Document xngBZEY78gX6gz9Y5wboZba1

P-1 y& TRADE SECRET 2 3 7DuPont-18318 tfa - A/S ? - $Q Study Title Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice 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 of Veterinary Medicine North Carolina State University 4700 Hillsborough Street Raleigh, North Carolina 27606 U.S.A. Laboratory Project ID: D uPont-18318 W ork Request N umber: 16160 Service Code Number: 1546 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Page 1 o f 281 in Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice PAGE RESERVED P-2 DuPont-18318 !\ 0 - ' 171 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice QUALITY ASSURANCE STATEMENT Work Request Number: Study Code Number: 16160 1546 DuPont-18318 Phase Audited Audit Dates Protocol: October 17, 2005 Conduct: November 11, 2005 November 17, 2005 May 30, 2006* June 14, 2006* June 27, 2006* July 24, 2006* October 25, 2006* Report/Records: February 2, 7, 2006 August 18, 21-24, 2006 November 28-29, 2006 * EPL QA Dates Date Reported to Study Director Date Reported to Management October 17, 2005 October 17, 2005 November 11,2005 November 18, 2005 October 31,2006* October 31,2006* October 31, 2006* October 31, 2006* October 31, 2006* November 11, 2005 November 18, 2005 November 2, 2006* November 2, 2006* November 2, 2006* November 2, 2006* November 2, 2006* February 7, 2006 August 25, 2006 November 29, 2006 February 8, 2006 October 19, 2006 January 8, 2007 Reported by: Joseph C. Hamill Quality Assurance Auditor <0 ( " F e b - ' I v o ? Date m Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice P-4 DuPont-18318 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 of this study and is based on the process chemistry provided by the sponsor. GLP characterization was performed concurrently during the course of the study. A pplicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: 1 Denise Hoban, B.A, MLT (ASCP) Staff Medical Technologist and Supervisor o ifcb o la o i Date Applicant/Sponsor: DuPont Representative Date Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice P-5 DuPont-18318 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Analytical Evaluation by: _> ----------- Z. Amanda Shcn, Ph.D. Research Chemist Date Clinical Pathology Evaluation by: m Nancy E, Everds, D.V.M., Diplfihatc A.C.V.P. Principal Research Clinical Pathologist and Manager 0/~ -F*.6-zco>7 Date Anatomic Pathology Evaluation by: a Gre^P/gykcs, VM.D.. Diplomate A.C.V.P., A.C.L.A.M.. A.B.T. Veterinary Pathologist 0 ( - f f b -2j2K>1 Date Anatomic Pathology /2 Evaluation Peer Review by: --fyXXiU/'Vv Steven R. Frame. D.V.M.. Ph.D., Diplomate A.C.V.P. Research Fellow and Manager 2.00^7 Date Reviewed and Approved by: J rty V ~~/TZv Scott E. Loveless. Ph.D. Research Manager and Director O i'fs t-'io o 'i- Date Issued by Study Director: w Denise Hoban, B.A, MLT (ASCP) Staff Medical Technologist and Supervisor O lfejy J V 7 Date m Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice P-6 DuPont-18318 TABLE OF CONTENTS Page PAGE RESERVED................................................................................................................................. 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..................................... 3 QUALITY ASSURANCE STATEM ENT.........................................................................................3 CERTIFICATION.................................................................................................................................. 3 LIST OF TABLES.................................................................................................................................. 3 LIST OF FIGURES................................................................................................................................3 LIST OF APPENDICES....................................................................................................................... 3 STUDY INFORMATION..................................................................................................................... 3 SUM M ARY..............................................................................................................................................3 INTRODUCTION................................................................................................................................... 3 STUDY DESIGN..................................................................................................................................... 3 A. Design Concentrations.................................................................................................................3 B. Study O verview ............................................................................................................................3 MATERIALS AND M ETHODS......................................................................................................... 3 A. Test G uidelines.............................................................................................................................3 B. Test Substance..............................................................................................................................3 C. Test System................................................................................................................................... 3 D. Animal Husbandry....................................................................................................................... 3 E. Pretest Period................................................................................................................................3 F. Assignment to Groups..................................................................................................................3 G. Dose Formulation Preparation and Administration................................................................. 3 H. Dose Formulation Sampling and A nalysis............................................................................... 3 I. Body Weights................................................................................................................................3 J. Food Consumption and Food Efficiency...................................................................................3 K. Clinical Observations...................................................................................................................3 L. Clinical Pathology Evaluation....................................................................................................3 M. Humoral Immune Function......................................................................................................... 3 N. Anatomic Pathology Evaluation................................................................................................ 3 O. Total Cell Counts......................................................................................................................... 3 P. Electron Microscopy Evaluation................................................................................................ 3 Q. Statistical Analyses...................................................................................................................... 3 'b Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 7 DuPont-18318 TABLE OF CONTENTS Page RESULTS AND DISCUSSION............................................................................................................3 Analytical E valuation............................................................................................................................. 3 A. Chromatography........................................................................................................................... 3 B. Recovery Samples.........................................................................................................................3 C. Concentration Verification, Uniformity o f Mixing, and 5-Hour Room Temperature Stability Sam ples..........................................................................................................................3 D. Concentration Verification and Uniformity o f Mixing Samples............................................3 E. Analytical Conclusions................................................................................................................ 3 In-Life M easurem ents............................................................................................................................ 3 A. Mean Body Weights and Body Weight G ains.......................................................................... 3 B. Food Consumption and Food Efficiency...................................................................................3 C. Clinical Observations and M ortality.......................................................................................... 3 Clinical Pathology Evaluation..............................................................................................................3 A. Hematology....................................................................................................................................3 B. Clinical C hem istry........................................................................................................................3 C. Clinical Pathology Conclusions.................................................................................................. 3 Immunotoxicity........................................................................................................................................ 3 A. Humoral Immune Function.........................................................................................................3 Anatomic Pathology Evaluation...........................................................................................................3 A. Cause o f D eath.............................................................................................................................. 3 B. Final Body and Organ Weight Data........................................................................................... 3 C. Gross Observations.......................................................................................................................3 D. Microscopic Findings................................................................................................................... 3 E. Untrastructural Findings.............................................................................................................. 3 F. Anatomic Pathology Conclusions...............................................................................................3 Total Cell C ounts.....................................................................................................................................3 A. Spleen Cell N um ber..................................................................................................................... 3 B. Thymus Cell Number................................................................................................................... 3 C O N C L U S I O N S ...................................................................................................................................... 3 RECORDS AND SAMPLE STORAGE.............................................................................................3 REFERENCES......................................................................................................................................... 3 TABLES..................................................................................................................................................... 3 FIGURES................................................................................................................................................... 3 APPENDICES.......................................................................................................................................... 3 Ammonium Perfluorooctanoate: p.8 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 Dosing Vehicle...............................................................................3 Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability of APFO in Dosing Solutions..........................................................................................3 Concentration Verification and Uniformity of Mixing of APFOin Dosing Solutions................. 3 Mean Body Weights of Male Mice...................................................................................................3 Mean Body Weight Gains of Male M ice......................................................................................... 3 Mean Daily Food Consumption by Male Mice................................................................................3 Mean Daily Food Efficiency of Male M ice..................................................................................... 3 Summary of Daily Animal Health Observations in Male M ice..................................................... 3 Summary of Detailed Clinical Observations in Male M ice............................................................3 Summary of Hematology Values for Male M ice.............................................................................3 Summary of Clinical Chemistry Values for Male M ice.................................................................3 Summary of Primary Humoral Immune Response to SRBC forMale Mice Dosed with APFO................................................................................................................................................. 3 Summary of Primary Humoral Immune Response to SRBC for Male Mice Dosed With Positive Control................................................................................................................................ 3 Mean Final Body and Organ Weights from Male Mice..................................................................3 Incidence of Gross Observations in Male M ice...............................................................................3 Incidence and Lesion Grades of Microscopic Observations in Male Mice..................................3 Summary of Total Cell Counts....................................................................................................... 3 Figure 1 Figure 2 Figure 3 LIST OF FIGURES Page Representative Analytical Calibration Curve...................................................................................3 Representative LC/MS/MS Chromatograms...................................................................................3 Mean Body Weights of Male Mice...................................................................................................3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice P-9 DuPont-18318 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 Appendix M Certificate of Analysis...................................................................................................................... 3 Individual Body W eights.................................................................................................................. 3 Individual Final Body and Liver Weights....................................................................................... 3 Individual Food Consumption......................................................................................................... 3 Individual Daily Animal Health Observations................................................................................3 Individual Detailed Clinical Observationsand Mortality Records................................................ 3 Individual Animal Clinical Pathology Data.................................................................................... 3 Individual Primary Humoral Immune Response D ata................................................................... 3 Individual Primary Humoral Immune Response Positive Control D ata...................................... 3 Individual Animal Final Body and Organ W eights....................................................................... 3 Individual Animal Pathology D ata.................................................................................................. 3 Individual Total Cell Counts.............................................................................................................3 Electron Microscopy Report from Experimental Pathology Laboratories, Inc............................ 3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 10 DuPont-18318 STUDY INFORMATION Substance Tested: Ammonium Perfluorooctanoate [APFO (linear)] 3825-26-1 (CAS Number) Haskell Number: 27308 Composition: Ammonium Perfluoroctanoate Solution 19.5% in water Purity: 19.5% Physical Characteristics: White to slightly opaque liquid Stability: The test substance was stable under the conditions of the study based on analytical results. Study Initiated/Completed: October 14, 2005 / (see report cover page) Experimental Start/Termination: October 19, 2006 / February 1, 2007 -10- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 11 DuPont-18318 SUMMARY The purpose of 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 20 male mice each were administered the test substance at daily levels of 0, 0.3, 1,10, 30, and 30/0 mg/kg. The group designated 30/0 mg/kg day was included to assess potential reversibility/recovery and was therefore administered the test substance for 23 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 24 and blood samples were collected from each mouse on test day 29. The serum samples were assayed for their concentration o f SRBCspecific IgM antibody to provide a quantitative assessment o f humoral immune response. Serum from animals similarly challenged with cyclophosphamide, a positive control immunosuppressive agent, was analyzed concurrently to provide confirmation that the assay performance was acceptable for detection of immunosuppression. Clinical pathology data were collected at test day 29 and assessed 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 o f the study. Test substance-related toxicity was observed during the in-life portion of 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 o f 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 of the study. Mice dosed with >1 mg/kg had decreased serum HDL cholesterol, increased serum albumin, and variable changes in serum globulin. Mice dosed with >10 mg/kg had increased neutrophils and monocytes, decreased eosinophils, icteric serum, decreased serum total cholesterol, non-HDL cholesterol, and triglycerides, and increased serum corticosterone. Mice dosed with 30 mg/kg also had decreased red cell mass parameters (red blood cell count, hemoglobin, and hematocrit), increased reticulocytes, and decreased lymphocytes. The only parameter with complete recovery in the 30/0 mg/kg group was non-HDL cholesterol. Partial recovery was observed for icteric serum, total and HDL cholesterol, triglycerides, and serum corticosterone. 'm p. 12 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Test substance-related organ weight effects were observed in the liver, spleen, and thymus. Mean liver weight parameters were increased at >0.3 mg/kg, mean spleen weight parameters were decreased at >1 mg/kg, and mean thymus weight parameters were decreased at >10 mg/kg. Test substance-related gross observations were observed at doses >10 mg/kg and included large and discolored livers, small spleens, and small thymuses. Microscopic examination of the liver demonstrated mild hepatocellular hypertrophy at 0.3 mg/kg; moderate to severe hepatocellular hypertrophy with secondary individual cell necrosis and focal necrosis at doses >1 mg/kg; and increased hepatocellular mitotic figures, hepatocellular fatty change, and bile duct hyperplasia at doses >10 mg/kg. Microscopic examination o f lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased granulocytic hematopoiesis in the bone marrow (>10 mg/kg) and increased erythrocytic hematopoiesis in the bone marrow and spleen (30/0 mg/kg). Test substance-related lymphoid depletion/atrophy was present in the thymus (>10 mg/kg) and spleen (30 mg/kg) o f less than half o f the mice at the respective dose levels. Mesenteric and popliteal lymph nodes had no test substance-related effects. There was test substance-related evidence o f immunosuppression in mice at 10, 30 and 30/0 mg/kg. The anti-SRBC titers for these groups were reduced 20, 28 and 30% when compared to the control group mean. There was no difference in mean primary humoral immune response between the 30 and 30/0 mg/kg, indicating that the shortened dosing period did not have an impact on this endpoint. No significant changes in total thymus or spleen cell number were noted in animals dosed with 0.3 or 1 mg/kg. Significant decreases were noted in animals dosed with >10 mg/kg. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male mice was 0.3 mg/kg and for immunosuppression was 1 mg/kg. - 12 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 INTRODUCTION The primary objective of this study was to evaluate the potential of ammonium perfluorooctanoate (APFO (linear)) to suppress the primary humoral immune response to sheep red blood cells (SRBC) when administered by oral gavage to male mice for up to 28 consecutive days. Additional endpoints of toxicity were also evaluated. The oral route of administration was selected because it is a potential route of human exposure. Ammonium perfluorooctanoate (APFO; FC-143, Cg; C7F15COONH/; 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% APFO 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 the 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/ Group3 20 20 20 20 20 20 Daily Dosage (mg/kg)b 0 (Control) 0.3 1 10 30 30/0d Dose Solution Concentration (mg/mL)c 0 0.03 0.1 1 3 3 a Mice were divided into sub groups A and B (10/sub group) because of limited sample volume. b Weight of test substance/kg or animal body weight, c Solutions were adjusted for purity (19.5%). d This group (XI) was dosed with 30 mg/kg of test substance through test day 23. Following injection of SRBC on test day 24, group XI was dosed with NANOpure water, at a volume of 10 mL/kg of body weight, until sacrifice. - 13 - no p. 14 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 B. Study Overview Study Parameters Frequency Body Weight Day 0, 3 (2 for subgroup B), 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 SRBC Injection Prior to dosing (test day 24) 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 of approved use as defined by the expiration date listed on the Certificate o f Analysis (COA) that is provided in Appendix A. In addition, no evidence o f instability, such as a change in color or physical state, was observed. C. Test System On October 6, 2005, 132 male Crl:CD(ICR) mice, with an assigned birth date o f August 22, 2005, were received from Charles River Laboratories, Raleigh, North Carolina. The Crl:CD(ICR) mouse was selected based on consistently acceptable health status and on extensive experience with this strain at Haskell Laboratory. By utilizing the Crl:CD(ICR) mouse, immunotoxicity studies can be conducted in the same strain that is used for other toxicology studies. p. 15 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 D. Animal Husbandry 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 o f 18-26C and a relative humidity of 30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. Excursions outside of these ranges were of insufficient magnitude and/or duration to have adversely affected the validity o f the study. 3. Feed and Water All mice were provided tap water ad libitum. All mice 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 of the study: Water samples are analyzed for total bacterial counts, and the presence of 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 of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity o f the study. E. Pretest Period Upon arrival at Haskell Laboratory, all mice were housed in quarantine. The mice 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 Mice DuPont-18318 observed with respect to weight gain and any gross signs of disease or injury. The mice were released from quarantine by the laboratory animal veterinarian or designee on the bases o f acceptable body weights and clinical signs o f all mice. F. Assignment to Groups Mice, 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. The weight variation o f selected mice did not exceed 20% o f the mean weight. At grouping, each mouse was assigned an animal number/cage identification number. Dose groups were subdivided into groups A and B, with 10 animals per group. The animal number/cage identification number were tattooed on the tail o f each mouse and included on the cage label. At study start (test day 0) the mice were approximately 8 weeks of age. G. Dose Formulation Preparation and Administration The dosing solutions were prepared in NANOpure water. The formulations were adjusted based on the percentage of APFO in the bulk test substance to achieve the desired concentrations. Dosing formulations were prepared on a daily basis. To accommodate the schedule of the laboratory, the initiation of dosing for group A mice was started one day prior to group B mice. Animals were dosed daily at approximately the same time ( 2 hours) by intragastric intubation at a dose volume of 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 mice were dosed with NANOpure water at a volume o f 10 mL/kg of body weight. The 30/0 mg/kg group (XI) was dosed with 30 mL/kg o f test substance through test day 23. Following injection of SRBC on test day 24, group XI was dosed with NANOpure water at a volume o f 10 mL/kg of body weight until sacrifice. In light o f marked body weight losses in some of the mice, a decision was made to suspend dosing for a few days with the intention of resuming dosing if the animals were sufficiently recovered. The table below lists the specific mice for which dosing was suspended as well as the test days on which the animals were not dosed. This protocol deviation did not adversely impact the study for several reasons: first, the suspension o f dosing was transient and affected some but not all of the animals dosed at 30 mg/kg/day. Second, the data collected from animals dosed on a daily basis combined with the data from the animals listed below are considered to provide sufficient data to meet the objectives of the current study. Third, if suspension of dosing had not been implemented, unscheduled mortalities may have precluded the collection of immune system data in these animals and, thus, reduced the amount of data available to assess potential immunotoxicity. - 16- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group IX (30 mg/kg) XI (30/0 mg/kg) XI (30/0 mg/kg) XI (30/0 mg/kg) XI (30/0 mg/kg) Animal Number 901 1108 1109 1117 1120 Test Days Not Dosed 9-11 9-11 9-11 8-10 8-10 p. 17 DuPont-18318 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 o f 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. 2. Dosing Solution Treatment Each dosing sample (1 mL) was initially diluted with NANOpure water to a nominal concentration o f 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 of 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 " /V Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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 of 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 o f the control sample. Before these aliquots were brought to the final volume, an 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 of PFOA dissociated from APFO measured by LC/MS/MS was used against the 370 m/z daughter ion of 1, 2-di-l3C PFOA internal standard to determine the concentrations of 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 of each sample to the calibration curve. Concentration verification of APFO in dosing samples was evaluated by the mean result o f 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 of the test substance throughout the solution. f i 5- 18 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Stability of 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 mice were weighed on test days 0, 3 (2 for subgroup B), and daily thereafter. J. Food Consumption and Food Efficiency During the test period, the amount of food consumed by each mouse 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 Flealth Observations Cage-site examinations to detect moribund or dead mice and abnormal behavior and/or appearance among mice were conducted at least once daily throughout the study. Abnormal behavior/appearance was recorded by exception. Moribund mice were sacrificed, and a gross examination performed. Tissues and blood were not collected from moribund mice. 2. General Clinical Observations An additional cage-site evaluation was conducted approximately 2 hours after dosing to detect acute clinical signs of systemic toxicity. 3. Detailed Clinical Observations At every weighing, each mouse was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all mice. The detailed clinical observations included (but were not limited to) evaluation o f fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of 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 surviving animals 29 days after initiation of the study. Animals from each dose group were divided into 2 groups, Group A and Group B with 10 animals in each (e.g., IA, IB, IIIA, IIIB). All animals were fasted for approximately 3 hours prior to the scheduled sacrifice. The fasting schedule was staggered so that the last animal -19- p. 20 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 was fasted for approximately the same amount o f time as the first animal. While the animals were under carbon dioxide anesthesia, the maximum amount of whole blood was collected from the abdominal vena cava. Samples were allocated as indicated below: Group A Hematology: 250 pL whole blood - EDTA Clinical Chemistry: remaining blood in serum tube for: total protein, albumin, globulin (calculated) Group B Not done Clinical Chemistry: all blood in serum tube for: Cholesterol, triglycerides, high-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol (calculated) In addition, for all animals, remaining sera were allocated for either humoral immune measurements or for serum corticosterone based on the optimal use of the serum volume remaining in the tube after the above tests were performed. Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were stained with Wright-Giemsa stain, but analysis was not necessary to support experimental findings. 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. Unless otherwise indicated, any historical control clinical pathology data referenced in the text is maintained in Haskell Notebook Number E 98560-AN. 1. Hematology (Group A Only) Complete blood counts, including reticulocytes, were determined on a Bayer Advia 120 hematology analyzer or determined from microscopic evaluation of the blood smear. WrightGiemsa-stained blood smears from all animals were examined microscopically for confirmation of automated results and evaluation of cellular morphology. Blood smears, stained with new methylene blue, were prepared from each animal undergoing a hematology evaluation, but were not needed for examination. The following parameters were determined: red blood cell count hemoglobin hematocrit mean corpuscular (cell) volume mean corpuscular (cell) hemoglobin mean corpuscular (cell) hemoglobin concentration red cell distribution width absolute reticulocyte count platelet count white blood cell count differential white blood cell count microscopic blood smear examination 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 \<\1- 20- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 (aspirating aqueous contents of 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 (group B) triglycerides (group B) total protein (group A) albumin (group A) globulin (calculated, group A) high-density lipoprotein cholesterol (group B) non-high-density lipoprotein cholesterol (calculated, group B) serum corticosterone (groups A and B) M. Humoral Immune Function On test day 24, animals were injected intravenously in the lateral tail vein with 0.2 mL of 1 x 109 SRBC/mL (Covance, Denver, Pennsylvania, U.S.A.). One mouse (716 in the 10 mg/kg test substance group) was inadvertently not injected with the appropriate amount of SRBC and the immune response for this mouse could not be evaluated. On test day 29, serum was collected from each mouse and frozen (see L.2.Clinical Chemistry). Serum was not collected from 6 mice (117 in the 0 mg/kg test substance group, 306 in the 0.3 mg/kg test substance group, 903, 904, and 906 in the 30 mg/kg test substance group, and 1112 in the 30 mg/kg (recovery) test substance group) due to sacrifice in extremis prior to test day 29 (117, 906 and 1112), insufficient serum sample volume (306 and 904), or no serum sample taken (903); therefore, the immune response for these mice could not be evaluated. Serum volume was insufficient for 3 mice (703 in the 10 mg/kg test substance group, 907 in the 30 mg/kg test substance group, and 1116 in the 30 mg/kg (recovery) test substance group) and the immune response for these mice could not be evaluated. 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 of the data was created and the linear portion of 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 of the serum dilution that had an OD value o f 0.5. If no points had an OD value o f 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 5 days with 90 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 and/or female cyclophosphamide monohydrate or vehicle serum samples were concurrently run. The pooled samples consisted of equal aliquots of serum taken from either the male or female rats dosed with cyclophosphamide monohydrate or vehicle. m Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 N. Anatomie Pathology Evaluation After 29 days on study, the surviving mice 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 sacrifice for scheduled deaths was stratified across groups. Mice were fasted for 3 hours before euthanasia. All mice, including 3 mice (mice 117, 906, and 1112) that were sacrificed in extremis during the study (test days 5, 9, and 5, respectively), were euthanized by carbon dioxide anesthesia and exsanguination. Gross examinations were performed for all mice. Final body weights and organ weights were recorded for all mice sacrificed by design on test day 29. The following tissues were collected from 120 mice (20/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 from 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). For mice sacrificed by design (SD) on test day 29, all tissues collected from control (0 mg/kg) and high-dose (30 and 30/0 mg/kg) mice were processed to slides and examined microscopically. In addition, the following organs were examined from all SD mice in order to determine a no observed-effect level for test substance-related microscopic findings: liver, thymus, spleen, and bone marrow. For the 3 mice sacrificed in-extremis (SE) before the scheduled sacrifice, all tissues were processed to slides and examined microscopically. Gross observations (recorded at necropsy) were examined microscopically for all animals. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 O. Total Cell Counts The following procedures were used for preparation of spleen and thymus single-cell suspensions for enumeration of total cell counts from each spleen or thymus: The weight o f the halved spleen or thymus (tissue) was documented, the half was placed in a labeled 15 mL centrifuge tube containing 3 mL Hank's Balanced Salt Solution (HBSS/H) and put on ice. The halved tissue/HBSS/H was poured into a small petri dish and cut into small pieces. 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 o f 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 mice (103 and 104) and 2 mice in the 1 mg/kg group (503 and 504) mice was placed in cassettes in a container of formalin, and shipped to Experimental Pathology Laboratories, Inc (EPL) for evaluation 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 o f the electron microscopic evaluation. More details are provided in Appendix M. Q. Statistical Analyses For all statistical analyses, significance was judged at p < 0.05. Comparisons were made of the dosed groups to the control group (Group I). Comparisons were also made between Group IX and Group XI. -23 - p. 24 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Parameter Body Weight Body Weight Gain Food Consumption Food Efficiency Humoral Immune Function Data" Clinical Pathology Organ Weights Total Cell Counts Preliminary Test Levene's test for homogeneity<8) and Shapiro-Wilk test<9) for normalityb Method of Statistical Analysis If preliminary test is not If preliminary test is significant significant One-way analysis of variance*l0) followed by Dunnett's test(MI2J3> Kruskal-Wallis test(l4) followed by Dunn's test(l5) a SRBC-specific serum IgM antibody titer data were transformed to Log2 to 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. -24- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 25 DuPont-18318 RESULTS AND DISCUSSION Analytical Evaluation A. Chromatography (Figures 1-2) PFOA dissociated from APFO and 1, 2-di-13C PFOA eluted together from the F1PLC column with a retention time of 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 of variation of 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% of nominal (mean percent recovery = 105.0% 5%, C.V. = 5%). The range o f the measured concentrations of 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 of the measured concentrations o f APFO for the 1 mg/mL level was 102.0% to 105.0% of 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 o f the dosing samples in the study. C. Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability Samples (Table 2) Dosing solutions prepared on October 17, 2005 were analyzed for concentration verification, uniformity o f mixing, and 5-hour room temperature stability, and results are shown in Table 2. The following table summarizes the results for concentration verification, uniformity of mixing, and 5-hour room temperature stability analyses. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Preparation Nominal Measured3 Average C.V. Stability5 Date mg/mL mg/mL % Nominal (%) % Nominal 17-October-O 5 0 NDC ------ 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) Dosing solutions prepared on November 15, 2005 were analyzed for concentration verification and uniformity o f mixing, and results are shown in Table 3. The following table summarizes the results for concentration verification and uniformity of mixing analyses. Preparation Date 15-November-0 5 Nominal mg/mL 0 0.03 0.1 1 3 Measured3 mg/mL NDb 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% of 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 analyses of the samples collected during the study indicate that the dosing formulations were at the targeted concentrations, mixed uniformly, and stable under the conditions of the study. Test substance was not found in the 0 mg/mL samples -26 703 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 In-Life Measurements A. Mean Body Weights and Body Weight Cains (Tables 4-5, Figure 3, Appendices B-C) Test substance-related and 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 14, 22, and 12% lower than the control group at 10, 30, and 30/0 mg/kg, respectively. Test substance-related increases in liver weights occurred and minimized the magnitude o f the effects on body weight. In an attempt to quantitatively separate the increased liver weights (see Appendix C) from the decreased body weights, an adjusted body weight (see Appendix C) was calculated for each animal by subtracting the weight of the liver from the final body weight. The means for the adjusted body weights were 28, 35, and 23% lower than controls at 10, 30, and 30/0 mg/kg, respectively. The reductions in mean body weight at 10 mg/kg and above resulted from overall body weight losses during the study; mice lost an average o f 3.8, 6.6, and 3.3 g during days 0 to 28 at 10, 30, and 30/0 mg/kg, respectively, whereas control group mice gained an average of 0.9 g. In general, the test substance-related effects on body weight parameters were dose-related relative to the magnitude o f the change and the onset of the reductions; effects on mice dosed at 30 mg/kg were more pronounced and evident sooner than effects at 10 mg/kg. The effects o f cessation o f dosing were evident in that the mean final body weights of mice at 30/0 mg/kg were 12% higher than those from the mice dosed at 30 mg/kg. Body weight and weight gain data for animals dosed at 0.3 and 1 mg/kg were generally comparable to controls. B. Food Consumption and Food Efficiency (Tables 6-7, Appendix D) Test substance-related effects on food consumption were evident at 10, 30, and 30/0 mg/kg. The food consumption data did not adhere to the hypothesis of a monotonic dose response. At 10 mg/kg, the mean food consumption was increased or significantly increased starting at the end o f the first week and persisting until the end o f the study. At 30 and 30/0 mg/kg, mean food consumed was usually lower or significantly lower than controls but for each group there was one interval with significantly increased food consumption. As a result of these somewhat erratic patterns of food consumption, overall food consumption during days 0 to 28 was generally comparable across the groups with the exception of the 10 mg/kg group for which mean food consumption was significantly increased. -27- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 C. Clinical Observations and Mortality (Tables 8-9, Appendices E-F) Test substance-related clinical signs observed at 1, 10, 30 and 30/0 mg/kg included: pallor, wet and stained fur, swollen penis, eye observations, prostrate, or yellow extremities. Swollen left shoulder observed in one mouse dosed at 10 mg/kg was possibly due to a dosing incident. Abnormal gait was observed in single animals dosed at 1 or 10 mg/kg. This observation was not considered test substance related because it was only observed in single animals and short in duration. Three mice were sacrificed in extremis prior to test day 29. Two mice were sacrificed on test day 5 due to dosing incidents from the control and 30/0 mg/kg groups. One mouse dosed at 30 mg/kg was sacrificed on test day 9 possibly due to a dosing incident, however, gross and microscopic pathology were unable to determine the exact cause of death Clinical Pathology Evaluation A. Hematology (Table 10, Appendix G) 1. Red Blood Cells Red cell mass parameters (red blood cell count, hemoglobin, and hematocrit) were minimally decreased in mice dosed with 30 mg/kg for 29 days. Mean values were 88-94% o f the control group means (not statistically significant). In addition, mean cell hemoglobin and mean cell hemoglobin concentration were decreased in mice dosed with 10 or 30 mg/kg for 29 days. Means at 10 and 30 mg/kg were 94-96% and 95-97% of the respective control group means for these 2 parameters (variable statistical significance). Some mice dosed with 1 or 10 mg/kg for 29 days had minimally lower reticulocyte counts compared to those dosed with 0 or 0.3 mg/kg. Mice dosed with 30 mg/kg either had higher (3/7) or lower (4/7) reticulocyte counts compared to control mice. Two of the 3 mice at 30 mg/kg with higher reticulocyte counts also had increased splenic extramedullary hematopoiesis. On an individual animal basis, there was no correlation between red cell mass and reticulocyte counts in this group. Effects on red cell mass were more pronounced (mildly decreased) in mice dosed with 30/0 mg/kg compared to those dosed with 30 mg/kg for the entire 29 days. At recovery, mean red blood cell count, hemoglobin, and hematocrit ranged from 82-86% of the respective control group means for these 3 parameters (all statistically significant). Decreased red cell mass parameters following recovery 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 -28- 2oC Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 decreased red cell mass parameters was not evident from in-life, clinical pathology, or anatomic pathology data. Therefore, the cause of the decreased red cell mass was not determined. Reticulocytes were mildly increased in mice dosed with 30/0 mg/kg. Mean reticulocyte count was 148% of the control group mean (not statistically significant). Consistent with the increase in reticulocyte counts, red cell distribution width was increased (mean was 109% o f the control group mean; not statistically significant), and mean cell hemoglobin concentration was decreased (97% of control group mean; statistically significant). Microscopically, this group had increased polychromasia (increased bluish staining o f red blood cells), a characteristic finding in blood with increased reticulocyte counts. The increases in reticulocytes and related parameters were considered to be in response to the decreased red cell mass described above. These changes also correlated with histologic evidence of increased splenic extramedullary hematopoiesis, which was observed in 15 o f 19 mice in the 30/0 mg/kg group. 2. White Blood Cells Neutrophils were increased in mice dosed with 10 or 30 mg/kg. Means were 236 and 296% of the control group mean, respectively (statistically significant). While all neutrophil counts for mice dosed with <1 mg/kg were between 0.00 and 2.00x103/pL, mice dosed with 10 mg/kg had neutrophil counts between 1.00 and 3.00x103/pL, while 2 o f 7 mice dosed with 30 mg/kg had neutrophil counts o f greater than 3.50xl03/pL (animal 901 and 904). Histologically, 2 o f 3 mice (mice 902 and 904) dosed with 30 mg/kg had minimal granulocytic hyperplasia of the bone marrow, corresponding to the observation o f increased neutrophils in the peripheral blood o f this group. The two 30 mg/kg mice with the highest neutrophil counts (901 and 904) also had higher lymphocyte and monocyte counts than other mice in this group, although the lymphocyte counts were similar to or lower than control values. Increased neutrophils may be due to stress (glucocorticoid-related; see corticosterone discussion below and anatomic pathology) or inflammation. The changes in neutrophils, in light of changes in other leukocyte types, are likely related to both o f these processes. Lymphocytes were generally decreased in mice dosed with 30 mg/kg (mean was 59% of the control group mean; not statistically significant). One mouse (animal 901) had increased neutrophil and unchanged lymphocyte counts compared to control mice. The decreases in lymphocyte counts in most mice dosed with 30 mg/kg were consistent with stress (see corticosterone discussion below and histology), and corresponded to increased serum corticosterone and lymphoid depletion/atrophy in the spleen, thymus, and lymph nodes in 30 mg/kg mice. The unchanged lymphocyte count in 1 mouse dosed with 30 mg/kg may have been due to a combination o f stress and inflammation. Monocytes were increased in mice dosed with 10 or 30 mg/kg. Means were 285 and 254% of the control group mean, respectively (variable statistical significance). In these 2 groups of mice, individual mice with increased monocytes tended to have increased neutrophil counts. Increased monocytes are observed with inflammation or stress. As discussed above, the combination of changes likely reflect both stress and inflammation. Eosinophils were decreased in mice dosed with 10 or 30 mg/kg. Means were 57 and 64% o f the control group mean, respectively (not statistically significant). A decrease in peripheral blood p. 30 Ammonium Pcrfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 eosinophils is consistently observed in response to stress. Therefore, this change was considered to be due to stress. Large unstained cells (LUC) were increased in mice dosed with 10 or 30 mg/kg. 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 of the total leukocyte population. The LUC count normally includes mostly lymphocytes and monocytes. In this study, the mice with the highest LUC counts usually had the highest lymphocyte and/or monocyte counts. Therefore, in this study, changes in LUC counts paralleled changes in lymphocytes and/or monocytes. In the 30/0 mg/kg group, neutrophil, lymphocyte, eosinophil, monocyte, and LUC counts were generally similar to those o f mice dosed at 30 mg/kg for the full 29 days (variable statistical significance compared to controls; lymphocyte counts statistically different from that of mice dosed with 30 mg/kg for 29 days). Therefore, there was little or no recovery for changes in differential white blood cell counts. The following statistically significant changes in mean hematology parameters were considered to be unrelated to treatment and non-adverse because they did not occur in a dose-related pattern: Decreased mean cell hemoglobin concentration in mice dosed with 10 mg/kg for 29 days Decreased red cell distribution width in mice dosed with 0.3 or 10 mg/kg for 29 days Increased total white blood cell count in mice dosed with 10 mg/kg for 29 days B. Clinical Chemistry (Table 11, Appendix G) Icterus was evident in serum of mice dosed with 10 or 30 mg/kg for 29 days. The incidences of icteric serum were 0/19, 0/19, 0/20, 16/20, and 17/17 in mice dosed with 0, 0.3, 1, 10, or 30 mg/kg for 29 days. Icterus is graded as none, trace, small, moderate, or large and in this study, grades o f trace, small and moderate were observed. The icterus grades were generally higher at 30 than at 10 mg/kg. Icterus is an indication of increased serum bilirubin and may result from either increased production of bilirubin from hemoglobin as a result o f increased red blood cell destruction or decreased processing and excretion o f bilirubin. There was no clinical or anatomic pathology evidence o f increased red cell destruction in mice dosed for 29 days. Histologically, minimal to mild hyperplasia o f the bile ducts were observed in mice dosed with 10 or 30 mg/kg, which, along with other hepatic changes, may have contributed to the presence o f icterus. In mice dosed in the 30/0 mg/kg group, the incidence and grades of icterus were lower than after 29 days o f treatment (trace to small icterus observed in 16/19 mice). Therefore, there was partial recovery from the finding of icteric serum. Total cholesterol was moderately decreased in mice dosed with 10 or 30 mg/kg for 29 days. Means were 69 and 51% o f the control group means (statistically significant). The decrease in cholesterol was due to decreases in HDL cholesterol at doses o f 1, 10 and 30 mg/kg (means were 71,61, and 44% of the control group mean, respectively, and were statistically significant), and - 30- p. 31 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 in non-HDL cholesterol at doses o f 10 and 30 mg/kg (means were 85 and 65% o f the control group mean, respectively; not statistically significant). Mean total and HDL cholesterol in the 30/0 mg/kg group were 80% and 69% of the control group mean (variable statistical significance compared to control but statistically different from mice dosed with 30 mg/kg for 29 days). Therefore, there was partial recovery of total and HDL cholesterol, and complete recovery for non-HDL cholesterol in mice dosed with 30/0 mg/kg. Triglycerides were moderately decreased in mice dosed with 10 or 30 mg/kg for 29 days. Means were 47 and 32% of the control group mean, respectively (statistically significant). There was partial recovery o f triglycerides, as the mean value in mice in the 30/0 mg/kg group was 57% of the control group mean (statistically different from control mice and mice dosed with 30 mg/kg for 29 days). Albumin was moderately increased in mice dosed with 1, 10, or 30 mg/kg for 29 days (means were 110, 145, and 131% o f the control group mean, respectively; variable statistical significance). Although albumin data were limited to 3 values at 30 mg/kg for 29 days, albumin concentrations for these 3 mice were greater than those of mice dosed with 0.3 or 1 mg/kg, suggesting a treatment-related increase. Increased albumin may result from dehydration (relative increase) or increased synthesis or decreased catabolism (absolute increase). There were no in life or clinical pathology data to support dehydration; however, urine concentration, an important sign o f dehydration, was not evaluated in this study. Some peroxisome proliferator-activated receptor (PPAR) agonists have been reported to cause increases in serum albumin concentration due to increased synthesis of albumin. Due to the lack of corroborative data, the cause of increased albumin in this study cannot be determined. In the 30/0 mg/kg group, albumin concentrations were similar to those o f mice dosed for 10 or 30 mg/kg for 29 days and were increased compared to those of control mice. Therefore, there was no recovery in albumin concentrations. Globulin concentrations were increased in the 30/0 mg/kg group compared to those of control mice and mice dosed with 30 mg/kg for 29 days (both statistically significant; mean was 119% of the control mean), suggesting a test substance-related effect. Total protein measurements include albumin and globulin, so changes in total protein are a function o f changes in these 2 components. Total protein concentration was increased at 10 mg/kg (due to increases in albumin) and similar to control values at 30 mg/kg (due to increases in albumin and decreases in globulin). Means for these 2 groups were 125 and 109% of the control group mean, respectively (variable statistical significance). In the 30/0 mg/kg group, total protein was 134% o f the control group mean (due to increases in both albumin and globulin). Serum corticosterone was moderately increased in several mice dosed with 10 or 30 mg/kg for 29 days (variable statistical significance). While serum corticosterone concentrations were between 0 and 400 ng/mL in all mice dosed with <1 mg/kg, concentrations greater than 400 ng/mL were observed in 7/10 and 6/10 mice dosed with 10 or 30 mg/kg, respectively, resulting in mean concentrations that were 229 and 231 % o f the control group mean for mice for these 2 groups. In the 30/0 mg/kg group, mouse serum corticosterone concentrations were still mildly increased (6/10 were greater than 400 ng/mL, and the mean was 137% o f the control -31 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 group mean; not statistically significant). These changes are consistent with partial recovery from stress. C. Clinical Pathology Conclusions Mice dosed with >1 mg/kg had decreased serum HDL cholesterol, increased serum albumin, and variable changes in serum globulin. Mice dosed with >10 mg/kg had increased neutrophils and monocytes, decreased eosinophils, icteric serum, decreased serum total cholesterol, non-HDL cholesterol, and triglycerides, and increased serum corticosterone. Mice dosed with 30 mg/kg also had decreased red cell mass parameters (red blood cell count, hemoglobin, and hematocrit), increased reticulocytes, and decreased lymphocytes. The only parameter with complete recovery in the 30/0 mg/kg group was non-HDL cholesterol. Partial recovery was observed for icteric serum, total and HDL cholesterol, triglycerides, and serum corticosterone. Immunotoxicity A. Humoral Immune Function (Tables 12-13, Appendices H-l) There was test substance-related evidence o f immunosuppression in mice at 10, 30 and 30/0 mg/kg. The anti-SRBC titers for these groups were reduced 20, 28 and 30% when compared to the control group mean. There was no difference in mean primary humoral immune response between the 30 and 30/0 mg/kg, indicating that the shortened dosing period did not have an impact on this endpoint. In contrast, mice injected for 5 days with 90 mg/kg of the immunosuppressive material, cyclophosphamide, demonstrated a 52% inhibition o f the IgM antibody response to SRBC. Anatomic Pathology Evaluation A. Cause of Death There were no test substance-related deaths. Only 3 of the 120 mice on study did not survive until the scheduled sacrifice on day 29. The 3 mice were sacrificed in extremis on test days 5 and 9. Mouse 117 (control) and mouse 1112 (30/0 mg/kg) were sacrificed on test day 5 due to dosing incidents. Both mice had ruptured esophagi identified on gross examination. Mouse 906 (30 mg/kg) was sacrificed on test day 9 because the mouse was clinically lethargic and pale. Gross and microscopic pathology did not reveal a cause of death. It is likely that this death was also due to a dosing incident. However, since the esophagus, lungs, and mediastinal tissue were not saved for microscopic examination, the cause of death was undetermined. -32- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 B. Final Body and Organ Weight Data (Table 14, Appendix J) Following 28 days of daily gavage administration of the test substance, test substance-related organ weight effects were observed in the liver, spleen, and thymus. Relative to controls, mean liver weight parameters were increased at >0.3 mg/kg, mean spleen weight parameters were decreased >1 mg/kg, and mean thymus weight parameters were decreased at >10 mg/kg. Text Table 1: Mean Absolute and Relative (% body weight) Organ Weights in Male Mice Group: Dose (mg/kg): Number of Mice/Sex: I 0 19 Final Body Wt. (g) 33.0 Liver absolute wt. (g) % body wt. (17) 1.782 5.421 Spleen absolute wt. (g) % body wt. (19) 0.117 0.355 Thymus absolute wt. (g) % body wt. (19) 0.050 0.153 III 0.3 20 33.4 (20) 2.407 7.196 (20) 0.116 0.346 (20) 0.045 0.137 V 1 20 33.8 (20) 3.272** 9.704** (20) 0.104 0.307* (20) 0.049 0.144 VII 10 20 28.4* (20) 6.061** 21.232** (20) 0.066** 0.232* (20) 0.025** 0.087** IX 30 19 26.0* (18) 5.899** 22.618** (19) 0.052** 0.195* (19) 0.025** 0.094** XI 30/0 19 30.5*A (18) 6.391** 21.209** (19) 0.076** 0.249* (19) 0.027** 0.088** 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 14%, 21%, and 8% 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. There was also a statistically significant 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 demonstrates 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. - 33- =2 /0 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 2. Liver Mean absolute liver weights were increased 35%, 84%, 240%, 231%, and 259% 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 (33%, 79%, 292%, 317%, and 291%, respectively). All increases were statistically significant, except for those in the 0.3 mg/kg dose group. The increased liver weights, at all dose levels, correlated with the microscopic finding o f mild to severe hepatocellular hypertrophy. It also correlated with the gross observation o f large livers at doses >10 mg/kg. 3. Spleen Mean absolute spleen weights were decreased 11%, 44%, 56%, and 35% in the 1,10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value. Mean relative (% body weight) spleen weights were similarly decreased (14%, 35%, 45%, and 30%, respectively). All decreases were statistically significant, except for the increase in the mean absolute spleen weight in the 1 mg/kg dose group. Mean spleen weight parameters in the 0.3 mg/kg dose group were similar to the control values. The decreased spleen weights, at >1 mg/kg, correlated with the microscopic finding of minimal to mild lymphoid depletion/atrophy in this organ. It also correlated with the gross observation of small spleens at doses >10 mg/kg. 4. Thymus Mean absolute thymus weights were decreased 50%, 50%, and 46% in the 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value. Mean relative (% body weight) thymus weights were similarly decreased (43%, 39%, and 42%, respectively). All decreases were statistically significant. Mean thymus weight parameters in the 0.3 and 1 mg/kg dose groups were similar to the control values. The decreased thymus weights, at >10 mg/kg, correlated with the microscopic finding of minimal to severe lymphoid depletion/atrophy in this organ. It also correlated with the gross observation of small thymuses at doses >10 mg/kg. 5. Other All other individual and mean organ weight differences were considered to be spurious or secondary to the decrease in final body weights. Mean absolute brain weights were decreased, while mean relative brain weights (% body weight) were increased only at doses (>10 mg/kg) that produced significantly decreased body weights. The lack of any gross or microscopic effects on the brain also suggests that the brain weight differences were a function of body weight and not indicative of a test substance-relate brain weight effect. -34- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 C. Gross Observations (Table 15 Appendix K) At the terminal sacrifice, test substance-relate gross observations were observed at doses >10 mg/kg and included large and discolored livers, small spleens, and small thymuses. Text Table 2: Incidences of Test Substance-Related Gross Observations in Male Mice Group: Dose (mg/kg): Number of Mice/Group: I 0 19** III V VII 0.3 1 10 20 20 20 IX 30 19** XI 30/0* 9** Liver Large Discoloration Spleen Small Thymus Small i 0 0 11 16 0 00 1 5 0 00 8 8 0 003 2 12 1 2 2 Underlined values were interpreted to be test-substance related increases, as compared to control values. * Not dosed with test substance following immunology challenge. ** Table excludes 3 mice that were euthanized on days 5 (mice #s 117 and 1112) and 9 (mouse #906). The observation o f large livers in mice given >10 mg/kg correlated with the microscopic finding o f severe hepatocellular hypertrophy in all mice at these dosages and greater than 200% increases in mean absolute liver weights, as compared to controls. Gross liver discoloration is also consistent with microscopic hepatocellular hypertrophy. Small spleens, which were observed in almost half o f the spleens from mice given 10 (8/20) and 30 (8/19) mg/kg of the test substance, correlated with decreased mean spleen weights at these same dose levels. Fewer small spleens (2/19) were observed in the recovery group (30/0 mg/kg), which was consistent with the partial recovery o f spleen weights in this dose group. Test substance-related microscopic lymphoid depletion was observed only at 30 mg/kg. Small thymuses were recorded in only a few thymuses at dose levels >10 mg/kg. These correlated with decreased mean thymus weights and microscopic lymphoid depletion at the same dose levels. D. Microscopic Findings (Table 16, Appendix K) Microscopic examination of the liver demonstrated mild hepatocellular hypertrophy at 0.3 mg/kg; moderate to severe hepatocellular hypertrophy with secondary individual cell necrosis and focal necrosis at doses >1 mg/kg; and increased hepatocellular mitotic figures, hepatocellular fatty change, and bile duct hyperplasia at doses >10 mg/kg -35- Z/2 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Microscopic examination o f lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased granulocytic hematopoiesis in the bone marrow (>10 mg/kg) and increased erythrocytic hematopoiesis in the bone marrow and spleen (30/0 mg/kg). Test substance-related lymphoid depletion/atrophy was present in the thymus (>10 mg/kg) and spleen (30 mg/kg) of less than half o f the mice at the respective dose levels. Mesenteric and popliteal lymph nodes had no test substance-related effects. Text Table 3: Incidences of Test Substance-Related Microscopic Findings in Male Mice Group: I III V VII IX XI Dose (mg/kg): 0 0.3 1 10 30 30/0* Number of Mice/Group: 20 20 20 j9** 19** Liver Hypertrophy, hepatocellular Necrosis, individual cell Necrosis, focal Mitotic figures, increased Hyperplasia, bile duct Fatty change, nonzonal Thymus Depl./Atr. Lymph. orNP. Spleen Depletion/Atrophy, lymphoid EMH, increased Bone Marrow Hyperplasia, granulocytic Hyperplasia, erythrocytic (19) 0 0 0 0 0 0 (19) 0 (19) 0 6(1.2) (19) 0 0 (20) 20 [2.0] 0 1 [1.0] 0 0 0 (20) 0 (20) 1 [1.0] 4 [1.5] 20} 0 0 (20) 20 [3.0] i i [1-1] 3 [1.0] 0 0 0 (19) 0 Offi 0 1 [1.0] 20} 0 0 (20) 20 [4.0] 20 [1.9] 4 [1.8] 10 [1.0] 6 [1.0] 9 [1.0] (19) 19 [4.0] 19 [2.0] 7 [1.9] 15 [1.0] 17 [1.2] 14 [1.0] 19} 8 [1.6] (17) 12 [2.9] (20) 0 3 [1.7] (19) 8 [1.1] 5 [1.2] (20) 3 [1.7] 0 (19) 4 [1.0] 0 (19) 19 [4.0] 19 [1.7] 3 [1.7] 19 [1.4] 12 [1.0] 4 [1.0] (19) 6 [2.8] (19) 2 [l.l] 15 [1.8] (19) 1 [1.7] 3 [2.0] [ ] = 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 weighed; 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. ** Table excludes 3 mice that were euthanized on days 5 (mice #s 117 and 1112) and 9 (mouse #906). . Includes lymphoid depletion/atrophy and thymus not present in mediastinal tissue (assumed grade 3 atrophy). 1. Liver a. Hepatocellular Hypertrophy Panlobular hepatocellular hypertrophy was observed in all surviving mice given the test substance and the severity was dose related. Hypertrophy was present in 0/19, 20/20, 20/20, 20/20, 19/19, and 19/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. The hypertrophy was graded as mild in all mice given 0.3 mg/kg, moderate in all mice given 1 mg/kg; and severe in all mice given >10 mg/kg. -36- 1 (3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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. At doses >1 mg/kg, the severity (moderate to severe) of the hepatocellular hypertrophy appeared to be responsible for increased individual cell necrosis and focal necrosis. At doses >10 mg/kg, the severity (severe) o f the hepatocellular hypertrophy was associated with nonzonal fatty change and hepatocellular regeneration (increased mitotic figures). Hepatocellular hypertrophy correlated with increased mean liver weight parameters at all doses and grossly large livers at doses >10 mg/kg. b. Individual Cell Necrosis Individual cell necrosis was observed in mice given >1 mg/kg o f the test substance; the incidence and severity were dose related. Individual cell necrosis was present in 0/19, 0/20, 11/20, 20/20, 19/19, and 19/19 in mice given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively, and was graded minimal to mild. A slight decrease in severity was apparent in the 30/0 mg/kg group as compared to the 30 mg/kg group. The individual cell necrosis was primarily due to the degeneration, necrosis, and lysis of enlarged hepatocytes in an individualized, non-zonal pattern. Although apoptotic cells were observed in most sections, the increased individual cell necrosis was usually not due to apoptosis. A slight secondary focal inflammatory cell inflitrate was often observed in association with necrotic hepatocytes. c. Focal Necrosis Test substance-related focal necrosis was also observed in mice given >1 mg/kg of the test substance. The incidence and severity were both mildly dose related. Focal necrosis was present in 0/19, 1/20, 3/20, 4/20, 7/19, and 3/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively, and was graded minimal to moderate. The single incidence of minimal focal necrosis in a 0.3 mg/kg mouse was considered incidental since this is sometimes a naturally occurring background lesion. A slight decrease in the incidence o f focal necrosis 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 of hepatocyte clusters is a common secondary effect o f hepatocellular hypertrophy and is most likely the result o f secondary focal ischem ia/l6) d. Increased Mitotic Figures Increased mitotic figures were observed in mice given >10 mg/kg o f the test substance; the incidence was dose related. Increased mitotic figures were present in 0/19, 0/20, 0/20, 10/20, 15/19, and 19/19 mice given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively, and was graded minimal to mild. A slight increase in the incidence and severity was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group. 2/ p. 38 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Increased mitotic figures were an apparent indication o f increased cell turnover in those mice with severe hepatocellular hypertrophy and subsequent individual cell necrosis. e. Bile Duct Hyperplasia Bile duct hyperplasia was observed in mice given >10 mg/kg and the incidence and severity were both dose related. Bile duct hyperplasia was observed in 0/19, 0/20, 0/20, 6/20, 17/19, and 12/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. The hyperplasia was graded as minimal in all mice except for three 30 mg/kg mice which were graded as mild. A slight decrease in the incidence o f bile duct hyperplasia was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group. The bile duct hyperplasia was characterized by a minimal to mild increase in the number of profiles of normal bile ducts. f. Fatty Change, Nonzonal Nonzonal fatty change was also present in mice given >10 mg/kg. The incidence was dose related but all lesions were graded as minimal. Fatty change was observed in 0/19, 0/20, 0/20, 9/20, 14/19, and 4/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. A decrease in the incidence of fatty change was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group. The fatty change was characterized by a minimal increase in the number of hepatocytes with small to medium size cytoplasmic fatty globules. The distribution of the affected hepatocytes was nonzonal as the fatty change was scattered throughout the liver. The fatty change was only observed in livers with severe diffuse hepatocellular hypertrophy and was considered to be a degenerative change secondary to the hypertrophy. 2. Thymus a. Lymphoid Depletion/Atrophy Minimal to severe thymic lymphoid depletion/atrophy was only observed in mice given >10 mg/kg. The lesion was present in 0/19, 0/20, 0/19, 6/19, 7/17, and 4/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. In addition, thymic tissue was not present in the mediastinal tissue section o f 9 other mice (given >10 mg/kg), suggesting that these animals also had moderate to severe thymic lymphoid depletion. Therefore, the combined incidence of lymphoid depletion and absent thymic tissue was 0/19, 0/20, 0/19, 8/19, 12/17, and 6/19 mice given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively. Both the incidence and severity were dose related. A slight decrease in the incidence and severity of thymic lymphoid depletion/atrophy was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group. Thymic lymphoid depletion/atrophy was characterized by decrease in the number of lymphocytes in the thymus in the absence of necrotic cells. As stated earlier, the absence of thymic tissue in the mediastinal tissue section was also interpreted to be indicative o f thymic depletion/atrophy. -38- p. 39 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 3. Spleen a. Lymphoid Depletion/Atrophy Minimal to mild splenic lymphoid depletion/atrophy was test substance related only at 30 mg/kg. The lesion was present in 0/19, 1/20, 0/20, 0/20, 8/19, and 7/19 mice given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively. The incidence and severity o f this effect was similar in the 30 and 30/0 mg/kg groups. As with the thymic lesion, splenic lymphoid depletion/atrophy was characterized by a decrease in the number of lymphocytes in the thymus in the absence of necrotic cells. b. Increased Extramedullary Hematopoiesis An increase in the incidence o f splenic extramedullary hematopoiesis (EMH) was considered test substance related only in high-dose mice allowed a recovery period (30/0 mg/kg dose group). Minimal to moderate increased EMH was observed in 6/19, 4/20, 1/20, 3/20, 5/19, and 15/19 mice given 0, 0.3, 1,10, 30, and 30/0 mg/kg, respectively. The increased splenic EMH was erythrocytic and correlated with both the bone marrow erythrocytic hyperplasia and the hematological finding of decreased red cell mass parameters and increased circulating reticulocytes (see Clinical Pathology) that were also observed only in the 30/0 mg/kg recovery mice. 4. Bone Marrow a. Granulocytic Hyperplasia A minimal to moderate increase in bone marrow granulocytic hyperplasia was observed in 0/19, 0/20, 0/20, 3/20, 4/19, and 3/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. The incidence and severity were not dose related in groups given >10 mg/kg o f the test substance. The increase in bone marrow granulocytic hyperplasia (>10 mg/kg) correlated with increases in the peripheral white blood cell and neutrophil counts (see Clinical Pathology). The granulocytic response was most likely secondary to the hepatocellular individual cell necrosis and focal necrosis observed in mice given >1 mg/kg o f the test substance. b. Erythrocytic Hyperplasia A mild increase in bone marrow erythrocytic hyperplasia was observed only in high-dose mice allowed a recovery period (30/0 mg/kg dose group). Erythrocytic hyperplasia was observed in 0/19, 0/20, 0/20, 0/20, 0/19, and 3/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively. As discussed for the spleen, the increase in bone marrow erythrocytic hyperplasia in recovery mice correlated with both microscopic splenic EMH and hematology findings (decreased red cell mass parameters and increased circulating reticulocytes (see Clinical Pathology)). -39tu*-' p. 40 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 5. Other All other microscopic observations in this study were consistent with normal background lesions in mice o f this age and strain. E. Ultrastructural Findings 1. Electron Microscopy Evaluation (Appendix M) At the 1 mg/kg dose of APFO (linear), an increase in peroxisomes was not observed. However, many organelles could not be clearly identified due to poor ultrastructural detail, which was likely the result of formalin fixation. Therefore, definitive conclusions on perosimal numbers in treated groups relative to controls could not be drawn. More details are provided in Appendix M. F. Anatomic Pathology Conclusions There were no test substance-related deaths. Only 3 o f the 120 mice on study did not survive until the scheduled sacrifice on day 29. The 3 mice were sacrificed in extremis on test days 5 and 9. Following 28-days of daily gavage administration of the test substance, test substance-related organ weight effects were observed in the liver, spleen, and thymus. Mean liver weight parameters were increased, mean spleen weight parameters were decreased, and mean thymus weight parameters were decreased. At the terminal sacrifice, test substance-relate gross observations were observed at doses >10 mg/kg and included large and discolored livers, small spleens, and small thymuses. Microscopic examination of the liver demonstrated mild hepatocellular hypertrophy at 0.3 mg/kg; moderate to severe hepatocellular hypertrophy with secondary individual cell necrosis and focal necrosis at doses >1 mg/kg; and increased hepatocellular mitotic figures, hepatocellular fatty change, and bile duct hyperplasia at doses >10 mg/kg Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased granulocytic hematopoiesis in the bone marrow (>10 mg/kg) and increased erythrocytic hematopoiesis in the bone marrow and spleen (30/0 mg/kg). Test substance-related lymphoid depletion/atrophy was present in the thymus (>10 mg/kg) and spleen (30 mg/kg) o f less than half o f the mice at the respective dose levels. Mesenteric and popliteal lymph nodes had no test substance-related effects. -40- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 41 DuPont-18318 Total Cell Counts A. Spleen Cell Number (Table 17, Appendix L) No significant changes in total spleen cell number were noted in animals dosed with 0.3 or 1 mg/kg. Significant decreases were noted in animals dosed with 10 mg/kg or greater, with the greatest suppression compared to vehicle-treated controls observed at 30 mg/kg (63% suppression). In the 30/0 mg/kg group, a rebound was seen (44% suppression), but this increase in cells compared to the 30 mg/kg was most likely due to the extramedullary hematopoiesis observed in 15 o f 19 mice in this treatment group. B. Thymus Cell Number (Table 17, Appendix L) No significant changes in total thymus cell number were noted in animals dosed with 0.3 or 1 mg/kg. Significant decreases were noted in animals dosed with 10 mg/kg or greater, with the greatest suppression observed at 30 mg/kg (82% suppression). In the 30/0 mg/kg group, a rebound was seen (51% suppression). CONCLUSIONS Under the conditions o f this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male mice was 0.3 mg/kg and for immunosuppression was 1 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. REFERENCES 1. Vanden Heuvel, J., Kuslikis, B., and Van Rafelghem, M. (1991). Tissue distribution, metabolism, and elimination o f 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. -41 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 3. Hansen, K.J., Clemen, L.A., Ellefson, M.E., and Johnson, H.O. (2001). Compound-specific, quantitative characterization of 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 of 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 o f 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, 6th edition, 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. 13. Tamhane, A.C. (1979). A comparison of 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 of variance. J. Amer. Statist. Assoc. 47, 583-621. 15. Dunn, O.J. (1964). Multiple contrasts using rank sums. Technometrics 6, 241-252. 16. Amacher, D.E., Schomaker, S.J., and Burkhardt, JE. (1998). The relationship among microsomal enzyme induction, liver weight and histological change in rat toxicology studies. Food Chem. Toxicol 36, 839-839. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 43 DuPont-18318 TABLES -43 - L/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice TABLES ABBREVIATIONS: EXPLANATORY NOTES 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 - - no data NC - not calculated or not calculable 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 DuPont-18318 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. -44 2/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 TABLES EXPLANATORY NOTES (Continued) NOTES: (Continued) Summary of Total Cell Counts Organ Weight as Percent of Body Weight Organ Weight (g) Final Body Weight (g) Total Number of o f Organ Cells (xlO8) Organ Weight (g) Elalf Organ Weight (g) Organ Cell Suspension Volume (mL) Number of Cells in Half Organ (x 106 cells/mL) - 100 -45 - p. 46 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice________________________________________________DuPont-18318 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 Mean 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 Mean 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 (B> 1.00 1.00 1.02 1.05 Mean 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 Mean 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. -462 ^ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 47 DuPont-18318 Table 2 Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability of APFO in Dosing Solutions Sample Date ____________ APFO (mg/mL)____________ Sample Typc(A) Nominal Measured 15-November-2005 Concentration Verification Control 0 ND(B) Percent Nominal -- #1 #2 #1 #2 #1 #1(C) #2(C) #1 #2 Stabil itv(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. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 48 DuPont-18318 Table 3 Concentration Verification and Uniformity of Mixing o f 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. -48 225 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DAYS ON TEST 0 7 14 21 28 Group I 0 mg/kg 32.4 1.7(20) 32.8 1.6(19) 33.0 1.8(19) 33.7 1.5(19) 33.4 1.5(19) Table 4 Mean Body Weights of Male Mice Group III 0.3 mg/kg MEAN BODY WEIGHTS (g) Group V Group VII 1 mg/kg 10 mg/kg 32.4 1.8(20) 32.6 1.8(20) 33.0 1.9(20) 33.8 2.0(20) 33.9 2.5(20) 32.6 1.8(20) 32.7 1.7(20) 33.1 2.0(20) 34.2 1.8(20) 34.2 1.8(20) 32.3 1.7(20) 31.8 2.3(20) 29.7* 2.3(20) 29.2* 2.0(20) 28.6* 2.1(20) Group IX 30 mg/kg 32.7 2.2(20) 28.1* 3.1(20) 28.6* 2.9(19) 26.0* 3.1(19) 26.2* 3.0(19) DuPont-18318 Group XI 30/0 mg/kg (Recovery) 32.7 1.6(20) 28.1* 3.1(19) 28.0* 2.7(19) 26.0* 2.8(19) 29.5* t 3.4(19) 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, 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. 49 -49- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 5 Mean Body Weight Gains of Male Mice DAYS ON TEST Group I 0 mg/kg 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 Group XI 30/0 mg/kg (Recovery) 0-7 0.2 0.3 0.0 -0.5 -4.6@ -4.7@ 0.7(19) 0.9(20) 1.0(20) 1.6(20) 3.0(20) 2.7(19) 7-14 0.3 0.3 0.4 -2.1@ 0.2 -0.0 0.8(19) 0.5(20) 0.8(20) 1.6(20) 3.0(19) 2.9(19) 14-21 0.7 0.7(19) 0.8 0.4(20) 1.1 0.9(20) -0.5@ 1.4(20) -2.6@ 3.5(19) -2.0@ 3.0(19) 21-28 -0.3 0.1 0.0 -0.6 0.3 3.4@ t rN} 0.7(19) 0.8(20) 0.8(20) 0.9(20) 1.7(19) 2.5(19) OVERALL 0-28 0.9 1.4(19) 1.5 1.8(20) 1.5 1.9(20) -3.8* 1.9(20) -6.6* 3.5(19) -3.3* 2.6(19) 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 111, 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. 50 - 50- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 DAYS ON TEST 7 14 21 28 OVERALL 0-28 Group I 0 mg/kg 5.1 0.4(19) 5.1 0.4(19) 5.1 0.4(19) 4.7 0.5(19) 5.0 0.3(19) Table 6 Mean Daily Food Consumption by Male Mice MEAN DAILY FOOD CONSUMED PER ANIMAL (g) Group III Group V Group VII Group IX 0.3 mg/kg 1 mg/kg 10 mg/kg 30 mg/kg 5.1 0.4(20) 5.2 0.4(20) 5.1 0.4(20) 4.9 0.4(20) 5.1 0.3(20) 5.2 0.4(20) 5.3 0.5(20) 5.1 0.5(20) 5.2 0.4(20) 5.2 0.3(20) 5.0 0.6(20) 5.8* 0.8(20) 5.3 0.9(20) 5.5@ 0.8(20) 5.4@ 0.5(20) 4.5 0.8(20) 5.6* 0.8(19) 4.4@ 1.2(18) 4.8 1.1(19) 4.9 0.9(18) Group XI 30/0 mg/kg (Recovery) 4.5* 0.7(19) 5.5 1.0(19) 4.6f 0.9(19) 5.5@ t 1.3(19) 5.0 0.7(19) Data arranged as: Mean Standard deviation (Number of values included in calculation) * Statistically significant differencefromcontrol at p < 0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant differencefromcontrol at p < 0.05 by Dunn's test, t Statistically significant differencefromGroup 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. 51 - 51 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 DAYS ON TEST 0-7 14 21 28 OVERALL 0-28 Group I 0 mg/kg Table 7 Mean Daily Food Efficiency of Male Mice 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 0.005 0.020(19) 0.006 0.023(19) 0.019 0.019(19) -0.010 0.021(19) 0.006 0.010(19) 0.008 0.026(20) 0.009 0.015(20) 0.023 0.011(20) 0.001 0.023(20) 0.010 0.012(20) 0.001 0.028(20) 0.010 0.020(20) 0.031 0.026(20) -0.000 0.022(20) 0.011 0.013(20) -0.020 0.053(20) -0.057@ 0.050(20) -0.014@ 0.040(20) -0.017 0.023(20) -0.026* 0.015(20) -0.161 @ 0.122(20) 0.004 0.082(19) -0.103@ 0.148(18) 0.008 0.056(19) -0.053* 0.032(18) Group XI 30/0 mg/kg (Recovery) -0.165@ 0.116(19) -0.003 0.071(19) -0.065@ t 0.091(19) 0.087@ 0.056(19) -0.025* 0.021(19) 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. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice ANIMAL COUNT: Enophthalmus Abnormal Gait Feces Absent Lethargic Not Eating Stained Cageboard Swollen Shoulder Swollen Penis Table 8 Summary of Daily Animal Health Observations in Male Mice Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 20 20 20 20 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 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%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Group IX 30 mg/kg 20 0 (0%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 1 (5%) Data arranged as: number of animals (percent of group) for which an observation was recorded DuPont-18318 Group IX 30/0 mg/kg (Recovery) 20 1 (5%) 0 (0%) 1 (5%) 1 (5%) 1 (5%) 2 (10%) 0 (0%) 0 (0%) p. 53 - 53 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice ANIMAL COUNT: Absent End of tail Eye Dark Enophthalmus Eye Partially Closed Wet Fur Prostrate Abnormal Gait Pale Feces Absent Labored Breathing Lethargic Not Eating Table 9 Summary of Detailed Clinical Observations in Male Mice Group I 0 mg/kg Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 mg/kg 20 20 20 20 0 (0%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 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%) 1 (5%) 0 (0%) 1 (5%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%) 0 (0%) DuPont-18318 Group IX 30 mg/kg 20 0 (0%) 0 (0%) 0 (0%) 1 (5%) 1 (5%) 0 (0%) 0 (0%) 4 (20%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) Group IX 30/0 mg/kg (Recovery) 20 0 (0%) 1 (5%) 2 (10%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) 1 (5%) 1 (5%) it? p. 54 -54- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Table 9 Summary of Detailed Clinical Observations in Male Mice (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 ANIMAL COUNT: 20 20 20 20 20 Misshapen Tail 0 (0%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) Stain Fur/Skin 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) Stained Cageboard 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Swollen Neck 1 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Swollen Face 1 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Swollen Shoulder 0 (0%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) Swollen Penis 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (5%) Data arranged as: number of animals (percent of group) for which an observation was recorded DuPont-18318 Group IX 30/0 mg/kg (Recovery) 20 0 (0%) 1 (5%) 2 (10%) 0 (0%) 0 (0%) 1 (5%) 0 (0%) >f ? p. 55 -55- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 10 Summary of Hematology Values for Male Mice 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 Group XI 30/0 mg/kg (Recovery) RBC (xlO%tL) DAY 29 HGB (g/dL) 10.23 0.72(10) 10.15 0.38(10) 9.30 1.43(10) 10.04 0.97(8) 9.64 0.89(7) 8.82@ 0.77(9) DAY 29 HCT (%) 16.0 1.1(10) 15.8 0.5(10) 14.3 2.2(10) 14.9 1.7(8) 14.1 1.9(7) 13.1@ 1.2(9) DAY 29 53.0 52.7 48.0 51.9 48.3 45.0@ MCV (fL) 3.3(10) 1.9(10) 7.4(10) 5.0(8) 6.1(7) 3.5(9) DAY 29 51.8 51.9 51.7 51.7 49.9 51.1 2.1(10) 1.4(10) 1.9(10) 2.6(8) 2.0(7) 2.2(9) MCH (pg) Uj DAY 29 15.6 15.6 15.4 14.9 14.6* 14.8 0.5(10) 0.6(10) 0.7(10) 1.0(8) 0.7(7) 0.6(9) MCHC (g/dL) DAY 29 30.1 0.7(10) 30.1 1.1(10) 29.8 0.8(10) 28.7* 1.0(8) 29.1 0.7(7) 29.1* 1.0(9) p. 56 - 56 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 10 Summary of Hematology Values for Male Mice (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 Group XI 30/0 mg/kg (Recovery) RDW (%) DAY 29 12.9 12.3@ 12.2 0.3(10) 0.3(10) 0.7(10) ARET (xlOVpL) DAY 29 326.5 342.3 274.8 27.9(10) 53.4(10) 65.3(10) PLT (xlOVpL) DAY 29 1177 1402 1176 NC(1) 202(2) 315(5) V ) WBC (xlOVpL) T s DAY 29 7.55 9.57 8.90 2.39(10) 2.84(10) 2.62(10) ANEU (xlOVpL) DAY 29 0.80 1.29 0.96 0.42(10) 0.46(10) 0.49(10) ALYM (xlOVpL) DAY 29 6.43 7.82 7.60 1.92(10) 2.66(10) 2.57(10) 12.1 @ 0.5(8) 248.2 62.1(8) - 11.14* 2.24(8) 1.89* 0.60(8) 8.67 2.08(8) 13.1 0.8(7) 350.4 160.1(7) - 6.75 2.61(7) 2.37* 1.14(7) 3.81 1.39(7) 14.0 2.2(9) 481.9 207.6(9) 1501 723(4) 7.29 1.06(9) 2.05* 0.57(9) 4.78f 0.77(9) p. 57 - 57- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 AMON (xlOVpL) DAY 29 AEOS (xlOVpL) DAY 29 ABAS (xlOVpL) DAY 29 ALUC (xlOVpL) DAY 29 Table 10 Summary of Hematology Values for Male Mice (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 0.13 0.09(10) 0.14 0.10(10) 0.01 0.02(10) 0.04 0.05(10) 0.16 0.11(10) 0.18 0.07(10) 0.02 0.01(10) 0.09 0.05(10) 0.11 0.07(10) 0.14 0.08(10) 0.02 0.01(10) 0.06 0.04(10) 0.37@ 0.18(8) 0.08 0.04(8) 0.02 0.02(8) 0.10 0.11(8) 0.33 0.23(7) 0.09 0.07(7) 0.02 0.02(7) 0.14 0.11(7) Group XI 30/0 mg/kg (Recovery) 0.23 0.14(9) 0.09 0.08(9) 0.02 0.04(9) 0.11 0.12(9) 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 differencefromcontrol at p < 0.05 by Dunn's test, t Statistically significant differencefromGroup 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. 5St p. 58 - 58 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group I 0 mg/kg Table 11 Summary of Clinical Chemistry Values for Male Mice Group III 0.3 mg/kg Group V 1 mg/kg Group VII 10 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 29 117 29(9) 167 37(9) 5.6 0.3(6) 2.9 0.2(6) 2.7 0.2(6) 77 19(9) 128 38(10) 148 41(10) 5.2 0.5(7) 2.8 0.3(7) 2.4 0.3(7) 77 13(10) 101 30(10) 157 41(10) 5.6 0.4(8) 3.2 0.1(8) 2.5 0.4(8) 55* 15(10) 81* 23(10) 78* 31(10) 0.3(7) 4.2* 0.3(7) 2.8 0.1(7) 47* 11(10) Group IX 30 mg/kg 60* 22(10) 53* 27(10) 6.1 0.8(3) 3.8* 0.3(3) 2.3 0.5(3) 34* 11(10) DuPont-18318 Group XI 30/0 mg/kg (Recovery) 94# 22(9) 96*# 23(9) 7.5*# 0.7(9) 4.3*# 0.4(9) 3.2*# 0.4(9) 53*# 11(9) p. 59 *o -59- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 NHDL (mg/dL) DAY 29 SCORT (ng/mL) DAY 28-29 Table 11 Summary of Clinical Chemistry Values for Male Mice (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) 40 10(9) 189 112(10) 51 27(10) 198 86(10) 46 18(10) 108 76(10) 34 14(10) 433* 156(10) 26 13(10) 437 278(10) 41# 13(9) 259 168(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 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. 60 - 60- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 LOG2a Table 12 Summary of Primary Humoral Immune Response to SRBC for Male Mice 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 9.027 0.587(19)b 8.819 0.787(19)b 8.310 0.619(20) 7.185@ 1.351 (18)c'd 6.513@ 1.037(16)b,c Group XI 30/0 mg/kg (Recovery) 6.277@ 0.680(18)b'c Data arranged as: Mean Standard deviation (Number of values included in calculation) a Mean log2 of the serum IgM titer data. b Serum was not collected from one or more animals, therefore, immune response could not be evaluated for these animals. c Serum volume was insufficient for one or more animals, therefore, immune response could not be evaluated for these animals. d One or more animal was not injected with the appropriate amount of SRBC, therefore, immune response could not be evaluated for these animals. @ Statistically significant difference from control at p < 0.05 by Dunn's test. 9SZ p. 61 - 61 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 13 Summary of Primary Humoral Immune Response to SRBC for Male Mice Dosed With Positive Control Saline3 Cyclophosphamide 90 mg/kga Saline*3 Cyclophosphamide 90 mg/kg*3 log2 8.603 0.685(10) 4.515 0.843(10) 8.662 5.129 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 Log2of the SRBC-specific serum IgM titer data for pooled samples. p. 62 - 62 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 14 Mean Final Body and Organ Weights from Male Mice Group I Omg/kg Group III Group V Group VII Group IX 0.3 mg/kg 1 mg/kg 10mg/kg 30 mg/kg _____________________________________________________ MEAN FINAL BODY AND ABSOLUTE ORGAN WEIGHTS (grams) LIVER 1.782 0.175(17)a 2.407 0.255(20) 3.272@ 0.231(20) 6.061 @ 1.320(20) 5.899@ 0.850(18)b SPLEEN 0.117 0.015(19) 0.116 0.032(20) 0.104 0.016(20) 0.066@ 0.019(20) 0.052@ 0.023(19) THYMUS 0.050 0.010(19) 0.045 0.010(20) 0.049 0.012(20) 0.025@ 0.009(20) 0.025@ 0.013(19) BRAIN 0.471 0.027(19) 0.478 0.029(20) 0.474 0.029(20) 0.446* 0.028(20) 0.440* 0.026(19) FINAL BODY WEIGHT (grams) 33.0 1.3(19) 33.4 2.5(20) 33.8 1.8(20) 28.4* 2.0(20) 26.0* 2.8(19) Group XI 30/0mg/kg (Recovery) 6.391 @ 1.505(18)' 0.076@ 0.022(19) 0.027@ 0.010(19) 0.442* 0.029(19) 30.5*t 3.7(19) p. 63 -63 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 14 Mean Final Body and Organ Weights from Male Mice (Continued) Group I Group III Group V Group VII Group IX Group XI 0 mg/kg 0.3 mg/kg 1 mg/kg 10 mg/kg 30 mg/kg 30/0 mg/kg ______________________________________________________________________________________________________ (Recovery) MEAN RELATIVE ORGAN WEIGHTS (% of body weight) LIVER/ FINAL BODY * 100 5.421 0.466(1 I f 7.196 0.418(20) 9.704@ 0.736(20) 21.232@ 3.715(20) 22.618@ 2.614( 18)b 21,209@ 4.835(18)' SPLEEN/ FINAL BODY * 100 0.355 0.045(19) 0.346 0.082(20) 0.307* 0.043(20) 0.232* 0.062(20) 0.195* 0.067(19) 0.249* 0.058(19) THYMUS/ FINAL BODY * 100 0.153 0.034(19) 0.137 0.034(20) 0.144 0.035(20) 0.087@ 0.031(20) 0.094@ 0.048(19) 0.088@ 0.027(19) 'G BRAIN/ 1.427 1.436 1.408 1.576* 1.703* 1.467t FINAL BODY * 100 0.080(19) 0.091(20) 0.103(20) 0.111(20) 0.132(19) 0.189(19) p. 64 - 64- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 14 Mean Final Body and Organ Weights from Male Mice (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 brain weight) LIVER/ BRAIN * 100 379.673 34.913(17)a 503.340 46.491(20) 691.370@ 55.479(20) SPLEEN/ BRAIN * 100 24.915 2.950(19) 24.226 6.087(20) 21.872* 3.312(20) THYMUS/ BRAIN * 100 10.758 2.438(19) 9.533 2.207(20) 10.199 2.245(20) 1357.057@ 275.001(20) 14.822* 4.260(20) 5.592@ 2.072(20) 1336.969@ 167.426(18)b 11.756* 4.890(19) 5.638@ 2.969(19) Group XI 30/0 mg/kg (Recovery) 1457.734@ 345,770( 18)b 17.267*f 4.669(19) 6.164@ 2.278(19) Data arranged as: Mean Standard deviation (Number of values included in calculation) a An error occurred while weighing livers for 2 animals in this group, and the liver weights were excluded from calculations, b Liver inadvertently not weighed from one animal in this group. * 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. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 15 Incidence of Gross Observations in Male Mice 1 1 1 LESIONS t. 1 1 LIVER 1 NO ABNORMALITY DETECTED ! LARGE. ! DISCOLORATION 1 j SPLEEN 1 NO ABNORMALITY DETECTED SMALL. 1 ! THYMUS ! NO ABNORMALITY DETECTED ! SMALL. ! 1 POPLITEAL LYMPH NODE NO ABNORMALITY DETECTED MESENTERIC LYMPH NODE 1 NO ABNORMALITY DETECTED SMALL. BRAIN NO ABNORMALITY DETECTED 1 TREATMENT I per day 1 LESION INCIDENCE (Numeric) 1 1 1 0 1 0.3 1 1 10 1 30 1 30/0 i 1 mg/kg| mg/kg| mg/kg j mg/kg| mg/kg 1 mg/kg | 1 1 1 1 1Recovery ! 1 I ! III 1 V 1 VII ! IX 1 XI ! t 1 (20) 1 19 11 1 (20) 1 20 ! 1 i (20) 1 20 ! 1 1 (20) 1 20 1 1 (20) i 20 j j (20) i 20 1 1 (20) 1 20 1 ! (20) 1 20 1 (20) 1 20 1 (20) 1 20 (20) 20 (20) 1 20 1 1 (20) 1 20 1 1 1 1 (20) 1 20 1 1 1 (20) ! 20 1 1 ! (20) 1 20 1 1 (20) 1 20 1 1 1 (20) ! 20 1 1 (20) 13 1 17 11 1 1 (20) ! 12 18 1 1 (20) j 17 13 1 (20) ] 20 1 (20) 20 1 (20) 1 20 1 I I (20) 13 1 16 16 ! 1 (20) 12 18 1 (20) ! 18 !2 1 1 (20) I 20 1 1 (20) 19 1 (20) 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 ! 1 1 1 1 ! (20) 3 17 1 (20) 18 2 (20) 18 2 (20) 20 (20) 20 (20) 20 ! 1 1 1 1 1 1 ! 1 ! 1 ! 1 1 ! 1 ! t I 1 1 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. 66 - 66- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 15 Incidence of Gross Observations in Male Mice (Continued) LESION INCIDENCE (Numeric) LESIONS | TREATMENT 1 per day ! 1 1 0 1 0.3 ! 1 1 10 1 30 1 30/0 1 mg/kg| mg/kg! mg/kg 1 mg/kg| mg/kg 1 mg/kg 111 1 1Recover y 1 I i III l V 1 VII 1 IX 1 XI FEMUR/KNEE JOINT NO ABNORMALITY DETECTED STERNUM NO ABNORMALITY DETECTED PENIS PARAPHIMOSIS. SKIN MASS, GREEN, AXILLA, LEFT. OTHER, ABSCESS AXILLA RIGHT. OTHER, abscess subcutaneous axilla right, subcutaneous air pocket, dorsal ne ck, right axilla. ESOPHAGUS RUPTURE. TRACHEA RUPTURE. 1 1 (20) 1 20 ! 1 (20) 1 20 1 1 1 1 (1) ! 1 11 1 1 1 1 (1) 11 1 i (U 11 1 1 ! (20) ! 20 1 t (20) ! 20 1 1 1 1 ! 1 1 1 1 1 1 1 I 1 1 ! 1 (20) 1 20 1 1 (20) 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 1 1 1 (20) 1 20 1 (20) ! 20 (1) 1 1 1 (20) 1 20 1 1 (20) 1 20 ! 1 (1) 11 i t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 ! 1 1 1 1 1 1 1 1 1 1 (20) 20 (20) 20 (1) 1 (1) 1 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. 67 - 67- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 16 Incidence and Lesion Grades of Microscopic Observations in Male Mice LESION INCIDENCE (NUMERIC) LESIONS | TREATMENT per day 1 1 ! 0 | 0.3 | 1 | 10 | 30 ! 30/0 1 mg/kg| mg/kg| mg/kg| mg/kg| mg /kg ! mg/kg 11! 1 1Recovery 1 I 1 Ill ! V I VII | IX ! XI LIVER NO ABNORMALITY DETECTED NECROSIS, INDIVIDUAL CELL, INCREASED. minimal mild Total observations per lesion NECROSIS, FOCAL. minimal mild moderate Total observations per lesion MITOTIC FIGURES, INCREASED, HEPATOCELLULAR. minimal mild Total observations per lesion INFLAMMATION, SUBACUTE/CHRONIC. minimal Total observations per lesion 1 1 (19) ! 17 1 1 1 1 ! 1 1 I 1 ! 1 1 1 | 1 1 | (20) | 1 1 ! ! 1 11 1 1 i1 1 1 1 1 1 11 1 ! | (20) ! 1 I 10 11 ! 11 ! |3 1 1 |3 1 1 1 ! 1 !1 |1 1 1 | (20) | | |3 | 17 1 20 | 12 !1 I1 14 1 1 10 ! 1 10 ! !4 14 ! 1 ! (19) ! ! 1 | 19 ! 19 i 14 | |3 17 1 | 15 1 | 15 1 11 !1 1 1 1 ! ! 1 1 I 1 1 ! I 1 ! 1 1 1 1 ! ! 1 (19) 6 13 19 2 1 3 12 7 19 5 5 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 Table excludes 3 mice that were euthanized on test days 5 (mice 117 or 1112) and 9 (mouse 906). Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 16 Incidence and Lesion Grades of Microscopic Observations in Male Mice (Continued) 1 1 1 LESIONS ! 1 ! 1 I 1 I ! 1 LIVER 1 HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR. mild 1 moderate i severe 1 Total observations per lesion 1 HYPERPLASIA, BILE DUCT. I minimal 1 mild 1 Total observations per lesion 1 HEMATOPOIESIS, EXTRAMEDULLARY. 1 minimal 1 Total observations per lesion 1 FATTY CHANGE, DIFFUSE. 1 minimal 1 mild 1 Total observations per lesion 1 FATTY CHANGE, NONZONAL. 1 minimal 1 Total observations per lesion ! TREATMENT per day ! LESION INCIDENCE (NUMERIC) ! ! 0 1 0.3 ! 1 | 10 | 30 ! 30/0 ! 1 mg/kg1 mg/kg| mg/kg! mg/kg| mg/kg 1 mg/kg j 1 1 1 1 1 IRecovery| 1 I j III ! V I VII | IX 1 XI | 11!11! 1 (19) | (20) ! (20) | (20) | (19) ! (19) 1!!111 1 1 20 | 1 ! ! ! 1 20 | 1 1 1 ! | 1 20 | 19 1 19 ! 1 20 | 20 | 20 | 19 1 19 11|!11 1 1 1 1 6 1 14 1 12 1 1 i 1 1 3! 1 ! 1 1 6 1 17 1 12 111111 1 1 1 1 1 11 1 1 1 1 1 11 111111 1 1| 1 1 1 ! 1 1| ! 1 1 i 1 21 | ! 1 1 1!|i11 1 1 1 1 9 ! 14 1 4 11! 9 1 14 1 4 1!1111 1 | 1 1 1 | | 1 | 1 ! 1 1 ! 1 ! 1 1 1 | | 1 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 Table excludes 3 mice that were euthanized on test days 5 (mice 117 or 1112) and 9 (mouse 906). p. 69 - 69- 'V i Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 16 Incidence and Lesion Grades o f Microscopic Observations in Male Mice (Continued) 1 I LESIONS 1 1 ! I TREATMENT 1 per day 1 1 LESION INCIDENCE (NUMERIC) ! 0 1 0.3 ! 1 ! 10 1 30 30/0 1 mg/kg| mg/kg 1 mg/kg 1 mg/kg 1 mg/ kg 1 mg/kg 1 1 | ! I 1 Ill 1 V 1 VII ! IX 1Recovery] 1 XI I ! 1 SPLEEN NO ABNORMALITY DETECTED HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY I minimal I mild I moderate 1 Total observations per lesion 1 DEPLETION/ATROPHY, LYMPHOID. minimal mild I Total observations per lesion i I THYMUS NO ABNORMALITY DETECTED I HYPERPLASIA, LYMPHOID, FOLLICULAR. | mild I Total observations per lesion 1 1 1 (19) 1 13 ! !5 11 1 16 1 1 1 1 1 (19) 19 1 1 1 ! 1 1 (20) 1 15 1 12 12 1 14 1 11 ! 11 1 1 (20) 1 20 1 1 1 1 (20) 1 19 1 11 1 1 !1 1 1 1 1 1 1 (19) 1 18 ! !1 11 1 (20) 1 17 1 11 !2 1 13 1 1 1 1 1 1 (19) 1 11 1 1 ! ! 1 (19) 19 ! !4 11 1 15 1 17 !1 18 1 1 (17) 15 1 1 ! 1 1 (19) 14 1 14 1 10 11 1 15 ! 16 11 17 1 1 (19) ! 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 1 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 Table excludes 3 mice that were euthanized on test days 5 (mice 117 or 1112) and 9 (mouse 906). p. 70 - 70- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 16 Incidence and Lesion Grades o f Microscopic Observations in Male Mice (Continued) LESION INCIDENCE (NUMERIC) LESIONS ! TREATMENT ! per day 1 1 !0 0.3 | 1 I 10 ! 30 ! 30/0 1 mg/kg1 mg/kg| mg/kg| mg/kg| mg/kg| mg /kg 1 1 1 1 1 1Recovery 1 I | H I 1 V I VII | IX | XI THYMUS DEPLETION/ATROPHY, LYMPHOID. minimal mild moderate severe Total observations per lesion CYST, EPITHELIAL. minimal Total observations per lesion NOT PRESENT IN MEDIASTINAL TISSUE. ECTOPIC THYROID. LYMPH NODE - POPLITEAL NO ABNORMALITY DETECTED NOT PRESENT IN TISSUE SECTION. MESENTERIC LYMPH NODE NO ABNORMALITY DETECTED DEPLETION/ATROPHY, LYMPHOID. mild Total observations per lesion 1 1 (19) 1 1 1 1 1 ! 1 1 1 ! 11 1 1 (10) 16 14 1 1 (19) 1 19 I 1 ! ! ! | (20) 1 1 1 1 1 1 1 1 1 ! 1 1 | ! 1 1 | (20) | 20 1 1 1 1 1 i (19) 1 | | 1 1 ! 1 1 1 1 ! ! 1 1 1 1 | (19) | 19 1 1 1 1 | (19) 5 1 6 2 1 | (20) | 19 | (17) 1 11 |2 11 13 17 1 ! 1 15 1 1 1 (18) f 14 14 | (19) | 16 1 !1 11 1 1 | (19) 1 11 1 |2 11 14 1 12 12 i2 1 1 ! (19) |5 ! 14 1 | (19) | 18 1 | | 1 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 Table excludes 3 mice that were euthanized on test days 5 (mice 117 or 1112) and 9 (mouse 906). Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 16 Incidence and Lesion Grades of Microscopic Observations in Male Mice (Continued) LESION INCIDENCE (NUMERIC) 1 I LESIONS I TREATMENT 1 0 1 0.3 | i i 10 | 30 30/0 | i I per day 1 mg/kg| mg/kg| mg/kg| mg/kg| mg/kg mg/kg | i 1 111I1 Recovery| ! 1 1 I 1 III I V I VII | IX XI | 1 1 11 11 1 I MESENTERIC LYMPH NODE 1 (19) 1 (20) | (19) | (20) | (19) (19) | ! NOT PRESENT IN TISSUE SECTION. 1 1 I 1 1| 2 1 1 1 | BONE MARROW 11 11 1 (19) 1 (20) | (20) | (20) ! (19) (19) | 1 NO ABNORMALITY DETECTED ! 19 ! 20 | 20 | 17 1 15 13 | | HYPERPLASIA, GRANULOCYTIC. 111!1 1 1 minimal | 1 1 ! 2t 4 2 1 ! moderate | 1 1 1 1| 11 I Total observations per lesion K HYPERPLASIA, ERYTHROCYTIC. 1 1 1 1 31 4 3 1 111 ! 1 I mild 111 1 31 I Total observations per lesion 111 ! 3! 1 11i11 1 | BRAIN 1 (19) | 1 l 1 (19) (19) | NO ABNORMALITY DETECTED 1 19 | 1 1 1 19 19 | 1!111 I FEMUR/KNEE JOINT 1 (19) | 1 i 1 (19) (19) | ! NO ABNORMALITY DETECTED 1 19 | 1 I 1 19 19 ! 1 1i1 1 1 | STERNUM 1 (19) | ! 1 1 (19) (19) i I NO ABNORMALITY DETECTED 1 19 ! i 1 1 19 19 | 1!i!1 1 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 Table excludes 3 mice that were euthanized on test days 5 (mice 117 or 1112) and 9 (mouse 906). p. 72 - 72- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 16 Incidence and Lesion Grades o f Microscopic Observations in Male Mice (Continued) 1 LESIONS 1 1 1 PENIS 1 EROSION/ULCER. 1 moderate 1 Total observations per lesion 1 PREPUTIAL GLANDS 1 ECTASIA. 1 mild 1 Total observations per lesion 1 SKIN ABSCESS. 1 moderate 1 Total observations per lesion 1 ! TREATMENT I per day 1 1 1 LESION INCIDENCE (NUMERIC) 1 1 ! 0 1 0.3 ! 1 | 10 | 30 [ 30/0 | 1 mg/kg| mg/kg| mg/kg| mg/kg| mg/kg| mg/kg | 1 1 1 1 1 1Recovery| ! I | III 1 V I VII | IX | XI | t1!11 1 1 i 1 G) 1 I!!111 1 1 | 1 1 11 1 1 1 1 1 11 11 111 11 1 ! (1) 1 111!I! ! 1 1 t 1| 1 1 ! 1 1 1| 111111 1 1 ! 1 G) 1 1 1!1 11 1 1 1 1 1| 1 1 1 1 1 1| 1 11!1! 1 1 1 1 1 1 1 1 1 1 1 ! ! 1 ! 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 Table excludes 3 mice that were euthanized on test days 5 (mice 117 or 1112) and 9 (mouse 906). p. 73 - 73- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 17 Summary of Total Cell Counts 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 Group XI 30/0 mg/kg (Recovery) Final Body Weight (g) SPLEEN Absolute Weight (g) Weight Ratio (% Body Weight) 33.02 1.33(19) 33.41 2.47(20) 33.77 1.81(20) 28.42 2.04(20) 25.99 2.84(19) 30.49 3.66(19) 0.117 0.015(19) 0.3553 0.0448(19) 0.116 0.032(20) 0.3457 0.0824(20) 0.104 0.016(20) 0.3066 0.0426(20) 0.066 0.019(20) 0.2317 0.0623(20) 0.052 0.023(19) 0.1949 0.0671(19) 0.076 0.022(19) 0.2491 0.0578(19) Half Weight J J ) (g) Cell Suspension Volume (mL) 0.058 0.008(19) 5.3 0.3(19) 0.055 0.016(20) 5.5 0.3(20) 0.051 0.009(20) 5.4 0.3(20) 0.031 0.011(20) 5.4 0.2(20) 0.026 0.010(19) 5.4 0.2(19) 0.038 0.009(19) 5.5 0.2(19) Number of Cells in H alf(x 106 cells/mL) 12.18 4.05(18) 11.27 4.79(20) 12.20 2.41(20) 5.92 2.35(20) 4.36 2.70(19) 6.45 2.88(18) Total Number of Cells (x 108) 1.29 0.34(18) 1.30 0.52(20) 1.34 0.25(20) 0.69* 0.25(20) 0.48* 0.35(19) 0.72* 0.34(18) p. 74 - 74- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Table 17 Summary of Total Cell Counts (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 Group XI 30/0 mg/kg (Recovery) THYMUS Absolute Weight (g) 0.050 0.010(19) 0.045 0.010(20) 0.049 0.012(20) 0.025 0.009(20) 0.025 0.013(19) 0.027 0.010(19) Weight Ratio (% Body Weight) 0.1532 0.0337(19) 0.1369 0.0339(20) 0.1439 0.0351(20) 0.0872 0.0309(20) 0.0942 0.0477(19) 0.0881 0.0266(19) Half Weight (g) '\T] Cell Suspension \\ V Volume (mL) 0.025 0.006(19) 5.5 0.2(19) 0.022 0.006(20) 5.4 0.2(20) 0.024 0.008(20) 5.5 0.2(20) 0.012 0.005(20) 5.5 0.2(20) 0.010 0.005(19) 5.4 0.2(19) 0.014 0.005(19) 5.4 0.2(19) Number of Cells in Half (x 106 cells/mL) 5.26 2.27(19) 5.36 2.14(20) 6.74 3.81(20) 2.18 2.36(20) 0.87 1.41(19) 1.05 1.37(18) Total Number of Cells (x 108) 0.57 0.22(19) 0.60 0.24(20) 0.75 0.38(20) 0.25@ 0.27(20) 0.10@ 0.16(19) 0.28@ 0.79(18) 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. 75 -75 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 FIGURES -76 3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Figure 1 Representative Analytical Calibration Curve DuPont-18318 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. -77- js-y Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Figure 2 Representative LC/MS/MS Chromatograms DuPont-18318 is approximately 4.5 minutes. -78- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Figure 2 Representative LC/MS/MS Chromatograms (Continued) p. 79 DuPont-18318 Figure 2c: Representative LC/MS/MS chromatogram of 0.0303 ppm APFO analytical standard (FI22703376) 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. -79< rv Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Figure 2 Representative LC/MS/MS Chromatograms (Continued) DuPont-18318 diluted with NANOpure* water after matrix correction to a nominal concentration of 0.0300 ppm. The measured concentration of the representative recovery sample is 1.02 mg/mL. -80- JS1 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Figure 3 Mean Body Weights of Male Mice DuPont-18318 0 mg/kg 0.3 mg/kg 1 mg/kg 10 mg/kg 30 mg/kg 30/0 mg/kg (Recovery) sr p. 81 -81 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 APPENDICES -82 05? Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 83 DuPont-18318 Appendix A Certificate of Analysis Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 84 DuPont-18318 Precise Research. Proven Results. 3 0 5 8 Research D rive State College, PA 16801 T: 814272.1039 exygt3i.com CERTIFICATE OF ANALYSIS This Certificate o f Analysis fulfills the requirement for characterization o f a test substance prior to a study subject to the GLP regulations. It documents the purity o f 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: 19.5% Last Date o f Analysis: Re-certification Date: G7-November-2005 07-November-2006 Origin of Certified Material: E.I. du Pont de Nemours and Company Wilmington, DE 19898 USA Testing Facllity/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 o f 1 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix B Individual Body Weights -85- GZ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice ABBREVIATIONS : g - grams INDIVIDUAL BODY WEIGHTS EXPLANATORY NOTES DuPont-18318 -86- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Body Weights Body Weight g Day 0 Body Weight g Day 2 Body Weight g Day 3 Body Weight g Day 4 Body Weight g Day 5 Body Weight g Day 6 Male, 1 0 mg/kg 101 32.4 102 34.3 103 32 .4 104 31. 1 10b 33.3 106 28 .8 107 30.9 108 32 .4 109 34.4 110 33.1 111 35.1 112 32.2 113 34.0 114 32 .4 115 32.0 116 31.7 117 29.4 118 33.3 119 30.8 120 34.2 34 .0 31 .4 33.9 32 .9 30.3 31.6 29.8 33.4 30.5 34 .1 32.1 34 .3 33.0 31 .7 33.0 29.0 31.0 31 .6 33.8 33.7 33.4 31.8 34.2 32.8 30.5 31. 5 26.3 33.4 30.5 34.2 31. 7 34 .1 32.4 31 .4 33.2 29.1 30.8 31.5 34.3 33.7 34.7 32.6 35.4 33.4 30.7 32.2 24 .6 34.4 31 .6 35.6 32 .0 34 .8 32 .5 32 .1 33.5 29.6 30.8 31.9 34 .6 34 .3 34 .6 33.2 35.3 33.4 31.5 32.5 24 .2 35.0 32.0 35.5 32.2 34 .4 33 .0 31 .3 33.2 29.5 30.7 32.0 34.0 34.4 33.6 32.5 35.1 33.0 30.9 32.8 34 .3 32 .1 35 .9 - 87- DuPont-18318 Body Weight g Day 7 Body Weight g Day 8 Body Weight g Day 9 3 1 ,. 8 3 4 ., 0 3 2 .. 7 3 1 .. 6 3 3 ., 7 2 9 .. 3 3 1 ., 2 3 1 ,, 4 3 4 ., 1 3 4 .. 3 3 4 ,, 6 3 3 .. 1 3 4 ., 6 3 2 ,, 8 3 0 .. 6 3 2 .. 5 3 4 .. 0 3 1 .. 6 3 4 .. 8 3 1 ,. 8 3 4 .. 8 3 2 .. 8 3 0 .. 5 3 2 ,, 4 2 9 ,. 7 3 1 ,. 0 3 1 .. 4 3 3 .. 9 3 4 ,. 0 3 5 .. 1 3 2 .. 0 3 4 .. 3 3 2 .. 2 3 0 ,. 5 3 2 .. 2 3 3 .. 1 3 1 ,. 8 3 5 .. 3 3 1 .. 6 3 4 .. 7 3 2 .. 5 3 1 .. 0 3 2 ., 7 2 9 .. 6 3 0 .. 4 3 1 .. 5 3 4 .. 4 3 4 ., 3 3 4 .. 9 3 1 .. 8 3 3 .. 5 3 1 .. 1 2 9 ,, 5 3 1 ., 8 3 3 .. 5 3 1 .. 8 3 4 .. 5 p. 87 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 0 Body Weight g Day 2 Body Weight g Day 3 Body Weight g Day 4 Body Weight g Day !5 Body Weight g Day 16 Body Weight g Day 7 Body Weight g Day 13 Body Weight g Day !3 M a l e , H I 0.3 mg/ kg 301 32. 6 302 34 .8 303 33. 9 304 32 .4 305 32 .2 306 30 .9 307 30. 5 308 31 .6 309 32 .9 310 33 .4 311 35. 6 312 30 .7 313 33 .5 314 30 .8 315 34 .0 316 32 .7 317 29. 0 318 32 .2 319 29. 4 320 34 .1 36. 1 31 .2 32 .8 31 .1 33 .4 32 .3 29. 8 31. 2 29. 7 33. 4 33 .0 34 .7 34 .6 33 .5 33. 4 31 .2 30 .0 32 .1 32 .1 33. 8 36 .1 31 .5 32 .8 31 .2 33. 4 32. 1 29. 5 31 .0 29. 5 33 .7 32. 9 34. 7 34 .9 33. 6 33. 1 30. 8 30. 1 32 .7 31. 8 33. 5 36. 6 31. 9 32 .8 31 .7 33. 8 32 .8 30. 1 31. 2 29. 8 34 .8 33 .5 35 .6 35 .5 34 .0 33 .4 31 .5 30 .9 33 .3 32 .4 34 .4 36 .4 32 .3 33 .0 31 .7 34 .6 33 .2 30 .1 31 .1 30 .1 34 .4 33 .8 35 .1 34 .9 34 .2 33 .3 32 .0 30 .3 33 .0 32 .4 34 .1 36 .6 31 .8 33 .2 31 .2 34 .2 33 .3 30 .3 31 .0 30 .1 34 .7 33.5 34 .4 35.3 34.0 33.1 31 .6 29.9 31 .8 31.5 33.5 36.4 31.9 32.6 32.1 33.8 32.9 29.8 30.4 30 .1 34 .4 33 .4 34 .2 35 .3 33 .9 32 .6 32 .2 29 .7 32 .0 31 .3 32 .9 36 .2 31 .7 32 .8 30 .7 34 .2 32 .6 29 .7 30 .3 29 .9 34 .1 33.,0 34 ,.6 35..2 33..7 32 .,7 31,,6 29 ,.8 32 .2 31 .4 32 .,9 36 .1 31 .5 32 .5 31 .0 33 ,7 33..2 29.,2 30..2 29.,8 33 .8 p. 88 - 88 - So CT\ Ammonium Perfluorooctanoate; 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 0 Body Weight g Day 2 Body Weight g Day 3 Body Weight g Day 4 Body Weight g Day 5 Body Weight g Day 6 Body Weight g Day 7 Body Weight g Day 8 Body Weight g Day 9 Male, V 1 mg /kg 501 32 .8 502 34 .6 503 33.8 504 31. 4 505 32.6 506 28 .6 507 31.0 508 32 .5 509 32.9 510 32 .9 511 36 .1 512 32.5 513 35 .1 514 31 .8 515 34 .3 516 31.6 517 29.6 518 31 .8 519 32.4 520 34 .1 35.2 31.4 33.4 31.6 33.2 30.9 28 .7 31 .2 30.7 33.6 33.3 34 .6 34.0 31.1 32 .9 29.5 30 .9 33.1 32.6 32.1 35.4 31.8 33.4 31.5 33.6 31 .1 29.0 31 .9 27.5 33.6 33.3 34 .6 33.6 31.3 32.1 29.7 31.0 32.8 32.9 32.3 36.0 32.5 34.5 32 .1 34 .8 32.4 29.4 32 .8 28.6 34 .5 34.3 35.9 34 .1 31.8 32 .8 30.2 31 .5 33.7 33.6 33.3 36.5 32 .4 34 .8 32.2 35.0 32.3 29.7 32.8 29.0 34 .9 34.0 35.0 33.8 31.5 32.5 30 .4 31.6 33 .6 33.5 32 .2 36.1 32.4 35.4 31. 9 35.2 32 .8 29.8 33.2 29.7 35.0 33.9 35.1 33.6 31.4 32.0 30.5 31.4 33.5 33.3 31 .6 35.5 31.9 34.7 31.6 34.5 32 .1 29.7 33.0 29.5 34 .3 34 .4 34 .3 33.6 31 .9 31 .7 30.5 31.2 33.6 32.4 31.4 35 .6 31 .2 34.2 31.6 35.2 32.1 29.7 32.5 28 .8 34 .0 33.9 33.8 33.8 30.8 32.1 30.4 30.8 33.5 32 .9 31 .1 35 .6 31 .9 33 .8 31.3 34 .5 31 .6 29.3 32 .4 28.6 34 .4 p. 89 - 89- >o 0^ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 0 Body Weight g Day 2 Body Weight g Day 3 Body Weight g Day 4 Body Weight g Day 5 Body Weight g Day 6 Body Weight g Day 7 Body Weight g Day 8 Body Weight g Day 9 Male, VII 10 mg/kg 701 33 .7 702 34.5 703 31.9 704 32.2 705 32 . / 706 29.2 707 31.2 708 32.8 709 34 .2 710 30.8 711 35.0 712 31.5 713 35.7 714 32.1 715 32.1 716 31 .5 717 29.5 718 31 .8 719 31.3 720 33.0 33.7 30.5 35.1 31 .3 32.3 31 .2 29.2 31 .8 31.2 32 .9 34.3 35.5 32.0 32 .9 32.5 29.9 31.5 33.1 35.0 32.1 33.8 30.4 36.3 31.5 32.3 31.2 29. 1 32 .9 31.5 33.1 34 .6 35.2 32 .9 33 .2 33 .3 30.7 31 .1 33.2 35.2 32.3 34.4 31 .2 37.4 28 .9 33.2 32.7 30.3 33.4 32.8 34 .5 34 .2 35.6 32 .2 34 .5 33.3 31.0 31 .0 33,7 35.4 32 .7 34.0 30.7 37 .8 28.7 33.0 32 .0 30.2 33.0 32.1 33 .7 33.4 34 .6 31.6 33.7 33.1 30 .8 30.2 32.8 35.1 31 .8 32.8 30.3 37.2 27 .4 32.1 29.9 29.5 32.7 30.5 33 .0 33.6 33.3 32.0 34 .4 33.4 30.3 28.6 33.0 34 .7 31 .2 32.1 29.7 36.8 27 .4 31.9 30 .4 28.9 31 .6 30.4 32 .3 33.0 32.7 32.0 32 .9 32 .9 29.9 28.7 31 .7 33.5 31 .2 31 .4 29.2 36 .2 27 .3 31 .9 30.4 28.4 31 .6 30.2 30.6 33.0 31.9 31.0 31 .9 32.5 29. 1 26.8 32.3 32 .9 30.4 31 .0 29.6 36.4 27 .8 30.1 28.2 27.7 31 .6 29.8 31.5 p. 90 - 90- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Qw K> C\ \X 1 -- Individual Body Weights Body Weight g Day 0 Male, IX 30 mg/kg Body Weight g Day 2 Body Weight g Day 3 Body Weight g Day 4 Body Weight g Day 5 Body Weight g Day 6 Body Weight g Day 7 Body Weight g Day 8 Body Weight g Day 9 901 902 903 904 905 906 90 / 908 909 910 911 912 913 914 915 916 917 918 919 920 33.7 35.9 32.3 31 .2 33.4 29.4 30.4 31.2 34 .2 33.7 37.5 33.1 33 .4 31 .3 34.7 31 .2 28 .7 32.4 31. 1 35.1 38 .9 33.0 33.2 31.2 35.8 31 .6 29.8 32 .8 31.7 35.2 33 .5 36.3 32 .1 30.1 34 .5 30 .1 30 .1 32 .6 34 .9 34 .4 38 .6 31 .6 33.3 32.1 36.0 31.8 29.3 33 .3 31.8 35 .9 31.4 33.8 30.8 27 .6 32.5 29.1 29.0 30.6 33 .6 33.4 37.9 32.6 33 .3 33.1 35.9 30.7 29.4 32.8 31.5 36.5 30.5 32 .3 29.8 28.6 29.3 28.8 27 .7 30.0 32.5 32.0 35.5 31.0 33.1 32 .5 35.1 29.9 30.0 32 .3 30 .9 35.7 28.4 30.1 28.8 24.4 26.5 26.1 26.3 27 .3 30.7 28.4 33.5 30 .3 31 .7 31 .1 34 .5 29.2 30.1 31 .2 30.2 35.0 25.5 27 .4 28.4 22.6 24 .2 24 .6 24 .4 25.7 28.5 26.2 30.2 29.5 31 .5 30 .9 32 .9 27 .8 28.9 29.9 29.0 34 .3 23.0 26.1 27.7 24 .1 23 .7 23.4 23.0 25.9 26.5 24 .8 30.0 28.6 31 .5 29.9 33.6 29.8 29.5 29.8 29.1 35.1 23 .6 25.0 27.2 25 .3 24.7 21.9 22.4 26.4 26.4 24.8 30 .1 27.8 30.7 29.3 32.4 28.3 29.2 29.9 29.0 34 .0 p. 91 -91 - \\ % Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 0 Body Weight g Day 2 Body Weight g Day 3 Body Weight g Day 4 Body Weight g Day 5 Body Weight g Day 6 Body Weight g Day 7 Body Weight g Day 8 Body Weight g Day 9 Male, XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 32 .4 36.0 32 .7 33.1 33.0 30.1 30.4 32.6 34 .4 31 .4 36.6 31 .4 33.3 31.7 32.3 32.5 32.2 32.5 31 .6 33.4 35.6 31.7 33.5 32.4 32.1 31 .8 31 .6 32.8 32.1 33.2 33 .1 36 .9 34 .7 33.6 34 .6 31 .4 31 .1 33 .7 36.1 33 .2 35.5 29.8 34 .4 32.1 31.5 31 .2 30.9 32.4 29.8 33.6 32.0 35.8 33.9 33.3 32 .8 2 9.7 29.8 32.0 33.3 31 .9 36.0 28 .4 33.1 32.2 31.4 30.6 30.7 31.6 28.1 33.9 32.5 35 .9 33.9 32 .6 31 .6 28 .1 29.9 31.8 32 .2 30.9 34 .3 27.5 32.7 31.9 30.6 28.0 29.0 31.4 26.3 29.4 30.0 35 .6 33.0 30.8 28 .6 26.2 26.9 29.3 29.7 30 .9 32 .9 30.8 30.8 30.7 25.0 25.9 29.0 24.1 27.9 30.2 34.5 33.6 29.6 27.6 24 .8 25.5 26.8 27 .3 29.9 30.6 30.2 28.6 29.4 23.4 23.4 28.1 24 .7 25.2 30.2 35.8 33.5 29.5 27.3 26.7 25.8 24 .7 27 .1 31 .9 30.5 32 .8 28.5 30.4 24 .6 21.3 29.0 25.4 23.9 29.4 35.4 33.5 29.2 29.7 27 .1 25.7 22.6 25.8 31.9 30.3 32.3 26.5 29.5 24 .9 20.8 29.3 24.3 24 .8 p. 92 - 92- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 10 Male, I 0 mg/kg Body Weight g Day 11 Body Weight g Day 12 Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Body Weight g Day 17 Body Weight g Day 18 101 31.4 102 34 .5 30.8 34 .6 30 .8 34 .7 30 .3 34 .6 30.9 35.0 30 .7 34.2 30.1 34 .4 30.7 34.7 31 .1 34 .6 103 32 .5 32.3 32.5 32.7 33.0 33 .5 32 .9 33.1 33 .9 104 31 .0 31.2 31.7 32.0 32.6 31.8 32 .0 32.8 33.2 105 32 .8 33.4 33.7 34.2 34.0 34.2 33.1 34 .1 34 .1 106 29.7 107 30.5 29.6 30.9 29.2 31.1 30.0 31.0 29.9 31.3 29.8 31.4 29.7 30.7 30.2 31 .6 30.4 31.6 108 31 .6 31 .9 31. 1 31.3 31 .3 31.7 31.0 31.9 31.4 109 34 .1 34 .0 34.7 34.3 34.0 34.2 33.4 34.4 34.4 110 34 .4 33.8 34.0 33.7 33.9 34 .1 33.8 34 .4 34.4 111 34.3 35.6 35.8 36.1 37.0 34 .7 35.7 36.4 36.0 112 32 .2 32 .5 32.5 32 .6 32.7 32.3 32.3 33.1 33.4 113 34 .1 34.1 33.7 33.6 34 .1 33.5 34 .1 33.9 34.4 114 31.2 31 .9 31.7 32.2 32 .0 32.2 32.4 32.2 32.7 115 30 .1 30.3 30.6 30.5 30.8 30.3 30.8 31 .0 32.1 116 31 .8 32 .2 32.4 32.2 32 .6 32.8 32.9 33.2 33.5 ' i O 117 \\ 118 33.6 33.8 33.5 33.6 34 .4 34 .3 34.7 34 .6 35.2 1 1 9 31.8 32.5 32 .6 32.5 33 .1 32.4 32 .8 32.9 33 .2 v> 120 35 .1 36.0 35.6 34.3 34 .9 35.2 35.4 35.7 35.7 p. 93 -93 - "N T * Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 10 Body Weight g Day 11 Body Weight g Day 12 Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Body Weight g Day 1 / Body Weight g Day 18 Male, III 0.3 mg/kg 301 302 303 304 305 306 30 1 308 309 310 311 312 313 314 315 316 311 318 319 320 33 .2 34 .5 35 .6 34 .1 32. 9 31 .,8 29,,9 32 ,8 31,.5 33,.5 35 .9 31 .6 33 .2 31 .5 33 .6 32 .7 30 .1 30 .3 29 .8 33 .4 33 .3 34 .6 35 .7 34 .0 32 .8 31 .8 30 ,3 32 ,2 31 ,.8 33 ,3 36 ,.1 31 .8 33 .6 32 .2 34 .1 33 .4 30 .5 30 .5 30 .2 34 .5 33 .6 34 .5 35 .4 34 .8 33 .3 32 .,0 30. 2 33 .,0 31 ,.8 33 .,8 36,,0 32 ,.0 34 ,.1 32,.0 33 .6 33 ,0 30 .2 30 .3 30 .5 34 .7 33 .7 35 .2 35. 9 34 .,6 33. 4 32 .,6 30 ,1 32 ,2 31 ,5 34 ,0 35,.9 31 .9 33,.4 31 .8 33 .7 32 .9 30 .2 30 .7 30 .0 35 .0 33 .8 35.,0 35 .8 34 ..6 33..2 32 .6 30 ,5 33.,0 31.,0 33 ,.9 36 .0 31 .9 33 .6 32 .9 34 .0 32 .3 29 .9 30 .1 30 .3 35 .3 33 .8 35 .0 35 .7 34 .8 33 .5 32 .5 30 .5 33 .,7 31..6 34 ,.3 35 .9 31 .6 33 .3 32 .2 33 .4 31 .7 30 .2 29 .6 29 .7 35 .4 34 .0 34 ..1 36 ,.1 34 ,.2 32 ,9 32 ,.3 29 .8 32 ,6 30..8 34 .0 36..6 32 .7 33 .5 32 .8 33 .7 32 .1 30 .3 30 .4 30 .4 36 .4 34 .3 35 .1 36 .7 34 .9 34 .0 33 .0 30 .7 33. 8 31..6 34 ,.4 37 .1 32 .6 33 .4 32 .4 33..9 32 .3 30 .3 30 .0 29 .7 36 .4 34 .3 35 .3 36 .7 35 .1 33 .9 33. 0 30 .5 33 .,1 31 .7 34 .1 36 .8 32 .8 33 .7 33..1 34 .2 32 .0 30 .7 29 .8 30 .7 36 .5 p. 94 - 94- 0\ \\ }J Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 10 Male, V 1 mg/kg Body Weight g Day 11 Body Weight g Day 12 Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Body Weight g Day 17 Body Weight g Day 18 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 51 / 518 519 520 33,,8 33,.6 34 ,3 31,,1 31 .9 30. 8 31 .0 33 .6 32 .6 30. 6 35 .2 32 .0 34 .4 31 .5 34 .8 31 .6 29. 6 33. 0 28. 5 34 .5 34 .1 33 .7 34 .4 31 .4 32 .5 31 ,4 31 ,.0 33 ,9 33 ,0 30 .,2 35 .,5 32 .1 35 .2 31 .7 35. 3 31. 9 30 .0 33 .3 28. 5 34 .8 34 .,3 33. 4 34 .2 31 .2 32 .3 31. 7 31. 1 33 .4 33. 2 30 .5 35. 4 32 .3 34 .7 32 .2 35. 1 32. 1 30. 2 33 .3 29. 1 35 .2 33.,8 33 ..8 34 .6 31 .6 32 .7 31 .5 30. 8 34 .0 33. 3 30. 4 35. 4 32 .5 35. 0 31. 7 35. 0 32 .2 30. 0 33. 3 29 .3 35. 5 34 .5 34 .4 34 .9 31 .3 32 .5 31 .9 30 .7 34 .2 33 .2 30 .6 35 .3 32 .9 35. 4 32 .0 35 .9 32 .2 30. 3 33 .4 29 .3 36 .1 34 .7 34 .1 35. 0 31. 9 32 .4 32 .6 31. 2 34 .5 33. 8 31 .1 34 .7 32. 8 34 .6 31. 3 35 .2 31 .7 30 .7 33. 2 28 .6 34 .9 34 ,2 33..5 34 .4 31 .2 32 .3 32 .5 30 .9 33 .9 33 .6 31 .0 35 .4 33 .2 34 .7 32 .0 35. 9 32 .3 30 .9 34 .0 29 .3 35. 6 34 .8 33. 9 35 .1 32 .4 32 .9 33 .3 31. 4 34 .6 34 .0 31 .9 35 .7 33 .4 35 .6 32 .2 36. 5 33. 5 31 .3 34 .2 29. 5 35 .8 34 .8 34 .0 34 .7 32 .7 33. 2 33. 7 31 .3 34 .8 34 .8 31 .8 35. 5 33 .9 35. 9 32. 4 36. 8 33. 5 31. 4 35 .2 30 .2 36 .3 p. 95 - 95- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 10 Body Weight g Day 11 Body Weight g Day 12 Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Body Weight g Day 17 Body Weight g Day 18 Male, VII 10 mg/kg 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 /17 718 719 720 32 .4 32 .8 30. 9 31. 4 32 .4 28 .9 26 .1 33. 7 32 .8 30 .0 30 ,2 29..6 35.,7 28 .0 30 .4 28 .2 27 .2 32 .1 30 .0 30 .0 32 .7 31 .8 31 .4 31 .2 30 .5 27 .2 25..4 31 .7 31 .7 29..6 29 .5 30 .,0 35 .6 28 .9 29 .9 29 .8 27 .1 32 .1 29 .5 31 .3 31 .1 32 .5 30 .9 31 .0 30. 7 27 .0 24 .8 31. 6 31 .3 30 .2 29 .1 29 .0 34 ,4 29 .0 29 .1 28 .5 27 .,9 30 .7 29 .2 29 .3 31 .2 32 .4 31 .4 29. 6 32 .1 28 .3 25 .1 32 .0 31 .9 29. 4 28 .7 29 .8 34 .6 30,.1 28 .,9 27 .5 27 .1 31 .1 30 .2 29 .0 32 .0 32 .2 30 .2 29. 1 30..7 28 .3 24 ..1 31 .,4 32 .6 29 .5 28 .8 29 .3 34 .4 29 .0 28 .8 26 .9 26 .8 31 .1 29 .9 28 .2 31 .8 32 .5 31. 1 28 .6 31 .2 28 .2 24 .9 31 .1 32 .3 30. 2 28 .5 29 .1 33 .4 29 .5 29..4 28..1 26..8 30..5 29..1 27 .9 31 .6 31 .7 29 .7 28 .3 31 .3 28 ..1 25 .4 30. 7 30 .5 29..6 27 .6 29,.4 33 .2 29 .8 29 .4 26 .3 26 .9 30 .8 29 .5 28 .1 31 .9 32 .8 29 .8 29 .4 30 .3 27 ..9 25 .1 30 .8 30 ..4 29 .,1 27 .,3 29 .3 33 ,2 30 .0 28 .9 26 .9 26 .9 30 .6 30 .9 27 .0 32 .1 32 .3 30 .3 29 .0 30 .8 27 .8 25 .0 30 .6 30 ..7 29..3 27 .1 29..9 32 ,2 29 .1 29 ,4 29 .1 26 .8 33 .5 30 .3 27 .8 ZLX p. 96 - 96- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 10 Body Weight g Day 11 Body Weight g Day 12 Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Body Weight g Day 17 Body Weight g Day 18 Male, IX 30 mg/kg 901 25 .4 902 25 .8 903 26 .9 901 26 .5 905 27 .5 906 29 .8 28 .5 26 .5 26 .5 28 .1 31 .8 30 .5 25 .9 26 .4 28 .9 32 .4 31 .4 25..2 26..1 29,,3 32 .5 31 .9 25 .2 25 .8 29 .3 31 .3 30 .5 24 .7 26 .4 28..1 29 .8 27 .8 25 .4 25 .1 26 .8 28 .9 26 .5 25 .1 25 .4 28 .0 26 .7 24 .8 24 .9 24 .9 25 .8 907 21 .9 21 .2 21,,6 22 .3 23 .0 22 .9 23 .4 23 .5 23 ,5 908 27 .2 26 .0 26..2 26.,4 25 .5 26 .5 25 .2 26 ,0 25 .9 909 28 .1 28 .9 29.,1 29 .3 29 .1 28 .2 26 .9 25 .6 25 .,0 910 25 .2 25 ,0 28 .1 29. 6 29 .4 30,,1 28 .9 28 .4 26 .2 911 29,,8 29 .,8 29 .3 28. 6 27 .8 28 ,1 28 .1 28 .4 27 .1 912 27 ,1 26 ,5 25 .5 25 .3 25 .0 24 .8 24 .6 24 .3 24 .0 913 30 .,7 30 ,6 30 .9 29. 5 29 .2 28 .9 29 .0 28 ,.8 28 .8 914 29 ,6 30 ,2 30. 1 29 .8 29 .6 29 .6 29 .4 28 ,8 29..2 V 915 31 .7 916 27 .5 32 ..3 28 .0 31 .9 27 .9 32 .1 27. 0 32 .4 25 .9 32 .6 26..6 33 .2 26 .5 32 .5 25..9 33 .0 25. 2 917 29. 3 29 .4 28. 6 28 .7 29,,4 29. 0 29.,9 29..7 30. 3 918 29 .2 29. 5 29. 1 28. 9 28 .8 28. 2 28 .2 28 .5 28 .8 Tv 919 29. 4 29 .3 29. 9 28 .9 29 .3 28. 5 28 .7 28 .2 28. 0 920 32 .6 33. 2 34 .1 34 .1 33..4 33. 5 33. 1 32 .3 31. 5 p. 97 - 97- \ %} '-4 V Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 10 Body Weight g Day 11 Body Weight g Day 12 Body Weight g Day 13 Body Weight g Day 14 Body Weight g Day 15 Body Weight g Day 16 Body Weight g Day 17 Body Weight g Day 18 Male, XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 30 .1 35.1 32.2 28.7 31 .1 27.4 24 .4 22.0 25.9 30.8 30.4 31.8 28.3 28.8 24.0 21.6 28 .1 24 .7 26 .8 28.8 33.4 31 .7 29.1 31.1 24 .9 25.2 22.3 27.0 31.3 30.5 31.4 27.9 29.0 24 .9 23.8 28 .1 25.8 28.7 28.4 33.0 31.2 29.6 31.7 26.0 24 .6 24.8 33.2 28.6 30.3 31.4 27.1 28.9 24.6 25.7 27 .2 24 .1 28.8 28.5 31.7 29.9 29.5 32.4 26.8 24.3 27.4 33.9 27 .2 29.7 30.0 27.0 28 .9 24.0 27 .3 26.9 23 .9 26.5 28 .1 30 .9 29.3 29.0 32 .0 26.0 23.9 28.8 34 .2 27.6 29.4 29.4 27 .4 28 .4 23.2 28 .0 25.8 25.2 26.2 29.0 32 .6 29.6 29.4 30.8 26.3 24.8 30.4 33.2 28.0 29.7 28.7 26.8 28.2 24.6 25.4 25.3 23.8 25.5 27.2 31 .2 28.4 27.8 30.4 25 .9 24 .2 28 .7 29.2 27 .0 29.5 28.9 27.7 28 .5 23.8 26.5 25.7 21 .9 25 .1 27 .6 32 .1 27 .7 27 .9 29.5 27 .5 23.6 28 .6 26.6 29.1 29.4 29.5 26.8 29.6 23.5 24.8 24 .9 22 .7 25 .3 27 .1 31 .6 27.3 27.9 29 .9 27 .6 23.0 26.4 24.2 30.2 29.4 30.3 26.5 29.3 23.4 25.0 24.8 23.2 24 .6 p. 98 - 98- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 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 Male, III 0.3 mg/ kg 301 34 .8 34 .8 35..0 35 .2 35 ,0 35. 4 36. 3 36 .0 36.4 302 36 .2 35 .6 35..8 36. 3 36.,3 36. 4 36. 9 35 .9 36.7 303 37 .0 36 .4 36 .6 36. 7 36..6 36. 3 37 .3 37 .2 37 .6 304 35,.8 35 .6 35. 7 36. 1 36 .3 36. 4 37 .1 37 .5 37.7 305 34 .,6 34 .3 34 .6 34 .1 35. 4 35 .0 35 .1 35..6 36.0 306 33..7 33,.3 33. 2 33 .5 33 .8 33 .2 33 .9 33 .7 33 .4 307 30 .8 30 .,7 31 .1 31 .3 31 .4 31 .1 31 .5 31 .,4 31.5 308 34 .0 34 ,.3 33. 9 34 .1 34 .0 34 .7 35. 1 34 .,7 35.1 309 31 .8 31 ..5 32. 2 31 .7 31 .7 31 .6 31 .5 31 .,6 31.6 310 34 .4 34 .2 34 .5 33. 7 34 .4 34 .8 34 .9 35 .1 35.1 311 36. 6 37 .0 37 .5 36 .9 37 .2 36. 7 37 .2 37 .3 37 .5 312 32 .4 33 .1 33. 3 32 .7 33 .2 33 .3 33 .8 33. 5 33.8 313 33. 3 33 .3 33. 8 33. 9 33 .8 34 .0 32 .9 32 .7 32 .9 314 32 .7 32 .9 32 .9 32. 6 32 .4 32 .5 33 .1 33 .1 32 .9 315 33. 6 34 .0 34 .5 33 .7 34 .3 34 .5 35 .1 34 .1 34 .9 316 31. 4 32 .0 32. 4 31 .5 32 .2 32 .5 32 .7 32 .2 31 .9 N} 317 30. 3 30 .9 31 .1 30. 3 30 .3 29. 9 30. 1 30 .1 30.3 318 29. 9 30. 0 30. 8 30. 2 30 .8 30 .9 31 .3 31. 1 31.2 319 30 .2 30 .8 31 .0 30. 9 31 .1 30 .8 31 .5 31. 2 31.4 320 35 .8 36 .5 36. 4 37. 1 36 .8 37 .0 37 .5 37 .2 36.8 p. 99 - 100- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight q Day 19 Body Weight g Day 20 Body Weight g Day 21 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 Male, I 0 mg/kg 1 0 1 31. 0 31 .2 31.2 31 .1 31 .5 31. 1 31 .2 31. 4 31.4 102 35.1 34 .9 35 .2 35.0 35.1 34 .9 35.6 35.8 36.4 103 34 .4 34.3 34 .1 33.7 33.4 34.0 34 .5 33.8 33.9 104 33.3 33.4 33.4 32.2 32 .0 32.5 32.7 32.4 33.3 105 34 .4 34 .0 34.4 33.6 33.6 34.5 34 .2 33.5 33.8 106 31 .0 30.8 30.7 31 .2 31.5 31.0 31.4 31.4 31.1 107 32.2 31.7 32.1 32.4 31.7 32.3 32.4 32.7 33.4 108 32.1 31.9 31 .8 32.2 31 .8 31.5 31.5 31 .1 31.2 109 35 .3 34.8 34.3 34.3 34.2 33.9 33.9 34 .3 33.8 110 35.0 34 .7 34.7 34 .9 34 .5 34.4 34.6 34 .6 35.2 111 36.5 36.4 35.7 35 .6 35.4 34.5 35.5 35.6 34.5 112 32.9 33.6 33.7 33.4 33.5 33.7 33.9 34 .2 34.4 113 33.9 34 .2 34 .4 33.6 34.5 34.5 34 .4 34.5 34.2 114 32 .1 32 . 9 33.7 33.4 33.6 33.7 34 . 2 33.6 33.7 115 31.2 32.0 32. 0 32. 1 31 . 9 31. 7 32. 6 32 . 4 31.3 1 1 6 33.5 34.0 34.3 33.9 34.1 33.7 33.8 33.4 33.7 117 118 35.0 35.0 35. 7 35.2 35.8 35. 4 35.8 35. 4 34. 4 119 3 3 . 3 33.8 33. 9 33.4 33 . 8 33.8 34 . 2 34 . 4 34 . 7 M 1 2 0 35.4 35. 7 35.5 34 . 9 35. 4 35. 0 35. 1 35.3 34 . 6 p. 100 - 99- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 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 Male, V 1 mg/kg 501 34 .7 502 34 .1 503 35 .1 504 32..7 505 32.,9 506 34 .3 507 31 .2 508 34 ,.6 509 34.,4 510 32..8 511 35. 4 512 33. 6 513 35. 2 514 32 .1 515 35. 3 516 32 .7 517 31 .1 518 34 .5 519 29. 5 520 35. 4 34 .5 33 .7 35 .0 32 .9 33 .2 34 .6 31 .3 34 .6 34 .9 32 .7 36..1 33 .,7 35 .4 32 .3 36 ,2 32 .8 31 .4 34 .9 30. 5 35 .7 35 .1 33 .9 34 .8 33 .3 33 .0 34 .,4 31 ,.0 34 .6 35 .0 32..8 36. 0 34 .2 36. 1 32 .2 37 .0 33. 4 32 .3 36. 2 31. 0 36. 9 34 .2 33 .5 34 .2 32 .9 32 .7 35 .0 30 .7 34 .6 34 .7 33..0 35..6 34 .1 35 .3 32 .6 36.,9 32..3 32 .0 36. 0 30. 9 35 .7 34 .3 33 .2 34 .5 33 .2 33 .0 35 .1 31 .,0 34 ,.9 34 .9 33.,3 36.,6 34 ,6 36..5 32 ,5 35..8 32 .6 32 .3 36. 2 31 .1 35 .8 34 .,7 33..5 34 .5 33..6 33 .2 35.,3 31 .4 34 ,.7 35..5 32..9 37 .0 34 .6 36. 2 32 .8 36 .0 32 .7 32 .1 35 .6 31 .2 36. 7 34 .4 33 .8 34 .7 33 .7 33 .8 35 .9 31 .6 35 .2 35 .5 33 .2 37 .4 35..7 36..5 32 .8 36..5 32 .8 32 ,7 36 .2 31 .3 37 ..1 34 .2 33 .6 34 .3 33 .9 33 .9 35 .3 31 .6 34 ,8 35,,0 32 .,5 37 .,5 35,,3 36..8 32 .. 0 36..4 32 .8 32 .5 36 .7 31 .6 36 .4 34 .0 34 . 0 34 .3 34 .,2 33..4 35..6 31 ,7 35,.2 35..3 33,. 0 38 ,. 0 35..4 36,.8 32 ..5 36. 4 32 .6 32 .7 36 .0 31 .6 35 .9 p. 101 3L7V - 101 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 Body Weight g Day 22 Body Weight g Day 23 Body Weight g Day 24 Body Weight g Day 25 Body Weight g Day 2 6 Body Weight g Day 27 Male, VII 10 mg/kg 701 702 703 704 705 706 707 /0 8 709 710 711 712 713 714 715 716 717 718 719 720 32 .4 32 .0 30 .3 29 .3 30. 0 27 .5 24 .6 30 .6 29 ,.9 29 .1 26..4 29 ,7 31 .6 28 .8 28 .4 27 .3 26 .5 29 .8 30 .6 27 .2 31 .8 32 .8 29 .5 28 .8 29 .6 27 .8 24 .3 31 .2 29 .7 28 .8 26 ,.0 29 .8 31 .6 28 .6 28 .6 27 .2 26 .3 29 .4 29 .9 27 .7 32 .0 32 .6 29. 2 28. 6 29. 7 27 ..3 24 ,.7 32 .1 29 .7 28 .6 27 .1 30 .7 31 .4 29 .2 28 .0 29 .1 26 .4 28 .6 30 .9 28 .1 31 .5 31. 7 29 .7 29. 1 30 .3 28 .2 25 .6 32 .3 30 .1 28 .9 27 .4 30 .3 31 .2 29..6 29 .3 27 .6 26.,6 28..3 31 .2 29 .1 30 .9 31 ..3 29 .3 28 ,.8 29..8 28 .3 26..0 32 .8 29 ,4 28 ,4 28 ,3 29 .8 31 .0 29 .1 29 .0 27 .1 27 .1 28 .5 30 .8 29 .1 31. 4 32 .7 29. 1 29 .0 29. 5 28 .6 25. 8 32 ..0 28 ,.6 28 .0 29 .0 30 .5 31 .2 30..2 28 .,9 28..0 26 .6 28 .6 30 .5 28 .8 31 .7 32 ,5 28 .9 28 ,.7 29 .4 28 .2 26 .0 31 .7 28 .4 27 .2 28 7 30 .2 30 .8 30 .0 28 .0 27 .7 25 .8 28 .0 29 .3 28 .3 31,.4 32 .,7 28 .6 28 .8 28 .6 27 .,8 25 .4 31 .1 28 .,1 27 .4 27 .4 30 .6 30 .3 30 .2 27 .7 28 .0 25 .6 29 .6 30 .1 28 .9 31 .8 31 .4 28 .9 28 .6 29. 1 27 .5 24 ..5 30 .6 28 ,.3 28 .0 27 .2 30 .1 30..3 29.,7 28 .2 27 .1 25 .7 30 .0 29 .5 26 .9 Ur p. 102 - 102 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 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 Male, IX 30 mg/kg 901 25.2 902 23.5 903 25.6 904 23.6 90b 24 .7 906 907 23.7 908 25.6 909 25.2 910 25.8 911 27.0 912 23.8 913 28.6 914 30.0 915 32.1 916 24 .9 917 29.8 918 27.9 919 27.8 920 30.3 23.3 22.9 25.0 26.3 24 .0 23.7 2 5.5 24.4 26.0 27.7 23.6 28.6 28.8 32.5 24 .8 29.8 28.1 28.1 29.5 21.5 22 .8 24 .7 23.7 22 .2 23.6 25.7 24 .0 24.7 27.8 22 .7 28.5 28.2 33.0 25.0 30.1 28.5 28.2 28.7 23.8 24.0 25 .1 24 .6 21.6 23.6 26.6 25.2 25.1 29.5 23 .2 29.4 29.9 33.4 25 .6 30.3 28.4 27.5 28 .4 24.8 23.3 24 .9 24 .1 23 .4 23 .5 26.3 26.7 25 .9 29.8 23 .6 29.3 29.3 32 .9 25.5 29.9 27.9 27.3 28.2 24.4 24.0 24.9 24.8 22 .5 23.4 26.3 26.3 26.4 29.5 22 .6 28.9 29.1 32.6 25.3 29.7 28.1 27.0 28.9 24 .9 24.0 24 .7 24 .0 23.7 23.1 26.3 26.1 26.9 28.4 22 .3 28 .6 28.2 32.5 24 .7 29.4 28.1 26.9 29.3 25 .9 25.0 24 .8 24 .6 24 .3 22 .4 26.5 25 .6 28 .1 27 .8 22 .1 28 .4 28.7 33.1 24 .8 29.8 28.0 26.6 29.0 26.7 24 .6 24 .4 24 .4 24 .3 21.9 25.7 25.3 28.4 26.4 22 .0 28.2 27.8 32.5 24.0 30 .4 28.5 26.9 28.9 09? p. 103 - 103 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Body Weights Body Weight g Day 19 Body Weight g Day 20 Body Weight g Day 21 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 M a l e , XI 30/0 mg/kg (Recovery) 1101 27.9 28 .1 27 .4 29.1 29.4 29.0 29.3 30 .1 30.2 1102 32.6 32 .9 32.6 33.1 33.8 32.7 34 .0 34 .2 36.8 1103 27 .0 25.9 26.1 28 .6 28 .7 28.5 28.9 30 .2 31 .4 1104 27 .1 27 .7 26.0 26.4 28 .1 27 .6 27 .3 27.9 28.7 1105 28.1 29.2 27.4 30.7 33 .2 31.4 32 .5 32.6 31 .8 1106 27.5 27.0 25 .9 25 .7 26.2 25.4 25.6 25.5 25.7 110 / 22.9 23.1 23.3 25.0 24 .8 23.9 23.6 23.6 24.3 1108 24 .8 24 .2 23.6 23.4 23.3 23 .6 25.2 26.0 28.4 1109 22 .5 22.9 22 .7 24.5 25.5 27.3 29.7 32 .6 34 .3 1110 29.8 28.4 26.0 23 .4 22.3 22.3 22.6 23.4 24 .6 1111 28 .4 28.8 28.5 28.5 28.3 28.5 28.5 29.2 29.4 1112 1113 31.1 30.8 30.7 31.3 31.1 30.4 30.3 31.2 32.2 1114 26.6 25.8 25.5 26.9 28.2 27 .6 26.5 28.2 29.5 Kj 1115 28.9 28 .9 29.2 29.2 29.3 29.3 29.1 29.9 29.7 CO 1116 22.8 23.7 22.9 23.2 24 .3 23.9 23.6 23.8 24 .1 1117 24.0 23.9 23.6 23.8 23.6 25.0 23 .6 26.0 25.4 1118 25.0 26.0 26.4 26.4 27 .8 26.6 26.9 27 .0 27 .7 1119 21.4 22.8 22 .4 23.2 23 .6 23.3 24 .5 24 .2 23.9 1120 25.1 24 .4 24 .2 25.0 25.5 25.7 26.6 27 .6 27 .9 p. 104 - 104- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Body Weight g Day 28 Male, I 0 mg/kg 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 31. 3 35.8 34.4 33 .6 33.7 30.9 32.3 30.7 33.1 34 .4 34.0 34 .0 34.5 33 .6 31.2 33.4 34.8 34.3 35.0 Individual Body Weights DuPont-18318 Ig? p. 105 - 105 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Body Weight g Day 28 M a l e , III 0.3 mg /kg 301 35. 7 302 36. 0 303 3 7.6 304 38. 0 305 3b. 2 306 33. 1 307 31 .1 308 33 .8 309 31 .4 310 34 .,7 311 37 ,5 312 34 .0 313 32 .5 314 32 .4 315 34 .6 316 31 .8 317 30 .0 318 31 .0 319 31 .0 320 36 .7 Individual Body Weights DuPont-18318 2 p. 106 - 106- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice -A Body Weight g Day 28 Male, V 1 mg/kg 501 33.8 502 34 .0 503 33.8 504 33.5 505 33.1 506 35.5 507 31. 9 508 34 .5 509 35.0 510 32 .6 511 37.0 512 35.8 513 36.8 514 32.2 515 36.3 516 32.2 517 32.7 518 35.7 519 30.7 520 36.2 Individual Body Weights DuPont-18318 p. 107 - 107 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Body Weight g Day 28 Male, VII 10 mg. 7 01 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 32.5 31 .4 28 .4 28.2 28.7 27.2 24 .4 31.1 27 .4 27 .2 26 .9 29.6 31. 9 29.4 27 .1 27 .5 26.1 30.2 29.2 26.9 Individual Body Weights DuPont-18318 p. 108 - 108- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice \ Body Weight g Day 28 Male, XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1 119 1 120 30.3 36.9 31.5 29.3 33.0 26.2 25.2 29.9 33.4 26.0 30.3 33.8 30.5 30.7 25.3 26.7 28.2 24.8 27.9 Individual Body Weights DuPont-18318 p. 109 - 110- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice n AJ *1 Body Weight g Day 28 Male, IX 30 mg/kg 901 26.2 902 24 .1 903 24.1 904 23 .6 905 24 .1 906 907 21.5 908 25.0 909 24 .5 910 28.9 911 26.2 912 22 .1 913 28 .2 914 27.9 915 33.2 916 23.5 917 29.7 918 28 .9 919 27 .9 920 29.0 Individual Body Weights DuPont-18318 p. 110 - 109- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 111 DuPont-18318 Appendix C Individual Final Body and Liver Weights m-111 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 112 DuPont-18318 INDIVIDUAL FINAL BODY AND LIVER WEIGHTS ABBREVIATIONS: FBW - final body weight na - not applicable NW - not weighed S.D. - standard deviation WE - weighing error EXPLANATORY NOTES FOOTNOTES: a Animal was sacrificed i n e x t r e m i s prior to this analysis. - 112- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Animal 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 MEAN S.D. 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 MEAN S.D. FBW (g) 31.0 34.7 33.4 32.5 33.0 31.2 31. 9 30.3 32.7 33.9 32.6 33 .9 34 .6 33.7 31.5 34 .2 34.5 34.3 33.5 33.0 1.33 35.1 36.1 37.4 37.2 34.5 33.1 31.3 33.7 29.9 32.8 36.6 33.4 31.5 32.2 33.8 31.6 30.2 30.1 30.9 36.7 33.4 2.47 Individual Final Body and Liver Weights Liver (g) 1.623 2.017 1.736 1.768 1.459 1.717 1.861 1 .727 1.711 1.634 1.801 WE WE 2.064 1.718 1.795 na 2 .172 1.859 1.639 1.782 0.18 FBW Liver (g) 29.4 32.7 31 .7 30.7 31 .5 29.5 30.0 28.6 31 .0 32 .3 30.8 na na 31.6 29.8 32.4 na 32 .3 32.4 31.9 31.1 1.26 2 .609 2.405 2.512 2.758 2.520 2.539 2.036 2.372 1.988 2.492 2.896 2.529 2.445 2 .206 2.514 2.294 2.053 2.054 2.199 2.715 2.407 0.25 32.5 33.7 34.9 34 .4 32.0 30.6 29.3 31.3 27 .9 30.3 33.7 30.9 29.1 30.0 31 .3 29.3 28.1 28.0 28.7 34 .0 31.0 2.26 p. 113 DuPont-18318 - 113 - 290 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Animal 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 MEAN S .D . 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 71 / 718 719 720 MEAN S .D . FBW (g> 33 .3 34 .0 33 .5 33 .3 33 .0 35 .1 31 .3 33 .9 33 .5 32 .3 36 .8 36 .0 35 .9 31 .4 35 .7 32 .1 32 .2 35 .0 30 .7 36 .4 33 .8 1.81 31 .8 30 .9 29 .1 28 .2 28 .4 27 .3 24 .7 29.,7 26..6 27 ..2 26..8 28 .9 31 ..6 29 .6 27 .4 27. 0 25. 2 31 .0 30. 0 26. 9 2 8 .4 2.04 Individual Final Body and Liver Weights Liver (q) 3 .274 3 .381 2 .978 3 .647 3 .095 3 .042 2 .992 2 .898 3 .490 3 .017 3 .191 3 .397 3 .539 3 .544 3 .535 3 .317 3 .300 3 .484 2 .996 3 .314 3 .272 0 .23 7 .969 7 .155 8 .574 6. 519 4 .627 4 .783 5 .517 5 .939 6. 075 5 .13 / 4 .228 5 .438 8 .346 5 .068 6 .320 4 .902 5 .396 8 .136 5 .884 5 .205 6 .061 1 .32 Liver (g) 30.0 30.6 30.5 29.7 29.9 32 .1 28 .3 31 .0 30.0 29.3 33.6 32 .6 32 .4 27.9 32.2 28.8 28 .9 31 .5 2 7.7 33.1 30.5 1.76 23.8 23.7 20.5 21.7 23.8 22.5 19.2 23.8 20.5 22 .1 22.6 23.5 23.3 24 .5 21 .1 22.1 19.8 22 .9 24 .1 21.7 22.4 1.53 p. 114 DuPont-18318 - 114- 2 ?/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Animal 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 MEAN S.D. 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 MEAN S.D. FBW (g) 26.2 24.3 23.7 24 .2 24.1 21.8 25.4 23.5 27.0 25.3 22.0 28.3 28.4 32.4 23.3 29.4 28.0 27.1 29.5 26.0 2.84 31.3 39.5 32.2 30.4 34.4 27.2 27.1 32 .3 34 .7 24.9 30.8 34 .7 30.4 31.6 26.9 27.5 28.6 26.2 28.7 30.5 3.66 Individual Final Body and Liver Weights Liver (g) 5.786 5.752 5.441 5.335 4 .810 na 5.128 5 .628 5.189 7.923 5.957 5.463 7.657 5.089 6.732 NW 6.316 5.574 5.895 6.499 5.899 0.85 FBW Liver (g) 20.4 18.5 18.3 18.9 19.3 na 16.7 19.8 18.3 19.1 19.3 16.5 20.6 23.3 25.7 na 23.1 22.4 21.2 23.0 20.2 2.46 8.009 8.361 5.329 6. 914 7.432 6.657 6.001 7.295 6.493 5.491 6.983 na 1 .670 6.359 NW 6.006 6.827 7.310 4 .637 7.271 6.391 1.51 23.3 31.1 26.9 23.5 27.0 20.5 21.1 25.0 28.2 19.4 23.8 na 33.0 24.0 na 20.9 20.7 21.3 21.6 21.4 24.0 3.84 p. 115 DuPont-18318 - 115- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix D Individual Food Consumption - 116- 5 3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice INDIVIDUAL FOOD CONSUMPTION EXPLANATORY NOTES ABBREVIATIONS : Cons . g/anm/day consumption grams of food consumed per animal per day p. 117 DuPont-18318 -117- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Food Consumption Food C o n s . Food C o n s . Food Cons. g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Food Cons g/anm/day Day 28 Male, I 0 mg/kg 101 4 .9 4 .7 4 .7 4.5 102 5 .6 5 .6 5.9 5.8 103 5.2 5.4 5.2 4 .5 104 4 .5 4 .7 4 .9 4 .2 105 5.3 4.9 5 .4 4.5 106 4.5 5.4 5.3 5.1 107 4 .3 5.0 5.1 4.9 108 4 .7 4 .6 4 .7 4 .1 o lO 109 5.1 4 .8 3 .9 110 5.5 5.5 5.2 4 .6 111 4.9 5.0 4 .8 4 .3 112 5.3 4 .7 5.0 4 .4 113 5.6 4 .9 4 .8 4.3 114 5.1 4 .5 4 .7 4 .8 115 4.5 4 .9 4 .9 4 .8 116 4 .8 5.2 5.2 5.0 118 5.0 5.3 5.6 5.3 119 5.4 5.7 5.6 5.1 120 5.9 5.3 5.4 5.1 M a l e , III 0.3 mg/kg 301 5.3 5.5 5.5 5.4 302 5.3 5.6 5.3 4 .9 303 5.1 6.0 5.3 5.2 304 5 .6 5.7 5.6 5.4 305 5.1 5.4 5.6 5.3 306 4.5 4 .9 5.1 4 .6 307 4.6 4 .8 4 .9 4 .9 308 4 .7 4 .6 4 .7 4 .5 309 5.2 5.2 5.0 4 .3 310 5.9 5.6 5.3 5.4 311 5.7 5.4 5.5 5.2 312 5.2 5.4 5.2 5 .1 313 5.5 5.8 5.3 4 .9 314 4 .9 5.2 5 .1 4 .7 315 5.1 5.2 4 .8 4 .9 316 4 .7 4 .5 3.9 4 .3 317 4 .9 5.0 4 .8 4 .4 318 4 .6 5.0 4 .6 5.1 319 5.1 4.9 4.9 4 .8 320 5.5 5.0 5.2 5.4 p. 118 DuPont-18318 - 118- 3& S Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Food Consumption Food C o n s . 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, V 1 mg/kg 501 5.9 5.8 6.2 5.0 502 6.2 4 .8 5.0 4 .6 503 5.0 5.6 5.0 4.5 504 4 .6 4 .5 4 .9 4 .9 505 5.4 5.3 5 .4 5.2 506 5.1 5.3 5.6 5.4 507 5.1 4 .7 5.2 4 .7 508 5.4 5.7 5.5 5.4 509 4 .6 6.5 3.8 5.0 510 4 .9 5.0 5.3 5.4 511 5.8 5.3 5 .1 5.7 512 5.2 5.1 4 .7 5.4 513 5.6 5.4 5.3 5.5 514 5.3 5.6 4 .9 4 .8 515 5.6 5.8 5.8 5.8 516 5.1 4 .9 5.2 5.0 517 4 .6 4 .7 4 .9 4.9 518 5.0 5.3 5.3 5.6 519 5.0 5.6 5 .1 5.5 520 5.2 5.7 4 .9 5.0 Male, VII 10 mg/kg 701 5.2 6.7 6.9 6.4 702 5.9 6.2 6.4 5.0 703 5.0 5.9 5 .1 4.5 704 5.8 6.0 6 .1 6.1 705 5.1 6.6 5 .7 5.1 706 5.0 5.4 5.2 5.2 707 4 .4 3.4 4 .4 4.3 708 4 .7 6.9 6.1 6 .1 709 5.7 6.9 6.5 5.7 710 5.3 5 .7 5.4 4 .5 711 3.9 5.3 4 .4 5.8 712 4 .4 6.5 5 .9 5 .4 713 5.4 6.3 4 .5 4 .1 714 3.8 5.9 5 .5 6.7 715 5.0 5.4 5.0 6.2 716 4.8 5.2 5.5 5.5 717 4 .7 5.0 4 .1 4 .7 718 5.5 6.2 4 .0 5.9 719 5.0 6.5 4 .1 5.9 720 5.6 4 .9 4 .2 6.9 p. 119 DuPont-18318 - 119- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Food Consumption Food C o n s . Food Cons. g/anm/day g/anm/day Day 7 Day 14 Food Cons. g/anm/day Day 21 Food Cons g/anm/day Day 28 M a l e , IX 30 mg/kg 901 3.7 5 .7 4 .5 4.8 902 3.7 5.4 2 .5 3.7 903 4 .7 4 .5 4 .0 4.4 904 3.3 5 .7 NM 3.5 905 3.3 5.5 3.8 4.0 906 3.9 907 3.2 3 .7 3.5 3.2 908 4 .7 6 .1 4 .2 5.6 909 4.8 5 .9 3.8 4 .1 910 5.0 5.2 4.5 4 .7 911 5.1 5.8 4 .4 5.7 912 5.3 4 .7 3.1 3.7 913 4 .8 4 .9 4 .1 3.9 914 5.1 5.7 4.7 5.1 915 5.7 6.7 7.2 7.3 916 3.9 6.1 4.2 4 .6 917 5.9 7 .0 7 .1 7.3 918 4 .9 6 .0 5.1 4.9 919 4 .3 5.6 4 .5 4 .6 920 5.3 6.4 3.9 5.2 M a l e , XI 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1113 1114 1115 1116 1117 1118 1119 1120 5.2 5.6 5.6 4 .1 5.1 3 .9 4 .4 4 .1 4.3 5.1 4 .1 4.3 5.0 4 .1 3.0 3.4 4 .4 4 .6 3 .9 6.4 5.7 5.4 5.3 7 .7 5 .1 4 .4 4 .8 7 .8 5.8 4.4 5.4 5.9 5.3 4.2 4 .1 5.8 6.3 5 .1 6 .1 5.1 3.9 4.6 6.2 5.5 3.6 5.2 4 .9 4.8 3.9 4 .7 5.5 5 .2 3.8 3.9 4 .1 3.0 3.4 6.1 5.4 5.7 4.0 6.0 4.2 4 .3 4 .4 7.5 3.3 4.5 5.1 6.7 5.5 5.0 5.2 6.8 8.8 5.9 Key: NM = Not Measurable p. 120 DuPont-18318 - 120- 2X1 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 121 DuPont-18318 Appendix E Individual Daily Animal Health Observations - 121 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Sex Group Animal MI MI M1 M1 MI MI MI M1 MI M1 M1 MI MI MI MI 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 M1 MI 116 117 MI MI MI M III M III M III M III M III M III M III M III M III M III M III M III M III M III M III M III M III M III M III M III 118 119 120 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 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 Abnormal Gait, Hindlimb,, Right, Severe General observation, No Abnormality Detected General observation. No Abnormality Detected Sacrificed i n e x t r e m i s 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 p. 122 DuPont-18318 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 0-2,7-28 3-6 0-28 0-5 5 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 - 122 2*?? Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Sex Group Animal MV MV MV MV MV MV MV MV MV MV MV MV MV MV MV MV MV MV MV MV M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 701 702 703 704 705 706 707 708 709 710 711 712 713 714 M VII M VII M VII M VII M VII M VII 715 716 717 718 719 720 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 Genera 1 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 Swollen Observations,, Shoulder, Left 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 co CM 1 o p. 123 DuPont-18318 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 0-28 0-28 0-4 5-28 0-28 0-28 0-28 0-28 0-28 0-28 - 123 - 3oo p. 124 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 M IX M IX M IX M IX M IX M IX M IX M IX M IX M IX M IX 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 M XI M XI M XI M XI M XI M XI M XI M XI M XI M XI M XI 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 Individual Daily Animal Health Observations General observation, No Abnormality Detected Comments, animal not dosed, pd obs not done General observation, No Abnormality Detected Comments, both ears, hindpaws/forepaws yellow Comments, animal not dosed, pd obs not done General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected Swollen Observations,, Shoulder, Left Sacrificed in extremis General observation, No Abnormality Detected General observation, No Abnormality Detected Comments, both ears/forepaws/hindpaws yellow Swollen Observations,, Penis General observation, No Abnormality Detected Abnormal Gait, Hindlimb, Right, Moderate General observation, No Abnormality Detected General observation, No Abnormality Detected Comments, both ears/hindpaws/forepaws yellow 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 Comments, both ears/forepaws/hindpaws yellow Comments, animal not dosed, pd obs not done Stained Cageboard, Yellow General observation, No Abnormality Detected Feces, Absent Comments, both ears/forepaws/hindpaws yellow Comments, animal not dosed, pd obs not done Stained Cageboard, Yellow Not Eating General observtion, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected Eye Observations, Enophthalmus, Bilateral Lethargic Sacrificed in extrem:Ls 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 Comments, animal not dosed, pd obs not done General observation, No Abnormality Detected General observation, No Abnormality Detected General observation, No Abnormality Detected Comments, animal not dosed, pd obs not done 0-8,12-28 9-11 0-8,12-22,28 23-27 9-11 0-28 0-28 0-28 0-6 7-8 9 0-28 0-21 22-27 22-28 0-16,19-28 17-18 0-28 0-27 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 0-8,12-21,28 22-27 9-11 22-27 0-8, 12-17, 19-21,28 18 22-27 9-11 22-27 18 0-28 0-28 0-2 3-5 3-5 5 0-28 0-28 0-28 0-28 0-7,11-28 8-10 0-28 0-28 0-7,11-28 8-10 - 124- 3Cf Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 125 DuPont-18318 Appendix F Individual Detailed Clinical Observations and Mortality Records SoU- 125 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 126 DuPont-18318 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal Observation M I 101 General observation, No Abnormality Detected Sacrificed by design M I 102 General observation, No Abnormality Detected Sacrificed by design M I 103 General observation, No Abnormality Detected Sacrificed by design M I 104 General observation, No Abnormality Detected Sacrificed by design M I 105 General observation, No Abnormality Detected Sacrificed by design M I 106 General observation, No Abnormality Detected Sacrificed by design M I 107 General observation, No Abnormality Detected Sacrificed by design M I 108 General observation. No Abnormality Detected Sacrificed by design M I 109 General observation, No Abnormality Detected Sacrificed by design M I 110 General observation, No Abnormality Detected Sacrificed by design M I 111 General observation, No Abnormality Detected Sacrificed by design M I 112 General observation, No Abnormality Detected Sacrificed by design M I 113 General observation, No Abnormality Detected Sacrificed by design M I 114 General observation, No Abnormality Detected Sacrificed by design M I 115 General observation, No Abnormality Detected Abnormal Gait, Hindlimb, Right, Severe Sacrificed by design M I 116 General observation, No Abnormality Detected Sacrificed by design M I 117 General observation, No Abnormality Detected Eye Observations, Enophthalmus, Bilateral Lethargic Breathing Observations, Labored Feces, Absent Comments, swollen thoracic ventral Swollen Observations, Face Swollen Observations, Neck Not Eating Sacrificed in extremis M I 118 General observation, No Abnormality Detected Sacrificed by design M I 119 General observation, No Abnormality Detected Sacrificed by design M I 120 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 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0,7-29 3 29 0-29 29 0 5 5 5 5 5 5 5 5 5 0-29 29 0-29 29 0-29 29 - 126- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 127 DuPont-18318 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal Observation M III M III M 111 M III M III M III M III M III M II [ M III M III M III M III M III M III M III M III M III M III M III 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 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 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 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 - 127- 3di~ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 128 DuPont-18318 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal Observation M V 501 General observation, No Abnormality Detected Sacrificed by design M V 502 General observation, No Abnormality Detected Sacrificed by design M V 503 General observation, No Abnormality Detected Sacrificed by design M V 504 General observation, No Abnormality Detected Sacrificed by design M V 505 General observation, No Abnormality Detected Abnormal Gait, Hindlimb,, Left, Severe Sacrificed by design M V 506 General observation, No Abnormality Detected Sacrificed by design M V 507 General observation, No Abnormality Detected Sacrificed by design M V 508 General observation, No Abnormality Detected Sacrificed by design M V 509 General observation, No Abnormality Detected Sacrificed by design M V 510 General observation, No Abnormality Detected Sacrificed by design M V 511 General observation, No Abnormality Detected Sacrificed by design M V 512 General observation, No Abnormality Detected Sacrificed by design M V 513 General observation, No Abnormality Detected Sacrificed by design M V 514 General observation, No Abnormality Detected Sacrificed by design M V 515 General observation, No Abnormality Detected Sacrificed by design M V 516 General observation, No Abnormality Detected Sacrificed by design M V 517 General observation, No Abnormality Detected Sacrificed by design M V 518 General observation, No Abnormality Detected Sacrificed by design M V 519 General observation, No Abnormality Detected Sacrificed by design M V 520 General observation, No Abnormality Detected Sacrificed by design Days 0-29 29 0-29 29 0-29 29 0-29 29 0,14-29 7 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 - 128 395 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 129 DuPont-18318 Individual Detailed Clinical Observations and Mortality Records Sex Group Animai Observation M VII M VII 701 702 M VII M VII 703 704 M VII M VII M VII M VII M VII M VII M VII M VII M VII M VII 705 706 707 708 709 710 711 712 713 714 M VII M VII M VII M VII M VII 715 716 717 718 719 M VII 720 General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Abnormal Gait, Hindlimb , Right, Moderate Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Pale 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 Absent, End of tail Swollen Observations, Shoulder, Left 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 Misshapen Observations, Tail Sacrificed by design General observation, No Abnormality Detected Sacrificed by design Days 0-29 29 0,14-29 7 29 0-29 29 0-7,28-29 14-21 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 7-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0 7-29 29 0-29 29 - 129 306 p. 130 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Individual Detailed Clinical Observations and Mortality Records Sex Group Animal Observation M IX M IX 901 902 M IX M IX M IX M IX 903 904 905 906 M IX M IX 90'/ 908 M IX M IX M IX M IX M IX M IX M IX M IX M IX M IX M IX M IX 909 910 911 912 913 914 915 916 917 918 919 920 General observation. No Abnormality Detected Pale Sacrificed by design General observation, No Abnormality Detected Pale Stain Fur/Skin, Perineum, Yellow Wet Fur, Perineum 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 Eye Observations, Partially Closed, Bilateral Lethargic Pale Sacrificed in extremis General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Comments, both ears/hindpaws/forepaws yellow Swollen Observations, Penis Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Comments, both ears/forepaws/hindpaws yellow Sacrificed by design General observation, No Abnormality Detected Pale 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 Days 0-14,28-29 21 29 0-14 21-28 21-29 21-28 29 0-29 29 0-29 29 0-29 29 0-7 9 9 9 9 0-29 29 0-21 28 28-29 29 0-29 29 0-29 29 0-21 28-29 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 - 130- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 131 DuPont-18318 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 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 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 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 Comments, both ears ;yellow Stained Cageboard, Yellow Sacrificed by design General observation, No Abnormality Detected Eye Observations, Enophthalmus, Bilateral Prostrate Comments, ears/extremities yellow Stained Cageboard, Yellow Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Eye Observations, Enophthalmus, Left Eye Observations, Dark, Bilateral Lethargic Feces, Absent Stain Fur/Skin, Perineum, Yellow Swollen Observations, Shoulder, Left Not Eating Sacrificed in extremis 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 Days 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-21 28 28-29 29 0-14 21 21 21 28-29 29 0-29 29 0-29 29 0 5 5 5 5 5 5 5 5 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 0-29 29 - 131 - p. 132 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Appendix G Individual Animal Clinical Pathology Data - 132- 3<J Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 133 DuPont-18318 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES ABBREVIATIONS : G en era l: Adeq CLOT or Clot Deer Mod NSR NP OK QNS UTD adequate sample clotted decreased moderate no sample received for testing not taken, not performed, or results not valid sample condition OK for testing sample quantity not sufficient for testing unable to determine I n d iv id u a l H em atology V a lu e s: 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 concentration I n d iv id u a l R ed B lo o d C e ll M orp h ology V a lu e s: ANIS - anisocytosis MIC - microcytes MAC - macrocytes POLY - p olychromas ia HYPO - hypochromasia ECHI - echinocytes ACAN - acanthocytes TARG - target cells RX - rouleaux HJB - Howell-Jolly body - - not observed l1 W h i t e B l o o d C e l l / P l a t e l e t M o r p h o l o g y SM - smudge white blood cells TOX - toxic neutrophils DB - Dohle bodies VC - vacuolated cytoplasm BC - basophilic cytoplasm PCE - platelet clumps / estimate GP - giant platelets BP - bizarre platelets - - not observed - 133 - 30 p. 134 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES (Continued) ABBREVIATIONS : (Continued) I n d iv id u a l C lin ic a l C h em istry V a lu es: 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 marked other. - 134- y Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice / iJJ 1 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 OK OK OK OK i RBC xloVpL 10.83 10.45 9.23 9 .99 11.04 10.68 8.80 10 .60 10.14 10.57 Group COND OK OK OK OK OK OK OK OK OK OK III RBC xIOVpL 10.00 10.13 10.36 10.67 9.84 9.86 9 .95 10.75 10.35 9.56 0 HGB g/dL 16.8 16.4 14 .3 16.0 16.2 17.0 13.8 17 .3 16.3 15.7 0.3 HGB g/dL 15 .9 15 .9 15.6 16.6 15.8 16.6 15.4 15.6 16.1 14 .8 mg /kg HCT 55.2 55.8 46.8 52.3 53 .5 56 .7 48 .4 55.6 54 .2 51 .5 mg/kg HCT 52.3 52.7 56.4 55.0 52.0 51.5 51.3 52.7 53.1 49.9 Day MCV fL 50.9 53.4 50.7 52 .4 48.4 53.1 54 .9 52 .5 53.4 48.7 Day MCV fL 52.3 52.0 54.4 51.6 52.8 52.3 51 .6 49.0 51 .2 52.2 29 MCH pg 15.5 15.6 15.4 16.0 14.7 15.9 15.7 16.3 16.1 14 .9 29 MCH pg 15. 9 15.7 15.1 15.5 16.1 16.8 15.5 14.5 15.5 15.5 MCHC g/dL 30.4 29.3 30.5 30.5 30 .3 30.0 28.6 31 .0 30.2 30.5 MCHC g/dL 30.4 30.2 27.7 30.1 30.5 32.2 30.0 29.6 30.3 29.7 RDW % 12.8 13.7 13 .1 12 .9 12 .9 12 .7 12 .4 12 .9 12 .7 12.6 ARET xIOVpL PLT xIOVpL 318.0 315.4 275 .1 333.4 320.8 369.1 330.3 367.1 333.5 302.5 NP NP NP NP NP NP NP NP NP 1177 RDW % 12.2 12.7 12.3 12.1 11.6 12.4 12.1 12.1 12 .6 12.4 ARET xIOVpL PLT xIOVpL 395.5 324 .4 344 .1 451.5 268.3 384.3 322.9 312.4 309.2 310.6 NP NP NP 1545 NP NP NP NP NP 1259 DuPont-18318 p. 135 - 135 - Osi Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 50 5 506 507 508 509 510 Group COND OK OK OK OK OK OK OK OK OK OK V RBC xlOVyL 9. 55 10,51 9.83 10.08 9. 63 9.28 5.37 9.52 9.37 9.82 1 HGB g/dL 15.9 15 .6 15.3 15.5 14 .6 15.3 8 .2 14 .3 14.3 14 .2 Male, Animal 701 702 703 / 04 705 / 06 707 708 709 710 Group COND OK OK OK OK OK CLOT OK OK QNS OK VII RBC xl06/pL 10.49 9.83 9. 92 8.09 10 .47 NP 11.34 10.69 NP 9.51 10 HGB g/dL 16.3 14 .9 16.7 11.9 14 .7 NP 16.3 15.7 NP 13.0 mg /kg HCT % 52.9 54 .8 49.0 50.9 48.5 50.6 27 .8 48.2 48.5 49.1 mg/kg HCT % 57 .6 51.7 55.2 41.7 52.8 NP 54.7 53.4 NP 47 .8 Day MCV fL 55 .4 52.2 49.9 50.5 50.3 54.5 51.7 50.6 51.7 50.0 Day MCV fL 54.9 52.6 55.7 51.5 50.5 NP 48.2 50.0 NP 50.2 29 MCH pg 16.6 14 .8 15.5 15.4 15.1 16.4 15.4 15.0 15.2 14.5 29 MCH pg 15.5 15.2 16.8 14 .7 14.0 NP 14.3 14 .7 NP 13.6 MCHC g/dL 30.0 28 .5 31 .2 30.4 30.1 30.2 29.7 29.7 29.4 29.0 MCHC g/dL 28 .2 28.8 30.1 28 .4 27 .8 NP 29.7 29.4 NP 27 .1 RDW % 11.2 12 .4 12.4 11 .7 12.5 13 .1 11 .2 11 .4 12 .9 12.7 ARET x l 0 J/pL PLT xlO'VpL 220.0 364.0 288.9 342.3 211.4 300.3 159.6 238 .3 330.2 293.3 NP NP 1453 NP NP NP 705 1125 1481 1114 RDW % 11.9 11.4 11 .9 12.2 12.7 NP 11 .6 12 .7 NP 12 .6 ARET xlOVpL 338.1 302 .9 248.3 162.0 171 .9 NP 278.5 271.3 NP 212.9 PLT xlOVpL NP NP NP NP NP NP NP NP NP NP DuPont-18318 p. 136 - 136- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, An imal 901 902 903 904 905 906 907 908 909 910 Group COND OK OK QNS OK OK NSR OK OK QNS OK IX RBC xlOVyL 9.65 8.12 NP 9.57 9.61 NP 10.72 10.68 NP 9.15 30 HGB g/dL 13.8 10.9 NP 14.0 13.4 NP 16.4 16.3 NP 13 .8 mg/ kg HCT 48.7 38.2 NP 47 .7 46.0 NP 54 .7 56.8 NP 46.0 Day MCV fL 50.4 47.0 NP 49.9 47.8 NP 51.1 53.1 NP 50.3 29 MCH pg 14.3 13.4 NP 14.6 13.9 NP 15.3 15.3 NP 15.1 MCHC g/dL 28.3 28.5 NP 29.3 29.1 NP 30.0 28 .8 NP 30.0 RDW % 14.5 13 .7 NP 12.3 12.7 NP 12.2 13.6 NP 12.9 ARET xloVyL PLT xlOVpL 521 .1 228.4 NP 181.0 245.7 NP 246.7 566.0 NP 4 63.7 NP NP NP NP NP NP NP NP NP NP Male, Group XI 30/0 mg /kg (Recovery) Day 29 RBC HGB HCT MCV MCH MCHC RDW ARET PLT u> Animal COND xlOVuL g/dL % fL p g g/dL % X l O V u L xlO'VpL 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 OK OK OK OK OK CLOT OK OK OK OK 9.58 8.13 9.57 8 .37 9.68 NP 9.64 8.34 7 .92 8.15 13.8 12 .6 13.7 13.3 14 .5 NP 14 .5 12.4 11.3 11 .5 48.4 43.3 47 .1 45.2 49.1 NP 48.0 43.2 42.1 38.4 50.5 53.3 49.2 54.0 50.7 NP 49.8 51.8 53.1 47 .1 14 .4 15.5 14 .3 15 .9 15.0 NP 15.0 14.9 14.3 14.1 28.5 29.0 29.0 29.5 29.5 NP 30.2 28.8 27.0 30.1 12 .6 13 .6 12 .4 12 .7 13.4 NP 12 .8 13.3 18.7 16.7 268.2 374 .0 377 .1 703.1 503.4 NP 347 .0 625.5 863.3 275.3 NP NP 1530 1360 NP NP NP NP 2434 679 DuPont-18318 p. 137 - 137- p. 138 3/5 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Animal 101 102 103 101 105 106 107 108 109 110 Group WBC xIOVpL 6.96 4 .12 5.04 10.08 6.20 12.10 7.98 6.21 9.05 7.75 I 0 mg/ kg ANEU xIOVpL ALYM xIOVpL AMON xIOVpL 0 .84 0.48 0.50 1.75 0 .69 1.32 0.71 0.43 0 .72 0.54 5 .84 3.47 4.54 7.79 5.26 10.17 6.94 5.53 7.79 6.98 0.21 0.04 0.00 0.16 0.06 0.20 0.09 0.19 0.27 0.08 Day 29 AEOS xIOVpL ABAS xIOVpL 0.07 0.07 0.00 0.26 0.11 0.25 0.11 0.06 0.27 0.16 0.00 0.02 0.00 0.04 0.01 0.05 0.02 0.00 0.00 0.00 ALUC xIOVpL 0.00 0.04 0.00 0.09 0.06 0.11 0.10 0.00 0.00 0.00 Male, Animal 301 302 303 304 305 306 307 308 309 310 Group III WBC xIOVpL ANEU xIOVpL 10.07 11.77 5.45 5 .80 11.77 10.72 9.50 10.05 6.48 14.07 1 .63 1 .72 1 .15 0 .83 1.16 1 .92 0 .99 1 .92 0 .77 0.84 0 .3 mg/ kg Day ALYM xIOVpL AMON xIOVpL AEOS xIOVpL 7 .90 9.45 3 .98 4.49 10.27 8.34 8.05 7.5 9 5.49 12 .67 0.23 0 .17 0 .10 0 .11 0.14 0.19 0.07 0.15 0.05 0.42 0.18 0.31 0.19 0.21 0.06 0.13 0.25 0.21 0.11 0.14 29 ABAS xIOVpL ALUC xIOVpL 0.03 0.04 0.01 0.02 0.02 0.03 0.03 0.03 0.01 0.00 0 .10 0.09 0.02 0 .14 0 .12 0 .10 0.10 0.14 0.05 0.00 - 138 - DuPont-18318 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 506 507 508 509 510 Group WBC x lo V p L 10.54 9.59 6. 94 12.33 6.87 12.44 7.76 6 .63 10.99 4 .93 V 1 mg/ kg ANEU x 10V pL ALYM xlOVpL AMON x loV pL 1.61 0.83 1.59 0.25 0.52 1 .24 0 .64 1.04 1.41 0.49 8.38 8.22 5.09 11.71 6.08 11.08 6.80 5.10 9.27 4.26 0.15 0.18 0.08 0.25 0.11 0.00 0.08 0 .15 0.11 0.03 Day AEOS xlo V p L 0.28 0.24 0.09 0 .12 0.07 0.12 0.12 0.23 0.09 0.07 29 ABAS xlo V p L 0.03 0.03 0.03 0.00 0.02 0.00 0.02 0.02 0.02 0.01 ALUC xlO'VpL 0.07 0.08 0.06 0.00 0.08 0.00 0 .10 0.10 0.09 0.06 Male, Animal 701 702 703 704 705 706 707 708 709 710 Group WBC x lo V p L 12.71 8.33 11 .31 9.79 15.71 NP 9.97 10.71 NP 10.55 VII ANEU xlo V p L 2.29 2.67 1 .81 1.20 2.57 NP 1.89 1 .71 NP 1.00 10 mg/ kg ALYM AMON X 10 '7pL x l O V p L 9.69 5.00 8.71 8 .12 12.52 NP 7.88 8.46 NP 8.94 0.34 0.58 0.68 0.19 0.23 NP 0.20 0.43 NP 0.30 Day AEOS xlOVpL 0.08 0.08 0.11 0.05 0.13 NP 0.00 0.11 NP 0.10 29 ABAS xlOVpL 0.06 0.00 0.00 0.03 0.05 NP 0.00 0.00 NP 0.03 ALUC x l 0 3/pL 0.25 0.00 0.00 0.20 0.21 NP 0.00 0.00 NP 0.17 - 139- DuPont-18318 p. 139 p. 140 317 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group WBC xl 0 V p L 11.69 6.02 NP 8 .98 4 .68 NP 5.31 5.05 NP 5.50 IX ANEU xlO V p L 3 .99 2.82 NP 3.70 1.02 NP 1. 70 1.67 NP 1.66 30 ALYM xIOVpL 6.83 2.83 NP 4.04 3.20 NP 3 .18 3.13 NP 3.43 mg/ kg AMON xlO'VpL 0.50 0.18 NP 0.75 0.25 NP 0.32 0.25 NP 0.04 Day AEOS xIOVpL 0.09 0.05 NP 0.16 0.03 NP 0.11 0.00 NP 0.18 29 ABAS xlO'VpL 0.07 0.01 NP 0.03 0.02 NP 0.00 0.00 NP 0.02 ALUC xIOVpL 0.21 0 .12 NP 0.30 0 .17 NP 0.00 0.00 NP 0.17 Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group WBC xIOVpL 7 .78 7.28 5.38 7.55 8.32 NP 8.24 7 .50 5 .67 7.86 XI ANEU xlO'/pL 1.40 1.72 1.26 2.34 2.50 NP 2.60 2 .10 1.66 2 .91 30/0 ALYM xIOVpL 6.22 5.03 3.74 4 .71 5.32 NP 4.85 4.87 3.71 4.56 mg/ kg (Recovery) Day AMON xIOVpL AEOS xIOVpL ABAS xIOVpL 0.16 0.28 0.14 0.09 0.50 NP 0.13 0.38 0.09 0.31 0.00 0.08 0.12 0.09 0.00 NP 0.27 0.15 0.06 0.08 0.00 0.01 0.01 0.05 0.00 NP 0.11 0.00 0.03 0.00 29 ALUC xIOVpL 0.00 0.15 0.11 0.27 0.00 NP 0.29 0.00 0.13 0.00 - 140- DuPont-18318 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Indivi Animal Clinical Pathology Data Male, Animal 101 102 103 104 105 106 107 108 109 110 Group ANIS _ - I MIC - - 0 MAC - - - mg/ kg POLY Few Few Few Few Few Few Few Few Few Trace Day HYPO _ - - - - 29 ECHI Trace ACAN - TARG RX HJB Male, Group Ill 0.3 mg/kg Day 29 Animal ANIS MIC MAC POLY HYPO ECHI ACAN TARG RX HJB u$ 301 - - - Few - 302 - - Few 303 : - - Few - 304 - - - Few - 305 - - - Trace - 306 - - - Few - 307 - - - Few - 308 - - - Trace - 309 - - - Few - 310 - - - Few - DuPont-18318 p. 141 - 141 - C\> N Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 506 507 508 509 510 Group ANIS Trace - - - Male, Animal 701 702 703 704 705 706 707 708 709 710 Group ANIS - - CLOT - - QNS - V MIC _ - - - VII MIC - - NP - - NP - 1 MAC Trace - - - 10 MAC - NP NP - mg/ kg POLY Trace Few Few Few Few Trace Trace Few Few mg/ kg POLY Few Few Trace NP Trace Trace NP - Day HYPO - Day HYPO - NP NP - 29 ECHI - - 29 ECHI - NP NP - ACAN - - - ACAN - NP NP - TARG - TARG - NP NP - RX - _ RX - ~ NP NP - HJB - HJB - NP NP - DuPont-18318 p. 142 - 142 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group ANIS - Trace QNS - NSR - QNS Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group ANIS - Trace - CLOT Trace Trace - IX MIC - NP - NP - NP " XI MIC Trace - NP Trace Trace - 30 MAC Trace NP - NP NP - 30/0 MAC - Trace - NP Trace Trace - mg/ kg POLY Mod Trace NP - Trace NP - Mod NP Few Day HYPO _ N_P - N_P - NP - 29 ECHI _ - N_P _ NP _ _ NP - mg/kg (Recovery) Day POLY Mod Mod Few Mod Mod NP Mod Many Many Few HYPO _ - NP - - - ECHI _ - _ - NP - - ACAN _ _ N_P _ N_P _ NP - 29 ACAN _ _ _ _ - NP _ TARG _ N_P _ N_P _ NP - TARG _ _ _ _ - N_P - _ _ RX _ N_P _ N_P _ NP - RX _ _ _ _ N_P _ _ HJB _ NP _ NP _ NP - HJB _ _ _ NP - _ _ DuPont-18318 p. 143 - 143 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Male, Animal 101 102 103 104 105 106 107 108 109 110 Group SM _ - - - I TOX _ - - - - 0 mg/ kg Day 29 DB VC BC PCE - - - Adeq - - - Adeq -- Adeq - - - Adeq - - - Adeq - - - Adeq - - ~ Adeq - - - Decr - - - Adeq Male, Group III 0.3 mg/ kg Day 29 Animal SM TOX DB VC BC PCE 301 - - - - - Adeq 302 - - - - - Adeq 303 - - - - - Adeq 304 V 305 - - - - - Adeq 306 - - - Adeq 307 - - - - - Adeq 308 - - - - - UTD 309 - - - - - Adeq 310 - - Clinical Pathology Data GP BP GP BP DuPont-18318 p. 144 - 144- Us Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Male, Animal 501 502 503 504 505 506 507 508 509 510 Group SM - Male, Animal 701 702 703 704 705 706 707 708 709 710 Group SM - _ - CLOT - QNS - V TOX _ * - VII TOX - - NP - NP - Individual Animal Clinical Pathology Data 1 mg/ kg Day 29 DB VC BC PCE _ - * Adeq - - - Adeq - --- - - Deer - - - Adeq - - Adeq - --- --- --- --- 10 mg/ kg Day 29 DB VC BC PCE - - - Adeq _ - - Deer - Deer - - - Adeq - - - Adeq NP NP NP NP - - - Deer - - - Deer NP NP NP NP - - - Deer GP - - - - GP NP NP - BP - - - - - BP NP - _ NP - DuPont-18318 p. 145 - 145 - ZZ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Male, Animal 901 902 903 904 905 906 907 908 909 910 Group SM - QNS NSR QNS - Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group SM - - - CLOT - IX TOX - NP NP NP - XI TOX - NP - - Individual Animal Clinical Pathology 30 DB - NP NP NP - 30/0 DB - - - NP - mg/ kg VC - NP NP NP - Day BC NP NP NP - 29 PCE Adeq Adeq NP Adeq Adeq NP Adeq Deer NP Adeq mg/kg (Recovery) Day VC BC PCE - - Adeq - - Adeq ---- - - UTD NP NP NP - - Adeq - - Adeq -- -- - GP NP NP NP - 29 GP - NP - BP - NP NP NP - BP - NP - - 146- DuPont-18318 p. 146 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Group I 0 mg/ kg Day 29 CHOL TRIG TP ALB GLOB HDL NHDL SCORT Animal HEM LIP ICT mg/dL mg/dL g/dL g/dL g/dL mg/dL mg/dL ng/mL 101 None None None NP NP NP NP NP NP NP NP 102 None None None NP NP NP NP NP NP NP NP 103 None None None NP NP 5.8 3.0 2 .8 NP NP 204 104 Trace None None NP NP 5.6 3.0 2 .6 NP NP NP 105 Small None None NP NP 5.5 3.0 2.5 NP NP NP 106 None None None NP NP 5.1 2 .6 2.5 NP NP NP 107 None None None NP NP NP NP NP NP NP NP 108 None None None NP NP 5.9 3.0 2.9 NP NP NP 109 None None None NP NP 5.7 3.0 2.7 NP NP NP 110 None None None NP NP NP NP NP NP NP NP 111 None None None 161 178 NP NP NP 106 55 391 112 None None None 128 156 NP NP NP 83 45 282 113 None None None 110 170 NP NP NP 78 32 214 114 None None None 142 214 NP NP NP 91 51 99 115 None None None 98 133 NP NP NP 64 34 64 116 None None None 121 234 NP NP NP 80 41 120 117 NSR NP NP NP NP NP NP NP NP NP NP 118 None None None 131 162 NP NP NP 87 44 92 K 119 None None None 101 141 NP NP NP 68 33 110 120 None None None 62 118 NP NP NP 37 25 323 DuPont-18318 p. 147 - 147- sts U) O p. 148 CM CO 00 CM CM CO m lO Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Group Animal 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 HEM None None None None None QNS Small None None None Small None None None None None None None None None III LIP None None None None None NP None None None None None None None None None None None None None None 0.3 ICT None None None None None NP None None None None None None None None None None None None None None mg/ kg CHOL mg/dL NP NP NP NP NP NP NP NP NP NP 132 121 101 72 140 148 122 123 104 215 Day TRIG mg/dL NP NP NP NP NP NP NP NP NP NP 190 164 112 121 187 116 141 92 136 219 29 TP g/dL NP 4 .4 5.5 5 .4 NP 5.6 NP 4 .7 NP NP NP NP NP NP NP NP NP NP ALB g/dL NP 3.1 NP 3.0 2.7 NP 2 .4 NP NP NP NP NP NP NP NP NP NP GLOB g/dL NP 1.8 2.5 2.7 2.6 NP 2 .6 NP 2.3 NP NP NP NP NP NP NP NP NP NP HDL mg/dL NP NP NP NP NP NP NP NP NP NP 89 72 68 52 77 87 78 80 69 96 NHDL mg/dL NP NP NP NP NP NP NP NP NP NP 43 49 33 20 63 61 44 43 35 119 SCORT ng/mL NP NP NP NP NP NP NP NP NP NP 170 212 262 236 78 231 220 360 139 76 DuPont-18318 - 148 - t. M Na v\ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinica- Male, Group Animal 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 HEM None None None None None None None None None None Trace None None None None None None None None None V LIP None None None None None None None None None None None None None None None None None None None None 1 ICT None None None None None None None None None None None None None None None None None None None None mg/ kg CHOL mg/dL NP NP NP NP NP NP NP NP NP NP 138 108 86 144 57 123 91 117 88 58 Day TRIG mg/dL NP NP NP NP NP NP NP NP NP NP 151 219 103 140 118 154 207 205 152 118 29 TP g/dL 5.4 5.4 6.6 NP 5.7 5.4 5.3 5.3 5.8 NP NP NP NP NP NP NP NP NP NP NP ALB g/dL 3.2 3.3 3.3 NP 3.2 3 .1 3.1 2 .9 3.2 NP NP NP NP NP NP NP NP NP NP NP - 149- p. 149 CM M3 CM CM L Pathology Data GLOB g/dL 2 .1 3.3 NP 2 .5 2.3 2.2 2 .4 NP NP NP NP NP NP NP NP NP NP NP HDL mg/dL NP NP NP NP NP NP NP NP NP NP 83 59 52 68 40 63 44 51 46 34 NHDL mg/dL NP NP NP NP NP NP NP NP NP NP 55 49 34 76 17 60 47 60 42 24 SCORT ng/mL NP NP NP NP NP NP NP NP NP NP 197 127 170 32 42 247 115 48 60 43 DuPont-18318 (a kj SI Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Group Animal 101 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 HEM None None Small None None None None None None None None None None None None None None None None None VII LIP None None None None None None None None None None None None None None None None None None None None 10 mg/ kg CHOL ICT mg/dL Trace Moderate None Trace None Trace Small Trace Small Trace Trace Trace Moderate None Trace None Small Trace Small Trace NP NP NP NP NP NP NP NP NP NP 63 60 77 113 87 66 96 100 44 106 Day TRIG mg/dL NP NP NP NP NP NP NP NP NP NP 60 99 63 65 35 131 103 110 47 64 29 TP g/dL 7 .1 7.2 7.3 7.0 NP 6.7 NP NP 7 .0 6 .4 NP NP NP NP NP NP NP NP NP NP ALB g/dL 4 .5 4 .4 4 .3 4.2 NP 3 .9 NP NP 4 .2 3.7 NP NP NP NP NP NP NP NP NP NP GLOB g/ dL 2.6 2 .8 3.0 2.8 NP 2.8 NP NP 2 .8 2 .7 NP NP NP NP NP NP NP NP NP NP HDL mg/dL NP NP NP NP NP NP NP NP NP NP 44 38 50 63 55 39 44 53 28 58 NHDL mg/dL NP NP NP NP NP NP NP NP NP NP 19 22 27 50 32 27 52 47 16 48 SCORT ng/mL NP NP NP NP NP NP NP NP NP NP 516 471 605 183 259 476 464 578 216 564 DuPont-18318 p. 150 - 150- ( V\ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Clinical Pathology Data Male, Group Animal 901 902 903 904 90b 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 HEM None None NSR QNS None NSR None None None None None None None None None None None None None None IX LIP None None NP NP None NP None None None None None None None None None None None None None None 30 mg/ kg CHOL ICT mg/dL Small Moderate NP NP Moderate NP Small Moderate Moderate Moderate Moderate Moderate Moderate Moderate Small Moderate Small Small Moderate Moderate NP NP NP NP NP NP NP NP NP NP 31 44 46 48 71 57 57 70 113 65 Day TRIG mg/dL NP NP NP NP NP NP NP NP NP NP 15 46 36 18 80 54 60 88 89 45 29 TP g/dL NP 6.1 NP NP NP NP NP 5.4 NP 6.9 NP NP NP NP NP NP NP NP NP NP ALB g/dL NP 4 .0 NP NP NP NP NP 3.4 NP 4.0 NP NP NP NP NP NP NP NP NP NP GLOB g/dL NP 2 .1 NP NP NP NP NP 2 .0 NP 2.9 NP NP NP NP NP NP NP NP NP NP HDL mg/dL NP NP NP NP NP NP NP NP NP NP 26 22 23 33 38 27 37 38 59 40 NHDL mg/dL NP NP NP NP NP NP NP NP NP NP 5 22 23 15 33 30 20 32 54 25 SCORT ng/mL NP NP NP NP NP NP NP NP NP NP 912 597 503 141 264 821 434 414 130 158 DuPont-18318 p. 151 - 151 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal ClinicaJ Male, Group Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 HEM None None None None None Small None None None None None NSR None None None Trace None None None None XI LIP None None None None None None None None None None None NP None None None None None None None None 30/0 ICT Sma 11 None None Trace Small Trace Small Small Small None Trace NP Trace Trace Trace Small Small Trace Trace Trace mg/ kg (Recovery) Day CHOL mg/dL TRIG mg/dL TP g/dL NP NP 7 .2 NP NP 7.3 NP NP 6 .4 NP NP NP NP 7 .9 NP NP NP NP NP NP NP 7 .8 NP NP 6. 9 NP NP 7.0 117 127 NP NP NP NP 93 111 NP 121 110 NP 121 115 NP 91 64 NP 77 77 NP 66 108 NP 96 76 NP 63 74 NP CO ro c o CO 29 ALB g/dL 4 .5 4 .2 4 .8 4 .6 4 .9 NP 4 .3 4 .1 3.6 NP NP NP NP NP NP NP NP NP NP bZE - 152- p. 152 CO CM _ Pathology Data GLOB g/dL 2.7 3 .1 2.6 3.6 3.3 3.7 NP 3.5 3.4 NP NP NP NP NP NP NP NP NP NP HDL mg/dL NP NP NP NP NP NP NP NP NP NP 64 NP 57 66 60 55 47 38 57 36 NHDL mg/dL NP NP NP NP NP NP NP NP NP NP 53 NP 36 55 61 36 30 28 39 27 SCORT ng/mL 240 25 NP NP NP NP NP NP NP NP 78 NP 136 325 433 NP 194 183 476 501 DuPont-18318 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 153 DuPont-18318 Appendix H Individual Primary Humoral Immune Response Data p. 154 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 INDIVIDUAL PRIMARY HUMORAL IMMUNE RESPONSE DATA EXPLANATORY NOTES FOOTNOTES : b Serum was not collected from this animal, therefore, immune response could not be evaluated, C Serum volume was insufficient for this animal, therefore, immune response could not be evaluated. d This animal was not injected with the appropriate amount of SRBC, therefore, immune response could not be evaluated. - 154- 33/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Primary Humoral Immune Response Data Animal Number SLOPE X Log2 Male, Group I - 0 mg/k.g 101 -1.0034 874 102 -0.9958 292 103 -0.9796 540 104 -0.8528 353 105 -0.9942 737 106 -0.8607 487 107 -0.8226 727 108 -0.9649 1018 109 -0.9366 417 110 -0.9625 777 111 -1.0109 340 112 -0.9652 784 113 -0.9896 537 114 -0.9866 4 97 115 -0.9834 384 116 -0.9607 689 117 a 118 -0.9716 215 119 -0.8730 457 120 -0.8733 549 9.771 8.190 9.077 8.464 9.526 8.928 9.506 9.992 8.704 9.602 8.409 9.615 9.069 8.957 8.585 9.428 7 .748 8.836 9.101 Male, Group III - 0..3 mg/kg 301 -1.0351 681 9.412 302 -0.9020 235 7.877 303 -0.9403 405 8.662 304 -0.9653 445 8.798 305 -0.9268 258 306 a 8.011 307 -1.0131 511 8.997 308 -0.9142 269 8.071 309 -0.9091 830 9.697 310 -0.9551 573 9.162 311 -0.9906 1205 10.235 312 -0.9573 645 9.333 313 -0.9484 417 8.704 314 -0.9460 793 9.631 315 -1.0128 401 8.647 316 -0.9552 552 9.109 317 -0.9239 132 7.044 318 -0.9189 57 9 9.177 319 -0.9709 211 7.721 320 -0.9938 622 9.281 p. 155 DuPont-18318 - 155- 33L Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Primary Humoral Immune Response Data Animal Number SLOPE X Log Male, Gr oup V - 1 mg/ kg 501 -1.0294 324 502 -1.0045 562 503 -0.9279 428 504 -0.9200 353 505 -1.0255 343 506 -1.0013 161 507 -0.9730 284 508 -0.9931 444 509 -0 .9765 223 510 -0.9230 331 511 -0.8852 673 512 -1.0076 222 513 -0.9627 317 514 -0.9910 363 515 -0.9622 734 516 -1.0029 351 517 -0.9352 205 518 -0.9065 151 519 -0.8622 253 520 -0.9383 220 8.340 9.134 8.741 8.464 8.422 7.331 8.150 8.794 7.801 8.371 9.394 7.794 8.308 8.504 9.520 8.455 7.679 7.238 7.983 7.781 Male, Group VII - 10 mg/kg 701 -0.8465 285 702 -0.9652 703 b 95 704 -0.9617 134 705 -1.0045 187 706 -0.9582 311 707 -1.0358 97 708 -1.0128 144 709 -1.0214 73 710 -0.9532 148 711 -0.7979 243 712 -0.9974 180 713 -1.0116 140 714 -0.9470 817 715 -0.8674 134 716 717 -1.0155 226 718 -1.0207 110 719 -0.9787 7 720 -0.7775 230 8.155 6.570 7.066 7 .547 8.281 6.600 7 .170 6.190 7.209 7 .925 7.492 7.129 9 .674 7.066 7 .820 6.781 2.807 7.845 p. 156 DuPont-18318 - 156 353 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Animal Number SLOPE Individual Primary Humoral Immune Response Data X Log:. Male, Group IX - 30 mg/kg 901 -0.9613 164 902 -0.9724 903 a 904 a 26 905 -1.0229 906 a 907 b 65 908 -1.0094 89 909 -0.9950 141 910 -1.0106 44 911 -0.9730 84 912 -1.0333 62 913 -0.8405 530 914 -0.9848 193 915 -0.9891 58 916 -1.0002 90 917 -0.9981 120 918 -1.0207 55 919 -0.9989 161 920 -0.9884 52 7.358 4 .700 6.022 6.476 7.140 5.459 6.392 5.954 9.050 7.592 5.858 6.492 6.907 5.781 7.331 5.700 Male, Group IX - 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 -0.9921 -1.0202 -0.9938 -1.0130 -0.9951 -0.9785 -0.9904 -0.9412 -0.9771 -0.8757 -1.0060 a -0.9806 -0.9696 -0.9458 b -0.9999 -0.9994 -0.9866 -0.9927 89 45 114 78 96 35 88 78 47 169 43 151 74 74 167 98 52 52 6.476 5.492 6.833 6.285 6.585 5 .129 6.459 6.285 5.555 7.401 5.426 7.238 6.209 6.209 7.384 6.615 5.700 5.700 p. 157 DuPont-18318 - 157 3 3 .4 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 158 DuPont-18318 Appendix I Individual Primary Humoral Immune Response Positive Control Data - 158- 335 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________ p. 159 DuPont-18318 AnimaI Number Individual Primary Humoral Immune Response Positive Control Data SLOPE X Log2 Male, Group Cl - Saline C101 C102 C103 C104 CIOS C106 C107 C108 C109 C110 -0.9624 -0.9535 -0.9312 -0.9892 -1.0066 -0.8598 -0.8395 -0.9841 -1.0023 -0.9946 578 747 417 505 268 410 140 485 429 271 9.175 9.545 8 .704 8.980 8.066 8.679 7.129 8.922 8.745 8.082 tie, Group CIII - 90 mg/kg Cyclophos] C301 C302 C303 C304 C305 C306 C307 C308 C309 C310 -0.9579 -0.9965 -1.0234 -0.8989 -1.0583 -0.9927 -1.0321 -0.9777 -0.8206 -1.0512 25 19 25 14 14 25 33 43 8 59 4.644 4 .248 4.644 3.807 3.807 4 .644 5.044 5.426 3.000 5.883 Male, Pooled Samples - Saline -1.0107 405 8.662 Male, Pooled Samples - 90 mg/kg Cyclophosphamide -0.9846 35 5.129 - 159- p. 160 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Appendix J Individual Animal Final Body and Organ Weights - 160- 3J?7 Ammonium Periluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 161 DuPont-18318 INDIVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS EXPLANATORY NOTES FOOTNOTES: a An error occurred while weighing livers for this animal, and the liver weight was excluded from calculations. b Liver inadvertently not weighed. Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Final Body and Organ Weights Group: IA Treatment: 0 mg/kg Sex: MALES ANIMAL FBW 1 BRAIN (Gms) 1 (Gms) %FBW (Gms ) LIVER %FBW %BRAIN SPLEEN (Gms) %FBW %BRAIN THYMUS (Gms) %FBW %BR A IN 101 31.00 1 0.409 1.3194 102 34 .70 1 0.460 1.3256 103 33.40 1 0.480 1.4371 104 32.50 1 0.453 1.3938 105 33.00 1 0.460 1.3939 106 31 .20 ! 0.425 1.3622 107 31.90 1 0.454 1.4232 108 30.30 1 0.462 1 .5248 109 32.70 1 0.495 1.5138 110 33.90 0.465 1 .3717 1.623 2.01/ 1.736 1.768 1.459 1.717 1.861 1 .727 1.711 1 .634 5.2355 5.8127 5.1976 5.4400 4.4212 5.5032 5.8339 5.6997 5.2324 4.8201 396.82 438.48 361.67 390.29 317.17 404 .00 409.91 373.81 345.66 351.40 0.106 0.110 0.124 0.111 0.091 0.114 0.128 0.107 0.112 0.119 0.341 9 25.917 0.3170 23.913 0.3713 25.833 0.3415 24.503 0.2758 19.783 0.3654 26.824 0.4013 28.194 0.3531 23.160 0.3425 22.626 0.3510 25.591 0.071 0.052 0.037 0.058 0.047 0.051 0.041 0.043 0.057 0.050 0.2290 0.1499 0.1108 0.1785 0.1424 0.1635 0.1285 0.1419 0.1743 0.1475 17.359 11.304 7.7083 12.804 10.217 12.000 9.0308 9.3074 11.515 10.753 Mean S .D . 32.46 I 0.456 1.4065 1.38 t 0.0250.0702 1.725 5.3196 378.92 0.147 0.4455 35.880 0.112 0.3461 24.634 0.010 0.0333 2.3978 0.051 0.1566 11.200 0.010 0.0325 2.6437 Group: IB Treatment: 0 mg/kg Sex: MALES ANIMAL i FBW BRAIN 1 i (Gms ) (Gms) %FBW 1 (Gms ) LIVER 1 SPLEEN 1 THYMUS %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 1 (Gms) %FBW %BRAIN i n i 32.60 0.505 1.5491 1 1 .801 5.5245 356.63 1 0.145 0.4448 28.713 1 0.052 0.1595 10.297 112 i 33.90 t 0.497 1.4661 1 a ! 0.122 0.3599 24 .547 1 0.074 0.2183 14.889 113 i 34.60 0.461 1.3324 1 a 1 0.117 0.3382 25.380 1 0.043 0.1243 9.3275 1 114 i 33.70 1 0.498 1.4777 1 2.064 6.1246 414.46 1 0.115 0.3412 23.092 1 0.035 0.1039 7.0281 115 i 31.50 1 0.496 1.5746 1 1.718 5.4540 346.37 0.107 0.3397 21 .573 1 0.061 0.1937 12.298 1 116 34 .20 1 0.447 1 .3070 1 1.795 5.2485 401.57 1 0.111 0.3246 24.832 1 0.044 0.1287 9.8434 1 118 34 .50 1 0.503 1.4580 1 2.172 6.2957 431.81 0.099 0.2870 19.682 1 0.053 0.1536 10.537 1 119 i 34 .30 0.492 1.4344 I 1 .859 5.4198 377.85 0.152 0.4431 30.894 1 0.044 0.1283 8.9431 ! 120 i 33.50 ! 0.487 1.4537 1 1 .639 4.8925 336.55 0.138 0.4119 28.337 1 0.045 0.1343 9.2402 1 Mean 1 S .D . 33 .64 1 0.487 1.4503 1 1.864 5.5657 380.75 i 1 .01 1 0.020 0,0873 0.190 0.4889 36.291 0.123 0.3656 25.228 0.050 0.1494 10.267 0.018 0.0552 3.5924 ! 0.012 0.0365 2.2340 FBW - Final Body Weight - 162- DuPont-18318 p. 162 (H?e Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Final Body and Organ Weights Group: IIIA Treatment: 0.3 mg/kg Sex: MALES ANIMAL FBW BRAIN 1 {Gms ) 1 (Gms) %FBW 1 (Gms ) LIVER I SPLEEN 1 THYMUS %FBW %BRAIN I (Gms ) %FBW %BRAIN 1 (Gms) %FBW %BRAIN 301 1 35 .10 1 0.516 1.4701 1 2 .609 7.4330 505.62 I 0.150 0.4274 29.070 ! 0.052 0.1481 10 .078 302 1 36. 10 ! 0.489 1.3546 2.405 6.6620 491.82 0.108 0.2992 22.086 ! 0.043 0.1191 8 .7935 303 1 37.40 1 0.486 1.2995 2.512 6.7166 516.87 j 0.104 0.2781 21.399 1 0.047 0.1257 9. 6708 304 1 37.20 1 0.526 1.4140 1 2.758 7.4140 524.33 I 0.188 0.5054 35.741 1 0.048 0.1290 9. 1255 305 34.50 1 0.516 1.4957 1 2 .520 7.3043 488.37 | 0.117 0.3391 22.674 1 0.038 0.1101 7 .3643 306 1 33.10 1 0.525 1.5861 ! 2 .539 7.6707 483.62 I 0.102 0.3082 19.429 1 0.034 0.1027 6. 4762 307 1 31.30 1 0.473 1.5112 1 2.036 6.5048 430.44 I 0.095 0.3035 20.085 1 0.060 0.1917 12 .685 308 1 33 .70 1 0.436 1.2938 1 2.372 7.0386 544.04 I 0.129 0.3828 29.587 1 0.049 0.1454 11 .239 309 1 29.90 1 0.441 1.4749 1 1.988 6.6488 450.79 | 0.112 0.3746 25.397 1 0.058 0.1940 13 .152 310 32 .80 1 0.455 1.3872 1 2.492 7.5976 547.69 | 0.130 0.3963 28.571 1 0.049 0.1494 10 .769 Mean S .D. 34.11 I 0.486 1.4287 2.45 I 0.034 0.0956 2.423 7.0990 498.36 I 0.124 0.3614 25.404 0.241 0.4378 37.749 | 0.028 0.0703 5.2317 0.048 0.1415 9.9352 0.008 0.0313 2.1325 Group: IIIB Treatment: 0.3 mg/kg Sex: MALES ANIMAL FBW (Gms) BRAIN (Gms ) %FBW 1 {Gms ) LIVER SPLEEN %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 311 36. 60 0.452 1.2350 1 2.896 7.9126 640.71 1 0.114 0.3115 25.221 312 33.40 0.478 1.4311 1 2.529 7.5719 529.08 ! 0.136 0.4072 28.452 313 31 .50 0.477 1.5143 1 2.445 7.7619 512.58 1 0.076 0.2413 15.933 314 32.20 0.471 1.4627 1 2.206 6.8509 468.37 1 0.135 0.4193 28.662 315 33.80 0.505 1.4941 1 2.514 7.4379 497.82 1 0.152 0.4497 30.099 316 31 .60 0.479 1.5158 1 2.294 7.2595 478.91 1 0.099 0.3133 20.668 317 30.20 0.458 1.5166 ! 2.053 6.7980 448.25 1 0.060 0.1987 13.100 318 30 .10 0.430 1.4286 2.054 6.8239 477 67 j 0.106 0.3522 24.651 319 30.90 0.459 1.4854 1 2.199 7.1165 479.08 1 0.060 0.1942 13.072 320 36.70 0.493 1.3433 1 2.715 7.3978 550.71 1 0.151 0.4114 30.629 Mean S.D. 32 .70 2.41 1 .4427 1 2.391 7.2931 508.32 1 0.109 0.3299 23.049 0.0908 1 0.280 0.3953 55.523 0.035 0.0940 6.9119 THYMUS (Gms) %FBW %BRAIN 0.033 0.040 0 . 022 0.045 0.049 0.054 0.057 0.031 0.051 0.048 0.0902 0.1198 0.0698 0.1398 0.1450 0.1709 0.1887 0.1030 0.1650 0.1308 7.3009 8.3682 4.6122 9.5541 9.7030 11.273 12.445 7.2093 11.111 9.7363 0.043 0.1323 9.1314 0.011 0.0375 2.3193 FBW - Final Body Weight - 163 - DuPont-18318 p. 163 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Final Body and Organ Weights Group: VA Treatment: 1 mg/kg Sex: MALES ANIMAL 501 502 503 504 505 506 507 508 509 510 Mean S.D. FBW BRAIN (Gms ) 1 (Gms) %FBW 1 (Gms ) LIVER 1 SPLEEN THYMUS %FBW %BRA 1N 1 (Gms ) %FBW %BRAIN 1 (Gms) %FBW %BRAIN 33 .30 1 0 .441 1.3243 1 3.274 9.8318 742.40 1 0.088 0.2643 19.955 1 0.037 0.1111 8 .3900 34.00 1 0.522 1.5353 1 3.381 9.9441 647.70 1 0.135 0.3971 25.862 1 0.074 0.2176 14 .176 33.50 1 0.479 1.4299 2 .978 8.8896 621.71 1 0.090 0.2687 18.789 ! 0.045 0.1343 9. 3946 33.30 1 0.502 1.5075 1 3 .647 10.952 726.49 ! 0.108 0.3243 21.514 1 0.069 0.2072 13 .745 33 .00 1 0 .444 1.3455 1 3.095 9.3788 697.07 1 0.092 0.2788 20.721 1 0.036 0.1091 8. 1081 35.10 1 0.451 1.2849 1 3.042 8.6667 674.50 1 0.115 0.3276 25.499 31. 30 ! 0.491 1.5687 1 2.992 9.5591 609.37 1 0.107 0.3419 21.792 33.90 1 0.509 1 .5015 1 2.898 8.5487 569.35 ! 0.103 0.3038 20.236 33.50 1 0.447 1.3343 1 3.490 10.418 780.76 1 0.112 0.3343 25.056 32.30 1 0.441 1.3653 1 3.017 9.3406 684.13 1 0.078 0.2415 17.687 0.062 0.056 0.054 0.032 0.040 0.1766 0.1789 0.1593 0.0955 0.1238 13 .747 11 .405 10 .609 7 .1588 9. 0703 1 33.32 1 0.473 1 .4197 3.181 9.5529 675.35 0.103 0.3082 21 .711 0.051 0.1514 10 .580 1. 01 0.032 0.1017 1 0.252 0.7635 64.982 ! 0.016 0.0462 2.8633 0.015 0.0429 2 .5811 Group: VB Treatment: 1 mg/kg Sex: MALES ANIMAL FBW (Gms) BRAIN (Gms) %FBW 1 (Gms ) LIVER 1 SPLEEN 1 THYMUS %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 511 512 513 514 515 516 517 518 519 520 Mean S.D. 36.80 0.497 1.3505 1 3.191 8.6712 642.05 l 0.081 0.2201 16.298 1 0.047 0.1277 9.4567 36.00 0.465 1.2917 1 3.397 9.4361 730.54 1 0.118 0.3278 25.376 1 0.049 0.1361 10.538 35. 90 1 0.480 1 .3370 l 3.539 9.8579 737.29 1 0.106 0.2953 22 .083 1 0.045 0.1253 9.3750 31 .40 1 0.508 1.6178 1 3.544 11.287 697.64 1 0.088 0.2803 17.323 1 0 .037 0 .1178 7.2835 35.70 ! 0.459 1.2857 1 3 .535 9.9020 770.15 1 0,124 0.3473 27 .015 1 0.032 0.0896 6.9717 32.10 0.502 1.5639 1 3.317 10.333 660.76 0.088 0.2741 17.530 1 0.046 0.1433 9.1633 32.20 1 0.443 1.3758 3 .300 10.248 744.92 1 0.093 0.2888 20.993 ! 0.055 0.1708 12.415 35.00 1 0.484 1.3829 1 3 .484 9.9543 719.83 ! 0.114 0.3257 23.554 1 0.059 0.1686 12.190 30.70 I 0.424 1.3811 1 2.996 9.7590 706.60 1 0.109 0.3550 25.708 0 .038 0.1238 8.9623 36.40 0.499 1.3709 1 3 .314 9.1044 664.13 1 0.122 0.3352 24.449 1 0.059 0.1621 11.824 1 34 .22 1 0.476 1.3957 3.362 9.8553 707.39 0.104 0.3050 22.033 1 0 .047 0.1365 9.8179 2 .34 1 0.028 0.1092 1 0 .177 0.7145 41.290 0.016 0.0412 3.8599 1 0.009 0.0254 1.9127 FBW - Final Body Weight - 164- DuPont-18318 p. 164 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Final Body and Organ Weights Group: VIIA Treatment: 10 mg/kg Sex : MALES ! ANIMAL 1 FBW 1 BRAIN 1 1 (Gms) 1 (Gms ) %FBW 1 1 (Gms) LIVER SPLEEN %FBW %BRAIN 1 (Gms) %FBW %BRAIN THYMUS (Gms ) %FBW %BRAIN ! 701 1 31.80 1 0 .471 1.4811 1 7.969 25.060 1691.9 1 0.078 0.2453 16.561 0.029 0.0912 6.1571 1 702 ! 30.90 1 0.477 1.5437 l 7 .155 23.155 1500.0 1 0.081 0.2621 16.981 0.039 0.1262 8.1761 1 703 1 29. 10 1 0.485 1.6667 I 8 .574 29.464 1767.8 1 0.042 0.1443 8.6598 0.016 0.0550 3.2990 1 704 1 28.20 1 0.388 1.3759 I 6.519 23.117 1680.2 1 0.053 0.1879 13.660 0.035 0.1241 9.0206 ! 705 1 28.40 ! 0.439 1.5458 1 4 .627 16.292 1054.0 1 0.058 0.2042 13.212 0.021 0.0739 4.7836 1 706 1 27 .30 f 0.454 1.6630 1 4 .783 17.520 1053.5 1 0.072 0.2637 15.859 0.035 0.1282 7.7093 1 707 ! 24 .70 l 0.414 1.6761 1 5.517 22.336 1332.6 1 0.054 0.2186 13.043 1 0.013 0.0526 3.1401 1 708 1 29.70 1 0.458 1.5421 1 5.939 19.997 1296.7 1 0.069 0.2323 15.066 0.030 0.1010 6.5502 1 709 1 26.60 1 0.465 1.7481 1 6.075 22.838 1306.5 1 0.045 0.1692 9.6774 0.010 0.0376 2.1505 1 710 1 27.20 1 0.389 1.4301 1 5.137 18.886 1320.6 1 0.076 0.2794 19.537 0.026 0.0956 6.6838 1 Mean 1 S .D. ! 1 28.39 1 0.444 1.5673 1 6.230 21.867 1400.4 1 0.063 0.2207 14.226 1 0.025 0.0885 5.7670 1 2.10 1 0.035 0.1193 1 1.331 3.8680 253.65 1 0.014 0.0442 3.3173 0.010 0.0328 2.3287 Group: VIIB Treatment: 10 mg/kg Sex: MALES ANIMAL 1 FBW 1 BRAIN 1 1 (Gms) 1 (Gms) %FBW 1 (Gms ) LIVER 1 SPLEEN 1 THYMUS tFBW %BRAIN 1 (Gms ) %FBW %BRAIN 1 (Gms ) %FBW %BRAIN 711 1 26.80 1 0.427 1 .5933 1 4 .228 15.776 990.16 1 0.066 0.2463 15.457 1 0.014 0.0522 3.2787 712 1 28.90 i 0.478 1.6540 1 5.438 18.817 1137.7 0.082 0.2837 1 /.155 1 0.037 0.1280 7.7406 713 1 31 .60 ! 0.466 1.4747 1 8.346 26.411 1791.0 ] 0.083 0.2627 17.811 1 0.017 0.0538 3.6481 714 1 29.60 1 0.455 1.5372 1 5.068 17.122 1113.8 1 0.128 0.4324 28.132 1 0.042 0.1419 9.2308 715 1 27.40 0.431 1.5730 1 6.320 23.066 1466.4 1 0.053 0.1934 12.297 1 0.020 0.0730 4.6404 716 1 27.00 0.436 1.6148 1 4.902 18.156 1124.3 1 0.052 0.1926 11.927 1 0.023 0.0852 5.2752 717 1 25.20 0.428 1.6984 1 5.396 21.413 1260.7 1 0.065 0.2579 15.187 1 0.026 0.1032 6.0748 718 1 31.00 0.436 1.4065 1 8.136 26.245 1866.1 1 0.064 0.2065 14.679 1 0.019 0.0613 4.3578 719 1 30.00 0.454 1.5133 1 5.884 19.613 1296.0 1 0.054 0.1800 11.894 1 0.027 0.0900 5.9471 720 1 26.90 0.477 1.7732 1 5.205 19.349 1091.2 1 0.046 0.1710 9.6436 1 0.019 0.0706 3.9832 Mean S.D. ! 28.44 1 2.09 t 0.449 1.5838 1 5.892 20.597 1313.7 ! 0.069 0.2427 15.418 1 0.024 0.0859 5.4177 0.020 0.1083 ! 1 .358 3.6430 301.90 1 0.024 0.0772 5.1497 1 0.009 0.0305 1.8903 FBW - Final Body Weight - 165 - DuPont-18318 p. 165 F ^F Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Final Body and Organ Weights Group: IXA Treatment: 30 mg/kg Sex: MALES I ANIMAL FBW 1 BRAIN 1 1 i (Gms) 1 (Gms ) %FBW 1 (Gms ) LIVER %FBW %BRAIN SPLEEN 1 THYMUS 1 (Gms ) %FBW %BRAIN l (Gms ) %FBW %BRAIN 1 1 901 i 26.20 1 0.469 1.7901 1 5.786 22.084 1233.7 1 0.066 0.2519 14.072 0.009 0.0344 1.9190 1 1 902 i 24 .30 1 0.414 1 .7037 1 5.752 23.671 1389.4 1 0.034 0.1399 8.2126 1 0.022 0.0905 5.3140 1 1 903 i 23.70 1 0.448 1.8903 1 5.441 22.958 1214.5 1 0.021 0.0886 4.6875 1 0.017 0.0717 3.7946 1 1 904 i 24 .20 1 0.420 1 .7355 1 5.335 22.045 1270.2 1 0.031 0.1281 7.3810 0.028 0.1157 6.6667 1 1 905 ! 24 .10 1 0.415 1.7220 1 4 .810 19.959 1159.0 ! 0.037 0.1535 8.9157 1 0.023 0.0954 5.5422 1 1 907 1 21.80 t 0.401 1.8394 1 5.128 23.523 1278.8 ! 0.026 0.1193 6.4838 ! 0.015 0.0688 3.7406 1 1 908 1 25.40 1 0.435 1.7126 1 5.628 22.157 1293.8 1 0.037 0.1457 8,5057 1 0.065 0.2559 14.943 1 1 909 1 23.50 1 0.416 1.7702 1 5.189 22.081 1247 .4 1 0.038 0.1617 9.1346 1 0.009 0.0383 2.1635 1 1 910 1 27.00 1 0.461 1.7074 1 7.923 29.344 1718.7 1 0.065 0.2407 14.100 1 0.027 0.1000 5.8568 1 I Mean 1 S .D. 1 24.47 1 0.431 1.7635 5.666 23.091 1311.7 1 0.039 0.1588 9.0548 0.024 0.0968 5.5489 1 1 1 55 1 0.024 0.0656 1 0.903 2.5850 164.98 1 0.016 0.0541 3.1653 1 0.017 0.0656 3.8830 1 Group : IXB Treatment : 30 mg/kg Sex: MALES 1 ANIMAL FBW 1 BRAIN 1 i (Gms) ! (Gms) %FBW 1 1 (Gms ) LIVER SPLEEN 1 THYMUS 1 %FBW %B RAIN 1 (Gms ) %FBW %BRAIN 1 (Gms ) cFBW %BRAIN 1 1 911 ! 25.30 1 0.466 1 .3419 1 5 .957 23.545 1278 .3 1 0.042 0.1660 9.0129 1 0.016 0.0632 3.4335 1 912 1 22.00 0.406 1.8455 1 5.463 24.832 1345.6 1 0.020 0.0909 4.92 61 1 0.014 0.0636 3.4483 1 1 913 1 28.30 1 0.435 1.5371 1 7 .657 27.057 1760.2 1 0.072 0.2544 16.552 ! 0.025 0.0883 5.7471 [ 1 914 28.40 1 0.431 1.5176 5.089 17.919 1180.7 1 0.082 0.2887 19.026 1 0.030 0.1056 6.9606 1 ! 915 1 32.40 1 0.445 1.3735 6.732 20.778 1512 .8 1 0.102 0.3148 22.921 1 0.039 0.1204 8.7640 l 1 916 1 23.30 1 0.419 1.7983 1 b 1 0.049 0.2103 11.695 1 0.023 0.0987 5.4893 1 1 917 ! 29.40 ! 0.462 1.5714 ! 6.316 21.483 1367.1 1 0.068 0.2313 14.719 i 0.039 0.1327 8.4416 1 ! 918 1 28.00 1 0.449 1.6036 1 5.574 19.907 1241 .4 1 0.067 0.2393 14.922 I 0.029 0.1036 6.4588 1 1 919 1 27 .10 1 0.459 1.6937 1 5.895 21.753 1284 .3 1 0.067 0.2472 14.597 ! 0.026 0.0959 5.6645 ! 920 1 29.50 1 0.504 1.7085 1 6.499 22.031 1289.5 0.068 0.2305 13.492 1 0.014 0.0475 2 .7778 1 1 Mean 1 S .D . 1 27.37 1 0.448 1.6491 6.131 22.145 1362.2 0.064 0.2274 14.186 1 3.09 1 0.028 0.1554 1 0.774 2.7075 175.82 1 0.022 0.0627 5.0057 0.026 0.0920 5.7185 1 0.009 0.0268 2.0510 1 FBW - Final Body Weight - 166- DuPont-18318 p. 166 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Final Body and Organ Weights Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES ANIMAL FBW BRAIN i (Gms ) 1 (Gms ) %FBW 1 (Gms ) LIVER %FBW %BRAIN SPLEEN (Gms ) %FBW %BRAIN THYMUS I (Gms ) %FBW %BRAIN I 1101 i 31.30 1 0.488 1.5591 1 8.009 25.588 1641.2 1102 ! 39.50 ! 0.418 1.0582 1 8.361 21.167 2000.2 1103 1 32.20 1 0.426 1.3230 ! 5.329 16.550 1250.9 1104 30.40 i 0.424 1.3947 6.914 22.743 1630.7 1105 1 34 .40 1 0.432 1.2558 I 7.432 21.605 1720.4 1106 1 27 .20 1 0.468 1.7206 1 6 .657 24.474 1422 .4 1107 1 27 .10 1 0.386 1.4244 1 6.001 22.144 1554 .7 1108 1 32.30 1 0.436 1.3498 1 7.295 22.585 1673.2 1109 I 34 .70 1 0.448 1.2911 1 6.493 18.712 1449.3 1110 24.90 1 0.446 1.7912 1 5.491 22.052 1231.2 Mean S.D. 1 31.40 1 0.437 1.4168 I 6.798 21.762 1557.4 1 4 .30 1 0.028 0.2203 1 1 .010 2.5973 230.63 0.091 0.107 0.069 0.086 0.081 0.058 0.060 0.067 0.104 0.091 0.2907 0.2709 0.2143 0.2829 0.2355 0.2132 0.2214 0.2074 0.2997 0.3655 18 .648 2b .598 16 .197 20 .283 18 .750 12 .393 15 .544 15 .367 23 .214 20 .404 0.081 0.2602 18.640 0.017 0.0510 3.9504 0.027 0.048 0.031 0.028 0.020 0.026 0.018 0.013 0.032 0.014 0.0863 0.1215 0.0963 0.0921 0.0581 0.0956 0.0664 0.0402 0.0922 0.0562 5.5328 11.483 7.2770 6038 6296 5556 6632 9817 7.1429 3.1390 0.026 0.0805 5.9009 0.010 0.0244 2.4641 Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES ANIMAL 1111 1113 1114 1115 1116 1117 1118 1119 1120 Mean S .D. FBW BRAIN (Gms ) (Gms) %FBW 30.80 34 .70 30.40 31 .60 26.90 27 .50 28.60 26.20 28.70 0.419 0.474 0.436 0.466 0.418 0.490 0.425 0.413 0.480 1.3604 1.3660 1 .4342 1.4747 1.5539 1 .7818 1.4860 1.5763 1.6725 29.49 2 .67 0.447 1.5229 0.030 0.1396 (Gms ) LIVER I SPLEEN %FBW %BRAIN I (Gms) %FBW %BRAIN 6.983 22.672 1666.6 0.064 0.2078 15.274 670 4.8127 352.32 0.133 0.3833 28.059 359 20.918 1458.5 0.048 0.1579 11.009 0.086 0.2722 18.455 006 22.327 1436.8 0.062 0.2305 14.833 827 24.825 1393.3 0.072 0.2618 14.694 310 25.559 1720.0 0.065 0.2273 15.294 637 17.698 1122.8 0.052 0.1985 12.591 7 .271 25.334 1514.8 I 0.055 0.1916 11.458 I 5.883 20.518 1333.1 I 0.071 0.2368 15.741 1.913 6.8645 435.92 I 0.026 0.0652 5.1490 THYMUS (Gms ) %FBW 1BRAIN 0.032 0.037 0.045 0.025 0.034 0.023 0.026 0.016 0 .020 0.1039 0.1066 0.1480 0.0791 0.1264 0.0836 0.0909 0.0611 0.0697 7.6372 7.8059 10.321 5.3648 8.1340 4.6939 6.1176 3.8741 4.1667 0.029 0.0966 6.4573 0.009 0.0278 2.1590 FBW - Final Body Weight - 167- DuPont-18318 p. 167 p. 168 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Appendix K Individual Animal Pathology Data - 168- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 169 DuPont-18318 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 .). - 169- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IA Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 101 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : FATTY CHANGE, DIFFUSE, THYMUS : ECTOPIC THYROID. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 102 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : SPLEEN : HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN. 102 Continued on the next page EXTRAMEDULLARY, minimal. p. 170 DuPont-18318 -170- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IA Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 102 Continued from previous page Histopathology : LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 103 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. minimal. No Microscopic Abnormality Observed : LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 104 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 .... p. 171 DuPont-18318 - 171 - 34-3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IA Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 104 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEAL 105 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEAL 106 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE jt)INT, STERNUM, POPLITEAL LYMPH NODE 106 Continued on the next page .... p. 172 DuPont-18318 - 172- 3H1 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IA Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 106 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 107 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 108 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 108 Continued on the next page .... p. 173 DuPont-18318 - 173 - 3^0 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IA Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 108 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEAL 109 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : FATTY CHANGE, DIFFUSE, CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 110 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 110 Continued on the next page .... p. 174 DuPont-18318 - 174- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IA Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 110 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEAL p. 175 DuPont-18318 - 175 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IB Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 111 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : SPLEEN : HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN. EXTRAMEDULLARY, mild. No Microscopic Abnormality Observed : LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 112 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 176 DuPont-18318 - 176- p. 177 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Individual Animal Pathology Data Group: IB Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 113 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 114 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : SPLEEN : HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN. EXTRAMEDULLARY, minimal. 114 Continued on the next page .... - 177- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IB Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 114 Continued from previous page Histopathology : No Microscopic Abnormality Observed : LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 115 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : SPLEEN : HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN. EXTRAMEDULLARY, minimal. No Microscopic Abnormality Observed : LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 116 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 116 Continued on the next page .... p. 178 DuPont-18318 - 178 - 3 -5*S Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IB Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 116 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 117 Unscheduled Sacrifice Duration of dosing-days: 5 Exposure Group : Early Deaths Animal is signed off from necropsy Gross Pathology : TRACHEA : RUPTURE. ESOPHAGUS : RUPTURE. SKIN : OTHER, abscess pocket, dorsal subcutaneous axilla neck, right axilla. r i g h t ,s u b c u t a n e o u s air No Macroscopic Abnormality Observed : LIVER Histopathology : MESENTERIC LYMPH NODE : DEPLETION/ATROPHY, LYMPHOID, minimal, (outer cortex and follicles). THYMUS : DEPLETION/ATROPHY, LYMPHOID, mild. ESOPHAGUS : INFLAMMATION, MYOFIBER, mild, (due to esophageal rupture). SKIN : Moderate, ABSCESS. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate. 117 Continued on the next page p. 179 DuPont-18318 - 179- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IB Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 117 Continued from previous page Histopathology : CAUSE OF DEATH : DOSING ACCIDENT. MEDIASTINUM : INFLAMMATION, CHRONIC, (due to esophageal LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. rupture). No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, TRACHEA, FEMUR/KNEE JOINT, STERNUM 118 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 119 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 119 Continued on the next page .... DuPont-18318 - 180- is '7 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IB Treatment: 0 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 119 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 120 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : SPLEEN : HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN. EXTRAMEDULLARY, minimal. No Microscopic Abnormality Observed : LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 181 DuPont-18318 - 181 - p. 182 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Individual Animal Pathology Data Group: IIIA Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 301 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 302 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with 302 Continued on the next page .... - 18? - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIA Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 302 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 303 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 304 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. 183 DuPont-18318 - 183 - 3 2 p. 184 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Individual Animal Pathology Data Group: IIIA Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 304 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 305 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 306 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 .... - 184 %I Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIA Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 306 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, mild, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 307 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 185 DuPont-18318 - 185 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: I1IA Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 308 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 309 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. 309 Continued on the next page .... HEPATOCELLULAR, stippling. mild, with p. 186 DuPont-18318 - 186- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIA Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 309 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 310 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, FOCAL, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 187 DuPont-18318 - 187- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIB Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 311 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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, mild, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 312 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. 312 Continued on the next page .... mild, with p. 188 DuPont-18318 - 188- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIB Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 312 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 313 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 314 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 314 Continued on the next page .... p. 189 DuPont-18318 - 189- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIB Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 314 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 315 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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, mild, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 190 DuPont-18318 -190- JL7 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIB Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 316 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 317 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. 317 Continued on the next page .... mild, with p. 191 DuPont-18318 - 191 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIB Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 317 Continued from previous page Histopathology : No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 318 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 319 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 319 Continued on the next page .... p. 192 DuPont-18318 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IIIB Treatment: 0.3 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 319 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. mild, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 320 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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, mild, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 193 DuPont-18318 - 193 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VA Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 501 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 502 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. 502 Continued on the next page .... p. 194 DuPont-18318 - 194- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VA Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 502 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 503 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 504 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 504 Continued on the next page .... p. 195 DuPont-18318 - 195 - 371 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VA Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 504 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. THYMUS : HYPERPLASIA, LYMPHOID, FOLLICULAR, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 505 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 196 DuPont-18318 - 196- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VA Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 506 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 507 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. 507 Continued on the next page .... p. 197 DuPont-18318 - 197- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VA Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 507 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 508 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, FOCAL, minimal. INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 509 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 509 Continued on the next page .... p. 198 DuPont-18318 - 198- 375 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VA Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 509 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 510 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 199 DuPont-18318 - 199- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VB Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 511 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 512 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, FOCAL, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MESENTERIC LYMPH NODE : NOT PRESENT. 512 Continued on the next page .... with p. 200 DuPont-18318 -200- 377 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VB Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 512 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, THYMUS, BONE MARROW 513 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 514 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 514 Continued on the next page .... p. 201 DuPont-18318 -201 - 378 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VB Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 514 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. NECROSIS, FOCAL, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 515 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 202 DuPont-18318 - 202 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VB Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 516 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. THYMUS : NOT PRESENT. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 517 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. 517 Continued on the next page .... p. 203 DuPont-18318 -203 - 360 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VB Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 517 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 518 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 CAUSE OF DEATH : SACRIFICE BY DESIGN. HEPATOCELLULAR, stippling. moderate, with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 519 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 519 Continued on the next page .... p. 204 DuPont-18318 -204 3s( Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VB Treatment: 1 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 519 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 520 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 205 DuPont-18318 -205- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 701 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. Minimal, FATTY CHANGE, NONZONAL. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 702 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NODE 702 Continued on the next page .... THYMUS, FEMUR/KNEE p. 206 DuPont-18318 -206 363 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref /02 Microscopic & Macroscopic Findings Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 703 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology 703 Continued LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, FOCAL, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERPLASIA, BILE DUCT, minimal. on the next page with DuPont-18318 - 207 - 3 3 if- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 703 Continued from previous page Histopathology : THYMUS : DEPLETION/ATROPHY, LYMPHOID, CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 704 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 208 DuPont-18318 - 208 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 705 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 706 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 706 Continued on the next page .... p. 209 DuPont-18318 -209- 38b Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 706 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 707 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. THYMUS : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. 707 Continued on the next page .... p. 210 DuPont-18318 -210- 387 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 707 Continued from previous page Histopathology : THYMUS : ECTOPIC THYROID. DEPLETION/ATROPHY, LYMPHOID, CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 708 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. THYMUS : DEPLETION/ATROPHY, LYMPHOID, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW p. 211 DuPont-18318 -211 - 38 7 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 709 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. THYMUS : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. Minimal, FATTY CHANGE, NONZONAL. THYMUS : DEPLETION/ATROPHY, LYMPHOID, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 710 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 710 Continued on the next page .... p. 212 DuPont-18318 -212- 3.S? Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIA Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 710 Continued from previous page Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 213 DuPont-18318 -213 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 711 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. Minimal, FATTY CHANGE, NONZONAL. THYMUS : NOT PRESENT. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 712 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 712 Continued on the next page .... p. 214 DuPont-18318 -214 3?! Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 712 Continued from previous page Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 713 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. DISCOLORATION, TAN, 1CM DIA. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, moderate. HYPERPLASIA, BILE DUCT, minimal. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. 713 Continued on the next page ... . p. 215 DuPont-18318 -215- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 713 Continued from previous page Histopathology : THYMUS : DEPLETION/ATROPHY, LYMPHOID, minimal. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE 714 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SKIN : MASS, GREEN, AXILLA, LEFT, 1.5CM DIA. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. SKIN : Moderate, ABSCESS. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate. 714 Continued on the next page p. 216 DuPont-18318 -216- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 714 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, THYMUS 715 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW p. 217 DuPont-18318 -217- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 716 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 717 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. THYMUS : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE POPLITEAL LYMPH NODE 717 Continued on the next page JOINT, STERNUM, p. 218 DuPont-18318 - 218 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 717 Continued from previous page Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. MESENTERIC LYMPH NODE : NOT PRESENT IN TISSUE SECTION. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, THYMUS, BONE MARROW 718 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. 718 Continued on the next page .... p. 219 DuPont-18318 -219- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 718 Continued from previous page Histopathology : LIVER : Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE OF DEATH : SACRIFICE BY DESIGN. minimal. No Microscopic Abnormality Observed : MESENTERIC LYMPH NODE, BONE MARROW 719 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW p. 220 DuPont-18318 -220- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: VIIB Treatment: 10 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 720 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, mild. INFLAMMATION, SUBACUTE/CHRONIC, minimal. THYMUS : DEPLETION/ATROPHY, LYMPHOID, minimal. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. with No Microscopic Abnormality Observed : SPLEEN, MESENTERIC LYMPH NODE p. 221 DuPont-18318 -221 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXA Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 901 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. NECROSIS, FOCAL, minimal, coagulative, subcapsular. HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). THYMUS : DEPLETION/ATROPHY, LYMPHOID, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 902 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. 902 Continued on the next page ... p. 222 DuPont-18318 - 222 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXA Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 902 Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. THYMUS : NOT PRESENT. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM 903 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. THYMUS : SMALL. 903 Continued on the next page .... p. 223 DuPont-18318 - 223 - HCO Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXA Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 903 Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 904 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. 904 Continued on the next page ... p. 224 DuPont-18318 -224 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXA Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 904 Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. NECROSIS, FOCAL, minimal, coagulative, subcapsular. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, lymphoid sheath). MESENTERIC LYMPH NODE : NOT PRESENT IN TISSUE SECTION. THYMUS : (periarteriolar NOT PRESENT IN MEDIASTINAL TISSUE. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEAL 905 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. 905 Continued on the next page ... p. 225 DuPont-18318 - 225 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 226 DuPont-18318 Individual Animal Pathology Data Group: IXA Animal Ref 905 Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Gross Pathology : SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . MESENTERIC LYMPH NODE : NOT PRESENT IN TISSUE SECTION. THYMUS : DEPLETION/ATROPHY, LYMPHOID, severe. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEAL 906 Unscheduled Sacrifice Duration of dosing-days: 9 Exposure Group : Early Deaths Animal is signed off from necropsy Gross Pathology : LIVER : DISCOLORATION, PALE, MOTTLED, DIFFUSE. 906 Continued on the next page .... - 226 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group: IXA Individual Animal Pathology Data Treatment: 30 mg/kg Sex: MALES Animal Ref 906 Microscopic & Macroscopic Findings Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, minimal, coagulative, subcapsular. FATTY CHANGE, PERIPORTAL, minimal. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, moderate, (periarteriolar lymphoid sheath). MESENTERIC LYMPH NODE : DEPLETION/ATROPHY, LYMPHOID, moderate, (inner cortex and outer cortex). THYMUS : DEPLETION/ATROPHY, LYMPHOID, severe. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate, (immature). CAUSE OF DEATH : UNDETERMINED. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. with left shift No Microscopic Abnormality Observed : BRAIN, FEMUR/KNEE JOINT, STERNUM 907 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. 907 Continued on the next page .. p. 227 DuPont-18318 -227- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXA Animal Ref 907 Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). MESENTERIC LYMPH NODE : DEPLETION/ATROPHY, LYMPHOID, mild, (inner cortex, outer cortex, and follicles). THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT. No Microscopic Abnormality Observed : BRAIN, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 908 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. 908 Continued on the next page ... p. 228 DuPont-18318 -228 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group: IXA Animal Ref 908 Individual Animal Pathology Data Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Gross Pathology : LIVER : DISCOLORATION, SPLEEN : SMALL. PENIS : PARAPHIMOSIS. TAN, MOTTLED. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, moderate, coagulative, subcapsular. HYPERPLASIA, BILE DUCT, mild. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, CAUSE OF DEATH : SACRIFICE BY DESIGN. PENIS : minimal. EROSION/ULCER, moderate. PREPUTIAL GLANDS : ECTASIA, mild. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. 908 Continued on the next page p. 229 DuPont-18318 -229- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXA Animal Ref 908 Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Histopathology : No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM 909 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. THYMUS : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. NECROSIS, FOCAL, minimal, coagulative, subcapsular. THYMUS : DEFLETIO N /ATROPHY, LYMPHOID, severe. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL p. 230 DuPont-18318 -230- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice________ Group: IXA Individual Animal Pathology Data Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 910 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. DISCOLORATION, SPLEEN : SMALL. TAN, MOTTLED, LEFT. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERPLASIA, BILE DUCT, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. DEPLETION/ATROPHY, LYMPHOID, mild, (periarteriolar lymphoid sheath) . THYMUS : NOT PRESENT. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL p. 231 DuPont-18318 -231 - W>8 Ammonium Periluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXB Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 911 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. THYMUS : DEPLETION/ATROPHY, LYMPHOID, severe. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 232 DuPont-18318 - 232 - m Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXB Animal Ref Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings 912 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. THYMUS : minimal. NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 913 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. DISCOLORATION, TAN, RIGHT, ACCESSORY. 913 Continued on the next page .... p. 233 DuPont-18318 - 233 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 234 DuPont-18318 Individual Animal Pathology Data Group: XB Treatment: 30 mg/kg Sex: MALES Animal Ref 913 Microscopic & Macroscopic Findings Continued from previous page Gross Pathology : MESENTERIC LYMPH NODE : SMALL. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. NECROSIS, FOCAL, moderate, coagulative, subcapsular. HYPERPLASIA, BILE DUCT, mild. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. THYMUS : DEPLETION/ATROPHY, LYMPHOID, moderate. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 914 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 914 Continued on the next page ... -234- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group: IXB Animal Ref 914 Individual Animal Pathology Data Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, HYPERPLASIA, BILE DUCT, minimal. CAUSE OF DEATH : minimal. SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 915 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. DISCOLORATION, LEFT, 1MM DIA. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. 915 Continued on the next page .... p. 235 DuPont-18318 - 235 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXB Animal Ref 915 Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 916 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : DISCOLORATION, TAN, CAUDATE, 0.3 CM DIA. LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. NECROSIS, FOCAL, moderate, coagulative, subcapsular. INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. THYMUS : DEPLETION/ATROPHY, LYMPHOID, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. 916 Continued on the next page ... p. 236 DuPont-18318 - 236 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice____________________ Group: IXB Individual Animal Pathology Data Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 916 Continued from previous page Histopathology : No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 917 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. NECROSIS, FOCAL, minimal, coagulative, subcapsular. HYPERPLASIA, BILE DUCT, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : . SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL p. 237 DuPont-18318 - 237 - p. 238 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Individual Animal Pathology Data Group: IXB Treatment: 30 mg/kg Sex: MALES Animal Ref Microscopic & Macroscopic Findings 918 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. HYPERPLASIA, BILE DUCT, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 919 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. 919 Continued on the next page .... - 238 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group: IXB Animal Ref 919 Individual Animal Pathology Data Treatment: 30 mg/kg Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. BONE MARROW : minimal. HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEAL 920 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 : HYPERPLASIA, BILE DUCT, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. 920 Continued on the next page .. .. p. 239 DuPont-18318 -239- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: IXB Treatment: 30 mg/kg Sex: MALES Animal Ref 920 Microscopic & Macroscopic Findings Continued from previous page Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). THYMUS : DEPLETION/AT R O P H Y , LYMPHOID, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL p. 240 DuPont-18318 -240- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice__________ Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1101 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, HYPERPLASIA, BILE DUCT, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : mild. DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 241 DuPont-18318 -241 - *H8 p. 242 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________________________________________ DuPont-18318 Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1102 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HYPERPLASIA, BILE DUCT, minimal. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. MESENTERIC LYMPH NODE : NOT PRESENT IN TISSUE SECTION. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, THYMUS, FEMUR/KNEE JOINT, STERNUM - 242 - H Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIA Animal Ref 1103 Treatment: 30/0 mg/kg (Recovery) Sex: MALES Microscopic & Macroscopic Findings Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. 1104 No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. 1104 Continued on the next page .... p. 243 DuPont-18318 -243 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic E'indings 1104 Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, EEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS : CYST, EPITHELIAL, minimal. BONE MARROW : HYPERPLASIA, ERYTHROCYTIC, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEAL 1105 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. 1105 Continued on the next page .... p. 244 DuPont-18318 -244- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Group: XIA Animal Ref Individual Animal Pathology Data Treatment: 30/0 mg/kg (Recovery) Sex: MALES Microscopic s Macroscopic Findings 1105 Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. BONE MARROW : HYPERPLASIA, ERYTHROCYTIC, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM 1106 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NODE 1106 Continued on the next page .... THYMUS, FEMUR/KNEE DuPont-18318 -245 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1106 Continued from previous page Histopathology : LIVER : MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. HYPERPLASIA, BILE DUCT, minimal. THYMUS : CYST, EPITHELIAL, minimal. DEPLETION/ATROPHY, LYMPHOID, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 1107 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. THYMUS : SMALL. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : 1107 LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. Continued on the next page .. with p. 246 DuPont-18318 -246- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1107 Continued from previous page Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. NECROSIS, FOCAL, minimal, coagulative, subcapsular. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1108 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. THYMUS : SMALL. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1108 Continued on the next page .... p. 247 DuPont-18318 -247- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1108 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. INFLAMMATION, SUBACUTE/CHRONIC, minimal. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1109 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. 1109 Continued on the next page .... severe, with p. 248 DuPont-18318 -248 - V25 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_______________ Individual Animal Pathology Data Group: XIA Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings Continued from previous page Histopathology : LIVER : HYPERPLASIA, BILE DUCT, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HEMATOPOIESIS, EXTRAMEDULLARY, minimal. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, moderate. DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). BONE MARROW : HYPERPLASIA, ERYTHROCYTIC, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM 1110 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. DISCOLORATION, TAN, LEFT, NECROTIC 6MM DIAM. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1110 Continued on the next page .... p. 249 DuPont-18318 -249- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 250 DuPont-18318 Individual Animal Pathology Data Group: XIA Animal Ref 1110 Treatment: 30/0 mg/kg (Recovery) Sex: MALES Microscopic & Macroscopic Findings Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, FOCAL, moderate, coagulative, subcapsular. HYPERPLASIA, BILE DUCT, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. SPLEEN : DE PLETION/ATROPHY, LYMPHOID, mild, (periarteriolar lymphoid sheath). HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. THYMUS : DEPLETION/ATROPHY, LYMPHOID, moderate. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM - 250- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_________ Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1111 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 1112 Unscheduled Sacrifice Duration of dosing-days: 5 Exposure Group : Early Deaths Animal is signed off from necropsy Gross Pathology : ESOPHAGUS : RUPTURE. SKIN : OTHER, ABSCESS AXILLA RIGHT. 1112 Continued on the next page .... p. 251 DuPont-18318 -251 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref 1112 Microscopic & Macroscopic Findings Continued from previous page Gross Pathology No Macroscopic Abnormality Observed : LIVER Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, minimal, with cytoplasmic eosinophilic stippling. FATTY CHANGE, DIFFUSE, minimal. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, moderate, (periarteriolar lymphoid sheath). MESENTERIC LYMPH NODE : DEPLETION/ATROPHY, LYMPHOID, mild. THYMUS : DEPLETION/ATROPHY, LYMPHOID, severe. BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate, with left shift (immature). CAUSE OF DEATH : DOSING ACCIDENT. No Microscopic Abnormality Observed : BRAIN, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEAL 1113 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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 1113 Continued on the next page ... p. 252 DuPont-18318 - 252 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1113 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1114 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. SPLEEN : SMALL. No Macroscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : 1114 LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling. HYPERPLASIA, BILE DUCT, minimal. Continued on the next page .... severe, with p. 253 DuPont-18318 - 253 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1114 Continued from previous page Histopathology : LIVER : NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. minimal. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1115 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. INFLAMMATION, SUBACUTE/CHRONIC, minimal. SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. 1115 Continued on the next page .... p. 254 DuPont-18318 -254V 5/ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1115 Continued from previous page Histopathology : CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1116 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD 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, severe, with cytoplasmic eosinophilic stippling. INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. NECROSIS, FOCAL, minimal, coagulative, subcapsular. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild. THYMUS : DEFLETIO N /ATROPHY, LYMPHOID, severe. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. 1116 Continued on the next page .... p. 255 DuPont-18318 - 255 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1116 Continued from previous page Histopathology : No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1117 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, mild. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild. HYPERPLASIA, BILE DUCT, minimal. INFLAMMATION, SUBACUTE/CHRONIC, minimal. Minimal, FATTY CHANGE, NONZONAL. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW p. 256 DuPont-18318 -256V J3 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1118 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. CAUSE OF DEATH : SACRIFICE BY DESIGN. LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION. No Microscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1119 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy Gross Pathology : LIVER : LARGE. 1119 Continued on the next page .... p. 257 DuPont-18318 -257- ^3^ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1119 Continued from previous page Gross Pathology : No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. DEPL E T ION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath). THYMUS : NOT PRESENT IN MEDIASTINAL TISSUE. CAUSE Or DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL 1120 Terminal Sacrifice Killed on Day : 29 Exposure Group : SD Animal is signed off from necropsy ` Gross Pathology : LIVER : LARGE. No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NODE 1120 Continued on the next page .... THYMUS, FEMUR/KNEE p. 258 DuPont-18318 - 258 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Animal Pathology Data Group: XIB Treatment: 30/0 mg/kg (Recovery) Sex: MALES Animal Ref Microscopic & Macroscopic Findings 1120 Continued from previous page Histopathology : LIVER : HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling. NECROSIS, INDIVIDUAL CELL, INCREASED, minimal. MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal. HYPERPLASIA, BILE DUCT, minimal. SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal. THYMUS : DEPLETION/ATROPHY, LYMPHOID, moderate. CAUSE OF DEATH : SACRIFICE BY DESIGN. No Microscopic Abnormality Observed : BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEAL p. 259 DuPont-18318 -259 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 260 DuPont-18318 Appendix L Individual Total Cell Counts -260- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 261 DuPont-18318 INDIVIDUAL TOTAL CELL COUNTS EXPLANATORY NOTES ABBREVIATIONS: NP - not taken or not performed FOOTNOTES: c Animal was sacrificed in extremis prior to this evaluation and tissue was not analyzed, d Count inadvertently not performed, e Unable to confirm results. NOTES : Organ Weight as Percent of Body Weight Organ Weight (g) Final Body Weight (g) Total Number of Organ Cells (xlO3) Organ Weight (g) Half Organ Weight (g) Organ Cell Suspension Volume (mL) 100 Number of Cells in Half X Organ (x 10 cells/mL) 100 -261 - *3$ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Animal Number Final Body Weight (g) Spleen Weight (g) Male, Group I - 0 mg /kg 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117d 118 119 120 31 .00 34 .70 33.40 32.50 33.00 31.20 31.90 30 .30 32.70 33 .90 32 .60 33 .90 34.60 33.70 31.50 34.20 NP 34.50 34.30 33.50 0.106 0.110 0.124 0.111 0.091 0.114 0 .128 0 .107 0.112 0.119 0.145 0.122 0.117 0.115 0 .107 0.111 NP 0.099 0.152 0.138 Individual Total Cell Counts Spleen Weight (% Body Weight) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 0.3419 0.3170 0.3713 0.3415 0.2758 0.3654 0.4013 0.3531 0.3425 0.3510 0.4448 0.3599 0.3382 0.3412 0.3397 0.3246 NP 0.2870 0.4431 0.4119 0.046 0.052 0.060 0.056 0.050 0.058 0.065 0.056 0.048 0.061 0.077 0.054 0.059 0.061 0.051 0.059 NP 0.051 0.076 0.061 5.5 5.5 5.3 5.2 5.5 5.3 5 .4 5 .4 5 .4 5.5 4 .4 5.5 5.0 5.5 5.5 4.5 NP 5.4 5.4 5.4 Number of Cells in Half Spleen (x 106 cells/mL) 10.06 7 .54 12 .04 16.00 10.89 8.86 10.94 8 .80 7 .37 13.97 25.08 13.42 14 .14 13.86 10.78 9.40 NP b 13.53 12.60 Total Number of Spleen Cells (xl0a) 1.27 0 .88 1 .32 1 .65 1.09 0.92 1.16 0.91 0 .93 1.50 2.08 1 .67 1.40 1 .44 1.24 0.80 NP b 1.46 1 .54 p. 262 - 262 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Total Cell Counts Animal Number Final Body Weight (g) Spleen Weight (g) Spleen Weight (% Body Weight) Male, Group III - 0.3 mg/kg Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 301 35.10 0.150 302 36.10 0.108 303 37.40 0.104 304 37.20 0.188 305 34 .50 0 .117 306 33.10 0.102 307 31.30 0.095 308 33.70 0.129 309 29.90 0.112 310 32.80 0.130 311 36.60 0.114 312 33.40 0.136 313 31.50 0.076 314 32.20 0.135 315 33.80 0.152 316 31. 60 0.099 317 30.20 0.060 318 30 .10 0 .106 319 30 .90 0.060 320 36.70 0 .151 0.4274 0.2992 0.2781 0.5054 0.3391 0.3082 0.3035 0.3828 0.3746 0.3963 0.3115 0.4072 0.2413 0.4193 0.4497 0 .3133 0.1987 0.3522 0.1942 0.4114 0.070 0.059 0.047 0.085 0.051 0.046 0.049 0.059 0.047 0.063 0.057 0.065 0.043 0.075 0.075 0.045 0.022 0.046 0.023 0.071 5.5 5.5 5.5 5.5 5.5 5.3 5.5 5.5 5.3 5.4 5.4 5.5 5.7 5.7 5.6 5.5 5.4 5.5 4 .5 6.0 Number of Cells in Half Spleen (x 106 cells/mL) 14 .74 10.94 11 .88 14.46 10.18 10.12 11.33 9 .62 8.20 6.88 16.34 18.20 6.16 21.84 17.32 7.10 3.02 8.25 5.56 13.20 Total Number of Spleen Cells (x 108) 1.74 1.10 1.45 1.76 1 .28 1.19 1.21 1.16 1 .04 0.77 1.76 2.09 0.62 2.24 1 .97 0.86 0.44 1 .05 0.65 1 .68 Oiri7 p. 263 -263 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Animal Number Final Body Weight (g) Spleen Weight (g) Male, Group V - 1 mg /kg 501 33.30 0.088 502 34 .00 0 .135 503 33.50 0.090 504 33.30 0.108 505 33.00 0.092 506 35 .10 0.115 507 31.30 0.107 508 33.90 0.103 509 33.50 0.112 510 32.30 0.078 511 36.80 0.081 512 36.00 0.118 513 35 .90 0.106 514 31.40 0.088 515 35.70 0.124 516 32.10 0.088 517 32.20 0.093 518 35.00 0 .114 519 30.70 0.109 520 36.40 0 .122 Individual Total Cell Counts Spleen Weight (% Body Weight) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 0.2643 0.3971 0.2687 0.3243 0.2788 0.3276 0.3419 0.3038 0.3343 0.2415 0.2201 0.3278 0.2953 0.2803 0.3473 0.2741 0.2888 0.3257 0.3550 0.3352 0.050 0.068 0.048 0.054 0.048 0.058 0.056 0.054 0.049 0.031 0.037 0.055 0.062 0.041 0.059 0.041 0.048 0.054 0.050 0.060 5.0 5.5 5.6 5.5 5.4 5.5 5.3 5.3 5.2 5.0 5.0 5 .6 5.7 5.7 5.7 5.5 5 .6 5.7 5.0 5.8 Number of Cells in Half Spleen (x 106 cells/mL) 16.50 16.66 7.42 13.26 11 .55 12 .60 14 .08 12 .43 13.42 9.02 13.80 10.06 12.16 11.72 11.22 8.08 11.06 13.04 11.55 14.36 Total Number of Spleen Cells (xl0s) 1.45 1.82 0.78 1.46 1.20 1 .37 1.43 1.26 1 .60 1.13 1 .51 1.21 1.19 1.43 1 .34 0.95 1 .20 1.57 1.26 1.69 p. 264 - 264 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Total Cell Counts Animal Number Final Body Weight <g> Spleen Weight (g) Spleen Weight (% Body Weight) Male, Group VII - 10 mg/kg Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 701 31. 80 0.078 702 30.90 0.081 703 29.10 0.042 704 28.20 0.053 705 28.40 0.058 706 27.30 0.072 707 24 .70 0.054 708 29.70 0.069 709 26.60 0.045 710 27.20 0.076 711 26.80 0.066 712 28.90 0.082 713 31 .60 0.083 714 29.60 0 .128 715 27.40 0.053 716 27.00 0.052 717 25.20 0.065 718 31.00 0.064 719 30.00 0.054 720 26.90 0.046 0.2453 0.2621 0.1443 0.1879 0.2042 0.2637 0.2186 0.2323 0.1692 0.2794 0.2463 0.2837 0.2627 0.4324 0.1934 0.1926 0.2579 0.2065 0.1800 0.1710 0.040 0.043 0.014 0.027 0.031 0.032 0.023 0.031 0.020 0.034 0.032 0.030 0.040 0.066 0.026 0.024 0.029 0.025 0.028 0.023 5.5 5.3 5.0 5.2 5.4 5.5 5.5 5.4 5.1 5.4 5.4 5.0 5.6 6.0 5.4 5.7 5.4 5.5 5.7 5.4 Number of Cells in Half Spleen (x 106 cells/mL) 11 .72 9.18 2.86 5 .61 5.17 4.51 3 .90 5.39 2 .97 5.88 7.15 6.93 8 .69 9.79 5.66 4.18 5.78 4.46 4 .29 4 .29 Total Number of Spleen Cells (xlO8) 1 .26 0.92 0.43 0.57 0.52 0.56 0.50 0.65 0.34 0.71 0.80 0.95 1 .01 1.14 0.62 0.52 0.70 0.63 0.47 0.46 p. 265 -265 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Animal Number Final Body Weight (g) Spleen Weight (g) Male, Group IX - 30 mg/kg 901 902 903 904 905 90 6a 907 908 909 910 911 912 913 914 915 916 917 918 919 920 26.20 24.30 23.70 24.20 24.10 NP 21.80 25.40 23.50 27 .00 25.30 22.00 28.30 28.40 32.40 23.30 29.40 28.00 27 .10 29.50 0.066 0.034 0.021 0.031 0.037 NP 0.026 0.037 0.038 0.065 0.042 0.020 0.072 0.082 0 .102 0.049 0.068 0.067 0.067 0.068 Individual Total Cell Counts Spleen Weight (% Body Weight) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 0.2519 0.1399 0.0886 0.1281 0.1535 NP 0.1193 0.1457 0.1617 0.2407 0.1660 0.0909 0.2544 0.2887 0.3148 0.2103 0.2313 0.2393 0.2472 0.2305 0.035 0.014 0.016 0.016 0.017 NP 0.013 0.034 0.015 0.036 0.020 0.013 0.038 0.036 0.041 0.026 0.032 0.029 0.031 0.033 5.0 5.5 5.7 5.5 5.5 NP 5.5 5.5 5.5 5.5 5.3 5.5 5.7 5.5 5.8 5.5 5.4 5.3 5.2 5.0 Number of Cells in Half Spleen (x 106 cells/mL) 4 .78 1.43 3.14 0.82 1 .04 NP 1 .48 3.85 2 .36 4 .73 1 .87 1 .92 6.44 9.84 7.04 4 .18 5.72 8.08 7 .15 7.04 Total Number of Spleen Cells (xlO'") 0.45 0.19 0.23 0.09 0.12 NP 0.16 0.23 0.33 0.47 0.21 0.16 0.70 1 .23 1.02 0.43 0.66 0.99 0.80 0.73 p. 266 - 266 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Total Cell Counts Animal Number Final Body Weight (g) Spleen Weight (g) Spleen Weight (% Body Weight) Male, Gr oup X X - 30/0 mg/kg (Recovery) Half Spleen Weight (g) Spleen Cell Suspension Volume (mL) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112a 1113 1114 1115 1116 1117 1118 1119 1120 31.30 39.50 32.20 30.40 34.40 27.20 27.10 32.30 34 .70 24.90 30.80 NP 34.70 30.40 31.60 26.90 27.50 28 .60 26.20 28.70 0.091 0.107 0.069 0.086 0.081 0.058 0.060 0.067 0 .104 0.091 0.064 NP 0.133 0.048 0.086 0.062 0.072 0.065 0.052 0.055 0.2907 0.2709 0.2143 0.2829 0.2355 0.2132 0.2214 0.2074 0.2997 0.3655 0.2078 NP 0.3833 0.1579 0.2722 0.2305 0.2618 0.2273 0.1985 0.1916 0.046 0.053 0.038 0.044 0.044 0.030 0.028 0.030 0.049 0.044 0.033 NP 0.053 0.026 0.043 0.030 0.039 0.031 0.029 0.024 5.5 5.5 5.5 5.5 5.5 5.5 5.6 5.5 5.5 5.0 5.5 NP 5.4 5 .7 5.5 5.6 5.4 6.0 5.5 6.0 Number of Cells in Half Spleen (x 106 cells/mL) 7 .04 10.84 5.56 10.84 9.13 4.02 5.06 4.29 11.33 8.36 5.06 NP 8 .96 2.42 7 .37 3 .63 4 .95 4.90 2.36 b Total Number of Spleen Cells (xlO5) 0.77 1.20 0.56 1.17 0.92 0.43 0.61 0.53 1 .32 0.86 0.54 NP 1.21 0.25 0.81 0.42 0.49 0.62 0.23 b p. 267 - 267 - v\ Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice 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 106 cells/mL) 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 11 73 118 119 120 0.071 0.052 0.037 0.058 0.047 0.051 0.041 0.043 0.057 0.050 0.052 0.074 0.043 O'. 035 0.061 0.044 NP 0.053 0.044 0.045 0.2290 0.1499 0.1108 0.1785 0.1424 0.1635 0.1285 0.1419 0.1743 0.1475 0.1595 0.2183 0.1243 0.1039 0.1937 0.1287 NP 0.1536 0 .1283 0.1343 0.035 0.023 0.020 0.032 0.024 0.026 0.021 0.024 0.028 0.028 0.017 0.036 0.019 0.019 0.032 0.020 NP 0.029 0.024 0.026 5.5 6.0 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.8 5.5 5.7 5.0 5.4 6.0 5.4 NP 5 .4 5.5 5.5 2.48 3.80 3.80 11.00 4.29 5.61 3.19 5.88 7.86 4 .34 3.30 6.66 2.42 4 .68 7.42 4.02 NP 8 .36 3.85 6 .93 Total Number of Thymus Cells (xl0s) 0.28 0.52 0.39 1 .10 0.46 0 .61 0.34 0.58 0.88 0.45 0.56 0.78 0.27 0.47 0.85 0.48 NP 0.83 0.39 0.66 DuPont-18318 p. 268 - 268 - A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity S tudy in M ale M ice Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight) Male, Group III - 0.3 mg/kg Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 106 cells/mL) 301 0,052 302 0.043 303 0.047 304 0.048 305 0.038 306 0.034 307 0.060 308 0.049 309 0.058 310 0.049 311 0.033 312 0.040 313 0.022 314 0.045 315 0.049 316 0.054 317 0.057 318 0.031 319 0.051 320 0.048 0.1481 0.1191 0.1257 0.1290 0.1101 0.1027 0.1917 0.1454 0.1940 0.1494 0.0902 0.1198 0.0698 0.1398 0.1450 0.1709 0.1887 0.1030 0.1650 0.1308 0.022 0.017 0.024 0.020 0.017 0.020 0.028 0.019 0.030 0.025 0.016 0.022 0.011 0.024 0.020 0.035 0.025 0.012 0.025 0.027 5.5 5.5 5.5 5.5 5.5 5.5 5 .5 5 .4 5 .5 5 .5 5.0 5 .5 5 .4 6.0 5.5 5.3 5.5 5.0 5.4 5.0 1.10 1.21 7.54 4 .12 3.58 7 .48 8.36 7.42 5.88 8.58 5.06 6.98 3 .14 5.34 3.08 5.50 5.50 6.32 5.88 5.12 Total Number of Thymus Cells (xlO8) 0.14 0.17 0.81 0.54 0.44 0.70 0.99 1.03 0.63 0 .92 0 .52 0.70 0 .34 0.60 0.42 0.45 0.69 0.82 0.65 0.46 D uP ont-18318 y -Mi p. 269 - 269- A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity Study in M ale M ice Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight) Male, Group V - 1 m g /kg Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 10 cells/mL) 501 0.037 502 0.074 503 0.045 504 0.069 505 0.036 506 0.062 507 0.056 508 0.054 509 0.032 510 0.040 511 0.047 512 0.049 513 0.045 514 0.037 515 0.032 516 0.046 517 0.055 518 0.059 519 0.038 520 0.059 0.1111 0.2176 0.1343 0.2072 0.1091 0.1766 0.1789 0.1593 0.0955 0.1238 0.1277 0.1361 0.1253 0.1178 0.0896 0.1433 0.1708 0.1686 0.1238 0.1621 0.018 0.039 0.022 0.041 0.016 0.035 0.027 0.026 0.009 0.020 0.017 0.025 0.024 0.019 0.016 0.029 0 .027 0.032 0.020 0.026 5.5 5.3 5.5 5.5 5.5 5.5 5.5 5.4 5.5 5.2 5.0 5.5 5.8 5 .8 5 .4 5 .7 5.5 6.0 5.0 5.3 2.58 10.67 6.00 16.00 5.34 7.59 11.44 7.81 3.02 5.17 5.83 5.34 9 .74 4 .84 4 .90 0.38 9.35 11 .22 1.38 6.16 Total Number of Thymus Cells (xl0a) 0.29 1 .07 0.68 1.48 0.66 0.74 1 .31 0.88 0.59 0.54 0.81 0.58 1.06 0.55 0 .53 0.03 1.05 1.24 0.13 0.74 D uP ont-18318 p. 270 - 270 - _ tv > r* A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity Study in M ale M ice Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight) Male, Group VII - 10 m g /kg Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 106 cells/mL) 701 0.029 702 0.039 703 0.016 704 0.035 705 0.021 706 0.035 707 0.013 708 0.030 709 0.010 710 0.026 711 0.014 712 0.037 713 0.017 714 0.042 715 0.020 716 0.023 717 0.026 718 0.019 719 0.027 720 0.019 0.0912 0.1262 0.0550 0.1241 0.0739 0.1282 0.0526 0.1010 0.0376 0.0956 0.0522 0.1280 0.0538 0.1419 0.0730 0.0852 0.1032 0.0613 0.0900 0.0706 0.014 0.021 0.009 0.019 0.012 0.015 0.006 0.009 0.005 0.009 0.007 0.019 0.012 0.020 0.009 0 .Oil 0.012 0.005 0.014 0.011 5.5 5.5 5.5 5.5 5.3 5.4 5.4 5.7 5.4 5.3 5.5 5.8 5.5 5.5 5.7 5.2 5.5 5.8 5.2 5.5 2.80 0.72 2.48 5.06 0.55 4 .51 0.11 0.82 0.16 1 .54 2 .20 6.71 1.26 8.08 0.00 4.24 0.94 0.22 1.21 0.06 Total Number of Thymus Cells (xlO8) 0.32 0.07 0.24 0.51 0.05 0 .57 0.01 0.16 0.02 0.24 0.24 0.76 0.10 0.93 0.00 0.46 0.11 0.05 0.12 0.01 D uPont-18318 p. 271 -271 - A m m onium Perfluorooctanoate: 28-D ay Im m unotoxicity Study in M ale M ice Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight) Male, Gr oup IX - 30 mg/kg Half Thymus Weight (g) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 10" cells/mL) Total Number of Thymus Cells (xl 0s) 901 902 903 904 905 906a 907 908 909 910 911 912 913 914 915 916 917 918 919 920 0 .009 0.022 0.017 0.028 0.023 NP 0.015 0.065 0.009 0.027 0.016 0.014 0.025 0.030 0.039 0.023 0.039 0.029 0.026 0.014 0.0344 0.0905 0.0717 0.1157 0.0954 NP 0.0688 0.2559 0.0383 0.1000 0.0632 0.0636 0.0883 0.1056 0.1204 0.0987 0.1327 0.1036 0.0959 0.0475 0.006 0.009 0.002 0.010 0.008 NP 0.006 0.013 0.003 0.009 0.008 0.009 0.016 0.018 0.019 0.011 0.019 0.014 0.013 0.006 5.5 5.5 5.3 5.0 5.5 NP 5.5 5.5 5.5 5.3 5.3 5.5 5.5 5.7 5.5 5.0 5 .8 5.4 5.7 5.5 0.06 0.06 0.00 0.06 0.00 NP 0.11 0.55 0.16 0.33 0.00 0.00 0.16 3.08 3.52 0.00 4.56 1.82 1.87 0.16 0.00 0.01 0.00 0.01 0.00 NP 0.02 0.15 0.03 0.05 0.00 0.00 0.01 0.29 0.40 0.00 0 .54 0.20 0.21 0.02 D uP ont-18318 p. 272 - 272 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Individual Total Cell Counts Animal Number Thymus Weight (g) Thymus Weight (% Body Weight} Half Thymus Weight (g) Male, Group XI - 30/0 mg/kg (Recovery) Thymus Cell Suspension Volume (mL) Number of Cells in Half Thymus (x 106 cells/mL) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112a 1113 1114 1115 1116 1117 1118 1119 1120 0.027 0.048 0.031 0.028 0.020 0.026 0.018 0.013 0.032 0.014 0.032 NP 0.037 0.045 0.025 0.034 0.023 0.026 0.016 0.020 0.0863 0.1215 0.0963 0.0921 0.0581 0.0956 0.0664 0.0402 0.0922 0.0562 0.1039 NP 0.1066 0.1480 0.0791 0.1264 0.0836 0.0909 0.0611 0.0697 0.012 0.019 0.018 0.016 0.010 0.014 0.012 0.007 0.012 0.006 0.016 NP 0.023 0.023 0.013 0.021 0.014 0.015 0.009 0.011 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.4 5.0 5.5 5.3 NP 5.5 5.5 5.0 5.0 5.5 5 .3 5.2 5.3 0.99 2.75 3.63 0.00 0.82 0.16 0.06 0.00 0.00 0.06 2.42 NP 3.19 0.00 3.52 0.06 0.77 0.50 0.00 c Total Number of Thymus Cells (xlO8) 0.12 0.38 0.34 0.00 0.09 0.02 0.00 0.00 0.00 0.01 0.26 NP 0.28 0.00 0.34 0.00 0.07 0.05 0.00 C DuPont-18318 QSf> p. 273 - 273 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 274 DuPont-18318 Appendix M Electron Microscopy Report from Experimental Pathology Laboratories, Inc. - 274 - ^5! Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice Experimental Pathology Laboratories, Inc. DUPONT/HASKELL LABORATORY DUPONT STUDY NUMBER: 18318 WORK REQUEST NUMBER: 16160 SERVICE CODE: 1546 AMMONIUM PERFLUOROOCTANOATE: 28-DAY IMMUNOTOXICITY STUDY IN MALE MICE ELECTRON MICROSCOPY PATHOLOGY REPORT EPL PROJECT NO. 129-080 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. 275 DuPont-18318 -275 - Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 276 DuPont-18318 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 -27656 Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 277 DuPont-18318 Experimental Pathology Laboratories, Inc. DUPONT/HASKELL LABORATORY DuPont-18318 DUPONT STUDY NUMBER: 18318 WORK REQUEST NUMBER: 16160 SERVICE CODE: 1546 EPL PROJECT NUMBER 129-080 AMMONIUM PERFLUOROOCTANOATE: 28-DAY IMMUNOTOXICITY STUDY IN MALE MICE ELECTRON MICROSCOPY PATHOLOGY SUMMARY The in-life phase of this study was conducted at Haskell Laboratory for Health and Environmental Sciences, E.l. duPont de 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 mice for at least 28 days. The table below summarizes the experimental design; Experimental Design Dose Solution Daily Dosage Concentration Group Number/Group (mg/kg)* (mg/mL)1' I 20 0 (Control) 0 III 20 0.3 0.03 V 20 1 0.1 VII 20 10 1 IX 20 30 3 XI 20 30 (Recovery)' 3 b3Weight of test substance/kg of animal body weight. Solutions will be adjusted for purity (20%) cThe recovery group (XI) will be dosed with 30 mg/kg of test substance through test day 23. Following injection of SRBC on test day 24, group XI will be dosed with NANOpure water, at a volume of 10 mL/kg of body weight, until sacrifice. 1 -277- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Experimental Pathology Laboratories, Inc._________________________ DuPont-18318 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 mice in Group I (Control) and two male mice in Group V (1mg/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 G06-403 G06-404 GQ6-405 G06-406 Tissue Liver Liver Liver Liver Animal ID 103 104 503 504 Group I (Control) I (Control) V (1mg/kg) V ___( 1 m9 ^ 9) TEM Negative Number (evaluated) 06-1906 to 06-1908 06-1909 to 06-1911 06-1912 to 06-1914 06-1915 to 06-1917 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 Spunepoxide 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 -278- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 279 DuPont-18318 Experimental Pathology Laboratories, Inc. RESULTS DuPont-18318 TEM #G06-403 (Animal 103, Control, Liver, TEM Neg # 06-1906 to 06-1908) Two low magnification images (06-1906 and 06-1907; 5.600X) show portions of adjacent hepatocytes as the primary cell type in the image. Electron-dense areas that are predominantly mitochondria and rough endoplasmic reticulum are separated by intervening areas that contain clustered electron-dense granules against an electron-lucent background. The electron-dense granules are glycogen depositis. A few fat vacuoles that appear as partially electron-lucent smooth contoured vaucuoles, are scattered in the cytoplasm of a few hepatocytes. The higher magnification image (06-1908; 22.400X) shows greater detail of mitochondria, rough endoplasmic reticulum, glycogen deposits, and a few membranous cytoplasmic profiles. TEM #G06-404 (Animal 104, Control, Liver, TEM Neg # 06-1909 to 06-1911) The low magnification images (06-1909 and 06-1910; 5.600X) show similar adjacent hepatocytes with electron-dense areas that are primarily mitochondria and endoplasmic reticulum and lighter (less electron-dense) areas with electron-dense granularity. The high magnification image (06-1911; 22.400X) shows the electron-dense granularity to be glycogen deposits. This image also shows greater detail of the mitochondria and rough endoplasmic reticulum that are relatively electron-dense as compared to the glycogen filled areas. All images also show a few smooth contoured lipid vacuoles within hepatocytic cytoplasm. Two deeply electron-dense membrane-bound bodies in the lower right quadrant are considered lysosomes. The core of these bodies have uniform electron-dense granularity compared to the prominent foldings of the cristae in mitochondria. TEM #G06-405 (Animal 503, Group V/1mg/kg, Liver, TEM Neg # 06-1912 to 06-1914) Both low magnification images (06-1912 and 06-1913; 5.600X) contain adjacent hepatocytes that have numerous mitochondria rather uniformly distributed in the cytoplasm. Pale granular areas surrounding mitochondria contain glycogen deposits. The glycogen deposits are best detailed in the portion of a hepatocyte in the lower portion of image 06-1913. The higher magnification image (06-1914; 22.400X) shows more detail of mitochondria, lipid vacuoles, glycogen and endoplasmic reticulum. No peroxisomes are clearly defined in the hepatocyte images from this animal. 3 -279- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice p. 280 DuPont-18318 Experimental Pathology Laboratories. Inc. DuPont-18318 TEM #G06-406 (Animal 504, Group V/1mg/kg, Liver, TEM Neg # 06-1915 to 06-1917) Electron-dense areas that are primarily mitochondria are the predominant feature other than the nucleus in the low magnification images (06-1915 and 06-1916; 5.600X). The paler and granular background is glycogen. A few small smooth-contoured lipid vacuoles are also scattered in a few hepatocytes. The high magnification image (06-1917; 22.400X) shows the detail of mitochondria, rough endoplasmic reticulum, and part of a nucleus. A few lipid vacuoles are also present. No structures that can be clearly defined as peroxisomes are noted. CONCLUSIONS At the 1 mg/kg dose of ammonium pefluorooctanoate an increase in peroxisomes was not observed. However, many organelles could not be clearly identified due to poor ultrastructural detail, which was likely the result of formalin fixation. Therefore, definitive conclusions on peroxisomal numbers in treated groups relative to controls could not be drawn. HGW/dc nHENNRR 'f G..WWALL, DVM, PhD Diplomate, ACVP Veterinary Pathologist Z Z Cstt*-* 2-^ Date 4 -280- Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice_________________ EPL Experimental Pathology Laboratories, Inc. p. 281 DuPont-18318 QUALITY ASSURANCE FINAL CERTIFICATION S tud y Title: Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice C lien t S tudy: DuPont-18318; Service Code 1546; Work Request 16160 EPL Project Num ber: 129-08 0 E PL Project C oo rd inator Dr. H enry W all E P L Pathologist: Dr. H enry W all 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 Draft Pathology Report Final Pathology Report June 14, 2006 June 27, 2006; July 24, 2006 October 25, 2006 June 14, 2006 June 27, 2006; July 24, 2006 October 25, 2006 Date reported to Study Director/Management: Date of last quarterly facility inspection: October 2 5 ,2 0 0 6 October 2006 ;PL Quality Assurance Unit < C < P S d-oob Date Form No. 6-2 (October 23. 2006) -281 -