Document ymGvydXw441qRdaEnog14E2zn

AR 2260532 Final Biological Phase Report Metabolism of N-Ethy! Perflusrooctane Sulfonamido Ethanol (Net-Fos<: T-6316) in Cynomolgus Monkeys Following Administration oaf Single Capsule Dose COVANCE> C4143 Sponsor: 3M Chemicals St. Paul, Minnesota Study Title: MetaCboylniosmmoolfguNs-EMtohnyklePyesrfFlouolrlooowcitnagneAdSmuilnfiosntraamtiidoonEothfaanSoilng(lNeetC-aFpossuel;eTD-6o3s1e6) in Data Requirement: None Authors: Frederic W. Thalacker, PhD Deborah K. Lee. Study Completed on: June 2, 1999 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: Covance 6329-226 Page 1 of 77 04144 2 -_ CoCouvannccee6s32i9o-2a26s STATEMENT OF NO DATA CONFIDENTIALITY CLAIM MetaCbyolniosmmoolfguNs-EMtohnyklePyesrfFloulolrooowcitnagneAdSmuilnfiosntraamtiidoonEothfaanSoilng(lNeetC-aFpossuel;eTD-o63s1e6) in No claim of confidentiality is made for any information contained in this biological phase on the basisofits falling within the scopeof FIFRA 10(d)(A), (B), or (C). `oTthheesretdhaatnacaormeptlhieanprcoepweirttyhoFfI3FMRACSheecmtiicoanls10a. ndSuabsmsiuscshiaorncofcotnhfeisdeendtaitaal ifnorcoamlppluirapnocsees with FIFRA does not constitute a waiver of any `under any other statute or in any other country. right to confidentiality that may exist Company: 3M Chemicals Company Representative: Andrew Seacat, PhD Title: Study Director Department: Codasee I Sone Signature ( [a [ge Date 04145 _ 3 CoCouvnanccee 6329-226 COMPLIANCE STATEMENT MetaCboylniosmmoolfguNs-EMtohnyklePyesrfFlouolrlooowcitnagneAdSmuilnfiosntraamtiidoonoEtfhaanSoilng(lNeetC-aFpossuel;eTD-o63s1e6) in APlrlotaescpteicotnsoAfgtehnicsybPieosltoigciicdael PprhoagsreawmesreGocoodndLuacbtoerdatinorayccPorracdtaincceeSwtiatnhdatrhdesE(n4v0irCoFnmRental 160). Andrew Seacat, PhD Date Study Director 3M Chemicals, Study Submitted by Date Sponsor Date 04146 4 QUALITY ASSURANCE STATEMENT Covance 6329226 `This report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Environmental Protection Agency (EPA) Good Laboratory Practice Standards, 40 CFR 160. The following inspections were conducted and findings reported to the study director and study director management. Written status reports of ionpsepreacttiinognpsraonceddufirnedsi.ngs are issued to Covance management according to standard Tnspection Dates From To. 04/22/98 05/13/98 06/02/98 10/09/98 11/06/98 11/06/98 11/06/98 05/19/99 06/01/99 04/22/98 05/13/98 06/02/98 10/09/98 11/16/98 11/16/98 11/16/98 05/19/99 06/01/99 Phase Protocol Review Test Article Administration Protocol Amendment Review Protocol Amendment Review Data Review Report Review Data Review Report Review Report Review Date Reported to Study Director and Study Director Management 05/04/98 05/13/98 06/03/98 10/09/98 04/06/99 04/06/99 04/06/99 06/02/99 06/02/99 Reprentat4 ive, Quali) Lownie -- Assurance Unit Date 42/9 04147 -_ Metabolic Chemistry Frederic W. Thalacker, PhD Biological Phase Investigator Deborah K. Lee Study Coordinator Philip J. Teitelbaum, PhD Associate Director LeAnn Eastman Supervisor Radiochemical Analysis Kuntal Sinha Study Chemist s KEY PERSONNEL CoCouvamncees63s20o-2a26s Toxicology Operations Peter Kong Manager Cara Himrich Supervisor Metabolism In-Life Quality Assurance Unit Nancy M. Centanni Manager Laboratory Animal Medicine Donna J. Clemons, DVM, MS Diplomate, ACLAM Supervisor 04148 6 SIGNATURES Covance 6329-226 -- WT Doli Frederic W. Thalacker, PhD Biological Phase Investigator Metabolic Chemistry Covance Laboratories Inc. 22,1979 fate Philip# Teitelbaum, PhD. Associate Director Metabolic Chemistry Covance Laboratories Inc. Date #2 nas TV Sova AStnuddryeDwirSeecactaotr, PhD 3M Chemicals C/9/94 Date 04149 7 -_-- CoCovvamnecee6u32e0-n22s6 TABLE OF CONTENTS STATEMENT OF NO DATA CONFIDENTIALITY CLAIM Page 2 COMPLIANCE STATEMENT 3 QUALITY ASSURANCE STATEMENT 4 KEY PERSONNEL 5 SIGNATURES 6 ABSTRACT 9 TITLE 10 OBJECTIVE 10 REGULATORY COMPLIANCE 10 QUALITY ASSURANCE 10 EXPERIMENTAL DESIGN AND PROCEDURES 10 Study Design 10 Justification for Dosing Route and Dose level n Test Substance Ii `Test Animals and Housing 2 Animal Selection 2 Justification 12 Surgical Procedure 2 Bile Replacement 13 Dose Preparation and Analysis 13 Dosing Procedure Antemortem Observations 13 13 Physical Examinations 1 Body Weights 1" Clinical Pathology 14 Clinical Pathology Tests 14 Antemortem Sample Collection Termination 15 16 Sample Identification, Retention, and Disposition 17 Blood, Plasma, Serum, and Cellular Fraction Preparation and Storage 17 `Sample Transfer Disposition of Raw Data, Records, Samples, and the Final Report 17 18 RESULTS AND DISCUSSION 18 Quarantine and Acclimation 18 04150 8 _-- CoCovvamnecee6n32e0-n22s6 Clinical Pathology 19 Body Weights and Doses Administered 19 Antemortem Observations 19 CONCLUSIONS 20 TABLES 21 `Table 1. Individual Kilograms Body Weights of Cynomolgus Monkeys Reported in 21 Table 2. Individual Body Weightsofand Dose Weights Administered in One Capsule to Cynomolgus Monkeys (50 mg/kg) Table 3. 1% Dextrose in Lactated Ringers Solution and Bile Salt Replacement 2 Solution Infusion Times 23 APPENDIX A 2 Protocol CMS Deviations 2267241, Protocol Amendment No. 1, and Protocol 2 APPENDIX B 2 SAbubmrmevairaytioofnAs,ntaendmoCrlitneimcaOlbPsaetrhvoatlioognys,DaCtlainTiacballePsathology Codes and 2 04151 -_ 9 cCoovanncea63n29226 ABSTRACT Tissue, plasma, serum, bile, feces, and urine specimens were provided to obtain information on the metabolismof N-ethyl perfluorooctane sulfonamido ethanol (Net-Fose; T-6316) in cynomolgus monkeys following administration of a single oral dose. dSioxsteedeancccyonrodminoglgtousthmeafloellaonwdinfgemsatluedymodenskiegyn:s from Covance Research Products were NandeSeex GGrroouupp _AnAniimomfaallss __SSwttass 1 66FMeimallee mac 2 22MFemale CBoinneuDlutetd DDoosee Somgkg SOmgkg CoCollelcecttoionnssPPosstudoossee UrTiisnseueasndffreocmessferxomon0D-1a2yasn2d, 2152,-a2n4dh0ou.rs, then ddaaiylsy. through 28 daanydwesek,ly through 90 B48lohoodurpsr,ed7dosaey,sa,nadnadtw1e,e2k,l4y,6t,hr1o2u,gh249,036d,ayasn.d BiTliessfureosmat02-812daaysn.d 12:24 hours, aBnldooddaiplryedhoesoeu,gahnd28atda1y,s2., 4,6, 12, 24, 36 and 48 hours, days, and weekly through 38 days pSerpeapraarteed bblyocoedntsraimfpulgeatsiowneroefctohlelbelcotoedd.foSreprreupmarwaatsiopnorfeppalraedsmbay aalnldowsienrgumt.hePbllaosomdatwoas clot and centrifuging it to separate the cellular debris. All specimens were shipped to the `Sponsor Sponsor. for analysis and the resultsofthese analyses will be reported separately by the 04152 10 Covance 6329-226 TITLE Metabolismof N-Ethyl Cynomolgus Monkeys Perfluorooctane Sulfonamido Following Administrationof a Ethanol (Net-Fosc; T-6316) Single Capsule Dose in OBJECTIVE "fTehmealpeurcpyonsoeomofltghuiss smtoundkyewyassatnodacdomlilneicsttsearmaplpeesrfflourordoeotcetramnienatteistonsoufbasbtsaonrcpettioomna,le and distribution, metabolism,andexcretion. REGULATORY COMPLIANCE "PTrhoetebcitoiloongiAcgalenpchyas(eEoPftAh),isGostouddyLawbaosractoonrdyucPtraecdtiicneaSctcaonrddaarndcse, w4i0thCFthRe E1n6v0iarnondmbeyntal CacocvoarndcaencLeabwoirtahtoCroiveasnIcnec.,Pr3o3t0o1coKliCnsMmSan22B6o7ul2eAv1ardda,teMdadAipsrioln,21W,is1c9o9n8s,iann,diCnovance Standard Operating Procedures deviations are in Appendix A. (SOPs). The protocol, protocol amendment, and protocol 9AlCl FprRoc3,edduarteesdiOnctthoibsesrt3u0d,y a1r9e89i,n caonmdpalsiamnocdeifwiietdhotnheMAanricmhal18,We1l9f9a1r.e IAnctthReeogpuilnatiioonnosf, the Sponsor work. and the study director, the study did not unnecessarily duplicate any previous QUALITY ASSURANCE `CTohveapnrcoetoQcoula,listtyudAysscuornadunccte,Udnaitta,(aQnAdUb)ioilnoagicccaolrdpahnacsee wfiintahlCroepvoarntcweerSOePasu.ditAeldl bsytutdhye activities performed Department. at 3M will be the responsibilityofthe 3M Quality Assurance EXPERIMENTAL DESIGN AND PROCEDURES Study Design Aotnhiemrawlassweinrteacta.ssTighneegdrtooutpwdoegsirgonuaptsi,ono,nenuwmabsetrhoefbainliemdaulcst,csapnneucliamteendcgorloleucptiaonnd, athned target dose level were as follows: 94153 -_-- TT aNned wSesx GrGroowpw_ofofAAnivmianlass_SaStwatsus I 6GFeMmaale Ina } 2 F2emaMle CBanimeulDauited --_-- i cCoovnancee s6329226 DeeRoeutme,esy eannddlLeevveel CoClollceictoinonssPPoostddoosee OneOCraaplmle SOmgkg TUirsisnueeasnfdrfoemcessefxroomn0-D13ayasn,d 21,3-a2n4dho90u.rs, then daily through 28 deys, and weekly through 9B48l0odhooaduyr.psr,ed7odsae,y,anadndaw1e,e2k,l4y,6t,hr1o2u,gh254,036d,ayasn.d OneOCmapisule TBiilsesufersoam20-812daasn.d 12.24 hours and daly Somghs tBhlroooudghpr2d8osaey,sanda 1,2, 4,6, 12, 24,36 and 4S8hhoouunrs7,dauyss, nndd weekklly trhroouugh228ddays Justification for Dosing Route and Dose level "The oral route has been used for evaluating systemic toxicityofthe compound in cnoynn-otmooxilcgussinmgloenkdeoysse.amTohuent50nmegce/sksgardyofsoerweacsovcehromseeansubreacbaulseecoitnpcrenotvriadteisotnhsoefmtahxeitmeustm substance in minor tssucs. `Test Substance The May t8e,st1s9u9b8stfarnocme,thTe-6C3o1v6a,nwceasDerepcaeritvmeednitnoftheToDxeipcaorltomgye.ntoStfabMieltitayboalnidcrCehteenmtiisotnrofy on a sample as Sponsor. specified in Information 40 on CFR 160.105ofthe test substance is the responsibility ofthe synthesis methods, composition, or other characteristics that define the test substance are on file with the Sponsor. `The following information was obtained from the label on the test substance container: Substance No. TCeosnttaSiunbesrtaNnoc.e:: Lot Nos. Date Combined: Purity/Concentration: SEtxopirraagteiRoenqDuaitre:ements: Physical State: 1084A3 6T5-6316 (Narrow Range N-Ethyl 3Pe0r0f3l5u,o3r0o0o3c7t,anaensdul3f0o0na3m9ido Ethanol, NEtFOSE) 07-31-1997 98.1% ORnoofmiletweimtphertahteurSeponsor Amber waxy solid, slight odor C4154 2 -_-- Test Animals and Housing CCoovvamncee e63u2s92n26e `The in-lfe portion of the study was conducted May 13, 1998, through August 11, 1998. CEolevvaenncemaRleeseaanrdchelPervoednufctesmawleerceyrneocmeoilvegdusonmoMnakrecyhs24(,no1n99h8u.maTnhperainmaitmeasl)sfwreorme young awdausltastsoiagdnueldt aa ntdemwpeoirgahreydiadpenptriofxiicamtaitoenlynu3mbteor4.kgBeaftoarreribveali.ngUpploanceadrroinvatle,ste,accahcahnimal naencikmatlagwaasttracahneddomtolythaescsoilglnaer.d a permanent identification number and identified with a Aaclclainmiamtailosn awnedrehhoouusesdedinininidnidviivdiduuaallm,estuasbpoelnidsemd,castgaeisnldeesssisgtneeeld,fwoirrteh-emseespharcaatgieosn daunrding collection of bile, urine, and feces while on test ``TwhheenprfiamsatteedsfowresruerfgeedryCearntdifpierdiPotrorimtaetset sDuibestt#an8c7e26adCmi(nHiasrtlraantiToenkltahdr)ouagdh laipbpirtoumx,imeaxtceelpyt 4wahsouprrsovpiodsetddofsree.shDdiaeitlsywaenrdeasduplipblietmume.nteEdnvwiirtohnmaeppnrtoaplricaotnetrforlusitfsoarntdhecearneailmsa.l rWoaotmer were set to maintain 18 light/12-hour dark cycle. t0 29C, 50% The 12-hour + 20% relative dark cycle was humidity, and interrupted, as a 12-hour necessary, to `accommodate study procedures, Animal Selection uClsiendictaolscehleecmtistetsrtyaaninmdalhse.matInoladodgiytitoens,t raensiumlatslsanwderpehysseilceaclteedxafmorintahteisounrgriecsaulltpsrwoceerdeure badamsiendisotnratthieonb,esatlaapbpoarraetnotryadajnuismtamlenvettteoritnhaerijaanckeevtalaunadtetdetthehre shyesatletmh.ofBtehfeoarneidmoasles and approved their use on the study. Justification `aSntiumdailess.usNiongalcicveepatnaibmlaelnsoanr-elievseseanntiimaallfomrocdhealrsacwteerreizaivnagiltahbeldei.spTohseitcioynnoofmoclhgeumsicals in minotnhkeeeyvahlausatbieoennoufsteedstarsetshuletnsoonb-traoidneendtisnpteocxieiscotlooegvyasltuuadtieestocxoicnidtuyc.teTdhiisn smtoundkyewyilslaanidd `winlulmbaelrsoopfraonviimdaelas bwaassisthfeormtihneiemxturmapnoluamtbieonrofnecseasfsetayrydattoapfrrovoimdaensicmiaelntsiftiochalulmyavnasl.idThe results. Surgical Procedure TPrhoecesduurrgeisc.al TprhoeceadnuirmealwsawsecroenfdausctteeddovienrancicgohrtdbaenfcoerewistuhrgCeorvyaanncdesSetdaantdeadrdwiOtpherating 04155 5 --- coCouvannccee63e2n02y26s kmeatianmtianien.edAunsianpgporoxpyrgieanteaanndtiibsiooftliucrawnaes. aLdamcitnaitsteedreRdi.ngeSru'rsgsioclaultiaonneswtahsesaidamwianisstered tstharfofuvegthearisnaarpihaenn.oAus cstaetrhielteesrutrhgricoaulghsocurtubthweassurpgeircfaolrmperdo.cedTuhreecoroamsmdoentebrilmeindeudctbaynad. `dwueoredeandmuimniwsatserceadnnauslraetecdomamnedntdheedgablylbtlhaeddstearffwavesterreimnaorvieand.anAddddiitsicoanradleda.ntiAbniaoltgicessics ahnadd/aotr lfelausitds10wedraeysadtmoirneicsotveerredfraosmrtehceosmumregincdaeldprboycethdeursetapfrfivoertetroidnaorsieana.dmiTnhiestarnaitmioanl.s Bile Replacement LBaecgtiantneidngRitnhgeerd'asysaofltuetrisounrwgearsyatdhmrionuigshte3reddayvsiapotshte dsuurogdeernya,la csaonlnuutliaonfoorfa1p%prdoexxitmraotseelyin 8fohuortuhrspopsetr sduarygiactalapdparyoxunitmialtsealcyri2f0icem,La/hboiluersatlotpsrseovleunttiodnehwyadsraitnifouns.edBveigaitnhneindguoodnentahle csoalnuntuiloan fwoarsappprreopxairemdatbeylyad8dhionugrs13p.e0rgdoafycahtolaipcpraocxiidmtaote1lyLo2f3 0m.L/9k%gs/oddayi.umTchhelobriildeesalts wsoalsutmiionx.edSaonddiutmhebpicHarabdojnuastteed(1t.o3 7g.)4 w10as7.a8dwdietdht0o.eIaNchHC1 ILoorfssoolduituimonh.ydTrhoexisdoel,utaison `appropriate. Infusion intervals and volumes administered were recorded (See Table 3). Dose Preparation and Analysis `The capsules (gelatin, No. 2, Torpac, Inc. on April 9, 1998. 0.37 mL) for the dose preparation were received from Tbahleantecsetacnodmapnouapnpdrowparsiahtoemaomgeonuinzteodf.tesAt Nsoub.st2acnacpes,ublaesweadsotnaarneidmoanlawneiangahltytaincdaldose lsecvienlt,ilwlaatsioandvdiealdsulsaibnegleadswpiattuhlat.he Tahneimcaalpsnuulmebsewre.reThseeaclaepdsaunledspwlearceedsitnorseedpaurnadteer20-mL ambient conditions. Dosing Procedure `Thheouarnsimpaosltsdwoseer.e fTahsetecdaopvseurlneigwhatspgriivorento10dotshee aandimimnailsstbraytiaodnetvhircoeusgphecaipfpircofxoirmcaatpesluyle administration. Antemortem Observations Moobrstearlviattyioannfdormogreinbeuranldihteyalcthheacnkds awpepreeadraonnceetwweirceeddaoinley (oan.cme. daanidlyp..mS.i)g.nsCoafgpesoiodrehealth or abnormal behavior were recorded as they were observed. 04156 1 -_-- Physical Examinations CCoovuanncec6e329226 oPnhycseicpaolstesxuarmgienraytiboefnowreertehepeirniftoiratmieodnotfwitcreeadtumreinntg. aAcncliimmaaltsiowne,roencaenepsrteh-estuirzgeedryw,itahnd kexeatmaimnianteiohny,drochloride according to Covance Standard Operating Procedures for the `Jwaecrkeetasnewsetrheetcihzeedckaesdaabcocvoerdfiorngthteo eCxoavmainncateiSont;anhdoawredvOepre,raatlionwgePrrdoocesdeuorfekse.taTmhienaenwimaasls administered for the limited examination. Body Weights fBoorddyoswienigghctaslcwulearteiotnask),enweweeekkllyyafdtuerridnogsaicncgl,iamnatdioonn,twhietdhainyo5fsdaacyrsiofifced.osing (to be uscd Clinical Pathology "aTdhmeinainsitmraaltsiownearsefnooltlofwass:ted.appBrlooxoidmawtaelsyco1lwleecetkedbveifaortehesufregmeorryalfrvoeminalblefaonriemadlosseand parpoprbolxeimmsadteevleyl4opaendd, 8bldoaoydswpaosstcsoullregcetreydffroormclciannincaullaptaetdhoalnoimgaylst.estsWthoeanssheesaslthhealth status as deemed necessary by the staffveterinarian. Clinical Pathology Tests `The following are the tests performed by the Clinical Pathology Department: Hematology Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Platelet count White blood cell count Differential blood cell count Blood cell morphology Prothrombin time Activated partial thromboplastin time 04157 1s -_ Glucose Clinical Chemistry Urea nitrogen Creatinine Total protein Albumin Globulin Total bilirubin Serum bile acids Cholesterol Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Creatine kinase `Gamma glutamyltransferase: Calcium Inorganic phosphorous Sodium Potassium Chloride cCovoanmce 6m32e0-2n26 . Antemortem Sample Collection No Teflon was used for this study. Blood (Groups `animals predose 1 and 2). Approximately 2 mLof blood was collected and approximately 2 mL. was collected at 1,2, 4, 6, 12, from Group 24, 36, and | 4`w8ashocuorlsl,ec7teddayfsr,oamnGdrwoeuepk2lyantihmraolusghpr9e0dodsaeysanpodstadpopsreo.xiAmaptperloyxi2mamtLelwya2s mcoLlolefctbeldoaotd 1, c2o,l4l,ec6t,e1d2,vi2a4a,3f6e,moarnadl4v8eihnouirnst,o haenpdaroinni7,ze1d4,tu2b1e,saanndd2p8ladcaeydsipmomsetddoisaet.elTyhoenbwleotodicew.as 2Ef-fmecLtibvleooJdunseam3,pl1e99w8a(sSctouldlyecDtaedyi2n1)taoangdlaaststhteubreewmiatihnoiuntghteipmaeripnoiantnsd,saent aadsdiidteiaotnarloom temperature to clot. iBnitloe p(lGasrtoicupco2n)t.aiBnielres wsausrrcooulnldcectdebdyfdrroym itchee pGrredoouse2p(afonrimatallseavsita1t2hehoiumrps)l,anatnedd acta0n-n1u2l,a 12-24, and continuing at 24-hour intervals through 28 days postdose. 0U-r1i2n,e1(2G-2r4o,uapnd1).conUtriinnueewdaast 2co4l-lheocutredinitnerpvlaasltsitchcroonutgahin2e8rsdasyusr,rotuhnednewdebeykldyryunitcielat 90 days postdose. 9415 16 Covance 6320-226 Finetceersva(lsGrthoruopug1h).28Fedcaeyss,wethreencwoleleekcltyeduantti0l-9102,da12y-s24p,osatnddosceo.ntSipneuceidmfeonrs2w4-ehroeur transferred into plastic containers at the timeofcollection. Termination SpecnhteodbaurlbeidtaSla.crCiefriecber.osAptintahle tfliumiedof(sCaScFr)ifwiaces,caonlilemcatlesdwoenrceeatnheesatnheitmiazledwawsitfhulsloydium anesthetized. CSF was placed on wet ice immediately after collection and collection hteimpeasriwne(raet lreeacsotrd2e0d.mLB)laonodd winatso cgollalsescttuebdevsiwaictahroduitacheppuanrcitnur(eatilnetaostgl2a0ssmtLu)b,esexwcietpht Animal No. 10541 that had a short sample obtained via vena cava. Following blood collection, the animals were sacrificed via exsanguination under sodium pentobarbital. Residual urine from the bladder was added to the last urine sample. Stomach, large and small intestinal contents were collected from the two per sex Group | animals that were sacrificed at Day 2 only. All tissues collected were excised, rinsed with water, blotted dry, weighed, and placed in a plastic container surrounded by wet ice. After the tissues were removed, the residual carcasses sacrifice, were discarded. The following except the gallbladder that was tissues were collected collected from Group from all 1 only: animals at Adrenal glands Aorta Bone (femur) Bone marrow (from femur) Brain Diaphragm Epididymis Esophagus Eyes Fat Gallbladder Heart Kidneys Large intestine: Liver Lungs Lymph nodes (cervical) Lymph nodes (mesenteric) Muscle (thigh) Ovaries Pancreas Prostate. Salivary glands Seminal vesicles Skin (dorsal, shaved) Small intestine Spinal cord Spleen Stomach Testes Thymus Thyroid/Parathyroid Tongue Trachea Urinary bladder Uterus 04159 1" --- Sample Identification, Retention, and Disposition CoCovvaanmcee s63u2e92m2s6 Specimens were identified with the study specimen number, and collection interval. number, sex, animal number, group, matrix, Blood, Plasma, Serum, and Cellular Fraction Preparation and Storage tBhleoionddiivnigdluaaslsttiumbeespowiintths,htehpearbilnoowdaswastsogreendtaltyaipnpvreorxteidmasteevleyra5ltCimpersiotrothooamnoagleysniisz.e,At tthiemne appoipnrto.xiAmtatseaclryif7i0ce0,Lappwraoxsitmaakteenlyahnadlfpolafctehdeibntloooadlwabaeslehdovmioaglefnoirzeeadchaninddpilvaidcueadl intoa labeled approximately vial. The subsamples were placed in a freezer 20C. The remaining bloodwascentrifuged at at atemperature of 2,400 rpm (1,300 x g) for ``waapsprtorxainmsaftererleyd1to0 msiepnaurtaetselayt laapbperloexdimviaatleslaynSdCp.lacTehdeirnefsrueletzienrgaptlataesmmpaearnadtcuerleluolafr fraction approximately 20C until they were shipped. "sTehreuamddwiatsioanlallo2w-emdLtobclloootdatcorloleocmtitoenmipnergaltasusretufboersawpiptrhooxuitmahteeplayri3n0ftoor6t0hemcionlulteecst.ionThoef aspapmrpolxeimwaatselthyeSnCc.entTrhifeusgeedruatmw2,a4s0r0ermpomve(1d,3a0n0dxplga)cfeodriapnptarooxliambaetleedlyvia1l0. mTinhuetecesllautlar debris was discarded and the serum was approximately -20Cto await shipment. stored in a freezer at atemperature of Sample Transfer All specimens 1998: were shipped on dry ice to the Sponsor on June 12, 1998, and August 18, Dr. Kris Hansen Environmental Technology and Safety Services 935 Bush Avenue Building 2-E3-09 St Paul, Minnesota 55133-3331 Phone: (612) 778-6018 Fax: (612) 788-6176 `The Sponsor completionof will the be responsible study. for further analysis, reporting, and archiving upon 94160 18 _-- CcCoovvauncmee6e32u9e2m26e Disposition of Raw Data, Records, Samples, and the Final Report wTihlel ifnocllluodwei,ngbudtatwialrlencootrdbsetloibmietmeaditno:taipnroetdoaconldatnradnsafmeernrdedmetonttsh;e asrtcuhdiyvecsoorfreCspoovnadnecnece; test material receipt; final report. `sTthuedyfodlaltoawwiinlglsiunpcplourdet,inbgutrewciolrldnsottobbeelriemtiatiendedtoa:t Cfeoevdaanncaelybsuits nroetcoarrdcsh;iwvaetdewriatnhaltyhseis rreeccoorrddss;; ainnistmraulmrenotomcaltiebmrpaetriaotnuarnedamnadihnutmeindaintcyerreeccoorrddss;; rUenfirtigeedraSttoart/efsreDeezperarttemmepnetraotfure Agriculture animal records; stock records. r`Tehpeorftowlillolwiinncglduadte,a rbeuctowridlsltnoobtebetrlainmsifteerdretdo:to the archivesof 3M upon completion of the Tn-life records: Animal receipt Acclimation Animal room maintenance Antemortem observations Body weights Physical examination records Clinical pathology Clinical pathology slides Dose administration Sample collection Bile salts replacement solution infusion times and amounts rAelslulctoorfrtehsipsonsdteundcye,widlolcbuemeanrtcahtiivoend,inretchoerdsst,orpargoetofcaocli,liatinedsoffinaClovreapnocret gfeonrearapteerdioadsoaf one oyfetahrefoalfloorweimnegntthieonseidgnmiantgeroiftahlsewifilnlablerespeonrtt.toOtnhee Sypeaornsaoftrearnsdiganirnegtuthmeffeienawlilrlepboert, all fochraargneda.ddiTthieonSalpopnesroiordomafytiemleectantdo hCaovveantcheemwaitlelrciahlasrgreetaaisnteodraignetfheee.CoIvfatnhceeSpAorcnhsiovres chooses to have Covance disposeofthe materials, a disposal fe will be charged. RESULTS AND DISCUSSION Quarantine and Acclimation A1l0l54a2n8i,maaplpseuanrdeedrcqluinaircaanltliynheeaanltdhaycacnldimwaetiroenrbeelgeiasnendinognMAaprriclh2244,,11999988., eAxnciemptalAn1i0m5a42l8 `weaxsamoibnsaetirovne,dotnoehafevcealdieaxrarmhienaaatinodnw,aasndmotnhireteorteudbeurnctuillinnotremsatls.weArte lpeearsftoornmeedphbyesfiocrael 94161 1 CCoovuanncce 6e329226 arcelcelaismeatferdomtoqutahreajnatcikneet. anWdeetketlhyerbsoydsytewmeirgehqtusirweedrfeorrebciolreddeudctancdanannuilmaatilosnw.ere Clinical Pathology Rheeaslutlhtsaobfnorpmraelsiurtgiiecsa.l AcllitnhiocaulghpastehvoelroaglymtaelsetssiinndGicraotuepd n1ohoabdvniootuasblgyrohuipghorleiunkdoicvyidtueal cAoufnetws,ftehmeasleesweirneGtrroiubpu1tehdadtomeixlcdiltyehmiegnht vaaslsuoecsifaoterdalwaintihnbelaomoidnocotlrlaencstfieornapsreoaccetdiuvrietsy. ccoynnsoimstoelngtuwsimthonHkepeaytsittihsatAdionefsecntoitona,davesrusbecllyineifcfaelctcolnidveirtifounncctoiomnmoornalnyiombaslehrevaeldth.in `Rceosmuplltiscoafttihones csleicnoicnadlarpyatthoolbiolgeydtuecsttscfaonlnulloawtiinogn.surFgoeurry ddaeymsonpsotsrtastuerdgenroy,criintcircaelased a`cgtaivmimtaiegslufotraamsypatrrtaantsefearmaisneo,traanndsfcerreaatsien,eaKliannaisnee ainmiGnrootruapns2faenraismea,lsalwkearlienecopnhsoisspthenattawsiet,h tmhaelessur(giAcnailmaprlocNeod.ur1e0.543T6h)e; taontadlobnieloifrutbhinewfaesmanloetsab(lAyniinmcarleaNsoe.d 1f0or54o4n1l)yeoxnheiobifttehde eevnizdyemneceaoctfibvlitoioeds gleonsesraflrloymetxhheibsiutregdicsailgpnirfoicceadnutrree.duEcitgihotnsdaiynsdipcoasttivseourfgecroyn,ttihneuehdigh ArenciomvaelryNof.ro1m05t4h3e6s.urPgeircsailstpernotc,edmuirled,lyantod mtootdaelrbaitleilryubeilnewvaatsednoacltoivnigteireselfoervaatlekdalfionre ppohsotspshuartgaesreyw(emraleecshaarnadctfeermiasltiecs)ofamnidlgdlaymminacrgelausteadmybliiliraarnysfperreassseur(emasleecsonodnalryy) taot 8bildeays hdeupcattciacnfnuulnacttiioonn,wbaustlniokrelmyalnovtalsiugensiffiocratnottlaylcboimliprruobminisaendd.serum bile acids indicated Body Weights and Doses Administered `ITnadbilveisdu1aalnbdod2.y AwcetiugahltsdaosnedsdaodsmeisniasdtmeirneidstrearnegdetdofGrroomup1s00.1 2an%d t2oa1re04p.r4es%oenfttehdein nominal doseof 50 mg/kg. Antemortem Observations e`Txhheibaintiemdanlso aopvepretarseidgnnsoorfmtaolxidcuirtiyntghdroosueghaodumtintihsetrstautdiyo.n aTnhdeanpopteeadreodbsheeravlatthiyonasndare listed in Appendix B. Noneofthese observations appeared to be test substance related. `OwnasMfaluysh2e2d, f1o9r98A,ninmoablil1e0f5l4o3w7waansdntohteebdilfeorstAanritemdaltosf[l0o5w43ag7aiann.d 1T0h5e44t1e.chnTihceiasnwifvoeulnd t`whaast cthoennsewcitveedl cfoorrrAenctilmyaaln1d0t5h4e41pwuamsphroesotkaretdedu.p Ainpcporrorxecitmlayteolnyth2ehporuervsiolautserd,atyh,erseo witas no bile flow, so the system was rechecked and bile began to flow. On May 26, 1998 and 04162 2 --- cCoovvanmcee 6e3a29d-2a26s mMoanyit2o9r,h1e9r98h,eablltohodstawtauss. dTrhaewnrefsruoltmsAonniMmaaly12095,4419198f,orweclrieniicnaldicchaetmiviesotfrycahnolaelsytsaissist,o but atlhseoreisnudlitcsatweedrtehaitmpthreovheepdatfircomdytshfeurnecstuilotns aopnpeMaaryed26m,in19i9m8a.l Tanhde cColinncilcuadlePdatvheorlyogliitslte hepatocellular degeneration or necrosis occurred. On June from the 4f,ec1e9s9.8,TAhneifmeacles1w0a54s4s9crdiadpendotfrhoamvethaescpraeneannadbcoovleletchteedp;ahnotwoevseepra,rastoemtehe urine cross-contamination may have occurred. Bile from flow slowed and Animal 105446. was noted However, on Study Day 17, 23, the animal exhibited naondsi2g4nstohfencostmopprpoemdiosnedDaheyal2t5h. Upon sacrifice it was noted that a providing an alternate pathofbile portionof flow. the bile duct had re-grown, evidently CONCLUSIONS StiasmspueledsiswtreirbeutpiroonvoifdeNd-ettohyolbtpaeirnfilnuofrooromcattainoen sounltfhoenapmhiardmoaectohkainnoelti(cNse,t-eFxocsree;tiTo-n6a3n1d6) aanfiemaarlssiwnegrlee cuasseudleindothsee sitnucdyy.noAmlollsgaumsplmeosnkweeyrse.shBiiplpee-ddutcot tchaennSuploantseodrafsowrealnlalayssiinstaacntd resultsofthese analyses will be reported effects were noted on the monkeys. separately by the Sponsor. No test substance 04163 - a TABLES cCoovnancce e632n9:226 Table 1. Individual Body Weightsof Cynomolgus Monkeys Reported inKilograms N1 u`NAmunmibbmeaelrr"F 1 3 5 9 9 Da1 ym mH 2 6 6 5 515H 5 105428 44 42 Gro1Mualpe 105430 39 3.9 105431 38 38 38 39 39 105432 42 42 42 42 42 105433 105435 4.1 40 40 4.0 39 41 4.4 43 43 43 44 44 43 43 105439 3.1 3.1 105440 31 3.1 Group 1 Femal 105442 3.0 29 28 3.0 29 105444 3.0 29 29 29 2.8 105447 32 31 32 33 33 105449 33 33 34 33 32 35 3.6 33 33 Gro2uMaple 105436 37 35 35 36 38 106437 37 37 36 38 36 Group2 Female: 105441 3.0 3.0 28 32 32 105446 42 4.1 3.8 42 4.1 2 E % % mm wm or Day 42 49 56 63 70 77 84 91 Group1 Male 105433 105435 45 46 46 47 48 4.1 42 42 42 42 48 4.9 49 42 43 43 Group 1 Female _os4o 105447 105449 3435 34 3435 34 3435 34 3435 34 3s36 35 35 3.6 35 3737 3.7 3136 37 04164 -_ Table 2. 2 CoCvovmacncees6332a9-2226 Individual Body Weightsofand Dose Weights Administered in One Capsule to Cynomolgus Monkeys (50 mg/kg) "Animal Number 105428 105430 105431 105432 105433 105435 105436 105437 105439 105440 105441 105442 105444. 105446 105447 J1s 05449 "Animal Group _ Weight (kg) T aq 1 39 1 38 1 42 1 a1 1 40 2 37 2 37 1 31 1 31 2 30 1 30 1 30 2 42 1 32 1 1 3333 Test Substance Administered @ (mg/kg) %ofNominal 02207 502 1003 0.1978 50.7 101.4 0.1930 508 101.6 02138 509 101.8 02082 508 101.6 0.2033 508 101.7 0.1884 509 101.8 0.1897 513 102.5 0.1573 507 101.5 0.1556 502 100.4 0.1522 507 1015 0.1508 503 100.5 0.1566 522 104.4 02108 502 100.4 0.1616 505 101.0 001.6165583 s5o0.r1 10100022 04165 --_-- Table 3. 23 CoCovvaamnccee6s32399-22266 1% Dextrose in Lactated Ringers Solution and Bile Salt Replacement Solution Infusion Times a Tne o es aaa e Dy TAM PM AM we PM AM PM AM AT rPeosutSwuggeeryy"SSanm FiFismshh SSunn FFsishh SSawin FFnshh SSaani FFhm 21 TSOT83I9SS 6s2s6o ISS 32 600 626 401 42 626 6s 15:03 14:15 43 661256 1I43s1 6612s 1400 0 61s 62 17 Hol 62 735 sol isu 56 o73n5 s1o5r2 7635 I14s3e6 76338s 1M53062 66337 1l3w6o 7 3 638 637 1440 1440 637 63 M0 128 63 62 lad) 42 623 700 1424 les 109 7620150#328206204 WS 2 700 62 WWs266324 ian as 112 662230 wiis e6ax is 47 623 ea 1s 449 Gaz ess jews 1538 1"1 is 660525 i14s4a5 s6asl 1538 1321 ess $21 1s 1323 sal 6s 1323 14:58 s21 B22 es 1438 6s) 48 616 1425 1167 651164M2S8 6S1466 11432306 6su1e6 11432556 6s4dss l13a8s6 1189 Si6d468 1W3S3L6 663498 14:51 aa 648 639 SL 1440 639 638 law 14:46 EF 2 639 638 ld M46 638 636 1446 M38 638 636 leds 1437 636 653 1436 so z 5 636 653 1436 10 653 617 1500 ws 653 617 4152000 s6s107 aBu4r 2 2 G78 5:50 347 SSO sa Bw 48 sso S80 1w3r48 ssaas a13:d48 2 2 S43 Sd6 1348 138 S46 6d BT 546 S364 11 1S) 649 639 14:53 14a 23 663499 14Sa3 6es3s9 led 57 639 65s lad 457 6s 636 sy laa 3301 665356 1M43557 663396 13s M3 636 63 W3s us 639 70 ul 14:59 2 3 76309 215M0301 2 Gus 1500 1946 202 Gus 144s46 6eaas 1144:3496 3 33s6 645 641 ade al 1440 641 140 444 61 Gul 1439 eal a4 Gus 444 1405 641 444 us 105 64s wos 101 14:38 37 338 645 701 os 8 zor sa ss 700 340 sa 1458 NAY 1340 NA Na NA sal 1340636 1447 636 ur NA NA aWo --_-- a2 663386 1e4d4s7 66s3t8 1448 "3s 638 6s 14:45 1435 NA NA 651 1435 NA NA es a OainpnfpurDsoaexydismwaiIttehtlhbyriol2eu0gshmalL3t/,sktaghte/aahpnopiurmrofaxolirsmaapwtpeerrleoyxi2in3mfaumtsLeedklywgi8hthoh1ouur%rfs,odreaaxnptdprrooosnexitihmneaLtraeeclmtyaaitnedihnoRugirnsdg.aeyrs'sthseoalnuitimoanlsatw. ere. b Not applicable. Since no bile was recovered, no bile salts were infused. 04166 2 -_-- CoCvoavnanccees6332992-2226 APPENDIX A Protocol CMS 22672A1, Protocol Amendment No. 1, and Protocol Deviations 04167 2 -_-- CCoouvamnceeo63n2p0-a226 PROTOCOL Sponsor 3M Chemicals St. Paul, Minnesota Protocol CMS 2267241 Study Title MetaCbyonliosmmologfuNs-EMtohnyklePyesrfFloulolroowocitnagnAedSmuilnfiostnraamtiidoonoEfthaanSoilng(lNeetC-aFpossuel;eTD-6o3s1e6) in Testing Guideline Environmental Protection Agency, Good Laboratory Practice Standards, 40 CFR 160 Date April 21, 1998 Performing Laboratory Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification Covance 6329-226 Page 10f 13 04168 2 _-- cCoovancme 63e29-2s26 STUDY IDENTIFICATION Metabolism ofN-Ethyl Perfluorooctane Sulfonamido Ethanol (Net-Fose; T-6316) in Cynomolgus Monkeys Following Administration ofa Single Capsule Dose Test Substance NE-tEhatnhoyll (PNeertf-lFuoosreo;ocTt-a6n3e1S6u)lfonamido Sponsor 3M Chemicals 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 Study Director Principal Analytical Investigator Andrew Seacat, PhD 3M Chemicals 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 Phone: (612) 575-3161 Fax: (612) 733-1773 Kris Hansen EnSveirrvoincmeesntal Technology andSafety 935 Bush Avenue Building 2-E3-09 St. Paul, Minnesota 55133-3331 Phone: (612) 778-6018 Fax: (612) 788-6176 Biological Phase Investigator CFroevdaenricce WL.aboTrhaatloarcikeesr,InPch. D P.O. Box 7545 Madison, Wisconsin 53707 Phone: (608) 242-2712 ext. 7370 Fax: (608) 241-7412 Biological Phase Location Proposed Study Timetable In-Life Experimental Start Date In-Life End Date Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 May 13,1998 August 11,1998 04169 2 Covance 6329-226 OBJECTIVE `fTehmealpeurcpyonsoeomfotlhgiuss smtoundkyeiyssadamnidnicsotlelrecat Psearmfpllueosrofoocrtdaenteetremsitnsautbisotnaonfceabtsoomrpatlieona,nd distribution, metabolism, and excretion. REGULATORY COMPLIANCE Environmental Protection Agency, Good Laboratory Practice Standards, 40 CFR 160 ARlelguplraotcieodnusr,e9sCinFtRhis3,pdraotteodcoOlcatroebeinrc3o0,mp1l9i8a9n,caenwdiatshmtohdeiAfniiemdaolnWMealrfacrhe 1A8c,t1991. In the opinion of the Sponsor and the study director, the study does not unnecessarily duplicate any previous work. `The protocol, study conduct, data, and biological phase final report will be audited by the Covance Laboratories Inc. Quality Assurance Unit in accordance with Covance Standard Operating Procedures. All study activities performed at 3M will be the responsibility of the 3M Quality Assurance Department. TEST SUBSTANCE Source The test substance will be provided by the Sponsor. The Sponsor will be responsible for analysisof the test substance. Storage Conditions The test substance will be stored at approximately -20C until dosing. Any test substance remaining will be stored at approximately -20C. Disposition Unused test substance will be retuned to the Sponsor or a recipient designated by the Sfopronrseotern,tioorndofisacarsdaemdplateotfhtehSepotnesstomrasterreqiuaelsta.s sTpheceiSfpioendsionr4w0iCllFRass16u0m.e19re5spiofntshiebisltiutdyy is longer than 4 weeks in duration. 04170 28 Safety Precautions Covance 6320-226 cSoanfseitdyeprraetciaountoifontshewSiplonbseotra'kseMnaatserrieaqluiSraefdetbyyDCaotvaaSnhceeetP,oloircoiteshearnrdelPervoacnetdusrafeest,yin information provided by the Sponsor. EXPERIMENTAL DESIGN Animals Species/Strain. Nonhuman primate/eynomolgus Source. Covance Research Products (Alice, Texas, or Denver, Pennsylvania). The sourceof the animals will be documented in the raw data. Weight at Arrival. 3 kg or greater AgeatArrival. Young adulvadult Number and Sex. Identification. Eight males and eight females on test, six male and six female intact and two male and two female bile duct cannulated. One emxatlrea acnandnounlaetfedemwaillel ebxetraavianitalcatbaalnsedptowsosimbalelereapnldactewmoenftemaanliemals. Individually numbered collar tags Housing Fthoerttehset paecrciloidm,attihoenapneirmiaolds, wtihlelabneimianlisndwiivlildbuealinmeitndaibvoildiuaslm,csatgaeinsldesessisgteneeldcfaogrest.heFor separation and collection of bile, urine, and feces. Food Csuerrgteirfyi.ed FPoriomdatwiellDiaeltso#8be72w6iCth(dHraarwlnanovTeerknliagdh)tapdrliiobrittou,ma,nedxcreeptturwnehdenapfparsotxeidmfaotrely 4 hours after dose administration. cereals. Diets will be supplemented with appropriate fruits and Water Ad libitum, provided fresh daily 04171 2 Covance 6320-226 Contaminants `Thereare no known contaminants in the food or ater that would interfere with this study. Environment 2En0v%irroenlmateinvteahlumcoindtirtoyl,safnodr tahe12a-nhiomuarllrioghotm/1w2i-lhloubredsaertktocymcalien.taTihne1182-thoo2ur9dCa,rk5c0y%cl+e may be interrupted to accommodate study procedures. Quarantine and Acclimation `phAynsiimcaalls,woinlel bfeecualndeexramqiunaartainotni,neanfodrtahtreleeatsutb3e0rcdualyisn.teAsttslweialslt boenedoqunaelibteyfocroentrreollease from quarantine. Weekly body weights will be taken. jackettether system required for bile duct cannulation. Animals will be acclimated to the. Animal Selection uCsliendictaolscehleecmtistetsrtyaannimdahlse.maAtnoilmogaylstewsitllrebsueltsselaencdtepdhfyosrictahle esxuragmiicnaaltpirooncreedsuurletsbwaisleldboen the best apparent adjustment to the jacket and tether system. In the case that all or most animals adjust well, animals will be selected in numerical order based on the individual cvoeltlearrintaargiannumwiblelresvaplrueavtieoutshleyhaesaslitghnoefd.theBeafnoirmealdsosaenaddamipnpirsotvreattihoeni,r ausleaboonratthoersytaundyi.mal Justification aSntiumdailess.usNiongalcicveepatnaiblmealnsona-rleievseseanntiimaallfomrocdhealrsacatreeriavzaiinlgabtlhee.diTshpoesictyinoonmooflcghuesmimcoanlkseiyn hevaaslbueateinounosfetdeassttrheesunlotns-orbotdaeinntedspienciteosxitcooelvoaglyuasttuedtioexsicciotny.duTchtiesd sitnumdoynwkiellysaiadnidn wtihlel `anlsuombperroovfiadneiambaalssisisfotrhethmeienxitrmaupomlantuimobnoefrsanfeceetsysadraytatofrpormovaindiemsaclisenttoifhiucmaallnys.valTidhe results. Surgical Procedure ``TOhpeersautrignigcaPlropcreodcuerdeus.re Twihlel baneicmoanldsuwcitleldbienfaacsctoedrdoavnecreniwgihtthbCeofovraencsuerSgetrayndaanrdd sedated b`weitmhakienttaamiinneed. usAinngaoppxryogperniaatnedanitsiobfiloutriacnew.illLabcetaatdemdinRiisntgeerre'ds. soSluurtgiiocnalwainllesbtehesiawill administered through a saphenous catheter throughout the surgical procedure or as 04172 30 BE Covance 6320-22)6 `dcetoemrmmoinnebdilbeyduacsttaafnfvdetdeuroidnaerniuamn.wiAllsbteericlaensnuurlgaitceadl asncdrutbhewiglallbleblpaedrdfeorrrmeedm.ovTedheand discarded. Additional Analgesics antibiotics will be administered and/or fluids may be as recommended by the staffveterinarian. administered as recommended by the staff veterinarian. The procedure prior to animals will have at dose administration. least 10 days to recover from the surgical Bile Replacement LBaecgtiantneidngRitnhgeerd'asysaofltuetrisounrwgielrlybtehraodumginhi3stdearyesdpvoisattshuergdeuroyd,enaaslolcuatninounloaf1fo%r dextrose in aBpepgrionxniimnagtoelnyth8ehfoouurrsthpeprosdtaysu(ragipcparloxdiamyatunetliyl 2s0acrmiLf/icheo,uarn)atpopprroepvreinattedeahmyodurnattioofn.mixed dbaiyl.esal`tTshesovloultuimoneowfiblillbeesianlftsusseodluvtiiaonthteodbueodademnianlicsatnenreudlawiflolr abepparpopxriomxaitmealtyel8yhours per 23 mL/kg/day. 0f 0.9% sodium Bile salts chloride solution solution. will be prepared by adding 18.0 gof Sodium bicarbonate (1.3 g) will be cholic acid to added to each 1I L L oofrssoloudtiiuonm.hyTdhroexisdoleu,taisonapwpirllopbreiamtei.xeIdnfaunsdiotnheinptHervaadljsusatneddvtool7u.m4etso a7d.8miwniitshte0r.e1dNwiHlClIbe recorded. Group Designations and Dose Level TDT exrquy, GGrrowp Number _a_nadndSSexx SuSamwss anadnRdloLueetveve,ell CoClollleetcitioonnssPossidossee To 6Male 6 Female mat One Capsule SOmgkg UTirsisnueeasnfdrofmecess efxroom0n-D1a2ysa2n,d 2182,:a2n4dho90u.rs, t9h0endadysa.ly through 28 days, and weekly through B48lohoodurpsr,ed7dosaey,s,anadndatw1e,e2k,l4y,6t,hr1o2u,g2h45,036d,ayasn.d 2 2Male 2Female CBoinlneuDluacetd ~~ OSnOeCmagpksugle TBiilsesufersoamt02-812daayns.d 12:24 hour, and daily tBhlroooudghpr2e8dodsaey,s.anda 1,2, 4,6, 12,24,36and --_-- d48hhoouusr7sddauyss,aanndweekkllyttrhorouugghh228 daayys.. sJuyssttiefmiiccattiooxnicfitoyr DofosthiengcoRmopuoteu/nDdoisnecLyenvoeml.olTghues omroanlkeryoust.e hTahsebe5e0nmugs/ekdgfdoorseevawlausating crehcoosveenrbmeecaasuusreabitlpercoovnicdeenstrtahteimonasxiinmmuimnonront-itssouxeisc. single dose amount necessary to 04173 3 -_-- Dosing Procedure CCoovancue 63n29-s226 PadrmeidnoissteraatnidonPotshrtoduogsheaFpeperdoixnigm.atAenliym4alhsouwrisllpobsetdfaosstee.d overnightpriorto dose Dose Administration. administration. The capsule will be administered by a device specific for capsule Observation of Animals (Aanmt.eamnodrtpemm).ObsCeargveastiidoenso.bseMrovrattailointyfoarndgemnoerrailbuhnedailtthy acnhdecakpspewialrlanbceedwoinle btweidcoendeaily once daily. observed. Signs of poor health or abnormal behavior will be recorded as they are PsuhrygseircyalbeEfxoarmeitnhaetiinointisa.tioTnwofitcreedautrmienngt.accAlniimmaatilosn,wiolnlcbeeparnee-sstuhregteirzye,dawnidthonkceetapmoisnte hydrochloride according to Covance Standard Operating Procedures for the examination. Jackets will replaced as nbeececshsaercyk.edTahcecoarndiimnaglstowCilolvbaencaeneSsttahnetdiazreddOapseraabtoivnegfPorrotcheedeuxraemsiannatdiwoinl;l be however, a lower doseofketamine may be administered for the limited examination. oBnodthyeWdeaiygohftss.acrWiefiecke.lyAddduirtiinognaaclcbliomdaytiwoeni,gohntsthmeadyabyoeftadkoesninagt,thweeedikslcyretthieorneoaffttehre,.and Biological Phase Investigator ora staff veterinarian. Clinical Pathology. The animals will not femoral vein before dose administration as be fasted. follows: Blood will be approximately collectedviathe 1 week before surgery Iffrhoemalatllh apnriomballesmasnddevaeplporpo, xbilmoaotdewli4yllabnedc8oldlaeycstepdosftorsculrigneircaylfpraotmhoclaongnyultaetstesdtaonaismsaelsss. ahfetaelrthmosdtiaftiuscaatsiodneoefmtehdenpeocretsssaforrycboylltehcetsitoanfofvfebtielreinwailrliabne. cAolnlyecbteldoobdefsoarmeptlhees taken administrationofthe bile salt replacement solution for the day. 04174 2 Covance 6320-226 Tests Clinical Chemistry Glucose Urea nitrogen Creatinine: Total protein Albumin Globulin Total bilirubin Serum bile acids Cholesterol Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Creatine kinase Gamma glutamyltransferase Calcium Inorganic phosphorous Sodium Potassium Chloride Hematology Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Platelet count White blood cell count Differential blood cell count Blood cell morphology Prothrombin time Activated partial thromboplastin time Antemortem Sample Collection NO TEFLON IS TO BE USED FOR THIS STUDY. Banliomoadls(Gprreoduopsse 1anadndap2p)r.oxAipmpartoexliym2atmelLyw2ilml Lboefcobllloeodctwaeildtl 1b,e2,co4l,l6e,ct1e2d,f2r4o,m36G,roanudp418 bhoeucrosl,l7ecdtaeydsf,raonmdGwreoeukply2tahnriomuaglhs9p0reddaoyssepaonsdtdaopsper.oxAipmpartoexliym2atmeLlyw2ilml Lbeocfobllleocotdedwialtl `1w,il2l,b4e,6c,ol1l2ec,t2e4d,v3i6a,aanfdem4o8rahlouvresi,nainndtoohnep7a,r1i4n,iz2e1d, taunbdes28anddaypslapcoesdtdiosmem.ediTahteelbylooondwet ice. The animals may be subcutaneously orviathe re-hydrated with lactated Ringer's solution given duodenal cannula at the discretionof the Biological Phase bInlvoeosdtivgoaltourmeosr cLoalbloercatteodryexAcneiemdatlheVerteecroimnmaerinadnatfioollnoswiinngCoblvoaondcecoSllteacntdiaornsd,Oipfetrhaetitontgal Procedures. Bcialnenu(lGarionutopp2l)a.stBiiclceonwtilalibneercsolsluercrtoeudndferdobmythderyGricoeuppr2edaonsiema(flosrvaitaltehaseti1m2plhaonutres)d, and at0-12, 12-24, and continuing at 24-hour intervalsthrough 28 days postdose. 04175 3 Covance 6329-226 Urine (Group 1). Urine will be collected in plastic containers surrounded by dry ice at 0-12, 12-24, and continuing at 24-hour intervals through 28 days, then weekly until 90 days postdose. Feces (Group 1). Feces will be collected at 0-12, 12-24, and continuing at 24 hour intervalsthrough 28 days, then weekly until 90 days postdose. transferred into plastic containers at the timeof collection. Samples will be Termination pUonsstcmhoerdtuelmeedxSaamcirniaftiicoesn afnordaDneyatgrhoss.s Tchhaengceasr.casFsuersthweirllebxeamrientaatinieodnamnadymbaeypuenrdfeorrgmoed upon Sponsor's request. Scheduled Sacrifice. At the timeofsacrifice, animals will be anesthetized with sodium pentobarbital. Cerebrospinal fluid (CSF) will be collected once the animal is fully anesthetized. times will be CSF will recorded. be placed Blood (at on wet ice immediately after collection and collection least 20 mL) will be collected via cardiac puncture into heparinized tubes. Following blood collection, the animals will be sacrificed via exsanguination under sodium pentobarbital. Residual urine from the bladder will be added to the last urine sample. Stomach, large and small intestinal contents will be collected from the two per sex Group 1 animals that are sacrificed at Day 2 only. Ainla tpilsassuteiscccoolnlteacitneedrwsiulrlrboeunedxecidsebdy,icrei.nsTedhewiftohlwlaotweirn,gbtlioststueeds dwriyl, wbeeicgohlelde,ctaenddfrploamcaeldl `animals at sacrifice, except the gallbladder will be collected from Group 1 only: 04176 -_ Adrenal glands (~0.5 g) Aorta (~0.5 g) Bone (femur) Bone Marrow (from femur) Brain Diaphragm Epididymis Esophagus Eyes Fat Gallbladder (~0.5 g) Heart Kidneys Large Intestine Liver Lungs Lymph nodes (cervical) Lymph nodes (mesenteric) 34 CCoovvanmcee6e32m0-a22s6 Muscle (thigh) Ovaries (~0.5 g) Pancreas Prostate Salivary Glands Seminal vesicles Skin (dorsal, shaved) Small Intestine Spinal Cord Spleen Stomach Testes Thymus Thyroid/Parathyroid (~0.75 g) Tongue Trachea Urinary Bladder Uterus Sample Identification, Retention and Disposition Specimens will be specimen number, identified with and collection the study interval. number, sex, animal number, group, matrix, Sample Preparation and Storage All specimens will be stored at approximately -20C until shipment. aBplporoodxismaamtpelleys 1wimllLbaetstthoereidndaitvaidpuparloxtiimmaetpeoliynt5saCnudntaiplpcreonxtirmiafutgealtyihoanl.foAftsuhbesasmacprlief,ice bbleocoedn,twriilflugbeedrteomsoevpaerdataendplpalsamcae.d iTnhae sbelpoaorda,tecellalbuelalredfrvaicalt.ionThoef trheemabilnoiondg(bRlBoCo)d,wainldl plasma will be frozen immediately and stored at approximately -20C. 04177 3s _-- Sample Transfer cCovoancme 63e29-2s26 All specimens will be shipped on dry to the following person and address: ice to the Sponsor. The specimens will be shipped Dr. Kris Hansen Environmental Technology and Safety Services 935 Bush Avenue Building 2-E3-09 St Paul, Minnesota 55133-3331 Phone: (612) 778-6018 Fax: (612) 788-6176 `The Sponsor will completion of the be responsible study. for further analysis, reporting, and archiving upon REPORT AbebsiuoblmoigitctaeldpthoatsheedSrpaoftnsroeprorftorirnecvluideiwnga,ndbuctonmomtelnitm.iteTdheton,,tahobsieoliotgeimcsallipshtaedsebefilnoawl will report will be provided. Experimental Design and Methods As defined by the protocol, protocol amendments, and any protocol deviations. Data and Results Animal receipt and acclimation Antemortem observations Body weights Physical examinations Clinical pathology Dose administration Sample collection Bile salts replacement solution infusion times and amounts 04178 36 Maintenance of Raw Data, Records, and Specimens Covance 6329-226 w`iTlhle finocllluodwei,ngbudtatwialrlencootrdbsetloibmietmedaitnot:ained and transferred to the archives of Covance Protocol and amendments Study correspondence Test material receipt Final report "The following study data will supporting records to include, but will not be be retained at limited to: Covance but not archived with the Feed analysis records Water analysis records Animal room temperature and humidity records Refrigerator/freezer temperature records Instrument calibration and maintenance records United States Department of Agriculture animal records Stock records The following data not be limited to: records to be transferred to the archives of 3M will include, but will Tn-life records: Animal receipt Acclimation Animal room maintenance Antemortem observations Body weights Physical examination records Clinical pathology Clinical pathology slides Dose administration Sample collection Bile salts replacement solution infusion times and amounts Arelslulctoorfrtehsipsonsdteundcye,widlolcbuemeanrtcahtiivoend,irnetchoerdsst,orpargoetofcaocli,liatinedsoffinaClovreapnocret gfeonaerrapteerdioadsoafone o`fyetahrefoalfloorweimnegntthieonseidgnmiantgeroiftalhsewfiilnlalberespeonrtt.toOtnhee Sypeaornsaoftrearnsdiganirnegtutrhneffeienawlilrlepboert, all cfohraragneda.ddiTthieonSalpopnesroiordomafytiemleectantdo hCaovveantcheemwaitlelricalhs raeatasritnoegrdaiegnetfheee.CoIvfatnhceeSpAornchsiovres chooses to have Covance dispose of the materials, a disposal fee will be charged. 04179 : amiznon PROTOCOL APPROVAL cCoovanmcem632e9-2n26 [Ta--ri SGuduDtilicer JU SSieet Mme BatAferfiz B oBreo .a PTeldgiood veTse righaie rlkr an. BAapih 21, pis2 MCeovtetsLCiobriroa FAMetamploLl iDCbheemaeiorneAy Fi 1 Core Libor 04180 3 Covance 6320-226 PROTOCOL AMENDMENT NO. 1 Covance 6329-226 MetaCbyonliosmmoolfguNs-EMtohnylkePyesrfFloulolrooowcitnagneAdSmuilnfiostnraamtiidoonoEfthaanSoilng(lNeetC-aFpossuel;eTD-o6s3e1.6) in Sponsor 3M Chemicals, 3M Center Building 220-2B-02 St. Paul, MN 55144-1000 Study Director Andrew Seacat, PhD 3M Center Building 220-2802 St. Paul, MN 55144-1000 Contractor Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Biological Phase Investigator Fred Thalacker, PhD Covance Laboratories Inc. P.0. Box 7545 Madison, Wisconsin 53707 `This amendment modifies the following portionsofthe protocol: Effective May 11,1998 I. Page 7. EXPERIMENTAL DESIGN, Observation of Animals, Body Weights. `To accurately reflect when the body weights will be taken, replace the first sentence with the following: Weekly during acclimation, on the calculations), weekly after dosing, dayofrandomization (to be and on the day of sacrifice. used for dosing 04181 39 Covance 6329-226 2. Page 9. EXPERIMENTAL DESIGN, Termination, Scheduled Sacrifice, To clarify the dispositionofthe animal carcass, please add a sentence following the first sentenceofthe third paragraph: After the tissues have been removed, the residual carcass will be discarded. 3. Page 10, EXPERIMENTAL DESIGN, Sample Preparation and Storage Since a subsample of less than 1 mLofwhole blood may needtobe removed, please replace. the second sentenceofparagraph two with the following: A subsample at the individual time points and approximately halfofthe sacrifice blood will be removed and placed ina separately labeled vial. Effective May 13,1998 4. Page, TEST SUBSTANCE, Storage Conditions Upon actual receiptof the test substance, it was noted that the storage was to be room temperature. Therefore, replace the first and second sentence with the following: The test substance and any remaining test substance will be stored at room temperature. Effective June 2, 1998 To indicate that an additional 2 mL blood sample will be removed for serum collection for the remaining time points, the following changes to the protocol are necessary. 5. Page, EXPERIMENTAL DESIGN, Antemortem Sample Collection, Blood (Groups 1 and 2). Add the following sentence after the third line: Effective June 3, 1998 (Study Day 21) and at the remaining time points, an additional 2 mL blood sample will be collected into a glass tube without heparin and set aside at room temperature to allow clotting 6. Page 10, EXPERIMENTAL DESIGN, Sample Preparation and Storage Add a new third paragraph as follows: Remove the serum and place it into a separately labeled vial. Discard the cellular debris and store the serum at approximately -20C for shipment. 94182 w Covance 6329226 To indicate that additional blood will be removed for serum collection at sacrifice, the following changes to the protocol are necessary. 7. Page 9, EXPERIMENTAL DESIGN, Antemortem Sample Collection, Scheduled Sacrifice. Replace the fourth line with the following: Blood will be collected via cardiac puncture into glass tubes with heparin (at least 20 mL) and into glass tubes without heparin (at least 20 mL). PROTOCOL AMENDMENT APPROVAL La, WtGut~ An Seacat, PhD Study Director 3M Chemicals 92s(93 Date cd tin 0 Noafo `red Thalacker, PhD Biological Phase Investigator Metabolic Chemistry Covance Laboratories Inc. s/1/19 Date <& 74.0O-- Philip $2 Teitelbaum, PhD Associate Director Metabolic Chemistry `Covance Laboratories Inc. sthz2/27 Date 94183 a Covance 6329-226 PROTOCOL DEVIATIONS Protocol "Actual -- Environmental controls for the animal room will be set to maintain a relative humidityof 50% 20% April 25, 1998, ranged from 80.1% to 86.2% between 2:27 and 8:00 May 28, 1998, ranged from 73.4% to 73.9% between 19:08 and 20:30 June 11, 1998, ranged from 71.5% 1073.6% between 7:30 and 10:00 and ranged from 75.3% to 82.6% between 17:19 and 21:00 June 20, 1998, ranged from 70.2% to 72.8% between 13:19 and 15:00 June 27,198, ranged from 71.3% to 78.1% between 6:38 and 9:00 At sacrifice, blood will be collected via cardiac puncture into glass tubes with heparin (at least 20 mL) and into glass tubes without heparin (at least 20 mL) A short sample was collectedviathe vena cava for serum and plasma. Body Weights. Weekly during `The body weights were not taken on the acclimation, on the dayofrandomization dayofrandomization, because no (10 be used for dosing calculations), randomization was needed. The animals weekly after dosing, and on the day of were selected by test results and sacrifice. adjustment to thejacket and tether system. The weights were taken within 5 daysofdosing to be used for dosing calculations. These deviations would not be expected ( have had an effect on the outcomeof the study. 04184 2 Covance 6329-226 APPENDIX B SummaryofAntemortem Observations, Clinical Pathology Codes and Abbreviations, and Clinical Pathology Data Tables 04185 Table BI. Animal 1D 105428 105430 105431 105432 105433 105435 105439 105440 105442 105444 a Summary ofAntemortem Observations Sacrifice Day and Sex Observation Noted Group 1 (Intact) 2 Day, Male Urine appears to contain water 2 Day, Male No observations noted 28 Day, Male Urine discolored, brown 28 Day, Male Urine appears to contain water Few feces 91 Day, Male No observations noted 91 Day, Male Urine appears to contain water Urine appears to contain water Urine discolored, red Low food consumption Low food consumption Low food consumption Low food consumption Low food consumption Low food consumption 2 Day, Female No observations noted 2 Day, Female Few feces 28 Day, Female Urine appears to contain water Urine appears to contain water Second container used for urine Low food consumption Low food consumption 28 Day, Female Urine appears to contain water Urine appears discolored, red Urine appears discolored, red Few feces Low food consumption Low food consumption Covance 6320-226 Study Day 1 2 10 2 19 64 66 7 7 74 75 76 kd 2 1" 19 1" n 12 19 9 10 2 1 12 04186 Table BI. Animal ID 105447 105449 Continued Sacrifice Day and Sex 91 Day, Female 91 Day, Female a Covance 6329-226 Study Observation Noted Day Urine contained feces. 2 Urine appears to contain water 10 Urine appears to contain water 2 Urine appears to contain water 13 Urine appears to contain water 15 Urine appears to contain water 16 Urine appears to contain water 17 Urine appears to contain water 18 Urine appears to contain water 19 Urine appears to contain water 23 Urine appears to contain water 3 Urine appears to contain water 64 Urine appears to contain water 78 Second container used for urine is Second container used for urine 16 Second container used for urine 17 Second container used for urine 64 Second container used for urine 8 Urine appears discolored, red 10 Discharge, appeared to be menstruating 10 No feces 2 Few feces 50 Low food consumption 7 Urine appears to contain water 10 Urine appears to contain water 43 Urine appears to contain water 50 Urine appears to contain water 57 Urine appears to contain water 64 Urine appears to contain water 71 Urine appears to contain water 78 Second container used for urine 57 Second container used for urine 6 Second container used for urine 78 Few feces 2 04187 as Covance 6320-226 Table BI. Continued Animal Sacrifice Day Study ID and Sex Observation Noted Day Group 2 (Bile Duct Cannulated) 105436 Bilecollected Discharge, vomitus containing food 3 28 Day Low food consumption 6 Male 105437 Bile collected `Small amountofbile 10 2Day Liquid feces 2 Male Liquid feces 13 Non-formed feces 6 Non-formed feces 7 Non-formed feces 2 Low food consumption 10 Low food consumption 1 Low food consumption 12 No feces 10 105441 Bile collected Small amount of bile 10 28 Day Non-formed feces 6 Female Non-formed feces 2 Non-formed feces 2 Low food consumption 10 Low food consumption 1 No feces 10 105446 Bile collected `Small amountofbile 17 2 Day `Small amountofbile 23 Female `Small amountofbile 24 No bile present 25 No bile present 26 Nobile present 27 Nobile present 28 No bile present 29 Non-formed feces 1 Non-formed feces 2 Non-formed feces 5 Non-formed feces 7 04188 NS QS/QNS NR FS sScH H SL L s1 1 u RE EE SE PC PD PI PL PA co HB PLASMO NO AGG FR 3 Covance 6320-226 Codes for Clinical Pathology GENERAL CODES No sample Quantity not sufficient No repeat (sample volume not sufficient for repeat analysis) Fibrin strands SSlaimgphltleychleotmioeldyzed Hemolyzed Slightly lipemic Lipemic Slightly icteric Ioteric Unscheduled/moribund bleed Recording error (recorded incorrect dat, e.g, wrong number, spelling error, incorrect date) Entry error (incorrect keyboard entry) Sampling error Platelets clumped Platelets decreased Platelets increased Platelets large Platelets appear adequate Color interferes with test Heinz bodies observed Plasmodium No aggregation Fractious CODES FOR BLOOD CELL MORPHOLOGY The following scale was used to measure the degreeofanisocytosis (ANISO), poikilocytosis (POIK), polychromasia (POLY), hypochromasia (HYPO), or basophilic stippling (BASTIP) or the presenceof Howell-Joily bodies (HIBODY), toxic neutrophils (TOXNEUT), or atypical lymphocytes (ATYPLYM): Scale Degree Presence - Normal for the species. 1 Slight 2 Moderate 43 NMoatrkaepdplicable Not present Rare Few MMoadneyrate 0418% a Covance 6329226 Abbreviations and Units for Clinical Hematology TeResdtblood cell count Hemoglobin Hematocrit Mean corpuscular volume MMeeaann ccoorrppuussccuullaarr hheemmoogglloobbiinn concentration Platelet count PAcrtoitvhartoemdbpianrttiiamlethromboplastin time: `White blood cell count DifNfuecrlenetaitaeldbrleododblcoelold ccoelulntcount, Segmented neutrophil count LByamnpdhnoecuytrtoephciolunctount Monocyte count BEoassionpohpihliclocuonutnt Anisocytosis Polychromasia Poikilocytosis Hypochromasia Toxic neutrophils ARbbBrCev(iEa6t/iUoLno(rUnXi1t0s%)4L) HGB (G/DL) HCT (%) MCV (FL) MCH (PG) MCHC (%) PLT (E3/UL or X10/uL)) PT (SEC) PWTBTC(S(EEC3)/UL or X10%/uL) NRBC (/100 WBC) N-SEG (E3/UL or X10%/uL) and % LN-YBMAPNHD((EE33//UULLoorrXX11007"u/LL))aanndd%% MONO (E3/UL or X10%4L) and % BEOASSION((EE33//UULLoorrXX1100%u/4LL))aanndd%% ANISO (1,23) POLY (1.2.3) POIK (-1,23) HYPO (12.3) TOXNEUT (11,234) Abbreviations and Units for Clinical Chemistry Test GUlruecaosnietrogen TCroetaatlinpirnoetein Albumin Globulin ChTooltaelstbeilriorlubin Triglycerides Aspartate aminotransferase: `Alanine aminotransferase `Alkaline phosphatase C`rGeaamtimnaegkliuntaasme:yltransferase Calcium Inorganic phosphorus Sodium Potassium Chloride Serum bile acids Abbreviation (Units) GLU (MG/DL) CURNE(AMTG/(DMLG)/DL) T PRO (G/DL) ALB (G/DL) GLOB (G/DL) TBILI (MG/DL) CHOL (MG/DL) TRIG (MG/DL) AST/SGOT (U/L) ALT/SGPT (IU/L) ALK PHOS (IU/L) GCGKT(U(LILL)) CA (MG/DL) IPHOS (MG/DL) NA (MMOL/L) K (MMOL/L) CL (MMOL/L) SBA (UMOL/L or MG/DL) 004120 g3 |:z zg net to we ies ss 3 omens mn . go 3 i5 5i = Be de HPCE Re Re A g i E 8 Ct . %2:32 :g 8 2 a32 :& i& # H 3 :i a@3  i 288 i $ 2 33 5i 5 ! e8 ii a 8 i 3g k g 3: ii : ! g i 8 8 Toone Em ] i "22 iI 8 mys vis tens Sin Comps tots30 mn ns mie . g 3 &5 28 ew TTT TINIE :i3 :i i .Hd i3235: 5:; || Comey wt ins stn stony ov | TL SURAT | ow Eas 2 om o8 or 1s Com otis fn is . 3 2|` 33 i 3 ii 3 : 32 ::i 3 3 2: 8 2 8 5 $3 ; R o i g. 2i 888 | so so iso | ELWR STRAT 3 F 2& Ho : gx 4& - | PE -- [RI g2 &8 - 4 H2 12 2 g B> | TTT TI i s i B2 8s &i >8 | mney we is ncn seston os | LL PS A SET | HE BRAT Se BE Eg Swe wen we | J 22 ifk 8% 8g fy -- oSaTon,re crS omensn asomsons rpraa rr coiz21