Document 82ZVx3xQV9kpd92VY3Xn787Va

c 0 V A At 'C-E*.'"@ THE DEVELOPMENT SERVICESCOMPANY Sponsor: 3M St.Paul,Nfimesota FINAL REPORT Study Title: 4-Week Range-FindingDietaryToxicityStudy with N-Methyl PerfluorooctanesulfonarniEdtohanol (N-MeFOSE, T-6314) inRats Author: PeterJ.Thomford, PhD Study Completion Date: July5, 2000 Performing Laboratory: Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Study Identification: Covance 6329-224 Sponsor ProjectIdentification-, 3M T-6314.2 Page I of 373 QUALITY ASSURANCE STATEM[ENT Covancc 6329-224 3M T-6314.2 F This report, with the exception of Appendix 7, has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations, 21 CFR 58. The data in Appendix 7 was generated by and is the responsibility of Pathology Associates InternationalT.he foflowinginspectionwsere conductedand findingsreportedtothe studydirectorand studydirectormanagement. Inspection Dates. From To 06/04/98 06/04/98 07/13/98 07/13/98 09/16/98 09/25/98 10/01/98 10/09/98 10/19/98- 10/22/98 06/02/99- 06/02/99 05/02/00 05/03/00 Phase ProtocolReview Postlife Data Review Data Review Report Review ProtocolAmendment ReportReview Review Date Reported to Study Directorand Study DirectorManagement 06/04/98 07/13/98 09/25/98 10/09/98 10/22/98 06/02/99 05/03/00 k at t4 A Ll A L" ogAILA RepresentaVve QualityAssuranceUnit i A 0%@,A@ T .2Doo Date 2 Covance 6329-224 3M T-6314.2 STUDY I]DENTUTCATTON 4-Week Range-FindinDgietaryToxicitSytudywithN-Methyl PerfluorooetanesulfonamiEdtohanol(N-MeFOSE, T-6314) inRats TestMaterial N-Methyl Perfluorooctanesulfonamido Ethanol(MEFOSE, T-6314) Sponsor 3M ToxicologyServices Building220-2E-02,3M Center St.Paul,Minnesota 55144-1000 StudyMonitor Andrew M. Seacat,PhD 3M ToxicologyServices 651.575.3161 AltemateStudyMonitor Study Location StudyDirector Study Timetable Study InitiatioDnate In-LifeStartDate In-LifeEnd Date Study CompletionDate Marvin T. Case,DVM, PhD 3M ToxicologyServices 651.733.5180 Covance LaboratoriesInc. 3301 Yinsman Boulevard Madison,Wisconsin 53704-2595 PeterJ.Thomford, PhD Covance LaboratoriesInc. P.O.Box 7545 Madison,Wisconsin 53707-7545 608.241.7207 June 8, 1998 June 12,1998 July 13, 1998 July5,2000 3 Covance 6329-224 3M T-6314.2 KEY PERSONNEL 4-Week Range-Fmding DietaryToxicityStudywithN-Methyl PerfluorooctanesulfonaniEitdhoanol(N-MeFOSE, T-6314) inRats Study Director Study Toxicologist Study Coordinator SupervisorS,mallAnimal Toxicology Supervisor,D*ose Formulation SupervisorC,hemisty/Tox Support SupervisorL,aboratoryAnimal Medicine ClinicaPlathologist Supervisor,ClinicaPlathology AnatomicalPathologist SupervisorA,natomicalPathology PeterJ.Thomford, PhD Thomas E. Ryan, BS Niivan Bruce-Konuah Nathan E.Snortum,BA, BT, ALAT DixieBushee,BS, LATG Brian SchoenficeB,S Donna J.Clemons, DVM Diplomate,ACLAM RobertL. Hall,DVM, PhD Diplomate,ACVP (ClinicPaalthology) RonaldMarkevitch,BS, MT (ASCP) RichardHamiltonBruner,DVM Diplomate,ACVP PathologyAssociatesInternational Deborah PirkelA,LAT 4 Covance 6329-224 3M T-6314.2 CONTENTS Page ABSTRACT ..............................................................8............ PURPOSE ...............................................................1.0.-........... REGULATORY COMPLIANCE ............................................1.0............ TEST MATERLKL AND VEHICLE ..........................................1.0............ TestMaterial...........................................................1.0........... Vehicle................................................................10............ Reserve(Archive)Samples...............................................I.I............ Dispositio.n...........................................................I.I............ TEST SYSTEM ...........................................................I.I............ TestAnimal ............................................................I.I............ Identificat.i.o.n........................................................I.I............ Acclimation............................................................I.I............ Housing and Maintenance................................................1.2............ Justificati.o.n.........................................................1.2............ PROCEDURES ..........................................................1.2............ Group Designationsand DietaryLevels.....................................1.3............ Dose Preparation................................i........................13............. RetentionSamples ......................................................1.4............ Dose Analyses.........................................................1.4............ Method ofAdministratio.n...............................................1.5............ CMnicalObservations....................................................1.6............ Body Weights..........................................................1.6............ Food Consumption ......................................................1.6............ Serum PerfluorooctanSeulfonicAcid Level(PFOS) Determination...............1.6........... ClinicaPlathology.......................................................1.6............ Necropsy ..............................................................1.7............ Organ Weights.........................................................1.8............ CellProliferatiToinssueCollectioannd ImmunohistocheniicaElvaluatio.n.........1.8....... Pahnitoyl-CoAOxidase TissueCollectionand Analyses.........................1.8............ LiverPFOS Analyses....................................................1.8............ TissuePreservatio.n.....................................................1.9............ StatisticAanlalyses......................................................2.0............. RECORD RETENTION ...................................................2.0............ 5 Covance 6329-224 3M T-6314.2 CONTENTS (Continued) Page RESULTS ........................................................................2.1................ Dose Analyses ............................................................2.1.............. ClinicalObservationsand Survival...........................................2.1............... Body Weights and Body Weight Changes .....................................2.2............... Food Consumption ........................................................2.2.............. Test MaterialConsumption .................................................2.3............... Serum PerfluorooctaneSulfonicAcid Level (PFOS) Determination................2.3.......... ClinicaPlathology.........................................................2.3.............. CellProliferatioTnissue Collectionand Immunohistochemical Evaluation...........2.4...... LiverPFOS Determination..................................................2.4.............. Anatomical Pathology......................................................2.4.............. CONCLUSIONS ............................................................25............... SIGNATURES .............................................................2.6............... REFERENCES .............................................................2.7............... PATHOLOGY REPORT ......................................................................2..8....................... COMMENTS ON THE DATA ................................................33............... CODES, ABBREVIATIONS, AND UNITS .....................................3.5............... General Codes and Abbreviation.s...........................................3.6............... Codes forClinicaPlathology................................................3.7............... Abbreviationsand UnitsforClinicaHlematology ...............................4.0.............. Abbreviationsand UnitsforClinicaClhemistry .................................4.2.............. Abbreviationsand UnitsforClinicaUlrinalysi.s................................4.4............... Codes forAnatomical Pa' thology.............................................4.5.............. TABLES 1 Resultsof Homogeneity Analyses(ppm) ...................................4.7.............. 2 Resultsof StabilitAynalyses(ppm) .......................................4.9............... 3 ResultsofDose PreparationAnalyses(ppm) ................................5.1.............. 4 Summary of ClinicalObservations....................I....................................5.3............... 5 Summary of Body Weight Data (g).......................................5.4............... 6 Summary of Body Weight Change Data (g)................................5.8............... 7 Summary of Food Consumption Data (g)..................................6.2............... 8 Summary of Test MaterialConsumption Data (mglkg/day)...................6.6.............. 9 Sumn-iaryof ClinicalHematology Data ....................................6.8............... 10 Summary of ClinicaClhemistryData ......................................7.2............... II Summary of ClinicaUlrinalysiDsata ......................................7.6............... 6 Covance 6329-224 3M T-6314.2 CONTENTS (Continued) Page TABLES 12 SunmiaryofAbsoluteOrgan Weight Data (g).............................7.8............ 13 Sumniary of Organ-to-BodyWeight Percentages...........................8.1............ 14 Summary of Organ-to-BrainWeight Ratios...............................8.4............ 15 IncidenceofMacrosco PIC Observations..................................8.7............ 16 Incidenceof MicroscopicObservation.s..................................8.9............ 17 Incidenceof Severityof SelectedMicroscopicObservation.s.................9.7............. APPENDIX 1............................................................9.8............ ProtocolDeviations.....................................................9.9............ Protoco.l.............................................................1.0.0............ ProtocolAmendment No. 1 ..............................................1.1.7............ APPENDIX 2...........................................................1.2.3............ IndividuaAlnimal FateData..............................................1.2.4............ IndividuaCllinicaOlbservation.s..........................................1.2.7............ APPENDIX 3...........................................................1.3.3............ IndividuaBlody Weight Data (g)..........................................1.3.4............ APPENDIX 4...........................................................1.3.8............ IndividuaFlood Consumption Data (g).....................................1.3.9............ IndividuaTlestMaterialConsumption Data (mg/kg/day.)......................1.4.3............ APPENDIX 5...........................................................1.4.6............ IndividuaCllinicaHlematology Data .......................................1.4.7............ IndividuaCllinicaClhemistryData .............................................1..5.9............ IndividuaCllinicaUlrinalysiDsata.........................................1.6.7............ APPENDIX 6...........................................................1.7.9............ IndividuaAlbsoluteOrgan Weight Data (g).................................1.8.0............ IndividuaOlrgan-to-BodyWeight Percentage.s..............................1.9.2............ IndividuaOlrgan-to-BrainWeight Ratios...................................2.0.4............ IndividuaAlnimal PathologyData.........................................2.1.6............ APPENDIX 7 ...........................................................3.5.9............ CellProliferatiRoenport ................................................3.6.0............ 7 ABSTRACT Covance 6329-224 3M T-6314.2 The purposeofthisstudywas toassessthetoxicitoyfthetestmaterial N-Methyl PerfluorooctanesulfonamiEdtohanol(N-MeFOSE, T-6314) when administered inthedietto ratsforatleast4 weeks. Six groupsofCrl:CD'O(SD)IGS BR ratswere exposedtoN-MEFOSE inthe dietforat least4 weeks. Each group containedsixnialesand sixfemales.The dose levelswere 0 (controglroup),1,10,30, 100,and 500 ppm. Food was providedad libitume,xceptwhen animalswere fasted.Water was provided ad libitum.The aninmlswere observedtwicedaily(anL and p.m.)formortalitaynd moribundity.At leastonce each week, each animalwas removed from itscage and examined forabnormalitieasnd signsoftoxicityB.ody weightsand food consumption k datawere collectedtwiceweekly forat least4 weeks. Blood and urinesampleswere, collectedforhematology,clinicaclhemistry,and urinalysitsestsfrom allsurvivinganimals after4 weeks of treatment.Afteratleast4 weeks of treatmentb,loodwas alsocollected from allsurvivinganimalsforserum perfluorooctanseulfoniaccid(PFOS) analyses.On Day 32,theanimalswere anesthetizedw,eighed,exsanguinateda,nd necropsied.At necropsy,macroscopic observationswere recorded,selectedorganswere weighed,and selectedtissueswere collecteadnd preserved.Samples of liverwere collectefdrom all survivinagnimalsforpahnitoyl-CoAoxidaseactivit(ybyCovance)and proliferaticoenll nuclearantigen(PCNA) evaluatioannd liverPFOS analysi(sbytheSponsor).The animal k thatdiedon testwas.alsonecropsiedb,ut organ weightswere notrecorded.Microscopic examinationswere done on tissuesfrom each animal. One animalgiven500 ppm N-MEFOSE diedon Day 25. Allotheranimalssurvivedto the scheduleds,acrificeN.o clinicaolbservationwsere notedthatwere consideredtorepresent primaryeffectsof thetestmaterial.Lower body weight,body weightgains,and food consumptionin animalsgiven500 ppm were testmaterial-related. DietaryadministratioonfN-MEFOSE ata dose levelof 500 ppm forapproximately 4 weeks was associatedwithseveraleffectosn clinicaplathologytestresultsincluding moderatelylowerred bloodcellcount,hemoglobin,and hematocritl;ower absolute eosinophiclount;mildlyhigherureanitrogenm;oderatelyhigheralbuminand mildlyto 8 k Covance 6329-224 3M T-6314.2 moderatelylower globulinm;arkedlylower cholesteromli;ddlytomoderatelyhighertotal bilirubians,partateaminotransferasea,nd al%aineaniinotransferasaen;d moderatelyhigher hepaticpahnitoyl-CoAoxidase.Of uncertairnelationshitpo administrationf N-MEFOSE were lower urinepH formales fed500 ppm and smalldifferencefsoranimalsfed 100 ppm includinhgigherureanitrogena,lbumin,and alanineaniinotramferas(emalesonly)and lower cholesterolO.f thedifferencefsoraninws fed 100 ppm onlythedifferencfeor alanineantinotransferawsaes statisticaslilgynificant. Mean absoluteand relativ(etobrainand body)liverweightsforallnialesand females given 100 and 500 ppm N-MEFOSE were significantilnycreasedovercontrolvalues,and thesevariationwsere regardedas treatrnent-relatAeldl.additionamlean organ weights, which variedsignificantflryom controlvalues,were consideredto be withintherangeor normal biologicvariationo,r were manifestationosf slighwteightlossassociatewdith physiologicstress. Treatment-relatemdicroscopicfindingwsere restrictetdo theliverand consistedof minimalto severecentrilobulhaerpatocellulahrypertrophy.Hypertrophicchangeswere slightlmyore severeinmales;and atthehigh-doselevellivercellswellinginseveralmales was associatedwithacutenecrosis.Necrosiswas attributetdorestrictebdlood supply (ischeniifar)om swollenlivercellsratherthandirecthepatocelluldaarmage. Additional microscopicfindingswere consistenwtithcommon, spontaneousalterationisnlaboratory ratsor changes associatedwithstressn,utritionadleprivationo,r tissueprocessingartifact. Based on theresultosf thisstudy,dietaryadministratioonfN-MEFOSE to Crl:CDO(SD) IGS BR ratsforatleast4 weeks resultedinadverseeffectasta dose levelbf 500 ppnl The no-observable-adverse-effleecvtelwas determinedto be 100 ppm. PURPOSE Covance 6329-224 3M T-6314.2 The purposeofthisstudywas toassessthetoxicitoyfthetestmaterial, N-Methyl PerfluorooctanesulfonarnEitdhoanol(N-MeFOSE, T-6314)when administered inthe dietto ratsforatleast4 weeks. REGULATORY COMPLL4,NCE The studywas conductedincompliancewiththeFood and Drug AdministrationGood LaboratoryPracticeRegulationsassetforthinTitle21 of theUS Code of Federal RegulationsP,art58,issuedDecember 22, 1978 (effectivJeune 20, 1979),and with any applicableamendments. TEST-MATERL4,L AND VEHICLE Test Material The testmaterialN,-Methyl PerfluorooctanesulfonamidEothanol(MEFOSE, T-6314), Lot No. F-11364,isan offwhitepowder. Itwas receivedatCovance on August 3, 1995. Informationon synthesimsethods,stabilitpyu,rity,composition,or othercharacteristics thatdefinethetestmaterialison filewiththeSponsor. The testmaterialwas storedatroom temperature. Vehicle The vehiclewas acetone(SpectrumQualityProductsInc.,Gardena,CaliforniaL)o,t No. LH0253 (expiratiodnate:June 2000). Itwas receivedatCovance on June 23, 1997. The vehiclewas storedatroom temperature. Informationon synthesimsethods,stabuityp,urity,composition,or othercharacteristics thatdefinetheacetoneison filweiththemanufacturer. 10 Covance 6329-224 3M T-6314.2 Reserve (Archive) Samples A reservesample (approximately5 g) of the testmaterialwas taken and storedat room temperature. These samples willbe transferredto the Sponsor afterauthorizationfrom the Sponsor. Disposition Remaining testmaterialwas retainedfor futureuse (seeProtocol Deviations page for exception). TEST SYSTEM Test Animal Male and female Crl:CDO(SD) IGS BR ratswere obtainedfrom the Raleigh,North Carolina,facilitoyf Charles River Laboratories,Inc.,on June 2, 1998. The aninmls were 31 to 37 days old at initiatioonf treatment. "lbenialesweighed from 144 to 194 g, and the females weighed from 114 to 158 g at initiatioonf treatment. Identification Each animal was assigneda temporary number upon arrival.Before initiatioonf treatment,a microchip identificatiodnevice was implanted intoeach animal. After randomization forplacement on test,each animal was assigned a permanent number, and the microchip was coded with the permanent number. AU data foran animal are recorded under these numbers. Acclimation Forty-one males and 41 females were received on June 2, 19.98,and acclimated in Animal Room 349 for 10 days before initiatioonf treatment. In general animalsin thisshipment appeared healthy. During acclimation,the animals were examined for abnormalities indicativeof healthproblem, and body weights were recorded forallanimalsat randomization. Covance 6329-224 3M T-6314.2 Housing and Maintenance Animal Room 349 was used for thisstudy. Envirommental controlsfor the animal room were setto maintain18 to 26*C, a relativheun-ddityof 30 to70%, and a 12-hourlight/12-houdrark cycle.Variationsfrom theseconditionsaredocumented inthe dataand areconsideredto have had no effecton the outcome of thestudy. The animalswere housed individuall(yexceptforthefirs7t days ofacclimationwhen animalswere group-housed)instainlesssteelscreen-bottomcages. Certifierdodent diet#5002 meal, (PNU NutritionInternationalw)as providedad libitum, exceptwhen animals were fasted.The dietisroutinelyanalyzedby the manufiicturefror nutritionaclomponents and environmentalcontaniinmts.The resultsare on filewith Covance-Madison. Water was provided ad libitum.Samples of thewater are analyzedfor specified microorganismsand environmentalcontaminants.The resultsare on filewith Covance-Madison. There were no known contaminantsinthedietor water atlevelsthatwould have interferewdith thisstudy. Animals not selectedforthe study(fivemales and fivefemales)were removed from the study room and used fortrainingprocedures. Jusfification Rats historicalhlayve been used insafetyevaluationstudiesand arerecommended by appropriateregulatoryagencies. PROCEDURES This study was conducted inaccordancewith the Protocoldated June 8, 1998, and ProtocolAmendment No. 1. The protocol,protocolamendment, and protocoldeviations are inAppendix 1. 12 Covance 6329-224 3M T-6314.2 The animals were examined by a laboratoryanimal veterinarianon June 10, 1998, and found to be suitableforstudy consideration.Selectionof aninmls forthe study was based on clinicaolbservations,body weights,and other data as appropriate.Anini& were assignedto treatment groups using a computerized blocldng procedure designed to achieve body weight balance with respectto treatmentgroup. At the time of randomization,the weight variationof theanimals foreach sex used did not exceed 2 standarddeviationsof the mean weight. Group man body weights were analyzed using Levene's testforhomogeneity of varianceat the 5.0% probabilityleveland found to be homogeneous. Animals were assignedto the study according to the followingdesign: Group Designations and Dietary Levels Group Number of Animals Male Female DietaryLevels (ppm MeFOS)a 1 (Control)" 6 6 0 2 (Low) 6 6 1 3 (Low-Mid) 6 6 10 4 (Mid) 6 6 30 5 (Nfid-High) 6 6 100 6 (Mgh) 6 6 500 a Dose levelswere expressed as ppm of MEFOS. b The controlanimalsreceivedthe basaldietonly. Dose Preparation Dietaryconcentrationswere based on the testmaterialas supplied.Dietswere prepared pretestforhomogeneity, and twice foruse in thein-lifpehase. Preparation of Diet for Group 1. The appropriateamount of dietwas weighed intoa labeledcontainer,and 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 Diet for Group 6. Each dose levelwas prepared independently.A specifiedamount of dietwas weighed intoa labeledHobart@ mixing bowl The required amount of testmaterialwas weighed and transferredintoa labeledcontainer. Approximately 5 mL of acetone was added to the containerand mixed manually. 13 Covance 6329-224 3M T-6314.2 Increments of approximately 5 mL acetone were added as necessary untilthe testmaterial had dissolved.To prepare a premix, the dietwas transferredintoa labeledHobart@ mixing bowl. The testmaterialand acetone were added to themixing bowl, overlaidwith a portionof dietfrom the mixing bowl and thoroughlymixed. A portionof dietfrom the mixing bowl was trawferredto a second mixing bowl mixed manually to recoverresidual testmaterialand returnedto the firstmixing bowl. The contentsof themixing bowl were thoroughly mixed. Preparation of Diets for Groups 2 through 5. Each dose levelwas prepared independently. A specifiedamount of dietwas weighed intoa labeledHobart@ mixing bowl and a specifiedamount of dietfor Group 6 was weighed foreach diet.A pocket was formed in the feed in the mixing bowl and the amount of dietforGroup 6 was transferred to the pocket. The contentsof the mixing bowl were thoroughly mixed for 10 minutes. Samples fordose analyseswere taken directlyfrom the mixing bowl The prepared testdietswere storedatroom temperature in covered containersuntil dispensed intofeedingjars. Retention Samples Samples (approximately 100 g) were taken from each dose preparationsampled for dose analysesand stored at room temperature. These samples were discarded on December 9, 1998 (see ProtocolDeviationspage forexception). Dose Analyses Analyses forthe concentrationof testmaterialin the dose preparationswere done by Covance using an analyticamlethod, MP-M324-MA, suppliedby the Sponsor and validatedby Covance. Homogeneity was determined foralltestmaterialdose preparationsonce pretest.One sample each (approximately100 g) from the top,middle,and bottom of the dose preparationsmixed forhomogeneity analyseswas collected,dividedintothreesubsamples forextractionand analyzedfortestmaterialcontent. Allsamples were storedat room 14 Covance 6329-224 3M T-6314.2 temperatureuntilanalyzed.In additiono,ne sampleeach (approximately100 g) from the top,middle,and bottom of the I-ppm dose preparatiomnixed forthein-lifpeortionof the studywas collectedd,ividedintothreesubsamplesforextractioannd analysisa,nd analyzedfortestmaterialcontent. To evaluatethestabiliotfythetestmaterialinthediet,foursetsof samples (approximately100 g each)were takenfrom thehighesttestmaterialconcentratiounsed forthestudy.One setwas analyzedon theday of mixing;a second sample ofthe high-dosepreparationwas storedatroom temperatureforatleast19 days,thenanalyzed. The thirdsample of thehigh-dosepreparationwas storedatroom temperaturefor 32 days,thenanalyzed.Another sample of thehigh-dosepreparationwas storedatroom temperaturefor62 days,thenanalyzed.The remainingsample of thehigh-dose preparatiownas storedin a freezersetto maintain-10 to -30*C for8 weeks,then analyzed.In additiont,hreesamples(approximately100 g each)were takenfrom the low-doselevelpreparatiomnixed forthein-lifpeortionofthestudyand analyzed. Homogeneity samplescollectefdrom themiddle of thelow-dosepreparationwere analyzedon theday ofmixing and used as thebaselinevalue. Samples (approximately100 g) from alldose preparationwsere analyzed.The homogeneitysamplecollectefdrom themiddleof thelow-doselevelpreparatiomnixed for Weeks I through4 was used fordose confirmation.Allsampleswere storedatroom temperatureuntilanalyzed. Method of Adxninistration Dietaryadmixturewas used becausethepotentiarlouteof exposureinhumans isoral. The dose preparationwsere administeredad libitumforatleast4 weeks, unlessotherwise specified. 15 Covance 6329-224 3M T-6314.2 CHnical Observations The animalswere observed twice daily(a.nLand p.rrl)formortalityand moribundity. Signs of poor healthor abnormal behavior were recorded as they were observed. At least once each week, each animalwas removed from itscage and examined. Any unusual or abnormal findingswere recorded. Body Weights Individualbody weight data were recorded on the firstday of treatmentand twice weekly thereafter. Food Consumption Individualfood consumption data were recorded twice weekly duringtreatment. Serum Perfluorooctane SuffonicAcid Level (PFOS) Determination Afterat least4 weeks of treatment,aninws were fastedovernight,anesthetizedwith carbon dioxide,and blood (asmuch as possible)was collectedviacardiacpuncture from allsurvivinganinmls. All samples were collectedwithout anticoagulanta,llowed to clot, at room temperature and centrifuged.Serum was harvestedand storedin a.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. ClinicalPathology After at least4 weeks of treatment,blood and urine samples were collectedfrom each animal. Animals were fastedovemight, and urinewas collectedchilled(approximately 16 hours before blood sampling);water was provided ad libitum.The anfinalswere anesthetizedwith carbon dioxide;blood was collectedviacardiacpuncture. Potassium EDTA was used as anticoagulantforhematology tests.Animals were bled in random order. The followingwere evaluated. 16 Covance 6329-224 3M T-6314.2 Hematology redblood cell(erythrocytec)ount hemoglobin hematocrit mean corpuscularvolume mean corpuscularhemoglobin mean corpuscularhemoglobin concentration platelectount whitebloodcell(leukocytec)ount differentiballood cellcount segmented neutrophilcount lymphocyte_count nionocytecount eosinophilcount basophilcount Reticulocytceount smears were made and heldforpossiblefutureexamination. glucose ureanitrogen creatinine totalprotein albumin globulin cholesterol totablimbin ClinicalChexnistry alaninearninotransferase gamma glutamylt-amferase aspartateaminotransferase calcium inorganicphosphorus sodium potassium chloride appearance volume specifigcravity pH protein urobilinogen Urinalysis glucose ketones bffimbin blood nucroscopicexaniinatioonf sediment The remainingurine(up to 10 mL) foreach animalwas storedina freezersetto maintain -10 to -30'C. Sarnpleswere packed on dry iceand shippedto theSponsor. Urine sampleswillbe storedby theSponsor forpossiblefutureanalysis. Necropsy A necropsywas done on theanimalthatwas found dead. Afteratleast4 weeks of treatment,allsurvivinganimalswere fastedovernight,anesthetizewdithcarbondioxide, bledforclinicaplathologytestsand serum PFOS samples,weighed,exsanguinateda,nd necropsied.Animalswere necropsiedinrandom order. .17 Covance 6329-224 3M T-6314.2 The necropsy includeda macroscopic examinationof the externalfeaturesof thecarcass; allexternalbody orificest;he abdominal,thoracica,nd cranialcavitieso;rgans;and tissues. Organ Weights At the scheduledsacrificet,hefollowingorgans (when present)were weighed; paired organs were weighed together(seeProtocolDeviationsforexception): adrenal(2) brain epididymis(2) kidney(2) liver ovary(2) spleen testi(s2) thymus thyroid(2)withparathyroid Organ-to-body weight percentagesand organ-to-braiwneight ratioswere calculated. CeU ProliferationTissue Collectionand hnmunohistochen3ical Evaluation At the scheduledsacrificer,epresentativseamples of leftlaterallobe of theEver were collectedfrom each animaland preservedinzincformalin.Afterfixations,ampleswere embedded inparaffinand shippedto Pathology AssociatesInternationaflorproliferation cellnuclearantigen(PCNA) evaluation.Resultsof theevaluationareinAppendix 7. Pahnitoyl-CoA Oiddase Tissue Collectionand Analyses At thescheduledsacrificae,sample (approximately500 mg) of therightlaterallobeof liverwas collectedfrom each animal flash-frozeinnliquidnitrogen,and storedina freezersetto maintain-60 to -80'C untilanalyzedforpahnitoyi-CoAoxidaseactivity. LiverPFOS Analyses A portionof theliverwas collectedfrom each animalatthe scheduledsacrificaend stored ina freezersetto maintain-60 to -80'C untilpacked on dry iceand shippedtothe Sponsor forPFOS and metabolitesanalyses.Resultsof theseanalyseswillbe reported separatelyby the Sponsor. 18 Covance 6329-224 3M T-6314.2 TissuePreservation The followingtissue(swhen present)from eachanimalwere couectedand preservedin 10% neutral-bufferfeodrmalin: adrenal(2) aorta brain cecum cervix colon duodenum esophagus epididymi(s2) eye(2) femur withbone marrow (articular surfaceof thedistaelnd) Harderiangland heart ileumwith Peyer'spatch(lymphoid aggregate) jejunum kidney(2) lesions liver lungwithmainstem bronchi lymph nodes (mesentericand mandibular) mammary glands(femalesonly) ovary(2) pancreas pituitary prostate rectum salivargyland[mandibular(2)] sciatincerve seminalvesicles skeletamluscle(thigh) skin spinalcord (cervicamli,d-thoracic, and lumbar) spleen sternumwithbone marrow stomach testi[s(2)preservedinBouin'sfixative] thymus thyroid(2)withparathyroid trachea urinarybladder uterus vagina Zymbal'sgland Tissueswere shippedto PathologyAssociatesInternationaflorprocessing.The adrenals, brain,eyes,kidneys,liverm,esentericlymph node,pancreas,spleen,testesa,nd ovaries from each animalwere embedded inparaffins,ectioneda,nd stainedwithhematoxylinand eosin(seeProtocolDeviations)T.he tissueslidewsere examined irAcroscopicaUby Dr. RichardH. Bruner,DVM, DAVCP ofPathologyAssociatesIntemationaL Bone marrow smears from thefemur of each animalatthescheduledsacrificweere prepared,stainedwithWright'sstain,and retainedforpossibleexaminatiorl 19 Covance 6329-224 3M T-6314.2 StatisticAanlalyses Levene'stestwas done totestforvariancehomogeneity.Inthecaseofheterogeneitoyf varianceatp -@0.05,transformationwsere used to stabiliztehevariance.Comparison teststook varianceheterogeneitiyntocx)nsideration. One-way analysiosfvariance(ANOVA) was used (ifapplicablet)oanalyzebody weights, body weightchanges,food consumption,continuousclinicaplathologyvalues,and organ weightdata.IftheANOVA was significanDtu,nnett'st-teswtas used forcontrolversus treatedgroup comparisons. Group comparisons(Groups 2 through6 versusGroup 1) were evaluatedatthe 5.0%, two-tailepdrobabilitlyevel.Only datacollecteodn or afterthefirsdtay of treatmentwas analyzedstatistically. RECORD RETENTION Allraw data,documentation,records,protocol and specimensgeneratedas a resultof thisstudywillbe archivedinthestoragefacilitioefsCovance-Madison fora periodof I year.One yearafterthesubmissionof thefinarleport,theSponsor willdeterminethe finaldispositioonf thematerials.AU raw datastoredon magneticmedia,theprotocoland protocolamendments, studycorrespondence,and an originaclopy of thefinarleportwill be retainedby Covance-Madison. PCNA evaluationdata,paraffibnlocks,and tissueslidesWM be retainedby Pathology AssociatesInternational. Lver, urine,and serum samplessenttothe Sponsor and analysidsatawillbe retainedby the Sponsor. 20 RESULTS Covance 6329-224 3M T-6314.2 Dose Analyses Resultsofthehomogeneity,stabilitayn,d dosepreparatioannalysesareinTables1 through3. Mean valuesof thehomogeneityanalysesranged from 102-268%, 95.9-102%, 93.7-97.0%,94.4-95.8%,and 94.0-101% of thetheoreticacloncentrationfsorthediets containing1, 10,30, 100,and 500 ppm N-MEFOSE, respectivelyB.ecausetheresultsof theanalysesof dietsmixed on June 11, 1998,forthedietcontainingI ppm were high (118%, 268%, and 129%), theywere reanalyzed.The resultosf thereanalyseswere 116%, 114%, and 102%. These resultisndicattehatthemixingprocedureproduced a homogeneous distributiofnthetestmaterialinthedose preparations. Resultsof stabilitaynalysesof samplesstoredfor19,32, and 62 daysatroom temperature and 8 weeks under frozenconditionsi,ndicatedthatthemean concentrations were 90.2%, 83.4%, 70.6% and 97.2%, respectiveloyf,thetheoreticacloncentrationosf 500 ppnl Resultsof stabiliatnyalysesof samplesfrom the I-ppm theoretical concentrationi,ndicatetdhatthemean concentrationwsere 132% and 72.6% forthediet preparationmsi,xed pretestand thein-lifpeortionof thestudy,respectivelyT.herefore, thedose preparationswere stablefor62 days atroom temperaturestorageand 8 weeks underfrozenconditionsformean concentratioonf 500 and Ippm, respectively. The mean concentrationosf thedose preparationanalysesforalllevelsrangedfrom 63.3% to 114% of thetheoreticacloncentrationsT.hese dataindicattehatthelevelsof N-methylperfluorooctanesulfonamiedtohanol(N-MeFOSE, T-6314)inthedose preparationwsere acceptableonlyfordose levelsof 10,30 100,and 500 ppm. ClinicalObservations and Survival Clinicaolbservationsaresummarized inTable 4;individuadlataareinAppendix 2. IndividuaalnimalfatedataarealsoinAppendix 2. 21 Covance 6329-224 3M T-6314.2 One animal given 500 ppm N-MEFOSE died on Day 25. This animal exhibitedsevere hepatocellularhypertrophy and moderately-severelivernecrosisat microscopic exarm AR other animals survived to the scheduled sacrifice. No clinicaolbservationswere noted thatwere considered effectsof the testmaterial. Body Weights and Body Weight Changes Body weight and body weight change data are summarized in Tables 5 and 6; individual data are inAppendix 3. Although not statisticalsliygnificanta,nimalsgiven 1, 10, 30, or 100 ppm N-MEFOSE had lower mean body weights than those of controlsformost of the study. Test material relatedlower body weight and body weight gains were noted foranimals given 500 PpnL For males and females given 500 ppm, statisticallloywer body weights were apparent beginning on Days 8 and 11,respectivelya,nd continuingthrough the end of study. Mean body weight gainsforanimals given 500 ppm were lower than those of controlanimals throughout the study. Food Consumption Food consumption data are summarized inTable 7; individualdata are inAppendix 4. Statisticaltleystmaterialrelateddecreasesinfood consumption were noted in nmles given 500 ppm throughout the study and in females given 500 ppm beginning on'Day 4 and continuingthroughout the study. The overallmean food consumption formales fed the 100- or 500-ppm concentrationwas significantlryeduced compared with thatof the controls,and the overallmean food consumption forfemales fed the 500-ppm concentrationwas significantlryeduced compared with thatof the controls. There were no significanftood consumption differencesnoted in animalsgiven 1, 10 or 30 ppm N-MEFOSE. 22 Covance 6329-224 3M T-6314.2 Test MaterialConsumption TestmaterialconsumptiondataaresummarizedinTable8. Individuadlataarein Appendix 4. Animalswere feddietscontaining1,10,30, 100,or 500 ppm N-Methyl PerfluorooctanesulfonamiEdtohanol(N-MeFOSE, T-6314). The mean amounts of testmaterialconsumed by theanimalsinthesegroupswere 0.087 to 0.140 mg/kg/day;0.816 to 1.325mglkgtday;2.432to 4.030niglkglday7;.768to 13.192niglkgldaya;nd 35.168to 62.612mg/kglday formales,respectivelyV.aluesfor femaleswere 0.096 to 0.147 mg/kglday;0.848 to 1.399 mgtkg/day;2.612 to 4.124 mg/kg/day;8.485to 13.802;and 38.527to 62.217,respectivelyT.he overalltest materialconsumptionranged from 0.105 to 44.590 mg/kglday forthemales and 0.111 to 45.050 mg/kg/day forthefemales. Serum PerfluorooctaneSuffonicAcid Level (PFOS) Determination Resultsof serum PFOS leveldetemiinaiiownillbe reportedseparatelbyy theSponsor. ClinicalPathology Hematology, coagulationc,linicaclhemistryand urinalysidsataaresummarized in Tables9 through11;individuadlataareinAppendix 5. The PathologyReportcontainsa discussionof the data. Dietaryadministrationf N-MEFOSE ata dose levelof 500 ppm forapproximately 4 weeks was associatedwithseveraleffectosn clinicaplathologytestresultisncluding moderatelylower redblood cellcount,hemoglobin,and hematocritl;owerabsolute eosinophiclount;mildlyhigherureanitrogenm;oderatelyhigheralbuminand mildlyto moderatelylower globulinm;arkedlylowercholesteromli;ldlytomoderatelyhighertotal bfflmbin,aspartateaniinotransferasaen,d alanineaniinotransferasaen;d moderatelyhigher hepaticpahnitoyl-CoAoxidase.Of uncertainrelationshtiopadministrationfN-MEFOSE were lower urinepH fornialesfed500 ppm and smalldifferencefsoranimalsfed 100 ppm includinghigherureanitrogena,lbumin,and alaninean-iinotransfer(amsaelesonly)and lowercholesterolO.f thedifferencefsoranimalsfed 100 ppm, onlythedifferencfeor alanineaniinotransferawsaes statisticaslilgynificant. 23 Covance 6329-224 3M T-6314.2 Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Results of cellproliferationand immunohistochemical evaluation provided by Pathology AssociatesInternationaalre inAppendix 7. Resultsof the cellproliferatioenvaluationwillbe provided by Pathology Associates International. Liver PFOS Determination Resultsof liverPFOS leveldeterminationwillbe reported separatelyby the Sponsor. Anatomical Pathology Terminal body weights,absoluteorgan weights,organ-to-body weight percentages,and organ-to-brainweight ratiosare summarized inTables 12 through 14; incidencesof macroscopic and microscopic observationsare summarized in Tables 15 and 16;the incidenceof severityof microscopic observationsissummarized in Table 17. Individual data arein Appendix 6. The Pathology Report containsa discussionof the data. Mean absoluteand relative(tobrainand body) liverweights forallmales and females given 100 and 500 ppm N-MEFOSE were significantliyncreasedover controlvalues,and thesevariationswere regarded as treatment-related.Alladditionalmean organ weights, which variedsignificantlfyrom controlvalues,were considered to be withinthe range or normal biologicvariationo,r were manifestationsof slightweight lossassociatedwith physiologicstress. Treatment-relatedmicroscopic findingswere restrictedto the liverand consistedof mftiinw to severe centrilobulahrepatocellulahrypertrophy. Hypertrophic changes were slightlmyore severein males;and at the high-doselevel livercellswellingin severaliiwes was associatedwith acute necrosis.Necrosis was attributedto restrictebdlood supply (ischemia)from swollenlivercers ratherthan directhepatocelluladramage. Additional nucroscopic findingswere consistentwith common, spontaneous alterationisn laboratory ratsor changes associatedwith stressn,utritionadleprivation,or tissueprocessingartifact. 24 CONCLUSIONS Covance 6329-224 3M T-6314.2 Based on the resultsof thisstudy,dietaryadministrationof N-MEFOSE, T-6314 to Crl:CDO(SD) IGS BR ratsfor at least4 weeks resultedin adverse effectsat a dose levelof 500 ppnl The no-observable-adverse-effectlevelwas determined to be 100 ppn-L 25 SIGNATURES Covance 6329-224 3M T-6314.2 Niivan Bruce-Konuah Study Coordinator Covance LaboratoriesInc. eteetaerrJi,,t@domrfodiad, Study b'l'rector Covance LaboratoriesInc. 5;, Da 26 Covance 6329-224 3M T-6314.2 REFERENCES Dunnett,C. W., "New TablesforMultipleConiparisonswitha ControL"Biometrics, 20:482-491(1964). Levene,H.,"RobustTestsforEqualityof Variances,C"ontributiontso ProbgbdLiZ Statistic(se,ds.)I.OlidnetaL,Ch. 25,pp. 278-292,StanfordUniversitPyress:Stanford, Californi(a1960). Winer,B. J.,"Designand Analysisof Single-FactoErxperiments,"StatisticParlincipleisn Exl2erimentaDlesi ,Second Ed.,Ch. 3,pp. 149-260,McGraw-HiIL- New York, New York (1971a). Winer,B. J.,"Analysisof Covariance,"StatisticParlincipleisnExperimentalDesig , Second Ed.,Ch. 10,pp.752-812,McGraw-Hill: New York,New York (1971b). 27 PATHOLOGY REPORT Covance 6329-224 3M T-6314.2 SUNEVL4,RY The purpose of thisstudy was to assessthe toxicityof thetestmaterial,N-Methyl PerfluorooctanesulfonaniidEothanol (N-MeFOSE, T-6314) when administeredinthe diet to ratsforat least4 weeks. The testmaterialwas administeredat dose levelsof 1, 10,30, 100, and 500 ppm Dietaryadministrationof N-MEFOSE at a dose levelof 500 ppm forapproximately 4 weeks was associatedwith severaleffectson clinicaplathology testresultsincluding moderately lower red blood cellcount,hemoglobin, and hematocrit;lower absolute eosinophilcount;mildlyhigherurea nitrogen;moderately higheralbun3inand mildlyto moderately lower globulin;markedly lower cholesterolm;ildlyto moderately higher total bdirubui,aspartateammommferase, and almne anunotrmferase; and moderately higher hepaticpahnitoyl-CoA oxidase. Of uncertainrelationshitpo administrationof N-MEFOSE were lower urine pH formales fed500 ppm and smalldifferencesforanimals fed 100 ppm includinghigherurea nitrogen,albumin, and alanineaminotransferase(males only) and lower cholesterol Of the differencesfor animalsfed 100 ppm, only the differencefor almine aminotrawferase was statisticalsliygnificant Treatment-relatedmicroscopic findingswere restrictetdo theliverand consistedof minimal to severe centrilobulahrepatocellulahrypertrophy. Hypertrophic changes were slightlmyore severein males;and at the high-doselevel liverceH swellingin severalmales was associatedwith acute necrosis.Necrosis was attributedto restrictebdlood supply (ischeniiaf)rom swollenlivercellsratherthan directhepatocerulardamage. Additional microscopic findingswere consistentwith common, spontaneous alterationisn laboratory ratsor changes associatedwith stress,nutritionadleprivation,or tissueprocessing artifact. 28 METHODS Covance 6329-224 3M T-6314.2 SixgroupsofCrl:CDO(SD) IGS BR rats(sixanin"sex/group) were feddietscontaining thetestmaterialata dose levelof 0 (controlgroup;fedbasaldietonly),1,10,30, 100,or 500 ppnl Beforetheschedulesacrificaend necropsyduringWeek 5,blood and urinewere collected forhematology,clinicaclhemistrya,nd urinalysitsests.At necropsy,macroscopic observationswere recorded,organ weightswere obtained,and tissueswere placedin fixativaes specifiebdy theprotocol.In additions,amplesof liverwere collectefdor evaluatioonf hepatocellulparolfferati(obnyPathologyAssociatesInternational), determinatioonfhepaticpdtnitoyl-CoAoxidaseactivit(yby Covance),and analysifsor PFOS and metabolite(sby theSponsor).The animalthatdiedon testwas also necropsied,but organ weightsand liversamplesforthespecialanalyseswere not obtained.Aficroscopicexaminationswere done on thefollowingtissuesfrom each animal: adrenals,brain,eyes,kidneys,liverm,esentericlymph node,pancreas,spleen,testes, ovaries,and lesions. StatisticalsliygnificandtifferencecsitedintheResultsand Discussionsectionarebased on comparisonsbetween thecontroland treatedgroups. RESULTS AND DISCUSSION Mortality Animal No. C94523 given500 ppm N-MEFOSE survivedto thescheduledsacrifice. diedon Day 25. The remaininganimals ClinicalPathology Individuavlaluesforhematology,clinicaclhemistry,urinalysiasn,d hepaticpaln-iitoyl-CoA oxidaseareinAppendix 5. Mean valuesforeach group and theresultsof statistical comparisonsareinTables9 through 11. 29 Covance 6329-224 3M T-6314.2 Week S. There were severalstatisticalsliygnificanotr otherwisenotabledifferencesfor clinicaplathology resultsbetween controland treatedanimals;only one of the statistically significandtifferencesaffectedanimalsfed lessthan 500 ppm Dfferences considered to be effectsof the testmaterialwere lower red blood cellcount,hemoglobin, and hematocrit;lower absoluteeosinophilcount;higherurea nitrogen;higheralbumin and lower globulin;lower cholesterolh;ighertotalbihmbin, aspartateaminotransferase,and alanineaminotransferase;and higherhepaticpahnitoyl-CoA oxidase. With theexception of lower globulinand higheraspartateaniinommferase forthefemales,these differences were statisticalsliygnificantO.f uncertainrelationshitpo administrationof N-MEFOSE were lower urinepH formales fed 500 ppm and smalldifferencesforaninmls fed 100 ppm . mc ludinghigherurea nitrogen,albumin,and alanineaminotramferase (nmles only) and lower cholesterol Of the differencesforanimals fed 100 ppm, only the minor difference foralanineaniinoft-ansferawsaes statisticalsliygnificantA.t dose levelsbelow 100 ppm, clinicaplathology resultswere unaffectedby treatment. The effectson the erythrocyteparameters (moderately decreased),eosinophilcount (decreased),and globulin(mildlyto moderately decreased)were consistentwith animalsin relativelpyoor health,failingto gain weight appropriately.Mildly higherurea nitrogen and moderately higheralbumin were most consistentwith mild dehydrationand indicated the effectson other parameters (e.g.,the erythrocyteparameters) might have been greater iftheanimals were normovolen-jic.The mild to moderate effectson alanine affmotransferase,aspartateanunotransferase,and totalbffimbinwere indicativeof the hepatoceflulardegeneration. The effecton cholesterolwas marked, but the mechanism for change was not apparent. Moderately higherhepaticpahnitoyl-CoA oxidase was indicativeof peroxisomal proliferation. AnatoiWcal Pathology Individualanatomicalpathology data arein Appendix 6. Individualabsoluteorgan weights,.organ-to-bodyweight percentages,and organ-to-braiiwieight ratiosare alsoin Appendix 6. Mean valuesforabsoluteand relativeorgan weights foreach group are in Tables 12 through 14. Incidencesununariesof the macroscopic and microscopic observationsare in Tables 15 and 16;the incidenceof severityof selectedmicroscopic observationsissununarized inTable 17. 30 4 Covance 6329-224 3M T-6314.2 UnscheduledDeath.AnhnalNo. C94523 given500 ppm N-MEFOSE diedon Day 25. NficroscopicaUy,thisanimal exhibitedseverehepatocerularhypertrophy and moderately-severe livernecrosis. Terminal Sacrifice Organ Weights. Mean absoluteand relative(to brainand body) liverweights for all males and females given 100 and 500 ppm N-MEFOSE were significantliyncreasedover controlvalues,and these variationswere regarded as treatment-related.Alladditional mean organ weights,which variedsignificantlfyrom controlvalues,were consideredto be withinthe range or normal biologicvariationo,r were manifestationsof slightweight loss associatedwith physiologicstress. Macroscopic Observations. Treatment-related,gross changes were restrictetdo the liverand stomach of nialesgiven 500 ppm N-MEFOSE. Animal Nos. C94521 and C94522 displayedenlarged liversa,nd inAnimal No. C94522, hepaticenlargement was associatedwith discoloration(dfffuselbyrown with lightareas).Microscopically, liverchanges were attributedto hepatocellulahrypertrophy and necrosis.In additionto liverchanges, dark or red mucosal areaswere presentin the glandularstomach of Animal Nos. C94521 and C94526 given 500 ppm N-MEFOSE and in theglandularstomach of one controlmale (Animal No. C94493). Gastricareaswere compatiblewith early"stress" erosionsor ulcers. Microscopic Observations. Treatment-relatedmicroscopic changes were restrictedto the liverof both sexesand consistedof hypertrophy (swelling)of Ever cellsin centrilobular regionsof the hepaticlobules.Occasionally,hypertrophiclivercellswere associatedwith acute hepaticnecrosis.HepatoceUular hypertrophy was not observed in controlsor in animals given I or 10 ppm N-MEFOSE. At higherdose levels,hypertrophicchanges were slightlmyore prevalentand severein niales,and at dose levelsof 30, 100, and 500 ppm N-MEFOSE, allmales displayedminimal to slightm,oderate to moderately-severe,and moderately-severeto severelivercellhypertrophy,respectively. Liver cellhypertrophy was not observed infemales given 30 ppm N-MEFOSE; however, at dose levelsof 100 and 500 ppm N-MEFOSE, hepatocehularhypertrophy in females ranged from slightto moderate and from moderate to moderately-severe,respectively. Hepatocellularhypertrophy in both sexes was compatible with theproliferatioonf 31 Covance 6329-224 3M T-6314.2 cytoplasmicsmooth endoplasmicreticulum Although at lower doses hypertrophy was restrictedto centrilobularregions,at the high-dose level hypertrophichepatocytes often extended intopanlobularregions. f In additionto hypertrophicchanges,moderate livercellnecrosiswas observed in two males given 500 ppim Necrosiswas attributedto localischeniiassociatedwith livercell sweffingand occlusionof regionalblood supplies.Nfinimalhepaticnecrosisin one of six nmles given 10 or 100 ppm and in one of sixfemales given 30 ppm was consistentwith common hepaticalterationisnlaboratoryrats.Allother microscopic findingswere consistentwith common spontaneous alterationisn laboratoryrats,includingchanges relatedto stress,nutritionasltatusor tissueprocessing artifact. Robert L Ha]L DVI@T PhD Diplomate, ACVP (ClinicaPlathology) Ric@wd@H. Br@uner.@DVM D Diplomate, ACVP Pathology AssociatesInternational 32 Covance 6329-224 3M T-6314.2 COMMENTS ON THE DATA Variousmodels of calculatorcso,mputers,and computerprogramswere used toanalyze datainthisstudy.Because differenmtodelsround offor truncatenumbers differently, valuesinsome tables(e.g.m,eans, standarddeviationso,r individuavlalues)May differ slightlfyrom thoseinothertablesf,rom individualclaylculateddata,or from statistical analysidsata.Neithertheintegritnyor theinterpretatioofnthedatawas affectedby these differences. The number of animalslisteidntheheadingof thesununarytablesforclinicaolbservations reflecttshenumber of animalsassignedto each group atthestartof thestudy. The sununarytableforclinicaolbservationisndicattehenumber of animalsforwhich a conditionwas observedwithoutregardto thespecifincature,severityr,eversibility, number of incidences/animaoLr thelengthof timetheconditionpersisted. Only observationostherdm normalareindicateodn thesummary clinicaolbservations tables. Each animalwithobservationrsecordedas "Normal" throughoutthe studyhas the comment "Animalhas no significafnitndingsi"ndicateodn theindividuacllinical observationstables. The specifidcetailfsorconunentsintheindividuacllinicaolbservationtsablesthatare indicatewdith a "C" can be found attheend of each group foreach sex. The day of initiatioofntreatmentis"Day 1,Week I." Body weightdataareenteredatthe startof a studyweek (e.g.a,body weightrecordedon Day I isconsidereda Week I body weight,a body weightrecordedon Day 8 isconsidereda Week 2 body weight).Body weightchangedataarecalculatefdrom thefirsdtay of thestudyweek to thefirsdtay of thefollowingstudyweek (e.g.W,eek I valuesarecalculatefdrom Day I through7) and areindicateidnthetableswiththeday beingthefirsdtay of thefollowingweek (e.g.W,eek I valuesareindicateads "Day 8").Weekly food consumptionand test materialconsumptionarecalculatefdrom thefirsdtay of thestudyweek to thefirsdtay of thefollowingstudyweek (e.g.W,eek 1 valuesarecalculatefdrom Day I through7). The valuesforsununaryand individuatlestmaterialconsumptionarereportedwithmore significafnitguresthanisappropriatfeorthedatadue to limitationosf thedatacollection and reportingsoftware. 33 Covance 6329-224 3M T-6314.2 COMMENTS ON THE DATA (Continued) Diettestmateriacloncentrationpsrovidedby thedatacollectiosnystem(PTS) are calculatetdo thenearest0.1 ppm. These valuesareused forcalculatioonftestmaterial consumption.For dietpreparatiopnurposes,thevaluesarerounded totheappropriate accuracyforthe balanceused. Food consumption valuesarereportedinwhole numbers;however,PTS carriefsood consumptionvaluesto one placeto therightofthedecimalfortestmaterialconsumption calculationsT.he differenceisnvaluesgenerateddo not influencteheinterpretatioofnthe calculation. The comment "SPILLED" on individuaflood consumptiondatatablesindicatetshatfood consumptionwas not recordeddue to spillagdeuringthe interval. The comment "NOT TAY.EN" on individuaflood consumptiondatatablesindicatetshe animaldiedbeforethe end of thefood consumptioninterval. Differencesinthe populationsize(N) on thesummary tablesforclinicaalnd anatomical pathologyareexplainedon theindividuadlatatablesor the codes sheets. The calculatiofnorindividuatlestmaterialconsumptionis: Testmaterialconsumption DietConcent-ationxFoodConsumption Food Consumption Intervalx[FirBsotdy Weight ofIntmal+ (DailyBody Weight GainxDay Fwtor)] where: DailyBody Weight Gain LastBody Weight of IntervalF-irstBody Weight of Interval Day ofLastBody Weight of IntervalD-ay of FirstBody Weight of Interval and: Day Factor= (Food Consumption StartDay- Day of FirstBody Weight ofInterval+) Food Consumption Interval 2 and: Food Consumption Interval Food Consumption End Day- Food Consumption StartDay On pages 4 and 7 of the protocol,thetestmateriailsindicateads MEFOS, T-6314 instead of MEFOSE, T-6314. 34 CODES9 ABBREVIATIONS9 AND UNITS Covance 6329-224 3M T-6314.2 GeneralCodes and Abbreviations Codes forClinicaPlathology Abbreviationasnd UnitsforClinicaHlematology Abbreviationasnd UnitsforClinicaClhemistry Abbreviationasnd UnitsforClinicaUlrinalysis Codes forAnatomicalPathology Note: The followinglistosfcodes,abbreviationasn,d unitsareusedby Covance. Some, but not necessarilaylLof thisinformationmay be needed forthisreport. 35 Covance 6329-224 3M T-6314.2 GeneralCodes and Abbreviations WK N Mean; MEAN SD; S.D.;STAND DEV; STANDARD DEV; sd NA p c UNSCBED DISPATCH TBW co Week. Number of measurements ina group. Arithmeticmean. Standarddeviatiom Group mean issignificantdlfyferentfrom themean ofthecontrolgroup (Group 1) atp .s0.05. No value;not applicablen;otpresent. Present. Comment found attheend of each group foreach sex. Unscheduled. Observationstransferrefdrom thein-life module of thedatacollectiosnystemto thenecropsymodule forreferenceduring necropsy.Observationsareduplicateosf thelastin-Iffoebservations. Terminalbody weight. Number. Clinicaolbservation. 36 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 6329-224 3M T-6314.2 Codes forClinicalPathology GENERAL CODES No sample Quantitynot sufficient No repeat(samplevolume not sufficienftorrepeatanalysis) Fibrinstrands Sample clotted Slightlhyemolyzed Hemolyzed Slightllyipernic Upeniic Slightliycteric Icteric Animal not fasted 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.w,rong number, spellinegrror,incorrecdtate) Entryerror(incorreckteyboardentry) Sampling error Plateletcslumped Plateletdsecreased Plateletisncreased Plateletlsarge Plateletasppear adequate Color interferewsithtest Heinz bodiesobserved Plasmodium No aggregation Fractious Unable to determine No coagulation 37 Covance 6329-224 3M T-6314.2 Codes for Clinical Pathology (Continued) RESULTS NOT INCLUDED IN STATISTICAL ANALYSES Hemolyzed clinicalchemistry or coagulation samples Samples from aninuls at unscheduled intervals Prothrombin times (PT) greaterthan 50 seconds Activatedpartialthromboplastintimes (PTf) greaterthan 110 seconds Bleed times (BLETTME) greaterthan 30 minutes CODES FOR BLOOD CELL MORPHOLOGY The followingscalewas used to measure the degree of anisocytosi(sANISO), poikflocytosi(sPOIK), polychroniasi(aPOLY), hypochromasia (HYPO), or basophilic stipplin(gBASTIP) or thepresenceof Howell-Jollybodies(HJBODY), toxicneutrophils (TOXNEUT), or atypicalymphocytes(ATYPLYM: Scale 1 2 3 4 Degree Normal forthe species Slight Moderate Marked Not applicable Presence Not present Rare Few Moderate Many URINE APPEARANCE Color Clarity Miscellaneous A Pale E Amber I Black J Clear M Debris t B Straw F Brown P Blue/green K Hazy 0 Feces C Yellow G Red Q Blue L Cloudy D Dark yellow B Green R Orange 38 Covance 6329-224 3M T-6314.2 Codes for Clinical Pathology (Continued) URINE CHEMISTRY MULTISTIXO Urine Glucose - Negative + 100 mg/dL ++ 250 nig/dL 500 mg/dL 1,000 mg/dL +++++ ;-,2,000mg/dL Urine Ketone - Negative + 5 mg/dL ++ 15 mg/dL i 1 40 mg/dL i i 80 mg/dL + 160 mg/dL STRIP Urine Blood - Negative + Small ++ Moderate .. Large Urine Urobilinogen - 0.2 mg/dL + I mg/dL ++ 2 mg/dL i 4 mg/dL 8 mg/dL (1 mg = approximately 1 Ehrlichunit) Urine Bilirubin + ++ +++ Negative Small Moderate Large URINE SEDIMENT Cells,Crystals,Casts, and Comments A Amorphous urates B Amorphous phosphates C Uric acid D Triplephosphates E Calcium oxalate F Calcium carbonate * Granular casts * Hyaline casts I Cellularcasts J Waxy casts K Unknown crystal P Mucous threads Q Sperm R Fecalcontamination S Pinworm ova found T Pinworrn 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 0 Not present I Few 2 Moderate 3 Many 39 Covance 6329-224 3M T-6314.2 Abbreviationsand Units forClinicalHeinatology Test Abbreviation (Units) Red blood cellcount RBC (E6/UL or X106//iL) Hemoglobin HGB (G/DL) Hematocrit HCT (%) .I- Mean corpuscularvolume MCV (FL) Mean corpuscularhemoglobin MCH (PG) i Mean corpuscularhemoglobinconcentration MCHC (%) Platelectount PLT (E3/LTLor X103/AL) Mean platelevtolume MTV (FL) Reticulocytceount RETIC (%) Absolutereticulocytceount RETIC (E3/UL orXLO'IAL) Heinz body count HEINZ (%) Erythrocytesedimentatiornate ESR (MM/HR) Prothrombm tmie PT (SEC) Activatedpartiatlhromboplasti6n= PTT (SEC) Thrombin time TT (SEC) Activatedcoagulationtime ACT (SEC) Fibrinogen Fibrin/fibrinogdeengradationproducts FBR (MG/DL) FDP (UGAffi) Plateleatggregation Collagen PAGG/COL Adenosine diphosphate PAGG/ADP Alpha 2-antiplasmin Bleedingfi= ANTTPLAS BLE TIME (SEC) Methemoglobin METHGB(%) Plasma hemoglobin PLA HGB (MG/DL) Myeloid/eqftoidratio M/E RATIO Estimatedmyeloid/erythroirdatio EST M/E RATIO White blood ceU count WBC (E3/TJLorX10'/,uL) Dfferentiabllood cellcount Nucleatedred bloodcellcount Correctedwhite blood cellcount Segmented neutrophilcount Band neutrophilcount Lymphocyte count NRBC (/100WBC) COR WBC (E3/UL orX103 /gL) N-SEG (E3/.TJoLr X1031AL) and % N-BAND (E3/UL orX103 IAL) and% LYNTH (E3[UL or X103 IAL)and % Monocyte count Eosinophilcount Basophflcount Anisocytosis MONO (E3[UL orXIO'IiiLa)nd % EOSIN (E3/UL orX103 //.iLa)nd % BASO (E3/IJLorX103 /IzL)and % ANISO (-,1,2,3) Polychromasia POLY (-1,,2,3) 40 Covance 6329-224 3M T-6314.2 Abbreviationsand Unitsfor ClinicalHematology (Continued) Test Poikilocytosis Hypochronasia Howell-Jollybodies Basophilicstippling Toxic neutrophils Atypicallymphocytes 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 (WBCIUL) 41 Covance 6329-224 3M T-6314.2 Abbreviationsand UnitsforCUWcal Cheniistry Test Glucose Urea nitrogen Urea Creatinine Totalprotein Albumin Globulin Albumin/globulirnatio Totalbilirubin Directbifirubin Indirecbtifirubin Cholesterol Triglyceride Urea.nitrogen/creafinrianteio Totallipids Phospholipids High-densitylipoproteicnholesterol Low-densitylipoproteicnholesterol Uricacid Aspartateaminotransferase Alanineaniinotransferase Alkalinephosphatase Ganum glutaimytlransferase Sorbitoldehydrogenase Lactatedehydrogenase Creatinekinase Amylase Lipase PalniitoyCloA oxidase Calcium Ionizedcalcium Inorganicphosphorus 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 BIM (MG/DL) D BELI (MG/DL) IBIU (MG/DL) CHOL (MG/DL) TRIG (MG/DL) UN/CREAT (RATIO) T LIEPIDS(MG/DL) P LIPIDS (MG/DL) HDL (MG/DL) LDL (MG/DL) UA (MG/DL) AST/SGOT (IUAL) ALT/SGPT (IUAL) ALK PHOS (IUAL) GGT (FJ/L) SDH (IITJ/L) LDH (IITJ/L) CK (FJ/L) AMYLASE (IUAL) LIPASE (][TJ/L) PCOAO (IU/G) CA (MG/DL) ION CA (MG/DL) IPHOS. (MG/DL) NA (MMOUL) K (MMOI-/L) CL (MMOUL) MG (MEQAL orMG/DL) ZN (MG/L orPPM) SR (MG/L orPPM) FE (tJG/DL) 42 Covance 6329-224 3M T-6314.2 Abbreviationsand Unitsfor CfinicalChemistry (Continued) Test Excess iron Totalironbindingcapacity Unbound ironbindingcapacity Percentironsaturation Plasma cholinesterase Red blood cellcholinesterase Braincholinestcrase Caudate putamcn Hippocampus Frontalcortex Cerebellum Bicarbonate Serum hemoglobin Serum bilcacids Fecalbilcacids Average fecalweight Fecalbileacids(calculation) Osmolality Electrophoresis Albumin Alpha-1-globulin Alpha-2-globulin Beta globulin Ganum globulin Mgh-densitylipoprotein Low-densitylipoprotein Very-low-densitlyipoprotein Insulin Adrenocorticotropihcormone Cortisol Glucagon Trfiodothyronine Thyroxine Creafinekinase Soenzymes BB MB mm Abbreviation (Units) EX FE (UG/DL) TIBC (UG/DL) UIBC (UG/DL) FE %SAT (%) CHEP (MU/ML) CHER (MU/ML) CHEB (MU/ML) CAUD PUT (UMOUG) HMPOCAM (UMOIJG) F CORTEX (UMOIJG) CEREBELL (UMOIJG) BICARB (MMOLJL) SER HGB (MG/DL) SBA (UMOLJL orMG/DL) FBA (UG/ML) FCC WGT (G) FBA (MG/Day) OSMO (MOSM/KG) E ALB (G/DL) E A-I (G/DL) E A-2 (G/DL) E BETA (G/DL) E GANOAA (G/DL) E-HDL (%) E-LDL (%) E-VLDL (%) INSULIN (UU/ML) ACTH (PG/ML) CORNSOL (UG/ML) GLUCAGON (PG/ML) T3 (NG/IDL) T4 (UG/I)L) CK-BB (U/L) CK-MB (U/L) CK-MM (UAL) 43 Covance 6329-224 3M T-6314.2 Abbreviations and Units for Clinical Urinalysis Test Urine volume 8 hour urinevolume Specificgravity Urine osmolality Quantitativeunnary/cerebrospmal fluidprotein Urine proteinexcretion Urine chemistryMultistix@ strip Urine pH Urine protein Urine glucose Urine ketones Urine bflirubin Urine blood Urine urobilinogen Urine reducing substances Microscopic examination of urinesediment Red blood cellsper high-power field VAiiteblood ceus per high-power field Epitheliaclellsper high-power field Bacteriaper high-power field Casts per low-power field Crystalsper low-power field Urine appearance Conxnents 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 UROBI]LI U RE SUB RBC (PER BPF) WBC (PER BPF) EPITH (PER BPF) BACT (PER BPF) CASTS (PER LPF) CRYSTALS (PER LPFI or PER LPF2) URINE APP I or URINE APP2 COMNENTS Miscellaneous Codes and Abbreviations for ClinicalPathology Fecal occultblood Fecal parasitedetection Hemolytic potential Osmolality Not applicable Not applicable Not applicable OSMO (MOSM/KG) 44 u Code T D Codes forAnatomical Pathology Definition AND4AL DEATH CODES Terminalsacrifice Found dead Covance 6329-224 3M T-6314.2 EX NOTTAKEN MISSING UNSUITABLE AUTOLYTIC EXCLUDE MACROSCOPIC CODES Indicatetshatorgan weightisexcludedfrom calculations Organ weightnot taken;explanationgiveninnecropsynotes Organ missingor lost Organ technicalluynsuitableforweighing Organ autolyzedand couldnot be weighed Weight was taken,but was excludedfrom allcalculations MICROSCOPIC CODES Codes BmNIx- Prefacing NeoplasticFindings Primary,benign neoplasm Primary,malignantneoplasm Metastaticneoplasm Locallyinvasiveneoplasm Other neoplasm Code Definition Distributionof Findings Focal Diff-use Multifocal 45 Covance 6329-224 3M T-6314.2 Codes for Anatoniiical Pathology (Continued) Grades I 2 3 4 5 for Severity or Amount Minimal - theleastamount of change thatcan be observed with the lightmicroscope 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 theaffectedcellsor organs Severe - a great amount of change with probable lossof functionof the affectedcellor organs and frequentlyinvolveslargeareas of the organ Other TL p MN Microscopic Codes Total Finding present Finding not present Mean TISSUE ABBREVIATIONS Abbreviation IN GL STOA4ACH, GL STOMACH, NONGL SALIV GL, MANDIB IN, ANT MES/PANC AUDITORY LACRIMAL SEB GL GLAND, EX HEMATO NEOPLASIA LACRIMAL GL, INT CAVITY, ABDOM SALJV GL,PAROTID LN, TRACBEOBRON CATHETER EXIT CATHETER ENTRANC CATHETER EXIT Definition Lymph node Gland Glandular stomach Nonglandular Mandibular salivarygland Anterior mesenteric/pancreatilcymph node Auditory sebaceous gland Exorbitallacdmal gland Hematopoietic neoplasia Internallacrimalgland Abdominal cavity Parotidsalivarygland Tracheobronchiallymph node Catheterizationsite:exitsitefrom the body Catheterizationsite:entrance siteintothe vessel Catheterizationsite:tissues(vascularor extravascular)associatedwith the catheternear its tip 46 c,i CV) \0 tn u 71 00 W'i b4 0 0 0 E$3 cl Z cn cn z CLO s tu 9 0 0 I.. 0 -'O=C,4 N W) r- m c:@ tn Itt ,itIt @t C,4 0 t-- @o r- W) It It Itt 00 (D r-L cn m \.o CD C,4 (z4% 00 m C,4 ci ci r-: 4 ,o wi tei efi c-i r-: 4 c@ C\ c@ C*i (ON c7N (3N C% C\ C71@ (7% (7N cn c7, o6 6 r-: c@ @6 o6 6 o6 C4 C4 M C4 Cq C4 N C4 M C4 W) 06 C-4 (ON C:> (=5 (0% 00 m ri C-i cl r 't -,tr--00 @c cr-, ;-@ C-,l C) (3% C7, CON c@ C5 c@ c@ 66 c@ 6 C,4 C-4 cn Cf) ,t t-- r- C,4 C,4 \@o in W) C,4 ON en -: 1:@ -i "i r,@)cri Ci er? Cl@ Cli c r. Cd - C,4 cn 1) cz , C,4 en 0-) C,4 cn tu CL ca tu cts > 03 CIM)I Table I (Continued) Resultsof Homogeneity Analyses (ppm) Mixed 6111/98 Covance 6329-224 3M T-6314.2 4-Week Range-Fmding DietaryToxicityStudy with N-Methyl PerfluorooctanesulfonamidoEthanol (N-MeFOSE, T-6314) inRats Sample Location Replicate T-6314 ( m) I Top 1 1.10 2 1.22 3 1.23 Mean 1.18(118)' Toe 1 1.23 2 1.15 3 1.10 Middle Mean 1 1.16(116) 2.91 2 1.40 3 3.73 Middle b Mean 1 2.68(268) 1.15 2 1.11 3 1.16 Bottom Mean 1 1.14(114) 1.20 2 1.45 3 1.21 BottoriP Mean 1 1.29(129) 1.01 2 0.986 3 1.06 Mean 1.02(102) a Each value inparenthesisisthe percentof theoretical. b Reassay. 48 Covance 6329-224 3M T-6314.2 Table2 Resultsof StabilitAynalyses(ppm) Mixed 5/19/98 4-Week Range-Fmding DietaryToxicityStudywithN-Methyl PerfluorooctanesulfonamiEdtohanol(N-MeFOSE, T-6314)inRats StorageConditions Replicate T-6314 (ppm) 1 500 Initial 1 1.36 511 2 1.35 482 3 1.25 522 Mean 1.32(132)" 505(101) At least19days, I - room temperature, 2 - Mean - 457 444 451(90.2) At least32 days, I - room temperature 2 - Mean - 420 414 417(83.4) 8 weeks, frozen I - 2 - Mean - 494 477 486(97.2) 62 days,room temperature 1 - 2 - Mean - 304 402 353(70.6) a Each valueinparenthesesisthepercentof theoretical. 49 Covance 6329-224 3M T-6314.2 Table 2 (Continued) Resuhs of StabilitAynalyses (ppm) Mxed 6/30/98 4-Week Range-Fmding DietaryToxicityStudy with N-Methyl PerfluorooctanesulfonaniidEothanol (N-MeFOSE, T-6314) in Rats T-6314 (ppm) StorageConditions Replicate 1 Initial 1 2 Mean 0.777 0.674 0.726(72.6)' a Each value inparenthesisisthe percent of theoretical 50 C,4 CD 00 00 00 ,Ct It 00 00 00 00 4. 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H w c@ CIO 0.0c, .@oci .0 C4 "D in -1 1 r-I rn C) C"D c@ CD o WNMiww oin HOOC@IWN WOWCD wooowo c@ c@ m m a @o L@ 0 WCDOOOOWI@ wowc@ Ln 1-04 0 IDci a cz C) 1 I rIq to ko I ii I ko I %D C4 -0 CD w r%D clq -0 0 W L- A A AA A lz 1 1-1C-4 .I w w E-4 z %D CD c@ 0 CD C) %D CD c@ w 0 0 CD CD C:) %D 0 A A A AA A AA 1 -4 clq(n -0 Ln -I p w CD %D 0 c@ @D 0 %D 0 c@ A AA A I li 14 El %0 c@ 0 (D ID 0 c; kooo0wo A A AA AA 1 HM-OOE p E- 0 z H ru lm -I zn H cn r@ C4 C!)E@ I @u : 0 094 @4 0 F4 m P4 r4 04 I I 0 4 Pd 11 rn cn @4 m E-4 pq @:) u 0 CQ Ul 141In, ZH .0 f3. 7 0 :H a4 @) r4 0@4 rA I Ec-n 0 94 r4 ; ".3 14 Eo@- ;@.3 DQ 141 U) r-4 4 @l a. zm O-Z r4 w w or4 I I .1co p H I PQ P4O 0 w u 0 E@ r4 0 H E@ 4 (n H U) 0 tn H 0 0 9 u u r4 0 I I APPENDIX I ProtocolDeviations Protocol ProtocolAmendment No. I Covance 6329-224 3M T-6314.2 k k 98 Covance 6329-224 3M T-6314.2 Protocol Deviations Protocol. Test material. Disposition of Test Material. After authorization from the Sponsor, any remaining testmaterialwillbe returned to the Sponsor. Actual Procedure. Remaining testmaterialwas retainedforfutureuse. Protocol. Dosing Procedures. Retention Samples. "Samples (approximately 100 g) will be taken from each dose preparationsampled fordose analysesand stored at room temperature. Unless used foranalyses,these samples willbe discardedapproximately I month aftercompletion of thein-Efephase." Actual Procedure. Retention samples were not discardedapproximatelyI month after completion of the in-lifpehase. Protocol. Termination. Organ Weights. At the scheduledsacrificteh,yroid(2) with parathyroidwas to be weighed. Actual Procedure. One thyroidwith parathyroid was not weighed for Animal No. C94539. Protocol. Experimental Design. Postmortem Procedures. "The followingtissues (when present)willbe collectedfrom each animal and preserved in 10% phosphate-buffered forrmlin."Gross lesionswere not to be processed and exan-dned rrficroscopically. Actual Procedure. Some tissuesr,equiredby the protocol,were not availablefor histopathologicexamination. Missing tissuesare listedwith appropriateconunents in the pathology data sheetsfor individuaalnimals. Summary tablesdo not includethem as having been examined. Gross lesionswere processed and examined microscopically. These deviationsare not expected to have affectedthe resultsof thestudy. 99 Protocol COVAW-'CE. THE DEMOPMI-'NT SERVICES COMPANY Sponsor: 3M St Paul,iMinnesota PROTOCOL Study Tlde: 4-Week Range-FindingDietaryToxicityStudy withN-Methyl PerfluorcoctanesulfonamiEdtohanol(N-MeFOSE, T-6314) inRats Date: June 8,1998 PerformingLaboratory: Covance LaboratorieIsnc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Study Identification: ProposalNo. 90545C Covance 6329-224 100 Covance6329-224 Page2 Study 4-WeekRange-FindiDnigetarTyoxici.SttyudywithN-Methyl Perfiuoro.octanesulfonam]iEdtohano(lN-MeFOSE, T-6314) inRats Purpose To assessthe-toxiriotfythetestr:nateriwahle'n-.administereidnthedietto rats for atleast@4w.eeks Sponsor 3M ToxicologyServices Building220-2E-02,3M Center St.p.aul,Minnesota 55144@-1000 Study Monit" Andrew M. Sencat,PhD 3M ToxicologyServices Telephone No.: 612.575.3161 FacsimileN.o.: 612.733.,1773 Altemate:Study Monitor Marvin T. Case,DVM, PhD 3M ToxicologyServices TelephoneNo.: 612.733.5190 FacsimilNeo.- 612.733,1773 Study!Location Covance LaboratoriesInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704-2595 mailingAddress: PO.Box:7545 Madison, Wisconsin 53707@-7545 101 Cova=e 6329-224 Page3 Study]DirtdorPeteJr.ThomfordP,hD Covance LaboratoriesInc. Telephone-No.:608.241-7207 Facsimne:No.: 608.242-2736 To3icologist T,homas E. Ryan, B-S Covance Laboratories Inc. Proposed -Study Timetable in-LifeStartDate: June 12, 1998 in-LffeEnd Date-...Jul1y3, 1998 Audited Draft Report Date: October 18,1998 Regulatory Compliance. This,study wnl be.conducted;in compliancc with the Food and Drug AdministrationGood Laboratory PracticeR.egulations as setforthinTitle 21 of the US Co de of Federal Replations,. Part;58-.i,ssu6d.December 22, 1978 (effectiveJune 20, 1979), and with any applicableamendnrnts. Animal Care and* Use Statement All procedures in thisprotocolare in compliance with the Animal Welfare Act Regulations,9 CFR 1-4. In the opinion of the Sponsor and study director,the study does not unnecessarilyduplicate,anypreviouswork. Qc"ty Amwance The protocol,study conduct, and finalreportwieibe.auditedby the Covance Quality Assurance Unit (QAU). The prolfferatiocne-llnuclearantigenevaluation,data,and report willbe audited by the QAU of Pathology AssociatesInternational.Liver and serum analyses,data, and reportwillbe audited.bythe QAU of 3M Envirommental Technology and Safety Sdrvices. 102 Covance6329-224 Page4 TestMaterial IdentMCgttion N-Methyplerfluorooctanes&onEatmhiadno(lMEFOS,T-6314) LotNumber Thelo@tnumbewrislblemaintainientdherawdata. .Purity ResponsibiolfitthyeS.ponsor Stabflity Responsibfoffitthye.Sponsor StoragCeonditions Atroomtemperature Characteristics Informatioonnsyntheismiesthodcso.m,positioorno,thecrharacteritshtaidtcesfintehe testniateri@iLslon filewiththeSponsor. Reserve (Archive)Sau*es A reservesample@(approxiimtel5y,g)-of.eaclhotwillbe taken and storedatroom ten3peratureT.hese,sampleswiuibe transferretdotheSponsor afterconvietionofthe in-lifpehase. Disposidon:of.Test.MaterW Afterauthorizatiofnr:om theSponsor,anyremainingtestmaterialwillbe returnedto: Andrew NL Seacat,PhD 3M ToxicologyServices Building.220-2E-023,M Center St.Paul,Minnesota 55144-1000 Telephone.No.:612.5,75.3161 FacsnnileNo.: 612@733;1@773 103 Covarice6329-224 Page 5_ Animab Species Rat Strain Crl:Ce(SD) IGS BR Source CharlesR.iverLaboratoriesI,nc.,Raleigh,North Carolina Age at@Initiation.@-T.r-eoaftment Preferablylessthan6 weeks of age,butnot mm-e.than8. weeks of age Weight- at lnifttionio,f'Treatment 100 to 300 g Number and'Sex 36 males and 36 females identifleation Implantablemicrochipi-dentificatidoenvice Husbandry Housing individua(lmay.begroup-housedduringacclh=ion). Anknalswffibe house(iin suspended, stainlesssteelcages. Diet CertifieRdodent Diet,#5002, imal (PMI@Nutrkion Internationala)d libitum,urdess otherwiseispecifiedT.he,.dieitsroutinelaynalyzedby -themanufacturerfornutritional components,and environmntalcontaminants.Specifiednutrienatnd contaminant analysesareon fileatCovance-Madison. 104 Covance6329-224 6 Water Ad ltbituSma.nVIeosfthewaterareroutinealnyalyzed.sfpoercifmiiecdrioargamms and environmentalcontaminants.The result@sareon fileatCovance-Madison. Contaminants There areno known contaminantsinthediet or water at levels that might interfere with thisstudy. Environment Environmental-controlfsor-thennitnalroom wiU-b,e setto mamtam 18 to 26'C, a relativehumidity of 30 to 70%, and a 12-hour light/12-hourdark cycle. Acclhmtion At least1 week Randomization body weights, clinicaolbservations, Selection of animalsfor the study wM be based on and other data as appropriate.Anfinals willbe assigned to treatment groups usmg a corwuterized blockingprocedure designed to.achievebody weight.balance with respect to treatn*cntg-roups@ At the time of randomization, the weight variationof the animals of eachisex used willnot exceed.Z standard deviationsof the mean weight, and the mean: body weight for each group of each sex win.not be statisticalldyffferent at the.5.0% probabihty@level. justification Rats historicallhyave been used in safetyevaluation studiesand are reconuneuded by appropriate regulatoryagencies. 105 Covance 6329-224 Page 7 Group! Designationsand DietaryLevels Group I (control)" 2 (Low): 3 (Low-Mid) 4 (NEd) 5 (Mid-Ifigh) 6-(IfflO)' a Dose- levels= Number ofAnhmls Male Fenmle 6 6 6 6 6 6 6 6 6 6 6 6 expressed*as ppm of DietaryLevels (ppm MEFOS) 0 1 10 30 100 500 S. b The controlanimals-willreceivethe basaldietOrlly- Dosi.ng Procedures Method:of Adnih"ration Dietary,7 days/week for at least4 weeks. Treatment willcontinue through the day before:.necropsy. .Reason for Dosing Route The @potential-routeiof exposure in humans- isOral. Dose Preparation the.study-specificmixing procedure All closeprepamtions willbe mixed accordhig to developed-by Covance. Dose concentrationswil].bebased on the MEFOS content as supplied..All dose.preparationswillbe stored at room temperature. Retention Samples Samples (approximately 1oo g) will-betaken from each dose preparation sampled for dose analyses and @storedat roomiteniperature. Unless used for analyses,these n of the in-lifpehase. samples.wir:be discardedapproximately 1 month afterCoMpletiO 106 Covance6329-224 Pag;8: DoseAnalyses By Covance, using a method supplied-bythe-Sponsor and validatedby Covance Homogeneity Homogeneity wffl@bedetermined.foarlltreateddose levelpreparationsonce pretest. one sample each (approximately,100 g).from the top, middle, and bottom of the dose preparations@nixed.for homogeneity analyseswillbe collected,divided into three subsaniplesfor extractionand analysis,and analyzed for testmaterialcontent. AD samples wffl.bes-tored.at room temperature untilanalyzed. In addition,one sample each (approximately 100 g) from the top, middle,and bottom.of the 1-ppm dose preparation mixed for -thein-lifpeortion of the study willbe collected,divided into three subsamples for extractionand,analysisa,nd analyzed for testmaterialcontent. Homogeneity analysiswillbe repeated ifbatch sizechanges by.more than 30%. Stabflity Four sets:of-samples (approxinately 100 g,each) willbe taken from the low- and high-dose levelconcentrationsof dietpreparationsmixed pretest.Homogeneity samples collectedfrom-the iniddl6.ofthe pretest@dosepreparations wffl.beanalyzed on the day of mixing.and used as the baselinevalue. The remaining samples from lowdose levelpreparation mixed pretestwillbe,discarded. One sample of the high-dose preparation willbe,stored at room temperature for-at.least19 days, then analyzed. The @thirdsample of the high-dose preparation wM. be stored at room temperature after at least32 days, then analyzed. The remaining sample of the high-dose preparation willbe stored in a freezersetto maintain .-10 to.-30'C for 8 weeks, then analyzed. In addition,three samples (approximately 100 @g each).wir bc taken from the low-dose levelpreparation n@,-,ed.fotrhe!in-lif@eport.ion of the study and analyzed according to the schedule.used.for the samples.of:pre-testhigh-dose preparation. Homogeneity samples collectedfrom the middle of the@low-dose levelpreparation willbe analyzed on;the day of mixing. and used @as the baseline value. Dose: conru-mation Samples (approxixmtely 100 g) from all,dosepreparations will@beanalyzed. The homogeneity sample collectedf:rom the middle of the low-dose levelpreparation mixed for!the in-Ufep.ortion of the study;willbe%used-fordose confirmation. All samples. will.b.e stored:at room ternperatureu.ntilanalyzed. 107 Covance 6329-224 Page 9 Observation-of Animals CHnical-ObservittionsEach animal willbe observed.twicedaily(ani and p.nL)formortalityand moribundity,Tecordingfindingsasi.theayreobserved.At leastonce weekly, each animalwillb,e observed(cagewillbe opened,and theanbiw willbe removed); abnormal@findingosr an:indicatioonfnomw:wfll be recorded.Additionalfindingswill be recorded as they areobserved. Body Weights@ Each.inimalm@iUb.e-weighed at@leastonce:priorto treatment,on the firsdtay of treatment,and twice weekly--thereafter. .Food:Consumption Food consunvtion willbe recordedforeach animaltwiceweekly duringtreatment. CHWeal Pathology Frequen,cy After atleast..w4e:eks of treatment Number of-Animals AU Method, of@CoRection Animals,will@bfeasted@ovemight;blood willbe collectedfrom:.a.jugulvaerin.The anticoagulanwtffl;be:potassiEuDmTA forhematology tests.Urine willbe collected chilleodvernight(appmximately16:hours). 108 Covance 6329-224 Pikge10 TESU RematolOgy red blood ceU (erythrocytec)ount platelecto.unt whiteblood cell(leukocytec)ount hemoglobin differentiballoodcellcount hematocrit bloodcellmorphology mean corpuscularvolume reticulocystmeear (made,butnot mean corpuscularhemoglobin mean corpuscuiarhemoglobinconcentration examined) CHnicalChemistry glucose ureanitrogen creathiine totalprotein albumin globulin cholesterol totalbilirubin alanineatninotransferase gamm. glutamyltransferase aspartatea,minotransferase calcium inorganicphosphorus sodium potassium chloride Uidnalysis appearanct volume specifigcravity pH, protein urobilinogen glucose ketones bilirubin blood microscopicexaminationof sediment The remainingurine(tipto 10 mL) foreachanimalwillstoredina freezersetto niaintain -10to-300C. Samples willb-e@packedon dryicea.nd shippedto: Kris J.IL@insenP@hD 3M:EnviromiientaTlechnology andSafetyServices 935 Bush Avenue -Buildin2g-3E-09 St.PauL Minnesota 55133-3331 TelephoneNo.: 612.778.6018 FacsimilNeo.- 612.779.6176 109 Covance 6129-224 Page 11 Urine samples willbe storedb3rthe Sponsor forpossiblefat= analysis. Serum Perfluorooetane Sulfomic Acid (PFOS) Analyses Frequency and Nmmbex of Amimab AR animals afteratleast4 weeks of treatment Method of Collection Lie r)-, L. Animals willbe fastedovernight;blood (approxanatrly2 mL) willbe collectedftm a jugularveirl Samples willbe collectedwithout anticoagulant 1-91vmr Saxnple Handling Blood samples willbe allowed to clotatroom temperatum and centrifuged.Serum samples willbe harvested and storedin a freezerset to maintain -60 to -80*C. Samples willbe packed orLdry ice and shipped to: Kris J.Hansen, PhD 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 k St.Paul, AE=esota 55133-3331 Telephone No.: 612.778.6018 Facsimile No.: 612.778.6176 Serum samples willbe analyzed for PFOS and metabolites by the Sponsor. Results willbe reported separatelyby the Sponsor. Termination Uiascheduled Sacrificesand ]Deaths Necropsies willbe done. Ani3nals to be sacrificedwM be anesthetized with carbon k dioxide,weighed, and exsanguinated. Scheduled Sacrifice After at least4 weeks of treatment allsurvivinganimals willbe fastedovernight,bled forclinicalpathology testsand PFOS samples, U=, anesthetizedwith carbon dioxide, weighed, exsauguinated, and necropsied. f k 110 A Postmortem Procedures Covance6329-224 Page12 Necropsy mal:featuresof the carcass;all The necropsy willinclude*an examination Of the exte external body orificest,h,e abdominal, thoracic,and cranialcavities;organs; and tissues. Organ Weights At the scheduled sacrfficet,he following organs (when present) willbe weighed; paired organs wiH be weighed together: adrenal(2) brain epididymis(2) kidney (2) liver ovary (2) spleen testi(s2) thymus thyroid(2) with:parathyroid Organ-to-body weight percentages and organ-to-brainweight ratioswfllbe calculated- Bone Marrow Smear From the femur of eachianimalat the scheduled sacrificeonly;made but not examined Cell ProliferationTissue Collection and. Immunohistochemical Evaluation At the scheduled@sacrificer,epresentativesamples of leftlateralobe of the.liverfrom each animal wiflbe collectedand preserved in zinc formalin. After fixation,each sample of liverwillbe embedded in paraffin,and the paraffin blocks will be shipped to: Sandra R. Eldridge,PhD Pathology AssociatesInternational 15 Worman@s Mill Court, Suite I Frederick,Maryland 21701 Telephone No.: 301.66"3.1644,ext..2201 Facsimile No: 301.663.8994 Covance 6329-224 Page 13 Prolfferaticoenll:nucleanrtigen(PCNA) evaluatiownillbe done on thesamples. Resultswillbe providedforinclusioinnthef.inarleport. Palniitoyl"CoAO.xidase TissueCoRectionand.AiW@@ At thescheduledsacrificae,sample(approximatel5y00 mg) oftherightlaterallobeof the:liverwilla.lso-be collectedfrom each.animal'andfluh-frozeninliquidnitrogen. The livertissuewillb.e storedina. freezersettomaintain-60to -80*C untilanalyzed by Covance forpaknitoyl-CoAoxidaseactivity. Liver PFOS.-Analysis At scheduledsacrificae,portionoftheliverfrom each animalwillbe storedina freezersetto nuintain-60.to-800C. Sampleswiu:bepacked on dryiceand shipped to@KdsJ.Hansen,,PhD,3.M EnviroranentaTlechnologyand SafetyServices.Liver samples wiU.be analyzed;foPrFOS. and metabolitesb@y theSponsor. Resultswillbe reportedseparatelbyy theSponsor. Tissue.Preservation The fbuowing.tissues(:when present)from each anbnw willbe preservedin 10% neutr-al-bufferefdo@rmaliv. adrenal(.2). aorta brain @cecum cervix colon duodenum esophagus epididymi(s2)) eye:(.2) femur withbone marrow (articular surfaceof the,distaelnd) Harderiangland heart ileum with,Peyeiespatch(lymphoid agogiregate) jejunum kidney(2) lesions liver lungwith-mainstem bronchi lymph nodes (rwsenteriacnd mandibular) mamniary glands(femalesonly) ovary(2) pancreas pituitary prostate rectum salivargyland[mandibula(r2)] sciatincerve seminalvesicles 112 Covance6329-224 Page14 skeletalnmsc-le(.thigh) skin spinalcord (cervicalm,id@thoracic, and lumbar) spleen sternum with bonelmarrow stomach thymus thyroid(2)with parathyroid trachea. urinarybladder uterus vagina Zymbars gland testis[(2)preserved inBouin's-fixative! Histopathology The adrenals,brain,eyes,k.id.neYS,t liverm,e:sentericlymph node, pancreas,spleen. testes,and-ovariesfrom each:animal wfflbe embedded in paraffin,sectioned,and stained.withhematoxylin and eosin. Tissue sh&s willbe -shipped to: Richard H'.Bruner, D.V.M. DAVCT Pathology Associates International Ohio Operations 6217. Centre@ Park Drive West Chester, Ohio 45069' Telephone No.: 513.7-79'.9600 . Facsimile No.: 513 .7-79.9603 Tissue slideswillbe examined microscopicallyby Dr. Bruner and-the resultsfrom his evaluation willbe included:inthe-finarleport. Aftercompletion of the exan3ination, slideswillbe returned to Covance-Madison. Reports One copy of the.&aft report willbe sent to the Sponsor. The report willincludethe following information: Experimental' Design-and @Metho& ResWts dose analyses mortality clinicalobservations body weights 113 Covance6329@-224 Page 15 body weight changes. food consumption test=terial consumption clinicaplathology results organ-weights organ-to-body weight percentages organ-toi-brainweigtlt;ratios. .palmitaylCoA oxidase-activities macroscopic observations microscopic observations ceB proliferationa-ssessments (provided by.the @Sponsor's designee) Statistical'Evaluation Levene's.testwillbe done-to testfor variance.homogeneity. In the case.of heterogeneity.of variance at@p -e0..05, transfo-nmtions willbe used to stabilizethe variance. .Comparison testswilltake varianceheterogeneity intoconsideration. One-way analysisof variance(ANOVA) willbe used (ifapplicable)to analyze body weights, body weight changes, food consumption, continuous clinicapla@thology values,and organ weight.data. Ifthe ANOVA is significantD,unnett's t-testwillbe used for.controlversus treatedgroup cornpahsons. Ifthe..ANOVA shows sipfficancefor body weights at Week 1, one-way analysisof covariance (ANCOVA) willbe used to analyzebody weights,with initiablody weights as.the covariate. Ifthe ANCOVA is significantc,ovariate-adjastedmeans willbe used for control versus treated@group comparisons. Group, comparisons (Groups 2 through 6 versus Group 1) willbe evaluated at the 4 5.0%, two@-tailedprobabilitylevel'O.nly.dita collectedon or afterthe firstday of treatment willbe@analyzed-stittistically. At the.end of 1 year afteri.ssuanceof the audited draft:reporti,fno -requested revisionsor instructionsto fmalizehave been.communicated by the Sponsor,,then the audited draft report;will.beconsi&red@'finala'nd issued:as the finalreport,signed by the study director, and submitted to the Sponsor. Any modifications or changes to the:audited@draftreport requested I year afterissuance willbe performed at-additionalc.ost;tot:he Sponsor. 114 Covance6329-224 Page16_ .of the-signedfinarleport(oneu.nbound @and one bound) willbe sentto the Two copies client. Record@Retention AU raw@data,d.ocumentation,.recordpsr,o.tocol,specimens,and finalreportgeneratedas a resultof thiss-tudywiE be archived@itnhestoragefaeffitioefsCovance-Madison,for a periodof I :yearfouowing.submissionofthe@f@W reporttotheSponsor. Allraw data storedon -magneticmedia,the:protocoalndprotocolamendments, studycorrespondence, and theoriginarleportwill@be.:retabiyneCdovance. One yearaftersubmissionof thefinal report,allof theaforementionedmaterialwsi.llbe sentitotheSponsor,and a returnfeewill be charged.The Sponsor may electtohavethematerialsretainedintheCovance.archives foran:additional!peri-oodftime,and Covance@willcharge.a storagefee..Ifthe Sponsor choosestohave Covance disposeof them.aterialsa,disposalf.eewillbe charged. PCNA evaluation.datpaa,raffibnlocks,and tissueslidewsi-llbe retainedby Pathology AssociatesI.ntemationaL Liver,urhie,.ands.erum samplessentto:theSponsor and analysisdatawillbe retainedby the Sponsor. 115 PROTOCOLAPPROVAL Covance 6329-224 Page 17 Andrew M. Seacat PhD Study Monitor 3M P&r'i. ord Study ector Covance LaboratoriesInc. Date /i 2hy Date"/ 116 ProtocoAlmendment No.I C 0 V A N -CE.*@' THE MMWPUENT.SMMM COMPMR PROTOCOL AMEENDMENT NO. 1 Covance 6329-224 4-Week Range-FindinDgietaryToxicitSytudy withN-Methyl PerfluorooctanesulfonanEitihdaonol(N-MeFOSE, T-6314)inRats Sponsor: 3M, St.Paul,Minnesota StudyMonitor: Andrew;M. Seacat,PhD TestingFacility:Covance LaboratorieIsnc-.M,adison,Wisconsin Study Director: PeterJ.Thomford,PbD This.amendment modifiesthefollowinpgortionosftheprotocol: EffectivJeune 8,1998 1 Page 4. To include,thvehicluesedtodissolvtehetestm.ateriableforemixing witht-hedieta,dd.thefollowinsgection. Vehicle Identification Acetone Lot Numbers The lot-numberswillbe maintaineidntheraw data. pmdty On filwei:ththemanufacturer Stabifity On filwei;ththemanufacturer Storage Conditions At room temperature 117 Covance6329-224 ProtocAomlenchnenNto.1 Page2 Chamctelistics Informatoinosnynthemseitshodsc,ompositioorno,thecrharacteristics thatd.efinethevehicleison:filewiththemanufacturer. EffectiveJuime19,1998 2. @Page 9,;ClinicalftthologyM,ethod of CoffectionTo indicatteh-ata dffcrent method ofcollectioinsrequiredtoprovideadditionabloocldeletethetextinthis sectionand:replacweith.thfebllowing: Animals wM,*bef:astedovernightand.anesthetizweidthcarbon dioxide;blood wfll be collectevdiacardiacpuncture.The anticoagulanwti-llbe potassiumEDTA for hematologytesm Urinew.illbe-collectecdhilleodvernight(approximately 16 hours). 3. Page 11 Serum:PerfluorooctanSeulfonicAcid (PFOS) Analyses,Method of Conectioim.To indicattehata differenmte:thod ofcollectioinsrequiredto provide additionablloodforPFOS analysesd,eletethet.extinthissectionand replacewith thefollowing: Animals willbe fasted..Ovemigahntd anesthetizewdithcarbondioxide;blood (as much aspossiblew)ffl,bceollectevdiac.ardiacp-uncture.Samples willbe collected without:anticoagulant. 4. Page 11@Termination@Scheduled Sacrifice.To indicatae change innecropsy proceduresrequiredforcollectioonfadditionablloodforPFOS analysisd,eletethe textinthissectionand replacewiththefbuowing: Afterat-lea4stweeks oftreatment0, survivinagnimalswillbe fastedovernight, thenanesthetizewdithcarbondioxideb,ledforclinicaplathologytestsand PFOS samples,weighed,-exsanguinateda,nd necropsied. 118 Covance6329-224 ProtocoAlmenchnentNo. I Page3 EffectiJvuely13,1998 5. Page 3, Qu[Wfty Assurance. TO indicatethatqualityassurancefortissue processing willbe done by:Pathology Associates Internationald.elete the text in thissectionand replacewith the foflowing: The protocol,study conduct,.and fmal reportwiU be audited by the Covance Quality Assurance@Unit'(QA-U). Tissue processing willbe audited by the QAU of Pathology Associates.IntemationaL OhiG Operations. The profferationceU nuclear antigen evahiation,dlata@and,:reportwM be audited by the QAU of Pathology Associates IntemationaL Frederick,M.aryland. Liver and serum analyses,data,and reportwillbe audited by the QAU of 3M Environmental Technology and Safety Services. 6. Page 12, Postmortem Procedures, EUstopathology. To reflectthe decision to ship tissuesto Pathology Associates Internationalfor processing, deletethe text in thissectionand-replacewith the-fohowing: Tissue will.be shipped:for;processing -to: Ms. Sheree Lovelace Pathology Associates International Ohio Operations 6217 Centre Park Drive West Chester,Ohio 45069 The adrenals,brain,eyes,kidneys, liver,n.iesentericlymph node, pancrcas@ spleer,, testes,and ovariesfrom each,animal wiu,be embedded inparaffin,sectioned,and stained with.:hematoxylinand eosin. Tissue slideswillbe examined microscopicauy by R@chard H. Bruner, D.V.M. DAVCP of Pathology AssociatesInternationaland the resultsfrom his evaluation wiU be included inthe-fmalreport. After completion of the examination, slidesand tissueswillbe-returned.toCovance-Madison.. 119 Covance 6329-224 ProtocolAmenchuent No. I Pagr.4 k Effective -July 20,.1998 7. Page 2,'.Study Monitor and@ Alternate -Study Monitor. To indicate the change intheiareacode,deletethe textinthesesectionand replacewith thefollowing: Study Monitor Andrew M','SeacatP,hD 3M .TelephoneN.o.: 65-1.575.3161 FacsimileN.o-.: 65.1.733.1773 Alternate:Stady:Mowtor Marvin T. Case, D.VK PhD 3M..Toxic(ylogy-Services Telephone No.: 651@733.5180 FacsimileNo.: 651.733.1773 8 Page l4,Disposition of Test@MateriaL. To indicatethe change in the area code, deletethe textin these sectionand replace with the following: After-authorizationf:tom the Sponsor,-any remaining testmaterialwillbe returned to: 4 Andrew M. Scacat,PhD 3M Toxicology Services f Building 220-2E-O2,:3M Center St. PauL Minnesota 55144-1000 Telephone No... 651.575 Facsimile No.: 651.733.1733 9: Page -10,.Clinicvd Pathology. 'Tests, Urinalysis Paragraph. 2. To indicate the change in:the area code, delete-thetext!in thisp.aragraph and replace with the followhig: The remaining urinc(up::to10 mL) foreach animal willstoredin a freezerset to maintain -10 to -30'C. Samples willbe packed on dry ice and shipped to: 120 m Covance-6329-224 ProtocolAmendment No. 1 Page 5 Kris:JH.mser. PhD 3M ]Environ ntalTechnology and SafetyServices 9@35:Bush Avenue Building-2-3E-09 St. p@tu-1I,N'@esota:: 55133:-3331 Telephone..No.:651.778-6018 FacsimileNo.: 65.1.2.778.'6.176 10, Page. ll,,Senun:pernuorooctane:Sulfonic Acid,(PFOS) Analysesq,SainPle Handling,'paragraph II.T.o:indicatethe change inthe area code, deletethe-text inthisparagraph and replacewith--thfeollowing: Blood. sanVIes willbe,allowed to clotat-room temperature and centrifuged. serum samples wLU.be;harvestedand stored in.a freezersetto maintain -6()!to-g(),*SCa.mples willbe -packedon dry iceand shippedto: K.ds:J.H,ansen, PhD 3M Environmmual Technology:and SafetyServices 935 Bush Avenue Building 2.;.3E-O9 St. paul,.Nlimesota55133-3331 Telephone No..:651.778:6018 FacshiiileNo@: 65:1.778-'.6176 Effective.December 15, 1998 Page.13i:Postwortem Pivcedures9,Cell!PtOlifemtiON Tissue CORecflOn and Immun6histochendcal:Eva-luation, Pamgraph 3. To includeexamination of liversectionswillbe stainedw:ithihematoxylin and,eosin as partof the cell proliferatioenv,aluationd,eletethisparagraph and replacewith the following: Proliferatiocnellnuclearantigen(PCNA) @evaluationwillbe done on-the samples. In addition,liversectionswillbe stainedwith hematoxyhn and eosinand examined microscopically-.Resultswillb,e providedzf6r inclusioninthe fmg report. 121 Covmm.6M-224 k ProtocolAmendment No. 1 Pay 6 AMMMBLENT'A"ROVAL Andww. X Sewat@PbD Study M63*or: 3M pt66f..;ii9alwPI)VF Study@DhJA& Covmwe:Laboratorie3..bc. Date Date(,, 122 APPENDIX 2 IndividualAnimal Fate Data IndividualClinicaOlbservations Covance 6329-224 3M T-6314.2 123 --pri C,3 r4 r4 m %D E@ U rri > 0 0 110, X 9 1 m 0 Ln Ln m tn Ln LnLn m nm 14 El cn N r4 NN NNNNN OB I E@ fn NNNNNN ul L,,,,,,n ....L,,L,,Ln 0 u, v,Ln Ln N NNNNN N NN Nc4N NN NNN ON @Z E@ F-t I E-I W6 @ 4,A -4 4 r-4 r4 rl H -4 -4,4 -4 -4,4 @4 H 4,-4 nH E@ r-r-r,c-r-r-r-r-r,-r-r-r-r-r-r@r@r-r-r-c-r-r-r@r-r-r-r-r-r@r-r-r-r-r- 0 CD 0 C) CD a C) c@ 0 C) (:)a 0 0 C) 0 0 c@p0 0 C) CD a 4=)0 0 4= CD a C) C) c@@CD CD E@ I rd w 4.J n U) fd @D C) &. 4. : rA (D 41 @. 1 ra Hz cla N Hu (a x 0 1 Ei 0 E- E@ P4 w 4 E-0 r40H I u u u u u u u u C) u u U L) U (i u u u u u u ci u u u u u u u u m u HHHHHHH 0 r4 rr4r@ r-I P4 C-4 06 r@ N N fU Er@ D4 W N r-.@4 rU C4 r4 C@ r.. r@. C4 r@ r.@ r.4@14 D'. P4 r4 r.@ rr4 H F4 H H H H H H H H H H H H H H 1-4H H H H H H H H " H H H H H H r4 H p@ 94 9 W. 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I u cn 0> L) 0 Lm tn to Lnno 0 mom 0 mmo m 0 MoLnoo MLM Lnm Mom w Mom Mtn cn E@ z m @.4 E- 21, z 14 rn E@ 0 E@ I I I om :W@: E@ W C,4C4 NNN NNNN c4NN NN N NCNNNNNN NNN NN NN NN Ncqc4 -1,4 V-4 '-4 @ri- Hr@ vr@ r- c- r@ r- '4 @4 r@ r- -4 C, -4 H'-i -4 r- r, r, r- -4 L- '4 L- @4 r, H r-4 '4 r@ r- r@ H r- H r- r- r, H r@ r@ r@ r- r- r- It Di E@ 4.J P U) (d @) 0 41 (($ E@Z Dr4 C,4 Hu IH td 80 N:HHHHHHHHHHH 0 N N PLI44 N r@ N N N Dr4 N l@4 04 rZ4 04 N N o@ C4 9 p@ 9 P4 9 9 9 z @4 g g g g uu@uuu@@uu@uuuuu@uuu@uuuuuu 4,C 4XC4 44 44444 cncnulcnEnLOM m m m m m m w m m m N I!r4Df4N N N N FX4 N rZ4N P4 r..r@ P4 r4 N HHHHHHH C4 g g g g g g P4 g g g g4 o@ o@ V4,4 z 444 4 uuc-)r-)Uu 1.9044404 @ .4 ag cc4 4 4 m m rncarnU)cncn(acncnEnrjc2nri)t/U3) -,4 E, z P4 r4 (d E4 0 1 0 H HH H H 9WI4 wi-4wo@C4 c4wgwg W94p@g 9 C499,499 C4 P El P E@ H E@E@ E@ ElEl E4 E- H E@ E4E- P P E- P P P P H P P E@ E4E@ E@ E@E- 10 z H Cr4 r., D3 Di w Di m m r4 Di w w r4 r4 C4 r4 w r4 pq w w w Da w N w w w w rA w w N r4 rA w cz .3 0 .1 0 .1 11 0 .4.3 @4 .1.4 4 0 0 @4 4.7.4 1-1 -1%.1.1 .1 0 @l 0 .4 .4 4 4 @a 4.4 0 z LD 0m r,::,z:,o @D::, D n tD B c:,-.-@,:,m m, =, 8 B 0 @:)B n @:,B 8 M, D t:, i-4t/) :w :MQQQQPQ on QPQQOQ on PQ oppRERE fZ4W D3 CEu E-4 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu I I 00 P4 90 w0 %4 114 : 1= PW 4P wo pu I I E@ E- E- P El P E@ E@ P E@ E- H E@ E@ E@ E@ E@ P E@ E@ E- E-4P E@ H H H P E@ P P E@ E@ P r4w w w WDIN N wir4 r4 W r4 WWWr4 WWr4 r4 WW W N rg Pq PQW W W W ww w r4 w rAw w w r4 r4 pq Do rar4 w w MN w ww w w wwr4 r4 C4 w w 14 t rT4 FX4N rT4 rtlr34 rMlDL4 OL4 PL441 44 OL4D4 fl4r'4 rl4 P4 CL4 PW P4 41 rX4 r,@N @L444 PE4 al PLI@l 44 r4 1 cn 0 0 P4 'i H _I _4 _I c,4c.4c.4(,4c] cq m m m rn m cn -o -vo -o -o -o Ln Ln Ln m ui m to %o @3 C4 1 Ln %D r- m m 0 H N m v 0 ko r- m m 0 H N m v m w r- m m a @4 N m v m w r@ m W : N N N N N m m m m m m m m m m v v -o v v -v vo v -o Ln m m m m m m m m cQ Ln Lo in Ln Ln @n Ln Ln Ln Ln Ln Ln Ln Ln m o Ln Ln Ln Ln Ln Ln Ln in Ln Ln Ln Ln in u) Ln Ln Ln Ln Zi o% a% a% aN a% a% aN cN a% m aN a% c% oN aN a) a% cn a% aN ch m aN a% aN a% a% aN aN a% CA a) C% a% UUUUUUC)UUUUUCJC)UUUUuuuuuuuuuuuuuuuu C.! CCCC4I'',,,mto I CJ en > 0 0 bw4 El ca Ln Ln Ln Ln to E@ 0n n E-4 tn C'3 C'g C4 r-I m m C" fn (3-NC-h z xE-1 In E@ pq 4J tn (d n E@ 0D4 0) 41 cd E-4Z cli H u x H @l (a x 0 r3 01 -,i E@ @- E@ rA 'd o ra n > 9.4 H rn rA w W rA r4 u rl4 H H 0 D,4DW r.,N IH H HH I Z I ix C4 C4 C4 : 0H U4 1 rn rn ul cn iE H 1 44.1.1 'L4) ta pa lz P4 C4 19 1 F4 w r4 r4 E@ E- P P o:a E4 w134 noon W FA Pq rA 4 4 .1 -q t:)0 0 0 rA pa w pq U L) U C) I ta to tn rj3 I @4 0 w :::) r4 .1 E=- 04 4M DQ 0 nu : E@ P E@ P 1 Da pq ra w D4 04 w r4 P4 N P4 t4 fl. I mm OD ko w CD In %D Ln @o o D Ln Ln in cn cn @m a% UU()U z 4 C,4 4 C'4 C,4 I E- -4 C4 > 0 0 r4 .1 cn pq m m0 Q r40 1 El I rn E@ U) z E@ Z C4 m E@ rn F-4 E-4 9Z 814 HE-4to u : 5 El' ul g : C4 cn - 0 C/I p0 0 ca Or, a) >, E- Z I C,4 rq 0 x .,4 ,a 00 ,d L) 0 .,q >,4 a) $'- 1 rn EPi 0 F-I 0 (d H 1 P, 1 93@ I z H n r-4 cn z .1 0 I @ II n r.,cn F I r4 0 u 0 0 z z zz zz z H F-I H H H " r@ r-4 P4 04 p@, r@ E@ E@ E@ H E@ r4 0 w @:) It .1 1 P4 @r ix w A4 1 0 1 D@ I rD I I u@ H H H r-4 F@_1i4 rH@ I H Iz z z I a 0 0 NH u u rH34 H@-I HF-I H F-i z 0 F-I cn cn ul 1% 0 0 0 0 0 00 zz zz F-i ul cn ca cn cn cn 0 0@ g ; .1 .4 .1 14 14 @4 0 C)i CA r4 @4 0 E@ In m m m Lo in o4 Q pq 0 E- E@ E- E@ E@ El aN cr@ (7% Ch u u 'C'i ("i L4), -0C,4 C"4 C,4kD C4 > 0 L) 11 rn mz rn, m0 P4 0 p rn H tn E-4 >@ r4 'Ell M3 U) E-4 Z U2 1 C', E@ %Io H EH 01 .0 rii C', 41 Cd U) 90 E- U, I >@ zi El 1 H 9 ci i x - >H4 '01 .@4 E4 E4@0 n L) 1 U) m 0- ccnli co 0 z0 Ip H rn CD E@ u w0 m I I (N :x : 4: tD 0 zz zzz z zzz z H tu H H H P4 r.. P4 PLI r-4 pH HH E@ uu H u H H C.. H H H D4 r@ r-I z 9 0 000 00 r4 z z z z z z 0 r4 @w C', PO@ EM4 Ln Ln tn m m o r4 0 N E@ H E, E@ H H m Ch m C) C) C) m m Ln u u u 0 u 0 v 00 -0cl r4 C4 .0 .MC,I4 'n %D C) 9 > u0 @i 1 0 0. 4 12, E-4 o 0 r-4 E- cn z Er4 z Em@ %f to I El I rtAn om I@n E- cz EH>--@'I I r4 0 u 1 CI" i V2 C,4r4 1-4 -04 0 H E-4 10 z F-i U, CD Ez8 90 g 'o C14 @ol 'F'l >- 04 10, 'E-' "0' E=, m z z H z z HH ZH ZZ ZzZ H HH @4 P E-@ @4 @H14 F-iH u0 Z0 Z0 9H 99 . "m m 0 0 0 a E@ E- Z Z Z 4M @:i@-I ul rn m 0(104 99 4 Pr4 D4 HH 6H Z0 Ln H U) 0 0 ZZ U) rn 9 o z 04 11 11 - Im, Ln Ln tn Ln Ln PQ 0 Ct,n H E- E@ H .0 Ln w c@ CD n r- 00 C) C:) Ln in n ON ON 0) m L) L) U 0 -0 cl C'4 cli C', ko I cn v rn 00 C4 Dk 090 E-4 rn 0 ul z ri) E-4H H Z'-i C" E@H m 0 C', .14 4J U3 1 0 H ,n z E4 U) 0- C,4 cn I x >H4 -ol cn OH E@ ra I m 1 P4 E@ 0 1 cn 100 zm D4 PQ 0 0 C4 bd 0 134 WM Z Z Z F-I zzz z P4 P4 oo oooo ;4 Z ZZ Z Z o E-4 No E=4 rA tn m m m m 0 wo nu z p E- E, El E- E-4 cm c;l rA CN cn -0 a 1-4 @4 H H H Ln m m m m n .0 v -0 as ON CYN v CN -* CYN -0 cn -0c@ C,3 C,4-o' 1 14 C,q %D 0m 9 > 0 L) Ln 1 co I cDn' C4 m Pq 0 a co 0 ptnp rn W E4 E- m U) I E, H z z U) E@ rn E@ rA m ul @:)o E-4 rr@ rn r4 Cl)- I m0-ci I c,4 1 C4 bq HH z I X -4 1 cil -,4 'a x 0 -.4 ,a u 0 1-4 -,i 04 r) E- 0 to cD :1 z zD 114 z ri n 00 134 1 .. 90 : 04 10 C4 0 r4 04 : x ru H @l zz z z z z zzzzz z PH4 rH@ [H@ rIH4 r-H4 f-@ E- H P H E@ E- HH H N r-4 H H z 0 H Hm Hm tn H H N PL, r'4 H H H z z 0 0 HH cn c/2 u3 00 000 0 zz z z z z m m El X H F-4 r4 0 E@ E24ri4 I:cQ r40 p ci g r4 M z E4 p E. p El Ln w c- m -4 H 1-1 14 m m Ln m -It -o -o (A c:) 11 N Ln Ln v .4 -0ci N C.4 m %0 I u cn w tri 0 ZO L) H 09 1 rn I co mo 004 Di 0 E@ tr3E- mz p z1-0 @4 i E- m F34 El I 0 C', .@i 4J cn 0 m @. @.1 E@ Z H0u X 4H xc 0 E@ r4 Eol x C-4 ZH tEn@ u, mu 0- CMI ul I E@ 0 z CD I H C/) CrDU 0 R @4 0 0 13 @4 W. w P4 0 I CD w I!r4 ZHZZZZ zzzzzz H HHH N 4. PW f.. P4 N E@ P P E@ P uuuu H H HH ry4 @4 H" D4 P4 HH zz z 00 0 H H H tn t/3 rn m 0000 zz zz u H rx4 D4 HH z a w HU3 00 zz r-4 0 E@ tn m E,4W* P4 r4 El El P E@ E@ El @4 C4 M " M W C,4 C,4 C,4 C,4 CN Lo 0 m m m Covance 6329-224 .I 3M T-6314.2 APPENDIX 3 IndividuaBlody Weight Data (g) 133 C,4 M1 4 m C4 %D rn I L) rn 14 0 C,4 t- 14 ON OD LO C4 Lr)OD m 1-4m 0 14 0 co .0 @D 0 C4 m 00 %0 -0 r, -0 OD In 0 en C" rn rn C-1cn Ln r4 (7% en -* m enm cn m cn tn Ln oD -o cn cD r. mmmrnmm 4 a% -4 r@ mc4mm Ch 0% kD C4 (n rwiw ko (D%0 14 (nmm.omm rn C,3t,-ko C4 in %D.0 0 -0tn -0 m en -0 cnm en %o cn w Ln q c4 %0 cn%D -0 N OD cnfnmmmm aD Ln m cD C,40 m a% cn m (vicn E-4 mz Z El a)(nr--o r- %D (n ko " r- -0 0 en m m m cn m @-Ico r- .0 @4 aD " C4 CD C4 -0 C4 mmmmcnm r- rn 0 (D r- (D 10 r- -0 en 0 w rnmmmmfn r-4-0 C,4Ln -4 co -4 w rn C4 m m inID0 cq-o rn Ln c4 cm m m m rnm C,3 cha Lno ena r4 4%D (Z)C4 -i rnmmmmcn -0 co(3)-0C4 rcn Ln14 -1a% .0 cn m cn m C4m a Ch C4 00 MC14mm (d 0 4J E4 rL. %o Ln cO (;NLn cYi (7%--1CD -4 M kO ()N(YN CD %D CO r-4 0) co rn r- m C)% Ln co a% %D c@ -o 0 clico co r- -1 Ch Ch .0 co r- 10 r- 14 clqrn en m C4 C*4 C,4C4 m clicq r4 m fn cq C,3C4 en C4 C4 C,3m z .W H rn H x k w E4 >- p 0 9w m -C &4 0 1-1 m Q ro H H : @ 04 L" cocn Ch (n r-Ln N " C4 m " N Ln co C-.CD vi m r@ C4 (4 m C4 C4 C,4 co r- Ch r-.0 m m c) w %oLn a% C,4 m C4 C4 C,4 C,4 r- m 1-1co %0 enw m C4 C4 C4 CN > z0 H rn I I CD u 0 I >- co I F.-4 a4 corn r- r@oco n %o Ln o c%i cq C-4C,4rl C4 cli c4mmimw Ln -o r- c,4Ln -o C,4 cq C,4 C4 C,4 cli m m -0 w -4 rn in r- m -o m w cq cla CN C4 CN rq Ln .0 co C) -0 -4 M C-- cq C4 C4 C,3 cwl-aM -* c-n--4 --1 claCN C,4 cliC,4 cli C-4C-4 wo c-nNm C-q clacliC4 H C4 C4 -M -M C-)--4C-DmC4 " CN C4 C4 CN C-4 C-01-4 -0 C4 1-1N C4 m -or-Lna)cn m a a m r@ m -IC4 C4 -4 r-4-4 13. a4 CDC40 coIqco aw CN 1-4ID a co r-4 -I " -IN H P4 @Dc)m a%aNvi m H r@ m r, 0 @4 C4 -4 r-4 -I r-4 m @4LnODr- MWE-O A -11-4N H r4 -1 r'-r-I-4 M M 4 W cDo Ln M V w m m r- kn ko W r- M -0 E- W 4 4 -f I @i @A 1-411 11 @i @H m M M M E- a r- CO Ln kD Ln 00 M"WM c- * Ln a) -4 r-4H 1 .1Pd 0p@ HNM-OMW 9 r- m m 0 H " 0P4 m v m @or-m 0p@ (7%(Dr-IN MMM(Dao 0 oooc:,oo 0 @p -o -* Ln Ln Ln tn Ln Ln Lo Ln Ln Ln Ln Ln Ln F-IZ 1 -0 --o-0 -0 --dl a% c)%aN m a) cm @v -0 --1I, -V -,$ ())0)m C))m m -0 -0 v -0 10 -V ONCN a% a% ONo@ -IF-@v-0 -0 m 0) cx@ON C) (L)C) C) u C) L) U r-)u u u u u C.)L) A " c@ C,4 C,4 cn m kD - E-' 0) m (n r4 > 0 L) k.0 to tn m Ch C) .0 r- r-iLn U) C', 1 -0%0 -0 0 -0cq C,4cn rn Ul 14m v C,414 v -0 @4 Ch cn m cn tn en (" m m cq CN rq N C,4 cq cq C,4cli C,4 @4 -0 OD -4r, Cn r- C4 r-i to cn kD to Ln 0% m r- cnm Ln Icn, ID r, Ln @4 w rn " Ln Ln r- rn Ln ID m rn Ln tn m c@@ mm mmm m rn(n C,4 C,4 C,4 C,4r4 C,4 C,4cq cliC4 cli C', cn I E- >4 mz E@ H w : @. c, It,:z@ : A r*l ZH E@ 1-4E- tm 0 m 4.J cp:lrTO4 CO ca w I Z: 4I1m E- Z I L) -r4 H @l (1) x 0 3: 0 E-4 10 E- r-I E-0 1 H >z 1-4 r. z m H 1-4(n El 0 I0 x 0:D r4T4 q Ln (n rn ko ID 0-0-0 rn (D -0cq r4 fli enenm m cnm a (D m C,4 C,4 tnr, C*4Lnko N r4 C,4clir4 C,4 ON @o m m r@ r4 @4-0 " C,3a) clicq clqcq cq 1-i r-iLn clirn o% cm o Ln co r-4 r-4OD M IM C> C4 en CN m cn m m rn r-4m Ln a) -o -0 %D en @4 --zLrO C,4C,4C,4N C4cli Ln H Ln cn@aN (n cD -ien en ioN C-4cliC,4r4 C,4@q 0 tn m CD c,4 r-i o a% r-i rn a% ko r-ir- OD ON %D @4 %D r-I C) 0% r4 Ln M N 10 m Ch m 14 14 r4 co m ()NCN -4 0 r- clqm cq C4 cn CN CN C,4 C,4C,4C,4C4 C,4C,4 H 4 C4 r4 C4 r-I r- 0% C4 C,4 ON L- Ln a% ID w rN CN clqC,4C4 N r,m OD" C)w 1-1r4 C4 -4 m C,4 C4 N C,4C,4C,4N m @4LnN o cn a% -icD c:)r14 r-iC,4 C,4 CN 1-1 rn r-I -0tD cliN m (n LnLn %D r-o c,4%orn m -o C*4r4 C,4C,4C,4cla 4 c,3cD r- Lnm C) cq @4 m 1411 r4 C4 CN 14 C,4CN co Ln r. i a%Ln r4 r@@m cn co w r-i11IH -4 r414 (3Nko -4 M C,4C,4 r-im m en Ln cliC) .0 li 14 r4 C-4C,3C,4eg C,4N aD en m m Ln cD m C) CD co cr%0 r-iC,4 N r-4r-ir4 r-ic)r- -ir,Ln, r, w c)oaD r-Ln -1 -4r-I 4 H i Ln c,4 coC) @qC,4 C) C) C-4r- Ln ON r-i 0) r, 'Ico r@ aN 4-4 C4 -I ir-I 1a344 C) (D Ln C) -0 N " m r, r@m 00 w r-r-11 1r-I4 1 -0 -M -W L-nr-I ri i i r-I-4 H L-nrw r-@W H H A H -4 H r, r-4r@.0 Ln -o %o @D Ln m H r-I@4 4 Ar-i Mo 0 ,.4H Ln r-m 0)0 0 rir4-4 -1,-"4 tn Ln LnLn Ln Ln ON C)Nm C)%CN 0 r-iN m -0m w g CN N C4 N " N LnLn Ln Ln LnLn as (n 0)m aN(3) r-m m a H N NN"Mmm Ln LnLn u) LnLn ch cn m cr,aN o@ C4 C4 I N ewn E@ cn > 0 (n m 1 C', 1 14 r@ tn en 1-414 14 0 C,4H cn 0 N CN C4 C,3cq C4 r- Ln Ln r4 co tD N co -0 Ln r, ()N C,4ri C,4C,414 @4 w OD Ln %D W Ln (3%C4 -0 M 4 (7% 14 clqN 14 C*414 CD 4 Ln fn 00 -1 4 Ch 1-1C*4C4 14 r@ 1-4 CN3 LnM Ln cliCN -0 C,4" @.4 C4 C4 C4 C4 C,3C*4 m OD r@ %D en kD -* a% W Ln CO (:) CN 14 (14r4 14 C,4 (7%N rn co 0 r-i (D -0 %D 0 m H C,4C,3C,4C,4C4 C4 Ch C,4 @4 OD Cq (n 0 14 C4 C4 N Ln rn z c,@ z1 -0 C'4 r-I E@ I H E-4 (d m 0 ii E4 P4 (d to m 0 T 4J E4 Z cn x0 0 E4 El N E4 0 c4 -o -o @D r- Ln r-ir-iC,4 r-4M (D C4 CN CN r4 N C,4 mmr--omcd cn ON Ln v r, 0% C,4 r4 Ck3 (14 ri r-I 14 (D c@ co r4 r-i C) (n Ln Ch C4 c@, C4 cliC4 rq C4 C4 cD Ln r- Ln OD r-ICN Ch 14 C4 C4 14 %o o% a% Ln Ln vD 0 0 H a C,4(3% C,4C4 C,4C,4C4 @-I Ln W r-4 %D " a% C*4co (n r@ co C,4 r-4 C,414 14 rq r- CD -10 %D ko m @4 .0 M r-ICh li C4 N ri C,4r-i ko OD CD@ CN r, C> r4 ON r-4 C,4C,4 r-A -a r, Ln co a% ko 0% ON 0 00 0 CID 1-H4 C,4-4C,4-4 C,4 r-I%D r@ %o 14 r-4OD N C4 %0 CO CNIr-4eq r4r-Ir-i -0 0 m CD m -0 OD0 clir-0 co r-ri4 C,4r-Ci4 1-4 .0 r-m 0 %0 CYN 1-1 co 14 -4 N r-i mn w m m crscoa r14 14 1-11-1C,41-1 m.mo" r, r-iM ko rr4 -4 clicq H r-i H r- CYN11 r, m m -4 -1Cq -I 1 4 OD Cn r, rtn a) cD r@ 14 li Cla r-i H to 04 @ rQ C.D loj 0 @4 0 m @3 04 ci %o a% i -o Ln r- r- r- r- co %D rA H IH 4 @4 m 0 14r4 r-iID 00 r- c@ CD Ln tn @4 r-ACN CN @4 ri (7N0 1-40 cn CD w co C:)%D OD r- I H CN i r-I 4 cn -0 -0 vOD 0) kO r-i14 11 r-I Ln " 00 r-ir- r@o ID r- %a r- U) -4 @q -4 @4 14 r-i cD r-3u) ko Ln OD C)D%0 aN r- -* -0 li 0 cD cm c,4criLn @O r, 00 Ln W Ln 14 @i H 14 c,;cn -o r-0 r- co Ln a Ch Z 0 ko wm -4 @i Iq 'A @ r-I 0 M M N r- r-M w .0 M LoN m @4 1-4 I-q r-I -iC@,r-4V 0 M il@ .0Lnr-cnLnm 134 1-4 -4 11 r-i 1-4 -4 CD 1-1. MMMN rQLnw w 14 @4 -4 C%l m ul >4 rA F4 m N w i a m W LnM m -4 N w -0 m CD mc@MHHW VWHO m M M " -0 N v m Ln -o i cq MMNN"" 4 -0 '_0ic'In li 11 @q r-I ri 14 @4 1-4 r4 @4 r-i -4 1 H 4 r-4 1-4 P@4 f'4 CIL. m -o Ln w r- co 1 0 mmmmmm tn Ln Ln Lf)Ln Ln -0 -0 -0 -0 1-@p.0 a%cn c)NaNa% oN z r)r.)u aN 0 --1C,4fn m .0,0 .0,0 -Ln0 0 Ln tn Ln LO Ln 'o -COC.NMcv"Ch.a0% -a@ap,@ C-) m%oc,- mmo 94 v " v -o o tn 0 Ln Ln kn Ln Ln Ln " @o -ZP-* -0 -0 cs m (n ONCh ON u u ci C-)u C.) -4 N m v in M Ln 0 Ln vi ul wi -0-0-0-0 a% ON ()NON L) U L)U -cl0ac@ C,4 %a CL) x cn 0 u -0 1 H 0 C,4 C4 C:)1-40 CD 11 r-I w %D -0co r@ r@ H r-I1-4 r-4 Ln Ch 0 ko r4 00co C*l 14 C*4 c- r- Ln Ch CO CO C,4 r4 H @i H r-4r-i r-I tn I C,4 I z H El H E- tn C) 0 4J P N En I I 4J Cl) Pz x .,1 4) x0 ol 1 El 0 4 P0 1 C'4 : co HI I Ln I > H z zD H H U) r 00 @4 0 Di I >@ co I -0 -H C4 cli (13'A Ch 14 C,4(IN r, .0 CF%00 C, r-ir-i @-4 r- CD 1-4 C,4 C4 cn a% -0 CF%CD 14 r-ir-ir-ir-i m Ln CO(Y' a%%D 0 m Ln%o -0 r- r-r- r-I -1 -4 -4 ID %D r- co v C-4 OD ko 1-1 ()N ko to cn ko r, %D %D tn w C, -4 4 Ln r, tD co m v Ln Lnm ul Lnko 1-4r-i -4 1-1 r-ir-i cq Ln r@ Cn r-40 Ln r-I Ln W -o Locn Ln tn L_ni r-ir-ir-i C, tn Ln co 14 1-1 r- C-4kD 04 M -0 N M -0 -0 1 -4 H r-f @q H H r-i @4 C) CD CD Ln v cy, C,4 m H @4 P4 --CiD .* 'D Ln 'r-oi-H .0Hr-ICIq4 Vr-ir-i tu P4 D tD 0 E41 o@ pq I a r4 Lnw 0 Ln Ln IX m Ln 0 -0-0 CA a% u r@ OD ()N(D '-1cN 0Ln Mu) LLnnWtn wLn %Lno -@p.0 -tr -@v .4 .@r a%(3N0)(7%CAcn u (-r)ic u APPENDICK 4 Covance 6329-224 3M T-6314.2 IndividuaFlood Consumption Data (g) IndividuaTlestMaterialConsumption Data (mg/kgtday) 138 -0 c@ C,4 cq -0 C,4 'n %D - E@' (Dx u cn > 0 n C4 to I I E@ z H r4 r4 E1--44EI4 I 4-Jd rn I I cd 0 E-@ 44 : -mi rn 0 4J ,C o 0 x0 0 El E, w 4 E-40 F-I >.4 Ln 1 n -4 : 1 ro > -,4 CD z H Q z t:) H U) El.Di 0 0 m1 Lnr@ m co ko -4 14 C,4 m I-q 0 14 -4 H 14 14 H ca ko LnLn %om ko C,3 0 m N @4 00 @4 i -1 4 -4 -4 C@ L-140 4 Cq r4 CD CD Cq H -4 -4 @4 14 H -4 co rnco o a% 4,r-3 -i -o r@ r- r4 a% %o CIDOD OD 0 co r, w c*4N m Ln r(ID00 0 OD OD M -0 C,4ON 14 co %D Ch ON co OD r, m Ln -0 0 OD r- r- co OD L" C4N C) -4 CD C4 H aN -1 H i 1 -4 ,o . . . H m cla11 CD a% H @i -4 -4 m o q c) a a% cD li r- r-C)%m a% a) C% 1-@ CD CD 14 .0 cq 0 MOD 00 ON 00 r, Ln C" I* A 0 C)D ()Dr- cn OD m r- c-4%ocqLnN r4 OD OD co t- r- m a) %D -1.0 w to r- co Ln-ooNririLn c@ a c@ rli-4 ON li @H 1-4-4 clcqlTi)viko-4 14 a N a C) C) 14 @4 1-4 1-1@i 1-1 cqenr--o kDLn @i 1-4c@ Ch CYNCD -4 @l @A @4 m .00 .0 m aNc) i H 14 D r@ m Ln -4 aN r- r- co OD 00 ko Lnrn li m Cl)r- CC)C- ()Nr, OD r- 14 r-a r- ,I@-H co co co r- r- m cn Ln oN -o r- r- r- m ON r- H 00 0) In aN(MI-aq CAON r, Ln D "1 -0 C)D 0 (3%1-@cn 0 C% Ln r- r, cn C,4 c) cDciNci)crNc co C,4 CO kD cnoD a% (D 04 a. a. a. I I CD P. P4 CD I Lnm . N w r- m r- r- r- r-m r-LnMM r- r- co ID El r- M Lnr-m m r, r- w kD @o r- 11 Lnm r- r- kD r- r- -1 94 1 w: 94 -4 C,4 cn -I:rtn %0 a mmmmmm pq v -0 -0 v .0 " v @o -0.0 aN a% a% (3)a% m u u ()r)ci r- OD m 0 -4 C,4 M m m m CD CD CD a -o-ov Lntntn -0 -0 -0 -@v-0 --o cn m m cn (n (YN r-C).())C) m -oLnw r-co a@ CD a n c@,C) c@ 0 LnLninLnLnLn --T.0 10 -0 -Im-0 m 0) a% a% aN 0% C)u u u u u m cD @H cq C) A -4 -4 LnLnLnLn 10 .1@-p* .0 aN aN aN m u C.C)-)r) C,4 C,4 -0 C,4 kD 2kD1 E4 C-4 mz z @-I C.) m %D E H E- >. c-i 0 Ec4n r.. I WH -0,4 >4 E@ x 0 ;04 01 E@ E@ 14 0 E@ 0 0 r4 n @4 H F-i >- Ln 1 co0 C@ 4 @4 m o Ln o -ir, 0 C) 4 CD H 0 4 4 -4 1 -4 C4 Lti Ch %D %D r- r- w w r- -0 C,3ai '3 0) OD OD ON C)D.4 r- w H rn 14 14 OD co OD C*4m -IV@4 @i r- r- OD r- 00 OD C4 CN Eq m CD Ln W .0 Ln W z r- -i-o Ln " m Ln %D ko r- W Ln c, CD (D Ch co cn ON CD @4 r-I m.m . . . ko r, w Ln kD r- to CD 14 -0 Ch -@p w OD co m r- r@ w m m r, -0 Ln m @4mvmmN L, r- w r, r, r-r- Ln%a Lr)LnLn w r- 0 0 m m Ln Ln%o @D u) Ln fcAD P4 'WI8 ,4 r.4 a CD c@ OD r@ fn r- (n (D ON OD 0 ON C) 4 14 14 C,4r. Ln cD r, C% r- ko r- OD r- r- m rn co -.IN, r, (IDco r- co r- r@ 'o "0 .0 C.4'D c- H C) co cq -o Ln C) .0 r-i@4 m -0 Ln to Ln uD W kO vi w @o %D Ln LO co cr@C) P4 ,-4 i N m %o H 0 0) m CD m cD m n m r, r, w r@ r- r@ r- C)4 m m m m Ln N ka r- OD r- co r- C:) C=) CD Ln I m m -4 m 14 @4 H w W Ln (7%C*4 .3 mr-MMMN w w r, r@ - r@ w w W.0 , "D U, w w Lr,w m DL4 LnM -0-a,0 a% to r, m m 0 Cd H r-i r-4 H N 0 Ul)U')Ln tn LnIn -0 -M -0 -0 -0 -0 m (A cr,Cl)(3%m C-)C.)r) u ci u H N m .0 kn w IM NNNNNN 0 kn LnLn tn LnLn -0 -0 -0 10 1* v chM o%manm Li u C) C.)u u r, m m C) -1 N NNNMMM in Ln m m wi m .0 .0 -41-0 -.0-0 ON (3%m ch cm a) u u C) "-4 F -0c@ cI I r4 w w El en cn > C', E@ mz c-@ r, z 14 m EH-1EI@ rA rjloa .0,i E@ IL4 to cloi I I E, I w @ Ln 0 0 rL4 r4 n H F-i 1 1 ,4 z H P4 p @l z Z) H U) Dk 0 0 P4 x0 P4 04 I I 1 1 >@ co I o -oLn N cocz) a) co Ch OD -M r- r-to ID ChC,3 OD %D a% ON %D r- %0 ID OD c- cor- cn Ch r, :Ir-w ID F-i a% CD %o co ()%rLn kD D %D koin C,4cn 14 10 clar%o Ln r, @o Lno ci% m Ln r, (,4 @i -o %o r- Ln w Ln OD .0 %D ON Ln kn Ln -o r- OD r- co OD r@ m c, r- kD aD r- %D Ln c@ cnC) Iq 0 r, w r@ C% r- co %0 Ln c:)a% rOD r@ VD kD %D en 1-4LO %D r, Ln %o %o Ln %o Ln a% r- r@ cn co 0 Lnvwwvv OC)CD -0 m 1-@N -o Lo %D Lr)Ln Ln r4 (11r-m Ln Ln Ln -o ko o r, ci Lnin C, OD 00 r- CD r- W C) OD kD ()N0 r, C,r- 0 tow 4 w r, r- C,4cq C,4 ko r-co r- m r, Ln cn r@ o ko r- r-r- to -4m r- 11 U) %D M Ln kD Ln ON (Vim m C=)C,4 Ln Ln r- Ln -o c co w Ln cs CZ) Lo m w Ln @D Ln r4 m m -0 Ln Ln Ln Ln r- m r, m riiw 1). -4 m w CD m ci. cn Ln r- a% C) CD -4 : cn C', N r, cD -icN Ln oD kor. w w o r- v w cq -@pm w m -o Ln @q Ln r- -o cD N o tD%D Ln tD Ln Ln -i m Ln %D%o Ln C-4 r4 I F-I 04 0n m -V Ln ID r- m @D m -@v mmmmmm LnLnLnLn Ln Ln -0 --@-p;r --ZP-@l@r c3Nm a% (7)CNm MV-O-OVV Ln Ln Ln Ln Lo Ln .0 10 @v -0 .0 10 m Ch m C)Nm cri u u u u C)L) UUUUUL) m w r- m CN a lox -IC-4(n @v v @o v --Zvv M 0 M LnLnLn Ln Ln Ln Ln tn Ln .0 10 -0 10 -;p" m Ch m m m CN Ln Ln Ln Ln -0 10-0 " MMMON u r-)u U L) C) C.)r-)riu cli r4 'M1 @4 en C,4 %a m El r)m 0 CM4 ca r, r- co o ko -o Ln %o Ln CN4 I m C.4 Ln tn -0 -0 en -0 -0 -0 Z @4 1 rn EH@ EI-4 1 1 0 E@ ru to I .0 m %D ko C4 kD co %0 1-4 -o Ln -o Ln Ln Ln .x, , x 0 100 00 0H 1 Ln i v@ i -ir4N w m N LnLn cn " cn -0 -0Ln 00 H o ZD F4 N > F-icn i m C,4 pq -@rcn -0 m CD OOVWW4 F-4 lnw m0 L-nt-n -0 -0 -0 C)m C, r, m m -.0CNw m v 0 m %0-0 --zr 00 C) Ln n 4 'nC, mw 14 mommw-o w .0 m .0 Ln C4 0 Ln o 0 Ln Ln g tLn n mLnDa 'w INko m Ln Ln Lo Ln u) Ln Ln (A Ch a% cn a% oN m cn u u u C-)L)u u C4 -P C,1 14 M, C4 %0 L)cn > 0 ii ul E- @m,z ,it E@ H C4 C,4 ,sg Zq cn E@ H E@ I ul 0 E- z E@ H CD I >4 -I : X to x a I -@4 0 ol 0 E- E@ 0 x CD z 4) ZF-4El) I rx4 (D E@ c) : 0 > 0 OD &4 : FX4 00 OD OD CO OD 00 c; r;,c; -o n 10 @n o, c@ 1-4-4 @4 14 -4 1-4 .. . * Cn OD 0) CO a% M c@ c; c; c; c; c@ Ln r-w tn v r@ c@ c@ r@ C4 r4, CD @4 C4 CIN .4@ .9 aD OD r-4 14 @4 @A H -4 c@ CD* c; co OD C)N OD OD 00 c; c; c@ c; c; 0* Ln Ln w Ln Ln c@ c@ C,4*c@ c@ CD r- m OD'(@ r@ c@ c; c@ 0' 1; c; c; r@ c@ c@ c@ c@ C-4 C@ L@ OD co 00 ON r-I 4 r-I@-Ir-4 4 c@ c; 0* c; -4 (n CD (3) 1-4r4 c; a% 1-4CD 0 0 r@ r@ C@ C@ CIN(A r-i @q r-i14 c@ c@ c; c@ c; 14 ri C4 r-ir-ir-i 14 14 r-Ir- Ln Cq C,4H cn*1; 1; 1; 1; Ln r-iW r, (@ _7-4 o CD C; o CD o C@ C@ m 14 P4 C',C@ r-i1-1 -1 C', c@ c@ 0* c; CD* M cnmm Ln Ln m Ln Ln -0 . . . . .. fn en en m m cn cla 0) (A r.. .. C-) C, (A C,4 Ch a% CD W Ln r- @o Ln r4 C4 r@ c@ rq clic@ 0 0 Z r, m a M M C:>a CD v Ln w r- m (DC) 0 0 0 0 0 iX m 0 r-IN m Z Ln to r- co a C) r-4-1 4 r4 -1 CD r-4 r-4 @A -4 .* -0 vi Ul Ln -lpv " -0 " -0 aN cn cy%(7,aN cn u u u u C) C) vi V) m vi V) Ln -* qw .0 -0 v q@r m a% ON CYNm CN Uri u () C) u Ln Ln Ln Ln Ln Ln .0 * -Iz-r0 -@,-0 m 0% a) 0% ON CN () L)u r-)u C) Ln Ln Ln Ln -*-q* -0 -0 ON ON m m Li L)U L) -0 c@ N 1 C,4 -0 mi m %o 10 4)z C,4 .@A Z @4 m cn t)l ID >@ cc,4 4J p 0 (a E-4N Ul ra 0 41 x lu $4 4J F-I X0 0 p >, p 94 o I: I @ ,li:I Ln i I @H i I I 4J H C4 ril 4) H E-4 en OD w Ln @4 -4 14 cm m m (D0 Ch o@ E4@ e@n c@ c; c; c@ c; c; E@ 0 z W! C@ OD OD C@ 19 OD co Ln C4 1; 1; en en cn en z C@ C@ @ r@ C@ ko -0 m C,4-0 ON en m m m cn en H 14 H 1-4H H c@ c; c; c; c@ ooc@1000 MOOMOOD c@ c; 1; c@ @4 c; o@ r@ oo(:)Ooo @4 C,4 %D m OD C-4 00 L@,@ en 'IV-0 1 r-4 ri H H 4 ON 0% 0 a% 0 co @@ @ 1; @ @ c@ C4 tn co 11 -i@4 (D r- Cn 0 14 rzc@ c@ 14u; -0cn -0 10 -0-0 @4 14 @-I r-I r-i@-I 0* c@c@ c@ c@c; 0 0 (7)C,4li (71 1; 0* > ,a x 0 r@ 1! C@ C@ L@ @4 CD cnr- Ln c*4c,4 0 C:) 0 CD (D c@ li IH -4 1-4 H (D -o Ln u) Ln I C@ C@ C@ N cq H mH z x Lnm m m Lnm C4 -4 'A a4 P4 04 P. C@ C@ C@ H -i H H P. Ln I cl@ %D I %D cn C,4a% -0 -0 ,4 -i H c,3 c@i .1 4 Ln 10 * 4 4 @ ; (@ (@ c@ r4 CN @o ko Ln Ln .4 mm Ln Ln m r3L, CT4 .1 P4 1 m CZ, -4 N M -0 M W cli-0 LO kD r, OD p@ MOHNMV 1 1-4 C,4 C,4N cliC,3cq C,4 C.D clim m m m en .0 rn --;-p0 -0 .0 " k I Ln Ln Ln Lo Ln LriLr)w) W) LO Lr)M LO Ln Ln Ln Ln Ln Ln Ln Ch a% CY%(na% (IN()NON u r-)ciU L) (3)a%()aNN alm u u u C-)u u aNasaNm a%a% u ()u C-u) u .0C.! 0, i r4 C,4 -0 I r-4 C.4 rn, cn %D I cn 0> 13, C4 cn E@ E-. H C', z r en E@ C-0 t-0 0 E@ 04 cn 0 z 41 p H xo R1 Ln uo I- 0) @. El r-4 P4 C4 E@ 0 $4 r4 P a) x CD z I Z0 1 J.i H ff@ to . 2 -M : E-@ F-4U2 0 >0 ro X 0 1 r4 @) : >@ OD @4 F-4 -o %D Q r- 00 W c@ C4 lol @ C14, cn LnCO 1-4 C:p()Dko c@ c@ 14 c@ c@ .0 Ln -0 a% C-4 9 ,@00*,.@ cr%-0 cr,C,4(A c@ @ 9 t-:@; 9 cn -0 m fn-0 m aN Ln.0 .0 -0ON c; c@ c@ c@ c@ o@ Ch C, ID m 0 C% en -0 -0 m -0 OD cr, Ch CA %D cq r-3 Cq Ln VD a a %D -0 C4 c@c@ c@ a;a; C@ C@ C@ L@ LnaD cn o o %D en m cn -0 -0 en C4 c@ c@ c@ c@ c@ c@ c@ r, c@ en c@ c@ -,*cn v; -o c@ -o CLDn ON 1-4li cn r4 1-4 C,4cn m en w C-.ko C,4r@ a c::; r; c@ c@ 1-4 -4 -4 11 cn Ln14W Ln c@ r; C@ L@ -0 .0 -4 -V 4 cn cD cn,,@pLn Ln %a @;14 14 CINOD .0 -4 C)N10 c; c@ r-i 1-4 14 @-411 o v cn c@ r@ c@ c@ c@ c@ a,,o H oo -o -o -o in Cn rq M M C4 Cn P4 P. cn I 14 N H m @P* cn, r@m c:)Lnc,4Ln 0 CD to C) r- C4 @4 i @4 Cq -4 CL. cn @o m -.0co -4 4 4 -4 -1-4 -1 p@ -o -o in Ln c,) -o CD CD UI) @i -0 m 0 1-1 r- H M-*4 r-Ir-Ali MH* w -0 N @i N.0 -4 .,ql m, c@ m r- w W Ln W 0 0 -oLn a inLnLnLnLnLn a% 0) aN M a) 0) u r) r-)r) L) U w P04 m LnLnLnLnLn Ln Ln Ln Ln Ln in C3NM (n 0) (7%a% u u C.)u r) u rL-nmLn Ch Ln (D 11 tD %D N %D Ln Ln Ln Lo Ln Ln aN c;)oN a% aN a% r-)u u () u u APPENDIX 5 IndividualClinicalHematology Data IndividualClinicalChemistry Data IndividualClinicalUrinalysisData Covance 6329-224 3M T-6314.2 146 c@ C,4 C4 V m1 C-4 cn C, ko d)z 0 4J 0 H0 4.) Ul li x Cd .14 r.) 10 -,1 omomww wmw m (Z)M H r- m w r- kD mw-@rNmr- vow =L NWVWWO mm %o r- -00 r- r- Ln Ln cn cn 0 1-@a% r- -0 @4 CN m 0) a% Ch OD m q M r-4(n cn m 0 CD 14 li c:@C4 CD co ON c@@ -4 C, 14 14 r-i @4 14 @4 I @4 I H 4 E- Z H I X dp C-) zI en El kD HI 3: &4 cn H0 U) D4 pq >4 Z E-4 1 H F-I x 00 p U gi. ru I I E4 r4 E@ 0 p cip rn m m -0 m cn cn (V)m m en m c@ ko cn cn U@ C@ ul ON CD a 1414 0 0 1 C-4 cq Cq C,4 C4 P4 @ r@ 10 a C4 " C4 CD.MM.M . . N .CO4D W c@ 1-4a rn CN @4 14 Ln %D w w %0 ko ID co r- C,4r- (n w ol C4 C@ a@ -!r@ @ li en en -0 (n cli(14 m m m C" fn cn CD @ 19 cn m a, C9 L@ O@ O@ W 0; v m en m m m m en m fn m cn en C@ C@ W rn CD r-iCD co Ch 0% CD 0 C,4C4C,4H i I C4 D4 (D Ln U3 CD 11 CD c@ (YAC) 0 0 C4 (,q C%4 C4 @-iCq C,4 @4 d) OMOONCD CD cw,t3o . .. . . 0 %D 0 0 41 %0 %D w Ul %D ko 0 c@ w til m -o w tnN (n c@ v; C,4 4 4 %o 4 c@ rn OD r-i(14(D Ch c@ @; 0; OD H 41 %D kD %D Ln tn %D C) 4) m co o cn en 0 -1 ci ol r@ .4 u@ 4 L@ %D 'ro-D a %0 CD C4 w zg pk cn i 0 a cn Ln cm ON CA (1) t_@q C4 14 C, OD L@ @o Ln* C@ Ln Ln Ln * @i -4.-01H C@ ir-nIV1-i co U@ W H cli a% Ln m C@ -! C, -o Ln Ln -4 -0 vi @4 14 14 @q 14 @4 0, m -"%a -0 li 0 0 x P4 w0 0 CX4 I1 lx =L 1 ren).- 1 > 0) C*4w r@ r-i11 m 0 wlnvmwm (L) 9 9 r@ m 0 n co (A -* w CN v r@ * > w mwmmwm 4 VOONWM .m0 in %D C,4 0 a) 9 r@9 r@ 9 c@ rn m 0 0 a n w koli -0 -01-1 cn a * v m r: rcliC, 'a H 91 Z1 C%l @4 C%im -0 Lr)@o a r, m m (D -4 " mmmmmo ON 0) a%a 0 0 -o -o @v-o -o -o @ 6 ;j o -o-o Ln Ln Ln 0 -o -,z-ro -o -o v 0 -0 v -0 .0 .0 .0 @4 a% Ch M ON Cn aN 0 r-r)iu u w- z rnz @4 a cyi 0) ON a% ON 0) u u u u r-u) rA zmz In F m -trm w r, m (=)a C) C) a 0 Ln Ln Ln Ln Ln Ln 0 v .0 1-:r@@ .0 .0 @.4 ON m cr%0) 0) C% 0 u r-u) ()L)ri z cnz -0c@ '1n 14 cn C4 %0 (%2D E1-4 u =L E-4 14 x L) E-4Z m cD r. @4 cq 4 4 C) r@ 0 r-414 m ri T-4r-ir-ir-4 W Lnr, CD r-i W-4 c@ C,4-0 m vmmvmm en (n cn m r- en mm r, ko m m w LntD H Lnc)No 0 a H C4 OD cn lillIH H "WW @i -o H -4 r-i C@ I! O@ 19 @ C@ fn1* rn cncq en cn cn m rn rn cq (3N C@ 11!10 m m C% 4m r@ ai C4 tn c% r@to m 0 OD kO 0 r-4 H r-i H r- m Ln %o OD ON r-I o!,=! c@ ovvmm mmcnmm n m z Ln El to 9 cn 0 @4 0 @q clq 10 C@ C! C@ -! r-iCq OD r@ 10 r-i ON 'D C@ It!@ 0! li Ln 41 x C, ril (Z)CD a 14 (D CD, c:l U) H C4 c@ 11 CD 0 CD eq c@.m C> 0 a rq up. 0 NNNNNN N 0 NNNNNN N 0 NNNNN N 04 PC 0 co DW r-i N 0 u-, r4 4J E4 I I .. .. . %D ko C4 H . 0% co 0 00 Lr)OD w cr,k-ol a -o Ln H o@ H M r_iC', 0 0 c@14c;c@ CD c@ UZ- Lnw m w m w w 4.) ko w w @o aw Ln w ii %DLnko ko%D w F-I Ln x r,., .ri u -H 00 p @- I Cg E-4 1 0 a c4 r- Lnc4w Ln c@ %D M ma%(AOO.O r-i-0 to 0 ko M ri Ln V) a,-4 0 (L) -,4 E-4 ta 0 H E-1 1 0 ap 0 -0 .0.0 .0 0 00 1-01-40 0 mmmmm m z fa F-40 -H Ma C,4 r@ @ 0! L@ 10 C@ -! c,4Ln cn c-i w liW Q@ U, @ t! C@ Ln CD Ln Ln Ln Ln" 10 Ln c) cq-wtm-oLn-o 10-4 (D r-fi@1-1r-Mi C,4 @4 @4 14 -4 -4 -4 -4 11 C) -4 -4 r-fH -4 -4 H CD 1-4-4 -4 14 14 M Ln 090 ::) @.4 I r., 04 u ixCD 1-1 x > m m m (n Ln m (,L) r. . . . . to 0 n ko (DLnw > > cn m a a% w 4 co a -4 %D OD r- m OD -0 0 cN m 0 0 &3 P4 I r4 (3Na liCN m .0 CD 4 -1 -1@ r-4 m Ln Ln m m Ln @4 .O0N .O0N.a0%.O0NO.MNC*h (D E)UC)UQU zwz Ln .. Ln %D r- oo (Y%o --1 4 -4,-4 -4 N Ln Lo Ln Ln Ln Ln 0 10-0-0 -0-0-0 @4 CA M 0) C)%a% M 0 uuuuuu %a r-iC4 LO kO " N N N C4 Ln Ln Ln Ln Ln 10 v -0 -* -0 6 Ch CYNa% ON CN w - mz 0 uuuuu zmz q 00 -t gC4,-@0 I r-i ON en r4 %D (n I a)z to 0 ap CD C) 0 (D 0 to ap z 1 tri 0 H li m 14 a N 14 14 w C) CD CD CD CD CD CD @o @i i @-I 0 -1 H -0 4W C) CD CD m 1-4CD CD w -0 A H ri C4 1 4 00wr-ODM-0 WM%O wvr-mwr- W r-IW r,-0 w %D %D r, .0ka ,)P 0 ul E0 E-@Z P, tn cn CD co cr,rco co co r- m r- C,3-0 w OD cnl-0 CD .0 r- 1-4 @4 .0 %D cc co co r@ co co OD OD m @@ CD C*4 cs r- CD 00 co w CD CD ON %0 co Z:;r@., x cn ca E@ ko ap 4.) E'- 0 Ul Z, E@ 0 0 rn N 0 4.) E@ i I go H z oc@a tn %D CD a% en m t@. ON ei LO 14 li m 14 N r@ %0 CD r4 w H H r-i H li.CD.0 .0 .C).c@. oko CD C@ CD 4 C@ C@ W %DC,r4n0 rr-i @qH C,4 14 C,q4 L@ko C! >4 Ln 00 1 -4 &@ 1 41 x .(d .H u r, -0 C,4 CD -4 1 =L 1-1 @q Ln C,4 C) C4 r-I 10 C@ C@ 10 C@ C@ C@ ID E-4 - I rj3 En r4 0 cn,(:,1 0 0 0 rJ H 0 0-4 1 DW r'4 (D n H PQ X : (L) r4 r4 CD 03C 1 ZH OZ .1 ri PL4 I c, w N > 0 -4 4 r-i fn 0 1 4.) pq 0-4 .14 F-4 v Ln c,, ID 4J -,4 1 .1 E@ 8 0 .I0 rl4 W4 1 x =L 1 a4 @ 1 C) id tn (11as m -0 -4 0 c@ c@ L@ %D --ov w c@ tn cn 0 C) r- m m w r- m Ln w 100 c@ C.4*c@ 00* c@ c@ tn 0m H 0 N .0 rA 'I@,@c@ *14 0N cn cq Ln w 4 =L C@ C@ r@ Ln M r4,@- : @4 11 rA Ul C) I C3 z n ID -4 C@, C@ C@ @-ir-IN -4 ID r-I li 11! 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E@ 00 x 11d -A C4 E-4 x r. 11 4 pa m d) .,A E- W0 H0 pH I @q li 11 r-i@4 -4 (14C4 14 r-f@4 r-i x r@4 to m E4 P. 0 0 z H= n 0 H0 134 ,4 FZ4 rA o@ 1 > pq i P4 -1,-10 4 r-4 r-i@4 @4 r-i CD 0 Pk tn rD r, U M I C3 -4 P4 I r4 No > (D Ln oo...o r4 r-)P4 N MX P4 w@ 1 1 I '0 0 a C)C)a a 0 rn 0 0 a a C) 0 0 C:) LO to 4 P4 I @4 cN rn -cmLn %o Ln Ln Ln m m m Ln Ln Ln Ln Ln Ln r- co Ch C) -4 CN m m in tD w ko m m m Ln m kn r@4 Va% "m .a%0 VaN .a%0 Vm 0@4 "a% c'% p"aN "at a@% Va%1 uuurjuu APPENDIX 6 Covance 6329-224 3M T-6314.2 IndividuaAlbsoluteOrgan Weight Data (g) IndividuaOlrgan-to-BodyWeight Percentages IndividuaOlrgan-to-BraiWneight Ratios IndividuaAlnimal PathologyData 179 v c@ C,4 C4 C,4ko a)= r > r-4%0 0 00 @4 N H m 0 @4 v I 0 r-rn kocn C*4 mv M r0 r@ %0 m a r- N H M M W 0%0 M I -0 Ch Ln (D Ln C" mN MM m %0 N CD 0,-4 M r-iCO CO U@ U@ L9 I'@li I L9 E@ 00(:)(DOCD I WOCD IOOOCDOO 1%000 1 OOCD wwwire- i mv m"ok0ovkohmwH i oN NLnw I Mor- li@4m I Lr.)0 00 Ln 1 r- m 14 Wwvmmrq Ln %D I Mc"oMWH w 1 -0 Ch I 0 r-i%n r@0 r,(A c@ c@ -! 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CIQI- mo 8 B Mp rj2 N N :,n,Ccho rA 'on 8 D-, mn4wm.-:M-ZH z E@ cn co co @iw C 1-4 w ricle r, cn 41 Z mmw ::,w: F4 0 9.4 E@ @ 0 En r4 i I C,4 P4 I Ln I'D 9 @m:lH zo - o z 0 M r4 -OW El m I I -P a% rz4 p@ Z 0- -:Z W r'4 C) @4 o w H>.Oox H>; Hz m 3: m N E,z H 04 -MD D pq E- P 4 9 Mz EH,3W: I .04@NHIngn El WU gm 00 @T4 ;4 4 rn p@ ik.r4 r'4 o H r4r4E-4 I E@ E@ En 40 APPENDIX 7 CellProliferatiRoenport Covance 6329-224 3M T-6314.2 Note: Thisappendixof thereportcontainsinfonmtionsuppliedby Pathology AssociatesInternationalnd has been reviewedby theQualityAssuranceUnit of PathologyAssociatesIntemationaL 359 Covance 6329-224 3M T-6314.2 CellProliferatioRneport PathologAyssockda kgomndomi A CompanyofScionmAppft@WnsInWmWOnalCorporadon MAL CELL pRoLnrERATION REPORT 4-WMK RANGFFUIMG DWARY TOXICITY STUDY WITH N-NWRHYL WMFLUOROOCRANZSULFONAMMO rnL4NOL (N-MeMSE, T-6314)IN RATS COVANCE STUDY NUMBER 6329-274 PREPARED FOR-. 3M TOXICOLOGY SERVICES BURDM 270-2EA2,3MCENTER ST. PAUX.,MN 55144-1000 PIMAREDRY: PATHOLO(;Y ASSOCIATES INTF.ITNATIONAL 15WORbL4" MRL COURT, SUITE I FRIKDERICK, MD 21701 MAY 12,2000 iswwmmes mm comt, suft I FroduiCI4Mw7k=d 21701 (301)663-1644 (301)66349% FAX 360 TABLZ OF CONTFNN 1. Cla-T PROLDTMA170N IOL TAX= M. SL(;NATUM PAGE IV. QUALM ASSURAMM V. *APPZNMI R"ORT 9TAIIMff= Covance 6329-224 3M T-6314.2 I-md COB PwHfaudn Rsput Cavan= StudyNumba 6329-224 Page2 361 Covance 6329-224 3M T-6314.2 CELL PRCffjFZRATlofq REMRT lrmdCeU PmUNnfin Report Cwmm Sb* Numbw 63296224 Pap3 4-W= RANM@NMMG DI"AIM TOMC= PZ"LUOROOCTANESULIFONAAGDO.F.Tg"OL COVANCE FRUVY-. NUMBER STMY W, fliN-bfL"IIM (N-MCMSE, T-6314) IN RATS 6329-224 PURPOBE Mm PWPOW' Of *0 sbWY wss tD8=as dw toxicitoyfftetmt nmtaw when.administmvdinthe diettoraftforatIng 4 wwkl Tb'snpmt@ mbw'ftd by PA&OBU Amwiaft H=BWOMI (PAI)to Ow studySpomw, 3K r%mmzk ft =B'FuHfcndm Bnd*p and btmpvtodw forCovmm StudyNmnber 6329-224 =ddod "4-Week Rmp Fblftg- Didwy Taxift Sady with NMeft, PC&= 16, . 11EdmuO R"WOSE, T-6314I)nRaW. Allaspmu oftheugb unwiftd:wM PAII poW= offtsstudywcm w3hcW inComPlia=ewft to Foodmd Drug dmkinftdm Good L*mtmy ftctim (GLP)RtrdadOns assetft* inTitle21 offt US Code of ]FedonlRqpkfiwp, Pwt A Wucd Dcc=bcr 22, 1979 (efefctiveJ=c 20, 1979), :andwi& any!xppficmc xacadmentl MATFJKLALS.AND HWMODS Tbm Coneedon forCen Prdff"Vd= Six:anim*h: per sm per in.IF' I dwough-6 wen swdficed during week 4 for b9. ' pofiftsfim A seedw of dw leftImad lobe of the liverftm ewh of 6 mts per S=Por.lp-m -I- (V=pe .1-6)wa @fimd md pwccsud: to pmwffin;block by Covmm per protocol rumn bkcks wm sb*ped to PAI, forswuamg and smmm& From each bbck, di& pvwed. for.H&EmaUWwn md - I dofttixmofprolifaating ceffnockw (PCNA),a I of6cH PmHhrftorL y forCA Prouteratim Sccfmm Of film= cutat.5,M mid I)Iamdon poswvcly cbaigedsh&m (-%qmrftQ4 Tub" S=ufftc;P,hbbmgk PA) to came ad on &mg p==.g fbr PCNA. -Stumind' mc&ods w= used to staintissue-sfbrPCNA (PArs. SOP for run BdaRY, fism sectionsw-ere inc@ with a momcbnal atitU3odtyo PnCNA (DAKO, lot#01r3P,AI No. A1723) umd reagentsrequiredforthe avidin-biotin pamddut (ABC Elhe ICitl,ot#?K-6100, PAI No. K 324) method forthe detectionof the complff- PCNA exprmsion incellsin allphasa of theecU cycle(G,,S.02 and M) was locahzedby thechmmagm 3,.3-dimnin@@e (DA]3;Sigma Chemical Co.,lot #19RS201). T'nm secflmswam Lowda with hi::tiwoicylAin.negativecontrolslide was inc@ in thesudming.ranand.consistedof smdy tissue*m was not ftkcubatewdith the pthaary moklk*. 362 Covance 6329-224 3M T-6314.2 Fiwd CeD PtoHfmdm Report Cmmm StmdyNwsba 6329L224 Pqp 4 CCU Prombradw For CallPoNfindm cvghmdms, dides wem fim pa=ed atlow mapification (IOOX) to judge qawhy of Nwidw nd smdmin& pattem of coU labeft (&g.,conuflobulwor mfifamdon,swh n KupffercoU bile duct epidielmin, andadwhm) =d. otphn -Cdumses @Cespwufaafionwas thenquantifieadt higber irla-pdkadm (2=(@ Mm pu=ntsp. of hqmwcytn in S phaseof tM cdl cycle(labehug buhxl.LI) detkmixwd by waring at Ing. 3000 hq*W" in10 randomlyseleded fid& perani=L Ffis'm-I;h-l- was fo6w amosed by evaluatintghesenalH&B slideforowh wimd cvduofad;foicrar.proffulfim ,nm Studentast-ted(two.@" =oqtud vuimw) was usedtotestf.orstatis&aslignificancien Iq betw= contol and ftsuctut:gmqp using 1.,EmsoftExcel-version5.0. A P value of 0.05 w= judppd to be dodwcdly upffiem'L RWSULTS cen --wernam bxhvi&W:mbnal.andpoW. - iccUpmAifizatimdmauepmeentedinS=tkmll(Tabics land 2). Mn penonmpof pmhfmmn hqmoxwta a dctwmmed by thelabelinigndex(I-Jd)idnot dlffcrmgn&wantly betwem controland Ucawd groupsofmale or femalerats. In ip - 1. 6 mm@les,. tbew vrm no P(NA, munwit vmm ivparted for this onsp. slide prcmd fdr ymirnal C94523@ 9 themfom five out Of six Ridapsa~ - Seetiom fiam &a mm timm bloeb'used for preparation of PCNA-staizted slides were stained witkhcmdczyhn and cosim,"M. forhid"athologiccmuation to facilitattheekmwpretifian Of ft' wsbined duka hWmduM ammal findmp and oroupsmnmanes we presentedin Appendix L The resuftssboww no chamn= in the:livw tisme of male and fanaleratsthatwould alterthe of thePCNA stminmgin Ob stady.Ilm liversfrom one und-highdose and two hlgt dow nale rgts@md two hsh do= fmale ros eachcontmned:fow ne=ns without a concomitantinnonnWM respoum lins nemas was not accompanied by a secondary c effecta-s I by an increaud PCNA Li. 363 Covance 6329-224 3M T-6314.2 FbidCoUPtoHftm*n Rqmt Cavmm StadyNunim 6329-224 Pop 5 lnftl digy,'ceH,proliferatiwoans nic&sm-edvhft the liverof male and fenwe ram fi,emomtml:(o ppo@ low dow.(lppo),low-inidon,(10ppm),mid dow (30ppm), mid-high don (100ppm), md.bigh don (500ppm) poups aft 4 wedm on stmdy.No stutijdcauy- dvifima iinceBprolifcmda%a determinebdy,*tbaPCNA WxHng'bxiu (LI)wcm,- abouved inmde or femak rib aiw 4 OfN-meft4 pan do edmal 364 Covance 6329-224 3M T-6314.2 FinalCCU ProliferadmRopmt Covame So* Nwaba 6329-224 PAP6 ILTABLES LeL=d SEM --StmmdardErrorofft Mom 365 Covance 6329-224 3M T-6314.2 Covum Sh* Table11-1C.eltProW*udon InLlwwofMaleRaftAfter4 We-elmofN-MEFOSE PGNA otam I -0 ppm (Comb" AMmal Laboik* sex Number Index m- cwoi =0.188% 2 -Ippm (Low) m C94M m C94403 m C94404 m C94495 M C94496 u@ sm m cs"07 m CD"oe m -C$4m. 1 0.136% NP 0.188% 1 1 0.407% 1 0.150% I ojwii@- -I..0-"B% 1 OA27% 0.082% M C94WO 0.170% m C"wl 0.05M M' CS-4W2 I Uie% Own 0-330% sm 0.110% 3 - 10 ppm (Low-MM) m co4m 0.377%@ M. C94504 0-328% M' OA79% m C945M Olw% M: C94507 0.0"% M. C94we 0.167% No.W.'&224 4- 30ppm P" 0.0"% m C24" 0.0=27r% m cusio O@064% m C94511 0.225% m cmii' O-U"9% m C9013 0.378% m 0281% Own IL216% 5 - ioD ppm OAWW4igh) m C94515 0-35M m C94516 1 0.337% m C94517 0253% m C94519 0295% m I cooig 0.394% m C94620 0.055% Nima 1 0.284% Sam 0.050% 8 - SW ppm (High) m C94521 0.196% m C94522 0.054% m C94523 NP m C94524 0.112% m C94525 1 0.085% m C94528 Mean 0or..119217%% SEM 0.028% NP; UssuenotPresoton Wiftfbr#C94493 NP; slidenotprese"tforOCS4523 Page.li-i 366 a ow Covance 6329-224 3M T-6314.2 CovanceStudyNo.6239-224 Table11-2C.oN ProllfW&ftInnUvw ofFenutoRatsAftW4-WeeksofN-M*FOSE I PCNA Animal I Labodne emruosm__@ 1 0 ppm confto numbw i Index F I C94527 I NO F C94528 1 0.082% r C94529 -1 0.138% F C94530 0.565% F C94531 0.084% F i CG"32 Mean 0.138% 0.201% SEM 0.012% 2 -I ppm (LOW) p C94533 0.135% F C94534 0.138% F C$4535 0-iii% F C94536 0.401% F i C94537 -0.347% d"sn I_ol?2% n 0.234% SEN 0.051% 3-@-i10pm@m ld F C9463U3@9 1 O.IEO% F C64540 1 0.054% A F C94541 1 0.080% F C94542 O.Iio% F C94543 0.168% F C--94-544 0.168% meow *;"M- BEN 0.020% 4 -30 ppm(MOI- F C94545- 0.137% F C94646 1 .110% F C94647 1 0.192% F 1 CV4545 1_ 0.112% F I -C94S49-1 0.161%; r- C94550 0.080% Mean 0.132% s - Ioa ppm @!"th) ............ ! 0.016% F C94551 -0.356% F C94562@ .090% F U94OW O-OS2% F C945" 0.081% F C94555 _L__R.053'7 F C$4556 I mean 1 0.123% SEM i 0.04T% 6 - Soo ppm (High) ....I...-FF F - CC945=57 56_-40.1-93% C 56 - 0.02T% C94559 1 0.081% (@6-@D l@O .193% F C94561 1 0.161% F 75@W62'i 0.142% Mean 0.133% SEM 0.027% ND; not_determindeude toinsumcietnitestamn slidIe Page 11-2 367 Covance 6329-224 3M T-6314.2 Fbat CCU PtcHfmw=.Ropm Covnm StudyNumber 6329@224 Pup 7 UL SKGNATURE PAGE Submittedby, projed him=W. Saxtra R. EldridykPh.D. projea 00 Date Carol@nMey-d, D.V-M.,D-*Immte, A-C.V.P. ELI D.%te 368 Covance 6329-224 3M T-6314.2 FbW Cdl ftbhndm Rgpmt Cava= StaV Numba 6329-224 Pap & IV.QUALXRY ASSURANCE STATEMENT 369 Pathology Assoclafts Intemation@at A Oxnnw ciSmMmA;Pkmfto adopmftmlC*PmWm Covance 6329-224 3M T-6314.2 CeB ProliferationReport 4-Wftk Paug*4%ding DieftryToxidtyStudy withN-Mdbyl Pwfluoroctanemffonamido lgtbanol(N-MeME, T-6314)in Rats Covmm SttodNyumbw. 6329-224 QUALXRY ASSURANCE STATENZNT Mds cog pwhtaboiikp*d hu be= irap@ a3bd"ted by thePAI Queft ,Ajootoa=Unk.(4QAIJ)=,Pqm;pplbytwGoW Pmetice(OLP)rtvduiom I ig- Iby theU.S.Foodmd Drug AdIWTA3tafi(cFoDA).ThoccUpeoUfamdon repartizan -P, I rdivedm aftber=wded dsitlThe followintgableisa reowd ofthe -An" petfionedmkd reportebdy dw QAU. Dates( iau2din W121" 03131;04104= 03t3lMAWOO 05/12tOO Pbm lummK4ed Daftnubap Reporbd toPAI managemantirreledMAW= StadyDmft.md Sq*oirdogDocomenodon Daft CoH NuGhnike Report Fmd CcH ProfiftudenReport W13/9S 04/WOD 04/04= 05/12100 2 Ka= E.BWa Date Quaft As=== Offm= 15 Wanw'S Mil CQkm% Subs I* Fredmid6 Maryland 21701 (3(116)53-1644 (301)663-e994FAX 370 Covance 6329-224 3M T-6314.2 IF@. .4r.411 Covence Study Number 6329-224 pap 9 ",]?F"10[- I@ 371 Covance 6329-224 3M T-6314.2 RW EhftCdlP.o*iksftR.eport Cmm So* No.6329-=4 bdividedAxMdlm - wpbdo* lftdkp InLtww: 4-Wuk Rmp ra 76 DKWY TaWk*Y,Sto*wkb N-Neftyl n Cl nounmomo:g*uW(N-MeFOWT-6314)in Rau AnimidNumAm@ Sam. DooGromp BkWb& FbdhW C94491, m C94492, m 1-0ppm (Cmftl) 1-0NO (Control)- No Sipincut Finftp No SignificanFtindings C94493' m- 1-0ppm (c4nw) Nosipiftwp%dbw t C94494 m I-OPPM'(CbF*CQ C344" m 1-0on (Cmaoo No-SipfficatFkdinp No SigafficatFindings C144% m I-Oppm (Cmirol) C94497 m 2-1ppa (Low) C34M. m .2-1p.pm (Low) No Sipfficant'F'ndinp No SignificanFtinding$ No SignificaFnitofto C344". m 2-1ppm (IAW) No &*Afic" IrmdiW C949M. m 2-1ppm (Low) No SiuAficutFm-diap C"= m 2@I'ppm (Low) No SipfflownFkidii2p C34= C"= m 2.1;ppm (Low) m 3-10ppm (Low-mid) No SimificunFinfto No SignificaFnbtdiqp C94SO4 m (W-9490: m 3-10ppin(Low-mid) 3@10 ppon(Low-mid) N*S*dficmtFkdinp No SignificaFnitxtinp C94M m 3-10ppm (Low-mid) No SignificaFnitnftp C"W.@ -M 3-10ppm%aw4n* No sipftmt Fmdkp C94M C9450 C94M m 3-10"m(Low-mid) m .4-30p.pm W m 4-30ppra(Mid) No SipfficauFtWbw No SignificaFnitnftp No SipffkmntFindings C"SILI m c9m2. m 4.@30pput (Bdkl) 4-30Mm No S%nfflmd Fmdkp No SignificaFnmtdiop CV4513 M. 4-30no No Sipi&=tFkmfinp C94514 m 4-30ppm(BAO No SipfficantFindbw C94515. m S-IODPPMW-IO& C94516' m 5-100ppm (btwwbigh) No Signi!fcentFindings No SignificanFtindings C9M7 m@ S-lWppm(MM-bigh) No-SipilicantFmdiap C%ng m 5-100ppm (NU-bigh) C!P4Sl9 m 5-1eo1*= (W"gh) FocalNecrosis No SignificaFnutdinp C94528 m 5-100ppn(bi"gh) No SigafficantFmdinp C9621 m C94522- m C945n M. 6-500%ppm(EEigh) 6-500,ppnffish) &500 ppm OUgh) No SignificaFnmtdimp No SignificanFtindings FocalNecrosis C94524 M. 6-5W ppm (High) FocalNcmusils C94=S m 6-500ppm(Ifigh) C94526 m 6-500ppm (High) No SignificanFtindings No SignificanFtindings 372 Covance 6329-224 3M T-6314.2 Ankmd Nmobw C94527 C94M C%M C3491 C"= cmm C34&Ul C94535 C945m: C94537 CV45M. C94539 C34W C94541 C94542 C94543 C9$544 C945" C94546 C94547 C94548 C9450 CV45" C94531 C9450 C9450 C94554 C94M C94556 C94557 c*wse C94559 C94SO C94561 C94562 bl&Vbkd Anind 4@Wa&R=pMbdlngDkM7 mw Ztbmd ruW DmftCellProlftWouRopot Covam Sb* No.6329-224 rodinp inLiva: Sbady wi& Zq.Metbo Q(-MEFOM T-014) InRats So& Dm Group p I-Oppm (CAmtrol) F I-Oppra (Contra]) p I-oppm (Control) F I-OPPM (c4nfti) F 1-0ppsn (Control) F 1-0ppm (Control) F 2-1ppm gow) F .24ppm (Low) p 2-1ppm (Low) F -2-1ppm (Low) F 2-1ppm (Low) F 2-1Mm (Low) F 3-19ppo (Low-ndd) F 3-10ppm (Low-nid) F 3-10ppn (Low-suid) F 3-10-ppa(Low-aiid) F' 3-10 ppm (Low-4mid) F 3-10ppm (Low-add) F 4-30ppm (bfkl) 'F 4-30ppm-04id) F 4--@30*ppWm4 F 4-30@ppm(W F 4-30ppa (Mid) F 4-30ppm (hfi(o 5-100ppon(bfi&ld&) F 5-100ppm(Mid-bigh) F S-IDOPPM(hW-high) F 5-100ppm 0"4h&) p 5-100ppm@Mi&bigh) F 5-100 ppm (hW-high) F 6-5,DOppm (Ffigh) p 6-500ppin(High) F 6-500ppm (Wigh) F 6-500ppm (Hi&) F 6-50Dppm (MO) F. 6-500"m (High) Bixtoio*FhAtup No SipfficanFtbdiap No SipfficanFtmdiap No SipfficauFtindings No Sipific= ]Fuiftw No S*dfieml Fkmftp No SignfficanFtindings No SipificwgFindings No SipWwwxt Fkdiap No-SigndficmtFindings No SipfficatFkwiinp No SigWfica*FftWinp No SigniracWFindinp No SipificatFtbiftp No SignifK=n@Ffiubngs: No SipfficwtFkdhW No SipificowFkmfinp No S*WficautFmdiap No SignificanFtindings No SignificaYnutwftp No sipfficanFk-Aina No Sig*ficamFin&w . No SignificanFtindings No SignificanFtindings No SipfficanFthKbnp No SignificanFtindings No Significant-Findings No SignificanFtftWEnp No SignificanFtindings NO significanFtindings No SignificanFtindings No SignificanFtindings No SignificaFnitndinp FocalNecrosis FocalNecrosis No SignificanFtindings No SignificanFtindings 373