Pathology Review of Reported Tumorigenesis
in a Two Year Study of FM-3924 in Rats

November 25,1998

BY
Pathology Associates International
6217 Centre Park Drive
West Chester, Ohio 45069

Telephone (513) 779-9600
Fax (513) 779-9603

003093US00004554

Pathology Reviewof Tumors Reported in Rats Given FM-3924

Introduction:

This report presents an independent assessment of tumorigenesis dataobtained from a study to determine the chronic toxicity and carcinogenic potential of thefluorochemical FM-3924 in rats. The study, entitled "Two Year (Diet) Toxicity /Carcinogenicity Study of Fluorochemical FM-3924 in Rats," was sponsored by the 3M
Company, St. Paul, Minnesota.
Biophase procedures were conducted at Riker
Laboratories, Inc., between April, 1981 and May, 1983 and were in compliance withFDA Good Laboratory Practice (GLP) guidelines. At Riker Laboratories, Leonard J.
Sibinski, BA, served as the Study Director. Following in-life procedures, all majororgans and tumors were processed into microslides and examined by Robert G. Geil,DVM, DACVP. Dr. Geil’s findings and interpretations were incorporated into the final
report for the study.
For the tumorigenesis review, the complete study report, including all relevant pathology
data was forwarded to Pathology Associates International (PAI), West Chester, Ohio for
examination by Richard H. Bruner, DVM, DACVP. The sponsor (3M) regarded original
pathology interpretations by Dr. Geil as adequate, and examination of microscopic tissue
sections was not included in the review process. Specific objectives of the review were toevaluate tumor data and to provide an opinion relative to the potential relationship ofreported neoplasms with the test material based upon: 1. The incidence and morphologyof observed tumors and associated proliferative and non-proliferative lesions. 2. A
literature review to examine the biologic behavior and carcinogenetic potential of similar
fluorochemicals, 3. Contemporary knowledge of tumor mechanistic data, especially withrespect to possible epigenetic pathways, 4. Consideration of chronic toxicity,
immunosuppression, hormonal modulation or ancillary biochemical interactions which
may serve as modulating factors in the development of tumors in this study, and
5. Personal experience in evaluating rodent carcinogenesis bioassays.

Review Procedures and Findings1. Reported tumorigenesis and ancillary pathologic changes for FM-3924:
Based upon reported pathology findings, the review pathologist concurred thattreatment-related changes were present at both the 1-Year (interim) and 2-Year(terminal) sacrifices as well as in some unscheduled deaths. At the 1-Year interim
sacrifice, treatment-related changes were generally restricted to the liver and werecharacterized by a dose-dependent increase in hepatocellular cytomegaly, vacuolationand necrosis along with slightly increased inflammatory cell infiltrates. Cytomegaly1and vacuolation were generally consistent with treatment-induced perturbation ofliver cell metabolism resulting in hypertrophy via proliferation of peroxisomes and/orsmooth endoplasmic reticulum (P-450 enzyme induction). Hepatocellular necrosis
was largely attributed to chronic liver cell swelling with compromised metabolism
and/or reduced blood perfusion (hypoxia).
Treatment-related microscopic findings in unscheduled deaths and animals continued
until the terminal (2-Year) sacrifice included persistent hepatocellular cytomegaly andvacuolation in both sexes. Additionally, cystoid degeneration ("spongiosis hepatis")of the liver was significantly increased in high dose males. Most notably,
hyperplastic nodules were increased in the liver of both sexes and hepatocellularadenomas and carcinomas were increased in females.
Based upon reported pathology data, dietary levels of 100 ppm FM-3924 for two
years resulted in unequivocal hepatocellular cytomegaly, vacuolation and cystoiddegeneration as well as increased hyperplastic nodules in both sexes and increased
adenomas and carcinomas in high dose females. Although increased hepatocellularneoplasms were not reported in males, it is likely that some of the "hyperplasticnodules" (in both sexes) would be regarded as hepatocellular adenomas if
contemporary diagnostic criteria were applied (1). It is noteworthy, also, that hepatic
cytomegaly was observed in males given 10 and 30 ppm of the test matereial
suggesting that a NOEL for hepatocytomegaly was not achieved for males assigned tothis investigation
2. Literature review of the biologic behavior and carcinogenic potential offluorochemicals
The reviewer regarded the test material (N-ethylperfluorooctanesulfonamido ethanol)
as a unique xenobiotic with unknown structure-activity relationships. A limited
literature review was conducted to determine if the biological behavior, including
tumorigenesis, of similar fluorochemicals had been reported. The literature review
included a survey of rodent carcinogenesis bioassays completed by the National
Toxicology Program (NTP) and select scientific journals and biological extracts.
Although no carcinogenesis bioassays with "complex" fluorochemicals werediscovered, several relevant reports concerning subchronic investigations werelocated. Additionally, several reports, including the NTP 2-year bioassay of sodium
fluoride were available to provide perspectives on the biological effects of long-term
fluoride exposure. Results of these studies are briefly summarized as follows:

1. Subchronic toxicity studies in rats with "complex" fluorochemicals haveidentified treatment-related hepatocellular hypertrophy (cytomegaly) similar tofindings with FM-3924. In studies conducted by Van Rafelghem et al, a single
intraperitoneal injection of perfluoro-w-decanoic acid (PFDA) in several rodent

2OOôloispecies resulted in persistent hepatocellular swelling which, ultrastructurally, wascharacterized by peroxisomal proliferation (12).
In subchronic studies sponsored by the 3M Company, Griffith and Long
administered ammonium perfluorooctanoate to rats, mice and monkeys via oralroutes (3). At doses of 30 ppm or greater, rats displayed hepatocellular
hypertrophy. This change was more prevalent and severe in males. Noteworthy
was the observation that all mice given doses of 1000 ppm ad libidum and allmonkeys given 100 mg/kg/day (gavage) died preterminally. Additionally, allmonkeys given 30 mg/kg/day displayed anorexia, emesis, black stools, facial
pallor, and prostration; and one monkey given 10 mg/kg/day displayed anorexia,
black stools and facial pallor. Liver effects, however, were not observed inmonkeys assigned to these studies. Furthermore, microbial assays using five
Salmonella stains and one Saccharomyces strain, with and without metabolic
activation, did not reveal mutagenic activity for the test material.
2. Review of studies relating to the chronic toxicity of fluoride and fluoridecontaining compounds was limited to the NTP 2-year carcinogenesis bioassay o fsodium fluoride and select environmental studies of fluoride in cattle. In the NTP
study, treatment-related pathologic changes were not observed in the liver o f rats
given dietary concentrations of up to 175 ppm sodium fluoride in the drinking
water for two years (11). Furthermore, in an extensive survey of cattle exposed tohigh environmental fluoride concentrations for lifetime periods, increased liverdisease or neoplasia was not reported although many animals exhibited dental andskeletal changes typical of advanced fluorosis (9).
3. Mechanisms resulting in increased hepatocellular nodular hyperplasia andneoplasia in rats given FM-3924:
Results of possible genotoxicity studies with FM-3924 were not provided to the review
pathologist. It was reasoned that this complex fluorochemical probably was not strongly
mutagenic and that most biologic effects were due to perturbation o f liver cell
metabolism, with peroxisomal proliferation and general disruption of multiple metabolic
pathways. Accordingly, it is likely that the development of liver cell tumors (and nodularhyerplasia) was associated with epigenetic mechanisms, possibly including peroxisomal
proliferation and genetic damage (ploidy) associated with oxidative stress and/or alteredregulation of the cell cycle. Following publication of the final report for this study(1988), numerous journal articles have been published which identify carcinogenesis inrodents following exposure to non-genotoxic test materials. Subsets of these chemicals
which induce liver cell tumors in rodents are characterized by antecendent hepatocytomegaly and peroxisomal proliferation (2,5,6,8).

ooaiosUS00004!

4. Effects of ancillary biochemical interactions which may have served as tumor
promoters:
Comments relative to all metabolic aberrations which may have influenced liver celltumorigenesis in this study would be largely speculative. It should be noted, however,that hepatic toxicity was unequivocally linked with ingestion of FM-3924, and at highdose levels liver cell alterations had resulted in necrosis. Correspondingly, accelerated
proliferation of hepatocytes would be expected to repair damaged tissue, and may have
served as a tumor promoter (10). Although it should be emphasized that increased liver
cell proliferation which may occur in rodents following exposure to many toxic materials
does not invariably result in increased tumor formation, most pathologists agree that cell
damage which promotes in increased mitotic activity may contribute to tumor formation(4,10,13).
5. Personal opinion relative to a relationship between proliferative lesions andFM-3924 observed in this study:
It is my opinion that distinct increases in hepatocellular neoplasms in high dose females,
combined with increased hyperplastic nodules in both sexes are clear indicators that thetest material should be regarded as a liver carcinogen in Sprague Dawley rats. Other
proliferative lesions and neoplasms were considered to be spontaneous alterations or
secondary to the systemic effects of altered hepatocellular metabolism (7). Based upon
histomorphologic changes observed in this study, it is likely that epigenetic mechanisms(especially peroxisomal proliferation, oxidative stress and other factors which deregulate
the cell cycle) were key factors in the development of hepatocellular proliferative lesions.
Ancillary data which would support epigenetic pathways for tumorigenesis in this study
would provide a rationale for selection of exposure thresholds in humans. Based uponreference literature available for the preparation of this review, liver damage, including
carcinogenesis, has not been reported in humans exposed to test materials that promote
hepatocytomegaly and neoplasia in rodents via peroxisomal proliferation and associatedmetabolic perturbations

Additional Information which might Contribute to the Safety
Assessment of FM-3924
1. Mutagenesis assays or ancillary procedures to establish the genotoxic potential of thetest material.
2. Cell proliferation studies to provide an index treatment-related increases in the cellcycle.
3. Ultrastructural analyses or contemporary analytical procedures to confirm thepossible peroxisomal proliferation and ancillary metabolic perturbations.
4. Recovery studies to establish the persistence of liver cell effects following subchronic
exposures to FM-3924.

US000I

Conclusions: Based upon review of the information available in the study report, it is
my opinion that dietary FM-3924 for 2 years resulted in chronic liver changes(megalocytosis) in males at all dose levels (10, 30 and 100 ppm) and for females at thehigh dose concentration (100 ppm). At the high dose level, hyperplastic nodules wereincreased in both sexes and hepatocellular tumors (adenomas and carcinomas) wereincreased in females. Incidence values for liver proliferative lesions indicated-that FM3924 should be regarded as a liver carcinogen for Sprague Dawley rats under theconditions of this study. The presence of persistent, dose-dependent liver cell cytomegalysuggested that epigenetic mechanisms were causative for tumorigenesis in this study, andthat safe exposure thresholds may be established providing that toxicokinetic andancillary data do not indicate additional adverse effects such as reduced excretion andbioaccumulation.

Richard H. Bruner, DVM, DACVP

Date'References
1. Goodman, D.G., Maronpot, R.R., Newbeme, P. M., Popp, J.A. and Squire,R.A.
Proliferative and Selected Other Lesions of the Liver in Rats (Draft Proposal).
SSNDC Guides fo r Toxicologic Pathology. STP/ARP/AFIP, Wash., D.C. (1993).
2. Green, S. Receptor-Mediated Non-Genotoxic Carcinogenesis: New Models for Risk
Assesment 1 Human Exp. Tox. (10): 390-395 (1991).
3. Griffith, F.D. and Long, L E. Animal Toxicity Studies with Ammonium
Perfluorooctanoate. Am. Ind Hyg. Assoc. J. (41): 576-583 (1980).
4. Huff, J Absence of Morphologic Correlation Between Chemical Toxicity andChemical Carcinogenesis. Environ. Health Perspect. 101 (Suppl. 5):45-54 (1993).
5. Lalwani, N.D., Dethloff, L.A., Haskins, J.R., Robertson, D.G., and De La Iglesia,
F. A. Increased Nuclear Ploidy, Not Cell Proliferation Is Sustained in the Peroxisome
Proliferator-Treated Rat Liver. Tox. Path. (25)No.2. 165-176 (1997).
6. McDonald, J.S., Lankas, G.R. and Morrissey, R.E. Toxicokinetic and Mechanistic
Considerations in the Interpretation of the Rodent Bioassay. Tox. Path, (22) No. 2.
124-140(1994).
7. McMartin, D.N., Sahota, P.S., Gunson, D.E., Han Hsu, H.and Spaet, R.H. Neoplasms
and Related Proliferative Lesions in Control Sprague-Dawley Rats from003104sCarcinogenicity Studies. Historical Data and Diagnostic Considerations. Tox. Path.
(20) No. 2,212-225 (1992).
8. Schafer, K.A. Cell Cycle: A Review. Vet. Path. (35) No. 6. 461-478 (1998).
9. Shupe. J.L., Bruner, R.H., Seymour, J.L. and Alden, C.L. The Pathology of Chronic
Bovine Fluorosis; A Review. Tox. Path, (20) No.2. 274-285 (1992).
10. Swenberg, J.A, Cell Proliferation and Chemical Carcinogenesis: Conference
Summary and Future Directions. Environ. Health Perspect. 101 (Suppl. 5) 153-158............... ...............
(1993).
11. US Department of Health and Human Services. NTP TR 393. NTP Technical Reporton Toxicology and Carcinogenesis Studies o f Sodium Fluoride in F-344 Rats and
B6C3F1 Mice (drinking water) (1990).
12. Van Rafelghem, M.J., Mattie, D.R., Bruner, R.H. and Anderson, M.E. Pathologicaland Hepatic Ultrastructural Effects o f a Single Dose of Perfluoro-n-decanoic Acid in
the Rat, Hamster, Mouse and Guinea Pig. Fund & Appi Tox. (9), 522-540 (1987).
13. Ward, J.M., Uno, H., Kurata, Y., Weghorst, C.M. and Jang, J. Cell Proliferation Not
Associated with Carcinogenesis in Rodents and Humans, Environ. Health
Perspect. 101 (Suppl. 5): 125-136 (1993).

003105;

6