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Anilin
(CAS-NR.: 62-53-3)

Ausgabe: Oktober 2002
Stand. Mai 2002

A) Genotoxicity:

In vitro investigations:

Studies in bacteria:

Studies evaluating the genotoxicity of aniline and other splenic carcinogens have shown negative or inconclusive results in standard Ames test protocols including Salmonella typhimurium strain Ta 102 (Jung et al., 1992; Rosenkranz and Poirier, 1979; Wilmer et al., 1981; Haworth et al., 1983; Bus and Popp, 1987; Simmon, 1979; DeFlora, 1981; Dunkel et al., 1984/85; Nakamura et al., 1987; Sakagami et al., 1988; Shahin, 1989).

In the presence of norharman, mutations in strain Ta 98 were observed after metabolic activation (Nagao et al., 1977; Sugimura and Nagao, 1981). The norharman modification of the Ames assay, however, is based on a chemical reaction product between aniline and norharman in the incubation medium (aminophenylnorharman and hydroxyaminophenylnorharman; Totsuka et al., 1998) and therefore artificial in nature.

a positive result was also reported from preincubation with human gastric juice (DeFlora et al., 1980/82).

a metabolite of aniline, phenylhydroxylamine, was shown to be mutagenic in Salmonella typhimurium strain Ta 100 when metabolically activated with S-9 mix preparation (Nohmi et al., 1984).

Urine samples (ether extracts) of rats receiving 300 mg aniline p.o. 24 hours before sampling showed mutagenicity in Ta 98 in the presence of S-9 mix (Tanaka et al., 1980).

In Bacillus subtilis recombination assays positive (Schiestl et al., 1989) and negative results (Mamber et al., 1983; McCarrol et al., 1981; Simmon et al., 1979) were obtained. DNa repair assay in E. coli Pol A+/A- was negative (Fluck et al., 1976; DeFlora et al., 1984; Rosenkranz et al., 1984), also DNa fragmentation (Rosenkranz et al, 1984).

Studies in mammalian cells:

UDS tests on hepatocytes of rats, mice, hamsters and humans showed negative results after autoradiographic evaluation; concentrations up to 1.0 mmol (93 µg/ml) were employed (Williams, 1980; McQueen et al., 1981; Yoshimi et al., 1988; Butterworth et al., 1989). In one assay employing a nonautoradiographic evaluation via BrdU incorporation, subsequent density shift gradient separation and liquid scintillation counting a positive result was generated (Andrae, 1986).

In an HGPRT assay on V79 cells Aniline exerted a weak activity at extreme concentrations (4,600 and 5,600 µg/ml) in the presence of metabolic activation (Fassina et al., 1990).

In mouse lymphoma cells, Aniline showed effects in the TK^+/- assay thymidine kinase locus with and without metabolic activation. Effective concentrations were in the range of 46.5 - 465 µg/ml with S-9 mix and 930 - 1,350 µg/ml without S-9 mix (Wangenheim and Bolesfoldi, 1988; McGregor et al., 1991; Mitchell et al., 1988). Myhr and Caspari (1988) found only weak effects at high concentrations, whereas Amacher et al. (1980) found a negative result (up to 1,100 µg/ml). Differentiations between large and small colonies were generally not recorded; therefore the effects may account for chromosome aberrations.

In mouse lymphoma cells and the presence of S-9 mix, a DNa fragmentation was obtained in a concentration of 21.5 mmol/l which is unusually high (ca. 2,400 µg/ml; Garberg et al., 1988).

No DNa fragmentation was found in V79 cells (Swenberg, 1976/81) or in human fibroblasts (Kozumbo, 1992).

Chromosome aberration assays in various hamster cell lines showed several positive results (Galloway et al., 1987: 1,600 and 5,000 µg/ml; Miltenburger et al., 1986: 4,300 µg/ml; Ishidate et al., 1988: 1,000 and 2,000 µg), that were typically obtained at high concentrations; other authors found negative results (up to 10-2 M; 1,400 µg/ml of aniline-HCl; Abe and Sasaki, 1977; Swenberg et al., 1976).

Weakly increased SCE rates were observed in hamster cells (Abe and Sasaki, 1977; Galloway et al., 1987) and a rat liver cell line (Cunningham and Ringrose, 1983). For the study of Abe and Sasaki (loc. cit.) the unusual length of incubation period (26 hours) has to be pointed out, which may have facilitated a spontaneous oxidation of the test material; furthermore, no doseresponse relation was apparent. Galloway et al. found 3,000 - 5,000 µg/ml as effective concentrations (with S-9 mix).

In human fibroblasts (465 and 930 µg/ml; Wilmer et al., 1981) or concanavalin Ainduced human lymphocytes (up to 93 µg/ml; Wilmer et al., 1984), increased SCE rates were recorded with aniline HCl. The authors also showed that this effect does not occur with purified lymphocytes and that the addition of hemoglobin (1,000 µg/ml) facilitates the expression of SCEs. The metabolites oaminophenol and N-phenylhydroxylamine were more potent SCE induces than aniline itself and produced a dosedependent increase in SCE rates in human fibroblasts (0.05 and 0.1 mmol/l; Wilmer et al., 1981). Other authors (Tohda et al., 1983; Takehisa and Kanaya) showed increased SCE rates after norharman addition which is an artificial modification according to today's knowledge (see above: studies in bacteria).

According to a poorly documented investigation, aniline did not produce micronuclei in SHE cells (Fritzenschaf et al., 1993).

In vitro cell transformation assays in SHE and BHK21 cells were negative (Dunkel et al., 1981; Pienta et al., 1981; Rosenkranz et al., 1984; Amacher et al., 1982; Styles, 1980). In the BALB/3T3 cell transformation assay a positive result in the absence of S-9 mix was obtained (Dunkel et al., 1981; Rosenkranz et al., 1984).

In vivo investigations:

In mice, induction of micronuclei after single administration was limited to the top dose levels only (tab. 1a) which were close to LD₅₀value and coincided with severe (hemato)-toxicity and a relative increase (in percentage) of polychromatic erythrocytes to normochromatic erythrocytes. Sometimes the micronuclei showed atypical morphology (Vlachos, 1989; Westmoreland and Gatehouse, 1991; Ashby et al., 1991). Negative results were obtained by Harper et al. (1984) and by BG (1985) at dose levels of 125 and 250 mg/kg and 610 mg/kg, respectively. More recently, a dose related increase of micronuclei rates was observed in B6C3F1 mice also after subchronic feeding with 500, 1,000 and 2,000 ppm (Witt et al., 2000).

The positive MNT-result was also confirmed with the acridine orange staining method (Ashby et al., loc. cit.), which is DNA-specific.