NTP Range-Finding Report on the Immunotoxicity of Perfluoro-N-decanoic acid in Female Harlan Sprague Dawley Rats (CAS No. 335-76-2)
Report Date: November 2012
The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.
Perfluoro-N-decanoic acid, or nonadecafluoro-n-decanoic acid, is a 10-carbon straight-chain fluorinated fatty acid belonging to a class of chemicals referred to as perfluoroalkyl acids. Because of their chemical and thermal stability, PFDA and other perfluorinated carboxylic acids have been used as plasticizers, lubricants, and water and oil surfactants (Harris et al., 1989). Accumulation of perfluorinated chemicals in the environment, identification of these chemicals in wildlife and humans, and their long half-lives in occupational workers (4-5 years) have raised considerable public health concerns on the effects of these chemicals (Guruge et al., 2005; Olsen et al., 2007).
The rationale for immunotoxicity testing of the PFAAs is based on a myriad of effects. PFAA exposure has been reported to produce bone marrow depression and thymic atrophy in mice, rats, hamsters, and guinea pigs (George and Andersen, 1986; Van Rafelghem et al., 1987; Nelson et al., 1992). PFAA levels have also been detected in human blood (Bloom et al., 2010; Joensen et al., 2009; Lindstrom et al., 2009) and breast milk (So et al., 2006). In addition, their presence has been detected in both wildlife (Yeung et al., 2009; Yoo et al., 2009), and the environment (Konwick et al., 2008; Zushi and Masunaga 2009). The structural resemblance of PFDA to other PFAAs such as perfluorooctane sulfonate and perfluorooctanoic acid, which have known effects on the immune system (Dewitt et al., 2008; DeWitt et al., 2009; Lefebvre et al. 2008; Son et al., 2008; Yang et al., 2002), suggests the potential for PFDA to be immunotoxic. PFDA has been shown to produce a spectrum of toxicological effects in laboratory animals including weight loss, hypophagia, thymic atrophy, liver damage, hypolipidemia, peroxisome proliferation, and tumor promotion (Harris et al., 1989; Olson et al., 1983). However, to date, no comprehensive immunotoxicological studies have been conducted using relevant doses and exposure routes of PFDA.
The National Toxicology Program requested that a dose range-finding study be performed to establish the potential effects of PFDA on the immune system. These studies were conducted in female Harlan Sprague Dawley rats. The in-life phase of these studies was carried out between 29, July 2010 and 26, April 2011. The doses selected for the study were 0.125, 0.25, 0.5, 1, and 2 mg/kg/day and were administered in deionized water containing 2% Tween 80 via oral gavage.
Female HSD rats exposed to PFDA demonstrated dose-dependent statistically significant decreases in both body weight and in body weight gain over the course of the 28-day exposure period. Significant decreases in body weights were observed beginning on day 22 for rats exposed to 1 mg/kg PFDA and beginning on day 15 for rats exposed to 2 mg/kg PFDA. By the end of the study, the average body weight of rats in the 2 mg/kg dose group was less than their average weight at the beginning of the study. In addition, significant decreases in body weight gain were also observed in rats exposed to 1 mg/kg and 2 mg/kg PFDA when compared to the vehicle control group. While the effects on the immune system at the 2 mg/kg PFDA dose are reported, the effects must be interpreted in light of the apparent overt toxicity at this dose.
PFDA exposure produced significant effects on the weights of the liver. When expressed as percent of body weight, the liver weights of PFDA-exposed animals were increased in a dose-dependent manner. Increases in spleen weights were only observed at doses ≥ 1 mg/kg. In contrast, in one of the three studies where thymus weights were evaluated, increased relative thymus weights were observed in the 0.125, 0.25, and 0.5 mg/kg PFDA dose groups, while no effects on thymus weights were observed in the other two studies.
Effects on hematological parameters were also observed. Erythrocyte counts in the peripheral blood were increased 12% at the 2 mg/kg, while reticulocyte counts were decreased 51% at the same dose level. Hematocrit was significantly increased at 1 mg/kg but not at 2 mg/kg, while significant decreases in mean corpuscular volume and mean corpuscular hemoglobin were observed at the 2 mg/kg dose. Mean corpuscular hemoglobin concentration was affected only at the 0.25 mg/kg dose, where a 6% decrease was observed. The absolute number and percentage of lymphocytes were both significantly increased in rats exposed to PFDA at 2 mg/kg, as was the percentage of neutrophils in the peripheral blood.
Spleen cell numbers were decreased at the 2 mg/kg dose in two studies and in one study at the 0.25 and 0.5 mg/kg dose levels. Spleen cell phenotypes were affected by PFDA exposure, but suppression was observed only at doses ≥ 1 mg/kg. When expressed as absolute values, decreases in all of the phenotypes evaluated, ranging from 38% to 62%, were observed at the 2 mg/kg dose. When evaluated as percent values, CD45+ (i.e., B lymphocytes) and natural killer cells were decreased, and the percentage of CD5+ cells (i.e., T lymphocytes) was increased.
The humoral immune response, as evaluated by the antibody-forming cell response to sheep erythrocytes, was statistically decreased only at the 2 mg/kg dose level. Serum antibody levels to two T-dependent antigens, sRBC and keyhole limpet hemocyanin, were unaffected by PFDA exposure. Cell-mediated immunity, as evaluated by the anti-CD3-mediated spleen cell proliferative response and the delayed-type hypersensitivity response to Candida albicans, was also unaffected at all dose levels. Innate immunity was evaluated by assessing the effects of PFDA exposure on the functional activity of the mononuclear phagocytic system and the activity of NK cells. When evaluated as specific activity (i.e., uptake per mg of tissue), the phagocytic activity of the liver was significantly decreased at doses ≥ 0.5 mg/kg. When evaluated in terms of percent uptake, no significant effects on liver phagocytosis were observed. No other effects on MPS activity were observed. The cytotoxic activity of NK cells was unaffected.
Bone marrow cell numbers, DNA synthesis, colony-forming units, and burst-forming units were unaffected by PFDA exposure at all dose levels. Similarly, bone marrow cell differentials were unaffected by PFDA exposure at all dose levels, with the exception of the percent values of CD3+ cells, which was increased in the animals treated with PFDA at doses ≥ 1 mg/kg. In the animals receiving the 0.25 mg/kg dose of PFDA, a significant decrease was observed in the percent values of the CD11b/c+ cells. However, at higher dose levels, the percent values were not significantly different than the control animals.
In conclusion, the majority of the toxicological and immunological effects observed following PFDA exposure by oral gavage in female HSD rats for 28 days occurred at doses levels that caused overt toxicity and a significant decrease in body weight. Minimal effects were observed on the innate, humoral, and cell-mediated components of the immune system in animals exposed to PFDA.
Harris M.W., Uraih L.C., & Birnbaum L.S. (1989). Acute toxicity of perfluorodecanoic acid in C57BL/6 mice differs from 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol. 13(4):723-36.
Guruge K.S., Taniyasu S., Yamashita N., Wijeratna S., Mohotti K.M., Seneviratne H.R., Kannan K., Yamanaka N., & Miyazaki S. (2005). Perfluorinated organic compounds in human blood serum and seminal plasma: a study of urban and rural tea worker populations in Sri Lanka. J Environ Monit. 7(4):371-7.
Olsen G.W., Burris J.M., Ehresman D.J., Froehlich J.W., Seacat A.M., Butenhoff J.L., & Zobel L.R. (2007). Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ Health Perspect. 115(9):1298-305.
George M.E., Andersen M.E. (1986). Toxic effects of nonadecafluoro-n-decanoic acid in rats. Toxicol Appl Pharmacol 85(2):169-180.
Van Rafelghem M.J., Mattie D.R., Bruner R.H., & Andersen M.E. (1987). Pathological and hepatic ultrastructural effects of a single dose of perfluoro-n-decanoic acid in the rat, hamster, mouse, and guinea pig. Fundam Appl Toxicol 9(3):522-540.
Nelson D.L., Frazier D.E., Ericson J.E., Tarr M.J., & Mathes L.E. (1992). The Effects of Perfluorodecanoic Acid (PFDA) on Humoral, Cellular, and Innate Immunity in F344 Rats. Immunopharm Immunotox 14:925-938.
Bloom M.S., Kannan K., Spliethoff H.M., Tao L., Aldous K.M., & Vena J.E. (2010). Exploratory assessment of perfluorinated compounds and human thyroid function. Physiology & behavior, 99:240-5.
Joensen U.N., Bossi R., Leffers H., Jensen A.A., Skakkebaek N.E., & Jorgensen N. (2009). Do perfluoroalkyl compounds impair human semen quality? Environ Health Perspect 117(6):923-927.
Lindstrom G., Karrman A., & van Bavel B. (2009). Accuracy and precision in the determination of perfluorinated chemicals in human blood verified by interlaboratory comparisons. Journal of chromatography. 1216(3):394-400.
So M.K., Yamashita N., Taniyasu S., Jiang Q., Giesy J.P., & Chen K., et al. (2006). Health risks in infants associated with exposure to perfluorinated compounds in human breast milk from Zhoushan, China. Environ Sci Technol 40(9):2924-2929.
Yeung L.W., Yamashita N., Taniyasu S., Lam P.K., Sinha R.K., & Borole D.V., et al. (2009). A survey of perfluorinated compounds in surface water and biota including dolphins from the Ganges River and in other waterbodies in India. Chemosphere 76(1):55-62.
Yoo H., Yamashita N., Taniyasu S., Lee K.T., Jones P.D., & Newsted J.L., et al. (2009). Perfluoroalkyl Acids in Marine Organisms from Lake Shihwa, Korea. Arch Environ Contam Toxicol.
Konwick B.J., Tomy G.T., Ismail N., Peterson J.T., Fauver R.J., & Higginbotham D., et al. (2008). Concentrations and patterns of perfluoroalkyl acids in Georgia, USA surface waters near and distant to a major use source. Environmental toxicology and chemistry / SETAC; 27(10):2011-2018.
Zushi Y., Masunaga S. (2009). First-flush loads of perfluorinated compounds in stormwater runoff from Hayabuchi River basin, Japan served by separated sewerage system. Chemosphere 76(6):833-840.
Dewitt J.C., Copeland C.B., Strynar M.J., & Luebke R.W. (2008). Perfluorooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice. Environ Health Perspect 116(5):644-650.
DeWitt J.C., Shnyra A., Badr M.Z., Loveless S.E., Hoban D., & Frame S.R., et al. (2009). Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha. Critical reviews in toxicology 39(1):76-94.
Lefebvre D.E., Curran I., Armstrong C., Coady L., Parenteau M., & Liston V., et al. (2008). Immunomodulatory effects of dietary potassium perfluorooctane sulfonate (PFOS) exposure in adult Sprague-Dawley rats. Journal of toxicology and environmental health. 71(23):1516-1525.
Son H.Y., Lee S., Tak E.N., Cho H.S., Shin H.I., & Kim S.H., et al. (2008). Perfluorooctanoic acid alters T lymphocyte phenotypes and cytokine expression in mice. Environmental toxicology.
Yang Q., Abedi-Valugerdi M., Xie Y., Zhao X.Y., Moller G., & Nelson B.D., et al. (2002). Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid. International immunopharmacology 2(2-3):389-397.
Harris M.W., Uraih L.C., & Birnbaum L.S. Acute toxicity of perfluorodecanoic acid in C57BL/6 mice differs from 2,3,7,8-tetrachlorodibenzo-p-dioxin. (1989). Fundam Appl Toxicol. 13(4):723-36.
Olson C.T., Andersen M.E. (1983). The acute toxicity of perfluorooctanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids. Toxicol Appl Pharmacol. 70(3):362-72.