Priority Substances List Assessment Report- 1,3-Butadiene

2.0 Summary of Information Critical to Assessment of "Toxic" under CEPA 1999 (continued)

2.4 Effects characterization

2.4.2 Toxicokinetics and metabolism

The database on the toxicokinetics and metabolism of butadiene in mammals is relatively extensive. The proposed metabolism is outlined in Figure 1, based on the pathways described by Henderson et al. (1993, 1996) and Himmelsteinet al.(1997). Available data for the pathways most extensively investigated indicate that metabolism is qualitatively similar among the various species studied, although there may be quantitative differences in the amount of butadiene absorbed as well as in metabolic rates and the proportion of metabolites generated. These differences appear to be in concordance with the observed variation in sensitivity to butadiene-induced toxic effects of the few strains of rodent species tested to date, in that mice appear to metabolize a greater proportion of butadiene to active epoxide metabolites than do rats. While less of these metabolites are also formed in samples of human tissues in vitro than in those of mice, available data are insufficient to characterize interindividual variability in humans. Although there are known genetic polymorphisms for a number of the enzymes involved in the metabolism of butadiene, information on genotype was not included in most investigations in humans.

Based on the metabolic pathways described in Figure 1, butadiene is first oxidized via cytochrome P-450 enzymes (primarily P-450 2E1, although other isoforms may also be involved, the relative contribution of which varies between tissues and species) to the monoepoxide 1,2-epoxy-3-butene, or EB, which is subsequently further oxidized via P-450 enzymes to the diepoxide 1,2,3,4-diepoxybutane, or DEB, or hydrolysed via epoxide hydrolase (EH) to butenediol (1,2-dihydroxy-3-butene). The monoepoxide, the diepoxide and the butenediol may all be conjugated with glutathione (GSH) to form mercapturic acids (the latter likely via oxidation to a reactive Michael acceptor), which are eventually eliminated in the urine. Hydrolysis of the diepoxide via epoxide hydrolase or oxidation of the butenediol via cytochrome P-450 will result in the formation of the monoepoxide diol (EBdiol). A small amount of butadiene may be converted to 3-butenal, which is subsequently transformed to crotonaldehyde (about 2-5% of the amount that is oxidized to the monoepoxide in human liver microsomes [Duescher and Elfarra, 1994] or microsomes of kidney, lung or liver of B6C3F₁ mice [Sharer et al., 1992]). However, this pathway has not been extensively investigated, nor was crotonaldehyde detected in a sensitive analysis (using nuclear magnetic resonance spectroscopy) of urinary metabolites of rats and mice exposed to ¹³C-butadiene (Nauhaus et al., 1996).

Metabolism of butadiene and subsequent conversion of EB to DEB may also take place to a more limited degree in the bone marrow (e.g., Maniglier-Poulet et al., 1995) by means other than P-450 oxidation (possibly via myeloperoxidase; Elfarra et al., 1996), based on in vitro observations and the detection of the epoxides in the bone marrow of rodents (Thornton-Manning et al., 1995a, 1995b), although this potential pathway has not yet been extensively investigated. EB may also react with both myeloperoxidase and chloride to form a chlorohydrin (1-chloro-2-hydroxy-3-butene) (Duescher and Elfarra, 1992). Metabolites arising from other possible pathways have been identified in the urine of mice exposed to butadiene (including metabolites known to be derived from metabolism of acrolein or acrylic acid) (Nauhaus et al., 1996), but no further research has yet been generated.

There is a substantial amount of evidence from in vitro and in vivo investigations that B6C3F₁ mice oxidize butadiene to the monoepoxide via P-450 (primarily 2E1, although 2A6 and other isoforms may also contribute) in the liver to a greater extent than do Sprague-Dawley rats and humans. Levels of EB in the blood and other tissues of mice were two- to eightfold higher than those in rats exposed to similar levels of butadiene (Bond et al., 1986; Himmelstein et al., 1994, 1995; Bechtold et al., 1995; Thornton-Manning et al., 1997).

Figure 1 Proposed metabolism of butadiene in mammals

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Available data also suggest that there are similar species differences in the amount of the diepoxide formed from oxidation of the monoepoxide. Levels of DEB were 40- to 160-fold higher in blood and other tissues of B6C3F₁ mice than in Sprague-Dawley rats exposed to the same concentration of butadiene (Thornton-Manning et al., 1995a, 1995b). While concentrations of EB at various sites were similar in male and female rats, levels of DEB were at least fivefold higher in females than in males, which correlates with the greater incidence of tumours in female rats. Although the mammary gland is a target tissue in rats, extended exposure to butadiene at 8000 ppm (17 696 mg/m³) for 10 days did not result in any accumulation of DEB at this site (Thornton-Manning et al., 1998), which suggests that DEB may not play a significant role in the induction of mammary tumours in rats. Available in vitro data in human liver and lung samples suggest that humans also form less of the active metabolites of butadiene than do mice (although somewhat varying results have been reported with respect to the magnitude of the differences between species) (Csanády et al., 1992; Duescher and Elfarra, 1994; Krause and Elfarra, 1997).

Although epoxide metabolites of butadiene are formed to a greater extent in mice than in rats or humans, they are also cleared via glutathione conjugation more rapidly in mice (Kreuzer et al., 1991; Sharer et al., 1992; Boogaard et al., 1996a, 1996b). Conversely, hydrolysis of EB and DEB is greater in humans than in rats, and hydrolysis of EB and DEB in rats is in turn greater than that in mice (Csanády et al., 1992; Krause et al., 1997). In both humans and monkeys, removal of EB via hydrolysis appears to predominate over conjugation with glutathione, based on analysis of urinary metabolites (Sabourin et al., 1992; Bechtold et al., 1994). Although hydrolysis of the epoxide metabolites is generally considered to be a detoxifying mechanism, it may also lead to the formation of the diolepoxide, EBdiol. However, no data were identified on species differences in the formation of EBdiol via metabolism of both epoxide metabolites.

The formation of stable adducts of both the monoepoxide and monoepoxide diol metabolites of butadiene with the N-terminal valine of hemoglobin has been observed in experimental animals and humans exposed to butadiene (Albrecht et al., 1993; Osterman-Golkar et al., 1993, 1996; Neumann et al., 1995; Sorsa et al., 1996b; Tretyakova et al., 1996; Pérez et al., 1997; Swenberg, 1998). Consistent with the greater formation of epoxide metabolites, greater concentrations of hemoglobin-EB adducts were measured in mice than in rats exposed to the same concentration of butadiene. However, levels of hemoglobin-EB adducts in butadiene-exposed workers, although significantly elevated compared with levels in non-exposed workers, were considerably less than would be expected on the basis of results of studies in mice and rats (Osterman-Golkar et al., 1993). Based on observations in rats and humans exposed to butadiene, levels of hemoglobin-EBdiol adducts are substantially greater than levels of hemoglobin-EB adducts (although it is noted that the same adduct can result from binding with DEB). Metabolites of butadiene may also form adducts with DNA (see Sections 2.4.3.4 and 2.4.4.4).

In addition to quantitative interspecies differences in the metabolism of butadiene, there is also evidence that there is significant variation within the human population. Indeed, although available data are inadequate to assess interindividual variation in metabolism, which has been observed in in vitro investigations in microsomes from a small number of subjects (Boogaard and Bond, 1996; Krause et al., 1997), there has been significant interindividual variability in the extent of formation of hemoglobin adducts with butadiene metabolites in human populations (Neumann et al., 1995; Osterman-Golkar et al., 1996). Such variability is not unexpected, in view of the complexity of the metabolic pathways involved in the biotransformation of butadiene: i.e., the three principal enzymatic processes that determine the extent of exposure to the putatively toxic epoxide metabolites, namely formation via cytochrome P-450-2E1 and removal via epoxide hydrolase and glutathione conjugation. For example, the inducibility of cytochrome P-450-2E1 by low molecular weight compounds such as ethanol is likely to contribute to interindividual variability in sensitivity. Moreover, genetic polymorphisms for glutathione-S-transferases and epoxide hydrolase might also contribute to considerable variation in sensitivity. While the influence of genotype for epoxide hydrolase has not been well investigated (although data indicate that hydrolysis of EB predominates over oxidation and glutathione conjugation in humans), interindividual sensitivity to the genetic effects of the epoxide metabolites in in vitro studies has been clearly related to genotype for the glutathione-S-transferases (see Section 2.4.4.4).

2.4.3 Experimental mammals and in vitro

2.4.3.1 Acute toxicity

Although few data are available, butadiene appears to be of low acute toxicity in experimental animals, with reported LC₅₀ values for rats and mice of >100 000 ppm (>221 000 mg/m³). Lowest LC₅₀ values for butadiene are reported for mice, at 117 000 ppm (256 000 mg/m³) (duration not specified) (Batinka, 1966) and 121 000 ppm (268 000 mg/m³) (2 hours) (Shugaev, 1969). Exposure to butadiene for 7 hours caused a concentration-dependent depletion of cellular non-protein sulphydryl content of liver, lung or heart in mice, with a Lowest-Observed-Effect Level (LOEL) of 100 ppm (221 mg/m³) (Deutschmann and Laib, 1989).

2.4.3.2 Short-term and subchronic toxicity

The majority of short-term and subchronic studies were designed as either range-finding studies preliminary to chronic bioassays or investigations of potential mechanisms of action for butadiene-induced cancer and are not adequate for determination of critical effect levels. Effects on body weight were observed in B6C3F₁ mice exposed to 625 ppm (1383 mg/m³) butadiene or more for 2 weeks; no histopathological changes were noted at any concentration at or below 8000 ppm (17 696 mg/m³) (NTP, 1984).

Hematological effects consistent with megaloblastic anemia and effects on bone marrow, including alterations in stem cell development, have been observed in two strains of mice (B6C3F₁and NIH Swiss) exposed to 1000 or 1250 ppm (2212 or 2765 mg/m³) butadiene for up to 31 weeks (Irons et al., 1986a, 1986b; Leiderman et al., 1986; Bevan et al., 1996). Other effects, including decreased survival and body weight gain (with males being more sensitive than females), altered organ weights and ovarian or testicular atrophy, have also been observed in B6C3F₁ mice exposed subchronically to similar or higher levels of butadiene (NTP, 1984; Bevan et al., 1996). In addition, an increased incidence of a variety of tumours has been observed in B6C3F₁mice exposed to 625 ppm (1383 mg/m³) butadiene for as little as 13 weeks (NTP, 1993) (see Section 2.4.3.3). Although histopathological changes and hematological effects were reported in early studies in rats exposed to low concentrations (3 or 10 mg/m³) (Batinka, 1966; Ripp, 1967; Nikiforova et al., 1969), these results were not confirmed in more recent investigations of rats exposed for up to 13 weeks to much higher concentrations (e.g., 17 600 mg/m³) (e.g., Crouch et al., 1979; Bevan et al., 1996). In view of the limitations of the studies in rats, it is not possible to draw any conclusions regarding species differences in response to subchronic exposure to butadiene.