reaction mass of 3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Endpoint summary

Additional information

The genetic toxicity of methylionone was analyzed in a bacterial reverse mutation assay according Ames et al. (Wagner, 1999). The bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were tested with and without metabolic activation by Aroclor-1254 induced rat liver S9 mix according to the plate incorporation method. Methylionone was used in concentration up to 6667 µg/plate dissolved in DMSO. Although some cytotoxicity in some strains at higher concentrations were noted, no mutagenic effect of methylionone was detected. It is worth to mention, that the result were presented in the RIFM database as well as by the FFHPVC Terpene Consortium and were sometimes different regarding concentrations given. However, in both cases the same negative result was stated.

In another test reported to RIFM, methylionone dissolved in DMSO was applied to bacteria S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 at concentrations of 0, 0.01, 0.1, 1, or 10 µl/well with and without metabolic activation (Quest, 1980). Since a slight increase in number of revertants was reported for strain TA1537at 0.01 µl/well in the presence of metabolic activation the strain, was tested again with concentrations of 0, 0.005, 0.01, 0.05, or 0.1 µl/well. Although cytotoxicity was observed in some strains at 1 and 10 µl/well, methylionone was found to be not mutagenic.

In addition, a study, where an test was performed with the isomer alpha-methyl-ionone (CAS 7779-30-8), could also taken for assessment (Wild, 1983). No mutagenic effects were noted in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation by rat liver microsome fraction S9 from Aroclor induced rats.

Methylionon 70 (CAS 1335-46-2) was tested in a HPRT test in CHO cells with and without metabolic activation (BASF SE, 2014). Cytotoxicity was found in the absence and presence of S9 mix, at least at hte highest applied concentrations. The test substance did not cause any relevant increase in the mutant frequencies in two independent experiments.

In a study using a chromosome aberration assay the genetic toxicity in vitro was analyzed in Chinese hamster ovary cells (Gudi, 2000). The cells were incubated for 4 h or 20 h with 12.5, 25, and 50 ug/ml methylionon dissolved in DMSO with and without metabolic activation. 0.1 and 0.2 ug/ml mitomycin C was used as positive control in the non-activated assay and 10 and 20 ug/ml cyclophosphamide in the activated assay. After harvesting the cells, frequencies of cells with structural and numerical aberrations were calculated by analyzing a minimum of 200 metaphase spreads and scoring for chromatid-type and chromosome-type aberrations. The resulting findings were ambiguous, as an induction of structural chromosome aberrations was found in the absence of S9 metabolic activation, but not in the presence of S9 metabolic activation. Furthermore, it was negative in both the absence and presence of S9 metabolic activation for the induction of numerical chromosome aberrations.

The genetic toxicity in vivo was analyzed in mammalian erythrocyte micronucleus test (Gudi, 2000). Five male and five female IRC mice per dose were intraperitoneally injected with 20 ml/kg bw at concentrations of 462.5, 925, or 1850 mg/kg body weight methylionone in corn oil. 24 h and 48 h after dosing, animals were sacrificed and bone marrow cells were isolated from the femurs.

Only a light to moderate reduction (up to 35%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the methylionone-treated groups relative to the respective vehicle controls. A statistically significant increase in micronucleated  polychromatic erythrocytes (8 MNPCE/10000 PCE) was observed in male mice 24 hrs after treatment with 925 mg/kg. However, this response is not considered biologically relevant (each of the five animals had no more than 3 MPCE, which are within the range of historical solvent control:  0-7 MN/2000 PCE/animal). Also, no significant increase and no dose responsiveness was observed in any other test article treated group regardless of dose level, sex, or bone marrow collection time. Thus, methhylionone could be regarded as not mutagenic in vivo.

 

Beside this study, a micronucleus test with the isomer alpha-methyl-ionone could also be used for assessment (Wild, 1980). In this study, male 2 – 6 male and female NMRI per does group mice were dosed with 0, 825, 1444 or 2063 mg/kg in olive oil by intraperitoneal injection. After 30 h, the mice were killed and bone marrow smears were prepared followed by determination of the mean number of micronucleated polycromatic erythrocytes (PE) / 1000 PE. Since the resulting values were from 0.7 – 1.9 which is in the range of the positive control (1.85), the test result was regarded as negative.

Thus, no genetic toxicity in vivo could be found with the isomer alpha-methyl-ionone.

 

In the same study, examination of number of sex-linked recessive lethal chromosomes in flies Drosophila were done and a concentration of 20 mM was analyzed (Wild, 1980). No increase in frequency of sex-linked recessive lethals was found, but the result was not taken for assessment as they have no relevancy for human.

 

Although ambiguous results were found in the chromosome aberration test in mammalian cells in vitro, no genetic toxicity could be detected in bacteria as well as in mammalian cells in vivo. Thus, methylionone could be regarded as not mutagenic.

Short description of key information:
Genetic toxicity:
- in vitro in bacteria: negative (Ames test)
- in vitro in mammalian cells: negative (HPRT)
- in vitro in mammalian cells: ambigious (chromosome aberration, CHO cells)
- in vivo: negative (MNT, SLRL)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Although ambiguous results were found in the chromosome aberration test in mammalian cells in vitro, no genetic toxicity could be detected in bacteria as well as in mammalian cells in vivo. Thus, due to overall negative results for genetic toxicity, no classification is required.