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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): Positive in particularly TA98 without metabolic activation in more than one assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Comet assay (OECD TG 489): Negative

Micronucleus test (OECD TG 474): Negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Two new Ames tests were performed with Allyl cinnamate, because a former Wild et al. (1983) study on Allyl cinnamate was considered insufficient for fulfilling the endpoint because not all strains were tested according to OECD TG 471 guideline. (Wild, D., King, M.T., Gocke, E., Eckhardt, K., 1983, Study of artificial flavouring substances for mutagenicity in the Salmonella/microsome, Basc and micronucleus tests., Food Chem Toxicol., 21, 707-19). The summaries of the two newly performed Ames tests are presented below. In view of the positive results in these Ames tests, further in vitro testing is required as is presented in ECHA guidance (2017). The in vitro gene mutation test in mammalian cells can be waived because the Ames test is positive. The in vitro cytogenicity test is waived because a relevant in vivo test has been performed within the flavour framework. EFSA requested a Comet assay (OECD TG 489), which includes an in vivo micronucleus test (OECD TG 474). These results are presented below.

First Ames test

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments in the absence and presence of S9 -mix. Adequate negative and positive controls were included. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 500 μg/plate in both the absence and presence S9-mix. Consequently, the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item was employed as the maximum dose in the second mutation test, depending on bacterial strain type and presence or absence of S9 -mix. In the first mutation test, the test item induced statistically significant increases in the revertant colony frequency of tester strains TA100, WP2uvrA, TA98 and TA1537 at sub-toxic dose levels in the absence of S9-mix only. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains dosed in the presence of metabolic activation (S9-mix) or for TA1535 dosed in the absence of S9-mix. In the second experiment, the test item induced statistically significant increases in the revertant colony frequency of tester strains TA100, TA98 and TA1537 at sub-toxic dose levels in the absence of S9 -mix only. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains dosed in the presence of metabolic activation (S9-mix) or for TA1535 (and WP2uvrA in this experiment) dosed in the absence of S9-mix. The test item was considered, therefore, to have induced substantial, dose-related and reproducible increases in the frequency of revertant colonies for Salmonella strains TA100, TA1537 and TA98 dosed in the absence of S9 -mix at sub-toxic dose levels. The individual revertant counts at the non-toxic, statistically significant dose levels for TA98 and TA1537 in particular, exceeded the in-house untreated/vehicle control counts for the strains. Based on the results, it is concluded that the substance is mutagenic (in the absence of S9-mix only) in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

Second Ames test

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments in the absence and presence of S9-mix. Adequate negative and positive controls were included.In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at the dose levels of 1600 and 5000 μg/plate in the presence of S9-mix in the tester strains TA1537 and TA98 and at the dose level of 5000 μg/plate in the absence and presence of S9-mix in the tester strain WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix except in tester strain WP2uvrA in the presence of S9-mix, where no toxicity was observed at any of the dose levels tested. In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in tester strain TA98. The increases observed in the tester strain were above the laboratory historical control data range and were up to 6.2- fold the concurrent control. In the presence of S9-mix, no increases in the number of revertants were observed at any of the five tester strains. In the second mutation assay, the test item was tested up to concentrations of 1600 μg/plate in the Salmonella typhimurium strains and up to 5000 μg/plate in tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at the dose level of 1600 μg/plate in the presence of S9-mix in the Salmonella typhimurium strains. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all Salmonella typhimurium tester strains in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed in the absence and presence of S9-mix. In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA1537 and TA98. The increases observed in the tester strains were above the laboratory historical control data range and were up to 8.3- fold the concurrent controls in both tester strains. In the presence of S9-mix, no increases in the number of revertants were observed at any of the five tester strains. Overall, based on the results of this study it is concluded that the substance is mutagenic (in the absence of S9-mix only) in the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the Escherichia coli reverse mutation assay.

In vivo assays

The in vivo Comet assay and the in vivo Micronucleus assay have been combined in one in vivo assay. In view of both tests having different endpoints these tests are discussed separately.

In vivo Comet assay

Method: The test article was evaluated for its genotoxic potential in liver and duodenum according to OECD TG 489 and in compliance with GLP. This test was combined with a Micronucleus assay performed according to OECD 474. Sprague-Dawley rats were exposed to the test substance by oral gavage on three consecutive days. Three to four hours after the last treatment, the animals were euthanized. Olive oil was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg bw/dose. In the dose range finding assay, the dose levels tested were 125, 250, 500 and 1000 mg/kg bw/day in 3 animals/sex. However, animals in the two high dose groups of 500 and 1000 mg/kg bw/day were not dosed on day 3 due to mortality in these two dose groups. Based upon the results, the high dose for the definitive assay was 250 mg/kg bw/day, which was estimated to be the maximum tolerated dose, because this dose piloerection, lethargy, hunched posture, crusty nose and irregular breathing was seen. At this dose there was also decrease in body weight (7.7%). As no differences in clinical observations were seen between the sexes, only males were used in the definitive assay. The definitive assay dose levels tested were 0 (olive oil), 62.5, 125 and 250 mg/kg bw/day in six male rats per dose. Ethyl methanesulfonate (EMS) was used as positive control. Two tests were performed because in the first test after the in vivo part, in the preparation of the slides, the voltage was possibly applied during the DNA denaturion instead of after denaturation. This may have caused the vehicle control the % tail DNA being outside the historical control for both liver and duodenum cells. And also the positive control did not result in positive result in the duodenum. Therefore the test was repeated, the results of this second valid test is presented in the result section.

Results in the liver: A statistically significant increase in % Tail DNA was observed at 250 mg/kg bw/day in liver cells compared to the vehicle control. However, the value was within the Testing Facility’s historical range, and thus, this increase was not considered biologically relevant. In order to confirm the absence of increase in % Tail DNA, the high dose and vehicle control slides were rescored with an additional 150 cells/animal. This additional scoring showed that there was no statistically significant or dose-dependent increase in % Tail DNA at 250 mg/kg bw/day in liver cells.

Results in duodenum: No statistically significant or dose-dependent increases in % Tail DNA were observed at any dose level in duodenum cells compared to the vehicle control.

Results controls: The positive controls in both liver and duodenum cells had a statistically significant increase in % Tail DNA compared to the vehicle control. The vehicle control % Tail DNA in both liver and duodenum cells was within the Testing Facility’s historical range. In order to confirm the increase in % Tail DNA observed in liver cells, the high dose and vehicle control slides were rescored with an additional 150 cells/animal. No statistically significant or dose-dependent increase in % Tail DNA was observed at 250 mg/kg bw/day in liver cells compared to the vehicle control. The vehicle control % Tail DNA in liver cells was within the Testing Facility’s historical range.

Conclusion: Under the conditions of this study, the administration of the test substance, at doses up to and including a dose of 250 mg/kg bw/day, did not induce a significant increase in DNA damage in liver or duodenum cells. Therefore, the test substance was concluded to be negative in the in vivo Comet assay.

In vivo Micronucleus Assay

Method: The test article was evaluated for its genotoxic potential in bone marrow according to OECD TG 474 and in compliance with GLP. Sprague-Dawley rats were exposed to the test substance by oral gavage on three consecutive days. Three to four hours after the last treatment, the animals were euthanized. Olive oil was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg bw/dose. In the dose range finding assay, the dose levels tested were 125, 250, 500 and 1000 mg/kg bw/day in 3 animals/sex. However, animals in the two high dose groups of 500 and 1000 mg/kg bw/day were not dosed on day 3 due to 100% mortality in these two dose groups. Based upon the results, the high dose for the definitive assay was 250 mg/kg bw/day, which was estimated to be the maximum tolerated dose because at this dose piloerection, lethargy, hunched posture, crusty nose and irregular breathing was observed. As no differences in clinical observations were seen between the sexes, only males were used in the definitive assay. The definitive assay dose levels tested were 0 (olive oil), 62.5, 125 and 250 mg/kg bw/day in six male rats per dose. The scoring positive control slides (fixed and unstained), generated from another study by the test lab, were included to verify scoring in the micronucleus assay. These slides were generated from male rats treated once with cyclophosphamide monohydrate (CP) at 40 mg/kg, and the bone marrow harvested 24 hours after treatment.

Results: Slight but significant decrease (-5%) in PCEs/EC ration was seen at the high dose of 250 mg/kg bw indicating that the substance has reached the bone marrow. No statistically significant increases in the incidence of MnPCEs were observed in the test article treated groups relative to the vehicle control group. Thus, the test article was negative (non-clastogenic). The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control group did not exceed the historical control range.

Conclusion: Under the conditions of this study, the administration of the test substance, at doses up to and including a dose of 250 mg/kg bw/day, did not induce a significant increase in the incidence of MnPCEs relative to the concurrent vehicle control. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.

Justification for classification or non-classification

The Ames test was positive and further in vivo testing was performed. Based on the negative effects in the Comet assay, which included a micronucleus test, the substance does not have to be classified for genotoxicity according to EU CLP (EC 1272/2008 and its amendments).