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Scientific Opinion on Flavouring Group Evaluation 204 (FGE.204): Consideration of genotoxicity data on representatives for 18 mono-unsaturated, aliphatic, α,β-unsaturated ketones and precursors from chemical subgroup 1.2.1 of FGE.19 by EFSA
The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to evaluate the genotoxic potential of 18 flavouring substances from subgroup 1.2.1 of FGE.19 in the Flavouring Group Evaluation 204. The Flavour Industry provided additional genotoxicity studies for two representative substances, 4-methylpent-3-en-2-one [FL-no: 07.101] and 7-methyl-3-octenone-2 [FL-no: 07.177], which were evaluated in this FGE.204. Based on these new data, the Panel concluded that the flavouring substance [FL-no: 07.101] does not present a safety concern with respect to genotoxicity and accordingly it can be evaluated using the Procedure. On the contrary, the Panel could not conclude on the in vivo genotoxicity of [FL-no: 07.177] and more appropriate in vivo genotoxicity tests, considering also first site of contact, should be performed. In addition, the substance 4-methyl-3-hepten-5-one [FL-no: 07.261] was now identified as a substance for which no representative substances could be identified in the present FGE, resulting in a need for additional data on the genotoxic potential of this flavouring substance. However, the Panel noted that the 2-methyl substituted α,β-unsaturated aldehydes in FGE.201Rev1 can be considered as structurally related to [FL-no: 07.261]. Thus, the final conclusion on [FL-no: 07.261] will be drawn based on the outcome of the evaluation of FGE.201Rev1.
© European Food Safety Authority, 2012
The European Food Safety Authority (EFSA) asked the Scientific Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (the Panel) to provide scientific advice to the Commission on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the Member States. In particular, the Panel was requested to consider the Joint FAO/WHO Expert Committee on Food Additives (the JECFA) evaluations of flavouring substances assessed since 2000, and to decide whether no further evaluation is necessary, as laid down in Commission Regulation (EC) No 1565/2000. These flavouring substances are listed in the Register, which was adopted by Commission Decision 1999/217/EC and its consecutive amendments.
The present Flavouring Group Evaluation 204 (FGE.204), corresponding to subgroup 1.2.1 of FGE.19, concerns 16 mono-unsaturated, aliphatic, α,β-unsaturated ketones and two precursors for such ketones, oct-3-en-2-ol [FL-no: 02.102], oct-2-en-4-ol [FL-no: 02.193], pent-3-en-2-one [FL-no: 07.044], 4-hexen-3-one [FL-no: 07.048], oct-2-en-4-one [FL-no: 07.082], 4-methylpent-3-en-2-one [FL-no: 07.101], hept-2-en-4-one [FL-no: 07.104], hept-3-en-2-one [FL-no: 07.105], 5-methylhex-3-en-2-one [FL-no: 07.106], oct-3-en-2-one [FL-no: 07.107], dec-3-en-2-one [FL-no: 07.121], 5-methylhept-2-en-4-one [FL-no: 07.139], 7-methyl-3-octenone-2 [FL-no: 07.177], non-2-en-4-one [FL-no: 07.187], non-3-en-2-one [FL-no: 07.188], trans-6-methyl-3-hepten-2-one [FL-no: 07.244], 6-methyl-3-hepten-2-one [FL-no: 07.258] and 4-methyl-3-hepten-5-one [FL-no: 07.261].
The α,β-unsaturated aldehyde and ketone structures are structural alerts for genotoxicity and the data on genotoxicity previously available did not rule out the concern for genotoxicity for these 18 flavouring substances.
The Panel identified two substances in subgroup 1.2.1 which will represent the other substances in this subgroup (4-methylpent-3-en-2-one [FL-no: 07.101] and 7-methyl-3-octenone-2 [FL-no: 07.177]). For these two substances the Panel requested genotoxicity data according to the test strategy worked out by the Panel.
According to the above requirements the Industry has submitted additional genotoxicity studies for 4-methylpent-3-en-2-one and 7-methyl-3-octenone-2.
Based on these new data, the Panel concluded that 4-methylpent-3-en-2-one and 7-methyl-3-octenone-2 do not induce mutations in Salmonella typhimurium when tested up to toxic concentrations in the absence and in the presence of metabolic activation.
4-Methylpent-3-en-2-one did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations in both the absence and presence of S9-mix metabolism. Contrary, 7-methyl-3-octenone-2 induced micronuclei in human peripheral blood lymphocytes in the absence of S9-mix metabolism following 24-hour treatment.
To clarify whether 7-methyl-3-octenone-2 was acting as a clastogen or an aneugen a fluorescence in situ hybridization (FISH) analysis was performed for 24 hours without S9-mix. Results obtained indicated that 7-methyloct-3-en-2-one is acting as a clastogenic compound.
In order to determine whether positive results obtained in vitro for 7-methyl-3-octenone-2 could be confirmed in vivo, a bone marrow micronucleus test in rats was performed. 7-Methyl-3-octenone-2 did not induce micronuclei in bone marrow polychromatic erythrocytes (PCE). However, this outcome was accompanied by complete absence of clinical signs of toxicity in all animals, in all treatment groups. The observed very weak dose-related decrease in mean % PCE was considered by the Panel not indicative of bone marrow exposure. Therefore, the absence of induction of micronuclei in bone marrow erythrocytes does not necessarily reflect absence of clastogenicity in vivo of the test compound.
On the basis of the available data, the Panel noted that no conclusions can be drawn about in vivo genotoxicity of 7-methyl-3-octenone-2 [FL-no: 07.177] and more appropriate in vivo genotoxicity tests, considering also first site of contact, should be performed. Alternatively, chemical analysis of the already available blood sample demonstrating target tissue exposure would also be suitable to make the results of the available micronucleus assay acceptable for the assessment of the hazard on genotoxic potential.
For 4-methylpent-3-en-2-one [FL-no: 07.101], the data available showed that it did not induce mutations in bacteria or micronuclei in human peripheral blood lymphocytes, neither in the presence nor in the absence of rat liver S9-mix metabolic activation. Based on these findings, the Panel concluded that 4-methylpent-3-en-2-one does not present a safety concern with respect to genotoxicity and accordingly the flavouring substance can be evaluated using the Procedure. Since this substance is considered to be representative for itself only (“stand-alone”), as it is the only substance with a methyl-substituent on the β-carbon atom of the double bond, this conclusion does not apply to any of the other candidate substances in this FGE.
Subsequent to the publication of the list of representatives (EFSA, 2008bc), the Panel noted that 4-methyl-3-hepten-5-one [FL-no: 07.261] differs structurally from all other substances in this subgroup owing to the presence of a methyl group in α-position of the double bond. Therefore, this substance is considered as a “stand alone” substance. The Panel noted that the 2-methyl substituted α,β-unsaturated aldehydes in FGE.201Rev1 (EFSA, 2012h) can be considered as structurally related to [FL-no: 07.261]. For the substances in FGE.201Rev1, 2-methylpent-2-enal [FL-no 05.090] has been selected as representative and further genotoxicity data were required. Thus, the final conclusion on [FL-no: 07.261] will be drawn based on the outcome of the evaluation of FGE.201Rev1.
FGE.19; subgroup 1.2.1, aliphatic, mono-unsaturated, α,β-unsaturated ketones.