Document vBKOp09zM3xzdvNXYyaV4dJy8

SREECSEEIVCEBDI.C qmFEp22 MN FINAL REPORT PROTOCOL 418-009 PERINCAOTMABLI/NPEODSTONRAATLAL(GRAEVPARGOE)DUFCERTTIIOLNITTY,OXDIECVITEYLOSPTMUEDNYTOAFL NA-NEDtFOSE IN RATS. SPONSOR'S STUDY NUMBER: 6316.5 FINAL REPORT DATE: 30 JUNE 1999 000002 PROTOCOL 418-009 PERINCAOTMABLI/NPEODSTONRAATLAL(GRAEVPARGOED)UFCETRTIIOLNITTYO,XDIECVITEYLOSPTMUEDNYTOAFLNA-NEDtFOSE INRATS SPONSOR'S STUDY NUMBER: 6316.5 TABLE OF CONTENTS SUBJECT PAGE I. SUMMARY AND CONCLUSION 1 A. Methods 1 B. Results 5 C. Conclusion 10 Il. DESCRIPTION OF TEST PROCEDURES 11 A. Conduct of Study 1 A. Sponsor 1 A2. Testing Facility 11 * A3. Study Number 1-1 Ad. Sponsor's Study Number 11 AS. Purpose of the Study 1 AS. Study Design [=] ii 000003 SUBJECT A7. Regulatory Compliance AB. Ownership of the Study AS. Study Monitor A10. Alternate Study Monitor A1. Study Director A12. Technical Performance A.13. Report Preparation A14. Report Review A.15. Date Protocol Signed A.16. Dates of Technical Performance A17. Records Maintained B. Test Article Information B.1. Description B2. Lot Number B.3. Date Received and Storage Conditions B.4. Special Handling Instructions BS. Analysis of Purity C. Vehicle Information C.1. Description C2. Lot Number C3. Dates Received and Storage Conditions C4. Special Handling Instructions i PAGE 2 2 1-2 2 2 2 2 3 3 "3 14 4 4 14 4 5 15 15 5 5 Is Is 000004 SUBJECT C5. Analysis of Purity D. Test Article Preparation D.1. Sample Information D.2. Analytical Results E. Test System E.1. Species E2. Strain E.3. Supplier (Source) Ed. Sex E.5. Rationale for Test System EB. Test System Data E.7. Method of Randomization E.8. F. System of Identification Husbandry F.1. Research Facility Registration F.2. Study Rooms F.3. Housing F.4. Lighting F.5. Sanitization F6. Feed F.7. Feed Analysis F.8. Water iv PAGE 1-5 11-6 1-6 1-6 1-6 1-6 7 7 7 7 n-7 7 1-8 9 1-9 n-9 1-9 I-10 1-10 I-10 I-10 1-10 000008 SuBJECT PAGE F.9. Water Analysis 1-10 F.10. Nesting Material 1-11 F.11. Bedding Analysis I-11 G. Methods I-11 G.1. Dosage Administration 1-11 G2. Assigned Rat Numbers 112 G.3. Rationale for Dosage Selection 112 G.4. Route of Administration I-12 G5. Rationale for Route of Administration I-12 G6. Frequency of Administration 112 G7. Length of Study 1-13 G8. Method of Study Performance 1-13 G.9. Gross Necropsy I-18 G.10 Statistical Analyses 1-21 Il. RESULTS - Fo GENERATION MALE RATS 1 A. Mortality and Clinical Observations 1 B. Body Weights and Body Weight Changes 1 . Absolute Values (g/day) and Relative (g/kg/day) Feed Consumption 1 D. Mating and Fertiity 2 E. Necropsy 2 F. OTerrgmainnaWleiBgohdtytWoeTiegrhmtisnaalndBoOdrygaWneiWgehitghts and Ratios (%) of 1-2 v 000006 SUBJECT PAGE IV. RESULTS - Fo F1 GENERATION GENERATION FEMALE LITTERS RATS/ vo A. Mortality and Clinical Observations vo AA. Mortality Iv-1 A2. Clinical Observations vA B. Body Weights [21 B.1. Precohabitation v-1 B2. Gestation v-2 B3. Lactation v-2 C. Absolute Values (g/day) and Relative (g/kg/day) Feed Consumption v3 C1. Precohabitation v3 C2. Gestation v-3 C3. Lactation v3 D. Estrous Cycling, Mating and Fertiity v4 E. Necropsy Observations v4 F. Caesarean-Sectioning and Litter Observations v4 G. Natural Delivery and Litter Observations vs H. Pup Clinical and Necropsy Observations v6 I. Reflex and Physical Development [2 11. Surface Righting 7 12. Pinna Unfolding v7 13. Eye Opening v7 vi 000007 SUBJECT 1.4, Acoustic Startle 15. Air Righting PAGE v-8 v-8 1.6. Pupil Constriction v-8 V. RESULTS - F1 GENERATION MALE AND FEMALE RATS V-1 A F1 Generation Male Rats V-1 A.1. Mortality and Clinical Observations V-1 A.2. Body Weights and Body Weight Changes V-1 A.3. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values V-1 A4. Sexual Maturation V-2 AS. Passive Avoidance Performance V-2 A6. Watermaze Performance v-2 A.7. Mating and Fertility v-2 A.8. Necropsy Observations Vv-3 A.9. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight Vv-3 B. F1 Generation Female Rats V-3 B.1. Mortality and Clinical Observations V-3 B.2. Maternal Body Weights and Body WeightChanges V4 B.3. Matemal Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values V-5 Bd. Sexual Maturation V-6 B.S. Passive Avoidance Performance V-6 B.6. Watermaze Performance V7 vit 000008 SuBJECT PAGE B.7. Mating and Fertility v7 B.8. Necropsy Observations v7 B.9. Natural Delivery and Litter Observations v7 B.10. Pup Clinical and Necropsy Observations v-8 REFERENCES vo APPENDIX A - REPORT FIGURES Figure 1. Body Weights -- Fo Generation Male Rats A Figure 2. Body Weights ~ Fo Generation Female Rats A2 Figure 3. Body Weights ~ F1 Generation Male Rats A3 Figure 4. Body Weights ~ F1 Generation Female Rats Ad APPENDIX B - REPORT TABLES ~ Fo GENERATION MALE RATS Table B1. Clinical Observations - Summary - Fo Generation Male Rats B-1 Table B2. Body Weights - Summary - Fo Generation Male Rats B-2 Table B3. Body Male Weight Rats Changes - Summary - Fo Generation B3 Table B4. Absolute Feed Fo Generation Consumption Male Rats Values (g/day) - Summary -- B4 Table BS. Relative Feed Fo Generation Consumption Male Rats Values (g/kg/day) - Summary -- BS Table BS. Mating and Fertility - Summary - Fo Generation Male Rats ~~ B-6 Table B7. Necropsy Rats Observations - Summary - Fo Generation Male B7 Table B3. Terminal Body Fo Generation Weights and Male Rats Organ Weights - Summary -- 88 hl 000009 SUBJECT PAGE Table BS. Ratios (%) Summary - oFfoOGregnaenraWteiiognhtMatlo eTeRramtisnal Body Weight Bo Table B10. Clinical Observations - Individual Data - FoGeneration Male Rats B-10 Table B11. Body Weights - Individual Data - Fo Generation Male Rats B-18 Table B12. Feed Consumption Values - Individual Data -- Fo Generation Male Rats B-23 Table B13. Mating and Fertility - Individual Data -- Fo Generation Male Rats B-28 Table B14. Necropsy Observations - Individual Data - FoGeneration Male Rats B-37 Table B15. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight -- Fo Generation Male Rats B-43 APPENDIX C ~ REPORT TABLES ~ Fo GENERATION FEMALE RATS Table C1 Clinical Observations - Summary - Fo Generation Female Rats C1 Table C2. Body Weights - Precohabitation - Summary- Fo Generation Female Rats c4 Table C3. Body Weight Changes - Precohabitation - `Summary -- Fo Generation Female Rats c-5 Table C4. Maternal Body Weights - Gestation - Summary - Fo Generation Female Rats C6 Table C5. FMaoteGrennaelraBtoiodny WFeeimgahlte CRhaatsnges - Gestation - Summary - cs Table C6. Maternal Body Weights - Lactation - Summary -- Fo Generation Female Rats c9 Table C7. Matemal Body Weight Changes - Lactation - Summary -- Fo Generation Female Rats c-10 000010 SUBJECT Table C8. PAGE APbrsecoolhuatbeitFaeteidonC-onSsuummmpatriyon- Values (g/day) Fo Generation Female Rats ~~ C-11 Table C9. PRreelcatoihvaebiFteaetidonCo-nSsuummpmtairoyn Values (glkg/day) - Fo Generation Female Rats C-12 Table C10. GMeastteamtaiolnA-bsSoulmumtearFyee-dFoCoGnesnuemrpattiioonn FVealmuaelse(Rga/tdsay) -- C13 Table C11. GMeastteartniaolnR-elSautmivmeaFreye-dFCoonGseunemrpattiioonnVFaelmuaelse(gR/atksg./day) -- C14 Table C12. Matemal Lactation A-bSsuolmumtaerFyee- dFoCoGnenseurmapttiioonn Values Female (g/day) Rats -- C15 Table C13. Matemal Lactation R-eSlautmimvearFyee-dFCooGnesnuemrpattiioonn Values Female (g/kg/day) Rats -- C16 Table C14. FEsotrGoeunserCaytciloinngF,eMmaatliengRaatnsd Fertility - Summar--y C17 Table C15. Necropsy Observations Female Rats - Summary - Fo Generation C19 Table C16. SCauemsmaarerayn--SFeoctGieonneirnagtiaonndFLeitmtaerleObRsaetrsvations -- c20 Table C17. Natural Female Delivery Rats Observations - Summary - Fo Generation c21 Table C18. Litter Observations (Naturally F1 Generation Litters Delivered Pups) - Summary -- c22 Table C19. Clinical Observations from Summary - F1 Generation Birth Pups to Day 21 Postpartum -- c2s Table C20. FR1eflGeexnearnadtiPohnysLiicttaelrsDevelopment - Summary -- C26 Table C21. Necropsy Observations - Summary - F1 Generation Pups C-33 Table C22. Clinical Female Observations Rats - Individual Data - Fo Generation C34 x 000011 SUBJECT Table C23. Body Weights Fo Generation - Precohabitation Female Rats - Individual Data ~ PAGE cas Table C24. MDaattear-naFloBGoednyerWaetiigohntsFe-mParleesuRmatesd Gestation - Individual C49 Table C25. Maternal Body Weights - Lactation - Individual Data -- Fo Generation Female Rats C-59 Table C26. FDaetead ~CoFnosuGemnpetriaotnioVnalFueemsal- ePrReactoshabitation - Individual C64 Table C27. Matemal Feed Consumption Values - Presumed Gestation - Individual Data - Fo Generation Female Rats C-69 Table C28. IMnadtievrinduaallFDeaetda C-oFnosuGmepnetriaotnioVnalFueemsa-lLeacRtaattsion -- C89 Table C29. EDsattrao-usFCoycGleinnegraatnidonDaFyesmailn eCoRhaatbsitation - Individual C94 Table C30. Necropsy Observations - Individual Data - FoGeneration Female Rats Cc-99 Table C31. Caesarean-Sectioning Observations - Individual Data -- Fo Generation Female Rats Table C32. Table C33. Litter Observations (Caesarean-Delivered Embryos) -- Individual Data - Fo Generation Female Rats Embryonal Vital Status - Individual Data - F1 Generation Litters/Embryos c-107 Cc-109 c-111 Table C34. Table C35. Natural Delivery, Implantation Sites, and Pup Viability and Sex - Individual Data ~ Fo Generation Female Rats/ F1 Generation Litters PPousptpBaordtyumWe-iIgnhdtiLviitdtuealr ADvaetraa-ge1s GfernoemrBaitritohntoLiDttaeyrs21 C-116 C21 Table C36. Pup Body Weights from Birth to Day 21 Postpartum -- Individual Data - F1 Generation Pups C-126 xi 000012 SUBJECT PAGE Table C37. PPousptpViatratluSmta-tIunsdiavnidduaSleDxaftraom- FB1irtGhentoerDaatyio2n1Pups C-156 Table C38. Clinical Observations from Birth Individual Data - F1 Generation to Day Pups. 21 Postpartum -- C161 Table C39. Surface Righting - Individual Data - F1 Generation Litters C-164 Table C40. Pinna Unfolding - Individual Data - F1 Generation Litters ~~ C-177 Table C41. Eye Opening - Individual Data - F1 Generation Litters c-187 Table C42. Acoustic Startle - Individual Data - F1 Generation Litters C-197 Table C43. Air Righting - Individual Data - F1 Generation Litters. c-207 Table C44. Pupil Constriction - Individual Data - F1 Generation Litters C-217 Table C45. Necropsy Pups Observations - Individual Data - F1 Generation c-227 APPENDIXD -- REPORT TABLES -- F1 GENERATION MALE RATS Table D1. Clinical Observations - Summary - F1 Generation Male Rats D-1 Table D2. Body Weights - Summary - F1 Generation Male Rats D2 Table D3. Body Weight Changes - Summary - F1 Generation Male Rats D-3 Table D4. Absolute Feed F1 Generation Consumption Male Rats Values (g/day) - Summary -- D4 Table DS. Relative Feed F1 Generation Consumption Male Rats Values (g/kg/day) - Summar--y D5 Table D6. Sexual Maturation - Summary - F1 Generation Male Rats D6 Table D7. Passive Avoidance Male Rats Performance - Summary - F1 Generation 07 Table D8. Watermaze Rats Performance - Summary - F1 Generation Male D8 xii 000013 SUBJECT Table D9. PAGE Mating and Fertility - Summary - F1 Generation Male Rats D-9 Table D10. Necropsy Observations- Summary - F1 Generation Male Rats D-10 Table D11. Terminal Body Weights and Organ Weights - `Summary -- F1 Generation Male Rats D-11 Table D12. Ratios (%) of Organ Weight to Terminal Body Weight -- Summary - F1 Generation Male Rats D-12 Table D13. Clinical Observations - Individual Data - F1 Generation Male Rats D-13 Table D14. Body Weights- Individual Data - F1 Generation Male Rats D-16 Table D15. Feed Consumption Values - Individual Data -- F1 Generation Male Rats D-22 Table D16. Sexual Maturation - Individual Data - F1 Generation Male Rats D-25 Table D17. Passive Avoidance Performance - Individual Data -- F1 Generation Male Rats D-26 Table D18. Watermaze Performance - Individual Data - F1Generation Male Rats D-29 Table D19. Mating and Fertility - Individual Data - F1 Generation Male Rats D-32 Table D20. Necropsy Observations - Individual Data - F1 Generation Male Rats D-35 Table D21. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight -- Individual Data - F1 Generation Male Rats. D-38 APPENDIX E -- REPORT TABLES ~ F1 GENERATION FEMALE RATS Table E1. Clinical Observations - Summary - F1 Generation Female Rats E-1 i 000014 BJECT PAGE Table E2. Body Weights - Precohabitation - Summary -- F1 Generation Female Rats E4 Table 3. Body Weight Changes F1 Generation Female - Precohabitation Rats - Summary -- ES Table E4. Matemal Body F1 Generation Weights - Gestation Female Rats - Summary -- E6 Table ES. Maternal Body F1 Generation Weight Female Changes Rats - Gestation - Summary =] Table E6. Matemal Body F1 Generation Weights - Lactation Female Rats - Summary -- E9 Table E7. Maternal Body F1 Generation Weight Female Changes Rats - Lactation - Summary -- E-10 Table E8. Absolute Feed Consumption Values (g/day) ~ Precohabitation - Summary - F1 Generation Female Rats ~~ E-11 Table 9. Relative Feed Consumption Values (g/kg/day) Prechabitation - Summary - F1 Generation Female Rats E12 Table E10. MGeastteartniaolnA-bsSoulmumtearFyee-dF1CoGnesnuemrpattiioonn FVealmuaelse (Rga/tdsay) -- E13 Table E11. Maternal Relative Feed Gestation - Summary - Consumption F1 Generation Values (g/kg/day) Female Rats -- E14 Table E12. Maternal Lactation Absolute Feed Consumption - Summary - F1 Generation Values Female (g/day) Rats E15 Table E13. Maternal Lactation Relative Feed Consumption - Summary - F1 Generation Values Female (g/kg/day) Rats. -- E-16 Table E14. Sexual Maturation - Summary - F1 Generation Female Rats E-17 Table E15. Passive Avoidance Performance - Summary - F1 Generation Female Rats E18 Table E16. Watermaze Performance Female Rats - Summary - F1 Generation E19 xiv 000015 SUBJECT PAGE Table E17. Mating Rats and Fer-tSiumlmatryy- F1 Generation Female E20 Table E18. Necropsy Observations Rats - Summar-y F1 Generation Female E21 Table E19. Natural Female Delivery Rats Observations - Summary - F1 Generation E22 Table E20. Litter Observations (Naturally F2 Generation Litters Delivered Pups) - Summary E23 Table E21. Clinical Observations from Summary - F2 Generation Birth Pups to Day 21 Postpartum -- E26 Table E22. Necropsy Observations - Summary - F2 Generation Pups E-27 Table E23. Clinical Female Observations Rats - Individual Data - F1 Generation E28 Table E24. Body Weights F1 Generation - Precohabitation Female Rats - Individual Data -- E32 Table E25. IMnadtievimdaulalBDoadtya W-eFi1ghGtesne-rPartieosnumFeedmaGleestRaattison -- E35 Table E26. Matemal Body F1 Generation Weights - Lactation Female Rats. - Individual Data -- E41 Table E27. Feed Data ~CoFn1sGuemnpetriaotnioVnalFueemsa-lePrReactoshabitation - Individual E44 Table E28. Matemal Individual Feed Data C-oFn1suGmenpetriaotnioVnalFueemsa-lPerReastusmed Gestation - E47 Table E29. Maternal Data - F1 Feed Consumption Values Generation Female Rats - Lactation - Individual E50 Table E30. Sexual Rats Maturation - Individual Data - F1 Generation Female E53 Table E31. FP1asGseinveerAavtoiiodnaFnecmeaPleerRfaotrsmance - Individual Data -- E54 w 000016 SUBJECT Table E32. Watermaze Performance - Individual Data - F1 Generation Female Rats PAGE E-57 Table E33. Days In Cohabitation - Individual Data - F1Generation Female Rats E-60 Table E34. Necropsy Observations- Individual Data - F1Generation Female Rats E-61 Table E35. Natural Delivery, Implantation Sites, and Pup Viability and Sex - Individual Data -- F1 Generation Female Rats/ F2 Generation Litters E-64 Table E36. Pup Body Weight Litter Averages from Birth to Day 21 Postpartum- Individual Data - F2 Generation Litters E-67 Table E37. Pup Body Weights from Birth to Day 21 Postpartum -- Individual Data - F2 Generation Litters E-70 Table E38. Pup Vital Status and Sex from Birth to Day 21 Postpartum Individual Data - F2 Generation Pups. E-88 Table E39. Clinical Observations from Birth to Day 21 Postpartum -- Individual Data - F2 Generation Pups E-91 Table E40. Necropsy Observations - Individual Data - F2 Generation Pups E-92 APPENDIX F - PROTOCOL AND AMENDMENTS F-1to F-55 APPENDIX G - DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY G-1t0G-2 APPENDIX H- TEMPERATURE AND RELATIVE HUMIDITY REPORTS H-1to H-12 APPENDIX |- STATEMENT OF THE STUDY DIRECTOR 1 APPENDIX J - QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT J-1t0J-9 i 000017 TITLE: 418-009:PAGE I-1 ACNODMBPIENREIDNAOTRAALL/P(OGSATVNAAGTEA)LFERRETPIRLIOTDYU,CDTEIVOENLOTOPXMIECNITTAYL STUDY OF N-E{FOSE IN RATS. ARGUS RESEARCH LABORATORIES, PROTOCOL NUMBER: 418-009 INC. SPONSOR'S STUDY NUMBER: 6316.5 I. SUMMARY AND CONCLUSION A. Methods Text Figure 1 provides a schematicof the study design. AA. Fo Generation Rats/F1 Generation Litters DOanwelehyu)ndrratesdwseerveenatsy-sfiigvneedmatloefiavne ddofsemaagleegCroru:pCsD(GBroRuVpAsF| /tPhlruosugh(VS)p,ra3g5uera-ts per sex per N-EtFOSE", dosage group. or the vehicle, The rats were 2.0% Tween administered the test article, 80, orally (via gavage). The male rats were dosed through the once daily day before beginning sacrifice. 28 days before cohabitation The female rats were dosed and continuing once daily absesgiingnniendgt2o 8Cadeasyasrebaenf-osreectcioohnaibnitga)t,ioDnGan2d4 continuing through DG 9 (rats (rats assigned to natural delivery that did not deliver a was 5 mL/kg. litter), or DL20 (rats that delivered a liter). The dosage volume AsltludFyoagnednefroarteifofnecrtastsofwethree toebsstearrtviecdleftowrivcieabdilaiitlyyadturleiansgttthweicdeosdaaigley dpuerriinogd the wbeeifgohretsaannddapfpereodxciomnatseulmyptoinoen hvoaulruepsosftordomsaalgee)raatsndweornetrheecdoardyesdacwreifeikcledy. duBroindgy rtheecodrodseadgweeepekrliyodtoacnodhaabtitsaactriiofni,ce.dailByodduyriwnegigthhtesgfeosrtatthieofnepmearlieodr,atosnwDeLrse 1, 4, 7,10 and 14 consumption (vraaltuseasswseigrneerdetcoorndaetudrawleedeklliyvetroyc)oahnabditaattsiaocnr,ifdiaciel.y Feed during the gestation delivery). period and on DLs 1,4, 7, 10 and 14 (rats assigned to natural a. aDreetapirloevdiddeesdcriinptthieonaspporfoapllripartoecesedcutrieosnsusoefdthiins trheepocrtonadnudctinofAPthPisEsNtDuIdyX F b. (PROTOCOL N-EIFOSE is AND AMENDMENTS), a metabolite of PFOS, the test article administered in Argus . RDeGsiesarucshedLaabsoraantoarbiberse,viInact.i,onPrfootrodcoaly 418-008. of (presumed) gestation). d. DLis used as an abbreviation for day of lactation or day postpartum. 000018 418-009:PAGE I-2 Tashseigfinrsetdtteon Cfaeemsaalreearant-ssepcetridoonsinaggeongrDoGup1w0i.thTahecornefmiarimneidngdafteemaoflemartaitsngwewreere opbesremirtvtaetdiotnosndauturrianlglypadretluirviteirolni,ttdeursr.atTihonesoef greasttsawtieorne, eivtaelrusaitzeedafnordcpliunpicavliability `atexbairmtih.nedMadtureimnagltbheeh2a1v-idoaroyfpotshtepdaartmusm was evaluated period. daily when the pups were Epoascthpalirttteurmwapesrieodv.aluPautpesd fionrevaicabhilliittyteartwleeraestctowuincteeedaocnhcdeadyaidlyu.rinPghytshieca2l1-sdiagyns ainndthoebpsueprsvewdernuerrsiencgorbdeehdavoinocrewdeariley rfeocr o2r1deddayosn pDoLstspa1 r(tbuimrt.h),P4u,p7,bo1d4yawnedig2h1.ts aSnurdfaaicrerirgihgthitnigngrreefflleexx,wepriennmaonuniftoolrdeidngd,ueriynegotpheeni2n1g-,daaycopuossttipcasrttarutmleperreisopdonunsteil walal spuepvsaliunattheed loitntecreroeancDhLed21t.heOcrnitDerLio4n,faorttahbelespoefcrifaincdtoesmt.unPituspiwlacsonustsreidcttioon coufllralintdteorsmtounfiotsurwmaasleusaenddtfoosuerlfeectma2l5empaulpes,awnhde2r5e fpeosmsailbele.pupOsninDGLr2o1u,pas tI,abIIle and Il for continued evaluation. pFeorigoednearnadtinoencrmoaplseierda;tsgwreosrse lseasciroinfiscwederaefterretcaoimnpedl.etiTohneotfestthees,coehpaibdiitdaytmiiodnes, prostate weighed and and seminal vesicles retained. (with and without fluid) were excised, individually FDoGg1e0nearnadtinoencfreopmsaileed;raptsreagsnsiagnnceydsttaotCuasewsaarsecaonn-fsiercmteido.niOnvgawreierse asnacdrigfriocesds on olfecsoiornpsorwaerleutreeataiinneeda.chTohvearryatasnwderiempelaxnatmaitnioend sfiotrest,heanndumvibaebrleaanndddinsotnrviibuatbiloen embryos. Embryos were discarded after examination. Fo generation female rats and necropsied. Ovaries assigned and gross to natural delivery were lesions were retained. sacrificed on DL The number and 21 distribution of implantation sites were recorded. Asitresdchleitdtuerlsoedf sdaacrmisficaellaoftweerdctoompnalteutriaolnlyodfetlhiveecroahalbiitttear)tiaonndpoenrioDdL(2m1al(efsemraaltes that rraatt)swaelrleowceodllteocntaetdufrarlolmy tdheeliivnefreraiolritvteer)nablcoaovdasfarmopmlefsive(arpaptsropxeirmasteexlype4rmdLospaegre egxrcoiuspeda,ndwesihgihpepde,datnodthaesSapmopnlseosrefcotriopnha(rlamtaecraolkilnoebte)icwaansalfyrsoisz.en The and liver was shipped to the Sponsor for analysis. nPeucprsopnsioetds.eleTchteedstfoormcaocnhticnounetdenetvsal(umaitlikocnurodn)DwLer4ewceorlleecstacerdiffircoemd aallndculled pups from five of the largest the Sponsor for analysis. litters from Groups I, Il and Ii, frozen and shipped to Pups not selected for continued evaluation were sacrificed on DL 21. 000019 418-009:PAGE I-3 Tsehleecltievedrsfoorfpthhaermpaucposkifnreotmitchesalmitptleres ocfoltlheectfiiovnewdearmesexinciGsreod,uppsoo| ltehdropuegrhliItVter, frozen and shipped to dosage group (Group the Sponsor for analysis. V) did not have surviving Dams in the 15 pups on DL 21. mg/kg/day A2. F1Generation Rats/F2 Generation Li sOenlcyontdhege0n(eVreahtiicolne)b, e1caaunsde5omf gs/ekvge/rdeapyudpomsoargtealigtryoduuprsiwngerleacctaotnitoinn. ueTdheirnteo wtehree 150 male and female F1 generation rats in the through 111), 25 rats per sex per dosage group. three dosage groups (Groups | Far1tigcleeneorraaltliyo(ngmaavlageea)nbdegfienmnailnegroatnsDwLer2e2gainvdencoanptpirnoupirnigattehrdoousgahgethseodfatyhebetfesotre sacrifice. Beginning at each dosage 2g4roduapysweorfeagtee,stoendeinmaalpeasrsaitvaenadvooniedafnecmealpearraadtifgmr.om each litter in Female rats were were evaluated evaluated for for the the age age of of vaginal patency beginning on DL preputial separation beginning on 28. DL Male rats 34. On epvoasltupaatretdumindaaywa7t0e,r-ofnieledmaMl-emaraztea.ndOonnaepfpermoxailmeartaetlfyrDoLm e90a,chthleittreartwsewriethin each dosage group were assigned to cohabitation. pFe1rigoednearnadtinoencrmoaplseierda,tsawseprreevsiaocursifliycdedesacfrtiebrecdomfporletthieoFnoofgetnheercaothiaobnitmaatlieonrats. tAlhlatF1delgievneerreadtiloitntefrsemwaelreersaatcsrwifeirceedpoenrmDitLte2d1 taosnaptruervailoluysdleyldievesrcrliitbteerds.foArll dams Fo generation female rats. On DL 21, lesions. all F2 generation pups were sacrificed and examined for gross 000020 BESTCOPY AVAILABLE Err { = TEER =, F = alES =I g = Ex I ---- HE CL am TH a - ) B. Results 418-009:PAGE I-5 B.1. Fo Generation Male Ra No Fo generation attributable to the male rats died during this study. test article was impaired righting The only clinical observation reflex in the 15 mg/kg/day dosage group rats. Groups reduced administered body weight 5 mg/kg/day gains for the and higher dosages entire dosage period. of the The test article 10 and had c1o5nmsgulmkpgt/idoanyvdaoluseasgefogrrtohuepesnthiared significantly reduced treatment period. absolute feed Dosages of the test article as fertilty parameters evaluated. high as 15 mg/kg/day did not affect any mating and sNiegcnirfoipcasnytloybsienrcvraetaisoendsncuomnbseirdeorfedmarleelartaetds to in the the test article included a 15 mg/kg/day dosage group with small seminal small prostate. vesicles and one 15 mg/kg/day dosage group male rat with a The 5, 10 and body weights. 15 mg/kg/day The absolute dosage weights gorfotuhpeslehftadepsiidgindiyfmiciasn,tlsyemriendaulcevdestiecrlmeisnawlith uTihde,raatnidosporfostthaetewewiegrhet osfigtnhieficleafnttlayndrerdiugchtedteisntetsheto15temrmgifnkagl/dbaoydydwoesiagghetsgrwoeurpe. significantly increased in the 5 (left only), 10 and 15 mg/kg/day dosage groups. B.2. Fo Generation Female Rats/F1 Generation Litters No Fo generation N-EtFOSE. female rats died during this study as a result of treatment with Observations 15 mg/kg/day of localized alopecia were dosage groups during the significantly increased in the 5, gestation and lactation periods. 10 and Gsirgoniufpiscanatdlmyinriesdtuecreedd b5omdgy/wkegi/gdhatygaanidnshfiogrhtehredeonstairgeesproefcothhaebitetsattiaortnicpleerihoadd (aDnSds*151mtg0/2k9g)./daGyesdtoastaiogne bgordoyupwseiognhtDGgasin0stwoe7reansdigniniftihceant1l5y mrge/dkugc/eddayindtohsea1g0e group on DGs reduced in the 18 15 to 20. As a result, mg/kg/day dosage bgordoyupwefiorghtthegaeintnisrewegresetasitginoinfipcearnitloyd (DGs 0 fo 20). dosage Absolute group on body DG weights were 0 through 17, significantly reduced and in the 10 and 15 in the 5 mg/kg/day mg/kg/day dosage groups groups honadalslidgnaiyfsicoafnttlhyergeedsutcaetdiobnopdeyriwode.ighTthseon10DaLnd1. 15 mg/kg/day dosage Lactation body weights continued to be significantly reduced in the 10 mg/kg/day dosage group on a. DSis used as an abbreviation for day of study. 000022 418-009:PAGE 1-6 dDoLssa4g,e7,gr1o0upasndon14D. LsBo1dtyow4.eigThhtel1os5smogc/ckugr/rdeadyindothseag1e0 garnodup15wamsg/pkrge/cdlauyded finrobmodfyurwtheeirghetvaglauiantioonn DbLecsa1u4setoal2l1puinptshdeie1d0 bmegf/okrge/dDaLy5d.osAsaiggenigfriocuanptwianscrease possibly associated production. with reduced litter size and reduced milk demand and Absolute and relative 10and 15 mg/kg/day feed consumption dosage groups for vtahleueenstiwreerperescigonhiafbiictaanttliyonrepderuicoed.d in the The a1b0saolnudte15anmdg/rkelga/tdiaveyfdeoesdacgoengsruomupptsiocnonvtailnuueesdetaorlhyaivnetshiegngiefsitcaatnitloyn rpeerdiuocd.ed A5bmsgo/lkugte/dfaeyeddocsoangseumgprtoiuopnovnalDuGessw0ertoe7a.lsAobssioglnuitfiecafneteldy rceodnuscuemdptiinotnhevalues cDoGnsti1nu0e1d0 t1o2baensdig1n2iftiocan1t5l,yarnedduicnetdhein1t5hmeg1/0kgm/gd/akyg/ddoasyagdeosgargoeupgrtohurpouognhout tt0he20r).emaAibnsdoelruotfe tahendgreesltaattiiveonfepeedricoodn(sDumGpsti10ontova1l2u,e1s2wteore15s,ig1n5iftioca1n6tlaynrded1u8ced for 15 the entire gestation period mg/kg/day dosage groups. (DGs 0 to 20) in the 5 (absolute only), 10 and A5b(saoblsuotleutaenodnlrye)laatinvde f1e0emdgc/okngs/udmapytdioonsavgaelugersowupesrefosritgnhiefiecnatnitrleylraecdtautcieodn pienrtihode. SoingnDiLfisca7nttore1d0ucintitohnes5inmagb/skogl/udtaeyadnodsaregleatgivreoufpe,edancdonfsourmalplttiaobnulvaatleudesinotcercvuarlrseidn wtheere10remdgu/ckegd/dian ythdeosoangeleitgtreoruwp.as Absolute available and for relative feed evaluation in consumption values the 15 mg/kg/day dosage group on DLs from further evaluation 1 bteoc4a. usTeheall1p5umpgs/dkige/ddabyefdoorse aDgLe group 5. was precluded Dosages in the 15 roaftsthpeertedstosaratgicelegraosuhpigthhaatswe1r5emegv/aklgu/adtaeyd.did not affect estrous cycling All necropsy article. observations were considered unrelated to treatment with the test Setmabtirsytoicsalolcycsuirgrniefdicianntthreed1u5ctmigo/nksg/indathyedaovseargaegegsrofuorp iamtpClaaenstaarteiaonn-saencdtivoianbilneg on DG 10. differences There in the lwitetrereanvoerbaigoelosgifcoarllcyoripmoproartlauntteaororstnaotinsvtiiacabllley significant embryos at Caesarean-sectioning on DG 10. The duration of gestation was 15 mg/kg/day dosage groups, significantly reduced in the an observation associated 5, 10 and with preimplantation lsiotsessipnetrheda1m5 wmga/skgsi/gdnaifyicdaonstlaygeregdruoceudp,(trheesualtvienrgagineansuimgnbiefricaonftilmyprleadnutacteidonlitter size). 000023 418-009:PAGE I-7 tPhueptevsiatbialrtiitcylew.asResfilgenciftiicnagnttlhyeasfefeefcfteecdtsb,ytthheev1ia0bialintdy a1n5dmlga/cktgat/idoanyidndoiscaegsewserofe significantly reduced in the 10 and 15 mg/kg/day dosage groups. A dosage-dependent pattem of reduced group administered the test article. The 1pumpg/bkogd/ydawyeidgohstsagweasgreovuipdetnetndien deatco h 1h5avmeg/rkegd/udcaeyd dpouspabgoedgyrwoeuipgshthsadonsiDgnLif4ic(apntrley-arneddupcosetdcuplulipngb)o.dyThweei5g,ht1s0 oanndall weighing days (no pups survived after DL 4 in the 15 mg/kg/day dosage group). Clinical and necropsy potential reduction in observations maternal care associated with occurred in the reduced pup 5, 10 and 15 viability and mg/kg/day dosage groups. were not nursing Cilnitnihceal5,ob1s0eravnadti1o5nsmgi/nkclgu/ddeady 1, 7 and dosage 3 litters groups, with pups that respectively; the incidence litters with was cold significant in the 10 to touch pups was mg/kg/day dosage group. also significantly increased The number in the of d1e0amdg/ikngcl/uddaeyddsoigsnaigfiecagnrtouipn.creNaescersoipnstyheobnsuermvbaetrioonfspiunppsuwpisththnaot wmielrkeifnound stomach in the 10 and 15 mg/kg/day dosage group litters. wRietvherbsoidbylewedieglhatysocincurrefrleedx ianntdheph5ysaincdal1d0emvge/lkogp/mdeanytdtohastaagreeghrioguhplsy.coSrurerlfaatceed righting was delayed in development for pinna the 5, 10 unfolding and was 15 mg/kg/day delayed in the dosage groups. The time 5, 10 and 15 mg/kg/day of dosage groups (gnroou1p5s.mgE/kyge/odpaeyndionsgawgaesgdreoluapypedupisn the 5 were and 10 mg/kg/day dosage evaluated for this parameter). T10hemgt/ikmeg/odfadyedvoesloapgmeegnrtooufpsth(enaoc1ou5smtigc/ksgta/rdtaley rdeoflseaxgweagsroduelpapyuepdsinsutrhveiv5eadntdo be 10 tested for this reflex). The mg/kg/day dosage groups ability (no 15 to air right was delayed in mg/kg/day dosage group the 5 pups and survived to be tested for this reflex). B3. F1Generation Male Rats No deaths occurred in the were considered unrelated F1 to generation male the test article. rats. All clinical observations The day 1 1 paonsdtw5emagn/ikngg/(dsaiygndifoiscaangteingrtohuep5s weighed less than mg/kg/day dosage the control group) and group body on Wweeiigghhttggaaiinnsswienrtehere1 dauncded5imngt/hkegs/edgaryoduopssatgherogurgohuopustwtehreeposisgtnwiefiacnainntlgy preerdioudc.ed (fsoirgtnhifeicparnetcoahta5bimtga/tikogn/dpaeyrioondlya).ndAfbosrodlautye1bpoodsytwweeaignhitnsg wteorteersmiignnaitfiicoanntly reduced period. in the 5 mg/kg/day dosage group throughout the postweaning dosage Absolute and relative feed were significantly reduced consumption values in the in the 5 mg/kg/day dosage F1 generation group on days male 8 to rats 15 000024 418-009:PAGE I-8 tphoisstdwoesanaigneg.groTuhpeoanbsdoalyuste1fteoed8 cpoonstswuemapntiinogn.vaRleuleatwivaesfseiegdnicfiocnasnutlmyptrieodunced in values were also significantly reduced on days 361043 in the 5 mg/kg/day dosage group. 15 to 22, 22 to 29, 29 to 36 and TF1hegeanveerraatgieondamyaloef rpartespuitniatlhese5pmarga/tkigo/ndwayasdossigangieficgarnotulypdveallauey,edbuftortthheeincrease was approximately one day and not considered toxicologically important There were no term retention, blioonlgo-gtiecralmlyreitmepnotritonanotrdriefsfeproennsceesinihnibtihteiovnalinuetshefoFr1legaemnienrga,tisohnort- male rats, as performance evaluated paradigm. by performance in a passive avoidance or watermaze Dosages of the test article as fertility parameters evaluated high as 5 in the F1 mg/kg/day generation did not affect male rats. any mating and All necropsy observations in unrelated to the test article. the F1 generation male rats Terminal body weights were were considered significantly reduced in the the 5 mg/kg/day dosage terminal body weight group. in the 5 Tmhge/krgat/idoasyofdotsheagleeftgarnodupriwgehtretesstiigsniwfeiciagnhttlsy to `iwnecirgehatsseodf. thNeoesptiadtiisdtyimcaildleyss,igtneisftiecsa,ntsedimfifnearlenvceessicolcecsu(rwrietdh ianntdhewiatbhsooultuftleuid) and lpriods)taaten,d apnrdostthaeterawteiiogshotfstthoetehpeidteirdmyimniadlesb,odsyemwieniaglhtveosfictlheesF(1wigtehnaenrdatwiiotnhomuatle rats. B.4. F1 Generation Female Rats/F2 Generation Litters AolblseFr1vagteinoenrsatdiuorninfgemtahleeprreatcsohsaubrivtiavteidonu,ntgilesstcahteiodnulaenddslaacrcitfaitcieo.n Al clinical periods were considered unrelated to the test article. The day 1 1 and 5 mg/kg/day postweaning (the droedsuacgteiognrwoaupssswigeniigfihceadntlienssthteha5nmtgh/ekcgo/ndtaryoldgorsoaugpeon gthrroouupg)haonutd tbhoedyprweecoihgahbtigtaaitniosnwpeerreiodg.eneBraoldlyywreeidguhctedgaiinnsthweesreegsriogunpifsicantly r[deadyuc1edpoisntbwoetahnitnesgttaortpirceiceo-thraebaitteadtgiornouapnsdfdoaryth1e teont5i7repporsetcwoehaabniitnagti(osingnpiefriicoadnt awter5emsgilgkngif/idcaanytloynlyr)e)d.ucAebdsotlhurtoeugbhooduyt wtehiegphrtescoinhatbhieta5timogn/kdgo/sdaagyedpoesriaogd.e group Mraetdeurcneadlibn otdhye w1eainghdt5gamign/skgd/udrianygdtohseaggeestgartoiuopnspeornioddaywser0etsoi7gnioffigceasnttaltyion. oBnodDyGwsei1gh4t0ga17i.nsAibnstohleut5embgo/dkyg/wdeaiyghdtosswaegreegsriogunipfiwcearntelysirgendifuicceandtloyniDncGrsea0sed through 18 in the 1 and 5 mg/kg/day dosage groups. Maternal body weights 000025 418-009:PAGE I-9 r10e,ma1i4naenddsi2g1niafincdanitnlythreed1ucmegd/kign/tdhaeydmogs/akgge/dgaroyudpoosnagDeLsgr1o,u7p on DLs and 14, 1, 4, 7, pFreeecdohcaobnitsautmipotniopenrivoadlu(edsaywser1etosi5gn7ifpiocsanttwleyarneidnugc)eidn fboortthhgereonutpirseadministered gthreoutpeswtearrteicslie.gniRfeilcaanttilvyeifnecerdeacsoendsuomnpdtaiyosn 8vatloue1s5,in15thteo522mga/nkdg/2d2aytod2o9sage dpoosstawgeeangirnogu.psAwbesorleustiegnfiefeidcanctolnysruemdputcieodn dvuarliunegstihnetfhierst1waenedk5ofmgt/hkeggiedsataytion period. Absolute feed were also significantly creodnuscuemdptoinonDGvaslu7etsofo10r.thTeh5emrge/lkatgi/vdeafyeeddoscaognseugmrpotuipon gvraoluupe. onThDeGasbs1o4ltuote17mawtaersnasilgnfiefeidcacntolnysruemdputcieodn ivnatlhuee 5wamsg/skiggn/idfaicyandtolsyargeeduced for the entire consumption lactation in value was the 5 mg/kg/day dosage group. significantly reduced on DLs 4 to The relative feed 7 and 7 to 10 in the 5 mg/kg/day dosage group, as compared to the control group values. oDfovsaaggiensalopfattheentceystinartthieclFe'1asgehniegrhataison5 mg/kg/day did not affect female rats. There were the no average day biologically iinmhpiobrittiaonntidnitfhfeerFe1ncgeesnienrtahteiovnalfueemsalfeorrlatesa,miansge,vlaolnuga-tteedrmbyrepteerntfioornmaorncreesipnoanse. passive avoidance or watermaze performance paradigm. Dosages of the test article as fertiity parameters evaluated high as 5 in the F1 mg/kg/day generation did affect any female rats. mating and All necropsy observations in unrelated to the test article. the F1 generation female rats were considered Avisabiolcictuyrirnetdhein5thmeg/pkrge/vdioauysdgoesnaegraetigroonu,pt.heTrehewansumabteernodfendcaymfsorwirtehdustcieldlboprunp ipnucprseawsaesd siingtnhiifsicdaontslaygiencgrreoauspe.d,Aasndalstoheocncuumrbreerd oinf tphueppdreeavtihosuswgaesnesriagtniifoinc,antly pup body days 1,4 weights (pre and were significantly reduced postculling), 7, 14 and 21 in the 5 mg/day dosage group on No clinical or necropsy observations dosages of the test article as high as in 5 the F2 generation mg/kg/day. were attributable to 000026 418-009:PAGE I-10 C. Conclusion On the basis of these data, the Fo generation matemal and paternal noohbisgehrevradbloes-aegffeesctc-aluesveeld(rNeOdEucLt)ioonfsNi-nEb{oFdOySwEeiisgh1tmgga/ink,g/tdheay1(05amngd/k1g5/dmagy/kagn/dday dosages also caused reduced feed consumption values). TonhemaFtoingge,neferrattiiltoynorreepsrtordouucsticvyeclNinOgEoLcciusrgrreeda.teTrhtehaNnO1E5Lmgfo/rkgvi/adbailyi;tynaonedffgercotwsth irnedtuhcetiFo1nsgeinnepruatpiobnodoyffwsepirignhgtigsai1nms,g/tkhge/1d0aya(ntdhe155 mmgg//kkgg//ddaayy ddoossaaggeescacuasuesded preimplantation survival). loss and reductions in litter size, pup viability, growth and T1 hmeg/Fk1glgednaeyra(ttihoen1maantdem5amlga/nkdg/pdaatyedmoaslaNgOesELcaoufsNe-dErteFduOcStEioinsslienssbothdaynweight gain and feed consumption). The F1 mating generation reproductive or fertility occurred. The NOEL NOEL is a dosage of5 mg/kg/day; no for viability and growth in the effects on sFt2illgbeinrtehrsaatniodnroefdfuscptriionngsisin1 limtgte/rksgi/zed,aypu(pthveia5bimligt/y,kgg/rdoawythdoasnadgseurcvaivuasl)e.d A 2-In7; red. Christian, Ph.D. Fellow, ATS Date Executive Director of Research Ho S$gu Loe 39 -J77 Alan MPHoberman, Ph.D., DABT Date Director of Research Pe sn ond G. York, Ph5.0) 8T Date Associate Director of Resadich and Study Director 000027 418-009:PAGE II-1 I. DESCRIPTIONOF TEST ED! A. ConductofStudy: A. Sponsor: 3M Corporate Toxicology, Minnesota 55144-1000 3M Center, Building 220-2-02, St. Paul, A2. Testing Facility: Argus Research Laboratories, Pennsylvania 19044-1297 Inc., 05 Sheehy Drive, Building A, Horsham, A3. StudyNumber: 418-009 A4. Sponsor's Study Number: 6316.5 AS. Purpose of the Study: NTh-eEtpFuOrSpoEsteroefattmheisntstoufdCyrw:aCsDtoBteRstVAfoFr/tPolxiucseffmeacltes/adnidstfurebmaanlceesrartessubletfionrgefrom cwoahsabdietsaitigonnedantdo ecvoanltuiantuienIgCtHhrHoaurgmhomnaitsiendg,TrgiepsatratittieonGuainddelliancteatsitoan.gesThAisthsrtouudgyh Ftoubfaltthreanrsepporrotd,ucitmipvleantpartoicoens,sgaesntdatsihoonu,lpdardteutreictitone,ffleaccttsatoinonthaendesmtarotuesmacylcle, behavior in and female female rats, on the development of the rats, and permit detection of functional offspring of the treated effects (e.g., effects on male libido or epididymal `examinations sperm maturation) that may not of male rat reproductive organs. be detected by histological Because manifestations of effects induced during this period may be delayed continued through production of F2 liters. in the offspring, observations were AS. Study Design: A modification of the (FDA)? was used as requirementsof the U.S. Food the basis for study design. and Drug Administration 000028 418-009:PAGE 11-2 A.7. RegulatoryCompliance: The study was conducted in compliance with (GLP) regulations of the U.S. Food and Drug the Good Laboratory Practice Administration (FDA)?, the CJoampamnuensietyMi(nEisEtCry).of HTehaelrtehwaenrdeWneolfdaerveia(tiMoHnWs)froamntdhethGeLEPurroepguelaantiEocnsontohamtic dafefreicvteedd ftrhoemqtuhaeliitnysoprecitniteognrsitdyuorfintghethsetucdoyn.duQcutaloiftythAiss ssutruadnycaereUndiotcfuimnedinntges d and have been Management. provided to the Study Director and the Testing Facility A8. Ownershipof the Study: `The Sponsor owns the study. tissues are the property of the All raw data, Sponsor. analyses, reports and preserved AS. Study Monitor: Marvin T. Case, D.V.M., Ph.D. A10. Alternate Study Monitor: AndreMw. Seacat, Ph.D. A11. Study Director: Raymond G. York, Ph.D., DABT (Associate Director of Research) A.12. Technical Performance: AJaorhonnF.J. BWaemieltetrs,tBei.nS,. B(.DSi.r(eRcetsoefrarLcahboArsastiosrtyanOtp)erations) Karen D. Klein, B.S. (Laboratory Scheduler) A13. Report Preparation: Raymond G. York, Ph.D., DABT JEroinAnHnagFarna,zeBe.,A.M.(SD.at(aStMuadnyaCgoeomrdeinntatSopre)cialist) Karen G. Parker, A.A. (Report Administrator) 000029 418-009:PAGE II-3 A.14.Report Review: MAilladnreMd. SH.oCbherrismtaina,n,PPhh..DD..,,DFAelBlTow,(DAirTeScto(rExoefcRuetsiveearDcihr)ectorof Research) A.15. DateProtocol Signed: 28 May 1998 A16. Datesof Technical Performance: A.16.2. Fo Generation Male Rats: Rat Arival Date DocsoantgienuPienrgitodhr(o2u8ghdaays14b-edfaoyrecochoahbaibtiattaitoinonp,earinodd and until day before sacrifice) Scheduled Sacrifice 02 JUN 98 08 JUN 98- 20 JUL 98 30JuL 98 A16.b. Fo Generation Female Rats: Rat Arrival Date DCoaseasgaerePaenr-iSoedc-tiFoenmianlge(R28atdsaAysssbiegfnoerde to DcooshaagbietaPteiroinodan- dFecmonatlienuRiantgstAhsrsoiugghneDdGto 9) Natural Delivery cohabitation and [28 days before continuing through DG 24 (rats that did not deliver a ltter) or DL*20 D(orsaatsgethaPterdieoldivEesrterdaousliCttyerc)l]e Evaluation Cohabitation Period Male 1 Male 2 DG 10 Caesarean-Sectioning DNaGtu2ra5lSDaeclriifviecrey(Preartisotdha(tDdLid1)not deliver alitter) DsLel2e1ctSeadcrfiofricceon(tdianmusedasntdudpyu)ps not 02 JUN 98 08 JUN 98 - 29 JUL 98 08 JUN 98 - 30 AUG 98 23 JUN 98 - 06 JUL 98 06 13 JUL JUL 98 98 P-M13 P-M20 JUL JUL 98 98 AM AM 17 JUL 98 - 21 AUG 98 28JUL 98 - 11 AUG 98 01AUG98-03 AUG 98 17 AUG 98 - 31 AUG 98 a. b. DDLGiiss uusseedd aass aann aabbbbrreevviiaattiioonn ffoorrddaayyooffl(apctraetsiuomn eodr)dgaeystpaotisotnp.artum. 000020 418-009:PAGE II4 A6.c. F1 Generation Rats: Dosage Period (Male Rats) Dosage Period (Female Rats) Passive Avoidance Testing Watermaze Testing Cohabitation Period Male 1 Male 2 Male Rats Sacrificed Natural Delivery Period DL 21 Sacrifice A17. Records Maintained: 18 AUG 98- 17 NOV 98 18 AUG 98 - 28 DEC 98 20 AUG 98 - 11 SEP 98 07 OCT 98 - 26 OCT 98 02 09 NOV NOV 98 98 PM PM - 09 16 NOV NOV 98 98 AM AM 18 NOV 98 24 NOV 98 - 09 DEC 98 14 DEC 98 - 29 DEC 98 The original report, raw vehicle components are data and retained reserve samples of the in the archives of Argus bulk test article and Research Laboratories, Inc. one Any year pafrteesretrhveedmatiilsisnugesofatrheerdertaafitnfeidnailn rtehpeoratr,cahfitveerswohficthhetTiemsettihneg Facility for Sponsor dwiilslcdaercdieddeatthetihrefTineasltidinsgpoFsaciitliiotny.. AUlnl uusneudsebdulpkretepsatraerdtifcolermwualsatiroentsumweedreto the. Study Monitor. B. TestArticle Information: B.A. Description: N-EtFOSE - a waxy solid B.2. Lot Number: FM-3929 [30035, 30037, 30039 (Expiration date: May 2000)] B3. Date Received and Storage Conditions: PTrheeptaersetdarstuisclpeewnsaisonrsecweeivreedsotnor2e0d May 1998, and frozen (-20C) stored at room temperature. 000031 418-009:PAGE 1-5 B.4. Special Handling Instructions: rSetsapnidraatrodrsaanfdetsyafperteycaguotgigolness)(uwseeroeftparkoetenctwihveenclhoathnidnigi,nggltohveesb,uldkustte-smtiasrtticle and prepared suspensions. B.5. Analysisof Purity: Information article is on regarding the identity, file with the Sponsor. composition, strength and purity of the test C. Vehicle Information: CAA. Description: 2.0% (RO. Tween 80 in reverse deionized water) osmosis membrane processed deionized water C2. LotNumber: MO3H0S C3. Dates Received and Storage Conditions: J`eSrhsiepym,enotns 2o2f TMwaye.en1Ju8l0y waenrde8rJeucleyiv1e9d98f,roamndJ.Ts.toBraekderat, rPhoiolmlitpesbmupregr,atNuerwe. FTahceiliRtyO.anddeiisonmiaziendtawianteedr is at ravoaoimlatbelmepferroamtuarec.ontinuous source at the Testing C.4. Special Handling Instructions: rSetsapnirdaatrodr,ssaaffeettyypgroegcgaulteisoonrs s(aufseetyofglparostseecstiavnedclaotfhaicneg-,shgileolvde)s,wedruset-tmaiksetn when handing the vehicle. C5. Analysis of Purity: Neither the Sponsor nor contaminants likely to be tphreeSsteuntdyinDtirheecvteohriwclaestahawtarweooufldainnyteprofteernetwiiatlh the results of this study. 000032 418-009:PAGE II-6 D. Test Article Preparation: aSnudsp3enmsgi/omnLs.ofTNh-eEttesFtOaSrEticwleerweasprceopnasrieddedraeidly1a0t0c%onpcuernetrfaotritohnes of 0, 0.2, 1, purpose of 2 dosage calculations. D1 Sample Information: `sampe Type Size | DRaetteained | CStoornatgeo/Sshipping |TSohipped | SDhaitoeped Concentration smi | 0B8aUNeSE | Frozen (20C) 0195.3J0U1N9988 2272006ECC 3880" 0044.AaNN 9999 26 DEC 08" 04 AN 99 VetTiwcieeeCnodm8p0onent Reserve sm [100NS8 | Room RO. Deionized Water _| Sm | 10 1uNS8_| temperature TFaecsitiitnyg | 20 ut 98 Archives | 20 JUL 08 2. AOonnsetyhraeilnifgqieusotwtda(as2yumpsLre)edpwatarosewds.ihtihEpdaprceahdwfsasoaanmmapplllyeessiwsfa.rsomdiTtvhhieedetodotph,inetrmoiqdwudoloeatlain(q3duombtios)t(t2woammLso.fraetthaeni3hneimdgdihi.estnroecsopTnseccesinvtieryat)i.on b- iFAhseayFiionggaeesnewaraabstaiucoksnuepad.ntdocwuirtihngratwhsafrmsptlaensddluarsitnwgetehkesofrfstdaonsdagsiextahdmwieneikstsraotfidoonsfaogreheadmFi1nigsetnrcattiaovnorfor . 0`sEh.ai0cph2pe.sdaamnfopd1rlmaengawlamyssi.sd.ciovinTdcheedenotntrthaoetriwaoolnisqaulioqtuo(t3sm(t2)mwLasanrdea3imnLe,d aretstpheectTiveesltyi)n.g FOancoiiaylg3uot8 (ba2cmkuwpat 2d.. 1O0m2mgmgmgccoocnnoccneecnnettnrrtaarttaiitooinn.on D.2. Analytical Results: SItnafboirlmitaytidoantaonfotrhpersetpaabirleidtyfoofrmtuhleabtuiloknstebsrtaacrkteitcliengistohne rfialnegweitohftchoencSepnotnrsaotri.ons and conditions of this study are concentration and homogeneity on file with the analyses were Sponsor. Results of the not available at the time of the writing of this report. E. TestSystem: EA. Species: Rat 000033 418-009:PAGE II-7 E2. Strain: Cr:CDBR VAF/Plus (Sprague-Dawley) E3. Supplier (Source): Charles River Laboratories, Inc., Raleigh, North Carolina E4. Sex: Male and female ES. Rationale for Test System: STyhsetCermib:eCcDauBsRe:VA1F)/tPhilsusstra(iSnporfargauteh-aDsawbleeeyn) dreatmownasstrsaetleecdtetod baes tsehnesiTteisvte to irnedpursotdruyctfiorvereapnrdodduecvteivleopamnedntdaelvetloxoipnmsenatnadlhtaosxicbietyenevwailduealtyiounss;ed2)thhrisotuogrhicoault pdahtaarmaancdoleoxgpicearlileyncaecteixviestinatthtehespTeecsiteisnganFadcisltriatiyn".; and 3) the test article is E6. Test System Data: Number of Rats. AApppprrooxxiimmaattee ADagteeastoAfrrBiivratlh WWeeiigghhtt ((gg))aotnStthuedyDaAyssaiftgenrmAernrtival E.7. Method of Randomization: Male Shipment 1 Rats Shipment 2 100 95 28MARSS 06APR98 67days 58days 223-331 223-336 Female Rats 205 30 MAR 98 65 days 179-229 193-216 E.7.a. Fo Generation Rats: oUfpcoonmparurtievarl-,gFeonegreanteerdatriaonndroamts were units. aAsfsteirgnaecdcltiomaitnidoinv,idmuaallehoaunsdinfgemoanlethreatbsasis rweecroerdseeldedcutreidngforacsctluimdaytoionn.theSebpasairsatoef pshhyispimceanltasppofeamraalnecreatasnwderbeodryawnediogmhitzsed aasssoingneegdrtooupfivteo denossuargeeagnroeuqpusiv(aGlrenotupdsist| rtihbruotiuognhoVf),bo3d5yrwaetisghptesr.seRxaptesrwdeorseage group, using a computer-generated (weight-ordered) randomization procedure. 000034 418-009:PAGE 11-8 aTshseigfinrestdtteon Cfaeemsaalreearnat-ssepcetridoonsinaggeongrDoGup 1w0.ithTahecornefmiarimneidngdafteemaoflemartatisngwewreere permitted to naturally deliver litters. aAmtoabnlge tohforsaentdhoatmsuuncictesswsfauslluysemdatteodsealefcetmafilvee rmaatlaessriatgsnepdertodonsataugrealgdreoluipvefrryom mfoertshcohdedwualsedusseacdritfoicseelaefctterficvoemfpleemtailoenroatfsptheercgorhoaubpitaftrioomn apemroiondg. tThhoseestahamte delivered alitter for scheduled for sacrifice on DL 21. E.7.b. F1 Generation Pups: On DL 4, a table were reduced to of random eight pups units each. was used to Whenever select pups to be culled, and litters possible, the same number of male and female pups per liter were continued on study. At weaning of used to select the 25 F1 generation pups male and 25 female pounpDsLin21e,acahtaobflGeroofurpasnId, oIlmaunnditIsll,was resulting postnatal in a total of evaluation. 150 At F1 generation least one male rats pup (75 and per sex) chosenforcontinued one female pup per litter, when ppouspsitbolxei,ciwtya(smosretlaelcittye)d.durGirnoguplacItVatwioans. not continued This decision ownasthemasdtuedyindcuoenstuolasteivoenre with the GroupV study veterinarian ater DL 5. and the Sponsor. There were no surviving pups in EB. System of Identification: E.8.a. Fo Generation Rats: Munailqeuaenpderfemmaanleentraitdsenwteirfiecaatsiosnignnuemdbteermspowrhaerny numbers assigned at to receipt and given the study before aiddemnitniifsiterdautsiionngofMothneelfirstsedlofs-paigeercoifngtheeartetsatgasrt(iGcleey. Rats Band were permanently and Tag Co., Inc., No. MSPT 20101). E.8.b. F1/F2 Generation Pups and Rats: ePvuaplsuawteerdeinnotterimndsivoifdutahlelylititdere.ntiAftiewdeadunriinngg,leaactcahtioFn1; galelnepraartaimoenterartssweelreceted for continued observation was identified with a Monel self-piercing ear tag. 000035 418-009:PAGE Il-9 F. Husbandry: FA. Research Facility Registration: USDA Registration et seq. No. 23-R-099 under the Animal Welfare Act, 7 U.S.C. 2131 F.2. Study Rooms: aThhealsltwuadyyarnodomisndweepernedemnatilnytasiunpepdliuenddewirtchoandmiitinoinmsuomf positive airflow of ten changes relative to per hour of 100% fresh temperature aairntdhahtumhiadditbyeweenrpeamsosneidtotrherdoucgohns9t9an.t9l7y%thHrEoPugAhofiulttetrsh.e Room study. hRuomoidmittyemwpaesrattaurrgeetwedasatta3r0g%etteod 7at0%6.4SFeteo 7A9PPFE(N1D8ICXtHo 2(6TEC)M;PrEeRlaAtiTvUe RE AND RELATIVE HUMIDITY REPORTS). F3. Housing: All cage sizes Care and Use aofndLahbooursaitnogrycAonndiimtailosns.were in compliance with the Guide for the F.3.a. Fo Generation Rats/F1 Generation Litters: cFaoggeesneerxacteipotndruartisngwetrhee icnodhiavbiidtuaaltliyonhaonudsepdosintpsataritnulmespsersitoedesl.wiDruer-ibnogttomed claothearbitthaatnioDn,G e2a0c,hFopaigrenoefrraattisownafsemhaolueseradtsinastsheigmnaeldetroatn'astucraagle.delBievgeirynnwienrgeno individually housed in in a common nesting nesting boxes. Each dam box during the postpartum and delivered period. litter were housed F.3.b. F1 Generation Rats/F2 Generation Litters: Acfothearbiwteaatinoinn,g,hotuhseeFd1ignepnaeirrast(ioonneramtaslweerraet pinedrivfiedmuaallleyrhato)udsuerdinbgefcoorhea.bitation, and individually as described for housedaftercohabitation. The same the Fo generation rats. Beginning no type of caging later than DG was 20, used F1 generation and delivered female rats were litter were housed individually housed in in a common nesting nesting boxes. box during the Each dam postpartum period. 000036 418-009:PAGE II-10 F4. Lighting: lAinghatu:t1o2m-ahtoiucraslldya-rcko,nwtirtohlleeadcfhludoarrekscpeenrtioldighbtecgyicnlneinwgaast m1a9i0n0tahionuerdsaEtS1T2.-hours F5. Sanitization: wCeargee cphaannglienderaspwperroexicmhaatenlgyedevaeprpyrootxhiemratweeleyk.thrBeeedtdiimnegs weaaschcwheaenkg.edCaasgeofsten as necessary to keep the rats dry and clean. F6. Feed: NRuattistiwoenreIngteirvneantiaodnalli,bSitt.umLoaucicse,sMsitsosoCuerrit)ifiinedinRdoivdiednuatl Diet #5002 feeders. (PMI F.7. Feed Analysis: Alenvaellysseexscweeedriengroutthienemlayxpiemrfuomrmceodncbenyttrhaetifoenedfosrucpeprltiiefri.ed Nfoeecdoonrtadmeviinaatnitosnsatfrom tehxepercestueldtsnuotfritthieonfaelerdeaqnuailryemseenstasrewearveaidleabtleectiendthbeyrtahwesdeataan.alyses. Copies of Neither the Study the feed that was kDniroewcntotronionrtetrhfeerSepwointshotrhewaressualwtas roef of any agent this stud. present in F.8. Water: mLoecmablrwaanteer(tRh.aOt. hwaadtebre)ewnapsraovcaeislsaebldeb1y0 pthaessraatgseatdhrloibuigthumafrreovmerasneaoustmoomsaitsic twhaeteprrioncgeascsceedswsastyerstaesmaanbdac/toerriionsdtiavti.dual water bottles. Chlorine was added to F.9. Water Analysis: cTohnetapmrioncaetsisoend(wLaatncearsitsearnLaalbyozreadtotrwiiecse, annually for possible chemical Lancaster, Pennsylvania) and monthly Pfoernnpsoyslsviabnleiab)a.cteCroipailecsonotfatmhienaretsiuolnts(AonfatlyhteiwcaalteLrabaonraaltoyrsieess,arIenca.,vaCihlaalbfloentin, the raw data. Neither the Study the water that was Director nor the Sponsor was aware known to interfere with the resultsof of any agent this study. present in 000037 418-009:PAGE II-11 F.10. Nesting Material: Bed-0'cobs was used Group, Maumee, Ohio). as nesting material (The Andersons Industrial Products F.11. Bedding Analysis: cNoenitthaemrinthaentSspolinkseloyrtonobretphreeSstenutdyinDtirheecbtoerddwiansg aware ofany potential that would interfere with the results of annually. this study. Copies of Analyses for the results of possible contamination are conducted the bedding analyses are available in the raw data. G. Methods: G.1. Dosage Administration": DGorsoaugpe|| (mDghoksgaigdeay)|| Conc(emngtirmaLt)ion| VDoolsuamgee || NuGemnbeerraotifoFno|| NuGemnbeerratoifoFnT (mikg) | Rats Per Sex| Rals Per Sex FTOehde)T--6 1 5 --T 5% 1%] rs T +Ter Tss T Ts s FV moTs LYTw sa] 1s1%1%] a. GmroorutpalIVdFu1rignegnlearcattatiioonn.pups were sacrificed at weaning on DL 21 because of severe pup b. There were no surviving Group V/ F1 generation pups after DL 5. The test article calculations. was considered 100% purefor the purpose of dosage a. SSeTeANAPDPAERNDDOIPXEGRA(TDEIVNIGATPIROONCSEFDRUORMESTHOEFPTRHOETTOECSOTLINAGNFDACILITY), items 1.and 2. 000038 418-009:PAGE I-12 G.2. Assi Rag tNn umbeerd s: Dosage AssigRnaetd NFuomGbeenresration Grow [Wale | Female| ResigRnaetd NFuTmGbeenresration Was [| Feraw | [TT 090913--99993750 | 1700017161-- 1100112140, 164007, | 1122010716--1122712050 ||1T22220226-1172262025| [Toei 70005 10126- 10145 | [VT 1o0v0a0t5-100007450 [10F 216-10 0250T . | Bb T-| T0 | 2515| a. b. G1GFr2euom1nua6gpl4el0IarV0catFa01t1iog0dne1.an2ye5r1aoetsfiscontapypue,psaanwdetrwheae srseamcqirusiesfsiitcneagfdtoavteewmeSiatgnuhietnygiDoinrrDaatLcw2a1sbexeclcudeaodfausnedsverereeplpacuepd mwoitrhtfaelmale .. There wereno sunning GroVuFp1 generation pups ater OL 5. G.3. Rationale for Dosage Selection: Dosages were selected by the Sponsor on the basis of previous studies conducted with the test article. G.4. Route of Administration: Oral (gavage) G.5. Rationale for Route of Administration: The oral (gavage) route was selected for use because: 1) in comparison with the odfietthaeryporsosutieb,lethreouetxeasctofdhousmagaen ceaxnpobseuraec.curately administered; and 2) it is one G.6. Frequency of Administration: G.6.a. Fo Generation Rats: The Fo vehicle generation once daily: male rats beginning were given appropriate dosages of the test article 28 days before cohabitation (which continued for or a maximum of 14 days) and continuing through the day before sacrifice after the completion of the cohabitation period. consecutive daily dosages. All male rats received a total of 52 a. See APPENDIX G, item 3. 000039 418-009:PAGE I-13 aTrhtieclFeoorgevneehriactleioonnfceemdaalielyrabtesgwinenrienggi2v8endathyesabpepfroorpericaotheabdiotsataigoens(owfhitchhe test acsosnitignnueeddtfooCraaesmaarxeainm-usemctoifo1n4indga)y,s)DGan2d4co(rnattisnuaisnsgigtnherdoutgohnDatGura9l(dreatlsivery that (d5idmLno/tkgd)elwivaesr aadljiutsert)e,dordaDilLy 2o0n (trhaetsbathsaitsdoefltihveermeodsatlritetcere)n.tlTyhreecdoorsdaegdebvoodlyume weight and given at approximately the same time each day. pDaartmursitiinont,heinporrodceersstoopfrdeeclliuvedreinpgospsuipblsewdeirsreupntoitodnoosfemdautnetimlaclombpelheatviioonr aofnd/or cannibalization of one daily dosage the pups. during the Consequently, delivery period. some dams were No dam missed not administered more than one daily dosage G.6.b. F1 Generation Rats: aFr1tigcelneeorratvieohnicmlealoencaenddafielymableegirnantsinwgeroen given day 1 appropriate dosages postweaning (DL 21) of the and test dciornetcitnlyuignigvetnhrtohueghtetshteardtiacyleb,ebfuotremasaycrihfaicvee. bFe2engepnoesrsiabtliyonexppuopssewdetroetnhoettest article during during the matemal the lactation period. gestation (in utero exposure) or via maternal milk G.7. Length of Study: Approximately seven months G.8. Method of Study Performance: G.8.a. Fo Generation Rats: AplelriFoodsgeonfetrhaetisotnudrya.ts Rwaetrsewoebrseeralvseod ofborseviravbeilditfyoratgelenaesrtaltwaipcepedaairlayndcueriantglealalst aeofpnfpcerceotxdsiuomrfaitnteghletyhteeosnatecacrhltoiicumlaret,aifaotbneorrptedirooisnosda,,geparnaednmdeaxtouanrmeithndeeedldiavfyeorrsiacelcsirniaifnciadcleddo.besaetrhvsatpriioonrstoofand `BaocdclyimwaetiigohntpserfioordF,owegeeknelryadtuiroinnmgatlhee rats were recorded dosage period and at at least once sacrifice. during Feed the consumption period. values for male rats were recorded weekly during the dosage a See APPENDIX G, items 4 and 5. 000040 418-009:PAGE I-14 Body weights for Fo generation female rats were recorded at least once during the acclimation period, weekly to cohabitation, daily during the gestationperiod, on DLs 1,4,7, 10 and 14 (rats assigned to natural delivery) and at sacrifice. Feed consumption values were recorded weekly to cohabitation, daily during the gestation period and on DLs 1, 4, 7, 10 and 14 (rats assigned to natural delivery). Feed consumption values were not recorded after DL 14, when it was expected that the pups would begin to consume matemal feed. A table of random units was used to select 15 female rats per dosage group for evaluation of estrous cycling by examination of vaginal cytology for 14 days before the start of the cohabitation period and continuing untiml ating. Within each dosage group, consecutive order was used to assign rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of 14 days. During cohabitation, all female rats were evaluateddaily until spermatozoa were observed in a smearof the vaginal contents and/oar copulatory plug was observed in situ (DG 0) and were assigned to individual housing. Female rats not mated within the first seven days of cohabitation were assigned alternate male rats that had mated (within the same dosage group) and remained in cohabitation for a maximum of seven additional days. during parturition, duration of gestation (DG 0 to the day the first pup was Rats allowed to naturally deliver litters were evaluated for clinical observations observed), litter size (all pups delivered) and pup viability at birth. Pups that either appeared stillborn or that died before initial examination of the litters for viability were examined for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Each behavior of litter was the dams subsequently examined daily for pup viability. Maternal was evaluated daily when the pups were examined during the 21-day postpartum period. Maternal behavior was recorded on DLs 1,4,7, 14 and 21. Variations from expected maternal behavior were recorded, if present, on allother days of the postpartum period. Fertility parameters were assessed for all dams assigned to natural delivery. Tinhperseegnpaanrcaimeest),ergsesitnactliuodnedinadefexrt(ilpietyrcienndteaxg(epoefrcpernetgangaencoifemsattihantgsretshualtterdesiunlttehde birth of live litters), number of offspring per litter (live and dead Pups), number of implantation sites, general condition of the dam andlitter during the postpartum paenrdiolda,ctvaitaibiolnitiyndiendxic(epser(cpeenrtcaegnetaogfepoufpspubposrbtohmattshuartvsiuvrevdiv2e1dd4ayasn)d. 7days), 000041 418-009:PAGE 1-15 G.8.b. FAIF2 Generation Pups ~ Preweaning Observations: Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day in which all pups in a litter were individually weighed (pup body weights were recorded after all pups in alitter were delivered and groomed by the dam). Vital status at birth was determined for pups that either appeared stillborn or that died before initial examination of the litter for viability. Pups that either appeared stillbom or that died before initial examination of the litter for viability were. examined for vital status at birth, as previously described. Each litter was evaluated for viability at least twice each day during the 21-day postpartum period. Pups in each litter were counted once daily. Physical signs (including variations from expected nursing behavior and gross extemal physical anomalies) in the pups were recorded once daily for 21 days postpartum. Dead pups observed at these times were removed from the nesting box. When not precluded by autolysis or cannibalization by the dam, any pup found dead was necropsied and examined for the cause of death. Pup body weights were recorded on DLs 1 (birth), 4, 7, 14 and 21 Reflex and physical development parameters in the F1 generation pups only `were monitored during the 21-day postpartum period. Surface righting reflex ability to right in 5 seconds (from DL 1)]. pinna unfolding (from DL 2), eye opening (from DL 12), acoustic startle response (from DL 13) and air righting reflex (from DL 14) were monitored daily until all pups (100%) in the litter reached the criterion for the specific test. Pupil constriction was evaluated once on DL 21; the number of pups per litter with this reflex present was recorded. G.8.c. F1 Generation Rats - Postweaning Observations: "Postweaning day" observations were recorded beginning on DL 22. All F1 generation rats were observed for viability at least twice daily during all periods. of the study. Rats were also observed for clinical observations of effects of the test article, abortions and premature deliveries prior to and approximately one hour after dosage and at sacrifice. Body weights of F1 generation male rats were recorded weekly during the dosage period and at sacrifice. Body weights for F1 generation female rats were recorded weekly to cohabitation, daily during the gestation period, on DLs 1, 4, 7, and 14 postpartum (rats assigned to natural delivery) and at sacrifice. Feed consumption values were recorded weekly except during cohabitation and on DGs 0, 7, 10, 14 and 20 and DLs 1, 4, 7. 10 and 14 (female rats only) Beginning at 23 to 25 days of age, one male rat and one female rat from each litter, where possible, were evaluated in a passive avoidance test for leaming, 000042 418-009:PAGE I-16 short-term retention and long-term retention. Each rat was tested on two days separated by a one-week interval, and the criterion for leaming was the same for both days of testing. The passive avoidance apparatus consisted of a twocompartment chamber with hinged Plexiglas lids. One compartment was fitted with a bright light and Plexiglas floor. The other compartment was fitted with a grid floor to which abrief (1 second) pulse of mild electric current (1 mA) could be delivered. The two compartments were separated by a sliding door. During each trial, the rat was placed into the "bright" compartment, the sliding door was opened and the light was tured on. The rat was allowed to explore the apparatus until it entered the "dark" compartment. The sliding door was then immediately closed, the light was tumed off and the brief puise of current was delivered to the grid floor. The rat was then removed from the apparatus and placed into a holding cage for 30 seconds before the start of the next trial. Trials were repeated until the rat remained in the "bright" compartment for 60 seconds on two consecutive trials (the criterion for leaming) or until 15 trials were completed. The latency to enter the dark compartment or the maximum 60-second interval was recorded for each tral. Dosage groups were compared for the following dependent measures: the number of trials to the criterion in the first session (overall leaming performance); the latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the first test session (activity level and exploratory tendency in a novel environment); the latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 2 in the first test session (short-term retention); the number of trials to the criterion in the second test session (long-term retention); and the latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the second session (long-term retention). Beginning at approximately 70 days postpartum, one male rat and one female rat from each litter were evaluated in a water-filed M-maze for overt coordination, swimming ability, leaming and memory. Each rat was tested in a watertight 16-gauge stainless steel modified M-maze. The maze was filled with water to a depth of approximately nine inches, and the water was monitored for temperature (range of 21C + 1C). On each test trial, the rat was placed into the starting position (base of the M-maze stem farthest from the two arms) and required to swim to one of the two goals of the M-maze, in order to be removed from the water. On the first tral, the rat was required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 was designated the incorrect goal during the remaining trials. Rats that failed to make a correct goal choice within 60 seconds in any given trial were guided to the correct goal and were then removed from the water. A 15-second intertrial interval separated each trial. Each rat was required to reach a criterion of five 000043 418-009:PAGE II-17 consecutive errorless trials to terminate the test session. The maximum number of trials in any test session was 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval was recorded for each trial, as is the number of errors (incorrect tums in the maze) during each trial. Each rat was tested twice. The test sessions were separated by a one-week interval, and the correct goal and the criterion were the same for both test sessions. Dosage groups were compared for the following dependent measures: the number of trials to criterion on the first day of testing (overall leaming); the average number of errors (incorrect tums in the maze) for each trial on the first day of testing (overall leaming); the latency (in seconds) to reach the correct goal on trial 2 of the first day of testing (short-term retention); the numberof rials to criterion on the second day of testing (long-term retention); the average number of errors for each trial on the second day of testing (longterm retention); and the latency (in seconds) to reach the correct goal on tial 1 of day 2 of testing (long-term retention). Female rats were evaluated for the age of vaginal patency beginning on day 28 postpartum'. Male rats were evaluated for the age of preputial separation beginning on day 39 postpartum. On DLs 84 to 97, the F1 generation rats within each dosage group were assigned to cohabitation, one male rat per female rat, based on computergenerated random units, with the exclusion of sibling matings. The cohabitation period consisted of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing. Female rats that did not mate within the first seven days of cohabitation were assigned alternate male rats from the same dosage group that had mated. Female rats were allowed to naturally deliver and maintain litters through a 21-day postpartum period. These rats were evaluated for clinical observations during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Pups that either appeared stillborn or that died before initial examination of the litters for viability were examined for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered livebon and to have died shortly after birth. Eachlitterwas subsequently examined daily for pup viability. Matemal behavior of the dams was evaluated daily when the pups were examined during the 21-day postpartum period. Maternal behavior was recorded on DLs1, 4, 7, 14 and 21. a. See APPENDIX G, item 6. 000044 418-009:PAGE 1-18 Variations from expected matemal behavior were recorded, if present, on all other days of the postpartum period. Fertiity parameters were assessed for all dams assigned to natural delivery. These parameters included a fertility index (percentage of matings that resulted in pregnancies), gestation index (percentage of pregnancies that resulted in the birth of live litters), number of offspring per litter (ive and dead pups), number of implantation sites, general condition of the dam and liter during the postpartum period, viability indices (percentage of pups bom that survived 4 and 7 days), and lactation index (percentage of pups bom that survived 21 days). G9. Gross Necropsy": G.9.a. Fo Generation Male an Sample Collection: le Rats Assi Pharmacokinetic At scheduled sacrifice after completion of the cohabitation period (male rats that sired litters of dams allowed to naturally deliver a litter) and on DL 21 (female rats allowed tonaturally deliver a litter) blood samples (approximately 4 mL per rat) were collected from five rats per sex per dosage group from the inferior vena cava into serum separator tubes and centrifuged. The resulting serum was immediately frozen on dry ice and maintained frozen (-70C) until shipment to the Sponsor for analysis. Theliverwas excised, weighed, and a sample section (lateral lobe) was frozen and retained at -70C until shipment to the Sponsor for analysis. The livers of the pups from the litters of the five dams in Groups | to IV selected for pharmacokinetic sample collection were excised, pooled per liter, frozen and retained at -70C until shipment to the Sponsor for analysis. The dams in the 15 mg/kg/day dosage group (Group V) did not have surviving pups on DL 21 G.9.b. Fo and F1 Generation Male Rats: Male rats were sacrificed by carbon dioxide asphyxiation after completion of the cohabitation period, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. Representative photographs of gross lesions are available in the raw data. The following organs were excised, individually weighed and retained for possible histologic evaluation: testes, a. Atable of random units was used to select one control group Fo and F1 generation rat of each sex from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions. 000045 418-009:PAGE 11-19 epididymides, prostate and seminal vesicles (with and without fluid). The testes were fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. The remaining organs were retained in neutral buffered 10% formalin. G.9.c. Fo Generation Female Rats Assigned to Caesarean-Sectioning: Female rats assigned to Caesarean-sectioning were sacrificed by carbon dioxide asphyxiation on DG 10, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Uteriof apparently nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites. All ovaries and gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. Representative photographsof gross lesions are available in the raw data. The rats were examined for placentae that appeared `abnormal (size, color or shape) and the numberofcorpora lutea in each ovary, implantation sites and viable and nonviable embryos. A viable embryo is oval or crescent shaped, pink and enclosed in an amniotic sac filed with clear fluid. A nonviable embryo is amorphous, small, pale pink to tan or deep red to black, soft and enclosed in an amniotic sac filled with clear, cloudy or opaque fluid. Embryos were discarded after examination. G.9.d. Fo Generation Female Rats Assigned Natural Delivery and F1 Generation Female Rats: After completion of the 21-day postpartum period, all dams that delivered litters. were sacrificed, and gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and distribution of implantation sites was recorded. Female rats assigned to natural delivery that did not deliver a litter were sacrificed on DG 25 and examined for gross lesions. To confirm the pregnancy status, uteri from rats that appeared nonpregnant were stained with 10% ammonium sulfide. Dams with no surviving pups were sacrificed after the last pup was found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. All ovaries were retained in neutral buffered 10% formalin for possible future evaluation. The Fo generation female rat in the 5 mg/kg/day dosage group selected for natural delivery that died was examined for the cause of death on the day the observation was made. The rat was examined for gross lesions. Pregnancy status and uterine contents were recorded. Delivered pups were examined to the extent possible. Ovaries were retained in neutral buffered 10% formalin. 000046 418-009:PAGE 11-20 G.9.e. F1/F2GenerationPups: Pups that died before examination of the litter for pup viability were evaluated for vital status at birth, as described previously. Pups found dead were examined for gross lesions and for the cause of death. Pups with gross lesions found on DLs 110 4 were preserved in Bouin's solution. Gross lesions of pups found on DLs 5 to 21 were preserved in neutral buffered 10% formalin. Representative photographs of pup gross lesions are available in the raw data. Pups not selected for continued evaluation on DL 4 were sacrificed by carbon dioxide asphyxiation and examined for gross lesions: pups with gross lesions. were preserved in Bouin's solution. Necropsy included a single cross-section of the head at the levelofthe frontal-parietal suture and examination of the crosssectioned brain for apparent hydrocephaly. The stomach contents (milk curd) were collected from culled pups from Groups |, Il and Ill. Samples were collected from all pups from fiveof the largest litters in these three dosage groups. Individual pup samples were combined by litter into polypropylene tubes and frozen at -20C. After completion of sample collection, samples were shipped (frozen on dry ice) to the Sponsor for analysis. On DL 21, F1 generation pups not continued on study and all F2 generation pups were sacrificed and examinedforgross lesions; gross lesions were preserved in neutral buffered 10% formalin. The 10 mg/kg/day F1 generation (Group IV) litters were sacrificed on DL 21 because of the severe pup toxicity during lactation (mortality and reduced body weights and delayed development). Necropsy procedures were the same as those used for pups culled on DL 4. 000047 418-009:PAGE II-21 G.10. Statistical Analyses: The following schematic represents the statistical analyses of the data: Type of Test! I. Parametric A. Bartlett's Test* Il. Nonparametric" A. Kruskal-Wallis Test (575% ties) Significant atps0.05 eres Not Significant | Analysis of Variance Significant atps0.05 Dunn's Test Not Significant Significant atps0.05 Not Significant B. Fisher's Exact Test (>75% ties) oesTest Iii. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilities are reported as either p<0.05 or ps0.01 b. Used only to analyze data with homogeneity of variance. c. Proportion data are not included in this category. d. Test for homogeneity of variance. 000048 418-009:PAGE 11-22 Proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g. body weights, body weight changes, feed consumption data and organ weights) were analyzed using Bartlett's Test of Homogeneity of VariancesTM and the Analysis of Variance?, when appropriate [i.e., Bartlett's Test was not significant (p>0.05)]. If the Analysis of Variance was significant (p=0.05), Dunnett's Test" was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate fi... Bartlett's Test was significant (p<0.05)], the Kruskal-Wallis Test" was used (s75% tes). In cases where the Kruskal-Wallis Test was statistically significant (050.05). Dunn's Method of Multiple Comparisons" was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test" was used. Data obtained at Caesarean-sectioning, natural delivery, preweaning reflex/physical developmental data involving discrete ata (e.g., number of corpora lutea, number of pups per litter trials to a criterion), were evaluated by the Kruskal-Wallis Test", as described above. One F1 generation dam (12228) in the 1 mg/kg/day dosage group hada liter consisting of only two pups. Because such occurrences can abnormally skew the distribution of the data", statistical analyses of gestation body weights, feed consumption values and natural delivery and litter data were made without the values for this dam and litter. 000049 418-009:PAGE Ill-1 Il. RESULTS - Fo GENERATION MALE RATS A. Mortality and Clinical Observations (Summary -Table B1; Individual Data - Table B10) No Fo generation male rats died during this study. The only clinical observation attributable to the test article was impaired righting reflex. This observation occurred in significant numbers (ps0.01) of 15 mg/kg/day dosage group rats, as compared with the control group values. All other adverse clinical observations were considered unrelated to the test article because the incidences were not dosage-dependent. These observations included localized alopecia on the limbs, dental problems (missing, broken or misaligned incisors), chromodacryorrhea, red peripenal substance and chromorhinorrhea. B. Body Weights and Body Weight Changes (Figure 1; Summaries - Tables B2 and B3; Individual Data - Table B11) Groups administered 5 mg/kg/day and higher dosages of the test article had reduced body weight gains. The values were significantly reduced (ps0.05 or p<0.01) in the 5 mg/kgiday dosage group on DSs 15 to 22, 22 to 29 and 29 to 36; in the 10 mg/kg/day dosage group on DSs 8 to 15, 15 to 22, 2210 29, 28 to 36, 36 to 43 and 50 to 53; and at all tabulated intervals in the 15 mg/kg/day dosage group. Reflecting these effects of the test article, body weight gains were significantly reduced (p<0.01) for the entire dosage period (DSs 1 to 53) in groups administered 5 mg/kg/day and higher of the test article. Absolute body `weights were significantly reduced (p<0.05 or p<0.01) in the 5 mg/kg/day dosage group on DSs 29, 36, 43, 50 and 53, and in the 10 and 15 mg/kg/day dosage groups on DSs 15, 22, 29, 36, 43, 50 and 53. Body weights and body weight gains were unaffected by the 1 mg/kg/day dosage of the test article. C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables B4 and BS; Individual Data - Table B12) Absolute (g/day) feed consumption values were significantly reduced (p<0.05 or p:0.01) in the 10 and 15 mg/kg/day dosage groups after the first week of treatment (DSs 8 to 15, 15 to 22, 22 to 29, 43 10 50 and 50 to 53). Reflecting these effects of the test article, the 10 and 15 mg/kg/day dosage groups had significantly reduced (ps0.01) absolute feed consumption values for the entire treatment period (DSs 1 t0 53). 000050 418-009:PAGE Ill-2 Absolute and relative (g/kg/day) feed consumption values were significantly reduced (ps0.05orps0.01) in the 15 mg/kg/day dosage group on DSs 15 to 22, 221029, 4310 50 and 50 to 53, as compared with the control group values. Feed consumption values were unaffected by the 5 mg/kg/day dosageofthe test article. D. Mating and Fertility (Summary -Table BS; Individual Data - Table B13) Dosages of the test article as high as 15 mg/kg/day did not affect any mating and fertiity parameters evaluated in the male rats. Values for the fertility and pregnancy indices (number of pregnancies per number of rats in cohabitation and rats that mated, respectively), the number of days to inseminate, the number of rats that mated and the number of rats with confirned mating dates during the first week of cohabitation were comparable among the five dosage groups. E. Necropsy Observations (Summary -Table BZ; Individual Data Table B14) A significantly increased (p<0.01) number of male rats in the 15 mg/kg/day dosage group had small seminal vesicles. One of these rats mated but did not impregnate a female rat; the other two rats impregnated female rats. One 15 mglkg/day dosage group male rat (10051) had a small prostate; this male rat was cohabited with and impregnated two female rats. Both of these gross lesions were considered related to the test article because: 1) the incidences were dosage-dependent; and 2) the absolute weights of the seminal vesicles with fluid and prostate were significantly reduced (p<0.01) in the 15 mg/kg/day dosage group (see section IILF). All other necropsy observations were considered unrelated to the test article because: 1) the observation occurred in a control group rat;or 2) the observation was considered a congenital malformation. One control group rat had a missing portion of a hindpaw digit. One 5 mg/kg/day dosage group male rat did not have a right testis and epididymis; this rat did not mate. F. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight (Summaries - Tables B8 and BS; Individual Data - Table B15) The 5, 10 and 15 mg/kg/day dosage groups had significantly reduced (ps0.01) terminal body weights. The absolute weights of the left epididymis, seminal vesicles with fluid, and prostate were significantly reduced (ps0.05 or ps0.01) in the 15 mg/kg/day dosage group. The absolute weights of the seminal vesicles 000051 418-009:PAGE Ill-3 without fluid and the right epididymis were also reduced in the 15 mg/kg/day dosage group, however not significantly (p>0.05). Testes weights were unaffected by dosages of the test article as high as 15 mg/kg/day. The ratios of the weight of the left and right testes to terminal body weights were significantly increased (p<0.05or p<0.01) in the 5 (left only), 10 and 15 mglkg/day dosage groups, as compared to the control group values. These observations were associated with the significantly reduced (ps0.01) terminal body weights in these dosage groups. The ratios of the weights of the epididymides, seminal vesicles and prostate were generally comparable among the five dosage groups. 000052 418-009:PAGE IV-1 IV. RESULTS - Fo GENERATION FEMALE RATS/F1 GENERATION ITTERS A. Mortality and Clinical Observations (Summaries - Table C1 and C15; Individual Data - Tables C22 and C30) AA. Mortality No deaths were caused by dosages of EtFOSE as high as 15 mg/kg/day. One dam (10170) in the 5 mg/kg/day dosage group died during delivery on gestation day (DG) 22. The event was considered unrelated to the test article because the incidence was not dosage-dependent. No other adverse clinical observations occurred in this dam, and its body weight and feed consumption values were unremarkable throughout the gestation period. Necropsy of the dam revealed external observations of red and wet perioral and perivaginal substance presumed to be evidence of bleeding during parturition. There were 15 implantation sites; eight normal pups were delivered and six dead fetuses were in utero. One conceptus was presumed cannibalized, because pup tissues were found in the stomach of the dam. All other dams survived to scheduled sacrifice. A2. Clinical Observations Relatively low incidences of localized alopecia occurred during the precohabitation, gestation and lactation periods only in groups treated with EtFOSE. These incidences of localized alopecia (total of all areas and limbs) were significant (p<0.05 or p<0.01) in the 5, 10 and 15 mg/kg/day dosage groups during the gestation and lactation periods. All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to the test article because: 1) the incidences were not dosage-dependent; and/or 2) the observation occurred in only one rat. These observations included dehydration, cold to touch, portion of tail black or missing, chromodacryorrhea, dental problems and chromorhinorrhea. B. Body Weights (Figure 2; Summaries - Tables C2 through C7; Individual Data - Tables C23 through C25) B.A. Precohabitation Groups administered 5 mg/kg/day and higher dosages of the test article had significantly reduced (p<0.01) body weight gains for the entire precohabitation period (DSs 1 to 29). During this period, values were significantly reduced (ps0.05or ps0.01) in the 5 mg/kg/day dosage group on DSs 8 to 16 and 23 to 000053 418-009:PAGE IV-2 29; and in the 10 and 15 mg/kglday dosage groups on DSs 110.8, 8 to 16, 16 to 23'and 23 to 29, as compared with the control group values. Body weights were significantly reduced (ps0.05 or p<0.01) in the 5 mg/kg/day dosage group on DSs 16 and 29, and in the 10 and 15 mg/kg/day dosage groups on DSs 16, 23 and 29, as compared with the control group values. Body weights and body weight gains during the precohabitation period were unaffected by the 1 mg/kg/day dosage of the test article. B.2. Gestation Gestation body weight gains tended to be reduced in the 1 and 5 mg/kg/day dosage groups and were significantly reduced (p<0.01) in the 10 and 15 mg/kg/day dosage groups on DGs 0 to 7. Weight gains generally continued to be reduced in these groups until DGs 10 to 12 (1 and 5 mg/kg/day dosage groups) or 12 to 15 (10 and 15 mg/kg/day dosage groups). Body weight gains were again significantly reduced (ps0.01) in the 15 mg/kg/day dosage group on DGs 18 to 20. Reflecting these effects of the test article, body weight gains. tended to be reduced in the 5 and 10 mg/kg/day dosage groups and were significantly reduced (p<0.01) in the 15 mg/kg/day dosage group for the entire gestation period (DGs 0 to 20) Body weights continued to be significantly reduced (p0.05 or p<0.01) in the 5 mglkglday dosage group on DGs 0 through 17, and in the 10 and 15 mglkg/day dosage groups on all days of the gestation period (DGs 0 through 20). Body weights and body weight gains during gestation were unaffected by the 1 mglkg/day dosage of the test article. B.3. Lactation The 10 and 15 mg/kg/day dosage groups had significantly reduced (p<0.01) body weights on DL 1. Lactation body weights continued to be significantly reduced (p<0.01) in the 10 mg/kg/day dosage group on DLs 4, 7, 10 and 14. Body weight loss occurred in the 10 and 15 mg/kg/day dosage groups on DLs 1 to 4. The 15 mg/kg/day dosage group was precluded from further evaluation because all pups died before DL 5. A significant increase (p<0.05) in body weight gain on DLs 14 10 21 in the 10 mg/kg/day dosage group was possibly associated with reduced litter size, milk demand and production. Body weights and body weight gains during lactation were unaffected by the 1.and 5 mg/kg/day dosage of the test article. 000054 418-009:PAGE IV-3 C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables C8 through C13; Individual Data - Tables C26 through C28) CA. Precohabitation Absolute (g/day) and relative (g/kg/day) feed consumption values were significantly reduced (ps0.05 or p<0.01) during the precohabitation period in the 10 and 15 mg/kg/day dosage groups on DSs 8 to 16, 16 to 23 and 23 to 29. Reflecting these effects of the test article, absolute and relative feed consumption values were significantly reduced (p0.05 or ps0.01) in the 10 and 15 mg/kg/day dosage group for the entire precohabitation period (DS 1 to 29) Feed consumption values during the precohabitation period were unaffected by the 5 mg/kg/day dosage of the test article. C2. Gestation The 10 and 15 mg/kg/day dosage groups continued to have significantly reduced (p<0.01) absolute and relative feed consumption values early in the gestation period (DGs 0to 7 and 7 to 10). Absolute feed consumption values were also significantly reduced (p<0.05) in the 5 mg/kg/day dosage group on DGs 010 7. Absolute feed consumption values continued to be significantly reduced (p<0.05 or p<0.01) in the 10 mg/kg/day dosage group on DGs 10 to 12 and 12 to 15, and in the 15 mg/kg/day dosage group throughout the remainder of the gestation period (DGs 1010 12, 1210 15, 15to 18 and 18 to 20). As a resultof these changes, absolute and relative feed consumption values were significantly r5e(daubcseodlu(tpe<o0n.l0y)5,o1r0p<a0n.d011)5 fmogr/tkhge/ednatyirdeogseasgtaetigornouppesr.iod (DGs 0 to 20) in the Feed consumption values during gestation were unaffected by the 1 mg/kg/day dosage of the test article. C3. Lactation Absolute and relative feed consumption values were significantly reduced (ps0.05 or ps0.01) in the 5 (absolute only) and 10 mg/kg/day dosage groups for the entire lactation period (DLs 1 to 14). Significant reductions (p=0.05 or p<0.01) in absolute and relative feed consumption values occurred on DLs 7 to 101n the 5 mg/kg/day dosage group, and for all tabulated intervals in the 10 mg/kg/day dosage group. Absolute and relative feed consumption values were reduced in the 15 mg/kg/day dosage group on DLs1 to 4, however the reductions were not statistically significant because only one litter was available for evaluation in this dosage group. The 15 mg/kg/day dosage group was precluded from further evaluation because all pups died before DL 5. 000055 418-009:PAGE IV4 Feed consumption values during lactation were unaffected by the 1 mg/kg/day dosage of the test article. D. Estrous Cycling, Mating and Fertility (Summ-aTarblye C14; individual Data - Table C29) Dosages of the test article as high as 15 mg/kg/day did not affect estrous cycling (number of estrus stages per 14 days) in the 15 rats per dosage group that were evaluated. A total of 34 or 35 female rats mated in each dosage group. The number of days in cohabitation, the number of rats that mated, the fertility and pregnancy indices (number of pregnancies per number of rats that mated and rats in cohabitation, respectively), and the number of rats with confirmed mating dates during the first and second week ofcohabitation were comparable in the five dosage groups. Pregnancy occurred in 35, 33, 32, 32 and 34 female rats in Groups | through V, respectively. E. Necropsy Observations (Summ-aTarblye C15; Individual Data = Table C30) All necropsy observations were considered unrelated to treatment with the test article. One, one and two rats in the 1, 10 and 15 mg/kg/day dosage groups, respectively, had moderate or marked dilation of the pelvis of one or both kidneys, observations that are common in this species and strain. Red and wet perioral and perivaginal substance occurred in the 5 mg/kg/day dosage group rat that died during delivery and was previously discussed. One rat in the. 5 mg/kgiday dosage group had localized alopecia at necropsy. F. Caesarean:Sectioning and Litter Observations (Summary = Table C16; Individual Data - Tables C31 through C33) Caesarean-sectioning observations on DG 10 were based on 10 pregnant dams in each of the five dosage groups. Statistically significant reductions (p<0.01) in the averages for implantations and viable embryos occurred in the 15 mg/kg/day dosage group, as compared to the control group. There were no biologically important or other statistically significant differences in the litter averages for corpora lutea or nonviable embryos. There were no dams with all nonviable embryos. 000056 418-009:PAGE IV-5 G. NCa1t8u;raIlndDievliidvuearlyDaantdaL-itTtaebrlOebsseCr3v4attihornosug(hSuCm3m7)aries-TableC17and There were 25 presumed pregnant rats assigned to natural delivery in each of the five dosage groups and 22 to 25 pregnant dams in each dosage group. One pregnant dam in the 5 mg/kg/day dosage group died during delivery on DG 22, as previously described. One dam in the 10 mg/kg/day dosage group was not observed to have delivered a litter but had one implantation site in utero at ngeencerroaplslyy oonccDurGs 2f5or, laitnteersveonftotnheatcomnacyeptinudsi.catAellroetshoerrptpiroengonfatnhterlaittsterd,ealisvseruecdha liter. The duration of gestation was significantly reduced (ps0.05 or ps0.01) in the 5, 10 and 15 mg/kg/day dosage groups, an observation associated with preimplantation loss in the 15 mg/kg/day dosage group [the average number of implantation sites per dam was significantly reduced (p<0.01), resulting in a significantly reduced (p<0.01) litter size] and an effect of the test article. Pup viability was significantly affected (p<0.05 or p<0.01) by the 10 and 15 mglkglday dosages of the test article, as described in the following information. The 10 mg/kg/day dosage group had a significantly increased (p<0.01) numberof dams with stillborn pups and the numbers of pups that died or were presumed cannibalized were significantly increased (p<0.01) on DLs, 1, 204,5107 and 8 to 14. The 15 mg/kg/day dosage group had postimplantation loss evident as reduced pup viability at birth [the gestation index (number of dams with liveborn pups per pregnant rats) was significantly reduced (<0.01), four dams in this dosage group had no livebor pups, the number of dams with stillborn pups was significantly increased (p<0.01), the litter average and number of stillborn pups was significantly increased (p<0.01) and the litter average and numberof liveborn pups was significantly reduced (p<0.01), as well as peripartum deaths [significant (p<0.01) numbers of dead and presumed cannibalized pups on DLs 1, 210 4 and 5 to 7 (all livebom pups died by DL 5) and significant (p<0.01) numbers of dams had all pups dead or presumed cannibalized on DLs 1 to 4]. Reflecting these effects, the viability and lactation indices were significantly reduced (p<0.01) in the 10 and 15 mg/kg/day dosage groups, as also were the averages for surviving pups (p0.01) in the 10 mg/kg/day dosage group on DLs 4 (pre- and postculling) and 7, and in the 15 mg/kg/day dosage group on DLs 1 and 4 (preculling). The live litter size at weighing was significantly reduced (p<0.05 or ps0.01) on DL1 in the 10 and 15 mg/kg/day dosage groups and on DLs 1, 4 (postculling), 7, 14 and 21 in the 10 mg/kg/day dosage group. "Significantly different from the vehicle control group value (p<0.01). 000057 418-009:PAGE IV-6 A dosage-dependent pattem of reduced pup body weights was evident in each group administered the test article. The 1 mg/kg/day dosage group tended to have reduced pup body weights on DL 4 (pre-and postculing). The 5, 10 and 15 mg/kg/day dosage groups had significantly reduced (p<0.01) pup body weights on all weighing days (no pups survived after DL 4 in the 15 mg/kg/day dosage group). The percentage of male pups was comparable across all five dosage groups. H. Pup Clinical and Necropsy Observations (Summaries = Tables C19 and C21; Individual Data -- Tables C38 and C45) Adverse clinical and necropsy observations associated with reduced pup viability and potential reduction in matemal care occurred in the 5, 10 and 15 mg/kg/day dosage groups. Adverse clinical observations included 1, 7 and 3 litters with pups that were not nursing in the 5, 10 and 15 mg/kg/day dosage groups, respectively; the incidence was significant (p<0.01) in the 10 mg/kg/day dosage group. The number of litters with cold to touch pups was also significantly increased (p<0.01) in the 10 mg/kg/day dosage group. Two litters in the 10 mg/kg/day dosage group had pups that were not nesting and one 10 mg/kg/day dosage group litter had dehydrated pups. The placentae and umbilical cords were not removed from pups in one 5 mg/kg/day dosage group liter. Necropsy observations in pups that were found dead included increases in the number of pups with no milk in the stomach in the 5, 10 and 15 mg/kg/day dosage group litters; the incidence was significant (p<0.01) in the 10 and 15 mg/kg/day dosage groups. All other clinical and necropsy observations in the F1 generation pups were considered unrelated to the test article because: 1) the incidences were not dosage-dependent;or2) the observation occurred in only one or two pups. Clinical observations included a black area on the right hindpaw, swollen left hindlimb, mass(es) in mouth, lesion on the neck and portion of tail missing or black. Necropsy revealed one 15 mg/kg/day dosage group stillborn pup with a short jaw and one 5 mg/kg/day pup with moderate dilation of the pelvis of the right kidney at necropsy on DL 21 I. Reflex and Physical Development (Summary = Table C20; Individual Data - Tables C39 through C44) Reversible delays in reflex and physical development that are highly correlated with body weight!" occurred in the 5 and 10 mg/kg/day dosage groups, as described below. 000058 418-009:PAGE IV-7 11. Surface Righting Surface righting was delayed in the 5, 10 and 15 mg/kg/day dosage groups, as. described below. The percentage of pups that could surface right was. significantly reduced (p<0.05or p<0.01) in the 5 mg/kg/day dosage group on DLs 2,3, 6, 9 and 10, in the 10 mg/kg/day dosage group on DLs2 through 18, and in the 15 mg/kg/day dosage group on DL 1." After DL 1, no pups in the 15 mg/kg/day dosage group surface righted; all pups were dead after DL 4. The average day that at least 50% of the pups in a dosage group had the ability to surface right was significantly increased (p<0.01) in the 10 mg/kg/day dosage group. All surviving pups ultimately attained the abilty to surface right. The ability to surface right was unaffected by the 1 mg/kg/day dosage of the test article. 1.2. Pinna Unfolding Pinna unfolding was delayed in the 1, 5, 10 and 15 mg/kg/day dosage groups. The effect was transient (evident only on one day) in the 1 mg/kg/day dosage group, and considered of no toxicological importance [the percentage of pups per liter with pinna unfolding was significantly reduced (p<0.05) on DL 3 only] The percentage of pups with an unfolded pinna was significantly reduced (ps0.05 or p<0.01) on DLs 3 and 4 in the 5 mg/kg/day dosage group, on DLs 3, 4,5 and 6 in the 10 mg/kg/day dosage group and on DLs 3 and 4 in the 15 mglkgiday dosage group. No pups in the 15 mg/kg/day dosage group had an unfolded pinna on DLs 2, 3 or 4; all pups in this group were dead after DL 4. The average day that at least 50% of the pups in a dosage group had an unfolded pinna was significantly increased (p<0.01) in the 5 and 10 mg/kg/day dosage groups. All surviving pups ultimately had unfolded pinnae. 1.3. Eye Opening Eye opening was delayed in the 5 and 10 mg/kg/day dosage groups (no 15 mg/kg/day dosage group pups were evaluated for this parameter). The percentage of pups with at least one open eye was significantly reduced (p<0.05 or p<0.01) in the 5 and 10 mg/kg/day dosage groups on DLs 14, 15 and 16. The day that at least 50% of the pups had at least one open eye was increased in the 5 and 10 mg/kg/day dosage groups; the value was significant (p<0.01) in the 5 mg/kg/day dosage group. All pups had open eyelids by DL 21, when pupil constriction was tested. The time to eye opening was unaffected by the 1 mg/kgiday dosage of the test article. 000059 418-009:PAGE IV-8 14. Acoustic Startle The time of development of the acoustic startle reflex was delayed in the 1, 5 and 10 mg/kg/day dosage groups; no 15 mg/kg/day dosage group pups survived to be tested for this reflex. The effect was transient (evident only on two days) in the 1 mg/kg/day dosage group, and considered of no toxicological importance [the percentage of pups perlitterwith this reflex was significantly reduced (ps0.05 or ps0.01) on DLs 14 and 15]. The percentage of pups in the 10 and 15 mg/kgiday dosage groups with this reflex was significantly reduced (p<0.05 or p<0.01) on DLs 13, 14 and 15. The day that at least 50% of the pups had the acoustic startle reflex was significantly increased (p<0.01) in the 5 and 10 mg/kg/day dosage groups. 15. Air Righting The ability to air right was delayed in the and 10 mg/kg/day dosage groups; no 15 mg/kg/day dosage group pups survived to be tested for this reflex. The percentage of pups that air righted was significantly reduced (p<0.05 or p<0.01) in the 5 mg/kg/day dosage group on DLs 14 through 18 and in the 10 mg/kg/day dosage group on DLs 14 through 20. The day that at least 50% of the pups could air right was significantly increased (ps0.05 or p<0.01) in the 5 and 10 mg/kg/day dosage groups. The ability to air right was unaffected by the 1 mg/kg/day dosage of the test article. 16. Pupil Constriction Alive pups in the 0 (Vehicle), 1, 5 and 10 mg/kg/day dosage groups had the pupil constriction response presenont DL 21. 000060 418-009:PAGE V-1 V. RESULTS-F1 GENERATION MALE AND FEMALE RATS A. F1 Generation Male Rats AA. Mortality andClinical Observations (Summary -Table D1; Individual Data - Table D13) All F1 generation male rats survived until scheduled sacrifice. All adverse clinical observations were considered unrelated to the test article because 1) the incidences were not dosage-dependent; and/or 2) the observation occurred in only one or two rats. These observations included dental problems (missing, broken and/or misaligned incisors), chromodacryorrhea, chromorhinorrhea and swollen and purple ears. Observations of emaciation, dehydration, brown perinasal discharge, labored breathing, brown or red perioral substance, excess salivation, gasping and rales occurred in one rat (12123) in the 1 mg/kg/day dosage group on days 70 to 85 postweaning and were considered related to a possible intubation injury on day 70 postweaning. This rat lost 101 g from days 71 to 78 postweaning but regained 88 g by day 85 postweaning. A2. Body Weights and Body Weight Changes (Figure 3; Summaries - Tables D2 and D3; Individual Data - Table D14) The 1 and 5 mg/kg/day dosage groups weighed less than the control group on day 1 postweaning [the reduction was significant (p<0.01) in the 5 mg/kg/day dosage group] and body weight gains were reduced in these groups throughout the postweaning period. Body weight gains were significantly reduced (p<0.01 and ps0.05) in the 1 and 5 mg/kg/day dosage groups on days 22 10 29 and 43 to 50 postweaning, and additionally in the 5 mg/kg/day dosage group on days 1 to 8,810 15 and 15 to 22 postweaning. Reflecting this pattern of weight gain, weight gains in the 1 and 5 mg/kg/day dosage groups were significantly reduced (p<0.05 and p<0.01) for the precohabitation period (day 1 postweaning to precohabitation) and for day 1 postweaning to termination (significant at 5 mglkglday only). Absolute body weights were significantly reduced (ps0.05) in the 1 mg/kg/day dosage group on days 36, 50 and 57 postweaning and significantly reduced (ps0.01) in the 5 mg/kg/day dosage group throughout the postweaning dosage period. A.3. Ab(sSoulmumtaeri(egs/d-aTya)balnedsRDe4laatinvdeD(5g;/kIgn/ddiaviyd)uFael eDdatCaon- sTuabmlpetiDo15n)Values Absolute (g/day) and relative (g/kg/day) feed consumption values in the F1 generation male rats were significantly reduced (ps0.01) in the 5 mg/kg/day dosage group on days 1 to 8 and 8 to 15 postweaning. Relative feed 000061 418-009:PAGE V-2 consumption values were also significantly reduced (p<0.01) on days 15 to 22, 221029, 2910 36, 36 to 43 and 1 to 57 in the 5 mg/kg/day dosage group. Feed consumption values were unaffected by the 1 mg/kg/day dosage of the test article. Ad. Sexu ration (Summary - Table D; Individual Data - Tabl The average day of preputial separation was unaffected by the 1 mg/kg/day dosage of the test article. The average day of preputial separation was significantly delayed (ps0.01) for the F1 generation male rats in the 5 mg/kg/day dosage group, as compared to the control group value, but the increase was approximately one day and not considered toxicological important. AS. Passive Avoidance Performance (Summary - Table D7: Individual Data - Table D17) There were no biologically important differences in the values for learning, shortterm retention, long-term retention or response inhibition in the F1 generation male rats, as evaluated by performance in a passive avoidance paradigm. No statistically significant differences occurred in the number of trials to criterion, trial latencies or numbers of rats that failed to leam. AG. Watermaze Performance (Summary - Table D8; Individual Data - Table D18) No biologically important dosage-dependent differences occurred in watermaze performance of the F1 generation male rats regarding leaming, short-term retention, long-term retention or response inhibition. No statistically significant differences occurred in the number of trials to criterion, trial latencies or numbers of rats that failed to learn. AZ. Mating and Fertility (Summary - Table D9; Individual Data - TableD19) Dosages of the test article as high as 5 mg/kg/day did not affect any mating and fertiity parameters evaluated. Values for the fertility and pregnancy indices (number of pregnancies per number of rats that mated and rats in cohabitation, respectively), the number of days to inseminate, the number of rats that mated and the number of rats with confirmed mating ates during the first week of cohabitation were comparable among the three dosage groups. Of the male rats assigned to cohabitation, 80.0%, 83.0% and 92.0% impregnated the cohort female rat. 000062 418-009:PAGE V-3 AS. Necropsy Observations (Summary - Table D10; Individual Data Table D20) Aulnlrneleactreodpstoy tohbesetrevstatairtoincsleinbetchaeuFs1e:gen1)ertahteiionncmiadelnecerastswewreerenoctondsoisdaegree-d dependent; and/or 2) the observation occurred in only one rat in a group. These observations included one control rat (12080) with testis and epididymis and one 1 mg/kg/day dosage group small male and flaccid rat (12123) left with a small left epididymis. 1 mg/kg/day dosage Control group rat 12080 sired a group did not mate, however this litter. Rat 12123 rat had clinical in the observations that suggested an intubation injury occurred two days before cohabitation, as previously described. AS. TWeerimgihntaltoBToedrymiWneailgBhotsdyanWdeiOgrhgtan(SWuemimgahrtisesan-dTaRbalteisosD(1%1) of Organ and D12; individual Data - Table D21) Terminal body weights were significantly reduced (ps0.01) in the 5 mg/kg/day dosage group, as compared to the control group value. The ratios of 5 mg/kg/day the left dosage and right testis weights to the terminal body weight in the group were significantly increased (ps0.01), as compared to trehfeleccotnstrtohlegsriognuipfivcaalnutesr.eduTchtiisonsiignnitfeircmainntalinbcordeaysweeiingthhtes rfeolratthiivse dteosstaisgewegirgohutps. `Twhiethoaubtsoflluuitd)e awnedigphrtosstoaftte,heaenpdidtihedyrmaitdieoss,oftetshteese,pisdeimdiynmaildevse,siscelmesin(awlitvheasincdles (with and without fluid) and prostate weights to the terminal body weight of the F1 generation male rats were comparable among the three dosage groups. The mean absolute weight of the left testis in the 1 mg/kg/day dosage group was significantly reduced (p<0.01), as compared to the control group value. This s1i)gntihfeicvaantlureedwuacstinoontwdaossangoet-cdoenpseinddeernetd;aannedff2e)ctaosfimtihleartersetdaurcttiicolen bdeidcanuosteo:ccur for the weight of the right testis. B. Fi Generation Female Rats B.A. Mortality and Clinical Observations (Summary -Table E1; Individual Data - Table E23 All F1 generation female rats survived until scheduled sacrifice. 000063 418-009:PAGE V4 All clinical observations during the periods were considered unrelated precohabitation, gestation and to the test article because: 1) lactation the incidences were not two rats. dTohseasgee-cdleinpiecanldeobnste;rvaantdi/oonrs2)intchleudoebdseprovrattiioonnofoctaciulrmriesdsiinngo,ndleynotanle to pswroolblleenmsan(dmipsuarlpilgeneeda,rsm,islsoicnalgiazendd/aolropbercoikaeonnintchiesobrasc),k,chhreoamdodaancdr/yoorrlrihmebas,, ptosis, urine-stained abdominal fur and scab or lesion on the head. EIntcFoOnStrEasttretaottehdeFsoliggehtnleyraitnicorneafseemdailneciradtesn,ctehseotfotlaolcailniczieddenacleopoefcilaocianlitzheed alopecia was significantly and 5 mg/kg/day dosage reduced (p<0.01) during the gestation period in groups, as compared to the control group. This the 1 sniogtnidfoiscaangte-reddeupcetnidoennwta;sannodt2c)otnhseidienrceiddetnrceeatomfeannt-aredlvaetresdebcelcianiucsael:obs1e)rivt awtaison is expected to increase, not decrease, in a toxicology study. B.2. Maternal Body Weights Summaries - Tables E2 and Body Weight Changes through E; Individual Data (Figure 4; - Tables E24 through E26) B.2.a. Precohabitation The day 1 1 and 5 mg/kg/day postweaning [the dosage groups weighed less than reduction was significant (p<0.01) the control group on in the 5 mg/kgiday dosage group] throughout the and body weight gains precohabitation period. were generally reduced in Body weight gains were these groups reduced in the 21 2m1g0/k2g9,/d3a6ytdoo4s3agaendgr4o3uptoto5086po%sttowe9a5n%inogfatnhde cboondtryowlegirgohutpgvaailnusewseorne days aSingdni8fictaon1t5lyprosetdwuecaendin(gp.s0.R0e1f)leicnttihneg 5thmegs/ekegf/fdeactysdoofstahgeetegsrtoaurptioclne,dbaoydsy1wteoig8ht gains were significantly reduced (p<0.05 and p<0.01) in both test article-treated pgrreocuophsabfiotratthieonenatnirde dparyecsoh1atboit5a7tipoonstpewreiaondin[gda(ysi1gnpiofsictawnetaanti5ngmgto/kg/day only). Avablsuoel)utien tbhoedy1 mwegilgkhgt/sdawyerdeossaliggehtlgyroruepdutcherdou(g9h7o%utttoh9e8p%reocfohtahbeitcoanttiroonlpgerroiuodp and were significantly reduced (p<0.05) on the first day of the cohabitation psiegrniiofdi.canAtblsyorleudteucbeodd(ypw<e0i.0g1h)tsthinrotuhgeh5oumtgt/hkeg/pdraeycodhaobsiatgaetigornoduopswaegreeperiod. B.2.b. Gestation Mraetdeurcneadl(bpo5d0y.0w5eiogrhpts0g.a0i1n)s dinurtihneg1thaendge5stmagt/iokng/pdearyioddowseargeesgirgnoiufpicsanotnlydays 0 to 7 of gestation (DGs 0 to 7), as compared to the control group values. Body 000064 418-009:PAGE V-5 weight gains in the 5 mg/kg/day dosage group were significantly increased (p<0.01) on DGs 14 to 17. B1o8diyn twheeig1hatsndwe5rmegs/ikggnilfdiacayntdloysraegdeucgerdou(ppss0a.n0d5ionrtph<e0.0m1g)/okng/DdGaysd0osthargoeugh group only on DGs 19 and 20. B.2.c. Lactation 5Mamtgelmkagl/dbaoyddyowseaiggehtgsrrouepmaoinneDdLssig1n,i4f,ic7a,nt1l0y,r1e4duacnedd 2(1psa0n.d05inorthpes01.0m1g)/kign/tdhaey dosage group on DLs 1 and 7. Materal (p<0.05) body in the weight 1 and gains during 5 mg/kg/day the lactation period were dosage groups on DLs 1 significantly to 21. reduced B.3. VMaatleurensa(lSAubmsmoalruitees(g-/Tdaayb)leasndE8Retlhartoiuvgeh (Ea1/3ka;/dIanyd)ivFiedueadlCDoantsau-mption Tables E27 through E29) B.3.a. Precohabitation The 1 mg/kg/day dosage of the test article reduction (ps0.05) in absolute (g/day) feed was associated with consumption values a significant on days 43 to 50 and 50 for the to 57 of the 5 mg/kg/day pdroescaohgaebigtraotuiponweperreiosdi.gniAfbiscoanltultyerfeedeudcecdon(spusm0p.t01i)ononvadlauyess 1 108, 36 0 43, group values. 43 to 50 and 50 to 57 postweaning, as compared to the control Reflecting these effects of the test article, feed consumption Vparlecuoehsawbietraetisoingnpifeirciaondtl(ydraeydsu1ce0d 5(7psp0o.s0t5waenadnipnsg0).0in1,bortehspgercotuivpeslya)dmfiornitshteeernetdire the test article. wReelraetisviegn(igf/ikcagn/tdlayy)infcereedasceodns(upm<p0t.0i5onorvapl<u0e.s01i)n tohned5amygs/8kgt/od1a5y, d1o5staog2e2garnodup22 to 29 postweaning, compared to the control group. B.3.b. Gestation Absolute feed consumption values in the 1 and 5 mg/kg/day dosage groups were significantly reduced (p0.05) during the first week of the gestation period (DGs 0 to 7), as compared to the control group. Absolute feed consumption values for the 5 mg/kgiday dosage group were also significantly reduced (p50.01) on DGs 7 to 10, as compared to the control group. 000065 418-009:PAGE V-6 The relative feed consumption value on DGs 14 to 17 was significantly reduced (p<0.05) in the 5 mg/kg/day dosage group, as compared to the control group value. B.3.c. Lactation The absolute matemal feed consumption value was significantly reduced (30.05 to p<0.01) for the entire lactation period (DLs 1 to 14) and at all tabulated intervals during the lactation period in the 5 mg/kg/day dosage group. The relative feed consumption value was significantly reduced (p<0.01) on DLs 4 to7 and 7 to 10 in the 5 mg/kg/day dosage group, as compared to the control group values. Absolute and relative feed consumption values during the lactation period were unaffected by the 1 mg/kgiday dosage of the test article. B.4. Sexual Maturation (Summary - Table E14; Individual Data - Table E30 The average day of vaginal patency was unaffected by the 1 and 5 mg/kg/day dosages of the test article. The average day of vaginal patency was significantly delayed (p<0.01) for the F'1 generation female rats in the 1 mg/kg/day dosage group, as compared to the control group value, but the delay was not dosagedependent and not considered toxicologically important. B.5. Passive Avoidance Performance (Summary - Table E15; Individual Data - Table E31 There were no biologically important differences in the values for learning, longterm retention or response inhibition in the F1 generation female rats, as evaluated by performance in a passive avoidance paradigm. No statistically significant differences occurred in the number of trials to criterion or numbers of rats that failed to leam. The latency of the second trial in the first testing session was significantly increased (p<0.05) in the 5 mg/kg/day dosage group, as compared to the control group. This observation was not considered an effect of the test article because a decrease in short-term memory,rather than an increase, would be expected as an indication of toxicity. 000066 418-009:PAGE V-7 B.6. Watermaze Performance (Summary -Table E16; Individual Data - Table E32) No biologically important dosage-dependent differences occurred in watermaze performance of the F'1 generation female rats regarding leaming, short-term retention, long-term retention or response inhibition. No statistically significant differences occurred in the number of trials to criterion, trial latencies or numbers of rats that failed to learn. B.7. Mating and Fertility (Summary - Table E17; Individual Data Table E33) Dosages of the test article as high as 5 mg/kg/day did not affect any mating and fertiity parameters evaluated. Values for the number of days in cohabitation, the number of rats that mated, the fertility and pregnancy indices (number of pregnancies per number of rats that mated and rats in cohabitation, respectively), and the number of rats with confirmed mating dates during the first and second week of cohabitation were comparable among the three dosage groups. B.S. Necropsy Observations Table E34) (Summary - Table E18; Individual Data - The only necropsy observation was moderate dilation of the pelvis of the right kidney in one 1 mg/kg/day dosage group female rat (12239). This observation was considered unrelated 10 the test article because it was a single occurrence of an observation that commonly occurs in this strain. B.9. Natural Delivery and Litter Observations (Summaries - Tables E19 `and E20; Individual Data - Tables E35 through E38 Pregnancy occurred in 23 (92.0%), 25 (100.0%) and 24 (96.0%) of the 25 female rats in each dosage group assigned to cohabitation in the 0 (Vehicle), 1 and 5 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters. The gestation index (the percentage of pregnant rats with live offspring) was comparable across the three dosage groups. As occurred in the previous generation, there was a tendency for reduced pup viability in the 5 mg/kg/day dosage group. The number of dams with stillborn pups was significantly increased (p<0.01) in this dosage group. The number of pups found dead or presumed cannibalized was significantly increased (ps0.01) on DLs 1, 210 4 and 8 to 14 and one 5 mg/kg/day dosage group dam (12255) had all pups die by DL 4. Asa result, the viability and lactation indices and the average number of surviving pups per litter on days 4 (preculing), 14 and 21 postpartum were significantly reduced (ps0.05) in this dosage group. 000067 418-009:PAGE V-8 As also occurred in the previous generation, pup body weights were significantly reduced (ps0.01) in the 5 mg/kg/day dosage group on days 1, 4 (pre and postculling), 7, 14 and 21, as compared to the control group values. There was not effect on pup viability or pup body weights in the 1 mg/kg/day dosage group. Administration of the test article at dosages as high as 5 mg/kg/day did not adversely affect any other parameter evaluated at natural delivery or during the 21-day lactation period (averages for implantations and live litter sizes and pup sex ratios). The duration of gestation was significantly reduced (ps0.05) in the 5 mg/kg/day dosage group. This slight reduction was considered unrelated to the test article because the magnitude of the change was small [0.3 day, or 99% ofthe 0 (Vehicle) and 1 mg/kg/day dosage group values). B.10. Pup Clinical and Necropsy Observations (Summaries - Tables E21 and E22; Individual Data -Tables E39 and E40) All clinical and necropsy observations were unrelated to dosages of the test article as high as 5 mg/kg/day because: 1) the incidences were not dosagedependent; and 2) the observation occurred in only oneortwo litters. These clinical observations included one pup in the 1 mg/kg/day dosage group and three pups in the 5 mg/kg/day dosage group that were not nursing; nine littermates in the 5 mg/kg/day dosage group that were cold to the touch; one control group pup with exophthalmos and a traumatized comea: and one control group pup with a portion of the tail black. No milk in the stomach occurred in 2 (100%), 2 (50.0%) and 13 (52.0%) of the pups that were found dead in the three respective dosage groups. The only necropsy observation at scheduled sacrifice was an absent left eye in one control group pup. 000068 418-009:PAGE V-9 REFERENCES 1. Study Design as Modification of: U.S. Food and Drug Administration (1994). Intemational Conference on Harmonisation; Guideline on detection of toxicity to reproduction for medicinal products. Federal Register, September 22, 1994, Vol. 59, No. 183 2. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. 3. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. 4. European Economic Community (1988). Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice. Official Journal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17. 5. Christian, M.S. and Voytek, P.E. (1982). In Vivo Reproductive and Mutagenicity Tests. Environmental Protection Agency, Washington, D.C. National Technical Information Service, U.S. Department of Commerce, Springfield, VA 22161. 6. Christian, M.S. (1984). Reproductive toxicity and teratology evaluations of naltrexone (Proceedings of Naltrexone Symposium, New York Academy of Sciences, Novembe7r, 1983), J. Clin. Psychiat. 45(9):7-10. 7. Lang, P.L.(1988). Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Cr:CD7BR Rat. Charles River Laboratories, Inc., Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 8. Institute of Laboratory Animal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 9. Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Arch. Pathol. Exp. Pharmakol. 247:367. 10. Snedecor, GW. and Cochran, W.G. (1967). Variance test for homogeneity of the binomial distribution. Statistical Methods, 6th Edition, lowa State University Press, Ames, pp. 240-241 000069 418-009:PAGE V-10 11. Sokal, RR. and Rohlf, F.J. (1969). Bartlett's test of homogeneity of variances. Biometry, W.H. Freeman and Co., San Francisco, Ppp. 370-371. 12. Snedecor, G.W. and Cochran, W.G. (1967). Analysis of Variance. Statistical Methods, 6th Edition, lowa State University Press, Ames, PP. 258-275. 13. Dunnett, CW. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1121 14. Sokal, RR. and Rohlf, F.J. (1969). Kruskal-Wallis Test. Biometry, W.H. Freeman and Co., San Francisco, pp. 388-389. 15. Dunn, OJ. (1964). Multiple comparisons using rank sums. Technometrics 6(3):241-252. 16. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, pp. 96-104. 17. Zambrama, MA. and Greenwald, G.S. (1971). Effects of fetal ovarian and placental weight of various number of fetuses in the rat. Biol. of Reprod. 4:216-223. 18. Lochry, EA. Hoberman, AM. and Christian, M.S. (1984). Positive correlation of pup body weight with other commonly used developmental landmarks. Teratology 29(2):44A. 000070 APPENDIAX REPORT FIGURES 000071 Fo GEBNOEDRAYTIWOENIMGAHLTESRATS Figur1e 3 | w| 1 2z ! vo | s" i g wm a ad ad 2 =a " a Pas --~ pd i * x x |v0(VENCLE) MGKGOAY onan oSMGKGOAY ve oT sd hEF goo py 4 X AF gE o.wen x Enfpe00s 2 3 7 3 wo CET oder ah a z' BODY WEIGHTS Fo GENERATIFiOgNuFr2EeMALE RATS || 4 | oncemona =| /, J aon =| A a gfe 5|" SSTVALY{| E 77{| wenacone go onVELA | FEBTT nul atan I SM ElRyE R viuans aa ERR g Gan REET gEET & 4 s=wans J x ffoosasos) : iswmm ois di be hunnuninnnd ii oun : DAYOF STUDY DAYOF GESTATION DAYOFLACTATION. 1 GEBNOEDRAYTIWOENIMGAHLTESRATS Figured - ;| wl|y|! fm g g- | g dow - A || so-mmanmrcon R aY T|| een ghmo v > snc A / yp. e5tal xs | 5 | ~ & | LE weexosmudnI r3 1 GENBEORDATYIWONEIFEGMHATLSE RATS - Figur4e EN ud a stoma - pemaet arm rE ntl Avaee d | 5Es ofoTn IEIa ErLTAIr NISOy NRY Eas sonar = g ouo"is | e0 005 oooh 2s i" .5 Sg oad 3 DAY POSTWEANING DAY OF + GESTATION ronsDAYOFLACTATION x APPENDIX B REPORT TABLES - Fo GENERATION MALE RATS 000076 J---- We we we we wen Te emChe okie. Ser aconeae 0.01 8g 3 2pi z :: i 2 3 E TLTODD DD onnomnnomin nin s 2 : i 3 2 Smmon men pee ma nen : 3 BE mnie $ : i 2 3 & z o RRR 28 3 & 8 8 sg& :ai 4 EEu Sianiticanely Sitterem from the vehicle comrel rows valve trie ob s 3 i3? z 3: i & z APPENDIX C REPORT TABLES - Fo GENERATION FEMALE RATS 000086 PRRCOAABITATION lokY 00 STIOYTOTHEOAToF ComabiTATION fn - - or Puiu uiE 5 s 3 3 g a r------ - EL wn ui an oss wn 2 rrsserens BIBL BLELEL LE nT . g2 5 i z 2 g So Wim. Linas. - LAS sss Goad owl.oyi ous Whois . g ptssirin druid] SSE A : : & 8 ro 41-45 mts So GH) AEA SEEAWEIA 0 EKASTA, RERRSCTON RII 50808 s S {3 3S 3 8 2 :5s ; BR 5: | Low gsEg a. 8 mee walneal INT Nn msoowrsn, mieninne imamies meine e en r nI es SS + BERLE a ccsco ar go f EEmlEELLALRE HRSII r= ota sernepon tc rn 3 Sr EEE na he cn my ve p:e ig 8 ; < een BD ov oa wo andi DER SGT whi ai RS Re le IN ESI I UL TU trier es ee toc gH FimiHcaniy ditiecen tron the Vhiciecontrol stow vton thes any "1 10 Fate 2 $ 3g : sQg a 8 I enve non 3 8 3 g 3oC LT Te ce . g or g 3 2 EEBs omen TT EE EE sc 5 g8 3 i $ 2 2 :gSs :a LOD me hee EE wl add Lf EERE RE es : 8 &$ S pe ar d He EE En encEe HteEsEero pTeEr erin en da 10 of estan. 2 ob So HRT HIE EE A LL TE mates sc cs penn tae cogs ee pe pu, 3 8BE RR or a ee 8g 2 SULDEDmmE Ta me me wpe CEREERmmaee 2g :i = PT < ree $ . VIRAL SR pv en g g3 3 2 : 8:i :; Z g g Ramer En g 82 3 2 5 : : i a 2 a :g in Loos ee RETIRE) rom the vehicle control group valve (pes. 01) LOSE eer ee eas aa 3 3 2|- o 8 , CEEem} 5 FR SH 8 2 TR SE 2 3 R 2 BRE yyy ge Bly HEME wm mo meow woo BE Le ni Je 8PR bF ESEIE TEES ta m -- nI tT oS 5 r rA eR A . SR & SE i8 2 2 biges TUE :a nom ou 8 Era : o mmm wom roRTION OFTAL MISSING wn oe oro oe a ee Ro ats ass JY. Dn hint on Berrien sean 1 SOSBMTION ra pane IE, wf oe 3 os 2 8  To omnomhnonnlofnnouho : i g :i; EE ERen tes ss er i5g + HR AEE g SE ge eevee i 8 2 8 5 + RY BR TR SEE g i g en Soi oocstr. omanes ass. on sss v TE | - rrr IE bBo was asm asee esa o Hn} SCR IDTE Sn a 2 8223 2 8 a & | BERL s gi a ig :: ` APPENDIX D REPORT TABLES - F1 GENERATION MALE RATS 000120  bu sou worane wo oe SA Mo nck SecEsEn We 5 Si 0 2 LPIA IOSSIMEINCIOEICE (1S3 WATERR C1 BTS TAINED $8 Chir iose ee i 3 2  meu mess olan all al eee SEI ESwiE e ces ecretrce on e . n e i R 2 Nv simi Glin rom he icles conrol Seock verona oh) 2 g Wan ws anes eran ar s po a= - EE 2 Se - tamrens Feeeohwicn fnSat wighis were Fecorded on the day cohabitation vas begun for the F1 generation rata; at that ti 3 : nN + SiamiEicamly different trom the vehicle control grant waren 050 28) gv BEEISERIENEEERRSram ---- g 33 2 | we mse masa ian g. g infST e iinL EG0 rEeI ie ESiEe ERaRnhsBn Ics sSsAapIne, a 0.50 pe 2i 8 8 a3 | a aa 8Sg 3 gg ? i 2 :gSg8 :a g S 3ig2 TERhte andSessions canton Muse 1 Seving vere seeLCedbys coon Bra 38 3 28 8 5 2 . . Ss 5 2 R g g2g :2 2 ERE ne ot men. s 2g g: RB ; 2 i n EySE ae RTE Sf ates ec 93H here kp deta s a 3 83 8 APPENDIX E REPORT TABLES ~ F1 GENERATION FEMALE RATS 000133 DE F---- hd Sith !oT D TRe AEp FEREE Z ee o : 3 ics ia * Wom _= i eo ia Si rs 2g a5 EE B winsome wia oug roue y tn . 2 g :833 T ST " o- 1 s2ecssass estan asespor ::. i:.: & 2 LEEse rss on ce oy nition ut of rain 80 4 tn o 8 HAIER mm ree wm i 2 2 & 7 :o BHE EETTRE IEEmeTshsotmhsaeiretccoemtongr2e1mgaroeetienntm onon mot 3 | me sn Tgg & 2 Hn i ou iI FY RE TE 0 EE TE 1.m seris ee ite epeevsr 5  i 2 3 g 8&  : 3 88 B 323 B mn : i J A A Te ve eeen 3s. &> 2 g 235g 32 mn  me TotrIoessoctk 8 SadSopp be vocoo. CorieForro oompee Dope $e 15:57 puatusaing wes 3 - a :3 5 mn TLEE, se 82 I Hmm afin hm dria Sn sm e- d a <a o 232 a m 2: EL i. 8 + Signiticancly dileremt (rom the vehicle control Jroup vive (52s 1) 2 o EME g aE : g a2 2 ~ 8  3CBecEaine i 1 preniona Chal A The bus Lente to commune maternal feed afcex day 10 of laceacion, mater) feud consemtion vatens 2 2SSR Sl 8 G2 Ria Sn m regu rolemiean) 3 382 @2g &g8 + Someieantly sitterent ronthe Sebcoebloh areup valve (so. 081 3 :: A Lr rt g 2 = sy alSg me g 8 m CyLliS miWoa of ede wr Sob 3 sek EE : i ~ mn g 2: 8 i : : 2 2 m wm, coun _ na : SU en or mises awnmaen ce z 3 ERICEIRA emi 3 4 ROTI nm pn na i R Boo ED WE mg Wa ERANRIRIEREEIRRAA i g nm oan an ak ; Bs orn ows wis sa see ne wh 2 EES pean g I 3g ora es.0. es 20 :ws. 2 i wh. DHTTTT 0 mpasiivtommen 2 Fre seaie iba eoaoteitnts _ weeerred OE - Ti Sianiticuntly aifferene" trom thevenSiomrrcelhproep value (p8.03. g . m EE dT pte a on oe s 2gs i83 i te is in 3 GE iii ei ian SA $ g R28s 22 m 8 APPENDIX F PROTOCOL AND AMENDMENTS 000161 418-009.PAGE F-1 neseanc Argus30R5esSehaerschhy LDarbiovrea,tBourlileds,inigncA. Fr Asonaronies HorTSs2h1a5m,usPaeTnnIsDylFv2a1n5i)a401598054847 -------------------------------------- STUDY TITLE PURPOSE: TESTING FACILITY: STUDY DIRECTOR: SPONSOR: PROTOCOL 418-009 SPONSOR'S STUDY NUMBER: 6316.5 PCeormibnaitnaeld/POorsatlna(taGlavRaegper)odFuercttiliiotyn, TDoxeivceiltoypSmteundtyalofand N-E{FOSE in Rats. d`Tihsetuprubrapnocsees orefstuhlitsisngtufdryomisNt-o EtetsFtOfSorEtotxriecatefmfeencttso!f Ccorh:abCiDtatBioRnVtAhFr/oPuglhusmatmiangl,e gaensdtafteimoanlaenrdatlsacbteatfioorne. This AsttuhdryoeuvgahluFaotfesthIeCrHepHraordmuocntiivseedprTorcipeasrstiatendGusihdoeullidnedestteacgtes effects on the estrous cycle, tubal transport, implantation, gfeesmtaalteiorna,tsp,arotnurtithieond,evlaecltoatpimoennatondf mthaeteofmfaslprbienghaovfitohrein fturnecattieodnamlalefefeacntds f(ee.mga.,leefrfaetcst.saonndlpibeirdmoitordeetpeicdtiidoynmaolf sperm maturation) that may not be detected by histological emaxnaimfiensattaitoinosnsofofmaeflfeecrtast irnedpurcodeudcdtuirvienogrgtahniss.perBieodcamuasye be tdherloauygehd pirnotdhuectoiffosnproifngF,2ogbesneerrvaattiioonnsfitwtierls.be continued Argus Research Laboratories, 905 Sheehy Drive, Building A Inc. THeolresphhaomn,e:Pen(n2s1y5i)va4n4i3a.671190044-1267 Telefax: (215) 443-8587 Raymond G. York, Ph.D., DABT Associate Director of Research 3M Toxicology Services 3M Center, Building 20-26-02 St. Paul, Minnesota 55144-1000 000162 418-009:PAGE F-2 Protocol 4P18a-g0e028 STUDY MONITOR: TMealneipnhoTn.e:Case(6,12D).V7.3M3.,.5P1h8.0D. Telefax: (612) 733-1773 ASTLUTDEYRNMAOTNEITOR: Andrew M. Seacat, Ph.D. Telephone: (612) 575-3161 Telefax: (612) 733-1773 REGULATORYCITATIONS: ISnttuedrynaDteiosniaglnCaosnfMeordeifnicceatoinonHaofr:moUn.iSs.atFiooond; aGuniddeDlriunge Aodnmidneitsetcrtaitoinonof(t1o9x9i4c)i.ty to reproduction No. 183. for medicinal products. Federal Register, September 22, 1994, Vol. 59, 2U1.S.CFFRooPdaratn5d8.Drug Administration. Good Laboratory Practice Regulations; Final Rule. Japanese for Safety Ministry Studies of on Health Drugs, aMnHd WWelOfradriena(n1c99e7)N.umGboeord21L,abMoarracthor2y6P,ra1c9t9i7c.e Standard `EaucrcoeppetaannceEcboyntohmeicEuCroompmeuanniEtcyo(n1o9m89i)c. CoCmoumnucniiltdyecoifsaionnOoEnC2D8 Jduelcyis1i9o8n/9roencotmh-e mendation on the European Ccoommmpulniiatniceesw:itLhepgrisilnactiipolne.sof32go(oNdo. lLab3o1r5a;to2r8y pOrcatcotbiecer.): Of1f-i1c7i.al Journal of REGULATORY COMPLIANCE: Trehgiuslsattiuodnyswcililtebdeacboonved.ucted in compliance with the Good Laboratory Practice (GLP) ADlilrecchtaonrgaensdotrhereSvpiosinosnosr,ofdtahtisedpraontodcomlaisnhtaallinbeeddwoicthumtehnetperdo,tosciolgned by the Study Tanhde wQiullaliintsypAecstsucrriatinccalepUhnaiste(sQAofU)thweilsltauuddyitinthaeccporortdoacnolc,etwhiethrtahwedSattaanadnadrdtOheperreaptoritn,g Procedures of Argus Research Laboratories, Inc. TachceurfaintaellyrerpeofrltecwtislltihneclruadwedaatsataotbetmaeinntedsidgunreidngbtyhtehpeeSrtfuodrymaDnicreecotforthtehastttuhdeyraenpdortthat aslilgnaipfpilciacntabdleeviGaLtPionrsegfurloamtiGonLsPwerergeulfaotliloonwsedocicnurt,heecaocnhduwicltl of be the study. described Should in detail, together with how the deviation might affect the quality or integrity of the study. 000163 418-009:PAGE F-3 Protocol 41P8a.g0e039 STUDYSCHEDULE: See ATTACHMENT 1 to the protocol. JA ESTRTIANC D VEL HICE LE: ifiat TestArticle: PNhaymsei:cal Description: ~~ SLpoevcBiafitcchGraNvuimtyb:er: Purity: Expiration Date: WNa-ExtyFsOoSliEd.. FM-3029 "17. (30035, 30037, 30038) 9Ma9y.,1%2000. wIintfhortmhaetiSopnonosnort.he identity, composition, strength and purity of the test article is on file Vehicle: D0e.i5o%niTzweedeWnater8)0.inSuRpepvleiresreanOdsmlootsiidsenMtiefmicbartiaonneofPTrowceeesnse8d0 DteoiboenidzoecduWmaetnetred(Ri.nO.the raw data. tNoeibteheprretsheenStpionntshoervneohrictlheetShattudwyouDlidreicnttoerrfiesrae wwairthe tohfearneyspuotsenotfitahliscosntutdaym.inaThnetrsefliokreel,y no analyses other than those mentioned in this protocol will be conducted. Safety Precautions: fGloorvmeusl,atimoanskpr,epaaprpartoiporniaatnedeydeospargoteecatdmiionniastnrdataiounn.ifoTrhmeilMaabtecroiaatlaSraefetotybDeawtaomShdeuerting (MSDS) is attached to the protocol (ATTACHMENT 2). Storage: VBeuhlikcTleesCtoAmrptiocnlee:nts: Prepared Prepared Vehicle: Formulations: RRoooomm tteemmppeerraattuurree.. FRrooozmente(m-p2e0raCt).ure. AJlullitaenstGaurltbiicnlseksih,iMpamnenatgsertootfhFeorTmeusltaitnigonFasc,ilaittytshehopurledviboeusaldydcrietsedseadddtroetshse aanttdention of telephone number. 000164 418-009:PAGE F4 Protocol 41P8a-g0e0s9 c`aSrhtiopnmsensthsouslhdoubled include labeled aipnpfroorpmraitaitoenlyc.onTcehrenirnecgipsiteonrtasgheocuolnddibteionnostiafinedd shipping in advance of shipment. EQRMULATION: Ereqouf Perepnaractiyon Formulations (suspensions) will be prepared daly at the Testing Facllty. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). AdjustfomrPeurnitty: The test article will be considered 100% pure for the purpose of dosage calculations. Testing Facility Reserve Samples: tThheecSopuornsseoorf will this reserve study. TahseaTmepsltein(g1 Fga)ociflteyawcihl lroetsoefrtvheeabuslakmtpelset a(r5timcLle) uosfeedacduhrliontgof tuhnedevrehtihceleprceovmipouosnleynctisteudsceodnddituiroinnsg.the courseof this study. Samples will be stored ANALYSES: `thSeamcpoluersseadodfittihoenasltutdoy.those described below may be takenif deemed necessary during Bulk Test Article Sampling: INnofoarnmaaltyisonesoonftthheesbtualbkiltteysotfartthieclbeuwliklltbesetcaortnidcuiectisedondufrilienwgitthhethceouSrpsoensoofrt.his study. Analyses of Prepared Formulations: Stability: cSotanbdiiltiityondsatofa tfhoirspsrteupdayraerdefoornmfuilleatwiiotnhstbhreaSckpeotnisnogr tahnedrawnilglenootf cboencdeentterramtiinoends adunrding the conduct of this study. Suspensions will be prepared daily at the Testing Facilfy. 000165 418-009:PAGE F-5 Protocol 41P8a-g0e0s9 `Homogeneity Analyses: `Hcoomuorsgeenofeithtiysosftutdhye. teAstsayrrtiincglee iwnillprbeepaurseedd suspensions to withdraw wil be verified samples (5 mL during each) the from the tEoapc,hmisdadmlpelaen(d5 bmoLt)towimllobfethdeivhiidgehdesitntcootnwceonatlriaqtuiootns,oonnteheof2firsmt Ldaaynodf oprneepaorfat3iomnL.. aOtntehealTieqsutoitn(g2FmacLi)liwtiyllasbeasbhaicpkpuedp fsoarmpalnea.lysiBsa;ctkhueposthaemrplaleisquwoiltl(b3emsLt)orweildl ubnedreertatihened previously Sponsor. cited conditions and discarded at the Testing Facility upon the request of the ConcentrationAnalyses: oCfontchiesntstruadtyi.onAofsytrhienpgerewpilalrebde tuessetdarttoicwlietshudsrpaewnssiaomnpslewsill(5bemLvereiafciehd)dfurroimngetahcehcourse c(5onmcLenteraacthi)onwildlurbiengditvhiedefdirsitntaondtwsoixatlhiqwueotesk, of dosage administration. one of 2 mL and one of 3 Each sample mL. One aliquot T(2esmtLi)ngwiFlalcibleitsyhaisppaebdafcokruapnaslyasmipsl;e.theBaotchkeurpalsiaqmupotle(s3 mwiLll)bweillstboererdetuanidneedr atthethe previously Sponsor. cited conditions and discarded at the Testing Facility upon the request of the Shipping Instructions: `Samples to be analyzed will be shipped (frozen on dry ice) to: Kris J. Hansen, Ph.D. 3M 935 Environmental Bush Avenue Technology and Safety Services Building 2-3E-08) TSte.lePpahuoln,eM:inn(e6s1o2t)a77585-163031-83331 Telefax: (612) 7786176 Both the recipient and the Study Monitor will be notified in advance of sample shipment DISPOSITION Prepared article will formulations be returned twoiltlhbeeSdtiusdcyarMdoenditaotrtahte tTheestpirnegviFoaucsillyyc.iteAdll remaining address. bulk test 000166 TEST SYSTEM: 418-009:PAGE F-6 Protocol 41P8a.g0e0t8 aTbenhcdeadCuesrveie::lCo1Dp)mteBhnRitaVslAtrFta/oixPnilonufssraant(dhSaphsraasbgebueeene-Dndaewwmilodeneyls)ytrruaastteweddatsthosrbeoleuegschetoneusdtitaiisnvdetuhstetorTryeefspotrroSdyucsttievme reproductive and developmental toxicity evaluations; 2) historical data and experience sexpiesctiaetstahnedTestsrtaiinn.g Facility"; and 3) the test article is pharmacologically active in the Number Initial population acclimated: 195 virgin male and 205 virgin female rats. Population selected for study: 175 male rats (35 per dosage group) and 175 female rats (35 per dosage group). Ten mated female rats per dosage group will be assigned to Caesarean-sectioning on dliatyers10 of presumed gestation; the remaining female rats will be permitted to deliver A total of 250 F1 generation pups (25 per sex per dosage group) will be selected at weaning on day 21 postpartum for continued postnatal observation. Body Weight and Age Male rats will be ordered to weigh from 300g to 325 g each at receipt, at which time they will be expected to be at least 60 days of age. Female rats will be ordered to `weigh from 200g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights will be recorded the day after receipt and will be documented in the raw data. The weight ranges will be included in the final report. Sex: mBaotlhe FaondanfedmFa1legerantesrawitlilobnemgailveenantdhefteemsatlaertriactlse will be evaluated. Only Fo generation Source: Charles River Laboratories, Inc., Raleigh, North Carolina. LTahbeorraattosriwielsl,bIencs.h,itpoptehdeiTnefsitlitenrgedFaccairlittoyn.s by air freight and/or truck from Charles River 000167 418-009:PAGE F-7 Protoca 41P8a.g0e0?8 Identification: EGoeneration: CRoa.t,sIanrc.e, pNeor.maMnSePntTly20i1d0e1nt)i.fieMdaulseiangndMofnemeallesrealtfs-paireercaisngsieganredtatgesm(pGoeryarByannudmabnedrsTaatg receipt before aadnmdingiisvtreantiuonniqoufethpeerfimrastndeonstaigdeenotifftihcaetitoenstnaurmtibceler.s when assigned to the study E1/F2 Generations: iPnutpesrmwisllonfotthebelititenrd.ivAidtuawlelayniidnegn,tifeiaecdhdurratinsgelleaccttaetdiofno;r aclolnptairnaumeedtoebrsserwivlaltbieonevwialllubaet.ed identified with a Monel self-piercing ear tag ANIMAL HUSBANDRY: All cage sizes and housing conditions and Useof Laboratory Animals. are in compliance with the Guide for the Care Housing: Eo Generation Rats/F1 Generation Litters: eFxocegepntedruartiinogn trhaetscwoihlabbietaitnidoinviadnudallpyoshtopuasretduminpsertiaoidnsl.essDsutreienlgwciorhea-bbiottattioomne,d ecaacghespair of rats will presumed be housed gestation, in Fo gtheenemraalteiornatf'semcaalgee.ratBsegaisnsniignngednoto lnaatetrurtahlandedliavyer2y0wiolfl be individually housed in `common nesting box dnuersitnigngthbeoxpeoss.tpaEratcuhm dam and period. delivered litter will be housed in a F1 Generation Rats/F2 Generation Litters: Ahfotuesrewdeainnipnagi,rst(heonFe1mgaelneerraattipoenr rfaetmsawlilel rbate) idnudriivnigduaclolhyabhiotuatsieodn,baefnodreincdoihvaibduiatlaltyion, hFoougseenderaafttieorncorhatasb.itaBteigoinn.niTnhgensoalmateertytpheanofdacyag2i0ngofwipllrbeesuumseedd gaesstdaetisocnr,ibed for the dFe1ligveenreerdatliitoenr fwiellmableehroatussweidllibneaicndoimvimdouanllnyehsotiunsgedboixn dnuersitnigngthboexpeso.stpEaarctuhmdpaermioad.nd 000168 418-009.:PAGE F-8 Protocol 41P6a-g0e0s9 NestingMaterial: Bedding delivery. material (bed-0'cobs) will be supplied to female rats assigned to natural ABneadldyisnegswiflolrbpeoscshibalnegecodnatsamoifntaetnioans anreececsosnadruyctteodkeanenpuatlhleyaaninmdaldsocdurmyeanntdedclienanthe raw data RTooemAimr,peraatnduHumridiety: The animal room fresh air that has biseienndeppaesnsdeednttlhyrosuugphpl9i9ed.9w7it%h HatEPleAasftiltteenrs c(hiarnogCelsepaenrhroouorm)o.f 100% Rcoonsotmanttleym.perRaotoumrehwuimlidbietymawiilnltaalisnoedbeatm6on4itFor(e1d8cCo)nsttoan7t9lyFa(n2d6mCa)inatnadinmeodniatto3r0ed% to 70%. Light Amaninatuationmeadt.icaElalcyhcodnatrrkolpleerdio1d2-whilolurbeiggihtn: 12-hour at 1900 dark fluorescent hours EST. light cycle will be Diet RaadtlsibwiitlumbefrgoimveinndiCveirdtuiafliefdeeRdoedres.nt Diet #5002 (PMI Nutrition Intemational) available: Water: Wanataeurtowimlaltbiec waavtaielraibnlgeaacdcelisbsitsuymsftreomm. iAnldlivwiadtuearl bwioltltlbees fartotmacaheldoctaol tshoeurccaegeasndorpfarsosmed tphrroocuegshsedrweavteerrseasosamobascitsermioesmtbatr;apnreocbeesfosreed uwsaet.erChisieorxipneectwieldl bteo caodndteaidntnoothmeore btahcatner1i.a2l pcopnmtacmhiincartinieonatatnhdettwiimceeoafnnanuaallylsyisf.or Wpoastseirblies acnhaelmyizceadl mcoonnttahmliynaftoiropno.ssible Contaminants: tNoeibteheprretsheenStpionntshoercenrotriftiheed Sditeut,dythDeirdercitnokrinigs aware water of or tahneynpeostteinntgiamlatceornitaalmaitnalenvteslslitkhealyt `rwoouutlindeilnytperefrefroerwmietdh tbhyetrheesufletesdosfuthpipslsitouedry.thTohseeremfeonrtei,onneodaninalthyissepsrootthoecrotlhwainll those be conducted. 000169 418-009:PAGE F-9 Protocol 41P8a-g0e0s9 RANDOMAINDZCOAHAT BITIATOION N: EGoeneration: Upon arrival, rats will be assigned to individual housing on the basis of computergenerated random units. After acclimation, male and female rats will be selected for study on the basis of physical appearance and body weights recorded during acclimation. The rats will be assigned to dosage groups based on computer-generated (weight-ordered) randomization procedures. Within each dosage group, consecutive order will be used to assign rats to cohabitation, one male rat per female rat. The cohabitation period will consist of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ wil be considered to be at day 0 of presumed gestation and assigned to individual housing. Female rats not mated within the first 7 days of cohabitation will be assigned alternate male rats that have mated (same dosage group) and will remain in cohabitation for a maximum of seven additional days. The first ten female rats per dosage group with a confirmed date of mating wil be: assigned to Caesarean-sectioning on day 10 of presumed gestation. The remaining female rats will be permitted to naturally deliver liters. Five rats per sex per dosage group wil be assigned to a pharmacokinetic sample collection male rats at scheduled sacrifice. per dosage group from A table among of random those that units will be successfully used to mated a select five female rat paesrsiiogdn)eadntdo tnoatsuerlaelctdefliivveefryem(aslceheradtuslepderfogrrsoaucpriffricoemaaftmeorncgomtphloesteiotnhaotfdtehleivceorheadbitaltititoern (scheduled for sacrifice on day 21 postpartum) E1IF2 Generation Pups: Day 1 which of all lpaucptsatiionna(lpiottsetrpaarretuimn)divisiddueaflilynewdeaisghtehde dayofbirth and is (pup body weights also will the first day be recorded on after all pups in a litter are delivered and groomed by the dam) Oannddlaiyte4rspwoilsltpbaerrtuemd,ucaedtatboleeoifghrtapnudposmeaucniht.s wWilhlebneevuseerdptoosssieblleec,ttphuepssatmoebenucmulbleerd,of male and female pups per litter will be continued on study. wAitllwbeeanuisnegdotfotsheeleFc1t g2e5nemraalteioanndpu2p5sfoenmadlaeyp2u1pspopsetrpagrrtouump,, aretsaubllteinogfirnaantdootaml uonfi2ts50 F1 generation rats (125 per sex) chosen pup and one female pup per liter, when for continued evaluation. possible, will be selected. At least one male 000170 418-009:PAGE F-10 Protocol 4Pa1g8e-0100 ADMINISTRATION: RR outee andasfoo r Chon ice: d`Tiheetaorryalro(ugtea,vatghee)erxoaucttedwoassagseelceacntebdefoarccuusreatbeelcyauasdem:ini1s)teirnecdo;mapnadri2s)oint iwsitohnetohef the possible routes of human exposure. Meat nd Fh reqo uend cy: Dosages will be adjusted for the most recently approximately the same time each day. recorded body weight and given at Eo Generation Male Rats: M(amlaexiramtusmwi1l4 bdeaygsi)veanntdhecotnetsitnauritnigcltehornocugehdtaihley dbaeygibnenfionrge 2s8acrdiafiycse.beMfoarlee croahtasbwiitlal tbieon sacrificed after completion of the cohabitation period. Eo Generation Female Rats: Female rats cohabitation wil be given the (maximum of 14 test article days) and once daily continuing tbehgrionungihndga2y89doafysprbeesfuormeed agsesstiagtnieodn t(orantastaursasligdneelidvetroyCtaheastardeoann-ostedcetliiovneirnag)l,ittdera)yor24daoyf p2r0epsousmtepdargteusmta(triaotsn t(hraatts delivera liter), E1 Generation: eFx1pgoesneedrattoiotnheptuepsts awritlilcnleotdubreindgirmecattleyrgniavlegnetshteattieosnt (airntiuctlee,robuetxpmoasyurbee) poorsvsiiablmyaternal milk during the lactation period. Rationale for Dosage Selection: Dosages wil with the test abrteiclsee.lected by the Sponsor on the basis of previous studies conducted 000171 418-009:PAGE F-11 ---- a DC osaogenLevcele s, ntratai nd o Voln ums es: my | com . C Co oTToeeT Ta TT iTremor ncmeem]n] CCvTeo|To eT T ToT[eT eveesmmuonmionm|n ere ter ---- ViEaSbTiSl,itAy-NMAaLloYSanEdSEAoNmDalMeERAaSt:UREMENTS - Fo GENERATION: All Periods: At least twice daily. Accimaton Paid: Clinical Observations and/or Atieastonce General Appearance - Male and Female Rats: Dosage Perot: Tuics daly. Piro dosage administration and once approximately one hour postdosage. Maternal Behavior: Days 1,4, `abnormal b7e, ha1v4ioarndwil2l1bpeosrtepcaorrtduemd. Any daily. observed Clinical observations may be recorded more frequently than cited above,if deemed AScoclcmaWteiiognhPtesri- dl: e Rats: Attesstonce. appropriate by the Study Director and/or Study Monitor. Sacre: Dosage Period: -- Weekly. 000172 418-009:PAGE F-12 Protocol 4Pa1g8e-0102 BW odyeig-Fh emat leRas ts: Acclimation Period: Atleast once. Dosage Period: Weekly to cohabitation. Daily during presumed gestation and on Days 1, 4, 7. 10 and 14 postpartum (rats assigned to natural delivery). Sacrifice: Terminal weigh. Feed Consumption Values - Male Rats (recorded and tabulated): Dosage Period Weekly Eeed Consumption Dosage Period Values -Fe (recorded and tabulated) Weekly to gestation. cohabitation. Days 1, 4. 7, Daily during presumed 10 and 14 postpartum (rats assigned to natural delivery). tFheaetdpucposnswuilmlpbteigoinnwtilolcnoontsbuemteabmualtaetrendalaffteeerd.day 14 postpartum, when it is expected Feed Consumption Values - Male and Female Rats Fneeceedsscaornysutmoprteipolnenviashlutehsemfaeeyd.beDurreicnogrdceodhamboitraetiforne,quwehnetlny ttwhoanractisteodcacbuopvyethifeitsiasme cage with one values will not fbeeedrejcaro.rdreedploernitasbhumleantetd.of the feed jars will be documented. Individual Estrous Cycling and Mating: A table of evaluation random units will be used to select of estrous cycling by examination 15 female rats per of vaginal cytology dosage group for for 14 days before the start of the cohabitation period. oDubrsienrgvecdohianbiatastmieoan,raolfl ftheemavlaegirnaatls cwiolnltbeentesvaanldu/aotredadcaoiplyuluanttiolrysppelrugmaits oozbosaeravreed in'situ Duration of Gestation The first pduurpatisioonbsoefrgveesdt.ation is calculated from day 0 of presumed gestation to the day the 000173 418-009:PAGE F-13 Protocol P4a1g8e00183 Eertility Parameters: Fertity Index (percentage of matings that result in pregnancies). Gestation Index (percentage of pregnancies that result in birth of live litters) Number of offspring per litter (ive and dead pups) Number of implantation sites. General condition of dam and liter during the postpartum period. Viability Indices (percentage of pups bom that survive 4 and 7 days). Lactation Index (percentage of pups bon that survive 21 days). Caesarean-Sectioning Observations: Rats will be Caesarean-sectioned on day 10 of presumed gestation. Placentae that appear abnormal (size. color or shape) will be noted in the raw data. The rats will be examined for number and distribution of Corpora Lutea Implantation Sites. Viable and Nonviable Embryos. (A viable embryo is oval or crescent shaped, pink. firm and enclosed in an amniotic sac pink to tan or filed with deep red clear fluid. A to black, soft nonviable embryo is amorphous. and enclosed in an amniotic sac small, pale filled with clear, cloudy, or opaque fluid.) Natural Delivery Female rats will be evaluated for: Clinical Observations During Parturition. Duration of observed). Gestation (day 0 of presumed gestation to the time the first pup is fLiersntgptuhpofdiPvairdteudritbiyonN-(1tipmeuposf dineleiavecrhy loitftelr)a.st pup minus the time of delivery of the. Litter Size (defined as all pups delivered). 000174 418-009:PAGE F-14 Protocol 4P1a8g.e00148 Pup Viability at Birth. E FICE: eRxaatmsinwialtliobne.sacrificed by carbon dioxide asphyxiation. Embryos will be discarded after NECROPSY. `Gesvreaoxlsfusartolimeoswniho(inacsthwaiballlllebtoeifsrsreuateansidenoexmdamuinininnteesudwtiralatllbnbeeucfurfsoeeprdseydtow1isl0le%lbeefcotrreomtanaleiinnceodfn,otrriopnloosgrsdrieobruleptofruapttruoorvfeiedaech cspoenctirfoilcatlilsysuceitsefdorbealnoyw,poasllsiobtlheerhitsitsospuaetshowillolgbiceadliesvcaalrudaetdi.ons of gross lesions). Unless Male and Female Rats Assigned to Pharmacokinstic Sample Collection gInroauddpiwtiilolnbteo atshseiagpnperdoptroiaatpeheavramlaucaotkiionnestdiecsscarmibpelde below, five collection. rMaatsleperratssewxillpebredosage dseellievcetrey.d ffermoamlaemroatnsgwitlhlobsee stehlatecstuecdcefsrsofmulalmyomnagtetdhoasefetmhaatlederlaitvaesrsediganelidftetro. naAttural s21chpeodsutlpeadrtsaucmri(ffiecemaalfeterractso)mpblleotoidosnaomfptlheesc(oahpapbriotaxtiimoantepleyri4odmL(mpaelre rraatt)s)wilalnbdeon day cruneotsliullletscihtneigdpsmfeerrnotummttohwietlhlienbfSeerpiioomnrmsveoedrinaaftocrelaayvnaaflryiosniztsoe.nseoTrnhudemrylsieviepcraerwaialntldobrmetaeuixbncetisasieandn,eddwcfeerinogtzrheienfdu,(ge7ad0.ndCT)ahe sample section for analysis. (lateral lobe) frozen and retained at -70C until shipment to the Sponsor After completion of sample (frozen on dry ice) to Kris J. collection, serum and Hansen, Ph.D. at the liver section samples will previously cited address be for shipped analysis Both the recipient and the Study Monitor will be notified in advance of sample shipment, Scheduled Sacrifice of Male Rats: nAfetcerrocposmyploefttihoentohofrtahceicc,ohaabbdiotmaitniaonl paenrdiopde,lvmiaclveisractesrawiwlilllbebesapcerriffoircmeedda.ndTahegrfooslslowing woeivratglhauonautstiowfnilul:ild)b.teesetTxehcs,eisetepedisdtaiensddywmiiilnlddeibsvei,dfupiaxrleoldsytiwaneteBiogauhniednd'ssaesnmodilnuratelitoavniefnsoeirdcl4fe8osrt(powo9se6siighbhloeeudrhswiisatthnoldaongtidhcen rreetmaaiinneidnginonreguatnraslwbilulffbeereredt1ai0n%edfoinrmnaeluitnraflorbpuofsfseirbelde1h0i%stofpoartmhaolliongical evaluation. The 000175 418-009:PAGE F-15 Protocol 4P1a8g-e00195 Scheduled Sacrific-e FemaleRatsAssi a ectioni aOnnddaaygr1o0ssofnepcrreospusmyedofgtehsetatthioorna,cifce,maabldeormaitnsawlilanbde psealcvriicfivciesdc,eCraaewsiallrebaen-pseerfcotrimoende.d, cUtoenrfiiromf athpepaarbesntelnycenoonfpirmepglnaantnattiraotns swiiltlebse. sAtlalionveadrwiietshwi1ll0b%earmemtaoinneidumin sulfide neutral to buffered 10% formalin for possible future evaluation. edule ifice - Femal i live eRaxtasmitnhaetddfoorngortodseslilveesrioans.lttUtweriilwbilel sbaecrsitfaiicneeddonwitdhay1205%oafmpmroensiuumemdsgulefsitdaetitooncoannfdirm the absence of implantation sites. 9Af1t0e5rScnoemcprloeptsioynofoftthheeth2o1r-adciacy, postpartum abdominal apnerdiopde,lvfiecmvailsecerraatswiwlilllbbeepesracfroirfmiecde.d, and The a `number and distribution of implantation sites will be recorded. Dams with No Surviving Pups: oDrapmrseswiutmhendocasunrnviibvailnigzepdu.psAwgilrlosbse nsaeccrriofpicseydoaffttehretthheorlaacsitc,puapbdisomfionuanld adneaddp,elmviiscsing viscera will be performed. summary tables. Postpartum data for these dams will be excluded from Rats Found Dead or Moribund `Reaxtasmtihnaetddfioer otrhearceausasceriofficdeedatbhecoarumsoeriobfumnodricboundnidticoonndointitohneodraaybotrhteioonbsweilrlvabteion is smeamdien.alTvheesircaltesswoilflmbaeleexraatmsiwnielldbfeoregxrcoisssedleasinodnsi.ndiTveisdtueasl,oerpgiadnidwyemiigdhetss,wiplrlobsteate and TBroheuceionrr'edsmeadsio(nlsuietnimgoinnoarflogravn4ess8iwctilole9ls6bweehroieugtrhaseidnaenwddiitnthhnaeennudtrreawtliatbihnuoefudftefirnleuidnde)1.ut0rT%alhfebourtmfeafsleitrnee.sdwi1Pllr0e%bgefnofarinmxacelydini.n dnseetluatitrvuaselraebndudfpfuueptreserdiwni1lel0cb%oenfteoexrnmatamlsiinon.fefdeUtmteoarlitehoefraeatxsptpewainrltlenpboteslsyriebncloeon.rdperdOe.vganraAinbeotsrrwtaietldls bfwieeltlrubesteeasisntaeandidnlieondrwith 10% ammonium sulfide to confirm the absenceof implantation sites. 000176 418-009:PAGE F-16 Protocol P1a8g.e00186 TM ESTSE ,ANAALYSS ESAU ND REME -F1N GENET RATS ION: Viability Preweaning Period Litters will be observed for dead pups at least twice ddaaiillyy.. The pups in each litter will be counted once Postweaning Period: Twice daily. ClOinicbal servanda /orGteneiralAoppenarasnce Preweaning Period: Once daily. Postweaning Period: Once weekly. Maternal Behavior: aDbanyosrm1.a4l,b7e,ha1v4ioarndwil2l1bpeosrtepcaorrtduemd.daAilnyy observed aCplipnriocparlioabtseebrvyatthieonSstumdayyDbireecrteocroradned/domrotrhee fSrteuqduyenMtolnyittohra.n cited above, if deemed BodyWeights: Preweaning Period: Postweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. Weekly. Presumed Gestation Period: Lactation Period: Days 0.7, 10, 14, 17 and 20 (female rats only). Days 1,4,7, 10 and 14 (female rats only). Sacrifice: Terminal weight. Feed Consumption Values (recorded and tabulated): Preweaning Period: Not recorded. Postweaning Period: Weekly except during cohabitation. Presumed Gestation Period: Lactation Period: Days 0,7, 10, 14, 17 and 20 (female rats only). Days 1,4, 7, 10 and 14 (female rats only). 000177 418-009:PAGE F-17 Protocol P41a8g-e00187 rFeepeldencioshnstuhmepfteiedo.n values During mcaohyabbietarteicoon,rdwehdemnortweofrreaqtsueonctclyupifyittihsenseacmesescaraygetowith one feed jar, values tabulated. will be documented when feed jars are filed. These intervals will not be. PreweaningDevelopmental Observations: cTohnetinnuumebseurntiolftphuepdsamyetehteincgrittehreiocnriitseraitotnaiinserdecboyradleldpuopnseiancthhedalyiteorf. testing. Testing Surface Righting Reflex (ability to right in 5 seconds): From day 1 postpartum. Pinna Unfolding: From day 2 postpartum. Eye Opening: From day 12 postpartum. Acoustic Startle Response: From day 13 postpartum. Air Righting Reflex: From day 14 postpartum. Pupil constriction is evaluated once, on day 21 postpartum. Postweaning lopmental Observations: `Sexual Maturation: Female rats postpartum. wMiallbeereatvsalwuialtbeed for the age of evaluated for vaginal the age poaftpernecpyu,tibaelgsienpnairnagtioonn,dbaeyg2in8ning on day 39 postpartum. Passive Avoidance Testing: wBhegeirneniponsgsiabtle2,4 w2il1l bdaeyevpaolsutaptaerdtuimn.aopnaessmiavleearvaotiadnadncoenetesftemfoarleleraartnifnrgo,msehaorcth-tleitremr, retention and long-term retention. PTlheexipgalsassivelidas.voiOdnaencceoampppaarrtamteunstcionsfiitstteds of a with atwbor-icghotmpigahrttmaenndt Pclheaximgblearswiftlohorh.inTgheed coutrhreerncto(m1pmaAr)tmceanntibs efdietlteidvewrietdh. aTgrhiedftlwooorctoompwahritchmeantbrsieafre(1sseepca)raptueldseboyfamislldideilnegctric idsooorp.enOend eaancdhttheestlitgrhatl,sttheumreatdisonp.laTchede irnattoitshaell"obrwiegdhtt"oceoxmpplaorrtemtehneta,ptphaerasltiudsinugntdioloitr tenutreerds tofhfea"nddartkh"ecobrmipeafrptumlesnet.of cTuhrerenstidiisndgeldiovoerreids tthoetnheimgmreiddifalotoer.lyTchloeserdat, itshtehleinght is srtearmtoovfetdhfernoemxtthteriaal.ppaTrriaatlussaraendreppleaacteeddiunnttoilathhoelrdaitngrecmaagiensfoirn3t0hes"ebcroignhdts" before the 000178 418-009:PAGE F-18 Protocol 4P1a8g-e00198 c1o5mtpriaarlstmheanvte fboere6n0csoemcpolnedtesdo.nTthweo claotnesneccyuttoiveenttreiraltsh(etdhearckricteormiponarfotrmleenatmoirngt)heor until maximum 60-second interval is recorded for each trial. tEhaechcrirtaetriisonteissttehdetswiacme.e fTorhebottehstdsaeysssoifontsestairneg.separated by a one-week interval, and Dosage groups are compared for the following dependent measures: t`oThceonmupmabreergorfoutrpisalsfotro otvheeraclrlitleeriaomnining the first session--this performance. measure will be used Tcohmeplaarttemnecynt(ionnsetrcioalnd1si)n ttoheenftiresrt ttehset"sdeasrski"onc-o--mthpiasrmtmeeansturferowmillthbee"ubrsiegdht"to `encvoimrpoanrmeegnrto.ups for activity levels and exploratory tendencies in a novel cTohmeplaarttemnecynt(ionnsetrcioalnd2si)n ttoheenftiestr ttehset"sdeasrski"ocn--otmhpiasrmtmeeansturferowmillthbeebursigehdt"to compare groups for short-term retention. `The number of trials be used to compare gtorotuhpescrfiotrerlioonngi-ntetrhme rseetcenotniodn.test session--this measure will `The latency (in seconds) compartment on trial 1 in to enter the the second "sdeasrski"onc~otmhpisarvtamleunetisfraonmotthheer "bright" indication of long-term retention. Watermatze Testing: eBaegcihnnliittnegr waitllabpepreovxailmuaatteeldy i7n0adwaaytserp-foisltlpeadrMtu-mm,azoenefomraolveerrtatcoaonrddionnateiofne,maslweirmamtifnrgom ability, learning and memory. iEsafcilhledrawtiisthtweastteedr itnoaa wdaetpetrhtiogfhtap1p6r-ogxaiumgaetesltyainnilneessinsctheeesl,moadnidfitehde Mwa-tmearzei.s moTnhietomraezde for temperature (range of 21C 1C). sOtnemeafcahrttheesstt tfrraol,mtthheertawtowiallrmbse)palnacderdeqinutioretdhetostsawrtiimngtopoosniteioonf t(hbeastewoofgtohaelsM-ofmtahzee eMn-tmearzbeo.thinaorrdmesrotfothbee removed from the maze before being water. On the removed from first the tial, the rat water. The is required inital arm to cfahilotsoenmaoknetraialco1rriescdtesgoiaglnactheodictehewiitnhcionrr6e0ctsgeocaolnddusriinngantyhegrievemnaitnriianlgartreialgsu. idReadtstotthhaet csoerpraercattgeoealacahndtriaal.re then Each removed from rat is required the water. to reach a Acri1te5r-isoencoofnfdivientceortnrsiealcuitnitevreval will `erforless trials to terminate the test session. The maximum number of trials in any test 000179 418-009:PAGE F-19 Protocol P41a8g-e00199 mseasxsiiomnumis 6150.-seLcaotenndciynt(emrevaalsuisrreedcionrdseedcofnordse)actho ctrhaolo,saestihsethceornruecmtbgeoraloforertrohres. (incorrect tums in the maze) during each trial. tEhaecchorrratecits tgeosatleadntdwitchee. cTrihteeritoenstasreestshieonssaamreefsoerpbaortahtetedstbyseassoinones-.week interval, and Dosage groups are compared for the following dependent measures: uTsheedntuomcboemrpoafrreiaglrsotuopscrfiotreroivoenroalnltlheearfniisntgdapeyrfofortmeasntcineg.--this measure will be fTihrset daavyeroafgteesntuinmgb--etrhiosfmeerarsoursre(iwniclolrraelcstotbuernussiendthteo cmaozmep)arfeorgeraocuhpstrfaolr oovnertahlel learning performance. The latency testing~this (in seconds) measure will to reach be used the correct to compare goal on groups tfroirals2hoorft-ttheermfirrsettdenatyioonf. bTeheusneudmbtoercoomfptaiarles gtorocurpitserfioornloonngt-hteersmecroetnedntdiaoyn of testing~-this measure will mTehaesauvreerawiglel anlusmobbeeroufseerdrtoorscofomrpeaarcehgtrroiaulposnfotrhelosnegc-otnerdmdraeyteontfitoen.sting-this The latency testing--this i(sinasneotchoenrdsi)ndtiocarteoarcohftlhoengc-ortreercmtrgeoteanltioonn.trial 1 of day 2 of ReproductiveCapacity: rbAteanaadpsposrmiogxunnieimdtastteoolfcyorh9aa0nbdidtoaamytisuonnoi,ft aotganebel,emst,ahleweiFt1rhatgtpeheneererxfactelimuoasnlieornartaostf,wsbiitabhsliienndgeaomcanthicnodgmosps.uatgeTerh-gegreonueprwaitled scophearbmiattatoizoona period will observed cinonasissmteoafraofmathxeivmaugminoafl 14 days. contents Female and/or a rats with copulatory plug c1o0obhsianebdriivtveaitdduiaiolnn shwiiotluulswibinelgl.absesFicegomnnaesdliedaelrrtaeetrdsnttahotaetbmedaoaltendorataytmsa0ftorefowmpirttehhisenusmtaehmedefgiedsstots7aatdgiaoeynsgaronofdupasthsaitgnheadve mated. 21-day pFoesmtapalreturamtspewriilodl.be allowed to naturally deliver and maintain ltters through a Mating Performance: As cited above for Fo generation rats. 000180 418-009:PAGE F-20 Protocol 4P1a8g.e02009 DuroafGtesitatoionn: As cited above for Fo generation rats. Fertility Parameters: As cited above for Fo generation rats. FG2eneraLitttierDoatna: Viability, as cited clinical observations and body above for F1 generation litters. weights for F2 generation pups will be recorded METHOD OF SACRIFICE - F1 GENERATION RATS/F2 GENERATION PUPS: As previously cited for Fo generation rats. NECROPSY - F1 GENERATION RATS: `Gervoaslusatlieosnio(nastwaiblleobef retained random uinnintesutwirlallbbeufufseerdedto1s0e%lefcotromanleincofnotrroplosgsriobulep future rat of each csoenxtrforlotmiwsshuiecshfaolrl atinsysupeossseixbalemihniestdopaattnheolcorgoipcsayl ewivlallbueatrieotnasinoefd,grionsosrdleesriotnos)p.rovUindleess specifically cited below, all ther tissues will be discarded. Scheduled Sacrifice - F1 Generation Male Rats: Rats will be necropsy of tsahceritfhiocreadciacf,tearbcdoommpilneatlioanndofptehlevic14v-idscaeyraclohwailblitbaetipoenrfpoerrmioedd.. A gross Testes, oeprigdaindywmeiidgehst.s wpirlolsbtaetereacnodrdseedmi(nsaemlivneaslicvleessicolfesmawleeigrhatesdwiwlilthbeanedxcwiistehdouatndidi)n.diviTdhueal ienpiBdoiudiny'msidseosluwtiillonbfeorre4t8aitnoed96inhnoeuurtsraalnbdutffheernerdet1a0i%nefdorimnanleinu.traTlhbeuftfeesrteeds wil be 10% fixed formalin. Scheduled Sacrifice - F1 Generation Female Rats: Female number rats and wdiisltrbiebustaicornifoifciedmpalfatnetractoiomnplseittiesonwiolf btheer2ec1o-rddaeyd.posRtaptasrtthuamt period. do not The deliver a litter 10% wailmlmboensiaucrmifsiuclefdidoentodacyon2f5iromftphreeasbusmeendcegeosftaitmipolnanatnadtiuotnersiitweilsl.beAstgarionsesd with necropsy without a coofntfhiermtheodramcaitci,nagbddaotmeintahlatadnod pelvic viscera will be not deliver a litter will performed. Female be sacrificed on an rats estimated day 25 of presumed gestation. 000181 418-009:PAGE F-21 Protocol 4P1a8g.e00t8 EG 1 ene RatsrFouandDt eadoirMooribn und: Rats that die or are sacrificed because of moribund condition or abortion will be Semexamadimeni.anleTvdheesfiorrcalttehssewoiclflamubaseleeeoxrfaadtmesianwtielhldobfreormeogxrrcoiisbssuendldeascnioodnndsii.ntdiiTovenisdtoueansl,toherepgidadaniydwyetmihiegdhoetbsss,ewirplvrloa5stti6aotneisand "rBToehuceionrr'desemdasio(nlsuietnmigoinnoarflogravn4ess8iwctiolle9ls6bweehroieugtrhaseidnaenwddiitnthhnaeennudtrrewatilatbihnuoefudfteifrnleuidnde).1ut0rT%alhfeborutmfeafsleitrnee.sdwi1Pllr0e%bgefnofarinmxcaeydliinn status and uterine contents of female rats will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible. Ovaries will be retained in neutral buffered 10% formalin. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites. E1 Generation Dams with No Surviving Pups: summary tables. Dams with no surviving pups or presumed cannibalized. A will be gross sacrificed after necropsyof the the last pup is found thoracic, abdominal dead, missing and pelvic viscera will be performed. Postpartum data for these dams will be excluded from E4IF2 Generation Pups Found Dead on Day 1 Postpartum: Pups that die status at birth. b efore examinationof The lungs wil be re mtohveeldittaenr dforimpmueprvsieabdiliintywawitlelr.be evaluated Pups with for vital lungs that sainndk twoillhabveeiddeinetdifsihedoratsy satfiltlebobrinr;thp. upPsupwisthwiltuhnggrsotshsatlefsloiaotnswilwlillbebeidpernteisfeiredveads ilnivBeobuoirnn',s esovlaultuiaotniofnosr,piotswsiillblbeefnutoutreed eivnaltuhaetnioenc.roSphsoyudladtapostmortem autolysis preclude these E1/F2 Generation Pups Found Dead or Moribund on Days 2 to 21 Postpartum: evaluations it wil be noted i the necropsy data Pups found dead or sacrificed due to moribund condition will be examined forgross. lesions and for the cause of the moribund condition or death. Pups with gross lesions. found on days 2 to 4 postpartum will be preserved in Bouin's solution for possible future `evaluation; gross lesions of pups found on days 5 to 21 postpartum will be preserved in neutral buffered 10% formalin. Should postmortem autolysis preclude these 000182 418-009:PAGE F-22 Protocol 4P1a8g-e00202 FAIF2 Generation Pups Not Selected for Continued Observation: F1 and F2 generation pups culled on day 4 postpartum will be sacrificed and examined for gross lesions; pups with gross lesions wil be preserved in Bouin's solution. Necropsy wil inciude a single cross-sectionofthe head at the levelofthe frontal-parietal suture and examinationof the cross-sectioned brain for apparent hydrocephaly. All F1 generation pups culled on day 21 postpartum will be sacrificed and examined for gross lesions; gross lesions will be preserved in neutral buffered 10% formalin. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examinationofthe cross-sectioned brain for apparent hydrocephaly. Scheduled Sacrifice - F2 Generation Pups: On day 21 postpartum, pups will be sacrificed and examined for gross lesions. Necropsy will include a single cross-section of the head at the level of the hfryodnrtoacle-ppahraileyt.al suture and examinationofthe cross-sectioned brain for apparent 000183  418-009:PAGE F-23 Protocol P4a18g-e00293 PROPOSEDSTATISTICALMETHODS'*: aApvperropargieastea.ndAdpdeirtcieonnatlagpersocwieldlubreescaalncdu/laotreda.nalLyitsteesr vmaalyuebsewiplelrbfeorumseedd, wifhaeprpreopriate. TypeofTest* I. Barametric A. Bartlett Test" Il. Nonparametric* A. Kruskal-Wallis Test (s75% ties) Significant at ps0.05 Not Significant Significant at ps0.05 Not Significant Nonparametric nlVariance J Test Significant at ps0.05 Dunnett's Test Not Significant B. Fisher's Exact Test (>75% ties) Il. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilties are reported as either p<0.05 or p<0.01 cb.. PUrsoepdorotniloyn tdoataanaalryezenodtaitnacwliutdhedhoinmothgiesnceaitteygoorfy.variance. d. Test for homogeneity of variance. 000184 418-009:PAGE F-24 Protocol 4P1a8g.e00284 DATAACQUISITION, VERIFICATIONANDSTORAGE: DDiarteactwoilrlabnedlhoarnadp-praonpdr/ioartceommapuntaegre-rmeecnotrdpeedr.soRneneclorwdisthwiinll21bedraeyvsiaefwteedr gbeynetrhaetiSotnu.dyAll original records will be stored in rbeequbeosutn.dParnedseirnvdeedxetdi.ssAuescowpily botfehaeslltaorrrcaehwdivdaeatsttaohfewitTlhleesbtTeienssgtuipFnpaglciiFeiadctiyltioattyt.hneoASlclphooarnrisggoienraflourpdooatnnae w il l year after mailing ofthe draft final report, after which time the Sponsor will be contacted to determine the dispositionof these materials. RECTOO BEMRAIND TAINSED: Protocol and Amendments. TARenasintmdaoAlmrtiAizccalqetu,iisVoienthiiSoccnlheedaunlde/so.r Reagent Receipt, Preparation and Use. Mating History. TGClreiennaeictramalelnOCbtos(meifrmveparntetsisco.rnisbaedndb/yorStGaefnfeVreatlerAinpapreiaanr)a.nce. Blood Sample Collection, Processing BFeoeddy WCeoingshutms.ption Values. and Shipment. (NLiaCttaeuerrsaaOlrbesDaeenrl-viSaveetcrityoinOosbn.sienrgvaOtbisoenrsv.ations. OGRerrfoglsaesnx NWaeencidrgohPphtysssyi(ciOabrlseqeDurievrvaeetdi)l.oonpsm.ent and Behavioral Observations - F1 Generation Pups. Photographs (f required). Study Maintenance (room and environmental records). Feed, Water and Bedding Analyses. Packing and/or Shipment Lists. KEY PERSONNEL: Executive Director of Research: Mildred S. Christian, Ph.D., ATS Director of Research: Alan M. Hoberman, Ph.D., DABT ADsirseocctioartoefDLiarbeoctroartoorfyROepseeraarticohnsa:ndJSothundyF. DiBreacteor,: BR.aS.ymond G. York, Ph.D., DABT MMaannaaggeerrooff ASntiumdaylCoOopredriantaitoionns:anVdalMeerimebeA.r,ShIanrsptietru,tiMo.naSl. Animal Care and Use Committee: Dena C. Lebo, V.M.D. Manager of Consultant, Regulatory Veterinary Compliance: Pathology: W . KRaathyleBernowAn., MDo.rVa.nM,.,M.PSh.. D. ACVP 000185 418-009:PAGE F-25 Protocol4P1a8g-e00285 EINALREPORT: A comprehensive draft finalreportwill be prepared on completion of the study and will be finalized following consultation with the Sponsor. The report will include the following: SExupmemriamreyntaanldDCeosnicglnusainond.Method. AEpvpaelnudaitcioens:of Test Results Figures, Summary and Individual Tables Summarizing the Above Data, Protocol and Assaciated Amendments and Deviations, Study Director's GQLAPU CSotmatpelmieanntc.e Statement, Reports of Supporting Data (if appropriate) and INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE STATEMENT: ITnhsetitpurtoiconeadluArneismadelsCcrairbeedanidn tUhsisepCroomtmociotltehea.veAbllepernorceevdiuerweesddbesyctrhiebeTdesitnitnhgisFapcrioltitoyc'osl that involve study animals will be conducted in a manner to avoid or minimize discomfort, distress or pain to the animals. nTehceesSspiotnysoforr'scosnidguncattiunrge tbheisloswtuddoycaunmdentthse ftahcetftahcatttthhaits iinsfnoortmaatniounnnceocnecsesranriinlgythe dpurpolciecdatuirveesswteurdey mavaayilbabeleobftoarimneeedtfirnogmtthheestSaptoendsopru.rpNosoeaslotfertnhaetisvetu(diyn.vitro) 000186 418-009:PAGE F-26 Protocol P41a8g-e02059 REFERENCES: 1. CThersitss.tianE,nvMi.rSo.nmaenndtaVloyPtreokt,ecPt.iEo.n (A1g9e82n)c.y,IWnaVsihviongRteopnr,odDu.cCt.ivNeatainodnaMluTteacghenniicciatly Information Service, U.S. Department of Commerce, Springfield, VA 22161 2. nCharlitsrteixaonn.eM.(SP.ro(c1e9e8d4i).ngsReopfrNoadlutcrteixvoenetoSxiycmiptyosainudmt,erNateowloYgoyrekvaAlcuaatdieomnsy ooff Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 3. CLoanntgr,olPLD.at(a19i8n8)t.he EChmabrrtyeos RainvdeFreCtrali:DCeDvelBoRpRmaetn.taClhaTroxliecsitRyiv(eTerrLaatboolroagtyo)ries, LInacb.o.raWtiolrmiiensg,toInn.c.)MA 01887-0630. (Data base provided by Argus Research 4. Institute of Laboratory Animal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 5. ISmaplleawnstkait,ioEn.ss(t1e9l6l4e)n. aFmarUbteemreusthdoedreRaztutem.maArkcrho.skPaotphiols.chEexnp.NaPchhawremiaskovlon 247.367 6. tShneedbeicnoormi,alGWdi.straibnudtioCno.chSrtaant,isWti.cGa.l M(e1t9h6o7d).s,V6atrhiaEndicteiotne,stlfoowrahSotmatoegUennieveirtosiyfty Press. Ames, pp. 240-241. 7. BSoikoamle,trRy.R.W.aH.ndFRrohelef,maFn.J.an(d19C6o9.,). SBaanrtFlreatntc'itsecsot,ofpph.o3m7o0g-e3n7e1ity of variances. 8 SMneetdheocdosr,,6tGhWE.ditainodn.CloocwharaSnt.atWe.GU.niv(e1r9s6i7t)y.PrAensasl,ysAimseosf,Vaprp.ian2c5e8.-275St.atistical . tDurnenaettmte,ntCs.wWi.th(1a95c5o)nt.rolA. muJ.ltAimpelre.coSmtapta.riAssosnocp.r5o0c:e1d0u8r6e-1fo1r29c.omparing several 10. WSoHk.al,FrReRe.maanndanRodhlCfo,..F.JS.an(1F9r6a9n)c.isKcrou,skpap.l-W3a8l8l-i3s89T.est. Biometry. 11. Dunn, .J. (1964). 6(3):241-252. Multiple comparisons using rank sums. Technometrics 12. MSiceGgreal.w-SH.il(1l95N6e).w YNoornkp,aprpa.me9t6r-1i0c4.Statistics for the Behavioral Sciences, 000187 PROTOCOL APPROVAL: FOR THE TESTING FACILITY 0. lol Alan M. Hoberman, Ph.D., DABT Director of Research A A Ramand G. York, Ph.0(, DABT Asst Director of Reseach Study Director 24S Lo o;de MDeemnbaeCr., Lienbsoti,tuVt.iMon.aDl.Animal Care and Use Committee FOR THE SPONSOR MSatruvdiynMTo.niCtaosre, D.V.M., Ph.D. 418-009:PAGE F-27 Protocol 4P1a8g.e02078 18 mm of Date 26-#1m) -95 _ Date 28 Nuss 95 Date Date 000188 418-009:PAGE F-28 ATTACHMENT 1 STUDY SCHEDULE 000189 ATTACHMENT 1 418-009:PAGE F-29 ProtocPoalg4e181-00r029 SCHEDULE 02JUN 88 08 JUN 98 08 JUN 98 - 20 JUL 98 08 JUN 98 - 01 SEP 98 23 JUN 98-06 JUL 98 06JULSBPM-13JUL9BAM 13JUL9BPM-20JUL9BAM o7JuL 38 204UL 98 03AUG 98 Animal Receipt - Acclimation Begins (Fo generation rats), Start of Dosage Period - Fo Generation Male Rats (28 days before cohabitation and continuing through a 14-day cohabitation period unti sacrifice after has been determined). successful mating Dosage Period - Female Rats Assigned to Caesarean-Sectioning (28 days before cohabitation and continuing through day 09 of presumed gestation). Dosage Period - Female Rats Assigned to Natural Delivery [28 days before cohabitation through day 24 of presumed gestation (rats that do not delivera litter) or day 20 postpartum (rats that deliver a ltter)] Dosage Period Estrous Cycle Evaluation Cohabitation Period Male 1(07 days) (Maximum of 14 days). Male 2 (07 days) First Last Possible Possible Day Day 0 0 of of Presumed Presumed Gestation. Gestation. FCoomGpelneetriaotnioofntMhaelCeohRaabtistaStaicorinfPiceerdioadft(eErarliest possible date) a. The study initiation date is the day the Study Director signs the protocol. 000190 ATTACHMENT 1 418-009:PAGE F-30 ProtocPoalg4e128.00i029 17JuL 98 300uL98 284UL 98 14 AUG 98 01AUG 98 14 AUG 98 17 AUG 98 03SEP 98 18 AUG 98 02 NOV 98- 16 NOV 98 30NOV 98 24 NOV 98 - 11 DEC 98 14 DEC 98 - 31 DEC 98 20 APR 99 First Possible Day 10 of Presumed Gestation Caesarean-sectioning. Last Possible Day 10 of Presumed Gestation Caesarean-sectioning, First Possible Delivery (Day 21 of presumed gestation). Last Possible Delivery (Day 25 of presumed gestation) First Possible Day 25 of Presumed Gestation Female Sacrifice. Last Possible Day 25 of Presumed Gestation Female Sacrifice First Possible Day 21 Weaning (Dams and F1 generation pups not selected for continued observation sacrificed). Last Possible Day 21 Weaning F1 Generation Postweaning Observations Begin (Details of tests cited in protocol) F1 Generation Cohabitation Period (Initiated when rats are approximately 90 days of age approximate inital date) F1 Generation Male Rats Sacrificed after Completion of Cohabitation Period Approximate Earliest Possible Date. Delivery Period - F1 Generation Dams/F2 Generation Litters (Approximate dates). Sacrifice of F1 Generation Dams and F2 G(eAnpeprraotxiiomnatLeitdtaetrseso)n Day 21 Postpartum Draft Final Report. 000191 413-009:PAGE F-31 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 000192 418-009:PAGE F-32 DMAATTAERISAHLEETSAFETY aM a Center N-E+FOSE 5St5.144P-au1l0,00Minnesota 1-800-364-3577 or (612) 737-6501 (24 hours) ACLoLpyrriigghhtt,s r1e9s98e,rveMdi.nnesCootpayinMginianngd/oarnddoMwannluofaadcitnugrinogf Company. this iisnfaolrlmoawteidonprfoorvidtehde tphuartp:ose of properly utilizing 3M products 1) pthreiorinfaogrremeamteinotn iiss ocobptiaeidnedinfrfoumllaMw,ithandno changes unless 2) dneiistthreirbuttehde wciotphy nthoer tihnetenotriiogninaolf iesarnriensgolda oprrofoitthertwhierseeon. TDIRVAIDSEIONNA:ME: 3M CHEMICALS 10FCN-U1M0BERF/LUU.OPR.ACD.:Brand Fluorochemical Alcohol 898--00221111--11517153.-77 0000--5511113355--0029144955--32 9988--00221111--16168230--06 2F-0002-0572-2 - . SIUSPSEUERDS:EDEJSa:nuaNroyvem29b,er 10959,8 1997 DOCUMENT: 10-3778-7 0000--5511113355--1009454329-.32 1. INGREDIENT PPEERRFFLLUUOORROOHOECXTAANNEESSUULLFFOONNAAMMIIDDOO AALLCCOOHHOOLL............ PPEERRFFLLUUOORROOBHUETPATNAENSEUSLUFLOFNOANMAIMDIODOALALCCOOWHOOLL........... PERFLUOROPENTANESULFONAMIDO ALCOMOL..... 2. PHYSICAL DATA C.A.S. NO. 314649515--9083--23 3648454595-.8793-.37 68555-72-6 PERCENT 80.0 3.0 - 90.0 - 7.0 2.0 2.0 - 6.0 . 6.0 1.0 - 3.0 BOILING POINT:................. ca. 118 C VAPOR PRESSURE:................ <101mmmmHgHg VAPOR DENSITY:................. >ca1l.c0 A@ir2=0t EVAPORATION RATE:.............. <ca1l.c0 B@uO2A0c=Ci. SSPOELCUIBFIILCITGYRAIVNITWYA:T.E.R.:...................... cnae.gli1g.7 Water=1 PERCENTVOLATILE:.............. 0(%of melt) VBHIISCOS.I.ToYv:u.i.i..l..i..l.i.0..e.0.0s.00 NNiIpA MELTINGPOINT: ....000000000000 wip 000193 Abbreviations: N/D - Not Determined N/A - Not Applicable CA- Approximately 418-009:PAGE F-33 MJSaDnSu:aryFC2-91,0 F1L99U8ORAD Brand Fluorochemical Alcohol PAGE 2 2. PHYSICAL DATA (continued) APPEARANCE AND ODOR: Amber waxy solid 3. FIRE AND EXPLOSION HAZARD DATA FFLLAAMSMHABPLOEINTL:I.M.I.T.S..= ..L.E.L.:................ > 148 N/A C Setaflash AFULTAOMIMGANBILETIOLNIMTIETMSPE-RAUTEULR:E.:............. N/A N/A EXTINGUISHING MEDIA: Water, Carbon dioxide, Dry chemical, Foam SPEWCeIarALfuFlIlREprFoItGeHcTtIiNvGePRcOlCoEtDhiUnRgE,S: including helmet, self-contained, panodsitpiavntes,prebasnsdusrearorounpdresasrumsr,e dwaeimsatndanbdrealteghsi,ngfaacpeparmaatsku,s, anbdunker coat protective covering for exposed areas of tne head. UNUSSeUeALHazFaIRrEdouAsNDDeEcXoPmLpOoSsIiOtNioHnAZAsReDcSt:ion for products of combustion. 4. REACTIVITY DATA STABILITY: Stable INCOMPATIBILITY - MATERIALS/CONDITIONS TO AVOID: Not applicable. HAZARDOUS POLYMERIZATION: Hazardous polymerization will not occur. HACZaArRbDoOnUSMoDnEoCxOiMdPeOSIaTnIdONCarPbRoOnDUCDTiSo:xide, Oxides of Nitrogen, Oxides of Sulfur, Hydrogen Fluoride, Toxic Vapors, Gases or Particulates. 5. ENVIRONMENTAL INFORMATION SPIRLeLferREStPoONoStEh:er sections of this MSDS for information regarding physical and health hazards, respiratory protection, ventilation, and preesrisdounea.l prPoltaceectiivneaeqU.uSi.pmeDnOtT.-appCroollveecdtcosnptialilneedr.material. Clean up 000194 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-009:PAGE F-34 MSDS: FC-10 FLUORAD Brand Fluorochemical Alcohol January 29, 1998 PAGE 3 5. ENVIRONMENTAL INFORMATION (continued) RECOMMENDED DISPOSAL: oIfncainecroamtbeustiinblaepemartmeirtitaeld. hazCaormdbouusstiownastperodiuncctisnerwailtlor ininclutdhee HpFr.esence wDaisstpeo.se of waste product in a facility permitted to accept chemical ' ENVIRONMENTAL DATA: Laboratory tests showed no biodegradation. 96-Hr. LDS0 Fathead Minnow (Pinephales promelas) - No mortality at water saturation. No statistically significant effect on % hatch, % survival, weight, and length in 30 day Fathead Minnow egg fry study. Lab tests showed 200 fold bioconcentration of FC-10 into muscle fillets of channel catfish. REGULATORY INFORMATION: Volatile Organic Compounds: N/A. VOC Less H20 & Exempt Solvents: N/A. This product complies With the chemical registration requirements of TSCA, EINECS, COSL, AICS and Korea. EPCRA HAZARD CLASS: FIRE HAZARD: No PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 6. SUGGESTED FIRST AID EYE CONTACT: Innediately flush eyes with large amounts of water. Get immediate medical attention. SKIN CONTACT: Immediately wash skin with soap and large amounts of water. Remove cWaosnhtacmoinnattaemdinactloetdhicnlg.othiInfg sibegfnosr/esymrpetuosmesanodccudri,spocsaellofa cphoynstiacmiianna.ted shoes. INHALATION: It signs/symptoms occur, remove person to fresh air. If signs/synptons continue, call a physician. IFCaSlWlALLaOWpEhDy:sician IMMEDIATELY. If swallowed, induce vomiting inmediately mouth to an as directed unconscious pbyersmoend.ical personnel. Never give anything by 000195 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-009:PAGE F-35 MSDS: FC-10 Jaruary 29, FLUORAD 1998 Brand Fluorochemical Alcohol PAGE 4 7. PRECAUTIONARY INFORMATION EYE PROTECTION: Avoid eye contact. Wear safety glasses with side shields. SKIN PROTECTION: mAavtoeirdiaslk.in Aconptaaicrt.of gWelaorvesappmradoeprifartoem tghleovefsollwohweninghamnadtleirnigalt(hsi)s are recommended: butyl rubber. Use one or more of the following cpoevresroanlalls.protection items as necessary to prevent skin contact: RECUsOeMMEwNiDtEhDapVEpNrToIpLrAiTaItOeN:local exhaust ventilation. Provide sufficient ventilation to maintain emissions below recommended exposure limits. If exhaust ventilation is not adequate, use appropriate respiratory protection. REASvPoIiRdATObRrYeatPhRiOnTgECToIfONa:irborne material. Select one of the following cNIoOnStHamianpapnrtosvedandreisnpiraactcoorrsdanbcaesedwitohn airborne concentration of OSHA regulations: half-mask dust respirator, full-face supplied air respirator. PREVENTION OF ACCIDENTAL INGESTION: Do not eat, drink or smoke areas thoroughly with soap ahnednwautseirn.g tHhaissh prhoadnudcst. afWtaesrh haenxdploisnegd and before eating. RECOMMENDED STORAGE: Store away from heat. Keep container closed when not in use. FIRE AND EXPLOSION AVOIDANCE: Nonflanmable. OTHNEoR smPoRkEiCnAgU:TIOSNmAoRkYingINwFhOiRMlAeTIuOsNi:ng this product can result in contamination of of the hazardous dtheecomtpoobsaicctoionandp/roorducstmsokemenatndionleedadinto stehcetiofnorm4atoifon this MSDS. HMIS HAZARD RATINGS: HEALTH: 1 FLAMMABILITY: 1 REACTIVITY: 0 PERSONAL PROTECTION: X (See precautions, section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT TYPE AUTH SKIN PPEERRFFLLUUOORROOHOECXTAANNEESSUULLFFOONNAAMMIIDDOO ALCOHOL... ALCOHOL... 0.1 0.1 MG/M3 MG/M3 THA aM TWA 3M Y Y PEARLFCLOUHOORLO.HEPTANuEvSULFONANIDO 0.1 MG/M THA aM vy Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 000196 418-009:PAGE F-36 MSDS: FC-10 FLUORAD Brand Fluorochemical Alcohol Jaruary 29, 1998 PAGE EXPOSURE LIMITS (continued) INGREDIENT VALUE UNIT TYPE AUTH SKIN PERFLUOROBUTANESULFONAMIDO ALCOHOL... 0.1 MG/M3 THA aM PERFLUOROPENTANESULFONAMIDO ALCOHOL. ee vvvnnnaneensnnneeaeeess 0.1 MG/M3 THA aM + SKIN NOTATION: Listed substances indicated with 'Y' under SKIN refer to the potential contribution to the overall exposure by the cutaneous route biyncdliurdeicntg mcuocnotuasctmeWmitbhrantehe ansdubseytea,ncee.ithVeerhibcyleasirbcoanrnealtore,r msokrien pabasrotripctuiloanr.ly, S- OU3MR:CE OF3MEXPROeScUoRmEmenLdIeMdITExDApToAs:ure Guidelines 8. HEALTH HAZARD DATA EYENoCOaNdTvAeCrTs:e health effects are expected from eye contact. SKIN CONTACT: Product is not expected to be irritating to the skin. May be absorbed through the skin and persist in the body for an extended time. INMHaAyLATbIeONa:bsorbed by inhalation and persist in the body for an extended time. IF SWALLOWED: Ingestion is not a likely route of exposure To this product. Illness may occur after a single swallowing of relatively large quantities of this material. MUTAGENICITY: Not mutagenic in in-vitro assays. RESPuRObDsUtCaTnIcVeE/wDaEsVEnLoOtPHtEeNrTaAtLogeTnOXiIcNSi:n the rat at doses as high as 30 milligrams per kilogram per day via oral route. OTHER HEALTH HAZARD INFORMATION: This product is not known to contain any substances regulated under California Proposition 65. A Product Toxicity Summary Sheet is available. 000197 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-003:PAGE F-37 JMSaDnSu:aryFC2-91,0 1F9L9U8ORAD Brand Fluorochemical Alcohol PAGE 6 SECTION CHANGE DATES HEADING SECTION CHANGED SINCE November 05, 1997 ISSUE Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately DTehecoirnrfeocrtmatasionofinthethidsateMatisesruieadl. Saf3MetyMAKDEaStaNOShWeAetRRA(NMTSIDESS), isEXPbReElSiSeEvDedORto MIEMRPLCIHEADN,TABIINLCILUTDYINOGR, FIBUTTNESNSOTFOLRIMIATEPDARTTOI,CULAANRY IMPLIED PURPOSE WARRANTY OF OR COURSE OF nPEeRtFhOeRrMANtChEe 3OMR pUSrAoGdEuctOFisTRAfDiEt. forUsearpairstirceuslpaornsipbulreposfeoranddetesrumiitnaibnlge for cuasner'asffemcetthotdheofuseuseandorapappplliiccaattiioonn.of aGiv3eMnprtoheducvta,riestomyeofoffaWchticohrsartehat tuhneiquuesleyr weivtahliunatethetheus3eMr'sprokdnuocwtledtgoedeantdermcionnetrowlh,ethietrisitesissenftitialforthaat particular purpose and suitable for user's method of use or application. D3Mueprtoovitdheesreimnoftoermaptoisosnibiilniteyletchtartonieclecftorromniacs tarasnesrfveircematyo hitasvecursetsoumletresd. rineprerersoernst,atioomnisssiaosnstooritsaltceormaptlieotnesnesisn otrhisacciunrfaocrym.atioInn, ad3dMitmiaokne,s no iinnffoorrmmaattiioonn oibntatihneedMSDfSromavaaildaabtlaebasdeiremcaytlynotfrobme aaMs. current as the 000198 418-009:PAGE F-38 ATTACHMENT 3 TEST ARTICLE PREPARATION PROCEDURE 000199 418-009:PAGE F-39 ATTACHMENT 3 Version: 418.P0r0o9to(c2o3lM4A18Y-9080)9 TEST ARTICLE PREPARATION PROCEDURE Page 1012 Test Article: N-EIFOSE. Vehicle: 0.5% Tween 80, in R.O. Water. A. Purpose: The purpose of this procedure is to provide a method for the preparation of dosage suspensionsof N-EIFOSE administrationto rats on Argus Study a41n8d-0t0h9e.control article for oral B. General Information: 1. All suspension containers willbe labeled and color coded. Each label will specify the protocol number, test article identification, Argus batch number, concentration, and storage conditions. dosage level, preparation date, expiration date 2. Suspensions wil be prepared: X_ Daily __ Weeky _For__daysofuse 3. Suspensions will be prepared at a final dosage volume of 5 mLikg. 4. Safety X_ Gloves, lab coat, goggles or safety glasses and faceshield XC Dust-Mist Respirator _ Half-Face Respirator Z FulkFace Respirator/Positive Pressure Hood Tyvek SuivApron 5. Dosage solutions Yes adjusted _X_ for No Free base and % Purity. (Calculations based on 100%) _ FreeBase __ Purity 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol, 000200 418-009:PAGE F-40 JR--, vem n aTeLnarss TEST ARTICLE PREPARATION PROCEDURE NOTE: Test aricle wil be prepared as a sera ton fom the igh dosage to tahceiolodwtdooseageG.ortaOinncerest; mn he final Shows oem aunt sting se volumes are achieved, stir bars are to be dosage administration. C. Test Article Suspension Preparation: 1 To prepare the 3-mg/mL, group V suspension, add the required amount of test article (See TEST ARTICLE CALCULATIONS) into an appropriately sized, labeled container. 2 Q. S. to the final desired value with the vehicle and mix by inversion. mio merson 3. To prepare the 2-mgimL, group IV suspension, remove therequired `amount of stock suspension (group V), add an equal volume of vehicle 4. To prepare the 1-mg/mL, group Ill suspension, remove the required amount of stock suspension (group IV), add an equal volumeofvehicle `and mix by inversion. pospitbponbon 8, To prepare the 0.2-mg/mL, group Il suspension, remove therequired amountof stock suspension (group III), add an equal volume of vehicle D. Preparation of the Control Group: L wansy Lf LyL y, Add the required amount of vehicle to an appropriate vessel. Approved by} are Date: _28 nr -9% carteaton: eFnotoo4 n atached creation form) bain Cal XBe nr fof 000201 418-009:PAGE F-41 axcos TEST A`RPTRIOCCELDEU/RSETECSLTAARNICFEICPARTEIPOANRATION Prococor: {17001 Venton: 415001 3m 18 CDlaacerifoifcasion Jselse J - [Sta-- gs L.5 Clarttteacion Que fo spillage. wore --gYtoocsuppzdeandecrkiered Yoobeog 3 See Calolation Ya. /57 as -- ---------------- S-------------- e-- r re-- -- i ---- T mesE hm - a ---------- er-------- -- i ------ e-- e ---------- J - ~e------------ i------ --er-------- e-- e ------ TT stgbimecter hate Reviewed by: ~C 0Da8t.e1:5T5T 1Ta-S-P3R-O14-502 000202 ~ PRIMEDXCA 418-009:PAGE F-42 Argue s ReShehsy earcv hDLraibvoer,at. oPBruAi1es9n5c4 TelTeepehotnaex:: ((2211%5))444433--84751807 PROTOCOL 418-009 COMBINED ORAL PERINATAUPOSTNATAL (RGEAPVRAOGDEU)CFTEIRTOINLITTOYX,ICDIETVYESLTOUPDMYENOTFANL-AENTDFOSE IN RATS. SPONSOR'S STUDY NUMBER: 6316.5 Amendment 1-July 17, 1998 1. Concentration Analyses (page 5 of the protocol): Samples last week (5 of mL the each) from F1 dosage each concentration will administration to verify be taken during the first concentrations of the and prepared test article formulations. ReaforsChoangne: This change was the F1 generation made male because dosage and female rats. administration was extended to include 2. Sex (page 6 of the protocol): The F1 generation male and female pups will be given the test article or vehicle Reafos rChoangne: Tinhfeosrmeactihoanngabeosuwtetrhee mefafdecetsatofthtehereteqsutesatrotifclethoenStphoensseocroinndorgdeneerrtaotiporno.vide 3. Method and Frequency (page 10 of the protocol) TorhaellyF1(ggaevnaegrea)tioonn dmaayle1 apnodstfweemaanliengputphsrowuilglhbteheaddmaiynibsetfeorreedsatchreiftiecse.t article eFx2pgoesneedrattoiotnheptuepstsawritlilclneotdubreindgirmecattley mgaivlengetshteattieosnt (airntiucltee,robuetxpmoasyurbee) poorsvsiiably maternal milk during the lactation period. 000203 418-009:PAGE F43 Pro`toAcmoeln4d1m8e.n0t081 Page 2 ReaforsChoangne: These changes were made at the request of the Sponsor and follow the method of exposure to Fo generation. 4. DCosaogenLecveles, ntrataindVoolnumses (page 11 of the protocol): Jer Lm cee| BE| ee M Ncumlber to |Numberof - y DoVsuagee. I I mn _ CeTeTe ee) [[ovoTe wT Ta oT Ts vT Tor T TeTTeevsnsmsecsoonnmmommvveenn]] [CovoTlweT Te oT Te wT [2 T 1 TeunmTesuouosnscmoenmmvoemmna)n]) ReafosrChoangne: This change was made because dosage administration was extended to include the F1 generation male and female rats. 5. Tests Analyses and Measurements - F1 Generation (page 16 of the protocol) Clinical Observations andlor General Apppearance: Preweaning Period: Once daily. Dosage Period: aTnwdicoendcaeilya.ppPrroixoirmtaotedloysaognee hadomuirnipsotsrtadtoisoange. Maternal Behavior: oDbasyesrv1,ed4,a7b,no1r4maanldb2e1hapvoisotrpwairltlubm.e rAecnoyrded daily. 000204 418-009:PAGE F-44 Prot"oAcmoaln4a1m8e.n0t081 Pages `WBodyeig- hMalteRasts: Preweaning Period: Dosage Period: Days 1 (birth), 4, 7. 14 and 21 postpartum. Weekly. Sacrifice: BWodyeig-FehmalteRasts: Terminal weight. Preweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. Dosage Period: Sacrifice: Weekly to cohabitation. Daily during presumed gestation and on Days 1, 4,7 and 14 postpartum (rats assigned to natural delivery). Terminal weight ReafosrChoangne: These changes were made because dosage administration was extended to include the F1 generation male and female rats. 6. F1/F2 Generation Pups Not Selected for Continued Observation (page 22 of the protocol). these three dosage groups. Individual pup samples will be combined by liter On day 4 postpartum, the stomach culled pups from Groups 1, Il and V contents (milk curd) (or highest dosage will be collected group available). from Samples will be collected from all pups from four to fiveof the largest litters in into polypropylene tubes and frozen at -20C. After completion of sample collection, samples will be shipped (frozen on dry ice) to Kris J. Hansen, Ph.D. at 3M Environmental Technology and Safety Services, 935 Bush Avenue, Building 2-3-09, St. Paul. Minnesota 55133-3331 for analysis. Both the recipient and the Study Monitor will be notified in advance of sample shipment. 000205 418-009:PAGE F-45 Prot`oAcmoeln4d18m.e0n01t8 Faget Reafos rCho angne: Cteosltleacrttiicolne iof mrielakchsianmgptlheespwueprsevrieaqluaecsttateidonby the Sponsor to determine if the : wl Alan M. Hoberman, Ph.D., DABT Director of Research fur Date Dena C. Lebo, V.M.D. Date Member, Institutional Animal Care and Use Committee Asm ry ond G. York, \P}.D., DABT Date Associate Director of Research and Study Director Pitoon 7 Goe 204 22 Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 000206 rr,PRIMED he ICA 418-009: PAGE F-46 Argus TReem lseepahrocnu he:LaGbvo1r. e9a)tPo4rBA4iue3ds-n1.3g59I8n14c40.3 Theres G13 443-3587 PROTOCOL 418-009 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF N-ETFOSE IN RATS `SPONSOR'S STUDY NUMBER: 6316.5 Amendment 2 - 13 August 1998 1. Necropsy (page 20ofthe protocol): On postpartum day 21, the 10 mg/kg/day dosage group (Group IV) dams and litters will be sacrificed. Reason for Change: Group IV was terminated at weaning because of the severe pup toxicity during lactation (mortality and reduced body weights and delayed development). 2. MaleandFemaleRatsAssignedtoPharmacokineticSampleCollection (page 14 of the protocol): On postpartum Day 21, the livers of the pups from five litters in each of the remaining groups will be excised, pooled per litter, frozen and retained at -70C until shipment to the Sponsor. Reason for Change This change was made at the request of the Sponsor for possible analysis. 3. Scheduled Sacrifice - Female Rats Assigned to Natural Delivery and Dams with No Surviving Pups (page 15 of the protocol): Ovaries will be retained in neutral buffered 10% formalin. 000207 418-009:PAGE F-47 Pro"toAcmoeln4d1m8e-n0t029 Page2 ReafosrChoangne: `This change was made to clarify the protoca 24 2 . 13-95 in M.foberman, Ph.D. rector of Research DABT Date Associate GD.irYeocrtko,r oPfh.ResearcBhTand Date - Study Director adil Godt (3g 1357 Tne 7 Bes tong bl / Dena C/Lebo, V.M.DU Member Institutional Animal Care Date Marvin T. Case, Study Monitor D.V.M., Ph.D. Date and Use Committee: 000208 iryrP ], RIMED ICA 418-009:PAGE F-48 ArcgushReSsheeaerhcyhDLraibvoer,a"tBBorulieis,nIngcA. TelTeopehtonaex (T21D9) 3437-415807 PROTOCOL 418-009 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF N-ETFOSE IN RATS SPONSOR'S STUDY NUMBER: 6316.5 `Amendment 3 - 24 November 1998 1. NaturalDelivery (page 13ofthe protocol): The Length of Parturition (time of delivery of last pup minus the time of delivery of the first pup divided by N-1 pups in each litter) will not be calculated. Reason for Change: A litter watch was not required by the Sponsor. Len A J T er 5s Alan M. Hoberman, Ph.D., DABT Date Director of Research nd G. York, Associate Director `Study Director 20-096 . DABT Date search and Lire Chl ify Dena C. Lebo, V.M.D. Member, Institutional Animal Care Date and Use Committee Pot Taw Akinssp Marvin T. Case, Study Monitor D.V.M., Ph.D. Date 000209 7 XCA rrP, RIMED! 418-009:PAGE F-49 Argus Research Laboratories, Inc. ri"sebi e> hs GI ale PROTOCOL 418-009 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAUPOSTNATAL REPRODUCTION TOXICITY STUDY OF N-ETFOSE IN RATS SPONSOR'S STUDY NUMBER: 6316.5 Amendment 4 - 25 January 1999 1. TestArticlePreparation Procedure (Attachment3 to the protocol). Reason for Change: The attached preparation procedure has been clarified to eliminatepreparation for Groups IV and V. Groups IV andV contained no animals in the second generation, therefore these dosage formulations were discontinued to reserve test articleusage. O deben som Alan M. Hoberman, Ph.D.. DABT Date Director of Research 0, ern) In sem ss Rayeabnd G. York(PhiD. DABT Date Associate Director of Research and `Study Director ee cht sd re ae Commis Dena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care Dn TC VEL 20 Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 000210 418-009:PAGE F-50 ATTACHMENT 3 Version: 418.P0r0o9to1ca6lD4E1C8-9080)0 Page to2 TEST ARTICLE PREPARATION PROCEDURE Test Article: N-EIFOSE. Vehicle: 0.5% Tween 80, in R.0. Water. A Purpose: Thepurposeofthisprocedureistoprovide amethodforthepreparation ofdosage suspensionsofN-EIFOSE and the control article for oral `administration to ratsonArgus Study 418-009. B. General Information: 1. All suspension containers will be labeled and color coded. Each label will specify the protocol number, test article identification, Argus batch number, concentration, dosage level, preparation date, expiration date and storage conditions. , 2. XSu_spenDsaiiolnys will be_pr_epareWde:eky _For__daysofuse 3. Suspensions will be prepared at a inal dosage volume of mL/kg. 4 sXa_fety:Gloves, lab coat, goggles or safety glasses and faceshield X__ HDuasltt:FMaicset RReessppiirraattoorr Z Full-Face Respirator/Positive Pressure Hood Tyvek SuitiApron 5. Dosage solutions adjusted for Free base and % Purity. Yes X_ No (Calculations based on 100%) __ FreeBase __ Pury 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. 000211 418-009:PAGE F-51 ATTACHMENT 3 Version: 41Pr8oto(-c1Po6alD0g4Ee108zC.o909(802)8 TEST ARTICLE PREPARATION PROCEDURE NOTE: tTheestfianratlicvloelwuilmlebseaprreeapcahrieedveads, asidrilbuatriosnafrreotmogbreouapddliel dtotgortohuep Il. Once containers; mixing should occur during sampling andor dosage administration. C. Test Article Suspension Preparation: 1. To prepare the 1-mgimL, group ll suspension, add the required amount of test article (See TEST ARTICLE CALCULATIONS) into an appropriately sized, labeled container. 2. QS. tothe final desired value with the vehicle and mix by inversion. 3. Toprepare the 0.2-mgimL, group If suspension, remove the required `amount of stock suspension (group Ii), add an equal volume of vehicle and mix by inversion. D. Preparation of the Control Group: 1. Add the required amount of vehicle to an appropriate vessel. Written by: 2. Approved by: Date: _2/-PT Clarification: _ No __Yes (See attached clarification form.) IniialiDate: _hetiXcRhegpooimt= /ffaq 000212 v.PRIMEDICA 418-009:PAGE F-52 ArgSuos3RSesheoarncHhoDLroaavbaeocrsBoPurAcks1,d9n5IAn4g.4 TelTphaonee: GG1I3) 444433-2478190 PROTOCOL 418-009 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAUPOSTNATAL REPRODUCTION TOXICITY STUDY OF N-ETFOSE IN RATS `SPONSOR'S STUDY NUMBER: 6316.5 `Amendment 5 - 8 February 1999 1. Test Article and Vehicle (page 3 of the protocol): [Effective Date: 7 June 1998] The vehicle was identified as 2.0% Tween 80 in R. O. deionized water, rather than 0.5% Tween 80 in R. O. deionized water. ReafosrChoangne: This change was made to obtain an adequate emuisionforthe test article. 2Ld _OGRLLS (ine [2 8 eke: 99 Alan M. Hoberman, Ph.D., DABT Date d G. York _PH.0., DABT Date Director of Research Associate Director of Research and `Study Director QuChle ihe Dena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and Use Committee I lovin TC 9/420 Marvin T. Case, D.V.M,, Ph.D. Date Study Monitor 000213 418-009:PAGE F-53 OPRIMEDICA ATR pepromLeGIS)EnR G30 e--r ----------------------------------T--ra--x G--13--43.4--989 PROTOCOL 418-009 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF N-EtFOSE IN RATS SPONSOR'S STUDY NUMBER: 6316.5 Amendmen6t - 19 May 1999 1. Concentration Analyses (page 5 of the protocol): The concentration samples were sent to the Sponsor. Sample analyses will be conducted at the discretion of the Sponsor and no report will be sent to the Testing Facility. Reason for Change: This change was made at the request of the Sponsor to clarify the protocol. 2. Male and Female Rats Assigned to Pharmacokinetic Sample Collection (page 14 of the protocol): The liver and serum samples were sent to the Sponsor. Samples will be analyzed at the discretion of the Sponsor. Reafos rChoangne: This change was made at the request of the Sponsor to clarify the protocol. 3. E1/F2 Generation Pups Not Selected for Continued Observation (page 22 and Amendment 1 of the protocol): The stomach contentsof these pups were sent to the Sponsor for analysis. Stomach contents will be analyzed at the discretion of the`Sponsor. 000214  418-009:PAGE F-54 Protaocnoilm41eT8na.0et0s8 Reason for Change: This change was made at the requestofthe Sponsor to clarify the protocol. L / Y, J / 1-77 forma /TE) (-mpy=19 bh fioberman, Ph.D. DABT Date Rayhond G. York, .0l, DABT Date Director of Research Associate Director of Research and Study Director aE Jena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and Use Committee Moves Te ee Marvin T. Case, D.V.M., Ph.D. Study Monitor ilsJory Date 000215 r PRIMEDICA 418-009:PAGE F-55 ps i TemenDr A houiL dee A Argus Research Laboratories, Inc. PROTOCOL 418-009 `COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF N-EtFOSE IN RATS SPONSOR'S STUDY NUMBER: 6316.5 Amendment 7 - 24 June 1999 1. `Sponsor (page 1 of the protocol): The Sponsor is 3M Corporate Toxicology, rather than 3M Toxicology Services. Reason for Change: The nameofthe Sponsor has changed. ae Algn Nf Hoberman, Ph.D., DABT Director of Research 7% Date G. Yor Associate Directot `Study Director 2.4 dow -95 D., DABT Date esearch and fd essneedLltinlid 23 srs PtrsnTon 280.00 na C. Lebo, V.M.D. Date Marvin T. Case, D.V.M., Ph.D. Date Chairperson, Institutional Animal Care Study Monitor and Use Committee 000216 APPENDIX G DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 000217 418-009:PAGE G-1 DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 1. From 16 June 1998 to 18 June 1998 [days 9 to 11 of study (DSs 9 to 11)), the Fo generation rats were administered the volume of test article based on the body weight recorded on DS 1 rather than DS 8. The following table summarizes the average percent of correct dosage that the rats were administered. Dosage Dosage Group (makgday Sex | O(Vehicle) FeMmalaele u 1 FeMmaalele " 5 FMeamlaele wv 10 FMeamlaele v 1s FMeamlaele Rathumbers 190900716--190913150 109191316--190917204, 10112664-0100,145 190917416--1100010850 1100108016.-1100024150 1100024116--1100027550 DGariolyup%AovferCoargreecotf Dosage Received 8294.870 5892.7480 89938633 99054533 9%.7156 `These deviations did not adversely affect the outcome or interpretation of the study because the male rats received approximately 90% of the intended dosage and the female rats received approximately 92% to 97% of the intended dosage. This is within the analytical variability (10%). 2. On 3 October 1998 (observation day 44), the following F'1 generation male rats were administered the test article dosage volume based on the previous weekly body weight rather than the current body weight. The following table summarizes the average percent of correct dosage that the rats were administered. Dosage Dosage GroIup (Om(aVkeghiicdleey)) "u 15 12R0a8l9Nu-m1b2e0r8s3 1122111325--1122114128 Group Average of% of Correct Do8s7a5g0e Received 8898.7437 These deviations did not adversely affect the outcome or interpretation of the study because the event occurred on a single day and all of the rats received most of the intended dosage (at least 85% of the intended dosage). 000218 418-009:PAGE G-2 3. On July 1998 (DS 32), ten Fo generation male rats in the 15 mg/kg/day dosage group (Group V) were administered the test article twice, once with the correct volume and once with an incorrect volume. Ra Paoertdo VCoomuemt EVowume VTolousme DTosoawge TAemwouAnutdoer STluomooboner Mis"vgVeeesssna ma2h o52d22 oE3E) ooloezz (Sweoonk illoooooksss vvNeeoss 22i1se 3ii2z 2aa ollooryze aa3nws olooo YYeess 3I2 230 23% wsss eairmr oTlooosokos vvveeesss iiisse 223) ai2 jHooess nmwoeerr tThheessteuddeyvbiaetciaounsseditdhenroetwaedrveernsoelayddafifteicotnatlheadovuetrcsoemcelionricialntoebrspreertvaattiioonnosf noted in these rats and the event occurred on a single day. 4. OF1ng2e0neSreaptitoenmbraetrs 1w9e9r8e (aDdmSisni20s,te2r7e,d2P8,FO2S9,,3t0h,e3t1e,st32ar,ti3c3leofror34a)n,oatlher sinttuedryp.reTtahtiisondeovfiatthieonstduiddynboetcaaduvseerstehleytaefsftecatrttihcleeoaudtmcinoimseteorred (PFOS) is sainmgelteabeovleintte. oTfhtehreetewsetraertincoleafdovretrhsise sctliundiycal(No-bEsterFvOaStEi)onasnfdorthtihsewraastsa administered the incorrect test article. 5. On 14 October 1888 (DS 53), one F1 generation female rat (12249) in the. m1 imsgslinkggifdraoym dtohseacgaegegraotutphe(Gtriomuepoflj)dowsainsg.notThdiossededvibaetciaonusdeidit nwoats aredcveeirvseedlysuaffffieccitenttheteostutarctoimclee toor einvtaelruparteetatthieonpoafratmheetesrt.udy because the rat 6. On 28 August 1998 (observation day 7), the vaginal patency observation was not performed for one F1 generation female rat (12223) in the 0affmegc/tktgh/edaoyutdcoosmaegeorgirnotueprp(reGtraotuiopnI)o. f tThheisstdeuvdiyatbieocnaudisdensoutffaidcvieenrtsedlayta were available to evaluate this parameter. Al deviations are documented in the raw data. Ell Nm JR ond G. York, 70) DABT Date Associate Director okResarch and Study Director 000219 APPENDIX H TEMPERATURE AND RELATIVE HUMIDITY REPORTS 000220 ARGUS 418-009:PAGE H-1 Temperature and Relative Humidity Report | Location: Room 04 Protocol Number: 418-009 Range of Dates: 02-Jun-1998 15:30 to 03-Jun-1998 09:30 || Target Range: Species: Rat TSemFpe1ra0t7ur9e | Rela3t0i%vetoHu7m0i%dity | TTToootttaaalll NNNuuummmbbeberreooorfffDDHaaoytusar:sP:aints: 171275 1712.5 Mean (25D): MeMdaixaimnu:m: Minimum: || NNumubemroobffPPoeoiinnrttss iHnigRhan(%g)e: (4): | Number of Points Low (%): 01 on | 53 24 7s2o3s 652535 695 sis lo19 (e0alg| 109 (e10o00) lo | o oo Report Generated: 09-Sep-1998 at 12:36 COMMENTS: REVIEWED BY: dA ote: ls fa 7 Cumulative by Location (04.01.57) 000221 ARGUS 418-009:PAGE H-2 Temperature and Relative Humidity Report Location: Room 02 Protocol Number: 418-009 | Range of Dates: 03-Jun-1998 09:30 to 27-Jul-1998 14:30 T`SapregceitesR:aRnagte: TToottaall NNuummbbeerr ooff HDoauyrss:: Total Number of Data Points: TSeRmpIerFs 13055075 128 || la3v0e%M0Hu7m0i%dity 130s0s75 1298 Mean (2 5D): MMMieandxiiiammnuu:mm:: 60 09 | 625 (50 66702.181 86s18is.a4 || NNuummbbeerr ooff PPooiinnttss HLiogwh((44)):: Report Generated: 09-Sep-1998 a 12:41 o ooo o || 1oo(o20 || COMMENTS: REVIEWED BY: HAD pate: lsh `Cumulative by Location (v04.01.97) 000222 ARGUS 418-009:PAGE H-3 Temperature and Relative Humidity Report Location: Room 02 Protocol Number: 418-009 | Range of Dates: 27-Jul-1998 14:30 to 17-Sep-1998 11:25 TSpaercgieetsR:aRnagte: TToottaall NNuummbbeerr ooffHDoauyrss:: | Tota NumobfDeatra Points: TFemlpearaTtuore 125434.74 1244 | Relative0H7um0i%dity i| 125434.74 | 1244 Mean ( SD): MMeadxiiamnu:m: | Minimum: NNuummbbeerr ooff PPooiinnttss iHnigRhan(%g)e: (%): Number of Points Low (%): 689 (209)| S77 (x44) 762848 57970 4 ees 7 | 124 (1000)|[ 121331 ) 0 (199g0) | 0) Report Generated: 28-Jan-1999 at 14:22 COMMENTS: REVIEWEDBY:_ co... . = A%- DATE: "cus `Cumulative by Location (v04.01.97) 000223 ARGUS 418-009:PAGE H-4 Temperature and Relative Humidity Report Location: Room 04 Protocol Number: 418-009 | Range of Dates: 27-Jul-1998 14:30 to 30-Jul-1998 18:00 || STpaercgieetsR:aRnagte: | EEE... Total Number of Days: Total Number of Hours: Temperature | Relative Humidity 84F 10 79F 30% to 70% 24 75.25 44 75.25 | Mean (2 SD): Maximum: Median: Minimum: Number of Points in Range (%): NumobfPoeinrts High (%): Number of Points Low (%): 688 (03)| 482 (x21) 696 533 1 688 | ae | 68.1 425 76 (100.0) | 76 (1000) 0 0.0) 0 0.0) 0 0) 0 .0) Report Generated: 28-Jan-1989 at 14:17 COMMENTS: REVIEWEDBY: _ "i... wo. --T DATE: _ ~2v-nn Cumulative by Location (v04.01.97) 000224 ARGUS 418-009:PAGE H-5 Temperature and Relative Humidity Report | Location: Room 14 Protocol Number: 418-009 Range of Dates: 17-Sep-1998 11:25 to 29-Dec-1998 12:00 Target Range: Species: Rat Total Total NNumubemroboffDeHaoyursr:s: `Total Number of Data Points: Temperature 84F 10 79F | Rela3t0i%vetHou7m0i%dity 104 2472.25 2481 104 247225 2481 Mean (+ SD): MMaexdiiamnu:m: Minimum; NNumubemroobffPPoeoiinnrttss in Range High (%): (%): Numobf Peoinrts Low (%): 693 (13) | 559 (36) 679254 6586.10 624 77 | 238 0 een | 281 | 0 (1000) .0) 2 .) 0 0) Report Generated: 26-Jan-1999 at 14:41 COMMENTS: REVIEWEDBY: Cove =. ce DATE: _[~>v/ <q Cumulative by Location (v04.01.97) 000225 418-009:PAHG-E6 e ee --------AR-- GUS tae-- Relative Humidity Deviations Report Location: Room 02 ----eer------Pr-- otoco-- l Num-- ber:-- 418--- 009 m------ meRnanegeOoFf RAREGn SR A RH SRN Dates: 03-Jun-1998 09:30 to 27-Jul-1998 14:30 Humidity Target Range: Species: Rat 30% to 70% uoGoiDnuuanntitteeessses T70i30m00e0 030 R7TH1B.0SMH 71TH 2ZvDuuanntteeosse T00i3100m00e 2uniese 0600 RTTHaa.s4hW Tis nnOwiineeiesse 0G095o00o00 1T70i24s2MhH ZZZuuunnneeiss 101100000000 7T7a2Hs1hm Tnoiiwnenisse 00100300000 TTTiooss2hhn nie O50 728M UnZnuiineei 111432000000 e7700s16mM nies 1500 744H HTniuinneeisss 11o71r00o00 70T1o34oHMn Hone 1800 T13H Uu2dnnni1ieessse 11760000 1800 0Te8eHn 74H dni 2000 727 HTHniiinneigsees Irene 2213900000 BE. 7T70a0172mnH Tab | RnZdinuiinnsEeeiEs 022h5200e000 B7T72r0s0o8MHk H=Value out ofrange - igh L = Value out of ange - Low Report Generated; 05-Sep-19R9.8H.at=1R2e:l4a8tive Humidity (%) v These deviations did not adversely affect the outcome or interpretation of he study The follwing devition(s) impacted on the outcome of the study as described: ESTs StudyDirector Jd---- A Oate: _ro/7% Deviations by Location (v04.01.97) 000226 418-009: PAGE H-7 ---- rerAR-- GUS -- Relative Humidity Deviations Report Location: Room 02 Protocol Number: 418-009 tees---------------- ee Range of Dates: A 03-Jun-1998 09:30 to IR1 27-Jul-1998 14:30 SHpuemciideist:y RTaatrget Range: 30% to 70% 134Dna1te998 T0i40m0e 13199 0500 RTHi.o 708k TuDna1t9e98 T10i:m0e0 1hn1ssE 1100 R7H2.9M 733H 1n3iune19s98 00760000 131998 1000 7T0i4oK T27H 1144dduunn11999988 11320000 1edunies 1700 773344MM 707M 1B3mn 1998 11210000 Bn1998 1300 7T0a1eKk T1SH TUdnniieesss 2190000 14dunioss 2300 770075HH 7041 BBnnii9e9s8 11490000 Tdi 1996 0000 T70091KH TIAH i1SSUuunn11eessss 00510000 Snes 0600 77032HH 731 1a4dduin1oosss 00320000 Tedniess 000 772265KK T24H S15iJnuni1ssse8 11620000 Sun1998 1800 77073H1 741M 1144di1n 919998 14dunioss 00650000 0800 STE BM 771490KM 7A20Mk iiSSuniess 15Juntess 21090000 2100 Vkeissaw 77i813HM 7077HM H= Value out of range - High L = Value out of range - Low R.H. = Relative Humidity (%) Report Generated: 05-Sep-1398 at 12:48 These deviations did not adversely affect the outcome of i. nterpretat'ion of the study. The following deviaton(s) impacted on the outcome of the study as described --- StudyDirector: ` Hw oate: 0/1/02 Deviations by Location (v04.01.97) 000227 418-009:PAHG-E8 --_--AR-- GUS ---- Relative Humidity Deviations Report Location: Room 02 er-- rer-- e er-- Protocol Number: 418-009 e20R5 ange CoTfTDaatSeRsi: C0o3-NJuunn-1R9E9S8 OM 09:30 toSdn 27-Jul-1998 ta14:30 Humidity Target Range: Species: Rat 30% to 70% iiSSDuuauntnieisss8 iownies T022i020m000e00 Rs7Haa.gaH 732H tToDodawntinesiees Ti716m000e0 iedinisss 1800 R77TH33a.53skkh oiowwnnigse ieunioss 0cr1e0o0 0300 aTsas7HH oak Tewnies 0400 737M ileodunniissesss unis 21090000 200 777371Mk 77H uni 2200 75en iieeuunniisssess evniees 0og5o000 0700 T7a6i8nH 705M ents 0800 702m nwiness nies 02500000 0100 a7roHm 75am nies 0200 09H eiedununeissess ieunien 10190000 1200 37443Hh Isak ieuniese 1300 T2oM nTiineise Tdnioss 0Gsa0o0) 0500 7r2oHm 753k nie 0600 7o1H Lein eddnnei0 ssese 11154900000 72T2a7T1HMH 1 Tnuniis ssss i10008000000 T7Ta4ed4aHhk H= Value out ofRrHan.ge= -ReHliagthive HuLm=idViatlyue(0o)ut of range - Low Report Generated; 09-Sep-1998 a 12:48 These deviations did not adversely fect te outcome o interpretation of the study, The following deviaion(s) impacted on the outcome of th study as described: ------------ I Study orton Ln RYken Yr Date: 1/702 Deviations by Location (404.0197) 000228 418-009:PAGE H-9 ARGUS _--_-- Relative Humidity Deviations Report Location: Room 02 -_-- Protocol Number: 418-009 _T -- Range of Dates: 03-Jun-199c 8 09:30 to 27-Jul-1998 14:30 Humidity Target Range: Species: Rat 30% to 70% 17-JDuant-e1998 T1i1m00e R7H3.2M 17un-1998 1200 705H 18uDna-t1e998 T0i80m0e 18-Jun-1998 07:00 1177--JJuunn--11999988 11340000 774823HK 17-Jun-1998 1500 715K 1188--JJuunn--11999988 0089::0000 18-Jun-1998 12:00 1177-uJunn1-9199988 11860000 77032HH 17-Jun-1998 19:00 76H 2201--JJuunn--11999888 2001::0000 21-Jun-1998 08:00 1177-0Juunn--11999988 2210:0000 775303HM 17-Jun-1998 2200 728K 2221-JJuunn--1199%988 0210:0000 22un-1998 05:00 T18JJuunn-iissssse 20300000 TTi660HH 1Bun1998 0100 713H 2232--JJuunn--11999988 0069:0000 23-Jun-1998 21:00 1188--JJuunn--11999888 00230000 772198MM _ 18-Jun-1998 0400 793M 2255-0Juunn--11999888 0101:0000 26-Jun-1998 02:00 T" Wom0 H = Value out of range RH. = - High Relative HLum=idViatlyue(%o)ut of range - Low Report Generated: 09-Sep-1998 at 12:48 R7H4.2M 2952HH 2775HH 772008HH 77289HK 27132HH 704TH 0011HH T7TS34.SH0HH "hese deviations did not adversely affect he outcome o interpretation of hestudy. `The following deviation(s) impacted on the outcome of the study as described: --_-- _-- Study Director: _ C 1 h Dae: _r0/r71 Deviations by Location (v04.01.97) 000229 ARGUS 418-009:PAGE H-10 Relative Humidity Deviations Report Location: Room 02 Protocol Number: 418-009 Range of Dates: 03-Jun-1998 09:30 to 27-Jul-1998 14:30 SHpuemciideist:y RTaatrget Range: 30% to 70% 2%uDna1te998 T0i60m0e R7H1.3K Znioes 0900 730k 24D1a9te08 T0i80m0e RTHi.SH Zooiintsieos 11050000 70449HK OiSuiiiee 00230000 7710S6H SSuuities 01830000 2001s 2100 772452H T2sH ahiisss 0os3o 00 iiss ore T7i0sHh 726m 2Z04i 1s 11620000 T7e8sHh i24i19o98 01080000 TS7e0H8H Zd2i0hii1esss8 o0e8e0o0 ve7e38wM H= Value out ofRrHan.ge= -ReHliagthive HLum=idViatlyue(%o)ut of range - Low Report Generated: 05-Sep-1998 at 1251 Ce These deviati" ons dd not adversely affect the outcome of interpretation of the study "The following deviaton(s) impacted on the outcome of the study as described: - nnn - nnn swayirectors_C H---------- oa [VN Deviations by Location (v04.01.87) 418-009:PAGE H-11 ARGUS Relative LHoucmaitdiiotny:DReovioamti0o2ns Report Protocol Number: 418-009 Range of Dates: 27-Jul-1998 14:30 to 17-Sep-1998 11:25 SHpuemciideist:y RTaatrget Range: TeAmDaGiteEs T01i33m0000e R0TH28.6HH AAAGgiIegs 110427000000 7774420HHM TTTAAAiGGgsSSe 11185000000 770564a2H ehAuuhgGosS 0Z21100000 777259660HK BAAgGs O00T0 776762HH 30% to 70% Date Time RH. H=Value out of RraHn.ge= R-Heilagthive HLum=idViatlyue(%o)utofrange - Low Report Generated: 28-Jan-1999 at 14:29 These deviations id not adversaly affectth outcome or interpretation ofthe study. The following deviation(s) impacted on the outcome of the study as described: Study Director: = tf 7 = Date: _18- [2h 99 Deviations by Location (v04.01.07) 60358~ 23) ARGUS 418-009:PAGE H-12 Temperature Deviations Report Location: Room 14 Protocol Number: 418-009 Range of Dates: 17-Sep-1998 11:25 to 29-Dec-1998 12:00 Temperature Target Range: Species: Rat 84F to 79F O7DDeact1eoss T0i5m00e T6e3m9p1. O077--DDeecc-11998988 2062:0000 6633B9LL 0087--DDeecc--11938888 0203:0000 663356LL 0088--DDeecc--11998888 0012:0000 663313LL 0O8e-DDeecc-11o99388 00340000 663208LL O08t-DDeecc-i1o9f9s8 00650000 66224710 0088--DDeecc--11998388 0078::0000 6633.51LL 0088--DDeecc--11998888 1112:0000 663359LL 0088--DDeecc--11998988 1143:0000 G63355LL 08-DDeact-e1998 T15i:m0e0 Te6m3p6.1 0088--DDeecc--11999988 1167::0000 663346LL 0088-DDeecc--11999988 1198::0000 6633981L 08-Dec-1998 23:00 63sL H = Value out of raTnegmep-.H=igThem_pLer=atuVraelu*eFout of range - Low Report Generated: 28-Jan-1999 at 14:48 + These deviations did not adversely affect the outcome or interpretation of the study. "The following deviation(s) impacted on the outcome of the study as described: Study Director: ) : . 0 7 = Date: \&- Fpi- 99 Deviations by Location (v04.01.97) 000226 13) APPENDIX | STATEMENT OF THE STUDY DIRECTOR "0007 233 418.000:PAGE I-1 2PRIMEDICA rSihn, PA 19064 TelTephhoenee: (O1133)444433..93751807 -------------------- PROTOCOL 418-009: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF N-EtFOSE IN RATS SPONSOR'S STUDY NUMBER: 6316.5 STATEMENT OF THE STUDY DIRECTOR performance of the study. No deviations from the U.S. Food and Drug "This final report accurately reflects the raw data obtained during the Administration (FDA) Good Laboratory Practice Regulations; Final Rule?, the Japanese Ministry of Health Standard for Safety Studies oanndDrWueglsf?araend(MtHheW)EuGroopoedaLnabEocroantoomriycPrCaocmtimcuenity (EEC) Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principlesof goodlaboratorypractice occurred that affected the quality or integrity of the study. y ~ A JU F0-c00-97 fond G. York, 70] DABT Date Associate Director of Research and Study Director Argus Research Laboratories, Inc. a. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. b. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. c. European Economic `Community (1988). Council decision on 28 July O19E8C9Dondetchiesiaocnc/erpetcaonmcmeebndyatthieonEuornopceoampnlEicanocneomwiitchCporimnmcuipnlietsoyfogfoaond lLaegbiosrlaattoiorny.pra3c2t(iNcoe.. LOf3f1ic5i;al2J8ouOrcntoablero)f:the1-E17u.ropean Communities: On APPENDIX J QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT 00027 2 PRIMEDICA 418-009:PAGE J-1 A Tagons Bene QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT Study Director: Raymond G. York, Ph.D., DABT Executive Director of Research: Mildred S. Christian, Ph.D., Fellow, ATS Protocol 418-009: Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of N-EtFOSE in Rats Sponsor's Study Number: 6316.5 The draft protocol for this study was audited for adherence to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations, Japanese Ministry of Health and Welfare (MHW); Good Laboratory Practice Standard for Safety Studies on Drugs, and European Economic Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice on 26 MAY 98. Critical phases of this study were inspected 29 times; study information and raw data were audited seven times (see tables 1 and 2for dates and phases/data). The draft final report and the raw data for this study were compared and audited for accuracy, for adherence to protocol requirements, and for adherence to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations, Japanese Ministry of Health and Welfare (MHW); GoodLaboratory Practice Standard for Safety Studies on Drugs, and European Economic Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice between 05 JAN 99 and 11 MAY 99, and 09 APR 99 and 19 MAY 99, and for revisions requested by the Sponsor 14 JUN 99 and 16 JUN 99 and for finalization on 30 JUN 99. 2% 418-009:PAGE J-2 Adminis`tTrhaistisotnud(yFDwAa)sGcooondduLcatbeodraatcocroyrdPirnagcttioceU.RSe.guFloaotdioansn,d Drug Japanese Ministry oSftuHdeiaelsthonanDdruWgesl,faarned (EMuHrWo)p;eaGnoEocdoLnaobmoircatCoormymPurnaicttiyce(1S9t8a9n)dacrodunfcoirlSdaefceitsyion OonE2C8DJduelyci1s9i8o9n/ornectohmemaecncdeapttiaonncoenbcyotmhpeliEaunrcoepewiatnh Epcrionncoipmliecs oCfogmomoudnity of an laboratory practice. Mlk iz 4NQauanlciytyJA. sGsounrgalniceewsMkainager Date Pia) - a QHueaaltihteyrALs.suRarbauntcteinSou,pMe.rSv.isor Dat and Principal Auditor 232 0002684 TABLE 1 CRITICAL PHASES INSPECTED 418-009:PAGE J-3 TAesdtAmrtiiclne istrat -Giavoan ge Dates of inspection: 16 JUN 98, 16 JUN 98, 25 AUG 98 Dates results reported to the Study Director and Management: 19 JUN 98, 19 JUN 98, 26 AUG 98 Estrous Cycle Evaluation Date of inspection: 25 JUN 98 Date results reported to the Study Director and Management: 25 JUN 98 Cohabitation Dates of inspection: 09 JUL 98, 03 NOV 98 Dates results reported to the Study Director and Management: 14 JUL 98, 06 NOV 98 Caesarean-Sectioning Date of inspection: 17JUL 98 Date 98 results reported to the Study Director and Management: 21 JUL Test Article Preparation Dates of inspection: 29 JUL 98, 28 AUG 98 Dates 31JUL 98, results 02 SEP r9e8ported to the Study Director and Management. 60282 238 418-009:PAGE J4 Natural Delivery and Litter Observations Dates of inspection: 29 JUL 98, 25 NOV 98 Dates results reported to the Study Director and Management: 31JUL 98, 02 DEC 98 Male Necropsy Dates of Inspection: 30 JUL 98, 18 NOV 98 Dates results reported to 31JUL 98, 23 NOV 98 the Study Director and Management: Mik Collection Date of inspection: 04 AUG 98 Date results reported to the Study Director and Management: 12 AUG 98 Surface Righting Reflex, Pinna Response, Air Righting Reflex, Unfolding, Eve Opening, Acoustic Startle Pupil Constriction, Sexual Maturation Dates of inspection: 04 18 AUG AUG 98, 98, 12 28 AUG AUG 98, 98, 12 14 AUG SEP 98, 98 12 AUG 98, 12 AUG 98, Dates results reported to the Study Director and Management: 07 19 AUG AUG 98, 98, 20 02 AUG SEP 98, 98, 14 15 AUG 98, SEP 98 14 AUG 98, 14 AUG 98, Culling Date of inspection: 04 AUG 98 Date results reported to the Study Director and Management 07 AUG 98 239 BOLE 418-009:PAGE J-5 Weaning Date of inspection: 19 AUG 98 Date results reported to the Study Director and Management: 20 AUG 98 Blood Collection Date of inspection: 19 AUG 98 Date results reported to the Study Director and Management: 20 AUG 98 Necropsy- Dam and Litter Sacrifice Dates of inspection: 19 AUG 98, 15 DEC 98 Dates results reported to the Study Director and Management: 20 AUG 98, 18 DEC 98 Passive Avoidance, Watermaze Dates of inspection: 27 AUG 98, 26 OCT 98 Dates results reported to the Study Director and Management: 28 AUG 98, 30 OCT 98 600284 240 : TABLE 2 418-000:PAGE J-6 RAW DATA AUDIT(S) "The following study information and raw data were audited on 15 SEP 98, 17 SEP 98, 18 SEP 98, 22 SEP 98 TO 26 SEP 98: Protocol. Protocol amendments. List of personnel and computer operator codes. Error codes and codes for clinical sign observations. AInn-ilimfaeltrraencseaipctt,iornanrdeocomridz.ation, physical examination and acclimation. Feed consumption. Necropsy. Organ weights. TKiesysufoerptaecstkiinnggfalicsitlsi.ty computer backup record abbreviations. Edit requests. Blood collection data and packing lists. The results of this audit were reported to the Study Director and Management on 28 SEP 98. fromT2h8e SfoElPlow9i8ngtost0u5dyOCinTfo9r8m;ation and raw data were audited In-life transaction record. on 1T3hNe OreVsu9l8ts of this audit were reported to the Study Director and Management 068R8STM 242 418-009:PAGE J-7 The following study information and raw data were audited from 29 SEP 98 to 04 OCT 98: Animal receipt, randomization, physical examination and In-lfe transaction record. Feed consumption, Estrous cycle evaluation. Cohabitation. Caesarean-sectioning. Maternal gross observations. Natural delivery observations. Litter observations, including reflex and development. Pup body weights and status. Table of random units. Organ weights. Tissue packing lists. General comments. Study maintenance records. Temperature and relative humidity reports. Feed, water and bedding analyses. Edit requests. Dosage volumes. Deviations. Data review pages. Blood collection data and packing lists. acclimation. on The results 05 OCT 98. of this audit were reported to the Study Director and Management The following study information and raw data were audited from 06 OCT 98 to 07 OCT 98 Vehicle receipt, preparation and use. Test article receipt, preparation and use. Test article packing lists. on The results 08 OCT 98. of this audit were reported to the Study Director and Management 666256 418-009:PAGE J-8 The following study information and raw data were audited from 18 JAN 99 to 21 JAN 99: In-life transaction record. Feed consumption. Passive avoidance. Watermaze. Genealogy chart. Necropsy. Organ weights Tissue packing lists. Edit requests, Sexual maturation. on The results 21 JAN 99. of this audit were reported to the Study Director and Management on The following 26 JAN 99: study information and raw data were audited Vehicle receipt, preparation and use. Test article preparation and use. Vehicle packing list. `The resultsof this audit were reported to the Study Director and Management on 28 JAN 98. M3 "600287 418-009:PAGE J-9 The following study information and raw data were audited from 21 JAN 99, 04 FEB 99, 05 FEB 99, 08 FEB 99, 09 FEB 99, 11 FEB 99. Randomization. In-life transaction record. Feed consumption Cohabitation. Natural delivery observations. Litter observations. Pup body weights and status. Passive avoidance. Watermaze. Table of random units. Necropsy. Tissue packing lists. General comments. Study maintenance records. Temperature and relative humidity Feed and water analyses. Edit requests. Dosage volumes. Data review pages. Sexual maturation reports. The results of this audit were reported to the Study Director and Management on 11 FEB 99. uy we.