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The overall data indicate that acetonitrile is not a point mutagen, but that high concentrations can interfere with chromosome segregation. Acetonitrile at concentrations up to 10,000 μg/plate was negative in S. typhimurium) strains TA1535, TA1537, TA97, TA98 and TA100 in the absence of S9 and in the presence of rat or hamster S9 induced with Aroclor 1254 (Mortelmans et al., 1986; NTP, 1996). Negative results were also obtained in a preincubation S. typhimurium assay and a reverse mutation assay in (S. cerevisiae) D7, conducted in the presence and absence of S9 from rats induced with acetonitrile or phenobarbitone (Schlegelmilch et al., 1988), although the bacterial assay was limited by the use of stationary cultures.

The IPH of Japan (1980) reported that acetonitrile was not mutagenic in bacterial strains S. typhimurium TA 100, TA 98, TA 1535, TA 1537, TA 1538 and E. coli WP2uvr A and WP2uvrA/pKM101, when tested at concentrations up to 5000 μg/plate with or without a metabolic activation system.

Mammalian cell point gene mutation was investigated in a mouse lymphoma assay. Under the experimental conditions reported acetonitrile did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Two mouse lymphoma studies and one HRPT assay all gave negative results for mamalian cell point mutations.

Sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells were significantly increased in the absence of S9 (only at 5000 μg/mL), but there was no effect in the presence of S9 (Galloway et al., 1987; NTP, 1996). Gene conversions in (S. cerevisiae) were increased in the absence of S9, but not in the presence of S9 (Schlegelmilch et al., 1988). Both of these assays measure repair of DNA damage, rather than persistent DNA damage.

Wu et al. (2009) report a positive response for acetonitrile in a comet assay performed in cultured human lymphocytes and HepG2 cells.


Chromosome aberrations were significantly elevated in CHO cells at 5000 μg/mL in the presence of rat S9 but the trend test was borderline (p= 0.016); there was no effect in the absence of S9 (Galloway et al., 1987; NTP, 1996). Although the types of chromosome aberrations were not reported, Galloway et al. (1987) reported that both simple and complex aberrations were elevated at the high dose. The ability of acetonitrile to induce mutations at the HGPRT gene locus in CHO cells in vitro was investigated both in the presence and absence of rat liver S9 (Bioassay Systems Corporation, 1984). There were no significant differences between treated and control cells over a concentration range of 0.1 to 30 mg/mL. Micronucleated normochromatic erythrocytes (NCEs) were slightly increased, without dose response, in the peripheral blood of male mice, but not in females, in a micronucleus assay conducted in conjunction with a 13-week inhalation toxicity experiment (NTP, 1996). Although the micronucleus assay is usually conducted in polychromatic erythrocytes (PCEs), it has been shown that micronucleated peripheral blood PCEs and NCEs are at steady state following dosing for 45-90 days. Schlegelmilch et al. (1988) found that intraperitoneal injection of mice with acetonitrile at 60% of the oral LD50 was weakly clastogenic, with micronucleated polychromatic erythrocytes significantly increased at 24 hours. When the mice were induced by injection of low doses of acetonitrile for 7 days, and then challenged with 60% of the oral LD50, an increase in micronucleated PCEs was not observed until 72 hours after the challenge dose. In a separate study, Jones et al (2001) reported that acetonitrile did not produce significantly increased frequencies of micronucleated polychromatic erythrocytes in the bone marrow or peripheral blood of mice following a single maximum tolerated dose by intraperitoneal injection.

Micronucleus formation can indicate clastogenic activity or interference with chromosome segregation. Acetonitrile (131 ppm for up to 70 minutes) induced aneuploidy (both chromosome gain and chromosome loss) in treated mature oocytes of Drosophila melanogaster females exposed either as larvae or as adults (Osgood et al., 1991a,b). Toxicity and sterility were induced by the 70-minute exposure. When S. cerevisiae was exposed to 5% acetonitrile, it induced mitotic aneuploidy (Zimmerman et al., 1985); the investigators suggested that the induction of aneuploidy by acetonitrile in the absence of point mutations or recombination resulted from interference with tubulin assembly and the formation of microtubules in the spindle apparatus. More recently, Sehgal et al. (1990) obtained in vitro evidence that acetonitrile does inhibit microtubule assembly in taxol-purified Drosophila or mouse microtubules, further indicating that acetonitrile has potential to interfere with chromosome segregation.


In summary, acetonitrile is negative in gene mutation assays and produces only marginal effects in chromosome aberration assays. The potential of high doses of acetonitrile to interfere with chromosome segregation both in vivo and in vitro have been demonstrated in D. melanogaster, which has been attributed to inhibition of microtubule assembly.


Justification for selection of genetic toxicity endpoint
A weight of evidence approach is taken to this endpoint.

Short description of key information:
A number of standard guideline and non-standard studies in vitro and in vivo are available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification of acetonitrile is proposed for genetic toxicity. Acetonitrile does not induce gene mutations in bacteria, and has produced only marginal effects in chromosome aberration assays in vitro. A positive response is also reported in a comet assay in vitro. Reliable in vivo micronucleus studies have shown marginal or negative results. The potential of acetonitrile to interfere with chromosome segregration in D. melanogaster has been demonstrated both in vitro and in vivo, which has been attributed to inhibition of microtubule assembly.