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 body tissue, mainly adipose, 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, 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.
Mixtures of polychlorinated biphenyls (PCBs) including 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118) were produced commercially before 1977 for the electric industry as dielectric insulating fluids for transformers and capacitors. Manufacture and use of these chemicals were stopped because of increased PCB residues in the environment, but they continue 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, during combustion of some waste materials, and during atmospheric recycling. This PCB mixture study was conducted as part of the dioxin TEF evaluation that 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. This study was originally a study of PCB 118 alone. However, midway through the study PCB 126 was identified as one of the minor contaminants (0.622%) of the bulk PCB 118 (98.5% pure). Given the 1,000-fold higher potency of PCB 126 for inducing dioxin-like effects (based on the TEFs for PCB 126 and PCB 118 of 0.1 and 0.0001, respectively), it was expected that the effects of administration of this compound would be due to the combined dioxin-like effects of both PCB 126 and PCB 118. Therefore, this study was reclassified as a mixture study of PCB 126 and PCB 118.
Groups of female Harlan Sprague-Dawley rats were administered the PCB mixture containing PCB 126 and PCB 118 by gavage in corn oil:acetone (99:1) or vehicle alone, 5 days per week for up to 104 weeks. Dose groups are referred to by the total levels of TCDD toxic equivalents (TEQ) provided by the PCBs in the mixture in each dose group. Groups of 81 female rats were administered 7, 22, 72, or 216 ng TEQ/kg; a group of 86 female rats was administered 360 ng TEQ/kg; and a group of 81 female rats was administered the corn oil:acetone vehicle alone. Up to 10 rats per group were evaluated at 14, 31, or 53 weeks. No animals in the 360 ng TEQ/kg group were examined at 53 weeks. A group of 50 female rats was administered 360 ng TEQ/kg for 30 weeks and then the vehicle alone for the remainder of the study. Nominal doses of PCB 118 and levels of PCB 126 in each dose group used were:
|7 ng TEQ/kg dose group:||62 ng/kg PCB 126 and 10 µg/kg PCB 118|
|7 ng TEQ/kg dose group:||62 ng/kg PCB 126 and 10 µg/kg PCB 118|
|22 ng TEQ/kg dose group:||187 ng/kg PCB 126 and 30 µg/kg PCB 118|
|72 ng TEQ/kg dose group:||622 ng/kg PCB 126 and 100 µg/kg PCB 118|
|216 ng TEQ/kg dose group:||1,866 ng/kg PCB 126 and 300 µg/kg PCB 118|
|360 ng TEQ/kg dose group:||3,110 ng/kg PCB 126 and 500 µg/kg PCB 118|
No animals in the 216 or 360 ng TEQ/kg core study groups survived to the end of the study, and survival in the 360 ng TEQ/kg stop-exposure group was significantly less than in the vehicle control group. Mean body weights of 72 ng TEQ/kg rats were less than those of the vehicle controls after week 33 of the study, and mean body weights of the 216 and 360 ng TEQ/kg core study rats and the 360 ng TEQ/kg stop-exposure group rats were less than those of the vehicle controls throughout most of the study. Clinical findings related to the administration of the binary mixture of PCB 126 and PCB 118 included abnormal breathing, thinness, and ruffled hair.
Thyroid Hormone Concentrations
Alterations in serum thyroid hormone levels were evaluated at the 14-, 31-, and 53-week interim evaluations. Total thyroxine (T4) and free T4 were significantly lower in most dose groups than in vehicle controls at the 14- and 31-week interim evaluations. Serum T3 was significantly lower in the 360 ng TEQ/kg group compared to vehicle controls at 31 weeks only. TSH levels were higher in the 216 and 360 ng TEQ/kg groups than in vehicle controls at 31 weeks only.
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. Labeling indices were elevated at doses above 216 ng TEQ/kg at 31 weeks and at doses above 72 ng TEQ/kg at 53 weeks.
Cytochrome P450 Enzyme Activities
CYP1A1-associated 7-ethoxyresorufin-O-deethylase (EROD) and CYP1A2-associated acetanilide-4-hydroxylase (A4H) activities were evaluated at the 14-, 31-, and 53-week interim evaluations to evaluate the expression of known dioxin-responsive genes. In addition, CYP2B-associated pentoxyresorufin-O-deethylase (PROD) activity was also analyzed. Hepatic and pulmonary EROD (CYP1A1) activity, hepatic A4H (CYP1A2) activity, and hepatic PROD (CYP2B1) activity were significantly greater in all dosed groups compared to the vehicle controls at weeks 14, 31, and 53.
Determinations of PCB 126 and PCB 118 Concentrations in Tissues
The tissue disposition of PCB 126 and PCB 118 was analyzed in the liver, lung, fat, and blood of up to 10 rats in each group at the 14-, 31-, and 53-week interim evaluations, except for the 360 ng TEQ/kg group at 53 weeks. The tissue disposition of PCB 126 and PCB 118 was also analyzed in 10 rats per group at the end of the 2-year study in the vehicle control, 7, 22, and 72 ng TEQ/kg core study groups and the 360 ng TEQ/kg stop-exposure group. Detectable concentrations of PCB 126 and PCB 118 were observed in the liver, fat, lung, and blood. The highest levels of PCB 126 were seen in the liver whereas the highest levels of PCB 118 were seen in the fat. In general, tissue concentrations increased with increasing doses of the mixture and increasing duration of exposure. Hepatic levels of PCB 126 and PCB 118 in the 72 ng TEQ/kg group at the end of the 2-year study were 284 ng/g and 3,769 ng/g, respectively. On a TCDD equivalents basis this corresponds to 28 ng TEQ/g and 0.4 ng TEQ/g for PCB 126 and PCB 118, respectively. Cessation of administration of the mixture in the stop-exposure group led to declines in the tissue concentrations of both PCB 126 and PCB 118 to levels comparable to those observed in the 7 ng TEQ/kg group at the end of the 2-year study.
Pathology and Statistical Analyses
At 14, 31, and 53 weeks, liver weights were significantly increased in treated groups with more pronounced effects occurring in the higher dose groups. At 14 weeks, hepatocyte hypertrophy and pigmentation were seen at doses less than 72 ng TEQ/kg. Exposure to the PCB mixture led to significant toxicity in the liver. At higher doses, the incidences of toxic hepatopathy were increased as indicated by increased incidences of multinucleated hepatocytes and diffuse fatty change. At 31 weeks, most rats in the 216 and 360 ng TEQ/kg groups had multiple hepatic nonneoplastic lesions. At 53 weeks all animals administered 216 ng TEQ/kg had multiple nonneoplastic lesions. The spectrum of effects and the severity of effects at the interim and 2-year time points increased with dose and duration of exposure. At the end of the 2-year study in all dosed groups, there were significantly increased incidences and severity of toxic hepatopathy characterized by hepatocyte hypertrophy, multinucleated hepatocytes, pigmentation, toxic hepatopathy, diffuse fatty change, nodular hyperplasia, centrilobular fibrosis, cholangiofibrosis, oval cell hyperplasia, bile duct cyst, bile duct hyperplasia, and portal fibrosis. There were also increased incidences of hepatocyte glandular structures, necrosis, centrilobular degeneration, eosinophilic focus, and metaplasia.
The incidences of cholangiocarcinoma (multiple and/or single) were significantly increased in groups administered 22 ng TEQ/kg or greater at 2 years. The incidences of hepatocellular adenoma were also significantly increased in the 216 and 360 ng TEQ/kg core study groups. In addition, single occurrences of hepatocholangioma, cholangioma, or hepatocellular carcinoma were observed in some dosed groups administered 72 ng TEQ/kg or greater.
In the lung at 53 weeks, the incidences of cystic keratinizing epithelioma and bronchiolar metaplasia were significantly increased in the 216 ng TEQ/kg group. Significantly increased incidences of cystic keratinizing epithelioma (single or multiple) occurred in groups administered 72 ng TEQ/kg or greater at 2 years. Nonneoplastic effects observed in the lung included bronchiolar metaplasia of the alveolar epithelium, squamous metaplasia, serosal fibrosis, and keratin cysts in the stop-exposure group.
Increased incidences of gingival squamous cell carcinoma of the oral mucosa were observed at the end of the 2-year study and were accompanied by increased incidences of gingival squamous hyperplasia of the oral mucosa.
Numerous nonneoplastic effects were seen in other organs at the interim time points including atrophy of the thymus, follicular cell hypertrophy of the thyroid gland, atrophy of the adrenal cortex and acinar cytoplasmic vacuolization of the pancreas. These responses were also affected by administration of PCB 126:PCB 118 at the end of the 2-year study and were accompanied by additional nonneoplastic responses in numerous organs including vacuolization of the adrenal cortex, acinar atrophy of the pancreas, and chronic active inflammation of the pancreatic acinus. In the kidney, severity of nephropathy was increased. Effects on the cardiovascular system were seen including cardiomyopathy of the heart, chronic active inflammation of the coronary artery, inflammation of the epicardium, and chronic active inflammation of the mesentric and pancreatic arteries. Other nonneoplastic lesions that were treatment related were hemorrhage of the brain and mandibular, mesenteric, and mediastinal lymph nodes; forestomach hyperplasia; hyperplasia of the nasal respiratory epithelium; metaplasia of the olfactory epithelium; and atrophy of the lymphoid follicle of the spleen.
Under the conditions of this 2-year gavage study there was clear evidence of carcinogenic activity of the mixture of PCB 126 and PCB 118 in female Harlan Sprague-Dawley rats based on increased incidences of cholangiocarcinoma and hepatocellular neoplasms (predominantly hepatocellular adenomas) of the liver, and cystic keratinizing epithelioma of the lung. The marginally increased incidences of gingival squamous cell carcinoma of the oral mucosa were also considered to be related to administration of the mixture of PCB 126 and PCB 118. Occurrences of cholangioma and hepatocholangioma of the liver may have been related to administration of the mixture of PCB 126 and PCB 118.
Administration of the mixture of PCB 126 and PCB 118 caused increased incidences of nonneoplastic lesions in the liver, lung, oral mucosa, thymus, thyroid gland, adrenal cortex, pancreas, kidney, heart, lymph nodes, mesenteric artery, brain, forestomach, spleen, and nose.