Methyl acetoacetate

Endpoint summary

Additional information

Key 1 – Bacterial reverse mutation assay

The assay was performed 1988 in accordance with OECD no. 471. The test was performed with and without liver microsomal activation and used Salm. typh. strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations: 10; 100; 500; 1000 and 5000 µg/plate (pre-experiment and main test).The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation. Precipitation was not observed. MAA was found to be non-mutagenic in this test system.

Support 1 – Bacterial reverse mutation assay

A study was performed according to OECD-guidelines 471/472 and Japanese Guidelines for Screening Mutagenicity Testing of chemicals. Test item concentrations of 313-5000 microg/plate (-S9 -mix) and 0.313-5000 microg/plate (+S9 -mix) were tested on the following five strains were used: TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2 uvrA. The study was performed in Japan and published in the JETOC Information sheet No. 43 of April 2000 - Special Issue no. 6. A complete English translation of the report is not available. MAA was found to be non-mutagenic in this test system.

Key 2 – In vitro chromosome aberration assay

The study was performed 1995/1996 under GLP. The test was performed with and without liver microsomal activation and used Chinese hamster ovary cells.For the first experiment, 9 dose levels, covering a wide concentration range, were tested. The highest dose was 5000 microg/ml. Subsequent dose levels were halving dilutions of this dose. Methyl acetoacetate was cytotoxic and non clastogenic in Test 1. In the second experiment the dose levels selected were: in presence of S9 mix: 313, 625, 1250, 2000, 2500 and 3000 microgr/ml and in absence of S9 mix: 313, 625, 1250, 2500, 3750 and 5000 microgr/ml. In the additional test (presence of S9 mix only) the dose levels selected were: 625, 1250, 2500 and 5000 microgr/ml. Cytotoxic effects were observed at higher concentrations. For details, please refer to the "Additional information on test results". In test 1, MAA was cytotoxic at concentrations of > 2500 microgr/ml with S9 -mix and at 5000 microgr/ml without S9 -mix. In test 2, cytotoxicity was similar to that of test 1 with S9 -mix, whereas concentrations above 2500 microgr/ml were cytotoxic without S9 -mix. In the additionally performed test with pH-adjustment, no toxicity was noted in any tested concentration from 625 -5000 microgr/ml.

In conclusion, the clastogenic response at higher concentrations in presence of S9 -mix might be rather a secondary effect due to possible degradation products than a substance specific property. At higher test substance concentrations showing toxicity, the pH changed significantly. It can be assumed, that hydrolysis of MAA to acetoacetic acid will occur at these concentrations, lowering the pH-value of the test medium.

It's a known phenomenon that changes of the pH may have an unspecific effect in vitro on chromosomes. As pH-shifts were observed only in presence of S9 -mix, it can further be assumed that hydrolysis of the test item is caused mainly by unspecific hydrolases of the S9 -mix. This assumption is confirmed by the additional experiment with pH-adjustment, where no clastogenic effects were observed up to 2500 microgr/ml (see also supporting study).

Support 2 – In vitro chromosome aberration assay

The study was performed in Japan according to OECD guideline no. 473 and Japanese Guidelines for Screening Mutagenicity Testing of chemicals on Chinese hamster lung fibroblasts (V79).As a result of chromosome analysis, cells with structural chromosomal aberrations and polyploid cells were statistically significantly increased only at the highest dose (1.2 mg/mL) for short-term treatment with S9 mix. Since color change of the medium to yellow was observed at the dose stated above just after the start and at the end of the treatment period, there were two possibilities on the cause of induced structural chromosomal aberrations. The one was medium acidification and the other was DNA damages directly caused by MAA. Therefore, a confirmation test was carried out at the dose stated above by adjusting the pH of the medium to neutral zone (7.0) just after addition of the test article to the medium. As a result, chromosomal aberrations or polyploid cells were not induced. Accordingly, it was shown that chromosomal aberrations induced by the treatment with MAA were caused by unphysiological condition through medium acidification, and the potency of MAA to induce chromosomal aberrations was judged to be negative. In conclusion, Methyl acetoacetate (MAA) did not induce chromosomal aberrations in CHL cells.

Key 3 – In vitro gene mutation assay

The study was performed 2011/2012 in accordance with OECD guideline no. 476. The test was performed with and without liver microsomal activation and used V79 cells.The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (1200 μg/mL) was equal to a molar concentration of about 10 mM. Neither genotoxicity nor cytotoxicity was observed.

Methyl acetoacetate is considered to be non-mutagenic in this HPRT assay.

Justification for selection of genetic toxicity endpoint
Guideline studies; Klimisch 1

Short description of key information:
The test item MAA was tested for genetic toxicity in three in-vitro studies in 1988, 1995 and 2012, all with and without metabolic activation. A reverse mutation assay (Ames test), a chromosomal aberration test and an HPRT-test (gene mutation) were performed, according to the corresponding OECD-guidelines. The tests were GLP-compliant and all assessed as Klimisch 1 studies.

The Ames test used Salmonella typhimurium and E.coli strains and showed a negative result (non-mutagenic). An additional Ames test published in literature (JETOC no.43) showed a negative result as well and confirmed the non-mutagenic result of the first study.
The chromosomal aberration test performed with Chinese hamster ovary cells showed clastogenic effects. However, this clastogenic response at higher concentrations in presence of S9 -mix might be rather a secondary effect due to possible degradation products than a substance specific property. This assumption is confirmed by the additional experiment with pH-adjustment, where no clastogenic effects were observed up to 2500 microgr/ml. In another chromosomal aberration test performed in Japan and published in literature; this study was performed with pH-adjustment and showed a non-clastogenic result, confirming the assumption explained above.

The HPRT-test was performed 2011/2012 under GLP in accordance with OECD guideline no. 476. The test was performed with and without liver microsomal activation and used V79 cells. Neither genotoxicity nor cytotoxicity was observed. MAA was considered to be non mutagenic in this test system.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the data available the substance is not classified or labeled according to Directive 67/548/EEC (DSD) or Regulation 1272/2008/EC (CLP).