Document LJX534YaNnDG4GE99O2NQ2dyd

P. 1 DuPont-18317 TRADE SECRE T Study Title Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats TEST GUIDELINES : U .S . EPA Health Effects Test Guideline s 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 HaskellsM Laboratory for Health and Environmental Sciences P.O. Box 5 0 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 50 0 Durham, North Carolina 27713 U .S .A . Laboratory for Advanced Electron and Light Optical Methods College of Veterinary Medicine No rth Carolina State University 4700 Hillsborough Street Raleigh, North Carolina 27606 U.S .A. LABORATORY PROJECT ID : DuPont- 18317 WORK REQUEST NUMBER : 16160 SERVICE CODE NUMBER : 154 5 C ~NTAlNS No r%n01. SPONSOR : E.I. du Pont de Nemours and Company Wilmington, Delaware 1989 8 U .S.A. Page 1 of 176 p. 2 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 PAGE RESERVE D -2- p. 3 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont- 18317 GOOD LABORATORY PRACTICE COMPLIANCE STATEMEN T 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 of 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 . Applicant / Sponsor : E .I . du Pont de Nemours and Company Wilmington, Delaware 1989 8 U .S .A . Study Director: Denise Hoban. B .A. MLT (ASCP) Staff Medical Technologist and Supervisor 0/ ~~ ~0'D ] Dat e Applicant /Sponsor. DuPont Representative Date -3/- Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats QUALITY ASSURANCE STATEMEN T Work Request Number : 16160 Study Code Number : 1545 p. 4 DuPont- 18317 Phase Audited Audit Dates Date Reported to Date Reported to Study Director Management Protocol : October 17, 2005 October 17, 2005 October 17, 2005 Conduct : November 11, 2005 November 11, 2005 November 11, 200 5 November 14, 2005 November 14, 2005 November 14, 2005 May 30, 2006* October 31, 2006* November 2, 2006* June 14, 2006* October 31, 2006* November 2, 2006* June 27, 2006* October 31, 2006* November 2, 2006* October 25, 2006* October 31, 2006* November 2, 2006 * Report/Records : February 2, 7, 2006 February 7, 2006 February 8, 2006 August 10, 11, 14-18, 2006 August 18, 2006 September 11, 2006 November 13-14, 2006 November 14, 2006 December 12, 2006 * EPL QA Dates Reported by: C " Joseph C . Hamill r D f- re Dat eQualityAsrncdo -4~ p. 5 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study . Analytical Evaluation by: ` o F" Z Amanda Shen, Ph.D. Date Research Chemist Clinical Pathology Evaluation by : 01- 7,m F~" 7 Nancy E, Evcrds, D.V .M.. Diplo A .C.V.P. Date Pri ncipal Research Clinical Pathologist and Manager Anatomic Pathology Evaluation by : Gre P ykcs, V . D., Diplomate A .C.V.P. . A.C.L.A.M., A.B.T.* Veterinary Pathologist 01 " G A`,~6 -2,=07 Dat e Anatomic Pathology Evaluation Peer Review by : .~~,cAML Steven R Frame, D .V.M., Ph .D ., Diplomatc A .C .V.P. Research Fellow and Manager Date Reviewed and Approved by: ca~tA 5cottE. Loveless, Ph.D. Research Manager and Directo r 61-Fr-4, -eb,07-- Date Issued by Study Director: L., <1/'~ 7 Denise Hoban, B .A. MLT (ASCP) Date Staff Medical Technologist and Supervisor -5S~ p. 6 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ................ ...................3 QUALITY ASSURANCE STATEMENT ............................................................... ....................4 CERTIFICATION .......................................................................................... .... ... ........................5 LIST OF TABLES .................... ........ ...... ........................ ................................ ...................... ... .. ....8 LIST OF FIGURES ......... ... ...................................................................... . ...........................--......8 LIST OF APPENDICES ...............................................................................................................9 STUDY INFORMATION ........................................................................................ ...................10 SUMMARY ........ .. ............................................................................................. ....... ....................11 INTRODUCTION ............................... .........................................................................................13 STUDY DESIGN ................... ...................................... ................................ ............................. ....13 A. Design Concentrations . . . . . . .. .. . . . . .. . . .. .... ........ .. .. . . . . . . . . . ........... .. . . . . . . . . . . . . . . . . . . . . . .. ........ . . .. .-- .-- . . . .13 B . Study Overview .. . . . . . . . . . . .. .. .. .. .................... . . . . . . . . . . . . ... .. . .. .. . . . . . . . . . . . . . . . . . . . . . .. . . .. ...... .. . . . . . . . . . . . . . . . .14 MATERIALS AND METHODS ........................... .....................................................................14 A . Test Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 B . Test Substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 C . Test System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 D . Animal Husbandry . . . . . . .-- .--- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 E . Pretest Period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 F . Assignment to Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .---- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-- .-- . . . . . . .16 G . Dose Formulation Preparation and Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 H . Dose Formulation Sampling and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 1 . Body Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 J . Food Consumption and Food Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 K . Clinical Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- . . . . . . . . . . . . .18 L . Clinical Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 M . Humoral Immune Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 N . Anatomic Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 0 . Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 P . Electron Microscopy Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . .- .-- . . . . . . . . . . . . . . . .--- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Q . Statistical Analyses . . . . . . . . . . . . . . . . .--- .--- . . . . . . . . . . . . . . . .--- . . . . . . . . . . .-- .-- .-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 -6- ~ p. 7 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 TABLE OF CONTENTS Page RESULTS AND DISCUSSION ......... ............................................ .............................................23 Analytical Evaluation .. ........... ............................................................... ......................................23 A. Chromatography ..... .... .......... .......... . . . . . . . . . . . . . . . . . . . . . .. .. .. .... ........... .. .. .. . . . . . . . . . . . . . . . . . . . . . .. .. .. ........23 B. Recovery Samples ... .... .... . . .. .... . .... ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ......... .. .. . . . . . . . . . . . . . . . . . . . . . . . ..23 C. Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability Samples . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. ............ .... .. .. .... . . .. . . . . .. . . . . . . . . . . . . . . . .. .... ........ ... .. . . .. . . . . .23 D. Concentration Verification and Uniformity of Mixing Samples .. . . . . . . . . . . . . . . . . . . . . . .. ......... .. .. .24 E. Analytical Conclusions ... .......... .... .. .. . . . . . . . . . . . . . . . ... ...................... .. .. . . . . . . . . . . . . . . . . . . . . . . . .. .. ......... .24 In-Life Measurements .............................................. ...................................................................25 A. Mean Body Weights and Body Weight Gains .. .. . . . . .. .. . . . . . . . . . . . . . . . . . . .. .................. .... . . . . . . . . . . . . .25 B . Food Consumption and Food Efficiency .. .... .................. .. . ... .. .. . . . . . . . . . . . . . . .. . ... .. ................ .. .25 C . Clinical Observations and Mortality . . . . . . . . . . . . . .. . . .. .. .... .................... .. .. . . .. . . . . . . . . . . . . .. .. .............26 Clinical Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. . ... . . . . . . . . . . .26 A . Hematology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Clinical Chemistryry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .............. .. . . . . .. . . . . . C . Clinical Pathology Conclusions . . . . . . . . . . . . . . . . . . .. .... ... .. .. .. .. . . .. .. . . . . .. . . . . . . . . . . . . . . . .. .... .................. . . .29 Immunotoxicity ..................................................... ..................................... ..................................29 A. Humoral Immune Function .... ........ .... .. ... . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. .. .. .... ....... . . .. . . . . . . . . . . . . . . . . . . . . . .. .29 Anatomic Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . .30 A . Cause of Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 B . Final Body and Organ Weight Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 C . Gross Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 D . Microscopic Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 E . Anatomic Pathology Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 3 Total Cell Counts .. ....... ............ ...... ...................................................... ........................................34 A. Spleen Cell Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .................... .. .. .. .. . . .. . . . . . . . . . . . . . . .. . . .. ................ ..34 B . Thymus Cell Number . . .. .... .. .... .. . . . . . . . . . . . . . . . . . . . . . .. . . .. ........................ .... . . . . . . . . . . . . . . . . .. . . . . .. .. ........3 4 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. .... .. .. . .34 RECORDS AND SAMPLE STORAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . .. . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..34 REFERENCES . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35 TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37 FIGURES . .... .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66 APPENDICES . . . .. .. .. . . .. . . . . . . .. . . .. . . .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . ..72 -7er p. 8 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 LIST OF TABLES Page Table 1 Recovery of APFO Added to Dosing Vehicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40 Table 2 Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperatur e Stability of APFO in Dosing Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Table 3 Concentration Verification and Uniformity of Mixing of APFO in Dosing Solutions . . . . . . . . . . . .42 Table 4 Mean Body Weights of Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43 Table 5 Mean Body Weight Gains of Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44 Table 6 Mean Daily Food Consumption by Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45 Table 7 Mean Daily Food Efficiency of Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46 Table 8 Summary of Daily Animal Health Observations in Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47 Table 9 Summary of Detailed Clinical Observations in Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48 Table 10 Summary of Hematology Values for Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49 Table 11 Summary of Clinical Chemistry Values for Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52 Table 12 Summary of Primary Humoral Immune Response to SRBC for Male Rats Dosed wit h APFO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Table 13 Summary of Primary Humoral Immune Response to SRBC for Male Rats Dosed With Positive Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Table 14 Mean Final Body and Organ Weights from Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55 Table 15 Incidence of Gross Observations in Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58 Table 16 Incidences and Lesion Grades of Microscopic Observations in Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60 Table 17 Summary of Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 LIST OF FIGURES Page Figure 1 Representative Analytical Calibration Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 Figure 2 Representative LC/MS/MS Chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68 Figure 3 Mean Body Weights of Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71 -8~ p. 9 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 LIST OF APPENDICES Page Appendix A Cert ificate of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73 Appendix B Individual Body Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75 Appendix C Individual Food Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 Appendix D Individual Daily Animal Health Observ ations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87 Appendix E Individual Detailed Clinical Obse rv ations and Mortality Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90 Appendix F Individual Animal Clinical Pathology Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94 Appendix G Individual Primary Humoral Immune Response Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Appendix H Individual Primary Humoral Immune Response Positive Control Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Appendix I Individual Animal Final Body and Organ Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Appendix J Individual Animal Pathology Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121 Appendix K Individual Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161 Appendix L Electron Microscopy Repo rt from Experimental Pathology Laborato ri es, Inc . . . . . . . . . . . . . . . . . . . . . . 169 -9- 16 P . 10 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont- 18317 STUDY INFORMATION Substance Tested : Ammonium Perfluorooctanoate [AFPO (linear)] 3825-26-1 (CAS Number) Haskell Number : 2730 8 Composition : Ammonium Perfluorooctanoate Solution 19 .5% in water Purity: 19 .5% Physical Characteristics : White to slightly opaque liqui d Stabili : 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 16, 2005 / February 1, 200 7 -10- // P . 11 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 SUMMARY The purpose of this study was to evaluate the potential of ammonium perfluorooctanoate [APFO (linear)] to suppress the primary humoral immune response following exposure via oral gavage for up to 28 consecutive days . Groups of 10 male rats 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 was included to assess potential reversibility/recovery and was therefore administered the test substance for 22 consecutive days followed by 6 consecutive days of vehicle (water) administration . Body weights, food consumption measurements, and clinical observations were recorded during the in-life period . Prior to sacrifice, the immune system was stimulated by injecting sheep red blood cells (SRBC) on test day 22 and blood samples were collected from each rat on test day 29 . The serum samples were assayed for their concentration of SRBCspecific IgM antibodies to provide a quantitative assessment of humoral immune response . Serum from animals dosed with cyclophosphamide, a positive control immunosuppressive agent, were analyzed concurrently to provide confirmation that the assay performance was acceptable for detection of immunosuppression . Clinical pathology data were collected at day 29 to assess effects on hematology and clinical chemistry . At sacrifice, each animal was examined grossly and selected organs were weighed (brain, spleen, and thymus) ; selected tissues (as outlined in the methods section) were retained and examined histologically . Thymus and spleen cells were manually counted from single-cell suspensions prepared from the collected tissue . Samples of the dosing formulations were chemically analyzed and the results indicated that the test substance was at the targeted concentrations, homogeneously mixed, and stable under the conditions of the study. Test substance-related toxicity was observed during the in-life portion 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 wer e accompanied by low incidences of clinical observations indicative of toxicity . Effects on body weight and food consumption parameters were detected at 1 mg/kg, but these reductions were not considered adverse . There were no test substance-related effects observed at 0 .3 mg/kg during the in-life portion of the study. Rats dosed with >0 .3 mg/kg had decreased serum total, HDL, and non-HDL cholesterol, and decreased triglycerides . Rats dosed with >_1 mg/kg had increased microscopic red cell morphologic changes and hemolyzed serum . Rats dosed with >_10 mg/kg had decreased hemoglobin, hematocrit, mean cell volumes, and mean cell hemoglobin concentrations ; increased reticulocyte counts and red cell distribution width, increased total white blood cell, neutrophil, monocyte, and LUC counts ; increased serum albumin and decreased serum globulin concentrations ; and increased serum corticosterone concentrations . Rats in the 30/0 mg/kg group had more pronounced red cell mass effects and red cell morphologic changes compared to those dosed with 30 mg/kg for 29 days . Parameters with complete recovery in rats dosed wit h 30/0 mg/kg were serum total, HDL, and non-HDL cholesterol, globulin, and corticosterone concentrations . -11- 1.2- p . 12 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 There was a test substance-related decrease in final body weights and increase in liver weights . Mean final body weights were decreased at dose levels >_10 mg/kg of the test substance . Mean liver weight parameters were increased at dose levels >0 .3 mg/kg . At the terminal sacrifice, test substance-related gross observations were limited to discoloration of the liver in a few rats at doses _>10 mg/kg . Microscopic examination of the liver demonstrated minimal to mild hepatocellular hypertrophy at 0 .3 and 1 mg/kg and moderate hepatocellular hypertrophy a t >_10 mg/kg . Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased hematopoiesis in the spleen of rats dosed with 30/0 mg/kg . No test substance-related evidence of immunosuppression was observed in male rats at any concentration tested ; the IgM titers were generally comparable across all groups . No significant changes in total spleen cell or thymocyte number compared to control rats were noted in any animals treated with any dose of APFO . Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male rats was less than 0 .3 mg/kg, whereas the NOAEL for immunotoxicity was 30 mg/kg . -12- ~3 p . 13 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 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 rats for at least 28 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 ; C7F15COO-NH4+ ; CAS Registry number 3825-26-1) is a surfactant used as a processing aid in the production of fluoropolymers . Perfluorooctanoate (PFOA ; C7F15COO-), the dissociation product of APFO, is not metabolized() 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 of mice to make antibodies to a T-cell dependent antigen . (5) The reported study employed a single 0 .02% PFOA in chow (approximately 30 mg/kg) for 16 days . In order to better characterize the immune response following exposure to this material, APFO was administered by oral gavage using a broad range of doses . Dosages for this study were selected based on results of a 14-day oral gavage study in male rats and mice . (6) STUDY DESIGN A. Design Concentration s Number/ Daily Dosage Dose Solution Concentration Group Group (mg/kg)a (mg/mL) b I 10 0 (Control) 0 III 10 0 .3 0 .03 V 10 1 0 .1 VII 10 10 1 IX 10 30 3 XI 10 30/0` 3 a Weight of test substance/kg or animal body weight . b Solutions were adjusted for purity (19 .5%) . c This group (XI) was dosed with 30 mg/kg of test substance through test day 22 . Following injection of SRBC on test day 23, group XI was dosed with NANOpure water, at a volume of 10 mL/kg of body weight, until sacrifice . -13- O f p . 14 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 B . Study Overview Study Parameters Frequency Body Weight Day 0, 3, 6, and daily thereafter Food Consumption Weekly Daily Animal Health Observation Twice daily General Clinical Observationa Day 0 and weekly thereafter Detailed Clinical Observation At each weighin g SRBC Injection Prior to dosing (test day 23) Clinical Pathology Evaluation Test day 2 9 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 Guideline s The study design complied with the following test guidelines : U .S . EPA, OPPTS 870 .7800 : Immunotoxicity, Health Effects Test Guidelines (1998) B . Test Substanc e (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 of Analysis (COA) that is provided i n Appendix A . No evidence of instability, such as a change in color or physical state, was observed . C. Test System On October 6, 2005, 66 male Crl :CD(SD) rats, with an assigned birth date of August 22, 2005, were received from Charles River Laboratories, Raleigh, North Carolina . The Crl :CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at Haskell Laboratory . By utilizing the Crl :CD(SD) rat, immunotoxicity studies can be conducted in the same strain that is used for other toxicology studies . -14- /5 p . 15 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 D. Animal Husbandry l . Housing All animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards . 2 . Environmental Conditions Animal rooms were maintained at a temperature of 18-26C and a relative humidity 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 of the study . 3 . Feed and Water All rats were provided tap water ad libitum . All rats were fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum . 4 . Animal Health and Environmental Monitoring Progra m 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 of the study . E. Pretest Period Upon arrival at Haskell Laboratory, all rats were housed in quarantine . The rats were : quarantined for 6 days . identified temporarily by cage identification . weighed at least 3 times during quarantine . -15- //c:7 p . 16 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 observed with respect to weight gain and any gross signs of disease or injury . The rats were released from quarantine by the laboratory animal veterinarian or designee on the bases of acceptable body weights and clinical signs of all rats . F. Assignment to Group s Rats, selected on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury, were distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there were no statistically significant differences among group body weight means within a sex . The weight variation of selected rats did not excee d 20% of the mean weight for each sex . At grouping, each rat was assigned an animal number/cage identification number . The animal number/cage identification number were tattooed on the tail of each rat and included on the cage label . At study start (test day 0) the rats were 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 . 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 rats were similarly dosed with NANOpure water at a volume of 10 mL/kg of body weight . The 30/0 mg/kg group (XI) was dosed with 30 mg/kg of test substance through test day 22 . Following injection of SRBC on test day 23, group XI was dosed with NANOpure water at a volume of 10 mL/kg of body weight until sacrifice . One rat from group XI (1109) was not dosed on test days 6 through 8 due to a decrease in body weight gain . Once body weight increases were observed for this rat, dosing resumed . H. Dose Formulation Sampling and Analysis 1 . Recovery Sample Analysi s Concurrent with dosing formulation analyses, recovery of APFO from spiked NANOpure water was tested at the low level (approximately 0 .03 mg/mL), the middle levels (approximately 0 .1 and 1 mg/mL), and the high level (approximately 3 mg/mL) to confirm the analytical method . A stock solution of APFO was prepared in NANOpure water . For all concentration levels, an appropriate aliquot of the stock solution was used to make the spiked solution upon further dilution with NANOpurea~ water. These spiked recovery samples were then processed and analyzed in the same manner as the dosing samples at similar concentrations . -16- !7 p . 17 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 2 . Dosing Solution Treatmen t Each dosing sample ( 1 mL) was initially diluted with NANOpure water to a nominal concentration of 0 .3, 1, 10, and 30 ppm APFO for the 0 .03, 0.1, 1, and 3 mg/mL dosing samples, respectively . The samples were fu rther 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 of the inte rnal standard in all dosing samples ; the 0 .1, 1, and 3 mg/mL samples were matrix corrected with the initial diluted solution of 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 m Flow Rate : 0 .4 mL/min Oven Temperature : 35C Injection Volume : 20 L Mobile Phase : A) 0 .15% Acetic acid in NANOpure water B) Acetonitri l e Gradient : Time (min) % Acetonit ri le 0 5 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 Quantitatio n The analytical reference of 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 of the stock solution were diluted with NANOpure water to make calibration standards that bracketed the target concentration of the diluted dosing samples after matrix correction with the initial diluted solution of the control sample. Before these aliquots were brought to the final volume, a n -17- /'Y p . 18 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 appropriate amount of 1, 2-di-13C PFOA internal standard was added to give an equivalent final concentration of 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- 13 C 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 of the duplicate analyses for each respective dosing level . Uniformity of mixing of 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 of the concentration verification samples . A coefficient of 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 . Stability of APFO in dosing samples was evaluated by using the mean result of the duplicate concentration verification analyses as the baseline for comparing the corresponding stability results . 1. Body Weights During the test period, all rats were weighed on test days 0, 3, 6, and daily thereafter . J. Food Consumption and Food Efficiency During the test period, the amount of food consumed by each rat over the weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of 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 Observation s 1 . Daily Animal Health Observation s Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least twice daily throughout the study . Abnormal behavior/appearance was recorded by exception . -18lf". P . 19 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 2 . General Clinical Observation s An additional cage-site evaluation was conducted approximately 2 hours after dosing to detect acute clinical signs of systemic toxicity . 3 . Detailed Clinical Observation s At every weighing, each rat was individually handled and examined for abnormal behavior and appearance . Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observations included (but were not limited to) evaluation of 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 animals approximately 29 days after initiation of the study . These animals were fasted after 3 p .m. for at least 15 hours. Blood samples for hematology measurements were collected from the orbital sinus of each animal while the animal was under carbon dioxide anesthesia . Blood samples for clinical chemistry and humoral immune system evaluations were collected at sacrifice from the abdominal vena cava of each animal while the animal was under carbon dioxide anesthesia . Bone marrow smears were prepared at sacrifice from all surviving animals . Bone marrow smears were stained with WrightGiemsa stain, but analysis was not necessary to support experimental findings . Blood smears, stained with new methylene blue, were prepared from each animal undergoing a hematology evaluation, but were not needed for examination . All blood samples were evaluated for quality by visual examination . Results were maintained in the study records and reported only if the sample was analyzed . 1 . Hematology Complete blood counts, including reticulocytes, were determined on a Bayer Advia 120 hematology analyzer or determined from microscopic evaluation of the blood smear . WrightGiemsa-stained blood smears from all animals were examined microscopically for confirmation of automated results and evaluation of cellular morphology . The following parameters were determined : red blood cell count red cell distribution width hemoglobin absolute reticulocyte count hematocrit platelet count mean corpuscular (cell) volume white blood cell coun t mean corpuscular (cell) hemoglobin differential white blood cell count mean corpuscular (cell) hemoglobin concentration microscopic blood smear examination -19- ~C./ p . 20 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 2 . Clinical Chemistry Routine serum clinical chemistry parameters were determined on an Olympus`~ AU640 clinical chemistry analyzer . Serum corticosterone was measured using a commercial RIA assay (Diagnostic Products Corporation, Los Angeles, CA ; Catalog #TKRC 1) . Corticosterone concentrations were determined according to the manufacturer's recommended procedure (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 of 5 and 10 ng/mL . The following parameters were determined : cholesterol globulin (calculated ) triglycerides high-density lipoprotein cholesterol total protein non-high-density lipoprotein cholesterol (calculated) albumin serum corticosteron e M. Humoral Immune Functio n On test day 23, animals were injected intravenously in the lateral tail vein with 0 .5 mL of 4 x 108 SRBC/mL (Covance, Denver, Pennsylvania, U .S.A.). On test day 29, serum was collected from each rat and frozen (see L .2. Clinical Chemistry) . Humoral immune function was evaluated by examining sera from individual animals for SRBCspecific IgM levels with an enzyme-linked immunosorbent assay (ELISA) .(7) The serum from each animal was assayed as 10 serial, 2-fold dilutions, with 1 replicate per dilution . The optical density (OD) of the contents of the reaction well was measured at the 405 nm wavelength with a MR 5000 Microplate Reader (Dynex Technologies) . SRBC-specific serum IgM titer data were analyzed with Revelation Software Version 2 .0 (Dynex Technologies) . For each serum sample, a semi-log graph 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 of 0 .5 was determined by regression analysis . The "titer" of each animal was defined as the reciprocal of the serum dilution that had an OD value of 0 .5 . If no points had an OD value of greater than or equal to 0 .5, the reciprocal of the starting dilution closest to an OD value of 0 .5 was used as the titer . Sera previously collected from rats injected with SRBC and dosed for 6 days with 20 mg/kg of the known immunosuppressive agent, cyclophosphamide monohydrate, or vehicle were run concurrently with the study samples to demonstrate that the assay functioned properly . For any test samples that needed to be rerun due to a poor curve fit or slope, pooled male or female cyclophosphamide monohydrate or vehicle serum samples were concurrently run . The pooled samples consisted of equal aliquots of serum taken from either the male or female rats dosed with cyclophosphamide monohydrate or vehicle . N . Anatomic Pathology Evaluatio n After 29 days on study, all rats from each dose group (0, 0 .3, 1, 10, 30, and 30/0 mg/kg body weight) were sacrificed and necropsied for evaluation of subchronic toxicity . The order of -20- ~/ p . 21 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 sacrifice for scheduled deaths was stratified across groups . Rats were fasted at least 15 hours before their scheduled sacrifice . All rats survived the duration of the study and were euthanized by carbon dioxide anesthesia and exsanguination . Gross examinations were performed and final body and organ weights were recorded . The following tissues were collected from all 60 rats (10/sex/group) on study . Digestive System stem Nervous Syste m livera braina'` (3 sections) Hematopoietic System Musculoskeletal System spleena femur/knee joint thymusa sternum popliteal lymph node mesenteric lymph node Other bone marrowb gross observations a Organs were weighed at necropsy . b Bone marrow was collected with the femur and sternum . c Including cerebrum, cerebellum, medulla/pon s Organ weight ratios (% final body weight, % brain weight) and group mean values for weighed organs were calculated . All tissues were fixed in 10% neutral buffered formalin . Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist . Microscopic findings were graded on a 4-point scale based on the severity or extent of the change (grade 1= minimal ; grade 2 = mild ; grade 3 = moderate ; grade 4 = severe) . All tissues collected from control (0 mg/kg) and high-dose (30 and 30/0 mg/kg) rats were processed to slides and evaluated microscopically . In addition, the following organs were examined from intermediate-dose rats in order to determine a no-observed-effect level for test substance-related microscopic findings : liver and spleen . Gross observations (recorded at necropsy) were examined microscopically for all animals . 0. 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 of the halved spleen or thymus (tissue) was documented ; the half was placed in a labeled 15 mL centrifuge tube containing 5 mL Hank's Balanced Salt Solution (HBSS/H) and put on ice . -21- p . 22 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 The halved tissue/HBSS/H was poured into a small petri dish and cut into small pieces . The tissue/HBSS/H was poured into a Stomacher 80 Lab System bag and placed into the Stomacher 80 Lab System on "high" setting for 120 seconds (spleen) or 60 seconds (thymus) . After the Stomacher 80 Lab System stopped, the cell suspension was pipetted back into the original centrifuge tube, rinsing the bag with 3 mL HBSS/H and adding that to the centrifuge tube . The centrifuge tube was inverted 2 or 3 times and left on ice for approximately 10 minutes to allow debris to settle to the bottom of the tube . The supernatant was transferred to a new centrifuge tube and the volume of the supernatant was documented . Total cell counts were determined from each tissue by hemacytometer . P. Electron Microscopy Evaluatio n A section of liver from 2 control rats (105 and 106) and 2 rats from the 30 mg/kg group (905 and 906) was placed in cassettes, in a container of formalin, and shipped to Experimental Pathology Laboratories, Inc (EPO) and evaluated by transmission electron microscopy . As a subcontractor to EPLO, the Laboratory for Advanced Electron and Light Optical Methods, College of Veterinary Medicine, North Carolina State University processed the tissues for electron microscopy and prepared electron photomicrographic images under the direction of Dr. Michael Dykstra. The printed electron photomicrographic images were provided to EPL for evaluation by an ACVP-certified veterinary pathologist who interpreted the images and prepared a final report of the electron microscopic evaluation . Q. Statistical Analyse s 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 . Method of Statistical Analysi s If preliminary test is not If preliminary test is Parameter Preliminary Test significant significant Body Weight Body Weight Gain Food Consumption Levene's test for Food Efficiency homogeneity (8) and One-way analysis of Kruskal-Wallis test (14) Humoral Immune Function variance('o) followed by 's test CDlaitn aaiSchaapilro( -W "i'lk'Z tes'ti(93) ) forfDo unlnleo tt'sw teset d by Dunn normality l Pathology s bOrganWeiht Total Cell Count s a SRBC-specific serum IgM antibody titer data were transformed to Logz 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 . -22- c;73 p . 23 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 RESULTS AND DISCUSSIO N Analytical Evaluation A. Chromatograph y (Figures 1-2 ) PFOA dissociated from APFO and 1, 2-di-13C PFOA eluted together from the HPLC 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 of 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 of 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 of 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 of 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 of the measured concentrations of APFO for the 3 mg/mL level was 101 .7% to 107 .0% of nominal (mean percent recovery = 104 .4 4%, C .V . 4%). Based on these data, the analytical method performed satisfactorily for the concentration range of the dosing samples in the study . C . Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability Sample s (Table 2) Analytical results from dosing solutions prepared on October 17, 2005 analyzed for concentration verification, uniformity of mixing, and 5-hour room temperature stability are shown in Table 2 . The following table summarizes the results for concentration verification, uniformity of mixing, and 5-hour room temperature stability analyses . -23~~ p . 24 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont- 18317 Preparation Nominal Measureda Average C .V . Stability Date mg/mL mg/mL % Nominal (%) % Nomina l 17-October-2005 0 ND C --- --- --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% of nominal), uniformly mixed (CV's = 0 .3%, 0 .9%, 3%, and 2%, respectively), and stable when held for 5 hours at room temperature in the vehicle . Test substance was not detected in the 0 mg/mL sample . D. Concentration Verification and Uniformity of Mixing Samples (Table 3) Analytical results from dosing solutions prepared on November 15, 2005 analyzed for concentration verification and uniformity of mixing are shown in Table 3 . The following table summarizes the results for concentration verification and uniformity of mixing analyses . Preparation Nominal Measureda Average C .V . Date mg/mL mg/mL % Nominal (%) 15-November-2005 0 ND --- --0 .03 0 .0276, 0 .0272 91 .3 1 0 .1 0 .0954, 0.0986 97 .0 2 1 1 .02, 1 .01 102 .0 0 .7 3 3 .21, 3 .23 107 .3 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 Conclusion s Data from the analysis of the samples during the study indicate that the test substance was 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 . -24C~5 p . 25 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-183I7 In-Life Measurement s A . Mean Body Weights and Body Weight Gains (Tables 4-5, Figure 3, Appendix B ) Test substance related adverse reductions in mean body weights and body weight gains were observed at 10, 30 and 30/0 mg/kg . Mean final body weights were 10, 25, and 21% lower than the control group at 10, 30, and 30/0 mg/kg, respectively, as a result of reduced body weight gains ; overall body weight gains during test days 0 to 28 were 26, 63 and 50% lower for the same respective doses . The magnitude and onset of the effects on body weight parameters were dose related in that the effects at 30 mg/kg were evident sooner and were more pronounced . There was no appreciable difference in the magnitude of the reduction between the 30 an d 30/0 mg/kg, indicating that the shortened dosing period did not have a significant impact on this endpoint . At 1 mg/kg, overall body weight gain during test days 0 to 28 was 10% lower, resulting in a 4% reduction in mean final body weight . These slight reductions appear to be test substance related ; however, they were not statistically significant nor were they considered to be adverse . Body weight data for animals dosed at 0 .3 mg/kg were generally comparable to control group data . B. Food Consumption and Food Efficiency (Tables 6-7, Appendix C ) Test substance related adverse reductions in mean daily food consumption and food efficiency were observed at 10, 30 and 30/0 mg/kg ; test substance-related effects on these parameters were also observed at 1 mg/kg but these effects at 1 mg/kg were not considered adverse based on the magnitude of the reductions . Mean daily food consumption was 4, 17 and 16% lower than controls at 10, 30, and 30/0 mg/kg, respectively, during test days 0 to 28 . The combined test substance-related reductions in mean body weight, weight gain, and food consumption resulted in test substance-related reductions in food efficiency . During test days 0 to 28, mean food efficiency was 23, 57 and 42% lower at 10, 30 and 30/0 mg/kg/day . The effects on food consumption parameters were similar to and consistent with the effects on body weight parameters in that the magnitude and onset of the effects on food consumption and efficiency were dose related terms of onset, severity, and duration of the effects . Additionally, there was no appreciable difference in the magnitude of the reduction of the overall mean daily food consumption between the 30 and 30/0 mg/kg, indicating that the shortened dosing period did not have a significant impact on this endpoint . -25- C:;~6 p . 26 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 At I and 10 mg/kg, mean daily food consumption and food efficiency was 3 and 7% lower than controls, respectively . These slight reductions appear to be test substance related ; however, they were not statistically significant nor were they considered to be adverse . Food consumption and food efficiency data for animals dosed at 0.3 mg/kg were generally comparable to control group data . C . Clinical Observa ti ons and Mortality (Tables 8-9, Appendices D-E) There was no test substance-related mortality at any level tested ; all animals survived to the scheduled sacrifice on test day 29 . Test substance related clinical observations were observed at 30 and 30/0 mg/kg and included wet and/or stained fur, absent or decreased feces, not eating, high carriage, and lethargy . These signs were reported for up to 3 animals in these groups and thus, the incidence was not overwhelming . However, the nature of the signs combined with the effects on body weight and food consumption discussed previously supported that these observations were test substancerelated and adverse . Hair loss was reported in up to 3 animals per group ; this unremarkable finding was not considered test substance related since the incidence was not dose-related . Clinical Pathology Evaluation A. Hematology (Table 10, Appendix F ) 1 . Red Blood Cell s Hemolysis was evident in serum of rats dosed with >1 mg/kg (see Clinical Chemistry section) . Hemoglobin and hematocrit were mildly decreased in rats dosed with 10 or 30 mg/kg for 29 days (means were 91-92% of the control group mean, respectively ; statistically significant), but there were no effects on red blood cell counts. The discordance between red blood cell count and hematocrit was likely due to decreased mean cell volume in rats dosed with 10 or 30 mg/kg for 29 days (hematocrit is the product of mean cell volume and red blood cell count) . Means for mean cell volumes were 97 and 95% of the control group mean ; (statistically significant at 30 mg/kg). Mean cell hemoglobin, which generally closely parallels mean cell volume, was also decreased in these 2 dose groups (means were 95 and 94% of the control group mean, respectively ; statistically significant) . A few rats dosed with 10 or 30 mg/kg for 29 days had increased reticulocytes, although there were no significant changes in mean reticulocyte counts (means were 109 and 112% of the control group mean) . Increased red cell distribution width generally correlated with increased reticulocytes in rats from these groups . Mean red cell distribution widths were 111 and 115 %, respectively, of the control group mean . Microscopically, some of the rats in these 2 groups ha d 7 -26- ~ p . 27 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 increased anisocytosis (variation in red cell size ; also obse rv ed in rats dosed with 1 mg/kg), macrocytosis ( increased numbers of larger cells), polychromasia ( increased bluish staining of red blood cells), and hypochromasia ( pale staining of red blood cells) . These changes were consistent with minimally increased reticulocytes in some animals . Effects on red cell mass parameters were present (red blood cell count) or more pronounced (hemoglobin, hematocrit) in the 30/0 mg/kg group of rats compared to those dosed wit h 30 mg/kg for 29 days . On test day 29, mean red blood cell count, hemoglobin, and hematocrit ranged from 86-88% of the respective control group means for these 3 parameters (all statistically significant) . Decreased red cell mass parameters on test day 29 could be due to one or more of the following processes : increased red cell destruction, red cell loss, or increased plasma volume . The mechanism for decreased red cell mass parameters was not evident from inlife, clinical pathology, or anatomic pathology data . Therefore, the cause of the decreased red cell mass was not determined . Reticulocytes were moderately increased in rats dosed with 30/0 mg/kg . Mean reticulocyte count was 197% of the control group mean . Consistent with the increase in reticulocyte counts, red cell distribution width was increased (mean was 123% of the control group mean) . Microscopically, this group had increased anisocytosis, macrocytosis, polychromasia, hypochromasia, and acanthocytosis (red blood cells with blunt surface projections) . All morphologic changes in red cells occurred at greater incidence or at higher severity grades in these rats compared to rats dosed with APFO for 29 days . These red blood cell changes also correlated with histologic evidence of increased extramedulla ry hematopoiesis, which was observed in 7 of ten 30/0 mg/kg rats, but in none of the 30 mg/kg rats after 29 days of dosing . 2 . White Blood Cell s White blood cell counts were minimally increased, p rimarily due to increases in lymphocytes, in some rats dosed with 10 or 30 mg/kg for 29 days (variable statistical significance) . Means were 130 and 137% (total white blood cell count), and 133 and 140% (lymphocyte count) of respective control group means . Individual rats dosed with 10 or 30 mg/kg with higher total white blood cell and lymphocyte counts generally had higher neutrophil, monocyte, and large unstained cell (LUC) counts as well, resulting in mean neutrophil, monocyte and LUC counts that were 114-147% of the control group means . Due to the normal range and variability of total and individual white blood cell counts, these changes did not result in statistically different means. LUCs are cells that cannot be identified as one of the 5 major leukocyte types by the Advia 120 automated hematology analyzer, and normally comp rise a small percentage of the total leukocyte population. The LUC count normally includes mostly lymphocytes and monocytes . Consistent with this obse rvation, in this study, the rats with the highest LUC counts usually had the highest lymphocyte and/or monocyte counts . The changes observed in total and individual white blood cell counts are consistent with inflammation . Histologically, there were no findings obse rved that correlated with these white blood cell changes . In rats that were dosed with 30/0 mg/kg, total white cell and lymphocyte counts were generally similar to their respective control group means, with the exception of a few rats with higher total white blood cell and lymphocyte counts (rats 1106 and 1107) . Monocyte and large unstained cell counts for rats dosed with 30/0 mg/kg were similar to those of rats dosed for 29 days with 1 0 -27- c'< p . 28 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 or 30 mg/kg in that the counts of most rats were similar to controls, but a few rats had higher monocyte and LUC counts . Therefore, there was no recovery . Mean eosinophil counts were minimally decreased in rats dosed with >_0 .3 mg/kg for 29 days (not statistically significant) . These decreases were the result of high eosinophil counts i n 3 control rats . There was no dose response in changes in eosinophil counts despite the 100-fold difference in dose administered in either terminal or recovery animals . In rats that were dosed with 30/0 mg/kg, eosinophil counts were similar to groups dosed with >_0 .3 mg/kg for 29 days . Therefore, the apparent decreases in eosinophil counts after 29 days of dosing at >_0 .3 mg/kg and in 30/0 mg/kg rats is of uncertain relationship to treatment . B . Clinical Chemistry (Table 11, Appendix F ) Hemolysis was evident in serum of rats dosed with >_1 mg/kg . Hemolysis is graded as none, trace, small, moderate or large . In this study, all samples had either none, trace, or small hemolysis . The incidence of serum graded trace to small for hemolysis was 1/10, 0/10, 3/10, 9/10, and 7/10 in rats dosed with 0, 0 .3, 1, 10, or 30 mg/kg, respectively . In rats that were dosed with 30/0 mg/kg, trace to small hemolysis was observed in 6/10 rats, and the severity was similar to that observed after 29 days of dosing at 30 mg/kg . Total cholesterol was decreased in rats dosed with 0 .3 or 1 mg/kg for 29 days . Means were 64 and 69% of the control group mean, respectively (statistically significant) . Cholesterol concentrations of rats dosed with 10 or 30 mg/kg, although higher than those dosed with lower doses, were still lower than controls (means were 81 and 84% of the control group mean, respectively ; not statistically significant) . The decreases in cholesterol were due to decreases in both HDL and non-HDL cholesterol . HDL cholesterol was decreased by a similar degree in all groups dosed with the test substance for 29 days ; means were 75-79% of the control group mean (variable statistical significance) . Non-HDL cholesterol, like total cholesterol, was lower in rats dosed with 0 .3 or 1 mg/kg (means were 58 and 63% of control group mean ; statistically significant) than in rats dosed with 10 or 30 mg/kg (means were 85 and 88% of control group mean ; statistically significant) . In rats that were dosed with 30/0 mg/kg, total, HDL, and non-HDL cholesterol concentrations were similar to controls, suggesting recovery for most rats . However, total, HDL, and non-HDL cholesterol concentrations were mildly higher in one recovery rat (1103), and lower in another recovery rat (1107) compared to other animals in the 30/0 mg/kg group . Triglyceride was decreased in rats dosed with >_0 .3 mg/kg for 29 days . The dose-response was flat across the doses tested ; means were 69, 75, 68, and 66% of control group means for rats dosed with 0 .3, 1, 10, or 30 mg/kg, respectively (variable statistical significance). Triglycerides were still decreased in rats that were dosed with 30/0 mg/kg (mean was 69% of the control group mean), indicating a lack of recovery for triglyceride concentrations . Albumin was increased in a few rats dosed with 1 mg/kg, and in most rats dosed with 10 or 30 mg/kg . Means were 106, 112, and 115% of the control group mean, respectively (variable statistical significance). In rats that were dosed with 30/0 mg/kg, albumin was similar to that o f -28- ~~ p . 29 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 rats dosed with 30 mg/kg for 29 days (with the exception of one male, rat 1109, with lower albumin) . Mean concentration for rats dosed with 30/0 mg/kg was 112% of the control group mean, indicating a lack of recovery for albumin concentration . Globulin was decreased in rats dosed with 10 or 30 mg/kg . Means were both 89% of the control group mean . In rats dosed with 30/0 mg/kg, globulin was similar to that of controls (with the exception of one male, rat 1109, with low globulin), indicating recovery for globulin concentrations . Serum corticosterone was increased in a few rats dosed with 10 or 30 mg/kg for 29 days . Concentrations greater than 300 ng/mL (approximate upper bound for corticosterone concentration in non-stressed rats) were observed in 0/10, 0/10, 0/10, 2/10, and 4/10 rats dosed with 0, 0 .3, 1, 10, or 30 mg/kg, respectively . The higher corticosterone concentrations in some rats dosed with 10 or 30 mg/kg resulted in mean concentrations that were 135 and 196% of controls, respectively . These changes are indicative of physiological stress . In rats that were dosed with 30/0 mg/kg, serum corticosterone concentrations were generally similar to controls, indicating recovery . C . Clinical Pathology Conclusion s Rats dosed with >0.3 mg/kg had decreased serum total, HDL, and non-HDL cholesterol, and decreased triglycerides. Rats dosed with _>1 mg/kg had increased microscopic red cell morphologic changes and hemolyzed serum . Rats dosed with >_10 mg/kg had decreased hemoglobin, hematocrit, mean cell volumes, and mean cell hemoglobin concentrations ; increased reticulocyte counts and red cell distribution width, increased total white blood cell , neutrophil, monocyte, and LUC counts ; increased serum albumin and decreased serum globulin concentrations, and increased serum corticosterone concentrations . Rats in the 30/0 mg/kg group had more pronounced red cell mass effects and red cell morphologic changes compared to those dosed with 30 mg/kg for 29 days . Parameters with complete recovery in rats dosed wit h 30/0 mg/kg were serum total, HDL, and non-HDL cholesterol, globulin, and corticosterone concentrations . Immunotoxicity A . Humoral Immune Functio n (Tables 12-13, Appendices G-H ) No test substance-related evidence of immunosuppression was observed in male rats at any concentration tested; the IgM titers were generally comparable across all groups . For the individual and pooled positive control sera, the primary humoral immune response to SRBC was decreased by 57 and 55%, respectively . Therefore, the SRBC-specific ELISA test system was valid for this study . -29- 36 p . 30 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Anatomic Pathology Evaluation A. Cause of Death There were no test substance-related deaths . All 60 rats on study survived until the scheduled sacrifice on test day 29 . B . Final Body and Organ Weight Data (Table 14, Appendix I ) Following 28-days of daily gavage administration of the test substance, there was a test substance-related decrease in final body weights and increase in liver weights . Mean final body weights were decreased at dose levels >10 mg/kg of the test substance . Mean liver weight parameters were increased at dose levels >0 .3 mg/kg . Text Table 1 : Mean Absolute and Relative (% body weight) Organ Weights in Male Rats Group: Dose (mg/kg) : Number of Rats/Sex : I III V VII IX XI 0 0 .3 1 10 30 30/0 10 10 10 10 10 10 Final Body Wt . (g) 423 .1 419 .7 410 .0 377 .0 * 314 .4 * 333 .8 * Liver (10) (10) (10) (10) (10) (10) absolute wt. (g) 13 .179 14 .379 17 .227 * 21 .469 * 18 .684 * 16.206 *A % body wt . 3 .113 3 .419 4 .194 5 .680 ** 5 .931** 4 .849 ** Spleen (10) (10) (10) (10) (10) (10) absolute wt. (g) 0.844 0 .872 0 .835 0 .808 0 .674 0 .780 % body wt . 0 .199 0 .208 0 .203 0 .215 0 .215 0 .232 Thymus (10) (10) (10) (10) (10) (10 ) absolute wt . (g) 0 .568 0 .604 0 .559 0 .581 0 .487 0 .639^ % body wt . 0 .133 0 .144 0 .136 0 .153 0 .153 0 .191 *^ wt . = weight ; O= 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 Weigh t Mean final body weights were decreased 11 %, 26%, and 21 % in the 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value . All decreases were statistically significant. Mean final body weights in the 0 .3 and 1 mg/kg dose groups were similar to the control values . -30J p . 31 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 There was a small, statistically insignificant increase in the mean final body weight of th e 30/0 mg/kg dose group, as compared to the 30 mg/kg dose group . This increase suggests partial recovery from the test substance-related final body weight decrease in the 6 recovery days following the injection of sheep red blood cells . 2. Liver Mean absolute liver weights were increased 9%, 31%, 63%, 42%, and 23% in the 0 .3, 1, 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value . Mean relative (% body weight) liver weights were similarly increased (10%, 35%, 82%, 91%, and 56%, respectively) . All increases were statistically significant, except for those in the 0 .3 mg/kg dose group and the mean relative liver weight in the 1 mg/kg dose group . The increased liver weights, at all dose levels, correlated with the microscopic finding of minimal to moderate hepatocellular hypertrophy. It also correlated with the gross observation of liver discoloration in a few rats at doses >10 mg/kg . 3 . Othe r All other individual and mean organ weight differences were considered to be spurious or secondary to the decrease in final body weights . Mean relative brain weights (% body weight) were increased only at doses (>10 mg/kg) that produced significantly decreased body weights . Similarly, small, statistically insignificant, decreases in mean absolute, and increases in mean relative (% body weight), spleen and thymus weights were interpreted to be secondary t o changes in final body weights . The lack of any gross or microscopic effects in the brain, spleen, and thymus further suggests that these organ weight differences were a function of body weight and not organ-specific effects . C. Gross Observations (Table 15, Appendix J) At the terminal sacrifice, test substance-relate gross observations were limited to discoloration of the liver in a few rats at doses >10 mg/kg. Text Table 2 : Incidences of Test Substance-Related Gross Observations in Male Rats Group : I III V VII IX XI Dose (mg/kg) : 0 0 .3 1 10 30 30/0 * Number of Rats/Group : 10 10 10 10 10 10 Liver Discoloration 0 0 0 1 2 1 Underlined values were interpreted to be test-substance related increases, as compared to control values . * Not dosed with test substance following immunology challenge . -31- ~ ~. p . 32 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 The gross liver discoloration observed in the 4 rats given >_10 mg/kg of the test compound was considered to be a result of the microscopic finding of hepatocellular hypertrophy . D. Microscopic Findings (Table 16, Appendix J ) Microscopic examination of the liver demonstrated minimal to mild hepatocellular hypertrophy at 0.3 and 1 mg/kg and moderate hepatocellular hypertrophy at >10 mg/kg . Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased hematopoiesis in the spleen of high-dose recovery rats (30/0 mg/kg) . The thymus, mesenteric lymph nodes and popliteal lymph nodes had no test substance-related effects . Text Table 3 : Incidences of Test Substance-Related Microscopic Findings in Male Rats Group : Dose (mg/kg) : Number of Rats/Group : I III V VII IX XI 0 0 .3 1 10 30 30/0 * 10 10 10 10 10 10 Liver (10) (10) (10) (10) (10) (10) Hypertrophy, hepatocellular 0 5 [1_0] 10 [1 .7] 10 [3 .0] 10 [3 .0] 10 [3 .0] Necrosis, focal 0 0 0 1 [1 .0] 4 [1 .0] 1[ 1 .0 ] Spleen (10) (10) (10) (10) (10) (10) EMH, increased 0 0 1 [1 .0] 0 0 7[1 .3] [] = Number in brackets is the average grade (grades 1- 4) when lesion is present (i .e ., sum of grades =# animals with lesion) . Grading scale : I = minimal ; 2= mild; 3 = moderate ; 4= severe . () = number in parenthesis is the number of organs examined ; EMH = Extramedullary hematopoiesis . Underlined values were interpreted to be test-substance related increases, as compared to control values . * Not dosed with test substance following immunology challenge. 1 . Live r a. Hepatocellular hypertroph y Panlobular hepatocellular hypertrophy was observed in all but 5 of the rats given the test substance and the incidence and severity were dose related . Hypertrophy was present in 0/10, 5/10, 10/10, 10/10, 10/10, and 10/10 rats given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The hypertrophy was graded as minimal in the 5 affected rats given 0.3 mg/kg, minimal in 3/10 and mild in 7/10 rats given 1 mg/kg, and moderate in all rats given >10 mg/kg . The hepatocellular hypertrophy was characterized by an increase in the size of all hepatocytes due to an increase in cytoplasmic volume . The cytoplasm had a uniformly eosinophilic granular appearance consistent with peroxisome proliferation . -32- w33 p . 33 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Hepatocellular hypertrophy correlated with increased mean liver weight parameters at all doses . Although the 30/0 mg/kg group still had moderate hypertrophy (grade 3 of 4), the decrease in mean liver weights, relative to the 30 mg/kg group suggests that there was some hepatocellular shrinkage and/or loss that was microscopically unapparent . b. Focal necrosis Focal necrosis was also observed in several rats given >10 mg/kg of the test substance . The incidence was mildly dose related . Focal necrosis was present in 0/10, 0/10, 0/10, 1/10, 4/10, and 1/10 rats given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . All were graded minimal . A decrease in the incidence was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group . Focal necrosis was characterized by the focal or multifocal coagulative necrosis of a cluster of hepatocytes . The distribution was usually subcapsular and the pattern was non-zonal . Focal coagulative necrosis of hepatocytes clusters is a common secondary effect of hepatocellular hypertrophy and is most likely the result of secondary focal ischemia . 2 . Spleen a . Increased extramedullary hematopoiesi s An increase in the incidence of splenic extramedullary hematopoiesis (EMH) was considered test substance related only in high-dose rats allowed a recovery period (30/0 mg/kg) . Minimal to mild increased EMH was observed in 0/10, 0/10, 1/10, 0/10, 0/10, and 7/10 rats given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The increased splenic EMH in the high-dose recovery rats was erythrocytic and correlated with the hematology findings, which included decreased red cell mass parameters and increased circulating reticulocytes (see Clinical Pathology) . 3 . Other All other microscopic observations in this study were consistent with normal background lesions in rats of this age and strain . E. Anatomic Pathology Conclusions There were no test substance-related deaths . All 60 rats on study survived until the scheduled sacrifice on test day 29 . Following 28-days of daily gavage administration of the test substance, there was a test substance-related decrease in final body weights and increase in liver weights . Mean final body weights were decreased at dose levels >_10 mg/kg of the test substance . Mean liver weight parameters were increased at dose levels >0 .3 mg/kg . At the terminal sacrifice, test substance-related gross observations were limited to discoloration of the liver in a few rats at doses >10 mg/kg . -33- ~~ p . 34 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Microscopic examination of the liver demonstrated minimal to mild hepatocellular hypert rophy at 0 .3 and 1 mg/kg and moderate hepatocellular hype rtrophy at >10 mg/kg . Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased hematopoiesis in the spleen of rats dosed with 30/0 mg/kg . The thymus, mesenteric lymph nodes and popliteal lymph nodes had no test substance-related effects . Total Cell Counts A. Spleen Cell Numbe r (Table 17, Appendix K ) No significant changes in total spleen cell number compared to control rats were noted in any animal treated with any dose of APFO. A 10% increase was observed at 10 mg/kg and a 16% decrease was observed at 30 mg/kg, but neither value was statistically different than vehicle control . B. Thymus Cell Number (Table 17, Appendix K) No significant changes in total thymocyte number compared to control rats were noted in any animals treated with any dose of APFO. For rats in the 30/0 mg/kg group, an increase in thymocyte number was observed, which was statistically greater than rats who continued to receive 30 mg/kg APFO, but not greater when compared to vehicle control . CONCLUSIONS Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male rats was less than 0 .3 mg/kg, whereas the NOAEL for immunotoxicity was 30 mg/kg . RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, the protocol, amendments (if any), and the final report will be retained at Haskell Laborato ry, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware . Laboratory-specific raw data such as personnel files, instrument, equipment, refrigerator and/or freezer raw data will be retained at the facility where the work was done . -34- .3,5 p . 35 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 REFERENCES 1 . Vanden Heuvel, J ., Kuslikis, B ., and Van Rafelghem, M . (1991) . Tissue distribution , metabolism, and elimination of perfluorooctanoic acid in male and female rats . Biochem . Toxicol. 6, 83-92. 2 . Taves, D ., Guy, W ., and Brey, W . (1976) . Organic fluorocarbons in human plasma : prevalence and characterization . Biochemistry Involving Carbon-Fluorine Bonds (R . Filler, Ed .), pp . 117-134 . American Chemical Society, Washington, D .C . 3 . Hansen, K.J., Clemen, L .A., Ellefson, M .E., and Johnson, H .O . (2001) . Compound-specific, quantitative characterization 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 of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferators, perfluorooctanoic acid . International lmmunopharmacology, 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 of variances . Contributions to Probability and Statistics (J . Olkin, ed .), pp 278-292 . Stanford University Press, Palo Alto . 9 . Shapiro, S .S . and Wilk, M .B . (1965) . An analysis of variance test for normality (complete samples) . Biometrika 52, 591-611 . 10. Snedecor, G .W . and Cochran, W .G. (1967) . Statistical Methods, 6`h 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 . -35~ p . 36 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 13 . Tamhane, A .C . (1979) . A comparison of procedures for multiple comparison of means with unequal variances . J. Amer. Statist. Assoc. 74, 471-480 . 14. Kruskal, W .H. and Wallis, W.A. (1952) . Use of 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 . -36- 3~ p . 37 Ammonium Perfluorooctanoate : 2 8-Day Imm uno t oxic ity S tu dy i n Male Rats DuPont-18317 TABLES -37,5 IF p . 38 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 TABLES EXPLANATORY NOTE S ABBREVIATIONS : Summary of Hematology Values RBC - red blood cell count HGB - hemoglobin HCT - hematocri t MCV - mean corpuscular (cell) volume MCH - mean corpuscular (cell) hemoglobi n 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 lymphocyt e AMON - absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophi l ALUC - absolute large unstained cell Summary of Clinical Chemistry Values CHOL - cholestero l TRIG - triglycerides TP - total protein ALB - albumin GLOB - globulin HDL - high-density lipoprotein cholesterol NHDL - non-high-density lipoprotein cholesterol SCORT - serum corticosterone NOTES : Summary of Hematology Values Summary of Clinical Chemistry Values Groups with identical values may vary in statistical significance, because tabulated statistics are rounded to fewer decimal places than the values used for statistical determination . -38- ~~ p . 39 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 TABLES EXPLANATORY NOTES (Continued ) NOTES : (Continued) Summary of Total Cell Count s Organ Weight as Percent of Body Weight - Organ Weight (g) Final Body Weight (g ) x 100 Total Number of Organ Cell Number o f Organ Cells Organ Weight (g) X Suspension x Cells in Half - 100 (x 108) Half Organ Weight (g) Volume Organ (mL) (x 106 cells/mL) -39- 'of A / (.,/ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 40 DuPont- 18317 Table 1 Recovery of APFO Added to Dosing Vehicl e Sample APFO (mg/mL) Percent Type Nominal Measured Nominal RECOVERY 0 .0302 0 .0327 108 .3 RECOVERY (B) 0 .0300 0 .0305 101 . 7 Mean 105.0 .5, C.V. S% RECOVERY (A) 0 .104 RECOVERY (B) 0 .100 0 .114 0 .104 Mean 109 .6 104 . 0 106.8 .4, CV. 4% RECOVERY (A) 1 .00 RECOVERY (B) 1 .00 1 .02 1 .05 Mean 102 .0 105 . 0 103.5 2, CV. 2% RECOVERY (A) 3 .00 RECOVERY (B) 3 .00 3 .05 3 .21 Mean 101 .7 107 . 0 104.4 4, CV. 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 . -40Z// p . 41 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Table 2 Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability of APFO in Dosing Solution s Sample Date APFO (mg/mL) Percent Sample Type (A) Nominal Measured Nominal 15-November-200 5 Concentration Ve ri fication Control 0 ND (B) ---- #1 #2 0.03 0 .0278 92 .7 0 .03 0 .0277 92 .3 Mean : 0.0278 t 0.0001 (92.7) #1 #2 CV. 0.3% 0.1 0.0966 96 .6 0 .1 0 .0979 97 .9 Mean: 0.0973 0.0009 (97.3) CV. 0.9% #1 1 0.979 97.9 # 1 (C) 1 1 .04 104 .0 #2 (c) 1 1 .03 103 . 0 Mean : 1 .02 f 0.03 (102.0) CV. 3 % #1 3 #2 3 3 .16 105 .3 3 .06 102 .0 Mean : 3.11 0.07 (103.7) Stability D) CV 2% 0.03 0 .0289 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 ve ri fication . Mean, S .D . and C .V . calculated to verify uniformity of mixing . (B) Denotes not detected . (C) Duplicate analyses from the re-diluted sample . (D) Samples held at room temperature for 5 hours . -41 4-2. p . 42 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Table 3 Concentration Verification and Uniformity of Mixing of APFO in Dosing Solutions Sample Type (A) APFO ( mg/mL) Percent Sample Date Nominal Measured Nominal Concentratio n Verification 1 I -October-200 5 Control 0 ND ( B ) ---- #1 #2 0 .03 0 .0276 92 .0 0 .03 0 .0272 90 . 7 Mean : 0.0274 0.0003 (91.3) C.V.1% #1 #2 0 .1 0 .1 Mean: 0 .0954 0 .0986 0.0970 0.002 CV. 2 % 95 .4 98 . 6 (97.0) #1 #2 1 1 .02 102 .0 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 veri fi cation . Mean, S .D . and C .V . calculated to verify uniformity of mixing . 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N I ~ L] I 4- 1 -ri U 1 I G.7 W 1 Zy C3 ~ I H E~ I N C N I I U U I 1-1 -ri .~ ~ E+ Ei I ro N U I I W W 1 (n N 071 ~ ~ I I I I 0 0 I (D Q I cn E 1 I ~ ~ I Iv ~ Cf) U 1 o~ I I Z 1 H I Ei E~ H I,~ C H H I .LJ a a I c ro o'U 1 1 0~C ~ I 4) o o X~ I I1 h~W I I I P: (1,' I rC{ O a~ 1 0 1 Z fYl w oz W ~C 1 z C < U Hc E 1 W 1 fZ O Z O I ua u~i Q 1 w z i ~4 ro 1 1 1 w ~ i b~ N Q N i - - - - - - ! - - - ---- I C.u H (0 p . 63 p . 64 00 --1 ~ r1 --' 0 r1 r1 E 00 't O Q"' 00 M M O 01 ~ ..i Vl O\ ~ U [~ - t N O O o0 M ~t ~ O ~~ CM ~O O O O O O O ~D O ~~O ~ N M M bA ~ C71,00 ~~n th 00 00 00 -- M C^ ) N O O [- N [- O N O M O ~n t M O [- O~ kn O O O O O O l~ -- ~ l~ " M - p ~_ O i . ~ ~ . .i 00 It N - "o ~ ~ 0~0 O iM O ~D N O O O O O O t~ O kr) ~ ~O N M M cn O ~ > O ~ ~O O ~ ~^ O 0 - N U M MIi O N N 00 ~ cd O O N 0 N O ~ O O ~ O - O M O O O O O O [ O (D 00 M M O kr1 Oll IZI' N O cd C/) k~ r) O p, N O r. N ON ~ O 0[ -0 001~ O O O O O C^ :)I ~^ O M M M N O~ N~D l~ O ~~O ~O c~1 .- . bA O 0 0~ O ~ -~ O^ O O 00 M G~ bA N Vl ~~O d1 M M 00 ~ 00 O y O --O o0 r , --~ O O O~ ~n ~n ~ ~ O O O O O O O ~D O ~~ ~ N ~ .~ .d 0 ~ 0 N ~bA bA O ^ ~ ~ O ct 3 uCl. E oo ~o ~ w _o E R ~ ~ ~ ~ -to -2as ? U> Zx F- U !o~ p . 65 s pp N `~ ~ O tnO^ O a p~ o o~ o ao, ~ cv n ~ t n O U ~O O M O M N O~ 00 O t~ Q 0~ O O O O O O l~ O O 00 ~~D t~ M , ....~ cz 0 3 7~ at ~ O U pA O ~ O ~ . ~~ 3 p~ t O O 00 I -- O O kn 00 . O O ~ ~ o - O N O N cM M--~ ~O N o E O O O O O O~ - O to 00 M --~ O ~ b > `~ -' -- ~ O ,-- O `-' O -- -- O O . ~ 00 M kn N 00 r1 -+ v~ - -- O N O N~ 00 O O o0 ~ ~ ~ O O O O O O O [~ O "O O W) O~ --~ O U -o ^ ~ ~~ o ~ o 0 0 .-- k!l M kr) N M M 00 O*N u O O O O O l~ O 00 00 U_ CC3 H ,..~ ~ Q 0 ~ 3 bb . O U U u c E d M M 00 N ;:3 L~ 7p~~`' o0 00 cn M 00 ~o r;~ 00 M N~n I-' vp) > V 4 o - - a 3 >G Y -- bA r . V~ ~ 00 . OOO 00 M M cn N 00 10 -- o X n - v CJ O O O O O O O I - O ~ N N cM ~ 0 -aI o ye ~ ~ ~ O bA bA _ O O cC q Un n v a3 ow U Cp ~.f'., bb 73 0 CIS ~ bUiD T x _ ~7;. -4 bb o Ll (n ` Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 66 DuPont- 18317 FIGURES -66~~ p . 67 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Figure 1 Representative Analytical Calibration Curve 1 .80Equation for the fitted line : Y = 0.0342697 + 33 .368 * X 1 .60- R-Sqared = 0 .99978 0 1 .401 .20.. 03 1 .000.800.600 .400 .2 .00 0 0.000 0.010 0 .020 0 .030 0 .040 0 .050 Concentration, pp m 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 . -671~99 p . 68 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Figure 2 Representative LC/MS/MS Chromatogram s c51017ZIM srr c rtWO M ~ 41 AFO 1 4 .4 8 2 sz46 18154 I anr2au ., ds, ES 3>3w 203oe~OX m 051 0] TM3 3rodt(n.1k3Ci) 7Rvbr2avrEIs .Eti FCO 415,370 1 306 4.46 6~1.5~ 003 rn 26 0 28D 3Q7 32) 340 3E0 383 4.00 4~ 4.40 44)- 4.8 6` 500 520 540 58) 580 600 62) 6 .40 66D 6.81 70 0 Figure 2a : Representative LC/MS/MS chromatogram of NANOpure water used as the diluent in the study . Retention time for PFOA is approximately 4 .5 minutes . 051017Mme sTna #*20 Q Mpt WM ,r R p 1 4 .47 15M 14272 2awnd s,~ 41 3>3E8 1 .637e+004 513 581 rrin 05101TIFm3 3rodh(A142C~ Mavtf2chardsES p M-t(t 415,370 SO_ 8619a+005 1 I 4.46 774~i70 863/82 o~ rnn 2E~30D 2 33)8 340 0 3~ 4 3.80 0 042D 4!40 '4.60 4.80 500 52) 540 560 580 600 620 640 6E0 68D 7A 0 Figure 2b : Representative LC/MS/MS chromatogram of 0 mg/mL control sample . Retention time for PFOA is approximately 4 .5 minutes . -68~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 69 DuP ont-18317 Figure 2 Representative LC/MS/MS Chromatograms (Continued ) 0510171671 9ra1t{MS2C3) MdvLf2dTrrHs,E~ sd5 413>3E8 p}~ 7.937eCQ5 1 4 .47 MMfl Z 79Z306 05101771Q1 stl5 m 9rodh(At42C3) AAdvtf2dands 6 415>370 7.531e+OC5 1 4 .46 67"M.19 7511- % - rri 2W 280 32J 340 3E0 380 4.Q~ 43) 4.40 4.~ 4.H1 SQ) 52) 5.40 5~ 580 600 620 fi40 66 6H7 ) 7 7.~ Figure 2c : Representative LC/MS/MS chromatogram of 0 .0303 ppm APFO analytical standard (H22703376) diluted with NANOpure water after matrix correction . 05101TRW 3rodh(A142o DAavtf2darids$ 1 .0rrgrrl, #1 413>369 AM 8382"006 1 4 .4s 74387.07 835454 r 0510177/kE0 9rodt(M%28) hfdvtf2darEs,ES 1 .0rtgYrt#1 41,5P370 1OD- 4.45 a146e'006 77197.9D 8123M % - ri 260 280 300 33) 340 360 380 400 410 4.40 4.6D 4.80 500 520 540 SEC) 580 fi00 7177 6E0 fi80 7.00 Figure 2d : Representative LC/MS/MS chromatogram of I mg/mL APFO dosing solution diluted to a nominal concentration of 0 .03 mg/mL . The measured concentration of the representative solution is 0 .979 mg/mL . -69749 p . 70 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Figure 2 Representative LC/MS/MS Chromatograms (Continued ) 051017ZTU) 9rndh(M,26) ~2dsndsE7 1.0 ncfrrf- sple 413>383 1 ,aRa 4.4 6 8361517 9`Q3 9.35Da+CO5 % nin 0510177EM 3rn(A1420 K#?vUF2dards,ES 1 .0 rpht, sple 41y370 1 IS1D a870e 005 4.45 79D7255 8BIft % 290 280 mn 33) 340 36O 3~ 4W 410 40 4.87 4.80 50D 51D 540 581 587 fi~ fi2) &40 &~ 68] 70 0 Figure 2e : Representative LC/MS/MS chromatogram of the 1 .00 mg/mL level recovery sample of APFO 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 . -70~~ 00 ~I O ~ ~ bD GO Y bq x 00 .`L ~ E E o0 O O - - M M i p . 71 N cd Cd ~I--1 O b,O w bb . 3 >1 o Cd ~ .~ ~ .~ 0 ~ o o 0 0 p V 7 7 7 C~ C D rM O oo ~O 7 N O x ~O M M rM M N N ~ Q ( 3 ) 14 2 !aM ,~ p OH uea K p . 72 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 APPENDICES -72- 73 p . 73 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix A Certificate of Analysis -73~~ p . 74 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 E na ~ 3058 Research Dive StapeCollege.PA 1680 1 T;814.272.1439 RE5EARCH ea~.mm Precise Research . Proven Rewlts. ;--,s= .-r~;~ .e~ ~-.,,~-- _~ ~ar ~ ~F:ri , ~~~I!:? w .~r CERTIFICATE OF ANALYSIS This Certificate of Analysis fulfills the requirement for characterization of a test substance prior to a study subject to the GLP regulations . It documents the purity of the test substance. This work was conducted under TSCA Good Laboratory Practice Standards (40 CFR 792) and FIFRA Good Laboratory Practice Standards (40 CFR 160) . Designation of the Certified Material : Compound : APF (Linear) Haskell Number: H27308 Analytical Data: The Purity of the Certified Material was Established by LC/MS/MS Purity: 19 .5 % Last Date of Analysis : 07-November-2005 Re-certification Date: 07-November-2006 Origin of Certified Material : E.I . du Pont de Nemours and Company Wilmington, DE 19898 USA Testing Facility/Performing Laboratory : Exygen Researc h 3058 Research Drive State College, PA 1680 1 Prepared By : Charles Si ns Study Director, Exygen Research de_~ Facili ty Management : ~,t t5- kSw -0 John Flaherty 'Pt"~~ ~VtC ~y Date Vice-President, Exygen Research I/ DuPont-18418 Exygen Research Study P0001843 Page 1 of 1 -74l5 p . 75 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix B Individual Body Weights -75- 76 p . 76 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 ABBREVIATIONS : g grams INDIVIDUAL BODY WEIGHT S EXPLANATORY NOTES -76l ~ p . 77 M 00 O ~J ~~ - .1 N o w n ~ N N 61 oJ rl lO N r CO r-i N 6~ ~fl o r 61 l0 ul :~ r1 VM 61 rl 61 .......... .. . . .. . . . . . . . . ., N ~ N N ~O .~ ~ N N N oJ O O I'D r. 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N . .N .61 r ul M . .m. O. :n . O . .N. ti N. O N~1. M. N ~ . n. :.n r-.1 M. l0. l0. N M M O~ O ul ~n O M N M O V' lO 61 ,-1 ~ N R' .-i r l0 10 CD ~O v' Ol M O 3 b~ ~+ v' M M~' Ol N N N~f1 N v' M tfl N O Cl N rl ct' r1 M .-~ V vO N .- 1 (tl M M M M N M M M M M f"1 M M M M M M M M M M M M M(`'1 M M("1 M M ?i u ~ O co Y (0 M M ~ ~ LP l0 O M r CV M(V M N O O rl 10 a0 m ~D ti N~O a' N . ... - .~ 01 61 O v' N v' \D M r 1 N l0 M O~ f~ O M :n Ol N M 10 rl ~~-1 N(`"1 :Il CO M r O CO ~ l0 10 O O~ --1 M :n 3 b~ T M N N NM ti O- d' - M N M N Ol M-+ (D (") O N C) M M Ol N O N :n O M M N M M M M M M M M f`7 N M M M M M ("1 M M M N t'r1 M M M(`"1 ?~ u Q) ~ 4J ~' - CT r 01 d' N .i M M W CO . O . 61 .. O . a0 ~fl .. 61 Ol . O . r1 c0 . .~ vCp T N 6~ lD :n r-1 O O r . . ... . N . ~-~ 3 U1 O~ N~~-1 N r N Ol O' r1 COOv' M M r w ~n O~O Vcp N r d' r T N M r b>rncorno1ocomcooo~ oaoorn1ornwr Or cor orn1ocomr n r0 ~ u . . . M N N N N M N M N M. .. . N M N N N M N N N N N M N ~ ~. 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N 00 [~ 6\ O n O~ 6l CO O N M N\O l0 > m v' O CO N v' Ol r ID 0 ~.n m r CO a' r tn Ifl In N N N M N N N N M N N N N N N N N N N N U N N N N N N N N N N o O .X ~ p 0 x ~ x' p, O M M rl N r ~O Q' ~ tf1 N~-1 W d' r ~O N O O ~ ~ O M M 61 Q' M 6~ r 6l v O N W In M M In c) O, Ul r~' ~ W O M r~fl ~O In co vr r r rn io ~o r r ui r m td .-1 N N N N N N N N N N O (V N N N N N N N N N O N ~ a M f' 1 Ol 61 r N ry O In r cn I n ~o ~o r ~ io N N N N N N N N N ~ ~r op Cl ^ ro ~ o 0 ~ N r-I N('1 v' iN lp f ~ 61 O o O ~ M v' In lO r o~ Q~ o f-I .-1 N M O' In ~ r CO 61 o r-1 0 0 0 0 0 0 0 0 0~ o 0 o O o ~ lo o c) o 0 o c) o 0 0~ ro - r+ - - ~ - - - ~-+ ~" r r r r r r r r r r E rn rn rn rn rn rn rn rn rn rn E ~ .~ --~ r+ , ~ 7A p . 79 00 0f..' ~ ~ CS .-1 .-i N N 3: CS r1 N m~ N .--I r1 Ln d' .......... M~~~ l0 N~--1 N v' a' ul Q' v' CV m~-i O O tfl r-1 r r N m r~fl m r ........ :tl O .~ ~ ~D l0 W (V M l0 O 61 M itl O f~ v' .-I LO lD N W O M v' ul .. .......... v' l0 O d' O l0 O W m t0 v' O ID O m v' O N Ol lO O r(V r r a ro ou mO ~ ~ CP r-1 m m V' ., .{ O . N N N r-I O W M a o 0 oa ?i C' a' Q' l' M ruo M O X Ln rl ~D ^ M m Ol - ifl .-1 61 l0 O M 6, lO O N ~O ul r1 O . . .... ... . .. . . M v` m O 6\ a' 6~ <t' .-i r N T r-I O N ~-1 O N d' M m r l0 l0 I~ m O M 01 n O N N n O m m m m d' m (V 01 t"1 t"1 ~O O r r+ lO r V' l"1 M~ OQ' a' a' Q' M Q' M M d' M ~ M Q' Q' M V' M~ P M ~~CP Ql m l0 OD v' N~1 O ~O M r Ln 7 i0 Ol r-+ ~--~ ~ N 61 0 l0 u~ o M M l0 v' it .~ rn . .. . . . . . . .. . . . . . . . ... 41 ~ co 61 1O u~ o lO O r ~D N r-~ m r r v' v' r rl r N O' O M ID O Ol Q' O r T CS M M C1 rl m m 61 61 cT O (V r1 Q' Cl m N 6l I~ M Ol .-i m M N~O 61 r ri l0 ?i c' v' v' Q' M M f~l M v' ~ P vvM M v' M M d' M Q' M V' v' M M M v' v' M ~ O O ~ ~ L ~n ZP ifl .~ m m O O\ .. a' . ~fl .. 01 . lO ID . .. 61 lD . .-1 6~ N N ri m O M N M Q` O r l0 O Q' l0 ~I) ....... ......... r . JJ Q) rl O M M T Ol N~O ~-1 M l0 lD 61 m M .-1 o T a' r. r. co l0 d' M M l0 cT ~ Ol W ~ 3: CP M("1 N O r Ol M Ol Q' Ol -1 -~ M m m N N r(V m O m v' N~ 61 r c) Ln ~D b~ ~+ O' O' ~' c' M M M M V' M ~ vvM f"1 V' M M~ M cT M O' v' M M M ct' v' M -~ T ro v zs u 3 0 W b O t~ m ~ 0~ b) V' (V w r l0 m r a' M co m l0 ~(V m N~ N O N O\ m m m d' M Ol rl m ~ ri - .-1 r . . . .. . .. .. .. .. l6 N ~ O m O ~ ~ rl rl' I D , ~D N M W O M O f~ m r1 ct` O lD M l0 ("1 O ti r N a 3 CP N rl .--1 O O m r m(") OJ O O N f~ r O m~ r-1 r O r r1 r-I v' m tD 61 m lD '~ ?~ v' Q' ~P cf' M("1 M f"1 v' f"1 ~~ vf"1 M c M M~ f") Q' M d' O' M M M M d' C') > "6 u -I O 'O W C ~ CP ~ lO 1) Ol f'1 01 ("7 v' m O m~D lO O m r~--1 Ol m l0 co ~-1 r1 m l0 r1 N r-1 N r .rl l0 .... .. .. . . .. .. . . . . . .. .. . .. N ~ O m M O r. Ou lD~ .-1 N ~1 O~-1 ~ftO(V v' v' O l0 M 61 .-1 O~l0 ~ O D O ~Oul 3: CT ~ rl O Ol ~ m ~D r N m O O N r lO O r ul r1 l0 61 lD rl (D t"1 f~ lD Ol MLr) a v O m ~ a' a' d' ("1 M M M M v' M vvvM M a' t"1 M v("1 M M d' v' M f7 M M d' M r uo ~ ~ CT M LO ~ o r M v' M m r ~fl OlO V' N- l0 61 r Q' m 6\ O rl m M r m r rl - . ... . .. .. .. U) ~ 1' Ln~D lO N Q' l0 '-i r r-I M M ~ M O N V' V' N Q' T(V r. N ID r 1~ M Q' l0 O O Ol CD N r l0 r .-1 m Ol 6l r-I r iD O r u~ o ~D m lD O O M r u) m N~1 Y ~ ro d' Q' M M M("1 M M O' M C) M V' t'7 M~ M M Q' M M("1 Q' V' M M M f"1 ~f' M R O u ~ Y . . .~ CP N N M Q' ~I' N N ~fl r~ m O N r Ol M rl m r O r O\ O Q' \O ul N .i <t' . .. . . . . . . . .......... .......... N ~ N ~O ~~ l0 m O N l' ~ ~ N v' ~fl Ml0 mNNum l ON ~ m Q' r ~M O m ~ .~ 3 tr~ rn rn m m r~ ~o ~o ,~ r m rn o lo n m ~o vrn~n r~n o rn M io ~n m,~ v~roM<"1M("1Mv'MV'Mt")<`1M M~'("1Mtf")v'M ~ ~ O u ~, . O O y..~ p y r,y' Q) X +~ \ C tn b~ o m m rn o o M M m ~ E M rn,~ -~ M x . .... . . ra \ M m v' M r v' v' r M N M ~-+ 6~ r N rn . r-I r o .. (V M,~ M 61 \O m . m b1 o m rn ,y n in ~o vN . f~ \ ' .f) r O O m r. O N 61 r T CO ~ M M M M M M(") M a' M O M M~ M M MO M t+l Mm M ~ M M M M(") M M fYl a' M -o 0 m 0 o H H ~ ,~ ' C [~ N G 1 ri ~-1 N f') l0 ^ m M O r-1 ~ ro 0 00000000~-+ m N ti N M V' ~~D r m M 0 00000000~ m 4 O rl .-1 N M v' ~Il ~O r m 61 O o000CD 0000~ Lfl ~ Ln ul L n 8~ p . 80 M ~ O a~ ~ b` l0 .~ H N N 3 IDI a ro -d o 0 m 14D N N v' N N o M 61 tIl ID ... .. Ul `,O N`-+ r~n m o r1 ..... . M O\ M 61 - r .-1 CD, m r N Sr r'n HM M In d' M C'1 I() m 61 l0 M r* N . N dl tn 61 61 61 M N Sr - - O M(") cl' r M C) M M M cI' V' M Q' M N N M N M M M M M M M M M 0 In v' ~D r r Ol M . . . ID W N NIn f-1 N O~ O N N r- 1 M M M M N N M ~ c iT O .{ p O' O M Ow . O 1~ N N Ln M v' . O . a' . O OM . . O 61 iO O O~' .. M a' ... M . a' Q) N r-~ rl ~ Wr M('M l0 r Ol CO lD M rl t0 rl 61 ;~ 61 ~D 6~ N T~fl N r Q' d' r ~fl ,'3 ~P lO 61 Ol lO M N ri l0 rl OJ ~' M CO O MI M r-i u7 O N M d` r N CO rl ~ 6~ ~--1 .'ti M M M t") M v' <t' M~T M N N M M M M M M M M M M M M M M M N .-1 M T CO o 0 0 m ~~~ \O ri m l0 r1 Ln 61 lO l~ .-1 M O r~. in r-~ LIl a 61 M~' N ~-I 10 61 ri 61 l0 N O r1 .~ ~ .... .. . .. ... . ......... N rl M~f) ~ Q' T' O N~n ~ .ti d' N r-1 -1 61 ifl ~' CU lp V' Q' r N d' ~' O' N M M Ol 3 b~ ~ N 61 lD M .-I '-1 lD C) N M O O~ O M rl - .-1 ID O N N a' r N~ O CO H C) ?i M M M M M~' a' M~ t+1 N M M M M M M M M M M CM M M M M M N N M ~ CO b ~ 0 ul JJ ~ CP O R' 61 N H O r Cl ~O l0 Sr rl Sr lD 6~ r1 r-1 r-1 N r v' N r oJ c"1 H N w r-1 O U) .i . . C . . . . O . . . . . . . . . . . . . . . . . ~ 4) rl ~ N l0 CO r h 61 N M 6~ (A CO CA ~-1 l0 In r Ln l0 r W r-I ~I) rl M ri O~ r ,C: 3: b` Ln N O~ tn M C) C) 19 O h O Ol r C. M .-1 a' r-1 l0 O r-1 N cT h Nm ri w H O LT ?i M M M M M Q' O' M O' M N N M M M(`"1 M M M("1 M M(`'1 M M M M(V (V M ~ ~ ro av -o c~ 3 0 W a '6 O a~ p W C C b) C) W 61 d' N m In v' H 61 oC CO CO H O~' N r N ~O v' M Ol 6~ V' oJ Ol 10 C) C ) rl .~ h . ...... . .. . ... .. .. ttl 4) f4 ~~ N O .-i L/l 'n N a' 7 30) Q' r l~ tt~ M 61 61 Ln O~ IO N a0 Ol l0 I~ C) N M l0 O N v' O Ol a' r 61 r1 r .-~ 61 ID N N O C) N rl M~O rl r Ol r--1 61 "(J ~ M M M M M M M M a' M N N M N M M M M M M M M M M M M N N N N >1 t6 > b Q ~ 0 O CQ G ~ ~ O d' 6~ d' r r-1 61 Ln O 6~ R' In O M M In .~ ~ .......... . ... .. 6l O 6~ if1 tV 10 N ri t~ M T ul .... .......... v r-I tn O~ W O l0 r C) 6l ~-1 01 W r v' W G' O ri ttl M N O1 N 61 6l Ol O N 61 l0 61 Ln 6l r N OJ lD 01 N ID ~' .-~ 'n O r1 (D MIn ri ~D O r O\ Ol .1 (`") M C') (") M M M("1 Cl M N N M N M M M M M M M C') M M M M f"1 N H N T ~ ~ O O CO cd ~ ~ b, m Ol IO r1 l0 M N a' ~ 0) N 6~ r~ Ln .~ In ...... . . . . N~' Sr Sr r-~ HIn Q' W Q' a' C~ r W'-1 b` vl0 f~ Sr M N W t1l W 'ID v' r ~n Q~ N M M M M Cl M M M M M N N M N M ~ T 0 ~ N 6~ CO M 61 ul CO M O C) C- N r 61 O . . . . ... N O a1 O W N N~ d' O W r r M M .-1 0' O ~n O r-i rl M ifl O ~O 61 f~ r 61 M M M M M M M M M M("1 N N rl N ~ ~ . .r .i q' ~ .-1 01 O O Q' r-1 ..... .1 ~O CV ~--1 r ul ~ V' 6~ r1 ~' ..... . . .-1 CO N O S1 In OJ O 61 r . ~ . .-~ N O r~' .-1 N f-I N O N~' W ti N~l l~ O 6~ rl ^ u~ ~D ' N N 6~ C1 N i .n .-i O N N 10 h V' CV r WWL1l ~O CO In 6) N 01 M O d' O O m CY) cV In HLf1 m r o J M M M M M M M f") f"1 M N N M N M N M M M M 0 N N M M M M CV CV rl N ~ ro -o Q 0 v a Z Os+ C) y~ -14' -C~ - (7l w M m O M O `++ ~ .. ~ 0 a a~ (S v r-I N O O N ul ~D ro rl M M m b -14 X b~ M rl m d' (V N rl 61 Ol CO M lO O H N I'D E m m N 61 V' N .... . . ~ i0 m[~ N N~ol 'n cV N O C) O Cl 1~ CO O M r In M .-1 N ~D o] r v' 6~ N 6~ N O V' O N . O Ol N- OIn m N m M M Cl M M M O (V (V M N M N M M M fl O M N M M M M N N r-~ N M M o C13 Q H > H x `~ ~ Q N U1 Q1 rI N M Sr In 10 h M 61 C) '-1 r-I N M Sr In ~D i~ ~~ O ri r-1 N M v' In CO 6l O r-I O O O O O C) O O O 0o b o000oooooti ro 000000ooo .-i ro f+~H~-+~ .--r+- H~ Q N '~ r r h r ~~ r r r r h E rn rn rn rn rn rn rn rn rn rn ',~ ,~ r-+ ,-~ ,-+ , i O i ~~ p . 81 00 0 u 4j .CP 6l ~ 61 M . . cl' T ... In 10 Y' M OJ r r v' N d' ..... .... .-1 d' O~ ~-+ M N o lD Q' C) In 10 Q' rl l 0 N N M ln l0 ~ r O O N Ol N N M N 61 r-I r cT N in IO O N V' N dl (") O M N N 3 O~ N r r ul rl v~ N M N c!' in ~D O ri N l0 M O r N ul .-1 l0 r N M , In .-i C) a b ~u O W ~ .C m C .V fP r O ~O M m IN ~' rl l0 In N M O N In l0 M O O l0 61 6~ N lp 'p M m O O 1~ U) N Q~ r1 l' r-I N O oJ 61 ~-1 N v' l0 lfl M lD ul t0 V' i0 t"1 M v' ~' r V' O r M r l0 .C 3 b~ r r r ~n rl M~ N N O' in ul 61 N`-1 ~D M O r N v~ ~ lD tp N("1 N M O O M Sr M Sr ul v' N ~O u 3 O a O O 4j P~ C -i LP rl ~ .~ ~ OJ Ir r O .Ir. r . v' CO .. d' r t~ r 61 .. V' o rm . CO l0 00 Ol T Q' . ul IT, M ..... In Sr l0 . N Q7 N O~D O l-I ri l") M c3, In* c:)* m N `.o In N N In M .-1 N to M O in m O In In ~ 3~ N r r In rl M r-1 M r Q' ~ in 61 N- ~D M O r N v' - l0 r oJ N O1 N O O T .1 3 > ,7., ~' v~ N m v~ v~ v~ v~ v~ v~ cr v~ v~ Sr - Sr - - - - m - Sr - Sr M v~ M v~ ~n ~r > a u -~ O "O W C J~ L CP N N v' ^ ul Ol l0 l0 OJ f~ M CO r ti Ol rl in O CO O M r1 ~ . .-i m t~ to Ol m r r M N ? ~ N O] uN N N W In 61 M ~n Ul ~n l0 V' O N .-1 N r M ~' v' v' Ol O l0 In N N r1 a' Sr - .--1 ~n rl M rl N M Ol l0 Q' O' N\D N N ~D cV O r N M r1 in If W N CO N O (D Sr Sr Sr Sr Sr Sr v' Sr Sr M Y' M Q' Ul Q' 0 CQ Y ?~ d' ~ L CP l0 -rl v' CO M O .. In . cV .. In . M O ... M . r-1 rl m d' lD in ul W~ lp (A NID M~D O N r1 l0 N Cl) N M N v' CO U) N O~ ID O O r r O r W r N In M lp rl O MM O ~ b~ l0 ~D In M O N O .ti r M M V' OJ .H O v' N Ol l0 ~ M In In r N CO N N IT, C' Q' Q' Q' V~ d' V~ d' Q~ Q' ci' Q' Q' O' p' O' M eT Q' Q' Q' Q' O' M d' M cT p~ M O Q _~ ~ --~ -i M . . lD . .Y'. O . OJ CO 61 ri N N . . . r to . O . lD lD ... O' . m . Ol . v' l0 N O' . In 61 CO In N GO O . N N v~ r ~n lID n Q1 In N r OJ ~ M~n W O r-I IT, f-1 O r-1 lD N M v' r O M ul N l0 O\ M 6l M ri M l0 M~~-1 I,r O O v' r-~ 6\ ~f) O M O~n to r r1 CO N OJ 61 ~va ~o u O X ~ ol ol E(] . "~' 3 L T C ~ ~~i -o C 0 ~ O ~ ~~ N N ~n V' .. lO M~D . o I . n. ~ ~l0 N M I n T W In M CP v' M r M M lO in ~tl r v' O M l0 O 61 r O M loLP In In ~ M 61 ,-~ O O . n N E Sr Sr Sr Sr M Sr Sr Sr Sr Sr O uO H H m H ti r1 in r r r 6~ r-i W N\p M("1 ~D O m I n (D Sr Q' Sr Q' f"1 Sr Sr C1 Sr Sr \ 61 ri In N N N O f~ N Ol P O ~ N O W N I~ O J t") M .~ > O N M P In lD ;~ ~ ~IY, o M~ In N lp f x 61 0 M a' In \D r CO 61 O ~ N ra o C) 0 C) o O o O o rl ru C) 0 0 0 0 0 0 o O to rp o 0 o O o O C) o O~ (') (r~ (") C'1 M M th M M("1 z Ul in In In in in J) in ~fl In 9~~ p . 82 M W 0 ~ ~~~ N M 6~ N T tf) l0 r tf) ~' ~ r O r N N 61 ~"t' H r t~ f ~ d' O Ol ,~ O ti N W ~ N O O l0 N t1) N O d' ra O N M CO 6l M M lD M r r N ~D l0 v' H ri N Q' ~ O 3 CS W N O N d t~ Ul OJ cf' r-! Ol r1 f-1 O tf) M iI) M l0 r-I t~ ~O \D N l9 N O' O O O' T M v' ~f M M V cl M d' a' N M T M!") M M M M M M M M a' M v' M M M M T ro -o 0 w0 ~~~ r OJ r-1 6l H r 10 .-i n 6~ r 61 ln -H J-J v r N O~ N .-i ti ifl tt1 \O r .-/ tP l0 M r~I O CO v' cl' v' - V' r-I Ol ~ T M V' d' M M d Q' M V' a' N M d' M ~' o CO M M H o M oO~MCO a' r~~T o M O~ .-1 r N r-1 ls) N N tb d O~ 6~ OJ ~' O r N r-1 ri O ul M ul M l0 ,-I tt) t~ iD .-1 t~ H M O 6~ ~' M M M M M M M M M Q' M a' f`"1 M N M H T ro v O u 3 0 m T ~ O N 13~ I~ r Ol \O ~~I) OJ N N r~' R' d' W T r-I r-1 M M-1 O H lD fr r-I 6l ul ~O I. O M .-t H m .......... .. . r N H r 6\ O\ O~ ftl Q) N tp N M N t~ H f:~ O Ol lf) r~-~ M CO M M M l0 M o r a 3ol r N Cl r Q' tn <T O 6\ O~--1 H tf1 M ttl M l0 r1 t~ Lfl ~--~ t~ N M 6~ [D M 71 T M a' d' M M d' a' M d` ~I' N M a' M M M M M M M M M M V' M~' t"1 N N M H > >v a r uo -H o O Om c ~ ~ ol -~ v r tn M M M M d' .-~ 10 O M~ ~ . .. . . . . . . . .. Ln N N r M l0 O . N t .f) M tO Ul V' M M ~O v' r1 N r ~D :3, c*. v' O r M l0 r-- r-1 O r d' M Q' r t"1 ~--1 ~ O O~ t11 M ttl N~o >~ M a' d' M M~t' V' M V' C' N M d' M M M M M M M M ID ~O 'I1 N M M~ M l9 l0 N lD r N r~ r V' V' (V H U) tn ~ O a' ~ N~ f M M M d M Q` M N N f") T v r uo 0 wj ~,~ CD ~~ N r U) Ln .-1 N'11 6~ O N OM O N .-1 un tn r M O v' 6~ ~P N r1 0~ ^ .~ ~ ........ . Q) N lD 6l M r ti l0 \O tf7 M M r1 N ri ~-1 r M W lD O 6~ CO ID r1 - ,i 6~ M l0 N N 3~ ~D 6~ O r V' tV M I- N O m O O O M N v Ln O M V' ~1 6~ ~1 O N CO tI1 N ~ T M M V M M T d M 4' a N M V' M M M M M M M M M M M M OM N N M T ro ou ~ . .-+ ~~ 3~ ~9 ~ v~o .-+rnr~raomrn r~o .-~aNrnmMO~M s+ omorNNMOr o -.i M N Q) N W O' O~ M M 61 r~ rl o rl o m 61 l0 Ol r rl M~O > OJ Nm m M o M~ r lO O O'~ v' N M r M O r O 6, 61 ~' N Q' N ~tl O O M M v' co m O .-i Y OT T M Q' d' M M<t' a' M cl' d~ N M M N M M M M M M C~ M M M M M Q' M N N M N tu tl R. bl X r x yO ~ N ol N OOO N O ~O tP \ O~ M N-1 ~ H O[~ M ~ M~l M M r M M .-i _ n d) Li (U N M N pp V' 61 a ,~ 3tp O lfl ol m ^ M N M r ~ T ro rl M M M M M tIl O' M E N 61 lfl O tr O' a M a Cl) H N tIl ID a r N O~ N~ O N p~ 61 6J O 61 Q` O t`l C~ Q' N Q' H'J1 O \ M M M I~ M O~~ M~ O N M M N M M M M M M O M M M M M M M N N M M M p 0~ J Q H H X O ~i,~ H ti ~ N td N o , C ~ ~ ~ N .-1 N M~T tf) lD rN~S O t`"1 Q' -~1 ~O r N 6~ O r-I H N Cl ~' ~fl U7 r OJ 6~ O -1 O O O O O O O O O~ 0 o O o 0 0 0 E r r r o r-I Id o o O O o O CD O o rl t6 r r r r r r r '~ rn rn rn rn rn rn rn a` a' ~ ~ H- H- 'H '~ r' '~ r' '-i - '-''''i-'-'~ '-~ ~~ p . 83 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont- 18317 Appendix C Individual Food Consumption -83~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats INDIVIDUAL FOOD CONSUMPTIO N EXPLANATORY NOTES ABBREVIATIONS : Cons . - consumption g/anm/day - grams of food consumed per animal per da y p . 84 DuPont-18317 -84~~ p . 85 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Food Consumptio n Food Cons . q/anm/day Day 7 Food Cons . Food Cons . Food Cons . g/anm/day g/anm/day g/anm/day Day 14 Day 21 Day 28 Male, I 0 mg/k g 101 102 103 104 105 106 107 108 109 110 30 .2 29 .3 30 .2 27 .6 24 .7 30 .0 27 .6 29 .6 30 .3 28 .1 30 .6 31 .5 28 .9 27 .9 25 .2 29 .1 27 .6 28 .1 33 .5 29 .2 31 .7 32 .1 32 .2 28 .1 26 .1 30 .8 28 .3 29 .1 32 .0 30 .1 31 .1 30 .5 31 .0 30 .8 26 .8 29 .9 28 .0 30 .2 31 .9 32 .2 Male, III 0 .3 mg/k g 301 302 303 304 305 306 307 308 309 310 29 .8 29 .0 30 .1 27 .5 25 .7 30 .2 25 .5 25 .3 30 .0 26 .9 29 .3 30 .9 31 .0 26 .8 26_1 32 .9 26 .1 26 .2 31 .1 26 .9 29 .2 29 .5 33 .6 27 .7 27 .3 32 .8 27 .5 25 .4 31 .1 28 .0 31 .2 30 .5 34 .9 27 .6 26 .6 30 .6 27 .9 26 .6 32 .3 28 .8 Male, V 1 mg/k g 501 502 503 504 505 506 507 508 509 510 28 .5 28 .3 29 .6 31 .4 27 .5 27 .4 24 .1 30 .1 31 .2 27 .3 28 .9 28 .8 30 .1 32 .9 26 .6 28 .4 24 .1 28 .8 32 .5 26 .2 29 .9 27 .8 29 .8 30 .6 25 .1 29 .3 22 .9 30 .0 33 .2 26 .7 28 .6 28 .4 27 .0 31 .8 26 .4 29 .1 23 .7 28 .2 33 .6 28 . 0 -85~~ p . 86 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Food Consumptio n Food Cons . g/anm/day Day 7 Food Cons . Food Cons . Food Cons . g/anm/day g/anm/day g/anm/day Day 14 Day 21 Day 28 Male, VII 10 mg/k g 701 702 703 704 705 706 7 07 708 709 710 27 .8 25 .5 26 .9 25 .3 24 .0 27 .2 29 .0 24 .4 26 .0 27 .4 26 .6 29 .8 30 .0 25 .9 29 .0 31 .1 31 .8 26 .8 29 .7 30 .3 27 .2 30 .2 30 .9 26 .6 26 .1 31 .2 34 .7 26 .9 28 .1 30_0 28 .8 30 .1 27 .1 26 .1 26 .7 30 .9 33 .9 27 .3 29 .3 31 .0 Male, IX 30 mg/k g 901 902 903 904 905 906 907 908 909 910 9 8 23 24 24 14 25 22 24 23 .2 .6 .4 .7 .7 .5 .0 .2 .5 .9 24 .6 27 .0 30 .5 23 .4 25 .3 35 .3 26 .2 29 .3 30 .9 26 .8 11 .9 23 .5 29 .2 22 .2 22 .9 30 .3 24 .2 25 .0 28 .7 22 .3 25 .7 24 .8 29 .7 23 .9 24 .0 29 .0 26 .4 28 .5 26 .5 26 .6 Male, XI 30/0 mg/kg (Recovery ) 1101 24 .2 1102 13 .9 1103 25 .8 1104 23 .7 1105 23 .0 1106 26 .9 1107 22 .1 1108 22 .8 1109 5 .1 1110 21 .4 23 .2 31 .9 28 .6 28 .6 25 .1 29 .2 22 .2 21 .0 15 .8 27 .1 26 .2 27 .2 26 .8 27 .0 22 .3 28 .9 24 .7 23 .5 20 .4 27 .9 24 .4 26 .1 25 .8 29 .7 25 .1 29 .2 25 .9 23 .1 32 .0 28 . 2 -86g 1-7 p . 87 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix D Individual Daily Animal Health Observations -87E8 p . 88 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Daily Animal Health Observation s Sex Group Animal Observation Days M 1 101 General observation, No Abnormality Detected 0-28 M I 102 General observation, No Abnormality Detected 0-28 M 1 103 General observation, No Abnormality Detected 0-28 M I 104 General observation, No Abnormality Detected 0-28 M I 105 General observation, No Abnormality Detected 0-28 M I 106 General observation, No Abnormality Detected 0-28 M I 107 General observation, No Abnormality Detected 0-28 M 1 108 General observation, No Abnormality Detected 0-28 M I 109 General observation, No Abnormality Detected 0-28 M 1 110 General observation, No Abnormality Detected 0-28 M III 301 General observation, No Abnormality Detected 0-28 M III 302 General observation, No Abnormality Detected 0-28 M III 303 General observation, No Abnormality Detected 0-28 M III 304 General observation, No Abnormality Detected 0-28 M III 305 General observation, No Abnormality Detected 0-28 M IiI 306 General observation, No Abnormality Detected 0-28 M 111 307 General observation, No Abnormality Detected 0-28 M III 308 General observation, No Abnormality Detected 0-28 M III 309 General observation, No Abnormality Detected 0-28 M iII 310 General observation, No Abnormality Detected 0-28 M V 501 General observation, No Abnormality Detected 0-28 M V 502 General observation, No Abnormality Detected 0-28 M V 503 General observation, No Abnormality Detected 0-28 M V 504 General observation, No Abnormality Detected 0-28 M V 505 General observation, No Abnormality Detected 0-28 M V 506 General observation, No Abnormality Detected 0-28 M V 507 General observation, No Abnormality Detected 0-28 M V 508 General observation, No Abnormality Detected 0-28 M V 509 General observation, No Abnormality Detected 0-28 M V 510 General observation, No Abnormality Detected 0-28 -88- ~! Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 89 DuPont-18317 Sex Group Individual Daily Animal Health Observation s Animal Observation Days M VII 701 General observation, No Abnormality Detected 0-28 M VII 702 General observation, No Abnormality Detected 0-28 M VII 703 General observation, No Abnormality Detected 0-28 M VII 704 General observation, No Abnormality Detected 0-28 M VII 705 General observation, No Abnormality Detected 0-28 M VII 706 General observation, No Abnormality Detected 0-2 8 M VII 707 General observation, No Abnormality Detected 0-28 M VII 708 General observation, No Abnormality Detected 0-28 M VII 709 General observation, No Abnormality Detected 0-28 M VII 710 General observation, No Abnormality Detected 0-2 8 M IX 901 General observation, No Abnormality Detected 0-3,8-17,19-28 Feces, Absent Comments, decreased feces 18 4-7 Not Eating 18 M IX 902 General observation, No Abnormality Detected 0-3,8-28 Comments, decreased feces 4- 7 Not Eating 4-- M IX 903 General observation, No Abnormality Detected 0-3,5-28 Not Eating 4 M IX 904 General observation, No Abnormality Detected 0-28 M IX 905 General observation, No Abnormality Detected 0-28 M IX 906 General observation, No Abnormality Detected 0-28 M IX 907 General observation, No Abnormality Detected 0-28 M IX 908 General observation, No Abnormality Detected 0-28 M IX 909 General observation, No Abnormality Detected 0-28 M IX 910 General observation, No Abnormality Detected 0-2 8 M XI 1101 General observation, No Abnormality Detected 0-3,5-28 Not Eating 4 M XI 1102 General observation, No Abnormality Detected 0-4,8-28 Comments, decreased feces 5- 7 M XI 1103 General observation, No Abnormality Detected 0-28 M XI 1104 General observation, No Abnormality Detected 0-28 M XI 1105 General observation, No Abnormality Detected 0-28 M XI 1106 General observation, No Abnormality Detected 0-28 M XI 1107 General observation, No Abnormality Detected 0-28 M XI 1108 General observation, No Abnormality Detected 0-2 8 M XI 1109 General observation, No Abnormality Detected 0-3,11-28 Feces, Absent 4-5 Stain Fur/Skin, Inguen, Brown 9-10 Stain Fur/Skin, Inguen, Red 5 Wet Fur, Inguen 5 Not Eating 4-5 M XI 1110 General observation, No Abnormality Detected 0-28 -89- .0 ~10 P . 90 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Appendix E Individual Detailed Clinical Observations and Mortality Records -90V Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats P . 91 DuPont-18317 Sex Individual Detailed Clinical Observations and Mortality Record s Group Animal Observation Days M I 101 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M I 102 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M 1 103 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M I 104 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M I 105 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M I 106 General observation, No Abnormality Detected 0- 7 Hair Loss, Forelimb, Bilateral 14-29 Sacrificed by design 2 9 M I 107 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M 1 108 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M I 109 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M 1 110 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M 111 301 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 302 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 303 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 304 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 305 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 306 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 307 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 308 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 309 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 310 General observation, No Abnormality Detected 0-29 M Sacrificed by design 29 M V 501 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 502 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 503 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 504 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 505 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 506 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 50'7 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 508 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 509 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 510 General observation, No Abnormality Detected Hair Loss, Forelimb, Bilateral Sacrificed by design 0 7-29 29 -91~~ p . 92 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Detailed Clinical Observations and Mortality Record s Sex Group Animal Observation Days M VII 701 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII "702 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 703 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M VII 704 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII 705 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 706 General observation, No Abnormality Detected 0-1 4 Hair Loss, Forepaw, Bilateral 21-29 Sacrificed by design 2 9 M VII 707 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 708 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M VII 709 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VI1 710 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 901 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M IX 902 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M IX 903 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 904 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M IX 905 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M IX 906 General observation, No Abnormality Detected 0 Hair Loss, Abdomen, Bilateral 7-29 Hair Loss, Forelimb, Bilateral 21-29 Hair Loss, Hindlimb, Sacrificed by Bilateral 21-29 design 2 9 M IX 907 General observation, No Abnormality Detected 0-7 Hair Loss, Forepaw, Bilateral 14-29 Sacrificed by design 2 9 M IX 908 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 909 General observation, No Abnormality Detected 0-1 4 Hair Loss, Forelimb, Bilateral 21-29 Hair Loss, Forepaw, Bilateral 21-29 Sacrificed by design 2 9 M IX 910 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 -92" --7 7 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 93 DuPont- 18317 Sex Individual Detailed Clinical Observations and Mortality Record s Group Animal Observation Days M XI 1101 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M XI 1102 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M XI 1103 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M XI 1104 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M XI 1105 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M X1 1106 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M XI 1107 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M XI 1108 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M XI 1109 General observation, No Abnormality Detected 0,11-29 Lethargic 6-7 Carriage, High 6_7 Feces, Absent 6-8 Stain Fur/Skin, Abdomen, Red 8 Stain Fur/Skin, Forepaw, Bilateral, Red 6-7 Stain Fur/Skin, Inguen, Red 8 Stain Fur/Skin, Perineum, Red 8 Stain Fur/Skin, Ventral body, Red 6-7 Stain Fur/Skin, Perinasal, Red 6-7 Stain Fur/Skin, Perioral, Red 6-7 Wet Fur, Not Ventral body, Ventral 6 Eating 6-7 Sacrificed by design 2 9 M XI 1110 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 -9391~ p . 94 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix F Individual Animal Clinical Pathology Data -94- ~~ p . 95 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTE S ABBREVIATIONS : General : Adeq - adequat e CLOT or Clot - sample clotted Decr - decreased Mod - moderat e NP - not taken, not performed, or results not valid OK - sample condition OK for testin g Individual Hematology Values : COND - sample condition RBC - red blood cell count HGB - hemoglobi n HCT - hematocri t MCV - mean corpuscular (cell) volume MCH - mean corpuscular (cell) hemoglobi n MCHC - mean corpuscular (cell) hemoglobin concentration RDW - red cell distribution widt h ARET - absolute reticulocyte count PLT - platelet coun t WBC - white blood cell coun t ANEU - absolute neutrophil (all forms) ALYM - absolute lymphocyt e AMON - absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophi l ALUC - absolute large unstained cell Individual Red Blood Cell Morphology ANIS - anisocytosi s MIC - microcytes MAC - macrocytes POLY - polychromasia HYPO - hypochromasia ECHI - echinocytes ACAN - acanthocytes TARG - target cell s RX - rouleau x HJB - Howell-Jolly body - - not observe d Values : Individual White Blood Cell / Platelet Morphology Values : SM - smudge white blood cell s TOX - toxic neutrophils DB - Ddhle bodie s VC - vacuolated cytoplasm BC - basophilic cytoplasm PCE - platelet clumps / estimate GP - giant platelet s BP - bizarre platelets - - not observed -95/6 p . 96 Ammonium Per fl uorooctanoate : 28-Day Immunotoxicity Study in Male Rats Du P ont-18317 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES (Continued ) ABBREVIATIONS : (Continued) Individual Clinical Chemistry Values : HEM - hemolysi s LIP - lipemia ICT - icterus CHOL - cholesterol TRIG - triglyceride s TP - total protein ALB - albumin GLOB - globuli n HDL - high-density lipoprotein cholesterol NHDL - non-high-density lipoprotein cholesterol SCORT - serum corticosteron e 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 marxed other . -96oil p . 97 00 0 Qr .a a ~ l0 6l rl r ~- N R' d' v' CD \D N 1 ~ a~ ao 0, ID a o..~` aT vOID mrnuno ~y O Z O Z . (V N Z Z~~ a O O~ r1 f-1 O O O O .-1 M ry rl .-i .~ rl .-I ri r-1 '-1 rl r~ .-1 rl x x a a M1oNM~n I o Ln .-+rn F 1o rnmCD vMr M ro o W O ~O m<l' O m a ct' N O 61 lD 61 N ornorn Iorn 2iomm ~oiom m n r iO t1 tD co m o y~ '~ N .-~ N r1 ri ri rl r-1 r-I <~-1 - ro x x .-1 ,-1 rl .-1 ,--I rl .-1 rl N 0 rn 0 co l0 O O co Ol r ~O 3 Ol m O m m 10 O q Qr. ti - ti - - - ti - - N a~ aro .-a b U a O m O\ r1 O M O v' M u 1-1 N~) N Ln cl' lD O~ m~D N "i U~ N ti r-1 M N N Z M(V N U~ N N N N(V N .-1 (V N N C ~` t"1 M M M M M M M M ~~ M M M t"1 M f") M M M M ~ U C ~ , ro W < o 6l~bl ~0 N. M . . m. N CL M 01 61 61 - ti - N . . M p, Q . '. m lD 61 ~~ . rn CP Ol C N a' a' 0 01 Z m 61 6~ O 61 cp W 6, Ol M* O, 6, o - - - - - - N ~ro 7 'O G Q > r l0 l0 o Ln T ~ > M'll U w N N~ CO M .-I Z m 6l m Ln ~o ~o u~ io ~o ~n n n l0 Ol OLn 0 ul ul q o m lD O r-~ 1 rO m lM 'o Lo Ln ~ X ~O ti U IAO M F ~Il v' ma 61 rl 0X 1 ~Il r m~ l0 u~ Z~1 ~~ U v vE - - - - - - - F M r r Ol 1o0 ul ~D v` l0 aw m a' f~ LO ~Il d' M V' v' u~ avv - - c - - m - ~ -O . o. . . . , p , CO O x b~ -~ .-~ ,~ rn -+ - -2 - -~ .-i ~ nCP -.-i- - - M - - - ti- - - N . .~L/'..-C C a "Cl MLn v' noN H O "7 m O Ol 1~ . . m . m. M V.' fM Z l~ H ~ lm 0 H i~ n~ m V' O~Il .~ O r-1 r ti n rl ~ ~fl M ~fl l0 a' rl o~ O a ti r r r^ r r r r t~ a r r m w r r r r r r x x aX 0 E Cl. Z3 ~ q F a' Q 0 O O O O O O a 000 ~ 0 0000000000 O U ~ U ~. -y ~ 0 CO G 41 e .-1 (N M - n 10 Ir m 01 O 41 E r-1 N M P~ lD r m 6~ o N C ~oo 00 0 0 0 0 0 0 0 0 00 ~ 0 ~ 0 0 o M Q p . 98 M ~ O ~ N ~D S O ~rl a- ~ N N OO f.-~ \ a N .-1 l0 p~ Ln N W M fL ~`p E\ `o ~ M P~ NMiRo+2N~C'Z1s.~NZ ~IDnMOM J x a wm t0 M S Ln f~ l0 aJ S ... .. . H 2 a ~n W~ r o r-~ N S u~ V' \ . . M r~ O f~ rl ~O O O[~ w'~ O r~ M N M I~ ~ v' W N m O ~ O Ln- S ~`y m < r--1 N~ - - - - - N~ O ,-1 M M ul N ~n S ~n <1' M < X (V N N ri ri N rl .~ N N ro x 0 O 3 r1 M M .-1 ~ 01o N N~~~~~~ O (~ rl ri e-i .~ rl oJ ID M o ... N r-~ ~ rl .-i ri rl rl f~ 3 r o\o N N N CO M~ ~0 61 ....... M N N M rl N ~-I rl .-1 rl rl .-a .-1 N'i r ... M N '-I ri rl W ro a. ~ tp U ..] d' N M ifl r N~ N M~1 U r-7 ~' u 1 ..'1 o d` fr ~ U7 N S c~ 'y 'O . . . . . . . . . . x -O .. . .. . .. . . ~ U\ r-i N N N ry N N rl N N U\ f-1 '-I C ~ M M M M M M M M M M 2: CP M M M M M M M M M M r{ ~ -i U r -i ~ x rl f~ N tt~ CO l0 C1 OJ rfl N S x~ N ~O .Il l0 .-i f~ r1 l0 6~ S ol . ..... . .. Q. 6\ 61 Ol o Ol OJ 61 Ol 6~ N N O. r~ CJ 61 OJ O] o CO [~ (W .-1 rl r-I N .-1 e-1 r-I r-1 ~-i r-I f-1 rl r-1 r-I rl r-1 N r-1 r1 .~ N N r-i ~ N N~~ r 00 ~ ro a ~"1 ~ ' r S~'1' S~ r o O' M 61 ~ ~ l~ N S M lO o r N v' Q` .1> rQ 3 S 6~ N ON,f~ , S N O r~ Q W ~T S l0 r-1 f~ 61 N r I~ S 71 lO l0 l0 n ~fl \O lD n LO n rn C M M 6l r-1 f~ M r .--I .-1 61 _X F+ .... \ U1 ~~ N~~ Q, ~. r Q. ~. \ LP x a' v' d' v' d` d' ct' d' ~' v' zjl U ~ lp O ~' T~--~ f~ 61 OJ M l0 .......... a1 M d' O r M(V ~) M M~ ~ 'x a' l' O' M V' ~' c1v' v' V' ~ W S O tfl M ~D O N~ ~ a . S~--1 O ul rtl ^ tV ~n M ~ ~\ M M M N M M O' ~T M M x ~ - - ,ti - - - - - .-, - - - - - - - - - - - cd ~ N (~ '~ ~- l0 c~ ~~ a a ~f) > M M o W OJ d' l-1 M ~ CO V' 61 o N a' e-~ 1f1 ~' o U\ 61 O N M M l0 ttl ul M O H S~' O~<t' N M ~O ~ O ~- m r r r r r r~ r o~ ~ x~ ~ r r ~o r r r r r r UO o ~ x x o k 0 o E E E p z a ~' x~' 0 000 x 0 x 0 x 0 x0x0^~ ?0L 0 ~ 0 z x~' 'x x x 0 0 0 0 0 0 Pl `x IL `x 0 0 0 x0 U U (5 Q) 0 ~ Q) ro rl N M Q' ul ~ r O J 6\ o - N(`") O' L n ~O r OJ 6~ o 0 0 0 o O o 0 0 0 0 o 0 0 o r+ E f--I ro c ~n L n ~n ~n ~n ~n ~n u~ ~n ~n ra r ^ r r r ~ r r- r~ Q N Q Ft P . 99 ~ LO a~+ O a a O ~0 6rn m v-1 ~n ' W N N W CO ~ r ti a 6~ `~ rl Ul r M(1~ r~D Ol LL rl o 2 0 o z~~ N N z aa o v,~ ,-a ~ 2 N~ M Z o X Fu M o O, l0 M Ol M t .f) '-1 M~Il Q' Ol l0 r u~ ~ W n O lD ~ N N~O M N .~ O~ W~\ lD lD tn lD N .ti Ol O M~t/ ~p O W O I~ ~Il M ul ,~ N N ul ~ O lO M W O N~' \D M l0 ,-1 yJ N N~ rl ri r 1 N N N N 4~ M M N d' O' N M M .Il M b X X u tp O 3 ^a p a Q' 3 u M r ~ m N r-1O r l vm w l m 0it . . . .. .. .. ow N N N N M M N Q' r Ol ul m m o . . . .. .. .. . u o\o ("1 ~T N cT Ln rl M N 61 M y~ ro w ~ b U i-7 N CO r~' N ti x o T N CO ~ rn U a r-1 N N x -o Q' ~ lO l0 Ol o OJ "'~ U~ f-1 N ti N~ N ti(V rl ti N U~ ~ N N~ ti N N rl rl -1 ,E b' M M M M th M f"1 (n M M E ZP tn M M t"1 M M M M M M ~ V fi ~ N O O N O O ~ x r ~fl O 10 l0 f~ vr ~fl 6~ ~ . p. r W 6~ r 6\ r N N N OJ ~ ~ a~i N 6\ CO 6~ 00 o O~ CO 6\ r < ~ ro a ~ y > a N 61 M ul m 10 d' l0 M Cl N ' NLn O Q' (D cl' \I ) u W r l0 rl N Ol ~ r ~D N r O U W O O l0 N 61 M 10 W M ID* "C7 tn Ln t0 ~~tl ~ ul u) ~t1 U ~ 10 lD ifl 10 ul ~o ul ul 1 ~ a~ 0 C -S4 O NE l061 l 1 ~O N tirWl ~ f~ v' cT r 6~ 10 \ U oW x N r-1 N ~ Ln N ~' W o V' N\ N o N . - O1 O Cl 01r O O 61 E Q' Q' V' d' Q' V' M Q' Q' ~' ~ Q' a' V' d' M M M Q' M a' ti 03 O a~ M ul M OJ ul v~-1 N v' ~ O . . . . . . . . . .~ m a .~ O M . 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M C') M M~ M vN V' M M O ,~ \ M M M N N N N M N( V bp - - - - - .-i - - ti .-i M b~ - - N ,--i ti - ,ti - - ti . .. -a a Q b U~ O' N Sl N M N ul ,-y N r ~ O] O N N W N r O NLn H V' N N r .-V O r~ N d' H ~ O 61 ~ N\fl lD N lD O N r l a ,~ r r ~o m r m~ r r r ,~ ~o ~ r 101~~o r ~o r p F X p r Q p. a C u Cy Z `.L `..4 `.4 `.L ,~ x ~Y L Y. ~`G 0 0000000000 t7 U ~ 0 u z 0000000000 U O^ N E ,~ N M V' ~t~ l0 r W 61 O N b r-+ N M V' ~fl l0 f~ O~ O~ O ~ Q < < f--1 -i o000CD 000~ o000000 0 ro "~ rnrnrnrnrnrnrnrnrnrn ~ ~ ~ ~ ~ ~ ~ N ~ ~~~~ p . 100 00 ro ar~o 0 ~ ~0 ~ 0 i~ Aro. -~ a -~ ro U~ ~ rn ~ co ~n .-i rn v~n U~ ~ N rn c~ ri r r rn .-i r+ U ~ G < ~ N O N O O- O O O~ ~ a Q o0000o zooo FC~ o00000000 0 x U ~ V7 71 l9 ~~("1 N~O l0 r ul V] ~ oJ N~ :n O(") v' l0 N f') ~ rn cC\ ooooCD oaooo rn aC,~ ooo(D ~CD oooo c: ~V < ~ O O O O O O 2 O O O N ~C c:) O O O O O O O O O O x x ~ m c N O~ x U n N l0 l0 N W l0 l0 co ~ l0 ~fl N .-1 f`"1 OJ l0 f~ O iD t"1 O f") O O r1 n+ O~ ~ O O O O O rl O oooooozoo ~ o000000000 x ~~ Z? 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Oa N N N N N N N 4/ N N H ~ N N N 47 N N N N N N G C G c G G G G G G C C C G G G G C G G O O O o o o o o o O O O O O O O O O O O Z Z Z Z Z Z Z Z Z Z ~ Z Z 2 Z Z Z Z Z 2 Z O ~, . O k a a, .-i u G G a E U U -{ U G~. G CO G G G w oG 0 0C 0 0`U tl `tl ` o 0 o w ro ro ro ro o ro ro ro ro ro 0. C7 E+ u Z Z ) Z Z ZE ~' r= - i' Z Z s-i V Z E E s, E E :-a F+ cn cn U) cn F+ cn cn C~ N ~ ~ N("1 V' ~fl lD r CO Ol o Q) rl N M~~f) l'J r W 6~ o /1 rl o 0 0 0 0 0 o O O r-~ r-1 o O 0 O o o O 0 O~--i ~- f0 if) ~rl ~n ~~ .n Ln ~f) ul ~n !p C r r r r~ f- -~ f~ ^ Q N ~ ~ C /t/ p . 110 W O F i-7 F .7 cc, E O\ M M- M r V, M M W N O \ U CP Q' r M N M N~-i U~ c cf) c u 1T M H N a a u ~ O a' N O 61 6~ N H r~ 5~ M v' M M M N O' M aa u~ M M ~~ 6~ CO ^ M l0 Ol O r in M~ V .f) M N~l v' M z z a ~ ~d u a 10 l0 N r~1l ~ N10 10 O Ol r-i N r~ rl N H N .-1 rl N x~ N N M E E M~ aN CV N N .fl ul O N N rl M N N ~ o ri O ~ N ro n. ,~ ~ io N N N N ~o .. N N ~o ~^ N N N . . . . .. . . 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M CV N N N N N N ~ U co r r m r s r o~ ~o r rn [q rn ID rn s r ~o r N io .......... .......... f'7 M M M M M M M M M N I.( ~ M M M M V' M("1 C) ("1 M ~ ri U H ro ~ y~ N M V' M,Il ~f o co s r ro ~~ OJ M o\O o V' M l0 cf' N c N E" ol lo lo lo lo lo lo lo ~ u F' ~ 1D lo r- ~o r 10 lo 10 v) ~o ~ ro a a -~ -rl ro 'O u c H ~ c7 b H ~Il CO ~f) ul r r~D N Q: \ ~fl a' ~ M M c,' N d' Q' F b' E v v 0 v' N 7 H ~f) T ~O 0 Q.' \ V' I^ U E r 6\ v' ~~ 1~ v' N ~I1 M l0 N M M O' Q' CT a a [T a a ID O O r~' r v' r M ul Q~ co ri N r v' v' ~O N N O~ ~ T~ ~t' rIl cr ~D vlD ~Ln Ln ~~ W ~O H ~D o~ l0 M N l0 tf1 E O E E O E ~ cz v v v v v v v v v o ~, v v v v v v v N N v o c G c c c c c c c c - c c c c G c c c c O O O O O O O O O O O O U o O O O O O O O OO . c C H z Z Z Z Z Z Z Z z z z z z z z z z z Z z .'. ~'O a v v v v v v v v v v a v v v v v v v v v v xHccccccccccHHccccccccG G 0 0 0 0 0 0 0 0 0 o x o 0 0 0 0 0 0 0 0 0 z z z z z z z z z z z z z z z z z z z z o~ . . ~ 0 o w m ro ro 0 .~. c~ y cn Z E~-~ v v v o. v v v U rl u U C 7 U C U ~-1 U c ro m m ro 0 w ro ro ro ro ro ro c uEi cn r ti Z ~j x cn ~ Z sa ~y ..Eii cn ~ z z 0 C13 Nr ti N o M V ~fl ~D r CO 61 O m I/'-'11 ~ E oOOOOOOOCD ~ .-~ F. .-1 N M V .Il l0 ^ m 61 O o00000000 ~ ~~~ p . 111 p . 112 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity S tudy in Ma l e Rats DuPont- 18317 Appendix G Individual Primary Humoral Immune Response Data - 112 /0 p . 113 Ammonium Perfluorooctanoate : 2 8- Day Im munotoxici ty Study in Mal e Rats DuPont-18317 Individual Primary Humoral Immune Response Dat a Animal Number SLOPE X Log~ Male, Group I - 0 mg/k g 101 102 103 104 105 106 107 108 109 110 -0 .8772 1648 10 .687 -0 .9165 812 9 .768 -0 .9442 5777 12 .496 -1 .0021 790 9 .626 -0 .9721 1355 10 .404 -1 .0214 2414 11 .237 -1 .0063 122 6 .928 -1 .0053 1586 10 .631 -1 .0377 394 8 .622 -1 .0022 1175 10 .198 Male, Group III - 0 .3 mg/k q 301 302 303 304 305 306 307 308 309 310 -1 .0141 1590 10 .635 -0 .9706 695 9 .441 -1 .0180 1237 10 .273 -0 .9477 1878 10 .875 -1 .0010 1854 10 .856 -0 .9884 1618 10 .660 -0 .9671 1605 10 .648 -0 .9972 1312 10 .358 -0 .9972 1095 10 .097 -0 .8896 914 9 .836 Male, Group V - 1 mg/k g 501 502 503 504 505 506 507 508 509 510 -0 .9758 1307 -0 .9001 2679 -0 .9786 1559 -0 .9388 111 -0 .9980 915 -1 .0325 1048 -0 .9969 3228 -0 .9742 791 -0_9732 227 -0 .9843 1409 10 .352 11 .387 10 .606 6 .794 9 .838 10 .033 11 .656 9 .628 7 .826 10 .460 -113~~ p . 114 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Primary Humoral I mm une Response Dat a Animal Number SLOPE X Log2 Male, Group VII - 10 mg/k g 701 702 703 704 705 706 707 708 709 710 -1 .0199 788 9 .622 -0 .9756 956 9 .901 -0 .9841 201 7 .650 -1 .0187 855 9 .740 -0 .9605 2116 11 .047 -0 .9560 7340 12 .842 -0 .9836 494 8 .948 -0 .9532 86 6 .426 -0 .9399 3660 11 .838 -0 .9843 2287 11 .159 Male, Group IX - 30 mg/kg 901 902 903 904 905 906 907 908 909 910 -1 .0014 1068 10 .061 -1 .0035 342 8 .418 -1 .0215 760 9 .570 -0 .9123 294 8 .200 -1 .0340 1788 10 .804 -0 .9742 2755 11 .428 -0 .9052 2078 11 .021 -0 .9060 457 8 .836 -0 .9504 1893 10 .886 -1 .0107 912 9 .83 3 Male, Group XI - 30/0 mg/kg (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 -1 .0040 543 9 .085 -0 .9456 3'70 8 .531 -0 .9969 392 8 .615 -0 .9935 460 8 .845 -0 .7713 1009 9 .979 -0 .9712 2410 11 .235 -0 .9856 314 8 .295 -0 .9682 2447 11 .257 -0 .9463 362 8 .500 -0 .9972 1844 10 .849 - 114 - t~`~ p . 115 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix H Individual Primary Humoral Immune Response Positive Control Data 115~~ p . 116 Ammonium Per fl uorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Primary Humoral Immune Response Positive Control Dat a Animal Numbei SLOPE X Log-, Male, Group CIX - Salin e C901 C902 C903 C904 C905 C906 C907 C908 C909 C910 -1 .0282 1215 10 .247 -1 .0269 1400 10 .451 -0 .9992 1249 10 .287 -1 .0116 977 9 .932 -0 .9398 820 9 .679 -0 .9457 109 6 .768 -0 .9708 554 9 .114 -0 .9619 1255 10 .293 -0 .9601 692 9 .435 -1 .0013 328 8 .358 Male, Group CXI - 20 mg/kg Cyclophosphamid e C1101 C1102 C1103 C1104 C1105 C1106 C1107 C1108 C1109 C1110 -0 .6474 4 -0 .9816 14 -0 .9912 18 -0 .9556 12 -0 .9067 23 -0 .8857 26 -1 .0035 55 -1 .0031 15 -0 .9962 14 -0 .9898 26 2 .000 3 .807 4 .170 3 .585 4 .524 4 .700 5 .781 3 .907 3 .807 4 .70 0 Male, Pooled Samples - 20 mg/kg Cyclophosphamide -0 .9859 29 4 .85 8 -0 .9832 12 3 .625 1~ p . 117 Ammonium Perfluorooctanoate : 2 8- Day Immun o t o xicity Stu dy in Mal e Rats DuP on t- 18317 Appendix I Individual Animal Final Body and Organ Weights - 117~/ p . 118 M ~ 1-- .' 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I 1 ~ 1 1 12-1 p . 121 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix J Individual Animal Pathology Data - 121 - / z~ p . 122 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 INDIVIDUAL ANIMAL PATHOLOGY DAT A 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, etc .) . - 122 - /23 p . 123 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : I Treatment : 0 mq/kq Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------101 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, srERNUM, POPLITEAL LYMPH NODE Histopatholoq y LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . minimal . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 102 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN 'PISSUE SECTION . 102 Continued on the next page . . . . -123- l2i~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 124 DuPont- 18317 Individual Animal Pathology Dat a Dose Group : I Treatment : 0 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 102 Continued from previous pag e Histopathology : No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 103 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE jOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . FATTY CHANGE, MEDIAN CLEFT, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 104 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 104 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E on the next page . . . . - 124 J~~ p . 125 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont- 18317 Individual Animal Patholoqy Dat a Dose Group : I Treatment : 0 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------- 109 Continued from previous pag e Histopatholoq y LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 105 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . minimal . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 106 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 106 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLII'EAL LYMPH NOD E on the next page . . . . - 12512& Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 126 DuPont- 18317 Individual Animal Pathology Dat a Dose Group : I Treatment : 0 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------106 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 107 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . BONE MARROW : FIBROSIS, FOCAL, minimal, (femur) . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM - 126- p . 127 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 individual Animal Pathology Dat a Dose Group : I Treatment : 0 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 108 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . BRAIN PIGMENT, FOCAL, minimal, hemosiderin (cerebellum) . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 109 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y 109 Continued LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . FATTY CHANGE, MEDIAN CLEFT, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . on the next page . . . . - 127 - /Z ~ Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats p . 128 DuPont- 18317 Individual Animal Pathology Dat a Dose Group : I Treatment : 0 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------109 Continued from previous pag e Histopathology : POPLITEAL LYMPH NOD E NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 110 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . minimal . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW - 128 - ~ ~~ p . 129 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : III Treatment : 0 .3 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------301 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with No Microscopic Abnormality Observed SPLEE N 302 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with 302 Continued on the next page . . . . - 129- p . 130 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : III Treatment : 0 .3 mg/kg Sex : Males --------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------- 302 Continued from previous pag e Histopathology : No Microscopic Abnormality Observed SPLEE N 303 Terminal. Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with No Microscopic Abnormality Observed SPLEE N 304 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 304 Continued on the next page . . . . -130- f31 p . 131 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : III Treatment : 0 .3 mg/kg Sex : Males -------------------------------------------------------------------------------- Ani :nal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------304 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observed SPLEE N 305 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . HEPATOCELLULAR, stippling . minimal, with No Microscopic Abnormality Observed SPLEE N 306 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 306 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE on the next page . . . . - 131 /~ ~ p . 132 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : III Treatment : 0 .3 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 306 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed SPLEE N 307 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observed SPLEE N 308 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 308 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE on the next page . . . . - 132 - 133 p . 133 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : III Treatment : 0 .3 mg/kg Sex : Males --------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 308 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observed SPLEE N 309 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholoq y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with No Microscopic Abnormality Observed SPLEE N 310 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 310 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E on the next page . . . . - 133 - /34~ p . 134 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : III Treatment : 0 .3 mg/kq Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 310 Continued from previous page Histopatholoqy LIVER INFLAMMATION, SUBACUTE/CHRONIC, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observed SPLEEN - I34 ~~~ p . 135 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : V Treatment : 1 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------501 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with No Microscopic Abnormality Observed SPLEE N 502 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, mild, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . - 135 ~ 3 16 p . 136 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : V Treatment : 1 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 503 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observed SPLEE N 504 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with 504 Continued on the next page . . . . - 136 - ~ 3-7 p . 137 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : V Treatment : 1 mg/kq Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------504 Continued from previous pag e Histopathology : No Microscopic Abnormality Observed SPLEE N 505 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observed SPLEE N 506 Terminal Sacrifice Killed on Day : 2 9 Animal is siqned off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE S06 Continued on the next page . . . . - 137 - J3)" p . 138 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : V Treatment : 1 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------506 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observed SPLEE N 507 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observed SPLEE N 508 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 508 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E on the next page . . . . -138- 2` ~ 1A F p . 139 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : V Treatment : 1 mg/kg Sex : Males --------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings ---------------------------------------------- ---------------------------------508 Continued from previous page Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal, with No Microscopic Abnormality Observed SPLEE N 509 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observed SPLEE N 510 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : 510 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E on the next page . . . . -139A(() p . 140 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : V Treatment : 1 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------510 Continued from previous pag e Histopathology LIVER IN'r'LAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observed SPLEEN -140W p . 141 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : VII Treatment : 10 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------701 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEE N 702 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with 702 Continued on the next page . . . . - 141 - / A(-. 2- p . 142 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : VII Treatment : 10 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------702 Continued from previous pag e Histopathology : No Microscopic Abnormality Observed SPLEE N 703 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERPLASIA, BILE DUCT, FOCAL, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEE N 704 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 704 Continued on the next page . . . . - 142 ~ ~~, ; Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats p . 143 DuPont- 18317 Individual Animal Pathology Dat a Dose Group : VII Treatment : 10 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------704 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEE N 705 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Patholog y LIVER DISCOLORATION, TAN, LEFT, LINEAR <3MM No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEEN - 143 A~' p . 144 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : VII Treatment : 10 mg/kq Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 706 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEE N 707 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y 707 Continued LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . NECROSIS, FOCAL, minimal, coagulative . MINERALIZATION, BILE DUCT, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . on the next page . . . . moderate, with - 144 - ~~S p . 145 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : VII Treatment : 10 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------707 Continued from previous pag e Histopathology : No Microscopic Abnormality Observed SPLEE N 708 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEE N 709 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 709 Continued on the next page . . . . -145~ItLCI~J p . 146 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : VII Treatment : 10 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------709 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observed SPLEE N 710 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPA'I'OCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with -146/~'l p . 147 Ammonium Per fluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : IX Treatment : 30 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------901 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . MESENTERIC LYMPH NODE : DEPLETION/ATROPHY, LYMPHOID, minimal, cortex, and follicles) . (inner cortex, outer CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 902 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . 902 Continued on the next page . . . . -147/ q-c? p . 148 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : IX Treatment : 30 mg/kg Sex : Males -------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------902 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 903 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E - 148 - C~j p . 149 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : IX Treatment : 30 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------904 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, minimal, coagulative, subcapsular . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 905 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Patholog y LIVER : DISCOLORATION, TAN, MOTTLED . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 905 Continued on the next page . . . . -149/<0 p . 150 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : IX Treatment : 30 mg/kg Sex : Males -------------------------------------------------------------------------- A-n--i-m-a-l---R-e-f--M-i--c-r-o-s-c-o--p-i-c--&---M-a-c-r-o--s-c-o-p-i-c---F-in-d-i--n-g-s---------------------------- 905 Continued from previous pag e Histopathology LIVER NECROSIS, FOCAL, minimal, coagulative, subcapsular . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 906 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E - 150/IS/ p . 151 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : IX Treatment : 30 mg/kg Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 907 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Patholog y LIVER LARGE DISCOLORATION, PALE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NOD E THYMUS, FEMUR/KNEE Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, minimal, coagulative, subcapsular . INFLAMMATION, SUBACUTE/CHRONIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 908 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . 908 Continued on the next page . . . . -151- 15~ p . 152 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont- 18317 Individual Animal Pathology Dat a D-o-s-e--G-r-o-u-p--:--I--X--T--r-e-a--t-m-e--n-t--:---3-0---m-g-/-k-a---S-e-x--:--M-a-l-e-s------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------908 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . HEPATOCELLULAR, stippling . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 909 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . NECROSIS, FOCAL, minimal, coagulative . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E - 152- I5 ~ p . 153 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : IX Treatment : 30 mg/kg Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------910 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E - 153 - I5~'- p . 154 Ammonium Perfl uorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kg (Recovery) Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings ------------------------------------------------------------- 1101 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Patholog y LIVER : DISCOLORATION, TAN, MOTTLED, LEFT . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, 'r'EMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal . BONE MARROW : FIBROSIS, FOCAL, minimal, (femur) . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 1102 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1102 Continued on the next page . . . . - 154 ~ 5~5 Ammonium Perfl uorooctanoate : 28-Day Immunotoxici ty Study in Male Rats p . 155 DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kg (Recovery) Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1102 Continued from previous paq e Histopathology LIVER MINERALIZATION, BILE DUCT, moderate, with fibrosis . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . HEMATOPOIESIS, EXTRAMEDULLARY, minimal . SPLEEN HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 1103 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stipplinq . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E - 155 fL. .~~j p . 156 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kq (Recovery) Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1104 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . with No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 1105 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y 1105 LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic Continued on the next page . . . . HEPATOCELLULAR, stippling . moderate, with -15615q p . 157 Ammonium Perfl uorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kg (Recovery) Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------- 1105 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . SPLEEN HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 1106 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . POPLITEAL LYMPH NODE : NOT PRESENT IN TISSUE SECTION . with No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW - 157 - /S*5 p . 158 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kg (Recovery) Sex : Males -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1107 Terminal Sacrific e Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 1108 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y 1108 LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . Continued on the next page . . . . moderate, with - 158 - l5? p . 159 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kg (Recovery) Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1108 Continued from previous pag e Histopathology No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 1109 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . FIBROSIS, FOCAL, minimal, subcapsular . SPLEEN HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E 1110 Terminal Sacrifice Killed on Day : 2 9 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE 1110 Continued on the next page . . . . - 159160 p . 160 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Individual Animal Pathology Dat a Dose Group : XI Treatment : 30/0 mg/kg (Recovery) Sex : Males -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1110 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELL"JLAR, moderate, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, minimal, coagulative . SPLEEN : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, POPLITEAL LYMPH NOD E - 160 - ~l~l p . 161 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 Appendix K Individual Total Cell Counts -161l(lJ~. p . 162 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 INDIVIDUAL TOTAL CELL COUNT S EXPLANATORY NOTES NOTES : Organ Weight as Percent of Body Weight Organ Weight (g ) Final Body Weight (q) 100 Number of Total Number of Organ Cel l Organ Weight (g) Organ Cells = Suspension Cells in Half (x 103) Half Organ Weight (q) rli) Volume Orga n (x 10' cells/mL) 100 - 162 - ~~~ p . 163 00 ~v ~ Sa ~ a v N a~ N r rl r M N~D l0 ~ d' M O M N N N O~ O Z a~ O ~ CO N N l0 N r-I rl ~ O v' .n V' l0 N CO 01 .--~ - r~ . .. . . . . .. .. . ~C wO U X ~ M r r N M~fl M ~f) r 61 M~ 61 M V' 7 V' N~' O O E- w a w rp O o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C~'~ ~ ul 6~ M O Q' N O rl 61 O Q' n O~ N~ N M N S~ ~ U rl r M N N -O un (V n m l0 l0 ~ l0 r ~fl N ~fl lO 1l ~ l~ r N~ ifl N N M N d' N l0 N r l0 N M V' M M M a 'i p N U x O Cf) O U ~ ~ V C C a ~7 CO O O N~ M N Ln L/l O oJ V' O O O O CO n .......... ...... ... 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DUPONT/HASKELL LABORATORY DUPONT STUDY NUMBER : 18317 WORK REQUEST NUMBER : 16160 SERVICE CODE: 1545 AMMONIUM PERFLUOROOCTANOATE: 28-DAY IMMUNOTOXiCITY STUDY IN MALE RAT S ELECTRON MICROSCOPY PATHOLOGY REPORT EPL PROJECT NO. 129-077 Submitted to : DuPont/Haskell Laboratory for Health and Environmental Science Stine Haskell Research Center 1090 Elkton Road Newark, DE 1971 1 Submitted by : Experimental Pathology Laboratories, Inc, P .O . Box 1276 6 Research Triangle Park, NC 27709 October 25, 2006 - 170 - 1 // p . 171 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 ORL Experimental Pathology Laboratories, Inc . TABLE OF CONTENTS Page PATHOLOGY SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ELECTROMICROGRAPHS /72 p . 172 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 W E P l@ Experimental Pathology Laboratories, Inc . DUPONTIHASKELL LABORATOR Y DUPONT STUDY NUMBER : 18317 WORK REQUEST NUMBER : 16160 SERVICE CODE : 154 5 EPL PROJECT NO. : 129-077 AMMONIUM PERFLUOROOCTANOATE : 28-DAY IMMUNOTOXICITY STUDY IN MALE RATS ELECTRON MICROSCOPY DuPont-18317 PATHOLOGY SUMMAR Y The in-life phase of this study was conducted at Haskell Laboratory for Health and Environmental Scien ces, 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 rats for at least 28 days . The table below summarizes the experimental design : Experimental Design Dose Solution Group Number/Group Daily Dosage Concentration m k a m mL 1 10 0 (Control) 0 III 10 0.3 0 .03 V 10 1 0 .1 VII 10 10 1 Ix 10 30 3 X! 10 30 Recove 3 8 Weight of test substanceJkg of animal body weight . b Solutions will be adjusted for purity (20%) `The recove ry group (XI) will be dosed with 30 mg/kg of test substance through test day 22 . Following injection of SRBC on test day 23, group XI will be dosed with NANOpure water, at a volume of 10 mUkg of body weight, until sacrifice . Electron microscopic evaluation of samples of liver from designated animals was added to clarify light microscopic histopathological findings in the liver . Samples of liver from two male 1 172- 73 p . 173 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 W E, PU Experimental Pathology Laboratories, Inc. DuPont-18317 rats in Group I (Control) and two male rats in Group IX (30 mg/kg) that were fixed in formaiin were submitted for transmission electron microscopy . The samples that were processed and evaluated are listed in the following table : TEM TEM Negative Num ber Tissue Animal ID Grou Number (evaluated) I G06-399 Liver 105 Control 06-1894 to 06-1896 1 G06-400 Liver 106 (Control) 06-1897 to 06-1899 Ix G06-401 Liver 905 (30 mg/kg) 06-1900 to 06-1902 IX G06 402 Liver 906 (30 mg/kg) 06-1903 to 06-1905 Samples, cut into small cubes, were preserved in formalin and shipped to Experimental Pathology Laboratories, Inc (EPL), Research Triangle Park, NC . The samples were transferred to the Laboratory for Advanced Electron and Light Optical Methods (LAELOM) at the College of Veterinary Medicine, North Caro lina State University, Raleigh, NC for further processing and examination by transmission elec tron microscopy . The samples were washed in buffer, post- fixed in 1% osmium tetroxide in the phosphate buffer, dehydrated in an ethanolic series culminating in acetone, and infiltrated with Spurr epoxide resin . The resulting blocks were t rimmed and semithin sections (approximately 0 .5 m thick) were cut, mounted on glass slides, and stained with 1% toluidine blue 0 in 1% sodium borate p rior to being examined with a light microscope . The slides of semithin sections were sent to Experimental Pathology Laborato ries for evaluation by the Pathologist, Dr. Henry Wall . When the slides were returned to the LAELOM, areas of interest for uftrathin sectioning were trimmed in the corresponding tissue blocks . Ultrathin (80-90 nm thick) sections were cut from the selected trimmed blocks and pla ced on 200 mesh copper grids before being stained with uranyt acetate and lead citrate . For each sample, two survey photographs ( final print magn ification 5,600x) were taken . One higher magnifi cation (fi nal print magn ifi cation 22,400x) was taken of each sample to show more cellular detail . 2 -173- 17~ p . 174 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Rats DuPont-18317 W E PU Experimental Pathology Laboratories, Inc . RESULTS Dupont-18317 TEM #G06-399 ( Animal 105, Control, Liver, TEM Neg # 06-1894 to 06-1896 ) Two low magnification images (06-1894 and 06-1895 : 5,60OX) depict portions of multiple hepatocytes . A few clear to moderately electron-lucent smooth-contoured lipid droplets are in the cytoplasm of most hepatocytes . The cytoplasm of all hepatocytes contain numerous wellformed mitochondria as the predominant cytoplasmic organelles . The higher magnification image (06-1896 ; 22,400X) shows greater detail of the hepatocytic mitochondria, lipid droplets, cistemae of rough endoplasmic reticulum, a few electron-dense membrane-bound peroxisomes, and electron dense clusters of cytoplasmic glycogen . No cell injury is apparent . TEM #G06-400 (Animal 106, Control, Liver, TEM Neg # 06-1897 to 06-1899 ) Both low magnification images (06-1897 and 06-1898 ; 5,600X) show multiple hepatocytes that have numerous mitochondria as their predominant cytoplasmic organelles . Aggregates of linearly arrayed rough endoplasmic reticulum are scattered in the cytoplasm of hepatocytes . The high magnification image (06-1899 ; 22,400X) shows greater detail of the several mitochondria, rough endoplasmic reticulum, and a few slightly electron-dens e lysosomes . A few of the smaller diameter electron-dense bodies may be peroxisomes, however, their structure is not optically resolved to the extent that their identity as peroxisomes can be confirmed . Irregular profiles of translucent smooth endoplasmic reticulum are interspersed between other organelles in the cytoplasm . A portion of a well formed nucleus is at the lower right of the image . No cell injury is present. TEM #G06 -401 (Animal 905, Group iXl30mgtkg, Liver, TEM Neg # 06-1900 to 06-1902) Both low magnification images (06-1900 and 06-1901 ; 5,600X) depict multiple hepatocytes with abundant densely arranged cytoplasmic mitochondria . Peroxisomes which appear as uniformly electron-dense bodies are prominent among the mitochondria . A few small clear smooth-contoured vacuoles are in most cells . These vacuoles are considered to be lipid vacuoles that lost their content during tissue processing . A few lysosomes are in some hepatocytes . Most lysosomes contain either lipid droplets or electron-dense residual bodies . In one image (06-1901) a cross section of a blood vessel contains electron-dense erythrocytes and shows the nucleus of an endothelial cell . The higher magnification image (06-1902 ; 3 - 174 - 1 75 p . 175 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 ,~,,,,r E P U Experimental Pathology Laboratories, Inc . DuPont-18317 22,400X) shows more detail of clear lipid vacuoles, numerous mitochondria, a lipid-laden lysosome and a few electron-dense peroxisomes . The peroxisomes are along the right border of the image and in the upper left quadrant of the image . TEM #G06-402 (Animal 906, Group IXl34mg/kg, Liver, TEM Neg # 06-1903 to 06-1905) The low magnification images (06-1903 and 06-1904 ; 5,600X) show numerous mitochondria as the predominant organelles in the cytoplasm of adjacent hepatocytes . Several uniformly electron-dense peroxisomes are scattered in the cells but are somewhat difficult to discern in the low magnification images . The high magnification image (06-1905 ; 22,400X) shows the detail of several peroxisomes in hepatocytic cytoplasm at the periphery of the endothelium of a blood vessel along the left border of the image. The peroxisomes are generally smaller in diameter than the more numerous mitochondria in the image . The peroxisomes are lined by a single membrane and have relatively uniform electron-dense matrix in this high magnification image . CONCLUSIONS Compared to hepatocytes from the two control rats, the hepatocytes from the two rats that received ammonium perfluorooctanoate have more abundant peroxisomes in their cytoplasm . HGW/dc ~ ,~~~ HENRY .WALL, DVM, PhD Diplomate, ACVP Veterinary Pathologist 2.Sk ~c~w- Zcc Date 4 -175- f /~ p . 176 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats DuPont-18317 E P1 Experimental Pathology Laboratories, Inc . QUALITY ASSURANCE FINAL CERTIFICATION Study Title : Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rats Client Study: DuPont-18317; Service Code 1545; EPL Project Coordinator : Dr. Henry Wall Work Request 16160 EPL Project Number 129-077 EPL Pathologist : Dr. Henry Wal l 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 Inspection Reporting EPL Project Sheets May 30, 2006 May 30, 2006 Data Review June 14, 2006 June 14, 2006 Draft Pathology Report June 27, 2006 June 27, 2006 Final Pathology Report October 25, 2006 October 25, 2006 Date repo rted to Study Director/Management : October 25, 2006 Date of last qua rterly facility inspection : October 2006 EP Quality Assurance Unit) Date 5 Form No . 6-2 (October 23, 2006) - 176 - ~~~