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DuPont! 3788 Reviewop Proliferative Lesions of the Exocrine Pancreas in Two Chronic Feeding Studies in Rats w ith A mmonium Perfluorooctanoate . Authors Steven R. Frame, D.V.M, PkD, DACVP Ernest E. McConnell, D.VJM, M.S. (Pathology), DACVP, DABT D ots October 16,2003 Laboratory Location E l. DuPont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, F.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project Identification DuPont-13788 Page lof8 DuPont-13788 Page Reserved STATEMENT OF CONFIDENTIALITY This document is the property o f E.L du Pont ds Nemours and Company and contains confidential and trade secret information. Except as required by law, this document should not bo partially or folly (1)photocopied or released in any form to an outside party without the prior written consent o f EX du Pont de Nemours and Company or its affiliates, or (ii) used by a registration authority to support the registration'ofany other product without foe prior written consent o f E.I. du Pont de Nemours and Company or its affiliates. 2. S ig n a tu re Pa g e DuPont-13788 A uthor Signatures A uthor Nam e! A uthor Position: A uthor Addressi D ate; M b / - q 3 Steven R. Frame, D.V.M., Eh.D., DACVP Principal Research Pathologist and Research Manager Haskell Laboratory for Health and Environmental Sciences EUrton Road, P.O. Box 50 Newark, Delaware 19714-0050 Author Signature; Author Nam et Author Position: A uthorAddress: if? *, D ate: Ernest E McConnell, D.V.M., M.S. (Palhology), DACVP, DABT President, Tox Path, Inc. Tox Path, Ino. Raleigh, North Carolina 3 DuPont-13788 Table of Contents Title Page.................................................................................................................. 1 Statement o f Confidentiality......... .......................................................................... 2 Signature Page..... !,.....................,........................ ..............................................,,,.,3 Table of Contents......................... ........... ................,,,,..........................................4 1.0 Introduction and Method*................. 5 2.0 Results and Discussion................................... ,, .................................... 6 '3.0 Conclmrfons................. ...................... 7 4.0 References.......................................................................................................8 4 DuPont-13788 Review of proliferative lesions of the Exocrine Pancreas in TwoC hronic Feeding Studies in Rats with Ammonium P erfluorooctanoate 1.0 Introduction and Methods compounds and tumorigenesis in the rodent liver, testis, and pancreas, male CD rats ware exposed to ammonium perfluorooctenoate (APFO) for 2-years at dietary concentrations o f300 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 fpancreatic 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 that study, no compoundrelated proliferative lesions o f the exocrine pancreas were identified. This latter study was sponsored by die 3M company and was conducted between 1981 and 1983. In an effort to address the apparent discrepancies between die two studies, the following activities were undertaken sequentially; * Slides o f 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 rats in the control and 300 ppm (high dose) groups of the 3M study. A t the request of 3M, pancreas slides (control and treated!) from tile DuPont study were reviewed by Dr. Ernest E. McConnell, a consulting veterinary pathologist Dr. McCmmell then reviewed all pancreas slides from the 3M study, Drs. Frame and McConnelljointly reviewed selected pancreas slides from both the 3M and DuPont studies. This review included pancreas slides from die 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 AFFO. 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 ofthese'reviews (see Results and Discussion below). 5 2.0 Results and Discussion DuPont-13788 Based on the 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 ofpancreatic acinar cell hyperplasia and fainTM o f the pancreas in male tats. Dr. McConnell confirmed all acinar cell lesions diagnosed as acinar cell neoplasia (adenoma or carcinoma). He also confirmed an increased incidence of hypoplasia in APFO-exposedrats, Based on thejoint 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 of carcinoma. Thus, APFO was not a pancreatic tumorigen in the 3M study. The reviews o fboth the 3M and DuPontstudies indicate that the results o f the two. studiesare more similar than dissimilar when considered in die context o f the pathogenesis and diagnostic criteria established for proliferative lesions o f fire exocrine pancreas of fire ra t Proliferative lesions o f the acinar pancreas are thought to exist along a continuum ofbyperplasta, adenoma and carcinoma. However, the likelihood o f progression or regression along that continuum is unknown inm ost cases, and typically there are no cytologjcal features that dearly distinguish hyperplasia from adenoma (Hansen et al., 1995; Eustis et al., 1990). Indeed, someauthors have used terms such as hyperplasia and adenomatous hyperplasia to describe nonmalignant acinar cell lesions (Greaves and Faccini, 1992). To achieve same uniformity in diagnoses across laboratories, diagnostic criteria have been established to differentiate acinar cell hyperplasia from tlimnitu, and the criterion most commonly employed is the somewhat arbitrary feature o f two-dimensional Sire of the lesion (Hansen et al., 1995; Eustis and Boorman, 1990), Thus, for many acinar cell proliferations, the difference between fiie diagnoses o f hypoplasia and adenoma reflects a difference in the twodimensional size o f a random section through the lesioh 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 o f the same rat strain and dietary concentration o f APFO (300 ppm) in the two studies is not known. However, there are a number of variables that could contribute to file differences observed. These studies were conducted approximately!0 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 fectors, a difference in the diets is fiie most likely factor influencing the different incidences o f 6 DuPont-13788 proliferative acinar cell lesions, Various forms o f dietary manipulation, mcludi-ng feeding raw soy diets or diets deficient in choline, are known to moderate the incidences o f acinar cell hyperplasia and neoplasia in the pancreas of rodents (Longnecksr 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), fir considering foe human relevance of acinar cell hyperplasia and neoplasia in the 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 o f human pancreatic neoplasms are o f the dnntular type (Longnecker and Millar, 1990). True ductular neoplasms o f the pancreas are rare in rats (Greaves and Faccini, 1992). In addition, pancreatic acinar cel] 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 o f the exocrine pancreas in humans. Thus, while the precise mechanism o f formation and significance ofpancreatic 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 fee two studies relative to compound-related lesions o f the exocrine pancreas; however, a formal pathology peer review o f fee pancreas from all study *whrut.Ta was not conducted. The results o f fee two studies were similar in feat AFFO produced proliferative lesions o fpancreatic acini in both studies at dietary concentrations- o f300 ppm. In fee 3M study, treatment-related changes were limited to slight increases in acinar cell hyperplasia. M fee DuPont study, increased incidences o f hyperplasia and adenoma were observed. Thus, these studies produced qualitatively similar responses in fee pancreas, with fee differences noted being quantitative-higher overall incidences of proliferative lesions and greater tendency for progression o f lesions to adenoma were observed in tiie DuPont study compared to fee 3M study. Since fee difference between pancreatic aCinar hyperplasia and adenoma in the rat is often a reflection of arbitrary diagnostic criteria (size) and not necessarily biological potential, fee results of the two studies are in reasonable concordance given fee experimental variables that exist between them. Although fee basts far the quantitative difference observed is not known, a number o f variables, especially diet, are known to modulate fee proliferative response of the acinar pancreas in rate and are likely operative in the differences in degree of response seen in the two chronic studies wife APFO. Despite these differences, target organ effects on fee pancreas, characterized by focal proliferative lesions o fpancreatic acini, were common to both fee 3M and DuPont studies. 7 4.0 References DuPont-13788 Biegel LB, H uitt ME, Fam e SR, O'Connor JC, and Cook JC (2001). CD rats. Toxicol Set. 60:44-55. Eustis, SL and Boorman, GA (1990). 8. Exocrine pancreas. In: Pathology o fthe Fischer R at (GA Boorman, SL Eustis, MR Elwell, CA Montgomery, Jr, and WF Mackenzie, eds.). Academic Press, Inc., San Diego, pp 95-108. Eustis SL and Boorman GA (1985). Proliferative lesions o f the exocrine pancreas: relationship to coin oil gavage in the National Toxicology Program. J N a tl Cancer Inst. 75:1067-73. Greaves, P (2000). Chapter VH, Digestive system 1. Exocrine pancreas, In: Histopathology ofP reclM cal Toxicity Studies, SecondE dition, Elsevier, Amsterdam, pp. 490-514. Greaves, P and Faccini, JM (1992). Digestive system. Pancreas. In: R at Histopathology, SecondEdition, Elsevier, Amsterdam, pp.118-126 Hansen, JF, 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 MiDar, PM (1990). Tumours of the pancreas. In: Pathology o fTumors in laboratory Animals. Volume/ - Tumours o fthe Rat, Second Edition (VS Tuiusov andJCJ Mohr, eds). International Agency for Research on Cancer, Lyon, pp. 241-249. McGuinness EE, Morgan RG, Levison DA, Frape DL, Hopwood D, and W onnsleyKG. (1980). The effects of long-term feeding o f soya flour on the rat pancreas. Scand J Gastroenterol, 15:497-502. 3M (1987). Two Year O ral (Diet) Toodcity/Carctnogemdty Study cfFC -143 in Rats. Ricker Experiment No. 0281CR0012. 8