Document 5bRBOE4Zb3b2b9aQL0GxaDgbz

FINAL REPORT ^ R X L6-055*J RECEIVED QPPT CBiC 2000FEB 22 AHIh3L PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 FINAL REPORT DATE: 10 JUNE 1999 000502 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 II. DESCRIPTION OF TEST PROCEDURES A. Conduct of Study A.1. Sponsor A.2. Testing Facility A.3. Study Number A.4. Sponsor's Study Number A.5. Purpose of the Study A.6. Study Design A.7. Regulatory Compliance A.8. Ownership of the Study A.9. Study Monitor i 000503 PAGE 1-1 1-1 I-5 1-10 11-1 11-1 11-1 11-1 11-1 11-1 11-1 11-1 II-2 II-2 II-2 SUBJECT A.10. Alternate Study Monitor A.11. Study Director A.12. Technical Performance A. 13. Report Preparation A.14. Report Review A. 15. Date Protocol Signed A. 16. Dates of Technical Performance A. 17. Records Maintained B. Test Article Information B.1. Description B.2. Lot Number B.3. Date Received and Storage Conditions B.4. Special Handling Instructions B.5. Analysis of Activity C. Vehicle Information C.1. Description C.2. Lot Numbers C.3. Dates Received and Storage Conditions C.4. Special Handling Instructions C.5. Analysis of Purity D. Test Article Preparation D.1. Sample Information PAGE II-2 II-2 II-2 II-2 II-3 II-3 II-3 II-4 lt-4 II-4 II-4 II-5 II-5 II-5 II-5 II-5 II-5 II-5 II-6 II-6 II-6 II-6 000504 SUBJECT D.2. Analytical Results E. Test System E.1. Species E.2. Strain E.3. Supplier (Source) E.4. 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 F.6. Feed F.7. Feed Analysis F.8. Water F.9. Water Analysis F.10. Bedding F.11. Bedding Analysis hi PAGE II-7 II-7 II-7 II-7 II-7 II-7 II-7 II-7 II-8 II-8 II-9 II-9 II-9 II-9 11-10 11-10 11-10 11-10 11-10 11-10 11-11 11-11 000505 SUBJECT G. Methods G. 1. Dosage Administration G.2. Assigned Rat Numbers G.3. Rationale for Dosage Selection G.4. Route of Administration PAGE 11-11 11-11 11-12 11-12 11-12 G.5. Rationale for Route of Administration G.6. Frequency of Administration 11-12 11-12 G.7. Length of Study G.8. Method of Study Performance 11-13 11-13 G.9. Gross Necropsy G.10. Statistical Analyses III. RESULTS - Fo GENERATION MALE RATS A. Clinical Observations 11-18 11-21 UI-1 111-1 B. Body Weights and Body Weight Changes C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values 111-1 111-1 D. Mating and Fertility E. Necropsy Observations III-2 III-2 F. Terminal Body Weights, Organ Weightsand Ratios (%) of Organ Weight to Terminal Body Weight IV. RESULTS - Fo GENERATION FEMALERATS III-3 IV-1 A. Clinical Observations IV-1 A.1. Mortality IV-1 IV 000506 SUBJECT A.2. Clinical Observations B. Body Weights and Body Weight Changes B.1. Precohabitation B.2. Gestation B.3. Lactation C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values C.1. Precohabitation C.2. Gestation C.3. 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 1.1. Surface Righting 1.2. Pinna Unfolding 1.3. Eye Opening 1.4. Acoustic Startle 1.5. Air Righting 1.6. Pupil Constriction PAGE IV-1 IV-1 IV-1 IV-2 IV-2 IV-3 IV-3 IV-3 IV-3 IV-4 IV-4 IV-4 IV-4 IV-5 IV-6 IV-6 IV-7 IV-7 IV-7 IV-7 IV-8 v 000507 SUBJECT PAGE V. RESULTS - F1 GENERATION MALE AND FEMALE RATS V-1 A. F1 Generation Male Rats V-1 A.1. Mortality and Clinical Observations V-1 A.2. Body Weights and Body Weight Changes V-2 A.3. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values V-2 A.4. Sexual Maturation V-2 A.5. Passive Avoidance Performance V-3 A.6. Watermaze Performance V-3 A.7. Mating and Fertility V-3 A.8. Necropsy Observations V-3 A. 9. Terminal Body Weights and Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight V-4 B. F1 Generation Female Rats V-4 B.1. Mortality and Clinical Observations V-4 B.2. Maternal Body Weights and Body Weight Changes V-5 B.3. Maternal Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values V-6 B.4. Sexual Maturation V-7 B.5. Passive Avoidance Performance V-7 B.6. Watermaze Performance V-7 B.7. Mating and Fertility V-7 B.8. Necropsy Observations V-8 B.9. Natural Delivery and Litter Observations wi V-8 000508 SUBJECT PAGE B.10. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations V-9 REFERENCES V-10 APPENDIX A - REPORT FIGURES Figure 1. Body Weights - Fo Generation Male Rats A-1 Figure 2. Body Weights - Fo Generation Female Rats A-2 Figure 3. Body Weights - F1 Generation Male Rats A-3 Figure 4. Body Weights - F1 Generation Female Rats A-4 APPENDIX B - REPORT TABLES - Fo GENERATION MALE RATS Table B1. Clinical Observations - Summary - Fo Generation Male Rats B-1 Table B2. Body Weights - Summary - Fo Generation Male Rats B-2 Table B3. Body Weight Changes - Summary - Fo Generation Male Rats B-3 Table B4. Absolute Feed Consumption Values (g/day) - Summary Fo Generation Male Rats B-4 Table B5 Relative Feed Consumption Values (g/kg/day) - Summary Fo Generation Male Rats B-5 Table B6. Mating and Fertility - 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 B-8 Table B9. Ratios (%) of Organ Weight to Terminal Body Weight Summary - Fo Generation Male Rats B-9 Table B10. Clinical Observations - Individual Data - Fo Generation Male Rats B-10 Table B11. Body Weights - Individual Data - Fo Generation Male Rats B-20 Vii 000509 SUBJECT PAGE Table B12. Feed Consumption Values - Individual Data Fo Generation Male Rats B-30 Table B13. Mating and Fertility - Individual Data - Fo Generation Male Rats B-40 Table B14. Necropsy Observations - Individual Data - Fo Generation Male Rats B-50 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 C-1 Table C2. Body Weights - Precohabitation - Summary - Fo Generation Female Rats C-4 Table C3. Body Weight Changes - Precohabitation - Summary Fo Generation Female Rats C-5 Table C4. Maternal Body Weights - Gestation - Summary Fo Generation Female Rats C-6 Table C5. Maternal Body Weight Changes - Gestation - Summary Fo Generation Female Rats C-8 Table C6. Maternal Body Weights - Lactation - Summary Fo Generation Female Rats C-9 Table C7. Maternal Body Weight Changes - Lactation - Summary Fo Generation Female Rats C-10 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 - Summary - Fo Generation Female Rats C-12 Table C10. Maternal Absolute Feed Consumption Values (g/day) Gestation - Summary - Fo Generation Female Rats vili 000510 C-13 SUBJECT PAGE Table C11. Maternal Relative Feed Consumption Values (g/kg/day) Gestation - Summary - Fo Generation Female Rats C-14 T a b le d 2. Maternal Absolute Feed Consumption Values (g/day) Lactation - Summary - Fo Generation Female Rats C-15 T a b le d 3. Maternal Relative Feed Consumption Values (g/kg/day) Lactation - Summary - Fo Generation Female Rats C-16 Table C14. Estrous Cycling, Mating and Fertility - Summary Fo Generation Female Rats C-17 T a b le d 5. Necropsy Observations - Summary - Fo Generation Female Rats C-19 Table C16. Caesarean-Sectioning Observations - Summary Fo Generation Female Rats C-20 Table C17. Natural Delivery Observations - Summary - Fo Generation Female Rats C-21 T a b le d 8. Litter Observations (Naturally Delivered Pups) - Summary - F1 Generation Litters C-22 Table C19. Clinical Observations from Birth to Day 21 Postpartum Summary - F1 Generation Pups C-25 Table C20. Reflex and Physical Development - Summary F1 Generation Litters C-26 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 C-40 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 000511 SUBJECT PAGE Table C26. Feed Consumption Values - Precohabitation - Individual Data - Fo Generation Rats C-75 Table C27. Maternal Feed Consumption Values - Presumed Gestation - Individual Data - Fo Generation Female Rats C-85 Table C28. Maternal Feed Consumption Values - Lactation Individual Data - Fo Generation Female Rats C-105 Table C29. Estrous Cycling and Days in Cohabitation - Individual Data - Fo Generation Female Rats C-110 Table C30. Necropsy Observations - Individual Data - Fo Generation Female Rats C-115 Table C31. Caesarean-Sectioning Observations - Individual Data Fo Generation Female Rats C-121 Table C32. Litter Observations (Caesaren-Delivered Embryos) Individual Data - F1 Generation Litters C-126 Table C33. Embryonal Vital Status - Individual Data - F1 Generation Litters C-131 Table C34. Natural Delivery, Implantation Sites, and Pup Viability and Sex - Individual Data - Fo Generation Female Rats/ F1 Generation Litters C-136 Table C35. Pup Body Weight Litter Averages from Birth to Day 21 Postpartum - Individual Data - F1 Generation Litters C-141 Table C36. Pup Body Weights from Birth to Day 21 Postpartum Individual Data - F1 Generation Pups C-146 Table C37. Pup Vital Status and Sex from Birth to Day 21 Postpartum - Individual Data - F1 Generation Pups C-176 Table C38. Clinical Observations from Birth to Day 21 Postpartum Individual Data - F1 Generation Pups C-181 Table C39. Surface Righting - Individual Data - F1 Generation Litters C-182 Table C40. Pinna Folding - Individual Data - F1 Generation Litters C-193 x 000512 SUBJECT PAGE Table C41. Eye Opening - Individual Data - F1 Generation Litters C-203 Table C42. Acoustic Startle - Individual Data - F1 Generation Litters C-213 Table C43. Air Righting - Individual Data - F1 Generation Litters C-223 Table C44. Pupil Constriction - Individual Data - F1 Generation Litters C-233 Table C45. Necropsy Observations - Individual Data - F1 Generation Pups C-243 APPENDIX D - REPORT TABLES - F1 GENERATION MALE RATS Table D1. Clinical Observations - Summary - F1 Generation Male Rats D-1 Table D2. Body Weights - Summary - F1 Generation Male Rats D-3 Table D3. Body Weight Changes - Summary - F1 Generation Male Rats D-4 Table D4. Absolute Feed Consumption Values (g/day) - Summary F1 Generation Male Rats D-5 Table D5. Relative Feed Consumption Values (g/kg/day) - Summary F1 Generation Male Rats D-6 Table D6. Sexual Maturation - Summary - F1 Generation Male Rats D-7 Table D7. Passive Avoidance Performance - Summary F1 Generation Male Rats D-8 Table D8. Watermaze Performance - Summary - F1 Generation Male Rats D-9 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 000513 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 D14. Body Weights - Individual Data - F1Generation 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 D-33 Table D19. Mating and Fertility - Individual Data - F1 Generation Male Rats D-36 Table D20. Necropsy Observations - IndividualData - F1 Generation Male Rats D-39 Table D21. Terminal Body Weights andOrgan Weights and Ratios (%) of Organ Weight to Terminal Body Weight - Individual Data - F1 Generation Male Rats D-43 APPENDIX E - REPORT TABLES - F1 GENERATION FEMALE RATS Table E1. Clinical Observations - Summary - F1 Generation Female Rats E-1 Table E2. Body Weights - Precohabitation - Summary - F1 Generation Female Rats E-4 Table E3. Body Weight Changes - Precohabitation - Summary F1 Generation Female Rats E-5 Table E4. Maternal Body Weights - Gestation - Summary F1 Generation Female Rats E-6 000514 SUBJECT Table E5. Maternal Body Weight Changes - Gestation - Summary F1 Generation Female Rats PAGE E-8 Table E6. Maternal Body Weights - Lactation - Summary F1 Generation Female Rats Table E7. Maternal Body Weight Changes - Lactation - Summary F1 Generation Female Rats Table E8. Absolute Feed Consumption Values (g/day) Precohabitation - Summary - F1 Generation Female Rats Table E9. Relative Feed Consumption Values (g/kg/day) Precohabitation - Summary - F1 Generation Female Rats E-9 E-10 E-11 E-12 Table E10. Maternal Absolute Feed Consumption Values (g/day) Gestation - Summary - F1 Generation Female Rats E-13 Table E11. Maternal Relative Feed Consumption Values (g/kg/day) Gestation - Summary - F1 Generation Female Rats E-14 Table E12. Maternal Absolute Feed Consumption Values (g/day) Lactation - Summary - F1 Generation Female Rats E-15 Table E13. Maternal Relative Feed Consumption Values (g/kg/day) Lactation - F1 Generation Female Rats E-16 Table E14. Sexual Maturation - Summary - F1 Generation Female Rats E-17 Table E15. Passive Avoidance Performance - Summary F1 Generation Female Rats E-18 Table E16. Watermaze Performance - Summary - F1 Generation Female Rats E-19 Table E17. Mating and Fertility - Summary - F1 Generation Female Rats E-20 Table E18. Necropsy Observations - Summary - F1 Generation Female Rats E-21 Table E19. Natural Delivery Observations - Summary - F1 Generation Female Rats E-22 XIII 000515 SUBJECT PAGE Table E20. Litter Observations (Naturally Delivered Pups) Summary - F2 Generation Litters E-23 Table E21. Clinical Observations from Birth to Day 21 Postpartum Summary - F2 Generation Pups E-26 Table E22. Necropsy Observations - Summary - F2 Generation Pups E-27 Table E23. Clinical Observations - Individual Data - F1 Generation Female Rats E-28 Table E24. Body Weights - Precohabitation - Individual Data - F1 Generation Female Rats E-34 Table E25. Maternal Body Weights - Presumed Gestation - Individual Data - F1 Generation Female Rats E-37 Table E26. Maternal Body Weights - Lactation - Individual Data F1 Generation Female Rats E-43 Table E27. Feed Consumption Values - Precohabitation - Individual Data - F1 Generation Female Rats E-46 Table E28. Maternal Feed Consumption Values - Presumed Gestation - Individual Data - F1 Generation Female Rats E-49 Table E29. Maternal Feed Consumption Values - Lactation - Individual Data - F1 Generation Female Rats E-52 Table E30. Sexual Maturation - Individual Data - F1 Generation Female Rats E-55 Table E31. Passive Avoidance Performance - Individual Data F1 Generation Female Rats E-56 Table E32. Watermaze Performance - Individual Data - F1 Generation Female Rats E-59 Table E33. Days in Cohabitation - Individual Data - F1 Generation Female Rats E-62 Table E34. Necropsy Observations - Individual Data - F1 Generation Female Rats 000516 xiv E-63 SUBJECT PAGE Table E35. Natural Delivery, Implantation Sites, and Pup Viability and Sex - Individual Data - F1 Generation Female Rats/ F2 Generation Litters E-67 Table E36. Pup Body Weight Litter Averages from Birth to Day 21 Postpartum - Individual Data - F2 Generation Litters E-70 Table E37. Pup Body Weights from Birth to Day 21 Postpartum Individual Data - F2 Generation Pups E-73 Table E38. Pup Vital Status and Sex from Birth to Day 21 Postpartum - Individual Data - F2 Generation Pups E-91 Table E39. Clinical Observations from Birth to Day 21 Postpartum Individual Data - F2 Generation Pups E-94 Table E40. Necropsy Observations - Individual Data - F2 Generation Pups E-95 APPENDX F - PROTOCOL AND AMENDMENTS F-1 to F-63 APPENDIX G - DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY G-1 APPENDIX H - TEMPERATURE AND RELATIVE HUMIDITY REPORTS H-1 to H-9 APPENDIX I - STATEMENT OF THE STUDY DIRECTOR 1-1 APPENDIX J - QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT J-1 to J-8 000517 xv TITLE: 418-008.PAGE 1-1 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS ARGUS RESEARCH LABORATORIES, INC. PROTOCOL NUMBER: 418-008 SPONSOR'S STUDY NUMBER: 6295.9 I. SUMMARY AND CONCLUSION A. Methods3 Text Figure 1 provides a schematic of the study design. A.1. Fo Generation Rats/F1 Generation Litters: One hundred seventy-five male and female Crl:CDBR VAF/Plus (SpragueDawley) rats were assigned to five dosage groups (Groups I through V), 35 rats per sex per dosage group. The rats were administered the test article, PFOS, or the vehicle, 0.5% Tween 80, orally (via gavage). The dosages were 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 before cohabitation and continuing through DG69 (rats assigned to Caesareansectioning), DG 24 (rats assigned to natural delivery that did not deliver a litter), or DLC20 (rats that did deliver a litter). The dosage volume was 5 mL/kg. All Fo generation rats were observed for viability at least twice daily during the study and for clinical signs of effects of the test article twice daily during the dosage period. Body weights and feed consumption values for male rats were recorded weekly during the dosage period and at sacrifice. Body weights for female rats were recorded weekly to cohabitation, daily during the gestation period, on DLs 1,4, 7, 10 and 14 (rats assigned to natural delivery) and at sacrifice. Feed consumption values were recorded weekly to cohabitation, daily during the gestation period and on DLs 1, 4, 7, 10 and 14 (rats assigned to natural delivery). a. Detailed descriptions of all procedures used in the conduct of this study are provided in the appropriate sections of this report and in APPENDIX 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. 000518 418-008:PAGE I-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, II and III 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 deliver a 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 litters in Groups I, II and III, frozen and shipped to the Sponsor for analysis. 000519 418-008:PAGE I-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 I 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. A.2. F1 Generation Rats/F2 Generation Litters: Only the 0.1 and 0.4 mg/kg/day dosage groups were continued into the second generation because of the excessive toxicity seen in the 1.6 and 3.2 mg/kg/day F1 generation pups. There were 150 male and female F1 generation rats in the three dosage groups (Groups I through III), 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. 000520 418-008:PAGE I-4 000521 Protocol 418-008: Combined Oral (Gavage) Fertility, Development and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rata Acclimation I 14 days | J5F.U. Rate/Doiaga Group Arriva al Totting Facility 1 Portoti ^P*0* r Rate Cohabitation 14 days FEMALE Estroua Acclimation I Evaluation Cohabitation 14 days I 15/oroup 14 days Pharmacokinetic Sandio Collection 5/group j Fo M ai* Rata Sacrificed DG 10 C- Sectioning 10/group D o tin g Parted End for Mala R ail 15 F# Female Dosing Rats/Dosage Period Group Arriva at Begin* for Taatteg Facility Famate Rate DG O G 10 li t Point*# D ali vary (DG 21) L a ti P olitela Oativery (DO 25) PARTURITION Lactation Day 1 (D l 1) Dosing Parted Ends tor Famala R ati (OL 20) Pharmacokinstic Sample Colaction S/group I Day of Sacrifica (0 L 2 t) P I GENERATION OosingPariod Begins for F! Ganaratten Rate Cohabitation ^ Indaga 1st Positela Delivery (DG21) Last Positela Datively (OG 25) OL 1 Culling: 2S mala and 25 SEXUAL MATURATION: temala pupi/group OL 21 Vaginal Patency continuad on itudy (OL 4) 4 REFLEX AND PHYSICAL DEVELOPMENT: Surface RghUng. P tw a Unfotemg. Eye Operung. Acovetc Starfte, A r R lghtng Reltex. Pup4 Conauction (X. 3# PASSIVE PieputM Separation Matamat Milk Cokectten (varfae tiam O L t ttva vg * OL 21 > AVOIDANCE: (Groupa I, li in d V) X 24*1 days 1 mala and 1 lamate pup/tittar Abbreviations. DG = Day o f (Presumed) Gestation DL Day of Lactation Dosage Perod WATERMA2E: DL 70. 1 mate and I lmete p u p M a DLM DG 0 Earnest day of sacrifice ter Ft mate rate PARTURITION Lactation Day 1 ( X t) Last Posatela Day of Doting for Mala and Famala Ft Rats ( X 20) F2 GENERATION SACRIFICE OF F I DAMS/ F t UTTERS: X 21 T1 CcO* cB B. Results 418-008.PAGE I-5 B.1. Fo Generation Male Rats: No Fo generation male rats died during this study as a result of treatment with PFOS. All 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 fertility 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). 000522 418-008'.PAGE I-6 Absolute and relative feed consumption values were reduced in the 1.6 and 3.2 mg/kg/day dosage groups during the precohabitation period. 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 maternal feed consumption values tended to be reduced in the 0.4 mg/kg/day dosage group and were significantly reduced in the 1.6 mg/kg/day dosage group for the entire lactation period, 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. Dosages of the test article as high as 3.2 mg/kg/day did not affect estrous cycling in the 15 rats per dosage group that were evaluated. Mating and fertility parameters were unaffected by the 3.2 mg/kg/day dosage of PFOS. All necropsy observations were considered unrelated to treatment. There were no biologically important or statistically significant differences in the litter averages for corpora lutea, implantations, viable embryos or nonviable embryos at Caesarean-sectioning on DG 10. The duration of gestation was significantly reduced in the 3.2 mg/kg/day dosage group, an observation associated with preimplantation loss [the average number of implantation sites per dam was significantly reduced, resulting in a significantly reduced litter size]. Pup viability was significantly reduced in the 1.6 and 3.2 mg/kg/day dosage groups. Reflecting these effects, the viability and lactation indices were significantly reduced in the 1.6 and 3.2 mg/kg/day dosage groups, the lactation index was also significantly reduced in the 1.6 mg/kg/day dosage group, as also were the averages for surviving pups in the 1.6 mg/kg/day dosage group beginning on DL 4 postculling, and in the 3.2 mg/kg/day dosage group on DL 1. A dosage-dependent pattern of reduced pup body weight was evident in each group administered the test article. The 0.1 mg/kg/day dosage group tended to have reduced pup weights on DL 4 (pre- and postculling), compared to the control group value. The 0.4 mg/kg/day dosage group tended to have reduced pup body weights on DLs 1 through 4 (postculling), compared to the control group value. However, none of the differences in the 0.1 and 0.4 mg/kg/day dosage groups were statistically significant and may represent normal biological variations. The 1.6 mg/kg/day dosage group had significantly reduced pup body weights on all weighing days. The 3.2 mg/kg/day dosage group had a significantly reduced pup body weight on DL 1 (no pups survived to subsequent scheduled weighings). Clinical and necropsy observations associated with reduced pup viability and potential reduction in maternal care occurred in the 3.2 mg/kg/day dosage group. 000523 418-008:PAGE I-7 Adverse clinical observations include three litters with pups that were not nursing (two of 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. Reversible delays in reflex and physical development that are highly correlated with body weight occurred in the 0.4 and 1.6 mg/kg/day dosage groups. Surface 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 the control pups) and in the opinion of the attending laboratory veterinarian, the rats were not suitable for further experimentation. It had been determined that the dosage of 1.6 mg/kg/day was a level which produced compound toxicity. Continued dosing of 1.6 mg/kg/day most likely would not result in any additional information and would subject the pups to additional distress. Therefore, consultation with the Sponsor lead to the decision not to continue the 1.6 mg/kg/day dosage group F1 generation pups. Only the 0 (Vehicle), 0.1 and 0.4 mg/kg/day dosage group pups were continued to a second generation. B.3. 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. All clinical observations were considered unrelated to the test article. 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 pattern of 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, 000524 418-008:PAGE I-8 respectively. Body weight gains in the 0.1 and 0.4 mg/kg/day dosage groups for day 1 to termination were 98.5% and 96.6% of the control group value, respectively. However, none of these body weight gain differences were statistically significant from control values. Absolute feed consumption values for the 0.1 and 0.4 mg/kg/day dosage groups were significantly reduced on postweaning days 1 through 8. Relative feed consumption values tended to be reduced in the 0.1 and 0.4 mg/kg/day dosage groups during the first week of intubation but none were statistically significant. 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. There were no biologically important differences in the values for learning, short-term retention, long-term retention or response inhibition in the F1 generation male rats, as evaluated by performance in a passive avoidance or watermaze performance paradigm. Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertility parameters evaluated in the F1 generation male rats. All necropsy observations in the F1 generation male rats were considered unrelated to the test article because. No statistically significant differences occurred in the absolute or relative values for the weights of the right or left testis, seminal vesicles, right epididymis or prostate. Terminal body weights tended to be reduced in the 0.1 and 0.4 mg/kg/day dosage groups but were not statistically significant from control values. 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 tended to be reduced in the 0.4 mg/kg/day dosage group on day 1 postweaning. 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. Significant maternal body weight loss occurred on DLs 1 to 4 in the 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 consumption values during the gestation period were unaffected by dosages of the test article as high as 0.4 mg/kg/day. The absolute and relative maternal 000525 418-008:PAGE I-9 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 biologically important differences in the values for learning, short-term retention, long-term retention or response inhibition in the F1 generation female rats, as evaluated by performance in a passive avoidance or watermaze performance paradigm. Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertility parameters evaluated in the F1 generation female rats. All necropsy observations in the F1 generation female rats were considered unrelated to the test article. 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 dosage groups, respectively. All pregnant dams delivered litters. The gestation index was comparable across the three dosage groups. As occurred in the previous generation, there was a tendency for reduced pup viability in the 0.4 mg/kg/day dosage group. The number of dams with stillborn pups was increased, and the number of pup deaths after DL 2 also were increased in this dosage group. As also occurred in the previous generation, pup body weights tended to be reduced in the 0.1 mg/kg/day dosage group on DL 4 (pre-and postculling) and DL 7. By DL 14, body weights were comparable to control group values. Pup body weights in the 0.4 mg/kg/day dosage group tended to be reduced, though not significantly, on DLs 4 through 21, with significant reductions present on DLs 7 and 14. No clinical or necropsy observations in the F2 generation pups were attributable to dosages of the test article as high as 0.4 mg/kg/day. 000526 C. Conclusions 418-008: PAGE 1-10 On the basis of these data, the Fo generation maternal and paternal noobservable-effect-level (NOEL) of PFOS is 0.1 mg/kg/day (0.4 mg/kg/day and higher dosages caused reductions in body weight gain and reduced feed consumption values). The Fo generation reproductive NOEL is greater than 3.2 mg/kg/day; no effects on mating, fertility or estrous cycling occurred. The NOEL for viability and growth in the F1 generation offspring 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). The F1 generation maternal and paternal NOEL of PFOS is 0.1 mg/kg/day (0.4 mg/kg/day dosage caused reductions in body weight gain and reduced feed consumption values). The F1 generation reproductive NOEL is greater than a dosage of 0.4 mg/kg/day; no effects on mating or fertility occurred. The NOEL for viability and growth in the F2 generation offspring is 0.1 mg/kg/day (0.4 mg/kg/day dosage caused stillbirths and reductions in litter size, pup viability, growth and survival). /o -V t* s fr Mildred ^/Christian, Ph.D., Fellow, ATS Date Executive Director of Research Aran IW Hoberman, Ph.D., DABT Director of Research Date 000527 II. DESCRIPTION OF TEST PROCEDURES 418-008:PAGE 11-1 A. Conduct of Study: A.1. 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 A.3. Study Number: 418-008 A.4. Sponsor's Study Number: 6295.9 A.5. Purpose of the Study: The purpose of this study was to test for toxic effects/disturbances resulting from PFOS treatment of Crl:CDBR VAF/Plus male and female rats before cohabitation through mating, gestation and lactation. This study was designed to evaluate ICH Harmonised Tripartite Guideline stages A through F of the reproductive process and detect effects on the estrous cycle, tubal transport, implantation, gestation, parturition, lactation and maternal behavior in female rats, on the development of the offspring of the treated male and female rats, and permit detection of functional 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. A.6. Study Design: A modification of the requirements of U.S. Food and Drug Administration (FDA)(1) were used as a basis for the study design. 000528 A.7. Regulatory Compliance: 418-008.PAGE H-2 The study was conducted in compliance with the Good Laboratory Practice (GLP) regulations of the U.S. Food and Drug Administration (FDA)<2), the Japanese Ministry of Health and Welfare (MHW)(3) and the European Economic Community (EEC)(4). 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. A.8. Ownership of the Study: The Sponsor owns the study. All raw data, analyses, reports and all preserved tissues are the property of the Sponsor. A.9. Study Monitor: Marvin T. Case, D.V.M., Ph.D. A.10. Alternate Study Monitor: Andrew M. Seacat, Ph.D. A.11. Study Director: Raymond G. York, Ph.D., DABT (Associate Director of Research) A.12. Technical Performance: John F. Barnett, B.S. (Director of Laboratory Operations) Kristen landola Sherer, B.S. (Research Associate/Fetal Evaluation) Joseph W. Lech, B.S. (Team Leader - General Laboratory) Sharon Adamski (Laboratory Technician) A.13. 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) 000529 A.14. Report Review: 418-008.PAGE II-3 Alan M. Hoberman, Ph.D., DABT (Director of Research) Mildred S. Christian, Ph.D., Fellow, ATS (Executive Director of Research) A.15. Date Protocol Signed: 21 May 1998 A-16, Dates of Technical Performance: Fo Generation Male Rats 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 31 JUL 98 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 DGa 9) 26 MAY 9 8 - 1 7 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 DLb20 (rats that delivered a litter) 26 MAY 98 - 30 AUG 98 Dosage Period Estrous Cycle Evaluation 09 JUN 98 - 06 JUL 98 ai .a DG is used as an abbreviation for day of (presumed) gestation. DL is used as abbreviation for day of lactation. 000530 (Fo Generation Female Rats Continued) 418-008:PAGE II-4 Cohabitation Period Male 1 Male 2 DG 10 Caesarean-Sectioning Natural Delivery Period (DL 1) DG 25 Sacrifice (rats that did not deliver a litter) DL 21 Sacrifice (dams and pups not selected for continued study) 06 JUL 98 PM - 13 JUL 98 AM 13 JUL 98 PM - 20 JUL 98 AM 17 JUL 9 8 - 3 0 JUL 98 28 JUL 9 8 - 1 0 AUG 98 01 AUG 9 8 - 0 7 AUG 98 17 AUG 9 8 - 3 0 AUG 98 F1 Generation Rats Dosage Period (Male Rats) 19 AUG 9 8 - 1 6 NOV 98 Dosage Period (Female Rats) 19 AUG 9 8 - 2 7 DEC 98 Passive Avoidance Testing 20 AUG 9 8 - 1 0 SEP 98 Watermaze Testing 07 OCT 9 8 - 1 9 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 98 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 9 8 - 2 8 DEC 98 A.17. Records Maintained: The original report, raw data and reserve samples of the bulk test article and vehicle components are retained in the archives of Argus 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 decide their final disposition. All unused test article suspensions were discarded at the Testing Facility. Unused bulk test article was returned to the Study Monitor. B. Test A rticle Inform ation: B.1. Description: PFOS - an off-white powder B.2. Lot Number: 217 (Expiration Date: May 2000) 000531 B.3. Date Received and Storage Conditions: 418-008.PAGE II-5 The test article was received on 20 May 1998, and stored at room temperature. Prepared suspensions were stored at room temperature overnight. B.4. Special Handling Instructions: Standard safety precautions (use of protective clothing, gloves, dust-mist respirator and safety goggles) were taken when handling the bulk test article and prepared suspensions. B. 5. Analysis of Activity: Information regarding the purity, identity, strength and composition of the test article is on file with the Sponsor. C. Vehicle Information: C.1. Description: 0.5% Tween 80 in reverse osmosis membrane processed deionized water (R.O. deionized water). C.2. Lot Numbers: M03H05, K03737, M29477 and L06662 C.3. Dates Received and Storage Conditions: The Tween 80 was received from J.T. Baker, Phillipsburg, New Jersey, on 15 August 1997 (Lot K03737), 22 May 1998 (Lot M03H05), 17 September 1998 (Lot M29477), 3 December 1998 (Lot M03H05) and 1 September 1998 (Lot L06662), 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. The vehicle was prepared weekly and stored at room temperature. 000532 C.4. Special Handling Instructions: 418-008:PAGE II-6 Standard safety precautions (use of protective clothing, gloves, dust-mist respirator, safety goggles or safety glasses and a face-shield) were taken when handling the vehicle. C. 5. 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 results of this study. D. Test Article Preparation: Suspensions of PFOS were prepared daily one day prior to the day of dosing at concentrations of 0, 0.02, 0.08, 0.32 and 0.64 mg/mL. The test article was considered 100% pure for the purpose of dosage calculations. D.1 Sample Information: Sample Type Size Date Retained Shipping/Storage Conditions Shipped To Date Shipped Concentration (all levels) Homogeneity 5mL* 5 mL 5 mL 5 mL 5 mLB 26 MAY 98 29 JUN 98 21 DEC 98 27 DEC 98 26 MAY 98 Room temperature Sponsor Room temperature Sponsor 26 MAY 98 29 JUN 98 04 JAN 98 04 JAN 98 26 MAY 98 Vehicle Component Reserve Tween 80 R.O. Water 5 mL 5 mL 25 MAY 98 10 SEP 98 10 NOV 98 25 MAY 98 Room temperature Testing Facility Archives 09 JUL 98 01 OCT 98 25 NOV 98 09 JUL 98 a. A syringe was used to withdraw samples from each concentration during the first and sixth week of dosage administration for the Fo generation and during the first* and last weeks of dosage administration for the F1 generation. Each sample was divided into two aliquots (2 mL and 3 mL, respectively). One aliquot (2 mL) was shipped for analysis. The other aliquot (3 mL) was retained at the Testing Facility as a backup. b. A syringe was used to withdraw samples from the top, middle and bottom of the highest concentration on the first day of preparation. Each sample was divided into two aliquots (2 mL and 3 mL, respectively). One aliquot (2 mL) was shipped for analysis. The other aliquot (3 mL) was retained at the Testing Facility as a 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), item 1. G00533 D. 2. Analytical Results: 418-008: PAG E II-7 Results of the concentration and homogeneity analyses were not available at the time of the writing of this report. E. Test System: E.1. Species: Rat E.2. Strain: Crl:CDBR VAF/Plus (Sprague-Dawley) E.3. Supplier (Source): Charles River Laboratories, Inc., Raleigh, North Carolina E.4. Sex: Male and Female E.5. 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 Facility15'7'; and 3) the test article is pharmacologically active in the species and strain. E.6. 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 000534 E.7. Method of Randomization: 418-008:PAGE II-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 I through V), 35 rats per sex per dosage group, using a computer generated (weight-ordered) randomization procedure. The first ten female rats per dosage group with a confirmed mating date were assigned to Caesarean-sectioning on DG 10. The remaining rats were permitted to naturally deliver litters. 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 litter). E.7.b. F1/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, II and III, resulting in a 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. E.8. 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. 000535 E. 8.b. F1/F2 Generation Pups and Rats 418-008P A G E II-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: F.1. Research Facility Registration: USDA Registration No. 23-R-099 under the Animal Welfare Act, 7 U.S.C. 2131 et seq. F.2. Study Rooms: The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of ten changes per hour of 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 o f Laboratory AnimalsTM. 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. Each dam and delivered litter were housed in a common nesting box during the postpartum period. 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 000536 418-008.PAGE 11-10 and delivered litter were housed in a common nesting box during the postpartum period. F.4. Lighting: An automatically-controlled fluorescent light cycle was maintained at 12-hours light: 12-hours dark, with each dark period beginning at 1900 hours EST. F.5. Sanitization: Cage pan liners were changed approximately three times each week. Cages were changed approximately every other week. Bedding was changed as often as necessary to keep the rats dry and clean. F.6. Feed: Rats were given ad libitum 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 aware of any agent present in the feed that was known to interfere with the results of this study. F.8. Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles. Chlorine was added to the processed water as a bacteriostat. F.9. 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. 000537 418-008:PAGE 11-11 Neither the Study Director nor the Sponsor was aware of any agent present in the water that was known to interfere with the results of this study. F. 10. Bedding: Bed-o'cobs was used as nesting material (The Andersons Industrial Products Group, Maumee, Ohio). P-11- BeddingAnalysis: Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to be present in the bedding that would interfere with the results of this study. Analyses for possible contamination are conducted annually and documented in the raw data. G. Methods: G.1. Dosage Administration: Dosage Group I II III IV V Dosage (mg/kg/day) 0 (Vehicle) 0.1 0.4 1.6 3.2 Concentration (mg/mL) Dosage Volume (mL/kg) Number of Fo Generation Rats Per Sex 05 0.02 5 0.08 5 0.32 5 0.64 5 35 35 35 35 35 Number of F1 Generation Rats Per Sex 25 25 25 _ - The test article was considered 100% pure for the purpose of dosage calculations. 000538 G.2. Assigned Rat Numbers: 418-008.PAGE 11-12 Dosage Group I II III IV V Assigned Fo Generation Rat Numbers Male Female 8101-8135 8801-8835 8136-8170 8171-8205 8206-8240 8836-8870 8871-8905 8906-8940 8241-8275 8941-8975 Assigned F1 Generation Rat Numbers Male Female 13101-13125 13201 -13225 13126-13150 13226-13250 13151 - 13175 13251 - 13275 aA bB a. On DL 21, all surviving pups in the 1.6 mg/kg/day dosage group (Group IV) were sacrificed because of severe toxic effects in the pups during lactation. b. There were no surviving pups after DL 2 in the 3.2 mg/kg/day dosage group (Group V). G.3. Rationale for Dosage Selection: Dosages were selected by the Sponsor on the basis of previous studies conducted with the test article. G.4. Route of Administration: Oral (gavage) G.5. Rationale for Route of Administration: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one of the possible routes of human exposure. G.6. Frequency of Administration: The Fo generation male rats were given appropriate dosages of the test article once daily beginning 42 days before cohabitation (which continued for a maximum of 14 days) and continuing through the day before sacrifice. The Fo generation female rats were given the appropriate dosages of the test article beginning 42 days before cohabitation (which continued for a maximum of 14 days) and continuing through DG 9 (rats assigned to Caesarean-sectioning), DG 24 (rats assigned to natural delivery that did not deliver a litter), or DL 20 (rats that did deliver a litter). The dosage volume (5 mL/kg) was adjusted daily on the basis of the most recently recorded body weight and given at approximately the same time each day3. a. See APPENDIX G, item 2. 000539 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 cannibalization of the pups. Consequently, some dams were not administered one daily dosage during the delivery period. No dam missed more than one daily dosage. F1 generation male and female rats were given appropriate dosages of the test article orally (gavage), beginning on day 1 postweaning (DL 22)aand continuing through the day before sacrifice. F2 generation pups were not directly given the test article, but may have been possibly exposed to the test article during gestation (in utero exposure) or via maternal milk during the lactation period. G.7. Length of Study: Approximately 7 months G.8. Method of Study Performance: G.8.a Fo Generation Rats All Fo generation rats were observed for viability at least twice daily during all periods of the study. Rats were also observed for general appearance at least once 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 Fo generation male rats were recorded at least once during the acclimation period, weekly during the dosage period and at sacrifice. Feed consumption values for male rats were recorded weekly during the dosage period. Body weights for Fo generation female rats were recorded at least once during the acclimation period, weekly to cohabitation, daily during the gestation period, on DLs 1, 4, 7, 10 and 14 (rats assigned to natural delivery) and at sacrifice. Feed consumption values were recorded weekly to cohabitation, daily during the gestation period and on DLs 1, 4, 7, 10 and 14 (rats assigned to natural delivery). Feed consumption values were not recorded after DL 14, when it was expected that the pups would begin to consume maternal feed. A table of random units was used to select 15 female rats per dosage group for evaluation of estrous cycling by examination of vaginal cytology for 28 days before the start of the cohabitation period. a. See APPENDIX G, item 3. 000540 418-008: PAGE 11-14 Within each dosage group, consecutive order was used to assign rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of 14 days. During cohabitation, all female rats were 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 group) and remained in cohabitation for a maximum of seven additional days. The first ten female rats per dosage group with a confirmed mating date were assigned to Caesarean-sectioning on DG 10. The female rats assigned to Caesarean-sectioning were examined for the number and distribution of corpora lutea, implantation sites and viable and nonviable fetuses. The remaining female rats were permitted to naturally deliver litters. These rats were evaluated for clinical observations during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Pups that either appeared stillborn or that died before initial examination of the litters for viability were examined for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Each litter was subsequently examined daily for pup viability. Maternal 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 (live 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 bom that survived 21 days). G.8.b F1/F2 Generation Pups - Preweaninq 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 000541 418-008: PAGE 11-15 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 during the 21-day postpartum period. Surface righting reflex [ability to right in 5 seconds (from DL 1)], pinna unfolding (from DL 2), eye opening (from DL 12), acoustic startle response (from DL 13) and air righting reflex (from DL 14) were monitored until all pups (100%) in the litter reached the criterion for the specific test. Pupil constriction was evaluated once on DL 21; the number of pups per litter with this reflex present was recorded. G.8.c. F1 Generation Rats - Postweaninq Observations "Postweaning day" observations were recorded beginning on DL 22. All F1 generation rats were observed for viability at least twice daily during all periods of the study. Rats were also observed for general appearance at least once 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 postweaning periods and at sacrifice. Feed consumption values were recorded weekly except during cohabitation and at sacrifice. Body weights for F1 generation female rats were recorded once weekly to cohabitation, daily during presumed gestation, on DLs 1, 4, 7 and 14 (rats assigned to natural delivery) and at sacrifice. Feed consumption values were recorded weekly to cohabitation, on DGs 0, 7, 10, 14, 17 and 20 and DLs 1, 4, 7, 10 and 14. Beginning at 24 days of age, one male rat and one female rat from each litter in each dosage group were tested for learning, short-term retention and long-term retention in a passive avoidance paradigm. Each rat was tested on two days, separated by a one-week interval; the criterion for learning was the same for both days of testing. The passive avoidance apparatus consisted of a twocompartment chamber with hinged Plexiglas lids. One compartment was 000542 418-008.PAGE 11-16 outfitted with a bright light and Plexiglas floor. The other compartment was outfitted with a grid floor to which a brief (1 second) pulse of mild electric 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 closed, the light was turned off, and the brief pulse of current was delivered to the grid floor. The rat was then removed from the apparatus and placed into a holding cage for 30 seconds before the start of the next trial. Trials were repeated until the rat remained in the "bright" compartment continuously for 60 seconds on each of two consecutive trials (the criterion for learning) or until 15 trials were completed. The latency to enter the dark compartment or the maximum 60-second interval was recorded for each trial. Dosage groups were compared on the following dependent measures: the number of trials to the criterion in the first session (overall learning performance); the latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 of the first test session (activity level and exploratory tendency in a novel environment); the latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 2 in the first test session (short-term retention); the number of trials to the criterion in the second test session (long-term retention); and the latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the second test session (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, learning and memory. Each rat was tested in a watertight 16-gauge stainless steel modified M-maze. The maze was filled with water to a depth of approximately nine inches, and the water was monitored for temperature (range of 21 C 1C). On each test trial, the rat was placed into the starting position (base of the M-maze stem farthest from the two arms) and required to swim to one of the two goals of the M-maze, in order to be removed from the water. On the first 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 each trial. Each rat was required to reach a criterion of five consecutive errorless trials to terminate the test session. The maximum number of trials in any test session was 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial. 000543 418-008P A G E 11-17 Each rat was tested twice. The test sessions were separated by a one-week interval; the correct goal and the criterion were the same for both test sessions. Dosage groups were compared for the following dependent measures: the number of trials to criterion on the first day of testing; the average number of errors (incorrect turns 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; the number of trials to criterion on the second day of testing; the average number of errors for each trial on the second day of testing; and the latency (in seconds) to reach the correct goal on trial 1 of day 2 of testing. 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 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 exclusion of sibling matings. The cohabitation period consisted of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing. Female rats that did not mate within the first 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 litters through a 21-day postpartum period. These rats were evaluated for clinical observations during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Pups that either appeared stillborn or that died before initial examination of the litters for viability were examined for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Each litter was subsequently examined daily for pup viability. Maternal 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 (live 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). 000544 G.9. Gross Necropsy*: 418-008: PAGE 11-18 G.9.a Fo Generation Male and Female Rats Assigned to Pharmacokinetic Sample Collection At scheduled sacrifice after completion of the cohabitation period (male rats that sired litters of dams allowed to naturally deliver a litter) and on DL 21 (female rats allowed to naturally deliver a litter) blood samples (approximately 4 ml_ per rat) were collected from five rats per dosage group from the inferior vena cava, into serum separator tubes and centrifuged. The resulting serum (approximately 2 mL) was immediately frozen on dry ice and maintained frozen (-70C) until shipment to the Sponsor for analysis. The liver was excised, weighed, and a sample section (lateral lobe) was frozen and retained a t-7 0 C until shipment to the Sponsor for analysis. The liver of each pup from the litters of the five dams in each of Groups I through IV that were selected for pharmacokinetic sample collection was excised, pooled per litter, frozen and retained at -70C, until shipment to the Sponsor for analysis. The dams in the 3.2 mg/kg/day dosage group (Group V) did not have surviving pups on DL 21. G.9.b. Fo and F1 Generation Male Rats Male rats were sacrificed by carbon dioxide asphyxiation after completion of the cohabitation period, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. Representative photographs of gross lesions are available in the raw data. The following organs were excised, individually weighed and retained for possible histologic evaluation: testes, epididymides, prostate and seminal vesicles (with and without fluid). The testes were fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. The remaining organs were retained in neutral buffered 10% formalin. G.9.c. Fo Generation Female Rats Assigned to Caesarean-Sectioning Female rats assigned to Caesarean-sectioning were sacrificed on DG 10, and a gross necropsy of the 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(9). All ovaries and gross lesions were a. A table of random units was used to select one control group Fo and F1 generation rat from each sex from which all tissues examined at necropsy were retained, in order to provide control tissues for possible histopathological evaluations of gross lesions. 000S45 418-008: PAGE 11-19 retained in neutral buffered 10% formalin for possible future evaluation. Representative photographs of gross lesions are available in the raw data. The rats were examined for the number and distribution of corpora lutea in each ovary and implantation sites, and viable and nonviable embryos. A viable embryo was oval or crescent shaped, pink, firm and enclosed in an amniotic sac filled with clear fluid. A nonviable embryo was amorphous, small, pale pink to tan or deep red to black, soft and enclosed in an amniotic sac filled with clear, cloudy, or opaque fluid. Embryos were discarded after examination. G.9.d. Fo Generation Female Rats Assigned to Natural Delivery and F1 Generation Female Rats At the completion of the 21-day postpartum period, all dams that delivered litters were sacrificed on DL 21, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and distribution of implantation sites was recorded. Female rats assigned to natural delivery that did not deliver a litter were sacrificed on DG 25 and examined for gross lesions. To confirm the pregnancy status, uteri from rats that appeared nonpregnant were stained with 10% ammonium sulfide(9). Dams with no surviving pups were sacrificed after the last pup was found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. All ovaries were retained in neutral buffered 10% formalin for possible future evaluation. Rats that died were examined for the cause of death on the day the observation was made. The rats were examined for gross lesions. Pregnancy status and uterine contents were recorded. Ovaries were retained in neutral buffered 10% formalin. G.9.e. F1/F2 Generation Pups On DL 21, F1 generation pups not continued on study and all F2 generation pups were sacrificed and examined for gross lesions. Necropsy procedures were the same as those used for pups culled on DL 4. Pups that died before examination of the litter for pup viability were evaluated for vital status at birth, as described previously. Pups found dead were examined for gross lesions and for the cause of death. Pups with gross lesions found on DLs1 to 4 were preserved in Bouin's solution. Gross lesions from pups found on DLs 5 to 21 were preserved in neutral buffered 10% formalin. Representative photographs of pup gross lesions are available in the raw data. Pups not selected for continued evaluation on DL 4 were sacrificed by carbon dioxide asphyxiation and examined for gross lesions. Necropsy included a single cross-section of the head at the level of the frontal and parietal suture and examination of the cross-sectioned head for apparent hydrocephaly. The 000546 418-008:PAGE II-20 stomach contents (milk curd) were collected from culled pups from Groups I, II and III. Samples were collected from all pups from five of the largest litters in these three dosage groups. Individual pup samples were combined by litter into polypropylene tubes and frozen at -20C. After completion of sample collection, samples were shipped (frozen on dry ice) to the Sponsor for analysis. 000547 G.10. Statistical Analyses: 418-008: PAGE 11-21 The following schematic represents the statistical analyses of the data: Type of Test3 I. Parametric13 A. Bartlett's Testd II. Nonparametricc A. Kruskal-Wallis Test (<;75% ties) Significant atps0.05 Not Significant Significant atp<;0.05 Nonparametric Analysis of Variance Dunn's Test Not Significant Significant at p^ 0.05 Not Significant B. Fisher's Exact Test (>75% ties) Dunnett's Test III. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilities are reported as either p<0.05 or 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 homogeneity of variance. 000548 418-008:PAGE II-22 Proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution(10). Continuous data (e.g., body weights, body weight changes and feed consumption data) were analyzed using Bartlett's Test of Homogeneity of Variances01' and the Analysis of Variance02', when appropriate [i.e., Bartlett's Test was not significant (p>0.05)]. If the Analysis of Variance was significant (p<;0.05), Dunnett's Test03' was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p<;0.05)], the Kruskal-Wallis Test04' was used (<75% ties). In cases where the Kruskal-Wallis Test was statistically significant (p^0.05), Dunn's Method of Multiple Comparisons05' was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test06' was used. Fisher's Exact Test06' was also used to evaluate necropsy data for the pups which were stillborn or found dead. Data obtained at Caesarean-sectioning, natural delivery, preweaning reflex/physical developmental data and postweaning behavioral data involving discrete data (e.g., number of corpora lutea, number of pups per litter, trials to a criterion), were evaluated by the Kruskal-Wallis Test04', as described above. 000549 III. RESULTS - Fo GENERATION MALE RATS 418-008:PAGE 111-1 A. Clinical Observations (Summary - Table B1: Individual Data Table B10) No Fo generation male rats died during this study. All 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, corneal opacity, dental problems (missing, broken or misaligned incisors), abrasion on the neck, hyperactivity, excess salivation, 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 body weight gains. The values were significantly reduced { p i 0.05 or p<0.01) in the 0.4 mg/kg/day dosage group on days 56 to 63 of study (DSs 56 to 63); 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 15 to 22, 29 to 36, 36 to 42 and 56 to 63. Reflecting these effects of the test article, body weight gains were significantly reduced (psO.OI) for the entire precohabitation period (DSs 1 to 42) in the 1.6 and 3.2 mg/kg/day dosage groups. The 0.4, 1.6 and 3.2 mg/kg/day dosage groups had significantly reduced (p<0.05 or p<0.01) body weight gains for the entire study, calculated from DSs 1 to 63 or from DS 1 to termination. Body weights were significantly reduced {p i0 .0 5 ) in the 1.6 mg/kg/day dosage group on DSs 56, 63 and at study termination. The 3.2 mg/kg/day dosage group had significantly reduced (p<0.01) body weights on DSs 36, 42, 56, 63 and at study termination. Body weights and body weight changes were unaffected by the 0.1 mg/kg/day dosage of the test article. C. Absolute (q/day) and Relative (q/kq/dav) Feed Consumption Values (Summaries - Tables B4 and B5: 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 relative (g/kg/day) feed consumption values were significantly reduced { p i0.01) in the 3.2 mg/kg/day dosage group on DSs 15 to 22, 29 to 36 and 36 to 42. As a result of these effects of the test article, absolute and relative feed consumption values were reduced and/or significantly reduced in these two dosage groups for ooosso 418-008.PAGE III-2 the entire precohabitation period (DSs 1 to 42). After the cohabitation period (DSs 56 to 63), absolute feed consumption values were significantly reduced (p<0.05 or p<0.01) in the 0.4 and 1.6 mg/kg/day dosage groups. Absolute and relative feed consumption values were significantly reduced in the 3.2 mg/kg/day 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 Table B13) Dosages of the test article as high as 3.2 mg/kg/day did not affect any mating and fertility parameters evaluated. Values for the fertility and pregnancy indices (number of pregnancies per number of rats in cohabitation and rats that mated, respectively), the number of days to inseminate, the number of rats that mated and the number of rats with confirmed mating dates during the first week of cohabitation were comparable among the five dosage groups. E. Necropsy Observations (Summary - Table B7: Individual Data Table B14) A significantly increased (p<0.01) 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 gross lesions of the liver were considered related to the test article because the incidences were dosage-dependent. One 1.6 mg/kg/day dosage group rat (8214) and three 3.2 mg/kg/day dosage group rats (8245, 8252, 8257) had small, flaccid and/or purple testes3. 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 female rat. The gross lesions observed in the testes and prostate were not considered test article-related as they are common findings in control group 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 incidences were not dosage-dependent. These observations included a large liver, a liver with a rough surface, constricted papillary process of the liver with a. In 2079 control group rats from 91 studies conducted at the Testing Facility from August, 1992 to April, 1999, there were 21 rats with small, flaccid and/or purple testes. 00GS51. 418-008:PAGE III-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 Body Weights. Organ Weights and Ratios (% \ of Organ Weight to Terminal Body Weight fSummaries - Tables B8 and B9: Individual Data - Table B15) The 1.6 and 3.2 mg/kg/day dosage groups had significantly reduced {p<. 0.05 and p<.0.01, respectively) terminal body weights. The absolute weights of the seminal vesicles with fluid and the prostate were significantly reduced (psO.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 (ps0.01) 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. 000552 IV. RESULTS - Fo GENERATION FEMALE RATS 418-008:PAGE IV-1 A. Clinical Observations (Summary - Table C1: Individual Data Table C22) A.1. 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 1.6 mg/kg/day dosage group was moribund sacrificed on study day (DS) 36 after administration of 35 daily dosages. Adverse clinical observations included chromodacryorrhea and a clear oral exudate on DSs 35 to 36, and ptosis, hunched posture and labored breathing on DS 36. 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 clinical observations indicated this rat was misintubated on or about DS 35. All other Fo generation female rats survived to scheduled sacrifice. A. 2. 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. All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to the test article because: 1) the incidences were not dosage-dependent; and/or 2) the observation occurred in only one rat. These observations included chromodacryorrhea and dental problems (missing, broken or misaligned incisors). Clear oral exudate, ptosis, hunched posture and 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.1. 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 000553 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 to 15, 22 to 29 and 29 to 36, as compared with the control group value. Body weights were significantly reduced (p^O.01) in the 3.2 mg/kg/day dosage group on DSs 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 0 of 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 remainder of gestation, beginning with DGs 10 to 12. Weight gains were significantly reduced in the 3.2 mg/kg/day dosage group on DGs 0 to 7 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 (p<0.01) 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 p<0.01) 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. B.3. 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 (psO.01) 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). 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. 000554 418-008.PAGE IV-3 C. Absolute (q/davl and Relative (q/kq/dav) Feed Consumption Values (Summaries - Tables C8 through C13: Individual Data - Tables C26 through C28) C.1. Precohabitation Absolute (g/day) and relative (g/kg/day) feed consumption values were reduced in the 1.6 and 3.2 mg/kg/day dosage groups during the precohabitation period. These effects were significant (p^0.05 or p^O.01) in the 1.6 mg/kg/day dosage group on DSs 22 to 29 and 29 to 36 (absolute only), and in the 3.2 mg/kg/day dosage group at most tabulated intervals. Reflecting these effects of the test article, the 3.2 mg/kg/day dosage group had significantly reduced (psO.01) absolute and relative feed consumption values for the entire precohabitation period (DSs 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. C.2. 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 orpsO.OI) 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 0 to 7, 7 to 10, 10 to 12 (absolute only). Significantly increased (p<.0.05 orp<0.01) 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 body weights and the later stages of gestation, when maternal feed consumption tends to increase. Despite the increases in relative feed consumption values, the 3.2 mg/kg/day dosage group had a 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. C.3. 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 (psO.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. 000555 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 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 in the five dosage groups. Pregnancy occurred in 33, 32, 28, 29 and 30 female rats in Groups I 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 C331 Caesarean-sectioning observations on DG 10 were based on 10 (100%), 7 (70.0%), 6 (60.0%), 9 (90.0%) and 9 (90.0%) pregnant dams in each of the five respective dosage groups. There were no biologically important or statistically significant differences in the litter averages for corpora ltea, implantations, viable embryos or nonviable embryos. No dam had litter in which there were no live embryos. G. Natural Delivery and Utter 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. 000556 418-008:PAGE IV-5 The duration of gestation was significantly reduced (p<0.01) in the 3.2 mg/kg/day dosage group, an observation associated with preimplantation loss [the average number of implantation sites per dam was significantly reduced (p<0.01), resulting in a significantly reduced (p<0.01), litter size] and an effect of the test article. Pup viability was significantly reduced (p<0.05 or p<0.01) in the 1.6 and 3.2 mg/kg/day dosage groups, as described in the following information. Although the gestation index (the percentage of pregnant rats with live offspring) was comparable across all five dosage groups, the 1.6 mg/kg/day dosage group had increased numbers of pups that died or were presumed cannibalized on DL 1, and significantly increased (p<0.05 or p<0.01) numbers of pups dead or presumed cannibalized on DLs 2 to 4 and 5 to 7. The 3.2 mg/kg/day dosage group had postimplantation loss evident as reduced pup viability at birth [one litter had no liveborn pups, the number of dams with stillborn pups was significantly increased (p<0.01), the litter average and number of stillborn pups were significantly increased (p<0.01), and the litter average and number of liveborn pups were significantly reduced (p<0.01)], as well as peripartum deaths [significant numbers (p<0.05 or p<0.01) of dead and presumed cannibalized pups on DLs 1 and 2 to 4 (all liveborn pups died by DL 2) and significant numbers (p<0.01) of dams had all pups dead or presumed cannibalized on DLs 1 to 4], Reflecting these effects, the viability index was significantly reduced (p<0.01) in the 1.6 and 3.2 mg/kg/day dosage groups, the lactation index was also significantly reduced (p<0.01) in the 1.6 mg/kg/day dosage group, as also were the averages for surviving pups (p<0.05 or p<0.01) in the 1.6 mg/kg/day dosage group beginning on DL 4 postculling, and in the 3.2 mg/kg/day dosage group on DL 1. A dosage-dependent pattern of reduced pup body weight was evident in each group administered the test article. The 1.6 mg/kg/day dosage group had significantly reduced (p<0.01) pup body weights on all weighing days. The 3.2 mg/kg/day dosage group had a significantly reduced (p<0.01) pup 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. Clinical Observations from Birth to Day 21 Postpartum and Necropsy Observations (Summaries - Tables C19 and C21: Individual Data - Tables C38 and C45) Adverse clinical and necropsy observations associated with reduced pup viability and potential reduction in maternal care occurred in the 3.2 mg/kg/day dosage group. Adverse clinical observations include three (p<0.01) litters with pups that were not nursing (two of these litters had pups from which the placenta had not been removed, and one had pups that were not nested). Apparent maternal 000557 418-008:PAGE IV-6 cannibalization was also evident in this dosage group (missing tail in one pup and a cannibalized forelimb in another pup), which may have occurred after 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 control 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. All other clinical and necropsy observations in the F1 generation pups were considered unrelated to the test article because: 1) the incidences were not dosage-dependent; or 2) the observation occurred in only one litter. Clinical observations included a domed head, portion of the tail missing, umbilical hernia, missing digits and bent tail. One 1.6 mg/kg/day dosage group stillborn pup had anasarca (edema of 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, and one pup from a different 0.4 mg/kg/day dosage group litter with slight dilation of the renal pelvis. I- Reflex and Physical Development (Summary - Table C2Q: Individual Data - Tables C39 through C44) Reversible delays in reflex and physical development that are highly correlated with body weight0 17) occurred in the 0.4 and 1.6 mg/kg/day dosage groups, as described below. 1.1. Surface Righting Surface righting was delayed in the 1.6 and 3.2 mg/kg/day dosage groups, as described below. The percentage of pups that could surface right was significantly reduced (p<0.05 or psO.01) in the 1.6 mg/kg/day dosage group on DLs 3, 4, 5, 6, 7, 8 and 10, and in the 3.2 mg/kg/day dosage group on DL 1, after which there were no surviving pups in this dosage group. The average day that at least 50% of the pups in a dosage group had the ability to surface right was significantly increased (p<0.05) in the 1.6 mg/kg/day dosage group. All surviving pups ultimately attained the ability to surface right. The ability to surface right was unaffected by the 0.4 mg/kg/day dosage of the test article. 000558 1.2. Pinna Unfolding 418-008:PAGE IV-7 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. Eve 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 litter with open eye lids were significantly reduced (p<0.05) 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 at least one 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. 1.4. Acoustic Startle The time of development of the acoustic startle reflex was delayed (p<0.05 or p<0.01) 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 p<0.01) 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 startle 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. 1.5. Air Righting 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 000559 418-008:PAGE IV-8 reflex. The effect was transient and not of toxicological importance in the 0.4 mg/kg/day dosage group, occurring only on DL 16. The percentage of pups in the 1.6 mg/kg/day dosage group that air righted was significantly reduced (p<0.01) on DLs 14 through 21, resulting in a significant increase d<0.01) in the day that at least 50% of the pups could air right. I.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. 000560 418-008:PAGE V-1 V. RESULTS - F1 GENERATION MALE AND FEMALE RATS A. F1 Generation Male Rats A.1. Mortality and Clinical Observations (Summary - Table D1; Individual Data - Table D131 A.1.a. Mortality No deaths in the F1 generation male rats were attributed to PFOS. Two control group rats were found dead on days 86 and 87 postweaning. These deaths 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 clinical 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 tissues appeared normal at necropsy. A.1.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 000561 418-008.'PAGE V-2 group was not considered treatment-related because the value was not dosagedependent. No other statistically significant increases in an adverse clinical observation occurred. Additional adverse clinical signs observed and considered unrelated to the test article included dental problems (missing, broken and/or misaligned incisors), chromorhinorrhea, abrasion on the head, near the eye, forelimb or back, chromodacryorrhea, laceration at the base of the tail, a swollen 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. A.2. Body Weights and Body Weight Changes (Figure 2: Summaries Tables D2 and D3: Individual Data - Table D14) 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, 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 precohabitation period (day 1 postweaning to precohabitation) were 98.4% and 97.6% of the control group value, respectively. Body weight gains in the 0.1 and 0.4 mg/kg/day dosage groups for day 1 to termination were 98.5% and 96.6% of the control group value, respectively. A.3. Absolute (q/dav) and Relative (q/kq/dav) Feed Consumption Values (Summaries - Tables D4 and D5; Individual Data - Table D15) Absolute (g/day) 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 unrelated to the test article because the value was not dosage-dependent. No other statistically significant differences occurred in the feed consumption values for this portion of the study. A.4, 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. 000562 418-008:PAGE V-3 A.5. Passive Avoidance Performance (Summary - Table D7: Individual Data - Table P i 7) There were no biologically important differences in the values for learning, short term 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. A.6. Watermaze Performance (Summary - Table D8: Individual Data Table D181 No biologically important dosage-dependent differences occurred in watermaze performance of the F1 generation male rats regarding learning, short-term retention, long-term retention or response inhibition. No statistically significant differences occurred in the number of trials to criterion, trial latencies or numbers of rats that failed to learn. A.7. Mating and Fertility (Summary - Table D9: Individual Data Table D19) Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertility 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. A.8. Necropsy Observations (Summary - Table D10: Individual Data Table D201 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. 000563 418-008: PAG EV-4 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-filled 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 rats that died as the result of an intubation error were described previously. A. 9. 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 or relative (to body weight) values for the weights of the 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. F1 Generation Female Rats B.1. 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 000564 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 adverse clinical observations occurred in this rat, although it lost weight on DLs 1 to 7. Its feed consumption values were unremarkable. This rat delivered 15 liveborn pups, of which seven were culled on DL 4 and eight were sacrificed after the death of the dam. Necropsy of the dam revealed gross lesions compatible with an intubation accident (white foam in the trachea, approximately 2 mL of a 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 0.4 mg/kg/day dosage group. These observations probably were associated with parturition, rather than an effect of the test article, and did not appear to 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 because: 1) the incidences were not dosage-dependent; 2) the observation occurred in only one or two rats; and/or 3) the observation is common in the 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 digits, perivaginal swelling, swollen forepaw, chromorhinorrhea, soft or liquid feces, missing tail sheath, tip of tail black or missing and abrasion on the chest. 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 precohabitation body weight gains or body weights occurred in the F1 generation female rats during the precohabitation period. 000565 B.2.b. Gestation 418-008:PAGE V-6 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 (p<;0.05) maternal body weight loss occurred on DLs 1 to 4 in the 0.4 mg/kg/day dosage group, as compared to the control group value, after which body weight gains were greater than, or weight loss less than the control group values. 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 (q/dav) and Relative (q/ka/dav) Feed Consumption Values /Summaries - Tables E8 through E13: individual Data Tables E27 through E291 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 (ps0.05) 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 (p>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. 000566 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. B.4. 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.5. Passive Avoidance Performance (Summary - Table E15: Individual Data -Table E31) There were no biologically important differences in the values for learning, short term 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 learn. 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 trials 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, trial latencies or numbers of rats that failed to learn. B.7. Mating and Fertility (Summary - Table E17: Individual Data Table E331 Dosages of the test article as high as 0.4 mg/kg/day did not affect any mating and fertility 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. 000567 418-008:PAGE V-8 B.8. Necropsy Observations (Summary - Table E18: Individual Data Table E34) All necropsy observations in the F1 generation female rats were considered unrelated to the test article because: 1) the observations occurred in rats in the control group; and 2) the observation commonly occurs in this strain of rat. These observations included rough and/or pitted cortex of the kidneys with calculi in the kidney and/or urinary bladder of two control group rats. One of these rats also had a tan granular material in the renal cortex and thickened walls of the urinary bladder. Necropsy observations for the 0.4 mg/kg/day dosage group dam that died as the result of an intubation error were described previously. B.9. Natural Delivery and Litter Observations (Summaries - Tables E19 and E20: Individual Data - Tables E35 through E38i 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 dosage groups, respectively. All pregnant dams delivered litters. The gestation index (the percentage of pregnant rats with live offspring) was comparable across the three dosage groups. As occurred in the previous generation, there was a tendency for reduced pup viability in the 0.4 mg/kg/day dosage group. The number of dams with stillborn pups was increased, albeit not significantly, and the number of pup deaths after DL 2 also were increased in this dosage group. As also occurred in the previous generation, pup body weights tended to be reduced in the 0.1 mg/kg/day dosage group on DL 4 (pre- and postculling) and DL 7, but by DL 14 were comparable to control values. Pup body weights in the 0.4 mg/kg/day dosage group tended to be reduced, though not significantly, on DLs 4 through 21, with significant reductions (ps0.05 and p<0.01, respectively) present on DLs 7 and 14, as compared to the control group values. F2 generation pups body weights were comparable among all groups. Administration 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 litter sizes, numbers of dams with all pups dying during lactation, viability and lactation indices, surviving pups per litter and pup sex ratios). 000568 418-008: PAG EV-9 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 had a raised tan area on the median and left lateral lobes of the liver at necropsy on DL 21. 000S69 REFERENCES 418-008.PAGE V-10 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. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. 4. European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice. Official Journal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17. 5. Christian, M.S. and Voytek, P.E. (1982). In Vivo Reproductive and Mutagenicity Tests. Environmental Protection Agency, Washington, D.C. National Technical Information Service, U.S. Department of Commerce, Springfield, VA 22161. 6. Christian, M.S. (1984). Reproductive toxicity and teratology evaluations of naltrexone (Proceedings of Naltrexone Symposium, New York Academy of Sciences, 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 Crl:CD7BR Rat. Charles River Laboratories, Inc., Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 8. Institute of Laboratory Animal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 9. Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Arch. Pathol. Exp. Pharmakol. 247:367. 10. Snedecor, G.W. and Cochran, W.G. (1967). Variance test for homogeneity of the binomial distribution. Statistical Methods, 6th Edition, Iowa State University Press, Ames, pp. 240-241. 000570 418-008:PAGE V-11 11. Sokal, R.R. and Rohlf, F.J. (1969). Bartlett's test of homogeneity of variances. Biometry, W.H. Freeman and Co., San Francisco, pp. 370-371. 12. Snedecor, G.W. and Cochran, W.G. (1967). Analysis of Variance. Statistical Methods, 6th Edition, Iowa State University Press, Ames, pp. 258-275. 13. Dunnett, C.W. (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, E.A., Hoberman, A.M. and Christian, M.S. (1984). Positive correlation of pup body weight with other commonly used developmental landmarks. Teratology 29(2):44A. 000571 APPENDIX A REPORT FIGURES 000572 575 550 525 500 X 475 450 425 400 375 O O O nI U BODY WEIGHTS Fo GENERATION MALE RATS Figure 1 0 (VEHICLE) MG/KG/DAY 0.1 MG/KG/DAY 0.4 MG/KG/DAY 1.6 MG/KG/DAY 3.2 MG/KG/DAY *p<0.05 **p<0.01 r 15 T' 22 TI ... I ................... ! 29 36 42a 49 db 63 DAY OF STUDY a. Last value recorded before cohabitation. b. First value recorded after cohabitation. c. Terminal body weight. 418-008: PAGE A-1 BODY WEIGHTS Fo GENERATION FEMALE RATS Figure 2 O a. Last value recorded before cohabitation. c/i 418-008:PAGE A-2 BODY WEIGHTS F1 GENERATION MALE RATS Figure 3 418-008: PAGE A-3 O Oo vi CA I I I ........... I I............. |------------| ------------ I | 15 22 29 36 43 50 57 64 a 71 78 b DAY POSTWEANING a. Precohabitation body weights. b. Terminal body weights. 425 375 325 275 0 11- BODY WEIGHTS F1 GENERATION FEMALE RATS Figure 4 418-008:PAGE A-4 000576 175 125 75 25 i 15 22 29 36 43 50 57 64 a DAY POSTWEANING DAY OF GESTATION DAY OF LACTATION a. Precohabitation body weight. APPENDIX B REPORT TABLES - Fo GENERATION MALE RATS 000577 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 B1 (PAGE I): CLINICAL OBSERVATIONS - SUMMARY Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE IMG/KG/DAY) I II II J iv V 0 (VEHICLE) 0.1 0.4 1.6 3.2 MAXIMUM POSSIBLE INCIDENCE 2345/ 35 23 45/ 35 2345/ 35 2345/ 35 2345/ 35 MORTALITY 0 0000 LOCALIZED ALOPECIA: LIMBS 102/ 4 54/ 2 37/ 2 100/ 4 3/ 2 LEFT EYE: CORNEAL OPACITY 0/ 0 0/ 0 0/ 0 0/ 0 34/ 1 INCISORS: TOTAL MISSING/BROKEN MISALIGNED 78/ 3 45/ 3 51/ 1 2/ 1 2/ 1 0/ 0 102/ 4 68/ 4 80/ 2 66/ 2 19/ 1 47/ 1 12/ I 12/ 1 0/ 0 NECK: ABRASION 0/ 0 0/ 0 0/ 0 0/ 0 3/ 1 HYPERACTIVITY 0/ 0 0/ 0 0/ 0 0/ 0 1/ 1 EXCESS SALIVATION 0/ 0 0/ 0 0/ 0 0/ 0 1/ 1 CHROMODACRYORRHEA 24/ 1 0/ 0 34/ 2 41/ 1 0/ 0 SNOUT : SWOLLEN 0/ 0 0/ 0 27/ 1 43/ 1 0/ 0 CHROMORHINRRHEA 0/ 0 1/ 1 0/ 0 4/ 1 0/ 0 DYSPNEA 0/ 0 0/ 0 0/ 0 4/ 1 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS x RATS!/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION. 418-008: PAGE B-1 000578 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POS(NATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 629S.9) TABLE B2 (PAGE 1): BODY WEIGHTS SUMMARY Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 111 0.4 RATS TESTED N 35 35 35 BODY WEIGHT (Gl DAY 1 MEAN+S.D 388.9 t 22.0 390.1 + 25.7 389.8 * 23.6 DAY a M E A N + S .D 417.2 t 25.8 419.8 24.9 417.1 x 24.0 DAY IS MEAN+S.D 441.0 t 30.1 441.1 28.4 438.1 + 28.4 DAY 22 M E A N t S .D 459.1 * 36.3 460.8 t 32.1 456.4 t 32.1 DAY 29 MEAN_+S .D 471.3 * 41.9 476.7 * 36.5 472.1 + 34.3 DAY 38 M E A N t S .D 488.4 + 42.4 488.5 + 36.4 482.3 x 36.1 DAY 42a M E A N t S .D SOI.7 + 45.3 S 3 .1 + 40.8 494.8 + 34.0 DAY 49 MEAN_*S.D 509.8 * 46.2 513.2 x 42.3 501.2 x 33.6 DAY 56b M E A N i S .D 520.2 t 48.8 518.6 + 45.0 507.9 + 37.2 DAY 63 M E A N * S .D 538.0 52.1 535.2 + 48.6 520.2 x 39.7 TERMINAL BODY WEIGHT MEANtS.D 542.6 + 52.3 539.3 * 49.2 522.6 + 43.2 DAY = DAY OF STUDY a. Last value recorded before cohabitation. b. First value recorded after cohabitation. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). IV V 1.6 3 .2 35 35 390.8 + 16.4 414.8 t 21.6 437.2 x 25.2 449.6 30.9 463.5 + 29.4 472.1 + 30.4 484.2 + 33.8 492.5 + 35.2 496.9 x 36.5* 510.9 x 39.4* 391.1 18.0 415.6 21.8 436.9 25.8 448.0 30.2 458.0 34.3 461.7 36.1** 468.8 + 37.7** 469.4 37.8** 473.2 37.9** 480.8 41.8** 512.8 * 40.5* 482.1 41.5** oo o cn --X 00 oo<ob il > Q m wCD PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNAIAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.91 TABLE B3 (PAGE 1): BODY WEIGHT CHANGES - SUMMARY - Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) 11 0 .1 I11 04 RATS TESTED N 35 3S 35 BODY WEIGHT CHANGE (G) DAYS 1 ' 8 MEAN+S.D. +28.2 + 16.1 29.7 _+ 11.4 +27.3 + 12.7 DAYS 8 - 15 MEAN*.D. +23.8 + 7.8 +21.3 + 6.6 +21.0 + 7.4 DAYS IS - 22 MEAN+S.D. +18.1 + 9.3 +19.8 + 7.8 +18.3 8.1 DAYS 22 - 29 MEANS .D . 12.2 + 17.2 +15.9 + 7.5 +15.7 + 6.5 DAYS 29 - 36 MEAN+S.D. 17.1 + IS .7 +11.8 +_ 5.6 +10.2 + 5 7 DAYS 36 - 42a MEAN+S.D. +13.3 7.8 +14.6 6.9 +12.4 12.1 DAYS 1 - 42a MEAN+S.D. +112.8 + 33 .7 +113.0 _ 25.8 +105.0 + 24.6 DAYS S6b- 63 MEAN+ S .D . +17.7 5.8 +16.6 + 7.2 +12.2 + 8.8* DAYS 1 - 63 MEANtS.D . +149.0 40.3 +145.1 + 33.6 130.3 + 30.8* DAY 1 TERMINATION MEAN+S.D. +153.6 41.5 +149.2 + 34 .S +132.8 + 34.0* DAY(S ) = DAY(S) OF STUDY a. Last value recorded before cohabitation. b. First value recorded after cohabitation. * Significantly different from the vehicle ** Significantly different from the vehicle control control group value group value (p<0.05). (pcO.Ol). IV V 1.6 3.2 35 35 +23.9 + 8.4 +22.5 + 6 .a +12.3 + 16.6 +13.9 + 8.8 +B .6 + 7.4** +12.1 + 7.2 +93.4 + 22.4** +14.0 +_ 6.6 +120.0 + 28.9** +24.5 +^ 6.5 +21.3 7.3 +11.1 10.7* *10.0 8.2 3.6 6.7** 7.2 * 6.3** +77.8 + 25.2** +7.7 + 11.4** +89.8 + 29.6** +121.9 + 30.2** +91.0 + 29.9** c o o cn Xo _x ooo00o O m 03 CO PROTOCOL 418-008: COMBINED ORAL (GAVAC.EI FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE B4 IPAGE 1) : ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) SUMMARY - Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 11 III IV 0 (VEHICLE) 0 .1 0.4 1.6 RATS TESTED N 35 35 35 35 FEED CONSUMPTION (G/DAY) DAYS 1 a MEAN*S.D . 26.6 + 3.0 26.7 t 2.2 26.8 + 2.1 26. 4 . 2.4 DAYS 8 15 DAYS 15 - 22 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 42b M E A N * S .D MEAN+S.D. MEAN+S.D. MEAN S .D . MEAN^S.D . 27.0 + 2.8 2 7 . 8 * 3.4 ( 34 1a 2 a.o . 3.6 29.5 . 3.7 27. 8 . 3 .2 27.0 + 2.2 1 34] a 2 7 . 9 * 3.0 28.5 . 2.4 29.2 2.4 1 34 )a 27 4 . 2.3 26.8 + 2.4 27.4 2.8 27.9 t 2.2 2 8 . 7 . 2.4 ( 341a 27.0 + 2.9 25.9 t 2.0 26.3 t 2.8 1 341a 26.8 . 2.4 27.8 + 2.6 1 33 )a 26.6 + 3.7 DAYS 1 42b MEAN *S .D . 27.8 2.8 27 8 . 2.1 27.4 + 1.9 26.6 + 2.0* DAYS 56c- 63 MEAN.S.D. 28.0. 2 .7 1 34]1a 27.6. 2.4 26.6 + 2.2* 2 5 .a + 2.3** DAYS * DAYS OF STUDY ( 1 = NUMBER OF VALUES AVERAGED a. Excludes values that were incorrectly recorded, as well as those associated with spillage or wet feed. b. Last value recorded before cohabitation. c. First value recorded after cohabitation * Significantly different Irom the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). V 3.2 35 26.8 + 2.3 25.8 + 2.3 25.1 + 3.4** 26.4 + 2.5 25.6 + 2.5** ( 341a 24.2 + 2.1** 25.7 + 2.0** 23.7 + 2.4** 418-008:PAGE B-4 000581 PROTOCOL 4)8-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PROS IN RATS (SPONSOR'S STUDY NUMBER 629' .9 TABLE B5 (PAGE 1): RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - SUMMARY - Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE IMG/KG/DAY) RATS TESTED N FEED CONSUMPTION (G/KG/DAY) DAYS 1 - 8 MEAN+S.D . DAYS 8 - 15 MEAN+S D . DAYS 15 - 22 MEAN+S.D, DAYS 22 - 29 MEAN+S.D . DAYS 29 - 36 MEAN+S.D. DAYS 36 - 42b MEAN+S.D . DAYS 1 - 42b MEAN+S.D. DAYS 56c- 63 MEAN+S D . 1 0 (VEHICLE) 35 65.8 + 5.4 63.0 + 4.0 61.5 + 4.4 [ 34) a 60.2 + 6.7 61.5 + 5.8 56.2 _+ 4 .2 61.4 + 3 .3 52.8 + 2.5 1 34)a 1I 0 .1 35 65.9 + 4.6 62.8 + 3.6 1 34) a 61.9 + 4.8 60.9 + 3.6 60.2 + 3.1 1 34]1a 55.2 + 2.4 61.2 + 2.4 52.4 + 3.4 III 0.4 35 66.4 + 4.6 62.7 + 3.9 61.4 + 4.9 60.2 + 4.0 59.9 + 3.3 [ 34 )a 55.3 + 5.7 61.0 + 2.7 51.7 + 3.3 IV 1.6 35 65.5 + 4.2 60.9 + 3.3* 59.2 + 5.2 l 34)a 58.9 + 4.4 59.3 + 4.7 ( 33) a 55.6 + 5.9 59.9 + 2.8* 51.2 + 3.3 DAYS = DAYS OF STUDY 1 ) * NUMBER OF VALUES AVERAGED a. Excludes values that were incorrectly recorded, as well as those associated with spillage or wet feed. b. Last value recorded before cohabitation. c. First value recorded after cohabitation. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). V 3.2 35 66.3 + 3.8 60.6 + 3.6* 56.7 + 6.3" 58.3 + 4.7 55.8 + 3.1** l 34)a 52.0 3.0" 58.4 + 2.3** 49.6 + 2.6" 418-008:PAGE B-5 000582 PROTOCOL 418 008: COMBINED ORAL IGAVAGE) FERTILITY. DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUOY NUMBER: 6295.9) TABLE B6 (PAGE 1): MATING AND FERTILITY SUMMARY Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II III IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 RATS IN COHABITATION N 35 35 35 34a 35 DAYS IN COHABITATION b MEAN+S.D 2 . 7 + 1.8 ( 34) ' 2 . 6 + 1.3 2.5 + 1.8 2.1 + 1.2 1 31) 2 . 7 + 1.0 RATS THAT MATED c N (% ) 35(100.0) 35(100.0) 33( 94.3) 34(100.0) 32( 91.4) FERTILITY INDEX d.e N/N (%) 33/35 ( 94.3) 32/35 ( 91.4) 27/33 ( 81.8) 29/34 ( 8S.3) 28/32 ( 87.5) RATS WITH CONFIRMED MATING DATES f N 34 35 33 31 32 RATS MATING g DAYS 1-7 N (%) 34 (100.0) 35 (100.0) 33(100.0) 31(100.0) 32 (100.0) RATS PREGNANT/RATS IN COHABITATION f N/N U) 33/35 ( 94.3) 32/35 ( 91.4) 27/35 ( 77.1) 29/34 ( 85.3) 28/35 < 80.0) l ) - NUMBER OF VALUES AVERAGED a. Excludes values for rat 8238, which was not assigned to cohabitation because there were no available female rats. b. Restricted to rats with a confirmed mating date on days 1 to 7 of cohabitation and rats that did not mate. c. Includes only one mating for each male rat. d. Number of pregnancies/number of rats that mated. e. Includes only one pregnancy for each rat that impregnated more than one female rat. f. Includes only one confirmed mating for each male rat. g. Restricted to rats with a confirmed mating date on days 1 to 7 of cohabitation. 418-008:PAGE B-6 000583 PROTOCOL 4 18 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER 1NATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUD, NUMBER: 6296.91 TABLE B7 lPAGE 1): NECROPSY OBSERVATIONS SUMMARY Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY I I 0 (VEHICLE) 11 0 .1 III 0.4 IV V 1 .6 3.2 RATS EXAMINED a N 16 16 35 35 35 MORTALITY N0 0 0 0 0 APPEARED NORMAL N 31 35 34 32 29 TESTES: BILATERAL, SMALL, PURPLE AND/OR FLACCID N 0 0 0 1 3 LIVER: LIGHT BROWN OR BROWN N 0 0 0 0 3** LARGE N1 0 0 1 0 ROUGH SURFACE N 0 0 0 \ 0 PAPILLARY PROCESS, CONSTRICTED AND NUMEROUS TAN, RAISED AREAS N 0 0 0 \ 0 PROSTATE: SMALL N1 0 0 0 1 EYES: LEFT, LENS OPAQUE N 0 0 0 0 1 KIDNEYS: BILATERAL, LARGE N 1 0 0 1 0 PARENCHYMA, CONTAINED MULTIPLE, PALE, FLUIDFILLED CYSTS N 0 0 0 1 0 RIGHT, PELVIS, SLIGHT DILATION N0 0 1 0 0 SPLEEN: LARGE N1 0 0 0 a. Refer to Che individual clinical observations table (Table BIO) for external observations confirmed at necropsy. 0 418-008:PAGE B-7 000584 PROTOCOL 418-008: COMBINED ORAL (GAVAGEI FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.V) TABLE B8 lPAGE 1) : TERMINAL BODY WEIGHTS AND ORGAN WEIGHTS SUMMARY - Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 11 III IV 0 (VEHICLE) 0.1 0.4 1.6 RATS TESTED N 35 35 35 35 TERMINAL BODY WEIGHT MEAN+S.D. S42.6 52.3 539.3 t 49.2 522.6 + 43.2 512.8 + 40.5* EPIDIDYMIS LEFT MEAN+S .D . 0.70 + 0.08 0.72 + 0.08 0.7 + 0.06 0.70 + 0.07 TESTIS LEFT SEMINAL VESICLES WITH FLUID SEMINAL VESICLES WITHOUT FLUID MEAN+S.D. MEAN+S.D. MEAN+S.D . 1,72 + 0.19 1 33) b 2.07 + 0.34 0.99 + 0.23 1.76 + 0.14 2.11 + 0.32 1.01 + 0.21 1.72 0.15 1.97 + 0.33 1 341b 0.95 + 0.18 1.74 + 0.13 ( 34) b 2.01 + 0.35 l 34) b 0.98 + 0.17 EPIDIDYMIS RIGHT MEAN+ S .D . 0.72 + 0.08 0.74 + 0.07 0.72 + 0.06 0 . 7 0 + 0.08 TESTIS RIGHT PROSTATE MEAN+S.D. MEAN+S.D. 1.75 + 0.16 1.18 4 0.27 1 34 )b 1.75 + 0.15 1.28 + 0.28 1.71 + 0.16 1.15 + 0.31 1 . 7 4 + 0.14 ( 34 Jb 1.13 0.29 ALL WEIGHTS WERE RECORDED IN GRAMS (G). ( J = NUMBER OF VALUES AVERAGED a. Excludes values for rats that had abnormal organs (weight affected) or organs damaged * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). (weight affected). V 3.2 35 482.1 + 41.5** 0.68 + 0.07 1.79 + 0.22 ( 321b 1.77 + 0.28** l 34)b 0.92 + 0.19 0.69 + 0.08 1.78 + 0.22 ( 321b 1 . 0 2 + 0.22* ( 34)b 418-008:PAGE B-8 000585 PROTOCOL 418-008: COMBINED ORAL (GAVAGEI FERTILITY. DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 629b 9) TABLE B9 (PAGE II: RATIOS (%) OF ORGAN WEIGH! TO TERMINAL BODY WEIGHT SUMMARY - Fo GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II Ill IV V 0 (VEHICLE) 0. 1 0.4 1.6 3.2 RATS TESTED N 35 35 35 35 35 TERMINAL BODY WEIGHT MEAN+S.D . 542.6 + 52.2 539.2 + 49.2 522.6 + 43.2 512.8 + 40.5* 482.1 41.5** EPIDIDYMIS LEFT MEAN+S.D. 0.131 + 0.018 0.134 + 0.016 0.134 + 0.016 0.136 + 0.016 0.141 + 0.018 TESTIS LEFT SEMINAL VESICLES WITH FLUID SEMINAL VESICLES WITHOUT FLUID MEAN+S.D. 0.319 t 0.043 MEAN+S.D. MEAN+S.D . 0.382 0.062 1 33 )b 0.183 + 0.046 0.327 + 0.032 0.391 + 0.054 0.186 0.036 0.331 + 0.037 0.380 + 0.067 ( 341 b 0.182 + 0.035 0.339 + 0.032 I 34) b 0.394 + 0.068 l 34 )b 0.192 + 0.035 0.369 + 0.046** 1 321b 0.368 + 0.061 l 34 1b 0.191 + 0.040 EPIDIDYMIS RIGHT MEAN^S.D. 0.134 + 0.018 0.138 + 0.016 0.137 + 0.015 0.137 + 0.016 0.142 + 0.016 TESTIS RIGHT PROSTATE MEAN+S.D. MEAN+S.D. 0.324 + 0 -038 0.218 0.053 1 34 )b 0.327 + 0.038 0.239 t 0.057 0.328 + 0.034 0.220 + 0.062 0.340 + 0.035 I 34 ]b 0.219 + 0.054 0.370 0.046** ( 321b 0.212 0.045 ( 34 3b ALL WEIGHTS WERE RECORDED IN GRAMS IG). RATIOS <%]1 = (ORGAN WEIGHT/TERMINAL BODY WEIGHT) X 100. 1 ) NUMBER OF VALUES AVERAGED a. Excludes values Cor rats that had abnormal organsi (weight affected) or organs damaged (weight affected). * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). 418-008:PAGE B-9 000586 APPENDIX C REPORT TABLES - Fo GENERATION FEMALE RATS 000587 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 629S.9) TABLE Cl (PAGE 1): CLINICAL OBSERVATIONS - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE IMG/KG/DAY) I II III IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 PRECOHABITATION (DAY 1 OF STUDY TO THE DAY OF COHABITATION): MAXIMUM POSSIBLE INCIDENCE 1470/ 35 1470/ 35 1470/ 35 1464/ 35 1470/ 35 MORIBUND SACRIFICED 0 0 0 la 0 LOCALIZED ALOPECIA: TOTAL LIMBS UNDERSIDE 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 83/ 4 83/ 4 0/ 0 43/ 3 32/ 2 11/ I 65/ 3 60/ 2 5/ 1 CHROMODACRYORRHEA 16/ 1 2/ 1 0/ 0 2/ la 3/ 1 INCISORS: MISSING/BROKEN 0/ 0 47/ 2 11/ 1 0/ 0 6/ 1 CLEAR ORAL EXUDATE 0/ 0 0/ 0 0/ 0 2/ la 0/ 0 PTOSIS 0/ 0 0/ 0 0/ 0 1/ la 0/ 0 HUNCHED POSTURE 0/ 0 0/ 0 0/ 0 1/ la 0/ 0 LABORED BREATHING 0/ 0 0/ 0 0/ 0 1/ la 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA HERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS X RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION, a. Rat 8938 was moribund sacrificed on day 36 of study. 418-008PAGE C-1 88S000 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 Cl (PAGE 2); CLINICAL OBSERVATIONS - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II I I I IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 PRESUMED GESTATION: a MAXIMUM POSSIBLE INCIDENCE 649/ 34 660/ 35 647/ 34 581/ 31 666/ 35 MORTALITY 0 0000 LOCALIZED ALOPECIA: TOTAL LIMBS BACK UNDERSIDE 0/ 0 0/ 0 0/ 0 0/ 0 19/ 1 19/ 1 0/ 0 0/ 0 15/ 4* 75/ 4 0/ 0 4/ 1 45/ 4** 83/ 8** 38/ 3 40/ 3 7/ 1 0/ 0 18/ 3 31/ 3 INCISORS: TOTAL MISSING/BROKEN MISALIGNED 0/ 0 0/ 0 0/ 0 22/ 1 22/ 1 0/ 0 2/ 1 2/ 1 0/ 0 0/ 0 0/ 0 0/ 0 33/ 2 21/ 1 12/ 1 CHROMODACRYORRHEA 4/ 2 0/ 0 0/ 0 0/ 0 12/ 1 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS x RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION, a. Restricted to rats with a confirmed mating date. * Significantly different from the vehicle control group value (p^O.OS). ** Significantly different from the vehicle control group value (p<0.01). 418-008.PAGE C-2 000589 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.0) TABLE Cl (PAGE 3): CLINICAL OBSERVATIONS - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II III IV 0 (VEHICLE) 0.1 0.4 1.6 LACTATION: MAXIMUM POSSIBLE INCIDENCE 483/ 23 525/ 25 462/ 22 384/ 20 MORTALITY 0 000 LOCALIZED ALOPECIA: TOTAL BACK UNDERSIDE LIMBS HEAD 41/ 3 0/ 0 0/ 0 21/ 2 20/ 1 22/ 2 0/ 0 0/ 0 22/ 2 0/ 0 65/ 4 0/ 0 21/ 1 65/ 4 0/ 0 65/ 5 21/ 1 1/ 1 43/ 3 0/ 0 INCISORS: TOTAL MISSING/BROKEN MISALIGNED 0/ 0 0/ 0 0/ 0 21/ 1 21/ 1 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS X RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION. ** Significantly different from the vehicle control group value (p<0.01). V 3.2 31/ 21 0 3/ 5 4/ 3`* 2/ 2 2/ 1 0/ 0 3/ 2 2/ 1 1/ 1 418-008PAGE C-3 000590 PROTOCOL 418-008: COMBINED ORAL (GAVAGE1 FERTILITY. DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RAIS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE C2 (PAGE 1) BODY WEIGHTS PRECOHABITATION SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) RATS TESTED BODY WEIGHT (G) DAY 1 DAY 6 DAY IS DAY 22 DAY 29 DAY 36 DAY 42a I 0 (VEHICLE) N 35 II 0.1 35 III 0.4 35 M E A N + S .D M E A N * S .D M E A N + S .D M E A N * S .D MEANtS.D MEAN*S .D M E A N + S .D 237.2 4 12.2 243.8 4 14.5 253.6 4 17.3 256.2 4 19.0 265.2 4 20.4 269.5 4 22.9 274.4 4 24.1 236.4 + 9.4 243.8 * 11.3 252.4 + 11.8 255.4 4 12.4 263.0 + 14.1 268.7 + 13.5 272.4 4 15.1 237.1 4 11.8 245.7 4 13.5 251.9 4 15.5 255.8 4 15.9 265.3 4 18.2 268.8 4 1 9 3 271.6 4 18.8 DAY = DAY OF STUDY ( 1 = HUMBER OF VALUES AVERAGED a. Last value recorded before cohabitation. b. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. ** Significantly different from the vehicle control group value (p<0.01). IV 1.6 35 238.4 * 11.7 243.9 4 13.6 251.5 4 13.6 252.1 + 17.8 259.2 t 18.0 259.5 + 19.9 263.6 t 19.1 l 34 l b V 3.2 35 236.0 4 10.2 239.4 4 12.3 239.6 4 13.6** 238.6 4 15.0** 239.9 4 15.6** 237.2 4 14.5** 241.4 4 15.7 `* 418-008:PAGE C-4 000591 PROTOCOL 418-008: COMBINED ORAL IGAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE C3 (PAGE 1): BODY WEIGHT CHANGES PRECOHABITATION - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) RATS TESTED BODY WEIGHT CHANGE (G) DAYS 1 - 8 DAYS 8 - 1 5 DAYS 15 - 22 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 42a DAYS 1 - 42a N MEANtS .D . M E A N + S .D. MEAN*S .D . MEAN+S.D . MEAN+S.D . MEAN+S.D. MEAN+S.D. I 0 (VEHICLE) 35 +6.6 _+ 7.3 +9.8 + 6.3 +2.6 + 6.7 +9.0 + 5.6 +4.3 + 6.0 4.8 + 6.8 +37.1 15.8 II 0.1 35 +7.5 + 6.4 +8.5 + 4.5 +3.0 + 5.7 +7.6 + 6.3 +5.8 t 5.1 +3.7 + 4.8 +36.0 10.5 III 0.4 35 +8.6 + 5.7 +6.2 + 10.6 +3.9 + 9.0 +9.5 + 6.6 +3.4 + 5.2 +2.8 + 4.1 +34.5 + 12.9 DAYS = DAYS OF STUDY l ) = NUMBER OF VALUES AVERAGED a. Last value recorded before cohabitation. b. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. ** Significantly different from the vehicle control group value (p<0.0l). IV V I .6 3. 2 35 35 +5.5 + 5.7 +7.6 + 4.5 +0.6 + 7.0 +7.0 + 7.0 +0.3 + 6-7** +2.9 + 4.5 t 341i b +25.0 + 11.9** I 34 1b +3.4 + 5.6 +0.2 +_ 5.2** -1.0 + 5.5 +1.3 5.8** -2.7 + 6.5** +4.3 + 4.7 +5.4 + 10.2** 69000 418-Q08:PAGE C-5 PROTOCOL 418008: COMBINED ORAL (GAVAGE! FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER : 6295.9) TABLE C4 (PAGE 1): MATERNAL BODY WEIGHTS - GESTATION SUMMARY Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 111 0.4 RATS TESTED N 35 35 35 PREGNANT N 33 32 28 INCLUDED IN ANALYSES N 32b 32 28 MATERNAL BODY WEIGHT (G! DAY 0 HEAN+S.D. 270.2 18.9 271.0 * 15.9 272.7 + 20.0 DAY 1 MEANrS.D. 277.1 * 18.2 276.5 + 15.4 278.2 20.4 DAY 2 MEANrS.D. 281.8 . 17.2 281.4 + 14.6 282.6 + 20.3 DAY 3 MEAN.SD. 284.3 * 18.3 285.0 + 16.0 286.2 . 20.9 DAY 4 MEAN.S.D. 287.9 . 18.1 288.1 . 15.9 286.8 t 21.4 DAY s MEAN.SD. 290 4 i 19.1 289.5 ^ 15.8 290.6 + 20.9 DAY 6 MEAN^S .D. 293.5 . 18.5 293.1 t 16.5 293.1 + 20.4 DAY 7 MEAN.S.D. 295.5 19.2 293.7 + 16.7 294.3 t 20.4 DAY a MEAN.S.D. 299.3 . 19.7 296.1 + 17.0 297.5 r 21.0 DAY 9 MEAN.S.D. 302.7 . 19.7 300.3 _* 17.1 301.2 . 20.9 DAY 10 MEAN.S.D. 307.8 . 19.3 305.8 . 18.0 306.2 t 20.0 DAY = DAY OF GESTATION a. Excludes values for rat 893B, which was moribund sacrificed on day 36 of study. b. Excludes values for rats that did not have a confirmed mating date. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<Q.0l|. c tn cs IV 1.6 34a 29 26b V 3.2 35 30 30 262.0 + 21.0 268.8 . 21.5 272.2 + 21.0 273.0 + 20.1* 276.3 21.3* 277.8 22.4* 280.9 . 21.9* 282.5 . 23.0* 285.4 t 22.2* 288.0 + 22.5** 293.5 + 22.7* 240.0 17.6** 244.2 16.0** 245.3 . 17.8** 246.0 18.3** 247.2 + 18.8** 250.1 + 18.5** 253.2 + 19.4** 254.6 19.3** 255.8 + 19.4** 258.3 . 18.9** 264.3 * 18.9** 418-008:PAGE C-6 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.91 TABLE C4 (PAGE 2): MATERNAL BODY WEIGHTS - GESTATION SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) 3 II III IV 0 (VEHICLE) 0.1 0.4 1.6 RATS TESTED N 35 35 35 34a PREGNANT N 33 32 28 29 INCLUDED IN ANALYSES N 22b, c 25c 22c I7b,c MATERNAL BODY WEIGHT (G) DAY 11 MEANtS.D. 312.5 t 19.2 313.5 t 15.9 313.4 f 19.5 298.5 t 21.4* DAY 12 MEANtS .D . 317.0 t 20.0 317.2 t 16.5 316.7 + 20.6 303.6 t 22.6 DAY 13 MEANtS .D . 320.2 t 19.8 320.4 + 16.7 320.6 19.4 306.9 t 21.3 DAY 14 MEANtS .D . 325.0 + 20.0 325.4 t 17.0 326.2 19.7 311.2 t 22.1 DAY 15 MEAN+S.D. 332.2 t 20.1 332.7 t 17.9 333.1 19.3 320.2 t 22.2 DAY 16 MEANtS.D. 342.0 i 21.9 342.7 t 17.6 343.9 +_ 19.0 330.6 t 22.8 DAY 17 MEANtS.D. 3S6.3 _* 23.5 357.6 t 18.2 358.4 18.5 345.4 t 24.9 DAY 18 MEANtS.D, 370.9 t 25.9 371.0 t 19.9 370.6 + 19.7 359.7 t 26.4 DAY 19 MEANtS .D . 383.5 t 26.0 383.1 t 18.6 384.5 19.7 373.2 _t 28.2 DAY 20 MEANtS.D. 396.4 t 27.3 397.5 t 19.8 397.8 + 21.7 386.1 t 29.6 DAY = DAY OF GESTATION a. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. b. Excludes values for rats that did not have a confirmed mating date. c. Excludes values for rats that were assigned to Caesarean-sectioning on day 10 of * Significantly different from the vehicle control group value (p<0.05), * Significantly different from the vehicle control group value (p^O.Ol). gestation. V 3.2 35 30 21c 266.0 t 20.9** 268.1 t 20.8** 271.9 t 21.7** 276.7 t 23.5** 283.4 t 23.1** 294.1 t 21.8** 308.6 t 21.9** 325.0 t 22.0** 339.6 t 22.4** 348.8 t 23.3** 418-008:PAGE C-7 000594 PROTOCOL 418- 008: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS [SPONSOR'S STUDY NUMBER: 6295 9) TABLE C5 (PAGE 1): MATERNAL BODY WEIGHT CHANGES GESTATION SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0 .1 III 0.4 IV 1,6 RATS TESTED N 35 35 35 34a PREGNANT N 33 32 28 29 INCLUDED IN ANALYSES N 32b 32 28 26b MATERNAL BODY WEIGHT CHANGE (G) DAYS 0 - 7 MEAN+S.D. +25.3 + 7.5 +22.7 + 7.4 +21.6 + 6.5 +20.5 + 7.1* DAYS 7 - 10 MEAN+S.D. + 1 2 . 2 + 4.4 +12.0 + 5.2 +12.0 + 5.6 +10.9 + 6.0 DAYS 10 - 12 DAYS 1 2 - 1 5 DAYS 15 - 18 DAYS 18 - 20 DAYS 0 - 20 MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. +10.2 + 3.4 1 22 ]c +15.3 + 4.2 l 22) c +38.7 + 7.8 ( 22) c +25.5 + 6.8 I 22)c +125.1 + 15.9 [ 22)c +10.3 + 4.6 [ 25) c +15.5 + 4.7 [ 25) c +38.2 + 7.4 I 25) c +26.5 + 5.5 1 25) C +123.8 + 13.3 l 25) c +8.8 + 4.1 ( 22) c +16.4 + 5.5 [ 22) C +37.4 + 8.8 ( 22) c +27.2 + 7.4 1 22) C +121.9 + 20.2 ( 22) C +10.5 + 5.5 1 17] c +16.6 + 4.8 1 17] c +39.5 + 8.0 l 17) c +26.4 + 7.4 I 17) C +123.1 + 18.3 1 17) c DAYS = DAYS OF GESTATION [ ) = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. b. Excludes values for rats that did not have a confirmed mating date. c. Excludes values for rats that were assigned to Caesarean-sectioning on day 10 of * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). gestation. V 3.2 35 30 30 +14.6 + 6.5** +9.7 + 7.6 +5.3 + 6.3** ( 21) c +15.3 + 6.7 I 21] c +41.5 + 6.2 i 21) c +23.8 + 4.2 ( 21) c +108.0 + 10.6** [ 21) c 418-008PAGE C-8 000595 PROTOCOL 4)8-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE C6 (PAGE 1): MATERNAL BODY WEIGHTS - LACTATION - SUMMARY Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) RATS ASSIGNED TO NATURAL DELIVERY N PREGNANT N DELIVEREDi LITTERS n MATERNAL BODY WEIGHT (G) DAY 1 MEAN+S.D. DAY 4 MEAN+S.D. DAY 7 MEAN+S.D. DAY 10 MEAN+S.D. DAY 14 MEAN+S.D. DAY 21 MEAN+S.D. I 0 (VEHICLE) 25 23 23 298.3 + 19.4 303.8 + 24.0 311.8 + 21.3 325.1 + 20.7 327.0 + 19.6 331.2 + 21.3 II 0.1 25 25 25 299.0 + 18.0 l 24]b 304.0 + 17.9 306.0 + 14.9 318.0 + 15.3 326.5 + 19.5 326.4 + 14.9 III 0.4 25 22 22 298.0 + 23.2 299.4 + 21.3 305.5 + 18.9 320.3 + 16.6 324.0 + 22.4 331.8 + 16.1 DAY = DAY OF LACTATION ( ) = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. b. Excludes values for dam 8842, which delivered one additional pup on day 2 of lactation. c. Excludes values that were not recorded. d. Excludes values for dams that had no surviving pups. ** Significantly different from the vehicle control group value (p^O.Ol). IV 1.6 24a 20 20 V 3.2 25 21 21 297.8 + 21.6 290.7 + 22.5 [ 181d 294.7 + 22.7 [ 18) d 306.4 + 20.2" ( 18) d 321.4 + 22.2 1 18) d 329.4 + 20.4 t 18) d 273.3 + 19.7" [ 19) c d 418-008:PAGE C-9 000596 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERI NATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE C7 (PAGE 1): MATERNAL BODY WEIGHT CHANGES LACTATION SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS ASSIGNED TO NATURAL DELIVERY N 25 25 25 PREGNANT N 23 25 22 DELIVERED LITTERS N 23 25 22 INCLUDED IN ANALYSES N 23 25 22 MATERNAL BODY WEIGHT CHANGE (G) DAYS 1 - 4 DAYS 4 - 7 MEAN+S.D. MEAN+S.D. +5.5 + 11.0 +8.0 + 10.8 +5.3 + 8.4 [ 241 c +2.0 + 7.6 +1.4 + 11.1 +6.1 + 11.5 DAYS 7 - 10 MEAN+S.D . +13.3 + 17.4 +12.0 + 6.0 +14.8 + 5.9 DAYS 10 - 14 MEAN+S.D. +1.9 + 18.4 +8.5 + 10.7 +3.6 + 15.2 DAYS 14 - 21 MEAN+S.D . +4.1 + 19.4 -0.1 + 10.0 +7.8 + 15.2 DAYS 1 - 21 MEAN+S.D. +32.8 + 19.7 +27.8 + 12.3 ( 24 c +33.8 + 17.8 DAYS = DAYS OF LACTATION ( ] = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. b. Excludes values for dams that had no surviving pups. c. Excludes values for dam 8842, which delivered one additional pup on day 2 of lactation. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). IV 1.6 24a 20 20 18b -6.7 + 11.0** +4.0 + 7.9 +11.7 + 6.4 +15.0 + 6.4* +8.0 + 9.3 +32.0 + 14.6 V 3.2 25 21 21 0b 418-008:PAGE C-10 000597 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERI NATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 0296.9) TABLE C8 (PAGE 1): ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) - PRECOHABITATION - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 36 35 35 FEED CONSUMPTION (G/DAY) DAYS 1 ' 8 DAYS e 15 DAYS 15 22 MEAN+S.D . MEAN+S.D . MEAN+S.D . 19.2 + 2.2 i 34 ]a 19.8 + 2.2 19.3 + 2.6 19.6 + 3.0 19.9 + 2.1 ( 3D a 19 7 + 1.8 19.3 + 2.6 18.8 + 2.4 ( 32)a 19.4 + 2.0 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 42b DAYS 1 - 42b MEAN+S.D . MEAN+S.D . MEAN +S .D . MEAN+S.D . 20.0 + 2.5 19.2 + 2.2 i 34 1a 19.3 2.0 19.S 1.9 19.5 + 2.0 19.1 + 2.0 [ 34 ]a 19.4 + 2.0 ( 34]1a 19.6 + 1.5 ( 34]1a 19.3 + 2.0 [ 34 )a 18.6 + 2.0 18.9 + 1.8 19.1 + 1.7 DAYS = DAYS OF STUDY ( ) = NUMBER OF VALUES AVERAGED a. Excludes values that were incorrectly recorded, as well as those associated with spillage. b. Last value recorded before cohabitation. c. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). IV 1.6 35 19.7 + 3.0 19.3 2.3 ! 34 )a 18.4 + 1.0 I 34 a 18.5 + 2.2** 18.0 + 2.7* 1 8 . 7 + 2.5 1 34 )c 1 8 . 9 + 2.1 l 34) c V 3.2 35 17.B + 1.9* 17.2 + 2.8** 16.0 + 2.0** 16.2 + 1.8** 14.7 + 1.7** 16.3 + 2.7** 16.4 + 1.6** 418-008:PAGE C-11 000598 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 C9 (PAGE 1 ) : RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - PRECOHABITATION - SUMMARY Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0 .4 RATS TESTED N 35 35 35 FEED CONSUMPTION (G/KG/DAY) DAYS 1 - 8 DAYS 8 - 15 DAYS 15 - 22 MEAN4S .D . MEANS .D. MEAN *S .D . 79.7 4 7.2 1 34 )a 79.8 7.8 75.7 + 7.6 81.5 4 11.5 79.8 4 8.5 ( 31) a 7 7 . 6 + 6.9 80.0 + 9.2 75.5 4 8.0 ( 321a 76.3 4 6.1 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 42b DAYS 1 - 42b MEAN 4S .D . MEANS.D . MEAN 4S.D . MEAN 4S .D . 76.6 4 7.1 71.54 5 2 [ 34)a 71.04 5 9 75.7 4 4 5 75.4 4 5.7 72.0 4 6.9 [ 34) a 7 1 . 9 + 5.8 1 34 ]a 76.5 4 4.4 l 34 )a 73.8 4 5.9 1 34 )a 69.6 4 4.7 6 9 . 9 4 4.7 74.3 4 4.3 DAYS = DAYS OF STUDY ( ) = NUMBER OF VALUES AVERAGED a. Excludes values that were incorrectly recorded, as well as those associated with spillage. b. Last value recorded before cohabitation. c. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). IV 1 .6 35 81.6 4 1 1 . 8 78.1 4 8.1 l 34) a 73.3 4 5.8 1 34 )a 72.4 4 6.3" 69.0 4 7.5 7 1 . 2 + 7.4 ( 34 )C 74.5 4 6.0 1 34 )c V 3.2 35 74.6 4 6.2* 71.6 4 10.2" 6 6 . 8 4 6 .0 " 67.8 4 5.5** 61.6 4 5.2** 68.2 4 10.4 68.5 4 4.3** 418-008:PAGE C-12 000599 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 CIO (PAGE 1): MATERNAL ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) - GESTATION - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0 .1 III 0.4 IV 1.6 RATS TESTED N 35 35 35 34a PREGNANT N 33 32 28 29 INCLUDED IN ANALYSES N 32b 32 28 26b MATERNAL FEED CONSUMPTION (G/DAY) DAYS 0 - 7 MEAN+S.D . 2 1 . 8 + 2.1 21.6 + 2.1 21.0 + 2.1 20.4 + 2.2* DAYS 7 - 10 MEAN +S .D . 23.4 + 2.2 23.6 + 2.4 22.6 + 3.0 21.0 + 2.8** DAYS 10 - 12 DAYS 12 - 15 DAYS 15 - 18 DAYS 18 - 20 DAYS 0 - 20 MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. 2 4 . 2 + 3.0 [ 22] c 23.8 + 2.4 I 22 ]c 2 4 . 8 + 2.8 ( 221c 22.2 + 2.6 [ 22] c 2 3 . 0 + 1.9 l 22] C 24.3 + 2.5 1 25] c 23.6 + 2.1 ( 25] c 24.4 + 2.3 i 25]Ic 22.4 + 2.3 [ 24]1c, d 22.9 + 1.2 1 24]1c, d 22.9 + 3.2 [ 22] c 22.8 + 2.5 [ 22] c 24.2 + 1.9 l 22] c 22.9 + 2.5 [ 22] c 2 2 . 4 + 2.0 [ 22] c 23.6 + 3.4 [ 171c 2 3 . 4 + 2.8 [ 17] C 2 6 . 6 + 2.9* I 17] C 23.6 3.3 [ 171 C 22.8 + 2.2 [ 17] C DAYS = DAYS OF GESTATION ( ] = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 ofstudy. b. Excludes values for rats that did not have a confirmed mating date. c. Excludes values for rats that were assigned to Caesarean-sectioning on day 10 of gestation. d. Excludes values thatwere associated with spillage or wet feed. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). V 3.2 35 30 30 17.0 + 2.1** 17.7 + 2.6** 1 8 . 6 + 3.3** l 211 c 19.6 + 2.7** f 21 ]C 23.3 + 2.7 [ 20]c,d 21.2 + 2.5 l 21] C 19.1 + 1.8** ( 21] C 418-008: PAGE C-13 000600 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RAIS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE Cll (PAGE 1); MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - GESTATION - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II III IV 0 (VEHICLE) 0.1 0.4 1.6 RATS TESTED N 35 35 35 34a PREGNANT N 33 32 28 29 INCLUDED IN ANALYSES N 32b 32 28 26b MATERNAL FEED CONSUMPTION (G/KG/DAY) DAYS 0 - 7 MEAN+S.D. 76.7 + 6.6 75.9 + 5.6 73.7 + 6.4 7 4 . 6 + 5.9 DAYS 7 - 10 MEAN+S.D. 77.7 + 5.4 78.8 + 5.4 75.3 + 9.2 72.8 + 7.4* DAYS IQ - 12 DAYS 12 - 15 DAYS 15 - 18 DAYS 18 - 20 DAYS 0 - 20 MEAN+S.D . MEAN+S.D. MEAN+S.D . MEAN+S.D. MEAN+S.D. 17.2 + 6.3 [ 22) c 7 3 . 5 + 5.6 1 22] c 70.7 + 6.1 1 22) c 5 8 . 1 + 6.1 [ 22) c 72.7 + 4.3 ( 22] c 77.8 + 7.3 t 251c 7 2 . 8 + 5.4 [ 25) c 69.4 + 6.3 ( 251 c 5 8 . 7 + 6.1 I 24)c,d 72.4 + 2.9 ( 241c ,d 73.2 + 9.7 l 22) c 70.4 + 6.6 [ 22] c 69.0 + 5.7 ( 221 c 59.7 + 6.7 [ 22] c 70.5 + 5.3 ( 22) c 78.7 + 7.4 l 17] C 75.4 + 6.2 [ 17] c 78.5 + 5.8** ( 17] c 63.4 + 8.0* [ 17] c 74.8 + 4.4 ( 17] c DAYS = DAYS OF GESTATION [ ) = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 ofstudy. b. Excludes values for rats that did not have a confirmed mating date. c. Excludes values for rats that were assigned to Caesarean-sectioning on day 10 of gestation. d. Excludes values thatwere associated with spillage or wet feed. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). V 3.2 35 30 30 68.8 + 6.0** 68.7 + 8.4** 70.1 + 12.1 1 21 ]c 71.3 + 8.5 ( 21] c 77.4 + 7.4** ( 20] c, d 6 2 . 8 + 6.9* ( 21] C 70.0 + 4.7 1 21 ]c 418-008.PAGE C-14 000601 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 C12 (PAGE 1) : MATERNAL ABSOLUTE FEED CONSUMPTION VALUES (G/DAYI LACTATION - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) 1 II III IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 RATS ASSIGNED TO NATURAL DELIVERY N 25 25 25 24a 25 PREGNANT N 23 25 22 20 21 DELIVERED LITTERS N 23 25 22 20 21 INCLUDED IN ANALYSES N 23 25 22 18b 0b MATERNAL FEED CONSUMPTION (G/DAY) DAYS 1 - 4 DAYS 4 - 7 DAYS 7 - 10 DAYS 10 - 14e DAYS 1 - 14e MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. 2 5 . 4 + 6.9 [ 22) c 3 7 . 5 + 7.5 4 7 . 2 + 5.2 l 22) c 5 6 . 9 + 5.1 1 22 1c 4 3 . 4 + 5.4 ( 22] C 24.0 + 4.4 [ 23]c,d 37.6 + 5.4 4 4 . 8 + 4.4 5 6 . 7 + 5.8 42.5 + 4.5 [ 24 ]d 22.0 + 4.5 [ 21 ]c 36.3 + 8.7 [ 20] c 4 5 . 3 + 5.1 53.6 + 9.2 4 1 . 0 + 6.9 16.7 + 4.0** 28.9 + 6.6** 36.6 + 1.6** 46.8 + 9.2** 33.4 + 6.5** DAYS = DAYS OF LACTATION [ ) = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. b. Excludes values for dams that had no surviving pups. c. Excludes values that were not recorded, as well as those associated with spillage or wet feed. d. Excludes values for dam 8842, which delivered one additional pup on day 2 of lactation. e. Because it is presumed that the pups begin to consume maternal feed after day 14 of lactation, maternal feed consumption values were not tabulated on days 14 to 21 of lactation. ** Significantly different from the vehicle control group value (p<0.01). 418-008:PAGE C-15 209000 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 Cl 3 (PAGE 1) : MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - LACTATION - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II III IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 RATS ASSIGNED TO NATURAL DELIVERY N 25 25 25 24a 25 PREGNANT N 23 25 22 20 21 DELIVERED LITTERS N 23 25 22 20 21 INCLUDED IN ANALYSES N 23 25 22 . 18b 0b MATERNAL FEED CONSUMPTION (G/KG/DAY) DAYS 1 - 4 DAYS 4 - 7 DAYS 7 - 10 DAYS 10 - 14e DAYS 1 - 14e MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. 84.6 + 22.2 l 22)c 122.2 + 24.7 147.9 + 15.6 [ 22) C 174.0 + 14.1 [ 22] C 137.8 + 16.2 [ 221 C 79.9 + 14.1 [ 23]C ,d 123.6 + 19.8 144.0 + 15.4 175.9 + 17.7 136.9 + 15.5 l 24 ]d 74.4 + 17.0 t 21JC 120.0 + 28.3 t 20] c 145.4 + 18.6 166.7 + 29.0 133.0 + 23.8 56.9 + 13.8" 98.6 + 20.0** 121.7 + 23.5** 148.9 + 25.9** 110.4 + 19.6** DAYS = DAYS OF LACTATION [ ] = NUMBER OF VALUES AVERAGED a. Excludes values for rat 8938, which was moribund sacrificed on day 36 of study. b. Excludes values for dams that had no surviving pups. c. Excludes values that were not recorded, as well as those associated with spillage or wet feed. d. Excludes values for dam 8842, which delivered one additional pup on day 2 of lactation. e. Because it is presumed that the pups begin to consume maternal feed after day 14 of lactation, maternal feed consumption values were not tabulated on days 14 to 21 of lactation. ** Significantly different from the vehicle control group value (p<0.01). 418-008:PAGE C-16 000603 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295 9) TABLE Cl 4 (PAGE 1): ESTROUS CYCLING, MATING AND FERTILITY - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) ESTROUS CYCLING RATS EVALUATED ESTROUS STAGES/ 28 DAYS RATS WITH 6 OR MORE CONSECUTIVE DAYS OF DIESTRUS RATS WITH 6 OR MORE CONSECUTIVE DAYS OF ESTRUS N MEAN+S.D . N N I II III IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 15 6.5 + 0.7 15 6.5 + 0.8 15 5.7 + 1.3 15 6.1 + 0.8 15 6.7 t 0.7 345 10 20 00 418-008PAGE C-17 000604 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 Cl4 (PAGE 2) : ESTROUS CYCLING, MATING AND FERTILITY - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) 0 (VEHICLE) II 0.1 III 0.4 MATING AND FERTILITY RATS IN COHABITATION N 35 35 35 DAYS IN COHABITATION b MEAN+S.D. 2.7 + 1.8 1 34) 2 . 6 * 1.3 2 . 9 + 3.1 RATS THAT MATED NIL 35(100.0) 35(100.0) 34( 97.1) FERTILITY INDEX c N/N <%) 33/35 ( 94.3) 32/35 ( 91.4) 28/34 ( 82.4) RATS WITH CONFIRMED MATING DATES N 34 33 34 MATED BY FIRST MALE d DAYS 1 7 imi 34(100.0) 33(100.0) 33( 97.0) MATED BY SECOND MALE d DAYS 8-14 Nil) 0( 0.0) 0( 0.0) 1( 2.9) RATS PREGNANT/RATS IN COHABITATION N/N m 33/35 ( 94.3) 32/35 ( 91.4) 28/35 ( 80.0) [ ] = NUMBER OF VALUES AVERAGED a. Excludes values for female rat 8938, which was moribund sacrificed on day 36 of study. b. Restricted to rats with a confirmed mating date and rats that did not mate. c. Number of pregnancies/number of rats that mated. d. Restricted to rats with a confirmed mating date. IV 1.6 34a 2 . 1 + 1.2 ( 31) 34(100.0) 29/34 ( 85.3) 31 31(100.0) 0( 0.0) 29/34 ( 85.3) V 3.2 35 3.2 + 3 35(100.0) 30/35 ( 85.7) 35 32( 91.4) 3( 8.6) 30/35 ( 85.7) 418-008:PAGE C-18 000605 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S S'lUDY NUMBER: 6295.9) TABLE Cl5 (PAGE 1): NECROPSY OBSERVATIONS SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II III IV 0 (VEHICLE) 0.1 0.4 1.6 RATS EXAMINED a N 35 35 35 35 MORIBUND SACRIFICED N 0 0 0 lb APPEARED NORMAL N 35 35 34 34b SPLEEN: ADHERED TO ABDOMINAL ADIPOSE TISSUE N 0 0 0 1 KIDNEYS: RIGHT, PELVIS, MODERATE DILATION N0 0 1 0 a. Refer to the individual clinical observations table (Table C22) for external observations confirmed at necropsy. b. Rat 8938 was moribund sacrificed on day 36 of study. V 3.2 35 0 35 0 0 418-008PAGE C-19 909000 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 Cl6 (PAGE 1) : CAESAREAN-SECTIONING OBSERVATIONS - SUMMARY Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) RATS ASSIGNED TO CAESAREAN SECTIONING N PREGNANT N(%) RATS PREGNANT AND CAESAR EAN-SECTION ED ON DAY 10 OF GESTATION N CORPORA LUTEA MEAN+S.D. IMPLANTATIONS MBAN+S.D. VIABLE EMBRYOS N MEAN+S.D . NONVIABLE EMBRYOS N MEAN+S.D. DAMS WITH ANY NONVIABLE EMBRYOS N(%) DAMS WITH ALL NONVIABLE EMBRYOS N DAMS WITH VIABLE EMBRYOS N (%) % NONVIABLE EMBRYOS/LITTER MEAN+S.D . I 0 tVEHICLE) 10 10(100.0) 10 16.6 + 3.6 13.5 t 4.0 133 13.3 t 4.1 2 0.2 + 0.6 1 ( 10.0) 0 10(100.0) 1 . 7 + 5.3 II 0.1 10 7( 70.0) 7 1 4 . 6 + 6.6 1 1 . 8 + 5.6 83 11.8 + 5.6 0 0.0 + 0.0 0( 0.0) 0 7(100.0) 0 . 0 + 0.0 III 04 10 6 ( 60.0) 6 14.8 + 4.3 12.2 + 5.6 73 12.2 + 5.6 0 0.0 + 0.0 0( 0.0) 0 6(100.0) 0.0 + 0.0 IV 1.6 10 9( 90.0) 9 15.7 + 4.6 12.9 + 4.2 115 12.8 + 4.1 1 0.1 + 0.3 i< li.i) 0 9(100.0) 0.6 + 1.8 V 3.2 10 9( 90.0) 9 16.2 + 3.2 13.1 + 4.0 118 13.1+ 4.0 0 0.0 + 0.0 0( 0.0) 0 9(100.0) 0.0 + 0.0 418-008`.PAGE C-20 000607 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 Cl 7 (PAGE 1): NATURAL DELIVERY OBSERVATIONS - SUMMARY - Fo GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II III IV V 0 (VEHICLE) 0.1 0.4 1.6 3.2 RATS ASSIGNED TO NATURAL DELIVERY N 25 25 25 24a 25 PREGNANT DELIVERED LITTERS N(%) N(%) 23( 92.0) 23(100.0) 25(100.0) 25(100.0) 22( 88. 0) 22(100.0) 20( 83.3) 20( 100. 0) 21( 84.0) 21( 100. 0) DURATION OF GESTATION b MEAN+S.D. 22.7 + 0.4 ( 22) 22.8 + 0.4 ( 24) 22.5 + 0.7 [ 22] 22.4 + 0.5 I 17) 22.2 + 0.4** ( 21) IMPLANTATION SITES PER DELIVERED LITTER N MEAN+S.D. 342 1 4 . 9 + 1.9 384 15.4 + 2.2 323 14.7+ 3.5 295 1 4 . 8 + 1.7 262 12.5 + 1.4** DAMS WITH STILLBORN PUPS N (%) 5 ( 21.7) 2 ( 8.0) S( 22.7) 4( 20.0) 15( 71.4) DAMS WITH NO LIVEBORN PUPS N (%) 0( 0.0) 0< 0.0) 0 ( 0.0) K 4.8) GESTATION INDEX c % N/N 100.0 23/ 23 100.0 25/ 25 100.0 22/ 22 100.0 20/ 20 95.2 20/ 21 DAMS WITH ALL PUPS DYING DAYS 1-4 POSTPARTUM N (%) 0( 0.0) o o o 2( 10.0) 20(100.0)** DAMS WITH ALL PUPS DYING DAYS 5-21 POSTPARTUM N 0 0 0 0 a. Rat 8938 was moribund sacrificed on day 36 of study. b. Calculated as the time (in days) elapsed between confirmed mating (arbitrarily defined as day 0) and the time (in days) the first pup was delivered. c. Number of rats with live offspring/number of pregnant rats. ** Significantly different from the carrier group value (p^O.Ol). 418-008:PAGE C-21 o o o o o o 000608 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 CIS (PAGE 1): LITTER OBSERVATIONS (NATURALLY DELIVERED PUPS) - SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP I II III IV MATERNAL DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0.1 0.4 1.6 DELIVERED LITTERS WITH ONE OR MORE LIVEBORN PUPS N 23 25 22 20 PUPS DELIVERED (TOTAL) N MEAN+S.D. 323 14 .0 + 2.1 350a 1 4 . 6 + 2.3a 303 13.8 + 3.4 260 13.0+ 2.5 LIVEBORN MEAN+S.D . N {%) 13 .6 + 2.3 313 < 96.9) 14.4 + 2.5a 346( 98.9)a 1 3 . 5 + 3.3 297( 98.0) 12.7 + 2.6 254( 97.7) STILLBORN MEAN+S.D. N(%) 0 .3 + 0.7 7 ( 2.2) 0.1 + 0.3a 2 ( 0.6)a 0 . 3 + 0.6 6( 2.0) 0 . 3 + 0.6 6( 2.3) UNKNOWN VITAL STATUS N 3 2a 0 0 PUPS FOUND DEAD OR PRESUMED CANNIBALIZED DAY 1 DAYS 2- 4 DAYS 5 - 7 DAYS 8-14 DAYS 15-21 N / N (%) N / N (%) N / N (%) N / N (%) N / N (%) 2/313( 2/311( 0/184( 0/184( 0/184( 0.6) 0.6) 0.0) 0.0) 0.0) 3/346( 3/358( 0/200( 0/200( 0/200( 0.9) a 0.8) 0.0) 0.0) 0.0) 1/2971 4/296( 0/169( 1/169( 2/168( 0.3) 1.4) 0.0) 0.6) 1.2) 27/254( 10.6) 59/227( 26.0)* 6/131( 4.6)** 1/125( 0.8) 0/124( 0.0) VIABILITY INDEX b % 98. 7 N/N 309/313 98 .3 340/346a 98 ,3 292/297 66.1* 168/254 LACTATION INDEX c % N/N 100..0 184/184 100..0 200/200 98 .2 166/169 94.6** 124/131 D A Y (S ) = DA Y (S) POSTPARTUM a. Excludes values for litter 8842; the dam delivered one additional pup on day 2 of lactation. b. Number of live pups on day 4 (preculling) postpartum/number of liveborn pups on day 1 postpartum. c. Number of live pups on day 21 (weaning) postpartum/number of live pups on day 4 (postculling) postpartum. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). V 3.2 20 200 10.0 + 3.4** 7.8 + 4.0** 156( 78.0)** 2.2 + 2.3** 44( 22.0)** 0 71/1561 45.5)** 85/ 85(100.0)** 0.0** 0/156 418-008:PAGE C-22 000609 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 C18 (PAGE 2): LITTER OBSERVATIONS (NATURALLY DELIVERED PUPS) - SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) DELIVERED LITTERS WITH ONE OR MORE LIVEBORN PUPS N SURVIVING PUPS/LITTER a DAY 1 b MEAN+S.D. DAY 4 PRECULLING MEAN+S.D . DAY 4 POSTCULLING MEAN+S.D . DAY 7 MEAN+S.D . DAY 14 MEAN+S.D . DAY 21 MEAN+S.D . I 0 (VEHICLE) 23 13.6 + 2.3 13.4 + 2.1 8.0 + 0.0 8.0 + 0.0 8.0 4- 0.0 8.0 4- 0.0 II 0.1 25 14.4 4- 2.4 ( 24) c 14.2 + 2.4 8.0 + 0.0 8.0 + 0.0 8.0 + 0.0 8.0 4- 0.0 III 0.4 22 13.5 4- 3.3 13.3 + 3.3 7.7 1.1 7.7 + 1.1 7.6 + 1.1 7.5 4- 1.2 IV 1 .6 20 12.7 4- 2.6 8.4 4- 4.0 6.6 4- 2.6* 6.2 4- 2.8** 6.2 4- 2.8** 6.2 4- 2.8** V 3.2 20 7.8 + 4.0** 0.0 + 0.0** 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 418-008: PAGE C-23 000610 PERCENT MALE PUPS PER NUMBER OF PUPS SEXED DAY 1 b DAY 4 PRECULLING DAY 4 POSTCULLING DAY 7 DAY 14 DAY 21 MEAN+S.D. MEAN+S.D . MEAN+S.D . MEAN+S.D . MEAN+S.D . MEAN+S.D. 46.3 + 17.0 46.8 + 16.9 48.9 + 11.2 48.9 + 11.2 48.9 + 11.2 48.9 + 11.2 S3.9 + 14.4 ( :24 1c 53.2 + 15.0 49.0 + 6.2 49.0 + 6.2 49.0 + 6.2 49.0 + 6.2 52.8 + 11.9 53.0 + 11.6 50.8 + 5.5 1 20) e 51.9 + 6.3 51.6 + 5.9 52.4 + 6.7 55.1 + 17.8 57.0 + 21.4 [ 18) d 53.8 + 19.7 l 18) d 51.7 + 23.2 t 18) d 51.8 + 23.0 [ 18) d 51.8 + 23.0 [ 18) d DAY = DAY POSTPARTUM [ ) = NUMBER OF VALUES AVERAGED a. Average number of live pups per litter, including litters with no surviving pups. b. Includes pups born alive, found dead day 1 postpartum. c. Excludes values for litter 8842; the dam delivered one additional pup on day 2 of lactation. d. Excludes values for litters that had no surviving pups. e. Excludes values for litters 8874 and 8899; a sexing error was found prior to weighing on day 1 postpartum. * Significantly different from the vehicle control group value (p^O.05). ** Significantly different from the vehicle control group value (p<0.01). 46.7 + 22.0 d 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 C18 (PAGE 3): LITTER OBSERVATIONS (NATURALLY DELIVERED PUPS) - SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP I II III IV MATERNAL DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0.1 0.4 1.6 DELIVERED LITTERS WITH ONE OR MORE LIVEBORN PUPS N 23 25 22 20 INCLUDED IN ANALYSES N 23 25 22 20 LIVE LITTER SIZE AT WEIGHING DAY 1 DAY 4 PRECULLING DAY 4 POSTCULLING DAY 7 DAY 14 DAY 21 MEAN+_S.D . MEAN+S.D. MEAN+S.D . MEAN+S.D. MEAN+S.D. MEAN+S.D. 13.6 + 2.2 13.4 + 2.1 8.0 + 0.0 8.0 + 0.0 8.0 + 0.0 8.0 0.0 14.3 + 2.4 l 24) b 14.2 + 2.4 8.0 + 0.0 8.0 + 0.0 8.0 + 0.0 8.0 + 0.0 13.4 + 3.4 13.3 3.3 7.7 + 1.1 7.7 + 1.1 7.6 + 1.1 7.5 + 1.2 11.6 + 3.5 9.3 + 3.0** [ 18) a 7.3 + 1.4 1 18) a . 9 + 1.9* [ 18J a 6.9 + 1.9** 1 18) a 6 . 9 + 1.9** l 18) a PUP WEIGHT/LITTER (GRAMS) DAY 1 DAY 4 PRECULLING DAY 4 POSTCULLING DAY 7 DAY 14 DAY 21 MEAN+S.D . MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+ S .D . MEAN+S.D . 6.6 + 0.6 9.2 1.2 9.4 + 1.2 14.0 + 1.9 31.1 + 3.3 49.8 + 5.1 6 . 6 + 0.5 ( 24) b 8.9 + 0.8 9.0 + 0.7 14.3 + 1.2 30.5 + 2.3 48.6 + 3.6 6.4 + 0.7 8.9 + 1.3 9.0 + 1.3 14.0 + 1.4 29.9 + 2.3 48.2 + 4.5 5.7 + 0.5** 7.3 + 1.1** [ 18) a 7.5 + 1.2** [ 18) a 11.0 + 1.8** ( 18) a 24.4 + 3.3** ( 18) a 40.3 + 4.3** I 18) a DAY = DAY POSTPARTUM [ ) * NUMBER OF VALUES AVERAGED a . Excludes values for litters that had no surviving pups. b. Excludes values for litter 8842; the dam delivered one additional pup on day 2 of lactation. Significantly different from the carrier group value (p<0.05). * * Significantly different from the carrier group value (p<0.01). V 3.2 20 11a 9.1 + 3.7** a 5.3 + 0.4** l io)a a 418-008.PAGE C-24 000611 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 Cl 9 (PAGE 1) : CLINICAL OBSERVATIONS FROM BIRTH TO DAY 21 POSTPARTUM - SUMMARY - FI GENERATION PUPS MATERNAL DOSAGE GROUP I II III IV MATERNAL DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0.1 0.4 1.6 LITTERS EXAMINED (N) 23 25 22 20 TRANSIENT CLINICAL OBSERVATIONS: a TOTAL FREQUENCY (DAYS x PUPS)/LITTERS WITH OBSERVATIONS NOT NURSING N/N 0/ 0 0/ 0 0/ 0 0/ 0 PLACENTAS NOT REMOVED N/N 0/ 0 0/ 0 0/ 0 0/ 0 NOT NESTING N/N 0/ 0 0/ 0 0/ 0 0/ 0 PERSISTENT CLINICAL OBSERVATIONS:: a TOTAL FREQUENCY (DAYS X PUPS)/LITTERS WITH OBSERVATIONS TAIL: MISSING N/N 0/ 0 0/ 0 0/ 0 0/ 0 LEFT FORELIMB: CANNIBALIZED N/N 0/ 0 0/ 0 0/ 0 0/ 0 HEAD: DOMED N/N 0/ 0 0/ 0 17/ 1 7/ 1 PORTION OF TAIL MISSING N/N 0/ 0 0/ 0 0/ 0 20/ 1 UMBILICAL HERNIA N/N 0/ 0 15/ 1 0/ 0 0/ 0 LEFT HINDPAW: DIGITS, MISSING N/N 21/ 1 0/ 0 0/ 0 0/ 0 TAIL: BENT N/N 8/ 1 0/ 0 0/ 0 0/ 0 STATISTICAL ANALYSES WERE RESTRICTED TO THE NUMBER OF LITTERS WITH OBSERVATIONS. N/N = TOTAL FREQUENCY (DAYS X PUPS)/LITTERS WITH OBSERVATIONS a. Tabulation restricted to adverse observations; all other pups appeared normal. ** Significantly different from the vehicle control group value (p<0.01). V 3.2 20 24/ 3** 22/ 2 2/ 1 1/ 1 1/ 1 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 418-008PAGE C-25 000612 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 C20 (PAGE 1): REFLEX AND PHYSICAL DEVELOPMENT - SUMMARY - FI GENERATION LITTERS MATERNAL, DOSAGE GROUP I II III IV V MATERNAL. DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0.1 0.4 1.6 3.2 SURFACE RIGHTING a N 23 25 22 20 lob DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY G DAY 7 DAY a DAY 9 DAY 10 DAY 11 DAY 12 DAY 13 DAY 14 MEAN+S.D. m e a n +s .d . MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. M E A N + S .D. MEAN+S.D. 46.0 + 20.0 38.7 + 24.7 47.8 + 27.6 71.0 + 31.4 85.9 + 21 .8 90.8 + 21.7 94.6 + 13.0 96.7 + 8.6 97.8 + 6 .1 99.4 + 2.6 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 51.6 + 24.3 ( 24) c 34.0 + 24.8 43.4 + 27.4 74.4 + 22.7 91.0 + 20.6 95.5 + 12.4 93.5 + 16.2 95.5 + 10.8 98.5 + 5.5 99.5 + 2.5 99.5 + 2.5 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 37.0 + 22.0 32.7 + 17.9 39.4 28.6 61.5 + 28.1 84.1 + 19.0 90.9 + 17.3 93.8 + 13.2 98.3 + 5.8 99.4 + 2.7 100.0 * 0.0 100.0 0.0 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 29.4 + 23.5 2 0 . 4 + 16.1 I 19) b 1 1 . 9 + 10.7** [ 191b 32.4 + 21.5** 1 I8]b 4 1 . 2 + 32.9** [ 18) b 47.1 + 30.1" [ 18) b 60.2 + 33.6" [ 18) b 77.6 + 29.3** [ 18) b 85.4 + 27.9 [ 18] b 89.6 + 28.5* [ 18) b 94.4 + 16.2 [ 18)b 93.0 + 24.0 [ 18] b 94.4 + 23.6 ( 18) b 94.4 + 23.6 [ 18 ]b 3.8 + b DAY = DAY POSTPARTUM 1 ) = NUMBER OF VALUES AVERAGED a. The average percentage of pups meeting the criterion for the developmental landmark tested on each day postpartum. b. Excludes values for dams that had no surviving pups. c. Excludes values for dam8842, which delivered one additional pup on day 2 of lactation. ` Significantly different from the vehicle control group value (p<0.05) . ** Significantly different from the vehicle control group value (p^O.Ol). 418-008:PAGE C-26 000613 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 C20 (PAGE 2): REFLEX AND PHYSICAL DEVELOPMENT - SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) SURFACE RIGHTING (Cont.)a N INCLUDED IN ANALYSES N DAY 15 MEAN+S.D. DAY 16 MEAN+S.D. DAY 17 MEAN+S.D . MEAN%+S.D.C I 0 (VEHICLE) 23 23 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 2.4 + 1.8 II 0.1 25 25 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 2.0 + 1.2 III 0.4 22 22 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 2.7 + 1.6 IV 1.6 20 18b 97.2 + 11.8 97.2 + 11.8 100.0 + 0.0 4.6 + 2.6* V 3.2 21 0b PINNA UNFOLDING a N 23 25 22 19b DAY 2 MEAN+S.D . 16.3 + 36.6 3.2 + 12.6 6.0 + 22.1 1 . 6 + 7.1 DAY 3 MEAN+S.D . 73.1 + 33.5 50.8 + 35.4* 43.9 + 38.8* 8.4 + 19.8** DAY 4 DAY 5 DAY 6 DAY 7 MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN%+S.D .c 99.2 + 3.7 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 3 .1 + 0.7 97.6 + 8.5 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 3.4 + 0.6 98.3 6.3 100.0 f 0.0 100.0 + 0.0 100.0 + 0.0 3.5 + 0.7 47.7 + 47.6** [ 18) b 8 1 . 9 + 32.1** l ie)b 93.0 + 20.7 [ 18) b 100.0 + 0.0 [ 18) b 4.7 + 1.0** { 18) b DAY = DAY POSTPARTUM [ ) = NUMBER OF VALUES AVERAGED a. The average percentage of pups meeting the criterion for the developmental landmark tested on each day postpartum. b. Excludes values for dams that had no surviving pups. c. The average day postpartum that at least 50% of the pups had the developmental measure present. * Significantly different from the vehicle control group value (p^O.OS). ** Significantly different from the vehicle control group value (p^O.Ol). 0b 418-008:PAGE C-27 000614 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE C20 (PAGE 3) : REFLEX AND PHYSICAL DEVELOPMENT SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP I il III IV V MATERNAL DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0.1 0.4 1.6 3.2 EYE OPENING a N 23 25 22 18b 0b DAY 12 MEAN+S-D . 0.0 + 0.0 0.5 + 2.5 0.6 + 2.7 3.5 + 12.0 DAY 13 MEAN+S.D . 7.1 + 14.0 3.5 + 11.1 3.9 + 9.2 4.2 + 12.9 DAY 14 MEAN+S.D . 55.4 + 32.6 34.0 + 31.1* 32.0 + 30.6* 13.9 + 21.4** DAY 15 MEAN+S.D . 93.5 + 9.9 86.0 + 25.6 83.5 + 24.2 37.9 + 38.1** DAY 16 MEAN+S.D . 100.0 + 0.0 iOO.O _+ 0.0 99.4 + 3.0 74.8 + 33.9** DAY 17 MEAN+S.D . 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 98.6 + 5.9 DAY 18 MEAN+S.D. 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 MEAN%+lS.D.C 14.3 + 0.6 14.8 + 0.7 14.9 + 0.5** 15.7 + 1.3** DAY = DAY POSTPARTUM a. The average percentage of pups meeting the criterion for the developmental landmark tested on each day postpartum. b. Excludes values for dams that had no surviving pups. c. The average day postpartum that at least 50% of the pups had the developmental measure present. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p^O.Ol). 418-008:PAGE C-28 000615 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 C20 (PAGE 4): REFLEX AND PHYSICAL DEVELOPMENT - SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP I II III IV V MATERNAL DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0.1 0.4 1.6 3.2 ACOUSTIC STARTLE a N 23 25 22 18b 0b DAY 13 MEAN+S.D . 75.5 + 31.9 83.0 + 20.4 69.7 + 21.0 39.3 + 27.4** DAY 14 MEAN+S.D . 84.2 + 25.1 87.0 + 18.2 78.6 + 23.7 52.3 + 40.8* DAY 15 MEAN+S.D. 88.6 + 21.9 78.5 + 34.1 77.1 + 27.7 68.7 + 29.6 DAY 16 MEAN+S.D. 95.1 + 14.5 81.5 + 32.9 80.9 + 29.7 67.3 36.0** DAY 17 MEAN+S.D . 98.9 + 5.2 91.5 + 20.6 89.2 + 22.9 76.6 + 35.1* DAY 18 MEAN+S.D. 100.0 + 0.0 95.5 + 14 .4 96.0 + 11.8 81.9 + 36.2** DAY 19 MEAN+S.D. 100.0 + 0.0 99.0 + 5.0 98.3 + 8.0 84.5 + 32.1** DAY 20 MEAN+S.D. 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 94.4 + 16.2* DAY 21 MEAN+S.D . 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 97.2 + 11.8 MEAN%+S.D.C 13.3 + 0.6 13.1 + 0.3 13.0 + 0.2 14.0 + 1.4 DAY = DAY POSTPARTUM a. The average percentage of pups meeting the criterion for the developmental landmark tested on each day postpartum. b. Excludes values for dams that had no surviving pups. c. The average day postpartum that at least 50% of the pups had the developmental measure present. * Significantly different from the vehicle control group value (p0.05). ** Significantly different from the vehicle control group value (p<0.01). 418-008:PAGE C-29 9T9000 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 C20 (PAGE 5): REFLEX AND PHYSICAL DEVELOPMENT - SUMMARY - FI GENERATION LITTERS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY1 AIR RIGHTING a N DAY 14 M E A N + S .D . DAY 15 MEAN+S.D. DAY 16 MEAN+S.D. DAY 17 MEAN+S.D. DAY 18 MEAN+S.D. DAY 19 M E A N + S .D . DAY 20 MEANtS.D. DAY 21 MEANtS.D. MEAN1+S.D.C I 0 (VEHICLE) 23 23.9 + 27.7 4 .4 + 28.8 64.7 + 26.8 79.3 + 25.7 97.8 + 8.1 100.0 0.0 100.0 + 0.0 100.0 + 0.0 15 5 * 1 .2 II 0.1 25 13.5 + 14 .4 33.5 + 22.2 66.0 + 21.5 77.0 + 19.0 90.5 + 1S.0 99.0 + 3.5 100.0 + 0.0 100.0 + 0.0 15.9 + 0.8 III 0.4 22 13.0 + 15.8 35.5 + 23.0 46.8 + 26.3* 73.0 + 21.9 93.9 + 13.3 98.3 + 8.0 100.0 + 0.0 100.0 0.0 16.1 + 1.1 IV 1.6 18b 3.5 + 8.4** 3.5 + 5.8** 21.3 + 25.0** 40.8 + 31.5** 60.6 + 22.5** 75.2 + 30.1** 93.8 + 16.2** 93.8 + 16.2** 17.5 + 0.8** V 3.2 Ob PUPIL CONSTRICTION a N 23 25 22 18b DAY 21 M E A N + S .D . 100.0 0.0 100.0 + 0.0 100.0 + 0.0 100.0 + 0.0 DAY = DAY POSTPARTUM [ ) = NUMBER OF VALUES AVERAGED a. The average percentage of pups meeting the criterion for the developmental landmark tested on each day postpartum. b. Excludes values for dams that had no surviving pups. c. The average day postpartum that at least 501 of the pups had the developmental measure present. * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p<0.01). 0b 418-008:PAGE C-30 000617 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 C21 (PAGE 1): NECROPSY OBSERVATIONS - SUMMARY - FI GENERATION PUPS MATERNAL DOSAGE GROUP I II III IV MATERNAL DOSAGE (MG/KG/DAY) 0 (VEHICLE) 0 .1 0.4 1.6 LITTERS EXAMINED (N) 23 25 22 20 TOTAL PUPS STILLBORN OR FOUND DEAD a NB 6 8 71 STILLBORN N5 1 4 6 FOUND DEAD N 3 5 4 65 NO MILK IN STOMACH b N(%) 2( 66.7) 0( 0.0) 3 ( 75.0) 49( 75.4) HINDLIMBS AND/OR PORTION OF TAIL: MISSING c N (%) 0( 0.0) 0 ( 0.0) 0( 0.0) 0( 0.0) WHOLE BODY EDEMA N (%) 0( 0.0) 0< 0.0) 0< 0.0) 1( 1.4) PUPS SACRIFICED AND NECROPSIED ON DAYS 4 OR 21 POSTPARTUM a LITTERS EVALUATED N 23 25 22 18d PUPS EVALUATED N 259 305 209 161 APPEARED NORMAL LITTER INCIDENCE PUP INCIDENCE N (%) N (%) 23(100.0) 259(100.0) 25(100.0) 305(100.0) 20 ( 90.9) 206( 98.6) 18(100.0) 161(100.0) BRAIN: LATERAL VENTRICLES, MODERATE TO SEVERE DILATION LITTER INCIDENCE N (%) 0( 0.0) PUP INCIDENCE N (%) 0( 0.0) 0( 0.0) 0( 0.0) 1( 4.5) 2( 1.0) 0( 0.0) 0< 0.0) KIDNEYS: RIGHT, PELVIS, SLIGHT DILATION LITTER INCIDENCE N(%) 0( 0.0) PUP INCIDENCE N (%) 0( 0.0) 0( 0.0) 0( 0.0) K 4.5) K 0.5) 0( 0.0) 0( 0.0) a. Complete necropsies were not performed on pups in which autolysis or cannibalization precluded evaluation. b. Analysis restricted to pups found dead or moribund sacrificed and necropsied. c. Presumed cannibalized by dam. d. Excludes values for litters that had no surviving pups. V 3.2 21 172 41 131 102( 77.9) 9( 5.2) 0( 0.0) 0d 0 418-008: PAGE C-31 000618 APPENDIX D REPORT TABLES - F1 GENERATION MALE RATS 0 0 0 1 ,1 1 ) 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 D1 (PAGE 1): CLINICAL OBSERVATIONS - SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II 0 (VEHICLE) 0.1 111 0.4 MAXIMUM POSSIBLE INCIDENCE 2207/ 25 2196/ 25 2215/ 25 FOUND DEAD 2a ,b 1c 0 INCISORS; TOTAL MISALIGNED MISSING/BROKEN 96/ 3 87/ 2 13/ 2 152/ a 108/ 4 76/ 7 97/ 4 94/ 3 17/ 2 CHROMORHINORRHEA 7/ lb 9/ 4 6/ 4 ABRASION d 0/ 0 61/ 4 32/ 2 CHROMODACRYORRHEA 58/ 1 50/ 4 38/ 1 BASE OF TAIL; LACERATION 0/ 0 0/ 0 11/ 1 SNOUT; SWOLLEN 0/ 0 8/ 2 3/ 1 RED PERIORAL SUBSTANCE 0/ 0 1/ 1 1/ 1 PENIS; RED SUBSTANCE 0/ 0 0/ 0 2/ 1 LOCALIZED ALOPECIA: TOTAL BACK LIMBS HEAD 6/ 1 0/ 0 6/ 1 0/ 0 66/ 4 16/ 3** 29/ 1 24/ 1 0/ 0 0/ 0 0/ 0 0/ 0 BACK: SCAB 14/ 1 8/ 2 0/ 0 URINE-STAINED ABDOMINAL FUR 0/ 0 4/ 1 0/ 0 SOFT OR LIQUID FECES 0/ 0 1/ 1 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS x RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION. a. Rat 13105 was found dead on day 87 postweaning. b. Rat 13116 was found dead on day 86 postweaning. c. Rat 13138 was found dead on day 66 postweaning. d. Located on the head,corner of left eye, left forelimb or back. ** Significantly different from the vehicle control group value (p<0.01). 418-008: PAGE D-1 000620 PROTOCOL 418 008: COMBINED ORAL IGAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS I.SPONSOR'S STUDY NUMBER: 6295.9) TABLE D1 (PAGE 2): CLINICAL OBSERVATIONS - SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II 0 (VEHICLE) 0.1 III 0.4 MAXIMUM POSSIBLE INCIDENCE 2207/ 25 2196/ 25 2215/ 25 FOUND DEAD 2a,b lc 0 EARS: SWOLLEN AND PURPLE 9/ 1 0/ 0 0/ 0 RALES 2/ 1 0/ 0 0/ 0 GASPING 1/ 1 0/ 0 0/ 0 EXCESS SALIVATION 1/ 1 0/ 0 0/ 0 EYES: DILATED PUPIL 1/ 1 0/ 0 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS X RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION. a. Rat 13105 was found dead on day 87 postweaning. b. Rat 13116 was found dead on day 86 postweaning. c. Rat 13138 was found dead on day 66 postweaning. 418-008:PAGE D-2 000621 PROTOCOL 418 008: COMBINED ORAL (GAVAGEI FERTILITY, DEVELOPMENTAL AND PERI NATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY'NUMBER: 629S.9) TABLE D2 (PAGE 1): BODY WEIGHTS - SUMMARY FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 ! TESTED N 25 25 25 ' WEIGHT (G) DAY 1 DAY 8 MEAN+S.D. MEAN+S.D. 54.0 5.8 101 .6 + 10.3 52.1 + 98.2 + 5.3 8.1 50.8 + 4.9 I 23 )a 96.4 + 8.5 DAY 15 MEAN+S.D. 161.8 + 13.6 156.2 + 11.6 154.5 + 11.8 DAY 22 MEAN+S.D. 223.7 + 17.1 219.6 _ 14.0 216.6 15.4 DAY 29 MEAN+S.D. 285.0 + 21.1 279.4 + 17.2 277.2 + 21.0 DAY 36 MEAN+S.D. 338.2 + 25.6 334.7 + 20.9 332.4 + 22.8 DAY 43 MEAN+S.D. 381 .1 30.3 377.9 t 21.6 373.3 26 -2 DAY 50 MEAN+S.D. 420.5 + 33.4 414.0 + 25.3 410.6 + 32.9 DAY 57 MEAN+S.D . 448.4 34 .2 437.0 t 29.5 436 .B + 37.4 DAY 64 MEAN+S.D. 472.2 36.9 465.1 + 34.3 462.6 44.0 PRECOHABITATION b TERMINAL BODY WEIGHT d MEAN+S.D. MEAN+S.D. 508.6 42.8 542.0 + 50.9 1 23]1c 499.2 t 39.0 ( 24]1c 533.0 + 43.6 l 24]lc 494.8 51. 1 522.3 _+ 54.6 DAY = DAY POSTWEANING ( ) = NUMBER OF VALUES AVERAGED a. Excludes values that were not recorded. b. Precohabitation body weights were recorded on the day that cohabitation began for the FI generation rats; at that time these rats were 85 to 97 days of age. c. Excludes values for rats that were found dead. d. Terminal body weights were recorded when the rats were 100 to 112 days of age. 418-008.PAGE D-3 000622 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE D3 (PAGE 1) : BODY WEIGHT CHANGES SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) 11 0.1 III 0.4 RATS TESTED N 25 25 25 BODY WEIGHT CHANGE (G) DAYS 1 6 DAYS 6 IS MEAN*S.D. MEAN+S.D. *47.7 5.5 60.2 6.8 *46.1 + 3.9 *58.0 + 5.3 +46.5 4.8 1 231a +58.1 4.9 DAYS IS 22 MEAN+S.D . 61.8 6.2 63.5 4.8 +62.2 + 5.7 DAYS 22 - 29 MEAN*S.D. *61.4 6.5 *59.7 + 4.7 +60.6 * 8.0 DAYS 29 - 36 MEANS .D . .53.2 10.8 .55.3 9.2 +55.2 + 5.0 DAYS 36 - 43 MEAhNS.D. .42.9 8.5 43.2 7.3 +40.9 + 8.9 DAYS 43 SO M E A N + S .0. *39.4 * 6.5 *36.1 + 10.9 +37.3 10.2 DAYS 50 S7 DAYS 57 64 M E A W * S ,D. MEAN+S.D . *27.8 i 8.7 .23.9 i 8.6 *23.0 * 22.9 +28.2 + 24.9 +26.3 7.2 +25.8 13.9 DAY 1 PRECOHABITATION b DAY 1 TERMINATION d MEAN+S.D . MEAN+S.D. *454.6 * 40.7 *486.0 48.6 i 231c +447.1 + 36.5 { 24]c 480.9 + 41.2 l 24 )c +443.5 + 51.4 ( 2311a +471.2 55.3 1 2311a DAY(S) = DAY(S) POSTWEANING l ) = NUMBER OF VALUES AVERAGED a. Excludes values Chat were not recorded. b. Precohabitation body weights were recorded on the day that cohabitation began for the FI generation rats; at that time these rats were 8S to 97 days of age. c. Excludes values for rats that were found dead. d. Terminal body weights were recorded when the rata were 79 to 91 days of age. 418-008:PAGE D-4 000623 PROTOCOL 418-OOB: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE D4 (PAGE 1): ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 RATS TESTED N 25 25 FEED CONSUMPTION (G/DAY) DAYS 1 - 8 MEAN+S .D . 14.7 + 1 .5 1 3 .a + 1.3* DAYS 8 - 15 MEAN+S.D. 21.7 + 1 .5 22.1 2.3 DAYS 15 - 22 DAYS 22 - 29 MEAN+S.D. MEAN+S.D . 26.0 + 2.2 f 24 1a 28.8 + 2.0 25.8 + 1 .4 29.2 1.6 DAYS 29 - 36 MEAN+S.D. 30.3 + 3.5 30.0 + 3.1 DAYS 36 - 43 MEAN+S.D . 32.2 + 2.8 32.6 + 2.9 DAYS 43 - so DAYS 50 - 57 MEAN+S.D. MEAN+S.D . 32.4 + 3 .4 ( 24 )a( 32.3 2.8 33.0 + 3.3 l 24] a 31.7 + 2.2 DAYS 57 - 64b DAYS 1 - 64b MEAN+S.D . MEAN+S.D. 3 0 . 5 + 2.7 1 22) a 27.3 t 1.7 I 22] ai 30.2 + 4 .1 27.6 1.5 DAYS = DAYS POSTWEANING l ) = NUMBER OF VALUES AVERAGED a. Spilled feed precluded the calculation of this value. b. Last value recorded before cohabitation. * Significantly different from the vehicle control group value ** Significantly different from the vehicle control group value (p<0.05). (p<0.01). m 0.4 25 13.4 + 2.9** 20.8 3.2 25.4 + 1.7 28.5 + 2.3 29.9 + 2.3 31.7 + 2.6 31.7 + 3 .1 31.5 3.1 30.9 + 3.2 27.1 + 1.9 418-008:PAGE D-5 000624 PROTOCOL 418 008: COMBINED 0RA1, (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RAIS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE DS (PAGE 1): RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 ! TESTED N 25 25 > CONSUMPTION (G/KG/DAY) DAYS 1 - 8 MEAN+S.D. 189.9 17.6 184.9 17.8 DAYS 8 - 15 MEAN+S .D . 165.5 + 11.0 174.5 + 16.3 DAYS 15 - 22 DAYS 22 - 29 MEAN+S.D . MEAN+S.D. 135.6 + 7.2 I 24) b 113.5 + 5.4 137.6 7.1 117.1 + 4.6* DAYS 29 - 36 MEAN+S.D. 97.3 + 0.9 97.7 + 8.1 DAYS 36 - 43 MEAN+S.D. 89.6 + 6.2 91.4 + 6.9 DAYS 43 - 50 DAYS 50 - 57 MEAN+S.D . MEAN+S.D. 80.7 + 5.5 1 24]lb 74.5 + 4.6 83.4 + 7.6 l 24]lb 74.7 + 4.1 DAYS 57 - 64c DAYS 1 - 64c MEANS.D . MEAN+S.D. 67.2 + 4 .7 l 22 lb 96.2 + 4.1 1 22 lb 67.1 +_ 8.4 97.4 + 3.8 DAYS DAYS POSTWEA1IING | ) = NUMBER OF VALUES AVERAGED a. Excludes values that were not recorded. b. Spilled feed precluded the calculation of this value. c. Last value recorded before cohabitation. * Significantly different from the vehicle control group value (p<0,05). III 0.4 25 183.6 +38.1 [ 231a 166.4 + 23.9 137.3 + 7.6 115.3 + 4.8 98.1 + 4.2 90.0 + 6.6 80.9 + 6.0 74.2 + 3.8 68.6 + 3.8 96.6 + 3.6 [ 23 |b 418-008:PAGE D-6 000625 PROTOCOL 4 18 008 : COMBINED ORAL (CAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295 9) TABLE D6 (PAGE 1) SEXUAL MATURATION - SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAG E (MG/KG/DAY) I 0 (VEHICLE) 11 0.1 MALE RATS N 25 25 PREPUTIAL SEPARATION a MEAN+S.D. 45.0 + 2.1 45.7 + 2.3 a. Average day postpartum that the prepuce was observed to be separated. III 0.4 25 45.1 1.8 418-008:PAGE D-7 929000 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE D7 (PAGE 1): PASSIVE AVOIDANCE PERFORMANCE SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0 .1 III 0.4 MALE RATS SESSION la N 23 25 22 TRIALS TO CRITERION LATENCY TRIAL lb LATENCY TRIAL 2b FAILED TO LEARN C MEANtS .D. MEAN *S .D . MEAN +S -D . N (%) 4.8 8.9 21.4 0( + 1.0 + 5.0 15.8 0 .0) 6.1 7.8 20.1 1( 2.9 4.8 f 19.7 4 .0) 4.8 10.5 23.9 0( + 1 .1 + .0 + 20.7 0 .0) SESSION 2a N 23 24 22 TRIALS TO CRITERION LATENCY TRIAL lb MEANtS.D. MEANtS.D. 3 .3 + 2.6 32.8 + 22.2 3.6 + 2.2 28.2 23.5 3.5 t 2.7 33.1 t 22.4 a. Sessions 1 (Learning Phase) and 2 (Retention Phase) of testing were separated by a one-week interval. b. The latency was recorded in seconds. c. Number of rats that did not meet the criterion in Session 1 (Learning Phase). 418-008:PAGE D-8 000627 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE D8 (PAGE 1): WATERMAZE PERFORMANCE - SUMMARY FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/OAY) I 0 (VEHICLE) II 0.1 III 0.4 MALE RATS SESSION la N 23 25 21 TRIALS TO CRITERION ERRORS PER TRIAL LATENCY TRIAL 2b FAILED TO LEARN c MEAN S . D . MEAN + S . D . MEAN + S . D . N <t) 9.1 0.37 11.7 0( 3.1 + 0.17 + 7.4 0 . 0) 9.4 * 3.0 0.(0 t 0.24 13.7 1 3 . 9 2( 8.0) 9 0 + 3.0 0.43 + 0.22 10.6 + 6.0 0( 0.0) SESSION 2a N 23 23 21 TRIALS TO CRITERION ERRORS PER TRIAL LATENCY TRIAL lb MEAN+S.D. MEAN+S.D. MEAN+S.D. 6.4 + 2.6 0.12+ 0.20 10.3 8.3 6.3 + 1.9 0.08+ 0.11 9.1 + 6.5 5.5 + 0.9 0.09 + 0.15 10.7 + 6.7 a. Sessions 1 (Learning Phase) and 2 (Retention Phase) of testing were separated by a one-week interval. b. The latency was recorded in seconds. c. Number of rats that did not meet the criterion in Session 1 (Learning Phase); Session 2 (Retention Phase) values for these rats were excluded from group averages and statistical analyses. 418-008:PAGE D-9 000628 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 D9 (PAGE 1): MATING AND FERTILITY SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS IN COHABITATION N 23a 24b 25 DAYS IN COHABITATION c MEANtS.D 3 . 2 + 2.1 3.4 +_ 1.9 2.6 + 1.5 RATS THAT MATED d N (% ) 20( 87.0) 22( 91.7) 25(100.0) FERTILITY INDEX e,f N/N <%) 19/20 ( 95.0) 20/22 ( 90.9) 24/25 ( 96.0) RATS WITH CONFIRMED MATING DATES g N 20 22 25 RATS MATING h DAYS 17 Nit) 20(100.0) 22( 92.0) 25(100.0) RATS PREGNANT/RATS IN COHABITATION g N/N (t) 19/23 ( 82.6) 20/24 ( 83.3) 24/25 ( 96.0) a. Excludes values for rats that were not assigned to cohabitation because therewere no available female rats. b. Excludes values for rat 13138, which was Found dead day 66 postweaning. c. Restricted to rats with a confirmed mating date on days 1 to 7 of cohabitation and rats that did not mate. d. Includes only one mating for each male rat. e. Number of pregnancies/number of rats that mated. f. Includes only one pregnancy for each rat that impregnated more than one female rat. g. Includes only one confirmed mating for each male rat. h. Restricted to rats with a confirmed mating date during days 1 to 7 of cohabitation. 418-008: PAGE D-10 000629 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE DIO (PAGE 1): NECROPSY OBSERVATIONS - SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAG E 1MG/KG/DAY) RATS EXAMINED a N I 0 (VEHICLE) 25 II 0.1 25 Ill 0.4 25 FOUND DEAD N 2b, c Id 0 APPEARED NORMAL N 23 21d 23 ABDOMINAL ADIPOSE TISSUE RED WITH FIRM AREAS N 0 0 1 KIDNEYS: BILATERAL, LARGE N 0 0 1 PARENCHYMA, NUMEROUS FLUID-FILLED CYSTS N 0 0 1 RIGHT OR LEFT, PELVIS, SLIGHT DILATION N 0 2 0 LEFT, PELVIS, SIX CALCULI N 0 1 0 TESTES; BILATERAL OR RIGHT, PURPLE AND/OR FLACCID N 0 3** 0 EPIDIDYMIDES: BILATERAL OR RIGHT, FLACCID N 0 3** 0 LEFT OR RIGHT, SMALL N 0 2 0 URETERS: LEFT, DILATED N 0 1 0 a. Refer to the individual clinical observations table (Table D13) for external observations confirmed at necropsy. b. Rat 13105 was found dead on day 87 postweaning. c. Rat 13116 was found dead on day 86 postweaning. d. Rat 13138 was found dead on day 66 postweaning. ** Significantly different from the vehicle control group value (p< 0.01) . 418-008.PAGE D-11 000G30 PROTOCOL 418-008: COMBINER ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RAIS (SPONSOR'S STUDY NUMBER: 6295 9) TABLE DIO (PAGE 2): NECROPSY OBSERVATIONS SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS EXAMINED a N 25 25 25 FOUND DEAD N 2b,c Id 0 URINARY BLADDER; WALLS, THICKENED AND NUMEROUS CALCULI N 0 1 0 THORACIC CAVITY: DARK RED CLOTTED MATERIAL N lc 0 0 LUNGS: ALL LOBES, DARK RED N lb 0 0 EXTERNAL OBSERVATIONS: RED PERINASAL SUBSTANCE N lc 0 0 a. Refer to the individual clinical observations table (Table D13) for external observations confirmed at necropsy. b. Rat 13105 was found dead on day 87 postweaning. c. Rat 13116 was found dead on day 86 postweaning. d. Rat 13138 was found dead on day 66 postweaning. 418-008:PAGE D-12 TC9000 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 Dll (PAGE 1): TERMINAL BODY WEIGHTS AND ORGAN WEIGHTS SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY)a 1 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a 24a 25 TERMINAL BODY WEIGHT EPIDIDYMIS LEFT TESTIS LEFT SEMINAL VESICLES WITH FLUID SEMINAL VESICLES WITHOUT FLUID EPIDIDYMIS RIGHT TESTIS RIGHT PROSTATE MEAN*S.D . M E A N + S .D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. 542.0 + 50.9 0.67 0.07 1.77 + 0.16 1.75 + 0 .33 1.04 + 0.17 0.67 + 0.06 1.78 + 0.18 0.93 + 0.19 5 3 3 . 0 + 43.6 0.65 + 0.06 l 23 ]b 1 . 8 0 + 0.16 t 231b 1 . 7 2 + 0.24 I 22) b 1.05 + 0.19 0 . 6 6 + 0.05 I 21) b 1.79 + 0.15 l 21]b 0.91 + 0.22 522.3 + 54.6 0.63 + 0.04 1.79 + 0.12 1.62 + 0.20 l 24) b 0.96 + 0.15 0.64 + 0.04 1.82 + 0.12 0.89 + 0.19 ALL WEIGHTS WERE RECORDED IN GRAMS (G). ( ) = NUMBER OF VALUES AVERAGED a. Excludes values for rats that were found dead. b. Excludes values for rats that had abnormal organs (weight affected) or organs damaged (weight affected) . 418-008:PAGE D-13 000632 PROTOCOL 418-000: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE D12 (PAGE 1): RATIOS (%) OF ORGAN WEIGHT TO TERMINAL BODY WEIGHT SUMMARY - FI GENERATION MALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY)a RATS TESTED N I 0 (VEHICLE) 25 II 0.1 25 III 0.4 25 INCLUDED IN ANALYSES N 23a 24a 25 TERMINAL BODY WEIGHT MEAN+S.D. 542.0 + 50.9 533.0 + 43.6 522.3 + 54.6 EPIDIDYMIS LEFT TESTIS LEFT SEMINAL VESICLES WITH FLUID SEMINAL VESICLES WITHOUT FLUID MEAN+S.D. MEAN+S.D . MEAN+S.D. MEAN+S.D . 0.124 + 0.017 0.329 + 0.041 0.325 + 0.068 0.193 t 0.037 0.122 + 0.014 ( 231b 0.332 0.043 l 23 )b 0.325 0.055 l 221b 0.198 + 0.037 0.121 0.014 0.346 + 0.037 0.316 + 0.055 ( 241b 0.184 + 0.032 EPIDIDYMIS RIGHT TESTIS RIGHT PROSTATE MEAN+S.D. MEAN+S.D . MEAN+S.D . 0.124 + 0.016 0.331 + 0.043 0.172 + 0.035 0.125 + 0.012 l 211b 0.339 + 0.042 ( 21Jb 0.171 0.043 0.122 + 0.015 0.351 + 0.037 0.170 t 0.035 ALL WEIGHTS WERE RECORDED IN GRAMS (G) I ) = NUMBER OF VALUES AVERAGED a. Excludes values for rats that were found dead. b. Excludes values for rats that had abnormal organs (weight affected) RATIOS (%) = (ORGAN WEIGHT/TERMINAL BODY WEIGHT) or organs damaged (weight affected). X 100. 418-008:PAGE D-14 000633 APPENDIX E REPORT TABLES - F1 GENERATION FEMALE RATS 000634 PROTOCOL 4 18 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE El (PAGE 1): CLINICAL OBSERVATIONS SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II 0 (VEHICLE) 0.1 hi 0.4 PRECOHABITATION (DAY 1 OF STUDY TO THE DAY OF COHABITATION): MAXIMUM POSSIBLE INCIDENCE 1721/ 25 1B44/ 25 1840/ 25 SACRIFICED FOUND DEAD la 0 lb 0 0 0 INCISORS: TOTAL MISALIGNED MISSING/BROKEN 29/ 3 29/ 3 14/ 2 32/ 4 9/ 1 23/ 3 77/ 6 52/ 4 25/ 2 CHROMODACRYORRHEA 6/ 1 9/ 1 19/ 3 EARS: SWOLLEN AND PURPLE LOCALIZED ALOPECIA: TOTAL BACK HEAD LIMBS 8/ 2 13/ 2 0/ 0 0/ 0 13/ 2 4/ 2 24/ 2 22/ 2 2/ 1 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 BACK: ABRASION 0/ 0 10/ 1 0/ 0 BACK: SCAB 0/ 0 1/ 1 0/ 0 URINE-STAINED ABDOMINAL FUR 10/ 2 0/ 0 0/ 0 LEFT HINDPAW: THIRD AND FOURTH DIGITS MISSING 74/ 1 0/ 0 0/ 0 PERIVAGINAL SWELLING 6/ 1 0/ 0 0/ 0 LEFT FOREPAW: SWOLLEN 1/ 1 0/ 0 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS x RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION. a. Rat 13205 was excluded from study on day 9 postweaning; the sex was incorrectly identified. b. Rat 13221 was found dead on day 28 postweaning. 418-008:PAGE E-1 000635 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE El (PAGE 2) ; CLINICAL OBSERVATIONS SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I II 0 (VEHICLE) 0.1 III 0.4 PRESUMED GESTATION: a MAXIMUM POSSIBLE INCIDENCE 509/ 23 539/ 24 54B/ 25 MORTALITY 000 INCISORS: TOTAL MISALIGNED MISSING/BROKEN 66/ 4 62/ 4 21/ 2 90/ 5 77/ 5 41/ 3 142/ 8 123/ 6 22/ 3 CHROMODACRYORRHEA 10/ 2 26/ 1 40/ 3 EARS: SWOLLEN AND PURPLE 44/ 2 75/ 4 23/ 2 RED PERIVAGINAL SUBSTANCE LOCALIZED ALOPECIA: TOTAL LIMBS HEAD UNDERSIDE 0/ 0 61/ 3 61/ 3 0/ 0 15/ 1 1/ i 32/ 3 27/ 2 5/ 1 0/ 0 2/ 2 21/ 2 15/ i 6/ 1 0/ 0 URINE-STAINED ABDOMINAL FUR 0/ 0 0/ 0 1/ 1 CHROMORHINORRHEA 14/ 2 1/ 1 0/ 0 SOFT OR LIQUID FECES 0/ 0 1/ 1 0/ 0 LEFT HINDPAW: THIRD AND FOURTH DIGITS MISSING 23/ 1 0/ 0 0/ 0 TAIL: SHEATH MISSING 12/ 1 0/ 0 0/ 0 TIP OF TAIL BLACK 12/ 1 0/ 0 0/ 0 TIP OF TAIL MISSING 10/ 1 0/ 0 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS x RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION, a. Restricted to rats with a confirmed mating date. 418-008:PAGE E-2 000636 PROTOCOL 418008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONOR'S STUDY NUMBER: 6295 9) TABLE El (PAGE 3): CLINICAL OBSERVAIIONS - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) LACTATION: I 11 0 (VEHICLE) 0.1 III 0.4 MAXIMUM POSSIBLE INCIDENCE FOUND DEAD INCISORS: TOTAL MISALIGNED MISSING/BROKEN RED PERIVAGINAL SUBSTANCE EARS: SWOLLEN AND PURPLE CHROMODACRYORRHEA LOCALIZED ALOPECIA; TOTAL LIMBS UNDERSIDE 462/ 22 441/ 21 493/ 24 0 0 la 65/ 4 44/ 3 42/ 2 43/ 3 42/ 2 34/ 3 116/ 6 116/ 6 18/ 3 0/ 0 0/ 0 5/ 5* * 45/ 3 84/ 4 42/ 2 21/ 1 7/ i 37/ 2 63/ 3 63/ 3 21/ i 42/ 2 42/ 2 0/ 0 21/ 1 21/ 1 13/ i CHROMORHINORRHEA CHEST; ABRASION TIP OF TAIL MISSING LEFT HINDPAW: THIRD AND FOURTH DIGITS MISSING URINE-STAINED ABDOMINAL FUR 6/ i 0/ 0 21/ 1 21/ 1 1/ 1 2/ 1 9/ 1 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 STATISTICAL ANALYSES OF CLINICAL OBSERVATION DATA WERE RESTRICTED TO THE NUMBER OF RATS WITH OBSERVATIONS. MAXIMUM POSSIBLE INCIDENCE = (DAYS X RATS)/NUMBER OF RATS EXAMINED PER GROUP. N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION, a. Dam 13257 was found dead on day 10 of lactation. ** Significantly different from the vehicle control group value (p<0.01). 418-008:PAGE E-3 000637 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 E2 (PAGE 1): BODY WEIGHTS PRECOHABITATION - SUMMARY FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0 .1 III 0.4 RATS TESTED INCLUDED IN ANALYSES N N 25 24a 25 25 25 25 BODY HEIGHT (G) DAY 1 MEAN+S.D . 51.1 + 6.1 50.5 + 4.2 48.7 + 5.1 DAY 8 DAY 15 DAY 22 MEAN*S.D . MEANtS .D . MEAN+S.D . 91.2 + 9.3 134.5 + 12.5 168.2 + 16.0 91.8 + 5.2 138.0 + 7.6 173.4 + 9.7 91.2 + 7.6 134.5 + 9.9 168.5 + 12.4 DAY 29 DAY 36 DAY 43 DAY 50 DAY 57 DAY 64 PRECOHABITATION c M E A N + S .D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D . MEAN+S.D. MEAN+S.D. 194.0 + 21.7 ( 23 )b 217.4 + 24.9 ( 23) b 234.9 + 25.2 [ 23) b 251.1 + 27.4 l 23) b 259.6 + 29.0 ( 23) b 273.2 + 31.4 l 23) b 262.6 + 33.6 1 23 1b 200.5 + 13.1 226.8 + 15.2 245.1 + 14.6 261.4 + 20.0 272.7 + 20.7 282.7 + 19.6 294.0 + 16.3 193.9 + 16.6 217.8 t 17.4 237.9 + 20.0 251.2 + 20.9 262.7 + 24.4 272.3 + 25.8 280.3 + 26.9 DAY = DAY POSTWEANING ( 1 = NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead day 28 postweaning. c. Precohabitation body weights were recorded on the day that cohabitation began for the FI generation rats; at that time these rats were 85 to 97 days of age. 418-008:PAGE E-4 000638 PROTOCOL 418-008: COMBINED ORAL (GAVAGEI FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE E3 (PAGE I): BODY' WEIGHT CHANGES - PRECOHABITATION SUMMARY FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) RATS TESTED INCLUDED IN ANALYSES N N I 0 (VEHICLE) 25 24a II 0.1 25 25 III 0.4 25 25 BODY WEIGHT CHANGE (G) DAYS 1 - 8 MEAN+S.D. +40.0 + 4.2 +41.4 + 2.9 +42.5 + 5.0 DAYS 8 - 15 MEANj+S .D . +43.3 + 6.3 +46.1 4.2 +43.3 + 4.5 DAYS 15 - 22 MEAN+S.D . 33.8 + 7.0 +35.5 + 5.2 34.0 + 6.8 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 43 DAYS 4 3 - 5 0 DAYS 50 - 57 DAYS 57 - 64 DAYS 1 - 64 DAY 1 PRECOHABITATION C MEAN+S.D . MEAN+S.D . MEAN+S.D . MEAN+S.D . MEAN+S.D . MEAN+S.D . MEAN+S.D. MEAN+S.D . +25.8 + 12.8 l 23]a,b +23.4 + 10.4 ( 23]a ,b +17.5 + 9.2 23 Ja ,b 16.2 + 6.6 1 23)a ,b 8.5 + 6.8 1 23]a ,b +13.6 + 7.6 t 23]a ,b +222.3 + 28.9 ( 23]a,b +231.9 + 30.7 ( 23]a ,b +27.0 + 5.8 +26.3 + 5.8 +18.3 + 5.1 +16.3 + 8.0 +11.3 + 9.3 +10.0 + 9.4 +232.2 + 20.3 +243.6 + 17.3 +25.4 * 8.7 +23.8 + 5.7 +20.1 + 7.4 13.3 + 5.9 +11.6 6.9 +9.6 + 7.8 +223.6 + 24.0 +231.6 25.1 DAY(S) = DAY(S) POSTWEANING ( ) NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 poatweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead day 28 postweaning. c. Precohabitation body weights were recorded on the day that cohabitation began for the FI generation rats; at that time these rats were 85 to 97 days of age. 418-008: PAGE E-5 000639 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 E4 (PAGE 1): MATERNAL BODY WEIGHTS GESTATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a ,b 25 25 PREGNANT N 22 21 24 MATERNAL BODY WEIGHT (G) DAY 0 MEAN*S.D . 284.4 i o .a 295.3 + 17.3 279.9 24.8 DAY 1 MEAN*S.D . 291 .4 + 31 .1 302.8 + 18.9 288.1 + 25.4 DAY 2 MEAN+S.D. 298.1 + 30.5 308.1 + 19.3 294.5 + 24.8 DAY 3 MEAN+_S .D. 303.1 + 31.0 312.8 * 19.3 297.5 25.9 DAY 4 MEANtS.D . 305.5 31 .1 315.3 19.1 300.2 *- 25.3 DAY 5 MEAN*S.D. 309.2 31.2 318.7 + 19.2 304.4 26.5 DAY 6 MEAN+S.D. 313.2 + 32.7 321.6 + 18.7 306.9 + 25.1 DAY 7 MEANtS .D . 317.4 + 33.2 322.8 + 19.9 309.0 * 26.0 DAY 8 MEAN+S.D. 321.6 + 32.8 328.0 + 19.9 313.2 f 27.4 DAY 9 MEAN t^S,D . 326.2 + 33.0 331.5 19.5 317.7 + 26.1 DAY 10 MEAN+S.D. 331.7 + 33.6 336.5 + 20.1 322.3 28.2 DAY = DAY OF GESTATION a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead on day 28 postweaning. 418-008:PAGE E-6 000640 PROTOCOL 118-008; COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PROS IN RATS (SPONSOR'S STUDY NUMBER; 6295.9) TABLE E4 (PAGE 2) ; MATERNAL BODY WEIGHTS - GESTATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a, b 25 25 PREGNANT N 22 21 24 MATERNAL BODY WEIGHT (G) DAY 11 MEAN+S.D. 337.0 32.3 341.0 + 21.5 329.0 27.1 DAY 12 DAY 13 MEAN*S.D. MEAN_S.D. 342.4 34.7 345.6 33.8 347.2 + 22.9 350.3 t 21.0 331.8 + 26.9 336.4 28.7 DAY 14 DAY 15 DAY 16 MEANS .D . MEAN*S.D. MEAN*S.D. 351 .9 i 34 .9 358.9 35.0 366.2 4 35.0 354.7 + 21.1 364.0 t 22.3 373.5 22.4 342.3 * 27.3 347.9 t 29.5 358.6 + 29.6 DAY 17 MEAN^S.D . 379.4 4 34 .4 385.8 * 25.3 372.3 + 30.2 DAY 18 MEAN^S.D. 394.6 4 32.8 401.6 + 27.4 388.1 _t 32.1 DAY 19 MEAN_S.D. 408.3 4 35.6 414.7 + 27.7 402.8 t 32.0 DAY 20 MEANS .D . 420.8 4 34 .4 428.0 t 30.3 417.7 + 34.4 DAY = DAY OF GESTATION a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning ; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead on day 28 postweaning. 418-008:PAGE E-7 000641 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 ES (PAGE 1) : MATERNAL BODY WEIGHT CHANGES - GESTATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 hi 0.4 RATS TESTED N 25 2S 25 INCLUDED IN ANALYSES N 23a, b 25 25 PREGNANT N 22 21 24 MATERNAL BODY WEIGHT CHANGE (G) DAYS 0 - 7 MEANtS .D . 33.0 + 6.9 +27.4 + 9.0 +29.2 + 7.1 DAYS 7 - 10 MEAN+S.D. +14.3 + 5.0 +13.8 + 6.9 +13.2 + 5.2 DAYS 10 - 14 MEAN+S.D. +20.2 + 5.9 +18.1 + 9.9 +20.0 + 6.2 DAYS 14 - 17 MEAN+S.D. +27.5 + 1 3 . 7 +31.2 + 9.4 +30.0 + 8.6 DAYS 17 - 20 MEANtS .D . +41.4 + 1 1 . 0 42.2 + 9.3 +45.4 + 9.0 DAYS 0 - 20 MEAN+S.D. +136.4 16.0 +132.7 + 22.7 +137.8 16.4 DAYS = DAYS OF GESTATION a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead on day 28 postweaning. 418-008:PAGE E-8 000642 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 E6 (PAGE 1): MATERNAL BODY WEIGHTS - LACTATION - SUMMARY FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a,b 25 25 PREGNANT N 22 21 24 DELIVERED LITTERS N 22 21 24 MATERNAL BODY WEIGHT (G) DAY 1 MEAN+S.D . 325.7 + 35.6 334.7 + 16.9 323.4 28.4 DAY 4 MEAN*S .D . 326.0 * 33.0 337.5 + 20.9 318.8 + 25.8 DAY 1 MEANtS.D. 331.5 i 31.2 340.2 t 19.1 324.2 + 23.2 DAY 14 DAY 21 MEANtS.D. MEAN+SD . 358.9 29.2 338.0 + 31.0 368.3 t 21.8 356.0 + 18.4 352.4 + 23.6 I 23) c 341.3 + 17.8 I 23 )c DAY DAY OF LACTATION 1 ) = NUMBER OF VALUES AVERAGED a . Excludes values for rat 13205, which was excluded from study on day 7 poatweaning; the sex was incorrectly identified, b. Excludes values for rat 13221, which was found dead on day 28 poatweaning. c . Excludes values for dam 13257, which was found dead on day 10 of lactation. 418-008:PAGE E-9 000643 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PF0S IN RATS (SPONSOR'S STUDY NUMBER: 6295 .9) TABLE E7 (PAGE 1): MATERNAL BODY WEIGHT CHANGES - LACTATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a,b 25 25 PREGNANT N 22 21 24 DELIVERED LITTERS N 22 21 24 MATERNAL BODY WEIGHT CHANGE (G) DAYS 1 - 4 MEAN+S.D . 2.3 + 13.6 *2 .B + 10.7 -4.6 8.4* DAYS 4 - 7 MEAN+S.D . 3.6 11.8 +2.7 + 10.7 +5.4 + 8.2 DAYS 7 - 14 DAYS 14 - 21 DAYS 1 - 21 MEAN+S.D . MEAN+S.D. MEAN+S.D . 27.3 _ 12.8 -20.9 + 17.1 12.3 + 19.1 28.0 + 15.6 -12.3 12.6 +21.3 + 15.3 +30.1 + 10.4 ( 23Jc -11.0 + 17.0 I 23 1C +21.2 + 18.7 1 231c DAYS = DAYS OF LACTATION 1 ) = NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead on day 28 postweaning. c. Excludes values for dam 13257, which was found dead on day 10 of lactation. * Significantly different from the vehicle control group value (p<0.05). 418-008: PAGE E-10 000644 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 E8 (PAGE 1): ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) - PRECOHABITATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 24a 25 25 FEED CONSUMPTION (G/DAY) DAYS 1 - 8 MEAN_*S.D. 1 3 . 6 + 1.3 13.7 + 1.2 12.6 + 1.7* DAYS 8 - 15 DAYS 15 - 22 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 43 DAYS 43 - 50 DAYS 50 - 57 DAYS 57 - 64d DAYS 1 - 64d MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. 1 8 . 9 + 1.4 ( 23) b 20.4 + 2.3 l 23 )b 2 0 . 5 + 2.8 [ 23 )c 2 0 . 5 + 2.7 l 23)c 22.4 + 3.8 ( 22)b ,c 22.9 + 3.2 [ 22]b,c 2 3 . 2 + 3.7 ( 23 ]C 2 1 . 5 + 2.3 [ 23) c 20.4 + 1.8 [ 23 )C 19.1 + 1.5 21.2 + 1.6 21.4 + 2.1 21.4 + 2.4 24.4 + 4.0 23.8 + 3.5 ( 24] b 23.5 + 3.0 21.9 + 2.0 l 24 ]b 21.2 + 1.7 1 24 )b 18.9 + 2.4 20.8 + 1.9 1 24]b 20.8 + 2.0 20.7 + 2.1 I 24 Jb 23.6 + 3.9 23.6 + 3.9 [ 24 ]b 22.3 + 2.9 21.5 + 3.0 [ 24 )b 20.6 + 1.9 1 241b DAYS = DAYS POSTWEANING ( ) = NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values that were associated with spillage or wet feed. c. Excludes values for rat 13221, which was found dead day 28 postweaning. d. Last value recorded before cohabitation. * Significantly different from the vehicle control group value (p<0.05). 418-008: PAGE E-11 000645 PROTOCOL 418-008: COMBINED ORAL (CAVAGEI FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.0) TABLE E9 (PAGE 1): RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - PRECOHABITATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 24a 25 25 FEED CONSUMPTION (G/KG/DAY) DAYS 1 - 8 MEAN+S.D. 191.9 + 18.7 192.8 + 14.1 180.9 + 2 3 . 4 DAYS 8 - 15 DAYS 15 - 22 DAYS 22 - 29 DAYS 29 - 36 DAYS 36 - 43 DAYS 43 - 50 DAYS 50 - 57 DAYS 57 - 64d DAYS 1 - 64d MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. MEAN+S.D. 167.9 + 15.3 l 23 )b 135.9 + 11.0 ( 23 )b 113.2 + 12.9 23) C 99.9 + 9.6 l 23)C 100.2 + 18.4 i 22)b,c 95.3 + 13.6 ( 22)b,c 91.5 + 14.7 ( 231c 8 0 . 9 + 6.4 ( 231c 109.6 + 8.8 l 231 c 166.7 + 11.4 136.4 + 7.6 1 1 4 . 2 + 6.8 100.3 + 7.8 103.4 + 16.7 93.9 t 11.6 ( 24 Jb 88.4 + 11.3 7 9 . 0 + 6.7 l 24 )b 109.2 + 6.6 ( 241 b 167.7 + 18.5 137.8 + 11.9 I 24 )b 114.6 + 6.8 100.9 + 7.8 l 241b 104.1 + 18.9 96.8 + 17.6 [ 24 lb 8 6 . 9 + 8.1 80.3 + 9.2 I 24 )b 109.5 + 9.1 l 24 ) b DAYS = DAYS POSTWEANING ( ) - NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values that were associated with spillage or wet feed. c. Excludes values for rat 13221, which was found dead day 28 postweaning. d. Last value recorded before cohabitation. 418-008:PAGE E-12 000646 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 E10 (PAGE 1): MATERNAL ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) GESTATION SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 Ill 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a, b 25 25 PREGNANT N 22 21 24 MATERNAL FEED CONSUMPTION (G/DAY) DAYS 0 1 DAYS 1 - 10 MEAN* S .D . MEAN*S.D . 25.2 * 2 2 27.2* 3 0 25.0 . 2.6 ( 20 1c 2 6 . 2 * 4.2 2 4 . 0 + 2.4 2 5 . 4 * 3.2 DAYS 10 - 14 DAYS 14 17 MEAN *S D . MEANS D . 26.8 3 2 27.8 *_ 3 9 25.6 * 3.1 2 6 . 0 * 4.1 25.0 2.6 I 231c 2 6 . 9 + 3.3 DAYS n 20 MEAN.S.D. 25.2 * 3.6 25.0 * 3.6 25.5 + 4.0 DAYS 0 - 20 MEAN.S D. 26.2* 2 4 25.5 i 2.5 25.1 * 2.4 DAYS * DAYS OF GESTATION ( 1 = NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning ; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found <dead on day 28 postweaning. c. Excludes values that were associated witli spillage. 418-008:PAGE E-13 00064 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERI NATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE Ell (PAGE 1): MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) GESTATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) 1 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a, b 25 25 PREGNANT N 22 21 24 MATERNAL FEED CONSUMPTION (G/KG/DAY) DAYS 0 - 7 DAYS 7 - 1 0 MEAN+S.D. MEAN+S.D . 83.6 + 5.8 84.1 + 6.9 80.0 + 5.4 [ 20] c 79.3 + 10.0 80.9 + 7.4 80.4 + 7.6 DAYS 10 - 14 DAYS 14 - 17 MEAN+S.D. MEAN+S.D . 78.8 + 7.8 76.7 + 10.2 74.0 + 7.6 70.3 + 10.0 75.8 + 7.1 l 23] c 75.7 + 6.8 DAYS 17 - 20 MEAN+S.D . 63.1 + 8.2 6i .3 + 7.6 64.5 + 8.8 DAYS 0 - 20 MEAN+S.D. 77.8 + 5.6 73.8 + 5.0 75.9 + 5.2 DAYS = DAYS OF GESTATION [ 1 = NUMBER OF VALUES AVERAGED a . Excludes values for rat 13205, which was excluded from study on day 7 postweaning,- the sex was incorrectly identified, b. Excludes values for rat 13221, which was found dead on day 28 postweaning. c. Excludes values that were associated with spillage. 418-008:PAGE E-14 GO0G48 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 E12 (PAGE 1): MATERNAL ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) - LACTATION SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a. b 25 25 PREGNANT N 22 21 24 DELIVERED LITTERS N 22 21 24 MATERNAL FEED CONSUMPTION1 (G/DAY) DAYS 1 - 4 DAYS 4 - 7 MEAN +S.D . MEAN+S.D. 29.2 + 6.9 3 6 . 9 + 4.8 30.1 + 5.7 ( 19) c 39.6 + 7.8 2 5 . 2 + 4.7* 3 5 . 9 + 6.4 DAYS 7 - 14d DAYS 1 - 14d MEAN+S.D. MEAN+S.D. 5 4 . 0 + 6.1 4 4 . 4 + 4.6 5 7 . 4 + 7.1 4 7 . 1 + 6.2 5 2 . 0 + 7.3 ( 23) e 42.2 + 5.5 [ 23]e DAYS = DAYS OF LACTATION ( ] = NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205. which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead on day 28 postweaning. c. Excludes values that were associated with spillage. d. Because it is presumed that the pups begin to consume maternal feed after day 14 of lactation, maternal feed consumption values were not tabulated on days 14 to 21 of lactation. e. Excludes values for dam 13257, which was found dead on day 10 of lactation. * Significantly different from the vehicle control group value (p<0.05). 418-008PAGE E-15 000649 PROTOCOL, 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE El3 (PAGE 1) : MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - LACTATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 Ill 0.4 RATS TESTED N 25 25 25 INCLUDED IN ANALYSES N 23a,b 25 25 PREGNANT N 22 21 24 DELIVERED LITTERS N 22 21 24 MATERNAL FEED CONSUMPTION (G/KG/DAY) DAYS 1 - 4 DAYS 4 - 7 MEAN+S.D. MEAN+S.D . 90.6 + 24.7 113.2 + 20.8 89.0 + 15.0 1 19) c 117.1 + 2 2 . 9 78.9 + 16.0 111.9 + 20.2 DAYS 7 - 14d DAYS 1 - 14d MEAN+S.D. MEAN+S.D. 157.6 + 21.9 133.4 + 19.9 162.3 + 2 0 . 2 136.6 + 17.6 154.1 + 17.7 I 23) e 128.8 + 15.1 ( 23)e DAYS = DAYS OF LACTATION [ ] = NUMBER OF VALUES AVERAGED a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13221, which was found dead on day 28 postweaning. c. Excludes values that were associated with spillage. d. Because it is presumed that the pups begin to consume maternal feed after day 14 of lactation, maternal feed consumption values were not tabulated on days 14 to 21 of lactation. e. Excludes values for dam 13257, which was found dead on day 10 of lactation. 418-008:PAGE E-16 000G50 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 14 (PAGE 1) : SEXUAL MATURATION - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) 11 0.1 III 0.4 FEMALE RATS N 25 25 25 INCLUDED IN ANALYSES N 24a 25 25 VAGINA PATENT b MEAN+S.D. 31.1 + 1.8 31.1 2.0 30.5 + 1.4 a. Excludes values for female rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Average day postpartum that the vagina was patent. PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS ( S P O N S O R S T U D Y NUMBER: 6295.9) TABLE El5 (PAGE 1): PASSIVE AVOIDANCE PERFORMANCE - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 FEMALE RATS SESSION la N 22b 25 22 TRIALS TO CRITERION LATENCY TRIAL lc LATENCY TRIAL 2c FAILED TO LEARN d MEAN+S.D . MEAN+S.D. MEAN+S.D. N <%) 6.4 8.0 23.2 1i 3 1 + 5.0 .18.1 4.5) 5 -2 7. 1 25.6 0( + 2.0 + 3.5 + 19.6 0 .0) 5.0 11.3 24.1 0( + 1.0 + 12.5 + 19.1 0. 0) SESSION 2a N 21 25 22 TRIALS TO CRITERION LATENCY TRIAL lc MEAN+S.D. MEAN+S.D. 4.0 1 2.9 26.0 + 21.3 3 .3 2.7 30.4 + 23.7 2.8 + 1.2* 40.3 + 24.6 a. Sessions 1 (Learning Phase) and 2 (Retention Phase) of testing were separated by a one-week interval. b. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. c. The latency was recorded in seconds. d. Number of rats that did not meet the criterion in Session 1 (Learning Phase). * Significantly different from the vehicle control group value (p<0.05). 418-008.PAGE E-18 ZS9000 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE E16 (PAGE 1): WATERMAZE PERFORMANCE - SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 HI 0.4 FEMALE RATS SESSION la N 21 2S 21 TRIALS TO CRITERION ERRORS PER TRIAL LATENCY TRIAL 2b FAILED TO LEARN c MEAN tS .D . MEAN *S.D . MEAN* S .D . n Tvi 11.2 2.7 0.51 4 0.28 12.2 t 7.4 3 ( 14 .3) 9.8 + 2.8 0.40 + 0.18 13.0 + 7.5 3 ( 12 .0) 11.8 0.51 14.5 2( 4 2.5 + 0.27 + 8.2 9 .5) SESSION 2a N 18 22 18d TRIALS TO CRITERION ERRORS PER TRIAL LATENCY TRIAL lb MEAN tS.D . MEAN.S.D. MEANrS.D. 7.3 4 3.2 0.16 4 0.18 14.4 *_ 12.1 7.5 4 3.0 0 . 1 5 4 0.14 10.4 * 5.6 6.3 4 1.8 0.17 + 0.20 10.7 4 3.5 a. Sessions 1 (Learning Phase) and 2 (Retention Phase) of testing were separated by a one-week interval. b. The latency was recorded in seconds. c. Number of rats that did not meet the criterion in Session I (Learning Phase); Session 2 (Retention Phase) values for these rats were excluded from group averages and statistical analyses. d. Excludes values for rat 13264, which was not tested during Session 2 (Retention Phase). 418-008:PAGE E-19 000653 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 E17 (PAGE 1): MATING AND FERTILITY SUMMARY - FI GENERATION FEMALE RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0 .I III 0.4 RATS IN COHABITATION N 23a, b 25 25 DAYS IN COHABITATION C MEAN+S.D 3.6 + 3.2 3 . 9 + 2.8 2 . 6 + 1.5 RATS THAT MATED N (% 23(100.0) 24( 96.0) 25(100.0) FERTILITY INDEX d N/N (%) 22/23 ( 95.6) 21/24 ( 87.5) 24/25 ( 96.0) RATS WITH CONFIRMED MATING DATES N 23 24 25 RATS MATING e DAYS 1- 7 DAYS 8-14 N<%) N (% ) 20( 87.0) 3( 13.0)f 22( 91.7) 2( 8.3) 25(100.0) 0( 0.0) RATS PREGNANT/RATS IN COHABITATION N/N (%) 22/23 ( 95.6) 21/25 ( 84.0) 24/25 ( 96.0) a. Excludes values for rat 13205, which was excluded from study on day 7 postweaning; the sex was incorrectly identified. b. Excludes values for rat 13121, which was found dead day 28 postweaning. c. Restricted to rats with a confirmed mating date and rats that did not mate. d. Number of pregnancies/number of rats that mated. e. Restricted to rats with a confirmed mating date. f. Includes data for rat 13228; rat was only in cohabitation six days with the first male because there were no available male rats on day 1 of cohabitation. 418-008PAGE E-20 000654 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PEP INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) T A B L E El 8 (PAGE 1) : N E C R O P S Y O B S E R V A T I O N S - S U M M A R Y - FI G E N E R A T I O N F E M A L E R ATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS EXAMINED a N 25 25 25 FOUND DEAD N lb 0 lc SACRIFICED APPEARED NORMAL N N Id 23b,d 0 25 0 24 THORACIC CAVITY: LIGHT ORANGE FLUID N 0 0 lc TRACHEA : WHITE FOAM PRESENT N 0 0 lc LUNGS: DARK RED N 0 0 lc URINARY BLADDER: ONE CALCULUS N 2 0 0 WALLS THICKENED N 1 0 0 KIDNEYS: BILATERAL OR LEFT, CORTEX PITTED AND/OR ROUGH N 2 0 0 RIGHT, PELVIS, ONE CALCULUS N 1 0 0 RIGHT, PELVIS, TAN GRANULAR MATERIAL N 1 0 0 a. R e f e r to the i n d i v i d u a l c l i n i c a l o b s e r v a t i o n s t able(Table E23) for externalobservations confirmed at necropsy. b. Rat 13221 was f o u n d d e a d o n d a y 28 p o s t w e a n i n g . c. D a m 13257 was f o u n d d e a d o n d a y 10 of lactation. d. Rat 13205 was e x c l u d e d f r o m s t u d y o n d a y 7 p o s t w e a n i n g ; the sex was i n c o r r e c t l y identified. 418-008:PAGE E-21 000655 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) T A B L E E l 9 (PAGE 1) : N A T U R A L D E L I V E R Y O B S E R V A T I O N S S U M M A R Y - FI G E N E R A T I O N F E M A L E RATS DOSAGE GROUP DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 RATS ASSIGNED TO NATURAL DELIVERY N 23a, b 25 25 PREGNANT DELIVERED LITTERS N (% ) N (% ) 22( 95.6) 22 (100.0) 21( 84.0) 21(100.0) 24( 96. 24(100 . D U R A T I O N O F G E S T A T I O N c M E A N + S .D . 2 2 . 9 + 0.5 22.9 + 0.4 22.8 + IMPLANTATION SITES PER DELIVERED LITTER N M E A N + S .D . 338 15.4 + 2.9 352 16.8+ 2.2 369 15.4 + DAMS WITH STILLBORN PUPS N (%) 4( 18.2) 3 ( 14.3) 9 ( 37. DAMS WITH NO LIVEBORN PUPS N 0 0 0 GESTATION INDEX d % N/N 100.0 22/ 22 100.0 21/ 21 100 24/ DAMS WITH ALL PUPS DYING DAYS 1-4 POSTPARTUM N 0 0 0 DAMS WITH ALL PUPS DYING DAYS 5-21 POSTPARTUM N 0 0 0 a. E x c l u d e s v a l u e s for rat 13205, w h i c h was e x c l u d e d f rom s t u d y on d a y 7 p o s t w e a n i n g ; the s ex was i n c o r r e c t l y i d entified. b. E x c l u d e s v a l u e s for rat 13221, w h i c h was found d e a d o n d a y 28 of study. c. C a l c u l a t e d as the time (in days) e l a p s e d b e t w e e n c o n f i r m e d m a t i n g ( a r b i t r a r i l y d e f i n e d as d a y 0) and the t ime (in days) the first pup was delivered. d. N u m b e r of rats w i t h live o f f s p r i n g / n u m b e r of p r e g n a n t rats. 418-008:PAGE E-22 000656 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 629S.9) T A B L E E20 (PAGE 1): L I T T E R O B S E R V A T I O N S (NATURALLY D E L I V E R E D PUPS) - S U M M A R Y F2 G E N E R A T I O N L I TTERS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 DELIVERED LITTERS WITH ONE OR MORE LIVEBORN PUPS N 22 21 24 PUPS DELIVERED (TOTAL) N M E A N + S .D . 316 14.4+ 3.5 325 15.5 + 2.5 357 14.9 + 2.6 LIVEBORN MEAN+S.D. N (% ) 14.1 + 3.5 310( 98.1) 15.1 + 2.4 318( 97.8) 1 4 . 3 + 2.5 344( 96.4) STILLBORN MEAN+S.D. N (%) 0.3+ 0.6 6( 1.9) 0.3 + 1.1 7( 2.2) 0 . 5 + 0.9 13( 3.6) PUPS FOUND DEAD OR PRESUMED CANNIBALIZED DAY DAYS DAYS DAYS DAYS 1 2- 4 5- 7 8-14 15-21 N / N (%) N / N (%) N / N (%) N / N (%) N / N (%) 2/310( 7/3081 0/171 ( 0/17M 0/171( 0.6) 2.3) 0.0) 0.0) 0.0) 2/3181 2/3161 2/1671 1/165( 0/1641 0.6) 0.6) 1.2) 0.6) 0.0) 2/344( 11/342( 5/191( 1/178( 0/177{ 0.6) 3.2) 2.6) 0.6)a 0.0)a VIABILITY INDEX b % N/N 97.1 301/310 98.7 314/318 96.2 331/344 LACTATION INDEX c % N/N 100.0 171/171 98.2 164/167 96.7 177/183 D A Y (S ) = D A Y (S ) P O S T P A R T U M a. E x c l u d e s v a l u e s for l i t t e r b. N u m b e r of live p u p s o n d a y c. N u m b e r of live p u p s o n d a y 13257; the dam was found dead on day 10 of lactation; eight pups were sacrificed. 4 (preculling) postpartum/number of liveborn pups on day 1 postpartum. 21 (weaning) postpartum/number of live pups on day 4 (postculling) postpartum. 418-008:PAGE E-23 000657 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY. DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6195.9) T A B L E E20 (PAGE 2): LITTER O B S E R V A T I O N S (NATURALLY DE L I V E R E D PUPS) - S U M M A R Y - F2 G E N E R A T I O N LITTERS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 hi 0.4 DELIVERED LITTERS WITH ONE OR MORE LIVEBORN PUPS N 22 21 24 SURVIVING PUPS/LITTER a DAY 1 b M E A N + S .D . 14.1 + 3.5 15.1 + 2.4 14.3 + 2.5 DAY 4 PRECULLING MEAN+S.D. 13.7 + 3.3 15.0 + 2.8 13.8 + 2.4 DAY 4 POSTCULLING MEAN+S.D. 7.8 + 0.8 8.0 + 0.2 8.0 + 0.2 DAY 7 M E A N + S .D . 7.8 + 0.8 7.8 + 0.5 7.8 + 0.7 DAY 14 D A Y 21 PERCENT MALE PUPS PER NUMBER OF PUPS SEXED MEAN+S.D. MEAN+S.D. 7.8 + 0.8 7.8 0.8 7.8 + 0.7 7.8 + 0.7 7.7 + 0.8 [ 24] c 7 . 7 + 0.8 [ 24 ]c DAY l b M E A N + S .D . 47.9 + 15.2 52.1 + 10.9 47.0 + 13.3 DAY 4 PRECULLING MEAN+S.D. 47.9 + 15.1 51.5 + 11.1 46.6 + 13.4 DAY 4 POSTCULLING MEAN+S.D. 47.0 + 10.0 4 9.6 + 1.6 49.1 + 5.7 DAY 7 MEAN+S.D. 47.0 + 10.0 48.9 + 3.9 48.7 + 6.4 D A Y 14 D A Y 21 M E A N + S .D . MEAN+S.D. 47.0 + 10.0 47.0 + 10.0 49.2 + 2.6 49.2 + 2.6 48.3 + 6.2 [ 24]1c 48.3 + 6.2 [ 24 ]c DAY = DAY POSTPARTUM l ] = NUMBER OF VALUES AVERAGED a. A v e r a g e n u m b e r of live p u p s p e r litter, i n c l u d i n g l itters w i t h no s u r v i v i n g pups. b. I n c l u d e s p u p s b o r n alive, f o u n d d e a d d a y 1 p o stpartum. c. E x c l u d e s v a l u e s for l i t t e r 13257; the d a m w as f ound d e a d o n d a y 10 of lactation; e i g h t pups were sacrificed. 418-008:PAGE E-24 000658 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERI NATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) TABLE E20 (PAGE 3); LITTER OBSERVATIONS (NATURALLY DELIVERED PUPS) - SUMMARY - F2 GENERATION LITTERS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 DELIVERED LITTERS WITH ONE OR MORE LIVEBORN PUPS N 22 21 24 LIVE LITTER SIZE AT WEIGHING DAY i M E A N + S .D . 14.0 + 3.5 15.0 + 2.6 14.2 + 2.5 DAY 4 PRECULLING MEAN+S.D. 13.7 + 3.3 15.0 + 2.8 13.8 + 2.4 DAY 4 P0STCULLING MEAN+S.D. 7.8 + 0.8 8.0 + 0.2 7.9 + 0.3 DAY 7 MEAN+S.D. 7.8 + 0.8 7.8 + 0.5 7.8 + 0.7 DAY 14 MEAN+S.D. DAY 21 MEAN+S.D. PUP WEIGHT/LITTER (GRAMS) 7 8 0.8 7 B 0.8 7.8 + 0.7 7.8 + 0.7 7.7 + 0.8 I 23) a 7.7 + 0.8 I 23] a DAY i MEAN+S.D. 6 .3 + 0.8 6.1 + 0.5 6.2 + 0.5 DAY 4 PRECULLING MEAN+S.D. 8 .7 1 .6 8.2 + 1.0 8.0 + 1.3 DAY 4 POSTCULLING MEAN+S.D. 8. a 1 .6 8.3 + 1.0 8.0 + 1.3 DAY 7 MEANTS.D. 14 .7 2.4 13.9 + 2.2 12.8 + 2.6* DAY 14 DAY 21 M E A N + S .D, MEAN+S.D. 32.0 3.5 50.1 + 5.1 31.8 + 3 .1 49.2 + 5.0 28.9 + 4.7** [ 23]1a 4 6 . 5 + 6.3 ( 23 ]a DAY = DAY POSTPARTUM ( ) = NUMBER OF VALUES AVERAGED a. Excludes values for litter 13257; the dam was found dead on day 10 of lactation; * Significantly different from the vehicle control group value (p<0.05). ** Significantly different from the vehicle control group value (p^O.Ol). eight pups were sacrificed. 418-008:PAGE E-25 000659 PROTOCOL 418 008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 629S.9) T A B L E E21 (PAGE 1): C L I N I C A L O B S E R V A T I O N S FROM BIRTH TO DAY 21 P O S T P A R T U M - S U M M A R Y F2 G E N E R A T I O N PUPS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 L I T T E R S E X A M I N E D (N) 22 21 24 TRANSIENT CLINICAL OBSERVATIONS: a TOTAL FREQUENCY (DAYS X PUPS)/LITTERS WITH OBSERVATIONS NOT NURSING OR NESTING DECREASED MOTOR ACTIVITY N/N N/N i/ 1 i/ i 0/ 0 0/ 0 0/ 0 0/ 0 COLD TO TOUCH N/N i/ 1 0/ 0 0/ 0 PERSISTENT CLINICAL OBSERVATIONS:: a TOTAL FREQUENCY (DAYS x PUPS)/LITTERS WITH OBSERVATIONS TIP OF TAIL MISSING N/N 21/ 1 0/ 0 1/ 1 UMBILICAL HERNIA N/N 0/ 0 0/ 0 15/ I STATISTICAL ANALYSES WERE RESTRICTED TO THE NUMBER OF LITTERS WITH OBSERVATIONS. N/N = TOTAL FREQUENCY (DAYS X PUPS)/LITTERS WITH OBSERVATIONS a. T a b u l a t i o n r e s t r i c t e d to a d v e r s e o b s e r v a t i o n s ; all o t h e r p u p s a p p e a r e d normal. 418-008:PAGE E-26 000660 PROTOCOL 418-008: COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PER INATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS (SPONSOR'S STUDY NUMBER: 6295.9) T A B L E E22 (PAGE 1): N E C R O P S Y O B S E R V A T I O N S S U M M A R Y - F2 G E N E R A T I O N PUPS MATERNAL DOSAGE GROUP MATERNAL DOSAGE (MG/KG/DAY) I 0 (VEHICLE) II 0.1 III 0.4 L I T T E R S E X A M I N E D (N) 22 21 24 TOTAL PUPS STILLBORN OR FOUND DEAD a N9 12 19 STILLBORN N4 7 12 FOUND DEAD N5 5 7 NO MIL K IN S TOMACH b N (%) 2 ( 40.0) 4 ( 80.0) 4( 57.1) P U P S S A C R I F I C E D A N D N E C R O P S I E D O N D A Y S 4, 10 O R 21 P O S T P A R T U M a L I T T E R S E V A L U A T E D N 22 21 24 PUPS EVALUATED N 301 311 325 APPEARED NORMAL LITTER INCIDENCE PUP INCIDENCE N (%) N (%) 2 I ( 95.4) 300( 99.7) 20 ( 95.2) 310 ( 99.7) 24(100.0) 325(100.0) LIVER: MEDIAN AND LEFT LATERAL LOBES, TAN RAISED AREA LITTER INCIDENCE N (%) 0( 0.0) PUP INCIDENCE N<%> 0( 0.0) M 4.8) 1< 0.3) 0( 0.0) 0( 0.0) BRAIN: HYDROCEPHALY, EXTREME DILATION OF ALL VENTRICLES LITTER INCIDENCE N (%) 1( 4.5) PUP INCIDENCE N (%) 1( 0.3) 0( 0.0) 0 ( 0.0) 0( 0.0) 0( 0.0) a. C o m p l e t e n e c r o p s i e s w e r e not p e r f o r m e d on p ups in w h i c h a u t o l y s i s or c a n n i b a l i z a t i o n p r e c l u d e d evaluation. b. A n a l y s i s r e s t r i c t e d to p u p s f o u n d d e a d a n d necro p s i e d . 418-008:PAGE E-27 000661 APPENDIX F PROTOCOL AND AMENDMENTS 000662 418-008P A G E F-1 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044 T: (215) 443-5710 F: (215) 443-5587 PROTOCOL 418-008 SPONSOR'S STUDY NUMBER: 6295.9 STUDY TITLE: Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats. PURPOSE: The purpose of this study is to test for toxic effects/ disturbances resulting from PFOS treatment of Crl:CDBR VAF/Plus male and female rats before cohabitation through mating, gestation and lactation. This study evaluates ICH Harmonised Tripartite Guideline stages A through F of the reproductive process and should detect effects on the estrous cycle, tubal transport, implantation, gestation, parturition, lactation and maternal behavior in female rats, on the development of the offspring of the treated male and female rats, and permit detection of functional 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 will be continued through production of F2 generation litters. TESTING FACILITY: Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044-1297 Telephone: (215)443-8710 Telefax: (215)443-8587 STUDY DIRECTOR: Raymond G. York, Ph.D., DABT Associate Director of Research SPONSOR: 3M Toxicology Services 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 000663 / 418-008.PAGE F-2 Protocol 418-008 Page 2 STUDY MONITOR: Marvin T. Case, D.V.M., Ph.D. Telephone: (612)733-5180 Telefax: (612) 733-1773 ALTERNATE STUDY MONITOR: Andrew M. Seacat, Ph.D. Telephone: (612) 575-3161 Telefax: (612)733-1773 REGULATORY CITATIONS: 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. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Community o f an OECD decision/recommendation on compliance with principles of good laboratory practice. Official Journal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17. REGULATORY COMPLIANCE: This study will be conducted in compliance with the Good Laboratory Practice (GLP) regulations cited above. All changes or revisions of this protocol shall be documented, signed by the Study Director and the Sponsor, dated and maintained with the protocol. The Quality Assurance Unit (QAU) will audit the protocol, the raw data and the report, and will inspect critical phases of the study in accordance with the Standard Operating Procedures of Argus Research Laboratories, Inc. The final report will include a statement signed by the Study Director that the report accurately reflects the raw data obtained during the performance of the study and that all applicable GLP regulations were followed in the conduct of the study. Should significant deviations from GLP regulations occur, each will be described in detail, together with how the deviation might affect the quality or integrity of the study. 000664 418-008:PAGE F-3 Protocol 418-008 Page 3 STUDY SCHEDULE: See ATTACHMENT 1 to the protocol. TEST ARTICLE AND VEHICLE: Identification: Test Article: Name: Physical Description: Lot/Batch Number: Specific Gravity: Purity: Expiration Date: PFOS. Light-colored powder. 217. - 0.6. 98.9%. May, 2000. Information on the identity, composition, strength and purity of the test article is on file with the Sponsor. Vehicle: 0.5% Tween 80 in Reversed Osmosis Membrane Processed Deionized Water (R.O. Deionized Water). Supplier and lot identification of Tween 80 to be documented in the raw data. Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the vehicle that would interfere with the results of this study. Therefore, no analyses other than those mentioned in this protocol will be conducted. Safety Precautions: Gloves, mask, appropriate eye protection and a uniform/lab coat are to be worn during formulation preparation and dosage administration. The Material Safety Data Sheet (MSDS) is attached to the protocol (ATTACHMENT 2). Storage: Bulk Test Article: Vehicle Components: Prepared Vehicle: Prepared Formulations: Room temperature. Room temperature. Room temperature. Frozen (-20C). All test article shipments to the Testing Facility should be addressed to the attention of Julian Gulbinski, Manager of Formulations, at the previously cited address and telephone number. G0G665 418-008:PAGE F-4 Protocol 418-008 Page 4 Shipments should include information concerning storage conditions and shipping cartons should be labeled appropriately. The recipient should be notified in advance of shipment. FORMULATION: Frequency of Preparation: Formulations (suspensions) will be prepared daily at the Testing Facility. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). Adjustment for Puritv: The test article will be considered 100% pure for the purpose of dosage calculations. Testing Facility Reserve Samples: The Sponsor will reserve a sample (1 g) of each lot of the bulk test article used during the course of this study. The Testing Facility will reserve a sample (5 mL) of each lot of the vehicle components used during the course of this study. Samples will be stored under the previously cited conditions. ANALYSES: Samples additional to those described below may be taken if deemed necessary during the course of the study. Bulk Test Article Sampling: No analyses of the bulk test article will be conducted during the course of this study. Information on the stability of the bulk test article is on file with the Sponsor. Analyses of Prepared Formulations: Stability: Stability data for prepared formulations bracketing the range of concentrations and conditions of this 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. 000666 418-008:PAGE F-5 Protocol 418-008 Page 5 Homogeneity Analyses: Homogeneity of the test article in prepared suspensions will be verified during the course of this study. A syringe will be used to withdraw samples (5 mL each) from the top, middle and bottom of the highest concentration on the first day of preparation. Each sample (5 mL) will be divided into two aliquots, one of 2 mL and one of 3 mL. One aliquot (2 mL) will be shipped for analysis; the other aliquot (3 mL) will be retained at the Testing Facility as a backup sample. Backup samples will be stored under the previously cited conditions and discarded at the Testing Facility upon the request of the Sponsor. Concentration Analyses: Concentration of the prepared test article suspensions will be verified during the course of this study. A syringe will be used to withdraw samples (5 mL each) from each concentration during the first and sixth week of dosage administration. Each sample (5 mL each) will be divided into two aliquots, one of 2 mL and one of 3 mL. One aliquot (2 mL) will be shipped for analysis; the other aliquot (3 mL) will be retained at the Testing Facility as a backup sample. Backup samples will be stored under the previously cited conditions and discarded at the Testing Facility upon the request of the Sponsor. Shipping Instructions: Samples to be analyzed will be shipped (frozen on dry ice) to: Kris J. Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone: (612)778-6018 Telefax: (612) 778-6176 Both the recipient and the Study Monitor will be notified in advance of sample shipment. DISPOSITION: Prepared formulations will be discarded at the Testing Facility. All remaining bulk test article will be returned to the Study Monitor at the previously cited address. 000667 418-008:PAGE F-6 Protocol 418-008 Page6 TEST SYSTEM: Species/Strain and Reason for Selection: The Cr1: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 Facility0'31; and 3) the test article is pharmacologically active in the species and strain. Number: Initial population acclimated: 195 virgin male and 205 virgin female rats. Population selected for study: 175 male rats (35 per dosage group) and 175 female rats (35 per dosage group). Ten mated female rats will be assigned to Caesarean-sectioning on day 10 of presumed gestation; the remaining female rats will be permitted to deliver litters. 250 F1 generation pups (25 per sex per dosage group) will be selected at weaning on day 21 postpartum for continued postnatal observation. Body Weight and Age: Male rats will be ordered to weigh from 300 g to 325 g each at receipt, at which time they will be expected to be at least 60 days of age. Female rats will be ordered to weigh from 200 g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights will be recorded the day after receipt and will be documented in the raw data. The weight ranges will be included in the final report. Sex: Both Fo and F1 generation male and female rats will be evaluated. Only Fo generation male and female rats will be given the test article. Source: Charles River Laboratories, Inc., Raleigh, North Carolina. The rats will be shipped in filtered cartons by air freight and/or truck from Charles River Laboratories, Inc., to the Testing Facility. 000668 418-008.PAGE F-7 Protocol 418-008 Page 7 Identification: Fo Generation: Rats are permanently identified using Monel self-piercing ear tags (Gey Band and Tag Co., Inc., No. MSPT 20101). Male and female rats are 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. F1/F2 Generations: Pups will not be individually identified during lactation; all parameters will be evaluated in terms of the litter. At weaning, each rat selected for continued observation will be identified with a Monel self-piercing ear tag. ANIMAL HUSBANDRY: All cage sizes and housing conditions are in compliance with the Guide for the Care and Use o f Laboratory Animals^. Housing: Fo Generation Rats/F1 Generation Litters: Fo generation-rats will be individually housed in stainless steel wire-bottomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of rats will be housed in the male rat's cage. Beginning no later than day 20 of presumed gestation, Fo generation female rats assigned to natural delivery will be individually housed in nesting boxes. Each dam and delivered litter will be housed in a common nesting box during the postpartum period. F1 Generation Rats/F2 Generation Litters: After weaning, the F1 generation rats will be 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 will be used as described for the Fo generation rats. Beginning no later than day 20 of presumed gestation, F1 generation female rats will be individually housed in nesting boxes. Each dam and delivered litter will be housed in a common nesting box during the postpartum period. C00669 418-008P A G E F-8 Protocol 418-008 Page 8 Nesting Material: Bedding material (bed-o'cobs) will be supplied to female rats assigned to natural delivery. Bedding will be changed as often as necessary to keep the animals dry and clean. Analyses for possible contamination are conducted annually and documented in the raw data. Room Air. Temperature and Humidity: The animal room is independently supplied with at least ten changes per hour of 100% fresh air that has been passed through 99.97% HEPA filters (Airo Clean room). Room temperature will be maintained at 64F (18C) to 79F (26C) and monitored constantly. Room humidity will also be monitored constantly and maintained at 30% to 70%. Light: An automatically controlled 12-hour light: 12-hour dark fluorescent light cycle will be maintained. Each dark period will begin at 1900 hours EST. Diet: Rats will be given Certified Rodent Diet #5002 (PMI Nutrition International) available ad libitum from individual feeders. Water: Water will be available ad libitum from individual bottles attached to the cages or from an automatic watering access system. All water will be from a local source and passed through a reverse osmosis membrane before use. Chlorine will be added to the processed water as a bacteriostat; processed water is expected to contain no more than 1.2 ppm chlorine at the time of analysis. Water is analyzed monthly for possible bacterial contamination and twice annually for possible chemical contamination. Contaminants: Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the certified diet, the drinking water or the nesting material at levels that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier or those mentioned in this protocol will be conducted. 000670 418-008:PAGE F-9 Protocol 418-008 Page 9 RANDOMIZATION AND COHABITATION: Fo Generation: Upon arrival, rats will be assigned to individual housing on the basis of computergenerated random units. After acclimation, male and female rats will be selected for study on the basis of physical appearance and body weights recorded during acclimation. The rats will be assigned to dosage groups based on computer-generated (weight-ordered) randomization procedures. Within each dosage group, consecutive order will be used to assign rats to cohabitation, one male rat per female rat. The cohabitation period will consist of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Female rats not mated within the first 7 days of cohabitation will be assigned alternate male rats that have mated (same dosage group) and will remain in cohabitation for a maximum of seven additional days. The first ten female rats per dosage group with a confirmed date of mating will be assigned to Caesarean-sectioning on day 10 of presumed gestation. The remaining female rats will be permitted to naturally deliver litters. A table of random units will be used to assign five rats per group to a pharmacokinetic sample collection at scheduled sacrifice after completion of the cohabitation period (male rats siring litters with dams allowed to naturally deliver a litter) or on day 21 postpartum (female rats allowed to naturally deliver litters). F1/F2 Generation Puds: Day 1 of lactation (postpartum) is defined as the day of birth and is also the first day on which all pups in a litter are individually weighed (pup body weights will be recorded after all pups in a litter are delivered and groomed by the dam). On day 4 postpartum, a table of random units will be used to select pups to be culled, and litters will be reduced to eight pups each. Whenever possible, the same number of male and female pups per litter will be continued on study. At weaning of the F1 generation pups on day 21 postpartum, a table of random units will be used to select 25 male and 25 female pups per group, resulting in a total of 250 F1 generation rats (125 per sex) chosen for continued evaluation. At least one male pup and one female pup per litter, when possible, will be selected. 000671 418-008PAGE F-10 Protocol 418-008 Page 10 ADMINISTRATION: Route and Reason for Choice: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one of the possible routes of human exposure. Method and Frequency. Dosages will be adjusted for the most recently recorded body weight and given at approximately the same time each day. Fo Generation Male Rats: 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 completion of the cohabitation period. Fo Generation Female Rats: Female rats will be given the test article once daily beginning 28 days before cohabitation (maximum of 14 days) and continuing through day 9 of presumed gestation (rats assigned to Caesarean-sectioning), day 24 of presumed gestation (rats assigned to natural delivery that do not deliver a litter) or day 20 postpartum (rats that deliver a litter). F1 Generation: F1 generation pups will not be directly 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. Rationale for Dosage Selection: Dosages will be selected by the Sponsor on the basis of previous studies conducted with the test article. 000672 418-008P A G E F-11 Protocol 418-008 Page 11 Dosage Levels. Concentrations and Volumes: Dosage Group Number of Rats Per Sex Dosage (mg/kg/day) Concentration (mg/mL) Dosage Volume (m U kg) Argus Batch Number 1 35 0 (Vehicle) II 35 0.1 III 35 0.4 IV 35 1.6 V 35 3.2 0 0.02 0.08 0.32 0.64 5 B-418-008-A(Day.Month.Year) 5 B-41 S-008-B(Day.Month. Year) 5 B-418-008-C(Day.M onth.Year) 5 B-418-008-D(Day.Month. Year) 5 B-418-008-E(Day.M onth.Year) The test article will be considered 100% pure for the purpose of dosage calculations. TESTS. ANALYSES AND MEASUREMENTS - Fo GENERATION: Viability - Male and Female Rats: All Periods: At least twice daily. Clinical Observations and/or General Appearance - Male and Female Rats: Acclimation Period: At least once. Dosage Period: Twice daily. Prior to dosage administration and once approximately one hour postdosage. Maternal Behavior: Days 1, 4, 7, 14 and 21 postpartum. Any observed abnormal behavior will be recorded daily. Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director and/or Study Monitor. Body Weights - Male Rats: Acclimation Period: At least once. Dosage Period: Weekly. Sacrifice: Terminal weight. 000673 418-008: PAGE F-12 Protocol 418-008 Page 12 Body Weights - Female Rats: 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. Feed Consumption Values - Male Rats (recorded and tabulated): Dosage Period: Weekly. Feed Consumption Values - Female Rats (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. Feed Consumption Values - Male and Female Rats: Feed consumption values may be recorded more frequently than cited above if it is necessary to replenish the feed. During cohabitation, when two rats occupy the same cage with one feed jar, replenishment of the feed jars will be documented. Individual values will not be recorded or tabulated. Estrous Cycling and Mating: A table of random units will be used to select 15 female rats per group for evaluation of estrous cycling by examination of vaginal cytology for 14 days before the start of the cohabitation period. During cohabitation, all female rats will be evaluated daily until spermatozoa are observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ. Duration of Gestation: The duration of gestation is calculated from day 0 of presumed gestation to the day the first pup is observed. 000674 418-008:PAGE F-13 Protocol 418-008 Page 13 Fertility Parameters: Fertility Index (percentage of matings that result in pregnancies). Gestation Index (percentage of pregnancies that result in birth of live litters). Number of offspring per litter (live and dead pups). Number of implantation sites. General condition of dam and litter during the postpartum period. Viability indices (percentage of pups bom that survive 4 and 7 days). Lactation Index (percentage of pups born that survive 21 days). Caesarean-Sectioning Observations: Rats will be Caesarean-sectioned on day 10 of presumed gestation. Placentae that appear abnormal (size, color or shape) will be noted in the raw data. The rats will be examined for number and distribution of: Corpora Lutea. Implantation Sites. Viable and Nonviable Embryos. (A viable embryo is oval or crescent shaped, pink, firm and enclosed in an amniotic sac filled with clear fluid. A nonviable embryo is amorphous, small, pale pink to tan or deep red to black, soft and enclosed in an amniotic sac filled with clear, cloudy, or opaque fluid.) Natural Delivery: Female rats will be evaluated for: Clinical Observations During Parturition. Duration of Gestation (day 0 of presumed gestation to the time the first pup is observed). Length of Parturition (time of delivery of last pup minus the time of delivery of the first pup divided by N-1 pups in each litter). Litter Size (defined as all pups delivered). 000675 418-008PAGE F-14 Protocol 418-008 Page 14 Pup Viability at Birth. METHOD OF SACRIFICE - Fo GENERATION: Rats will be sacrificed by carbon dioxide asphyxiation. Embryos will be discarded after examination. NECROPSY - Fo GENERATION: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation (a table of random 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 evaluations of gross lesions). Unless specifically cited below, all other tissues will be discarded. Male and Female Rats Assigned to Pharmacokinetic Sample Collection: At scheduled sacrifice after completion of the cohabitation period (male rats siring litters with dams allowed to naturally deliver a litter) and on day 21 postpartum (female rats allowed to naturally deliver a litter), five rats per group will be assigned to a pharmacokinetic sample collection. In addition to the appropriate evaluations described below, blood samples (approximately 4 ml_ per rat) will be collected from the inferior vena cava into serum separator tubes and centrifuged. The resulting serum (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. After completion of sample collection, serum and liver section (lateral lobe) samples will be shipped (frozen on dry ice) to Kris J. Hansen. Ph.D., at the previously cited address for analysis. Both the recipient and the Study Monitor will be notified in advance of sample shipment. 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 without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. The remaining organs will be retained in neutral buffered 10% formalin. G0676 418-008:PAGE F-15 Protocol 418-008 Page 15 Scheduled Sacrifice - Female Rats Assigned to Caesarean-Sectionina: On day 10 of presumed gestation, female rats will be sacrificed, Caesarean-sectioned, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites<5). Uteri of nonpregnant rats and all ovaries 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. Uteri will be stained with 10% ammonium sulfide to confirm the absence of implantation sites<5). After completion of the 21-day postpartum period, female rats will be sacrificed, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The number and distribution of implantation sites will be recorded. Dams with No Surviving Pups: Dams with no surviving pups will be sacrificed after the last pup is found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Postpartum data for these dams will be excluded from summary tables. Rats Found Dead or Moribund: Rats that die or are sacrificed because of moribund condition or abortion will be examined for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Testes, epididymides, prostate and seminal vesicles of male rats will be excised and individual organ weights will be recorded (seminal vesicles weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. The remaining organs will be retained in neutral buffered 10% formalin. Pregnancy status and uterine contents of female rats will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible. Ovaries will be retained in neutral buffered 10% formalin. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites<5). 000677 418-008: PAGE F-16 Protocol 418-008 Page 16 TESTS. ANALYSES AND MEASUREMENTS - F1 GENERATION: Viability: Preweaning Period: Litters will be observed for dead pups at least twice daily. The pups in each litter will be counted once daily. Postweaning Period: Twice daily. Clinical Observations and/or General Appearance: Preweaning Period: Once daily. Postweaning Period: Once weekly. Maternal Behavior: Days 1, 4, 7, 14 and 21 postpartum. Any observed abnormal behavior will be recorded daily. Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director and/or the Study Monitor. Body Weights: Preweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. Postweaning Period: 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. Feed Consumption Values (recorded and tabulated): Preweaning Period: Not recorded. Postweaning Period: Weekly except during cohabitation. 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). 000678 / 418-008:PAGE F-17 Protocol 418-008 Page 17 Feed consumption values may be recorded more frequently if it is necessary to replenish the feed. During cohabitation, when two rats occupy the same cage with one feed jar, values will be documented when feed jars are filled. These intervals will not be tabulated. Preweaning Developmental Observations: The number of pups meeting the criterion is recorded on each day of testing. Testing continues until the day the criterion is attained by ail pups in the litter. Surface Righting Reflex (ability to right in 5 seconds): From day 1 postpartum. Pinna Unfolding: From day 2 postpartum. Eye Opening: From day 12 postpartum. Acoustic Startle Response: From day 13 postpartum. Air Righting Reflex: From day 14 postpartum. Pupil constriction is evaluated once, on day 21 postpartum. Postweaninq Developmental Observations: Sexual Maturation: Female rats will be evaluated for the age of vaginal patency, beginning on day 28 postpartum. Male rats will be evaluated for the age of preputial separation, beginning on day 39 postpartum. Passive Avoidance Testing: Beginning at 24 1 day postpartum, one male rat and one female rat from each litter, where possible, will be evaluated in a passive avoidance test for learning, short-term retention and long-term retention. The passive avoidance apparatus consists of a two-compartment chamber with hinged Plexiglas lids. One compartment is fitted with a bright light and Plexiglas floor. The other compartment is fitted with a grid floor to which a brief (1 sec) pulse of mild electric current (1 mA) can be delivered. The two compartments are separated by a sliding door. On each test trial, the rat is placed into the "bright" compartment, the sliding door is opened and the light is turned on. The rat is allowed to explore the apparatus until it enters the "dark" compartment. The sliding door is then immediately closed, the light is turned off and the brief pulse of current is delivered to the grid floor. The rat is then removed from the apparatus and placed into a holding cage for 30 seconds before the start of the next trial. Trials are repeated until the rat remains in the "bright" 000679 418-008:PAGE F-18 Protocol 418-008 Page 18 compartment for 60 seconds on two consecutive trials (the criterion for learning) or until 15 trials have been completed. The latency to enter the dark compartment or the maximum 60-second interval is recorded for each trial. Each rat is tested twice. The test sessions are separated by a one-week interval, and the criterion is the same for both days of testing. Dosage groups are compared for the following dependent measures: The number of trials to the criterion in the first session-this measure will be used to compare groups for overall learning performance. The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the first test session-this measure will be used to compare groups for activity levels and exploratory tendencies 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-this measure will be used to compare groups for short-term retention. The number of trials to the criterion in the second test session-this measure will be used to compare groups for long-term retention. The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the second session-this value is another indication of long-term retention. Watermaze Testing: Beginning at approximately 70 days postpartum, one male rat and one female rat from each litter will be evaluated in a water-filled M-maze for overt coordination, swimming ability, learning and memory. Each rat is tested in a watertight 16-gauge stainless steel modified M-maze. The maze is filled with water to a depth of approximately nine inches, and the water is monitored for temperature (range of 21 C 1C). On each test trial, the rat will be placed into the starting position (base of the M-maze stem farthest from the two arms) and required to swim to one of the two goals of the M-maze, in order to be removed from the water. On the first trial, the rat is required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 is designated the incorrect goal during the remaining trials. Rats that fail to make a correct goal choice within 60 seconds in any given trial are guided to the correct goal and are then removed from the water. A 15-second intertrial interval will separate each trial. Each rat is required to reach a criterion of five consecutive errorless trials to terminate the test session. The maximum number of trials in any test 000G80 418-008: PAGE F-19 Protocol 418-008 Page 19 session is 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval is recorded for each trial, as is the number of errors (incorrect turns in the maze) during each trial. Each rat is tested twice. The test sessions are separated by a one-week interval, and the correct goal and the criterion are the same for both test sessions. Dosage groups are compared for the following dependent measures: The number of trials to criterion on the first day of testing-this measure will be used to compare groups for overall learning performance. The average number of errors (incorrect turns in the maze) for each trial on the first day of testing-this measure will also be used to compare groups for overall learning performance. The latency (in seconds) to reach the correct goal on trial 2 of the first day of testing-this measure will be used to compare groups for short-term retention. The number of trials to criterion on the second day of testing-this measure will be used to compare groups for long-term retention. The average number of errors for each trial on the second day of testing-this measure will also be used to compare groups for long-term retention. The latency (in seconds) to reach the correct goal on trial 1 of day 2 of testing-this is another indicator of long-term retention. Reproductive Capacity: At approximately 90 days of age, the F1 generation rats within each dosage group will be assigned to cohabitation, one male rat per female rat, based on computer-generated random units or random unit tables, with the exclusion of sibling matings. The cohabitation period will consist of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Female rats that do not mate within the first 7 days of cohabitation will be assigned alternate male rats from the same dosage group that have mated. Female rats will be allowed to naturally deliver and maintain litters through a 21-day postpartum period. Mating Performance: As cited above for Fo generation rats. 000681 418-008:PAGE F-20 Protocol 418-008 Page 20 Duration of Gestation: As cited above for Fo generation rats. Fertility Parameters: As cited above for Fo generation rats. F2 Generation Litter Data: Viability, clinical observations and body weights for F2 generation pups will be recorded as cited above for F1 generation litters. METHOD OF SACRIFICE - F1 GENERATION RATS/F2 GENERATION PUPS: As previously cited for Fo generation rats. NECROPSY - F1 GENERATION RATS: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation (a table of random 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 evaluations of gross lesions). Unless specifically cited below, all other tissues will be discarded. Scheduled Sacrifice - F1 Generation Male Rats: Rats will be sacrificed after completion of the 14-day cohabitation period. A gross necropsy of the thoracic, abdominal and pelvic visceral will be performed. Testes and epididymides of male rats will be excised and individual organ weights will be recorded. The epididymides will be retained in neutral buffered 10% formalin. The testes will be 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 deliver a 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(S). A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Female rats without a confirmed mating date that do not deliver a litter will be sacrificed on an estimated day 25 of presumed gestation. 000682 418-008: PAGE F-21 Protocol 418-008 Page 21 F1 Generation Rats Found Dead or Moribund: Rats that die or are sacrificed because of moribund condition or abortion will be examined for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Testes, epididymides, prostate and seminal vesicles of male rats will be excised and individual organ weights will be recorded (seminal vesicles weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. The remaining organs will be retained in neutral buffered 10% formalin. Pregnancy status and uterine contents of female rats will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible. Ovaries will be retained in neutral buffered 10% formalin. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites(5). FI Generation Dams with No Surviving Puds: Dams with no surviving pups will be sacrificed after the last pup is found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Postpartum data for these dams will be excluded from summary tables. F1/F2 Generation Pups Found Dead on Day 1 Postpartum: Pups that die before examination of the litter for pup viability will be evaluated for vital status at birth. The lungs will be removed and immersed in water. Pups with lungs that sink will be identified as stillborn; pups with lungs that float will be identified as liveborn, and to have died shortly after birth. Pups with gross lesions will be preserved in Bouin's solution for possible future evaluation. Should postmortem autolysis preclude these evaluations, it will be noted in the necropsy data. F1/F2 Generation Puds Found Dead or Moribund on Davs 2 to 21 Postpartum: Pups found dead or sacrificed due to moribund condition will be examined for gross lesions and for the cause of the moribund condition or death. Pups with gross lesions found on days 2 to 4 postpartum will be preserved in Bouin's solution for possible future evaluation; gross lesions of pups found on days 5 to 21 postpartum will be preserved in neutral buffered 10% formalin. Should postmortem autolysis preclude these evaluations it will be noted in the necropsy data. 000683 / 418-008:PAGE F-22 Protocol 418-008 Page 22 F1/F2 Generation Pups Not Selected for Continued Observation: F1 and F2 generation pups culled on day 4 postpartum will be sacrificed and examined for gross lesions; pups with gross lesions will be preserved in Bouin's solution. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. All F1 generation pups culled on day 21 postpartum will be sacrificed and examined for gross lesions; gross lesions will be preserved in neutral buffered 10% formalin. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Scheduled Sacrifice - F2 Generation Puds: On day 21 postpartum, pups will be sacrificed and examined for gross lesions. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. 000684 418-008:PAGE F-23 Protocol 418-008 Page 23 PROPOSED STATISTICAL METHODS^ 12': Averages and percentages will be calculated. Litter values will be used where appropriate. Additional procedures and/or analyses may be performed, if appropriate. Type of Test* I. Parametric18 II. Nonoarametric6 A. Bartlett's Test0 A. Kruskal-Wallis Test (75% ties) Significant at p<;0.05 Not Significant Significant at ps0.05 Not Significant Nonparametric Analysis of Variance Dunn's Test Significant at p<;0.05 Not Significant B. Fisher's Exact Test (>75% ties) Dunnett's Test III. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilities are reported as either p;0.05 or psO.01. b. Used only to analyze data with homogeneity of variance. c. Proportion data are not included in this category. d. Test for homogeneity of variance. 000685 418-008:PAGE F-24 Protocol 418-008 Page 24 DATA ACQUISITION. VERIFICATION AND STORAGE: Data will be hand- and/or computer-recorded. Records will be reviewed by the Study Director and/or appropriate management personnel within 21 days after generation. All original records will be stored in the archives of the Testing Facility. All original data will be bound and indexed. A copy of all raw data will be supplied to the Sponsor upon request. Preserved tissues will be stored at the Testing Facility at no charge for one year after mailing of the draft final report, after which time the Sponsor will be contacted to determine the disposition of these materials. RECORDS TO BE MAINTAINED: Protocol and Amendments. Test Article, Vehicle and/or Reagent Receipt, Preparation and Use. Animal Acquisition. Randomization Schedules. Mating History. Treatment (if prescribed by Staff Veterinarian). General Comments. Clinical Observations and/or General Appearance. Blood Sample Collection, Processing and Shipment. Body Weights. Feed Consumption Values. Caesarean-Sectioning Observations. Natural Delivery Observations. Litter Observations. Reflex and Physical Development and Behavioral Observations - F1 Generation Pups. Gross Necropsy Observations. Organ Weights (if required). Photographs (if required). Study Maintenance (room and environmental records). Feed, Water and Bedding Analyses. Packing and/or Shipment Lists. KEY PERSONNEL: Executive Director of Research: Mildred S. Christian, Ph.D., ATS Director of Research: Alan M. Hoberman, Ph.D., DABT Associate Director of Research and Study Director: Raymond G. York, Ph.D., DABT Director of Laboratory Operations: John F. Barnett, B.S. Manager of Study Coordination: Valerie A. Sharper, M.S. Manager of Animal Operations and Member, Institutional Animal Care and Use Committee: Dena C. Lebo, V.M.D. Manager of Regulatory Compliance: Kathleen A. Moran, M.S. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP 000686 418-008PAG E F-25 Protocol 418-008 Page 25 FINAL REPORT: A comprehensive draft final report will be prepared on completion of the study and will be finalized following consultation with the Sponsor. The report will include the following: Summary and Conclusion. Experimental Design and Method. Evaluation of Test Results. Appendices: Figures, Summary and Individual Tables Summarizing the Above Data, Protocol and Associated Amendments and Deviations, Study Director's GLP Compliance Statement, Reports of Supporting Data (if appropriate) and QAU Statement. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE STATEMENT: The procedures described in this protocol have been reviewed by the Testing Facility's Institutional Animal Care and Use Committee. All procedures described in this protocol that involve study animals will be conducted in a manner to avoid or minimize discomfort, distress or pain to the animals. The Sponsor's signature below documents the fact that information concerning the necessity for conducting this study and the fact that this is not an unnecessarily duplicative study may be obtained from the Sponsor. No alternative (in vitro) procedures were available for meeting the stated purposes of the study. 000687 418-008:PAGE F-26 Protocol 418-008 Page 26 REFERENCES: 1. Christian, M.S. and Voytek, P.E. (1982). In Vivo Reproductive and Mutagenicity Tests. Environmental Protection Agency, Washington, D.C. National Technical Information Service, U.S. Department of Commerce, Springfield, VA 22161. 2. Christian, M.S. (1984). Reproductive toxicity and teratology evaluations of naltrexone (Proceedings of Naltrexone Symposium, New York Academy of Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 3. Lang, P.L. (1988). Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Cr1:CDBR Rat. Charles River Laboratories, Inc., Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 4. Institute of Laboratory Animal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 5. Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Arch. Pathol. Exp. Pharmakol. 247:367. 6. Snedecor, G.W. and Cochran, W.G. (1967). Variance test for homogeneity of the binomial distribution. Statistical Methods, 6th Edition, Iowa State University Press, Ames, pp. 240-241. 7. Sokal, R.R. and Rohlf, F.J. (1969). Bartlett's test of homogeneity of variances. Biometry, W.H. Freeman and Co., San Francisco, pp. 370-371. 8. Snedecor, G.W. and Cochran, W.G. (1967). Analysis of Variance. Statistical Methods, 6th Edition, Iowa State University Press, Ames, pp. 258-275. 9. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1129. 10. Sokal, R.R. and Rohlf, F.J. (1969). Kruskal-Wallis Test. Biometry, W.H. Freeman and Co., San Francisco, pp. 388-389. 11. Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6(3):241-252. 12. Siegel, S. (1956). Nonparametnc Statistics for the Behavioral Sciences, McGraw-Hill, New York, pp. 96-104. 000688 PROTOCOL APPROVAL: FOR THE TESTING FACILITY U - George E. Dearlove, Associate Director of Research 418-008.PAGE F-27 Protocol 418-008 Page 27 -2--------- ^ I --- h Date ___ u Date "4% XLv' rbara J. Patte B.A. Chairperson, Institi ional Animal Care and Use Committee LJ ^ x Date FOR THE SPONSOR Marvin T. Case, D.V.M., Ph.D. Study Monitor 1ULtL Date 000689 418-008.PAGE F-28 ATTACHMENT 1 STUDY SCHEDULE 000690 ATTACHMENT 1 418-008: PAGE F-29 Protocol 418-008 Page 1 o f2 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 22 JUN 98 PM - 29 JUN 98 AM 29 JUN 98 PM - 06 JUL 98 AM 23 JUN 98 06 JUL 98 20 JUL 98 Animal Receipt - Acclimation Begins (Fo generation rats). Start of Dosage Period - Fo Generation Male Rats (28 days before cohabitation and continuing through a 14-day cohabitation period until sacrifice after successful mating has been determined). Dosage Period - Female Rats Assigned to Caesarean-Sectioning (28 days before cohabitation and continuing through day 09 of presumed gestation). Dosage Period - Female Rats Assigned to Natural Delivery [28 days before cohabitation through day 24 of presumed gestation (rats that do not deliver a litter) or day 20 postpartum (rats that deliver a litter)]. Dosage Period Estrous Cycle Evaluation. Cohabitation Period (Maximum of 14 days). Male 1 (07 days) Male 2 (07 days) First Possible Day 0 of Presumed Gestation. Last Possible Day 0 of Presumed Gestation. Fo Generation Male Rats Sacrificed after Completion of the Cohabitation Period (Earliest possible date). a. The study initiation date is the day the Study Director signs the protocol. 000691 ATTACHMENT 1 418-008:PAGE F-30 Protocol 418-008 Page 2 of 2 03 JUL 98 16 JUL98 14 JUL 98 31 JUL 98 18 JUL 98 31 JUL 98 03 AUG 98 20 AUG 98 04 AUG 98 19 OCT 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 Presumed Gestation Caesarean-sectioning. Last Possible Day 10 of Presumed Gestation Caesarean-sectioning. First Possible Delivery (Day 21 of presumed gestation). Last Possible Delivery (Day 25 of presumed gestation). First Possible Day 25 of Presumed Gestation Female Sacrifice. Last Possible Day 25 of Presumed Gestation Female Sacrifice. First Possible Day 21 Weaning (Dams and F1 generation pups not selected for continued observation sacrificed). Last Possible Day 21 Weaning. F1 Generation Postweaning Observations Begin (Details of tests cited in protocol). F1 Generation Cohabitation Period (Initiated when rats are approximately 90 days of age approximate initial date). F1 Generation Male Rats Sacrificed after Completion of Cohabitation Period Approximate Earliest Possible Date. Delivery Period - F1 Generation Dams/F2 Generation Litters (Approximate dates). Sacrifice of F1 Generation Dams and F2 Generation Litters on Day 21 Postpartum (Approximate dates). Draft Final Report. 000692 418-008:PAGE F-31 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 000693 418-008: PAGE F-32 MATERIAL SAFETY DATA SHEET 3M 3M Center St. Paul, Minnesota 55144-1000 1-800-364-3577 or (612) 737-6501 (24 hours) Copyright, 1998, Minnesota Mining and Manufacturing Coapany. All rights reserved. Copying and/or downloading of this inforaation for the purpose of properly utilizing 3M products is allowed provided that: 1) the inforaation is copied in full with no changes unless prior agreeaent is obtained froa 3M, and 2) neither the copy nor the original is resold or otherwise distributed with the intention of earning a profit thereon. DIVISION: 3M CHEMICALS TRADE NAME: FC-95 FLUORAD Brand Fluorocheaical Surfactant ID NUMBER/U .P .C .: 98-0207-0103-7 00-51135-09054-1 98-0207-0104-5 98-0211-0888-5 00-51135-09362-7 98-0211-3916-1 ZF-0002-1044-1 ISSUED: January 29, 1998 SUPERSEDES: November 05, 1997 DOCUMENT: 10-3796-9 00-51135-09055-8 00-51135-02311-2 1 . INGREDIENT C.A.S. NO. PERCENT POTASSIUM PERFLUOROALKYL SULFONATE.... POTASSIUM PERFLUOROALKYL SULFONATE.... POTASSIUM PERFLUOROALKYL SULFONATE.... POTASSIUM PERFLUOROALKYL SULFONATE.... POTASSIUM PERFLUOROALKYL SULFONATE.... 2795-39-3 3871-99-6 29420-49-3 60270-55-5 3872-25-1 82 3 3 2 1 - 86 -8 -7 -6 -3 2. PHYSICAL DATA BOILING POINT:..... VAPOR PRESSURE:.... VAPOR DENSITY:..... EVAPORATION RATE:... SOLUBILITY IN WATER: SPECIFIC GRAVITY:... PERCENT VOLATILE:... p H : ....................................... VISCOSITY:......... MELTING POINT:..... N/A N/A N/A N/A slight ca. 0.6 Water=1 (Bulk) 0\ 7 -8 (0.1% Aqueous) N/D N/D APPEARANCE AND ODOR: Light colored, free flowing powder. Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 000G94 MSDS: FC-95 FLUORAD Brand Fluorochemical Surfactant January 29, 1996 418-008:PAGE F-33 PAGE 2 3. FIRE AND EXPLOSION HAZARD DATA FLASH POINT:................... None FLAMMABLE LIMITS - LEL:.. N/A FLAMMABLE LIMITS - UEL:.. N/A AUTOIGNITION TEMPERATURE:..... N/A EXTINGUISHING MEDIA: Mater, Carbon dioxide, Dry cheaical, Foaa SPECIAL FIRE FIGHTING PROCEDURES: Mear full protective clothing, including helaet, self-contained, positive pressure or pressure deaand breathing apparatus, bunker coat and pants, bands around arms, waist and legs, face mask, and protective covering for exposed areas of the head. UNUSUAL FIRE AND EXPLOSION HAZARDS: See Hazardous Decomposition section for products of combustion. 4. REACTIVITY DATA STABILITY: Stable INCOMPATIBILITY - MATERIALS/CONDITIONS TO AVOID: Not applicable. HAZARDOUS POLYMERIZATION: Hazardous polymerization will not occur. HAZARDOUS DECOMPOSITION PRODUCTS: Carbon Monoxide and Carbon Dioxide, Oxides of Sulfur, Hydrogen Fluoride, Toxic Vapors, Gases or Particulates. 5. ENVIRONMENTAL INFORMATION SPILL RESPONSE: Observe precautions from other sections. Vacuum, use wet sweeping compound or water to avoid dusting. CAUTION! A vacuum cleaner could be an ignition source. Clean up residue with water. Place in an approved metal container. Seal the container. RECOMMENDED DISPOSAL: Do not release to waterways or sewer. Do not use in products or processes that could result in aquatic concentrations greater than 1/10 of the lowest EC50 or LC50 concentration. Incinerate in an industrial or commercial facility in the presence of a combustible material. Combustion products will include HF. Disposal alternative: Dispose of waste product in a facility permitted to Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately HSOS: FC-95 FLUORAD Brand Fluorochemical Surfactant January 29, 1998 418-008:PAGE F-34 PAGE 3 5. ENVIRONMENTAL INFORMATION (continued) accept chemical waste. ENVIRONMENTAL DATA: 96-Hr. Aquatic Fish LC50, Fathead Minnow(Pimephales promelas)=38 mg/1, Bluegill Sunfish(Lepois aacrochirus)*68 ag/1, Rainbow Trout(Salmo gairdneri)-11 ag/1; 48-Hr. EC50, Daphnia Magna * 50 ng/1; COO*.004 g/g; B0020 * Nil. REGULATORY INFORMATION: Volatile Organic Coapounds: N/A. VOC Less H20 & Exempt Solvents: N/A. Since regulations vary, consult applicable regulations or authorities before disposal. U.S. EPA Hazardous Waste Number * None (Not U.S. EPA Hazardous). This product complies with the chemical registration requirements of TSCA, EINECS, CDSL, AICS, MITI and Korea. EPCRA HAZARD CLASS: FIRE HAZARD: No PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 6. SUGGESTED FIRST AID EYE CONTACT: Immediately flush eyes with large amounts of water for at least 15 minutes. Get immediate medical attention. SKIN CONTACT: Immediately flush skin with large amounts of water. Remove contaminated clothing. If irritation persists, call a physician. Mash contaminated clothing before reuse. INHALATION: If signs/symptoms occur, remove person to fresh air. If signs/symptoms continue, call a physician. IF SWALLOWED: Drink two glasses of water. Call a physician. 7. PRECAUTIONARY INFORMATION EYE PROTECTION: Avoid eye contact. Wear vented goggles. Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 000696 MSOS: FC-95 FLUORAD Brand Fluorochaaical Surfactant January 29, 1998 418-008:PAGE F-35 PAGE 4 7. PRECAUTIONARY INFORMATION (continued) SKIN PROTECTION: Avoid skin contact. Wear appropriate gloves uhen handling this aaterial. A pair of gloves Bade froa the following aaterial(s) are reconaended: butyl rubber. Use one or aore of the following personal protection iteas as necessary to prevent skin contact: head covering, coveralls. Protective garments (other than gloves) should be made of either of the following materials: polyethylene/polyvinylidene chloride (Saranex). RECOMMENDED VENTILATION: Use with appropriate local exhaust ventilation. Use in a wellventilated area. Provide sufficient ventilation to maintain emissions below recommended exposure liaits. If exhaust ventilation is not adequate, use appropriate respiratory protection. RESPIRATORY PROTECTION: Avoid breathing of dust. Select one of the following NIOSH approved respirators based on airborne concentration of contaminants and in accordance with OSHA regulations: half-mask dust and mist respirator, half-mask supplied air respirator, full-face dust and mist respirator, full-face supplied air respirator. PREVENTION OF ACCIDENTAL INGESTION: Do not eat, drink or smoke when using this product. Wash exposed areas thoroughly with soap and water. Nash hands after handling and before eating. RECOMMENDED STORAGE: Keep container dry. Keep container closed when not in use. FIRE AND EXPLOSION AVOIDANCE: Nonflammable. OTHER PRECAUTIONARY INFORMATION: No smoking: Smoking while using this product can result in contamination of the tobacco and/or smoke and lead to the formation of the hazardous decomposition products mentioned in section 4 of this MSDS. HMIS HAZARD RATINGS: HEALTH: 2 FLAMMABILITY: 0 REACTIVITY: 0 PERSONAL PROTECTION: X (See precautions, section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT TYPE AUTH SKIN POTASSIUM PERFLUOROALKYL SULFONATE... 0.1 MG/ M3 POTASSIUM PERFLUOROALKYL SULFONATE... 0.1 MG/M3 POTASSIUM PERFLUOROALKYL SULFONATE... 0.1 MG/M3 POTASSIUM PERFLUOROALKYL SULFONATE... 0.1 MG/M3 TWA 3M TWA 3M TWA 3M TWA 3M Y Y Y Y Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 00697 USDS: FC-95 FLUORAO Brand Fluorochemical Surfactant January 29, 1998 418-008PAGE F-37 PAGE 6 8. HEALTH HAZARD DATA (continued) REPRODUCTIVE/DEVELOPMENTAL TOXINS: Not teratogenic in the rat at oral doses below maternally toxic levels. OTHER HEALTH HAZARD INFORMATION: This product is not known to contain any substances regulated under California Proposition 65. A Product Toxicity Summary Sheet is available. SECTION CHANGE DATES HEADING SECTION CHANGED SINCE November 05, 1997 ISSUE Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately The information in this Material Safety Data Sheet (MSDS) is believed to be correct as of the date issued. 3M MAKES NO WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR COURSE OF PERFORMANCE OR USAGE OF TRADE. User is responsible for determining whether the 3M product is fit for a particular purpose and suitable for user's method of use or application. Given the variety of factors that can affect the use and application of a 3M product, some of which are uniquely within the user's knowledge and control, it is essential that the user evaluate the 3M product to determine whether it is fit for a particular purpose and suitable for user's method of use or application. 3M provides information in electronic form as a service to its customers. Due to the remote possibility that electronic transfer may have resulted in errors, omissions or alterations in this information, 3M makes no representations as to its completeness or accuracy. In addition, information obtained from a database may not be as current as the information in the MSDS available directly from 3M. 000699 418-008:PAGE F-36 USDS: FC-95 FLUORAO Brand Fluorochemical Surfactant January 29, 199B EXPOSURE LIMITS (continued) PAGE 5 INGREDIENT VALUE UNIT TYPE AUTH SKIN* POTASSIUM PERFLUOROALKYL SULFONATE... 0.1 MG/M3 TWA 3M Y * SKIN NOTATION: Listed substances indicated with 'Y' under SKIN refer to the potential contribution to the overall exposure by the cutaneous route including mucous eenbrane and eye, either by airborne or, ore particularly, by direct contact with the substance. Vehicles can alter skin absorption. SOURCE OF EXPOSURE LIMIT DATA: - 3M: 3M Recommended Exposure Guidelines 8. HEALTH HAZARD DATA EYE CONTACT: Mild Eye Irritation: signs/symptoms can include redness, swelling, pain, and tearing. SKIN CONTACT: Mild Skin Irritation (after prolonged or repeated contact): signs/symptoms can include redness, swelling, and itching. May be absorbed through the skin and persist in the body for an extended time. INHALATION: May be harmful if inhaled. May be absorbed by inhalation and persist in the body for an extended time. Single overexposure, above recommended guidelines, may cause: Irritation (upper respiratory): signs/symptoms can include soreness of the nose and throat, coughing and sneezing. IF SWALLOWED: Ingestion is not a likely route of exposure to this product. Illness may result from a single swallowing of a moderate quantity of this material. 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 000698 418-008: PAG E F-38 ATTACHMENT 3 TEST ARTICLE PREPARATION PROCEDURE 000700 / 418-008:PAGE F-39 ATTACHMENT 3 Protocol 418-008 Version: 418-008 (21 MAY 981 Page 1 of 3 TEST ARTICLE PREPARATION PROCEDURE Test Article: PFOS. Vehicle: 0.5% Tween 80 in R.O. Deionized Water. A. Purpose: The purpose of this procedure is to provide a method for the preparation of dosage suspensions of PFOS and the control article for oral administration to rats on Argus Study 418-008. B. General Information: 1. All suspension containers will be labeled and color coded. Each label will specify the protocol number, test article identification, Argus batch number, concentration, dosage level, preparation date, expiration date and storage conditions. 2. Suspensions will be prepared: X Daily __ Weekly _For___ days of use 3. Suspensions will be prepared at a final dosage volume of 5 mL/kg. 4. Safety: X Gloves, lab coat, goggles or safety glasses and faceshield X Dust-Mist Respirator _ Half-Face Respirator _ Full-Face Respirator/Positive Pressure Hood _ Tyvek Suit/Apron 5. Dosage solutions adjusted for Free base and % Purity. __ Yes X No (Calculations based on 100%) __ Free Base __ Purity 6. Samplingrequirements: Cited in protocol. 7. Storage: Cited in protocol. 000701 418-008:PAGE F^O ATTACHMENTS Protocol 418-008 Version: 418-008 (21 MAY 981 Page 2 of 3 TEST ARTICLE PREPARATION PROCEDURE NOTE: Test article will be prepared as a serial dilution from the high dosage to the low dosage. Stir bars are to be added to the containers; mixing should occur during sampling and/or dosage administration. C. Preparation of the 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. To prepare the 0.64-mg/mL, (group V) solution, add 160 mg of test article into an appropriately sized, labeled container. 2. Q. S. to 250 mL with the vehicle and mix by inversion. 3. Add a stir bar and heat the preparation to 80 C in a water bath for approximately 30 minutes (or until the test article has dissolved). 4. Remove the solution from the water bath, and slowly spin over night while the solution equilibrates to room temperature. 5. To prepare group IV, (the 0.32-mg/mL solution), add 100 mL of group V solution to an appropriately labeled container, then q.s. to 200 mL with vehicle. Add a stir bar and mix until uniform. 6. To prepare group III, (the 0.08-mg/mL solution), add 50 mL of group IV solution to an appropriately labeled container, then q.s. to 200 mL with vehicle. Add a stir bar and mix until uniform. 000702 418-008:PAGE F-41 ATTACHMENTS Protocol 418-008 Version: 418-008 (21 MAY 981 TEST ARTICLE PREPARATION PROCEDURE Page 3 of 3 7. To prepare group II, (the 0.02-mg/mL solution), add 50 mL of group III solution to an appropriately labeled container, then q.s. to 200 mL with vehicle. Add a stir bar and mix until uniform. _ <)MC Clarification: ^ N o V Yes (See attached clarification form.) Initial/Date : R C I --U ^ 000703 418-008-.PAGE F-42 argus TEST ARTICLE/SUBSTANCE PREPARATION PROCEDURE CLARIFICATION Protocol : HiZ'OO S' Version: V ISf-Ooft (n ."*r.if) Data of Preparation Clarification Step t V^'A-r Clarification 1 Co^ ^ c ! <? ? J > c 1 r >5 7 d d f t - i *> p r -c r c} r-w p /? y / / S' A/*" Study Director Date Study Director Date Reviewed by Me ( & r r t * / / r A s Study Director Date Date ; ~ I^ ^ 08.15.97 TASPPM-01-02 000704 418-008:PAGE F-43 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044 T: (215) 443-5710 F: (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 1 - June 2, 1998 1 Storage (page 3 of the protocol): [Effective Date: May 26, 1998] Prepared formulations will be stored at room temperature overnight, rather than frozen. Reason for Change: This change was made to clarify the protocol and facilitate dose formulation preparation. 2. Freouencv of Preparation (page 4 of the protocol): [Effective Date: May 26, 1998] The vehicle (0.5% Tween 80 in R.O. Deionized Water) will be prepared weekly by adding 15 mLs of Tween 80 to 2985 mL of R.O. Deionized Water. Reason for Change: This change was made to clarify the protocol and facilitate dose formulation preparation. 3. Stability (page 4 of the protocol): [Effective Date: May 27, 1998] The Sponsor has confirmed a 48-hour stability on the test article in 0.5% Tween 80 solutions. This allows for preparations to be made one day prior to the day of dose administration. Due to the lengthy 000705 418-008:PAGE F-44 Protocol 418-008 Amendment 1 Page 2 preparation procedure, dose solutions may be made one day prior to the day of dosing. Reason for Change: This change was made to clarify the protocol and facilitate dose formulation preparation. L TV-O-- ') Alan M. Hoberman, Ph.D., DABT Date Director of Research Ryrrtnd G. York, Ph.D.,/DABT Associate Director of Research and Study Director Date sd. Dena C. Lebo, V.M.D. Date Member, Institutional Animal Care and Use Committee Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 000706 418-008PA G E F-45 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044 T: (215) 443-8710 F: (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 2 - June 11,1998 1. Body Weights - Female Rats (page 12 of the protocol): [Effective Date: May 26, 1998] Body weights and feed consumption values will also be collected on Fo generation females on postpartum day 10. Reason for Change: This information was added to the protocol to match data collection on F1 generation females. 2. Scheduled Sacrifice of Male Rats (page 14 of the protocol): [Effective Date: May 26, 1998] At scheduled sacrifice of Fo male rats, all organs will be weighed individually. Reason for Change: This change clarifies the protocol. 3. Scheduled Sacrifice - Female Rats Assigned to Caesarean-Sectioning (page 15 of the protocol): [Effective Date: May 26, 1998] Uteri of non-pregnant rats will not be retained. 000707 418-008.PAGE F-46 Protocol 418-008 Amendment 2 Page 2 Reason for Change: This change was made at the request of the Sponsor. 4. Scheduled Sacrifice - F1 Generation Male Rats (page 20 of the protocol): [Effective Date: May 26, 1998] Seminal vesicles (with and without fluids) and prostates, will be weighed at scheduled sacrifice of all F1 generation male rats. Reason for Change: This information was added to the protocol to match data collection on Fo generation males. ' - --------------- _ _ / / -f f Alan M. Hoberman, Ph.D., DABT Date Director of Research (ayraond G. York, Associate Director o' and Study Director s_ , DABT search Date C _____ //J'lu.,, Dena C. Lebo, V.M.D. Date Member, Institutional Animal Care and Use Committee Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 000708 418-008:PAGE F-47 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044 T: (215) 443-8710 F: (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 3 - June 25, 1998 1. Administration. Method and Frequency (Page 10 of the protocol): [Effective Date: June 19, 1998] Due to a programming error in dosage calculation the dosage period will be extended for an additional 2 weeks and the cohabitation period will occur two weeks later than originally scheduled. The error in dosage calculations caused the male rats to receive approximately 89% to 90% of the targeted dosage and female rats to receive approximately 95% to 99% of the targeted dosage. This occurred for six of the seven days in the second and third weeks and two days of the fourth week of the dosage period. Reason for Change: This change was made to ensure that all rats receive the correct dosage before cohabitation. 000709 418-008:PAGE F-48 Protocol 418-008 Amendment 3 Page 2 2. Schedule (Attachment 1 of the protocol): SCHEDULE 12 MAY 98 Animal Receipt - Acclimation Begins (Fo generation rats). 26 MAY 98 26 MAY 98 - 29 JUL 98 26 MAY 98 - 02 SEP 98 09 JUN 98 - 06 JUL 98 06 JUL 98 PM - 13 JUL 98 AM 13 JUL 98 PM - 20 JUL 98 AM 07 JUL 98 20 JUL 98 03 AUG 98 17 JUL 98 30 JUL 98 Start of Dosage Period - Fo Generation Male Rats (42 days before cohabitation and continuing through a 14-day cohabitation period until sacrifice after successful mating has been determined). Dosage Period - Female Rats Assigned to Caesarean-Sectioning (42 days before cohabitation and continuing through day 09 of presumed gestation). Dosage Period - Female Rats Assigned to Natural Delivery [42 days before cohabitation through day 24 of presumed gestation (rats that do not deliver a litter) or day 20 postpartum (rats that deliver a litter)]. Dosage Period Estrous Cycle Evaluation. Cohabitation Period (Maximum of 14 days). Male 1 (07 days) Male 2 (07 days) First Possible Day 0 of Presumed Gestation. Last Possible Day 0 of Presumed Gestation. Fo Generation Male Rats Sacrificed after Completion of the Cohabitation Period (Earliest possible date). First Possible Day 10 of Presumed Gestation Caesarean-sectioning. Last Possible Day 10 of Presumed Gestation Caesarean-sectioning. 000710 418-008:PAGE F-49 Protocol 418-008 Amendment 3 Page 3 28 JUL 98 14 AUG 98 01 AUG 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 First Possible Delivery (Day 21 of presumed gestation). Last Possible Delivery (Day 25 of presumed gestation). First Possible Day 25 of Presumed Gestation Female Sacrifice. Last Possible Day 25 of Presumed Gestation Female Sacrifice. First Possible Day 21 Weaning (Dams and F1 generation pups not selected for continued observation sacrificed). Last Possible Day 21 Weaning. F1 Generation Postweaning Observations Begin (Details of tests cited in protocol). F1 Generation Cohabitation Period (Initiated when rats are approximately 90 days of age approximate initial date). F1 Generation Male Rats Sacrificed after Completion of Cohabitation Period Approximate Earliest Possible Date. Delivery Period - F1 Generation Dams/F2 Generation Litters (Approximate dates). Sacrifice of F1 Generation Dams and F2 Generation Litters on Day 21 Postpartum (Approximate dates). Draft Final 000711 418-008:PAGE F-50 Protocol 418-008 Amendment 3 Page 4 Reason for Change: This schedule was changed due to the extension of the dosing period by two weeks and the cohabitation period occurring two weeks later than scheduled. Alan M. Hoberman, Ph.D., DABT Date Director of Research z 5-<3av Ryfiond G. Yc(rk, Hh.D., DABT Date Associate Directbr-of Research and Study Director r/ vTL*.- 9 ^ Dena C. Lebo, V.M.D. Date Member, Institutional Animal Care and Use Committee Marvin T. Case, D.V.M., Ph.D. Date Study Monitor 000712 O P rimedica 418-008:PAGE F-51 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-8710 Telefax: (215) 443-8587 PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAUPOSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS. SPONSOR'S STUDY NUMBER: 6295.9 Amendment 4 - July 14, 1998 1. Test Article Preparation Procedure (Version 29 JUN 98 of Attachment 3 to the protocol): [Effective Date: June 29, 1998] The volume of the test article solutions prepared each day will be increased. The new Test Article Preparation Procedure is attached to this amendment. Reason for Change: It is necessary to increase the volume of the test article solutions prepared each day^because of the increasing body weights of the rats. / y - 3lLL -9/fr ^/G e o rg e E. DearloveV^p.D., DABT Date RayrnoHd G. York, PH^p., DABT Associate Director of Research Associate Director of Research Study Director ./ j --I * -----^ /Barbara J. Poerson. B.A. Date Chairperson, Institutional Animal Care and Use Committee J -u b . 9 Marvin T. Case, D.V.M., Ph.D. Study Monitor Date GOO 418-008:PAGE F-52 ATTACHMENTS Protocol 418-008 Version: 418-008 (29 JN 981 TEST ARTICLE PREPARATION PROCEDURE Page 1 of 3 Test Article: PFOS. Vehicle: 0.5% Tween 80 in R.O. Deionized Water. A. Purpose: The purpose of this procedure is to provide a method for the preparation of dosage suspensions of PFOS and the control article for oral administration to rats on Argus Study 418-008. B. General Information: 1. All suspension containers will be labeled and color coded. Each label will specify the protocol number, test article identification, Argus batch number, concentration, dosage level, preparation date, expiration date and storage conditions. 2. Suspensions will be prepared: X Daily __ Weekly _For____days of use 3. Suspensions will be prepared at a final dosage volume of 5 mL/kg. 4. Safety: X Gloves, lab coat, goggles or safety glasses and faceshield X Dust-Mist Respirator _ Half-Face Respirator _ Full-Face Respirator/Positive Pressure Hood _ Tyvek Suit/Apron 5. Dosage solutions adjusted for Free base and % Purity. __ Yes X No (Calculations based on 100%) __ Free Base ___ Purity 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. ?|M 418-008:PAGE F-53 ATTACHMENTS Protocol 418-008 Version: 418-008 (29 JUN 981 TEST ARTICLE PREPARATION PROCEDURE Page 2 of 3 NOTE: Test article will be prepared as a serial dilution from the high dosage to the low dosage. Stir bars are to be added to the containers; mixing should occur during sampling and/or dosage administration. C. Preparation of the 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. To prepare the 0.64-mg/mL, (group V) solution, add 224 mg of test article into an appropriately sized, labeled container. 2. Q. S. to 350 mL with the vehicle. 3. Add a stir bar and heat the preparation to 80 C in a water bath for approximately 30 minutes (or until the test article has dissolved). 4. Remove the solution from the water bath, and spin while the solution equilibrates to room temperature. 5. To prepare group IV, (the 0.32-mg/mL solution), add 150 mL of group V solution to an appropriately labeled container, then q.s. to 300 mL with vehicle. Add a stir bar and mix until uniform. 6. To prepare group III, (the 0.08-mg/mL solution), add 65 mL of group IV solution to an appropriately labeled container, then q.s. to 260 mL with vehicle. Add a stir bar and mix until uniform. ^15 OOOS 418-Q08:PAGE F-54 ATTACHMENTS Protocol 418-008 Version: 4 1 8 -0 0 8 (29 JUN 981 Page 3 of 3 TEST ARTICLE PREPARATION PROCEDURE 7. To prepare group II, (the 0.02-mg/mL solution), add 50 mL of group III solution to an appropriately labeled container, then q.s. to 200 mL with vehicle. Add a stir bar and mix until uniform. '\ initial/Date ii-aflo *>9 1 *> 0 0 0 i33: G P rim edica 418-008:PAGE F-55 Argus Research Laboratories, Inc. 90S Sheehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-8710 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 5 - July 17, 1998 1. Concentration Analyses (page 5 of 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 6 of the protocol): The F1 generation male and female pups will be given the test article or vehicle. Reason for Change: These changes were made at the request of the Sponsor in order to provide information about the effects of the test article on the second generation. 3. Method and Frequency (page 10 of the protocol): The F1 generation male and female pups will be administered the test article orally (gavage) on day 1 postweaning through the day before sacrifice. F2 generation pups will not be directly 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. V* G023 \ 418-008:PAGE F-56 Protocol 418-008 Amendment 5 Page 2 Reason for Change: These changes were made at the request of the Sponsor and follow the method of exposure to Fo generation. Dosaoe Levels. Concentrations and Volumes (page 11 of the protocol): Dosage Group Number of Fo Generation Rats Per Sex I 35 II 35 III 35 IV 35 V 35 Number of F1 Generation Rats Per Sex Dosage (mg/kg/day) 25 0 (Vehicle) 25 0.1 25 0.4 25 1.6 25 3.2 Concentration (mg/mL) 0 0.02 0.08 0.32 0.64 Dosage Volume (mL/kg) Argus Batch Number 5 B-418-008-A(Day.Month. Year) 5 B-418-008-B(Day.Month. Year) 5 B-418-008-C(Day.Month.Year) 5 B-418-008-D(Day. Month.Year) 5 B-418-008-E(Day. Month.Year) Reason for Change: This change was made because dosage administration was extended to include the F1 generation male and female rats. Tests. Analyses and Measurements - F1 Generation (page 16 of the protocol): Clinical Observations and/or General Appearance: Preweaning Period: Once daily. Dosage Period: Twice daily. Prior to dosage administration and once approximately one hour postdosage. Maternal Behavior: Days 1, 4, 7 , 14 and 21 postpartum. Any observed abnormal behavior will be recorded daily OOO' 418-008:PAGE F-57 Protocol 418-008 Amendment 5 Page 3 Body Weights - Male: Preweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. Dosage Period: Weekly. Sacrifice: Terminal weight. Body Weights - Female: Preweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. 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. Reason for Change: These changes were made because dosage administration was extended to include the F1 generation male and female rats. F1/F2 Generation P uds Not Selected for Continued Observation (page 22 of the protocol): On day 4 postpartum, the stomach contents (milk curd) will be collected from culled pups from Groups I, II and V (or highest dosage group available). Samples will be collected from all pups from four to five of the largest litters in these three dosage groups. Individual pup samples will be combined by litter into polypropylene tubes and frozen at -20C. After completion of sample collection, samples will be shipped (frozen on dry ice) to Kris J. Hansen, Ph.D. at 3M Environmental Technology and Safety Services, 935 Bush Avenue, Building 2-3E-09,, St. Paul, Minnesota 55133-3331 for analysis. Both the recipient and the Study Monitor will be notified in advance of sample shipment. 000734 418-008:PAGE F-58 Protocol 418-008 Amendment 5 Page 4 Reason for Change: Collection of milk samples were requested by the Sponsor to determine if the test article is reaching the pups via lactation. 'rp 'GVeordtje E. Dearlove| l.D., DABT Date Associate Director orResearch Raymond G. York>0h.D., DABT Associate Director of Research Study Director Date N '' ; , s -* -/st ~ ^ Barbara J. Patterson,~B.A. Date Chairperson, Institutional Animal Care and Use Committee Marvin T. Case, D.V.M., Ph.D. Study Monitor Date J IO 0 0 0 ,5`8 S O P rimedica 418-008.PAGE F-59 Argus Research Laboratories. Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-871C 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 6 -13 August 1998 1. Necropsy (page 20 of the protocol): On postpartum day 21, the 1.6 mg/kg/day dosage group (Group IV) dams and litters will be sacrificed. Reason for Change: Group IV was terminated at weaning because of the severe pup toxicity during lactation (mortality and reduced body weights and delayed development). 2. Male and Female Rats Assigned to Pharmacokinetic Sample Collection (page 14 of the protocol): On postpartum Day 21, the livers of the pups from five litters in each of the remaining groups will be excised, pooled per litter, frozen and retained at -70C until shipment to the Sponsor. Reason for Change: This change was made at the request of the Sponsor for possible analysis. 3. Scheduled Sacrifice - Female Rats Assigned to Natural Delivery and Dams with No Surviving Pups (page 15 of the protocol): Ovaries will be retained in neutral buffered 10% formalin. 3X\ OOG'236 418-008: PAG E F-60 Amendment 6 Page 2 Reason for Change: was made to clarify the protoo E. Dearlove, Ph.D., DABT Date Associate Director of Research Rycnnd G. York, Associate Director Director Il s & S Date /.w j-jL trf BarbaraKj/Pattersoni(b |a . Date Chairperson, Institutional Animal Care Marvin T. Case, D.V.M., Ph.D. Study Monitor and Use Committee Date r* OOOiffZ G P rimedica 418-008:PAGE F-61 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-8710 Telefax: (215) 443-8587 PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAUPOSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 7 - 2 4 November 1998 1. Natural Delivery (page 13 of the protocol): The Length of Parturition (time of delivery of last pup minus the time of delivery of the first pup divided by N-1 pups in each litter) will not be calculated. Reason for Change: A litter watch was not required by the Sponsor. r Dearlove, Ph.D., DABT Date Associate Director of Research ~A RaiV^ir^^LLd G. York, P Associate Director of Study Director DABT arch Date -py/Af).vy-P Dena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and Use Committee Marvin T. Case, D.V.M., Ph.D. Study Monitor ? (> Date ^23 0007-38 O Primedica 418-008:PAGE F-62 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-8710 Telefax: (215) 443-8587 PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAIVPOSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 8 - 1 8 May 1999 1. Concentration Analyses (page 5 of the protocol): The concentration samples were sent to the Sponsor. Sample analyses will be conducted at the discretion of the Sponsor and no report will be sent to the Testing Facility. Reason for Change: This change was made at the request of the Sponsor to clarify the protocol. 2. Male and Female Rats Assigned to Pharmacokinetic Sample Collection (page 14 of the protocol): The liver and serum samples were sent to the Sponsor. Samples will be analyzed at the discretion of the Sponsor. Reason for Change: This change was made at the request of the Sponsor to clarify the protocol. 3. F1/F2 Generation Puds Not Selected for Continued Observation (page 22 and Amendment 5 of the protocol): The stomach contents of these pups were sent to the Sponsor for analysis. Stomach contents will be analyzed at the discretion of the Sponsor. 000739 418-008:PAGE F-63 Reason for Change: Protocol 418-008 Amendment 8 Page 2 This change was made at the request of the Sponsor to clarify the protocol. X L,(. /c u l l C Q ^o rg^E ^e arlove , Ph.D., DABT Date Associate Director of Research Raymond G. York, F^KD), DABT Associate Director ofResearch Study Director 29 ate LU Lzu it if ena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and Use Committee Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 000740 APPENDIX G DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 0007^ 418-008:PAGE G-1 DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY On 8 August 1998, the 5 mL concentration sample was not taken during the first week of dosage administration for the F1 generation rats. This deviation did not adversely affect the outcome or interpretation of the study because sufficient data were collected to accurately evaluate this parameter. On 3 to 8 June 1998 [study days (DSs) 9 to 14], 10 to 15 June 1998 (DSs 16 to 21), 17 June 1998 (DS 23) and 18 June 1998 (DS 24), all Fo generation male and female rats were dosed with the dosage volumes based on body weights recorded on DS 1 (26 May 1998), rather than on weekly body weights. As a result of this error, male rats were administered approximately 89% to 90% of the targeted dosages 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 period was extended for an additional two weeks to ensure that all rats were administered the correct dosages before cohabitation. On 21 August 1998 (postweaning day 1), F1 generation male rats 13158 and 13159 in the 0.4 mg/kg/day dosage group were neither weighed nor dosed. The dosage volume for the rest of the week was 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 evaluate this parameter, and the dosage volumes were based on the day 2 postweaning weight were presumed to be very similar to dosage volumes based on the day 1 postweaning weight. All deviations ajpe^documented in the raw data. Raymond G. York/Ph.D., DABT Associate DirectorVofjxesearch and Study Director Date to OOi'K APPENDIX H TEMPERATURE AND RELATIVE HUMIDITY REPORTS ? 0 0 0 ? - 418-008:PAGE H-1 ARGUS Tem perature and R elative Hum idity Report Location: Room 17 Protocol N um ber 418-008 Range o f Dates: 12-M ay-1998 12:00 to 12-Jun-1998 09:46 Target Range: Species: Rat Total Number of Days: Total Number of Hours: Total Number of Data Points: Temperature 64*F to 79*F 32 741.5 742 Relative Humidity 30% to 70% 32 741.5 742 Mean ( SD): Maximum: Median: Minimum: Number of Points in Range (%): Number of Points High (%): Number of Points Low (%): 69.6 (1.1) 46.2 ( 3.9) 71.9 61.2 69.7 46.5 66.4 37.9 742 (100.0) 742 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Report Generated: 04-Sep-1998 at 13:18 COMMENTS: REVIEWED BY: DATE: Cumulative by Location (v04.01.97) ?2l 000^44" ARGUS 418-008.PAGE H-2 Tem perature and R elative Hum idity R eport Location: Room 03 Protocol N um ber 418-008 Range o f Dates: 12-Jun-1998 09:46 to 21-S ep-1998 13:40 Target Range: Species: Rat Total Number of Days: Total Number of Hours: Total Number of Data Points: Temperature 64*F to 79*F 102 2427.74 2423 Relative Humidity 30% to 70% 102 2427.74 2423 Mean ( SD): Maximum: Median: Minimum: Number of Points in Range (%): Number of Points High (%): Number of Points Low (%): 69.0 75.3 69.1 62.0 2405 0 18 Report Generated: 31-Mar-1999 at 09:02 ( 2.0) (99.3) (0.0) (0.7) 61.6 72.3 63.1 45.7 2404 19 0 (4.8) (99.2) (0.8) (0.0) COMMENTS: REVIEWED BY: DATE: 'h l - Cumulative by Location (v04.01.97) >30 OOOSiSL ARGUS 418-008:PAGE H-3 Tem perature and R elative Hum idity R eport Location: Room 05 Protocol Num ber. 418-008 Range o f Dates: 27-Jul-1998 12:50 to 28-D ec-1998 08:29 Target Range: Species: Rat Total Number of Days: Total Number of Hours: Total Number of Data Points: Temperature 64*F to 79*F 155 3691.26 3677 Relative Humidity 30% to 70% 155 3691.26 3677 Mean (1 SD): Maximum: Median: Minimum: Number of Points in Range (%): Number of Points High (%): Number of Points Low (%): 69.3 82.7 69.7 64.6 3661 16 0 (1.8) (99.6) (0.4) (0.0) 56.3 71.3 58.1 34.3 3667 10 0 (6.7) (99.7) (0.3) (0.0) Report Generated: 31-Mar-1999 at 09:22 COMMENTS: REVIEWED BY: DATE: Cumulative by Location (v04.01.97) ?3l 000 ARGUS 418-008:PAGE H-4 Tem perature and Relative H um idity Report Location: Room 35-37 Protocol N um ber 418-008 Range o f Dates: 21-Sep-1998 13:40 to 28-Dec-1998 08:29 Target Range: Species: Rat Total Number of Days: Total Number of Hours: Total Number of Data Points: Temperature 64*F to 79*F 99 2346.52 2336 Relative Humidity 30% to 70% 99 2346.52 2336 Mean ( SD): Maximum: Median: Minimum: Number of Points in Range (%): Number of Points High (%): Number of Points Low {%): 69.7 73.0 69.7 67.8 2336 0 0 ( 0.7) (100.0) (0.0) (0.0) 52.5 69.5 51.7 23.6 2332 0 4 Report Generated: 31-Mar-1999 at 09:52 (4.0) (99.8) (0.0) (0.2) COMMENTS: REVIEWED BY Cumulative by Location (v04.01.97) G O O "? - ARGUS 418-008:PAGE H-5 Tem perature D eviations Report Location: Room 03 Protocol Number: 418-008 Range o f Dates: 12-Jun-1998 09:46 to 21-Sep-1998 13:40 Tem perature Target Range: Species: Rat 64F to 79F D a te 17-Jun-1998 17-Jun-1998 17-Jun-1998 17-Jun-1998 17-Jun-1998 28-Jun-1998 28-Jun-1998 08-Jul-1998 08-JuM998 08-JuM998 09-Jul-1998 09-JUM998 09-JuM 998 09-Jul-1998 09-JuM 998 09-UI-1998 09-JuM 998 Tim e 03:00 04:00 05:00 06:00 07:00 06:00 07:00 21:00 22:00 23:00 00:00 01:00 02:00 03:00 04:00 05:00 06:00 Temp. 63.9 L 63.5 L 63.2 L 62.9 L 62.7 L 63.9 L 63.7 L 63.6 L 63.2 L 62.8 L 62.6 L 62.5 L 62.3 L 62.2 L 62.1 L 62.0 L 62.0 L D a te Tim e 09-Jul-1998 07:00 Temp. 62.4 L H Value out of range - High L = Value out of range - Low Temp. Temperature F Report Generated: 31-Mar-1999 at 09:09 These deviations did not adversely affect the outcome or interpretation of the study. The following deviation(s) impacted on the outcome of the study as described: Deviations by Location (v04.01.97) ARGUS 418-008:PAGE H-6 Relative H um idify Deviations Report Location: Room 03 Protocol N um ber 418-008 Range o f Dates: 12-Jun-1998 09:46 to 21-S ep-1998 13:40 H um idify Target Range: Species: Rat 30% to 70% D a te 26-Jun-1998 26-Jun-1998 09-Jut-1998 09-Jul-1998 09-JuH998 21-JUI-1998 24-Jul-1998 16-Aug-1998 16-Aug-1998 18-Aug-1998 24-Aug-1998 25-Aug-1998 27-Aug-1998 30-Aug-1998 31-Aug-1998 15-Sep-1998 16-Sep-1998 Tim e 11:00 12:00 08:00 11:00 12:00 12:00 10:00 17:00 19:00 01:00 15:00 17:00 15:00 13:00 11:00 21:00 14:00 R .H . 70.7 H 71 .OH 70.1 H 70.2 H 70.6 H 70.2 H 70.1 H 72.0 H 71.0 H 70.2 H 70.3 H 70.6 H 72.3 H 70.6 H 71.2 H 70.2 H 72.1 H D a te Tim e H = Value out of range - High L = Value out of range - Low R.H. = Relative Humidity (%) Report Generated: 31-Mar-1999 at 09:15 These deviations did not adversely affect the outcome or interpretation of the study. The following deviation^) impacted on the outcome of the study as described: Deviations by Location (v04.01.97) OOO'^Jife ARGUS 418-008:PAGE H-7 Tem perature Deviations Report Location: Room 05 Protocol Number. 418-008 Range o f Dates: 27-Jui-1998 12:50 to 28-D ec-1998 08:29 Tem perature Target Range: Species: Rat D a te 19-Sep-1998 19-Sep-1998 19-Sep-1998 19-Sep-1998 19-Se(>1998 19-Sep-1998 19-Sep-1998 19-Sep-1998 20-Sep-1998 20-Sep-1998 20-Sep-1998 20-Sep-1998 20-Sep-1998 20-Sep-1998 20-Sep-1998 20-Sep-1998 Tim e 16:00 17:00 18:00 19:00 20:00 21:00 22:00 23:00 00:00 01:00 02:00 03:00 04:00 05:00 06:00 07:00 Temp. 80.8 H 81.7 H 82.4 H 82.7 H 82.4 H 82.1 H 81.9 H 81.8 H 81.6 H 81.5 H 81.3 H 81.2 H 81.1 H 81.0 H 81.1 H 81.1 H 64F to 79F D a te Tim e Tem p. H = Value out of range - High L = Value out of range - Low Temp. = Temperature F Report Generated: 31-Mar-1999 at 09:36 These deviations did not adversely affect the outcome or interpretation of the study. The following deviation(s) impacted on the outcome of the study as described: Deviations by Location (v04.01.97) T35 OOGi^T 418-008:PAGE H-8 ARGUS Relative Humidity Deviations Report Location: Room 05 Protocol Num ber 418-008 Range o f Dates: 27-Jul-1998 12:50 to 28-Dec-1998 08:29 Humidity Target Range: Species: Rat Date 26-Sep-1998 26-Sep-1998 26-Sep-1998 30-Sep-1998 0&-Oct-1998 14-Oct-1998 14-Oct-1998 03-NOV-1998 07-060-1998 Tim e 14:00 16:00 19:00 17:00 02:00 07:00 11:00 16:00 08:00 R.H. 70.7 H 70.1 H 70.1 H 70.1 H 70.5 H 70.4 H 70.1 H 70.3 H 71.3 H 30% to 70% Date Tim e H - Value out of range - High L = Value out of range - Low R.H. = Relative Humidity (%) Report Generated: 31-Mar-1999 at 09:45 These deviations did not adversely affect the outcome or interpretation of the stL The following deviations) impacted on the outcome of the study as described: Deviations by Location (v04.01.97) ?3C 418-008.PAGE H-9 ARGUS R elative Hum idity D eviations Report Location: Room 35-37 Protocol N um ber 418-008 Range o f Dates: 21-Sep-1998 13:40 to 28-Dec-1998 08:29 Hum idity Target Range: Species: Rat Date 23-Sep-1998 23-Sep-1998 23-Sep-1998 23-Sep-1998 Tim e 10:00 11:00 12:00 13:00 R .H . 29.3 L 27.3 L 28.2 L 23.6 L 30% to 70% D a te Tim e R.H. H = Value out of range - High L = Value out of range - Low R.H. a 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 outcome of the study as described: Deviations by Location (v04.01.97) 000716 APPENDIX I STATEMENT OF THE STUDY DIRECTOR G O (S 418-008: PAGE 1-1 Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044 T: (215) 443-8710 F: (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 STATEMENT OF THE STUDY DIRECTOR This final report accurately reflects the raw data obtained during the performance of the study. No deviations from the U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations; Final Rule3, the Japanese Ministry of Health and Welfare (MHW) Good Laboratory Practice Standard for Safety Studies on Drugsband the European Economic Community (EEC) Council decision on 28 July 1989 on the acceptance by the European Economic Community o f an OECD decision/recommendation on compliance with principles o f good laboratory practice0occurred that affected the quality or integrity of the study. Associate Director of Research and Study Director i Argus Research Laboratories, Inc. a. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. b. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard fo r 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 o f an OECD decision/recommendation on compliance with principles o f good laboratory practice. Official Journal of the European Communities: Legislation. 32(No. L 315; 28 October): 1-17. ?3<| GOO APPENDIX J QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT -9M 0 G074 O P rim edica 418-008:PAGE J-1 Arg9u0s5TReSelhTseeepeaelherhocHfyanhoxeDrL::sra((hi22bva11eom,55r,a))BtP44uoA44rili33de--1si88n,9750gI18n4A0c74. 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: Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats Sponsor's Study Number: 6295.9 The draft protocol for this study was audited for adherence to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations, Japanese Ministry of Health and Welfare (MHW); Good Laboratory Practice Standard for Safety Studies on Drugs, and European Economic Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice on 13 MAY 98. Critical phases of this study were inspected 24 times; study information and raw data were audited six times (see tables 1 and 2 for dates and phases/data). The draft final report and the raw data for this study were compared and audited for accuracy, for adherence to protocol requirements, and for adherence to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations, Japanese Ministry of Health and Welfare (MHW); Good Laboratory Practice Standard for Safety Studies on Drugs, and European Economic Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice between 05 DEC 98 and 29 APR 99, and for revisions requested by the Sponsor 18 MAY 99, 7 JUN 99, 9 JUN 99, and for finalization on 10 JUN 99. 7m 0007, 418-008:PAGE J-2 This study was conducted according to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations, Japanese Ministry of Health and Welfare (MHW); Good Laboratory Practice Standard for Safety Studies on Drugs, and European Economic Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice. Nancy J. Gongliewski Date Quality Assurance Manager Heather L. RaButtino, M.S. Quality Assurance Supervisor and Principal Auditor Date O O O TS- TABLE 1 CRITICAL PHASES INSPECTED 418-008:PAGE J-3 Test Article Administration - Gavaae 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 Estrous Cycle Evaluation 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 - Dav 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: 14 AUG 98, 02 DEC 98 Blood Collection Z IX Q GOGS 418-008:PAGE J-4 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 ConstrictionSexual Maturation Dates of inspection: 31 JUL 98, 13 AUG 98, 18 AUG 98, 28 AUG 98, 11 SEP 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 OCT 98 COO! 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: Protocol. Protocol amendments. List of personnel and computer operator codes. Error codes and codes for clinical sigh observations. Animal receipt, randomization, physical examination and acclimation. In-life transaction record. Feed consumption. Necropsy. Organ weights. Tissue packing lists. Edit requests. Deviations. 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. 7M5 G O G &l 418-008:PAGE J-6 The following study information and raw data were audited on 06 OCT 98, 12 OCT 98, 02 NOV 98 TO 06 NOV 98: Animal receipt, randomization, physical examination and acclimation. In-life transaction record. Feed consumption. Estrus cycle evaluation. Cohabitation. Caesarean-sectioning. Maternal gross observations Natural delivery observations. Litter observations. Pup body weights 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. The results of this audit were reported to the Study Director and Management on 08 NOV 98. 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 98. The following study information and raw data were audited on 26 JAN 99 to 28 JAN 99: In-life 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 99. OOQZ2G 418-008:PAGE J-8 The following study information and raw data were audited on 28 JAN 99 to 01 FEB 99: In-life transaction record. Feed consumption. Cohabitation. Natural delivery observations. Litter observations. Pup body weights and status. Passive avoidance. Watermaze. Table of random units. Necropsy. Tissue packing lists. General comments. Sexual maturation. Study maintenance records. Temperature and relative humidity reports. Feed, water and bedding analyses. Edit requests. Dosage volumes. Data review pages. The results of this audit were reported to the Study Director and Management on 01 FEB 99. The following study information and raw data were audited on 25 JAN 99: 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 28 JAN 99. 00072*?