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Mutagenicityin bacteria

No key study has been defined addressing mutagenic potential of 4-methoxybenzaldehyde in bacteria. However, a variety of Ames tests have been taken into consideration in a weight of evidence. The test substance was negative in bacterial reverse mutation assays in the presence or absence of a metabolic activation system at concentration levels up to 5000 µg/plate. No relevant increases in revertants were observed in S. typhimurium strain TA1537 in a standard plate test (BASF AG, 1987; 85/49) and in S. typhimurium strains TA1535, TA97, TA98 and TA100 in a preincubation test (NTP, 2002). In line, 4-methoxybenzaldehyde was found negative in S. typhimurium strains TA100, TA1535, TA98, TA1537 and E. coli WP2uvrA (Chemicals Investigation Promoting Council, 2001) and in TA98, TA100, TA1535, TA1537, TA1538 plus E. coli. WP2uvrA (JETOC, 1996-2008). Furthermore, Ames tests in literature with limited documentation confirmed the absence of a mutagenic potential of 4-methoxybenzaldehyde in bacteria.

Mutagenicityin mammalian cells

In the chosen key study, a gene mutation assay in chinese hamster V79 cells in vitro (V79/HPRT) test acc. to OECD 476 and GLP, no substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration of 4-methoxybenzaldehyde (10 mM) with and without metabolic activation (BASF, 2012; 50M0538/11X266). The mutation frequency observed remained well within the historical range of respective solvent controls. Therefore, 4-methoxybenzaldehyde is considered to be non-mutagenic in this HPRT assay.

Further contradicting data from literature with limited documentation are available. However, indications of a mutagenic potential of 4-methoxybenzaldehyde in mammalian cells could not be confirmed by the chosen key study, which was performed according to current OECD protocol under GLP.

Frequencies of 6-thioguanine resistant mutations in chinese hamster V79 induced by UV or x-rays were decreased by treatment with 10, 33 or 100 µM 4-methoxybenzaldehyde (Imanishi, 1990).

In a mouse lymphoma assay (results for 50 substances were reported in a summary, no information on putative confounding factors) using heterozygous L5178Y TK +/- cells, 4-methoxybenzaldehyde was reported to lead to an dose dependent, up to 4-fold increase in the mutation frequency in the absence of a metabolic system (not tested with metabolic activation). Mutation frequency increases were significant at 3.56, 4.04, 4.56 and 5.08 mM 4-methoxybenzaldehyde, resulting also in evident decreases in total growh (Wangenheim,1988).

In a DNA alkaline unwinding assay (results for 78 substances were reported in a summary), 4-methoxybenzaldehyde induced increases of single strand DNA at cytotoxic concentrations (7.02 and 8.03 mM) in mouse lymphoma cells, being indicative for DNA strand breaks under the given testing conditions (Garberg, 1988).

Clastogenicity in mammalian cells

No key study is available for clastogenicity of 4-methoxybenzaldehyde. However, study data from literature were considered for the assessment in a weight of evidence.

In a chromosomal abberation test in vitro with a Chinese hamster cell line (CHL/IU), according to OECD TG 473 and GLP (reported from secondary source), no increase in chromosomal aberrations was observed after 6 hour (with/without metabolic activation) or 24 hour treatment (without metabolic activation) up to 10 mM 4-methoxybenzaldehyde (Chemicals Investigation Promoting Council, 2001).

In a chromosomal abberation test in vitro with a Chinese hamster cell line B241, reported from literature with limited documentation, a significant increase in chromosomal abberations has been reported for 4-methoxybenzaldehyde at a non-cytotoxic concentration, i.e. 50 nM (Kamasaki, 1982).

In a chromosomal abberation test in vitro with Chinese hamster lung cells, reported from literature with limited documentation, no dose-related increase in the percentages of polyploid cells or cells with structural chromosome aberrations was observed up to 500 µg/ml (approx. 4 mM). Under the test conditions, 4-methoxybenzaldehyde was not mutagenic in the chromosome aberration test (Ishidate, 1983).

In a sister chromatid exchange assay in vitro with human lymphocytes, reported from literature with limited documentation, 4-methoxybenzaldehyde induced a concentration dependent increase of SCE frequencies, when tested up to 2 mM (no data on cytotoxicity available; Jansson, 1988).

In a sister chromatid exchange assay in vitro with Chinese hamster ovary cells, reported in literature with limited documentation, 4-methoxybenzaldehyde did not induce increases in SCE frequencies at concentrations up to 100 µM (no data on cytotoxicity available ; Sasaki 1987). However, a dose dependent increased frequency of SCEs was observed for 4-methoxybenzaldehyde, when pretreated with MMC (0.15 µM).

An in vivo micronucleus test is reported in literature with limited documentation (Sasaki, 1990). Male ddY mice were irradiated with X-rays (200 rad) prior to single oral treatment with 250, 313 or 500 mg/kg bw 4-methoxybenzaldehyde. Based on the authors, none of the flavouring compounds (including 4-methoxybenzaldehyde) did affect the frequency of PCEs (only shown for vanillin). Furthermore, X-ray-induced chromosome aberrations were suppressed by 4-methoxybenzaldehyde. The frequency of polychromatic erythrocytes with micronuclei was dose-dependently decreased, leading to a 60% decrease in the 500 mg/kg bw dose group.

Taken from literature with limited documentation, the structurally related 4-ethoxybenzaldehyde has been tested in a micronucleus assay using NMRI mice (Wild 1983). Four animals per dose group have been treated twice intraperitoneally (0, 24 hours) with 335, 670 or 1005 mg/kg bw 4-ethoxybenzaldehyde and bone marrow was assessed after 30 hours. Two of 4 animals in the high dose group died during the course of the study. 4-ethoxybenzaldehyde was found to induce no significant increase in micronuclei in polychromatic erythrocytes under the chosen testing conditions.The tested substance is considered sufficiently similar for a read across to 4-methoxybenzaldehyde since it possesses a high similarity concerning its functional groups. The respective structures only differ between an ethoxy- vs. an methoxy-group in para-position. 

Overall in a weight of evidence, 4-methoxybenzaldehyde was not mutagenic in bacteria under the given testing conditions. Based on the chosen key study according to current OECD protocol and GLP, 4-methoxybenzaldehyde was not mutagenic in mammalian cells. Contradicting results concerning clastogenic effects of 4-methoxybenzaldehyde in mammalian cells in vitro have been reported in literature, however, these findings were not confirmed in animal studies.


Endpoint Conclusion:

Justification for classification or non-classification

Overall, the present data on genetic toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008 and therefore, a non-classification is warranted.