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 adipose tissue resulting in chronic lifetime human exposure.
Since human exposure to DLCs always occurs as 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 that 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.
3,3',4,4',5-Pentachlorobiphenyl (PCB 126) was produced commercially before 1977 for the electric industry as a dielectric insulating fluid for transformers and capacitors. Manufacture and use of the chemical was stopped because of increased PCB residues in the environment, but it continues to be released into the environment through the use and disposal of products containing PCBs, as by-products during the manufacture of certain organic chemicals, and during combustion of some waste materials. Bioaccumulation of PCB 126 results in persistent levels in animal and human tissues and the biological responses to PCB 126 are similar to those of TCDD, a known human carcinogen. PCB 126 was selected for study by the National Toxicology Program as a 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 DLCs, structurally related PCBs, and mixtures of these compounds. PCB 126 was included since this is the most potent coplanar PCB that has dioxin-like activities. 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 PCB 126 study are presented and discussed. Female Harlan Sprague-Dawley rats were administered PCB 126 (99% pure) in corn oil with acetone by gavage for 14, 31, or 53 weeks or 2 years.
Groups of 81 female rats were administered 30, 100, 175, 300, 550, or 1,000 ng PCB 126/kg body weight in corn oil:acetone (99:1) by gavage, 5 days per week, for up to 104 weeks; a group of 81 vehicle control female rats received the corn oil/acetone vehicle alone. A group of 28 rats received 10 ng/kg for up to 53 weeks only. Up to 10 rats per group were evaluated at 14, 31, or 53 weeks. A stop-exposure group of 50 female rats was administered 1,000 ng/kg PCB 126 in corn oil:acetone (99:1) by gavage for 30 weeks then the vehicle for theremainder of the study.
Mean body weights of 30 and 100 ng/kg rats were similar to those of the vehicle controls during most of the study, mean body weights of 175 and 300 ng/kg rats were less than those of the vehicle controls during year 2 of the study, and mean body weights of 550 ng/kg, 1,000 ng/kg core study, and 1,000 ng/kg stop-exposure rats were less than those of the vehicle controls after week 17.
Thyroid hormone concentrations
Alterations in serum thyroid hormone levels were evaluated at the 14-, 31- and 53- week interim evaluations. In the 550 and 1,000 ng/kg rats, total thyroxine (T4) and free T4 were significantly lower than vehicle controls and serum triiodothyronine (T3) and thyroid stimulating hormone (TSH) levels were significantly higher than vehicle controls at the 14-week interim evaluation. Serum T3 was also significantly higher in the 300 ng/kg rats compared to vehicle controls at 14 weeks. At 31 weeks, T3 was significantly higher at doses of 100 ng/kg or greater compared to vehicle controls. TSH levels were higher in 550 and 1,000 ng/kg rats than in vehicle controls. At 53 weeks, significantly lower serum concentrations of total T4 and free T4 were observed compared to vehicle controls in groups administered 175 ng/kg or greater and 30 ng/kg or greater, respectively. Serum T3 levels were significantly higher at doses of 175 ng/kg or greater compared to vehicle controls. No changes in TSH were observed between vehicle controls and dosed rats at 53 weeks.
Hepatic cell proliferation data
To evaluate hepatocyte replication, analysis of labeling of replicating hepatocytes with 5-bromo-2'-deoxyuridine was conducted at the 14-, 31-, and 53-week interim evaluations. The hepatocellular labeling index was significantly higher at doses of 300 ng/kg or greater at 14 weeks and 175 ng/kg or greater at 31 weeks compared to vehicle controls. No statistically significant differences were observed between vehicle controls and PCB 126 dosed rats at 53 weeks. However at 53 weeks, a 5.8-fold increase above the vehicle controls was observed in the 1,000 ng/kg group.
Cytochrome P450 enzyme activities
To evaluate the expression of known dioxin-responsive genes, CYP1A1 associated 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A2-associated acetanilide 4-hydroxylase (A-4-H) activity were evaluated at the 14-, 31-, and 53-week interim evaluations. In addition, CYP2B associated pentoxyresorufin-O-deethylase (PROD) activity was also analysed. Hepatic PROD (CYP2B1) and hepatic and pulmonary EROD (CYP1A1) activity were significantly greater in all dosed groups than in vehicle controls at weeks 14, 31, and 53. Hepatic A-4-H (CYP1A2) activity was significantly greater in the 30, 100, 175, 300, 550, and 1,000 ng/kg groups compared to vehicle controls at weeks 14, 31, and 53.
Determinations of PCB 126 concentrations in tissues
The tissue disposition of PCB 126 was analyzed in the liver, lung, fat, and blood of all rats in vehicle controls and all dosed groups at the 14-, 31-, and 53-week interim evaluations and in 10 rats per group including vehicle controls at the end of the 2-year study (104 weeks). Detectable concentrations of PCB 126 were observed in the liver, fat, lung, and blood. Measurable concentrations of PCB 126 were present in the liver and fat at weeks 31, 53, and 104. Hepatic and fat concentrations increased with increasing doses of PCB 126. Measurable concentrations of PCB 126 were present in vehicle control lung tissue at 53 and 104 weeks. No PCB 126 was observed in the blood from the vehicle control rats. Lung and blood concentrations tended to increase with increasing doses of PCB 126, with a few exceptions. In the stop-exposure group, PCB 126 concentrations in liver and fat were lower than the levels observed in the 30 ng/kg group. In the stop-exposure group, lung tissue PCB 126 concentrations were equivalent to the levels observed in the 30 ng/kg group. In blood from the stop-exposure group, PCB 126 concentrations were equivalent to the levels observed in the 100 ng/kg group.
Pathology and statistical analyses
Absolute and relative liver weights were significantly increased at all time points and correlated with increased incidences of hepatocellular hypertrophy. At 2 years, there were significant treatment-related increases in the incidences of cholangiocarcinoma and hepatocellular adenoma. Three hepatocholangiomas were seen in the 1,000 ng/kg core study group and a single incidence of cholangioma each occurred in the 550 and 1,000 ng/kg core study groups.
At 2 years, a significant dose-related increase in hepatic toxicity was observed and was characterized by increased incidences of numerous lesions including hepatocyte hypertrophy, multinucleated hepatocytes, diffuse fatty change, bile duct hyperplasia, bile duct cyst, oval cell hyperplasia, necrosis, pigmentation, inflammation, nodular hyperplasia, portal fibrosis, cholangiofibrosis, and toxic hepatopathy. The incidences of these lesions were generally decreased in the 1,000 ng/kg stop-exposure group compared to the 1,000 ng/kg core study group.
The lung weights of 1,000 ng/kg rats were generally significantly increased at weeks 14, 31, and 53. At 2 years, treatment related increases in the incidences of cystic keratinizing epithelioma and squamous cell carcinomas were observed. In addition, dose-related increases in the incidences of bronchiolar metaplasia of the alveolar epithelium and squamous metaplasia were also observed.
The incidence of gingival squamous cell carcinoma of the oral mucosa was significantly increased in the 1,000 ng/kg core study group at 2 years. Gingival squamous cell carcinoma, although reduced in incidence as compared to the 1,000 ng/kg core study group, was still present in the 1,000 ng/kg stop-exposure group.
At 2 years, adenomas and/or carcinomas were present in the adrenal cortex of most core study groups and in the 1,000 ng/kg stop-exposure group. Dose-related effects on the incidences of adrenal cortex atrophy and cytoplasmic vacuolization were also seen.
There were dose-related increases in the incidences of numerous nonneoplastic responses including: chronic active inflammation, acinar atrophy, and acinar cytoplasmic vacuolation of the pancreas and chronic active inflammation of the pancreatic arteries; nephropathy; cardiomyopathy; follicular cell hypertrophy of the thyroid gland; thymic atrophy; clitoral gland cystic ducts; chronic active inflammation of the mesenteric artery; and, lymphoid follicular atrophy of the spleen.
Under the conditions of this 2-year gavage study there was clear evidence of carcinogenic activity of PCB 126 in female Harlan Sprague-Dawley rats based on increased incidences of cholangiocarcinoma of the liver, squamous neoplasms of the lung (cystic keratinizing epithelioma and squamous cell carcinoma), and gingival squamous cell carcinoma of the oral mucosa. Hepatocellular adenoma and hepatocholangioma of the liver were also considered to be related to the administration of PCB 126. Neoplasms of the adrenal cortex and cholangioma of the liver may have been related to administration of PCB 126.
PCB 126 administration caused increased incidences of nonneoplastic lesions of the liver, lung, adrenal cortex, pancreas, kidney, heart, thyroid gland, thymus, spleen, clitoral gland, and mesenteric artery in female rats.