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In the key study similar to OECD TG 471, different ranges of 4-methylanisol concentrations (i.e. 0.01, 0.1, 1 and 10 µl/plate or 0.0001, 0.001, 0.01, 0.1 µl/plate in DMSO) were tested in an Ames test and no mutagenic potential in the Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 with/without metabolic activation was shown (Givaudan ULR/59/791049). Toxicity to bacteria was found as absence or incomplete formation of the background bacterial lawn from 1 µl/plate onward.

In a supportive study, 4-methylanisol was found negative in an Ames test at concentrations of 0.1, 0.5, 1, 2.5, 5, 10, 25 and 50 µl/plate using Salmonella strain TA1535, TA1537, TA1538, TA98 and TA100 with/without metabolic activation (Lorillard1984).

In a supportive study, 4-methylanisol was reported to be negative in an Ames test using Salmonella typhimurium strain TA 1535, TA 1537, TA 98 and TA 100 with/without metabolic activation, at a single test substance concentration, i.e. 366.5 µg/plate (Florin1980).

In vitro gene mutation of 4-methylanisol in bacteria was tested in a further Ames test specified as plate incorporation assay, being reported as short database summary. 4 -methylanisol was found negative, using Salmonella strain TA1535, TA1537, TA1538, TA98 and TA100 with/without metabolic activation (Heck1989A). The test substance concentration of 50000 µg/plate was reported as highest concentration tested.

 

Taken together, 4-methylanisol is not mutagenic in bacteria with or without metabolic activiation.

 

Several in vitro unscheduled DNA synthesis assays similar to OECD TG 482 using primary rat hepatocytes and a chromosomal aberration test in Chinese hamster ovary cells are available for 4-methylanisol, however, data on test substance specification, i. e. purity, are lacking.

In an UDS test using hepatocytes from Fischer 344 rats, a range of 5-500 µg/ml 4-methylanisol in DMSO has been tested in the first experiment and 10-250 µg/ml 4-methylanisol in a second experiment (Hazleton1988). No effects have been observed up to 25 µg/ml. A relevant increase in the percentage of nuclei containing at least 6 grains has been observed in 1 of 2 trials at 50 µg/ml, showing no evident cytotoxicity (97% survival based on trypan blue exclusion). Higher concentrations tested, i.e. 100, 188, 250 or 500 µg/ml resulted in evident cytotoxicity (down to 68% survival) and unscheduled DNA synthesis in terms of relevant increases in net nuclear grains per nucleus versus controls and percentages of nuclei containing at least 6 or 20 grains.

In an UDS test using hepatocytes from Sprague Dawley rats, p-methylanisol did not cause a significant increase in mean numbers of net nuclear grain counts at a concentration range of 0.003, 0.01, 0.03, 0.1 and 0.3 µl/ml in DMSO (Microbiological Associates 1989). Cytotoxicty, assessed by LDH release, has been observed from 0.1 µl/ml (approx. 0.1 µg/ml) onward (down to 80% survival), ensuring the absence of genotoxic effects of 4-methylanisol up to cytotoxic concentrations.

Positive effects were reported in an in vitro UDS test using 188 µg of 4-methylanisol as the lowest active concentration tested (Heck1989B). No further information on the protocol or results are available, since this reference is derived from a secondary source.

Overall, conflicting data from UDS in vitro tests are present in terms of cytotoxicity and the genotoxic potential of p-methylanisol, mainly observed at cytotoxic concentrations. 

In an in vitro chromosomal aberration test in Chinese hamster ovary cells similar to OECD TG 473, several concentrations of 4-methylanisol in DMSO (93 - 504 µg/ml) were incubated with and without metabolic activation (Hazleton 1989). No significant increases in chromosomal abberations have been observed in the 10 hour and 20 hour test setup without metabolic activation, whereas cytotoxicity was observed at the highest concentration assessed in the 20 hour setup (504 µg/ml). In the presence of a metabolic system, no significant increases in chromosomal abberations have been observed in the 10 hour setup, whereas concentration dependent and significant increases in the number of cells with chromosomal abberations were detectable in the 20 hour setup with and without concomitant overt cytotoxicity.

Based on the equivocal findings from the in vitro genotoxicity tests in mammalian cells, an unscheduled DNA synthesis assay in rats and a micronucleus test in mice have been conducted to assess the mutagenic and/or clastogenic potential of 4-methylanisole in vivo.

In the key study, i.e. an unscheduled DNA synthesis assay in Wistar rats according to OECD TG 486 and GLP, 4-methylanisol, emulsified in corn oil, was administered once orally to male animals at dose levels of 1000 and 2000 mg/kg body weight (BASF80M0506/094419). Hepatocytes were harvested by in situ liver perfusion 3 and 14 hours after administration of the test substance and scored for DNA repair activity (incorporation of radiolabeled thymidine). The administration led to clinical signs of toxicity at both sacrifice intervals. However, no reduced viability of hepatocytes as indication for test substance induced toxicity was observed. Administration of 4-methylanisol did not lead to a biologically relevant increase in the mean net nuclear grain counts at any dose level at both sacrifice intervals, being close to the range of the concurrent vehicle controls and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, 4-methylanisol did not induce DNA-damage leading to increased unscheduled DNA synthesis in hepatocytes of male Wistar rats in vivo.

In the key study, i.e. a micronucleus assay in male NMRI mice according to OECD TG 474 and GLP, 4-methylanisol was formulated in corn oil and administered orally at 500, 1000, and 2000 mg/kg bw (24 h preparation interval) or 2000 mg/kg bw (48 h preparation interval; BASF26M0506/099093). 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. Clinical signs of toxicity, i.e. ruffled fur, were observed in all test substance dose groups and reduction of spontaneous activity was observed in the high dose animals. After treatment, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control, indicating that 4-methylanisol did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant increase in the frequency of the detected micronuclei at any preparation interval and dose level tested. In conclusion, it can be stated that under the experimental conditions, 4-methylanisol did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, 4-methylanisol is considered to be non-mutagenic in this micronucleus assay.

Overall, on the basis of the present studies in vivo, 4-methylanisol is not considered to have a genotoxic potential.

 

 

 


Short description of key information:
Ames test: negative (Givaudan ULR/59/791049)
Unscheduled DNA synthesis assay in vivo (according to OECD TG 486 and GLP): negative (BASF80M0506/094419)
Micronucleus assay in vivo (according to OECD TG 474 and GLP): negative (BASF26M0506/099093)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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.