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 tissues, 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, 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.
PCBs, including 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 2,2',4,4',5,5'- hexachlorobiphenyl (PCB 153), were commercially produced between 1929 and 1977 for the electric industry as dielectric insulating fluids for transformers and capacitors. PCBs were also produced for use in hydraulic fluids, solvents, plastics, and paints. The manufacture and use of PCBs in the United States was stopped in 1977 after PCB residues increased in the environment in the 1960s and 1970s. However, PCBs 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, and during combustion of some waste materials (USEPA, 2000a). PCBs were 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 polychlorinated biphenyls (PCBs). The dioxin TEF evaluation includes conducting multiple 2-year rat bioassays to evaluate the relative chronic toxicity and carcinogenicity of dioxin-like compounds, structurally related PCBs, and mixtures of these compounds. Female Harlan Sprague-Dawley rats were administered a binary mixture of PCB 126 and PCB 153 (at least 99% pure) in corn oil:acetone (99:1) by gavage for 14, 31, or 53 weeks or 2 years. While one of the aims of this study was a comparative analysis of effects seen with PCB 126 and the mixture of PCB 126 and PCB 153, in this Technical Report only the results of the present study of PCB 126 and PCB 153 are presented and discussed.
The 2-year study of a binary mixture of PCB 126 and PCB 153 was designed to assess the carcinogenicity of a constant ratio mixture of PCB 126 and PCB 153. In addition, varying ratio mixture groups were used to assess the impact of increasing PCB 153 on the carcinogenicity of PCB 126. Dose groups were divided into two study arms (Figure 1). TCDD equivalent (TEQ) doses are based on the PCB 126 doses after adjustment for the PCB 126 TEF of 0.1.
Groups of 81 (Groups 2, 3, 5, and 7) or 80 (Groups 4 and 6) female rats received a mixture of PCB 126 and PCB 153 in corn oil:acetone (99:1) by gavage 5 days per week for up to 105 weeks; a group of 81 female rats received the corn oil:acetone (99:1) vehicle only and served as the vehicle control (Group 1). Up to 10 rats per group were evaluated at 14, 31, and 53 weeks.
Survival of all dosed groups was similar to that of the vehicle controls. The mean body weights of Groups 4 and 5 were generally less than those of the vehicle controls after week 25. The mean body weights of Group 6 were less after week 12, and those of Group 7 were less after week 8.
Group 1: Vehicle Control
Group 2: 10 ng/kg PCB 126 plus 10 µg/kg PCB 153 (1 ng TEQ/kg)
Group 3: 100 ng/kg PCB 126 plus 100 µg/kg PCB 153 (10 ng TEQ/kg)
Group 5: 300 ng/kg PCB 126 plus 300 µg/kg PCB 153 (30 ng TEQ/kg)
Group 7: 1,000 ng/kg PCB 126 plus 1,000 µg/kg PCB 153 (100 ng TEQ/kg)
Varying ratio mixture groups:
Group 4: 300 ng/kg PCB 126 plus 100 µg/kg PCB 153 (30 ng TEQ/kg)
Group 5: 300 ng/kg PCB 126 plus 300 µg/kg PCB 153 (30 ng TEQ/kg)
Group 6: 300 ng/kg PCB 126 plus 3,000 µg/kg PCB 153 (30 ng TEQ/kg)
Study Arms and Dose Groups in the 2-Year Gavage Study of the Binary Mixture of PCB 153 and PCB 126 [TCDD equivalent (TEQ) doses are shown in parentheses]
Thyroid Hormone Concentrations
Alterations in serum thyroid hormone levels were evaluated at the 14-, 31-, and 53-week interim evaluations. In the constant ratio groups, serum total thyroxine (T4) and free T4 generally showed a treatment-related decrease relative to controls. Serum total triiodothyronine (T3) exhibited a treatment-related increase at the 14-, 31-, and 53-week interim evaluations, but serum thyroid stimulating hormone (TSH) levels were increased at the 14-week time point only. In the varying ratio groups, the decrease in total and free T4 was more pronounced in those groups dosed with the increasing proportion of PCB 153 at the 31- and 53-week time points.
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. At 31 and 53 weeks, a significant increase in the hepatocellular labeling index occurred in Group 7. In the varying ratio groups, the labeling index at the 53-week interim time point was significantly higher in Group 6, which had the highest proportion of PCB 153 compared to the other varying ratio groups.
Cytochrome P450 Enzyme Activities
To evaluate the expression of known PCB 126-responsive genes, 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. In addition, PCB 153-inducible CYP2B-associated 7-pent-oxyresorufin-O-dealkylase (PROD) activity was analyzed. In the constant ratio Groups 2, 3, 5, and 7, hepatic and pulmonary EROD (CYP1A1) activities, hepatic A4H (CYP1A2) activities, and hepatic PROD (CYP2B) activities were significantly greater in all dosed groups compared to the vehicle controls at weeks 14, 31, and 53. In the varying ratio groups, hepatic EROD, A4H, and PROD activities at 14 weeks were higher in groups receiving a greater proportion of PCB 153 in the PCB mixture. At 31 and 53 weeks, hepatic CYP1A1 and CYP1A2 enzyme activities in Group 6 were generally lower than in Groups 4 and 5.
Determinations of PCB 126 and PCB 153 Concentrations in Tissues
Concentrations of PCB 126 and PCB 153 were determined in fat, liver, lung, and blood at the 14-, 31-, and 53-week interim evaluations and at the end of the 2-year study (105 weeks). PCB 126 was not detectable in vehicle control animals, but increased with increasing dose of PCB 126 and duration of exposure; the highest concentrations were found in liver and fat, and lower levels were seen in lung and blood. Increasing the proportion of PCB 153 in the mixture relative to PCB 126 led to a general decrease in the amount of PCB 126 in liver and lung at the later time points, whereas in fat and blood, there was generally either no effect of PCB 153 on the disposition of PCB 126, or there was an increase in the amount of PCB 126 in the tissue. In vehicle control animals, PCB 153 was detectable in the fat at all time points, in the lung at all time points except 53 weeks, and in the liver and blood at 2 years. PCB 153 was measurable in all examined tissues of treated animals, with the highest concentrations found in fat at the end of the 2-year study in groups administered the highest doses of PCB 153.
Pathology and Statistical Analyses
Constant Ratio Mixture of PCB 126 and PCB 153
At 14, 31, and 53 weeks, the absolute and relative liver weights of all dosed groups were generally greater than those of the vehicle controls.
Exposure to the PCB mixture led to significant toxicity in the liver. At 14 weeks, the incidences of several nonneoplastic liver lesions were increased compared to the vehicle controls including hepatocyte hypertrophy, pigmentation, multinucleated hepatocytes, and diffuse fatty change. The spectrum and severity of effects increased with dose and duration of exposure. At the end of the 2-year study, there were significantly increased incidences and severities of toxic hepatopathy characterized by hepatocyte hypertrophy, multinucleated hepatocytes, pigmentation, diffuse and focal fatty change, eosinophilic focus, nodular hyperplasia, cholangiofibrosis, oval cell hyperplasia, bile duct cysts, bile duct hyperplasia, necrosis, and portal fibrosis.
Significantly increased incidences of hepatocellular adenoma, cholangiocarcinoma, and hepatocholangioma were observed in the study. In addition, two animals in the highest dose group had hepatocellular carcinoma. The incidences of these lesions generally exceeded the historical vehicle control ranges.
At 2 years, a significantly increased incidence of cystic keratinizing epithelioma of the lung was observed in Group 7. In addition, single occurrences of squamous cell carcinoma were seen in the top two dose groups. Nonneoplastic effects whose incidences were increased in the lung included bronchiolar metaplasia of the alveolar epithelium and squamous metaplasia.
Significantly increased incidences of squamous cell carcinoma (gingival) of the oral mucosa were seen at the end of the 2-year study and were accompanied by increased incidences of gingival squamous hyperplasia.
In the pancreas at 53 weeks, the incidence of acinar cytoplasmic vacuolization was significantly increased in the highest dose group. At 2 years, increased incidences of acinar atrophy and acinar cytoplasmic vacuolization were seen in addition to pancreatic acinar neoplasms in dosed groups. In Groups 5 and 7, these incidences exceeded the historical vehicle control ranges.
In the uterus at 2 years, there was a marginal increase in the incidence of squamous cell carcinoma in Group 5.
Numerous nonneoplastic effects were seen in other organs at the interim time points including atrophy of the thymus and follicular cell hypertrophy of the thyroid gland. These responses were also affected by administration of the mixture of PCB 126 and PCB 153 at the end of the 2-year study and were accompanied by additional nonneoplastic responses in numerous organs including atrophy of the adrenal cortex and cortical hyperplasia, severity of nephropathy, and incidences of pigmentation of the kidney. Other nonneoplastic lesions that were treatment related were forestomach hyperplasia, hyperplasia of the nasal respiratory epithelium, metaplasia of the olfactory epithelium, and ectasia of the mandibular lymph node.
Varying Ratio Mixture of PCB 126 and PCB 153
An effect of increasing the proportion of PCB 153 in the PCB mixture was seen in several tissues, most notably in the liver. Treatment-related nonneoplastic effects seen across the varying ratio groups were generally the same as those seen in the constant ratio groups. In general there was a positive effect of PCB 153 in the mixture on the incidences and severities of these lesions with higher incidences and higher severities being seen in Group 6, which had the highest proportion of PCB 153. A significant positive effect of increasing the proportion of PCB 153 in the PCB mixture was seen for hepatocyte hypertrophy, cholangiofibrosis, eosinophilic focus, clear cell focus, basophilic focus, diffuse and focal fatty change, bile duct hyperplasia, and hematopoietic cell proliferation. In contrast, the incidences of pigmentation decreased with increasing proportions of PCB 153.
At 2 years, there was a significant positive effect of increasing PCB 153 in the mixture on the incidences of hepatocellular adenoma and cholangiocarcinoma. In addition, hepatocholangiomas were observed only in Groups 5 and 6.
A significant effect of increasing the proportion of PCB 153 in the PCB mixture was also seen for nonneoplastic lesions in the lung (bronchiolar metaplasia of alveolar epithelium), pancreas (acinar cytoplasmic vacuolization), thyroid gland (follicular cell hypertrophy) and kidney (pigmentation and pelvic inflammation of the kidney).
Under the conditions of this 2-year gavage study there was clear evidence of carcinogenic activity of a constant ratio binary mixture of PCB 126 and PCB 153 in female Harlan Sprague-Dawley rats based on increased incidences of cholangiocarcinoma, hepatocholangioma, and hepatocellular neoplasms (predominantly adenomas) of the liver, squamous neoplasms of the lung (predominantly cystic keratinizing epithelioma), and gingival squamous cell carcinoma of the oral mucosa. Increased incidences of pancreatic acinar neoplasms were also considered to be related to administration of the binary mixture of PCB 126 and PCB 153. The increased incidences of uterine squamous cell carcinoma may have been related to administration of the binary mixture of PCB 126 and PCB 153.
Administration of the binary mixture of PCB 126 and PCB 153 caused increased incidences of nonneoplastic lesions in the liver, lung, oral mucosa, pancreas, adrenal cortex, thyroid gland, thymus, kidney, nose, and forestomach.
Constant Ratio Mixture
|Varying Ratio Mixturea
(Groups 4, 5, and 6)
|Doses in corn oil/acetone by gavage||Group 1: Vehicle control;
Group 2: 10 ng/kg PCB 126 plus 10 µg/kg PCB 153;
Group 3: 100 ng/kg PCB 126 plus 100 µg/kg PCB 153;
Group 5: 300 ng/kg PCB 126 plus 300 µg/kg PCB 153;
Group 7: 1,000 ng/kg PCB 126 plus 1,000 µg/kg PCB 153
|Group 4: 300 ng/kg PCB 126 plus 100 µg/kg PCB 153;
Group 5: 300 ng/kg PCB 126 plus 300 µg/kg PCB 153;
Group 6: 300 ng/kg PCB 126 plus 3,000 µg/kg PCB 153
|Body weights||Groups 5 and 7 were less than Group 1 (vehicle controls)||Groups 4, 5, and 6 were less than Group 1 (vehicle controls)|
|Survival rates||22/53, 21/53, 22/53, 24/53, 24/53||28/50, 24/53, 27/51|
|Nonneoplastic effects|| Liver:
hepatocyte, hypertrophy (1/53, 7/53, 17/52, 33/52, 50/51)
hepatocytes, multinucleated (0/53, 0/53, 14/52, 46/52, 48/51)
pigmentation (2/53, 5/53, 38/52, 50/52, 50/51)
fatty change, diffuse (3/53, 1/53, 9/52, 31/52, 38/51)
fatty change, focal (3/53, 4/53, 7/52, 1/52, 12/51)
eosinophilic focus (includes multiple) (14/53, 16/53, 30/52, 40/52, 18/51)
toxic hepatopathy (0/53, 2/53, 34/52, 48/52, 49/51)
bile duct, cyst (4/53, 3/53, 1/52, 5/52, 23/51)
bile duct, hyperplasia (8/53, 2/53, 9/52, 29/52, 46/51)
necrosis (4/53, 8/53, 5/52, 4/52, 20/51)
oval cell, hyperplasia (2/53, 2/53, 15/52, 39/52, 46/51)
portal fibrosis (0/53, 0/53, 0/52, 7/52, 34/51)
hyperplasia, nodular (0/53, 0/53, 2/52, 24/52, 42/51)
cholangiofibrosis (0/53, 1/53, 0/52, 7/52, 39/51)
metaplasia, squamous (0/53, 0/53, 1/52, 2/53, 11/52)
alveolar epithelium, metaplasia, bronchiolar (0/53, 6/53, 23/52, 34/53, 32/52)
gingival, hyperplasia, squamous (8/12, 8/11, 18/25, 22/30, 24/36)
acinus, vacuolization cytoplasmic (0/53, 0/53, 0/52, 7/52, 40/50)
acinus, atrophy (0/53, 2/53, 1/52, 1/52, 8/50)
atrophy (0/53, 0/53, 0/52, 3/52, 35/51)
hyperplasia (11/53, 18/53, 23/52, 25/52, 18/51
follicular cell, hypertrophy (14/53, 17/53, 34/51, 35/52, 42/52)
atrophy (33/53, 33/50, 43/48, 42/50, 49/51)
severity of nephropathy (1.2, 1.0, 1.1, 1.3, 2.2)
pigmentation (0/53, 1/53, 3/52, 7/52, 35/51)
respiratory epithelium, hyperplasia (10/53, 5/53, 7/53, 11/53, 20/53) olfactory epithelium, metaplasia (4/53, 3/53, 5/53, 6/53, 15/53)
hyperplasia, squamous (1/53, 1/53, 2/52, 7/52, 8/51)
hepatocyte, hypertrophy (22/50, 33/52, 47/51)
pigmentation (50/50, 50/52, 44/51)
fatty change, diffuse (28/50, 31/52, 47/51)
fatty change, focal (4/50, 1/52, 11/51)
basophilic focus (5/50, 3/52, 18/51)
eosinophilic focus (includes multiple) (27/50, 40/52, 45/51)
clear cell focus (5/50, 3/52, 11/51)
bile duct, hyperplasia (20/50, 29/52, 40/51)
hematopoietic cell proliferation (18/50, 19/52, 29/51)
cholangiofibrosis (5/50, 7/52, 13/51)
alveolar epithelium, metaplasia, bronchiolar (39/50, 34/53, 30/50)
acinus, vacuolization cytoplasmic (3/49, 7/52, 44/49)
follicular cell, hypertrophy (28/49, 35/52, 44/50)
pigmentation (2/48, 7/52, 17/51)
pelvis, inflammation (1/48, 3/52, 8/51)
|Neoplastic effects|| Liver:
cholangiocarcinoma (0/53, 0/53, 1/52, 9/52, 30/51)
hepatocholangioma (includes multiple) (0/53, 0/53, 0/52, 2/52, 6/51)
hepatocellular adenoma (0/53, 0/53, 3/52, 5/52, 27/51)
hepatocellular carcinoma (0/53, 0/53, 0/52, 0/52, 2/51)
cystic keratinizing epithelioma (0/53, 0/53, 0/52, 1/53, 11/52)
squamous cell carcinoma (0/53, 0/53, 0/52, 1/53, 1/52)
squamous cell carcinoma (gingival) (0/53, 0/53, 2/53, 5/53, 9/53)
adenoma (0/53, 1/53, 1/52, 3/52, 1/50)
adenoma or carcinoma (0/53, 1/53, 1/52, 4/52, 2/50)
cholangiocarcinoma (7/50, 9/52, 25/51)
hepatocellular adenoma (2/50, 5/52, 21/51)
|Level of evidence of carcinogenic activity||Clear evidence|
a Effects shown for the varying ratio mixture groups are those data where there was a significant effect of varying ratio on the incidence. Not all effects in Groups 4, 5, and 6 that were related to treatment are shown.