Scientific Opinion on Flavouring Group Evaluation 68 (FGE.68):

Transcription

1 SCIENTIFIC PININ Scientific pinion on Flavouring Group Evaluation 68 (FGE.68): Consideration of cinnamyl alcohol and related flavouring agents evaluated by JECFA (55 th meeting) structurally related to aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives evaluated by EFSA in FGE.15Rev1 (2008) 1 EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) 2, 3 European Food Safety Authority (EFSA), Parma, Italy SUMMARY The Scientific Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (the Panel) was asked 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 FA/WH 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 JECFA has evaluated 55 substances in the group of cinnamyl alcohol and related substances at their 55 th meeting. Twenty-six of these substances are alpha,beta-unsaturated aldehydes or precursors for such, which the Panel considers to be a structural alert for genotoxicity. The following 25 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , and ] have initially been considered in FGE.214 with respect to genotoxicity. The Panel concluded that for these 25 substances the genotoxicity data available do not preclude their 1 n request from the Commission, Question No EFSA-Q T, adopted on 25 March Panel members: Arturo Anadon, David Bell, Mona-Lise Binderup, Wilfried Bursch, Laurence Castle, Riccardo Crebelli, Karl-Heinz Engel, Roland Franz, Nathalie Gontard, Thomas Haertle, Trine Husøy, Klaus-Dieter Jany, Catherine Leclercq, Jean Claude Lhuguenot, Wim Mennes, Maria Rosaria Milana, Karla Pfaff, Kettil Svensson, Fidel Toldra, Rosemary Waring, Detlef Wölfle. 3 Acknowledgement: The Panel wishes to thank the members of the Working Groups on Flavourings for the preparation of this pinion: Ulla Beckman Sundh, Vibe Beltoft, Wilfried Bursch, Angelo Carere, Riccardo Crebelli, Karl-Heinz Engel, Henrik Frandsen, Jørn Gry, Rainer Gürtler, Frances Hill, Trine Husøy, John Christian Larsen, Catherine Leclercq, Pia Lund, Wim Mennes, Gerard Mulder, Karin Nørby, Gerard Pascal, Iona Pratt, Gerrit Speijers, Harriet Wallin. Suggested citation: EFSA ; Scientific pinion on Flavouring Group Evaluation 68 (FGE.68): Consideration of cinnamyl alcohol and related flavouring agents evaluated by JECFA (55th meeting) structurally related to aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives evaluated by EFSA in FGE.15Rev1 (2008). EFSA Journal 2009; 7(11):2009. [51 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 evaluation through the Procedure. For the remaining substance (of the 26 alpha,beta-unsaturated aldehydes or precursors for such), allyl cinnamate [FL-no: ], which may be metabolised to allyl alcohol and further to acrolein, considered with respect to genotoxicity in subgroup of FGE.19, a final conclusion as to its genotoxic properties could not be reached and additional data were requested. Accordingly, this substance will not be considered in the present FGE. This consideration therefore only deals with the 54 JECFA evaluated substances. The Panel concluded that the 54 substances in the JECFA flavouring group of cinnamyl alcohol and related flavouring substances are structurally related to the group of nine aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives evaluated by EFSA in the Flavouring Group Evaluation 15, Revision 1 (FGE.15Rev1). The Panel agrees with the way the application of the Procedure has been performed by the JECFA for the 54 substances considered in this FGE. However for six substances [FL-no: , , , , and ] the JECFA evaluation is only based on MSDI values derived from production figures from the USA. EU production figures are needed in order to finalise the evaluation of these substances. For all 54 substances use levels are needed to calculate the mtamdis in order to identify those flavouring substances that need more refined exposure assessment and to finalise the evaluation. In order to determine whether the conclusion for the 54 JECFA evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications. Adequate specifications including complete purity criteria and identity are available for 13 of the JECFA evaluated substances considered in this FGE. Information on stereoisomerism is lacking for 41 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ] and compositional information of mixture is lacking for four substances [FL-no , , and ]. Thus, in total, for 46 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ] the Panel has reservations (no European production volumes are available, preventing them to be evaluated using the Procedure, and/or missing data on stereoisomerism and/or compositional information of mixture). For the remaining eight substances in the group of the JECFA evaluated cinnamyl alcohol and related substances [FL-no: , , , , , , and ] the Panel agrees with JECFA conclusion No safety concern at estimated levels of intake as flavouring substances based KEY WRDS Cinnamyl alcohol, cinnamyl derivatives, JECFA 55 th meeting, FGE.15Rev1, aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives, food safety 2

3 TABLE F CNTENTS Summary... 1 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment... 5 History of the Evaluation of the Substances in the present FGE Presentation of the Substances in the JECFA Flavouring Group Description JECFA Status EFSA Considerations Isomers JECFA Status EFSA Considerations Specifications JECFA Status EFSA Considerations Intake Estimations JECFA Status EFSA Considerations Genotoxicity Data Genotoxicity Studies Text Taken from the JECFA (JECFA, 2001b) Genotoxicity Studies - Text taken from EFSA FGE.15Rev1 (EFSA, 2008q) Genotoxicity Studies and Conclusion on Genotoxicity and Carcinogenicity - Text taken from FGE.214 (EFSA, 2009y) EFSA Considerations Application of the Procedure Application of the Procedure to 54 Cinnamyl Alcohol and Related Substances by JECFA (JECFA, 2001b) Application of the Procedure to Nine Aryl-Substituted Saturated and Unsaturated Primary Alcohol/Aldehyde/Acid/Ester Derivatives by EFSA (EFSA, 2008q) EFSA Considerations Conclusion Table 1: Specification summary Table 2: Genotoxicity data Table 3: Summary of Safety Evaluations References Abbreviations

4 BACKGRUND Regulation (EC) No 2232/96 of the European Parliament and the Council (EC, 1996) lays down a Procedure for the establishment of a list of flavouring substances, the use of which will be authorised to the exclusion of all other substances in the EU. In application of that Regulation, a Register of flavouring substances used in or on foodstuffs in the Member States was adopted by Commission Decision 1999/217/EC (EC, 1999a), as last amended by Commission Decision 2009/163/EC (EC, 2009a). Each flavouring substance is attributed a FLAVIS-number (FL-number) and all substances are divided into 34 chemical groups. Substances within a group should have some metabolic and biological behaviour in common. Substances which are listed in the Register are to be evaluated according to the evaluation programme laid down in Commission Regulation (EC) No 1565/2000 (EC, 2000a), which is broadly based on the pinion of the Scientific Committee on Food (SCF, 1999). Commission Regulation (EC) No 1565/2000 lays down that substances that are contained in the Register and will be classified in the future by the Joint FA/WH Expert Committee on Food Additives (the JECFA) so as to present no safety concern at current levels of intake will be considered by the European Food Safety Authority (EFSA), who may then decide that no further evaluation is necessary. In the period , during its 55 th, 57 th, 59 th, 61 st, 63 rd, 65 th 68 th and 69 th meetings, the JECFA evaluated about 1000 substances, which are in the EU Register. TERMS F REFERENCE EFSA is requested to consider 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 (EC, 2000a). These flavouring substances are listed in the Register which was adopted by Commission Decision 1999/217 EC (EC, 1999a) and its consecutive amendments. 4

5 ASSESSMENT The approach used by EFSA for safety evaluation of flavouring substances is referred to in Commission Regulation (EC) No 1565/2000 (EC, 2000a), hereafter named the EFSA Procedure. This Procedure is based on the opinion of the Scientific Committee on Food (SCF, 1999), which has been derived from the evaluation procedure developed by the Joint FA/WH Expert Committee on Food Additives (JECFA, 1995; JECFA, 1996a; JECFA, 1997a; JECFA, 1999b), hereafter named the JECFA Procedure. The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (the Panel) compares the JECFA evaluation of structurally related substances with the result of a corresponding EFSA evaluation, focussing on specifications, intake estimations and toxicity data, especially genotoxicity data. The evaluations by EFSA will conclude whether the flavouring substances are of no safety concern at their estimated levels of intake, whether additional data are required or whether certain substances should not be put through the EFSA Procedure. The following issues are of special importance. Intake In its evaluation, the Panel as a default uses the Maximised Survey-derived Daily Intake (MSDI) approach to estimate the per capita intakes of the flavouring substances in Europe. In its evaluation, the JECFA includes intake estimates based on the MSDI approach derived from both European and USA production figures. The highest of the two MSDI figures is used in the evaluation by the JECFA. It is noted that in several cases, only the MSDI figures from the USA were available, meaning that certain flavouring substances have been evaluated by the JECFA only on the basis of these figures. For Register substances for which this is the case the Panel will need EU production figures in order to finalise the evaluation. When the Panel examined the information provided by the European Flavour Industry on the use levels in various foods, it appeared obvious that the MSDI approach in a number of cases would grossly underestimate the intake by regular consumers of products flavoured at the use level reported by the Industry, especially in those cases where the annual production values were reported to be small. In consequence, the Panel had reservations about the data on use and use levels provided and the intake estimates obtained by the MSDI approach. It is noted that the JECFA, at its 65 th meeting considered how to improve the identification and assessment of flavouring agents, for which the MSDI estimates may be substantially lower than the dietary exposures that would be estimated from the anticipated average use levels in foods (JECFA, 2006c). In the absence of more accurate information that would enable the Panel to make a more realistic estimate of the intakes of the flavouring substances, the Panel has decided also to perform an estimate of the daily intakes per person using a modified Theoretical Added Maximum Daily Intake (mtamdi) approach based on the normal use levels reported by Industry. As information on use levels for the flavouring substances has not been requested by the JECFA or has not otherwise been provided to the Panel, it is not possible to estimate the daily intakes using the mtamdi approach for the substances evaluated by the JECFA. The Panel will need information on use levels in order to finalise the evaluation. Threshold of 1.5 Microgram/Person/Day (Step B5) Used by the JECFA The JECFA uses the threshold of concern of 1.5 microgram/person/day as part of the evaluation procedure: The Committee noted that this value was based on a risk analysis of known carcinogens which involved several conservative assumptions. The use of this value was supported by additional 5

6 information on developmental toxicity, neurotoxicity and immunotoxicity. In the judgement of the Committee, flavouring substances for which insufficient data are available for them to be evaluated using earlier steps in the Procedure, but for which the intake would not exceed 1.5 microgram per person per day would not be expected to present a safety concern. The Committee recommended that the Procedure for the Safety Evaluation of Flavouring Agents used at the forty-sixth meeting be amended to include the last step on the right-hand side of the original procedure ( Do the condition of use result in an intake greater than 1.5 microgram per day? ) (JECFA, 1999b). In line with the pinion expressed by the Scientific Committee on Food (SCF, 1999), the Panel does not make use of this threshold of 1.5 microgram per person per day. Genotoxicity As reflected in the pinion of SCF (SCF, 1999), the Panel has in its evaluation focussed on a possible genotoxic potential of the flavouring substances or of structurally related substances. Generally, substances for which the Panel has concluded that there is an indication of genotoxic potential in vitro, will not be evaluated using the EFSA Procedure until further genotoxicity data are provided. Substances for which a genotoxic potential in vivo has been concluded, will not be evaluated through the Procedure. Specifications Regarding specifications, the evaluation by the Panel could lead to a different opinion than that of JECFA, since the Panel requests information on e.g. isomerism. Structural Relationship In the consideration of the JECFA evaluated substances, the Panel will examine the structural relationship and metabolism features of the substances within the flavouring group and compare this with the corresponding FGE. HISTRY F THE EVALUATIN F THE SUBSTANCES IN THE PRESENT FGE At its 55 th meeting the JECFA evaluated a group of 55 flavouring substances consisting of cinnamyl alcohol and related substances. Twenty-six of these substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , , and ] are alpha,beta-unsaturated aldehydes or precursors for such. As the alpha,beta-unsaturated aldehyde and ketone structures are considered by the Panel to be structural alerts for genotoxicity (EFSA, 2008b), they have been given special considerations in the Flavouring Group Evaluation 19 (FGE.19). FGE.19 contains 360 flavouring substances from the EU Register being alpha,beta-unsaturated aldehydes or ketones and precursors which could give rise to such carbonyl substances via hydrolysis and / or oxidation (EFSA, 2008b). The alpha,beta-unsaturated carbonyls were subdivided into 28 subgroups on the basis of structural similarity (EFSA, 2008b). In an attempt to decide which of the substances could go through the Procedure, a (quantitative) structure-activity relationship ((Q)SAR) prediction of the genotoxicity of these substances was undertaken. The Panel took note of the (Q)SAR predictions by using two ISS Local Models (Benigni & Netzeva, 2007a; Benigni & Netzeva, 2007b) and four DTU-NFI MultiCASE Models (Gry et al., 2007; Nikolov et al., 2007) and the fact that there are available data on genotoxicity, in vitro and in vivo, as well as data on carcinogenicity for several substances. The Panel decided that 11 subgroups (1.1.2, 1.1.3, 1.1.4, 2.4, 2.6, 2.7, 3.1, 3.3, 4.1, 4.2 and 4.4) (EFSA, 2008b) should be further examined to determine whether evaluation through the Procedure is feasible. Corresponding to these 11 subgroups, 11 Flavouring Group Evaluations (FGEs) were established (FGE.201, 202, 203, 210, 212, 213, 214, 216, 217, 218 and 220). 6

7 The present FGE.68 includes the consideration of 54 flavouring substances of which 25 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , and ] were allocated to FGE.19 subgroup 3.1 and evaluated in FGE.214 (EFSA, 2009y) with respect to genotoxicity. For the remaining substance, allyl cinnamate [FL-no: ] (precursors of an alpha,beta-unsaturated aldehyde), considered with respect to genotoxicity in subgroup of FGE.19 (EFSA, 2008b) as it may be metabolised to allyl alcohol and further to acrolein (prop-2-enal, [FL-no: ]), a final conclusion as to its genotoxic properties could not be reached and additional data were requested. 1. Presentation of the Substances in the JECFA Flavouring Group 1.1. Description JECFA Status The JECFA has evaluated a group of 55 flavouring substances consisting of cinnamyl alcohol and related substances EFSA Considerations Twenty-six of the 55 substances in the group of cinnamyl alcohol and related substances evaluated by the JECFA (JECFA, 2001b) are alpha,beta-unsaturated aldehydes or precursors for alpha,betaunsaturated aldehydes. As the alpha,beta-unsaturated aldehyde and ketone structures are considered by the Panel to be structural alerts for genotoxicity, these 26 substances have initially been considered with respect to genotoxicity (EFSA, 2008b). Twenty-five of the alpha,beta-unsaturated cinnamyl derivatives [FL-no: , , , , , , , , , , , , , , , , , , , , , , , and ] have been considered with respect to genotoxicity in FGE.214 (EFSA, 2009y), corresponding to subgroup 3.1 of FGE.19. For these substances the Panel concluded that although they have a structural alert for genotoxicity, the data available do not preclude to evaluate them through the Procedure. For the remaining substance, allyl cinnamate [FL-no: ] considered with respect to genotoxicity in subgroup of FGE.19, and which may be metabolised to allyl alcohol and further to acrolein a final conclusion as to its genotoxic properties could not be reached and additional data were requested. The present flavouring group consideration therefore only deal with 54 JECFA evaluated substances. The Panel concluded that these 54 substances in the JECFA flavouring group of cinnamyl alcohol and related substances are structurally related to the group of aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives evaluated by EFSA in the Flavouring Group Evaluation 15 Revision 1 (FGE.15Rev1), (EFSA, 2008q) Isomers JECFA Status Three of the substances in the group of the JECFA-evaluated cinnamyl alcohol and related substances have a chiral centre [FL-no: , and ] and 40 substances [FL-no: , , , , , , , , , , , , , , 7

8 09.018, , , , , , , , , , , , , , , , , , , , , , , , and ] can exist as geometrical isomers due to presence and position of a double-bond EFSA Considerations Information is lacking on the stereoisomerism for 41 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ] Specifications JECFA Status The JECFA specifications are available for all substances (JECFA, 2002d; JECFA, 2001c). See Table EFSA Considerations Specifications including complete purity criteria and identity are available for 13 substances. Information on the stereoisomerism is missing for 41 substances (see Section 1.2) and compositional information of mixture is lacking for four substances [FL-no , , and ]. 2. Intake Estimations JECFA Status For 48 of the 54 substances evaluated through the JECFA Procedure intake data are available for the EU, see Table 3.1. For the remaining six substances [FL-no: , , , , and ] production figures are only available for the USA EFSA Considerations As production figures are only available for the USA for the following six substances [FL-no: , , , , and ], MSDI values for the EU cannot be calculated for these. 3. Genotoxicity Data 3.1. Genotoxicity Studies Text Taken 4 from the JECFA (JECFA, 2001b) In vitro Cinnamaldehyde (trans- and unspecified stereochemistry), cinnamyl alcohol [FL-no: ] (transand unspecified stereochemistry), cinnamic acid [FL-no: ], alpha-methylcinnamaldehyde [FLno: ], cinnamyl acetate [FL-no: ], benzyl cinnamate [FL-no: ], cyclohexyl cinnamate [FL-no: ], alpha-amylcinnamaldehyde [FL-no: ], alpha-hexylcinnamaldehyde [FL- 4 The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE has been removed. 8

9 no: ], para-methoxy-alpha-methylcinnamaldehyde [FL-no: ], and 3-phenylpropionaldehyde [FL-no: ] generally did not cause reverse mutation in Salmonella typhimurium strains TA92, TA94, TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538, and TA2637. The assays were performed at concentrations up to the level of cytotoxicity, both in the absence and presence of metabolic activation obtained from the livers of Aroclor or methylcholanthreneinduced Sprague-Dawley rats or Syrian hamsters (Dunkel & Simmon, 1980; Eder et al., 1980; Florin et al., 1980; Lijinsky & Andrews, 1980; Lutz et al., 1980; Eder et al., 1982a; Eder et al., 1982b) and (Kasamaki et al., 1982; Lutz et al., 1982; Prival et al., 1982; Sekizawa & Shibamoto, 1982; Neudecker et al., 1983; Wild et al., 1983; Ishidate et al., 1984) and (Huang et al., 1985; Marnett et al., 1985a; Mortelmans et al., 1986; Fujita & Sasaki, 1987; Tennant et al., 1987) and (Kato et al., 1989; Eder et al., 1991b; Dillon et al., 1992; Azizan & Blevins, 1995). Weakly positive or positive results were reported for cinnamaldehyde [FL-no:05.014] in S. typhimurium strain TA100 with the pre-incubation method (Dillon et al., 1992; Ishidate et al., 1984), but most other studies in this strain, including a recent study with a prolonged pre-incubation time (120 min.) and others in which the standard plate incorporation method was used gave no evidence of mutagenicity (Sasaki & Endo, 1978; Lijinsky & Andrews, 1980; Eder et al., 1982a; Eder et al., 1982b; Kasamaki et al., 1982) and. Negative or weakly positive results were obtained in S. typhimurium with pre-incubation with orthomethoxycinnamaldehyde [FL-no: ] (Eder et al., 1991b; Mortelmans et al., 1986). The weakly positive results in strain TA100 with metabolic activation were obtained with two different activation systems (Mortelmans et al., 1986). Negative results were obtained in strains TA1535, TA1537, and TA98 both with and without metabolic activation. In the study with strain TA100, negative results were reported in the absence of metabolic activation (Eder et al., 1991b). No standard plate incorporation assay was available for ortho-methoxycinnamaldehyde, which might be expected to behave similarly to the other cinnamyl compounds on the basis of structural and metabolic similarities. The results of assays for mutation in Escherichia coli strains WP2uvrA, PQ37, and Sd-4-73, including several in which the pre-incubation method was used, were negative with cinnamaldehyde [FL-no: ], cinnamyl alcohol [FL-no: ], cinnamic acid [FL-no: ], alphamethylcinnamaldehyde [FL-no: ], and alpha-amylcinnamaldehyde [FL-no: ] (Szybalski, 1958; Sekizawa & Shibamoto, 1982; hta et al., 1986b; Yoo, 1986; Kato et al., 1989; Eder et al., 1991b; Eder et al., 1993). In the rec assay in Bacillus subtilis, positive results were reported with cinnamaldehyde [FL-no: ] and cinnamyl alcohol [FL-no: ], whereas cinnamic acid [FLno: ], ethyl cinnamate [FL-no: ], methyl cinnamate [FL-no: ], and benzyl cinnamate [FL-no: ] gave negative results in all such tests (da et al., 1979; Sekizawa & Shibamoto, 1982; Kuroda et al., 1984a; Yoo, 1986). Assays with isolated mammalian cells gave mixed but generally positive results for cinnamyl esters overall. Equivocal to positive results were obtained for cinnamaldehyde [FL-no: ] in the assay for forward mutation in L5178Y mouse lymphoma cells with and without metabolic activation, but the reports of these tests did not provide sufficient detail of the method, concentrations tested, or cytotoxic effects to allow adequate evaluation of the results (Rudd et al., 1983; Palmer, 1984). In L1210 mouse lymphoma cells, DNA strand breaks were observed only at cytotoxic concentrations of cinnamaldehyde (Eder et al., 1993). The results of tests for the induction of sister chromatid exchange in Chinese hamster ovary cells exposed to cinnamaldehyde [FL-no: ] were negative at low concentrations and weakly positive at concentrations approaching cytotoxic levels, suggesting only weak activity (Galloway et al., 1987a; Sasaki et al., 1989). A dose-dependent increase in the frequency of sister chromatid exchange was reported only when cultures were pre-treated with mitomycin C (Sasaki et al., 1989); however, the activity in conjunction with mitomycin contributes little to an evaluation of potential sister chromatid exchange activity. Cinnamaldehyde at concentrations < 15 µg/ml was reported to induce chromosomal aberrations in Chinese hamster fibroblasts and B241 cells tested with and without metabolic activation 9

10 (Kasamaki et al., 1982; Ishidate et al., 1984; Kasamaki & Urasawa, 1985). However, higher concentrations did not induce chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of metabolic activation in a well-conducted, repeated assay (Galloway et al., 1987a). The results of assays for cell transformation with cinnamaldehyde [FL-no: ] were positive at near-cytotoxic concentrations or after multiple generations of growth and negative in human HAIN-55 cells (Kasamaki et al., 1987; Matthews et al., 1993). Subcutaneous injection of the transformed cells into nude mice led to the formation of nodules at the site of injection and neoplastic growth in the spleen (Kasamaki et al., 1987). Negative results were obtained with cinnamaldehyde [FL-no: ] (No. 656) in Chinese hamster V79 cells (Fiorio & Bronzetti, 1994), while a weak increase in the incidence of micronucleated Hep-G2 cells was reported by (Sanyal et al., 1997). The results obtained with the other cinnamyl compounds in isolated mammalian cells were, in general, comparable to those obtained with cinnamaldehyde [FL-no: ]. Sister chromatid exchange was not observed in Chinese hamster ovary cells exposed to cinnamyl alcohol [FL-no: ], cinnamic acid [FL-no: ], ethyl cinnamate [FL-no: ], methyl cinnamate [FL-no: ], cinnamyl acetate [FL-no: ], or 3-phenylpropionaldehyde [FL-no: ]. Pretreatment with mitomycin C increased the incidence of sister chromatid exchange in assays with cinnamic acid [FL-no: ], methyl cinnamate [FL-no: ], and ethyl cinnamate [FL-no: ] but not cinnamyl alcohol [FL-no: ], cinnamyl acetate [FL-no: ], or 3-phenylpropionaldehyde [FL-no: ] (Sasaki et al., 1989; Palmer, 1984) reported reproducible, dose-related increases in the incidence of reversions in L5178Y mouse lymphoma cells, with and without metabolic activation, after treatment with cinnamyl alcohol [FL-no: ], cinnamic acid [FL-no: ], cinnamyl cinnamate [FL-no: ], and ortho-methoxycinnamaldehyde [FL-no: ]. The results of assays in L5178Y mouse lymphoma cells at the Tk +/ locus have yielded equivocal results. The positive results were seen at near-lethal concentrations in studies in which this was reported. The results of assays with simple aliphatic and aromatic substances were not consistent with the results of other, standard assays for genotoxicity (Tennant et al., 1987; Heck et al., 1989). Culture conditions of low ph and high osmolality, which may pertain with substances that have a potentially acidifying effect on the culture medium (aldehydes, carboxylic acids, lactones, and hydrolysed esters), have been shown to produce false-positive results in this and other assays (Heck et al., 1989). ther reports of positive responses in the mouse lymphoma cell assay lacked information on the concentration tested and on cytolethality (Rudd et al., 1983; Palmer, 1984). In vivo Most of the results of tests of the administration of cinnamyl compounds in vivo pertains to cinnamaldehyde [FL-no: ]. An increase in the frequency of sex-linked recessive lethal mutations was reported when Drosophila melanogaster were injected with cinnamaldehyde at mg/kg of diet, but no increase in the frequency of mutations was seen when D. melanogaster were fed 800 mg/kg of diet for 3 days. Reciprocal translocations were not observed in either assay (Woodruff et al., 1985). No increase in the frequency of unscheduled DNA synthesis was found in the hepatocytes of rats or mice given cinnamaldehyde at 1000 mg/kg bw by oral gavage (Mirsalis et al., 1989). The frequency of micronuclei was not increased when rats or mice were given 1700 mg/kg bw or 1100 mg/kg bw, respectively, of cinnamaldehyde by oral gavage (Mereto et al., 1994) or when mice were given 500 mg/kg bw by intraperitoneal injection (Hayashi et al., 1984; Hayashi et al., 1988). The frequency of micronucleated bone-marrow cells in mice that had been exposed to X-rays decreased after injection of 500 mg of cinnamaldehyde (Sasaki et al., 1990b). An increase in the frequency of micronucleated cells was reported in rat and mouse hepatocytes and in rat (but not mouse) forestomach cells after the animals had received up to 1100 (rats) or 1700 (mice) mg/kg bw of cinnamaldehyde by oral gavage. No increase in the frequency of micronuclei in liver or forestomach was observed at doses 850 mg/kg bw, and no DNA fragmentation was observed in rat hepatocytes or gastric mucosal cells. The incidence and size of gamma-glutamyl transferase-positive 10

11 foci were increased in hepatocytes of rats pretreated with N-nitrosodiethylamine and then given cinnamaldehyde at 500 mg/kg bw per day by oral gavage for 14 days (Mereto et al., 1994). The positive findings with cinnamaldehyde [FL-no: ] in rat forestomach and in the livers of both rats and mice treated in vivo are not consistent with the results of the standard assays in bone marrow and were observed at doses that far exceeded those resulting from intake of cinnamaldehyde in foods. Cinnamaldehyde given at oral doses 500 mg/kg bw depleted hepatocellular glutathione concentrations (Swales & Caldwell, 1991; Swales & Caldwell, 1992; Swales & Caldwell, 1993), and the increases in micronucleus frequency were found at doses that appeared to affect cellular defence mechanisms, such as glutathione depletion. As the micronucleus formation was dose-dependent, induction of micronuclei may be a threshold phenomenon which occurs at intakes orders of magnitude greater than that of cinnamaldehyde as a flavouring agent. Furthermore, the bolus doses resulting from gavage probably resulted in much greater exposure of both the forestomach and the liver than administration in a dietary admixture. The author of the study in which these results were obtained (Mereto et al., 1994) acknowledged these facts and concluded that their data did not justify the conclusion that cinnamaldehyde is clastogenic. In view of the apparent threshold for micronucleus induction and the lack of activity in other studies in vivo, the effects induced by the bolus dose in the liver and forestomach are considered irrelevant to the evaluation of the safety of cinnamaldehyde when used as a flavouring agent. Wild et al. (1983) reported negative results in tests for sex-linked recessive lethal mutation in D. melanogaster and in an assay for micronucleus formation in mouse bone-marrow cells after administration of alpha-methylcinnamaldehyde [FL-no: ], alpha-amylcinnamyl alcohol [FL-no: ], alpha-amylcinnamaldehyde [FL-no: ], or alpha-hexylcinnamaldehyde [FL-no: ] (Wild et al., 1983). Conclusion Cinnamyl alcohol [FL-no: ] and related compounds lack direct mutagenic or genotoxic activity, as indicated by the negative results obtained in bacterial test systems. The mixed results in the assay for DNA repair and in various studies of antimutagenicity were associated with cytotoxicity, as noted by (Sekizawa & Shibamoto, 1982). Evidence of genotoxic activity was found in isolated mammalian cells, the cinnamyl compounds inducing chromosomal aberrations and/or mutations in the presence or absence of metabolic activation; however, the reported activity in vitro was not seen as mutagenic, clastogenic, or genotoxic activity in vivo. For a summary of in vitro / in vivo genotoxicity data considered by JECFA see Table Genotoxicity Studies - Text taken 5 from EFSA FGE.15Rev1 (EFSA, 2008q) In vitro / in vivo Limited in vitro genotoxicity data are available for only two candidate [FL-no: and ] and for six supporting substances [FL-no: , , , , and ]. The mutagenicity studies available on the candidate substances 4-hydroxy-3,5-dimethoxycinnamic acid [FL-no: ] and 4-hydroxy-3-methoxycinnamic acid [FL-no: ] are considered to provide little useful information regarding the genotoxicity of the candidate substances. 4-Hydroxy-3-methoxycinnamic acid [FL-no: ] was tested for its influence on spontaneous as well as induced sister chromatid exchange (SCE) in cultured Chinese hamster ovary (CH) cells only in the absence of metabolic activation. The result was negative. 5 The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE has been removed. 11

12 The five supporting substances [FL-no: , , , and ] have been tested for their ability to induce mutations in various strains of Salmonella typhimurium (e.g. TA92, TA94, TA98, TA100, TA1535, TA1537 and TA1538), in the presence or absence of an exogenous metabolic activation system. None of the compounds was mutagenic when tested at concentrations up to 5000 microgram/plate. Four of the substances, cinnamic acid [FL-no: ], methyl cinnamate [FL-no: ], ethyl cinnamate [FL-no: ] and 3-phenylpropionaldehyde [FL-no: ] were tested for induction of spontaneous SCEs in cultured CH cells only in the absence of metabolic activation. For all the four substances no influence on cell cycle and SCE was observed. Ethyl cinnamate [FL-no: ] in a study carried out in the absence of S9 activation did not induce chromosomal aberrations in Chinese hamster fibroblasts. There are no in vivo genotoxicity data available for the candidate and supporting substances in the present flavouring group evaluation. Conclusion on genotoxicity verall, the data available are not sufficient to evaluate the genotoxicity adequately and no in vivo genotoxicity data are available for the candidate or for the supporting substances, but the various studies carried out with supporting substances give no indication of a mutagenic activity in bacterial cells or of a direct clastogenic effect on mammalian cells. The limited genotoxicity data available do not preclude evaluating the nine candidate substances, using the Procedure. For a summary of in vitro / in vivo genotoxicity data considered by EFSA see Table 2.2 and Genotoxicity Studies and Conclusion on Genotoxicity and Carcinogenicity - Text taken 6 from FGE.214 (EFSA, 2009y) In subgroup 3.1 there are studies available for six of the substances. For cinnamaldehyde [FL-no: ] 19 in vitro studies (in total 27 tests) and four in vivo studies (5 different end points) have been evaluated. nly in one of the valid studies for reverse mutations in bacterial cells a positive result was obtained. However, the same test in the same strain provided negative results in other valid studies. Some positive results were obtained in bacterial tests for DNA repair (Rec and SS-chromo assays), but these tests are not considered relevant for the evaluation of genotoxicity. A gene mutation study in mammalian cells provided also a negative result, but was considered too limited to be considered valid. In contrast, two studies which were considered as valid provided indications that cinnamaldehyde may induce chromosomal aberrations in vitro in Chinese hamster fibroblast or B241 cells. For the same endpoint also a valid negative study has been reported but a study in Hep-G2 cells provided a limited indication that cinnamaldehyde might induce micronuclei. Several studies reported cinnamaldehyde-induced Sister Chromatic Exchanges (SCEs), but this endpoint is considered of very limited relevance. A study with limited validity indicated induction of DNA strand breaks in mouse lymphoma cells at very high concentrations, which were clearly cytotoxic. With several other candidate substances [FL-no: , , , and ] data from mutation tests with S. typhimurium have been reported. These studies did not indicate a mutagenic potential for these substances. However, for one substance a positive result has been reported [FL-no: ]. In two of the in vivo studies with cinnamaldehyde an increase in hepatocellular micronuclei has been observed in rats and mice after gavage dosing. Although the tests were appropriately performed, the relevance of this effect is not clear as it was obtained in animals that had undergone 2/3 hepatectomy 6 The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE has been removed. 12

13 and received the substance at 50% of the LD 50. In these animals, no DNA fragmentation was observed in hepatocytes and in stomach mucosal cells. Similarly, no increase in micronuclei frequency was observed in bone marrow cells from these animals. In another valid in vivo bone marrow assay with i.p. injection no increase in bone marow micronuclei formation was observed, either. From the few studies available with other substances [FL-no: , and ] also no indication of genotoxicity in vivo was obtained. For validation and study results see Table 2.4 and 2.5. Conclusion on Genotoxicity and Carcinogenicity Some concern could be raised by studies carried out with cinnamaldehyde [FL-no: ], showing an ability to induce chromosomal damage in vitro, and by the positive result obtained for 2- methoxycinnamaldehyde [FL-no: ] in an Ames test. For cinnamaldehyde the concern was not confirmed in in vivo studies. Thus it is concluded that cinnamaldehyde does not have a genotoxic potential in vivo. In addition, the carcinogenicity studies with trans-cinnamaldehyde did not indicate a carcinogenic potential. The ring substituents (4-methyl, 4-hydroxy, 4-methoxy, 3-or 5-methoxy or 2-methoxy) are anticipated not to increase but rather decrease the reactivity of the alpha,beta-unsaturated aldehyde group. Therefore, the Panel concluded that the seven ring-substituted cinnamyl derivatives [FL-no: , , , , , and ] like the un-substituted cinnamyl derivatives were not of concern with respect to genotoxicity EFSA Considerations Twenty-five of the 54 substances in FGE.68 are alpha,beta-unsaturated aldehydes or precursors of such aldehydes which are considered by the Panel to be structural alerts for genotoxicity. This was discussed in FGE.214, where the Panel concluded that data available do not preclude to evaluate the substances through the Procedure. For the remaining 29 of the 54 substances from the JECFA group of cinnamyl alcohol and related substances not being alpa,beta-unsaturated substances, the Panel also concluded that the genotoxicity data available do not preclude to evaluate these substances through the Procedure. 4. Application of the Procedure 4.1. Application of the Procedure to 54 Cinnamyl Alcohol and Related Substances by JECFA (JECFA, 2001b) According to the JECFA 48 of the substances belong to structural class I, and six to structural class II using the decision tree approach presented by Cramer et al. (Cramer et al., 1978). The JECFA concluded for cinnamyl alcohol and 49 related substances at step A3 in the JECFA Procedure i.e. the substances are expected to be metabolised to innocuous products (step 2) and the intakes for all substances are below the thresholds for their structural classes I and II (step A3). For four substances it was concluded at step A5 i.e. the intakes are above the thresholds for their structural classes, the substances are not endogenous, but a NAEL is available that can provide an adequate margin of safety to the estimated intake of the substances [FL-no: , , and ]. In conclusion, the JECFA evaluated all substances as to be of no safety concern at the estimated levels of intake as flavouring substances based 13

14 The evaluations of the 54 substances are summarised in Table 3.1: Summary of Safety Evaluation of Cinnamyl Alcohol and Related Substances (JECFA, 2001b) Application of the Procedure to Nine Aryl-Substituted Saturated and Unsaturated Primary Alcohol/Aldehyde/Acid/Ester Derivatives by EFSA (EFSA, 2008q) All nine candidate substances evaluated in FGE.15Rev1. are classified into structural class I using the decision tree approach presented by Cramer et al. (Cramer et al., 1978). The nine substances were concluded at step A3 i.e. the substances are expected to be metabolised to innocuous products (step 2) and the estimated daily intake is below the threshold for the structural class (step A3). In conclusion, the Panel evaluated all nine substances as to be of no safety concern at the estimated levels of intake as flavouring substances based The stepwise evaluations of the nine substances are summarised in Table 3.2: Summary of Safety Evaluation Applying the Procedure (EFSA / FGE.15Rev1) EFSA Considerations The Panel agrees with the way the application of the Procedure has been performed by the JECFA for all 54 substances in the group of cinnamyl alcohol and related substances. However, the structural class have, based on EFSA considerations, been changed for the following flavouring substances: [FL-no: , and and ] from structural class I to class II, [FL-no: ] from structural class II to class I, [FL-no: ] from structural class II to class III. These changes in structural classes do not give rise to change in the outcome of the application of the Procedure. For six substances [FL-no: , , , , and ] no European production figures were available and consequently no European exposure estimates could be calculated. Accordingly, the safety in use in Europe could not be assessed using the Procedure for these six substances. 5. Conclusion The JECFA has evaluated 55 substances in the group of cinnamyl alcohol and related substances at their 55 th meeting. Twenty-six of these substances are alpha,beta-unsaturated aldehydes or precursors for such, which the Panel considers to be a structural alert for genotoxicity. The following 25 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , and ] have initially been considered in FGE.214 with respect to genotoxicity. The Panel concluded that for these 25 substances the genotoxicity data available do not preclude their evaluation through the Procedure. For the remaining substance (of the 26 alpha,beta-unsaturated aldehydes or precursors for such), allyl cinnamate [FL-no: ], which may be metabolised to allyl alcohol and further to acrolein, considered with respect to genotoxicity in subgroup of FGE.19, a final conclusion as to its genotoxic properties could not be reached and additional data were requested. 14

15 Accordingly, this substance will not be considered in the present FGE. This consideration therefore only deals with 54 JECFA evaluated substances. The Panel concluded that the 54 substances in the JECFA flavouring group of cinnamyl alcohol and related flavouring substances are structurally related to the group of nine aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives evaluated by EFSA in FGE.15Rev1. The Panel agrees with the way the application of the Procedure has been performed by the JECFA for the 54 substances considered in this FGE. However for six substances [FL-no: , , , , and ] the JECFA evaluation is only based on MSDI values derived from production figures from the USA. EU production figures are needed in order to finalise the evaluation of these substances. For all 54 substances use levels are needed to calculate the mtamdis in order to identify those flavouring substances that need more refined exposure assessment and to finalise the evaluation. In order to determine whether the conclusion for the 54 JECFA evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications. Adequate specifications including complete purity criteria and identity are available for 13 of the JECFA evaluated substances considered in this FGE. Information on stereoisomerism is lacking for 41 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ] and compositional information of mixture is lacking for four substances [FL-no , , and ]. Thus, in total, for 46 substances [FL-no: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ] the Panel has reservations (no European production volumes are available, preventing them to be evaluated using the Procedure, and/or missing data on stereoisomerism and/or compositional information of mixture). For the remaining eight substances in the group of JECFA evaluated cinnamyl alcohol and related substances [FL-no: , , , , , , and ] the Panel agrees with JECFA conclusion No safety concern at estimated levels of intake as flavouring substances based 15

16 TABLE 1: SPECIFICATIN SUMMARY Table 1: specifications summary for the JECFA evaluated substances in the present group (JECFA, 2001c; JECFA, 2000d) Table 1: Specification Summary of the Substances in the JECFA Flavouring Group of Cinnamyl Alcohol and Related Substances (JECFA, 2001c) FL-no JECFAno EU Register name Structural formula FEMA no CoE no CAS no Cinnamyl alcohol 6) alpha-pentylcinnamyl alcohol 6) 3-Phenylpropan-1-ol 5-Phenylpentan-1-ol Cinnamaldehyde 6) alpha-butylcinnamaldehyde 6) alpha-pentylcinnamaldehyde 6) alpha-hexylcinnamaldehyde 6) 2-Methoxycinnamaldehyde 6) H H H H Phys.form Mol.formula Mol.weight Solid C 9 H C 14 H C 9 H C 11 H C 9 H C 13 H C 14 H C 15 H Solid C 10 H Solubility 1) Solubility in ethanol 2) to slightly soluble Moderately soluble Slightly soluble Slightly soluble Boiling point, C 3) Melting point, C ID test Assay minimum % 141 (7 hpa) 95 % % 155 (26 hpa) 98 % % 265 MS 98 % % (20hPa) 95 % (16 hpa) % Refrac. Index 4) Spec.gravity 5) n.a. n.a n.a. n.a. EFSA comments According to JECFA: Min. assay value is "94 (min. 95% combined aldehyde and corresponding acid)" and 16