(epoxyethyl)benzene

Endpoint summary

Additional information

In vitro assays

Induction of mutation in bacteria

In several studies, styrene oxide was mutagenic in Salmonella typhimurium strain TA100 without metabolic activation at concentrations ranging from 0.6 μg/mL to 12,000 μg/mL. Styrene oxide also was mutagenic in various studies without metabolic activation in strain TA1530 at a concentration of 768 μg/mL, strain TA104 at a concentration of 120 μg/mL, and strain TA1535 at concentrations ranging from 0.6 μg/mL to 5000 μg/mL. The mutagenic activity of styrene oxide was reduced by the presence of glutathione or S9 liver homogenate. The R enantiomer of styrene oxide was found to be more mutagenic in S. typhimurium strain TA100 than the S enantiomer. In different studies, styrene oxide was not found to be mutagenic in S. typhimurium strains TA1537, TA98, or TA97 with metabolic activation when tested over a concentration range of 250 to 6,000 μg/mL. It is remarkable that styrene oxide was predominantly tested positive in S.typhimurium strains sensitive for base pair mutations, suggesting this to be the predominant mechanism of mutagenicity.

Furthermore styrene oxide was found to be mutagenic in Escherichia coli strain WP2 uvrA without metabolic activation at concentrations ranging from 480 μg/mL to 720 μg/mL. This suggests that a crosslinking effect is involved in the mechanism of mutagenicity.

 

Induction of mutation in yeasts

Styrene oxide caused mitotic gene conversions in Saccharomyces cerevisiae at a concentration of 1,200 μg/mL without metabolic activation and induced forward mutations in Schizosaccharomyces pombe at a concentration of 600 μg/mL (Loprieno et al. 1976).

 

Induction of mutation in mammalian cells

Styrene oxide induced a positive response in L5178Y (TK+/-) cells in the mouse lymphoma assay at a concentration of 13.80 μg/mL without metabolic activation. Metabolic activation reduced the mutagenic activity of styrene oxide in this study (Amacher and Turner 1982, cited in IARC 1994a).Styrene oxide induced forward mutations at the hprt locus in Chinese hamster V79 cells at concentrations ranging from 100 μg/mL (Nishiet al. 1984, cited in IARC 1994a) to 1,020 μg/mL without metabolic activation (Loprieno et al. 1976, cited in IARC 1994a).

In human T-lymphocytes treated with styrene oxide for 24 hours or 6 days at concentrations of 0.2 to 0.4 mM, the maximal dose-dependent mutation frequency at the hprt locus was10 to 20 mutants per 106 clonable cells. This is approximately fourfold higher than background in human T lymphocytes. No increase in hprt mutation frequency was seen at the lowest concentration tested (0.05 mM) (Bastlova et al. 1995).A subsequent, similarly conducted study found that styrene oxide induced mutations at the hprt locus at a frequency 3.6 to 4.8 times higher than background in human T-lymphocytes (Bastlova and Podlutsky 1996).

 

 

Chromosomal aberration

Styrene oxide induced an increased frequency of chromosomal aberrations without metabolic activation in Chinese hamster V79 cells at a concentration of 90 μg/mL(Turchiet al.

1981, cited in IARC 1994a)and in human lymphocytes at concentrations ranging from 3.00 μg/mL to 80.00 μg/mL(Pohlova and Sram 1985;(Linnainmaaet al. 1978; Fabry et al 1978).

 

Micronuclesus test

Styrene oxide induced increased micronucleus formation in cultured human cells at a concentration of 80 μg/mL without metabolic activation(Linnainmaa et al. 1978a,b, cited in IARC1994a).

 

Sister chromatid exchanges

In a mammalian cell cytogenetics (SCE) assay, primary lymphocyte cultures were exposed to styrene oxide dissolved in DMSO at concentrations of 0, 10, 20, 50, 100, and 200 µM without metabolic activation. There was a concentration related positive response of SCE induced over background (Laffon B et al 2001).

 

Styrene oxide induced an increased frequency of sister chromatid exchanges (SCE) without metabolic activation in Chinese hamster ovary (CHO) cells at a concentration of 50.00 μg/mL (de Raat 1978),and in cultured human lymphocytes at concentrations ranging from 1.00 μg/mL to 8.4 μg/mL (Linnainmaa et al. 1978).

 

Exposure of cultured human lymphocytes to a styrene oxide concentration of 100 μM for 22, 36, 48, or 72 hours resulted in a six-fold increase in the induction of SCE at 22 hours of exposure. However, there was a clear and significant inverse relationship between exposure time and SCE frequency (r = -0.9337, P = 0.0018). No relationship between the replication index and the frequency of SCE was seen (r = -0.36, P > 0.05), although cell viability was decreased 74% relative to the control(Chakrabartiet al. 1997).

 

A study was conducted with cultured lymphocytes from human donors to determine the influence of glutathione S-transferase M1 (GSTM1) genotype on SCE induction by styrene oxide. In cultured human lymphocytes treated with styrene oxide at concentrations of 50 or 150 μM for 48 hours, the frequency of SCE was significantly increased (P < 0.001). The GSTM1 genotype had no influence on SCE induction by styrene oxide (Uuskula et al. 1995).

 

A subsequent, similarly conducted study to determine the influence of glutathione S-transferase T1 (GSTT1) genotype on SCE induction by styrene oxide also found increased frequency of SCE induction following treatment with styrene oxide. In lymphocytes from individuals lacking the GST1 gene, the mean numbers of SCE/cell were 1.7 and 1.4 times the control values at styrene oxide concentration of 50 μM (2.78 and 4.83) and 150 μM (13.74 and 18.98), respectively. In lymphocytes from individuals with the GST1 gene, the mean numbers SCE were 2.78 and 13.74 times the control values at concentrations of 50 μM and 150 μM, respectively (Ollikainen et al. 1998).

 

DNA single-strand breaks

In a mammalian single cell gel (Comet) assay, primary lymphocyte cultures were exposed to styrene oxide dissolved in DMSO at concentrations of 0, 10, 20, 50, 100, and 200 µM without metabolic activation. Tail length, percentage of DNA in the comet tail, and tail moment are all increased in those cultures exposed to styrene oxide concentrations equal to or higher than 50mM (Laffon B et al 2001, Laffon B et al 2002).

Styrene oxide induced DNA single-strand breaks in human lymphocytes and calf thymus cells in culture in a Comet assay. Styrene oxide was tested at concentrations of 0.05 to 0.6 mM for periods ranging from 1 to 24 hours (in a series of six experiments) and at concentrations of 0.1 or 0.2 mM for 6 days (in a series of three experiments). Overall, styrene oxide treatment decreased the survival of clonable cells. Styrene oxide formed O6-guanine DNA adducts at a level of 1 to 4 adducts per 108 nucleotides at concentrations of 0.2 to 0.6 mM in 24 hours. Styrene oxide -induced single-strand DNA breaks occurred at all concentrations tested; the breaks in DNA were repaired within 24 hours(Bastlova et al. 1995).

 

 

In vivo assays

 

Germ cell mutagenicity

Styrene oxide did not induce dominant lethal mutations or reciprocal translocations in meiotic germ cells of male BALB/c mice administered styrene oxide by intraperitoneal injection at a dose of 250 mg/kg (Fabry et al. 1978).

 

Styrene oxide induced an increased frequency of sex-linked recessive lethal mutations in

Drosophila melanogaster when administered as a vapor at a concentration of 200 ppm

(980 mg/m3), six hours per day for four days, or orally at a dose of 200 mg/kg in the feed

for 24 hours without metabolic activation (Donner et al. 1979)

 

Micronucleus test

Styrene oxide administered by intraperitoneal injection at a dose of 250 mg/kg had no effect on the frequency of micronuclei in polychromatic erythrocytes of BALB/c mice (Fabry et al. 1978). Intraperitoneal injection of 100, 200, or 400 mg/kg (single dose for evaluation of peripheral blood, two injections for evaluation of bone marrow), did not increase the number of micronuclei in erythrocytes of male ICR mice. (Hagiwara et al, 1996)

The same result was obtained by Morita et al. (1997) after intraperitoneal injection of 100, 200 and 400 mg/kg of styrene oxide to male CD-1 mice.

Chromosomal aberrations

Gavage treatment of CD-1 mice with 50, 500, or 1,000 mg/kg of styrene oxide resulted in increased incidences of chromosomal aberrations (CA) in bone marrow cells at all dose levels tested (Lopreino et al. 1978, cited in IARC 1985, 1994a).

In vivo inhalation exposure to styrene oxide (25, 50,75 and 100 ppm) for 2, 4 or 20 days (25 ppm only) had no effects on chromosomal aberration rates or sister chromatid exchange frequencies in the bone marrow cells of Chinese hamsters. The only positive response in aberration frequency was obtained when styrene oxide was injected in lethal concentration (500 mg/kg body weight, i.p.) into the animal. (Norppa et al, 1979).

Sinsheimer et al (1993) found, that only the S-enantiomer revealed a statistically significant effect on chromosome aberrations and sister chromatid exchanges if injected intraperitoneally at a dose of 100 mg/kg to male CD-1 mice.

 

Sister chromatid exchanges

Inhalation exposure of mice to SO vapor at a concentration of 50 ppm (245 mg/m3) induced a slight increase in SCE in regenerating liver cells and alveolar cells, but not in bone marrow cells (Conner et al. 1982, cited in IARC 1985, 1994a). However, no increases in the incidence of SCE were observed in the bone marrow cells of male Chinese hamsters exposed to SO vapor by inhalation at concentrations of 25, 50, 75, or 100 ppm (122, 245, 368, or 490 mg/m3) for 2, 4, and 21 (25 ppm only) days (Norppaet al. 1979).

 

 

DNA damage/repair

Peripheral blood lymphocytes, liver cells, and kidney cells obtained from mice exposed to SO showed evidence of DNA damage (DNA single-strand breaks) upon analysis with the alkaline version of the single cell gel electrophoresis (Comet) assay. In the study, female C57BL/6 mice were given intraperitoneal injections of SO (in corn oil) at doses of 50, 100, 150, or 200 mg/kg four to six hours before sacrifice. Increased DNA damage, though not statistically significant (P < 0.05) in a one-tailed Kolmogorov-Smirnov two sample test was observed in all cell types tested from the 50-mg/kg dose level.

Statistically significant (P < 0.001) damage in DNA occurred in lymphocytes, liver, and kidney cells at doses ≥ 100 mg/kg. Statistically significant increases in the frequency of DNA damage in the bone marrow were seen only at the two highest doses tested (Vaghef

and Hellman 1998).

Intraperitoneal injection of a single dose of 400 mg/kg styrene oxide to male mice caused chromosome strand breaks in stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow in a single cell gel electrophoresis (Comet) assay (Tsuda et al, 2000).

Intraperitoneal injection of a single dose of 400 mg/kg styrene oxide to male CD1 mice resulted in a significant increase of chromosome strand breaks as shown by the means of a comet assay. (Sasaki et al, 1997).

 

Styrene oxide caused single-strand DNA breaks in the liver, lung, kidney, testis, and brain of male mice administered SO by intraperitoneal injection at doses of 1.8 to 7.0 mM/kg (Walles and Orsen 1983, cited in IARC 1985).

 

Host-mediated assay

Gavage doses of 100 mg/kg of SO to male Swiss albino mice increased the frequencies of gene conversion in Saccharomyces cerevisiae and of forward mutations in Schizosaccharomyces pombe in a host-mediated assay (Loprieno et al. 1976).

 

 

 

Conclusion

As cited in RoC 11^thedition, styrene oxide was mutagenic to bacteria, yeast and insects; it induced chromosomal aberrations and micronuclei in plants. The compound was mutagenic to mammalian cells in vitro;it induced DNA damage in mammalian cells both in vivo and in vitro,chromosomal aberrations and sister chromatid exchanges in vitro.In several studies in mice and hamsters in vivo,no dominant lethal mutations, chromosomal aberrations, micronuclei or sister chromatid exchanges were induced; however, in one study in mice, styrene oxide induced chromosomal aberrations.

 

Short description of key information:
Styrene oxide is genotoxic in a variety of test systems, including prokaryotic, plant, eukaryotic, and mammalian (including human) in vitro and in vivo systems.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

Negative data for in vivo heritable germ cell mutagenicity in mammals are available from a dominant lethal test in mice and a translocation assay in pre-meiotic male mice.

In-vivo studies were both positive and negative. Styrene oxide induced DNA damage in mammalian cells in vivo (positive UDS and Comet assay). In several studies in mice and hamsters in vivo, no chromosomal aberrations, micronuclei or sister chromatid exchanges were induced; however, in one study in mice, styrene oxide induced chromosomal aberrations in bone marrow cells after gavage application. Therefore there is some evidence that styrene oxide has the potential to cause mutations to germ cells.

Styrene oxide was positive in various in-vitro test systems. It was mutagenic to bacteria, yeast and insects; it induced chromosomal aberrations and micronuclei in plants. The compound was mutagenic to mammalian cells and induced DNA damage in mammalian cells.

In conclusion positive results from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells require classification for germ cell mutagenicity for styrene oxide. Category 1B is justified according to GHS Regulation EC No 1272/2008 and category 2 (R46) according to directive 67/548/EEC.