Document 3NNEbX0rMgYpnd6nQpwDvzN1n

P. 1 DuPont-18318 TRADE SECRE T Study Title Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice 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, 200 7 PERFORMING LABORATORY : E .I . du Pont de Nemours and Company HaskellsM Laborato ry for Health and Environmental Sciences P.O . Box 5 0 Newark, Delaware 19714 U .S .A . Exygen Researc h 3058 Research Drive QA , State College, Pennsylvania C 16801 ,y~q//yS U .S .A . NQ Experimental Pathology Laboratories, Inc . 615 Davis Drive, Suite 500 Durham, No rt h Carolina 27713 U .S .A . Laboratory for Advanced Electron and Light Optical Methods College of Veterina ry Medicine North Carolina State University 4700 Hillsborough Street Raleigh, No rth Carolina 27606 U .S .A . LABORATORY PROJECT ID : DuPont- 18318 WORK REQUEST NUMBER : 16160 SERVICE CODE NUMBER : 154 6 SPONSOR : E .I . du Pont de Nemours and Company Wilmington, Delaware 1989 8 U .S.A. Page 1 of 28 1 /7? p. 2 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 PAGE RESERVE D V 4t 00 -2- (7 ~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice QUALITY ASSURANCE STATEMEN T Work Request Number : 16160 Study Code Number : 1546 p. 3 DuPont- 18318 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 17, 2005 November 18, 2005 November 18, 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* July 24, 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 18, 21-24, 2006 August 25, 2006 October 19, 2006 November 28-29, 2006 November 29, 2006 January 8, 2007 * EPL QA Date s Reported by: C- Q r'Fe fa -2Q-O? Joseph C. Hamill Date Quality Assurance Auditor -4lU o Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p. 4 DuPont- 18318 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 t A:,6 o20 7 Date Applicant/ Sponsor; DuPont Representative Date -3t6l p. 5 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study . Analytical Evaluation by : Z Amanda Shen, Ph.D. Research Chemist Date Clinical Pathology Evaluation by: 49L-a -0100 6 Nancy E, Everds, D.V .M.. Dip mate A .C.V.P. Dat e Principal Research Clinical Pathologist and Manager Anatomi c Pathology Evaluation by : (~~P~2~ Cme ykes, V .D., Diplomate A.C.V.P., A.C.L .A.M., A .B .T. Veterinary Pathologist Q t R Fc b - lPTd7 Date Anatomic Pathology: =,~ Evaluation Peer Revie w by Steven R. Frame, D .V .M.. Ph .D., Diplomate A.C.V.P . Date ~ Research Fellow and Manager Reviewed and Approved by: Scott E. Loveless. Ph.D. Research Manager and Director Flu - Z4 D-4-Date Issued by Study Director : .D" enise Hobanl, B .Au . ML ~ T (-ASCP)QD IQat-e260 7 Staff Medical Technologist and Supervisor -5to ? p. 6 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 TABLE OF CONTENTS Page PAGE RESERVED . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . .2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . .3 QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . .3 CERTIFICATION . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. .. . . . .3 LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .3 LIST OF FIGURES . . . . . . . . . . . . . . . . .. . . .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . .. .. .. . . . .3 LIST OF APPENDICES . . . . . . . . .. . . . . .. .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . .3 STUDY INFORMATION .. . . . . . . . . .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . .3 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . .. .. . . .. .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . .3 INTRODUCTION . . . . . . . . . . . . . .. .. .. .. .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .3 STUDY DESIGN . . . . . . . . . . . . . .. .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. . . . . . . . . . . 3 A . Design Concentrations . . . . . . . . . . . . . . .. .. .. .... .. . . . . . . . . . . . . . . . .. .. ... .. . . . . . . . . . . . . . . . . . . . . . . . .. .. ...... .. . . .. . . . . . . . . . . . . . . ..3 B. Study Overview . . . . . . . . . . . . . . . . . . . . . . . . .. ......... . .. . . . . . . . . . . . .. ... . .. . .. .. . . . . . . . . . . . . . . . . . . . . . .... ...... .. . . . . . . . . . . . . . . . . . . ..3 MATERIALS AND METHODS ......................................................... ... .. ............................ ........3 A . Test Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 B . Test Substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 C . Test System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 D . Animal Husbandry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 E . Pretest Period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 F . Assignment to Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 G . Dose Formulation Preparation and Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 H . Dose Formulation Sampling and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 1 . Body Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 J . Food Consumption and Food Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 K . Clinical Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 L . Clinical Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 M . Humoral Immune Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 N . Anatomic Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 0 . Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 P . Electron Microscopy Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Q . Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 -6- ~~ 3 p. 7 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuP ont-18318 TABLE OF CONTENTS Page RESULTS AND DISCUSSION ...... ... .. .. ...................................................................................... .3 Analytical Evaluation .. ....... .. .... ...................................... .... .. .... ............................ .......... .. .. ..... .... ..3 A . Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 B . Recovery Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 C . Concentration Verification, Uniformity of Mixing, and 5-Hour Room Temperature Stability Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ---- . -- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 D . Concentration Verification and Uniformity of Mixing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 E . Analytical Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .- .-- .-- .-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 In-Life Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 A . Mean Body Weights and Body Weight Gains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 B . Food Consumption and Food Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 C . Clinical Observations and Mo rtality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Clinical Pathology Evaluation . . . . . . . . . . . .. .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 A . Hematology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 B . Clinical Chemist ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 C . Clinical Pathology Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Immunotoxicity . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .3 A . Humoral Immune Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -- . -- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Anatomic Pathology Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 A . Cause of Death . . . . . . . . . . . . . . . -- . --- . -- . -- . . . . . . . . . . . . . . . . . . . .--- . -- . -- . . . . . . . . . . . . . . . . .----- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 B . Final Body and Organ Weight Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 C . Gross Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 D . Microscopic Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 E . Untrastructural Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 F . Anatomic Pathology Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 A . Spleen Cell Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 B . Thymus Cell Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..3 RECORDS AND SAMPLE STORAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . ..3 TABLES . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. .. . . . .3 FIGURES . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . .3 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .3 -7- ~ ~-`~ p. 8 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 LIST OF TABLES Page Table I Recovery of APFO Added to Dosing Vehicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Table 2 Concentration Ve ri fication, Uniformity of Mixing, and 5-Hour Room Temperature Stability of APFO in Dosing Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 3 Concentration Veri fi cation and Uniformity of Mixing of APFO in Dosing Solutions . . . . . . . . . . . . . . 3 Table 4 Mean Body Weights of Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Table 5 Mean Body Weight Gains of Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Table 6 Mean Daily Food Consumption by Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 7 Mean Daily Food Efficiency of Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 8 Summary of Daily Animal Health Obse rv ations in Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 9 Summary of Detailed Clinical Obse rvations in Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 10 Summary of Hematology Values for Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 11 Summary of Clinical Chemistry Values for Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 12 Summary of Pri ma ry Humoral Immune Response to SRBC for Male Mice Dosed wit h APFO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Table 13 Summary of P rimary Humoral Immune Response to SRBC for Male Mice Dosed With Positive Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Table 14 Mean Final Body and Organ Weights from Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 15 Incidence of Gross Observ ations in Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Table 16 Incidence and Lesion Grades of Microscopic Obse rv ations in Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Table 17 Summary of Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 LIST OF FIGURES Page Figure 1 Representative Analytical Calibration Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Figure 2 Representative LC/MS/MS Chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Figure 3 Mean Body Weights of Male Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 -8[~~ p. 9 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice D uP ont-18318 LIST OF APPENDICES Page Appendix A Certificate of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix B Individual Body Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix C Individual Final Body and Liver Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix D Individual Food Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Appendix E Individual Daily Animal Health Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix F Individual Detailed Clinical Observations and Mortality Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix G Individual Animal Clinical Pathology Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix H Individual Primary Humoral Immune Response Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Appendix I Individual Primary Humoral Immune Response Positive Control Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Appendix J Individual Animal Final Body and Organ Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix K Individual Animal Pathology Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix L Individual Total Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Appendix M Electron Microscopy Report from Experimental Pathology Laboratories, Inc . . . . . . . . . . . . . . . . . . . . . . . . . .3 -9` L76 P . 10 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 STUDY INFORMATIO N Substance Tested : Ammonium Perfluorooctanoate [APFO (linear)] 3825-26-1 (CAS Number ) Haskell Number : 2730 8 Composition : Ammonium Perfluoroctanoate Solution 19 .5% in water Purity : 19 .5% Physical Characteristics : White to slightly opaque liqui d Stability : The test substance was stable under the conditions of the study based on analytical results . Study Initiated/Completed : October 14, 2005 /(see report cover page) Experimental Start/Termination : October 19, 2006 / February 1, 200 7 -10- ~ Z) ~ P . 11 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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 20 male mice each were administered the test substance at daily levels of 0, 0 .3, 1, 10, 30, and 30/0 mg/kg . The group designated 30/0 mg/kg day was included to assess potential reversibility/recovery and was therefore administered the test substance for 23 consecutive days followed by 6 consecutive days of vehicle (water) administration . Body weights, food consumption measurements, and clinical observations were recorded during the in-life period . Prior to sacrifice, the immune system was stimulated by injecting sheep red blood cells (SRBC) on test day 24 and blood samples were collected from each mouse on test day 29 . The serum samples were assayed for their concentration of SRBCspecific IgM antibody to provide a quantitative assessment of humoral immune response . Serum from animals similarly challenged with cyclophosphamide, a positive control immunosuppressive agent, was analyzed concurrently to provide confirmation that the assay performance was acceptable for detection of immunosuppression . Clinical pathology data were collected at test day 29 and assessed effects on hematology and clinical chemistry . At sacrifice, each animal was examined grossly and selected organs were weighed (brain, spleen, and thymus) ; selected tissues (as outlined in the methods section) were retained and examined histologically . Thymus and spleen cells were manually counted from single-cell suspensions prepared from the collected tissue . Samples 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 were 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 . Mice dosed with >1 mg/kg had decreased serum HDL cholesterol, increased serum albumin, and variable changes in serum globulin . Mice dosed with >10 mg/kg had increased neutrophils and monocytes, decreased eosinophils, icteric serum, decreased serum total cholesterol, non-HDL cholesterol, and triglycerides, and increased serum corticosterone . Mice dosed with 30 mg/kg also had decreased red cell mass parameters (red blood cell count, hemoglobin, and hematocrit), increased reticulocytes, and decreased lymphocytes . The only parameter with complete recovery in the 30/0 mg/kg group was non-HDL cholesterol . Partial recovery was observed for icteric serum, total and HDL cholesterol, triglycerides, and serum corticosterone . -11- 1~ p . 12 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Test substance-related organ weight effects were observed in the liver, spleen, and thymus . Mean liver weight parameters were increased at >_0 .3 mg/kg, mean spleen weight parameters were decreased at >1 mg/kg, and mean thymus weight parameters were decreased at >10 mg/kg . Test substance-related gross observations were observed at doses >10 mg/kg and included large and discolored livers, small spleens, and small thymuses . Microscopic examination of the liver demonstrated mild hepatocellular hypertrophy at 0.3 mg/kg ; moderate to severe hepatocellular hypertrophy with secondary individual cell necrosis and focal necrosis at doses >_1 mg/kg ; and increased hepatocellular mitotic figures, hepatocellular fatty change, and bile duct hyperplasia at doses >10 mg/kg . Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased granulocytic hematopoiesis in the bone marrow (>10 mg/kg) and increased erythrocytic hematopoiesis in the bone marrow and spleen (30/0 mg/kg) . Test substance-related lymphoid depletion/atrophy was present in the thymus (>I 0 mg/kg) and spleen (30 mg/kg) of less than half of the mice at the respective dose levels . Mesenteric and popliteal lymph nodes had no test substance-related effects. There was test substance-related evidence of immunosuppression in mice at 10, 30 and 30/0 mg/kg . The anti-SRBC titers for these groups were reduced 20, 28 and 30% when compared to the control group mean . There was no difference in mean primary Immoral immune response between the 30 and 30/0 mg/kg, indicating that the shortened dosing period did not have an impact on this endpoint . No significant changes in total thymus or spleen cell number were noted in animals dosed with 0.3 or 1 mg/kg . Significant decreases were noted in animals dosed with >10 mg/kg. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male mice was 0 .3 mg/kg and for immunosuppression was 1 mg/kg . -12I~,11 p . 13 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 INTRODUCTION The primary objective of this study was to evaluate the potential of ammonium perfluorooctanoate (APFO (linear)) to suppress the primary humoral immune response to sheep red blood cells (SRBC) when administered by oral gavage to male mice for up to 28 consecutive days . Additional endpoints of toxicity were also evaluated . The oral route of administration was selected because it is a potential route of human exposure . Ammonium perfluorooctanoate (APFO ; FC-143, C8 ; C7F15COO-NH4+ ; CAS Registry number 3825-26-1) is a surfactant used as a processing aid in the production of fluoropolymers . Perfluorooctanoate (PFOA ; C7F1SCOO-), 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% APFO in chow (approximatel y 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 the 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 Groupa (mg/kg)b (mg/mL) c I 20 0 (Control) 0 III 20 0.3 0 .03 V 20 1 0 .1 VII 20 10 1 IX 20 30 3 XI 20 30/0d 3 a Mice were divided into sub groups A and B (10/sub group) because of limited sample volume . b Weight of test substance/kg or animal body weight . c Solutions were adjusted for purity (19 .5%) . d This group (XI) was dosed with 30 mg/kg of test substance through test day 23 . Following injection of SRBC on test day 24, group XI was dosed with NANOpure water, at a volume of 10 mL/kg of body weight, until sacrifice . -13~ i0 p . 14 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont- 18318 B. Study Overvie w Study Parameters Frequency Body Weight Day 0, 3 (2 for subgroup B), and daily thereafter Food Consumption Weekl y Daily Animal Health Observation Twice daily General Clinical Observationa Day 0 and weekly thereafter Detailed Clinical Observation At each weighing SRBC Injection Prior to dosing (test day 24) Clinical Pathology Evaluation Test day 2 9 Serum Collection for Antibody Determination At sacrifice (test day 29) Anatomic Pathology Evaluation Test day 2 9 a A check for acute signs of toxicity was conducted approximately 2 hours post-dosing . MATERIALS AND METHOD S 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 . In addition, no evidence of instability, such as a change in color or physical state, was observed . C. Test Syste m On October 6, 2005, 132 male Crl :CD(ICR) mice, with an assigned birth date of August 22, 2005, were received from Charles River Laboratories, Raleigh, North Carolina . The Crl :CD(ICR) mouse was selected based on consistently acceptable health status and on extensive experience with this strain at Haskell Laboratory . By utilizing the Crl :CD(ICR) mouse, immunotoxicity studies can be conducted in the same strain that is used for other toxicology studies . -14~~ p . 15 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 D . Animal Husbandr y 1 . Housing All animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards . 2. Environmental Condition s 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 mice were provided tap water ad libitum . All mice were fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum . 4. Animal Health and Environmental Monitoring 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 Perio d Upon arrival at Haskell Laboratory, all mice were housed in quarantine . The mice were : quarantined for 6 days . identified temporarily by cage identification . weighed at least 3 times during quarantine . 15/-.p p . 16 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont- 18318 observed with respect to weight gain and any gross signs of disease or injury . The mice were released from quarantine by the laboratory animal veterinarian or designee on the bases of acceptable body weights and clinical signs of all mice . F. Assignment to Group s Mice, selected on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury, were distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there were no statistically significant differences among group body weight means . The weight variation of selected mice did not exceed 20% of the mean weight . At grouping, each mouse was assigned an animal number/cage identification number . Dose groups were subdivided into groups A and B, with 10 animals per group . The animal number/cage identification number were tattooed on the tail of each mouse and included on the cage label _ At study start (test day 0) the mice were approximately 8 weeks of age . G. Dose Formulation Preparation and Administratio n The dosing solutions were prepared in NANOpure water . The formulations were adjusted based on the percentage of APFO in the bulk test substance to achieve the desired concentrations . Dosing formulations were prepared on a daily basis . To accommodate the schedule of the laboratory, the initiation of dosing for group A mice was started one day prior to group B mice . Animals were dosed daily at approximately the same time ( 2 hours) by intragastric intubation at a dose volume of 10 mL/kg body weight for at least 28 consecutive days ; individual dose volumes were calculated based on the most recently collected body weight data . Control mice were dosed with NANOpure water at a volume of 10 mL/kg of body weight . The 30/0 mg/kg group (XI) was dosed with 30 mL/kg of test substance through test day 23 . Following injection of SRBC on test day 24, group XI was dosed with NANOpure water at a volume of 10 mL/kg of body weight until sacrifice . In light of marked body weight losses in some of the mice, a decision was made to suspend dosing for a few days with the intention of resuming dosing if the animals were sufficiently recovered . The table below lists the specific mice for which dosing was suspended as well as th e test days on which the animals were not dosed . This protocol deviation did not adversely impact the study for several reasons : first, the suspension of dosing was transient and affected some but not all of the animals dosed at 30 mg/kg/day . Second, the data collected from animals dosed on a daily basis combined with the data from the animals listed below are considered to provide sufficient data to meet the objectives of the current study . Third, if suspension of dosing had not been implemented, unscheduled mortalities may have precluded the collection of immune system data in these animals and, thus, reduced the amount of data available to assess potential immunotoxicity . / 9 3 -16- p . 17 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Animal Test Day s Group Number Not Dosed IX (30 mg/kg) 901 9- 11 XI (30/0 mg/kg) 1108 9- 11 XI (30/0 mg/kg) 1109 9- 11 XI (30/0 mg/kg) 1117 8- 10 XI (30/0 mg/kg) 1120 8- 10 H . Dose Formulation Sampling and Analysi s 1 . Recovery Sample Analysis 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 I 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 NANOpure water. These spiked recovery samples were then processed and analyzed in the same manner as the dosing samples at similar concentrations . 2. Dosing Solution 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 further diluted to a final expected concentration of 0 .03 ppm with NANOpure water for analysis . The 0 mg/mL sample followed the 0 .03 mg/mL sample dilutions . Before the final dilution, the internal standard (1, 2-di-13C PFOA) was added to each sample to give an equivalent final concentration of the internal 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) I 100 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) Acetonitrile 17~~r~ p . 18 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Gradient : Time (min) % Acetonitril e 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 k V Cone Voltage : 15 V Source Temperature : 120C Desolvation Temperature : 350C Scan Function : PFOA : 413 m/z (parent) to 369 m/z (daughter ) 1, 2-di-13C PFOA : 415 m/z (parent) to 370 m/z (daughter) 4. Calibration and Quantitation The analytical reference 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, an 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-13C 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 . -18- f q5 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice P . 19 DuPont- 18318 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 mice were weighed on test days 0, 3 (2 for subgroup B), and daily thereafter . J . Food Consumption and Food Efficiency During the test period, the amount of food consumed by each mouse over the weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount 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 mice and abnormal behavior and/or appearance among mice were conducted at least once daily throughout the study . Abnormal behavior/appearance was recorded by exception . Moribund mice were sacrificed, and a gross examination performed . Tissues and blood were not collected from moribund mice . 2. General Clinical 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 mouse was individually handled and examined for abnormal behavior and appearance . Detailed clinical observations in a standardized arena were also evaluated on all mice . The detailed clinical observations included (but were not limited to) evaluation 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 Evaluatio n A clinical pathology evaluation was conducted on all surviving animals 29 days after initiation of the study . Animals from each dose group were divided into 2 groups, Group A and Group B with 10 animals in each (e .g ., IA, IB, IIIA, IIIB) . All animals were fasted for approximately 3 hours prior to the scheduled sacrifice . The fasting schedule was staggered so that the last anima l -19// ~7 p . 20 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 was fasted for approximately the same amount of time as the first animal . While the animals were under carbon dioxide anesthesia, the maximum amount of whole blood was collected from the abdominal vena cava . Samples were allocated as indicated below : Group A Group B Hematology : 250 L whole blood - EDTA Not don e Clinical Chemistry : remaining blood in Clinical Chemistry : all blood in serum tube for : serum tube for : total protein, albumin, Cholesterol, triglycerides, high-density globulin (calculated) lipoprotein cholesterol, non-high-densit y lipoprotein cholesterol (calculated) In addition, for all animals, remaining sera were allocated for either humoral immune measurements or for serum corticosterone based on the optimal use of the serum volume remaining in the tube after the above tests were performed . Bone marrow smears were prepared at sacrifice from all surviving animals . Bone marrow smears were stained with Wright-Giemsa stain, but analysis was not necessary to support experimental findings . All blood samples were evaluated for quality by visual examination . Results were maintained in the study records and reported only if the sample was analyzed . Unless otherwise indicated, any historical control clinical pathology data referenced in the text is maintained in Haskell Notebook Number E 98560-AN . 1 . Hematology (Group A Only) Complete blood counts, including reticulocytes, were determined on a Bayer Advia 120 hematology analyzer or determined from microscopic evaluation of the blood smear . WrightGiemsa-stained blood smears from all animals were examined microscopically for confirmation of automated results and evaluation of cellular morphology . Blood smears, stained with new methylene blue, were prepared from each animal undergoing a hematology evaluation, but were not needed for examination . The following parameters were determined : red blood cell count red cell distribution width hemoglobin absolute reticulocyte count hematocrit platelet count mean corpuscular (cell) volume white blood cell count mean corpuscular (cell) hemoglobin differential white blood cell count mean corpuscular (cell) hemoglobin concentration microscopic blood smear examination 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 procedur e -20- 1V p . 21 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 (aspirating aqueous contents of the assay tube rather than decanting) . If necessary, the standard curve was extended at the low end of the range by including standards of 5 and 10 ng/mL . The following parameters were determined : cholesterol (group B) globulin (calculated, group A ) triglycerides (group B) high-density lipoprotein cholesterol (group B ) total protein (group A) non-high-density lipoprotein cholesterol (calculated, group B) albumin (group A) serum corticosterone (groups A and B ) M . Humoral Immune Functio n On test day 24, animals were injected intravenously in the lateral tail vein with 0 .2 mL of 1 x 109 SRBC/mL (Covance, Denver, Pennsylvania, U .S .A .) . One mouse (716 in the 10 mg/kg test substance group) was inadvertently not injected with the appropriate amount of SRBC and the immune response for this mouse could not be evaluated . On test day 29, serum was collected from each mouse and frozen (see L .2 .Clinical Chemistry) . Serum was not collected from 6 mice (117 in the 0 mg/kg test substance group, 306 in the 0 .3 mg/kg test substance group, 903, 904, and 906 in the 30 mg/kg test substance group, and 1112 in the 30 mg/kg (recovery) test substance group) due to sacrifice in extremis prior to test day 29 (117, 906 and 1112), insufficient serum sample volume (306 and 904), or no serum sample taken (903) ; therefore, the immune response for these mice could not be evaluated . Serum volume was insufficient fo r 3 mice (703 in the 10 mg/kg test substance group, 907 in the 30 mg/kg test substance group, and 1116 in the 30 mg/kg (recovery) test substance group) and the immune response for these mice could not be evaluated . Humoral immune function was evaluated by examining sera from individual animals for SRBCspecific IgM levels with an enzyme-linked immunosorbent assay (ELISA) .(7) The serum from each animal was assayed as 10 serial, 2-fold dilutions, with I 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 5 days with 90 mg/kg of the known immunosuppressive agent cyclophosphamide monohydrate or vehicle were run concurrently with the study samples to demonstrate that the assay functioned properly . For any test samples that needed to be rerun due to a poor curve fit or slope, pooled male and/or female cyclophosphamide monohydrate or vehicle serum samples were concurrently run . The pooled samples consisted of equal aliquots of serum taken from either the male or female rats dosed with cyclophosphamide monohydrate or vehicle . -21- /9 _5 p . 22 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 N . Anatomic Pathology Evaluatio n After 29 days on study, the surviving mice from each dose group (0, 0 .3, 1, 10, 30, and 30/0 mg/kg body weight) were sacrificed and necropsied for evaluation of subchronic toxicity . The order of sacrifice for scheduled deaths was stratified across groups . Mice were fasted for 3 hours before euthanasia . All mice, including 3 mice (mice 117, 906, and 1112) that were sacrificed in extremis during the study (test days 5, 9, and 5, respectively), were euthanized by carbon dioxide anesthesia and exsanguination . Gross examinations were performed for all mice . Final body weights and organ weights were recorded for all mice sacrificed by design on test day 29 . The following tissues were collected from 120 mice (20/sex/group) on study . Digestive System Nervous Syste m livera brain" (3 sections) Hematopoietic System Musculoskeletal System spleena femur/knee joint thymusa sternum popliteal lymph nod e 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/pons . Organ weight ratios (% final body weight, % brain weight) and group mean values from weighed organs were calculated . All tissues were fixed in 10% neutral buffered formalin . Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist . Microscopic findings were graded on a 4-point scale based on the severity or extent of the change (grade 1= minimal ; grade 2 = mild ; grade 3 = moderate ; grade 4 = severe) . For mice sacrificed by design (SD) on test day 29, all tissues collected from control (0 mg/kg) and high-dose (30 and 30/0 mg/kg) mice were processed to slides and examined microscopically . In addition, the following organs were examined from all SD mice in order to determine a noobserved-effect level for test substance-related microscopic findings : liver, thymus, spleen, and bone marrow . For the 3 mice sacrificed in-extremis (SE) before the scheduled sacrifice, all tissues were processed to slides and examined microscopically . Gross observations (recorded at necropsy) were examined microscopically for all animals . -22- ~ ~~ p . 23 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 0 . Total Cell Count s 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 3 mL Hank's Balanced Salt Solution (HBSS/H) and put on ice . The halved tissue/HBSS/H was poured into a small petri dish and cut into small pieces . The centrifuge tube was inverted 2 or 3 times and left on ice for approximately 10 minutes to allow debris to settle to the bottom 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 Evalua ti o n A section of liver from 2 control mice (103 and 104) and 2 mice in the 1 mg/kg group (503 and 504) mice was placed in cassettes in a container of formalin, and shipped to Experimental Pathology Laboratories, Inc (EPL) for evaluation by transmission electron microscopy . As a subcontractor to EPL, the Laboratory for Advanced Electron and Light Optical Methods, College of Veterinary Medicine, North Carolina State University processed the tissues for electron microscopy and prepared electron photomicrographic images under the direction of Dr . Michael Dykstra . The printed electron photomicrographic images were provided to EPL for evaluation by an ACVP-certified veterinary pathologist who interpreted the images and prepared a final report of the electron microscopic evaluation . More details are provided in Appendix M . 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 . - 23 Z00 p . 24 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Method of Statistical Analysi s If preliminary test is not If preliminary test is Parameter Prelimina Test significant significan t Body Weight Body Weight Gain Food Consumption Levene's test fo r Food Efficiency homogeneity (8) and One-way analysis of Kruskal-Wallis test (ia) Humoral Immune Function variance(") followed by (is) Dataa Shapiro-Wilk test(q) for Dunnett's test (11,12,13) followed by Dunn's test Clinical Pathology normality b Organ Weights Total Cell Counts a SRBC-specific serum IgM antibody titer data were transformed to Log2 to obtain normality or homogenous variances . b If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version of Dunnett's test was used. If the Shapiro-Wilk test was significant, Kruskal-Wallis test was followed by Dunn's test . -24- 1 ~ ~ p . 25 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 RESULTS AND DISCUSSION 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 recove ry 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 recove ry = 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 f 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) Dosing solutions prepared on October 17, 2005 were analyzed for concentration verification, uniformity of mixing, and 5-hour room temperature stability, and results are shown in Table 2 . The following table summarizes the results for concentration verification, uniformity of mixing, and 5-hour room temperature stability analyses . -25~~.~~ p . 26 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont- 18318 Preparation Nominal Measured' Average C .V . Stability Date mg/mL mg/mL % Nominal (%) % Nominal 17-October-05 0 ND` --- --- --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) Dosing solutions prepared on November 15, 2005 were analyzed for concentration verification and uniformity of mixing, and results 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-05 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 analyses of the samples collected during the study indicate that the dosing formulations were at the targeted concentrations, mixed uniformly, and stable under the conditions of the study. Test substance was not found in the 0 mg/mL sample s -26- Z03 p . 27 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 In-Life Measurement s A . Mean Body Weights and Body Weight Gains (Tables 4-5, Figure 3, Appendices B-C ) Test substance-related and adverse reductions in mean body weights and body weight gains were observed at 10, 30 and 30/0 mg/kg . Mean final body weights were 14, 22, and 12% lower than the control group at 10, 30, and 30/0 mg/kg, respectively . Test substance-related increases in liver weights occurred and minimized the magnitude of the effects on body weight . In an attempt to quantitatively separate the increased liver weights (see Appendix C) from the decreased body weights, an adjusted body weight (see Appendix C) was calculated for each animal by subtracting the weight of the liver from the final body weight . The means for the adjusted body weights were 28, 35, and 23% lower than controls at 10, 30, and 30/0 mg/kg, respectively . The reductions in mean body weight at 10 mg/kg and above resulted from overall body weight losses during the study ; mice lost an average of 3 .8, 6 .6, and 3 .3 g during days 0 to 28 at 10, 30, and 30/0 mg/kg, respectively, whereas control group mice gained an average of 0 .9 g . In general, the test substance-related effects on body weight parameters were dose-related relative to the magnitude of the change and the onset of the reductions ; effects on mice dosed at 30 mg/kg were more pronounced and evident sooner than effects at 10 mg/kg . The effects of cessation of dosing were evident in that the mean final body weights of mice at 30/0 mg/kg were 12% higher than those from the mice dosed at 30 mg/kg . Body weight and weight gain data for animals dosed at 0 .3 and 1 mg/kg were generally comparable to controls . B. Food Consumption and Food Efficiency (Tables 6-7, Appendix D ) Test substance-related effects on food consumption were evident at 10, 30, and 30/0 mg/kg . The food consumption data did not adhere to the hypothesis of a monotonic dose response . At 10 mg/kg, the mean food consumption was increased or significantly increased starting at the end of the first week and persisting until the end of the study . At 30 and 30/0 mg/kg, mean food consumed was usually lower or significantly lower than controls but for each group there was one interval with significantly increased food consumption . As a result of these somewhat erratic patterns of food consumption, overall food consumption during days 0 to 28 was generally comparable across the groups with the exception of the 10 mg/kg group for which mean food consumption was significantly increased . -27- 2tJ q" p . 28 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 C . Clinical Observations and Mortalit y (Tables 8-9, Appendices E-F) Test substance-related clinical signs observed at 1, 10, 30 and 30/0 mg/kg included : pallor, wet and stained fur, swollen penis, eye observations, prostrate, or yellow extremities . Swollen left shoulder observed in one mouse dosed at 10 mg/kg was possibly due to a dosing incident . Abnormal gait was observed in single animals dosed at I or 10 mg/kg. This observation was not considered test substance related because it was only observed in single animals and short in duration . Three mice were sacrificed in extremis prior to test day 29 . Two mice were sacrificed on test day 5 due to dosing incidents from the control and 30/0 mg/kg groups . One mouse dosed at 30 mg/kg was sacrificed on test day 9 possibly due to a dosing incident, however, gross and microscopic pathology were unable to determine the exact cause of deat h Clinical Pathology Evaluation A . Hematology (Table 10, Appendix G ) l . Red Blood Cells Red cell mass parameters (red blood cell count, hemoglobin, and hematocrit) were minimally decreased in mice dosed with 30 mg/kg for 29 days . Mean values were 88-94% of the control group means (not statistically significant) . In addition, mean cell hemoglobin and mean cell hemoglobin concentration were decreased in mice dosed with 10 or 30 mg/kg for 29 days . Means at 10 and 30 mg/kg were 94-96% and 95-97% of the respective control group means for these 2 parameters (variable statistical significance) . Some mice dosed with 1 or 10 mg/kg for 29 days had minimally lower reticulocyte counts compared to those dosed with 0 or 0 .3 mg/kg . Mice dosed with 30 mg/kg either had higher (3/7) or lower (4/7) reticulocyte counts compared to control mice . Two of the 3 mice at 30 mg/kg with higher reticulocyte counts also had increased splenic extramedullary hematopoiesis . On an individual animal basis, there was no correlation between red cell mass and reticulocyte counts in this group . Effects on red cell mass were more pronounced (mildly decreased) in mice dosed wit h 30/0 mg/kg compared to those dosed with 30 mg/kg for the entire 29 days . At recovery, mean red blood cell count, hemoglobin, and hematocrit ranged from 82-86% of the respective control group means for these 3 parameters (all statistically significant) . Decreased red cell mass parameters following recovery could be due to one or more of the following processes : increased red cell destruction, red cell loss, or increased plasma volume . The mechanism fo r -28- ~~~~ p . 29 Ammonium Perfluorooctanoate : 2 8-Day Imm u n o t oxicity Study in Male Mice DuPont-18318 decreased red cell mass parameters was not evident from in-life, clinical pathology, or anatomic pathology data . Therefore, the cause of the decreased red cell mass was not determined . Reticulocytes were mildly increased in mice dosed with 30/0 mg/kg . Mean reticulocyte count was 148% of the control group mean (not statistically significant) . Consistent with the increase in reticulocyte counts, red cell distribution width was increased (mean was 109% of the control group mean ; not statistically significant), and mean cell hemoglobin concentration was decreased (97% of control group mean; statistically significant) . Microscopically, this group had increased polychromasia (increased bluish staining of red blood cells), a characteristic finding in blood with increased reticulocyte counts. The increases in reticulocytes and related parameters were considered to be in response to the decreased red cell mass described above. These changes also correlated with histologic evidence of increased splenic extramedullary hematopoiesis, which was observed in 15 of 19 mice in the 30/0 mg/kg group . 2. White Blood Cell s Neutrophils were increased in mice dosed with 10 or 30 mg/kg. Means were 236 and 296% of the control group mean, respectively (statistically significant) . While all neutrophil counts for mice dosed with <1 mg/kg were between 0 .00 and 2 .00x103/L, mice dosed with 10 mg/kg had neutrophil counts between 1 .00 and 3 .00x103/L, while 2 of 7 mice dosed with 30 mg/kg had neutrophil counts of greater than 3 .50x 103/L (animal 901 and 904) . Histologically, 2 of 3 mice (mice 902 and 904) dosed with 30 mg/kg had minimal granulocytic hyperplasia of the bone marrow, corresponding to the observation of increased neutrophils in the peripheral blood of this group . The two 30 mg/kg mice with the highest neutrophil counts (901 and 904) also had higher lymphocyte and monocyte counts than other mice in this group, although the lymphocyte counts were similar to or lower than control values . Increased neutrophils may be due to stress (glucocorticoid-related ; see corticosterone discussion below and anatomic pathology) or inflammation. The changes in neutrophils, in light of changes in other leukocyte types, are likely related to both of these processes . Lymphocytes were generally decreased in mice dosed with 30 mg/kg (mean was 59% of the control group mean ; not statistically significant) . One mouse (animal 901) had increased neutrophil and unchanged lymphocyte counts compared to control mice . The decreases in lymphocyte counts in most mice dosed with 30 mg/kg were consistent with stress (see corticosterone discussion below and histology), and corresponded to increased serum corticosterone and lymphoid depletion/atrophy in the spleen, thymus, and lymph nodes i n 30 mg/kg mice . The unchanged lymphocyte count in 1 mouse dosed with 30 mg/kg may have been due to a combination of stress and inflammation . Monocytes were increased in mice dosed with 10 or 30 mg/kg . Means were 285 and 254% of the control group mean, respectively (variable statistical significance) . In these 2 groups of mice, individual mice with increased monocytes tended to have increased neutrophil counts . Increased monocytes are observed with inflammation or stress . As discussed above, the combination of changes likely reflect both stress and inflammation . Eosinophils were decreased in mice dosed with 10 or 30 mg/kg . Means were 57 and 64% of the control group mean, respectively (not statistically significant) . A decrease in peripheral blood -29- ~` ~. J{ ~ p . 30 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 eosinophils is consistently observed in response to stress . Therefore, this change was considered to be due to stress . Large unstained cells (LUC) were increased in mice dosed with 10 or 30 mg/kg . LUCs are cells that cannot be identified as one of the 5 major leukocyte types by the Advia 120 automated hematology analyzer, and normally comprise a small percentage of the total leukocyte population. The LUC count normally includes mostly lymphocytes and monocytes . In this study, the mice with the highest LUC counts usually had the highest lymphocyte and/or monocyte counts . Therefore, in this study, changes in LUC counts paralleled changes in lymphocytes and/or monocytes . In the 30/0 mg/kg group, neutrophil, lymphocyte, eosinophil, monocyte, and LUC counts were generally similar to those of mice dosed at 30 mg/kg for the full 29 days (variable statistical significance compared to controls ; lymphocyte counts statistically different from that of mice dosed with 30 mg/kg for 29 days) . Therefore, there was little or no recovery for changes in differential white blood cell counts . The following statistically significant changes in mean hematology parameters were considered to be unrelated to treatment and non-adverse because they did not occur in a dose-related pattern : Decreased mean cell hemoglobin concentration in mice dosed with 10 mg/kg for 29 days Decreased red cell distribution width in mice dosed with 0 .3 or 10 mg/kg for 29 days Increased total white blood cell count in mice dosed with 10 mg/kg for 29 day s B . Clinical Chemistry (Table 11, Appendix G) Icterus was evident in serum of mice dosed with 10 or 30 mg/kg for 29 days . The incidences of icteric serum were 0/19, 0/19, 0/20, 16/20, and 17/17 in mice dosed with 0, 0 .3, 1, 10, o r 30 mg/kg for 29 days . Icterus is graded as none, trace, small, moderate, or large and in this study, grades of trace, small and moderate were observed . The icterus grades were generally higher at 30 than at 10 mg/kg . Icterus is an indication of increased serum bilirubin and may result from either increased production of bilirubin from hemoglobin as a result of increased red blood cell destruction or decreased processing and excretion of bilirubin . There was no clinical or anatomic pathology evidence of increased red cell destruction in mice dosed for 29 days . Histologically, minimal to mild hyperplasia of the bile ducts were observed in mice dosed with 10 or 30 mg/kg, which, along with other hepatic changes, may have contributed to the presence of icterus . In mice dosed in the 30/0 mg/kg group, the incidence and grades of icterus were lower than after 29 days of treatment (trace to small icterus observed in 16/19 mice) . Therefore, there was partial recovery from the finding of icteric serum . Total cholesterol was moderately decreased in mice dosed with 10 or 30 mg/kg for 29 days . Means were 69 and 51 % of the control group means (statistically significant) . The decrease in cholesterol was due to decreases in HDL cholesterol at doses of 1, 10 and 30 mg/kg (means were 71, 61, and 44% of the control group mean, respectively, and were statistically significant), an d C:. Oq ~' ' `~ p . 31 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 in non-HDL cholesterol at doses of 10 and 30 mg/kg ( means were 85 and 65% of the control group mean, respectively ; not statistically significant) . Mean total and HDL cholesterol in the 30/0 mg/kg group were 80% and 69% of the control group mean (variable statistical significance compared to control but statistically different from mice dosed with 30 mg/kg for 29 days) . Therefore, there was pa rtial recove ry of total and HDL cholesterol, and complete recovery for non-HDL cholesterol in mice dosed with 30/0 mg/kg . Triglycerides were moderately decreased in mice dosed with 10 or 30 mg/kg for 29 days . Means were 47 and 32% of the control group mean, respectively (statistically signi ficant). There was partial recove ry of triglycerides, as the mean value in mice in the 30/0 mg/kg group was 57% of the control group mean (statistically different from control mice and mice dosed with 30 mg/kg for 29 days) . Albumin was moderately increased in mice dosed with 1, 10, or 30 mg/kg for 29 days (means were 110, 145, and 131% of the control group mean, respectively ; variable statistical signi ficance) . Although albumin data were limited to 3 values at 30 mg/kg for 29 days, albumin concentrations for these 3 mice were greater than those of mice dosed with 0 .3 or 1 mg/kg, suggesting a treatment-related increase . Increased albumin may result from dehydration (relative increase) or increased synthesis or decreased catabolism (absolute increase) . There were no inlife or clinical pathology data to support dehydration ; however, urine concentration, an important sign of dehydration, was not evaluated in this study . Some peroxisome proliferator-activated receptor ( PPAR) agonists have been report ed to cause increases in serum albumin concentration due to increased synthesis of albumin . Due to the lack of corroborative data, the cause of increased albumin in this study cannot be determined . In the 30/0 mg/kg group, albumin concentrations were similar to those of mice dosed for 10 or 30 mg/kg for 29 days and were increased compared to those of control mice . Therefore, there was no recovery in albumin concentrations . Globulin concentrations were increased in the 30/0 mg/kg group compared to those of control mice and mice dosed with 30 mg/kg for 29 days (both statistically significant ; mean was 119% of the control mean), suggesting a test substance-related effect . Total protein measurements include albumin and globulin, so changes in total protein are a function of changes in these 2 components . Total protein concentration was increased at 10 mg/kg ( due to increases in albumin) and similar to control values at 30 mg/kg (due to increases in albumin and decreases in globulin) . Means for these 2 groups were 125 and 109% of the control group mean, respectively (variable statistical signi fi cance) . In the 30/0 mg/kg group, total protein was 134% of the control group mean (due to increases in both albumin and globulin) . Serum co rticosterone was moderately increased in several mice dosed with 10 or 30 mg/kg for 29 days (variable statistical significance) . While serum corticosterone concentrations were between 0 and 400 ng/mL in all mice dosed with <1 mg/kg, concentrations greater tha n 400 ng/mL were observed in 7/10 and 6/10 mice dosed with 10 or 30 mg/kg, respectively, resulting in mean concentrations that were 229 and 231 % of the control group mean for mice for these 2 groups . In the 30/0 mg/kg group, mouse serum co rt icosterone concentrations were still mildly increased ( 6/10 were greater than 400 ng/mL, and the mean was 137% of the contro l ~-3~1-`~1 p . 32 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 group mean ; not statistically significant) . These changes are consistent with partial recovery from stress . C. Clinical Pathology Conclusion s Mice dosed with >1 mg/kg had decreased serum HDL cholesterol, increased serum albumin, and variable changes in serum globulin . Mice dosed with >10 mg/kg had increased neutrophils and monocytes, decreased eosinophils, icteric serum, decreased serum total cholesterol, non-HDL cholesterol, and triglycerides, and increased serum corticosterone . Mice dosed with 30 mg/kg also had decreased red cell mass parameters (red blood cell count, hemoglobin, and hematocrit), increased reticulocytes, and decreased lymphocytes . The only parameter with complete recovery in the 30/0 mg/kg group was non-HDL cholesterol . Partial recovery was observed for icteric serum, total and HDL cholesterol, triglycerides, and serum corticosterone . Immunotoxicity A . Humoral Immune Functio n (Tables 12-13, Appendices H-I ) There was test substance-related evidence of immunosuppression in mice at 10, 30 and 30/0 mg/kg . The anti-SRBC titers for these groups were reduced 20, 28 and 30% when compared to the control group mean . There was no difference in mean primary humoral immune response between the 30 an d 30/0 mg/kg, indicating that the shortened dosing period did not have an impact on this endpoint . In contrast, mice injected for 5 days with 90 mg/kg of the immunosuppressive material, cyclophosphamide, demonstrated a 52% inhibition of the IgM antibody response to SRBC . Anatomic Pathology Evaluation A . Cause of Deat h There were no test substance-related deaths . Only 3 of the 120 mice on study did not survive until the scheduled sacrifice on day 29 . The 3 mice were sacrificed in extremis on test days 5 and 9 . Mouse 117 (control) and mouse 1112 (30/0 mg/kg) were sacrificed on test day 5 due to dosing incidents . Both mice had ruptured esophagi identified on gross examination . Mouse 906 (30 mg/kg) was sacrificed on test day 9 because the mouse was clinically lethargic and pale . Gross and microscopic pathology did not reveal a cause of death . It is likely that this death was also due to a dosing incident . However, since the esophagus, lungs, and mediastinal tissue were not saved for microscopic examination, the cause of death was undetermined . -32- ~~~ p . 33 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 B . Final Body and Organ Weight Data (Table 14, Appendix J) Following 28 days of daily gavage administration of the test substance, test substance-related organ weight effects were observed in the liver, spleen, and thymus . Relative to controls, mean liver weight parameters were increased at >0 .3 mg/kg, mean spleen weight parameters were decreased >1 mg/kg, and mean thymus weight parameters were decreased at >10 mg/kg . Text Table 1 : Mean Absolute and Relative (% body weight) Organ Weights in Male Mice Group : Dose (mg/kg) : Number of Mice/Sex : I III V VII IX XI 0 0 .3 1 10 30 30/0 19 20 20 20 19 19 Final Body Wt . (g) 33 .0 33 .4 33 .8 28 .4* 26.0* 30 .5*^ Liver (17) (20) (20) absolute wt . (g) 1 .782 2 .407 3 .272 ** /a body wt . 5 .421 7 .196 9 .704 ** (20) 6 .061 ** 21 .232 ** (18) 5 .899 ** 22 .618 ** (18 ) 6 .391 ** 21 .209 ** Spleen (19) (20) absolute wt . (g) 0 .117 0 .116 % body wt . 0 .355 0 .346 (20) (20) 0 .104 0 .066 ** 0 .307 * 0 .232 * (19) 0 .052 ** 0 .195 * (19 ) 0 .076 ** 0.249 * Thymus (19) (20) (20) (20) (19) (19 ) absolute wt . (g) 0 .050 0 .045 0 .049 0 .025 ** 0 .025 ** 0 .027 ** % body wt . 0 .153 0 .137 0 .144 0 .087 ** 0 .094 ** 0 .088 ** 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 14%, 21 %, and 8% in the 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value . All decreases were statistically significant. Mean final body weights in the 0 .3 and 1 mg/kg dose groups were similar to the control values . There was also a statistically significant increase in the mean final body weight of th e 30/0 mg/kg dose group, as compared to the 30 mg/kg dose group . This increase demonstrates partial recovery from the test substance-related final body weight decrease in the 6 recovery days following the injection of sheep red blood cells . -33- J_~ ~) p . 34 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 2 . Live r Mean absolute liver weights were increased 35%, 84%, 240%, 231%, and 259% in the 0 .3, 1, 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value . Mean relative (% body weight) liver weights were similarly increased (33%, 79%, 292%, 317%, and 291 %, respectively) . All increases were statistically significant, except for those in the 0 .3 mg/kg dose group . The increased liver weights, at all dose levels, correlated with the microscopic finding of mild to severe hepatocellular hypertrophy. It also correlated with the gross observation of large livers at doses >10 mg/kg . 3. Spleen Mean absolute spleen weights were decreased 11%, 44%, 56%, and 35% in the 1, 10, 30, and 30/0 mg/kg dose groups, respectively, as compared to the control value . Mean relative (% body weight) spleen weights were similarly decreased (14%, 35%, 45%, and 30%, respectively) . All decreases were statistically significant, except for the increase in the mean absolute splee n weight in the 1 mg/kg dose group . Mean spleen weight parameters in the 0 .3 mg/kg dose group were similar to the control values . The decreased spleen weights, at >_1 mg/kg, correlated with the microscopic finding of minimal to mild lymphoid depletion/atrophy in this organ. It also correlated with the gross obse rvation of small spleens at doses >10 mg/kg . 4. Thymu s Mean absolute thymus weights were decreased 50%, 50%, and 46% in the 10, 30, an d 30/0 mg/kg dose groups, respectively, as compared to the control value . Mean relative (% body weight) thymus weights were similarly decreased (43%, 39%, and 42%, respectively) . A ll decreases were statistically significant . Mean thymus weight parameters in the 0 .3 and 1 mg/kg dose groups were similar to the control values . The decreased thymus weights, at >10 mg/kg, correlated with the microscopic finding of minimal to severe lymphoid depletion/atrophy in this organ . It also correlated with the gross observation of small thymuses at doses >10 mg/kg . 5 . Other All other individual and mean organ weight differences were considered to be spurious or secondary to the decrease in final body weights . Mean absolute brain weights were decreased, while mean relative brain weights ( % body weight) were increased only at doses (>10 mg/kg) that produced significantly decreased body weights . The lack of any gross or microscopic effects on the brain also suggests that the brain weight differences were a function of body weight and not indicative of a test substance-relate brain weight effect . VI p . 35 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity S tudy in Mal e Mic e DuP o nt- 18318 C . Gross Observation s (Table 15 Appendix K ) At the terminal sacrifice, test substance-relate gross observations were observed at doses >10 mg/kg and included large and discolored livers, small spleens, and small thymuses. Text Table 2 : Incidences of Test Substance-Related Gross Observations in Male Mice Group : I III V VII IX XI Dose (mg/kg) : 0 0 .3 1 10 30 30/0 * Number of Mice/Group : 19** 20 20 20 19** 19** Liver Large 1 0 0 17 16 17 Discoloration 0 0 0-1- 5 - 1 Spleen Small Thymus 0 0 0 8 8 2 Small 0 0 0 3 2 2 Underlined values were interpreted to be test-substance related increases, as compared to control values . * Not dosed with test substance following immunology challenge . ** Table excludes 3 mice that were euthanized on days 5 (mice #s 117 and 1112) and 9 (mouse #906) . The observation of large livers in mice given >_10 mg/kg correlated with the microscopic finding of severe hepatocellular hypertrophy in all mice at these dosages and greater than 200% increases in mean absolute liver weights, as compared to controls . Gross liver discoloration is also consistent with microscopic hepatocellular hypertrophy . Small spleens, which were observed in almost half of the spleens from mice given 10 (8/20) and 30 (8/19) mg/kg of the test substance, correlated with decreased mean spleen weights at these same dose levels . Fewer small spleens (2/19) were observed in the recovery group (30/0 mg/kg), which was consistent with the partial recovery of spleen weights in this dose group. Test substance-related microscopic lymphoid depletion was observed only at 30 mg/kg . Small thymuses were recorded in only a few thymuses at dose levels >10 mg/kg . These correlated with decreased mean thymus weights and microscopic lymphoid depletion at the same dose levels . D . Microscopic Findings (Table 16, Appendix K ) Microscopic examination of the liver demonstrated mild hepatocellular hypertrophy at 0.3 mg/kg ; moderate to severe hepatocellular hypertrophy with secondary individual cell necrosis and focal necrosis at doses >_1 mg/kg ; and increased hepatocellular mitotic figures, hepatocellular fatty change, and bile duct hyperplasia at doses ?10 mg/k g -35- ,?12 p . 36 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased granulocytic hematopoiesis in the bone marrow (>10 mg/kg) an d increased erythrocytic hematopoiesis in the bone marrow and spleen (30/0 mg/kg) . Test substance-related lymphoid depletion/atrophy was present in the thymus (>10 mg/kg) and spleen (30 mg/kg) of less than half of the mice at the respective dose levels . Mesenteric and popliteal lymph nodes had no test substance-related effects. Text Table 3 : Incidences of Test Substance-Related Microscopic Findings in Male Mice Group : I III V VII IX XI Dose (mg/kg) : 0 0 .3 1 10 30 30/0 * Number of Mice/Group : 19** 20 20 20 19** 19** Liver (19) (20) (20) 20 19 19 Hypertrophy, hepatocellular 0 20 [2 .0] 20 [3 .0] 20 [4 .0] 19 [4 .0] 19 [4 .0] Necrosis, individual cell 0 0 11 [1 .1] 20 [1 .9] 19 [2 .0] 19 [1 .7] Necrosis, focal 0 1 [1 .0] 3[1 .0] 4[1 .8] 7[1 .9] 3[1 .7] Mitotic figures, increased 0 0 0 10 [1 .0] 15 [ 1 .0] 19 [1 .4] Hyperplasia, bile duct 0 0 0 6[1 .0] 17 [1 .2] 12 [1 .0] Fatty change, nonzonal 0 0 0 9 [1 .0] 14 [1 .0] 4[1 .0] Thymus (19) (20) Depl ./Atr . Lymph. or NP . 0 0 0 19 (19 ) 17 (19) 8[1 .6] 12 [2 .9] 6[2 .8] Spleen (19) (20) 20 (20) (19 ) 1 9 Depletion/Atrophy, lymphoid 0 1 [1 .0] 0 0 8[1 .1] 2 [ 1 .1 ] EMH, increased 6(1 .2) 4 [1 .5] 1 [1 .0] 3 [1 .7] 5 [1 .2] 15 [1 .8] Bone Marrow 19 (20) (20) 20 (19 ,) (19 ) Hyperplasia, granulocytic 0 0 0 3[ 1 .7] 4[ 1 .0] 3[ 1 .7] Hyperplasia, erythrocytic 0 0 0 0 0 3[2 .0] [] = Number in brackets is the average grade (grades 1- 4) when lesion is present (i .e ., sum of grades =# animals with lesion) . Grading scale : 1= minimal ; 2 = mild ; 3= moderate ; 4 = severe . O number in parenthesis is the number of organs weighed ; EMH = Extramedullary hematopoiesis . Underlined values were interpreted to be test-substance related increases, as compared to control values . * Not dosed with test substance following immunology challenge . ** Table excludes 3 mice that were euthanized on days 5 (mice #s 117 and 1112) and 9 (mouse #906) . Includes lymphoid depletion/atrophy and thymus not present in mediastinal tissue (assumed grade 3 atrophy) . 1 . Live r a . Hepatocellular Hypertrophy Panlobular hepatocellular hypertrophy was observed in all surviving mice given the test substance and the severity was dose related . Hypertrophy was present in 0/19, 20/20, 20/20, 20/20, 19/19, and 19/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The hypertrophy was graded as mild in all mice given 0 .3 mg/kg, moderate in all mice give n 1 mg/kg ; and severe in all mice given >10 mg/kg. -36- 2 L3 p . 37 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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 . At doses >_1 mg/kg, the severity (moderate to severe) of the hepatocellular hypertrophy appeared to be responsible for increased individual cell necrosis and focal necrosis . At doses >_10 mg/kg, the severity (severe) of the hepatocellular hypertrophy was associated with nonzonal fatty change and hepatocellular regeneration (increased mitotic figures) . Hepatocellular hypertrophy correlated with increased mean liver weight parameters at all doses and grossly large livers at doses >10 mg/kg . b. Individual Cell Necrosi s Individual cell necrosis was observed in mice given >_1 mg/kg of the test substance ; the incidence and severity were dose related . Individual cell necrosis was present in 0/19, 0/20, 11/20, 20/20, 19/19, and 19/19 in mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively, and was graded minimal to mild . A slight decrease in severity was apparent in the 30/0 mg/kg group a s compared to the 30 mg/kg group . The individual cell necrosis was primarily due to the degeneration, necrosis, and lysis of enlarged hepatocytes in an individualized, non-zonal pattern . Although apoptotic cells were observed in most sections, the increased individual cell necrosis was usually not due to apoptosis . A slight secondary focal inflammatory cell inflitrate was often observed in association with necrotic hepatocytes . c . Focal Necrosi s Test substance-related focal necrosis was also observed in mice given >1 mg/kg of the test substance . The incidence and severity were both mildly dose related. Focal necrosis was present in 0/19, 1/20, 3/20, 4/20, 7/19, and 3/19 mice given 0, 0.3, 1, 10, 30, and 30/0 mg/kg, respectively, and was graded minimal to moderate . The single incidence of minimal focal necrosis in a 0.3 mg/kg mouse was considered incidental since this is sometimes a naturally occurring background lesion . A slight decrease in the incidence of focal necrosis was apparen t 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 hepatocyte clusters is a common secondary effect of hepatocellular hypertrophy and is most likely the result of secondary focal ischemia .(16 ) d. Increased Mitotic Figures Increased mitotic figures were observed in mice given >-I 0 mg/kg of the test substance ; the incidence was dose related . Increased mitotic figures were present in 0/19, 0/20, 0/20, 10/20, 15/19, and 19/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively, and was graded minimal to mild . A slight increase in the incidence and severity was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group . 37- 2,1,~4 p . 38 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Increased mitotic figures were an apparent indication of increased cell turnover in those mice with severe hepatocellular hypertrophy and subsequent individual cell necrosis . e . Bile Duct Hyperplasi a Bile duct hyperplasia was observed in mice given >_10 mg/kg and the incidence and severity were both dose related. Bile duct hyperplasia was observed in 0/19, 0/20, 0/20, 6/20, 17/19, and 12/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The hyperplasia was graded as minimal in all mice except for three 30 mg/kg mice which were graded as mild . A slight decrease in the incidence of bile duct hyperplasia was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group . The bile duct hyperplasia was characterized by a minimal to mild increase in the number of profiles of normal bile ducts . f. Fatty Change, Nonzonal Nonzonal fatty change was also present in mice given >10 mg/kg . The incidence was dose related but all lesions were graded as minimal . Fatty change was observed in 0/19, 0/20, 0/20, 9/20, 14/19, and 4/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . A decrease in the incidence of fatty change was apparent in the 30/0 mg/kg group, as compared to th e 30 mg/kg group . The fatty change was characterized by a minimal increase in the number of hepatocytes with small to medium size cytoplasmic fatty globules . The distribution of the affected hepatocytes was nonzonal as the fatty change was scattered throughout the liver . The fatty change was only observed in livers with severe diffuse hepatocellular hypertrophy and was considered to be a degenerative change secondary to the hypertrophy . 2 . Thymu s a . Lymphoid Depletion/Atrophy Minimal to severe thymic lymphoid depletion/atrophy was only observed in mice give n >_10 mg/kg . The lesion was present in 0/19, 0/20, 0/19, 6/19, 7/17, and 4/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . In addition, thymic tissue was not present in the mediastinal tissue section of 9 other mice (given >10 mg/kg), suggesting that these animals also had moderate to severe thymic lymphoid depletion . Therefore, the combined incidence of lymphoid depletion and absent thymic tissue was 0/19, 0/20, 0/19, 8/19, 12/17, and 6/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . Both the incidence and severity were dose related . A slight decrease in the incidence and severity of thymic lymphoid depletion/atrophy was apparent in the 30/0 mg/kg group, as compared to the 30 mg/kg group . Thymic lymphoid depletion/atrophy was characterized by decrease in the number of lymphocytes in the thymus in the absence of necrotic cells . As stated earlier, the absence of thymic tissue in the mediastinal tissue section was also interpreted to be indicative of thymic depletion/atrophy . -38- 2 ~ ~-,, -~ p . 39 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity S tudy in M al e M i ce DuP ont-18318 3 . Spleen a. Lymphoid Depletion/Atrophy Minimal to mild splenic lymphoid depletion/atrophy was test substance related only at 30 mg/kg . The lesion was present in 0/19, 1/20, 0/20, 0/20, 8/19, and 7/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The incidence and severity of this effect was similar in the 30 and 30/0 mg/kg groups . As with the thymic lesion, splenic lymphoid depletion/atrophy was characterized by a decrease in the number of lymphocytes in the thymus in the absence of necrotic cells . b . Increased Extramedullary Hematopoiesi s An increase in the incidence of splenic extramedullary hematopoiesis (EMH) was considered test substance related only in high-dose mice allowed a recovery period (30/0 mg/kg dose group) . Minimal to moderate increased EMH was observed in 6/19, 4/20, 1/20, 3/20, 5/19, and 15/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The increased splenic EMH was erythrocytic and correlated with both the bone marrow erythrocytic hyperplasia and the hematological finding of decreased red cell mass parameters and increased circulating reticulocytes (see Clinical Pathology) that were also observed only in the 30/0 mg/kg recovery mice . 4 . Bone Marrow a. Granulocytic Hyperplasi a A minimal to moderate increase in bone marrow granulocytic hyperplasia was observed in 0/19, 0/20, 0/20, 3/20, 4/19, and 3/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . The incidence and severity were not dose related in groups given > I 0 mg/kg of the test substance . The increase in bone marrow granulocytic hyperplasia (>10 mg/kg) correlated with increases in the peripheral white blood cell and neutrophil counts (see Clinical Pathology) . The granulocytic response was most likely secondary to the hepatocellular individual cell necrosis and focal necrosis observed in mice given >1 mg/kg of the test substance . b . Erythrocytic Hyperplasi a A mild increase in bone marrow erythrocytic hyperplasia was observed only in high-dose mice allowed a recovery period (30/0 mg/kg dose group) . Erythrocytic hyperplasia was observed in 0/19, 0/20, 0/20, 0/20, 0/19, and 3/19 mice given 0, 0 .3, 1, 10, 30, and 30/0 mg/kg, respectively . As discussed for the spleen, the increase in bone marrow erythrocytic hyperplasia in recovery mice correlated with both microscopic splenic EMH and hematology findings (decreased red cell mass parameters and increased circulating reticulocytes (see Clinical Pathology)) . -39- -7 p . 40 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 5 . Othe r All other microscopic observations in this study were consistent with normal background lesions in mice of this age and strain . E . Ultrastructural Finding s 1 . Electron Microscopy Evaluation (Appendix M ) At the 1 mg/kg dose of APFO (linear), an increase in peroxisomes was not observed . However, many organelles could not be clearly identified due to poor ultrastructural detail, which was likely the result of formalin fixation . Therefore, definitive conclusions on perosimal numbers in treated groups relative to controls could not be drawn . More details are provided in Appendix M . F. Anatomic Pathology Conclusion s There were no test substance-related deaths . Only 3 of the 120 mice on study did not survive until the scheduled sacrifice on day 29. The 3 mice were sacrificed in extremis on test days 5 and 9 . Following 28-days of daily gavage administration of the test substance, test substance-related organ weight effects were observed in the liver, spleen, and thymus . Mean liver weight parameters were increased, mean spleen weight parameters were decreased, and mean thymus weight parameters were decreased . At the terminal sacrifice, test substance-relate gross observations were observed at doses >10 mg/kg and included large and discolored livers, small spleens, and small thymuses . Microscopic examination of the liver demonstrated mild hepatocellular hypertrophy at 0.3 mg/kg ; moderate to severe hepatocellular hypertrophy with secondary individual cell necrosis and focal necrosis at doses >_1 mg/kg ; and increased hepatocellular mitotic figures, hepatocellular fatty change, and bile duct hyperplasia at doses >10 mg/k g Microscopic examination of lymphohematopoietic organs (spleen, thymus, bone marrow, lymph nodes) revealed increased granulocytic hematopoiesis in the bone marrow (>10 mg/kg) and increased erythrocytic hematopoiesis in the bone marrow and spleen (30/0 mg/kg) . Test substance-related lymphoid depletion/atrophy was present in the thymus (>10 mg/kg) and spleen (30 mg/kg) of less than half of the mice at the respective dose levels . Mesenteric and popliteal lymph nodes had no test substance-related effects. -40- 2 17 p . 41 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Total Cell Counts A. Spleen Cell Number (Table 17, Appendix L ) No significant changes in total spleen cell number were noted in animals dosed with 0 .3 or 1 mg/kg . Significant decreases were noted in animals dosed with 10 mg/kg or greater, with the greatest suppression compared to vehicle-treated controls observed at 30 mg/kg (63% suppression) . In the 30/0 mg/kg group, a rebound was seen ( 44% suppression), but this increase in cells compared to the 30 mg/kg was most likely due to the extramedulla ry hematopoiesis observed in 15 of 19 mice in this treatment group . B. Thymus Cell Number (Table 17, Appendix L ) No significant changes in total thymus cell number were noted in animals dosed with 0 .3 o r 1 mg/kg . Significant decreases were noted in animals dosed with 10 mg/kg or greater, with the greatest suppression observed at 30 mg/kg ( 82% suppression) . In the 30/0 mg/kg group, a rebound was seen ( 51 % suppression) . CONCLUSIONS Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for APFO for systemic toxicity in male mice was 0 .3 mg/kg and for immunosuppression was 1 mg/kg . RECORDS AND SAMPLE STORAGE Specimens ( if applicable), raw data, the protocol, amendments (if any), and the fi nal report will be retained at Haskell Laborato ry , Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware . Laboratory-speci fic raw data such as personnel files, instrument, equipment, refrigerator and/or freezer raw data will be retained at the facility where the work was done . REFERENCES 1 . Vanden Heuvel, J ., Kuslikis, B ., and Van Rafelghem, M . (1991) . Tissue distribution, metabolism, and elimination 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 . -41- ~~~ p . 42 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 3 . Hansen, K .J ., Clemen, L .A ., Ellefson, M .E ., and Johnson, H .O . (2001) . Compound-specific, quantitative characterization of organic fluorochemicals in biological matrices . Environ. Sci . Technol. 35, 766-770 . 4 . Olsen, G .W ., Burris, J.M., Burlew, M .M., and Mandel, J .H. (2000) . Plasma cholecystokinin and hepatic enzymes, cholesterol and lipoproteins in ammonium perfluorooctanoate production workers . Drug Chem. Toxicol. 23, 603 620. 5 . Yang, Q ., Abedi-Valugerdi, M ., Xie, Y ., Zhao, X ., Moller, G ., Nelson, B .D ., and DePierre, J .W . (2002) . Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferators, perfluorooctanoic acid . International Immunopharmacology, 2 (2002), 3 89-397 . 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. 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 . 16 . Amacher, D .E ., Schomaker, S .J ., and Burkhardt, JE . (1998) . The relationship among microsomal enzyme induction, liver weight and histological change in rat toxicology studies . Food Chem . Toxicol 36, 839-839. -42~ ~~'~ p . 43 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 TABLES -43~~ ~ p . 44 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 TABLES EXPLANATORY NOTE S ABBREVIATIONS : Summary of Hematology Values 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 width 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 - - no data NC - not calculated or not calculable 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 corticosteron e NOTES : Summary of Hematology Values Summary of Clinical Chemistry Value s Groups with identical values may vary in statistical significance, because tabulated statistics are rounded to fewer decimal places than the values used for statistical determination . -44- ~~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 45 DuPont- 18318 TABLES NOTES : (Continued) EXPLANATORY NOTES (Continued ) Summary of Total Cell Count s Organ Weight as Percent of Body Weight - Organ Weight (g) Final Body Weight (g) X 100 Organ Cell Number of Total Number of = Organ Weight (g) Suspension Cells in Half 100 of Organ Cells (x108) Half Organ Weight (g) X Volume X Orga n (mL) (x 106 cells/mL) - 45 - c~o~-2- p . 46 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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, CV. 5% 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, C.V. 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. -46+.)-JI -~ p . 47 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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 Verification Control 0 ND(B) ---- #1 0.03 0 .0278 92.7 #2 0 .03 0 .0277 92 .3 Mean : 0.0278 0.0001 (92.7) C.V. 0.3% #1 0 .1 0.0966 96.6 #2 0 .1 0 .0979 97 .9 Mean : 0.0973 f 0.0009 (97.3) #1 1 CV 0.9% 0.979 97.9 # 1 (C) 1 1 .04 104 .0 #2(c) 1 1 .03 103 .0 Mean: 1 .02 0.03 (102.0) CV. 3 % #1 3 3 .16 105 .3 #2 3 3 .06 102 .0 Mean : 3 .11 0.07 (103.7) Stability(n) 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 verification . Mean, S .D . and C .V . calculated to verify uniformity of mixing . (B) Denotes not detected . (C) Duplicate analyses from the re-diluted sample . (D) Samples held at room temperature for 5 hours . -47- Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 48 DuPont- 18318 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 Concentration Verifi cation 11-October-2005 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 1 #2 1 1 .02 102 .0 1 .01 101 .0 Mean : 1.02 t 0.008 (102.0) CV 0.7 % #1 3 3 .21 107 .0 #2 3 3 .23 107 .7 Mean : 3.22 f 0.01 (107.3) C. V. 0.4% (A) Duplicate analyses per level performed for concentration verification . Mean, S .D . and C .V . calculated to verify uniformity of mixing . (B) Denotes not detected . -48- 2 G- -5, p . 49 00 o ~ o rn a o o C) ~ r- I'D - ~ ot~ Qoo ~n~ .n M, Q N-- oo M M N 06 N ~O N O~ M N N N a3 0 U ~ N bp x ~" O O O~ a\ d~ Q, bA ~ -x ~ ~ ~ ~ ~ ~ ~ N N o0 M 00 N ~D M M to E M N N N N O ~ CZ ~-~i bA O C/) >~ o 0 0 0 M t~ 00 M I~ M N Q ~O --~ o b 7~ ~ N~ -- N a1 N O~ N OO N u WUMMNNN Cd o > o 0 0 0 0 w r~ ~~ v ~ 'lO 00 t - l - --~ Q (11 C,0 N 00 -- N -- N - M N ~-- ~- M M M M M U u C~. c ~ cd ~ U.d N YYN ~_ O ~ Q 3 G >-, V3 O O O O Q n ::3 E ) cC O 00 0 U Q 0O~ Q 00 0' 1 v p 4> V cM N cM N o V ~ v O M M M M M o O : ~ O U V U bjD 44 O Q~ O~ ON O~ > w w U O ce 00 \ O Q 00 r- kn ch V'~ O C c.' > +U, `.J Q N--~ N--~ M--~ M --~ M--~ Cd sU, sU. 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CO (x (~.' frl Z CQ C~i I C U~--i I W r~C r~C W W FC ^.G ~C ~ FC I-r-1 r- O E I v7 I H H a) k E ~- I a I W I Z O w O?1 I w z z x x H ?+ Z O C4 O z ~~ a 2 Z O I (f) U (1) 1 NA CIS I I U) 2 r~ ~ w I S-a S 4J I I w ~~1 I ~ CQ E~ I cV 1 - - - - I i ~~ ro o (x w E-+ W G cn I b~ 41 .Q - - - - - - - - E+ E- + ~ ~ AO% ,.~ ~~ W O a Q I - - I I I `- b~ ~ I I O -1 G I \\ I O fT U H I I Q I I C4 I ~ I I > I I O 1 I I ) I I I I I - - - - I - - - - - - - - - - - - - - - - I ~D I~ I IS I O I U I -14 I H I \ I H H H H ~-i H I I I CD CT Q) U ~ I---- 1------ - 1 ) o i U 4) 0 z I bi I 1 x I ~ ~ I \ H I H H H I ~ W I O b~ H I I 1 U I rl ~ > I co m I -rl U z I I I ,~ 71 I W I - - - - I - - - - - - - - - - - - - - - - I J-1 G U Ca 1 CT~ I I r~ I H I .~G I I S-I zU i I \ 1 CI P -I I 4 N 0 U I H I rl E I I -0 'CS H CC 1 z I - - - - I - - - - - - - - - - - - - - - - 1 Ill N r-I 1 U 1 iT I I G--1 1 H I ~ I 1 - .-I 4-+ S-I 1 U) I M\ H 1 I ~-ri O .^' I W I ~ H 1 I ro J-1 cn I i-7 I D E H 1 I >C G I- 1 I I I U G H O I I - - - - I - - - - - - - - - - - - - - - - I i'~ H y 1 I ~ I I ~ -~ Cz , I I I -k I I \ I I rl N I .-I 1J U U I I ~S I I O I ~ I H 10 O - rl I I U G~ ~ I I I - I - - - - I - - - - ~ I I 1 I I I I -r-I - - I I m ~a4 CO O Ln U 3 Q ~ O 1 I I Z~6 I I W'~ I O'LS I S-I 1 7 Id ~ 1 I 0 1 I El 14 ~ I 1 I I U-rl TS -rl 4-1 I < N I ~ I 1 Cc] Q., 1 I I ~-ri 4 l co I I E~ I I rl R, UcoN N 1, N ~ cl I I I ro In I 1 I ~ G I I - I I - .-1 G O I I I G O U I 7~ I I I I ro-~-1 '~ I In O I 1 I I G I G G 1 4-I 0 0 II -rl co -H In N N 0 1 0 -- - ~ I -rl I V) roG ~4 W Q I I Q) 4) N I N,~ ,~ I f rl r-1 c~ n I I ~ I S-I Sa r i I ,Q +~ iJ I ~ a S-i I G n N f I N 4) N I G N ~." I I 1 a o. 1 11 N N ro 14 Cd I I U) co co 1 'C~ U N U I I G C C I t-1 -rl 3 U ~" I U I 1 ~ I I I 0 0 0 I 'O I - .i -ri I N G 1~ 1~ L1 1-1 I ~4 -r-i ro I ro ro ro I ro x ~4 4J I ~4 Sa >a I co U ~-~ ~ I I U) co co I u1 ~ U C L^Y I ~ N-Q -Q (D -Q I 4) G-rl O co 0 41 O I ~ G E I I Cx7 10 Ll rr} I 1~ O~ I I U ~4 ri z H 14 .--i I G ~d M N r0 < 'CS ro N (6 I N Q I I ~-0 -w -0 TI 1J I la 44 U) I \ O O U -ri o O O I t0 O N I co I zE E-A < E E-+ cn ~ Ei I C2, ZJ 0 I a I z I o o 1 a H H U) yU) 41 a -1 1 H I (n < r ' U 1 -rl G U I u] I O El E-+ U) I N>C I w I U1 0.,' D U M 1 tl) u) (D -~ "" I `-7 I H w W fi] z< I N S] p~ I I z f~] H I S-I r0 4) Q I I w f1,' ~ I ~ .-i I I UL UL U~ I bi N .~ Q N I I --- ~S~~' p . 73 ~ p . 74 o Q p j ` o~ -00 O N ~~O O'~ t~ N O O N O 0 It v0 l~ oO Mo C N )tnV N ) o~ O M o 0 0 o o o 00 00 t0000 N [ - mI:t .c CD 0 i--~ p~ ~~.O N~ o 00 kn 01 00 O O O O O N M I~ ~ M N N O O O O o o o -zt N o 0 .-~ bA O oN -~ oN ~--. O O CA R:t C) k n O~ tn O O N O O O N O~ M O N ~ o 00 N O O o 0 o O vl O ~n N o 0 N O U > U f~, 44 tiD N~O ~_ rq rq 00 ~ O M O O O ~ M N~ M N M-+ O O O O O O In O N N -O ~ 4-~, O N O W') kn N N_ rM 0N0 nI p M ~ ~t ~ o M O O O In M N l~ m~n M N O O O O O O V.) ~ Ep . O ,y, bb N M M - O M 00 In V'1 00 00 --~ --V') o 00 O O O M M O 00 00 I n O~ N M U` o Mo 0 0 0 0 0 ~ o N~ ~ o .~ o 0 E E E C,~ En e4 x .a.'0 U> 2~ E-~ U p . 75 ~ Q ~o 00 00 cz 'b > 00 IC Q" N~ 0 0 (V ~ o ~ v ) t-- o0 00 00 ~N oM 00 O~ CIA c~ ~o~ o0 00 00 ~no oo ~ CO O U !~^1~ ~ ~_ ~ ^~ ~/ n~r bJ) kn M N ~/ 41 r1 41 3 x ~ O O O O O O ~ N 00 o 0 0 0 0 o tn o .-- .--o O-- o o bp O pp aq V) o N . ~ N~ .N ~ l- o kr) o ~-~ ~^ O rq N O 00 M O ~ 00 v) [- O O O O O O kr) N --~ M N N ~ ~ o 0 0 0 0 0 ~n O N N o o o ~ cz O 7i :G O U cn y ^ Cd -~ a,, O ap E ~ O to 00 ~ U O O ~ O O O v'~ N ~ 00 ~ 0 kn O \~c o 0Oo O ~ kn U a3 C rl -.~ en o N o ~ Q Q 73 to t O O N N o lzt a~ -O -lf n v O M o o ---o 0 0 ~ N M --~ ~ N CO o 0 kr) N o o p V V -o E~ CO 0~ O o o >G OO CN E E x c~i bA in ' vl M N o > v ~ ayi o o --o O O ke) N N N ~n N d U a> ~'" CJ o O o 0 0 o O vl O kn N o o > =o C~ O O Cc r- -- v to bJ) O to bA O ^ v n ~ C 1= .~ oo_ Cd on 7:; Z cz C13 o :5 74 p . 76 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 FIGURES -76- Z~~ p . 77 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Figure 1 Representative Analytical Calibration Curve 1 .80 Equation for the fitted line : Y = 0.0342697 + 33 .368 * X 1 .60 R-Sqared = 0 .99978 0 1 .401 .20at 04 1 .00 Cd a> 0 .800 .600 .400 .200 .00 0 .000 0 .010 0 .020 0 .030 0 .040 0 .05 0 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 . -77~~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 78 DuPont- 18318 Figure 2 Representative LC/MS/MS Chromatogram s 051017Z4M sToc"nh iP HO 100, M?v ,c PM 4.4 8 28246 18154 tr2d-er~ s, LI> 413>369 209Da+004 rri n 0510173e3 Bnzit(IUS20 tiHvtf2dgnds,Ei FM 415>370 1CO. 303 4.46 6g61 .549e+OQi rin 260 2E0 3.00 32) 3.40 367 380 4.N 42 ) 40 460 4.8J 500 520 540 5.63 5.80 600 620 640 40 680 7.00 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 . c61017Ffl70 3rth(*20 bfdvkf2dards,E'i 0 1 rrzjrrL 413>369 pFFD 1.697e+OD3 4.47 152R 14212 % L-_513 581 rrin 061017~ 3roctl(M1At2Ci) Mdvkf2ctards,E> 0 rrzpt 415>370 103- ~p 4.46 861~Ot3`'i 7749670 899782 % 01 260 28 1 300 32) 340 36) 386 4.7 - rrin 43) 4.40 4.6) 4.80 50J 52D 540 590 580 fi00 6i0 640 660 68D 7.0 0 Figure 2b : Representative LC/MS/MS chromatogram of 0 mg/mL control sample . Retention time for PFOA is approximately 4 .5 minutes . -78e7?'56 p . 79 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Figure 2 Representative LC/MS/MS Chromatograms (Continued ) 0510 1TAXO srnce(~ Wv ts2der ds, Er~ sti5 41 3>369 1 AM 7.9T7eC 5 4.47 7IH3H~B 79Li06 rrin 051017ZP621 8rodf(R/426) MdAf2dar E s,Ei skJ5 415>370 ~p 7 .531e+ 005 IOD- 4.4 6 6752419 751139 % rrin 26D 2 8D 30D 3iD 340 3~ 38D 47 431 4.40 4.60 4.87 50J 520 540 5 0 580 60D 62D 4 660 680 7 " Figure 2c : Representative LC/MS/MS chromatogram of 0 .0303 ppm APFO analytical standard (H22703376) diluted with NANOpure water after mat ri x correction . 0510173EBD 3rorlh(Ith2(3) torrg'nt, 1 lfia _ 4.46 74387.07 R361.r1 NRvlf2dar ds,ES #1 413>369 a383 006 - m n 05101T/1~0 97odh(PA~2,Ci) 1 .0rrzJ ~ #1 ~ 2dvnds,ES 415>370 1 ISfD _ a146e +OQi 4.45 72297.90 812389 % nin 260 280 300 32 ) 340 3~ 38D 4~ 431 4 .40 4.~ 4.80 500 52) 540 560 58D 600 62D 6.40 680 680 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 . -79- 2~~ p . 80 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Figure 2 Representative LC/MS/MS Chromatograms (Continued ) 05i017zPM9 s m"*acs> 1 .o myRt,sP ~e pqFO 100, 4.4 6 8~15.17 9= r ffvts2au"5 ES 413>369 9.350a+005 rrin 051017Z1ffi) 8rodh(A" M?hf2dgnds,6 1 .0 rrgYrl, sPle 4151370 ~p aSiik 006 100, 4.45 79(J71~i 8B4ffi8 rri n 267 28 1 3AD 32J 340 3~ 380 4~0 42 ) 440 467 480 500 5a) 540 57587 600 62J 640 68] 68l 7.' 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 . -80C-,~q ~ p . 81 ~ O ~3Qr C-0 -0 , Y ^.D Y bA Y Y GO an E en E E ~ "' ~ o 0 0 O O - - M M 00 -~/ N ct 4-~ O M ~ w MO bA - o0 y O N ct .. ti .d ~ U N C". ~ ~--r---- - ---- - --- ~---- o V1 V M N - O Ol JO l~ ~p V1 O M M M M M M N N N N N (21 ) a42i aM Cpog ueayV Q N ~~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 82 DuPont- APPENDICES -82- a5q p . 83 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix A Certi ficate of Analysis -83~ ~-~ p . 84 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 3058 Research Drrv PA 1eScaOColg, 680 1 RESEARCH T:814.272.1039 exygm .am Precise Resea rch . Proven Results. - CCERRTIFICATE OF ANALYSIS This Certifi cate of Analysis fulfills the requirement for characteriza tion 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 : APFO (Linear) Haskell Number. H27308 Analytical Data : The Purity of the Certi fied Materi al was Established by LC/MS/MS Purity: 19 .5% Last Date of Analysis : 07-November-2005 Re-cert ification Date : 07-November-2006 O rigin of Certified Material : E.I. du Pont de Nemours an d Company Wilmington, DE 19898 USA Testing Facility/Performing Laboratory : Exygen Research 3058 Research D rive State College, PA 1680 1 Prepared By: ~f a Charles Si ns D e Study Director, Exygen Research Facility Management : `L~"Zw 4 1 John Flaherty 'ju a 4I- 4'y' Dat e Vice-President, Exygen Research DuPont-18418 Exygen Research Study P0041843 Page 1 of 1 -84- p . 85 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix B Individual Body Weights -85- ~ 62 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice ABBREVIATIONS : g - grams INDIVIDUAL BODY WEIGHT S EXPLANATORY NOTES p . 86 DuPont- 18318 -86CU3 p . 87 00 .~.' 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M M M M M M N M M M M M M M M M N M ro ~. o 0 .. O Q' M M m ~ s~ b` ~ ~ x rl Ol N M O M O~' .-1 lO oJ N N N M M O ri r . . . . . .. . . L N M tn ~' Q' (`'1 N O M N v' ~O ra M r-1 d' M CD, ID, d' ~ ,'~ CP ?i M M M M M M M M M M M M M M M M M M M M CS N .1 7, O U) "O 3 0 a7 ~ O O a~ ~ W ~ _C Ln lD tn O d' ul 61 M d' ~' d' M O I~ ~O N rl r1 rl Q' p C ~ .~ ~ . . . . .. . . . . . 1 rtl (1) M~ Lc) v' M ri O M N V' ~ N M r-I O' M O .-1 O ct ' ~ 3(7~ ?i M M M M M c"1 M M M Cl m M M M M M M('"1 M Cl) ro rt J 'O C ~ 0 Oa ~ O~ Ol f~ Ol lO .-i m r-1 f m n l0 6l m r~ OJ ri N W N .,~ d. . .... . ... .. . . . . . . . . . N N d' Q' M M O O N r~ M lO rl N r1 M N O .-1 6~ Q' M M M M M M M M M M M M M M M N M ~ oN ~0 CO U ',~ ~ L ~ O r ~O ~ a' cV O-1 r-1 CO ~-1 ~ CU N d' '-1 ul O~ r -,-1 M .. .. .. ... . ~ M Q' Q' M M rl O N N M ~O r-1 N ri M N C~ r-I 6~ M ~?M ("1 M M M M M M M M M(`l Cl M M M N M N M U_ ar Qo 4) [d O ~ ~. (7~ ~-1 . .w r1 N N N rl ~ M~ N ..... r V ..... 4) \O .-+ N r-1 M N~~~ M (~ ',3 ~ T M M M M M M N M N M ae cC C/) O a "O O m O ~3 0 b~ E ~ocorn~rNrnin~ornv~or ~nmoroNV,~ L ~ .. .. .. . . ~ Q) M N Q' M N N O O rl N M~ O M O d (V 6l N 61 Q' a .~ ',3 O~ ? M M M M M M(`"l M M M M M M M M Cl (V M N M d O O O C ~ Q p0 Q (d O N O O N r-I rl N M d' ~ ~D 1~ W 61 O--I N M Q' ~ l0 r OJ Ol O O O O O O O rl ~,' M M M M M M M M M M M M M M M M M M M M ~~ p . 89 0 0_ M 00 O a~ ~ O~ 61 N N N .-~ V' N~fl 6~ ti ~O 6~ cA M ul lD M v' i0 v .{ Ol . .. .. . .. . . IN a) M M M O N O O M N~~ rl M r-1 ~' .-1 61 N W a' M M M M("1 M Cl M("t M M f') M M M M(V M ro 0~ "O Q ~ ~ ~ V' M l0 Ol [~ ~ N~ Q' Q' ~ N N l0 .~ ~ .. .... .. .. . N O' ~' ,3 IT ?i M ro ~ o ~0 w M r-1 M M M M (n .-+ O .~ M N r 1 ~Il rl f'/ l'1 ("1 M M M N rl [~ ~ O~ O .. . .. . a' rl ~ N 61 N N Q ' M M Cl M N M N M ~ .b ,~ ~` 6 , r-1 \D v' O~fl v' .fl In ~D :n O~ f~ lD ~fl rl !~ O~Il M .. . ...... . N M N lf) M r-I N o ri M M .-! n rl O` .-1 V' N 6\ M 61 v' M M M M -M t"1 -(n f`l M(`") M M M M N M N M ~ 0 CQ 41 ~ O~ O O OJ tf) ul d' l0 ~O 7 N r1 ~' ~' 61 N W N N f~ O U) - .i l0 . . . .. . . . . . . .. . . . . .. . 1~ U) V' ~ M~-V N O .-~ M M N l0 N~ .~ ~ N 61 M Ol iIl M M M M M M M M M M M M M M("1 M N M N M b~ N I ~v C~ v b 3 O Pa T O W C: CP M r-I ri ul ~tl N O' LO d' -o ro JJ ~ 6\ .. ri W m .... .. N~ f~ ... . lD . M . rl N O ti f") M M tD N v' N~fl N~ N 61 V' M M M C') M M M M M M M M M M M(M N M N("1 N v' > -d d c m Ij ~ CP M l0 lO M .--1 I~ O N Ol M O ul ul r1 W CU ~D ~Il .,{ v . .. .... ... 0) M v' M r1 N~~ N N N ~O N V` N a' N 6~ N(A v' 3~~ M M M M M N M M("1 f"1 Cl (`"1 C') M M M N M N M ro -0o oa ~~O~ M l0 O r-1 O\ u~ 61 ~--~ lO .,1 M .... . .. co V' ul l0 r1 O 61 :n l0 . . .. N t"1 M O M M(`"1 M M M N M f`") f'1 M M M M M M M N M N f`") ro 0 a> C0 W N O M N O 01 00 I . .+ C~ .~ --i N ,~ CP 7+ M M M M M .. M 10 N O\ Ir N Ir l0 ..... .. M O N t i O M N M f"1 Cl L Y ~, q 71 O cQ ~U 0 ~ N E E L b, b~ .i O \ (V a o b 0 coioWo1o~oCD ~nrn .X T M .-1 N W~ N N N~ bl M fM M M t"l N M M M M M ~ > n ~mM1o1om N~ r-1 Q' rl 6~ ti(V V' M M M M M Nm t"1 M Q 0o ro Q N a N M v' ~Il l0 OJ Ol O~ N M tfl lD ~'~ ~ 61 O o00000000 .-~ E In In ~n In Ln ~V V 00 0 ~~ rP O -~ ~ 6, c, m .. . n r-1 CU ("1 Ol d' O l0 d' W . . . . .-i N r lD ~ ul . . . v c`~1 rl N rl N 61 l0 (V N O r-1 61 lb r O W r~ 6~ f-I ~ b` ~r c"1 M tn t+l (+1 N N<`"1 fh (`"1 f`"1 (V ("1 (V ~`"1 (V N f~ (V ("1 ~ ~ u ~ O Qa JJ ~ tP O r O 61 61 Ol r r n N d' N N M Ol d' O' ~O N i 0 -'{ CO ........ ........ .. N ~"1 3: CP > uro ~0b N T N N(V 61 t`'1 l'7 Cl (h CO r1 f'1 .-+ .-1 61 l0 r (`1 N N mm M(+1 N f`"1 N .-a O N r1 O O M M N(+1 M M m a~ ~ b~ lO fh O~t' d' f`l lD O r N rl Ir N Q' 61 V' Ol ID -r{ r .. ...... .. . . . ~ f"1 M N Q' ("1 O OJ <'l V' rl N 01 l0 [~ rl O W ti O N .~ b~ ~ ("1 f~ ('~1 t-~1 (~'1 ("1 N C~1 C~1 fr1 f"1 N<"1 CV f") ('~1 N(h (n (") > uro ~O w ~ ~ bI m .i l0 v' l0 t0 r r-~ N N~ r-i W N(") N v' rl 6~ :fl r ul O .. .. . .. . . . . 11 ~ ("1 Q' r1 (`"1 Cl O O N~ r-~ N O r 1~ N bl Ol N O f'"1 .C 3 b' ~v [`l (~-] (") l*1 M(~'1 (h M t+l f7 M M l+) N(") N N(") <~ f"1 ~ ro .i ,r u 3 O oa T ~ m r CP N lO N!ll M O O r d' r O -i -rl ~ll ... .. . . .. m O r N(+1 ~ 3 b~ 7. M<+1 (+t C~1 f"1 f`~ c`"1 (`"1 t`~ 'O N .i ? Ca f~ O~ r O O N O .~ .......... cV O .N ~ r`l M f'~1 N("1 ("1 M t"t t"1 r . M f`l -i O T7 CTl C JJ ~ ET l0 N Ol N M r rl N N f`l a' N d' Ol N r f"1 O' ~ ~ -~ ~ . ... ... ....... N 1' .3 b~ ~ c`"1 (`"1 f`-1 b ~ u a O W :n c`l O r-1 t") ~/1 M f`"1 ("1 f'1 ["1 N V' f`'1 M ~-1 r N C'1 M (`'1 N f') f') N O(") N l") f'1 f'7 d' t"1 41 -C b~ f'~1 Lf~ O 61 un 61 Ln '-1 O r-I o] v' f') N .~ 61 Lf) ~ -~ ~ . .. . . ... . CP ?i c+l t~ (+1 M (~'1 N (`~ M (n (~"1 f~'1 (~'1 [~"1 f~l ("1 ("1 N t"1 (+1 t+l ro >, u 0 a cz r ~ r~ r-I . .r ~ .i N f"1 M N N QJ N O .......... QJ (`"1 O~ r-1 N r-~ 6~ N .~ N cC 3 b~ ?i <n ri c7 r~ c+~ r~ N ri ri ri ~ u 0 _ ~ ~ ~ CP b~ r~ O N^ N N o~ N CO O iIl r f-1 r-~ ~~ O~ ('~1 O r O ~ Q1 ("1 d' rl N N 61 f-1 N V' O O Cl t"1 m (n f"1 N t") (+1 t+l n'1 !~ [`l (h ('1 ('"I C~1 N (~ r) ~ 0 E ~] Ob N 7?5 u O ~6 O O O ~ r r^ r ~ ti > a-~ ~Nriv~~n~rmrno~Nr-~~~n~ormrnID O O O O O O~ .-1 -+ r1 r' r-1 r-~ rl r~ .-1 N r r r r r r r r r r r r r^ ; r 1 '20 P . 90 ~ P . 91 ~ 0.r. ' -~M 4--I 41 -C~ LP ~O O N M r Ol d' ~ vOJ e--1 N r M Q' M N Ol O O . .~ Q~ .................... 4) M u) r~fl v' r-1 N lO lO v' O r O 6~ N N 6l 61 Ol m ,~ CP .'h N N N N N N N N N N M N M N M N N N N M ro ~. n d 0 w ~ ~ b~ CD ,~r -+r oorn Lnmorn~ocn~n m .~ ~ . ..... ... .. N M ~D r Q' M M M .n lD a' o W . 61 M M 61 6l 6l ifl ,~ CP ~ N N N N N N N N N N M N M N(") N (N N(V M ro >, u ~0 w ~~tP ul Q' V' l0 N lD a' r~ N N~~fl Ol 61 W Ol 61 o M . .~ r ...... . ... Ql u~ r OJ N d' v' d' ~ OJ l0 0 6\ r1 o N 1 . OJ 6\ 61 V' ~ ZP ~ N N N N N N N N N N(7 N M M M N N N N M ro a u ~0 W ~ ~ u) rl ~ co d' N ti M M f~ O~f) M r~ tN N ry N N O . ...... . 1-j Q) m O N d' lD lD lD r O cA M O~-1 .-1 vOl O r-1 On .G 3: CP 7r N M N N N N N N M (N M M M M M N M("1 f"1 M ~ ro Q) 'o 3 0 ~ aa o o w .c IP rl ul .n M N l0 t+l Cp r o .fl .. . . . .. o~ .. . o . ti~ N N O N T O] (5, CO r O N NLn r-1 M N~1 6~ O N O tn ~ CO Ol >~ M M N N N N N M M M M M f`l M M N M M M M >. u -rl T G F o CO 1j I -rl d' d/ rl M ,3 LP T N >, u b ~ Qa O M CO o7 t0 ul ti O l0 m lO M r1 m r q' co ul Ln . .. .. . . f~ N M 61 O M M r N M M~fl O 01 N r-I \O M M N M N N M t"1 M Cl M M fM M M N M M M r1 61 o M 61 r ~ G) cc A~ ~ CP ~ M,-I N M l0 N O Q' 3 CS ?i M f') M M rl ~~ .-1 l0 6~ o' lD l0 M~-1 O CO M M~ O l . . ... . O O N v' v' oJ (N~D~ M M r-1 U-) M M M M M M M("1 M M M(`'1 N M M M ~ u O ~ .-~ ~ on .i N ~ rP W T M t`l 01 O N NM ID W O~ r N .... . ... M M .~ n ti Cl N rl ~ M M M M N M t"1 M T uro o ~p . ~ C) O X c tP - .~ O~ x ~ r 6~ o tr~ E c) M N d' Q' a' N N r~ .-1 P M :~ N r Q' rl rl .. .. . . .. . M M* M M M M M N MO M M M M M M M M M NM M M M f..,'" ~ x p7 H Q N ~.-. -i ri N M V' Jl lD ~~ 61 O r-~ N M V' ~ i0 r N Ol O ro o00000000~ .-~,~ ~ rn rn rn rn rn rn rn rn rn rn rn rn rn rn rn rn rn m rn nr b ~ ~+} ~ C.' P-~ ~ ~ .~ ~ n N r1 ^ l0 N 61 M t") Lnn Ol N t"1 M N . ... . . .. ~ 61 ~f7 l`l Ol Ol f~ ~) N'1) ,-I O N lO Ol d' O 61 3~~ N M M N N N N N N~"1 M M N N N N N(V N ro ?i u "O O CT~ ~ ~ O~ N .~ ~ Q) O ~~~ f~l ~ ~ u T1 O CO O~ ifl -n MOJ ... . ... ~n M 6~ ~ ~i ' n f ~ f`"1 M N N N N N N M .. . O N Q' M M N ID ("1 O a' Ol ...... ~ OD, O01 ~f1 M M N N N(V N ~ ~ b~ N~I) lD ~O l0 CU ~n CU M 6~ ~O N l0 V' .~ ~ . .. . .. .. . a' v' rl. f~ N Q) oM 61 1~ fr m O CD* CO 61 I')' M, oJ a' Ifl 3(Ji ~ t") M(`l N N(V (V N N IN M M N N N N N N N >. uro b0 w ~~~ O `Z' CD M~ N rl ~ yJ Q) O ~n ~ rl ?i N TS 3 0 Oa ~ ~ o i ro u N 6l t"l I~ dl 61 07 N[~ OM O r-1 O ~ .. . .. .... . m O OJ ~O l0 6\ 61 O N O O O In ~f) 61 <l' f~ In C7 N N N N N(`"1 M M(`l ("1 N N N N N ~ Ln Ol 61 z, ~O ~-1 Ol ~ N 61 f"1 '.n 6l l0 O O a' M d' .-i -rl ul . . ... .. .. .. . . ~ fd 4) N u) M(N .-1 N 6~ rl N O IN .-1 O OJ m rl ,D 6' a ,'3 ~~ M M M f'1 (`l N N M M Cl M N M(") M N N M N N ~ ro rl 7+ u ~ TJ -I O o oa c ~ ol O .~ ~ 4) N Ul ~`l WM . . M N O\ M WM O M 61 O V' rl N d' ~O . . . . . 6l N M rl l0 ~ M N r-1 O O rl CO (") (`"/ N N t"1 f'1 (`l M N M M("1 M M M N M >. uro ~ 0 m ~ ~ rl .,.~ M 61 Ir l0 l0 . v' .. .~ fr r1 Nn N v' r1 Lf) N 6~ ~' O~ lO . .. . . . N M l0 V' f") d' r-1 r1 f") l0 Mn 61 d' N!-1 rv O N Ol Cl 3~ 7. ("1 M<"1 t"1 ("1 ("1 Cl f'1 M M('M N f`") M M M M M(V M ? u O U) ~ ~ T N ~ In .-1 M N IN* IN* M 3 ZDI ~, O M M M Cl M f'1 M M(") ("1 N O ~ u N -uo O p V 4) a ,~ "S O ~~ c IN ~ t~ zT .~ o IN M M M f"1 M M r-i~nNIn`Z)~ . ..... .. ... .. . O O N Q' r-i ~O .-~ M r-1 N N N N- M M M M M M M M M(m M M Cl f~ Ol M T o ~- + m x Q Q ~ rl N N ~ M Q' n ~O r N 61 0~ N M ~r ~ o 0 0 0 0 0 0 0 ID ~-+ - - I - I - - - - - - .a .- .-i - - - ~ - -i r-+ - - ~n ~o r co rn o - - - - - - - ,-i - - .-i p . 92 N 00 + 0 ~J ~ tS f-I .~ ~ lO 61 .. (V .-I ~' lO a' Q' cf' O d' a' f ti~ N N r . ... ... Q) .~ --I v' M M O' O rl rl Q' a' l0 M Q' N N M ~Il M~ ~ b~ M M M("1 M f"1 M M M M M M M M M M M("1 f'1 T ~ ro -o 0 0 cq ~ ~ ~ r r rl OJ r-a N ~O 6~ O' d' ~' rl 61 N O N l0 Ol r .. ...... ... .. N rv O~' M N Q' O r-1 r-1 V' ~' ~D M M N r-1 M v' N~ 3 b~ M M M M M f'l M M Cl M M M t"1 M M M M M M ~ ~ b o a O m ~ ~ .~ ~ 61 O rl fr Ir O Q' co r m r1 v' N 6l Ir c0 O ' .... . . ... . U) ~ O V' N(V M m O rl M M m N~' N c D* T N Ul ,3 YT M M M M M N M M M M M M t`-1 M M M M M M ~ a ro u a 0 w ~ ~ CP ~ N~1 m .i ifl J-~ N r-' O d' .G ,'3 bi M M M OJ N W~' r N~ r M~ N M .... . .. .. M ry V' 61 r1 ri ~' ~~ N(`"1 M M(V M M("1 M M M M M CO M iY' N . . .. N O N a' N~ M M M M M -rt ~. ro 3 O x] a -O o W .C to 61 O O 10 O 01 M M O M O O N i0 a' -I 01 -i .-1 v . .. ... ...... ... N N O ul M N V' 61 .-1 ~~' M f~ N Q' N O N v' M~ a ~ b~ ~ M M M M M N M M M M M M M M M(`"1 M M M -~ T ro > -d a ~ o v m c ~ ~ b` M IQ r ( D O O M M I- rl l0 l0 N n (V ~~1 M . .i M .. . ... . .. ~ CP M ~ ~ ro -o a 0 w f'7 M M N N Y' O~ rl d' M ID* N M M 'm M M Cl M M M M N O N M N a' M M M(`-) M U ,~ CP M ~ ~ CP CO Ir ~ r r N~-/ .-1 Ir O CU n r r l0 Q' . .1 N - . . . ... . . n \O ~D 0) .~ O o' tV r1 M 6~ ri f-I ~t' d' ~ N M~ c D, M(V ~tl M M M M N M M M M M M(`-1 f"l M M f') M M O N S3 4J ,ry' ~ . .-. CS' aW ~O ("1 N a' lD 6l 61 O CO l0 ul r-I 61 M N w un O ..i .-1 ................ ... M Ol O rl Q' M~ N Q' ~-i O N M N ~O O .~ ,3 b' ~ M M M f'1 M N(M Cl M M("1 M M M M M M fM M C c o T a ro o ~ G ~. . o X ~ O Q) r.y' N CP ~ <1' ./1 ~fl o N r ~fl l0 rl Q' M N,--i N r-1 W l0 ~ rl --~ o x ..... . . .. . .. . .. rl \ ti VN rl N 01 O~-1 d' a' V' N~' r1 O .-i M ti~f ) zy cn M M c"1 t"1 N M M M M M M M M Cl M M M M o a o W H E F~ N Q O O N N O f-1 N M V' n i0 r CO Ol O O O O O O O O .~ ~ ry ~-1 N ti .-a ~,ti N E .~ .-+ - - - - - ti - - ,-r - '~.~ 1 p . 93 M C.'. O ~ ~ ~ ~ f"l f") r rl 61 0~ rl r r-I OJ CO Ir e-i N o r W r~ Q) r-1 N' ~fl l0 ul ("1 <") O("1 lD N f'1 f`") N' N C), M, O l0 '~ (T ("1 f"1 ("1 Cl ("1 M f`'1 ("1 C7 <"1 f"1 ("1 f"1 f'"7 f'l (") M N C') f`"1 ~ >, -d ruo 0 w ~ .~ ~ r-i~r a~oorm~o~~~oa~v~rnririor v ~ . .... . .. . . .. Q) ri d~ tt~ l0 ("1 0("1 rl d' Ir N f`"1 N In N o o ,'3 ZT Cl (`l ("1 M("1 ("1 (`") f'1 (`") M M M f') C) f'1 C) f"1 f"1 N f"1 a >, -o ruo 0 w ~i(P o ti r-I N 61 (`"1 OJ 10 O] o lD r m co r r1 M .~ 10 . ... . . .. . .. p) rl . N Nm N O d' ~O N In N M N C O O ~O ~ ( P f"1 f') f`"1 M f"1 c"1 N f"1 C'1 M f`'1 M M(") f`l <"1 f`l Cl M M ~ N ~O u 0 m i~ ~ CP N O[" ~ n ul Ln r~O ('7 61 lD In N~P f^ N l0 r d ' N . . .. . . .. J-~ N r-I ("1 ~Ln Q' Cl N O f"1 r1 v' In r-I f"1 N f') r-~ m O 61 61 ~ ,C: '3 (7l t3) - .1 ~ a~ o 30 ~m -d o ~ s ~ r-I ~ .i rd ~ c"1 f7 c"1 In f`"1 f"1 In c"1 (") C) f'1 In M M f'7 ~ ro u N OIn~ N l0 Ln O O bl O Ol l0 6~ O("1 ~' . . .. . .. .. rl (`l f"1 (V O M rl f"1 lD .~ f`l N- In N N<'l 61 .-1 f'1 In . .. NOO ~ a f'l c`'1 f'1 ('l f`l M f`"1 M c"1 mm In M("1 M c"1 N f`"1 M["1 > ~d u ., o "C3 m ~ ~ ~ fr .~ m N m . v ri <+1 In ill d' 3 b' l+l f"1 In f"1 <`"1 f'"l ID r-1 N N o 61 Q' CO r Ol N Ir o 0 . . . . .. . . . . (`"1 (V O IN ~~t' M(V O O O ~f) ("1 f"1 C') f"1 c`") ("1 <`') f"1 <"1 f`l M(`') In Cl ~ roT -o u 0 m~j ~ ~ .,-1 N l0 In T N f"1 o N CD OJ OJ o CD .-I o W o N(`"1 N r . . . . . f"1 (M ) N O~"1 r1 t"1 IO N a' N M t`"1 O O O ~ ,'3 bl In l"l In M("1 t`'1 ("1 f'l t"1 M M M In t"1 M("1 <`') f'1 f"1 M ? b d u O N cz ZT ." -,~ ,~ C b 'd u O In ~o r o cn o~ Cl W f'1 f-1 N l0 N~--1 v' If1 ~f7 NI n ...... . cn ~~ T<V r1 O cV ~~"1 'O. r-1 ("1 N I' M C), D ,OlInriInrncnInInInririInrncnCl)mrnr > a ' ~ ~ 0 N Li N ~ ,3 ~ O G (P E N~ l0 rl 61 oJ 6l N ul ul 61 ~O N tn 10 .~ ~ . . . ... . ~ ("1 Cl Q' ~ V` N rl 6l N rl Ct(ll .-~ 1 <"-1 -I i [+') t"1 t"1 f"1 M f~l N('1 M~`l I`"1 I'"1 <`"1 `'1 O ro u "OO co ~a (. M I'1 ri M oJ v' .... N O O 61 f~l lM In (`"1 N t") Q N ~ -I r-I N M T' ~fl l9 ^ W 6~ O .~ N t`") O' ~~O [~ ~ 61 O o00ID ooooorl- - - - - - - - - N In M("1 (`") f`"1 f'1 (`"1 M t`"1 (`") (`") ('~1 In Cl M f"l (`l Cl l`") (`" 1 ~ ~71 ~~ p . 94 a M O a~ CP W O I~ r N f~ M N CO OJ ..f) O~ O\ C Cp ~fl d' N N M ..i N .... .... . U7 ~ d' d' d' N M C) r-~ v' a' r-I ul M In N 1O M .~ in O ~O 3: bl M M M M M M M M M M 1"1 M M M M M M M M M ~ o ruo 0 m ~ ~ - .~ r ~ ro o a 0 m N. 6) e-1 ~.' 61. (". 1 Q' . l. 0 O . 61 . r T . lO IN In ~n M N .. .. In CD . I n N N M rl ~~ rl ill M I n N lD M ti V' 61 in M M(`'1 M M M M M f") M M M M M M M M(V M ~ ~ CT .r/ l0 N . N ~ cT M ,~ CT M M M M ~ ~ o rou 0 m In . O' M M N M~ 61 M l0 Cl Q' N ........ f-I IN IN O M M .~ In M M CO M f"1 l`"1 M M ~O .. O' N M M O1 M 6~ o M ID ... U) N O V' 61 tf) CM M M N M ~ ~ m -rl ul 0 61 ~' \O N L ) OJ ~--1 r~~O CO N r r N l0 Ol .. ..... .. .. .. l.~ N N Q' Q' ~fl 1--1 N N r1 v' M r1 V' N ci' .~ ~n ry O M m V .C 3~ M M M M M M M M M c"7 M M M M M M M M N M ~ T -~ T ro N 'O u 3 0 m ~ ro o co .c ~ ul ~ -~ ~ 61 M Ln 61 r N IN ~D M 0 6\ N M v' M .- 1 . . . . . . . .. . . . .. . . ~0 U) ~ 4' ~' d' r1 N r1 O a' M Ot) N ~n N ul N O M 6l l0 ~:l 2z: CP M M M f~l ("1 f'1 M M M M C) M M M M M M M N("1 ~ T ro > 'd0 u is m c .~ ~T N N lD ..a M 4) r-1 f"1 M l0 .. In OJ CD M~ P Ln CD Ir C) N CD M M un .. . . . . o Q' M O tn NLn rl In N O M m ~n ~ O~ M M M M M M M M M M M M M M M M M M N M ~ >, rt1 T3 u 0 m ~ ~ CP M d' N N M r r1 d' IN ~1 V' M r N ri .-1 N("1 r1 N .fi N . ..... ... 0) r-I v' M V' rl N r~ r-1 M M O Ln N T N ul N c D* 6l ul Q ~ Ln ~ M M M M M M M M M M M M M M M M M M(V M a> ~ ~ -o ruo 0 m 4j Ol r-1 r Y' d' Ln Q' 0 61 o N'n r1 N Ir n 61 cm ul O~ ~ ^+ .-I r1 . . . . . . . . . . . . . . . .. .. . N ~ d' M a' ~-1 IN M M O :n N U1 r1 I n C cD -ICC '1O O .~ ,3 O~ ~ M M M M M M M M M M M fM M M M M M M(V M ca V) -d uro o m Q x ,~ ~ Q rl J]~ ;5 3 YP Cl bl CP O .X fM f`-1 M CO l0 M .-1 .. M M v' r M M M 61 m c) l0 lD l0 N O cT tfl N l0 l0 O ~n Ln . . ..... . ~-1 O .~ M(V O In (V Q' .-1 <t' ti 61 M W v M M M M M f'/ M M M N f'1 N M G~ > ro ~E a o .-+ C ~ m > o E C] a E ~ N O O Cl O ,n ~O r~ pl o r-~ N M Q' :fl lO '~ OJ O~ O O OO -.n In In I n ~n ui .n Ln ~n Ln ~ ~ ~ --~ a p . 95 n p . 96 ~ O ~ r ~ rl M M O~ W O l0 I oo . . v ~ N N O 61 O r ,3 ~ M M M N M N N M T >-o ruo 0 f M r-I 6~ N ~ O o 61 r 61 N M N N N M N .-1 ~' 6\ M N N r-1 OJ ~ M W . Ol l0 M C), 1 N M M N ~ ~ (7~ 6~ .,i r v rl rl O~ N Q' M 6~ r1 W d' r-1 M M N O dl 61 6l 10 m O .. . .. . . . I~N O M, M 6 O l url O61f"1 OoI~ D I OO O r 2z (Jl M M(V N M N N M M N N N M M N N N M M N ~ ~ b ~ u 0 m ~ ~ .~ ~ lO r r M M ry Q' r ul l0 \O O' N N v' f') 61 N~ ~ .. . .. . . . . 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QJ .~ ~ r1 co r o~ v' ~ 6\ r 61 N r OJ M l0 ~ .-1 ~f1 ol N C`1 N N M N N N N N N N N N N N N N N ~ ~ ro 0 m fn ~~2T O~ ~O v' OJ M N v' N O r r CO N l0 OJ tP rl L( 1 . . . . . . . . . ... . . 1~ 47 .-1 Ol N 61 61 O l0 Y' O M M M oJ ~O O] v' ttl n M ,n ~ ~~ (V M N N M N N f`"1 ("l N N N N N N N N N N ol ~ 3: o a oa ~ o m ol r-1 ~ ~ .-i 61 M O .. . 7 N ri OJ O 61 61 N l0 a ol N(") (V N~`l N N ~ O O 61 W N~O Q' ~' cf' R' N O OJ N N . .... .. ... f"l N V` r 61 61 r W Cl N u~ ~ l 0 N M N(V N N N N N N N N a T ~ ~ ro > 0 a fn c ~ ~ ol .,1 M n f ~~O V' CU M~' Ol N r O o 61 O M M M I-) . .... ... v ~ CO ~-+ Ol O~ N~~' r('1 r 61 o r O] d' r~O M\D 3ul N M N(V (`") N N N M N N M N N N N(V N N T 7. m 'O 0 0 m JJ ~ ~ v' ~ N O N ID r O l0 W N ~9 M ~' r-1 m lO r N rl N ...... .. . ... . . O ,~ CO t`~ fi 6rn ri ~o v' v' M cU O .-~ Ir CD d' ~ r v' CU ~ ',3 ~ N M(`"1 N M N N N r1 N(`") f'1 N N N N N N N y ~ ro 'O O 0 a ~ a ~ s~ mor ~--~~rnNMOC~'~ .n ~rrnornm .-imr ......... . .~ ~ ~ O ,-~~ ' o~ M~ 6l r-1 d' ~') N r'-1 o rl r 61 ~I' C'1 N ~n W ~ 'T 3 ZT 0 N M(+~ cV M N N N N t"1 M M N N N N N N N Q >. U y ~ N N O m O ~ O O c b .,..~ o r, .~N~,-+vvornmvcoriwo~o .-+r o~ . . . .. . . . . . . ........ ~~ v ~ O O~fl N CU ~ r V' N~ O O ~ W N V' rl ~O d' ~O a ,~ '~ ~ \ c7 M M N M N N N N('~1 ~`"1 M N N N N N N N 0 li a o r0o ~ o ~+ co x c Q N ~ N M~' ~'l l0 r N 61 O~-1 N("1 v~n ~o ~~ m rn O Q N E ,~ - - .-a -.i ~- -- --- -,- ,--r --,i-- -- -- ,i-- -ti I o0 M O r b~ ~' r\O r OLn r1 lO ri ~n co Ol 61 61 m m N d' N . .i r .. . . .... . N N l0 ~D r r l0 M .-1 ln r-1 in r M N N~' rl O rl rl l 0 ~ CP mm m" mM M M M M M M M M M M M M M ~ T N a o O m ~ r CP O 6l .i ~D N N ,3 2T ~ ~ ro -o ca w0 N ul l0 r V' r ~O r1 M 7 r rl r-I cV .-1 rl N N .. .. .. .. . . .. . lD m I'~ mr in M rl d` rl tn r M N M q` (V O r1 rl ^ Mm mm M M M M M M Cl f'1 M M M M M M .~ ~ CP .~ ~n M m m. .-1 rl Ol in ri ~Il 6l .... .. N W 6~ ,-1 r-I ..... r ti . m. ul . Ln. N N l0 lD fr r in M r1 ifl rl O' r M N M~fl N o~ rl r '.~ CT M M mm mm M M M M M M M M M M M M M M ~ ~ b 'O O 0 m ~ C P ~f' d' M O N r 1 f~ l0 W r M O~n ~fl ul M MN O ...... .......... l~ N N ~n lD lD l0 ~fl M ri o' .-1 d ~O M v' N V' N Ol O O r ,C , ~ CP M M M M M M M M M("1 M M M M M M N M M M ~ ro 3 o Q m ~ a o .j m G LS O M l0 M v' CO cT O .-i .i M . .. . 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U N ~ M vl0 ^ ~n c1M O v' r 1 m lD N M N M rl a, ^~ ~ LP M M M M M M M M M M M M M M M M M Ol N OO N O dM ~ OM tn E E a ro o 0 rl ti ~ N N o O o lO Ir m m O rl N M a' ul iD r W 61 O o O O o oO N E M M f~l (") M M M M M M M M M M M M M M M f") ~ ~~ P . 99 p . 100 M 00 0 ~ a~ ~ ~ v' ~' Ol M~~ d' N N N~ O' N!~ M Ir Q' [~ ~D .~ r . . . ... ... N N ~ ~O M M M rl M e-1 M N~' ~' O' M rl M ~' d' Q' 3~ M M M M M M Cl (`") M M M M M M M M M(+1 C') ~ ~ ro '~ u O ~ ~ (JV' OJ ~ d' ~ v' f~ r-I M lD ~O N ~fl l0 Od' ~' ~I' M .~ ~ . ........ .. .. .. Q) (V ~~ M N M r-I N~ T V' t~ a O' M N M ~ d Ln 3(J~ M M M M(`") M M M Cl M M M M M M f'"1 M M M ~ -o uro 0 oa s~CT N l0 ~f1 [~ N d' ~' ~ 61 l0 ifl 61 O' N ID OJ OJ N .~ -,~ N . ...... ...... . 4) N rl ~ v' N d' r-1 N r~ C1 ~' ~ M v' v' N M tn ~' ~ 3 ( 7 ~ ("1 M M M M M M M M M M M M M mm M M M ~ T ro O u O '.,Aj ~ t7` r1 6~ O~ n O MU-) 61 a' ~Il r tn r[' Ir O m . .i d' ....... .. . iJ QJ N -1 ~' ~P N a' ~(V r-I M v' ~' M~t' M r-1 M Ln MU-) L 3 b~ M M M M M M M M f"1 M M f') M M M M (`l M M ~ T ~ ~ v -o ruo 3 O ~ ~ ~ O a~ o L17 ~ O ~ N rv v' O lD M f~ OJ N un d' ~Ln lD Ol .-I CO oJ cT .-i .i M . ........ . .. . ftl 4) N M Lo M u) a M M M M M M c") M f'1 M M M M M M M M mm -O ~. -fl T N J '~ u "O 2] C ~ ~ pi rl .,~ N v N 3~ M O I~ N~ N ct' N M 6l ~O <t' ~O v' rl 61 (V V' ~ . .. . ... . . .. .-~ ~ M N M .-1 N N d' d' M M M M N M ~ M a' M M M M M M M C') M f"1 M M M M M M M M T ro ~ u O W ~ L ~7N N .~ ~ f-1 a' . d' f . ~ O] . M[~ .. l~ . Ir . ~I' r. O M . !. - 6~ -_ n . d) N -1 .I1 v' f"/ d' O M N d' ~1 M ~tl ~ 3(f ~ l"1 M M M M M M M M M M M M f~/ M M M M M l0 -O Ll O cfl cd .~.i ~ N . .r ~,,,~ 3~ +~ 61 M a' O oJ f~ .i O ~ N .-1 T M M M M M M M M M ~ ro -d u o 61 d' M M ID NM O O O N M V' O M MM M M M M M M M M ~ . . ~, 0 p p~ O~ o~ vM vo N .-i M o u~ m rn~ N u~ o M m .~ rn x . . . ... . .. ... Q~ ) rl \ .~ ~ Q' M M d' ~ N N Ul ~ ~ lD N M N~ M ~T ,.~. 3 ~ M M M M M M M M M M M M Cl M(`l M M M M ~ C) C's m ro O ~ . ri ~-1 N M d~ lD r CO 6~ O .-1 N M Q' ~fl ~D I~ O~ 6~ O O O O O O O O O O N - - r+ - - - - - - N rl - - .-1 e-1 - .-i - .ti - - - f-1 N r-1 .-1 r-1 rl r1 .- i r,r-C7- 71 7 p . 101 ~ O ~ ~ ZT o o M N d' lD r N M o o Q' N~~' \O r o 10 61 -'{ r .... . . . M In .~ ul In M WIf) \O N IO N N QO ti LIl 3 CS M M M M M M M M M t") M M M M M M M M t"/ M T ~ a rou 0 ~ ,.~ L ~ N l0 M 61 6~ M\O N o~fl N M N o O' W~fl r l0 ~t' .~ ~ ........ .. . .. . 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N N v' M v' N N ~f1 O M~ v' ~Il (V ~ N N ~O O ul 3 CP M M M M M M M M M M M M M M<`"1 M M M M M T T b "O u O CQ v' lD N~ N N~~~ ~ V c JJ -C O~ rl 6~ N -rl r-1 N N ul M~' >1 M M M O Q' O l0 o CO O N r1 . ... M M orl v' ul N ~D Q' lO M f"7 M M M M M M M M M N O V' M .. N r M N M M M M N O . ~O M M 6~ M a RS 'O u 0 c~ . .-, 4j 1 - .i o In r o Ol N lD t`") l0 61 r rl r ~T M N W Q' O\ ~ r .................... N N v' M~Il IN MLll N ~O N .~ d' O Lft CC .~ 3~ M M M M M M M M M M M M M M M M (`"1 M M M ~ a r o ou o .~ o X ,~ ~ p tT b~ - .~ rn r~~ r rn M N io vw m ~o N~ M Ln ~n cr x .. . .. . . Ir) N In t"1 I n ' N ul N r 1 d' 61 I n ' CP M M M M M M M M M M M M M M M(~l M M N M ~, ro E 'd u ,-~ O J E Q ~ N ~tl o 0 0 N rl rl N M Q' ifl l0 r N O~ O rl N M ]' Ln ~O r OJ Oi O 0 0 0 0 0 CD .-1 ,-4,--4,--4,-4 .-~ rl -i r~ rl N in~n~n~nui~n~n~n~n~nLn nn Ln ~~~ W a~+ ~~~ W~' .~ r 61 .. l0 .-a \9 t") O N~ M r N ri r ..... 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(V CO N C'l Q' Ol OO lp 6~ 2:~ N N N N N N N N N N N N N("1 N N N N N ~ T ro -a Q 0 m ~ ~ tP 61 .f{ ~n N N - ~ T ro 'O n 0 am O r O r . .. . . rl f"1 f-1 Ol d' (") ~O N~n fr Q' .-1 61 (`l . ... .. . O` f"1 m lp \p ID CO N(A N N d' Ol CO ip m N (\j N N N N N N N N N N N l`"1 N N N N N ~ b~ vo rn m Ln - ri m v~n ~o rn,~ ~ ri r~ o nr m .~ v ..... . . . J~ N N a' ~' O' O' N f`"1 \D l0 \D 6l N W Ol N ~n O~ (A r CU ,~ ,~ CP N N N(V N N N N N N N N N f`"1 N N N N N N 'O q 3 0 P~ T ~ O 4 1 O~ w f'1 6~ t"1 Ir 6~ W l0 f'1 t") T Ln . . .. . . . . ro 4) N v' f`"1 f7 t") l0 lD ~n 61 t"1 61 01 N u-) a ~ CP N N N N N N N N N N N N N("1 N N T~ ~ 1 ~v > -o r 0o --+ o -o w c m Q~ f"1 N .. m r r N N N ~ ~ ~ W .,.~ N O rl l0 . lp lp .. \p N ti'.f) N V' 61 ~' ~O M a' ~fl Q' . . N N Cl ~ u~ v' ,-I M l0 ~Il ~n 61 ["1 6~ al t`1 Ln Om r W ~ CP N N(V N N N(V N N(V N N N- N(`-1 N N N ~ T N 0 m ~ ~ z3l .~ ~ Lc) W r f~ N ~O I~ O r W r ul N O O ti~ N r . .. . . .. . N N .-1 N Q' f~l N C1 ~ p' V` r N CO N f"1 Lo O CD N OJ ~ 3 CP N N(V N N N N N N N(V N N(") N f"1 N(V N c C 7. N 'O q O C) co cz Cr (Na) c) f"1 O r'.n Q' O r l0 lD OJ n Co m rl r1 ~fl ri O N N f'~1 N ul l0 V' m ~n 1' lD r C'1 N CO N Q' Ol OJ N m (~ .~ '3 CP cV N N N N N N N N N N N N f"1 N N N N N 0 ~ Y r x y~ v ~ E a ,~ LP N(V N N N N N 1) ~o ID r ^ ~o N o~ o m ~o orn co rn m r~ 'nri Lnrivrn~n .n~nrricooN~rrnrr o N N N N N N(`-1 f~l N N N N M ~ ao o x Q + ~ f`-1 V' -n lD r W ( DM O ti N f'1 O' in lD r O] 61 O ~ ro O O o 0 0 0 0 0 0~-I rl - r-1 r--1 '-I rl N Q N ~ rnrnrnrna~rnrnrnrnrnrnrnrnrnrnrnrnrnrnrn ~ ~4 . .~ p . 103 00 C0O ~ ~ ~ CP N N V' r N r M V' f`") l0 c!' N~ r r~ v' r Ol 61 .~ r . ... .. . . . . ... ~ N O l0 r-I O~ r-I ~'l O' W a` v' 6~ N 6~ 61 ~ tn r("1 r 3 O~ ('1 I'1 ('1 (`"1 N N N(`") N N M N N N N N N(V a ma M0 ~ ~ b~ .~ r1 ~ 3N: aNl O a a ro b ~ 0 w N N 6~ ~O ~n l0 O~ a' (V N N O\ O~ O O N ~D . .. .. . . ... a' O r N ~n ("1 ("1 Cl M~O N N(`"1 N N IN M 6~ N f'l ri N 6~ N ("1 N M~D r d' N N N N N r N ~~ CJ~ ('1 0 61 Cl In ~D l0 N r l0 u~ ("7 ltl rl In ... . .. Ul N 61 ~' C O r NLn (`l Ln O) N CO c), I . 3 CP N(`"1 N N(`"1 N N N N N N (`"1 N a a m -O o 0 ~ rl lD l0 6~ ~n lD .. m f`"1 M l0 O' (O N N N IN IN J-1 ~ tP o r ~n ~o vvrn ~o ri (n ~n v~o (n rn o ~o (-~ r N rl ~ ........... ........ JJ 4) N ~ N O~ r r-I ul ("1 M r N CO O f^ dl ("1 it) l0 (`'1 ul tT cV (`"1 (N N('l N N N (N N N ('1 N N N N N N N ol a i a N N O C] 3 0 ~ a O 0 r~, zT V' CO r ti N N W f~l ~n C) f1 ~ rl rl ......... t0 Ql N 6~ (~') N W("1 lD d' f"1 ~ N CO ~3 0) N('"1 N N('"1 N N N N N N (`l _0 rl a > 'd rl 0 -0 m c a ro O O .1 N(`l f"l l0 QJ l0 ~ .. . . . .-i (U 61 O' M[- (") ul N N N N N N N ~~ol .-I r1 iD v' r 1~ O~' Ln <t' tn () 6~ N N OJ d' N O N . . . . . . . ... .... . .. . N N 61 (") OJ l0 O In ~n (`l a' Cl OJ rl l0 6~ C7 f`"I lO r'1 ~n tr N('1 N N(`"1 N N N N N N M N N N N N N N a a ~0 ~ U O C'Oj ~ ~ ~' lD r1 O V' 6~ M iD r O in r ul N 6~ l0 v' v' N .ry ry . . . . . . . .. .. ... .. .. . Q) N I~ N~~~'~ tn C'1 f`I N ~O W O N 6~ N f") ~O N d' ~ 3(7~ N(`"1 N N N N N N N N N f`l N N N N N N N N 'O O 0 m ~ ~ ~ a ~+ b~ Sa r-1 m Ol f~ N O r-1 N 6~ d' N OJ co Ol r m O W d' . . .i O N ....... . .. . .. . . . U N ' W(V ~n r~ r(`"1 Q' N W N O n m M("1 ~O N V' C~ .~ 3 C7` O N(`"1 N(V N(V N N N N N M N N N N N(V (V ~ a U a ro v -0 d o a ~ ry, x o +' \ O ~ ~+ QI r 1 a ^~ 3 CP \ O N O~ E In nrnm~nIn ~r ,~~ornmo ^ N r I~ OJ r N Q' N Ol f'1 N N N N N N N N ID ro -C1 q 0 xl X ~ e-1 \O OJ N f`"7 N N N~' Ln N N N rl ~n N N G Q N r1 N C) O' ( ri O O O O O ~n l9 ^ OJ 61 CI .~ N!`") V' (n l0 f ~ T O O O O O- r-1 - - rl rl r-I - r-1 .~ N - - .-i r-a - '-+ - .-i - - - - - - - ti ~ - - - - - - ti - - - - - - r-- + ( oa ! p . 104 p . 105 OC L.' O O ~ ~ r N ~ ~ O ~ m O ~ ro a o ~ c ~ U -/-~ 4) C~ .'. OY O Z2:' O p X O Q) (,~" Qy '~ a~ C2T b~ M CO ~' l0 [~ 6~ M!~ r1 v CD CD 10 lO N a' 00 M O U/ N 3; b` \ .-1 ~tl P M M O N O M V' d' Q' V' M r-I M a' a' in (J` M M M M M M M M M M M M M M M M M M M '0d o 0 C] 1 N rl ~O N O O O O Q N - - rl N M Q' ~fl l0 f M M CD O O O O O~ - ti - - - - ra - .~ N M ~t' u~ l0 f~ O~ 61 O ~ ~~~ p . 106 ~ .s.' O P;3. ~ ~ ~ N O rao ~ c ~ c~ ~ .~ .b C L~ o o X x F: r O ~O O N - - N - r~ o~ ~t' lO o~ O O O f O c ~ ~+ 4) N M ~fl lD ~ O~ n M rl M .-1 ~tI'n r Q' N N T ri O-I r1 lO .~- 3 tJ~ CO M M M M M M M M CO CO M M In M CO M In M CD E 0 EQ G~ N ~ O N O O O N M I n l0 r CO 61 o rl N(`"1 ~~ r N Ol O OOOOO---------- N M M M In M M M M t"1 c"1 M M M t"1 (`"1 M M Cl f'l M ,~~'~ p . 107 c+ 1 00 0L'. U) ~ tr~ ~ N T 'O o ~ ro a 'U .{ O G C) U !-. d) (13 ~ .'. OZ O p X ~ Oy b~ yy O (U r,y' v ~,y CP .C ,~OmO x N . n. r. 1 In m . In O ,O O N m N M N I~ f~ f~ . . . N N \ M Cl M M n rl v~ Ifl N ~fl 0 N ~O N N~ O ~O ,3 CP Q~ M M M M M M M M M M M M M M - M M M M M ~ ~ ~ N O Q N v -1 --i N M - In l0 f~ ~ 61 O ti N M O' ~fl iD (~ N 61 O Cl O O O O Cl O E In - M n In In In In un In ~n .n ~n n In In In Ln ~4' 4 p . 108 _C M W O G-~ ~ ~ ~ ~ N O 0 0 p7 O ~ ro a ~ ~ 0 c H Cd ~ .'. CC .d f..' ~ ~ cn O LZ ~' O ~ ~ ~~ O' N[~ N Q' .-1 d' N 61 l0 61 ~' rl ~~--I N N Ol .rl ~ ~ . .. . ..... . . .. . Gy .~ ~ N N ti CO N CO f~ V' ti f ~ lD 61 ~--1 6~ f r~D O 61 ~ O 3~ O ("1 f`"1 N N N N N(`"1 N N N N- N N N N f`"i N N > o ~ ri .~ N("1 V' ~ l0 ^ N 6~ O ti N M a' n 10 [~ OJ T O pQ ([3 O O O O O O 0 O O - .-1 ~- - r1 r 1 - ri rl N p . 109 00 O ~ , ~ ~ O W p ro v, > c ~ ~ c~ a~ ~ a3 o v O p X O ~ p aj ~' N x a~ \ C o b.~~ ~ E m~~n rn o .N N .CO vo rn co In r r.i . r N . m. rn. N o o l0 ,ti 6~ Cl l0 u~ 61 fn lp o f~l o o ~n l~ (D P r 3 CP \ M Cl t~ N M N N N("1 N(`"1 f"1 f~l M N N N N N c+ GE T O~ a N O o Q W X E Q) .~ N C'1 v' ~f/ lD fr OJ ~ -I O O O O O O O O 0o ro ~ ~ .-, ~ - r, Q (V E .-+ .-~ ti .~ .- ,~ - 61 C. r-I N f"1 I n \O r m 61 O O .-~ .,-~-I r,1 ~ ,r~-I - ., ,r~-1 .~ .-i rl rl r ~-i .-1 N .- .-~ - ti .- ,- ~ - - p . 110 ~ O N ~ ~ ~ N T ~ O ro a { ~ c a) G) ai .~ . CS O OU t. X c b x M N r-1 N 61 O N -f.+ N N ~ ~D ~' d' t+l a' -1 ~f) V' CO l0 N~ I~ ("1 ("1 Ql OJ I~ Ol a ~~-+ ~~ N N N N N N N N N N N N N("1 N N N N N CD E o0 o x ~p OJ Q' N E td O rn rn rn O v rl r1 N M d' ~~O I~ ~ 61 O~ N O O O O O O O rl - ri ("1 V' ~fl lD [~ N 6~ O .-1 .-1 f-1 - rl rl - N rn rn rn rn rn rn rn rn rn rn rn rn rn rn rn rn rn Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 111 DuPont- 18318 Appendix C Individual Final Body and Liver Weights - 111 - 2~? p . 112 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 INDIVIDUAL FINAL BODY AND LIVER WEIGHT S ABBREVIATIONS : FBW - final body weight na - not applicable NW - not weighe d S .D . - standard deviation WE - weighing error EXPLANATORY NOTES FOOTNOTES : a Animal was sacrificed in extremis prior to this analysis . -112ey"', I p . 113 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Final Body and Liver Weight s Animal FBW FBW Liver Liver (g) (g) (g ) 101 31 .0 1 .623 29 .4 102 34 .7 2 .017 32 .7 103 33 .4 1 .736 31 .7 104 32 .5 1 .768 30 .7 105 33 .0 1 .459 31 .5 106 31 .2 1 .717 29 .5 107 31 .9 1 .861 30 .0 108 30 .3 1 .727 28 .6 109 32 .7 1 .711 31 .0 110 33 .9 1 .634 32 .3 111 32 .6 1 .801 30 .8 112 33 .9 WE na 113 34 .6 WE na 114 33 .7 2 .064 31 .6 115 31 .5 1 .'/18 29 .8 116 34 .2 1 .795 32 .4 117 na no 118 34 .5 2 .172 32 .3 119 34 .3 1 .859 32 .4 120 33 .5 1 .639 31 . 9 MEAN 33 .0 1 .782 31 .1 S .D . 1 .33 0 .18 1 .26 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 MEAN S .D . 35 .1 36 .1 37 .4 37 .2 34 .5 33 .1 31 .3 33 .7 29 .9 32 .8 36 .6 33 .4 31 .5 32 .2 33 .8 31 .6 30 .2 30 .1 30 .9 36 .7 33 .4 2 .47 2 .609 2 .405 2 .512 2 .758 2 .520 2 .539 2 .036 2 .372 1 .988 2 .492 2 .896 2 .529 2 .445 2 .206 2 .514 2 .294 2 .053 2 .054 2 .199 2 .'715 2 .407 0 .25 32 .5 33 .7 34 .9 34 .4 32 .0 30 .6 29 .3 31 .3 27 .9 30 .3 33 .7 30 .9 29 .1 30 .0 31 .3 29 .3 28 .1 28 .0 28 .7 34 . 0 31 .0 2 .26 - 113 - 42c?o Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice Individual Final Body and Liver Weight s Animal FBW FBW Liver Liver (9) (q) (g ) 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 ME AN S .D . 33 .3 34_0 33 .5 33 .3 33 .0 35 .1 31 .3 33 .9 33 .5 32 .3 36 .8 36 .0 35 .9 31 .4 35 .7 32 .1 32 .2 35 .0 30 .7 36 .4 33 .8 1 .81 3 .274 3 .381 2 .978 3 .647 3 .095 3 .042 2 .992 2 .898 3 .490 3 .017 3 .191 3 .397 3 .539 3 .544 3 .535 3 .317 3 .300 3 .484 2 .996 3 .314 3 .272 0 .23 30 .0 30 .6 30 .5 29 .7 29 .9 32 .1 28 .3 31 .0 30 .0 29 .3 33 .6 32 .6 32 .4 27 .9 32 .2 28 .8 28 .9 31 .5 27 .7 33 .1 30 .5 1 .76 70l 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 71/ 718 719 720 MEAN S .D . 31 .8 30 .9 29 .1 28 .2 28 .4 27 .3 24 .7 29 .7 26 .6 27 .2 26 .8 28 .9 31 .6 29 .6 27 .4 27 .0 25 .2 31 .0 30 .0 26 .9 28 .4 2 .04 7 .969 7 .155 8 .574 6 .519 4 .627 4 .783 5 .517 5 .939 6 .075 5 .137 4 .228 5 .438 8 .346 5 .068 6 .320 4 .902 5 .396 8 .136 5 .884 5 .205 6 .061 1 .32 23 .8 23 .7 20 .5 21 .7 23 .8 22 .5 19 .2 23 .8 20 .5 22 .1 22 .6 23 .5 23 .3 24 .5 21 .1 22 .1 19 .8 22 .9 24 .1 21 . 7 22 .4 1 .53 p . 114 DuPont- 18318 -114~~ p . 115 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Final Body and Liver Weight s Animal FBW FBW Liver Liver (q) (g) (g ) 901 26 .2 5 .786 20 .4 902 24 .3 5 .752 18 .5 903 23 .7 5 .441 18 .3 904 24 .2 5 .335 18 .9 905 24 .1 4 .810 19 .3 906 na na 907 21 .8 5 .128 16 .7 908 25 .4 5 .628 19 .8 909 23 .5 5 .189 18 .3 910 27 .0 7 .923 19 .1 911 25 .3 5 .957 19 .3 912 22 .0 5 .463 16 .5 913 28 .3 7 .657 20 .6 914 28 .4 5 .089 23 .3 915 32 .4 6 .732 25 .7 916 23 .3 NW na 917 29 .4 6 .316 23 .1 918 28 .0 5 .574 22 .4 919 27 .1 5 .895 21 .2 920 29 .5 6 .499 23 . 0 MEAN 26 .0 5 .899 20 .2 S .D . 2 .84 0 .85 2 .46 1101 31 .3 8 .009 23 .3 1102 39 .5 8 .361 31 .1 1103 32 .2 5 .329 26 .9 1104 30 .4 6 .914 23 .5 1105 34 .4 7 .432 27 .0 1106 27 .2 6 .657 20 .5 1107 27 .1 6 .001 21 .1 1108 32 .3 7 .295 25 .0 1109 34 .7 6 .493 28 .2 1110 24 .9 5 .491 19 .4 1111 30 .8 6 .983 23 .8 1112 na na 1113 34 .7 1 .670 33 .0 1114 30 .4 6 .359 24 .0 1115 31 .6 NW na 1116 26 .9 6 .006 20 .9 1117 27 .5 6 .827 20 .7 1118 28 .6 7 .310 21 .3 1119 26 .2 4 .637 21 .6 1120 28 .7 7 .271 21 .4 MEAN 30 .5 6 .391 24 .0 S .D . 3 .66 1 .51 3 .84 - 115 - ~.~2 p . 116 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix D Individual Food Consumption -116al~l Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice INDIVIDUAL FOOD CONSUMPTIO N EXPLANATORY NOTES ABBREVIATIONS : Cons . - consumptio n q/anm/day - grams of food consumed per animal per day p . 117 DuPont- 18318 -II7~~~ p . 118 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Food Consumptio n Food Cons . g/anm/day Day 7 Food Cons . Food Cons . Food Cons . q/anm/day q/anm/day q/anm/day Day 14 Day 21 Day 28 Male, I 0 mg/k q 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 118 119 120 4 .9 5 .6 5 .2 4 .5 5 .3 4 .5 4 .3 4 .7 5 .1 5 .5 4 .9 5 .3 5 .6 5 .1 4 .5 4 .8 5 .0 5 .4 5 .9 4 .7 5 .6 5 .4 4 .7 4 .9 5 .4 5 .0 4 .6 5 .0 5 .5 5 .0 4 .7 4 .9 4 .5 4 .9 5 .2 5 .3 5 .7 5 .3 4 .7 5 .9 5 .2 4 .9 5 .4 5 .3 5 .1 4 .7 4 .8 5 .2 4 .8 5 .0 4 .8 4 .7 4 .9 5 .2 5 .6 5 .6 5 .4 4 .5 5 .8 4 .5 4 .2 4 .5 5 .1 4 .9 4 .1 3 .9 4 .6 4 .3 4 .4 4 .3 4 .8 4 .8 5 .0 5 .3 5 .1 5 .1 Male, III 0 .3 mg/k g 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 5 .3 5 .3 5 .1 5 .6 5 .1 4 .5 4 .6 4 .7 5 .2 5 .9 5 .7 5 .2 5 .5 4 .9 5 .1 4 .7 4 .9 4 .6 5 .1 5 .5 5 .5 5 .6 6 .0 5 .7 5 .4 4 .9 4 .8 4 .6 5 .2 5 .6 5 .4 5 .4 5 .8 5 .2 5 .2 4 .5 5 .0 5 .0 4 .9 5 .0 5 .5 5 .3 5 .3 5 .6 5 .6 5 .1 4 .9 4,'1 5 .0 5 .3 5 .5 5 .2 5 .3 5 .1 4 .8 3 .9 4 .8 4 .6 4 .9 5 .2 5 .4 4 .9 5 .2 5 .4 5 .3 4 .6 4 .9 4 ,5 4 .3 5 .4 5 .2 5 .1 4 .9 4 .7 4 .9 4 .3 4 .4 5 .1 4 .8 5. 4 118~~ p . 119 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Food Consumptio n Food Cons . Food Cons . Food Cons . Food Cons . g/anm/day g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Day 28 Male, V 1 mg/k g 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 5 .9 6 .2 5 .0 4 .6 5 .4 5 .1 5 .1 5 .4 4 .6 4 .9 5 .8 5 .2 5 .6 5 .3 5 .6 5 .1 4 .6 5 .0 5 .0 5 .2 5 .8 4 .8 5 .6 4 .5 5 .3 5 .3 4 .7 5 .7 6 .5 5 .0 5 .3 5 .1 5 .4 5 .6 5 .8 4 .9 4 .7 5 .3 5 .6 5 .7 6 .2 5 .0 5 .0 4 .9 5 .4 5 .6 S .2 5 .5 3 .8 5 .3 5 .1 4 .7 5 .3 4 .9 5 .8 5 .2 4 .9 5 .3 5 .1 4 .9 5 .0 4 .6 4 .5 4 .9 5 .2 5 .4 4 .7 5 .4 5 .0 5 .4 5 .7 5 .4 5 .5 4 .8 5 .8 5 .0 4 .9 5 .6 5 .5 5 .0 Male, VII 10 mg/k q 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 5 .2 5 .9 5 .0 5 .8 5 .1 5 .0 4 .4 4 .7 5 .7 5 .3 3 .9 4 .4 5 .4 3 .8 5 .0 4 .8 4 .7 5 .5 5 .0 5 .6 6 .7 6 .2 5 .9 6 .0 6 .6 5 .4 3 .4 6 .9 6 .9 5 .7 5 .3 6 .5 6 .3 5 .9 5 .4 5 .2 5 .0 6 .2 6 .5 4 .9 6 .9 6 .4 5 .1 6 .1 5 .7 5 .2 4 .4 6 .1 6 .5 5 .4 4 .4 5 .9 4 .5 5 .5 5 .0 5 .5 4 .7 4 .0 4 .1 4 .2 6 .4 5 .0 4 .5 6 .1 5 .1 5 .2 4 .3 6 .1 5 .7 4 .5 5 .8 5 .4 4 .1 6 .7 6 .2 5 .5 4 .7 5 .9 5 .9 6.9 - 119Z~~ p . 120 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Food Consumptio n Food Cons . Food Cons . Food Cons . Food Cons . g/anm/day g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Day 28 Male, IX 30 mg/k g 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 3 .'7 3 .7 4 .7 3 .3 3 .3 3 .9 3 .2 4 .7 4 .8 5 .0 5 .1 5 .3 4 .8 5 .1 5 .7 3 .9 5 .9 4 .9 4 .3 5 .3 5 .7 5 .4 4 .5 5 .7 5 .5 3 .7 6 .1 5 .9 5 .2 5 .8 4 .7 4 .9 5 .7 6 .7 6 .1 7 .0 6 .0 5 .6 6 .4 4 .5 2 .5 4 .0 NM 3 .8 3 .5 4 .2 3 .8 4 .5 4 .4 3 .1 4 .1 4 .7 7 .2 4 .2 7 .1 5 .1 4 .5 3 .9 4 .8 3 .7 4 .4 3 .5 4 .0 3 .2 5 .6 4 .1 4 .7 5 .7 3 .7 3 .9 5 .1 7 .3 4 .6 7 .3 4 .9 4 .6 5 .2 Male, XI 30/0 mg/kg (Recovery ) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1113 1114 1115 1116 1117 1118 1119 1120 5 .2 5 .6 5 .6 4 .7 5 .1 3 .9 4 .4 4 .1 4 .3 5 .1 4 .1 4 .3 5 .0 4 .1 3 .0 3 .4 4 .4 4 .6 3 .9 6 .4 5 .7 5 .4 5 .3 7 .7 5 .1 4 .4 4 .8 7 .8 5 .8 4 .4 5 .4 5 .9 5 .3 4 .2 4 .1 5 .8 6 .3 S .1 6 .1 5 .1 3 .9 4 .6 6 .2 5 .5 3 .6 5 .2 4 .9 4 .8 3 .9 4 .7 5 .5 5 .2 3 .8 3 .9 4 .1 3 .0 3 .4 6 .1 5 .4 5 .7 4 .0 6 .0 4 .2 4 .3 4 .4 7 .5 3 .3 4 .5 5 .1 6 .7 5 .5 5 .0 5 .2 6 .8 8 .8 5 .9 Key : NM = Not Measurable - 120- ~~ ~ p . 121 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix E Individual Daily Animal Health Observations -121- ~~~~` p . 122 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Daily Animal Health Observation s Sex G--oup Animal Observation Days M 1 101 General observation, No Abnormality Detected 0-28 M 1 102 General observation, No Abnormality Detected 0-28 M 1 103 General observation, No Abnormality Detected 0-28 M 1 104 General observation, No Abnormality Detected 0-28 M I 105 General observation, No Abnormality Detected 0-28 M 1 106 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 I 111 General observation, No Abnormality Detected 0-28 M 1 112 General observation, No Abnormality Detected 0-28 M I 113 General observation, No Abnormality Detected 0-28 M I 114 General observation, No Abnormality Detected 0-2 8 M I 115 General observation, No Abnormality Detected 0-2,7-28 Abnormal Gait, Hindlimb, Right, Severe 3- 6 M 1 116 General observation, No Abnormality Detected 0-28 M I 117 General observation, No Abnormality Detected 0-5 Sacrificed in extremis 5 M I 118 General observation, No Abnormality Detected 0-28 M I 119 General observation, No Abnormality Detected 0-28 M 1 120 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 ill 305 General observation, No Abnormality Detected 0-28 M III 306 General observation, No Abnormality Detected 0-28 M III 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 III 311 General observation, No Abnormality Detected 0-28 M 1I : 312 General observation, No Abnormality Detected 0-28 M III 313 General observation, No Abnormality Detected 0-28 M III 314 General observation, No Abnormality Detected 0-28 M III 315 General observation, No Abnormality Detected 0-28 M III 316 General observation, No Abnormality Detected 0-28 M III 317 General observation, No Abnormality Detected 0-28 M ill 318 General observation, No Abnormality Detected 0-28 M III 319 General observation, No Abnormality Detected 0-28 M III 320 General observation, No Abnormality Detected 0-28 - 122 - :291 r' p . 123 Ammonium Per fluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont- 18318 Sex Group Individual Daily Animal Health Observation s Animal Observation Days 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 M V 511 General observation, No Abnormality Detected 0-28 M V 512 General observation, No Abnormality Detected 0-28 M V 513 General observation, No Abnormality Detected 0-28 M V 514 General observation, No Abnormality Detected 0-28 M V 515 General observation, No Abnormality Detected 0-28 M V 516 General observation, No Abnormality Detected 0-28 M V 517 General observation, No Abnormality Detected 0-28 M V 518 General observation, No Abnormality Detected 0-28 M V 519 General observation, No Abnormality Detected 0-28 M V 520 General observation, No Abnormality Detected 0-28 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 0-28 M VII 707 General observation, No Abnormality Detected 0-28 M VII 708 General observation, No Abnormality Detected 0-28 M VII /09 General observation, No Abnormality Detected 0-28 M VII 710 General observation, No Abnormality Detected 0-28 M VII 711 General observation, No Abnormality Detected 0-28 M VII 712 General observation, No Abnormality Detected 0-28 M VII 713 General observation, No Abnormality Detected 0-28 M VII 714 General observation, No Abnormality Detected 0- 4 Swollen Observations, Shoulder, Left 5-28 M VII 715 General observation, No Abnormality Detected 0-28 M VII 716 General observation, No Abnormality Detected 0-28 M VII 717 General observation, No Abnormality Detected 0-28 M VII 718 General observation, No Abnormality Detected 0-28 M VII 719 General observation, No Abnormality Detected 0-28 M VII 720 General observation, No Abnormality Detected 0-28 -123- J61:) p . 124 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-I8318 Individual Daily Animal Health Observation s M IX 901 General observation, No Abnormality Detected 0-8,12-28 Comments, animal not dosed, pd obs not done 9-1 1 M IX 902 General observation, No Abnormality Detected 0-8,12-22,28 Comments, both ears, hindpaws/forepaws yellow 23-27 Comments, animal not dosed, pd obs not done 9-1 1 M IX 903 General observation, No Abnormality Detected 0-28 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- 6 Swollen Observations, Shoulder, Left 7-8 Sacrificed in extremis 9 M IX 907 General observation, No Abnormality Detected 0-28 M IX 908 General observation, No Abnormality Detected 0-21 Comments, both ears/forepaws/hindpaws yellow 22-27 Swollen Observations, Penis 22-2 8 M IX 909 General observation, No Abnormality Detected 0-16,19-28 Abnormal Gait, Hindlimb, Right, Moderate 17-1 8 M IX 910 General observation, No Abnormality Detected 0-28 M IX 911 General observation, No Abnormality Detected 0-27 Comments, both ears/hindpaws/forepaws yellow 2 8 M IX 912 General observation, No Abnormality Detected 0-28 M IX 913 General observation, No Abnormality Detected 0-28 M IX 914 General observation, No Abnormality Detected 0-28 M IX 915 General observation, No Abnormality Detected 0-28 M IX 916 General observation, No Abnormality Detected 0-28 M IX 917 General observation, No Abnormality Detected 0-28 M IX 918 General observation, No Abnormality Detected 0-28 M IX 919 General observation, No Abnormality Detected 0-28 M IX 920 General observation, No Abnormality Detected 0-28 M XI 1101 General observation, No Abnormality Detected 0-28 M XI 1102 General observation, No Abnormality Detected 0-28 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 0-2 8 M XI 1108 General observation, No Abnormality Detected 0-8,12-21,28 Comments, both ears/forepaws/hindpaws yellow 22-27 Comments, animal not dosed, pd obs not done 9-11 Stained Cageboard, Yellow 22-2 7 M XI 1109 General observation, No Abnormality Detected 0-8, 12-17, 19-21,2 8 Feces, Absent 18 Comments, both ears/forepaws/hindpaws yellow 22-27 Comments, animal not dosed, pd obs not done 9-11 Stained Not Cageboard, Yellow Eating 22-27 18 M XI 1110 General observation, No Abnormality Detected 0-28 M XI 1111 General observation, No Abnormality Detected 0-28 M XI 1112 General observation, No Abnormality Detected 0- 2 Eye Observations, Enophthalmus, Bilateral 3-5 Lethargic 3-5 Sacrificed in extremis 5 M XI 1113 General observation, No Abnormality Detected 0-28 M XI 1114 General observation, No Abnormality Detected 0-28 M XI 1115 General observation, No Abnormality Detected 0-28 M XI 1116 General observation, No Abnormality Detected 0-2 8 M XI 1117 General observation, No Abnormality Detected 0-7,11-28 Comments, animal not dosed, pd obs not done 8-1 0 M XI 1118 General observation, No Abnormality Detected 0-28 M XI 1119 General observation, No Abnormality Detected 0-2 8 M XI 1120 General observation, No Abnormality Detected 0-7,11-28 Comments, animal not dosed, pd obs not done 8-1 0 -I24~ .4,i p . 125 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix F Individual Detailed Clinical Observations and Mortality Records - 125 - Z 3(? p . 126 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Detailed Clinical Observations and Mortality Record s Sex 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 I 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 0-2 9 Sacrificed by design 29 M I 107 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M I 108 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M I 109 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M I 110 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M I 111 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M I 112 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M I 113 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M I 114 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M I 115 General observation, No Abnormality Detected 0,7-29 Abnormal Gait, Hindlimb, Right, Severe 3 Sacrificed by design 2 9 M I 116 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M I 117 General observation, No Abnormality Detected 0 Eye Observations, Enophthalmus, Bilateral 5 Lethargic 5 Breathing Observations, Labored 5 Feces, Absent 5 Comments, swollen thoracic ventral 5 Swollen Observations, Face 5 Swollen Observations, Neck 5 Not Eating 5 Sacrificed in extremis 5 M I 118 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M I 119 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M I 120 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 -126- ~~~~ p . 127 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Detailed Clinical Observations and Mortality Record s Sex Group Animal Observation Days M III 301 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 302 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 303 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 304 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 305 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 306 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 307 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 308 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M II[ 309 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 310 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 311 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M II .[ 312 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 313 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 314 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 315 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M IiI 316 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 317 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M III 318 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M III 319 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M III 320 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 - 127- ~~~~ ~ Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 128 DuPont- 18318 Individual Detailed Clinical Observations and Mortality Record s Sex Group Animal Observation Days M V 501 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 502 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 503 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 504 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 505 General observation, No Abnormality Detected 0,14-29 Abnormal Gait, Hindlimb, Left, Severe 7 Sacrificed by design 2 9 M V 506 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 507 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 0-2 9 Sacrificed by design 29 M V 511 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 512 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 513 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 514 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 515 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 516 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 517 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M V 518 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M V 519 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M V 520 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 - 128 - ~o Jr., p . 129 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Sex Individual Detailed Clinical Observations and Mortality Record s 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 0,14-29 Abnormal Gait, Hindlimb, Right, Moderate 7 Sacrificed by design 2 9 M VII 703 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII 704 General observation, No Abnormality Detected 0-7,28-29 Pale 14-21 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 0-2 9 Sacrificed by design 29 M VII 707 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII 708 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 709 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M VII 710 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII 711 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 712 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M VII 713 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII 714 Absent, End of tail 0-29 Swollen Observations, Shoulder, Left 7-29 Sacrificed by design 2 9 M VII 715 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 716 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M VII 717 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M VII 718 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M VII 719 General observation, No Abnormality Detected Misshapen Observations, Tail 0 7-29 Sacrificed by design 29 M VII 720 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 ^ -129- .116 p . 130 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Sex Individual Detailed Clinical Observations and Mortality Record s Group Animal Observation Day s M IX 901 General observation, No Abnormality Detected 0-14,28-29 Pale Sacrificed by 21 design 2 9 M IX 902 General observation, No Abnormality Detected 0-14 Pale 21-28 Stain Fur/Skin, Perineum, Yellow 21-29 Net Fur, Perineum 21-28 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-/ Eye Observations, Partially Closed, Bilateral 9 Lethargic 9 Pale 9 Sacrificed in extremis 9 M IX 907 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 908 General observation, No Abnormality Detected 0-2 1 Comments, both ears/hindpaws/forepaws yellow 28 Swollen Observations, Penis 28-29 Sacrificed by design 2 9 M IX 909 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 910 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M IX 911 General observation, No Abnormality Detected 0-21 Comments, both ears/forepaws/hindpaws yellow 28-29 Sacrificed by design 2 9 M IX 912 General observation, No Abnormality Detected 0-14 Pale 21-29 Sacrificed by design 2 9 M IX 913 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 914 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M IX 915 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M IX 916 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 917 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M IX 918 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M IX 919 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M IX 920 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 - 130 - .w" :r p . 131 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 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 XI 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 0-21 Comments, both ears yellow 28 Stained Cageboard, Yellow 28-29 Sacrificed by design 2 9 M XI 1109 General observation, No Abnormality Detected 0-14 Eye Observations, Enophthalmus, Bilateral 21 Prostrate 21 Comments, ears/extremities yellow 21 Stained Cageboard, Yellow 28-29 Sacrificed by design 2 9 M XI 1110 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M XI 1111 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M XI 1112 General observation, No Abnormality Detected 0 Eye Observations, Enophthalmus, Left 5 Eye Observations, Dark, Bilateral 5 Lethargic 5 Feces, Absent 5 Stain Fur/Skin, Perineum, Yellow 5 Swollen Observations, Shoulder, Left 5 Not Eating 5 Sacrificed in extremis 5 M XI 1113 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M XI 1114 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M XI 1115 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M XI 1116 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M XI 1117 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 M XI 1118 General observation, No Abnormality Detected 0-29 Sacrificed by design 2 9 M XI 1119 General observation, No Abnormality Detected 0-29 Sacrificed by design 29 M XI 1120 General observation, No Abnormality Detected 0-2 9 Sacrificed by design 29 - 131 - 3n p . 132 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix G Individual Animal Clinical Pathology Data E-132- ~ 67 p . 133 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTE S ABBREVIATIONS : General : Adeq - adequat e CLOT or Clot - sample clotted Decr - decreased Mod - moderat e NSR - no sample received for testin g NP - not taken, not performed, or results not valid OK - sample condition OK for testin g QNS - sample quantity not sufficient for testing UTD - unable to determin e 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 ALYM - absolute lymphocyt e forms) AMON - absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophi l ALUC - absolute large unstained cell Individual Red Blood Cell Morphology Values : 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 Individual White Blood Cell / Platelet Morphology SM - smudge white blood cell s TOX - toxic neutrophils DB - Dohle bodie s VC - vacuolated cytoplasm BC - basophilic cytoplas m PCE - platelet clumps / estimate GP - giant platelet s BP - bizarre platelets - - not observed Values : - 133 - ~ /t/-) p . 134 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 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 marked other . ~~! -134- p . 135 M 00 O r tn o. a m a a a a a, n. a a a r a m a a a c w a w 0. n . tn O z z z z z z z z z .~ a O z z z in z z z z z N x x F,a a o vr+ vco ,-t tn F, tn m- tn rn m rn vN~o ....... ro O M O 61 O rM, N. a ul v~ c .-1 oJ ON N Ol c .-I .-1 r M N lO M l0 M O O m N O' Ln l0 N N .-I r-1 y~ X M M N M f~ M M M M M a X M M M d' N M M M("1 M ro n ~~ 3 W r r-1 61 61 r Q' 6\ ,'~ \D 3 N r M .-I l0 O' rl r-1 l0 . . . . .. .. .. .......... 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O N~--1 O O U~ O O r O O N O 61 c)* m ~ N*~CPMN"1c`f~ltPMNco Vl ~ M U ~ < tn lo 0 rn m .-~ rn m un m tn Ln Ln n ol ol N - - - - lo ti~ N -~ ~ -N - ~ ~ - ~ ~t tn iN ~ ~ 0 > O~ v' r V' v' r1 m ~n cT r > M O d' ID N M lO O N N -~ ro U 'O Q ~c W tn O M O N w M - N M CO ro U W N N v' tn tn tn v~n LO ,n .n va tn tn Ln un r1 N N .-1 6~ ti N ~n tn ~n - Ln ~ F N N~ M ~n ~~ l0 N~Il ~ E f"1 r~' o c) ~n M!r rl Ol ~ tn tn a tnn ul l0 0 N ) M ~l0t~ D O .n vN ' ri N l0 ~n N rl r1 N M 61 tn tn ~r tn ~n tn ~ u~ u~ tn tn ~n Ln tn tn ~n o N U_ rJ o C7 r i-] CO Q' M o N O()J M M fr m l0 l0 OJ l0 d` l0 r1 N M r ~o tn (D d in ~n tn ~o in D m Ln tP - - - ,ti .ti .-i ti .ti r+ .-i o ~ - - - ti - - ti ,-t - Gi F+ H ~ 00 v' N~ O tD ~ l0 ~ ~n H u~ O,--i c") l0 N CO Ol ~n M u-CD"oL l Y V) o P;o OOO~ooo H a0 OOOID x x 00 r n y 0 0 z a E a x x :3x x x 0 0 C7 a x x x~ x 0 0 0 0 0 0 U q z x x x x 0 0 0 o 0 0 ~ U x x x 0 0 0 0 x x x 0 0 0 0 ~ CG N ro r-~ N M V' tn l0 r OJ 61 o N rl o000ooooor-+ -1 -~ O N M oo p'0tonol0 p 0 r 0W~6-1+O < ""v~~~ p . 136 W O F\ Q. 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Fa Qo:- N war aaM acw rlwv n' r-1 0' OO CO U bl z Z Z Z Z Z Z Z Z.wZw ,-rrnnnv~u mn NM co,-~M~,-, v] G F7 `~ E P~ .0i W n+ (1+ LL L4 (L LL 0.~ ~ N M~ M O O N T ul z z Z Z Z Z Z 2 Z N N ~ M M N MIn N ro a a~ a a a a CL CL f1 0.i CL a ~O N M M m r r co rn o w Z Z Z Z Z 2 Z Z Z Z N N N M M N M fM Ln ro n ~ ' o 0 J~ ro w ~ ro a d a~ aaaa wa aaawn,awaaw ZNz2Zz zN Z N Z Z zz ZZZZzz u va a a a n a a a a a a a a w a a w -C <~ Z ~, Z Z Z Z Z~ Z Z Z Z Z Z Z Z Z Z z tT U ~ ro r~ rn w o w aan a awawwn,www a E+ ~ Z~ Z Z Z Z Z Z~ Z Z Z Z Z Z Z Z Z Z < ~ ro a > o wao.wwn,aaaw om~ o~rn ro q tr, ZR:Z Z Z Z Z Z Z Z z .-+ a' M~ w Ln lo co co v c ~ O~ aa O~ a, cL a, n, w n, a~ 0.1 n, a, ti v lo m,~ r m Z Z Z Z Z Z Z Z Z Z M cr m vr in Ln d) ..U. N N d) U 41 N O N N N N N N N w aj y j ro ro m m ro m ro m ro ro ro ro ~tl N Z CL SJ CL '~ la i~ S1 S4 S1 1 .1 3-i Si Z Z ro N N N N Ol N N ro N ro ro Q7 N -a -0 O o O O O O O O O O O ~ .nC.. ~"d ~ N N N N N Ol N N N N N N 4) N N N N x H c c rw w c a c c c c c c c c c c c c c c o o z z o z O o 0 0 0 0 0 0 0 0 0 0 0 0 z Z z Z Z Z z z ZZzZz z Zz z z O k o ~ 0 0 c c~ z c~ c c c c c c c a ~ m o 0 0 0 0 0 0 0 ccccccc 0 0 0 0 0 0 Z Z Z Z Z z z z z z z z Z z z 2 Z N .f'~y 1 ftl 6~ a N ~ ~ 6~ .-1 N M V 11 iD f ~ 61 O rl (V M P~ l0 r W 6~ O O O O O O O O O - .-1 ti rl e-1 r-1 ,-I - r/ r1 N 6~ 6~ Ol T Ol 61 O~ Q~ 61 p~ pl T Ol Ol Ol 61 Oi O~ p . 151 p . 152 O P-~ Fa rz E o ~n a w w a w w w a m a~o ~n r~ a~r r~ ~o ,~ Uri, N N z Z Z Z Z Z 2 2 r Z~~ ~ vl C u a a a a a a a n a a r w o n~ o o m rn r 2 b , Z Z Z 2 2 2 Z Z Z Z~n 2 ri ~n io c+~ c~7 N c`"i N z E ro ~ ro u o aaw l wwaw m awn .- wr r oo n o r u ~ Z Z Z 2 Z Z 2 Z Z Z ~ Z ~ ~ o n v ' r"i nm E y a O 071 r,-+ lo rA ~o r~ r a~n m m n, CL , C4 R, W W W P. CL - N m N t"1 M t"1 z ' ("1 N M Z Z Z z~Iz z Z z Z Ul JJ d ro ri U 6~ ~'d t.f) N CO N l0 6l a fl r1 l0 .r1 W Q~ Q, W W(1 Q, 0+ "c N 6~ v~ v~ ri v~ ~~ Z' v~ ~r ri 2 Z Z Z Z Z'z Z Z Z ~ ~ U ~ ro E C J. a N , a -0M~' Q' 6. 1, l9 OJ 6~ O waaaaa~n.a .ww lo u c `" ~ r r~ m r co z r~ r z Z Z Z 2 2 Z Z Z Z z3l -1 rt ~ O U H C d) H an.CW aaal aa a .a, 'i'v rCO o 2 Z Z Z? Z 2 Z Z 2 Z~~~~ ID r r ~ aa ~ 'S ~, z Z Z Z z z z Z Z Z~ z rn~ rn r ~ rn~ EU E 4) ~" ~ N~ N-1 F-i N N N v ri ~1 Ol N N F U-1 rt - U U U~-i U U U y U m C C ro ro m m m m 4f m m m ro ro m m m ~ r~ ~ z Z F c/) U UEE c-~ u) E E U~+ u) m Z N~+ ~+ 'Z H F F E E .EMs4U)~F+F~H + . .~'.. T N N N 41 O~ H n' C C C N N N N N N N N N N N N N N N C C C C C C C G W C C C C C C CC O O o O O O O o O O O Z O O O O O O O O Z z z 2 Z Z Z Z Z Z z z Z 2 z 2 Z Z Z O L~' O a ai ai a> a~ m'-' a~ v a~ a~ ai a~ a~ v~ a~ a~ ai a~ s o o W 0 c0c0c 0c 0 ccc oc 0 c c 0 c ~0 o00 oor- rco oc 0 c0 c0 a ~ y 2 Z 2 2 Z Z ZZZZZZZZZZZ ZZ ZZ 2Z ZZ cZ~Z,H2'Z.z Z Z p ra ~ N~'l ~' in lo f~ OJ rn o rl N rl V' i1l \O Ir cn 61 0 o00000000- - - ,-+- - - - r+- - - - - - - - - - - - - - - - .. - r, - - .-, - - - - - - - - - Q N -327 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 153 DuPont- 18318 Appendix H Individual Primary Humoral Immune Response Data ~~ -153- 9 p . 154 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 INDIVIDUAL PRIMARY HUMORAL IMMUNE RESPONSE DATA EXPLANATORY NOTE S FOOTNOTES : b Serum was not collected from this animal, therefore, immune response could not be evaluated . c Serum volume was insufficient for this animal, therefore, immune response could not be evaluated . d This animal was not injected with the appropriate amount of SRBC, therefore, immune response could not be evaluated . -154- ~~~ p . 155 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Primary Humoral Immune Response Dat a Animal SLOPE X Log2 Numbe r Male, Group I- 0 mg/kg 101 -1 .0034 874 102 -0 .9958 292 103 -0 .9796 540 104 -0 .8528 353 105 -0 .9942 737 106 -0 .8607 487 107 -0 .8226 727 108 -0 .9649 1018 109 -0 .9366 417 110 -0 .9625 777 111 -1 .0109 340 112 -0 .9652 784 113 -0 .9896 537 114 -0 .9866 497 115 -0 .9834 384 116 -0 .9607 689 117 '' 118 -0 .9716 215 119 -0 .8730 457 120 -0 .8733 549 9 .771 8 .190 9 .077 8 .464 9 .526 8 .928 9 .506 9 .992 8 .704 9 .602 8 .409 9 .615 9 .069 8 .957 8 .585 9 .428 7 .748 8 .836 9 .101 Male, Group III - 0 .3 mg/k g 301 302 303 304 305 30 6 307 308 309 310 311 312 313 314 315 316 317 318 319 320 -1 .0351 -0 .9020 -0 .9403 -0 .9653 -0 .9268 681 235 405 445 258 9 .412 7 .877 8 .662 8 .798 8 .011 -1 .0131 511 8 .997 -0 .9142 269 8 .071 -0 .9091 830 9 .697 -0 .9551 573 9 .162 -0 .9906 1205 10 .235 -0 .9573 645 9 .333 -0 .9484 417 8 .704 -0 .9460 793 9 .631 -1 .0128 401 8 .647 -0 .9552 552 9 .109 -0 .9239 132 7 .044 -0 .9189 579 9 .177 -0 .9709 211 7 .721 -0 .9938 622 9 .281 - 155 - ,~27- p . 156 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Primary Humoral Immune Response Dat a Animal SLOPE X Log2 Numbe r Male, Group V - 1 mg/k g 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 -1 .0294 324 8 .340 -1 .0045 562 9 .134 -0 .9279 428 8 .741 -0 .9200 353 8 .464 -1 .0255 343 8 .422 -1 .0013 161 7 .331 -0 .9730 284 8 .150 -0 .9931 444 8 .794 -0 .9765 223 "1 .801 -0 .9230 331 8 .371 -0 .8852 673 9 .394 -1 .0076 222 7 .794 -0 .9627 317 8 .308 -0 .9910 363 8 .504 -0 .9622 734 9 .520 -1 .0029 351 8 .455 -0 .9352 205 7 .679 -0 .9065 151 7 .238 -0 .8622 253 7 .983 -0 .9383 220 7 .781 Male, Group VII - 10 mg/k g 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 71 6 717 718 719 720 -0 .8465 285 -0 .9652 95 b 8 .155 6 .570 -0 .9617 134 -1 .0045 187 -0 .9582 311 -1 .0358 97 -1 .0128 144 -1 .0214 73 -0 .9532 148 -0 .7979 243 -0 .9974 180 -1 .0116 140 -0 .9470 817 -0 .8674 134 7 .066 7 .547 8 .281 6 .600 7 .170 6 .190 7 .209 7 .925 7 .492 7 .129 9 .6"74 7 .066 -1 .0155 226 7 .820 -1 .0207 110 6 .781 -0 .9787 7 2 .807 -0 .7775 230 7 .845 -156- ~w~~,~ p . 157 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Primary Humoral Immune Response Dat a Animal SLOPE X Log Numbe r Male, Group IX - 30 mg/k g 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 -0 .9613 164 -0 .9724 26 ` ' -1 .0229 65 a b -1 .0094 89 -0 .9950 141 -1 .0106 44 -0 .9730 84 -1 .0333 62 -0 .8405 530 -0 .9848 193 -0 .9891 58 -1 .0002 90 -0 .9981 120 -1 .0207 55 -0 .9989 161 -0 .9884 52 7 .358 4 .700 6 .022 6 .476 7 .140 5 .459 6 .392 5 .954 9 .050 7 .592 5 .858 6 .492 6 .907 5 .781 7 .331 5 .70 0 Male, Group IX - 30/0 mg/k(g (Recovery) 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 -0 .9921 89 -1 .0202 45 -0 .9938 114 -1 .0130 78 -0 .9951 96 -0 .9785 35 -0 .9904 88 -0 .9412 78 -0 .9771 47 -0 .8757 169 -1 .0060 43 ' -0 .9806 151 -0 .9696 74 -0 .9458 74 b -0 .9999 167 -0 .9994 98 -0 .9866 52 -0 .9927 52 6 .476 5 .492 6 .833 6 .285 6 .585 5 .129 6 .459 6 .285 5 .555 7 .401 5 .426 7 .238 6 .209 6 .209 7 .384 6 .615 5 .700 5 .700 - 157 - ~3)5L p . 158 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix I Individual Primary Humoral Immune Response Positive Control Data - 158 - '335 p . 159 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Primary Humoral Immune Response Positive Control Dat a Animal Number SLOPE X Log, Male, Group CI - Salin e C101 C102 C103 C104 C105 C106 C107 C108 C109 C110 -0 .9624 -0 .9535 -0 .9312 -0 .9892 -1 .0066 -0 .8598 -0 .8395 -0 .9841 -1 .0023 -0 .9946 578 747 417 505 268 410 140 485 429 271 9 .175 9 .545 8 .704 8 .980 8 .066 8 .679 7 .129 8 .922 8 .745 8 .082 Male, Group CIII - 90 mg/kg Cyclophosphamid e C301 C302 C303 C304 C305 C306 0307 C308 C309 C310 -0 .9579 25 -0 .9965 19 -1 .0234 25 -0 .8989 14 -1 .0583 14 -0 .9927 25 -1 .0321 33 -0 .9777 43 -0 .8206 8 -1 .0512 59 4 .644 4 .248 4 .644 3 .807 3 .807 4 .644 5 .044 5 .426 3 .000 5 .883 Male, Pooled Samples - Salin e -1 .0107 405 8 .66 2 Male, Pooled Samples - 90 mg/kg Cyclophosphamide -0 .9846 35 5 .129 - 159 - -33 g p . 160 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix J Individual Animal Final Body and Organ Weights -160- ~~ r,7 p . 161 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 INDIVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS EXPLANATORY NOTE S FOOTNOTES : a An error occurred while weighing livers for this animal, and the liver weight was excluded from calculations . b Liver inadvertently not weighed . -161- .~~ . .'"~ p . 162 ~ O I - - I - - - - - - - - - - - - - - - 1 ----------- - 2 O\ M V' Ir O N d' ul M O r 1 Z I r m In r-I OJ V' fr r1 N r o I H ttl o OJ o~-1 0 o r~-1 n o M 1-1 1 61 CO r CO 6l M M M O ~O V ' I M M O~ N O f"1 CD ~1 r N V' I I N'o N N N V' tn V' a' N M I 'L~ 1 r O M l0 I I ~ I M O N Ol N N I m I r .-I N O N rl O rv I I m I O d' N O O I o1o I ~-1 r-~ r ~-1 r-1 r-+ 61 m ri ~ rl N I o\ I r1 ~ 61 r .-1 61 ~-1 M 61 r1 N I 1 co I O dl MLn v' In Ln 61 M 7 l0 ~n I V) I I W,'3 I 6~ 6~ O N N M N .-1 V' r ~O IN I ~,3 I I ~ f3-0 I N V' rl 1- V' ~O N V' r v' ~n M I ,'J+ f~-, I N~~ rl rl r~ r-1 ry .-1 N ti O I I I W o~o I . . . . . . . . . . .. 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N V' O~ ~n V' I . . . . x 1 I tP ul ul Ln ul V' in (D I x I I In 10 In 1) l0 ul V' u~ o I v I I I v I I U (!] I I M'~ \D CO Ol r~-V .N rl O' 7~ 1 (n I 1 ~ V' O~ ~ N 61 6\ V' C) I I N r-1 M 10 Vl r-~ l0 IN ~-1 M N v' 1 I ~ I O ID ~ Cl r'Jl m iD m I I N I~O O[r Ir V' r OJ ^ Ir 10 Ir r-I I I U) I W C) Ir r~ CU l0 OJ - I ~ I I 1 ~ I { I I (') I ti N rl rl r-V N r-1 .-1 .-1 .-i r1 CD, I I L7 I ti ro ro N~~ N .-1 ~-1 r1 O 1 " = ' ZT ~ X 1 - - I - - - - - - - - -- - - - - 1 ,X - - I - - - - - - - - - - - - C~ ~ 1 I V' ~O .-i OJ In N N N N fr In cv 1 ol 1 I .-1 ~-I V' Ir ~9 0 o V' r M M 1 ~G ~ 1 3 I T ~n r M M N M V' M rl l0 o I ~ I 3 I T ID N r V' r CO -/-~ I fLW 1 r-I N M 6~ 61 l0 N N~ f O r 1 I m I V' lfl M r r O ul M~n ~n w I ~ O I Z 1++ I M M V' M M M M Q' O I O I 2 Gw I Ul d' M V' ifl M O I ." i H o\ 1 .......... . . ~ 1 1~ ew I ......... . .1 < I o (~ . .~ I W' 1 1 I [L I 1 O 1~ 1 m 1 ~ o C) M o~n V' N~Jl In ~O L(l 1 AU I 0.1 I ~n r rl (KI lO f M N Ir fr O 1 ~' ,~ {~ I .~ 1 O ~O O~ tn lD NLf-) ~O 61 \O ~n (V I C I ~ I O 6\ ~O 6\ 61 V' C ) OJ N N C~ ~ N I ul I v' v' V' V' d' V' a' - O I N 1 U1 I ul In .......... . ......... O I . .1 O I O O C) C) O O ID O o o O o 1 11 1 I C) O ID O CD O o O O O O 1 f6 1 =' I 1 ro 1 ~ I 1 I 11 .~ 0) .r l N ,~ Ili 1 - - 1 - - - - - - - - - - - - - I N 1 - - I - - - - - - - - - - - - 1 ~ x .~ Q LI I 1 1 1J 1 I ~ H 1 ~ 1 O O O O O O o O o O lD N I H 1 I O O O O O ID O C) O V' I 1 Y . 3 Vr I I O r V' ~n C) N 6\ M r 6i V' M I I ~ Ul I 1.0 6~ l0 r LfI N ul M~n l0 O 1 'cl I m ~ I [i~ (`J I I I W ~ I 1 O 1 ~-i V' M N M~ r-1 O N M N r-i I I [*a C~ I N M V' M .-1 V' V' V' M M~-1 I '.T1 a =S I =' I M M M M M M M M M fM t`") I I I M(`") M m M M["1 Cl M M 1 ~+ I I I 1 I 1 -i EG - - r -1 - -I --- - - - I - - - - I m I - - I - - - - - - - - - -_ ___ - I 3 H I I I H 1 I I C I ~l I .-i (V M V' ~ l0 r N 6\ O I 1 ~l I .-1 N M O' ~Jl l0 OJ 61 O I ~rl ~~+ 1 r~ 1 O o o CCO Q G~ 7 C1. 1 i-; 1',Z O O O O O O .-1 I 1 ~ I r-1 r-t rl r+ ~-1 r-I r-1 rl ,~ ,~ ,-+ ti ,- ,- ,-i - ~-- ti C ~ I 1 ~ I .-1 - .- - ,~ ,~ r+ ,~it 1 ro C] 1 f1, i H I rtl N q I N I ~ 1 z I N I '~~ 1 1 1 0 ~p Sa 1 1 FC 1 1 I ~ cn I Sa o 1 1 < I E I I cn 1 3 '11 Q N (NI--F --- ---- ------ 1 C7 1-- I--- 1 14 331? p . 163 M 00 I - - - - - - - 1 I - - -------------- N- : Z i N~ CO ~n M N Ul 01 N~ 1 I Z I O\ --- M ul M ri f`"1 a' M I H 1 r M O Ifl v' ~O N M ul l0 IA N I H I O N N~ M r v Ol .-1 l0 rl 6~ ~ I O 61 r N~ r ~O N r1 r M M I ~Q I (D ' O - in O N a' O .-~ M M r~ I f1.' 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C7 C), 0 ro o l 1 ~ I ... 0 I 0 0 ..... .. 0 I 0 00 i ( n , 0 09 I I 1 ~ . 0 0 n I N O O O O O O O O O O O O I I !~ O O O O O O O O O O O O 1 . . . . . . . .. 0 0 0 0 0 0 0 0 in ni in p , - - , - - - - - - _ , - - ------------- .~ I z lD 61c ) rl v 01 in r r .-1 cT fr I Z I rl N M N in CO in r-1 N 61 61 m 1 C I H t in N 61 ~' r(V CO W m r O~-1 I I H I N tIl M in 61 in O In r N in H I ro I Q O O M r in v' O Ln M in ct' M I I N v' in in O ~D rl ~D c> 'D O rl I I (L' I N I 1 ~ I 61 ~ I m I 6~ N ry ~n N 61 O 01 tf-) W in I I m I IIl DJ in CO O O MM O M I .~ o\o I N N N M N H N N N N N tP o\0 1 N N in N M N~-+ N .~ M N ~O m Z ct' N~-1 V' H N~fl (A ~D M v' M I I 2 I Ifl N M M r M r N N~' 6\ O 1 W 3 1 r in CO in m m M N v' lO .-i O 1 I W ',~ 1 r1 r .-i O~ 6~ M N H I W CO N M r O M O O CO r in l0 r I W m I H O in in in r-I 01 1f1 61 N~' ro 1 14 in (N a ow 1 .N. in. M . .M. M . M . . M. M .M. O I I 1 1 ~- .] Cv w ow , I M . v' . N . V' . ~' . M . rl . M . r1 ~' .. 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I . . . . . . 1 -~ I U 1 O O O O O O O O O O O O I 1 U' I O O O O O O O O O O O O I C I ~ I I `-' I , - - , - - - - - 1 , - - 1 - - - - - - - - Cn Z N N r M r N~' O' T Ol lD 6~ 1 (n Z H W W r N rl ~f1 [h N .-i N M a W I H ~ N00 M M l0 v' O r lD M Q' 1 W I H I r O~ M CO in N in O r M N I ,-~ ~] I F~ I . . . . r I 17 I < I . . . . . in I .~ /~ I (1v' I in .-1 ,O ~ W M o v' O r co I /L I o in N O] r~~ r 61 in N 1 ~ I m I O 61 ~-+ N W W M v' In d' in f I ~ 1 m I v' N r1 lO 61 r cT f r in O in 1 in a In IIl in Q' ~n O' M I I o\o I lD In iI1 in tn 1 ~ [ Q: O ID ID O M r in in O CO C.." in 61 in 61 tn O in in Co ti M x I W in 1 M NlO V' ~' o a' N QJ r in r 1 X I W 3 1 N r1 rl O fr 61 in M l0 r In I v I > m M~D rl rl o r O M o' Ol 61 M i N I > m I r1 r l0 LO M in m N r-1 61 61 m I V1 I H Ca4 I Q' ~D r v' M l0 ~Il in lD In o v' I in I H[a .~ 1 6\ ~n r~~ N r~ r-1 M N M I I i-7 0\0 1 . . . . 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M . v' .O. ,I ~ .~ JJ I r~ C 1 I ti~-1 .-1 !x .-~ rl r-1 rl r-1 .-1 rl I I C rl O 1 I l~ FZ I rl ri r-1 rl ri r-I (2,' I .~ rl i-1 f-I , N O I O Q) 1 O ~ pa 1 l0 I ~ I N 1 C.7 14 I Ol l0 l0 ID in M~O rl in 'D in I N 1 m I N CO fr ~-1 If) 61 m c) 61 M o .-1 I in CO N ri N r M O' In m M 1 ~ I - I In in r Ir in r in M in in r N 1 JJ In LO In ~t' O 1 J~ I .......... . . I 41 I Ol 1 v' O' v v' in d v' Q' cT ~' v' O I C I .......... . . I tP I in o O O O in o O O O o o I Q) I (9 ,Elo O O in O O O in in O O in I -H ~' I I ~4 I I N p I 1 - - - - - - I - - I H 1 - I I - - - - - - - - - - - - - I ~ O 1 ~ I o 0 in O O o o O O O tiLn t I ~, O O O o in O O in O O ni I ~mU~7II rl. .- . ~. O . '. N . .In. r. l .M!~ . . OI~I N mI i~ n Iv' i. n. N . ~ . l. O . N . . ti. in . r .r . aII O "(j I Ga (') I ~n ~O r r a M H M 61 N V' (V I I w(~ 1 l~ f`") r-I N M rl O O O ~D (V N I m /-:; I ~ I M M M M(-'1 M M M N M M I m 1 ~ I Cl M M M M M t"1 Cl M M M I H H I I i_--- - -I - H _- - - 1 - - H - _ - I - H I I ID m l t rl N M Q' ~Il r W O I I ~l O O o 0 o O O O r1 , I a I 1 , y -----In ---m-m----- .-V N M in in r O I - ~-I in rl ry in in in in in in - ~ 03 I ~ I M M M M M M M M M M C I i ~ I M M M M M M M M M M C I I/-~-I a 1 H I ro 0 I Q. 1 I td p I I ,^ ~ o i rtZ. ~O LI I I i ~ ~(!) 1 1 7 1 z I ~. O I rr^ I E U7 I I in CN (f) I - - i _ - - - - - -- I-~ _ I _ i - - - - - _ 1I Wm ~~ ~ p . 164 00 0 ~ - - 1 - - - - - - - - - - - - - 1 f - - 1 f 2 1 CD ~O l9 -n .H r- In Ol N M o .-1 1 f Z 1 I O] o -n r M itl O M d' 6\ Ir 1 H I O r Q' a' NO O GO O W rl H I ~ M tn M r1 M ri 6l N(V r N 1 1 61 ~-1 61 r o r d lD ul r ~ O~ 1 ul In [~ m r ~O v' r-1 ~O N r1 rl 1 1 LG 1 M M .-~ rl O ~n I 1 V' M N 6~ H Ol CU 6~ 1 W I d' M M f-1 O O - 0.l O N N I I o\ 1 N~-1 6l r1 OJ rl i r-V I~ 6~ r-1 N 1 0\ 1 6l '-1 61 [~ ~ 61 rl r-1 N--~ 6~ ~-I 1 1 1 (n I .-1 ~O M N .H ID T M In m d' 61 1 V) I r r-1 M N ~D M OJ ~O O~ rl In d' 1 ~~ I f-1 r Q' r 6~ l0 CO 6~ In M rl N I ) 3 r l0 tn f~ m M O M In (V ,O _0 1 ~ I ~ p7 I ~ I >>+ [ti 1 .-1 r1 M O O r r ~n Ol N ~ a 1 1 ~ Oa 1 N Cl N r-1 N Q' r~ N ~D M N 1 .-1 N~ N r-1 .-I .-i r-~ O rl .~ O 1 1 ,>+ [.U I r~ rl rl r~ O rl f-1 ti rl H .H O 1 ......... . I 1 ~ o\0 1 .... .... 1 I H O O O O O O O O O O O O 1 H O O O O O O O O O O O O 1 ~ ro I r d' ul bl lO N l0 v' N O rl u~ I 1 [~ 6~ N i~ N ~O tn 61 OJ T r 6~ 1 -~ I M r v ~O M~O tn ~n M v' tn f-1 I 1 -~ 1 Q' d' v' M M-n In M Ln v' O 1 I tn I O O O O O O O O O O O O I I V] I O O O O O O O O O O O O I I E 1 . ........ . .II~1 .. . . . . . . . . . .1 ~ C7 1 0 0 0. Co O O O C)* O o o O 1 1 (' 1 o O O O CD O O CO O O O o 1 4 f ~ ~ - - - - - - - - - - - - - - - - - ------------- In N 61 V r-1 61 N~~ r rl M 1 1 ',Z 1 W ~O M M un c, M M 61 1 ~ I H In 'D N rl Nm M M tn OJ r1 M 1 1 H 1 6) r N N'-1 M M ~n O d' M 61 I ro 1 /f 1 m m r ln r d' r N O ~O r~O 1 I I N M O M O n M ul r v' O tll i 1 a' I N 1 I I OJ 1 1 LY~ 1 .~ I o\ I M In M r-1 CD ~Il f-1 OIn r .-' 1 1 fY1 1 ~ in N r r r O M irl d' N I .-~ N~-+ N N N N N N rV N N I I \ I .~ N N rl N .-1 N N N N N M I ~ 2 1 M rl r M W l0 dl co M Lf) N N Z ~-1 N M M M .-1 N t~ O N O N 1 W,'3 1 - r cc Q' N r r-I M V ~-i W lD 1 1 W'~ 1 O Ir ~ o r d' W~In In I n H L] fY1 M ~ N r N V' O M v' OI 1 W ff1 1 N N~ CO d r O~ N ul M O v' 1 ~D ro ~-7 C+~ I N M N M N M M M M N M O 1 I ~l C+-i I N M N N M N N M M M M O I -" I W o~ I .......... 1 I '1~ o\ I ~ (n O O O O O C. O O O O O O 1(n I O O O O O O O O O O O O 1 1 I w I I O~ ul O OJ N-n r f"1 Nco M ID I -- m m O 61 ri O O r1 l- O rl ro Ul 1 O rl o r-I O r-1 r-I rf rl O E 1 ~ 1 ........ .-1 I I 1 rl N~D CU I N M d' O~ N v' l0 1 I -- I N r1 O CO N N 6~ rl O N O--1 1 0 1 Ul 0~ r1 o r-1 0 0 .-1 .H r-1 rl C. 1 . . _~ U 0 0 0 0 0 0 0 0 0 0 0 0 .1 i 11U E 1 1 o O o 0 0 0 0 0 0 o O O C 1 ~ 1 1 t 1 i- - - - - - - - - - - - - - - - f - - N ,U,,~ ro Z 1 0 o rl 61 r o r~1 lD M n N 1 Z ul M v n l0 N M o M 61 C .n N~ r~ r 6~ N~~ M 61la1-Q'rdotnM1JHot 1 .~ 1 Q i ~ I ~ I N ~ :!] I ',.i.' N r rl lO r V' 6\ 6~ O d' ul I 1 N O l~ 1~ O O d' Ol l0 Q' r '.Il I R' v' N N 61 1~ O ~D ~ W I~ ~1' V1 I W d' M M 61 r~D v' rl O l0 O r 1 _~ W 1 d 1 r ~D lO r ~O ~O lO tIl Ir 10 ~O lO f R7 1 0A0 1 ~O r r~ r I'D r Ir r~O r d' f .~ ~l f F-1 1 f C r~ 'y' CO r-1 ~O N W r ri fr CU l0 O~ Ltl f ~ 1 CLf N H~ r O M m M o d MItl f H '~ 1 f+] 3 1 rl V 61 N~ l0 Ol OJ ~--I o N M f ~ 1 W 3 f rl l0 ~^ N N M v' ~f' 61 v' ~n V f > W 1 M v' N Ol r~O ~n Q' ~' ~' ~n ~ 1 1 ~ W f [~ M Ul N O M N Ul Ln O ~n rl 1 H[z~ 1 ~ ~1 O~ M ~O tn tn M ~n r 1 1 Iti [ .a 1 ~O a' OJ 61 - 6~ r-1 ~ r 1 O o . 1 1 ~-7 0\0 1 rl o o . 1 x ~ 1 61 61 N .-1 Ol N Ol 00 .H 61 Ol 0 I x I I N 61 Ol rl 61 ri ,-~ 61 6l 61 bl 0 1 ~ 1 1 1 ~ 1 1 cn I I ~' .H N t~ ~I1 N N CO O r ~ N I (n I I ~ r 6~ V' ul r O I -~ I t~ N r v' O~ v' 61 M m r1 N 11l I I -~ I 61 6~ M a' M r-1 O~ 6~ r-I lD r I 1 I Ul I N M 61 ID O O 61 O] v` O E 1 .......... .-1 N 1 1 f<7 1 . .IIEI .-1 MIn Ln In M[n d' dl M M r1 1 . . . . .. .. .. . .1 I U' I M M N M M M N N M M M O I 1 U' I M M("1 t`") M f") Cl M N Cl f'l O 1 1 1 1 1 1 ~` I I I C~ 1 I 1 - - - - - - - - - - - - - 1 Y - - - - - (~ ~. I ~~ ~~ (~ U) b` H 4) 1 ~ f I I M M 61 ifl In In M M f~ r i CP I I ~n r O W I~ 6~ ~ 6~ .~ 6~ r N 1 E 1 3 1 ~' In 61 r Ln co t>7 01 r~ 1 E 1 ,'3 0~-i r r In M -Il N .-1 O I W 1 N M N O d oJ l0 O M~O rl O I 1 R7 1 ~~ M rl o~ ~O r N CO r O~ O 1 1 M Ln d' t!7 M N Ln In M M I 2 G-1 1 M N M O N IP M M M M M rl I aW 1 I f H o\ I I Q I r1 .-I f-1 ,-i r-I rl .--1 r1 r-1 r1 .H CD, I 1 ~ 1 .-1 !-1 ~-1 rl .H rl ,-1 `-1 .~ H .-1 0 1 .. 1 ~ 1 1 . . 1 C. 1 LJ I '.. I .-1 N 61 N d' ~i '-1 61 r r-I M N 1 1~ I 07 1 I~ I/1 O N 61 N M v' v' 61 lD W 1 O a' In 61 O b' Q' f M I G I I 61 'D w O In O~t' W N 6l f N I 1~ m 1 d' ~ O' ln d' d` ~' ln Q' Q' d' O 1 (1) 1 !n ~. 1 ~ 1 1 E 1 ~ 1 . n . I ~T I O o 0 o O O o 0 o O O O I J-~ f U' 1 O O o O O O ID O O O O O I -i ~ 3~ -. . .-Vi S ro a ~ i 1 - v f 1 -- - 1 - 1 - 1 - - 1 1 - -~ 1 - 1 1 1 ro - f -fv - 1 ~ - 1 1 ~-'~ ~ ~ I ~ .~~-! CN 1 -' I O O O O O O O O O O N rl I E+ 1 ~ 1 O O O. O . 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I 1 U) I O O O O O O O O O O O O I 1 U) I O O O O (D O O O O O O I W (n I I I U) I I I W I I H 1~ Ol l9 H~ O - v' r-I ry - I W I I V' M(A lO N N ~n N t .f) r1 lO I H r-7 1 ^- . I 61 O lO CO GO ~n l0 l0 O 61 N rl 1 1-7 I -~ I lD M R' w l0 I~ l0 1)~ f~ N I b ~ I U] I O rl O O O O O O r1 O O O I rf I U1 I O H O O O O O O O O O I .......... . .1 ........ . . O O O O O O O O O O O O I O O oO O O o O O O i ~-' < ~-' 1 I x ~ - - I - - - - - - - - - - - - - I X" I - - I - - ~ N 1 (/) I 2 N("1 N 7 I H I I ~ 1 1 O' M I U) I 2 I iD N~l OJ M O l0 I I H I M N OJ .-i N 1 6~ I .~ I < I H O O O O N v' M O~ ti t~ I I a I ID ~ l0 M O N v' M -~ I f1 .' I v' O in M N N ~n f~ V' M ttl O I I (L' I ~O N ~n M m N N r-1 M In I I p~ I ~O O N~ f~ a' ~n ~O ~' N ul M I I CO 1 lO itl ct' ~ M I~ ~--i ~Il M M I _~ oW I f-1 N .-1 .-1 .-~ rl r1 ri r1 rl r-1 cV I ~. I o\o I .-1 M r1 ~-V .-1 r1 ,-1 rl .-1 0~ I .~ ~ I I I T I I I 3-1 I f1.' 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F I ... ..... . .I ~ I U' I o~ N Ln lD f~ ~O ~O Ir ~O Ln lO ~ I ~ I (-J' I ~O r-I l0 A~~ f Q' fr ~n ~ I C~ ~ ~ I v i i ~ E 1 I v I I tli - - I - - - - - - - - - - - - - I - - - - - - - o O C3 ~ I I r-1 N O f W lD a' ~ .~ N W M I ~ I I V' O N fr 61 N o M in 6l 10 I O I ~ I T W M v' 11) O a' 6~ .-1 rl l0 O I O I ',3 I O ' O d' M r-1 lD l0 N N 6\ I M I W 1 ln itl N 61 in N N v' m o~ f-I N I M I W I l0 lo m 1' ul OJ N h 1~ N M I . .-r H Z C~+ O M N f~ Q' M N I~ v' N I Z 14 M M a\o .......... . . I o\o .. n .. .. Un l0 ... In rl .. .~ I ~' } I rl 11 .-1 ~-1 f-1 r-~ .-1 r-1 r-I rl rl r-+ O I [Y~ I I I I I ry rI r-1 ~-I r-1 rl ~-1 ,-1 ,-1 ,-I O I 1J I ~ I I C 1 CO I CO cD l0 V' N N lO l0 W l0 fr N I C 1 W I 61 d' ~O 10 OJ O ~n M o f~ O I N I -~ I OJ ri N N M ~D W(`"1 v' V' M N I Q) I -~ I ti 1^ M~D rl 61 N r1 OJ V' M I JJ C/~ ~ i ~ i v ~r v~ v~ a v~ M v m<r v~ O i ~ i ~ i - m - v v - - - - ~r o i ~ O. N R1 I C9 1 O O O CD O O O O O CD, O O I ro I (7 I O O O O O O o O O o O I I " 1 I 4) 1 -' I I ~ p X 14 I F - - I - - - - - - - - - - - - - I S~ I - - I - - - - - - - - - - - - I ~ I I I F+ ~ I ~ (,Oy' O O O O O O O O O O o o I ~ --, I O O O O O O O O O 61 r I ~ I ~ tl) I M\n N V' d' N ti M f~ 61 ~' M 1 1 3 U1 w Ir O' l0 6~ ~n l0 N 1 W E I .......... . .1IWE1 ......... . .10 i [.ti (J' I r-1 6l N O Q' [~ Ir N~' O' r1 v' I I [a, C7 I O v' o r-+ ~D fr N lD ~ 6~ N 1 fYW ~ I ~ I M M M M M N N M M N M I I " I M M M M[V CV N N N N I ~ HI - I I - - - - - I _ _ -I - - H I W I1 - I - - - -I - - - - - -I - - r - Id H ,.~~ ~ 'x I I I x i I I ~ I i-] t'-1 N M T ~n l0 f ~ 61 O I I .] I ,-~ M Q ~n \O Ir N 6~ O I -rl O O O O O O O O~ I I r~ I - - H,-I ,-1 ti r-1 - ~ I [L p ~ I I .-1 rl ti H rl ~-1 .~ rl c ~ I I~ I~-1 rl ri rl r-1 rl .-1 rl C I C C1. I H I rl ~-1 r-I rl fi rl .-i ~i ti N ~6 ~ 1 CL 1 H I r-~ r+ rl ~I ti,ti r-1 rl ,H N ~ I I -r-i Q a 1 Z I N I 7 1 2 I 4) . ~+ O 1 r-~ I E V) I O I FC I ~ 4 I I I ~ I I ~ M I ~ Q N U' I - - 1 - - - - - - - - - - - - - I - - - - - -_ -_- _- I I W w ,~ 4,11 p . 168 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix K Individual Animal Pathology Data - 168 - Ammonium Perfl uorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 169 DuPont- 18318 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 b, c, d, etc .) . (i .e ., a, - 169 .~~C'j Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 170 DuPont- 18318 Individual Animal Pathology Dat a Group : IA Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 101 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 FATTY CHANGE, DIFFUSE, THYMUS ECTOPIC THYROID . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 102 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 102 Continued SPLEEN HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN . on the next page . . . . EXTRAMEDULLARY, minimal . - 170 '~ ~ p . 171 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IA Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------102 Continued from previous pag e Histopathology LYMPH NODE - POPLITEA L NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 103 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 104 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 NODE on the next page . . . . - 171 3+e p . 172 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IA Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------104 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEA L 105 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEA L 106 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 106 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JbINT, STERNUM, POPLITEAL LYMPH NOD E on the next page . . . . -172- ~9 -j p . 173 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IA Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------106 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 107 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 108 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 108 Continued No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE on the next page . . . . -173- 3,-Yo Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 174 DuPont- 18318 Individual Animal Pathology Dat a Group : IA Treatment : 0 mg/kg Sex : MALES --------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 108 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEA L 109 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 FATTY CHANGE, DIFFUSE, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 110 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 110 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 . . . . -174J-) p . 175 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-I8318 Individual Animal Pathology Dat a Group : IA Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------110 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEAL - I75 - 3,..AL Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 176 DuPont- 18318 Individual Animal Pathology Dat a Group : IB Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1ll Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 SPLEEN HEMATOPOIESIS, INCREASED, CAUSE OF DEATH : SACRIFICE BY DESIGN . EXTRAMEDULLARY, mild . No Microscopic Abnormality Observe d LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 112 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W - I76 - .~~~~ p . 177 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IB Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 113 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 114 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . 114 Continued on the next page . . . . - 177- ~~ ~ p . 178 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IB Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------114 Continued from previous pag e Histopathology No Microscopic Abnormality Observe d LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 115 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNJM, BONE MARRO W 116 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 116 Continued on the next page . . . . - 178 ~~~ p . 179 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IB Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------116 Continued from previous pag e Histopathology CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 117 Unscheduled Sacrifice Duration of dosing-days : 5 Exposure Group : Early Deaths Animal is signed off from necropsy Gross Patholog y TRACHEA RUPTURE . ESOPHAGUS : RUPTURE . SKIN : OTHER, abscess subcutaneous axilla right,subcutaneous air pocket, dorsal neck, right axilla . No Macroscopic Abnormality Observed LIVE R Histopathology MESENTERIC LYMPH NOD E DEPLETION/ATROPHY, LYMPHOID, minimal, (outer cortex and follicles) . THYMUS DEPLETION/ATROPHY, LYMPHOID, mild . ESOPHAGUS : INFLAMMATION, MYOFIBER, mild, (due to esophageal rupture) . SKIN : Moderate, ABSCESS . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate . 117 Continued on the next page . . . . - 179 IS6 p . 180 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IB Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------117 Continued from previous page Histopathology : CAUSE OF DEATH DOSING ACCIDENT . MEDIASTINUM : INFLAMMATION, CHRONIC, (due to esophageal rupture) . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, TRACHEA, FEMUR/KNEE JOINT, STERNUM 118 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 119 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 119 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 . . . . - 180 - ~~ ~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 181 DuPont- 1 8318 Individual Animal Pathology Dat a Group : IB Treatment : 0 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------119 Continued from previous pag e Histopathology CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 120 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMED'JLLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d LIVER, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW -181- 3 1.r-~r. Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 182 DuPont- 18318 Individual Animal Pathology Dat a Group : IIIA Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 301 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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, mild, with cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 302 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . mild, with 302 Continued on the next page . . . . -1R~- p . 183 Ammonium Perfluorooctanoate : 2 8- D ay Immunoto xi city S tudy in Male Mice DuP ont-18318 Individual Animal Pathology Dat a Group : IIIA Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------302 Continued from previous page Histopathology : No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 303 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observe d LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, =EMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, mild, with cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 304 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . . . . - 183 3 p . 184 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IIIA Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------304 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . HEPATOCELLULAR, stippling . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 305 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 306 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 NOD E on the next page . . . . - 184 - 47 ._ ~ p . 185 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IIIA Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------306 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, mild, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 307 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARRO W -185- ~~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 186 DuPont- 18318 Individual Animal Pathology Dat a Group : IIIA Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------308 Terminal Sacrific e Killed on Day : 29 Exposure Group S D 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 . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 309 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 309 Continued LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . on the next page . . . . HEPATOCELLULAR, stippling . mild, with - 186 - ~ 6,3 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 187 DuPont- 18318 Individual Animal Pathology Dat a Group : IIIA Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 309 Continued from previous pag e Histopathology : No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 310 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, FOCAL, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW . ~~ J 187- , p . 188 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IIIB Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 311 Terminal Sacrific e Killed on Day : 29 Exposure Group S D 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, mild, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 312 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 312 Continued LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . on the next page . . . . HEPATOCELLULAR, stippling . mild, with - 188 - ~~5 p . 189 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IIIB Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 312 Continued from previous pag e Histopatholoqy : No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 313 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 314 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 314 Continued on the next page . . . . -189~~~ p . 190 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IIIB Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 319 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . HEPATOCELLULAR, stippling . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 315 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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, mild, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESiS, INCREASED, EXTRAMEDULLARY, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS, BONE MARROW - 190 - 3C7 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 191 DuPont- 18318 Individual Animal Pathology Dat a Group : IIIB Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 316 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . mild, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 317 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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, mild, with cytoplasmic eosinophilic stippling . SPLEEN DEPLETION/ATROPHY, LYMPHOID, CAUSE OF DEATH : minimal . SACRIFICE BY DESIGN . 317 Continued on the next page . . . . -191- 3~ ? Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 192 DuPont- 183 I 8 Individual Animal Pathology Dat a Group : IIIB Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 317 Continued from previous pag e Histopathology : No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 318 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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, mild, with cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 319 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 319 Continued on the next page . . . . - I92 - . . ~~ ~ p . 193 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IIIB Treatment : 0 .3 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 319 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, mild, with cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN _ No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 320 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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, mild, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS, BONE MARRO W - 193 - ~ 71-~) p . 194 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VA Treatment : 1 mg/kg Sex : MALES --------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------501 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 502 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 502 Continued LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . on the next page . . . . with - 194- .3,~~ p . 195 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VA Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------502 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 503 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 504 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 504 Continued No Macroscopic Abnormality Observed LIVER, SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E on the next page . . . . -195- `37Z p . 196 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VA Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------504 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . THYMUS HYPERPLASIA, LYMPHOID, FOLLICULAR, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 505 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARRO W - 196 - 3 73 p . 197 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VA Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------506 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 507 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 507 Continued LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . on the next page . . . . with -197- 3" ~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 198 DuPont- 18318 Individual Animal Pathology Dat a Group : VA Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------507 Continued from previous pag e Histopathology : No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 508 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, FOCAL, minimal . INFLAMMATION, SUBACUTE/CHRONIC, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 509 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 509 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 . . . . -198- ,37-6 p . 199 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VA Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 509 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, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 510 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW -199- ,376 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 200 DuPont- 18318 Individual Animal Pathology Dat a Group : VB Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 511 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 512 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 512 Continued LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MESENTERIC LYMPH NOD E NOT PRESENT . on the next page . . . . with -200- '377 p . 201 Ammonium Per fluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VB Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 512 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observed SPLEEN, THYMUS, BONE MARROW 513 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 514 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 514 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 . . . . - 201 - 3T8 p . 202 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VB Treatment : 1 mq/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------514 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . NECROSIS, FOCAL, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 515 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARRO W -202- 37 ~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 203 DuPont- 18318 Individual Animal Pathology Dat a Group : VB Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------516 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . THYMUS NOT PRESENT . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 517 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . 517 Continued on the next page . . . . - 203 - 3 ~~ ~,? Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 204 DuPont-18318 Individual Animal Pathology Dat a Group : VB Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 517 Continued from previous pag e Histopathology : CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 518 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 NODF. Histopatholog y LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic CAUSE OF DEATH : SACRIFICE BY DESIGN . HEPATOCELLULAR, stippling . moderate, with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 519 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 519 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 . . . . -204- ~~P p . 205 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VB Treatment : 1 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------519 Continued from previous page Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 520 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW - 205 '30 ?,- p . 206 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------701 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . Minimal, FATTY CHANGE, NONZONAL . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 702 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . 702 Continued No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH JOINT, STERNUM, POPLITEAL LYMPH on the next page . . . . NODE, NOD E THYMUS, FEMUR/KNEE -206- 363 p . 207 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- /02 Continued from previous pag e Histopathology LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 703 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . SPLEE N SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology 703 Continued LIVER INFLAMMATION, SUBACUTE/CHRONIC, minimal . NECROSIS, FOCAL, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERPLA IA, BILE DUCT, minimal . on the next p ge . . . . with -207- 3,5> ~- p . 208 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------703 Continued from previous pag e Histopathology THYMUS DEPLETION/ATROPHY, LYMPHOID, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 704 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEEN SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARRO W -208- -2v~7, p . 209 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------705 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NOD E THYMUS, FEMUR/KNEE Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 706 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NODE THYMUS, FEMUR/KNEE 706 Continued on the next page . . . . - 209 - 39& Ammonium Perfluorooctanoate : 2 8- Day Immunotoxi c i ty S tudy in Male Mice p . 210 DuPont- 18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------706 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 707 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . SPLEEN SMALL . THYMU S SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology 707 Continued LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, wit h cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . on the next page . . . . -210- 3 87 p . 211 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------707 Continued from previous pag e Histopathology THYMUS ECTOPIC THYROID . DEPLETION/ATROPHY, LYMPHOID, CAUSE OF DEATH : SACRIFICE BY DESIGN . minimal . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 708 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . THYMUS DEPLETION/ATROPHY, LYMPHOID, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARRO W -211 - 39 F Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 212 DuPont- 18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 709 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEE N SMALL . THYMUS SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . Minimal, FATTY CHANGE, NONZONAL . THYMUS DEPLETION/ATROPHY, LYMPHOID, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 710 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 710 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 . . . . - 212 -~ Fq Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 213 DuPont- 18318 Individual Animal Pathology Dat a Group : VIIA Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 710 Continued from previous pag e Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW -213 - 3~a p . 214 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 711 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . Minimal, FATTY CHANGE, NONZONAL . THYMUS : NOT PRESENT . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARROW 712 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 712 Continued on the next page . . . . - 2I4 - -34 p . 215 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES --------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------- 712 Continued from previous pag e Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 713 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology LIVER LARGE . DISCOLORATION, TAN, 1CM DIA . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology 713 Continued LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, moderate . HYPERPLASIA, BILE DUCT, minimal . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . on the next page . . . . -215- 377- p . 216 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kq Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------713 Continued from previous pag e Histopathology THYMUS DEPLETION/ATROPHY, LYMPHOID, minimal . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NOD E 714 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SKIN : MASS, GREEN, AXILLA, LEFT, 1 .3CM DIA . No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . SKIN : Moderate, ABSCESS . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate . 714 Continued on the next page . . . . 3473 - 216 - p . 217 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------714 Continued from previous pag e Histopathology CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, THYMUS 715 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEEN SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, BONE MARRO W -217-D Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 218 DuPont- 18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 716 Terminal Sacrific e Killed on Day 29 Exposure Group : S D 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 NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARROW 717 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEE N SMALL . THYMUS SMALL . 717 Continued No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE POPLITEAL LYMPH NOD E on the next page . . . . JOINT, STERNUM, -218- 3~ ..~ p . 219 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont- 18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES --------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------717 Continued from previous pag e Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . MESENTERIC LYMPH NODE : NOT PRESENT IN TISSUE SECTION . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observed SPLEEN, THYMUS, BONE MARRO W 718 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology LIVER LARGE . SPLEEN SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology 718 Continued LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . on the next page . . . . - 219 - 3 76 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 220 DuPont- 18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------718 Continued from previous pag e Histopathoiog y LIVER Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed MESENTERIC LYMPH NODE, BONE MARROW 719 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, cytoplasmic eosinophilic stippling . CAUSE OF DEATH : SACRIFICE BY DESIGN . with No Microscopic Abnormality Observe d SPLEEN, MESENTERIC LYMPH NODE, THYMUS, BONE MARRO W 5q -220- ~ p . 221 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : VIIB Treatment : 10 mg/kg Sex : MALES --------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 720 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEE N SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, mild . INFLAMMATION, SUBACUTE/CHRONIC, minimal . THYMUS DEPLETION/ATROPHY, LYMPHOID, minimal . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observed SPLEEN, MESENTERIC LYMPH NOD E -221- 31719 p . 222 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 901 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . NECROSIS, FOCAL, minimal, coagulative, subcapsular . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS DEPLETION/ATROPHY, LYMPHOID, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 902 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 902 Continued on the next page ... . - 222 - 3~i~ p . 223 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kq Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings --------------------------------------------------------------------------------- 902 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . THYMUS NOT PRESENT . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNU M 903 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEE N SMALL . THYMUS SMALL . 903 Continued on the next page . . .. - 223 YX p . 224 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------- Ani :nal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------903 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 904 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEEN SMALL . 904 Continued on the next page . . . . - 224 - p . 225 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALE S ---Animal Ref Microscopic & Macroscopic Finding s ---------- --------------------- ---904 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . NECROSIS, FOCAL, minimal, coagulative, subcapsular . Minimal, FATTY CHANGE, NONZONAL . SPLEEN DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . MESENTERIC LYMPH NOD E NOT PRESENT IN TISSUE SECTION . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEAL 905 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 905 Continued on the next page . . .. - 225 - -M~- p . 226 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------905 Continued from previous pag e Gross Pathology SPLEEN SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . MESENTERIC LYMPH NOD E NOT PRESENT IN TISSUE SECTION . THYMUS DEPLETION/ATROPHY, LYMPHOID, severe . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE POPLITEA L 906 Unscheduled Sacrifice Duration of dosing-days : 9 Exposure Group : Early Deaths Animal is signed off from necropsy Gross Patholog y LIVER : DISCOLORATION, PALE, MOTTLED, DIFFUSE . 906 Continued on the next page . . . . -226- // 3 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 227 DuPont- 18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES ---------------------------Animal Ref Microscopic & Macroscopic Finding s 906 Continued from previous pag e ---- ------ Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, moderate, wit h cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, minimal, coagulative, subcapsular . FATTY CHANGE, PERIPORTAL, minimal . SPLEEN DEPLETION/ATROPHY, LYMPHOID, moderate, (periarteriolar lymphoid sheath) . MESENTERIC LYMPH NOD E DEPLETION/ATROPHY, LYMPHOID, moderate, (inner cortex and outer cortex) . THYMUS DEPLETION/ATROPHY, LYMPHOID, BONE MARROW : severe . HYPERPLASIA, GRANULOCYTIC, moderate, with left shift (immature) . CAUSE OF DEATH : UNDETERMINED . LYMPH NODE - POPLITEA L NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observed BRAIN, FEMUR/KNEE JOINT, STERNUM 907 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEEN : SMALL . 907 Continued on the next page . . . . -227ly-6 4 p . 228 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------907 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopatholoay LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . MESENTERIC LYMPH NOD E DEPLETION/ATROPHY, LYMPHOID, mild, (inner cortex, outer cortex, and follicles) . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL NOT PRESENT . No Microscopic Abnormality Observe d BRAIN, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 908 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 908 Continued on the next page ... . - 228 y)~g p . 229 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES ----------------------------------Animal Ref Microscopic & Macroscopic Finding s ----908 Continued from previous pag e ----- ----- Gross Pathology LIVER DISCOLORATION, TAN, MOTTLED . SPLEE N SMALL . PENIS PARAPHIMOSIS . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, moderate, coagulative, subcapsular . HYPERPLASIA, BILE DUCT, mild . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . PENIS : EROSION/ULCER, moderate . PREPUTIAL GLANDS : ECTASIA, mild . LYMPH NODE - POPLITEA L NOT PRESENT IN TISSUE SECTION . 908 Continued on the next page . . . . -229ft p . 230 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------908 Continued from previous pag e Histopathology No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM 909 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEEN SMALL . THYMUS SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NODE Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, wit h cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . NECROSIS, FOCAL, minimal, coagulative, subcapsular . THYMUS DEPLETION/ATROPHY, LYMPHOID, severe . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L -230A/ /1 p . 231 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXA Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Finding s 910 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off -----------------------------from necropsy Gross Patholog y LIVER : LARGE . DISCOLORATION, TAN, MOTTLED, LEFT . SPLEE N SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERPLASIA, BILE DUCT, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . DEPLETION/ATROPHY, LYMPHOID, mild, (periarteriolar lymphoid sheath) . THYMUS NOT PRESENT . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L -231~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 232 DuPont- 18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 911 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . SPLEEN SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . THYMUS DEPLETION/ATROPHY, LYMPHOID, severe . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW -232(-V Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 233 DuPont- 18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES Animal Ref Microscopic & Macroscopic Findings 912 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy ------------------- -------------- Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . THYMUS : minimal . NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 913 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y 913 Continued LIVER : LARGE . DISCOLORATION, TAN, on the next page . . . . RIGHT, ACCESSORY . - 233 - 4 / 0, p . 234 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------913 Continued from previous pag e Gross Pathology : MESENTERIC LYMPH NODE SMALL . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, wit h cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . NECROSIS, FOCAL, moderate, coagulative, subcapsular . HYPERPLASIA, BILE DUCT, mild . Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . THYMUS DEPLETION/ATROPHY, LYMPHOID, moderate . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 914 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology : 914 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 . . . . - 234 - q1 ~ p . 235 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES --------------------------------- Animal Ref Microscopic & Macroscopic Finding s -------------- ------- 919 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, wit h cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 915 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . DISCOLORATION, LEFT, 1MM DIA . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . 915 Continued on the next page . . . . - 235 4 fv p . 236 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------915 Continued from previous pag e Histopathology CAUSE OF DEAT H SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 916 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : DISCOLORATION, TAN, CAUDATE, 0 .3 CM DIA . LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . NECROSIS, FOCAL, moderate, coagulative, subcapsular . INFLAMMATION, SUBACUTE/CHRONIC, minimal . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . THYMUS DEPLETION/ATROPHY, LYMPHOID, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . 916 Continued on the next page . . . . -236- ~/3 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 237 DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES Animal Ref Microscopic & Macroscopic Findings ------------ 916 Continued from previous pag e ----- ------- -- Histopathology : No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 917 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . NECROSIS, FOCAL, minimal, coagulative, subcapsular . HYPERPLASIA, BILE DUCT, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L -237~ / Af p . 238 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------918 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . HYPERPLASIA, BILE DUCT, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . SPLEEN : DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 919 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . 919 Continued on the next page . . .. -238- ~~~ ~ p . 239 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALE S --------------------- - Animal Ref Microscopic & Macroscopic Finding s ----------------------------------- 919 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEA L 920 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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 HYPERPLASIA, BILE DUCT, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . 920 Continued on the next page . . . . -239- !i 6 p . 240 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : IXB Treatment : 30 mg/kg Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------920 Continued from previous pag e Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS : DEPLETION/ATROPHY, LYMPHOID, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNJM, BONE MARROW, LYMPH NODE - POPLITEA L - 240 - ,p 7 p . 241 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES Animal Ref Microscopic & Macroscopic Finding s ------------ 1101 Terminal Sacrific e ------------------- -- Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, JOINT, STERNUM, POPLITEAL LYMPH NOD E FEMUR/KNEE Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, HYPERPLASIA, BILE DUCT, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN : mild . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW - 241 */ 9 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 242 DuPont- 18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kq (Recovery) Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1102 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 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 . HYPERPLASIA, BILE DUCT, minimal . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild . SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . MESENTERIC LYMPH NODE : NOT PRESENT IN TISSUE SECTION . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, THYMUS, FEMUR/KNEE JOINT, STERNU M - 242 - T19 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 243 DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Finding s 1103 Terminal Sacrific e ---- - ------ Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM 1104 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 1104 Continued on the next page ... . - 243 ~ p . 244 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1109 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS CYST, EPITHELIAL, minimal . BONE MARROW : HYPERPLASIA, ERYTHROCYTIC, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEA L 1105 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y 1105 LIVER : LARGE . SPLEEN SMALL . Continued on the next page .. . . -244- ~~?1 p . 245 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES ----------------------------------- Animal Ref Microscopic & Macroscopic Finding s --------------- ---------- 1105 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . BONE MARROW : HYPERPLASIA, ERYTHROCYTIC, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM 1106 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 1106 Continued No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH JOINT, STERNUM, POPLITEAL LYMPH on the next page . . . . NODE, NOD E THYMUS, FEMUR/KNEE - 245 ~G-~ p . 246 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1106 Continued from previous pag e Histopathology LIVER MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . HYPERPLASIA, BILE DUCT, minimal . THYMUS CYST, EPITHELIAL, minimal . DEPLETION/ATROPHY, LYMPHOID, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 1107 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . THYMUS SMALL . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . 1107 Continued on the next page . . . . - 246 - ~/--3 p . 247 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES --------------------- -- Animal Ref Microscopic & Macroscopic Finding s ---------------------------------------- 1107 Continued from previous pag e Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . NECROSIS, FOCAL, minimal, coagulative, subcapsular . Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . THYMU S NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 1108 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . THYMUS : SMALL . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 1108 Continued on the next page . . . . -247- ~2~ p . 248 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1108 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MITOTIC FIG-ORES, INCREASED, HEPATOCELLULAR, minimal . INFLAMMATION, SUBACUTE/CHRONIC, minimal . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1109 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology 1109 LIVER HYPERTROPHY, PANLOBULAR, cytoplasmic eosinophilic Continued on the next page . . . . HEPATOCELLULAR, stippling . severe, with - 248 - ~ 2 '5 p . 249 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALE S ------------- ------- Animal Ref Microscopic & Macroscopic Finding s -------------------------------------------------------------------------------1109 Continued from previous pag e Histopathology LIVER HYPERPLASIA, BILE DUCT, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HEMATOPOIESIS, EXTRAMEDULLARY, minimal . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, moderate . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . BONE MARROW : HYPERPLASIA, ERYTHROCYTIC, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNU M 1110 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . DISCOLORATION, TAN, LEFT, NECROTIC 6MM DIAM . No Macroscopic Abnormality Observed : SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E 1110 Continued on the next page . . . . -249- q-76 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 250 DuPont- Individual Animal Pathology Dat a Group : XIA Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------- Ani :nal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1110 Continued from previous page Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, FOCAL, moderate, coagulative, subcapsular . HYPERPLASIA, BILE DUCT, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . SPLEEN DEPLETION/ATROPHY, LYMPHOID, mild, (periarteriolar lymphoid sheath) . HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . THYMUS DEPLETION/ATROPHY, LYMPHOID, moderate . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNU M -250- q-Z I p . 251 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont- 1 8318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mq/kq (Recovery) Sex : MALES ---- Animal Ref Microscopic & Macroscopic Finding s -------------------------------------------------------------------------------1111 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN : HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 1112 Unscheduled Sacrifice Duration of dosinq-days : 5 Exposure Group : Early Deaths Animal is signed off from necropsy Gross Pathology : ESOPHAGUS RUPTURE . SKIN : OTHER, ABSCESS AXILLA RIGHT . 1112 Continued on the next paqe . . . . - 251 - q--, Z-9 p . 252 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-I8318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1112 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observed LIVE R Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . FATTY CHANGE, DIFFUSE, minimal . minimal, wit h SPLEEN DEPLETION/ATROPHY, LYMPHOID, moderate, (periarteriolar lymphoid sheath) . MESENTERIC LYMPH NODE DEPLETION/ATROPHY, LYMPHOID, mild . THYMUS DEPLETION/ATROPHY, LYMPHOID, severe . BONE MARROW : HYPERPLASIA, GRANULOCYTIC, moderate, with left shift (immature) . CAUSE OF DEATH : DOSING ACCIDENT . No Microscopic Abnormality Observe d BRAIN, FEMUR/KNEE JOINT, STERNUM, LYMPH NODE - POPLITEAL 1113 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D 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 1113 Continued on the next page . . . . - 252 - , ~~~ Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 253 DuPont-18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1113 Continued from previous pag e Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 1114 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . SPLEE N SMALL . No Macroscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology 1114 LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, cytoplasmic eosinophilic stippling . HYPERPLASIA, BILE DUCT, minimal . Continued on the next page . . . . severe, with - 253 ~I"~~ p . 254 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1114 Continued from previous page Histopathology LIVER NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 1115 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Pathology LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . INFLAMMATION, SUBACUTE/CHRONIC, minimal . SPLEEN DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . 1115 Continued on the next page . . . . -254, ~ / Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice p . 255 DuPont- 18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1115 Continued from previous pag e Histopathology CAUSE OF DEAT H SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 1116 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D 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, severe, with cytoplasmic eosinophilic stippling . INFLAMMATION, SUBACUTE/CHRONIC, minimal . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . NECROSIS, FOCAL, minimal, coagulative, subcapsular . SPLEE N HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, mild . THYMUS DEPLETION/ATROPHY, LYMPHOID, severe . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . 1116 Continued on the next page . . . . -255- *-32 p . 256 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1116 Continued from previous pag e Histopathology No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARRO W 1117 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, mild . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild . HYPERPLASIA, BILE DUCT, minimal . INFLAMMATION, SUBACUTE/CHRONIC, minimal . Minimal, FATTY CHANGE, NONZONAL . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW -256- 33 'l' p . 257 Ammonium Per fluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mq/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1118 Terminal Sacrific e Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, JOINT, STERNUM, POPLITEAL LYMPH NOD E THYMUS, FEMUR/KNEE Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . CAUSE OF DEATH : SACRIFICE BY DESIGN . LYMPH NODE - POPLITEAL : NOT PRESENT IN TISSUE SECTION . No Microscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, BONE MARROW 1119 Terminal Sacrifice Killed on Day : 29 Exposure Group : S D Animal is signed off from necropsy Gross Patholog y LIVER LARGE . 1119 Continued on the next page . . . . - 257 - '~-3+ p . 258 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------- Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------1119 Continued from previous pag e Gross Pathology : No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH NODE, THYMUS, FEMUR/KNEE JOINT, STERNUM, POPLITEAL LYMPH NOD E Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, mild . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . DEPLETION/ATROPHY, LYMPHOID, minimal, (periarteriolar lymphoid sheath) . THYMUS NOT PRESENT IN MEDIASTINAL TISSUE . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, STERNUM, BONE MARROW, LYMPH NODE - POPLITEA L 1120 Terminal Sacrifice Killed on Day : 29 Exposure Group S D Animal is signed off from necropsy Gross Patholog y LIVER : LARGE . 1120 Continued No Macroscopic Abnormality Observe d SPLEEN, BRAIN, MESENTERIC LYMPH JOINT, STERNUM, POPLITEAL LYMPH on the next page . . . . NODE, NOD E THYMUS, FEMUR/KNEE - 258 - ~~ ~ p . 259 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Individual Animal Pathology Dat a Group : XIB Treatment : 30/0 mg/kg (Recovery) Sex : MALES -------------------------------------------------------------------------------Animal Ref Microscopic & Macroscopic Findings -------------------------------------------------------------------------------- 1120 Continued from previous page Histopathology LIVER HYPERTROPHY, PANLOBULAR, HEPATOCELLULAR, severe, with cytoplasmic eosinophilic stippling . NECROSIS, INDIVIDUAL CELL, INCREASED, minimal . MITOTIC FIGURES, INCREASED, HEPATOCELLULAR, minimal . HYPERPLASIA, BILE DUCT, minimal . SPLEEN HEMATOPOIESIS, INCREASED, EXTRAMEDULLARY, minimal . THYMUS DEPLETION/ATROPHY, LYMPHOID, moderate . CAUSE OF DEATH : SACRIFICE BY DESIGN . No Microscopic Abnormality Observe d BRAIN, MESENTERIC LYMPH NODE, FEMUR/KNEE JOINT, BONE MARROW, LYMPH NODE - POPLITEA L STERNUM, - 259 4 -~ 6 p . 260 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-I8318 Appendix L Individual Total Cell Counts - 260 - ~~ ~ p . 261 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 INDIVIDUAL TOTAL CELL COUNT S EXPLANATORY NOTES ABBREVIATIONS : NP - not taken or not performed FOOTNOTES : c Animal was sacrificed in extremis prior to this evaluation and tissue was not analyzed . d Count inadvertently not performed . e Unable to confirm results . NOTES : Organ Weight as Percent of Body Weight - Organ Weight (g ) 0 Final Body Weight (g) " 10 Total Number of Organ Cell Number of Organ Weight (g) Cells in Hal f Organ Cells - x Suspension Volume x - 100 (x10a) Half Organ Weight (g) (mL) Orga n (x 10 cells/mL) -261- C'l 00 O ~4 .A ~ a~~ m r W N N 61 N lD r-I M O WO O Z a~ O N W M lD O 6~ ri Ol 61 ~ O~ O' d' N W`.L U X rl o rl rl rl o rl o r-I N rl rl ' -1 .-1 O bw v 0 H ~ E w ro O ~ N C'~ v -,~ v N l0 v' O 61 Q' [~ W N d' l0 W o"' o o co 1O rn r rn o o~ W . ... .WM . . . . . . M O ~ Ul ~ ~"'~ O, . N~D O ~ O ~ ~ M~ M V' M O Z M N f-I . ~-y W W r rl N rl rl rl r-I o' O 2 H ,+ U X ~c ~ o - .1 U) n ~ M N n m ul Ln O ul ul ul ............... . 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O O O o N U N N N M Q' ~Il l0 1~ O~ 61 O rl N th Q' ~~O [~ N m O ~ z o o O o 0 0 0 0 o r1 r-i ry rl rl -1 rl .-1 .1 N ~ .-1 rl rl - r-I e-1 rl rl rl e-I r. ~ N rl ti~-1 1 .-1 rl rl ra .-a ,~ r- oC) zt', o x o ~Q Q N ~ v ~ p . 273 p . 274 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 Appendix M Electron Microscopy Report from Experimental Pathology Laboratories, Inc . -274~~ p . 275 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 LEM: Experimental Pathology Laboratories, Inc. DUPONT/HASKELL LABORATORY DUPONT STUDY NUMBER : 18318 WORK REQUEST NUMBER : 1616 0 SERVICE CODE : 1546 AMMONIUM PERFLUOROOCTANOATE: 28-DAY IMMUNOTOXICITY STUDY IN MALE MIC E ELECTRON MICROSCOPY PATHOLOGY REPORT EPL PROJECT NO. 129-080 Submitted to : DuPonUHaskell Laboratory for Health and Environmental Science Stine Haskell Research Center 1090 Elkton Road Newark, DE 1971 1 Submitted by: Expe ri mental Pathology Laborato ri es, Inc . P .O . Box 12766 Research Tri angle Park, NC 27709 October 25, 2006 - 275 - J~4 ~ p . 276 Ammonium Perfluorooctanoate : 28-Day Immunotoxici ty Study in Male Mice DuPont-18318 E P10 Experimental Pathology Laboratories, Inc . TABLE OF CONTENTS Page PATHOLOGY SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 RESULTS . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 QUALITY ASSURANCE STATEMENT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ELECTROMICROGRAPHS -276- '`~,~ p . 277 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 E P I Experimental Pathology Laboratories, Inc . DuPont-18318 DUPONT/HASKELL LABORATORY DUPONT STUDY NUMBER : 18318 WORK REQUEST NUMBER : 16160 SERVICE CODE : 1546 EPL PROJECT NUMBER 129-080 AMMONIUM PERFLUOROOCTANOATE : 28-DAY IMMUNOTOXICITY STUDY IN MALE MIC E ELECTRON MICROSCOPY PATHOLOGY SUMMARY The in-life phase of this study was conducted at Haskell Laboratory for Health and Environmental Sciences, E .I . duPont de Nemours and Company, Newark, Delaware . The objective of this study is to evaluate the potential of ammonium perfluorooctanoate to suppress the prima ry humoral immune response to sheep red blood cells (SRBC) when administered by oral gavage to male mice for at least 28 days . The table below summarizes the experimental design: Experimental Design Dose Solution Daily Dosage Concentration Group Number/Group (mg/kg)" (mq/ml_) 1 20 0 (Control) 0 III 20 0.3 0 .03 V 20 1 0.1 Vll 20 10 1 IX 20 30 3 XI 20 30 (Recovery` 3 ' Weight of test substan ce/kg 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 substan ce through test day 23. Following injection of SRBC on test day 24, group XI will be dosed with NANOpure water, at a volume of 10 mtJkg of body weight, until sacrifi ce . - 277 - J~-"~q Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice p . 278 DuPont- 18318 WEPL~ Experimental Pathology Laboratories, Inc. DuPont-18318 Electron microscopic evaluation of samples of liver from designated animals was added to clarify light microscopic histopathological findings in the liver . Samples of liver from two male mice in Group I (Control) and two male mice in Group V (1mglkg) that were fixed in formalin were submitted for transmission electron microscopy . The samples that were processed and evaluated are listed in the following table: TEM Negative TEM Number Tissue Animal ID Grou Number (evaluated) I G06-403 Liver 103 (Control) 06-1906 to 06-1908 1 G06-404 Liver 104 (Control) 0 6 -1909 to 06-191 1 V G06-405 Liver 503 _C1 m /k 06-1912 to 06- 1 914 V G06-406 Liver 504 (1 mg/kg) 06-1915 to 06-1917 Samples, cut into small cubes, were preserved in formalin and shipped to Experi mental Pathology Laboratories, Inc (EPL), Research Triangle Park, NC . The samples were transferred to the Laborato ry for Advanced Electron and Light Optical Methods (LAELOM) at the College of Veterinary Medicine, North Carolina State University, Raleigh, NC for further processing and examination by transmission electron microscopy . The samples were washed in buffer, post-fixed in 1% osmium tetroxide in the phosphate buffer, dehydrated in an ethanolic series culminating in acetone, and infiltrated with Spurr epoxide resin . The resulting blocks were trimmed and semithin sections ( approximately 0 .5 m thick) were cut, mounted on glass slides, and stained with 1% toluidine blue 0 in 1k, sodium borate prior to being examined with a light micro scope . The slides of semithin sections were sent to Experimental Pathology Laboratories for evaluation by the Pathologist, Dr. Henry Wall . When the slides were returned to the LAELOM, areas of interest for ultrathin sectioning were trimmed in the corresponding tissue blocks . Uftrathin (80-90 nm thick) sections were cut from the selected trimmed blocks and placed on 200 mesh copper grids before being stained with uranyl acetate and lead citrate . For each sample, two survey photographs (final print magnification 5, 600x) were taken . One higher magnification (fi nal print magnification 22,400x) was taken of each sample to show more cellular detail . 2 - 278 ~ ~~ p . 279 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 WE PLO Experimental Pathology Laboratories, Inc . RESULTS DuPont-18318 TEM #G06-403 (Animal 103, Control, Liver, TEM Neg # 06-1906 to 06-1908 ) Two low magnification images (06-1908 and 06-1907 ; 5,600X) show portions of adjacent hepatocytes as the p rima ry cell type in the image . Electron-dense areas that are predominantly mitochondria and rough endoplasmic reticulum are separated by intervening areas that contain clustered electron-dense granules against an electron-lucent background . The electron-dense granules are glycogen depositis . A few fat vacuoles that appear as partially electron-lucent smooth contoured vaucuoles, are sca ttered in the cytoplasm of a few hepatocytes . The higher magnification image (06-1908 ; 22,400X) shows greater detail of mitochondria, rough endoplasmic reticulum, glycogen deposits, and a few membranous cytoplasmic profiles . TEM #G06-404 (Animal 104, Con trol, Liver, TEM Neg # 06-1909 to 06-1911 ) The low magnification images (06-1909 and 06-1910 ; 5,600X) show similar adjacent hepatocytes with electron-dense areas that are primarily mitochondria and endoplasmic reticulum and lighter (less electron-dense) areas with electron-dense granularity . The high magnification image (06-1911 ; 22,400X) shows the electron-dense granulari ty to be glycogen deposits . This image also shows greater detail of the mitochond ri a and rough endoplasmic reticulum that are relatively electron-dense as compared to the glycogen filled areas . All images also show a few smooth contoured lipid vacuoles within hepatocytic cytoplasm . Two deeply electron-dense membrane-bound bodies in the lower right quadrant are conside red lysosomes. The core of these bodies have uniform electron-dense granularity compared to the prominent foldings of the cristae in mitochondria . TEM #G06-405 (Animal 503, G ro up Y/1mglkg, Liver, TEM Neg # 06-1912 to 06-1914) Both low magnification images (06-1912 and 06-1913 ; 5,600X) contain adjacent hepatocytes that have numerous m itochondria rather uniformly distributed in the cytoplasm . Pale granular areas surrounding mitochondria contain glycogen deposits . The glycogen deposits are best detailed in the po rtion of a hepatocyte in the lower portion of image 06-1913 . The higher magnification image ( 06-1914 ; 22,400X) shows more detail of mitochondria, lipid vacuoles, glycogen and endoplasmic reticulum . No peroxisomes are clearly defined in the hepatocyte images from this animal . 3 - 279 - 4?~56 p . 280 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 WEPLe Experimental Pathology Laboratories, Inc . DuPont-18318 TEM #G06-406 ( Animal 504, Group V11mg/kg, Liver, TEM Neg # 06-1915 to 06-1917) Electron-dense areas that are prima ri ly mitochondria are the predominant feature other than the nucleus in the low magnifi cation images (06-1915 and 06-19 1 6; 5,600X) . The paler and granular background is glycogen . A few small smooth-contoured lipid vacuoles are also scatt ered in a few hepatocytes . The high magnification image ( 06-1917; 22,400X) shows the detail of mitochondria, rough endoplasmic reticulum, and part of a nucleus . A few lipid vacuoles are also present . No s tructures that can be clearly defined as peroxisomes are noted . CONCLUSION S At the 1 mg/kg dose of ammonium pefluorooctanoate an increase in peroxisomes was not observed . However, many organelles could not be clearly identified due to poor ultrastructural detail, which was likely the result of formalin fixation . Therefore, defi nitive conclusions on peroxisomal numbers in treated groups relative to cont ro ls could not be drawn . HGW/dc :4-i-R' "N' ~~~ HEN RY G .INALL, DVM, PhD Diplomate, ACVP Veterinary Pathologist 2S d~~ Date ~~1a~ 4 -280- ~s~ p . 281 Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mice DuPont-18318 WEPLq) Experimental Pathology Laboratories, Inc. QUALITY ASSURANCE FINAL CERTIFICATION Study Title : Ammonium Pertluorooctanoate : 28-Day Immunotoxicity Study in Male Mice Client Study : DuPont-18318 ; Service Code 1546; EPL Project Coordinator. Dr. Henry Wall Work Request 16160 EPL Project Number : 129-080 EPL Pathologist : Dr. Henry Wall The following aspects of this study were inspected by the Quality Assurance Unit of Experimental Pathology Laboratories, Inc . Dates inspections were performed and findings repo rted 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 ; July 24, 2006 July 24, 2006 Final Pathology Report October 25, 2006 October 25, 2006 Date reported to Study Director/Management: October 25, 2006 Date of last quarterly facility inspection: October 2006 tQ-~'p, a5 PL uaiify Assurance Unit Date 5 'goo(' Form Na . 6-2 ( October 23, 2006) -281~ 1~