Chronic dietary toxicity and carcinogenicity study with ammonium perfluorooctanoate in Sprague–Dawley rats

doi:10.1016/j.tox.2012.04.001

Toxicology

Volume 298, Issues 1–3, 16 August 2012, Pages 1-13

https://doi.org/10.1016/j.tox.2012.04.001 Get rights and content

Abstract

In order to assess the potential chronic toxicity and tumorigenicity of ammonium perfluorooctanoate (APFO), a 2-year dietary study was conducted with male and female rats fed 30 ppm or 300 ppm (approximately 1.5 and 15 mg/kg). In males fed 300 ppm, mean body weights were lower across most of the test period and survival in these rats was greater than that seen either in the 30 ppm or the control group. Non-neoplastic effects were observed in liver in rats fed 300 ppm and included elevated liver weight, an increase in the incidence of diffuse hepatocellular hypertrophy, portal mononuclear cell infiltration, and mild hepatocellular vacuolation without an increase in hepatocellular necrosis. Mean serum activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were elevated up to three times the control means, primarily at the 300 ppm dose. A significant increase in Leydig cell tumors of the testes was seen in the males fed 300 ppm, and tumors of the liver and acinar pancreas, which are often observed in rats from chronic exposure to peroxisome proliferating agents, were not observed in this study. All other tumor types were those seen spontaneously in rats of this stock and age and were not associated with feeding of APFO.

Highlights

► A 2-year dietary toxicity and cancer bioassay was conducted with APFO in rats. ► Non-neoplastic effects were observed in liver in rats fed 300 ppm. ► The predominant liver effect was diffuse hepatocellular hypertrophy. ► Leydig cell tumors of the testes were increased in males fed 300 ppm. ► No other increases in benign or metastatic tumors were observed.

Introduction

The ammonium salt of perfluorooctanoic acid (APFO, CASRN 3825-26-1) has been used commercially as a surface-active agent in the production of various fluoropolymers. The toxicology of this chemical has been reviewed (Kennedy et al., 2004, Lau et al., 2007) covering both short and longer-term exposure studies as well as the standard toxicological endpoint studies. One of the key studies for hazard determination is a 2-year chronic toxicity and carcinogenicity bioassay in rats which has, to this point, only been available as a four-volume report on the United States Environmental Protection Agency public docket, Administrative Record AR-226. The study was conducted from April 1981 through May 1983 by the Pathology and Toxicology Department at Riker Laboratories, then a subsidiary of the 3M Company, St. Paul, MN. After this study was initially reported, Biegel et al. (2001) reported on a 2-year dietary bioassay of APFO in male rats which was conducted at a single dietary dose (300 ppm) equivalent to the high dose of the study reported herein. The Biegel et al. study was designed to further investigate the mode of action of APFO with emphasis on changes in response over a chronic dosing period.

Although the 3M study is an older study, the increased attention given to the potential health hazards of APFO in the scientific literature in recent years has prompted this detailed summary of the study to make the key findings and conclusions of the study more accessible. The study materials were audited in recent years and found to be complete and available. In addition, representative tissues from the study have been subjected to pathology peer review. These have included the pancreas (Frame and McConnell, 2003), the uterus and ovaries (Mann and Frame, 2004), and female mammary tissues (Hardisty et al., 2010).

Section snippets

Test material

Ammonium perfluorooctanoate (APFO, FC-143, Lot 37, 97.2% pure (total of linear and branched isomers)) was supplied by 3M Company, St. Paul, MN. The APFO sample was determined to be stable over the course of the study based on analysis prior to the start of the study, approximately 1-year later, and at the termination of the exposure period.

Laboratory animals and husbandry

Three-hundred and sixty Sprague–Dawley rats (Crl:COBS@ CD(SD)BR, Charles River Company, Portage, MI) were obtained and quarantined for 10–14 days during

Test article consumption

The APFO concentration measured as ppm in the diet was determined at 3 month intervals with a duplicate analysis performed when aberrant values were detected. The mean deviations from the target concentration of the low and high dose APFO groups were less than 3%. Actual APFO doses were determined for each 2 week period for each sex and each group and expressed as mg/kg per day. The mean test article consumption was 1.3 and 14.2 mg/kg for males and 1.6 and 16.1 mg/kg for females in the 30 and 300

Discussion

This article covers the experimental details and results of a 2-year chronic toxicity and oncogenicity study of APFO in the rat. To date, only the laboratory report and subsequent pathology peer-reviews have been available since the study's completion in 1983. All of the study materials and records were audited and found to be available and reliable prior to conducting additional pathology reviews on mammary glands, ovaries, and pancreas. Presented in this article are the original findings as

Conflict of interest

John L. Butenhoff, Shu-Ching Chang and Geary W. Olsen are employees of 3M Company, a former manufacturer of ammonium PFOA and the company supporting the work reported on in the article. Gerald L. Kennedy, Jr. represents DuPont Company, a current manufacturer and user of ammonium PFOA.

Acknowledgement

This project was funded in its entirety by 3M Company.

References (52)

D.E. Amacher
Serum transaminase elevations as indicators of hepatic injury following the administration of drugs
Regul. Toxicol. Pharmacol.
(1998)
P. Bentley et al.
Hepatic peroxisome proliferation in rodents and its significance for humans
Food Chem. Toxicol.
(1993)
L.B. Biegel et al.
Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies
Toxicol Appl Pharmacol
(1995)
J.A. Bjork et al.
Multiplicity of nuclear receptor activation by PFOA and PFOS in primary human and rodent hepatocytes
Toxicology
(2011)
J.L. Butenhoff et al.
The reproductive toxicology of ammonium perfluorooctanoate (APFO) in the rat
Toxicology
(2004)
J. Doull et al.
A cancer risk assessment of di(2-ethylhexyl)phthalate: application of the new U.S. EPA Risk Assessment Guidelines
Regul. Toxicol. Pharmacol.
(1999)
P. Gervois et al.
Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate
J. Biol. Chem.
(2004)
Y. Hirose et al.
Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes
Toxicology
(2009)
N. Johansson et al.
EFSA's risk assesment on PFOS and PFOA in the food
Toxicol. Lett.
(2009)
R.C. Leonard et al.
Retrospective cohort mortality study of workers in a polymer production plant including a reference population of regional workers
Ann. Epidemiol.
(2008)

R.C. Liu et al.

The direct effect of hepatic peroxisome proliferators on rat Leydig cell function in vitro

Fundam. Appl. Toxicol.

(1996)

R.C. Liu et al.

Effect of the peroxisome proliferator, ammonium perfluorooctanoate (C8), on hepatic aromatase activity in adult male Crl:CD BR (CD) rats

Fundam. Appl. Toxicol.

(1996)

T. Nakamura et al.

Microgram-order ammonium perfluorooctanoate may activate mouse peroxisome proliferator-activated receptor α, but not human PPARα

Toxicology

(2009)

J. Whysner et al.

Phenobarbital mechanistic data and risk assessment: enzyme induction, enhanced cell proliferation, and tumor promotion

Pharmacol. Ther.

(1996)

V.L. Anderson et al.

Design of Experiments: a Realistic Approach

(1974)

L.B. Biegel et al.

Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats

Toxicol. Sci.

(2001)

J.A. Bjork et al.

Structure-activity relationships and human relevance for perfluoroalkyl acid-induced transcriptional activation of peroxisome proliferation in liver cell cultures

Toxicol. Sci.

(2009)

l. Boone et al.

Selection and interpretation of clinical pathology indicators of hepatic injury in preclinical studies

Vet. Clin. Pathol.

(2005)

B.D. Car et al.

Clinical pathology of the rat

W.J. Conover

Practical Nonparametric Statistics

(1971)

W.J. Conover et al.

Rank transformations as a bridge between parametric and nonparametric statistics

Am. Stat.

(1981)

C.W. Dunnett

New tables for multiple comparisons with a control

Biometrics

(1964)

C.R. Elcombe et al.

Hepatocellular hypertrophy and cell proliferation in Sprague–Dawley rats following dietary exposure to ammonium perfluorooctanoate occurs through increased activation of the xenosensor nuclear receptors PPARα; and CAR/PXR

Arch. Toxicol.

(2010)

E.A. Emmett et al.

Community exposure to perfluorooctanoate: relationships between serum levels and certain health parameters

J. Occup. Environ. Med.

(2006)

K.T. Eriksen et al.

Perfluorooctanoate and perfluorooctanesulfonate plasma levels and risk of cancer in the general Danish population

J. Natl. Cancer Inst.

(2009)

S.L. Eustis et al.

Proliferative lesions of the exocrine pancreas: relationship to corn oil gavage in the National Toxicology Program

J. Natl. Cancer Inst.

(1985)

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Chronic dietary toxicity and carcinogenicity study with ammonium perfluorooctanoate in Sprague–Dawley rats

Abstract

Highlights

Introduction

Section snippets

Test material

Laboratory animals and husbandry

Test article consumption

Discussion

Conflict of interest

Acknowledgement

Regul. Toxicol. Pharmacol.

Food Chem. Toxicol.

Toxicol Appl Pharmacol

Toxicology

Toxicology

Regul. Toxicol. Pharmacol.

J. Biol. Chem.

Toxicology

Toxicol. Lett.

Ann. Epidemiol.

Fundam. Appl. Toxicol.

Fundam. Appl. Toxicol.

Toxicology

Pharmacol. Ther.

Design of Experiments: a Realistic Approach

Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats

Toxicol. Sci.

Structure-activity relationships and human relevance for perfluoroalkyl acid-induced transcriptional activation of peroxisome proliferation in liver cell cultures

Toxicol. Sci.

Selection and interpretation of clinical pathology indicators of hepatic injury in preclinical studies

Vet. Clin. Pathol.

Clinical pathology of the rat

Practical Nonparametric Statistics

Rank transformations as a bridge between parametric and nonparametric statistics

Am. Stat.

New tables for multiple comparisons with a control

Biometrics

Hepatocellular hypertrophy and cell proliferation in Sprague–Dawley rats following dietary exposure to ammonium perfluorooctanoate occurs through increased activation of the xenosensor nuclear receptors PPARα; and CAR/PXR

Arch. Toxicol.

Community exposure to perfluorooctanoate: relationships between serum levels and certain health parameters

J. Occup. Environ. Med.

Perfluorooctanoate and perfluorooctanesulfonate plasma levels and risk of cancer in the general Danish population

J. Natl. Cancer Inst.

Proliferative lesions of the exocrine pancreas: relationship to corn oil gavage in the National Toxicology Program

J. Natl. Cancer Inst.