Document yk76Vw09LDq03ez5rLymB3Jp2

Sponsor: 3M St. Paul, Minnesota CHARLES RIVER LABORATORIES Discovery a n d Lkvehpment Svnkes Paihobg- Asoat<s> Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA; 3M T-7091.1) in Rats Study Number: TRC 1132-100 Performing Laboratory: Gene Logic, Inc. (formerly, Therlmmune Research Corporation) 15 Firstfield Road Gaithersburg, Maryland 20878 Prepared by: Pathology Associates Division of Charles River Laboratories, Inc. (formerly, Pathology Associates International) 15 Worman's Mill Court, Suite I Frederick, MD 21701 Date: Original Final Report: August 9, 2000 Amended Final Report: November 8, 2004 15 Worman's Mill Court, Suite I * Frederick, Maryland 21701 * (301) 663-1644 * (301) 663-8994 FAX TABLE OF CONTENTS Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 2 ___________________________________________________________Section Report Narrative I Summary Tables II Individual Animal Data Tables III Appendix 1 - In-life Report IV Appendix 2 - Electron Microscopy Report V Appendix 3 - Palmitoyl CoA Oxidase Report VI Appendix 4 - Study Protocol and Amendments VII Signature Page VIII Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 3 I. REPORT NARRATIVE STUDY REPORT Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 4 Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA; 3M T-7091.1) in Rats Study Number: TRC 1132-100 SPONSOR: 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 SPONSOR REPRESENTATIVE: Andrew Seacat, Ph.D., (previously, Marvin T. Case, D.V.M, Ph.D., who retired prior to final report) Sponsor contact: Melinda Mitchell 3M Corporate Toxicology Phone No.: 651 736-0443 Fax No.: 651 733-1773 Email: mmitchell@mmm.com TEST FACILITY: Gene Logic (formerly, Therlmmune Research Corporation) 15 Firstfield Road Gaithersburg, Maryland 20878 STUDY DIRECTOR: Gary W. Wolfe, Ph.D., D.A.B.T. Therlmmune Research Corporation Phone No.: 301 330-3723 Fax. No.: 301 330-3738 Email: gwolfe@lab.row.com PRINCIPAL INVESTIGATOR: Sandra R. Eldridge, Ph.D. Pathology Associates Division of Charles River Laboratories, Inc., (formerly, Pathology Associates International) Phone No. 301 624-2036 Fax No. 301 663-8994 Email: seldridge@criver.com Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 5 HISTOPATHOLOGIST: Carolyn Moyer, D.V.M., Diplomate, A.C.V.P. Pathology Associates International No longer with Pathology Associates at the time report finalized ULTRASTRUCTURAL PATHOLOGIST: James B. Nold, D.V.M., Ph.D., Diplomate, A.C.V.P. Pathology Associates International No longer with Pathology Associates at the time report finalized STUDY TIMELINE: Initiation: January 12, 1999 Completion of in-life phase: April 6,1999 Final Report: August 9, 2000 Amended Final Report: November 8, 2004 REGULATORY COMPLIANCE This study was conducted in the spirit of Good Laboratory Practice (GLP) regulations. PURPOSE The objective of this study was to assess cell proliferation and peroxisome proliferation in rats administered test material in the diet. INTRODUCTION This study was designed to assess cell proliferation and peroxisome proliferation in rats administered N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA; 3M T-7091.1). In addition, rats were administered Wy-14,643 as a positive control for cell proliferation and peroxisome proliferation. The endpoints evaluated for test article effect are listed in Text Table 1. Because this study was a multidisciplinary team effort, the results are summarized in Sections I, II and III of this report, and individual studies are reported in the Appendix. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 6 Text Table 1. Experimental Endpoints Examined Endpoint Examined Cell proliferation Histopathology Clinical chemistry Body and liver weights Electron microscopy Palmitoyl CoA oxidase Performing Laboratory Pathology Associates Pathology Associates LabCorp Therlmmune Pathology Associates Covance Location of Report Herewithin Sections I, II, III Sections I, II, III Sections I, II, III Sections I, II, IV - Appendix 1 Sections I, II, V - Appendix 2 Sections I, II, VI - Appendix 3 FINAL REPORT AMENDEMENTS The study report was originally finalized August 9, 2000. 'Fhe present study report (amended final report dated November 8, 2004) has been revised to reflect a correction in the test article abbreviation for N-ethyl perfluorooctanesulfonamide from "PFOSA" to N-EtFOSA, and in the concentration of Wy-14,643 from " 1000 ppm" to 100 ppm. These corrections have been made to all Sections of this report, except for Section IV (Appendix 1), which is the report from the testing facility (Therlmmune Research Corporation; presently, Gene Logic) that was only obtainable in pdf format and therefore, unable to be revised. Additionally, due to the inability to obtain signatures from all participating investigators involved in this study, this amended final report is signed only by the principal investigator, Dr. Sandra Eldridge, on behalf of original signatures from Dr. Carolyn Moyer (study pathologist, Pathology Associates; originally signed August 9, 2000), Dr. James Nold (EM pathologist, Pathology Associates; originally signed October 4, 1999), Dr. Gary Wolfe (study director, Therlmmune; originally signed May 2, 2000), and Dr. Ron Markevitch (Palmitoyl CoA Oxidase activity, Covance; originally signed December 1, 1999). The study protocol (original dated January 12, 1999) and all protocol amendments have also been revised to reflect the corrections noted above, and are included in this amended final study report unsigned. MATERIALS AND METHODS Experimental Design The experimental design was as follows in Text Table 2. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 7 Text Table 2. Group Designations, Dietary Levels and Scheduled Sacrifice Time Points >lumber of Male Rats Group Number Control N-EtFOSE PFOS N-EtFOSA Wy-14,643 Total No. (Time Point) (Oppm) 300 100 30 ppm 20 ppm 100 ppm 100 ppm of Animals 1 (48 hrs) 10 10 10 10 10 10 5 65 2 10 5 5 5 5 5 5 40 (7 days) 3 10 5 5 5 5 5 5 40 (14 days) 4 10 5 5 5 5 5 5 40 (1 wk recovery) 5 (4 wk recovery) Total No. of Animals 10 50 In-life Portion of Study 55 30 30 5 30 5 30 5 30 5 40 25 .. 225... The in-life portion of this study was conducted at Therlmmune Research Corporation. Methods for the in-life portion of this study, including test article formulation and administration, test animals and husbandry, observations, termination and tissue collection are reported in Section IV, Appendix 1. Cell Proliferation Staining and Evaluation Representative samples of the left lateral lobe of the liver and any macroscopic lesions were collected and preserved in formalin. After fixation, each sample of liver was processed and stained proliferation cell nuclear antigen (PCNA). In addition, liver sections prepared from the same tissue blocks were stained with hematoxylin and eosin (H&E) and examined microscopically. Sections of paraffin-embedded tissues were cut at approximately 5 pm and placed on positively charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) to ensure adhesion during processing for PCNA. Standard immunohistochemical methods for PCNA were used to stain tissues. Briefly, tissue sections were incubated with a monoclonal antibody to PCNA (DAKO, Sponsor: 3M Study Number: TRC i 132-100 Amended Final Report Page 8 Carpintera, CA) and reagents required for the avidin-biotin peroxidase (ABC Kit, Vectastain, Burlingame, CA) method for the detection of the antigen-antibody complex. PCNA expression was localized by the chromagen 3,3'-diaminobenzidine (DAB; Sigma Chemical Co., St. Louis, MO). Tissue sections were counterstained with hematoxylin. The percentage of hepatocytes in S-phase (labeling index, LI) was determined by scoring at least 3000 hepatocytes in 10 fields of liver. A negative control slide was included in the staining run and consisted of study tissue that was not incubated with the primary antibody. For cell proliferation evaluations, slides were first perused at low magnification (100X) to judge quality of staining, processing and sectioning, potential patterns of cellular proliferation, and histomorphologic changes. Cell proliferation was then quantified at higher magnification (200X) as described above. Histomorphology was further assessed by evaluating the H&E slide prepared from the same tissue block for each animal evaluated for cell proliferation. Clinical Chemistry Animals were fasted overnight before animal's scheduled necropsy; blood was collected from a jugular vein into an EDTA-coated tube. Serum enzyme levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), cholesterol and triglycerides were determined by LabCorp, and reported separately to Pathology Associates. Tissue Collection for Electron Microscopic Evaluation Sections of liver from all animals were collected, minced to approximately one millimeter cubes, placed in McDowell-Trump fixative, and submitted to Pathology Associates's North Carolina laboratory for processing, sectioning and evaluation. Electron microscopy was performed on select animals as described in Section V, Appendix 2. Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately 500 mg) of the right lateral lobe of the liver was collected from select animals and flash-frozen in liquid nitrogen. The liver tissue was stored in a freezer set to maintain -60 to -80 C until analyzed by Covance for palmitoyl-CoA Oxidase activity. The liver samples analyzed included all study animals, except for the Wy-14,643 animals and all animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study will be analyzed for palmitoyl-CoA Oxidase activity and reported separately; these samples consist of liver samples from 35 rats and 35 guinea pigs. Remaining Liver Tissue The remaining liver tissue was frozen and is being stored at -60 to -80 C for possible future analysis. Statistical Analysis Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 9 The Student's /-test (two-sided, unequal variance) was used to test for statistical significance in LI and clinical chemistry between control and treatment groups using Microsoft Excel version 5.0. A P value of less than 0.05 was judged to be statistically significant. Analysis of variance using Dunnett's procedure was used to analyze body and organ weight data with a P value of less than 0.05 judged to be statistically significant. Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the storage facilities of Pathology Associates for a period of 1 year following submission of the final report to the Sponsor. One year after submission of the final report, all of the aforementioned materials will be sent to the Sponsor and a return fee will be charged. All raw data stored on magnetic media will be retained by Pathology Associates. RESULTS Cell Proliferation and Histopathology Group mean PCNA labeling indices are presented in Section II, Table 1. The severity and incidence of cell proliferative responses are presented in Section II, Table 2. The severity is defined as the fold increase in PCNA LI compared to controls. The incidence represents the number of animals within a treatment group with a LI greater than the highest concurrent control value. Individual animal cell proliferation data are presented in Section III, Table 1. Statistically significant increases in cell proliferation were revealed in only one treatment group: 100 ppm N-EtFOSA at the 7 day time point. Although statistically significant, the labeling indices for individual animals were within the range seen in the control group; therefore, this response was not biologically significant. Biologically significant increases in cell proliferation, as determined by a severity of at least 2-fold and an incidence of at least one animal in the treatment group, were identified only in the positive control group (100 ppm W y-14,643) during the treatment period at 48 hours and 7 days. During the recovery period, biologically significant increases in cell proliferation were seen in 300 and 30 ppm N-EtFOSE, 20 ppm PFOS, and 100 ppm N-EtFOSA, but not in 100 ppm N-EtFOSE or 100 ppm Wy-14,643, at the 4 week recovery period. Histologic findings are presented in Section III, Table 3. Examination o f the H&E slides revealed no significant changes in the liver of rats sacrificed at 48 hours or 7 days. At 14 days, and 1 and 4 week recovery time points, lipid vacuolization was observed in animals from all groups, including controls. In addition, livers from Wy-14,643 treated animals exhibited mild Kupffer cell hypertrophy and multifocal, minimal hepatocellular necrosis at 14 days. At the 1 and 4 week recovery time points, no significant treatment related findings were noted, except for Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 10 the mild Kupffer cell hypertrophy seen in Wy-14,643 treated animals which was absent 4 weeks post-dosing. Clinical Chemistry Group mean clinical chemistry parameters are presented in Section II, Table 3. Individual animal clinical chemistry data are presented in Section III, Table 2 Statistical differences in ALT, ALP and AST were seen sporadically among treatment groups and time points, but were within the normal range for each parameter. Triglycerides were significantly decreased as well as outside the nonnal range in the following treatment groups: 300 ppm N-EtFOSE at 7 days and 1 week recovery, and 100 ppm N-EtFOSA at the same time points. Cholesterol was significantly decreased as well as outside the normal range in all treatment groups at one or more time points during the dosing period. At the 1 week recovery period, but not the 4 week recovery period, cholesterol remained decreased in all groups except 20 ppm PFOS and 100 ppm Wy-14,643. Body and Liver Weight Group mean body weights, liver weights and liver to body weight ratios are presented in Section II, Table 4. Individual animal body and organ weight data are presented in Section IV, Appendix 1. Body weights were not affected during the dosing period. Mean body weight was reduced only at the 1 week recovery period in groups 300 ppm N-EtFOSE, 100 ppm N-EtFOSA and 100 ppm Wy-14,643. Liver weights were significantly elevated at one or more time points during the dosing period in all treatment groups except 30 ppm N-EtFOSE and 20 ppm PFOS. At the 48 hour time point, liver weights were elevated in groups 100 ppm N-EtFOSE and 100 ppm Wy14,643. At the 7 day time point, liver weights were elevated in groups 100 ppm N-ETFOSA and 100 ppm Wy-14,643. At the 14 day time point, liver weights were elevated in group 300 ppm NEtFOSE. At both the 1 and 4 week recovery periods, liver weight was elevated in the 300 ppm N-EtFOSE group. Liver to body weight ratios were significantly increased at one or more time points during the dosing period in all treatment groups except 20 ppm PFOS. At both the 1 and 4 week recovery time points, liver to body weight ratios were significantly increased in treatment groups 300 ppm N-EtFOSE and 100 ppm N-EtFOSA, whereas Wy-14,643 was elevated at the 1 week recovery period, but not the 4 week. Peroxisome Proliferation Electron microscopy (EM) and analysis of palmitoyl CoA oxidase activity were used to assess peroxisome proliferation. Palmitoyl CoA oxidase group summary data are presented in Section Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 11 II, Table 5. The EM report is presented in Section V, Appendix 2, and individual animal palmitoyl CoA oxidase data are presented in Section VI, Appendix 3. At 48 hours, Wy-14,643 induced a doubling in the number of peroxisomes compared to controls. None of the other treatment groups examined (100 ppm N-EtFOSE, 20 ppm PFOS or 100 ppm N-EtFOSA) revealed an increase in mean number of peroxisomes per hepatocyte. Palmitoyl CoA oxidase activity did not differ remarkably among controls and all treatment groups at all time points examined. DISCUSSION In the present study, multiple endpoints were examined to assess cell proliferation and peroxisome proliferation in the liver of rats given N-EtFOSE, PFOS, N-EtFOSA, or Wy-14,643 as a positive control test material. A summary of the findings during the dosing period and recovery period are presented in Text Tables 3 and 4, respectively. As expected, the positive control test material, 100 ppm Wy-14,643 produced the anticipated increase in hepatocellular proliferation and liver weight without a concomitant increase in liverassociated serum enzymes. Cholesterol was lowered as expected. These changes were reversed upon cessation of dosing. In contrast, none of the test materials induced cell proliferation during the dosing period, whereas, during the recovery period, hepatocellular proliferation was increased in the NEtFOSE, PFOS and N-EtFOSA groups. This apparent response may have been an aberration due to the low control group mean labeling index seen at the week 4 recovery time point. These animals, which were 13 weeks of age at this time, exhibited a control LI of only 0.016%. A PCNA labeling index of 0.17% has been reported for control rats of the same age (Eldridge and Goldsworthy, 1996). Thus, the control labeling index value seen in this study at the week 4 recovery time point is approximately 10-fold below that which has been previously reported. Furthermore, the response was not dose-related in the N-EtFOSE treatment groups. Like Wy-14,643, the test materials did not affect liver-associated serum enzyme levels, but did cause a decrease in cholesterol. N-EtFOSE (300 ppm) and N-EtFOSA (100 ppm) also caused a decrease in triglycerides that was reversible within 4 weeks following cessation of dosing. It is interesting to note that Wy-14,643 only lowered triglycerides at 7 days of dosing, but not after 14 days of dosing. Palmitoyl CoA oxidase activity and peroxisomal proliferation were not elevated in any test materials evaluated besides the positive control Wy-14,643. Taken together, these findings do not support N-EtFOSE, PFOS or N-EtFOSA to be peroxisome proliferators in rats. Although these test materials produced an apparent cell proliferative response in the liver of rats, the proliferative response did not occur upon initial dosing as seen Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 12 with Wy-14,643, rather it appeared during the recovery period following 14 days of dosing and was not associated with overt cytotoxicity. The cell proliferative response seen at the week 4 recovery time point may have been an aberration due to the abnormally low control mean labeling index. Like Wy-14,643, these materials decreased cholesterol in rats, but the lowering effect was reversible up to 4 weeks post-dosing as it was with Wy-14,643. Text Table 3. Summary of Findings to Assess Hepatocellular Proliferation and Peroxisome Proliferation During the Dosing Period2 N- N- N- N- Wy- EtFOSE EtFOSE EtFOSE PFOS EtFOSA 14,643 Endpoint Controls 300 ppm 100 ppm 30 ppm 20 ppm 100 ppm 100 ppm Cell proliferation - - - - - - Increase Body weight - - - - - - - Liver weight - Increase Increase - - Increase Increase Liver/body wt. ALT - Increase Increase Increase - Increase Increase - - -- - - - ALP - - - - - - - AST - - - - - - - Triglycerides Decreas * - Decreas - ee Cholesterol " Decreas Decreas Decreas Decreas Decreas Decreas eeeeee Palmitoyl CoA " - - " - - ND oxidase Peroxisomes -- - -- - - aAn increase or decrease identified statistically and/or descriptively (judged to be biologically relevant) at any time during the 14 day dosing period. ND, not determined Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 13 Text Table 4. Summary of Findings to Assess Hepatocellular Proliferation and Peroxisome Proliferation During the Recovery Period3 Endpoint N- N- NEtFOSE EtFOSE EtFOSE Controls 300 ppm 100 ppm 30 ppm PFOS 20 ppm N- WyEtFOSA 14,643 100 ppm 100 ppm Cell proliferation - Increase - Increase Increase Increase - Body weight - Decreas - - - Decreas Decreas e ee Liver weight - Increase - - - - - Liver/body wt. - Increase - - - Increase Increase ALT -- - -- -- ALP -- - -- -- AST -- - -- - - Triglycerides - Decreas - - - Decreas - ee Cholesterol - Decreas Decreas Decreas - Decreas - eee e Palmitoyl CoA " "- " ND oxidase Peroxisomes ND ND ND ND ND ND ND aAn increase or decrease identified statistically and/or descriptively (judged to be biologically relevant) at any time during the recovery period. ND, not determined SUMMARY N-EtFOSE, PFOS and N-EtFOSA are not peroxisome proliferators, do not have overt hepatocellular proliferative effects, but do exhibit a cholesterol lowering effect in rats that is reversible within 4 weeks following cessation of exposure. LITERATURE CITED Eldridge, S. R., and Goldsworthy, S. M. Cell proliferation rates in common cancer target tissues of B6C3F1 mice and F344 rats: effects of age, gender, and choice of marker. Fund. Appl. Toxicol. 32: 159-167, 1996. II. SUMMARY TABLES Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 15 Table II-l. Group Summary of Cell Proliferation Data Treatm ent Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,S43 100 ppm 48 Hr. PCNA Labeling Index Mean 0.080% 0.052% 0.093% 0.047% 0.066% 0.063% 0.536% 48 Hr. PCNA Labeling Index SEM 0.012% 0.006% 0.030% 0.014% 0.013% 0.010% 0.256% Treatm ent Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm 1 W eek Recovery 1 W eek Recovery PCNA PCNA Labeling Index Labeling Index Mean SEM 0.050% 0.017% 0.047% 0.007% 0.092% 0.022% 0.065% 0.025% 0.052% 0.032% 0.063% 0.011% 0.042% 0.025% "Statistically greater than controls, P<0.05 7 Day PCNA Labeling Index Mean 0.046% 0.031% 0.026% 0.030% 0.038% 0.105%" 0.102% 7 Day PCNA Labeling Index SEM 0.013% 0.013% 0.008% 0.004% 0.012% 0.012% 0.036% 4 W eek Recovery 4 W eek Recovery PCNA PCNA Labeling Index Labeling Index Mean 0.016% SEM 0.007% 0.078% 0.046% 0.023% 0.006% 0.064% 0.030% 0.050% 0.028% 0.049% 0.016% 0.030% 0.024% 14 Day PCNA Labeling Index Mean 0.118% 0.058% 0.040% 0.113% 0.060% 0.054% 0.110% 14 Day PCNA Labeling Index SEM 0.028% 0.016% 0.012% 0.038% 0.014% 0.011% 0.048% Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 16 Table II-2. Severity and Incidence of Proliferative Responses in Rat Liver T reatm en t Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm W y-14,643 100 ppm 48 Hr. PCNA Labeling I ndex S e v e rity * - 0.7 1.2 0.6 0.8 0.8 6.7 48 Hr. PCNA Labeling Index In cidence6 - 0/9 2/10 1/10 1/10 0/10 4/5 7 Day PCNA Labeling Index S everity - 0.7 0.6 0.7 0.8 2.3 2.2 7 Day PCNA Labeling Index In c id e n c e - 0/5 0/5 0/5 0/5 0/5 1/3 14 Day PCNA Lab elin g Index S everity . 0.5 0.3 1.0 0.5 0.5 0.9 14 Day PCNA Labeling Index In c id e n c e . 0/5 0/5 0/5 0/5 0/5 0/5 Treatm ent Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm W y-14,643 100 ppm 1 W eek Recovery 1 W eek Recovery PCNA PCNA Labeling I ndex Labeling In dex S e v e rity In c id e n c e -- 0.9 0/5 1.8 0/5 1.3 1/5 1.0 1/5 1.3 0/5 0.8 0/5 4 W eek Recovery 4 W eek Recovery PCNA PCNA Labeling Index Labeling In dex S e v e rity In c id e n c e -- 4.9 2/5 1.4 0/5 4.0 2/5 3.1 2/5 2.9 3/5 1.9 1/5 aFold increase over controls bNumber of animals with a PCNA labeling Index greater than the highest control value Treatment Group Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm Treatment Group 1 Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm Treatment Group Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm Table II-3. Group Summary of Clinical Chemistry Values ALANINE AMINOTRANSFERASE (Normal Range: 20 - 60) 48 hr 7 days 14 days 1 week recovery' 31.8 34.8 38.0 36.4 32.8 43.0 42.6 38.8 32.1 34.2 44.4 44.4 31.0 34.4 38.8 38.4 32.7 33.2 46.4* 41.0 30.2 31.8 44.4 47.6 36.3 33.2 60.6* 93.2 ALKALINE PHOSPHATASE (Normal Range: 150 - 380) 48 hr 7 days 14 days 1 week recovery 296.8 263.9 287.6 249.8 208.3 193.0 184.1 199.0 281.3 297.4 286.3 316.5 306.0 239.8 244.4 251.6 294.4 287.2 198.4 227.0 265.0 273.2 326.0* 151.6 233.4* 195.6 176.4 171.0 ASPARTATE AMINOTRANSFERASE (Normal Flange: 70 - 125) 48 hr 7 days 14 days 1 week recovery 144.8 123.6 155.6 135.4 131.3 133.6 120.4 112.4 127.3 127.6 139.2 139.4 145.8 136.9 130.9 150.0 132.6 134.6 132.6 119.4* 141.8 152.2 121.4 173.2 123.4 115.0 129.4 164.8 4 week recovery 40.3 49.6 50.6* 41.6 42.4 77.4 38.8 4 week recovery 137.4 140.8 130.4 117.8 129.8 136.8 118.2 4 week recovery 154.4 159.2 178.0 187.0 176.8 185.2 133.0 Table II-3, continued Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 18 Treatment Group Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm 48 hr 41.6 30.3 37.5 47.4 34.4 30.6 45.4 TRIGLYCERIDES (Normal Range: 25 - 120) 7 days 14 days 1 week recovery 50.8 37.0 46.5 18.2* 23.4 24.0* 29.6* 30.0 33.6 37.4 37.2 30.4* 36.4 31.0 27.6* 35.4 19.8* 23.6* 35.2* 43.6 41.0 4 week recovery 36.1 34.6 48.6 45.0 38.8 34.2 70.0 CHOLESTEROL (Normal Range: 50 - 150) Treatment Group 48 hr 7 days 14 days 1 week recovery Control 67.3 68.3 59.8 54.5 N-EtFOSE 300 ppm 56.1 37.0* 40.0* 30.4* N-EtFOSE 100 ppm 67.3 39.4* 41.4* 37.8* N-EtFOSE 30 ppm 60.3 48.2* 50.6 43.0* PFOS 20 ppm N-EtFOSA 100 ppm 61.1 58.4 58.4* 53.6* 42.0* 34.8* 47.2 38.6* Wy-14.643 100 ppm 36.8* 61.0 87.2* 82.8* *Statistically different from controls, P<0.05 4 week recovery 48.3 49.4 47.4 52.6 50.2 48.6 66.6 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 19 Table II-4. Group Summary of Body and Liver Weights MEAN BODY WEIGHT Treatment Group 48 hr 7 days 14 days 1 week recovery Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm 207.6 g 208.7 g 221.1 g 248.1 g 235.1 g 258.6 g 297.4 g 287.7 g 284.4 g 339.3 g 293.3 g * 318.0 g N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm 207.9 g 208.9 g 213.7 g 217.2 g 244.2 g 243.4 g 257.5 g 253.6 g 300.0 g 285.8 g 268.1 g 283.1 g 321.9 g 320.5 g 309.8 g* 289.2 g * MEAN LIVER WEIGHT Treatment Group 48 hr 7 days 14 days 1 week recovery Control 7.514 g 8.475 g 9.795 g 11.321 g N-EtFOSE 300 ppm 7.878 g 9.642 g 14.148 g* 14.118 g* N-EtFOSE 100 ppm 8.515 g* 9.665 g 11.272 g 11.966 g N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm 7.788 g 7.748 g 7.982 g 8.541 g 8.830 g 10.193 g * 11.053 g 10.002 g 10.766 g 10.549 g 11.095 g 12.681 g W y-14,643 100 ppm 9.555 g * 16.087 g* 19.975 g* 11.991 g MEAN LIVER WEIGHT TO BODY WEIGHT RATIO Treatment Group 48 hr 7 days 14 days 1 week recovery Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm W y-14,643 100 ppm 3.620% 3.769% 3.850% 3.749% 3.703% 3.731% 4.400%* 3.409% 4.105%* 3.740% 3.496% 3.630% 3.949%* 6.349%* 3.290% 4.903%* 3.967%* 3.684%* 3.495% 4.013%* 7.058%* 3.320% 4.794%* 3.764% 3.278% 3.460% 4.094%* 4.142%* *Statistically different from controls, P<0.05 4 week recovery 407.3 g 395.5 g 385.0 g 393.6 g 401.9 g 382.7 g 392.7 g 4 week recovery 12.800 g 17.146 g* 12.785 g 13.103 g 13.457 g 15.152 g 13.480 g 4 week recovery 3.115% 4.333%* 3.316% 3.346% 3.351% 3.956%* 3.417% Table II-5. Group Summary of Palimitoyl CoA Oxidase Activity Treatment Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Treatment Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm 48 Hr. PCOAO' Mean 8 9 8 9 8 7 48 Hr. PCOAO S.D. 1.8 2.9 2.3 2.1 1.9 1.9 48 Hr. PCOAO Range 5-11 6-16 4-12 6-13 6-11 5-11 48 Hr. Sample Size 10 10 10 10 10 10 14 Day PCOAO Mean 6 3 3 5 4 3 14 Day PCOAO S.D. 1.4 0.8 1.6 0.8 0.4 1.1 14 Day PCOAO Range 5-9 2-4 1-5 4-6 4-5 2-4 14 Day Sample Size 10 5 5 5 5 5 7 Day PCOAO Mean 5 6 7 6 7 6 7 Day PCOAO S.D. 1.3 2.5 1.9 1.9 1.5 2.1 7 Day PCOAO Range 4-8 3-10 4-9 4-9 5-9 3-8 7 Day Sample Size 10 5 5 5 5 5 1 Week Recovery PCOAO Mean 7 4 5 6 6 6 1 Week Recovery PCOAO S.D. 2.3 1.3 0.8 1.9 1.8 0.4 1 Week Recovery PCOAO Range 3-10 3-6 4-6 4-8 3-8 6-7 1 Week Recovery Sample Size 10 5 5 5 5 5 "Palmitoyl CoA Oxidase activity, IU/g Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 21 III. INDIVIDUAL ANIMAL DATA TABLES Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 22 Table III-l. Individual Animal Cell Proliferation Data 48 Hour, 7 Day and 14 Day Sacrifices 48 H our Interim S acrifice PCNA Treatm ent G roup Anim al N um ber Labeling Index Controls R10381 R10382 0.145% 0.052% R10383 0.081% R10384 0.078% R10385 0.113% R10386 R10387 0.086% 0.105% R10388 0.080% R10389 0.027% R 10390 0.029% N-EtFOSE 300 ppm R10391 R10392 R10393 0.053% 0.052% 0.057% R 10394 0.058% R10395 R10396 ND 0.059% R10397 R10398 0.026% 0.085% R10399 0.053% R10400 0.026% N-EtFOSE 100 ppm R10401 R10402 0.056% 0.087% R10403 0.190% R10404 0.323% R10405 0.029% R10406 0.103% R10407 0.000% R10408 R10409 0.055% 0.055% N-EtFOSE 30 ppm R10410 R10411 R10412 0.029% 0.152% 0.000% R10413 0.084% R10414 0.055% R10415 0.059% R10416 0.030% R10417 R10418 0.000% 0.029% R10419 0.030% R10420 0.028% PFOS 20 ppm R10421 0.153% R10422 0.053% R10423 R10424 R 10425 0.052% 0.086% 0.027% R10426 0.053% R 10427 R10428 0.027% 0.027% R10429 R10430 0.077% 0.109% N-EtFOSA 100 ppm R10431 0.089% R10432 0.059% R10433 R10434 R10435 0.084% 0.054% 0.000% R 10436 0.029% R10437 0.085% R10438 0.059% R10439 0.114% Wy-14,643 100 ppm R10440 R10441 0.058% 1.543% R10442 0.274% R10443 R10444 0.326% 0.117% R10445 0.422% 7 D ay Interim Sacrifice PCNA A nim al N um ber Labeling Index R10446 0.071% R10447 0.058% R10448 0.030% R10449 0.000% R 10450 0.027% R10451 0.136% R10452 0.058% R 10453 0.050% R10454 0.027% R10455 0.000% R10456 0.000% R10457 0.077% R 10458 0.027% R10459 0.024% R10460 0.027% R10461 R10462 R10463 R10464 R10465 0.024% 0.026% 0.053% 0.000% 0.025% R10466 R10467 R10468 R10469 R10470 0.026% 0.025% 0.047% 0.024% 0.026% R10471 R 10472 R10473 R10474 R10475 0.029% 0.085% 0.026% 0.026% 0.026% R10476 R10477 R10478 R10479 R10480 0.130% 0.076% 0.134% 0.077% 0.106% R10481 R10482 R10483 R10484 R10485 ND ND 0.078% 0.173% 0.054% 14 D ay In terim Sacrifice PCNA A nim al N u m b er Labeling Index R 10486 0.106% R10487 0.190% R10488 0.332% R10489 0.056% R10490 R10491 0.082% 0.133% R10492 0.056% R10493 0.086% R10494 0.026% R10495 0.117% R 10496 0.119% R10497 0.029% R10498 0.056% R 10499 0.031% R10500 0.055% R10501 R10502 R10503 R10504 R10505 0.028% 0.058% 0.059% 0.000% 0.057% R 10506 R10507 R10508 R10509 R10510 0.152% 0.240% 0.088% 0.057% 0.029% R10511 R10512 R10513 R10514 R10515 0.090% 0.030% 0.093% 0.030% 0.059% R10516 R10517 R10518 R10519 R10520 0.031% 0.031% 0.057% 0.090% 0.059% R10521 R10522 R10523 R10524 R10525 0.290% 0.029% 0.029% 0.115% 0.089% ND, not determined due to lack of tissues on slide or suboptimal staining Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 23 Table III-l. Individual Animal Cell Proliferation Data 1 and 4 Week Recovery 1 W eek R eco very S acrifice PCNA Treatm ent G roup A nim al N u m b er Labeling In dex Controls R10526 R10527 0.152% 0.026% R 10528 0.028% R10529 R10530 0.028% 0.027% R10531 0.150% R10532 0.029% R10533 R10534 0.029% 0.000% R10535 0.027% N-EtFOSE 300 ppm R10536 0.059% R10537 0.056% R10538 0.058% R10539 R10540 0.030% 0.031% 4 W eek R ecovery S acrifice PCNA A nim ai N um ber Labeling In dex R 10566 0.058% R10567 0.000% R10568 0.028% R10569 0.000% R10570 0.000% R10571 ND R10572 0.028% R10573 0.000% R10574 0.000% R10575 0.027% R10576 0.000% R10577 0.000% R10578 0.241% R10579 0.030% R10580 0.117% N-EtFOSE 100 ppm R10541 R10542 R10543 R10544 R10545 0.146% 0.089% 0.062% 0.137% 0.028% R10581 R10582 R10583 R10584 R10585 0.028% 0.029% 0.030% 0.000% 0.029% N-EtFOSE 30 ppm R10546 R 10547 R10548 R10549 R10550 0.154% 0.029% 0.087% 0.029% 0.028% R10586 R10587 R10588 R10589 R10590 0.175% 0.030% 0.000% 0.059% 0.058% PFOS 20 ppm R10551 R10552 R10553 R10554 R10555 0.060% 0.000% 0.000% 0.173% 0.027% R10591 R10592 R10593 R10594 R10595 0.077% 0.028% 0.146% 0.000% 0.000% N -B F O S A 100 ppm R 10556 R10557 R10558 R10559 R10560 0.028% 0.090% 0.059% 0.083% 0.057% R 10596 R10597 R10598 RI 0599 R10600 0.031% 0.060% 0.000% 0.061% 0.094% Wy-14.643 100 ppm R10561 R10562 R 10563 R10564 R10565 0.000% 0.000% 0.054% 0.026% 0.132% R10601 R10602 R10603 R10604 R10605 ND. not determined due to lack of tissues on slide or suboptimal staining 0.026% 0.000% 0.126% 0.000% 0.000% Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 24 Table III-2. Individual Animal Clinical Chemistry 48 Hour Interim Sacrifice Treatm ent Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm A nim al N u m b er R10381 R 10382 R 10383 R 10384 R10385 R 10386 R10387 R 10388 R10389 R 10390 R10391 R 10392 R 10393 R 10394 R 10395 R 10396 R 10397 R 10398 R 10399 R10400 R10401 R 10402 R 10403 R 10404 R 10405 R 10406 R 10407 R10408 R 10409 R10410 R10411 R10412 R10413 R10414 R10415 R10416 R10417 R10418 R10419 R10420 R10421 R10422 R 10423 R 10424 R 10425 R10426 R 10427 R 10428 R 10429 R 10430 R10431 R 10432 R 10433 R 10434 R 10435 R 10436 R 10437 R 10438 R 10439 R10440 R10441 R 10442 R 10443 R 10444 R10445 A L T (IU /L) 33 29 38 33 30 29 37 33 20 36 33 27 30 39 33 37 29 25 33 42 35 44 37 36 32 33 31 22 28 23 31 35 51 36 23 21 30 31 26 26 36 48 30 39 31 30 29 27 35 22 34 39 30 34 36 27 26 28 25 23 41 45 33 37 27 A LP (IU /L) 259 349 250 293 266 205 316 293 346 391 399 251 318 226 166 322 244 144 322 247 212 273 277 367 275 291 342 255 294 227 297 352 301 268 373 184 306 296 337 260 338 369 256 232 189 303 245 235 208 488 421 435 194 252 397 426 210 307 334 189 443 254 319 256 258 A S T (IU /L) 138 131 148 163 130 127 218 137 148 108 126 134 134 153 115 102 90 99 132 151 120 137 143 171 169 101 126 92 117 97 153 125 241 123 98 111 182 205 112 108 144 155 103 219 99 149 141 122 127 110 123 171 114 124 123 171 120 121 146 96 147 187 151 133 132 TR IG (m g/dl) 22 57 25 32 60 34 65 41 30 50 22 21 12 51 43 28 27 41 17 41 27 47 37 40 39 49 35 31 40 30 39 19 51 33 98 37 21 46 72 58 33 34 31 40 22 42 17 54 48 23 23 16 27 44 30 27 49 34 23 33 33 43 39 37 75 C H O L (m g/dl) 85 70 65 49 68 66 56 72 85 57 43 60 43 55 73 52 67 71 38 59 58 64 75 64 75 81 51 72 67 66 71 30 62 75 64 75 61 57 57 51 68 51 38 65 68 93 41 61 84 42 57 45 75 60 43 44 81 61 41 77 35 33 44 36 36 Table III-2. Individual Animal Clinical Chemistry 7 Day Interim Sacrifice Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 25 Treatm ent Group C ontrols N -E tFO S E 300 ppm N -E tFO S E 100 ppm N -E tFO S E 30 ppm PFOS 20 ppm N -E tF O S A 100 ppm W y-14,643 100 ppm Animal Num ber R 10446 R10447 R10448 R10449 R10450 R10451 R10452 R10453 R10454 R10455 R 10456 R 10457 R10458 R 10459 R10460 R10461 R 10462 R 10463 R 10464 R10465 R10466 R10467 R10468 R10469 R10470 R10471 R10472 R10473 R10474 R10475 R10476 R 10477 R10478 R10479 R10480 R10481 R10482 R10483 R10484 R 10485 A LT (IU/L) 41 40 29 30 32 31 31 30 37 47 30 39 43 67 36 38 34 32 33 34 31 32 34 37 38 33 35 40 29 29 39 32 27 27 34 35 26 32 35 38 ALP (IU/L) 315 279 214 373 237 313 370 203 289 283 181 311 353 228 176 204 245 214 335 201 208 188 261 242 323 166 216 373 188 315 297 414 180 232 349 254 254 275 322 331 AST (IU/L) 216 137 152 118 138 178 144 216 134 123 121 130 121 168 137 172 132 85 145 104 125 116 103 128 191 126 137 123 138 149 156 177 77 124 129 108 127 131 85 146 TRIG (m g/dl) 67 35 34 38 32 83 59 52 61 47 13 15 18 18 27 27 19 44 17 41 47 38 45 23 34 47 19 25 48 43 29 24 76 24 24 21 34 39 38 44 CHO L (m g/dl) 71 66 71 79 55 65 61 56 72 87 36 33 36 41 39 36 34 56 34 37 60 49 48 33 51 53 60 67 60 52 55 44 69 58 42 49 71 65 51 69 Table III-2. Individual Animal Clinical Chemistry 14 Day Interim Sacrifice Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 26 Treatm ent Group C ontrols N -E tFO S E 300 ppm N -E tFO S E 100 ppm N -E tFO S E 30 ppm PFOS 20 ppm N -E tFO S A 100 ppm W y-14,643 100 ppm Animal Num ber R 10486 R10487 R10488 R10489 R10490 R10491 R 10492 R 10493 R 10494 R10495 R10496 R10497 R 10498 R10499 R10500 R10501 R10502 R10503 R10504 R10505 R10506 R 10507 R 10508 R10509 R10510 R10511 R10512 R10513 R10514 R10515 R10516 R10517 R10518 R10519 R 10520 R10521 R 10522 R 10523 R 10524 R 10525 A LT (IU/L) 39 45 39 32 36 34 35 37 41 42 37 33 34 57 52 51 33 62 31 45 35 51 45 29 34 50 42 50 43 47 48 63 48 34 29 54 62 63 49 75 ALP (IU/L) 169 166 212 226 212 229 282 196 176 215 226 134 134 208 263 284 201 154 148 205 286 149 267 219 214 206 340 230 280 269 220 249 419 238 240 313 234 354 375 354 AST (IU/L) 133 132 110 158 141 164 128 88 112 136 128 97 119 167 157 118 121 174 169 114 151 206 119 121 112 113 146 197 195 110 149 148 92 114 104 137 161 242 160 166 TRIG (m g/dl) 53 34 50 25 29 48 37 37 23 34 35 19 19 17 27 19 31 18 48 34 54 41 31 43 17 36 16 34 17 52 20 22 25 15 17 22 35 57 55 49 CHOL (m g/dl) 74 77 58 49 57 72 39 66 52 54 48 31 42 35 44 27 40 39 55 46 50 50 46 54 54 54 21 39 46 50 36 36 36 27 39 73 107 93 87 76 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 27 Table III-2. Individual Animal Clinical Chemistry 1 Week Recovery Sacrifice Treatm ent Group C ontrols N -E tFO S E 300 ppm N -E tFO S E 100 ppm N -E tFO S E 30 ppm PFO S 20 ppm N -E tFO S A 100 ppm W y-1 4,643 100 ppm Anim ai Num ber R10526 R10527 R 10528 R10529 R10530 R10531 R10532 R10533 R10534 R 10535 R10536 R10537 R10538 R10539 R10540 R10541 R10542 R10543 R 10544 R 10545 R 10546 R10547 R 10548 R10549 R10550 R10551 R10552 R10553 R10554 R10555 R 10556 R 10557 R 10558 R10559 R10560 R10561 R10562 R10563 R10564 R10565 ALT (IU/L) 57 34 59 27 34 27 30 32 29 35 29 46 47 29 43 65 32 50 37 38 34 32 40 43 43 48 47 35 42 33 37 39 52 40 70 57 112 52 70 175 ALP (IU/L) 158 274 176 150 166 162 271 118 226 140 199 197 215 191 193 123 142 159 190 144 250 228 233 252 204 191 249 135 214 189 150 178 207 186 161 157 141 150 186 221 AST (IU/L) 243 114 187 105 106 96 98 71 94 90 158 123 112 78 91 269 119 94 108 107 115 154 127 118 103 125 114 145 111 80 125 138 112 138 134 162 148 123 150 241 TRIG (m g/dl) 45 81 52 39 18 27 52 74 29 48 6 31 20 24 39 51 26 23 32 36 29 19 30 37 37 23 31 21 26 37 24 20 17 22 35 51 55 28 44 27 CHOL (m g/dl) 63 61 62 67 40 42 53 50 48 59 17 23 32 33 47 49 35 28 28 49 47 39 48 32 49 33 50 53 38 62 39 32 43 31 48 105 98 70 78 63 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 28 Table III-2. Individual Animal Clinical Chemistry 4 Week Recovery Sacrifice Treatm ent Group C ontrols N -E tFO S E 300 ppm N -E tFO S E 100 ppm N -E tFO S E 30 ppm PFO S 20 ppm N -E tFO S A 100 ppm W y-14,643 100 ppm Animal Num ber R 10566 R 10567 R10568 R10569 R10570 R10571 R10572 R10573 R10574 R10575 R10576 R10577 R10578 R10579 R10580 R10581 R 10582 R10583 R10584 R10585 R10586 R10587 R10588 R10589 R10590 R10591 R10592 R10593 R10594 R 10595 R10596 R10597 R10598 R10599 R10600 R10601 R10602 R10603 R10604 R10605 ALT (IU/L) 40 38 40 58 49 ND 34 32 37 35 51 43 47 47 60 55 46 47 57 48 54 31 40 35 48 46 36 49 38 43 51 103 125 35 73 42 33 38 43 38 ALP (IU/L) 160 108 133 125 167 ND 137 135 135 137 133 113 140 155 163 142 138 126 152 94 120 154 84 124 107 147 127 139 115 121 133 173 137 95 146 144 105 123 118 101 AST (IU/L) 170 171 153 155 139 ND 125 156 202 119 187 136 161 158 154 181 222 169 164 154 207 137 206 225 160 235 183 186 148 132 143 256 242 120 165 168 168 104 124 101 TRIG (m g/dl) 51 32 25 39 30 ND 37 32 51 28 36 32 33 24 48 40 33 104 31 35 21 49 28 58 69 25 39 29 36 65 43 28 35 40 25 51 132 51 52 64 CHOL (m g/dl) 47 62 47 53 54 ND 41 43 49 39 63 38 58 27 61 39 38 64 44 52 54 44 47 45 73 43 55 35 45 73 48 49 38 60 48 60 89 50 67 67 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 29 Table III-3. Individual Animal Histopathology Findings 48 Hour Interim Sacrifice Treatm ent Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm^ N-EtFOSA 100 ppm W y-14,643 100 ppm H istopatho log y F in d in g s A nim al N um ber Liver R10381 R10382 R10383 No significant findings No significant findings No significant findings R10384 No significant findings R10385 R 10386 No significant findings No significant findings R10387 No significant findings R 10388 R 10389 R10390 No significant findings No significant findings No significant findings R10391 R10392 R10393 No significant findings No significant findings No significant findings R10394 No significant findings R10395 R10396 No significant findings No significant findings R10397 No significant findings R10398 R10399 No significant findings No significant findings R10400 R10401 No significant findings No significant findings R10402 No significant findings R10403 R10404 R10405 No significant findings No significant findings No significant findings R10406 R10407 R10408 No significant findings No significant findings No significant findings R 10409 R10410 R10411 No significant findings No significant findings No significant findings R10412 No significant findings R10413 R10414 No significant findings No significant findings R10415 R10416 No significant findings No significant findings R10417 No significant findings R10418 R10419 R10420 No significant findings No significant findings No significant findings R10421 No significant findings R 10422 R10423 R10424 No significant findings No significant findings No significant findings R10425 No significant findings R 10426 No significant findings R10427 No significant findings R10428 R 10429 R 10430 No significant findings No significant findings No significant findings R10431 R10432 R 10433 No significant findings No significant findings No significant findings R 10434 R10435 R 10436 No significant findings No significant findings No significant findings R10437 R 10438 R 10439 No significant findings^ No significant findings No significant findings R 10440 R10441 R 10442 No significant findings No significant findings No significant findings R10443 R10444 R 10445 No significant findings No significant findings No significant findings Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 30 Table III-3. Individual Animal Histopathology Findings 7 Day Interim Sacrifice Treatm ent Group C o n tro ls N -E tF O S E 300 ppm N -E tF O S E 100 ppm N -E tF O S E 30 ppm PFOS 20 ppm N -E tF O S A 100 ppm W y-1 4,643 100 ppm H istopathology F in d in g s Anim al Num ber L iv e r R 10446 N o s ig n ifica n t fin d in g s R 10447 N o s ig n ifica n t fin d in g s R 10448 N o s ig n ifica n t fin d in g s R 10449 N o s ig n ifica n t fin d in g s R10450 N o s ig n ifica n t fin d in g s R 10451 N o s ig n ifica n t fin d in g s R10452 N o s ig n ifica n t fin d in g s R 10453 N o s ig n ifica n t fin d in g s R 10454 N o s ig n ifica n t fin d in g s R10455 N o s ig n ifica n t fin d in g s R 10456 No s ig n ifica n t fin d in g s R10457 N o s ig n ifica n t fin d in g s R 10458 N o s ig n ifica n t fin d in g s R 10459 N o sig n ifica n t fin d in g s R 10460 N o sig n ifica n t fin d in g s R10461 No sig n ifica n t fin d in g s R 10462 N o sig n ifica n t fin d in g s R 10463 N o sig n ifica n t fin d in g s R 10464 N o sig n ifica n t fin d in g s R 10465 N o sig n ifica n t fin d in g s R 10466 N o sig n ifica n t fin d in g s R 10467 N o sig n ifica n t fin d in g s R 10468 N o sig n ifica n t fin d in g s R 10469 N o sig n ifica n t fin d in g s R10470 N o sig n ifica n t fin d in g s R10471 N o sig n ifica n t fin d in g s R 10472 N o sig n ifica n t fin d in gs R10473 N o sig n ifica n t fin d in g s R 10474 N o sig n ifica n t fin d in g s R10475 N o sig n ifica n t fin d in g s R10476 N o sig n ifica n t fin d in g s R 10477 N o sig n ifica n t fin d in g s R 10478 N o sig n ifica n t fin d in g s R10479 N o s ig n ifica n t fin d in g s R 10480 N o s ig n ifica n t fin d in gs R10481 N o s ig n ifica n t fin d in g s R 10482 N o s ig n ifica n t fin d in g s R10483 N o sig n ifica n t fin d in g s R 10484 N o s ig n ifica n t fin d in g s R10485 N o sig n ifica n t fin d in gs Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 31 Table III-3. Individual Animal Histopathology Findings 14 Day Interim Sacrifice Treatm ent Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm H istopathology Findings Animal Num ber Liver R10486 lipid vacuolization, lipid, minimal R10487 lipid vacuolization, lipid, minimal R10488 No significant findings R10489 lipid vacuolization, lipid, minimal; aggregates, mixed inflammatory cell, multifocal, minimal R 10490 lipid vacuolization, lipid, minimal; aggregates, mixed Inflammatory cell, multifocal, minimal R10491 No Significant Findings R10492 lipid vacuolization, lipid, minimal R10493 lipid vacuolization, lipid, minimal; aggregates, mixed inflammatory cell, multifocal, minimal R10494 lipid vacuolization, lipid, minimal R10495 lipid vacuolization, lipid, minimal; aggregates, mixed inflammatory cell, multifocal, minimal R10496 lipid vacuolization, lipid, mild R10497 lipid vacuolization, lipid, mild R 10498 lipid vacuolization, lipid, mild R 10499 lipid vacuolization, lipid, mild R10500 lipid vacuolization, lipid, mild R10501 lipid vacuolization, lipid, mild; aggregates, mixed inflam m atory cell, multifocal, m inim al R10502 lipid vacuolization, lipid, mild R 10503 lipid vacuolization, lipid, mild; aggregates, mixed inflam m atory cell, multifocal, m inim al R10504 No significant findings R10505 lipid vacuolization, lipid, mild; aggregates, mixed inflam m atory cell, multifocal, m inim al R10506 lipid vacuolization, lipid, mild R10507 lipid vacuolization, lipid, mild R10508 lipid vacuolization, lipid, mild R10509 lipid vacuolization, lipid, minimal R10510 lipid vacuolization, lipid, minimal R10511 lipid vacuolization, lipid, minimal; aggregates, mixed inflam m atory cell, multifocal, m inim al R10512 No significant findings R10513 No significant findings R10514 No significant findings R1515 lipid vacuolization, lipid, minimal R10516 lipid vacuolization, lipid, mild R10517 lipid vacuolization, lipid, minimal R10518 lipid vacuolization, lipid, minimal R10519 lipid vacuolization, lipid, minimal R10520 lipid vacuolization, lipid, minimal R10521 hypertrophy, Kupffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, minimal R10522 hypertrophy, Kupffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, minimal R10523 hypertrophy, Kupffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, minimal R 10524 hypertrophy, Kupffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, minimal R10525 hypertrophy, Kupffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, minimal; aggregates, mixed inflam m atory cell, multifocal, m inim al Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 32 Table III-3. Individual Animal Histopathology Findings 1 Week Recovery Sacrifice Treatm ent Group C ontrols N -EtFO SE 300 ppm N -EtFO SE 100 ppm N -EtFO SE 30 ppm PFOS 20 ppm N -E tFO S A 100 ppm W y-14,643 100 ppm Histopathology Findings Animal Num ber Liver R10526 N o significant findings R10527 lip id va cu o liza tio n , lipid, m ild R10528 lip id va cu o liza tio n , lipid, m ild; a g gregates, m ixed in fla m m a to ry cell, m u ltifo ca l, m inim al R10529 lip id va cu o liza tio n , lipid, m ild R10530 lip id va cu o liza tio n , lipid, m ild; a g gregates, m ixed infla m m a to ry cell, m u ltifo ca l, m inim al R10531 lipid va cuolization, lipid, m inim al R10532 lipid va cuolization, lipid, m inim al R10533 lipid va cuolization, lipid, m inim al R10534 lipid va cuolization, lipid, m inim al R10535 lipid va cu o liza tio n , lipid, m ild R10536 lipid va cu o liza tio n , lipid, m ild; a g g re g a te s, m ixed in fla m m a to ry cell, m u ltifo ca l, m inim al R10537 lipid va cu o liza tio n , lipid, m ild R10538 lipid va cu o liza tio n , lipid, m ild R10539 lipid va cu o liza tio n , lipid, m ild R10540 lipid va cuolization, lipid, m inim al R10541 lipid va cu o liza tio n , lipid, m in im a l R10542 lipid vacuolization, lipid, m inim al; aggregates, m ixed in fla m m a to ry cell, m ultifocal, m inim al R10543 lipid va cuolization, lipid, m ild R10544 lipid va cuolization, lipid, m inim al R10545 lipid va cuolization, lipid, m inim al R10546 lipid va cu o liza tio n , lipid, m ild R10547 lipid va cuolization, lipid, m inim al R10548 lipid va cuolization, lipid, m inim al R10549 lipid va cuolization, lipid, m inim al R10550 lipid va cu olization, lipid, m inim al R10551 lipid va cu olization, lipid, m inim al R10552 lipid va cu olization, lipid, m inim al R10553 lipid va cu o liza tio n , lipid, m in im a l R10554 lipid va cu o liza tio n , lipid, m inim al; a g g re g a te s, m ixed in fla m m a to ry cell, m u ltifo ca l, m inim al R10555 lip id va cu o liza tio n , lipid, m inim al R10556 lip id va cu o liza tio n , lipid, m ild R10557 N o significant findings R10558 N o significant findings R10559 lip id va cu o liza tio n , lipid, m in im a l R10560 lip id va cu o liza tio n , lipid, m in im a l R10561 hypertrophy, K upffer cell, m ild; hep a to ce llu la r necrosis, m ultifocal, m inim al; lipid vacuolization, lipid, m inim al; spleen, fo llicu la r hyperplasia, m oderate R10562 hypertrophy, K u p ffe r cell, m ild; lipid va cu o liza tio n , lipid, m inim al R 10563 hypertrophy, K u p ffe r cell, m ild; lipid va cu o liza tio n , lipid, m inim al R 10564 hypertrophy, K u p ffe r cell, m ild; lip id va cu o liza tio n , lipid, m in im a l R10565 hypertrophy, K u p ffe r cell, m ild; lip id va cu o liza tio n , lipid, m inim al Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 33 Table III-3. Individual Animal Histopathology Findings 4 Week Recovery Sacrifice Treatm ent Group C o n tro ls N -E tF O S E 300 ppm N -E tF O S E 100 ppm N -E tF O S E 30 ppm PFO S 20 ppm N -E tF O S A 100 ppm W y-14,643 100 ppm H istopathology Findings Anim al Num ber L iv e r R10566 lip id v a cu o liza tio n , lipid, m ild lip id v a cu o liza tio n , lipid, m ild; R10567 a g g re g a te s , m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10568 lip id va cu o liza tio n , lipid, m in im a l R10569 lip id v a cu o liza tio n , lipid, m ild R10570 lip id va cu o liza tio n , lipid, m in im a l R10571 a g g re g a te s, m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10572 lip id va cu o liza tio n , lipid, m in im a l R10573 a g g re g a te s , m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10574 lip id va cu o liza tio n , lipid, m in im a l lip id va cu o liza tio n , lipid, m in im a l; R10575 a g g re g a te s , m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10576 lip id va cu o liza tio n , lipid, m ild R 10577 lip id v a cu o liza tio n , lipid, m ild R10578 lip id va cu o liza tio n , lipid, m in im a l lip id v a cu o liza tio n , lipid, m ild; R10579 a g g re g a te s , m ixe d in fla m m a to ry ce ll, m u ltifo c a l, m in im a l R10580 lip id va cu o liza tio n , lipid, m in im a l R10581 lip id v a cu o liza tio n , lipid, m ild R 10582 lip id v a cu o liza tio n , lipid, m in im a l R10583 N o sig n ifica n t fin d in gs R 10584 N o sig n ifica n t fin d in gs lip id v a cu o liza tio n , lipid, m ild; R10585 a g g re g a te s, m ixe d in fla m m a to ry ce ll, m u ltifo c a l, m in im a l lip id v a cu o liza tio n , lipid, m in im a l; R 10586 a g g re g a te s, m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R 10587 N o sig n ifica n t fin d in gs R10588 lip id va cu o liza tio n , lipid, m in im a l R10589 lip id v a cu o liza tio n , lipid, m in im a l R 10590 N o sig n ifica n t fin d in g s R10591 a g g re g a te s , m ixe d in fla m m a to ry ce ll, m u ltifo c a l, m in im a l R 10592 lip id va cu o liza tio n , lipid, m in im a l R10593 lip id va cu o liza tio n , lipid, m in im a l lip id va cu o liza tio n , lipid, m in im a l; R10594 a g g re g a te s , m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10595 a g g re g a te s, m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10596 a g g re g a te s, m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10597 lip id v a cu o liza tio n , lipid, m in im a l R10598 N o sig n ifica n t fin d in gs R 10599 N o sig n ifica n t fin d in gs lip id va cu o liza tio n , lipid, m in im a l; R10600 a g g re g a te s, m ixe d in fla m m a to ry cell, m u ltifo c a l, m in im a l R10601 N o sig n ifica n t fin d in gs R 10602 N o sig n ifica n t fin d in g s R10603 N o sig n ifica n t fin d in gs lip id va cu o liza tio n , lipid, m in im a l; te ste s, sp e rm g ra n u lo m s; R10604 e p id id y m is , sp e rm g ra n u lo m a R 0 6 0 5 N o sig n ifica n t fin d in gs Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 34 IV. APPENDIX 1 - IN-LIFE REPORT Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 35 V. APPENDIX 2 - ELECTRON MICROSCOPY REPORT ANCILLARY PATHOLOGY REPORT Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 36 ELECTRON MICROSCOPIC EVALUATION OF LIVER IN CRL:CD(SD)IGS BR RATS) CELL PROLIFERATION STUDY WITH N-ETHYL PERFLUOROOCTANESULFONAMIDO ETHANOL (N-EtFOSE; 3M T-6316.11), PERFLUOROOCTANE SULFONIC ACID POTASSIUM SALT (PFOS; 3M T-6295.16), AND N-ETHYL PERFLUOROOCTANESULFONAMIDE (N-EtFOSA 3M T-7091.1) IN RATS TRC STUDY NUMBER 1132-100 PAI EM PROJECT NUMBER EM 99.62 SUMMMARY An increase in the mean number of peroxisomes per hepatocyte was detected by electron microscopy in male Crl:CD@(CD) IGS BR rats given 100 ppm Wy-14,643 by diet after 48 hours of treatment. The mean number of peroxisomes was approximately doubled over control values. The mean numbers of peroxisomes per hepatocyte were not remarkably different for animals given 100 ppm N-EtFOSE, 20 ppm PFOS, or 100 ppm NEtFOSA for 48 hours when compared with control values. PROCEDURES The purpose of this study was to examine by electron microscopy the livers from selected rats administered the test materials to assess peroxisome proliferation in hepatocytes. Male Crl:CD(CD) IGS BR rats were given the test material in the diet according to Text Table 1. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 37 Text Table 1. Group Designations, Dietary Levels and Scheduled Sacrifice Time Points Niamber of Male Rats Group Number1 (Time Point) Contro 1 (0 ppm) N-EtFOSE 30 10 30 0 0 ppm PFOS 20 ppm NEtFOSA 100 ppm W y -14,643 100 ppm Total No. of Animals 1 10 10 10 10 10 10 5 65 (48 hrs) 2 10 5 5 5 5 5 5 40 (7 days) 3 (14 days) 10 5 5 5 5 5 5 40 4 (1 wk recovery) 10 5 5 5 5 5 5 40 5 (4 wk 10 5 5 5 5 5 5 40 recovery) Total No. of 50 30 30 30 Animals 30 30 25 225 1 Animals in Groups 1 through 3 received the test diet for 48 hours, 7 days, and 14 days, respectively. Animals in Groups 4 and 5 received the test diet for 14 days followed by a 1 or 4 week recovery period, respectively. After the prescribed dosing or recovery period, the animals were fasted overnight, bled for serum samples, anesthetized with CO2, weighed, and exsanguinated. Postmortem procedures included weighing the liver and collecting samples of liver for cell proliferation studies, routine histopathology, palmitoyl-CoA Oxidase activity analysis, and electron microscopy. Liver samples for cell proliferation and routine histopathology were collected in zinc formalin and submitted to Pathology Associates International (PAI) Maryland for evaluation. Liver samples for palmitoyl-CoA Oxidase activity analysis were flash frozen in liquid nitrogen and submitted to Covance Laboratories for analyses. Liver samples for electron microscopy evaluation were thin-sliced and placed in McDowell-Trump fixative (McDowell EM, 1976), and submitted to this laboratory (PAI North Carolina) for electron microscopy processing and evaluation. Per sponsor request, only liver samples from selected rats were processed and evaluated ultrastructurally (Text Table 2). Only liver samples from animals dosed for 48 hours were examined by electron microscopy. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 38 Text Table 2. Animals Selected for Electron Microscopic Evaluation Treatment Group Control N-EtFOSE 100 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm Animal Number R10384 R10388 R 10403 R 10404 R10421 R 10430 R10431 R10439 R10441 R 10443 Timepoint 48 hour 48 hour 48 hour 48 hour 48 hour The selected livers were then processed into Spurr's resin for ultrastructural examination. Thick (lp ) sections were cut, stained with toluidine blue, and examined to select representative areas for further electron microscopy processing. Thin sections (approximately 90 nm) were cut, mounted on 200-mesh copper grids, stained with 5% methanolic uranyl acetate and Reynold's lead citrate, and examined on a Zeiss 900 transmission electron microscope. Centrilobular hepatocytes, where clearly identifiable in liver sections, were preferentially examined. Five representative electron photomicrographs of hepatocytes were taken and significant ultrastructural features were summarized for each photograph and animal on a designated transmission electron micrograph interpretation form. The number of peroxisomes in hepatocytes was manually counted for each photographed hepatocyte and recorded. The mean number of peroxisomes per hepatocyte was manually calculated by summing the number of peroxisomes counted in five hepatocytes per animal and dividing by five. RESULTS AND DISCUSSION Individual interpretations of electron micrographs for each animal selected for evaluation follow this report narrative. The original signed/dated raw data sheets and photomicrographs are maintained in the archived study file at Pathology Associates' North Carolina facility. After 48 hours of treatment, the numbers of peroxisomes appeared significantly increased over control values only for animals given 100 ppm of Wy-14,643 (Text Table 3). The mean number of peroxisomes was approximately double the control values. The mean numbers of peroxisomes per hepatocyte, however, were not remarkably different for animals given 100 ppm N-EtFOSE, 20 ppm PFOS, or 100 ppm N-EtFOSA when compared with control values. Figures 1 through 4 show hepatocytes from control and non-affected treated animals. Some normal ultrastructural features are illustrated in these figures. Figure 5 shows a hepatocyte from an animal given 100 ppm Wy-14,642, illustrating the increased numbers of peroxisomes. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 39 Text Table 3. Quantitation of Hepatocellular Peroxisomes Treatment Group Timepoint Control N-EtFOSE 100 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm 48 hour 48 hour 48 hour 48 hour 48 hour Animal Number R10384 R10388 R 10403 R 10404 R10421 R10430 R10431 R 10439 R10441 R 10443 Mean Number of Peroxisomes per Hepatocyte 11.4 13.6 16.6 11.4 15.2 3.8 17.8 5.0 29.6 29.4 No other ultrastructural abnormalities were identified in the samples evaluated. CONCLUSION As evaluated by electron microscopy, hepatocellular peroxisomes were increased in male Crl:CD(CD) IGS BR rats given 100 ppm Wy-14,643 by diet after 48 hours of treatment. The mean number of peroxisomes was approximately doubled over control values. The mean numbers of peroxisomes per hepatocyte were not remarkably different for animals given 100 ppm N-EtFOSE, 20 ppm PFOS, or 100 ppm N-EtFOSA for 48 hours when compared with control values. REFERENCES McDowell, EM and Trump, BF: Histologic fixative for routine diagnostic light and electron microscopy. Arch. Pathol. Lab. Med., 100:405-414, 1976. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 40 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION study No.: .1 132-1W (LMW.02I Amina! No : -- .............. Treatment Group: Control______ Sex:__ Male_________________ Spcocs/SUain:i Tissue: Ltvcr_____________ Block Nofsk: 99 62-4________ Z 17346 - Photo/Negaiive Not*).: ZI73SO Significant Lesions (check one): Yes X No Interpreting Palhuiogisi i signature an*, d ato A etEsj- A ---- XL"!! - i. : |-bl Features: Z17346. Liver. Hpatocyte. 10.320X. This hpatocyte contains a central oval nucleus and is bordered by adjacent hcpatocytes on two sides and sinusoids ventrally and dorsally. The lighter staining endothelial cell cytoplasm is evident. MiUxhondria and rough endoplasmic reticulum (RER) profiles arc frequent through the cytoplasm of the hcpatocytc. The intervening cytosol appears finely granular and contains ribosomes, glycogen and smooth endoplasmic reticulum (SERi. Bile canaltculi arc present on the left and upper right margins. Peroxisomes are infrequent; four (5) peroxisomes counted. Z17347. Liver. Hcpatocytc. 10.320X. This hcpatocytc is bordered by endothelial cells on both the right and the left. Mitochondria and rough endoplasmic reticulum <RER> profiles aie fiequent tluuugli lire cytoplasm uf the liepatoeyie. A prominent microvillous border is evident at the top right margin of this cell. Nine (9) mitochondria counted. Z17348. Liver. Hcpatocytc. 10.320X. This hcpatocytc is surrounded by two adjacent hcpatocytes and a sinusoid along the right margin. The ceil has abundant RER intermixed with mitochondria and other organelles. Several mitochondria are irregularly- shaped and contain cytoplasmic invagination. Eight (8) peroxisomes counted. Z 17349. Liver. Hcpatocytc. 10.320X. This hepatocyte contains several lipid droplets, of which one contains a dark round membranous inclusion. An endothelial cell is present at the top right, and on the left some lamellar debris is present in the extracellular space. Thirteen (13) peroxisomes counted. Z17350. Liver Hcpatocytc. 10.320X. Similar toZ17349. several medium-sized lipid droplets arc present in this iKpalixytc. There arc abundant mitochondria and numerous RER profiles evident. Twenty-two (22) peroxtsomes counted_____________________ nclusHins: Nm .a! u-| ,n>. vii Mi nuiiiK-i p.-nno... ties pci licjut'H yu- f. I i .4 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 41 TRANSMISSION KI.KCTRON MICROGRAPH INTKRPRKTATION Study No. 1132 100 tEM99A2) Animal No ,, ,W M ___ ____ Treatment Group: Control S e x : ___ Mills________________ Species/Stramm O aiSU iJ DKHat Tissue Liver_____________ Block Note) `W.62-8_ _ _ ZI735I - Pboto/Ncgutivc Note)-: Z P355 Significant Lesions (check one}: __ Yes X No Intetpieting Patologtst (signature and dale): V ^ J L u &_ Sent. IW) F iatures: 7.17351 Live? Uepixyir IDJ2UX Hus hepaioeyhr ha*a central roundnucleus with tsse ptominera ncleo) The wopbsm s finely gnmid.u aiHlenotatns many mitoIwsndria and ULR prohLc. There arc al*o abunekuM Useseme and sonte peroxisome. Scvcia hile cunalie uh are present between Hus cel) and adjacent ocpatocytea. Al the right manan is a dark* -.taill::: In p a h u > le. :n .1 a sii;;.x>ttl.-il * r \r h r .\\le iv pr. sonl tr. a nisi-, mi li h il l m ! Hi, phottsgntph. Twelve fill svkwm vutmted / P ' 1* fiver Wrpaiecytr l(i.'2i:*N. Ninnermts mitoeh.uidfi anc li: l? profiles arc present in his hep.'iTi'cyte Its nucleus i>polygonal and eentmlly inenred. Ai the right tmitt are two darker hepatceyto. that appear to have s.irr.cn h.r bree.- and more nursevsnt ntiteolioiva ia Rantloin dark pcmxisame'iton) Iv m im >u*s an* present in the* cytoplasm Hlteen <15* per.ixxi cs cm: ik *.I Liver Hcpahvytc OmHv k'fi u ttiin I* *.let of cncinhcMevil Itriine u sinUMtkd is presetn. The !icp;.i< . sic lias i n - m o l : an nucleus and be cvc-iI.imii contains mans iiillociuindtci. KIR pmiik#. etcIseveral palc-sbimny lipid droplets Some peres is. mes .im' lyso'.onu's .i calsi i*resent. ImvcP peroMsomes e>unktl / liver Hejxiuwxte. KJ.320X. IVvViNtal hrtpsUoeyir t boftfetedb? liepai.Kryt.rNon must margin., .ihimuch .i Mtgtne t ttl sinusoid i-. p . nt *i1 1 e !*ti issilli viyihuteylo. and bsittoiii. Sc vgt a! bile eunulisuh an. piese it tenecu the ccstual and beide; iuv Itepaioeytc- Scveial small Iioki dtopkts are present \ . . neicus si,ul. vm 'v e- ami pcnrxis i.-- ,m- present Txcnty-thtee *.2i) petoxt.** .miiKed. Pcitns<ttics, K ur.-s ...a <-ate tniuseltomhia .veiv sets difficult lodiilcrrnlul? . i llus photograph 717355 I ver. Hfpatucyte, Ks.e'dX, fliis licpsitvesic soiilams I nn >n il-lo !r.edatn-ice<i lipid .hopleiv S im iv * ate nvsent .it the i p. r l > !'. in o ! i pfin ciapii. I:;J by paler lamme .ri-lh.'lial eels t j-.leen t 13} pcrosisome- sonntc-J. ________________________ Conclusions: Normal bepatocyii*. Mean o 136 peroxisomes pci hepntocyte counted Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 42 TRANSMISSION ELECTRON MJ< KOOK U 'U INTERPRETATION Study No.: I H2 KX>lEMW.fiJ) Animal No. R10403 Treatment Group: N l.tlOSI-, 100pom Sex:...... E U S , . - ___ . __________ Spec ics/vStrain:<.t|CL)?'(S[ l<JSBRKat Tissue:......... Ljvgr_____________ Block No(s>.: 62-23 Z 17356 - Photo/Negativc No(s).:__717360 Significant Lesions (check one): _________ Yes X No Interpreting Pathologist {signature and date) ...Sept. 29. 1999 Features: /17336. Liver. Hcpatocylc. 10.320X. This hepatocyte contains approximately ten small- to mediurn-si/ed lipid droplets Tlie cytoplasm has numerous mitochondria and RER profiles present. Bile canaliculi are present between this cell and adjacent hepatocytes on the left and the right. Erythrocytes in sinusoids are present on three margins. Some stain debris/artifact is present on the photograph. Eight (8) peroxisomes counted. 7.1735?. I.ivcr. Hcpatocytc. 1Q.320X. This hcpalocytc is binuclcatcd. An endothelial cell is present at the top. hut all other margins are adjacent hepatocytes. The cytoplasm contains numerous mitochondria and RER profiles. One distinct lipid droplet is present, and there arc numerous small to medium-sized dark peroxisomes and/or lysosomes. Twenty-three (23yperoxisomes counted. Z 17358. Liver. Ilepalocyte. 10.320X. A nucleus is mil evident in this plane of section for this hepatocyte. Four medium-sized lipid droplets are present, and the other organdies appear very* similar to Z17357. Some lysosomes contain clear vacuoles within them and many arc irregularly shaped. Thirteen (13) peroxisomes counted. 7.17357, Liver. Hcpatocytc. 10.320X. Hcpalocytc contains one large lipid droplet. Twenty-three (23) peroxisomes counted. Some stain dcbns/arUlaet present on the photograph. ZI7360. Liver Hepatocyte. 10.320X. Multiple small- to medium-sized lipid droplets arc present. Sixteen 116) peroxisomes counted ( 'inclusions: N.-rnvl liepn'.vyu- m ? .n r age v<omm.,tes rvi hci c. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 43 TRANSMISSION KI.KOTRON MICRO.RAIMI INTERPRETATION Study No. Animal No. BJi&iM _____ Treatment Group: N-F.(pOSE lOQppm Sex:___Mate--------------------- Specics/Sti ain;CtLCU2Sii>Ulltili UKRat Tissue: _ Liycx_____________ Block No(s').: w 67 ?4______ / t / ii'l Photo/Negative NofsE: Z 17365 Significant l iio n s (check one): Yes _____X No Interpreting PathoJogn.1 (signature and date}: September 30. 1000 Features: 7.17361 Liver, Hpatocyte. KU2UX. 11ns central bepatocyte exhibits a microvillous border adjacent to .sinusoid* on thiee margins and borders with three adjacent bepatocytes on three other margins Bile camdiculi aie vident with two of the adjacent hpatocytes. The hpatocyte has around central nucleus and quire a few smalt- io medium-sized lipid droplets. Ihere aie numerous mitochondria and RER proliics. The remaining cytosol is finely granular and contains scattered lysosomes and peroxisomes. Sixteen ( I6i peroxisomes counted. Zl<362, Liver. Hcputocytc. 10.32X. This hpatocyte appears somewhat diamond-shaped in this photograph, with a sinusoid ai the top left and bottom. Part of a sinusoidal lining cell and its nucleus is evident at the top left of the photograph. The bepatocyte has a round central nucleus and abundant RER profiles and mitochondria. Also, numerous lysosomes and a lew- peroxisomes are present. Six (6) peroxisomes counted 717.363. Liver Hpatocyte. 1.32UX. This bepatocyte appears morphologically similar to those desenbed above. Several small lipid droplets arc also present. Eleven (11}peroxisomes counted. 2 17364. Liver. Hpatocyte. I0.32OX. Must of two hepatocytes are present in this picture A long profile of a bile canaliculus is present between the two cells. The complete heparocyte on the right contains several medium-si/ed and one large lipid droplet. Eight (8) peroxisomes counted 717365. Liver. Hepatocvie. 1U.320X This is a large bepatocyte next to a sinusoid at the top right of the photograph. Two erythrocytes are present in ihc sinusoid. The bepatocyte has a large round nucleus and numerous milfichondtu and RER piotites. Also, there an: multiple lysosomes and peroxisomes in the cytoplasm. (Tear distinction between some peioxisoines and lysosomes is difficult Sixteen (16} peroxisomes counted. Conclusions; Noiiiutl hcpalucyie Mean of 11.4 peroxisomes per hcpatocyte Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 44 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No.: R 10421___________ Treatment Group: PFOS 20ppm Sex: Male__________________ Species/Strain:Crl:CDtsm 1GS BR Rat Tissue:_____ Liver_____________ Block No(s).: 99.62-41______ Z17366- Photo/Negative No(s).: Z17370 Significant Lesions (check one): ________ Yes X No Interpreting Pathologist (signature and date): ___________________ Sept. 30. 1999 Features: Z17366. Liver. Hepatocyte. 10,320X. This hepatocyte has a centrally located polygonal nucleus surrounded by cytoplasm rich in mitochondria and RER profiles. Multiple small- to medium-sized lipid droplets are also present. The remaining cytoplasm appears finely granular and contains some lysosomes and peroxisomes. Twenty-seven (27) peroxisomes counted. Z17377. Liver. Hepatocyte. 10,320X. This hepatocyte has adjacent hepatocytes both dorsally and ventrally. To the left is a sinusoid lined by an endothelial cell and containing an erythrocyte. An endothelial-lined sinusoid is also present along the right margin. Approximately nine small lipid droplets are present in the cytoplasm. Fifteen (15) peroxisomes counted. Z 17368. Liver. Hepatocyte. 10,320X. In this hepatocyte the mitochondria appear smaller and more condensed. Profiles of RER and SER are easily evident in the cytoplasm. Five (5) peroxisomes counted. Z 17369. Liver. Hepatocyte. 10,320X. Several small and one large lipid droplet are present in this hepatocyte. At the top right and bottom left margins o f the photograph are erythrocytes within sinusoids. The hepatocyte's nucleus is not present in this plane o f section. Numerous mitochondria and RER profiles are visible. The intervening cytosol is finely granular and contains SER and other organelles, including some peroxisomes and lysosomes. Some crystalloid structures appear very prominent in some peroxisomes. Fifteen (15) peroxisomes counted. Z17370. Liver. Hepatocyte. 10,320X. Hepatocyte appears very similar to Z17369. A nucleus is not evident in the plane o f section photographed. Several small- to medium-sized lipid droplets are present. Sixteen (16) peroxisomes counted.___________________________________________ Conclusions: Normal hepatocyte. Mean of 15.2 peroxisomes per hepatocyte. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 45 TRANSMISSION KI.KCTRON MK'IWNKAPII INT! RI*ROTATION Study No.;. Ammtil No,: RUH30 Treatment Group:. PFOS 20 onm Sex:---- Mais-------------------------- Spec tes/Sn .> llfSJIft Km Tissue:_____ LLysT____________ Block N o(s).:__ 99.62 5 0 ____ Z1737I - Photo/Negative No<s).: ZI7375 Significant Lesions (check onci: Yes No Interpreting Pathologist (signature and date): Sept, 30. 1999 Features: Z17371. Hcpatocyte. 10.320X. This hepatocytc has an eccentrically located oval nucleus. It is bordered by an sinusoid at the top and right and another hepatocytc on the lett. Numerous mitochondria, RER profiles, finely granular cytosol, and a few situili lipid droplets are present in the cytoplasm. Some small lysosomcs are scattered in the cytoplasm Five <5 peroxisomes counted Z17372 Liver. Hcpaiocyic. 10.320X. This hcpaiocvtc is surrounded by hepaiocylcs on oil sides except ihc top. which is a sinusoid Tliccdl has abundant RF:R profiles ami mitochofuli la in the cytoplasm, as well as multiple irregularly-shaped small to medium-sired lipid droplets. Some lysosomes and a few peroxisomes are present. Four <4>peroxisomes counted /.I7373. Liver. Hepatocytc. 10.320X. The hepntocyte appears essentially similar to previously dessi died licpalucyles. touLminig a laigc round nucleus and numcious uutoclioudtia ami KHR profiles Some lipid droplets ate preve'U Lysosumes and peroxisomes are rare. Two (2j probable peroxisomes counted. 2 17374, Liver. Hcpatocyte. I0.320X. This hepatocytc is somewhat lighter staining than most of the previously examined cells were, and is located beneath a cemrilohular vein along the right. Some collagen fibers ate evident in the Space of Disse beneath the endothelial cell margin Peroxisomes in the hepatocytc are better defined than in most oiher cells, and have prominent crystalloid structures. Eight (8) peroxisomes counted. 7.17375, Liver, Hcpatocyte, 10.320X. Tins hcpatocyte o t*urdeted by a sinusoid along the top margin The cell's microvillous border is next to ;hc endothelial lined sinusoid, which contains several erythrocyte*. Adjacent hepatocytes are cm the right and left of the central heputocylc. A nucleus is not present in this plane of section The cytoplasm is finely granular and contniris inulliplt* tllUUi liU! dll.l and RI U ptldties /out (1)1 petoxonmes air clearly dtsn-rnitde__________ Conclusions: Normal hcpatocyte Average 3.8 peroxisomes per hepatocytc. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 46 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No.: R10431___________ Treatment Group: N-EtFOSA lOOppm Sex: Male__________________ Species/Strain:Crl:CD(Sm 1GS BR Rat Tissue: Liver_____________ Block No(s).: 99.62-51________ Z17376 - Z17380 Photo/Negative No(s).: Z17396 Significant Lesions (check one): ________ Yes X No Interpreting Pathologist (signature and date): __________________ Sept. 30. 1999 Features: Z17376. Liver. Hepatocyte. 10,320X. This light-staining hepatocyte has a central polygonal nucleus surrounded by cytoplasm containing numerous mitochondria. RER and SER profiles are present and there are greater than a dozen small- to medium-sized lipid droplets. Nine (9) peroxisomes counted. Z 17377. Liver. Hepatocyte. 10,320X. This hepatocyte stains somewhat darker than the preceding example. The cytoplasm is finely granular and mitochondria appear denser. Peroxisomes and some lysosomes are generally smaller and darker than the mitochondria, and are often difficult to differentiate from each other. Twenty-two (22) peroxisomes counted. Z17378. Liver. Hepatocyte. 10,320X. The top margin of this cell shows a microvillous border adjacent to a sinusoid. The nucleus is not evident in this plane o f section for this hepatocyte. The cytoplasm contains abundant SER between the mitochondria and other organelles, including some lysosomes and peroxisomes. Sixteen (16) peroxisomes counted. Z17379. Liver. Hepatocyte. 10,320X. Mitochondria and RER profiles are prominent in this hepatocyte's cytoplasm. A single medium-size lipid droplet is present near the top border o f this cell. A sinusoid lined by an endothelial cell is present along the left margin. Seventeen (17) peroxisomes counted. Z17380. Overexposed negative; not printed. Z17396. Liver. Hepatocyte. 10,320X. The centered hepatocyte is bordered by adjacent hepatocytes on the wide and bottom. At the top is a sinusoid lined by an endothelial cell and containing some granulocytes and erythrocytes. The hepatocyte has a central polygonal nucleus surrounded by numerous mitochondria and RER profiles. Scattered darker peroxisomes are present. Twenty-five (25) peroxisomes counted.___________________________________________ Conclusions: Normal hepatocyte. Average 17.8 peroxisomes per hepatocyte. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 47 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No.: R10439__________ Treatment Group: N-EtFOSA lOOppm Sex: Male__________________ Species/Strain:Crl:CD(SD) IGS BR Rat Tissue: Liver__________ __ Block No(s).: 99.62-59_______ Z17381 - Photo/Negative No(s).: Z17385 Significant Lesions (check one): ________ Yes X No Interpreting Pathologist (signature and date): ____________________ October 1, 1999 Features: Z17381. Liver. Hepatocyte. 10,320X. A medium-sized round nucleus is present in this hepatocyte. It is surrounded by numerous mitochondria and RER profiles. Five lipid droplets are evident in the cytoplasm. Some mitochondria have dark inclusions; this may be some stain precipitate. Several lysosomes are present, but zero (0) peroxisomes are clearly discernible. Z17382. Liver. Hepatocyte. 10,320X. This hepatocyte is somewhat lighter staining than Z17381. The round nucleus is surrounded by numerous mitochondria and RER profiles. The cytoplasm contains several small- to medium-sized lipid droplets. Similarly, several lysosomes are present, but zero (0) peroxisomes are clearly discernible. Z17383. Liver. Hepatocyte. 10,320X. This slightly darker-staining hepatocyte has a sinusoid lined by an endothelial cell at its upper right margin. The cytoplasm contains numerous mitochondria and RER profiles. Several lipid droplets are present. Nine (9) peroxisomes counted. Z17384. Liver. Hepatocyte. 10,320X. Erythrocyte containing sinusoids are evident on three sides of this hepatocyte. Several medium- to large-sized lipid droplets are present in the cytoplasm of this hepatocyte. There are numerous mitochondria and RER profiles in the cytoplasm. Five (5) peroxisomes counted. Z17385. Liver. Hepatocyte. 10,320X. The cytoplasm of this hepatocyte contains numerous RER profiles and mitochondria. Several lipid droplets, one with a lamellar body, are present. Some scattered lysosomes and peroxisomes are present, and are difficult to clearly differentiate from each other. Eleven (11) peroxisomes counted. Conclusions: Normal hepatocyte. Average 5.0 peroxisomes per hepatocyte. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 48 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No.: R10441__________ Treatment Group: Wy-14,643 lOOppm Sex: Male__________________ Species/Strain:Crl:CDtSD) 1GS BR Rat Tissue: Liver_____________ Block No(s).: 99.62-61________ Z17386- Photo/Negative No(s).: Z17390 Significant Lesions (check one): X Yes ________ No Interpreting Pathologist (signature and date): ____________________ Oct. 01, 1999 Features: Z17386. Liver. Hepatocyte. 10,320X. This plate of three hepatocytes has sinusoids on both the right and left margins. The central hepatocyte has an oval nucleus which is surrounded by numerous mitochondria and finely granular cytoplasm. Peroxisomes are the darker staining organelles. Twenty-two (22) peroxisomes counted. Z17387. Liver. Hepatocyte. 10,320X. Four medium-sized lipid droplets are present in this hepatocyte. The cytoplasm is finely granular and contains numerous mitochondria and some RER profiles. Three erythrocytes are present in the sinusoid at the top of the photograph. Thirty-seven (37) peroxisomes counted. Z17388. Liver. Hepatocyte. 10,320X. This hepatocyte appears ultrastructurally similar to those described above. It has a central round nucleus surrounded by cytoplasm containing numerous mitochondria, finely granular cytosol, RER profiles, and some lipid droplets. Additionally some variably-sized dark-staining peroxisomes are present. An erythrocyte in a sinusoid is present at the top right margin of the photograph. Twentytwo (22) peroxisomes counted. Z17389. Liver. Hepatocyte. 10,320X. Hepatocyte appears ultrastructurally similar to Z17388. Forty-one (41) peroxisomes counted. Z17390. Liver. Hepatocyte. 10,320X. Only a small segment of nucleus is present in this hepatocyte. This hepatocyte is bordered by adjacent hepatocyte on three sides and by an endothelial-lined sinusoid on the left. Its cytoplasm contains numerous mitochondria, RER profiles, scattered dark peroxisomes, and some lysosomes. Twenty-six (26) peroxisomes counted. Conclusions: Increased peroxisomes. Average 29.6 peroxisomes per hepatocyte. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 49 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 (EM99.62) Animal No.: R 10443_________ Treatment Group: Wv-14,643 lOOppm Sex: Male__________________ Species/Strain:Crl:CD(SDl 1GS BR Rat Tissue: Liver_____________ Block No(s).: 99.62-63________ Z17391- Photo/Negative No(s).: Z17395 Significant Lesions (check one): ____X___Yes _____ ___ No Interpreting Pathologist (signature and date): ___________________ Oct. 01, 1999 Features: Z17391. Liver. Hepatocyte. 10,320X. A polygonal-shaped hepatocyte is centered in the photograph. At the top is a sinusoid containing an endothelial cell and erythrocyte on the left and a perisinusoidal lipid-storing cell at the right. Another sinusoid is present at the bottom of the photograph. The hepatocyte contains numerous mitochondria and RER profiles. The intervening cytosol is finely granular and also contains scattered dark lysosomes and peroxisomes. Thirty (30) peroxisomes counted. Z17392. Liver. Hepatocyte. 10,320X. This hepatocyte contains greater than a dozen small- to medium-sized lipid droplets. Numerous RER profiles and mitochondria are present in the cytoplasm. Twenty-six (26) peroxisomes counted. Z17393. Liver. Hepatocyte. 10,320X. This somewhat lighter-staining hepatocyte is bordered by adjacent hepatocytes on all margins in this photograph. A nucleus is not evident in this plane of section. Several lipid droplets are present. Forty-one (41) peroxisomes counted. Z17394. Liver. Hepatocyte. 10,320X. This hepatocyte appears morphologically similar to Z17393, except it has a large central polygonal nucleus. Thirty-eight (38) peroxisomes counted. Z17395. Liver. Hepatocyte. 10,320X. This round hepatocyte appears essentially similar to Z17391. The hepatocyte contains numerous mitochondria and RER profiles. The intervening cytosol is finely granular and also contains scattered dark lysosomes and peroxisomes. Twelve (12) peroxisomes counted. Conclusions: Increased peroxisomes. Average 29.4 peroxisomes per hepatocyte. Sponsor: 3M Study Number: IR C 1132-100 Amended Final Report Page 50 VI. APPENDIX 3 - PALMITOYL CoA OXIDASE REPORT Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 51 Table VI-1. Individual Animal Palmitoyl CoA Oxidase Activity Treatment Group Controls N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS20 ppm N-BFOSA 1Q0 ppm 48 Hour Interim Sacrifice Anim al Number PCOAO* Activity R10381 R10382 R10383 R10384 9 9 6 6 R1038S R10386 R10387 5 6 8 R10388 11 R10389 R10390 R10391 R 10392 7 8 7 101 R 10393 R10394 R10395 R10396 10 16 6 7 R10397 7 R10398 R10399 10 8 R 10400 R10401 R 10402 R10403 10 5 8 10 R10404 R10405 R10406 R10407 9 9 4 7 R10408 R 10409 R10410 R10411 8 12 9 10 R10412 R10413 R10414 R10415 9 13 9 6 R10416 R10417 7 9 R10418 R10419 8 12 R10420 R10421 R10422 R10423 9 11 8 8 R10424 R 10425 R10426 6 8 6 R10427 R10428 R10429 R10430 6 10 11 8 R10431 R 10432 R10433 R10434 6 5 6 R 10435 R10436 R10437 R 10438 6 5 11 7 R10439 R 10440 6 6 7 Day Interim Sacrifice Animal Number PCOAO* Activity R10446 R10447 8 4 R10448 5 R10449 5 R10450 R10451 5 5 R10452 4 R 10453 5 R10454 R10455 5 7 R 10456 6 R10457 3 R 10458 6 R 10459 6 R10460 10 14 Day Interim Sacrifice Animal Number PCOAO* A ctivity R10486 5 R10487 6 R10488 5 R10489 5 R 10490 8 R10491 6 R 10492 7 R 10493 9 R10494 5 R 10495 7 R10496 R 10497 2 4 R 10498 2 R10499 3 R10500 3 R10461 6 R10501 2 R10462 7 R10502 5 R10464 4 R10504 3 R10465 8 R10505 4 R10466 5 R10506 5 R10467 6 R10507 6 R10468 4 R10508 4 R10469 7 R10509 4 R10470 9 R10510 5 R10471 8 R10511 4 R10472 7 R10512 4 R10473 5 R10513 5 R10474 8 R10514 4 R10475 9 R10515 4 R 10476 R10477 R10479 R10480 7 3 6 ------- r 4 R10516 R10517 R10519 R10520 2 4 2 4 2 1 Week Recovery Sacrifice Anim al Number PCOAO* Activity R10526 6 R 10527 9 R 10528 7 R10529 5 R10530 10 R10531 10 R10532 7 R 10533 8 R10534 3 R10535 5 R 10536 3 R 10537 5 R 10538 5 R 10539 3 R 10540 6 R10541 R 10542 R 10543 R10544 R10545 6 4 4 5 5 R 10546 R 10547 R10548 R10549 R10550 5 5 8 4 8 R10551 R10552 R10553 R10554 R10555 6 6 5 8 3 R 10556 R 10557 R10559 R10560 6 6 6 7 Palmitoyl CoA Oxidase activity. IU/g Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 52 VII. APPENDIX 4 - STUDY PROTOCOL AND AMENDMENTS Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 53 Sponsor: 3M St. Paul, Minnesota CHARLES RIVER LABORATORIES O s tw a y and Development Services PdthokjgyAsuxites PROTOCOL Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Date: January 12,1999 Performing Laboratory R.O.W. Sciences 15 Firstfield Road Gaithersburg, Maryland 20878 Laboratory Study Identification: Study Number: (1132-100) PAI Project Number: (Histology number to be assigned by PAI by protocol amendment) 15 Worman's Mill Court, Suite 1 * Frederick, Mary land 21701 * (301) 663-1644 * (301) 663-8994 FAX Study Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Purpose To assess cell proliferation and peroxisome proliferation in rats administered test material in the diet. Sponsor 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 Study Representative Marvin T. Case, D.V.M, Ph.D. 3M Corporate Toxicology Phone No.: 651 733-5180 Fax No.: 651 733-1773 Email: mtcase@mmm.com Alternative Study Representative Andrew M. Seacat, Ph.D. 3M Corporate Toxicology Phone No.: 651 575-3161 Fax No.: 651 733-1773 Email: amseacat@mmm.com Test Facility Therlmmune Research Corporation (formerly, R.O.W. Sciences) 15 Firstfield Road Gaithersburg, Maryland 20878 Study Monitor Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: srepaisaic@aol.com Study Director Gary W. Wolfe, Ph.D., D.A.B.T. R.O.W. Sciences Phone No.: 301 330-3723 Fax. No.: 301 330-3738 Email: gwolfe@lab.row.com Principal Investigator Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@aol.com Study Pathologist Carolyn Moyer, D.V.M., Diplomate, A.C.V.P. Pathology Associates International Phone No. 301 624-2928 Fax No. 301 663-8994 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 55 Proposed Study Timetable In-life Start Date: To be added by protocol amendment; Day 0 In life End Date: To be added by protocol amendment Audited Draft Report Date: To be added by protocol amendment Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 56 Regulatory Compliance This study will be conducted in the spirit of Good Laboratory Practice (GLP) regulations. Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance Not applicable. Test Materials Test Material: N-EtFOSE (completed by 3M) Identification: N-EtFOSE Lot Number: Purity: FM 3929 Lots 30035, 30037, 30039 99.2% Stability: > 5 years Storage room temp. Conditions: Characteristics: waxy solid PFOS (completed by 3M) PFOS 217 99% > 5 years room temp. white powder N-EtFOSA (to be added by protocol amendment) N-EtFOSA Lot 541 W y-14,643 (to be added by protocol amendment) Wy > 5 years room temp. amber waxy solid Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 57 Reserve (Archive) Samples A reserve sample (approximately 5 g) o f each lot will be taken and stored at room temperature. These samples will be transferred to the Sponsor after completion of the in-life phase to be retained in accordance with 40 CFR 792.195. Disposition of Test Material After authorization from the Sponsor, any remaining test material will be returned to: Marvin Case, D.V.M., Ph.D. 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Phone No.: 651-733-5180 Fax No.: 651-733-1773 Animals Species: Strain: Source: Age at Initiation of Treatment: Weight at Initiation of Treatment: Number and Gender: Identification: Rat Crl:CD(SD) IGS BR Charles River Laboratories, Inc., Raleigh, NC Preferable 6 weeks of age, but not more than 8 weeks of age 150 to 300 g males unique identification by individual car tags and cage cards Husbandry Housing: Diet: Water: Contaminants: Environment: Acclimation: Randomization: Justification: Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 58 Single housed in hanging stainless steel wire cages Teklad 7012 Certified Rodent Diet. Fresh food will be provided weekly. Feed is analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinate hydrocarbons, organophosphates, and specified nutrients. Specified nutrients analyses are on file at R.O.W. Sciences. Tap water, provided ad libitum via an automatic watering system or water bottles. The water is analyzed at least two times per year for contaminants and specific microbes. The results of these analyses are on file at R.O.W. Sciences. The study director and/or the Sponsor have considered possible interfering substances potentially present in animal feed and water, including the test material itself or possible structurally related materials as well as the items listed in (2) and (3) above. None of these contaminants are reasonably expected to be present in animal feed or water at levels sufficient to interfere with this study. The targeted temperatures are between 64 and 79F with a relative humidity between 30% and 70%. Temperature and humidity are monitored continuously. A 12-hour light/12-hour dark cycle will be maintained. Ten or greater air changes/hour will be maintained. Animals will be acclimated to the facility for a minimum o f 7 days prior to the start of dosing. Animals will be observed for general health and suitability for testing during this period. Animals that are diseased or unsuitable for testing will be removed from the study. Using computer-generated random numbers wit assignment to groups, At the time of randomization, the weight variation of the animals o f each sex used should not exceed 2 S.D. of the mean weight, and the mean body weights for each group of each sex will not be statistically different. Rats will be used because of the extensive historical data base, and the FDA requirements for a rodent species. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 59 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points Number of Male Rats Group Number Control N-EtFOSE PFOS N-EtFOSA Wy- (Time Point) (0 ppm) 300 100 30 ppm 20 ppm 100 ppm 14,643 100 ppm Total No. of Animals 1 10 10 10 10 10 10 5 65 (48 hrs) 2 10 5 5 5 5 5 5 40 (7 days) 3 10 5 5 5 5 5 5 40 (14 days) 4 10 5 5 5 5 5 5 40 (1 wk recovery) 5 10 5 5 5 5 5 5 40 (4 wk recovery] Total No. of Animals 50 30 30 30 30 30 25 225 Dosing Procedures Method of Administration Dietary. Animals in Groups 1 through 3 will receive test diet for 48 hours, 7 days, and 14 days, respectively. Animals in Groups 4 and 5 will receive test diet for 14 days followed by a 1 or 4 week recovery period, respectively. Reason for Dosing Route The potential human exposure is by the oral route. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 60 Dose Preparation Before initiation of treatment, dose preparation of each test material will be mixed. All dose preparations will be stored at room temperature. Dose preparation will be documented and reported. See Attachment I for test diet preparation procedures. Retention Sample Samples (approximately 100 g) will be taken from the dose preparation and stored at room temperature. Unless used for analyses, these samples will be discarded at least 1 month after completion of the in-life phase. Observation of Animals Clinical Observations Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity; findings will be recorded as they are observed. Body Weights Prior to treatment (at randomization), weekly for Week 1 through 4 weeks of recovery. Food Consumption Weekly for Week 1 through 4 weeks of recovery. Clinical Chemistry Animals will be fasted overnight before animal's scheduled necropsy; blood will be collected from a jugular vein into an EDTA-coated tube. Serum enzyme levels of alanine aminotransferase (ALT), alkaline phosphatase, aspartate aminotransferase (AST), cholesterol and triglycerides will be determined. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 61 Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with CO2, weighed, and exsanguinated. Scheduled Sacrifices Interim Sacrifices At 48 hrs, 7 days, and 14 days, animals will be fasted overnight, bled for serum samples, anesthetized with CO2, weighed, and exsanguinated. NOTE: Two serum samples will be needed, (1) a 0.5 ml sample for clinical chemistry and (2) a 1.5 ml sample for compound level analysis. The abdominal cavity of each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection. Terminal Sacrifices After 1 and 4 weeks of recovery, animals will be fasted overnight, bled for serum samples, anesthetized with CO2, weighed, exsanguinated, and necropsied. NOTE: Two serum samples will be needed, (1) a 0.5 ml sample for clinical chemistry and (2) a 1.5 ml sample for compound level analysis. Postmortem Procedures Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 62 Necropsy The necropsy will include an examination of the external features of the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Representative samples of the left lateral lobe of the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin. After fixation, each sample of liver will be delivered to: Sandra R. Eldridge, Ph.D. Pathology Associates International 15 Worman's Mill Court, Suite I Frederick, Maryland 21701 Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition, liver sections prepared from the same tissue block will be stained with hematoxylin and eosin and examined microscopically. Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately 500 mg) o f the right lateral lobe of the liver will also be collected from select animals and flash-frozen in liquid nitrogen. See Attachment II for procedure. The liver tissue will be stored in a freezer set to maintain -60 to -80 C until analyzed by Covance for palmitoyl-CoA Oxidase activity. The liver samples to be analyzed will include all study animals, EXCEPT for the Wy-14,643 animals and all animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study will be analyzed for palmitoyl-CoA Oxidase activity; these samples consist of liver samples from 35 rats and 35 guinea pigs. Tissue Collection for Electron Microscopic Evaluation Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 63 Sections of liver from all animals will be collected, minced to approximately one millimeter cubes and placed in a fixative appropriate for electron microscopy. The containers and fixative will be provided by PAI. Electron microscopy will be performed on one animal per treatment group exhibiting the highest cell proliferative response as well as one control animal at the discretion of the Sponsor, from one time point as well as the 4-week recovery. Thus, EM will be performed on one animal from the control, N-EtFOSE (one dose only to be determined), PFOS, N-EtFOSA, and Wy groups at one of the time points, as well as the 4 week recovery, for a total of 10 animals. Remaining Liver Tissue The remaining liver tissue will be frozen and stored at -60 to -80 C for possible future analysis. Organ Weights At the scheduled sacrifices, the liver will be weighed. Histopathology Liver from each animal that is examined for cell proliferation will be stained with hematoxylin and eosin, and examined microscopically for histopathologic changes. Reports One copy of the draft report will be sent to the Sponsor. The report will include the following information: Experimental Design and Methods Results dose analyses mortality clinical observations body weights body weight changes food consumption test material consumption clinical pathology results palmitoyl-CoA oxidase activities macroscopic observations microscopic observations ultrastructural observations cell proliferation assessments Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 64 Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the storage facilities of PAI for a period of 1 year following submission of the final report to the Sponsor. One year after submission of the final report, all of the aforementioned materials will be sent to the Sponsor and a return fee will be charged. All raw data stored on magnetic media will be retain by PAI. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 65 PROTOCOL APPROVAL Marvin Case, D.V.M., Ph.D. Study Representative 3M Corporate Toxicology Gary W. Wolfe, Ph.D., D.A.B.T Study Director R.O.W. Sciences Date Date Sandra R. Eldridge, Ph.D. Principle Investigator Pathology Associates International Date Attachment: I Test Diet Preparation Procedures Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 66 1) Determine the amount of test diet (feed) that is to be prepared and weigh out that amount of feed. 2) Calculate the amount of test article that is needed to prepare the test diet at the desired concentration. 3) Accurately weigh out the necessary amount of test article. 4) Transfer the weighed test article to a container and add a small volume of acetone to container. Manually mix to dissolve the test material adding acetone as necessary (typical ratio of test material to acetone is 1 g: 15-20 ml acetone). Visually inspect test material/acetone for solubility of test material. 5) Prepare a pre-mix by transferring the dissolved test material into 4 kg o f feed in a Hobart mixing bowl. Mix for 10 minutes. Transfer the premix to a larger mixer, add remaining amount of weighed diet, mix for 30 minutes. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 67 Attachment: II Collection of Tissue Samples for Biochemical and Molecular Analysis Because of the extreme instability of certain enzymes and biomolecules, it is essential that tissues be harvested as soon after death as possible and flash frozen immediately in liquid nitrogen. Failure to follow these procedures may lead to loss o f the entire sample and all the energies and resources that were invested into generating the samples. Therefore, make every effort to comply with the following: 1) Harvest the tissue samples as soon as possible after death. Delays may allow for biodegradation and/or inactivation o f the desired endpoint. 2) Immediately submerse the tissue sample directly into liquid nitrogen. Dry ice or other alternatives will not suffice. It is important that the tissue be immersed directly in liquid nitrogen; transferring it to a dry vessel (or sample container) suspended in liquid nitrogen will not suffice. The tissue may freeze to the vessel wall and will then be impossible to remove without completely destroying the vessel (or sample container). 3) Be absolutely sure to maintain the tissue frozen. It should be stored in a sealed container at 70 C and shipped or transferred on dry ice. If needed, the frozen sample can be fractured (broken into portions for different applications) by placing in a crucible which contains liquid nitrogen to keep the sample frozen while grinding/fracturing. PROTOCOL AMENDMENT #1 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 68 Date: February 24,1999 Study Number: 1132-100 Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perflurooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Change/replace the following: a) Title page: PAI Project Number: 99-1233 b) Page 3: In-life Start Date: 2/23/99 In-life End Date: 4/6/99 Draft Report Date: 5/18/99 c) Page 4: Test Materials Test Material: Identification: Lot Number: Purity: Stability: Storage Conditions: Characteristics: N-EtFOSA N-EtFOSA 541 Not provided > 5 years room temp. amber waxy solid Wy-14,643 (Cayman Chemical) Wy 11990 Not provided > 1 year room temp. white powder d) Page 5: Animal identification is by ear tag e) Page 8: Observation of Animals; add, Physical Examinations Weekly at each weigh interval. f) Entire protocol: Change R.O.W. Sciences to Therlmmune Research Corporation g) Page 8: Clinical Chemistry ...blood will be collected from a jugular vein into a serum-separator tube. Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 69 h) Page 9: Scheduled Sacrifices Serum samples for clinical chemistry will be performed by PAI at Chevy Chase, MD. Serum samples for compound level analysis will be stored in a freezer set to maintain -60 to -80C and packed on dry ice and shipped to: Dr. Kris J. Hansen 3M E.T. & S Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Telephone: 612 778-6018 Fax: 612 778-6176 For both Interim and Terminal Sacrifices: NOTE: Two serum samples will be needed, (1) at least a 0.25 ml sample for clinical chemistry and (2) the remaining sample for compound level analysis. i) Page 10: Palmitoyl-CoA Oxidase Tissue Collection and Analysis Liver samples for palmitoyl-CoA oxidase activity will be shipped on dry ice to: Dr. Ron Markevitch Covance Laboratories Inc. 3301 Kingsman Boulevard Madison, WI 53704 Telephone: 608 242-2712 ext. 2337 Fax: 608 241-7227 j) Page 10: Tissue Collection for Electron Microscopic Evaluation Liver samples for EM should be shipped to: Sharon Ambrose PAI-NC 4915 D Prospectus Drive Durham, NC 27713 k) Page 10: Remaining Liver Tissue The remaining liver tissue will be frozen and stored at -60 to -80C in PAI's long-term archive for possible future analysis. l) Page 10: Cell Proliferation Tissue Collection and Immunohistochemical Evaluation ...liver will be collected and preserved in formalin. m) Page 10: Tissue Collection for Electron Microscopic Evaluation Sections of the left lateral lobe o f the liver from all animals... Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 70 n) Page 7: Change dose of Wy-14,643 from 1000 ppm to 100 ppm o) Page 13, Item #5: Prepare a pre-mix by transferring the dissolved test material into feed. Mix until homogeneous.... Reason for Amendment: a) Assigned after protocol approved b) Dates determined after protocol signed; study is non-GLP, therefore report will not be audited c) Information obtained after protocol signed d) Spelling error e) Physical exams should be conducted in addition to body weights f) Testing facility changed name g) No anticoagulant is used for clinical chemistry h) Provide shipping instructions and specify minimum amount of serum needed for clinical chemistry i) Provide shipping instructions j) Provide shipping instructions k) Provide storage instructions m) Specify lobe of liver to be taken for EM n) 100 ppm Wy, 14,643 is adequate for inducing a cell proliferative response, increase in liver weight and increase in peroxisomes o) Accurate description of test diet preparation procedure Approvals: Marvin Case, D.V.M., Ph.D. Study Representative 3M Corporate Toxicology Date Gary W. Wolfe, Ph.D., D.A.B.T Study Director Therlmmune Research Corporation Date Sandra R. Eldridge, Ph.D. Principle Investigator Pathology Associates International Date PROTOCOL AMENDMENT #2 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 71 Date: April 16,1999 Study Number: 1132-*100 Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perflurooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Add the following: a) Page 9: Necropsy Gross lesions will be collected, processed and stained with H&E for microscopic evaluation. Reason for Amendment: a) For histopathologic examination of gross lesions. Approvals: Marvin Case, D.V.M., Ph.D. Study Representative 3M Corporate Toxicology Date Gary W. Wolfe, Ph.D., D.A.B.T Study Director Therlmmune Research Corporation Date Sandra R. Eldridge, Ph.D. Principle Investigator Pathology Associates International Date Date: August 9, 1999 PROTOCOL AMENDMENT #3 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 72 3M Study Number: TRC 1131-100 Title: "Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perflurooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats" Original: Page 10: Tissue Collection for Electron Microscopic Evaluation Sections of liver from all animals will be collected, minced to approximately one millimeter cubes and placed in a fixative appropriate for electron microscopy. The containers and fixative will be provided by PAI. Electron microscopy will be performed on one animal per treatment group exhibiting the highest cell proliferative response as well as one control animal at the discretion of the Sponsor, from one time point as well as the 4-week recovery. Thus, EM will be performed on one animal from the control, N-EtFOSE (one dose only to be determined), PFOS, N-EtFOSA, and Wy groups at one of the time points, as well as the 4 week recovery, for a total of 10 animals. Change/replace the following: Page 10: Tissue Collection for Electron Microscopic Evaluation Sections of liver from all animals will be collected, minced to approximately one millimeter cubes and placed in a fixative appropriate for electron microscopy. The containers and fixative will be provided by PAI. Electron microscopy will be performed on two animals per treatment group from the 48-hour time point. Thus, EM will be performed on two animals from the control, N-EtFOSE (100 ppm dose group only), PFOS, N-EtFOSA, and Wy-14,643 groups at the 48-hour time point for a total of 10 animals. Reason for Amendment: Animals selected on the basis of cell proliferation results. Approvals: Marvin Case, D.V.M., Ph.D. Study Representative 3M Corporate Toxicology Date Gary W. Wolfe, Ph.D., D.A.B.T Study Director R.O.W. Sciences Date Sandra R. Eldridge, Ph.D. Principle Investigator Pathology Associates International Date Date: May 30, 2000 PROTOCOL AMENDMENT #4 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 73 Study Number: TRC 1132-100 Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perflurooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA; 3M T-7091.1) in Rats Change/replace the following: a) Globally replace PFOSA with N-EtFOSA because the acronym for N-Ethyl Perfluorooctanesulfonamide that is currently accepted is N-EtFOSA, rather than PFOSA. b) Page 4: Revise table because 3M provided the lot numbers, and purity information is changing with ongoing analysis at 3M. Test Material: Identification: N-EtFOSE (provided by 3M) N-EtFOSE PFOS (provided by 3M) PFOS PFOSA (provided by 3M) PFOSA W y-14,643 (Cayman Chemical) Wy Lot Number: Purity: Stability: Storage Conditions: Characteristics: 30035,30037, 30039 combined Responsibility of 3M > 5 years room temp. waxy solid 217 Responsibility of 3M > 5 years room temp. white powder 541 11990d Responsibility of 3M > 5 years room temp. 98% > 2 years room temp. amber waxy solid white powder Approvals: Marvin Case, D.V.M., Ph.D. Study Representative 3M Corporate Toxicology Date Gary W. Wolfe, Ph.D., D.A.B.T Study Director Therlmmune Research Corporation Date Sandra R. Eldridge, Ph.D. Principle Investigator Pathology Associates International Date VIII. SIGNATURE PAGE Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 74 Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (N-EtFOSA 3M T-7091.1) in Rats Study Number: TRC 1131-100 / / ACy Sandra R. Eldridge. Ph.D L-' Dale Principle Investigator Pathology Associates Division of Charles River Laboratories. Ine.