Polyhalogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have the ability to bind to and activate the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Structurally related compounds that bind to the AhR and exhibit biological actions similar to TCDD are commonly referred to as "dioxin-like compounds" (DLCs). Ambient human exposure to DLCs occurs through the ingestion of foods containing residues of DLCs that bioconcentrate through the food chain. Due to their lipophilicity and persistence, once internalized, they accumulate in body tissues, mainly adipose, resulting in chronic lifetime human exposure.
Since human exposure to DLCs always involves a complex mixture, the toxic equivalency factor (TEF) methodology has been developed as a mathematical tool to assess the health risk posed by complex mixtures of these compounds. The TEF methodology is a relative potency scheme that ranks the dioxin-like activity of a compound relative to TCDD, which is the most potent congener. This allows for the estimation of the potential dioxin-like activity of a mixture of chemicals, based on a common mechanism of action involving an initial binding of DLCs to the AhR.
The toxic equivalency of DLCs was nominated for evaluation because of the widespread human exposure to DLCs and the lack of data on the adequacy of the TEF methodology for predicting relative potency for cancer risk. To address this, the National Toxicology Program conducted a series of 2-year bioassays in female Harlan Sprague-Dawley rats to evaluate the chronic toxicity and carcinogenicity of DLCs and structurally related polychlorinated biphenyls (PCBs) and mixtures of these compounds.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) are not manufactured commercially other than for scientific research purposes. The main sources of TCDD and PeCDF releases into the environment are from metal smelting, refining, and processing; combustion and incineration sources; chemical manufacturing and processing; biological and photochemical processes; and existing reservior sources that reflect past releases. PCB mixtures were commercially produced and used in the electric power industry as dielectric insulating fluids in transformers and capacitors and used in hydraulic fluids, plastics, and paints. TCDD, PeCDF, and PCB 126 were selected for study by the National Toxicology Program as part of the dioxin TEF evaluation to assess the cancer risk posed by complex mixtures of polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and PCBs. The dioxin TEF evaluation includes conducting multiple 2-year rat bioassays to evaluate the relative chronic toxicity and carcinogenicity of DLC's, structurally related PCBs, and mixtures of these compounds. Female Harlan Sprague-Dawley rats were administered a mixture of TCDD, PeCDF, and PCB 126 (henceforth referred to as the TEF mixture) in corn oil:acetone (99:1) by gavage for 14, 31, or 53 weeks or 2 years. While one of the aims of the dioxin TEF evaluation was a comparative analysis across studies, in this Technical Report only the results of the present study of the mixture of TCDD, PeCDF, and PCB 126 are presented and discussed.
Groups of 81 female rats were administered 10, 22, 46, or 100 ng toxic equivalents (TEQ)/kg body weight in corn oil:acetone (99:1) by gavage, 5 days per week, for up to 105 weeks; a group of 81 vehicle control female rats received the corn oil/acetone vehicle alone. Actual doses used for each compound in the mixture were: for 10 ng TEQ/kg: 3.3 ng/kg TCDD, 6.6 ng/kg PeCDF, and 33.3 ng/kg PCB 126; for 22 ng TEQ/kg: 7.3 ng/kg TCDD, 14.5 ng/kg PeCDF, and 73.3 ng/kg PCB 126; for 46 ng TEQ/kg: 15.2 ng/kg TCDD, 30.4 ng/kg PeCDF, and 153 ng/kg PCB 126; and for 100 ng TEQ/kg: 33 ng/kg TCDD, 66 ng/kg PeCDF, and 333 ng/kg PCB 126. Up to 10 rats per group were evaluated at 14, 31, or 53 weeks. Survival of all dosed groups of rats was similar to that of the vehicle control group. Mean body weights of the 22 and 46 ng TEQ/kg groups were less than those of the vehicle control groups after week 69 of the study. Mean body weights of the 100 ng TEQ/kg group were less than those of the vehicle control group after week 37 of the study.
Alterations in serum thyroid hormone concentrations were evaluated at the 14-, 31-, and 53-week interim evaluations. At 14, 31, and 53 weeks, there were dose-dependent reductions in total serum and free thyroxine concentrations. There were dose-dependent increases in serum triiodothyronine concentrations at 14 and 31 weeks. No changes in serum thyroid stimulating hormone concentrations were observed at any time point.
To evaluate hepatocyte replication, analysis of labeling of replicating hepatocytes with 5-bromo-2'-deoxyuridine was conducted at the interim evaluations. At 14 weeks, no effects on the hepatocellular labeling index were observed in the dosed groups compared to the vehicle controls. At 31 and 53 weeks, the hepatocellular labeling index was significantly higher in the 46 and 100 ng TEQ/kg groups compared to the vehicle controls.
To evaluate the expression of known dioxin-responsive genes, CYP1A1-associated 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A2-associated acetanilide-4-hydroxylase (A4H) activity were evaluated at the interim time points. Liver and lung EROD (CYP1A1) activities and hepatic A4H (CYP1A2) activities were significantly greater in all dosed groups than in the vehicle controls at all interim evaluations (14, 31, and 53 weeks).
Tissue concentrations of TCDD, PeCDF, and PCB 126 were analyzed in the fat, liver, lung, and blood at each interim evaluation and at the end of the 2-year study (105 weeks). The highest concentrations of TCDD, PeCDF, and PCB 126 were observed in the liver followed by fat. Liver and fat concentrations of TCDD, PeCDF, and PCB 126 at each interim evaluation and at 105 weeks were higher in groups with increasing doses of the mixture and generally increased with duration of dosing. In the lung, PeCDF was present at detectable concentrations in the 46 and 100 ng TEQ/kg groups at 14 and 31 weeks. Measurable concentrations of TCDD and PCB 126 were observed at 14 and 31 weeks in the lung of rats in all dosed groups with the highest concentrations observed in the 100 ng TEQ/kg group. At 53 weeks, concentrations of TCDD, PeCDF, and PCB 126 in the lung generally increased with increasing dose. At 105 weeks, detectable concentrations of TCDD, PeCDF, and PCB 126 in the lung were observed in all dosed groups. In blood, TCDD and PCB 126 concentrations at 14 and 31 weeks generally increased with increasing dose. Blood concentrations of PeCDF were detectable in the 46 and 100 ng TEQ/kg groups at 14 weeks and at 22 ng TEQ/kg or greater at 31 weeks. At 53 and 105 weeks, concentrations of TCDD, PeCDF, and PCB 126 in blood generally increased with increasing dose and duration of dosing.
Relative liver weights were significantly increased in all dosed groups at 14, 31, and 53 weeks and correlated with increased incidences of hepatocellular hypertrophy. Increasing duration of exposure led to an increase in the spectrum, incidence, and severity of nonneoplastic effects. The only significant effect at 14 weeks was increased incidences of hepatocellular hypertrophy. At 53 weeks, there was a significant effect on the incidences of hepatocellular hypertrophy, multinucleated hepatocytes, pigmentation, focal fatty change, bile duct hyperplasia, and toxic hepatopathy.
At 2 years, there were significant increases in the incidences of hepatocellular adenoma and cholangiocarcinoma of the liver. There was an increase in hepatic toxicity characterized by increases in the incidences of numerous nonneoplastic lesions including hepatocyte hypertrophy, multinucleated hepatocytes, pigmentation, inflammation, diffuse fatty change, bile duct hyperplasia, oval cell hyperplasia, nodular hyperplasia, eosinophilic focus, cholangiofibrosis, bile duct cysts, necrosis, portal fibrosis, mixed cell focus, and toxic hepatopathy.
In the lung, there were dose-dependent increases in the incidences of bronchiolar metaplasia of the alveolar epithelium at 53 weeks and at 2 years and squamous metaplasia at 2 years. At 2 years, there was a dose-dependent increase in the incidences of cystic keratinizing epithelioma.In the pancreas, there were increases in the incidences of numerous nonneoplastic lesions including arterial chronic active inflammation, acinar cytoplasmic vacuolization, acinar atrophy, chronic active inflammation, and duct dilatation. At 2 years, incidences of acinar adenoma or acinar carcinoma that exceeded the historical control ranges were seen in all dosed groups except the 100 ng TEQ/kg group.
Treatment-related increases in the incidences of nonneoplastic lesions were seen in other organs including hyperplasia, cystic degeneration, atrophy, and cytoplasmic vacuolization of the adrenal cortex; gingival squamous hyperplasia of the oral mucosa; squamous metaplasia of the uterus; atrophy of the thymus (incidence and severity); chronic active inflammation of the ovary; nephropathy of the kidney (incidence and severity); cardiomyopathy; bone marrow hyperplasia; transitional epithelium of the urinary bladder; chronic active inflammation of the mesenteric artery; and follicular cell hypertrophy of the thyroid gland.
Under the conditions of this 2-year gavage study, there was clear evidence of carcinogenic activity of the mixture of TCDD, PeCDF, and PCB 126 in female Harlan Sprague-Dawley rats based on increased incidences of hepatocellular adenoma and cholangiocarcinoma of the liver and cystic keratinizing epithelioma of the lung. Neoplasms of the pancreatic acinus may have been related to administration of the mixture of TCDD, PeCDF, and PCB 126.
Administration of the mixture of TCDD, PeCDF, and PCB 126 caused increased incidences of nonneoplastic lesions of the liver, lung, pancreas, adrenal cortex, oral mucosa, uterus, thymus, ovary, kidney, heart, bone marrow, urinary bladder, mesenteric artery, and thyroid gland in female rats.
TCDD: Dioxin; dioxine; TCDBD; 2,3,7,8-TCDD; 2,3,7,8-tetrachlorodibenzo(b,e)(1,4)dioxin; 2,3,7,8-tetrachlorodibenzo-p-dioxin; 2,3,6,7-tetrachlorodibenzo-p-dioxin; 2,3,7,8-tetrachlorodibenzo-1,4-dioxin; tetrachlorodibenzodioxin; 2,3,6,7-tetrachlorodibenzoidioxin; tetradioxin
PeCDF: Dibenzofuran, 2,3,4,7,8-pentachloro-; 2,3,4,7,8-PeCDF; 2,3,4,7,8-PnCDF; 2,3,4,7,8-penta-CDF
PCB 126: 1,1'-Biphenyl, 3,3',4,4',5-pentachloro-(9Cl)
Summary of the 2-Year Carcinogenesis Study of the TEF Mixture in Female Sprague-Dawley Rats
Doses in corn oil/acetone by gavage
0 ng TEQ/kg
10 ng TEQ/kg (3.3 ng/kg TCDD, 6.6 ng/kg PeCDF, 33.3 ng/kg PCB 126)
22 ng TEQ/kg (7.3 ng/kg TCDD, 14.5 ng/kg PeCDF, 73.3 ng/kg PCB 126)
46 ng TEQ/kg (15.2 ng/kg TCDD, 30.4 ng/kg PeCDF, 153 ng/kg PCB 126)
100 ng TEQ/kg (33 ng/kg TCDD, 66 ng/kg PeCDF, 333 ng/kg PCB 126)
22, 46, and 100 ng TEQ/kg groups less than the vehicle control group
16/53, 23/53, 24/53, 23/53, 8/53
hepatocyte hypertrophy (1/53, 27/53, 34/53, 46/53, 50/51)
multinucleated hepatocyte (0/53, 12/53, 10/53, 39/53, 51/51)
pigmentation (4/53, 35/53, 41/53, 48/53, 51/51)
inflammation (36/53, 50/53, 45/53, 50/53, 50/51)
diffuse fatty change (3/53, 5/53, 14/53, 34/53, 36/51)
bile duct hyperplasia (2/53, 3/53, 5/53, 25/53, 42/51)
oval cell hyperplasia (0/53, 1/53, 1/53, 26/53, 42/51)
nodular hyperplasia (0/53, 1/53, 3/53, 11/53, 38/51)
eosinophilic focus (5/53, 9/53, 11/53, 20/53, 19/51)
cholangiofibrosis (0/53, 2/53, 3/53, 4/53, 17/51)
bile duct cyst (1/53, 3/53, 3/53, 4/53, 9/51)
necrosis (3/53, 1/53, 9/53, 3/53, 15/51)
portal fibrosis (0/53, 0/53, 0/53, 0/53, 11/51)
mixed cell focus (21/53, 32/53, 35/53, 36/53, 17/51)
toxic hepatopathy (0/53, 5/53, 14/53, 38/53, 47/51)
alveolar epithelium, metaplasia, bronchiolar (0/53, 20/53, 33/53, 41/53, 40/53)
squamous metaplasia (2/53, 0/53, 2/53, 8/53, 11/53)
arterial chronic active inflammation (0/52, 6/53, 3/53, 8/53, 14/51)
acinar cytoplasmic vacuolization (1/52, 0/53, 3/53, 15/53, 30/51)
acinar atrophy (3/52, 2/53, 7/53, 7/53, 20/51)
chronic active inflammation (3/52, 1/53, 6/53, 7/53, 16/51)
duct dilatation (0/52, 0/53, 0/53, 0/53, 5/51)
hyperplasia (12/52, 26/53, 23/53, 25/53, 21/51)
cystic degeneration (9/52, 15/53, 19/53, 25/53, 16/51)
atrophy (0/52, 3/53, 0/53, 0/53, 18/51)
cytoplasmic vacuolization (6/52, 13/53, 11/53, 7/53, 15/51)
gingival squamous hyperplasia (8/53, 17/53, 18/53, 26/53, 30/53)
squamous metaplasia (21/52, 32/53, 32/53, 35/53, 30/51)
atrophy (32/52, 43/48, 45/50, 50/53, 48/50)
severity of atrophy (2.3, 2.9, 3.3, 3.7, 3.9)
chronic active inflammation (0/52, 2/52, 1/53, 2/53, 5/51)
nephropathy (26/52, 41/53, 40/53, 47/53, 49/51)
severity of nephropathy (1.1, 1.2, 1.3, 1.3, 2.1)
cardiomyopathy (11/53, 26/53, 31/53, 30/52, 32/53)
hyperplasia (36/53, 36/53, 34/53, 41/53, 48/53)
transitional epithelial hyperplasia (0/52, 0/52, 1/53, 3/53, 4/50)
arterial chronic active inflammation (0/52, 1/53, 0/53, 3/53, 6/51)
follicular cell hypertrophy (4/53, 13/53, 12/51, 18/52, 23/51)
hepatocellular adenoma (0/53, 1/53, 1/53, 1/53, 11/51)
cholangiocarcinoma (0/53, 0/53, 2/53, 7/53, 9/51)
cystic keratinizing epithelioma (0/53, 0/53, 0/53, 2/53, 20/53)
acinus adenoma or carcinoma (0/52, 1/53, 2/53, 2/53, 0/51)
Level of evidence of carcinogenic activity
Report Date: September 2006