Document LJR782eygN68dg86Vdy3LnqqQ

m Z 'O ift Pathology Associates International A Company of Science Applications International Corporation SECOND DRAFT CELL PROLIFERATION REPORT 104-WEEK DIETARY CHRONIC TOXICITY AND CARCINOGENICITY STUDY W ITH PERFLUOROOCTANE SULFONIC ACID POTASSIUM SALT (PFOS;T-6295) IN RATS COVANCE STUDY NUMBER 6329-183 PREPARED FOR: 3M TOXICOLOGY SERVICES BUILDING 220-2E-02, 3M CENTER ST. PAUL, MN 55144-1000 PREPARED BY: PATHOLOGY ASSOCIATES INTERNATIONAL 15 WORMAN'S M ILL COURT, SUITE I FREDERICK, MD 21701 AUGUST 24, 1999 5 Worman's Mill Court, Suite I * Frederick, Maryland 21701 * (301) 663-1644 * (301) 663-8994 FAX 002375 TABLE OF CONTENTS Contents Narrative Tables Figures Signature Page Quality Assurance Statement Appendix Second D raft Cell Proliferation Report C ovance Study N um ber 6329-183 Section I n m IV V VI Page 1-2 002376 Second Draft Cell Proliferation Report Covance Study Number 6329-183 CELL PROLIFERATION REPORT 104-W EEK DIETARY CHRONIC TOXICITY AND CARCINOGENICITY STUDY WITH PERFLUOROOCTANE SULFONIC ACID POTASSIUM SALT (PFOS;T6295) IN RATS COVANCE STUDY NUMBER 6329-183 PURPOSE The purpose of the study was to assess the chronic toxicity and carcinogenicity of the test material when administered in the diet to rats for at least 104 weeks. This report, submitted by Pathology Associates International (PAI) to the study Sponsor, 3M, represents the cell proliferation findings and interpretation for Covance Study Number 6329-183 entitled " 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Rats". All aspects of the tasks associated with PAI's portion of this study were conducted in compliance with the Environmental Protection Agency Good Laboratory Practice (GLP) Regulations as set forth in Title 40 of the US Code of Federal Regulations, Part 792, issued November 29,1983 (effective December 29,1983), and any applicable amendments with the following exception. An expired reagent was used in the BrdU immunohistochemistry staining procedure (mouse IgG, Vector, lot # PK-4002, PAI No. K351, exp. 4/9/99). The staining was judged to be satisfactory; therefore, this GLP deviation does not impact the scientific validity of the study. MATERIALS AND METHODS Proliferating Cell Nuclear Antigen (PCNA) Staining and Evaluation Tissue Collection for Cell Proliferation Five animals per sex per group in groups 1 through 5 were sacrificed at weeks 4 and 14 for hepatocellular proliferation. A section of the left lateral lobe of the liver from each of 5 rats per group (groups 1-5) was fixed in zinc formalin, processed, and embedded to paraffin block by Covance per protocol specifications. Tissue blocks were shipped to PAI for sectioning and staining. From each block, a slide was prepared for H&E evaluation and immunohistochemical detection of proliferating cell nuclear antigen (PCNA), an endogenous marker of cell proliferation. Immunohistochem istry for Cell Proliferation Sections of paraffin-embedded tissues were cut at 5 (im and placed on positively charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) to ensure adhesion during processing for PCNA. Standard immunohistochemical methods were used to stain tissues for PCNA [PAI's Standard Operating Procedure for Immunohistochemical Staining (SOP #707)]. Briefly, tissue sections were incubated with a monoclonal antibody to PCNA (DAKO, lot #016; PAI No. A1899) and reagents required for the avidin-biotin peroxidase (ABC Kit; Vector, lot #PK-6100; PAI No. K 327) method for the detection of the antigen-antibody complex. PCNA expression in cells in all phases of the cell cycle (G,, S, G2and M) was localized by the chromagen 3,3'-diaminobenzidine (DAB; Sigma Chemical Co., lot #18H8201). Tissue sections were counterstained with hematoxylin. Page 1-3 002377 Cell Proliferation M easurements Second Draft Cell Proliferation Report Covance Study Number 6329-183 The percentage of proliferating cells (proliferating index, PI) was determined by scoring at least 2000 hepatocytes in at least 6 fields of liver. In most cases, at least 3000 hepatocytes were scored in 10 fields, but in four animals there was insufficient tissue present on the slide to be able to score as many cells. Slides were first perused at low magnification (100X) to judge quality of staining, processing and sectioning, and pattern of cell labeling (e.g., centrilobular or panlobular, extrahepatocellular proliferation, such as Kupffer cells, be duct epithelium, endothelium), and histomorphologic changes. The latter was further assessed by evaluating the serial H&E slide for each animal evaluated for cell proliferation. Cell proliferation was then quantified at higher magnification (200X) as described above. A negative control slide was included in the staining run and consisted of study tissue from animal #C93008 that was not incubated with the primary antibody. Statistical Analysis The Student's f-test (two-sided, unequal variance) was used to test for statistical significance in PI between control and treatment groups using Microsoft Excel version 5.0. A P value of 0.05 was judged to be statistically significant. Bromodeoxvuridine (BrdU) Staining and Evaluation Tissue Collection for Cell Proliferation Seven days before the week 52 interim sacrifice, osmotic pumps containing 20 mg/ml BrdU were surgically implanted in five animals per sex per group in groups 1 and 5 for hepatocellular proliferation. A section of the liver and duodenum from each of 5 rats per group (groups 1 and 5 only) was taken at necropsy and embedded to paraffin block by Covance per protocol specifications. Tissue blocks were shipped to PAI for sectioning and staining. From each block, a slide was prepared for H&E evaluation and immunohistochemical detection of BrdU, an exogenous marker of cell proliferation. Im m unohistochem istry for Cell Proliferation Sections of paraffin-embedded tissues were cut at 5 |im and placed on positively charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) to ensure adhesion during processing for BrdU. Standard immunohistochemical methods were used to stain tissues for BrdU [PAI's Standard Operating Procedure for Immunohistochemical Staining (SOP #707)]. Briefly, tissue sections were incubated with a monoclonal antibody to BrdU (Becton Dickenson, lot #81604; PAI No. A2089) and reagents required for the avidin-biotin peroxidase (ABC Kit; Vector, lot #PK6100; PAI No. K351) method for the detection of the antigen-antibody complex. BrdU incorporation into cells in S phase of the cell cycle was localized by the chromagen 3,3'diaminobenzidine (DAB; Sigma Chemical Co., lot #108H8210). Tissue sections were counterstained with hematoxylin. Cell Proliferation M easurements The percentage of BrdU-labeled cells (labeling index, LI) was determined by scoring at least 2000 hepatocytes in 10 fields of liver. Slides were first perused at low magnification (100X) to judge quality of staining, processing and sectioning, pattern of cell labeling (e.g., centrilobular or panlobular, extrahepatocellular proliferation, such as Kupffer cells, bile duct epithelium, endothelium), and histomorphologic changes. The latter was further assessed by evaluating the Page 1-4 002378 Second Draft Cell Proliferation Report Covance Study Number 6329-183 H&E slide for each animal evaluated for cell proliferation. Cell proliferation was then quantified at higher magnification (200X) as described above. A negative control slide was included in the staining run and consisted of study tissue from animal #C92534 that was not incubated with the primary antibody. Statistical Analysis The Student's /-test (two-sided, unequal variance) was used to test for statistical significance in LI between control and treatment groups using Microsoft Excel version 5.0. A P value of 0.05 was judged to be statistically significant. RESULTS PCNA Cell Proliferation Individual animal and group mean PCNA cell proliferation data are presented in Section II (Tables 1-4) and graphically in Section HI (Figures 1 and 2). The negative control tissue was judged to be free from background staining. At 4 and 14 weeks on study, the percentage of proliferating hepatocytes (PI) in treated animals did not differ significantly from control animals for either sex. BrdU Cell Proliferation Individual animal and group mean BrdU cell proliferation data are presented in Section II (Table 5) and graphically in Section HI (Figure 3). The negative control tissue was judged to be free from background staining. The percentage of labeled hepatocytes (LI) in treated animal groups did not differ significantly from control animals for either sex at 52 weeks on study. H isto p ath o lo g y Sections from the same tissue blocks used for preparation of PCNA- and BrdU-stained slides were stained with hematoxylin and eosin (H&E) for histopathologic evaluation to facilitate the interpretation of the immunostained slides. Individual animal findings and group summaries are presented in Appendix I. The results showed that no significant changes were observed in the liver from male and female rats, which would alter the interpretation of the PCNA or BrdU staining in this study. D IS C U S S IO N In the present study, cell proliferation was measured by PCNA immunohistochemistry within the liver of male and female rats from control (Group 1), low dose (Group 2), mid dose (Group 3), mid-high dose (Group 4), and high dose (Group 5) groups after 4 and 14 weeks on study, and by BrdU immunohistochemistry in the liver from control (Group 1) and high dose (Group 5) male and female rats after 52 weeks on study. The decision to use BrdU labeling to detect proliferating cells at 52 weeks was based on the increased sensitivity of continuous labeling with BrdU compared to PCNA in detecting a proliferative response in the liver of rats. In this case, BrdU was administered via osmotic pump continuously for 7 days. A statistically significant increase in cell proliferation, as determined by the PCNA proliferating index (PI) at 4 and 14 weeks, or by the BrdU labeling index at 52 weeks, was not observed in the liver of male or female rats. Although statistical analysis did not reveal a significant cell proliferative response to the test material, biological significance was evident in individual animals. Biological significance for an individual animal is defined as a doubling in PI or LI over controls Page 1-5 002379 Second Draft Cell Proliferation Report Covance Study Number 6329-183 and greater than the highest control value. Thus, the data are presented in terms of severity (fold increase over controls) and incidence (number of animals showing biological significance). As seen in Text Table 1, biologically significant cell proliferation was evident in 2/5 male rats at the high dose after 4 weeks on study and in 1/5 male rats at the mid-high dose and mid dose after 4 weeks on study. This apparent and minimal proliferative response seen in male rats at 4 weeks was less than the control PI at 14 weeks, and was not sustained as evidenced by the lack of a response at week 14. Furthermore, none of the animals exhibited a proliferative response at 52 weeks. Therefore, the proliferative responses seen in these individual male animals was a possible mild response evident only at 4 weeks. Text Table 1. Biologically Significant Cell Proliferation in Liver of Male Rats Group Week 4 Week 14 Week 52 Severity1 Incidence2 Severity1 Incidence2 Severity1 Incidence2 Low dose <1 0/5 <1 0/5 - - Mid dose 1.6 1/5 <1 0/5 - - Mid-high dose 1 .0 1/5 <1 0/5 - - High dose 1.5 2/5 <1 0/5 <1 0/4 `Fold increase over controls -- 2Number of animals with a PI or LI double the mean control value and greater than the highest control animal In contrast to male rats, biologically significant cell proliferation was evident in female rats at the mid-high dose (2/5 rats) and at the high dose (2/5 rats) after 4 weeks, and at the low dose (2/5 rats) and mid dose (1/5 rats) after 14 weeks on study, as seen in Text Table 2. The degree of the proliferative response observed in females was greater than that seen in the males. The proliferative response observed at week 4 was not sustained after 14 weeks, nor was a proliferative response observed at 52 weeks. Based on the severity and incidence of the proliferative response seen in the high dose females, and the dose response relationship seen at 4 weeks, the test material is judged to induce a transient proliferative response in the liver of female rats at the high dose. Text Table 2. Biologically Significant Cell Proliferation in Liver of Female Rats Group Week 4 Week 14 Week 52 Severity1 Incidence2 Severity1 Incidence2 Severity1 Incidence2 Low dose 1 .0 0/5 2.6 2/5 - - Mid dose 1.1 0/5 1.2 1/5 - - Mid-high dose 1.8 2/5 <1 0/5 - - High dose 3.5 2/5 <1 0/5 <1 0/5 `Fold increase over controls 2Number of animals with a PI or LI double the mean control value and greater than the highest control animal Interestingly, a rare pattern of PCNA-labeling was observed in high dose female rats at week 14, which was not seen in any other groups, nor at the earlier time point. The staining was characterized by a granular cytoplasmic staining pattern localized to the hepatocytes around central veins of the liver. The significance of this type of labeling pattern is not known; however, it has been speculated that it may represent mitochondrial DNA synthesis (unpublished observation). Recently, it has been shown that PCNA can cross-react with peroxisomal multifunctional protein (Mayer, et al., 1998). Thus, it is possible that the centrilobular staining pattern observed in the high dose animals may be associated with peroxisomes. Page 1-6 002380 Second Draft Cell Proliferation Report Covance Study Number 6329-183 The cell proliferation rates in the week 14 male rats were all higher than those in the week 4 male rats. In the female rats, cell proliferation rates at week 14 were higher than at week 4 in the control, low dose and mid dose groups, but not in the mid-high or high dose groups. The reason for the consistently higher cell proliferation rates in the week 14 animals is not known. However, all slides from the 4 and 14 week time points were stained together, so variation in staining between runs can be ruled out. One possibility is that the tissues were handled differently in terms of fixation and processing, which may have affected the staining. Despite this difference, comparisons can be made between each treatment group and the control group for a specific time point. SUMMARY The cell proliferation seen in the liver of male rats at 4 weeks was not dose-related, was minimal in severity (high dose was less than two-fold greater than controls), occurred in two of five animals in the high dose group, and was transient in nature (i.e., was not sustained at 14 weeks). In female rats, cell proliferation in the liver was dose-related at week 4 but not at week 14, greater in severity compared to male rats (greater than two-fold at the high dose), and occurred in two of five animals in both the high and mid-high dose groups. Based on these findings, cell proliferation effects seen at 4 weeks in the rats were considered to be related to test material. At 52 weeks, there was no evidence of hepatocellular proliferation in male or female rats at the high dose. LITERATURE CITED Mayer, D., Forstner, K., Beier, K., and Volkl, A. (1998). Monoclonal antibodies against proliferating cell nuclear antigen cross-react with the peroxisomal multifunctional protein. Anal Biochem256: 135-137. Page 1-7 002381 Second Draft Cell Proliferation Report Covance Study Number 6329-183 II. TABLES Legend BrdU, bromodeoxyuridine LI, labeling index, percentage of cells in S phase of the cell cycle as determined by BrdU staining ND, not determined PCNA, proliferating cell nuclear antigen PI, proliferating index, percentage of cells in S, G l, G2 and M phases of the cell cycle as determined by PCNA staining SEM, standard error of the mean 002382 COVANCE STUDY NO. 6329-183 TABLE 1. PCNA CELL PROLIFERATION IN LIVER OF MALE RATS AFTER 4 WEEKS ON STUDY G roup 1 - 0 ppm PFOS (Control) 1 - 0 ppm PFOS (Control) 1 - 0 ppm PFOS (Control) 1 - 0 ppm PFOS (Control) 1 - 0 ppm PFOS (Control) 2 - 0.5 ppm PFOS (Low) 2 - 0.5 ppm PFOS (Low) 2 - 0.5 ppm PFOS (Low) 2 - 0.5 ppm PFOS (Low) 2 - 0.5 ppm PFOS (Low) 3 - 2.0 ppm PFOS (Mid) 3 - 2.0 ppm PFOS (Mid) 3 - 2.0 ppm PFOS (Mid) 3 - 2.0 ppm PFOS (Mid) 3 - 2.0 ppm PFOS (Mid) 4 - 5.0 ppm PFOS (Mid-High) 4 - 5.0 ppm PFOS (Mid-High) 4 - 5.0 ppm PFOS (Mid-High) 4 - 5.0 ppm PFOS (Mid-High) 4 - 5.0 ppm PFOS (Mid-High) 5 - 20.0 ppm PFOS (High) 5 - 20.0 ppm PFOS (High) 5 - 20.0 ppm PFOS (High) 5 - 20.0 ppm PFOS (High) 5 - 20.0 ppm PFOS (High) Sex M M M M M M M M -M M M M M M M M M M M M M M M M M j j Anim al I Number | C92506 C92524 C92526 C92529 C92546 Mean SEM C92574 C92575 C92610 C92613 C92618 Mean SEM C92646 C92650 C92652 C92668 C92678 Mean SEM C92715 C92718 C92731 C92734 C92744 Mean SEM C92752 C92759 i C92779 ! C92793 C92798 Mean SEM PCNA P ro life ra ting Index* 0 .0 2 7 % 0.000% 0.130% 0.000% 0.053% 0 .042% 0 .0 2 4% 0.000% 0.027% 0.054% 0.080% 0 .0 2 7 % 0 .038% 0 .014% 0 .0 2 6 % 0.027% 0.027% 0.098% 0 .1 6 6 % 0 .0 6 9 % 0 .028% 0.136% 0 .0 5 3 % 0.000% 0 .0 2 7 % 0 .0 0 0 % 0 .0 4 3 % 0 .0 2 5 % 0.027% 0.027% 0.000% 0.136% 0.133% 0 .065% 0 .029% 'P roliferating cell nuclear antigen; percentage of cells in G 1, S, G2 and M phases Page 11-1 0023S3 COVANCE STUDY NO. 6329-183 TABLE 2. PCNA CELL PROLIFERATION IN LIVER OF FEMALE RATS AFTER 4 WEEKS ON STUDY G roup | Anim al Sex I Num ber PCNA P ro life ra ting Index* 1 - 0 ppm PFOS (Control) F i C92861 0 .0 8 0 % 1 - 0 ppm PFOS (Control) F C92864 0 .1 3 2 % 1 - 0 ppm PFOS (Control) F C92914 0 .0 0 0 % 1 - 0 ppm PFOS (Control) 1 - 0 ppm PFOS (Control) F C92919 ND F C92926 0 .0 0 0 % Mean 0.0 5 3% SEM 0 .0 3 2% 2 - 0.5 ppm PFOS (Low) F C92941 0 .0 2 7 % 2 - 0.5 ppm PFOS (Low) F C92945 0 .0 5 6 % 2 - 0.5 ppm PFOS (Low) F C92950 0 .0 2 7 % 2 - 0.5 ppm PFOS (Low) F C92969 0 .1 1 0 % 2 - 0.5 ppm PFOS (Low) F C92982 0 .0 5 3 % Mean 0 .0 5 5% SEM 0.0 1 5% 3 - 2.0 ppm PFOS (Mid) F C92996 0 .0 5 4 % 3 - 2.0 ppm PFOS (Mid) F C93006 0 .0 5 3 % 3 - 2.0 ppm PFOS (Mid) F C93008 0 .1 0 8 % 3 - 2.0 ppm PFOS (Mid) F C93031 0 .0 2 8 % 3 - 2.0 ppm PFOS (Mid) F C93045 0 .0 5 4 % Mean 0 .0 5 9% SEM 0 .0 1 3% 4 - 5.0 ppm PFOS (Mid-High) F I C93058 0 .1 8 6 % 4 - 5.0 ppm PFOS (Mid-High) F C93067 0 .1 6 1 % 4 - 5.0 ppm PFOS (Mid-High) F C93070 0 .0 2 7 % 4 - 5.0 ppm PFOS (Mid-High) F C93075 0 .0 0 0 % 4 - 5.0 ppm PFOS (Mid-High) F C93084 0 .1 1 0 % Mean 0.0 9 7% SEM 0.0 3 6% 5 - 20.0 ppm PFOS (High) F C93114 0 .0 5 6 % 5 - 20.0 ppm PFOS (High) F C93123 0 .0 2 7 % 5 - 20.0 ppm PFOS (High) F C93142 0 .3 4 9 % 5 - 20.0 ppm PFOS (High) F C93144 0 .4 3 0 % 5 - 20.0 ppm PFOS (High) F C93159 0 .0 5 4 % I Mean 0.1 8 3% i SEM | 0.0 8 5% Proliferating cell nuclear antigen; percentage of cells in G1, S, G2 and M phases Page II-2 0023S4 COVANCE STUDY NO. 632-0-183' TABLE 3. PCNA CELL PROLIFERATION IN LIVER OF MALE RATS AFTER 14 WEEKS ON STUDY PCNA G ro u p Anim al P ro life ra ting Sex N um ber Index* 1 - 0 ppm PFOS (Control) M C92509 0 .3 2 3 % 1 - 0 ppm PFOS (Control) M C92511 0 .5 2 1 % 1 - 0 ppm PFOS (Control) M C92521 0 .5 8 8 % 1 - 0 ppm PFOS (Control) M C92528 0 .1 3 7 % 1 - 0 ppm PFOS (Control) M C92532 0 .2 4 7 % Mean 0.3 6 3% SQM 0 .0 8 4% 2 - 0.5 ppm PFOS (Low) M C92593 0 .0 8 4 % 2 - 0.5 ppm PFOS (Low) M C92600 0 .0 8 1 % 2 - 0.5 ppm PFOS (Low) M C92616 0 .2 4 0 % 2 - 0.5 ppm PFOS (Low) M C92621 0 .2 9 6 % 2 - 0.5 ppm PFOS (Low) M C92627 0 .1 8 8 % Mean 0.1 7 8% SEM 0 .0 4 2% 3 - 2.0 ppm PFOS (Mid) M C92640 0 .1 3 6 % 3 - 2.0 ppm PFOS (Mid) M C92645 0 .5 6 1 % 3 - 2.0 ppm PFOS (Mid) M C92662 0 .2 4 2 % 3 - 2.0 ppm PFOS (Mid) M C92676 0 .1 6 1 % 3 - 2.0 ppm PFOS (Mid) M C92684 0 .1 0 8 % Mean 0.2 4 2% SEM 0 .0 8 3% 4 - 5.0 ppm PFOS (Mid-High) M C92713 0 .1 3 7 % 4 - 5.0 ppm PFOS (Mid-High) M C92714 0 .4 6 6 % 4 - 5.0 ppm PFOS (Mid-High) M C92719 0 .1 0 7 % 4 - 5.0 ppm PFOS (Mid-High) M C92722 0 .5 4 2 % 4 - 5.0 ppm PFOS (Mid-High) M C92728 0 .4 2 1 % Mean 0.3 3 5% SEM 0 .0 8 9% 5 - 20.0 ppm PFOS (High) M C92765 0 .5 6 0 % 5 - 20.0 ppm PFOS (High) M C92777 0 .3 5 2 % 5 - 20.0 ppm PFOS (High) M C92789 0 .0 2 7 % 5 - 20.0 ppm PFOS (High) M C92799 0 .0 5 6 % 5 - 20.0 ppm PFOS (High) M C92812 0 .0 5 3 % Mean 0.2 1 0% SEM 0.1 0 6% ;i I' ' Proliferating cell nuclear antigen; percentage of cells in G1, S, G2 and M phases Page II-3 0023S5 COVANCE STUDY NO. 6329-183' TABLE 4. PCNA CELL PROLIFERATION IN LIVER OF FEMALE RATS AFTER 14 WEEKS ON STUDY A nim a l G roup Sex ! Num ber 1 - 0 ppm PFOS (Control) F C92880 1 - 0 ppm PFOS (Control) F C92887 1 - 0 ppm PFOS (Control) F C92898 1 - 0 ppm PFOS (Control) F C92903 1 - 0 ppm PFOS (Control) F C92905 Mean SEM 2 - 0.5 ppm PFOS (Low) F C92944 2 - 0.5 ppm PFOS (Low) F C92962 2 - 0.5 ppm PFOS (Low) F C92967 2 - 0.5 ppm PFOS (Low) F C92978 2 - 0.5 ppm PFOS (Low) F C92987 Mean SEM 3 - 2.0 ppm PFOS (Mid) F C92993 3 - 2.0 ppm PFOS (Mid) F I C93000 3 - 2.0 ppm PFOS (Mid) F i C93018 3 - 2.0 ppm PFOS (Mid) F I C93023 3 - 2.0 ppm PFOS (Mid) F C93035 Mean SEM 4 - 5.0 ppm PFOS (Mid-High) F C93051 4 - 5.0 ppm PFOS (Mid-High) F C93064 4 - 5.0 ppm PFOS (Mid-High) F C93071 4 - 5.0 ppm PFOS (Mid-High) F C93085 4 - 5.0 ppm PFOS (Mid-High) F C93109 Mean SEM 5 - 20.0 ppm PFOS (High) F C93111 5 - 20.0 ppm PFOS (High) F i C93127 5 - 20.0 ppm PFOS (High) F C93143 5 - 20.0 ppm PFOS (High) F C93155 5 - 20.0 ppm PFOS (High) F C93169 Mean SEM PCNA P ro life ra tin g Index* 0 .0 8 2 % 0 .1 6 1 % 0 .1 3 7 % 0 .0 2 8 % 0 .0 8 5 % 0.0 9 9% 0.0 2 3% 0 .1 1 0 % 0 .8 2 6 % 0 .2 1 5 % 0 .0 8 0 % 0 .0 5 4 % 0.2 5 7% 0.1 4 5% 0 .0 8 1 % 0 .0 5 4 % 0 .0 5 4 % 0 .3 5 6 % 0 .0 5 3 % 0 .1 2 0% 0 .0 5 9% 0 .0 5 4 % 0 .0 8 2 % 0 .0 5 7 % 0 .0 2 7 % 0 .0 5 3 % 0 .0 5 5% 0 .0 0 9% 0 .0 2 8 % 0 .1 4 0 % 0 .0 8 1 % 0 .0 0 0 % 0 .0 8 4 % 0.0 6 7% 0 .0 2 4% ' Proliferating cell nuclear antigen; percentage of cells in G 1, S, G2 and M phases Page II-4 0023S6 COVANCE STUDY NO. 6329-183 TABLE 5. BRDU CELL PROLIFERATION IN RAT LIVER AFTER 52 WEEKS ON STUDY Treatm ent Group 1 1 1 1 1 5 5 5 5 1 1 1 1 1 5 5 5 5 5 Gender Male Male Male Male Male Male Male Male Male Female Female Female Female Female Female Female Female Female Female Anim al Number C92504 C92512 C92534 C92562 C92570 Mean SEM C92778 C92795 C92796 C92817 Mean SEM C92876 C92889 C92899 C92923 C92930 Mean SEM C93112 C93120 C93154 C93163 C93171 I Mean SEM B rdll Labeling Index* 0.51% 0.19% 0.09% 0.26% 0.24% 0 .2 6 % 0 .0 7 % 0.08% 0.31% 0.09% 0.38% 0 .2 2 % 0 .0 8 % 6.94% 1.23% 1.64% 1.40% 0.11% 2 .2 6 % 1 .2 0 % 0.26% 0.11% 0.03% 0.27% 0.13% 0 .1 6 % 0 .0 5 % Brom odeoxvuridine; percentage of cells in S phase Page II-5 002387 Second Draft Cell Proliferation Report Covance Study Number 6329-183 III. FIGURES 0023S8 0.5 y0.45 - 0.4 FIGURE 1. PCNA CELL PROLIFERATION IN LIVER OF MALE RATS Group 1 Group 2 Group 3 Group 4 Group 5 5 0 ppm 0.5 ppm 2.0 ppm 5.0 ppm 20.0 ppm 0B Page 111-1 0.5 j 0.45 - 0.4 - XLU 0.35 Q Z 0.3 0z 5 0.25 - ffi 0.2 O GC CL 0.15 - FIGURE 2. PCNA CELL PROLIFERATION IN UVER OF FEMALE RATS Week 4 Week 14 O O Group 1 Group 2 Group 3 Group 4 Group 5 10 CO 0 ppm 0.5 ppm 2.0 ppm 5.0 ppm 20.0 ppm ioO Page III-2 FIGURE 3. BRDU CELL PROLIFERATION IN LIVER OF RATS AFTER 52 WEEKS Page HI-3 Second Draft Cell Proliferation Report Covance Study Number 6329-183 IV. SIGNATURE PAGE Submitted by: PAI Project Manager: Sandra R. Eldridge, Ph.D. PAI Project Pathologist: Date Carolyn Moyer, D.V.M., Diplomate, A.C.V.P. Date 002392 Second Draft Cell Proliferation Report Covance Study Number 6329-183 V. QUALITY ASSURANCE STATEMENT 002393 Pathology Associates International A Company of Science Applications International Corporation AnEmptoym OwntdCctrtpany Cell Proliferation Report 104-Week Dietary Chronic Toxicity and Carcinogenicity Study With Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Rats Covance Study No.: 6329-183 QUALITY ASSURANCE STATEMENT This cell proliferation project has been inspected and audited by the PAI Quality Assurance Unit (QAU) as required by the Good Laboratory Practice (GLP) regulations promulgated by the U. S. Environmental Protection Agency (EPA-TSCA). The cell proliferation report is an accurate reflection of the recorded data. The following table is a record of the inspections/audits performed and reported by the QAU. Date of Inspection Phase Inspected Date Findings Report to PAI Management/ Project Manager/Pathologist 08/18/98 10/07/98 10/07/98 05/10/99 08/20,24/99 Labeling (100% slide/block match) Study Data and Supporting Documentation Draft Cell Proliferation Report Staining and Coverslipping Second Draft Cell Proliferation Report 08/18/98 10/08/98 10/08/98 05/10/99 08/24/99 Karen E. Butler Quality Assurance Specialist Date 002394 4 15 Worman's Mill Court, Suite I Frederick, Maryland 21701 (301) 663-1644 (301) 663-8994 FAX Second Draft Cell Proliferation Report Covance Study Number 6329-183 VI. APPENDIX 002395 Covancc Study Number 6329-183 Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 4 Weeks on Study Tissue: Liver Animal Number C92506 C92524 C92526 C92529 C92546 C92574 C92575 C92610 C92613 C92618 C92646 C92650 C92652 C92668 C92678 C92715 C92718 C92731 C92734 C92744 C92752 C92759 C92779 C92793 C92798 Sex Dose G roup M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) Histologic Findings Liver No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings Page VI-1 002396 Covanec Study Number 6329-183 Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 4 Weeks on Study Tissue: Liver Animal Number C92861 C92864 C92914 C92919 C92926 C92941 C92945 C92950 C92969 C92982 C92996 C93006 C93008 C93031 C93045 C93058 C93067 C93070 C93075 C93084 C93114 C93123 C93142 C93144 C93159 Sex Dose Group F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) Histologic Findings Liver No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings Page VI-2 002397 ' Covance Study Number 6329-183 Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 14 Weeks on Study Tissue: Liver Animal Number C92509 C92511 C92521 C92528 C92532 C92593 C92600 C92616 C92621 C92627 C92640 C92645 C92662 C92676 C92684 C92713 C92714 C92719 C92722 C92728 C92765 C92777 C92789 C92799 C92812 Sex Dose Group M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 1-0 ppm PFOS (Control) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 2-0.5 ppm PFOS (Low) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 3-2.0 ppm PFOS (Mid) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 4-5.0 ppm PFOS (Mid-high) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) M 5-20.0 ppm PFOS (High) Histologic Findings Liver No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings Page VI-3 002398 Covance Study Number 6329-183 Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 14 Weeks on Study Tissue: Liver Animal Number C92880 C92887 C928J8 C92903 C95905 C92944 " C92962 C92967 C92978 C92987 C92993 C93000 C93018 C93023 C93035 C93051 C93064 C93071 C93085 C93109 C93111 C93127 C93143 C93155 C93169 Sex Dose Group F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 1-0 ppm PFOS (Control) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 2-0.5 ppm PFOS (Low) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 3-2.0 ppm PFOS (Mid) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 4-5.0 ppm PFOS (Mid-high) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PFOS (High) F 5-20.0 ppm PpQS (High) Histologic Findings Liver No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings Page VI-4 002399 Covance Study Number 6329-183 Individual Animal Findings 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS;T-3295) in Rats Histopathologic Findings after 52 Weeks on Study Tissue: Liver Animal Number C92562 C92512 C92534 C92504 C92570 C9276 C92899 C92923 C92930 929 C92795 C92817 C92778 C92796 C93171 C93112 C93120 C93154 C$3163 Sex Dose Group M 1-0 ppm PFOSE (Control) M 1-0 ppm PFOSE (Control) M 1-0 ppm PFOSE (Control) M 1-0 ppm PFOSE (Control) M 1-0 ppm PFOSE (Control) F 1-0 ppm PFOSE (Control) F 1-0 ppm PFOSE (Control) F 1-0 ppm PFOSE (Control) F 1-0 ppm PFOSE (Control) F 1-0 ppm PFOSE (Control) M 5-20.0 ppm PFOSE (High) M 5-20.0 ppm PFOSE (High) M 5-20.0 ppm PFOSE (High) M 5-20.0 ppm PFOSE (High) F 5-20.0 ppm PFOSE (High) F 5-20.0 ppm PFOSE (High) F 5-20.0 ppm PFOSE (High) F 5-20.0 ppm PFOSE (High) F 5-20.0 ppm PFOSE (High) Histologic Findings Liver No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings No Significant Findings Page VI-5 002400