Document 2NzOgEOjvqxgV2E8eXmML217g

FINAL REPORT RECEIVED 0PPT CBIC 20FEB 22 A411: 34 PROTOCOL 418-014 ORAL (GAVAGE) CROSS-FOSTERING STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: T-6295.13 FINAL REPORT DATE: 23 JULY 1999 245 40975 um PROTOCOL 418-014 ORAL (GAVAGE) CROSS-FOSTERING STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: T-6295.13 SUBJECT TAOFBCONLTENETS ' SUMMARY AND CONCLUSION A. Methods B. Results Cc. Conclusion I! DESCRIPTION OF TEST PROCEDURES A. `Conduct of Study A.1. Sponsor A.2. Testing Facility A3. Study Number A4. Sponsor's Study Number A5. Purpose of the Study AB. Study Design A.7. Regulatory Compliance PAGE = 1 4 1-6 1 1-1 1 1-1 1 1 n-1 1 1-1 : "000290ne SUBJECT A8. Ownership of the Study A9. Study Monitor A.10. Altemate Study Monitor A.11. Study Director A.12. Technical Performance A.13. Report Preparation A.14. Report Review A.15. Date Protocol Signed A.16. Dates of Technical Performance A.17. Records Maintained B. Test Article Information B.1. Description B.2. Lot/Batch Number B.3. Date Received and Storage Conditions B.4. Special Handling Instructions B.5. Analysis of Activity Cc. Vehicle Information C.1. Description C.2. Lot Numbers C.3. Dates Received and Storage Conditions C.4. Special Handling Instructions C.5. Analysis of Purity ! PAGE 1-2 1-2 1-2 2 2 2 1-2 1-2 n-3 1-3 3 1-3 1-3 1-3 n-4 4 1-4 11-4 14 n-4 n-4 1-4 wu? VOUT hg SUBJECT PAGE D. TestArticle PrepaandSrtoaragteCoindiotionns 1-5 D.1. Sample Information 15 D2. Analytical Results 5 E. TestSystem 5 EA. Species 5 E2. Strain 5 E3. Supplier (Source) 16 Ed. Sex 16 E5. RatiforoTenstaSylsteem 16 6. Test System Data [3 E7. BreMaeledRateDarta 6 E8. Meot fRanhdomo izatd ion 6 E9. SyosfIdtentie ficam tion wr F. Husbandry 8 FA. ResFaeciltayRergisctrathion 8 F2. Study Rooms 8 F3. Housing 1-8 Fa. Lighting 1-8 F5. Sanitization 1-8 F6. Feed 1-8 F7. Feed Analysis 19 F8. Water 19 ii SUBJECT F.9. Water Analysis F.10. Bedding F.11. Bedding Analysis G. Methods G.1. Dosage Administration G.2. Rationale for Dosage Selection G.3. Route of Administration G4. Rationale for Route of Administration G5. Frequency of Administration G.6. Length of Study G.7. Method of Study Performance H. Gross Necropsy H.1. Fo Generation Rats H.2. F1 Generation Pups 1. Statistical Analyses mn. RESULTS A. Mortality, Clinical and Necropsy Observations B. Body Weights C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values D. Natural Delivery and Litter Observations D.1. Natural Delivery Observations D.2. Cross-Fostering Litter Observations v PAGE 1-9 1-9 I-9 1-10 1-10 1-10 1-11 11 1-11 1-11 11 1-15 1-15 . 1-16 I-17 n-1 n-1 n-1 n-2 n-2 n-2 n-2 006293 ~ SUBJECT PAGE E. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations "3 F. Electron Microscopic Evaluation of the Liver and Lung from Da1y Postpartum Pups <4 FA. Liver <4 F2. Lung 4 REFERENCES 5 APPENDIX A - REPORT FIGURES Figure 1. Matemal Body Weights A Figure 2. Matemal Body Weights - Rats Assigned to Cross Fostering ~~ A-2 APPENDIX B - REPORT TABLES Table 1. Clinical Observations - Summar-y Fo Generation Female Rats B81 Table 2. Necropsy Observations - Summary - Fo Generation Female Rats B4 Table 3. Body Weights-Precohabitation - Summary- Fo Generation Female Rats B5 Table 4. Body Weight Changes - Precohabitation - Summary - Fo Generation Female Rats B6 Table 5. Matemal Body Weights - Gestation - Summary - Fo Generation Female Rats B87 Table 6. Matemal Body Weight Changes - Gestation - Summary - Fo Generation Female Rats B8 Table 7. Matemal Body Weights - Lactation - Summary - Fo Generation Female Rats B-9 Table 8. Matemal Body Weight Changes - Lactation - Summary - Fo Generation Female Rats 8-10 v 60029 250 SUBJECT Table 9. Absolute Feed Consumption Values (g/day-) Precohabitation - Summary - Fo Generation Female Rats PAGE B-11 Table 10. Relative Feed Consumption Values (g/kg/day) - Precohabitation -- Summary - Fo Generation Female Rats B-12 Table 11 Maternal Absolute Feed Consumption Values (g/day) Gestation - Summary - Fo Generation Female Rats B-13 Table 12. Maternal Relative Feed Consumption Values (g/kg/day) Gestation - Summary - Fo Generation Female Rats B-14 Table 13. Matemal Absolute Feed Consumption Values (g/day) - Lactation - Summary - Fo Generation Female Rats B-15 Table 14. Matemal Relative Feed Consumption Values (g/kg/day) Lactation - Summary - Fo Generation Female Rats B-16 Table 15. Natural Delivery Observations - Summary - Fo Generation Female Rats B-17 Table 16. Litter Observations (Naturally Delivered Pups) - Summary F1 Generation Litters B-20 Table 17. Clinical Observations from Birth to Day 21 Postpartum Summary - F1 Generation Pups B-24 Table 18. Necropsy Observations - Summary - F1 Generation Pups B-25 Table 19. Clinical Observations - Individual Data - Fo Generation Female Rats B-26 Table 20. Necropsy Observations - Individual Data - Fo Generation Female Rats B-30 Table 21. Body Weight-s Precohabitation - Individual Data - Fo Generation Female Rats B-33 Table 22. Matemal Body Weights - Presumed Gestation - Individual Data - Fo Generation Female Rats B45 Table 23. Maternal Body Weights - Lactation - Individual Data -- Fo Generation Female Rats B-53 25 wv 666295 wold SUBJECT Table 24. Feed Consumption Values - Precohabitation - Individual Data - Fo Generation Female Rats PAGE B-59 Table 25. Maternal Feed Consumption Values - Presumed Gestation - Individual Data - Fo Generation Female Rats B-63 Table 26. Maternal Feed Consumption Values - Lactation - Individual Data - Fo Generation Female Rats B-71 Table 27. Natural Delivery, Implantation Sites, and Pup Viability and Sex - Individual Data - Fo Generation Female Rats/ F1 Generation Litters B-74 Table 28. Duration of Gestation and Average Duration of Delivery (Minutes) Per Pup Per Litter - Individual Data - Fo Generation Female Rats B-77 Table 29. Pup Body Weight Litter Averages from Birth to Day 21 Postpartum - Individual Data - F1 Generation Litters B-80 Table 30. Pup Body Weights from Birth to Day 21 Postpartum Individual Data - F1 Generation Pups B-83 Table 31. Pup Vital Status and Sex from Birth to Day 21 Postpartum Individual Data - F1 Generation Pups B-99 Table 32. Clinical Observations from Birth to Day 21 Postpartum Individual Data - F1 Generation Pups B-102 Table 33. Necropsy Observations - Individual Data - F1 Generation Pups B-104 APPENDIX C- PROTOCOL AND AMENDMENT C-1to C41 APPENDIX D- DEVIATIONS FROM THE PROTOCOL AND THE `STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY D-1 APPENDIX E - TEMPERATURE AND RELATIVE HUMIDITY REPORTS E-1toE-7 APPENDIX F - PATHOLOGY REPORT F-1to F-46 APPENDIX G - STATEMENT OF THE STUDY DIRECTOR G-1 vii 06296 weg 252 suBJECT APPENDIX H- QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT PAGE H-1to H-5 vii 600797 wigy 418.014:PAGE I-1 TITLE: ORAL (GAVAGE) CROSS-FOSTERING STUDY OF PFOS IN RATS ARGUS RESEARCH LABORATORIES, INC. SPPROONTSOOCRO'LSNSUTMUBDEYR:NU4M1B8E-R01:4 T-6295.13 IL SUMMARY AND CONCLUSION A. Methods wSeevreentays-seiiggnhetdvtiorgtiwnofedmoaslaegCergir:oCuDp@sB(RGrVoAuFp/sPl| aunsd (IlS,p4ra2gruaet-sDianwtlheey)cornattrsol garrtoiculpe,aPndFO3S6 r(aFtCs-9i5n)t,heortrtehaetevdehgicrloeu,p.0.T5h%eTrwatesewnere80a,domrianlilsyt(evrieadgatvhaegtee)s,t once d`aTihleydboesgaignenivnogl4u2medawyassb5efmoUrkegc.ohaDboitsaatgieons twherroeugahdjduasyte2d1 dpaoilsytpfaorrtbuomdy(DwLei2g1h)t. changes and given at approximately the same time each day. dReaptsenwdeirneg aosnsitghneeodrdteorcorfosdse-lfiovsetreyrianngdotrhpehaavramialcaboiklitnyetoficrastas mfporlecrcooslsl-efcotsitoenring. TTwheentlyit-tferisvewelriteerasspseirgndeodsasgucehgtrhoautpfowuerrseuabssseitgsnweedrteo ocbrtoassi-nfeodstaemroinnggotnheDLtw1o dosage groups as described in the table below. Dosage| MaNtaerdmbFeermoafle| Dosage | NaLimtebresorf | ResssiTgynpeed Lier Group | Rais | (mghgday)| Reassignes| (Dom Treatment) [ I II ToNvTr ehde)[18 | 7 s Trees | a | [0Tw 17 T | Uweaes |0| gErigohutpfweemraeleasrsatisgnferdomtothpehacronmtarcoolkginreotuipcasnadmtpwloe fceolmlaelcetiroantsfoflrloowmitnghedetlrievaetreyd. These rats were not cross-fostered. a. aDreetapirloevdiddeesdcriinptthieonaspporfoapllripartoecseedcutrieosnsusofedthiins the conduct of this study report and in APPENDIX C (PROTOCOL AND AMENDMENT). 154 oooz=s W@W 418-014:PAGE |-2 AA. Fo Generation Rats Assigned to Cross-Fostering: The Fo generation female rats were observedforviability at least twice each day of the study and for clinical observations of effects of the test article, abortions, premature deliveries and/or deaths before postdosage. Body weights were recorded and approximately one daily during the dosage hour period 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. The female rats were evaluated for durationofgestation, length of parturition, liter sizes and pup viability at birth. Pups that either appeared stillborn or that died before initial examination of the liter for viability were examined for vital pstuaptsusweatrebiretxh.amMianteedrndaulribneghatvhieo2r1o-fdtahyepdoastmpsarwtausm epvearliouda.teOdbdsaeilryvewdhemnattheemal behaviorwas recorded on DLs 1,4, 7, 14 and 21. Twenty-four-hour urine and fecal samples were collected from DLs 21 to 22 and biood samples were collected on DL 22, via the inferior vena cava, from the maternal rats. The blood samples were centrifuged. The urine and fecal samples and the serum were shipped to the Sponsor. All Fo generation rats assigned to cross-fostering were sacrificed on DL 22 and a gross necropsy of the thoracic, The number and distribution of abdominal and pelvic viscera was implantation sites were recorded. performed. A liver section (tight lateral lobe) from each dam was collected and shipped to the Sponsor. Rats that did not deliver a litter were sacrificed on DG 25 and examined for gross lesions. A blood sample was collected prior to sacrifice via the inferior vena cava; samples were centrifuged and serum samples were shipped to the Sponsor. A liver section (right lateral lobe) from each dam was retained and shipped to the Sponsor. Dams with no surviving pups were sacrificed after the last pup was found dead, missing or presumed cannibalized. A2. FiGeneration Pups Assigned to Cross-Fostering: Day 1 of lactation (postpartum) was defined as the day of birth. Each litter was evaluated for viability at least twice each day of the postpartum period. The pups present in each litter were counted once each day. Physical signs were recorded for the pups once each weights were recorded on DLs 1 day during the (birth), 4, 7, 14 postpartum and 21 and period. Pup at sacrifice. body Pups found dead or sacrificed because of moribundity were examined for gross lesions and for the cause of death or the moribund condition. For all pups found dead on DLs 2 to 4, the lungs and the liver were preserved. Pups with gross 155 000380 ee 418-014:PAGE I-3 lesions found on DLs 2 to 4 were preserved and for pups found dead on DLs 5 to 21, the lungs, liver and gross lesions were preserved. On DL 4, cross-fostered F1 generation litters were culled to five male and five female pups per litter, where The lungs and the liver were possible. preserved. Pups The with gross lungs and lesions were preserved. the liver were collected feraocmhtdhoesfairgset tgernocuuplloend epaucphs dfaryom(ceoanctrholdopsooalgeonglryoounp (DiLrr1e)s.peTcthieveluonfglsittaenr)dftrhoem liver were retained. Remaining culled pups were sacrificed and discarded without evaluation. On DL 21, all pups were sacrificed via decapitation and examined for gross lesions. Gross lesions were retained. Six litters per subset were randomly selected for collection of pooled samples (per litter) of blood and liver. Blood samples were collected via the inferior vena cava from each pup, pooled (per litter) and the samples were centrifuged. The serum samples were shipped to the Sponsor. A.3. Fo Generation Rats Assigned to Pharmacokinetic Sample Collection: Viability and clinical observations, body weights, feed consumption values and delivery observations were performed as previously described for Fo generation dams assigned to cross-fostering. Pharmacokinetic samples were collected from eight and two Fo generation rats ianppGrrooxuipmsat|ealnydtwIIo, rteosspiexcthiovuerlys,poonstDdLos1a4.ge MoinlkDsLam14p.leTshweerseamcpollelesctweedre shipped to the Sponsor. Following completion of milk sample collection, female rats were sacrificed and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and distribution of implantation sites was recorded. Blood samples were collected via the inferior vena cava and centrifuged and the serum milk-secreting glands from was shipped to the Sponsor. A the axillary, thoracic, abdominal liver and section inguinal and the regions. of each dam (left side only) were collected and shipped to the Sponsor. A.4. F1 Generation Pups Assigned to Pharmacokinetic Sample Collection: Viability and clinical observations, body weights and litter observations were performed as previously described for F1 generation pups assigned to cross- fostering. On DL 14, litters assigned to the pharmacokinetic sample collection were sacrificed and blood samples were collected via the inferior vena cava from `each pup, pooled (per litter), centrifuged and the serum was shipped to the Sponsor. Theliver from each pup was collected, pooled (per litter), and shipped to the Sponsor. ooozmm 156 418-014:PAGE 14 B. Results AalnldFonegcernoeprsaytioobnsefrevmaatlieonrsatwsesruervciovnesditdoersecdheudnurleeladtseadcrtiofitchee. aAdlmlinaidsvterrasteiocnlionfical PFOS. Bproedcyohwaebiigthattigoaniansndwegresetarteidouncepderiinodtsh,e a1s.6comgm/pkagr/eddaytodtohseacgoentgrroolugprdouurpinvaglutehse. bMoadtyerwneailghbtodgyaiwnesigfohrttghaeiennstiwreerleacvtaartiiaobnlepetrhirooduwgehoruetgtehneerlaalcltyatcioonmppaerriaobdleand aremdouncgedthien ftohuer1c.r6omssg-/fkogs/tdeareyddsousbasgeetsg.roAubpsobleugtiennbiondgyawtetihgehtesndweorfethselightly precohabitation period and continuing through the gestation and lactation periods. Absolute and administered relative the 1.6 feed consumption mg/kg/day dosage values were generally reduced for rats of PFOS during the precohabitation, gestation and lactation periods, as compared to the control group values. Tgrhoeupd.uraAtsioonbosfergveestdatinioanptreenvdieoudstloy bceonrdeudcutceeddsitnutdhye(1A.r6gumsg/Rkegs/edaarychdosage Lparbeoirmaptloarniteast,ioInncl.o,ssP;rotthoecoalve4r18a-g0e08n)u,mbtheirs roefdiumcptliaonntawtaisonassistoecsipaetreddwaimthwmaisnimal osflidghetlliyverreydupceerdp,urpes(uilntmiingnuitneas)sltieghntdleydretodubceedreddeulcievedreind tlhiteter1.s6izmeg./kTgh/edaduyration dosage dosage group. group The live was also litter size before slightly reduced. cross-fostering The gestation in the index 1.6 was mg/kg/day 100% in each dosage group; all pregnant rats delivered live offspring. Pup mortality was greatest in pups from test article-treated dams fostered by test article-treated dams (subset C). Pup mortality was also increased in pups from test article-treated dams fostered by vehicle-treated dams (subset A). As a rpeuspusltoonf tDhLes4epirneccruelalsiensg/innupmubpermoofrtpaluiptsy,ctrhoessv-ifaobisltietryeidndDiLc1e)s (wenruembreerduocfeldi,veand onnumDbLer4sporfecsuulrlviinvgining tphuepsse ptewrolsitutberseatnsd. liPvue plitmtoerrtsailzietsy aitn wpeuipgshifnrgomweverheicrleed-uced treated dams fostered by test article-treated dams (subset D) was comparable to that in pups from vehicle-treated dams fostered by vehicle-treated dams. (issurbesseptonBs).iblTehfeosrepuopbsmeorrvtaatliitoyn,sainndditchaatte ptohtaetntiniaulteerxopoesxuproesutroethtoe tthesettaerstticalreticvliea: maternal milk did not adversely affect the viability of the pups. Pup body weights wanedreD)r,edauscceodmopnarDeLd 1toinpbuopthbosduybsweetisghotfsdianmtsheadsmuibnsiesttseroefddaPmFsOSad(msiunbissettesreCd the vehicle (subsets A and B). Potential exposure to the test article via maternal milk after DL1 reduced pup body weights, regardless of in utero exposure to the mu. WN 15% 418-014:PAGE I-5 test article or vehicle. After DL 1, pup body weights were somewhat reduced in subsets D (pups from vehicle-treated dams fostered by test article-treated dams) and A (pups from test article-treated dams fostered by vehicle-treated dams), as `compared to the pups from vehicle-treated dams cross fostered to vehicletreated dams (subset B). After DL 1, pup body weight reductions were greatest in subset C (pups from test article-treated dams fostered by test article-treated dams). "Two litters in subset A (pups from test article-treated dams cross-fostered to vehicle-treated dams) and one litter in subset C (pups from test article-treated dams cross-fostered with test article-treated dams) had pups that did not nurse; pup mortality was greatest in these two subsets. There was no milk in the stomach of 57.1%, 100% and 87.0% of the pups that were found dead and necropsied in subsets A, C and D, respectively. Evaluation of the livers of pups that died or were sacrificed on DL 1 by electron microscopy revealed increased numbers of peroxisomes within hepatocytes.of pups from dams Quantitation of p treated with eroxisomes 1.6 mg/kg/day PFOS, indicated slightly more compared than twice with the controls. number of peroxisomes in treated pups. Differences in cellular membranes or mitochondria were not discernible between control and treated pups in the samples examined. Evaluation of the lungs of pups that died or microscopy revealed Type Il pneumocytes were sacrificed on containing lamellar DL 1 with bodies in electron both control and 1.6 mg/kg/day dosage group pups, with an increased number of Type Il pneumocytes and lamellar bodies in lungs from treated pups. Although some lamellar material (surfactant) was identifiable within alveolar lumina, no significant difference was noted between the control group and 1 6 mg/kg/day dosage group. 15% --- 418-014:PAGE 1-6 C. Conclusion rTprehedevuic1o.eu6dsmfmgeu/elkdtgic/godennaesyruadmtopistoainogsnetvuoadflyPuwFeisOthSfotrchtaehuteseseFtdoargrteeidncuelcreta(itAoirnogsnuidsnaRbmeossd,eyaawrseciohgchcturgraeidn iannad Laboratories, Inc., Protocol 418-008). As previously observed. the 1.6 mglkg/day dosage of PFOS administered to the Fo generation dams in this study also reduced the durations of gestation and parturition, caused preimplantation loss and reductions in litter size and pup growth. Cross-fostering of pups from test article treated dams and vehicle treated dams croevnedaulcetdedthasttutdhye (e4f1f8e-c0ts08o)n, cpaunp mboertaatltirtiybuttheadt tooccinururfeedroinextpheospurreevitooustlhye test article, and that potential exposure to the test article via maternal milk did not autdevreorseexlpyoasfufreectatnhde mviaatbielmitaylofmitlhke cpaupuss.ed Erxepdousctuiroenstointhpeutpesgtroawrttihc.le via both in Mlbd otafo Mildred S. Christian, Ph.D., Fellof, ATS Executive Director of Research 2a Date Gothelbor Alan M. Hoberman, Ph.D., DABT Director of Research 2n50 y Date 4g. Al 25-29. Asasyodchioantde GD.irYeocrtokr, o"f 0Re,eDaArcBhTand Date Study Director Il. DESCRIPTIONOFTEST URS 418-014:PAGE II-1 A. CondofuStcudty: AA. Sponsor: 3M Corporate Toxicology, 3M Center Building 220-2E-02, St. Paul, Minnesota 55144-1000 A2. TestingFacility: Argus Research Laboratories, Inc., 905 Sheehy Drive, Building A, Horsham, Pennsylvania 19044-1297 A3. Study Number: 418-014 Ad. Sponsor's Study Number: T-6295.13 AS. Puropfthoe Sstudey: The purposeof following PFOS this study treatment was to evaluate survival of of Crl:CDBR VAF/Plus F1 Fo generation generation pups. female rats during premating, gestation and lactation. F1 generation pups were cross-fostered during the lactation period to differentiate between effects on pups exposed to the test article in utero and pups exposed to the test article via maternal milk. Selected pharmacokinetic samples were collected from Fo generation female rats and F1 generation pups. AS. Study Design: The requirements of the U.S. Food and Drug Administration (FDA) were used as the basis of study design. A7. Regulatory Compliance: The study was conducted in compliance with Good Laboratory Practice (GLP) regulations of the Ministry of Health U.S. and WFeolofdaraen(dMDHrWu)gAdamnidnitshteraEtuironop(eFaDnAE)c?otnhoemiJcapCaonmemsuenity (EEC). There were no deviations from the GLP regulations that affected the quality or integrity of the study. Quality Assurance Unit findings derived from the a 160 418-014:PAGE II-2 inspections during the conduct of this study are documented and have been provided to the Study Director and the Testing Facility Management. A.8. Ownershipof the Study: The Sponsor owns the study. All raw data, analyses, reports and preserved tissues are the property of the Sponsor. A.9. Study Monitor: Marvin T. Case, D.V.M., Ph.D. A.10. Alternate Study Monitor: Andrew M. Seacat, Ph.D. A.11. Study Director: Raymond G. York, Ph.D., DABT (Associate Director of Research) A.12. Technical Performance: John F. Bamett, B.S. (Director of Laboratory Operations) Margaret M. Martin (Research Associate) Corrie L. Pabst (Quality Control Associate) A.13. Report Preparation: Raymond G. York, Ph.D., DABT Jo Ann Frazee, M.S. (Study Coordinator) Susan K. Bradshaw, B.S. (Data Management Specialist) Karen G. Parker, A.A. (Report Administrator) A.14. Report Review: Alan M. Hoberman, Ph.D., DABT (Director of Research) Mildred S. Christian, Ph.D., Fellow, ATS (ExecutiveDirector of Research) A.15. Date Protocol Signed: 23 October 1998 Gotacs oo-m 418-014:PAGE 13 A. of Technical Performance: Rat Arrival 270CT 98 Dosage Period (42 days before cohabitation until DL* 21) 02 NOV 98 - 28 JAN 99 Cohabitation Period Day 0 of Presumed Gestation (DG 0) 13 DEC 98 PM - 19 DEC 98 AM 14 DEC 98 - 19 DEC 98 Delivery Period 04 JAN 99 - 09 JAN 99 DL 1 Culling (Control Pool) DL 4 Culling (Cross-Fostered Litters) 04 JAN 99 - 09 JAN 99 08 JAN 99 - 11 JAN 99 DG 25 Sacrifice DL 14 Pharmacokinetic Group Sacrifice 08 JAN 99- 12 JAN 99 17 JAN 99 - 22 JAN 99 F1 Generation Pups Sacrifice (DL 21) 25 JAN 99 - 28 JAN 99 Fo Generation Dams Sacrifice (DL 22) 26 JAN 99 - 29 JAN 99 AA7. Records Maintained: 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 preserved tissues are after the mailing of the retained in draft final the archives of the Testing Facility report, after which the Sponsor will for decidetheir final discarded at the disposition. All Testing Facility. uUnnuusseeddprbuelpkarteedsttaerstticalretiwcalesforremtuulmaetdiotnos were the Study Monitor. B. Test Article Information: B.1. Description: PFOS (FC-95) - a light-colored powder B.2. Lot/Batch Number: 217 (Expiration date: May 2000) B.3. Date Received and Storage Conditions: The test article temperature. was received on 21 October 1998, and stored at room a. DLis used as an abbreviation for day of lactationor day postpartum. a 418-014:PAGE 14 B.4. Special Handling Instructions: Standard safety precautions (use of protective clothing, gloves, dust-mist respirator and safety goggles) were taken when handing the bulk test article and prepared formulations. B.5. Analysis of Activity: Information regarding the identity, strength, composition and purity of the test article is on file with the Sponsor. C. Vehicle Information: Ca. Description: 0.5% (RO. Tween 80 in Reverse Deionized Water) Osmosis Membrane Processed Deionized Water C.2. LotNumbers: Tween 80 - MO3HOS, LO6662 and M29477 C3. Da eived and Storage Conditions: The Tween 80 was received on 3 December 1998 (lot MO3HOS), J1.TS.eBpatkeemrb,ePrhi1l9l9ip8sb(ulortg,L0N6e6w62J)erasnedy,1a7nSdewpatesmsbteorre1d9a9t8r(olootmMt2e9m4p7e7r)a,tufrer.om The R.O. deionized water is available from a and is maintained at room temperature. continuous source at the Testing Facilty C.4. Special Handling Instructions: rSetsapnidraatrodr,sasfaefteytypgroegcgaulteisonors s(aufseetyofglpraostseecstiavnedclaotfhaicneg-,shgileolvde)s,wedruset-tmaiksetn when handling the vehicle. C.5. Analysis of Purity: cNoenitthaemrintahnetSspolinkseloyrtonobretphreeSstenutdyinDitrheecvteohriwcalestahawtarweouolfdainnyteprofteernetiwailth the results of this study. 00305 gup 418-014:PAGE II-5 D. Test Article Preparation and Storage Conditions: Suspensions were prepared daily at the Testing Facility at concentrations of 0 and 0.32 mg/mL. Records are maintained in the raw data to document how the test article formulations were prepared. Prepared suspensions were stored D.1. Sample Information: [ameree Ton Joma [E22 -- EEa n aon lO =Eo [ae LotLOBEE2 SmL | 280CT98 empervie Joon Josetes =| ll |g on Aushives. 25NOV98. Tp ep D.2. Analytical Results: Data verifying the stability of the test article in the vehicle for 48 hours under the conditions of administration are on file with the Sponsor. Homogeneity of the prepared formulations is on file with the Sponsor. Results of the concentration analyses have not been received. BE Test System: E.1. Species: Rat E.2. Strain: Crl:CDBR VAF/Plus (Sprague-Dawley) CoesesTe 418-014:PAGE II-6 E3. Supplier (Source): Charles River Laboratories, Inc., Portgage, Michigan Ed. Sex: Female (Male rats of the same source and strain were used only as breeders and were not considered part of the Test System). ES. Rationale for Test System: The Cr:CD@BR VAF/Plus (Sprague-Dawley) rat was selected as the Test System because: 1) this strain of rat was used in the reproductive and developmental toxicity studies; 2) historical data and experience exist at the Testing Facility"; and 3) the test article is pharmacologically active in the species and strain. E6. TestSystemData: Number of Rats Approximate Date of Birth Approximate Age at Arrival Weight (g) on the Day after Arrival Weight (g) at Study Assignment 86 23AUG 98 66 days 179-226 200-244 E7. Breeder Male Rat Data: Number of Rats Approximate Date of Birth Approximate Age at Arrival Weight (g) on the Day After Arrival Weight (g) at Cohabitation 110 13 JAN 98 77 days 317-352 514-866 E.8. Method of Randomization: Upon arrival, male and female rats were assigned to individual housing on the basis of computer-generated random units. After acclimation, 78 virgin female rats were selected for study on the basis of physical appearance and body weights recorded during acclimation. The female rats were assigned to dosage groups (42 in the control group and 36 in the treated group) based on computergenerated (weight-ordered) randomization procedures. Within each dosage group, consecutive order was used to assign female rats to cohabitation with breeder male rats, one male rat per female rat. Rats were 5 assigned to cross-fostering or pharmacokinetic collection depending on the order LE. 418-014PAGE 117 tofhedecloinvterroyl agnrdoutpheanadvaitlwaobifletmyaolferraattssfofrrocrmotssh-efotsrteeartiendg.groEuipghwtefreemadleesirgantsatferdomfor pharmacokinetic sample collection following delivery; these rats were not crossfostered", Twenty-five liters per dosage group were cross-fostered on DL 1. The liters were assigned such that four subsets were obtained among the two dosage groups, as described in the table below. Subset assignments were made based on order of deliveries. Dosage|MaWtoerdmFeermoafle| Dosage | WoLimtebresorr | RessaTgynpeed Lier Subset Group| Rais | (nghgiday) | Reassgned| (Dam Treatment) | Number [TT Towedo| 1 [ Treas [| A| | T| 72 Towed| v2 | uweaes | 5| |r nvT | v6T [ T v2 | Tneemesds[[5c]| On DL 4, cross-fostered liters were culled to five male and five female pups per liter, where possible, using a table of random units. E9. System of identification: E.9.a. Fo Generation Rats: Rats were permanently identified using Monel self-piercing ear tag (Gey Band iadnedntiTfaigcatCioon, nIuncm.b,eNro.s uMpSoPnTas2s01i0g1n)m.entMatloethreatTsewsetrineggFiavceinltuyn'isqbureeepdeerrmamnaelnetrat pUonpiuqluaetiopne.rmaFneemnatleidreanttsifwicearteionasnsuimgbneedrstewmhpeonraarsysingunmebdertos tahte receipt study. and given Cage tiadegnstiwfeicraetimonaraknedddwoistahgtehelesvtelu.dy number, permanent rat number, sex, test article E.9.b. F1 Generation Pups: Pups were not individually identified during lactation; all parameters were evaluated in terms of the ler. a See APPENDIX D (DEVIATIONS FROM THE PROTOCOL AND THE SitTeAmsND1 AanRdD2.OPERATING PROCEDURES OF THE TESTING FACILITY), b. See APPENDIX D, item 3. 160 242 418-014:PAGE II-8 F. Husbandry: FA. Research Facili istration: USDA Registration No. 23-R-099 under the Animal Welfare Act, 7 U.S.C. 2131 et seq F2. Study Rooms: Tahhealsltwuadyyarnodomisndweeprenedmeanitnltyasiunpepdliuenddewirtchoandmiitinoinmsuomf opofstietnivcehaainrfgleosw rpeelrathioveurtoof 100% freshair that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 64F to 79F (18C to 26C); room humidity was targeted at 30% to 70%. See APPENDIX E (TEMPERATURE AND RELATIVE HUMIDITY REPORTS). F.3. Housing: Fo generation rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During the cohabitation period, each pair of rats was housed in the male rat's cage. Beginning no later than DG 20, Fo generation female rats were individually housed in nesting boxes, except during the collection interval for urine and fecal samples. During this collection interval (DLs 21 to 22), the female rats were housed housed individually in in a common metabolism nesting box cages. Each dam and assigned during the postpartum period. All liter were cage sizes and housing conditions were ofLaboratory Animals. in compliance with the Guide for the Care and Use. F.4. Lighting: An automatically-controlled fluorescent light cycle was maintained at 12-hours light:12-hours dark, with each dark period beginning at 1900 hours EST. F.5. Sanitization: wCeargeecphaanngliendersapwperroexicmhaatenlgyedevaeprpyrootxhiemratweeleyk.thrBeeedtdiimnegs weaaschcwheaenkg.edCaasgeosften as necessary to keep the rats dry and clean. F6. Feed: Rats were given ad /ibitum access to Certified Rodent Nutrition International, St. Louis, Missouri) in individual Diet #5002 feeders. (PMI 16% QoOLTO 43 418-014:PAGE 11-9 F.7. Feed Analysis: Analyses were routinely performed by the feed supplier. No contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were detected by these analyses. Copies of the results of the feed analyses are available in the raw data Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to have been present in the feed that would have interfered with the results of this study. FS. Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles. Chlorine was added to the processed water as a bacteriostat F.9. WaterAnalysis: The processed water is analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, Pennsylvania) and monthly for possible bacterial contamination (Analytical Laboratories, Inc., Chalfont, Pennsylvania). Copies of the resus of the water analyses are available in the raw data. Neither the Sponsor nor the StudyDirector was awareof any potential contaminants likely to with the results of this have been study. present in the water that would have interfered F.10. Bedding: Bed 0'cobs was used as the nesting material (Andersons Industrial Products Groups, Maumee, Ohio). F.11. Bedding Analysis: Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to have been present in the bedding that would have interfered with the results of this study. Analyses for possible contamination are conducted annually and documented in the raw data. G. Methods: pr G.1. Dosage Administration: [+[owe 418-014:PAGE 11-10 ToT & a. The test arice was considered 100% pure forthe purpose ofdosage calcuatons. | Jo]wee[TSE] Se|Solf | Te Eratston Sirean |ee] C wom= To we] 10901 - 18942 1691o5, a189251.8514e002r. Dosage| Susser EE [dT 1s8a5s2e4z, 16529. 16934. 16935, 16637, 16541,| 1i8o9s7e2s. 18950. 16959. 19977. 8956, 16951. [ro[samge. tsosz2.a18535.e166d36.v10e532. 19931|| e10915.T1801e7. T1o2012. r0o006.t8e004.m16r001| LTTeTT1Eo0S9T6E.7 11l86997843.-116i69956d35..v11o0690n7501.t110o6607s43S,s 11096529. 68|e1188996043.:v1186n506007,-t11659e0587..o1180e050406.o1T805s94176..211065%4E30| [EERERREEERas ST95ers006fsoe a red Conse sesi2 G2. Rationale for Dosage Selection: Dosages were selected on the basis of a previous two-generation study conducted with the test article (Argus Research Laboratories, Inc. Protocol 418-008). In this study the Fo generation maternal and paternal noobservable-effect-level (NOEL) of PFOS was 0.1 mg/kg/day (0.4 mg/kg/day and higher dosages caused reductions in body weight gain and reduced feed consumption values). The Fo generation reproductive NOEL was greater than 3.2 mg/kg/day; no effects on mating, fertility or estrous cycling occurred. The NOEL for viability and growth in the F1 generation offspring was 0.4 mg/kg/day (1.6 mg/kg/day and higher dosages viability, growth caused preimplantation and survival). loss and reductions in litter size, pup 169 O0ROT mg 418-014:PAGE I-11 The F1 generation maternal and patemal NOEL of PFOS was 0.1 mg/kg/day (0.4 mg/kg/day dosage caused reductions in body weight gain and reduced feed consumption values). The F1 generation reproductive NOEL was greater than a dosage of 0.4 mg/kg/day; no effects on mating or fertility occurred. The NOEL for viability and growth in the F2 generation offspring was 0.1 mg/kg/day (0.4 mg/kg/day dosage caused stillbirths and reductions in litter size, pup viability, growth and survival). G3. Routeof Administration: Oral (gavage) G4. Rationale for Route of Administration: The oral (gavage) route was selected for use because: 1) this was the route of administration in the developmental and reproductive toxicology studies; and 2) it is one of the possible routes of human exposure. G5. Erequency of Administration: G.5.a. Fo GenerationFemale Rats: Female rats were given the test article or the vehicle once daily beginning 42 days prior to cohabitation through DL 21. Dosages were adjusted daily for body weight changes and given at approximately the same time each day. G.5.b. F1 Generation Pups: F1 generation pups were not directly given the test article, but may have been possibly exposed to the test article during maternal gestation (in utero exposure) or via maternal milk during the lactation period. G6. Length of Study: Approximately 3 months G7. Method of Study Performance: G.7.a. Cohabitation and Cross-Fostering Procedures: Twenty-five rats assigned to the control group were cohabited one day prior to cohabitation for rats assigned to the treated group and the cohabitation period consisted of a maximum of six days". The cohabitation period for the remaining a. See APPENDIX D, item 4. 418-014:PAGE I-12 matasxiinmtuhme ocfonftirvoeldagyrso.upDaunrdintghecorhaatbsitaastsiiognn,ebdottoh tchoenttrroelaatenddgPrFoOuSp-twraesataed fgermoaulpes wraet.reFceom-ahloeusraetds wwiitthh usnpterremaattedozmoaaleobbsreerevdeedrsi,n oansemmeaalreorfatthpeevraognineal contents andlor a copulatory plug observed DG 0 and assigned to individual housing in situ were considered to be at pSaervteurriatliodnaomcscuarsrseidgnweedretoaltlheowceodnttrooldeglrioveurpatnhadtrdeetlaiinvetrheedironnattuhrealfipstupdsayunotfil anveaeidlaebdlefofrorcriomsmse-fdoisatteericnrgospsu-rfpoossteesr.ingThoifsppurposvifdreodm aPFpOoSo-ltorfecaotnetdrodladmasm,sthus preventing these pups from nursing from their PFOS-treated dams. Srteamrotivnegdwiitmhmetdhieadteelliyve(raisessoofonthaesdpaamrtsurointiotnhewasseccoonmdpldeaty,eda)llfpruopmsthweeirre respective group or a dPFaOmSs-tarnedatpeldacdeadmw.ithWheietnhera adnaotmhearssdiagnmedastsoitghneedcotnotrtohle gcroonturpolwas cnoonttraovlaiplaobolle,dathmenwaasdtahmenfrsaocmritfhiececdonvtiraoldepcoaoplitwaatisonu.sed. The liter from that Ti.he.i,sCcornostsr-oflolsPteFrOiSng(SpurbosceetduAr),e Croensturlotle/dCoinntfrooulr g(SruobuspestofB),12PoFrO1S3/PdaFmOsS/pups, (bSeumbsaedteC)beatnwdeePnFOpSr/enCaotnatlroalnd(SpuobsstneattaD)l.efTfheictssporfoctheeducroentailnluoowuesd mdaistteimncatlions to PFOS treatment G.7.b. Fo Generation Rats Assigned to Cross-Fostering: oTfhetheFostguednyeraantdiofnorfgeemnaelrealraatsppweearreaonbcseeartveledasftorovnicabeildiutyriantgletahsetatcwciicmeaetaicohn day tpeesrtioadr.ticTleh,eabroarttsiwoensr,eparlesmoaetxuarmeidneeldivefroirecsliannicda/loorbdseeartvhastiboenfsoorfeefafnedcts of the approximately one hour postdosage. wBeoidgyhtwseiwgehrtes rweecroerdreedcodradileyddautrilneagstthoendcoesdaugreinpgertihoedacacnldimaattsiaocnripfeicrei.od.FeBeoddy cweoenksluympttoicoonhavbailtuaetsiown,erdeailryecdourrdiendg attheleparstesounmceeddguersitnagttihoen apcecrliiomdaatinodnopnerDioLds, 1, e4x,p7e,c1t0edantdhat14t.heFpeuepdscboengsaunmpttoicononwsausmenomtattaebmulaaltefdeeadf.ter DL 14, when it was oTbhseerfveamtailoensradtusrwiengrepacrotnutriitniuoanll[ypeevraflouramteeddu(n2d4erhoruerdslipgehrt day) for clinical conditions during the dark period (ime each pup was observed was recorded), duration of gestation (DG 0 to the time the first pup was delivered), length of partuition (time of 13H "e 418-014:PAGE I-13 observation of last pup minus the time of observation of the first pup divided by N-1 pups in each litter), litter sizes (defined as all pups delivered) and pup veixaabimliintyataitobnirotfh.thPeulpisterthfaotr eviitahbeirltaypwpeeraereedxastmiilnlbeodrnfoorr tvihtaatl dsiteatdubseaftorbeithin.itiaTlhe 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. Matemal behavior of the dams was evaluated daily when the pups were examined during the 21-day postpartum period. Observed maternal behavior was recorded on DLs 1, 4, 7, 14 and 21. Deviations from expected maternal behavior were recorded, if and when present, on all other days during the postpartum period cFoollgeecnteiornatoifounrifneemaalnedrfaetcsawl esraemphloeussefdroimndDiLvisdu2a1lltyoin22m. etFaoblolloiwisnmgctahgee2s4f-ohrour cicoel,lescttoiroendifntreorzvealn,(s-a7m0pCleosr wbeerloewc)oallnedctsehdiipnpteodcetnottrhiefuSgpeotnusboesr,foprlaancaeldysoins.dry On DL 22, blood samples (approximately 4 mL each) were collected from the maternal rats via the inferior vena cava and transferred into serum separator tubes after removal from metabolism caging. The samples were spun in a refrigerated centrifuge. The serum was transferred into polypropylene tubes labeled with the study number, rat identification, date of collection, study day and collection timepoint. All samples were immediately frozen on dry ice, stored frozen (-70C or below) and shipped to the Sponsor for analysis. G.7.c. E1GenerationPupsAssignedtoCross-Fostering: Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups ina litter were individually weighed. Each litter was evaluated for viability at least twice each day of the postpartum period. Dead pups observed at these times were removed from the nesting box. The pups present in each litter were counted once each day. Physical signs (including gross external physical anomalies) were recorded for the pups once each day during the postpartum period. Pup body weights were recorded on DLs 1 (birth), 4, 7, 14 and 21 and at sacrifice. On DL 4, cross-fostered F1 generation litters were culled to five male and five female pups per litter, where possible. oozes WI 418-014:PAGE II-14 G.7.d. Fo Generation Rats Assigned to Pharmacokinetic Sample Collection: Viability and clinical observations, body weights, feed consumption values and delivery observations were performed, as previously described for Fo generation dams assigned to cross-fostering. Blood, milk and liver samples were collected from eight and two Fo generation craotlsleicntiGornopueprsd|oasnadgeIl,grreosuppecotniveDlLy,1d4.esiMgilnkatseadmpfolrepshwaermraeccooklilneecttiecd sample approximately two to six hours postdosage on DL 14. Each dam was removed from the nesting box and individually housed for approximately four hours. The dam was injected intravenously with one unit of oxytocin (20 USP units/mL, Lot Number 52064, expiration date July, 2000, manufactured by Osborn) approximately five minutes before milk samples (at least 100 ul per sample) were collected. The samples were immediately frozen on dry ice, stored frozen (-70C or below) and shipped to the Sponsor. Following milk sample collection, blood samples (approximately 4 mL each) were collected from the maternal rats via the inferior vena cava and transferred into serum separator tubes. The samples were spun in a refrigerated centrifuge. The serum was transferred into polypropylene tubes labeled with the study number, rat identification, date of collection, study day and collection timepoint. All samples were immediately frozen on dry ice, stored frozen (-70C or below) and shipped to the Sponsor. Following collection of milk and blood samples, a liver section (right lateral lobe) and the milk-secreting glands from the axillary, thoracic, abdominal and inguinal regions of each dam (left side only) were collected, stored frozen (-70C or below) and shipped to the Sponsor. G.7.e. F1 Generation Pups Assigned to Pharmacokinetic Sample Collection: Viability and clinical observations, body weight and litter observations were performed as previously described for F1 generaton pups assigned to crossfostering Caps and labeled tubes were weighed (combined weight, to the nearest 0.001 g) before and after retention of pooled pup samples for subsequent use in pharmacokinetic analyses. Blood samples were collected via the inferior vena cava from each pup on DL 14, pooled (per litter) and transferred into serum separator tubes. The samples were spun in a refrigerated centrifuge. The serum was transferred into 113 polypropylene tubes labeled with the study number, pup identification, date of my 418-014:PAGE II-15 collection, study day and collection timepoint. All samples were immediately frozen on dry ice, stored frozen (-70C or below) and shipped to the Sponsor for analysis. The liver from each pup was collected, pooled (per litter), stored frozen (-70C or below) and shipped to the Sponsor for analysis. H. Gross Necropsy: All Fo generation rats were sacrificed by carbon dioxide asphyxiation, 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 maternal gross lesions are available in the raw data. H.1. Fo Generation Rats: The female rats were sacrificed on DL 22. The number and distribution of implantation sites were recorded. A liver section (right lateral lobe) from each `dam was collected, stored frozen (-70C or below) and shipped to the Sponsor for analysis. Following completion of milk sample collection on DL 14, female rats assigned to pharmacokinetic collection were sacrificed. Blood and liver samples were collected as previously described. The number and distribution of implantation sites was recorded. Rats that did not deliver alitterwere sacrificed on DG 25 and examined for gross. lesions. Uteri were stained with 10% ammonium sulfide to confirm the absence of implantation sites. Dams with no surviving pups were sacrificed after the last pup was found dead, missing or presumed cannibalized. A blood sample (approximately 4 mL) was collected, prior to sacrifice, via the inferior vena cava and transferred into serum separator tubes. The samples were spun in a refrigerated centrifuge. The serum was transferred into a polypropylene tube labeled with study number, rat identification, date of collection, study day and collection timepoint. The samples were immediately frozen on dry ice, and stored frozen (-70C or below) and a. A table of random units was used to select one Fo generation control group female rat and one F1 generation control group male and female pup from which all tissues examined at necropsy were retained, in order to provide control tissues for potential comparative histopathological evaluations. 1M Leona L. 418-014:PAGE I-16 SdhaimppweadstocotlhleecStpedo,nssotorrefodrfarnoazleynsi(s-.7A0Cliovrerbseelcotwi)ona(nrdigshthilpatpeerdaltlootbhee) fSrpoomnseoarch for analysis H2. Fi Generation Pups: vPiutaplssttahtautsdiatedbibrethf,oraes epxraemviionuastliyondeosfcrtihbeeidt.erPfuoprspuwipthvigarboistsylweesrieonsevwaelrueated for pprreesseerrvveedd iinn BBoouuiinn''ss Ssoolluuttiioonn,faorndpotshseibllievefruwtaurse pervaelsueartvieond.inTnheuetrlaulngbsufwfeerreed 10% formalin for possible future evaluation. pPuuppss cfurlolmeddoanmsDLas4siwgenreedstaocrtihfeicceodntvrioaldpeocoalpiatantdiocnu.lleTdhoenlDunLgs1 aannddtahleoftihveerr lwiettrere) caonldletchteedfrfsrtotmenthpeufpisrsthtaetn wceulrleedfopuunpdsdferaodm t(hneo caounttorloylsips)olfr(oimeesapcechtive of dliovserasgweegrreorueptaoinneDdLin1.neTuhreallubnugfsfewreedre1r0e%tafionremdalininBofourinp'ossssioblulteifonu,tuarend the eevvaalluuaattiioonn.. Remaining culled pups were sacrificed and discarded without lPeuspisonsfoaunndd fdoeratdheorcasaucsreifoifceddeabtehcaoursteheofmmoorriibbuunnddictoyndwiteiroen.exFaomrianleldpufporsgfroousnsd dweaasdpornesDerLvsed21i0n 4n,eutthreallbuunfgfsewreedre1p0r%esfeorrmvaeldini,n fBooruipno'sssisbolleutfiuotnu,reanevdaltuhaetifoinv.er fPourppsoswsiitbhlegrfoustsurleeseivoanlsuaftoiuonn.d oFnorDalLl p2upfos4fowuenrdedperaedseornveDdLisn 5Botuoin21',s tshoelultuinogns, fpiovsesriabnled fgurtousrse elveasliounastiwoenre preserved in neural buffered 10% formalin for rOenspDecLti1v4e, dliattmerss wasesriegnsaecdritfoictehde bpyhacramrabcoonkidnieotxiicdesaasmpphlyxeiactoilolne.ctioInndainviddutahle,ir dpeosoclreidbesda.mpTlhees p(upeprsiweerr)eoefxbalmoiondeadnfdorligvreorswserleesicoonlsl.ected as previously lOensioDnLs.21G,raollsspulpessiwonesrewesraecrirfeitcaeidnevdiaindenceauptirtaaltbiuofnfearnedde1x0a%mifnoermdalfionr.grSoisxs liters pbleorosdubasnedtlwiveerr.e Brlaonoddomslaympselleesctweedreforcoclollelcetcetdiovniaoftpheooilnefedrisoarmvpelneasc(apvera lfirtoerm) of Seaamcphlpeusp,weproeolsepdu(npeinr aitreerf)riagnedrattreadnCsefnetrrriefdugien.to Tsheerusmesreupmarwaatsortrtaunbsefse.rrTedheto cpoolllyepctrioopny,lsetnuedtyudbaesy laanbdelceodllweictthiotnhetimsetpuodiyntn.umAblelrs,amraptliedsenwtiefriecatiimomne,ddiaatteeloyf fozen onary ic, sored fozen (70Co blow) and hipped 0 the Sponsor for 3 2. analysis. TY 860738 IM 418-014:PAGE 11-17 All pups selected for continued observation were sacrificed via decapitation and examined for gross lesions. I. Statistical Analyses: Averages and percentages were calculated. Litter values were used where appropriate. 6 252 i. RESULTS 418-014:PAGE l-1 A. Mortality, Clinical and Necropsy Observations (Summaries -Tables 1 and 2; Individual Data - Tables 19 and 20) All rats survived to scheduled sacrifice. All adverse clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to the test article because the incidences were not dosage-dependentand/or the observations are commonly seen in rats in the laboratory environment. These observations included chromorhinorrhea, scaly tail, abrasion on the head, neck, tail and/or forelimb, missing, broken and/or misaligned incisors, localized alopecia on the head, limbs, neck and/or back, chromodacryorrhea, scab on the head or tail dehydration, perineal hernia, broken or swollen forepaw digit and protruding xiphoid. The only necropsy observation was an inguinal mass (presumed to be a galactocele) in one control group dam assigned to pharmacokinetic sample collection on lactation day 14 (DL 14). B. Body Weights (Figures 1 and 2; Summaries - Tables 3 through 8; IndividualData -- Tables21 through23 Body weight gains during the precohabitation period were reduced as compared to the control group values, beginning on days 15 to 22 of the study (DSs 15 102) and continuing through DSs 29 to 36. Rats in both dosage groups lost weight on DSs 36 10 43; the body weight loss was greater in the 1.6 mg/kg/day dosage group. Although calculated for a seven-day interval, the weight losses generally occurred on days 42 to 43 of the study and were associated with movement of the male breeder rats into the animal room and mating activity in the animal room when the rats were cohabited on day 42. As a result of these changes, body weight gains for the entire precohabitation period (DSs 1 to 43) were reduced and absolute body weights were slightly reduced on DS 43 (96.6% of control) in the 1.6 mg/kg/day dosage group. Matemal body weight gains continued to be reduced in the 1.6 mg/kg/day dosage group in the first two weeks of the gestation period (days 0 10 14 of gestation, DGs 0 to 14). As a result, body weight gains were reduced for the entire gestation period (DGs 0 to 20) in the 1.6 mg/kg/day dosage group. Maternal body weights tended to be reduced in the 1.6 mg/kgiday dosage group throughout gestation. Reduced maternal body weights persisted in the two subsets of rats administered 1.6 mg/kg/day PFOS during the lactation period (subsets C and 0), 17 LL. 418-014:PAGE III-2 as compared to the rats in the subsets administered the vehicle (subsets A and B). Lactation body weights had the expected degree of variability but were `comparable between subsets A and B (vehicle control groups) and between subsets C and D(test article-treated groups). Maternal body weight gainsfor the `entire lactation period (DLs 1 to 22) were essentially comparable among the four subsets. Cc. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables 9 through 14; Individual Data - Tables 24 through 26) Absolute (g/day) and relative (g/kg/day) feed consumption values were generally reduced for rats administered the 1.6 mg/kg/day dosage of PFOS during the precohabitation, gestation and lactation periods, as compared to the control group values. D. Natural Delivery and Litter Observations (Summaries - Tables 15 and 16; Individual Data - Tables 27 through 31 D.1. Natural Delivery Observations The duration of gestation tended to be reduced in the 1.6 mg/kg/day dosage group, both when the duration of gestation was calculated in whole days and to the nearest tenth of a day. As previously observed, this reduction was associated with minimal preimplantation loss; the average number of implantation sitesper dam was slightly reduced, resulting in a slightly reduced delivered litter size. The duration of delivery per pup (in minutes) tended to be reduced in the 1.6 mg/kg/day dosage group. The live litter size before crossfostering in the 1.6 mg/kg/day dosage group was also slightly reduced, an observation related to total litter size and associated with stillbirths. The gestation index was 100% in each dosage group; all pregnant rats delivered live offspring. D.2. Cross-Fostered Litter Observations After cross-fostering on DL 1, live litter sizes were comparable among the four subsets. Pup mortality was greatest in subset C (pups from test article-treated dams fostered by test article-treated dams); 19.2% of the pups were found dead or presumed cannibalized on DLs 2 to 4. Pup mortality was also increased in subset A (pups from test article-treated dams fostered by vehicle-treated dams) on DLs 2 to 4; 9.0% of these pups were found dead or presumed cannibalized. As a result of these increases in pup mortality, the viability indices (number of 217% ceozer MWR 418-014:PAGE III-3 live pups on DL 4 preculling/number of pups cross-fostered on DL1) were reduced, and numbers of surviving pups per litter and live litter sizes at weighing were reduced on DL 4 preculing in these two subsets. Pup mortality in subset D (pups from vehicle-treated dams fostered by test article-treated dams) was vceohmipcaler-atbrleeatteod tdhaamtsi)n.suTbhseetseBo(bpsueprsvaftrioomnsveihnidcilcea-tteretahtaetdinduatmesrofeoxstpeorseudrebyto the test article is responsible for pup mortality, and that potential exposure to the test article via maternal milk did not adversely affect the viability of the pups. aPdumpinbiosdtyewreeidghPt/FlOitSte(rswuebrseetsreAduacnedd Co)n, aDsL c1oimnpbaortehdstuobspeutpsboofdpyuwpesifghrtosm dinatmhse subsets of pups from dams administered the vehicle (subsets B and D). Potential exposure to the test article via maternal milk after DL1 reduced pup DboLd1y, wpeuipghbtso,dyrewgeairgdhltesssweofreinsuotmeerowheaxptorseudruecteodtihnestuebssteatrsticDle(oprupvsehifcrloem. After vehicle-treated dams fostered by test article-treated dams) and A (pups from test article-treated dams fostered by vehicle-treated dams), as compared to the pups from vehicle-treated dams fostered by vehicle-treated dams (subset B). After DL 1, pup body weight reductions were greatest in subset C (pups from test article-treated dams fostered by test article-treated dams). Sex ratios and the lactation index (number of live pups on DL 21/number of live pups on DL 4 postculling) were comparable among the four cross-fostered subsets. E. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations (Summaries - Tables 17 and 18; Individual Data - Tab 32l ane d3s 3) Two litters in subset A (pups from test article-treated dams fostered by vehicle treated dams) and one litter in subset C (pups from test article-treated dams fostered by test article-treated dams) had pups that did not nurse; pup mortality was greatest in these two subsets. There was no milk in the stomach of 57.1%, C1a00n%d aDn,dre8s7pe.c0t%iveolfyt.he pups that were found dead and necropsied in subsets A, All other clinical and necropsy observations in the F1 generation pups were considered unrelated to the test article because: 1) the incidences were not dobosseargvea-tdieopnesnidnecnltu;deodr b2l)atchketiopbsoefrtvaialt, iloanboorcecdurbrreedatihninogn,lydaornke ilnitcteorl.orC,limniiscsailng hindpaw digit and bent tail. One pup in subset A had slight dilation of the renal pelvis at necropsy on DL 21 1M coor WW ans 418-014:PAGE Ill-4 F. le i opic Evaluation of Liver and Lung from Day 1 Postpartum Pups (APPENDIX G) FA. Liver Ultrastructurally, there were increased numbers of peroxisomes within hepatocytes of pups from dams treated with 1.6 mg/kg/day PFOS, compared with controls. Quantitation of peroxisomes indicated slightly more than twice the number of peroxisomes in treated mitochondria were not discernible bpueptsw.eeDnifcfoenrtreonlceasnidn tcreelalutleadr pmuepmsbrinatnhees or `samples examined. F.2. Lung Type Il pneumocytes containing lamellar bodies were identified in both control ainncdre1a.s6emdgn/kugm/bdearyodfoTsyapgee Igl rponuepumpuopcsy;tehsowaenvderl,amtehlelraer abpopdeieasreidn ltuonbges farnom treated pups. Although some lamellar material (surfactant) was identifiable `within alveolar lumina, no significant difference was noted between the control group and 1.6 mg/kg/day dosage group. osoees WA. REFERENCES 418-014:PAGE III-5 1. U.S. Food and Drug Administration (1994). Intemational Conference on Harmonisation; Guideline on detection of toxicity to reproduction for medicinal products. No. 183. Federal Register, September 22, 1994, Vol. 59, 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 (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Community of an ECD decision/recommendation on compliance with principles ofgood 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, November 7, 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 Cri:=CDBR 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. GOO2e4 APPENDIX A REPORT FIGURES 2% awezes ll wed MATERNAL BODY WEIGHTS | Ile | Tle Figur1e ! ///V]/A LEP | g5 , -- - / | JA So2"0 | || =5 g 3 onvorsnow onvor estan z 3 MATERNAL BODY WEIGHTS RATS ASSIGNED TO CROSS-FOSTERING "0 J Figure 2 wm | Am 0 - rd v 4 ggm o | - - -v oe mowo -- w| v v or a wl| a o - A wo J :" b; orucmnol: . . $ HR EEE T R a E Sr _vsonseran |-- mieo"rce -- . 3:g z8 APPENDIX B REPORT TABLES oevzes 145 =- : a8 : FA] RT :; 0: | $2 : mm nonomn ER Rtsimote ot ve I g; z= ; 3 ; Ez : FI E A TLSe Ey : ! SE no ly con dover heh 1. oat done areup Moser. Z z :3? I 2 i, : 2 iz min un FERRE Sa :t a | i= i ] - 22 i ; ix : | bP EEEE EE E EEsSnSeBe a ee : Baran poralanFsSinToon Stnrel fee after day 10 of Jactation, saernal feed coomegon values 3 i ic: : Se I Ft : f. : | | |= 2 B Ty BY WHY Zz I : I... li ih EN 3 SETE sa oi m tn n Syaadsga e eeeee . bE TE ER Fr sa een g 2S z ENER:: i $ & 3s | | Ex : i 3 : 32 i SgmS0H0ew BE TM pap sg ES) ERLE ASO BEST LIGJHTOREPMTHLND DIGIT Sin + wou gare i or 0ONof sheGs asmarion Co 5 z ig or 8 gs frEeBd EEaEpEa BEeee.E gir ererrsasir aoree ins _ :g E HEISE... ei a ports . &2 g 8i EERE 3 LT es se crore. z . . :8 :5 2  r E rme E ml RheeE ne onSCRE SF ET 33 ILI SA I Tv 2 a Z ] no, a vn, co nn sm rs ss. rns sn 5 gg i& g gg8 J mmooooS EErsOEoa1TEB5 HEmrnismEEmEbbMEmEeREEEEa 3gzg 2 wn ws . o SERA, Au Tissues women 28 EL LT ey og2 HEIN. 3 : a 2 & | 1 ie vorvis tan iin on- mtn rout : i 3 3 : HEE ; :5 :i s z  z a 8 3 B a 8 g:2 HEERlEeoEnEemRiD. 38&3E z g : : 8 : Fe ATT RR TR Bede mroBe ome ome me ne mcm he he ne i he en g A Seid winRecor 1m cue (6). ) or 2i :3 g 5 : 8g 38 5 g: if Ne ELT VERE ECOROED I GaaRS (1 s8 3 ~ ? FE ee en g : :2 i i g 5 28 :I & 3$o HER EERE E TI cy os se ss ees fi eia tr 5 ot gi :i 8 3 23 | wy ener win ee eins A = & 3g 3: 8 & 5 " TTI TTIN ony neound tet wn eset stonsdy 3 a sean 2 8g 8 3 3 8 5 Eg 2g 8 & = 3 g 2: 2$ 3g & i i o ionsvkmeconctanees 61 3 8g : 8 3 ' 5: ; 8 2 & & o EMM OBOE om OB OB om om ou B 2 H I E etrae vcces it 1. uooray oes ven itero . r ) BT dr eete st esa g C38 a ] BOF EEE REE EEE EEG :g EHRETLRE om nSnSeo. wvie.nity.bets cmeeet Tg3 g mie QF MLPI IIE LI lI hs I eI sonar drew ditvers : 5 z we EI ST SE Ca) wT :g Ss EEE mm Bm Sons Bae ALL MEIGNTS MERE RECORDED GAAKS (6) ) " BN :i52 8 i ; 8 i g : f 2S 33 > ? : : gSb@) m Valum e wasmnot"p recorded. e Th. 22 : 3 g g i gSb BValRue CappEearIed EincLorrEectAly SM, recorded and was saciudes from group averages. g x3 8 3 > 2 EEE SL 38g 3ga 3 & 2 : g5 2: g4 i3 8 3 8 3  2 g i g2 i a i 2:8 g : g S 3 8 &: 3  g g3 3 a R & HR s 8 3 8 8 3 z Hs g 3&g33 g 8S H MEEiRm EnIa ITIa I ee crn ce rt tetear 10 -- 53 eer am aieldfe SS ee z33 @ + WITLI @8  a Bo sRailBe. EB5 ar ll EeEur sot upto mon 33 J. eT Ln ns en Saree peers 8 g |] : 8 S g2:i z g ggS Eomm oXmeo:PoopEBp8op mem EHEERE BRO SSMSdAoirGgoadAatRiooAprpi riodpibiliioiiirioooiEiiiE ios38RF yr Cm rs. atsorLows sorts sors 31 -- oS omEEmmm oE 8H fE iopn Bnoooommm HFBEFo Gfvpeaondgdioooirriirooorirrioraiaipiiiiii osg3f DSQ EEE w$7iImRMA rOFeFaUPiSDsYIW PRIORTO CAOSS-FOS-TERING GWDAY 1 POSTRANTIN nl -- i i LySEER ce vt wry oe ce. g# < g 2 2 8 ig g8 =um ine 21 3i" 2 BE ined eb sare sco ad - . 83 sms olHoon is veces ofon hos set aco. avian by ns on 2 2 g g E um 7BH ozi gat 3g GSFb$ R STiEthT I Sh nA EeLnICLa Ht Ite hto tee ds by.21spp - 83z + EgmERE gg 2 en i LL a i:2 3 z gSHEEC mm E E Hi nER HEEHEHIE ME HDDEESSERiLmEa E aRiEmEi R E EEGE 5gi Bhi FELL LR eR EEShen ce tn 4 trrn hBmeee ns : gI & od oEuE RUE EEHEHEEEE df hEeEDTalTE.AE Ee om imor Sm robweirs men 3 Liv Forseaay UF SE EE Iare - 3 g . : 8g :: 8 | ei i in - -- g mmmi HmaondniiBdbnouononouonaannnnh,ia 2 Sie D Eevn se fienoTnineanssn1sve, - ovum,pan, - oecones MLE essesans &2z I Sr ee Tatt So g | asts, on nn, x ran on or Jo E mn HwnmmmHBHHuELHOUBEHuBH uLsE nR nE n, 3 : J BFE BE ' 38 g g3 m mi Em i Hmuua mHEuHeEuHmHHHIuLnAHnLuLuLnnnAiLuIinAiIg], Hi 3 b2fiS Pon,a, cE oI n,tne, rsSs oens 8i gi pmmampn RHEE EEL ee 8ghmsi Sraatatsta ro HsoSm peREH o1r0e2c2oD ns i, :3 bE i aya SE RAR TLR : LICKarS vere not cross fomtered: values sxciuded from grou p sversge7e | VEUT 8 g gmm hmh piaHR BHHAMHAMSE Y E :i I HER na - - Te 8 i EE pe o mmna mn SwHiTafATHHEEHDHRHIITHAMIENAERIRMAEGDLSGWARaTnHEaR, es i. : de ow ) ) ) z ha EE EL i | Tre cL S g gi mom EHBEEBHIEEEIENEIIIHmISERIHImMMIEMSIIELGEIHR mTiS ENiEE i,ieEr, &32 Sg iEmEni ammIEpRIIiMEIIEaEIIEBIEETBRIEEIRGIEEIIERIIRRIIRIIRAIMR I aLLLe , A :an sees c=ommas ore 33 Ep aSIeaehiret , LiS nenSh rele CoTT ntLe ee 5 8 R gg oumh MRL IEMIEEIEEIEEEMIHEERIBEOIEHNENmEinNimEa 3 : o i Phi cove , comm,atete coon 8g ggS BoEm mBiEmilmmEimmBEIERIIRHIBRIEBBI EAIR RIRID RINS IEL LL, ey Q N aeF IrmamnE R n s wanE wE eoonoesein cannE es e16) ,Asroe mme- s conti 2v#3 Cross: fostered: Values excluded from Group sverapes' g 8 g3 EaEr IMEg ImmsiEimiminioRI) EELDT Hi E TH ve,con, teeecmon : 3i +E SEE he ee tee i 8  3 Bm mi EBIEEm IEITEIESIEISIE Bi MIRCRIRIRISIRIL,, 3 :2 h NEISiy a RE i Rp ASa R L ee 23 OE HEE nnaseaen 5 :g ;3 i g: ;ia & EEE MBE GaR ~ gg 3:2 ; ; g 8 g im MIELE AHR BmmS TL a, ConFen, 5 tecose : 2 R 1 ie cEoAm en Ee E TE e i S00 22 8 io Stren me ee a a aw o$88 EE MREm A CRm IHLE NL eIXe NL OE R8 IRI IRI-IL LL ) 33:5 G IT mL inmeI a aba rol 3 s2 :: 2 ;3 z : : gQF E NETRA EIN II UD INNS ruehv ars mm em, on ie f3? 3 8 g g 32 2 i 2 8  2 8 88 &5 2 : =I I: I .c.cpi n. 8 F Se LC TB e o 3 @ wo Sp Se :. 2 a 1 008seve om. ee Sa . :5 3 7 8 | hn igo on Sn, mers mesos PEFEIREEEEe g i RE $ CRE ee : 8 CR g 28 Ta I : 3 8 APPENDIX C PROTOCOL AND AMENDMENT | 000395 418-014:PAGE C-1 rv,PRIMED!ICA -_--_---- Hsis ResSeharechyLSaibvoar:atoSriues,iIn:c. `TelTeeplheoHfnaoexr::sh((22a11m55,))4P444A33-1.698705148047 STUDYTITLE: PURPOSE: TESTING FACILITY: STUDY DIRECTOR: `SPONSOR: STUDY MONITOR: ALTERNATE STUDY MONITOR: PROTOCOL 418-014 SPONSOR'S STUDY NUMBER: T-6295.13 Oral (Gavage) Cross-Fostering Study of PFOS in Rats Trahtes fpoulrlpoowisnegoPfFthOiSs sttruedaytmisentto oefvaClruia:teCDpBuBpRsVurAvFi/vaPlloufsF1 female rats Pups will be dcurroisnsg-fporsetmearteidngd,urgiensgtatthieonlaactnadtiloanctpaetriioon.d F1 to edixfpfoesreunrteiaatendvieaxmpaotseumraelthmrilokugthhemdaitfefmeraelncmeislk.forSienleuctetreod pFhoagremnaecroaktiinoentifcemsaalmepsleasndwiFll1bgeenceolrlaetcitoend pfurposm. Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A THeolresphhaomn,e:Pen(n2s1y5l)va4n4i3a-871190044-1297 Telefax: (215) 443-8587 Raymond G. York, Ph.D., DABT Associate Director of Research 3M Corporate Toxicology 3M St. Center, Building Paul, Minnesota 220-2-02 55144-1000 Marvin T. Case, D.V.M,, Ph.D. Telephone: (651) 733-5180 Telefax: (651) 733-1773 Andrew M. Seacat, Ph.D, Telephone: (651) 575-3161 Telefax: (651) 733-1773 000396 . 418-014:PAGE C-2 Protocol 41P8a.g0e124 REGULATORY CITATIONS: GU.uSi.deFlionoedoannddetDercutgioAndmoifntiosxtircaittyiotno r(1e9p9r4o)d.uctIinotnemfaotrimoendailcCinoanlfeprreodnuccetso.n HFaerdmeornailsation; Register, September 22, 1984, Vol. 59, No. 183. 2U1.S.CFFRooPdaratn5d8.Drug Administration. Good Laboratory Practice Regulations; Final Rue. Japanese for Safety Ministry Studies of on Health Drugs, aMnHd WWelOfradriena(n1c99e7)N.umGboeord21L,abMoarracthor2y6P,ra1c9t9i7c.e Standard `EaucrcoeppetaannceEcboyntohmeiEcuCroompmeuanniEtcyo(n1o9m8i9)c.CoComumnucniiltdyeocfisainonOoEnC2D8 dJuelcyis1i9o8n9/roencotmh-e mendation on the European Ccoommmpulniiatniceesw:itLhepgriisnlcatiipolne.sof32go(Nood. laboratory L 315; 28 practice. October): Official 1-17. Journal of REGULATORY COMPLIANCE: rTehgiuslsattiuodnyswicliltebdeacboonvdeu.cted in compliance with the Good Laboratory Practice (GLP) All changes Director and otrhereSvpiosinosnosr,ofdtahtisedpraontdocmolaisnhtalalinbeed dwoicthumtehnetperdo,tocsoilg.ned by the Study Tanhde wQiulalliintsypAecstsucrriatinccalepUhnaiste(sQoAfUt)hweilsltauuddyitinthaeccporortdoacnolc,etwhiethrathwedSattaanadnadrdthOeperreapotritn,g Procedures of Argus Research Laboratories, Inc. `aTchceurfaitnaellyrerpeofrltecwtisltihneclruadwedaatsataotbetmaeinntedsidgunreidngbtyhtehpeeSrtfuodrymaDnicreecotforthtehastttuhdeyraenpdortthat aslilgnaipfpilciacnatbdleeviGaLtPionrsegfurloamtiGonLsPwerergeulfaotliloonwsedocicnutrh,eecaocnhdwuicltl of be the study. described Should in detail, together with how the deviation might affect the quality or integrity of the study. `SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE: `See ATTACHMENT 1 to the protocol. 000397 418-014 PAGE C3 Protocol 41P8a-g0e134 TA ESTRTIANC DVEL HICE LE: Identification: Test Article: NPhaymsei:cal Description: SLpoetcBiafitcchGrNavuimtby:er: Pury: Expiration Date: LPiFghOtS-co(lSoyrendonpyomw:derF.C-95) 201576.. 9M8a.y9%2.000. wIintfhortmhaetiSopnonosnort.he identity, composition, strength and purity of the test article is on file Vehicle: 0De.i5o%niTzweedeWnater8)0. inSuRpepvleiresreanOdsmlootsiidsenMtiefmicbartiaonnoefPTrwoeceesnsed80DetioonbiezdedocWuamteenrt(eRd.0i.n . the raw data tNoeibteheprretsheenStpionntshoervneohritchleecSotmupdoynDeinrtescttohratiswaowualrdeionftearnfeyrepowtietnhtitahle croensutlatmsionfantthiss likely cstounddyu.ctTehde.refore, no analyses other than those mentioned in this protocol wil be Safety Precautions: Gfolromvuelsa,timoanspkr,epaaprpartoiporniaatnedeyademipnriosttercattiioonn.anTdhae uMnaitfeorrima/llaSbafceotayt DaarteatoShbeeewt o(mMSdDuSr)ingis attached to the protocol (ATTACHMENT 2). Storage: VBeuhkicTleesCtoAmrptiocnlee:nts: Prepared Prepared Vehicle: Formulations: RRoooomm tteemmppeerraattuurree.. RRoooomm tteemmppeerraattuurree.. 000398 418-014:PAGE C4 Protocol 41P8a.g0e1s4 All test article shipments to the Testing Facility should be addressed to the attention of Julian Gulbinski, Manager telephone number. of Formulations, at the previously cited address and `Shipments should include information conceming storage conditions and shipping schairptmoennsts.hould be labeled appropriately. The recipient should be notified in advance of FORMULATION: EreqoufPerepnaractioyn: Formulations (suspensions) will be prepared daily at the Testing Facility. Data verifying the stabilityofthe test article in the vehicle for 48 hours under the conditions of `administration are on file with the Sponsor. Adiustment for Purity: Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). Testing Facility Reserve Samples: The test article will be considered 100% pure for the purpose of dosage calculations. The Sponsor will reserve a sample (1 g) of each lot of the bulk test article used during the course of this study. The Testing Facility will reserve a sample (5 mL) of each lot of the vehicle components used during the course of this study. Samples will be stored under the previously cited conditions. ANALYSES: tShaempcoluersseadodfittihoenasltutdoyt.hose described below may be taken if deemed necessary during Bulk Test Article Sampling: No analyses of the bulk test article will be conducted during the courseofthis study. Information on the stability of the bulk test article is on file `with the Sponsor. Analyses re} yd For lations: Prepared. 000399 Homogeneity and stability of prepared formulations is on file with the Sponsor. However, records will be maintained to document how the test article formulations were 418-014:PAGE C-5 Protocol 4P1a8g0e'1s `ConcoefTensttArtriclae Fotrmuilatoionns: Concentrationofthe prepared formulations will be verified during the course of this study. Duplicate samples (2 mL each) will be taken from the first and lastpreparation on the day prepared. One sample of each set will be shipped for analysis; the remaining samples will be retained at the Testing Facility as backup samples. Backup samples will be stored frozen (-70C or below) and discarded at the Testing Facility upon requestofthe Sponsor. `Shipping Instructions: Samples to be analyzed will be shipped (frozen on dry ice) to: Kis J. Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone: (612) 778-6018 Telefax: (612) 778-6176 The recipient will be notified in advance of sample shipment. DISPOSITION: Prepared formulations will be discarded at the Testing Facility. All remaining bulk test article will be retumed to the Study Monitor at the previously cited address. upon completion of all work with the test article. EST SYSTEM: `Species/Strain and Reason for Selection: The Cri:CDBR VAF/Plus (Sprague-Dawley) rat was selected as the TestSystem because: 1) this strain of rat was used in the reproductive and developmental toxicity studies; 2) historical data and experience exist at the Testing Facility; and 3) the test article is pharmacologically active in the species and strain. 000400 418-014:PAGE C6 Protocol 18Pa.g0es14 Number: Initial population acclimated: Population selected for study: Population selected for pharmacokinetic sample collection (day 14 postpartum): BWodey iganhdAgte: 86 virgin female rats. 54 mated female rats (30 in the control group and 24 in the treated group). 12 mated female rats (six per dosage group). Female rats will be ordered to have body weights of 200 g 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 braenrgeecwoirlldebdetihneclduadyedafitnerthreecfeiinpalt arenpdortw.ill be documented in the raw data. The weight Sex: Female rats will be given the test article. Male rats of the same source and strain will be used only as breeders and are not considered part of the Test System. Source: Charles River Laboratories, Inc. The rats will be shipped in filtered cartons by air freight andor truck from Charles River Laboratories, Inc., to the Testing Facility. Identification: Eo Generation: Rats are permanently identified using Monel `self-piercing ear tags (Gey Band and Tag Co., Inc., No. MSPT 20101). Male rats are given unique permanent identification nFeummableersrautpsoanreasasssiiggnnmeednttetomptohreaTreystniunmgbeFarcisliatty'rsebceriepetdaenrdmagliveenratupnoipquuleatpieornm.anent identification numbers when assigned to thestudy. E1 Generation: iPnutpesrmwisllofnotthebelititenrd.ividually identified during lactation; all parameters will be evaluated 000401 . 418-014:PAGE C-7 Protocol 41P8a.g0e1?4 ANIMAL HUSBANDRY: AalnldcaUgseeosfizLesabaonrdathooruysAinnigmcaolnsdi.tions are in compliance with the Guidefor the Care Housing: FGo eneRatrs/Fa1Get nerai tionoLittn ers: `Feoxcgeepntedruartiinogntrhaetscwoihlalbbietaitnidoinviadnuadllpyoshtopuasretduminpsetraiiondlse.ssDsutreienl,g wciorhaeb-ibtoatttioomne,decaacghespa.ir opfrerastusmweildl bgeesthaotuiosne,dFion tgheenemraalteiornatf'esmcaalgee.ratBsegwiilnlnbienginndoivildautearlltyhhanoudsaeyd2i0n noefsting cboolxleesc,tieonxcienptetrvdaulrsi,ntghceolfleemcatlioen rianttsewrivlallsbefohr ouursineedainnddifveidcuaallslyamipn lmeest.abDoulriisnmg ctahgeesse. Each dam and assigned postpartum period. litter will be housed in a common nesting box during the Nesting Material Bedding material (bed-o'cobs) wil be provided. ABneadldyisnegswfilolrbpeoscshibalnegceodntaasmoifntaetnioans anreececsosnadruyctteodkeaennputahlelyaaninmdadlsocdurmyeanntdedcleiann.the raw data. Room Air, Temperature and Humidity: Tfrheeshanaiirmtahlatrohoasm biseeinndeppaesnsdeednttlhyrosuugphpl9i9ed.9w7it%hHatEPleAasftiltteenrsc(hAiarnogeCslepaenrhroouormo)f. 100% cRoonsotmanttleym.peRraotoumrehwuimllidbietymawiilnltaalisnoedbeatmo6n4itFor(e1d8cCo)nsttoan7t9lyFa(n2d6mCa)inatnadinmeodnaitto3r0e%d to 70%. Light: Amaninatuationmeadt.icaElalcyhcodnatrrkolpleerdi1od2-whilolurbleiggihnt:a1t2-1h9o0u0r dark fluorescent hours EST. light cycle wil be Diet: aRadtlsibwiiltlumbfergoimveinndCievritdiuafliefdeRedoedresn.t Diet #5002 (PMI Nuttition Intemational) available 000402 418-014:PAGE C8 Protocol 41Pa8g.e0s14 Water: aWnataeurtowimlaltbiec waavtaielraibnlgeaacdcelisbsitsuymsftreomm. iAnldlivwiadtuearl wbioltltbleesfartotmacaheldoctaol tshoeurccaegeasnodrpfarsosmed pthrroocuegshseadrweavteerrseasosamobsacitsermioesmtbatr;apnreocbeesfosreed uwsaet.erCihsioerxipneectwieldl btoecaodndtaeidntnoothmeore btahcatner1i.a2l pcopnmtacmhilnoartinieonatatnhdettwiimceeoafnannuaallylsyisf.orWpoastseirblies acnhaelmyizceadl cmoonnttahmliynaftoiropno.ssible Contaminants: tNoeibteheprretsheenStpionntshoernceorrtiftiheed dSiteut,dythDeirdercitnokrinigs awawtaerreoorftahneynpeostteinntgiamlatceornitaalmaitnalnevteslslitkhealyt `rwoouutlindeilnytperefrefroerwmietdh bthyetrheesufletesdofstuhpipslisetruodry.thoThseermefeonrtei,onneodainnalthyissepsroottohceorltwhialnlbteh.ose. conducted. RANDOMIZATION, COHABITATION AND CROSS-FOSTERING PROCEDURES: cUopmopnutaerrri-vagle,nemraalteeadnrdafnedmoamleunriattss. wiAlfltebreaacsclsiimganteidont,o7i8ndviivrigdiunalfehmoaulseinrgatosnwitlhlebebasis of dsuerliencgteadccfloirmsattuidony.onThtheefbeamsailseorfaptshywsiillcableaapspseiagrnaendcteo adnodsabgoedygrwoeuipgsht(s42reicnotrhdeed ocorndterroeld)grroaunpdoamnidza3t6ioinn tphreocteredautreedsgarnodupt)rebaatseeddwiotnh ceoitmhpeurttehre-gveehniecrlaeteordt(hweeitgehstt-article: for 42 days prior to cohabitation. cWoihtahbiinteataicohn dwoitshagbreegerdoeurp,macloensreactsu,tiovneeomradlerewrialtl pbeerufseemdalteo arasts.igTnhefermaatlseasrastisgntoed to ttrheeactoendtrgorlougpr.ouTphweilcobheabciothaatibointepderainoed dwialylpcroinosritsotocfohaabmiatxatiimounmfoorfrfaitvseadsasyisg.neFdetmoalthee praltusgwoibtshesrpveerdmiantsoiztouawiollbsbeercvoendsiindearsedmetaorbeofatthdeavyag0inoaflpcroenstuemnetds agneds/toatriaoncoapnudlatory assigned to individual housing. fMoaltloewds:feomfatlheer4at2sf(etmhaolsee wriattsh acsosnifginremdedtoevtihdeecnocnetroofl mgartoiunpg,)3w0ilmabteeadssfiegmnaelde arasts will absesaigsnseidgnteodtthoetthreeactoendtrgorlogupr,ou2p4fmoractreodssf-efmosatleerirnagtspwuirlpobseesa;sosfitghneed3t6o tfheematlreeartaetds dgorosuapgefogrrcoruopssw-iflolsbteerdiensgipgunraptoesdesf.or Aphnaradmdaictoikoinnaeltsiicx smaamtpeldefceomlalleectiroants(farsodmesecarcihbed rbeemlaoiwn)ifnogllfoewmianlgederlaitvserwyi,thbuctontfhiersmeedratesviwidlelnncoetobfemcartoisnsg-ftohsattewreedr.e Faisnsaillgyn,eadntyo the control group prior to cohabitation will be assigned to the control poolforcoss-fosteging 10 418-014:PAGE C-9 Prt srnpaore purposes. Any remaining female rats with confirmed evidence of mating that were assigned to the treated groupprior to cohabitation will be sacrificed anddiscarded without further evaluation at the discretion of the `Study Director and the Study Monitor. Day 1 of lactation (postpartum) is defined as the day of birth and is also the first day on which all pups in a litter are individually weighed (pup body weights will be recorded after all pups ina litter are delivered and groomed by the dam). Twelve dams and their respective litters will be selected and assigned to the pharmacokinetic sample collection (six dams and litters per dosage group). These litters will not be cross-fostered. Up to 24 litters per dosage group will be reassigned for cross-fostering on day 1 postpartum. The litters will be assigned such that four subsets are obtained `among the two dosage groups as described in the table below. [CeT merTome CT Tove Ln oreT [ervmem T[oeae]] aTT] Detailed procedures for reassignment of litters for cross-fostering are attached to this protocol (ATTACHMENT 4). On day 1 postpartum, pups from dams assigned to the control pool will be culled, sacrificed via decapitation and handled as described in ATTACHMENT 4. dosage Group rospacive of Hie) deremined 1s bo ot do 4 oreo On day 4 postpartum, cross-fostered litters will be culled to five male and fivefemale Psaucprsifpiceerdlvititaerd, ewchaepirteatpioosns;ibtlhee. lPunugpss(nsoatvesdelienctBeoduifno'rscsoonltuitniuone)d aenvdaltuhaetiloinvewril(lsbaveed in neutral buffered 10% formalin) will be collected from the first ten culled pups from each preserved for possible future histopathological evaluation. Remaining culled pups will be sacrificed via decapitation and discarded without necropsy evaluation. 000404 418-014:PAGE C-10 Protocol 4P1a8g.e01140 ADMINISTRATION: RR outee andasfoo r Chon ice: The oral (gavage) route was selected for use because: 1) this was the route of administration in the developmental and reproductive toxicology studies; and 2) it is one of the possible routesof human exposure. Method and Frequency: EoGeneration FemaleRats: Female rats will be given the test article or the vehicle once: daily beginning 42 days bproidory twoeicoghhatbcithaatnigoenstharnodugghivdeanya2t1appporsotxpiamrattueml.y tDhoesasgaemse wtiillmebeeaacdhjudsatye.d daily for E1 Generation Pups: F1 generation pups exposed to the test will not be di article during rectly given the test matemal gestation (airntiuctlee,robuetxpmoasyurbee)oprosvsiiablmyatemal milk during the lactation period. Rationale for Dosage Selection: (DAorsgaugseRsewseeraercshelLeacbtoerdatoonritehse, bIancs.i,soPrfotaopcorlev4i1o8u-s00s8t)u.dy conducted with the test article Dosage Levels, Concentrations and Volumes: Lfeous| "Lona| Caircmmmrseern| V|in|e poe | [1 wee Tovman] 0 1%lovsoronuomven] Bhae atSserticako,wrtrThbeescssoaonrs2dm00aaska rnhvi 4p peoseofosea eobcaatsc wokshfse ch esan 14 TESTS, ANALYSES AND MEASUREMENTS - Fo GENERATION: Viability: All Peerriods: At least twii ce daily. 000405 418-014:PAGE C-11 Protocol 4P1a8g.e01141 ClO inib cal serv anda /orGt enei ral Ao ppen arans ce: Acclimation Period: Atleast once. Dosage Period: a`Tpwpircoexidamialtye.lyPrainorethooaudrmipnoissttdroastaigoen.and once. Matemal Behavior: aDbanyosrm1a,l4,b7e,ha1v4iaorndwil2l1bpeorsetpcaorrtduemd.daiOlby.served `CalpipnircoaplrioabtseerbvyatthieonSstumdayyDbireecrteocroarndde/domroSrteudfyreMqouneinttolry.than cited above,if deemed BodyWeights: Acclimation Period: Atleast once. Dosage Period: Daiy. Sacrifice: Terminal weight. Feed Consumption Values (recorded and tabulated): Acdlimation Period: Atleast once. Dosage Period: Weekly to cohabitation. gestation and days 1, 4. Daily 7. 10 during and 14 presumed postpartum. pFoesetdpacrotnusmu,mwphtieonniwtiilslenxoptebcetetdabtuhlaattpeudpasftewrildl abyegi1n4 10 consume matemal feed. Feed consumption replenish the feed. values These may be recorded intervals will not bmeotraebuflraetqeud.ently if itis necessary to MatingPerformance: oMbasteirnvgatwiilolnboefesvpaelrumaatetdozdoaailyindaursinmgeatrheofcothhaebivtaagtiinoanl pceornitoedntasnadncdo/nofriarmceodpublyatory plug observed in situ. 000406 . 418-014:PAGE C-12 Protocol P41a8g.e01142 NaturalDelivery: Female rats will be evaluated for: cCoinndiictailoOnsbsdeurrviantgitohnes dDaurrkinpgerPiaordtu(rtiitmieone[atcohbepupperisfoorbmseedrvuenddewirllrebde lrieghctorded). Duration of observed) Gestation (day of presumed gestation to the time the first pup is `LoebnsgetrhvaotfioPnarotfurtihteiofinrs(ttpimuepodfiovbidseedrvbaytiNo-n1opfulpasstipnuepacmhinluitsert),he time of Liter Size (defined as all pups delivered). Pup Viability at Birth. Pharmacokinetic Sample Collection: bCeafposreananddlaafbteelredrettuenbteisonwilolfbpeoowleeidgpheudp (scaommpbliensefdorwesiugbhste,qtuoetnhteunseearienst .001 gram) cpohpairemsacoofktihneesteicweainaglhytsseswi.ll bTeheisneclwuediegdhwtisthwiltlhebepadcokciungmelnistt epdrioirn the raw data, to shipment. and Blood, Mik and Liver Samples - Fo Generation Rats: opBfhlaosoradmm,apcmloieklikcnoaelnltedicctliisovaenmrsps(labemlpocolodlelsaecnwtdiilomnbilepke)crowldilolescbateegderefgcrrooormudpseidox nimnadttaheyemr1aa4lwprdoaasttstapd.aerstiugmn.atTehdefotrime fpoMoirslaktppspararomtxpuilmm.eastEweaillcylhbfeoduacromhlolwueirclst.ebdeTarhpepemrodoxvaiemmdawtifelrlloybmettwhionejtenocetssetidxinihgnotubrroasxvepanonosdutsdilnoydsiwavigitdehuaoolnnlyedhauonyiuts1o4efd (aox-ry7et0coocClilonercatbpeepdlr.oowxT)ihmeuanttsieallmysphfliievpesmmewinilntlutbtoeetsihembmeSefpdoorienatsmeoilrlykffsorraomaznpealnleyossnis(da.tryleiacseta1n0d0 mwal.inptearinseadmpflreo)zen scFeoolpllalerocawttieondrgftmruioblmeksts.haemTpmhlaeetsecamomlalplelcretaistosnw,ivlilbalboteohdespisunafnmerpiinloreasvre(enfarapipgcrearovaxatieamdnacdteenttl4rryainfmsufLgeer.eraeTcdhhi)entwsoilelsreburemuwmill iibmdeemnettdriifaainctsaefteliryornfe,rddoiaznteteonopofonlcoydlprlryeioccptyieolnea,nnesdttumudabyiendstaalyianbaeenlddefdcrowolizlteehnctt(ih7oen0sttiCumdeoypronibunetml.boewAr)l,lusnatanimlipmsalhleispmweilnltbteo the Sponsor for analysis. 000407 418-014:PAGE C-13 Protocol 4P1a8g.e01143 10 the Sponsor for analysis. Following collection of milk and blood samples, a liver section (right lateral lobe) and the `milk-secreting glands from the axillary, thoracic, abdominal and inguinal regionsof each dam (left side only) will be collected, frozen and stored (70C or below) until shipment Blood and Liver Samples - F1 Generation Pups: analysis. Blood samples will be collected via the inferior vena cava from each Pup on day 14 postpartum, pooled (per litter) and transferred into serum separator tubes. The samples will be spun in a refrigerated centrifuge. The serum will be transferred into Polypropylene tubes labeled with the study number, animal identification, date of collection, study day and collection timepoint. All samples will be immediately frozen on dry ice and maintained frozen (-70C or below) until shipment to the Sponsor for Tbehleowi)veurntfirlosmhiepamcehnptutpo twihlel bSpeocnoslloerctoerd.anpaoloylseids (per ter), frozen and stored (70C or `Shipping Instructions: shipment. All samples will be maintained frozen (-70C or below) until shipment for analysis. Samples will be shipped on frozen on dry ice via `overnight mail. A packing list will be included with the samples andsent to Kris J. Hansen, Ph.D., at thepreviouslycited address. Both the recipient and the Study Monitor will be notified in advance of sample rine and Fecal Sample Collection: `Shipping Instructions: Fo generation female rats will be housed individually in metabolism cages for collection of urine and fecal samples from days 21 to 22 postpartum. Following the 24-hour csotlolreecdtifornozinetner(v-al7,0sCamoprlbeeslowwil)l ubnetilcoslhliepctmeedntinftoorcaennatlryisfiusg.e tubes, placed on dry ice and shipment. All samples will be maintained frozen (-70C or below) until shipment for analysis. Samples will be shipped on frozen on dry ice via ovemight mail. A packing list will be included with the samples and sent to Kris J. Hansen, Ph.D., at the previously cited address. Both the recipient and the Study Monitor wil be notified in advance of sample METHOD OF SACRIFICE - Fo GENERATION RATS: Rats wil be sacrificed by carbon dioxide asphyxiation. 000408 418-014:PAGE C-14 Protocol 4P1a8g-e01144 NECROPSY - Fo GENERATION RATS: Gevraolsusatlieosnio(nastwaibllleboefrreatnaidnoemd iunnintesuwtirlallbbeufufseerdedto1s0e%lefcotromnaleincofnotrrpoolsgsriobulep fruattufrreom wtihsiscuhesalflotriasnsuyepsoesxsiabmlienheidstaotpnatehcorloopgsicyawlilelvbaeluraettiaoinnseodf, ignroosrsdelrestioonpsr)o.viUdnelceosnst.rol specifically cited below, all other tissues will be discarded. Rats Not Delivering a Litter: `Reaxtasmtihnaetdfdoorngortosdsellievsieaornsl.itteUrtewriilwbilel sbaecrsitfaiicendedownitdhay1205%oafmpmroensiuumemdsuglefsitdaetitooncoannfdirm the absence of implantation sites, Scheduled Sacrifice -Rats Asi Pharmacokis a lection: tOhne dmialyk-1s4ecproesttipnagrgtluamn,dsmiflrkoamndthbelaoxoildlasraym,ptlheosra,ciac,liavebrdsoemcitniaoln (arnidghtinlgautiernaall lroebgei)onasndof (`e7a0chCdaormb(elelfotws)iduentiolnlsyh)iwpimllenbte tcooltlheectSepdoanssodresfcorriabnealdyspirse.vioFuoslllyo,wfirnogzetnheacnodmpslteotrieodn oafgprhoasrsmnaeccokrionpestyoicftshaemptlheorcaoclilce,ctaibodn,omFionaglenaenrdatpieolnvifcemvaislceerraatwsilwlilblebepesrafcorirfmiecde.d, Tahned number and distribution of implantation sites will be recorded. `Scheduled Sacrifice - Day 22 Postpartum: On rats dfaoyll2ow2inpgosrtepmaorvtaulm,frbolmoomdestaabmoplliessmwiclalgibneg,collected from the Fo generation female Bthleooidnfseraimorplveesna(acpapvraoxainmdatterlayns4femrLredeaicnhto) swielrl ubme sceoplalrecatteodr ftruobemst.heThmaetseammapllerastswivlliabe stupbuensilnaaberleefdriwgietrhattehdecsetnutrdiyfunguem.beTrh,easniemraulmiwdielnltibfeictartaionns,fedrarteed oifntcoolploelcytpiorno,pyslteundey day amanidnctoalilneecdtifornotziemnep(o-i7n0t.CAolrl sbealmopwl)esuntwiilllsbheipimmemnetditaottehleySfprooznesnoronfodrraynailceysainsd Tanhde pfeelmvailcevirsatcserwailwliltlhebne bpeersfaocrrimfeidc.ed,Thaendnuamgbreosrsannedcdriosptsriybuotfiotnheofthiomrpalcainct,aatibodnomsiitneasl fwirlolzbeen arencdorsdteodr.edA(-l7i0verCsoerctbieolno(wr)iguhnttillatsehrailplmoebnet)ftorotmheeSapcohnsdoarmfowirllanbaelysciosl.lected, 000409 418-014PAGE C-15 Protocol 4P1a8g-e0114,5 DS amu swirthv Noivin Pupgs: oDrapmrseswiutmhednocasunrnviibvailnigzepdu.ps wil be sacrificed after the last pup is found dead, missing sPmraaimtoperlmteaolwsialrclartibfveiicase,pthuaenbiilnnofoeardiroesrfarvmiepgnelareact(eaadvpapceranotnxridimfaturtgaeenl.syfTe4rhrmeeLds)eiwnritluolmsbweeirlcluomblelseetcprtaaerdnasftfroeorrmetdutbheei.nst.o aThe ocponollldyercpytrioiopcney,lasentnduedtmyuadbiaenytlaaaibnnedeldecdoflrwloiezchetnitoh(ne-t7si0mteuCpdoyoirnntub.emlbToewhr)e, usanatniimlmpsallheiiwpdimelneltnibtfeictiaotmtimohened,iSdapatotenelsyooffrfroozren analysis. (lAi7vge0rrosCsescotrnieobcnerl(oorpiwgs)hytuonltfaitltehrsaehlitbhpomerea)nctifctr,ooamtbhedeaoSmcpihondnalasomarnwfdilolrpebalnevailccyoslvilises.ccteerPdao,swftirploazbreetnupamenrddfaotsratmoferoder.dthAese dams will be excluded from summary tables. Rats FoundDead or Meribund: doRebalstiesvrevtrhayattiwiodlnliebiseormeaaxdraeem.siancTerhidfefiocrreatdthsbeweilcclaabuuesseeeoxofafmmdioenraetidhbufonordrmgcorornoidsbistuilnoedns,icooannbsdo.irAttiioolnnivooenrr spterhceetmidaoatnyu(rtrehieg.ht rslaahttiseprwamilellnlotbbeeto)retfchroeormdSepedoa.ncshoArbdfoaormtrewadinlaflebytesuissce.osllaePncrtdee/dgo,rnafdrneoclziyevnesrtaeandtudpssuatpnosdrewuidltl(er-bi7en0eexCcaoonmrtiebnneetldsowtoo)f tufhenemlale `exatmemnotnpioussmibsluel.fidUetteoricoofnfaiprpmartehnetalybsneonncpereogfniamnptlarnattastwiiolnl be stained sites. with 10% `Shipping Instructions: Ai`lnSlcalmsupadlmeepdslwewiistllhwibltelhebseshiampmappielndetsafiranonezddensfeornontztednroy(Ki-rc7ie0svJiC.aoHoravnbesemeliongw,h)tPuhmnat.ii.ll..shaAitptpmhaeecnkptirnefgovriloiasuntsalwlyiyslcisib.teed sahdidrpemsesn.t. Both the recipient and the Study Monitor will be notified in advance of sample TESTS, ANALYSES AND MEASUREMENTS - F1 GENERATION: Viability: Postpartum Period: dLaiitltye.rs Twihlel bpeuposbsienrevaecdhfolritderewaidllpbuepscoautnlteeadstotnwciece dail. 000410 418-014:PAGE C-16 Protocol P41a8g.e01146 COlinibcal servanda /orGteneiralAoppenaransce: Postpartum Period: Once daily. Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the StudyDirector and/orthe Study Monitor. BodyWeights: Postpartum Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. `Sacrifice: Terminal weight. METHODOFSACRIFICE -F1GENERATRIAOTSN: As previously cited for Fo generation rats. Culled pups will be sacrificed via decapitation. NECROPSY - 1 GENERATION RATS: Gross lesions wil be retained in neutral buffered 10% formalin for possible future seveaxlufartoimownh(iacthaballletoifssruaensdeoxmamuniintesdwialtl nbeecursoepdsytowisllelbeectroetnaeinceodn,trionolrgdreorutporaptroovfiedaech scpoenctirfoilcatlilsysuceistefdorbealnoyw,poaslsiobtlheerhitsitsospuaetshowliolgbicealdiesvcaalrudaetdi.ons of gross lesions). Unless As described previously, caps and labeled tubes will be weighed (combined weights, to the nearest .001 gram) before and after retentionofpooled pup samples. These weights will be documented in the raw data, and copies of these weights will be included with the packing list prior to shipment. Pups Found Dead on Day1 Postpartum: Pups that die before examination of thelitter for pup viability will be evaluated for vital status at birth. The lungs will be removed and immersed in water. Pups with lungs that sink will be identified as stillbom; pups with lungs that float will be identified as livebom, and to have died shortly after birth. Pups with gross lesions will be preserved in Bouin's `solution for possible future evaluation. The lungs will be preserved in Bouin's solution and the liver will be preserved in neutral buffered 10% formalin for possible future evaluation. 000411 . 418-014:PAGE C-17 Protocol 4P1ag8e01147 Pups Found Dead or Moribund on Days 2 to 21 Postpartum: lPeuspisonfsoaunndd fdoeratdheorcasaucsreifoifceddeabtehcoarutsheeofmomorriibbuunnddictoyndwiitlilobn.e ePxuapmsinweitdhfgorrogsrsoslsesions efvoaulnudatoinond;agyrso2sstole4sipoonsstopfaprtuupms wfiollunbde opnredsaeyrsvedtion 2B1oupions'tspsaorltuutimownilflorbepopsrseibsleervfeudtuirne neutral buffered 10% formalin. BFoourianl'lspsuoplsutfioonunadnddetahdeloivnerdwaiylsl b2etop4repsoesrtvpeadrtinumn,eutthreallubnugffsewrield be preserved 10% formalin in for lpousnsgisbalnedfutthuerelievvearlwuialtliboen.prFeosreralvlepduipnsnefuoturnadl dead on buffered days 10% f5otroma2l1inpfoosrtppaorstsiubml,etfhueture evaluation, Pups Not Selected for Continued Observation - Days 1 and 4 Postpartum: dAllap4yuppsosctupllaerdtuomn (dalalyot1 hpeorsctuplalredtupmup(sp)upwsillfrboemsdacarmifsicaesdsviiganededcatpoitthaeticoonn.troTlhpeoollu)ngasnd and the liver `each dosage wil be group collected on each dfaryom(ctohnetrfoilrstpotoeln pups culled only on day 1(iproresstppeacrttiuvme)o;ftlhieterl)unfgrsomwill be retained formalin for in Bouin's solution, and the possible future evaluation. Rlievmearsinwiilnl gbecurlelteadipneudpsinwnilelutbrealsabcurfiffeirceedd 10% and discarded without evaluation. Scheduled Sacrifice - Litters Assigned to Pharmacokinetic Sample Collection: sOancridfaiyce1d4wiptohsttphaeirrturme,spleictteirvseadsasimgsn.edIntdoivtihdeuaplh.apromoacloekdisnaemtipclessam(ppelrelcitotlelr)ecotfiobnlowioldl be and liver will lesions. collected as described previously. The pups will be examined for gross Scheduled Sacrifice - Litters Assigned to Sacrifice on Day 21 Postpartum: On day pooled 21 postpartum, six samples (per litter) litters per subset will of blood and liver. be randomly selectedfor collection of lBilttoeor)dasnadmptrlaenssfweirllrebdeicnotollseectreudmvsiaeptahreatinofrertiuobrevse.naThceavsaamfprloemsewaicllh pup, pooled be spun in a (per Wrietfhritgheerastetdudcyennturmibfuegre,. aTnhiemasleirduenmtiwfiilcabtieont,radnastfeerorfecdolilnetcotpioonl,ypsrtoupdyyldeaney taunbdescollalebcetlieodn timepoint. (-70C or bAellloswa)mupnlteisshwiilpl mbeentimtmoetdhieatSelypfroozfneonrsoannaoldyrsyrisi.ce and maintained frozen 000412 418-014:PAGE C-18 Protocol 4P1a8g.e01148 `pTohoelepdup(spewrilllitbeer),efxraomzienneadnfdorsgtorroesds (l-es7i0onCs.orTbheelolwiv)erunftrilomsheiapcmhenptuptowitlhlebSepcoonlsloecrtefdo,r analysis. All other remaining pups will be sacrificed and discarded without further evaluation. Shipping Instructions: ASlalmspalmepslewisllwiblel bsehimpapiendtafirnoezdenfroonzednry(i-c7e0viCaoorvebrenliogwh)tumnatiill.shAippmaecnktinfgorlaisntawliylslibs.e iandcdlruedsesd. wBiothththtehesarmecpilpeisenatnadndsetnhtetSotKurdiys MJ.onHiatonrsewni,lPbhe.Dn.o,tiaftietdhienpardevviaonucselyofcistaedmple shipment. STATISTICAL EVALUATION: A`avpeprroapgreiastea.ndAdpdeirtcieonnatlagpersocweildlubreescaalncdu/laotreda.nalLyitsteesr vmaalyuebsewiplelrbfeorumseedd, wifhdeereemed appropriate. DATA ACQUISITION, VERIFICATION AND STORAGE: DDiarteactwoilrlabnedoharnadp-paronpdr/ioartecommapuntaegr-ermeecnortdepde.rsoRneneclorwditshwinil2b1edraeyvsiaefwteedr gbeynetrhaetiSotnu.dyAll original records will be be bound and indexed. stAorceodpiynotfhaellarrcahwivdeastaofwitlhlebTeessutpipnlgiFeadcitlotty.he All original data Sponsor upon will ryeeqauresatf.terPmraeisleinrgveodf ttihsesudreasftwiflinablerseptoorrte,d aafttetrhewhTiecshtitnigmeFatchieltSypaotnsnoorcwhiallrgbee fcoornotnaec.ted to determine the disposition of these materials. 000413 418-014:PAGE C-19 Protocol 4P1a8g.e01149 REC TO O BE MR AIND TAINS ED: TPerosttoAcrotlicalen,dVAemhiecnldemaenndt/so.r Reagent Receipt, Preparation and Use. ARnainmdaolmiAzcaqutiisointiSocnh.edules. Maing History. GTerneeartamlenCto(mifmpernetsscr.ibed by Staff Veterinarian) CTliisnsiucaelaOnbdseSravmaptlioenCsolalnedc/toironG,enPerroacelsAspipnegaarnadncSeh.ipment. CBoadpyaWnedigLhatbse.led Tube Weights. FNaeteudraCloDnesluimveprtyioObnseVravlauteiso.ns. LGirtotesrsObNseecrrvoaptisoynOs.bservations. OPhrogtaongrWaepihgsh(sif (rief qrueiqrueidr)e.d). Study Feed, Maintenance (room Water and Bedding Aannadlyesnevsi.ronmental records). Packing and/or Shipment Lists. KPEEYRSONNEL: Executive Director of Director of Research: RAelsaenarMc.h:HoMbielrdmraedn,S.PhC.hDri.s,tiDaAn,BPTh.D., Fellow, ATS DiArsescotcoiraotef DLiarbeoctroartoorfyROepseeraartciohnas:ndJSothundyF.DiBraemcettort:, Raymond B.S. G. York, Ph.D., DABT MMaannaaggeerr of of ASntiumdaylCoOopredriantaitoinosn:anVdalMeerimebeA.r,ShIanrsptietru,tioMn.aSl.Animal Care and MaUnsaegeCromoiftRteeguel:atDoreynaCoCm.plLieabnoc,eV:.MB.aDr.bara J. Patterson, B.A. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP 000414 418-014:PAGE C-20 Protocol P41a8g.e01240 EINAL REPORT: A comprehensive draft final report will be finalized following consultation with be prepared on completion of the study and the Sponsor. The report will include the will following: `Summary and Conclusion. Experimental Design and Method. AEpvpaleunadtiicoens:of TFiegsutreRse,suSltusm.mary and Individual Tables Summarizing the Above Data, Protocol and Associated Amendments and Deviations, Study Director's GLP Compliance Statement, Reports of `Supporting Data (if appropriate) and QAU Statement. INSTITUTIONALANIMAL CAREAND USE COMMITTEESTATEMENT: thatinvolve study animals wil be conducted in a manner to avoid or minimize. The procedures described in this protocol have been reviewed by the Testing Facility's Institutional Animal Care and Use Committee. All Procedures described in this protocol discomfort, distress or pain to the animals. The Sponsor's signature below documents the fact that information conceming the necessity for conducting this study and the fact that this is not anunnecessarily duplicative study may be obtained from the Sponsor. No alternative (invitro) procedures were available for meeting the stated purposes of the study. 000415 418-014:PAGE C-21 Protocol 4P1a8ge01241 REFERENCES: 1. CThersitsst.iaEn,nvMi.rSo.nmaenndtaVloyPtreokt,ecPt.iEo.n (A1g9e6n2c).y,IWnaVsihviongRetporno,dDu.cCt.ivNeatainodnaMluTteacghenniiccailty Information Service, U.S. Department of Commerce, Springfield, VA 22161. 2. nCahrlitsrteixaonn,eM(.PSr.o(c1e9e8d4i)n.gsReofprNoadlutcrteixvoenetoSxiycmiptyosainudmt,erNateowloYgoyrekvaAlcuaatdieomnsyooff Sciences, November 7, 1983), J. Clin. Psychiat, 45(9):7-10. 3. CLoanntgr,olP.DLa.t(a19i8n8t)h.e EChmabrriyeosRainvderFeCtrail:DCeDveBlRopRmaetn.taClhaTrolxiecsitRyiv(eTrerLaatboolroagtyo)ries, LInacb.o,raWtiolrmiiensg,toInn,c.)MA 01887-0630. (Data base providedby Argus Research 4. LInasbtoirtuatteoorfyLAanbiomraaltso.ryNaAtniiomnaall RAecsaoduermcyesPr(e1s9s96,)W.asGhuiindgetfoonr,thDe.CC,are and Use of 5. SImaplleawnstaktiioEn.ss(t1e9l6l4e)n. aFmrUbteemruesthdoedreRaztutme.mAarkcrh.oskPaotphiosl.chEexnp.NaPchhawremiaksovlo.n 247:367. 000416 . BROTOCOLAPPROVAL: FOR THE TESTING FACILITY Qf DAilraenctMo.r oHfobReersmeaanr,chPh.D., DABT A ond G. York, . DABT Associate Director of Research Study Director Leos foslor Dena C. Lebo, V.M.D. Use Commitee Member, Institutional Animal Care and FOR THE SPONSOR Dp: a Marvin T. Case, D.V.M., Ph.D. Study Monitor 418-014:PAGE C-22 Protocol P41a8g.e0r1s6 22 Ctl Date 23-Oct-98 Date 2a DF 97 Date 270.4 2g Date 000417 418-014:PAGE C-23 ATTACHMENT 1 SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE 000418 ATTACHMENT 1 `STUDYSCHEMATIC CROSS-FOSTERING STUDY* 418-014:PAGE C-24 ProtocPola4g1e8.t0o1r42 Sof Endof Schedued Saree' Femde Rais Pend | Cotati| pruned | Popa Period Period Gestation Lactation (420ays) (5daps) Period Perin ME a. b. c. d. Dosage Period. For additional protocol. details see "Tests, Analyses and Measurements" section of the continuing unt F1 generation pups will be cross-fostered beginning on day 1 postpartum and il day 21 postpartum. F1 generation pups will be sacrificed on will be sacrificed onday22postpartum. day 21 postpartum. Fo generation dams 000419 `r`weiSlsal pmbepecltesisavcefroilrfitiatcneeardslyofsnoilsdlaoowyfinp1gh4aspraommsatpcploaekritcnuoemltl.eiccTtsihoweni.lslebleitcteorlslweicltlendotfrboem csrioxsds-afmosstaerneddtahneidr 418-014:PAGE C-25 ATTACHMENT 1 ProtoPcag41e82.00i124 : 270CT 98 02NOV 98 13DEC9814PMD-EC1998DEC98 AM 19 DEC 98 04 JAN 99 13 JAN 99 04 JAN 99 12 JAN 99 08 JAN 99 16 JAN 99 08 JAN 99 13 JAN 99 17 JAN 99 26 JAN 99 24 JAN 99 - 02 FEB 99 25 JAN 99- 03 FEB 99 08 JUN 99 SCHEDULE* Animals Arrive -Acclimation Begins. cDoohsaabigteatPieornioundti-l Fdeamya2l1e pRoasttspa(r4t2umd)a.yspriorto Cohabitation Period. LFiarsstt PPoossssiibbllee DDaayy 00ooffPPrreessuummeedd GGeessttaattiioon.n gFierssttatPioons)s.ible Delivery (Day 21 of presumed gLeassttatPioosns).ible Delivery (Day 25 of presumed CFiornsttroPlosPsoioblleOnDlay.y 1 Postpartum Culling CLoansttroPlosPsoioblleOnDlayy. 1 Postpartum Culling - CFirrosstsP-oFsossitbelreedDaLyitt4erPso.stpartum Culing CLraostssP-oFsossitbelreeDd aLity4terPso.stpartum Culling - First Possible Day Female Sacrifice. 25 of Presumed Gestation FLaesmtalPoessSaicbrlieficDea.y 25 of Presumed Gestation PFihrasrtmPaocsoskiibnleetDicaySa1cr4ifPiocset.partum PLhaastrmPaocsoskiibnleetDicaySa1cr4ifPiocse.tpartum Scheduled Sacrifice (Day 21 postpartum). F1 Generation Pups Scheduled Sacrifice (Day 22 postpartum). Fo Generation Dams Draft Final Report. a. The study initiation date is the day the StudyDirectorsigns the protocol. 000420 418-014:PAGE C-26 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 000421 . 418-014:PAGE C-27 DMAATTEARISAHLEETSWFETY Center sSts.1eaP-a1ul0,00Minnesota 1-800-364-3577 or (612) 737-8501 (24 hours) Copyright, 1998, Minnesota Mining and Manufacturing Coapany. iiAsLnLfaolrrlaiogawhtstidsonprrfeoosvreirdvteehdde. tphuarCtpo:opsyeingof apnrdo/poerrldyownUltoilaidziinngg oSfN thpirsoducts 1) 2) pntreheiiotrhienrafgotrrheseeamtceionoptnyiisnsoroSbottphaieiendeodriingfifrnouamlll,wiistahrnedsnooldchoarngeostheurnwliesses distributed With the intention of earning a profit thereon. TDRIAVDISEIOWNA:KE: 3CHEMICALS 10FGM-M9B5ERF/LUU.ORPA.DC.:Brand Fluorochesical Surfactant 9988--00221017--00818083-75 20002 104d 1 0000--o551111a3355.-0099036642-.17 9988.-00221017--30911064.-15 0000..5511113355.-0039901515.-58 DSIUOSPCSEUURMESEDEN:DTE:JSa:n1u0aN-ro3yv7e9m62b9-,e5r19059,8 1997 1. INGREDIENT CAS. Wo. PPPOOOTTTAAASSSSSSIIIUUUMMN PPPEEERRRFFFLLLUUUOOORRROOOAAALLLKKKYYYLLL SSSUUULLLFFFOOORNWAAATTTEEE...............|.. PPOOTTAASSSSIIUUNM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONNAATTEE............ 32709751..3095..385 5209247200.-8455..53 3872-2801 2. PHYSICAL DATA VVBAAOPPIOOLRRINppGrePseOsIuNrTwe::..1s1.11..11T.I1.I1.II.1I.1.IY1.I.1.1:... MWyAa WA SSEPoVELACuPIeOIFRLAIITCITGOYANVRIInATTYwE::a.t..e...n.:1..11111111I11111l1] Mslyiagne Ga. o.6 Materet BPEHR:CENI TVOLATILI E: ......I ........ 0(%Bulk) MvELTIINGSpoiSnt:O11S11ETYw:wiip0p.1% AAque)ous; APPLEiAgRhAtNCcEoloArNeDd,GDOfRr:ee flowing powder. PeRCENT 82 T-s ss 53 sor Ts 000422 Abbreviations: N/D - Not Datersined N/A - Not Applicable Ch - Approximately 418-014:PAGE C-28 MJSaDnSu:aryFG2-99,5 F1L9U9O8RAD Brand Fluorocheaical Surfactant PAGE 2 3. FIRE AND EXPLOSION HAZARD DATA FFLLAASHBLPEOINLTI:N.I.T.S...- .C...E..1I.L.I..IE..T. NNoAne AFULTAOMTAGAHBILTEIOLNINTITESMPEUREALT:U.R0E1S1.1..] WNI/AA EXWTaItNeGrU,ISHCIaNrGbonMEDdIiAo:xide, Dry chemical, Foss SPWPEoeCsIaiArtLifvuFelIlREprperFsoIstGueHrcTetIiNvoGerPRcpOlrCoeEtsDhsiUunRrgEe,S:deimnacnldudibnrgeathhelisnegt,Appsaerlaft-ucso,ntabiunnekde,r cost aPnrOdTepcatnitsv,e bcoavnedrsinagrofuonrd aerxmpso,sedwaiasretasanodfletghse, hfeaadc.e mask, and UNSUeSeUALHaFzIaRrEdouAsNDDeEcXoPmLpOSoIsOiNtiHoAnZAsReDcSt:ion for products of combustion. a ReAcTIVITY oaATA STABILITY: Stable INNCoOtMPAaTpIpBlIiLcaIbTlYe.- MATERIALS/CONDITIONS TO AVOID: HAZARDOUS POLYMERIZATION: Hazardous polymerization Will not occur. HAZCaArRbDoOnUSMoDnEoCxOiWdPeOSIaTndIOCNarPbRoOnDUCDTiSo:xide, Oxides of Sulfur, Fluoride, Toxic Vapors, Gases or Particulates. Hydrogen 5. ENVIROWENTAL INFORMATION rr SPOIbLsLerRvEeSPOpNrSeEc:autions from other sections. Vacuum, use wet sweeping cbeompaonunigdniotriownatesorurtcoe.avoiCdleadnustuipngr.esiCdAuUeTIOwNi!thAwavtaecru.um Pcllaecaeneirncoaunld approved metal container. Seal the container. REDCoOMMnEotNDEreDleDaIsSPeOStAoL:Waterways or sewer. Do not use in products or p1r7o7c0esosfesthethaltowecsotuldECSr0esoulrtLiOnSaqcuoantciecntrcaotnicoenn.tratIinocnisnrgarteeateirn atnhan mailanttdeeurrsnitaartlii.avle:CoormbDcuiossmtpmioeosrneciapoflrodfwauasccttieslitwpyrioldliuncitnthceilnudpaerefsHaFe.cnicleiDtioysfpoapsearclaoimtbtuesdtitbole (0 0423 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-014:PAGE C-29 JaSnOSu:ary1C2-99,5 1F9L9U8ORAD Brand Fluorocheaical Surfactant PAGE 3 S. EWIROWENTAL INFORMATION (continued) accest chemical waste. EN9V6I-RHOrM.ENATqAuLatiDcATAF:ish LCSD, Fathead Minnou(Pisspheles proslas)s=38 mg/l, gBaliuredgnielrli)S=u1n1fiwsgh/(1L;ep4o8n-iHsr.saEcCrSo0c,hirDuasp)h=n6i3a Hmga/gln,a =RaSiOnbaogw/1T;rouCtO(DS=a.l0m0o4 0/0; 80020 = Wil. REVGoUlLaAtTiOlReY OIrNgFaOnRMiAcTICOoNm:pounds: N/A. VOC Less H20 & Exempt Solvents: W/A. bSeifnocreerdeigsuploastailo.ns Uv.aSr.y, EPcAonMsauzlatrdaopupslicWaabsltee Nruemgbuelrat=ionsNonoer a(uNtohtorUi.St.ies EPA Hazardous.) TTShCiAs, pErIoNdEuCcSt, cCoOmSpLl,ieAsICWSi,thMItThIe acnhdemiKcoraela.registration requirements of EFPICRREA WWAZZAARRDD: CLNAoSS:PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 5. SUGGESTED FIRST AID eveTamceodmimaatce:ly flush eyes with large amounts of water for at least 15 minutes. Get immediate medical attention. SKTIaNmeCdOiNaTtACeTl:y flush skin with large amounts of water. Remove ccoonnttaamsiinnaatteedd ccllootthhiinngg. beIfforierrirteuastei.on persists, call a physician. Hash INIHfALAsTiIgOnNs:/sysptoms occur, remove person to fresh air. If signs/sysptoms continue, call a physician. IFOrSiHAnLkLOtWwEoD:glasses of ater. Call a physician. 7. PRECAUTIONARY INFORMATION or EYAEvoPiROdTEeCyTeIOcNo:ntact. Wear vented goggles. 000424 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-014:PAGE C-30 MSDS: FC-95 January 29, FLUORAD 1998 Brand Fluorochesical Surfactant LE 7. PRECAUTIONARY INFORMATION (continued) SKAIvNoiPdROsTkEiCnTIOcNo:ntact. Wear appropriate gloves when handling this mraetceormieanld.ed:A pbauitrylofrugblboevre.s made Use ofnreomorthemofroslolofwitnhge fmaotlelroiwailn(gs) are cpoevresroinnagl, pcroovteercatlilos.n" itePmrsoteacstinveecegsasramreynttso p(roetvheenrtthsakninglcoovnetsa)ct:shouhleadd Pboelymeatdheyloefnse/iptohleryviofnyltihdeenfsollcohwlionrgidemat(eSrairaalnse:x). REUCsOMeMEwNiDtEhDaVpEpNrToIpLrAiTaItOeN:local exhaust ventilation. Use in a wellevmeinstsiiloantsedbearleoaw. recPormomveinddeedsufefxpiocsiuernet vliemnittisl.atioInf etxohamuasitntavienntilation is not adequate, use appropriate respiratory protection. REASvPoIRiAdTObRrYeatPhRiOnTgECToIfONd:ust. Select one of the following NIOSH approved arcecsopridraantcoerswibtahseOdSHonA raeigrubloartnieoncso:ncenhtarlaft-imoanskodfusctonatnadminmiasnttsraensdpiriantor, fhualllf--fsaacsek ssuupppplliieedd aaiirr rreessppiirraattoorr., full-face dust and mist respirator, PRDEoVENnToItONeatO,F dArCiCnIkDENoTrALsmoIkNeGESWThIeOnN:using this product. Wash exposed baerfeoarsetheoatrionugg.hly With soap and Water. Wash hands after handling and RECKOeMeMpENcDoEnDtaSinTeOrRAdGrE:y. Keep container closed when not in use. FINRoEnfAlNaDssEaXbPlLeO.SION AVOIDANCE: OTHNoER snPoRkEiCnAgU:TISOmNoAkRYingINwFhOiRMlAeTIuOsNi:ng this product can result in coofnttahmeinhaatziaorndouosf dtheecomtpoobsaictcioonanpdr/oodrucstwsokemeanntdionleedadintosetchteiofnor4matofion this MSDS. HIS HAZARD RATINGS: PHEEARLSTOHN:AL2PROFTLEACMTMIAOBNI:LITXY:(S0ee RprEeAcCaTuItViIoTnYs:, 0section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONRAATTEE...... 0.1 0.1 MG/M3 MG/M3 PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONNAATTEE...... 0.1 0.1 MG/M3 MG/M3- Abbreviations: N/O - Not Daterainad N/A - Not Applicable TYPE AUTH SKIN TMTMoowwm Y Y HA aM vy aY CA - Approximately 000425 418-014:PAGE C-31 JMaSnDSu:aryFC2-0,6 1F9L9U8ORAD Brand Fluorochesical Surfactant races | EXPOSURE LIMITS (continued) MGREDIENT VALUE NIT TIPE ATH SKIN POTASSIMN PERFLUOROALKYL SULFONATE... 0.1 Ma/k3 THA av t=heSKpIoNteNnOtTiAaTlIONc:ontrLiibsutteidonsutbostathnecesovairnadlilcaetxepdoswuirteh b'yY'thuendceurtaSnKeINousrefreourteto biyncdluidriencgt mcuocnotuasctmweimtbhranteheansdubsetyae,ncee.ithVeernibcyleasirbcoarnnealtore,r smkoirne apbasrotripctuiloanr.ly, S"O3UMR:CE OF3MEXRPeOcSoUmRsEenLdIeMdITExDApToAs:ure Guidelines 5. HEALTH HAZARD DATA EveWilcodntEaycet:Irritation: pain, and tearing. signs/syaptoss can include redness, selling, SKIMNildcoNSTkAiCnT:Irritation (after prolonged or repeated signa/sysptons can include redness, swelling, and ictocnhtiancgt.): eMaxytenbdeedabtsiomreb.ed through the skin and persist in the body for an THMHaAyLATbIeONh:araful if inhaled. TMaiyne.be absorbed by inhalation and persist in the bady for an extended Single overexposure, above recommended guidelines, may cause: SIorrreinteastsionof (tuhpepenrosreesapnidrattohrryo)a:t, sicgonusg/hsiynsgptaonmdssnceaenziinngc.lude TFInsWgAeLsLtOiNoEnD:is not a likely route of exposure to this product. tIhlilsnesmsatemrayialr.esult from a single swallowing of a moderate quantity of May be haraful if sualloed. MUTMAuGtEaNgIeCnIiTcYi:ty assays indicate the product is not mutagenic. 000426 Abbreviations: N/D - Not Determined N/A - Not Applicable GA - Approximately 418-014:PAGE C-32 MSDS: FC-95 FLUGRAD Brand Fluorocheaical Surfactant January 29, 1998 PAGE & 8. HEALTH WAZARD OATA (continued) REPNROOTDUtCeTrIaVtEo/gDeEnViEcLOiPnHEtNhTeALratTOaXItNSo:ral Levels. doses below maternally toxic OTTHhEiRs WpErAoLdTuHctHAZisARDnotINkFnOoRwMnATItOoN:contain California Proposition 65. any substances regulated under A Product Toxicity Summary Sheet is available. SECTION CHANGE DATES HEADING SECTION CHANGED SINCE November 0S, 1957 ISSUE Abbreviations: NID - Not Determined NIA - Not Applicable Ga Approximately TbIehMePLcIoiErnDrf,eocrtmINaCtaLsiUoDnoIfNiGtn,hetBhUidTsateNMOaTtiseLsruIieaMdlI.TESDaIfHTeOt,yWAAKDENaStYaNIONSPhLWeIAeERtDRA(WNMTASIRDESR)SA,NTiYsEXOPbFRelEiSeSvEeDdORto NPwEEhRReCFtHOhAReNMrTAANtBhCeIELIOSRMTYUprSOoARdGuEFcItOTFNiEsSTSRAfDiFEtO.RfoArUPsAeaRrTpaIirCstUiLrcAeuRslpaoPrnUsRiPpbOulSreEposfOeRoraCndOdeUtReSsrEumiitOnaFibnlge for cuuansnieqrua'efslfyemcetwtihtothdhienoufstehueseaunsdoerra'pasppplklinicocaawttliieoodnng.eof anaGdiv3ceonnptrtrohodelu,cvta,irtiesotimsye oefsofsfeWnahtciitcaohlrsatrhteahtat tphaertiucsuelrarevapluuraptoesethaned3sMuiptraobdluectftoor duesteerr'asinmeethwohdethoferusiet iosr faiptplifcoartiaon. D3iunMeeprtrrooovritsdh,eesormeiimnsofstoieromnapstoisoosnribaiillnitteeyrlaettchitoarntosniecliencftotrrhoiansiacsinfatorrsasenarstvfiioorcne,matySoHhimatavskeecsursentsooumletresd. riiennpffrooerrsmmaeatntitioaontniooinnbstatihaensedtWoSDfSriotmsavaacioldmaapbtllaeebtaesdnieersemscatyolrynoatfcrcoubmreacSayHs..curIrnenatddiastiotnh,e 000427 418-014:PAGE C-33 ATTACHMENT 3 TEST ARTICLE PREPARATION PROCEDURE 000428 418-014:PAGE C-34 ATTACHMENT 3 Vesion418.0P1ro4t(oc2o2l O4C18T-90814) TEST ARTICLE PREPARATION PROCEDURE Page of2 Test Article: PFOS. Vehicle: 0.5% Tween 80 in R.0. Deionized Water. A. Purpose:ofThdeopsuargpeosseuosfpetnhssiopnrsocoefdPuFreOSis atondprtohveidveehaicmleetfhoordorfaolratdhmeinpirsetpraartaitoinonto rats on Argus Study 418-014. B. General Information: 1. Aslplecsiufsyptehnesipornotcoocnotlaniunemrbserw,illtebset laratbieclleedidaenndticfoilcaotricono,dAerdg.usEbaacthchlabel will anunmdbsetro,racgoencceonntdriattiioonsn., dosage level, preparation date, expiration date 2. Suspensions will be prepared: X_ Daily -- Weekly _For__daysofuse 3. Vehicle will be prepared: Daily X_ Weekly _For__daysofuse 4. Suspensions will be prepared at a final dosage volume of Uk, 5 Safety: X_ X_ DGulsotv:eMsi,stlaRbecsopaitr,atgoorggles or safety glasses and faceshield _ -- Half-Face Full-Face Respirator Respirator/Positive Pressure Hood Tyvek SuitApron 6. --DosageYessolutions adjuXs_ted Nfoor (FCraelecublaasteioansndba%sePduriotny.100%) _ FreeBase __ Purity 7. Sampling requirements: Cited in protocol. 8. Storage: Cited in protocol. 000429 418-014:PAGE C-35 ATTACHMENTS Protocol 418.014 Version;418-014 (2P2aOgCezTo9l82) TEST ARTICLE PREPARATION PROCEDURE NOTE: cOonnctaeintehresf;inmailxvionglusmheosuladreocaccuhriedvuerdi,ngstsirambpalrisnagreantdo/bore aaddmdienidsttroatthieon C. Preparation of Vehicie: 1 Add the required amount of R.O. deionized water to anappropriately labeled container. Heat the water to 50C, +5C, add therequired amount of Tween 80 and mix until uniform. (See TEST ARTICLE `CALCULATIONS for exact quantities.) D. Test Article Suspension Preparation: 1. To prepare the suspension for the high dosage group, add the required amount of test article (See TEST ARTICLE CALCULATIONS) into an appropriately sized, labeled container. QS ad to the required amount with vehicle, add a stir bar and heat the mixture to 80C, 5C, in a water bath for approximately 30 minutes or until the test article dissolves. day before use.) 2 Once the test article has dissolved, remove the suspension from the water bath and spin the suspension while it cools. (Be sure there is avisible vortex, this will achieve the desired emulsion. This `may be prepared the Cle al Written by: f ons 4 VI Approved Clarification: Ch Date: 23:ccr%5 / No __ Ys (See attached clarification form.) initaVDate: Choe 2 op 0600430 418-014:PAGE C-36 ATTACHMENT 4 CCROSS-FOSTERING PROCEDURE 000431 418-014:PAGE C-37 ATTACHMENT & CROSS-FOSTERING PROCEDURE ProtocPola4g1e8.10of141 gdTerhloeiuvpec.rohaaDnbadimtawsitlilaosrnestpiaegirnnieotddhetwioirltlnhabeteucgroiannltropounlpegsdrauonuytpielatnrhleaiteedrmeafdotreftoohrencrrtaohtsess-faifsrossstitdgearniyendwgitlploubtrhepeoascleolsno.twreoTdlhitos wdPiaFlmlOsSp..rtorviedaeteadpdoaolmso,f ctohnutsroplredvaemntsinavgatilhaebsleepfuorpsimfmreodmianutresicnrgosfsr-foomstteheriirngPFtOoSp-utprseaftreodm Ocon-htohuesreedmawiintihnmgadlaeybsroeefdceorhsa,biotnateiomna,lbeortaht ctoonotrnoel faenmdalPeFOraSt-. trSetaartteidnggrwiotuhptshiwisllsebteof gdferrlooiumvpetrhoieerisra,raPelFsOppeSuc-pttisrveweialdtleabdmesdraaemnm.dovpiefldaacdiemadmmewdiatishasteieigltnhyee(rdaastnosotothhoeenrcoadnstarpmoalratgusrrsioitugiponneidsisntcooottmahpvelaeictloeanbt)lreo,l tthheenn abedsaamcriffriocmedthveiacdoenctraoplitpaotoilonwil be used. The liter from that control pool dam will (Tpsohasevtepfidarrsittnutnemenuwtpilrulaplbsebfudrfeofcemarpteihdtea1tc0eond%t,rfoaolnrdmpaotloihlne)(liwurinrllegssbpee(csctaoivlveledeocfitneldiBttoaeurn)idnd'epstreesorslemuritvnieeondd)tfaoornbdpeotsashtiebdllaeiv.yer1 fbuetusraecrhiifsitcoepdatvihaoldoegciacpailtaevtailounatainodn.diTschaerdreedmawiintihnogutpunepcsrforposmy tehvealcuoanttiroonl pool dams will TCPohFniOtsrSoc/lr/oPsCFso-OnftSorso(tleSru(ibSnsugebtsperDto)c.Ae).dTuhCriosenwptirlrolocrleePdsuuFlrOteSiwn(iflSlouuabrlslgeotrwoBud)ip,sstPioFnfOctS1i/2oCndosantmtosr/bapelum(pSasu,dbesice.bt.eCt)weaennd prenatal and postnatal effects of the continuous maternal PFOS treatment. 000432 418-014:PAGE C-38 r~PRIMED ICA - ArSsTReLseaarbchoLpaA borSatoiriees,gIc. WW teehew PROTOCOL 418-014 Oral (Gavage) Cross-Fostering Study of PFOS in Rats `SPONSOR'S STUDY NUMBER: T-6295.13 - Amendment 1 -- 4 January 1999 1. CrossFosaring rocedute (Paragraph 3 ofAtachment 4 ote rose) Tpuhpesfitrhsattteanrepfuopusnfdrdoematdhe(ncoonaturtoollpysoiosl p(rierrseesnpte)ctpievre doofslaitgteer) garnodupthdeetfiersrtmtienned to be at day 1 postpartum will be decapitated at approximately 1 to 3 hours after birth, and the lungs and the liver will be retained. Several (minimum of two) rTcTooocrnatdaousea cpaopevdno ediblhewe Srpainmaset opSaencepao. Snevperraalb(eerrviwiluebme[roreefpmaworveae)tdion refrigeration in scintillation vials) (see attached) for future evaluation. The atameeadr0n% mwaanoivnrmoropmpseSnrdooraeprorras orUeSslailoinl,orThe remaining pups from the control group dams will be sacrificed viadecapitation The liver sections in McDowell-Trump's Fixative will be shipped (on cold packs), for evaluation by electron microscopy, to: Jeanne deWard Pathology Associates International 4915 D Prospectus Drive Durham, North Carolina 27713 Telephone: (919) 544-5257 Telefax: (919) 544-3218 The remaining samples will be shipped (ambient conditions) to the Kris J. Hansen, Ph.D., at the address cited in the protocol forhistopathological and biochemical evaluation. 000433 418-014:PAGE C-39 : Reafosr Choannge Promceonld4m1e8n.0t161 Page? The Sponsor has requested that these additional samples be retained for continued evaluationofthe reasons for reduced pup survival observed in previous studies. 2. Cross-FosteringProcedure (Paragraph 4 of Attachment 4 to the protocol): rTehaed:fourth paragraphofthe Cross-Fostering procedure has been changed to PFOS treatment. This cross-fostering procedure will result in four groups of 12dams/pups, ie., Control/Control (Subset B), Control/PFOS (Subset A), PFOS/Control (Subset D) and PFOS/PFOS (Subset C). This Procedure will allowdistinctions to be made between prenatal and postnatal effectof the continuous maternal ReaforsChoangne: This corrects the protocol. Ge - AW OFTess Alan M. Hoberman, Ph.D. DABT Date fr Raydkbnd G. J York, Gh.9., 043-59 DABT Date Director of Research As`sSotcuidaytDeirDeicrteocrtor ol Research and Diane Clube o4lnss Use Commitee Dena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and ornTore 0482 Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 000434 418-014:PAGE C40 : Protocol 418.014 FIXATIVE PREPARATION Version: PROCEDURE 4182.F3ag0Dee11c094182) Eixative: McDowell-Trump'sFixative A Purpose: oTfhMecDpourwpeolsle-Torfutmhpis'sprFoicxeadtuirveesfiosr ttoispsruoevisdaempalmeestfhoordstfuodryth4e18p-r0e1p4a.ration B. General Information: 1. Aplrlotsoocloultinounmcboenrt,aifniexratsivwiellidbenetilfaibcealteido.n,Eparcehpalraabteilonwildlatsep,eceixfpyirtahteion date and storage conditions. 2. Fixative wil be prepared: _ Day _ Weekly _x Once 3 Safety. X_ X GDulsotv-eMsi,stlaRbecsopaitr,atgoorggles or safety glasses and face shield XC -- FHuallfl-FFaacceeRReessppiirraattoorr/Positive Pressure Hood X_ Tyvek Other: SuitiApron Chemical Fume Hood 4. Dosage Factor: suspensions adjusted for % Activity/Purity or Correction -- Yes X_ No (Calculations based on 100%) 5. Sampling requirements: Cited in protocol 7. Storage: Room temperature 000435 418-014:PAGE C41 Protocol 418.014 Version: 418(-29D0e1 c94 8) Faces ols FIXATIVE PREPARATION PROCEDURE Cc. Preparation of Fixative: McDowell-Trump's Fixative 1 lAadbdele8d60comntLasinoefr.R.0. Deionized water into an appropriately 2 Add 100mLs of 37% Formaldehyde to the container and stir until uniform. 3 u`Andidfo4rm0.mLs of Gluteraldehyde to the container and stir until 4. Add 11.6gofSodium Phosphate Monobasic to the container and stir until uniform. 5. Add 2.70gof Sodium Hydroxide (0 the vessel and si uni uniform, NOTE: The final solution shoud have a pH ofapproximately 7.3] Wiiten By: (lee2l0:Jt,hA on Approved wll ue: RINE Clarification: /No __ Yes (see atached clasiication form] tialsDate: aLo gbiXK:rPopes 2/rcfry 000436 APPENDIX D DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 000437 APPENDIX E TEMPERATURE AND RELATIVE HUMIDITY REPORTS 000438 418-014:PAGE E-1 ARGUS Temperature and Relative Humidity Report Location: Room 04 Protocol Number: 418-014 Range of Dates: 27-Oct-1998 13:15 to 23-Nov-1998 15:49 ee |Temperature aRelative Humidity Mean ( SD): 69.1 (19) 83.2 (8.0) `Number of Points High (%): 0 (0.0) 0 (0.0) -- -- -- -- REVIEWED BY: a Hai..- DATE: 2.399 000439 418-014:PAGE E-2 ARGUS TemperatureLoacnadtiRoenl:atRioveoHmu2m7idity Report Protocol Number: 418-014 Range of Dates: 23-Nov-1998 15:49 to 31-Dec-1998 14:10 STpaarcgieetsR:aRnagte: TToottaall NNuummbbeerr ooff HDoauyrss:: Total Number of Data Points: Mean ( 5D): MMiMenadixiimamnuu:mm:: NNuummbbeerr ooff PPooiinnttss iHnigRhan(%g)e: (%): Number of Points Low (%): Report Generated: 04-Feb-199a9t 17:28 COMMENTS: TSemFpe0ra7tuare Rela3t0i%ve0Hu7m0i%dity 9130901 9130901 913 913 09 @on| 1 wen 771208 568532 0 60 9o3 (1e0o00)|| 9o13 (`0e0o0) o 0.0) 0 (0.0 ~~: REVIEWED BY: mn Had. DATE: 57] 000440 418-014:PAGE E-3 ARGUS Temperature and Relative Humidity Report Location: Room 35-37 Protocol Number: 418-014 Range of Dates: 31-Dec-1998 14:10 to 18-Jan-1999 08:45 `TSapregceitesR:aRnagte: TToottaall NNuummbbeerr ooff DHoauyrss:: Total Number of Data Points: Mean (+ SD): MMeadxiiamnu:m: Minimum: NNumubemroobffPPoeoiinnrttss iHnigRhan(%g)e: (%): Number of Points Low (%): Temperature S4F 0 79F | Rela3t0i%ve1H0u7m0i%dity 4216824 4216924 427 421 756 @1n | S01 (7.4 776843 5561.81 68.5 50 421 0 ((01.000)0) | 47 0 @1 0) 0) 10 @3 Report Generated: 04-Feb-1999 at 17:30 COMMENTS: -- -_-- -_-- REVIEWED BY: hn. Mod... DATE: 2.897 000441 418-014:PAGE E-4 ARGUS Temperature and Relative Humidity Report Location: Room 27 Protocol Number: 418-014 Range of Dates: 18-Jan-1999 08:45 to 29-Jan-1999 09:57 TSpaercgieetsR:aRnagte: TToottaall NNuummoobbffDHeaeoyursrr:s: Total NuomfDabtaePoinrts: TSemFpe0ra7t9ure 21520 26 Rela3ti0ve%toHu7m0i%dity 261250 268 Mean ( 5D): MMeadxiiamnu:m: Minimum: NNumubemroobffPPaaeiinnrttss HinigRhan(g%)e (%): Number of Points Low (%): 4 @05| sa3 ey 770240 6044 69.1 31 2o6 o(o1000)|| 264 5 (09.946)) 0 (0.0) 0 (0.0) Report Generated: 04-Feb-1999 at 17:33 COMMENTS: r--e ---- e------eeeremrm-- REVIEWED BY: Nad. ATE: 2.691 000442 418-014:PAGE E-5 -_-- ARGUS Relative LHoucmaitdiiotny:DReovioamti0o4ns Report ree e Protocr ol Numbe er: 418s -014 seems - Theres Range of Dates: 27-Oct-1998 13:15 to 23-Nov-1998 15:49 Humidity Target Range: Species: rat 30% to 70% 03NDowa-t1e998 T1i4m00e R2H8s. Date Time RH. eee Hi= Value outofRraHn.ge= R-eHliagthive HuLm=idViatlyue(%o)utofrange - Low Report Generated: 13-May-1999 at 10:45 LL These deviations did not adversely affect the outcome or interpretation of the study. The following deviaton(s) impacied on the outcome of the study as described: Study Director: Z - = : z Date: _/T~ M8 -39 000443 418-014:PAGE E6 ARGUS --_--_-- Relative Humidity Deviations Report Location: Room 35-37 Protocol Number: 418-014 --_----RangeofDates: 31-Dec-1998 14:10 to 18-Jan-1999 08:45 Humidity Target Range: Species: rat 30% to 70% 05vDana-t1e999 0055--JJaann--11998999 0066--JJaann--11999988 0066--JJaann--11998889 0066--JJaann--11999999 06-Jan-1993 T21i:m0e0 RsH8.L 2232.0000 ssoiLL 0010::0000 ssiic 0032.0000 ss23LL 0054::0000 ss56LL 06:00 s6L Date Time RH. H = Value out ofRrHan.ge= R-eHliagthive HLum=idViatlyue(%o)ut of range - Low Report Generated: 13-May-1999 at 10:50 / hes deitons i ot acersaty affect tv tome o protonof esc. "The following deviation(s) impacted on the outcome of the study as described: Study Director: it zi = Date: _13 41-9 000444 418-014:PAGE E-7 ARGUS -_-- Relative Humidity Deviations Report Location: Room 27 Protocol Number: 418-014. -_-- _-- Range of Dates: 18-Jan-1999 08:45 to 29-Jan-1999 09:57 ss Humidity Target Range: Species: rat 30% to 70% Date Time RH. 24-Jan-1999 06:00 04H Date Time RH. H =Value out of range R.H. = - High Relative L = Value outof Humidity (%) range - Low Report Generated: 13-May-1999 at 10:54 + These doviations id not adversely affect the outcome or interpretation of the stu, `The following deviations) impacted on the outcome of the study as described: `Study Director: Z 2 IS Date: _ (3m G99 000445 418-014:PAGE D-1 DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 1. Two exira female from the treated group (Group Il) were used for pharmacokinetic sample collection on (DL 14), 22 January 1999. These two day 14 female of lactation rats in tum received all acfoflelcetcttihoensouatncdowmeereofstahcerisftiuceddy obnecDaLus1e4.thTehipshadremvaicaotkioinnedtiidcnostamapdlveerssweelyre not analyzed. 2. wEiegrhet ufseemdalfeorrapthsarramtahceorktihnaentiscixsfaemmpalleecroaltlsecftrioomn othneDcLont1r4o,l1g7rJoaunpu(aGrryou19p9I9), 21 all January 1999 and sample collections 22 January 1999. The and were sacrificed on two DL extra female rats received 14. This deviation did not adversely affect the outcome samples were not analyzed. of the study because the pharmacokinetic 3. wAteortealcroofss2-5folisttteerrsedfrroamthtehretchoanntr2o4l (liGttreorsu.pTIh) iasnddetvrieaattieodn d(iGdronuopt aIld)vgerrosueplsy affect the outcome of the study because only one additional litter was added to each dosage group. 4. Twenty-five female rats rather than all of the rats in the control group c(oGhraobuiptaIt)iwoenrpeerpiloadcefdoritnhtoesceohraabtistlaatsitoendosnix1d3ayDsecreatmhbeerrth1a9n88f.iveTdhaeys. This deviation provided did not adversely affect sufficient mated rats for the outcome of cross-fostering. the study because it All deviations are documented in the raw data. Lt R York, PRD. DABT Associate Director(of Resefrch and Study Director: 23-3 Date 000446 APPENDIX F PATHOLOGY REPORT 000447 PZ.7) 418-014:PAGE F-1 P4aCtohmpoalnyoofgSycaAnscesAoppciicaattnsesvemIantotnearinCaotrioonnal = re= : PATHOLOGY REPORT (ANCILLARY STUDY) ELECTRON MICRCORSLC:OCPDICBERVVAALFU/APTLIUOSNORFATLSI)VER AND LUNG IN ORAL (GAVAGE) CROSS-FOSTERING STUDY OF PFOS IN RATS CLIENT STUDY NUMBER 418-014 PAISTUDY NUMBER EM 99.46 Prepared by: James B. Nold, D.V.M., Ph.D., Diplomate, A.C.V.P. Patho4l9o1g5y.ADsPsorcoisapteecstuIsntDerrinvaetional Durham, NC 27713 April 5, 1999 PREPARED FOR 3M CORPORATE TOXICOLOGY, 3M CENTER, ST. PAUL, MN 55144-1000 000448 4915D Prospectus Drive + Durham, North Carolina 27713 + (919) 544.5257 + (919) 544-3278 FAX Pathology Narrative `Transmission Electron Micrograph Interpretation Forms Transmission Electron Micrographs `Quality Assurance Statement 418-014:PAGE F-2 Section 1 4 m v 000449 418-014:PAGE F-3 L Pathology Narrative 000450 418-014:PAGE F4 Sp PATHOLOGY REPORT (ANCILLARY STUDY) ELECTRON MICROSCOPIC EVALUATION OF LIVER AND LUNG IN CRL:CDBR VAF/PLUS RATS) ORAL (GAVAGE) CROSS-FOSTERING STUDY OF PFOS IRN ATS CLIENT STUDY NUMBER 418-014 PAISTUDY NUMBER EM 99.46 SUMMMARY E e rt rT e ee setS seS B t eEe r cneoomnpaatraledrawtiptuhpcolnitvreorlssf.roQmuadntaimtsattiroenatoefd pweirtohxi1.s6ommegs/kign/ddicaayte(dhisglhi-gdhtolsye)moPrFeOSthan twice PROCEDURES PFOS. In addition to a general qualitative evaluation for differences between control and shuerpfaatcotcaynttesl,ammeellmabrrbaondeieasnidnmtihteolcuhnogn.drial changes in hepatocytes, and the quantitation of EEE TEEE eeer [e rmT 30.6 E ER Ee Lace 1 TT oen6 ielT y o" p -- p 1--g 5----|] E . 000451 Ne 418-014:PAGE F-5 -- ESrEmeEr fe Trump fixative (McDowell EM, 1976),and submittedtothis laboratory for electron suultbrmaistttreucdtusraamlpllye(sTewxetrTeaibdleent2i)f.iePdubpynduammbenrusmwbeerreoanlsys.igTnehdebfyivtehidsigliatbnoruamtboeryr biesctahuese CommiTbe ema] A, (saelpepcrtoxreipmraetseelnyta9t0ivnema)rewaesrfeorcuftu,rtmhoeurnetleecdtroonn 2m0i0c-rmoescsohpcyopprpoecresgsriindgs., sTtahiinnedsewcittihon5s% methanolic uranyl acetate and Reynolds lead citrate, and examined on a Zeiss 900 Se EEyE re RESULTS AND DISCUSSION er -- ye evident in the liver compared with the lung. Lung is inherently less dense thanliverand ocarhlgalaronawecsltleberesit,zteedrdilibanytfaidtlitirosanctoionoftnoeifnntduoohupeslapfsilxmaaistcimvareemtieintmcoublrtuhameneatisns,dsutleho.essoSnuufbclodepeattraiimelaonlvfeflcioyxptaeot,ipolananswdmaidsiclatation 000452 418-014:PAGE F-6 TT the presence of peroisomes in the hepatocytes and lamellar bodies in the lung and to LIVER Control (0 mg/kg/day). Subcellular features ofcontrol `hepatocytes are demonstrated in EH E r RE a reticulum and the nuclear envelope were additional artifacts of poor fixation. Fine SE a were identified and quantitated in from representative hepatocytes. Forthe control livers, peroxisomes per cell (Text Table 3). Fetal and neonatal rat liver is a hematopoietic tissue, and numerous hematopoietic cells were evident within the liver sinusoids, CeomnpCeo|mo imme | Qmphgley 1 onbow emma MewCommo 0Tobe or] Hwiagshh-iDgohsere i(n1.t6hemgh/ikghg-/ddoasye).aniTmhaelsacvoermapgaernedumwbietrh coofnptreorlosxi(sToemxtesTpaebrlhee3p).atoOctyhteerw(i1s4e.7,) I E BEE e eome ne pneumocytes.were idenified by the presence of lamellar bodies, which are cytoplasmic 000453 418-014:PAGE F-7 PrimedAnccilplaurysSuPdastoNles 000 lrvaeampceruleolslaeenrstmcatothneetrmaiaaitlneirwniigatlehliseneccatrirerotwne-addiyensntpsoaetchleaesmwianlavasetoevldearmryesmrpabarcreelasynaiedselsniutkrieffaipcertdoafniitnl.ecso.SnutrLrfoaalmceltlualnnagtsr.obroTdhiees tthyipnemIaprngeiunmoofccyyttoep(lPaNs1m).i TychpaeraIcptneeriusmtoccalyltyesladiieffneerdenatinadtelifnreosmthteypaelvIeoplnarcusmpoasceyiaessa nrMeeaponrneaysteapnlnteaaundmaonhcsayidttenosontabpsetpaeegnaerfebudlelttyoweebxeepnacPunNbdo2eisddaawnlidtwhiPatNihrL.osu,Addedissiptteiicnoicnatalillvlyeys.liasnmcoeelmlteahrecsubebodoliiuednsag,lsaewpniedtrhmeealiyal cells represented terminal bronchiolar epithelium. wlTueynrpgeesaIalnspdorpncrouemsroeencltiyatwbeliesthaqinunadnctalipatimaletllialoranyr clbououmdliidneasb.eweasrseesisnefrdeoqfuetnhtelmy.idSenotmifeieldamienltlhaers-elickoentbroodlies oeHsfistgeynhpt-eiDaolIlsypens(ie1mu.im6loamcrgy/tkegcso/ndatanrydo)ll.asmaUemllprllaaesrst.broudcTitheuesrr;aelaappalpeteahsaotr,leoidgd,yeonhftoitwfeyhvienrgh,itgh0he-mbdeofsoiernlciurllneuagsssterwdaatnisvuembers wapinutdrhpisonespeciasapwimlaalsadreeyasdlieuefmriintnihtaaivnaenidqnuocaonnttniartoaslltmisoaanylmilpmlpnerusar.catbiTechraelw.ceorSleolamspeeseenldawnmiaettlhuliranerabolofvdemoioleiss.twoFefirtgehureiedseanl9tvi-efo1il2eid illusrate some representative lung fields from the high-dose animals CONCLUSION (Uclotnrtarsotlr)ucotrur1a.l6emvga/lkuagt/idoanyo(fhliigvhe-rdaosned)lPunFgOsoSfirdaetntpifuipesd ifnrcormeadsaemdsnturmebaeterdswoifth 0 Dipinefdrfiocexaritesenodcmeseslsiignwhticlteyhllimunolahrreepmatethmoabcnyrttawenisecoesftohhreigmnhiu-tdmoobcsheeornrodartfippaeurpwoelxriiveesrnos.omtesdQiuisancnettrrinetiaabttleiedonbaneoitfmwpaeelersnoxciosnotmreosl. lpaarnmedeulumleoaacrtybetodedasineaisnmwadellrsaemineildtlehanetrisbfaoimdepdileiesnswbeaoxtsahmecivoannletudra.toeldsI.nanlTduynhpgiesghaI-mdppolrseeesumatnohieemypatrleesss:echnoocnweteaovifenrti,ynpgteheIIre `faeprxpoetmarrwaeecadetleldubasleanamadrpnlweiisnt.chrienSaocsaempdeilnlluaamrmybelellruaomrifnbtaoy.dpieeAslItTahnpodnuegluahmmoeslcolymateremslyaeamlneidlnl-lalarimmkealetlefaririgbuaorldes(isewusrefriaectlaunntg)s cwoanstriodlenatnidfiuabalteewditshaimnpallevseolar lumina, no significant difference was noted between SIGNATURE OF AUTHOR ect) NA DaimpleosmaBt.eN,oAldC,.VD..PV.M. PhD, 1 lope) S, ladg Dae 000454 418-014:PAGE F-8 PrimedicaAAnrciglulsarSytuPdaythoNloo.gy41R8e.p0o1r4t. REFERENCES McDowell, EM and Trump, electron microscopy. Arch. BF: Histologic fixative for routine diagnostic Pathol. Lab. Med. 100:405-414, 1976. light and 000455 5 418-014:PAGE F-9 IL Transmission Electron Micrograph Interpretation Forms' "iFnoarrmcshiavreedtsytpougdryafpihlie.cally corrected versions of signeddated rawdatasheets maintained 000456 418-014:PAGE F-10 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No:418-014,EM.90.46 AnimalNo: 180041 Treatment Group: _0mg/kgControl Sex: non applicable Specics/Strain:CfLmCDRBR VAFPus Tissue: Liver Block No(s).: _99.46-1 Photo/Negative2N1o5(6s8)3.,:_2Z1155668846,-88. Sigaificant Lesions (check one): _____ Yes _X No Interpreting Pathologist (signature and date): pFreoabtaubrlees:endotZh1e5l6ia8l3:c-ellHeopnatroigchyttes,idseoevferpahloetroygtrharpohc.yteSsixin(6s)inpuesrooidx,isaonmdensuecvlieduesntofin glarragneulcaerntmraatlerhieapla,togclyytceo.genDi,liantecdytbolsoelb.s iDneRnEsReamrietopcshcounddor-ipaerwoixtihssomoemse.grAabnuulnadradnetpofsiinets 2a1n5d6d8i4l:ateSdecvreirsatlae.hepPaotoorcydteifcinniutciloenoifpreensdeontt,hebluitalpoceolrl.cytoplasmic margin definition. cSoommeer.swTelwloin(g2)ofpemriotoxcihsoonmdersiai.n cDeinltaratledcebll.ebsIninadRjaEc.entBtiwleoccaenllasliocnullyussiinnglleower left 2p1e5ro6x8i6s:omSeisngalreehcelepaartloycyetviedseunrt.rounded by erythrocytes. No clear definition of menidtootchheolnidalricaelalsn.d RDEiRs.tendMeidtoccyhtoonsodlriwaithhavgelyscoogmeen-glriakneulgarranduenlsairtpiaerstiacnldesd.ilAabteudndcrainsttae. Dilated Z15687: bleSbisnignleREhRep.atoTceynte(.10G)rpaenruoloxciystoimcesceelvli(dhenetm.atopoietic) present in bottom left gcroamneurl.arDdiesptoseintdse.d cTywtooso(l2)wpietrhogxriasnoumleasr emavtiedreinatl.inDceenntsrealmihetpoacthoocnydter.ia with some R21E5R6.88C:ytoHseaplateoxcpytaen.dedErwyitthhrofiidneprgercanuurlsaorrmparteesreinatl,inprloobwaebrllyegtlsyicnougseoni.d.TDhirleaete(d3)blebs in peroxisomes present. P`Cloanscmlausmieomnsb:raNnoersmpaolorhleypadteofciynteeds.. D2e3n/s5e=m4i.t6ocpheornodxriisaomweisthpesromheepagtroacnyultaeredveidpeonsti.ts and dilated cristae. Suboptimal fixation. 000457 418-014:PAGE F-11 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_418:014;EM-09.46 Animal No: 18904: Treatment Group:_0mg/kgControl Sex: __non applicable Species/Strain:aC.CDOBR VAF/Plus Tissue: Lung Block No(s): __99.46:2 Photo/Negative No(s).ZZ1516586993 Significant Lesions (check one): _____ Yes _X__ No Interpreting Pathologist (signature and date): aFleaamteulrleasr:-liZkIe56bo8d9y;. CSaepviellraarlytwyipteh1tphrneeeuemaodcotybteelsia(lPcNel)l pnruecsleein.t sIunrlroaumnedninogfccaappiillllaarryy. is ZN1o56p9n0e:umToycyptee2lpanmeelulmaorcbyotdeie(sPpNr2e)sepnrte.sent, containing multiple lamellar bodies (7). Beneath the nuclei. PNI PN2 is a to right sciadpeiolflapryhoctoongtraianpihngwainthermyotrherotcryatnes,lupcleanttelceyt,taopnldaesnmd.othelial cell rZo1u5n6d91n:uclCeoulsl.apOstehderalcveelloslalresssepctlae.arlOyndetfoipinesd.a bPuNt1apwpietahrftloatbteenperdimcayrtiolpylaPsNm1sanidn large 2co1l5l6a9p2s:ed sCeopltla.apsNeod paloveeuomloarcystepetal.amOelnlatropboidsiaestheivni-dleanyte.red PN1, and in the bottom left lisamlealrlgaerrdperborfiilseaondfatPwNo1.leuCceonctyrtaelsl.yAlasppoeasrosmetobleipiad cdaepbirlilsariy plruemseenntcionnttahiencianpgilsloamrey. plunmeeunm.ocEyntdoethlealmiealllacrelblocdiyetsopildeanstmifiisede.vident, but no endothelial cell nucleus. No 215693: Beneath itCioslalatphsiend-laalyveeroeldarPsNe,ptaa.ndAttotthopeils eafprsoifdieolefophfaotPoNi1s in the a PN1. alveolar space. Subjacent nuclei caroellparposbeadballyveeonldiotahnedlitahlinc-ellilnaeuPciNei.. NAot pbnoetutommoclyeftteislaamneelrlyatrhrboocdyitees. idJeunsttiftioedt.he right is a `Conclusions: Normal lung tissue. Fixation adequate. Only one PNZ identified. 000458 418-014:PAGE F-12 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_418-014; EM-09.46 AnimalNo: 180042 `Treatment Group: 0mg/kg/Control Sex: __non applicable `Species/Strain: ECaULCVOABFPAlusR. Tissue: Liver Block No(s): _99.46-3 Photo/Negative No(s).2:1_5Z61596498- Significant Lesions (check one): _____ Yes _X No Interpreting Pathologist (signamre and date): __ rFoeuatnudreedsb:lebZso15f6R9E4:R. HeCpyattoopclyatsem.fuMlullotfigprleanmuiltaorchgolnydcroigae,n.soPmoeorREenRdoatnhedlimaulltciepllle wdeiftihnistoimoneingrsainnuulsaoriddse.posNiutcsleaantddeidleartyetdhcrroicsytaec.inTbhorteteom(3l)efpte.rMoixtioscohmeosndirdienatiafpipeed.ar dense d2e1p5o6s9i5t:s andHedpialtaotceydtcer.istDaiel.atCeydtoRpElRa.smMiucltmiaprlgeindsenasnedmeintdootchheolnidarlicaelwlisthpgoroarnluyldaerfined. 2S1ix56(966):perHoexpiastoocmyetse.ideMntainfiyedd.ilated blebs of RER. Dense mitochondria with granular `dTewpoeslivtes(a1n2)d pdeilraotxeidscormisetsae.ideTntoipfieldef.t hepatocyte has swollen, less dense, mitochondri. w2h1i5c6h97a:reSdeivfefircaulltpotoordliyf-ffeirxeentdihateepaftroocmytpeesr.oxMiasonmyesl.ighFtiavned(d5)arpkerdiolxaitseodmbelsebisdeonftRifEieRd,in singlemorecircumscribed hepatocyte. wZiLt5h69gl8y:cogeHne.patFociyvtee(.5)Lpaerrgoexcilseoamreasreeavsideinntc.ell contain fine granular material consistent cCeolnl.clusions: Normal hepatocytes. Suboptimal fixation. 31/5 = 6.2 peroxisomes per 000459 418-014:PAGE F-13 `TRANSMISSION ELECTRON MICROGRAPHINTERPRETATION Study No: 418.014 EM.99.46 Animal No. _189042 Treaument Group: _Qmg/kgControl Sex: __onapplicable Species/Strain: CULCDRBR VAFPID Rat Tissue: Lug Block No(s): 90.46145699 Photo/Negative No(s)._Z15704 Significant Lesions (checkone): ____ Yes _X No Interpreting Pathologist (signaancddwatre)e: lcFeaelralgteauprcpueebsao:risdaZtl1o5cc6eol9nl9tsa:dionAlancvoeotouclpounlsteacsionmnatalanliynlilanamgemlesllealvraerrbaboloddicieuesbs,o,ipdbrauoltbaapbprlpeyeuaamrPotNco2yb.ee Tcpenhlleesu.omtoThcehyretteTwsoower cawonhnditcabhionrhstaoavemsnea'gtrmfeclanapttitloelfnaerldiueomsuitlniainlnetdsoubrPyfaeNcnedowotirhteahlriesaotlemcreemlidsne.anlsTebhreeolnefcclhtairotolenan-reddeenpPisNteh!emlaaittae,trhieAaltb,toShtoetmolmeet aZdin1ld5a7tr0eid0g;hc.riAsTltvoaeepoillnaemrtistpponacechuoemnwodirctiyaht.ceacpoinltlariynsletrhyrheero"ceymtptaty"levtacaunodlepsn.eupmooscssiybtlyesexatrrouudnedd top CZfaLapmSie7ll0llLaa:rryboMadtuilletsoi.wpelrePiNPg1Ihtastwriliitnghihnetgnsdaosltlvheieogllhiatarylsmcpeoalclr.ede.leNcotrolnamdeenlslea.bodies or PN2s evident. p2b1re5eo7uu0mt2os:icdyetCeetlhloenctayrppiieglhlctaoranyntadanilndoiewnnegdroctecnhetenrilarilaalllaccmeeelll.llaaTrhtibosopdilsyefantn,otaburcteleacaoorfmlypcrodelisslcasepersdneidbbleaetl.vweeIoetlnaaprtphseeeaprtsa.to ai2ln1v5cee7on0lt3aer:rsoeAfpltvpaehooitlnoubgsortaatpnohdl.mincCeiannptgieclrellaalrnsyd.arnLigdahrteg.nedDoPitlNhaetleadipucpmreiaastrtsaeefttoincofospnohtmoateinomgaisrtiaonpcghhl.oendClroailmale.lalpasredbody ceZoLmuSpp7tl0ye4:vcalceAaulrovlveeaoccluouonslteawsii,ntiahlntlghionsuiognhmgeprtlheaiumsmecloelclaylrtimesaslt.ienreiCdaell.bly iDaneflnlosawetertrecnleeedlftaPtcNcoIemnetrercolnettaicnosntpaairntsalaly- Conclusions: Normal lung and alveoli. A few lamellar|bodies identified. 000460 418-014:PAGE F-14. TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: _418-014; EM-09.46 Animal No.: __18912-1 Treatment Group:_0me/kg,Control Sex: _nonapplicable Species/Strain:xCALCDVAERBusRe Tissue: iver Block No(s): _99.46-5 ZI5773 Photo/NegativeNo(s). ZISTT7 Significant Lesions (check one Yes _X No Interpreting Pathologist (signarure and date): Ftheeatuuprpeesr:riZg1h5t7c7o3m;er.HepEantdooctyhteel.ialScoemlledseteagimlesnatrseoifndhiestmiantcotp,oainedtifcixcaetlilosnairsepporoers.enTtheirne piserdiolxaitsioonmeasndcobulnetbebdi.ngofthe RER and some dilationofthe nuclear envelope. Six (6) 2b1l5e7b7b4i:ng ofHeRpEaRt.ocyFtoeursu(r4r)opuenrdoexdibsyomseosmceouhnetmeadt.opoietic cells. Marked dilation and r2i1g5h7t.75E:ndoHtehpealitaolcycteelswiptohorclayn-adlefiicnueldauatsndthfeibxaottitoonmisansudbospitnuismoaild.alElereyvtehnro(c1y1t)e at top peroxisomes counted. 2RE1R5.776M:itHoecphaotnodcryitaeairsepdoeonrsley-afnixdecdoanntadidnissroumpteedg.ranMualrarkeddepdoisliattsioannadnddilbaiteebdbcirnisgtoafe.the. 2S1e5v7e7n7(:7)Hpeepraotxoicsyotmeesapcpoeuanrtseds.imilar to those described above. It is poorly fixed and RER and nuclear envelopeare dilated. Seventeen (17) peroxisomes counted. Conclusions: Suboptimal fixation. Nine (9) peroxisomes/hepatocyte (average of5cells) 000461 418-014:PAGE F-15 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014; EM-99.46 AaimalNo.: 18912: `Treaument Gr_o 0mg/ukg,pCont:rol Sex: __non applicable Species/Strain:RC.xCDOBR VAFPlu Tissue: Lung Block No(s).: __99 46:6 Photo/Negative Nofs).2:1_5Z71567872 Sigaificant Lesions (check one): _____ Yes _X No Interpreting Pathologist (signature and date): bTFoehdaeiteucser,netssru:aglgZse1sm5ta7ilv6lee8r:oceaAllPf rcNoo2wn.toafiSncossumebeovieddriaasllteeepnmidtpehtdeylciravilasctcaueelosli,enspmriwothboiacchbholnymdabrriyoan.hcahvieolbaerenepiltahmeelliluamr. d`2Ta1hr5ek7et6rw9co:elcleAngatapripanel.acrsesellvtseorcbaolentcaauibbnaosiaadlfalceewelplliatmheellilaalrcbeoldlsi,escoannsidstaernetpwriotbhabblryonPcNh2iso.larTheepisthmealliluemr. PdZaNLrISksT.l0i:ningAlcveellolcuosntwaiitnhsLfionuirnlgacmeellllsaarnbdoduinedseralnydinigs caoPlNl2ap.seTdhaelvoetohlearrtsewpota,linTinhgeceslmlasllare sfZlqLauSteTtee7zn1ee;diAnPvbNeeats(weleoeewnmesrtwrtiooghcbtue)b.oaiCtdeeanrltmieenpriattlhoebplriiosanlacchPeilNolls2e. jwAuitntchtthfieiovnreiignlhgatmweailntldhaarlnebfaotldaivreeeso,claapIritlaalparrpeieaeaswr.istThthoea. right one 215772; contains a Two large lcaumbeolildaarlbeopidtyheilniailtsclelulmseonr. pneumocytesand a PN1 lining cell (bottom). Some dilated cristae in mitochondria noted. Conclusions: ~Appears normal. A few random PN2s with lamellar bodies noted. 000462 418-014:PAGE F-16 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_418-014;EM-09.46 Animal No.: __I8915: Treatment Group: 0mg/kg,Control Sex:__nonapplieable Species/Surain:ECx.CDOBRVAFRlu Tissue: ver Block No(s): _99.46.7 Photo/Negative No(s).2:1_5Z716537-67 Significant Lesions (check one): _____ Yes _X__ No Interpreting Pathologist (signanwre and date): aFebautnudraenst:gl2y1c5og7e6n3-:likHeepmaatteorciaylt.e iPnlpahsomtaogmreampbhrhanaesas laanrdgeenadroetahoeflciayltoceplllassamrefiploloerdlwyi:th adenfdindieldatdeudectroisptaoeo.rTfhiexraetioins.diMlaittioocnohofntdhreiaRaEreRdesnhsoewianngd acsonrtoauinndsbolmeebsdoennscerodsesp-osescitsion. 2A15bi7l6e4c:anHaelpicautloucsyties,prseismenitliatnrolZoIwSe7r6l3ef.t. FFiovuer(t5e)epne(r1o4x)ipseormoexsicsooumnetsedc,ounted. sZiInSuTs6oSi:ds. HEeipgahttoc(y8t)e.perPooxoirsofimxeasticoonunctleedar.ly evidenced by lossof endothelial cells lining gZrLaSn7u6le6s:, pSrioxb(a6b)lpyemroosxtilsyomgelsyccooguennt,edb.utMaulscohcoofntsihsetecntytwoiptlhapsomocronfitxaaitnison.fine dispersed EZlLe5v7e6n7;(1H1e)ppaetroocxyitsesoimmeislcaorutnotetdh.ose described above. Many dilated blebs of RER. 8C.o8n/ccelluls(ifoinvse:coSuunbteodp)t.imal fixation of tissue. Number of peroxisomes counted: 000463 418-014:PAGE F-17 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014; EM-99 46 Animal No.: 18915: `Treatment Group: 0mg/kg,Control Sex: _nonapplicsble Species/Strain:Ci CDOBRVAFPu Ra Tissue: Lug Block No(s): _ 99.468 Photo/Negative No(s)2.1.5_71558762___ Significant Lesions (check one): _____ Yes __X No Interpreting Pathologist (signatureand date): ______ eTFleweaocttucrraoepnsi-l:dlea1nr5siee7s5ciy8nt:otphleCaocslomllialcpaspmesadeardlgvaierneoacloacnrotsnaetipanitinan.gsAoalmfreeswlpaoamrcegelalnaaterlbslotertsutacotnmudriesasilfanrreegede wbdiiytlhaaitnetdhtibhnleeb. ZlleuLfmSti7an5pap9,e:LaraAsmlevtloeolclaounrstbaoaidnnideassleapwimiaet.lhlimanorspbtrloeyduycmowolilctayhpistensdt.ohre fePnrdeoNtihntelhliienaealiacrierslpslpacacyceetsso.palraCesampn.oitllSeamvraiydlelatnetrtop OZdfLar5Pk7N6nL0u;clSeAuurbsejaaiocneftnhcteoltcoleanttpiessresdappsapecepeairai,ss wtaioctabhpeaiilarnlausrcpyalcceoeunsttoaoilnefafit.nPgITa.hlearsgpeaclaemselllianrebdbodyytwhiitnhimnartghien clyutmoepnl.asmA.toTpheigchletariscelcleiln,tphreocbeanbtleyraapPpNe2a,rswhtoicbheacopnntaciunmsocaystien,gle lamellar body in is ZacyLtSto7hp6elLba:ostm.tPohmAoitcroognsrtpaaaipcnhescissonoltmianeiendpsablsyee-vfseltraaatiltnecinanepgdillPliapNridiemcsayittneorptilhaaelscmio.nltlNeaorpmsilexadmesedelpwltiaatr.hboTednhideoetslhanerlogiteaelldueimeneln cp2yn1te5ou7pm6lo2ac:symt.CeesnF.tlraaTtlheidnsaerdpkrPocNfeli1ll acipynptleouaprmlseanstommblaeiyeasbPetNhte2wbrwueilmatarhisnaufdreefrawcotflaantmtheel(laliaarrmesblpoladaciree.bsodTisnw).iots capillary llaummeilnlaa,rdmoartsearliatlo aisndinttohethairrwaiyghtolfutmheins.PN2, contain some lamellar material. A focus of la`COmotenhlcellrualsraimmoaentlsel:raiaNlliowkramssatrluprcfesetentutarlesliuwnnegar.ierOwrnaeeyePlwNiu2tmhewinna.scaipdielnltairfyiledumwiintah,saonmdeaIsaimnegllleafrobcoudsicoef. 000464 418-014:PAGE F-18 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014;EM-09.46 Animal No.: __18915:2 Treatment Group:0me/ke,Control Sex: _nonapplicable `Species/Strain:RCa.sCDBR VAF/Plus Tissue: __ Liver BlockNo(s): 9924 157563 0 Photo/Negative No(s). 215757 Significant Lesions (check one): Yes _X No Interpreting Pathologist (signature and date): em Fdielaattuartieosn:s nZo1t5e7d5i3n;nucHleepaartcoacvyetleopwei.thAnbuumnedraonutsgdrialnualeadrbgllycooegfebRnEisnRcyprteospelnats.m.Some (M9i)topcehroonxdirsioameasppceoaunrtdeedn.se with some granular densities and some dilated cristae. Nine 2ee1l5s75a4re; pHoeorplaytodceyftienesdurdruoeutnodepdoobryfeirxyattihorno.cytHeespaatnodcyhteemactoonptoaiientsimcacenlylsd.iElnadtoetdhseelgiamlents 2a1n5d7b5l5e:bsoLafrRgeERcenatnrdalnhuecplaetaorceyatveelwoipteh. Two (2) peroxisomes counted. Five (5) abundant gpleyrcooxgeins.omeBslecbobuintnegdo.fRER abundant. 2csw1oa5mi7pn5li6neg:t.e FcSeiolelm.ldeCwdeiitllhastsehedavscerrniausltmaheeerpnoaouttosecdydtiilenastm,eidhtoobwcieheovbnesodrr,fiaoR.nElRyF,oounsreo(ma4te)tdpoaeprrckoexnaitnsedormsweoismthceonluuincgthletedu.s is a The cell counting, at lower right appears to have more peroxisomes, but is an incomplete cell for 2hTa1ws5e7ld5ve7en:s(e1S2me)ivtpeorecarhlooxhniedsproaimtaoecswyitcteohsu,ngtraealdntu.hloaurgdhecnseinttieerscaelnldwniutmhenruoculseudsiliastoendlbylceobsmpolfeRteEcRell, It f`iCxoanticolnu.sions: Average number of peroxisomes = 6.4 (5 cells counted). Suboptimal 000465 418-014:PAGE F-19 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No:418-014:EM-9946 Animal No: __18915:2 Treatment Gr_o0mg/ukg,pcont:rol Sex: __non applicable `Species/Strain:2CUaLCDOBR VAERI Tissue: __Lung Block No(s).: _99.46:10 Photo/Negative No(s).2:1_5Z71547852- Significant Lesions (check one): Yes _X No Interpreting Pathologist (signature and date): sFeepattuumr,eas:ndZt1h5e7r4e8:is aTlwaorgealcveelolliatwtiatchheidntteortvheenirnigghst.epTthuims and cell chaapsilmliarnyi.maPlINcTyStolpinleastmhiec coarpgialnlealrlyesl,ummeing.htbubteias lnyomtpphroecsyetnet.in Sproemuemolcamyetlelsarinmattheirsipahloitsogprreaspehn.t within the ls2ia1nm5ge0lle4l9alr:ambeoAldllivaeersoblowuisdt,yhlciiynnittnosgpccleyaltslsom,polafanpsdrmcojobulaslbtalbpyseenPdeNa2stseph.tat.he Lniucnlienugs.pnAetumcoecntyrtael ocopntaariensseaveral ZL5750; bodies. Cuboidal epithelium of probably bronchiole. Centrally is a PN2 with lamellar ltZaLh5me7e5ll1ul:amrenbAoodlfitveehso.elucsapailnldarlyi.niAngtPtMopIle.ft,Aitnlocowlelraprsiegdhstesptaalcaaprielal,ariys awiPtMha2lawmietlhlsaervebroadly in ciZsaLpSai1lsl5ia2nrg;ileesAcaletvlleceownlituteshroatnhnrdleeefltovwwaeicrtuhorlilegihsnti.wnhgipcoheuapmpoecayrtteoshasvfereleaomfelllaamrelsltarrucbtoudrieess.in tAhetmo.pTrwiog.ht cCaopniclllaursyiolnusm:inaS.everal PN2 and lamellar bodies identified. Some lamellar bodies were In 000466 418-014:PAGE F-20 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_418-014; EM-99.46 Animal No.: 18952: `Treatment Group: 1.6mg/kg.high-dose Sex: _nonapplicable Species/Strain: aCUCDOBRVAFPus Tissue: _ Liver Block No(s).: _994Z16572-2-15 Photo/Negative No(s).;_Z15726 Significant Lesions (check one): __X Yes _____ No Interpreting Pathologist (signature and date Features; glycogen. SZo15m7e22di:lateHdepbalteobcsoytfeR.ECRy.toMpiltaoscmhocnondtraiiansapapbeuanrdadenntsfeinweitghragnrualnaurlamartdeerpiaols,its iasnpdosorolmye-ddeiflianteedd (crpiostoare.fixTawtieonn)t.y (20) peroxisomes identified. Adjacent endothelial cel pZrLe5s7e2n3t;inSbiomtitlaormto0f2p1h5o7t2o2g.rapNhu.meTreonu(s10r)oupnedrodxiilsatoemdesblceobusntoefd,RER. Bile canaliculus pg2ro1ao5nr7ul2ly4ofc:iyxteSidic.nuceTslowliodincitononcptoamliepfntl.ientgEensdheovetephraeatlloiceayrltyetcsherlooncayrntiedgsh.lt.ipSiAed-gcfmoeenwatatpieonrifonhgxeipmseaortmiosepisoniuaesroteiindcoatlecde,llbaatre 201 counted. NZuLSmTe2r5o:usHdeiplaattoecdybtlee.bsBoifleRcEaRn.aliFciufliuesenat(1t5op);pseirnouxsiosiodmienslcoowuenrterdi.ght of photograph. d2i1l5a7t2ed6:bleHbsepoaftoRcEyRt.e. NBiinleetceaennal(i1c9u)lipeartotxoipsroimgehst aconudntleedt.. Numerous, variably-sized (`4Cocneclllsuscioounnst:ed)I.ncrSeuabsoepdtinmuamlbefrisxoatfiopne.roxisomes. Average 16 peroxisomes/hepaiociye 000467 418-014:PAGE F-21 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014;EM-99.46 AnimalNo.:_18952.1 Treatment Group: 1.6 mg/kg,high-dose Sex: __non applicable. Species/Strain:ECa.CDVOAFPBluRs Tissue: Lug Block No(s): __992.1457617-16 Photo/Negative Nofs).:_Z1572 Significant Lesions (checkone): __X Yes _____ No Interpreting Pathologist (signature and dare); hsFiaenvautesuoariedPs.N:2Se1bv5ee7rt1aw7le.elanAmltevhleelomal.rubsHodowiwieteshveairrne,inpagrePcsNeel1nltsm.aasTrsgwoicoinalstateirdlglewsiPetpNhaIrtsahteaersPteNhp2er,ePsaeNnnd?t safonrmdoemapoifpeetahresm 2ipipseacryttobpelaesxmt,raccoenlsliusltaer.ntTwhiethPglNycoognent.he left contains abundant ine granular materia] ZtnhoLrr7em1ea8(l:3p)uiClnomolonlneaa,prsayenddarascrheeivateeoncfta(ul7r)veeioinslutanhrcesleoetpahterarb..ecTBawouoshecocefenlttlrshaelacpceolpllsleatcpooasnbetrdeaisPntaNtl2easm,elallatrhbooudgihes, mtZhuLelSt7cie1pn9lte:ri(oT)lwesloaamcreeellllasasrswoibcotidhaiteaesbd.uwnTidhtaihnstciccleielanl.traCipeopnleetasrraaslpl0pyeb aarreattoPe wrNoep.cruebsAoeiadtlaablrrgceoenlllcash.mieolllOaanrrecbeclolodsny,taaiisnnds `agsrsaoncuilaatremdatweirtihalt,hecolnefstiscteelln,tbwuitthitmglayycbogeene.xtracellular. Both cells contain abundant cZ2iI1g5Sh7Tt22i0Ln; thAAellovvteehooellru.uss,Safonimvdeellilinnaiimnnegglclecalerllssm.,atTaenrwdioaalccaeaplplipslelcaoarnrstytaoiinnbteloapemxertlirlgahacterloblfoudlpaihreo.st,ogsrixapihn.onTehiasndis lpaomseslilbalry baojduinecst,iaonndwiatphpeatretmoibnealPNb2rson.chiole. Two ofthe pacumocytes contain multiple ``SCoonmceleuxstiroancse:lluTlhaerrleamaeplpleaarrmtaotebreiailncnroetaesde.d numbers of PNZs containing lamellar Bodies 000468 418-014:PAGE F-22 TRANSMISSION ELECTRON MICROGRAPHINTERPRETATION Study No.:_418:014; EM-99 46 AnimalNo. 189522 Treatment Group: 1.6ma/ks.high-dose: Sex: _nonapplicable Species/Strain:BCaHLCVDABRPRSRD Tissue Live Block No(s): 9__9426157-121- 7 Photo/Negative No(s).._Z157_16 Significant Lesions (check one): __X Yes. -- No Interpreting Pathologist (signanure and date); faFiennadetbguorratenstu:olmaZroIfmSap7the1ort2ioaglraHcpeohpn.saitsoTtcehynetteh.weiptPahotogorlclyyyct-oedgecefyni.tooeMpdiltasiosncmuhsaoopnipddersairaasadavpoeplneudamoritdnheoenulssieuwmaint3dh hoaabpvuen.Fdigahntt pseormoexigsroanmuelsariddeenptiofsiietds. and dilated cristae. Some dilated biebs of RER. Eight (8) nfZiIugShmt7b.1e3rA;sbuofnHdeppaeanrtotoxcfiyisntoeem.gersaH.neumTlawateronpmtaoytie-erstieiavcle,cnegll(ly2tc7oo)rgpiegenhr,to.ixniBcsiyoltmeoecpsalnpaarstemis.ceuAnltpupseparresteonbteinmleoawsered cZyLt5o7p1l4as:m.HeFpoautrotcyeteen.(1M4)apneyrodxiliastoemdebsieibdseontfifRiEedRinatnhdisacbeulln.dant glycogen particles in ZLIS715: Hepatocyte. Many dilated blebsof RER and abundant glycogen particles in pcyetroopxliassomm.esMiidteoncthiofineddriina twhiitshcesllo,me granular deposits and dilated cristae. Nineteen (19) hO`2he1fep5Rpa7aEt1tRo6oc:cyyattreeSessipnacruoeesnsotenianodtit.nwiimntMaphunhlloytitinompigilnrtegaoppechnhe,doronotdxhrieislaio,amleRscEelaRlr.eanpCdre.elsglelunyltca,rogbfeuintx-anltoiitkoencopisaurnpttoioecrld.esb.eAcdDajiualscaeetnewdthoblleebs LCo7nmcelpuastioocnyst:e fixation. H(e4pcaetlloscyctouensteadp)p.earCetlolscocnotnatianininacbruenadsaedn tnuglmybceorgse no.f pSeurbooxpitsiommaels,c ealvleurlaege 000469 418-014:PAGE F-23 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014; EM-09.46 AnimalNo.: 18952: `Treatment Group:16mg/kg,high-dose: Sex: __non applicable. Species/Strain:CBaCtDOBRVAFPIu Tissue: Lung Block No(s): 99426157-071- 8 Photo/Negative Nofs).:_ZL57! Sigaificant Lesions (check one): __X Yes ____ No Interpreting Pathologist (signature and date): em nFuecalteuurse,sa:nd1a5l7s0o1m;ulStiinpglleecPleNaZr vlaicniunogleasl,vewohliusc.h aCreellmocsonttlayiensmpmtuyliopflceonmtietntosc.honTdhreisaeawreoruend the bpryoabsaibnlgylelammeelmlbarrabnoed,iewshewrheiacshtwheermeietxotcrhuodneddriianthoatvhee3aldvoeuobllaer mspeamcber.anThee.se vacuoles are lined e1n5d7o0t8h:eliCaolllcealplsewditahlvmeaonlayrpsienpioac.ytAottchivnleasyiecrleodePns,Nasicnogvleresltamhelsluarrfabcoed.y.BeCneenattrhalilsyainsan area oifn ftlheaiuepnpeedrbraigshetm,ewnhticmhemcbonrtaaniensmmataenryialmictooncthaoinndirniga.maTnoytchoelleafgteonffitbhriislcs.elAardearktwcoellaimseplrleasrent ZboLdiSe7s,0a9Al:thonuucglhetahteeyd aPpNp2eacrotnotabieniinngamcuylttoippllaeslmaimcelelxatrenbsoidoinesof(a5)naupnpiedaernstifibede coevlle,rlain by a PN lining the alveolus. cZoUvSe7r1e0d:byAliv'esolcayrtospplaacseimnistaonp erxitgrhtaciesllluilnaerd abreaatywhiitnhPseNvercaelll.(3)Adlajmaeclelnatrtboodtiheiss.PNA1caapnidllary is cdloerasrallycidoentnifitablaaeniaesrntyothicrelonlcytgytpee.and endothelial cel. Several cels within collapsedsepaare not `ZeImSpTrLyLv:acAuolseisn,glwehdiacrhk hPaNv2e aisppsaqrueenetzleydebxettruwdeeednltawmoePlNl1asb.odiItess.cyTtooplthaesmricgohnttoafintshissecveelrlaalre PwNo.lamragcerloapmhealglear,boordimeasywibtehienxtrsaocmelelullapri.d-iAkesmmaaltelrciaalp.illTahryesies ibnotdhieesowmaeyr rbieghitn mthaergaidnjaocfentthe photograph. ``mCoorneclluasmieolnlsa:r bTohdeireeswaeprpeesacteor bienitnhcisresaasmepdlne.umbOtehresrowfitsyep,eceIllluplraerummoorcpyhioelso(gPyN2asp)p,eaarnedd essentially normal. Fixation appeared satisfactory. 000470 418-014:PAGE F-24 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No: 818.014: EM-99.46 AnimalNo: 189561 Treatment Group:_L6mg/kg.high-dose. Sex: anponplicable Species/Strain: C:CDVRAFBPluRs Rat Tissue: __ Liver Block No(s): __99.46:13 215732 Photo/NegativeNo(s). Z1S736 Significant Lesions (checkone): _X Yes _____ No Interpreting Pathologist (signature and date): mFeaattuurreeesr:ytZhrIoSc7y5t2es;. HSeopamteiclasmienlulsaoriddecbornitsaiisnipnrgesneunctlieattehdeesriynutshoriody.iTewaonden3dCotohuepllieal cell 2nu1c5l7e3i3a:re Hpreepsaetnot.cytTehecroenatraiennsoabcuonmdpalnettegrhaenpualtaorcypatretsicilneshicsonpshiostteongtrawpiht.h glycogen, and annudmedriloaucsedRcEriRstapea.lisEaldeevse.nM(i1t1o)cpheornodxriisaoampepsecaorudnetneds.eAandbihlaevceansaolmiecuglursaniuslparredseepnotsits ZbIeStw7e3e4n: twHoephaetpoactyotcey.teSsi.milar to Z15733. Numerous dilated blebs ofRER are present. 2Tw1e5n7t3y5-:foHuerpa(t24o)cypteerowxiitshomseimsi.lar peroxisomes counted. morphology as described for above cells. Sixteen (16) Z15736; Hepatocyte. DilatedblebsofRERpresent. Mitochondrairae dense with granular deposits and dilated cristae. Eighteen (18) peroxisomes. pCeornchleupsaitooncsyt:e (Inacvreeraasgeedonfufmobuerrcsouonftpeedrcoexlilss)o.meSsubpoeprthiempaaltofciyxtatei.on.17.25 peroxisomes 000471 418-014:PAGE F-25 `TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014; EM-09.46 AnimalNo.: _18956-1 Treatment Group: _1.6mg/kg,high-dose Sex: __nonapplicshle Species/Strain: CHL.CVDAFORRusRe Ru Tissue: Lung Block Nos): __99Z.1457627-14 Photo/Negative Nos): 21573 Significant Lesions (check one): _X_ Yes ___ No Interpreting Pathologist signature and date): FareeatPuNr2esw:ith15l7a27m;ellAalrvebooldiuess.witShoamdejoafctenhtebcoodliaepssedareseppatrat.iaTlhlyeetmwporby,oaondminpoRneewcemlol ceys alaprpgeeaPrN1L0b.eAetxtthreudliefntgimsaatecraipaillleaxrtyrawcietlhlualanrulcylewaittehdoetrhyetrhrloicpyitdem.aterial. At the top is a 2la1m5e7l2l8ar: bCoodlylaappspeedaraslvteooblearwsietphtian. tSheevleurmalencoefllas ccaopnitlalianryl.amellar bodies. Atleftone e2n1d5o7t2he9l:iaSlocmelelwnhuactleTu-ssihsavpeendtrcaalpitlolatrhye ceorynttharionciyntge.anWeiltehcitnrotnh-edelnusmeeenryotfhrtohceyctaep.ilTlahrey oatrheesrecveelrlaslalpapmeealrltaobboediseesc.tiAontsltefhtroofugfhieplndeiusmtohecytthienscayttvoaprliaosumsicanlgilneisn.g of a BNL. The s2m1a5l7l30l:amelAllavreoboldairelsunmeexntatnoditlsinnuicnlgecuesl.ls.ThCeendtarrakl-lsyitainisnmgaclyltoPpNl2ascmonttoatihneinrgigahtcooufptlheis cpenleluampopceyatress,toabnedantheeyxtceonnstiaoinnfarcoomupalneallavmeeolllaarrmbaocdrioeps.hage. Totheleftare a couple bZoUdSiTes3.1:A rAilgvhetocleanrtrlalu,mepanlea-nsdualiinniinngg,cpenllesu.mTocoyplteefatclseollcoinstaaiPnNs2sewvietrhaltswmoalllamlealmlealrlar bodies. The septa are collapsed inthisarea. l`aCmoenlcllaursiboondsi:esInnoctreedasiendcnapuimlblaerrieos.f pacumocytes containing lamellar bodies. Some 000472 418-014:PAGE F-26 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.:_418-014;EM-0946 Animal No. _18973.1 Treatment Group:_.6mek,high-dose. Sex: _nonapplicable Species/Strain:ECiuLCDBR VAFPI Tissue: L __ iver Block No(s): __9946:11 Photo/Negative No(s).2.1_5Z71547247 Significant Lesions (check one): __X__ Yes ____ No Interpreting Pathologist (signawre and date): gFleyactourgeesn:iZntIh5e7c4y2t:oplHaespma.tocTyhteerecoanrteainnusmearbuonudsadnitlaftiende bglreabnsuloafrRmaEtReroianlccroonsssissteecnttiownith pMeirtooxcihsoonmdersiacoaupnpteeadr.deSnosmeeanldamhealvlearsmoamteerigarlannuoltaerddeepxotsriatcse.lluSlearvleyntaenednp(o1s7s)ibly within ZclLe5a7r4s3p:aceHsepiantothceytlee,ft opvoerrtailolnosifmtihlaer hepatocyte. to 215742, although fixation appears better. Nine 2(19)57p4e4r;oxiHseopmaetsoccyoutnet.edA.tlLeafmteisllaarhemmaatteorpioalieattitcocpealppienasrisnutsoobied eaxntdraactelulpuplearr right Naipnpeeatresen0(1b9e)apdeirloaxtiesdobmielesccaonuanltiecdu.lus. Abundant area of glycogen in cytoplasm. C2e1n5t7r4a5ll:y Sisinaucsloeiadr,ceyntdoopthlealsimailccaerlelaawnidtihnmcaomnpyledtielaptoerdtbiloenbssooaff RcEoRu.pleAhfepeawtocytes. Zp1e5r4o6x:isomHeespaatroecyprteesecnotn.taNinospaebruonxdiasnotmfeinceogurnatnmualadre.material consistent with glycogen in `tahpepceyartodpelnassem.anTdhhearveeasreomneugmrearnouulsarddileaptoesditbsleabnsodfsRomEeRdiolnatcerdoscsrissetcaet.ionE.leMvietnoc(h11o)ndria 2pe1r5o7x4i7s;omNeos cpohuonttoegdr.aph. evaluated. its not necessary Negative was to retake or too ry dark. With to priat this five photographs imag already c`eClolnscclouusnitoends.: ASupbpoepatrimtoalbefiixnactrioena.sed peroxisomes per hepatocyte. Average 147cell (four 000473 418-014:PAGE F-27 "TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 418-014;EM-0946 Animal No: _18973:1 Treatment Group:L6 ma/ke.high-dose: Sexi _nonspplicable Ea Species/Strain: C.CDBR VAF/Plus Tissoe: _Lung Block No(s): __99.46-12 Phow/Negative No(s):2Z115S731741 Significant Lesions (check one): __X Yes ______ No Interpreting Pathologist (signaantdduer)e: Features: Z15737; Collapsed alveolar septa. Centrally is a PNZ with approximately eecxiotgnrhtatacilenallmsueollmalrearslpbaaomcdeeil.leasr,Vbeonoftdrwayhlilvcyahcaursoeelvaeelsrs.aolAsaepgcpmoeeuaaprlttesoPobfNe1arscellaerlae'sipncrgeystteohnpetilraacsdojmnattcheeanntttsaopitpnehtaoertsPhNeto2, pahnodtoacgarpaiplhl.ary with erythrocyte and endothelial cell ispresentin lower left central area of cZyItSoTp3l8a:smAllivneeosltuhsewaiirthsplaicnei.ng Bceelnlcs.atThhtehilsacreglelnauncdlejuustbteoltohnegsri0ghat aPrNe1sewvehroaslelamellar beoydhiress,cyatlethtohuagthdioe'sicnoantsauibnjascoemnet claapmiellllaarry.deTboritsheinmithdedlleumleenf. i a capillary with an Z10Sb7e39wi:thiAnlvtehoelluusmaenndocfolalcaappisleldarsye.ptaP. NSi1millianer tthoeZa1lSv7e3o8la,rssopmacee.lamellar bodies appear 2ex1t5e7a4s0i:on, LparrogbeabclaypiallPaNry2,witthhatscevoenrtaalinesrydtihsrtoicnyctetsltamoetlhlearribgohtd.y.LeAft toiftnhliasyiesraofcePlNluIlar lZiInSe7s4t1h:e aAllvveeoloalrarspsapcaec.e contains abundant lamellar material. Several PN1s are present lluomnegntahnedliinninsgo,mbeutinntoersPtNit2ialarspeacespresenitnt.hLearmieglhltasridbeoodfietshearephporteosgeranpihn.one capilzy sCaomnpclleu,siaolntsh:ougAhppmeaanrytwoebreeiinncrceapaisleldalryamleulmliarnaboadnidessoinmethientailsvseuoelcaormapnadriendtewrsittihticalontrol spaces. 000474 418-014:PAGE F-28 IL Transmission Electron Micrographs 000475 418-014:PAGE F-29 NOTE: D`rTh.e oMrairgivnianlTg.lCosassyefwiigturhethpheodtroagfrtappahtsho(lFoiggyurreespo1r-t12on)Mwaerrechdel1i9v,e1r9e9d9t,o 000476 418-014:PAGE F-30 : PrimedicaAnAcriglluasrySePuadbyoNloogy41R5e.p0o1r4t Figure Legends mEiigtuorceho1.ndrLiivaerr.eprHeespenattoscoymtee sdihloawtaitnigononofrcmraisltcaey.topSloamsmeicpeorrogxanieslolmeess. aWrehiitdeentsipfoitesd.wiTthoin t1h.e0rmiggh/tkgis/adnaeynd(cootnhterloil)a,l 1ce1l,l2a0n0dXemraygtnhirfoiccyatteisoon.fadjacent sinusoid. Animal No. 18904. Eriegtuirceu2lu.m (Libvleerb.binHge)paitnodciyctatee.s sDiulbaotpattiimoanloffinxuactiloena.r Menivteolcohpoenadnridaraoruegdhenesnedoapnldassmhiocw Hseommaetogproaineultaircdceeplolssiatrseapnrdesdeinltatiendscirniusstaoei.d aStoumpeperreprriegshtenotfatpihvoetpoegrraopxhi.soAmneismaarle Nlaob.eled. 18912-1, 0 mg/kg/day (contro), 11,200X magnification. Eilgabuerleed3,.anLdivaeprp.eaCrelmlourlaernmuomreprhooulsogtyhains isnimciolnatrrotlos.FiAgurbeil2e.caSnoalmiecupleursoixsipsroemseesntarien `uApnpiemralriNghot.o1f8p9h5o2t2og,r1a.p6hmagn/dkgh/edmaatyo(phoiigeht-idcoscee)l,lsa1r1e,p2r0e0sXemnatgniinfsiicnautisooni.d at upper left. pEiegruorxeis4o.meLsi,veri.ncrDeialsaetdatiinonnuomfbreoru,gahreenldaobpelleads.miAcniremtailcNuolu.m8i95ev6i-d1e,nt1..6 mSgo/mkeg/day (high-dose), 11.200X magnification. . dEiisgpuerresi5.on Loifveorr.ganHeelpleast.ocyCtleeadremsopnascteraattitonpg osfocmeelw,hwahtbiecthecrofnitxaiantsiosnowmiethmemmobrreaenvoeuns mdegb/rkigs,/daapype(ahrisght-odobseee)x,tr1a1c,e2ll0u0lXarmiangntihfeicpeartiisoinn.usoidal space. Animal No. 8973-1, 1.6 pEingeuurmeoc6.yteL.ungL.amTelylpaer I pneumocyte bodies within tihsepcreysteonptlaaslmonigdetnhieifaylvtehoilsacrewllaslla,s adjacent a type II to type I preumocyte. Animal No. 18904-1, 0 mg/kg/day (contro), 15,680X magnification. pEingeuurmeo7.cyLeunigs.evTihdreentecaulboongidtahelvpenneturmalocpyotrteisolnionfetthhies aaiirrwwaayyssppeaccee.. TAwoflaswmeanlledlatmyeplelIar obfodtiseslaimneollnaercbuobdoiiedsalancdellissduegsgteisntedthtios biseacotympeeaIItyppneeuI mpoaceyutmeocwyhtiec.h AhnasimsaelcrNetoe.d 1m8o9s0t42.0 mg/kg/day (control), 11,200X magnification. bEoidgiuerse,8.appLeuanrgs.sqAuemeozreed dbeetnwseeelnyt-wsotatiytnpyiIepnegpInelupmnoceyutmeosc.yteS,ocmoentlaaimneilnlgartwmoatlearmieallliasr talhseotpyrpeeIseTnptnweiutmhoicnytthee.caApnililmaarly lNuo.men1o8f91t5h-e1,s0epmtagl/kwagl/ldabyot(hcoanbtroovle),an1d1,t2o0t0hXe right of magnification, 000477 418-014:PAGE F-31 PrimedicaAAncriglulasryStPuadtyhoNloo.gy18Re.p0o1r4t IFipgnueruemo9.cytLeunsg..FoDuenrsdei-ssttianicntilaagmeclelllaribaodtiyepse aIrIeppnreeusmenotcyitntehpeosciyttioopnleadsbmoefttweheen ttywpoetIyTpe pthreeurmoicyogtfeht,hitasltchelolugahretthewoladmaerlklalrammaetlelraira-ltyipsepbaordtialelsyswuarrsohuenddeodutbiynstohemsee lviapcidu.olTehs.isTo aApnpiemaarls Ntoo.be18w9i5t2h-i2n,ac1.6elmlg/inkag/codlalyap(sheidgha-rdeoasoef),se1p1ta,.20a0nXd mmaagynifbiecaatmioanc.rophage. Fliagmeulrlear1b0.odLiuesn.g.ACotylpleapIspendaeruemaoocfytseepistaoshthoewilenfgt otftwhoetsyepeceIlflsp.aeCuampoilclyatreiesscaonntdaaidnjiancgent alveolar cells are 1, 1.6 mg/kg/day (phriegshe-adtodsoer)s,al1l1y.a2n0d0Xtomatghneifriigchattoifotn.he photograph. Animal No. 18952- Eiagppueraer|s . bLeunagt.ypCeeInTtrpaulcluymoiscyacteel.lcGolnytcaoigneian-gliskeevemraatlerliaamellalparp-elairksetsotrfuilcltmuruecshwohfitchhe cytoplasm. bronchiolar Adjacent cells to the right contain cells. Cuboidal epithelialcellsare centrioles to the left and represent andventralto ciliated the type II preumocyte. Animal No. 18952-1, 1.6 mg/kg/day (high-dose), 11,200X magnification. cFoingsuirseten12t. wLiutnhgs.urfAalcvteaonltasrecsrpeatceedcfornotamitnyspaebIuInpdnaenutmolcamyetlelsa.r-lAiketympaeteIripanle,uwmhoicchytislines tmhaetesrpiaacleapopnetahretloefbteapnnderuimgohtc.ytTews,opcruobboaibdlayl tcyeplelsIclos nwthaiicnihnhgagvleycroegleena-sleidktehegirranlaumlealrlar bodies. Animal No. 18973-1, 1.6 mg/kg/day (high-dose), 11.200X magnification. 000478 Abbreviation A BC BE ca Cap oN cp DDD pc DN DR EC ER G HC L LB NmM P PNL PN2 PM RER 418-014:PAGE F-32 PrimedicsAAncriglulasrySuPadtyhoNloo.gy41R5e.p0o14r IndexofLabels for Ultrastructural Structures Steueture BAillveeoclaanralsipcaucleus CBarpoinlclhairoylalrumeepinthelium CCeapnitlrloalreys CDuebsomiodaslompereumocyte DDielnasteeddcerpiosstiates DDiillaatteedd nRuEcRlear envelope EEnrdyotthhreolciyatle cell Giycogen LHiepmiadtodproopileettic cells MLiatmoeclhloanrdbroiday Nucleus TPyerpoexIisponmeeamocyte TPlyapsemIa mpercmubmroacnyete Rough endoplasmic reticulum 000479 SESTUOPY AvAABE mh RE * par at TbfSTE Nw CviToY lRiEnE. Ayx GeAtE cSn PL wcy onf ) Se No ST 2 Fa Slit: SBA UXT hos Pl U< RIu BEE Se RE c, OTM R E = EE a CSR CCCOEe ee DENIAL LR 3 T&h aTtn e ihd : 3 Be NL doc Ra GE eT w tie rE ANEEET apo = C"omef Tg aRTT s REar= 2 Lo grt CL ~BET eR fat7 ` - 2 ul ; y : Hs ) 0O04a80 BESTCOPY AVAILABLE A 22 ETRA Nye %. La S18.016PorAeGE F34 A aE SE R W TERY a BDRRTThtAargpeW R MmST 2i Sh RE Shon < ba figs Cs ag eB Se gh 3 = Ek GE Sou). so a oo v5 FSR,O hAeR S afme rTya TS liN e lyfo 3: ' : 3: rS E ieW e cVCA. a iA IAC2 AaNGEpoSELeahsoo ~ 41 BORN ER SEEOLES Ci OTRaYA RET owi R Rly ad es 4 % OFFane5 aLnCRNLRe MR eRtAyag TAe R E FE T eid foeesa.e T gheEttNo SBaEie, Nad en JOR aie a Fe. o 1 RT LL re RDEENEG F FE5IANP Harty SPN CL eR Md Sa 600481 BESTCOPY AVAILABLE 418-014:PAGE F-35 ro - Aig Rit , ETT WR Ce aN 8 - 2 9 a Fe TN i bo ars E R Rne e RE RR KA CTE AACR A FT acy 5YEARR C SEga HF Ad An oy e-S ow ae Ta ake Xo Ne k(g&. c TE eR adfA 2 CEre SE a Br - BpeN. A:}3 WNWP RBAIL o on T Tl RL Aey ji iA ge CRs EAt N gy rR NS Pin La TEeo YoTEN o A 4 A BY" |OCAa RE NR RPE ye S Ra Lag. ON ERR . NE SS CN Bh oh p.- Cm BEw ax = A A E27y ERT => S0 RoEp VEB REER A 3 : < y . CE EpPce a SeBd RT hNEeELl TER 0, a%e hg Po oye oe,ie TE : HE RN AR NEaO, me iY ) igs PR GRE af a . Ca EE]5 :, Pa , A 5 Lm 000482 BEST COPY AVAILABLE A1BO1EPAGE 30 | BB Re | ua Sa TO. Era |B aaron eA iiS.saSEN iE 4 25 wy |op ie Ba Ea 5 BN add a L.-T= a SE SASia T el" Jesaige , TUS RE Pea% EvesoR BOR ORE BR 5 oa vEl Y SdTE aE b= i a gor % pyEe) Tiaar FraHde . GbRaEss EE AE ) " . 33 PE EREERE: tisea oF Be Taailna Sr.RAEe =ebyk oF =x 2 NE RSBOehE a T y is i BRE pea) 5 000483 REST COPY AVAILABLE C PEMOwsT so ae 418016A0E 37 mY pps $7FaR neEtITRS ey BREiE . ely pr or Re VR eBEl el 3 ages Carpe ae 25 Ta BE Sp E A ok TER BL a sioeal 4 Se la A oh es FD RR Eek eRle NEeoyn Bh. 00S DG Be lG A euotea WORE NAS g SaR FoWeESSaTnas El Seley ol Se 2 oats: B(uEEkR Tai FREI BE = SL o A fog celeTlhasl Sir Ean Stade 8 Nay he a a] 2 > I, 2 CUR, i SRE E a Gae pec Eda iN gi A Sad ; ld fo RSba Rr SE COge 3 + RGEhia IRR aeR agh,T SSEET pias ELSTEM C2 r . B SEoAmE TE R Pa AA veS sER oe sTAiSsai4 e 0 OTCaNi s 000484 Vi eo BESTCOPY avaiLape BO AMEE . BiTFr h.ASIJE EoE plcaPn, GE. o 418.014 PAGE F-38 CCTAL SAE oi oy SE HR E Lik Ehicaed elias o df R i 2 I g |||T( EOS Eo l Es fBay a l sI MoaO T l s / "iA L o Lrg aya Lp No [I evy mS a I a e 3 a R Ld TC RC oo 8a B Eo o ) pf o 1 Are = Na 74 rt Chi FoRkX' ql GE SL ER OE Ea "2 Parl pa EA 0o0ass ESTCOPY AVAILABLE HovePagE 28 : Fay AE E R Lag Gl FRE $5 ei sh $k SRE x hdT heBn Ey FOR LE Ent XR PY oh EARS S50 PR gs Fa rh ES) 23 PTO TAREae Bet PT YE EASAT GER SSIN NGSE Le eee Ooh eg Beywoeka Sl hoo LEEPER Sa 5 a 8 3 ga LCIR S CEATLRs x SS E TNg T Bi A E W R fs C DBERAT Es, 900 SH DRA Le. EeEohN TEER on J-- 2) GO04AKE6 BEST COPY AVAILABLE seorerace rao iesy NEL OE CALE | BleyFi SN Eh SL ON TRE Ay Gh TA Se an I yr. . Bt) sg * RAENA 4 R RRsCCTNRA lr | TL i ' Mi I AAEERN ES,ErAeN ok HE Sd Seay sd Snail "a CES BeS an R Fe T=e P ee Lon EE SRR wh ie Ee A J BEST TN SEW 0004ARY BESTCOPY AvanLapy 418014.PAGE Ft1 e SaddANtE a GApA oe A vi Os Hy i @5 AN BE Nw DRT 1 I i, ON vs lS . BE ore RR ; 2a 4 Cong, ava CSBERaSnRIA N R Ng RFer Cs Ne ELE LCEa R STARET N Yi FI hax] Eo SEI L Fak EY 04) ogi > EFrodsh mTia MY eS oe: I> He RE a Ar oo? aEEE | FRR33 Ir S ARRREgSo ea1d] 3d Gig Oy EE Ra a SO {aid F N x2 MEMl ESr Lg LOcov A ERE 5: 8 aINS Ri 08 frit t EL SE te LO aeNH F Ck T lH TTE A IRL R Y alee N Cn TIT hr OeNE EB HL S00 NR Aotode RR ReeREGEooany BEST COPY AVAILABLE #18014 PAGE Fz EA E SuS , bse N Eg fiBi NEaALSi gS enOsIA=W R , E + g r EiE sEhd S e C 3forAiRESEi-E GENER NE PIN A PELE Sp ARE Se E 2E ' - a Er eSEa Sah.Bs & ENACT SR Bo SS ty SSEma NE Ne eB s ae s Frgee FNplage i Or ir SES Fr: onB pn YE. Bk Gi Bs 5s ees Nn {Xi e fs A Eranpe.y at, hE Ae a: gu Ea EROR ) Mea > A REA A 4, rsPIbPR bEERTa N EAi B e ER r ANRsRTe SSeS AF BS TSA ELFa Goa r A) =ASA EE T8FN F ApR L a EE ae a EitLaty [7 oP a aa RR SR TinCR Sh ami ResaL SEa e PEL Ll IN RN REE ra0lR A S ieTA 0 VRP Re A iC TaRE p Y L G00489 BEST COPYvg gy FE * Ry RE ke ent i] WY= Ler aa ye5 =Ys ppExt4 8 ERA | 8 oJza "a g he t Fiat Waki. Ne, B RST E R Ea Eolen S = Pa en a Sb ET a EEy E Te a> 58 3 a, C Ee o cul T ah A es R EEoO bin ee aA SE 2 Ran fsat cB n be e oa SIRE Wel pa8s La unR ge ' SER ena or pe E20 Ehren Be REA Se et 000499 BEST COPY AVAILABLE ers pat Tm STL UE EL TWEE TL E ficeTaEeRR WaganEO GE Lh a 4 5 a CRAJee, ESAe ESSV Q TSR 9 SAv or RS ES ASREE ie Bre alE SNE SaN gr. 3 CREe S4EE gESdaa } a iJF ak or NS Lg Eo RT of i & hd iE aa MB ig 2 d Barn 2$4d Pi 2} DETREI POA LE oo 3 AC pEo YY al bo r= Sa Pidar a [POi SE e TY | CR EekeatG,Y ensBL RN Ras 5 EA EEER 5 J grPaCRtCeniBnP aSES l faa S% EG: TX SL3 id /on ES GUE a PCRg SoNE sygg 0 Pe fal LAesRBCENYA eIN SRvl E i d h8y EA i N RaSd pyhw sh ) p faig aETEJMR i k iw 2o T lh Ae S S ah 006491 418-014:PAGE F-45 IV. Quality Assurance Statement 000492 P77 418-014:PAGE F46 P4aCtohmpoalnyoogfyScAarscseAopscciataotnseseImnatcesrnCaatrisoonnal = Te r . QUALITY ASSURANCE STATEMENT QsueaultihrteaynUAT.csShs.siuUFrlnoaeionctdtcaresaonUnrndeiqmtDuiricwrurhegoidscAbchdoymrpieiynpnioessrttGtursocaytdoiidhroeanLc.stalbbPyoeArteIoantmhoiaarnsnysapaPgerfcautcneicdcieaosnnidFnageuagdinutdeodrnbesyspheemoQ.uarltey of inspections/aucils and their resuting reports: ement. Tne alosien record DaIotseeeoction PhaseInspected 1DM2aatneFaigndeinmgesnegored and StudyDirec 216,199 Processing and Embedding 2109 em Data Audit i789 imme Dratt Report Aucit 1789 sen Final Report Aucit 5199 &ty AGsoswuarradn,ceBSSp.eciRaOliAstPGLP Ge Sponsor: 3M Corporate Toxicology Test Aricte: PFOS Study Number: 418.014, PAI Stuy EM-95.46 000493 49150 Prospectus Dive + Dumam. Neri Carolina. 27713 + (919) 544-5257 (979) 544-3278 Fx APPENDIX G STATEMENT OF THE STUDY DIRECTOR 000494 PRIMEDICA 418-014:PAGE G-1 SparnobrsyBie T _-- ether PROTOCOL 418-014: ORAL (GAVAGE) CROSS-FOSTERING STUDYOF PFOS IN RATS SPONSOR'S STUDY NUMBER: T-6295.13 STATEMENT OF THE STUDYDIRECTOR This final report accurately reflects the raw of the study. No deviations from the U.S data obtained during theperformance Good Laboratory Practice Regulations; . Food and Drug Administration (FDA) Final Rule, the Japanese Ministry of SHteuadlithesaonnd DWreulgfsarean(dMHthWe)EGuoroopdeLaanboErcaotnoormyiPcraCcotimcmeunStiatnyd(aErdECf)orSCaoufnectiyl decision on `Community 28 July 1989 on the acceptance of an OECDdecision/r by the European Economic osftugdoy.od laboratory practice occurreedctohmamteanfdfeacttieodntohne qcuoamlpitlyiaonrcientweigtrihtpyrionfctihpeles tz; ot 2ea -99 nd G. York, . DABT Associate Director of Research and Study Director Date a. RUe.gSu.lFatoioodnsa;ndFinDarluRguAldem.in2i1strCaFtiRonP.artGo58o.dLaboratoryPractice b. Japanese Ministry of Health and Welfare (1988). GoodLaboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997, c. E1u9r8o9poenanthEecaocncoempitca`nCcoembmyuntihetyEu(1r9o8p9e).an CEocuonnciolmidcecCisoimomnuonnit2y8oJfualny OECDdecision/recommendation `on compliance laboratory practice. Official Journal of the Europ with principles of good Legislation. 32(No. L 315; 28 October): 1-17. ean Communities: 000495 APPENDIX H QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT 000496 r~,PRIFRIMEDICA -_-- 418-014:PAGE H-1 Am50s5 RSehseeaerhcyhDLiabvo.ratBouriteasn,gIncx. TelTeeplheofHnaoex::rs((h22a11m55,)) 4P444A33.-1889750184074 QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT Study Director: Raymond G. York, Ph.D., DABT ExecutiveDirectorof Research: Midred S. Christian, Ph.D., Fellow, ATS Protocol 418-014: OSrpaolns(oGra'vsaSgteu)dCyrNosusm-bFeors:terTi-n6g29S5t.u1d3y of PFOS in Rats aMinndisDtrryuTghofeAHddemriaanlftitshptraroanttdoicoWonell(ffFoarDrAteh)i(sGMsoHtoWud)dy;LwaGbaoosroadatuodLriaytbeoPdrraaftcoortriaycdePhrReaercgetunilccaeetiStotonsU,.SJ. apFaonoedse dSeacfiestiyonStoundi2e8s JounlyDr1u9g8s9, oanndthEeuarcocpeepatnanEcceonboymtihce CEoumrmoupenaintyE(c1o9n8o9ma)nidccoaurndciflor oCfogmomoudniltayboorfataonryOpEraCcDticdeecoinsi1o9nOirCeTco9m8m.endation on compliance with principles and rawCridtaitcaalwpehraeseausdoiftetdhitswsitcued(ysweeerteabilnesspe1catnedd nine times; 2 for dates study information and phases/data) faoudUi.tS.edTFfhooeroddarcaacfuntrdaficDnyra,lugrfeoAprdoamrditnhaiensrtdernatctheeirotnaow(pFrdDoaAtto)acoGfloorroetdhqiuLsiarsbetomuredanyttowsre,yraencdofmopr aardehderaanndce RPreagcutliacteioSntsa,nJdaarpdanfeorseSaMfientiystSrtyudofieHseaolnthDraungds,WealnfdarEeur(oHpWe)an;EPGrcoaocontdiLocamebeoratory cCEouomrmpomlpuienaainntceyEcw(oi1tn9h8o9mp)riiccnocCuinopclmeimsluodnfeicgtiosyoidoofnlaoanbnoO2ra8EtCoJruDylypdre1ac9ci8ts9iicoeonnb/etrtheewceoaemcnmceenpdtaatnicoenbaynthe 2nd 10 22 JUL JUN 99, 99, and and for revisions for finalization on requested by 23 JUL 99. the Sponsor on 21 20 MAY 99 JUL 99, 000497 418-014:PAGE H-2 9AdfmHienailstTthhriaastnisdotnuW(deFylDfwaAar)seG(coMooHndWd)uL:ca;tbeGodroaaotcdocroLyradbPiornragcatttioocreUy.RPSer.gaucFltoaiotcideonSastn,adnJDdaarpruadgnfeosreSaMfientisytry Studies on on 28 July Drugs, and 1989 on the European Economic `Community (1989) acceptance by the European Economic council decision `Commun OECD decision/recommendation on compliance with principles of good ity of an laboratory practice. QNuaanlcityypAsfsurlanece lManagerTone HeQuaatlihteyrALs.suf ranca e Supemrsvi.s"or bate and Principal Auditor 000498 TABLE 1 CRITICAL PHASES INSPECTED 418-014:PAGE H-3 Test Article Administration -Gavage Date of inspection: 03 NOV 98 0D6atNeOrVesu9l8ts reported to the Study Director andManagement: Test ArticlePreparation Date of inspection: 03 NOV 98 0D6atNeOrVesu9l8ts reported to the Study Director and Management: Cohabitation Date of inspection: 17 DEC 98 D1a8tDeErCesu9l8ts reported to the StudyDirector andManagement: Natural Delivery/Litter Observations Date of inspection: 06 JAN 99 Date results 19 JAN 99 reported to the Study Director andManagement: Blood Collection - Dams and Pups Dates of inspection: 28 JAN 99, 28 JAN 99 0D2atFeEsBre9s9u,lt0s2rFepEoBrt9e9d to the Study Director andManagement: 000499 Eecal and Urine Collection 418-014:PAGE H-4 Date of inspection: 28 JAN 99 Date results 02 FEB 99 reported to the Study Director andManagement: Scheduled Sacrifice - Dams and Pups Dates of inspection: 28 JAN 99, 28 JAN 89 Dates results reported 02 FEB 99, 02 FEB 99 to the Study Director andManagement: 000500 : TABLE 2 418-014:PAGE H-5 RAW DATA AUDIT(S) from`T2h6e FfoElBlow9i9ng10st1u0dMy AinRfo9r9m:ation and raw data were audited AInn-ilimfae ltrraencseaicptt,iornanredcoomridz.ation, physical examination and acclimation. Feed consumption. Cohabitation, Litter observations. Natural delivery observations. Pup body weights and status. Table of random units. Milk collection data and packing lists. Blood and liver collection data and packing lists. UNercirnoepasny.d fecal collection data and packing lists. TMiaslseuberpeaecdkeirngcolliostnsy. records. General comments. STteumdpyermaatiunrteenaanndcreelraetciovredsh.umidity reports. Feed, water and bedding analyses. Edit requests. Dosage volumes. Data review pages. on The results 12 MAR 99. of this audit were reported to the Study Director and Management The from 26 following study information FEB 99 to 10 MAR 99: and raw data were audited Vehicle receipt, preparation and use. Test article receipt, preparation and use. Test article packing lists. on The results 12 MAR 99. of this audit were reported to the Study Director andManagement 000501