[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Delta-damascone gave negative results in the Ames test (OECD 471) with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli strain WP2 uvrA, with and without metabolic activation.

In the presence of metabolic activation, the substance induced an increase in the frequency of structural chromosome aberrations in the chromosome aberration test (OECD 473) with human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo, the substance was negative in a mouse bone marrow micronucleus test (OECD 474) after a single oral (gavage) exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro: reverse mutation assay:

An in vitro mutagenicity test using microorganisms was performed according to a protocol equivalent or similar to OECD Guideline 471, and according to GLP. The ability of the substance to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2uvrA using a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). In the absence of the S9, the highest dose tested was 78.1 µg/plate, while in the presence of S9 mix it was 313 µg/plate based on a dose-range finding test. No precipitation was observed at any dose level with and without S9 mix. Bacterial growth inhibition was only observed at 78.1 µg/plate without S9 mix and at 156 and 313 µg/plate with S9 mix. The numbers of revertant colonies in the substance treatment groups were less than two times that observed in each negative control in all test strains and therefore, the mutagenicity of the substance was judged negative. As such, it is concluded that the substance has no ability to induce mutations under the present test conditions.

In vitro: chromosome aberration test:

An in vitro chromosome aberration study according to OECD Guideline 473 and in compliance with GLP was performed. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers for three hours followed by 18 hours recovery. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. The dose levels selected for the metaphase analysis were 2.5, 20 and 45 μg/mL in the presence of S9 mix and 10, 20 and 25 μg/mL in the absence of S9 mix.

In the absence of S9-mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control. In the presence of S9 mix, the test substance caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at dose levels of 20 μg/mL (p<0.01: including gaps only) and 45 μg/mL (p<0.001: both including and excluding gaps), when compared with the solvent control. No statistically significant increases in the proportion of polyploid cells were seen. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that the test substance has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the presence of S9 mix only, in this in vitro cytogenetic test system, under the experimental conditions described.

In vivo: micronucleus assay:

In an in vivo micronucleus test, performed according to OECD 474 and in compliance with GLP, ICR mice were exposed to the test substance in corn oil by oral gavage. In the dose range-finding study, three male mice were exposed to the test substance at 1100, 1250 or 1500 mg/kg bw and three female mice were exposed to 1500, 1750 or 2000 mg/kg bw. Based upon the results of the dose range finding study, a dose of 1250 mg/kg bw was estimated to be the maximum tolerated dose for male mice. Furthermore, since a two fold difference in the MTD dose between male and female mice was not observed, the micronucleus test was conducted using male mice only. In the micronucleus test, five male mice per group were treated with the vehicle, positive control (cyclophosphamide monohydrate) or 437.5, 875 or 1250 mg/kg bw of the test substance and were euthanized 24 hours after treatment. In addition, five male mice per group were treated with the vehicle or 1250 mg/kg bw of the test substance and were euthanized 48 hours after treatment. At the time of euthanasia, femoral bone marrow was collected and bone marrow smears (slides) were prepared and stained with Acridine orange stain (a nucleic acid specific stain). Bone marrow cells (polychromatic erythrocytes (PCEs); 2000 PCEs/animal) were examined microscopically for the presence of micronuclei (micronucleated PCEs; MPCEs). A statistical analysis of data was performed using one way analysis of variance (ANOVA, significant level of p≤0.050) and Dunnett’s t-test (post hoc). In addition, the ratio of polychromatic erythrocytes to total of 1000 erythrocytes (PCEs/ECs ratio) was determined as an indicator of bone marrow exposure to the test substance and subsequently as a measure of test substance cytotoxicity. Additional 6 satellite groups per each test substance dose (each composed of 3 mice) were assigned for blood collection. These animals were dosed with the test substance at 437.5, 875 or 1250 mg/kg bw and were bled at 15 minutes, 30 minutes, 1, 2, 4, and 8 hours post-dose. Blood from the 3 main study animals, dosed with the vehicle or test substance, was also collected at 24 hours post-dose just prior to bone marrow collection. Plasma was analyzed as a confirmation of systemic exposure to the test substance.

Measurable concentrations of the test substance were detected in the plasma samples of animals dosed with the test substance indicating systemic exposure. No mortality was observed in any of the treatment groups, but clinical symptoms and weight loss (about 6 -10% relative to pretreatment weight) were observed in mice of the 875 and 1250 mg/kg bw dose groups. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the bone marrow were observed in the test substance groups relative to the respective vehicle control groups suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration. The positive control induced a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met.

Under the conditions of this test, a single oral administration of the test substance at doses up to and including 1250 mg/kg bw (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male ICR mice. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.

Justification for classification or non-classification

Based on the available data the substance is not genotoxic and therefore, is not classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).