TCDD administration caused increased incidences of nonneoplastic lesions of the liver, lung, oral mucosa, pancreas, thymus, adrenal cortex, heart, clitoral gland, kidney, forestomach, mesentery, and thyroid gland in female rats.
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 bio-concentrate 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 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.
TCDD is not manufactured commercially other than for scientific research purposes. The main sources of TCDD releases into the environment are from combustion and incineration; metal smelting, refining, and processing; chemical manufacturing and processing; biological and photochemical processes; and existing reservoir sources that reflect past releases. TCDD (dioxin) 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 TCDD results are presented and discussed. TCDD was included because it is the reference compound for the dioxin TEF methodology. Female Harlan Sprague-Dawley rats were administered TCDD (at least 98% pure) in corn oil:acetone (99:1) by gavage for 14, 31, or 53 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse bone marrow cells.
Groups of 81 or 82 female rats were administered 3, 10, 22, 46, or 100 ng TCDD/kg body weight in corn oil:ace-tone (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, or 53 weeks. A stop-exposure group of 50 female rats was administered 100 ng/kg TCDD in corn oil:acetone (99:1) by gavage for 30 weeks and then just the vehicle for the remainder of the study.
Survival of dosed groups was similar to that of the vehicle control group. Mean body weights of 100 ng/kg core study and stop-exposure groups were less than those of the vehicle control group after week 13 of the study. Mean body weights of 46 ng/kg rats were less than those of the vehicle controls during year 2 of the study, and those of 22 ng/kg rats were less than those of the vehicle controls the last 10 weeks of the study.
Thyroid hormone concentrations
Alterations in serum thyroid hormone levels were evaluated at the 14-, 31- and 53-week interim evaluations. At 14 weeks, there were significant decreases in serum total and free thyroxine (T4) levels and increases in serum total triiodothyronine (T3) and thyroid stimulating hormone (TSH). At 31 weeks, there were significant decreases in serum total and free T4 levels and increases in serum total T3 but no significant effect on TSH. At 53 weeks, there were significant decreases in serum total T4 levels and increases in serum total T3. There were no significant effects on total T4 or TSH levels.
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 in the 22 ng/kg group compared to vehicle controls at 14 weeks. At the 31-week interim evaluation, the labeling indices in hepatocytes were significantly higher in all dosed groups than in the vehicle controls. At 53 weeks, labeling indices were significantly higher in the 46 and 100 ng/kg groups than in the vehicle controls.
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 (A4H) activity were evaluated at 14, 31, and 53 weeks. In addition, pentoxyre-sorufin-O-deethylase (PROD) activity was analyzed. Hepatic EROD, PROD, and A4H activities were significantly higher in all dosed groups relative to vehicle controls at the 14-, 31-, and 53-week interim evaluations. Pulmonary EROD was also significantly higher in all dosed groups compared to vehicle controls at 14, 31, and 53 weeks.
Determinations of TCDD concentrations in tissues
The tissue disposition of TCDD was analyzed in the liver, lung, fat, and blood of all animals in each group 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). The highest concentrations of TCDD were observed in the liver, followed by fat tissue. Liver and fat tissue concentrations of TCDD increased with increasing doses of TCDD. No measurable concentrations of TCDD were observed in blood from vehicle control or treated rats at any of the interim evaluations. Mean levels of TCDD in the liver and fat in the 100 ng/kg group at the end of the 2-year study were 9.3 and 3.2 ng/g, respectively. In liver tissue from the stop-exposure group, TCDD concentrations were slightly higher than those observed in the 3 ng/kg group. In the stop-exposure group, TCDD concentrations in fat were below the limits of quantitation.
Pathology and statistical analyses
Absolute and/or relative liver weights were increased at 14, 31, and 53 weeks, with more severe effects occurring in the higher dosed groups. Increased liver weights correlated with increased incidences of hepatocyte hypertrophy at 14, 31, and 53 weeks.
Exposure led to a treatment-related increase in hepatic toxicity with a broad spectrum of lesions. Incidences and severities of lesions increased at higher doses and longer durations of exposure. The earliest effects were increased incidences and severities of hepatocyte hypertrophy at 14 weeks. At 2 years, there was a significant increase in toxic hepatopathy characterized by increased incidences of numerous nonneoplastic liver lesions including hepatocyte hypertrophy, multinucleated hepatocytes, altered hepatocellular foci, inflammation, pigmentation, diffuse fatty change, necrosis, portal fibrosis, oval cell hyperplasia, bile duct hyperplasia, bile duct cysts, cholangiofibrosis, and nodular hyperplasia
At 2 years, the incidence of hepatocellular adenoma was significantly increased in the 100 ng/kg core study group. Dose-related increased incidences of cholangiocarcinoma were seen in core study rats administered 22 ng/kg or greater. The highest incidence of cholangiocarcinoma was seen in the 100 ng/kg core study group and included a significant number of animals with multiple cholangiocarcinomas. Two cholangiocarcinomas and two hepatocellular adenomas were seen in the 100 ng/kg stop-exposure group. Two hepatocholangiomas were seen in the 100 ng/kg core study group, and one cholangioma was seen in the 100 ng/kg stop-exposure group.
In the lung, the incidence of cystic keratinizing epithelioma of the lung was significantly increased at 2 years in the 100 ng/kg core study group. Nonneoplastic effects in the lung included increased incidences of bronchiolar metaplasia.
The incidence of gingival squamous cell carcinoma of the oral mucosa was significantly increased in the 100 ng/kg core study group at 2 years and was accompanied by an increased incidence of gingival squamous hyperplasia.
At 2 years, the incidence of squamous cell carcinoma of the uterus in the 46 ng/kg group was significantly increased, and there were two squamous cell carcinomas in the 100 ng/kg stop-exposure group.
At 2 years, one acinar adenoma and two acinar cell carcinomas of the pancreas were seen in the 100 ng/kg core study group; one acinar carcinoma was seen in the 100 ng/kg stop-exposure group. The incidences of acinar cell adenoma or carcinoma (combined) exceeded the historical vehicle control range. Nonneoplastic effects in the lung included acinar cytoplasmic vacuolization, chronic active inflammation, acinar atrophy, and arterial chronic active inflammation.
Numerous nonneoplastic effects were seen in other organs including thymic atrophy, adrenal cortex atrophy, adrenal cortex hyperplasia, cardiomyopathy, mesenteric artery inflammation, clitoral gland cysts, nephropathy, squamous hyperplasia of the forestomach, and thyroid gland follicular cell hypertrophy. A decrease in the incidence of ovarian atrophy was also observed.
TCDD was not mutagenic in any of several in vitro and in vivo short-term tests. No induction of gene mutations was seen in S. typhimurium strains TA98, TA100, TA1535, or TA1537 exposed to TCDD with or without S9 activation enzymes. No induction of trifluorothymidine resistance (gene mutations) was observed in L5178Y tk+/- mouse lymphoma cells tested with or without S9 activation. In cytogenetic tests with cultured Chinese hamster ovary cells, no consistently reproducible induction of sister chromatid exchanges or chromosomal aberrations were noted after culturing with TCDD with or without S9. In vivo tests in male D. melanogaster showed no induction of sex-linked recessive lethal mutations in germ cells following treatment of adult flies by injection with TCDD. In male B6C3F1 mice, no increases in the frequency of chromosomally aberrant cells were seen in bone marrow samples taken at two different posttreatment sampling times.
Under the conditions of this 2-year gavage study there was clear evidence of carcinogenic activity of TCDD in female Harlan Sprague-Dawley rats based on increased incidences of cholangiocarcinoma and hepatocellular adenoma of the liver, cystic keratinizing epithelioma of the lung, and gingival squamous cell carcinoma of the oral mucosa. The increased incidence of squamous cell carcinoma of the uterus was also considered to be related to TCDD administration. The marginally increased incidences of pancreatic acinar neoplasms and occurrences of hepatocholangioma and cholangioma of the liver may have been related to TCDD administration.