Document GK9zMXOQ589djLebeyYJQey6n

COVAW'C-E-'-*'\ THE DEVELOPMENT SERVICES COMPANY Sponsor: 3M St.Paul,Ntinnesota FINAL REPORT Study Title: 13-Week DietaryToxicityStudywithN-Methyl Perfluorooctanesulfonamido Ethanol(N-MeFOSE, T-6314)inRats Author: PeterJ.Thomford, PhD Study Completion Date: June 30,2000 PerformingLaboratory: Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704-2595 LaboratoryStudy Identification: Covance 6329-225 Sponsor Study Identification: StudyNo. 3M T-6314.1 Page I of560 QUALITY ASSURANCE STATEmyNT Covance 6329-225 3M T-6314.1 Thisreport,withtheexceptionofAppendix 7,hasbeenreviewedby theQuality Assurance Unit of Covance LaboratoriesInc.,inaccordancewiththeFood and Drug Administratio(nFDA) Good LaboratoryPracticReegulations2,1 CFR 58. The datain Appendix 7 isgeneratedby and isthe responsibiliotfytheSponsor.The following inspectionwsere conductedand findingsreportedto thestudydirectoarnd study director management. Inspection Dates From To 08/31/98 08/31/98 08/31/98 08/31/98 04/12/99 04/29/99 04/02/99 04/07/99 06/02/99 06/02/99 04/19/00 04/20/00 04/28/00 04/28/00 04/28/00 04/28/00 Phase ProtocolReview MaterialDispensation ReportReview Data Review ProtocolAmendment Review Report Review ProtocolAmendment Review ProtocolAmendment Review Date Reported to StudyDirectorand StudyDirectorManagement 08/31/98 08/31/98 04/29/99 05/04/99 06/02/99. 04/20/00 04/28/00 04/28/00 Representative QualityAssurance Unit io Dat 2 STUDY IDENT]IFICATION Covance 6329-225 3M T-6314.1 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) inRats Test Material N-Methyl Per-fluorooctanesulfonamido Ethanol (MEFOSE, T-6314) Sponsor 3M Toxicology Services Building 220-2E-02, 3M Center St.Paul, Minnesota 55144-1000 Study Monitor Andrew M. Seacat, PhD 3M Toxicology Services 651.575.3161 Alternate Study Monitor Marvin T. Case, DVK PhD 3M Toxicology Services 651.733.5180 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Study Director Peter J.Thomford, PhD Covance Laboratories Inc. P.O. Box 7545 Madison, Wisconsin 53707-7545 608.241.7207 Study Timetable Study InitiationDate In-Life StartDate In-LifeEnd Date Study Completion Date August 27, 1998 September 1, 1998 December 2, 1998 June 30, 2000, 3 KEY PERSONNEL Covance 6329-225 3M T-6314.1 Study Director StudyToxicologist StudyCoordinator Supervisor,SmallAnimal Toxicology SupervisorD,ose Formulation SupervisorC,hemistry/ToxSupport SupervisorL,aboratoryAnimal Medicine ClinicaPlathologist SupervisorC,linicaPlathology AnatomicalPathologist AnatomicalPathologist SupervisorA,natomicalPathology PeterJ.Thomford, PhD Thomas E. Ryan, BS NiiVan Bruce-Konuah Nadian E. Snortum,BA, BT, ALAT DixieBushee,BS, LATG BrianSchoenike,BS Donna J.Clemons,DVM Diplomate,ACLAM RobertL.HalL DVM, PhD Diplomate,ACVP (ClinicaPlathology) RonaldMarkevitchB,S, Mr (ASCP) Sandra R. Eldridge,PhD PathologyAssociatesInternational JohnnieJ.Eighmy,DVM, MS Diplomate,ACVP Diplomate,ABT Deborah PirkeLALAT 4 Covance 6329-225 3M T-6314.1 CONTENTS Page ABSTRACT ..............................................................8............ PURPOSE ...............................................................1.1............ REGULATORY COMPLIANCE ............................................1.1............ TEST MATERIAL ........................................................I.I............ TestMaterial..........................................................I.I............ Reserve(Arcbive)Sample................................................I.I............ Dispositio.n...........................................................1.1............ ANIMALS AND HUSBANDRY ............................................1.2............ TestAnimal ...........................................................1.2............ Identificat.i.o.n........................................................1.2............ Acclimation...........................................................1.2............ Housing and Maintenance................................................1.2............ Justificati.o.n.........................................................1.3............ PROCEDURES ..........................................................1.3............ Group Designationsand Dose Levels.......................................1.3............ Dose Preparation.......................................................1.4............ RetentionSamples ......................................................1.5............ Method ofAdministratio.n...............................................1.5............ Dose Analyses.........................................................1.5............ ClinicaOlbservations....................................................1.6............ Body Weights..........................................................1.6........... Food Consumption ......................................................16............ Ophthalmology.........................................................1.7........... ClinicaPlathology......................................................................1.7....................... Serum PerfluorooctanSeulfonicAcid Level(PFOS) Determination 18 Necropsy ..............................................................i.s............ Organ Weights.............................................:..........................1.9..................... CellProliferatiToinssueCollectioannd ImmunohistochemicalEvaluation..............1.9... Pahnitoyl-CoAOxidase TissueCollectionand Analyses.........................1.9........... LiverPFOS Analyses.............................4..................................1.9........... TissuePreservatio.n.....................................................20............ Histopathology.........................................................2.0........... StatisticAanlalyses......................................................21............ RECORD RETENTION ...................................................2.1............ 5 Covance 6329-225 3M T-6314.1 CONTENTS (Continued) Page RESULTS ............................................................................2.2................ Dose Analyses ..............................................................2.2............... ClinicalObservations and Survival.............................................2.3............... Ophthalmology ..............................................................2.3.............. Body Weights ...............................................................2.3............... Food Consumption ..........................................................2.3............... Test MaterialConsumption ...................................................2.4............... ClinicalPathology ............................................................2.4.............. Serum PerfluorooctaneSulfonicAcid Level (PFOS) Determination ................2.5.......... CellProliferatioTnissue Collectionand Immunohistochemical Evaluation...........2.5...... LiverPFOS Determination....................................................2.5............... Anatomic Pathology .......6...............I...................................2.-.5.............. CONCLUSIONS SIGNATURES ..............................................................2.6............... ................................................................2.7............... REFERENCES ................................................................28............... OPHTHALMOLOGY REPORT .................................................2.9.............. PATHOLOGY COMMENTS REPORT ........................................................30............... ON THE DATA ..................................................3.7............... CODES, ABBREVIATIONS, AND UNITS .......................................3.9............... General Codes and Abbreviations..............................................4.0............... Codes forClinicalPathology ..................................................4.1............... Abbreviationsand Units forClinicalHematology .................................4.4.............. Abbreviationsand Units forClinicalChemistry ..................................4.6............... Abbreviationsand Unitsfor ClinicalUrinalysis...................................4.8............... Codes forAnatomic Pathology ................................................4.9............... TABLES I Resultsof Homogeneity Analyses (ppm) ....................................5.1................ 2 Resultsof StabilitAynalyses (ppm) .........................................5.2............... 3 Resultsof Dose PreparationAnalyses (ppm) .................................5.5............... 4 Summary of ClinicalObservations..........................................5.6............... 5 Sumnwy of Ophthalmic Observations.......................................5.8............... 6 Summary of Body Weight Data (g).........................................5.9............... 7 Summary of Body Weight Change Data (g)..................................6.1............... 8 Summary of Food Consumption Data (g)....................................6.3............... 6 Covance 6329-225 3M T-6314.1 CONTENTS (Continued) Page TABLES 9 Summary ofTestMaterialConsumptionData (mg/kg/day)...................6.5............ 10 Summary of ClinicaHlematology Data -Week 5...........................6.7............ 11 Summary of ClinicaHlematology Data-Week 14...........................71............ 12 Summary of ClinicaClhemistryData - Week 5 .............................7.5........... 13 Summary of ClinicaClhemistryData - Week 14............................8.1........... 14 Summary of ClinicaUlrinalysiDsata -Week 14 ............................8.7........... 15 Sumniary of Organ Weight Data - Week 5 Sacrific.e........................89............ 16 Summary of Organ Weight Data - Week 14 Sacrific.e.......................91............ 17 Incidenceof Macroscopic Observations.................................1.0.9............ 18 Incidenceof MicroscopicObservations..................................1.1.2........... 19 Incidenceof Severityof SelectedMicroscopicObservation.s................1.2.1............ APPENDIX I...........................................................1.2.4............ ProtocolDeviations....................................................1.2.5............ Protoco.l.............................................................1.2.7........... ProtocolAmendment No. I..............................................1.4.4........... ProtocolAmendment No. 2 ..............................................1.4.6........... ProtocolAmendment No. 3 ..............................................1.4.9........... APPENDIX 2...........................................................1.5.1............ IndividuaAlnimal FateData..............................................1.5.2........... IndividuaCllinicaOlbservation.s..........................................1.5.7........... IndividuaOlphthalmicObservations.......................................1.7.3............ APPENDIX 3...........................................................1.8.9............ IndividuaBlody WeightData (g)..........................................1.9.0........... APPENDIX 4...........................................................2.0.1............ IndividuaFlood Consumption Data (g).....................................2.0.2........... IndividuaTlestMaterialConsumptionData (mg/kg/day.)......................2.1.3............ APPENDIX 5...........................................................2.2.3............ IndividuaCllinicaHlematology Data .......................................2.2.4........... IndividuaCllinicaClhemistryData .........................................2.4.8............ IndividuaCllinicaUlfinalysiDsata.........................................2.7.6............ APPENDIX 6 ...........................................................2.8.8........... IndividuaAlnatomic PathologyData .......................................2.8.9........... APPENDIX 7 ...........................................I ................5.4.6........... CellProliferatiRoenport................................................5.4.7........... 7 ABSTRACT Covance 6329-225 3M T-6314.1 'Ihepurpose of thisstudywas to assessthetoxicitoyf thetestmaterial N-Methyl PerfluorooctanesulfonaniEitdhoanol(N-MeFOSE, T-6314) when administered inthedietto ratsforatleast13 weeks. Four groups ofCrl:CDO(SD) IGS BR ratswere exposedtoN-MEFOSE inthedietforat least13 weeks. Each group contained20 males and 20 females.The dietary concentrationwsere 0 (controglroup),3,30,and 100 ppm. Food was providedad libitume,xceptwhen anfinalwsere fasted.Water was provided ad libitum.The animalswere observedtwicedaily(a.rmand p.irlf)ormortalitaynd moribundity.At leastonce weekly,each animalwas removed from itscage and examined forabnormalitieasnd signsof toxicityB.ody weightdatawere collecteodn thefirsdtay of treatmentand weekly thereafterF.ood consumptiondatawere collectewdeekly. Ophthaln-Acexaminationswere done beforeinitiatiofntreatmentand duringWeek 14. Blood sampleswere collectefdorhematology and clinicaclhemistrytestsduringWeeks 5 and 14. Urinesamplesforurinalysitsestswere collecteduringWeek 14. During Weeks 5 and 14,blood was collectefdrom fiveanimals/sex/groufporserum perfluorooctanseulfonicacid(PFOS) analyses.At theWeek 5 sacrificsea,mplesof the liverwere collectefdrom fiveanimals/sex/groufporpalmitoyl-CoAoxidaseactivitaynd liverPFOS analysis.AninmlssacrificeadtWeek 5 were notnecropsied.During Week 14, theremaininganimalswere anesthetizedw,eighed,exsanguinateda,nd necropsied.At necropsy,macroscopic observationswere recorded,selectedorganswere weighed,and selectedtissueswere collected and preserved.Samples ofliverwere collectedfrom all survivinganimalsforpahnitoyl-CoAoxidaseactivitaynd liverPFOS analysisS.amplesof theliverwere collectefdrom fiveanimaWsex/group forproliferaticoenllnuclearantigen (PCNA) evaluatioannd microscopicexamination.Nhcroscopicexaminationswere done on tissuesfrom each animalinthecontroland high-dosegroups(terminaslacrificoenly) and from thelow-doseanimalthatwas sacrificeadtan unscheduledintervalN.ficroscopic examinationswere alsodone on theEver and stomach from each terminalsacrificaenimal inthemid-dosegroup,and on theliverfrom each terminalsacrificaenimalinthelow-dose group- 8 Covance 6329-225 3M T-6314.1 One femalegiven3 ppm N-MEFOSE was sacrificeodn Day 59 due to severelaceratioonf thetail.There were no macroscopicor nucroscopicchangesto indicattelidittsmoribund conditionwas testmaterial-relateAdU. otheranimalssurvivedto thescheduledsacrifice. No clinicaolbservationswere notedthatwere consideredtorepresentprimaryeffectsof thetestniaterialStatisticaslilgynificantlloywermean body weightswere notedbeginning atWeek 4 and continuingthroughtheend of studyforanimalsgiven 100 pprm Statisticalsliygnificantlloywer mean body weightswere noted beginningatWeek 7 and continuingthroughtheend of studyformalesgiven30 ppm. Body weightchangeswere statisticaslilgynificantlloywer ina sporadicmanner foraniniangiven30 or,100 ppHL Overallbody weightchanges(Weeks 1-14)were statisticaslilgynificantlloywerforall groupsgivenN-MEFOSE. Although statisticaslilgynificaonntlyatWeek 13,food consumptionwas ingenerallowerformalesgiven100 ppnl For femalesgiven100 ppm, statisticallolwyer food consumptionwas apparentatWeeks 3,4, 6,7, 11,and 12. Dietaryadministrationf N-MEFOSE was associatewdithlower hematocritformalesand femalesfed 100 ppm; higherureanitrogenformales and femalesfed100 ppm; higher albuminfornialesfed 100 ppm; lowerglobulinformales fed30 or 100 ppm; lower cholesteroalnd triglyceridfeosrmales fed30 or 100 ppm and femalesfed 100 ppm; higher alanineaminotransferasfeormales fed100 ppm; and higherhepaticpahnitoylCoA oxidaseformales and femalesfed100 ppn-L The effectwsere relativelmyud withthe exceptionof thoseon cholesteroalnd triglyceridfeosrthehigh-dosenialest;hoseeffects were consideredto be of moderate severityA.llof thetestmaterial-relatceldinical pathologyfindingwsere more prominentatWeek 14 thanatWeek 5. Statisticalsliygnificanatnd testmaterial-relatcehdangesinterminalbody weightsand liver weightswere seeninmales and/orfemalesinthetwo highestdose groups. Statistically significandtecreaseswere seen inthetern-iirbwody weightsof males fed 30 ppm and in both males and femalesfed100 ppm of N-MEFOSE. Statisticalsliygnificainntcreases were seeninthemean hver-to-bodyweightpercentageand mean hver-to-braiwneight ratioinnialesfed30 ppm, inmean absoluteliverweight and mean liver-to-bodwyeight percentageinfemalesfed30 ppm, and inallliverweightparametersinmales and females fed100 ppm. 9 Covance 6329-225 3M T-6314.1 Test material-relatemdacroscopic findingswere noted at a low incidencein the liverand glandularstomach of nialesand femalesin the 100 ppm dose group only. Liver findings includedorgans thatwere largeand dfffuseldyark. In the glandularstomach, dark focior areas were sometimes seen in the gastricmucosa. Test material-relatemdicroscopic findingswere seen inthe liverof anftnalsfed 30 and 100 ppm of N-MEFOSE and inthe glandularstomach of males and femalesin the 100 ppm group only. No testmaterial-relatecdhanges were seen in animals fed 3 ppm of N-MEFOSE. In the livero,ne or more testmaterial-relatefdindingsaffectedallaninws fed 100 ppm and includedslight-to-moderatecentrflobulaorr diffusehepatocellular hypertrophy,min@-to-sHght coagulativenecrosis,mhiinw hepatocerularpigment, and mhinml-to-shght centrilobulatro nfidzonalor diffusehepatocerularvacuolation.In animalsfed 30 ppm, centrilobulahrepatocerularhypertrophy was alsoseen,although the incidenceand severitywas decreased at thisdose. In addition,a low incidenceof minimal hepatoceffularvacuolationoccurred in30 ppm dose group males,and theliverof a single female fed 30 ppm had minimal coagulativenecrosis.A possibletestmaterial-related change in the glandularstomach of animals fed 100 ppm was a relativellyow incidenceof mucosal erosions.The erosionswere of minimal to slightseverityand involveda very small portion of the mucosal surfacein allcases. Based on anatonficpathology,clinicaplathology,and body weight effectsat 30 and 100 ppm the no-observable-adverse-effecltevelwas determined to be lessthan 30 ppm when N-MEFOSE isadministeredat concentrationsof 0, 3, 30 and 100 ppm to Crl:CD"'(SD) IGS BR ratsforat least13 weeks through dietaryadministrationB.ased on thebody weight effectsat 3, 30, and 100 ppm, theno-observable-effecltevelwas determined to be lessthan 3 ppm when N-MEFOSE isadministeredat concentrationsof 0, 3, 30 and 100 ppm to Crl:CDO (SD) IGS BR ratsforat least13 weeks through dietary administration.In the absence of correlativcehanges in anatomic pathology and clinical pathology the body weights effectsat 3 ppm were not consideredto be biologically adverse. 10 PURPOSE Covance 6329-225 3M T-6314.1 The purpose of thisstudy was to assess the toxicityof the testmaterial, N-Methyl PerfluorooctanesulfonamidoEthanol (N-MeFOSE, T-6314) when administered in thedietto ratsforat least13 weeks. REGULATORY COMPLIANCE Allaspectsof thisstudy were in accordance with the Food and Drug AdministrationGood Laboratory PracticeRegulations as setforthin Title21 of the US Code of Federal Regulations,Part 58, issuedDecember 22, 1978 (effectivJeune 20, 1979),and with any applicableamendments. TEST MATERIAL Test Material The testmaterial,N-Methyl PerfluorooctanesulfonamidoEthanol (MEFOSE, T-6314), Lot No. F-1 1364, isan offwhite powder. Itwas receivedat Covance on August 3, 1995. The testmaterialwas storedat room temperature. Information on synthesismethods, stabilityp,urity,composition, or other characteristics thatdefinethe testmaterialison filewith the Sponsor. Reserve (Archive)Sample A reserve sample (approximately5 g) of thetestmaterialwas taken and storedat room temperature. This sarnplewillbe transferredto the Sponsor aft-earuthorizationfrom the Sponsor. Disposition Remaining testmaterialwillbe returned afterauthorizationfrom the Sponsor. ANIMALS AND HUSBANDRY Covance 6329-225 3M T-6314.1 Test Animal Male and female Crl:CDO(SD) IGS BR ratswere obtained from the Portage, Michigan, facilitoyf Charles River Laboratories,Inc.,on August 17, 1998. The animalswere 39 to 45 days old at initiatioonf treatment.The nialesweighed from 184 to 236 g, and the females weighed from 140 to 179 g at initiatioonf treatment. Identification Each aninml was assigned a temporary number upon arrival.Before initiatioonf treatment,a microchip identificatiodnevicewas implanted intoeach anhual After randomization for placement on test,each animal was assigneda permanent number, and the microchip was coded with thatnumber. Alldata foran animal are recorded under these numbers. Accffimtion Ninety-two males and 92 females were received on August 17, 1998, and acclimated in Animal Room 348 for 15 days beforeinitiatioonf treatment. In general,aninialsin this shipment appeared healthy.During acclimation,the animals were examined for abnormalitiesindicativeof healthproblems, an ophthahnic examination was done, and body weights were recorded forallanimalsat randomization. Housing and Maintenance Animal Room 348 was used forthisstudy. Environmental controlsforthe aninialroom were setto maintain 18 to 26'C, a relativehumidity of 30 to 70%, and a 12-hour light/12-hourdark cycle. The animals were housed individuall(yexceptforthe first7 days of acclimationwhen animals were group-housed) in stainlessteel,screen-bottom cages. 12 Covance 6329-225 3M T-6314.1 Certifierdodentdiet#5002 meal,(PNE NutritioInnternationawla)s provided(see ProtocolDeviationsforexceptionsa)d libitume,xceptwhen animalswere fasted.The dietisroutinelaynalyzedby themanufacturerfornutritionaclomponents and environmentalcontaminants.The resultasreon filewithCovance-Madison. Water was providedad libitum.Samples of thewater areroutinelaynalyzedforspecified nucroorganismsand environmentalcontaminants.The resultasreon filewith Covance-Madison. There were no known contaminantsinthedietor water atlevelsthatwould have interferewdiththisstudy. Animalsnot selectedforthestudy(12 malesand 12 females)were removed from the studyroom and used fortraininpgrocedures. Justification Historicallryatshave been used insafetyevaluationstudiesand arerecommended by appropriateregulatoryagencies. PROCEDURES This studywas conducted inaccordancewith theProtocoldatedAugust 27, 1998,and ProtocolAmendment Nos. 1,2,and 3. The protocol,protocolamendments, and protocol deviationsareinAppendix 1. Group Designationsand Dose Levels The animalswere examined by a laboratoryanimalveterinariaonn August 31, 1998,and foundto be suitablfeorstudyconsiderationS.electionof animalsforthe studywas based on clinicaolbservationsb,ody weights,ophthalmicexaminationsa,nd otherdataas appropriate.Animalswere assignedto treatmentgroupsusinga computerizedblocking proceduredesignedto achievebody weightbalancewithrespectto treatmentgroup. At thetimeof randomizationt,heweightvariatioonf theanimalsforeach sex used didnot 13 Covance 6329-225 3M T-6314.1 exceed 2 standard deviationsof the mean weight. Group mean body weights were analyzed using Levene's testforhomogeneity of varianceat the 5.0% probabilityleveland found to be homogeneous. Animals were assignedto the study accordingto the following design: Number of Animals DietaryLevels Group I (Control)b Male 20 Feniale 20 (ppm MeFOSE)a 0 2 (Low) 20 20 3 3 (Nfid) 20 20 30 4 (Ifigh) 20 20 100 a Dose levelswere expressedas ppm of MEFOSE. b The controlanimalsreceivedthe basaldietonly. Dose Preparation Dietaryconcentrationswere based on thetestmaterialas supplied.Before initiatioonf treatment,dietsof 1, 3, 30, 100, and 500 ppm concentrationswere mixed. Diets were prepared forstabilitaynalysesat the I-ppm and 500-ppm concentrationsand for homogeneity analysesat the 3, 30, and 100 ppm concentrations.Diets were prepared once every 4 weeks for use in the in-hfephase. Preparation of Diet for Group 1. The appropriateamount of dietwas weighed intoa labeledcontainerand acetone was added at the same concentrationas the high-dose concentration.The combination of the dietand acetone was then mixed for 15 minutes. Preparation of Diets for Groups 2 through 3. Each dose levelwas prepared independently. A specifiedamount of dietwas weighed intoa labeledcontainerand a specifiedamount of dietforGroup 4 was weighed foreach diet. A trenchwas fon-nedin the feed in the mixing bowl and the amount of dietforGroup 4 was transferredto the trenck The contentsof themixing bowl were thoroughlymixed for 15 minutes. Preparation of Diet for Group 4. A specifiedamount of dietwas weighed intoa labeled container.The required amount of testmaterialwas weighed and transferredintoa labeledcontainer.Approximately 5 mL of acetone was added to the containerand mixed manually. Increments of approximately 5 mL acetone were added as necessaryuntilthe testmaterialhad dissolved.To prepare a premix, the dietwas transferredintoa labeled 14 Covance 6329-225 3M T-6314.1 Hobartomixingbowl. The testmaterialand acetonewere added tothemixingbowl, overlaidwitha portionofdietfrom thenixingbowl,and thoroughlymixed.A portionof dietfrom themixingbowl was transferretdo a secondmixingbowl mixed manuallyto recoverresidualtestmaterialand returnedto thefirsmtixingbowl. The contentsofthe mixingbowl were thoroughlymixed. Samples fordose analyseswere takendirectlfyrom themixingbowl The preparedtestdietswere storedatroom temperatureincoveredcontainerusntil dispensedintofeedingj'ars. Retention Samples During thein-lifpehase,samples(approximately100 g) were taken from each dose preparationfordose analysesand storedatroom temperature.These sampleswere discardedon May 11,1999 (seeProtocolDeviationsforexceptions). Method of Administration Dietary admixture was used because the potential route of exposure in humans is oral The dose preparations were administered ad libitum for at least 13 weeks, unless otherwise specified. Dose Analyses Analyses for the concentration of testmaterialin the dose preparations were done by Covance using an analyticalmethod, W-M324-MA, supplied by the Sponsor and validatedby Covance. Homogeneity was determined for the 3-ppm 30-ppm, and 100-ppm dose preparations once pretest.One sample (approximately 100 g) each from the top, middle, and bottom of the dose preparations mixed for homogeneity analyses were collected,divided into three subsamples for extractionand analysis,and analyzed for testmaterialcontent. All samples were stored at room temperature untilanalyzed within 7 days of mixing. 15 Covance 6329-225 3M T-6314.1 Four samples(approximatel1y00 g each)were takenfrom the I-ppm dose level concentratioonf dietpreparationmsixed pretest.One sample was analyzedon theday of mixing and usedas thebaselinevalue.One samplewas storedatroom temperatureforat least19 days,thenanalyzed.A thirdsample was storedatroom temperatureforatleast 32 days,thenanalyzed.The remainingsamplewas storedina freezersettomaintain -10 to-30'C for8 weeks,thenanalyzed.Inadditiont,wo samples(approximatel1y00 g each)were takenfrom the500-ppm dosepreparationmixed pretest.One samplewas analyzedon theday of mixing.The second sample was storedatroom temperatureafter atleast32 days,thenanalyzed.Two additionaslamples(approximatel1y00 g each)were takenfrom the3-ppm dose preparatiomnixed forWeeks 9 through12. One sample was storedatroom temperatureforatleast19 days,thenanalyzed.The second sample was storedatroom temperatureforatleast32 days,thenanalyzed.The sample collectefdor dose confimiationanalysiswas used as thebaselinesample. During thein-lifpehase,samples(approximatel1y00 g) from alldosepreparationwsere collecteadnd analyzedfordose confirmationA.U sampleswere storedatroom temperatureuntilanalyzed. ClinicalObservations The animalswere observedtwicedaily(a.nLand p.nL)formortalityand moribundity(see ProtocolDeviationsforexceptions)S.ignsofpoor healthor abnormalbehaviorwere recordedas theywere observed.At leastonce weekly,each animalwas removed from its cage and exan3ined.Any unusualor abnormalfindingwsere recorded. Body Weights Individuablody weightdatawere recordedon thefirsdtay of treatment,and weekly thereafter. Food Consumption Individualfood consumption datawere recordedweekly duringtreatment.Food consumption was not measured duringWeek 5 foranimalsscheduledforsacrificdeuring Week 5. 16 Covance 6329-225 3M T-6314.1 Ophthalmology Ophthahnicexaminationswere done beforeinitiatioofntreatmentand duringWeek 14. The pupilswere dilatedwith0.5% Mydriacyl@,and theeyeswere examined by a veterinariawnith an indirecotphthalmoscope. ClinicalPathology Blood sampleswere collectefdrom 10 animaLs/sex/groudpuringWeeks 5 and 14. Urine sampleswere collectedfrom 10 animaL-/sex/groudpuringWeek 14. Animalswere fasted overnight(,seeProtocolDeviationsforexception)and urinewas collectecdhilled. (approximately16 hoursbeforebloodsampling)w;aterwas providedad libitum.Blood was collectefdrom a jugularvein.PotassiumEDTA was used as theanticoagulanftor hematology tests.No anticoagulanwtas used fortheclinicaclhemistrysamples.Animals were bledinrandom order.The followingwere evaluated: Hematology red bloodcell(erythrocytec)ount hemoglobin hematocrit mean corpuscularvolume mean corpuscularhemoglobin mean corpuscularhemoglobin concentration platelectount whitebloodcell(leukocytec)ount differentiballood cellcount segmented neutrophilcount lymphocytecount monocyte count eosinophiclount basophilcount bloodcellmorphology Reticulocytceount smearswere made and heldforpossiblefutureexamination. glucose urea nitrogen creatftiine totalprotein albumin globulin totalbuirubin cholesterol triglycerides ClinicalChemistry aspartateaniinotransferase alanineaniinotransferase gamnia glutamyltransferase calcium inorganicphosphorus sodium potassium chloride 17 volume specificgravity pH protein glucose ketones Covance 6329-225 3M T-6314.1 Urinalysis bflirubin blood urobilinogen microscopic examination of sediment appearance Serum Perfluorooctane Suffonic Acid Level (PFOS) Determination During Weeks 5 and 14, fiveanimals/sex/goup were fastedovernight,(seeProtocol Deviationsforexception)anesthetizedwith carbon dioxide,and blood (approximately 4 mL) was collectedfrom a jugularvein. Allsamples were collectedwithout anticoagulanta,llowed to clotat room temperature,and centrifuged.Serum was harvestedand storedina freezersetto maintain -60 to -80'C untilpacked on dry iceand shipped to the Sponsor foranalyses.The samples were analyzed forPFOS. Resultsof analyseswillbe reported separatelyby the Sponsor. Necropsy A necropsy was done on the Group 2 fenmle thatwas sacrificedat an unscheduled interval.During Week 5, fiveanimaLVsex/group were fastedovernight(seeProtocol Deviationsforexception),anesthetizedwith carbon dioxide,bled forserum PFOS samples,weighed, and exsanguinated. These animals were discardedwithout necropsy. The liverwas removed from these animals,weighed, and collectedfor pahidtoylCoA oxidase and PFOS analysis.During Week 14,the renuahiinganimalswere fastedovernight,anesthetizedwith carbon dioxide,bled forclinicaplathology tests (10 anfinals/sex/grouwpith the exceptionof Group 2 female) and serum PFOS samples (fiveanimals/sex/group)w,eighed, exsanguinated,and necropsied. Liver samples were collectedforpalinitoyCloA oxidase,PCNA, and PFOS analysis.Anfimls were necropsied in random order. The necropsy includeda macroscopic examination of the externalfeaturesof the carcass; a externalbody odfices;theabdominal, thoracic,and cranialcavitieso;rgans;and tissues. 18 Covance 6329-225 3M T-6314.1 Organ Weights After13 weeks oftreatmentt,hefollowingorgans(when present)were weighed;paired organswere weighed together: adrenal(2) brain kidney(2) liver ovary(2) spleen testi(s2) thymus thyroid(2)withparathyroid Organ-to-bodyweightpercentagesand organ-to-braiwneightratioswere calculated. CellProliferatioTnissue Collectionand b=unohistochemical Evaluation At theterminalsacrificree,presentativseamplesof theleftlaterallobeof theliverwere collectefdrom fiveanimaLs/sex/grouapnd preservedinzincformalin.Afterfixation, sampleswere embedded inparaffinand shippedto PathologyAssociatesInternational (FTederickM,aryland)forproliferaticoenllnuclearantigen(PCNA) evaluationI.n additionl,iversectionwsere stainedwithhematoxylinand eosinand exanfined microscopicallyR.esultsof theevaluatioanreinAppendix 7. Pahnitoyl-CoA Oiddase TissueCollectionand Analyses At thescheduledsacrificetsh,erightlaterallobeoftheliverwas collectefdrom each animal,flash-frozeinnliquinditrogena,nd storedina freezersetto maintain-60 to -80'C untilanalyzedby Covance forpahnitoylCoA oxidaseactivity. LiverPFOS Analyses At thescheduledsacrificeas,portionof theliverfrom each animalwas flash-frozeinn liquidnitrogenand storedina freezersetto maintain-60 to,-80'Cuntilpacked on dry ice and shippedto theSponsor forPFOS and metabolitesanalyses.Resultsof theseanalyses willbe reportedseparatelbyy theSponsor. 19 Covance 6329-225 3M T-6314.1 Tissue Preservation The following tissues(when present) from the aninialthat was sacrificedat an unscheduledintervalor from each anfta sacrificeadtthe terminalsacrificweere collectedand preservedin 10% neutral-bufferefdormalin,unlessotherwisespecified: adrenal(2) aorta brain cecum cervix colon duodenum epididymis(2) esophagus eye (2) femur withbone nmffow (articular surfaceof the distalend) Harderian gland heart ileumwith Peyer'spatch(lymphoid aggregate) jejunum Iddney(2) lesions Ever lung with mainstem bronchi lymph nodes (mesentericand mandibular) mammary glands(femalesonly) ovary(2) pancreas pituitary prostate rectum salivargyland[mandibular(2)] sciaticnerve seminalvesicle(2) skeletamluscle (thigh) skin spinalcord (cervicalmid-thoracica,nd lumbar) spleen sternum with bone marrow stomach testi(s2) thymus thyroid(2)withparathyroid trachea urinarybladder uterus vagina Zynibal'sgland lestopathology Tissues(asappropriate)were embedded inparaffins,ectioned,stainedwith hematoxylin and eosin,and examined microscopicallfyrom each animal inthe controland high-dose groups and from the low-dose anftnalthatwas sacrificeadtan unscheduled intervalD.ue tomicroscopicfindingsnoted inthe high-dosegroup, the liverand stomach from animals inthe mid-dose group, and the liverfrom animalsinthe low-dose group were also embedded inparaffins,ectioned,stainedwith heniatoxylinand eosin,and examined microscopically. 20 Covance 6329-225 3M T-6314.1 Macroscopic lesionsfrom animalsinthelow- and mid-dosegroups were embedded in paraffins,ectioned,stainedwithhematoxylinand eosin,and examined microscopically. Bone marrow smearsfrom thefemur of each animalwere preparedatthescheduled sacrificaefter13 weeks oftreatment,stainedwithWright'sstain,and retainedforpossible examination. StatisticaAlnalyses Levene'stest(Levene,1960) was done to testforvariancehomogeneity. In thecaseof heterogeneitoyf varianceatp :50.05,transformationwsere used to stabiliztehevariance. Comparison teststook varianceheterogeneitiyntoconsideration. One-way analysiosf variance[ANOVA (Winer,1971a)]was used to analyzebody weights,body weight changes,food consumption,continuousclinicaplathologyvalues; organweights,organ-to-bodyweightpercentagesa,nd organ-to-braiwneightratios.If theANOVA was significanDtu,nnett'st-tes(tDunnett,1964)was used forcontrolversus treatedgroup comparisons. Group comparisons(Groups2 through4 versusGroup 1)were evaluatedatthe 5.0%, two-tailepdrobabilitlyevel Only datacollecteodn orafterthefirsdtay of treatmentwas analyzedstatistically. RECORD RETENTION Allraw data,documentation,records,protocol,and specimensgeneratedas a resultof thisstudywillbe archivedinthestoragefacilitioefsCovance-Madison fora periodof at leastI year.At leastI yearafterthesubmissionofthefinalreport,the Sponsorwill determinethefinaldispositioonf thematerialsA.llraw datastoredon magneticmedia, theprotocoland protocolamendments, studycorrespondence,and an originaclopy ofthe finalreportwillbe retainedby Covance-Madison. PCNA evaluationdata,paraffibnlocks,and tissueslideswillbe retainedby Pathology AssociatesInternational. 21 Covance 6329-225 3M T-6314.1 Liver and serum samples sent to the Sponsor and analysisdata generated from these samples willbe retainedby the Sponsor. RESULTS Dose Analyses Results of the homogeneity, stabilitya,nd dose preparationanalysesare inTables I through 3. Mean valuesof the homogeneity analysesranged from 74.0-78.0%, 84.0-96.7%, and 77.5-84.2% of the theoreticacloncentrationsforthe dietscontaining3, 30,and 100 ppm N-MEFOSE, respectively.These resultsindicatethattheniaing procedure produced a homogeneous distributioonf the testmaterialin thedose preparations; although variabilitgyenerallyappeared slightlgyreaterat the 3 and 30-ppm dietary concentrations. Results of stabilitaynalysesof samples stored for 19 and 32 days atroom temperature and 8 weeks under frozenconditionsindicatedthatthe mean concentrationsranged from 54.4-62.5% of the initiacloncentrationsof 1 ppm fordietmixed pretest.Samples stored for32 days atroom temperature indicatedthatthe mean concentrationwas 98.4% of the initiacloncentrationsof 500 ppnl Resultsof stabilitaynalysesof samples storedfor 19 and 32 days atroom temperature indicatedthatthe mean concentrationsranged from 93.2-94.0% of the initiacloncentrationsof 1 ppm fordietmixed at Week 4. Samples storedfor 19 days at room temperature indicatedthatthe mean concentrationwas 89.0% of the initiacloncentrationsof 3 ppm. The mean concentrationsof thedose preparationanalysesforalllevelsranged from 63.7 to 110.0% of the theoreticacloncentrations.Inherentvariabilitaynd lackof sufficienstensitivitayt low levelsin theanalyticalmethod formeasuring N-MEFOSE resultedin homogeneity, stabilitaynd routine analysisdata thatwere in many cases outside of the standardlimitsof +/- 15%. These resultswillbe reevaluatedin conjunctionwith the analyticarlesultsfrom the blood and liverleveldeterminationswhen they areprovided by the sponsor. 22 Covance 6329-225 3M T-6314.1 ClinicalObservations and Survival ClinicaolbservationasresununarizedinTable4;individuadlataareinAppendix 2. IndividuaalninialfatedataarealsoinAppendix 2. One femalegiven 3 ppm N-MEFOSE was sacrificeodn Day 59 due to severelaceratioonf thetailThere were no macroscopicor microscopicchangesto indicattehatitsmoribund conditionwas testmaterial-relateAdR. otheranimalssurvivedtothescheduledsacrifice. No clinicaolbservationwsere noted thatwere consideredeffectosf thetestmaterial Ophthalmology Ophthahnicobservationasresummarized inTable 5;individuadlataareinAppendix 2. The Ophthalmology Report containsa discussionof thedata. Animals selectedforthestudyhad no lesionsattheprestudyexamination.No test material-relatoepdhthahnicobservationwsere noted attheWeek 14 examination. Body Weights Body weight and body weightchange dataaresummarized inTables6 and 7;individual body weight dataareinAppendix 3. Testmaterial-relatleodwer body weightswere noted formales given30 and 100 ppm and formales and femalesgiven 100 ppn-L Statisticalsliygnificantlloywer mean body weights were noted beginningatWeek 4 and continuingthroughtheend of studyformales and femalesgiven100 ppnl Statisticaslilgynificantlloywermean body weightswere noted beginningatWeek 7 and continuingthroughtheend of studyformales given30 ppn-L Body weight changeswere statisticaslilgynificantlloywer ina sporadicmanner fornmles given30 ppm and foranimalsof bothsexesgiven 100 pprm Overallbody weight changes (Weeks 1-14)were statisticaslilgynificantlloywerforallgroupsgivenN-MEFOSE. Food Consumption Food consumptiondataaresummarizedinTable8;individuadlataareinAppendix 4. 23 Covance 6329-225 3M T-6314.1 Although statisticalsliygnificanotnly atWeek 13 fornialesgiven 100 ppm, food consumption was lower fornialesgiven 30 and 100 ppm when compared to those of controlsformost of the study. Females given 100 ppm had statisticalsliygnificantlyower food consumption than thoseof controlsat Weeks 3, 4, 6, 7, 11,and 12. The overall mean food consumption for femalesfedthe 100-ppm concentrationwas significantly lower by 17.5% when compared with thatof the controls. Test Material Consumption Test materialconsumption data aresummarized in Table 9; individualdata are in Appendix 4. Animals were fed dietscontaining3, 30,or 100 ppm N-MEFOSE, T-6314. The mean amounts of testmaterialconsumed by the animals in these groups were 0.148 to 0.304 mg/kg/day; 1.390 to 3.094 mgtkg/day; and 5.054 to 10.291 mg/kg/day formales, respectively.Values forfemales were 0.184 to 0.319 mg/kglday; 2.204 to 3.395; and 6.494 to 10.378,respectively.The overallmean testmaterialconsumption formales fed dietscontaining3, 30, or 100 ppm N-MEFOSE, T-6314 were 0.203, 2.041, and 6.844 mg/kglday, respectively.The overallmean testmaterialconsumption for females fed dietscontaining3, 30, or 100 ppm N-MEFOSE, T-6314 were 0.240,2.588, and 7.702, respectively. ClinicalPathology Hematology, clinicalchemistry,and urinalysidsata are summarized inTables 10 through 14;individualdata arein Appendix 5. The Pathology Report containsa discussionof the data. Dietaryadministrationof N-MEFOSE was associatedwith lower hematocritformales and females fed 100 ppm; higher urea nitrogenformales and females fed 100 ppnr, higher albumin formales fed 100 ppm; lower globulinfornialesfed 30 or 100 ppm; lower cholesteroland triglyceridefsormales fed 30 or 100 ppm and females fed 100 ppm; higher alanineaniinotransferasfeor males fed 100 ppin; and higher hepaticpah-nitoyCloA oxidase formales and females fed 100 ppm. The effectswere relativelny-&d with the exception of those on cholesteroland triglyceridefsorthe high-dose males;those effects 24 Covance 6329-225 3M T-6314.1 were consideredtobe ofmoderate severityA.R of thetestmaterial-relatceldinical pathologyfindingwsere more prominentatWeek 14 thanatWeek 5. Serum PerfluorooctaneSulfonicAcid Level (PFOS) Determination Resultsof serum PFOS leveldeterminatiownillbe reportedseparatelbyy the Sponsor. CeH ProliferatioTnissue Collectionand Immunohistochenilcal Evaluation Resultsof cellproliferatiaonnd immunohistochemicalevaluatiopnrovidedby Pathology AssociatesInternationaalreinAppendix 7. Liver PFOS Determination Resultsof liverPFOS leveldeterminatiownin be reportedseparatelbyy theSponsor. Anatomic Pathology Terminalbody weights,absoluteorgan weights,organ-to-bodyweightpercentagesa,nd organ-to-braiwneightratiosaresummarized inTables 15 and 16;incidencesof macroscopic and microscopicobservationasresummarized inTables17 and 18;the incidenceof severityof microscopicobservationisssununarizedinTable 19. Individual dataareinAppendix 6. The PathologyReport containsa discussionof thedata. Statisticalsliygnificanatnd testmaterial-relatcehdanges interminalbody weightsand liver weightswere seeninmales and/orfemalesinthetwo highestdose groups. Statistically significadnetcreaseswere seenintheterminal.bodyweightsof males fed30 ppm and in both males and femalesfed100 ppm ofN-MEFOSE. Statisticalsliygnificainntcreases were seeninthemean liver-to-bodwyeightpercentageand mean hver-to-braiwneight ratioinmales fed30 ppM inmean absoluteliverweightand mean hver-to-bodyweight percentagein femalesfed30 ppni,and inallliverweightparametersinmales and females fed 100 ppm. 25 Covance 6329-225 3M T-6314.1 Testmaterial-relatmeadcroscopicfindingwserenotedata low incidencienthelivearnd glandularstomach ofmalesand femalesinthe100ppm dosegroup only.Liverfindings includedorgansthatwere largeand diffuseldyark.Intheglandulasrtomach,darkfocior areaswere somed=s seeninthegastrimcucosa. Testmaterial-relatmeidcroscopicfindingwsere seenintheliverofanimalsfed30 and 100 ppm ofN-MEFOSE and intheglandulasrtomach ofnialesand femalesinthe 100 ppm group only.No testn-iaterial-reclhaatnegdeswere seeninanimalsfed3 ppm of N-MEFOSE. Inthelivero,ne ormore testmaterial-relaftienddingasffecteadllanimals fed100ppm and includedslight-to-moderacteentrilobuloarrdiffusheepatocellular hypertrophym,ftiimal-to-slicgohatgulativneecrosism,inimalhepatoceflulpairgment,and n@@-to-slight centrilobultaormidzonalor diff-uhseepatocellulvaarcuolationI.n animalsfed30 ppm, centrilobulhaerpatocellulhayrpertrophywas alsoseen,althoughthe incidenceand severitwyas decreasedatthisdose. Inadditiona,low incidenceofminimal hepatocerulavracuolatioonccurredin30 ppm dosegroup nialesa,nd theEverofa single fenialfeed30 ppm had minimalcoagulativneecrosis.A possibletestmaterial-related changeintheglandularstomach ofaninmlsfed100 ppm was a relativeloyw incidencoef mucosalerosions.The erosionswere ofminimaltoslighsteveritaynd involveda very smallportionofthemucosal surfaceinallcases. CONCLUSIONS Based on anatomicpathology,clinicaplathology,and body weighteffectast30 and 100 ppm theno-observable-adverse-eflfevcetlwas determinedtobelessdm 30 ppm when N-MEFOSE isadministereadtconcentrationosf0,3,30 and 100 ppm to Crl:CD'O(SD)IGS BR ratsforatleast13 weeks throughdietaraydministratioBna.sedon thebody weighteffectast3,30,and 100ppm, theno-observable-effleecvtelwas determinedtobe lessthan3 ppm when N-MEFOSE isadministereadtconcentrationosf 0,3,30 and 100 ppm toCrl:CDO(SD)IGS BR ratsforatleast-13weeks throughdietary administratioInn.theabsenceofcorrelaticvheangesinanatomicpathologyand clinical pathology the body weights effectsat 3 ppm were not consideredto be biologically adverse. 26 SIGNATURES Covance 6329-225 3M T-6314.1 Nii'@anBruce-Konuah Date Study Coordinator Covance LaboratoriesInc. 'PeteJr.'YlKmf6rd,PW Dat Study Director Covance LaboratoriesInc. 27 REFERENCES Covance 6329-225 3M T-6314.1 Dunnett,C. W., "New TablesforMultipleComparisons witha ControL"Biometrics, 2@0:482-491(1964). Levene,H., "RobustTestsforEqualityof Variances,C"ontributiontso Prob&ffityand Statistic(se,ds.)I.OlkinetaL,Ch. 25,pp. 278-292,StanfordUniversitPyress:Stanford, Californi(a1960).. Winer, B. J.,"Designand Analysisof Single-FactoErxperiments,"StatisticParlincipleisn ExperimentalDesi Second Ed.,Ch. 3,pp. 149-260,McGraw-Hill: New York, New York (1971a). 28 OPHITL4,LMOLOGY REPORT Covance 6329-225 3M T-6314.1 Ophthahnic examinationswere done beforeinitiatioofntreatmentand duringWeek 14. Animalsselectedforthestudyhad no lesionsattheprestudyexamination.No test material-relatoepdhtbahnicobservationwsere noted attheWeek 14 examination. L C-QQ-l@ @Donna J.Cle@ions,DVM, MS Viplomate,A@@ \ Lk",o k @ ,0-o oo Dt 29 PATHOLOGY REPORT Covance 6329-225 3M T-6314.1 SUNMARY The purpose of thisstudywas to assessthetoxicitoyf thetestmaterialN,-Methyl PerfluorooctanesulfonamiEdtohanol(N-MeFOSE), when administereidnthediettorats foratleast13 weeks. The testmateriawlas administeredatdose levelsof3, 30,and 100 PpnL DietaryadministratioonfN-MEFOSE was associatedwithlower henmtocritformalesand femalesfed 100 ppm; higherureanitrogenformalesand femalesfed 100 ppm; higher albuminformales fed 100 ppm; lowerglobulinformales fed30 or 100 ppm; lower cholesteroalnd triglyceridfeosrmalesfed 30 or 100 ppm and femalesfed 100 ppm; ligher alanineaniinotransferafsoermales fed 100 ppm; and higherhepaticpahnitoylCoA oxidaseformales and femalesfed 100 ppm. The effectwsere relativelmyildwiththe exceptionof thoseon cholesteroalnd triglyceridfeosrthemales fed 100 ppm; those effectwsere consideredto be of moderateseverityA.llofthetestimterial-relatceldinical pathologyfindingswere more prominentatWeek 14 thanatWeek 5. Statisticalsliygnificanatnd testmaterial-relatcehdangesinterminalbody weightsand liver weightswere seeninmales and/orfemalesinthetwo highestdose groups. Statistically significandtecreaseswere seen intheterminalbody weightsof males fed30 ppm and in bothmales and femalesfed 100 ppm. Statisticalsliygnificainntcreasewsere seeninthe mean liver-to-bodwyeightpercentageand mean liver-to-brawienightratioinmales fed 30 ppm, inmean absoluteliverweightand mean liver-to-bodwyeightpercentagein femalesfed30 ppm and inallliverweightparametersinmales and femalesfed 100 ppm. Testniaterial-relamtaecdroscopicfindingwsere noted ata low incidenceintheliverand glandularstomach of males and femalesinthe 100 ppm dose group only.Liverfindings includedorgansthatwere largeand diffuseldyark.In theglandularstomach,dark focior areaswere sometimes seen inthegastricmucosa. Test material-relatmeidcroscopicfindingwsere seenintheliverof animalsfed30 and 100 ppm of N-MEFOSE and intheglandularstomach of nmles and fen3aleisnthe 100 ppm group only. No testmaterial-relatcehdangeswere seeninanimalsfed3 ppm of 30 Covance 6329-225 3M T-6314.1 N-MEFOSE. In thelivero,ne or more testmaterial-relatfeidndingsaffecteadllanimals fed 100 ppm, and includedslight-to-moderacteenvflobulaorr diffiihsepatocerular hypertrophy,mftlimal-to-slicgohatgulativneecrosism,inimalhepatoceflulpairgment,and rnininml-to-slicgehnttrilobultaorniidzonaolr diffusehepatocerularvacuolationI.n animalsfed 30 ppm, centrflobulhaerpatocerulahrypertrophywas alsoseen,althoughthe incidenceand severitwyas decreasedatthisdose. In additiona, low incidenceofminimal hepatocehularvacuolationoccurredin30 ppm dose group males,and theliverofa single femalefed30 ppm had niniirwcoagulativneecrosis.A possibletestmaterial-related change intheglandularstomach of animalsfed100 ppm was a relativeloyw incidenceof mucosal erosions.The erosionswere of minimalto slightseveritaynd involveda very smallportionof themucosal surfaceinallcases. METHODS Four groups of Crl:CDO(SD) IGS BR rats(20 animaLs/sex/groupw)ere feddiets containintghetestmaterialata dose levelof 0 (controlgroup;fedbasaldietonly),3,30, or 300 ppm. During Week 5,fiveanimals/sex/grouwpere sacrificetdo obtainliver samplesfordeterminatioonf hepaticpahnitoylCoA oxidaseactivit(ybyCovance) and analysifsorPFOS and metabolite(sby3M). One femalefed3 ppm was sacrificeadnd necropsiedduringWeek 9 becauseof poor health(unscheduledsacrificeT)h.e remaining animalswere sacrificeadnd necropsiedduringWeek 14 (terminaslacrifice). Blood was collectedforhematologyand clinicaclhemistrytestsduringWeeks 5 and 14. Urinewas collectefdorurinalysitsestsduringWeek 14. At necropsy,macroscopic observationwsere recorded,organweightswere obtained(terminaslacrificoenly),and tissuewsere placedinfixativaes specifiebdy theprotocol.In additions,amplesof liver were collecteadttheterminalsacrificfeorevaluationofproliferaticoenU nuclearantigen (PCNA; by PathologyAssociatesInternationafli;veanimals/sex/groupa)n,d both scheduledsacrificefsordeterminatioonf hepaticpalrnitoyCloA oxidaseactivit(yby Covance) and analysifsorPFOS and metabolite(sby3M). Microscopicexaminations were done on protocol-requiretdissuefsrom allcontroland high-doseaninulssacrificed duringWeek 14 and the3 ppm dose group femalesacrificeidna moribund condition duringWeek 9 (unscheduledsacrificeI)n.additionE,vers,stomachs,and macroscopic 31 Covance 6329-225 3M T-6314.1 lesionswere examined from allterminalsacrificaenimals fed 30 ppm, and macroscopic lesionsand liversonly were examined from animals fed 3 ppm. Statisticalsliygnificandtifferencescitedin the Results and Discussion sectionare based on comparisons between the controland treatedgroups. RESULTS AND DISCUSSION Mortality One fenxilefed 3 ppm (Animal No. C96016) was sacrificeduring Week 9 (Day 59) because of poor health.Allother animalssurvived to therespectivescheduled sacrifice. ClinicalPathology Weeks 5 and 14. There were severalstatisticalsliygnificanotr otherwisenotable differencesforclinicaplathology resultsbetween controland treatedanimals. Differences considered to be effectsof thetestmaterialwere lower hermtocritformales and females fed 100 ppm; higherurea nitrogenformales and females fed 100 ppm; higheralbumin for males fed 100 ppm, lower globulinformales fed 30 or 100 ppm; lower cholesteroland triglyceridefsor males fed 30 or 100 ppm and females fed 100 pprn;higherah-inine aniinotransferasfeormales fed 100 ppm; and higherhepaticpahnitoylCoA oxidase for males and females fed 100 ppnl The effectswere relativelmyild with the exceptionof those on cholesteroland triglyceridefsorthe males fed 100 ppm those effectswere consideredto be of moderate severity.All of the testniaterial-relatceldinicaplathology findingswere more prominent at Week 14 than at Week 5. The mild effectson hematocritand globulinwere consistentwith aninws failintgo gain weight appropriately.Although mildlyhigherurea nitrogenand albumin suggested a relativedehydrationforanimals given 100 ppm, therewere no effectson urinevolume or urme specificgravityconsistentwith dehydration. The mild effecton alanine aminotransferasewas indicativoef the hepatocellulardegeneration,and mildlyhigher hepaticpahnitoylCoA oxidase was indicativeof peroxisomal proliferationT.he effectson cholesteroland triglyceridewsere themost prominent findings,and suggested lipid metabolism by the liverwas altered.. 32 Im Covance 6329-225 3M T-6314.1 Of uncertairnelationshtiopadministratioofnthetestmateriawlas statisticallolwyermean corpusculavrolume forfemalesfed100 ppm The differencfeormean corpuscular volume became smallerovertime(i.ef.r,om Week 5 toWeek 14).Otherstatistically significadnitfferencewsere consideredincidentalnd unrelatedtoadministratiofnthe testmaterial. Anatomic Pathology Unscheduled Deaths. The one fenialfeed3 ppm thatwas sacrificeidna moribund conditioonn Day 59 (AnimalNo. C96016) had no macroscopicor microscopichangesto indicattehatitsmoribund conditiownas testmaterial-related. Terminal Sacrifice Terminal Body Weights and Organ Weights. Testmaterial-relactheadngeswere seen in theterminalbody weights and in absoluteand relativeliverweights in animals fed 30 and 100 ppm N-MEFOSE. Mean terminalbody weights were statisticalsliygnificantldyecreased inmales and females thatwere fed 100 ppm of N-MEFOSE and inmales only fed 30 ppm. The decreased mean body weights at both dose levelswere considered to be testmaterial-related. Statisticalsliygnificantand testmaterial-relateidncreaseswere seen in the mean liver-to-bodyweight percentage and themean liver-to-braiwneight ratioin nialesfed 30 ppni,in mean absoluteliverweight and mean liver-to-bodyweight percentage in females fed 30 ppm, and in allliverweight parameters in males and females fed 100 pprrl The liverweight increasescorrelatedmacroscopicallywith largeliversin 3 of 15 nialesin the 100 ppm dose group and microscopicallywith the presence of hepatocehular hypertrophy in allmales and females in the high-dose group. 33 Covance 6329-225 3M T-6314.1 Additionalstatisticalsliygnificanatbsoluteor relativeorgan weight changes affected severalother organs in both sexes and inmultipledose groups. Because therewere no macroscopic or microscopic findingsthatcorrelatedwith the weight changes,the remaining organ weight variationswere consideredto be withintherange of normal biologicvariationor were attributedto the lower body weights in treatedversuscontrol animals. Macroscopic Flndings. Test rmterial-relatemdacroscopic findingswere noted at a low incidenceinthe liverand glandularstomach of nmles and females fed 100 ppm N-MEFOSE. Liversthatwere largewere seen in 3 of 15 nialesand in 0 femalesfed 100 ppm, and diffuselydark liversoccurred in 2 of 15 males and I of 15 females fed 100 ppm. No liverchanges occur-redin animalsfed thecontrolmaterialor in animalsfedlower doses of N-MEFOSE. The largeand dfffuselydark liverscorrelatedmicroscopically with hepatocellularhypertrophy inthe affectedanimals. In the glandularstomachs, dark focior areas occurred at an incidenceof 0 of 15, 1 of 15,0 of 15,and 2 of 15 innudes, and at an incidenceof 0 of 15,0 of 14,0 of 15,and 3 of 15 in females inthe contr-ol 3 ppm 30 ppm, and 100 ppm groups respectively.The dark focior areas generally correlatedmicroscopicallywith smallerosionsin the glandularmucosa. Diffusereddening and mottlingof thethymus and mandibular lymph nodes as well as red focior areas inthe thymus were relativelcyonnnon findingsand frequentlycorrelated nucroscopicallywith hemorrhage in the affectedorgans. Because these findingsdid not occur in a dose-relatedmanner and because ratsinmultipledose groups, includingcontrol nmles, were sometimes affected,the findings,wereconsidered to be agonal changes that were not associatedwith testmaterialadministration.AR remaining macroscopic findings were considered to be spontaneous and incidentaalnd not relatedto testmaterial administration. Microscopic Findings. Test material-relatemdicroscopic findingswere seen inthe liver of animalsfed 30 and 100 ppm of N-MEFOSE and in the glandularstomach of males and females in the 100 ppm group only. There were no testmaterial-relatefdindingsin the liversof animals fed 3 ppnl 34 Covance 6329-225 3M T-6314.1 Test material-relatfeidndingsintheliverincludedhepatocellulahrypertrophyc,oagulative necrosish,epatocellulaprigment,and hepatoceuularvacuolation.The incidenceand severitoyf thesefindingstendedto be greaterin 100 ppm animals.The incidenceof pertinenltiverfindingisspresentedinText Table 1. TextTable I Incidenceof SelectedMcroscopic FindingsintheLiver Group Number/Sex Number Examined IM 2M 3M 4M 15 15 15 15 IF 2F 3F 4F 15 14 15 15 Hypertrophy,hepatocelluladri,ff-use 0 0 0 0 Hypertrophy,hepatocellular, Centrilobular Necrosis,coagulative Pigment,hepatoceflular 0 0 14 15 00 03 00 02 Vacuolation,hepatocellular, centrilobultaormidzonal 00 27 Vacuolation,hepatocelluladri,ffuse 0 0 2 2 00 0 1 0 0 7 14 00 10 0 007 0 002 0 000 As noted intheabove table,hepatoceflulhayrpertrophygenerallayffectedcentrilobular areasof theliverinanimalsfed30 and 100 ppm but theliverwas more diffusely hypertrophiedinone fernalefed100 ppn-L The hypertrophichepatocyteswere enlarged withabundantcytoplasmthatoftenhad a granularto globulareosinophiliacppearance.In some animalsinthehigh-dosegroup (two nialesand sevenfemales)t,hecytoplasmof hepatocytesalsocontainedminimalwnounts of a brown granularpigment-attheperiphery ofthecell.Inadditionl,iverceflsfrequentlwyere vacuolated(seeText Table1), particularliynmales given100 ppm, withthevacuolationgenerallayffectinhgepatocytes incentrilobularnd midzonalareas.Diffusehepatoceflulavracuolationwas alsoseenin two males ineach of the30 and 100 ppm dose groups,but inno otheraninmls.Lastly, minimalto slightcoagulativneecrosisseenin3 of 15 nmles fed.100 ppm and I of 15 femalesfed 30 ppm was likelryelatedto dietaryadministratioonfthetestmaterial sinceno controlor 3 ppm dose group animalswere similarlayffected. A possibletestniaterial-relactheadnge intheglandularstomach was thepresenceof mucosal erosions.The erosionswere of minimalto slightseveritaynd involveda very smallportionof themucosal surfaceinallcases.The erosionswere seenwithan 35 Covance 6329-225 3M T-6314.1 incidenceof 0 of 15,0 of 15,and 2 of 15 in males,and at an incidenceof 0 of 15,0 of 15, and 3 of 15 in females in thecontrol,30 ppm, and 100 ppm groups respectively.The presence of erosionsngcroscopicaffygenerallycorrelatedwith thedark focior areasseen in the gastricmucosa at necropsy. One nmle fed 3 ppm alsohad a slightgastricerosion thatwas collectedas a gross lesionatnecropsy. Since no erosions were seen in any of the anftnals'stomachs exan3inedfrom animals fed 30 ppm, the lesionin the 3 ppm male was considered unlikelyto be testmaterial-related. Allremaining microscopic findingswere considered to be spontaneous and incidental changes and typicalof ratsof thisage and str Robert L. Hall,DVM, PhD Diplomate, ACVP (ClinicaPlathology) *nnie J.Eiony, OVW MS Diplomate, ACVP Diplomate, ABT d5late Date 36 Covance 6329-225 3M T-6314.1 COMMENTS ON THE DATA Variousmodels of calculatorcso,mputers,and computer programs were used to analyze datainthisstudy.Because differenmtodels round offor truncatenumbers differently, valuesinsome tables(e.g.m,eans, standarddeviationso,r individuavlalues)may differ slightlfyrom thoseinothertablesf,rom individualclaylculateddata,or from statistical analysidsata.Neithertheintegritnyor theinterpretatiofnthedatawas affectedby these differences. The number of animalslisteidntheheadingof thesummary tablesforclinicaolbservations reflecttshenumber ofaninmlsassignedto each group atthestartof thestudy. The summary tableforclinicaolbservationisndicattehenumber of anfinalfsorwhich a conditionwas observedwithoutregardto thespecifincature,severityr,eversibility, number of incidenceslanimaolr thelengthof timetheconditionpersisted. Only observationostherthannormalareindicateodn thesummary clinicaolbservations tables. Each animalwith observationsrecordedas "Normal" throughout the studyhas the comment "Animalhas no significafnitndingsi"ndicateodn theindividuacllinical observationstables. The specifidcetailfsorconnnentsintheindividuacllinicaolbservationtsablesthatare indicatedwitha "C" can be found attheend of eachgroup foreach sex. The day of initiatioofntreatmentis"Day 1,Week I." Body weightdataareenteredatthe startof a studyweek (e.g.a,body weightrecordedon Day I isconsidereda Week 1 body weight,a body weightrecordedon Day 8 isconsidereda Week 2 body weight).Body weight change dataarecalculatefdrom the firsdtay of thestudyweek to thefirsdtay of thefollowingstudyweek (e.g.W,eek 1 valuesarecalculatefdrom Day 1 through7) and areindicateidnthetableswiththeday beingthefirsdtay of thefollowingweek (e.g.W,eek I valuesareindicateads"Day 8.").Weekly food consumptionand test materialconsumptionarecalculatefdrom thefirsdtay of thestudyweek tothefirsdtay of thefollowingstudyweek (e.g.W,eek I valuesarecalculatefdrom Day I through7). The valuesforsummary and individuatlestmaterialconsumptionarereportedwithmore significafnitguresthanisappropriatfeorthedatadue to limitationosf thedatacollection and reportingsoftware. 37 Covance 6329-225 3M T-6314.1 COMMENTS ON THE DATA (Continued) Food consumption values are reported in whole numbers; however, PTS carriesfood consumption values to one place to therightof the decimal forfood efficiencaynd test materialconsumption calculations.The differencesin valuesgenerated do not influence the interpretatioonf the calculation. The comment "SPIILLED" on individualfood consumption data tablesindicatesthatfood consumption was not recorded due to spillageduring the interval. The comment "NOT TAKEN" on individualfood consumption data tablesindicatesthe animal died before the end of the food consumption interval Differencesin the populationsize(N) on thesununary tablesforclinicaalnd anatomic pathology are explained on the individualdata tablesor the codes sheets. The calculationforindividualtestmaterialconsumption is: Test materialconsumption DietConcenaafionxFoDd Consumption Food ConsumptionIntervalx[FiBrosdty Weight of InWval+ (DailyBody Weight GainxDay F=or)] where: Daffy Body Weight Gain LastBody Weight ofIntervalF-irstBody Weight of Interval Day ofLastBody Weight ofIntervalD-ay of FirstBody Weight of Interval and: Day Factor= Food Consumption Interval (Food Consumption St= Day@ Day of FustBody Weight of Interval+) 2 and: Food Consumption Interval Food Consumption End Day- Food Consumption StartDay 38 CODES, ABBREVIATIONS, AND UNITS Covance 6329-225 3M T-6314.1 General Codes and Abbreviations Codes for ClinicalPathology Abbreviations and Units for ClinicalHematology Abbreviations and Units for ClinicalChemistry Abbreviations and Units for ClinicalUrinalysis Codes forAnatomic Pathology Note: The following listsof codes, abbreviations,and unitsare used by Covance. Some, but not necessarilyalL of thisinformation may be needed for thisreport. 39 Covance 6329-225 3M T-6314.1 General Codes and Abbreviations V@K N Mean; MEAN SD; S.D.;STAND DEV; STANDARD DEV; sd NA p c UNSCHED DISPATCH TBW co Week. Number ofmeasurementsina group. Arithmeticmean. Standarddeviation. Group mean issignificantdliyfferenftrom themean ofthecontrolgroup(Group 1) atp :50.05. No value;not applicablen;otpresent. Present. Comment found attheend of eachgroup foreach sex. Unscheduled. Observationstransferrefdrom thein-life module of thedatacollectiosnystemto thenecropsymodule forreferenceduring necropsy. Observationsare duplicateosf thelastin-lifoebservations. Terminalbody weight. Number. Clinicaolbservation. Animal Death Codes: I T m Interimsacrific1e. Terminalsacrifice. Sacrificeadtan unscheduledinterval 40 NS QS/QNS NR FS sc SH H SL L .si I NF u DT/DOT DB Ti TE RE EE SE PC PD Pi PL PA co HB PLASMO NO AGG FR UTD NOCOAG Covance 6329-225 3M T-6314.1 Codes for Clinical Pathology GENERAL CODES No sample Quantitynotsufficient No repeat(samplevolume not sufficiefnotrrepeatanalysis) Fibrin strands Sample clotted Slightlhyemolyzed Hemolyzed Slightllyipernic Lipemic Slightliycteric Icteric Animal notfasted Unscheduled/moribundbleed Animal diedon test Died duringbleeding Technicianjudgment to repeattest Technicalerror(instrumenotr techniciaenrrorthatresultisn unacceptabledata,e.g.,unacceptableinstrumentoutput,sample spillede,ntryof invalidata) Recordingerror(recordedincorrecdtata,e.g.,wrong number, speflinegrror,incorrecdtate) Entryerror(incorreckteyboardentry) Sampling error Plateletcslumped Plateletdsecreased Plateletisncreased Plateletlsarge Plateletasppear adequate Color interferewsithtest Heinz bodiesobserved Plasmodium No aggregation Fractious Unable to determine No coagulation 41 Covance 6329-225 3M T-6314.1 Codes for Clinical Pathology (Continued) RESULTS NOT INCLUDED IN STATISTICAL ANALYSES Hemolyzed clinicalchemistry or coagulation samples Samples from animals at unscheduled intervals Prothrombin times (PT) greaterthan 50 seconds Activated partialthroniboplastitnimes (PTT) greaterthan I10 seconds Bleed times (BLETIME) greaterthan 30 minutes CODES FOR BLOOD CELL MORPHOLOGY The followingscalewas used to measure thedegree ofanisocytosis(ANISO), poikilocytosi(sPOIK), polychromasia(POLY), hypocbroniasia(HYPO), or basophilic stipplin(gBASTEP) or thepresenceof Howell-Jollybodies(HJBODY), toxicneutrophils (TOXNEUT), or atypicalymphocytes(ATYPLYM): Scale I 2 3 4 Degree Normal forthe species Slight Moderate Marked Not applicable Presence Not present Rare Few Moderate Many URINE APPEARANCE A Pale B Straw C Yellow D Dark yellow Color E Amber F Brown G Red B Green I Black P Blue/green Q Blue R Orange Clarity lear K Hazy L Cloudy Miscellaneous M Debris 0 Feces 42 Covance 6329-225 3M T-6314.1 Codes for Clinical Pathology (Continued) URINE CHEMISTRY MULTNTIXO STRIP Urine Glucose + ++ ... .... ..... Negative 100 mg/dL 250 mg/dL 500 mg/dL 1,000 mg/dL @!2,000 mg/dL Urine Ketone - + ++ ... .... Negative 5 mg/dL 15 mg/dL 40 mg/dL 80 mg/dL ..... 160 mg/dL Urine Blood - Negative + Small ++ Moderate Large Urine Urobilinogen 0.2 mg/dL + 1 mg/dL ++ 2 mg/dL 4 mg/dL 8 mg/dL (I mg = approximately1 Ehrlichunit) Urine Bflirubin + ++ ... Negative Small Moderate Large URINE SEDIEMENT Ceffs,Crystals,Casts,and Comments A Amorphous urates B Amorphous phosphates C Uric acid D Triplephosphates E Calcium oxalate F Calcium carbonate G Granular casts H Hyaline casts I Cellularcasts J Waxy casts K Unknown crystal P Mucous threads Q Sperm R Fecalcontamination S Pinworm ova found T Pinworm larvaefound U Parasiteova found 0 Not present 1 1-5 per field 2 6-10 per field 3 11-20 per field 4 >20 per field Bacteria 6-Not present I Few 2 Moderate 3 Many 43 Covance 6329-225 3M T-6314.1 Abbreviations and Units for Clinical Hematology Test Red blood cellcount Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Plateletcount Mean plateletvolume Reticulocytecount Absolute reticulocytecount Heinz body count Erythrocyte sedimentationrate Prothrombin time Activated partialthrornboplastitnime Thrombin time Activatedcoagulationd= Fibrinogen Fibrin/fibrinogednegradationproducts Plateletaggregation Collagen Adenosine diphosphate Alpha 2-antiplasmin Bleeding time Methemoglobin Plasma hemoglobin Myeloid/eqftoid ratio Estimated myeloid/erythroidratio Vy'ffibtleood cellcount Differentiballood cellcount Nucleated red blood cellcount Corrected white blood ceR count Segmented neutrophilcount Band neutrophilcount Lymphocyte count Monocyte count Eosinophfl count Basophid count Anisocytosis Polychromasia Abbreviation (Units) RBC (E6/UL or X106/,uL) HGB (G/DL) HCT (%) MCV (FL) MCH (PG) MCHC (%) PLT (E3/UL or X103 /IzL) MTV (FL) RETIC (%) X103 RETTC (E3/UL or IgL) HEINZ (%) ESR (MM/HR) PT (SEC) PTr (SEC) TT (SEC) ACr (SEC) FBR (MG/DL) FDP (UG/ML) PAGG/COL PAGG/ADP ANTIPLAS BLE TDAE (SEC) METHGB(%) PLA HGB (MG/DL) NI/E RATIO EST M/E RATIO WBC (E3[UL or X103 IgL) NRBC (/100WBC) X103 COR WBC (E3[UL or /,uL) N-SEG (E3/UL or X103 /yL) and % N-BAND (E3[UL X103 or lAiL)and % X103 LYNTH (E3/UL or //iL)and % MONO (E3/UL or X103/gL) and % EOSIN (E3/LJLor X103 IgL) and % BASO (E3/IJLor X103//.iLa)nd % ANISO (-,1,2,3) POLY (-,1,2,3) 44 Covance 6329-225 3M T-6314.1 Abbreviationsand UnitsforClinicalHematology (Continued) Test Poikilocytosis Hypochromasia HoweU-JoUy bodies Basophilicstippling Toxic neutrophils Atypicallyniphocytes Aqueous whitebloodcellcount(righetye) Aqueous whitebloodcellcount(lefetye) Abbreviation (Units) POIK (-1,,2,3) HYPO (-,1,2,3) HJBODY (-,1,2,3,4) BASTIP (-,1,2,3) TOXNEUT (-,1,2,3,4) ATYPLYM (-,1,2,3,4) R EYE (WBCFUL) L EYE (WBCFUL) 45 Covance 6329-225 3M T-6314.1 Abbreviations and Units for Clinical Cheniistry Test Glucose Urea nitrogen Urea Creadnine Total protein Albumin Globulin Albumin/globulinratio Total bffimbin Directbffimbin Indirectbirmbin Cholesterol Triglyceride Urea nitrogen/creatininreatio Totallipids Phospholipids legh-densitylipoproteincholesterol Low-density lipoproteincholesterol Uric acid Aspartate aminotrawferase Alanine aniinotransferase ARmEne phosphatase Gamma glutamyl transferase Sorbitoldehydrogenase Lactate dehydrogenase Creatine kinase Amylase Lipase PalmitoylCoA oxidase Calcium Ionized calcium Inorganic phosphoms. Sodium Potassium Chloride Magnesium Zinc Strontium Iron Abbreviation (Units) GLU (MG/DL) UN (MG/DL) UREA (MG/DL) CREAT (MG/DL) T PRO (G/DL) ALB (G/DL) GLOB (G/DL) A/G RATIO T BILI (MG/DL) D BILI (MG/DL) I BELI (MG/DL) CHOL (MG/DL) TRIG (MG/DL) UN/CREAT (RATIO) T LIPIDS (MG/DL-) P LIPIDS (MG/DL) HDL (MG/DL) LDL (MG/DL) UA (MG/DL) AST/SGOT (IUIL) ALT/SGPT (FJIL) ALK PHOS (IU/L) GGT (IU/L) SDH (IU/L) LDH (IU/L) CK (FJ/L) AMYLASE (IU/L) LIPASE (IU/L) PCOAO (IU/G) CA (MG/DL) ION CA (MG/DL) I PHOS (MG/DL) NA (MMOUL) K (MMOLIL) CL (MMOUL) MG (NEQ/L or MG/DL) ZN (MG/L or PPM) SR (MG/L or PPM) FE (UG/DL) 46 Covance 6329-225 3M T-6314.1 Abbreviationsand UnitsforClinicalChemistry (Continued) Test Excess iron Totalironbindingcapacity Unbound ironbindingcapacity Percentironsaturation Plasma cholinesterase Red blood cellcholinesterase Braincholinesterase Caudate putamen Hippocampus Frontalcortex Cerebellum Bicarbonate Serum hemoglobin Serum bileacids Fecalbileacids Average fecalweight Fecalbileacids(calculation) Osmolality Electrophoresis Albumin Alpha-I-globulin Alpha-2-globulin Beta globulin Gamma globulin High-densitlyipoprotein Low-densitylipoprotein Very-low-densitlyipoprotein Insulin Adrenocorticotropihcormone Cortisol Glucagon Triiodothyronine Thyroxine Creatinekinaseisoenzymes BB MB mm Abbreviation (Units) EX FE (UG/DL) TIBC (UG/DL) UIBC (UG/DL) FE %SAT (%) CHEP (MU/ML) C]HER (MU/ML) CHEB (MU/ML) CAUD PUT (UMOLIG) HMPOCAM (UMOUG) F CORTEX (UMOUG) CEREBELL (UMOIJG) BICARB (MMOIJL) SER HGB (MG/DL) SBA (UMOL/L or MG/DL) FBA (UG/ML) FCC WGT (G) FBA (MG/Day) OSMO (MOSWKG) E ALB (G/DL) E A-I (G/DL) E A-2 (G/DL) E BETA (G/DL) E GAMMA (G/DL) E-HDL (%) E-LDL (%) E-VLDL (%) INSULIN (UU/ML) ACTH (PG/ML) CORTISOL (UG/ML) GLUCAGON (PG/ML) T3 (NG/DL) T4 (UG/DL) CK-BB (U/L) CK-MB (U/L) CK-MM (U/L) 47 Covance 6329-225 3M T-6314.1 Abbreviations and Units for Clinical Urinalysis Test Urine volume 8 hour urinevolume Specificgravity Unne osmolahty Quantitativeurinary/cerebrospinal fluidprotein Urine proteinexcretion Urine chemistryMultistixostrip UrinepH Urineprotein Urine glucose Urine ketones Urine bihmbin Urine blood Urine urobilinogen Urine reducing substances Microscopic examinationof urinesediment Red blood cellsper high-power field VvNte blood cersper high-powerfield Epitheliaclellsper high-powerfield Bacteriaper high-power field Castsper low-power field Crystalsper low-power field Urine appearance Conmients Abbreviation (Units) U VOL (ML) 8 HR VOL (ML) SP GR U OSMO (MOSM/KG) QUAN PRO (MG/DL) PRO EXC (MG) U PH U PRO (MG/DL) U GLU U KET U BILI U BLOOD UROBILI U RE SUB RBC (PER HPF) WBC (PER BPF) EPITH (PER BPF) BACT (PER BPF) CASTS (PER LPF) CRYSTALS (PER LPFI PER LPF2) URINE APPI or URINE COMNMNTS or APP2 MiscellaneousCodes and Abbreviations forClinicalPathology Fecal occultblood Fecalparasitedetection Hemolytic potential Osmolality Not applicable Not applicable Not applicable OSMO (MOSM/KG) 48 Code T m Codes forAnatoniicPathology Definition ANIMAL DEATH CODES InterimsacrificeIs Terminalsacrifice Sacrificeadtan unscheduledinterval Covance 6329-225 3M T-6314.1 EX NOT TAKEN MISSING UNSUITABLE AUTOLYTIC EXCLUDE MACROSCOPIC CODES Indicatetshatorgan weightisexcludedfrom calculations Organ weightnot taken;explanationgiveninnecropsynotes Organ missingor lost Organ technicalluynsuitableforweighing Organ autolyzedand could notbe weighed Weight was taken,but was excludedfrom allcalculations MICROSCOPIC CODES Codes PrefacingNeoplasticFindings B- Primary,benignneoplasm m- Primary,malignantneoplasm N- Metastaticneoplasm I- Locallyinvasiveneoplasm x- Other neoplasm Code Definition Distributionof Findings Focal Diffuse Multifocal 49 Covance 6329-225 3M T-6314.1 Codes for Anatomic Pathology (Continued) Grades I 2 3 4 5 for Severity or Amount Mhiinal - the leastamount of change thatcan be observed with the lightn3icroscope Slight- lessthan average amount of change, but readilydiscernible as abnormal Moderate - the average amount of change thatisexpected fora lesion Moderately severe(marked) - a marked amount of change with possiblelossof functionof the affectedcellsor organs Severe - a greatamount of change with probable lossof functionof the affectedcellor organs and frequentlyinvolveslargeareasof the organ Other Microscopic TL p MN Codes Total Finding present Finding not present Mean TISSUE ABBREVIATIONS Abbreviation J-N GL STOMACH, GL STOMACH, NONGL SALIV GL, MANDIB LN, ANT NES/PANC AUDITORY SEB GL LACRIMAL GLAND, EX BEMATO NEOPLASIA LACRIMAL GL, INT CAVITY, ABDOM SALIV GL,PAROTID LN, TRACHEOBRON Definition Lymph node Gland Glandular stomach Nonglandular Mandibular salivarygland Anteriormesenteric/pancreatilcymph node Auditory sebaceous gland Exorbitallacrfinaglland Hematopoietic neoplasia Internallacrinw gland Abdonunal cavity Parotidsalivarygland Tracheobronchiallymph node 50 Table I Covance 6329-225 3M T-6314.1 ResultsofHomogeneity Analyses(ppm) Mixed 8/20/98 13-WEEK DIETARY TOXICITY STUDY WITH N-METHYL PERFLUOROOCTANESULFONANUDO ETHANOL (N-MeFOSE, T6314) IN RATS Samples Location Replicate 3 T6314 (ppm) 30 100 Top 1 2.19 33.7 79.4 2 2.43 27.8 76.5 3 2.09 25.6 76.7 Mean 2.24(74.7)' 29.0(96.7) 77.5(77.5) Middle 1 2.24 25.2 75.0 2 2.27 25.3 79.7 3 2.14 25.1 89.5 Mean 2.22(74.0) 25.2(84.0) 81.4(81.4) Bottom 1 2.03 23.7 83.2 2 3.04 27.6 81.6 3 1.94 29.4 87.8 Mean 2.34(78.0) 26.9(89.7) 84.2(84.2) a Each valueinparenthesiissthepercentof theoretical 51 Covance 6329-225 3M T-6314.1 Table 2 Resultsof StabilitAynalyses (ppm) Mxed 8/20/98 13-WEEK DIETARY TOXICITY STUDY WITH N-METHYL PERFLuoRoocrANESULFONAMIDO ETHANOL (N-MeFOSE, T6314) IN RATS Storage Conditions T6314 (ppm) Replicate 1 500 Initial 1 2 Mean 1.88 0.954 1.42(142) 415 468 442(88.4)a 19 day, room temperature 32 day, room temperature 1 2 Mean 1 2 Mean 0.733 0.811 0.772 (77.2) - - 444 425 - 435(87.0) 32 day, room temperature' 1 2 Mean 8 week,' frozen 1 2 Mean 0.709 0.850 0.780 (78.0) - 0.779 - 0.995 - 0.887 (88.7) - a Each value in theparenthesisisthe percent of theoretical b Reinjected 52 Covance 6329-225 3M T-6314.1 Table2 (Continued) Resultsof StabilitAynalyses(ppm) Nfixed9/24/98 13-WEEK DIETARY TOXICITY STUDY WITH N-MEETHYL PERFLUOROOCTANESULFONAMIDO ETHANOL (N-MeFOSE, T6314)IN RATS T6314 (ppm) StorageConditions Replicate 500 1 Initial I - 2 - Mean - 0.788 0.656 0.722(72.2)a 19 day, room temperature I - 2 - Mean - 0.707 0.651 0.679(67.9) 32 days, room temperature I - 2 - Mean - 0.688 0.657 0.673(67.3) a Each valueintheparenthesiissthepercentof theoretical. 53 Table 2 (Continued) Covance 6329-225 3M T-6314.1 Resultsof StabilitAynalyses (ppm) Mixed 10/22/98 13-WEEK DIETARY TOXICITY STUDY WITH N-METHYL PERFLUOROOCTANESULFONA@ODO ETHANOL (N-MeFOSE, T6314) IN RATS T6314 (ppm) StorageConditions Replicate 3 Initial 1 2 Mean 2.24 2.49 a 2.37(79.0) 19 day, room temperature 1 2 Mean 1.89 2.33 2.11(70.3) a Each value in the parenthesisisthe percent of theoretical. 54 Covance 6329-225 3M T-6314.1 Table 3 ResultsofDose PreparationAnalyses(ppm) 13-WEEK DIETARY TOXICITY STUDY WITH N-METHYL PERFLUOROOCTANESULFONAMIDO ETHANOL (N-MEFOSE, T-6314)IN RATS Week 1-4 1-4c 1-4d 5-8 9/12 13/14 Replicate 0 1 a 2 a Mean - I - 2 - Mean - I - 2 - Mean - 1 a 2 a Mean a 1 a 2 a Mean a 1 a 2 a Mean a T-6314(ppm) 3 30 100 2.16 29.7 87.3 2.12 27.0 85.2 2.14(71.3)b 28.4(94.7) 86.3(86.3) 1.98 1.84 1.91(63.7) 2.48 2.28 2.38(79.3) 3.55 3.05 3.30(110) 27.9 89.3 27.0 82.3 27.5(91.7) 85.8(85.8) 2.24 27.6 89.7 2.49 26.8 87.2 2.37(79.0) 27.2(90.7) 88.5(88.5) 2.51 26.2 98.1 2.42 26.4 98.7 2.47(82.3) 26.3(87.7) 98.4(98.4) a Below thelimitofquantitati(o<n0.5ppm). b Each valueinparenthesiissthepercentoftheoretical. c Retest. d Retention. 55 Ln C,4 .0 cnI C,q rn I 4 r) to ril z CC,) C) rA @q 0 CD CD c@ 0 C) CD CN m C4 %D riifn0 0 4 cn C,4 0 c@ m 00 CD c@, 0 CD CD 0 rA Z E' I I pq oNq C,4m C) 0 (n c@ 0 c@-0 00 0 0 C4 N x cn E@ 0 H r4 U) 140 n rq ri CD 0 lia 0 C) 0 0 CL) 3: r4 .4 CIT IV -0 H 1-4 C4 0 li 00 C) @q r-i @4 CL) id ,4 V E-4 C-) 44 0 E@ F-4 U E@ F4 1010 E@ 0 C" CD 0 1 IWX r.41 M C4 1 C,4m 0 1 Z cq c:lcn 0 C) c@ C) 0%0 (D 0 CD -4 C) (0 (D 0 0 (D 0 0 rd E@ rA ik, cn co x r4 w p CJ riP48 a c:l C,4 w w m cn r4 En 0 0 ixA I 0 C) m CD 0 0 C) r-i CD (D c@ 4 p E@ N rz4 C4 r4 1 E4 .1 CA H mn El C4 00 w 4 .4 a 0a 19 m .1 cnz H ul H C)H tn FA P4 NM mm Hp zz u OgH oun I 4CD I wm m 4 F-I F-I m Co H m ON r4M r-)Z I E-4 pq 04 F-4 E- .1 0 C4 P Z 8 F-IF-4Pa @4 X C4 E@ i@ 14 n i"@w 0a @ C)i E44Z4F-4F-i CtAnE04IE53404 4HH z 4= HZ wP@ 4(n ;P@4 0 N 9F-IpHnq 4 D3 w El t-4 r4 F-4 H 3: = Ur4 4 En W a, 4 V4 rJ) E@ rJ]0 ;2@ r4 Pg H 04 Ln li C4 C,4 -0 cy1 , 1-4 C,4 cn 'n . Ell C,4 CL)z rd 0 > rn z H 1 00 1 1-4 C,4 1 cn E4l kD E- XI E'@ I C) pg rz4 z 04 : cli .01 r4 rX4 EI ron 0 : C,4 a) fd C) E-4 1-4 u 44 0 El 0 1 r.. >4 0 E@ L) E-4 H >4 co E@ 0 E@ r4 44 14 c,4 1 m cli (D cD clq w 04 r4 r4 r4 m cn N ul0 0 m C4 0 C4 0 134 0 C) HCD a 0 c) cD (D cD cD c@ o 0 c@ c) c) c) cD Hn z rn H " go x iz 4 f-4 cli N rn riX rn 0 tnE,4 o 'D @ a : "' '4'4p oz 4& 1 114 cn Z.4 0 rA >4 Cn >@ 1 tn Le) C,4 C*4 -0 1 -MI Cr,n4 10 E'@ 0)x um to 0 rn z 00 L. H 0 .,i 4.) .0 EM %o E'@ z1 @ pa m ril El0 H r.. 0 Z I Ln u E- -0,4,4 rn 4 0 E@ 44 0 HU E@@'. 00 E- Q H E4 r@4 Lel cn PQ E4 U r4 41 DN C,4m Ln Ni @q (D Ln F4 I c@ r4m,@ Ln cq cl@ in -I -4(3 Ln pq r4 0894 1 CD .1 r.. pq gk4 x ra r4 rn w to0 0 m (=I 0 C> 0 a 0 C) C) CD 0 r-i r-iC) c@ CD c@, c@ u 0 114 rn H U) E-4 H 1>-4 F-I F-I Dr4 014 E@ H 0 0o@g : 0 0 E@ E@ 9 4 u E@ r4 El V) CD H F-4 rA m 4 00 .4 Dr4 lx 0 E-49 1- rg i 00 4 tn C4, m1 @A c.q m en %D . 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Lno " 11 @i H @4 (@ H I c) Ln Lno c) Lnc@ Ln cD tn C4 r-I @4 r-t H -10 Ln Ln a cD Ln r-i r-fCC!, cq r-4o r-icq c:- -o-000-09 -4 ri r-i IM Z cn 4 1 0 X m F4 co 0 A A AA A I r-t C,4 @l N E4 z A AA A 1 -4 @4 E@ A AA A A 1 @4 C4 .4 w z Cf) ri E@ F4 w 1 44 x Oo 0 r4 r4 @4 El W rS4 cn 44 9 r4 r4 0 E@ 0 14 0 FY4 P4 r4 pq U) Pi occ 411 E@ r4 cn @4 PQ r4 @D E4 cn r4 CJ 0 E@ I m r4 In,Hz Cl)0 r@ W IX @ .1 It z 9c F-I 11 E@ x4 0H4 1 1 (H:) E4 1 0 E@ m : H -4 > .1 W;;o rn I 14 n to gn4.1 C) E@ 10 0 r4 0 H 9 m 4 r4 u 0 0 cn E@ - p -C z U2 14 0 F-I 0 .4 F-I n a U) L) 0 0 C4 El rn APPENDIX 1 ProtocolDeviations Protocol ProtocolAmendment No. 1 ProtocolAmendment No. 2 ProtocolAmendment No. 3 Covance 6329-225 3M T-6314.1 124 Protocol Deviations Covance 6329-225 3M T-6314.1 Protocol. Housing. Diet. "CertifiedRodent Diet #5002, meal (PMI Nutrition Internationala)d libitun4unlessotherwise specified." Actual Procedure. The animals were given Teklad 8728C pelletedfood from August 17, 1998 to August 23, 1998. Protocol. Dose Preparation. Retention Samples. "During the in-lifephase, samples (approximately 100 g) willbe taken from each dose preparation sampled for dose analyses and stored at room temperature. Unless used for analyses,these samples willbe discarded approximately I month afterc,ompletion of the in-lifephase." Actual Procedure. Retention samples were discarded afterapproximately I month of the completion of the in-lifpehase. Protocol. Observation of Aninmls. ClinicalObservations. 'Each animal willbe observed twice daily(am. and p.m.) formortalityand moribundity, recording findingsas they are observed." Actual Procedure. On Days 16 through 30, the a.nL and p.nL mortalityand moribundity checks were not documented. Protocol. Termination. Scheduled Sacrifices."After at least4 weeks of treatment, fiveanimals/sex/group willbe fastedovernight,bled for serum samples, anesthetizedwith carbon dioxide, weighed, and exsanguinated." Actual Procedure. Fasting of animals at the Week 5 scheduled sacrificewas not documented. 125 ProtocolDeviations(Continued) Covance 6329-225 3M T-6314.1 Protocol.ExperimentalDesign. PostmortemProcedures."The followingtissue(swhen present)or representativseampleswillbe collectefdrom each animaland preservedin 10% phosphate-bufferefdormalinu,nlessotherwisespecified." Actual Procedure. Some tissuesr,equiredby theprotocol were not availablfeor histopathologiecxaniinationM.issingtissuesarelistedwithappropriatecomments inthe pathologydatasheetsforindividuamlirnals.Summary tablesdo not includethem as having been examined. These deviationsarenotexpectedto have affectedtheresultosf thestudy. 126 C 0 V AA-CE. THE DEMWPUENT SERVMS COMPANY Sponsor: 3M St.Paul,Minnesota PROTOCOL Study Title: 13-Week DietaryToxicityStudywithN-Methyl Perfluorooctanesulfonanlido Ethanol(N-MeFOSE, T-6314)inRats Date: August 27,1998 PerformingLaboratory: Covance LaboratorieIsnc. 3301 Kinsman Boulevard Madison,Wisconsin 53704-2595 Laboratory Study Identification: ProposalNo. 90545D Covance 6329-225 SponsorPr01jectIdentification: 3M T-6314.1 127 Covance 6329-225 Page 2 Study 13-Week DietaryToxicityStudy with N-Methyl PerfluorooctanesulfonarrfiEdtohanol (N-MeFOSE, T-6314)inRats Purpose To assessthetoxicitoyfthetestmateriawlhen administereidnthediettoratsforatleast 13 weeks Sponsor 3M ToxicologyServices Building220-2E-02,3M Center St.Paul,Minnesota 55144-1000 Study Monitor Andrew M. Seacat,PhD 3M ToxicologyServices Telephone No.: 651.575.3161 FacsimileNo.: 651.733.1773 AlternateStudy Monitor Marvin T. Case,DVM, PhD 3M ToxicologyServices TelephoneNo.: 651.733.5180 FacsimileNo.: 651.733.1773 Study Location Covance LaboratorieIsnc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 MailingAddress: PO Box 7545 Madison,Wisconsin 53707-7545 128 StudyDirector PeterJ.ThomfordP,hD Covance LaboratoriesInc. TelephoneNo.: 608.241.7207 FacsimileNo.: 608.242.2736 Covance6329-225 Page3 Toidcologist Thomas E. Ryan, BS Covance LaboratorieIsnc. Proposed Study Timetable In-LffeStartDate: September 1,1998 In-LifeEnd Date: December 3, 1998 AuditedDraftReportDate: May 26, 1998 Regulatory Compliance Thisstudywillbe conductedincompliancewiththeFood and Drug AdministratioGnood LaboratoryPracticeRegulationsas setforthinTide 21 of theUS Code of Federal RegulationsP,art58,issuedDecember 22, 1978 (effectivJeune 20, 1979),and withany applicableamendments. Animal Care and Use Statement AR proceduresinthisprotocolareincompliancewith theAnimal WelfareAct Regulations9, CFR 1-4.In theopinionof theSponsorand studydirectort,hestudydoes not unnecessarildyuplicataeny previouswork. Quality Assurance The protocol,studyconduct,and finarleportwillbe auditedby theCovance Quality AssuranceUnit(QAU). The proliferaticoenllnuclearantigen(PCNA) evaluationd,ata, and reportwillbe auditedby theQAU ofPathologyAssociatesIntemationaLLiverand serum analysesd,ata,and reportwillbe auditedby theQAU of 3M Environmental Technology and SafetyServices. 129 Test Material Covance 6329-225 Page 4 Identification N-Methyl Perfluorooctanesulfonainido Ethanol (MEFOS, T-6314) Lot Number The lot numbers will be maintained in the raw data. Purity Responsibility of the Sponsor Stability Responsibility of the Sponsor Storage Conditions At room temperature Charactexistics Information on synthesis methods, composition, testmaterialison filewith the Sponsor. or other characteristicsthat define the Reserve (Archive) Samples A reservesample (approximately5 g) of each lotwillbe taken and storedat room temperature. These samples willbe ft-ansferretdo the Sponsor aftercompletion of the in-Iffpehase. Disposition of Test Material After authorizationfrom the Sponsor, any remaining testmaterialwillbe returned to: Andrew M. Seacat,PhD 3M Toxicology Services Building220-2E-02, 3M Center St.Paul,Nfimesota 55144-1000 Telephone No.: 651.575.3161 FacsimileNo.: 651.733.1773 130 Animals Covance 6329-225 Page 5 Species Rat Strain Crl:CDO(SD) IGS BR Source Charles River Laboratories, Inc.,Portage, Mchigan Age at Initiation of Treatment Preferably less than 6 weeks of age but not more than 8 weeks of age Weight at Initiation of Treatment 100 to 300 g Number and Sex 80 niales and 80 females Identification Implantable microchip identificationdevice Husbandry Housing Individual (may be group-housed during acclimation). Animals will be housed in suspended, stainlessteelcages. Diet CertifieRdodent Diet #5002, meal (PNU NutritionInternationala)d libitumu,nless otherwise specified.The dietisroutinelyanalyzedby themanufacturer fornutritional components and environmentalcontaminants. Specifiednutrientand contaniinant analysesare on fileat Covance-MadisorL 131 Covance 6329-225 Page 6 Water Ad libitum.Samples of thewater areroutinelyanalyzedforspecifiedmicroorganisms and environmentalcontaminants.The resultasreon fileatCovance-Madison. Contaminants There areno known contaminantsinthedietor water atlevelsthatmight interferweith thisstudy. Environment Environmentalcontrolsfortheanimalroom willbe setto maintain18 to 26'C, a relativheumidityof30 to70%, and a 12-hourlight/12-houdrarkcycle.The light/dark cyclemay be interruptetdoaccommodate in-lifperocedures. Acclimation At leastI week Randomization Selectionof animalsforthestudywillbe based on clinicaolbservationso,phthalmic examinationsa,nd otherdataasappropriate.Animals willbe assignedto treatment groups usinga computerizedblocldngproceduredesignedto achievebody weight balancewith respectto treatmentgroups. At thetimeofrandomizationt,heweight variatioonf theanimalsofeach sexused willnot exceed 2 standarddeviationosf the mean weight,and themean body weightforeach group of each sexwillnotbe statisticadlilfyferenattthe5.0% probabilitlyevel Justification Rats historicalhlayve been usedinsafetyevaluatiosntudieasnd arerecommended by appropriateregulatoryagencies. 132 Covance 6329-225 Page 7 Group Designations and Dietary Levels Number of Animals DietaryLevels Group Male Female (ppm MEFOS)' I (Control)" 20 20 0 2 (Low) 20 20 3 3 (Md) 20 20 30 4 (High) 20 20 100 a Dose levelsareexpressedas ppm of MEFOS. b The controlanimalswillreceivethebasaldietonly. Dosing Procedures Method ofAdministration Dietary,7 days/weekforatleast13 weeks. Treatmentwillcontinuethroughtheday beforenecropsy. Reason forDosing Route The potentiarlouteof exposureinhumans isoral. Dose Preparation AR dose preparationwsillbe mixed accordingto thestudy-specifmiicxingprocedure developedby Covance. Dose concentrationwsillbe based on theMEFOS contentas supplied.AR dose preparationwsillbe storedatroom temperature. Before initiatiofntreatmentd,osepreparationosf 1 ppm, 3 ppm 30 ppn-4100 ppm and 500 ppm willbe mixed. Dose preparationwsillbe mixed atleastonce every4 weeks duringthein-Iffpehase. Retention Samples During thein-lifpehase,samples(approximately100 g) willbe taken from each dose preparationsanipledfordose analysesand storedatroom temperature.Unlessused for analysest,hesesampleswillbe discardedapproximatelyI month aftercompletionof the in-lifpehase. 133 Covance 6329-225 Page 8 Dose Analyses By Covance, using a method supplied by the Sponsor and validated by Covance Homogeneity Homogeneity will be determined for 3-ppm, 30-ppni, and 100-ppm dose preparations once pretest.One swnple (approximately100 g) each from the top,middle,and bottom of the dose preparationsmixed forhomogeneity analyseswillbe collected,dividedinto threesubsamples forextractionand analysis,and analyzed fortestmaterialcontent. All samples willbe storedat room temperature untilanalyzed within7 days aftermixing. Homogeneity analysiswillbe repeated ifbatch sizechanges by more than 30%. Stability Four samples (approximately 100 g each) willbe taken from the I-ppm dose level concentrationof dietpreparationsn-dxedpretest.One sample willbe analyzed on the day of mixing and used as the baselinevalue. One sample willbe storedat room temperature forat least19 days,then analyzed. A thirdsample willbe storedat room temperature afterat least32 days, then analyzed. The remaining sample willbe stored in a freezersetto maintain-10 to -30*C for 8 weeks, then analyzed. In addition,two samples (approximately100 g each) willbe taken from the 500-ppm dose preparationmixed pretest.One sample willbe analyzed on the day of mixing. The second sample willbe willbe storedatroom temperature afterat least32 days, then analyzed. Dose Confirxnation During the in-lifpehase, samples (approximately 100 g) from alldose preparationswill be analyzed. Allsutles willbe storedatroom temperatureuntilanalyzed. 134 ObservationofAnimals Covance6329-225 Page9 ClinicalObservations Each animalwillbe observedtwicedaily(am and p.m) formortalitaynd moribundity, recordingfindingsas they areobserved. At leastonce weekly, each animal willbe observed (cage willbe opened, and the animal willbe removed); abnormal findingsor an indicationof.normal willbe recorded. Additionalfindingswillbe recorded as they are observed. Body Weights Each animal willbe weighed at leastonce priorto treatment,on the firstday of treatment,and weekly thereafter. Food Consumption Food consumption willbe recorded weekly duringtreatment.Food consumption Will not be measured duringWeek 5 foranknalsscheduledforsacrificdeuringWeek 5. Ophthahnic Examinations Ophthalmic examinations willbe done foreach animal beforeinitiatioonf treatment and before the Week 14 scheduledsacrifice.The eyes of each animal willbe examined by a veterinarianusing an indirectophthalmoscope. A mydriaticagent willbe instilleidnto the eyes priorto examination. ClinicalPathology Frequency Hematology and clinicaclhemistryduring Weeks 5 and 14;urinalysidsuring Week 14 Number of Animals Ten animaLs/sex/group(thesame anftnalswillbe used at each intervalifpossible) Method of Collection Animals willbe fastedovemight; blood willbe collectedfrom a jugularvein. The anticoagulantwillbe potassium EDTA forhematology tests.Urine willbe collected chilledovernight(approximately16 hours). 135 Covance 6329-225 Page 10 Tests Hematology red blood cell(erythrocytec)ount hemoglobin hematocrit mean corpuscularvolume mean corpuscularhemoglobin mean corpuscularhemoglobin concentration platelectount whitebloodcell(leukocytec)ount differentiballood cellcount blood cellmorphology reticulocytsemear (made,butnot examined) glucose urea nitrogen creafinine totalprotein albumin globulin cholesterol triglycerides totalbierubin CMcal Chemistry alanineamino=wferase gamma glutamyltransferase aspartateaminotransferase calcium inorganicphosphorus sodium potassium chloride appearance volume specifigcravity pH protein urobilinogen Urinalysis glucose ketones bibmbin blood microscopicexaniinatioonf sediment Serum PerfluorooetaneSuffonicAcid (PFOS) Analyses Frequency and Number of Animals FiveanimaLs/sex/grouapfteratleast4 and 13 weeks of treatment.Samplesafter 4 weeks of treatmentwillbe collectefdrom animalsselectedfortheinterim sacrifice. 136 Covance 6329-225 Page II Method of Collection Animals willbe fastedovernight;blood (approxiimtely4 mL) willbe collected from a jugularvein. Samples willbe collectedwithout anticoagulant. Sample Handling Blood samples willbe allowed to clotat room temperatureand centriffiged. Serum samples willbe harvestedand storedin a freezerset to maintain -60 to -80'C. Samples willbe packed on dry iceand shipped to: Kris J.Hansen, PhD 3M Environmental Technology and SafetyServices 935 Bush Avenue Building2-3E-09 St.Paul,Minnesota 55133-3331 Telephone No.: 651.778.6018 FacsimileNo.: 651.778.6176 Serum samples willbe analyzed forPFOS and metabolitesby theSponsor. Results willbe reported separatelyby the Sponsor. Termination Unscheduled Sacrificesand Deaths Necropsies willbe done. Animals to be sacrificewdillbe anesthetizedwith carbon dioxide,weighed, and exsanguinated. Scheduled Sacrifices After at least4 weeks of treatment,fiveanimaLs/sex/groupwillbe fastedovernight,bled forserum samples,anesthetizedwith carbon dioxide,weighed, and exsanguinated. The abdominal cavityof each animalwillbe opened, the liverwillbe removed and weighed, and liversamples willbe collectedforpahrnitoylCoA and PFOS analysis.Aninmls will be discardedafterlivercollection. 137 Covance 6329-225 Page 12 Afteratleast13 weeks oftreatment,allsurvivinganimalswillbe fastedovernight,bled forserum samples (fiveanimaL-,/sex/grouapn)d clinicaplathologytests(10/sex/group), anesthetizedwith carbon dioxide,weighed,exsanguinated,and necropsied.Liver samples willbe collectedforpalirnitoyCloA, PCNA, and PFOS analysis. Postmortem Procedures Necropsy The necropsy willincludean examinationof theexternalfeaturesof the carcass;all externalbody orificest;heabdominal thoracica,nd cranialcavitieso;rgans;and tissues. Organ Weights At the scheduledsacrificaefter13 weeks of treatment,the followingorgans (when present)willbe weighed; pairedorgans willbe weighed together: adrenal(2) brain kidney(2) liver ovary(2) spleen testi(s2) thymus thyroid(2)withpamthyroid Organ-to-body weight percentagesand organ-to-braiwneight ratioswillbe calculated. Bone Marrow Smear From the femur of each animalatthe scheduledsacrificaefter13 weeks oftreatment only;made but not examined CellProliferatioTnissueCollectionand hnmunohistochemical Evaluation At the scheduled sacrificaefter13 weeks oftreatment,representativseamples of left laterallobe of the liverfrom each animalwillbe collectedand preservedinzinc fonnalin. Afterfixatione,ach sample of liverwillbe embedded inparaffina,nd the paraffibnlocks willbe shippedto: 138 Covance 6329-225 Page 13 Sandra R. Eldridge,PhD Pathology AssociatesInternational 15 Worman's MR Court, SuiteI Frederick,Maryland 21701 Telephone No.: 301.663.1644, ext.2201 FacsimileNo: 301.663.8994 Proliferatiocnellnuclearantigen(PCNA) evaluationwillbe done on the samples. Resultswillbe provided forinclusionin the finalreport. Pahnitoyl-CoA Oiddase Tissue Collection and Analyses At the scheduled sacrificest,herightlateralobe of the liverwillbe collectedfrom each animal and flash-frozeninliquidnitrogen.The livertissuewillbe storedin a freezerset to maintain-60 to -80'C untilanalyzedby Covance forpahnitoyl-CoA oxidase activity. Liver PFOS Analysis At scheduled sacrificest,heremaining portionof the liverfrom each animalafterother requiredcollectionwsillbe storedina freezersetto maintain-60 to -80'C. Samples willbe packed on dry iceand shipped to Kris J.Hansen, PhD, 3M Environmental Technology and SafetyServices.Liver samples willbe analyzed forPFOS and metabolitesby the Sponsor. Results wiU be reported separatelyby the Sponsor. Tissue ]?reservation The followingtissues(when present)from each animal thatisfound dead, sacrificedat an unscheduled intervalo,r sacrificedat the scheduled sacrificaefter13.weeks of treatmentwillbe preservedinlo% neutral-bufferefdormalin: 139 Covance 6329-225 Page 14 adrenal(2) aorta brain cecum cervix colon duodenum epididymis(2) esophagus eye (2) femur with bone marrow (articulasrurface of the of the distalend) Hardenan gland heart ileum with Peyer'spatch (lyrnphoid aggregate) jejunum kidney(2) lesions liver lung with mainstem bronchi lymph nodes (mesentericand mandibular) mammary glands (femalesonly) ovary(2) pancreas pituitary prostate rectum salivarygland [mandibular(2)] sciaticnerve seminalvesicle(2) skeletamluscle (thigh) sldn spinalcord (cervicalrnid-thoracica,nd lumbar) spleen sternum with bone marrow stomach testi(s2) thymus thyroid(2)with parathyroid trachea urinarybladder uterus vagina Zymbal's gland I-listopathology Tissues(asappropriate)from each animal inthe controland high-dose groups and each aninml thatdiesor issacrificeadt an unscheduled intervalwillbe embedded inparaffin, sectioned,stainedwith hematoxylin and eosin,and examined microscopically Macroscopic lesionsfrom animalsin thelow- and mid-dose groups willbe embedded in paraffin,sectioned,stainedwith hematoxylin and eosin,and examined microscopically. Suspected targetorgans noted at the high dose willbe examined microscopicallyfrom each animal (atthe Sponsor'srequestand added by amendment). Reports One copy of the draftreportwillbe sent to the Sponsor. The reportwillincludethe followinginformation: 140 Experimental Design and Methods Covance 6329-225 Page 15 Results dose analyses mortality clinicaolbservations body weights body weight changes food consumption testmaterialconsumption ophthahnic findings clinicaplathology results pahnitoylCoA oxidase activities macroscopic observations microscopic observations ce.Uproliferatioanssessments (providedby the Sponsor's designee) StatisticalEvaluation Levene's testwillbe done to testfor variancehomogeneity. In the case of heterogeneity of varianceatp -50.05, transfonnationswillbe used to stabiliztehe variance. Comparison testswilltake varianceheterogeneityintoconsideration. One-way analysisof variance(ANOVA) willbe used (ifapplicable)to analyzebody weights,body weight changes,food consumption, continuous clinicaplathology values, and organ weight data. Ifthe ANOVA issignificantD,unnett'st-testwillbe used for controlversustreatedgroup comparisons. Ifthe ANOVA shows significancfeorbody weights at Week 1, one-way analysisof covariance(ANCOVA) wiU be used to analyzebody weights,with initiablody weights as the covariate.Ifthe ANCOVA issignificantc,ovariate-adjustemdeans willbe used for controlversus treatedgroup comparisons. Group comparisons (Groups 2 through 4 versus Group 1) willbe evaluatedat the 5.0%, two-toed probabilitlyevel.Only data collectedon or afterthe firsdtay of treatmentwillbe analyzed statistically. 141 Covance 6329-225 Page 16 At the end of 1 year afterissuanceof the auditeddraftreport,ifno requestedrevisionsor instructiontso finalizheave been communicated by the Sponsor, then theauditeddraft reportwillbe considered 'finarand issuedas the finalreport,signedby the study director, and submitted to the Sponsor. Any modificationsor changes to theaudited draftreportrequested I year afterissuance willbe performed at additionalcost to the Sponsor. Two copies of the signed finalreport(one unbound and one bound) willbe sentto the client. Record Retention AR raw data,documentation, records,protocol,specimens, and finalreportgenerated as a resultof thisstudy willbe archivedinthe storagefacilitioefs Covance-Madison fora period of I year followingsubmission of the finalreportto theSponsor. One year after submission of the finalreport,the aforementioned materialswillbe sentto theSponsor, and a returnfeewillbe charged; allraw data storedon magnetic media, protocol study correspondence, and the originalfinalreportwiU be retainedby Covance. The Sponsor may electto have thematerialsretainedin the Covance archivesforan additionalperiod of time,and Covance willcharge a storagefee. Ifthe Sponsor chooses to have Covance dispose of the materials,a disposalfee wiU be charged. PCNA antigenevaluationdata willbe retainedby Pathology AssociatesInternational. Liver and serum samples sentto the Sponsor and analysisdata willbe retainedby the Sponsor. 142 PROTOCOL APPROVAL Covance 6329-225 Page 17 IIJ@AZ)4 Ai@@rew?@.Seacat,PhD Study Monitor 3M P ete'rJ.T rd,P D StudyDirec r Covance LaboratodesInc. Date Date 143 COVAW-'C@--E.@ THEDRELOPAIgUrSUM= OOMPANY PROTOCOL AMENDMENT NO. I Covance 6329-225 13-Week DietaryToxicityStudywithN-Methyl Perfluorooetanesulfonarrjido Ethanol(N-MeFOSE, T-6314)inRats Sponsor: 3M, St.PauL Minnesota StudyMonitor: Andrew M. Seacat,PhD TestingFacility: Covance LaboratorieIsnc.,Madison,WLsconsin StudyDirector: PeterJ.Thomford, PhD Thisamendment modifiesthefollowingportionof theprotocol: EffectiveAugust 27, 1998 1. Page 3, Proposed Study Timetable. To correcttheauditeddraftreportdate, deletethetextinthissectionand replacewiththefollowing: In-LffeStartDate: September 1,1998 In-LifeEnd Date: December 3, 1998 AuditedDraftReportDate: May 26, 1999 EffectiveOctober 1,1998 2. Page 12,Postmortem Procedures,CellProliferatioTnissueCollectionand ImmunohistocheniicalEvaluation,Paragraph 1. To reflectthedecisiotno performproliferaticoenllnuclearantigenevaluationforfiveanimaLVsex/group only,deletethisparagraphand replacewiththefollowing: At thescheduledsacrificaefter13 weeks of treatment,representativseamplesof theleftlaterallobeoftheliverfrom fiveanimals/sex/grouwpillbe collecteadnd preservedinzincformalin. 144 AMENDMENT APPROVAL Covance 6329-225 ProtocolAmendment No, 1 Page 2 Andiew M. Seacat,PhD Date StudyMonitor 3M Pever ,Orrdd-,,PhD" Date Study D' r Co)rancf,aboratorieIsnc. 145 C 0 V Alt C'E--.'--' THE DEVELOPMENT SERVICESCOMPANY PROTOCOL AMEENDMIENT NO. 2 Covance 6329-225 13-Week DietaryToxicityStudywithN-Methyl Perfluorooetanesulfonamido Ethanol(N-MeFOSE, T-6314)inRats Sponsor: 3M, St.Paul,Minnesota StudyMonitor: Andrew M. Seacat,PhD TestingFacility: Covance LaboratorieIsnc.,Madison,Wisconsin StudyDirector: PeterJ.Thomford,PhD Thisamendment modifiesthefollowingportionof theprotocol: EffectiveOctober 21, 1998 I. Page 8,Dose Preparation,Dose Analysis,StabilityT.o reflectthe decisionto performadditionasltabiliatnyalysisa,dd thefollowingto thissection: Two additionaslamples(approximately100 g each)willbe takenfrom the3-ppm dose preparationmixed forWeek 9 through12. One samplewillbe storedat room temperatureforatleast19 days,thenanalyzed.The second sample willbe storedatroom temperatureforatleast32 days,thenanalyzed.The -sample collectefdordose confirmatioannalysiwsillbe used asthebaselinesample. 146 Covance 6329-225 ProtocolAmendment No. 2 Page 2 EffectiveDecember 4, 1998 2. Page 13,Postmortem Procedures, Liver PFOS Analysis,Sentence 1. To clarify collectioonf liversectionsforPFOS analysisd,eletethissentenceand replacewith thefollowing: At scheduledsacrificetsh,eremainingportionof theliverfrom eachanimalafter otherrequiredcollectionwsillbe flash-frozeinnliquidnitrogenand storedina freezersetto maintain-60 to -80*C. EffectiveDecember 15,1998 3. Page 13,Postmortem Procedures,CellProliferatioTnissue Collectionand Immunohistochemical Evaluation,Paragraph 3. To includeexaminationof liversectionswillbe stainedwithhematoxyhn and eosinas partof thecen proliferatieovnaluationd,eletethisparagraphand replacewiththefollowing: ProliferaticoenR nuclearantigen(PCNA) evaluationwillbe done on thesamples. Inadditionl,iversectionswillbe stainedwithhermtoxylinand eosinand examined microscopicallyR.esultswillbe providedforinclusioinnthefinal report. EffectiveJanuary 25, 1999 4. Page 14,Postmortem Procedures,Histopathology,Paragraph 3. Liverand stomach were identifieads targetorgansinanimalsinthehigh-dosegroup and will be examined inanimalsfrom themid-dosegroup. Thereforedeletethisparagraph and replacewiththefollowing: Liverand stomach from eachanimalinthe ryfid-dosgeroup wM be embedded in paraffins,ectioned,stainedwith hematoxyhn and eosin,and examined microscopically. 147 Covance 6329-225 ProtocolAmendment No.2 Page 3 EffectivFeebruary 8,19" 5. Page 14,PostxnortemProcedures,HistopathologyP,aragraph 3. liverand was identifieadstargetorgan inanfinalisnthe mid-dose group and will be examined inanimalsfrom the low-dose group. nerefore deletethisparagraph and replacewith the following: Liverand stomach from each animalinthe mid-dose group and liverfrom each animalinthe low-dose group willbe embedded inparaffm,sectioned,stainedwith hematoxylinand eosin,and examined microscopically. AMENDMENT APPROVAL Andrew M. Seacat,PhD Study Monitor 3M Petwj-.TbAOrd, @hD Study Dirdgt@or Covance LaboratoriesInc. Date Date 148 c 0 V A-Nr-C'E-.-"' THE DENLOPMENT SERVICESCOMPANY PROTOCOL AMENDMIENT NO. 3 Covance6329-225 13-Week DietaryToxicityStudywith N-Methyl Perfluorooctanesulfonamido Ethanol(N-MeFOSE, T-6314) inRats Sponsor: 3M, St.Paul,Minnesota Study Monitor: Andrew M. Seacat,PhD TestingFacility: Covance LaboratoriesInc.,Madison, Wisconsin Study Director: PeterJ.Thomford, PhD This amendment modifiesthe followingportionofthe protocol: EffectiveAugust 28,1998 1 . Page 4. To includethevehicleused to dissolvethetestmaterialbeforemixing with the diet,add the followingsection. Vehicle Identification Acetone Lot Numbers The lotnumbers willbe maintainedintheraw data. Purity On filewiththemanufacturer Stability On filewiththemanufacturer 149 Storage Conditions At room temperature Covance 6329-225 ProtocolAmendment No.-e- Page 2 Characteristics Informationon synthesismethods,composition,or othercharacteristitchsat definethevehicleison filewiththemanufacturer. AMIENDNMNT APPROVAL A) Andrew M. SeacatP,hD Study Monitor 3M Datc6/ PeiVrf fh or rrdP,hD' Study Dire,1?1(9or@( Covance LaboratoriesInc. 150 APPENDIX 2 IndividuaAlnimal FateData IndividuaCllinicaOlbservations IndividuaOlphthalmicObservations Covance 6329-225 3M T-6314.1 151 Ln 4 C4 -0 1 @4 ()Nrn C,3 %D 2%D1E4 0 v v -ov -o-o-o-o-o tnviviLno-@vv v v v v v v 1-4 cn v v vv m nm o 00 M M M M M M,-ir4H,-4H M M M M M M M M M M M M M M M H H H @4 >@ E4 ca cn I Z F-i >4 I x rn [I @o 0 Cq NNNN NNN MM : (D cn CD c@.c@,C) a a CD C) CD CD CD c@,c> C) C"Dc@ C"DC"DC) CD C:)CD C"D"a CD CD c> c"@C-) C) :D CD E-4 0 1I C,4Cq Cr-,I3CH ,4@H @ C,4C,4C,4C4 C,4C) CD c@,C> a C.4C,4C.4C,4C,4N cq C.4cq C4 " C4 cq C4 ND (D CD c@ @4 r-i r-4 H r-i H r-i ri H r-i H 14 14 14 14 '1 'A H r-I r-I r-I 4 4 r-i H 'I I 0 C,q 4J (d 1-1 E- tn E-0 z rg pqr4 D4 m UUUUL) H H H H F-I N Pl.P4 r..N w pqw Dq U L)L)L) HHHF4 N N Dr44. w Di r4 w 04 m w r4 r4 r49 PH44:04 p@w w pqw DQ w 41 w w Di N rAN r4r4 OH@HwH9 C4 U L)UUU U UC-) u ur)u UoL)UUF-)U Li U u UL) L)L) L) uuu u uuu 0 I N r-4P4 N 04 r@ N N r-Il@-M. 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RZHU- a,- :c ou @m m u "Ir.z, z m u cn 0 PQ " @r4 @ < 0 >1 ORDO@cHis5mg: :4 C4 u m 4 H >1 - H 1-4 - - o@ X 1 3: i@:@wo]O@4pzz>g w El H 1 4 N r4 4 w : 14 z -1 -,< r4 z H Z> w u z " r4 a@ @ 4 H :r @ 0Z .1a - 04 Z E-Z E- H H .1 z H z z z w H3 I OON M u)z 1:1 4% 4 a.cnZ:) 00 OL,M 04 W mZ 0 @l 4 H fe> i .4 0 @L4 I x E@ E@ C4 W E,' I E- E- ca APPENDIX 7 CeU ProliferatioRneport Covance 6329-225 3M T-6314.1 Note: This appendix of the reportcontainsinfonmtion suppliedby Pathology AssociatesInternationaalnd has been reviewed by the QualityAssurance Unit of Pathology AssociatesIntemationaL 546 Covance 6329-225 3M T-6314.1 PathologyAssociatesInternational wpm,go A Company ofScience ApplicationsIntemationalCorporation An frAPA*ve-OwnedCoeripany FINAL CELL PROLIFERATION REPORT 13-WEEK DIETARY PERFLUOROOCTANESULFONAMIDO COVANCE TO)ACITY STUDY ETHANOL STUDY NUMBER WITH N-METHYL (N-MeFOSE, T-6314)IN RATS 6329-225 PREPARED FOR: 3M TO)ACOLOGY SERVICES BUILDING 220-2E-02,3M CENTER ST. PAUL, MN 55144-1000 PREPARED BY: PATHOLOGY ASSOCIATES INTERNATIONAL 15 WORMAN'S MILL COURT, SUITE I FREDERICY,., MD 21701 MAY 12,2000 '5Worman's MillCourt,SuiteI Frederick,Maryland 21701 * (301)663-1644 (301)663-8994FAX 547 Covance 6329-225 3M T-6314.1 FinalCell ProliferatiRoenport Covance Study Number 6329-225 Page 2 TABLE OF CONTENTS CELL PROLIFERATION REPORT TABLES SIGNATURE PAGE IV.. QUALITY ASSURANCE STATEMENT V. APPENDIX I 548 CELL PROLIFERATION REPORT Covance 6329-225 3M T-6314.1 FinalCellProliferatioRneport Covanee Study Number 6329-225 Page 3 13-WEEK DIETARY PERFLUOROOCTANESULFONAMIDO COVANCE TOMCITY STUDY STUDY ETHANOL NUMBER WITH N-METEIYL (N-MeFOSE, T-6314) 6329-225 IN RATS PURPOSE The purpose of the study was to assess the toxicity of the test material when administered in the dietto ratsforat least13 weeks. This report,submitted by Pathology AssociatesInternational(PAI) to the study Sponsor, 3M, representsthe cellproliferationfindingsand interpretatiofnor Covance Study Number 6329-225 entitled" 13-Week Dietary Toxicity Study with N-Methyl PerfluorooctanesulfonamidoEthanol (N-MeFOSE, T-6314) In Rats". All aspectsof the tasks associatedwith PAI's portion of this study were conducted in compliance with the Food and Drug AdministrationGood Laboratory Practice(GLP) Regulations as setforthinTitle21 of the US Code of Federal Regulations, Part 58,issued December 22, 1978 (effectivJeune 20, 1979), and with any applicableamendments. MATERIALS AND METHODS Tissue Collection for Cell Proliferation Five animals per sex per group in groups I through 4 were sacrificedduring week 13 for hepatocellulaprroliferation.A sectionof the leftlaterallobe of the liverfrom each of 5 ratsper group (groups 1-4) was fixed and processed to paraffinblock by Covance per protocol specificationsT.issue blocks were shipped toPAI for sectioningand staining.From each block, a slidewas prepared for H&E evaluationand immunohistochemical detectionof proliferating cellnuclearantigen(PCNA), a marker ofcellproliferation. Immunohistochemistry for CellProliferation Sectionsof paraffin-embedded tissueswere cut at 5 gm and placed on positivelycharged slides (SuperfrostPlus,Fisher ScientificP,ittsburgh,PA) to ensure adhesion during processing for PCNA. Standard immunohistochemical methods were used to staintissuesfor PCNA (PAI's SOP for immunohistochemistry). Briefly,tissuesectionswere incubatedwith a monoclonal antibodyto PCNA (DAKO, lot#0 16, PAI No. A 1723) and reagentsrequiredforthe avidin-biotin peroxidase(ABC Kit, lot#PK-6100, PAI No. K 324) method f6r the detectionof the antigenantibodycomplex. PCNA expressionin cellsin allphases of the cellcycle(G,, S, G2 and M) was localizedby the chromagen 3,3'-diaminobenzidine(DAB; Sigma Chemical Co., lot #18H8201). Tissue sectionswere counterstainedwith hematoxylin. A negativecontrolslide was included in the stainingrun and consistedof study tissuethatwas not incubated with the primary antibody. 549 Covance 6329-225 3M T-6314.1 FinalCellProliferatiRoenport Covance Study Number 6329-225 Page 4 Cell Proliferation Measurements For cell proliferation evaluations, slides were firstperused at low magnification (IOOX') to judge qualityof staining,processing and sectioning,patternof cell labeling(e.g.,centrilobulaorr panlobuiar,extrahepatocellulaprroliferations,uch as Kupffer cells,bile duct epithelium, endothelium),and histomorphologic changes. Cell proliferatiownas then quantifiedat higher magnification(20OX). The percentage of hepatocytesin S phase of the cellcycle (labeling index,LI) was determined by scoringat least3000 hepatocytesin 10 randomly selectedfields per animal. Histomorphology was furtherassessedby evaluatingthe serialH&E slidefor each animal evaluatedfor cellproliferation. r Statistical Analysis The Student's t-test (two-sided, unequal variance) was used to test for statistical significance in Ll between controland treatmentgroups using Microsoft Excel version 5.0. A P value of less than 0.05 was judged to be statisticalsliygnificant. RESULTS CellProliferation Individualanimal and group mean cellproliferatiodnata are presentedin SectionII (Table 1). The percentage of proliferatinhgepatocytes,as determined by the labelingindex (LI),was not significantliyncreasedin treatedgroups compared to thatof controlsin eithermale Or female rats.The Ll was significantllyessthan controlsin female ratsfrom groups 3 and 4. Histopathology Sections from the same tissueblocks used forpreparationof PCNA-stained slideswere stained with hematoxylin and eosin (H&E) for histopathologicevaluationto facilitattehe interpretation of the immunostained slides.Individualanimal findingsand group sunnnariesare presented in Appendix I. k The resultsshowed no changes in the livertissueof male and female ratsthatwould alterthe interpretatioonf the PCNA stainingin thisstudy. 4 k 550 4 SUMNLARY Covance 6329-225 3M T-6314.1 FinalCellProliferafioRneport Covance Study Number 6329-225 Page 5 In the present study, cellproliferatiownas measured within the liverof male and female rats from control(0 ppm), low dose (3 ppm), mid dose (30 ppm) and high dose (100 ppm) after13 weeks on study. No statisticallsyignificanitncreasesin cellproliferationa,s determined by the PCNA labelingindex (LI) were observed in male or female ratsafter13 weeks of N-methyl perfluorooctanesulfonamidoethanol.Although cell proliferatiownas statisticalllyess than controlsin mid dose (30 ppm) and high dose (100 ppm) females,the decrease was not doserelated,and the individualanimal values were within the range observed in control animals. Therefore,thisdecrease was not considered to be biologicallyrelevant. 551 Covance 6329-225 3M T-6314.1 FinalCeH ProliferatiRoenport Covance StudyNumber 6329-225 Page 6 ][IT.ABLES Legend SEM Standard Error oftheMean 552 Table 11-1.Cell ProliferatioinRat Liver Covance 6329-225 3M T-6314.1 Covance Study No. 6329-225 Dose Group 1 (C ntrol- 0 ppm) 1 (Control- 0 ppm) I (Contro-l 0 ppm) I (Contro-l0 ppm) 1 (Control- 0 ppm) 2 (Low -3 ppm) 2 (Low -3 ppm) 2 (Low - 3 ppm) 2 (Low -3 ppm) 2 (Low -3 ppm) 3 (Mid- 30 ppm) 3 (Mid-30 ppm) 3 (Mid-30 ppm) 3 (Mid-30 ppm) 3 (Mid- 30 ppm) 4 (High- 100 ppm) 4 (High- 100 ppm) 4 (High- 100 ppm) 4 (High- 100 ppm) 4 (High- 100 ppm) PCNA Animal Labeling Sex Number 1 Index m I C95902 0.387 m C95905 0.246 m C95906 0.227 m I C95909 0.153 m C95918 0.583 Mean 0.319 SEM 0.076 m C95924 0.190 m C95925 0.208 m C95927 0.235 m C95936 M C95937 0.063 0.224 Mean 0.184 SEM 0.031 C95942 0.162 M C95947 0.160 m- C95948 0.186 m C95950 0.065 m C95958 0.160 Mean 0.147 SEM 0.021 M C95962 0.130 m Cl 0-163 m C95967 0.881 c-95968 0.096 M I C95977 mean 0.162 0.286 SEM 0.149 Page 11-1 553 Table 11-1.CellProliferatiionRat Liver Covance 6329-225 3M T-6314.1 Covence Study No. 6329-225 PCNA Animal Labeling Dose Group Sex Number Index 1 (Control- 0 ppm) F C95985 0.179 1 (Control- 0 ppm) F C95987 0.153 1 (Control- 0 ppm) F C95988 0.298 1 (Control- 0 ppm) F C95997 0.215 1 (Control- 0 ppm) F C96000 0.181 k Mean 0.205 SEM 0.025 2 (Low -3 ppm) F C96002 0.244 2 (Low -3 ppm) C96003 0.749 2 (Low -3 ppm) 2 (Low - 3 ppm) 2 (Low -3 ppm) iF F F c-i C960C)8 C9601 0 0.253 0.122 mean 1 -0.330 0.108 3 (Mid- 30 ppm) F C96021 0.088 3 (Mid- 30 ppm) F C96023 0.063 3 (Mid- 30 ppm) F C96026 0.099 3 (Mid- 30 ppm) 3 (Mid-30 ppm) _:@F C96038 0.181 0.062 Mean 0.099 SEM 1 0.022 (High-100 ppm) 96045 0.032 4 (High-100 ppm) 4 (High- 100 ppm) 4 (High-100 ppm) F C96046 0.128 F C96048 0.121 F C9604 9 0.152 4 (High- 100 ppm) F C96056 i 0.128 k mean L 0.112 SEM 0.021 Page 11-2 554 Covance 6329-225 3M T-6314.1 FinalCellProliferafioRneport Covance Study Number 6329-225 Page 7 III.SIGNATURE PAGE Submitted by: ProjectManager: Sandra R. Eldridge,Ph.D. ProjectPathologist: 00 Date - Carolyn Moy(W, D.V.M., Di'plomate,A.C.V.P. Date 555 Covance 632 9-225 3M T-6314.1 FinalCellProliferatiRoenport Covance Study Number 6329-225 Page 8 IV. QUALITY ASSURANCE STATEMENT 556 t-ovance6329-225 3M T-6314.1 Pathology Associates lntemational aw A Company ofScienceApplicatioInnsternatioCnoarlporation A@ Empiyooown#d Coffpny CellProliferatioRneport 13-Week DietaryToxicityStudy withN-Methyl PerfluoroctanesulfonamidEothanol (N-MeFOSE, T-6314) inRats Covance Study Number: 6329-225 QUALITY ASSURANCE STATEMENT This cellproliferatiopnrojecthas been inspectedand auditedby the PAI Quality AssuranceUnit (QAU) as requiredby the Good LaboratoryPractice(GLP) regulations promulgatedby the U.S. Food and Drug Administratio(nFDA). The cellproliferation reportisan accuratereflectioonftherecordeddata.The followingtableisa recordofthe inspections/audiptesrformed and reportedby theQAU. Date of Inspection 12/23/98 04/03/00 04/03/00 @05/12/00 Phase Insi)ected Date FindingsReported to PAI Manar-ement/ProiectManaaer Microtomy Study Data and SupportingDocumentation DraftCellProliferatioRneport FinalCellProliferatiRoenport 12/23/98 04/03/00 04/03/00 05/12/00 Karen E. Butler QualityAssuranceOfficer /c c-.., Date 557 15Worman's MillCourt,SuiteI FrederickM,aryland2170!1o (301)663-:1!64o4(301)663-8994 FAX Covance 6329-225 3M T-6314.1 FinalCellProliferatiRoenoort Covance Study Number 6329-225 Page 9 APPENDIX I 558 ANIMAL NUMBER C96027 C96026 C96021 C96023 C96038 C95948 C95950 C95942 C95947 C95958 C95965 C95967 C95968 C95962 C95977 C96048 C96045 C96056 C96049 C96046 C95985 C95997 C95988 C95987 C96000 C95909 C95902 C95918 C95905 C95906 IndividuaAlniM21 Findings Covance 6329-225 3M T-6314.1 COVANCE #6329-225 13-Week Diet2ryToxicityStudy withN-Methyl PerfluorooctanesulfonamidEothanol (N-MeFOSE, T-6314)inRats Tissue:Liver SEX DOSEGROUP HISTOLOGIC FINDINGS F 3 F 3 F 3 F 3 F 3 m 3 m 3 m 3 m 3 m 3 m 4 m 4 m 4 m 4 m 4 F 4 F 4 F 4 F 4 F 4 F I F I F I F I F I m I m I m I m I m I No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings 559 ANEWAL NUMMER C95925 C95937 C95936 C95924 C95927 C96010 C96003 C96002 C96006 C96008 C96003 C96002 C96006 C96008 IndividualAnimal Findings Covance 6329-225 3M T-6314.1 COVANCE #6329-225 13-Week DietaryToxicityStudy with N-Methyl PerfluorooctanesulfonaniidEothanol (N-MeFOSE, T-6314) in Rats Tissue: Liver SEX DOSE GROUP HISTOLOGIC FTNDINGS m 2 m 2 m 2 m 2 m 2 F 2 F 2 F 2 F 2 F 2 F 2 F 2 F. 2 F 2 No SignificanFtindings No SignificanFtindings No SignificantFindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings 560