Document pppojrx91JaVwOVm2EO2kEBJ7

Final BiologicalPhase Report Metal-)oli.@o;fiiNi"-[iii,, -,-I"tjklrooctane (Net-Fose, : iiiiCyn oi-,,i 11-ldmin isti-atioioiI-1-@siIiI PREPARED ["OR: 3M Cliciiiicals COVANCE SI-L;L)YNUMBER: 6329-226 C 0 V Alg-OOC@Ell@ THE DEVELOPMENT SERVICES COMPANY Sponsor: 3M Chemicals St.Paul,Minnesota Study Title: Metabolism ofN-EthylPerfluorooctanSeulfonamidoEthanol(Net-FoseT;-6316)in Cynomolgus Monkeys FollowingAdministratioonfa SingleCapsuleDose Data Requirement: None Authors: FredericW. ThalackerP,hD Deborah K. Lee Study Completed on: June 2, 1999 Performing Laboratory: Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704 Laboratory ProjectIdentification: Covance 6329-226 Page 1 of 77 2 Covance 6329-226 STATEMIENT OF NO DATA CONFIDENTIALITY CLAIM Metabolismof N-EthylPerfluorooctanSeulfonamidoEthanol(Net-FoseT;-6316) in Cynomolgus Monkeys FollowingAdministratioonf a SingleCapsuleDose No claimof confidentialiitsymade forany informationcontainedinthisbiologicaplhase on thebasisofitsfallinwgithinthescopeofFIFRA I0(d)(A)(,B),or(C). These dataarethepropertyof 3M Chemicals and assuch areconfidentiaflorallpurposes otherthancompliancewithFIFRA Section10. Submissionof thesedataincompliance withFIFRA does notconstitutaewaiverof any rightto confidentialitthyatmay exist under any otherstatutoer inany othercountry. Company: 3M Chemicals Company RepresentativeA:ndrew Seacat,PhD Title:StudyDirector Department: Signature Date 3 COMPLIANCE STATEMENT Covance 6329-226 Metabolism ofN-EthylPerfluorooctanSeulfonamidoEthanol(Net-Fose;T-6316)in Cynomolgus Monkeys FollowingAdministratioonfa SingleCapsuleDose All aspectsofthisbiologicaplhase were conductedinaccordancewiththeEnvironmental ProtectionAgency PesticidPerograms Good LaboratoryPracticeStandards(40 CFR 160). Andrew Seacat,PhD Study Director 3M Chemicals Study Submittedby Sponsor Date Date Date 4 Covance 6329-226 QUALITY ASSURANCE STATEMENT Thisreporthasbeenreviewedby theQualityAssuranceUnitofCovance Laboratories Inc.,inaccordancewiththeEnvironmentalProtectioAngency (EPA) Good Laboratory PracticeStandards,40 CFR 160. The followinginspectionwsere conductedand findings reportedtothestudydirectoarnd studydirectomranagement. Writtenstatusreportsof inspectionasnd findingsareissuedto Covance management accordingto standard operatingprocedures. Inspection Dates From To Phase Date Reportedto Study Directorand Study DirectorManagement 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 ProtocolReview TestArticleAdministration ProtocolAmendment Review ProtocolAmendment Review Data Review ReportReview Data Review ReportReview ReportReview 05/04/98 05/13/98 06/03/98 10/09/98 04/06/99 04/06/99 04/06/99 06/02/99 06/02199 Keprv(sentatiOva'lei,6 AssuranceUnit Date 5 Covance6329-226 KEY PERSONNEL Metabolic Chemistr-y FredericW. Thalacker,PhD BiologicalPhase Investigator Toxicology Operations PeterKong Manager Deborah K. Lee Study Coordinator PhilipJ.Teitelbaum,PhD Associate Director LeAnn Eastman Supervisor Radiochemical Analysis Kuntal Sinha Study Chemist Cara Himrich Supervisor Metabolism In-Life Quality Assurance Unit Nancy M. Centanni Manager Laboratory Animal Medicine Donna J.Clemons, DVM, MS Diplomate,ACLAM Supervisor 6 SIGNATURES Covance 6329-226 FredericW. Thalacker,PhD BiologicalPhase Investigator MetabolicChemistry Covance LaboratorieIsnc. >7 Philip@'@TeitelbauPmh,D AssociateDirector MetabolicChemistry Covance Laboratories Inc. ]Wate Date Andrew Seacat,PhD Study Director 3M Chemicals Date 7 Covance 6329-226 TABLE OF CONTENTS STATEMENT OF NO DATA CONFIDENTIALITY CLAIM COMPLIANCE STATEMENT QUALITY ASSURANCE STATEMENT KEY PERSONNEL SIGNATURES ABSTRACT TITLE OBJECTIVE REGULATORY COMPLIANCE QUALITY ASSURANCE EXPERIMENTAL DESIGN AND PROCEDURES Study Design Justificatiofnor Dosing Route and Dose level Test Substance Test Animals and Housing Animal Selection Justification Surgical Procedure Bile Replacement Dose Preparation and Analysis Dosing Procedure Antemortem Observations Physical Examinations Body Weights ClinicalPathology ClinicalPathology Tests Antemortem Sample Collection Termination Sample IdentificationR,etention, and Disposition Blood, Plasma, Serum, and CellularFraction Preparation and Storage Sample Transfer Disposition of Raw Data, Records, Samples, and the Final Report RESULTS AND DISCUSSION Quarantine and Acclimation Pap,e 2 3 4 5 6 9 10 10 10 10 10 10 II II 12 12 12 12 13 13 13 13 14 14 14 14 15 16 17 17 17 18 18 18 8 Covance 6329-226 ClinicalPathology 19 Body Weights and Doses Administered 19 Antemortem Observations 19 CONCLUSIONS 20 TABLES 21 Table 1.IndividuaBlody Weights of Cynomolgus Monkeys Reportedin Kilograms 21 Table2.IndividuaBlody Weightsofand Dose WeightsAdministeredinOne CapsuletoCynomolgus Monkeys (50mg/kg) 22 Table 3. 1% DextroseinLactatedRingersSolutionand BileSaltReplacement SolutionInfusionTimes 23 APPENDIX A 24 ProtocolCMS 22672AI, ProtocolAmendment No. 1,and Protocol Deviations 24 APPENDIX B 42 Summary ofAntemortem ObservationsC,linicaPlathologyCodes and Abbreviationsa,nd ClinicaPlathologyData Tables 42 9 Covance 6329-226 ABSTRACT Tissue,plasma,senun,bile,feces,and urinespecimenswere providedto obtain informationon themetabolismof N-ethylperfluorooctanseulfonamidoethanol (Net-Fose;T-6316)incynomolgus monkeys followingadministratioonfa singleoral dose. Sixteencynomolgus male and femalemonkeys from Covance ResearchProductswere dosed accordingtothe followingstudydesign: Group 1 Number and Sex of Animals 6 Male 6 Female Status Intact 2 2 Male BileDuct 2 Female Cannulated Dose 50 mg/kg 50mg/kg CollectionPsostdose Tissuesfrom2/sexon Days 2,28,and 90. Urineand fecesfrom 0-12 and 12-24hours,then dailythrough28 days,andweeklythrough90 days. Bloodpredose,and at1,2,4,6,12,24,36,and 48 hours,7 days,andweeklythrough90 days. Tissuesat28 days. Bilefrom 0-12and 12-24hours, and dailythrough28 days. Blood predosea,nd at1,2,4,6,12,24,36 and48 hours,7days,and weeklythrough28 days. Separateblood sampleswere collectefdorpreparatioonf plasma and serum. Plasma was preparedby centrifugatiofntheblood. Serum was preparedby allowingthebloodto clotand centrifuginigttoseparatethecelluladrebris.Allspecimenswere shippedto the Sponsorforanalysisand theresultosf theseanalyseswillbe reportedseparatelbyy the Sponsor. 10 Covance 6329-226 TITLE Metabolism of N-Ethyl PerfluorooctaneSulfonamido Ethanol(Net-Fose;T-6316) in Cynomolgus Monkeys Following Administrationof a SingleCapsule Dose OBJECTIVE The purpose of thisstudywas to administera perfluorooctanetestsubstanceto male and female cynomolgus monkeys and collectsamples fordeterminationof absorption, distributionm,etabolism, and excretion. REGULATORY COMPLIANCE The biologicalphase of thisstudy was conducted in accordance with theEnvirom-nental ProtectionAgency (EPA), Good Laboratory PracticeStandards,40 CFR 160 and by Covance LaboratoriesInc.,3301 Kinsman Boulevard, Madison, Wisconsin, in accordance with Covance ProtocolCMS 22672A I dated April 21, 1998, and Covance Standard Operating Procedures (SOPs). The protocol,protocolamendment, and protocol deviationsarein Appendix A. All proceduresin thisstudy arein compliance with theAnimal Welfare Act Regulations, 9 CFR 3, dated October 30, 1989, and as modified on March 18, 199 1. In the opinion of the Sponsor and the study director,the study did not unnecessarilyduplicateany previous work. QUALITY ASSURANCE The protocol,study conduct,data,and biologicalphase fmal reportwere auditedby the Covance QualityAssurance Unit (QAU) in accordance with Covance SOPS. All study activitiepserformed at3M willbe theresponsibilitoyf the3M QualityAssurance Department. Study Design EXPERIMENTAL DESIGN AND PROCEDURES Animals were assigned to two groups, one was the bileduct cannulated group and the otherwas intact.The group designation,number of animals,specimen collectiona,nd targetdose levelwere as follows: Covance 6329-226 Group Number and Sex ofAnimals Status Dose Frequency, Route, and Level CollectionPsostdose 1 6 Male Intact One Capsule Tissuesfrom 2/sexon Days 2,28, and 90. 6 Female Oral Urineand fecesfrom 0-12 and 12-24hours, 50 mg/kg thendailythrough28 days,and weekly through 90 days. Blood predose,and at 1,2,4,6, 12,24,36,and 48 hours,7 days,and weekly through90 days. 2 2 Male BileDuct One Capsule Tissuesat28 days. 2 Female Cannulated Oral Bilefrom 0-12 and 12-24hours,and daily 50 mg/kg through28 days. Blood ptedose,and at1,2,4,6, 12,24,36 and 48 hours,7days,and weekly through28 days. Justification for Dosing Route and Dose level The oral route has been used for evaluating systemic toxicity of the compound in cynomolgus monkeys. The 50 mg/kg dose was chosen because itprovidesthe maximum non-toxicsingledose amount necessaryto recover measurable concentrationsof the test substance in minor tissues. Test Substance The testsubstance,T-6316, was received inthe Department of Metabolic Chemistry on May 8,1998 from the Covance Department of Toxicology. Stabilitaynd retentionof a sample as specifiedin 40 CFR 160.105 of the testsubstanceisthe responsibilitoyf the Sponsor. Informationon synthesismethods, composition,or othercharacteristictshat definethe testsubstance are on filewith the Sponsor. The followinginformationwas obtainedfrom thelabelon thetestsubstancecontainer: Substance No.: ContainerNo.: Test Substance: Lot Nos.: Date Combined: Purity/Concentration: ExpirationDate: Storage Requirements: Physical State: 1084A3 65 T-6316 (Narrow Range N-Ethyl PerfiuorooctanesulfonamidoEthanol,NETFOSE) 30035, 30037, and 30039 07-31-1997 98.1% On filewith theSponsor Room temperature Amber waxy solid,slightodor 12 Covance 6329-226 Test Animals and Housing The in-lifpeortionof thestudywas conductedMay 13,1998,throughAugust 11,1998. Eleven mate and elevenfemalecynomolgus monkeys (nonhuman primates)from Covance ResearchProductswere receivedon March 24, 1998. The animalswere young adultto adultand weighed approximately3 to4 kg atarrivalU.pon arrivale,achanimal was assigneda temporaryidentificatinounmber. Beforebeingplacedon teste,ach animalwas randomly assigneda permanentidentificatinounmber and identifiewditha neck tagattachedtothecollar. Allanimalswere housed inindividuals,uspended,stainlesssteelw,ire-mesh cagesduring acclimationand housed in individuamletabolismcagesdesignedfortheseparatioannd collectioonfbile,urine,and feceswhileon test. The primateswere fedCertifiePdrimateDiet#8726C (HarlanTeklad)ad libitume,xcept when fastedforsurgeryand priortotestsubstanceadministratiotnhroughapproximately 4 hours postdose.Dietswere supplementedwith appropriatferuitasnd cereals.Water was providedfreshdailyand ad libitum.Environmentalcontrolsfortheanimalroom were setto maintain18'to29'C, 50% 20% relativheumidity,and a 12-hour lightI/2-hourdark cycle.The 12-hourdark cyclewas interrupteda,snecessary,to accommodate studyprocedures. Animal Selection Clinicalchemistryand hematology testresultasnd physicalexaminationresultwsere used toselecttestanimals.In additiona,nimalswere selectedforthesurgicalprocedure based on thebestapparentadjustmenttothejacketand tethersystem.Beforedose administratioan,laboratoraynimalveterinariaenvaluatedthehealthof theanimalsand approvedtheiruse on thestudy. Justification Studiesusing liveanimalsareessentiaflorcharacterizintghedispositioonf chemicalsin animals.No acceptablenon-liveanimalmodels were available.The cynomolgus monkey has been used asthenon-rodentspeciesto evaluatetoxicityT.hisstudywillaid intheevaluationof testresultosbtainedintoxicologystudiesconductedinmonkeys and willalsoprovidea basisfortheextrapolatioonf safetydatafrom animalstohumans. The number ofanimalswas theminimum number necessarytoprovidescientificavlallyid results. SurgicalProcedure The surgicalprocedurewas conducted inaccordancewith Covance StandardOperating Procedures.The animalswere fastedovernightbeforesurgeryand sedatedwith 13 Covance 6329-226 ketamine.An appropriataentibiotiwcas administered.Surgicalanesthesiwaas maintainedusingoxygen and isofluraneL.actatedRinger'ssolutionwas administered througha saphenouscathetetrhroughoutthesurgicalprocedureoras determinedby a staffveterinarianA.sterilseurgicaslcrubwas performed.The common bileductand duodenum was cannulatedand thegallbladdewras removed and discarded.Analgesics were administeredas recommended by thestafvfeterinarianA.dditionalantibiotics and/orfluidswere administeredasrecommended by thestaffveterinarianT.he animals had atleast10 days torecoverfrom thesurgicalprocedurepriortodose administration. BileReplacement Beginningtheday aftersurgerythrough3 days postsurgery,a solutionof 1% dextrosein LactatedRinger'ssolutionwas administeredviatheduodenalcannulaforapproximately 8 hoursperday atapproximately20 mL/hour topreventdehydration.Beginningon the fourthpostsurgicadlay untilsacrificae,bilesaltsolutionwas infusedviatheduodenal cannulaforapproximately8 hoursperday atapproximately23 mL/kg/day. The bilesalts solutionwas preparedby adding18.0g of cholicacidto I L of 0.9% sodium chloride solution.Sodium bicarbonat(e1.3g)was addedto each 1 L of solution.The solution was mixed and thepH adjustedto 7.4to7.8with0.IN HCI orsodium hydroxide,as appropriateI.nfusionintervalasnd volumes administerewdere recorded(SeeTable3). Dose Preparationand Analysis The capsules(gelatinN,o. 2,0.37mL) forthedose preparatiownere receivedfrom Torpac,Inc.on April9,1998. The testcompound was homogenized. A No. 2 capsulewas taredon an analytical balanceand an appropriateamount oftestsubstance,based on animal weight and dose levelw,as added usinga spatula.The capsuleswere sealedand placedinseparate20-mL scintillativoinalslabeledwiththeanimalnumber. The capsuleswere storedunder ambientconditions. Dosing Procedure The animalswere fastedovernightpriorto doseadministratiotnhroughapproximately 4 hourspostdose.The capsulewas giventotheanimalsby a devicespecififcorcapsule administration. Antemortem Observations Mortalityand moribunditycheckswere done twicedaily(a.m.and p.m.).Cageside observationforgeneralhealthand appearancewere done once daily.Signsofpoor health or abnormal behaviorwere recordedas theywere observed. 14 Covance 6329-226 PhysicalExaminations Physicalexaminationwere performedtwiceduringacclimationo,nce pre-surgerya,nd once postsurgerybeforetheinitiatiofntreatment.Animals were anesthetizewdith ketaminehydrochlorideaccordingto Covance StandardOperatingProceduresforthe examination. Jacketswere checked accordingtoCovance StandardOperatingProcedures.The animals were anesthetizeadsabove fortheexamination;however, a lower dose of ketaminewas administeredforthelimitedexamination. Body Weights Body weightswere takenweekly duringacclimationw,ithin5 daysof dosing(tobe used fordosingcalculationsw)e,ekly afterdosing,and on theday ofsacrifice. ClinicalPathology The animalswere not fasted.Blood was collectevdiathefemoralveinbeforedose administratioans follows:approximatelyI week beforesurgeryfrom allanimalsand approximately4 and 8 dayspostsurgeryfrom cannulatedanimals.When health problems developed,blood was collectefdorclinicaplathologyteststo assesshealth statusas deemed necessaryby thestaffveterinarian. ClinicalPathology Tests The followingarethetestsperformed by theClinicalPathologyDepartment: Hematology Red blood cellcount Hemoglobin Hematocrit Mean corpuscularvolume Mean corpuscularhemoglobin Mean corpuscularhemoglobin concentration Platelectount White bloodcellcount Differentiballoodcellcount Blood cellmorphology Prothrombintime Activatedpartiatlhromboplastitnime 15 Covance6329-226 ClinicalChemistry Glucose Urea nitrogen Creatinine Totalprotein Albumin Globulin Totalbilirubin Serum bileacids Cholesterol Aspartate aminotransferase Alanine aininotransferase Alkaline phosphatase Creatine kinase Gamma glutamyltransferase Calcium Inorganicphosphorous Sodium Potassium Chloride Antemortem Sample Collection No Teflon was used forthisstudy. Blood (Groups I and 2). Approximately 2 mL of blood was collectedfrom Group I animals predose and approximately 2 mL was collectedat 1,2, 4, 6, 12, 24, 36,and 48 hours,7 days,and weekly through 90 days postdose. Approximately 2 mL of blood was collectedfrom Group 2 animals ptedose and approximately2 mL was collectedat 1, 2,4, 6,12,24, 36, and 48 hours,and on7, 14,21, and 28 days postdose. Thebloodwas collectedvia a femoral vein intoheparinizedtubes and placed immediately on wet ice. EffectiveJune 3,1998 (Study Day 21) and atthe remaining time points,an additional 2-niL blood sample was collectedintoa glasstube without heparin and setasideatroom temperatureto clot. Bile(Group 2). Bile was collectedfrom the Group 2 animals via the implanted cannula intoplasticcontainerssurrounded by dry icepredose (foratleast12 hours),and at 0-12, 12-24,and continuingat 24-hour intervalsthrough 28 days postdose. Urine (Group 1). Urine was collectedin plasticcontainerssurrounded by dry ice at 0-12, 12-24, and continued at24-hour intervalsthrough 28 days, then weekly until 90 days postdose. 16 Covance 6329-226 Feces (Group 1).Feceswere collecteadt0-12,12-24,and continuedfor24-hour intervaltshrough28 days,thenweekly until90 days postdose.Specimens were transferreidntoplasticontainersatthetimeof collection. Termination Scheduled Sacrifice.At thetime ofsacrificaen,imalswere anesthetizewdithsodium pentobarbitalC.erebrospinaflluid(CSF) was collecteodnce theanimalwas fully anesthetizedC.SF was placedon wet iceimmediatelyaftercollectioannd collection timeswere recorded.Blood was collectevdiacardiacpunctureintoglasstubeswith heparin(atleast20 mL) and intoglasstubeswithoutheparin(atleast20 mL), except Animal No. 10541 thathad a shortsample obtainedviavena eava. Followingblood collectiont,heanimalswere sacrificevdiaexsanguinatiounnder sodium pentobarbital. Residualurinefrom thebladderwas added tothelasturinesample. Stomach, largeand smallintestincaolntentswere collectefdrom thetwo persexGroup I animalsthatwere sacrificeadtDay 2 only. Alltissuescollectewdere excised,rinsedwithwater,blotteddry,weighed,and placedin a plasticontainersurroundedby wet ice.Afterthetissuewsere removed,theresidual carcassewsere discarded.The followingtissuewsere collectefdrom allanimalsat sacrificex,ceptthegallbladdetrhatwas collectefdrom Group I only: Adrenalglands Aorta Bone (femur) Bone marrow (fromfemur) Brain Diaphragm Epididymis Esophagus Eyes Fat Gallbladder Heart Kidneys Large intestine Liver Lungs Lymph nodes(cervical) Lymph nodes(mesenteric) Muscle(thigh) Ovaries Pancreas Prostate Salivaryglands Seminal vesicles Skin (dorsals,haved) Small intestine Spinalcord Spleen Stomach Testes Thymus Thyroid/Parathyroid Tongue Trachea Urinarybladder Uterus 17 Covance 6329-226 Sample IdentificatioRne,tention,and Disposition Specimenswere identifiewdiththestudynumber,sex,animalnumber,group,matrix, specimen number, and collectioninterval. Blood,Plasma,Serum,and CellulaFrractioPnreparatioannd Storage Blood in glasstubes with heparin was stored atapproximately 5'C priorto analysis.At the individualtime points,the blood was gently inverted severaltimes to homogenize, then approximately 700 @tL was taken and placed into a labeled vialfor each individual time point. At sacrificea,pproximately halfof the blood was homogenized and placed into a labeled vial.The subsamples were placed in a freezerat a temperature of approximately -20'C. The remaining blood was centrifugedat2,400 rpm (1,300 x g) for approximately 10 minutes atapproximately 5'C. The resultingplasma and cellularfraction was transferredto separatelylabeled vialsand placed in freezerat a temperature of approximately -20'C untilthey were shipped. T'he additional2-mL blood collectionin glasstubes without heparin for the collectionof serum was allowed to clotat room temperature for approximately 30 to 60 minutes. The sample was then centrifugedat 2,400 rpm (1,300 x g) for approximately 10 minutes at approximately 5'C. The serum was removed and placed into a labeledvial.The cellular debris was discarded and the serum was stored in a freezerat a temperature of approximately -20'C to await shipment. Sample Transfer All specimens were shipped on dry iceto the Sponsor on June 12,1998, and August 18, 1998: 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 be responsible forfurtheranalysis,reporting,and archiving upon completion of the study. 18 Covance 6329-226 Disposition of Raw Data, Records, Samples, and the Final Report The following data records to be maintained and transferred to the archives of Covance willinclude,but willnot be limitedto: protocoland amendments; study correspondence; testmaterialreceipt;finalreport. The followingsupportingrecordsto be retainedat Covance but not archivedwith the studydata willinclude,butwillnot be limitedto: feed analysisrecords;water analysis records;animal room temperatureand humidity records;refrigerator/freezteermperature records;instrumentcalibrationand maintenance records;United StatesDepartment of Agricultureanimal records;stockrecords. The followingdata recordsto be transferredto thearchivesof 3M upon completion of the reportwillinclude,but willnot be limitedto: In-lifreecords: Animal receipt Acclimation Animal room maintenance Antemortem observations Body weights Physicalexamination records Clinicalpathology Clinicalpathology slides Dose administration Sample collection Bile saltsreplacement solutioninfusiontimes and amounts All correspondence,documentation, records,protocol,and finalreportgeneratedas a resultof thisstudy willbe archivedin the storagefacilitieosf Covance fora period of one year followingthe signingof thefinalreport.One year aftersigningthe finalreport,all of theaforementioned materialswillbe sentto the Sponsor and a returnfeewillbe charged. The Sponsor may electto have the materialsretainedin the Covance Archives foran additionalperiodof time and Covance willcharge a storagefee. Ifthe Sponsor chooses to have Covance dispose of the materials,a disposalfee willbe charged. RESULTS Quarantine and Acclimation AND DISCUSSION All animals under quarantineand acclimationbeginning March 24,1998, except Animal 105428, appeared clinicallhyealthyand were releasedon April 24, 1998. Animal 105428 was observed to have diarrheaand was monitored untilnormal. At leastone physical examination,one fecalexamination, and threetuberculintestswere performed before 19 Covance 6329-226 releasefrom quarantine.Weekly body weightswere recordedand animalswere acclimatedtothejacketand tethersystemrequiredforbileduct cannulation. ClinicalPathology Resultsofpresurgicacllinicaplathologytestsindicatedno obviousgroup orindividual healthabnormalitiesA.lthough severalmales inGroup I had notablyhighleukocyte counts,thesewere attributetdoexcitementassociatewdith blood collectiopnrocedures. A few femalesinGroup I had mildlyhigh valuesforalanineaminotransferasaectivity consistenwtithHepatitiAs infectiona,subclinicaclonditioncommonly observedin cynomolgus monkeys thatdoes notadverselyeffectliverfimctionor animalhealth. Resultsoftheclinicaplathologytestsfollowingsurgerydemonstratedno critical complicationsecondarytobileductcannulation.Four days postsurgery,increased activitiefsoraspartataeminotransferasea,laninearninotransferasael,kalinephosphatase, gamma glutamyltransferasaen,d creatinekinaseinGroup 2 animalswere consistenwtith thesurgicaplrocedure.The totalbilirubiwnas notablyincreasedforonlyone ofthe males (AnimalNo. 105436);and one ofthefemales(AnimalNo. 105441)exhibited evidenceof blood lossfrom thesurgicaplrocedure.Eightdays postsurgery,thehigh enzyme activitigeesneralleyxhibitedsignificanrteductionisndicativoef continued recoveryfrom thesurgicaplrocedure,and totalbilirubiwnas no longerelevatedfor Animal No. 105436.Persistenmti,ldlytomoderatelyelevatedactivitifeosralkaline phosphatase(malesand females)and gamma glutarnyltransfer(amsaelesonly)at8 days postsurgerywere characteristoifcmildlyincreasedbiliarpyressuresecondarytobile ductcannulationb,utnormal valuesfortotalbilirubiannd serum bileacidsindicated hepaticfunctionwas likelnyot significantcloympromised. Body Weights and Doses Administered Individuablody weightsand dosesadministeretdoGroups I and 2 arepresentedin TablesI and 2. Actualdosesadministeredrangedfrom 100.2% to 104.4% of the nominal doseof 50 mg/kg. Antemortem Observations The animalsappearednormal duringdoseadministratioannd appearedhealthyand exhibitendo overtsignsoftoxicittyhroughoutthestudy.The notedobservationasre listeidn Appendix B. None of theseobservationasppearedtobe testsubstancerelated. On May 22,1998,no bileflowwas notedforAnimals 105437 and 105441. The swivel was flushedforAnimal 105437 and thebilestartetdoflow again.The technicianfound thattheswivelforAnimal 105441was hooked up incorrectloyn thepreviousday,so it was connectedcorrectlaynd thepump restartedA.pproximately2 hours latert,herewas no bileflow,so thesystem was recheckedand bilebegan to flow. On May 26,1998 and 20 Covance 6329-226 May 29,1998,bloodwas drawn from Animal 105441 forclinicaclhemistryanalysisto monitorherhealthstatus.The resultosn May 29,1998,were indicativoef cholestasibsu,t theresultwsere improved from theresultosn May 26,1998.The ClinicaPlathologist alsoindicatetdhatthehepaticdysfimctionappearedminimal and concludedvery little hepatocelluladregenerationornecrosisoccurred. On June 4,1998,Animal 105449 didnothave a screenabove thepan to separatetheurine from thefeces.The feceswas scrapedfrom thepan and collectedh;owever,some cross-contaminatiomnay have occurred. Bileflow slowed and was notedon Study Day 17,23,and 24 thenstoppedon Day 25 from Animal 105446. However, theanimalexhibitendo signsof compromised health. Upon sacrificietwas notedthata portionofthebileducthad re-grown,evidently providingan alternatpeathof bileflow. CONCLUSIONS Samples were providedto obtaininformatioonn thepharmacokineticse,xcretionand tissuedistributiofnN-ethylperfluorooctanseulfonamidoethanol(Net-Fose;T-6316) aftera singlecasuledose incynomolgus monkeys. Bile-ductcannulatedas wellas intact animalswere usedinthestudy.Allsampleswere shippedtotheSponsorforanalysisand resultosf theseanalyseswillbe reportedseparatelbyy theSponsor. No testsubstance effectwsere noted on themonkeys. 21 Covance 6329-226 Table 1. TABLES IndividualBody Weights of Cynomolgus Monkeys Reported in Kilogrwns Animal Day Number 1 3 8 9 16 22 23 31 35 105428 105430 105431 105432 105433 105435 GroUp I Male 4.4 4.2 3.9 3.9 3.8 3.8 3.8 3.9 3.9 4.2 4.2 4.2 4.2 4.2 4.1 4.0 4.0 4.4 4.3 4.0 3.9 4.1 4.3 4.3 4.4 4.4 4.3 4.3 105439 105440 105442 105444 105447 105449 GroMp 1 Female 3.1 3.1 3.1 3.1 3.0 2.9 2.8 3.0 2.9 3.0 2.9 2.9 2.9 2.8 3.2 3.1 3.2 3.3 3.3 3.3 3.3 3.4 3.3 3.2 3.5 3.6 3.3 3.3 Group 2 Male 105436 3.7 3.5 3.5 3.6 3.8 106437 3.7 3.7 3.6 3.8 3.6 Grogp 2 Female 105441 3.0 3.0 2.8 3.2 3.2 105446 4.2 4.1 3.8 4.2 4.1 Day 42 49 56 63 70 77 84 91 105433 105435 Groa I Male 4.5 4.6 4.6 4.7 4.8 4.8 4.9 4.9 4.1 4.2 4.2 4.2 4.2 4.2 4.3 4.3 105447 105449 GropS I Female 3.5 3.5 3.5 3.5 3.6 3.4 3.4 3.4 3.4 3.5 3.6 3.7 3.6 3.5 3.7 3.7 Table 2. 22 Covance 6329-226 IndividualBody Weights of and 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 105449 Group 1 1 1 1 1 1 2 2 1 1 2 1 1 2 1 1 Animal Weight (kg) 4.4 3.9 3.8 4.2 4.1 4.0 3.7 3.7 3.1 3.1 3.0 3.0 3.0 4.2 3.2 3.3 Test Substance Administered (g) (mg/kg) % of Nominal 0.2207 0.1978 0.1930 0.2138 50.2 50.7 50.8 50.9 100.3 101.4 101.6 101.8 0.2082 0.2033 0.1884 50.8 50.8 50.9 101.6 101.7 101.8 0.1897 0.1573 0.1556 0.1522 0.1508 0.1566 0.2108 0.1616 51.3 50.7 50.2 50.7 50.3 52.2 50.2 50.5 102.5 101.5 100.4 101.5 100.5 104.4 100.4 101.0 0.1653 50.1 100.2 23 Covance 6329-226 Table 3. 1% Dextrose inLactatedRingers Solutionand BileSaltReplacement SolutionInfusionTimes Day PostSurgery 105436 AM Start PM Finish Animal Identification 105437 105441 Time AM PM AM PM Start Finish Start Finish 105446 AM Start PM Finish 1 7:07 14:55 5:59 13:58 6:00 14:01 6:26 15:03 2 5:58 13:59 6:26 14:32 6:26 14:32 6:15 14:15 3 6:26 14:31 6:15 14:00 6:15 14:17 6:22 14:01 4 6:15 14:18 6:22 14:01 6:22 14:01 7:35 15:42 5 6:22 14:01 7:35 15:42 7:35 15:42 6:38 14:36 6 7:35 15:42 6:38 14:36 6:38 14:36 6:37 14:40 7 6:38 14:40 6:37 14:40 6:37 14:40 6:23 14:24 8 6:37 14:40 6:23 14:24 6:23 14:24 7:00 14:57 9 6:23 14:28 7:00 14:57 7:00 14:57 6:24 14:12 10 7:00 15:03 6:24 14:12 6:24 14:12 6:23 14:15 11 6:24 14:12 6:23 14:15 6:23 14:15 6:42 14:44 12 6:23 14:15 6:42 14:47 6:42 14:49 6:55 15:38 13 6:42 14:44 6:55 15:38 6:55 15:38 5:21 13:23 14 6:55 15:38 5:21 13:21 5:21 13-.23 6:51 14:58 15 5:21 13:22 6:51 14:58 6:51 14:58 6:16 14:25 16 6:51 14:58 6:16 14:24 6:16 14:25 5:46 13:56 17 6:16 14:24 5:46 13:56 5:46 13:56 6:48 14:51 18 5:46 13:56 6:48 14:51 6:48 14:51 6:39 14:41 19 6:48 14:51 6:39 14:41 6:39 14:40 6:38 14:46 20 6:39 14:41 6:38 14:46 6:38 14:46 6:36 14:36 21 6:38 14:46 6:36 14:38 6:36 14:37 6:53 15:01 22 6:36 14:36 6:53 15:01 6:53 15:01 6:17 14:14 23 6:53 15:01 6:17 14:18 6:17 14:20 5:50 13:47 24 6:17 14:18 5:50 13:47 5:50 13:47 5:43 13:48 25 5:50 13:47 5:43 13:48 5:43 13:48 5:46 13:44 26 5:43 13:48 5:46 13:47 5:46 13:47 6:49 14:53 27 5:46 13:48 6:49 14:53 6:49 14:53 6:39 14:41 28 6:49 14:53 6:39 14:41 6:39 14:41 6:55 14:57 29 6:39 14:41 6:58 14:57 6:55 14:57 6:36 14:42 30 6:55 14:57 6:36 14:35 6:36 14:35 6:39 14:31 31 6:36 14:35 6:39 14:31 6:39 14:31 7:02 14:59 32 6:39 14:31 7:02 15:00 7:02 14:59 6:45 14:46 33 7:02 15:00 6:45 14:46 6:45 14:46 6:41 14:39 34 6:45 14:46 6:41 14:40 6:41 14:39 6:41 14:44 35 6:41 14:40 6:41 14:44 6:41 14:44 6:45 14:05 36 6:41 14:44 6:45 14:05 6:45 14:05 7:01 14:58 37 6:45 14:05 7:01 14:58 7:01 14:58 NA' NA 38 7:01 14:58 5:41 13:40 5:41 13:40 NA NA 39 5:41 13:40 6:36 14:47 6:36 14:47 NA NA 40 6:36 14:47 6:38 14:48 6:38 14:45 NA NA 41 6:38 14:45 6:51 14:35 6:51 14:35 NA NA 42 6:51 14:35 a On Days I through3,theanimalswere infusedwith 1% dextroseinLactatedRingees solutionat approximately20 mL/kg/hourforapproximately8 hours,and on the remainingdays theanimalswere infusedwithbilesaltsatapproximately23 mL/kg/hour forapproximately8 hours. b Not applicableS.inceno bilewas recovered,no bilesaltswere infused. 24 Covance 6329-226 APPENDIX A ProtocolCMS 22672AI, ProtocolAmendment No. 1,and ProtocolDeviations 25 Covance 6329-226 PROTOCOL Sponsor 3M Chemicals St.Paul,Minnesota ProtocolCMS 22672AI Study Title Metabolism of N-Ethyl PerfluorooctaneSulfonamido Ethanol (Net-Fose;T-6316) in Cynomoigus Monkeys Following Administrationof a SingleCapsule Dose Testing Guideline Environmental ProtectionAgency, Good Laboratory PracticeStandards,40 CFR 160 Date April 21, 1998 Performing Laboratory Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identificaflon Covance 6329-226 Page 1 of 13 26 Covance 6329-226 STUDY IDENTIFICATION Metabolism ofN-Ethyl PerfluorooctanSeulfonarnidoEthanol(Net-Fose;T-6316) in Cynomolgus Monkeys FollowingAdministratioonf a SingleCapsuleDose Test Substance N-EthylPerfluorooctanSeulfonamido Ethanol(Net-FoseT;-6316) Sponsor 3M Chemicals 3M Center Building220-2E-02 St.Paul,N4N 55144-1000 Study Director Andrew Seacat,PhD 3M Chemicals 3M Center Building220-2E-02 St.Paul,NlN 55144-1000 Phone: (612)575-3161 Fax: (612)733-1773 PrincipaAlnalyticaIlnvestigator KrisHansen EnvironmentalTechnology and Safety Services 935 Bush Avenue Building2-E3-09 St.Paul,Minnesota 55133-3331 Phone: (612)778-6018 Fax: (612)788-6176 BiologicalPhase Investigator FredericW. Thalacker,PhD Covance LaboratoriesInc. P.O.Box 7545 Madison,Wisconsin 53707 Phone: (608)242-2712ext.7370 Fax: (608)241-7412 BiologicalPhase Location Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704 Proposed Study Timetable In-LifeExperimentalStartDate In-LifeEnd Date May 13,1998 August 11,1998 27 Covance 6329-226 OBJECTIVE The purpose of thisstudy is administer a Perfluorooctane testsubstance to male and female cynomolgus monkeys and collectsamples for determination of absorption, distributionm,etabolism, and excretion. REGULATORY COMPLIANCE Environmental Protection Agency, Good Laboratory Practice Standards, 40 CFR 160 All procedures in thisprotocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 3, dated October 30, 1989, and as modified on March 18, 1991. In the opinion of the Sponsor and the study director,the study does not unnecessarily duplicateany previous work. The protocol,study conduct, data,and biologicalphase finalreportwill be audited by the Covance Laboratories Inc.Quality Assurance Unit in accordance with Covance Standard Operating Procedures. All study activitiepserformed at3M willbe the responsibilityof the 3M Quality Assurance Department. TEST SUBSTANCE Source The testsubstance willbe provided by the Sponsor. The Sponsor willbe responsible for analysisof the testsubstance. Storage Conditions The testsubstance will be stored at approximately -20*C untildosing. Any testsubstance remaining willbe stored at approximately -20"C. Disposition Unused testsubstance will be returnedto the Sponsor or a recipientdesignated by the Sponsor, or discarded at the Sponsor's request. The Sponsor will assume responsibility forretentionof a sample of the testmaterial as specifiedin 40 CFR 160.195 ifthe study is longer than 4 weeks in duration. 28 Covance 6329-226 SafetyPrecautions Safetyprecautionswillbe takenasrequiredby Covance Policiesand Procedures,in consideratioonftheSponsor'sMaterialSafetyData Sheet,or otherrelevantsafety informationprovidedby theSponsor. EXPERIMENTAL DESIGN Animals Species/Strain. Nonhuman primate/eynomolgus Source. Covance ResearchProducts(Alice,Texas,or Denver, Pennsylvania)T.he sourceof theanimalswillbe documented in theraw data. Weight atArrival. 3 kg or greater Age at Arrival. Young adult/adult Number and Sex. Eightmales and eightfemaleson test,sixmale and sixfemale intactand two male and two femalebileductcannulated.One male and one femaleextraintactand two male and two female extracannulatedwillbe availableaspossiblereplacementanimals. Identification. Individuallnyumbered collartags Housing For theacclimatiopneriod,theanimalswillbe in individuals,tainlesssteelcages.For thetestperiod,theanimalswillbe inindividuamletabolismcagesdesignedforthe separatioannd collectioonfbile,urine,and feces. Food CertifiePdrimateDiet#8726C (HarlanTeklad)ad libitume,xceptwhen fastedfor surgery.Food willalsobe withdrawnovernightpriorto,and returnedapproximately 4 hours afterdose administrationD.ietswillbe supplementedwithappropriatferuitasnd cereals. Water Ad libitump,rovidedfreshdaily 29 Covance 6329-226 Contaminants Thereareno known contaminantisnthefoodorwaterthatwould interfewrieththis study. Environment Environmentalcontrolsfortheanimal room willbe settomaintain18'to29'C, 50% + 20% relativheumidity,and a 12-hourlight/12-houdrark cycle.The 12-hourdark cycle may be interruptetdo accommodate studyprocedures. Quarantine and Acclimation Animals willbe underquarantineforatleast30 days. At leastone qualitycontrol physicalo,ne fecalexamination,and threetuberculitnestswillbe done beforerelease from quarantine.Weekly body weightswillbe taken.Animals willbe acclimatedtothe jacket/tethseyrstemrequiredforbileductcannulation. Animal Selection Clinicaclhemistryand hematologytestresultasnd physicalexaminationresultwsillbe usedto selectestanimals.Animals willbe selectefdorthesurgicaplrocedurebased on thebestapparentadjustmenttothejacketand tethersystem.In thecasethatallormost animalsadjustwell,animalswillbe selectedinnumericalorderbasedon theindividual collartagnumbers previouslyassigned.Beforedose administratioan,laboratoraynimal veterinariawnillevaluatethehealthof theanimalsand approvetheiruseon thestudy. Justification Studiesusingliveanimalsareessentiaflorcharacterizintghedispositioonf chemicalsin animals.No acceptablenon-liveanimal models areavailableT.he cynomolgus monkey hasbeen used asthenon-rodentspeciestoevaluatetoxicityT.hisstudywillaidinthe evaluationoftestresultosbtainedintoxicologystudiesconductedinmonkeys and will alsoprovidea basisfortheextrapolatioonf safetydatafrom animalstohumans. The number ofanimalsistheminimum number necessarytoprovidescientificalvlaylid results. SurgicalProcedure The surgicalprocedurewillbe conducted inaccordancewith Covance Standard OperatingProcedures.The animalswillbe fastedovernightbeforesurgeryand sedated withketainineA.n appropriataentibiotiwcillbe administered.Surgicalanesthesiwaill be maintainedusingoxygen and isofluraneL.actatedRinger'ssolutionwillbe administeredthrougha saphenouscatheterthroughoutthesurgicaplrocedureoras 30 Covance 6329-226 determinedby a stafvfeterinarianA.sterilseurgicaslcrubwillbe performed.The common bileductand duodenwn willbe cannulatedand thegallbladderremoved and discarded.Analgesicswillbe administereads reconunendedby thestafvfeterinarian. Additional antibioticsand/or fluidsmay be administered as recommended by the staff veterinarian.The animals willhave atleast10 days to recover from the surgical procedurepriortodose administration. Bile Replacement Beginning the day after surgery through 3 days post surgery, a solution of 1% dextrose in LactatedRinger'ssolutiownillbe administeredviatheduodenalcannulafor approximately 8 hours per day (approximately 20 niL/hour) to prevent dehydration. Beginning on the fourth post surgicalday untilsacrifice,an appropriate amount of mixed bilesaltssolutionwillbe infused via the duodenal cannula for approximately 8 hours per day. The volume of bilesaltssolutionto be administered willbe approximately 23 niL/kg/day. Bile saltssolutionwill be prepared by adding 18.0 g of cholic acidto I L of 0.9% sodium chloridesolution. Sodium bicarbonate (1.3 g) will be added to each I L of solution.The solutionwill be mixed and the pH adjusted to 7.4 to 7.8 with 0.IN HCI or sodium hydroxide, as appropriate. Infusion intervalsand volumes administered will be recorded. Group Designations and Dose Level Group 1 2 Number and Sex 6 Male 6 Female 2 Male 2 Female Status Dose Frequency, Route, and Level CollectionsPostdose Intact One Capsule 50 mg/kg Tissuesfrom 2/sex on Days 2, 28, and 90. Urine and fecesfrom 0-12 and 12-24 hours, thendailythrough 28 days,and weekly through 90 days. Blood predose,and at 1,2, 4, 6, 12,24, 36, and 48 hours,7 days,and weekly through 90 days. BileDuct Cannulated One Capsule 50 mg/kg Tissues at28 days. Bilefrom 0-12 and 12-24 hours,and daily through 28 days. Blood predose,and at 1,2,4, 6, 12,24, 36 and 48 hours,7days,and weekly through 29 days, Justification for Dosing Route/Dose Level. The oral route has been used for evaluating systemic toxicityof the compound incynomoigus monkeys. The 50 mg/kg dose was chosen because itprovides the maximum non-toxic singledose amount necessary to recover measurable concentrations in minor tissues. 31 Dosing Procedure Covance 6329-226 Predose and Postdose Feeding. Animals willbe fastedovernight priorto dose administrationthrough approximately 4 hours postdose. Dose Administration. The capsule will be administered by a device specificforcapsule administration. Observation of Animals Antemortem Observations. Mortality and moribundity checks will be done twice daily (a.m. and p.m.). Cageside observation forgeneral healthand appearance willbe done once daily. Signs of poor health or abnormal behavior willbe recorded as they are observed. Physical Examinations. Twice during acclimation, once pre-surgery, and once post surgery before the initiatioonf treatment. Animals willbe anesthetizedwith ketainine hydrochloride according to Covance Standard Operating Procedures for the examination. Jackets will be checked according to Covance Standard Operating Procedures and will be replaced as necessary. The animals will be anesthetizedas above for the examination; however, a lower dose of ketaininemay be administered forthe limitedexamination. Body Weights. Weekly during acclimation,on the day of dosing, weekly thereaftera,nd on the day of sacrifice.Additional body weights may be taken atthe discretionof the Biological Phase Investigatoror a staffveterinarian. Clinical Pathology. The animals will not be fasted. Blood willbe collectedvia the femoral vein before dose administrationas follows: approximately I week before surgery from allanimals and approximately 4 and 8 days post surgery from cannulated animals. Ifhealth problems develop, blood will be collectedfor clinicalpathology teststo assess health statusas deemed necessary by the staffveterinarian.Any blood samples taken aftermodification of the ports forcollectionof bilewill be collectedbefore the administrationof the bilesaltsreplacement solutionfor the day. Tests 32 Covance 6329-226 ClinicaClhemisLry Glucose Urea nitrogen Creatinine Totalprotein Albumin Globulin Totalbilirubin Serum bileacids Cholesterol Aspartateaminotransferase Alanineaminotransferase Alkalinephosphatase Creatinekinase Gamma glutamyltransferase Calcium Inorganicphosphorous Sodiwn Potassium Chloride Hematology Red blood cellcount Hemoglobin Hematocrit Mean corpuscularvolume Mean corpusculahremoglobin Mean corpusculahremoglobin concentration Platelectount NVhitebloodcellcount Differentibaloodcellcount Blood cellmorphology Prothrombintime Activatedpartiatlhromboplastitnime Antemortem Sample Collection NO TEFLON IS TO BE USED FOR THIS STUDY. Blood (Groups 1 and 2). Approximately2 mL of blood willbe collectefdrom Group I animalspredoseand approximately2 mL willbe collecteadt 1,2,4,6, 12,24,36,and 48 hours,7 days,and weeklythrough90 dayspostdose.Approximately2 mL of bloodwill be collectefdrom Group 2 animalspredoseand approximately2 mL willbe collecteadt 1,2,4,6, 12,24,36,and 48 hours,and on 7, 14,21,and 28 days postdose.The blood willbe collectevdiaa femoralveinintoheparinizedtubesand placedimmediatelyon wet ice.The animalsmay be re-hydratewdithlactateRdinger'ssolutiongiven subcutaneouslyor viatheduodenalcannulaatthediscretioonf theBiologicalPhase Investigator LaboratoryAnimal Veterinariafnollowingbloodcollectionisf,thetotal blood volumes collecteedxceedtherecommendationsin Covance StandardOperating Procedures. Bile(Group 2). Bilewillbe collectedfrom the Group 2 animalsvia the implanted cannulaintoplasticontainersurroundedby dryicepredose(foratleast12 hours),and at0-12,12-24,and continuingat24-hourintervaltshrough28 days postdose. 33 Covance 6329-226 Urine (Group 1). Urine will be collected in plastic containers surrounded by dry ice at 0-12, 12-24, and continuing at24-hour intervalsthrough 28 days, then weekly until90 days postdose. Feces (Group 1). Feces will be collectedat 0-12, 12-24, and continuing at 24 hour intervalsthrough 28 days, then weekly until90 days postdose. Samples will be transferredintoplasticcontainersat the time of collection. Termination Unscheduled Sacrificesand Deaths. The carcasseswillbe retainedand may undergo postmortem examination for any gross changes. Further examination may be performed upon Sponsor's request. Scheduled Sacrifice. At the time of sacrifice,animals will be anesthetized with sodium pentobarbital.Cerebrospinal fluid(CSF) willbe collectedonce the animal isfully anesthetized.CSF willbe placed on wet ice immediately aftercollectionand collection times willbe recorded. Blood (atleast20 mL) willbe collectedvia cardiacpuncture into heparinizedtubes. Following blood collection,the animals willbe sacrificedvia exsanguination under sodium pentobarbital. Residual urine from the bladder willbe added to the lasturine sample. Stomach, large and small intestinalcontents willbe collectedfrom the two per sex Group I animals that are sacrificedat Day 2 only. All tissuescollectedwillbe excised,rinsedwith water, blotteddry, weighed, and placed in a plasticcontainersurrounded by ice. The following tissueswillbe collectedfrom all animals atsacrificee,xcept the gallbladderwillbe collectedfrom Group I only: 34 Covance 6329-226 Adrenal glands(-0.5g) Aorta(-0.5g) Bone (femur) Bone Marrow (from femur) Brain Diaphragm Epididymis Esophagus Eyes Fat Gallbladder(-0.5g) Heart Kidneys Large Intestine Liver Lungs Lymph nodes (cervical) Lymph nodes (mesenteric) Muscle (thigh) Ovaries (~0.5g) Pancreas Prostate SalivaryGlands Seminal vesicles Skin (dorsal,shaved) Small Intestine SpinalCord Spleen Stomach Testes Thymus Thyroid/Parathyroi(d-0.75 g) Tongue Trachea Urinary Bladder Uterus Sample Identification,Retention and Disposition Specimens willbe identifiedwith the study number, sex,animal number, group, matrix, specimen number, and collectioninterval. Sample Preparation and Storage All specimens willbe storedat approximately -20*C untilshipment. Blood samples willbe storedatapproximately 5*C untilcentrifugationA. subsample, approximately I mL attheindividualtime pointsand approximatelyhalfof thesacrifice blood,willbe removed and placed in a separatelabeledvial.The remaining blood will be centrifugedto separateplasma. The blood,cellularfractionof theblood (RBC), and plasma willbe frozenimmediately and storedatapproximately -20'C. 35 Covance 6329-226 Sample Transfer All specimens willbe shipped on dry iceto the Sponsor. The specimens willbe shipped to the followingperson and address: Dr. Kris Hansen Environmental Technology and SafetyServices 935 Bush Avenue Building2-E3-09 St Paul,Minnesota 55133-3331 Phone: (612) 778-6018 Fax: (612)788-6176 The Sponsor willbe responsibleforfurtheranalysis,reporting,and archivingupon completion of the study. REPORT A biologicalphase draftreportincluding,but not limitedto,those items listedbelow will be submittedto the Sponsor forreview and comment. Then, a biologicalphase final reportwillbe provided. Experimental Design and Methods As definedby theprotocol,protocolamendments, and any protocoldeviations. Data and Results Animal receiptand acclimation Antemortem observations Body weights Physical examinations Clinicalpathology Dose administration Sample collection Bilesaltsreplacement solutioninfusiontimes and amounts 36 Covance 6329-226 Maintenance of Raw Data, Records, and Specimens The followingdatarecordstobe maintainedand transferretdothearchivesof Covance willincludeb,ut willnotbe limitedto: Protocoland amendments Study correspondence Testmaterialreceipt Finalreport The followingsupportingrecordstobe retainedatCovance butnotarchivedwiththe studydatawillincludeb,utwillnotbe limitedto: Feed analysisrecords Water analysisrecords Animal room temperatureand humidityrecords Refrigerator/freetzemrperaturerecords Instnimentcalibratioannd maintenancerecords UnitedStatesDepartmentof Agriculturaenimalrecords Stock records The followingdatarecordstobe transferretdothearchivesof 3M willinclude,butwill not be limitedto: In-lifreecords: Animal receipt Acclimation Animal room maintenance Antemortem observations Body weights Physicalexaminationrecords Clinicaplathology Clinicaplathologyslides Dose administration Sample collection Bilesaltrseplacementsolutioninfusiontimesand amounts All correspondenced,ocumentation,records,protocol,and finalreportgeneratedas a resultofthisstudywillbe archivedinthestoragefacilitioefsCovance fora periodof one yearfollowingthesigningofthefinalreport.One yearaftersigningthefinalreporta,ll oftheaforementionedmaterialwsillbe senttotheSponsorand a returnfeewillbe charged.The Sponsor may electtohave thematerialsretainedin theCovance Archives foran additionapleriodof time and Covance willchargea storagefee.IftheSponsor choosestohave Covance disposeofthematerialsa,disposalfeewillbe charged. April21, 1998 37 PROTOCOL APPROVAL Covance 6329-226 Covancc 6329-226 Page 13 of 13 A-ndrew Seacat, PhD Study Director 3M Chemicals FredericW. Thalacker, PhD BiologicalPhase Investigator Metabolic Chemistry Covance Laboratories Inc. Philipqeitilbaum, PhD AssociateDirector Metabolic Chemistry Covanr-e LaboratoriesInc. Date Dako 38 Covance 6329-226 PROTOCOL AMENDMENT NO. 1 Covance 6329-226 Metabolism ofN-EthylPerfluorooctanSeulfonarnidEothanol(Net-FoseT;-6316)in Cynomolgus Monkeys FollowingAdministratioonfa SingleCapsuleDose SRonso 3M Chemicals 3M Center Building220-2E-02 St.Paul,MN 55144-1000 Study Direct Andrew Seacat,PhD 3M Center Building220-2E-02 St.Paul,MN 55144-1000 Contractor Covance LaboratorieIsnc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 BiologicalPhase Investivator Fred Thalacker,PhD Covance LaboratoriesInc. P.O. Box 7545 Madison, Wisconsin 53707 Thisamendment modifiesthefollowingportionsof theprotocol: EffectivMeay 11,1998 1. Paye 7, EXPERIMENTAL DESIGN, Observation of Animals, Body Weights To accuratelryeflecwthen thebody weightswillbe taken,replacethefirsstentence withthefollowing: Weekly duringacclimationo,n theday ofrandomization(tobe used fordosing calculationsw)e,ekly afterdosing,and on theday of sacrifice. 39 Covance 6329-226 2. Patze9, EXPERIMENTAL DESIGN, Termination,ScheduledSacrificeT.o clariftyhedispositioonf theanimal carcassp,leaseadd a sentencefollowingthefirst sentenceof the thirdparagraph: Afterthetissuehsave been removed, theresidualcarcasswillbe discarded. 3. Page 10, EY.PERIMENTAL DESIGN, Sample Preparation and Stor?.ge Since a subsampleof lessthan I mL ofwhole blood may need to be removed, pleasereplace thesecond sentenceofparagraphtwo withthefollowing: A subsampleattheindividuatlimepointsand approximatelyhalfof thesacrifice blood willbe removed and placedina separatellyabeledvial. EffectivMeay 13,1998 4. Page 3, TEST SUBSTANCE, StorageConditions Upon actualreceiptof thetest substancei,twas notedthatthestoragewas tobe room temperature.Therefore, replacethefirsatnd second sentencewiththefollowing: The testsubstanceand any remainingtestsubstancewillbe storedatroom temperature. EffectiveJune 2,1998 To indicattehatan additiona2l mL blood sample willbe removed forserum collection forthe remainingtime points,thefollowingchanges totheprotocolarenecessary. 5. Page 8, EXPERIMENTAL DESIGN, Antemortem Sample CollectionB,lood (Groups I and 2).Add thefollowingsentenceafterthethirdline: EffectivJeune 3,1998(StudyDay 2 1)and attheremainingtimepoints,an additional 2 mL blood sample willbe collecteidntoa glasstubewithoutheparinand setasideat room temperatureto allowclotting. 6. Page 10, EXPERIMENTAL DESIGN, Sample Preparation and Storage Add a new thirdparagraphasfollows: Remove theserum and placeitintoa separatellyabeledvial.Discardthecellular debrisand storetheserum atapproximately-20'C forshipment. 40 Covance 6329-226 To indicattehatadditionablloodwillbe removed forserum collectioantsacrificteh,e followingchanges to theprotocolarenecessary. 7. Pa2e 9, EXPERIMENTAL DESIGN, Antemortem Sample Collection, Scheduled SacrificeR.eplacethefourthlinewiththefollowing: Blood willbe collectevdiacardiacpunctureintoglasstubeswithheparin(atleast 20 mL) and intoglasstubeswithoutheparin(atleast20 nlL). PROTOCOL AMENDMENT APPROVAL @@@Seacat, PhD Date Study Director 3M Chemicals Vred Thalacker,PhD BiologicaPlhase Investigator MetabolicChemistry Covance LaboratorieIsnc. ZI-I . Date < Philip@Teitelbaum,PhD Date AssociateDirector MetabolicChemistry Covance LaboratorieIsnc. 41 Covance 6329-226 PROTOCOL DEVIATIONS Protocol Actual Environmentalcontrolsfortheanimal room willbe settomaintaina relative humidityof50% 20% April25,1998,rangedfrom 80.1% to 86.2% between 2:27 and -8:00 May 28,1998,rangedfrom 73.4% to 73.9% between 19:08and 20:30 June 11,1998,ranged from 71.5% to73.6% between 7:30and 10:00and rangedfrom 75.3% to82.6% between 17:19 and 21.-00 June 20,1998,rangedfrom 70.2% to 72.8% between 13:19and 15:00 June 27,1998,rangedfrom 71.3% to 78.1% between 6:38 and 9:00 At sacrificbel,oodwillbe collectevdia cardiacpunctureintoglasstubeswith heparin(atleast20 mL) and intoglass tubeswithoutheparin(atleast20 mL). A shortsample was collectevdiathevena cava forserum and plasma. Body Weights. Weekly during The body weightswere nottakenon the acclimationo,n theday ofrandomization day ofrandomization,becauseno (tobe usedfordosingcalculations), randomizationwas needed. The animals weekly afterdosing,and on theday of were selectedby testresultsand sacrifice. adjustmentto thejacketand tether system.The weightswere takenwithin 5 days of dosingtobe used fordosing calculations. These deviationwsould notbe expectedtohave had an effecton theoutcome of the study. 42 Covance 6329-226 APPENDLX B Summary of Antemortem Observations, ClinicalPathology Codes and Abbreviations,and ClinicalPathology Data Tables Table B 1. Animal ID 105428 105430 105431 105432 105433 105435 105439 105440 105442 105444 43 Covance 6329-226 Summary of Antemortem Observations SacrificDeay and Sex ObservationNoted Study Day Group I (Intact) 2 Day, Male Urine appearstocontainwater 1 2 Day, Male No observationnsoted 28 Day, Male Urine discoloredb,rown 21 28 Day, Male Urine appearstocontainwater 10 Few feces 2 91 Day, Male No observationnsoted 91 Day, Male Urine appearstocontainwater 19 Urine appearstocontainwater 64 Urine discoloredr,ed 66 Low food consumption 72 Low food consumption 73 Low food consumption 74 Low food consumption 75 Low food consumption 76 Low foodconsumption 77 2 Day, Female No observationnsoted 2 Day, Female Few feces 2 28 Day, Female Urine appearstocontainwater 17 Urine appearstocontainwater 19 Second containerused forurine 17 Low food consumption II Low food consumption 12 28 Day, Female Urine appearstocontainwater 19 Urine appearsdiscoloredr,ed 9 Urine appearsdiscoloredr,ed 10 Few feces 2 Low food consumption II Low food consumption 12 Table B 1. Animal ID 105447 105449 Continued SacrificDeay and Sex 91 Day, Female 91 Day, Female 44 Covance 6329-226 ObservationNoted Urine containedfeces Urine appearsto containwater Urine appearsto containwater Urine appearstocontainwater Urine appearsto containwater Urine appearsto containwater Urine appearsto containwater Urine appearsto containwater Urine appearsto containwater Urine appearsto containwater Urine appearstocontainwater Urine appearsto containwater Urine appearstocontainwater Second containerused forurine Second containerused forurine Second containerused forurine Second containerused forurine Second containerused forurine Urine appearsdiscoloredr,ed Discharge,appearedto be menstruating No feces Few feces Low foodconsumption Study Day 22 10 12 13 15 16 17 18 19 23 43 64 78 15 16 17 64 78 10 10 2 50 73 Urine appearstocontainwater 10 Urine appearstocontainwater 43 Urine appearstocontainwater 50 Urine appearsto containwater 57 Urine appearsto containwater 64 Urine appearstocontainwater 71 Urine appearsto containwater 78 Second containerused forurine 57 Second containerused forurine 64 Second containerused forurine 78 Few feces 2 45 Covance 6329-226 Table B 1. Animal ID 105436 105437 105441 105446 Continued SacrificDeay and Sex ObservationNoted Group 2 (BileDuct Cannulated) Bilecollected 28 Day Male Discharge,vomituscontainingfood Low foodconsumption Bilecollected 2 Day Male Bilecollected 28 Day Female Bilecollected 2 Day Female Small arnountof bile Liquidfeces Liquidfeces Non-formed feces Non-formed feces Non-forrnedfeces Low food consumption Low food consumption Low food consumption No feces Small amount of bile Non-formed feces Non-formed feces Non-fonned feces Low food consumption Low food consumption No feces Small amount ofbile Small amount ofbile Small amount ofbile No bilepresent No bilepresent No bilepresent No bilepresent No bilepresent Non-formed feces Non-formed feces Non-forrnedfeces Non-formed feces Study Day 8 6 10 12 13 6 7 23 10 II 12 10 10 6 22 23 10 II 10 17 23 24 25 26 27 28 29 1 2 5 7 NS QS/QNS NR FS sc SH H SL L si I u RE EE SE PC PD Pi PL PA co HB PLASMO NO AGG FR 46 Covance 6329-226 Codes forClinicalPathology GENERAL CODES No sample Quantitynot sufficient No repeat(samplevolume notsufficienftorrepeatanalysis) Fibrinstrands Sample clotted Slightlhyemolyzed Hemolyzed Slightllyipemic Lipemic Slightliycteric Icteric Unscheduled/moribundbleed Recordingerror(recordedincorrecdtata,e.g.w,rong number, spellinegrrori,ncorrecdtate) Entryerror(incorreckteyboardentry) Sampling error Plateletcslumped Plateletdsecreased Plateletisncreased Plateletlsarge Plateletasppearadequate Color interferewsithtest Heinz bodiesobserved Plasmodium No aggregation Fractious CODES FOR BLOOD CELL MORPHOLOGY The followingscalewas used tomeasure thedegreeof anisocytosi(sANISO), poikilocytos(iPsOIK),polychromasia(POLY), hypochromasia(HYPO), orbasophilic stipplin(gBASTIP) orthepresenceofHowell-Jollbyodies(HJBODY), toxicneutrophils (TOXNEUT), oratypicallymphocytes(ATYPLYM): Scale Degre Presence Normal forthespecies I Slight 2 Moderate 3 Marked 4 Not applicable Not present Rare Few Moderate Many 47 Covance 6329-226 Abbreviations and Units for ClinicalHematology Test Red blood cellcount Hemoglobin Hematocrit Mean corpuscularvolume Mean corpusculahremoglobin Mean corpusculahremoglobin concentration Platelectount Prothrombintime Activatedpartiatlhromboplastitnime ViMte blood cellcount Differentiballoodcellcount Nucleatedredblood cellcount Segmented neutrophilcount Band neutrophilcount Lymphocyte count Monocyte count Eosinophilcount Basophilcount Anisocytosis Polychromasia Poikilocytosis Hypochromasia Toxic neutrophils Abbreviation (Units) RBC (E6/IJLorX10'/,uL) HGB (G/DL) HCT (%) MCV (FL) MCH (PG) MCHC (%) PLT (E3/UL orX IO'lAzL)) PT (SEC) PTT (SEC) WBC (E3/TJLorXl 0'/izL) NRBC (/100WBC) N-SEG (E3/UL orXIO'/gL)and % N-BAND (E3/UL orXIO'/AL)and % LYMPH (E3AJL orX10'/ML)and % MONO (E3/TJLorX I0'/i2La)nd % EOSIN (E3/UL orXIO'/,uL)and % BASO (E3/LJLorXIO'/,uL)and % ANISO (-,1,2,3) POLY (-,1,2,3) POIK (-1,,2,3) H-Y?O (-,1,2,3) TOXNEUT (-,1,2,3,4) Abbreviationasnd UnitsforClinicaClhemistry Test Glucose Urea nitrogen Creatinine Totalprotein Albumin Globulin Totalbilirubin Cholesterol Triglycerides Aspartateaminotransferase Alanineaminotransferase Alkalinephosphatase Gamma glutamyltrmsferase Creatinekinase Calcium Inorganicphosphorus Sodium Potassium Chloride Serum bileacids Abbreviation(!Jnits) GLU (MG/DL) UN (MG/DL) CREAT (MG/DL) T PRO (G/DL) ALB (G/DL) GLOB (G/DL) T BILI(MG/DL) CHOL (MG/DL) TRIG (MG/DL) AST/SGOT (IU/L) ALT/SGPT (IU/L) ALK PHOS (IU/L) GGT (IU[L) CK (IU/L) CA (MG/DL) IPHOS (MG/DL) NA (MMOL/L) K (MMOL/L) CL (MMOL/L) SBA (UMOL/L orMG/DL) ANIMAL NUMBER ------ Group: 1 105428 105430 105431 105432 105433 105435 MEAN S.D. N Group: 2 105436 10543'1 MEAN S.D. N Extra 105429 105434 105438 MEAN S.D. N Summary and Individual Clinical Chemistry Data Males Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE GLU UN CREAT T PRO ALB GLOB T BILI CHOL SBA MG/DL MG/DL MG/DL G/DL G/DL G/DL MG/DL MG/DL umol/L -------- -------- -------- -------- -------- -------- -------- -------- -------- Dose Level: 50 134 16 77 17 80 20 124 20 99 17 lie 13 105 23.7 6 17 2.6 6 Dose Level: 50 100 17 106 11 103 4.2 2 17 .0 2 Dosage Unit: mg/kg 1.1 8.8 1.0 8.0 1.0 8.4 1.1 9.1 1.0 8.6 1.1 8.8 1.0 .05 6 8.6 .38 6 Dosage Unit: mg/kg 1.3 8.4 1.0 8.4 1.2 .21 2 8.4 .00 2 4.6 4.4 4.6 4.8 4.6 4.7 4.6 .13 6 4.8 4.6 4.7 .14 2 4.2 3.6 3.8 4.3 4.0 4.1 4.0 .26 6 3.6 3.8 3.7 .14 2 .2 85 13 .2 95 10 .2 150 8 .3 128 7 .4 94 10 .3 137 12 .3 115 10 .08 26.9 2 6 6 6 .3 138 14 .1 146 11 142 12 .14 5.7 2 2 2 2 124 105 103 ill 11.6 3 19 16 18 18 1.5 3 1.3 1.2 1.3 1.3 .06 3 8.4 9.3 7.8 8.5 .7 5 3 4.8 4.7 4.7 4.7 .06 3 3.6 4.6 3.1 3.8 .76 3 .2 172 11 .2 133 17 .4 113 8 .3 139 12 .12 30.0 4 3 3 3 ANIMAL NUMBER ------ Group: 1 105428 105430 105431 105432 105433 105435 MEAN S.D. N Group: 2 105436 105437 MEAN S.D. N Extra 105429 105434 105438 MEAN S.D. N Summary and Individual Clinical Chemistry Data males Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTAME SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE AST/SGOT IU/L -------- ALT/SGPT IU/L -------- ALK PHOS IU/L -------- GGT IU/L -------- CK IU/L -------- CA MG/DL -------- I PHOS MG/DL -------- NA MMOL/L -------- K MMOL/L -------- --- Dose Level: 50 32 58 62 28 56 33 56 116 39 78 35 24 47 12.8 6 56 35.8 6 Dose Level: 50 39 38 35 25 37 2.8 2 32 9.2 2 Dosage Unit: mg/kg 413 76 421 94 547 ill 604 189 452 43 566 15'7 500 81.8 6 112 53.5 6 Dosage Unit: ing/kg 370 53 295 82 332 53.0 2 68 20.5 2 361 571 761 316 125 367 417 220.5 6 340 348 344 5.7 2 9.9 9.5 9.0 10.2 9.4 10.6 9.8 .58 6 10.9 10.2 10.6 .49 2 6.2 3.6 6.2 7.9 5.8 6.5 6.0 1.40 6 6.8 4.4 5.6 1.70 2 155 147 146 155 154 152 152 4.0 6 160 152 156 5.7 2 4.3 4.6 4.9 5.7 4.2 4.8 4.0 .54 6 5.1 4.3 4.7 .57 2 so 48 34 44 8.7 3 34 120 28 61 51.5 3 607 297 416 440 156.4 3 138 122 ill 124 13.6 3 1055 1019 255 776 451.8 3 9.6 11.1 10.4 10.4 .75 3 6.4 5.9 5.5 5.9 .45 3 151 159 156 155 4.0 3 5.2 4.8 5.2 5.1 .23 3 ANIMAL NUMBER ------ Group: 1 105439 105440 105442 105444 105447 105449 MEAN S.D. N Group: 2 105441 105446 MEAN S.D. N Extra 105443 105445 105448 MEAN S.D. N Summary and Individual Clinical Chemistry Data Females Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETRANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE GLU MG/DL -------- UN MG/DL -------- CREAT MG/DL -------- T PRO G/DL -------- ALB G/DL -------- GLOB G/DL -------- T BILI MG/DL -------- CHOL MG/DL -------- SBA =ol/L -------- Dose Level: 50 112 17 116 18 128 19 ill 18 78 22 114 26 110 16.8 6 20 3.4 6 Dose Level: 50 98 16 91 16 94 4.9 2 16 .0 2 Dosage Unit: mg/kg 1.1 8.4 1.2 9.7 .9 8.5 1.0 9.0 1.0 8.8 1.0 9.4 1.0 .10 6 9.0 .51 6 Dosage unit: mg/kg 1.2 9.3 .8 8.3 1.0 .28 2 8.8 .71 2 4.6 4.7 4.4 4.6 3.6 4.5 4.4 .40 6 4.7 4.1 4.4 .42 2 3.8 5.0 4.1 4.4 5.2 4.9 4.6 .55 6 4.6 4.2 4.4 .28 2 .2 122 26 .4 182 49 .3 123 9 .3 148 9 .2 126 12 .3 141 13 .3 140 20 .08 23.0 15 6 6 6 .2 181 11 .3 201 7 .2 191 9 .07 14.1 2 2 2 2 88 94 114 99 13.6 3 16 14 12 14 2.0 3 .9 .9 1.1 1.0 .12 3 7.7 9.2 8.4 8.4 .75 3 4.0 4.3 4.4 4.2 .21 3 3.7 4.9 4.0 4.2 .62 3 .4 202 10 .2 155 20 .6 136 5 .4 164 12 .20 34.0 7 3 3 3 ANIMAL NUMBER ------ Group: 1 105439 105440 105442 105444 105447 105449 MEAN S.D. N Group: 2 105441 105446 MEAN S.D. N Extra 105443 105445 105448 MEAN S.D. N Summary and Individual Clinical Chemistry Data Females Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE AST/SGOT ALT/SGPT ALK PHOS GGT CK CA I PHOS NA K IU/L IU/L IU/L IU/L IU/L MG/DL MG/DL MMOL/L MMOL/L -------- -------- -------- -------- -------- -------- -------- -------- -------- mm Dose Level: 50 Dosage Unit: mg/kg 57 134 95 199 27 25 38 94 55 233 42 216 52 23.7 6 150 80.8 6 Dose Level: 50 363 98 233 90 215 44 192 87 284 52 407 69 282 86.2 6 73 21.9 6 Dosage Unit: mg/kg 218 160 104 256 202 239 196 56.1 6 10.2 10.7 9.2 10.1 9.1 10.0 9.9 .62 6 6.3 6.2 4.7 6.0 4.3 4.6 5.4 .91 6 155 157 153 156 149 158 155 3.3 6 4.8 4.2 5.1 4.3 4.0 4.3 4.4 .41 6 46 34 40 8.5 2 34 37 36 2.1 2 296 184 240 79.2 2 78 92 85 9.9 2 1061 134 598 655.5 2 10.5 9.4 10.0 .78 2 6.2 3.9 5.0 1.63 2 161 151 156 7.1 2 5.0 5.1 5.0 .07 2 31 28 44 34 8.5 3 B2 95 118 98 18.2 3 160 308 195 221 77.3 3 76 64 108 83 22.7 3 147 205 652 335 276.3 3 9.1 11.1 9.5 9.9 1.06 3 3.1 4.5 4.2 3.9 .74 153 159 152 155 3.8 3 3.7 4.5 4.5 4.2 .46 3 ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Surmary and Individual Clinical Chemistry Data Males - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE GLU MG/DL -------- UN MG/DL -------- CREAT MG/DL -------- T PRO G/DL -------- ALB G/DL -------- GLOB G/DL -------- T BILI MG/DL -------- CHOL MG/DL -------- SBA umol/L ------- Dose Level: 50 Dosage Unit: mg/kg 75 27 1.2 7.8 3.7 4.1 2.4 181 33 99 18 .9 8.1 4.2 3.9 .5 81 83 87 22 1.0 8.0 4.0 4.0 1.4 131 58 17.0 6.4 .21 .21 .35 .14 1.34 70.7 35 2 2 2 2 2 2 2 2 2 84 15 1.1 8.3 3.9 4.4 2.1 148 245 ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Summary and individual Clinical Chemistry Data Males - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE AST/SGOT IU/L -------- ALT/SGPT IU/L -------- ALK PHOS IU/L -------- GGT IU/L -------- CK IU/L -------- CA MG/DL -------- I PHOS MG/DL -------- NA MMOL/L -------- K MMOL/L -------- mm --- Dose Level: 50 Dosage Unit: mg/kg 175 194 184 13.4 2 316 131 224 130.8 2 1231 453 842 550.1 2 267 228 248 27.6 2 1652 2328 1990 478.0 2 9.6 10.0 9.8 .28 2 5.5 4.0 4.8 1.06 2 153 149 151 2.8 2 4.9 4.5 4.7 .28 2 397 364 1278 237 3267 9.8 5.1 150 5.3 ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and individual Clinical Chemistry Data Females - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE GLU MG/DL -------- UN MG/DL -------- CREAT MG/DL -------- T PRO G/DL -------- ALB G/DL -------- GLOB G/DL -------- T BILI MG/DL -------- CHOL MG/DL -------- SBA umol/L -------- Dose Level: 50 Dosage unit: mg/kg 91 15 1.1 6.9 3.5 3.4 .4 103 6 108 25 .9 8.5 3.8 4.7 .4 153 2 100 20 1.0 7.7 3.6 4.0 .4 128 4 12.0 7.1 .14 1.13 .21 .92 .00 35.4 2 2 2 2 2 2 2 2 2 2 90 14 .8 7.3 3.6 3.7 .9 139 1 ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Chemistry Data Females - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE AST/SGOT IU/L -------- ALT/SGPT IU/L -------- ALK PHOS IU/L -------- GGT IU/L -------- CK IU/L -------- CA MG/DL -------- I PHOS MG/DL -------- NA MMOL/L -------- K MMOL/L -------- Dose Level: 50 Dosage Unit: mg/kg 248 123 186 88.4 2 232 123 178 77.1 2 258 246 252 8.5 2 48 67 58 13.4 2 1802 2832 2317 728.3 2 9.6 10.2 9.9 .42 2 3.9 4.2 4.0 .21 2 156 159 158 2.1 2 4.6 4.9 4.8 .21 2 153 150 145 63 2669 9.2 3.7 155 4.6 ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Summary and Individual Clinical Chemistry Data Males - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE GLU MG/DL -------- UN MG/DL -------- CREAT MG/DL -------- T PRO G/DL -------- ALB G/DL -------- GLOB G/DL -------- T BILI MG/DL -------- CHOL MG/DL -------- SBA umol/L -------- Dose Level: 50 Dosage Unit: mg/kg 85 24 1.2 7.8 3.9 3.9 .6 121 4 98 le 1.0 7.6 4.1 3.5 .3 76 5 92 21 1.1 7.7 4.0 3.7 .4 98 4 9.2 4.2 .14 .14 .14 .28 .21 31.0 2 2 2 2 2 2 2 2 2 110 14 1.1 8.5 3.9 4.6 3.8 240 932 ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Summary and Individual Clinical Chemistry Data Males - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE AST/SGOT ALT/SGPT ALK PHOS IU/L IU/L IU/L -------- -------- -------- GGT IU/L -------- CK IU/L -------- CA I PHOS NA MG/DL MG/DL MMOL/L -------- -------- -------- K MMOL/L -------- Dose Level: 50 Dosage Unit: mg/kg 36 27 32 6.4 2 141 63 102 55.2 2 1141 690 916 318.9 2 281 260 270 14.8 2 468 214 341 179.6 2 9.5 10.3 9.9 .57 2 5.5 3.0 4.2 1.77 2 149 146 148 2.1 2 4.7 4.4 4.6 .21 2 345 444 4815 364 533 10.2 4.2 153 4.5 ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Chemistry Data Females - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE GLU UN MG/DL MG/DL -------- -------- Dose Level: 50 CREAT T PRO ALE MG/DL G/DL G/DL -------- -------- -------- Dosage Unit: mg/kg GLOB G/DL -------- T BILI MG/DL -------- CHOL MG/DL -------- SBA Umol/L ------- 114 10 1.2 7.4 3.8 3.6 .2 63 5 115 19 .8 8.2 3.8 4.4 .4 152 5 114 14 1.0 7.8 3.8 4.0 .3 108 5 .7 6.4 .28 .57 .00 .57 .14 62.9 2 2 2 2 2 2 2 2 2 74 6 .7 7.1 3.6 3.5 1.6 135 382 ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Chemistry Data Females - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE AST/SGOT ALT/SGPT ALK PHOS GGT CK CA I PHOS NA K IU/L -------- IU/L -------- IU/L -------- IU/L -------- IU/L -------- MG/DL -------- MG/DL -------- MMOL/L -------- MMOL/L -------- K Dose Level: 50 Dosage Unit: mg/kg 38 35 36 2.1 2 101 58 80 30.4 2 377 229 303 104.7 2 52 66 59 9.9 2 88 1048 568 678.8 2 10.7 10.1 10.4 .42 2 3.5 3.7 3.6 .14 2 156 144 150 8.5 2 5.8 5.7 5.8 .07 2 396 374 841 262 617 9.1 3.0 148 4.1 ANIMAL NUMBER ------ Group: 1 105428 105430 105431 105432 105433 105435 MEAN S.D. N Group: 2 105436 105437 MEAN S.D. N Extra 105429 105434 105438 MEAN S.D. N Summary and Individual clinical Hematology Data Males Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION ETHANOL (NET-FOSE; T-6316) IN OF A SINGLE CAPSULE DOSE RBC X10'/ML -------- HGB G/DL -------- HCT % -------- mcv FL -------- MCH PG -------- MCHC % -------- PLT X10'/juL -------- PT SEC -------- PTT SEC -------- Dose Level: 50 Dosage unit: mg/kg 6.55 5.97 5.35 5.90 6.14 6.41 12.8 11.6 9.9 12.4 11.1 12.7 6.05 .426 6 11.8 1.12 6 Dose Level: 50 43.2 37.9 32.6 40.9 38.9 43.4 65.9 63.5 60.9 69.3 63.3 67.8 39.5 4.03 6 65.1 3.13 6 Dosage Unit: mg/kg 19.6 19.4 18.5 21.1 18.0 19.8 19.4 1.08 6 29.7 30.6 30.4 30.4 28.5 29.2 29.8 .83 6 754 352 529 375 371 653 506 169.0 6 9.6 0.7 9.3 8.9 8.6 9.7 9.1 .47 6 15.9 13.9 18.6 13.4 15.3 15.9 15.5 1.8 6 6.80 12.9 44.9 66.0 19.0 28.8 547 9.3 12.9 6.21 11.5 38.5 62.1 18.5 29.8 383 9.3 14.3 6.50 .417 2 12.2 .99 2 41.7 4.53 2 64.0 2.76 2 18.8 .35 2 29.3 .71 2 465 116.0 2 9.3 .00 2 13.6 .9 2 7.85 6.29 6.05 6.73 .977 3 15.1 12.6 11.5 13.1 1.84 3 48.7 42.6 38.7 43.3 5.04 3 62.0 67.7 63.9 64.5 2.90 3 19.3 20.1 19.0 19.5 .57 3 31.0 29.7 29.8 30.2 .72 3 491 545 390 475 78.7 3 9.3 9.4 9.6 9.4 .15 3 15.1 12.6 13.3 13.7 1.2 3 ANIMAL NUMBER ------ Group: 1 105428 105430 105431 105432 105433 105435 MEAN S.D. N Group: 2 105436 105437 MEAN S.D. N Extra 105429 105434 105438 MEAN S.D. N Summary and Individual Clinical Hematology Data Males Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFOMAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE WBC X10'/,uL -------- N-SEG X10'/,uL -------- LYMPH X10'/pL -------- MONO X10'/,uL -------- EOSIN X10'/,uL -------- BASO X10'/ML -------- N-SEG% -------- LYMPH% -------- MONO% -------- EOSIN% -------- Dose Level: 50 Dosage Unit: mg/kg 35.6 16.4 14.2 2.2 2.8 .1 46 40 6 8 29.9 17.2 9.9 2.0 .8 .1 57 33 7 3 12.7 5.5 5.6 1.2 .3 .0 43 44 10 2 26.8 13.0 11.6 1.8 .2 .0 49 43 7 1 27.0 14.6 10.2 1.8 .5 .1 54 38 7 2 25.3 14.3 8.9 .9 1.1 .0 57 35 4 4 26.2 13.5 10.1 1.6 1.0 .0 51 39 7 3 7.56 4.20 2.86 .50 .96 .05 6 6 6 6 6 6 5.9 4.4 1.9 2 6 6 6 6 Dose Level: 50 Dosage Unit: mg/kg 15.3 10.2 4.1 .9 .1 .0 67 27 6 1 17.6 10.0 5.6 1.6 .5 .0 57 32 9 3 16.4 10.1 4.8 1.2 .3 .0 62 30 8 2 1.63 .14 1.06 .49 .28 .00 7.1 3.5 2.1 1 2 2 2 2 2 2 2 2 2 2 19.1 10.5 6.7 1.2 .7 .0 55 35 7 3 16.5 7.2 8.1 .8 .4 .0 44 49 5 2 16.7 9.8 4.7 1.8 .5 .0 56 28 11 3 17.4 9.2 6.5 1.3 .5 .0 52 37 6 3 1.45 1.74 1.71 .50 .15 .00 7.4 10.7 3.1 3 3 3 3 3 3 3 3 3 3 Individual Clinical Hematology Data Males Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE ANIMAL NUMBER ------ ANISO -------- POLY -------- POIK -------- HYPO -------- TOXNEUT -------- Group: 1 105428 105430 a 105431 105432 105433 105435 Dose Level: 50 - - - - - - - - - - - - Dosage Unit: mg/kg - Group: 2 105436 105437 Dose Level: 50 - - - - Dosage Unit: mg/kg - - - Extra 105429 - - - - 105434 - - - - 105438 - - - - a Plasmodium was observed. ANIMAL NUMBER ------ Group: 1 105439 105440 105442 105444 105447 105449 MEAN S.D. N Group: 2 105441 105446 MEAN S.D. N Extra 105443 105445 105448 MEAN S.D. N Summary and Individual Clinical Hematology Data Females Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE RBC X10'/,uL -------- HGB G/DL -------- HCT % -------- mcv FL -------- MCH PG -------- MCHC % -------- PLT X10'/,uL -------- PT SEC -------- PTT SEC -------- Dose Level: 50 Dosage Unit: mg/kg 7.08 6.97 5.29 6.52 6.07 7.01 12.9 11.9 11.0 11.5 11.7 13.1 6.49 .702 6 12.0 .82 6 Dose Level: 50 44.3 41.9 35.4 39.7 39.2 45.0 62.5 60.2 66.8 60.9 64.7 65.3 41.1 3.77 6 63.4 2.61 6 Dosage Unit: mg/kg 18.3 17.1 20.8 17.6 19.2 18.7 18.6 1.31 6 29.3 28.4 31.2 29.0 29.7 28.6 29.4 1.01 6 556 421 531 291 324 444 428 106.7 6 9.5 10.2 9.6 9.1 9.4 9.5 9.6 .36 6 14.7 18.1 14.3 14.0 15.9 15.5 15.4 1.50 6 7.64 5.58 6.61 1.457 2 14.1 10.4 12.2 2.62 2 50.8 33.6 42.2 12.16 2 66.6 60.2 63.4 4.53 2 18.5 18.7 18.6 .14 2 27.8 31.1 29.4 2.33 2 449 446 448 2.1 2 10.0 9.6 9.8 .28 2 17.3 13.2 15.2 2.90 2 6.37 6.29 5.99 6.22 .200 3 12.2 11.3 11.5 11.7 .47 3 37.8 38.0 38.9 38.2 .59 3 59.3 60.5 64.9 61.6 2.95 3 19.2 17.9 19.2 18.8 .75 3 32.3 29.6 29.6 30.5 1.56 3 392 402 402 399 5.8 3 9.9 9.3 9.7 9.6 .31 3 14.2 13.2 16.0 14.5 1.42 3 ANIMAL NUMBER ------ Group: 1 105439 105440 105442 105444 105447 105449 MEAN S.D. N Group: 2 105441 105446 MEAN S.D. N Extra 105443 105445 105448 MEAN S.D. N Summary and Individual Clinical Hematology Data Females Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE WBC X103 /,uL -------- N-SEG X103 /ML -------- LYMPH X103 li4L -------- MONO X10'/,uL -------- EOSIN X10'11AL -------- BASO X103 /,uL -------- N-SEG% -------- LYMPH% -------- MONO% -------- EOSIN% -------- Dose Level: 50 Dosage Unit: mg/kg 14.7 6.4 6.4 1.5 .4 .0 43 43 10 3 9.7 2.4 6.5 .5 .2 .0 25 67 5 2 21.0 6.4 13.4 .6 .6 .0 30 64 3 3 12.2 5.0 5.4 1.6 .2 .0 41 44 13 2 1110..48 54..03 55..74 ..66 ..51 ..00 4440 5500 65 51 13.3 4.13 6 4.9 1.49 6 7.1 3.11 6 .9 .3 .0 37 53 .51 .20 .00 7.8 10.2 6 6 6 6 6 7 3 3.7 1 6 6 DoSe Level: 50 Dosage Unit: mg/kg 15.2 7.6 5.5 1.2 .9 .0 50 36 8 6 12.3 5.7 5.7 .6 .3 .0 46 46 5 2 13.8 6.6 5.6 .9 .6 .0 48 41 6 4 2.05 1.34 .14 .42 .42 .00 2.8 7.1 2.1 2 2 2 2 2 2 2 2 2 2 2 14.3 4.3 8.2 1.1 .6 .1 30 58 8 4 12.5 5.4 5.5 1.5 .2 .0 43 44 12 1 10.8 3.0 5.5 1.9 .3 .0 28 51 18 2 12.5 4.2 6.4 1.5 .4 .0 34 51 13 2 1.75 1.20 1.56 .40 .21 .06 8.1 7.0 5.0 1. 3 3 3 3 3 3 3 3 3 3 Individual Clinical Hematology Data Females Presurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE ANIMAL NUMBER ------ ANISO -------- POLY -------- POIK -------- HYPO -------- TOXNEUT -------- Group: I 105439 105440 105442 105444 105447 105449 Dose Level: 50 - - - - - - - - - - - - Dosage Unit: mg/kg - - - - - - - - - - - - - - - - - - Group: 2 105441 105446 Dose Level: 50 - - - - Dosage Unit: mg/kg - - - - - - Extra 105443 - - - - - 105445 - - - - - 105448 - - - - - ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. 'N Extra 105434 Summary and Individual Clinical Hematology Data Males - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE RBC X10'/,uL -------- HGB G/DL -------- HCT % -------- mcv FL -------- MCH PG -------- MCHC % -------- PLT X10'/,uL -------- PT SEC -------- PTT SEC -------- Dose Level: 50 Dosage Unit: mg/kg 6.46 6.62 6.54 .113 2 11.9 11.9 11.9 .00 2 39.7 39.8 39.8 .07 2 61.4 60.1 60.8 .92 2 18.4 17.9 18.2 .35 2 30.0 29.9 30.0 .07 2 @679 519 599 113.1 2 9.0 8.9 9.0 .07 2 15.8 14.1 15.0 1.2 2 5.81 11.2 37.7 64.9 19.2 29.6 498 9.7 15.3 ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Summary and Individual Clinical Hematology Data Males - 4 Days Postsurgery WBC X10'/,uL -------- METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION N-SEG X10'/ML -------- LYMPH X101/juL -------- MONO X103 /,uL -------- EOSIN X103 /,uL -------- BASO X10'/,uL -------- ETHANOL (NET-FOSE; T-6316) IN OF A SINGLE CAPSULE DOSE N-SEG% LYMPH% MONO% EOSIN% -------- -------- -------- -------- Dose Level: 50 Dosage Unit: mg/kg 14.7 10.2 3.6 .8 .1 .0 70 24 5 1 9.4 5.2 3.1 .9 .3 .0 55 32 9 3 12.0 7.7 3.4 .8 .2 .0 62 28 7 2 3.75 3.54 .35 .07 .14 .00 10.6 5.7 2.8 1 2 2 2 2 2 2 2 2 2 2 10.3 3.6 5.7 .7 .2 .1 35 55 7 2 Individual Clinical Hematology Data Males - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE ANIMAL NUMBER ------ ANISO POLY POIK HYPO TOXNEUT -------- -------- -------- -------- -------- Group: 2 105436 105437 Dose Level: 50 - Dosage Unit: mg/kg Extra 105434 - ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Hematology Data Females - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE RBC X10'/,uL -------- HGB G/DL -------- HCT % -------- mcv FL -------- MCH PG -------- MCHC % -------- PLT X101/,uL -------- PT SEC -------- PTT SEC -------- Dose Level: 50 Dosage Unit: mg/kg 3.77 5.76 4.76 1.407 2 7.3 10.7 9.0 2.40 2 25.8 35.6 30.7 6.93 2 68.6 61.7 65.2 4.88 2 19.5 18.5 19.0 .71 2 28.4 30.0 29.2 1.13 2 458 598 528 99.0 2 9.3 8.7 9.0 .42 2 16.2 12.6 14.4 2.5 2 6.37 11.3 37.3 58.5 17.7 30.2 522 9.1 13.5 ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Hematology Data Females - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE WBC X10'/,uL -------- N-SEG LYMPH XIOI/juL X10111AL -------- -------- MONO X10'/,uL -------- EOSIN X10'/,uL -------- BASO X10'/,uL -------- N-SEG% LYMPH% -------- -------- MONO% -------- EOSIN% ------- Dose Level: 50 Dosage Unit: mg/kg 12.4 5.3 5.4 1.4 .2 .2 43 44 11 15.3 7.7 5.6 1.7 .2 .1 50 36 11 13.8 6.5 5.5 1.6 .2 .2 46 40 11 2.05 1.70 .14 .21 .00 .07 2 2 2 2 2 2 4.9 5.7 .0 2 2 2 10.4 3.8 5.5 .8 .2 .0 37 53 8 Individual Clinical Hematology Data Females - 4 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROC>CTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE ANIMAL NUMBER ------ ANISO POLY POIK HYPO TOXNEUT -------- -------- -------- -------- -------- Group: 2 105441 105446 Dose Level: 50 1 1 - - Dosage Unit: mg/kg Extra 105443 - ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Summary and Individual Clinical Hematology Data Males - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE RBC X10'/gL -------- HGB G/DL -------- HCT % -------- mcv FL -------- MCH PG -------- MCHC % -------- PLT X10'/,uL -------- PT SEC -------- PTT SEC -------- Dose Level: 50 6.10 6.28 11.5 11.5 6.19 .127 2 11.5 .00 2 Dosage Unit: mg/kg 38.0 37.5 62.2 59.7 37.8 .35 2 60.9 1.77 2 18.9 18.3 18.6 .42 2 30.4 30.6 30.5 .14 2 651 424 538 160.5 2 9.1 8.7 8.9 .28 2 15.2 14.0 14.6 .8 2 5.91 11.5 38.4 65.0 19.5 30.0 516 9.6 14.9 ANIMAL NUMBER ------ Group: 2 105436 105437 MEAN S.D. N Extra 105434 Summary and Individual Clinical Hematology Data Males - 8 Days Postsurgery WBC X103/,uL -------- METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION N-SEG XIO'/,uL -------- LYMPH XIO'/,uL -------- MONO X1031juL -------- EOSIN X103/juL -------- BASO X10'/,uL -------- Dose Level: 50 Dosage Unit: mg/kg ETHANOL (NET-FOSE; T-6316) IN OF A SINGLE CAPSULE DOSE N-SEG% -------- LYMPH% -------- MONO% -------- EOSIN% -------- 13.5 7.9 4.4 1.0 11.6 5.9 .2 .1 58 33 7 4.2 .9 .6 .0 51 36 12.6 6.9 4.3 1.0 .4 .0 1 a 5 1.34 2 1.41 .14 .07 54 34 .28 .07 4.9 2.1 6 3 2 2 2 2 2 2 2 .7 2 2 2 7.6 2.5 4.4 .6 .1 .0 33 58 8 1 Individual Clinical Hematology Data Males - 8 Days POstsurgery METABOLISM OF N-ETRYL PERFLUOROOCTANE SULFONAMIDO ETHANOL CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGL(ENETC-AFPSOUSLEE; TD-o6316) IN ANIMAL NUMBER ------ SE ANISO POLY POIK HYPO TOXNEUT -------- -------- -------- -------- -------- Group: 2 105436 Dose Level: 50 - Dosage Unit: mg/kg 105437 - Extra 105434 - ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Hematology Data Females - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE RBC HGB X10'/,uL G/DL -------- -------- Dose Level: 50 HCT t -------- mcv FL -------- MCH PG -------- Dosage Unit: mg/kg MCHC % -------- PLT X101/,uL -------- PT SEC -------- PTT SEC ------- 4.80 5.91 5.36 .785 2 9.4 10.8 10.1 .99 2 33.9 35.8 34.9 1.34 2 70.7 60.5 65.6 7.21 2 19.5 18.3 18.9 .85 2 27.6 30.2 28.9 1.84 2 773 652 712 85.6 2 9.0 15. 8.9 13. 9.o 14. .07 1. 2 5.76 10.7 34.4 59.7 18.6 31.2 497 10.0 16. ANIMAL NUMBER ------ Group: 2 105441 105446 MEAN S.D. N Extra 105443 Summary and Individual Clinical Hematology Females - 8 Days Postsurgery Data METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE CAPSULE DOSE WBC X101//jL -------- N-SEG X10'/14L -------- LYMPH X103 /juL -------- MONO X10-'/ML -------- EOSIN X10'/,uL -------- BASO X10'/juL -------- N-SEG% -------- LYMPH% -------- MONO% -------- EOSIN% -------- Dose Level: 50 Dosage Unit: mg/kg 14.6 5.8 6.7 1.8 .2 .1 40 46 12 2 16.5 6.5 7.6 1.3 1.0 .1 39 46 8 6 15.6 6.2 7.2 1.6 .6 .1 40 46 10 4 1.34 .49 .64 .35 .57 .00 .7 .0 2.8 2 2 2 2 2 2 2 2 2 2 2 9.9 3.3 5.5 .9 .2 .0 33 55 9 2 Individual Clinical Hematology Data Females - 8 Days Postsurgery METABOLISM OF N-ETHYL PERFLUOROOCTANE SULFONAMIDO ETHANOL (NET-FOSE; T-6316) IN CYNOMOLGUS MONKEYS FOLLOWING ADMINISTRATION OF A SINGLE-CAPSULE DOSE ANIMAL NUMBER ------ ANISO -------- POLY -------- POIK -------- HYPO -------- TOXNEUT -------- Group: 2 105441 105446 Dose Level: 50 - Dosage Unit: mg/kg Extra 105443 - Covance isan independent,publiclyheld company operatingin over 15 countriesand over30 offices worldwide,with headquartersin Princeton,New Jersey,USA. Covance isthemarketingname forCovance Inc. and itssubsidiarieasround theworld,the principal ones of which arelistedon thispage.The use of "Covance" in thisreportrefersto one or more of theseand other subsidiaries. THE AMERICAS Princeton 1.800.773.0011 EUROPE Brussels +32.2.773.29.10 ASIA/PACIFIC Singapore +65.7747233 COVANCE LABORATORIES Madison, Wi, USA 888-541-LABS (5227) Vienna,VA, USA 800-742-8378 Harrogate,UK Oll 44 1423 500011 MOnster, Germany Oll 49 251 97980 C 0 V AW-""CE@@ THE DEVELOPMENT SERVICES COMPANY Shaping Solutions PRIN7EO IN U.S.A.