn,g w3iMthE.Et.he&coPi.Cl.e,cteBdldg. rceotrernetsipoonndianngd wdeiisgphotssitioornvoolfumtehse. samTphleesS.ponsHoWIr diosesresnoptonsaicbclepet faornythe nroerspoanresibtihleisteyrefsourlttshepraensaelnytseids oifn tthhiestirespsouret.samples collected in this study Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Repor rTheetairnaewddaitna,thereacorrcdhsi,vesandofanHAIoriigninaaclcorsdiagnnceed cwoiptyh HoNfIthSeOP.final report will be 000369 00001S Page 13 of 42 Body Weights RESULTS Individual and mean body weights are in Table 1. weight gains from Day 1 to Day 29. HWI 6329-130 All animals exhibitedbody Clinical Observations tIhnrdoiuvigdhuoault cthleinisctauldy swiigtnhs atrhee fionllToawbilneg 2.exceAlpltioannsi:mals appeared normal + One Group 2 female (No. F53398) treated with T-6049 at & mg/kg appeared to have injured its back at the time of the 24-hour blood collection. This animal was sacrificed, necropsied, and replaced with No. F52999. The replacement animal appeared normal throughout the study. + OpnreiorGrotuopco3mpfleemtailoen (Noof. thFe533e5x5p)osutrreeatpeedriowdithwitTh-60i4ts9 alteg 1c00augmhgt/kginwatshe fcoaugned floor, appeared to have injured its back, and was sacrificed. Since this animal did not receive its full test material exposure, the animal was discarded without a gross necropsy examination. The animal wasreplaced wsittuhdy.No. F53351. The replacement animal appeared normal throughout the DermalIrritation Individual dermal irritation scores are in Table 3. The control material produced no dermal irritation. No dermal irritation was observed in the animals treated with T-6049 at any of the dose levels. Pathology Individual animal pathology comments are presented in Table 4. Individual animal tissue weights and bile volumes are in Table 5. The treated skin in two animals dosed at 0 mg/kg, two animals dosed at 5 mg/kg, and one animal dosed at 500 mg/kg was diffusely red or had multiple red areas of variable wseizree. noThleesseionfsindoibnsgesrveadre innotanyconosfidtehreedremtaoinbiengtesatnimmaaltse.rial-related. There Page 15 contains a pathology report by the study pathologist. 000370 00001 Page 14 of 42 HHT 6329-130 DISCUSSION Twheere aceuvtaeluastyesdtemiincmaalbesoarnpdtiofne/matloexicailtbyinoandrabrbeiltastivwehenskiandmiinrirsittearnecdy of as T-6049 a single dweeringahlt gaapipnlicoartiionn-.lifeThecrleiniwcearle nfoindtiensgtsmaatterainayl-orfeltahteeddoscehanlgeeveslsi.n bNoodytest mnaotetreisatl-mraetleartieadl-rdeerlmaatledirlersiitoantsionobwsaesrveodbseartvendecrdouprsiyn.g the study. There were SIGNATURE AE NW SStteuvdeynDMi.recGtlaozra Acute Toxicology Na = 9s Date REFERENCE 1. Dorfaitzhee',SaJf.etHy.', of"ACcuhteemiDcearlmsalinToFoxoidcsi,tyDr(uSgisnglaendECxopsomseutriec)s, - IDn:ermaAlppraisal T(o1x95i9c)i.ty, Association of Food and Drug Officials of the U.S., pp. 54-56 000371 | 000020 Page 15 of 42 HHT 6329-130 PATHOLOGY REPORT Tlthheeevreeltserwmeoirfnea0t,siiox5n, rao1fb00b,itthseands(ttu5hd0ry0e.emgm/aOklngeesoffeanmdabloedtyhr(ewAeeniimgfaheltmaleNeou.st)haFn5ei3az3c9eh8d)fradnodomsefndoeucrraotpds5oisemedg/kagt oTmhafitsebroidaaynli,mwaelidgohswteaswlaersveeplls,aaccerddiayfiwcioetfdhdeaoanntohtD,haeyran2dfebgmeracolaesusse(oAbonisfmearalvnaNtaoip.opnasrFe5n2r9te9c9ob)rr.doekdeTnhfeobractkea.scth animal are in the Individual Pathology Comments that follow this report. Amtultniepclreopsrye,d tarheeastreofatevdarisakbilne insizseo.me Tahniismalisncwlausdeddiftfwousealnyimarlesd odrosehdadat 0Themrge/kgw,eretwnooavniismiablsledoTseesdionast 5inmga/nkyg,ofatndheonreemaaniinmianlg daonsiemdalsa.t 5T0h0emgl/ikvger., beialceh, ofanthaepsperoxainmimaatles,1-acnndxbo1t-hcmkisdencetyisonfroofmthoenedmearlmael anadpploinecatfieomnalesitien efarcohm gfroorupbilwee)r,e at 5 mg/kg cfroolzleenc,teda.nd sTheentttiossutehesaSpmopnlseosr.wereTheweitgihsesdues(voflruommethoenlyfedmeatleermdionseedd and sacrificed on Day 2 were collected but were not weighed. After necropsy, the animals were discarded. Twhiethiannimtahle sskaeclreitfailcedmusocnleDayof2thheadlmuunlbtairp-lseacrdaalrkrergeidonareoafsthoef vsapriniaalblecolsuimzne. Tbahceks.e findings correlated with the clinical observation of an apparent broken J PathologiGstoamer, = = 0(36/32229/-91350.51h) rEAzfs 000372 0000221 Animal Number F53358 F53359 F53353 Mean F53402 F53364 F53352 Mean F53360 F53400 F53346 Mean F53365 F53354 F53347 Mean Page 16 of 42 Table 1 Individual and Mean Body Weights (g) Male Randomization Day -4 Day TT 29 Animal Number Female Random- ization Day-4 Group Control) - Distilled Water (0 mg/kg HWI 6329-130 Day 29 2,066 2,030 2,136 2,077 2,231 2,093 2,312 2,816 2,670 3,012 F53404 F53405 F53356 2,200 2,426 2,110 2,212 2,833 2,245 Group 2 - T-6049 (5 mg/kg 2,286 2,474 2,224 2,328 2,997 2,911 2,685 2,864 2,288 2,244 2,102 2,211 2,382 2,364 2,018 2,938 2,923 2,622 2,255 Group 2,828 3 - T-i F53399 F53349 F53398 F52999 00 me 2,095 2,376 2,331 2,485 2,319 2,122 2,492 2,419 2,625 2,413 2,580 2,919 3,272 2,924 2,157 2,076 2,115 2,116 2,194 2,122 2,211 2,943 2,696 2,694 F53350 F53355 F53397 F53351 2,320 2,280 2,145 2,059 2,176 2,718 2,175 Group 4 - T-6049 (500 mg/kg! 2,409 2,297 2,245 2,253 2,302 2,99 - 2,706 2,606 2,769 2,126 2,325 2,103 2,185 2,216 2,478 2,152 2,302 2,951 3,008 2,646 2,868 F53403 F53000 F53345 2,170 2,241 2,306 2,239 2,270 2,268 2,341 2,293 2,860 2,914 2,951 2,908 2 Animal No. F53398 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificed after completion of the exposure period due to a possible broken back and was replaced with No b AFn5i2m9a9l9. No.TheFS3b3o5d5y wwaesighotrsigifnorallNoy. sFe5l3e3c9t8ed arbey tnohte rinacnlduodmeidzatiinontheprgorgroaump mfeoarnsu.se in the study and was treated. This animal was sacrificed before completion of the exposure period due to a possible broken back and was replaced with No. F53351. The body weights for No. F53355 are not included in the group means. - Not applicable. 000373 Ve 0000222 Page 17 of 42 HAI 6329-130 - IndividualTabClleini2cal Signs Sex Animal 1-4 Hours Day Number _O_bservation (Day1) 2 3throug2h3 Group1 (Control) - Distilled Water (0 ma/ka) Male ~~ F53358 Appeared normal vv v F53359 Appeared normal /v v F53353 Appeared normal vv v Female F53404 Appeared normal vv v 53405 Appeared normal /v v F53356 Appeared normal vv v Group 2 - T-6049 (5 ma/kg Male F53402 Appeared normal vv v F53364 Appeared normal vv v F53352 Appeared normal vv v Female 53399 Appeared normal vv v 53349 Appeared normal vv v 53398 Appeared normal v- Possible broken back b(lateedtiinmge oifnte2r4v-ahlo)ur - v Moribund sacrifice -v F52999 Appeared normal vv v /~ CCoonnddiittiioonn enoxtisteevdident. a Animal No. F52999 replaced Animal No. F53398. 000374 00100002233 Page 18 of 42 Table 2 (Continued) Individual Clinical Signs HWI 6329-130 Sex. Animal Number Observation Group 3 - T-6049 1-4 Hours Day Day. 2 3 through 29 (100 mg/kg. Male F53360 Appeared normal 7 7 7 F53400 Appeared normal 2 v Jv Female F53346 F53350 Appeared normal Appeared normal 7 Jv v v7 v 7 F53355 ALpegpeacraeudghtnorimnalcage floor (possible broken back) Moribund sacrifice Jv - - 7 - Jv F53397 Appeared normal 2 J 7 F53351 Appeared normal 2 7 v Group 4 - T-6049 (500 mg/kg Male F53365 Appeared normal 4 J 7 F53354 Appeared normal J v Jv F53347 Appeared normal Jv Jv Jv Female F53403 Appeared normal 7 2 J F53000 Appeared normal v LJ J F53345 Appeared normal Jv Jv 7 / Condition existed. - Condition not evident. a Animal No. F53351 replaced Animal No. F53355. 000375 01000zx32, 4 Page 19 of 42 Table 3 Individual Dermal Irritation Scores HWI 6329-130 Group 1 (Control) - Distilled Water (0 mg/kg) Dermal Reaction Males I 2Study 4Day & Females I 2Study 4Day & Animal No. F53358 Anima) No. F53404 Erythema Edema DAetsoqnuiaamation : Coriaceousness Fissuring 0 0 0 0 0 0 [ 0 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 0 0 0 I 0 0 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F53359 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F3405 0 0 0 0 0 0 0 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 Erythema Edema Atonia Desquamation FCoirsisaucreionugsness Animal No. F53353 [ 0 0 0 0 0 [1 0 0 0 0 0 0 0 0 [ 00 00 00 00 Animal No. F53356 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [J 00 00 00 0[ 000376 00005 Page 20 of 42 IndividuaTlablDeerm3al(CoInrtriintuaetdi)on Scores Group 2 - T-6049 (5 mg/kg) HUI 6329-130 Dermal Reaction EEdreymtahena ADtesoqnuiaanation FiCsorsiuarcienogusness EEdryetnhaena ADtesoqnuiaanation CFoirsisaucreionugsness EErdyetnhaema ADteosnqiuaaation CFoirsisaucreionugsness EEdryetnhaema ADtesoqnuiaamation CFoirsisaucreionugsness Males StudyDay 1248 Animal No. F53402 00 00 00 00 00 00 00 00 0o 00 00 00 Animal No. F53364 00 00 00 00 00 00 00 00 00 00 00 00 Aninal No. F53352 0 00 00 00 00 00 00 00 00 00 00 o0o -- Females -- StudDy ay 1 2 4 FE Anina) No. F53399 00 00 00 00 00 00 00 00 00 00 00 00 Aninal No. F53349 00 00 00 00 00 00 00 00 00 00 00 00 Aninal No. F53398* [0I 0 |o J o. 0oo0 .- Animal No. F52999 [I I 00 00 00 00 00 00 00 00 ba RAneipmlaalcemreenptlacaendimawlithforNo.No.F52F959393.98. - Not applicable. 000377 000026 Page 21 of 42 HHI 6329-130 Table 3 (Continued) Individual Dermal Irritation Scores Group 3 - T-6049 (100 mg/kg) Dermal Reaction 1TSt2Mualevs4Da&y Animal No. F53360 S I t 2Femau lD&eas yy& Animal No. F53350 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Animal No. F53400 Anima) No. F53355% Erythema Edema Atonia 0 0 0 0 [1] 0 0 0 0 0 0 0 Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 [J = - - 0 - - - 0 - - - 0 - - - 0 - - - 0 - - - Animal No. F53346 Animal No. F53397 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Anima) No. 0 0 0 0 0 0 0 0 0 0 0 I F5335] Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [J 0 0 0 0 a Animal replaced with No. F53351. Replacement animal for No. F53355. - Not applicable. 000378 000027 Page 22 of 42 IndividuaTlablDeerm3al(CoInrtriintuaetdi)on Scores Group 4 - T-6049 (500 mg/kg) HHT 6329-130 Dermal Reaction EEdreymtahema ADetsoqnuiaamation CFoirsisaucreionugsness EErdyetnhaema ADtesoqnuiaamation CFoirsisaucreionugsness EEdreytnhaena ADetsoqnuiaamation FiCosrsiuarcienogusness Males StudyDay 1. 2 4 8 Aninal No. F53365 00 00 00 00 00 00 00 00 00 00 00 00 Aninal No. F53354 00 00 00 00 00 00 00 00 00 00 00 00 Aninal No. F53347 00 0 0 0 0 0 0 00 00 00 00 0c 00 00 00 -- Females -- StuDd ay y 1 2 aE Aninal No. F53403 00 00 00 00 00 00 00 00 00 00 00 00 Aninal No. F53000 00 00 00 00 00 00 00 00 o 0 o0 00 0oo Anina) No. F53345 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 000379 0000225 Page 23 of 42 Individual Table 4 Pathology Comments HUI 6329-130 AMnuimmbaelr Sex DT ied eSas Dcariyft iced Necropsy Observation Group1 (Control) - Distilled Water (0 ma/kg F53358 Mo - 29 No visible lesions. F53359 Mo - 2 No visible lesions. F53353 Mo - 29 Tdehrenaslkinapphlasicamtuilotniplseiter,eduparetaos3wictmhixn2thcne. Collected with routine section. F504 F - 29 No visible Tesions. 53405 F - 29 No visible lesions. 53356 F - 29 Tdhaerktrreeda.tedCoslkilnectaerdeawiatphpearrosutdinieffusseecltyion. Group 2 - T-6049 (5 maka) F53802 HW - 29 No visible lesions. F53364 NM - 29 No visible lesions. F532 Mo - 29 No visible lesions. 53399 F - 29 Tthhee esnkitnirehasdordsiaflfusTeulmybardarakndretdhoraarceiacs along region. Collected with routine section. F53349 FF - 29 TahpeplisckaintiohnassmiutletiuppletoreIdcmareiansdionamettheer. Collected with routine section. F53398' F - 2 Tthheereskealreetamlulmtuispclleedaofrkthreedlaurmebaasr wsiatchrailn r1e.g5i%on2.s0urrcno.unding the spinal column, up to F52999 F - 29 No visible lesions. a- ANnoitmaalpplriecpalbalcee.d with No. F52999. b Replacement animal for No. F53398. . 000380 00002y Page 24 of 42 IndiviTdauballe P4at(hCoolnotginyueCdo)mments AMnuinmbaelr Sex DiedTestSaDcaryificed Necropsy Observation F53360 M 53400 MH F53346 HM F53350 F 53397 F P5335 F - GT roup- 3 - 60 (104 0ma9 /ka) 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible Tesions. 29 No visible lesions. 2 No visible lesions. HI 6329-130 53365 MH F53354 HM F347 Mo 53403 F 53000 F - F53345 F - Group 4 - T-6049 (500 ma/ke) 29 No visible lesions. 29 No visible lesions. 29 No visible Tesions. 29 No visible lesions 29 Tahpeplisckaintiohnassimtuel,tiuppletored1 camreaxs3oncn.the Collected with routine section. 29 No visible lesions. - Not applicable. 000381 000030 Sex Male Female Male Female Page 25 of 42 HHT 6329-130 Individual Animal Table Tissue Weights and Bile Volumes ANnuimmbaelr WeiahtDer( mal qApp) TiLiver Kidneys cation Site VoluBmiele (ni Group1 (Control) - Distilled Water (0 ma/ka) 53358 81.381 - 0.783 1.4 F53359 73.857 - 0.761 0.8 F53353 77.411 15.761 0.644 0.6 F53404 70.277 12.585 0.620 1.8 53405 78.270 - 0.775 11 F53356 74.687 - 0.915 0.5 Group 2 - 1-6049 (5 ma/ka F53402 78.731 14.611 0.468 2.3 FS53364 84.099 - 0.641 1.8 F53352 67.613 - 0.706 1.8 F53399 65.818 - 0.546 0.8 F53349 77.595 14.211 0.489 0.9 F53308" - - - - F52999 87.510 - 0.660 0.5 a- MNoortibaupnpdlicsaabclrei.fice on Day 2 (01/08/95) due to possible broken back. wTaisssuneost wtearkeencoslilneccetetdh,ishorweeqvueirr,emetnhte bceocrarmeespoenfdfiencgtivweeigohnt01o/r24v/o9l5u.me b ARneipmlaalcemreenptlacaendimawlithfoNro.No.F52F959393.98. 000382 000031 Sex Male Female Page 26 of 42 HAI 6329-130 Individual AnimaTlablTeiss5ue(CWoenitginhutesd)and Bile Volumes Animal Number Liver Weight (g Dermal AppTi- Kidneys cation Site Group 3 - T-6049 (100 ma/kq Bile Volume (mL FS3360 76.347 15.873 0.802 0.9 FS53400 74.958 - 0.395 1.6 F53346 75.414 - 0.570 13 F53350 74.050 15.120 0.784 1.4 53397 74.882 - 0.801 0.9 F53351 80.686 - 0.279 0.5 Group 4 - T-6049 (500 ma/ka Male F53365 82.598 - 0.441 1.8 53354 70.968 15.234 0.540 13 FS3347 83.6% - 0.716 2.0 Female F53403 70.624 15.427 0.910 1.4 F53000 79.617 - 0.714 0.8 53345 81.341 - 0.633 1.8 - Not applicable. 000383 0000332 Page 27 of 42 APPENDIX A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 HWI 6329-130 000384 000033 Page 28 of 42 Protocol Deviations HUI 6329-130 Protoco CP.ageObs7,erv7.atiEoxnperoifmeAnntimaallsD,esi(g1n), CSleinntiecnacle. ObsFeorrvactliionnisc,al Fisrisgtns baedfnoirniesttreasttioonr canodntrfoolr mcaltienirciaall aspipgnrsoxiamnadtemloyrta1,lit2y.5,atand 4 haodumrisnisatfrtaetriotnest(Dmayate1r)iaalnd Dsiaginlsy, tahnedretawfitceer dfaoirlycli(nai.cma.l aTnedastp.m28.") dafyosr. mortality for at Actual Procedure mOonrtDaalyit4yacnhdec6,k wtahse afternoon ifonradvtheertetwnotlyrepnoltacecmoenndtucted aanndinaGlrsoup(G3r,oupNo.2, FN5o3.351F)5.2999, CP.agOebs7,erv7.atiEoxnperoifneAnntiamallsD,esi(g3n), BroadnydomWieizgahttiso.n, bFeorfore test or c(oDnatyro1l), maonterDiaayl 29a,pplaindcataiton usunrsvcihveadluleedxcedeedasth1 (dwahye)n. Tanhiemablodythawteigwahst osfacrtiheficGerdoupon2 dDoacyum2enwtaesd inadvertently not Tohuetcsoenedevoifattihoensstuadrye. not considered to have had an adverse effect on the 000385 000034 @HazLEToN ) RRS Page 29 of 42 commmacaa, Sponsor: 3M St. Toxicology SPearuvli,ceMiMnendeiscoatlaDepartment PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Date: ) December 30, 1994 Perforning Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 aboratory Project Identification: HWI 6329-130 J) 000386 EPXhPoRnEeSS sMoAsN24O1fauaE ny 10 rmswan sive Far sox 000035 arorras watson an LR Page 30 of 42 ) PTaPg3e0162 .A8 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits HWI No. 6329-130 Test Material T-6049 Sponsor 3MDeTpoaxritcnoelnotgy Service Medical 3M Center, Bldg. 220-2-02 P.0. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD . 3M Toxicology Service Medical Department. 3M Center, Bldg. 220-2-02 P.0. Box 33220 ) S(t6.12)Pau7l3,3-1MN9.62 55133-3220 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date January 7, 1995 February 4, 1995 March 18, 1995 000387 000036 Page 31 of 42 . ) TP3016.A8 Page 3 1. sStiungglye-Dose Dernal Absorption/Toxicity Study in Rabbits 2. pTuorpaossseess irritancy otfhe a styessttemmicatearbisaolrptwihoenn aanpdpliteodxitcoitytheandskirneloaftivreabbsfkiisn 3. ReTLhaaiubsolraastttoourrdyyyCwoPirmlaplcltiibaecneccoRenedguucltaedtiionnsa/ccSotradnadnacerdwsi/tGhuiitnhdeees:lfollowing Good [[X1] 2C1ondCuFRct58as(aFDAN)onregulated Study [[1] 4400 CCFFRR 716902 ((EEPPAA--TFSICFAR)A) 1{[1] ]15NC9(o8t1iN)fo3ih0csaant(iFoNinon.alN)o3.85(03O1E3C(DJ)a(pJaanpeasneeseMAFHFO)H) dAWNie)rlefcaptrroeorc,AecdtutrheeRsesgtuiulndayttihodinosse.sprontoIotncoutlhneneaorcepeisnsiianroincloymofpldiutaphenlciecSaptwoeintshoarnythaepndrAenvsiitmouaudlsy work. ) 4. QTthhueealipQtruyoatloiActsoyslu,ArsasnsuctrueadnycecoUndnuictt,inaancdcotrhdeanFcienalwitrhepoHratzlewitloln Wbiescaoundsiitned by (HW1) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. ITd-e60n4t9ification B. P(hTyosibcealdocDuemsecnrtiepdtioinn the raw data) C. dTpehteeurSmpioarnnnasdtoiriSotnaasstbsiu(lmiyientscylurdeisnpgonsunidbeirlittyestforconpduirtiitoynsa)n.d stability 0. SRotoomragteemperature 000388 000037 Page 32 of 42 ) . Reserve Samples PTa9g3e0146.48 Reserve sample(s) of each batch/lot of test and control materials will be taken for this study. F. Retention The HWI test in a and control freezer set material reserve samples will be stored at to maintain a temperature of -20*C 10C for 10 years 10 years for per HNI SOP. The Sponsor disposition in accordance will with be contacted after the appropriate regulatory Good Laboratory Practices. Any unused completion test material will be returned to the of the in-life phase of the study. Sponsor after 6. SaAfserteyqupirreecdaubtyiHoHnIs SOPs and policies 6. Contro) Materia A. Identification Distilled water B. PhysicalDescriotion ) Clear, colorless liquid C. Purity and Stability The purity 0. SRtooormagteempCeornadtiutrieons considered and stability of this manufactured to be adequate for the purposes of material is this study. E. ReserveSamples See Section 5. E. Reserve Samples F. Retention Any: renaining control material may be used for other testing G. Safety Precautions and will not be discarded after issuance of the final report. As required by HWI SOPs and policies 7. BxperinentalDesian A. fnimals (1) RSapbebciites ) (2) Strain/Source Hra: (NZW)SPF/HRP, Inc. 000389 000035 Page 33 of 42 - D (3) AiAgdenulaitttiation Tp3016.48 Page 5 (4) W2e.i0ghtt03a.t0kIngitiation (5) N1u2nbmaelresandandSex12 females (6) IInddeinvtiidfuizc]atniuonmbered ear tag (7) Husbandry (a) HIonudsiviindgually, (heavy gauge} in screen-botton stainless steel cages (b) FAt(ohPeoHmIedmaasFnueurefdeasdc,taunrIoenrcu'.n)t.foorfThnLeuatbrfoiortoaidtoonriaysl rRcaoobumbtpiiotnneelDnyitestanHaFlyz#e5d326by environmental contaminants. and ) (c) w4adattee1rribairteunanfarloymzeadn bayutWoWmIatifcorstyosttaelm.disSsaomlpvleeds soof itdhse, ohfroagrradnnsoeesplshe,ocstpeahdnadteelSsep,meecnaitnfsdi,ecdhhlmeoiacrvriyonbamietoteladolgsh,iycdarlocacrobnotnesn.t and (9) TtChhoeantrteawmoiaunrlaedntnsointkneorwfnerecownittahmintahnitssstiundyt.he food or water )) EsEnetviErtoonEmmaein)nttaincoanttroelmsperfaotrurteheofani1m9alC trooom23wCi,ll abe rTeilgahtti/v1e2-hhouumrididtayrkocfyc5l0e%. 20%, and a 12-hour (f) AAtcclTienaasttio7ndays (8) SnBaoeadlseeeqadcuntianitoaeonlnnheuoinamflbteoThbrevsitoaofnudAsenlxbiytomrdaayplosowarneiimghaheltasltahwciclolrdibnegputorchWaWIsedSOPsso. thaAtn ) dsaantyirsnaatloisffiewsidtluldbyodbyeinwipetliiaagctheitdonri.anntodomsitzuadtyiognriosupprpsolagcrueasdminwgointahtienst.nineThe 000390 000039 Page 34 of 42 J PTaPg3e0166.A8 (9) HiJsutsotirfiiccaaltliyo,n the animal of "ftohre NSepwecZieeaslaSnedleWchtiitoen choice because of the alalrbgienoamroaubnbtitofhas been background information on this species. B. DAosdeministration (1) TestGroups Group Test Material Dose Level _(no/ka_) Number of Animals Males -- Femajes 21 (Control) DisTt-il6l0e4d9water 3 T-6049 or 100See 3 33 3 33 : T-6049 500 3 3 2*,* TToo bbee aaddmmiinniisstteerreedd aatt aa ddoossee vvoolluunnee ooff 2..010 mmlL//kkgg ) (2) OPifrnetpnhaeercaetdsiasoyanrybeoff(otroEexopbottseasuitrnemauAtnreberlaieanlishaepdplisckaitni)o,n,thetheflabnakcsk oanfd, Wseiaulcrlhfaccreaobnbsaitrteiat.uwtilelThaepbpetrrocexlaiitpmmpaeetndetlyfsr2iet0ee%sooff(hiantithrea.cttotTsakhlien)bsohwdaiyvleld area be tainhniasmtpaelcwstoeudwlidlflporrbeecilncutldeierpfpeterhdeingUasselneesoeifdoneasdn,ythoirfrorutightheaotiuaotnni,mtahleosr.stduedTfyhe.ects (3) DoAATsdTemianniimasltsrwaitlliorneceive a respective test or control msaitnegrlieala.dmiTnhiestrdaatyioonf otfreatthmeent wGaitrloluapdboe2sedwielvsloilgunbmaeeteddiolfuats.e0d1Damywli/t1kh.g.diTsheTthiedlolseredespwfeaoctrteirevaecahnddoasaneipmpaifloire.din AweeaTicghhdtoasneijsmuasltinbineGfrooGurrpeosupasd1m-4i3niwaisnltdlra4tbiewoinlblasaenbdde oanppltiheed aunnidmiallustedb.ody will be spread onto at1rh0ee-acnaorxfea1ao0pf-pclniecxgapatouiszouenreb(aGinnrdouaapgsethis1ne-4c)uarnedwdilulwniitfbheorpcmaopvaeerrleadytearwp.eitharTohauend, batelalpecoeldtlgoaesrperdoavnidddueroivneagrnnrotahcepcple2ud4si-vwheiotuhdrreaSspasprilannigc.aWrtaipoTnheapnerdraibEobdli.atsstowpilla)st (4) RhTeuhamesaondsne.rfnoarlRroouutteeoifsAdampiontiesnttriaaltiornoute of exposure in ) 000391 000040 Page 35 of 42 _) TPP3a0g1e6s.48 (5) AaReppmppolrvioacxlaitmiaootfnelTytehset23bMaahnotduearrgsieaslaftanedr ctoelsltarosr wciolnltroble rmaetmeorvieadland atnheaprpesriodpurailatetesstolmvaentte,riaIlf wnielclessbaeryr.emoved using water or C. ObservoaftAniimoalns (1) ClFaidoarniinccilaisntlirOcabatslieornsviagatnnisdobfnoesrfocrleintiecsatl orsigcnonstraonld mmoartterailailty at saaidpgmpnirsno,ixsitanrnadatteiltoywnic1,e(Dda2ay.5i,l1y) aan(ndad.m4.dahiaonludyrspt.hamef.rt)eearfftotreemrsotrfmotarat)eelrtiiyanlfcsaolr daitrelcetaesdt 2by8 dtahyes.studOybsedrirveacttioorn.s may be extended wich (2) dRBeeerafndoairlnegiterosrftitDaoetrrmiaoclnontrreIoarldriitnmagattieworinillal beadnmaidneistarndatiroencortdheedinaietiianle ) aDifartyreirt1atrbieaoannddiangrgeeadr(ieAnmtogtvsaaclwhimle(lnDtaybe12)).nadaneAdddaioptnpirooSntxauildmyatdeDeralmyyas)30 mainndutses FIonrdiveiadchualanidmearln.al irritation records will be maintajued (3) aBFopodpryliFcHaaentidigoohnntisza(tDiaoyn,1),beofnoreDayte2s9t,orandconattroulnsmcahteedruilaeld death (when survival exceeds 1 day) (4) sampleCollections (a) FBreefqorueenciynitiation post-dose (Day 2), (DDaayys 14)., 9a.ppr16o,xim23a,telaynd24at hours experimental termination (Day 29) (b) ANunmber of Animals 000392 000041 Page 36 of 42 0 PPa3g0e168.A8 (c) MBflerotoonhdodthseoafmmpalCreogslilne(acaltpipoeranorxivmeaitnelyof4eintL)herwilelarboencboalylsect1.ed 2(,act4u,al9, vo1l6,umeandto 23b.e be obtained fron the dpooAcsputpmereronixtoiermdatveeilnnaytch2ae0vanrLaowfodfeaatbcalh)oowdill sanaicnrailficseadcriatfictehde tiinmea samples will be stored omforinbeucnrdopscyond(iDtaiyon29)o.r The at roon temperature and then cfernatcrtiifoungseds,toraendd itnheasferpeareazteer sseetruntoamnadinctealilnular Sfp2ro0ancsCtoiro4n1s0afCtw.eilrl Tehxbeeperssieemnpetanrtafatrleodzetnesremorinunmadtraiynodni,cceelltoulathre Samples will be shipped to: JManEesE0. LJPoChn.son B9l3d5g.Bus2h-3A6v-0e9nue St. Paul, HN 55106 ) tJaemleesphoD.neJoathns(o6n12)or77a8l-t5e2r3n4atepriwiolrl tboethneotsihfiipedmenbty of the samples. D. Pathology (1) AmnoyriUnbasuncnhiedmdaullceoddnydiitnigondSacriufcesrwiilnlgabntedhesusbtjuedcyteodr stoacranifiacbebdreviinataed graerncoeossrstdheendte.iczreodApnswiyimtaehlxsamsioinndaituainomnopreniatbnoudbnadarlblciotnaadblintoir(omvanialiwtiilinlejsecbwetiiloln be in 0et.xhseaPnmagaturhigonilaontgaeyld,. ea(3r)TivsSesiaunme)ps,l,e blCaseodldleevcsitcairoitnbh,eedwvieilnnla sbceeacvtaic,oonllaencdted. After necropsy, the animals will be discarded. (2) ScAshtuervdtieuvrlimenigdnSataaincoirnmiafloisfcewtihlel exbeperainmeesntthaeltizpehdasewit(hDayso2d9i)u,m bpalebenbdtroebvvaiiraabtiettdhaelgrvoe(snvsaiancaeivcnarj,oepcsetyxiosnaenxgaiumniinntaahtteeidom,na.rgainndTahlesuaebanjriemcatvleesdin)w1,i6llan bbee nreeccroorpdseided in random order and all abnormalities will 000393 000042 Page 37 of 42 ) PTaPg3e0169.28 (3) swTaahmsephlesediCtweosiltlhoefcltutikeosetwnaranndtacpontwraotlermaptreiroiraltoaptphelinceatciroonpswyill be ap1nr-iocncnaeldssue,rcet.iaonndThboefothtwhheoklieddenrelmyiasvlerf,arpopmblilitech,eatifaoninrsatpsipmtraeolxeifmraoatmndealf1le.mcamlex pnleaccaepdsieidn ainferaecehzergrosuept 10C. After necropsy, twiollmaibnetacionlleactteedmpearndatuirnemeodfiat2e0lyC the animals will be discarded. TKahfietdenretyises)xspuewerislilm(elnbitevaelrs,enttbeirlmfeir,noaztedineornmo.anl draThypepliisccaeamtptilooensthwesiiltSe)p,obnesor Ssphoinpspoerd tiso rtheesppoenrssiobnlelfiosrtedtheinreSteecntitoinon7.aCn.d(4d)i.s(pco)s.itioTnhe of the samples. E. StHaotistsattiicsatlicAanlalaynsaleysses are required. 8. RAepfoirnatl report including those items listed below will be submitted. ) DDeessccrriippttiioonn ooff tthhee tteesstt asnydstecmontrol materials DPartoecseduorfesexperimental initiation and termination DTeasbcurliapttiioonnooffmoanrytaltiotxiycdaetfafecbtys/sdeexrmaanld diorsreitalteiveoln GTarbouslsatpiaotnholoofgymeafnindbiondygsw/geriogshstspabythoselxogyandrepdoorste level 9. LoOfrcaicagitilinioatlnatoedfataRaua,duitoDirantgcao,ptRiheeescsottrhudedsrye,odafun,rdiFnwiiglnliatlsbeRperapovogarrieltsasbleandatbeHMfIoreto raalecltcaeiopnrtieagdnicneianlofthpeatphaeerrcfhdiinaatvlae,s roeifpnocrHltHu.IdiangcWcheotnrhdoistenhge ittfoeinmaHlHlIisrtSeOepPdo.rtbelioswcwoimlplletbeed, DPorsoetocporlepaarnadtipornotorceoclordasmendments InB-oTdiyfeweriegchotrsds DOobsseervaadtniionnisstration SAnaamtpolmeiccaolllepcattihoonlogreycorredcsords SSthuidpypincgorrreecsoprodnsdence ) Final report (original signed copy) 000394 000043 Page 38 of 42 ) PTa0g3e01160.8 Tnohte bfeolalrocwhiinvgedsuwpiptohrtitnhge rsteucdoyrdsdatwai.ll be retained at HNI but will MAnaitmearl arneacleyispits/acreelcoirndastion records ARInenifsmrtairlgaeerrnaottoonrcatalenimdbprearftariteouenrzeeranadntdemmaphieunrntaietdnuiartneycerreercceoocrroddrssds ' 000395 000044 Page 39 of 42 ) TPaPg3e01611.A8 PROTOCOL APPROVAL gE 2 &latory John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department /-5=9 Date ) Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. sentative Lek . Quality Assurance Un Hazleton Wisconsin, Inc. (6329-130.protdsk2) vos . Date a /l- ale7 t 000396 000045 Page 40 of 42 | TP3016.A8 ) Page 12 Attachment 1 Scoring Scale for Acute Dermal Reactions Erythema 01-- NSolnieght 23 -- HSoedveerreate Edema 01 -- SNToingeht (barely perceptible to well defined by definite raising) 32 -- SMeovdeerreate(ra(irsaeidsemdoraepptrhoaxnima1taeml)y 1 mn) Atonia ) 01 -- NSolnieght (slight impairment of elasticity) 23 -- MMoadrekreadte(no(sleolwastriectiutryn)to normal) Desquamation 01 -- NSolnieght (slight scaling) 32 -- MMoadrekreadte(pr(osncoaulnecsedandflafkliankges)with denuded areas) Coriaceousness. 01 -- SNloingeht (decrease in pliability) 32 -- MMaordkeerdate(to(ulgehathaendrybrtietxttluer)e) Eissuring 01 -- NSolnieght (definite cracks in epidermis) ) 23 -- MMoadrekreadte(cr(accrkasckswitihn dernis) bleeding) 000397 000016 Page 41 of 42 + @HAzLETON ABBA a +commacorany PROTOCOL TP3016.A8 Sponsor Single-Dose Dermal Absorption Toxicity Study of T-6049 in Rabbits WNL 6329-130 Contractor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2-02 P.0. Box 33220 St. Paul, MN 55133-3220 Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Study Director = --_-- John L. Butenhoff, PhD Steven M. Glaza Anendnent No. 1 This amendment modifies the following portions of the protocol: J Effective January 10, 1995 1. Page 6.7. Experinental Desian; 8, Dose Adainistration: (3) ose Administration. Animal Nos. F53355 (Group 3 female) and F53398 broken (Group 2 backs). female) were sacrificed on Day These animals will be replaced 2 due to injury in this study. (apparent Replacement collection) animals at the will same be dosed, time as the bled, and animals necropsied (includes tissue in another 3M sponsored section: study (6329-137). Add the following as the second paragraph to this Due to the sacrifice on Day 2 of one Group 2 female (Animal No. F53398) and one Group 53355), replacement animals will 3 female (Animal No. be treated at the respective dose Tevels sitrurdiyt.atioTnh,e in the same manner as for the other animals in the oabndserbvoadtyiownesigh(tcsl)iniacnadl thoebsepravtahtoiloongsy, ofretahdeinganiomfaldsermal (unscheduled sacrifices sample collection) will and deaths, scheduled sacrifice, and be conducted in the same manner as for the other Will animals be in the in the same study. manner as The for method of the other blood sample animals in collection the study. The frequency of blood collection will be as follows: J) Before initiation (Day ((bDaayy 22)5,). Days 4, 8, 15, 1), 22, approximately 24 hours post-dose and at experimental termination eEXnPoRnEeSS sMoAsIL241SEsLaVryERY 0 xisuan Biv0 MaisFoorn 8a0n020t1i0i000047 000398 Page 42 of 42 3 Anendnent No. 1 PHaLge63229-130 Effective January 24, 1995 At of the request of bile collected the Sponsor, the weights of tissues will be documented in the raw data. collected and the volume These weights and volumes will be included with the sample shipment. Modify thefollowing sections of the protocol to include these additions. 2. Page 9, 7. Experimenta) Designs D. Patholoay: (3) Sample Collection Modify the second sentence in the first and second paragraphs of this section with the following underlined additions: collected, weiched (volune only deternined for bile). and The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female necropsied in each group will be -i2m0meCdia1t0elCy. placed in a freezer set to maintain a temperature of The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.C.(4).(c). 3. Page9,8.Report. Add the following to this section: ) Individual animal tissue weights and bile volumes PROTOCOL AMENDMENT APPROVAL Fb 7. Luiilondirtf John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Ne WW. . Steven M. Glaza Study Director AHaczulteetoTnoxWiicsocloongsyin, Inc. 2/9 /ae Date pats Date hel Representativs Quality Assurance Unit J Hazleton Wisconsin, Inc. Date 000399 202.95 (6329-nl1.3ds0k2.) 000035 9.1.2 Analytical protocol AMDT-020895.1 000400 00004y 3M Environmental Laboratory Protocol - Analytical Study Single-dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits In-Vivo Study Reference Number: HWI#6329-130 Study Number: AMDT-020895.1 Test Substance: FC-95 (T-6049) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Addressof 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 Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement Ton Analyzer by Means of an Orion EA940 Expandable AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochenmicals Using Electrospray Mass Spectrometry Author: James D. Johnson Approved By: es D. Johfigon ftudy Diregbr 1fofas Date Pom L. BGM John Butenhoff, PhD Sponsor Representative off Date 000401 000050 1 1.0 PURPOSE -------------------------- `The purpose ofthis study is to assess the dermal absorption of FC-95 (T-6049) in rabbits after a single dermal dose ofFC-95. 2 EST MATERIALS 2.1 Test, Control, and Reference Substances and Matrices 2.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 22..11..23 AAnnaallyyttiiccaall RCeofnetrreonlcSeuMbasttarnicxe:: BoNvoinnee liver and bovine serum 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source of Materials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.14 liver), Sigma Chemical Company (2.1.2, 2.1.4 serum) 2.3 Number of Test and Control Samples: Tissues and fluids from 18 test animals and 6 control animals. One animal was replaced on day 2 (animal F53398 in the 5 mg/kg dose group replaced by animal F52999). Tissues and fluids include tlihveers, tkiisdsnueeys,wsilelrubme,actetlhleuldairsfcrraecttiioonn,ofdetrhemaSltuadpyplDiicraetcitoonr.site and bile. Analysis of 2t.h4e HIdWenItiafniicmaatlioindeonftiTfeisctataionndnCuomnbterrolwhSiacmhplceosns:isTtsheofasamlpetlteesr aanred ifdievnetdiifgiietd using number, plus the tissue identity and day identity (serum). 2.5 Purity and Strength of Reference Substance: To be determined by Sponsor. 2.6 Stability ofReference Substance: To be determined by Sponsor. 22.07 +Sto1r0agCe(C2.o1n.d2i,t2i.o1.n4s).foTreTsetsatnMdaCtoenrtiraolls:saRmpoloems wtielmlpebrearteucreeiv(2e.d1.a1c)c,ording to AMDT-S-10-0. 2.8 DispositionofSpecimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required studies longer than 28 days. 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 000402 000051 2 3.0 EXPERIMENTAL - Overview Rabbits were dosed with T-6049 in a control group, 5 mg/kg, 100 mg/kg, and 500 mg/kg dermally. (See HWI#6329-130 for dosing, animal weights, tissue collection, etc) Tissues and serum are available from these animals for analysisoftotal organic fluorine content. At the discretion of the Study Director samples will be analyzed to the extent necessary to provide information to assess the extentofdermal absorption. 4.0 EXPERIMENTAL - Methods 4.1 Liver and Serum screening methods: (attached) 4.1.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Means ofaModified 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 Fluorochenmicals Using Electrospray Mass Spectrometry 5.0 DATA ANALYSIS 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. Statistics used, at the discretionofthe Study Director, may include serum averages and standard deviationsof concentrations for different dose groups. If necessary, simple statistical tests such as Student'ts test may be applied to determine statistical difference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS 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. 000403 000052 3 ZOREFERENCES 7.1 AMDT-S-10-0, Sample Tracking System 8A 0 TTACHMENTS 8D.o1hArMmaDnTn-MD-X12-000,0 TOhregramnailc EHxatlriadcetAionnaloyfzeFrlu-oLriivdeer by Means ofa Modified 8E.x2paAnMdDaTbl-eM-Io2n-A0n,alFylzuoerride Measurement by Means of an Orion EA940 8.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver E8.l4ecAtrMoDsTpr-aMy-M5a-s0s,SApneacltyrsoimsetorfyRabbit Liver Extract for Fluorochenmicals Using 4 00040: 000053 3M Environmental Laboratory Method Thermal Extraction ofOFrlguaonriicdeHablyidMeeaAnnasloyfzearM-oLdiivfeired Dohrmann DX2000 Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: /g- #-9< Revision Date: None e-- ser---------------- Author: Rich Youngblom Approved by: foup Leade / ---- i,phz i Jae Quality Assurance lof3/7< Date 2-4-7 Date Software: MS Word 5.12 Affected Documents: AMDTE-xMp-a2ndaFbllueorfiodneAMneaalsyuzerrement by Meansof an Orion EA940 AMDT-EP-3 Organic RHoaultiideneAnMaaliynzteernanceof a Modified Dohrmann DX2000 000405 000051 3 SCOPE , APPLICABLE COMPQUNDS, AND MATRIC ES L.1 Scope: This method is for the operation ofa Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution foranalysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological tissues, particularly liver. 2.0 KEYWORDS 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, 'DX2000, fluorochemicals. 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 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them todry down before starting run. 3e.q5uiPpotmeennttial biohazardsdueto the biological matrices. Use appropriate personal protective 4.0 SUPPLIES AND MATERIALS 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSL 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 4.6 SOarimopnle94c0ol9l9e9ctTiootnavliaIlosn,icHDStPrEe.ngth Adjustment Buffer (TISAB II ) or equivalent. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 _EQUIPMEN 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 5.4 DX2000 software, Excel Spreadsheet, version version 15..000,ormogrdeiaftieerd for fluoride extraction. 6.0 INTERFERENCES m6.a1y Svaamrpylferosimzemaitsrliixmittoedmattoraixpproximately 150 mg, depending on sample moisture content. This 000406 2 000055 7.0 SAMPLE HANDLING 7s.a1mpSlaem.plUesseafroerncoetpstoorbeprhoabnedlteodtrwaintshfebratriesshuaensd.s. Fluoride may leach from the skin to the c7.l2eaSnasmcpallpeeslofalnidvceurttairnegcbuotafrdr.omTfhreozceunttliinvgerbaoanrddpalnacdesdcailnpaeltasrheoduladndbelacbleelaendedwewiigthhbwoaatte.r,Use a `methanol, or methanol-water solution after each liver is cut 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparationof Calibration Standards R8.e1f.e1rTthoeprsottaoncdoalrdfsorrtehqautirpreodjefcotr.each project will need to be appropriate for that individual project. 88..11..32 TFyoprircaablbliyt 5li0v-e5r0s0tupdipems,FuCs-eb9e5eifnlmiveetrhaasnotlhestmaatnrdiaxr.dsCaurteauspeid.ece of frozenbeefliver (100 150 mg) and weigh it in a labeled and tared weigh boat. 8.2 Calibration - Overview `cTuhrevenoirsmraelquciarleidbrtahteiponroicsetdhuerfeluliosrtieddebceulrovwe (isAuMsDeTd.-M-2). However,ifan optional spiked liver a8.n2d.1coAmbcuasltiibrnagtitohnecmuursvienfgorthtehesaDmXe2m0e0t0hoisdgseannedramtaetdrbixytsyppiekiasngthseamspalmepslewsittho kbenotewsnteds.tandards 8.2.2 Typically, spiked three replicatesof each standard and five concentrationsof standards will be: R8.e2c.o3veSrteadndFar(dugc)uornvethwielyl-abxeisp.loGtteenderaastMeaassreSgrpeiskseidonFc(uurgv)e on the x-axis and calculate and the SeqtuaantdiaorndfoMratshse line and the r2 value. 8.2.4 Mass SpikedF (ug) *FC-95 is = (Amount spiked 60.04% F therefore in mL) 0.6004 xis(tChoenfca.ctoorf sutsaenddtaordcoinnvperptm)FCx-9(50.t6o00F4)* 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 Start Up b8.e3.u1s.e1diRfutnhe2porrevmioorues sCalmepalneCsyccolnetsaiwnheednhisgtahrtcionngceinntsrtartuimoennstofefalcuhordaiyd.e. More clean cycles may 8.32 Blanks 8.3.2.1 8.3.2.2 PFroerpraarbebistasmtpuldeiesu,siunsgebteheefslaimveermaesththoedsmaatnridx.typPeroepfamraetratixleaasstt3hesatemsptlseasmopflbee.ef liver 8(.130.02.-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 ion selective electrode analysis. 000407 3 000056 8b.e3f.o2r.e4prFoocrereadbibnigt wsitutdhietsh,e tcahleibmreatteiorn.reaBduirnng sfoarmpalbelsanukntislatmhpilselismhiotuilsdrebaec0h.e0d3, oprpumntoirl linowtheer jhouurdsg).emenotf the operator the reading is stable with respectto historical readings (previous 48 83.2.5 before For non-rabbi proceeding with stthuediceasl,ibtrhaetiboln.ank readings should reach a predetermined ion concentration 8.3.2.6 It may combustion. be necessary to mix approximately 50 mg of charcoal with the sample to aid 833 Standard Curve 83.3.1 labeled Weigh out at weigh boats. least 15 matrix samples (5 For rabbit studies, weigh 1s0t0a-n1d5a0rdmsgwbiethef3lirvepelricsaatmepslecsa.ch)Reincotarrdedweaingdhts in s8.t3u.d3y.2dataP.lacSetowreeitghheemdatbreiexf samples on dry liver sample in ice or ice packs to Dohrmann sample keep boat. them frozen until used. 8qu3a.n3ti3tySotfartthweistthantdhaerldoowensotrsitnantdhaermdatcroinxc.entFroartiroanb.bitUssitundgieas,Haumsiel4tounLosfysritnagned,aerjdectaandfeijxeecdt it on or in the beef liver. 8.3.3.4 may be Atleast 3 replicates should be used used at the discretionofthe analyst. for the lowest standard concentration; more replicates 8.3.3.5 83.3.6 RCuonmablulst1Sthsetasnadamrpdlse. Iasfdoensecrriebpeldicianteseisctsiiognni9f.i3caanntldyadniaflfyerzeentacfcroormditnhge toothAeMrDtTwo-M-2. roelpdlirceaptleisc,atreuannadnotthhaterthseamnpelwerfeoprlitchaatteswtialnldabred.usIendditcoactaelciunldaatteaththeatretghreesnseiwonrceuprlviec.ate replaces the l8e3a.s3t.07.95W,hceonnsaullltswtiatnhdasrudpserhvaivseorb.een run, calculate the 12. r2 must be at least 0.95. If it is not at 8.33.8 A changed. new New standard standard curve curve mshaoyuladlsboebreunruwnhaetnthtehdeicsocrmebtuisotnoifotnhteubaenaolrysstample matrix is 8.4 Storage Conditions for Standards 8T.y4p.i1caSltloy,rasgteanrdeaqrudisreamreensttsorfeodr sattarnodoarmdsteamrpeedreatpuernedeinntploansttihcesicnrdeiwvitdoupalbosttlaensd.ards used. 8.4.2 New FC-95 standards should be prepared at least onceamonth. 9.0 PROCEDURES 9.1 Typical Operating Conditions: 9.1.1 9.1.2 Combustion Oxygen and tHuebleituemmfpleroawt=ur5e0=cc9/5m0inCu.te. 9.1.3 9.1.4 VBaapkoeritziamtei=on3/0Dr0ysiencgontdism.e = 240 seconds. 99..22.1StIafrttheUpprPorgorcaemdiusrneo:t started, start the EOX program on the PC. 99..22..32 POuptetnhethfeumSaYceSTmoEdMulSeEaTnUdPthweicnedllowin.the READY mode. 9.2.4 Close the SYSTEM SETUP window. 000408 0004057 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.1apOppreonxitmhaeteSlAyM5P0LmEgoHfcAhTaCrcHoaalndwiptlhactehethseamspalmeplteo aiindthceomBbOusAtTi.on.ItImfathyisbeisndeocnees,sacrhayrtcooal should also be mixed in while establishing the baseline and when generating the standard curve. 9.3.2 Close SAMPLE HATCH. 9l.a3b.e3ledAdsadmapplperocporlilaectteiovnolviualm.e oTfypTiIcaSlAlBy 0s.o6lumtiLontoor151:m1 LTIaSrAeBu:sMedi.llFio-rQrTMabwbaittesrtumdiixest,urueseto1.a0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9in.c3h.e4s.PlGaacseetsherevliaelasseodtdhuartitnhge ptyiproolfystihse mCuOsMt BbuUbSblTeItOhNrouTgUhBtEhe iTsIiSnAtBh,e TISAB at least 0.25 9.3.5 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 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTTM. 9r.e3m.o8vReinasney tThIeSeAndBofretmhaeincionmgbuosnttihoenttuubeb.e with Milli-QTM water and wipe with a KIMWIPE to 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed `with a cotton tipped applicator or vacuumed out. It may be necessary to scrap particles off the bottom with a spatula or other similar device. A drop of Milli-QTM water may be added to the boat to aid in the Clean Cycle. 9.3.10 Close the hatch. 99..33..1112 SRaumnpltheeisCrLeEadAyNfoBr OanAalTyspirsobgyraimo.n selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 9.4.2 USsaemptlhee sMtaasndsaRrdeccouvrevreetdo cFal(cuugl)at=e(tTheISsAamBplvoelvianlume.L) x(Orionreadinginporn-intercept) (Slope) 10.0 VALIDATION. Bi Bo pono 10.1 Quality Control a1n0a.l1y.1siDsaiislsytaSrttaerdtbUypanCahlyezcikngSaQmCplseasm:plOesn.ceTthhee QstCansdaamrdplceusrvareeistoesbteabtlhieshseadm,eeaacshthdeayloowfest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% of the standard curve, then a third F1l0u.o2riPdreeNciostieobnooaknsd 2A,c3,cuarnadc5y. : PSreeceismieotnhaondddaecvceulroapcmyevnatriaensalwyhsiesnaanndalsyazmipnlgesaanmapllyessiosfidnifferent matrices and different reference compounds. 10.3 Other Validation Parameters: NA 000409 000s055 11.0 DATA ANALYSIS 11.1 Calculations 1111 For the standard curve, use regression analysis in Excel, version 5.0 or greater. 11.1.2 To calculate the fluoride contraction in the sample, see method AMDT-M-2. 11.2 Analyzing the Data 11.2.1 12 must be at least 0.95 or greater, "Outliers" may be excludedif two of the three. replicates are within 20%ofeach other and the outlier is greater than 200%ofthe average of those two or less than 50%ofthe average ofthose two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. None 13.0 REFERENCES 31439.1, rReovsiesimoonuBn,t DDeochermmbaenrn1D9X932)000 Organic Halide Analyzer Operator's Manual (Manual 915- 13.2 AMDT-M-2 Fluoride Measurement by Means ofan Orion EA940 Expandable lon Analyzer 13.3 AMDT-EP-3 Routine Maintenance ofa Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS Revision Number ReasonforChange. Revision Date 000410 6 00005 3M Environmental Laboratory Method Fluoride Measurement by Meansof an Orion EA940 Expandable Ion Analyzer Method Identification Number: AMDT-M-2 Revision Number: 0 Adoption Date: [0-445 Revision Date: None Author: Rich Youngblom Approved By: Gro] Leader 1L/2s Date Le ABAL Quality Assurance [o-#35 Date Software: MS Word 5.12 DAfofhercmtaednnDoDcXu2m0e0n0tsO:rgAanMiDcTH-aMli-d1e ATnhaleyrzmearl Extraction of Fluoride by Means ofa Modified wu0411 1 000060 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES L1 Ton SCOPE: Analyzer. This method is for the calibration and operationof an Orion EA940Expandable 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3 APPLICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred pH of 6.0. 2.0 KEYWORDS 2.1 Fluoride, fluorine, ion selective electrode 30 0PRECAUTION0 S 00 3.1 No hazards identified with this method. 4.0 SUPPLIES AND MATERIALS 4.1 4.2 Orion Orion 940999 Total onic Strength Adjustment Buffer II (TISABII) Model 900001 electrode filling solution (AgCl) or equivalent. or equivalent. 4.3 Orion 940907 100 ppm fluoride standard or 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. 50 0EQUIPMENT 00 5.1 Orion Model EA940 Expandable lon Analyzer or equivalent. 5.2 Orion Model 960900Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 386 or 486 computer (only neededifusing Orion 3E software). 5.5 Orion RS232 interface cable (only neededif using Orion 3E software). 5.6 Microsoft Excel 5.0 (only neededifusing Orion 3E software). 6.0 INTERFERENCES 6.1 It is recommended that the pH be at or near 6.0. QM water will generally bring sample to pHof 6.0. A 1:1 mixture of TISAB andsample/Milli- 6.2 Sample temperature may effect fluoride measurement. at room temperature as the standards were when the meter It is recommended was calibrated. that the sample be 6.3 The rate the samples are stirred at should be consistent with the rate the standards were stirred. 000412 0020061 u6.n4deArirelbeucbtrboldees. trapped under electrode can give erroneous readings. Make sure noair is trapped 2.0 SAMPLE HANDLING 7.1 No special handling necessary. 88..11.1PrMeepaasruarteio5n0 mofLCoaflTibIrSatAiBonITStinatnodar1d0s0 mL plastic volumetric flasks. 8.1.2 8.1.3 PLaibpeelteth0e.0f5l,a0s.ks1,a05.50,.051,.0,0.a1n,d0.15.,5 1.0, mL and 1S of 100 ppm ppm fFl-u,oarlidoengstwaintdhartdheindtaotethaenadpyproouprriinaitteilalys. labeled flasks. 8.1.4 8.1.5 SAhdadkeaptphreofxliamsaktsetloy m3i0xmtLheosfolMuitliolnis-. QTM water to each flask. t8.h1e.f6laEslkismionvaetrethaierabiurbbbulbebslefsrom the flasks by tipping the flasks ontheir sides and rolling the air in 8.1.7 8.1.8 Bring Invert the and vsohlaukmeethien tflhaeskfslafsokrs tuhpe tfointahlemi1x0i0ngm.L mark with Milli-QTM water. 8.1.9 Record standards in Standards Log Book. 88..22.1CaIlinebcreastsiaorny, remove tape from electrode filling hole. 8.2.2 8.2.3 EIjnevcetrtapfreowbedrtoopwseotf top seal filing solution from bottomof electrodtoe wet lower seal 88..22..54 TFihlletmheeteelrecatnrdodtehewiFt-helfeiclitnrgodseaolruetitoynp.ically calibrated by direct measurement with no blank tcohrermeacntuiofna,ctuusrienrg'sstiannsdtarurcdtsiownist.h concentrations of 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, following 8.2.6 8.2.7 Record the slope in Clean the electrode the by appropriate rinsing with log book. Milli-QTM waterand wiping the sides down with lab tissues 88..33.1SCtaolriabgreatCioonndsittanidoanrsdsfoarreStstaonrdedaradtsroom temperature, 9.0 PROCEDURES 99..11.1CaTlhiebrsaatmipolneatnodbeMemaesausrureemdennte,edSsttaonbdearmdixmeedthwiotdh:TISAB using the proportions 9r.e1c.2omPmlaecnedaedstibrybatrheinTIthSeAsBammpalneufaancdtuprlearc.e the sample on the stir plate. e9l.e1.c3trAoldeloiswitnhseersteadm,plmeaktoemsiuxreftohrearefaerwesneoacoinrdbsubbebfloersetirnaspepretdinugntdheeretlheceterloedcet.rodWe.hen the 9.1.4 same The rate sample should be as the calibration 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. 000413 0030062 9.2 Calibration And Measurement, Using Orion 3E Software: 9.2.1 Calibration: 9.2.1.1 Follow steps 8.2.1 0 8.2.4 9.2.1.2 Press Function Key #8 (FS). c9o.n2.c1e.n3trTahteiocnoomfputhteesrtsancdraeredns,wialnldaswkheytohuertoorconnoftiarmbltahnekniusmtboebreofinsctlaunddeadridnstthoe cbaeliubsreadti,on. M9.a2k.1e.4aPnlyacneectehsesaelreycctrhoadenignettsohetfhiersitnsftoarnmdaatridononprtehseesnitredplaantde aclnidckcloinckCoOnNCTOINNTUIEN,UE. s9t.a2b.i1l.i5zeOdb,seprrevsestAheCrCeEadPiTngsRoEnADthIeNgGr.aphic display on the computer. When the readings have, 9.2.1.6 9.2.1.7 Repeat step 9.2.1.4 and After the final standard, 9.2.1.5 for the remaining standards. the computer will display the slope of the curve, as well as the. ainntderccleipctkaonndCcoOrNreTlaItNioUn.E.ReTchoerdcatlhiebrsaltoipoen,dianttaericseaptu,toamnadticcoarlrlelyactioopnieidn ttoheC:a\pOprrioopnr\iDaattea\lCoaglibbo.otkxt. 9.2.2 Data Spreadsheet: 9.2.2.1 to store Select either data in. NEW or OPEN from the FILE menu to open a neworexisting spreadsheet 9.2.2.2 Record the nameofthe spreadsheet used in the appropriate log book. 9.2.3 Fluoride Measurement: 9.2.3.1 Follow steps 9.2.1 through 9.2.4 9.2.3.2 9.23.3 Enter Click the on tnhaemNeEofWthSeAsMamPpLleE ibnutthteonappropriateplace on the screen. 9.2.3.4 When the readings appropriate log book have stabilized, click on the RECORD button and write the result in the 10.0_VALIDATION 10.1 Quality Control: _ eee 102 Precision and Accuracy 103 ppm fOltuohreirdeVuaplitdoaatisoantuPraatreadmseotleurtsionAocfcofrludoirnidge.to Reference 13.2, the rangeofdetection is 0.02 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHMENTS None 3.0 REFERENCES 000414 4 000063 13.1 Orion Model EA940 Incorporated, 1991. Expandable fon Analyzer Instruction Manual, Orion Research 13.2 Orion Model 960900 Solid Research Incorporated, 1991 State Combination Fluoride Electrode Instruction Manual, Orion 10 40REVIS0 IONS 00000 ReNvuimsbieorn ReafosrChoannge D Reviesion 000415 s 000063 Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 eer Author: Dave Christenson/Cynthia Weber Approved By: ip Leader a7f, ff Quality Assurance Adoption Date: /o-7/-55 Revision Date: None ressre---------- Jo --3/-25 Dae 33s Dac Software: MS Word, 6.0 Affected Documents: Mass Spectroscopy. M-5, AnaloyfRsabibist Extract for Fluorochemicals Using Electrospray 000416 0000165 1.1 Scope: This method is for the extraction offluorochemicals from rabbit livers, eEltehcytlraocseptraatyemias sussesdpetcoterxotsrcaocptyf.luorochemicals from the livers for `analysis by 1.2 Applicable Compounds: Fluorochemicals or other fluorinated`compounds. 1.3 Matrices: Rabbit Livers. 2.1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated `compounds, extraction. 4.1 Supplies 44..11..12 44..11..43 4.1.5 44..11..76 ESpyrpienngdeo,rcfaptaybpleeoorfdmiesapsosuarbilnegp1ip0e0tpsL. PGllaosvtiecsgrinding tubes Plastic centrifuge tubes, 15 mL LNaibweolgsen 4.1.8 Timer 44..11..910 AFnilatleyrtsi,cTailtpainpnesy:lognlassysrivnogleumfeitltreircs,p0i.p2esp.m. 4.1.11 4.1.12 Disposable plastic 3 cc Crimp cap autovials. syringes. 4.2 Reagents 4.2.1 pAqpumeoaquuseAoumsmsoonluituiomn Aocfeatmamteon(Aildurmicahc)e,taatpepbroyxa.d2d5i0ngpp2m5:0 Pmrgepaarmemaon2i50u0m acetate to a 100mLvolumetricflaskand dilute to volume with Milli-Q water. Dilute this solution 1:10 foar 250 ppm solution. 4.2.2 (SoNdai,uCmO/cNarGbHoCnOa,te)/S0o2d5iuMm:BiWceairgbhon2at6eBgufofferso(dJ.iTu.Bmackaerrb)o,nate (Na,CO,) abrnidng21.0to vgoolfumseodwiituhm Mbiilclair-bQonwaatteer.(NaHCO) intao 1 L volumetric flask and 4.2.3 Dilute acetonitrile solution, dilute acetonitrile 1:1 with Milli-Q water. 4.2.4 Ethyl Acetate 4.2.5 Methanol 4.2.6 Milli-Q water 4.2.7 1H,1H,2H,2H - perfluorooctanesulfonic acid(Aldrich) 4.2.8 FC-95 (3M Specialty Chemical Division) 000.417 0000626 SOEQUIPMENT 5.1 Ultra-Turrax T25 Grinder for grinding liver samples. 55..23 VCoerntmeixfumgiexer 5.4 Shaker 5.5 Analytical Evaporator 6I 0 NTERFERENCES 6.1 There are no known interferences at this time. ZOSAMPLEHANDLING 7.1 Tpehrefroarbmbeidt.livers are received frozen, and must be kept frozen until the extraction is 8S 0CT ALIBARATNIONDANA D RDIZATION 8.1 Preparation of Internal Standards 8.1.1 Pperrefplauroeroaoncitnatneersnuallpshtoannidcaarcdiodftaopbperoaxdidmeadtteoleya1c2hplipvmer 1sHam,p1lHe,.2H,2H8.1.2 Weigh atleast 0.1 g of 1H,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 Toa 250mL volumetric flask, add 3 mLsofthe stock standard and bring to volume with Milli-Q water. Calculate the actual concentration of the standard. actual phone scid mg perfluoroctane- X Aml = 0.1L 250 mL. acwal concentatpopnm, 8.2 Prepare FC-95 Anion Standards 8.2.1 Prepare FC-95 standards for the standard curve. 8.2.2 Weigh approximately 100 mg of FC-95into a 100 mLvolumetric flask. Record the actual weight. 8.2.3 Bring up to volume with dilute acetonitrile. 8.2.4 Dilute the solution with dilute acetonitrile1:10for a solution of aapsporlouxtiimonatoeflyap1pr0o0x.ppm1.0 pDpimlu.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 5.0 ppm, 1.0 ppm and 500 ppb. 8.3 Prepare Beef Liver Homogenate to Use for Standards 8.3.1 Weigh 40g of Bovine liver into a 250 mL Nalgene bottlecontaining 200 mLs Milli-Q water. Grind to a homogenous solution. 8.3.2 A1 dmdL1amliLquootfsothfetshoelustoilounttioonai1n51m5 LmLcencternitfruigfuegteubteu.bePs.reBpaeresuaretottoaloref-eight suspend solution by shaking it between aliquots. 000418 000067 8.3.3 blank Spike seven of the 1 mL aliquots with the following amounts owforking standards in step 9.12 of the procedure. One 1 mL aliquot serves as the forking Standard oT (Approximate Conc.) Approximate fir concentration of FC-95 in liver 8.4 Calculate the actual value of the standards: of standard x concentration (in ppm) = final concentration (ppm) 171 mg liver / 1 ml homogenate ofFC -95 in liver *Average weight of bovine liver in solution as determined by weighing F1Cm-L95hiosmroegpeonrattedesaosfeq4u0ivmaglelnitvserofinF2C0-095mpLotoafsMsiilulmi-sQal.water. The amount of Spectrometer. 8.5 Calibration 8.5.1 Extract the spiked beef liver homogenate following 9.13 t0 9.23ofthis method. Use these standards to establish your curve on the mass 8.5.2 Alternatively, a standard curve may be generated using ratios ofresponses of the perfluorooctansulfonate anion and the internal standard anion versus concentration of the perfluorooctanesulfonate anion. 8.6 Storage Conditions for Standards 8.6.1 cNoevwersetdapnldaasrtdisc caernetprriefpuageretdubweisthunetialchthaenaalnyasliyss.isSotanntdhaerdmsasasresspteocrterdoimneter is performed. 8.7 Storage Conditions for Standards 8.7.1 Beef liver homogenates may be frozen after preparation. 9.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been 9.2 PaUcsekaadgiesdse`cwtitihngotshcearlpteilssauensd. cut off approximately 1 g of liver. 9.3 9.4 Weigh Record the the sample directly liver weight in into the a tared plastic grinding study note book. tube. 9.5 Puta label on the vial with the study number, weight, rabbit ID, date and analyst initials. 000419 000065 4 99..67 Add 2.5 mLs water. Grind the sample. Put the grinder probe in the sample and grind for about 2 9.8 mRiinnusteesth,enpirlobethoefsfaimnptolethies asahmopmloegweinteho2u.s5 smoLlustwiaotnewriutshinnog laarpgiepecth.unks. 9.9 TAaMkDeT-tEheP-gr2i2n,der apart and clean it with methanol after each sample. Follow 99..1110 PCiappetth1emsLamipnlteoaan1d5mvoLrtceexntfroirf1u5gesetcuboen.dsL.abelthecentrifuge tube with the identical irnefmoarimnaitnigonstaespst.h)e grinding tube. (See AMDT-M-4 Worksheet for documenting the 9.12 aSspidkeesctrhiebbeedefinli8v.e3r.homogenates with the appropriate amount of FC-95 standard 99..1143 ASpdidke1mthLesoafmptlheessaonddiubmeceafrlbiovneratheo/msoogdeinumatbeiscawribtohna1t0e0buufLfoefrainndtem1 amlLstaanmdmarodn.ium 9.15 Uacseitnatge.an analytical pipet, add 5 mL ethyl acetate. 99..1176 CPuatptthheemsainmptlhee sahnadkevrofrotrex2200mtion.30 seconds. 9.18 Coenntthreifcuegneuifforug2e0tt0o2255, minutes, until the layers are well separated. Set the power 9.19 RmeLmgorvaedu4amteLdsgolfastshepipteotp. oTrrgaannsifcerlatyheerltaobealftroesthhe15frmesLh tcuebnet.rifuge tube witha 9.20 Baplporwoxtihmeastealmypl3e0dtoow4n0 omnintuhteesa.nalytical evaporator to near dryness with nitrogen, 99.:2212 BVroirnigexth1e5rseemcaoinndisn.g sample up in 1 mL dilute acetonitrile with an analytical pipet. 9.23 Tthreanssafmeprlteheinstaomapflreestho cae3ntmriLfusgyertinugbee.orAtataacuthovaia0l.,2 uLambenlylthoentmuebsehorfivlitaerl,waintdh ftihleter 9.24 Cstaupdyanndumhboledrfaonrdanaanliymsails nbuymebleerc.trospray mass spectroscopy. 9.25 Complete AMDT-M-4 workshees and attach to page of sway notebook 0 VALIDATION 1100..12 QPuraecliistiyonCoanntdroAlc-cunrotacayp-plnioctabalpeplicable 10.3 Other Validation Parameters- not applicable 11.1 None, 1A 2.0 12T .1 WorT ksheet AA MDT-M-4C . HM ~~E ~ N ~~T ~ S 13.1 AMDT-EP-22 Routine Maintenanceof Ulira-Turrax T-25 R1ev4is0iRonEVISIONS ~~~ Number ReasonforChange 000000 Revision Dae 000420 000069 5 Worksheet AMDT-M-4 Sar NSuimmbper | appTroxC0s5ppm | approTxTLSppm | appCr5$5opxom. | InDiieoakrnduac TLE ee[ET [aw r r rr r an r r r r r r r r r rr r r r r r r r r r r tt r r t r rr rr r tr [TSomE e err phheeteeoinlpe--nheteSoda dpdalacea dont 1---- [oe Daelik [ r fi----s-- e] RR, E-- E ------| f-- r2 a AT Raat [Mr] [ee------ [ee] ---------- pee [oe [ome es [hee] pe 000421 000070 6 Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M- Adoption Date: - -45 Revision Number: 0 Revision Date: None --r-- e -------------------------------- Author: Dave Christenson/Cynthia Weber Approved By: roup Leader l/es Date 4 Ce Quality Assurance Z4 (f Dae Software: MS Word, 6.0 Affected Documents: M-4, Extraction of Fluorochemicals from Rabbit Livers 000422 000071 1 Loscope 1.1 fSlcuoipdse:fTorhifslumoertohcohedmiiscaflosr tuhseinagnatlhyesiesloefcterxotsrparcatysomfasrsabsbpietctlriovmeretoerr.otThehretainsasluyessiosr isstapnedrafrodrmMe/dZb=y 4s2i7ng,laenidonotmhoenritapoprrionpgroiaftFeCm-a9s5seasn.ion, M/Z= 499, the intemal 11..23 AMpaptrliicceasb:leRaCbobmitpoLiuvnedrss:(sFamlpuloerso)c,hBemeiecfaLlisvoerr o(tshtaenrdafrlduso)r,inoattheedrciosmspueosunadnsd. fluids, 20KEYWORDS 00000000 2.1 sFpleucotrroocmheetmeirc,alrasb,biftluloirveirnsa.ted compounds, electrospray mass spectroscopy, mass 30 PRECAUTIONS 3.1 iUnsteo tchaeutpiroonbewiDthOthNeOvoTltTaOgeUcCabHleTfHorEtPheRpOrBoEbe,.tWhehreenistrhieskvoofletlaegcetcraibclael isshpoclku.gged 3.2 sDtoonpoatndrurneltehaeseppurmespsuarbe,ovTehiet'specaapkatcuibtiynogfm4a0y00bepspi.luIgfgperde.ssTurroeubgloeesshoovoetr b4a0c0k0topsi 3.3 Dfionndottherpulnugthaenpdurmepplatcoedrtyhneespsl.ugged tubing. Sec AMDT-EP-15 4.0 SUPPLIES AND MATERIALS ~~~ 4.1 S4.u1p.p1liNeistrogen gas regulated to 140 psi. 44..11..23 F1l0u0ofuiLx ocrol2u5m0nuoLrfelqauitviaplseynrti.nge for sample injection. 4.2 Reagents 44..22..12 DMiillultie-Qacweattoenri,trialllewmaotbeirleuspehdasien,tdhiislumteetahceotdonsihtroiulled1b:1e wMiitlhliM-iQllwait-eQr.water. S0EQ5.U1 IPVMGETrNioT2000 Electrospray Mass Spectrometer or equivalent. 55.:32 SIpSeCctOraSpyhryisnigcesPAuSm3p00 Autosampler 55..45 A10u0 tuLoAvsosriecmaabpllpyesd centrifuge tubes 0000 6.0 INTERFERENCES 6.1 There are no known interferences at this time. 7.0 SAMPLE HANDLING 7.1 uKnetielprtehaedeyxtfroracatneadlyssaimsples in capped 15 mL centrifuge tbes or in capped autovials 000423 000072 2 8.1 8P.r1e.p1araSetxetivroeannctbioeofnefCprlaoilvcieerbdursrateat.nido(anSredSestaAanMndDdoTanr-edMsb-l4a,nksbeecetifolniv8e.r0)are prepared during the 8.2 Calibration 8.2.1 Run the seven beef liver standards twice, starting with the lowest standard 8.2.2 T1y0poibctaalilnytohenestsatnadnadradrdcuirsvre.un after each to 7 samples. Choose a standard in the same range of concentration as the samples. 8.3 Storage Conditions for Standards 8.3.1 cForveeshresdtaplnadsatridcscaernetrpirfeupgaerteudbewsituhnielacthheanaanlaylsyissi.s Sotnanthdearmdassasrespsetcotroemdeitner is performed. Samples and standards are NOT refrigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8.4.1 Beef liver homogenates may be frozen after preparation. 90PROCEDURE 9.1 Initial Set-up 991.11..21 RSeetcosrodftbwaarcekitnog"pOrpeesrsautree oinn"t,heIoinnsMtorudmeenEtS".log, 9.1.3 Fill the solvent cylinder with mobile phase. 9.1.4 `Sceotmtihnegopuutmopfttoh"eRtuinp"o.f Stehte tphreobfel.oTwhteo p1r0e0s0suurLe/msihno.ulOdbsbeeravtes1.d7r0o0pltoet1s800 9.1.5 Bheck the fused silica at the end of the probe. Use an eye piece to check for cchuitpso.ffTthhee ttiipposfhothueldsibliecaflwatitwhitahcnoolujmanggceudtteedrgaesn.d Ipfulalntyhechsiipliscaartehrfoouugnhd to the appropriate length 9.1.6 Cnihtercokgenyoluerakniintgroagreonunsudpptlhye.tiTpuofntohenptrhoebeni.tArogfeinn.e Tmhiestreshsohuoludlbdebeconmoing, 9.1.7 Coaurteoffultlhyegtuipi.de the probe into the opening. Insert it until it won't go any 9.1.8 fGarothetro.thCeon"nEdeicttort"hepavgoel,taagnedcasbetleTotnoitzhaetipornobMeo.de to ES, and the appropriate masses to 427 and 499. the log book. 9.1.9 If itis not in single ion mode go to "Option" and set SIR. 9.1.10Start Acquisition. Assigna file name, MO-DAY-YR + letter. Record it in 9.1. SE ee 11bReugnintnhiengbeoefftlhieverruns..a.mpRluensafiQrsCt, cruhnencikngbyearcuhnnsitnagndoanred sttwaincdeaartd tahfeter every 9.2 Manual Injection 9.2.1 Dinrjaecwti1on50pourtl. oIfnjseacmtpslleowilnyt.o aRescyroirndget.heInsjaemctplteheIsDaminplteheilntoog tbhoeork,heodyne 99..220.32 WTauirtn tthweovmailnvuetteos,"Oann"d.inject the next sample. 9.2.4 Record the scan numberfor each sample in the logbook. 000424 000073 9.3 9U.s3i.n1g StehteupAustaomsplaemprlaeyrA, B, or C. 9.3.2 Rvieaclosrmdatyhebesaimnpelaecsharnudn.their positions in the instrument log book. Up to 17 9.3.3 9S.e3t.u3p.t1he samplPeurs:h the sample bution 99..33..33..32 SSeettisnajmepclteslaomopplsei=z2e = 100 ul 99..33..33..45 SNeatmCeytchleeftilie:meLi=v0ers 99..33..33..67 AIddedntitfhye tshaemptlraeysutsoeQdueue by pressing "Enter" 9.3.3.8 Press "Run" tostart 10.0VALIDATION 10.1 Q1u0a.l1.itIyRuCnonatsrtoalndard every 5 to 7 samples. If a significant change(t 50%) in s`pteaankdahredigwhitllocbceurussesdt.opThteherreumn.aiOnnilngy stahempslaemsplweilslbbeeforreeantahleyzleasdt.acceptable 10.2 P1r0e.c2i.s1iSoeneaMnedthoAdccVualriadcaytion Report number AMDT-M-5.0.V1 1100..43 ORetfheerrtoVMaeltihdaotdiVoanliPdaatriaomneRteeprosrt Number AMDT-M-5.0.V1 110DATAANALYSIS 1111.:12 CPlaoltcutlhaetisotnasndard curve, using the mean of the two values obtained for each s1t1a.n2d.ar1dRead peak heights or areas for the samples from the printout. Use linear 11.2.2rCeaglrceuslsaitoentthoemdegteorfmiFnCe-t9h5e asnaimopnl,eocroontcheenrtrfaltuioornosc.hemical in the total rabbit liver: mg FC-95 anion in the totalrabbit liver = mggmFsCo-f9l5ivaenriuosnefdrfoomr astnda.lycsuirsve x Total mass of liver, gms 1111..43 PMraiknteaa crhersoulmtasttoagbrleamanfdoreentaecrhist aimnptlhee,swtiutdhy tbhoeokp.eaks labeled with the sample or ssttaunddyafrodldIeDr.. WSrtiatpleetahlel scthurdoymnatuomgbrearmosnttohgeeptrhienrtoauntd,innuimtbale,rdpaatgee,sa.nd puittin the 000425 000073 4 12A .0 TTA ~C ~~ HMENTS None 13.0REFERENCES ~~~ 0000 13.1 AMDT-EP-17 140REVISIONS ~~~ 0000 RNeuvimsbieorn Reafosrchoannge Due Revision 000426 000075 5 9.3 Quality Assurance Unit Statement 000427 000076 AachmentD GLP Study Quality Assurance Statement Study Tide: Single-dosTe-6D0e4r9mailn RAabbsobripttsion/Toxicity Study of Study Number: AMDT-020895.1 Nameof Auditor: Kari Rambo "`TThhiesfsitnuddiynhgasswebreeenreipnosrpteecdtoed tbhyetshteuQduyadliirteyctAosrsaunrdanmcaenaUgnietmeansti.ndicated in the following table. Inspection Dates Eom To Phase 101395 10/1995 Final Report DMaatneaIgnespmeecntiSonuRdepyorDtierdecttoor 101905 10195 QSAaU Audbitoor i Le4D5at-e95 000428 000077 9.4 Key Personnel Involved in the Study 000: 429 000078 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 000430 00007y 9.11 Data 000431 000050 9.11.1 Summary and raw data; ug F" in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 000432 000051 Summary of Combustion Data - Liver AMDT-020895.1, BWI 6329-130 As Referenced in Final Report section 6.0 DATA ANALYSIS Total pg Fluoride in Whole Liver Mean per Dose Group* i Control Group 5.0 mg/kg dose (T6049) (0.003 mg/kg)** 100 mg/kg dose (T6049) (0.06 mg/kg)** 500 mg/kg dose (T6049) (0.30 mg/kg) ug Std. Dev. 22.4 + 5.7 17.6 + 4.9 15.8 + 2.6 22.3 + 2.2 "Calculated as the mean of triplicate samples from each of three male and three female rabbits. **Test material is a 0.06% solution of T6049, actual dose in parenthesis. 000433 000082 RPTI30LXLS Fess AB pAcptmuFal. AvpeprmaFg-e ver Wlhiovelre TotwahloFl-ein re%wy i(nWlWve)r i(nWliWv)er bumed (grams) weight (grams) Liver bink-1 0835 0.1138 liver (ug) Dosage (mg/kg) LUivveerrsbpiniki.2 106% 01.218470 00..11430437 F5U3v4e0r5s.p1k2 99% 01427040 00.11124418 FF553344050.523 00320815 033 001132202 782 2768 0 FFssa333s566-21 00224192 0.1431 0220 01458 7489 1640 o FeF533345046-31 [01358 00.11237697 F5F35430440.432 00225444 034 00.11823s0 7028 2768 0 FF5s33335533.12 00220352 0208 00.1140891 7741 1593 0 LFi5v3e3r5b3i.k31 00128712 00..11138555 LUivveerrsbpkk12 91% 01.112101 00..11253570 UF5v3e3rssep-1k2 102% 01.04092 00..11344068 FFs533335586..32 00238230 0335 o0i1t3e7s0 813 27.27 o FFs533335500--21 00230%0 0267 0011117443 7886 1982 o FF5533430520-.13 00.119479 00.11154128 FF553344002223 0011537 0147 0o.t1e4d3s4 7873 11st 5 FF5533336644.-12 00228755 028 00.11104807 8410 2500 5 FFsa33a64s.3 0023537 00..11608855 FFss3335522..23 0022617 025 00.11441018 e161 1728 5 FF5s33334409.21 00.2820 0284 001.01130% 7760 1815 5 F5F35330304.913 0022830 00..11041347 FF5s13330990..32 o02s0s9 0200 00.11022010 6562 1320 5 FFesa334466-21 00.117627 0222 00.113640 7541 75 100 FF5533335406-13 00238248 00..1122803 FF553333550023 00127646 0261 00.11437775 7405 1787 100 FFss3a9e77.-12 0022031 0253 00.1132773 7488 1883 100 Fsa07:3 0230 01128 Page 000434 0000x873 RPTI30LXLS Fees AB pAcptuaml ApvpemraPg-e Iver Wlhiovelre TotwahloFl-ein % inlver inlver bumed weight ver Dosage F5s3a40I00D02 roviy (0W15W7) ots (WW) (0g.r1a4m1s8) (grams) ont 0123 Tess (ug) 1280 (mgikg) 100 F5533246000.3 ooietess 0011632508 FF5s3a300600-32 001s9t oes 0011035078 763s 1288 100 LLiivveerr bbiikk 21 o03as oolorr LUvveerrssppkk1z 9955%% 11.236889 0011103328 FsF3a234477:-21 Fs3047:3 003248s 020 001120271 8370 28 0195 01415 50 FFs5a30e65s.21 F5a3653 0022098 026 0011320112 6260 1788 015 o'1s8s 50 FsFas%a4ass-12 00226511 022 o01u43s8 8134 208 50 FF5a3s35s4-1 0043230s a0i1r32502 FFss3a0a5s4e32 0023148 033 0o1102921 7087 2088 50 FFss3a400a342 0odsz ome 001112ss 7062 AT 50 FsF5a3400003-1 00334007 0o1a4r0s7 FF553a000000.32 00320112 0203 0012157 7962 238 50 FsF258209000:-21 002:663 02:0 001130609 e751 013 5 FsF2s0a9519-.13 00211513 00112s1t8 FsFaasst1.2 00126083 0195 0011241409 8080 1571 100 IIvveerrssppkkedd 8994%% 01.909778 o0t1338t52 IIvveerrssppkkse 11TI9%% 5.a9d2s07 001122518 verso? 100% 4330 o'1s01 000435 Pag2e 0000853 9.11.2 Summary and raw data; analysisof liver extracts using electrospray mass spectrometry. 000436 000087 HWI 6329130 TsProwoantyoH:caoltaNruem:ber: MRaSiqsuared Value: RRoetsypoantse Factor Amount: oMnot,nod: In(sArBuBmAeSnEtsFe: STTiiPonIoldeNs-GDiAnosSReabDbairtmsa(lCAb3s6o)rption Daogv9erss o1 i2e7E.05 aNnDs T sFistonsoVnG 2000 lecrospray MS A Dail uidhacson 0-31-95 [=ws ] ov rou Tr loDmigekgd water | FFFsea3i4ss0s4 FFsswusoss Fes lGraomuaps: + Fae FFoaxse FFouxees oooumsor5as | Fsaeo FFose Fue 0o0umsan4g sum FFsweoo FFaeaess Faas Tor wCoun eEreeed Deetrer NNNDDO 1T1.10z70232 111 eNnD ztoimes i1 ND toms i sesss Tuansk i' wWoD Tvos7a ii mms 10289 ' vusns oGmosy 'i sNDs 11235179 ii Wo Vzs0 3 sNesDs troosw '1 aemms iirzee 1i NNDD rToesa ii Conacgeernto NnNOoD NooDste ND ooosseo7 NNDD ozs cootues Noloen no oNsOs oadcosr NNOo Tootmhears 776302.011095336 T7c1a6s4a 7501s eevaras 76.o00s8 sees a73s s T72sae 73083 eaao0n02 77727s zss raris FTooRsTsapmeoruineSr nNNoDD 0N0D0s ND S0o0a0rs unoo oote 00000087 0W0O10 no oN0io0 a0o0a0 NnOo * Rabbits F52995 (G2) and F522 (G3) were not analyzed = T{ahcetocroniscethntrraetsiuolntowfas&cmailsccuallactueldabtiyonusininagpptlhyeinsgtafnodramrudlacu8r.v4eiannMdetmhulotdipAlMyDiTng-Mt-hoA0re.sul1t37bmyg45o.fTihvoer&5was used in Shihsouctalbuet.atBiyonmrualthleir nhgatnh)e camloc.uTlahteedccoonncceennttrraattiioonnInntthheessttaannddaarrddccuurrvveebayr4o h5or,heelocroorr4ectarresurltth1sanabtihaeiyned. 000437 000056 es FE 24e3isA 4% 20 Feoas Ceplot Method DLCLIV Run date 05-09-SS1ea3am3rp5alero0r6D:L4CG6LL:CI5V2 Version: 11 Printed on 05-09-1395 AT 06:47:07 Straight Line Fit forced through Origin. -amon: 3 11 sen 7 AEH - om mn: A J 4pd / Pp , ~ AL oLhy 2 ~ =z 2. AKAHT EXTERNCAoLmpoSnTeAnNtDARD1 CALIBRATION ever amount AREA :i 5.3050000 2823432707 a3 11.52900000 1234503292 Y. sioeE + x + umencEer Amouhnetss = 1+ .2677.0a53-2045E-0x4 + AArmeoaunt ++ 50..0000n000ER+r0050 2 squared + 0.3355 000438 000057 Wapto ane Bete ME Adin s 2 One Sle wibedbich (scan 2636, 55353)ashied Guia muon nncnin oly os ou z Cons pags A3 hei Aro FSialep:l0o4-8C3A9RB5O0POL ABD IVLAEBR-BTAHSTERA~CTTShe MSoiDnaittsa SOyus.tem roo sr 290 . I ren azoe 23 g 83/84/1995 89:06 2 ag ae 376 353 saan |[ 592 - bors RE anKeZ 9 oy P 59686g 13BE3 pre ge wR wn JH Haigh: :< Z = El g fffree nds 8 iFiSal5m1pe8l:e a:dG0A39R5B0OPOLRADDILAFB--BIEArXSTEIRVA--CETTRSh-e MS10D/at1a56Syosc.tem Ce "opps Sopp p 83/04/1995 09:66 --Tey hers. T x +t 9% - TE 15308 Le q5g65 io , wFeae T | 8 \ Ise? 85 abe 560 4a 5b 7 oo i166 126% 4 g 33 FSi aimplsioen`Canmp1soiLoABDrnB-ABSeoEEnn~teTTSho NS Data Syston 63/04/1995 oe 09:06 -- 8S ee ro . 401g 136 3 [100; "ng oF We 4 382: eof TE e NN T 3 TT) 3 g a83g FiliceCs amassp oLILsBRAoEIDAiGTThEeI.FSeOpatnSaSuseEten SmI e306 5 1B0A0B3Hm9Hesowoe mShes oToOr EWaE Soone nreemy -H groasn3eas - oe eet 3 TR me 3a8 rT Re EE t g 23 FSiaSmlploeT:C aE4a3As5n TRBABBIOT ILVP APB-BEAOXSTEIAL -CITSh.o|Mh S1oD/aitaaese Syston e/04/19% 09:86 " 3g 100) w1d2, " F ET s FgSnT GE2e moeHme a2iagy burs: 2p 3942 hoo) EES? [rs A lscR255a Joa 3766 SWea 7] gp 34395 3deo 3cae sees dead I [3 * FEe ireT mamansctr -sper e LOBn E - Te NG"betts Steen Ta ese or TEave aecatain5 vung TUE ue EB Toe re ET re asa 5291 3Iz WBS ome 8 SLiJd ang Zdarn,hee 8oe S738 fe : wr qa a3Ph 1 EE un < g 22 FSilPEoiosCoaagsmnaoross-iLunAmDmBEOsaSEtnRi~CTsTShoo|1SnDIaStIa hSystobn 0/0N/19%5 69:06 - 8Sg roosn csageamt fo. vs [sf 5560 pvoen cea 2p5r HWi E |2 AY @ #90 6050 |6100|6150 |zee|6250 c3ea ' 35 od 1$ :: Efnlicearmausmonnbso.ipwpia-isLtAaB-rBoASaEmc- iTshe MS oe ata Systeene roo sa a ap fe er - ood 0 EEoz JU H\@ t 3 g 83/84/1995 10:53 ner aie) igeOWp r gL 3 - anges 1HE z Sa gir serHDeepNe3g3)\ p-o opege mSp5s8 mim| Ins" Te 5io or D1a7f6ED ide ie 38, Te