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Key value for chemical safety assessment

Additional information

In Vitro Studies

 

The key studies are considered to be bacterial mutation assays (Araki et al 1994, Madhusree et al 2002) and a mammalian cell cytogenetic assay (Asakura et al 2008).  These are two recognised core assay types for investigating mutation in vitro. 

1,3-Butadiene was tested in a standard Ames test and with exposure to the chemical contained in gas bags. S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli (WP2 uvrA and WP2P uvrA) were treated with 1,3-butadiene both with and without auxiliary metabolic activation from rat liver (S9) at concentrations up to 50% in the atmosphere. Positive results were obtained in Salmonella strain TA1535 in the presence of S9 in both studies, and a limited response in TA100 in one study (Araki et al 1994). Negative results were obtained in the absence of S9. Similar positive responses in the presence of S9 have been reported by other investigators (EU RAR, 2002). 1,3-Butadiene is mutagenic in bacterial gene mutation assays. Asakura et al (2008) examined 1,3-butadiene in an in vitro cytogenetic assay with CHL cells in both the absence and presence of S9. The test system was designed to allow a contained exposure to gaseous materials and concentrations up to 20% 1,3-butadiene were used. An increase in the frequency of chromosomal aberrations was observed, and a positive response reported both in the absence and presence of S9. The positive response in the absence of S9 is not consistent with the Ames test results but nevertheless the overall result indicates that 1,3-butadiene is clastogenic in mammalian cells in vitro.

 

There are additional reports of positive responses in mammalian cell gene mutation assays, but due to protocol or reporting deficiencies these are considered unreliable (EU RAR 2002). The REACH requirement for an adequate in vitro gene mutation study in mammalian cells is waived, as adequate data are available from in vivo gene mutation tests (Column 2 adaptation). Conflicting responses have been reported in studies examining for the endpoint of sister chromatid exchange (EU RAR 2002). 1,3-butadiene is considered to be mutagenic in vitro.

 

 

In Vivo Studies – Non-Human Information 

 

The key studies are considered to be somatic cell cytogenetic and gene mutation studies in the mouse (Adler et al 1994, Cunningham et al 1986, Cochrane and Skopek 1994), and dominant lethal studies in the mouse (Adler et al 1994, Brinkworth et al 1998) and the rat (Hughes et al 1996). These are recognised core assay types for investigating mutation in vivo.

 

The induction of micronuclei in bone marrow and peripheral blood erythrocytes was investigated in mice by Adler et al 1994). Adult (102/E1xC3H/E1)F1 mice were exposed to 0, 50, 200, 500 or 1,300 ppm (110, 442, 1106 or 2876 mg/m3) 1,3-butadiene, 6 hours/day for 5 days and bone marrow and blood samples taken 18-24 hours after the last exposure. There was a statistically significant increase in the frequency of micronucleated cells in the bone marrow and peripheral blood at all exposure concentrations. It was also observed that male mice were more sensitive than females at the higher exposure levels. Similar positive results in the mouse bone marrow micronucleus test were obtained by Cunningham et al (1986). Male B6C3F1 mice were exposed to 1,3-butadiene at concentrations of from 10 to 10,000 ppm (22-22126 mg/m3) (6h/day for 2 consecutive days). A significant dose-dependent increase in micronuclei induction was observed at concentrations of 100 ppm (221 mg/m3) and above.

The ability of 1,3-butadiene to cause gene mutation in vivo was investigated at the hprt locus in splenic T cells in male B6C3F1 mice (Cochrane and Skopek 1994). Animals were exposed to 0 or 625 ppm (1383 mg/m3) 1,3-butadiene for 6 hours/day, 5 days/week for 2 weeks and sacrificed 2 weeks later. Splenic T cells were isolated and cultured for 10 days to allow growth of mutant hprt- colonies. A statistically significant increase in mutation frequency to 5 times the control value was observed. It was concluded that repeated exposure to 1,3-butadiene causes gene mutations in mice. Age and gender dependent differences in 1,3-butadiene-induced mutagenicity were investigated at the hrtp locus in splenic T cells in mice (Meng et al, 2007; Walker et al., 2009a). Values obtained in this study were compared with those obtained in previous studies (Meng et al 1998, 1999, Walker and Meng 2000). To investigate age differences, female mice aged 8-9 weeks or 4-5 weeks were exposed to 1250 ppm (2765 mg/m3)(6 h/day, 5 days/week) of 1,3- butadiene for 2 weeks. 1,3- butadiene was mutagenic to female mice, with higher peak Hprt mutant frequencies achieved in the 4 -5 week old mice compared to the 8 -9 week old mice. However when the mutation potency (the mutation response over time) was compared between the two groups, there was no significant difference. To investigate sex differences, mice aged 4-5 weeks were exposed to 1250 ppm of 1,3-butadiene for 2 weeks. Mutation frequencies in treated males were 6.2-fold greater than in control males whilst the figure in female mice was 2.3-fold higher than that in males. Female mice are therefore more susceptible to 1,3-butadiene-induced hrpt mutations than male mice. Exposure to 3 ppm BD for 2 weeks also induced a weak mutagenic response in female mice, with an increase of 1.6 fold Hprt mutant frequency over control female mice (Walker et al., 2009a).

 

There are a number of further reports of positive results for 1,3-butadiene in mice including both cytogenetic studies and gene mutation studies (EU RAR 2002). Adler et al 1994 and Cunningham et al (1986) provide examples. In a positive mouse spot test, pregnant female mice were exposed to 1,3-butadiene at 500 ppm (1106 mg/m3), 6h/day from days 8 -12 of gestation. An increased incidence of coat colour spots of genetic relevance were found in offspring (Adler et al 1994). Male B6C3F1mice were exposed to 1,3-butadiene by inhalation, at concentrations from 10 to 10,000 ppm (22-22,126 mg/m3), 6h/day, for 2 consecutive days in a sister chromatid exchange assay. There was a significant increase in sister chromatid exchanges in mouse bone marrow cells at concentrations ≥ 100 ppm and the test was judged to be positive (Cunningham et al, 1986).

A limited number of studies has been conducted in the rat, and 1,3-butadiene has been found to be non-mutagenic in this species. Cunningham et al (1986) exposed male B6C3F1 mice and male Sprague-Dawley rats to 0 or 10 – 10,000 ppm (22-22126 mg/m3)1,3-butadiene for 6 hours/day on two consecutive days. Animals were sacrificed 24 hours after the second exposure and the bone marrow sampled to examine for the presence of micronuclei. A dose-related increase in the incidence of micronuclei was seen at 1,3-butadiene concentrations of 100 ppm and higher in the mouse, but there was no difference in the incidence of micronuclei between control and exposed rats at any concentration. Cunningham et al (1986) conducted a sister chromatid exchange assay in rats. Male Sprague-Dawley rats were exposed to 1,3-butadiene by inhalation, at concentrations from 10 to 10,000 ppm (22-22126 mg/m3), 6h/day, for 2 consecutive days. Bone marrow cells were evaluated for the induction of sister chromatid exchanges. No significant increases in sister chromatid exchanges occurred.

Gene mutation at the hrpt locus has also been investigated in rats (Meng et al., 2007; Walker et al., 2009b). Mutation frequencies in splenic T cells were determined in male rats exposed to 1,3- butadiene at 1250 ppm (2765 mg/m3)(6 h/day, 5 days/week) for 2 weeks and compared to previous results in female rats (Meng et al 1998, 1999, Walker and Meng 2000). 1,3-Butadiene was weakly mutagenic in rats, mutation frequencies in treated male rats were 1.9-fold greater than in control males whilst the figure in female rats was 1.9-fold higher than that in males. Hrpt mutations in female rats were also seen after exposure to the lower dose of 62.5 ppm (138mg/m3)1,3- butadiene for 4 weeks (Meng et al., 2007). A small but statistically significant increase in mutation frequency over controls occurred

1,3-butadiene is therefore mutagenic in somatic cells in the mouse but not in the rat in standard cytogenetic and gene mutation studies. A weak positive response was seen for hrpt mutations in the rat. Studies on the oxidative metabolites of 1,3-butadiene—but-3-ene-1,2-diol (i.e. butadiene monoepoxide diol) and 2-(oxiran-2-yl)oxirane (i.e. butadiene diepoxide)—in mice and rats indicate that these metabolites are mutagenic in both species (Walker et al., 2009c,d), and indicate that the substantial differences in mutagenic response to 1,3-butadiene between the species are largely the result of toxicokinetic differences in the systemic burden of toxic metabolites.

 

1,3-Butadiene has also been examined for germ cell mutagenicity. Adler et al (1994) reported a dominant lethal assay in which male (102/E1xC3H/E1)F1 mice were exposed to 0 or 1,300 ppm (2876 mg/m3) 1,3-butadiene 6 hours/day for 5 days. Each male was then mated with pairs of unexposed females for a period of 4 consecutive weeks. Pregnant females were sacrificed on days 14-16 of gestation and the uterine contents were examined for the presence of live and dead implants. A positive result for dominant lethal mutations was based on post-implantation losses seen primarily in the second week post-exposure. Male mice were exposed to 1,3-butadiene (12.5 or 125 ppm; 27 or 276 mg/m3) for 10 weeks or for a single 6 h period. 1,3-Butadiene caused a statistically significant increase in dominant lethality at 125 ppm but not 12.5 ppm. No significant increase in testicular DNA repair was found with either dose level or exposure period while only 6 h exposure to 125 ppm caused a small but significant increase in DNA damage as detected by the Comet assay. These effects demonstrate the genotoxicity of 1,3-butadiene to germ cells at 125 ppm (276 mg/m3) in the mouse but do not confirm its ability to cause abnormalities in the offspring via the sperm (Brinkworth et al, 1998).

Pacchierotti et al (1998) however, showed cytotoxic genetic effects in the sperm of male mice exposed to 1,3-butadiene (up to 1300 ppm; 2876 mg/m3) and chromosome-type structural aberrations in first-cleavage embryos conceived by these males (see Section on Toxicity to Reproduction).Other studies have reported positive results for 1,3-butadiene in the dominant lethal assay in the mouse (EU RAR 2002).

The potential for 1,3-butadiene to induce dominant lethal mutations in the rat was investigated by BIBRA (1996). Male Sprague-Dawley rats were exposed to 0, 65, 400 or 1,250 ppm (143, 885 or 2765 mg/m3) 1,3-butadiene for 6 hours/day, 5 days/week for 10 weeks. Each male was then allowed to mate with two untreated females over a 10 day period. Females were sacrificed on day 20 of pregnancy and numbers of corpora lutea, live implantations, early deaths, late deaths and dead fetuses were recorded. 1,3-Butadiene had no effect on the parameters measured.

 

1,3-butadiene is therefore mutagenic in germ cells in the mouse but not in the rat.

 

 

In Vivo Studies – Human information

 

There is a significant body of genotoxicity information from human occupational studies; these studies have been reviewed by Albertini et al (2010). BD-exposed workers from both BD monomer production and the polymerization plants were examined for both gene mutation endpoints and cytogenetic analysis. Internal exposure and production of epoxide metabolites did occur in these workers based on biomarkers of exposure. Most of the studies did not show any association between BD exposure and increased gene mutations, primarily HPRT mutations. However, using a different method to measure HPRT mutations, one group of investigators showed a relationship in workers exposed to BD in monomer production and in the styrene-butadiene rubber industry. Co-exposures in these studies may not have been adequately accounted for. A recent study from these investigators using their same method have shown that reduced exposures to all potential genotoxic agents in these facilities have resulted in negative findings (Wickliffe et al, 2009). Sram et al (1998) reported increased chromosomal aberrations in BD-exposed Czech workers, but subsequent analyses of the same blood samples failed to show any positive relationship between adducts and chromosome changes (Zhao et al, 2001). Increased micronuclei were reported in workers exposed to very high levels of BD at a Chinese facility (Wang et al, 2010). In the review by Albertini et al (2010), it was concluded that, with the exception of the recent Chinese study, all other studies have failed to find an association between chromosome-level mutations and BD.

 

Conclusions

 

1,3-Butadiene has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown positive results for mutagenicity in vitro in both bacterial and mammalian cell systems, and in vivo in both somatic cells and germ cells in the mouse. A more limited, but nevertheless adequate evaluation in rat somatic and germ cells using comparable endpoints has given negative results. There is therefore evidence for species differences in regard to the genotoxicity of 1,3-butadiene. It is known to require metabolism in order to produce genotoxic entities, and it is likely that the response of species to the material will in part depend on the nature and extent of metabolism (see Section on Toxicokinetics, Metabolism and Distribution). Studies on several groups of BD-exposed workers, both in BD monomer production and in the BD polymerization plants, have not found an association between chromosome-level mutation and BD, with the exception of a recent study in China showing increased micronuclei in workers exposed to very high levels of BD.

It is concluded that the available data indicate that 1,3-butadiene is genotoxic in vitro and in vivo in both somatic and germ cells in the mouse but is not genotoxic in vivo in both somatic and germ cells in the rat. Similar conclusions have been published in the EU RAR (2002) and SCOEL (2007). A weak positive response was seen for hrpt mutations in the rat.

 

 

Additional References

EU RAR (2002). European Union Risk Assessment Report for 1,3-butadiene. Vol. 20. European Chemicals Bureau (http://ecb.jrc.ec.europa.eu/DOCUMENTS/Existing-Chemicals/RISK_ASSESSMENT/REPORT/butadienereport019.pdf)

 

SCOEL. (2007). Recommendation from the Scientific Committee on Occupational Exposure Limits: risk assessment for 1,3-butadiene. SCOEL/SUM/75 final (updated Feb 2007).

 


Short description of key information:
In non-human studies, 1,3-butadiene is genotoxic in vitro and is genotoxic in vivo in both somatic and germ cells in the mouse. The available data on several groups of 1,3-butadiene-exposed workers did not show any association between 1,3-butadiene exposure and increased gene mutations, primarily HPRT mutations. No 1,3-butadiene-related chromosome aberrations have been demonstrated in humans.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

1,3 -Butadiene is genotoxic in vitro and in vivo in both somatic and germ cells in the mouse. It therefore warrants classification of Muta.Cat 2: R46 (May cause heritable genetic damage) under Dir 67/548/EEC and Germ Cell Mutagenicity Cat1B: H340 (May cause genetic defects) under GHS/CLP.