Abstract

Despite the cessation of its production and use in many parts of the world, hexachlorobenzene (HCB) remains highly persistent in the environment, and chronic, low-dose exposure to HCB in humans continues. Its structural resemblance to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), ability to activate the aryl hydrocarbon (Ah) receptor, TCDD-like toxicities, and bioaccumulative nature suggest HCB be included in the toxic equivalency factor (TEF) methodology. Consequently, the National Toxicology Program conducted this subchronic study of HCB, including measurement of a variety of toxicological and biochemical endpoints, to allow comparison to TCDD data obtained in a previous 2-year bioassay.

Groups of 10 female Sprague Dawley (Hsd:Sprague Dawley^® SD^®) rats (core study) were administered doses of 0.03, 0.1, 0.3, 1, 3, 10, or 25 mg HCB/kg body weight/day (mg/kg/day) in corn oil:acetone (99:1) by gavage, 5 days/week for 13 weeks (3 months). Elimination of HCB after repeated dosing was evaluated in a group of 10 female rats (elimination study) administered 0.03 or 25 mg/kg/day for 13 weeks (3 months) and then held without dosing until necropsy at 21 (5 rats) or 29 (5 rats) weeks. Vehicle control female rats (10 core study and 10 elimination study) were administered the corn oil:acetone vehicle only. Genotoxicity in Salmonella typhimurium was also evaluated.

No significant effect of HCB on survival occurred in either the core or elimination study. In core study animals, the mean body weight gains of 10 and 25 mg/kg/day groups and the final mean body weight of the 25 mg/kg/day group were significantly increased compared to the vehicle control group. At 3 months, the absolute and relative spleen weights of the 3, 10, and 25 mg/kg/day groups, liver weights of the 10 and 25 mg/kg/day groups, and lung weights of the 25 mg/kg/day group were significantly increased compared to the vehicle control group. The absolute kidney weight of the 25 mg/kg/day group was also significantly increased compared to the vehicle control group.

Free and total serum thyroxine (T₄) were significantly decreased in the 10 and 25 mg/kg/day groups, and total triiodothyronine (T₃) was significantly decreased in the 25 mg/kg/day group. There were no significant changes in serum thyroid stimulating hormone (TSH). Hepatic cytochrome P450 (CYP) enzyme activity (acetanilide-4-hydroxylase [A4H], 7-ethoxyresorufin-O-deethylase [EROD], and pentoxyresorufin-O-deethylase [PROD]) was increased in all dosed groups; pulmonary EROD activity was increased only at the 25 mg/kg/day dose. The incidences of hepatocyte hypertrophy and lymphoid hyperplasia of the spleen in the 10 and 25 mg/kg/day groups, chronic inflammation of the lung in the 3, 10, and 25 mg/kg/day groups, and mammary gland hyperplasia, suppurative inflammation of the skin, and ulcer of the skin in the 25 mg/kg/day group were significantly increased compared to the vehicle control group. The teeth of all groups of rats administered 1 mg/kg/day or greater showed maxillary incisor degeneration.

Thyroid hormone levels, hepatocyte proliferation, and hepatic and pulmonary CYP activity data were used to determine relative potency factors (RPFs) for consideration of inclusion of HCB in the TEF methodology. Due to dissimilar maximal responses and lack of parallel dose curves of HCB and TCDD, an RPF derived from a median effective dose (ED₅₀) could not be determined using the thyroid hormone and CYP activity values. These data suggest that although the toxicological effects are similar to those seen with TCDD administration, HCB acts through different or additional mechanisms than activation of the Ah receptor and should not be included in the TEF concept.

HCB was not mutagenic in any of the four strains of Salmonella typhimurium, with or without exogenous metabolic activation.

In the elimination study, the half-life of HCB was estimated to be between 48 and 53 days in blood.

Under the conditions of this 3-month gavage study, oral administration of HCB in female Sprague Dawley rats resulted in dose-related lesions in the liver, lung, spleen, mammary gland, skin, thymus, and teeth. HCB administration decreased total T₃, free T₄, and total T₄; there were no compensatory increases in TSH and no thyroid histological changes observed. Consistent with literature indicating that HCB acts through other mechanisms in addition to activation of the Ah receptor, these data indicate that HCB should not be included in the TEF methodology.

Contents

• Foreword
• Collaborators
• Contributors
• Peer Review
• Publication Details
• Acknowledgments
• Introduction
  • Chemical and Physical Properties
  • Production, Use, and Human Exposure
  • Absorption, Distribution, Metabolism, and Excretion
  • Toxicity
  • Reproductive and Developmental Toxicity
  • Carcinogenicity
  • Genetic Toxicity
  • Role of the Aryl Hydrocarbon Receptor in the Toxicity of Hexachlorobenzene
  • Study Rationale
• Materials and Methods
  • Procurement and Characterization
  • Preparation and Analysis of Dose Formulations
  • Animal Source
  • Animal Welfare
  • Core Study
  • Elimination Study
  • Analysis of Hexachlorobenzene in Blood and Tissue Samples
  • Modeling
  • Statistical Methods
  • Quality Assurance Methods
  • Genetic Toxicology
• Results
  • Data Availability
  • Core Study
  • Determination of Relative Potency Factors
  • Genetic Toxicology
  • Elimination Study
• Discussion
  • Core Study
  • Relative Potency Factor Determination
  • Elimination Study
• References
• Appendix A. Chemical Characterization and Dose Formulation Studies
• Appendix B. Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat Ration
• Appendix C. Sentinel Animal Program
• Appendix D. Toxicokinetic Data
• Appendix E. Genetic Toxicology
• Appendix F. Supplemental Data