Document zQvV1N8p3g6E4yE9RYRwyE7dR

A m & -1105 Sponsor: 3M St. Paul, Minnesota CHARLES RIVER LABORATORIES Discovery und Ltemiopment Samoa fbthabgy/bsacixcs 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 our is I\S4iiV 'J:s PP P , ! 5 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 Appendix 4 - Study Protocol and Amendments VI VII Signature Page VIII 2V 2 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 3 I. REPORT NARRATIVE <&3 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.: 30 1 330-3738 Email: gwolfe@lab.row.com PRINCIPAL INVESTIGATOR: Sandra R. Eldridge, Ph.D. Pathology Associates Division of Charles River Laboratories, Inc., (formerly, Pathology Associates International) J V t/ 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. 2S 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. The 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 o f 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 dumber of Male Rats Group Number Control N-EtFOSE PFOS N-EtFOSA Wy-14,643 Total No. (Time Point) (0 ppm) 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 10 5 5 5 5 5 5 40 (4 wk recovery) Total No. of Animals 50 30 30 30 30 30 25 225 In-life Portion of Study The in-life portion o f 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 o f the liver and any macroscopic lesions were collected and preserved in formalin. After fixation, each sample o f 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 o f 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, 3</7 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 8 Carpintera, CA) and reagents required for the avidin-biotin peroxidase (ABC Kit, Vectastain, Burlingame, CA) method for the detection o f 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 o f 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 o f 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 t-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 o f 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 Wy-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 normal 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 =2 s o 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 o f 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 o f rats given N-EtFOSE, PFOS, N-EtFOSA, or Wy-14,643 as a positive control test material. A summary o f 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 o f only 0.016%. A PCNA labeling index of 0.17% has been reported for control rats o f 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 =35/ 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 Period" Endpoint N- N- NEtFOSE EtFOSE EtFOSE Controls 300 ppm 100 ppm 30 ppm PFOS 20 ppm N- Wy- EtFOSA 14,643 100 ppm 100 ppm Cell proliferation - - - - - - Increase Body weight - - - - - - - Liver weight - Increase Increase - - Increase Increase Liver/body wt. - Increase Increase Increase - Increase Increase ALT -- --- - - ALP -- --- - - AST -- --- - - Triglycerides - Decreas - - " Decreas - ee Cholesterol " Decreas Decreas Decreas Decreas Decreas Decreas eeeeee Palmitoyl CoA - "--- - ND oxidase Peroxisomes ------- "An 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 3SZ 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 Period" 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 "An 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 o f 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 o f age, gender, and choice of marker. Fund. Appl. Toxicol. 32:159-167,1996. 053 II. SUMMARY TABLES <3S</ Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 15 Table II-l. Group Summary of Cell Proliferation Data Treatment Group Controls N -EtFO SE 300 ppm N -EtFO SE 100 ppm N -EtFO SE 30 ppm P F O S 20 ppm N -EtFO SA 100 ppm Wy-14,643 100 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% Treatment Group Controls N -EtFO SE 300 ppm N -EtFO SE 100 ppm N -EtFO SE 30 ppm P F O S 20 ppm N-EtFOSA 100 ppm Wy-14,643 100 ppm 1 Week Recovery 1 Week 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 Week Recovery 4 Week Recovery PCNA PCNA Labeling Index Labeling Index Mean SEM 0.016% 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 R at Liver Treatment Group Controls N -EtFO SE 300 ppm N -EtFO SE 100 ppm N -EtF O SE 30 ppm P F O S 20 ppm N -EtFO SA 100 ppm Wy-14,643 100 ppm 48 Hr. PCNA Labeling Index Severity* - 0.7 1.2 0.6 0.8 0.8 6.7 48 Hr. PCNA Labeling Index I n c id e n c e 11 - 0/9 2/10 1/10 1/10 0/10 4/5 7 Day PCNA Labeling Index Severity - 0.7 0.6 0.7 0.8 2.3 2.2 7 Day PCNA Labeling Index Incidence - 0/5 0/5 0/5 0/5 0/5 1/3 14 Day PCNA Labeling Index Severity - 0.5 0.3 1.0 0.5 0.5 0.9 14 Day PCNA Labeling Index Incidence - 0/5 0/5 0/5 0/5 0/5 0/5 Treatment Group Controls N -E tF O SE 300 ppm N -E tF O SE 100 ppm N -E tF O SE 30 ppm P F O S 20 ppm N -E tF O S A 100 ppm W y-14,643 100 ppm 1 Week Recovery 1 Week Recovery PCNA PCNA Labeling Index Labeling Index Severity Incidence -- 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 Week Recovery 4 Week Recovery PCNA Labeling Index PCNA Labeling Index Severity Incidence -- 4.9 2/5 1.4 0/5 4.0 2/5 3.1 2/5 2.9 3/5 1.9 1/5 "Fold increase over controls "Num ber of animals with a P C N A 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 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 h r 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 36.3 33.2 44.4 60.6* 47.6 93.2 ALKALINE PHOSPHATASE (Normal Range: 150 - 380) 48 hr 7 days 14 days 1 week recovery 296.8 263.9 281.3 297.4 286.3 316.5 306.0 287.6 249.8 239.8 244.4 251.6 294.4 287.2 208.3 193.0 198.4 227.0 265.0 273.2 326.0* 184.1 199.0 151.6 233.4* 195.6 176.4 171.0 ASPARTATE AMINOTRANSFERASE (Normal I lange: 70 -125) 48 hr 7 days 14 days 1 week recovery 144.8 155.6 131.3 120.4 123.6 127.3 145.8 136.9 130.9 150.0 135.4 127.6 132.6 134.6 132.6 119.4* 133.6 139.2 141.8 152.2 121.4 173.2 112.4 139.4 123.4 115.0 129.4 164.8 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 I Control N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm j 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 Cform al Range: 50 - 150) I Treatment Group 48 h r 7 days 14 days 1 week recovery I 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 61.1 58.4* 42.0* 47.2 N-EtFOSA 100 ppm 58.4 53.6* 34.8* 38.6* 1Wy-14,643 100 ppm 36.8* 61.0 87.2* 82.8* *Statistically different from controls, P<0.C 5 4 week recovery 48.3 49.4 47.4 52.6 50.2 48.6 66.6 I 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 207.6 g 208.7 g 248.1 g 235.1 g 297.4 g 287.7 g 339.3 g 293.3 g * N-EtFOSE 100 ppm N-EtFOSE 30 ppm 221.1 g 207.9 g 258.6 g 244.2 g 284.4 g 300.0 g 318.0 g 321.9 g PFOS 20 ppm 208.9 g 243.4 g 285.8 g 320.5 g N-EtFOSA 100 ppm Wy-14,643 100 ppm 213.7 g 217.2 g 257.5 g 253.6 g 268.1 g 283.1 g 309.8 g * 289.2 g * MEAN LIVER WEIGHT Treatment Group 48 h r 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 7.514 g 7.878 g 8.515 g* 7.788 g 7.748 g 7.982 g 9.555 g * 8.475 g 9.642 g 9.665 g 8.541 g 8.830 g 10.193 g * 16.087 g * 9.795 g 14.148 g * 11.272 g 11.053 g 10.002 g 10.766 g 19.975 g * 11.321 g 14.118 g * 11.966 g 10.549 g 11.095 g 12.681 g 11.991 g MEAN LIVER W EIGHT TO BODY W EIGHT RATIO I Treatment Group 48 h r 7 days 14 days 1 week recovery IControl N-EtFOSE 300 ppm N-EtFOSE 100 ppm N-EtFOSE 30 ppm PFOS 20 ppm N-EtFOSA 100 ppm Wy-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 aPalmitoyl CoA Oxidase activity, IU/g e2 ^ 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 HI-1. Individual Animal Cell Proliferation Data 48 Hour, 7 Day and 14 Day Sacrifices 48 H our Interim Sacrifice PCNA Treatment Group ! Animal Number Controls 1 R10381 R10382 Labeling Index 0.145% 0.052% R10383 0.081% R10384 0.078% R10385 0.113% R10386 0.086% R10387 0.105% R10388 0.080% R10389 0.027% R10390 0.029% N -E tF O SE 300 ppm R10391 R10392 R10393 0.053% 0.052% 0.057% R10394 0.058% R10395 ND R10396 0.059% R10397 0.026% R10398 0.085% R10399 0.053% R10400 0.026% N -E tF O SE 100 ppm R10401 R10402 0.056% 0.087% R10403 0.190% R10404 0.323% R10405 R10406 0.029% 0.103% R10407 0.000% R10408 0.055% R10409 0.055% N -EtFO SE 30 ppm R10410 R10411 R10412 0.029% 0.152% 0.000% R10413 0.084% R10414 0.055% R10415 0.059% R10416 0.030% R10417 0.000% R10418 0.029% R10419 0.030% R10420 0.028% P F O S 20 ppm R10421 R10422 0.153% 0.053% R10423 0.052% R10424 R10425 R10426 0.086% 0.027% 0.053% R10427 R10428 0.027% 0.027% R10429 0.077% R10430 0.109% N -EtFO SA 100 ppm R10431 R10432 0.089% 0.059% R10433 R10434 0.084% 0.054% R10435 0.000% R10436 R10437 0.029% 0.085% R10438 0.059% R10439 0.114% R10440 0.058% W y-14,643 100 ppm R10441 R10442 1.543% 0.274% R10443 0.326% R10444 0.117% R10445 0.422% 7 Day Interim Sacrifice PCNA Animal Number Labeling Index R10446 0.071% R10447 0.058% R10448 0.030% R10449 0.000% R 10450 0.027% R10451 0.136% R10452 0.058% R10453 0.050% R10454 0.027% R10455 0.000% R10456 0.000% R10457 0.077% R10458 0.027% R10459 0.024% R 10460 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 R10472 R10473 R10474 R10475 0.029% 0.085% 0.026% 0.026% 0.026% R10476 R10477 R10478 R 10479 R10480 R10481 R10482 R10483 R10484 R10485 i ! 0.130% 0.076% i 0.134% 0.077% 0.106% ! ! ND ND 0.078% 0.173% 0.054% 14 D ay Interim Sacrifice PCNA Animal Number Labeling Index R 10486 0.106% R10487 0.190% R 10488 0.332% R10489 0.056% R 10490 0.082% R10491 0.133% R10492 0.056% R10493 0.086% R10494 0.026% R10495 0.117% R 10496 0.119% R10497 0.029% R10498 R10499 0.056% 0.031% R 10500 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 0.290% R10522 ! 0.029% R10523 ! 0.029% R10524 I 0.115% R10525 0.089% ND, not determined due to lack of tissues on slide or suboptimal staining D4.Z- 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 Recovery Sacrifice PCNA Treatment Group Animal Number Labeling Index Controls R10526 0.152% R10527 0.026% R10528 0.028% R10529 0.028% R10530 0.027% R10531 0.150% R10532 0.029% R10533 0.029% R10534 0.000% R10535 0.027% N -EtFO SE 300 ppm R10536 0.059% R10537 0.056% R10538 0.058% R10539 0.030% R10540 0.031% 4 W eek Recovery Sacrifice PCNA Anim ai Number Labeling Index R10566 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 -EtFO SE 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 -EtFO SE 30 ppm R10546 R10547 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% P F O S 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 -EtFO SA 100 ppm R10556 R10557 R10558 R10559 R10560 0.028% 0.090% 0.059% 0.083% 0.057% R10596 R10597 R10598 R10599 R10600 0.031% 0.060% 0.000% 0.061% 0.094% W y-14,643 100 ppm R10561 R10562 R10563 R10564 R10565 i 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 3 0.026% 0.000% 0.126% 0.000% 0.000% Table III-2. Individual Animal Clinical Chemistry 48 Hour Interim Sacrifice Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 24 Treatment Group Anim al Num ber Controls R10381 R10382 R10383 1 R10384 I R10385 R10386 I R 10387 I R10388 R 10389 R 10390 N -E tF O S E 300 ppm R10391 R10392 R 10393 R10394 R10395 R10396 R 10397 R10398 R10399 R10400 N -EtF O SE 100 ppm R10401 R10402 R10403 R10404 R10405 R10406 R10407 R10408 R10409 R10410 N -E tF O S E 30 ppm R10411 R10412 R10413 R10414 R10415 R10416 R10417 R10418 R10419 R10420 P F O S 20 ppm R10421 R10422 R 10423 R 10424 R10425 R10426 R10427 R10428 R10429 R10430 N -E tF O S A 100 ppm R10431 R 10432 R10433 R 10434 R10435 R10436 R10437 R10438 R10439 R10440 W y-14,643 100 ppm R10441 R10442 R10443 R10444 ! R10445 A L T (IU/U :A L P (IU/D 33 ! 259 29 349 38 250 33 293 30 266 29 205 37 316 33 293 20 346 36 391 33 399 27 251 30 318 39 226 33 166 37 322 29 244 25 144 33 322 42 247 35 212 44 273 37 277 36 367 32 275 33 291 31 342 22 255 28 294 23 227 31 297 35 352 51 301 36 268 23 373 21 184 30 306 31 296 26 337 26 260 36 338 48 369 30 256 39 232 31 189 30 303 29 245 27 235 35 208 22 488 34 421 39 435 30 194 34 252 36 397 27 426 26 210 28 307 25 334 23 189 41 443 45 254 33 319 37 256 27 258 A S T (IU/L) T R IG (mg/dl) 138 22 131 57 148 25 163 ; 32 130 60 127 34 218 65 137 41 148 30 108 50 126 22 134 21 134 12 153 51 115 43 102 28 90 27 99 41 132 17 151 41 120 27 137 47 143 37 171 40 169 39 101 49 126 35 92 31 117 40 97 30 153 39 125 19 241 51 123 33 98 98 111 37 182 21 205 46 112 72 108 58 144 33 155 34 103 31 219 40 99 22 149 42 141 17 122 54 127 48 110 23 123 23 171 16 114 27 124 44 123 30 171 27 120 49 121 34 146 23 96 33 147 33 187 43 151 I 39 133 I 37 132 i 75 I C H O L (mg/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 Treatment G roup Controls N -E tF O SE 300 ppm N -E tF O S E 100 ppm N -E tF O SE 30 ppm P F O S 20 ppm N -E tF O S A 100 ppm Wy-14,643 100 ppm I Anim al Num ber R10446 R10447 R10448 R10449 R10450 R10451 R10452 R10453 R10454 R10455 R10456 R10457 R10458 R10459 R10460 R10461 R10462 R10463 R10464 R10465 R10466 R10467 R10468 R10469 R10470 R10471 R10472 R10473 R10474 R10475 R10476 R10477 R10478 R10479 R10480 R10481 R10482 R10483 R10484 R10485 A L T (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 A L P (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 A S T (IU/L) T R IG (mg/dl) 216 67 137 35 152 34 118 38 138 32 178 144 59 216 52 134 61 123 47 121 13 130 15 121 18 168 ! 18 137 27 172 27 132 19 85 44 145 17 104 41 125 47 116 38 103 45 128 23 191 34 126 47 137 19 123 25 138 48 149 43 156 29 177 24 77 76 124 24 129 24 108 21 127 34 131 39 85 38 146 44 tCoO CCOO C H O L (mg/dl) 71 66 71 79 55 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 2 Table HI-2. Individual Animal Clinical Chemistry 14 Day Interim Sacrifice Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 26 Treatment Group Controls N -E tF O S E 300 ppm N -E tF O S E 100 ppm N -E tF O SE 30 ppm P F O S 20 ppm N -E tF O SA 100 ppm Wy-14,643 100 ppm A n im al N u m b e r !A L T (IU/L) A L P (IU/L) R10486 39 169 R10487 45 166 R10488 39 212 R10489 32 226 R10490 36 212 R10491 34 229 R10492 35 282 R10493 37 196 R10494 41 176 R10495 42 215 R10496 37 226 R10497 33 134 R10498 34 134 R10499 57 208 R10500 52 263 R10501 51 284 R10502 33 201 R10503 62 154 R10504 31 148 R10505 45 205 R10506 35 286 R10507 51 149 R10508 45 267 R10509 29 219 R10510 34 214 R10511 50 206 R10512 42 340 R10513 50 230 R10514 43 280 R10515 47 269 R10516 48 220 R10517 63 249 R10518 48 419 R10519 34 238 R10520 29 240 R10521 54 313 R10522 62 234 R10523 63 354 R 10524 49 375 R 10525 75 ! 354 A S T (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 T R IG (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 C H O L (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 Table II1-2. Individual Animal Clinical Chemistry 1 Week Recovery Sacrifice Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 27 Treatm ent G rou p A n im al N u m b e r A L T (IU/L) Controls R10526 57 R10527 34 R10528 59 R 10529 27 R10530 1 34 R10531 27 R10532 30 R10533 32 R10534 29 R10535 35 N -E tF O SE 300 ppm R10536 29 R10537 46 R10538 47 R10539 29 R10540 43 N -E tF O SE 100 ppm R10541 65 R10542 32 R10543 50 R10544 37 R10545 38 N -E tFO SE 30 ppm R10546 34 R10547 32 R10548 40 R10549 43 R10550 43 P F O S 20 ppm R10551 R10552 48 47 R10553 35 R10554 42 R10555 33 N -E tF O SA 100 ppm R10556 37 R 10557 39 R10558 52 R10559 40 R10560 70 Wy-14,643 100 ppm I R10561 57 R 10562 112 R10563 52 R10564 70 R10565 175 A L P (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 A S T (IU/L) ! T R IG (m g/dl) 243 45 114 81 187 52 105 39 106 18 96 27 98 52 71 74 94 29 90 48 158 6 123 31 112 20 78 24 91 39 269 51 119 26 94 23 108 32 107 36 115 29 154 19 127 30 118 37 103 37 125 23 114 31 145 21 111 26 80 37 125 24 138 20 112 17 138 22 134 35 162 51 148 55 123 28 150 44 241 I 27 C H O L (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 27 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 28 Table III-2. Individual Animal Clinical Chemistry 4 Week Recovery Sacrifice Treatment Group Controls N -E tF O SE 300 ppm N -E tF O S E 100 ppm N -E tF O SE 30 ppm P F O S 20 ppm N -EtF O SA 100 ppm Wy-14,643 100 ppm An im al N um ber A L T (IU/L) R10566 40 R10567 R10568 , 38 40 R10569 58 R10570 49 R10571 ND R10572 34 R10573 32 R10574 37 R10575 35 R10576 51 R10577 43 R10578 47 R10579 47 R10580 60 R10581 55 R10582 46 R 10583 47 R10584 57 R10585 48 R10586 54 R10587 31 R10588 40 R10589 35 R10590 48 R10591 46 R10592 36 R10593 49 R10594 38 R10595 43 R10596 51 R10597 103 R10598 125 R10599 35 R10600 73 R10601 42 R10602 33 R10603 38 R10604 43 R10605 38 A L P (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 A S T (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 T R IG (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 C H O L (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 1II-3. Individual Animal Histopathology Findings 48 Hour Interim Sacrifice H isto p a th o lo gy F in d in g s Treatment Group Animal Number Liver Controls R10381 No significant findings R10382 No significant findings R 10383 No significant findings R10384 N o significant findings R10385 No significant findings R10386 No significant findings R10387 No significant findings R10388 No significant findings R10389 No significant findings R10390 No significant findings N -E tF O SE 300 ppm R10391 No significant findings R10392 No significant findings R10393 No significant findings R10394 No significant findings R10395 No significant findings R10396 No significant findings R10397 No significant findings R10398 No significant findings R10399 No significant findings R10400 No significant findings N -E tF O SE 100 ppm R10401 No significant findings i R10402 No significant findings R10403 N o significant findings R10404 N o significant findings R10405 R10406 N o significant findings N o significant findings R10407 N o significant findings R10408 N o significant findings R10409 No significant findings R10410 No significant findings N -E t F O S E 30 ppm R10411 No significant findings R10412 N o significant findings R10413 No significant findings R10414 No significant findings R10415 N o significant findings R10416 N o significant findings R10417 N o significant findings R10418 N o significant findings R10419 No significant findings R10420 No significant findings P F O S 20 ppm R10421 No significant findings R10422 R10423 No significant findings No significant findings R10424 No significant findings R10425 No significant findings R10426 No significant findings R10427 No significant findings R10428 No significant findings R10429 N o significant findings R 10430 N o significant findings N -EtF O SA 100 ppm R10431 N o significant findings R10432 N o significant findings R 10433 N o significant findings R10434 N o significant findings R10435 N o significant findings R10436 No significant findings R10437 No significant findings R10438 N o significant findings R10439 No significant findings R10440 No significant findings W y-14,643 100 ppm R10441 No significant findings R10442 No significant findings R10443 No significant findings R10444 No significant findings R10445 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 G roup Controls N -E tF O SE 300 ppm N -E tF O S E 100 ppm N -E tF O SE 30 ppm P F O S 20 ppm N -E tF O S A 100 ppm W y -14,643 100 ppm H istopathology F in d in g s Anim al Num ber i L iv e r R 10446 i N o significant findings R 1 0447 |N o sign ifican t fin d in gs R 1 0448 N o significant findings R10449 No significant findings R 10450 N o significant findings R10451 N o significant findings R 10452 |N o sign ifican t fin d in gs R 10453 N o significant findings R10454 N o significant findings R 10455 N o significant findings R 10456 N o significant findings R 10457 N o significant findings R10458 N o significant findings R10459 N o significant findings R10460 N o significant findings R10461 N o significant findings R10462 No significant findings R10463 N o significant findings R10464 N o significant findings R10465 N o significant findings R 10466 N o significant findings R10467 N o significant findings R10468 N o significant findings R 10469 N o significant findings R10470 N o significant findings R10471 No significant findings R10472 N o significant findings R10473 N o significant findings R10474 N o significant findings R10475 N o significant findings R10476 N o significant findings R 10477 N o significant findings R10478 N o significant findings R10479 No significant findings R10480 N o significant findings R10481 N o significant findings R 10482 N o significant findings R 10483 N o significant findings R10484 R 10485 N o significant findings i N o significant findings 3-70 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 31 Table HI-3. Individual Animal Histopathology Findings 14 Day Interim Sacrifice Treatm ent G rou p Controls N -EtF O SE 300 ppm N -EtFO SE 100 ppm N -EtFO SE 30 ppm P F O S 20 ppm N -EtFO SA 100 ppm W y-14,643 100 ppm H istopathology Fin d ings Anim al N um ber L iv e r R10486 lipid vacuolization, lipid, m inim al R10487 lipid vacuolization, lipid, m inim al R10488 N o significant findings R10489 lipid vacuolization, lipid, m inim al; a g g re g a te s, m ixe d inflam m ato ry cell, multifocal, m inim al R10490 lipid vacuolization, lipid, m inim al; a g g re g a te s, m ix e d inflam m ato ry cell, multifocal, m inim al i R10491 N o Significant Findings R10492 lipid vacuolization, lipid, m inim al R10493 lipid vacuolization, lipid, m inim al; a g g re g a te s, m ix e d inflam m ato ry cell, multifocal, m inim al R10494 lipid vacuolization, lipid, m inim al R10495 lipid vacuolization, lipid, m inim al; a g g re g a te s, m ixe d inflam m ato ry cell, multifocal, m inim al R10496 lipid vacuolization, lipid, mild R10497 lipid vacuolization, lipid, m ild R10498 lipid vacuolization, lipid, mild R10499 lipid vacuolization, lipid, mild R10500 lipid vacuolization, lipid, mild R10501 lipid vacuolization, lipid, mild; a g g re g a te s, m ixe d inflam m ato ry cell, multifocal, m inim al R10502 lipid vacuolization, lipid, mild R10503 lipid vacuolization, lipid, mild; a g g re g a te s, m ixe d inflam m ato ry cell, multifocal, m inim al R10504 N o significant findings R10505 lipid vacuolization, lipid, mild; a g g re g a te s, m ix e d inflam m ato ry cell, multifocal, m inim al R10506 lipid vacuolization, lipid, mild R10507 lipid vacuolization, lipid, mild R10508 lipid vacuolization, lipid, mild R10509 lipid vacuolization, lipid, m inim al R10510 lipid vacuolization, lipid, m inim al R10511 lipid vacuolization, lipid, m inim al; a g g re g a te s, m ixe d inflam m ato ry cell, multifocal, m inim al R10512 N o significant findings R10513 N o significant findings R10514 N o significant findings R10515 lipid vacuolization, lipid, m inim al R10516 lipid vacuolization, lipid, mild R10517 lipid vacuolization, lipid, m inim al R10518 lipid vacuolization, lipid, m inim al R10519 lipid vacuolization, lipid, m inim al R10520 lipid vacuolization, lipid, m inim al R10521 hypertrophy, K up ffe r cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, m inim al R10522 hypertrophy, K up ffe r cell, mild; hepatocellular ne crosis, multifocal, minimal; lipid vacuolization, lipid, m inim al R10523 hypertrophy, K upffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, m inim al R10524 hypertrophy, K u p ffe r cell, mild; lepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, m inim al R10525 hypertrophy, K upffer cell, mild; hepatocellular necrosis, multifocal, minimal; ipid vacuolization, lipid, m inim al; a ggre ga te s, m ixe d inflam m atory cell, multifocal, m inim al 2*7/ Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 32 Table IH-3. Individual Animal Histopathology Findings 1 Week Recovery Sacrifice Treatment Group Controls N -EtFO SE 300 ppm N -E tF O S E 100 ppm N -E tF O SE 30 ppm P F O S 20 ppm N -E tF O S A 100 ppm Wy-14,643 100 ppm 1 Histopathology Findings ; Anim al Num ber Liver R10526 No significant findings R10527 lipid vacuolization, lipid, mild R10528 lipid vacuolization, lipid, mild; aggregates, mixed inflammatory cell, multifocal, minimal I R10529 ! R10530 lipid vacuolization, lipid, mild lipid vacuolization, lipid, mild; aggregates, mixed inflammatory cell, multifocal, minimal R10531 lipid vacuolization, lipid, minimal R10532 lipid vacuolization, lipid, minimal R10533 lipid vacuolization, lipid, minimal R10534 lipid vacuolization, lipid, minimal R10535 lipid vacuolization, lipid, mild R10536 lipid vacuolization, lipid, mild; aggregates, mixed inflammatory cell, multifocal, minimal R10537 lipid vacuolization, lipid, mild R10538 lipid vacuolization, lipid, mild R10539 lipid vacuolization, lipid, mild R10540 lipid vacuolization, lipid, minimal R10541 lipid vacuolization, lipid, minimal R10542 lipid vacuolization, lipid, minimal; aggregates, m ixed inflammatory cell, multifocal, minimal R10543 lipid vacuolization, lipid, mild R10544 lipid vacuolization, lipid, minimal R10545 lipid vacuolization, lipid, minimal R10546 lipid vacuolization, lipid, mild R10547 lipid vacuolization, lipid, minimal R10548 lipid vacuolization, lipid, minimal R10549 lipid vacuolization, lipid, minimal R10550 lipid vacuolization, lipid, minimal R10551 lipid vacuolization, lipid, minimal R10552 lipid vacuolization, lipid, minimal R10553 R10554 lipid vacuolization, lipid, minimal lipid vacuolization, lipid, minimal; aggregates, m ixed inflammatory cell, multifocal, minimal R10555 lipid vacuolization, lipid, minimal R10556 lipid vacuolization, lipid, mild R10557 No significant findings R10558 No significant findings R10559 lipid vacuolization, lipid, minimal R10560 lipid vacuolization, lipid, minimal R10561 hypertrophy, Kupffer cell, mild; hepatocellular necrosis, multifocal, minimal; lipid vacuolization, lipid, minimal; spleen, follicular hyperplasia, moderate R10562 hypertrophy, Kupffer cell, mild; lipid vacuolization, lipid, minimal R10563 hypertrophy, Kupffer cell, mild; lipid vacuolization, lipid, minimal R10564 hypertrophy, Kupffer cell, mild; lipid vacuolization, lipid, minimal R10565 hypertrophy, Kupffer cell, mild; lipid vacuolization, lipid, minimal cSLZA Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 33 Table III-3. Individual Animal Histopathology Findings 4 Week Recovery Sacrifice T reatment G roup Controls N -E tF O SE 300 ppm N -E tF O SE 100 ppm N -E tF O S E 30 ppm P F O S 20 ppm N -E tF O SA 100 ppm W y-14,643 100 ppm H istopath ology F in din gs Anim ai Num ber Liver R10566 lipid vacuolization, lipid, mild lipid vacuolization, lipid, mild; R10567 aggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R 10568 lipid vacuolization, lipid, m inim al R 10569 lipid vacuolization, lipid, mild R10570 lipid vacuolization, lipid, m inim al R10571 a g g re g a te s, m ixed inflam m atory cell, m ultifocal, m inim al R10572 lipid vacuolization, lipid, m inim a! R10573 a ggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R10574 lipid vacuolization, lipid, m inim al lipid vacuolization, lipid, m inim al; R10575 a g gre ga te s, m ixed inflam m atory cell, multifocal, m inim al R 10576 lipid vacuolization, lipid, mild R10577 lipid vacuolization, lipid, mild R10578 lipid vacuolization, lipid, m inim al lipid vacuolization, lipid, mild; R10579 a ggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R10580 lipid vacuolization, lipid, m inim al R10581 lipid vacuolization, lipid, mild R10582 lipid vacuolization, lipid, m inim al R10583 No significant findings R 10584 No significant findings lipid vacuolization, lipid, mild; R 10585 a ggre ga te s, m ixed inflam m atory cell, multifocal, m inim al lipid vacuolization, lipid, m inim al; R10586 a ggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R 10587 No significant findings R 10588 lipid vacuolization, lipid, m inim al R 10589 lipid vacuolization, lipid, m inim al R 10590 No significant findings R10591 a g g re g a te s, m ixed inflam m atory cell, multifocal, m inim al R10592 lipid vacuolization, lipid, m inim al R 10593 lipid vacuolization, lipid, m inim al lipid vacuolization, lipid, m inim al; R10594 aggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R10595 a g g re g a te s, m ixed inflam m atory cell, multifocal, m inim al R 10596 a ggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R10597 lipid vacuolization, lipid, m inim al R10598 N o significant findings R10599 N o significant findings lipid vacuolization, lipid, m inim al; R10600 aggre ga te s, m ixed inflam m atory cell, multifocal, m inim al R10601 N o significant findings R10602 N o significant findings R10603 N o significant findings lipid vacuolization, lipid, m inim al; testes, sperm granulom s; R10604 epididymis, sperm granulom a R10605 N o significant findings c2 7 3 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 34 APPENDIX 1 - IN-LIFE REPORT <27 / Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 35 V. APPENDIX 2 - ELECTRON MICROSCOPY REPORT Z7S 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 W ITH 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 PRO JECT NUMBER EM 99.62 SUMMMARY An increase in the mean number o f 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 o f 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. 274 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) 1 Contro 1 (0 ppm) 10 N-EtFOSE PFOS 30 10 30 20 0 0___ PPm .. ppm 10 10 10 10 NEtFOSA 100 ppm 10 Wy-14,643 100 ppm 5 Total No. of Animals 65 (48 hrs) 2 (7 days) 10 5 5 5 5 5 5 40 3 (14 days) 10 5 5 5 5 5 5 40 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 50 30 30 30 Animals 30 30 25 225 Anima s in Groups 1 through 3 received the test diet 'or 48 hours, 7 days, and 14 days, respectively. 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 o f 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. 2 .7 ? 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 R10403 R10404 R10421 R10430 R10431 R10439 R10441 R10443 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 o f 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 o f 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 o f 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 o f peroxisomes. 2 -7 $ Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 39 Text Table 3. Quantitation of Hepatocellular Peroxisomes Treatment Group Timepoint Control 48 hour N-EtFOSE 100 ppm 48 hour PFOS 20 ppm 48 hour N-EtFOSA 100 Ppm Wy-14,643 100 PP 1 48 hour 48 hour Animal Number R10384 R10388 R10403 R10404 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 o f 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 1132-100 iiitapriisng Faiholrfgtti i M g rtM W ;>4 ilute Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 41 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: j m i t f f i M M Animal No,: Treatment Group:..Control.,...... Sex:-- U ilf----- ---------------- S p ecie^S ttiin :& | iiS am ilA i Tissue Liver Block NX) 217351 * PboifVNcgative NofsL:...Z 17355...... laesiOMS{cheek *): _____ _ Yes ...... X......No Interpreting PMhologtst (signature and dale): Features: ZI735I. Lis*: HegMOcyie. IU.32UX, Tin*hepawcjrte'l*a entailrosndnucleus with tvm ptmiiitcfit uelmlt Tie cytoplasm is finely granular andcontains many itodwudrii and HER profile* There aw also abiatdaat lysosonic* and some peroxisome. Several bile carutlu-uli arc prosent between ibis 11 tnd adpsenl bcpatocytcs. At the right matgts is dark* staining fcepaiocyle, and a dnusotrltil mtbrycyie is peeaent in a sinusoid at the bottom of the photograph. Twelve (I2tpe>xi.*iitt 011011**1 K17352, Liver, Hepatoqrte. 10,32i>?i. Nittncrou* miiocbodria awl HER profiles arc present 'n tits hepatocyre. Its mwfats is polyp#! and centrally Incared. At the right margin are two darker hepatoeyws that appear m have soinewhm latter and mat nttroerons mltochondrisi.. Random lari peroxvxMttts ami ($$41*0* are present in the eyiplsni. Fifteen i JS) ptfuxi*AMtes emjtueJ, Z11ATV Uvcf. Hepaiocyie 1CL321IK. O# die kft a linn botde.r tif emimhelia! cell lining mbum ! Is present. 11 twpwocyw let* ,1 veaiml msmi use lews aftd the eycoptam contain many niiwelfrsdm, R68 profite. and several pale-staining lipal droplet*. S<tm {lerosisonsestmd lysoMnne* a also pteseiti. Five <5s peroxisome* counted. 71T314, Liver. Hejxwytie. UL320X. 11 oemral tepatoeyte- A bptdeted by )wputney* 0 . most margin* aifliiMtgh a sugmeot of simmnil ts pres on the felt iwjth erythrocyte) and bottom, Scverai bile vuiiaHasii ate prevent Imwwm the centta! and botJetiug hepatccyie* Several small lipkl ihojjfeb arc prase. NtBiwnt* dark aJ fxwxuHMuoase .prevent. Twcaty-thnee i2 i) pern*isoares ewanied. ?srmmMie% Jysomme* and some mfuchcmdris were wi> ditTicub toUd'fc!?i.,;v ;-it.;* t-lKnograpn 7X1211, f n . r . Hetv.iyvUr. HU'bX. litis L e p ro tic 4u:au:rs t..i 1 ttredlirev-vieed lipid Jreplctv SsmisauA .. - w : at ds t.tf .n 4 hi 4 ?i. p?H<`<.>ei.rei!. tilled by paler .u-im.;.*n:l<^l>,,liiil cells (i,ii.:.n t.*`-p:rusi*<-ini-s-..i,.kJ, _______ Cnwditsteiws! Normal hepatocyte. Mean of 13.6 peroxisomes pet bepetocyte camited. ^/ Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 42 TRANSMISSION ELECTRON M IC R O APH INTERPRETATION Study No.: 1132-100 (EM99.62L... Animal No. RI403.............. ...... Treatment Group: J tiM S S - M m Set; ,, Timm: _ JLfaffl Block m m .: 9.62-23 zm m Photo/Ncgative Note),:....217360..... Significant Lesions (cheek one): ............... Yes .... X .... No interpreting Pathologist (signature m date): 4 ----- Sept- 29. 1999 .. li ^ ^ This bcpatocyte contains approximately ten small- to mediuni-si2ed lipid droplets. The cytoplasm has numerous mitochondria and RER profiles present. Bile canalcult are present between this cell and adjacent hepatocytes on the left and the right. Erythrocytes in sinusoids are present on three margins. Some stain dfc&rls/artifact is present on the photograph. Eight (8 ) peroxisomes counted. Z 17337. Liver. Hpatocyte. 10.320X. This bepatocyte is himielealcd, An endothelial cell is present at the top, but all other margins are adjacent hpatocytes. The cytoplasm contains numerous mitochondria and RER profiles. One distinct Jtpid droplet is present, and there arc numerous smalt to medium-sited dark peroxisomes and/or lysosomes. Twenty-three (23) peroxisomes counted. Z 17358. Liver. Hpatocyte. I0.320X. A nucleus is not evident in this plane of section for this hepatocyte. Four tnediuro-sbed lipid droplets are present, and the other organelles appear very similar to Z17357. Some lysosomes contain clear vacuoles within them and many are irregularly shaped. Thirteen (13) peroxisomes counted. Z17357. Liver, Hpatocyte. I0.320X. Hpatocyte contains one large lipid droplet. Twenty-throe <23) peroxisomes counted. Some nain debos/artifact present on the photograph. Z 17360. Liver. Hepatocyte. 1032OX. M u ltiple sm all- to m edium -sixed lip id droplets are present. Sixteen ( 16) peroxisomes counted. Conclusions: Normal hepanxyto. 0.0 average tvrcv\i\*roes per hepatocyte. K. 8F Study Number: TRC 1132-100 Amended Final Report Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 44 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No. : 1132-100 (EM99.62) Animal No.: R10421_________ Treatment Group: PFOS 20t>pm Sex: Male________________ Species/S train :Cr1:CDiSDl IGS 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 lysosom es 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. Z17368. Liver. Hepatocyte. 10,320X. In this hepatocyte the mitochondria appear smaller and more condensed. Profiles o f RER and SER are easily evident in the cytoplasm. Five (5) peroxisomes counted. Z17369. 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. Study Number: TRC 1132-100 Amended Final Report Features: Z17S11. Hepatoeyte. 10,32fiX, Iltb hcpatocyie has an eccentrically located oval nucleus, It is bordered by an sinusoid at the top id right and another hepatoeyte on the left. Numerous mitochomlria, RER profiles, finely granular cytosol, ami a few small lipid droplets sue present in the cytoplasm. Some smiUI lysosotttesarc scattered in the cytoplasm Five (5) Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 46 TRANSMISSION ELECTRON MICROGRAPH INTERPRETATION Study No.: 1132-100 OEM99.62) Animal No.: R10431___________ Treatment Group: N-EtFOSA lOOppm Sex: Male_________________ _ Species/S train :Crl:CDfSDl IGS BR Rat Tissue: Liver_____________ Block No(s).: 99.62-51________ Z17376 -Z17380 Photo/Negative NofsL: 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. Z17377. Liver. Hepatocyte. 10,320X. This hepatocyte stains somewhat darker than the preceding example. The cytoplasm is finely granular and mitochondria appear denser. Peroxisomes and som e lysosom es 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 o f 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/S train :Crl:CD(SDl 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 1EM99.621 Animal No.: R10441__________ Treatment Group: Wv-14.643 lOQppm Sex: Male__________________ S p ecies/S train :Crl:CD(SDt IGS BR Rat Tissue: Liver_____________ Block No(s).: 99.62-61________ Z17386- Photo/Negative No(sb: Z17390 Significant Lesions (check one): X Yes ________ No Interpreting Pathologist (signature and date): ____________________ Oct. 01,1999 Features: Z17386. Liver. Hepatocyte. 10,320X. This plate o f 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 o f 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 o f 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 (EM99.621 Animal No.: R10443________ Treatment Group: Wv-14.643 lOQppm Sex: Male__________________ Species/Strain:Crl:CD(SDt IGS BR Rat Tissue: Liver_____________ Block No(s).: 99.62-63________ Z17391 - Photo/Negative NotsV: 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 o f 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: TRC 1132-100 Amended Final Report Page 51 Table VI-1. Individual Animal Palmitoyl CoA Oxidase Activity Treatm ent Group Controls N-EtFOSE 300 ppm N -B FO SE 100 ppm N-EtFOSE 30 ppm PFO S 20 ppm N-EtFOSA 100 ppm A 48 nim al HN uomu rbInetre riPmCSOaAcOri*ficAec tiv ity R10381 9 R10382 9 R10383 6 R10384 6 R10385 5 R10386 6 R10387 8 R103B8 11 R10389 7 R10390 8 R10391 7 R10392 101 R10393 10 R10394 16 R10395 6 R10396 7 R10397 7 R10398 10 R10399 8 R10400 10 R10401 5 R10402 8 R10403 10 R10404 9 R10405 9 R10406 4 R10407 7 R10408 8 R10409 12 R10410 9 R10411 10 R10412 9 R10413 13 R10414 9 R10415 6 R10416 7 R10417 9 R10418 8 R10419 12 R10420 9 R10421 11 R 10422 8 R10423 8 R10424 6 R10425 8 R10426 6 R10427 6 R10428 10 R10429 11 R10430 8 R10431 6 R10432 5 R10433 9 R10434 6 R10435 6 R10436 5 R10437 11 R10438 7 R10439 6 R10440 6 A n im 7 al DNauymInbteerrimPCSOaAc Orifi*cAe c tiv ity 14 Day Interim Sacrifice Anim al Number PCOAO* A ctivity R10446 8 R10486 5 R10447 4 R10487 6 R10448 5 R10488 5 R10449 5 R10489 5 R 10450 5 R1Q490 8 R10451 5 R10491 6 R10452 4 R10492 7 R10453 5 R10493 9 R 10454 5 R10494 5 R10455 7 R10495 7 R10456 R10457 _______ _______ 3 R10496 R10497 2 4 R10458 6 R10498 2 R10459 6 R10499 3 R10460 10 R10500 3 R10461 6 R10501 2 R10462 7 R10502 5 R10463 9 R10503 R10464 4 R10504 3 R10465 8 R10505 4 R10466 5 R10506 5 R10467 6 R 10507 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 R10476 7 R10516 2 R10477 3 R10517 4 R10478 8 R10518 2 R10479 6 R10519 4 R10480 4 R10520 2 1 Week Recovery Sacrifice Anim al Number I PCOAO* A ctivity R10526 i 6 R10527 9 R10528 7 R10529 5 R10530 10 R10531 10 R10532 7 R10533 8 R10534 3 R10535 5 R 10536 3 R10537 5 R10538 5 R10539 3 R10540 6 R10541 R10542 R10543 R10544 R10545 6 4 4 5 5 R10546 R10547 R10548 R10549 R10550 5 5 8 4 8 R10551 R10552 R10553 R10554 R10555 6 6 5 8 3 R10556 R10557 R10558 R10559 R 10560 6 6 6 6 7 Palmitovf CoA Oxidase activity, IU/q =27/ 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 Dim sw y md ikmikipimmi Services Iktihoiogy Amtiaies 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 W o m a n 's Mill Court, Suite I * Frederick, Maryland 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 ms 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 o f 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) of 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 -277 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 58 Husbandry Housing: Diet: Water: Contaminants: Environment: Acclimation: Randomization: Justification: 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 svstem or water bottles. The water is analyzed at least two times per year for contaminants and specific microbes. The results o f 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 o f 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 of 7 days prior to the start o f 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 o f randomization, the weight variation o f the animals of 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 o f the extensive historical data base, and the FDA requirements for a rodent species. 2 .9 8 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 59 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points dumber 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. 27? 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 o f recovery. Food Consumption Weekly for Week 1 through 4 weeks o f 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. 300 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 3o/ 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 o f the liver and any macroscopic lesions o f 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 o f 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 o f liver samples from 35 rats and 35 guinea pigs. Tissue Collection for Electron Microscopic Evaluation 3oZ 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 o f the time points, as well as the 4 week recovery, for a total o f 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 o f 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 363 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 o f 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. 3a V 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 30S 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 o f 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 o f weighed diet, mix for 30 minutes. 336 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 of 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 70C3C 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. 3o7 Date: February 24,1999 PROTOCOL AMENDMENT #1 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 68 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. 36 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 of the liver from all animals... 3oj 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 l) 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 3/6 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 3 // 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 o f 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 o f 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 3/2 Date: May 30,2000 PROTOCOL AMENDMENT #4 Study Number: TRC 1132-100 Sponsor: 3M Study Number: TRC 1132-100 Amended Final Report Page 73 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 Responsibility of 3M > 5 years room temp. amber waxy solid 11990d 98% > 2 years room temp. 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 3 /3 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 /.s y. / / Sandra R. Eklndar. Ph.O .V Date Principle Investigator Pathologv Associates Division of Charles River Laboratories, Inc. Y 314-