Research Thyroid-Hormone–Disrupting Chemicals: Evidence for Dose-Dependent Additivity or Synergism Kevin M. Crofton,1 Elena S. Craft,1* Joan M. Hedge,1 Chris Gennings,2 Jane E. Simmons,3 Richard A. Carchman,2 W. Hans Carter Jr.,2 and Michael J. DeVito3 1Neurotoxicology and 3Experimental Toxicology Divisions, National Health and Environmental Effects Research Laboratory, Ofﬁce of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; 2Solveritas LLC, Richmond, Virginia, USA Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and poly- brominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gav- age with 18 different polyyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose–response functions (based on seven to nine doses per chemical) were determined for individ- ual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmen- tal concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a ﬂexible single-chemical-required method applicable to chemicals with differing dose thresholds and maxi- mum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimen- tally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the ﬁrst results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs. Key words: additivity, cumulative risk, polyhalogenated aromatic hydrocarbons, synergism, thyroid hormone disruptors. Environ Health Perspect 113:1549–1554 (2005). doi:10.1289/ehp.8195 avail- able via http://dx.doi.org/ [Online 21 July 2005] Thyroid-disrupting chemicals (TDCs) are xenobiotics that alter the structure or function of the thyroid gland, alter regulatory enzymes associated with thyroid hormone (TH) home- ostasis, or change circulating or tissue concen- trations of THs. TDCs include a wide range of chemical structures. Chemicals such as per- chlorate inhibit the uptake of iodide into the thyroid gland, with subsequent decrease in iodine-based TH synthesis (Wolff 1998). Other chemicals (e.g., thionamides, amitrole, and ethylenethiourea) decrease TH synthesis by inhibition of thyroid peroxidase (Capen 1997, 1998; Hill et al. 1998; Hurley 1998; McClain 1995). Many classes of xenobiotics alter TH levels by altering catabolic pathways. Polyhalogenated aromatic hydrocarbons (PHAHs) represent one such class of chemicals that induce uridine diphosphoglucuronosyl transferases (UGTs). UGTs glucuronidate THs, and induction of these enzymes increases the elimination of THs (Hill et al. 1998; Hood and Klaassen 2000a; McClain et al. 1989; Oppenheimer et al. 1968). A major uncertainty regarding the endocrine-disrupting ability of environmental xenobiotics is the potential for additive or syn- ergistic (i.e., greater-than-additive) effects of exposure to mixtures (Daston et al. 2003; International Programme on Chemical Safety 2002). Solving the problem of predicting the effects of chemical mixtures is a daunting task. There are limited studies in the peer-reviewed literature that examine mixtures of TDCs (Desaulniers et al. 2003; Teuschler et al. 2002; Wade et al. 2002). Desaulniers et al. (2003) found that use of 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD) toxic equiv- alents predicted the additive effects of a mixture of coplanar polychlorinated biphenyls Environmental Health Perspectives • VOLUME 113 | NUMBER 11 | November 2005 (PCBs), polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans on circu- lating thyroxine (T4) concentrations in neo- natal rats. Wade et al. (2002) subchronically exposed adult male rats to a complex mixture of 16 organochlorines, lead, and cadmium. Effects on thyroid histopathology and hor- mone concentrations were underpredicted based on assumptions of additivity using pub- lished health advisories [e.g., reference doses (RfDs), acceptable daily intakes (ADIs)]. These previous efforts investigated the effects of mixtures without concurrent experimental characterization of the effects of the individual chemicals. This type of approach is useful on a case-by-case basis but does not help answer global issues in the arena of mixtures risk assessments (LeBlanc and Olmstead 2004). Our present study tested the hypothesis that a mixture of 18 PHAHs acts in a dose- additive manner. The hypothesis was tested using a flexible single-chemical-required (FSCR) method of analysis (Gennings et al. Address correspondence to K.M. Crofton, Neurotoxicology Division, MD-105-04, National Health and Environmental Effects Laboratory, U.S. EPA, Research Triangle Park, NC 27711 USA. Telephone: (919) 541-2672. Fax: (919) 541-4849. E-mail: crofton.kevin@epa.gov *Current address: Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708 USA. Supplemental Data Table 1 lists the chemicals tested, doses (µg/kg/day), group mean serum total thyroxine (T4) concentrations expressed as percent- age of control, standard deviations, and group sam- ple sizes. These data are available on the EHP website (http://ehp.niehs.nih.gov/docs/2005/8195/ supplemental.pdf). Raw data ﬁles can be obtained by contacting the corresponding author. We thank T. Grim at Cambridge Isotope Laboratories for help with the custom synthesis of the mixture. This work would not have been possible without the invaluable technical assistance of T. Zhou and D. Ross. G. We acknowledge G. LeBlanc and S. Padilla for commenting on a previous version of the manuscript. C.G., R.A.C., and W.H.C. were supported by U.S. EPA contract RFQ-RT-03-00298. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. C.G., R.A.C., and W.H.C. have a ﬁnancial inter- est in Solveritas LLC. All other authors declare they have no competing ﬁnancial interests. Received 11 April 2005; accepted 21 July 2005. 1549