Document 2rpV743jBkn3km3N5B567xMN

C O V /H H > THE DEVELOPMENT SERVICES COMPANY Sponsor: APME Ad-Hoc APFO Toxicology Working Group FINAL REPORT Study Title: 4-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys Author: Peter J. Thomford, PhD Study Completion Date: December 18, 2001 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Project Identification: Covance 6329-230 Sponsor Project Identification: 3M Study No. T-6889.2 Page 1 o f 159 Covance 6329-230 3M T-6889.2 COMPLIANCE STATEMENT 4-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys All aspects of this study were in accordance with the Environmental Protection Agency Good Laboratory Practice Standards (GLPs), 40 CFR 792, except that bile acid determinations done by the University of Dundee were not done in compliance with GLPs. 2 Covance 6329-230 3M T-6889.2 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Environmental Protection Agency (EPA) Good Laboratory Practice Standards, 40 CFR 792. The following inspections were conducted and findings reported to the study director and study director management. Inspection Dates From To 06/07/98 06/07/98 07/16/98 07/16/98 09 22 98 10/01/98 10/01/98 10/01/98 09/15/98 10/02/98 06/10/99 06/10/99 12/07/99 12/07/99 02 23 01 02/23/01 11/27/01 11/27/01 Phase Protocol Review Clinical Laboratory Inspection Report Review Protocol Amendment Review Data Review Protocol Amendment Review Protocol Amendment Review Report Review Report Review Date Reported to Study Director and Study Director Management 06/08/98 07/16/98 10/01/98 10/01/98 10/02/98 06/10/99 12/07/99 02/23/01 11/28/01 L tii... a-1*-_____ i___ A -- i- *- / v \ ____________ - Representative Quality Assurance Unit 3 Covance 6329-230 3M T-6889.2 STUDY IDENTIFICATION 4-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys Test Material Ammonium Perfluorooctanoate (APFO) Sponsor APME Ad-Hoc APFO Toxicology Working Group Study Monitor Paul Lieder, PhD, DABT 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 612.737.2678 Alternate Study Monitor John Butenhoff, PhD, DABT 3M Toxicology Services 612.733.1962 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Study Director Peter J. Thomford, PhD Covance Laboratories Inc. P.O. Box 7545 Madison, Wisconsin 53707-7545 608.241.7207 Study Timetable Study Initiation Date In-Life (Experimental) Start Date In-Life Termination Date Experimental Termination Date June 11, 1998 June 17, 1998 July 16, 1998 December 18, 2001 4 KEY PERSONNEL Covance 6329-230 3M T-6889.2 Study Director Study Toxicologist Study Coordinator Supervisor, Large Animal Toxicology Supervisor, Dose Formulation Supervisor, Laboratory Animal Medicine Clinical Pathologist Supervisor, Clinical Pathology Anatomical Pathologist Supervisor, Anatomical Pathology Peter J. Thomford, PhD Dale Aldridge, BS Patricia K. McKee Pesik, BS, LAT Meechelle Bordeaux, LAT Dixie Bushee, BS, LATG Donna J. Clemons, DVM, MS Diplomate, ACLAM Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) Ronald Markevitch, BS, MT (ASCP) James L. Carter, DVM, PhD Diplomate, ACVP Laurie J. Schuller, BA, LAT 5 Covance 6329-230 3M T-6889.2 CONTENTS Page ABSTRACT........................................................................................................................ 9 PURPOSE.......................................................................................................................... 11 REGULATORY COMPLIANCE..................................................................................... 11 TEST MATERIAL............................................................................................................ 11 Test Material..................................................................................................................11 Reserve (Archive) Samples............................................................................................11 Disposition.....................................................................................................................11 TEST SYSTEM................................................................................................................ 12 Test Animal................................................................................................................... 12 Identification..................................................................................................................12 Acclimation....................................................................................................................12 Housing and Maintenance.............................................................................................12 Justification....................................................................................................................13 PROCEDURES................................................................................................................. 13 Group Designations and Dose Levels........................................................................... 13 Dose Preparation........................................................................................................... 14 Dose Analyses............................................................................................................... 14 Method of Administration.............................................................................................14 Clinical Observations.................................................................................................... 15 Body Weights................................................................................................................ 15 Blood Hormone Determination..................................................................................... 15 Serum APFO Level Determination............................................................................... 16 Clinical Pathology......................................................................................................... 16 Additional Blood Collection..........................................................................................17 Necropsy........................................................................................................................ 17 Palmitoyl CoA Oxidase Determinations.......................................................................17 Cell Proliferation Evaluation.........................................................................................18 Receptor Level Determination...................................................................................... 18 Bile Acid Determination................................................................................................18 Liver APFO Determination............................................................................................18 Tissue Preservation........................................................................................................ 19 Histopathology.............................................................................................................. 19 Statistical Analyses........................................................................................................ 19 RECORD RETENTION...................................................................................................20 6 Covance 6329-230 3M T-6889.2 CONTENTS (Continued) Page RESULTS......................................................................................................................... 20 Clinical Observations....................................................................................................20 Body Weights................................................................................................................ 21 Food Consumption........................................................................................................21 Blood Hormone Analyses............................................................................................. 21 Clinical Pathology.........................................................................................................21 Cell Proliferation Evaluation........................................................................................ 22 Anatomical Pathology...................................................................................................22 CONCLUSIONS............................................................................................................... 22 SIGNATURES.................................................................................................................. 23 REFERENCES.................................................................................................................. 24 PATHOLOGY REPORT...................................................................................................25 COMMENTS ON THE DATA........................................................................................ 28 CODES, ABBREVIATIONS, AND UNITS.................................................................... 29 General Codes and Abbreviations................................................................................. 30 Codes for Clinical Pathology........................................................................................ 31 Abbreviations and Units for Clinical Hematology........................................................ 33 Abbreviations and Units for Clinical Chemistry........................................................... 35 Codes for Anatomical Pathology.................................................................................. 37 TABLES 1 Summary of Clinical Observations - A.M./Weekly................................................38 2 Summary of Clinical Observations - 30 Minutes Postdose.................................... 39 3 Summary of Clinical Observations - 60 Minutes Postdose.................................... 40 4 Summary of Clinical Observations - 90 Minutes Postdose.................................... 41 5 Summary of Body Weight Data (kg)...................................................................... 42 6 Summary of Clinical Hematology Data - Day -8....................................................43 7 Summary of Clinical Hematology Data - Day -5....................................................45 8 Summary of Clinical Hematology Data - Day 30...................................................47 9 Summary of Clinical Chemistry Data - Day -8.......................................................49 10 Summary of Clinical Chemistry Data - Day -5....................................................... 52 11 Summary of Clinical Chemistry Data - Day 2 .......................................................55 12 Summary of Clinical Chemistry Data - Day 30...................................................... 58 13 Incidence of Macroscopic Observations................................................................. 61 14 Incidence of Microscopic Observations.................................................................. 62 7 Covance 6329-230 3M T-6889.2 CONTENTS (Continued) Page APPENDIX 1.................................................................................................................... 63 Protocol..........................................................................................................................64 Protocol Amendment No. 1 .......................................................................................... 81 Protocol Amendment No. 2 .......................................................................................... 86 Protocol Amendment No. 3 .......................................................................................... 88 Material Safety Data Sheet........................................................................................... 91 Certificate of Analysis...................................................................................................98 APPENDIX 2 ...................................................................................................................100 Individual Animal Fate Data........................................................................................101 Individual Clinical Observations................................................................................. 102 APPENDIX 3...................................................................................................................107 Individual Body Weight Data (kg).............................................................................. 108 APPENDIX 4 ...................................................................................................................109 Individual Clinical Hematology Data.......................................................................... 110 Individual Clinical Chemistry Data............................................................................. 119 APPENDIX 5...................................................................................................................131 Individual Animal Pathology Data.............................................................................. 132 APPENDIX 6...................................................................................................................140 Quality Assurance Statement.......................................................................................141 Summary and Individual Hormone Analyses Data..................................................... 142 APPENDIX 7...................................................................................................................144 Cell Proliferation Report.............................................................................................145 8 ABSTRACT Covance 6329-230 3M T-6889.2 The purpose of this study was to provide data for determining an estimated maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study. In addition, the effect on critical enzyme levels, hormones, and other selected biochemical parameters was determined. Male cynomolgus monkeys were assigned to three groups (two animals in Group 1, three animals each in Groups 2 and 3). Animals in Groups 2 and 3 received gelatin capsules containing 2 and 20 mg Ammonium Perfluorooctanoate (APFO)/kg of body weight/day (mg/kg/day), respectively. Animals in Group 1 received empty gelatin capsules. Food was provided once or twice daily. Water was provided ad libitum. The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. At least once daily, animals were examined for abnormalities and signs of toxicity, and food consumption was assessed qualitatively. In addition, animals were observed approximately 30, 60, and 90 minutes postdose for signs of poor health or abnormal behavior. Body weight data were collected before initiation of treatment, on the first day of treatment, and weekly thereafter. Blood samples for hormone analyses [estradiol, estrone, estriol, thyroid stimulating hormone, total and free triiodothyronine (T3), and total and free thyroxin (T4)] were collected before initiation of treatment and on Day 30. Serum was harvested, and the samples were sent to Ani Lytics Inc., for analyses. Blood samples for cholecystokinin analyses were collected before initiation of treatment and on Day 30. Plasma was harvested, and the samples were sent to the DuPont Haskell Company for possible analyses. Blood samples for serum APFO concentration analyses were collected before initiation of treatment and on Day 30. Serum was harvested, and samples were sent to 3M for analyses. Blood samples were collected for hematology and clinical chemistry tests twice before initiation of treatment and on Day 30. In addition, blood for clinical chemistry tests was collected on Day 2. Additional blood was collected at the time of exsanguination, and samples were shipped to 3M for possible future analysis. On Day 30, the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, and selected tissues were collected and preserved. In addition, a sample of liver was collected from each animal for palmitoyl CoA oxidase activity analyses. Representative samples of liver, testes, and pancreas were collected from each animal and sent to Pathology Associates, A Charles River Company for proliferation cell nuclear antigen evaluation. A sample from 9 Covance 6329-230 3M T-6889.2 the liver and pancreas was collected from each animal and sent to the DuPont Haskell Company for possible receptor level determinations. Bile was collected from each animal and sent to the University of Dundee for bile acid determination. A sample of liver was collected from each animal and sent to 3M for APFO concentration analyses. Microscopic examinations were done on the adrenals, liver, pancreas, spleen, and testes from each animal. All animals survived to the scheduled sacrifice. There were no apparent test material-related effects on estradiol, estriol, thyroid stimulating hormone, total and free triiodothyronine, and total and free thyroxin. Estrone levels were notably lower for males given 2 and 20 mg/kg/day. There were no apparent test material-related effects on clinical observations, body weights, food consumption, clinical pathology parameters, or macroscopic or microscopic findings. There did not appear to be a cell proliferative response to the test material in the liver, testes or pancreas, as determined by proliferating indices which were similar in all animals. Based on the results of this study, daily capsule administration of Ammonium Perfluorooctanoate to cynomolgus monkeys for 29 days at dose levels of 2 and 20 mg/kg/day was well tolerated and produced no apparent evidence of toxicity. 10 PURPOSE Covance 6329-230 3M T-6889.2 The purpose of this study was to provide data for determining an estimated maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study. In addition, the effect on critical enzyme levels, hormones, and other selected biochemical parameters was determined. REGULATORY COMPLIANCE All aspects of this study were done in accordance with the Environmental Protection Agency Good Laboratory Practice Standards (GLPs), 40 CFR 792, except that bile acid determinations done by the University of Dundee were not done in compliance with GLPs. TEST MATERIAL Test Material The test material, Ammonium Perfluorooctanoate (APFO), Lot No. 332 (expiration date: December 15, 2001), is a white powder and is 95.2% pure. It was received at Covance on June 10, 1998. The test material was stored at room temperature. Information on synthesis methods, composition, or other characteristics that define the test material is on file with the Sponsor. The Certificate of Analysis is in Appendix 1. Reserve (Archive) Samples A reserve sample (1 g) of the test material was taken before initiation of treatment and stored at room temperature. This sample was transferred to the Sponsor on May 2, 2001. Disposition The remaining test material was returned on May 2, 2001. 11 TEST SYSTEM Covance 6329-230 3M T-6889.2 Test Animal Young adult to adult cynomolgus monkeys were obtained from Covance Research Products Inc. (Denver, Pennsylvania and Alice, Texas), on March 10, 1998. The animals weighed 2.1 to 3.6 kg at initiation of treatment. Identification Each animal was assigned a permanent number upon arrival and identified with collar tag before initiation of treatment. All data for an animal are recorded under this number. Acclimation Nine males were received on March 10, 1998, transferred from the Covance in-house stock colony on June 3, 1998. All animals were acclimated in Animal Room 2015; animals were acclimated at least 30 days before initiation of treatment (duration includes days animals were in the stock colony). In general, animals in this shipment appeared healthy. During acclimation, the animals were examined for abnormalities indicative of health problems. In addition, tuberculosis tests were done on each animal. Results of the tuberculosis tests were negative for all animals. One male was eliminated from study consideration because it had the highest body weight. There were eight males in the randomization for assignment to groups (two males in Group 1 and three males each in Groups 2 and 3). The animal not selected for the study was returned to the Covance stock colony. Housing and Maintenance Animal Room 2015 was used for this study. Environmental controls for the animal room were set to maintain 18 to 29oC, a relative humidity of 30 to 70%, and a 12-hour light/12-hour dark cycle. The animals were housed individually in stainless steel cages. Certified primate diet (#8726C, Harlan Teklad) was provided once or twice daily. The lot numbers are recorded in the data. The diet is routinely analyzed by the manufacturer 12 Covance 6329-230 3M T-6889.2 for nutritional components and environmental contaminants. Results of specified nutrient and contaminant analyses are on file with Covance-Madison. Fruit or additional supplements were provided, but did not require analysis. Water was provided ad libitum. Samples of the water are analyzed for specified microorganisms and environmental contaminants. The results are on file with Covance-Madison. There were no known contaminants in the diet or water at levels that would have interfered with this study. Justification APFO is a known hepatic peroxisome proliferator (PP) in the rat. When exposed to PP, nonhuman primates (such as the cynomolgus monkey) respond similarly to humans (i.e., low to no hepatic response) and therefore are an appropriate human surrogate species. PROCEDURES This study was conducted in accordance with the Protocol dated June 11, 1998, and Protocol Amendment No. 1 through 3. The protocol and protocol amendments are in Appendix 1. Group Designations and Dose Levels Selection of animals for the study was based on clinical observations, body weights, and other data as appropriate. Animals were assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment group. 13 Covance 6329-230 3M T-6889.2 Dose Level Total Material Dose Level Group (mg/kg/day) (mg/kg/day) Number of Males 1 (Control) 0a 0a 2 2 (Low-dose) 2 2 3 3 (High-dose) 20 20 3 a The control group (Group 1) received the same size and number of empty gelatin capsules as used for Group 3. Dose Preparation Gelatin capsules (Torpac, Inc., Fairfield, New Jersey), Size No. 2, Lot Nos. 122630 (expiration date June 26, 2002) and 122932 (expiration date June 12, 2003), were used for dose administration. The test material was dispensed into capsules weekly. The dose levels were based on the test material as supplied. For Groups 2 and 3, the specified amount of test material was weighed and transferred into the gelatin capsules. The top and bottom halves of each capsule were joined, and the capsules were placed into the appropriate container labeled with study number, group number, animal number, and dose level. The prepared capsules were stored at room temperature. Dose Analyses Because the test material was not being mixed with a vehicle, dose analyses were not required. Method of Administration Gelatin capsules were used for test material administration to compare with previously conducted toxicology studies using the oral route. The dose preparations were administered orally in gelatin capsules once daily (7 days/week) for 29 days. The dosing tubes were flushed with 5 mL of reverse osmosis water. Individual doses were calculated based on the most recently recorded body weights, with the exception of body weight collection days when the previous body weight was used. Animals were dosed at approximately the same time each day. 14 Covance 6329-230 3M T-6889.2 Clinical Observations The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Each animal was observed daily, and food consumption was assessed qualitatively; abnormal findings were recorded. Animals were observed approximately 30, 60, and 90 minutes postdose for signs of poor health or abnormal behavior. Effects were recorded as they were observed. Animals were observed once weekly; abnormal findings or an indication of normal was recorded. Body Weights Individual body weight data were recorded before initiation of treatment, on the first day of treatment, and weekly thereafter. An additional body weight was recorded on Day -1 for the Day 1 dose calculations. Blood Hormone Determination Blood was collected from a femoral vein of each animal before initiation of treatment (Day -8) and on Day 30 (after 29 days of treatment). Blood was collected between 07:00 and 09:00. Animals were fasted overnight before collections. Approximately 3 mL (Day -8) or 6 mL (Day 30) of blood for plasma samples were collected into tubes with potassium EDTA at the anticoagulant. Approximately 5 mL (Day -8) or 6 mL (Day 30) of blood for serum samples were collected without anticoagulant and allowed to clot for serum samples. Blood samples for plasma (Day 30) were maintained chilled until plasma was harvested. Blood samples collected on Day -8 for serum and plasma and blood samples collected on Day 30 for serum were maintained at room temperature until serum and plasma were harvested. Samples for serum were centrifuged within 1 hour after collection, and serum was harvested. Serum was divided into two approximately equal aliquots and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to Ani Lytics Inc., for analyses. The serum samples were analyzed for estradiol (E2), estrone (E1), estriol (E3), thyroid stimulating hormone (TSH), total and free triiodothyronine (T3 and FT3, respectively), and total and free thyroxin (T4 and FT4, respectively). Results of these analyses provided by Ani Lytics Inc., are in Appendix 6. Samples for plasma were centrifuged within 1 hour after collection. Plasma was harvested and stored in a freezer set to maintain -60 to -80C until packed on dry ice and 15 Covance 6329-230 3M T-6889.2 shipped to DuPont Haskell Company for possible future analyses. Results of the analyses for cholecystokinin, if any, will be reported separately. Serum APFO Level Determination Approximately 2 mL of whole blood were collected from a femoral vein of each animal before initiation of treatment (Day -8) and on Day 30 (after 29 days of treatment). Animals were fasted overnight before collections. All samples were collected without anticoagulant, maintained at room temperature, and allowed to clot. Samples were centrifuged within 1 hour after collection, serum was harvested and stored in a freezer set to maintain -10 to -30C until packed on dry ice and shipped to 3M for analyses. The samples were analyzed for APFO. Results of analyses will be reported separately by 3M. Clinical Pathology Blood samples were collected from each animal twice before initiation of treatment (Days -8 and -5) and on Day 30 (after 29 days of treatment) for hematology and clinical chemistry tests. In addition, blood for clinical chemistry tests was collected from each animal on Day 2 (approximately 24 hours after the first dose). Animals were fasted overnight; water was provided ad libitum. Blood was collected from a femoral vein. The following were evaluated: Hematology red blood cell (erythrocyte) count hemoglobin hematocrit mean corpuscular volume mean corpuscular hemoglobin mean corpuscular hemoglobin concentration platelet count white blood cell (leukocyte) count differential blood cell count segmented neutrophil count lymphocyte count monocyte count eosinophil count basophil count blood cell morphology reticulocyte count 16 glucose urea nitrogen creatinine total protein albumin globulin total bilirubin cholesterol triglycerides aspartate aminotransferase alanine aminotransferase alkaline phosphatase Covance 6329-230 3M T-6889.2 Clinical Chemistry gamma glutamyl transferase sorbitol dehydrogenase creatine kinase calcium inorganic phosphorus sodium potassium chloride bile acids amylase lipase pancreatic-specific amylase Additional Blood Collection Whole blood (approximately 15 mL) was collected from the vena cava of each animal at the time of exsanguination. Approximately equally sized samples (approximately 5 mL each) of serum (collected without anticoagulant) and whole blood and plasma using potassium EDTA as the anticoagulant were transferred into containers and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to 3M for possible future analysis. Necropsy On Day 30, animals that were fasted overnight were anesthetized with ketamine and xylazine, weighed, bled for the additional blood collection, exsanguinated, and necropsied. Animals were necropsied in random order. The necropsy included a macroscopic examination of the external surface of the body; all orifices; the cranial cavity; the brain and spinal cord; the nasal cavity and paranasal sinuses; cervical tissues and organs; and the thoracic, abdominal, and pelvic cavities and viscera. Palmitoyl CoA Oxidase Determinations Representative samples of the right lateral lobe of liver were collected from each animal at the scheduled sacrifice. The sample was weighed, flash-frozen in liquid nitrogen, and 17 Covance 6329-230 3M T-6889.2 stored in a freezer set to maintain -60 to -80oC until analyzed for palmitoyl CoA oxidase activity. Cell Proliferation Evaluation Representative samples of the left lateral lobe of the liver, left and right testes, and pancreas were collected and preserved in zinc formalin. After fixation, samples for proliferation cell nuclear antigen (PCNA) evaluation were embedded in paraffin and maintained at ambient temperature (with slides stained with hematoxylin and eosin) until shipped to Pathology Associates, A Charles River Company for PCNA analyses. Results of the evaluation provided by Pathology Associates, A Charles River Company are in Appendix 7. Receptor Level Determination Samples (approximately 2 g) of liver and pancreas was collected from each animal at the scheduled sacrifice, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to the DuPont Haskell Company for possible analysis. Results of the receptor level determination, if any, will be reported separately. Bile Acid Determination Approximately 1.0 to 2.0 mL of bile were collected from each animal at the scheduled sacrifice, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to the University of Dundee for bile acid determination. Results of the bile acid determination will be reported separately by the University of Dundee. Liver APFO Determination A section of liver (approximately 20 g) was collected from each animal at the scheduled sacrifice, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped with plasma samples to 3M for APFO analysis. Results of these analysis will be reported separately by 3M. 18 Covance 6329-230 3M T-6889.2 Tissue Preservation The following tissues (when present) or representative samples were collected and preserved in 10% neutral-buffered formalin, unless otherwise specified, for possible future microscopic examination: adrenal (2) aorta brain cecum colon duodenum epididymis (2) esophagus eyes [preserved in Davidson's fixative(2)] femur with bone marrow (articular surface of the distal end) gallbladder heart ileum jejunum kidney (2) lesions liver lung lymph node (mesenteric) mammary gland muscle (thigh) pancreas pituitary prostate rectum salivary gland [mandibular (2)] sciatic nerve seminal vesicle (2) skin spinal cord (cervical, thoracic, and lumbar) spleen sternum with bone marrow stomach testis (2) thymus thyroid (2) with parathyroid trachea urinary bladder Histopathology The adrenals, liver, pancreas, spleen, and testes were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from each animal. Statistical Analyses Only data collected on or after the first day of treatment were analyzed statistically. One-way analysis of variance [ANOVA (Winer, 1971a)] was used to analyze initial body weights; palmitoyl CoA oxidase activities, and continuous clinical pathology values. Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations were used to stabilize the variance. 19 Covance 6329-230 3M T-6889.2 ANOVA was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t-test (Dunnett, 1964) was used for control versus treated group comparisons. One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyze body weights, with initial body weights as the covariate. Although Levene's test for variance homogeneity was done (see above), no transformations were used because covariance adjustment removed extraneous heterogeneity. If the ANCOVA was significant, least squares means t-test was used for control versus treated group comparisons. Groups 2 and 3 were compared with Group 1 (control). Group comparisons were evaluated at the 5.0%, two-tailed probability level. RECORD RETENTION All raw data, documentation, records, protocol, and specimens generated as a result of this study will be archived in the storage facilities of Covance-Madison for a period of 1 year. One year after the submission of the final report, the Sponsor will determine the final disposition of the materials. All raw data stored on magnetic media and an original copy of the final report will be retained by Covance-Madison. Within 1 year after submission of the final report, all the aforementioned materials from the Sponsor's designees (Ani Lytics Inc., DuPont Haskell Company, Pathology Associates International, and the University of Dundee) will be sent to the Sponsor (Paul Lieder, PhD, DABT, 3M). Pathology Associates, A Charles River Company (PAI) is responsible for maintenance of any raw data or specimens produced by PAI. RESULTS Clinical Observations Clinical observations are summarized in Tables 1 through 4; individual data are in Appendix 2. Individual animal fate data are also in Appendix 2. All animals survived to the scheduled sacrifice. There were no test material-related clinical observations. 20 Covance 6329-230 3M T-6889.2 On Day 23, Animal No. I05419 (Group 2) was observed with a 4-cm laceration on the inner left thigh. The animal was anesthetized with ketamine, and the wound was cleaned and sutured. This condition was not considered to be test material-related. Body Weights Body weight data are summarized in Table 5; individual data are in Appendix 3. There were no test material-related effects on body weights. Food Consumption Food consumption data are summarized in Table 1 (Summary of Clinical Observation - A.M./Weekly); individual data are included in the individual clinical observations in Appendix 2. Low or no food consumption was noted for one animal (Animal No. I05235) given 20 mg/kg/day. For this animal, no food consumption was noted on Day 12, and low food consumption was noted on Days 5, 7, 11, 14, 16, 17, and 24. The decrease in food consumption for this animal may be test material-related. Blood Hormone Analyses Summary and individual hormone analyses data are in Appendix 6. There were no apparent test material-related effects on estradiol, estriol, thyroid stimulating hormone, total and free triiodothyronine, and total and free thyroxin. Estrone levels were notably lower for males given 2 and 20 mg/kg/day. Clinical Pathology Hematology and clinical chemistry data are summarized in Tables 6 through 12; individual data are in Appendix 4. The Pathology Report contains a discussion of the data. Administration of APFO had no effects on clinical pathology test results. 21 Covance 6329-230 3M T-6889.2 Cell Proliferation Evaluation Results of cell proliferation evaluation provided by Pathology Associates, A Charles River Company are in Appendix 7. Cell proliferation, as determined by measuring the proliferating index, was not increased in the liver, testes or pancreas of monkeys. Thus, there did not appear to be a cell proliferative response to the test material in the liver, testes or pancreas, as determined by proliferating indices which were similar in all animals. Anatomical Pathology Incidences of macroscopic and microscopic observations are summarized in Tables 13 and 14. Individual data are in Appendix 5. The Pathology Report contains a discussion of the data. Administration of APFO had no effects on anatomical pathology test results. CONCLUSIONS Based on the results of this study, daily capsule administration of Ammonium Perfluorooctanoate to cynomolgus monkeys for 29 days at dose levels of 2 and 20 mg/kg/day was well tolerated and produced no apparent evidence of toxicity. 22 SIGNATURES Covance 6329-230 3M T-6889.2 23 Covance 6329-230 3M T-6889.2 REFERENCES Dunnett, C. W., "New Tables for Multiple Comparisons with a Control," Biometrics, 20:482-491 (1964). Levene, H., "Robust Tests for Equality of Variances," Contributions to Probability and Statistics, (eds.) I. Olkin et al., Ch. 25, pp. 278-292, Stanford University Press: Stanford, California (1960). Winer, B. J., "Design and Analysis of Single-Factor Experiments," Statistical Principles in Experimental Design, Second Ed., Ch. 3, pp. 149-260, McGraw-Hill: New York, New York (1971a). Winer, B. J., "Analysis of Covariance," Statistical Principles in Experimental Design, Second Ed., Ch. 10, pp. 752-812, McGraw-Hill: New York, New York (1971b). 24 PATHOLOGY REPORT Covance 6329-230 3M T-6889.2 SUMMARY The purpose of this study was to provide data for determining an estimated maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study and to assess the effect of the test material, Ammonium Perfluorooctanoate (APFO), on critical enzyme levels, hormones, and other selected biochemical parameters. Administration of APFO had no effects on clinical or anatomical pathology test results. METHODS Three groups of male cynomolgus monkeys were administered the test material orally via capsule at a dose level of 0 (control group; received empty gelatin capsules), 2, or 20 mg/kg of body weight/day (mg/kg/day) for 29 days. There were two animals in the control group and three animals in each of the groups receiving APFO. All animals survived to the scheduled sacrifice. Blood was collected for hematology and clinical chemistry tests twice before initiation of treatment (Days -8 and -5) and on Day 30. In addition, blood for clinical chemistry tests was collected on Day 2. The animals were sacrificed and necropsied on Day 30; macroscopic observations were recorded, and tissues were preserved in fixative as specified by the protocol. Microscopic examinations were done on adrenals, liver, pancreas, spleen, and testes from all animals. Samples of liver were collected and frozen for determination of hepatic palmitoyl CoA oxidase activity and for determination of APFO content (done by 3M). Samples of liver, testes, and pancreas were collected and preserved in zinc formalin for evaluation of proliferation cell nuclear antigen (PCNA; done by Pathology Associates, a Charles River Company). Samples of liver and pancreas were collected and frozen for possible receptor level determination (done by the DuPont Haskell Company). Samples of bile were collected and frozen for bile acid determination (done by the University of Dundee). Statistically significant differences cited in the Results and Discussion section are based on comparisons between the control and treated groups. 25 RESULTS AND DISCUSSION Mortality All animals survived to the scheduled sacrifice. Covance 6329-230 3M T-6889.2 Clinical Pathology Individual values for hematology and clinical chemistry tests are in Appendix 4. Mean values and the results of statistical comparisons are in Tables 6 through 12. Days -8 and -5. Clinical pathology test results indicated no obvious group or individual health abnormalities. Day 2. Administration of the test material had no effects on clinical chemistry test results at this collection interval. There were only two statistically significant differences for clinical chemistry results between the control and treated groups; lower glucose for the low-dose group and lower total bilirubin for the high-dose group. These differences were considered incidental because similar differences were present before treatment was initiated. There were no other notable differences for clinical chemistry results between the control and treated groups. Day 30. Administration of the test material had no effects on hematology or clinical chemistry test results at Day 30. The only statistically significant differences between the control and treated groups was lower glucose for the high-dose males. This difference was considered incidental because a similar difference was present before treatment was initiated. There were no other notable differences for clinical pathology results between the control and treated groups. Anatomical Pathology Individual animal pathology data are in Appendix 5. Incidence summaries of the macroscopic and microscopic observations are in Tables 13 and 14, respectively. 26 Covance 6329-230 3M T-6889.2 Macroscopic Findings. There were no test material-related gross findings noted at necropsy. A few commonly occurring macroscopic findings frequently observed as spontaneous lesion in monkeys were present. Microscopic Findings. There were no test material-related microscopic findings. The only microscopic findings were minimal mineralization of the adrenals for one male given 2 mg/kg/day and immature testes for one male given 2 mg/kg/day and one male given 20 mg/kg/day. There were no differences in the appearances of the testes of the other two males given 2 mg/kg/day or the other two males given 20 mg/kg/day. 27 Covance 6329-230 3M T-6889.2 COMMENTS ON THE DATA Various models of calculators, computers, and computer programs were used to analyze data in this study. Because different models round off or truncate numbers differently, values in some tables (e.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or from statistical analysis data. Neither the integrity nor the interpretation of the data was affected by these differences. Some tabular data were compiled using Excel Version 7.0 software. The summary tables for clinical observations indicate the number of animals for which a condition was observed without regard to the specific nature, severity, reversibility, number of incidences/animal, or the length of time the condition persisted. Only observations other than normal are indicated on the summary clinical observations tables. The day of initiation of treatment is "Day 1." Animal No. I05419 (Group 2) was observed and treated by the laboratory animal veterinary staff, and the data were recorded on the day the examination was done. These data are referenced in the Request for Veterinary Services and are archived with the raw data but do not appear on the summary or individual clinical observations tables. 28 CODES, ABBREVIATIONS, AND UNITS Covance 6329-230 3M T-6889.2 General Codes and Abbreviations Codes for Clinical Pathology Abbreviations and Units for Clinical Hematology Abbreviations and Units for Clinical Chemistry Codes for Anatomical Pathology Note: The following lists of codes, abbreviations, and units are used by Covance. Some, but not necessarily all, of this information may be needed for this report. 29 Covance 6329-230 3M T-6889.2 General Codes and Abbreviations WK N Mean; MEAN CAM SD; S.D.; STAND DEV; STANDARD DEV; sd * -; NA P UNSCHED DISPATCH MIN OBS Week. Number of measurements in a group. Arithmetic mean. Covariate-adjusted mean. Standard deviation. Group mean is significantly different from the mean of the control group (Group 1) at p < 0.05. No value; not applicable; not present. Present. Unscheduled. Observations transferred from the in-life module of the data collection system to the necropsy module for reference during necropsy. Observations are duplicates of the last in-life observations. Minute. A.M. daily/weekly observation. Animal Death Codes: T Terminal sacrifice. 30 NS QS/QNS NR FS SC SH H SL L SI I NF U DT/DOT DB TJ TE RE EE SE PC PD PI PL PA CO HB PLASMO NO AGG FR UTD NO COAG Covance 6329-230 3M T-6889.2 Codes for Clinical Pathology GENERAL CODES No sample Quantity not sufficient No repeat (sample volume not sufficient for repeat analysis) Fibrin strands Sample clotted Slightly hemolyzed Hemolyzed Slightly lipemic Lipemic Slightly icteric Icteric Animal not fasted Unscheduled/moribund bleed Animal died on test Died during bleeding Technician judgment to repeat test Technical error (instrument or technician error that results in unacceptable data, e.g., unacceptable instrument output, sample spilled, entry of invalid data) Recording error (recorded incorrect data, e.g., wrong number, spelling error, incorrect date) Entry error (incorrect keyboard entry) Sampling error Platelets clumped Platelets decreased Platelets increased Platelets large Platelets appear adequate Color interferes with test Heinz bodies observed Plasmodium No aggregation Fractious Unable to determine No coagulation 31 Covance 6329-230 3M T-6889.2 Codes for Clinical Pathology (Continued) RESULTS NOT INCLUDED IN STATISTICAL ANALYSES Hemolyzed clinical chemistry or coagulation samples Samples from animals at unscheduled intervals Prothrombin times (PT) greater than 50 seconds Activated partial thromboplastin times (PTT) greater than 110 seconds Bleed times (BLETIME) greater than 30 minutes CODES FOR BLOOD CELL MORPHOLOGY The following scale was used to measure the degree of anisocytosis (ANISO), poikilocytosis (POIK), polychromasia (POLY), hypochromasia (HYPO), and toxic neutrophils (TOXNEUT): Scale 1 2 3 4 Degree Normal for the species Slight Moderate Marked Not applicable Presence Not present Rare Few Moderate Many 32 Covance 6329-230 3M T-6889.2 Abbreviations and Units for Clinical Hematology Test Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Platelet count Mean platelet volume Reticulocyte count Absolute reticulocyte count Heinz body count Erythrocyte sedimentation rate Prothrombin time Activated partial thromboplastin time Thrombin time Activated coagulation time Fibrinogen Fibrin/fibrinogen degradation products Platelet aggregation Collagen Adenosine diphosphate Alpha 2-antiplasmin Bleeding time Methemoglobin Plasma hemoglobin Myeloid/erythroid ratio Estimated myeloid/erythroid ratio White blood cell count Differential blood cell count Nucleated red blood cell count Corrected white blood cell count Segmented neutrophil count Band neutrophil count Lymphocyte count Monocyte count Eosinophil count Basophil count Anisocytosis Polychromasia Abbreviation (Units) RBC (E6/UL or X106/pL) HGB (G/DL) HCT (%) MCV (FL) MCH (PG) MCHC (%) PLT (E3/UL or X103/pL) MPV (FL) RETIC (%) RETIC (e 3/UL or X103/pL) HEINZ (%) ESR (MM/HR) PT (SEC) PTT (SEC) TT (SEC) ACT (SEC) FBR (MG/DL) FDP (UG/Ml ) PAGG/COL (%) PAGG/ADP (%) ANTIPLAS (%) BLE TIME (SEC) METHGB (%) PLA HGB (MG/DL) M/E RATIO EST M/E RATIO WBC (E3/UL or X103/pL) NRBC (/100 WBC) COR WBC (E3/UL or X103/pL) N-SEG (E3/UL or X103/pL) and % N-BAND (E3/UL or X103/pL) and % LYMPH (E3/UL or X103/pL) and % MONO (E3/UL or X103/pL) and % EOSIN (E3/UL or X103/pL) and % BASO (E3/UL or X103/pL) and % ANISO (-,1,2,3) POLY (-,1,2,3) 33 Covance 6329-230 3M T-6889.2 Abbreviations and Units for Clinical Hematology (Continued) Test Poikilocytosis Hypochromasia Howell-Jolly bodies Basophilic stippling Toxic neutrophils Atypical lymphocytes Aqueous white blood cell count (right eye) Aqueous white blood cell count (left eye) Abbreviation (Units) POIK (-,1,2,3) HYPO (-,1,2,3) HJBODY (-,1,2,3,4) BASTIP (-,1,2,3) TOXNEUT (-,1,2,3,4) ATYPLYM (-,1,2,3,4) R EYE (WBC/UL) L EYE (WBC/UL) 34 Covance 6329-230 3M T-6889.2 Abbreviations and Units for Clinical Chemistry Test Glucose Urea nitrogen Urea Creatinine Total protein Albumin Globulin Albumin/globulin ratio Total bilirubin Direct bilirubin Indirect bilirubin Cholesterol Triglyceride Urea nitrogen/creatinine ratio Total lipids Phospholipids High-density lipoprotein cholesterol Low-density lipoprotein cholesterol Uric acid Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Gamma glutamyl transferase Sorbitol dehydrogenase Lactate dehydrogenase Creatine kinase Amylase Lipase Palmitoyl CoA oxidase Calcium Ionized calcium Inorganic phosphorus Sodium Potassium Chloride Magnesium Zinc Strontium Iron Abbreviation (Units) GLU (MG/DL) UN (MG/DL) UREA (MG/DL) CREAT (MG/DL) T PRO (G/DL) ALB (G/DL) GLOB (G/DL) A/G RATIO T BILI (MG/DL) D BILI (MG/DL) I BILI (MG/DL) CHOL (MG/DL) TRIG (MG/DL) UN/CREAT (RATIO) T LIPIDS (MG/DL) P LIPIDS (MG/DL) HDL (MG/DL) LDL (MG/DL) UA (MG/DL) AST/SGOT (IU/L) ALT/SGPT (IU/L) ALK PHOS (IU/L) GGT (IU/L) SDH (IU/l ) LDH (IU/L) CK (IU/L) AMYLASE (IU/L) LIPASE (IU/L) PCOAO (IU/G) CA (MG/DL) ION CA (MG/DL) I PHOS (MG/DL) NA (MMOL/L) K (MMOL/L) CL (MMOL/L) MG (MEQ/L or MG/DL) ZN (MG/L or PPM) SR (MG/L or PPM) FE (UG/DL) 35 Covance 6329-230 3M T-6889.2 Abbreviations and Units for Clinical Chemistry (Continued) Test Excess iron Total iron binding capacity Unbound iron binding capacity Percent iron saturation Plasma cholinesterase Red blood cell cholinesterase Brain cholinesterase Caudate putamen Hippocampus Frontal cortex Cerebellum Bicarbonate Serum hemoglobin Serum bile acids Fecal bile acids Average fecal weight Fecal bile acids (calculation) Osmolality Electrophoresis Albumin Alpha-1-globulin Alpha-2-globulin Beta globulin Gamma globulin High-density lipoprotein Low-density lipoprotein Very-low-density lipoprotein Insulin Adrenocorticotropic hormone Cortisol Glucagon Creatine kinase isoenzymes BB MB MM Pancreatic-specific amylase Abbreviation (Units) EX FE (UG/DL) TIBC (UG/DL) UIBC (UG/DL) FE %SAT (%) CHEP (MU/ML) CHER (MU/ML) CHEB (MU/ML) CAUD PUT (UMOL/G) HIPPOCAM (UMOL/G) F CORTEX (UMOL/G) CEREBELL (UMOL/G) BICARB (MMOL/L) SER HGB (MG/DL) SBA (UMOL/L or MG/DL) FBA (UG/ML) FCC WGT (G) FBA (MG/Day) OSMO (MOSM/KG) E ALB (G/DL) EA-1 (G/DL) E A-2 (G/DL) E BETA (G/DL) E GAMMA (G/DL) E-HDL (%) E-LDL (%) E-VLDL (%) INSULIN (UU/ML) ACTH (PG/ML) CORTISOL (UG/ML) GLUCAGON (PG/ML) CK-BB (U/L) CK-MB (U/L) CK-MM (U/L) P AMYL (U/L) 36 Covance 6329-230 3M T-6889.2 Codes for Anatomical Pathology MICROSCOPIC CODES Distribution of Findings Focal Diffuse Multifocal Grades for Severity or Amount 1 Minimal - the least amount of change that can be observed with the light microscope 2 Slight - less than average amount of change, but readily discernible as abnormal 3 Moderate - the average amount of change that is expected for a lesion 4 Moderately severe (marked) - a marked amount of change with possible loss of function of the affected cells or organs 5 Severe - a great amount of change with probable loss of function of the affected cell or organs and frequently involves large areas of the organ Abbreviation STOMACH, GL TISSUE ABBREVIATIONS Definition Glandular stomach 37 DAYS 1-30 CATEGORY KEYWORD QUALIFIER *** TOP OF LIST *** EXCRETION NO FECES SKIN & PELAGE BROKEN SKIN THIGH-LEFT SCAB(S) LIMB-HIND-LEFT QUALITATIVE FOOD CONSUMPTION LOW NONE *** END OF LIST *** Table 1 Summary of Clinical Observations A.M./Weekly 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS NUMBER OF ANIMALS AFFECTED SEX GROUP DOSE -MALE--123 0 2 20 NUMBER : 2 3 3 001 010 010 111 001 Covance 6329-230 3M T-6889.2 PAGE: 1 38 DAYS 1-29 CATEGORY KEYWORD QUALIFIER *** TOP OF LIST *** SKIN & PELAGE BROKEN SKIN DIGIT(S)-HIND-RIGHT THIGH-LEFT SCAB(S) LIMB-HIND-LEFT *** END OF LIST *** Table 2 Summary of Clinical Observations 30 Minutes Postdose 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS NUMBER OF ANIMALS AFFECTED SEX: GROUP : DOSE: -MALE123 0 2 20 NUMBER : 2 3 3 100 010 010 Covance 6329-230 3M T-6889.2 PAGE: 1 39 DAYS 1-29 CATEGORY KEYWORD QUALIFIER *** TOP OF LIST *** SKIN & PELAGE BROKEN SKIN DIGIT(S)-HIND-RIGHT THIGH-LEFT SCAB(S) LIMB-HIND-LEFT *** END OF LIST *** Table 3 Summary of Clinical Observations 60 Minutes Postdose 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS NUMBER OF ANIMALS AFFECTED SEX: GROUP : DOSE: -MALE123 0 2 20 NUMBER : 2 3 3 100 010 010 Covance 6329-230 3M T-6889.2 PAGE: 1 40 Table 4 Summary of Clinical Observations 90 Minutes Postdose 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DAYS 1-29 CATEGORY KEYWORD QUALIFIER *** TOP OF LIST *** APPEARANCE UNDER EFFECTS OF ANESTHESIA SKIN & PELAGE BROKEN SKIN DIGIT(S)-HIND-RIGHT THIGH-LEFT SCAB(S) LIMB-HIND-LEFT *** END OF LIST *** NUMBER OF ANIMALS AFFECTED SEX GROUP DOSE -MALE--123 0 2 20 NUMBER : 2 3 3 010 100 010 010 Covance 6329-230 3M T-6889.2 PAGE: 1 41 Table 5 Summary of Body Weight Data (kg) 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS WEEK SEX: GROUP: DOSE: UNITS: 1 0 MG/KG/DAY --MALE----2 2 MG/KG/DAY 3 20 MG/KG/DAY -2 N MEAN S.D. a -1 N MEAN S.D. b -1 N MEAN S.D. 1N MEAN S.D. 2N CAM MEAN S.D. 3N CAM MEAN S.D. 4N CAM MEAN S.D. 5N CAM MEAN S.D. a Day -7. b Day -1. 2 3.0 0.71 2 3.0 0.71 2 3.1 0.78 2 3.1 0.71 2 2.9 3.1 0.57 2 2.9 3.2 0.64 2 2.9 3.1 0.64 2 2.9 3.2 0.64 3 2.8 0.70 3 2.9 0.75 3 2.8 0.75 3 2.9 0.75 3 2.9 2.9 0.70 3 3.0 2.9 0.70 3 2.9 2.9 0.70 3 3.0 3.0 0.75 3 2.7 0.50 3 2.8 0.50 3 2.8 0.50 3 2.8 0.55 3 2.9 2.8 0.56 3 2.9 2.8 0.56 3 2.9 2.8 0.50 3 3.0 2.9 0.56 42 Covance 6329-230 3M T-6889.2 PAGE: 1 Table 6 Summary of Clinical Hematology Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE RBC HGB HCT MCV MCH MCHC PLT RETIC RETIC mg/kg/day X106/p,L G/DL % FL PG % X103/p,L % X103/p,L Covance 6329-230 3M T-6889.2 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 5.96 2.157 2 6.85 1.196 3 6.39 .925 3 12.6 .92 2 13.6 .57 3 13.3 .96 3 40.5 6.65 2 44.2 1.21 3 42.3 .72 3 70.4 14.28 2 65.8 11.04 3 67.2 10.84 3 22.2 6.43 2 20.3 4.63 3 21.2 4.78 3 31.3 2.83 2 30.7 1.80 3 31.4 2.22 3 430 75.7 2 422 41.1 3 522 131.7 3 .4 .42 2 .6 .57 3 .3 .23 3 19 17 2 34 30 3 20 10 3 43 DOSE WBC mg/kg/day X103/p,L Table 6 Summary of Clinical Hematology Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS N-SEG LYMPH X103/ iL X103/ iL MONO EOSIN BASO X103/fJ,L X103/ iL X103/ iL N-SEG% LYMPH% MONO% EOSIN% Covance 6329-230 3M T-6889.2 BASO% 0 MEAN 10.4 1.8 7.4 .6 .6 .0 17 72 6 6 0 S.D. 1.63 1.13 .64 .28 .71 .00 8.5 17.0 2.1 6.4 .0 N 22222222222 2 MEAN 9.4 2.6 5.9 .9 .1 .0 27 63 9 1 0 S.D. 1.01 1.51 1.40 .42 .06 .00 15.5 18.1 3.2 .6 .0 N 33333333333 20 MEAN 9.6 2.8 6.0 .5 .3 .0 28 63 62 0 S.D. 1.10 1.83 1.38 .15 .15 .06 16.6 18.1 1.2 1.5 .6 N 33333333333 44 Table 7 Summary of Clinical Hematology Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE RBC HGB HCT MCV MCH MCHC PLT RETIC RETIC mg/kg/day X106/p,L G/DL % FL PG % X103/p,L % X103/p,L Covance 6329-230 3M T-6889.2 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 5.47 1.711 2 5.91 1.061 3 5.75 1.057 3 11.5 .35 2 11.8 .49 3 11.9 .42 3 37.3 4.45 2 38.4 1.17 3 37.9 1.48 3 70.6 14.00 2 66.3 10.81 3 67.3 10.68 3 22.0 6.29 2 20.6 4.93 3 21.2 4.83 3 31.0 2.83 2 30.8 2.22 3 31.4 2.20 3 404 61.5 2 395 22.1 3 500 101.4 3 .6 .85 2 .9 .61 3 .2 .12 3 26 36.1 2 51 24.5 3 9 4.4 3 45 DOSE WBC mg/kg/day X103/p,L Table 7 Summary of Clinical Hematology Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS N-SEG LYMPH X103/ iL X103/ iL MONO EOSIN BASO X103/fJ,L X103/ iL X103/ iL N-SEG% LYMPH% MONO% EOSIN% Covance 6329-230 3M T-6889.2 BASO% 0 MEAN 11.1 2.0 7.8 .7 .6 .0 18 72 64 0 S.D. 1.56 1.13 .49 .14 .64 .07 7.8 14.1 .7 4.9 .7 N 22222222222 2 MEAN 10.7 4.3 5.6 .7 .2 .0 40 52 62 0 S.D. 3.18 1.81 1.76 .12 .10 .00 12.1 12.6 .6 .6 .0 N 33333333333 20 MEAN 10.5 4.1 5.4 .6 .2 .0 38 54 52 0 S.D. 3.10 1.89 .76 .32 .21 .00 9.0 12.7 2.1 1.7 .0 N 33333333333 46 Table 8 Summary of Clinical Hematology Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE RBC HGB HCT MCV MCH MCHC PLT RETIC RETIC mg/kg/day X106/p,L G/DL % FL PG % X103/p,L % X103/p,L Covance 6329-230 3M T-6889.2 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 5.96 1.457 2 6.74 .824 3 6.24 .814 3 12.4 .14 2 13.6 1.19 3 13.0 1.11 3 40.8 2.26 2 44.3 1.51 3 41.9 1.31 3 70.1 13.44 2 66.5 11.02 3 68.1 10.91 3 21.6 5.52 2 20.5 4.52 3 21.3 4.78 3 30.5 2.05 2 30.6 1.63 3 31.1 2.25 3 461 66.5 2 407 4.2 3 489 116.0 3 .2 .21 2 .2 .21 3 .2 .21 3 14 9 2 10 11 3 14 12 3 47 DOSE WBC mg/kg/day X103/p,L Table 8 Summary of Clinical Hematology Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS N-SEG LYMPH X103/ iL X103/ iL MONO EOSIN BASO X103/fJ,L X103/ iL X103/ iL N-SEG% LYMPH% MONO1 0 MEAN 9.8 1.6 7.2 .6 .4 S.D. .07 .64 .78 .21 .35 N 22222 2 MEAN 8.0 2.2 5.4 .3 .0 S.D. 2.29 .79 1.60 .15 .06 N 33333 20 MEAN 8.3 3.1 4.4 .6 .1 S.D. .85 1.11 1.31 .10 .12 N 33333 0 16 74 6 07 7.1 7.8 2.1 2222 0 27 67 5 00 4.6 6.6 2.9 3333 0 38 53 7 00 12.7 14.6 1.5 3333 Covance 6329-230 3M T-6889.2 BASO% 40 3.5 .7 22 10 .0 .0 33 21 .6 .6 33 48 DOSE mg/kg/day Table 9 Summary of Clinical Chemistry Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS GLU MG/DL UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA umol/L CHOL MG/DL Covance 6329-230 3M T-6889.2 TRIG MG/DL 0 MEAN S.D. N 74 20.5 2 22 7.8 2 1.0 .21 2 8.7 2.40 2 5.2 1.13 2 3.5 1.27 2 .4 .14 2 9 146 5.7 9.2 22 34 7 2 2 MEAN S.D. N 76 14.2 3 18 2.0 3 1.1 .26 3 8.4 1.30 3 5.1 .30 3 3.3 1.00 3 .3 .12 3 9 174 2.5 19.9 33 36 6 3 20 MEAN 64 23 1.2 8.0 4.8 3.3 .2 12 141 31 S.D. 5.1 2.3 .25 .55 .21 .75 .06 .6 12.7 6 N 3333333333 49 Table 9 Summary of Clinical Chemistry Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE AST/SGOT ALT/SGPT ALK PHOS mg/kg/day IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Covance 6329-230 3M T-6889.2 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 41 1.4 2 48 14.7 3 52 6.7 3 45 9.9 2 53 22.2 3 59 26.7 3 847 420.0 2 1013 106.1 3 948 65.5 3 122 11.3 2 159 45.1 3 136 35.5 3 2 348 492 .0 244.0 70.0 222 5 369 522 4.4 159.3 174.5 333 5 195 496 1.2 40.3 32.0 333 43 1.4 2 46 23.4 3 43 8.5 3 252 33.2 2 250 41.7 3 239 37.3 3 50 Table 9 Summary of Clinical Chemistry Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE mg/kg/day CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 11.4 1.70 2 11.0 .35 3 10.9 .53 3 7.2 .99 2 7.5 1.11 3 7.0 .29 3 158 11.3 2 158 4.6 3 154 1.5 3 6.6 .71 2 5.6 .35 3 6.1 .40 3 112 1.4 2 108 1.2 3 108 .6 3 Covance 6329-230 3M T-6889.2 51 DOSE mg/kg/day Table 10 Summary of Clinical Chemistry Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS GLU MG/DL UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA umol/L CHOL MG/DL Covance 6329-230 3M T-6889.2 TRIG MG/DL 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 84 41.0 2 66 6.5 3 67 14.4 3 18 9.2 2 15 1.0 3 20 5.1 3 1.2 .14 2 1.1 .21 3 1.1 .15 3 8.4 1.70 2 8.0 .85 3 8.0 .57 3 5.0 .78 2 4.8 .10 3 4.7 .25 3 3.4 .92 2 3.2 .90 3 3.4 .75 3 .6 .07 2 .3 .17 3 .4 .31 3 4 138 2.1 26.2 22 5 155 4.0 12.3 33 1 141 1.0 13.5 33 28 .7 2 34 3.8 3 39 11.0 3 52 Table 10 Summary of Clinical Chemistry Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE AST/SGOT ALT/SGPT ALK PHOS mg/kg/day IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Covance 6329-230 3M T-6889.2 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 38 4.9 2 39 13.3 3 48 9.8 3 43 12.7 2 49 15.2 3 51 14.5 3 788 393.9 2 930 83.5 3 896 54.7 3 112 6.4 2 137 39.7 3 121 38.2 3 3 494 495 .0 478.7 76.4 222 3 279 520 1.0 227.6 174.8 333 3 171 466 1.0 29.3 15.0 333 40 2.8 2 56 9.2 3 57 13.5 3 252 26.2 2 252 37.2 3 229 18.4 3 53 Table 10 Summary of Clinical Chemistry Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE mg/kg/day CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 10.9 1.27 2 10.4 .61 3 10.4 .51 3 6.8 1.27 2 6.4 1.04 3 6.2 .17 3 156 9.2 2 155 3.5 3 152 2.6 3 5.4 .92 2 5.2 .75 3 5.0 .58 3 108 2.1 2 110 2.6 3 107 1.7 3 Covance 6329-230 3M T-6889.2 54 DOSE mg/kg/day Table 11 Summary of Clinical Chemistry Data Males Day 2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS GLU MG/DL UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA umol/L CHOL MG/DL Covance 6329-230 3M T-6889.2 TRIG MG/DL 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 76 4.2 2 65 * 4.2 3 68 2.0 3 21 7.1 2 20 4.9 3 23 1.0 3 1.0 .21 2 1.0 .21 3 1.0 .12 3 9.0 2.19 2 8.4 .65 3 8.3 .51 3 5.2 .92 2 4.9 .10 3 4.8 .21 3 3.9 1.27 2 3.5 .75 3 3.5 .70 3 .4 .21 2 .3 .06 3 .1 * .06 3 10 4.9 2 9 3.0 3 14 4.4 3 152 7.8 2 169 22.3 3 132 10.0 3 28 7 2 52 8 3 45 11 3 55 Table 11 Summary of Clinical Chemistry Data Males Day 2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE AST/SGOT ALT/SGPT ALK PHOS mg/kg/day IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Covance 6329-230 3M T-6889.2 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 47 2.8 2 53 32.7 3 55 4.9 3 53 25.5 2 52 26.8 3 56 22.4 3 872 355.7 2 1029 113.3 3 921 124.2 3 125 5.7 2 148 44.9 3 126 39.1 3 1 225 .0 62.2 22 2 794 1.5 1173.2 33 1 165 1.0 44.4 33 477 59.4 2 483 144.3 3 479 40.1 3 29 1.4 2 44 7.5 3 33 12.2 3 249 29 2 244 42 3 234 32 3 56 Table 11 Summary of Clinical Chemistry Data Males Day 2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE mg/kg/day CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 11.6 1.63 2 10.5 .57 3 10.7 .83 3 8.8 .28 2 8.0 1.30 3 7.4 .90 3 162 12.0 2 155 2.5 3 156 4.2 3 6.2 .99 2 5.3 .44 3 5.5 .67 3 110 4.9 2 109 3.8 3 109 1.5 3 Covance 6329-230 3M T-6889.2 57 DOSE mg/kg/day Table 12 Summary of Clinical Chemistry Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS GLU MG/DL UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA umol/L CHOL MG/DL Covance 6329-230 3M T-6889.2 TRIG MG/DL 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 84 9.2 2 76 3.2 3 65 * 2.1 3 19 5.7 2 17 2.1 3 20 3.5 3 1.0 .07 2 1.1 .06 3 1.2 .35 3 8.6 1.77 2 8.3 .60 3 7.8 .47 3 5.1 .71 2 5.0 .17 3 4.5 .23 3 3.4 1.06 2 3.3 .75 3 3.3 .70 3 .5 .28 2 .1 .06 3 .1 .15 3 4 150 3.5 31.8 22 4 147 1.5 9.2 33 6 138 4.5 31.5 33 32 .7 2 43 3.2 3 52 10.6 3 58 Table 12 Summary of Clinical Chemistry Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE AST/SGOT ALT/SGPT ALK PHOS mg/kg/day IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Covance 6329-230 3M T-6889.2 PCOAO IU/G 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 52 .7 2 50 19.7 3 55 11.5 3 59 24.0 2 75 33.3 3 83 50.0 3 917 335.2 2 1111 88.9 3 704 87.8 3 110 11.3 2 136 35.8 3 101 33.2 3 2 511 471 1.4 446.9 28.3 222 2 324 500 1.0 220.4 155.0 333 2 207 441 1.5 9.0 71.8 333 42 16.3 2 38 7.8 3 39 11.6 3 242 6.4 2 244 49.9 3 218 52.8 3 2 .7 2 1 1.7 3 6 3.1 3 59 Table 12 Summary of Clinical Chemistry Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS DOSE mg/kg/day CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L 0 MEAN S.D. N 2 MEAN S.D. N 20 MEAN S.D. N 10.7 .71 2 10.8 .53 3 10.2 .70 3 7.2 1.77 2 6.7 1.06 3 5.5 .31 3 153 5.7 2 155 2.1 3 151 5.1 3 5.4 .49 2 5.2 .90 3 5.2 .66 3 106 2.8 2 109 2.9 3 109 2.1 3 Covance 6329-230 3M T-6889.2 60 TABLE 13 Incidence of Macroscopic Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 1 TABLE INCLUDES: SEX=ALL;GROUP=ALL;WEEKS=ALL DEATH=ALL;SUBSET=ALL -- N U M B E R - O F - A N I M A L S - A F F SEX: -MALE- --- GROUP: -1- -2- -3- ORGAN AND KEYWORD(S) OR PHRASE NUMBER: 2 3 3 ** TOP OF LIST ** GENERAL COMMENT (GC) ..................... ............ NUMBER EXAMINED: 2 3 3 EYES - DAVIDSONS NO MACROSCOPIC LESIONS 233 031 KIDNEY (KD) ............................... ............ NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 1 3 3 LIGHT FOCUS(I)/AREA(S) 100 LUNG (LU) ................................. ............ NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 2 3 2 ADHESION(S) 001 STOMACH, GL (ST) ......................... ............ NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 1 3 1 RED FOCUS(I)/AREA(S) ** END OF LIST ** 1 02 ECTED 61 TABLE 14 Incidence of Microscopic Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 1 TABLE INCLUDES: SEX=ALL;GROUP=ALL;WEEKS=ALL DEATH=ALL;FIND=ALL;SUBSET=ALL -- N U M B E R - O F - A N I M A L S - A F F E SEX: -MALE- --- GROUP: -1- -2- -3- ORGAN AND FINDING DESCRIPTION NUMBER: 2 3 3 ** TOP OF LIST ** LIVER (LI) ....................... .................... NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 2 3 3 CTED SPLEEN (SP) ...................... .................... NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 2 3 3 PANCREAS (PA) .................... .................... NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 2 3 3 TESTIS (TE) ...................... .................... NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 2 2 2 --IMMATURE 011 ADRENAL (AD) ..................... .................... NUMBER EXAMINED: 2 3 3 NOT REMARKABLE: 2 2 3 --MINERALIZATION 010 ** END OF LIST ** 62 APPENDIX 1 Protocol Protocol Amendment No. 1 Protocol Amendment No. 2 Protocol Amendment No. 3 Material Safety Data Sheet Certificate of Analysis Covance 6329-230 3M T-6889.2 63 C O V JV 1 tfci> THE DEVELOPMENT SERVICES COMPANY Sponsors: APME Ad-Hoc APFO Toxicology Working Group PROTOCOL Study Title: 4-Week Capsule Toxicity Study with Ammonium Perfluorooctonoate (APFO) in Cynomolgus Monkeys Date: June 11, 1998 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Study Identification: Proposal No. w6806a Covance 6329-230 64 Covance 6329-230 _____________________________________________________ ________________ Page 2 Study 4-Week Capsule Toxicity Study with Ammonium Perfluorooctonoate (APFO) in Cynomolgus Monkeys Purpose To provide data for determining an estimated maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study. In addition, the effect on critical enzyme levels, hormones, and other selected biochemical parameters will be determined. Sponsors APME Ad-Hoc APFO Toxicology Working Group Study Monitor Paul Lieder, PhD, DABT 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota, 55144-1000 Telephone No.: 612.737.2678 Facsimile No.: 612.733.1773 Alternate Study Monitor John Butenhoff, PhD, DABT 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota, 55144-1000 Telephone No.: 612.733.1962 Facsimile No.: 612.733.1733 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 65 Covance 6329-230 _________________________________________________________________________________ Page 3 Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7454 Study Director Peter J. Thomford, PhD Covance Laboratories Inc. Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 Toxicologist Dale Aldridge, BS Covance Laboratories Inc. Proposed Study Timetable Study Initiation Date: June 11, 1998 In-Life (Experimental) Start Date: June 17,1998 In-Life Termination Date: July 17, 1998 Experimental Termination Date: To be determined Audited Draft Report Date: November 3, 1998 Regulatory Compliance This study will be conducted in compliance with the Environmental Protection Agency Good Laboratory Practice Regulations as set forth in Title 40 of the US Code of Federal Regulations, Part 792, issued November 29, 1983 (effective December 29, 1983), and with any applicable amendments. Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. 66 Covance 6329-230 ________________________________________________________________ _________________ Page 4 Quality Assurance The protocol, study conduct, and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen evaluation data and report will be audited by the QAU of Pathology Associates International. The serum and liver APFO analysis and report will be audited by the QAU of 3M Environmental Laboratories. The blood hormone determinations and report will be audited by the QAUs of Ani Lytics Inc and DuPont, as appropriate. The receptor level determinations and report will be audited by the QAU of the University of Dundee. Test Material Identification Ammonium Perfluorooctonoate (APFO) Lot Number The lot number will be maintained in the raw data. Purity Responsibility of the Sponsor Stability Responsibility of the Sponsor Storage Conditions At room temperature Characteristics Information on synthesis methods, composition, or other characteristics that define the test material is on file with the Sponsor. Gelatin Capsules Capsules (Size No. 2) obtained from Torpac, Inc. (Fairfield, New Jersey); lot number will be supplied by the manufacturer. Gelatin capsules will be stored at room temperature. A copy of the Certificate of Analysis provided by the manufacturer will be maintained in the data. 67 Covance 6329-230 __________________________________________________________________________________ Page 5 Reserve (Archive) Samples A reserve sample (1 g each) of each lot of test material will be taken and stored at room temperature. These samples will be transferred to the Sponsor after completion of the in-life phase. Disposition of Test Material Any remaining test material will be returned after authorization from the Sponsor. Animals Species Cynomolgus monkey Source Covance Research Products Inc. Age at Initiation of Treatment Young adult/adult Weight at Initiation of Treatment 2 to 4 kg Number and Sex Eight males Identification Collar tag Husbandry Housing Individual. Animals will be housed in suspended, stainless steel cages. 68 Covance 6329-230 ________________________________________________________________________ Page 6 Diet Certified primate diet (#8726C, Harlan Teklad) once or twice daily. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Results of specified nutrient and contaminant analyses are on file at Covance-Madison. Fruit, vegetables, or other supplements may be provided but will not require analysis. Water Ad libitum. Samples of the water are routinely analyzed for specified microorganisms and environmental contaminants. The results are on file at Covance-Madison. Contaminants There are no known contaminants in the diet or water at levels that might interfere with this study. Environment Environmental controls for the animal room will be set to maintain 18 to 29C, a relative humidity of 30 to 70%, and a 12-hour light/12-hour dark cycle. Acclimation Minimum of 4 weeks Randomization Animals will be weighed, stratified by body weight, and allocated to the number of blocks equal to the number of animals to be selected for each group. Animals in each block will then be assigned to groups using computer-generated random numbers. Justification APFO is a known hepatic peroxisome proliferator (PP) in the rat. When exposed to PP, nonhuman primates (such as the cynomolgus monkey) respond similarly to humans (i.e., low to no hepatic response) and therefore are an appropriate human surrogate species. 69 Group Designations and Dose Levels Covance 6329-230 rage / Group Dose Level 'Total Material Dose Level (mg/kg/day) (mg/kg/day) Number of Males 1 (control) 2 (low-dose) 3 (high-dose) 0a 2 20 0a 2 20 2 3 3 a The control group (Group 1) will receive empty gelatin capsules Dosing Procedures Method of Administration Orally by gelatin capsules, daily for at least 28 days Reason for Dosing Route To compare with previously conducted toxicology studies using the oral route, which is the route of most likely exposure. Dose Preparation Capsules will be prepared at least weekly. The size and number of capsules will depend on the physical characteristics of the test material, the dose level, and the weight of the monkey. Individual daily doses will be based on the most recent individual body weights, with the exception of body weight collection days when the previous body weight will be used. Dose levels will be based on the test material as supplied. All dose preparations will be stored at room temperature until dosed. Dose Analysis Because the test material is not being mixed with a vehicle, dose analyses are not required. Observation of Animals Clinical Observations Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity; findings will be recorded as they are observed. 70 Covance 6329-230 _______________________________________________________________________________ Page 8 Each animal will be observed daily, and food consumption will be assessed qualitatively; abnormal findings will be recorded. Once weekly, each animal will be observed; abnormal findings, or an indication of normal will be recorded. During treatment, each animal will be observed for signs of poor health or abnormal behavior approximately 30, 60, and 90 minutes postdose. Additional findings will be recorded as they are observed. Body Weights Weekly before initiation of treatment, Day -1 (for the Day 1 dose calculations), on the first day of treatment, and weekly thereafter. Clinical Pathology Frequency Unscheduled Collections When possible, blood will be collected for clinical pathology tests from animals sacrificed at unscheduled intervals Scheduled Collections Hematology and clinical chemistry will be done twice before initiation of treatment and after at least 28 days of treatment; clinical chemistry will also be done on Day 2 (approximately 24 hours after the first dose). Method of Collection Animals will be fasted overnight; blood will be collected from a femoral vein (an alternative vein may be used if necessary). Anticoagulant will be potassium EDTA for hematology tests. No anticoagulant is used for clinical chemistry tests. 71 Tests Covance 6329-230 __________ Page 9 Hematology red blood cell (erythrocyte) count hemoglobin hematocrit mean corpuscular volume mean corpuscular hemoglobin mean corpuscular hemoglobin concentration platelet count white blood cell (leukocyte) count differential blood cell count blood cell morphology reticulocyte count Clinical Chemistry glucose urea nitrogen creatinine total protein albumin globulin total bilirubin direct bilirubin (if total bilirubin is greater than 2.0 mg/dL) cholesterol triglycerides alanine aminotransferase alkaline phosphatase aspartate aminotransferase gamma glutamyltransferase sorbitol dehydrogenase creatine kinase calcium inorganic phosphorus sodium potassium chloride bile acids amylase lipase pancreatic-specific amylase Blood Hormone Determination Frequency Before initiation of treatment and after 28 days of treatment; blood will be collected between 7:00 a.m. and 9:00 a.m. Number of Animals All 72 Covance 6329-230 ________________________________________ _______________________ _____________ Page 10 Method of Collection Animals will be fasted overnight; blood will be collected from a femoral vein (an alternative vein may be used if necessary). Approximately 5 mL of blood for plasma samples will be collected with EDTA as anticoagulant. Approximately 5 mL of blood for serum samples will be collected without anticoagulant and allowed to clot. Serum Sample Handling Samples for serum will be centrifuged within 1hour after collection, and serum will be harvested. Serum will be divided into two approximately equal aliquots and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to: Dr. Saroj Das Ani Lytics Inc. 200 Girard Street, Suite 200 Gaithersburg, Maryland 20877 Telephone No.: 301.921.0168 Facsimile No.: 301.977.0433 Ani Lytics Inc. will be notified regarding shipment of samples. Serum Tests Samples will be analyzed by Ani Lytics Inc., for estradiol, estrone, estriol, thyroid stimulating hormone, triiodothyronine (T3), and thyroxin (T4). Results will be provided for inclusion in the final report. Plasma Sample Handling Samples for plasma will be centrifuged within 1hour after collection. Plasma will be harvested and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to: 73 Covance 6329-230 __________________________________________________________ ______________________ Page 11 Jon C. Cook, PhD, DABT E. I. du Pont de Nemours & Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Building 14 Elkton Road Newark, Delaware 19714-0500 Telephone No.: 302.366.5495 Facsimile No.: 302.366.5003 DuPont will be notified regarding shipment of samples. Plasma Analysis Samples will be analyzed by DuPont Haskell Company for cholecystokinin. Results will be provided for inclusion in the final report. Serum APFO Level Determination Frequency Once before initiation of treatment and after at least 28 days of treatment Number of Animals All Method of Collection Animals will be fasted overnight; blood (approximately 2 mL) will be collected from a femoral vein (an alternative vein may be used if necessary) without an anticoagulant. Sample Handling Samples will be centrifuged within 1 hour after collection, and serum will be harvested. Serum samples will be stored in a freezer set to maintain -10 to -30C. Sample Shipping Serum samples will be packed on dry ice and shipped to: 74 Covance 6329-230 ____________________________________ ____________________ _______________________ Page 12 Kris J. Hansen, PhD 3M E.T. & S Bldg. 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55106 Kris J. Hansen, PhD or her designee will be notified regarding the shipment of the samples. Results will be provided for inclusion in the final report. Additional Blood Collection At scheduled and unscheduled necropsies, blood (as much as possible, up to 20 mi,) will be collected from animals at the time of exsanguination. Blood from each animal will be transferred into containers (approximately 5 mL each) and stored in a freezer set to maintain -60 to -80C until shipped to the Sponsor for possible future analysis. Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with ketamine and xylazine, weighed, bled for the additional blood collection, and exsanguinated. Scheduled Sacrifice After at least 4 weeks of treatment, all surviving animals will be fasted overnight, then anesthetized with ketamine and xylazine, weighed, bled for the additional blood collection, exsanguinated, and necropsied. Postmortem Procedures Necropsy The necropsy will include examination of: all orifices cranial cavity external surface of the brain; the external surface of the spinal cord and cut surfaces of the brain and spinal cord will be examined whenever tissue trimming is performed 75 Covance 6329-230 ______________________________________ _______________________ _________________ Page 13 cervical tissues and organs thoracic, abdominal, and pelvic cavities and viscera external surface of the body nasal cavity and paranasal sinuses Palmitoy! CoA Oxidase Determinations Representative samples of the right lateral lobe of liver will be collected from each animal at the scheduled sacrifice, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until analyzed for palmitoyl CoA oxidase activity. Cell Proliferation Evaluation Representative samples of the left lateral lobe of the liver, left and right testes, and pancreas will be collected and preserved in zinc formalin. After fixation, samples for proliferation cell nuclear antigen (PCNA) evaluation will be embedded in paraffin and shipped to: Sandra R. Eldridge, PhD Pathology Associates International 15 Worman's Mill Court Suite I Frederick, Maryland 21701 Telephone No.: 301.663.1644 ext. 2201 Facsimile No: 301.663.8994 Pathology Associates International will be notified regarding shipment of samples. Results of PCNA analyses will be provided for inclusion in the final report. Receptor Level Determination All available bile (up to 5 mL) will be collected from each animal at the scheduled sacrifice, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C. Samples (approximately 2 g) of liver and pancreas will be collected from each animal at the scheduled sacrifice, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C. Bile, liver, and pancreas samples will be packed on dry ice and shipped to: 76 Covance 6329-230 __ _______________________________________ ___________________ _________________ Page 14 Dr. Cliff Elcombe Biomedical Research Center University of Dundee Dundee, Scotland Telephone No.: Oil 44 1382 632641 Facsimile No.: Oil 44 1382 668278 Results of analyses will be provided for inclusion in the final report. Liver APFO Determination A section (approximately 20 g) of liver will be collected from each animal at the scheduled sacrifice, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until shipped with plasma samples to 3M (Kris Hansen) for analysis. Results will be provided for inclusion in the final report. Tissue Preservation The following tissues (when present) from each animal will be preserved in 10% neutral-buffered formalin, unless otherwise specified, for possible future microscopic examination: adrenal (2) aorta brain cecum colon duodenum epididymis (2) esophagus eye [to be preserved in Davidsoris fixative for sacrificed animals only (2)] femur with bone marrow (articular surface o f the distal end) gallbladder heart ileum jejunum kidney (2) lesions liver lung mammary gland mesenteric lymph node pancreas pituitary prostate rectum salivary gland [mandibular (2)] sciatic nerve seminal vesicle (2) skeletal muscle (thigh) skin spinal cord (cervical, thoracic, and lumbar) spleen sternum with bone marrow stomach testis (2) thymus thyroid (2) with parathyroid trachea urinary bladder 77 Covance 6329-230 ___________________________________________ __________________ ___________________ Page 15 Histopathology The pancreas, liver, adrenals, spleen, and testes from each animal will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Reports A draft report that includes the following information will be prepared and submitted: Experimental Design and Methods Results mortality clinical observations body weights food consumption clinical pathology results palmitoyl CoA oxidase activities hormone analyses (provided by Ani Lytics and DuPont Haskell Company) receptor determination (provided by the Sponsor's designee) serum and liver APFO levels (provided by 3M) macroscopic observations microscopic observations cell proliferation evaluations (provided by the Sponsor's designee) Statistical Evaluation Levene's test will be done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations will be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration. One-way analysis of variance (ANOVA) will be used (if applicable) to analyze initial body weights, continuous clinical pathology values, and palmitoyl CoA oxidase activities. If the ANOVA is significant, Dunnett's t-test will be used for control versus treated group comparisons. One-way analysis of covariance (ANCOVA) will be used to analyze body weights, with initial body weights as the covariate. If the ANCOVA is significant, covariate-adjusted means will be used for control versus treated group comparisons. 78 Covance 6329-230 ____________ ________________________________________________________________ Page 16 Group comparisons (Groups 2 and 3 versus Group 1) will be evaluated at the 5.0% two-tailed probability level. Only data collected on or after the first day of treatment will be analyzed statistically. At the end of 1 year after issuance of the audited draft report, if no requested revisions or instructions to finalize have been communicated by the Sponsor, then the audited draft report will be considered 'final' and issued as the final report, signed by the study director, and submitted to the Sponsor. Any modifications or changes to the audited draft report requested 1 year after issuance will be performed at additional cost to the Sponsor. Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the storage facilities of Covance-Madison for a period of 1 year following submission of the final report to the Sponsor. One year after submission of the final report, all of the aforementioned materials will be sent to the Sponsor, and a return fee will be charged. The Sponsor may elect to have the materials retained in the Covance archives for an additional period of time, and Covance will charge a storage fee. If the Sponsor chooses to have Covance dispose of the materials, a disposal fee will be charged. All raw data stored on magnetic media will be retained by Covance. 79 80 C O V /H tfC > THE DEVELOPMENT SERVICES COMPANY PROTOCOL AMENDMENT NO. 1 Covance 6329-230 4-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys Sponsor: Study Monitor: Testing Facility: Study Director: APME Ad-Hoc APFO Toxicology Working Group Paul Lieder, PhD, DABT Covance Laboratories Inc., Madison, Wisconsin Peter J. Thomford, PhD This amendment modifies the following portions of the protocol: Effective June 11,1998 1. Page 1, Study Title and all other occurrences of the name of test material in the protocol. To correct the spelling of the test material, delete the name of the test material and replace with the following: Ammonium Perfluorooctanoate (APFO) l. Page 3, Regulatory Compliance. Because bile acid determinations done by the University of Dundee will not be in compliance with GLPs, delete the text in this section and replace with the following: This study will be conducted in compliance with the Environmental Protection Agency Good Laboratory Practice Regulations as set forth in Title 40 of the US Code of Federal Regulations, Part 792, issued November 29, 1983 (effective December 29, 1983), and with any applicable amendments except that bile acid determinations done by University of Dundee will not be done in compliance with GLPs. 81 Covance 6329-230 Protocol Amendment No. 1 ___________________________________________________ __ _________________________ Page 2 3. Page 4, Quality Assurance, Sentence 5. Because receptor level analysis will be done by DuPont Haskell Company, delete this sentence and replace with the following: The receptor level determinations and report will be audited by the QAU of the DuPont Haskell Company. Bile acid determinations done by the University of Dundee will not be audited. 4. Page 7, Dosing Procedures, Reason for Dosing Route. To correct the reason for the dosing route, delete the text in this section and replace with the following: To compare with previously conducted toxicology studies using the oral route 5. Page 10, Blood Hormone Determination, Method of Collection. To increase the volume of the blood collected for serum and plasma samples, to specify the use of serum separation tubes, and to specify the storage conditions of blood samples before serum or plasma samples are prepared, delete the text in this section and replace with the following: Animals will be fasted overnight; blood will be collected from a femoral vein (an alternative vein may be used if necessary). Approximately 6 mL of blood for plasma samples will be collected into tubes with potassium EDTA as the anticoagulant. Approximately 6 mL of blood for serum samples will be collected without anticoagulant and allowed to clot. Blood samples will be maintained at room temperature until serum is harvested. Blood samples will be maintained chilled until plasma is harvested. 6. Page 10, Blood Hormone Determination, Serum Tests, Sentence 1. To specify total and free triiodothyronine (T3) and total and free thyroxin (T4) tests that will be done, delete this sentence and replace with the following: Samples will be analyzed by Ani Lytics Inc., for estradiol, estrone, estriol, thyroid stimulating hormone, total and free triiodothyronine (T3), and total and free thyroxin (T4). 82 Covance 6329-230 Protocol Amendment No. 1 _______________________________________________ _______________________ Page 3 7. Page 11, Blood Hormone Determination, Plasma Sample Handling, Paragraph 2. To correct the shipping address, delete this paragraph and replace with the following: Jon C. Cook, PhD, DABT E. I. du Pont de Nemours & Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Building 11 Room 1325 1090 Elkton Road Newark, Delaware 19711-0500 Telephone No.: 302.366.5495 Facsimile No.: 302.366.5003 8. Page 12, Additional Blood Collection, Sentence 2. To clarify the processing and disposition of the additional blood samples, delete this sentence and replace with the following: Approximately equally sized samples of serum (collected without anticoagulant) and whole blood and plasma (both collected using potassium EDTA as the anticoagulant) from each animal will be transferred into containers (approximately 5 mL each). Whole blood, serum, and plasma samples will be stored in a freezer set to maintain -60 to -80C until shipped to the Sponsor for possible future analysis. 9. Page 13, Receptor Level Determination. To correct and clarify the process, delete this section and replace with the following: Receptor Level Determination Samples (approximately 2 g) of liver and pancreas will be collected from each animal at the scheduled sacrifice, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C. Liver and pancreas samples will be packed on dry ice and shipped to: 83 Covance 6329-230 Protocol Amendment No. 1 _________________________________________________________________________ Page 4 Jon C. Cook, PhD, DABT E. I. du Pont de Nemours & Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Building 11 Room 1325 1090 Elkton Road Newark, Delaware 19711-0500 Telephone No.: 302.366.5495 Facsimile No.: 302.366.5003 Results of receptor level determinations will be provided for inclusion in the final report. Bile Add Determination All available bile (up to 5 mL) will be collected from each animal at the scheduled sacrifice, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C. Bile samples will be packed on dry ice and shipped to: Biomedical Research Centre Level 5 Ninewells Hospital and Medical School University of Dundee Dundee DD1 9SY UK Dr Cliff Elcombe Biomedical Research Centre Ninewells Hospital and Medical School University of Dundee Dundee DD1 9SY, UK Telephone: +44 (0)1382-632641 Fax: +44 (0)1382-425586 Results of bile acid determinations will be provided for inclusion in the final report. 10. Page 15, Reports, Results. To include bile acid determinations in the list of items to be reported, add the following: Bile Acid Determinations (provided by the University of Dundee) 84 Covance 6329-230 Protocol Amendment No. 1 ___________________________________________________________________________ Page 5 11. Page 16, Record Retention. To clarify the record retention requirements for the Sponsor's designees, add the following: Within 1 year after submission of the final report, all of the aforementioned materials from the Sponsor's designees (Ani Lyrics Inc., E. I. du Pont de Nemours & Company, Inc., 3M E.T. & S, Pathology Associates International, and the University of Dundee) will be sent to the Sponsor (Paul Lieder, PhD, DABT, 3M). AMENDMENT APPROVAL 85 COVA'fJ'CT'' THE DEVELOPMENT SERVICES COMPANY PROTOCOL AMENDMENT NO. 2 Covance 6329-230 4-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys Sponsor: Study Monitor: Testing Facility: Study Director: APME Ad-Hoc APFO Toxicology Working Group Paul Lieder, PhD, DABT Covance Laboratories Inc., Madison, Wisconsin Peter J. Thomford, PhD This amendment modifies the following portions of the protocol. Effective December 15,1998 1. Page 13, Postmortem Procedures, Cell Proliferation Evaluation, Paragraph 3, Sentence 2. To include evaluation of slides stained with hematoxylin and eosin in the PCNA evaluation, delete this sentence and replace with the following. PCNA evaluation (including examination of slides stained with hematoxylin and eosin) will be done on the samples and results will be provided for inclusion in the final report. Effective April 8,1999 2. Page 16, Record Retention, Paragraph 2. To modify the record retention requirements for Pathology Associates International, delete this paragraph and replace with the following. Within 1 year after submission of the final report, all of the aforementioned materials from the Sponsor's designees (Ani Lytics Inc., E. I. du Pont de Nemours & Company, Inc., 3M E.T. & S, and the University of Dundee) will be sent to the Sponsor (Paul Lieder, PhD, DABT, 3M). Pathology Associates International (PAI) is responsible for the maintenance of any raw data or specimens produced by PAL 86 87 CO V/VftfCT^ THE DEVELOPMENT SERVICES COMPANY PROTOCOL AMENDMENT NO. 3 Covance 6329-230 4-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys Sponsor: Study Monitor: Testing Facility: Study Director: APME Ad-Hoc APFO Toxicology Working Group Paul Lieder, PhD, DABT Covance Laboratories Inc., Madison, Wisconsin Peter J. Thomford, PhD This amendment modifies the following portions of the protocol. Effective October 21,1999 1. Page 11, Blood Hormone Determination, Plasma Analysis. To reflect the Sponsor's decision to retain these samples for possible future analysis, delete the text in this section and replace with the following. Plasma samples for cholecystokinin determination will be stored in a freezer set to maintain -60 to -80C for possible future analysis. 2. Page 11, Serum APFO Level Determination, Sample Shipping, Paragraph 2, Sentence 2. To reflect the Sponsor's decision to report the results of specified analyses separately, delete this sentence and replace with the following. Results will be reported separately. 3. Page 13, Postmortem Procedures, Receptor Level Determination, Paragraph 2, Sentence 1. To reflect the Sponsor's decision to retain these samples for possible future analysis, delete this sentence and replace with the following. 88 Covance 6329-230 Protocol Amendment No. 3 _________________________________ _________________ ________ ________________ Page 2 Liver and pancreas samples for receptor level determination will be stored in a freezer set to maintain -60 to -80C for possible future analysis. 4. Page 14, Postmortem Procedures, Bile Acid Determination, Paragraph 2, Sentence 1. To reflect the Sponsor's decision to report the results of specified analyses separately, delete this sentence and replace with the following. Results will be reported separately. 5. Page 14, Postmortem Procedures, Liver APFO Determination, Sentence 2. To reflect the Sponsor's decision to report the results of specified analyses separately, delete this sentence and replace with the following. Results will be reported separately. 6. Page 15, Report, Results. To reflect the Sponsor's decision to report the results of specified analyses separately, delete this sentence and replace with the following. mortality clinical observations body weights food consumption clinical pathology results palmitoyl CoA oxidase activities hormone analyses (provided by Ani Lytics) macroscopic observations microscopic observations cell proliferation evaluations (provided by the Sponsor's designee) 89 90 Covance 6329-230 3M T-6889.2 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 Company. All rights reserved. Copying and/or downloading of this information for the purpose of properly utilizing 3M products is allowed provided that: 1) the information is copied in full with no changes unless prior agreement is obtained from 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-143 FLUORAD Brand Fluorochemical Surfactant ID NUMBER/U.P.C.: 98-0211-0008-0 00-51135-09138-8 98-0211-0891-9 98-0211-5489-7 00-51135- 0 2 9 4 1 -1 98-0211-6581 -0 98-0211-7394-7 00-51135-10762-1 ZF-0002-0378-4 ISSUED: January 29, 1998 SUPERSEDES: November 05, 1997 DOCUMENT: 10-3808-2 00-51135-09365-8 00-51135-10405-7 --- ;. INGREDIENT C.A.S. NO. PERCENT a...iONIUM PERFLUOROOCTANOATE....... .... AMMONIUM PERFLUOROHEPTANOATE...... .... AMMONIUM PERFLUOROPENTANOATE....... .... AMMONIUM PERFLUOROHEXANOATE........ ..... 3825-26-1 6130-43-4 68259-11-0 21615-47-4 93 1 1 0.1 - 97 -3 -3 -1 2. PHYSICAL DATA BOILING POINT:..... VAPOR PRESSURE:.... VAPOR DENSITY:..... EVAPORATION RATE:... SOLUBILITY IN WATER: SPECIFIC GRAVITY:... PERCENT VOLATILE:... pH: ................. VISCOSITY:......... MELTING POINT:..... N/A N/A N/A N/A apprec. 0.4 - 0.5 Water=1 (Bulk) N/A ca. 5 (0.5% Aqueous) N/D N/A PEARANCE AND ODOR: -T* ight colored ponder; slight odor. 91 MSDS: FC-143 FLUORAD Brand Fluorochemical Surfactant January 29, 1998 Covance 6329-230 3M T-6889.2 PAGE 2 3. FIRE AND EXPLOSION HAZARD DATA FLASH POINT:................... Non-flammable FLAMMABLE LIMITS - LEL:...... N/A FLAMMABLE LIMITS - U E L : ...... N/A AUTOIGNITION TEMPERATURE:..... N/A EXTINGUISHING MEDIA: Water, Carbon dioxide, Dry chemical, Foam SPECIAL FIRE FIGHTING PROCEDURES: Wear full protective clothing, including helmet, self-contained, positive pressure or pressure demand 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 Nitrogen, Hydrogen Fluoride, Ammonia. 5. ENVIRONMENTAL INFORMATION SPILL RESPONSE: Observe precautions from other sections. Collect spilled material. Use wet sweeping compound or water to avoid dusting. Clean up residue. Place in a closed container. RECOMMENDED DISPOSAL: 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 accept chemical waste. 92 . FC-143 FLUORAD Brand Fluorocheraical Surfactant .nuary 2 9 , 1998 Covance 6329-230 3M T-6889.2 PAGE 3 5. ENVIRONMENTAL INFORMATION (continued) ENVIRONMENTAL DATA: SUPPORTING DATA: BIODEGRADATION: Theoretical Oxygen Demand (ThOD): 0.320 g/g Chemical Oxygen Demand (COD): 0.0007 g/g 20-Day Biochemical Oxygen Demand (BOD20): Nil AQUATIC TOXICITY: Fathead minnow (Pimephales promelas) 96-hr LC50: 766 mg/L Water flea (Daphnia magna) 48-hr EC50: 632 mg/L Bluegill sunfish (Lepomis macrochirus) 96-hr EC50: 569 mg/L Green Algae (Selenastrum capricornutum) 14-Day EC50: >1000 mg/L Microtox (Photobacterium phosphoreum) 30-min EC50: 730 mg/L BIONCONCENTRATION: BCF = 1.8 REGULATORY INFORMATION: Volatile Organic Compounds: N/A. VOC Less H20 & Exempt Solvents: N/A. jince regulations vary, consult applicable regulations or authorities before disposal. U.S. EPA Hazardous Waste Number = None (Not U.S. EPA Hazardous). The components of this product are in compliance with the chemical registration requirements of TSCA, EINECS, CDSL, AICS, MITI and KTCCL. OTHER ENVIRONMENTAL INFORMATION: This substance did not degrade significantly in a ready biodegradation test. This compound is completely fluorinated (perfluorinated), or it contains perfluorinated portions. Perfluoroalkyl groups resist degradation in most natural environments. This substance has minimal toxicity to aquatic organisms (100 mg/L < Lowest LC50, EC50, or IC50 < or = 1000 mg/L). No data are available on the toxicity effects of this substance on wastewater treatment system organisms. This substance has been found to not bioconcentrate or accumulate significantly in one or more living organisms. EPCRA HAZARD CLASS: FIRE HAZARD: No PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 93 MSDS: FC-143 FLUORAD Brand Fluorochemical Surfactant January 29, 1998 Covance 6329-230 3M T-6889.2 PAGE 4 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: Flush skin with large amounts of water. If irritation persists, get medical attention. INHALATION: If sign s / s y m p t o m s occur, remove person to fresh air. If signs/symptoms continue, call a physician. IF SWALLOWED: Do not induce vomiting. Drink two glasses of water. Call a physician 7. PRECAUTIONARY INFORMATION " " PROTECTION: ivoid eye contact. Wear vented goggles. SKIN PROTECTION: Avoid skin contact. Wear appropriate gloves when handling this material. A pair of gloves made from the following material(s) are recommended: butyl rubber. Use one or more of the following personal protection items 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. Provide sufficient ventilation to maintain emissions below recommended exposure limits. If exhaust ventilation is not adequate, use appropriate respiratory protection. RESPIRATORY PROTECTION: Avoid breathing of airborne material. Select one of the following NIOSH approved respirators based on airborne concentration of contaminants and in accordance with OSHA regulations: full-face high efficiency filter respirator, full-face supplied air respirator. PREVENTION OF ACCIDENTAL INGESTION: 3 not eat, drink or smoke when using this product. Wash exposed areas thoroughly with soap and water. Wash hands after handling and before eating. 94 . FC-143 FLUORAD Brand Fluorochemical Surfactant unuary 29, 1998 Covance 6329-230 3M T-6889.2 PAGE 5 7. PRECAUTIONARY INFORMATION (continued) RECOMMENDED STORAGE: Do not store containers on their sides. Store at room temperature. Keep container dry. Keep container closed when not in use. **PREVENT MOISTURE CONTAMINATION TO KEEP POWDER FREE FLOWING.** FIRE AND EXPLOSION AVOIDANCE: Keep container tightly closed. No smoking while handling this material. HMIS HAZARD RATINGS: HEALTH: 3 FLAMMABILITY: 0 REACTIVITY: 0 PERSONAL PROTECTION: X (See precautions, section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT TYPE AUTH SKIN* AMMONIUM PERFLUOROOCTANOATE...... ... AMMONIUM PERFLUOROHEPTANOATE..... .... AMMONIUM PERFLUOROPENTANOATE...... ... IONIUM PERFLUOROHEXANOATE....... ... 0.01 0.1 0.1 0.1 MG/M3 MG/M3 MG/M3 MG/M3 TWA ACGIH Y TWA 3M Y TWA 3M Y TWA 3M Y ^KIN NOTATION: Listed substances indicated with 'Y' under SKIN refer to potential contribution to the overall exposure by the cutaneous route including mucous membrane and eye, either by airborne or, more particularly, by direct contact with the substance. Vehicles can alter skin absorption. SOURCE OF EXPOSURE LIMIT DATA: - ACGIH: American Conference of Governmental Industrial Hygienists - 3M: 3M Recommended Exposure Guidelines 8. HEALTH HAZARD DATA EYE CONTACT: Moderate Eye Irritation: signs/symptoms can include redness, swelling, pain, tearing, and hazy vision. Airborne product may cause eye injury consisting of corneal opacity. SKIN CONTACT: Product is not expected to be irritating to the skin. Mild Skin Irritation (after prolonged or repeated contact): signs/symptoms can include redness, swelling, and itching, r .ay be absorbed through the skin and persist in the body for an extended time. 95 Covance 6329-230 3M T-6889.2 MSDS: FC-143 FLUORAD Brand Fluorochemical Surfactant January 29, 1998 PAGE 6 8. HEALTH HAZARD DATA (continued) INHALATION: Illness requiring medical attention may result from a single exposure by inhalation to moderate quantities of this material. 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. Prolonged or repeated overexposure, above recommended guidelines, may cause: Liver Effects: signs/symptoms can include yellow skin(jaundice) and tenderness of upper abdomen. Repeated inhalation of airborne product above the exposure guideline can result in elevated organofluoride levels in the blood. r 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. CANCER: A mixture of ammonium perfluorooctanoate, ammonium perfluoroheptanoate, ammonium perfluoropentanoate and ammonium perfluorohexanoate, that was 93 to 97% AMMONIUM PERFLUOROOCTANOATE (3825-26-1) was fed to albino rats for 2 years, no compound induced carcinogenicity was found in the study. There were statistically significant compound related benign testicular tumors. In a second two-year study there were statistically significant compound related benign tumors in the liver, pancreas, and testis when compared to ad libitum and pair-fed controls. Based on the current knowledge, these findings have no human health implications. (3825-26-1) (1983 and 1993 studies conducted jointly by 3M and DuPont). MUTAGENICITY: Not mutagenic in invitro mutagenicity assays. Did not cause cell transformation in a mammalian cell transformation assay. PRODUCTIVE/DEVELOPMENTAL TOXINS: Not teratogenic in rabbits by oral administration. Not teratogenic to rats by gavage or inhalation exposures. 96 Covance 6329-230 3M T-6889.2 FC-143 FLUORAD Brand Fluorochemical Surfactant anuary 29, 1998 PAGE 7 8. HEALTH HAZARD DATA (continued) 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. ,A provides information in electronic form as a service to its customers r -. to the remote possibility that electronic transfer may have resulted _ 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. 97 Covance 6329-230 3M T-6889.2 Centre Analytical L aboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 Purity1 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-033 3M Product: Ammonium Perfluorooctanoate Test Control Reference #: TCR-99131-37, Lot #: 332 Purity: 95.2% Test Name Specifications Result 95.2% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total %Impurity (NMR) Total %Impurity (LC/MS) Total %Impurity (GC/MS) Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate ,5. Nitrite 6. Phosphate 7. Sulfate Organic Acids1(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis1: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Ammonium Analysis1 Ion Selective Electrode ' White, crystalline solid -i. ;:V: Theoretical Value = 22.3% Theoretical Value = 0.935% Theoretical Value = 3.25% Theoretical Value = 0% Theoretical Value = 66.1% Theoretical Value = 4.18% Conforms Positive 1. 0.001wt./wt.% 2. <0.001 wt./wt.% 3. 0.005 wt./wt.% 4. <0.001 wt./wt.% !: 5. <0.001 wt./wt.% 6. <0.001 wt./wt.% 7. <0.001 wt./wt.% 0.34 wt./wt.% 4.49 wt./wt.% None Quantified None Detected 99.7% 1. <0.015 wt./wt.% 2. <0.005 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt/wt.% 5. <0.006 wt./wt.% 6. <0.006 wt./wt.% 7. <0.040 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. <0.1 wt./vrt.% 4. <0.25 wt./wt.% 1. 18.9wt./wt.% 2. 1.31 wt./wt.% 3. 3.75 wt./wt.% 4. 4.34 wt./wt.% 5. 63.2 wt./wt.% 3.49 wt./wt. % COA023033-37.doc Page 1 o f 2 98 Covance 6329-230 3M T-6889.2 C entre A nalytical L ab oratories, Inc. 3048 Research Drive ' State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-033 3M Product: Ammonium Perfluorooctanoate Test Control Reference #: TCR-99131-37, Lot #: 332 Date o f Last Analysis: 12/15/00 Expiration Date: 12/15/01 Storage Conditions: < -10 C Re-assessment Date: 12/15/01 lPurity = 100% - (Total Metal impurities, 0.006% + Total NMR impurities, 0.34% Total LC/MS impurities, 4.49%) Total impurity from all tests = 4.84% Purity = 100% - 4.84% = 95.2% 2 TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 3Theoretical value calculations based on the empirical formula, CgFisC^'^NH^ (MW==431.1) LC/MS Purity Profile: Peak# 1 2 3 Total Retention Time (min) 12.140 13.504 14.099 - Mass(s) 269 331,319 369 - Identity c6 C7 h o m o lo g u e/F i3 PFDA - Area 167596 860991 21861700 22890287 % Area 0.73 3.76 4.49 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Date ytical Laboratories //fA Date Laboratory Manager, Centre Analytical Laboratories COA023033-37.doc Page 2 of 2 99 APPENDIX 2 Individual Animal Fate Data Individual Clinical Observations Covance 6329-230 3M T-6889.2 100 APPENDIX 2 Individual Animal Fate Data Covance 6329-230 3M T-6889.2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS PAGE: 1 ANIMAL DOSE NUMBER GROUP I05353 I05417 I05231 I05352 I05419 I05235 I05351 I05416 1 1 2 2 2 3 3 3 SEX MALE MALE MALE MALE MALE MALE MALE MALE DEATH CODE T T T T T T T T TYPE OF DEATH SCHEDULED SCHEDULED SCHEDULED SCHEDULED SCHEDULED SCHEDULED SCHEDULED SCHEDULED DESCRIPTION OF DEATH TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE TERMINAL SACRIFICE DATE OF DEATH 07/16/98 07/16/98 07/16/98 07/16/98 07/16/98 07/16/98 07/16/98 07/16/98 DAY OF STUDY 30 30 30 30 30 30 30 30 WEEK OF STUDY 5 5 5 5 5 5 5 5 101 Appendix 2 Individual Clinical Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 1 CATEGORY GROUP: Ml DOSE: 0 MG/KG/DAY 'C' - COMMENTS LISTED AT END OF OBSERVATIONS FOR SEX/GROUP ANIMAL DEATH WK OF KEYWORD OBS 30 MIN 60 MIN 90 MIN DISPATCH NUMBER CODE DEATH QUALIFIER DAYS 1-30 DAY I05353 T 5 SKIN & PELAGE BROKEN SKIN DIGIT(S)-HIND-RIGHT NORMAL NO REMARKABLE OBSERVATIONS I05417 T 5 NORMAL NO REMARKABLE OBSERVATIONS QUALITATIVE FOOD CONSUMPTION LOW 18 - P P P - 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 21 P - - - - 102 Appendix 2 Individual Clinical Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 2 CATEGORY GROUP: M2 DOSE: 2 MG/KG/DAY 'C' - COMMENTS LISTED AT END OF OBSERVATIONS FOR SEX/GROUP ANIMAL DEATH WK OF KEYWORD OBS 30 MIN 60 MIN 90 MIN DISPATCH NUMBER CODE DEATH QUALIFIER DAYS 1-30 DAY I05231 T 5 NORMAL NO REMARKABLE OBSERVATIONS I05352 T 5 NORMAL NO REMARKABLE OBSERVATIONS I05419 T 5 APPEARANCE UNDER EFFECTS OF ANESTHESIA SKIN & PELAGE BROKEN SKIN THIGH-LEFT SCAB(S) LIMB-HIND-LEFT NORMAL NO REMARKABLE OBSERVATIONS 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 23 - - - P - 24 P P P P 25 - P P P - 26 P P P P 27 P P P P 28 P P P P 29 P - - - 30 P - - - P 1P - - - - 103 Appendix 2 Individual Clinical Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 3 CATEGORY GROUP: M2 DOSE: 2 MG/KG/DAY 'C' - COMMENTS LISTED AT END OF OBSERVATIONS FOR SEX/GROUP ANIMAL DEATH WK OF KEYWORD OBS 30 MIN 60 MIN 90 MIN DISPATCH NUMBER CODE DEATH QUALIFIER DAYS 1-30 DAY (CONTINUED FROM PREVIOUS PAGE) I05419 T 5 NORMAL NO REMARKABLE OBSERVATIONS QUALITATIVE FOOD CONSUMPTION LOW 8P 15 P - - - 22 P - - - - 24 P 104 Appendix 2 Individual Clinical Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 4 CATEGORY GROUP: M3 DOSE: 20 MG/KG/DAY 'C' - COMMENTS LISTED AT END OF OBSERVATIONS FOR SEX/GROUP ANIMAL DEATH WK OF KEYWORD OBS 30 MIN 60 MIN 90 MIN DISPATCH NUMBER CODE DEATH QUALIFIER DAYS 1-30 DAY I05235 T 5 EXCRETION NO FECES NORMAL NO REMARKABLE OBSERVATIONS QUALITATIVE FOOD CONSUMPTION LOW I05351 T NONE 5 NORMAL NO REMARKABLE OBSERVATIONS 17 P - - - - 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 5P - - - 7P - - - 11 P - - - 14 P - - - 16 P - - - 17 P - - - 24 P - - - 12 P - - - - 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 105 Appendix 2 Individual Clinical Observations 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 5 CATEGORY GROUP: M3 DOSE: 20 MG/KG/DAY 'C' - COMMENTS LISTED AT END OF OBSERVATIONS FOR SEX/GROUP ANIMAL DEATH WK OF KEYWORD OBS 30 MIN 60 MIN 90 MIN DISPATCH NUMBER CODE DEATH QUALIFIER DAYS 1-30 DAY I05416 T 5 NORMAL NO REMARKABLE OBSERVATIONS 1P - - - 8P - - - 15 P - - - 22 P - - - 29 P - - - 30 P - - - P 106 APPENDIX 3 Individual Body Weight Data (kg) Covance 6329-230 3M T-6889.2 107 Appendix 3 Individual Body Weight Data (kg) 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER WEEK -2 a WEEK -1 GROUP: I05353 I05417 GROUP: I05231 I05352 I05419 GROUP: I05235 I05351 I05416 MALE 1 - 0 MG/KG/DAY 2.5 2.5 3.5 3.5 MALE 2 - 2 MG/KG/DAY 2.7 2.9 2.1 2.1 3.5 3.6 MALE 3 - 20 MG/KG/DAY 2.7 2.8 2.2 2.3 3.2 3.3 a Day -7. b Day -1. b WEEK -1 2.5 3.6 2.8 2.1 3.6 2.7 2.3 3.3 WEEK 1 2.6 3.6 2.9 2.1 3.6 2.8 2.2 3.3 WEEK 2 2.7 3.5 2.9 2.2 3.6 2.7 2.3 3.4 WEEK 3 2.7 3.6 3.0 2.2 3.6 2.7 2.3 3.4 WEEK 4 2.6 3.5 2.9 2.2 3.6 2.8 2.3 3.3 WEEK 5 2.7 3.6 3.0 2.2 3.7 2.8 2.4 3.5 Covance 6329-230 3M T-6889.2 PAGE: 1 108 APPENDIX 4 Individual Clinical Hematology Data Individual Clinical Chemistry Data Covance 6329-230 3M T-6889.2 109 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Hematology Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS RBC HGB HCT MCV MCH MCHC PLT RETIC RETIC X106/fJ,L G/DL FL PG X103/p,L X103/fJ,L Dose Level: 0 Dosage Unit: mg/kg/day 4.44 7.49 11.9 13.2 35.8 45.2 80.5 60.3 26.8 17.7 33.3 29.3 376 483 .7 31 .1 7 5.96 2.157 2 12.6 .92 2 40.5 6.65 2 70.4 14.28 2 22.2 6.43 2 31.3 2.83 2 430 75.7 2 .4 .42 2 19 17.0 2 Dose Level: 2 Dosage Unit: mg/kg/day 7.08 13.1 43.5 61.5 18.5 30.0 455 .4 5.56 14.2 43.5 78.3 25.6 32.7 376 1.2 7.92 13.4 45.6 57.5 16.9 29.3 435 .1 28 67 8 6.85 1.196 3 13.6 .57 3 44.2 1.21 3 65.8 11.04 3 20.3 4.63 3 30.7 1.80 3 422 41.1 3 .6 .57 3 34 30.0 3 Dose Level: 20 Dosage Unit: mg/kg/day 7.18 5.37 6.61 12.4 14.3 13.1 42.7 42.8 41.5 59.4 79.6 62.7 17.2 26.5 19.9 29.0 33.4 31.7 530 649 386 .2 14 .6 32 .2 13 6.39 .925 3 13.3 .96 3 42.3 .72 3 67.2 10.84 3 21.2 4.78 3 31.4 2.22 3 522 131.7 3 .3 .23 3 20 10.7 3 Covance 6329-230 3M T-6889.2 110 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N WBC X103/fJ,L Appendix 4 Individual Clinical Hematology Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS N-SEG LYMPH X103/p,L X103/fJ,L MONO EOSIN X103/fJ,L X103/fJ,L BASO N-SEG% X103/|iL LYMPH% MONO% EOSIN% Covance 6329-230 3M T-6889.2 BASO% Dose Level : 0 9.2 11.5 1.0 2.6 10.4 1.63 2 1.8 1.13 2 Dose Level : 2 9.3 8.5 10.5 4.0 1.0 2.8 9.4 1.01 3 2.6 1.51 3 Dose Level : 20 9.0 9.0 10.9 3.2 .8 4.4 9.6 1.10 3 2.8 1.83 3 Dosage Unit: mg/kg/day 7.8 .4 .1 6.9 .8 1.1 7.4 .6 .6 .64 .28 .71 222 Dosage Unit: mg/kg/day 4.3 1.0 7.0 .4 6.3 1.2 .1 .1 .2 5.9 .9 .1 1.40 .42 .06 333 Dosage Unit: mg/kg/day 5.0 .4 .4 7.6 .5 .1 5.5 .7 .3 6.0 .5 .3 1.38 .15 .15 333 0 11 0 23 84 60 4 10 7 10 0 0 17 72 6 6 0 00 8.5 17.0 2.1 6.4 .0 2 2 2 2 22 0 43 46 10 0 12 82 5 0 27 60 11 10 10 20 0 27 63 9 10 00 15.5 18.1 3.2 .6 .0 3 3 3 3 33 0 35 56 5 4 0 1 9 84 5 1 1 0 40 50 7 20 0 28 63 6 20 06 16.6 18.1 1.2 1.5 .6 3 3 3 3 33 111 Appendix 4 Individual Clinical Hematology Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER ANISO POLY POIK HYPO TOXNEUT Group: 1 I05353 I05417 Group: 2 I05231 I05352 I05419 Group: 3 I05235 I05351 I05416 Dose Level: 0 -_ -- Dose Level: 2 -_ -_ -_ Dose Level: 20 -_ -_ -_ Dosage Unit: mg/kg/day --- Dosage Unit: mg/kg/day ---- Dosage Unit: mg/kg/day ---- Covance 6329-230 3M T-6889.2 112 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Hematology Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS RBC HGB HCT MCV MCH MCHC PLT RETIC RETIC X106/fJ,L G/DL FL PG X103/p,L X103/fJ,L Dose Level: 0 4.26 6.68 11.3 11.8 5.47 1.711 2 11.5 .35 2 Dose Level: 2 6.27 4.72 6.75 11.5 12.4 11.6 5.91 1.061 3 11.8 .49 3 Dose Level: 20 6.61 4.57 6.07 11.4 12.2 12.0 5.75 1.057 3 11.9 .42 3 Dosage Unit: mg/kg/day 34.2 40.5 80.5 60.7 26.5 17.6 37.3 4.45 2 70.6 14.00 2 22.0 6.29 2 Dosage Unit: mg/kg/day 38.9 37.1 39.3 62.0 78.6 58.3 18.3 26.3 17.3 38.4 1.17 3 66.3 10.81 3 20.6 4.93 3 Dosage Unit: mg/kg/day 39.2 36.3 38.3 59.3 79.4 63.1 17.3 26.6 19.7 37.9 1.48 3 67.3 10.68 3 21.2 4.83 3 33.0 29.0 31.0 2.83 2 29.5 33.4 29.6 30.8 2.22 3 29.2 33.6 31.3 31.4 2.20 3 360 447 404 61.5 2 1.2 .0 .6 .85 2 418 393 374 395 22.1 3 .8 1.6 .4 .9 .61 3 516 593 392 500 101.4 3 .1 .3 .1 .2 .12 3 51 0 26 36.1 2 50 76 27 51 24.5 3 7 14 6 9 4.4 3 Covance 6329-230 3M T-6889.2 113 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N WBC X103/fJ,L Appendix 4 Individual Clinical Hematology Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS N-SEG LYMPH X103/p,L X103/fJ,L MONO EOSIN X103/fJ,L X103/fJ,L BASO N-SEG% X103/|iL LYMPH% MONO% EOSIN% Covance 6329-230 3M T-6889.2 BASO% Dose Level : 0 10.0 12.2 1.2 2.8 11.1 1.56 2 2.0 1.13 2 Dose Level : 2 9.0 8.8 14.4 4.6 2.4 6.0 10.7 3.18 3 4.3 1.81 3 Dose Level : 20 11.1 7.1 13.2 4.8 2.0 5.6 10.5 3.10 3 4.1 1.89 3 Dosage Unit: mg/kg/day 8.2 .6 .1 7.5 .8 1.0 7.8 .7 .6 .49 .14 .64 222 Dosage Unit: mg/kg/day 3.7 .6 .2 5.8 .6 .1 7.2 .8 .3 5.6 .7 .2 1.76 .12 .10 333 Dosage Unit: mg/kg/day 5.1 .7 .3 4.9 .2 .0 6.3 .8 .4 5.4 .6 .2 .76 .32 .21 333 1 12 82 6 1 1 0 23 62 7 80 0 18 72 6 4 0 07 7.8 14.1 .7 4.9 .7 2 2 2 2 22 0 51 40 7 2 0 0 27 65 6 10 0 42 50 6 20 0 40 52 6 20 00 12.1 12.6 .6 .6 .0 3 3 3 3 33 0 44 46 7 3 0 0 28 69 3 00 0 43 48 6 3 0 0 38 54 5 2 0 00 9.0 12.7 2.1 1.7 .0 3 3 3 3 33 114 Appendix 4 Individual Clinical Hematology Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER ANISO POLY POIK HYPO TOXNEUT Group: 1 I05353 I05417 Group: 2 I05231 I05352 I05419 Group: 3 I05235 I05351 I05416 Dose Level: 0 -_ -- Dose Level: 2 -_ -_ -_ Dose Level: 20 -_ -_ -_ Dosage Unit: mg/kg/day --- Dosage Unit: mg/kg/day ---- Dosage Unit: mg/kg/day ---- Covance 6329-230 3M T-6889.2 115 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Hematology Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS RBC HGB HCT MCV MCH MCHC PLT RETIC RETIC X106/fJ,L G/DL FL PG X103/p,L X103/fJ,L Dose Level: 0 4.93 6.99 12.5 12.3 5.96 1.457 2 12.4 .14 2 Dose Level: 2 7.03 5.81 7.38 13.2 14.9 12.6 6.74 .824 3 13.6 1.19 3 Dose Level: 20 6.95 5.35 6.41 12.0 14.2 12.8 6.24 .814 3 13.0 1.11 3 Dosage Unit: mg/kg/day 39.2 42.4 79.6 60.6 25.5 17.7 40.8 2.26 2 70.1 13.44 2 21.6 5.52 2 Dosage Unit: mg/kg/day 43.6 46.0 43.2 62.0 79.1 58.5 18.7 25.6 17.1 44.3 1.51 3 66.5 11.02 3 20.5 4.52 3 Dosage Unit: mg/kg/day 42.0 43.1 40.5 60.5 80.6 63.2 17.3 26.6 20.0 41.9 1.31 3 68.1 10.91 3 21.3 4.78 3 32.0 29.1 30.5 2.05 2 30.3 32.4 29.2 30.6 1.63 3 28.6 33.0 31.6 31.1 2.25 3 414 508 461 66.5 2 412 404 406 407 4.2 3 489 605 373 489 116.0 3 .4 .1 .2 .21 2 .1 .4 .0 .2 .21 3 .3 .4 .0 .2 .21 3 20 7 14 9. 2 7 23 0 10 11. 3 21 21 0 14 12. 3 Covance 6329-230 3M T-6889.2 116 ANIMAL NUMBER WBC X103/fJ,L Appendix 4 Individual Clinical Hematology Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS N-SEG LYMPH X103/p,L X103/fJ,L MONO EOSIN X103/fJ,L X103/fJ,L BASO N-SEG% X103/fJ,L LYMPH% MONO% EOSIN% Covance 6329-230 3M T-6889.2 BASO% Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 151 I05416 MEAN S.D. N Dose Level : 0 Dosage Unit: mg/kg/day 9.9 1.1 7.8 .7 .2 .1 11 79 9.8 2.0 6.7 .4 .7 .0 21 68 9.8 1.6 7.2 .6 .4 .0 16 74 .07 .64 .78 .21 .35 .07 7.1 7.8 22222222 Dose Level : 2 Dosage Unit: mg/kg/day 6.2 1.9 3.8 .5 .0 .0 30 61 7.3 1.6 5.4 .2 .0 .0 22 74 10.6 3.1 7.0 .3 .1 .0 30 66 8.0 2.2 5.4 .3 .0 .0 27 67 2.29 .79 1.60 .15 .06 .00 4.6 6.6 33333333 Dose Level: 20 Dosage Unit: mg/kg/day 9.2 8.2 7.5 4.1 1 .9 3.3 4.3 5.8 3.2 .6 .5 .7 .2 .0 .2 .0 .0 .0 45 23 45 47 70 43 6 8.3 3.1 4.4 .6 .1 .0 38 53 .85 1.11 1.31 .10 .12 .00 12.7 14.6 33333333 7 4 6 2.1 2 8 3 3 5 2.9 3 7 1 9 7 1.5 3 2 7 4 3.5 2 1 1 1 1 .0 3 2 2 2 .6 3 1 0 0 .7 2 0 0 0 0 .0 3 0 1 1 1 .6 3 117 Appendix 4 Individual Clinical Hematology Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER ANISO POLY POIK HYPO TOXNEUT Group: 1 I05353 I05417 Group: 2 I05231 I05352 I05419 Group: 3 I05235 I05351 I05416 a Dose Level: 0 -_ -- Dose Level: 2 -_ -_ -_ Dose Level: 20 -_ -_ -_ Dosage Unit: mg/kg/day --- Dosage Unit: mg/kg/day ---- Dosage Unit: mg/kg/day ---- a Plasmodium was observed. Covance 6329-230 3M T-6889.2 118 ANIMAL NUMBER GLU MG/DL Appendix 4 Individual Clinical Chemistry Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA UMOL/L CHOL MG/DL Covance 6329-230 3M T-6889.2 TRIG MG/DL Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN .D. Dose Level: 0 59 28 88 17 74 20.5 2 22 7.8 2 Dose Level: 2 60 16 85 18 84 20 76 14.2 3 18 2.0 3 Dose Level: 20 63 24 70 20 60 24 64 5.1 3 23 2.3 3 Dosage Unit: mg/kg/day .9 7.0 1.2 10.4 4.4 6.0 1.0 .21 2 8.7 2.40 2 5.2 1.13 2 Dosage Unit: mg/kg/day .9 8.3 5.1 1.0 7.1 4.8 1.4 9.7 5.4 1.1 .26 3 8.4 1.30 3 5.1 .30 3 Dosage Unit: mg/kg/day 1.4 8.3 4.6 .9 7.4 5.0 1.2 8.4 4.7 1.2 .25 3 8.0 .55 3 4.8 .21 3 2.6 4.4 3.5 1.27 2 3.2 2.3 4.3 3.3 1.00 3 3.7 2.4 3.7 3.3 .75 3 .3 .5 .4 .14 2 .2 .4 .2 .3 .12 3 .2 .1 .2 .2 .06 3 5 13 9 5.7 2 152 139 146 9.2 2 29 40 34 7 2 11 6 9 9 2.5 3 157 170 196 174 19.9 3 40 28 39 36 6 3 12 11 12 12 .6 3 126 149 147 141 12.7 3 39 27 28 31 6 3 a en 119 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Chemistry Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS AST/SGOT ALT/SGPT ALK PHOS IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Dose Level: 0 40 38 42 52 41 1.4 2 45 9.9 2 Dose Level: 2 43 33 65 77 37 50 48 14.7 3 53 22.2 3 Dose Level: 20 45 89 58 38 54 50 52 6.7 3 59 26.7 3 Dosage Unit: mg/kg/day 1144 550 130 114 2 2 847 420.0 2 122 11.3 2 2 .0 2 Dosage Unit: mg/kg/day 1122 910 1007 209 122 145 10 2 3 1013 106.1 3 159 45.1 3 5 4.4 3 Dosage Unit: mg/kg/day 985 116 986 115 872 177 4 4 6 948 65.5 3 136 35.5 3 5 1.2 3 520 175 348 244.0 2 307 550 250 369 159.3 3 210 225 149 195 40.3 3 442 541 492 70.0 2 415 427 723 522 174.5 3 461 524 502 496 32.0 3 42 44 43 1.4 2 228 275 252 33.2 2 51 21 67 46 23.4 3 223 229 298 250 41.7 3 44 51 34 43 8.5 3 207 280 230 239 37.3 3 Covance 6329-230 3M T-6889.2 120 Appendix 4 Individual Clinical Chemistry Data Males Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level : 0 10.2 12.6 7.9 6.5 11.4 1.70 2 7.2 .99 2 Dose Level : 2 11.3 10.6 11.0 8.7 7.4 6.5 11.0 .35 3 7.5 1.11 3 Dose Level : 20 11.3 11.1 10.3 7.2 7.2 6.7 10.9 .53 3 7.0 .29 3 Dosage Unit: mg/kg/day 150 6.1 111 166 7.1 113 158 11.3 2 6.6 .71 2 112 1 2 Dosage Unit: mg/kg/day 159 5.6 109 153 6.0 107 162 5.3 109 158 4.6 3 5.6 .35 3 108 1 3 Dosage Unit: mg/kg/day 156 6.1 108 154 6.5 109 153 5.7 108 154 1.5 3 6.1 .40 3 108 3 Covance 6329-230 3M T-6889.2 121 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N GLU MG/DL Appendix 4 Individual Clinical Chemistry Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA UMOL/L CHOL MG/DL Covance 6329-230 3M T-6889.2 TRIG MG/DL Dose Level: 0 55 24 113 11 84 41.0 2 18 9.2 2 Dose Level: 2 66 14 60 15 73 16 66 6.5 3 15 1.0 3 Dose Level: 20 78 14 51 24 73 21 67 14.4 3 20 5.1 3 Dosage Unit: mg/kg/day 1.1 7.2 4.4 1.3 9.6 5.5 1.2 .14 2 8.4 1.70 2 5.0 .78 2 Dosage Unit: mg/kg/day 1.0 8.1 4.9 .9 7.1 4.8 1.3 8.8 4.7 1.1 .21 3 8.0 .85 3 4.8 .10 3 Dosage Unit: mg/kg/day 1.1 8.2 4.4 .9 7.4 4.9 1.2 8.5 4.7 1.1 .15 3 8.0 .57 3 4.7 .25 3 2.8 4.1 3.4 .92 2 3.2 2.3 4.1 3.2 .90 3 3.8 2.5 3.8 3.4 .75 3 .6 .7 .6 .07 2 .1 .4 .4 .3 .17 3 .1 .7 .3 .4 .31 3 6 156 3 119 4 138 2.1 26.2 22 1 141 5 160 9 164 5 155 4.0 12.3 33 1 128 2 155 0 140 1 141 1.0 13.5 33 28 29 28 .7 2 31 32 38 34 3.8 3 30 51 35 39 11.0 3 122 ANIMAL NUMBER Appendix 4 Individual Clinical Chemistry Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS AST/SGOT ALT/SGPT .ALK PHOS IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level: 0 42 34 35 52 38 4.9 2 43 12.7 2 Dose Level: 2 31 35 54 65 31 46 39 13.3 3 49 15.2 3 Dose Level: 20 37 66 54 37 54 51 48 9.8 3 51 14.5 3 Dosage Unit: mg/kg/day 1066 509 117 108 3 3 788 393.9 2 112 6.4 2 3 .0 2 Dosage Unit: mg/kg/day 1026 872 893 182 108 120 4 3 2 930 83.5 3 137 39.7 3 3 1.0 3 Dosage Unit: mg/kg/day 945 106 906 92 837 164 2 3 4 896 54.7 3 121 38.2 3 3 1.0 3 833 156 494 478.7 2 141 542 155 279 227.6 3 181 194 138 171 29.3 3 441 549 495 76.4 2 395 446 720 520 174.8 3 451 467 481 466 15.0 3 42 38 40 2.8 2 233 270 252 26 2 64 46 58 56 9.2 3 220 244 293 252 37 3 57 71 44 57 13.5 3 213 249 224 229 18 3 Covance 6329-230 3M T-6889.2 123 Appendix 4 Individual Clinical Chemistry Data Males Day -5 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level : 0 10.0 11.8 7.7 5.9 10.9 1.27 2 6.8 1.27 2 Dose Level : 2 11.1 10.3 9.9 6.9 7.1 5.2 10.4 .61 3 6.4 1.04 3 Dose Level : 20 10.8 10.5 9.8 6.1 6.4 6.1 10.4 .51 3 6.2 .17 3 Dosage Unit: mg/kg/day 149 4.8 107 162 6.1 110 156 9.2 2 5.4 .92 2 108 2 2 Dosage Unit: mg/kg/day 159 5.6 113 153 5.6 109 153 4.3 108 155 3.5 3 5.2 .75 3 110 2 3 Dosage Unit: mg/kg/day 154 5.3 108 153 5.3 108 149 4.3 105 152 2.6 3 5.0 .58 3 107 1 3 Covance 6329-230 3M T-6889.2 124 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Chemistry Data Males Day 2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 GLU MG/DL UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA UMOL/L CHOL MG/DL TRIG MG/DL Dose Level : 0 Dosage Unit: mg/kg/day 73 26 .8 7.5 4.5 3.0 79 16 1.1 10.6 5.8 4.8 .3 13 157 .6 6 146 23 34 76 4.2 2 21 7.1 2 1.0 .21 2 9.0 2.19 2 5.2 .92 2 3.9 1.27 2 .4 10 152 28 .21 4.9 7.8 7.8 2222 Dose Level : 2 Dosage Unit: mg/kg/day 64 17 .8 8.4 4.9 3.5 70 26 .9 7.7 5.0 2.7 62 18 1.2 9.0 4.8 4.2 .4 6 143 .3 9 183 .3 12 180 49 45 61 65 4.2 3 20 4.9 3 1.0 .21 3 8.4 .65 3 4.9 .10 3 3.5 .75 3 .3 .06 3 9 169 3.0 22.3 33 52 8.3 3 Dose Level : 20 Dosage Unit: mg/kg/day 68 24 1.1 8.4 4.6 3.8 70 22 .9 7.7 5.0 2.7 66 23 .9 8.7 4.7 4.0 .1 11 121 .2 12 136 .1 19 140 35 57 43 68 23 1.0 8.3 4.8 3.5 .1 14 132 45 2.0 1.0 .12 .51 .21 .70 .06 4.4 10.0 11.1 3333333333 125 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Chemistry Data Males Day 2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS AST/SGOT ALT/SGPT ALK PHOS IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L Dose Level: 0 45 35 49 71 47 2.8 2 53 25.5 2 Dose Level: 2 33 31 91 82 36 42 53 32.7 3 52 26.8 3 Dose Level: 20 49 80 58 36 57 51 55 4.9 3 56 22.4 3 Dosage Unit: mg/kg/day 1123 620 129 121 1 1 872 355.7 2 125 5.7 2 1 .0 2 Dosage Unit: mg/kg/day 1151 927 1009 200 119 126 1 4 2 1029 113.3 3 148 44.9 3 2 1.5 3 Dosage Unit: mg/kg/day 1062 827 875 110 98 171 2 0 1 921 124.2 3 126 39.1 3 1 1.0 3 181 269 225 62.2 2 113 2149 121 794 1173.2 3 175 203 116 165 44.4 3 435 519 477 59.4 2 366 438 644 483 144.3 3 433 503 502 479 40.1 3 28 30 29 1.4 2 228 270 249 29.7 2 45 36 51 44 7.5 3 204 239 289 244 42.7 3 41 39 19 33 12.2 3 205 269 227 234 32.5 3 Covance 6329-230 3M T-6889.2 126 Appendix 4 Individual Clinical Chemistry Data Males Day 2 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level : 0 10.4 12.7 9.0 8.6 11.6 1.63 2 8.8 .28 2 Dose Level : 2 11.0 10.7 9.9 8.1 9.3 6.7 10.5 .57 3 8.0 1.30 3 Dose Level : 20 11.0 11.4 9.8 7.5 8.3 6.5 10.7 .83 3 7.4 .90 3 Dosage Unit: mg/kg/day 153 5.5 106 170 6.9 113 162 12.0 2 6.2 .99 2 110 4 2 Dosage Unit: mg/kg/day 158 5.5 113 155 5.6 106 153 4.8 107 155 2.5 3 5.3 .44 3 109 3 3 Dosage Unit: mg/kg/day 159 5.7 110 157 6.1 107 151 4.8 109 156 4.2 3 5.5 .67 3 109 1 3 Covance 6329-230 3M T-6889.2 127 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Chemistry Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 GLU MG/DL UN MG/DL CREAT MG/DL T PRO G/DL ALB G/DL GLOB G/DL T BILI MG/DL SBA UMOL/L CHOL MG/DL TRIG MG/DL Dose Level : 0 Dosage Unit: mg/kg/day 77 23 .9 7.3 4.6 2.7 90 15 1.0 9.8 5.6 4.2 .7 .3 6 172 1 127 33 32 84 9.2 2 19 5.7 2 1.0 .07 2 8.6 1.77 2 5.1 .71 2 3.4 1.06 2 .5 .28 2 4 150 3.5 31.8 22 32 .7 2 Dose Level : 2 Dosage Unit: mg/kg/day 75 15 1.1 8.4 5.1 3.3 80 16 1.2 7.7 5.1 2.6 74 19 1.1 8.9 4.8 4.1 .2 .1 .1 2 137 4 149 5 155 45 39 44 76 3.2 3 17 2.1 3 1.1 .06 3 8.3 .60 3 5.0 .17 3 3.3 .75 3 .1 .06 3 4 147 1.5 9.2 33 43 3.2 3 Dose Level : 20 Dosage Unit: mg/kg/day 64 16 1.5 8.0 4.4 3.6 63 22 1.2 7.3 4.8 2.5 67 22 .8 8.2 4.4 3.8 .1 10 107 50 .0 1 138 63 .3 6 170 42 65 2.1 3 20 3.5 3 1.2 .35 3 7.8 .47 3 4.5 .23 3 3.3 .70 3 .1 .15 3 6 138 4.5 31.5 33 52 10.6 3 128 ANIMAL NUMBER Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Appendix 4 Individual Clinical Chemistry Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 AST/SGOT .ALT/SGPT ALK PHOS IU/L IU/L IU/L GGT IU/L SDH IU/L CK IU/L AMYLASE LIPASE IU/L IU/L P AMYL U/L PCOAO IU/G Dose Level : 0 52 42 51 76 52 .7 2 59 24.0 2 Dose Level : 2 35 53 72 113 42 58 50 19.7 3 75 33.3 3 Dose Level : 20 43 140 66 60 55 48 55 11.5 3 83 50.0 3 Dosage Unit: mg/kg/day 1154 680 118 102 1 3 917 335.2 2 110 11.3 2 2 1.4 2 Dosage Unit: mg/kg/day 1155 1009 1170 177 115 115 1 2 3 1111 88.9 3 136 35.8 3 2 1.0 3 Dosage Unit: mg/kg/day 799 80 626 83 686 139 3 2 0 704 87.8 3 101 33.2 3 2 1.5 3 827 195 511 446.9 2 145 570 256 324 220.4 3 206 216 198 207 9.0 3 451 491 471 28.3 2 344 501 654 500 155.0 3 382 521 420 441 71.8 3 53 30 42 16.3 2 238 247 242 6.4 2 36 32 47 38 7.8 3 186 273 272 244 49.9 3 47 45 26 39 11.6 3 175 277 202 218 52.8 3 2 1 2 .7 2 0 0 3 1 1.7 3 5 3 9 6 3.1 3 129 Appendix 4 Individual Clinical Chemistry Data Males Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS ANIMAL NUMBER CA MG/DL I PHOS MG/DL NA MMOL/L K MMOL/L CL MMOL/L Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level : 0 10.2 11.2 8.4 5.9 10.7 .71 2 7.2 1.77 2 Dose Level : 2 11.2 11.0 10.2 7.1 7.5 5.5 10.8 .53 3 6.7 1.06 3 Dose Level : 20 10.3 10.9 9.5 5.8 5.6 5.2 10.2 .70 3 5.5 .31 3 Dosage Unit: mg/kg/day 149 5.0 104 157 5.7 108 153 5.7 2 5.4 .49 2 106 2 2 Dosage Unit: mg/kg/day 157 5.2 112 156 6.1 107 153 4.3 107 155 2.1 3 5.2 .90 3 109 2 3 Dosage Unit: mg/kg/day 155 5.1 111 152 5.9 107 145 4.6 108 151 5.1 3 5.2 .66 3 109 2 3 Covance 6329-230 3M T-6889.2 130 APPENDIX 5 Individual Animal Pathology Data Covance 6329-230 3MT-6889.2 131 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 1 ANIMAL NUMBER: I05353 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 2500.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 11:10 PROSECTOR: KEVIN BILLINGS RECORDER: JILL PAUS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY KIDNEY (KD) : -LIGHT FOCUS(I)/AREA(S); HILUS: SINGLE TAN AREA, ROUTINE SECTION GENERAL COMMENT (GC) : -EYES - DAVIDSONS RIGHT ON CORTICAL SURFACE, NEAR 0.2 CM IN DIAMETER; COLLECTED ON KIDNEY (KD) : >NOT REQUIRED TO BE EXAMINED FOR ANIMAL THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), STOMACH, GL (ST), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), ADRENAL (AD) 132 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 2 ANIMAL NUMBER: I05417 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 3325.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 11:15 PROSECTOR: JERALD W. JOHNSON RECORDER: JAMES T. CALLEN POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY STOMACH, GL (ST) : -RED FOCUS(I)/AREA(S); MUCOSA: MULTIPLE RED AREAS, UP TO 0.8 X 0.5 CM; COLLECTED ON ROUTINE SECTION GENERAL COMMENT (GC) : -EYES - DAVIDSONS STOMACH, GL (ST) : >NOT REQUIRED TO BE EXAMINED FOR ANIMAL THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), ADRENAL (AD) 133 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 3 ANIMAL NUMBER: I05231 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 2795.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 12:43 PROSECTOR: JERALD W. JOHNSON RECORDER: JAMES T. CALLEN POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY GENERAL COMMENT (GC) : -EYES - DAVIDSONS -NO MACROSCOPIC LESIONS ADRENAL (AD) : -MINERALIZATION,-MINIMAL, MULTI-FOCAL THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), STOMACH, GL (ST), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE) 134 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 4 ANIMAL NUMBER: I05352 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 1980.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 12:40 PROSECTOR: KEVIN BILLINGS RECORDER: JILL PAUS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY GENERAL COMMENT (GC) : -EYES - DAVIDSONS -NO MACROSCOPIC LESIONS TESTIS (TE) : -IMMATURE,-MODERATE, DIFFUSE THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), STOMACH, GL (ST), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), ADRENAL (AD) 135 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 5 ANIMAL NUMBER: I05419 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 3435.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 11:57 PROSECTOR: JERALD W. JOHNSON RECORDER: JAMES T. CALLEN POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY GENERAL COMMENT (GC) : -EYES - DAVIDSONS -NO MACROSCOPIC LESIONS GENERAL INFORMATION (XX) : >NOTE:>LEFT HIND LIMB, NEAR STIFLE JOINT: ANIMAL HAS SIX SUTURES; POSSIBLY CONSISTENT WITH CLINICAL OBSERVATIONS OF SCABS (HIND LEFT) THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), STOMACH, GL (ST), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), ADRENAL (AD) 136 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 6 ANIMAL NUMBER: I05235 SEX: MALE DOSE GROUP: 3 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 2635.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 13:09 PROSECTOR: JERALD W. JOHNSON RECORDER: JAMES T. CALLEN POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY STOMACH, GL (ST) : -RED FOCUS(I)/AREA(S); MUCOSA: MULTIPLE RED AREAS, UP TO 0.6 X 0.4 CM; COLLECTED ON ROUTINE SECTION GENERAL COMMENT (GC) : -EYES - DAVIDSONS STOMACH, GL (ST) : >NOT REQUIRED TO BE EXAMINED FOR ANIMAL THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), ADRENAL (AD) 137 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 7 ANIMAL NUMBER: I05351 SEX: MALE DOSE GROUP: 3 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 2185.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 11:56 PROSECTOR: KEVIN BILLINGS RECORDER: JILL PAUS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY GENERAL COMMENT (GC) : -EYES - DAVIDSONS -NO MACROSCOPIC LESIONS TESTIS (TE) : -IMMATURE,-MODERATE, DIFFUSE THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), LUNG (LU), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), STOMACH, GL (ST), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), ADRENAL (AD) 138 APPENDIX 5 Individual Animal Pathology Data 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 PAGE: 8 ANIMAL NUMBER: I05416 SEX: MALE DOSE GROUP: 3 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 07/16/98 STUDY DAY OF DEATH: 30 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: 3120.0 GRAMS DATE AND TIME OF NECROPSY: 07/16/98 13:05 PROSECTOR: KEVIN BILLINGS RECORDER: JILL PAUS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. JAMES L. CARTER WEIGHER: NOT AVAILABLE NECROPSY OBSERVATIONS HISTOPATHOLOGY LUNG (LU) : -ADHESION(S); ALL LOBES TO EACH OTHER, PLEURA AND CAUDAL LOBES TO DIAPHRAGM: MULTIPLE TAN GRAY FIBROUS ADHESIONS; COLLECTED INTACT STOMACH, GL (ST) : -RED FOCUS(I)/AREA(S); MUCOSA: SINGLE AREA, 0.5 X 0.2 CM GENERAL COMMENT (GC) : -EYES - DAVIDSONS LUNG (LU) : >NOT REQUIRED TO BE EXAMINED FOR ANIMAL STOMACH, GL (ST) : >NOT REQUIRED TO BE EXAMINED FOR ANIMAL THE FOLLOWING ORGANS WERE UNREMARKABLE AT NECROPSY: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), BONE, FEMUR (FE), MARROW, FEMUR (FM), MARROW, STERNUM (SE), BONE, STERNUM (SB), EYE (EY), BRAIN (BR), KIDNEY (KD), HEART (HT), TRACHEA (TR), ESOPHAGUS (ES), THYROID (TY), PARATHYROID (PT), THYMUS (TH), LN, MESENTERIC (MS), AORTA (AO), PITUITARY (PI), SALIV GL, MANDIB (SG), MUSCLE, SKELETAL (SM), SPINAL CORD (SC), NERVE, SCIATIC (SN), DUODENUM (DU), GALLBLADDER (GB), JEJUNUM (JE), ILEUM (IL), CECUM (CE), COLON (CO), RECTUM (RE), SKIN (SK), MAMMARY, MALE (MM), URINARY BLADDER (UB), PROSTATE (PR), SEMINAL VESICLES (SV), EPIDIDYMIDES (EP), ADRENAL, CORTEX (AC), ADRENAL, MEDULLA (MA) THE FOLLOWING TISSUES WERE UNREMARKABLE AT MICROSCOPIC EXAMINATION: LIVER (LI), SPLEEN (SP), PANCREAS (PA), TESTIS (TE), ADRENAL (AD) 139 APPENDIX 6 Covance 6329-230 3M T-6889.2 Quality Assurance Statement Summary and Individual Hormone Analyses Data Note: The data included in this appendix were supplied by Ani Lytics Inc. and have been reviewed by the Quality Assurance Unit of Ani Lytics Inc. 140 Covance 6329-230 3M T-6889.2 I v f JL JLV-/W-I 200 Girard Street, Suite 200, Gaithersburg, MD 20877 I N C O R P O R A T E D 301-921-0168 800-237-2815 The reports listed below was reviewed for compliance with the FDA Good Laboratory Practices and with the EPA Good Laboratory Practices. The final report and all associated raw data were reviewed for accuracy and consistency and the findings were reported to management. The methods used were the methods described and the report accurately reflects the data. Therefore, these studies were done in compliance with the FDA and EPA Good Laboratory Practices. Franklin B . Newman QA Auditor SPONSOR: COV^A/dK $65 STUDY: 9 2 7 -, 2 3 # REPORT TYPE: r3j'TLi T$i)f Ft3i f r F AUDIT DATE: REPORT TO MGMT: AUDIT #: management c\}74F X 141 Appendix 6 Summary and Individual Hormone Analyses Data Day -8 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 ANIMAL NUMBER THYROID TOTAL TOTAL FREE FREE ESTRONE ESTRADIOL ESTRIOL STIMULATING TRIIODOTHYRONINE THYROXIN TRIIODOTHYRONINE THYROXIN pg/mL pg/mL ng/mL jiIU/mL ng/dL jig/dL pg/mL ng/dL Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level: 0 163.53 236.08 12.39 39.04 199.81 51.301 2 25.72 18.844 2 Dose Level: 2 195.55 187.55 191.95 23.04 30.44 30.63 191.68 4.007 3 28.04 4.328 3 Dose Level: 20 183.99 186.13 213.49 9.10 14.61 12.69 194.54 16.449 3 12.13 2.797 3 Dosage Unit: mg/kg/day 0.00 0.00 1.40 1.61 205.95 159.89 0.00 0.000 2 1.51 0.148 2 182.92 32.569 2 Dosage Unit: mg/kg/day 0.00 0.00 0.00 1.17 1.18 1.64 187.06 174.59 166.70 0.00 0.000 3 1.33 0.269 3 176.12 10.265 3 Dosage Unit: mg/kg/day 0.00 0.00 0.00 1.50 0.74 1.10 145.43 176.53 172.10 0.00 0.000 3 1.11 0.380 3 164.69 16.823 3 5.11 4.28 4.70 0.587 2 3.41 5.71 3.12 4.08 1.419 3 3.73 5.99 3.69 4.47 1.317 3 5.08 4.77 4.93 0.219 2 7.61 2.96 5.58 5.38 2.331 3 5.92 3.75 3.44 4.37 1.351 3 1.08 1.14 1.11 0.042 2 1.21 1.17 0.97 1.12 0.129 3 1.41 2.06 0.67 1.38 0.695 3 142 Appendix 6 Summary and Individual Hormone Analyses Data Day 30 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTANOATE (APFO) IN CYNOMOLGUS MONKEYS Covance 6329-230 3M T-6889.2 ANIMAL NUMBER THYROID TOTAL TOTAL FREE FREE ESTRONE ESTRADIOL ESTRIOL STIMULATING TRIIODOTHYRONINE THYROXIN TRIIODOTHYRONINE THYROXIN pg/mL pg/mL ng/mL jiIU/mL ng/dL jig/dL pg/mL ng/dL Group: 1 I05353 I05417 MEAN S.D. N Group: 2 I05231 I05352 I05419 MEAN S.D. N Group: 3 I05235 I05351 I05416 MEAN S.D. N Dose Level: 0 182.68 48.21 13.13 34.20 115.45 95.085 2 23.67 14.899 2 Dose Level: 2 29.31 30.04 21.10 25.88 16.03 17.89 26.82 4.964 3 19.93 5.233 3 Dose Level: 20 14.55 28.80 26.67 10.34 11.50 11.45 23.34 7.687 3 11.10 0.656 3 Dosage Unit: mg/kg/day 0.00 0.00 1.68 1.55 188.20 137.22 0.00 0.000 2 1.62 0.092 2 162.71 36.048 2 Dosage Unit: mg/kg/day 0.00 0.00 0.00 1.28 1.17 0.98 173.12 150.56 193.34 0.00 0.000 3 1.14 0.152 3 172.34 21.401 3 Dosage Unit: mg/kg/day 0.00 0.00 0.00 1.71 1.06 1.06 138.52 143.31 129.89 0.00 0.000 3 1.28 0.375 3 137.24 6.801 3 5.50 4.31 4.91 0.841 2 3.84 3.60 5.44 4.29 1.000 3 4.71 5.00 2.62 4.11 1.298 3 4.93 4.72 4.83 0.148 2 5.91 4.98 6.82 5.90 0.920 3 4.55 4.67 3.78 4.33 0.483 3 1.66 1.43 1.55 0.163 2 0.99 1.14 1.69 1.27 0.369 3 1.12 1.19 0.68 1.00 0.276 3 143 APPENDIX 7 Cell Proliferation Report Covance 6329-230 3M T-6889.2 Note: This appendix of the report contains information supplied by Pathology Associates, A Charles River Company and has been reviewed by the Quality Assurance Unit of Pathology Associates, A Charles River Company. 144 Covance 6329-230 3M T-6889.2 145 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 1 CELL PROLIFERATION REPORT 4-WEEK CAPSULE TOXICITY STUDY WITH AMMONIUM PERFLUOROOCTONOATE (APFO) IN CYNOMOLGUS MONKEYS COVANCE STUDY NUMBER 6329-230 PURPOSE The purpose of this study was to provide data for determining an estimated maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study. In addition, the effect on critical enzyme levels, hormones, and other selected biochemical parameters, such as cell proliferation, were determined. This report, submitted by Pathology Associates -A Charles River Company (PAI) formally Pathology Associates International to the study Sponsor, APME Ad-Hoc APFO Toxicology Working Group, represents the cell proliferation findings and interpretation for Covance Study Number 6329-230 entitled "4-Week Capsule Toxicity Study with Ammonium Perfluorooctonoate (APFO) in Cynomolgus Monkeys". All aspects of the tasks associated with PAI's portion of this study were conducted in compliance with the Environmental Protection Agency Good Laboratory Practice (GLP) Regulations as set forth in title 40 of the US Code of Federal Regulations, Part 792, issued November 29, 1983 (effective December 29, 1983), and any applicable amendments. MATERIALS AND METHODS Tissue Collection for Cell Proliferation Two animals in group 1, and three animals each in groups 2 and 3 were sacrificed at the end of week 4. A section of the left lateral lobe of the liver, left and right testes, and pancreas from each animal was fixed in zinc formalin, processed and embedded to paraffin block by Covance per protocol specifications. Tissue blocks were shipped to PAI for sectioning and staining. From each block, a slide was prepared for H&E evaluation and immunohistochemical detection of proliferating cell nuclear antigen (PCNA), a marker of cell proliferation. Immunohistochemistry for Cell Proliferation Sections of paraffin-embedded tissues were cut at = 5 pm and placed on positively charged slides (Sueprfrost Plus, fisher Scientific, Pittsburgh, PA) to ensure adhesion during processing for PCNA. Standard immunohistochemical methods were used to stain tissues for PCNA [PAI's Standard Operating Procedure for Immunohistochemical Staining (SOP #707]. Briefly, tissue sections were incubated with a monoclonal antibody to PCNA (DAKO, lot #016; PAI No. A1899) and reagents required for the avidin-biotin peroxidase (ABC Elite Kit; Vector, lot #PK6100; PAI No. K 327) method for the detection of the antigen-antibody complex. PCNA expression in cells in all phases of the cell cycle (Gi, S, G2 and M) was localized by the 146 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 2 chromagen 3,3'-diaminobenzidine (DAB; Sigma Chemical Co., lot #18H8201). Tissue sections were counterstained with hematoxylin. Cell Proliferation Measurements The percentage of proliferating cells (proliferating index, PI) was determined by scoring at least 3000 hepatocytes in 10 fields of liver, at least 500 Leydig cells of the testis (except for animal #105417 in which 250 cells were scored), and at least 2000 acinar cells of the pancreas per animal. Although both testes presented on the slide were perused, only one testis was scored for proliferation; no attempt was made to distinguish left from right testis. A negative control slide was included in the staining run and consisted of study tissue from animal #10516 that was not incubated with the primary antibody. For cell proliferation evaluations, slides were first perused at low magnification (100X) to judge quality of staining, processing and sectioning, potential patterns of cellular proliferation, and histomorphologic changes. Cell proliferation was then quantified at higher magnification (200X for liver, and 400X for testes and pancreas) as described above. Histomorphology was further assessed by evaluating the H&E slide prepared from the same tissue block for each animal evaluated for cell proliferation. Statistical Analysis Due to the small sample size, statistical analysis was not performed. RESULTS Cell Proliferation Individual animal and group mean cell proliferation data are presented in Section II (Table 1) and graphically in Section III (Figures 1-3). Histopathology Sections from the same tissue blocks used for preparation of PCNA-stained slides were stained with hematoxylin and eosin (H&E) for histopathologic evaluation to facilitate the interpretation of the immunostained slides. Individual animal findings are presented in Appendix I. The results showed that no significant changes were observed in either the liver, pancreas, or testicular tissues from male monkeys, which would alter the interpretation of the PCNA staining in this study. SUMMARY Although a glance at the cell proliferation data suggest a "dose-response" in terms of cell proliferation in the liver and testes, the sample size is extremely small. Furthermore, in the liver, only one PCNA-labeled cell was detected in those animals with a proliferating index greater than 147 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 3 zero percent. In the testes, only one control animal was able to be evaluated due to immaturity of the only other animal in that group, and the proliferating indices in the two treatment groups overlapped. Therefore, the data are considered to be inconclusive and do not support a cell proliferative response to the test material. 148 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 II. TABLE 149 COVANCE STUDY NO. 6329-230 TABLE 1. CELL PROLIFERATION IN MONKEYS Covance 6329-230 3M T-6889.2 Dose Group 1 - 0 mg/kg/day (Control) 1 - 0 mg/kg/day (Control) 2 - 2 mg/kg/day (Low dose) 2 - 2 mg/kg/day (Low dose) 2 - 2 mg/kg/day (Low dose) 3 - 2 0 mg/kg/day (High dose) 3 - 2 0 mg/kg/day (High dose) 3 - 2 0 mg/kg/day (High dose) Anim al Number I05353 105417 Mean SEM 105231 I05352 105419 Mean SEM I05235 105351 105416 Mean SEM ND, not determined due to immaturity S E M , standard error of the mean | Proliferating Index Liver 0.000% 0.000% 0.000% 0.000% 0.000% 0.033% 0.033% 0.022% 0.011% 0.030% 0.033% 0.033% 0.032% 0.001% Proliferating Index Testes ND 9.6% 9.6% Unable to calculate 19.6% ND 11.6% 15.6% 4.0% 20.4% ND 15.6% 18.0% 2.4% Proliferating Index Pancreas 50.5% 51.5% 51.0% 0.5% 49.7% 49.6% 42.5% 47.3% 2.4% 43.9% 35.6% 49.7% 43.1% 4.1% 150 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 III. FIGURES 151 Covance 6329-230 3M T-6889.2 152 Covance 6329-230 3M T-6889.2 153 FIGURE 3. CELL PROLIFERATION IN PANCREAS 60 i------------------------------------------------ Covance 6329-230 3M T-6889.2 COVANCE STUDY NO. 6329-230 Animal number shown above bars 154 Covance 6329-230 155 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 V. QUALITY ASSURANCE STATEMENT 156 Covance 6329-230 3M T-6889.2 PATHOLOGY ASSOCIATES A CHARLES RIVER COM PANY Cell Proliferation Report 4-Week Capsule Toxicity Study with Ammonium Perfluorooctonoate (APFO) in Cynomolgus Monkeys Covance Study Number 6329-230 QUALITY ASSURANCE STATEMENT This cell proliferation project has been inspected and audited by the PAI Quality Assurance Unit (QAU) as required by the Good Laboratory Practice (GLP) regulations promulgated by the U.S. Environmental Protection Agency (EPA-TSCA). The cell proliferation report is an accurate reflection of the recorded data. The following table is a record of the inspections/audits performed and reported by the QAU. Date of Inspection Phase Inspected Date Findings Reported to PAI Management/Proiect Manager 08/12/98 10/05/98 10/05/98 07/25/01 Microtomy Study Data and Supporting Documentation Draft Cell Proliferation Report Final Cell Proliferation Report 08/12/98 10/06/98 10/06/98 07/25/01 15 Worman s Mill Court, Suite I, Frederick, M D 21701 301.663.1644 FAX: 301.663.8994 www.paicriver.com 157 Covance 6329-230 3M T-6889.2 Final Cell Proliferation Report Covance Study Number 6329-230 APPENDIX I 158 Covance 6329-230 3M T-6889.2 Covance Study Number 6329-230 Individual Animal Histopathologic Findings 4-Week Capsule Toxicity Study with Ammonium Perfluorooctonate (APFO) in Cynomolgus Monkeys Tissues Examined: Liver, Testes, Pancreas Animal Number 105353 105417 105231 105352 105419 105235 105351 105416 Sex Dose Group M 1-0 mg/kg/day (Control) Histologic Findings (Liver, Testes, Pancreas) No Significant Findings M 1-0 mg/kg/day Some tissue distortion in testis, (Control) changes do not preclude evaluations M 2-2 mg/kg/day No Significant Findings (Low Dose) M 2-2 mg/kg/day (Low Dose) No Significant Findings M 2-2 mg/kg/day (Low Dose) No Significant Findings M 3-20 mg/kg/day (High Dose) No Significant Findings M 3-20 mg/kg/day (High Dose) No Significant Findings M 3-20 mg/kg/day Some tissue distortion in testis, (High Dose) changes do not preclude evaluations 159