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In vitro: several good quality in vitro gene mutation studies (Klimisch 1 and 2) in bacteria, yeast and mammalian cells are available for different grades of the reaction mass of divinylbenzene and ethylstyrene. All studies reported negative results for gene mutation. One in vitro chromosomal aberration study (Klimisch 1) is available for DVB-96 reporting negative results.

In vivo: The in vivo genotoxic potential of DVB-55 (a mixture of diethenylbenzene and ethenylethylbenzene) was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. In a previously published study, Kligerman et al. (1996, Mutation Research, 370, 107-113) showed a weak positive effect for the induction of micronuclei following inhalation exposure of mice to DVB-55. In the current study, male B6C3F1/Crl mice were whole-body exposed to actual concentrations of 0, 35.0 ± 3.0, 74.1 ± 4.3, or 142.9 ± 7.9 ppm DVB-55 six hours/day for three days. The exposure concentrations were selected to overlap those employed by Kligerman et al. with the highest concentration representing the maximum tolerated exposure concentration. Peripheral blood from exposed mice was sampled 2 and 48 hours after the final exposure. The first sampling time was selected to replicate the timing of the Kligerman et al. study and the second time was consistent with the OECD test guidelines. All animals were observed for clinical signs prior to the exposure, immediately following the exposure, approximately three hours following each exposure, and daily after the final exposure. Groups of mice, five/exposure/timepoint, except the highest exposure where six/timepoint, were sacrificed approximately 2 and 48 hours after the final exposure for the collection of peripheral blood and evaluation of RET (approximately 5,000/animal) for MN by flow cytometry. The proportion of RET was determined based upon 5,000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single oral gavage dose and sacrificed 48 hours later served as positive controls. All animals survived to the end of the observation period. Treatment-related clinical signs including decreased activity, decreased feces, and decreased body temperatures were observed in all test material exposure groups. There were no statistically significant increases in the frequencies of MN-RET in groups exposed to the test material and sampled at approximately 2 and 48 hours after the final exposure. There were statistically significant decreases in the percent RET in all test material exposure groups. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group. Under the experimental conditions used, DVB-55 was considered to be negative in the mouse peripheral blood micronucleus test via inhalation exposure.

In the more recent in vivo micronucleus study with DVB-55 (Dow 2010) a decrease in body temperature 3 hours after the initial exposure was observed in the high dose groups. Hypothermia may lead to an increase in micronuclei as an indirect result of this physiological change and it has been hypothesized that clastogenic injury may be caused by interference with microtubule assembly and spindle function (Asanami, S., Shimono, K., (1997) Mutation Research, 390:70-83 and Asanami, S., Shimono, K., Kaneda, S., (1998) Mutation Research, 413:7-14). Although this effect has not been observed in this recent study it may have caused the slight increase in micronuclei reported by Kligerman et al (1996). No body temperature data has been reported by Kligerman. Hence, it cannot be excluded that the slight positive effects reported by Kligerman were artefacts due to hypothermia.

This is also supported by the results of an in vivo micronucleus test conducted with DVB-80 as part of an NPT bioassay (2006) were no increase in the frequency of micronuclei was seen in peripheral blood of male or female B6C3F1 mice exposed to up to 200 ppm DVB-80 by inhalation for 3 months.


Short description of key information:
GLP-studies equivalent to OECD guideline 471 are available for DVB-96 and DVB-HP. For DVB-55, non-GLP studies equivalent to OECD guideline 471 and 480 are available. This data is supported by a publication reporting Ames test results for an unknown grade of the reaction mass. In addition, a GLP-study equivalent to OECD guideline 473 is available for DVB-96. For DVB-55 and DVB-HP GLP-studies according to OECD guideline 476 are available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No genotoxicity of DVB/EVB was reported in any of the in vitro gene mutation studies conducted with several grades of the reaction mass of divinylbenzene and ethylstyrene. In a weight of evidence evaluation of the 3 in vivo cytogenicity studies conducted with DVB-55 and DVB-HP it has been concluded that the different grades of the reaction mass do not have the potential to induce clastogenic effects in vivo. Hence, no classification for genotoxicity is required for any grade of the reaction mass of divinylbenzene and ethylstyrene.

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