Dioxin Toxic Equivalency Factor Evaluation Overview
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 human 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.
2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) is not manufactured commercially other than for scientific research purposes. The main sources of PeCDF releases into the environment are from combustion and incineration sources. PeCDF 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. 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 PeCDF study are presented and discussed. Female Harlan Sprague-Dawley rats were administered PeCDF (at least 97% pure) in corn oil:acetone (99:1) by gavage for 14, 31, or 53 weeks or 2 years.
2-Year Study in Rats
Groups of 81 female rats were administered 6, 20, 44, 92, or 200 ng PeCDF/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. Up to 10 rats per group were evaluated at 14, 31, and 53 weeks. A stop-exposure group was administered 200 ng/kg PeCDF in corn oil:acetone (99:1) by gavage for 30 weeks and then the vehicle for the remainder of the study. The PeCDF in this study was at least 97% pure. Survival of dosed groups was similar to that of the vehicle control group. Mean body weights of the 200 ng/kg core and stop-exposure groups were less than those of the vehicle controls during year 2 of the study.
Thyroid Hormone ConcentrationsAlterations in serum thyroid hormone levels were evaluated at the 14-, 31- and 53-week interim evaluations. There were significant decreases in total serum thyroxine (T4) levels at the 14-week interim evaluation. There were no significant differences observed in serum free T4, total triiodothyronine (T3), or thyroid stimulating hormone (TSH) at 14 weeks. At both 31 and 53 weeks, there were treatment-related decreases in free and total T4 concentrations and increases in serum T3 levels. Serum TSH levels in dosed groups at 31 and 53 weeks were not significantly different than in the vehicle controls.
Hepatic Cell Proliferation Data
To evaluate hepatocyte replication, analysis of labeling of replicating hepatocytes with 5-bromo-2'-deoxyuridine (BrdU) was conducted at the 14-, 31-, and 53-week interim evaluations. At 14 and 53 weeks, hepatocyte BrdU-labeling indices were significantly higher in the 200 ng/kg groups compared to time-matched vehicle controls. No significant differences were observed between the dosed groups and vehicle controls at 31 weeks.
Cytochrome P450 Enzyme ActivitiesTo 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 14-, 31-, and 53-week interim evaluations. Hepatic EROD and A4H activities were significantly higher in all groups administered PeCDF relative to the vehicle controls at all three interim evaluations. Pulmonary EROD was also significantly higher in all dosed groups compared to vehicle controls at 14, 31, and 53 weeks.
Determinations of PeCDF Concentrations in Tissues
The tissue disposition of PeCDF was analyzed in the liver, lung, fat, and blood of all animals at the 14-, 31-, and 53-week interim evaluations, and in 10 animals per group at the end of the 2-year study (105 weeks). In the liver of vehicle controls, PeCDF concentrations were detectable at 105 weeks. Measurable concentrations of PeCDF were not detected in fat or lung from vehicle control rats at any of the interim evaluations or at 105 weeks. Hepatic and fat concentrations were higher in groups with increasing doses of PeCDF, demonstrating a dose-related increase in tissue burden of PeCDF at each time point. No measurable concentrations of PeCDF were detected in the lungs of vehicle controls or any of the dosed groups at 14 weeks or in the lungs of the vehicle control group at 31, 53, and 105 weeks, or the 6 ng/kg group at 31 and 53 weeks. In groups with measurable levels, PeCDF concentrations were higher with respect to increasing doses. Mean levels of PeCDF in the liver, fat, lung, and blood in the 200 ng/kg group at the end of the 2-year study were 500 ng/g, 7.75 ng/g, 0.28 ng/g and 0.04 ng/mL, respectively. Negligible PeCDF concentrations were observed in blood of the 200 ng/kg group at 53 weeks and the 92 and 200 ng/kg groups at 105 weeks. In liver and fat from the stop-exposure group, the PeCDF concentrations were between the levels observed in the 6 and 20 ng/kg groups. In the stop-exposure group, PeCDF concentration in lung was comparable to levels observed in the 6 ng/kg group. No measurable concentrations were observed in blood from the stop-exposure group.
Pathology and Statistical Analyses
There were dose-dependent increases in both absolute and relative liver weights at 4, 31, and 53 weeks, and these tended to correlate with increased incidences of hepatocellular hypertrophy. In the liver at 14 weeks, the only significant effect was an increase in the incidences of hepatocellular hypertrophy. At 53 weeks, there were significant increases in the incidences of hepatocellular hypertrophy and pigmentation.
At 2 years, there were significant dose-dependent trends for increased incidences of hepatocellular adenoma and cholangiocarcinoma of the liver. A significant dose-dependent increase in hepatic toxicity was observed and was characterized by increased incidences of numerous nonneoplastic lesions including hepatocellular hypertrophy, multinucleated hepatocytes, oval cell hyperplasia, diffuse fatty change, pigmentation, nodular hyperplasia, eosinophilic foci, hepatocellular necrosis, bile duct hyperplasia, bile duct fibrosis, cholangiofibrosis, and toxic hepatopathy.At 2 years, three gingival squamous cell carcinomas of the oral mucosa were seen in the 200 ng/kg core and stop-exposure groups, two occurred in the 6 ng/kg group, and one occurred in each of the vehicle control, 20 ng/kg, and 92 ng/kg groups. Gingival squamous hyperplasia occurred in all groups including the vehicle controls, with increasing incidences in groups administered 44 ng/kg or greater.
The incidence of carcinoma of the uterus was marginally increased in the 92 ng/kg group at 2 years. Increased incidences of chronic active inflammation of the uterus were observed in all dosed groups, and the incidence in the 200 ng/kg stop-exposure group was greater than those in the vehicle control and 200 ng/kg core study groups. Increased incidences of squamous metaplasia of the uterus occurred in all dosed groups. In the 200 ng/kg stop-exposure group, the incidence of squamous metaplasia was significantly greater than that in the vehicle controls, but was lower than that in the 200 ng/kg core study group.
At 14-weeks, lung weights were significantly increased in the 200 ng/kg group compared to the vehicle controls. A single occurrence of a multiple cystic keratinizing epithelioma of the lung was observed in the 200 ng/kg core study group. There were increases in the incidences of bronchiolar metaplasia of the alveolar epithelium and sporadic incidences of squamous metaplasia.
One pancreatic acinar adenoma and one pancreatic acinar carcinoma were each observed in the 92 ng/kg group and in the 200 ng/kg stop-exposure group at 2 years. Significantly increased incidences of acinar cytoplasmic vacuolization and arterial chronic active inflammation and increased severity of chronic active inflammation were observed in the 200 ng/kg core study group.Numerous nonneoplastic effects were seen in other organs including thyroid follicular cell hypertrophy, thymic atrophy, adrenal cortex cystic degeneration, nephropathy, cardiomyopathy, and squamous hyperplasia of the forestomach.
There were significantly increased incidences of mammary gland carcinoma in the 6 and 20 ng/kg groups at 2 years, with a trend to lower adjusted incidences in higher dose groups, although there was no clear dose-response pattern. Similarly, there was significantly increased incidence of pituitary gland adenoma in the 6, 20, 44, and 92 ng/kg groups. Mammary gland fibroadenoma, a spontaneous lesion in female rats, occurred at a high incidence in all groups, but was not considered treatment related.
Under the conditions of this 2-year gavage study, there was some evidence of carcinogenic activity of PeCDF in female Harlan Sprague-Dawley rats, based on increased incidences of hepatocellular adenoma and cholangiocarcinoma of the liver and gingival squamous cell carcinoma of the oral mucosa. Occurrences of cystic keratinizing epithelioma of the lung, neoplasms of the pancreatic acinus, and carcinoma of the uterus may have been related to administration of PeCDF.
PeCDF administration caused increased incidences of nonneoplastic lesions of the liver, oral mucosa, uterus, lung, pancreas, thyroid gland, thymus, adrenal cortex, kidney, heart, and forestomach in female rats.