Document pY6mn3byjkj9Ry8qOnrLgGzd

PathologyAssociateslntemational A Company ofScienceApplicatioInnsternatioCnoarlporation AA Empbya&Owrad CaMwy SECOND DRAFT CELL PROLIFERATION REPORT 104-WEEK DIETARY PERFLUOROOCTANE CHRONIC TOXICITY AND CARCINOGENICITY STUDY WITH SULFONIC ACID POTASSIUM SALT (PFOS;T-6295) IN RATS COVANCE STUDY NUMBER 6329-183 PREPARED FOR: 3M TOXICOLOGY SERVICES BUILDING 220-2E-02,3M CENTER ST. PAUL, MN 55144-1000 PREPARED BY: PATHOLOGY ASSOCIATES INTERNATIONAL 15 WORMAN'S MILL COURT, SUITE I FREDERICK, MD 21701 AUGUST 24, 1999 5 Worman's Mill Court, Suite I * Frederick, Maryland 21701 * (301) 663-1644 (301) 663-8994 FAX TABLE OF CONTENTS Contents Narrative Tables Figures SignaturePage QualityAssurance Statement Appendix Second DraftCellProliferatioRneport Covance Study Number 6329-183 Section I n m IV v vi Page 1-2 Second DraftCellProlifemtioRneport CoVance Study Number 6329-183 CELL PROLIFERATION REPORT 104-WEEK DIETARY CHRONIC WITH PERFLUOROOCTANE COVANCE TOXICITY AND CARCINOGENICITY SULFONIC ACID POTASSIUM SALT 6295) IN RATS STUDY NUMBER 6329-183 STUDY (PFOS;T- PURPOSE The purposeofthestudywas toassestshechronitcoxiciatnyd carcinogenicoifttyhetestmaterial when administeredinthediettoratsforatleast104 weeks. This report,submittedby PathologyAssociatesInternation(aPlAI)to thestudySponsor,3M, representtshecellproliferatifoinndingsand interpretatifoonrCovance Study Number 6329-183 entitle"d104-Week DietaryChronicToxicityand CarcinogenicitSytudy with Perfluorooctane SulfonicAcid PotassiumSalt(PFOS; T-6295)inRats". Allaspectsof thetasksassociatewdith PAI's portionof thisstudywere conductedin compliancewith theEnviroranentaPlrotection Agency Good LaboratoryPractic(eGLP) Regulationass setforthinTitle40 of theUS Code of FederalRegulationsP,art792,issuedNovember 29,1983 (effectivDeecember 29, 1983),and any applicablaemendments with thefollowingexception.An expiredreagentwas used in theBrdU inununohistochemistsrtyainingprocedure(mouse IgG, Vector,lot# PK-4002, PAI No. K3 5 1, exp. 4/9/99).The staininwgas judgedto be satisfactortyh;ereforet,hisGLP deviatiodnoes not impactthescientifviacliditoyfthestudy. MATERIALS AND METHODS ProliferatinCgellNuclear Antigen (PCNA) Stainingand Evaluation Tissue Collectionfor Cell Proliferation Five animalsper sex per group in groups I through5 were sacrificeadtweeks 4 and 14 for hepatocellulparroliferatioAn.sectionof theleftlaterallobeoftheliverfrom each of 5 ratsper group (groups1-5)was fixedin zincformalin,processed,and embedded to paraffibnlockby Covance per protocolspecificationsT.issueblockswere shippedto PAI for sectioninagnd staining.From each block,a slidewas preparedforH&E evaluatioannd inimunohistochemical detectioonf proliferaticnegllnuclearantigen(PCNA), an endogenousmarkerof cellproliferation. Immunohistochemistry for Cell Proliferation Sectionsof paraffin-embeddetdissueswere cutat5 gm and placedon positivelcyharged slides (SuperfrosPtlus,FisherScientifiPci,ttsburghP,A) to ensureadhesionduringprocessingfor PCNA. Standardimmunohistochemicamlethods were used to staintissuesforPCNA [PAI's StandardOperatingProcedureforImmunohistochemicalStaining(SOP #707)]. Briefly,tissue sectionwsere incubatedwitha monoclonalantibodytoPCNA (DAKO, lot#0 16;PAI No. A 1899) and reagentrsequiredfortheavidin-biotpienroxidase(ABC Kit;Vector,lot#PK-6100; PAI No. K 327)method forthedetectioonf theantigen-antibocdoymplex. PCNA expressionin cellsinall phasesof thecellcycle(G,,S,G2and M) was localizebdy thechromagen 3,3'-diaminobenzidine (DAB; Sigma CherrdcalCo., lot#18H8201). Tissue sectionswere counterstainewdith hematoxylin. Page 1-3 SecondDraftCellProlifemtRieopnort CovanceStudyNumber6329-183 CellProliferatiMoenasurements The percentagoef proliferaticneglls(proliferatindgex,PI) was determinedby scorinaatleast 2000 hepatocytesinatleast6 fieldsofliver.Inmost cases,at least3000 hepatocyteswere scored in 10 fieldsb,utinfouranimalstherewas insufficietnitssuepresenton theslidetobe ableto score as many cells.Slideswere firspterusedatlow magnificatio(n10OX) tojudge qualityof staining, processingand sectioninga,nd patternof celllabeling(e.g.,centrilobuloar panlobular, extraepatocellulparroliferatiosnu,ch as Kupffercellsb,ileductepitheliume,ndothelium)a,nd histomorphologicchanges.The lattewras furtherassessedby evaluatintgheserialH&E slidefor each animalevaluatedforcellproliferationC.ellproliferatiwoans then quantifieadt hicher magnificatio(n20OX) asdescribedabove. A negativecontrolslidewas includedinthestaininrgun and consistedof study tissuefrom animal #C93008 thatwas notincubatedwiththeprimaryantibody. StatisticalAnalysis The Student'st-tes(ttwo-sidedu,nequalvariance)was used to testforstatisticsailgnificancienPI betweencontroland treatmengtroupsusingMicrosoftExcelversion5.0. A P valueof 0.05 was judged tobe statisticaslilgynificant. Bromodeoxyuridine (BrdU) Staining and Evaluation Tissue Collection for Cell Proliferation Seven daysbeforetheweek 52 interimsacrificoes,moticpumps containing20 mgtrliBlrdU were surgicalliymplantedin fiveanimalsper sex per group in groups I and 5 for hepatoceuular proliferatioAn.sectionoftheliverand duodenum from each of 5 mts per group (groupsI and 5 only) was takenat necropsyand embedded to paraffinblock by Covance per protocol specificationTsi.ssueblockswere shippedtoPAI forsectioninagnd stainingF.rom each block,a slidewas preparedfor H&.E evaluatioannd inununohistocherridiectaelctionof BrdU, an exogenous markerof cellproliferation. Immunohistochemistry for Cell Proliferation Sectionsof paraffin-embeddedtissueswere cutat5 pm and placedon positivelcyhargedslides (SuperfrosPtlus,FisherScientifiPci,ttsburghP,A) to ensureadhesionduringprocessingfor BrdU. Standardimmunohistochen-dcmaelthods were used to staintissuesfor BrdU [PAI's StandardOperatingProcedureforInimunohistochemicSatlaining(SOP #707)]. Briefly,tissue sectionswere incubatedwitha monoclonalantibodytoBrdU (BectonDickenson,lot#81604; PAI No. A2089) and reagentsrequiredfortheavidin-biotpienroxidase(ABC Kit;Vector,lot#PK6100; PAI No. K351) method for the detectioonf the antigen-antibocdoymplex. BrdU incorporatiointocellsin S phase of thecellcyclewas localizebdy the chromagen 3,3diaminobenzidin(eDAB; Sigma Chemical Co., lot #108H8210). Tissue sectionswere counterstainewdith hematoxylin. Cell ProliferationMeasurements The percentageofBrdU-Iabeledcells(labelinigndex,LI)was determinedby scoringatleast2000 hepatocyteisn 10 fieldsof liver.Slideswere firspterusedatlow magnificatio(nIOOX) tojudge qualityof stainingp,rocessingand sectioningp,atternof celllabelin(ge.g.,centrilobuloarr panlobulare,xti-ahepatocellpurloalriferatiosnu,ch as Kupffer cells,bile duct epithelium, endothelium)a,nd histomorphologicchanges. 'Me lattewras furtherassessedby evaluatingthe Page 1-4 Second Dmft CellProlifemtionReport Covance Study Number 6329-183 H&E slidefor each animal evaluated forcellproliferation.Cellproliferationwas then quantifiedat higher magnification (20OX) as described above. A negative controlslidewas included in thestainin0-run and consisted of study tissuefrom animal #C92534 thatwas not incubated with the primary antibody. Statistical Analysis The Student's t-test(two-sided,unequal variance)was used to testfor statisticaslignificancein LJ between controland treatment groups using Ntcrosoft Excel version 5.0. A P value of 0.05 was judged to be statisticalsliygnificanl RESULTS PCNA Cell Proliferation Individualanimal and group mean PCNA cellproliferatiodnata are presented in Section Il (Tables 1-4)and graphicallyin Section M (Figures I and 2). The negative controltissuewas judged to be free from background staining. At 4 and 14 weeks on study, the percentage of proliferating hepatocytes (PI)in treatedanimals did not differsignificantlfyrom controlanimals for eithersex. BrdU Cell Proliferation Individualanimal and group mean BrdU cellproliferatiodnataare presented in Section II (Table 5) and graphicallyin Section M (Figure 3). The negativecontroltissuewas judged to be freefrom background staining.The percentage of labeledhepatocytes (LI) in treatedudmal groups did not differsignificantlyfrom controlanimals foreithersex at52 weeks on study. Histopathology Sections from the same tissueblocks used for preparationof PCNA- and BrdU-stained slideswere stained with hematoxylin and eosin (H&E) for histopathologicevaluation to facilitattehe interpretatioonf the immunostained slides. Individualanimal findings and group summaries are presented in Appendix I. The resultsshowed thatno significantchanges were observed in the liverfrom male and female rats,which would alterthe interpretatioonf thePCNA or BrdU stainingin thisstudy. DISCUSSION In the present study, cellproliferationwas measured by PCNA immunohistocheniistry within the liverof male and female ratsfrom control(Group 1),low dose (Group 2), mid dose (Group 3), mid-high dose (Group 4), and high dose (Group 5) groups after4 and 14 weeks on study, and by BrdU immunohistochernistryin the liverfrom control(Group 1) and high dose (Group 5) male and female ratsafter52 weeks on study. The decision to use BrdU labelingto detectproliferating cellsat 52 weeks was based on the increased sensitivitoyf continuous labeling with BrdU compared to PCNA in detectinga proliferativreesponse inthe liverof mts. In thiscase,BrdU was administered via osmotic pump continuously for 7 days. A statisticalsliygnificantincreasein cellproliferationa,s determined by the PCNA proliferating index (PI)at4 and 14 weeks, or by theBrdU labelingindex at52 weeks, was not observed in the liverof male or female rats. Although statisticalnalysis did not reveal a significantcell proliferativreesponse to the testmaterial,biologicalsignificancewas evident in individualanimals. Biologicalsignificancefor an individualanimal isdefined as a doubling in PI or LI over controls Page 1-5 SecondDraftCellProliferatRieopnort CovanceStudyNumber 6329-183 andgreattehranthehichesctontrovlalueT.hus,thedatarepresentiendtermsofseveri(tfyold increaosveercontrolasn)dinciden(cneumberofanimalsshowingbiologicsailgnificancAse). seen in TextTable 1, bioloaicalsliycnificacnetllproliferatiwoans evidentin 2/5male ratsatthe highdose after4 weeks on studyand in 115male ratsatthemid-highdose and n-dddose after4 weeks on study. This apparent and minimal proliferative response seen in male rats at 4 weeks was lessthan the control PI at 14 weeks, and was not sustained as evidenced by the lack of a response at week 14. Furthermore, none of the animals exhibiteda proliferativeresponse at 52 weeks. Therefore, the proliferativeresponses seen in theseindividualmale animals was a possible mild response evident only at 4 weeks. Text Table 1. Biologically Significant Cell Proliferation in Liver of Male Rats Group Low dose Mid dose -high dose High dose Week Severity' <1 1.6-' 1.0 1.5 4 2 Incidence 015 115 115 2/5 Week 1 Severity' <1 <1 <1 <1 14 2 Incidence 015 015 015 015 'Fold increaseover controls .'Number of animals with a PI or LI double the mean controlanimal control value and Week 52 Severity I 2 Incidence - - <1 0/4 greaterthan the highest In contrastto male rats,biologicallysignificantceH proliferatiownas evident in fenialeratsat the mid-high dose (2/5 rats)and atthe high dose (2/5 mts) after4 weeks, and atthe low dose (2/5 rats) and mid dose (115 rats)after14 weeks on study, as seen in Text Table 2. The, degree of the proliferativreesponse observed in females was greater than that seen in the males. The proliferativreesponse observed at week 4 was not sustained after14 weeks, nor was a pmliferafive response observed at 52 weeks. Based on the severityand incidence of the proliferativeresponse seen in the high dose females, and the dose response relationshipseen at4 weeks, the testmaterial isjudged to induce a transientproliferativreesponse in the liverof female ratsat the high dose. Text Table 2. Biologically Significant Cell Proliferation in Liver of Female Rats Group Week 4 Severity1 Incidenc62 Week 14 1- Week 52 2 Severity' I Incidence "verity' Incidence Low dose 1.0 015 2.6 2/5 Mid dose 1.1 015 1.2 115 I -high dose 1.8 2/5 <1 015 - - High dose 3.5 215 <1 015 <1 015 'Fold increase over controls 2Number of animals with a PI or LI double the mean controlanimal control value and greater than the highest Interestin-lya, rarepatternof PCNA-labeling was observed in high dose female ratsat week 14, which was not seen in any other groups, nor at the earliertime point. The staining was characterizedby a granular cytoplasmic stainingpatternlocalizedto the hepatocytes around central veins of the liver.The significanceof thisqW of labelingpatternisnot known; however, ithas been speculated thatitmay representmitochondrialDNA synthesis (unpublished observation). Recently, ithas been shown thatPCNA can cross-reactwith peroxisomal multifunctionalprotein (Mayer, et al.,1998). Thus, itispossiblethatthe centrilobulasrtainingpatternobserved in the hic,hdose animals may be associated with peroxisomes. Page 1-6 Second DraftCellProliferatioRneport Covance Study Number 6329-183 The cellproliferatiroantesintheweek 14 male ratswere allhigherthanthosein theweek 4 male rats.In thefemalerats,cellproliferatiroantesat week 14 were higherthanatweek 4 in the controll,ow doseand mid dose groups,butnotinthe mid-highor high dose groups. The reason fortheconsistenthliyghercellproliferatiroantesintheweek 14 animalsisnot known. However, allslidesfrom the4 and 14 week timepointswere stainedtogether,so variatioinn staining between runscanbe ruledout.One possibiliitsythatthetissuewsere handleddifferentliyn term of fixationand processing,which may have affectedthe staining.Despitethisdifference, comparisonscan be made between each treatmengtroup and thecontrolgroup fora specififci= point. SUMMARY The cellproliferatisoenenintheliveorf male ratsat4 weeks was notdose-relatewda,s minimalin severity(highdose was lessthantwo-foldgreaterthancontrols)o,ccurredin two of fiveanimals inthehighdosegroup,and was transienitnnature(i.e.w,as not sustaineadt 14 weeks). In femaleratsc,ellproliferatiionntheliverwas dose-relateadtweek 4 butnot atweek 14, greaterin severitcyompared tomale rats(greatetrhantwo-foldatthehighdose),and occurredin two of five animalsinboth thehigh and mid-highdose groups. Based on thesefindings,cellproliferation effectseen at4 weeks intheratswere consideredtobe relatedtotestmaterial.At 52 weeks, there was no evidenceofhepatoceuularproliferatiionnmale orfemale ratsatthehigh dose. LITERATURE CITED Mayer, D., Forstner,K., Beier,K., and Volkl, A. (1998). Monoclonal antibodiesagainst proliferaticnegllnuclearantigencross-reacwtiththeperoxisomalmultifunctionaplrotein.Anal Biochem 256: 135-137. Page 1-7 Second DraftCellProliferatioRneport Covance Study Number 6329-183 111.TABLES lzgmd BrdU, bromodeoxyuridine LI,labelingindex,percentageof cellsinS phaseofthecellcycleas determinedby BrdU staining ND, notdetermined PCNA, proliferaticnegllnuclearantigen PI,proliferatiinngdex,percentageofcellsinS,Gl, G2 and M phasesof thecellcycleas detenninedby PCNA staining SEM, standarderrorofthemean COVANCE STUDY NO. 6329-18i TABLE 1.PCNA CELL PROLIFERATION IN LIVER OF MALE RATS AFTER 4 WEEKS ON STUDY Group Sex 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) M 2 - 0.5 ppm PFOS (Low) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid) m 4 - 5.0 ppm PFOS (Mid-High) M 4 - 5.0 ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m PCNA Animal i Proliferating Number Index* C92506 0.027% C92524 0.000% C92526 0. 13 0 0,@la C92529 0.000% C92546 0.053% mean 0.042% SBA 0.024% C92574 0.000% C92575 0.027% C9261 0 0.054% C92613 0.080% C92618 0.027% Mean 0.038% SBA 0.014% C92646 0.026% C92650 0.027% C92652 0.027-/. C92668 0.098% C92678 0.166% Mean 0.069% SEM 0.028% C9271 5 0.136% C92718 0.053% C92731 0.000% C92734 0.027% C92744 0.000% Mean 0.043'vco SBA 0.025% C92752 0.027% C92759 0.027% C92779 0.000% C92793 0.136% C92798 0.133% Mean 0.065% SEM 0.029% i*Proliferaticnegllnuclearantigen:percentage of cellsinGl, -S,G2 and M phases Page 11-1 COVANCE STUDY NO. 6329-183' TABLE 2.PCNA CELL PROLIFERATION INLIVER OF FEMALE RATS AFTER 4 WEEKS ON STUDY Group Sex 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 4 - 5.0 ppm PFOS (Mid-Hi h) F 4 - 5.0 ppm PFOS (Mid-High) F 4 - 5.0 ppm PFOS (Mid-High) F 4 - 5.0 ppm PFOS (Mid-High) F 4 - 5.0 ppm PFOS (Mid-High) F 5 - 20.0 ppm PFOS (H@gh) F 5 - 20.0 ppm PFOS (High) F 5 - 20.0 ppm PFOS (High). F 5 - 20.0 ppm PFOS High) F 5 - 20.0 ppm PFOS (High) F Animal Number C92861 C92864 C92914 C9291 9 C92926 Mean SBA C92941 C92945 C92950 C92969 C92982 Mean SBA C92996 C93006 C93008 C93031 C93045 Mean SBA C93058 C93067 C93070 C93075 C93084 mean SEM C93114 C93123 C93142 C93144 C93159 Mean SEM PCNA Proliferating Index* 0.080% 0.132% 0.000% ND 0.000% 0.053% 0.032% 0.027% 0.056% 0.027% 0.110% 0.053% 0.055% 0.015% 0.054% 0.053% 0.108% 0.028% 0.054% 0.059% 0.013% 0.186% 0.161% 0.027% 0.000% 0.110% 0.097% 0.036% 0.05 % 0.027% 0 3AQ% 0.430% 0.054% 0.183% o.oes% I*Proliferatingcell nuclear antigen; percentage of cellsin Gl, S. G2 and M phases Page 11-2 COVANCE STUDY NO. 632-9-189 TABLE 3.PCNA CELL PROLIFERATION IN LIVER OF MALE RATS AFTER 14 WEEKS ON STUDY Group Sex 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 1 - 0 ppm PFOS (Control) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) m 2 - 0.5 ppm PFOS (Low) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid) m 3 - 2.0 ppm PFOS (Mid m 3 - 2.0 ppm PFOS (Mid) m 4 -5.0ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-High) m 4 - 5.0 ppm PFOS (Mid-Hiqh) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (High) m 5 - 20.0 ppm PFOS (Hiah) m PCNA Animal Proliferating Number Index* C92509 0.323% C92511 0.521% C92521 0.588% C92528 0.137% C92532 0.247% Mean 0.363% SEM 0.084% C92593 0.084% C92600 0.081% C92616 0.240% C92621 0.296% C92627 0.188% Mean 0.178% SEM 0.042% C92640 0.136% C92645 0-561% C92662 0.242% C92676 0.161% C92684 =0.108% Mean 0.242% SEM 0.083% C92713 0.137% C92714 0.466% C9271 9 0.107% C92722 0.542% C92728 0.421% Mean 0.335% SEM o.osg% C92765 0.560% C92777 0.352% C92789 0.027% C92799 0.056% i C92812 0.053% Mean 0.210% SEM 0.106% I*Proliferaticnegllnuclear antigen;percentage of cellsin Gl, S, G2 and M phases Page 11-3 COVANCE STUDY NO. 632Q-183" TABLE 4.PCNA CELL PROLIFERATION INLIVEROF FEMALE RATS AFTER 14WEEKS ON STUDY Group Sex 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 1 - 0 ppm PFOS (Control) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 2 - 0.5 ppm PFOS (Low) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 3 - 2.0 ppm PFOS (Mid) F 4 - 5.0 ppm PFOS (Mid- F 4 - 5.0 ppm PFOS (Mid-Hiah) F 4 - 5.0 ppm PFOS (Mid-High) F 4 - 5.0 ppm PFOS (Mid-High) F 4 - 5.0 ppm PFOS (Mid-High) F 5 - 20.0 ppm PFOS (Hig F 5 - 20.0 ppm PFOS (High) F 5 - 20.0 ppm PFOS (High) F 5 - 20.0 ppm PFOS (Hiah) F 5 - 20.0 ppm PFOS (High) F Animal Number C92880 C92887 C92898 C92903 C92905 Mean SEM C92944 C92962 C92967 C92978 C92987 Mean SEM C92993 C93000 C93018 C93023 C93035 Mean SEM C93051 C93064 C93071 C93085 C931 09 Mean SEM C93111 C93127 C93143 C93155 C93169 Mean SEM PCNA Proliferating Index* 0.082% 0.161% 0.137% 0.02-8% 0.085% 0.0-99% 0.023% 0.110% 0.826% 0.215% 0.080% 0.054% 0.257% 0.145% 0.081% 0.054% 0.054% 0.356% 0.053% 0.120% 0.059% 0.054% 0.082% =0.057% 0.027% 0.053% 0.055% 0.009% 0.028% 0.140% 0.081% 0.000% 0.084% 0.067% 0.024% I*Proliferaticnegllnuclearantigen-p,ercentageofcellsinGi, S, G2 and M phases Page 11-4 COVANCE STUDY NO. 6329-183 TABLE 5.BRDU CELL PROLIFERATION IN RAT LIVER AFTER 52 WEEKS ON STUDY Treatment Group 1 1 1 1 1 5 5 5 5 1 1 1 1 1 5 5 5 5 5 Gender Male Male Male Male Male Male Male Male Male Female Female Female Female Female Female Female Female Female Female Animal Number C92504 C92512 C92534 C92562 C92570 Mean SEM C92778 C92795 C92796 C92817 Mean SEM C92876 C92889 C92899 C92923 C92930 Mean SBA C93112 C93120 C93154 C93163 C93171 Mean SEM BrdU Labeling Index* 0.51% 0.19% 0.09% 0.26% 0.24% 0.26% 0.07% 0.08% 0.31% 0.09% 0.38% 0.22% 0.08% 6.94% 1.23% 1.64% 1.40% 0 11% 2.*26% 1.20% 0.26% 0.11% 0.03% 0.27% 0.13% 1 0.16% @@0-05% @*Bromodeoxyuridine;percentage of cellsin S phase Page 11-5 Second DraftCellProlifemtioRneport Covance Study Number 6329-183 III.FIGURES FIGURE 1.PCNA CELL PROLIFERATION IN UVER OF MALE RATS 0.5 0.45 0.4-- 0.35 x w 0z 0.3 Z 0.25 r4-c cc UUJ 0.2 =I 0 c0c. 0.15 0.1 0.05 0 Group 1 0 ppm Group 2 0.5 ppm Group 3 2.0 ppm Group 4 5.0 ppm Group 20.0 pr Page111-1 FIGURE 2.PCNA CELL PROLIFERATION IN LIVER OF FEMALE RATS 0.5 0.45 0.4 OZO@, 0.35 Z- 0.3 z 4r- 0.25 im ui u- 0.2 0 cr. 0.15 0.1 0.05 - 0 mi Group 1 0 ppm 11 Group 2 0.5 ppm @l Group 3 2.0 ppm in Group 4 5.0 ppm Group 20.0 p Page111-2 2.5 2 1 .5 z z :1 w to FIGURE 3.BRDU CELL PROLIFERATION IN LIVER OF RATS AFTER 52 WEE 0.5 0 Week 52-Males Week 52-Females Page 111-3 Second DraftCellProlifemtionReport Covance Study Number 6329-183 IV. SIGNATURE PAGE Submittedby: PAI ProjectManager: SandraR. Eldridge,Ph.D. PAI ProjectPathologist: Date Carolyn Moyer, D.V.M., Diplomate, A.C.V.P. Date Second DraftCellProliferatiRoenport Covance Study Number 6329-183 V. QUALITY ASSURANCE STATEMENT -n iff All I PathologyAssociateslntemational A Company ofScienceApplicatiolnnstemationCaolrporation ANEMOW," -O~Conpeny CellProliferatRieopnort 104-Week Dietary Chronic Toxicityand Carcinogenicity Study With PerfluorooctaneSulfouicAcid Potassium Salt(PFOS; T-6295) in Rats Covance StudyNo.:6329-183 QUALITY ASSURANCE STATEMEENT This cellproliferatipornojecthas been inspecteadnd auditedby the PAI Quality AssuranceUnit(QAU) asrequirebdy theGood LabomtoryPractic(eGLP) regulations promulgatedby theU. S.Enviro=entalProtectioAngency (EPA-TSCA). The cell proliferatiroenportisan accuratreeflectiofntherecordeddata.The followingtableisa recordoftheftispections/aupdeirtfsormedand reportebdy theQAU. Date of Inspection Phase Inspected Date FindingsReport to PAI Management/ ProjectManaaer/Patholoeis 08/18/98 10/07/98 10/07/98 05/10/99 08/20,24/99 Labeling(100% slidelblomcaktch) StudyData and SupportingDocumentation DraftCellProliferatiRoenport Stainingand Covershpping Second Dmft CeR ProliferatiRoenport 08/18/98 10/08/98 10/08/98 05/10/99 08/24/99 2,-o@, Karen E. Butler QualityAssuranceSpecialist D'ate 4 15Worman's MillCourt,SuiteIa Frede6ck,Maryland21701 (3016)63-1644 (301)663-8994FAX Second DraftCellProliferatiRoenport Covance Study Number 6329-183 VI. APPENDIX Covance Study Number 6329-183 IndividualAnimal Findinfa3s 104-Week Dietary Chronic Toxicityand CarcinogenicityStudy with PerfluorooctaneSulfonicAcid Potassium Salt (PFOS;T-3295) in Rats HistopathologicFindings after 4 Weeks on Study Tissue: Liver Animal --Number C92506 F C92524 C92526 C9252FC92546 C92574 C92575 C92610 C92613 C92618 C92646 C92650 C92652 C92668 C92678 C92715 Sex I - m m M M m m m m m m m m m m m m C92718- C92731 m C92734 m C92744 m C92752 M C92759 M C92779 m C92793 m ,F-C9-2718 m Dose Group I 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (ConFol)- 2-0.5p m PFOS (Low) 2-0.5ppm PFOS (LOW) 2-0.5ppm PFOS (LFw)- 2-0.5ppm PFOS (Low) 2-0.5ppm PFOS (LOW) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (MFd)- 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid)- 3-2.0ppm PFOS (Mid) 4-5. ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-hi-h) 4-5.0ppm PFOS (Mid-high) 5-20.0ppm PFOS (Hiah) 5-20.0ppm PFOS (Hi.ah) 5-20.0ppm PFOS (High) 5-20.0ppm PFOS (HTgh)- 5-20.0ppm PFOS (HFgh)- Histologic Find--ings Liver No SicrnificaFnitndings No SignificanFtindings No SignificanFtindings No SionificanFtindings No SignTfica-nFt-indings No SignificanFtindinaas No SignificanFtindings No SignificanFtindings No SignificanFtindincs No Si.-nificaFnitndings No SignificanFtindings No SignificanFtindincs No SignificanFtindings No SignificanFtindinas No SignificanFtindings No SignificanFtindings No SignificanFtindinas No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindinas Page VI- I Covance Study Numbcr 6329-183 IndividualAnimal Findings 104-Week Dietary Chronic Toxicityand CarcinogenicityStudy with PerfluorooctaneSulfonicAcid Potassium Salt (PFOS;T-3295) in Rats HistopathologicFindings after4 Weeks on Study Tissue: Liver Animal Number C92861 TSex F C92864 F C92914 F C92919 F .C92926 F C92941 F C92945 F C92950 F C92969 F C92982 F C92996 F C93006 F C93009-- F C93031 F C93045 F C93058 F C93067 F C93070 F C93075 F C93084 F C93114 F C9312T- F C93142 F F C93159 F Dose Group T 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 2-0.5ppm PFOS (Low) 2-0.5ppm PFOS (Low) 2-0.5ppm PFOS (Low) 2-0.5ppm PFOS (LOW) 2-0.5ppm PFOS (LOW) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid) 4-5.0ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-hi,-,h) 4-5.0ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-high) 5-20.0ppm PFOS (Hicrh) 5-20.0ppm PFOS (High) 5-20.0ppm PFOS (High) 5-20.0ppm PFOS (High) 5-20.0ppm PFOS (Hi-h) HistologicFindings Liver No Sio:,,nificFaintdings No SignificanFtindings No Siiill'-flcant-Findings No SignificanFtindincs No SignificanFtindings No SignificanFtindinas No SignificanFtindings No SianificanFtindings No SignificanFtindings No SignificanFtindinas No SignificanFtindings No SignificanFtindinas No SicrnificaFnitndings No SignificanFtindings No SianificanFtindings No SignificanFtindings No SignificanFtindings No Si,,.naificanFtindings No SignificanFtindincs No SianificanFtindincs No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings Pace VI-2 * Covance Study Number 6329-183 IndividualAnimal Findings 104-Week Dietary Chronic Toxicityand CarcinogenicityStudy with PerfluorooctaneSulfonicAcid Potassium Salt (PFOS;T-3295) in Rats HistopathologicFindings after 14 Weeks on Study Tissue: Liver Animal Number -C92509 C925 11 C92521 C92528 C92532 C92593 C92600 C92616 C92621 C92627 C92640 C92645 C92662 C92676 C92684 C92713 C92714 C92719 C92722 C92728 C92765 C92777 C92789 C92799 C9-2112 SETT Mm m m m m m m m m m m m m m m m m m m m M m m m Dose Group 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 1-0ppm PFOS (Control) 2-0.5ppm PFOS (LOW) 2-0.5ppm PFOS (Low) 2-0.5ppm PFOS (Low) 2-0.5ppm PFOS (LOW) 2-0.5ppm PFOS (Low) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Nfid) 3-2.0ppm PFOS (Mid) 3-2.0ppm PFOS (Mid) 4-5.0ppm PFOS (Mid-high) 4-5.0 ppm PFOS (Nfid-high) 4-5.0ppm PFOS (Mid-high) 4-5.0ppm PFOS (Mid-high) 4-5.0 ppm PFOS (Mid-high) 5-20.0ppm PFOS (High) 5-20.0ppm PFOS (High) 5-20.0ppm PFOS (HighT5-20.0ppm PFOS (High) 5-20.0ppm PFOS (High) Histologic Findings Liver No SignificanFtindings No SignificanFtindings No Si,-,nificFainntdings No SiomificanFtindings No SignificanFtindings No SignificanFtindings No SianificaFnitnTingsNo Sic,,nificaFnitndings No SignificanFtindings No SignificanFtindings No SionificanFtindings No SignificanFtindings No SignificanFtinrm.-SNo SignificanFtindings No SignificanFtindinas SignificanFtindin(ys No SignificanFtindings No SignificanFtindings No SignificanFtiiFdin.-s- No Si,-,nificFainntdings No Si.,-nificanFtindings No SignificanFtindings No SianificanFtindings No SignificanFtindings No SignificanFtindings Page VI-3 Covance Study Number 6329-183 IndividualAnimal Findings 104-Week Dietary Chronic Toxicityand CarcinogenicityStudy with Pernuorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats HistopathologicFindings after 14 Weeks on Study Tissue: Liver Animal Number C92880 Sex D os-e 6 r-uo-p -fifstologicFindings Liver F 1-0ppm PFOS (Control) No Sian!Ficantkindings C92887 F 1-0ppm PFOS (Control) No Significant-Fi-nFi@-nis C92899- F I--op-p-mPFOS (Control) No SignificanFtindings C92903 F 1-0ppm PFOS (Control) No SignificanFtindings C95905 F 1-0ppm P.FOS (Control) No SignificanFtFnding-s C92944 F 2-0.5ppm PFOS (Low) No SignificanFtindings C92962 2-0.5ppm PFOS (Low) No SignificanFtindings C92967 F 2-0.5ppm PFOS (Low) No SignificanFtiWdings- C92978 F 2-0.5ppm PFOS (Low) No SignificanFtindings C92987 F 2-0.5ppm PFOS (Low) No SignificanFtinTin.-s- C92993 F 3-2.0ppm PFOS (Nfid) No SignificanFtindings C93000 F 3-2.0ppm PFOS (Mid) No Si,.,,anificaFnitndings C93018 F 3-2.0ppm PFOS (infid) No Sir.a,nificaFnitndings C93023 F 3-2.0ppm PFOS (NEd) No Si,,anificaFnitndine.,s C93035 F 3-2.0ppm PFOS (Mid) No Si,-,nificFainntdincs C93051 C93064 C93071 C93085 C93109 C93111 F 4-5.0ppm PFOS No Significant (Mid-high) F 4-5.0ppm PFOS No SignificanFtindings (Mid-high) F 4-5.0ppm PFOS No SignificanFtindings (Mid-high) F 4-5.0ppm PFOS No SicrnificaFnitndings (Mid-high) F 4-5.0ppm PFOS No SignificanFtindings (Mid-high) F 5-20.0ppm PFOS (High) No SicmificanFtindings C93127 F 5-20.0ppm PFOS (High) N-o-'qignificaFnitndings C93143 F 5-20.0ppm PFOS (High) No SignificanFtindings C93155 F 5-20.0ppm PFOS (High) No SignificanFtindings C93169 F 5-20.0ppm PFOS (High) No SignificanFtinUin-gzs' Page VI-4 Covance Study Number 6329.183 IndividualAnimal Findings 104-Week Dietary Chronic Toxicityand CarcinogenicityStudy with PerfluorooctaneSulfonicAcid Potassium Salt (PFOS;T-3295) in Rats HistopathologicFindings after 52 Weeks on Study Tissue: Liver Animal Sex Number C92562 M C92512 M C92534 m C92504 C92570 m C9287V-- F CT2-PT- F C92923 F C9293T---F- C92889 F C92795 M C92817- M C92778 M C92796 M C93171 F C93112 F C93120 F C93154 r C93163 F Dose Group I 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 1-0ppm P SE (Control) 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 1-0ppm PFOSE (Control) 5-20.0ppm PFOSE .(High) 5-20.0ppm PFOSE (High) 5-20.0ppm PFOSE (High) 5-20.0ppm PFOSE (High) 5-20.0ppm PFOSE (High) 5-20.0ppm PFOSE (High) 5-20.0ppm PFOSE (High) j-20.0ppm PFOSE (High) 5-20-0Fp-mP-FOSE (High) istologicFindings Liver No SignificanFtindings No SianificantFindi-nc-,,s No SignificanFtindings No SignificantFFnding-s No S i,&,nific-aFnitndings No SignificanFtindings No SignificanFtinji@n-gsNo SignificanFtindings No SignificanFtindings No SignificanFtiiTdincrsNo SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No SignificanFtindings No Si.-,nificFainntdings No SignificanFtindings No SignificanFtindings No SignificanFtindings Page VI-5