Document e13GR1JNx9EM2Vb6w4KMR7YDe

DuPont-18317 TRADE SECRET Study Title Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Test Guidelines: U.S. EPA Health Effects Test Guidelines OPPTS 870.7800(1998) Author: Denise Hoban, B.A, MLT (ASCP) Study Completed on: February 1, 2007 Performing Laboratory: E.I. du Pont de Nemours and Company HaskellSMLaboratory for Health and Environmental Sciences P.O. Box 50 Newark, Delaware 19714 U.S.A. Exygen Research 3058 Research Drive State College, Pennsylvania 16801 U.S.A. Experimental Pathology Laboratories, Inc. 615 Davis Drive, Suite 500 Durham, North Carolina 27713 U.S.A. Laboratory for Advanced Electron and Light Optical Methods College o f Veterinary Medicine North Carolina State University 4700 Hillsborough Street Raleigh, North Carolina 27606 U.S.A. Laboratory Project ID: DuPont-18317 Work Request Number: 16160 Service Code Number: 1545 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats_____________ PAGE RESERVED P-2 DuPont-18317 3- 2 P-3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats _____________________________________________ DuPont-18317 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA FIFRA (40 CFR part 160) Good Laboratory Practice Standards, which are compatible with current OECD and MAFF (Japan) Good Laboratory Practices, except for the item documented below. The item listed does not impact the validity of the study. A non-GLP characterization was performed prior to the initiation o f this study. The accuracy of the composition at the concentrations documented in this report is considered sufficient for the purpose o f this study and is based on the process chemistry provided by the sponsor. GLP characterization was performed concurrently during the course o f the study. Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: Denise Hoban. B.A, MLT (ASCP) Staff Medical Technologist and Supervisor i fkh<2oo7 Date Applicant / Sponsor: DuPont Representative Date Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats___________ QUALITY ASSURANCE STATEMENT Work Request Number: Study Code Number: 16160 1545 P-4 DuPont-18317 Phase A udited A udii Dates Date Reported to Study Director Date Reported to Management Protocol: October 17, 2005 October 17, 2005 October 17, 2005 Conduct: November 11, 2005 November 14, 2005 May 30, 2006* June 14, 2006* June 27, 2006* October 25, 2006* November 11,2005 November 14, 2005 October 31, 2006* October 31, 2006* October 31, 2006* October 31,2006* November 11, 2005 November 14, 2005 November 2, 2006* November 2, 2006* November 2, 2006* November 2, 2006* Report/Records: February 2, 7, 2006 August 10, 11, 14-18, 2006 November 13-14, 2006 February 7, 2006 August 18, 2006 November 14, 2006 February 8, 2006 September 11, 2006 December 12, 2006 * EPL QA Dates Reported by: ih - J U Joseph C. Hamill Quality Assurance Auditor QI z . ' 2 DO 7 Date Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats___________ P-5 DuPont-18317 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Analytical Evaluation by: Z. Amanda Shen, Ph.D. Research Chemist Date Clinical Pathology Evaluation by: /% ______ Nancy E, Evcrds, D.V.M., Diplogfatd A.C.V.P. Principal Research Clinical Pathologist and Manager Date Anatomic Pathology Evaluation by: GrejfP^&ykcs, VJiLD., Diplomalc A.C.V.P., A.C.L.A.M., A.B.T. Veterinary Pathologist (P/~ ~2jbo? Date Anatomic Pathology Evaluation Peer Review by: Steven R- Framc.'D.V.M., PhD., Diplomalc A.C.V.P. Research Fellow and Manager 2 .0 0 7 Date r Reviewed and Approved by: Scott R Loveless, PhD. Research Manager and Director Issued by Study Director: Denise Hoban, B.A. MLT (ASCP) Staff'Medical Technologist and Supervisor Date 7Olfe h le n Date Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats__________ P-6 DuPont-18317 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT.................................. 3 QUALITY ASSURANCE STATEMENT................................................................................. 4 CERTIFICATION....................................................................................................................... 5 LIST OF TABLES....................................................................................................................... 8 LIST OF FIGURES..................................................................................................................... 8 LIST OF APPENDICES............................................................................................................. 9 STUDY INFORMATION..........................................................................................................10 SUMMARY................................................................................................................................ 11 INTRODUCTION...................................................................................................................... 13 STUDY DESIGN........................................................................................................................ 13 A. Design Concentrations............................................................................................................... 13 B . Study Overview..........................................................................................................................14 MATERIALS AND METHODS...............................................................................................14 A. Test Guidelines...........................................................................................................................14 B. Test Substance............................................................................................................................14 C. Test System................................................................................................................................. 14 D. Animal Husbandry......................................................................................................................15 E. Pretest Period.............................................................................................................................. 15 F. Assignment to Groups................................................................................................................ 16 G. Dose Formulation Preparation and Administration............................................................... 16 H. Dose Formulation Sampling and Analysis............................................................................. 16 I. Body Weights.............................................................................................................................. 18 J. Food Consumption and Food Efficiency.................................................................................18 K. Clinical Observations................................................................................................................. 18 L. Clinical Pathology Evaluation.................................................................................................. 19 M. Humoral Immune Function....................................................................................................... 20 N. Anatomic Pathology Evaluation.............................................................................................. 20 O. Total Cell Counts....................................................................................................................... 21 P. Electron Microscopy Evaluation.............................................................................................. 22 Q. Statistical Analyses.................................................................................................................... 22 6- 7 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p.7 DuPont-18317 TABLE OF CONTENTS Page RESULTS AND DISCUSSION.......................................................................................... Analytical Evaluation.......................................................................................................... A. Chromatography................................................................................................................. B. Recovery Samples............................................................................................................... C. Concentration Verification, Uniformity o f Mixing, and 5-Hour Room Temperature Stability Samples.................................................... ....................................................... D. Concentration Verification and Uniformity o f Mixing Samples.................................. E. Analytical Conclusions...................................................................................................... In-Life Measurements......................................................................................................... A. Mean Body Weights and Body Weight Gains................................................................ B. Food Consumption and Food Efficiency......................................................................... C. Clinical Observations and Mortality................................................................................ Clinical Pathology Evaluation........................................................................................... A. Hematology......................................................................................................................... B. Clinical Chemistry............................................................................................................. C. Clinical Pathology Conclusions....................................................................................... Immunotoxicity.................................................................................................................. A. Humoral Immune Function............................................................................................. Anatomic Pathology Evaluation........................................................................................ A. Cause of Death................................................................................................................... B. Final Body and Organ Weight Data................................................................................ C. Gross Observations........................................................................................................... D. Microscopic Findings........................................................................................................ E. Anatomic Pathology Conclusions................................................................................... Total Cell Counts............................................................................................................... A. Spleen Cell Number.......................................................................................................... B. Thymus Cell Number........................................................................................................ CONCLUSIONS................................................................................................................ RECORDS AND SAMPLE STORAGE.......................................................................... REFERENCES................................................................................................................... TABLES.............................................................................................................................. FIGURES............................................................................................................................ APPENDICES.................................................................................................................... 23 23 23 23 .23 ,24 ,24 .25 .25 .25 .26 .26 .26 .28 .29 .29 .29 .30 .30 .30 .31 ..32 ..33 ..34 ..34 ..34 ..34 ..34 ..35 ..37 ..66 ...72 -7 - X Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p.8 DuPont-18317 Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 LIST OF TABLES Page Recovery of APFO Added to Dosine Vehicle Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability of APFO in Dosing Solutions. Concentration Verification and Uniformity of Mixing of APFO in Dosing Solutions ...... 42 Mean Body Weights of Male Rats Mean Body Weight Gains of Male Rats Mean Daily Food Consumption bv Male Rats Mean Daily Food Efficiency of Male Rats Summary of Daily Animal Health Observations in Male Rats Summary of Detailed Clinical Observations in Male Rats Summary o f Hematology Values for Male Rats Summary of Clinical Chemistry Values for Male Rats Summary of Primary Humoral Immune Response to SRBC for Male Rats Dosed with A P F O .................... Summary of Primary Humoral Immune Response to SRBC for Male Rats Dosed With Positive Control.................. Mean Final Body and Organ Weights from Male Rats Incidence of Gross Observations in Male Rats Incidences and Lesion Grades of Microscopic Observations in Male Rats Summary of Total Cell Counts......... ..... 60 LIST OF FIGURES Figure 1 Figure 2 Figure 3 Representative Analytical Calibration Curve Representative LC/MS/MS Chromatograms Mean Body Weights of Male Rats..... Page P-9 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats____________ ____________________________________ DuPont-18317 LIST OF APPENDICES Page Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Appendix G Appendix H Appendix I Appendix J Appendix K Appendix L Certificate of Analysis..................................................................................................................... 73 Individual Body Weights.................................................................................................................73 Individual Food Consumption........................................................................................................ 83 Individual Daily Animal Health Observations...............................................................................87 Individual Detailed Clinical Observations and Mortality Records..............................................90 Individual Animal Clinical Pathology Data................................................................................... 94 Individual Primary Humoral Immune Response D ata................................................................ 112 Individual Primary Humoral Immune Response Positive Control D ata....................................115 Individual Animal Final Body and Organ W eights.....................................................................117 Individual Animal Pathology D ata............................................................................................... 121 Individual Total Cell Counts.......................................................................................................... 161 Electron Microscopy Report from Experimental Pathology Laboratories, Inc.......................169 -9 - /6 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats____________________ p. 10 DuPont-18317 STUDY INFORMATION Substance Tested: Ammonium Perfluorooctanoate [AFPO (linear)] 3825-26-1 (CAS Number) Haskell Number: 27308 Composition: Ammonium Perfluorooctanoate Solution 19.5% in water Purity: 19.5% Physical Characteristics: White to slightly opaque liquid Stability: The test substance was stable under the conditions o f the study based on analytical results. Study Initiated/Completed: October 14, 2005 / (see report cover page) Experimental Start/Termination: October 16, 2005 / February 1, 2007 - 10// Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 11 DuPont-18317 SUMMARY The purpose o f this study was to evaluate the potential o f ammonium perfluorooctanoate [APFO (linear)] to suppress the primary humoral immune response following exposure via oral gavage for up to 28 consecutive days. Groups o f 10 male rats each were administered the test substance at daily levels o f 0, 0.3, 1,10, 30, and 30/0 mg/kg. The group designated 30/0 mg/kg was included to assess potential reversibility/recovery and was therefore administered the test substance for 22 consecutive days followed by 6 consecutive days of vehicle (water) administration. Body weights, food consumption measurements, and clinical observations were recorded during the in-life period. Prior to sacrifice, the immune system was stimulated by injecting sheep red blood cells (SRBC) on test day 22 and blood samples were collected from each rat on test day 29. The serum samples were assayed for their concentration o f SRBCspecific IgM antibodies to provide a quantitative assessment o f humoral immune response. Serum from animals dosed with cyclophosphamide, a positive control immunosuppressive agent, were analyzed concurrently to provide confirmation that the assay performance was acceptable for detection o f immunosuppression. Clinical pathology data were collected at day 29 to assess effects on hematology and clinical chemistry. At sacrifice, each animal was examined grossly and selected organs were weighed (brain, spleen, and thymus); selected tissues (as outlined in the methods section) were retained and examined histologically. Thymus and spleen cells were manually counted from single-cell suspensions prepared from the collected tissue. Samples o f the dosing formulations were chemically analyzed and the results indicated that the test substance was at the targeted concentrations, homogeneously mixed, and stable under the conditions of the study. Test substance-related toxicity was observed during the in-life portion o f the study at 1 mg/kg and higher. Adverse reductions in body weights, weight changes, food consumption, and food efficiency occurred at 10 mg/kg and higher; at 30 and 30/0 mg/kg, these reductions were accompanied by low incidences o f clinical observations indicative of toxicity. Effects on body weight and food consumption parameters were detected at 1 mg/kg, but these reductions were not considered adverse. There were no test substance-related effects observed at 0.3 mg/kg during the in-life portion o f the study. Rats dosed with >0.3 mg/kg had decreased serum total, HDL, and non-HDL cholesterol, and decreased triglycerides. Rats dosed with >1 mg/kg had increased microscopic red cell morphologic changes and hemolyzed serum. Rats dosed with >10 mg/kg had decreased hemoglobin, hematocrit, mean cell volumes, and mean cell hemoglobin concentrations; increased reticulocyte counts and red cell distribution width, increased total white blood cell, neutrophil, monocyte, and LUC counts; increased serum albumin and decreased serum globulin concentrations; and increased serum corticosterone concentrations. Rats in the 30/0 mg/kg group had more pronounced red cell mass effects and red cell morphologic changes compared to those dosed with 30 mg/kg for 29 days. Parameters with complete recovery in rats dosed with 30/0 mg/kg were serum total, HDL, and non-HDL cholesterol, globulin, and corticosterone concentrations. -11 - p. 12 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 There was a test substance-related decrease in final body weights and increase in liver weights. Mean final body weights were decreased at dose levels >10 mg/kg of the test substance. Mean liver weight parameters were increased at dose levels >0.3 mg/kg. At the terminal sacrifice, test substance-related gross observations were limited to discoloration of the liver in a few rats at doses >10 mg/kg. Microscopic examination o f the liver demonstrated minimal to mild hepatocellular hypertrophy at 0.3 and 1 mg/kg and moderate hepatocellular hypertrophy at >10 mg/kg. Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased hematopoiesis in the spleen o f rats dosed with 30/0 mg/kg. No test substance-related evidence of immunosuppression was observed in male rats at any concentration tested; the IgM titers were generally comparable across all groups. No significant changes in total spleen cell or thymocyte number compared to control rats were noted in any animals treated with any dose o f APFO. Under the conditions o f this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male rats was less than 0.3 mg/kg, whereas the NOAEL for immunotoxicity was 30 mg/kg. - 12- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 INTRODUCTION The primary objective o f this study was to evaluate the potential o f ammonium perfluorooctanoate [APFO (linear)] to suppress the primary humoral immune response to sheep red blood cells (SRBC) when administered by oral gavage to male rats for at least 28 days. Additional endpoints o f toxicity were also evaluated. The oral route o f administration was selected because it is a potential route of human exposure. Ammonium perfluorooctanoate (APFO; FC-143, Cg; C7F15COO N H /; CAS Registry number 3825-26-1) is a surfactant used as a processing aid in the production o f fluoropolymers. Perfluorooctanoate (PFOA; C7F15COO), the dissociation product o f APFO, is not metabolized(1) and has been identified in blood samples from exposed workers and the general population.(2,3,4) PFOA has been reported to inhibit the ability o f mice to make antibodies to a T-cell dependent antigen.(5) The reported study employed a single 0.02% PFOA in chow (approximately 30 mg/kg) for 16 days. In order to better characterize the immune response following exposure to this material, APFO was administered by oral gavage using a broad range o f doses. Dosages for this study were selected based on results o f a 14-day oral gavage study in male rats and mice.(6) STUDY DESIGN A. Design Concentrations Group I III V VII IX XI Number/ Group 10 10 10 10 10 10 Daily Dosage (mg/kg)a 0 (Control) 0.3 1 10 30 30/0c Dose Solution Concentration (mg/mL)b 0 0.03 0.1 1 3 3 a Weight of test substance/kg or animal body weight, b Solutions were adjusted for purity (19.5%). c This group (XI) was dosed with 30 mg/kg of test substance through test day 22. Following injection of SRBC on test day 23, group XI was dosed with NANOpure water, at a volume of 10 mL/kg of body weight, until sacrifice. -13 / p. 14 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats B. Study Overview DuPont-18317 Study Parameters Frequency Body Weight Day 0, 3, 6, and daily thereafter Food Consumption Weekly Daily Animal Health Observation Twice daily General Clinical Observation3 Day 0 and weekly thereafter Detailed Clinical Observation At each weighing SRJBC Injection Prior to dosing (test day 23) Clinical Pathology Evaluation Test day 29 Serum Collection for Antibody Determination At sacrifice (test day 29) Anatomic Pathology Evaluation Test day 29 a A check for acute signs of toxicity was conducted approximately 2 hours post-dosing. MATERIALS AND METHODS A. Test Guidelines The study design complied with the following test guidelines: U.S. EPA, OPPTS 870.7800: Immunotoxicity, Health Effects Test Guidelines (1998) B. Test Substance (Appendix A) APFO (linear), was supplied by the sponsor as a white to slightly opaque liquid in a 19.5% aqueous solution. The bulk test substance was used within the period o f approved use as defined by the expiration date listed on the Certificate o f Analysis (COA) that is provided in Appendix A. No evidence o f instability, such as a change in color or physical state, was observed. C. Test System On October 6, 2005, 66 male Crl:CD(SD) rats, with an assigned birth date o f August 22, 2005, were received from Charles River Laboratories, Raleigh, North Carolina. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at Haskell Laboratory. By utilizing the Crl:CD(SD) rat, immunotoxicity studies can be conducted in the same strain that is used for other toxicology studies. -14- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats D. Animal Husbandry DuPont-18317 1. Housing All animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. 2. Environmental Conditions Animal rooms were maintained at a temperature of 18-26C and a relative humidity o f 30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. Excursions outside o f these ranges were of insufficient magnitude and/or duration to have adversely affected the validity o f the study. 3. Feed and Water All rats were provided tap water ad libitum. All rats were fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum. 4. Animal Health and Environmental Monitoring Program As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity o f the study: Water samples are analyzed for total bacterial counts, and the presence o f coliforms, lead, and other contaminants. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations o f key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence o f these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity o f the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation o f these data did not indicate any conditions that affected the validity of the study. E. Pretest Period Upon arrival at Haskell Laboratory, all rats were housed in quarantine. The rats were: quarantined for 6 days. identified temporarily by cage identification. weighed at least 3 times during quarantine. -15- p. 16 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 observed with respect to weight gain and any gross signs o f disease or injury. The rats were released from quarantine by the laboratory animal veterinarian or designee on the bases o f acceptable body weights and clinical signs of all rats. F. Assignment to Groups Rats, selected on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury, were distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected rats did not exceed 20% o f the mean weight for each sex. At grouping, each rat was assigned an animal number/cage identification number. The animal number/cage identification number were tattooed on the tail o f each rat and included on the cage label. At study start (test day 0) the rats were 8 weeks o f age. G. Dose Formulation Preparation and Administration The dosing solutions were prepared in NANOpure water. The formulations were adjusted based on the percentage o f APFO in the bulk test substance to achieve the desired concentrations. Dosing formulations were prepared on a daily basis. Animals were dosed daily at approximately the same time ( 2 hours) by intragastric intubation at a dose volume o f 10 mL/kg body weight for at least 28 consecutive days; individual dose volumes were calculated based on the most recently collected body weight data. Control rats were similarly dosed with NANOpure water at a volume of 10 mL/kg o f body weight. The 30/0 mg/kg group (XI) was dosed with 30 mg/kg o f test substance through test day 22. Following injection o f SRBC on test day 23, group XI was dosed with NANOpure water at a volume o f 10 mL/kg o f body weight until sacrifice. One rat from group XI (1109) was not dosed on test days 6 through 8 due to a decrease in body weight gain. Once body weight increases were observed for this rat, dosing resumed. H. Dose Formulation Sampling and Analysis 1. Recovery Sample Analysis Concurrent with dosing formulation analyses, recovery o f APFO from spiked NANOpure water was tested at the low level (approximately 0.03 mg/mL), the middle levels (approximately 0.1 and 1 mg/mL), and the high level (approximately 3 mg/mL) to confirm the analytical method. A stock solution of APFO was prepared in NANOpure water. For all concentration levels, an appropriate aliquot o f the stock solution was used to make the spiked solution upon further dilution with NANOpure water. These spiked recovery samples were then processed and analyzed in the same manner as the dosing samples at similar concentrations. - 16- p. 17 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats________ DuPont-18317 2. Dosing Solution Treatment Each dosing sample (1 mL) was initially diluted with NANOpure water to a nominal concentration of 0.3, 1, 10, and 30 ppm APFO for the 0.03, 0.1, 1, and 3 mg/mL dosing samples, respectively. The samples were further diluted to a final expected concentration o f 0.03 ppm with NANOpure water for analysis. The 0 mg/mL sample followed the 0.03 mg/mL sample dilutions. Before the final dilution, the internal standard (1, 2-di-13C PFOA) was added to each sample to give an equivalent final concentration o f the internal standard in all dosing samples; the 0.1, 1, and 3 mg/mL samples were matrix corrected with the initial diluted solution o f the control sample. 3. Chromatographic Conditions LC Parameters Instrument: Agilent (Hewlett-Packard) 1100 liquid chromatograph Column: Zorbax RX-C8, 2.1 x 150 mm, 5 pm Flow Rate: 0.4 mL/min Oven Temperature: 35C Injection Volume: 20 pL Mobile Phase: A) 0.15% Acetic acid in NANOpure water B) Acetonitrile Gradient: Time (min) % Acetonitrile 05 0.9 5 1.0 80 5.0 80 5.1 5 7.0 5 MS Parameters Instrument: Waters (Micromass) Quattro Micro Ionization Mode: Electrospray (ESI), negative ion Capillary Voltage: 2.7 kV Cone Voltage: 15 V Source Temperature: 120C Desolvation Temperature: 350C Scan Function: PFOA: 413 m/z (parent) to 369 m/z (daughter) 1, 2-di-13C PFOA: 415 m/z (parent) to 370 m/z (daughter) 4. Calibration and Quantitation The analytical reference o f APFO (H-22703-376, 100%) was used for quantitation o f this study. A stock solution was prepared in NANOpure water. This stock solution was mixed to ensure that all material was dissolved in solution. Before analysis, appropriate aliquots o f the stock solution were diluted with NANOpure water to make calibration standards that bracketed the target concentration o f the diluted dosing samples after matrix correction with the initial diluted solution of the control sample. Before these aliquots were brought to the final volume, an - 17 /? p. 18 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 appropriate amount o f 1, 2-di-13C PFOA internal standard was added to give an equivalent final concentration o f the internal standard in all standard solutions. The 369 m/z daughter ion o f PFOA dissociated from APFO measured by LC/MS/MS was used against the 370 m/z daughter ion of 1 ,2-di-13C PFOA internal standard to determine the concentrations o f the dosing samples. Peak area ratios (369 m/z peak versus 370 m/z peak) of these standards were used to construct a calibration curve by least square regression (see Figure 1 for a representative calibration curve). Measured concentrations for dosing solutions were determined by applying the peak area ratios from replicate injections o f each sample to the calibration curve. Concentration verification o f APFO in dosing samples was evaluated by the mean result of the duplicate analyses for each respective dosing level. Uniformity of mixing o f APFO in dosing samples was evaluated by calculating the coefficient of variation (C.V. = standard deviation/mean x 100) of the measured concentration in the duplicate analyses o f the concentration verification samples. A coefficient o f variation of less than or equal to 10% is the standard criterion at Haskell Laboratory for acceptable distribution o f the test substance throughout the solution. Stability o f APFO in dosing samples was evaluated by using the mean result o f the duplicate concentration verification analyses as the baseline for comparing the corresponding stability results. I. Body Weights During the test period, all rats were weighed on test days 0, 3, 6, and daily thereafter. J. Food Consumption andFood Efficiency During the test period, the amount o f food consumed by each rat over the weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount o f spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined. From the food consumption and body weight data, the mean daily food efficiency of test substance was calculated for each animal. K. Clinical Observations 1. Daily Animal Health Observations Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least twice daily throughout the study. Abnormal behavior/appearance was recorded by exception. - 18- p. 19 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 2. General Clinical Observations An additional cage-site evaluation was conducted approximately 2 hours after dosing to detect acute clinical signs o f systemic toxicity. 3. Detailed Clinical Observations At every weighing, each rat was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observations included (but were not limited to) evaluation o f fur, skin, eyes, mucous membranes, occurrence o f secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence o f clonic, tonic, stereotypical, or bizarre behavior. Any abnormal clinical signs noted were recorded. L. Clinical Pathology Evaluation A clinical pathology evaluation was conducted on all animals approximately 29 days after initiation o f the study. These animals were fasted after 3 p.m. for at least 15 hours. Blood samples for hematology measurements were collected from the orbital sinus o f each animal while the animal was under carbon dioxide anesthesia. Blood samples for clinical chemistry and humoral immune system evaluations were collected at sacrifice from the abdominal vena cava of each animal while the animal was under carbon dioxide anesthesia. Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were stained with WrightGiemsa stain, but analysis was not necessary to support experimental findings. Blood smears, stained with new methylene blue, were prepared from each animal undergoing a hematology evaluation, but were not needed for examination. All blood samples were evaluated for quality by visual examination. Results were maintained in the study records and reported only if the sample was analyzed. 1. Hematology Complete blood counts, including reticulocytes, were determined on a Bayer Advia 120 hematology analyzer or determined from microscopic evaluation o f the blood smear. WrightGiemsa-stained blood smears from all animals were examined microscopically for confirmation of automated results and evaluation o f cellular morphology. The following parameters were determined: red blood cell count red cell distribution width hemoglobin absolute reticulocyte count hematocrit platelet count mean corpuscular (cell) volume white blood cell count mean corpuscular (cell) hemoglobin differential white blood cell count mean corpuscular (cell) hemoglobin concentration microscopic blood smear examination -19- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 2. Clinical Chemistry Routine serum clinical chemistry parameters were determined on an Olympus AU640 clinical chemistry analyzer. Serum corticosterone was measured using a commercial RIA assay (Diagnostic Products Corporation, Los Angeles, CA; Catalog #TKRC1). Corticosterone concentrations were determined according to the manufacturer's recommended procedure (aspirating aqueous contents o f the assay tube rather than decanting). If necessary, the standard curve was extended at the low end of the range by including standards o f 5 and 10 ng/mL. The following parameters were determined: cholesterol triglycerides total protein albumin globulin (calculated) high-density lipoprotein cholesterol non-high-density lipoprotein cholesterol (calculated) serum corticosterone M. Humoral Immune Function On test day 23, animals were injected intravenously in the lateral tail vein with 0.5 mL of 4 x 108 SRBC/mL (Covance, Denver, Pennsylvania, U.S.A.). On test day 29, serum was collected from each rat and frozen (see L.2. Clinical Chemistry). Humoral immune function was evaluated by examining sera from individual animals for SRBCspecific IgM levels with an enzyme-linked immunosorbent assay (ELISA).(7) The serum from each animal was assayed as 10 serial, 2-fold dilutions, with 1 replicate per dilution. The optical density (OD) of the contents of the reaction well was measured at the 405 nm wavelength with a MR 5000 Microplate Reader (Dynex Technologies). SRBC-specific serum IgM titer data were analyzed with Revelation Software Version 2.0 (Dynex Technologies). For each serum sample, a semi-log graph o f the data was created and the linear portion o f the curve was identified by using a log-log curve fit. A slope between -0.600 and -1.200 was obtained. The serum dilution expected to produce an OD o f 0.5 was determined by regression analysis. The "titer" o f each animal was defined as the reciprocal o f the serum dilution that had an OD value of 0.5. If no points had an OD value of greater than or equal to 0.5, the reciprocal o f the starting dilution closest to an OD value o f 0.5 was used as the titer. Sera previously collected from rats injected with SRBC and dosed for 6 days with 20 mg/kg of the known immunosuppressive agent, cyclophosphamide monohydrate, or vehicle were run concurrently with the study samples to demonstrate that the assay functioned properly. For any test samples that needed to be rerun due to a poor curve fit or slope, pooled male or female cyclophosphamide monohydrate or vehicle serum samples were concurrently run. The pooled samples consisted of equal aliquots o f serum taken from either the male or female rats dosed with cyclophosphamide monohydrate or vehicle. N. Anatomic Pathology Evaluation After 29 days on study, all rats from each dose group (0, 0.3, 1,10, 30, and 30/0 mg/kg body weight) were sacrificed and necropsied for evaluation of subchronic toxicity. The order of Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Smdy in Male Rats DuPont-18317 sacrifice for scheduled deaths was stratified across groups. Rats were fasted at least 15 hours before their scheduled sacrifice. All rats survived the duration o f the study and were euthanized by carbon dioxide anesthesia and exsanguination. Gross examinations were performed and final body and organ weights were recorded. The following tissues were collected from all 60 rats (10/sex/group) on study. Digestive System liver3 Nervous System brain3,c (3 sections) Hematopoietic System spleen3 thymus3 popliteal lymph node mesenteric lymph node bone marrowb Musculoskeletal System femur/knee joint sternum Other gross observations a Organs were weighed at necropsy, b Bone marrow was collected with the femur and sternum, c Including cerebrum, cerebellum, medulla/pons Organ weight ratios (% final body weight, % brain weight) and group mean values for weighed organs were calculated. All tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist. Microscopic findings were graded on a 4-point scale based on the severity or extent o f the change (grade 1 = minimal; grade 2 = mild; grade 3 = moderate; grade 4 = severe). All tissues collected from control (0 mg/kg) and high-dose (30 and 30/0 mg/kg) rats were processed to slides and evaluated microscopically. In addition, the following organs were examined from intermediate-dose rats in order to determine a no-observed-effect level for test substance-related microscopic findings: liver and spleen. Gross observations (recorded at necropsy) were examined microscopically for all animals. O. Total Cell Counts The following procedures were used for preparation o f spleen and thymus single-cell suspensions for enumeration of total cell counts from each spleen or thymus: The weight of the halved spleen or thymus (tissue) was documented; the half was placed in a labeled 15 mL centrifuge tube containing 5 mL Hank's Balanced Salt Solution (HBSS/H) and put on ice. -21 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 The halved tissue/HBSS/H was poured into a small petri dish and cut into small pieces. The tissue/HBSS/H was poured into a Stomacher 80 Lab System bag and placed into the Stomacher 80 Lab System on "high" setting for 120 seconds (spleen) or 60 seconds (thymus). After the Stomacher 80 Lab System stopped, the cell suspension was pipetted back into the original centrifuge tube, rinsing the bag with 3 mL HBSS/H and adding that to the centrifuge tube. The centrifuge tube was inverted 2 or 3 times and left on ice for approximately 10 minutes to allow debris to settle to the bottom of the tube. The supernatant was transferred to a new centrifuge tube and the volume o f the supernatant was documented. Total cell counts were determined from each tissue by hemacytometer. P. Electron Microscopy Evaluation A section o f liver from 2 control rats (105 and 106) and 2 rats from the 30 mg/kg group (905 and 906) was placed in cassettes, in a container o f formalin, and shipped to Experimental Pathology Laboratories, Inc (EPL) and evaluated by transmission electron microscopy. As a subcontractor to EPL, the Laboratory for Advanced Electron and Light Optical Methods, College of Veterinary Medicine, North Carolina State University processed the tissues for electron microscopy and prepared electron photomicrographic images under the direction of Dr. Michael Dykstra. The printed electron photomicrographic images were provided to EPL for evaluation by an ACVP-certified veterinary pathologist who interpreted the images and prepared a final report of the electron microscopic evaluation. Q. Statistical Analyses For all statistical analyses, significance was judged at p < 0.05. Comparisons were made o f the dosed groups to the control group (Group I). Comparisons were also made between Group IX and Group XI. Parameter Body Weight Body Weight Gain Food Consumption Food Efficiency Humoral Immune Function Data3 Clinical Pathology Organ Weights Total Cell Counts Preliminary Test Levene's test for homogeneity(8) and Shapiro-Wilk test(9) for normal ityb Method of Statistical Analysis If preliminary test is not If preliminary test is significant significant One-way analysis of variance*10*followed by Dunnett's test*11'12'13* Kruskal-Wallis test*14* followed by Dunn's test*15* a SRBC-specific serum IgM antibody titer data were transformed to Log2to obtain normality or homogenous variances. b If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version of Dunnett's test was used. If the Shapiro-Wilk test was significant, Kruskal-Wallis test was followed by Dunn's test. - 2273 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 RESULTS AND DISCUSSION Analytical Evaluation A. Chromatography (Figures 1-2) PFOA dissociated from APFO and 1, 2-di-I3C PFOA eluted together from the HPLC column with a retention time o f approximately 4.5 minutes. The mixture was separated into 2 resolved peaks at 369 m/z and 370 m/z, respectively, by MS/MS detection. Representative LC/MS/MS chromatograms are shown in Figures 2(a - e). Test substance was not detected in the 0 mg/mL samples. B. Recovery Samples (Table 1) Detailed analytical results of recovery samples are summarized in Table 1. The variability o f the analytical method was demonstrated by the coefficients o f variation o f the recovery results at each targeted dosing concentration (approximately 0.03, 0.1, 1, and 3 mg/mL) over the course of the study. The range o f the measured concentrations of APFO for the 0.03 mg/mL level was 101.7% to 108.3% o f nominal (mean percent recovery = 105.0% 5%, C.V. = 5%). The range o f the measured concentrations o f APFO for the 0.1 mg/mL level was 104.0% to 109.6% of nominal (mean percent recovery = 106.8% 4%, C.V. = 4%). The range o f the measured concentrations o f APFO for the 1 mg/mL level was 102.0% to 105.0% o f nominal (mean percent recovery = 103.5 2%, C.V. = 2%). The range o f the measured concentrations o f APFO for the 3 mg/mL level was 101.7% to 107.0% o f nominal (mean percent recovery = 104.4 4%, C.V. = 4%). Based on these data, the analytical method performed satisfactorily for the concentration range of the dosing samples in the study. C. Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability Samples (Table 2) Analytical results from dosing solutions prepared on October 17, 2005 analyzed for concentration verification, uniformity o f mixing, and 5-hour room temperature stability are shown in Table 2. The following table summarizes the results for concentration verification, uniformity o f mixing, and 5-hour room temperature stability analyses. -23- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Preparation Nominal Measured2 Average C.V. Stability13 Date mg/mL 17-0ctober-2005 0 mg/mL NDC % Nominal (%) % Nominal -- ---- 0.03 0.0278, 0.0277 92.7 0.3 96.3 0.1 0.0966, 0.0979 97.3 0.9 99.0 1 0.979, 1.04, 1.03d 102.0 3 96.9 3 3.16, 3.06 103.7 2 102.0 a Duplicate samples analyzed. b Stability samples held for 5 hours at room temperature, c Denotes not detected. d Data obtained from one of the duplicate initial analyses and 2 repeats from the re-diluted sample. The data for samples submitted on October 17, 2005 show that the test substance was at the targeted levels ( 7.3% o f nominal), uniformly mixed (CV's = 0.3%, 0.9%, 3%, and 2%, respectively), and stable when held for 5 hours at room temperature in the vehicle. Test substance was not detected in the 0 mg/mL sample. D. Concentration Verification and Uniformity of Mixing Samples (Table 3) Analytical results from dosing solutions prepared on November 15, 2005 analyzed for concentration verification and uniformity o f mixing are shown in Table 3. The following table summarizes the results for concentration verification and uniformity of mixing analyses. Preparation Date 15-November-2005 Nominal mg/mL 0 0.03 0.1 1 3 Measured3 mg/mL 0.0276, 0.0272 0.0954, 0.0986 1.02, 1.01 3.21,3.23 Average % Nominal -- 91.3 97.0 102.0 107.3 C.V. (%) -- 1 2 0.7 0.4 a Duplicate samples analyzed, b Denotes not detected. The data for samples submitted on November 15, 2005 show that the test substance was at the targeted levels ( 8.7% o f nominal) and uniformly mixed (CV's = 1%, 2%, 0.7%, and 0.4%, respectively). Test substance was not detected in the 0 mg/mL sample. E. Analytical Conclusions Data from the analysis o f the samples during the study indicate that the test substance was at the targeted concentrations, mixed uniformly, and stable under the conditions o f the study. Test substance was not found in the 0 mg/mL samples. -24 2$ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 In-Life Measurements A. Mean Body Weights and Body Weight Gains (Tables 4-5, Figure 3, Appendix B) Test substance related adverse reductions in mean body weights and body weight gains were observed at 10, 30 and 30/0 mg/kg. Mean final body weights were 10, 25, and 21% lower than the control group at 10, 30, and 30/0 mg/kg, respectively, as a result o f reduced body weight gains; overall body weight gains during test days 0 to 28 were 26, 63 and 50% lower for the same respective doses. The magnitude and onset o f the effects on body weight parameters were dose related in that the effects at 30 mg/kg were evident sooner and were more pronounced. There was no appreciable difference in the magnitude o f the reduction between the 30 and 30/0 mg/kg, indicating that the shortened dosing period did not have a significant impact on this endpoint. At 1 mg/kg, overall body weight gain during test days 0 to 28 was 10% lower, resulting in a 4% reduction in mean final body weight. These slight reductions appear to be test substance related; however, they were not statistically significant nor were they considered to be adverse. Body weight data for animals dosed at 0.3 mg/kg were generally comparable to control group data. B. Food Consumption and Food Efficiency (Tables 6-7, Appendix C) Test substance related adverse reductions in mean daily food consumption and food efficiency were observed at 10, 30 and 30/0 mg/kg; test substance-related effects on these parameters were also observed at 1 mg/kg but these effects at 1 mg/kg were not considered adverse based on the magnitude o f the reductions. Mean daily food consumption was 4, 17 and 16% lower than controls at 10, 30, and 30/0 mg/kg, respectively, during test days 0 to 28. The combined test substance-related reductions in mean body weight, weight gain, and food consumption resulted in test substance-related reductions in food efficiency. During test days 0 to 28, mean food efficiency was 23, 57 and 42% lower at 10, 30 and 30/0 mg/kg/day. The effects on food consumption parameters were similar to and consistent with the effects on body weight parameters in that the magnitude and onset o f the effects on food consumption and efficiency were dose related terms o f onset, severity, and duration o f the effects. Additionally, there was no appreciable difference in the magnitude o f the reduction o f the overall mean daily food consumption between the 30 and 30/0 mg/kg, indicating that the shortened dosing period did not have a significant impact on this endpoint. -25- p. 26 Ammonium Peril uorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 At 1 and 10 mg/kg, mean daily food consumption and food efficiency was 3 and 7% lower than controls, respectively. These slight reductions appear to be test substance related; however, they were not statistically significant nor were they considered to be adverse. Food consumption and food efficiency data for animals dosed at 0.3 mg/kg were generally comparable to control group data. C. Clinical Observations and Mortality (Tables 8-9, Appendices D-E) There was no test substance-related mortality at any level tested; all animals survived to the scheduled sacrifice on test day 29. Test substance related clinical observations were observed at 30 and 30/0 mg/kg and included wet and/or stained fur, absent or decreased feces, not eating, high carriage, and lethargy. These signs were reported for up to 3 animals in these groups and thus, the incidence was not overwhelming. However, the nature o f the signs combined with the effects on body weight and food consumption discussed previously supported that these observations were test substancerelated and adverse. Hair loss was reported in up to 3 animals per group; this unremarkable finding was not considered test substance related since the incidence was not dose-related. Clinical Pathology Evaluation A. Hematology (Table 10, Appendix F) 1. Red Blood Cells Hemolysis was evident in serum of rats dosed with >1 mg/kg (see Clinical Chemistry section). Hemoglobin and hematocrit were mildly decreased in rats dosed with 10 or 30 mg/kg for 29 days (means were 91-92% of the control group mean, respectively; statistically significant), but there were no effects on red blood cell counts. The discordance between red blood cell count and hematocrit was likely due to decreased mean cell volume in rats dosed with 10 or 30 mg/kg for 29 days (hematocrit is the product of mean cell volume and red blood cell count). Means for mean cell volumes were 97 and 95% o f the control group mean; (statistically significant at 30 mg/kg). Mean cell hemoglobin, which generally closely parallels mean cell volume, was also decreased in these 2 dose groups (means were 95 and 94% of the control group mean, respectively; statistically significant). A few rats dosed with 10 or 30 mg/kg for 29 days had increased reticulocytes, although there were no significant changes in mean reticulocyte counts (means were 109 and 112% o f the control group mean). Increased red cell distribution width generally correlated with increased reticulocytes in rats from these groups. Mean red cell distribution widths were 111 and 115%, respectively, o f the control group mean. Microscopically, some o f the rats in these 2 groups had - 26- p. 27 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 increased anisocytosis (variation in red cell size; also observed in rats dosed with 1 mg/kg), macrocytosis (increased numbers o f larger cells), polychromasia (increased bluish staining o f red blood cells), and hypochromasia (pale staining o f red blood cells). These changes were consistent with minimally increased reticulocytes in some animals. Effects on red cell mass parameters were present (red blood cell count) or more pronounced (hemoglobin, hematocrit) in the 30/0 mg/kg group o f rats compared to those dosed with 30 mg/kg for 29 days. On test day 29, mean red blood cell count, hemoglobin, and hematocrit ranged from 86-88% o f the respective control group means for these 3 parameters (all statistically significant). Decreased red cell mass parameters on test day 29 could be due to one or more o f the following processes: increased red cell destruction, red cell loss, or increased plasma volume. The mechanism for decreased red cell mass parameters was not evident from in life, clinical pathology, or anatomic pathology data. Therefore, the cause o f the decreased red cell mass was not determined. Reticulocytes were moderately increased in rats dosed with 30/0 mg/kg. Mean reticulocyte count was 197% o f the control group mean. Consistent with the increase in reticulocyte counts, red cell distribution width was increased (mean was 123% o f the control group mean). Microscopically, this group had increased anisocytosis, macrocytosis, polychromasia, hypochromasia, and acanthocytosis (red blood cells with blunt surface projections). All morphologic changes in red cells occurred at greater incidence or at higher severity grades in these rats compared to rats dosed with APFO for 29 days. These red blood cell changes also correlated with histologic evidence o f increased extramedullary hematopoiesis, which was observed in 7 o f ten 30/0 mg/kg rats, but in none o f the 30 mg/kg rats after 29 days o f dosing. 2. White Blood Cells White blood cell counts were minimally increased, primarily due to increases in lymphocytes, in some rats dosed with 10 or 30 mg/kg for 29 days (variable statistical significance). Means were 130 and 137% (total white blood cell count), and 133 and 140% (lymphocyte count) o f respective control group means. Individual rats dosed with 10 or 30 mg/kg with higher total white blood cell and lymphocyte counts generally had higher neutrophil, monocyte, and large unstained cell (LUC) counts as well, resulting in mean neutrophil, monocyte and LUC counts that were 114-147% o f the control group means. Due to the normal range and variability o f total and individual white blood cell counts, these changes did not result in statistically different means. LUCs are cells that cannot be identified as one o f the 5 major leukocyte types by the Advia 120 automated hematology analyzer, and normally comprise a small percentage o f the total leukocyte population. The LUC count normally includes mostly lymphocytes and monocytes. Consistent with this observation, in this study, the rats with the highest LUC counts usually had the highest lymphocyte and/or monocyte counts. The changes observed in total and individual white blood cell counts are consistent with inflammation. Histologically, there were no findings observed that correlated with these white blood cell changes. In rats that were dosed with 30/0 mg/kg, total white cell and lymphocyte counts were generally similar to their respective control group means, with the exception o f a few rats with higher total white blood cell and lymphocyte counts (rats 1106 and 1107). Monocyte and large unstained cell counts for rats dosed with 30/0 mg/kg were similar to those o f rats dosed for 29 days with 10 -27- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 or 30 mg/kg in that the counts o f most rats were similar to controls, but a few rats had higher monocyte and LUC counts. Therefore, there was no recovery. Mean eosinophil counts were minimally decreased in rats dosed with >0.3 mg/kg for 29 days (not statistically significant). These decreases were the result o f high eosinophil counts in 3 control rats. There was no dose response in changes in eosinophil counts despite the 100-fold difference in dose administered in either terminal or recovery animals. In rats that were dosed with 30/0 mg/kg, eosinophil counts were similar to groups dosed with >0.3 mg/kg for 29 days. Therefore, the apparent decreases in eosinophil counts after 29 days of dosing at >0.3 mg/kg and in 30/0 mg/kg rats is of uncertain relationship to treatment. B. Clinical Chemistry (Table 11, Appendix F) Hemolysis was evident in serum of rats dosed with >1 mg/kg. Hemolysis is graded as none, trace, small, moderate or large. In this study, all samples had either none, trace, or small hemolysis. The incidence o f serum graded trace to small for hemolysis was 1/10, 0/10, 3/10, 9/10, and 7/10 in rats dosed with 0, 0.3, 1, 10, or 30 mg/kg, respectively. In rats that were dosed with 30/0 mg/kg, trace to small hemolysis was observed in 6/10 rats, and the severity was similar to that observed after 29 days o f dosing at 30 mg/kg. Total cholesterol was decreased in rats dosed with 0.3 or 1 mg/kg for 29 days. Means were 64 and 69% o f the control group mean, respectively (statistically significant). Cholesterol concentrations o f rats dosed with 10 or 30 mg/kg, although higher than those dosed with lower doses, were still lower than controls (means were 81 and 84% o f the control group mean, respectively; not statistically significant). The decreases in cholesterol were due to decreases in both HDL and non-HDL cholesterol. HDL cholesterol was decreased by a similar degree in all groups dosed with the test substance for 29 days; means were 75-79% of the control group mean (variable statistical significance). Non-HDL cholesterol, like total cholesterol, was lower in rats dosed with 0.3 or 1 mg/kg (means were 58 and 63% o f control group mean; statistically significant) than in rats dosed with 10 or 30 mg/kg (means were 85 and 88% of control group mean; statistically significant). In rats that were dosed with 30/0 mg/kg, total, HDL, and non-HDL cholesterol concentrations were similar to controls, suggesting recovery for most rats. However, total, HDL, and non-HDL cholesterol concentrations were mildly higher in one recovery rat (1103), and lower in another recovery rat (1107) compared to other animals in the 30/0 mg/kg group. Triglyceride was decreased in rats dosed with >0.3 mg/kg for 29 days. The dose-response was flat across the doses tested; means were 69, 75, 68, and 66% o f control group means for rats dosed with 0.3, 1, 10, or 30 mg/kg, respectively (variable statistical significance). Triglycerides were still decreased in rats that were dosed with 30/0 mg/kg (mean was 69% of the control group mean), indicating a lack o f recovery for triglyceride concentrations. Albumin was increased in a few rats dosed with 1 mg/kg, and in most rats dosed with 10 or 30 mg/kg. Means were 106, 112, and 115% of the control group mean, respectively (variable statistical significance). In rats that were dosed with 30/0 mg/kg, albumin was similar to that of - 28- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats______________ p. 29 DuPont-18317 rats dosed with 30 mg/kg for 29 days (with the exception o f one male, rat 1109, with lower albumin). Mean concentration for rats dosed with 30/0 mg/kg was 112% o f the control group mean, indicating a lack of recovery for albumin concentration. Globulin was decreased in rats dosed with 10 or 30 mg/kg. Means were both 89% of the control group mean. In rats dosed with 30/0 mg/kg, globulin was similar to that o f controls (with the exception of one male, rat 1109, with low globulin), indicating recovery for globulin concentrations. Serum corticosterone was increased in a few rats dosed with 10 or 30 mg/kg for 29 days. Concentrations greater than 300 ng/mL (approximate upper bound for corticosterone concentration in non-stressed rats) were observed in 0/ 10, 0/ 10, 0/ 10, 2/ 10, and 4/10 rats dosed with 0, 0.3, 1, 10, or 30 mg/kg, respectively. The higher corticosterone concentrations in some rats dosed with 10 or 30 mg/kg resulted in mean concentrations that were 135 and 196% of controls, respectively. These changes are indicative o f physiological stress. In rats that were dosed with 30/0 mg/kg, serum corticosterone concentrations were generally similar to controls, indicating recovery. C. Clinical Pathology Conclusions Rats dosed with >0.3 mg/kg had decreased serum total, HDL, and non-HDL cholesterol, and decreased triglycerides. Rats dosed with >1 mg/kg had increased microscopic red cell morphologic changes and hemolyzed serum. Rats dosed with >10 mg/kg had decreased hemoglobin, hematocrit, mean cell volumes, and mean cell hemoglobin concentrations; increased reticulocyte counts and red cell distribution width, increased total white blood cell, neutrophil, monocyte, and LUC counts; increased serum albumin and decreased serum globulin concentrations, and increased serum corticosterone concentrations. Rats in the 30/0 mg/kg group had more pronounced red cell mass effects and red cell morphologic changes compared to those dosed with 30 mg/kg for 29 days. Parameters with complete recovery in rats dosed with 30/0 mg/kg were serum total, HDL, and non-HDL cholesterol, globulin, and corticosterone concentrations. Immunotoxicity A. Humoral Immune Function (Tables 12-13, Appendices G-H) No test substance-related evidence o f immunosuppression was observed in male rats at any concentration tested; the IgM titers were generally comparable across all groups. For the individual and pooled positive control sera, the primary humoral immune response to SRBC was decreased by 57 and 55%, respectively. Therefore, the SRBC-specific ELISA test system was valid for this study. -29- 3 p. 30 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats_______________________________________________ DuPont-18317 Anatomic Pathology Evaluation A. Cause of Death There were no test substance-related deaths. All 60 rats on study survived until the scheduled sacrifice on test day 29. B. Final Body and Organ Weight Data (Table 14, Appendix I) Following 28-days o f daily gavage administration of the test substance, there was a test substance-related decrease in final body weights and increase in liver weights. Mean final body weights were decreased at dose levels >10 mg/kg of the test substance. Mean liver weight parameters were increased at dose levels >0.3 mg/kg. Text Table 1: Mean Absolute and Relative (% body weight) Organ Weights in Male Rats Group: Dose (mg/kg): Number of Rats/Sex: I 0 10 Final Body Wt. (g) 423.1 Liver absolute wt. (g) % body wt. (10) 13.179 3.113 Spleen absolute wt. (g) % body wt. (10) 0.844 0.199 Thymus absolute wt. (g) % body wt. (10) 0.568 0.133 III 0.3 10 419.7 (10) 14.379 3.419 (10) 0.872 0.208 (10) 0.604 0.144 V 1 10 410.0 (10) 17.227* 4.194 (10) 0.835 0.203 (10) 0.559 0.136 VII 10 10 377.0* (10) 21.469* 5.680** (10) 0.808 0.215 (10) 0.581 0.153 IX 30 10 314.4* (10) 18.684* 5.931** (10) 0.674 0.215 (10) 0.487 0.153 XI 30/0 10 333.8* (10) 16.206*A 4.849** (10) 0.780 0.232 (10) 0.639A 0.191*A wt. = weight; ( ) = number in parenthesis is the number of organs weighed. Underlined values were interpreted to be test-substance related effects, as compared to control values. * = statistically significant (Dunnett/Tamhane-Dunnett parametric pairwise test), compared to control value. ** = statistically significant (Dunn's non-parametric pairwise test), compared to control value. A = statistically significant (Dunn's non-parametric pairwise test) change in Group XI value compared to Group IX value. 1. Final Body Weight Mean final body weights were decreased 11%, 26%, and 21% in the 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value. All decreases were statistically significant. Mean final body weights in the 0.3 and 1 mg/kg dose groups were similar to the control values. - 30- 3/ p. 31 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 There was a small, statistically insignificant increase in the mean final body weight o f the 30/0 mg/kg dose group, as compared to the 30 mg/kg dose group. This increase suggests partial recovery from the test substance-related final body weight decrease in the 6 recovery days following the injection o f sheep red blood cells. 2. Liver Mean absolute liver weights were increased 9%, 31%, 63%, 42%, and 23% in the 0.3, 1,10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value. Mean relative (% body weight) liver weights were similarly increased (10%, 35%, 82%, 91%, and 56%, respectively). All increases were statistically significant, except for those in the 0.3 mg/kg dose group and the mean relative liver weight in the 1 mg/kg dose group. The increased liver weights, at all dose levels, correlated with the microscopic finding of minimal to moderate hepatocellular hypertrophy. It also correlated with the gross observation of liver discoloration in a few rats at doses >10 mg/kg. 3. Other All other individual and mean organ weight differences were considered to be spurious or secondary to the decrease in final body weights. Mean relative brain weights (% body weight) were increased only at doses (>10 mg/kg) that produced significantly decreased body weights. Similarly, small, statistically insignificant, decreases in mean absolute, and increases in mean relative (% body weight), spleen and thymus weights were interpreted to be secondary to changes in final body weights. The lack o f any gross or microscopic effects in the brain, spleen, and thymus further suggests that these organ weight differences were a function o f body weight and not organ-specific effects. C. Gross Observations (Table 15, Appendix J) At the terminal sacrifice, test substance-relate gross observations were limited to discoloration of the liver in a few rats at doses >10 mg/kg. Text Table 2: Incidences of Test Substance-Related Gross Observations in Male Rats Group: I III V VII IX Dose (mg/kg): 0 0.3 1 10 30 Number o f Rats/Group: 10 10 10 10 10 Liver Discoloration 000 I 2 XI 30/0* 10 I Underlined values were interpreted to be test-substance related increases, as compared to control values. * Not dosed with test substance following immunology challenge. p. 32 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 The gross liver discoloration observed in the 4 rats given >10 mg/kg of the test compound was considered to be a result o f the microscopic finding o f hepatocellular hypertrophy. D. Microscopic Findings (Table 16, Appendix J) Microscopic examination of the liver demonstrated minimal to mild hepatocellular hypertrophy at 0.3 and 1 mg/kg and moderate hepatocellular hypertrophy at >10 mg/kg. Microscopic examination o f lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased hematopoiesis in the spleen o f high-dose recovery rats (30/0 mg/kg). The thymus, mesenteric lymph nodes and popliteal lymph nodes had no test substance-related effects. Text Table 3: Incidences of Test Substance-Related Microscopic Findings in Male Rats Group: Dose (mg/kg): Number of Rats/Group: I 0 10 Liver Hypertrophy, hepatocellular Necrosis, focal (10) 0 0 Spleen EMH, increased (10) 0 III 0.3 10 (10) 5 [1.0] 0 (10) 0 V 1 10 (10) 10 [1.7] 0 (10) 1 [1-0] VII 10 10 (10) 10 [3.0] I [1.0] (10) 0 IX 30 10 (10) 10 [3.0] 4 [1.0] (10) 0 XI 30/0* 10 (10) 10 [3.0] I [1.0] (10) 2[1.3] [ ] = Number in brackets is the average grade (grades 1 - 4) when lesion is present (i.e., sum of grades ^ # animals with lesion). Grading scale: 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. ( ) = number in parenthesis is the number of organs examined; EMH = Extramedullary hematopoiesis. Underlined values were interpreted to be test-substance related increases, as compared to control values. * Not dosed with test substance following immunology challenge. 1. Liver a. Hepatocellular hypertrophy Panlobular hepatocellular hypertrophy was observed in all but 5 of the rats given the test substance and the incidence and severity were dose related. Hypertrophy was present in 0/10, 5/10, 10/10, 10/10, 10/10, and 10/10 rats given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively. The hypertrophy was graded as minimal in the 5 affected rats given 0.3 mg/kg, minimal in 3/10 and mild in 7/10 rats given 1 mg/kg, and moderate in all rats given >10 mg/kg. The hepatocellular hypertrophy was characterized by an increase in the size o f all hepatocytes due to an increase in cytoplasmic volume. The cytoplasm had a uniformly eosinophilic granular appearance consistent with peroxisome proliferation. 33- 32- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Hepatocellular hypertrophy correlated with increased mean liver weight parameters at all doses. Although the 30/0 mg/kg group still had moderate hypertrophy (grade 3 o f 4), the decrease in mean liver weights, relative to the 30 mg/kg group suggests that there was some hepatocellular shrinkage and/or loss that was microscopically unapparent. b. Focal necrosis Focal necrosis was also observed in several rats given >10 mg/kg o f the test substance. The incidence was mildly dose related. Focal necrosis was present in 0/10, 0/10, 0/10, 1/10, 4/10, and 1/10 rats given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively. All were graded minimal. A decrease in the incidence was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group. Focal necrosis was characterized by the focal or multifocal coagulative necrosis o f a cluster of hepatocytes. The distribution was usually subcapsular and the pattern was non-zonal. Focal coagulative necrosis o f hepatocytes clusters is a common secondary effect o f hepatocellular hypertrophy and is most likely the result o f secondary focal ischemia. 2. Spleen a. Increased extramedullary hematopoiesis An increase in the incidence o f splenic extramedullary hematopoiesis (EMH) was considered test substance related only in high-dose rats allowed a recovery period (30/0 mg/kg). Minimal to mild increased EMH was observed in 0/10, 0/10, 1/10, 0/10, 0/10, and 7/10 rats given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. The increased splenic EMH in the high-dose recovery rats was erythrocytic and correlated with the hematology findings, which included decreased red cell mass parameters and increased circulating reticulocytes (see Clinical Pathology). 3. Other All other microscopic observations in this study were consistent with normal background lesions in rats o f this age and strain. E. Anatomic Pathology Conclusions There were no test substance-related deaths. All 60 rats on study survived until the scheduled sacrifice on test day 29. Following 28-days o f daily gavage administration o f the test substance, there was a test substance-related decrease in final body weights and increase in liver weights. Mean final body weights were decreased at dose levels >10 mg/kg o f the test substance. Mean liver weight parameters were increased at dose levels >0.3 mg/kg. At the terminal sacrifice, test substance-related gross observations were limited to discoloration o f the liver in a few rats at doses >10 mg/kg. - 33- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Microscopic examination of the liver demonstrated minimal to mild hepatocellular hypertrophy at 0.3 and 1 mg/kg and moderate hepatocellular hypertrophy at >10 mg/kg. Microscopic examination o f lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased hematopoiesis in the spleen of rats dosed with 30/0 mg/kg. The thymus, mesenteric lymph nodes and popliteal lymph nodes had no test substance-related effects. Total Cell Counts A. Spleen Cell Number (Table 17, Appendix K) No significant changes in total spleen cell number compared to control rats were noted in any animal treated with any dose o f APFO. A 10% increase was observed at 10 mg/kg and a 16% decrease was observed at 30 mg/kg, but neither value was statistically different than vehicle control. B. Thymus Cell Number (Table 17, Appendix K) No significant changes in total thymocyte number compared to control rats were noted in any animals treated with any dose o f APFO. For rats in the 30/0 mg/kg group, an increase in thymocyte number was observed, which was statistically greater than rats who continued to receive 30 mg/kg APFO, but not greater when compared to vehicle control. CONCLUSIONS Under the conditions o f this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male rats was less than 0.3 mg/kg, whereas the NOAEL for immunotoxicity was 30 mg/kg. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, the protocol, amendments (if any), and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Laboratory-specific raw data such as personnel files, instrument, equipment, refrigerator and/or freezer raw data will be retained at the facility where the work was done. -34- p. 35 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats________________________________________________ DuPont-18317 REFERENCES 1. Vanden Heuvel, J., Kuslikis, B., and Van Rafelghem, M. (1991). Tissue distribution, metabolism, and elimination of perfluorooctanoic acid in male and female rats. Biochem. Toxicol. 6, 83-92. 2. Taves, D., Guy, W., and Brey, W. (1976). Organic fluorocarbons in human plasma: prevalence and characterization. Biochemistry Involving Carbon-Fluorine Bonds (R. Filler, Ed.), pp. 117-134. American Chemical Society, Washington, D.C. 3. Hansen, K.J., Clemen, L.A., Ellefson, M.E., and Johnson, H.O. (2001). Compound-specific, quantitative characterization o f organic fluorochemicals in biological matrices. Environ. Sci. Technol. 35, 766-770. 4. Olsen, G.W., Burris, J.M., Burlew, M.M., and Mandel, J.H. (2000). Plasma cholecystokinin and hepatic enzymes, cholesterol and lipoproteins in ammonium perfluorooctanoate production workers. Drug Chem. Toxicol. 23, 603 620. 5. Yang, Q., Abedi-Valugerdi, M., Xie, Y., Zhao, X., Moller, G., Nelson, B.D., and DePierre, J.W. (2002). Potent suppression o f the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferators, perfluorooctanoic acid. International Immunopharmacology, 2 (2002), 389-397. 6. DuPont Haskell Laboratory (2005). APFO (Linear/Branched), APFO (Linear), and APFO (Branched): 14-Day Oral Gavage Study in Male Rats and Mice. Unpublished report, DuPont-14162. 7. Temple, L., Kawabata, T.T., Munson, A.E., and White, K.L., Jr. Comparison o f ELISA and plaque-forming cell assays for measuring the humoral immune response to SRBC in rats and mice treated with benzo(a)pyrene or cyclophosphamide. Fundam. Appl. Toxicol. 1993:21, 412-419. 8. Levene, H. (1960). Robust test for equality of variances. Contributions to Probability and Statistics (J. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alto. 9. Shapiro, S.S. and Wilk, M.B. (1965). An analysis o f variance test for normality (complete samples). Biometrika 52, 591-611. 10. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6thedition, pp 246-248 and 349-352. The Iowa State University Press, Iowa. 11. Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482-491. 12. Dunnett, C.W. (1980). Pairwise multiple comparisons in the unequal variance case. J. Amer. Statist. Assoc. 75, 796-800. -- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 13. Tamhane, A.C. (1979). A comparison o f procedures for multiple comparison o f means with unequal variances. J. Amer. Statist. Assoc. 74, 471-480. 14. Kruskal, W.H. and Wallis, W.A. (1952). Use o f ranks in one-criterion analysis o f variance. J. Amer. Statist. Assoc. 47, 583-621. 15. Dunn, O.J. (1964). Multiple contrasts using rank sums. Technometrics 6, 241-252. -36 57 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 TABLES -37- J57 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats TABLES EXPLANATORY NOTES ABBREVIATIONS: Summary of Hematology Values RBC - red blood cell count HGB - hemoglobin HCT - hematocrit MCV - mean corpuscular (cell) volume MCH - mean corpuscular (cell) hemoglobin MCHC - mean corpuscular (cell) hemoglobin concentration RDW - red cell distribution width ARET - absolute reticulocyte count PLT - platelet count WBC - white blood cell count ANEU - absolute neutrophil (all forms) ALYM - absolute lymphocyte AMON - absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophil ALUC - absolute large unstained cell DuPont-18317 Summary of Clinical Chemistry Values CHOL - cholesterol TRIG - triglycerides TP - total protein ALB - albumin GLOB - globulin HDL - high-density lipoprotein cholesterol NHDL - non-high-density lipoprotein cholesterol SCORT - serum corticosterone NOTES: Summary of Hematology Values Summary of Clinical Chemistry Values Groups with identical values may vary in statistical significance, because tabulated statistics are rounded to fewer decimal places than the values used for statistical determination. - 38 3? Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 TABLES EXPLANATORY NOTES (Continued) NOTES: (Continued) Summary of Total Cell Counts Organ Weight as Percent o f Body Weight Organ Weight (g) Final Body Weight (g) Total Number o f Organ Cells (x 108) Organ Weight (g) Half Organ Weight (g) Organ Cell Suspension Volume (mL) Number of Cells in Half Organ (x 106 cells/mL) - 100 -39- 4 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 40 DuPont-18317 Table 1 Recovery of APFO Added to Dosing Vehicle Sample Type R e c o v e r y (A) R e c o v e r y (B) APFO (mg/mL) Nominal Measured 0.0302 0.0327 0.0300 0.0305 M ean Percent Nominal 108.3 101.7 105.0 5 , C.V. 5% R e c o v e r y (A) R e c o v e r y (B) 0.104 0.100 0.114 0.104 M ean 109.6 104.0 106.8 4, C. V. 4% R e c o v e r y (A) R e c o v e r y '8 ' 1.00 1.00 1.02 1.05 M ean 102.0 105.0 103.5+2, C. V. 2% R e c o v e r y (A) R e c o v e r y (B) 3.00 3.00 3.05 3.21 M ean 101.7 107.0 104.4 4 , C. V. 4% (A> Processed with dosing samples submitted October 17, 2005 for concentration verification, uniformity of mixing, and 5-hour room temperature stability analyses. (B> Processed with dosing samples submitted November 15, 2005 for concentration verification and uniformity of mixing analyses. -40- f/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 41 DuPont-18317 Table 2 Concentration Verification, Uniformity o f Mixing, and 5-Hour Room Temperature Stability of APFO in Dosing Solutions Sample Date ____________ APFO (mg/mL)____________ Percent Sample Type(A)_________Nominal___________ Measured___________Nominal 15-November-2005 Concentration Verification Control 0 ND(B) -- #1 #2 #1 #2 #1 #1(C) #2(C) #1 #2 iability(D) 0.03 0.03 Mean: 0.1 0.1 Mean: 1 1 1 Mean: 3 3 Mean: 0.0278 0.0277 0.0278 0.0001 C. V. 0.3% 0.0966 0.0979 0.0973 0.0009 C.V. 0.9% 0.979 1.04 1.03 1.02 0.03 C. V. 3% 3.16 3.06 3.11 0.07 C. V. 2%> 0.03 0.0289 92.7 92.3 (92.7) 96.6 97.9 (97.3) 97.9 104.0 103.0 (102.0) 105.3 102.0 (103.7) 96.3 0.1 0.0990 99.0 1 0.969 96.9 3 3.06 102.0 (A) Duplicate analyses per level performed for concentration verification. Mean, S.D. and C.V. calculated to verify uniformity of mixing. (B) Denotes not detected. (C) Duplicate analyses from the re-diluted sample. (D) Samples held at room temperature for 5 hours. -41 - 4 -J L p. 42 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats________________________________________________ DuPont-18317 Table 3 Concentration Verification and Uniformity of Mixing of APFO in Dosing Solutions Sample Type(A) Sample Date Concentration Verification 11-October-2005 Control APFO (mg/mL) Nominal Measured 0 ND(B) Percent Nominal -- #1 0.03 0.0276 92.0 #2 0.03 0.0272 90.7 Mean: 0.0274 0.0003 (91.3) C.V. 1% #1 0.1 0.0954 95.4 #2 0.1 0.0986 98.6 Mean: 0.0970 0.002 (97.0) C. V. 2% #1 1 1.02 102.0 #2 1 1.01 101.0 Mean: 1.02 0.008 (102.0) C.V. 0.7% #1 3 3.21 107.0 #2 3 3.23 107.7 Mean: 3.22 0.01 (107.3) C. V. 0.4% (A) Duplicate analyses per level performed for concentration verification. Mean, S.D. and C.V. calculated to verify uniformity of mixing. (B) Denotes not detected. 5- 42- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 DAYS ON TEST 0 7 14 21 28 Group I 0 mg/kg 269.1 11.6( 10) 329.8 15.8(10) 378.8 20.1( 10) 424.0 24.4(10) 453.4 26.5(10) Table 4 Mean Body Weights of Male Rats Group III 0.3 mg/kg MEAN BODY WEIGHTS (g) Group V Group VII 1 mg/kg 10 mg/kg Group IX 30 mg/kg 270.3 11.3(10) 328.5 15.2(10) 375.0 20.5(10) 419.7 24.4(10) 446.6 30.7(10) 270.6 13.7(10) 328.2 18.4(10) 373.7 25.5(10) 412.6 34.5(10) 437.2 38.7(10) 270.5 14.1(10) 314.7 17.9(10) 358.0 21.2( 10) 387.5 30.1(10) 407.5* 34.8(10) 271.3 8.9(10) 278.0@ 37.4(10) 3 10.7@ 28.3(10) 322.0* 38.0(10) 339.6* 36.1(10) Group XI 30/0 mg/kg (Recovery) 268.4 8.7(10) 275.0@ 47.0(10) 298.5@ 49.9(10) 322.2* 45.4(10) 359.8* 39.8(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p < 0.05 by Dunn's test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; no significant differences between IX and XI were detected. p. 43 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 DAYS ON TEST 0-7 7-14 14-21 21-28 OVERALL 0-28 Group I 0 mg/kg 60.7 7.8(10) 49.0 8.5(10) 45.3 8.6( 10) 29.3 3.9(10) 184.3 21.2( 10) Table 5 Mean Body Weight Gains of Male Rats Group III 0.3 mg/kg MEAN BODY WEIGHT GAINS (g) Group V Group VII 1 mg/kg 10 mg/kg Group IX 30 mg/kg 58.2 9.4(10) 46.5 7.2(10) 44.7 6.6( 10) 26.9 7.4(10) 57.6 8.7(10) 45.5 7.5(10) 38.9 10.7(10) 24.7 9.5(10) 44.2 10.2( 10) 43.3 10.0( 10) 29.5* 9.5(10) 20.0 7.7(10) 6.7@ 35.4(10) 32.7 19.5(10) 11.3* 13.6(10) 17.6@ 8.8( 10) 176.3 25.7(10) 166.6 28.6(10) 137.1* 30.9(10) 68.3* 34.3(10) Group XI 30/0 mg/kg (Recovery) 6 .6@ 45.0(10) 23.5* 16.9(10) 23.7*t 11.9(10) 37.5t 19.0(10) 91.4* 38.3(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p < 0.05 by Dunn's test, t Statistically significant difference from Group IX at p < 0.05 by Dunn's test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; significant differences between IX and XI were detected. p. 44 - 44- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 DAYS ON TEST 7 14 21 28 OVERALL 0-28 Group I 0 mg/kg 28.8 1.8( 10) 29.2 2.3(10) 30.0 2.1( 10) 30.2 1.7(10) 29.5 1.8( 10) Table 6 Mean Daily Food Consumption by Male Rats MEAN DAILY FOOD CONSUMED PER ANIMAL (e) Group III Group V Group VII Group IX 0.3 mg/kg 1 mg/kg 10 mg/kg 30 mg/kg 28.0 2.1( 10) 28.7 2.6( 10) 29.2 2.6( 10) 29.7 2.7(10) 28.9 2.4(10) 28.5 2.2( 10) 28.7 2.7(10) 28.5 3.0(10) 28.5 2.7(10) 28.6 2.5(10) 26.4 1.6( 10) 29.1 2.0( 10) 29.2 2.7(10) 29.1 2.4(10) 28.4 2.0( 10) 2 0 . 1@ 6.7(10) 27.9 3.6(10) 24.0* 5.2(10) 26.5* 2.0( 10) 24.6* 3.2(10) Group XI 30/0 mg/kg (Recovery) 2 0 .9@ 6.6( 10) 25.3* 4.8(10) 25.5* 2.7(10) 27.0* 2.7(10) 24.7* 2.9(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p < 0.05 by Dunn's test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; no significant differences between IX and XI were detected. p. 45 - 45- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 DAYS ON TEST 0-7 14 21 28 OVERALL 0-28 Group I 0 mg/kg Table 7 Mean Daily Food Efficiency o f Male Rats MEAN DAILY FOOD EFFICIENCY (g weight gain/g food consumed) Group III Group V Group VII Group IX 0.3 mg/kg 1 mg/kg 10 mg/kg 30 mg/kg 0.301 0.029(10) 0.240 0.033(10) 0.215 0.033(10) 0.139 0.018(10) 0.222 0.018(10) 0.296 0.035(10) 0.231 0.024(10) 0.218 0.022( 10) 0.128 0.027(10) 0.217 0.019(10) 0.287 0.029(10) 0.226 0.025(10) 0.192 0.037(10) 0.122 0.038(10) 0.207 0.019(10) 0.238 0.047(10) 0.211 0.039(10) 0.143* 0.038(10) 0.096@ 0.032(10) 0.171* 0.029(10) -0.070@ 0.401(10) 0.164 0.096(10) 0.054* 0.080(10) 0.096@ 0.049(10) 0.096* 0.042(10) Group XI 30/0 mg/kg (Recovery) -0.209@ 1.003(10) 0.127* 0.081(10) 0.135* 0.078(10) 0.193t 0.076(10) 0.129*f 0.044(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p < 0.05 by Dunn's test, t Statistically significant difference from Group IX at p < 0.05 by Dunn's test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; significant differences between IX and XI were detected. p. 46 - 46- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats ANIMAL COUNT: Wet Fur Feces Absent Decreased Feces Not Eating Stain Fur/Skin Table 8 Summary of Daily Animal Health Observations in Male Rats Group 1 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 10 10 10 10 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Group IX 30 mg/kg 10 0 (0%) 1 ( 10%) 2 (20%) 3 (30%) 0 (0%) Data arranged as: number of animals (percent of group) for which an observation was recorded DuPont-18317 Group IX 30/0 mg/kg (Recovery) 10 1 ( 10%) 1 ( 10%) 1 ( 10%) 2 (20%) 1 ( 10%) p. 47 -47 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats ANIMAL COUNT: Wet Fur Carriage, High Feces Absent Hair Loss Lethargic Not Eating Stain Fur/Skin Table 9 Summary of Detailed Clinical Observations in Male Rats Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 10 10 10 10 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 ( 10%) 0 (0%) 1 ( 10%) 1 ( 10%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Data arranged as: number of animals (percent of group) for which an observation was recorded DuPont-18317 Group IX 30 mg/kg 10 0 (0%) 0 (0%) 0 (0%) 3 (30%) 0 (0%) 0 (0%) 0 (0%) Group IX 30/0 mg/kg (Recovery) 10 1 ( 10%) 1 ( 10%) 1 ( 10%) 0 (0%) 1 ( 10%) 1 ( 10%) 1 ( 10%) p. 48 -48 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats RBC (x l0 6/pL) DAY 29 HGB (g/dL) DAY 29 HCT (%) DAY 29 MCV (fL) DAY 29 MCH (pg) DAY 29 MCHC (g/dL) DAY 29 Group I 0 mg/kg 7.66 0.20(9) 14.9 0.3(9) 46.2 1.0(9) 60.3 2.0(9) 19.5 0.5(9) 32.3 0.5(9) Table 10 Summary of Hematology Values for Male Rats Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 7.61 0.33(10) 14.7 0.5(10) 45.4 1.5(10) 59.7 2 .0( 10) 19.3 0.6( 10) 32.4 0.3(10) 7.60 0.32(10) 14.7 0.6( 10) 45.6 1.8( 10) 60.1 2 .2( 10) 19.4 0.7(10) 32.2 0.4(10) 7.28 0.50(10) 13.5@ 0.7(10) 42.6* 2.3(10) 58.5 2.3(10) 18.6* 0.8( 10) 31.8 0.5(10) Group IX 30 mg/kg 7.47 0.60(10) 13.6@ 0.7(10) 42.7* 2 .0( 10) 57.3* 2.4(10) 18.3* 0.7(10) 31.9 0.5(10) DuPont-18317 Group XI 30/0 mg/kg (Recovery) 6.75*# 0.34(10) 12.8@# 0.3(10) 40.2*# 1.2( 10) 59.6 2.8( 10) 19.0 0.9(10) 31.8 0.6( 10) p. 49 - 49- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Table 10 Summary of Hematology Values for Male Rats (Continued) Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg Group IX 30 mg/kg RDW (%) DAY 29 ARET (x l0 3/|iL) DAY 29 PLT (xlOVpL) DAY 29 WBC(xlOVpL) DAY 29 ANEU (x l0 3/pL) DAY 29 ALYM (x l0 3/|iL) DAY 29 11.5 0.3(9) 187.3 16.5(9) 1090 125(6) 12.49 3.48(9) 1.46 0.58(9) 10.40 2.80(9) 11.4 0.5(10) 170.5 19.7(10) 1058 76(10) 11.41 2.83(10) 1.38 0.49(10) 9.56 2.54(10) 11.7 0.3(10) 177.5 24.1(10) 1094 111(6) 13.28 2.83(10) 1.55 0.71(10) 11.19 2.47(10) 12.8@ 0.7(10) 204.5 46.2(10) 1044 344(7) 16.26 2.69(10) 1.66 0.53(10) 13.87* 2.41(10) 13.2@ 0.8( 10) 208.9 40.8(10) 1196 174(7) 17.07* 2.93(10) 1.79 0.59(10) 14.53* 2.67(10) DuPont-18317 Group XI 30/0 mg/kg (Recovery) 14.2@ 2.1( 10) 369.8@# 88.6( 10) 1207 162(8) 13.91 4.42(10) 1.46 0.43(10) 11.81 4.07(10) p. 50 - 50- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 AMON (xlOVpL) DAY 29 AEOS (xlOVpL) DAY 29 ABAS (xlOVpL) DAY 29 ALUC (xlOVpL) DAY 29 Table 10 Summary of Hematology Values for Male Rats (Continued) Group I Group III Group V Group VII Group IX Group XI Omg/kg 0.3 mg/kg 1 mg/kg 10mg/kg 30 mg/kg 30/0 mg/kg __________________________________________________ _____________________________ (Recovery) 0.25 0.09(9) 0.17 0.11(9) 0.05 0.02(9) 0.15 0.08(9) 0.22 0.09(10) 0.08 0.04(10) 0.05 0.03(10) 0.11 0.03(10) 0.26 0.12( 10) 0.09 0.04(10) 0.07 0.04(10) 0.13 0.04(10) 0.33 0.17(10) 0.13 0.07(10) 0.07 0.04(10) 0.20 0.11( 10) 0.35 0.15(10) 0.11 0.07(10) 0.07 0.04(10) 0.22 0.12( 10) 0.29 0. 11( 10) 0.10 0.06(10) 0.06 0.05(10) 0.19 0.17(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p < 0.05 by Dunn's test. # Statistically significant difference from Group IX at p < 0.05 by t-test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; significant differences between IX and XI were detected. 'Z 'S p. 51 -51 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Group I 0 mg/kg Table 11 Summary of Clinical Chemistry Values for Male Rats Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg Group IX 30 mg/kg CHOL (mg/dL) DAY 29 TRIG (mg/dL) DAY 29 TP (g/dL) DAY 29 ALB (g/dL) DAY 29 GLOB (g/dL) DAY 29 HDL (mg/dL) DAY 29 64 17(10) 68 19(10) 6.1 0.2( 10) 3.3 0.1( 10) 2.8 0.1( 10) 24 4(10) 41@ 10( 10) 47@ 17(10) 6.1 0.2( 10) 3.4 0.1( 10) 2.8 0.2( 10) 18* 3(10) 44@ 8( 10) 51 20( 10) 6.2 0.3(10) 3.5 0.2( 10) 2.7 0.2( 10) 19* 3(10) 52 10( 10) 46@ 15(10) 6.1 0.4(10) 3.7@ 0.2( 10) 2.5* 0.2( 10) 18* 3(10) 54 9(10) 45@ 11( 10) 6.2 0.3(10) 3.8@ 0.1( 10) 2.5* 0.3(10) 19 4(10) DuPont-18317 Group XI 30/0 mg/kg (Recovery) 73t 23(10) 47@ 16(10) 6.5 0.5(10) 3.7@ 0.3(10) 2.7# 0.2( 10) 25# 5(10) p. 52 - 52- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Smdy in Male Rats DuPont-18317 NHDL (mg/dL) DAY 29 SCORT (ng/mL) DAY 29 Table 11 Summary o f Clinical Chemistry Values for Male Rats (Continued) Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg Group IX 30 mg/kg 40 14(10) 137 95(10) 23@ 8( 10) 167 73(10) 25@ 6( 10) 147 69(10) 34 7(10) 185 91(10) 35 5(10) 268 217(10) Group XI 30/0 mg/kg (Recovery) 47f 18(10) 131 90(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p < 0.05 by Dunn's test. t Statistically significant difference from Group IX at p < 0.05 by Dunn's test. # Statistically significant difference from Group IX at p < 0.05 by t-test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; significant differences between IX and XI were detected. p. 53 -53 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 LOG2a Table 12 Summary of Primary Humoral Immune Response to SRBC for Male Rats Dosed with APFO Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg Group IX 30 mg/kg 10.060 1.503(10) 10.368 0.466(10) 9.858 1.503(10) 9.917 1.928(10) 9.906 1.142(10) Group XI 30/0 mg/kg (Recovery) 9.519 1.198(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) a Mean log2 of the serum IgM titer data. There were no statistically significant differences from control at p < 0.05. v j > Table 13 Summary of Primary Humoral Immune Response to SRBC for Male Rats Dosed With Positive Control Saline3 Cyclophosphamide Cyclophosphamide 20 mg/kga 20 mg/kgb log2 9.456 1.147(10) 4.098 0.978(10) 4.241 0.872(2) Data arranged as: Mean Standard deviation (Number of values included in calculation) a Mean log2 of the SRBC-specific serum IgM titer data for individual samples, b Log2 of the SRBC-specific serum IgM titer data for pooled samples. p. 54 - 54- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 14 Mean Final Body and Organ Weights from Male Rats Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg MEAN FINAL BODY AND ABSOLUTE ORGAN WEIGHTS (grams) LIVER 13.179 1.397(10) 14.379 1.604(10) 17.227* 2.860(10) SPLEEN 0.844 0.167(10) 0.872 0.209(10) 0.835 0.144(10) THYMUS 0.568 0.126(10) 0.604 0.123(10) 0.559 0. 121( 10) BRAIN 2.012 0.088(10) 2.111 0.117(10) 2.086 0.087(10) FINAL BODY WEIGHT (grams) 423.1 26.0(10) 419.7 25.0(10) 410.0 35.2(10) 21.469* 2.864(10) 0.808 0.126(10) 0.581 0.134(10) 1.999 0.123(10) 377.0* 32.8(10) Group IX 30 mg/kg 18.684* 2.866( 10) 0.674 0.085(10) 0.487 0.171(10) 1.992 0.108(10) 314.4* 35.1(10) Group XI 30/0 mg/kg (Recovery) 16.206*f 2.170(10) 0.780 0.182(10) 0.6391 0. 110( 10) 1.913 0.129(10) 333.8* 36.1(10) p. 55 - 55- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Table 14 Mean Final Body and Organ Weights from Male Rats (Continued) Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg Group IX 30 mg/kg MEAN RELATIVE ORGAN WEIGHTS (% of body weight) LIVER/ FINAL BODY * 100 3.113 0.229(10) 3.419 0.250(10) 4.194 0.524(10) SPLEEN/ FINAL BODY * 100 0.199 0.033(10) 0.208 0.047(10) 0.203 0.021( 10) THYMUS/ FINAL BODY * 100 0.133 0.024(10) 0.144 0.028(10) 0.136 0.024(10) BRAIN/ FINAL BODY * 100 0.477 0.028(10) 0.504 0.030(10) 0.511 0.038(10) 5.680@ 0.385(10) 0.215 0.030(10) 0.153 0.029(10) 0.533* 0.044(10) 5.931@ 0.503(10) 0.215 0.020( 10) 0.153 0.046(10) 0.639* 0.059(10) DuPont-18317 Group XI 30/0 mg/kg (Recovery) 4.849@ 0.345(10) 0.232 0.039(10) 0.191 *f 0.019(10) 0.577*1 0.052(10) p. 56 - 56- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 14 Mean Final Body and Organ Weights from Male Rats (Continued) Group I Group III Group V Group VII Group IX Group XI 0 mg/kg 0.3 mg/kg 1 mg/kg 10mg/kg 30 mg/kg 30/0 mg/kg ____________________________________________________________________________ (Recovery) MEAN RELATIVE ORGAN WEIGHTS (% of brain weight) LIVER/ BRAIN * 100 654.981 61.796(10) 681.399 71.841(10) 825.207* 128.554(10) 1073.082* 119.548(10) 937.602* 129.373(10) 846.704* 95.980(10) SPLEEN/ BRAIN * 100 41.814 7.212(10) 41.120 8.849(10) 39.940 6.035(10) 40.554 6.671(10) 33.805 3.600(10) 40.732t 8.641(10) THYMUS/ BRAIN * 100 28.191 5.790(10) 28.563 5.440(10) 26.750 5.543(10) 29.072 6.473(10) 24.424 8.255(10) 33.3191 4.511(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant differencefrom control at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant differencefrom control at p < 0.05 by Dunn's test, t Statistically significant differencefrom Group IX at p < 0.05 by Dunn's test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; significant differences between IX and XI were detected. p. 57 - 57- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 15 Incidence of Gross Observations in Male Rats LESION INCIDENCE (Numeric) LESIONS LIVER NO ABNORMALITY LARGE DISCOLORATION DETECTED SPLEEN NO ABNORMALITY DETECTED THYMUS NO ABNORMALITY DETECTED POPLITEAL LYMPH NODE NO ABNORMALITY DETECTED MESENTERIC LYMPH NODE NO ABNORMALITY DETECTED BRAIN NO ABNORMALITY DETECTED TREATMENT 10 1 0.3 1 1 1 10 1 30 1 30/0 ! 1 mg/kg 1 mg/kg| mg/kg| mg/kg| mg/kg| mg/kg | 1I 1 III 1 V 1 VII 1 IX ! XI 1 1 1 1 1 1 1R e c o v e r y ] 11!11! ! (10) 1 G O ) 1 (10) 1 G O ) 1 G O ) ! (10) ! 10 1 10 1 10 1 9 ! 8 1 9 1 ! ! 1 i 11 1 ! ! 1 11 21 1 11i 1 GO) ! GO) ! GO) 1 GO) i GO) (10) 1 10 1 10 ! 10 1 10 1 10 1 10 11111! 1 G O ) 1 G O ) 1 (10) 1 (10) 1 G O ) (10) 1 10 1 10 1 10 1 10 1 10 1 10 11111 1 G O ) 1 (10) 1 G O ) 1 (10) 1 G O ) (10) ! 10 1 10 1 10 1 10 1 10 1 10 111i1 1 (10) 1 (10) 1 (10) i (10) 1 G O ) (10) ! 10 ! 10 1 10 1 10 1 10 1 10 1I111 ! G O ) ! (10) 1 (10) 1 (10) 1 G O ) (10) 1 10 ! 10 ! 10 1 10 1 10 10 1!!111 1 1 ! i ! 1 1 1 1 1 ! 1 1 1 1 1 I 1 1 Figures in p a re nt he se s are the nu m b e r of an im al s g r o s s l y e x a m i n e d for this tissue The absence of a number indicates the finding specified was not identified p. 58 - 58- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 15 Incidence of Gross Observations in Male Rats (Continued) LESIONS LESION INCIDENCE (Numeric) 1 TREATMENT 1 per day 1 1 10 | 0.3 | 1 ! 10 | 30 | 30/0 | 1 mg/kg| mg/kg| mg/kg| mg/kg| mg/kg| mg/kg | 1I 1 III 1 V I V I I | IX | XI | 11 1 1 1R e c o v e r y | ! FEMUR/KNEE JOINT I NO ABNORMALITY DETECTED STERNUM NO ABNORMALITY DETECTED 1 1 GO) 1 10 I 1 GO) 1 10 1 1 ! (10) | 10 I | GO) | 10 1 1 | (10) | 10 1 | (10) 1 10 1 1 | (10) | 10 i | (10) | 10 1 1 1 GO) | 10 ! | (10) | 10 1 1 | | 1 ! | 1 GO) 10 GO) 10 Figures in parentheses are the number of animals grossly examined for this tissue The absence of a number indicates the finding specified was not identified | | ! | p. 59 - 59- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 16 Incidences and Lesion Grades of Microscopie Observations in Male Rats LESION INCIDENCE (NUMERIC) LESIONS I TREATMENT I per day I | 0 I 0.3 I 1 I 10 ! 30 I 30/0 I mg/kg! mg/kgl mg/kgl mg/kgi mg/kgl mg/kg II I III I V ! VII I IX ! XI jR e c o v e r y LIVER NO ABNORMALITY DETECTED NECROSIS, FOCAL, minimal Total observations per lesion MINERALIZATION, BILE DUCT, minimal moderate Total observations per lesion INFLAMMATION, SUBACUTE/CHRONIC. minimal mild Total observations per lesion HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, minimal mild moderate Total observations per lesion HYPERPLASIA, BILE DUCT, FOCAL, minimal Total observations per lesion 111 GO) I (10) I GO) 1 GO) GO) 1 GO) 1I 11 11 11 41 1 1 11 41 1 1 I1 I1 11 ! 1 1 11 ! 11 11 1 1! 9I 9 91 91 91 8 11 11 11 2 9I 9 10 1 10 ! 10 I 10 11 5 3! ! ! 71 1 ! ! 10 1 10 1 10 5 10 1 10 ! 10 1 10 j1 11 1 1 1! ! 1! 1 Figures in parentheses are the number of animals m i cr os co pi ca ll y examined for this tissue The absence of a number indicates the lesion specified was not identified 1 t1 (1 11 1 1 p. 60 - 60- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 16 Incidences and Lesion Grades of Microscopic Observations in Male Rats (Continued) LESIONS TREATMENT per day LESION INCIDENCE (NUMERIC) 0 mg/kg I 10 mg/kg VII 30 I 30/0 mg/kg| mg/kg IX XI Recovery LIVER HEMATOPOIESIS, EXTRAMEDULLARY, minimal Total observations per lesion FIBROSIS, FOCAL, minimal Total observations per lesion 5 FATTY CHANGE, MEDIAN CLEFT, minimal Total observations per lesion SPLEEN NO ABNORMALITY DETECTED HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal mild Total observations per lesion THYMUS NO ABNORMALITY DETECTED GO) (10) ( 10) ( 10) ( 10) GO) 11 ! 11 11 11 11 11 11 11 21 21 1 1 1 1 1 G O ) 1 G O ) 1 G O ) 1 (9) 1 G O ) 1 G O 10 1 10 1 9 1 9 1 10 1 3 1 1 11 1 11 ! 1 1 11 1 5 2 7 GO) ! 1 I GO) 1 G O 10 1 1 1 10 ! 10 Figures in parentheses are the number of animals microscopically examined for this tissue The absence of a number indicates the lesion specified was not identified p. 61 -61 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Table 16 Incidences and Lesion Grades o f Microscopic Observations in Male Rats (Continued) LESIONS TREATMENT per day LESION INCIDENCE (NUMERIC) 0 10.3 mg/kg| mg/kg 1 I III ! 1 I 10 mg/kgI mg/kg V I VII 30 mg/kg IX 30/0 mg/kg XI Recovery POPLITEAL LYMPH NODE NO ABNORMALITY DETECTED NOT PRESENT IN TISSUE SECTION. MESENTERIC LYMPH NODE NO ABNORMALITY DETECTED DEPLETION/ATROPHY, LYMPHOID. minimal Total observations per lesion BONE MARROW NO ABNORMALITY DETECTED FIBROSIS, FOCAL, minimal Total observations per lesion BRAIN NO ABNORMALITY DETECTED PIGMENT, FOCAL, minimal Total observations per lesion GO) 2 8 I (10) I 10 I I I GO) 9 1 1 GO) 9 1 1 GO) ! 7I 3! (10) 8 2 G O ) GO) 9 10 1 1 GO) ! (10) 10 I 9 I !1 I1 I ! I I i GO) GO) 10 10 Figures in parentheses are the number of animals mi cr os co pi ca ll y examined for this tissue The absence of a number indicates the lesion specified was not identified p. 62 - 62 - SX X Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Table 16 Incidences and Lesion Grades o f Microscopic Observations in Male Rats (Continued) DuPont-18317 LESION INCIDENCE (NUMERIC) LESIONS 1 1 1 FEMUR/KNEE JOINT ! NO ABNORMALITY 1 1 STERNUM 1 NO ABNORMALITY 1 DETECTED DETECTED TREATMENT per day I0 10.3 | 1 10 30 | 30/0 | mg/kg| mg/kg| mg/kg| mg/kg| mg/kg| mg/kg 1 I 1 III I V 111 1 VII 1 1 IX 1 1 XI 1 IR e c o v e r y | 1 111 I 1 G O ) 1 1 1 1 G O ) 1 (1 0 ) | 1 10 1 1 1 1 10 1 10 | 1111!1 1 1 (1 0 ) ! 1 ! 1 G O ) 1 G O ) | 1 10 1 1 1 1 10 1 10 | 11!111 1 Figures m parentheses are the number of animals microscopically examined for this tissue The absence of a number indicates the lesion specified was not identified p. 63 63 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Final Body Weight (g) SPLEEN Absolute Weight (g) Weight Ratio (% Body Weight) Half Weight (g) Cell Suspension Volume (mL) Number of Cells in Half (x 106 cells/mL) Total Number of Cells (x 108) Group 1 0 mg/kg 423.12 26.05(10) 0.844 0.167(10) 0.1988 0.0330(10) 0.435 0.089(10) 6.9 0.5(10) 41.58 17.41(10) 5.65 2.60(10) Table 17 Summary of Total Cell Counts Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 419.74 24.95(10) 410.03 35.20(10) 376.95 32.76(10) 0.872 0.209(10) 0.2078 0.0469(10) 0.443 0.110(10) 7.1 0.3(10) 44.22 16.99(10) 6.23 2.65(10) 0.835 0.144(10) 0.2026 0.0210(10) 0.421 0.081(10) 7.4 0.9(10) 43.89 24.34(10) 6.45 3.29(10) 0.808 0.126(10) 0.2146 0.0296(10) 0.416 0.062(10) 7.1 0.5(10) 45.43 16.31(10) 6.24 2.10(10) Group IX 30 mg/kg 314.42 35.14(10) 0.674 0.085(10) 0.2147 0.0198(10) 0.348 0.038(10) 7.5 1.4(10) 34.32 17.07(10) 4.72 1.97(10) Group XI 30/0 mg/kg (Recovery) 333.79 36.15(10) 0.780 0.182(10) 0.2323 0.0390(10) 0.401 0.095(10) 6.8 0.3(10) 44.44 16.55(10) 5.79 2.05(10) p. 64 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 THYMUS Absolute Weight (g) Weight Ratio (% Body Weight) Half Weight (g) Cell Suspension Volume (mL) Number of Cells in Half (x 106 cells/mL) Total Number of Cells (x 108) Group I 0 mg/kg Table 17 Summary of Total Cell Counts (Continued) Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 0.568 0.126(10) 0.1335 0.0238(10) 0.284 0.069(10) 7.2 0.3(10) 85.03 23.22(10) 12.34 3.34(10) 0.604 0.123(10) 0.1439 0.0281(10) 0.292 0.063(10) 7.0 0.3(10) 83.16 36.67(10) 12.44 5.92(10) 0.559 0.121( 10) 0.1357 0.0238(10) 0.272 0.057(10) 7.2 0.4(10) 88.66 28.23(10) 13.18 4.49(10) 0.581 0.134(10) 0.1531 0.0290(10) 0.289 0.066(10) 7.2 0.4(10) 96.80 40.05(10) 14.03 5.81(10) Group IX 30 mg/kg Group XI 30/0 mg/kg (Recovery) 0.487 0.171(10) 0.1529 0.0463(10) 0.247 0.086(10) 7.2 0.3(10) 80.30 39.10(10) 11.67 6.26(10) 0.639 0.110( 10) 0.1909 0.0190(10) 0.325 0.055(10) 7.3 0.2( 10) 120.95 38.87(10) 17.471 6.40(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) t Statistically significant difference from Group IX at p < 0.05 by Dunn's test. NOTE: All data from groups III, V, VII, IX and XI were compared with the control (I) group data. In addition, data from group IX were compared with data from group XI; significant differences between IX and XI were detected. p. 65 -65- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 66 DuPont-18317 FIGURES Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Figure 1 Representative Analytical Calibration Curve DuPont-18317 Figure 1: Calibration curve showing linear fit (line) to replicate peak area ratio measurements (squares) for matrix matched calibration solutions of APFO diluted over a concentration range of 0.00505 to 0.0505 ppm. -67- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Figure 2 Representative LC/MS/MS Chromatograms p. 68 DuPont-18317 is approximately 4.5 minutes. -68- p. 69 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats________________________________________________ DuPont-18317 Figure 2 Representative LC/MS/MS Chromatograms (Continued) Figure 2c: Representative LC/MS/MS chromatogram of 0.0303 ppm APFO analytical standard (H22703376) diluted with NANOpure water after matrix correction. nominal concentration of 0.03 mg/mL. The measured concentration of the representative solution is 0.979 mg/mL. -69 70 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats___________ Figure 2 Representative LC/MS/MS Chromatograms (Continued) p. 70 DuPont-18317 ppm. The measured concentration of the representative recovery sample is 1.02 mg/mL. -70- 7/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Smdy in Male Rats Figure 3 Mean Body Weights of Male Rats DuPont-18317 0 mg/kg -Q- 0.3 mg/kg l i r 1 mg/kg 10 mg/kg 30 mg/kg 30/0 mg/kg (Recovery) p. 71 - 71 - p. 72 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats________________________________________________ DuPont-18317 APPENDICES - 72 73 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix A Certificate of Analysis 77- 7 3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 v a c i\ o c 0 iu i. Proven Restas. 3058 Research Drive S ta College, R M 6801 T :8 1 4 .2 7 2 .1 0 3 9 ygen.com CERTIFICATE OF ANALYSIS This Certificate of Analysis fulfills the requirement for characterization o f a test substance prior to a study subject to the GLP regulations. It documents the purity of the test substance. This work was conducted under TSCA Good Laboratory Practice Standards (40 CFR 792) and FIFRA Good Laboratory Practice Standards (40 CFR 160). Designation of the Certified Material: Compound: APFO (Linear) Haskell Number. H27308 Analytical Data: The Purity o f the Certified Material was Established by LC/MS/MS Purity: Last Date o f Analysis: Re-certification Date: 19.5% 07-November-2005 07-November-2006 Origin of Certified Material: E.I. du Pont de Nemours and Company Wilmington, DE 19898 USA Testing Faciiity/Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Prepared By: Study Director, Exygen Research Date DuPont-18418 Exygen Research Study P0001843 Page 1 of 1 -74- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 75 DuPont-18317 Appendix B Individual Body Weights Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats ABBREVIATIONS : INDIVIDUAL BODY WEIGHTS EXPLANATORY NOTES DuPont-18317 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Bod Body Weight g Day 0 Body Weight g Day 3 Body Weight g Day 6 Body Weight g Day 7 Body Weight g Day 8 Male, I 0 m g /kg 101 274 .9 102 258.4 103 276.7 104 280.8 105 248.1 106 270.4 107 267 .3 108 264.3 109 287.6 110 262.2 299.7 282 .9 299.4 301.2 262.3 287.8 282.8 289.0 304.9 281.0 336.0 323. 9 329.7 327.2 290.3 319.2 305 .9 319.8 342.0 313.0 343.3 333.2 339.5 340.5 295.5 325.8 320.2 324.2 351.9 323.7 351.4 342.2 344.2 342.7 301.2 333.3 325.2 331.6 358.0 328.6 Male, III 0.3 m g / kg 301 275.5 302 262.8 303 283.5 304 277 .4 305 24 7.4 306 272.3 307 272 .5 308 265.4 309 284 .9 310 261.5 300.8 288.5 304 .9 293.9 269.0 297.1 286.8 275.1 304.4 276.8 336. 1 321.6 338.8 321. 9 298.6 333.1 315.2 308.6 339.4 307.2 342.5 333.3 350.9 324.0 306.5 339.0 321.2 311.1 342.2 314.0 350.5 338.7 358.6 326.9 309.4 340.8 324 .1 317 .6 351.9 316.7 Male, V 1 mg/kg 501 266. 9 502 259.7 503 276.2 504 283.6 505 245.0 506 271.9 507 274 .5 508 271 .3 509 295.2 510 261.8 284.9 278.2 302.9 297.6 264.5 287.1 284 .0 2 92.0 313.7 277.2 320 .1 308.9 336.9 336.0 296.4 320.2 309.4 321 .6 353.3 305.1 331.2 317.5 346.3 346.2 300.5 326.5 314 .1 329.3 359.6 310.6 337.0 327.4 354.1 354.8 300.8 326.4 322.5 335 .9 370.1 317.3 - 77- DuPont-18317 y Weights Body Weight g Day 9 Body Weight g Day 10 Body Weight g Day 11 Body Weight g Day 12 361 .0 354 .6 354 -.4 353..8 311 .9 341 .5 331..7 338..2 372 .1 340,,3 372 .4 366.,8 364 .,3 357 ,9 316..7 348 .0 336.,0 341,,6 376.,7 346.,7 375 _7 374 .5 369..9 358 .,7 322 .2 348 .8 343 .0 351 .,2 381 .9 353 ,1 382 .1 382 .0 376. 8 371 .5 331 .1 362 .2 352 .2 352 ..9 388 .8 360. 1 361.0 353.3 371.9 340.3 323.1 356.7 336.0 328.0 366.2 329.2 364.7 360.7 373.1 339.9 328 .4 361 .6 340 .1 332.0 365.6 330 .6 374 .1 366.9 386.4 347.4 333.6 371.4 340.7 333 .9 373.2 336 .8 380.6 376.2 397.7 353.8 340.1 377.8 353.9 339.5 389.0 346.7 349 .5 332 .2 365.,1 367 .5 311 ,7 341 .7 32 7.,7 347 .4 379 ,8 327 ,.4 349 .4 341 .4 369,,1 372 .7 316..0 34 3 .6 332 .,7 352 .0 386.,1 327 ,.2 360 .1 341 .8 375..2 376 .6 318 .6 347 ,.8 336 .1 359 .4 396.,5 334 .2 375..9 353. 6 385. 5 385. 7 326 .1 356. 4 342 .3 366. 9 406 .1 340 .9 p. 77 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Body Weights Body Weight g Day 0 Body Weight g Day 3 Body Weight g Day 6 Body Weight g Day 7 Body Weight g Day 8 Body Weight g Day 9 Body Weight g Day 10 Body Weight g Day 11 Body Weigl g Day 12 M a l e , VII 10 mg/kg 701 702 703 704 705 706 707 708 709 710 270.1 252.6 278.4 285.3 243.3 273 .1 275.2 270.7 290.6 265.4 284.6 274 .2 298.0 302.5 257.6 296.0 295.5 287.1 307.5 280.3 305.9 305.9 322.2 306.3 270.4 321.9 325.0 307.0 328.1 315.3 308.8 306.3 323.0 314.0 273.3 325.2 334.7 309.3 336.3 316.0 313.9 313.8 334.5 324.5 273.9 334 .5 338.4 320.3 346.7 317.9 320.2 323.6 342.6 333.5 289.0 346.2 349.9 323.8 354.0 331.5 325.7 333.8 349.9 330.2 298.6 353.1 356.4 336.5 364.9 337.9 328.0 340.4 346.9 327.1 302.3 358.2 360.0 340.9 369.0 340.6 326.7 348 .1 354.6 332.2 313.9 372 .8 370.6 345.3 377.1 349.6 M a l e , IX 30 mg/kg 901 269.5 902 261.2 903 278.1 904 277.8 905 254.4 906 277.7 907 274.6 908 267.2 909 284.0 910 268.0 233.8 225.3 268.2 270.6 262.0 261.0 261.7 249.8 278 .1 249.2 201.9 186.8 304.0 293 .9 298.4 217.9 294.5 279.2 315.6 281.7 223.0 212.2 307 .6 293.0 301.6 246.5 309.4 284.0 317 .1 285.2 242.9 231.4 315.5 295.7 305.7 257.5 312 .0 288.4 316.6 291.4 250.8 241.0 328.1 299.6 312 .9 267.4 318.5 290.6 322.1 300.8 2 44 .5 254.4 335.8 298.6 315.1 276.0 320.5 297.2 327 .5 303.1 264.6 257.3 337.0 296.1 315.4 280.8 326.8 306.4 335.8 297 .8 2 75.1 263.3 349.3 296.9 322.0 291.5 330.7 310.8 339.5 304 .0 Male, XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 263 .1 25 7.0 275.3 286.3 259.9 269.4 267.3 272.9 2 71.7 260.9 254.2 238.0 274 .1 280.8 248.8 272.4 254 .7 259.5 227.5 256.7 284 .3 225.8 307 .1 313.0 270.9 314 .6 287.1 279.5 173.0 263.3 287.6 243.3 313 .2 325.2 276.6 319.2 284.2 273.0 162.8 264.9 286.5 255.1 315.4 324 .4 277.0 327.1 285.1 270.9 171 .5 275.9 273.9 260.8 316.0 320.2 284 .0 335.7 290.0 274.6 193.5 283.0 275.1 267.2 322.5 322 .3 287 .7 346.0 290.4 267.8 213.0 280.9 287.5 286.6 324 .1 329.8 293.4 346.0 285.6 261.2 228.1 276.2 293 .6 289. 9 326.7 342.8 301.8 354 .8 287.4 266.8 225.8 283.0 p. 78 - 78 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Male, I 0 mg/kg 101 393.0 102 388.3 103 384.8 104 373.9 105 337.0 106 364 .0 107 354.3 108 357.3 109 403.8 n o 372 .1 392.2 396.2 385.3 385.4 346.4 369.2 360.2 362.5 414.7 375.5 404.3 405.5 396.5 386.0 351.7 374.3 366.4 371.3 417.8 381.7 410.1 419.6 403.5 390.9 357.3 380.9 366.3 375.4 421.8 382.0 Male, III 0.3 mg/kg 301 381 .3 302 379.9 303 397 .1 304 352 .9 305 341.7 306 382.0 307 359.3 308 346.1 309 389.3 310 347.8 385.8 392.0 404 .2 3 65.7 353.9 388.4 362.6 348.6 396.3 352.1 393.5 393.0 414.6 373.4 360.2 402.1 374 .6 354.9 402.7 364 .4 400.8 405.6 421 .6 375 .0 360.8 402.7 374 .1 354.9 410.9 366. 6 Male, v 1 mg/kg 501 375.0 502 357.8 503 390.9 504 390.7 505 328.1 506 367.5 507 350.5 508 372 .6 509 409.4 510 337.2 375.8 359.7 402.0 399.7 334.9 367.0 355.4 380.6 413.5 348.2 389.8 362.9 407.0 402.1 336.8 377.3 357.7 383.8 424,7 356.1 393.8 369.1 411 .1 406.9 339.6 376.1 360.2 390.1 436.2 355.7 DuPont-18317 Individual Body Weights Body Weight g Day 17 Body Weight g Day 18 Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 420.4 418.2 410.8 405.7 361.6 383.8 375.7 381 .4 431.3 386.8 430.5 433.8 428.0 404 .9 379.4 392.5 386. 9 391 .6 44 3.6 396.9 438.9 439.8 436.6 415.8 380.4 396.2 390.5 397.0 446.6 402.3 4 42.1 441.8 440.8 418.4 383.9 403.0 394 .8 398.9 450.5 409.1 453.1 445.2 445.8 425.1 386.2 412.1 401 .1 402.5 4 54.4 414.7 406.3 402.9 429.6 378.5 370.2 409.8 380.2 357 .6 418.2 371.0 416.6 419.1 438.9 393.2 381.5 420.1 389.8 374 .0 427.3 387.2 421.7 418.5 4 47.5 397 .6 384 .9 424 .7 397 .1 371 .1 437.5 392.2 4 24.6 429 .1 454.3 401.8 387 .9 432.1 399.5 381.1 440.9 392.6 425.7 430.2 461.8 405.7 396.5 436.8 408.7 384 .5 44 6.0 400.7 404.5 370.9 416.8 413.8 346.3 383.4 360.3 391 .9 437.1 362.8 408.3 386.4 444.0 423.7 353.6 396.0 374.4 405.6 459.5 368.7 414.9 390.0 433.6 426.5 360.0 399.3 374 .3 410.6 467.4 379.1 421.0 390.3 432.9 434.6 363.0 409.2 377.6 416.5 4 66.1 377.0 424.4 390.1 446.5 440.6 368.2 408.6 376.0 42 4.3 470.4 376.5 p. 79 - 79- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Male, VII 10 m g /kg 701 328.8 702 350.8 703 362.0 704 336.3 705 319.0 706 375.3 707 372.1 708 342.8 709 380.4 710 360.2 337.9 364.0 371.0 342 .4 321.1 378.1 380.6 342.2 384.1 358.7 342.3 364 .9 374.5 344.1 331. 6 381 .4 385.3 354 .2 388.4 364 .6 345.0 370.4 375.9 348.4 330.7 396.1 387 .9 350.5 399.0 371. 9 M a l e , IX 30 m g /kg 901 281. 1 902 272.9 903 348.9 904 296.8 905 324.3 906 297 .6 907 329.0 908 309.1 909 341.1 910 304.6 261.5 282.5 355.4 297.9 320.1 299.4 331.1 30 7.8 345.2 306.0 24 4.9 279.2 357.9 298.1 321.5 312.2 340.9 309.8 350.3 308.9 229.4 288.5 367.0 294.3 328.3 304.5 340.9 311.0 355.9 303.6 Male, XI 30/0 m g /kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 300.9 296.9 329.2 353.9 307.4 358.8 290.6 263.9 197.7 285.2 298.5 298.4 329.4 355.8 312.0 355.9 291.7 2 7 0.1 181 .2 292.0 312.5 312.8 339.3 354.0 309.0 368.7 297.2 277 .7 173 .9 293.0 312.9 309.5 332.2 359.6 319.2 369.1 300.7 272.3 199.9 296.5 DuPont-18317 ndividual Body Weights Body Weight g Day 17 Body Weight g Day 18 Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 345.0 375.8 378.9 350.4 331.2 395.8 394 .5 355.4 402 .1 364 .9 355.0 382.4 396.9 358.2 337.1 407 .9 409.7 362.3 403.6 379.6 353.6 385.1 395.8 364.6 334 .1 410.5 418.9 365.6 408.7 381 .1 361.0 391.4 397.0 369.3 338.0 423.8 413.0 366.7 417.8 389.2 358.6 392.6 398.4 369.2 333.2 419.0 424.3 367 .9 421.5 390.6 215..8 292 .8 368. 8 289..1 326..0 307 ,4 340 .2 312 ,.7 363 .2 306 .6 208 .4 298 .1 378. 4 301. 6 336. 9 315. 1 34 9. 1 317 ,.1 365..2 306 ,.7 234 .,3 302 .0 381 .1 301..5 339..1 315 .5 344 .0 318 .9 366 .8 304 .4 248 ,5 296 .6 383 ,.5 301 .9 336.,4 321 .0 339..4 317 .0 359..9 306 .9 253 .1 297 .4 392 ,.5 294 .6 337 ,2 325 .1 341 ,7 319 .1 353 ,5 305 .3 320.4 312 .3 334 .9 360.9 319.4 374 .8 297.9 279.6 211.0 297.0 317.4 328.8 341.7 375.8 321.3 383.1 311.2 280.8 219.1 307.0 324 .8 327.1 342.6 374.9 324.1 384 .9 308.6 283.2 213.0 309.1 329.0 338.9 344 .6 375.0 328.0 387.4 314 .3 284.4 197.3 315.4 334.0 333.1 343.0 370.5 324 .4 388.6 308.7 282 .7 225.9 311.3 p. 80 - 80 - k% Vv Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Body Weight 9 Day 22 M a l e , I 0 m g /kg Body Weight g Day 23 Body Weight g Day 24 Individual Body Weights Body Weight g Day 25 Body Weight g Day 26 Body Weight g Day 27 Body Weight g Day 28 101 454.7 102 452 .1 103 44 6.2 104 430.5 105 393.4 106 416.6 107 400.3 108 409.6 109 457.0 110 420.5 Male, III 0.3 m g / kg 464.8 457.7 455.6 426.4 399.0 417.8 408.8 414.9 4 63.1 425.2 463.6 462.8 454 .3 438.0 405.5 422.2 409.5 416.3 470.0 430.3 4 7 8.2 472.2 468.4 4 4 8.7 405.5 429 .9 413.6 425.6 475.8 435.7 480.1 476.8 470.7 451.7 411.0 433.7 413.7 430.4 479.8 440.4 479.0 471.6 474.3 451.9 412.2 430.4 418.1 429.6 481.2 442.5 483.9 475.9 476.3 454,4 417.4 440.5 420.6 432.4 489.3 442.8 301 431.6 302 435.2 303 467.3 304 407.5 305 397 .9 306 439.8 307 411.1 308 388 .1 309 44 8.5 310 406.3 Male, V 1 mg/kg 438.7 440.5 471.0 407.6 400.6 4 4 3.4 415.9 392.9 456.4 409.6 437.1 447 .1 480.3 417.4 408.6 447.5 422.5 394.8 460.4 416.6 4 44.3 454.8 489.7 420.1 416.9 461.1 425.5 401.0 473.8 421.0 4 4 7.7 453.7 492.7 426.9 415.4 4 62.0 432.7 405.8 473.8 421 .6 454.2 456.3 496.0 423.2 416.5 465.6 436.3 404.0 476.0 423.6 452.7 463.7 502.4 419.8 421.4 467.1 434.4 402 .9 475.1 426.3 501 429.4 502 401.3 503 44 5.7 504 448.3 505 372.3 506 412.6 507 380.5 508 427 .5 509 478 .7 510 389.4 433.2 409.4 453.2 451.5 373 .9 416.8 383.5 425.2 481.8 391.0 431.8 406.2 455 .6 458.3 376.6 421.0 380.2 423 .1 488.6 390.2 438.3 413.1 459.9 4 66.7 384.5 424 .9 388.9 426.7 502.7 402.3 442.8 415.9 4 63.9 470.4 385.5 428 .9 390.3 429.5 505.4 405.6 443.9 414.9 464.8 467.6 384 .6 430.3 387.8 433.0 507.0 406.7 450.2 418.0 464.6 472.4 389.0 433.3 390.6 433.4 512 .1 408.6 81 DuPont-18317 p. 81 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Body Weights Body Weight g Day 22 Body Weight g Day 23 Body Weight g Day 24 Body Weight g Day 25 Body Weight g Day 26 Body Weight g Day 27 Body Weight g Day 28 Male, VII 10 mg/kg 701 363 .8 702 392.4 703 398.0 704 374.6 705 339.6 706 425.8 707 434.2 708 372.0 709 424.6 710 393.5 368 .4 4 04.6 408.1 3 73.9 343.7 429.4 437.4 378.0 431.8 400.9 366.0 399.6 403.1 377 .2 341.7 426.5 436.5 375.1 423.8 403.5 375.7 413.5 406 .3 375.3 344.3 433.3 4 4 3.4 376.1 434.6 411.0 378 .7 422.9 403.6 372 .5 345.5 451.8 4 47.2 377.2 440.7 409.4 378.7 422 .8 401 .1 381 .9 345.1 445.7 446.6 377.1 441.5 415.9 380.2 420.3 406.9 382 .2 345.9 452.5 450.6 384.7 441.5 410.4 Male, IX 30 mg/kg 901 261.0 902 302.8 903 395.3 904 296.8 905 34 4.1 906 327.5 907 342.1 908 318.0 909 358.7 910 306.3 271.7 300.6 399.1 299.4 346.2 329.9 347 .8 321.3 353.8 306.3 281.9 302.0 401.2 301.0 337 .8 323.0 348.2 326.1 350.5 309.5 292.3 307 .6 406.7 310.5 350.2 334.2 350.7 327 .3 363.6 316.0 295.4 307.4 416.8 311.4 353.1 338.1 353.3 333.3 363.1 316.0 296.7 313.9 413.4 308.0 351.8 337.3 352.3 331. 1 366.0 312.3 292.6 313.7 418.0 309.7 353.2 333.2 356.9 333.4 367.1 317.7 M ale, XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 338.3 338.5 339.3 378.3 330.7 399.3 314 .3 284 .1 233.5 315.9 338.0 338.4 348.0 389.7 333.2 400.2 313.3 288 .0 247 .7 316.0 338.7 346.3 351.0 394 .4 341.9 409.4 323.2 286.1 258.8 322.7 352.6 356.6 357 .5 408.2 347.9 416.3 327 .9 2 94.3 274.6 332 .6 353.1 360.6 361.7 417.1 352 .9 421 .5 337 .6 299.4 289.0 339,3 352.0 358.9 364 .8 418.4 359.8 418.4 3 3 4.7 300 .6 297.4 342 .0 352 .5 366.7 366.1 424 .4 361 .0 421.9 342.1 304 .0 309.1 349.8 - 82- DuPont-18317 p. 82 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix C Individual Food Consumption - 83- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats INDIVIDUAL FOOD CONSUMPTION EXPLANATORY NOTES ABBREVIATIONS : Cons. - consumption g/anm/day - grams of food consumed per animal per day DuPont-18317 - 84- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats__________ Individual Food Consumption Food Cons . Food C o n s . Food C o n s . Food Cons g/anm/day g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 2 1 Day 28 Male, I 0 mg/kg 101 102 103 104 105 106 107 108 109 110 30.2 29.3 30.2 27.6 24 .7 30.0 27.6 29.6 30.3 28.1 M a l e , III 0.3 mg/kg 30.6 31.5 28.9 27.9 25.2 29.1 27.6 28.1 33.5 29.2 31 .7 32.1 32.2 28.1 26.1 30.8 28.3 29.1 32.0 30.1 31 .1 30.5 31.0 30.8 26.8 29.9 28.0 30.2 31.9 32.2 301 29.8 302 29.0 303 30.1 304 27.5 305 25.7 306 30.2 307 25.5 308 25.3 309 30.0 310 26.9 Male, V 1 mg/kg 29.3 30.9 31.0 26.8 26.1 32.9 26.1 26.2 31.1 26.9 29.2 29.5 33.6 27.7 27.3 32.8 27.5 25.4 31.1 28.0 31.2 30.5 34.9 27.6 26.6 30.6 27.9 26.6 32.3 28.8 501 28.5 28.9 29.9 28.6 502 28.3 28.8 27.8 28.4 503 29.6 30.1 29.8 27.0 504 31.4 32.9 30.6 31.8 505 27.5 26.6 25.1 26.4 506 27.4 28.4 29.3 29.1 507 24 .1 24.1 22.9 23.7 508 30.1 28.8 30.0 28.2 509 31.2 32.5 33.2 33.6 510 27.3 26.2 26.7 28.0 p. 85 DuPont-18317 -85& Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Food Consumption Food Cons. g/anm/day Day 7 Food Cons. g/anm/day Day 14 Food Cons . Food Cons, g/anm/day g/anm/day Day 21 Day 28 M a l e , VII 10 mg/kg 701 27.8 702 25.5 703 26.9 704 25.3 705 24.0 706 27.2 707 29.0 708 24 .4 709 26.0 710 27.4 26.6 29.8 30.0 25.9 29.0 31 .1 31.8 26.8 29.7 30.3 27.2 30.2 30.9 26.6 26.1 31.2 34 .7 26.9 28.1 30.0 28.8 30.1 27.1 26.1 26.7 30.9 33.9 27.3 29.3 31.0 M a l e , IX 30 mg/kg 901 9.2 24 .6 902 8.6 27.0 903 23.4 30.5 904 24.7 23.4 905 24.7 25.3 906 14 .5 35.3 907 25.0 26.2 908 22.2 29.3 909 24.5 30.9 910 23.9 26.8 Ma l e , XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 24.2 13.9 25.8 23.7 23.0 26.9 22.1 22.8 5.1 21.4 23.2 31.9 28.6 28.6 25.1 29.2 22.2 21.0 15.8 27.1 11.9 23.5 29.2 22.2 22.9 30.3 24 .2 25.0 2 8.7 22.3 26.2 27.2 26.8 27.0 22.3 28.9 24.7 23.5 20.4 27.9 25.7 24.8 29.7 23.9 24 .0 29.0 26.4 28.5 26.5 26.6 24.4 26.1 25.8 29.7 25.1 29.2 25.9 23.1 32.0 28.2 DuPont-18317 - 86 87 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats__________ p. 87 DuPont-18317 Appendix D Individual Daily Animal Health Observations Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Sex Group Animal MI MI M1 MI MI MI MI MI MI M1 M III M III M III M III M III M III M III M III M III M III MV MV MV MV MV MV MV MV MV MV 101 102 103 104 105 106 107 108 109 110 301 302 303 304 305 306 307 308 309 310 501 502 503 504 505 506 507 508 509 510 Individual Daily Animal Health Observations Observation General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation. No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation. No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected DuPont-18317 Days 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-28 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Daily Animal Health Observations Sex Group Animal Observation M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII M IX 701 702 703 704 705 706 707 708 709 710 901 M IX M IX M IX M IX M IX M IX M IX M IX M IX M XI M XI M XI M XI M XI M XI M XI M XI M XI 902 903 904 905 906 907 908 909 910 1101 1102 1103 1104 1105 1106 1107 1108 1109 M XI 1110 General observation, General observation, General observation, General observation, General observation, General observation, General observation, General observation, General observation, General observation, General observation, Feces, Absent No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected No Abnormality Detected Comments, decreased feces Not Eating General observation, No Abnormality Detected Comments, decreased feces Not Eating General observation, No Abnormality Detected Not Eating General observation, General observation, General observation, General observation, General observation, General observation, General observation, General observation, Not Eating No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality Detected Detected Detected Detected Detected Detected Detected Detected General observation. No Abnormality Detected Comments, decreased feces General observation, General observation, General observation, General observation, General observation, General observation, General observation, Feces, Absent No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality No Abnormality Detected Detected Detected Detected Detected Detected Detected Stain Fur/Skin, Inguen, Brown Stain Fur/Skin, Inguen, Red Wet Fur, Inguen Not Eating General observation, No Abnormality Detected CO CM 1 o DuPont-18317 Days 0-28 0-28 0-28 0-28 CO CO CM 1 o 0-28 0-28 0-28 0-28 0-28 18 4-7 18 9-28 0 1 LO CO 1 00 CM 1 ro I o 4-7 4-7 0-3,5-28 4 0-28 0-28 0-28 0-28 0-28 0-28 0-28 0-3,5-28 4 0-4,8-28 5-7 0-28 0-28 0-28 0-28 0-28 0-3,11-28 4-5 9-10 5 5 4-5 0-28 -89- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 90 DuPont-18317 Appendix E Individual Detailed Clinical Observations and Mortality Records -90- f/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal Observation MI MI MI MI MI MI 101 102 103 104 105 106 MI M1 MI MI M III M III M III M III M III M III M III M III M III M III M MV MV MV MV MV MV MV MV MV MV 107 108 109 110 301 302 303 304 305 306 307 308 309 310 501 502 503 504 505 506 507 508 509 510 General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Hair Loss, Forelimb, Bilateral Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Hair Loss, Forelimb, Bilateral Sacrificed by design Days 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-7 14-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0 7-29 29 -91 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal M VII 701 M VII 702 M VII 703 M VII 704 M VII 705 M VII 706 M VII M VII M VII M VII M IX M IX M IX M IX M IX M IX 707 7 08 709 710 901 902 903 904 905 906 M IX M IX M IX 907 908 909 M IX 910 Observation General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Hair Loss, Forepaw, Bilateral Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Hair Loss, Abdomen, Bilateral Hair Loss, Forelimb, Bilateral Hair Loss, Hindlimb, Bilateral Sacrificed by design General observation. No Abnormality Detected Hair Loss, Forepaw, Bilateral Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Hair Loss, Forelimb, Bilateral Hair Loss, Forepaw, Bilateral Sacrificed by design General observation, No Abnormality Detected Sacrificed by design Days 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-14 21-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0 7-29 21-29 21-29 29 0-7 14-29 29 0-29 29 0-14 21-29 21-29 29 0-29 29 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal Observation M XI M XI M XI M XI M XI M XI M XI M XI M XI M XI 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Lethargic Carriage, High Feces, Absent Stain Fur/Skin, Abdomen, Red Stain Fur/Skin, Forepaw, Bilateral, Red Stain Fur/Skin, Inguen, Red Stain Fur/Skin, Perineum, Red Stain Fur/Skin, Ventral body. Red Stain Fur/Skin, Perinasal, Red Stain Fur/Skin, Perioral, Red Wet Fur, Ventral body, Ventral Not Eating Sacrificed by design General observation, No Abnormality Detected Sacrificed by design Days 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 6-7 6-7 6-8 8 6-7 8 8 6-7 6-7 6-7 6 6-7 29 0-29 29 05 CM 1 c"H O Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix F Individual Animal Clinical Pathology Data Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES ABBREVIATIONS : General: Adeq CLOT or Clot Deer Mod NP OK adequate sample clotted decreased moderate not taken, not performed, or results not valid sample condition OK for testing Individual Hematology Values: COND - sample condition RBC - red blood cell count HGB - hemoglobin HCT - hematocrit MCV - mean corpuscular (cell) volume MCH - mean corpuscular (cell) hemoglobin MCHC - mean corpuscular (cell) hemoglobin RDW - red cell distribution width ARET - absolute reticulocyte count PLT - platelet count WBC - white blood cell count ANEU - absolute neutrophil (all forms) ALYM - absolute lymphocyte AMON - absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophil ALUC - absolute large unstained cell concen Individual Red Blood Cell Morphology Values : ANIS - anisocytosis MIC - microcytes MAC - macrocytes POLY - polychromasia HYPO - hypochromasia ECHI - echinocytes ACAN - acanthocytes TARG - target cells RX - rouleaux HJB - Howell-Jolly body - - not observed Individual White Blood Cell / Platelet Morphology Values: SM - smudge white blood cells TOX - toxic neutrophils DB - Ddhle bodies VC - vacuolated cytoplasm BC - basophilic cytoplasm PCE - platelet clumps / estimate GP - giant platelets BP - bizarre platelets - - not observed Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES (Continued) ABBREVIATIONS : (Continued) Individual Clinical Chemistry Values: HEM - hemolysis LIP - lipemia ICT - icterus CHOL - cholesterol TRIG - triglycerides TP - total protein ALB - albumin GLOB - globulin HDL - high-density lipoprotein cholesterol NHDL - non-high-density lipoprotein cholesterol SCORT - serum corticosterone NOTES: When individual animal data are not reported, it may be due to one of the following reasons or other reasons, all of which are explained in the study records: the sample was clotted (CLOT) there was insufficient sample for testing (QNS) a valid result could not be obtained (RNV) the sample was not suitable for testing the animal died prior to sample collection no sample was available for testing (NSR) Only positive findings were recorded for special observations (e.g., additional cell types) or observations marred other. N0 V Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 101 102 103 104 105 106 107 108 109 110 Male, Animal 301 302 303 304 305 306 307 308 309 310 Group COND OK OK OK OK OK OK CLOT OK OK OK i RBC x l 0 6/pL 7.77 7.53 7 .85 7.84 7.32 7.48 NP 7 .90 7.59 7.67 Group COND OK OK OK OK OK OK OK OK OK OK ni RBC xl O'/pL 7.87 7.40 8.15 8.11 7.50 7.31 7.57 7 .61 7.45 7 .17 0 HGB g/dL 14 .6 15.0 15.4 15 .1 14 .8 14 .8 NP 15.1 14 .6 14 .8 0.3 HGB g/dL 15.6 14 .5 15.4 14 .9 14.6 14 .5 13.9 14 .6 14 .5 14 .7 mg/kg HCT % 45.6 47.1 48.3 45 .5 46.4 45.8 NP 45 .9 45.1 45.9 mg/kg HCT % 48 .3 44.7 47 .7 45.9 45.0 44.6 43.5 44 .6 44 .4 45.6 Day MCV fL 58.7 62.6 61 .6 58.0 63.4 61.2 NP 58.1 59.5 59.9 Day MCV fL 61 .4 60.3 58.5 5 6.6 59.9 61.0 57.5 58.6 59.5 63.5 29 MCH pg 18.8 19.9 19.6 19.2 20.3 19.8 NP 19.2 19.3 19.4 29 MCH pg 19.8 19.6 18 .9 18 .4 19.4 19.9 18 .4 19.2 19.4 20.4 MCHC g/dL 32.0 31.8 31 .9 33.1 32.0 32.3 NP 33.0 32.4 32.3 RDW % 11.5 11.8 11 .6 11.0 11.0 11.8 NP 11 .9 11.7 11.6 ARE T xloVpL 200.3 196.6 208.2 194.3 160.5 198.6 NP 164.5 182.1 180.9 PLT x l O '/p L NP 1006 NP 1119 1261 1207 NP NP 968 976 MCHC g/dL 32.2 32.5 32.2 32.5 32.4 32 .6 31 .9 32.8 32 .6 32.2 RDW % 11.9 12.1 11.4 11. 8 11.2 11.0 10.8 10.7 11 .8 11.6 ARET xloVpL 169.0 152.6 166.7 189.9 148 .8 158.0 147 .4 180.3 185.7 206.3 PLT xloVpL 1042 940 1104 1114 1064 1080 1096 1052 1167 923 DuPont-18317 p. 97 - 97- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 506 507 508 509 510 Group COND OK OK OK OK OK OK OK OK OK OK V RBC xloVpL 7.9 6 8.05 7.23 7.33 7 .30 7.68 7.58 7.54 7 .31 8.03 Male, Animal 701 702 703 704 705 706 707 708 709 710 Group COND OK OK OK OK OK OK OK OK OK OK VII RBC xlOVpL 7 .98 7 .44 7.09 6. 10 7 .48 7.24 7.31 7.65 7.54 7.00 1 HGB g/dL 15.2 15.8 13.9 15.0 14.5 14.3 14 .6 15.0 14.2 14 .6 10 HGB g/dL 13 .7 13 .9 13.1 12.0 13 .5 13.5 14.7 14 .2 13.5 13 .3 mg /kg HCT % 48.3 48.3 42.9 46.1 45.7 44 .3 44.7 47 .1 44 .1 44.9 mg/ kg HCT 43.6 44.0 40.4 37.4 43.1 42 .7 45.9 43.8 43 .3 41.6 Day MCV fL 60.7 59.9 59.4 62.9 62 .6 57.7 59.0 62.4 60.3 55.9 Day MCV fL 54.7 59.2 56. 9 61.3 57.6 59 .0 62.7 57.2 57 .4 59.4 29 MCH pg 19.1 19.7 19.2 20.5 19.8 18.6 19.3 19.8 19.5 18 .2 29 MCH pg 17 .2 18.6 18.5 19.6 18 .1 18 .7 20.1 18.6 17.9 18.9 MCHC g/dL 31 .4 32.8 32.3 32.5 31.7 32.2 32 .6 31.8 32.3 32.5 MCHC g/dL 31 .4 31. 5 32.5 32 .0 31 .4 31.7 32 .1 32.5 31.2 31 .9 RDW % 12.1 12.3 11.3 11.5 11 .6 11.8 11.6 11.3 12 .0 11.7 ARET xloVpL 163.1 207.6 158.2 140.3 197.9 161.5 166.7 170.6 210.8 197.9 PLT xlOVpL NP 872 1112 1166 NP 1159 1120 NP 1135 NP RDW 12 .7 12.2 13.8 12.3 12 .4 13.6 11.9 12 .2 13.7 12.7 ARET xl0J/pL PLT xlOVpL 210.5 235.8 233.8 152 .7 123.0 257,1 195.8 154.9 248.5 232 .4 981 1038 NP 352 NP 1182 NP 1022 1350 1380 DuPont-18317 p. 98 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 90 5 90 6 907 908 909 910 Group COND OK OK OK OK OK OK OK OK OK OK IX RBC xloVpL 7 .44 7 .22 6.85 8 .72 7 .13 8.08 6.75 7.81 7.22 7 .47 30 HGB g/dL 13.3 13.5 13.3 14 .8 13.5 14.4 12 .1 14.2 13.4 13.5 mg/ kg HCT % 42.6 41.1 42.0 45.8 42.6 44.9 38.7 44.2 42 .1 42.8 Day MCV fL 57.2 56.9 61.3 52.5 59.8 55.6 57 .4 56.6 58.3 57.3 29 MCH pg 17.9 18 .7 19.4 17.0 19.0 17 .8 18.0 18.2 18.5 18.0 MCHC g/dL 31 .2 32.8 31 .7 32.4 31 .8 32 .1 31 .4 32.2 31. 8 31 .5 RDW % 14 .6 13.2 12 .1 12.7 12.4 12 .8 13.8 13 .1 12 .8 14 .1 ARET xloVpL PLT xloVpL 280.3 206.0 178 .4 152.4 198.0 156.9 213.6 222 .3 221.9 259.3 NP 1027 1081 NP 1280 998 1278 1217 NP 1491 Male, Group XI 30/0 mg/kg (Recovery) Day 29 RBC HGB HCT MCV MCH MCHC RDW ARET PLT D Animal COND xlOVpL g/dL % fL pg g/dL % xlO V p L xlO V p L 1101 1102 1103 1104 1105 110 6 1107 1108 1109 1110 OK OK OK OK OK OK OK OK OK OK 6.98 6.80 7.22 6.52 6 .68 6.22 6. 67 7.00 6.28 7 .15 13.0 13.3 13.0 12.8 12.4 12.8 12.3 13 .1 12.3 12 .8 42.0 41.1 40.5 40.7 39.4 39 .4 37.7 40 .9 39.6 40.3 60.2 60.5 56.0 62.4 59.0 63.4 56.5 58.4 63 .1 56.3 18.7 19.5 18.0 19.6 18.6 20.7 18 .4 18.7 19.5 17.9 31.1 32.2 32.2 31.4 31.5 32 .6 32.6 31 .9 31 .0 31. 8 13.7 14.3 12.9 14 .6 15.7 11.9 13 .5 12 .8 19.3 13.0 366.7 396.5 285.1 406.3 422.5 241.4 369.9 330 .6 563.7 315.5 1410 1156 1179 963 NP 1278 1264 1391 NP 1015 DuPont-18317 p. 99 - 99 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 101 102 103 104 105 106 107 108 109 110 Group I WBC xlO'VyL ANEU xlOVpL 16.73 12.13 17.69 10.15 6.35 10 .92 NP 11.56 14.79 12.06 2 .54 1 .03 1 .59 1 .22 0.45 1 .27 NP 1 .61 1 .86 1.58 0 AL YM xl 0 V y L 13.11 10.69 15.23 8 .63 5.65 9 .16 NP 9.14 12 .13 9.83 mg /kg AMON xlOVyL 0.42 0.22 0.28 0 .15 0.12 0.20 NP 0.28 0.32 0.28 Day AEOS xlOVpL 0.34 0.05 0.31 0.06 0.06 0.12 NP 0.28 0.16 0.16 29 ABAS x10 '/yL ALUC x l 0 ?/yL 0.06 0.05 0.08 0.03 0.02 0.06 NP 0.06 0.07 0.05 0.26 0.09 0.21 0.06 0.05 0 .11 NP 0.19 0.24 0.15 Male, Animal 301 302 303 304 305 306 307 308 309 310 Group WBC xlO'/uL 12 .65 12.13 15.31 16.29 11.09 8.30 7 .40 11.29 10.11 9 .52 III ANEU xlO'VyL 1.84 1 .78 1 .87 1 .14 1 .30 0 .67 1.01 2.14 1.04 1.04 0.3 ALYM xlOVpL 10.21 9.90 12.73 14.70 9.29 7 .33 6.10 8.65 8.64 8.07 mg/kg AMON xlOVyL 0.19 0.26 0.41 0.1/ 0.24 0.11 0 .12 0.29 0.20 0.21 Day AEOS xlOVyL 0.16 0.05 0.12 0.11 0.03 0.08 0.06 0.07 0.10 0.06 29 ABAS xlO'VyL 0.08 0.02 0.05 0.05 0.10 0.03 0.04 0.06 0.02 0.03 ALUC xlOVpL 0.17 0 .12 0 .13 0.13 0 .13 0.07 0.07 0.09 0 .11 0.11 DuPont-18317 p. 100 xo/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 50 6 50 7 508 509 510 Group WBC xIOVpL 17.11 14.81 10.66 9 .63 12.87 11.28 14 .91 16.44 9.61 10.45 V ANEU xIOVpL 1 .54 2 .13 0.95 0.99 1 .29 1 .42 1.38 1.28 1 .18 3.33 1 ALYM xIOVpL 14.87 12.15 9.24 8.26 11.07 9.39 13.15 14.54 8.04 11.14 mg/kg AMON xIOVpL 0.37 0.24 0.28 0.20 0.24 0.11 0.14 0.31 0.20 0.52 Day AEOS xIOVpL 0.08 0.08 0.07 0.02 0.05 0.13 0.06 0.09 0.09 0.18 29 ABAS xIOVpL 0 .12 0.08 0.04 0.03 0.06 0.13 0.05 0.05 0.02 0.07 ALUC xIOVpL 0.13 0 .14 0 .10 0.13 0 .14 0 .10 0.11 0.16 0.08 0.21 Male, Animal 701 702 703 704 705 706 707 708 70 9 710 Group WBC xIOVpL 16.43 15.57 15.38 13.51 12.35 14.85 21.39 15.73 19.09 18 .32 V I I 10 ANEU x 10 V p L ALYM x l O 3/ p L 1 .44 1 .66 1.85 0.84 1.70 2.43 2.30 1.03 1.25 2.13 14.29 13.11 13.38 12.30 9.95 11.64 17.99 13.84 16.99 15.20 mg/kg AMON xIOVpL 0.26 0.34 0.00 0.19 0.34 0.38 0 .62 0.39 0.26 0.47 Day AEOS xlO'VpL 0.16 0.11 0.15 0.04 0.12 0.29 0.10 0.05 0.13 0.11 29 ABAS xIOVpL 0.08 0.10 0.00 0.04 0.08 0.03 0.10 0.12 0.11 0.07 ALUC xIOVpL 0.20 0.24 0.00 0.11 0 .16 0.09 0.28 0.29 0.34 0.33 101 DuPont-18317 p. 101 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group WBC xlOVpL 13 .64 18 .92 17 .38 15.26 13.29 18.04 18.41 22.08 19 .68 13 .97 IX ANEU xloVpL 1.76 0.76 2 .24 1 .58 1.15 2 .60 1.54 2.53 1.67 2 .10 30 ALYM xlO 7 pL 11.35 17.59 14.57 12.86 11.61 14.51 16.05 18.42 16.97 11.35 mg/ kg AMON xl07pL 0 .32 0.57 0.28 0.32 0.22 0.35 0.24 0.51 0.57 0.16 Day AEOS xloVpL 0.02 0.00 0.12 0.16 0.08 0.25 0.07 0.15 0.11 0.12 29 ABAS xloVpL 0 .07 0.00 0.05 0.05 0.06 0.08 0.10 0.13 0.11 0.05 ALUC xlOVpL 0.12 0.00 0.12 0.29 0 .17 0.26 0.41 0.34 0.25 0.19 Male, Group XI 30/0 mg /kg (Recovery) Day 29 Animal WBC xlOVpL ANEU xlOVpL ALYM xlOVpL AMON xlOVpL AEOS xloVpL ABAS xloVpL ALUC xloVpL 1101 13 .64 1.09 12.14 0.27 0.14 0.00 0.00 1102 10.03 1.31 8.31 0.23 0.06 0.03 0.09 o 1103 1104 15.43 11.94 0.93 1 .68 13.41 9.78 0.29 0.26 0.17 0.04 0.07 0.03 0.56 0.14 1105 11.44 2 .30 8 .74 0.17 0.09 0.05 0.08 1106 18.93 1.38 16.78 0.31 0.09 0.12 0.26 1107 23.15 1.89 19.98 0.53 0.21 0.16 0.39 1108 14 .53 1.64 12.20 0.42 0.10 0.05 0.12 1109 8.10 0.99 6.71 0.25 0.03 0.04 0.09 1110 11.89 1. 37 10.02 0.21 0.02 0.07 0.20 - 102- DuPont-18317 p. 102 N Q. Tv Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 101 102 103 104 10b 106 107 108 109 110 Group ANIS Trace Trace Few - CLOT - - Male, Animal 301 302 303 304 305 306 307 308 309 310 Group ANIS Trace - Trace - Few Trace I MIC NP - - III MIC - 0 MAC Trace Trace Few NP - - 0.3 MAC Trace - - Trace - - - Few Trace mg/kg POLY Trace Trace Trace Trace - Trace NP - _ _ mg/kg POLY - - Trace - ~ - Trace Trace Day HYPO _ Few Trace - NP - - _ Day HYPO Few _ - - - Few - 29 ECHI Few Trace Few - Mod NP - Mo_d 29 ECHI _ _ - Few - ACAN Mod Trace Few Trace - Few N_P Mo_d ACAN _ - Trace _ _ - - Few - TARG _ - N_P - _ TARG _ _ _ _ _ _ - - RX _ _ - N_P _ _ RX _ _ _ _ - - _ HJB _ - _ - _ N_P _ HJB _ _ _ _ _ _ _ _ DuPont-18317 p. 103 - 103 - CO Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 506 50 / 508 509 510 Group ANIS Trace Trace Trace Trace Trace Trace - ['race Trace - Male, Animal 701 702 703 704 705 706 707 708 70 9 710 Group ANIS Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace V MIC - - - - - VII MIC _ - - - - 1 MAC Trace Trace Trace Trace Trace Trace - Trace Trace - 10 MAC Trace Trace Trace Trace Trace Trace Trace Trace Trace Trace mg/ kg POLY - Trace - Trace - - Trace Trace mg/ kg POLY Trace Few Trace Trace Few Trace Trace Trace Day HYPO Few - Mod - Mod Trace - Few Few - Day HYPO Mod Mod Many Mod Many Few Few Trace Few Few 29 ECHI Few - Trace - - - 29 ECHI - ACAN Trace - Trace - Trace Trace - Few - ACAN Few Trace Trace Trace Few Few - TARG - - - - TARG _ RX - - - - RX - * HJB - - - - HJB - - DuPont-18317 p. 104 - 104- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Male, Animal 901 902 903 904 903 906 907 908 909 910 Group ANIS Trace Trace Trace Trace Trace Trace Trace Trace Male, Animal 1101 1102 1103 1104 1105 1106 110 / 1108 1109 1110 Group ANIS Trace Few Trace Few Few Trace Trace Trace Few Trace IX MIC - Trace Trace XI MIC - Trace Trace Trace 30 MAC Trace Trace Trace Trace Trace Trace Trace Trace 30/0 MAC Trace Few Trace Few Few Trace Trace Trace Few Trace mg/kg POLY Few Trace Trace - Trace - Trace Trace Trace Few Day HYPO Mod Trace Many - Few - Few Trace Mod Few 29 ECHI _ - - - - - mg/ kg (Recovery) Day POLY HYPO ECHI Mod Few Few Mod Mod Few Few Few Mod Few Few Mod Mod Mod Many Few Few Few Many Mod Trace - Trace - Few / & Clinical Pathology Data ACAN Trace Trace Trace Trace Few Few 29 ACAN Few Trace Few Trace Few Few Few Trace Few Few TARG TARG RX RX HJB HJB DuPont-18317 p. 105 - 105 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Male, Animal 101 102 103 104 105 106 107 108 109 110 Group SM _ - - - CLiOT - - I TOX _ - - - NP - - 0 mg/ kg Day 29 DB VC BC PCE - - - Decr - - Decr - NP NP NP NP Decr -- - Male, Group in 0.3 mg/ kg Day 29 Animal SM TOX DB VC BC PCE 301 _ _ 302 - - o 303 - - 304 305 : : -- 306 - 307 - - _ _ 308 - - 309 - - 310 - - Clinical Pathology Data GP BP NP NP GP BP DuPont-18317 p. 106 - 106- X) Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Male, Animal 501 502 503 504 505 506 50 / 508 509 510 Male, Animal 701 702 703 704 705 706 707 708 70 9 710 Group SM - - Group SM _ - - V TOX - - - - -_ - - VII TOX _ - -- - Individual Animal 1 mg/ kg Day 29 DB VC BC PCE --- _- _ Adeq --- -- - - _- - - Adeq -- - -- - - - - - Adeq --- - - - - Adeq 10 mg/ kg Day 29 DB VC BC PCE ____ ---- - - Decr - - -- - - - Decr -- -- - - - Adeq -- - ------- Clinical Pathology Data GP BP GP BP DuPont-18317 p. 107 - 107- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Male, Animal 901 902 903 904 905 906 907 908 909 910 Group SM - - - - - - - Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group SM IX TOX _ - - - - XI TOX Individual 30 DB - - - - - 30/0 DB mg/ kg VC - - - Day BC _ - - - mg/ kg (Recovery) VC BC Animal Clinical Pathology Data 29 PCE Adeq - - Deer - - Adeq GP - - - - - BP - - - Day PCE - Ad_eq Adeq 29 GP - - BP - - DuPont-18317 p. 108 - 108 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Male, Group I Animal 101 102 103 104 105 106 107 108 109 110 HEM Small None None None None None None None None None Male, Gr oup LIP None None None None None None None None None None III Animal 301 302 303 304 305 306 307 308 309 310 HEM None None None None None None None None None None LIP None None None None None None None None None None 0 ICT None None None None None None None None None None 0.3 ICT None None None None None None None None None None mg/kg CHOL mg/dL 95 67 70 58 46 56 91 55 57 43 mg/kg CHOL mg/dL 32 51 37 27 29 54 36 43 53 49 Day TRIG mg/dL 57 92 65 50 36 59 88 66 94 69 Day TRIG mg/dL 48 86 40 54 26 51 29 34 53 46 29 TP g/dL 6.4 6.1 6 .4 5.9 5.9 6.1 6.4 6.2 5.9 6.1 29 TP g/dL 6 .7 6.2 6.3 5 .9 6.0 6.2 6.1 5.9 5.9 6.0 Clinical Pathology Data u> U) ALB g/dL 3.4 3.3 3.4 3.3 3.2 3.5 3.3 3 .1 3 .3 GLOB g/dL 3.0 2.8 3.0 2.6 2.6 2.9 2.9 2 .9 2 .8 2 .8 HDL mg/dL 29 27 25 22 19 21 31 23 22 19 NHDL mg/dL 66 40 45 36 27 35 60 32 35 24 SCORI ng/mL 213 32 104 60 109 218 50 251 283 46 ALB g/dL 3.5 3.4 3.3 3 .4 3 .4 3.4 3.4 3.3 3.2 3.3 GLOB g/dL 3.2 2.8 3.0 2.5 2 .6 2.8 2 .7 2 .6 2 .7 2 .7 HDL mg/dL 16 21 18 14 15 21 17 17 22 20 NHDL mg/dL 16 30 19 13 14 33 19 26 31 29 SCORI' ng/mL 64 190 106 88 113 227 214 258 262 148 DuPont-18317 p. 109 - 109- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Male, Group Animal 501 502 503 504 505 506 507 508 509 510 Male, HEM None None None None None Trace Small Trace None None Group Animal 701 702 703 /'04 705 706 707 708 709 710 HEM Trace Trace Small Small None Small Small Trace Small Small V LIP None None None None None None None None None None VII LIP None None None None None None None None None None 1 ICT None None None None None None None None None None 10 ICT None None None None None None None None None None mg/kg CHOL mg/ dL 64 43 34 44 48 42 46 34 42 42 mg/kg CHOL mg/dL 55 48 74 52 53 46 55 41 36 59 Day TRIG mg/dL 99 54 44 47 51 43 26 30 56 56 Day TRIG mg/dL 47 47 59 25 42 44 81 32 42 40 29 TP g/dL 6.4 6.7 6.0 5.8 6.4 6.5 6.0 5.7 6.5 6.3 29 TP g/dL 5.9 6.5 5.8 5 .7 6.0 6.3 6.8 6 .3 6.5. 5 .6 ALB g/dL 3.6 3.7 3.5 3.5 3.6 3.6 3.4 3.2 3.6 3.7 ALB g/dL 3.5 3.8 3.4 3.5 3.5 3.7 4 .0 3 .8 3 .8 3 .5 -no - Pathology Data GLOB g/dL 2.8 3.0 2.5 2.3 2.8 2.9 2.6 2.5 2 .9 2.6 HDL mg/dL 26 22 15 19 21 18 18 15 19 18 NHDL mg/dL 38 21 19 25 27 24 28 19 23 24 SCORT ng/mL 221 10 107 155 128 50 271 208 116 14 7 CO lO CM CM GLOB g/dL 2 .4 2.7 2.4 2.2 2.5 2 .6 2 .7 2 .1 HDL mg/dL 20 18 24 19 19 15 21 16 12 20 NHDL mg/dL 35 30 50 33 34 31 34 25 24 39 SCORT ng/mL 174 354 176 158 302 60 88 119 236 182 DuPont-18317 O CM Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Clinical Pathology Data Male, Group IX Animal 901 902 903 904 905 906 907 908 909 910 HEM Small None Trace Small None Trace Small Trace Trace None Male, Group LIP None None None None None None None None None None XI Animal 1101 1102 1103 1104 1105 1106 110 1 1108 1109 1110 HEM Small Trace None Trace None Small Small Trace None None LIP None None None None None None None None None None 30 ICT None None None None None None None None None None 30/0 ICT None None None None None None None None None None mg/ kg CHOL mg/dL 46 70 50 47 64 47 64 47 53 55 Day TRIG mg/dL 55 48 55 35 37 47 26 42 44 62 29 TP g/dL 5.8 6.3 6.4 6.3 6.5 6.4 6.0 5.9 6.7 mg/ kg (Recovery) Day CHOL mg/dL TRIG mg/dL TP g/dL 88 45 6 .8 61 79 6.3 122 57 7 .0 67 39 6.6 84 64 7.0 64 25 6.4 36 35 6.3 82 37 6.6 62 44 5 .4 59 42 6.2 ALB g/dL 3.7 3.7 3.8 3.7 3.9 3.7 3.8 3.6 3.7 3.9 29 ALB g/dL 3 .9 3.6 3.9 3.9 4 .2 3 .7 3 .6 3.7 3.2 3.6 GLOB g/dL 2 .1 2.6 2 .6 2 .6 2 .6 2 .7 2.2 2.2 2.2 2.8 HDL mg/dL 16 26 18 17 25 18 22 16 16 20 NHDL mg/dL 30 44 32 30 39 29 42 31 37 35 SCORT ng/mL 436 736 370 238 76 43 331 38 287 124 GLOB g/dL 2 .9 2 .7 3 .1 2.7 2 .8 2 .7 2 .7 2 .9 2 .2 2 .6 HDL mg/dL 29 23 35 24 28 25 15 30 22 22 NHDL mg/dL 59 38 87 43 56 39 21 52 40 37 SCORT ng/mL 71 317 90 198 68 54 89 95 245 85 DuPont-18317 p. 111 co lO - Ill - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 112 DuPont-18317 Appendix G Individual Primary Humoral Immune Response Data - 112- u3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Animal Number SLOPE Individual Primary Humoral Immune Response Data X Log^ Male, Group I - 0 mg/kg 101 -0.8772 1648 102 -0.9165 872 103 -0.9442 5777 104 -1.0021 7 90 105 -0.9721 1355 106 -1.0214 2414 107 -1.0063 122 108 -1.0053 1586 109 -1.0377 394 110 -1.0022 1175 10.687 9.768 12.496 9.626 10.404 11.237 6.928 10.631 8.622 10.198 Male, Group III - 0.3 mg/kg 301 302 303 30 4 305 306 307 308 309 310 -1.0141 -0.9706 -1.0180 -0.9477 -1.0010 -0.9884 -0.9671 -0.9972 -0.9972 -0.8896 1590 695 1237 18 78 1854 1618 1605 1312 1095 914 10.635 9.441 10.273 10.875 10.856 10.660 10.648 10.358 10.097 9.836 Male, Group V - 1 mg/kg 501 -0.9758 1307 10.352 502 -0.9001 2679 11.387 503 -0.9786 1559 10.606 504 -0.9388 111 6.794 505 -0.9980 915 9.838 506 -1.0325 1048 10.033 507 -0.9969 3228 11.656 508 -0.9742 791 9.628 509 -0.9732 22 7 7 .826 510 -0.9843 1409 10.460 p. 113 DuPont-18317 - 113 - //f p. 114 Ammonium Periluorooctanoate: 28-Day Immunotoxicity Study in Male Rats________________________________________________ DuPont-18317 Individual Primary Humoral Immune Response Data Animal Number SLOPE X Log2 Male, Group VII - 10 mg/kg 701 -1.0199 788 9.622 702 -0.9756 956 9.901 703 -0.984/ 201 7.650 704 -1.0187 855 9.740 705 -0.9605 2116 11.047 706 -0.9560 7340 12.842 707 -0.9836 494 8 .948 708 -0.9532 86 6.426 709 -0.9399 3660 11.838 710 -0.9843 2287 11.159 Male, Group IX - 30 mg/kg 901 -1.0014 1068 10.061 902 -1.0035 342 8.418 903 -1.0215 760 9.570 904 -0.9123 294 8.200 905 -1.0340 1788 10.804 906 -0.9742 2755 11.428 907 -0.9052 2078 11.021 908 -0.9060 457 8.836 909 -0.9504 1893 10.886 910 -1.0107 912 9.833 Male, Group XI - 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 -1.0040 -0.9456 -0.9969 -0.9935 -0.7713 -0.9712 -0.9856 -0.9682 -0.9463 -0.9972 543 370 392 460 1009 2410 314 2447 362 1844 9.085 8.531 8.615 8.845 9.979 11.235 8.295 11.257 8.500 10.849 - 114 - 115 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 115 DuPont-18317 Appendix H Individual Primary Humoral Immune Response Positive Control Data - 115 - // Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 116 DuPont-18317 Individual Primary Humoral Immune Response Positive Control Data Animal Numbe r SLOPE X Log2 Male, Gr oup CIX - Saline C901 C902 C903 C904 C905 C906 C90 7 C908 C909 C910 -1.0282 -1.0269 -0.9992 -1.0116 -0.9398 -0.9437 -0.9708 -0.9619 -0.9601 -1.0013 1215 1400 1249 977 820 109 554 1255 692 328 10.247 10.451 10.287 9.932 9.679 6.768 9.114 10.293 9.435 8.358 Male, Group CXI - 20 mg/kg Cyclophosphamide CUOI Cl 102 Cl 103 C 11 04 Cl 105 C1106 Cl 107 C 1108 Cl 10 9 C1110 -0.6474 -0.9816 -0.9912 -0.9556 -0.9067 -0.8837 -1.0035 -1.0031 -0.9962 -0.9898 4 14 18 12 23 26 55 15 14 26 2.000 3.807 4 .170 3.585 4.524 4.700 5.781 3.907 3.807 4.700 o CM 1 Maie, Pooled Samples mg/kg Cyclophosphamide -0.9859 -0.9832 29 12 4 .858 3.625 -116- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 117 DuPont-18317 Appendix I Individual Animal Final Body and Organ Weights - 117- //J -___, Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Final Body and Organ Weights Group: 1 Treatment: 0 mg/kg Sex: MALES ANIMAL 1 FBW 1 BRAIN 1 1 {Gms) 1 (Gms) %FBW 1 (Gms ) LIVER 1 SPLEEN 1 THYMUS %FBW %BRAIN 1 (Gms ) %FBW %BRAIN (Gms ) %FBW %BRAIN 101 1455.90 1 2.163 0.4744 1 16.208 3.5552 749.33 I 1 .143 0.2507 52 .843 1 0.691 0.1516 31.946 102 1441.60 1 1.995 0.4518 1 13.249 3.0002 664.11 1 0.761 0.1723 38.145 1 0.651 0 .1474 32.632 103 445.00 1 2 .122 0.4769 1 14.415 3.2393 679.31 1 0.976 0.2193 45.994 1 0.775 0.1742 36.522 104 1427.00 1 1 .959 0.4588 1 13.245 3.1019 676.11 1 0.771 0.1806 39.357 1 0 .690 0.1616 35.222 10b 390.80 2.072 0.5302 11.388 2.9140 549.61 1 0.806 0.2062 38.900 1 0.425 0.1088 20.512 106 1404.70 1 1.949 0.4816 1 12.348 3.0511 633.56 1 0.669 0.1653 34.325 0.499 0.1233 25.603 107 13 8 T .90 1 2.004 0.5166 1 12.113 3.1227 604.44 0.617 0.1591 30.788 0.429 0.1106 21.407 108 405.90 1 1 .948 0.4799 12.243 3.0163 628.49 1 1 .008 0.2483 51.745 1 0.435 0.1072 22.331 109 1457 .80 1 2 .038 0.4452 12.617 2.7560 619.09 1 0.944 0.2062 46.320 1 0 .544 0.1188 26.693 110 414.60 1.873 0.4518 13.968 3.3690 745.76 1 0 . /44 0.1795 39.722 1 0.544 0.1312 29.044 Mean S .D. i423. 12 1 2.012 0.4767 13.179 3.1126 654.98 1 0.844 0.1988 41.814 1 0.568 0.1335 28.191 I26.047 1 0.088 0.0280 1 1.397 0.2286 61.796 0.167 0.0330 7.2116 1 0.126 0.0238 5.7896 Group: III Treatment: 0.3 mg/kg Sex: MALES 1 ANIMAL ! FBW 1 BRAIN 1 (Gms) 1 (Gms) %FBW (Gms ) LIVER I SPLEEN 1 THYMUS 1 %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 1 ! 301 1 302 303 1 304 t 305 1 306 1 307 1 308 1 309 1 310 1 1 Mean 1 S .D . 420.90 !434.90 458.60 1403.80 395.90 1437.60 414.00 1380.00 448.60 1403 .10 I 1419.74 !24.955 2 .301 ! 2 .100 1 2.196 1 2.021 2.140 2 .286 i 2 .062 1 1 .977 1 2 .009 2.019 1 2.111 1 0.117 0.546/ 0.4829 0.4788 0.5005 0.5405 0.5224 0.4981 0.5203 0.4478 0.5009 0.5039 0.0299 15.614 3.7097 678.57 1 0.990 0.2352 43.025 1 0.704 0 .1673 30.595 16.174 3.7190 770.19 0.692 0.1591 32.952 1 0.828 0.1904 39.429 1 15.448 3.3685 703.46 1 1.254 0.2734 57.104 1 0.504 0.1099 22.951 1 14.184 3.5126 701.83 1 1.073 0.2657 53.093 1 0 .652 0.1615 32.261 1 12.639 3.1925 590.61 0 .880 0.2223 41.121 0.619 0.1564 28.925 14 .947 3.4157 653.85 1 1.018 0.2326 44.532 0.689 0.1574 30.140 [ 13.090 3.1618 634.82 1 0 .683 0.1650 33.123 0.421 0.1017 20.417 1 11.256 2.9621 569.35 0.763 0.2008 38.594 0.457 0.1203 23.116 1 15.946 3.5546 793.73 1 0.582 0.1297 28.970 1 0.577 0.1286 28.721 1 14.488 3.5941 717.58 0.781 0.1937 38.683 ! 0 .587 0.1456 29.074 1 14.379 3.4191 681.40 0.872 0.2078 41.120 j 0 .604 0.1439 28.563 1 1 .604 0.2497 71.841 ! 0 .209 0.0469 8.8488 1 0.123 0.0281 5.4395 1 r'BW - Final Body Weight - 118 - DuPont-18317 p. 118 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Final Body and Organ Weights Group: V Treatment: 1 mg/kg Sex: MALES i ANIMAL 1 FBW 1 BRAIN 1 t 1 (Gms) 1 (Gms) %FBW 1 (Gms ) LIVER %FBW %BRAIN SPLEEN I THYMUS (Gms ) %FBW %BRAIN I (Gms) %FBW %BRAIN 1 501 419.60 ! 2.036 0.4852 1 23.052 5.4938 1132.2 1 502 1395.30 I 2.090 0.5287 1 16.936 4.2843 810.33 1 503 439.20 1 2.031 0.4624 1 16.885 3.8445 831.36 [ 504 1441.30 2.108 0.4777 16.810 3.8092 797.44 [ 505 1364.70 2.086 0.5720 14.998 4.1124 718.98 50 6 1408.80 507 1369.90 1 .987 0.4861 1 15.668 3.8327 788.53 2.096 0.5666 1 14.125 3.8186 673.90 508 1406.10 2.093 0.5154 1 15.830 3.8981 756.33 509 1476.20 2 .307 0.4845 1 21.644 4.5451 938.19 510 1379.20 2.028 0.5348 1 16.321 4.3041 804.78 Mean S.D. 1410.03 135.195 2.086 0.5113 17.227 4.1943 825.21 0.087 0.0383 1 2.860 0.5238 128.55 0.725 0.762 0.907 1.004 0.673 0 .836 0.660 0 .938 1 .085 0.758 0.1728 0.1928 0.2065 0.2275 0.1845 0.2045 0.1784 0.2310 0.2278 0.1999 35.609 36.459 44.658 47.628 32.263 42.073 31.489 44.816 47.031 37.3 77 0.835 0.2026 39.940 0.144 0.0210 6.0349 0.427 0.561 0 .634 0.745 0.410 0.569 0.521 0 .691 0 .633 0.396 0.1018 0.1419 0.1444 0.1688 0.1124 0.1392 0.1408 0.1702 0.1329 0.1044 20.972 26.842 31.216 35.342 19.655 28.636 24.857 33.015 27.438 19.527 0.559 0.1357 26.750 0.121 0.0238 5.5431 Group: VII Treatment: 10 mg/kg Sex : MALES ANIMAL ! FBW BRAIN 1 (Gms) 1 (Gms) %FBW 1 (Gms ) LIVER %FBW %BRAIN 701 702 703 704 705 706 707 708 709 ;io t354.30 1393.60 1382.60 1355.70 1317.70 1409.10 1421.10 134/.60 1409.00 !378.80 ! 1.933 0.5456 f 1.826 0.4639 2.053 0.5366 1 .910 0.5370 1 .965 0.6185 2.285 0.5585 2 .029 0.4818 1.949 0.5607 2.057 0.5029 1 .983 0.5235 1 20.606 1 22.561 1 21.889 1 17.564 ! 16.749 1 25.831 1 23.567 1 19.401 1 23.440 1 23.086 5.8160 5.7320 5 .7211 4.9379 5.2720 6.3141 5.5965 5.5814 5.7311 6.0945 1066.0 1235.5 1066.2 919.58 852.37 1130.5 1161.5 995.43 1139.5 1164.2 Mean S.D. 1376.95 132.760 1 .999 0.5329 21.469 5.6797 1073.1 0.123 0.0438 1 2.864 0.3849 119.55 SPLEEN (Gms ) %FBW %BRAIN 0.776 0.760 0.750 0 .927 0.652 0.767 0.912 0.625 0.898 1 .017 0.2190 0.1931 0.1960 0.2606 0.2052 0.1875 0.2166 0.1798 0.2196 0.2685 40.145 41.621 36.532 48.534 33.181 33.567 44.948 32.068 43.656 51.286 0.808 0.2146 40.554 0.126 0.0296 6.6706 THYMUS (Gms ) %FBW %BRAIN 0.679 0. 1916 35 .127 0.547 0. 1390 29 .956 0.674 0. 1762 32 .830 0.420 0. 1181 21 .990 0.319 0. 1004 16 .234 0.569 0. 1391 24 .902 0.717 0. 1703 35 .338 0.528 0. 1519 27 .091 0.734 0. 1795 35 .683 0.626 0. 1653 31 .568 0.581 0.1531 29.072 0.134 0.0290 6.4726 FBW - Final Body Weight DuPont-18317 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Final Body and Organ Weights Group: IX Treatment: 30 mg/kg Sex: MALES ANIMAL I FBW I BRAIN I (Gms) (Gms) %FBW LIVER 1 SPLEEN (Gms ) %FBW %BRAIN ! (Gms) %FBW %BRAIN THYMUS I (Gms) %FBW sBRAIN I 901 I272.60 1 1.814 0.6654 902 294.00 1 1.946 0.6619 903 I394.10 1 2.086 0.5293 904 1284.00 1 2.135 0.7518 905 1329.20 1 1.961 0.5957 906 1303.40 1 2.043 0.6734 907 1334.00 1 1.979 0.5925 908 1303.80 1 1.908 0.6280 909 t335.00 I 2.144 0.6400 910 294.10 1 1.904 0.6474 15.544 5.7021 856.89 1 0.498 0.1827 27.453 15.920 5.4150 818.09 1 0.633 0.2153 32 .528 22.466 5.7006 1077.0 0.779 0.1977 37 .344 15.315 5.3926 717.33 1 0.673 0.2370 31 .522 18.034 5.4781 919.63 1 0.673 0.2044 34.319 18.366 6.0534 898.97 1 0.713 0.2350 34.900 23.114 6.9204 1168.0 1 0.762 0.2281 38.504 18.002 5 .9256 943.50 1 0.734 0.2416 38.470 21.838 6.5188 1018.6 1 0.691 0.2063 32.229 18.242 6.2027 958.09 1 0.586 0.1993 30.777 0.299 0.1097 16.483 0.668 0.2272 34.327 0.765 0.1941 36.673 0.191 0.0673 8.9461 0.563 0.1710 28.710 0.582 0.1918 28.488 0.487 0.1458 24.608 0.372 0.1224 19.497 0.487 0.1454 22.715 0.453 0.1540 23.792 Mean S.D. 1314 .42 [ 1.992 0.6385 135.139 1 0.108 0.0590 18.684 5.9309 937.60 1 0.674 0.2147 33.805 2.866 0.5033 129.37 1 0.085 0.0198 3.6004 0.487 0.1529 24.424 0.171 0.0463 8.2546 Group: XI Treatment: 30/0 mg/kg (Recovery) Sex : MALES ANIMAL 1 FBW BRAIN 1 (Gms) 1 (Gms) %FBW (Gms ) LIVER SPLEEN 1 THYMUS %FBW %BRAIN 1 (Gms ) %FBW %BRA IN 1 (Gms ) %FBW %BRAIN 1101 1102 1103 1104 1105 1106 1107 1108 1 1109 1 1110 1 1 Mean 1 S.D. [329.40 1343.20 344.00 !393.00 I336.60 386.50 1312.50 283.60 !28/.60 1321.50 333.79 [36.149 1 .808 0.5489 1 1 .964 0.5723 1 1 .993 0.5794 2 .056 0.5232 1 1 .999 0.5939 1 1 .914 0.4952 i 1 .782 0.5702 1 1 .641 0.5786 1 .969 0.6846 1 2 .003 0.6230 1 .913 0.5769 1 0.129 0.0521 17.005 5.1624 940.54 1 0.596 0.1809 32.965 1 0.607 0.1843 33.573 17.783 5.1815 905.45 1 0.782 0 .2279 39.817 0.619 0.1804 31.517 16.745 4.8677 840.19 ! 0.722 0.2099 36.227 1 0.739 0.2148 37.080 19.117 4.8644 929.82 1 1.076 0.2738 52.335 1 0.834 0.2122 40.564 17.024 5.0576 851.63 1 0 .665 0.1976 33.267 1 0.571 0.1696 28.564 17.552 4.5413 917.03 1 1.013 0.2621 52.926 1 0.776 0.2008 40.543 16.575 5.3040 930.13 0.841 0.2691 47.194 1 0.534 0.1709 29.966 11.859 4.1816 722.67 I 0 .624 0.2200 38.026 I 0.483 0.1703 29.433 13.520 4.7010 686.64 1 0 .548 0.1905 27.831 0 .624 0.2170 31.691 14.881 4.6286 742.94 1 0.936 0.2911 46.730 1 0,606 0.1885 30.255 16.206 4.8490 846.70 0 .780 0.2323 40.732 2 .170 0.3449 95.980 1 0 .182 0.0390 8.6409 0 .639 0.1909 33.319 0 .110 0.0190 4.5108 FBW - Final Body Weight - 120- DuPont-18317 p. 120 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 121 DuPont-18317 Appendix J Individual Animal Pathology Data - 121 - H2 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 122 DuPont-18317 INDIVIDUAL ANIMAL PATHOLOGY DATA KEY TO APPENDIX LESION GRADING: Histopathology changes are described according to their morphologic character, distribution and severity. The distribution (extent of tissue involvement) is indicated, where appropriate, by modifiers such as focal, multifocal, diffuse, unilateral, bilateral, etc. A severity score, if appropriate, is also assigned as follows: MINIMAL: The amount of change present barely exceeds that which is considered to be within normal limits. MILD: In general, the lesion is easily identified but of limited severity. The lesion probably does not produce any functional impairment. MODERATE: The lesion is prominent but there is significant potential for increased severity. Limited tissue or organ dysfunction is possible. SEVERE : The degree of change is either as complete as considered possible or great enough in intensity or extent to expect significant tissue or organ dysfunction. COMMENT: Grades minimal through severe represent progressive involvement/severity along a continuum with minimal lesions being the least severe and severe lesions being the most severe. While the grades refer to the morphologic characteristics of lesions, they also indicate their relative biologic significance. Gross observations listing multiple masses for a tissue are distinguished with letters (i.e., a, b, c , d, e t c .). - 122- 123 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: I Treatment: 0 mg/kg Sex: Males Animal Ref 101 Microscopic & Macroscopic Findings Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. minimal. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 102 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. 102 Continued on the next page .... p. 123 DuPont-18317 - 123 - p. 124 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats_______________________________________________ DuPont-18317 Individual Animal Pathology Data Dose Group: I Treatment: 0 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 102 Continued from previous page Histopathology : No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 103 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. FATTY CHANGE, MEDIAN CLEFT, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 104 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 104 Continued on the next page .... - 124- IlS Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: I Treatment: 0 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 104 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 105 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. minimal. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 106 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 106 Continued on the next page .... p. 125 DuPont-18317 - 125 - no Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: I Treatment: 0 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 106 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 107 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. BONE MARROW : FIBROSIS, FOCAL, minimal, (femur). CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM p. 126 DuPont-18317 - 126- rl 17 f Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: I Treatment: 0 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 108 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. BRAIN : PIGMENT, FOCAL, minimal, hemosiderin (cerebellum). CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 109 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. FATTY CHANGE, MEDIAN CLEFT, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. 109 Continued on the next page . p. 127 DuPont-18317 - 127 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: I Treatment: 0 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 109 Continued from previous page Histopathology : POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 110 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 128 DuPont-18317 - 128- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: III Treatment: 0.3 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 301 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, minimal, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN 302 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal, with 302 Continued on the next page .... p. 129 DuPont-18317 - 129- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: III Treatment: 0.3 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 302 Continued from previous page Histopathoiogy : No Microscopic Abnormality Observed : SPLEEN 303 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathoiogy : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal, with No Microscopic Abnormality Observed : SPLEEN 304 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 304 Continued on the next page .... p. 130 DuPont-18317 -130- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats individual Animal Pathology Data Dose Group: III Treatment: 0.3 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 304 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : SPLEEN 305 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal, with No Microscopic Abnormality Observed : SPLEEN 306 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 306 Continued on the next page .... p. 131 DuPont-18317 - 131 /3Z Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: III Treatment: 0.3 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 306 Continued from previous page Hi stopathology LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN 307 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Hi stopathology LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN 308 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 308 Continued on the next page .... p. 132 DuPont-18317 - 132- 133 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: III Treatment: 0.3 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 308 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN 309 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal, with No Microscopic Abnormality Observed : SPLEEN 310 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 310 Continued on the next page .... p. 133 DuPont-18317 - 133 - iH Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: III Treatment: 0.3 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 310 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN p. 134 DuPont-18317 -134- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: V Treatment: 1 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 501 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRON1C, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, minimal, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN 502 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, mild, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. p. 135 DuPont-18317 - 135- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: V Treatment: 1 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 503 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN 504 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal, with 504 Continued on the next page ... p. 136 DuPont-18317 - 136- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: V Treatment: 1 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 504 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN 505 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN 506 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 506 Continued on the next page p. 137 DuPont-18317 - 137- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: V Treatment: 1 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 506 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN 507 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE UOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN 508 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 508 Continued on the next page .... p. 138 DuPont-18317 - 138 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: V Treatment: 1 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 508 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal, with No Microscopic Abnormality Observed : SPLEEN 509 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN 510 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 510 Continued on the next page .... p. 139 DuPont-18317 - 139- 40 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: V Treatment: 1 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 510 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLGBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN p. 140 DuPont-18317 - 140- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animai Pathology Data Dose Group: VII Treatment: 10 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 701 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN 702 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with 702 Continued on the next page .... p. 141 DuPont-18317 - 141 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: VII Treatment: 10 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 702 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN 703 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERPLASIA, BILE DUCT, FOCAL, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN 704 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 704 Continued on the next page .... p. 142 DuPont-18317 - 142- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: VII Treatment: 10 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 704 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN 705 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : LIVER : DISCOLORATION, TAN, LEFT, LINEAR <3MM . No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN p. 143 DuPont-18317 - 143 - //f Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: VII Treatment: 10 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 706 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Hi stopathology LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN 707 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, FOCAL, minimal, coagulative. MINERALIZATION, BILE DUCT, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. 707 Continued on the next page .... moderate, with p. 144 DuPont-18317 - 144- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose G r o u p : VII Treatment: 10 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 707 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN 708 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRON1C, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN 709 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 709 Continued on the next page ... p. 145 DuPont-18317 - 145- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: VII Treatment: 10 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 709 Continued from previous page Histopathology LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN 710 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with p. 146 DuPont-18317 - 146- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 901 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. MESENTERIC LYMPH NODE : DEPLETION/ATROPHY, LYMPHOID, minimal, (inner cortex, outer cortex, and follicles) . CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 902 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. 902 Continued on the next page .... p. 147 DuPont-18317 - 147- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 902 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. moderate, with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 903 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE p. 148 DuPont-18317 - 148- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 904 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, minimal, coagulative, subcapsular. CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 905 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : LIVER : DISCOLORATION, TAN, MOTTLED. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 905 Continued on the next page p. 149 DuPont-18317 - 149- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 905 Continued from previous page Histopathology : LIVER : NECROSIS, FOCAL, minimal, coagulative, subcapsular. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 906 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE p. 150 DuPont-18317 - 150- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 907 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : LIVER : LARGE DISCOLORATION, PALE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, minimal, coagulative, subcapsular. INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 908 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. 908 Continued on the next page .... p. 151 DuPont-18317 - 151 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 908 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 909 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, FOCAL, minimal, coagulative. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE p. 1 5 2 DuPont-18317 - 152- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: IX Treatment: 30 mg/kg Sex: Males Animal Ref Microscopic & Macroscopic Findings 910 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE p. 1 5 3 DuPont-18317 - 153 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animal Ref Microscopic & Macroscopic Findings 1101 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : LIVER : DISCOLORATION, TAN, MOTTLED, LEFT. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal. BONE MARROW : FIBROSIS, FOCAL, minimal, (femur). CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1102 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1102 Continued on the next page .... p. 1 5 4 DuPont-18317 - 154- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animal Ref Microscopic & Macroscopic Findings 1102 Continued from previous page Histopathology : LIVER : MINERALIZATION, BILE DUCT, moderate, with fibrosis. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. HEMATOPOIESIS, EXTRAMEDULLARY, minimal. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 1103 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE p. 1 5 5 DuPont-18317 - 155- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animal Ref Microscopic & Macroscopic Findings 1104 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. with No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1105 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic 1105 Continued on the next page .... HEPATOCELLULAR, stippling. moderate, with p. 1 5 6 DuPont-18317 - 156- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animai Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animal Ref Microscopic & Macroscopic Findings 1105 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 1106 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION. with No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 1 5 7 DuPont-18317 - 157- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animai Ref Microscopic <& Macroscopic Findings 1107 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 1108 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : 1108 LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. Continued on the next page moderate, with p. 158 DuPont-18317 - 158- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animal Ref Microscopic & Macroscopic Findings 1108 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 1109 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. FIBROSIS, FOCAL, minimal, subcapsular. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE 1110 Terminal Sacrifice Killed on Day : 29 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1110 Continued on the next page .... p. 1 5 9 DuPont-18317 - 159- !(,D Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Animal Pathology Data Dose Group: XI Treatment: 30/0 mg/kg (Recovery) Sex: Males Animal Ref Microscopic & Macroscopic Findings 1110 Continued from previous page Hi stopathology LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, minimal, coagulative. SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NODE p. 1 6 0 DuPont-18317 - 160- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 1 6 1 DuPont-18317 Appendix K Individual Total Cell Counts - 161 - IQ- A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity S tudy in M ale R ats p. 1 6 2 DuPont 18317 INDIVIDUAL TOTAL CELL COUNTS EXPLANATORY NOTES NOTES : Organ Weight as Percent of Body Weight Organ Weight (g) Final Body Weight (g) Total Number of Organ Cells (x 103) Organ Weight (g) If Organ Weight (g) Organ Cell Suspension Volume (mL) 100 Number of Ceils in Half Organ <x 10' cells/mL) 100 A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity S tudy in M ale R ats D uP ont-18317 Individual Total Cell Counts Animal Number Final Body Weight (g> Spleen Weight (g) Mal.e, Group I - 0 mg/ kg Spleen Weight (% Body Weight) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 101 455.90 1. 143 102 441.60 0.761 103 445.00 0.976 104 427.00 0.771 105 390.80 0.806 106 404 .70 0.669 107 387.90 0.617 108 405.90 1.008 109 457.80 0.944 110 414.60 0.744 0.2507 0.1723 0.2193 0.1806 0.2062 0.1653 0.1591 0.2483 0.2062 0.1795 0.589 0 .379 0.511 0.411 0.439 0.346 0.303 0.506 0.499 0.370 7.8 7.0 7.0 7 .2 6.5 6.3 7.2 7.2 6.5 6.5 Male, Group III - 0.3 mg/kg 301 420.90 0.990 302 434.90 0.692 303 458.60 1.254 304 403.80 1.073 305 395.90 0.880 306 437.60 1.018 307 414.00 0 .683 308 380.00 0 . 763 309 448.60 0.582 310 403.10 0.781 0.2352 0.1591 0.2734 0.2657 0.2223 0.2326 0.1650 0.2008 0.1297 0.1937 0.485 0.365 0.645 0.556 0.468 0.514 0.349 0 .384 0.281 0.380 7.0 7.0 7 .8 7.4 7.0 7.0 7.0 7.0 6.8 6.5 Number of Cells in Half Spleen ( X 106 cells/mL) 71.50 27.50 53.90 58.30 22 .00 26.40 35.20 22.00 45.10 53.90 66.00 26.40 71.50 66.00 27 .50 35.20 42 .90 35.20 36.30 35.20 Total Number of Spleen Ceils (xlO9) 10 .82 3.87 7.21 7 .87 2.63 3.22 5.16 3.16 5.55 7.04 9.43 3.50 10.84 9.43 3.62 4 .88 5.88 4 .90 5.11 4.70 p. 163 - 163 - A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity S tudy in M ale R ats D uP ont-18317 Individual Total Cell Counts Animal Number Final Body Weight (g) Spleen Weight (g) Male, Group V - 1 mg/kg Spleen Weight (% Body Weight) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 501 419.60 0.725 502 395.30 0.762 503 439.20 0.907 504 441.30 1.004 505 364 .70 0 .673 506 408.80 0 .836 507 369.90 0.660 508 406.10 0.938 509 476.20 1 .085 510 379.20 0.758 0.1728 0.1928 0.2065 0.2275 0.1845 0.2045 0.1784 0.2310 0.2278 0.1999 0.366 0.364 0.459 0.545 0.366 0.416 0.301 0.497 0.521 0.375 9. 9 7.5 7.5 7 .2 7.0 6.5 7.0 7.2 7 .5 7.0 Male, Group VII - 10 mg/kg 701 702 /0 3 704 705 706 707 708 709 710 354.30 393.60 382.60 355.70 317.70 409.10 421.10 347.60 409.00 3/8.80 0.776 0.760 0.750 0.927 0 .652 0.767 0.912 0 .625 0.898 1.017 0.2190 0.1931 0.1960 0.2606 0.2052 0.1875 0.2166 0.1798 0.2196 0.2685 0.397 0.399 0.430 0.453 0.374 0.368 0.455 0.304 0.448 0.530 8 .0 7 .0 6.5 6.8 7.0 7 .8 6.9 7.0 6 .5 7 .4 Number of Cells in Half Spleen (x 106 cells/mL) 38.50 42 .90 18 .70 105.60 22 .00 30.80 52.80 34 .10 44 .00 49.50 52 .80 16.50 79.20 36.30 45.10 48.40 39 .60 50.60 33.00 52 .80 Total Number of Spleen Cells (x 108) 7 .55 6.74 2 .77 14.01 2.83 4 .02 8.10 4 .63 6 .87 7.00 8.26 2.20 8.98 5.05 5.50 7.87 5.48 7 .28 4 .30 7 .50 p. 164 - 164 - A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity Study in M ale R ats D uP ont-18317 Individual Total Cell Counts Animal Number Final Body Weight (g) Spleen Weight (g) Male, Group IX - 30 mg/kg Spleen Weight (% Body Weight) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 901 272.60 0.498 902 294.00 0.633 903 394 .10 0.779 904 284.00 0.673 905 329.20 0.673 906 303.40 0.713 907 334.00 0.762 908 303.80 0.734 909 335.00 0 .691 910 294.10 0 .586 0.1827 0.2153 0.1977 0.2370 0.2044 0.2350 0.2281 0.2416 0.2063 0.1993 0.293 0.297 0.367 0.340 0.368 0.334 0.416 0.388 0.346 0.330 10.0 10.2 7 .5 6.8 7.3 7.0 6.3 6.5 7.0 6.5 Male, Group XI - 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 329.40 343.20 344.00 393.00 336.60 386.50 312.50 283.60 287.60 321.50 0.596 0.782 0 . 722 1.076 0 .665 1.013 0.841 0.624 0.548 0 .936 0.1809 0.2279 0.2099 0.2738 0.1976 0.2621 0.2691 0.2200 0.1905 0.2911 0.317 0.392 0.357 0.529 0.353 0.552 0.436 0.360 0.248 0.470 7 .1 6.5 6 .8 6.8 7 .0 7 .0 6.5 6.5 7.2 6.5 Number of Cells in Half Spleen (x 106 cells/mL) 25.30 12.10 46.20 26.40 36.30 23.10 33.00 70.40 20.90 49.50 58 .30 2 9.70 33 .00 66 .00 36 .30 58.30 48.40 61 .60 15.40 37.40 Total Number of Spleen Cells (xlO") 4 .30 2 .63 7.35 3.55 4.85 3.45 3.81 8 .66 2 .92 5.71 7.78 3 .85 4 .54 9.13 4.79 7.49 6.07 6.94 2.45 4 .84 p. 165 - 165 - A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity Study in M ale R ats Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight) Male, Group I - 0 mg/kg Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x IQ6 cells/mL) 101 0.691 102 0.651 103 0 ,775 104 0 .690 105 0.425 106 0.499 107 0.429 108 0.435 109 0.544 110 0.544 0.1516 0.1474 0.1742 0.1616 0.1088 0.1233 0.1106 0.1072 0.1188 0.1312 0.343 0.331 0.388 0.371 0.219 0.266 0.197 0.223 0.269 0.232 7 .5 7.5 7.5 7 .0 6.5 7 .0 7 .2 7.5 7 .2 7.5 77.00 112.75 121.00 111.10 65.45 72.05 85.25 78.10 80.30 47 .30 Male, Group 111 - 0.3 mg/kg 301 0.704 302 0.828 303 0.504 304 0.652 305 0.619 306 0 .689 307 0.421 308 0.457 309 0.577 310 0 .587 0.1673 0.1904 0.1099 0.1615 0.1564 0.1574 0.1017 0.1203 0.1286 0.1456 0.367 0.388 0.266 0.330 0.293 0.324 0.225 0.192 0.238 0.292 6 .5 7.5 7.5 7 .0 7 .0 7 .0 6.8 7 .0 7 .0 7 .0 18.70 137.50 68.75 84.15 116.60 132.00 72.05 72.60 78.10 51.15 Total Number of Thymus Cells (xlO8) 11.63 16.63 18 .13 14.46 8.26 9.46 13.37 11.43 11,69 8.32 2.33 22.01 9.77 11.64 17.24 19.65 9 .17 12.10 13.25 7 .20 D uP ont-18317 p. 166 - 166 - <5^ A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity S tudy in M ale R ats Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight) M a l e , Gr oup V - 1 mg/kg Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 106 cells/mL) 501 0.427 502 0.561 503 0.634 504 0.745 505 0.410 506 0.569 507 0.521 508 0 .691 509 0.633 510 0.396 0.1018 0.1419 0.1444 0.1688 0.1124 0.1392 0.1408 0.1702 0.1329 0.1044 0.237 0.277 0.307 0.341 0.199 0.278 0.264 0.354 0.291 0.172 6.6 7.5 7.8 7.0 7.0 7.0 7.0 7 .5 7.4 7 .3 67 .10 70.40 135.85 89.10 59.40 111.65 108.90 112.75 84 .70 46.75 Male, Group VII - 10 mg/kg 701 0.679 /02 0.547 703 0 .674 704 0.420 705 0.319 706 0.569 707 0.717 708 0 .528 709 0.734 710 0.626 0.1916 0.1390 0.1762 0.1181 0.1004 0.1391 0.1703 0.1519 0.1795 0.1653 0.341 0.268 0.353 0.237 0.143 0.295 0.303 0.260 0.366 0.323 7 .7 7.2 7.5 6.8 7 .5 6.5 6.8 7.3 7 .0 7 .4 123.20 81.40 107.25 39.05 49.50 157.30 145.20 81 .95 119.35 63 .80 Total Number of Thymus Cells (xl0?) 7.98 10.69 21.88 13.63 8.57 16.00 15.04 16.51 13.63 7.86 18.89 11.96 15.36 4.71 8.28 19.72 23.36 12.15 16.75 9.15 D uP ont-18317 p. 167 - 167- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Individual Total Cell Counts Animal Numbe r Thymus Weight (g) Thymus Weight (1 Body Weight) Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 10 cells/mL) Male, Gr oup IX - 30 mg/kg 901 0.299 902 0.668 903 0.765 904 0.191 905 0.563 906 0.582 907 0.487 908 0.372 909 0.487 910 0.453 0.1097 0.2272 0.1941 0.0673 0.1710 0.1918 0.1458 0.1224 0.1454 0.1540 0.153 0.333 0.401 0.116 0.280 0.269 0.227 0.167 0.269 0.254 7 .4 7 .5 7.6 6 .8 7.0 7 .5 7.3 7 .0 7 .0 7 .0 46.20 110.50 102.85 21.45 105.05 80.30 152.90 39.05 72 .60 72.05 Total Numbe of Thymus Cells (xl 0" ) 6 .68 16.62 14 .91 2.40 14.79 13.03 23.95 6.09 9.20 8 .99 Male, Group XI - 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 0.607 0.619 0.739 0.834 0.571 0.776 0 .534 0.483 0.624 0.606 0.1843 0.1804 0.2148 0.2122 0.1696 0.2008 0.1709 0.1703 0.2170 0.1885 0.307 0.330 0 .378 0.365 0.296 0.428 0.268 0.238 0 .315 0.320 7.4 7.5 7.0 7.5 7 .3 7 .3 7.3 7.0 7 .0 7.3 127.60 112.20 148.50 177.65 121.00 169.40 130.35 66.00 92.95 63.80 18 .67 15.78 20.32 30.44 17.04 22.42 18 .96 9.38 12.89 8 .82 DuPont-18317 p. 168 - 168 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 169 DuPont-18317 Appendix L Electron Microscopy Report from Experimental Pathology Laboratories, Inc. - 169 170 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats Experimental Pathology Laboratories, Inc. DUPONT/HASKELL LABORATORY DUPONT STUDY NUMBER: 18317 WORK REQUEST NUMBER: 16160 SERVICE CODE: 1545 AMMONIUM PERFLUOROOCTANOATE: 28-DAY IMMUNOTOXICITY STUDY IN MALE RATS ELECTRON MICROSCOPY PATHOLOGY REPORT EPL PROJECT NO. 129-077 Submitted to: DuPont/Haskell Laboratory for Health and Environmental Science Stine Haskell Research Center 1090 Elkton Road Newark, DE 19711 Submitted by: Experimental Pathology Laboratories, Inc. P.O. Box 12766 Research Triangle Park, NC 27709 October 25, 2006 p. 170 DuPont-18317 - 170- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 171 DuPont-18317 Experimental Pathology Laboratories. Inc. TABLE OF CONTENTS Page PATHOLOGY SUMMARY..................................................................................................... 1 R E S U L T S ................................................................................................................................. 3 C O N C L U S IO N S ....................................................................................................................... 4 QUALITY ASSURANCE STATEMENT................................................................................ 5 ELECTROMICROGRAPHS - 171 - /72 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Experimental Pathology Laboratories, Inc._________________________ DuPont-18317 DUPONT/HASKELL LABORATORY DUPONT STUDY NUMBER: 18317 WORK REQUEST NUMBER: 16160 SERVICE CODE: 1545 EPL PROJECT NO.: 129-077 AMMONIUM PERFLUOROOCTANOATE: 28-DAY IMMUNOTOXICITY STUDY IN MALE RATS ELECTRON MICROSCOPY PATHOLOGY SUMMARY The in-life phase of this study was conducted at Haskell Laboratory for Health and Environmental Sciences, E.l. duPontde Nemours and Company, Newark, Delaware. The objective of this study is to evaluate the potential of ammonium perfluorooctanoate to suppress the primary humoral immune response to sheep red blood cells (SRBC) when administered by oral gavage to male rats for at least 28 days. The table below summarizes the experimental design: Experimental Pesian Dose Solution Daily Dosage Concentration Group Number/Group (mg/kg) ` (mg/mL)0 1 10 0 (Control) 0 III 10 0.3 0.03 V 10 1 0.1 VII 10 10 1 IX 10 30 3 XI 10 30 (Recovery)" 3 3Weight of test substance/kg of animal body weight. 6Solutions will be adjusted for purity (20%) cThe recovery group (XI) will be dosed with 30 mg/kg of test substance through test day 22. Following injection of SRBC on test day 23, group XI will be dosed with NANOpure water, at a volume of 10 mL/kg of body weight, until sacrifice. Electron microscopic evaluation of samples of liver from designated animals was added to clarify light microscopic histopathological findings in the liver. Samples of liver from two male 1 - 172 - 173 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 173 DuPont-18317 Experimental Pathology Laboratories, Inc._________________________ DuPont-18317 rats in Group I (Control) and two male rats in Group IX (30 mg/kg) that were fixed in formalin were submitted for transmission electron microscopy. The samples that were processed and evaluated are listed in the following table: TEM Number G06-399 G06-400 G06-401 G06-4Q2 Tissue Liver Liver Liver Liver Animal ID 105 106 905 906 Group I (Control) I (Control) IX (30 mg/kg) IX (30 mg/kg) TEM Negative Number (evaluated) 06-1894 to 06-1896 06-1897 to 06-1899 06-1900 to 06-1902 06-1903 to 06-1905 Samples, cut into small cubes, were preserved in formalin and shipped to Experimental Pathology Laboratories, Inc (EPL), Research Triangle Park, NC. The samples were transferred to the Laboratory for Advanced Electron and Light Optical Methods (LAELOM) at the College of Veterinary Medicine, North Carolina State University, Raleigh, NC for further processing and examination by transmission electron microscopy. The samples were washed in buffer, post-fixed in 1% osmium tetroxide in the phosphate buffer, dehydrated in an ethanolic series culminating in acetone, and infiltrated with Spurr epoxide resin. The resulting blocks were trimmed and semithin sections (approximately 0.5 pm thick) were cut, mounted on glass slides, and stained with 1% toluidine blue O in 1% sodium borate prior to being examined with a light microscope. The slides of semithin sections were sent to Experimental Pathology Laboratories for evaluation by the Pathologist, Dr. Henry Wall. When the slides were returned to the LAELOM, areas of interest for ultrathin sectioning were trimmed in the corresponding tissue blocks. Ultrathin (80-90 nm thick) sections were cut from the selected trimmed blocks and placed on 200 mesh copper grids before being stained with uranyl acetate and lead citrate. For each sample, two survey photographs (final print magnification 5,600x) were taken. One higher magnification (final print magnification 22,400x) was taken of each sample to show more cellular detail. 2 - 173 - !7f- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 174 DuPont-18317 Experimental Pathology Laboratories, Inc. RESULTS DuPont-18317 TEM #G06-399 (Animal 105, Control, Liver, TEM Neg # 06-1894 to 06-1896) Two low magnification images {06-1894 and 06-1895; 5.600X) depict portions of multiple hepatocytes. A few clear to moderately electron-lucent smooth-contoured lipid droplets are in the cytoplasm of most hepatocytes. The cytoplasm of all hepatocytes contain numerous wellformed mitochondria as the predominant cytoplasmic organelles. The higher magnification image (06-1896; 22.400X) shows greater detail of the hepatocytic mitochondria, lipid droplets, cistemae of rough endoplasmic reticulum, a few electron-dense membrane-bound peroxisomes, and electron dense clusters of cytoplasmic glycogen. No cell injury is apparent. TEM #G06-400 (Animal 106, Control, Liver, TEM Neg # 06-1897 to 06-1899) Both low magnification images (06-1897 and 06-1898; 5,600X) show multiple hepatocytes that have numerous mitochondria as their predominant cytoplasmic organelles. Aggregates of linearly arrayed rough endoplasmic reticulum are scattered in the cytoplasm of hepatocytes. The high magnification image (06-1899; 22.400X) shows greater detail of the several mitochondria, rough endoplasmic reticulum, and a few slightly electron-dense lysosomes. A few of the smaller diameter electron-dense bodies may be peroxisomes, however, their structure is not optically resolved to the extent that their identity as peroxisomes can be confirmed. Irregular profiles of translucent smooth endoplasmic reticulum are interspersed between other organelles in the cytoplasm. A portion of a well formed nucleus is at the lower right of the image. No cell injury is present. TEM #G06-401 (Animal 905, Group IX/30mg/kg, Liver, TEM Neg # 06-1900 to 06-1902) Both low magnification images (06-1900 and 06-1901; 5.600X) depict multiple hepatocytes with abundant densely arranged cytoplasmic mitochondria. Peroxisomes which appear as uniformly electron-dense bodies are prominent among the mitochondria. A few small clear smooth-contoured vacuoles are in most cells. These vacuoles are considered to be lipid vacuoles that lost their content during tissue processing. A few lysosomes are in some hepatocytes. Most lysosomes contain either lipid droplets or electron-dense residual bodies. In one image (06-1901) a cross section of a blood vessel contains electron-dense erythrocytes and shows the nucleus of an endothelial cell. The higher magnification image (06-1902; 3 - 174- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 175 DuPont-18317 Experimental Pathology Laboratories, Inc. DuPont-18317 22.400X) shows more detail of clear lipid vacuoles, numerous mitochondria, a lipid-laden lysosome and a few electron-dense peroxisomes. The peroxisomes are along the right border of the image and in the upper left quadrant of the image. TEM #G06-402 (Animal 906, Group IX/30mg/kg, Liver, TEM Neg # 06-1903 to 06-1905) The low magnification images (06-1903 and 06-1904; 5.600X) show numerous mitochondria as the predominant organelles in the cytoplasm of adjacent hepatocytes. Several uniformly electron-dense peroxisomes are scattered in the cells but are somewhat difficult to discern in the low magnification images. The high magnification image (06-1905; 22.400X) shows the detail of several peroxisomes in hepatocytic cytoplasm at the periphery of the endothelium of a blood vessel along the left border of the image. The peroxisomes are generally smaller in diameter than the more numerous mitochondria in the image. The peroxisomes are lined by a single membrane and have relatively uniform electron-dense matrix in this high magnification image. CONCLUSIONS Compared to hepatocytes from the two control rats, the hepatocytes from the two rats that received ammonium perfluorooctanoate have more abundant peroxisomes in their cytoplasm. HGW/dc HENRY G.WALL, DVM, PhD Diplomate, ACVP Veterinary Pathologist A S'tx . 'tc'b Date 4 - 175 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats p. 176 DuPont-18317 Exprimentai Pathology Laboratories, Inc. QUALITY ASSURANCE FINAL CERTIFICATION S tud y Title: Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats C lient Study: DuPont-18317; Service Code 1545; Work Request 16160 EPL Project Number 129-077 E PL Project Coordinator: Dr. Henry W all EPL Pathologist: Dr. Henry Wall The following aspects of this study were inspected by the Quality Assurance Unit of Experimental Pathology Laboratories, Inc. Dates inspections were performed and findings reported to the EPL Project Coordinator and Management are Indicated below. Dates Area Inspected EPL Project Sheets Inspection May 30,2006 Reporting May 30, 2006 Data Review June 14, 2006 June 14,2006 Draft Pathology Report June 27, 2006 June 27, 2006 Final Pathology Report October 25, 2006 October 25, 2006 Date reported to Study Director/Management: October 25, 2006 Date of last quarterly facility inspection: October 2006 ERL Quality Assurance U n it) o?S o iw 'C - Date 5 Form No. 6-2 (October 23, 2006) - 176 177