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FINAL REPORT PROTOCOL 418-008 PERCIONMABTIANLE/DPOOSRTANLAT(AGLAVRAEGPER)OFDEURCTITLIIOTYN,TDOEXIVCEILTOYPSMTEUNDTYALOFANPDFOS INRATS SPONSOR'S STUDY NUMBER: 6295.9 FINAL REPORT DATE: 10 JUNE 1999 50133 PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 TABLE OF CONTENTS SUBJECT I. SUMMARY AND CONCLUSIONS A Methods B. Results C. Conclusion nn DESCRIPTION OF TEST PROCEDURES A. Conduct of Study A.1. Sponsor A.2. Testing Facility A3. Study Number Ad. Sponsor's Study Number AS. Purposeof the Study AG. Study Design A. Regulatory Compliance AB. Ownershipof the Study AS. Study Monitor ' PAGE 1 =} 1-5 1-10 1-1 14 1 I-1 1 11 1-1 1 2 2 2 000134 SUBJECT A10. Atemate Study Monitor A11. Study Director A12. Technical Performance A.13. Report Preparation A.14. Report Review A15. Date Protocol Signed A16. Datesof Technical Performance A.17. Records Maintained B. Test Article Information B.1. Description B.2. Lot Number B.3. Date Received and Storage Conditions B.4. Special Handling Instructions B.5. Analysis of Activity GC. Vehicle Information G1. Description C.2. Lot Numbers C3. Dates Received and Storage Conditions C4. Special Handing Instructions C.5. Analysis of Purity D. TestArticle Preparation D.1. Sample Information i PAGE 2 2 2 n-2 1-3 3 3 4 1-4 n-4 1-4 1-5 1-5 5 Is Is 5 Is [3 1-6 [i I 000135 SUBJECT D.2. Analytical Results E Test System E.1. Species E2. Strain E.3. Supplier (Source) Ed. Sex E.5. Rationale for Test System E.6. Test System Data E.7. Method of Randomization E.8. System of Identification F. Husbandry F.1. Research Facility Registration F.2. Study Rooms F.3. Housing F.4. Lighting F.5. Sanitization F6. Feed F.7. Feed Analysis F.8. Water F.9. Water Analysis F.10. Bedding F.11. Bedding Analysis it PAGE 7 I-7 7 7 7 m7 7 7 1-8 -8 1-9 1-9 1-9 -9 11-10 I-10 : 1-10 1-10 1-10 I-10 11 I-11 000136 SUBJECT PAGE G. Methods 1-11 G.1. Dosage Administration 11 G2. Assigned Rat Numbers 12 G.3. Rationale for Dosage Selection I-12 G4. Route of Administration 1-12 G.5. Rationale for Route of Administration 112 G6. Frequencyof Administration 1-12 G.7. Length of Study 13 G8. Method of Study Performance 113 G.9. Gross Necropsy 1-18 G.10. Statistical Analyses 21 Il. RESULTS -- Fo GENERATION MALE RATS 1 A. Clinical Observations 1 B. Body Weights and Body Weight Changes n-1 C. VaAblsuoelsute (g/day) and Relative (g/kg/day) Feed Consumption 1 D. Mating and Fertiity 2 E. Necropsy Observations 2 F. Terminal Body Weights, Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight 0-3 IV. RESULTS -- Fo GENERATION FEMALE RATS vA A. Clinical Observations : V1 Al. Mortality vA v 0137 SUBJECT A2. Clinical Observations B. Body Weights and Body Weight Changes B.A. Precohabitation B2. Gestation B3. Lactation C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values C.1. Precohabitation C2. Gestation C3. Lactation D. Estrous Cycling, Mating and Fertility E. Necropsy Observations F. Caesarean-Sectioning and Litter Observations G. Natural Delivery and Litter Observations H. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations I. Reflex and Physical Development 11. Surface Righting 12. Pinna Unfolding 13. Eye Opening 14. Acoustic Startle 15. Air Righting 16. Pupil Constriction v PAGE v-1 vet v-1 v2 v2 v3 v3 v3 v3 v4 v4 v4 v4 v5 ve Iv-6 v7 v7 7 v7 v-8 60138 SUBJECT V. RESULTS - F1 GENERATION MALE AND FEMALE RATS A. F1 Generation Male Rats A.1. Mortality and Clinical Observations A2. Body Weights and Body Weight Changes A3. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values A4. Sexual Maturation AS. Passive Avoidance Performance AB. Watermaze Performance A.7. Mating and Fertility AS. Necropsy Observations AS. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight B. B.1. F1 Generation Female Rats Mortality and Clinical Observations B.2. Maternal Body Weights and Body Weight Changes B.3. Maternal Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values B.4. Sexual Maturation B.5. Passive Avoidance Performance B.6. Watermaze Performance B.7. Mating and Fertility B.8. Necropsy Observations B.S. Natural Delivery and Litter Observations ` PAGE V-1 V-1 V-1 V2 V-2 V2 V-3 V-3 V-3 va V4 V4 v4 V-5 V6 V7 v7 V7 V7 V-8 V-8 00139 SUBJECT PAGE B.10. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations vo REFERENCES V-10 APPENDIX A - REPORT FIGURES Figure 1. Body Weights ~ Fo Generation Male Rats A Figure 2. Body Weights -- Fo Generation Female Rats A2 Figure 3. Body Weights -- F1 Generation Male Rats A3 Figure 4. Body Weights ~ F1 Generation Female Rats Ad APPENDIX B - REPORT TABLES ~ Fo GENERATION MALE RATS Table B1. Clinical Observations - Summary -- Fo Generation Male Rats B-1 Table B2. Body Weights ~ Summary -- Fo Generation Male Rats B2 Table B3. Body Weight Changes -- Summary ~ Fo Generation Male Rats B-3 Table B4. Absolute Feed Fo Generation Consumption Male Rats Values (g/day) -- Summary -- B4 Table BS Relative Feed Consumption Values (g/kg/day) ~ Summary -- Fo Generation Male Rats B-5 Table B6. Mating and Fertiity -- Summary ~ Fo Generation Male Rats ~~ B-6 Table B7. Necropsy Observations -- Summary - Fo Generation Male Rats B-7 Table B8. Terminal Body Weights and Organ Weights -- Summary -- Fo Generation Male Rats B88 Table BY. Ratios (%) of Organ Weight to Terminal Body Weight -- `Summary - Fo Generation Male Rats B-9 Table B10. Clinical Observations Male Rats - Individual Data ~ Fo Generation B10 Table B11. Body Weights- Individual Data -- Fo Generation Male Rats ~~ B-20 vii 601410 SUBJECT PAGE Table B12. FFeoeGdenCeornastuimopntMiaolneVRaaltuses - Individual Data - B30 Table B13. Mating and Male Rats Fertity ~ Individual Data -- Fo Generation B40 Table B14. MNaelceroRpastysObservations -Individual Data -- Fo Generation 850 Table B15. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight -- Individual Data -- Fo Generation Male Rats B-58 APPENDIX C- REPORT TABLES -- Fo GENERATION FEMALE RATS Table C1. Clinical Observations ~ Summary -- Fo Generation Female Rats C1 Table C2. Body Weights ~ Precohabitation -- Summary -- Fo Generation Female Rats c4 Table C3. Body Weight Changes Fo Generation Female -- Precohabitation Rats -- Summary - cs Table C4. Matera Body Weights -- Gestation -- Summary -- Fo Generation Female Rats C6 Table C5. Matemal Body Weight Changes ~ Gestation -- Summary -- Fo Generation Female Rats c-8 Table C6. Maternal Body Weights -- Lactation -- Summary -- Fo Generation Female Rats cs Table C7. FMaoteGrennaelraBtoidony FWeeimgahlte CRhaatsnges -- Lactation -- Summary -- c10 Table C8. Absolute Feed Consumption Values (g/day) -- Precohabitation ~ Summary -- Fo Generation Female Rats c-11 Table C9. Relative Feed Consumption Values (g/kg/day) -- Precohabitation -- Summar--y Fo Generation Female Rats c-12 Table C10. Maternal Absolute Feed Consumption Values (g/day) -- Gestation -- Summary --- Fo Generation Female Rats vii c-13 000141 SUBJECT PAGE Table C11. Maternal Relative Feed Consumption Values (g/kg/day) -- Gestation -- Summar--y Fo Generation Female Rats C14 Table C12. Matemal Absolute Feed Consumption Values (g/day) Lactation -- Summary -- Fo Generation Female Rats C-15 Table C13. Maternal Relative Feed Consumption Values (g/kg/day) -- Lactation -- Summary -- Fo Generation Female Rats C-16 Table C14. Estrous Cycling, Mating and Fertiity -- Summary Fo Generation Female Rats C17 Table C15. Necropsy Observations ~ Summary -- Fo Generation Female Rats c-19 Table C16. Caesarean-Sectioning Observations -- Summary -- Fo Generation Female Rats c20 Table C17. Natural Delivery Observations -- Summary -- Fo Generation Female Rats c-21 Table C18. Litter Observations (Naturally Delivered Pups) - Summary F1 Generation Litters. c22 Table C19. Clinical Observations from Birth to Day 21 Postpartum -- Summary - F1 Generation Pups c25 Table C20. Reflex and Physical Development -- Summary -- F1 Generation Litters C26 Table C21. Necropsy Observations -- Summary ~ F1 Generation Pups c-31 Table C22. Clinical Observations Individual Data -- Fo Generation Female Rats C-32 Table C23. Body Weights ~ Precohabitation -- Individual Data -- : Fo Generation Female Rats C40 Table C24. Maternal Body Weights -- Presumed Gestation -- Individual Data -- Fo Generation Female Rats C-50 Table C25. Maternal Body Weights -- Lactation -- Individual Data -- Fo Generation -- Fo Generation Female Rats C-70 ix 000142 SUBJECT Table C26. Table C27. Table C28. Table C26. PAGE Feed Data -CoFnosuGemnpetriaotnioVnalRuaetss -- Precohabitation -- Individual c75 Maternal Feed Individual Data C--onFsouGmepnteiroantiVoanluFeesma--lPerReastusmed Gestation -- C85 Maternal Feed Consumption Values -- Lactation Individual Data -- Fo Generation Female Rats C-105 FEsotGreounserCaytciloinngFeamndalDeaRyastsin Cohabitation -- Individual Data -- C110 Table C30. Table C31. Necropsy Observations ~ Individual Data -- Fo Generation Female Rats CFaoesGaerneeraant-iSoenctFieomnainlge ORbastesrvations Individual Data -- C115 c-121 Table C32. Litter Observations (Caesaren-Delivered Embryos) -- Individual Data -- F1 Generation Litters C-126 Table C33. LiEtmtberrysonal Vital Status ~ Individual Data ~ F1 Generation Table C34. Natural Delivery, Implantation Sites, and Pup Viability and FS1exGe--nIenrdaitviiodunalLiDttaetras -- Fo Generation Female Rats/ c-131 c-136 Table C35. Table C36. Table C37. Table C38. Pup Body Weight Litter Averages from Birth to Day 21 Postpartum -- Individual Data -- F1 Generation Litters C-141 IPnudpiviBdoudaly DWaetiagh--tsF1frGoemneBriratthitoon DPauyps21 Postpartum -- c-146 IPnudpivViidtuaallSDtaattaus--aFn1dGSeenxerfartoimonBiPrtuhptso Day 21 Postpartum -- C176 IClnidniivciadluaOlbsDeartvaa--tiFo1nsGefrnoemraBtiirothn tPouDpasy 21 Postpartum c-181 Table C39. Surface Righting ~ Individual Data -- F1 Generation Litters ~ C-182 Table C40. Pinna Folding -- Individual Data -- F1 Generation Liters C-193 * 60143 SUBJECT PAGE Table C41. Eye Opening ~ Individual Data -- F1 Generation Litters C203 Table C42. Acoustic Startle -- Individual Data -- F1 Generation Litters ~~ C-213 Table C43. Air Righting ~ Individual Data ~ F1 Generation Litters c223 Table C44. Pupil Constriction ~ Individual Data ~ F1 Generation Litters C-233 Table C45. Necropsy Observations ~ Individual Data -- F1 Generation Pups C243 APPENDIX D - REPORT TABLES -- F1 GENERATION MALE RATS Table D1. Clinical Observations ~ Summar--y F1 Generation Male Rats D1 Table D2. Body Weights ~ Summary -- F1 Generation Male Rats D3 Table D3. Body Weight Changes -- Summary -- F1 Generation Male Rats D4 Table D4. Absolute Feed Consumption Values (g/day) -- Summary -- F1 Generation Male Rats Ds Table DS. Relative Feed Consumption Values (g/kg/day) ~ Summar--y F1 Generation Male Rats 06 Table D6. Sexual Maturation -- Summary -- F1 Generation Male Rats ~~ D-7 Table D7. Passive Avoidance Performance -- Summary -- F1 Generation Male Rats D8 Table D8. Watermaze Performance ~ Summary ~ F1 Generation Male Rats D9 Table D9. Mating and Fertility ~ Summary ~ F1 Generation Male Rats D-10 Table D10. Necropsy Observations -- Summary -- F1 Generation Male Rats D-11 Table D11. Terminal Body Weights and Organ Weights ~ Summary -- F1 Generation Male Rats D-13 xi 00144 SUBJECT PAGE Table D12. Ratios (%) of Organ Weight to Terminal Body Weight -- Summary - F1 Generation Male Rats D-14 Table D13. Clinical Observations ~ Individual Data ~ F1 Generation Male Rats. D-15 Table Di4. Body Weights -- Individual Data ~ F1 Generation Male Rats D-20 Table D15. Feed Consumption Values ~ Individual Data ~ F1 Generation Male Rats D-26 Table D16. Sexual Maturation ~ Individual Data ~ F1 Generation Male Rats D-29 Table D17. Passive Avoidance Performance -- Individual Data - F1 Generation Male Rats D-30 Table D18. Watermaze Performance -- Individual Data ~ F1 Generation Male Rats 0-33 Table D19. Mating and Fertility -- Individual Data -- F1 Generation Male Rats D3 Table D20. Necropsy Observations - Individual Data -- F1 Generation Male Rats D-39 Table D21. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight -- Individual Data -- F1 Generation Male Rats D43 APPENDIX E- REPORT TABLES -- F1 GENERATION FEMALE RATS Table E1. Clinical Observations ~ Summary -- F1 Generation Female Rats 1 Table E2. Body Weights -- Precohabitation ~ Summary - 1 Generation Female Rats E4 Table E3. Body Weight Changes --Precohabitation -- Summary -- F1 Generation Female Rats ES Table E4. Maternal Body Weights -- Gestation -- Summary -- F1 Generation Female Rats E6 xi 0014S SUBJECT Table ES. Table E6. Table E7. Table E8. Table E9. Table E10. Table E11. Table E12. Table E13. Table E14. Table E15. Table E16. Table E17. Table E18. Table E19. PAGE Maternal Body Weight Changes ~ Gestation -- Summary -- F1 Generation Female Rats ES Maternal Body Weights ~ Lactation -- Summary -- F1 Generation Female Rats E9 Maternal Body Weight Changes ~ Lactation -- Summary -- F1 Generation Female Rats E-10 Absolute Feed Consumption Values (g/day--) Precohabitation ~ Summary -- F1 Generation Female Rats E-11 Relative Feed Consumption Values (g/kg/day) -- Precohabitation -- Summary ~ F1 Generation Female Rats ~~ E-12 Maternal Absolute Feed Consumption Values (g/day) -- Gestation -- Summary -- F1 Generation Female Rats E13 Matemal Relative Feed Consumption Values (g/kg/day) ~ Gestation -- Summary -- F1 Generation Female Rats. E14 Maternal Absolute Feed Consumption Values (g/day) -- Lactation -- Summary F1 Generation Female Rats E15 Maternal Relative Feed Consumption Values (g/kg/day) -- Lactation ~ F1 Generation Female Rats E-16 Sexual Maturation -- Summary -- F1 Generation Female Rats E-17 Passive Avoidance Performance ~ Summar--y F1 Generation Female Rats E-18 Watermaze Performance -- Summary ~ F1 Generation Female Rats E19 Mating and Fertility ~ Summary ~ F1 Generation Female Rats E20 Necropsy Observations -- Summary -- F1 Generation Female Rats E21 Natural Delivery Observations -- Summary ~ F1 Generation Female Rats E22 xii 60146 SUBJECT PAGE Table E20. Litter Observations (Naturally Delivered Pups) -- Summary ~ F2 Generation Litters E23 Table E21. Clinical Observations from Birth to Day 21 Postpartum -- `Summary -- F2 Generation Pups E26 Table E22. Necropsy Observations -- Summary - F2 Generation Pups E-27 Table E23. Clinical Observations ~ Individual Data -- F1 Generation Female Rats E28 Table E24. Body Weights -- Precohabitation -- Individual Data -- F1 Generation Female Rats E34 Table E25. Matemal Body Weights -- Presumed Gestation -- Individual Data -- F1 Generation Female Rats E37 Table E26. Table E27. Table E28. Table E29. Maternal BodyWeights -- Lactation ~ Individual Data -- F1 Generation Female Rats E43 Feed Consumption Values ~ Precohabitation -- Individual Data - F1 Generation Female Rats E46 Maternal Feed Consumption Values ~ Presumed Gestation -- Individual Data -- F1 Generation Female Rats E49 Maternal Feed Consumption Values ~ Lactation ~ Individual Data -- F1 Generation Female Rats E52 Table E30. Sexual Maturation -- Individual Data ~ F1 Generation Female Rats E55 Table E31. Passive Avoidance Performance ~ Individual Data -- F1 Generation Female Rats E56 Table E32. Watermaze Performance -- Individual Data -- F1 Generation Female Rats E59 Table E33. Days in Cohabitation -- Individual Data -- F1 Generation Female Rats E-62 Table E34. Necropsy Observations ~ Individual Data - F1 Generation Female Rats E63 xiv 00147 SUBJECT Table E35. Table E36. Table E37. Table E38. PAGE Natural Delivery, Implantation Sites, and Pup Viability and Sex Individual Data -- F1 Generation Female Rats/ F2 Generation Litters E67 Pup Body Weight Litter Averages from Birth to Day 21 Postpartum -- Individual Data -- F2 Generation Litters E-70 Pup Body Weights from Birth to Day 21 Postpartum -- Individual Data ~ F2 Generation Pups E73 Pup Vital Status and Sex from Birth to Day 21 Postpartum -- Individual Data -- F2 Generation Pups E-91 Table E39. CIlnidniivciadluaOlbsDeartvaa~tiFo2nsGefnreormaBtiirothn tPouDpasy 21 Postpartum -- E94 Table E40. Necropsy Observations -- Individual Data -- F2 Generation Pups E-95 APPENDX FAPPENDIX G - APPENDIX H - PROTOCOL AND AMENDMENTS DEVIATIONS FROM THE PROTOCOL AND THE STETSATNIDNAGRFDACOIPLEIRTAYTING PROCEDURES OF THE TEMPERATURE AND RELATIVE HUMIDITY REPORTS Foto F-63 G1 H-1to H-9 APPENDIX |- APPENDIX J - STATEMENT OF THE STUDY DIRECTOR QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT 1 J-1t0 J-8 xv 00148 418-008:PAGE I-1 TITLE: ACNOMDBPIENREIDNOATRAALLP(OGSATVNAAGTEA)LFERRETPILRIOTDYU,CDTEIVOENLOTOPXMIECNITTAYL STUDY OF PFOS IN RATS ARGUS RESEARCH LABORATORIES, INC. SPPROONTSOOCRO'LSNSUTMUBDEYR:NU4M1B8E-R00:8 6295.9 I. SUMMARY AND CONCLUSION A. Methods" Text Figure 1 provides a schematicof the study design. AA. FG o eneRatrs/Fa 1Genteratiiono Littn ers: DOanwelehyu)ndrratesdwseerveenatsy-sfiigvneedmatloefiavne ddofseamagleegCroru:pCsD(GBrRouVpAsF|/tPhlruosugh(VS)p,ra3g5urea-ts tpheer vseehxicpleer, d0o.s5a%gTewgereounp.80T,heorraaltlsy w(veiraegaadvmaignei)s.teTrheed dthoesategsetsarwteicrlee, PFOS, or 0 (Vehicle), 0.1, 0.4, 1.6 and 3.2 mg/kg/day. The male rats were dosed once daily beginning 42 days before cohabitation and continuing through the day before sacrifice. The female rats were dosed once daily beginning 42 days. sbeecftoiroenicnogh)a,biDtGati2o4n (arnatds caosnstiignnueidngtothnraotuurgahl DdGeliv9er(yratthsaatsdsiidgnnoetd dteoliCvaeersaarliettaenr)-, 5ormDULk* g20 (rats that did delivera litter). The dosage volume was All Fo generation rats were observed for viability at least twice daily during the dstousdaygaenpderfioord.clinBiocadlyswiegingshotfseaffnedctfseoefd tchoentseusmtpatritiocnlevtawliuceesdfaoirlymadlureinragtsthweere freemcaorldeerdatwseweeklrye druercionrgdetdhewdeoesklaygetopceorhiaobditaantdioant,sdaacriilfyicdeu.rinBgodthyewgeeisgthattsiofnor period, on DLs 1, 4, 7, 10 and 14 (rats assigned to natural delivery) and at dsaucrriinfigcet.he FgeesetdatcioonnsupemrpitoidonanvdalounesDLwser1e, 4r,ec7o,r1d0edanwede1k4ly(rtaotscoahsasbiitganteidont,o daily natural delivery). a. aDreetapirloedviddeesdcriinptthieonaspporfoapllripartoecseedcutrieosnsusofedthiins trheepocrotnadnudctinofAPthPisEsNtDuIdyX F (PROTOCOL AND AMENDMENTS). b. DG is used as an abbreviation for day of (presumed) gestation. c. DL is used as an abbreviation for day of lactation or day postpartum. 60149 418-008:PAGE 1-2 The first ten female rats per dosage group with a confirmed date of mating were assigned to Caesarean-sectioning on DG 10. The remaining female rats were. permitted to naturally deliver litters. These rats were evaluated for clinical observations during parturition, duration of gestation, litter size and pup viability at birth. Maternal behavior of the dams was evaluated daily when the pups were examined during the 21-day postpartum period. Each litter was evaluated for viability at least twice each day during the 21-day postpartum period. Pups in each litter were counted once daily. Physical signs in the pups were recorded once daily for 21 days postpartum. Pup body weights and observed nursing behavior were recorded on DLs 1 (birth), 4, 7, 14 and 21 Surface righting reflex, pinna unfolding, eye opening, acoustic startle response and air righting reflex were monitored during the 21-day postpartum period until all pups in the litter reached the criterion for the specific test. Pupil constriction was evaluated once on DL 21. On DL 4, a table of random units was used to cull litters to four male and four female pups, where possible. On DL 21, a table of random units was used to select two male and two female pups from each litter from Groups I, Il and Il for continued evaluation Fo generation male rats were sacrificed after completion of the cohabitation period and necropsied; gross lesions were retained. The testes, epididymides, prostate and seminal vesicles (with and without fluid) were excised, individually weighed and retained Fo generation female rats assigned to Caesarean-sectioning were sacrificed on DG 10 and necropsied; pregnancy status was confirmed. Ovaries and gross lesions were retained. The rats were examined for the number and distribution of corpora lutea in each ovary and implantation sites, and viable and nonviable embryos. Embryos were discarded after examination. Fo generation female rats assigned to natural delivery were sacrificed on DL 21 and necropsied. Ovaries and gross lesions were retained. The number and distribution of implantation sites was recorded. At scheduled sacrifice after completion of the cohabitation period (male rats that sired litters of dams allowed to naturally delivera litter) and on DL 21 (female rats allowed to naturally deliver a litter), blood samples (approximately 4 mL per rat) were collected from the inferior vena cava from five male and five female rats per dosage group and shipped to the Sponsor for pharmacokinetic analysis. Pups not selected for continued evaluation on DL 4 were sacrificed and necropsied. The stomach contents (milk curd) were collected from all culled pups from five of the largest liters in Groups I, Il and Ii, frozen and shipped to the Sponsor for analysis. Ce150 418-008:PAGE 1-3 The liver from each rat was excised, weighed, and a sample section (lateral lobe) was frozen and shipped to the Sponsor for analysis. The livers of the pups from the litters of the five dams in Groups | through IV selected for pharmacokinetic sample collection were excised, pooled per litter, frozen and shipped to the Sponsor for analysis. Dams in the 3.2 mg/kg/day dosage group (Group V) did not have surviving pups on DL 21 A2. F1Generation Rats/F2 Generation Litters: Only the 0.1 and 0.4 mg/kg/day dosage groups were continued into the second generation because F1 generation pups. of the excessive toxicity seen in the 1.6 and 3.2 mg/kg/day There were 150 male and female F1 generation rats in the three dosage groups. (Groups | through lil), 25 rats per sex per dosage group. F1 generation male and female rats were given appropriate dosages of the test article orally (gavage) beginning on DL 22 and continuing through the day before sacrifice. Beginning at 24 days of age, one male rat and one female rat from each litter in each dosage group were tested in a passive avoidance paradigm. Female rats were evaluated for the age of vaginal patency beginning on DL 28. Male rats `were evaluated for the age of preputial separation beginning on DL 34. On postpartum day 70, one male rat and one female rat from each litter were evaluated in a water-filled M-maze. On approximately DL 90, rats within each dosage group were assigned to cohabitation. F1 generation male rats were sacrificed after completion of the cohabitation period and necropsied, as previously described for the Fo generation male rats. All F1 generation female rats were permitted to naturally deliver litters. All dams that delivered litters were sacrificed on DL 21 and necropsied, as previously described for the Fo generation female rats. On DL 21, all F2 generation pups were sacrificed and examined for gross lesions. Necropsy procedures were the same as those described for the F1 generation pups. cCa51 aril LT TILL B. Results 418-008:PAGE I-5 B.A. Fo Generation Male Rats: No Fo generation male rats died during this study as a result of treatment with PFOS. Al clinical observations (other than normal) were considered unrelated to the test article and not signs of compound toxicity. Groups administered 0.4 mg/kg/day and higher dosages of the test article had reduced body weight gains for the entire study. Absolute and relative feed consumption values were reduced in the 1.6 and 3.2 mg/kg/day dosage groups for the entire precohabitation period. After the cohabitation period, absolute feed consumption values were significantly reduced in the 0.4 and 1.6 mg/kg/day dosage groups. Dosages of the test article as high as 3.2 mg/kg/day did not affect any mating and fertiity parameters evaluated A significantly increased number of male rats in the 3.2 mg/kg/day dosage group had a light brown or brown liver, an observation attributed to effects of PFOS. The 1.6 and 3.2 mg/kg/day dosage groups had significantly reduced terminal body weights. The absolute weights of the seminal vesicles with fluid and the prostate were significantly reduced in the 3.2 mg/kg/day dosage group. B.2. Fo Generation Female Rats/F1 Generation Litters: No Fo generation female rats died during this study as a result of treatment with PFOS. Observations of localized alopecia were increased during the precohabitation, gestation and lactation periods in the 0.4, 1.6 and 3.2 mg/kg/day dosage groups. Groups administered 1.6 mg/kg/day and higher dosages of the test article had significantly reduced body weight gains for the entire precohabitation period. The 1.6 and 3.2 mg/kg/day dosages of PFOS continued to reduce body weights and body weight gains during gestation. The 3.2 mg/kg/day dosage group had significantly reduced body weight on DL 1. Body weight gains tended to be. reduced in the 0.4 mg/kg/day dosage group on DLs 1 to 4, when significant weight loss occurred in the 1.6 mg/kg/day dosage group (the 3.2 mg/kg/day dosage group was precluded from further evaluation because all pups died before DL 2). Co153 418-008:PAGE 16 A3b.2somlgu/tkega/dnadyrdeloastiavgeefegerdoucposnsduurmipntgiothnevparleuceoshawbeirteatrieodnupceerdiodi.n the 1.6 and The 1.6 and f3e.2edmgc/okngs/udmapytidoonsvaagleuegsroeuaprlsycionntthienugeedsttaotihoanveperrieoddu.ceAdbsaoblsuotleutaenadnrdelraetliavteive maternal feed dosage group consumption values tended to and were significantly reduced be in reduced in the 0.4 mg/kg/day the 1.6 mg/kg/day dosage group for 3.2 the entire lactation mg/kg/day dosage period, group and was at most intervals within this period. precluded from evaluation because The there were no surviving pups after DL 1. Dcyocslainggeisn otfheth1e5tersattsarpteircldeoassahgieghgraosup3.t2hamtg/wkegr/edeavyaldiudatneodt.afMfaetcitnegstarnodusfertlty parameters were unaffected by the 3.2 mg/kg/day dosage of PFOS. All necropsy observations were considered unrelated to treatment. Tlihteerreavweerraegensofboirolcoogripcoarllayliumtepao,rtiamnptlaonrtasttaitoinsst,icvalilayblseigenimfbircaynotsdiofrfneornevncieasblien the. embryos at Caesarean-sectioning on DG 10. gTrhoeupd,uraantioobnseorfvgaetsitoantiaosnswocaisatseidgnwiifitchanptrleyimrpeldauncteadtiionntlhoess3.[2thmeg/akvge/rdaagye dosage number orfediumpcleadntlaitteironsiszei]t.esPpuepr dviaambilwitayswasisgnsiifginciafnitclayntrleydurceeddu,cerdesiunlttihneg 1i.n6aasnidgnificantly 3la.c2tamtgi/okngi/nddaicyedsowseargeesgirgnoiufpisc.antRleyfrleecdtuicnegdtihnesteheef1f.e6ctasn,dth3e.2vimagbi/lkigty/daanyd dosage dgroosuapgse, gtrhoeulpa,ctaastiaolnsoinwdeerxewathseaalvseorsaiggenisfifcoarnstluyrvrievdinugcepdupinstihneth1e.61m.6g/mkgg//kdga/yday dosage group beginning group on DL 1. on DL 4 postculling, and in the 3.2 mg/kg/day dosage Agrdooupsaagdem-idneipsteenrdeedntthpeatttesetrnarotifclree.duTcheed 0p.u1pmbgo/dkyg/wdeaiyghdtowsaagseegvriodeunpttiennedaecdhto hcoanvterorlegdruocuepd vpaulupe.weiTghhets0.o4nmDgL/k4g/(dparey-daonsdagpoestgcruolluipngt)e,ncdoedmptaorheadvetortehdeuced Pgruopubpovdayluwee.igHhotwseovnerD,Lnso1nethorfotuhgeh d4if(fpeorsteenuclelsinign),thceo0m.p1aarnedd 0t.o4tmheg/ckognt/rdoaly dvaorsiaatgieongsr. ouTphse w1e.r6 emgst/aktgis/tdicaaylldy ossigangiefigcarnotuapnhdadmasyignriefpirceanstelnytrneodrumcaeld bpiuolpogbiocdaly `siwgeniigfhitcsanotnlyalrlewdeuicgehdinpgupdabyso.dy Twheeig3h.t2omng/DkLg/1d(anyodpouspasgesugrrvoivuepdhtaodsaubsequent scheduled weighings). Cploitneinctailalanredduncetciroonpsinymoabtseermvaalticoanrse aoscscoucriraetdedinwtihteh 3r.e2dumcge/dkgp/udpayvidaboisliatgy eangrdoup. 00154 418-008:PAGE I-7 Adverse clinical observations include three ltters with pups that were not nursing (twoof these litters had pups from which the placenta had not been removed, and one had pups that were not nested). Apparent maternal cannibalization was also evident in this dosage group (missing tail in one pup and a cannibalized forelimb in another pup). It was not possible to determine whether these occurred before or after these pups had died. Necropsy observations in pups that were stillborn or found dead indicated many pups with no milk in the stomach in the 1.6 and 3.2 mg/kg/day dosage group pups. The 3.2 mg/kg/day dosage group also had evidence of increased maternal cannibalization at necropsy of stillborn and found dead pups. Nine of these pups had missing hindlimbs and/or portion of the tail. wRietvherbsoidbylewedieglhatysocincurrefrleedx ianntdheph0y.s4icaanldd1e.v6elmogp/mkegn/tdatyhadtoasraegehigghrloyupcso.rreSluartfeadce righting was delayed in the 1.6 and 3.2 mg/kg/day dosage groups. The time of development for pinna unfolding, eye opening, acoustic startle reflex and ability to air right were delayed in the 1.6 mg/kg/day dosage group (no 3.2 mg/kg/day dosage group pups were evaluated for this parameter). All live pups in the 0(Vehicle), 0.1, 0.4 and 1.6 mg/kg/day dosage groups had the pupil constriction response when testing was done on DL 21 (end of lactation). Upon weaning of the litters (DL 21), a decision was made regarding the F1 generation pups in the 1.6 mg/kg/day dosage group. The 1.6 mg/kg/day dosage group pups were small (they had gained 20% less weight during lactation than trahtescwoentrreolnoptupssui)taabnlde ifnortfhuerothpeirnieoxnpeorfitmheentaatttieonnd.ingIt lhaabdorbaetoernydveetteerrimnianreiadn,thtahte the dosageof 1.6 mg/kg/day wasa level which produced compound toxicity. Continued dosing of 1.6 mg/kg/day most likely would not result in any additional cionnfsourlmtaattiioonnawnidthwtohueldSpsounbjseocrt ltehaedptuoptshetodaedcdiistiioonnanlotditsotrceosnst.inTuheertehfeore, 1.6 0.4 mg/kg/day mg/kg/day dosage dosage group group F1 generation pups. pups were continued Only the O (Vehicle), 0.1 to a second generation. and B3. F1 Generation Male Rats: No deaths in the F1 generation male rats at 0.1 and 0.4 mg/kg/day dosages were attributed to PFOS. the test article. All clinical observations were considered unrelated to The 0.1 and 0.4 mg/kg/day dosage groups tended to weigh less than the control group on day 1 postweaning and generally gained one or two grams less body weight per week than the control group rats. Reflecting this patternof weight gain, weight gains in the 0.1 and 0.4 mg/kg/day dosage groups for the precohabitation period were 98.4% and 97.6% of the control group value, CC155 418-008:PAGE I-8 dreasype1cttiovteleyr.minBaotdiyonwweeirghet9g8a.i5n%s in the 0.1 and and 96.6% of 0.4 the cmogn/tkrogl/dgaryoudposvaaglee.groups for srteastpiesctticiavlellyy.sigHnoifwiecvaentr,frnoomnceonotfrotlhevsaelubeos.dy weight gain differences were Awbesroelusitgeniffeiecdanctloynsreudmupcteidononvaplousetswfeoarntihneg0d.1ayasnd1 0t.h4romugg/hkg8./dRaeyladtoisvaegfeeegdroups gcroonuspusmpdtuiroinngvtahleuefsirsttewnedeekd to of bineturbeadtuiconedbuitn the non 0.1 and e were s0ta.t4ismtgic/aklgly/ day dosage significant. dbDiaooysloaoggficepasrlelopyfutitimhapelortsetesaptnatarradttiiicflofeneraiesnntchehiseghiFn1atshgee0.nv4earlmaugte/iskongf/omrdaallyeeadrirnaditnsng.,otTsahhfoefrrectet.weterhrmeearevnteoernatigoen, long-term retention or response inhibition in the F1 generation male rats, as peavraalduiagtme.d by performance in a passive avoidance or watermaze performance Dosagesof the test article as high as 0.4 mg/kg/day did not affect anymating and fertility parameters evaluated in the F1 generation male rats. oAulcnlcreunlrearcteredodpistnoytthohebesaetbressvotaltaurittoeincosleinbreetlchaaetuivFse1e.vgaelNnueoersasttfaoitroinstthimecaawllleeyigsriahgttnssiwfoiefcratenhtecdorinifgsfhietdroeerrnecldeefst testis, seminal vesicles, right epididymis or prostate. Terminal body weights tended to be reduced statistically significant ifnrotmheco0n.t1raolndva0lu.e4s.mg/kg/day dosage groups but were not B.4. F1 Generation Female Rats/F2 Generation Litters: No deaths in the F1 generation female rats were attributed to PFOS. All adverse clinical observations that occurred during the precohabitation, gestation and lactation periods were considered unrelated to the test article. Body weights postweaning. tended to Matemal be reduced in body weights the and b0.o4dymgw/ekigg/hdtagyaidnossadugreinggrotuhpe on day 1 gestation pSiegrniiofdicwaenrtemautnearfnfaelctbeoddbyywdeoisghatgelsososfotchceurtersetd aortnicDleLsas1 high to 4 as in 0.4 the mg/kg/day. 0.4 mg/kg/day dosage group. The 0.4 mg/kg/day dosage of the test article was associated with a significant reduction in absolute feed consumption and a tendency for reduced relative feed consumption on days 1 to 8 postweaning. Absolute and relative feed tchoenstuesmtpatritoicnlevaalsuheisgdhuarisn0g.t4hmegg/eksgt/adtaiyo.n pTehrieodabwseorluetuenaanffdecretleadtibvye mdaotseargneasl of 00156 41R8E-V0I0S8E:PDAPGAE G1-E9 feed consumption values were reduced during lactation in the 0.4 mg/kg/day dosage group. Dosages of the test article as high as 0.4 mg/kg/day did not affect the average day of vaginal patency in the F1 generation female rats. There were no lbioonlgo-gtiecralmlyreitmepnotriotnanotrdriefsfeproennsceesinihnibtihteiovnalinuetshefoFr'1legaemnienrga,tisohnofrte-mtaelrem rraettse,ntasi.on, evaluated by performance in a passive avoidance or watermaze performance paradigm. aDnodsafgeretsitoyfptahreatmeesttearrsticelveaalsuahtiegdhiansth0e.4F1mgg/eknge/rdaatyiodnidfenmoatlaeffreactts.any mating Aulnlrneleactreodpstoytohbesetresvtataritoincsle.in the F1 generation female rats were considered 2P5refgenmaanlceyroatcscuarsrseidgnien d22to(c9o5h.a6b%i)t,at2i1on(8in4.t0h%e)0a(nVdeh2ic4le()9,6.00.)1 oafntdhe0.243,m2g5/kagn/dday dosage groups, respectively. Al pregnant dams delivered liters. The gestation index was comparable across the three dosage groups. Viability and growth of tthhee shiegchoensdt gdeonseargaetitoenstoefdf,sp0r.i4ngmg(/Fk2gp/duapys.) tTohweeraenwienrgewneoretoaxliscooluongiacfaflelcyted by oibmspeorrvtaanttiodnisffienrtehneceFs2frgeonmetrhaeticoonntpruolpsgrwoeurpevaatltureisb.utaNbolectloindicoaslaogrenseocfrotphesytest article as high as 0.4 mg/kg/day 0157 418-008:PAGE I-10 REVISED PAGE C. Conclusions On the basis of these data, the Fo generation matemal and patemal noobservable-effect-level (NOEL) of PFOS is 0.1 mg/kg/day (0.4 mg/kg/day and hciognhseurmpdtoisoangevsalcuaesu)s.ed reductions in body weight gain and reduced feed TonhemaFtoingge,neferrattiiitoynorreepsrtordouucsticvyeclNiOngEoLcciusrgrerde.ateTrhtehaNnO3E.L2 mfogr/kviga/bdilaiyt;y annodegffreocwttsh in the F1 generation offspring is 0.4 mg/kg/day (1.6 mg/kg/day and higher dosages caused preimplantation loss and reductions in litter size, pup viability, growth and survival). T(0h.e4 mF1gkgge/nderaaytidoonsmaagteecmaaulseadndrepdautcetmiaonlsNiOn EboLdyofwPeFigOhSt giasi0n.1anmdg/rkegd/udcaeyd feed consumption values). T0.h4emFg1/kgge/ndearya;tinoonerfefpercotsduocntimvaetNinOgEoLr ifsergtriietaytoecrctuhrarneda. dTohseagNeOoEfL for viability and growth in the F2 generation offspring is also 0.4 mg/kg/day. There were no toxicologically important effects on pup survival or growth at the highest dosage tested, 0.4 mg/kg/day. Mildred S. Christian, Ph.D., Fellow, ATS Date Execytive Director of Research Hoon Dolo ze M. floberman, Ph.D., DABT Date Director of Research Lema Se IN TTT 3k ve Raymond G. York,7b.pre Date Associate Director of Redearch and Study Director 060158 I. DESCI F TEST PROCEDURE! A. ConductofStudy: 418-008:PAGE II-1 A. Sponsor: 3M Toxicology Services, 3M Center, Building 220-2E-02, St. Paul, Minnesota 55144-1000 A:2. Testing Facility: Argus Research Laboratories, Inc., 905 Sheehy Drive, Building A, Horsham, Pennsylvania 19044-1297 A3. StudyNumber: 418-008 A.4. SponSs tudoy Nrum'bes r: 6295.9 AS. Purpose ofthe Study: The purpose of this study was to test for toxic effects/disturbances resulting from PFOS treatment of Cr: CDBR VAF/Plus male and female rats before cohabitation through mating, gestation and lactation. This study was designed to evaluate ICH Harmonised Tripartite Guideline stagesA through F of the reproductive process and detect effects on the estrous cycle, tubal transport, implantation, gestation, parturition, lactation and maternal behavior in female ats, on the development of the offspringof the treated male and female rats, and permit detectionoffunctional effects (e.g., effects on libido or epididymal sperm maturation) that may not be detected by histological examinations of male rat reproductive organs. Because manifestations of effects induced during this period may be delayed in the offspring, observations were continued through production of F2 generation litters. AS. StudyDesign: A modification of the requirements of U.S. Food and Drug Administration (FDA) were used as a basis for the study design. 60159 418-008:PAGE Il-2 A. Regulatory Compliance: The study was conducted in compliance with the Good Laboratory Practice (GLP) regulations of the U.S. Food and Drug Administration (FDA), the Japanese Ministry of Health and Welfare (MHW) and the European Economic Community (EEC). There were no significant deviations from the GLP regulations that affected the quality or integrity of the study. Quality Assurance Unit findings derived from the inspections during the conduct of this study are documented and have been provided to the Study Director and the Testing Facility Management. AB. Ownership of the Study: The Sponsor owns the study. All raw data, analyses, reports and all preserved tissues are the property of the Sponsor. AS. Study Monitor: Marvin T. Case, D.V.M., Ph.D. A.10.AlternateStudyMonitor: Andrew M. Seacat, Ph.D. AA1. Study Director: Raymond G. York, Ph.D., DABT (Associate Director of Research) AA12. Technical Performance: John F. Bamett, B.S. (Director of Laboratory Operations) Kristen landola Sherer, B.S. (Research Associate/Fetal Evaluation) Joseph W. Lech, B.S. (Team Leade--r General Laboratory) Sharon Adamski (Laboratory Technician) AA13. Report Preparation Raymond G. York, Ph.D., DABT Michelle R. Rzaca, B.S. (Study Coordinator) Heidi M. Green, M.S. (Study Coordinator) Erin Hagan, B.A. (Data Management Specialist) Karen G. Parker, A.A. (Report Administrator) 060160 418-008:PAGE 1-3 A.14. Report Review: Alan M. Hoberman, Ph.D., DABT (Director of Research) Midred S. Christian, Ph.D., Fellow, ATS (Executive Director of Research) A.15. DateProtocolSigned: 21 May 1998 A16. Dates of Technical Performance: Fo Generation Male R Rat Arrival Date Dosage Period (42 days before cohabitation, through a 14-day cohabitation period and until sacrifice) Scheduled Sacrifice 12 MAY 98 26 MAY 98 - 30 JUL 98 31JuLe8 Fo Generation Female Rats Rat Arrival Date 12 MAY 98 Dosage Period ~ Female Rats Assigned to Caesarean-Sectioning (42 days before cohabitation and continuing through DG 9) 26 MAY 98 -- 17 JUL 98 Dosage Period ~ Female Rats Assigned to Natural Delivery [42 days before cohabitation through DG 24 (rats that did not deliver a litter) or DL20 (rats that delivered alitter) 26 MAY 98 - 30 AUG 98 Dosage Period Estrous Cycle Evaluation 09 JUN 98-06 JUL 98 a. DGis used as an abbreviation for day of (presumed) gestation. b. DLis used as abbreviation for day of lactation. 000161 (Fo Generation Female Rats Continued) 418-008:PAGE I14 Cohabitation Period Male 1 Male 2 DG 10 Caesarean-Sectioning Natural Delivery Period (DL 1) DG 25 Sacrifice (rats that did not deliver alliter) DL 21 Sacrifice (dams and pups not selected for continued study) 06 JUL 98 PM 13 JU9L8AM 13JUL 98 P--M 20 JUL 98 AM 17 JUL 98 - 30 JUL 98 28 JUL 98 - 10 AUG 98 01 AUG 98 - 07 AUG 98 17 AUG 98 - 30 AUG 98 F1 Generation Rats Dosage Period (Male Rats) 19 AUG 98 - 16 NOV 98 Dosage Period (Female Rats) 19 AUG 98 - 27 DEC 98 Passive Avoidance Testing 20 AUG 98 - 10 SEP 98 Watermaze Testing 07 OCT 98 - 19 OCT 98 Cohabitation Period (Initiated when the rats are approximately 90 days of age) Male 1 02 NOV 98 PM -- 09 NOV 98 AM Male 2 09 NOV 88 PM -- 16 NOV 98 AM Male Rats Sacrificed 17 NOV 98 Natural Delivery Period (DL 1) 24 NOV 98 - 08 DEC 98 DL 21 Sacrifice 14 DEC 98 - 28 DEC 98 AA7. Records Maintained: The original report, raw vehicle components are data and retained reserve samples in the archives of of the Argus bulk test article and Research Laboratories, Inc. Any preserved tissues are retained in the archives of the Testing Facility for one year after mailing the draft final report, after which time the Sponsor will adtectihdeeTethsetiirnfginFaalcidliistpyo.sitUinonu.sedAllbuulnkutseesdt atretsitclaertwicalse sruestuprennesdiotnosthweerSteuddiyscarded Monitor. B. TestArticleInformation: B.A. Description: PFOS - an off-white powder B.2. LotNumber: 217 (Expiration Date: May 2000) 06C162 B.3. Date Received and Storage Conditions: 418-008:PAGE II-5 TPrheeptaersetdarstuisclpeewnsaisonrescweievreedsotnor2e0d aMtaryo1o9m9t8e,mapnedrasttuorreedovaetrnriogohtm. temperature. B.4. Special Handling Instructions: Standard respirator safety precautions (use of and safety goggles) were protective clothing, gloves, dust-mist taken when handling the bulk test article and prepared suspensions. BS. Analysis of Activity: Information article is on regarding the purity, identity, file with the Sponsor. strength and composition of the test C. VehicleInformation: C1. Description: 0.5% Tween 80 in reverse osmosis membrane processed deionized water (R.O. deionized water). C.2. LotNumbers: MO3HOS, K03737, M29477 and L06662 C3. Dates Received and Storage Conditions: The Tween 80 was received from J.T. Baker, Phillipsburg, New Jersey, on 15 August 1997 (Lot M29477), 3 (Lot K03737), 22 May 1998 (Lot MO3H05), 17 September December 1998 (Lot MO3HO5) and 1 September 1998 1998 (Lot L0BB62), and was stored at room temperature. R.O. deionized water is. available from a continuous source at the Testing Facility and is maintained at room temperature. temperature. The vehicle was prepared weekly and stored at room 60163 C.4. Special Handling Instructions: 418-008:PAGE 11-6 Standard safety precautions (use of protective clothing, gloves, dust-mist respirator, safety goggles or safety glasses and a face-shield) were taken when handing the vehicle. C5. Analysis of Purity: Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to be present in the vehicle that would interfere with the. resultsofthis study. D. TestArticle Preparation: Suspensions of PFOS were prepared daily one day prior to the day of dosing at concentrations of0, 0.02, 0.08, 0.32 and 0.64 mg/mL. The test article was considered 100% pure for the purpose of dosage calculations. [CeT=[or D.1 SampleInformation: Tom [oe [or] [omy Tome[wiv [rere |esr| VericeComponentReserve iSmn || ZBoiEoCescs 25UAYSS | Roomtempersue| Tsing GElaisneg | 090s Ehav is 5505s ASsmyiiantgsetwoansourse(dhetoFwoigiornearwaisoanmpainedsuffom asncehrcotnacnaidraasttownedeurkisnogfth4e03fr2s9t8a3ndisinxtsh tweaektooif odohnesag1e oGenerpatiforneanEaalydcshiss.amTplheewtahserdiavikdeud n(o3 iwL)owaalsoerst(2asiathnndT3eeis.tdesFpacekctiye2)..3 Oancekuapiqudt (2 mi) was 5. oAfsyrroipnagaetwoans. uEsaechtoswamiplae wwassaampleisfoidomwihoeeS(oep,tdmsid(e2a.ndanbd 3omofteahsepheicr gihees)t.oOnncaeontot(nat2het)fwriesstodany Snpped for analysis The ther uot (3 iL) was eained a he Testing Faciy 35 3 backup. Stability data for prepared formulations bracketing the range of concentrations are on file with the Sponsor. The Sponsor confirmed a 48-hour stability on the test article in 0.5% Tween 80 solutions. a. See APPENDIX G (DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY), ite 1. CCCiG4 D.2. Analytical Results: 418-008:PAGE I1-7 Results of the concentration and homogeneity analyses were not available at the time of the writing of this report. E. TestSystem: EA. Species: Rat E2. Strain: Cr:CDBR VAF/Plus (Sprague-Dawley) E.3. Supplier(Source): Charles River Laboratories, Inc., Raleigh, North Carolina E4. Sex: Male and Female E5. Rationale for Test System: The Crl:CDBR VAF/Plus (Sprague-Dawley) rat was selected as the Test System because: 1) this strain of rat has been demonstrated to be sensitive to reproductive and developmental toxins and has been widely used throughout industry for reproductive and developmental toxicity evaluations; 2) historical data and experience exist at the Testing Facility"; and 3) the test article is pharmacologically active in the species and strain. E6. Test System Data: Male Rats Female Rats Number of Rats Approximate Date of Birth Approximate Age at Arrival Weight (g) on the Day after Arrival Weight (g) at Study Assignment 195 12 MAR 98 62 days 282-338 334-396 205 12 MAR 98 62 days 172-239 209 - 242 CC165 E7. Method of Randomization: 418-008:PAGE 1-8 E.7.a. Fo Generation Rats Upon arrival, Fo generation rats were assigned to individual housing on the basis of computer-generated random units. After acclimation, male and female rats were selected for study on the basis of physical appearance and body weights recorded during acclimation. Rats were assigned to five dosage groups (Groups | through V), 35 rats per sex per dosage group, using a computergenerated (weight-ordered) randomization procedure. The first ten female rats per dosage group with a confirmed mating date were. assigned to Caesarean-sectioning to naturally deliver liters. on DG 10. The remaining rats were permitted A table of random units was used to assign five rats per group to pharmacokinetic sample collection either at scheduled sacrifice after completion of the cohabitation period (male rats siring litters with dams allowed to naturally deliver a litter) or on DL 21 (female rats allowed to naturally deliver a fitter). E.7.b. FA/F2 Generation Pups On DL 4, a table of random units was used to select the pups to be culled and litters were reduced to eight pups each. Whenever possible, the same number of male and female pups per litter were continued on study. At weaning of the F1 generation pups on DL 21, a table of random units was. used to select 25 male and 25 female pups in each of Groups I, Il and Ill, resulting ina total of 150 F1 generation rats (75 per sex) chosen for continued evaluation. At least one male pup and one female pup per litter were selected. There were no surviving pups in Group V after DL 2; pups in Group IV were not continued on the study because of severe toxic effects in the pups (death and retarded growth) during lactation. This decision was made in consultation with the study veterinarian and the Sponsor. E8. System of Identification: E.8.a. Fo Generation Rats were permanently identified using Monel self-piercing ear tags (Gey Band and Tag Co., Inc., No. MSPT 20101). Male and female rats were assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study before administration of the first dosage of the test article. 0CC166 E.8.b. F1/F2 Generation Pups and Rats 418-008:PAGE Il-9 Pups were not individually identified during lactation; all parameters were evaluated in terms of the litter both before and after culling on DL 4. At weaning, each F1 generation rat selected for continued observation was identified with a Monel self-piercing ear tag. F. Husbandry: FA. Research Facility Registration: USDA Registration No. et seq. 23-R-099 under the Animal Welfare Act, 7 U.S.C. 2131 F.2. Study Rooms: Tahhealsltwuadyyarnodomisndweepernedmeanitnltyasiunpepdliuenddewirtchoandmiitinoinmsuomf poofstietnivcehaainrfgleosw rpeelrathiovuertoof 100% fresh air 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); relative humidity was targeted at 30% to 70%. See APPENDIX H (TEMPERATURE AND RELATIVE HUMIDITY REPORTS). F.3. Housing: All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals. F.3.a. Fo Generation Rats/F1 Generation Litters Fo generation rats were individually housed in stainless steel wire-bottomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of rats was housed in the male rat's cage. Beginning no later than DG 20, Fo generation female rats assigned to natural delivery were individually housed in nesting boxes. in a common nesting box during the Each dam postpartum and delivered period. litter were housed F.3.b. F1 Generation Rats/F2 Generation Litters After weaning, the F1 generation rats were individually housed before cohabitation, housed in pairs (one male rat per female rat) during cohabitation, and individually housed after cohabitation. The same type of caging was used as described for the Fo generation rats. Beginning no later than DG 20, F1 generation female rats were individually housed in nesting boxes. Each dam C0167 418-008:PAGE 11-10 and delivered period litter were housed in a common nesting box during the postpartum 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. F5. Sanitization: wCeargeecphaanngliendersapwperroexicmhaatenlgyedevaeprpyrooxtihemratweeleyk.thrBeeedtdiimnegs weaaschcwheaenkg.edCaasgeofsten as necessary to keep the rats dry and clean. F.6. Feed: Rats were givenadlibitum access to Certified Rodent Diet #5002 (PMI Nutrition International, Inc., St. Louis, Missouri) in individual feeders. F.7. Feed Analysis: Analyses were routinely performed by the feed supplier. No contaminants at levels exceeding the maximum concentrations 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 Study Director nor the Sponsor was awareof any agent present in the feed that was known to interfere with the results of this study. F8. 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. FS. Water Analysis: `The processed water is analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Inc., Lancaster, Pennsylvania) and monthly for possible bacterial contamination (Analytical Laboratories, Inc., Chalfont, Pennsylvania). Copies of the results of the water analyses are available in the raw data. CCei68 418-008:PAGE 11-11 Neither the Study Director the water that was known tnooirnttehrefeSrpeownistohrthweasresauwiatrseofotfhaisnystaudgye.nt present in F.10. Bedding: Bed-0'cobs was used Group, Maumee, Ohio) as nesting material (The Andersons Industrial Products F.11. Bedding Analysis: cNoeintthaemrintahentSsploinkesloyrtnoorbethpereSsteuntdyinDtirheectboerddwiansg aware of any potential that would interfere with the results of this annually and dsotcuduym.enAtneadlyisnetsheforrapwosdsaitbal.e contamination are conducted G. Methods: G1. Dosage Administration: Dosage| Dosage | Concantaton| Dosage Voiume| NumberofFo | NumberotF1 1 | Sroovee|| ((mry otate (mgm) Ug) | GenePreatriSoenxRats| GeneProartiSoenxRats [T [0[ o 5Tw o1n % | [Evw1TseeT 1oow[ |o 5T 1w 0 T 7} The test article was considered 100% pure for the purpose of dosage calculations erry169 G.2. Assigned Rat Numbers: 418-008:PAGE Il-12 [oes OoeanGrow [Wak Toor | | wsFooemuwin| [|eovrWwe | [| anemmmavs]]| [ov eowmwmo[womwvewms |oei0-rovs mre|| Team |wee [0Te a. sOenveDrLe2l1o,vaellefsfuercvtisviinnghpeuppsupinstdhueri1.n6 gmlgac/tatdioan,y dosage group (Group IV) were sacrificed because of b. There were no surviving pups afer DL2 inthe 3.2 mgaiday dosage group (Group V. G3. Rationale for Dosa ion: Dosages were selected by the Sponsor on the basisof previous studies conducted with the test article. G4. RAoudtmeionfistration: Oral (gavage) G.5. Rationale for Route of Administration: dTiheetaorryalro(ugtea,vatghee)erxoaucttedwoassagseelceacntebdefoarccuusreatbeelcyauadsmei:nis1)teirnecdo;mapnadri2s)onitiwsitohnet.he of the possible routes of human exposure. G6. Frequency of Administration: oTnhceeFdoaigleynbeergaitninoinnmga4l2e draatysswbeerfeorgeivceonhaabpiptraotipornia(twehdiochsacgoenstionfuetdhefotresat article maximum of 14 days) and continuing through the day before sacrifice. The Fo generation female rats were given the appropriate dosages article beginning 42 days before cohabitation (which continued for a of the test maximum of D14Gd2a4ys()raatnsdascsoingtinneudintog ntahtruoruaglhdeDlGive9ry(rtahtastadsisdingonteddetloivCearesaalriettaenr)-,soerctDiLon2i0ng), (rats that did deliver a liter). The dosage volume (5 mLkg) was adjusted daily on the basis of the most `approximately the same recently recorded time each day". body weight and given at a. See APPENDIX G, item 2. C0170 418-008:PAGE 11-13 Dams in the process of delivering pups were not dosed until completion of parturition, in order to preclude possible disruption of maternal behavior and/or ocannenidbaailliyzdatoisoanogef dtuhreinpguptsh.e dCeolnisveerqyuepnetrlioyd,. sNoomedadammmsiswseerde mnootreadtmhiannisotneered daily dosage. F1 generation male and female rats were given appropriate dosages of the test tarhtrioclueghortahlleyd(agyavbaegfeo)r,ebseagcirinfniicne.g on F2 dgaeyne1raptoisotnwpeuapnisnwge(rDeLn2o2t)d*iraencdtlycognitviennuitnhge gteessttaatrtiiocnle,(inbuuttemraoyexhpaovseurbee)eonrpvoisasimbaltyerenxaplosmieldk tdourtihnegttehste alratcitcalteidonurpienrgiod. G.7. LenofgSttudhy: Approximately 7 months G.8. Method of Study Perfor ce: G.8.a Fo Generation Rat All Fo generation rats were observed for viabilty at least twice daily during all openrcieoddsuroifntghtehsetaudcyc.limRaattisonweperreioadlsaonodbfsoerrcvleidnicfaolr ogbesneerrvaaltiaopnpseaorfaenffceectast olfeatshte test article, abortions, premature deliveries, prior to and approximately one hour after dosage and on the day sacrificed. `BaocdclyimwaetiigohntpserfioordF,owgeeenkelryadtuiroinngmatlhee rdaotssawgeereperreicoodradnedd at at slaecarsitfiocnec.eFdeueridng the `consumption values for male rats were recorded weekly during the dosage period. tBhoedayccwleiimgahttisonfopreFrioodg,enweereaktliyontofecmohaalbeitraattisown,erdeairlyecdourrdiendg atthelegaessttaotnicoen dpuerriinogd, on DLs 1,4,7, 10 and 14 (rats assigned to natural delivery) and at sacrifice. gFeesetdatcioonnspuemrpitodioanndvaolnueDsLwser1,e4r,e7c,or1d0edanwdee1k4ly(rtaotscaohsasbiigtnaetdiotno, ndaatiulryadluring the delivery). expected Feed consumption that the pups would values begin were not recorded after DL to consume maternal feed. 14, whenit was A table of random units was used to select 15 female rats per dosage group for evaluation of estrous cycling by examination of vaginal cytology for 28 days before the start of the cohabitation period. a See APPENDIX G, item 3. cCe171 418-008:PAGE II-14 cWoihtahbiintaetaicohn,doonseagmealgeroruapt,pceornfseemcaultievreato.rdTehrewcaoshaubsiteadtitoonapsesriigondrcatosnstiosted of a maximum of 14 days. During cohabitation, all female rats were evaluated daily until observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug in situ (DG 0), following which they were assigned to individual housing. Female rats that did not mate within the first seven days of cohabitation were assigned alternate male rats that had mated (within the same dosage days. group) and remained in cohabitation for a maximum of seven additional The first ten female rats per dosage group with a confirmed mating date were assigned to Caesarean-sectioning on Caesarean-sectioning were examined DG for 10. the The female number and rats assigned distribution of to corpora lutea, implantation sites and viable and nonviable fetuses. The remaining female rats were permitted to naturally deliver clinical observations during parturition, litters. These rats were evaluated duration of gestation (DG 0 to the for day the first pup was observed), litter size (all pups delivered) and pup viability at birth Pups that either appeared stillborn or that died before initial examination of the litters for viability were examined for vital status at birth. The lungs were srteilmlobovren;d paunpdsiwmimtherlsuendgsitnhwaattfelro.atPedupwserweitcholnusnigdsertehdatlsivaenbkorwneraendcotonshiadveereddied shortly after birth. Each liter was subsequently examined daily for pup viability. Maternal behavior of the dams was evaluateddaily when the pups were examined during the 21-day postpartum period. Observed maternal behavior wbeahsavrieocrorwdeerdeornecDoLrsde1d,,4i,f a7,nd14whaendn 2p1r.eseVnatr,iaotnioanlsl oftrhoemredxapyesctoefdthmeatpeorsntaplartum period. Fertility parameters were assessed for all dams assigned to natural delivery. These parameters included the fertiity index (percentage of matings that resulted resulted in in pregnancies), gestation index (percentage the birth of live litters), number of offspring of pregnancies that per litter (ive and dead Pups), number of implantation sites, general condition of the dam and litter during the postpartum period, viability indices (percentage of pups born that survived 4 and 7 days), and lactation index (percentage of pups born that survived 21 days). G.8b F1/F2 Generation Pups - Preweaning Observations Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Vital status at birth was determined for pups that either appeared stillborn or that died before initial examination of the litter for viability. Pups that either appeared 6CL72 418-008:PAGE II-15 stillborn or that died before initial examination of the litter for viability were `examined for vital status at birth, as previously described. Each litter was evaluated for viability at least twice each day during the 21-day postpartum period. Pups in each litter were counted once daily. Physical signs (including variations from expected nursing behavior and gross external physical anomalies) in the pups were recorded once daily for 21 days postpartum. Dead pups observed at these times were removed from the nesting box. When not precluded by autolysis or cannibalization by the dam, any pup found dead was necropsied and examined for the cause of death. Pup body weights were recorded on DLs 1 (birth), 4, 7, 14 and 21 Reflex and physical development parameters in the F1 generation pups only `were monitored {ability to right in during the 21-day 5 seconds (from DpLos1t)p},arptiunnmapeurnifoodl.dinSgur(ffarcoem rDiLght2)i,ngeyreeflex opening (from DL 12), acoustic startle response (from DL 13) and air righting reflex (from DL 14) were monitored until all pups (100%) in the litter reached the tcrhietenruiomnbfeorr tohfepsuppescipfiecr tleitstte.r wPiutphilthciosnrsetfrliecxtipornewseanst ewvaasluraetceodrdoendc.e on DL 21; G.8.c. F1 Generation Rats -- Postweaning Observations "Postweaning day" observations were recorded beginning on DL 22. All F1 ogfentehreatsituodny.ratRsawtesrweeorbesaelrsvoeodbfsoerrvvieabdilftory gaetnleeraaslt tawpipceeadraainlyceduartinlgeaasltl opnercieods during the acclimation period and for clinical observations of effects of the test article, abortions, premature deliveries prior to and approximately one hour after dosage and on the day sacrificed. Body weights for F1 generation female rats were recorded weekly during the pwoesetkwleyaenxicnegptpedruiroidnsg acnodhaabtitsaatciriofnicaen.dFaetesdacrciofincseu.mption values were recorded Bcoohdaybiwteatiigohnt,s dfaoirlyF1dugreinnegrpartieosnumfeemdagleestraattisowne, roen rDeLcsord1,ed4,o7ncaendwe1e4kl(ryattso assigned to natural delivery) and at sacrifice. Feed consumption values were 1re0c.oarndded14w . ete ocohk abil tatiy on, on DGs 0, 7, 10, 14, 17 and 20 and DLs 1, 4, 7, Beginning at 24 days of age, one male rat and one female rat from each litter in each dosage retention in a group were tested passive avoidance for learning, paradigm. short-term retention Each rat was tested and long-term on two days, separated by a one-week interval; the criterion for leaning was the same for both days of testing. The passive avoidance apparatus consisted of a two- compartment chamber with hinged Plexiglas lids. One compartment was 00173 418-008:PAGE II-16 outfitted outfitted with with a a bright light and Plexiglas grid floor to which a brief (1 floor. The other compartment second) pulse of mild electric was current (1 mA) was delivered. The two compartments were separated by a sliding door. During each trial, the rat was placed into the "bright" compartment, the sliding door was opened and the light was turned on. The rat was allowed to explore the apparatus until it entered the "dark" compartment. The sliding door was then immediately delivered to closed, the grid the light was turned off, floor. The rat was then and the brief pulse of current removed from the apparatus was and placed into a holding cage were repeated until the rat for 30 seconds before the start of the remained in the "bright" compartment next trial. Trials continuously for 6105 tsreiaclosnwdesreoncoemapclhetoefdt.woThcoensleatceuntciyvetotreinaltser(tthheecdraitrekricoonmfporarlteamrennitng)orotrheu.ntil maximum 60-second interval was recorded for each tral. Dosage groups were compared on the following dependent measures: the number of trials to the criterion in the first session (overall learning performance); tchoemplaatretnmceyn(tinosnetcroianld1s)oftotheentfeirrstttheest"dsaersks"icoonm(paactritvimteynltevferloamntdheex"pblroirgahtt"ory tendency in a novel environment); the latency (in seconds) to enter the "dark" `compartment from the "bright" compartment on trial 2 in the first test session (short-term retention); the number of rials to the criterion in the second test cseosmspiaorntm(leonntg-ftreormm trheete*nbtriiognh)t;"acnodmptahertlmaetnentcyon(itnrisael c1onindsth)etsoeecnotnerd ttheest"sdaersks"ion (long-term retention). On postpartum day 70, one male rat and one female rat from each litter were evaluated in a water-filled M-maze for overt coordination, swimming ability, slteeaerlnimnogdiafnidedmeMm-omrazye.. EaTchhe rmaatzweaswatesstfieldledinwaitwhawteartteirghtto a16d-egpatuhgeofstainless 0afpp2r1oxCima+t1elCy).ninOeniencahcehs,teasntdtrtiahle, twhaeterratwwaassmpolnaicteodreidntofotrhteesmtpaerrtaintgurpeos(irtiaonnge (baseof the M-maze stem farthest from the two arms) and required to swim to one of the two goalsof the M-maze, in order to be removed from the water. On the first trial, the rat was required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 was designated the incorrect goal during the remaining trials. Rats that failed to make a correct goal choice within 60 seconds in any given trial were guided to the correct goal and were then removed from the water. A 15-second intertrial interval separated teraiaclhs ttroiatle.rmEiancahteratthewatesstresqeusisrieodn.toTrheeacmhaaxicrmiutemrionnuomfbfeirveofcotnrisaelcsuitniavneyetrersotrless session was 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial. 0roa74 418-008:PAGE 1-17 iEnatcerhvarla;ttwhaescotrersetcetdgtowailce.andThtehetcersittsereisosniwoenrsewethreessaepmaerafotredbobtyh ateostnes-eswseieokns. Dosage groups were compared for the following dependent measures: the number of trials to criterion on the first day of testing; the average number of errors (incorrect tums in the maze) for each trial on the first day of testing; the latency (in seconds) to reach the correct goal on trial 2 of the first day of testing; otfheernruormsbeforrofetarcihaltsritalo ocnrittehreiosneocnontdhedasyecoofntdesdtainyg;ofantedsttihneg;ltahteenacvye(rian gseecnounmdbse)r to reach the correct goal on trial 1of day 2 of testing. Female rats were evaluated for the age of vaginal patency, beginning on DL 28. Male rats DL34. were evaluated for the age of preputial separation, beginning on On DLs 85 to 98, the F1 generation rats within each dosage group were assigned to cohabitation, one male rat per female rat, based on random unit tables, with the exclusionof sibling of a maximum of 14 days. Female matings. rats with The cohabitation period consisted spermatozoa observed in a smear of the vaginal to be at DG contents and/or 0 and assigned a copulatory to individual plug observed in housing. Female situ were rats that considered did not mate within the first 7 days of cohabitation were assigned alternate male rats from the same dosage group that had mated. Female rats were allowed to naturally deliver and maintain liters through a 21-day postpartum period. These rats were gestation (DG 0 evaluated to the day for the clinical observations during parturition, duration first pup was observed), litter size (all pups of delivered) and pup viability at birth. Pups that either appeared stillborn or that sdtiaetdusbeaftorbierthin.itiTalheexalmunignsatwieorneofrethmeovlietderasnfdorivmiambeilristyedweirn ewaetxear.minPeudpsfowritvihtal lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Each litter was subsequently examined daily for pup viability. Matemal behavior of the dams was evaluated daily when the pups were examined during the 21-day postpartum period. Observed maternal behavior was recorded on DLs 1, 4, 7, 14 and 21. Variations from expected maternal behavior were recorded, if and when present, on all other days of the postpartum period Fertility parameters were assessed for all dams assigned to natural delivery. These parameters included the fertility index (percentage of matings that resulted in pregnancies), gestation index (percentage of pregnancies that resulted in the birth of live litters), number of offspring per litter (ive and dead pups), number of implantation sites, general condition of the dam and litter during the postpartum period, viability indices (percentage of pups born that survived 4 and 7 days), and lactation index (percentage of pups born that survived 21 days). 0eL7s G9. Gross Necropsy: 418-008:PAGE Il-18 G.9.a Fo Generation Male and Female Rats Assigned to Pharmacokinetic Sample Collection Asitrsedchleidteurlseodf sdaacrmisficaellaofwteerd ctoompnalteutriaolnloy fdetlhievecroahalbitittear)tiaonndpoenrioDdL(2m1al(efermaatslethat rraatt)s waelrleowceodllteocntaetdurfarlolmy fdievleivreartas pleirttedro) sbalogoedgsraomupplfersom(atphperoixnifemraitoerlvye4namcLavpae,r 2intmoLs)ewrausm ismempeadriaatotreltyubferoszaenndoncedntrryifiucgeead.ndTmhaeinrtesauilnteidngfrsoezreunm((7a0ppCr)oxuinmtailtely shipment to the Sponsor for analysis. The liver was excised, weighed, and a t`hseamSppleonsseocrtifoonr (alnaatleyraslisl.obe) was frozen and retained at ~70C until shipment to ITVhethalitvwereorfeesaeclhecptuepd fforropmhtahremalictotekrisnoefttihcesfaimvepldeamcoslliencteiaocnhwoafsGerxocuispesd,| tphorooluegdh per liter, frozen and retained at 70C, until shipment to the Sponsor for analysis. surviving pTuhpesdoanmDsLin21t.he 3.2 mg/kg/day dosage group (Group V) did not have Gob. F Generation Male Rats cMoahlaebirtaattsiwoenrpeersiaocdr,ifaincdedabgyrcoasrsbnoencdrioopxsiydeofastphheytxhioartaicoicn,afatbedrocmoimnpalletainodn poefltvhice viscera was performed. Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. Representative photographs of gross lesions are available in the raw data. The following organs were excised, individually weighed and retained for possible histologic evaluation: testes, wepeirdeidfyimxeiddeisn,Bopuroisnt'astseolauntdiosnefmoirn4a8l vteos9i6clehsou(rwsitahnadndthweinthroeuttaifnlueidd).in nTehuetrtaelstes oburfgfaenrsedwe1r0e%rfeotramianleidninfonrepuotsrsalibblueffheirsetodpa1t0h%olofgoircmaallien.valuation. The remaining G.9.c. Fo Generation Female Rats Assigned toCaesarean-Sectioning Female rats assigned to Caesarean-sectioning were sacrificed on DG 10, and a gross necropsyofthe thoracic, abdominal and pelvic viscera was performed. Uteri of apparently nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites. All ovaries and gross lesions were a. A table of random units was used to select one control group Fo and nF1ecgreonpesryatwieorne rraettfarionemd,eaicn horsdeexr ftroopmrowvhiidcehcaolnlttrioslstuiessseuexsamfoirnpeodssaitble histopathological evaluations of gross lesions. reive 418-008:PAGE II-19 Rreetparienseedntinatnievuetrpahlobtuofgfrearpedhs1o0f%grfoosrmsalleisnifoonrspaorsesiabvlaeilfaubtluereinevtahleuartaiwond.ata. The oravtasrywearnedeixmapmlainnteadtifoonrstihteesn,uamnbdevriaabnlde dainsdtrinbountviioanoblfecoermpborryaosl.utAea viinabelaech feimllbedrywiothwacsleaorvaflluiodr. crAesncoennvtiasbhlaepeedm,brpyinok,wafisrmamaonrdpehnoculso,sesdmalinl,anpaalmenipoitnikctosac tan or deep red to cloudy, or opaque black, fluid. soft and Embryos enclosed in an amniotic sac filled were discarded after examination. with clear, G.9.d. Fo Generation Female Rats Generation Female Rats Assigned to Natural Delivery and F1 wAtertehesaccormipfliectedioonnoDfLth2e1,21a-nddayapgorsotspsanretcurmoppesryioodf,tahlel dthaomrsacitch,atadbedloimvienraedl laintders pelvic viscera was recorded. was performed. The number and Female rats assigned to natural distribution of implantation delivery that did not deliver sites a plirtteegr nwaenrceyssatcartiufsi,ceudteorni fDrGom2r5atasntdhaetxaapmpienaerdedfornognrposrseglneasinotnsw.erTeo sctoanifnierdm wtihteh l1a0st%paupmmwoansifuomunsduldfeidaed",.miDsasminsgwoirthprneossuumrevidvicnagnnpiubpasliwzeedr.e Asacgrriofsiscendeacfrtoerpstyhe rofettahienetdhoirnacniecu,traabldboumfifnearledan1d0%peflovrimcavliisnceforra pwoasssipbelreffourtmuerde.evAallluaotviaorni.es were wRaatssmtahdate.dieTdhweerraetsewxearmeineexdamfiornethdefcoraugsreososf ldeesaitonhs.onPtrheegdnaaynctyhestoabtsuesrvaantdion uterine contents were recorded. formalin Ovaries were retained in neutral buffered 10% G.9.e. F1G/eF2neratiPoupns On DL 21, pups were F1 generation sacrificed and pups not continued examined for gross on study lesions. and all F2 Necropsy generation procedures were the same as those used for pups culled on DL 4. vPiutaplssttahtautsdiatedbibrethf,oraesedxeasmcirniabteidopnroefvitouhselyl.ittePrufporspfuopunvdiabdieliatdy wweerree eevxaalmuianteedd ffoorr gross DLs1 lesions and for the cause of death. Pups with gross lesions to 4 were preserved in Bouin's solution. Gross lesions from found on pups found on DLs 5 to 21 were preserved in neutral photographs of pup gross lesions are buffered available 10% formalin. Representative in the raw data. dPiuopxsidneoatsspehlyexcitaetdiofnoracnodnteixnaumeidneevdalfuoartgiroonsosnleDsiLon4s.weNreecsraocprisfyiciendclbuydecdarabon single cross-sectionof the head at `examination of the cross-sectioned the level head for of the frontal and parietal apparent hydrocephaly. suture The and Cer? 418-008:PAGE II-20 satnodmIallc. hScaomnptleentssw(emrielkccoulrlde)ctweedrferocmolallelcpteudpsfrformomcuflilveedopfutphse flrarogmesGtrloiuttpesrs |i,nIl tpohleyspertohpryeleendeostaubgeesgaronudpsf.rozIenndiavtid~u2a0lpCu.pAsfatemrplcoemspwleetrieoncoofmbsianmepdlebycollitlteecrtiinotno, samples were shipped (frozen on dry ice) to the Sponsorforanalysis [elalob irk] G.10. Statistical Analyses: 418-008:PAGE Il-21 `The following schematic represents the statistical analyses of the data: Type of Test I. Parametric" A. Bartlett's Test" Il. Nonparametric A. Kruskal-Walis Test (575% ties) Significant at ps0.05 Nonparametric Not Significant | Analysis of Variance Significant atps0.05 Dunn's Test Not Significant Significant atp<0.05 | Dunnett's Test Not Significant B. Fisher's Exact Test (>75% ties) Ill. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilities are reported as either p<0.05 or p<0.01. b. Used only to analyze data with homogeneity of variance. c. Proportion data are not included in this category. d. Test for homogeneityofvariance. reL79 418-008:PAGE 11-22 Proportion data were analyzed Binomial Distribution". using the Variance Test for Homogeneity of the Continuous data (e.g., body weights, body weight consumption data) were analyzed using Bartlett's changes and feed Test of Homogeneity of TVeasrtiawnacsenso"t asnigdnitfhiecaAnntal(py>s0i.s05o)f]V. aIrfitahencAena,lywshiesnofapVparroiparnicaetewa[ise.s,iBganritfliectatn'ts i(npdsi0v.i0d5u)a,l Dgurnounpest.t'sIfTtehsetATMnawlayssisusoefdVatroiiadnencteifwyatshenosttataipsptricoaplrisaitgnei[fii.ec.a,nBcaertolfettth'es Test was significant (p<0.05)], the In cases where the Kruskal-Wallis Kruskal-Wallis Test" was used Test was statistically significant (575% ties). (p<0.05), sDiugnnni'fsicManectehoofdtohfeMiunldtiivpildeuaClogmropuapsr.isIof tnhserweawseruesegdretaotiedrentthiafny t7h5e%sttaiteiss,tical eFvisahleura'tseEnxeaccrtoTpseysdtatwaafsorutsheed.pupFisshwehri'cshEwxearcte Tsteislltb"ornwaorsfaolusnodudseeadd.to rDeaftlaexo/bpthyasiinceadl adtevCealeosaprmeeannt-aslecdtaitoanainngd,pnoasttuwraelandielnigvebreyh,apviroerwaelandaitnag involving discrete data (e.g., number of corpora lutea, number of criterion), were evaluated by the Kruskal-Wallis Test", pups per liter, trials as described above. to a r01S0 Hl. RESULTS -Fo GENERATION MALE 418-008:PAGE Ill-1 A. Clinical Observations (Summary - Table B1: Individual Data ble B10) No Fo generation male rats died during this study. Al clinical observations were considered unrelated to the test article because the incidences were not dosage-dependent and/or the observation occurred in only one or two rats. These observations included localized alopecia on the limbs, comeal opacity, abrasion on the dental problems (missing, broken or misaligned incisors), neck, hyperactivity, excess salvation, chromodacryorrhea, swollen snout, chromorhinorrhea and dyspnea. B. Body Weights and Body Weight Changes (Figure 1: Summaries Tables B2 and B3; Individual Data - Table B11 Groups administered 0.4 mg/kg/day and higher dosages of the test article had reduced ps0.01) body in the weight gains. The values were significantly 0.4 mg/kg/day dosage group on days 56 to reduced (p<0.05 63 of study (DSs or 56 t3o.263m)g;/ikngt/hdeay1.d6omsga/gkeg/gdraoyupdoonsaDgSesgr1o5utpoo2n2,D2S9st2o936t,o 3366;taon4d2ianntdhe56 to 63, Reflecting these effects of the test article, body weight gains weresignificantly arenddu3c.e2d m(gp/<k0g./0d1)ayfodrotshaegeentgirroeuppsr.ecoThahbeit0a.4t,io1n.6pearnidod3.(2DSmsg/1kgto/d4a2y) dinosthaege1.6 groups had significantly reduced (p<0.05 or p<0.01) body weight gainsfor the. entire study, calculated from DSs 1 to 63 or from DS 1 to termination. gBrooduypwoenigDhStssw5e6,re63siagnnidfiacatnsttlyudryedteurcmeidnat(iposn0..05T)hein 3t.h2e m1g.6/kmgg//dkagy/ddaoysadgoesaggreoup shtauddysitgenrifmiicnaanttiloyn.reduced (p<0.01) body weights on DSs 36, 42, 56, 63 and at Body weights and body weight changes were unaffected by the 0.1 mg/kg/day dosageofthe test article. Cc. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables B4 and BS; Individual Data - Table B12) Relative feed consumption values were significantly reduced (p<0.05) in the 1.6 and 3.2 mg/kg/day dosage groups on DSs 8 to 15. Absolute (g/day) and irneltahteiv3e.(2g/mkgg//kdga/y)dafyeeddoscaongseugmrpotuipononvaDlSuess1w5etroe2s2i,gn2i9fitcaon3tl6y arneddu3c6edto(4p2s.0.0A1s) a vreasluuletsofwetrheesreeedfufceecdtsaonfdt/hoer tseisgtniafritciacnltel,yarbesdoulucteed ainn dthreesleattivweofdeoedsacgoensgurmoputpisofnor ccecist 418-008:PAGE ll2 the entire precohabitation period (DSs 1 0 42). After the cohabitation period ((pDsS0s.0556otrop6s30),.0a1b)soinluttheef0e.e4dacnodns1u.6mpmtgi/okng/vdaalyuedsowseargee sgirgonuipfsi.cantAlbysorleudtueceadnd relative feed consumption values were significantly reduced in the 3.2 mg/kglday dosage group during this period. Absolute and relative feed consumption were unaffected by the 0.1 mg/kg/day dosage of the test article. D. Mating and Fertility (Summary - Table B6; Individual Data - TableB13) Dosages of and fertiity the test article as high parameters evaluated. aVsal3u.2esmgfo/rktgh/edafeyrtdiiidtynaotndafpfrecetgannanycmyatinidnigces. (number of pregnancies per numbeorf rats in cohabitation and rats that mated, respectively), the number of days to inseminate, the number of rats that mated and the number of rats with confirmed mating dates during the first week of cohabitation were comparable among the five dosage groups. TableB14) E. Necropsy Observations (Summary - Table B7; Individual Data - A significantly increased (p<0.01) number of male rats in the 3.2 mg/kg/day dosage group hada light brown or brown liver, an observation attributed to effects of PFOS. The gross lesions of theliver were considered related to the test article because the incidences were dosage-dependent. gOrnoeup1.r6atmsg(/8k2g4/5d,ay82d5o2s,a8g2e57g)rohuapd rat (8214) and three small, flaccidand/or 3.2 mg/kg/day dosage purple testes". All four of these male rats mated but did not impregnate a female rat. One 3.2 mg/kg/day dosage group male rat (8243) had a small prostate; this rat impregnated a fceomnsaildeerraetd: tTehsteagrrtoicslse-lreesliaotneds oabsstehrevyedarein ctohemmteosntesfianndidngpsroinstcaotnetrwoelrgeronoutp male rats and did not vary from Historical Control incidence at the Testing Facility. No other male rats had gross lesions of the reproductive organs. All other necropsy observations were considered unrelated to the test article because: 1) the observation occurred in only one rat in a group; or 2) the ilinvceri,deanlcievserwweirteh anortoduogshasguer-fdaecep,encdoensnttr.ictTehdespaepiolblsaerryvpartoicoensssinofcltuhdeedlivaerlawrigteh a. In 2079 control group rats from 91 studies conducted at the Testing fFlaaccicliitdyafnrdo/morAupguursptl,e t1e9st9e2s.to April, 1999, there were 21 rats with small, 060182 418-008:PAGE IIl-3 pinpoint raised tan areas, opaque lens of the eye, large kidneys, kidneys with cysts in the parenchyma, dilation of the renal pelvis and a large spleen. F. Terminal ights, Ore ightsand Ratios (%) of Organ Weight to Terminal Body Weight (Summaries - Tables BS and BS; Individual Data - Table B15) The 1.6 and 3.2 mg/kg/day dosage groups had significantly reduced (p<0.05 and ps0.01, respectively) terminal body weights. The absolute weights of the seminal vesicles with fluid and the prostate were significantly reduced (p<0.05 or p<0.01) in the 3.2 mg/kg/day dosage group, compared to the control group. Epididymides and testes weights and weights of the seminal vesicles without fluid were unaffected by dosages of the test article as high as 3.2 mg/kg/day. The ratios of the weight of the left and right testes to terminal body weights were significantly increased (p<0.01) in the 3.2 mg/kg/day dosage group, as compared to the control group values. These observations were associated with the significantly reduced (p<0.01) terminal body weights in this dosage group. The ratios of the weights of the epididymides, seminal vesicles and prostate were generally comparable among the five dosage groups. 00183 IV. RESULTS - Fo GENERATION FEMALE RATS 418-008:PAGE IV-1 A. Clinical Observations (Summary - Table C1; Individual Data - TableC22) A. Mortality No deaths were attributable to PFOS. The only death occurred in a 1.6 mg/kg/day dosage group rat, an event attributable to an intubation accident Female rat 8938 in the study day (DS) 36 after 1.6 mg/kg/day administration dosage group was moribund sacrificed of 35 daily dosages. Adverse clinical on observations included chromodacryorrhea and a clear oral exudate t0 36, and ptosis, hunched posture and labored breathing on DS 36. on DSs 35 Observations of chromodacryorrhea and ptosis were confirmed at necropsy; all other tissues appeared normal at necropsy. This rat lost body weight and had reduced feed consumption on DSs 29 to 36. The ciinical observations indicated this rat was misintubated on or about DS survived to scheduled sacrifice. 35. All other Fo generation female rats A2. Clinical Observations Observations of localized alopecia were increased during the precohabitation, gestation and lactation periods in the 0.4, 1.6 and 3.2 mg/kg/day dosage groups. The incidence of localized alopecia at any area was significantly increased (p<0.05 or p<0.01) in the 0.4, 1.6 and 3.2 mg/kg/day dosage groups during the gestation period. The incidence of localized alopecia on the back was significantly increased (p<0.01) in the 3.2 mg/kg/day dosage group during the. lactation period. Aplelroitohdesrwcelrineiccalonosbisdeerrveadtiuonnrseldautreidngtothteheprteesctohaarbtiictlaetiboenc,augseset:at1i)onthaendinlcaicdteantcioens were not dosage-dependent; and/or 2) the observation occurred in only one rat. Tbrhoekseen oobrsemrivsaatliiognnsedinicncliusdoersd).chrColmeaordaocrarlyoerxruhdaetae,anpdtodseisn,tahlupnrcohbeldempsos(tmuirsesianng,d labored breathing occurred during the precohabitation period in the 1.6 mg/kg/day rat that was moribund sacrificed, as previously described. B. Body Weights and Body Weight Changes (Figure 2:Summaries - Tables C2 through C7; Individual Data - Tables C23 through C25) B.A. Precohabitation Groups administered 1.6 mg/kg/day and higher dosages of the test article had significantly reduced (p<0.01) body weight gains for the entire precohabitation 000184 418-008:PAGE IV-2 period (DSs 1 to 42). During this period, values were significantly reduced (p<0.01) in the 1.6 mg/kg/day dosage group on DSs 29 to 36; and in the 3.2 mg/kg/day dosage group on DSs 8 10 15, 22 to 29 and 29 to 36, as compared with the control group value. Body weights were significantly reduced (p<0.01) in the 3.2 mg/kg/day dosage group on DS 15, 22, 29, 36 and 42, as `compared with the control group values. Body weights and body weight gains during the precohabitation period were unaffected by dosages of the test article as high as 0.4 mg/kg/day. B.2. Gestation The 1.6 and 3.2 mg/kg/day dosages of PFOS continued to reduce body weights and body weight gains during gestation. Reflecting effects that occurred during the precohabitation period, the 1.6 mg/kg/day dosage group tended to weigh less and the 3.2 mg/kg/day dosage group weighed significantly less (p<0.01) than the control group on day 0of gestation (DG 0). Weight gain was significantly reduced (p<0.05) in the 1.6 mg/kg/day dosage group on DGs 0 to 7 and then essentially comparable to the control group values at all intervals. throughout the remainderof gestation, beginning with DGs 10 to 12. Weight gains were significantly reduced in the 3.2 mg/kg/day dosage group on DGs 0 107 and 10 to 12, after which this group also had weight gains comparable to the control group. Reflecting these effects, body weight gains were significantly reduced (ps0.01) in the 3.2 mg/kg/day dosage group for the entire gestation period (DGs 0 to 20) and body weights were significantly reduced (p<0.05 or ps0.01) in the 1.6 and 3.2 mg/kg/day dosage groups on DGs 3 through 11 and 0 through 20, respectively. Body weights and body weight gains during gestation were unaffected by dosages of the test article as high as 0.4 mg/kg/day. B3. Lactation The 3.2 mg/kg/day dosage group had significantly reduced (p<0.01) body weight on DL 1, as compared with the control group value. Body weight gains tended to be reduced in the 0.4 mg/kg/day dosage group on DLs 1 to 4, when significant weight loss (ps0.01) occurred in the 1.6 mg/kg/day dosage group (the 3.2 mg/kglday dosage group was precluded from further evaluation because all pups died before DL 2). A significant increase (p<0.05) in body weight gain on DLs 10 to 14 in the 1.6 mg/kg/day dosage group was possibly associated with reduced litter size and associated with reduced milk demand and production. Body weights and body weight gains during lactation were unaffected by the 0.1 mg/kg/day dosage of the test article. 000185 418-008:PAGE IV-3 C. A{bSsuomlmuatreie(sg/-daTya)balensd CR8elatthirvoeug(gh/Ck1a/3d;ayI)ndFieveidduaCloDnastuamp-tTiaobnleVsaCl2ue6s throughC28) CA. Precohabitation Ainbstohleu1t.e6(agn/dday3).2amngd/rkegl/adtiavye d(og/skagg/edagy)rofuepesd dcuornisnugmtphteioprnevcoahlaubeistawteiroen rpeerdiuocd.ed gTrhoeuspeoenffDecStss w2e2r0e s2i9gnaifnidca2nt9 (tpo<306.0(5abosroplsu0t.e0o1n)lyi)n, tahned1i.n6 tmhge/3k.g2/dmagy/kdgo/dsaayg.e dosage group at most tabulated intervals. Reflecting these effectsof the test aarbtsioclleu,ttehaen3d.2remlga/tikvge/fdeaeyddcoosnasguempgtrioounp vhaalduessigfnoirfitchaentelnytirreedpurceecdoh(apb<i0t.a0t1i)on period (DS 1 to 42). Feed consumption values during the precohabitation period were unaffected by dosages of the test article as high as 0.4 mg/kg/day. C2. Gestation The 1.6 and 3.2 mg/kg/day dosage groups continued to have reduced absolute and relative feed consumption values early in the gestation period. Absolute and relative feed consumption values were significantly reduced (p<0.05 or p<0.01) in the 1.6 mg/kg/day dosage group on DGs 0 to 7 (absolute only) and 7 to 10, and in the 3.2 mg/kg/day dosage group on DGs Oto 7, 7 to 10, 10 to 12 (absolute only). Significantly increased (ps0.05 or ps0.01) feed consumption values occurred in the 1.6 and 3.2 mg/kg/day dosage groups on DGs 15 to 18 (absolute and relative) and DGs 18 to 20 (relative only), observations interrelated with reduced consumption body tends weights and to increase. the later Despite stages of gestation, when maternal the increases in relative feed feed consumption values, the 3.2 mg/kg/day dosage group hada significantly reduced (p<0.01) absolute feed consumption value for the entire gestation period (DGs 0 to 20). Feed consumption values during gestation were unaffected by dosages of the test article as high as 0.4 mg/kg/day. C3. Lactation Absolute and relative maternal feed consumption values tended to be reduced in the 0.4 mg/kg/day dosage group and were significantly reduced (p<0.01) in the 1.6 mg/kg/day dosage group for the entire lactation period (DLs 1 to 14), and at most intervals within this period. The 3.2 mg/kg/day dosage group was precluded from evaluation because there were no surviving pups after DL 1. 0186 418-008:PAGE IV-4 Feed consumption values during lactation were unaffected by the 0.1 mg/kg/day dosage of the test article. D. Estrous Cycling, Mating and Fertility (Summary - Table C14; individual Data - Table C29) Dosages of the test article as high as 3.2 mg/kg/day did not affect estrous cycling (number of estrus stages per 28-days) in the 15 rats per dosage group that were evaluated. All female rats mated except one in the 0.4 mg/kg/day dosage group. The number of days in cohabitation, the fertiity and pregnancy indices (numberofpregnancies per number of rats that mated and rats in cohabitation, respectively), and the number of rats with confirmed mating dates during the first and second week of cohabitation were comparable in the five dosage groups. Pregnancy occurred in 33, 32, 28, 29 and 30 female rats in Groups | through V, respectively. E. Necropsy Observations (Summary - Table C15; Individual Data - Table C30) All necropsy observations were considered unrelated to treatment. One rat in the 0.4 mg/kg/day dosage group had moderate dilation of the pelvis of the right kidney, an observation that is common in this species and strain. One rat in the 1.6 mg/kg/day dosage group had adhesion of abdominal adipose tissue and the spleen. No other gross lesions occurred in the female rats in this study. F. Caesarean-Sectioning and Litter Observations (Summary - Table C16; Individual Data -Tables C31 through C33) Caesarean-sectioning observations on DG 10 were based on 10 (100%), 7 (70.0%). 6 (80.0%), 9 (90.0%) and 9 (30.0%) pregnant dams in eachofthe five respective dosage groups. There were no biologically important or statistically significant differences in the litter averages for corpora lutea, implantations, viable embryos or nonviable embryos. No dam had litter in which there were no live embryos. G. Natural Delivery and Litter Observations (Summaries - Tables C17 and C18; Individual Data - Tables C34 through C37 Although 25 presumed pregnant rats in each dosage group were assigned to natural delivery, because one rat (1.6 mg/kg/day dosage group) was moribund sacrificed during the premating period (on DS 36), as previously described, this dosage group had only 24 rats assigned to natural delivery. Of these rats, 20 to 25 were pregnant and delivered litters. C0187 418-008:PAGE IV-5 d`Tohseadgueragtrioounp,ofagnesotbasteirovnatwiaosn saisgsnoifciicaatnetldywriethdupcreedim(pplsa0n.t0a1t)ioinn ltohses3[.t2hemga/vekrga/gdea.y number of implantation sites per dam was significantly reduced (p<0.01), resulting article. in a significantly reduced (p<0.01), litter size] and an effect of the test Pup viability was significantly reduced (p<0.05 3.2 mg/kg/day dosage groups, as described in or p<0.01) in the following the 1.6 and information. wAlatshocuogmhpatrhaebgleestaactrioonssinaldlefxiv(ethdeopseargceegnrtoaugpes,oftphree1g.n6anmtg/rkatgs/dwiatyhdloivseaogfefsgprrionugp) DhaLd1,inacnrdeassiegdninfiucmanbtelrysinocfrpeuaspesdth(apt<0d.i0e5d oorrpw<e0r.e01p)rensuummbeedrscaonfnpiubaplsizdeedadonor gprroeuspumheadd cpaonsntiibmaplliaznteadtoionnDlLosss2e0vid4enatndas5rteod7u.ceTdhpeu3p.2vimagbi/lktgy/adtabyirdtohs[aognee liter had no significantly livebom pups, the number of dams with stillborn pups was increased (p<0.01), the litter average and number of stilloom pups lwievreebosringnpiufipcsanwtelyreinscirgeniafsiecdan(tpl<y0r.e01d)u,ceadnd(pt<h0e.0l1i)t]te,r aasvewreallgeasanpderniupamrbteurmodfeaths [significant numbers (p<0.05 or ps0.01)ofdead and presumed cannibalized npuupmsbeornsD(Lps<01.0a1n)dof2dtaom4s(ahlladliavllebpourpspudpesaddioerd pbryeDsLum2e)dacnadnnsiigbnailfiizceadnton DLs 1 to (p<0.01) 4]. Reflecting in the 1.6 and t3.h2esmeg/efkfge/cdtsa,ytdhoesvaiagbeiligtryouipnsd,exthweaslacstiagntiifoincainntdleyxrweadusced also significantly reduced (ps0.01) in the 1.6 mg/kg/day dosage group, as also dwoesraegtehegraovueprabgeegsinfnoirnsgurovnivDinLg 4pupopsstc(upl<li0n.g0,5 aonrdp<i0n .t0h1e)3i.n2 tmhge/1k.g6/dmagy/kdgo/sdaagye group on DL 1. A dosage-dependent pattern of reduced group administered the test article. The pup body weight was evident 1.6 mg/kg/day dosage group in each had significantly reduced (p<0.01) pup body weights 3.2 mgkgiday dosage group had a significantly on all weighing days. reduced (p<0.01) pup The body weight on DL 1 (no pups survived to subsequent scheduled weighings). The percentage of male pups was comparable across all five dosage groups. H. CNleicnricoaplsOybOsbesrevravtaitoinosnfsro(mSuBmirmtahritoesDa- yTa2b1lePsosCt1p9aratnudmCa2n1d; Individual Data - Tables C38 and C45) Aadnvdeprosteenctliianlicraeldauncdtionnecirnompastyeombaslercvaarteiooncscuarsrseodciianttehdew3i.t2h mrge/dkugc/eddaypudposviaagbielity group. Adverse clinical observations include three (p<0.01) litters with pups that were been not nursing (two of removed, and one these litters had pups from which the placenta had not had pups that were not nested). Apparent maternal 60188 418-008:PAGE IV-6 acanndniabaclainznaitbiaolnizweadsfaolrseoliemvbidinenatnointhtehirspduop)s,agwehigcrhoumpay(mhisasviengoctcaiulrirneodnaeftperup these pups died. Necropsy observations in pups that were stillborn or found dead included significant increases (p<0.05 or p<0.01) in the numbers of pups with no milk in the stomach in the 1.6 and 3.2 mg/kg/day dosage group pups. No pups (p<0.01) in the 0.1 mg/kg/day dosage group had this observation, as compared with two iconnctrreoalsgerdomuapteprunpasl. cTanhneib3a.l2izmagt/ikong/adtanyedcorsoapsgye ogfrsotuipllablosmoahnaddfeovuinddendceeadofpups. Nine of these pups had missing hindlimbs and/or portion of the tail, Aclolnostihdeerrecdlinuincralelaantdednteocrtohepstyesotbasretricvlaetbioencsauisnet:he1F)1thgeenienrcaitdieonncepsuwpesrweernoet odbosseargvea-tdieopnesnidnecnltu;deodr a2)dtohmeeodbsheeravda,tipoonrtoicocnuorfretdheintaoilnlmyisosniengl,ittuemr.bilCilcianlichaelrnia, missing digits and bent tail. One 1.6 mg/kg/day dosage group stillborn pup had anasarca (edemaof the entire body). Necropsy on DL 21 revealed two 0.4 mg/kg/day dosage group littermates with moderate or severe dilation of the lateral ventricles of the brain, grouplitterwith slight dilation and one pup from of the renal pelvis. a different 0.4 mg/kg/day dosage I. Reflex and Physical Development (Summary - Table C20: Individual Data - Tables C3 through C: Reversible delays in with body weight" reflex and physical occurred in the 0.4 development that are highly correlated and 1.6 mg/kg/day dosage groups, as described below. 11. SurfaceRighting dSeusrcfraicbeerdigbhetlionwg.waTshedeplearyceedntinagteheof1.p6uapsndth3a.t2cmogu/lkdgs/udrafyacdeosriagghtewgarsoups, as significantly reduced (p<0.05or ps0.01) in the 1.6 mg/kg/day dosage group on `DwLhsich3,t4he,r5e,w6e,r7e,8noansdur1v0i,viangndpuinpsthien 3t.h2ismdgo/skagg/edagyroduops.agTehegraovueproangeDdLay1,tahfatter at least 50% of the pups in a dosage group had the ability to surface right was significantly increased (p<0.05) in the 1.6 mg/kg/day dosage group. pups ultimately attained the ability to surface right. All surviving `The ability to surface right was unaffected by the 0.4 mg/kg/day dosage of the test article. 00489 418-008:PAGE IV-7 12. PinnaUnfolding Pinna unfolding was delayed in the 0.1, 0.4 and 1.6 mg/kg/day dosage groups. (no 3.2 mg/kg/day dosage group pups were evaluated for this parameter). The effect was transient (evident on only one day) in the 0.1 and 0.4 mg/kg/day dosage groups, and considered of no toxicological importance [the percentages of pups per litter with pinna unfolding were significantly reduced (p<0.05 or p<0.01) in the 0.1 and 0.4 mg/kg/day dosage groups on DL 3 only]. The percentage of pups with an unfolded pinna was significantly reduced (p<0.01) on DLs 3, 4 and 5) in the 1.6 mg/kg/day dosage group, and the average day that at least 50% of the pups in a dosage group had an unfolded pinna was significantly increased (p<0.01) in this dosage group. All surviving pups ultimately had unfolded pinnae. 1.3. Eye Opening Eye opening was delayed in the 0.1, 0.4 and 1.6 mg/kg/day dosage groups (no. 3.2 mg/kg/day dosage group pups were evaluated for this parameter). The effect was transient (evident on only one day) in the 0.1 and 0.4 mg/kg/day dosage groups, and considered of no toxicological importance [the percentages of pups per liter with open eye lids were significantly reduced (00.05) in the 0.1 and 0.4 mg/kg/day dosage groups on DL 14 only]. The percentage of pups with at least one open eye was significantly reduced (p<0.01) in the 1.6 mg/kg/day dosage group on DLs 14, 15 and 16. The day that at least 50% of the pups had atleast ane open eye was significantly increased (p<0.01) in the 0.4 and 1.6 mg/kg/day dosage groups. All pups had open eyelids by DL 21, when pupil constriction was tested. I4. AcousticStartle The time of development of the acoustic startle reflex was delayed (p<0.05 or ps0.01) in the 1.6 mg/kg/day dosage group; no 3.2 mg/kg/day dosage group pups survived to be tested for this reflex. The percentage of pups in the 1.6 mg/kg/day dosage group with this reflex was significantly reduced (p<0.05 or ps0.01) on DLs 13, 14, 16, 17, 18, 19 and 20, resulting in a tendency for an increase in the average day that at least 50% of the pups had the acoustic startie reflex. `The development of the acoustic startle reflex was unaffected by the 0.1 and 0.4 mg/kg/day dosages of the test article. 15. AiRrighting The ability to air right was delayed (p<0.01) in the 0.4 and 1.6 mg/kg/day dosage groups; no 3.2 mg/kg/day dosage group pups survived to be tested for this 0190 418-008:PAGE IV-8 0re.f4lemxg./kTgh/edaefyfedcotswaagse tgrraonuspi,enotccaunrdrinnogtoonfltyoxoincoDlLogi1c6.al Timhpeorptearncceentiangteheof pups. i(np<t0h.e011).6omngD/kLgs/d1a4ytdhroosuagghe2g1r,oruepsutlhtaitngaiirn righted was significantly reduced a significant increase (00.01) in the day that at least 50% of the pups could air right. 1.6. Pupil Constriction All live pups in the 0 (Vehicle), 0.1, 0.4 and 1.6 mg/kg/day dosage groups had the pupil constriction response present on DL 21 000191 418-008:PAGE V-1 V. RESULTS - F1 GENERATION MALE AND FEMALE RATS A. FiGeneration Male Rats AA. Mortality and Clinical Observations (Summary -Table D1; Individual Data - Table D13) A.1.a. Mortality gNrooudpeartahtss winertheefFo1ungdendeeraadtioonn dmaaylse8r6atsanwder8e7aptotsritbwuetaenditnog.PFTOhS.eseTdweoatcohnstrol were attributed to intubation errors. One 0.1 mg/kg/day dosage group rat was found dead on day 66 postweaning; this death was considered unrelated to the. test article because it was a single, nondosage-dependent effect. Observations for these rats that died are provided below. Control group male rat 13105 was found dead on day 87 postweaning, one hour and 11 minutes after the 87th daily dosage. No other adverse clinical observations occurred in this rat, and its body weight gains and feed consumption values were comparable to other rats in this dosage group. Necropsy of the rat revealed that all lung lobes were dark red, which in association with the time death occurred, is compatible with an intubation accident Control group male rat 13116 was found dead on day 86 postweaning, after it had been administered 85 daily dosages. Additional adverse ciinical observations in this rat were limited to chromorhinorrhea (days 67 to 72 and 79 postweaning). Body weight gains and feed consumption values in this rat were comparable to those of other rats in this dosage group. Necropsy of the rat revealed dark red clotted material (presumed to be blood) in the thoracic cavity and a red perinasal substance, observations compatible with an intubation accident. Male rat 13138 (0.1 mg/kg/day dosage group) was found dead on day 66 postweaning, after it had been administered 65 daily dosages. No other adverse clinical observations occurred in this rat, and its body weight gains and feed consumption values were comparable to those of other rats in this dosage group. All issues appeared normal at necropsy. A.b. Clinical Observations All adverse clinical observations were considered unrelated to the test article because: 1) the incidences were not dosage-dependent; and/or 2) the observation occurred in only one or two rats. A significant increase (p<0.01) in the incidence of localized alopecia on the back in the 0.1 mg/kg/day dosage 000192 418-008:PAGE V-2 dgerpoeunpdewnats. noNtocoontsheirdesrteatdisttriecaaltlmyesnitg-nriefliactaentd ibneccraeuasseestihneavnalaudevwerassencoltindicoaslageoubnsreerlvataetdiotno otchceutrersetd.artAidcldeitiinocnlauldeaddvdeernstealclpirnoicballesmisgn(smiosbssiengr,vebdroaknedn caonnds/iodrered fmoirsealliimgbneodr biancciks,orcsh),rocmhordoamcorryhoirnrohrerahe,al,acaebrraatsiioonnaotnthtehebahseeado,f ntehaertatilh,eaesyew,ollen snout, a red perioral substance, a red substance on penis, localized alopecia on the back, limbs and/or head, a scab on the back, urine-stained abdominal fur, soft or liquid feces, swollen and purple ears, rales, gasping, excess salivation and dilated pupils. A2. Body Weights and Body Weight Changes (Figure 2; Summaries - Tables D2 and D3; Individual Data - Table D14) The 0.1and 0.4 mg/kg/day dosage groups tended to weigh less than the control group on day 1 postweaning and generally gained one or two grams less body weight per week than the control group rats, with the exception of days 71 to 78, when these groups had significant reductions (p<0.05 or p<0.01) in body weight gains, as compared with the control group value. Reflecting this pattern of weight gain, weight gains in the 0.1 and 0.4 mg/kg/day dosage groups for the 9pr7e.c6o%hoabfittahteiocnontpreorliogdro(duapyva1lpueo,strweespaencitnigvetloy.prBecoodhyabwietiagthiotng)awinesrein9t8h.e40%.1anadnd 0.4 mg/kg/day dosage groups for day 1 to termination were 98.5% and 96.6% of the control group value, respectively. A3. Absolute (a/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables D4 and D5; Individual Data - Table D15) Absolute (glday) feed consumption values were significantly reduced (p<0.05 and p<0.01, respectively) and relative (g/kg/day) feed tended to be reduced in the 0.1 and 0.4 mg/kg/day dosage groups during the first week of intubation (days 1 to 8 postweaning), as compared to the control group values. These observations commonly occur and were associated with the tendency for reduced body weight in these two dosage groups on day 1 postweaning. A significant increase (p<0.05) in the relative feed consumption value in the 0.1 mg/kg/day dosage group on days 22 to 29 postweaning was considered outnhreerlastteadtisttoictahlelytessitgnairftiiccalnet bdeicfafeurseenctehseovcacluurerewdasin ntohte dfoeseadgceo-ndseupmepntdieontn.vaNluoes for this portion of the study. Ad. Sexual Maturation (Summary - Table D6: Individual Data - Table D16) Dosages of the test article as high as 0.4 mg/kg/day did not affect the average day of preputial separation in the F1 generation male rats. No significant differences occurred among the three dosage groups. 000193 418-008:PAGE V-3 AS. Passive Avoidance Performance (Summary - Table D; Individual Data - Table D17) There were no biologically important differences in the values for learning, shortterm retention, long-term retention or response inhibition in the F1 generation male rats, as evaluated by performance in a passive avoidance paradigm. No statistically significant differences occurred in the number of trials to criterion, trial latencies or numbers of rats that failed to learn. AS. Watermaze Performance (Summary -Table D8; Individual Data - Table D18) No biologically important dosage-dependent differences occurred in watermaze. performance of the F1 generation male rats regarding leaming, short-term retention, long-term retention or response inhibition. No statistically significant differences occurred in the number of trials to criterion, tral latencies or numbers of rats that failed to learn. A7. Mating and Fertility (Summary - Table D; Individual Data - Table D19) Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertity parameters evaluated in the F1 generation male rats. Values for the fertility and pregnancy indices (number of pregnancies per number of rats that mated and rats in cohabitation, respectively), the number of days to inseminate, the number of rats that mated and the number of rats with confirmed mating dates during the first week of cohabitation were comparable among the three dosage groups. Of the male rats assigned to cohabitation, 82.6%, 83.3% and 96.0% impregnated the cohort female rat. A8. Necropsy Observations (Summary - Table D10; Individual Data - Table D20) All necropsy observations in the F1 generation male rats were considered unrelated to the test article because: 1) the incidences were not dosagedependent; and/or 2) the observation commonly occurs in this strain of rat. Gross lesions of male reproductive organs included flaccid, small and/or purple testes and epididymides in three (p<0.01) 0.1 mg/kg/day dosage group male rats (13134, 13145, 13148), each of which sired a litter). These observations were considered unrelated to the test article because: 1) the incidences were not dosage-dependent; and 2) these findings are known to be genetically mediated `and common in this rat strain. No other gross lesion occurred at a statistically significant incidence. 00194 418-008:PAGE V4 Additional necropsy observations considered unrelated to the test article included red abdominal adipose tissue with firm areas (one 0.4 mg/kg/day dosage group male rat; 13162), and urinary tract lesions, as described in the following information. One 0.1 mg/kg/day dosage group male rat (13143) had a dilated ureter; another rat in this dosage group (13134) had slight dilation of the renal pelvis. One 0.4 mg/kg/day dosage group male rat (13173) had large kidneys with numerous fluid-filed cysts in the parenchyma; the left kidney had slight dilation of the pelvis, in which there were six calculi, and thickened urinary bladder walls associated with the presence of numerous calculi. Necropsy observations in the control group fats that died as the result of an intubation error were described previously. AS. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight (Summaries - Tables D11 and D12; individual Data - Table D21 No statistically significant differences occurred in the absolute of relative (to body weight) values for the weightsofthe right or left testis, seminal vesicles (with and without fluid), right epididymis or prostate. Terminal body weights tended to be reduced in the 0.1 and 0.4 mg/kg/day dosage groups; the terminal body weights in the 0.1 and 0.4 mg/kg/day dosage groups were 98.3% and 96.4%, respectively, of the control group value. B. F1GenerationFemaleRats B.A. Mortality and Clinical Observations (Summary -Table E1; Individual Data - Tables E23) B.1.a. Mortality No deaths in the F1 generation female rats were attributed to PFOS. One control group rat was sacrificed on day 7 postweaning, because it was a male rat that had been incorrectly identified as a female rat. One control group rat was found dead on day 28 postweaning; no cause of death was identified for this rat. One rat in the 0.4 mg/kg/day dosage group was found dead on day 10 of lactation (DL 10) as the result of an intubation accident. Observations for these rats are provided below. No gross lesions were revealed by necropsy of control group rat 13205 on day 7 postweaning The control group female rat (13221) that was found dead on day 28 postweaning, after it had been administered 27 daily dosages did not have any other adverse clinical observations, and its body weight gains and feed 00495 418-008:PAGE V-5 consumption values were comparable to other rats in this dosage group. All tissues appeared normal at necropsy. Female rat 13257 (0.4 mg/kg/day dosage group) was found dead on the morning of DL 10, after it had been administered a total of 106 daily dosages. No other 1a0d7v.ersItes fcleiendicaclonosbsuemrpvtaitoinonvsaloucecsurwreerdeinuntrhiesmarartk,aabllteh.ouTghhisit rlaotstdweleiivgehrtedon15DLs 1 liveborn pups, of which seven were culled on DL 4 and eight were sacrificed after wtihtehdaenatihntoufbatthieodnaamc.cidNeenctr(owphsiyteoff otahem dinatmheretvreaaclheeda,garpopsrsolxeismiaotneslcyo2mpmaLtiboflea light orange fluid in the thoracic cavity, and dark red lungs). B.1.b. Clinical Observations A red perivaginal substance occurred in one and two dams in the 0.1 and 0.4 mg/kg/day dosage groups, respectively, on days 21 or 22 of gestation, and red perivaginal substance was also observed on DL 1 in five (ps0.01) dams in the with 0.4 mg/kg/day dosage group. Th parturition, rather than an effect eofsethoebtseesrtvaarttiicolnes, parnodb adibdlynowteraeppaesasroctioat ed adversely affect the health of the dams or their offspring. No other adverse clinical observation occurred at a statistically significant incidence. All other adverse clinical observations that occurred during the precohabitation, gestation and lactation periods were considered unrelated to the test article obceccuarurseed: in1)ontlhye oinneciodrentcweas rwaetsr;eannodt/odros3)agteh-edoebpseenrdveantti;on2i)stcheomombsoenrvianttihoen laboratory environment. These observations included dental problems (misaligned, missing and/or broken teeth), chromodacryorrhea, swollen and purple ears, localized alopecia on the back, head, limbs and/or underside, abrasion or scab on the back, urine-stained abdominal fur, missing hindpaw dfiegcietss,, pmeirsisvianggintaaill sswheelaltihn,g,tispwooflltaeinl fbloarcekpaowr,mcihsrsoimnogrhainndorarbhreaas,iosnofotnorthleiqcuhiedst. B.2. Maternal Body Weights and Body Weight Changes (Figure 4; Summaries - Tables E2 through E7; Individual Data- Tables E24 through E26) B.2.a. Precohabitation Body weights tended to be reduced in the 0.4 mg/kg/day dosage group on day 1 postweaning. No statistically significant or dosage-dependent differences in fperemcaolheabriattsatdiuorninbgodthyewperiegchothagbaiitnastioornbpoedryiowde.ights occurred in the F1 generation 0CC196 418-008:PAGE V-6 B.2.b. Gestation Maternal body weights and body weight gains during the gestation period were. unaffected by dosages of the test article as high as 0.4 mg/kg/day. All values were comparable among the three dosage groups and did not significantly differ. B.2.c. Lactation Significant (50.05) maternal 0.4 mg/kg/day dosage group, body weight loss occurred on DLs as compared to the control group 1 to 4 value, in the after which group body weight values. gains were greater than, or weight loss less than the control Maternal body weights and body weight gains during the lactation period were unaffected by the 0.1 mg/kg/day dosage of the test article. B.3. Maternal Absolute (g/day) and Relative (g/ke/day) Feed Consumption Values (Summaries - Tables E8 through E13; Individual Data Tables E27 through E29) B.3.a. Precohabitation The 0.4 mg/kg/day dosage of the test article was associated with a significant reduction (p<0.05) in absolute (g/day) feed consumption and a tendency for reduced relative (g/kg/day) feed consumption on days 1 to 8 postweaning, observations that were associated with the tendency for smaller body weight in this group on day 1 postweaning. Absolute and relative feed consumption values for the remainder of the precohabitation period were comparable and did not significantly differ among the three dosage groups. B.3.b. Gestation Absolute and relative feed consumption values during the gestation period were unaffected by dosages of the test article as high as 0.4 mg/kg/day. All values were comparable among the three dosage groups and did not significantly differ. B.3.c. Lactation The absolute maternal feed consumption value was significantly reduced (p<0.05) on DLs 1 to 4 in the 0.4 mg/kg/day dosage group, as compared to the control group value. The relative feed consumption value was also reduced, however not significantly (9>0.05). These observations were associated with the significant weight loss that occurred in this group on DLs 1 to 4 and attributed to the test article. cei97? 418-008:PAGE V-7 Absolute and relative feed consumption values during the lactation period were. unaffected by the 0.1 mg/kg/day dosage of the test article. B4. Sexual Maturation (Summary - Table E14; Individual Data - Table E30 Dosages of the test article as high as 0.4 mg/kg/day did not affect the average day of vaginal patency in the F1 generation female rats. No significant differences occurred among the three dosage groups. B.S. Passive Avoidance Performance (Summary - Table E15; Individual Data -Table E31) There were no biologically important differences in the values for learning, shortterm retention, long-term retention or response inhibition in the F1 generation female rats, as evaluated by performance in a passive avoidance paradigm. No statistically significant differences occurred in the trial latencies or numbers of rats that failed to lean. The number of trials to criterion was significantly reduced (p<0.05) for the female rats in the 0.4 mg/kg/day dosage group, as compared to the control group value. This observation was not considered an effect of the test article because an increase in the number of rials to criterion, rather than a decrease, would be expected as an indication of toxicity. B.6. Watermaze Performance (Summary - Table E16; Individual Data - Table E32) No biologically important dosage-dependent differences occurred in watermaze performance of the F1 generation female rats regarding learning, short-term retention, long-term retention or response inhibition. No statistically significant differences occurred in the number of trials to criterion, tral latencies or numbers of rats that failed to learn. B.7. Mating and Fertility (Summary - Table E17; Individual Data - Table E33) Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertiity parameters evaluated in the F1 generation female rats. Values for the number of days in cohabitation, the number of rats that mated, the fertility and pregnancy indices (number of pregnancies per number of rats that mated and rats in cohabitation, respectively), and the number of rats with confirmed mating dates during the first and second week of cohabitation were comparable among the three dosage groups. C0198 418-008:PAGE V-8 REVISED PAGE B.8. Necropsy Observations (Summary - Table E18; Individual Data Table E34) All necropsy unrelated to observations in the F1 generation female rats were considered the test article because: 1) the observations occurred in rats in the Tcohnetsroelogbrsoeurpv;atainodns2)itnhcleuodbesderrvoautgihonancdo/mormopintlteydoccocrutresx oinf tthhies kstirdanienyosfwriatth, tcahlecsuleiriantsthaelsKoidhnaedyaantda/nogrruarniunlaarrymbaltaedrdiealr oifn tthweorceonnatlroclorgtreoxuapnrdatst.hiOckneeneodf walls of the urinary bladder. Necropsy observations for the 0.4 mg/kg/day dosage group previously. dam that died as the result of an intubation error were described B.9. Natural Delivery and Litter Observations (Summaries - Tables E19 and E20; Individual Data - Tables E35 through E38) Pregnancy occurred in 22 (95.6%), 21 (84.0%) and 24 (96.0%) of the 23, 25 and 25 female rats assigned to cohabitation in the 0 (Vehicle), 0.1 and 0.4 mg/kg/day dinodseaxge(thgeropueprsc,enretsapgeectoifveplrye.gnAalntprreagtsnawnitthdlaivmesofdfeslpirvienrg)edwlaitstecrso.mpTahreagbelsetation across the three dosage groups. The viability of the second generation offspring (F2 pups) was unaffected at the highest dosage tested, 0.4 mg/kg/day. Small increases in the number of dams with stillborn pups and in the number of pup deaths after DL 2 in the. 0.4 mg/kg/day dosage group were not toxicologically important because: 1) the values were not significantly different from the control group values; 2) the average for live litter sizes delivered and surviving to DL 21 were greater than or comparable to the control group values; and 3) neither the viability of lactation indices for the group values. 0.4 mg/kg/day dosage group remarkably differed from the control Pup body weights of the second generation offspring (F2 pups) were unaffected at the highest dosage tested, 0.4 mg/kg/day. Small reductions in pup body weights in the 0.1 and 0.4 mg/kg/day dosage groups were not toxicologically important because: 1) the small differences between the pup body weights for athnedcwoentrreolasasnodci0a.t1emdgw/iktgh/tdhaeyldaorgsearglevegrloituteprssiwzeerseonfotthesta0t.i1stmicga/lklyg/sidganyifdicoasnatge group on DLs1 through 4, as compared with the control group values, and the random selection of pups for continued observations on DL 4; 2) the small reductions in pup body weights in the 0.4 mg/kg/day dosage group were associated with a minimally larger live litter size at birth (DL 1) and on DL 4 preculling, as compared with the control group values, and the random selection of pups for continued observation on DL 4; the statistically significant reductions 50199 418-008:PAGE V-9 REVISED PAGE (p<0.05 and p<0.01, respectively) present on DLs 7 and 14, as compared to the control group values, were transient and disappeared by DL 21 Adminisration of the test article at dosages as high as 0.4 mg/kg/day did not adversely affect any other parameter evaluated at natural delivery or during the 21-day lactation period (duration of gestation, averages for implantations and live fitter sizes, numbers of dams with all pups dying during lactation, viability and lactation indices, surviving pups per litter and pup sex ratios). B.10. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations (Summaries - Tables E21 and E22; Individual Data - Tables E39 and E40) No clinical or necropsy observations were attributable to dosages of the test article as high as 0.4 mg/kg/day because: 1) the incidences were not dosagedependent; and/or 2) the observation occurred in only one or two pups. These clinical observations included one control group pup that was not nesting or nursing, had decreased motor activity and was cold to the touch, one pup in each of the control and 0.4 mg/kg/day dosage groups that had a missing tip of tail and one 0.4 mg/kg/day dosage group pup that had an umbilical hernia. No milk in stomach occurred in 2, 4 and 4 pups that were found dead in the three respective dosage groups. One control group pup had hydrocephaly and one 0.1 mg/kg/day dosage group pup hada raised tan area on the median and left lateral lobes of the liver at necropsy on DL 21. CCZ0o0 418-008:PAGE V-10 REFERENCES 1. Study Design as Modification of: U.S. Food and Drug Administration (1994). International Conference on Harmonisation; Guideline on detection of toxicity to reproduction for medicinal products. Federal Register, September 22, 1994, Vol. 59, No. 183. 2. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. 3. JPraapcatniceeseStMainndisatrrdy foofrHSeaafletthyaSntdudWieelsfoanreD(r1u9g9s7,).MHGoWoOdrLdaibnoarnacteory Number 21, March 26, 1997. 4. European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Communityof an OECD decision/recommendation on compliance with principles of good laboratory practice. Legislation. 32 (No. Official Journal of the European L 315; 28 October): 1-17. Communities: 5. Christian, M.S. and Voytek, P.E. (1982). In Vivo Reproductive and Mutagenicity Tests. Environmental Protection Agency, Washington, D.C. National Technical Information Springfield, VA 22161 Service, U.S. Department of Commerce, 6. Cnharlitsrteixaonn,eM.(SP.ro(c1e9e8d4i)n.gsReopfrNoadlutcrteixvoenetoSxiycmiptyosaindumt,erNateowloYgoyrekvaAlcuaatdieomnys of 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:CD7BR Rat. Charles River Laboratories, Inc., Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 8. Institute of Laboratory Animal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 9. Salewski, E. (1964). Implantationsstellen Farbemethode zum am Uterus der Ratte. makroskopischen Nachweis von Arch. Pathol. Exp. Pharmakol. 247:367. 10. Snedecor, GW. and Cochran, W.G. (1967). Variance test for homogeneity of the binomial distribution. Statistical Methods, 6th Edition, lowa State University Press, Ames, pp. 240-241. CCZo1 418-008:PAGE V-11 11. Sokal, R.R. and Rohlf, F.J. (1969). Bartlett's test of homogeneity of vpapr.i3an7c0e-s3.71Biometry, W.H. Freeman and Co., San Francisco, 12. Snedecor, GW. and Cochran, W.G. (1967). Analysisof Variance. Statistical Methods, 6th Edition, lowa State University Press, Ames, pp. 258-275, 13. Dunnett, CW. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1121. 14. Sokal, R.R. and Rohlf, F.J. (1969). Kruskal-Wallis Test. Biometry, W.H. Freeman and Co., San Francisco, pp. 388-389. 15. Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6(3):241-252. 16. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, pp. 96-104. 17. Lochry, EA. Hoberman, A.M. and Christian, M.S. (1984). Positive cloarnrdemlaartikosn. ofTepruaptobloodgyy w29e(i2g)h:t44wAi.th other commonly used developmental ccozo2 APPENDIAX REPORT FIGURES 0203 | wo - J- :ega] BODY WEIGHTS Fo GENERATFIiOguNrM1eALE RATS ----- arene arco 0||a--avn-cow =o= y |esencxconr iA ? Sa pleon oo Q3 UTTARoavofEsuoE wa ww g Te B[o=s)o 3 : Fo GENBEORDATYIFOaWuNErFIe?EGMHATLSE RATS : : 4 FA 7\ N a . 01 MONGDAY og EF : i oe as i oot" - FT et { Bid ant = 2i EERE ER 7 |orenaon WS |= ; SR :iE ; F==erl i : 3 ea SET: ean 3 -~ = TThada STITT Van ab hhh amd 11} hdd 2 3 & + transom za F1 GEBNOEDRAYTWIEOINGMAHLTESRATS, Figur3e I. - el anencun arco --[v--R-- ' = 5 c-asera A 2 CTR ER eeu 2 S8$ Betas 7 rr o--t BODY WEIGHTS ; Lo os - i sR Gus |i 4 en a\d A er `CsE n i: 3 2 Camb en dl EE I A Th baum hhhhwnd 1) aa 38g DAYPOSTWEANING "DAYOF GESTATION venaDAYOFLACTATION 22 z APPENDIX B REPORT TABLES - Fo GENERATION MALE RATS 0208 Jen 223 $ weilw ouiwmey a wou ul 3 g T+L1 SEtElesCa SSrtUtaTrIenNt fGmIthe va eers cmomednt p gros r value (ppent. 03). g8 2 32 25 ?2 iQ 3 B2 8B 2 3 & B8 t p------ $32 3 BI g? ++ BEITEEESEMINesinn wm et it es 12 soitLScSos sEspRio onie, EE rem . .332 g & 2 2 3 J ap . . . s '. .: 33 BB g 3 ummm m G.C iin, HEH iS.e er . Soin . tine 5 wipeene . ~g g : TL iO MGS. 0s 80 Swan ea mies ea ames es was ase 1 eo Sanioncs u i TH 2 2 TEL Toarios 3 Gncan wkiour reise apt VEiGHx 10. 0 rn) oan des Ca ata 3 3 883 2g S 33 FwErEL, ORR es - $3 5a 3 2 g2 itz bs rHR H wn BIiTneO sr . +. chservationconirmedat necropay. 23 88 - -- 32 a3 e3 3;88 5 2z sd SHES 3 a " 5IE] a. "ebaervation confirmed at necropsy. - " i ote 2 3  3 & 888 z 3 g B8 owE J lin ESEEREER mma -- a 3 2g Rr 32 323 i&B z 2g :i 38 = 2 "333 280 2 8~ 2 i2 8 g EE a TT in wi ee a 22 2 8i8 2 eT :a sro 2 z8 388 z8 3 2 g 388 324 Rr 8 g | eH Ei i g g 8 . : 3 8 8& 8 a 3 8 8 8 iig222 &g3 - 28 288 8 & 238g2 :3 2 ovat 3 233 8 g 88@ 8 PRTRTEAREE 2 22 a 388 g FEitimenn, 5g23 Z:i . i; 2: 2: B 3 3& 28 % 2 338a3 2R?8 : frie, i . 3 Viiv Tope Tacorrectly recorded aod wis ovchoded from rou averages sed acaisticel anaiyses & o23g :8$ a & Q82 f MESSI we 3 3 3 7 8 : i :  ~ . oon 2 5 Bt eesaT I rE habitation period and vas conapiced sith a second female at. & 22 38 %85 z kor) va as wan eet #g3 FERRER CONFINED NC +MOT CoMpimNED. Se wm - 3i3 32 i2? #3 i 3 J i Pe OR fA oc 82 bBEOrdnERaIEE tums rts nt vo comms wi ce ets3 on 3 ? & 2 2 ag i 3 i FE -- <. oy BN 3 2B i s Era poo rr rescresseress - VARY onsie vs 3 BEEE RE tones cote i ot ri 8 3 i sr omm ow re 22 aFf EEBE 0&0 Mnmu imme 32 8 3[I Hp1 ERGELwEommumEE -- & I ELI rit china cmon aie te ef ser ose meso scion 33i z i22 ig f 32 BiHETiEff Ii p ELmmmm rmmmes mmm g3i 2B 8 wFBiIma cr cmon ve a ot tor eet ones: tit re, & Tn de it wie ames oo 3-2 B8BE EYBEfE i E ESomep msshe mEEns msmm 2:z 8a sien Be TOO. = - a mefer to the individual clinical observations table (Table B10) for external cbaervations confirsed at mecropey. 23 2B EF i pigs : mew BvenwReEonEo.WmEm sNm warn 38 8 in rr 0 FEET we 2 82: ; B80 Mumm & -3 WHOEERS mMpiemeeeoE 23 33 H2E.R5 4i p MEmn RNEw ERE :2 Bn tan cites secon bie ve 101 to ert soon cote secre a3388 g853 L:BE BOEEHEEA R E EEe E BHr EEiL GH z 2 ALVes WERE RBCORDED I GANS 10) AMS. WE. + ORGANWEIGHT. ne To = (ORGANWEIGHT TERMINAL BobY WEIGHT] % 160. NL oT ns Recon Th GRAN (3. Abe. WE oa u n RRL 3 Tees RE o ica x 100 in3: i3 ~ oF iu . i3 fe rr asorn wei iL ee onan wera Team boy WEIGHT) x 160 ai i ici es wi + .2 32 3&g 2 &5 BE HH Egg EEEaEy BTERSIS EAEEREEREAEEENREEY | 5 : i POT on So CO,onI.St, RARE, ern Tn nn 3 2 f HOW Eom 2 2 NLVaiGHTS Whe SECORORD TH Gane 6 es WE. Oho EIGHT. MEL. \ TI (ORGANVElGHT TAAL BOY WEIGHT 4 100 g i 2 3 235 Z& ao 3 BBOeBmO WWoEHS oOAR E oi MfHmoommmonomomoHoH 33 #BE se erm rir mT 8 2g i2 a 3 g2 nd anal 3 a haan weanSoo,ic x i :3 :z FS MOIR ou a I i oa a Bd on 2 3 A Bel a. a vs 3 DErr A eaves crm spanish ret veneers Eur ae a @ noSTAY, exstulist 000 vp Wh WR AIH SES, a i 38 - BE ne 20. pIe uee 2g aaa &8 "nv va--" wa vw 22 8 zi 3 3i" . 2g&: er a i a THES Vm wom wm WTR wm ale BoE OHH EH EHEH EEE 2 2 NLLWEIGHTS WERE RBCORDRD. IN GRANS (0). ARG. WT. .ONAN WEIGHT. REL. \ TEN + (ONGAM WETGHT/TERMINAL BOON WEIGHT) x 160, . nEc RindiTvidualMnIeVcrIopsy obpsuerrpvlaetitonnsd tfalbalcecid(Tadbele Bc1o4)s.ts values excludedtromgroup averages 4nd statistical amalyses. See the 0 LS wi 2828 3? 8 P 33 F ET wor mr EEm T , SEp ey ae op t schmg itt ET 2 2 gi&o 3 337 ? APPENDIX C REPORT TABLES - Fo GENERATION FEMALE RATS 6CR86 rpm---- wonworn zo ows ers ens a se de we me ow ow rJe -- FE R E REIS 32 8 52 e2 EE arn II rss. osraemores cmmemcronns STATTore RARER Fhsv oRS ARS0 118 1TH oRSATIOn Gvgoeooowmwl wa we UwyLaouwu se u wwai we we wa 22 Ro3 8@ 8 -] ion s . - hb, > 2 ern -- pre iT ve mows owe ou: heii a a re agree sa, A PLR Lo i ens z3- 3 8g 3 2:8 3g En cdg ut a 3 3 a? g8 g EE 4 2 + Significantly different from the vehicle control geoup valve (ped. 91). 3 285 8 32i ;g &  SIE TRE EG grr of wo. ERE EEREy 2 28 8g $ ; &2388 g EL 3 z e382 3 8 i -- al JM SEL wan ae 3 8o> = 5 & Fr SIE a oisst of vo RN 328 a . 2g } EE a Sr 2 EL i 228i8 ig ween ain NERS TRAST TOR i.wee men Ta he h The RST Th ACTeteT Eo EL SeSn nab ot gion a 3 3288 o = Ss 233g ii322 g one meme elles wearme mime veesmee { EEERHE ESrTtU,E fHaer iA R t hekoRtE erSeHihofote es cin : 2 3 R 3 g 8 . -.2 $ 882 gg Sy rn oid ee nt PE cores pesesmsornss Samar ns san as ape sus b | ERE R ST a .i 2 38 oeg2a 2 a ET -33 33 gg 2g& 22 38 3g 88 CE 333&3 3 wom CR wt 24 we oh LL 2 8ag EE HEME ERE WEI MH EE E w or i on rs er i ne , ERR TER ER ay -- gg 3 32 g 3 g23 RB ee mn aviwa wanes wii, LnddT 2 3 DEERE vin em 2 3 of ERE AI ey te, itydr iho soso. ) Excludes values for litter 8a43; the dem delivered one assitional pup on day 2 of lactation. 8 3 PER TE BETAS B L a ttuni tly itoerceLnitlFireomvitshee bveahsicleerecortsieolteatoTuopovralwuee(fposusnt05]prior to velghing on deydar 3 pestoncpaste. 58bEbRdEDEAR ERa EaEno sE estonE ee "g rma wae oe ve oe wr rr EE ve oe oe FN woo. sm wae ve ws ve ov JrIBi T ar i iE id, ER ISm ve a a sg 3 3 i23 a22 5  4.) Tavern Thetacobfgspups sscing th criterion or the developmental Hnduch Sted on exch day pstpurn, 2 Eh CI or ash, thnsete ehasdicionst op on dn 3 of Bacestion. ' 33 IEEE Ren Te B8 Y 23 He --" 3 EL HEY ---- g2 QI Siero fom th viele Sorel Ses vas Ese on zgE3S 0i - g 8  2 i3 3282 } i 8: 3 BET 5 - 32 33 2& 25 PE EE RL etm. I) BE BE ii SiEE EE I EE 32 325 g Eow.. SEBS. . oifiHoon EbEEHrEEMoEEnER ee 3i3 3385 VERE ee o i i2ra BaO E==E NoAovmaes rivormes 0 Bo. ees i 8 FELLA Ee se ea ' or  233 82 B7RdB EA BEES -- Ferwen 0G + DAY OF PAESINEDGESTATION OL + DAY OFLACTATION or 3 2 / Sa8 &<Q Bom CECE me 3 2 D 8 F mFa BNemaim we 3 ii} S o 8 SE mnt i 8 3 88 i i 33 g 8 33 32 2 8Hy & 332 ;: 5 s Eman. i 2 & 3 8 2 23 38 & R 8 2 33 R 88 5 or So ems np es -3 2 2 88 2 s 8 8g88 2 : 5 8 2 RL veiacnsaabrCoRsoED Inones (G1 2$ggg 2z8 : sg :8 g 8 EE sh i i<5 ii 3Z3 :; 83 . 2 2 g S8 3 58 888 4 iQ BA. 8 8 : g & g ne awr ncra i 2 =2g re 3 2 89? ' i: : a rnin vr+ wor phirvais ei a Een 2 - a 33 23 o i 3 '3 8 2 : 3 o 333 2 32 23 25 33 8 : s i g g s wae gl te Tt : 3222 gg$ 2 2 i; i : 2 e 2 8 8 2 328 23g HERAT 5 . 3 2g & g2 i g 33 a NLL Secs MERE RECORDED TH GRANS. (0) i 3 2 g g = EE nT 42 08 son op 97 pr eok fsmg ppn 0 s g 8 3 4 sgg&g Z3 2 32 s82a $ : g i 8a2 bY : o 2 sf em FERRER a 2888 - - 3 *& 3 so 8 '8 g 3e & i i g : i 5 3  sg ggg i 3 8 8 83 f . i 3g g LTE SR . 223 g2822 & i i 2 3 HOE RERE 8 8g 3 8 :3 4 8 g g 3 LT 00 1, tors en,nn ee LL . 322 3 &$ 3 i g i 8g 3B aFae 2 2 ] 1 ; :5 ig aE Go nes J x rni i -s gg3 eerie nell -- g NT. - a 3& g i 5 i @ aWEiia Fo EL 2g 3 EC. 3 i 8 & wR EE EEE E EERE i : 1 2& z 8g ST eai eo ss i : 22 : 38 2 3 g 38o8& 22@2 i: 8 rt .2 i Cg 23 3 2 3 483 o8 2 RE Sn, 2g g2 & 4 TITIES Td retteuSuicssaiaion o thin wae - 3 3 22 2 8&a 8 BE REAR er i Tg 2 g i :2i :3 8 gg :8 i 8 R 8 : 8 s 8 3gs 83 DLMA mgmt B. 5pilled feed preciuded the calculation of this value. Stele diy shot eturion, 2 8 8 TUTE 8 22 2 8 HEELERS1 em oncnt tn te sro ion : 2g2 5 283 88 28 FE ram es ere mt men i 2 2g 2%8 g2 ' 2? 2 ors. Sst,o, sem Rs en Ss FERITERISI CEcove re ir -- g:z & g2 8 Q 2 g8 gg om Lo Da sixormoreconsecudtaiyvoef diestive chaerved. BL RTI a of nten chuee o~n Co ' 2i or - - s wre ERE en wy SEED ann g . . 2S8 m wm iTR " s -- . _-- i. .: 38 -- : - .: ps :: g I Lo - Co : 8 i ETEEEhmrmmae ; oe : : 2 - . B 3 BL o Mo contirmedamiatrineg da ate. sof dissin sbserad or - me er ws 3 ' 2 or Bh - Co a = : no ; gn : is : 2 2 iN pwimeooEwnhoob0 I HHoo NMCETREE Nmreawe no Nem te on 33 8 Qg Em Eo nie cerior oo Gein Te A ITE II et conervacions canble (Table CE for external cbarunons contrac ac necrepny. 8 2 8a HOE 1p Bimmer 5B gs EEEdEoiRsEsuOPOP OEFE MmihmITEimEmRmRm ah gz }8 MEETS ividual clinical observations table (Table C32) for external pmobservations conticaad at necrapey. *@ s Eo pEEmm= HE NE oe 2 8 BwooOOgEpR fRf n mMimsmsmhmmeeeomm Tos lor or smn 00 Laur or rassmin) olson 04 ONY 07 iacrarion or or g 8 w"e oe3 - =v moogh mooEn oF pF MMuimsmihmaelSm wr 82 8 wis LLtrie Se " oir aporsana Ak TI ANTES vase 2 B 32 L TIEcus cosrmcios cate fate co fox sacan comevtions snes a econ. g 2 2&S 2 3 Bos} Ymmem 2 3 Bog 7 mimmimmem 2 5 - moarmomnsr wp +y8b6or+sorhF u d ws se, HR - 4. meter to the individual clinical obaervatioss cable (Table C32) for external sbasrvations contirmed at necroper. 2 8 : 3 4 2 g8 g B g2 $8 5g: i: 2 : i 32 22 8 g g23i : 8 3 :i 8 8  3 g 3Z: 23 s g28 38&8g2 2i 3 g : 3 8 5es 38 WeUs Wows A A a a aa ax aa iaaaaah haiaa aaaaaa i gg 2 R 2 3 2 2 2 ;: g i a Ee a Dano banores sosirion on camvix 3 > TT Ce i2 2 8 MINE A A A A A A A A AJA A A aA a a tr $g ga 3 2 EER aed DAS 23 g& Ll 8  {EEE 8 SI oR ELI LRT acces me sermono vases 3 8 EERE nN 8 Flolid LH 8 EEE LB esse J. PhS tit lB, 8 EB o T Ln nLamass A vexing ervor "ua found prior to weighing on day 7 postpartum, viLaluteLos gfonrtedeays4spwSosotpearsteumesposstoS ee.ullienT gsvseree,vie a22 eh BIE PRALIL va an on : obF SEE RREE i 8 2 2 8 dso dd debe gg IEEE HLEEER AT meme monn rnin i: 3 5 g 2 i 8 z an Bi i TL oF ER mn LttS e i LEs ii meosr g : 08 2 : oF Erm rs a rs i 3 bE 2 4. 2- Oanrbdiatyhar1i3iypodsitpsatrrtiubau.tedthbeetpuupcsenftrhoem tluiottderm80497ndwaerleoedsiscooilv]erebde Iinnchtuhseedlitbtner31boouxp wSvietrhaslietesr sad$s6.caTehetn iSaseiyetcs 52 3 :2 e Biet LL oe 82 82 HR ASRLEABBHsa A 8 8g foe . BERR BE 5 Eh rmmma 3 L 3TrLet Tr Tol RHE LATE RLL AT 8gg s & 3 32 | ih g 8 3a 2 3 8 4 San 8043deliveroende additional pup on dey 3 OF LactACion; Values for day postpartum excluded from group averages and 2 2 z 2 < m 8g8 i 8 3 g 3 2o $ 8 8 & 2 z & re TT esses sooner NARS POLLOUING "SECOND LETTER" INOICATES TNE DAY FOSTIRRIN THE SVEN GECoRmTRoD. CI ALIZED) 3: . 3 8 2 2 g8 PHOT 0-58 CROMECADGE) RTI. EYOTA. APATA ROFROTAL SRGTiOn TORIC TY 0 2 8 3S a 82 23o 2 a Teton "oesLETT WOKATTSh on os Eo seen OIE 3 5a 22 e8 & EE AN is AN Son, cout, wns(aso omits g g gg a8 I@] 3g : 3 2 momar on se . & Sg22 iQe I g228 Sn - or Zg :5 H 30 2S g4 8 ne pa LE 3 : 2 TLRS TEAE Ot I coe, nEonien(neeesscsoroammscrim pctimwe 83 nciuted from 3roup avecsges and sat aCion) Mnayees. 2 2 2 222 8 ats orLouth Sons ESTEROS TCATThESSETPAR To Eo Sees TEOED CAMIIALIZEO) 3 g38 8 zg 8 : : EY EE Bar co, ty me ov 233 2 hi ics ane consi anes (61 - - 8 Suton Torre "NAL orLiaoin0:TRCRRTAIL 0160. C.CULLED NNISSING (PAESNED CHRIBALEEED) B 288 23g 253 & een Pointe"SRC LHTLRINTEAHTEESSh MSTRAREON HE EUSeco, MISES! 2 28 2 8 2%  RS Foiours eon rr barTeEsSH SoS HEBe Seen ASIII) 3 s8 e8 2a a oT PRE ' PELE 2 2 iBtRmTorri ALI, 5SvALSOR .NCHAALN: nan OID C.COLLED. WNIESING (RESONED CAMIIALEED) gg 3# 45 2 .2:2 me AMER Foun SAGORS LETTER TNOLCATES TE a TOSTTARTONTScece i r-- CE) 3 :2 g 2 2 8 g hie ha g 2 R a& 8 2 38 2 Ga5 z8 28 a g222 2 aaa 3[ o & iv ShonLETTER wOTCATES HE SAYPBA Bo seep, ED CAALIZEOL 3 2 g 5 3 BEeR m EoT n A : : 3 3 ER ye 3 A 8 2 27& EE rr eee PE FLERE BM Sr ie ttTe, : $3 3 8 <g 3 :3 2 piigbik z g8g R323 32 : z: 2 3 ? BOARIIEIEIE HITE Re, i 5 Bn A a a SRAI ELF pron oho nar ces 1 $: 2 / 8 g 28 3 g |bon HER 5 day1atputr he ptui(naLikes TE LI TeLmPciL $e) veredisconarad bn he Bitter box wich ister ane. The pope were te 2i 3 2 a i g a 2 23 8 oF ner a 310 ors, rica wens 1 GhmmlciLSSIS I Sse sh oer mes spaces secs 5R2 2 2a 3 Po A ST RT - ERE GREE - Eman, w we R ROHRSRRWROwrBEwL wMEOwWe oaa oa -rr MI SiAe TAIA TAAIE 3 ae saerns saeoins evaeire ee . N/M <WNAEROF UPS IhRACKLITTERWITH DEVELOMWENTAL WEASURE/TOTALWAGER OF SUS 1hTHELITTER 2$232 :i $23 - 4$ 8 oi GT oo - ws ule Sa San ain 0 sos A ATE Vin hveormen WERE Wa 07 rs ivr wesarerits Tr z 3 2 8 3 ATERAL/LITTE8R warenoosaaechou 11 osama or ww BE UWRW oom EEE ER URED un er ae wr owe wo IIR R IEW ae oan gw HE YH Ba 35 IL ER i 3 ; IN ara0 Pons I BACH LETTER Vi DEVRLOMENTALNEASORR/TOTAL WAGER OFPUPTSh THE Lire o '32 g2 8a B3 - BASS saa wo IEICE emo oar omnownoa au vw BE RY E AE um 3 2 w-w RBEL ZLELG, 83 Ss Sn ain is see ms eee 33TWN anorPUPS In BACKLITTER WITH DRVELOPENTALNEAGURE/TOTALINBEROFFURS INTHE LiTTER 8 ia i . 3i :38S - 2 g wT i aa - noon BOEHNER aon wmn eGEoEESy Go4R BERBBBEBEE w RRO RGagEESa wmmoe esm BROOWB E RMRE OWE BSARAEE MRuEtOW a 23: 8 re o heir ieSd SEo E A ii " ---- a 3 8 pent 7 aa aa -- ees evensrerpti mvoe WARUEERIE E E OEE NMA SOWAROEWL aonh oun 3 g a 8 e 33 8 2 gg Ri 3 wn ee---------- wT e------ sor sn 8 wn BR w mmmmmi tntsn ate i g 3 TR i i oa a a g::3 i: willl LJ : wot a 2 Win awER OF FURS TN BACKLITTER WITH DEVELOPMENTAL NENEURE/TOTAL WNOGF PEUPSR1H THE LITTER 2 332 8 A Ar $38 g2:3 "areas + areas coset cious 1 woe or wn um we oi esmaroar Co ce - Co wa ie a em ws ww tm ha WEEE da nm ame 3 Ya Wn ae %2 Vin Iinnosr ons I EACH LETTER ETH DEVELOPRNTAL NEASURE/TOTAL WAGER OF PUPS IN THE LITTER : &? or & - Roa 2 g23E 2 8 ws iA ene - FEE . w-o d GhaaaNmE aWRlEAe we R23 - da Aa Q ino ir i Sn Lien WH SHLAA MASHERROMER 5FO BYTH LTTER 8 2?8 Ro riea SAL SRT i or hn : 3 8g '$8 2&2 - BEEN - BEE -- BBORpB AaA n 2 NL Wh OF ups 1X EACH LITTER WITHGRVRLOMENTAL NEASURE/TOTAL WAGER OF PUPS I TH LiTTRR g&3S :3 or Q2g a a ol on we ERE ER g 8 :58 gmmmm mm m 22 a Bot TIL.0. 3 -- G0 WL AER OF PUBS I BAGH LITTER WITH DEVELOPMENTAL NEASURE/TOTAL INGER OF FURS4 Ti 8 = 3 g2: 8 2 iE - BL, wg w - g Ea g32 - AAA H 22 LG OF UPS TH ACH TIER TH ORVELOENTAL NEBR TOTALWAGER OF PS 1H THE LITTER - or 2 38 ELL SE - BUY. 8 2 82 23 mE Ea mo Ea moo g gw m E EEh T aa i o wes. VOWS WS & ~ erie era ee asie;i Wh WAR oF PS. I BACK LITTER WITH ORVELOMENTAL NEMEURR/TOTAL WAGEROFUPS1.4 THE LITTER - wn oir n 2 a si EE : 3g3: 2 2 ww dna HN WEounn GN FEE I -- E TL a : a WON we & &BE EE TE ep, 3 "- da aa 2 WR Wa AOnWdaaya 1o5 rporsetvpsartIuhs.EACtHheLpIuTTpEsRfWrIoTmHDliEtVteErLO$N83T7AvLerNeEdAiSsUcRoEv/eOrTedLIWntBheE JOiFttFeUrFSboxTNvTiHtEh iliteterr 8096. Thepups vere =g ; 2 ig i 8 = Stu WN ue on shennan un ee os ahhh NS, I. wpe we we sa owe or NEN ew i or da Ya da a dd ale 2 Hr EE ve Sapemp a,aa ll] sAL SA 5i Rr YT TE SE 25 2 f H a : : 2 8 PO % E Bwo mm--R--R ma ws 8 on 90 SURVIVING FUP OW DAY 2 OF LACTATION & FE Sr I g :8 2i@ 3 ig moa RE 8 WIN WNBER OF PUPS IN EACH LITTER WITH DEVELOPMENTAL NEASURE/TOTAL WONDER OF PUBS 1h THE LITTER i&: - - Sa8s 2gi 8 Ce -aEBEo EEa aT gE Ta RATg Co !TT w Ho zg =wo 4Hw ana 2 23 gQ g R a i SE Ss A 3 g 2 a 2 8 3 wT aT - BE a i Bh " E -- SR own - oon wT we wy gr we wa SAU we ae wy : -wR HBRAWER 2 & g mda 2 CIENHREREESEIEE : w-r aAoer are BAS o WN we in ain 0 or IA LT i SERS TaNOld T du a NEARSTri WoF Fe ors oe ren Te 22 2 2 {a 22 8a wo EER aoa owe oa I a w BAS ummm - hth Chit Sa allel 2 3 = < To ~ N SE gqH g2 WIN +WAR OF PUPS IN EACH LITTER ITH DEVELOPMENTAL NEASURE/TOTAL WNBER OF FURS IN THE LITTER 8 5 : 2 3 2 8 ATAL/LITIE|R ari boda a oenoue 3.3 arvajonr J 2 I 20 nv sos oon 3 or sacarcn aSQE Lm 30 ein wo ox ov 3 cr acon " . 2 8 2 3 .g : i -- hG h d Ed wm wa - Hod, g3g: - hod 0 WNL NER or rs Te ACK LITTERWITHDRVELOMENTAL MEAGURE/TOTALWAMEROF FURS TH THELITTER or i2 moO A ge g . :8 ig "es SON we wa EL wo WA WH -PO BuIu Ee ww Aad IJ CAS 2 mm IAT a lis 22 CMA WanoF PUPS TH BACH LETTER WITH ORVELTEVTAL REASOVEITOTAL WER OFPOPS TNTHE LETTER a i 3 8 or 8 ee AWE CU ne wer vO ONE we ON Wh ue a 8 '3 2 2a a mom w wma ENE wo REE BA 3 " Wn am me em ee 9 &8 . i: i 3i 5 moo EEE a aH Es 85H uh Eh US Uh ua un e FhOSn uh Lh Gn un eee mooE EHH . - Yada la ala dW daa 2 en SOU N wa we we we S 3:gg?S - ey WN NBER Gp bobs IHBACKLITTERWITH OEVELOENTALNEAGURR/TOTAL WNBA OFPUPS INTHE LETTER co 8 4 2? 22 $8 2a orm 8 reaGoer crow 5.2 wpm oo or a oe 0 smtp on ok 3on ican 2BE g3e wm wm 0 mvt son ou a 3 or acraTion FE -- oo8n EU Tdi 5 So 3 a Livre ve REAL KERR TAL WA GF Fk ive gg 8g 2 8 @ - a0 - cd we i 3 we in & :agg Wi ne 0 urs In BAGH LITTER WITH DRVELOMENTAL NEASURR/TOTAL WNBER OF PUPS IX THE LITTER oo 2 ag orm novesoakermow 11 ol sera - we ix] ey ut wo : ne sion 2 gi8 ; aL oan or urs I EACHLITER WITHDEVELOPMENTAL WEASURR/TOTAL WaaeR oF pons 1% TE Livres 3 2g 2iii 3g8 8 rr a i mi - -: wo : ws g wn 8 1B G M EE NG OR r FURS INBACHLIT TRTEmR WITHDEEVELEOMMENTALmNEeASURE/TOTALMETNEMERTOFAFURSEINSETHEELIT0TER eenprrw,e < TY a 2 3R a88 28 -2 : i RR A g2 g 3 f::i i er on i es srt i ne 40 SURVIVING POPS Cu DAY 3 OF LACTATION 3 eg iL nen oF PURS 1h ACK LIFTERWITHDEVELOPKENTAL WEKGURE/TOTAL INGER OF PUPS. IN THE LITTER = Pr 1 Ss it 1,SE, OAR, To nT 5 nrmaiuirren + aria onescon 5.2 waiaron - - - 3 g & f = 5g hot o mr cose es so om i 8g wzw i} Ae i me e mm 3B B 8 E e L i SrER g BEE SAE men meme sommes Rr Bi Sy Sr SE SR 3 8 Sg8s & g fni2a iai AEAL reRoi ARErvaenEneED moma ER 3ea 8B & i r rI r a mRA J TT 5 ; < i g 2 J BI E ll |2 &gFin EsT is aE wpe fe pS SS r i Lsirai rinerparsi .Sa =i 5 0 g BE 3 8 22 a 32 R  susrans Aer rn 2 Tw Te eLrn caheer e atrioinosdSabsle" p(Tabl4e aC3i0efsorhoexatelrsnal3cciionincealtSaoeaeirvoatipornsiceondf} uroedsotumeCESaPRiYe 0 he a o g8 8 @4@ ER en g22& 88 8 fi . TT adi vow wos a rRSl ReSR a EE LE, as z 88 *8 3 g @ b sanimPaLiiHeckuTL is BhA AAT Sthe S a8 eiet oF2 g& - : prominin . on pT 3 s8gg C. E E mmiom r iiem la i ress entsssaaneam A s egeb to0  2 2 2a23 2 APPENDIX D REPORT TABLES - F1 GENERATION MALE RATS 000541 mctsons, jr Wm ows E gE [I ---- we wa we i 82 a. Fat 13105 as found dead onday #7 postweaning. mn 2 hat 1212s fae found duns om & oveeteto: 2 [Ket AEC HEyd A SO ove osswns ax J-- emu worn wa we ws Va we we Va we we va we us g g8 85 a 2 g 3F EBRS eS 0BE erTT T rT et deecn chray ch er cobtaion tno vpn a. fo he mccin ne 2 on cence 83 e2aa ?2 2 i Eh BE g bE ELSE aa8 a Cu Cotaon bepn or he FY eerie ch 8 Ch hoe hee t 3  BREiET0 Ee Snooenero un pa a 33 2 a 8&23 2 i pert) ot cain wate. 2 i ianitieunly itierensfron ehe vehicle control areup valve (55.651 i 2 g 8 2 2S i 8 g I fon fi nS rsnmsansssnss Sn SE Deni en an fa wi ind wi fan 2 2 R 8 2 2 ores tm. sc cs er, en, a en iT : 3$ 2& g ELIE wn - un 23 2aa8 3 8 arrow, so 0 . : . g ors, 8 err, orate 1 . 2 o i aie teh nian Hien ericn hic Tabi 513) for externa) ohaarvations conticeed a secropey. 8 S mHiOumRiSmaEme arom re 23 PEERS Ln 8 g ag Es + when a i | EE TET STs see acs arn st is ste 83 82 3g aa .2gg E. 2iigt g4 BE ma SEE pm, wLn RBe E EEEm 32 3 Sate BE Se as 0 Kovense pivommas. za g a Eon 2 LE vm wt cer 22 82 aSSa 5I - Bo EEE Dee orl ny SOFTOKLiguIDFECES. 3 32aag Re 85 3 2 PTL 00 Stn,otEI, SE, OAR, emir ncn mn 3 2g 833 2 a a8 "conan roost RL -- EICaI EE go Ee mma 2 pr oellval SE g: 2 o cio the to cotton be 1 he 1 gion rcs: co hs z 23? 2 a2a 8 CEE : 2 i2 af eaitso orgs wan Gop "HAL POSINTL REBOOT Tecan se of gE J EEERERERER NE 2 g & g NLL lcs WERE RESOUSED 1 Guang (0) i ;: :: 2 ' i EE Trmmaotaotn oy whseto e Fcoa Rdad on Che dy GobADCaCion vn Beg En the 73 usarsion Fat 48 Chat Lime these z b. Seay "eign san TecorobnBeyd 2 postuesning and ws excluded from grow averages and scatiscica ansiyee. g 22 a&a 2 33i 8a28 8 Fas --. i 2g 2& 1 BIRSILITELI SU a vee 8 5 ggg i 8 Tt toe ona icson. 3 2 g8 g2 8 a i : BER [a Ea s . ii a g 2 3 3 :: ii Sh sa J : if stony J -- swe tre or i ogo EEE on i me g SIT Hi ms 2 raion hse of ton srs stad br + ose cer i 2 38 S$a ?8 $&3 ev i et : i g7g g2 z : : Spin Genin man 2082 Retention Phas) of Casting vere separated by 4 ane-ve ncerval g a "a iers zune ergs mm amma 1 i 2z &2 EE g R8 EBEa E ar aahPUsess 8gE & 3 3gSFSiE e 1IIIERShy IR Ihe fink Shanicacion berics Td 1 SIME UIE 1 etme 2& g g g fg og% & EomsiEsm : goa Ee ress soins vest EE Wa Bh wie ours S I Ei TE Eien GHcaaAn.ln.i.tce an 1S mmtr T ion ees eire 38 2 i en 8g : 3a 2 zis Wao Bprisnte i iEERmmIuRnAeHn 53 ot sere Serio tit ee ier, ii ; LTTEou niessa erie ners 2 rs i ga88 go EPE RE I r oLrLm me rari ee $3 g222 z RE CS i : 2 & Sr HEB RHUE EEE ERSeen hm TR lll SED MOTTA 3 og iEraiR biialE nLLOMEEIEEREGi SEShlI I TE ee g8 gG8F ETRE B ftI hp ies tts te sr rcs sd sin rR na EER o g 38 i 83 EC] z I ET RIA ect TTE E Tr pn eho LELR Ey we i i g 8 2g 5 aa g 3 A Waa z APPENDIX E REPORT TABLES -- F1 GENERATION FEMALE RATS 000590 eoiscssunisnariosn Gmhetosr sr o Her anv oF comin onT we r or E p at se-- n r-- a wn. vin wwiowown owwee wo wa we earGINsve "a we we 2 tn aga _ gr r seer Sued & QE MELE LL ey itis 3 g T 8 ate rss wcamce rn, orm os onbeannna [E-------- sor wae srs ayo ery www wow wa ve wa wa J---- wwe we i 2S2gINIRNJ TIEA TIR I ON NaGiL r oF iS iiviIvAemATal sis WSed PMmri RIICES To TE KLo Lor 0d Si e8 se BE 32 n z 8 Petite HAY or " o cess EI 95 ot onsen os eonssrortas Pi cern TIEIL LL ee " g2 nn mae te pond cde oat . een asossoestii 3 2 i?mn SE en + wen 4 Fo ) ) RET I 2- + EEL E Eh a Re Le ; 2gsg T er ons SEE. co, sp,ss a Ae PO REISS 8 + wehen a bi or a 3 8agg & fh EE es 2 7a 5 RSI EE 2g 8 gm nn Ly - ; 5 FE RI TRC UID EER SAL Leen: te sex san cores deities i 2 8 ' n 2a - - < 3 23 8Z 2 . IIIT ee 2 3g 3 m3n ag8 winnwmae eins meee aE] LE + din i on i emt oho fe a's ty a1 ions dh Bn ro ty te LaniEicanciy Gitfareat trom the venice control Srovp valor. ipo 45) 2 g g 8 : i gg 3 ae A woe wes wa wn Lh EEEne bt rare ty is 2 REBE LL 3# 8g2g n% EEE Ram eesm es : sg52: 2Z En wd nee EEE : ive' Yalues EEBL for cat 13305, which eS wae excluded (rom study on day 7 postueanion; the sex was incorrectly identified. .2gg 382Tz 22 $I ER ai nsem srnay pet, a co ry emit 32 R 2 n S a Z 3 8g822a m5 TCL 01. om co.to,as. sibeenTSs AN EPRI TY 7 8 ALE C1 01 TU RAT 1 Cin nS AEN CTR Sot oR EEBS wer mse mass whl, he EL to stn tr 14 tn, re fon cmv se 8 2 283 mn a 8 fe a on hy lo a i eslnics 2 3- 37 2 3g iEi i 223 @8 I RL SLI ery entries zR az rn + mine a o 3 aise aries peea 3 aeincicn as SE i ue sed V+ eh FR or or 2gg :&n73 g8 =2 8S3sS SRR sm ss sm ohn aioe for at 108 AIC ar cies Eroey or ty 3 asiaSnh eswn ncnrelcy nine Rentt eTutesvirth Een mes eatinenedSa S18 te . : HLEE :Bg (Table no#23) for external chservations contiteed ot mecropey. - i2 fe nei RR REI Aee atreaym vmivnee i E &3 g n 8 R 2 acahese forTs iteer 13157 he dun was found dend on day 10 of Bacon ight pups vere sacrificed a AE SEE TR In on 1 eeiear someartuaminsof18 Top ohSar toioen ptotpaervn g 2 22g ' z2 2 nTT1oS L ne TroS shasR wens pe titer. dnctutio ters sieh 0 suvivins up. 3f2a 23 TETT ITE EE Ss on aay 10 of nection; cian pops vere saceitices n2 S22 > rns nr we Chin an FLE ETiRe E ee o| em . i om REET EES re et ) 23 a 2g32 8 a 2 . g 2;38 g5  i 2:g g g2B SBiid EEr EERETT -- 3| , Bd To cn aur 2 ostesntess the vex sas Incorecely Aeasiied TET ET g FEN Emm i 5 re 3 amon ou oa ortacTaTion Cr or - 3!B or iBE mSeu e mBBmAe iEseR 223 g33 ISIN. 22 28R 3 vPiEas nies eT wn 3 g 2 i roe 50 LGA GNF otra 1 on orBeran - 2g 28& oe 32 m8 ELI Miu uso sr msn 0 5 ss EL STEiR ao in : 2 % 3 8 TE Sa o lTg re ree coded cn te dy comtaon an ben In he 1 gearsion xs Sh. on Seve 2 amn 3g 8 88@ EE ER Bsre het ction se ho 1 ion rt os si che g 8883 i& a aSalmautnhTI at aT in arpa: oa 4 poscveaning 3 mmm i A... : aCuies tro esd on doy 7 oncvenni: the sex van dncorsaely. demos. g ] 232 4 238 a a eek rom etoyn iy 3 potvesnio; che sox van Incorrectly idntifind, 2 2 ' 3 z8 228 * BEARER & 3 8 8 2 - ]m 383 & 88 EEL 8g g2 g 2gg8 f; 3i 7z gg a corso -- 33 2 8 n &&2 2 : R 2328&a m& ED B cosas car - L ens wien DE ri lm min swe sey sense R3N &2 8 82 5 FREE Rr ws ae. i k Z 5 8 : z: i 8 E AE RIce ay posse: the sex wan canst enties g3 3 g 2 m 5 83 a E ER 23 o g 8nm 3 3 2g22g a8 EAL A LT I g 32] 2$ m8 g2& I LlELI TIT et iter dar 2 of vacation 2 23 &mn a 382 PPR pra x, Be eomasse toot SL 3 IIRL reete sent fe sce eo nin Z 2& 882 % 2 ET cattr tr on 47 psoion to se vo ore omen g & a2322 22 27 Srl Tals sas 4083 tension Phase) of Senin ser separa by ees nce z rT ees 2 m8 32@ CE o . _-- % 3.11 Pog s g |SEELLE EElEy mas 1ctt ore tas 4 sem pent cn een mF g3 3g 3 5 EL a, & g3: 28g nz& 23 mo th. Stsct,r,s RS SA I TLor oven vans tsi mn wavesses wo : g22 2&:&2 5 Es. gii32 3i 2 3& 82&8 m& i I Sha rpierietide go Eelgbro a 3 RTI ds on car 0 pte 2 83 33 8a8 g BOOED Pb Humimmm 3 Ee sare Boro vosmeianns 55 +onyor (peesiniD)GeTATION a0r1e+ osnyooFiacsarviotn tr an ot mcs - 28 EE a i ER SE ren or 2 2 i g aa 2 2 232 2&g8 8 Dae own r ih ALi Tiss ArrEAReD bosmat. a : EEnoRE BE 4 EEEEER :2 2g 8 g2 Et PRIRr EEER woos pBein, on rns sevens stn ii8 a4g & 2 i EEE 2 2: g82 3 2 FERR3ERsUtTae esiLLop etn i2 g2g8 me mscn,ps ee RET i de Eo SR RELB 1 TET cite tem sa cn ty 3 pecans te se wn corey sce. i 8 3 g28 22R 23 m3n 8&8 RB Er or en ou dL a z 2 2B hn oun se on aa 10 of acacion ean pup wee mcebtices on dy 10 entre. 2 2g a 28 8 Hr FE : 2 SAS TS SETTLE SE miei 3 22 2a 82? 8 vu ---- . EE om sw tpg wes 3 & 23g222 n# : 28g8 2"i3 wo a eC Te & @a2 TT A LT RIT LSS aenetcsen srssserssssaton 2 2 R8 @m ES rey hin] rtmame oWIsSEaRo51b.iCSLkD. NSALeSa(SAD COMTALIED) 23 23g ]n3 2@3 i 2 Sg82& 0.10.20, _-- 3 a-- 8 83? T8 D FE e N OB l, oe . 2 22g oe vwa] Tgg 58 @n SEs oi MELEE HN. i RSE Foi "aco hr BACAR Te hyPOA Eo Sea ISN COMIMIZID) i 8 8m g3 Er g -3 23 a g3 m&2n 3HN CR --J -2gg28 . &n83z 3. BAC 13205 was excluded from study on day 7 postweaning; the sex vas incorrectly identified. 3 a 5 38 22 &a8 EEE Em cove, ne es cor i 32 ii 3 IL TREESRRR Senti i 28 2& &2& rr Eo Seon. 3 g m g 8 &S R53: i z EBE Ei ty LE ERFe,tm ms amiss 4 BAC 13305 was excluded {com study on day 7 postweaning; the sex was incorrectly identified. - g 3a - gg 33 m & :Eman EE : :S 2 g 3 2 a 22@ 28 8 2 Cae an st aay" ateiesion to en evs Scoreedty deities 22g g 22 R3 m 2 2 3 : g 8 8 g 2 = cseterrertieaaten rel ees AAG SLANT | roririn a 2 23 : m 27] 2 o wn Ale ARcm 3 S ans on ALL PUPS. APPEARED WORNAL mn 28 7 LI RS SrR a R m ml LeT 8 g .2g gg iz i 2mg o son S AR L GrE uR T158veI s AI rPEAS AED N, OL |, 8&2 a8 pie eo ini-Ca 3 HS iii SERP RE eR iMLL GfIEoiRestTnsISSUeES AsPPEARED MOARAL. g2 " a i Sh il ai peices si is 3 28 Q 2&@2 m8 . et soree TiEe A rns. 3 Q B DbeSdt I rosin ars hSkI iporriotraLeTIhke 3rai )tSoeLLesIntTotmtci hieatSSnhserionelons cotcremneieesaoe ecwcoere si 3 - 2 m87 22 : gzs E R E Ea A Si iepi ae in 273 3 8 APPENDIX F PROTOCOL AND AMENDMENTS 000691 418-008:PAGE F-1 neseancy x Argus90R5esSehaerechhyLDarbiovrea,tBouriiledsi,nigncA. Asonuronis Horsh21a5m),UsPaeTnIn0sylFv2a1ni5ae1s980474 ---------------------------------------------------- STUDYTITLE: PURPOSE: TESTING FACILITY: STUDYDIRECTOR: SPONSOR PROTOCOL 418-008 SPONSOR'S STUDY NUMBER: 6295.9 CPeormibnaitnaeld/PoOsrtanla(taGlavRaegper)odFuecrttiiliotny,TDoxeivceiltoypSmteundtyalofaPnFdOS in Rats. dTihsetuprubrapnocseesorfetshuiltsisngtufdryomisPtoFOteSsttfroerattomxeinctefoffeCctrsl!:CDBR tVhArFo/uPglhumsatimnagl,egaesntdatfieomnalaendraltasctbaetifoonr.e cohabitation This study tehvraoluugatheFsofItCHheHarremporondiuscetdivTerippraortcietsesGauinddelsihnoeulsdtadgeetsecAt gefefsetcattsioonn, ptahretuersittiroonu,slcaycctlaet,iotnubaanldtrmaantsepomratl, ibmephlaavnitoartiionn, tfreemaatleed rmaatsl,eoanndthfeedmeavleelroaptsm,enatnodfptehremiotffdseptreicntgioofn otfhe mfuantcutriaotniaoln)eftfheacttsm(ae.yg.notefbfeectdsetoenctleibdidboyohriestpoildoigdiycamlal sperm meaxnaimfiensattaitoinosnsofofmaeflfeecrtastirnedpurocdeudcdtuirvienogrgtahinss.perBieocdamuasye be delayed through pirnotdhuectoiffosnproifnFg,2 ogbesneerrvaattiioonnslitweirlsl. be continued Argus Research Laboratories, 05 Sheehy Drive, Building A Inc. THeolresphhaomn,e:Pen(n2s1y5l)v4an4i3a-671190044-1297 Telefax: (215) 443-8587 ARsasyocmioantde GD.irYecotrokr, oPfh.RDe.s,eDarAcBhT 3M 3M CTeonxtiecro,loBguyilSdeirnvgi2c2es0-2E-02 St. Paul, Minnesota 55144-1000 000692 . ` 418-008:PAGE F-2 Protocol 41P8a.g0e028 STUDYMONITOR: SATLUTDEYRMNOANTIETOR: Marvin T. Case, DV.M., Ph.D. Telephone: (612) 733-5180 Telefax: (612) 733-1773 Andrew M. Seacat, Ph.D. Telephone: (612) 575-3161 Telefax: (612) 733-1773 REGULATORYCITATIONS: ISnttuedrynaDtieosniaglnCaosnfMeordeifnicceatoinonHaofr:moUn.iSs.atFiooond; aGuniddeDlriunge Aodnmidneitsetcrtaitoinoonft(o1x9i9c4i)t.y to Nroe.pr1o8d3u.ction for medicinal products. Federal Register,September22, 1994, Val. 59, 2U1.S.CFFRooPdaratn5d8)Drug Administration. Good Laboratory Practice Regulations; Final Rule. Japanese for Safety Ministry Studies of on Health Drugs, aMnHd WWelOfradriena(n1c9e97N).umGboeord21L,abMoarracthor2y6P,ra19c9t7i.ce Standard aEcucreoppteaannceEcboyntohmeicEuCroompmeuanniEtcyo(n1o9m8i9)c.CoCmoumnucniiltdyecoifsaionnOoEnC2D8 Jduelcyis1i9o8n/9roencotmh-e tmheendEautrioopneaonn Ccoommmpulniiatniceesw:itLhepgriisnlcaitpiloens.of32go(oNod.lLab3o1r5a;to2r8ypOrcatcotbiecer.): Of1f-i1c7i.al Journal of REGULATORYCOMPLIANCE: `rTehgiuslsattiuodnyswciiltebdeacboovned.ucted in compliance with the Good Laboratory Practice (GLP) All changes Director and othereSvpisoinosnosr,ofdtahtisedpraontdocmolaisnhtaallinbeed dwoicthumtehnetperdo,tocsoilg.nedby the Study aTnhde wQiullaliintsypAesctsucrriatinccalepUhnaitse(sQoAfUt)hewilsltauuddyitinthaeccporortdoacnolc,etwhiethrathwedSattaanadnadrdthOeperreapotritn,g Procedures of Argus Research Laboratories, Inc. aaTlchlceauprfpailtniaecllayrbelrpeeofrlGteLcwtPislltriehngeculrluaadtweiodanastsatwaeotrbetemaefionnlteldsoiwdgeundreiidnngbttyhheetchpoeenSrdtfuuocdrtymoafDnitcrheeecotsfotrtuthdheya.sttStuhhdeoyuralendpdortthat stioggneitfhiecranwtidtehvhiaotwiotnhsefdreovmiaGtiLoPn mreigguhltataifofencstotchceurq,uaelaitcyhowrililnbteegrditeyscorfitbheedsitnuddye.tail, 000693 418-008:PAGE F-3 Protocol 41P8a-g0e038 STUDYSCHEDULE: See ATTACHMENT 1 to the protocol. TA ESTRTIANC DVEL HICE LE: Identification: TestArticle: Name: PFOS. PLohty/sBiactaclhDeNsucmribpetrio:n: ~~ 2Li1g7ht-colored powder. PuSrpietcyi.fic Gravity: ~08. 98.9%. Expiration Date: May, 2000. Information on the with the Sponsor. identity, composition, strength and purityof the test article is on file Vehicle: 0De.i5o%niTzweedeWnat8e0r)i.n RSeuvpeplriseerdaOnsdmloostiisdenMteifmibcartaionneofPrTowceeesnse8d0DteoiboneidzeodcuWmaetnetre(dR.iOn.the raw data. tNoeibteheprretsheenStpionntshoervneohirctlheetShattudwyouDlidreicnttoerrfiesraewwairtheotfhearneysuplottseonfttihaliscosntutdaym.inTahnetrsefliokreel,y no analyses other than those mentioned in this protocol will be conducted. Safety Precautions: fGolromvuelsa,timoanskpr,epaaprpartoiporniaatnededyeospargoteecatdimoinniasntrdataiounn.ifTohrmefMaabtecroiaatal rSeafteotybeDawtaomShdeuerting (MSDS) is attached to the protocol (ATTACHMENT 2). Storage: VBeuhlikcTleesCtoAmrtpiocnlee:nts: PPrreeppaarreedd VFoerhmiucllaet:ions: RRoooomm tteemmppeerraattuurree.. RFrooozment(e-m2p0erCa)t.ure. AJlullitaenstGaurltbiicnlseksi,hiMpamnenatgsertootfhFeorTmeusltaitnigonFasc,ilaittytshehopurledviboeusaldydcrietsedseadddtroetshseaanttdention of telephone number. 000694 418-008:PAGE F4 - Protocol 1P8a0g0es8 `Shipments should include information conceming storage conditions and shipping csahritpomnesnts.hould be labeled appropriately. The recipient should be notified in advance of FORMULATION: Ereqouf Perepnaractioyn: Formulations (suspensions) will be prepared daly at the Testing Facility. Detailed preparation procedures are attached to this protocol (ATTACHMENT3). AdjustfomrPeurnitty The test article will be considered 100% pure for the purpose of dosage calculations. Testing Facility ReserveSamples: The Sponsor will reserve a sample (1 g) of each lot of the bulk test article used during the courseof this study. The Testing Facility wil 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: Samples additional to those described below may be takenif deemed necessary during the courseof the study. Bulk Te licle Sampling: No analyses of the bulk test article wil be conducted during the course of this study. Information on the stabilityofthe bulk test article is on file with theSponsor. AnalysesofPrepared Formulations: --_-- Stability data for prepared formulations bracketing the rangeofconcentrations and conditionsofthis study are on file with the Sponsor and will not be determined during the conduct of this study. Suspensions will be prepared daily at the Testing Facility. 000695 418-008:PAGE F-5 Protocol 41P8a-g0e0s8 HomogeneityAnalyses: cHooumrosgeenofeithtiys softutdhye.teAstsyarrtiincglee wiinllprbeepaurseeddstuoswpietnhsdiroanwsswailmlpbleesve(r5ifmieLd deuarcihn)gftrhoem the tEoapc,hmisdadmipelean(d5 bmoLt)towimllobfethdeivhiidgehdesitntcootnwcoenatlriaqtuiotosn,oontneheofif2rstmLdaaynodf oprneepaoraf3timoLn.. aOtntehealTieqsutoitn(g2FmaLci)liwtiyllasbeasbhaicpkpuepd sfoarmapnlael.ysiBsa;ctkhuepostahemrplaleisquwoiltl(b3emsLt)orweidl ubnedreerttahineed previously Sponsor. cited conditions and discarded at the Testing Facilty upon the request of the Concentration Analyses: oCfontchiesntsrtuadtyi.onAofsytrhienpgreewpialrbedeteussteadrttiocwleitshudsrpaewnssiaomnpslewisll(5bemLvereiafciehd)dfurroimngetahcehcourse (c5onmcLenteraacthi)onwildlurbiengditvhiedefdirsitntaontdwsoixatlhiqwueotesk, oofnedoosaf2gemLadmainndistornaetioofn.3 Each sample mL. One aliquot T(2esmtLi)ngwiFlalcbiletyshaisppaebdafcokruapnaslaysmipsl;e.theBoatchekrupalsiaqumoptle(s3 mwiLll)bweillstboererdetuanidneedr tathethe previously Sponsor. cited conditions and discarded at the Testing Facility upon the requestofthe `Shipping Instructions: Samples to be analyzed will be shipped (frozen on dry ice) to: Kris J. Hansen, Ph.D. 3M 935 EBnuvsirhoAnvmeenntuael Technology and Safety Services Building 2-35-08 TSte.lePpauhlo,neM:inn(e6s1o2t)a77585-163031-83331 Telefax: (612) 7786176 Both the recipient and the Study Monitor will be notified in advanceof sample shipment. DISPOSITION: aPrrteicplaerweildlfboermrueltatuimoendsowillthbeeSdtiusdcyaMrodneidtaotrathtetTheestpirnegviFoaucsilliytyc.itAedlardedmraeisnsi.ng bulk test 000696 418-008:PAGE F-6 Protocol 418-008 Pages JESTSYSTEM: SpeciaendsRe/asSont forrSea leci tionn: bTehceauCs:eC: D1)BthRisVsAtrFa/iPnolfursat(hSapsrabgeueen-Ddaewmloenys)trraattweadstosbeleescetnesditaisvethteo TreesptroSdyucsttievme raenpdroddeuvcetliovpemaenndtadlevtoexlionpsmaenntdahlatsoxbiceietny ewviadleulaytiuosnesd; t2)hrhoiustgohroiuctalidnadtuastaryndfoerxperience exist at species tahnedTsetsrtaiinn.g FaciltyTM; and 3) the test article is pharmacologically active in the Number: Initial population accimated: Population selected for study: 117955 mviarlgienrmaatsle(3a5npde2r0d5osviarggeingfreomuapl)earnadts.175 female rats (35 per dosage group). pTernesmuamteeddgfesetmaatlieonr;attshewilrlebmaeiansisniggfneemdatloerCaatesswairlelabn-espeecrtmiiotntiendgtoondedlaivyer10liotefrs. 250 day F1 21 generation postpartum pfourpcso(n2t5inpueerdspeoxstpneartadloosbasgeervgartioounp.) will be selected at weaning on BWodyeiganhdAgte: Male they wrilaltsbewilelxbpeecotredderteodbeto weigh from 300 at least 60 days g to 325 g each of age. Female at receipt, at which rats will be ordered time to `lweeaistgh6f0rdoamy2so00fgagteo.22A5ctguaelabcohdaytwreeciegihptts, watilwhbiecrhetciomredetdhetyhewildlabyeafetxeprercetceeidpttoabned awtill be documented in the raw data. The weight ranges will be included in the final report. Sex: mBaolthe FaondanfdemFa1legerantesrawitlilobnemgailveenantdhefteemsatlaertriactles.will be evaluated. Only Fo generation Source: Charles River Laboratories, Inc., Raleigh, North Carolina. The rats will be shipped Laboratories, Inc., to the iTnefsittienrgedFaccairlittoyn.s by air freight and/or truck from Charles River 000697 ` 418-008:PAGE F-7 Protocol 41P8a-g0e0?8 Identification: EGoeneration: CRoa.t,sIanrc.e, pNeor.maMnSePntTly20i1d0en1t)i.fieMdaulseinagndMofnemeallesrealtfs-paireercaisnsgieganredtatgesm(pGoeryarByannudmbaenrdsTaatg receipt before aadnmdingiisvterantuionnioqfutehpeerfimrastndeonstaigdeeontfitfihceatitoenstnaurtmibclee.rs when assigned to the study E1/F2Generations: iPnutpesrmwislol fntoht ebeitienrd.ivAidtuwalelayniidnegn,tiefiaecdhdurratinsgelleaccttaetdiofno;r aclol nptairnaumeedtoebrsserwivlal tbieonevwialllubaeted identified with a Monel self-piercing ear tag. ANIMALHUSBANDRY: aAlndcUasgeeosfizeLsabaonrdathooruysAinnigmaclosndi.tions are in compliance with the Guideforthe Care Housing: FG o eneRar ts/Fa 1Get nerai tioo nLittn ers: eFxocgeepntedruartiinogn trhaetscowihlalbbiteatinidoinviadnuadllpyoshtopuasretduminpsertiaoidnsl.essDsutreienlgwciorhea-bbiottattioomne,d ecaacghespair pofrerastusmweildl gbeesthaotuiosne,dFion tgheenemraalteiornatf'esmcaalgee.ratBsegaisnsniignngednotolnaatterurtahlandedliavyer2y0wiolfl be. cinodimvmidounallnyeshtoiunsgebdoxindnuersitnigngthbeopxoess.tpaEratcuhmdpearmioda.nd delivered litter will be housed in a EG 1 eneRatrs/Fa 2Get nerai tiono Littn ers: Ahfotuesrewdeainnipnagi,rst(hoenFe1mgaelneerraattipoenr rfaetmsawlilel rbaet)idnudriivnidguaclolhyabhiotuatsieodn,baefnodreincdoihvaibduiatlaltyion, Fhoougseenderaatteironcorhaatbs.itaBteigoinn.niTnhgensoalmateerttyhpaenofdcaayg2i0nogfwipllrbeesuumseedd gaesstdaetsiconr,ibed for the dFe1ligveenreerdatliitoenr wfielmlableehroatussewidlibneaicndoimvimdouanllnyehstoiunsgebdoixn dnuersitnigngthbeoxpeoss.tpEaarcthumdpaemrioadn.d 000698 418-008:PAGE F-8 Protocol 41P8a.g0e0s8 NestingMaterial: dBeeldidveirnyg. material (bed-c'cobs) will be supplied to female rats assigned to natural ABneadldyisnegswiflolr bpeoscshibalnegecodntaasmoifntaetnioans anreececsosnadruyctteo dkeanenpuatlhleyaaninmdadlsocdurmyeanntdedcleinant.he raw data. RTooemAimr, peraatnduHumridiety: fTrheeshanaiimtahlatrohoams biseienndeppaesnsdeednttlhyrosuugphpl9i9ed.9w7it%hHatEPleAasftiteernsc(hAiarnogeCslepaenrhroouormo)f. 100% 7cRo0on%sot.manttleym.peRraotoumrehwuimllidbietymawiilnltaalisnoedbeatm6on4itFor(e1d8cCo)nsttoan7t8lyFa(n2d6mCa)inatnadinmeodnaitto3r0ed% to Light mAaninatuationmeadt.icaElalcyhcodnatrrkolpleerdio1d2-whiolurbleiggihnt:a1t2-1h9o0u0rhdoaurrksflEuSoTr.escent light cycle will be Diet: aRadtlsibwiiltlumbfergoimveinndiCveirdtuiafliefdeRedoedresn.t Diet #5002 (PMI Nutrition Intemational) available Water: aWnataeurtowimlaltbiec wavaatielraibnlge aacdcleisbsitsuymsftreomm. iAnldlivwiadtuearl wbioltltlbees fartotmacaheldoctaol tshoeurccaegeasndorpfarsosmed pthrroocuegshseadrweavteerrseasosamobascitsermioesmtbatr;apnreocbeesfsoered uwsaet.erCihsleorxipneectwieldl btoe caodndteaidntnoothmeore btahcatner1i.a2l pcopnmtacmhilnoaritnieonatatnhdettwiimceeoafnannuaallylsyisf.or Wpoastseirblies acnhaelmyizceadl mcoonnttahmliynfaotripoonssible. Contaminants: `0NweoibutlehdeprirnettsheeerfneStrpeionwnitsthohertncehorertitrfehiseeudltSdsiteuto,dfytthhDieisrdsertciutndokyri.nigsTawhwaetareerfreooorfre,tahnneyonpaeosnttaeilnnytgisaemlsatcoeotrnhiteaarlmtaihtnaalnnevtteshloslsitekhealyt croountdiuncetlyedp.erformed by the feed supplier or those mentioned in this protocol wil be 00699 418-008:PAGE F-9 Protocol 41P8a-g0e0s8 RANDOMAINDZCOAHAT BITIATOION N: FGoeneration: gUepnoenraatrreidvarl,anradtosmwiulnlitbse. aAsfstiegrnaecdcltiomaintdiiovni,dumaallheoausnidnfgeomnalteherabtasswiisllobfecosmepleuctteerd-for asctculdiymaotniont.he bTahseisraotfspwhiylsibcealasaspipgenaerdantocedoasnadgbeogdryouwepisgbhatsserdecoonrdceodmpduutreirng-generated (weight-ordered) randomization procedures. Within each dosage group, consecutive order will be used to assign rats to mcoahxabiimtautmioonf, 1o4nedamysa.leFreatmaplerefreatmsalweitrhats.peTrhmeatcoohzaobaitoabtsieonrvpeedriiondawislml ecaonrsoifstthoef a vaginal contents and/oar copulatory plug observed insituwill be day 0 of presumed gestation and assigned to individual housing. considered to be Female rats not at hmaavteedmawittehdin(tshaemfeirdsto7sadgaeysgroofucpo)haabnidtawtiilol nrewimlal ibneianscsoihganbeitdaatilotnemfaotrea mmaalxeirmatusmthoaft seven additional days. a`Tshseifginrsetdtteon Cfaeemsaalreearnat-ssepcetridoonisnaggeongrdoauyp 1w0itohfaprcoensfuimremdedgedsattaetoiofnm.atTihnegrweilmlabineing female rats will be permitted to naturally deliver ters. sAatmapbllee ocfolrlaecntdioonmautnistcshewidlullbeedussacerdiftioceasasfitegrncfoimveplreattsiopnerofgtrhoeupcothoabaitpahtairomnacpoekriinodetic (male rats siring liters with dams allowed to postpartum (female rats allowed to naturally naturally deliver deliver litters). a litter) or on day 21 EAIF2 Generation Pups: wDhaiych1oaflllpauctpastiionna(lpiottsetrpaarretuimnd)ivisiddueaflilynewdeaisghtehde (dpauypobfobdirythweaingdhtissawlilslobtehereficrsotrddeady on after all pups in a litter are delivered and groomed by the dam). aOnnddlaityte4rspwoilsltpbaertruemd,ucaedtatboleeiogfhrtapnudposmeuanciht.s wWilhlebneevueserdpotsossieblleec,ttphuepssatmoebenucmulbleedr,of male and female pups per iter will be continued on study. wAitllwbeeanuisnegdotfotsheeleFc1t g2e5nemraalteioanndpu2p5sfoenmadlaeyp2u1pspopsetrpagrrtouump,, aretsaubltlienogfirnaantdootaml uonfit2s50 pFu1pgeannedraotnieonfermatasle(1p2u5pppeerrsleixtt)erc, hwohseennfpoorscsiobnltei,nwuieldbeevasleulaetcitoedn.. At least one male 00700 418-008:PAGE F-10 - Protocol 4P1a8g.e00180 ADMINISTRATION: Route and Reason forChoice: `The oral (gavage) route was selected for use because: 1) in comparison with the dpioestsairbyleroruotuet,etsohefehxuacmtadnoesxapgoesucraen. be accurately administered; and 2) it is one of the MeatndFhreqouendcy: Dosages will be adjusted for the most recently recorded body weight and given at approximately the same time each day. EGo eneraMtalieRoatns: Male rats will be given the test article once daily beginning 28 days before cohabitation (maximum 14 days) and continuing through the day before sacrifice. Male rats will be sacrificed after completionofthe cohabitation period. Eo GenerationFemaleRats: delivera iter), Female rats will be given the test article once daily beginning 28 days before cohabitation (maximum of 14 days) and continuing through day 9of presumed gestation (rats assigned to Caesarean-sectioning), day 24of presumed gestation (rats assigned to natural delivery that do not delivera litter) or day 20 postpartum (rats that EG1eneration: F1 generation pups will not be directly given the test article, but may be possibly exposed to `milk during the the test article during lactation period. maternal gestation (in utero `exposure) or via maternal RatfoirDoosangeaSelelctieon: Dosages will be selected by the Sponsor on the basisofprevious studies conducted with the test article. 00701 DC osaogenLevcele s, ntratai nd Vooln umess: 418-008:PAGE F-11 Protocol P41a8g-e00181 loTsTarI ow [5[ecucossommennen CoTs ocTom | 5[ocsomcommmnven] [wv[os [ 6| ow [os [euscosommuommn] LvTs[ soTou [os [ecwooseommmn] Thetest ricewilbeconsdare 100% ureforth purposeofdosagecaluaions. TM ESTSE ,ANA ALYSS ESAU ND REME -FoN GENET RATS ION: Via- MbaleiandlFemialteRayts: Al Periods: Atleast twice daily. Clinical Observations andl e - Male and Female Rats: Acclimation Period: Atleast once. Dosage Period: Twice daily. Prior to dosage administration and once approximately one hour postdosage. Maternal Behavior: aDbanyosrm1a,l4,b7e,ha1v4iaorndwi2l1bpeosrtepcaorrtduemd.daiAlny.y observed aCplipnriocparlioabtseerbvyatthieonSstumdayyDbireecrteocroradnedldomroSrteudfyreMqouneinttolry.than cited above, if deemed Body Weights - Male Rats: Acclimation Period: Atleast once. Dosage Period: Weekly. Sacrifice: Terminal weight. 000702 . ` 418-008:PAGE F-12 Protocol P41a8g-e00182 BW odyeig-Fh emalteRas ts: Acclimation Period: At least once. Dosage Period: Weekly to cohabitation. Daily during presumed gestation and on Days 1, 4, 7 and 14 postpartum (rats assigned to natural delivery). Sacrifice: Terminal weight. ECeedonsumVapluets-iMaloeRants (recorded and tabulated): Dosage Period: Weekly. FCeedonsumValpuest-FeimaloeRants (recorded and tabulated): Dosage Period: Weekly to cohabitation. Daily during presumed gestation. Days 1, 4, 7 and 14 postpartum (rats assigned to natural delivery). Feed consumption not tabulated after day 14 postpartum, when it is expected that pups will begin to consume maternal feed. Eeed Consumption Values - Male and Female Rats: Feed consumption values may be recorded more frequently than cited aboveifit is necessary to replenish cage with one feed jar, the feed. During cohabitation, replenishomfetnhet feed jars when two rats occupy the same will be documented. Individual values will not be recorded or tabulated. EstrousCyclingand Mating: ceAoshttarabboiluetsaoctfyicorlnainnpgedrobimoyd.eunxiatmsiwnialltiboenoufsveadgtionaslelceyctto1l5ogfyefmoarl1e4radtasypsebregfrooruepthfeorsetvaartloufattihoen of During observ cohabitation, all female ed in a smearofthe vagi rats nal will be evaluated contents and/oar daily until copulator s y perm plug atozoa are is observed in situ. DuraoftGesitaotionn: The durationof gestation is calculated from day 0of presumed gestation to the day the first pup is observed. 0703 418-008:PAGE F-13 Protocol P41a8g-e00183 Fertility Parameters: Fertiity Index (percentage of matings that result in pregnancies). Gestation Index (percentage of pregnancies that rest in birth of ive liters) Numofboffesprring per liter (ive and dead pups). Number of implantation sites. General conditionof dam and liter during the postpartum period. Viability Indices (percentage of pups born that survive 4 and 7 days). Lactation Index (percentage of pups born that survive 21 days). Caesarean-SectioningObservations: aRaptpseawrillabbneoCrameaslar(seiazne,-sceoclotrioonresdhaopned)awyill10boefnpotreedsuinmethdegreastwadtiaotna.. Placentae that The rats will be `examined for number and distribution of: Corpora Lutea, Implantation Sites. V(iAavbilaeblaendemNbornyvoiaibsloevaElmborrycorse.scent shaped, pink, fim and enclosed in an paimnnkiottoitcasnaocrfdileleedpwirtehd ctloebalrafclku,ids.ofAt annodnveinacblleoseemdbirnyaonisamanmioortpihcosuasc,fsimlaeldlw,itphale clear, cloudy, or opaque fluid.) NaturalDelivery: Female rats will be evaluated for: Clinical Observations During Parturition. DurationofGestation (day 0 of presumed gestation to the time the first pup is observed). fLiersntgptuhpodfiPvairdteudribtiyonN-(1tipmueposf dineleiavecrhyolifttlera).st pup minus the timeofdeliveryof the Liter Size (defined as all pups delivered). 00704 418-008:PAGE F-14 Protocol P41a8g.e00184 Pup Viability at Birth. MS ETHA ODOC F RI-FFoI GENEC RATIE ON: Rats will be sacrificed by carbon dioxide asphyxiation. Embryos will be discarded after examination. NEC-RFoOGENPERASTIOYN: s`Geervxoafslrusaotlmieoswniho(inascthwaiballlllebtoeifsrsreuatenasidenoexmdamuinniinnteesudwtiralatllbnbeeucfurfsoeeprdsedytow1is0le%llbeefcotrroemtnaaleiinnceodfn,otrriopnloosgrsridobuelpterofruapttruoorvfeiedaech control tissues for any possible histopathological evaluationsof gross lesions). Unless specifically cited below, all other tissues will be discarded. At scheduled sacrifice after completionof the cohabitation period (male rats siring litters. `with dams allowed to naaltluorwaeldlytdoenlaitvuerraallylitdteerl),ifvieavre rlaitttserp) earngdroonupdwaiyll 2b1e paossstipganretdumto(afemale rats pharmacokinetic sample collection. In addition to the appropriate evaluations described vbeelnoaw,cabvlaooidntsoasmeprluems s(eappaprraotxoirmattuebleys4anmdLcpeenrtrriaftu)gweidl.l bTehceolrleescutletdinfgrsoemrtuhme inferior (approximately 2 mL) will be immediately frozen on dry ice and maintained frozen (70C) until shipment to the Sponsor for analysis. The liver will be excised,weighed, and a sample section (lateral lobe) will be frozen and retained at -70C until shipment to the Sponsor for analysis. sample shipment. Aft be er completion shipped (froz of en sa on mple collection, dry ice) to Kris serum and J. Hansen, liver section Ph.D., at the (lateral lobe) samples will previously cited address for analysis. Both the recipient and the Study Monitor will be notified in advance of Scheduled Sacrifice of Male Rats: After completion of the cohabitation period, male rats will be sacrificed and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The following organs will be excised and weighed and retained for possible histologic evaluation: testes, epididymides, prostate and seminal vesicles (weighed with and withoutfluid). The testes will be fixed in Bouin's solutionfor 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. Theremaining organs will be retained in neutral buffered 10% formalin. 00705 418-008:PAGE F-15 Protocol P4a18g-e00185 uled - Fe ts Asign arean-Se : aOnnddaaygr1o0ssofnepcrreospusmyeodftghesetatthioorna,cifce,maabldeormaitnsawlilalnbde psealcvriicfivciesd,ceCraaewsiallrebaen-pseerfcotrimoende.d, cUtoenrfiiromf tahpepaarbenstelnycenoofnpirmepglnaanntattriaotnswsiiltlebse. sUttaienreodfwnitohnp1r0eg%naanmtmorantisuamndsuallfliodveatroies will be retained in neutral buffered 10% formalin for possible future evaluation. Scheduled Sacrifice - Female Rats Assigned to Natural Delivery: Rats that do not deliver a litter will be sacrificed on day 25 of presumed gestation and examined for gross lesions. the absence of implantation Uteri will sites. be stained with 10% ammonium sulfide to confirm After completion of the 21-day gross necropsyofthe thoracic, postpartum abdominal apnerdiopde,lvfiecmvailsecerraatswiwlillbbeepsearcfroirfmiceedd., aTnhde a number and distributionof implantation sites will be recorded. Dams with No Surviving Pups: oDrapmrseswiutmhednocasnunrivbiavliinzgedp.upAs wgilrlosbse nsaeccrriofpicseydoaffttehretthheorlaacsitc,puapbdios mfionuanld adenadd,pelmviiscsing viscera will be performed. summary tables. Postpartum data for these dams will be excluded from Rats Found Dead or Moribund: `Reaxtasmtihnaetddfioerorthaerceausasceriofficdeedatbhecoarumsoeriobfumnodricboundnidticoonndointitohneodraaybotrhteioonbsweilrlvabet:ion is smeamdien.alTvheesircaltesswoilflmbaeleexraatmsiwnielldbfeoregxrcoisssedleasniodnsi.ndiTveisdtueasl,orepgiadnidwyemiigdhetss,wiplrlobsteate and Broeucionr'dsedso(lsuetmioinnaflorv4es8ictloe9s6wehoiugrhsedanwditthhaenndrewtiatihnoeudt ifnlunide)u.trTalhebutfefsetreesdwi1l0l %befofrimxaeldini.n s`Tthaetursemaanidniuntegrionregacnonstweinltsboeffreetmaailneedriantsnewuiltlrablebruefcfoerrdeedd.10A%bofrortmeadlifn.etuPsersegannadn/ocry ndeeultirvaelrebdufpfueprsedwi1ll0b%efeoxrmaamliinn.edUtteortiohefeaxptpeanrtenpotslsyinbloen.prOevganrainets rwaitls bwielrbeteasinteadiniend with 10% ammonium sulfide to confirm the absenceof implantation sites. 000706 418-008:PAGE F-16 ~ Protocol P1a8g.e00186 TM ESTSE ,ANA ALYSS ESAU ND REME -F1N GENET RATIS ON: Viability: Preweaning Period: dLaiitltye.rs Twihllebpeuposbsienrevaecdhfolritderewaidllpbuepscoautnlteeadstotnwcicee. daily. Postweaning Period: Twice daily. ClOinib cal servanda lor t GeneiralAoppen aransce: Preweaning Period: Once daily. Postweaning Period: Once weekly. Maternal Behavior Dabanyosrm1,al4,b7e,ha1v4ioarndwi2ll1bpeosrtepcaorrtduemd. daAilnyy observed CalpipnriocparlioabtseerbvyatthieonSstumdayyDibreecrteocroarndde/domrotrhee fSrteuqduyenMtolnyittohra.n cited above,if deemed BodyWeights: Preweaning Period: Postweaning Period: Days 1 (bith), 4,7, 14 and 21 postpartum, Weekly Presumed Gestation Period: Days 0, 7, 10, 14, 17 and 20 (female rats only). Lactation Period: Days 1, 4,7, 10 and 14 (female rats only). Sacrifice: Terminal weight. FeedConsumptionValues (recorded and tabulated) Preweaning Period: Not recorded. Postweaning Period: Presumed Gestation Period: Weekly except during cohabitation. Days 0, 7, 10, 14, 17 and 20 (female rats only). Lactation Period: Days 1,4, 7, 10 and 14 (female rats only). 00707 ' 418-008:PAGE F-17 Protocel 4P1a8g-e00178 Feed consumption replenish the feed. values During may be recorded more frequently fit is cohabitation, when two rats occupy the necessary to same cage with one feed jar, values tabulated. will be documented when feed jars are filled. These intervals will not be Preweaning Developmental Observations: `cTohnetinnuumebseurntiolftphuepdsamyetehteincgrittehreiocnriitseraitotnaiinserdecboyradleldpuopnseiancthhedalyitoerf.testing. Testing Surface Righting Reflex (abilty to right in 5 seconds): From day 1 postpartum. Pinna Unfolding: From day 2 postpartum. Eye Opening: From day 12 postpartum. Acoustic Startle Response: From day 13 postpartum. Ai Righting Reflex: From day 14 postpartum. Pupil constriction is evaluated once, on day 21 postpartum. PostweaningDevelopmentalObservations: SexualMaturation: Fpoesmtaplareturmat.s wiMlallbeereavtsalwuialtbede feovratlhueataegdefoorftvhaegiangaleopfatpernecpyu,tibaelgsienpnairnagtioonn,dbaeygi2n8ning on day 39 postpartum. PassiveAvoidanceTesting: Beginning at 24 `where possible, 1 will bdeayevpaolsutaptaerdtuimn,aopnaessmiavleearvaotiadnadncoenetesftemfoarlelreaatmfirnogm, sehaorcth-tleitremr, retention and long-term retention. TPlheexipgalsassivelidasv.oiOdnaencceoampppaarrtamteunstcisonfsiitsttesd woiftah tawbor-icghotmpigahrttmaenndtPclheaximgblearswifltohorh.inTgheed coutrhreerncto(m1pmaArt)mceannt bsefditetleidvewrietdh.aThgreidtfwlooorctoomwphaircthmeantbrsieafr(e1 sseepca)raptueldseboyf amislldideilnegctric disooorp.enOendeaancdhttheestlirgahlt,isttheumraetdisonp.laTcehde irnattoitshaell"borwiegdhtt"oceoxmpplaorrtemtehneta,ptphaerastiudisnugntdioloitr etnutmeerds tohffea"nddartkh"ecbormipeafrptumlesneto.f cTuhrreesntidiisndgeldiovoerreids tthoetnheimgmreiddifalotoer.lyTchloeserdat, itshtehleinght is rsteamrtoovfedthfernoemxtthteriaalp.paTrriaatlussaraendreppleaacteedduinnttoiathheolrdaitngrecmaagiensfoirn3t0hese"bcroinghdts" before the 000708 418-008:PAGE F-18 Protocol P41a8g.e00188 `1c5otmrpiaalrsthmaevntefboere6n0csoemcpolentdesd.onTthweo lcaotnesneccyuttioveentteiralth(etdhearckricteormiponarfotrmleenatrnoirngt)heor until maximum 60-second interval is recorded for each trial. tEhaecchrirtaetriionteissttehdetswaicmee. fTorhebottehstdsaeysssiofontsesatirneg.separated by a one-week interval, and Dosage groups are comparedforthe following dependent measures: t`oThceonmupmabreergorfoutrpisalsfotrootvheeraclrlitleeriaomniningtpheerffiorrstmasnecses.ion--this measure will be used cTohmeplaarttemnecynt(ionnsetcrioalnd1si)nttoheenftiestr ttehset"sdeasrski"onc--otmhpiasrmtemaensturferowmillthbee"ubrsiegdht"to e`cnovmipraornemegnrtoups for activity levels and exploratory tendencies in a novel cTohmeplaarttemnecynt(ionnsetrcioalnd2si)n ttoheenftiestr ttehset"sdeasrski"ocno~tmhpiasrmtemaensturferowmillthbee"ubrsiegdht"to `compare groups for short-term retention bTeheusnedutomcobofmtepriaarrles gtorotuhpescrfiotrerlioonng-intetrhme rseetcenotnidont.est session--this measure will cTohmepalarttemnecnyt(ionnsetrcioalnd{si)n ttoheensteecrotnhde s"edsasrki"onc-otmhpisarvtamleuentisfarnomotthheer"ibnrdiigchatt"ion of long-term retention. Watermaze Testing: a`Bbeeialgciihtny,lniiltneegrarwanitillnagbpeparneodvxaimlmeuamattoeerldyy.i7n0adwaaytserp-ofsitlpedarMt-umm,azoenefomraolveerrtatcoaonrddionnateiofne,maslweimramtifnrgom isEafcihledrawtiithtweastteerdtion aawdaetpetrhtiogfhatpp1r6o-xgiamuagteesltyainnilneessinsctheeesl,maodnidfitehde wMa-tmearzeis.moTnhietomraedze for temperature (range of 21C * 1C). sOtnemeafcahrthteesstt tfrriaol,mtthheertawtowiallrmbse)palnadcerdeqinutioretdhetostsawrtiimngtopoosniteioofnt(hbeastewoofgtohaelsMo-fmtahze.e eMn-tmearzbeo,thinaomrdseorfttohbeemraezmeovbeedfofrreobmetihnegwraetmero.veOdnfrtohme tfihrsetwtaitae,r.theThraet iinsitraelquaimred to fcahilotsoemnaoknetraialco1rriescdtegsoiaglnactheodictehewiitnhcionrr6e0ctsgeocaolnddusriinngantyhegrievmeanitnrianlgartreiaglsu.idReadttsotthhaet escroerrporarerlcaettsgseotearaliaclahsntdtoriaatlr.eermEtiahnecanhterraetthmeiosvtreeesdtqufsierrsoesmdiottnho.erweTaathceehr.maacAxrii1tem5r-uisomneocnfofnuidvemintcoeborfntrseireailcarulistnitievnrevaanlywitlelst 00709 418-008:PAGE F-19 Protocol P41a8g-e00189 `semsasxiiomnuims 6105.-sLeactoenndciynt(emrevaalsuisrerdecionrdseedcofnordse)actho ctrhiaolo,saestihsethceornruecmtbgeoralofoerrtrhoers (incorrect tums in the maze) during each trial. Each rat s the correct gteosatleadntdwitchee. cTrihteertieonstasreestshieonssaamree fsoerpbaortahtetedstbyseassoinones-.week interval, and Dosage groups are comparedforthe following dependent measures: u`Tsheedntuomcboemrpoafrtreiaglrsotuopscriftoerroivoenroalnltlheeafmiirsntgdapeyroffortmesatnicneg.--this measure will be fTihrset daavyeorfagteesntinug~tmhoibsfemreeraosrrusre(inwciolrraelcstotbuernussiendthteo cmaozmep)arfeorgeraocuhpstrfioalroonvertahlel leaming performance. t`eTshteilnagt--ethnicsy m(ienasseucroendwsi)l tboeruesaecdh ttoheccoomrpraecrteggoraoluopns tfroirals2hoorft-ttheerfmirrsettdeantyioonf. b`Teheusneudmbtoercoomfptrairales gtorocuriptserfioornloonngt-hteersmecroetnedntdiaony.oftesting--this measure will `mTehaesauvreerawiglel anlusmobbeeruosfeedrrtoorscofomrpeaarcehgtrroaulposnftorhelosnegc-otnedrmdraeyteonftitoens.ting--this The latency testing~this i(sinasneoctohnedrsi)ndtiocarteoarcohftlhoengc-ortreercmtrgeoteanltioonn.trial 1 of day 2 of ReproductiveCapacity: bAteaapspsriogxniemdatteolcyoh9a0bidtaaytsioonf, aognee, mtahleeF1ratgepneerrfateimoanlerartast,wibtahsiendeoanchcdomopsuatgeer-ggreonueprwaitlled `rcaonhadboimtatuinointspoefriroadnwdilolmcounnististtabolefas, mwaitxhitmhuemexocfl1u4sidoanyosf.sibFleimnaglmeatriantgssw.ithThe o`sbpseerrmvaetdozinoasiotbusweilrlvbeed cionnasisdmeeraerd otfo tbheeavtagdianyal0coofnpternetssuamnedd/ogresatactoipounlaatnodryaspsluiggned to individual cohabitation hwiolulsbinega.ssFiegmnaeldealrattesmathtaet mdaolenortatmsaftreowmitthhienstahmeefirdsot s7adgaeysgroofup that have mated. 21-day pFoesmtaplaretruamtspewrililodb.e allowed to naturally deliver and maintain liters through a MatingPerformance: As cited above for Fo generation rats. 00710 418-008:PAGE F-20 DuraoftGesitaotionn: Protocol P41a8g.e02008 As cited above for Fo generation rats. Fertility Parameters: As cited above for Fo generation rats. EG2eneraLitttierDoatna: Viability, clinical observations and body weights for F2 generation pups will be recorded as cited above for F1 generation litters. ME \CRIFICE - 'S) UPS: As previously cited for Fo generation rats. NEC- FR 1GEO NERAPTIOSNRAYTS: Gross lesions wil be retained in neutral buffered 10% formalin for possible future evaluation (a tableofrandom units will be used to select one control group rat of each sex from which all tissues examined at necropsy will be retained, in order to provide control tissues for any possible histopathological evaluationsof gross lesions). Unless `specifically cited below, all other tissues will be discarded. `Scheduled Sacrifice - F1 Generation Male Rats: Rats will be sacrificed after completionof the 14-day cohabitation period. A gross necropsy of the thoracic, abdominal and pelvic visceral will be performed. Testes and epididymidesofmale rats will be excised and individual organ `weights will be recorded. The epididymides will be retained in neutral buffered 10% formalin. The testes willbe fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. Scheduled Sacrifice - F1 Generation Female Rats: Female rats will be sacrificed after completion of the 21-day postpartum period. The number and distribution of implantation sites will be recorded. Rats that do not delivear litter will be sacrificed on day 25 of presumed gestation and uteri will be stained with 10% ammonium sulfide to confirm the absence of implantation sites, A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Female rats `without a confirmed mating date that do not delivera litter will be sacrificed on an estimated day 25 of presumed gestation. 00711 418-008:PAGE F-21 Protocol P41a8g.e02018 EG 1 ene Ratsr Foua ndDt eadoi r Moo ribn und: Reaxtasmitnheatdfdoiertohrearceausasceriofficdeedatbhecoarumsoeroifbumnodricboundnidticoonndointitohneodraaybotrhteioonbsweilrlvabteion is smeamdien.alTvheesircaltesswoilflmbaeleexraatmsinwieldfboergerxcoisssedleasinodnsi.ndiTveisdtueasl,oerpgiadnidwyemiigdhetss,wiplrlosbteate and Broeucionr'dsedso(lsuetmiionnaflorv4es8ictloe9s6wehoiugrhsedanwditthhaenndrewtiatihnoeudt ifnlunide)u.trTalhebutfefsetreesd wi1ll0%befofrimxaeldinin. s`Tthaeturseamanidniuntegrionregacnonstweinltsboeffreetmaailneedriantsnewuiltlrablebruefcfoerrdeedd.10A%bofrortmeadlifne.fuPsresegannadn/ocry ndeeultirvaelrebdufpfueprsedwi1ll0b%efeoxrmaamliinn.edUtteorithoefeaxptpeanrtenpotslsyibnloen.prOevganrainets rwaitls bwiellrbeteaisnteadiniend with 10% ammonium sulfide to confirm the absence of implantation sites". EG 1 ene Damr swia thNot Suri vivio ngPun ps: oDrapmrseswiutmhednocasnunrviibvailnigzepdu.psAwgilrlosbse nsaeccrriofpicseydofafttheer tthheorlaacsitc,puapbdiosmfionuanld adneaddp,elmviiscsing viscera will be performed. summary tables. Postpartum data for these dams will be excluded from EAIF2GenerationPupsFoundDeadonDay1Postpartum: sPtuaptsustahtatbidrtihe. beTfhoereleuxnagmsiwnialltiboenroefmtohveeldittaenr dforimpmueprvsieabdiliintywwaitlelrb.ePeuvpalsuwaittehdlfuonrgvsitatlhat asinndk twoilhl abveeiddeinetdifsihedoratsysatfitlerbobmi;rthp.upPsupwisthwiltuhnggrsotshsatlefsioiaotnswilwilllbebeidpenrteisfeiredveadsilniBvoeubionm',s esovlaultuiaotniofnosr,poitswsiillblbeefnutoutreedeivnaltuhaetinoenc.roSphsoyudladtap.ostmortem autolysis preclude these E1/F2 Generation Pups Found Dead or Moribund on Days 2 to 21 Postpartum: Pups found lesions and fdoeratdheorcasaucsreiofifcetdhedumeortiobmuonrdicbounndditcioonndiotridoenatwhi.l be examined for gross Pups with gross lesions efvoaulnudatoinond;agyrsos2stole4sipoonsstopfaprtuupmswfiolulnbde opnredsaeyrsve5dtion2B1oupions'tspsaorltuutimonwilflorbepopssriebsleervfeutdurien: `neevuatlruaaltibounfsfeirt ewidll1b0e%nfootremadliinn.theShnoeuclrdoppsoystdamtoar.tem autolysis preclude these 00712 ' 418-008:PAGE F-22 - Protocol 4P1a8g.e00282 EAIF2Generation PupsNotSelectedforContinuedObservation: F1 for and F2 generation pups gross lesions; pups with culled gross on da4y postpartum will lesions will be preserved be sacrificed and examined in Bouin's solution. Necropsy will include asingle cross-section of the headatthe level ofthe fhryodnrtoacle-ppahraileyt.al suture and examination of the cross-sectioned brain for apparent All F1 generation pups culled on day 21 postpartum will be sacrificed and examined for gross lesions; gross lesions will be preserved in neutral buffered 10% formalin. Necropsy will include a single cross-sectionof the head at the levelofthe frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. `ScheduledSacrifice-F2 GeneratPiuposn: On day 21 postpartum, pups will be sacrificed and examined for gross lesions. Necropsy wil include a single cross-section of the head at the levelofthe frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. 00713 418-008:PAGE F-23 Protocol P41a8g-e02038 PROPOSEDSTATISTICALMETHODSTM": Aapvperroaprgieastea.ndAdpdeirtcieonnatlagpersocwieldlubreescaalncdu/loarteadn.alLyitsteesr vmaalyuebsewiplelrbfeorumseedd,iwfhaeprpreopriate. TyofpTee st I. Parametric A. Bartlett's Test' II. Nonparametric A. Kruskal-Wallis Test (s75% ties) Significant at p<0.05 Not Significant Significant at ps0.05 Not Significant _ nl Variance JTest Significant at ps0.05 Dunnett's Test Not Significant B. Fish(e5r7's5%Extaicest)Test Il. TPesrtfoorportiDoatna oVfartihaenBcienToemsiatlfoDrisHtorimbougtieonneity a. b. UStsaetidstoincallylytosiagnnailfiyczaentdpartoabwaibtihlthioesmoagreenreeiptoyrtoefdvaasrieaintchee.r p<0.05 or p0.01. c. d. TPreosptofrotriohnomdoagteanaerietnyootfvianrciluadnecde.in this category. 00714 418-008:PAGE F-24 Protocol 4P1a8g-e00284 DATAACQUISITION,VERIFICATION ANDSTORAGE: DDiarteactwoilrlabned/hoarnadp-paronpdr/ioartceommapuntaegre-rmeecnotrdpeedr.soRnneeclorwditshiwinll21bedraeyvsiaefwteedrbgyenetrhaetSiotnu.dyAl obreigbionaulnrdecaonrddsinwdilelxebde.stAarceodpiyn otfhaelarrcahwivdeastoafwtihllebTeesstuipnpgliFeadcitlotyt.heASlpoornisgoinralupdoatna will yreeqauresaftt.erPmraeisleirngveodf ttihsesudreasftwiflilnablersetpoorrte,daafttetrhewhTiecshtitnigmeFatchieltSypaotnnsoorchwiallrgbeefcoornotnaected to determine the disposition of these materials. REC TOO BEMR AINTD AINS ED: TPerosttoAcrotlicalen,dVAemhiecnldemaenndt/so.r Reagent Receipt, Preparation and Use. ARnainmdaolmiAzcaqutiisointioSnc.hedules. Mating History. TGerneeartamlenCto(mifmeprnetssc.ribed by Staff Veterinarian). CBllionoidcalSaOmbspelrevaCtoliloencstiaonnd,/PorroGceensesrianlg AapnpdeaSrhainpmceen.t. Body Feed WCeoingshutmsp.tion Values. CNaateusraarleaDnei-iSveecrtyioOnbsienrgvaOtbisoenrsv.ations. LRietftleerxObasnedrvPahtyisoincsa.l Development and Behavioral Observations - F1 Generation Pups. GOrrogsasn NWeecirgohptssy(iOf brseequrivraetdi)o.ns. PSthuodtyogMraaipnhtsen(iaf nrceqeui(rreodo).m and environmental records). PFaecekdi,nWgaatnedr/oarndShBiepdmdeinntg LAinstasl.yses. KPEEYRSONNEL: DEixreeccuttoirvoefDRiersecetaorrchof:RAelsaenarMc.h:HoMbielrdmreadn,S.PhC.hDri.s,tiDaAn,BPTh.D., ATS DiArsescotcioarotefDLiarbeocrtoartoorfyROepseeraartciohnsa:ndJSothundyF.DiBraemcettort:, BR.aSy.mond G. York, Ph.D., DABT MMaannaaggeerrooffASntiumdaylCoOopredriantaitoinosn:anVdalMeerimebeAr,ShIanrsptietru,tiMo.naSl.Animal Care and Use MaConmamgietrteoef:ReDguelnaatoCr.yLCeobmop,liV.aMn.cDe.: Kathleen A. Moran, M.S. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP 00715 418-008:PAGE F-25 Protocol 4P1a8g-e02058 EINALREPORT: Abecfoimnpalriezhedenfsoillvoewidnrgafctofnisnualltraetpioornt will with bteheprSepapnasroerd.onThcoemrpelpeotritonwilolfitnhcelusdteudtyheand will following: `ExSpuemrmiamreyntaanldDCeosnicglnusainodn.Method. AEpvapleunadtiicoens:of Test Results. Figures, Summary and Individual Tables Summarizing the Above GDaLtPa,CPormoptloicoalncaendStAastseomceinatt,edReApmoertnsdmofenSutpspoarntdinDgevDiaattaio(nisf,apSptruodpyriDaitree)ctaonrd's QAU Statement. NAL ANI Mi IT `ITnshteitpurtoiocneadluArneismdaelsCcrairbeedanidn tUhsisepCroomtomciotltheea.veAlbleeprnorceevdiuerweesddbeysctrhieveTdesitnitnhgisFapcrioltitoyc'osl tdhiastcoimnfvoorltv,e dsitsutdryesasnoirmaplasinwitlol be the caonnidmaulcst.ed in a manner to avoid or minimize TnehceesSspiotnysoforr'scosnidguncattiunrge tbheilsoswtuddoycaunmdentthse ftahcetftahcatttthhaits iinsfnoortmaatniounnnceocnecsesranriinlgythe. dpurpolciecdautirveesswteurdey mavaayilabbeleobftoarimneeedtfirnogmtthheestSaptoendsopru.rpNosoeasltofemtahteivsteu(diyn.vitro) 00716 418-008:PAGE F-26 Protocol 4P1a8g-e0s0.8 REFERENCES: 1. CThersitsst.ianE,nvMi.rSo.nmaenndtaVloyPtreokt,ecPt.iEo.n (A1g8e6n2c)y.,IWnaVsihviongRteopnr,odDu.cCt.ivNeatainodnaMluTteacghenniiccailty Information Service, U.S. Departmentof Commerce, Springfield, VA 22161 2. Cnharlitsrteixaonn,eM.(SP.ro(c1e9e8d4i).ngsReopfrNoadlutcrteixvoenetoSxiycmiptyosainudmt,erNateowloYgoyrekvaAlcuaatdieomnsyooff Sciences, November 7, 1983), J. Ciin. Psychiat. 45(8):7-10. 3. LCoanntgr,olP.DLa.t(a19i8n8t)h.e EChmabrrlyeos RainvdeFreCtarl:DCeDveBloRpRmaetn.taClhaTrolxiecsitRyiv(eTerrLaatboolroagtyo)ries, Inc., Wilmington, MA Laboratories, Inc.) 01887-0630. (Data base provided by Argus Research 4. LaIbnsotritauttoeorfyLAanbiomraaltso.ryNaAtniiomnaall RAecsaoduermcyesPr(1e9s9s6,).WasGhuiindgetfoonr,tDh.eCC.are and Use of 5. ISmaplleawnstkait,ioEn.ss(t19e6l4l)e.n aFmarUbteemruesthdoedreRaztutem. maArkcrho.skPaotphiols.chEexnp.NaPchhawrmeaiksolv,on 247:367. 6. tShneedbeicnoormi,aGl.dWis.traibnudtioCno.chSrtaant,isWti.cGal.M(e1t9h67o)d.s,V6atrhiaEndicteiotne,stlfoowrahSotmaotgeeUnneiivteyrsoifty Press, Ames, pp. 240-241. 7. BSiookmale,trRy,R.W.aHn.dFRroehlefm,aFn.Ja.n(d19C69o).. SBaanrtFlreatntcitsecsot,ofpp.ho3m7o0g-e3n7e1i.ty of variances. 8. SMneetdheocdosr,,6tGh.WE.ditainodn,CloocwharaSnt,atWe.UGn.iv(e1r9s6i7t)y.PrAensasl,ysAimseosf,Vaprpi.a2n5c8e-.27S5t.atistical 9. Dunnett, C.W. (1855). A treatments with a control. muJ.ltAimpelre.coSmtpata.rAisssooncp.r5o0c:e1d0u8r6e-f1o1r2c9o.mparing several 10. SWo.kHa.l,FrReRe.maanndanRodhlCfo,.,F.SJ.an(1F96r9a)n.cisKcrou,skpapl.-W3a8l8l-i3s88T.est. Biometry, 11. 6D(u3n)n2,41O.-J2.52(.1964). Muliiple comparisons using rank sums. Technometrics 12. SMiceGgrealw,-HS.il(l1,95N6e).w YNoornkp,aprpa.me9t6r-i1c04S.tatistics for the Behavioral Sciences, 00717 PROTOCOLAPPROVAL: FOR THE TESTING FACILITY eden A George E. Dearlove, Ph.D., iT Associate Directorof Research oi G. York, Ph.D. DABT Assdviate Director of h Study Director rbara J. Pattesori, BA. hairperson, Institutional Animal Care and Use Committee 418-008:PAGE F-27 Protocol 4Pa1g8e028r 2m Date 2/- wy 94 Date Mue z tro Date FOR THE SPONSOR Pre: Tos SMtaurvdiynMTo.niCtaosre, D.V.M., Ph.D. 29 they ace Date 060718 418-008:PAGE F-28 ATTACHMENT 1 STUDY SCHEDULE 0719 ATTACHMENT 1 418-008:PAGE F-29 ProtocPoalg4e181001028 SCHEDULE" 12 MAY 98 26 MAY 98 26 MAY 98- 15 JUL 98 26 MAY 98- 19 AUG 98 09 JUN 98 - 22 JUN 98 2229JJUUNNSSBBPPMM--2096JJUULNSIBBAAMM 23 JUN 88 06.JUL 98 200uL 98 A(nFiomgaelneRreacteioinpt-ratAsc),climation Begins Start Rats (o2f8DdoasyasgebePfeorrieodco-haFboitGaetnieornaatnidon Male pceornitoidnuuinntgiltsharcoruifgihceaa1ft4e-rdsauycccoehsasbfiutlatmiaotning has been determined). CDaoessaagreeaPne-rSieocdti-oFneimnagl(e28RadtasyAssbseifgonreed to pcorheasbuitmaetdiognesatnadtiocno)n.tinuing through day 09 of DNaotsuaragleDPeelriivoedry- [F2e8mdaalyesRbaetfsorAessciohganbeidtattoion through day 24 of that do not deliver presumed a liter) or gestation day 20 (rats postpartum (rats that deliver a ltter)) Dosage Period Estrous Cycle Evaluation. Cohabitation Period Male 1 (07 days) (Maximum of 14 days). Male 2 (07 days) FLiarsstt PPoossssiibbllee DDaayy 00ooff PPrreessuummeedd GGeessttaattiioonn.. CFoomGpelneetriaotnioofnthMealCeohRaabtistaStaicorinfPiceerdioadft(eErarliest possible date). a. The study initiation date is the day the Study Director signs the protocol. C00720 ATTACHMENT 1 418-008:PAGE F-30 ProtocPoalg4e182-001028 03JUL 98 16 JUL 98 14.JUL98 310uL98 184UL 98 31JuL98 03AUG 98 20AUG 98 04 AUG 98 19. 0CT 98-02 NOV 98 16 NOV 98 10 NOV 98 - 27 NOV 98 30 NOV 98 - 17 DEC 98 06 APR 99 First Possible Day 10 of Caesarean-sectioning. Presumed Gestation Last Possible Day 10 of Caesarean-sectioning. Presumed Gestation First Possible Delivery (Day 21 of presumed gLeassttatPioosns)i.ble Delivery (Day 25 of presumed gestation). First Possible Day Female Sacrifice. 25 of Presumed Gestation FLaesmtalPoessSaicbrlieficDea.y 25 of Presumed Gestation FFi1rsgtePnoesrsaitbiloen Dpauyps21noWtesaenliecntged(Dfoarmcsonatnidnued LoabssterPvoastsiiobnlesacDraifyic2e1d).Weaning. F1 Generation Postweaning Observations Begin (Detailsof tests cited in protocol). wF1heGnenreartastairoenaCpophraobxiitmaattieolnyP9e0ridoady(sInoiftiaagteed approximate inital date). CF1omGpelneetriaotnioofnCoMhaalbeitRaattisonSaPcreirfiiocded- after Approximate Earliest Possible Date. GDeelnievreartyioPenrLiiotdte-rsF1(AGpepnreorxaitmiaotne Ddaatmess)/.F2 GSaecnreirfaicteioonf LFi1ttGeersneornatDiaoyn2D1aPmosstapnadrtFu2m (Approximate dates) Draft Final Report. 00721 418-008:PAGE F-31 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 00722 WDaATTEARISAHLEETSAFETY an0 canter SStS.iasPa1u0l0,0 Wimesota 1-800-364-3577 or (612) 737-6501 (24 hours) 418-008:PAGE F-32 ACiLonLpfyorrriaigaghthtit,sonr1fe9os98re,rvtehMdei.nnpeusCropotopsayeinMgiofniannpgdro/poaernrdldyoMwaWnntluioflaaidczitinungrginogf tChopimrspoadnuyc.ts 51)2 pTahrleiloorwiendsfgorrpemrsaostveiinodtnediissthcoaob:ptiaeidnedinfrfoumlloHW,ithandno changes unless 2) anlessttrhiebruttehde wciotphy tnhoer tihnetenotriiogninaolf issarnriensgolda oprrofoitthertwhiesreean. TDRIAVGIESIONNA:HE: am CHEMICALS 10FoNO-WsB5ERF/LUU.DRF.ACD. Brand Fluorochemical Surfactant 9980.-00221017.-00818083..57 260002. 104d 1 0000-.5511113355-.0099035642-.17 9988.-00221017--9091106.41.50000-.5511113955-.0092035151..83 S1USPSUEERDS:EDEJSa:nuaNroyvem29b,er10558, 1997 DOCUMENT: 10-5796-5 1. increotent CAs Ho. Percent PPPOOOTTTAAASSSSSSIIIUUUMMM PPPEEERRRFFFLLLUUUGOORRROOOAAALLLKKKYYYLLL SSSUUULLLFFFOOOWNNAAATTTEEE.................. 2S27e99745i2.-039.904-.536.3 832 3 |- a?s PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOOWNAATTEE............ @308z7702..552.55 .112 is4 2. PYSIOAL BATA vVBaAOpPIoOLrRIPNRpGEeSPnSsUTORrE::.IL1N111T1111.1 .. WNIAA Mia SSEPOVELACUPIBOFIRILACITTIYGORNATRVnAWITTAEYT;:E1RS.11.1.1.1010110010010] NaiAagne aa. 0.6 Hateres PERCENT VOLATILE: ............. 0'%(Bulk) VIwSeSLTOISNIGTYP:oro e: L aL nL . HI(D0.1% Ne Aqueous) APLPiEgAnRtANcCoEloArNeDd,ODOfRr:es flowing powder. Abbreviations: N/D - Not Dsterained Wi - Not Applicable Ga . Approximately 000723 JSaDnSu:aryFG-299,5 1F9L9U8ORAD Brand Fluorochemical Surfactant 418-008:PAGE F-33 PAGE 2 3. FIRE AND EXPLOSION HAZARD DATA FFLLAASMHMABPLOEINTL:I.M.I.T.S....L.E.L.:.1..1.0.0.0.0. NN/oAne AFULTAOMIMGANBILTEIOLNIMTIETMSPE.RAUTEULR:E.:............. NN//AA EXMTaItNeGrU,ISHCIaNrbGonMEdDIiAo:xide, Dry chemical, Foam SPWEeCaIrALfuFlIlREprFoItGeHcTtIiNvGePcRlOoCtEhDiUnRgE,S:including helmet, self-contained, apnodsitpiavntes,prbeasnsdusrearoorunpdresarsmusr,e wdaeimsatndanbdrelaetghsi,ngfaacpeparmaatsuk,s,anbdunker coat protective covering for exposed areas of the head. UNSUeSeUALHazFaIrRdEouAsNDDeEcXoPmLpOoSsIiOtNioHnAZAsReDcSt:ion for products of combustion. 4. REACTIVITY DATA STABILITY: Stable . INNCoOtMPaApTpIlBiIcLaIbTlYe.- MATERIALS/CONDITIONS TO AVOID: HAZARDOUS POLYMERIZATION: Hazardous polymerization will not occur. HACZaArRbDoOnUSMoDnEoCxOiMdPeOSIaTndIONCarPbROoDnUCDTiSo:xide, Oxides of Sulfur, Hydrogen Fluoride, Toxic Vapors, Gases or Particulates. 5. ENVIRONMENTAL INFORMATION a SPOILbLserRvEeSPOpNrSeEc:autions from other sections. Vacuum, use wet sweeping bceompanounidgniotrioWnatesrourtcoe.avoiCdleadnustuipngr.esCiAdUuTeIOwNi!thAwavtaecru.um cPllaecaenerincoanuld approved metal container. Seal the container. RECD0OMMnoEtNDErDeleDaIsSePOStAoL:waterways or sewer. Do not use in products or p1r/o10cesosfesthethaltowecsotuldEC5r0esuolrtLCi5n0aqcuoantciecntrcaotnicoenn.tratIinocnisnegrrateeateirn atnhan miantdeursitarli.al CorombcuosmtmieornciaplrodfuacctisliwtiyllinintchleudperesHFe.nceDiosfpoasaclombustible alternative: Dispose of waste product in a facility permitted to Abbreviations: NID - Kot Determined NIA - Not Applicasie Ca Approximately C0074 , ` 418-008:PAGE F-34 JMaSnDSu:aryFC2-99,5 1F9L9U8ORAD Brand Fluorochemical Surfactant PAGE 3 S. ENVIRONMENTAL INFORMATION (continued) BE accept chemical waste. EN9V6I-RHOrN.MENATqAuaLtiDcATAF:ish LOSO, Fathead Minnow(Pimephales prosslas)=3s mg/l, BGgilagui;erdgni8e0lr0ls2)0S=u1n=1fiNsinlhg.(/1L;epo4m8i-sHr.maEcCrSoOc,hiDraupsh)n=i6a8 mMga/gln,a R=ai50nb8o3w/1T;roGuOtD(=S.a0l0m4o REVGoUlLaAtTiOlReY OIrNgFaOnRiMcATICOoNm:pounds: N/A. VOC Less H20 & Exempt Solvents: N/A. Sbienfcoererdeigsuploastailo.ns vU.aSr.y, EPcAonHsauzltardaopupsliWcaasbtlee Nreugsublearti=onsNonoer a(uNtothorU.iSt.ies EPA Hazardous.) TTShCiAs, prEoINdEuCcSt, coCOmSpLl,iesAICwSi,thMItThIe canhdemiKocraela.registration requirements of EPFICRREA HHAAZZAARRDD: GLNAoSS:PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 6. SUGGESTED FIRST AID or EYEInCmOeNdTiAaCtTe:ly flush eyes ith large amounts of water for at least 15 minutes. Get immediate medical attention. SKIINmmCeOdNiTaAtCeTl:y flush skin with large amounts of water. Remove cCoonnttaammiinnaatteedd ccllootthhiinngg. beIfforierrrietuastei.on parsists, call a physician. Wash INIHfALA_TsIiOgnNs:/sysptoms occur, resove person to fresh air. If signs/syaptons continue, call a physician. 1FD_rSiHnALkLOtWwEoD:glasses of water. Call a physician. 7. PRECAUTIONARY INFORMATION Tm EEAvoPiRdOTEeCyTeIOcNo:ntact. Wear vanted goggles. Abbreviations: N/D - Not Deterained N/A - Not Applicable GA - Approximately ~COO7RS 418-008:PAGE F-35 JMSaDnSu:aryFC-299,5 F1L9U98ORAD Brand Fluorochemical Surfactant PAGE 4 7. PRECAUTIONARY INFORMATION (continued) SKAIvNoiPdROTsEkCiTnIOcNo:ntact. Wear appropriate gloves when handling this mraetceormimaeln.ded:A pabiutrylofrugblboevre.s maUdsee ofnreomorthemorfeollofowitnhge fmaotlelroiwailn(gs) are cpoevresroinnagl, pcroovteercatlilosn. itePmrsoteacstinveecesgsaarrsyenttso p(roetvheenrtthsakninglcoovnetsa)ct:shouhledad pboelymeatdheyloefneeiptohleyrvionfyltihdeenefollcohwlionrigdemat(eSrairzalnse:x). REUCsOeMMwEiNtDhEDapVpErNToIpLrAiTaItOeN:local exhaust ventilation. Use in a well evmeinstsiiloantsedbearleoaw. recPormomviednededsufefxipcoiseunrte lviemnittisl.atioInf etoxhamuasitntavienntilation is not adequate, use appropriate respiratory protection. REASvPoIiRdATObRrYeatPhRiOnTgECToIfONd:ust. Select one of the following NIOSH approved arcecsopridraantcoerswibtahsedOSHonA raeigrubloartnieoncso:ncenhtarlaft-imoanskofdusctontaanmdinmaisnttsreasnpdiriantor, fhualllf--fmaacsek ssuupppplliieedd aaiirr rreessppiirraattoorr., full-face dust and mist respirator, PREDVoENnToItONeatO,F AdCrCiInDkENoTrALsmoIkNeGESWThIeOnN:using this product. Wash exposed bareefaosretheaotrionugg.hly With soap and Water. Hash hands after handling and REKCeOeMpMENcDoEnDtaiSnTeOrRAGdEr:y. Keep container closed when not in use. FINRoEnfAlNaDmmEaXbPlLeO.SION AVOIDANCE: OTHNEoRsmPoRkEiCnAgU:TISOmNoAkRiYngINwFhOiRlMeATIuOsNi:ng this product can result in ocfonttahmeinhaatziaorndouosf tdheecomtpoobsaictcioonandp/roorducstmsokemenatndionleedadintosethcetiofnorm4atoifon this MSDS. HMIS HAZARD RATINGS: PHEEARLSTOHN:AL2PROFTLEACMTMIAOBNI:LITXY:(Se0e pRErAeCcTaIuVtIiToYn:s, 0section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT TYPE AUTH SKIN PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONNAATTEE...... 00..11 MMGG//MM33 TMTMHAA 3aMM vY PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONRAATTEE...... 00..11 MMGG//MM33 TMTMAoow YY Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 000726 418-008:PAGE F-36 MSDS: FC-95 FLUORAD Brand Fluorochemical Surfactant January 29, 1998 PAGE 5 EXPOSURE LIMITS (continued) INGREDIENT VALUE UNIT TYPE AUTH SKIN POTASSIUM PERFLUOROALKYL SULFONATE... 0.1 MG/M3 A aM Y + SKIN NOTATION: Listed substances indicated With 'Y' under SKIN refer to itnheclupdoitnegntimaulcoucsontmreimbburtaineonantdo the overall eye, either ebyxpoasiurrbeornbey the or, cmourteanepaorutsicruoluatrely, by direct contact with the substance. Vehicles can alter skin absorption. S- OU3MR:CE OF3MEXRPeOcSoURmEmenLdIeMdITEDxApToAs:ure Guidelines 8. HEALTH HAZARD DATA EYEMilCdONTEAyCeT:Irritation: signs/symptoms can include redness, swelling, pain, and tearing. SKIMiNldCONSTkAiCnT:Irritation (after prolonged or repeated contact): signs/symptoms can include redness, swelling, and itching. Meaxytenbdeedabtsiomreb.ed through the skin and persist in the body for an INMHaAyLATbIeONh:armful if inhaled. May be tine. absorbed by inhalation and persist in the body for an extended Single overexposure, above recommended guidelines, may cause: sIorrreinteastsionof (tuhpepernorseespainrdattohrryo)a:t, sicgonusg/hsiyngmptaonmdssnceaenziinngc.lude IF InSgHAeLsLtOiKoEnD:is not a likely route of exposure to this product. Illness may result this material. from a single swallowing of a moderate quantity of May be harmful if swallowed. MUTAGENICITY: Mutagenicity assays indicate the product is not mutagenic. Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-008:PAGE F-37 MJSaDnSu:aryFO-299,5 F1L8U98ORAD Brand Fluorochesical Surfactant PAGE 8. WEALTH HAZARD DATA (continued) RENPoRtODUtCeTrIaVtEo/gDeEnViEcLOiPnHEtNhTeALratTOXaItNSo:ral doses below maternally toric Levels. OTTHhEiRsHEprAoLdTuHctHAZ1sARDnotINkFnOoRwMnATItOoN:contain any substances regulated under California Proposition 65. A Product Toxicity Susmary Sheet is available. SECTION CHANGE DATES HEADING SECTION CHANGED SINCE November 05, 197 ISSUE Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately TbIeMhPeLcIoiErnDrf,eocrtmIaNtaGsiLoUDnoIfNiGnt,hethBiUdsTateMNOaTtiessrLuiIeaMdlI.TESDaf3eTMOt,yMAAKDNEaYStaNISOMhPeWLeAItRERDA(NWMTASIDRESRS)A,NTiYsEXOPbFReElSieSvEeDdORto HPwhEEeRRtFChOHeRArMNATNAtChBeEILO3IRMTYUprSOoRAdGuEFcItOTFNEiSTsSRATDiFEtO.RfAorUsPeaArRpTIaiCsrUtLirAceRuslpaoPrnUsRiPpbOulSreEposOfeRorCanOddUetReSsrEumiitOnFaibnlge for cuasner'asffemcetthotdheofuseuseandorapappplliiccaattiioonn.of GaivIeMnprtohdeucvta,riestoymeofoffWahcitocrhsartehat uthneiquuesleyr Weivtahliunatethteheuse3rM'sprokdnuocwtledtgoedeatnedrmcionnetrowlh,ethiterisitesisseTnittialforthaat particular purpose and suitable for user's method of use or application. 3DinMueperrtrooovritsdh,eesormieinmsfostoieromnapstoisosornibiaillnitteeyrlaetctithorantosnieclinecftotrrhoiasniacsintfarorasmneasrtfvieiorcne,matyo3Mhiamtvasekecsursetnsooumletresd. irnefporremsaetnitoatnioonbstaiansedtofriotms acodmaptlaebtaesneesmsayornoatccubreacays.curIrnenatddiatsiotnh,e information in the MSOS available directly from 3M. 000728 418-008:PAGE F-38 ATTACHMENT 3 TEST ARTICLE PREPARATION PROCEDURE 000729 3 ` 418-008:PAGE F-39 ATTACHMENT 3 Version:418.P0r0o8toc(2o1lM4A1Y8.0980)8 `TEST ARTICLE PREPARATION PROCEDURE Page tof3 Test Article: Vehicle: PFOS. 0.5% Tween 80 in R.O. Deionized Water. A. Purpose: TofhdeopsuargpeosseuosfpetnhsisiopnrsocoefdPuFreOiSs taondprtohveidceonatrmoeltahrtoidclfeofrotrhoeraplreparation `administration to rats on Argus Study 418-008. B. General Information: 1. All suspension containers will be labeled and color coded. Each label will specifythe protocol number, test article identification, Argus batch number, concentration, dosage level, preparation date, expiration date. and storage conditions. 2. Suspensions will be prepared: X_ Daily _ Weekly _For__daysofuse 3. Suspensions will be prepared at a final dosage volume of milkg. 4. XSa_fety:Gloves, lab coat, gogglesor safety glasses and faceshield X_C HDuasltf--MFiascte RReessppiirraattoorr Z TFyuvleFkaScueitR/eAsppriornator/Positive Pressure Hood 5. Dosage solutions adjusted for Free base and % Purity. -- Yes X_ No (Calculations based on 100%) _ FreeBase __ Purity 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. 090730 418-008:PAGE F-40 ATTACHMENT 3 Version: 418-P0r0o8to(c2o1lM41A8Y.0906)8 TEST ARTICLE PREPARATION PROCEDURE Page 2013 NOTE tTheestloarwtidcolseawgiel.beSiprrebpaarrseadreastoabseeraiadlddeidluttoiotnhfercoomnttahienehrisg;h mdioxsiangge to should occur during sampling and/or dosage administration. C. Preparationofthe Control Group: 1. Add the required amount of vehicle to an appropriate vessel. (See TEST ARTICLE CALCULATIONS for exact quantities.) D. Test Article Solution Preparation: 1. Tinotporaenpaaprperotphreia0t.e6l4y-smigz/emdL,,la(bgerloeudpcVo)ntsaoilnuetrio.n, add 160 mg of test article 2. Q.S.t0250 mL with the vehicle and mix by inversion. 3. `Aadpdpraosxtiimratbealrya3n0dmhienauttetshe(oprrenptairatthieontetsot8a0rticCleinhaaswdaitsesorlbveadt)h.for 4. Remove the solution from the solution equilibrates to the water bath, and room temperature. slowly spin over night while 5. sTooluptrieopnatroeagnroapupproIVp,ri(attheel0y.l3a2be-lmegd/mcoLntsaoliunteiro,n)t,heandd.s1.00tom2L00ofmgLrowuipthV vehicle. Adda stir bar and mix until uniform. 6. sTooluptrieopnatroeagnroapupprolplri(attheel0y.l0a8b-emlegd/mcLonstoaliunteiro,n)t,haednd4.55.0tmoL20of0gmrLouwpitIhV Vehicle. Adda stir bar and mix until uniform. 00731 418-008:PAGE F-41 ATTACHMENT 3 Version: 416.0P0r8o.to(c2o1lM41A8Y.05088 TEST ARTICLE PREPARATION PROCEDURE Pagesars 7. Written by: STooluptrieopnatroeagnroapupproI,pr(itahteel0y.0l2ab-emlgeidmcLonstolauitnieorn,),thaednda5.0. mtLo.2o0f0grmoLupwiItih vehicle. Add a stir bar and mix unt uniform. ; Le (sete & eel Aoproved be ed] Eh, vate: 22-916 oc rn) Clarification: _No _Y_Yes (See attached clarification form.) Initial/Date : MC 1-11-49 00732 418-008:PAGE F42 axcos : TEST APRRTOICCELDEU/RSEUBCSLTAARNICFEICPARTEIPOANRATION Protocol: _Y/-00% CDaltaestfoifcacion shia Eben rip PrSeptaacnascion ct Version: 4/-00% (21 mw.ay Clastsicatson 200 0s of ohio os odeled ce prepens Contre] Gocen cp lish Teds Dive hams --- -- c-- e ---------------- BErl EE EE Reviewed by: deML ee . CE er lish, 0D8a.c1e57:5_7_FT-AS1P5P-E9-081-02 LL0733 418-008:PAGE F-43 A iF Sr neseancy Argus Research Laboratories, inc. B x LABORATOC RIES , HovrTshaaem,siPeennnlssyl.vaneiysa e1s30s6?4 PROTOCOL 418-008 COMBAINNEDDPOERRAILN(AGTAAVLAPGOES)TNFEARTTAILLIRTYE,PRDOEDVUECLTOIPOMNENTAL TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 1- June 2, 1998 r--m--------eeetee-------------------------------- 1. Storage (page 3ofthe protocol) t[EefmfpeecrtiavteurDeatoev:ernMiagyht2,6r,at1he9r98t]hanPrefproazreen.d formulations willbe stored at room `Refoar s Chaongne: "This change preparation. was made to clarify the protocol and faciitate dose formulation 2. EreqoufPe repnaractiy on (page 4 of the protocol): W[Eaftfeerc)tiwviellDabtee:preMpaayre2d6,we1e9k8l8y] bTyhaeddveihnigcl1e5(m0L.s5%ofTwTeweenen80808intoR.2O9.8D5emioLniozfed RO. Deionized Water. Reafos rCho angne: This change preparation. was made to clarify the protocol and facilitate dose formulation 3. Stability (page 4ofthe protocol): o[EnfftehcetitveestDaarttei:cleMiany02.75,%1T9w9e8]enT8h0eSpsoolnustoiornsh.asTchoisnfailrlmoewds afo4r8p-rheopuarrasttiaboinlsittyo be made ane daypriorto the day of dose administration. Due to the lengthy 000734 418-008:PAGE F44 - Prot`oAcmoeln4d1m8e-n0t081 Page2 preparation dosing. procedure, dose solutions may be made one day prior to the day of Rea fos rCho angne: `pTrheipsarcahtaionng.e was made toclarify the protocol and faciltate dose formulation Co obi pres, Ut peu ADliarnecM.toHfrobReersmeaanr,chPh.D., DABT Date AsRsaoycmiiatned GD.irYeoctrokr, oPfh.DR.e[spDaArcBhT Date and Study Director 2Cot Co;ho thew TGow fuetr DMeemnbaeCr.,LIenbsoti,tuVt.iMon.aDl.Animal Care Date MSatruvdiynMTo.niCtoars,e, DVM.PhD. Date and Use Committee 0073S 418-008:PAGE F-45 esearch Argus Research Laboratories, Inc. ----------------------A--S----C--s----------------------------Ho--rTers--ha--m,s--aPsa--enrn--soy--l.v--aa1n--isa--a1--s90s--44 PROTOCOL 418-008 COMBIANNEDDPOERRAILNA(TGAAUVAPGOES)TNFAERTTAILLIRTEY,PRDOEDVUECLTOIPOMNENTAL TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 2 - June 11, 1998 1. Body Weights - Female Rats (page 12of the protocol): a[lEfsfoecbteivceolDlaetec:tedMoanyF2o6,ge1n9e9r8a]tiBonodfyemwaeliegshtosnapnodstfpeaerdtucmondsauymp1t0i.on values will Reafos rCho angne: TFh1isgeinnefroartmiaotniofnemwaalses.added to the protocol to match data collection on 2. Scheduled Sacrifice of Male Rats (page 14ofthe protocol): w[iElflfbecetiwveeiDgahteed: iMndaivyid2u6a,lly1.998] At scheduled sacrifice of Fo male rats, all organs ReafosrChoangne: "This change clarifies the protocol. 3. Sc(phaegdeul1e5odfStachreifpirocteo-caFle)m:aleRatsAssignedtoCaesarean-Sectioning [Effective Date: May 26, 1988] Uteri of non-pregnant rats wil not be retained. 000736 418-008:PAGE F-46 Pro`toAcmoeln1d8me.n0t082 Page2 Reason for Change: This change was made at the requestof the Sponsor. 4. Scheduled Sacrifice - F1 Generation Male Rats (page 20 of the protocol): [pErfofsetcattievse,Dawtiel:beMawyei2g6h,e1d99at8]scSheemdiunlaeld svaecsriicfliecseo(fwiatlhl aFn1dgweintehroauttioflnuimdas)leanrdats. Rea forsCho angne; gTheinseriantfioornmamtailoens.was added to the protocol to match data collection on Fo Atle nse Ue LL re Alan M. Hoberman, Ph.D., DABT Date Director of Research Raykdond ASsociate G. York, Director fol. DABT oMRsearch Date and Study Director WANNA Dena C. Member, LIenbsoti,tuVt.iMon.aDl. Animal Ca/re Date and Use Commitiee i The 115 see MSatnuvdiynMTo.niCtaosre, DVM. PhD." Date 000737 . 418-008:PAGE F-47 research RK Argus90R5esSehaerechhyLDarbiovrea,tBouriiledsi,nIgncA. E did m HorTsahiagm,uPesnnrsylFvaanria ae13s044 PROTOCOL 418-008 COMBAINNDEDPOERRAILN(AGTAAVUAPGOES)TNFEARTTAILLIRTYE,PRDOEDVUECLTOIPOMNENTAL TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 3 - June 25, 1998 1. AdminiMsetthordaandtFireqouennc,y (Page 10 of the protocol): c[aElfcfuecltaitvieonDatthee:dJousnaege19p,er1i9o9d8]wilDlubeeteoxatepnrdoegdrafomrmainngadedrirtoironinald2oswaegeeks and the ercroohrabiintadtoiosnagpeerciaoldcuwillaltioocncsurcatuwsoewdetehkesmlaalteerrtahtasntoorriegicneailvley ascphperdouxliemda.telTyh8e9% to 90% 99% of tohfethteartgaertgeedteddosdaogsea.geThainsdofcecmuarlreedraftosr stioxroefcetihveesaepvpernoxdiamyasteilnyth9e5% to second and third weeks and two daysofthe fourth weekof the dosage period. Reason for Change: This change cohabitation. was made to ensure that all rats receive the correct dosage before 000738 418-008:PAGE F-48 Prot`oAcmolen4d1m8e-n03t8 Page 2 2. Schedule (Attachmen1tofthe protocol): 12 MAY 98 SCHEDULE A(nFoimgaelneRreacteiiopntr-atAsc)c.iimation Begins 26 MAY 98 26 MAY 98 - 29 JUL 98 26 MAY 98 - 02 SEP 98 09 JUN 98 - 06 JUL 98 0163JJUULL9SBBPPMM--1230JJUULLI9B8AAMM 07.JuL 98 20JUL 98 03AUG 98 17JuL 98 304uL98 RStaatrsto(f42DodsayasgebePfeorrieocdo-haFboitGaetnieornaatnidon Male. pcoenrtiionduuinntgiltsharcoruifgihceaa1ft4e-rdsauycccoehsasbfiutlatmiaotning has been determined). DCaoessaagreeaPne-rSieocdti- oFneimnagl(e42RadtasysAsbseifgonreed to cohabitation and continuing presumed gestation) through day 09 of NDaotsuargale DPeelriivoedry- (F4e2mdaalyesRbaetfsorAesscioghanbeidtattoion through day 24 of that do not deliver presumed a litter) or gestation day 20 (rats postpartum (rats that delivear litter). Dosage Period Estrous Cycle Evaluation. Cohabitation Period Male 1(07 days) (Maximum of 14 days). Male 2 (07 days) First Last Possible Possible Day Day 0 0 of of Presumed Presumed Gestation. Gestation. CFoomGpelneetriaotnioofnthMealCeohRaabtistaStaicornifiPceerdioadft(eErarliest possible date). First Possible Day 10of Caesarean-sectioning. Presumed Gestation Last Possible Day 10 Caesarean-sectioning, of Presumed Gestation 00739 28JUL98 14 AUG 98 01AUG 98 14 AUG 98 17 AUG 98 03 SEP 98 18.AUG 98 02 NOV 98 - 16 NOV 98 30 Nov 98 24 NOV 98- 11 DEC 98 14 DEC 98 - 31 DEC 98 06 APR 99 418-008:PAGE F49 Pro`toAcmoeln4d1m8e.n0t083 Page 3 First Possible gestation) Delivery (Day 21 of presumed Last Possible gestation). Delivery (Day 25 of presumed FFiermstalPeosSsaicbrliefiDcea.y 25 of Presumed Gestation Last Possible Day Female Sacrifice. 25 of Presumed Gestation FFi1rsgtePnoesrsaitbiloen Dpauyps21noWtesaenliecntged(Dfoarmcsonatnidnued observation sacrificed). Last Possible Day 21 Weaning. BFe1gGienn(eDreattaiilosn oPfotsetswtesacniitendg iOn bpsreotrovcaotl)i.ons wF1heGnenreartsatairoenaCpophraobxiitmaattieolny P9e0ridoadys(Inoiftiaagteed `approximate initial date). F1 Generation Male Rats Sacrificed after ACpopmrpolxeitmiaontoefECaorlhiaebsittPaotsisoinblPeerDiaotde.- `DGeelnievreartyiPoenrLiiotdte-rsF1(AGpepnreorxaitmiaotne Ddaatmess)/.F2 Sacrifice of F1 Generation Dams and F2 (GAepnperroaxtiimoantLeitdtaetresso).n Day 21 Postpartum Draft Final 000740 418-008:PAGE F-50 -" Prot`oAcomlen4d18m.e0n03t8 Page + Rea forsCho angne: `wTeheisksscahneddutlheewcaohsabcihtaantgioendpdeureiotdootchceurerxitnegnstiwoonowfetehkes dlaotseirntghpaenrisocdhebdyultewdo. (VonZo AS en[25 pws $e, ADliraenctMo.r HofobReersmeaanr,chPh.D., DABT Date 2355p 958 ARsasyocmioantde GDi.recYtdiok,r-f fRh.eD.s,eDarAcBhT Date and Study Director Lave Chale 25disr Dena C. Lebo, V.M.D. Date ManedmbUesre,CIonmstmiitutttieoneal Animal Care Pons TC 13d 5 Marvin T. Case, `Study Monitor D.V.M., PhD. Date 00741 7.,PRIMED]ICA 418-008:PAGE F-51 Argu56s5S Researh chDeLavbeoe r,aBtooriiey sn,gInc. TelpO1h3)on47s10 Telefax: (215) 443-8587 PROTOCOL 418-008 `COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS. SPONSOR'S STUDY NUMBER: 6295.9 Amendment 4 - July 14, 1998 1 TpersottoAcrotli)c:lePreparationProcedure (Version 29 JUN 98of Attachment 3 to the p[Erfefpeactrievde eDaatceh:daJyunwiell29b,e 1998] The increased. volumeof The new tThesettAersttiacrlteicPlreespoalruattiioonns Procedure is attached to this amendment. Reafos rChoangne: Itis necessary to increase the volume of the test article solutions prepared each d ecause of the increasing body weightsof the rats. A= Sf an . A 19-quc-9; ~#/GAsesoorcgieatEe.DDieraerlcotvoerlof ResPeh.Da.,rDcAhBT Date RAsasyoicoialtdeGD.irYeocrtko,r oPhf.D.ReseDaArcBhT Date `Study Director Jarbara J. P: on, BA. Date Chairperson, Inftitutional Animal Care and Use Committee Wlewon Tle 278 Marvin T. Case, D.V.M., Ph.D. Date Study Monitor 00742 . 418-008:PAGE F-52 ATTACHMENT 3 Version: 418-P0r0o8to(co2lJ94U1N8.5080)8 TEST ARTICLE PREPARATION PROCEDURE Page 103 Test Article: PFOS. Vehicle: 0.5% Tween 80 in R.O. Deionized Water. A. Purpose: oTfhdeopsuargpeosseusopfetnhssiopnrsoocfedPuFrOeSis taondprtohveidceonatrmoeltahrtoidclfeofrotrhoeraplreparation `administration to rats on Argus Study 418-008. B. General Information: 1. sAlplecsiufsypethnesipornotcoocnoltaniunmerbserw,illtebset laratbieclleedidaenntdifcioclaotirocn,odAerdg.usEbaacthchlabel will number, concentration, and storage conditions. dosage level, preparation date, expiration date 2. Suspensions wil be prepared: _X_ Daily Weekly _For__daysofuse 3. Suspensions will be prepared at a final dosage volume of mLiks. 4. Safety: X_ XC Gloves, lab coat, goggles Dust-Mist Respirator or safety glasses and faceshield _ Half-Face Respirator Z Z TFuylvleFkaScueitRieAsppriornator/Positive Pressure Hood 5. Dosage solutions -- Yes adjusted _X_ for No Free base and % Purity. (Calculations based on 100%) Z_ FreeBase __ Pury 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. 00743 418-008:PAGE F-53 ATTACHMENT 3 Version: 418.P0r0o8to(co2l0418JU-N 0980)8 `TEST ARTICLE PREPARATION PROCEDURE Page2013 NOTE: Ttheestloarwtidcolseawgilel.beStpirrebpaarrseadreastoabseeriaadlddeidluttoiotnhfercoomnttahienehrisg;h mdioxsiangge to should occur during sampling andor dosage administration. C. Preparationofthe Control Group: 1. AAdRdTItCheLEreCqAuiLrCedULamAoTuInOtNofSvfeohriecxlaecttoqauanntaiptpireos.p)riate vessel. (See TEST D. Test Adicle Solution Preparation: 1. Tinotoparnepaaprperotphreia0t.e6l4y-smigz/emdL,,la(bgerloeudp V) solution, container. add 224 mg of test article 2. Q.S.1t0350mLwith the vehicle. 3. A`adpdpraoxsitmiratbealrya3n0dmhienauttethse(oprreupntairlattihoentetsot8a0rticCleinhaaswdaitsesrolbvaetdh).for 4. Remove the solution from the water equilibrates to room temperature. bath, and spin while the solution 5. TsooluptrieopnatroeagnraopupproIpV,ri(attheely0.l3a2be-lmegd/mcoLntsaoliunteiro,n)t,heanddq.s1.50tom3L0o0fmgLrowuipthV/ vehicle. Add a stir bar and mix until uniform. 6. Tsooluptrieopnatroeagnroapupprolipl,ri(attheel0y.l0a8b-emlegd/mcLonstoaliunteiro,n)t,haenddq.6s5. tmoL26of0gmrLouwpitIhV vehicle. Add a stirbar and mix until uniform. 00744 418-008:PAGE F-54 ATTACHMENT 3 Version: 418Pr-o0to0co8l2418JU.N090B8 TEST ARTICLE PREPARATION PROCEDURE Page 3013 7. SToolpurtieopnatroeagnroauppproI,pr(itahteel0y.0l2ab-emlge/dmcLonstoaluitnieorn,),thaedndq5.50. mtoLo20f0gmroLupwiitlh vehicle. Add a stir bar and mix until uniform. Witten by: `b < Approved by: Date: /3-FUes8 Clarification: 2" No ___Yes (See attached clarification form.) InitiaiDate = ) 200 99 00745 418-008:PAGE F-55 "2 PRIMED]ICA AT STnTuemGeA ne - Whew PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS. `SPONSOR'S STUDY NUMBER: 6295.9 Amendment 5-July 17, 1998 1. ConcentrationAnalyses (page 5of the protocol): `Samples (5 mL each) from each concentration will be taken during the first and last week of the F1 dosage administration to verify concentrations of the prepared test article formulations. Reason for Change: This change was made because dosage administration was extended to include the F1 generation male and female rats. 2. Sex (page 6ofthe protocol): The F1 generation male and female pups will be given the test article or vehicle. Rea forsCho angn e: These changes were made at the requestofthe Sponsor in orderto provide information about the effectsof the test article on the secondgeneration. 3. Method and Frequency (page 10ofthe protocol): The F1 generation male and female pups will be administered the test article orally (gavage) on day 1 postweaning F2 generation pups will not be directly through the day before sacrifice. given the test article, but may be possibly exposed to the test article during maternal gestation (in utero exposure) or via `maternal milk during the lactation period. : 000746 418-008:PAGE F-56 ProtAomcoeln4d1m8.e0n0t58 Page2 `Refaor sChaongne: o`fTheexspeocshuarengteosFwoegreenemraadtieonat the requestofthe Sponsor and follow the method 4. DoCsaogenLevcele s, ntratai ndVooln umess (page 11 ofthe protocol): [oe]DCEoRsEs|| comtSeTE n I parser tm | commen | narha| answer | mon C|oe |To [om |[5][64010-a5o0r 0ve8n|| Reason for Change: "tThheisFc1hgaenngeerawtaiosnmmaadlee because dosage and female rats. administration was extended to include 5. Tests Analyses and Measurements - F1 Generation (page 16of the protocol): ClO inib cal serav nd/a orGt enei ralAo ppean rans ce: Preweaning Period. Once daily. Dosage Period: Tawnidcoendcaeya.ppPrroixoirmtaotedloysoangee ahdomuirnipsotsrtadtoisoange. Maternal Behavior: `Doabysser1v,ed4,ab7n,or1m4alanbdeh2a1vipoorstwiplalrbteumr.ecAonrdyed daily ' 00747 418-008:PAGE F-57 `WBodey igh-tMalse: Preweaning Period: Dosage Period: Sacrifice: ProAtomceonld4m1e8.n0t058 Page 3 Days 1 (birth), 4, 7, 14 and 21 postpartum. Weekly. Terminal weight. Preweaning Period: Dosage Period: Sacifice: Days 1 (birth), 4, 7, 14 and 21 postpartum. WegeesktaltyitonoacnodhaobnitDataiyosn.1,D4a,il7yadnurdin1g4 presumed postpartum (rats assigned to natural delivery). Terminal weight. ReaforsCho angne: iTnhcelsuedecthhaenFg1esgewneerreatmiaondemableecaaunsdefedmoaslaegeratasd.ministration was extended to 6. EpAr/otFoc2oGle).nerationPupsNotSelectedforContinuedObservation (page 22 of the cOunlldedaypu4ppsosftrpoamrGturmo,uptshe|,sItl oamnadcVh c(oorntheingthses(tmidlokscaugred)gwrilolupbeavcaoilllaebcltee)d. from itSnhateomsppelotelhysrpewreiollpdyoblseeancgeoeltlguerbcoetuespdsa.fnrdoImfnrdaolilzveipdnuupaaltsp-f2ur0pomCs.famoApfulttreoesrfcwiiovlemlpoblfeettchieoomnlobarifgnesesadtmlbpiylteelristtiern 3coMlleEcntviionr.onsmaemnptlaelsTweiclhlnboelsohgiypapendd (frozen Safety on dry ice) to Services, 935 Kris J. Bush Hansen, Avene, Ph.D. at rBeuciidpiienngt2a-n3dE-t0h9e,,StStu.dyPaMuol,niMtionrnweilslobtea n5o5t1if3i3e-d3i3n3a1dvfoarnacnealoyfsissa.mpBloethshtihpement. 000748 418-008:PAGE F-58 Pro"toAcmoeln4d1m8e.n0t08 Page Reafos rCho angne: tCeosltleacrttiicolne oifs mrielakchsianmgpltheespwueprse vrieaqluaecstatteidon.by the Sponsotro determineif the ~rGBeorfee cE. kDearlovelAPh.fDe. DABA_T17-DB 3a0-t9e RayminI d G. York\@h., a DABT Date Associate Directoorf Research Associate Director of Research Study Director Cams Cte fo rminive Barbara J. Patterson B.A. Date Chairperson, Institutional Animal Care and Use Committee en TCar 200114 Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 00749 r ~ PRIMEDICA --_--_--mmmmm . 418-008:PAGE F-59 Argu0s5ReSsheeacrhHcyohrDLsrahibavocers.a.tBoPruAieis1i.9n04g44 TelTeeplheofnaex:: ((221153)) 444433--8857817C PROTOCOL 418-008 COMBINED ORAL PERINATALPOSTNATAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 6 - 13 August 1998 1. Necropsy (page 20ofthe protocol) On postpartum day 21, litters will be sacrificed. the 1.6 mg/kg/day dosage group (Group IV) dams and ReaforsChoangne: IGarcotautpioInV(wmoarstatleitrymiannadterdeadtucweedanbiondgy bweeciaghutsse aonfdthdeelsaeyveedredepvueplotpomxiecnitt)y.during 2. Male and Female of the protocol): Rats Assigned to Pharmacokinetic Sample Collection (page 14 OrenmapionsitnpgargtruomupDsawyil2l1b,etehxecliisveedr,sopfotohleedpuppers lfitrtoerm, ffirvoezelinttaernsdinreetaacihneodfatthe-70C until shipment to the Sponsor. Reason for Change: This change was made at the requestofthe Sponsor for possible analysis. 3. Schedul - Femal Natural Delivery NoSurvivingPups (page 15 of the protocol): Ovaries will be retained in neutral buffered 10% formalin. andDamswith 000750 418-008:PAGE F-60 AmendPmaegnet2 ReaforsChoangne: `This change was matodclareify the protoc Lis. eordh E. Dearlove, Ph.D., DABT Date Associate Diroe; fcRetseoarrch lads Jo Bana terson, BJA Date Chair ,Institutional Animal Care and Use Committee foe Prvg98 nd G. York, #h.D./DABT Date AStsudsyociaDitreecDtiorrector of Redearch Tens 27dey 9 SMatruvdiynMTo.niCtaosre, D.V.M,, Ph.D. Date 000751 o PRIMED]ICA _-- 418-008:PAGE F-61 Argu7sHSRehseearychDLeabvoerstBurBiuildcs, Inc TelTeopheotnae:cGG1199) 444334-8751857 PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS `SPONSOR'S STUDY NUMBER: 6295.9 --rr-- r -- Amendmen-- t 7 - 24 Nov-- ember 1998~ eer - 1 NaturalDelivery (page 13ofthe protocol): The Length of Parturition (timeof deliveryof last pup minus the time ofdelivery ofthe first pup divided by N-1 pups in each litter) will not be calculated. Reafosr Choangne: A litter watch was not required by the Sponsor. A Zan 71) Hr [Gorge . Deariove, Ph.D., DABT Date Associate Director of Research Ray G. York, Pi Associate Director of Study Director ABT arch Date Decline Pir Dena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and Use Committee Pre Tl 30m 7e Marvin T. Case, D.V.M., Ph.D. Study Monitor Date Co752 SPRIMEDICA -_ 418-008:PAGE F-62 PRobTb aghuyeoe rEeRBAi ihlEads eee PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS INRATS `SPONSOR'S STUDY NUMBER: 6295.9 - `Amendment 8 - 18 May 1999 1 Concentration Analyses (page 5of the protocol): The concentration samples were sent to the Sponsor. Sample analyses will be conducted at the discretion of the Sponsor and no report will be sent to the Testing Facility. Reason for Change: 2 This change was made at the request of the Sponsor to clarify the protocol. Finueslepaontdorcan: s Asigned to Pha ic Sample Colson (age 14 The liver and serum samples were sent to the `Sponsor. Samples will be analyzed at the discretionofthe Sponsor. Rea forsCho angne: This change was made at the request of the Sponsor to clarify the protocol. 3. EA/F2GenerationPupsNotSelectedfor ContinuedObservation (page 22 and `Amendment 5 of the protocol): STthoemsatcohmacocnhtceonnttsewnitllsobfethaneasleyzpeudpsatwtehreedissecnrtettiootnohfe `tShpeonSspoornfsoorr.analysis. 000753 418-008PAGE F-63 Reason for Change: ProtAomceolnd41m8e.n0t0s8 Page2 This change was made at the request of the Sponsor to clarify the protocol. . hi org E_Dearlove, Ph.0., DABT Date Associate Director of Research = Ja ("ope 1 \ emmns EMI: 29 Rayinond G. York, #0), DABT Gn ASstsuodcyiDaitreecDtiorrector of Research q rise Alone srmnyss Itunes Toe lrg 70 ChfairrapCe.rsLoenb,o,InVst.iMt.utDi.onal Animal CaDraete SMtaundiynMTo.niCtaosre, DVM, PhD. Date and Use Committee 0075% APPENDIX G DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 000755 418-008:PAGE G-1 DEOVPIEARTAITOINNSGFPRROOMCTEHDEURPERSOTOOFCTOHLEATNEDSTTIHNEGSFTAACINLDIATRYD 1. On 8 August 1998, the 5 mL concentration sample was not taken during the first week of dosage administration for the F1generation rats. This deviation did not adversely affect the outcome or ianctceurrparteetlaytieovnaolfutahtee tshtiusdpyabreamceatuesre. sufficient data were collected to 2. On3to8 June 1998 [study days (DSS) 9 to 14], 10to 15 June 1998 (FDoSgsen1e6rattoi2o1n),ma1l7eJaunnde Volumes based on body fw1ee9im9ga8hlte(sDrSartes2c3ow)redarenedddoo1ns8eDJdSunw1iet(h1296t9hMe8ad(yoDsS1a92g948e)),, all wraetrheeratdhmainniosntewreeedkalpyprbooxdiymawteeiglhyt8s.9%Astoa9r0e%sulotfotfhtehtisaregrerotre,d mdaolseagraetss and female rats were administered approximately 95% to 99% of the targeted dosages. This deviation did not adversely affect the outcome or interpretation of the study because the precohabitation dosage wpeerrieodadwmaisniesxtteerenddetdhefocrorarnecatdddiotsioangaelstwboefwoereekcsohatboietantsiuorne. that all rats 3. On 21 August 1898 (postweaning day 1), F1 generation male rats w1e3i1g5h8eadnndor13d1os5e9di.n tThhee0d.4osmagg/ekgv/odlauymdeofsoargtehegrreosutpowfetrhee wneeietkhewras based on the postweaning day 2 body weight. This deviation did not adversely affect the outcome or interpretation of the study because a sufficient number of postweaning day 1 body weights were collected to edvvaoaylluu2maeptsoesbtthawisseeapdnaironangmetwtheeeirgd,hataynw1deprtoehseptdrwoeesasanugimneegdvwoteloiugbhmete,svewreyrseimbialasredto odnosthaege All deviations age documented in the raw data. Gin C4) zu 99 ARsasyoicioantde GDi.reYctoolro(kf onsBe.seDarAcBhT Date and Study Director 000756 APPENDIX H TEMPERATURE AND RELATIVE HUMIDITY REPORTS 000757 418-008:PAGE H-1 ARGUS Temperature and Relative Humidity Report Location: Room 17 Protocol Number: 418-008 Range of Dates: 12-May-1998 12:00 to 12-Jun-1998 09:46 STpaercgieetsR:aRnagle: TTToootttaaalll NNNuuummmbbbeeerrr ooofff HDDaoatuyarss:P:oints: T6e4mpFetroa1t9urFe | Rela3t0i%vetHou7m0i%dity 7432125 1734522 Mean (& SD): MeMdaixainmu:m: Minimum: `Number of Points in Range (1%): Number of Points High (%): Number of Points Low (%): 68 (etn) | 462 (39) 679s7 a61s2 664 373 nz 0g | 2 (000 0 (0.0) 0 (0.0) (0.0) 0 (0.0) Report Generated: 04-Sep-1998 at 13:18 COMMENTS: REVIEWED BY: \ 44.Le) DATE: 9/4/57 `Cumulative by Location (v04.01.97) 000758 ARGUS 418-008:PAGE H-2 Temperature and Relative HumidityReport Location: Room 03 Protocol Number: 418-008 Range of Dates: 12-Jun-1998 09:46 to 21-Sep-1998 13:40 pectic Target Range: TTToototaal NNNuoemmmoboopfffDbehaeaeoyurrr:sF:oie: Tomparaturs 64F to 79F 2po1702 | Relative Humidity 30% to 70% wp2ians M= eaMexnainme(usS:D): Numberofpoimsinfange 6 | ror Number ofpons Points HhHighG4(%): ReportGenerated: 31Mart368 1002 comments: a57695.30 (20) 205 wom| Oe 0 (0.0) 2a7621336 (24.8) 2 woz Y19 8a0.8) _ REVEWEDBY: cle AT pare _b-2c an CTR Ron Ty 00759 ARGUS 418-008:PAGE H-3 TemperatureLoacnadtiRoenl:atRioveomHu0m5idity Report Protocol Number: 418-008 Range of Dates: 27-Jul-1998 12:50 to 28-Dec-1998 08:29 STpaercgieetsR:aRnagte: TToottaall NNuummbbeerr ooff DHoauyrss:: Total Number of Data Points: TSempFeratTure | Rela3t0i%v1e0Hu7m0i%dity 39115526 369115.28 Ed 3677 Mean(50): MMeadxiiamnu:m: Minimum: NNuummbbeerr ooff PPooiinnttss HinigRhan(4g)e: (%): Number of Paints Low (4): 603 ey | 3 en 86277 7S831 643 u3 ,61 @0e4s) || 6C70 w03n o ) | o o Report Generated: 31-Mar-1999 at 09:22 COMMENTS: r ---- --mn REVIEWEDBY: __ = oo Yot=" DATE: 3-3-1 Cumulative by Location (v04.01.97) 00760 418-008:PAGE H-4 ARGUS Temperature and Relative Humidity Report Location: Room 35-37 Protocol Number: 418-008 Range of Dates: 21-Sep-1998 13:40 to 28-Dec-1998 08:29 rTaartgietosRaRngte: Tremaperraeturre || Rotaate Hmumidity TTTootoaal NNNuummubbeemrrooobfffoeDeuakyrsrs:P:oints: PrT99e iSn9o9is Man 450): WWeNadistianmnnu:m:: NNumebemrobfPPeooiinnrttss HinigRhanGgre (x): Number of Points Low (%): wr o79a0 | 238 o on| sms wo H8ae7s (G10o0|0) | mB 2 (es (0.0) 4 2) Report Generated: 440-1099 80952 comments: _ revieweo av: GCafet (nAngon A b ov pare: 33f1o Cumulative by Location (v04.01.97) 000761 418-008:PAGE H-5 ARGUS Temperature Deviations Report Location: Room 03 Protocol Number: 418-008 Range of Dates: 12-Jun-1998 09:46 to 21-Sep-1998 13:40 Temperature Target Range: Species: Rat B4F 0 79F 7JuDnat1e998 T0i3m00e Te6m3p9.1 11773unn11999988 00450000 663352L0 71J7uunn1999988 00670000 6622791 2u80nmi19o98 00780000 6e3397LL 006811999988 2212:0000 663352L0 000860-JJu1ukl9-1199999888 203O0:T000000 6662228850L0 o0Jouuiuiigseess 00230000 66223200 006UJuukk11999988 00450000 662210L0 00uk1998 06:00 620L OsuDla-t9e98 T07i:m0e0 Te6m2p4.1 H=Valus out of raTngeemp-.H=igThe_mpeLra=tVuarleu*eFout of range - Low. Report Generated: 31-Mar-1999 at 08:09 +These deviationsdi not adversely afect the autcame o interpretationof the study. The following deviation(s) impacted on the outcomeofthe study as described: Study re ` ; F-- Date: _3/-Mip 99 000762 DeviationsbyLocation (v04.01.97) 418-008:PAGE H-6 ARGUS Relative Humidity Deviations Report Location: Room 03 Protocol Number: 418-008 Range of Dates: 12-Jun-1998 09:46 to 21-Sep-1998 13:40 SHpuemciideist:y RTaatrget Range: 30% to 70% 26vDuan-t1e998 T1i1m00e R0H7.H 2085Juu-n1i9o9s88 10820000 T0110HH 003B-uuFk11999988 11120000 7T0026HH 2241JJuukF119998%88 11200000 7T002HH 1166--AAuugg--11999988 1187:0000 2T010HH 2148--AAuugg--11998988 0115:0000 0T023HH 225AAuuggi1o0s8e 11750000 TT0283HH 3310-AAWuGg-11999988 11310000 TT1O2SHH 1165--SSeepp--11999988 2114:0000 770221HH Date Time RH. H= Value out ofRrHan.ge= -ReHliagthive HuLm=idViatlyue(%o)ut of range - Low Report Generated: 31-Mar-1999 at 09:15 Tress deviationsdo sryatttoctcoms opr fh sa. The following deviation(s) impacted on the outcome ofthe study as described: ef : z Hi Date: _3/-mte. 99 Deviations by Location (v04.01.97) 000763 418-008:PAGE H-7 ARGUS Temperature Deviations Report Location: Room 05 Protocol Number: 418-008 Range of Dates: 27-Jul-1998 12:50 to 28-Dec-1998 08:29 Temperature Target Range: Species: Rat 19-SDeapt-e1998 T1i60m0e 1199--SSeepp--11999988 11780000 1190-.SSeepp-11999988 21090000 1199SSeepp11990988 22120000 2190-SSeepp1-1998988 2003:0000 222000SSSeeeppp111099899888 000123000000 20-Sep-1998 04:00 222000-5SSeeeppp-111999899888 000567:000000 Te8m0pH. BB2L4THH 8B2247HH BB1291HH B18IHBH 8BL1S3HH B12H 81.1 H 8L1B1.101HHH 64F to 79F Date Time Temp. H=Value out of raTnegmep-.H=igThem_peLra=tVuarleu*eFout of range - Low Report Generated: 31-Mar-1999 at 09:36 Aandi tvsaf hts armartin of i. The following deviation(s) impacted on the outcome of the studyasdescribed: `Study iZ- L 1 -- Date: J/-4ifp 59 000764 Deviations by Location (v04.01.97) 418-008:PAGE H-8 ARGUS Relative HLoucmaitdiiotny:DReovioamti0o5ns Report Protocol Number: 418-008 Rangeof Dates: 27-Jul-1998 12:50 to 28-Dec-1998 08:29 HSpuemciideist:yRTaatrget Range: 30% to 70% 20SDeapt1eos8 T1im0e RTH0.TH 2260SSeppiiooss8s 1198000 TT001IHH 3000S0e-p1i9o9s88 01270000 TTO0S1HH 11440Occk1io99988 01710000 TTOOAI O037.NDoevc1199%988 01860000 7T1033HH Dats Time RH. . | | 1 eeere-------------------- H=VoutaofrRlaHn.gue=-RHeeliagthive HLum=iVdailtuye(o%u)tofrange -Low ReportGenerated: 31-Mar-1989at09:45 . These devdidnaotadtveriselyoaffnoc stheoutcomeorinteropftrheesttation1 Thefollowingdevition(s)impactedontheoutcomeofth studyes describe: StudyDirector: " He Date: 3rsrpsie 29 | DeviationsbyLocation(v04.01.97) 00765 418-008:PAGE H-9 ARGUS A, Relative Humidity Deviations Report Location: Room 35-37 Protocol Number: 418-008 r Range of Dates: 21-Sep-199e 8 13:40 to 28-Dece-e1998 08:2c9 ee Humidity Target Range: Species: Rat 30% to 70% 235Deapt1e908 T1i00m0e R2H8.3L 2233SSeeppi1soees8 11210000 22783200 23Sepises 1300 z36L Date Time RH. H =Value outof range - High L = Value out of range - Low RH.= Relative Humidity (%) Report Generated: 31-Mar-1999 at 10:12 These deviations did not adversely affect the outcome or interpretation of the study. "The following deviation(s) impacted on the outcomeofthe stasudescdribyed: StudyDirector: i : 1% = Date: _g/-ua 5) Deviations by Location (v04.01.97) 000766 APPENDIX | STATEMENT OF THE STUDY DIRECTOR 000767 418-008:PAGE 1-1 peseancs Argus Research Laboratories, Inc. _--_-- Tehw onioe ns ereme HorTs2e h1a9m,uPae ean7nisoylFs .va2n18i)a44s 1358054647 PROTOCOL 418-008: CDOEMVBEILNOEPDMEONRTAALL(AGNAVDAPGEE)RIFNEARTTIALLIPTYO,STNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 STAOT FTHE E SM TUDE YDIRN ECTT OR "This final report accurately reflects the raw data obtained during the performance of the study. No deviations from the U.S. Food andDrug Administration (FDA) Good Laboratory Practice Regulations; Final Rule?, the JStaapnadnaersdefMoirnSiastfreytyofStHuedailetsh oanndDrWueglsfaraend(MtHheW)EuGroopoedaLnabEocroantoomriycPrCaocmtimcuenity E(cEoECn)omCiocunCcoimlmduenciistiyoonfonan28OEJuClyD 1d9e8c9isoinont/hreeaccocmempetnadantcieobnyotnhecoEmuprloipaenacne with principlesofgood laboratory practice* occurred that affected the quality or integrity of the study. 7 atin Z] Ze 103647 ARsasyowciioantde GD.irYeocrtko,r ofh.RDe.s,eaDrAcBhT Date and Study Director | Argus Research Laboratories, Inc. a. U.S. Food and Drug Administration. Good Laboratory Practice b. JReagpualnaetsioensM;inFiisntarly RofulHee.al2t1h CanFdRWPealrftar58e. (1987). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. c. European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratorypractice. Official Journal of the European Communities: Legislation. 32(No. L 315; 28 October): 1-17. 000768 APPENDIX J QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT 00769 r7r;, DICA _-- 418-008:PAGE J-1 Argu90s5RSehseeearhHcyohDrLrsaihbvaoerm,a,tBoPruAiles1a,9g0In3cA.4 TelTeeplheofnaex:: ((221155))444433--88578170 QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT Study Director: Raymond G. York, Ph.D., DABT Executive Director of Research: Mildred S. Christian, Ph.D., Fellow, ATS Protocol 418-008; CPeormibnaitnaeld/PoOsrtanlat(aGlavRaegper)odFuecrttiiliotny,ToDxeivceiltoypSmteundtyalofaPnFdOS in Rats Sponsor's Study Number: 6295.9 and The draft protocol Drug Administration (foFrDtAh)isGsotouddyLwaabosraatuodriytePdrafocrtiacdehReergeunlcaetitoonsU,.SJ.aFpoanoedse SMainfiesttyryStoufdHieeasltohn aDnrudgWse,lafnardeE(uMrHoWp)ea;nGEocoodnoLambiocraCtoomrmyuPnriatctyic(e19S8t9a)ncdoaurndciflor Cdeocmimsuionnitoyn o2f8aJnulOyE1C98D9doencitshieoanc/creepctoammnecnedbaytitohne oEnurcoopmepalniaEnccoenwoimtihcprinciples of good laboratory practice on 13 MAY 98. and rawCridtaitcaalwpehraeseausdoifttehdissisxttuidmyeswe(rseeeintasbpleecste1da2n4dt2imfeosr;dsattuedsyainndformation phases/data) auditedTfhoer adrcacfutrfaicnyal, rfeoproardthaenrdentchee rtoawprdoattoacoflorrtehqiusirsetmuednytwse,raendcofmorpaardehderaenndce tRoegUu.lSa.tiFoonosd, JaanpdaDnreusgeAMdimniinsitsrtyroaftiHoenal(tFhDaA)ndGWoeoldfaLraebo(rMaHtWor)y; PGroacotdicLeaboratory CProamcmtuicneitStya(n1d9a8r9d)fcooruSnacfieltdyecSitsuidoinesonon28DrJuuglsy,1a9n8d9 EounrtohpeeaanccEecpotnaoncmeicby the cEoumrpolpieaannceEcwiotnhompriicncCioplmemsuonfigtoyodoflaanboOraEtCorDy pdreaccitsiicoenb/ertewceoemnme0n5daDtiEoCn 9o8n and 29 APR 7 JUN 99, 9 99, and JUN 99, faonrdrefvoirsfiionnaslirzeatqiuoensotned1b0yJtUhNe Sponsor 88. 18 MAY 99, 00770 418-008:PAGE J-2 AdminisTthriastisotnud(yFDwAa)sGcooondduLcatbeodraatcocroyrdPirnagcttioceU.RSe.guFloaotdionasn,d Drug Japanese Ministry SoftuHdeiaelsthonanDdruWgesl,faarned (EMuHrWo)p;eaGnoEocdoLnaobmoircatCoormymPurnaicttiyce(1S9t8a9n)dacrodunfcoirl Sdaefceitsyion oOnEC28DJduelyci1s9i8o9niornectohmemaecncdeapttiaonncoenbcyotmhpeliEaunrcoepewiatnhEpcrionncoipmliecs Cofomgmooudnityof an laboratory practice. Farry)Lrigllvak.seer dlpatsosL.fot Lou 93 QNuaanlcityyJA/sGsnugrlainecweskMianager Dat QHueaaltihteyrAsL.suRrabauntcieinSou,pMer.vSisor Date and Principal Auditor 000771 TABLE 1 CRITICAL PHASES INSPECTED 418-008:PAGE J-3 `est Article Administration - Gavage Dates of inspection: 28 MAY 98, 02 JUN 98, 03 SEP 98 Dates results reported to the Study Director and Management * 15 JUN 98, 15 JUN 98, 18 SEP 98 EstrousCycleEvaluation Date of inspection: 11 JUN 98 Date results reported to the Study Director and Management: 11 JUN 98 Cohabitation Dates of inspection: 08 JUL 98, 04 NOV 98 Dates results reported to the Study Director and Management: 14 JUL 98, 12 NOV 98 `Scheduled Sacrifice - Day 10 Date of inspection: 17 JUL 98 Date results reported to the Study Director and Management: 03 AUG 98 Test Article Preparation Dates of inspection: 29 JUL 98, 16 SEP 98 Dates results reported to the Study Director and Management: 14 AUG 98, 24 SEP 98 Natural Delivery Dates of inspection: 31 JUL 98, 25 NOV 98 Dates results reported to the Study Director and Management: Blood aS: 02 DEC 98 000772 418-008:PAGE J4 Dates of inspection: 31 JUL 98, 18 AUG 98 Dates results reported to the Study Director and Management: 14 AUG 98, 21 AUG 98 Physical and Reflex Development - Surface Righting, Pinna Unfolding, Eve Opening, Auditory Startle, Air Righting, Pupil Constriction. Sexual Maturation Dates of inspection: 31 JUL 98, 13 AUG 98, 18 AUG 98, 28 AUG 98, 11SEP 98 Dates results reported to the Study Director and Management: 14 AUG 98, 03 SEP 98, 08 SEP 98, 11 SEP 98, 12 SEP 98 Male Necropsy Date of inspection: 31 JUL 98 Date results reported to the Study Director and Management: 14 AUG 98 Necropsy - Dam and Litter Sacrifice Dates of inspection: 18 AUG 98, 15 DEC 98 Dates results reported to the Study Director and Management: 21 AUG 98, 18 DEC 98 Day 21 Weaning Date of inspection: 18 AUG 98 Date results reported to the Study Director and Management: 08 SEP 98 Behavioral Testing - Passive Avoidance, Watermaze, Dates of inspection: 25 AUG 98, 08 OCT 98 Dates results reported to the Study Director and Management: 12 SEP 98, 12 0CT 98 000773 418-008:PAGE J-5 TABLE 2 RAW DATA AUDIT(S) The following study information and raw data were audited on 29 SEP 98, 01 OCT 98, 04 OCT 98 TO 05 OCT 98: PPrroottooccooll. amendments. ELirsrtorofcpoedressonannedlcaonddescofmorpuctlienricaolpesriagthoorbcsoedrevsa.tions. AInn-ifmfealtrraencseaicptt,iornanrdecoomridz.ation, physical examination and acclimation. Feed consumption. Necropsy. Organ weights. Tissue packing lists. DEedviitartieoqnuse.sts Data review page/pages. Blood collection data and packing lists. Key for testing facility computer backup record abbreviations. The results of this audit were reported to the Study Director and Management on 06 OCT 98. 00774 418-008:PAGE J-6 on "The following study 06 OCT 98, 12 OCT information and 98, 02 NOV 98 TrOaw0d6atNaOwVer9e8:audited Animal receipt, randomization, physical examination and acclimation. In-lfe transaction record. Feed consumption. Estrus cycle evaluation. Cohabitation. Caesarean-sectioning. Maternal gross observations. Natural delivery observations. PLiuttperboobdsyerwveaitgihotnss.and status. Table of random units. Necropsy. Tissue packing lists. General comments. Reflex and physical development. Study maintenance records. Temperature and relative humidity reports. Feed, water and bedding analyses. Edit requests. Data review page. Blood collection data and packing lists. Pup stomach contents. on The results 08 NOV 98 of this audit were reported to the Study Director and Management Coo07T75 418-008:PAGE J-7 The following study information and raw data were audited on 08 NOV 98 to 09 NOV 98: Vehicle receipt, preparation and use. Test article receipt, preparation and use. Test article packing lists. The results of this audit were reported to the Study Director and Management on 12 NOV 88. The following study information and raw data were audited on 26 JAN 99 to 28 JAN 99: In-lfe transaction record. Feed consumption. Passive avoidance. Watermaze. Genealogy chart. Necropsy. Organ weights. Tissue packing lists. Sexual Maturation. Edit request. : The results of this audit were reported to the Study Director and Management on 28 JAN 98. 000776 418-008:PAGE J-8 on 2T8hJeAfNol9lo9witnog0s1tuFdEyBin9f9o:rmation and raw data were audited IFne-elfde ctornanssuamcpttiioonn.record. Cohabitation. Natural delivery observations. Litter observations. Pup body weights and status. Passive avoidance. Watermaze. Table of random units. Necropsy. TGiesnseurealpaccokmimnegnltisst.s. Sexual maturation. Study maintenance records. Temperature and relative humidity reports. . FEdeietdr,ewqauetsetrsa.nd bedding analyses. Dosage volumes. Data review pages. on 0T1hFeErBes9ul9t.softhis audit were reported to the Study Director and Management The following study information and raw data were audited on 25 JAN 98: VTeehsitcalretircelceeirpetc,eipptr,epparreaptairoantiaonnd aunsde. use. Test article packing lists. on 2T8hJeArNes9u9l.ts of this audit were reported to the Study Director and Management ere