Aniline

Endpoint summary

Additional information

The genotoxic potential of aniline has been extensively assessed in bacteria and cultured mammalian cells as well as in rodents and has been discussed in detail by Bomhard and Herbold (2005), by MAK (2007) and in the EU risk assessment report (ECB, 2004).

In the routine bacterial mutation assay aniline was not mutagenic in all common Salmonella typhimiurium and Escherichia coli tester strains in concentration up to 6.6 mg/plate the absence and presence of metabolic activation (Dunkel et al., 1984; Haworth et al., 1983, De Flora, 1981; Simmon, 1979).

Controversial findings have been reported for gene mutation in cultured mammalian cells in the mouse lymphoma thymidine kinase assay. Aniline was tested negative for gene mutation in concentrations up to 15 mM in the presence of metabolic activation (Amacher et al., 1980). In the remaining four studies positive results were reported. A clear positive effect was observed when tested at concentrations up to 15 mM and 5 mM in the absence and presence of metabolic activation, respectively (Wangenheim and Bolcsfoldi, 1988). In contrast, a less pronounced positive effect was reported in three studies at concentrations of up to 22 mM, exceeding the recommended limit concentration of 10 mM in the OECD test guideline 476 (Mitchell et al., 1988; Myhr and Caspary, 1988; McGregor et al, 1991). In addition, a positive result in a HPRT test in cultured V79 at 60 mM in the presence of metabolic activation was reported, whereas in the absence of metabolic activation no increase in the number of mutants was observed at concentrations up to 20 mM.

Studies on chromosomal aberration in cultured mammalian cells gave positive results in the presence of metabolic activation for high aniline doses, i.e. 11 mM – 54 mM (Ishidate, 1988; Galloway et al., 1987; Miltenburger, 1986). In the absence of metabolic activation an increase in the percentage of chromosomal aberration was observed only in one study at a concentration of 47 mM (Miltenburger, 1986).

No unscheduled DNA synthesis was observed in primary human and rat hepatocytes (Butterworth et al., 1989; Yoshimi et al., 1988) in doses up to 1.0 mM. An increase in DNA strand breaks at was reported in mouse lymphoma cells at a aniline dose of 21.5 mM and (Garberg et al., 1988) only in the presence of metabolic activation.

In a host mediated assay, 10 rats were given a aniline dose of 300 mg/kg p. o. and the urine collected during the first 24 hours. The ether extracts of the urine were tested for mutagenicity in Salmonella typhimurium TA 100 and TA98 in the presence and absence of metabolic activation. A clear dose-dependent effect was obtained in the tester strain TA98 in the presence of S9-mix (Tanaka et al., 1980).

Several appropriate in vivo studies to assess the potential of aniline to induce micronuclei in bone marrow cells of rats or mice have been performed according to or similar to the OECD test guideline 474. In mice, positive bone marrow micronucleus tests were reported after intraperitoneal (Vlachos, 1989; Ashby et al., 1991; Westmoreland and Gatehouse, 1991) and oral administration of aniline (Westmoreland and Gatehouse, 1991). After intraperitoneal administration the effective doses were 2 x 300 mg/kg (Vlachos, 1989) and 2 x 380 mg/kg (Ashby et al., 1991; Westmoreland and Gatehouse, 1991), the latter corresponding to 80% of the LD-50 (Ashby et al., 1991). After oral administration a positive response was found after administration of 1000 mg/kg by Westmoreland and Gatehouse (1991). Furthermore, a positive result for micronucleated erythrocytes in peripheral blood has been reported in mice feed doses of approx. 360 mg aniline/kg bw/d for 90 days (Witt et al., 2000). In the rat a dose dependent increase in micronucleated polychromatic erythrocytes has been observed after oral application of approximately 290 to 360 mg aniline/kg (according to 400 and 500 mg anline hydrochloride/kg bw) at the 24 h sampling time. At the 48 h sampling time no increase in micronucleated erythrocytes was observed (Bomhard, 2003).

In addition, two appropriate in vivo studies on aniline induced chromosomal aberration in bone marrow cells in mice and rats are available. In the mouse no increase in chromosomal aberration has been observed at intraperitoneal aniline doses of approx. 273 mg/kg (Jones and Fox, 2003). In rats a statistically significant increase in chromosomal aberration in bone marrow cells has been observed at the highest orally administered aniline dose of approx. 360 mg/kg at the 18 h but not at the 30 h sampling time (Bomhard, 2003).

Induction of DNA strand breaks determined by the alkaline elution technique has been observed in various tissues of rats after a single intraperitoneal administration of aniline. In rats dosed with 52.5, 105, 210, 420 or 840 mg/kg positive findings were reported for rat livers (from 105 mg/kg upwards) and kidney (210 mg/kg) but a negative finding for spleen (210 mg/kg). In mice dosed with 210 or 420 mg/ aniline kg no induction of DNA strand breaks were observed in the liver, kidney and bone marrow (Parodi et al., 1982).

In rats dosed with 50 mg/kg bw/d for seven days and a final dose of 250 mg [14C]aniline/kg on day 8 a statistically significant increase in DNA binding was observed primarily in kidney but also in spleen and large intestine. of the highest dose group, pre-dosing No substantial DNA binding was found in rat livers and in mice treated similarly (McCarthy et al., 1985).

In a dominant lethal assay Wistar-derived rats were dosed with 47, 150 or 200 mg/kg aniline/day i.p. for 5 consecutive days. The males were mated 1:1 with females of the same strain for ten consecutive one-week periods. Observed severity of clinical signs of toxicity and a decrease in bodyweight were treatment related. No consistent time and dose dependent decrease in the number of live implants and increase in the number of early deaths were observed (CTL, 1998).

Furthermore, no increase in sperm head abnormalities was observed in mice receiving approx. aniline doses of 12 to 144 mg/kg intraperitoneally on five consecutive days (Topham, 1980).

In conclusion, the genotoxicity of aniline has been investigated in numerous experiments including a considerable number of in vivo studies.

Several studies in rats and mice pointed to a clastogenic potential, which was however expressed at high, erythrotoxic dose levels only (Ashby et al., 1991; Bomhard, 2003; George et al., 1990; Ress et al., 2002; Vlachos, 1989; Westmoreland and Gatehouse, 1991). While in rats an increased micronucleus rate in the bone marrow was accompanied by an increase in chromosome aberrations (Bomhard, 2003) this was not the case with mice (Jones and Fox, 2003). The wide database on aniline studies and specific investigations point to a low potency and an indirect mechanism of clastogenicity, mediated by erythrotoxicity followed by spleenic toxicity and compensatory extramedullary hematopoesis in the bone marrow of rats. The consequential increased cell turnover and iron cycling in the bone marrow is discussed to encourage chromosomal damage (Greim, 2007).

Endpoint Conclusion:

Justification for classification or non-classification

Aniline is negative in routine bacterial mutation tests. In cultured mammalian cells positive effects were obtained primarily with respect to chromosomal effects, generally at concentrations exceeding the recommended limit dose of 10 mM and in the presence of an exogenous metabolic activation system. In vivo aniline induced micronuclei and chromosomal aberrations in the bone marrow of rats and micronuclei in the bone marrow of mice. However, the doses in these studies were high and caused toxicity/erythrotoxicity.

The wide database on aniline studies and specific investigations point to a low potency and an indirect mechanism of clastogenicity, mediated by erythrotoxicity followed by spleenic toxicity and compensatory extramedullary hematopoesis in the bone marrow of rats. The consequential increased cell turnover and iron cycling in the bone marrow is discussed to encourage chromosomal damage (Greim, 2007). Overall, the genotoxicity of aniline appears to be very low, if any (SCOEL, 2010).

Positive results in in vivo mutagenicity/genotoxicity tests conducted with aniline were generally confined to high doses that were accompanied with signs of hematotoxicity. Aniline is therefore classified as a category 3 mutagen according to Annex VI to 67/548/EEC. According to the classification criteria in Annex I of the CLP regulation (EC) 1272/2008 aniline is classified into category 2 for germ cell mutagenicity. This classification is in accordance with the entries in the Annex VI to the CLP regulation.