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Key value for chemical safety assessment

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Gene mutation in bacteria

The key study was an Ames test in bacteria according to a method similar to OECD guideline 471 (Ames test). The plate incorporation assay was performed with concentrations of 0.125, 0.25 and 0.5 µl/plate, with and without metabolic activation. Negative and positive controls were included as well. The frequency of revertant colonies was recorded.

The positive and negative control were valid: all positive control chemicals induced an increase in frequency of revertant colonies for the relevant strains. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Based on the results of this Ames test, the test item Citrus bergamia Risso was considered to be non-mutagenic under the conditions of the test.

 

In vitro mammalian chromosome aberration test

Two in vitro chromosomal aberration tests performed according to OECD guideline 473 for the test substances Lime oil and Lemon oil were available. These results are evaluated in a weight of evidence approach and read across to Bergamot oil, see the read across justification document.

In the in vitro chromosomal aberration test with Lime oil Chinese hamster fibroblast cells (CHL) were continuously exposed to three different doses (with 0.04 mg/ml as maximum) for 24 and 48 hours without metabolic activation. Lime oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations in the absence of metabolic activation and was therefore considered not clastogenic in this test.

In the in vitro chromosomal aberration test with Lemon oil Chinese hamster fibroblast cells (CHL) were continuously exposed to three different doses (with 0.125 mg/ml as maximum) for 24 and 48 hours without metabolic activation. Lemon oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations in the absence of metabolic activation and was therefore considered not clastogenic in this test.

Based on the data for Lemon and Lime oil no cytogenicity is expected for Bergamot oil.

 

In vitro mammalian cell gene mutation test

The key study was an in vitro mammalian cell gene mutation test performed according to OECD guideline 476 for the test substances Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae), in short Lemon oil. The result of this test is read across to Bergamot oil, see the read across justification document.

In the in vitro mammalian cell gene mutation test mouse L5178Y lymphoma cells were exposed to various concentrations of Lemon Oil Cold Pressed 1-Fold-Lemon, ext. Two experiments were performed: 1) experiment testing Lemon oil at concentrations of 70 and 450 µg/ml with and without 8% S9 mix during incubation for 3 hours; and 2) experiment testing Lemon oil at concentrations of 55 and 500 µg/ml with and without 12% S9 mix during incubation for 3 hours (with S9) and 24 hours (without S9).

In the absence and in the presence of S9-mix the test substance did not induce a significant increase in the mutation frequency in both experiments. It is concluded that Lemon Oil does not induce gene mutations under the experimental conditions described in the report.

Based on these data for Lemon oil no mutagenicity is expected for Bergamot oil.


Justification for selection of genetic toxicity endpoint
No selection is made as a Weight of Evidence approach was followed which is described below.

Short description of key information:
- Gene mutation in bacteria: (Bacterial Reverse Mutation Assay/Ames) (according to OECD 471): not mutagenic.
- In vitro Mammalian chromosome aberration test (equivalent or similar to OECD 473): not clastogenic without metabolic activation.
- In vitro Mammalian cell gene mutation test (according to OECD 476): negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the Ames test, Bergamot oil was considered to be non-mutagenic under the conditions of the test. Data on in vitro cytogenicity and mutagenicity in mammalian cells were obtained for Lemon oil and Lime oil and indicate no cytogenicity. These results are read across to Bergamot oil.

Considering the outcome of the available genotoxicity studies, it can be concluded that Bergamot oil is not genotoxic and does not need to be classified for genotoxicity in accordance with the criteria as outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).