Page 9: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Benzene

Part II. Science and Technical Considerations - Continued

8.0 Kinetics and metabolism

Oral exposure to benzene at low concentrations in animals has been shown to result in complete absorption. Sabourin et al. (1987) administered radiolabelled (14C) benzene orally (through corn oil gavage and intraperitoneally) to Sprague-Dawley and F344/N rats and B6C3F1 mice and analysed urine and faeces at 4, 8, 16, 24, 32, and 48 hours after dosing for radiolabelled benzene (and/or benzene metabolites). The percentages of the dose excreted by each route were similar following gavage or intraperitoneal injection. The absorption of benzene in F344/N rats, Sprague-Dawley rats, and B6C3F1 mice was determined by comparing excretion routes following administration (by gavage or intraperitoneal injection) of benzene at 0.5 or 150 mg/kg bw; it was found that the absorption of benzene was essentially 100% for all three test species.

Results from the Sabourin et al. (1987) study are supported by a study on rats, mice, and hamsters by Mathews et al. (1998). Animals treated by oral gavage (in corn oil) with a range of benzene doses that overlapped those in the Sabourin et al. (1987) study displayed complete absorption from the gastrointestinal tract (in all three species); however, excretion routes were influenced by dose. For example, at a high dose of 100 mg/kg bw, a significant portion of benzene was eliminated by exhalation (from 22% in mice to 50% in rats). Both studies reported a greater proportion of metabolites excreted in urine at the low doses, with a shift to greater amounts of unmetabolized benzene excreted in exhaled air at the high doses. These results suggest that saturation of metabolism occurs at doses greater than approximately 100 mg/kg bw. At oral doses that could be found in drinking water, however, animal results suggest a linear increase in total metabolite production with exposure level.

No relevant animal studies are available that allow a comparison of absorption between gavage and drinking water administration. In theory, ingesting drinking water or food containing benzene may result in some loss from the stomach through volatilization, whereas administration by gavage using an oil vehicle may limit benzene volatilization. It is also possible that a greater proportion of benzene from large bolus doses would escape absorption and pass through into the faeces, while smaller doses would be better absorbed. Since essentially complete absorption has been observed even at high gavage doses in animals, in the absence of human data, it is postulated that complete absorption of benzene by ingestion can be expected in humans as well.

Absorption of benzene through inhalation, like absorption following ingestion, depends on the dose. As seen in oral exposure studies, a larger proportion of benzene is retained at lower exposures versus higher exposures. Humans experimentally exposed to low to moderate levels of benzene (1.7-32 ppm) absorbed on average 50% of the benzene inhaled. Pekari et al. (1992) exposed three males to both 1.7 and 10 ppm benzene for 4 hours, during which six samples of exhaled air and blood were taken from each subject. After exposure, phenol was measured in exhaled air, blood, and urine. The average absorption was found to be 52% ± 7.3% at 1.7 ppm and 48% ± 4.3% at 10 ppm.

Nomiyama and Nomiyama (1974) exposed three females and three males to benzene levels ranging from 52 to 62 ppm for 4-hour periods. At 1-hour intervals during exposure, exhaled air was sampled. The average absorption at the 1-hour exposure period was found to be approximately 60% for women and 45% for men. After 2 hours of exposure, absorption was approximately 43% for women and 35% for men. The average absorption over the 3- to 4-hour time periods was reported at 30.2%. In general, absorption was higher for both sexes during early exposure, approaching a steady state only after 3 hours.

Studies measuring exhaled air from occupational and environmental exposures further support a 50% absorption of benzene following inhalation exposure. In an occupational study by Perbellini et al. (1988), exhaled air from subjects who had low background exposure to benzene (median 19 ng/L in air, or 0.019 ng/m3) showed an average absorption of 55%. Another study by Wallace et al. (1993) found 70% absorption of benzene from measurements of exhaled air for non-smokers. In most studies of this sort, exhaled air samples are collected in the post-exposure period, with the concentration of benzene in exhaled air falling rapidly following removal from exposure; therefore, post-exposure samples would be expected to predict a lower absorption. In general, however, experimental, occupational, and environmental exposure studies suggest that an absorption fraction of 50% is a good estimate.

Human and animal studies have shown that benzene is readily absorbed through the skin from both the liquid and vapour phases (Franz, 1975; Maibach and Anjo, 1981; Franz, 1984; Susten et al., 1985). Absorption of benzene through the skin, however, depends on several factors, including skin permeability, which increases with increasing temperature (Nakai et al., 1997). Susten et al. (1985) estimated the amount of benzene absorbed through the skin of tire industry workers by conducting a series of in vivo studies in hairless mice. Percutaneous absorption, following single dermal applications of [14C]benzene contained in rubber solvent at a concentration of 0.5% (v/v) benzene, was calculated directly from the sums of radioactivity found in excreta, expired breath, and the carcass. Data from the study suggested that benzene absorption via the skin could contribute from 20% to 40% of the total benzene dose of these workers.

Although animal studies show that exposure to oral doses to which humans are likely exposed suggest a linear increase in total metabolite production with exposure level, the doserelated production of benzene metabolites in humans is not well understood, particularly at low levels of exposure. Kim et al. (2006) investigated unmetabolized benzene in urine and all major urinary metabolites (phenol, E,E-muconic acid, hydroquinone, and catechol), as well as the minor metabolite, S-phenylmercapturic acid, in 250 benzene-exposed workers and 139 control workers in Tianjin, China. Metabolite concentrations in urine were found to be consistently elevated when the median air benzene levels were at or above the following: 0.2 ppm for E,Emuconic acid and S-phenylmercapturic acid, 0.5 ppm for phenol and hydroquinone, and 2 ppm for catechol. The dose-related production of E,E-muconic acid, phenol, hydroquinone, catechol, and total metabolites reportedly declined by 2.5- to 26-fold as the median air benzene levels increased from 0.027 to 15.4 ppm. Reductions in metabolite production were found to be most pronounced for catechol and phenol at levels below 1 ppm, indicating that metabolism favoured the production of the toxic metabolites, hydroquinone and E,E-muconic acid, at low exposures. Another study by Rappaport et al. (2005) investigated the production of benzene oxide and 1,4- benzoquinone in 160 Chinese workers exposed to benzene at levels ranging from 0.074 to 328 ppm. Both benzene oxide and 1,4-benzoquinone levels plateaued at approximately 500 ppm benzene, suggesting that cytochrome P4502E1 (CYP2E1) (which is responsible for oxidizing benzene to benzene oxide, the first step in benzene metabolism) became saturated at this point. These results indicate that benzene metabolism may be much more effective at low levels of benzene and that exposure to levels of benzene above 50 ppm may have a diminished impact on the human health risk of leukaemia, since benzene metabolism becomes substantially saturated at this level. On the other hand, these results suggest that exposure to levels of benzene below 50 ppm may produce the maximum amount of metabolites per unit of benzene exposure.

Scientific evidence suggests that metabolism plays an important role in benzene toxicity (Snyder and Hedli, 1996). As an example, competitive inhibition of metabolism by toluene (at levels much higher than found in drinking water) decreases benzene toxicity. Valentine et al. (1996) reported that transgenic mice lacking CYP2E1 expression had lower benzene metabolism, cytotoxicity, or genotoxicity compared with wild-type mice; there is no indication, however, that the route of exposure has an effect on the metabolites formed (IPCS, 1993). Two major pathways are proposed as being responsible for benzene toxicity. The first involves the metabolites phenol, catechol, and hydroquinone, and the second pathway involves open ring forms of benzene. Benzene is primarily metabolized in the liver by CYP2E1 (Johansson and Ingelman-Sundberg, 1988) to form benzene oxide, which spontaneously rearranges to phenol. Catechol is formed by the oxidation of phenol, or it can be formed by the conversion of benzene oxide to benzene-1,2-dihydrodiol in the liver by epoxide hydrolase, with subsequent conversion to catechol by dehydrogenases. It is believed that catechol formation from phenol oxidation may be significant only during high-dose exposures. Hydroquinone is formed from the oxidation of phenol by mixed-function oxidases.

It is suggested that benzene-induced haematotoxicity, such as aplastic anaemia, pancytopenia, thrombocytopenia, granulocytopenia, lymphocytopenia, and carcinogenesis, involves the metabolism of phenolic metabolites of benzene, in particular the metabolism of hydroquinone to benzoquinone, semiquinones, and free radicals (Smith, 1996; Snyder and Hedli, 1996; Smith and Fanning, 1997). Blood transports phenolic metabolites (phenol, hydroquinone, catechol, and 1,2,4-trihydroxybenzene) to bone marrow, where they can be converted to reactive species by peroxidases and other enzymes. The redox reactions that accompany these reactions generate oxygen free radicals, lipid peroxidation products, and other free radicals (Subrahmanyam et al., 1991). Bone marrow contains approximately 3% dry weight of myeloperoxidase in addition to other peroxidases, such as eosinophil peroxidase and prostaglandin synthetase (Smith, 1996). The primary biological function of a peroxidase enzyme is to oxidize hydrogen donors at the expense of peroxide or molecular oxygen. Snyder and Kalf (1994) found that NADPH-dependent quinone oxidoreductase, an enzyme that efficiently reduces (detoxifies) quinones, is found in low concentrations (relative to other tissues) in bone marrow, which may explain in part why the bone marrow is a target tissue for benzene toxicity. Glutathione conjugates of hydroquinone and 1,2,4-benzenetriol readily auto-oxidize to quinone species, which may react with cellular macromolecules directly or generate free radical species (Snyder and Hedli, 1996). In a review by Witz et al. (1996), it was reported that some researchers have hypothesized that metabolites of benzene where the aromatic ring has been broken may also significantly contribute to benzene haematotoxicity. Trans,trans-muconaldehyde co-administration with hydroquinone, for example, is very potent in damaging bone marrow cells.

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