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For delta-damascone, an Ames test, an in vitro chromosome aberration test and an in vivo micronucleus test were available.

The following results were observed in the studies:

Ames test

An in vitro mutagenicity test using microorganisms with delta-damscone was performed according to a protocol equivalent or similar to OECD Guidelines 471 and 472, and according to GLP (Hita Laboratory, 2006). The ability of delta-damascone to induce mutations was investigated usingSalmonella typhimuriumstrains TA100, TA1535, TA98 and TA1537 andEscherichia colistrain 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 selected was 78.1 µg/plate, while in the presence of S9 mix it was 313 µg/plate based on the observed bacterial growth inhibition. No precipitation was observed at any dose level with and without S9 mix.

The numbers of revertant colonies in the test substance treatment groups were less than two times that observed in each negative control in all test strains and therefore, the mutagenicity of the test substance was judged negative. As such, it is concluded that delta-damascone has no ability to induce mutations under the present test conditions.

In vitro chromosomal aberrations in culturedhuman lymphocytes

A study according to OECD Guideline 473 and according to GLP was performed to assess the ability of delta-damascone to induce chromosomal aberrations in human lymphocytes cultured in vitro (Huntingdon Life Sciences Ltd., 2009b).

In the presence of S9 mix, delta-damascone 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. As a clear statistically significant response was displayed, no further testing was conducted. It is concluded that the test substance delta-damascone 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

To evaluate the genotoxic potential of delta-damascone as measured by its ability to induce micronucleated polychromatic erythrocytes (PCE) in mouse bone marrow after a single oral (gavage) exposure, a GLP-compliant in vivo micronucleus assay was performed according to OECD Guideline 474 (BioReliance, 2009).

In the dose range finding study (toxicity study), male mice were exposed to delta-damascone at dose levels of 1100, 1250 or 1500 mg/kg bw, whereas female mice were exposed to 1500, 1750 or 2000 mg/kg bw. As no significant differences in the toxicity between male and female mice were observed in the dose range finding study, only male mice were used in the micronucleus test.

In the micronucleus test, corn oil served as a vehicle control and cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg bw, was used as a positive control. Male mice treated with the vehicle, positive control or 437.5, 875 or 1250 mg/kg bw delta-damascone were euthanized 24 hours after treatment. Five male mice treated with the vehicle or 1250 mg/kg bw delta-damascone were euthanized 48 hours after treatment. 

Measurable concentrations of delta-damascone were detected in the plasma samples of animals dosed with the test article indicating systemic exposure. Piloerection and diarrhea were observed at 875 and 1250 mg/kg, and lethargy, hunched position and palpebral closure were observed in mice at 1250 mg/kg, showing that the substance has become systemically available. No mortality was observed in any of the treatment groups.

No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the bone marrow were observed in the test article groups relative to the respective vehicle control groups, suggesting that the test article did not inhibit erythropoiesis.

No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test article groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (ANOVA, Dunnett’s t-test p>0.050).

CP, the positive control, induced a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control (p ≤ 0.050, Dunnett’s t-test).

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 as specified in the protocol.

Under the conditions described in this report, a single oral administration of delta-damascone 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, delta-damascone was concluded to be negative in the in vivo micronucleus assay.

Overall evaluation of genotoxicity of delta-damascone

In summary, for delta-damascone a negative Ames test (gene mutation), a positive in vitro chromosome aberration test, and a negative in vivo chromosome aberration test were available. According to chapter R.7A, pp. 374-402 and in accordance with Table R.7.7-5 of the REACH Guidance chapter R.7A, no further testing on genotoxicity is required in this case. Furthermore, according to Table R.7.7 -5, the conclusion to be drawn on genotoxicity is "not genotoxic". Thus, based on the data available for delta-damascone, it is valid to conclude that delta-damascone is negative for genotoxicity.


Short description of key information:
Delta-damascone does not induce gene mutations in a bacterial in vitro system. In vitro tests on the induction of chromosomal aberrations (human lymphocytes) has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the presence of S9 mix only.
In vivo, delta-damascone was negative in a mouse bone marrow chromosomal aberration test after repeated a single oral (gavage) exposure.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data and in accordance with Directive 67/548/EEC and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for mutagenicity.