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AR226-2811 DuPont-13788 Trade S ecret Review of Proliferative Lesions of the Exocrine Pancreas in Two Chronic Feeding Studies in Rats with Ammonium Perfluorooctanoate Authors Steven R. Frame, D.V.M, Fh.D, DACVP Ernest E. McConnell, D.V.M., M.S. (Pathology), DACVP, DABT Date October 16,2003 Laboratory Location EJ . DuPont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project Identification DuPont-13788 Page 1 o f 8 Company Sanitized. Does not contain TSC A CBI DuPont-13788 Page Reserved S tatement of Confidentiality This document is the property o f E.I. du Pont de Nemours and Company and contains confidential and trade secret information. Except as required by law, this document should not be partially or fully (i) photocopied or released in any form to an outside party without the prior written consent o f E.I. du Pont de Nemours and Company or its affiliates, or (ii) used by a registration authority to support the registration of any other product without the prior written consent o f E.I. du Pont de Nemours and Company or its affiliates. 2 Company Sanitized. Does not contain TSCA CBI S ignature Page DuPont-13788 Author Signature: Author Name: Author Position: Author Address: Date: J Steven R. Frame, D.V.M., Ph.D., DACVP Principal Research Pathologist and Research Manager Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.0. Box 50 Newark, Delaware 19714-0050 ?3 Author Signature: Author Name: Author Position: Author Address: Date. / y @03 Ernest E. McConnell, D.V.M., M.S. (Pathology), DACVP, DABT President, Tox Path, Inc. Tox Path, Inc. Raleigh, North Carolina 9 ---------------------------------------------------------------------------------- ----------------------------------- 3 Company Sanitized. Does not contain T SC A C81 DuPont-13788 Table of Contents Title Page..................................................................... 1 Statement of Confidentiality....................................................................................... 2 Signature Page.............................................................................................................. 3 Table o f Contents......................................................................................................... 4 1.0 Introduction and Methods...................................................................................5 2.0 Results and Discussion....................................................................................... 6 3.0 Conclusions......................................................................................................... 7 4.0 References........................................................................................................... 8 Company Sanitized. Does not contain TSCA CBI 4 DuPont-13788 Review of Proliferative Lesions of the Exocrine Pancreas in Two Chronic Feeding S tudies in Rats with A mmonium Perfluorooctanoate 1.0 Introduction and Methods As part o f study investigating the relationship between peroxisome-proliferating compounds and tumorigenesis in the rodent liver, testis, and pancreas, male CD rats were exposed to ammonium perfluorooctanoate (APFO) for 2-years at dietary concentrations of 300 ppm (Biegel et al., 2001). This study was conducted at DuPont's Haskell Laboratory during 1991 and 1992. Among the findings in that study were increased incidences o f pancreatic acinar cell hyperplasia and adenoma in APFO-treated rats. These results were in apparent contrast to a previous chronic feeding study with APFO in which the same rat strain was exposed to the same dietary concentration of 300 ppm (3M, 1987). In^hat study, no compoundrelated proliferative lesions of the exocrine pancreas were identified. This latter study was sponsored by the 3M company and was conducted between 1981 and 1983. In an effort to address the apparent discrepancies between the two studies, the following activities were undertaken sequentially: Slides of the pancreas from the 3M study were reviewed by Dr. Steven R. Frame, a veterinary pathologist at DuPont (the study pathologist for the DuPont Study). This review focused on slides from male tats in the control and 300 ppm (high dose) groups o f the 3M study. At the request of 3M, pancreas slides (control and treated) from the DuPont study were reviewed by Dr. Ernest E. McConnell, a consulting veterinary pathologist. Dr. McConnell then reviewed all pancreas slides from the 3M study. Drs. Frame and McConnell jointly reviewed selected pancreas slides from both the 3M and DuPont studies. This review included pancreas slides from the DuPont study stained with 5-bromo-2'-deoxyuridine (BrdU) to detect increased cell proliferation. The slide reviews outlined above were conducted to help clarify the findings relative to pancreatic acinar cell lesions in the two chronic feeding studies with APFO. These reviews were not, however, designed as exhaustive pathology peer reviews. Thus, not all slides from all groups were examined but only those deemed necessary to assess and compare the effects on the exocrine pancreas in the two studies. The reviewing pathologists were in agreement regarding the general conclusions of these reviews (see Results and Discussion below). Company Sanitized. Does not contain TSCA CBI 5 2.0 Results and Discussion DuPont-13788 Based on die review of Dr. McConnell, the conclusions o f the DuPont study were confirmed. Specifically, chronic exposure to 300 ppm APFO was associated with increased incidences o f pancreatic acinar cell hyperplasia and adenoma o f the pancreas in male rats. Dr. McConnell confirmed all acinar cell lesions diagnosed as acinar cell neoplasia (adenoma or carcinoma). He also confirmed an increased incidence o f hyperplasia in APFO-exposed rats. Based on the joint review by Drs. McConnell and Frame, exposure o f male rats to 300 ppm APFO in the 3M study was associated with a slight increase in acinar cell hyperplasia but not adenoma or carcinoma. Thus, APFO was not a pancreatic tumorigen in die 3M study. The reviews of both the 3M and DuPont studies indicate that die results o f the two studies are more similar than dissimilar when considered in the context o f the pathogenesis and diagnostic criteria established for proliferative lesions of the exocrine pancreas of the rat. Proliferative lesions o f the acinar pancreas are thought to exist along a continuum o f hyperplasia, adenoma and carcinoma. However, die likelihood o f progression or regression along that continuum is unknown in most cases, and typically there are no cytological features that clearly distinguish hyperplasia from adenoma (Hansen et al., 1995; Eustis et al., 1990). Indeed, some authors have used terms such as hyperplasia and adenomatous hyperplasia to describe nonmalignant acinar cell lesions (Greaves and Faccini, 1992). To achieve some uniformity in diagnoses across laboratories, diagnostic criteria have been established to differentiate acinar cell hyperplasia from adenoma, and the criterion most commonly employed is the somewhat arbitrary feature of two-dimensional size o f the lesion (Hansen et al., 1995; Eustis and Boorman, 1990). Thus, for many acinar cell proliferations, the difference between the diagnoses of hyperplasia and adenoma reflects a difference in the two dimensional size of a random section through the lesion rather than known differences in biological potential. Based on these considerations it is apparent that the results of the DuPont and 3M studies are qualitatively similar, as APFO produced increased incidences of proliferative acinar cell lesions o f the pancreas in both studies. The differences observed were quantitative, that is, more and larger focal proliferative acinar cell lesions were produced in the DuPont study compared to the 3M study. The reason for this quantitative discordance despite use of die same cat strain and dietary concentration o f APFO (300 ppm) ip the two studies is not known. However, there are a number of variables that could contribute to the differences observed. These studies were conducted approximately 10 years apart and in different laboratories. It is expected that these temporal and locational variables were associated with some differences in animal husbandry, including diet, as well as potential genetic drifts within the rat strain. O f all possible factors, a difference in the diets is the most likely factor influencing the different incidences o f 6 Company Sanitized. Does not contain TSCA CBI DuPont-13788 proliferative acinar cell lesions. Various forms of dietary manipulation, including feeding raw soy diets or diets deficient in choline, are known to moderate die incidences o f acinar cell hyperplasia and neoplasia in the pancreas of rodents (Longnecker and Millar, 1990; McGuinness et al., 1980). The incidences o f acinar cell hyperplasia and adenoma were increased five fold in control male F344 given com oil by gavage compared to rats not gavaged (Eustis and Boorman, 1985). In considering the human relevance of acinar cell hyperplasia and neoplasia in die rodent, it is noteworthy that the histological type o f tumor seen in the rodent is distinctly different from tumors o f the exocrine pancreas most commonly observed in humans. While rodent tumors are typically derived from acinar cells, the majority of human pancreatic neoplasms are of the ductular type (Longnecker and Millar, 1990). True ductular neoplasms o f the pancreas are rare in rats (Greaves and Faccini, 1992). In addition, pancreatic acinar cell hyperplasia and adenoma have been produced in rats by a number o f compounds which, like APFO, produce peroxisome proliferation in the liver. These include pharmaceuticals widely used in humans, such as the hypolipidemic agent, clofibrate. There is no known association between these compounds and tumors of the exocrine pancreas in humans. Thus, while the precise mechanism of formation and significance of pancreatic acinar cell proliferations in rats are not known, they probably do not indicate a cancer hazard for humans (Greaves, 2000) . 3.0 Conclusions Microscopic lesion observed in the exocrine pancreas in two chronic feeding studies in rats, conducted by 3M and DuPont respectively, were reviewed by two veterinary pathologists. These reviews were conducted to clarify apparent differences between the two studies relative to compound-related lesions of the exocrine pancreas; however, a formal pathology peer review of the pancreas from all study animals was not conducted. The results o f the two studies were similar in that APFO produced proliferative lesions of pancreatic acini in both studies at dietary concentrations of 300 ppm. In the 3M study, treatment-related changes were limited to slight increases in acinar cell hyperplasia. In the DuPont study, increased incidences of hyperplasia and adenoma were observed. Thus, these studies produced qualitatively similar responses in the pancreas, with the differences noted being quantitative-higher overall incidences o f proliferative lesions and greater tendency for progression o f lesions to adenoma were observed in the DuPont study compared to the 3M study. Since the difference between pancreatic acinar hyperplasia and adenoma in the rat is often a reflection of arbitrary diagnostic criteria (size) and not necessarily biological potential, the results of the two studies are in reasonable concordance given the experimental variables that exist between them. Although the basis for the quantitative difference observed is not known, a number of variables, especially diet, are known to modulate the proliferative response of die acinar pancreas in rats and are likely operative in the differences in degree of response seen in the two chronic studies with APFO. Despite these differences, target organ effects on the pancreas, characterized by focal proliferative lesions o f pancreatic acini, were common to both the 3M and DuPont studies. 7 Company Sanitized. Does not contain TSCA CBI 4.0 References DuPont-13788 Biegel LB, Hurtt ME, Frame SR, O'Connor JC, and Cook JC (2001). Mechanisms o f extrahepatic tumor induction by peroxisome proliferators in male CD rats. Toxicol Sci. 60:44-55. Eustis, SL and Boorman, GA (1990). 8. Exocrine pancreas. In: Pathology o fthe Fischer Rat (GA Boorman, SL Eustis, M R Elwell, CA Montgomery, Jr, and WF Mackenzie, eds.). Academic Press, Inc., San Diego, pp 95-108. Eustis SL and Boorman GA (1985). Proliferative lesions of the exocrine pancreas: relationship to com oil gavage in the National Toxicology Program. JN atl.C ancer Inst. 75:1067-73. Greaves, P (2000). Chapter VII. Digestive system 1. Exocrine pancreas. In: Histopathology o fPreclinical Toxicity Studies, Second Edition, Elsevier, Amsterdam, pp. 490-514. Greaves, P and Faccini, JM (1992). Digestive system. Pancreas. In: Rat Histopathology, Second Edition, Elsevier, Amsterdam, pp.l 18-126 Hansen, IF, Ross, PE, Makovec, GT, Eustis, SL, and Sigler, RE (1995). Proliferative and other selected lesions o f the exocrine pancreas in rats. GI-6. In: Guidesfo r ToxicologicPathology. STP/ARP/AFIP, Washington, DC. Longnecker, DS and Millar, PM (1990). Tumours o f the pancreas. In: Pathology o f Tumors in Laboratory Animals. Volume I - Tumours o fthe Rat, Second Edition (VS Turusov and U Mohr, eds). International Agency for Research on Cancer, Lyon, pp. 241-249. McGuinness EE, Morgan RG, Levison DA, Frape DL, Hopwood D, and Wormsley KG. (1980). The effects o f long-term feeding o f soya flour on the rat pancreas. Scand J Gastroenterol, 15:497-502. 3M (1987). TVvo Year Oral (Diet) Toxicity/Carcinogenicity Study ofFC-143 in Rats. Ricker Experiment No. 0281CR0012. Company Sanitized. Does not contain TSCA CBS 8