Report Date: August 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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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.
The World Health Organization and the United States Environmental Protection Agency regulate polychlorinated dibenzo-p-dioxins and dibenzofurans using the Toxic Equivalency Factor methodology. A number of studies have reported that the polybrominated dibenzo-p-dioxins and dibenzofurans are present in sediment (Takigama et al., 2005), fish (Hayward et al., 2007; Fernandes et al., 2008), and in human serum (Schecter et al., 2005). There are in vitro studies (Behnisch et al., 2003) suggesting that some of the brominated analogs may be more potent than 2,3,7,8-tetrachlorodibenzodioxin. It has been reported that the TEFs for the PBDD/Fs are greater than for the PCDD/F analogues (Ma et al., 2009). However, there are few in vivo studies that have evaluated the relative potencies of the PBDD/Fs. The brominated dibenzofurans have been reported to be more abundant than brominated dioxins in shellfish (Fernandes et al, 2009) and also in incinerated wastes containing brominated flame retardants (Ma et al., 2009). It has been reported that the biologic and toxic activity of dibenzo-p-dioxins is reduced following the addition of a non-lateral halogen group to those compounds having either Cl or Br in each of the lateral (2, 3, 7, and 8) positions (Mason, et al., 1987), however this was not studied for the dibenzofurans. The WHO has assigned a TEF of 0.5 to 2,3,4,7,8-pentachlorodibenzofuran (Van den Berg et al., 1998). However, in vitro studies have suggested the relative potency of this chlorinated dibenzofuran may be as high as 0.93 (Behnisch et al., 2003). The relative potency of its brominated analogue, 2,3,4,7,8-pentabromodibenzofuran was significantly lower, with in vitro studies suggesting a relative potency of 0.14 (Behnisch et al., 2003).
As part of the National Toxicology Program evaluation of the TEF methodology for use in dioxin risk assessments, the relative potencies of the brominated dioxins and their chlorinated analogs have been evaluated by investigating the effects of these compounds on humoral immunity in mice. Halogenated aromatic hydrocarbons have well-documented effects on the immune system, and a TEF approach to examining the relative potencies of dioxins, polychlorinated biphenyls, and dibenzofurans demonstrated that TCDD is one of the most potent inhibitors of immune function in the mouse (Davis and Safe, 1988). Inhibition of the antibody-forming cell response to sheep red blood cells in mice following an acute single dose of dioxins has been shown to be one of the most sensitive indicators of exposure to these compounds (Harper et al., 1993; Johnson et al., 2000).
The NTP requested that an evaluation of two HAHs, 2,3,4,7,8-PeBDF and 2,3,4,7,8-PeCDF, be conducted to assess their immunosuppressive effects following a single oral administration. These studies were conducted in female B6C3F1/N mice. Five 2,3,4,7,8-PeBDF dose levels were utilized (3, 9, 15, 30, and 90 µg/kg), given in a single oral administration. Five 2,3,4,7,8-PeCDF dose levels were utilized (3, 9, 15, 30, and 90 µg/kg), given in a single oral administration. Corn oil was used as the vehicle for 2,3,4,7,8-PeBDF and 2,3,4,7,8-PeCDF administration.
A single exposure to 2,3,4,7,8-PeBDF had no effect on body weight or body weight gain over the entire experimental period. Absolute liver weights were increased as compared to vehicle control mice at day 3 following exposure to 2,3,4,7,8-PeBDF in a dose-related manner, reaching the level of significance at the 90 µg/kg dose only. By day 11, however, absolute liver weights were increased in mice treated with 15, 30, and 90 µg/kg 2,3,4,7,8-PeBDF. 2,3,4,7,8-PeBDF exposure resulted in decreased spleen weight at the 90 µg/kg dose, while the weights of the other major immune organ, the thymus, were unaffected. Overall, hematological parameters were unaffected, while spleen cell numbers and humoral immune responses were decreased. Specifically, total spleen cell number was significantly decreased at the 90 µg/kg dose. The AFC response was significantly decreased at all 2,3,4,7,8-PeBDF doses, while significant decreases in serum anti-sRBC IgM antibody titers were observed at doses ≥ 9 µg/kg.
2,3,4,7,8-PeCDF exposure had no effect on periodic body weights, although significant increases in body weight gain were observed for the 9 and 30 µg/kg dose groups on day 7 of the 11 day study. These increases were not dose-responsive, and they were not present at study termination on day 11. Absolute liver weights were not affected following exposure to 2,3,4,7,8-PeCDF on day 3, although relative liver weights were increased at the 9 and 90 µg/kg exposure levels. By day 11, absolute and relative liver weights were increased at doses ≥ 15 µg/kg. 2,3,4,7,8-PeCDF exposure did not affect the absolute or relative weights of the major immune organs, the spleen and the thymus. In addition, hematological parameters were unaffected by 2,3,4,7,8-PeBDF exposure. Leukocyte numbers were increased at the 3 µg/kg dose only, while absolute leukocyte differentials were unaffected, with the exception of an increase in absolute lymphocyte numbers at the 3 and 9 µg/kg doses. When expressed as percent of total leukocyte counts, leukocyte differentials were also unaffected, with the exception of decreased percentages of monocytes at all dose levels except for 30 µg/kg. No effects were observed on spleen cell number, however, the T-dependent antibody response was significantly decreased in a dose-related manner following the single 2,3,4,7,8-PeCDF exposure. Specifically, the numbers of AFC/106 Spleen Cells (Specific Activity) and the numbers of AFC/Spleen (Total Spleen Activity) were decreased at both 30 µg/kg and 90 µg/kg. Total Spleen Activity was also significantly decreased at the 15 µg/kg exposure level. Serum anti-sRBC IgM antibody levels were decreased at the 90 µg/kg dose only.
Pairwise multiple comparisons between these compounds demonstrated that 2,3,4,7,8-PeBDF produced greater suppression of the humoral immune response (AFC, sRBC ELISA) than did 2,3,4,7,8-PeCDF, suggesting that the brominated dibenzofuran evaluated in these studies is more potent than its chlorinated analogue.
In conclusion, when administered orally in a single dose at levels up to 90 µg/kg, both 2,3,4,7,8-PeBDF and 2,3,4,7,8-PeCDF produced significant decreases in the TDAR) (i.e., AFC, serum IgM antibody titers), although the brominated analogue produced significant decreases at lower doses than the chlorinated compound. The suppression observed following 2,3,4,7,8-PeBDF exposure appears to be greater than that following 2,3,4,7,8-PeCDF exposure, suggesting that 2,3,4,7,8-PeBDF is a more potent immunosuppressive agent than 2,3,4,7,8-PeCDF.
Takigama H., Sakai S., & Brouwer A. (2005). Bio/chemical analysis of dioxin-like compounds in sediment samples from Osaka Bay, Japan. Environ Technol 26(4):459-69.
Hayward D., Wong J., & Krynitsky A.J. (2007). Polybrominated diphenyl ethers and polychlorinated biphenyls in commercially wild caught and farm-raised fish fillets in the United States. Environ Res 103:46-54.
Fernandes A., Dicks P., Mortimer D., Gem M., Smith F., Driffield M., White S., & Rose M. (2008). Brominated and chlorinated dioxins, PCBs and brominated flame retardants in Scottish shellfish: methodology, occurrence, and human dietary exposure. Mol Nutr Food Res 52:238-249.
Schecter A., Papke O., Tung K.C., Joseph J., Harris T.R., & Dahlgren J. (2005). Polybrominated diphenyl ether flame retardants in the U.S. population: current levels, temporal trends, and comparison with dioxins, dibenzofurans, and polychlorinated biphenyls. J Occup Environ Med 47:199-211.
Behnisch P.A., Hosoe K., & Sakai S. (2003). Brominated dioxin-like compounds: in vitro assessment in comparison to classical dioxin-like compounds and other polyaromatic compounds. Environment Int 29:861-877.
Ma J., Addinik R., Yun S., Cheng J., Wang W., and Kannan K. (2009). Polylbrominated dibenzo-p-dioxins/dibenzofurans and polybrominated diphenyl ethers in soil, vegetation, workshiop-floor dust, and electronic shredder residue from an electronic waste recycling facility and in soils from a chemical industrial complex in eastern China. Environ Sci Technol 43:7350-7356.
Fernandes A., Mortiimer D., Gem M., Dicks P., Smith F., White S., & Rose M. (2009). Brominated dioxins (PBDD/Fs) and PBDEs in marine shellfish in the UK. Food Additives and Contaminants 26(6):918-927.
Mason G., Zacharewski T., Denomme M.A., Safe L., & Safe S. (1987). Polybrominated dibenzo-p-dioxins and related compounds: quantitative in vivo and in vitro structure-activity relationships. Toxicology 44:245-255.
Van den Berg M., Bernbaum L., Bosveld A.T., Brunstrom B., Cook P., Feeley M., Giesy J.P., Hanberg A., Hasegawa R., Kennedy S.W., Kubiak T., Larsen J.C., van Leeuwen F.X., Liem A.K., Nolt C., Peterson R.E., Poellinger L., Safe S., Schrenk D., Tillitt D., Tysklind M., Younes M., Waern F., & Zacharewski T. (1998). Toxic equivalency factors for PCBs, PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 106:775-792.White K.L., Musgrove D.L. and Brown R.D. (2010). The Sheep Erythrocyte T-Dependent Antibody Response. Methods Mol Biol, 598:173-84.
Harper N., Connor K., Steinberg M., & Safe S. Immunosuppressive activity of polychlorinated biphenyl mixtures and congeners: nonadditive (antagonistic) interactions. Fundam Appl Toxicol. 1995 Aug;27(1):131-9.
Johnson C.W., Williams W.C., Copeland C.B., DeVito M.J., & Smialowicz R.J. Sensitivity of the SRBC PFC assay versus ELISA) for detection of immunosuppression by TCDD and TCDD-like congeners. Toxicology. 2000 Dec 7;156(1):1-11.