Repeated dose toxicity and relative potency of 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for induction of CYP1A1, CYP1A2 and thymic atrophy in female Harlan Sprague–Dawley rats

Abstract

In this study we assessed the relative toxicity and potency of the chlorinated naphthalenes 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Chemicals were administered in corn oil:acetone (99:1) by gavage to female Harlan Sprague–Dawley rats at dosages of 0 (vehicle), 500, 1500, 5000, 50,000 and 500,000 ng/kg (PCN 66 and PCN 67) and 1, 3, 10, 100, and 300 ng/kg (TCDD) for 2 weeks. Histopathologic changes were observed in the thymus, liver and lung of TCDD treated animals and in the liver and thymus of PCN treated animals. Significant increases in CYP1A1 and CYP1A2 associated enzyme activity were observed in all animals exposed to TCDD, PCN 66 and PCN 67. Dose response modeling of CYP1A1, CYP1A2 and thymic atrophy gave ranges of estimated relative potencies, as compared to TCDD, of 0.0015–0.0072, for PCN 66 and 0.00029–0.00067 for PCN 67. Given that PCN 66 and PCN 67 exposure resulted in biochemical and histopathologic changes similar to that seen with TCDD, this suggests that they should be included in the WHO toxic equivalency factor (TEF) scheme, although the estimated relative potencies indicate that these hexachlorinated naphthalenes should not contribute greatly to the overall human body burden of dioxin-like activity.

Introduction

Polychlorinated naphthalenes (PCNs) are a family of two-ringed aromatic compounds, which contain one to eight chlorines per naphthalene and form 75 possible congeners (Falandysz, 2003). PCN products are generally mixtures and were used in a variety of commercial applications including cable insulation, wood preservation, engine oil additives, electroplating masking compounds, capacitors, and refractive index testing oils and as a feedstock for dye production (WHO, 2001). Production of PCNs has ceased due to substitutions of less toxic chemicals (Hayward, 1998). PCNs are also formed during production of technical mixtures of chlorobiphenyls and can be found in in various polychlorinated biphenyl formulations (Falandysz, 2003). A variety of PCN mixtures were sold in the United States under the trade name Halowaxes; while in Europe they were sold under the trade names Nibren Waxes, Seekay Waxes and Clonacire Waxes, similar to PCB mixtures, the PCNs were sold as mixtures with different degrees of chlorination.

PCN exposure can occur through oral, inhalation, and dermal routes. Non-occupational exposure can result from air contamination near manufacturing sites, incineration of waste, and disposal of PCN containing items at landfills. PCNs have been detected in both urban and rural soils (Krauss and Wilcke, 2003), waterway sediment (Brack et al., 2003, Kannan et al., 2001), aquifer water samples (Espadaler et al., 1997) and urban air (Helm and Bidleman, 2003). As with other polychlorinated diaromatic hydrocarbons, PCNs are lipophilic compounds that persist in the environment and bioaccumulate in biological tissues (Falandysz, 2003). Chlorinated naphthalenes have also been identified in fish (Ofstad et al., 1978), whale and seal tissue (Helm et al., 2002) representing potential dietary sources of these compounds.

Few studies examining human tissue concentrations of PCNs are available. PCN concentrations determined in pooled human milk from Sweden collected from 1972 and 1992 decreased from 3081 to 483 pg/g lipid over this 20 year period (Lunden and Noren, 1998). In Sweden, concentrations of total PCNs ranged from 1000 to 4000 pg/g lipid in human adipose samples collected at autopsy with PCN 66 and 67 making up approximately 25–50% of the total (Weistrand and Noren, 1998). More recently, PCN concentrations were measured in adipose tissue samples collected from 43 (14 male and 29 female) patients undergoing liposuction procedures in New York City (Kunisue et al., 2009). PCN congeners 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) made up approximately 16% of the total PCNs present at concentrations of approximately 100 and 47 pg/g lipid in males and females respectively. The PCN congeners 1,2,3,5,7- and 1,2,4,6,7-pentachloronaphthalene made up approximately 30% of the PCNs present in the adipose tissue from these tissues.

Occupational exposure to PCNs has been shown to produce illness similar to that caused by dioxin-like compounds. Workers from a cable manufacturing plant developed a high incidence of chloracne and liver disease associated with PCN exposure. Additional symptoms associated with PCN exposure include eye irritation, fatigue, headache, anemia, hematuria, impotency, anorexia, vomiting, and abdominal pain (HSDB, 2011). Fatal cases of PCN toxicity have been associated with jaundice and hepatotoxicity (Hayward, 1998). The carcinogenicity of PCNs has not been well studied, however, a cohort exposed to Halowax was noted as having an increased incidence of gastrointestinal and respiratory neoplasms (Hayward, 1998).

PCN exposure in animals results in toxicity similar to that reported in humans. Hyperkeratosis of rabbit ears and the skin of hairless mice was observed after PCN exposure (HSDB, 2011). Fatal liver necrosis occurred in rabbits subcutaneously injected with a 15-ppm mixture of penta- and hexachloronaphthalene for up to 26 days (Hayward, 1998). Rats fed mixtures of penta- and hexachloronaphthalenes every other day for 26 days developed swollen, vacuolated, and necrotic liver cells (HSDB, 2011). Inhalation exposure to penta- and hexachloronaphthalene mixture to rats for 6 weeks resulted in hyalinization, swelling and slight granulation of the liver (Hayward, 1998).

PCNs were sold as a variety of mixtures that ranged from those containing predominately low chlorinated mono and dichloronaphthalenes to mixtures containing predominately the octachloronaphthalenes. The more toxic mixtures are those containing predominately the penta- and hexachlorinated naphthalenes. These higher chlorinated mixtures induce biological effects similar to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) including induction of the liver cytochrome P450-associated enzyme activities ethoxyresorufin-O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH). In a rat hepatoma H-4-II cell line, the relative potency value of a mixture of PCN 66/67, compared to TCDD, was 0.002 and 0.003, based on measurement of cytochrome P450-associated EROD and AHH, respectively (Hanberg et al., 1991). In another in vitro study relative potency estimates based on EROD induction were 0.00063 for PCN 66 and 0.00029 for PCN 67 (Villeneuve et al., 2000). Hexachlorinated naphthalenes can also induce the Aryl hydrocarbon (Ah) receptor dependent reporter gene activities (Behnisch et al., 2003).

Given this profile of activity, ability to persist in the food chain (Falandysz, 2003), and ability of PCN mixtures to produce toxicities similar to that of dioxins, PCNs have been considered for inclusion in the World Health Organization's (WHO) dioxin toxic equivalency factor (TEF) scheme (Van den Berg et al., 2006). While there have been studies of in vitro potency of individual congeners (Behnisch et al., 2003), there are no in vivo data available to estimate the relative potency for individual PCN congeners. The aim of this present study was to investigate the relative toxicity and potency of PCN 66 and PCN 67 for induction of toxicologic and biochemical endpoints in female Harlan Sprague–Dawley rats, compared to that of TCDD, after subacute (14 day) repeated exposure. Although production of PCNs has ceased in recent years due to substitutions of less toxic chemicals (Hayward, 1998), the environmental contamination of PCNs and the detection of PCN 66 and PCN 67 in human tissue warrant further investigation of the in vivo toxicity and relative potency of these dioxin-like compounds. These data will provide for a better evaluation of the possible inclusion of PCN 66 and PCN 67 in the TEF scheme (Van den Berg et al., 2006).