(PCB-138); 2,2´,4,4´,5,5´-hexachlorobiphenyl (PCB-153); 2,3,3´,4,4´,5-hexachlorobiphenyl (PCB-156); 3,3´,4,4´,5,5´-hexachlorobiphenyl (PCB-169); and 2,2´,3,4,4´,5,5´-heptachloro- biphenyl (PCB-180). The dose ranges and number of dose groups are provided in Table 1. The PHAH mixture was custom syn- thesized by Cambridge Isotope Laboratories (Andover, MA) and delivered to the U.S. Environmental Protection Agency (EPA) in corn oil at 3 times the highest tested concentra- tion. Concentrations of individual chemicals in the mixture (Table 2) were verified by chro- matographic/mass spectrophotometric analyses (Accustandard Corp., New Haven, CT ). The concentrations used in our present study were determined analytically and varied only slightly from the target concentrations. The ratio of chemicals in the mixture was based on the ratios of PHAHs found in breast milk, fish, and other sources of human exposure (Giesy et al. 1994; Larsen et al. 1994; Patterson et al. 1994; Schecter et al. 1994a, 1994b). All dosing solutions were prepared by dilution of the stock solutions with corn oil (Sigma Chemical Co., St. Louis, MO). Animals and dosing. We obtained female Long Evans rats (23 days of age) from Charles River Laboratory (Raleigh, NC) and allowed them to acclimate for a minimum of 4 days in an animal facility approved by the Association for Accreditation of Laboratory Animal Care before being treated. Two animals were housed per plastic cage (45 cm × 24 cm × 20 cm), with heat-treated pine shaving bedding. They were maintained at 21 ± 2°C with 50 ± 10% humidity on a 12/12-hr light/dark cycle (lights on 0600–1800 hr). Feed (Purina Rodent Chow 5001, Barnes Supply Co., Durham, NC) and tap water were provided ad libitum. All animal procedures were approved by the U.S. EPA Institutional Animal Care and Use Committee. Table 1. Chemicals tested, dose ranges, and number of doses, for individual chemicals. Dose range (µg/kg/day) No. of dosesa Chemical TCDD PCDD TCDF 1-PCDF 4-PCDF OCDF PCB-28 PCB-52 PCB-77 PCB-101 PCB-105 PCB-118 PCB-126 PCB-138 PCB-153 PCB-156 PCB-169 PCB-180 Crofton et al. 2004). This model assumes that the effects of the mixture will be predicted by the constraint of Berenbaum’s definition of additivity (Berenbaum 1985). In addition this model allows the calculation of the predicted mixture outcome for chemicals with differing dose thresholds and maximum asymptotes (Gennings et al. 2002, 2004). A short-term oral exposure model (Craft et al. 2002) was used to estimate the impact of 18 PHAHs, both alone and as dilutions of an 18-chemical mixture, on serum T4 concentrations. This exposure paradigm allowed for an economic approach to deriving extensive dose–response information (seven to nine doses per chemical) for 18 individual chemicals. Doses associated with a 30% TH decrease from control (ED30 estimates) were calculated for each chemical, rather than ED50 estimates because some chemicals had asymptotic responses at a 50% decrease. We then tested a mixture of these 18 PHAHs in which the chemical ratios were based on a rough average of concentrations found in breast milk, fish, and other food sources of human exposure (Giesy et al. 1994; Larsen et al. 1994; Patterson et al. 1994; Schecter et al. 1994a, 1994b). Concentrations of the individual chemicals in the undiluted mixture were at least an order of magnitude lower than those found to have significant biologic activity (with the exception of PCB-126, where there was an ~ 16% decrease in T4 at the dose found in the highest concen- tration of the mixture). Last, the exposures ranged from approximately background human body burdens to body burdens similar to some highly exposed populations (DeVito et al. 1995; Liem et al. 2000; Longnecker et al. 2003; Lorber 2002). We used this approach to decrease uncertainty in low-dose extrapolation in mixture testing (Feron and Groten 2002). Materials and Methods Chemicals. All individual PHAHs were obtained from Accustandard Corporation (New Haven, CT) or Radian Corporation (Austin TX) at purities > 99%. Non-coplanar PCBs were custom synthesized for 99.9% purity. The following chemicals were tested: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD); 2,3,7,8-tetrachlorodibenzofuran (TCDF); 1,2,3,7,8-pentachlorodibenzofuran (1-PCDF); 2,3,4,7,8-pentachlorodibenzofuran (4-PCDF); 1,2,3,4,6,7,8,9-octachlorodibenzo- furan (OCDF); 2,4,4´-trichlorobiphenyl (PCB-28); 2,2´,5,5´-tetrachlorobiphenyl (PCB-52); 3,3´,4,4´-tetrachlorobiphenyl (PCB-77); 2,2´,4,5,5´-pentachlorobiphenyl (PCB-101); 2,3,3´,4,4´-pentachlorobiphenyl (PCB-105); 2,3´,4,4´,5-pentachlorobiphenyl (PCB-118); 3,3´,4,4´,5-pentachlorobiphenyl (PCB-126); 2,2´,3,4,4´,5´-hexachlorobiphenyl 1550 We dosed rats by oral gavage for 4 consec- utive days with each individual chemical to establish dose–effect functions. This paradigm was previously shown to result in dose-related decreases in T4 concentrations after exposure to PCB-126 and PCB-153 (Craft et al. 2002). Dose ranges and numbers of dose groups are shown in Table 1. We spaced doses at one-half and one-third log units with the aim to have two to three doses with no measurable response, three to four doses closely spaced around the no-effect level, and two to three doses on the descending portion of the dose response. We used two to three separate blocks of animals (separate groups of animals ordered, dosed, and sampled at different dates) to map the dose response for each chemical. Blocks were used to enable testing of large dose ranges. Numbers of animals per group were not simi- lar for each block and ranged from 4 at some of the highest concentrations to 14 in some low-dose and control groups. Control animals (n = 8–14) were dosed with the vehicle (1.0 mL/kg corn oil) only. The mixture study was conducted with serial dilutions of the mix- ture (n = 12/dose). We recorded body weights daily and adjusted dosing volumes daily. Rats were semirandomly assigned to treatment groups by counterbalancing for body weights. On the day following the last dose, animals were randomly sacriﬁced by decapitation (no anesthesia) between 0800 and 1000 hr. Trunk blood was harvested and allowed to clot on ice for 45–90 min. Serum was obtained by cen- trifugation of clotted blood at 2,500 rpm at 4°C for 20 min and stored at –80°C until analysis. T4 assays. We measured serum total T4 by standard radioimmunoassay assay kits (Diagnostic Products Corporation, Los Angeles, CA) and analyzed all samples in duplicate. Intraassay and interassay coefﬁcients of variance for all assays were below 10%. Table 2. Chemical composition of the mixture. Chemical TCDD PCDD TCDF 1-PCDF 4-PCDF OCDF PCB-28 PCB-52 PCB-77 PCB-101 PCB-105 PCB-118 PCB-126 PCB-138 PCB-153 PCB-156 PCB-169 PCB-180 Concentrationa (µg/mL) 0.013 0.013 0.019 0.006 0.026 0.065 78.600 155.200 2.000 153.800 76.700 381.100 0.610 380.900 382.200 13.100 0.400 377.900 Ratio (TCDD) 1.0 1.0 1.4 0.4 1.9 4.6 5,605.3 11,074.7 141.1 10,973.4 5,468.9 27,186.0 43.1 27,168.7 27,265.9 934.4 28.1 26,957.1 Ratio (total mass) 0.000007 0.000007 0.000010 0.000003 0.000013 0.000032 0.039237 0.077523 0.000988 0.076814 0.038282 0.190302 0.000302 0.190181 0.190861 0.006541 0.000197 0.188700 0.0001–10 0.003–10 0.3–100 0.03–100 0.03–90 0.1–300 100–90,000 100–90,000 100–30,000 50–30,000 90–90,000 10–10,000 0.001–100 100–90,000 100–90,000 10–10,000 1–1,000 100–90,000 10 10 7 7 9 8 9 9 8 9 8 9 10 9 9 8 8 8 aIncludes a control group. aChemical concentration in the highest dose of the mixture administered. VOLUME 113 | NUMBER 11 | November 2005 • Environmental Health Perspectives