SCIENTIFIC OPINION. Scientific Opinion of the Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)

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1 The EFSA Journal (2009) 880, 1-27 SCIENTIFIC PININ Flavouring Group Evaluation 214: alpha,beta-unsaturated aldehydes and precursors from chemical subgroup 3.1 of FGE.19: Cinnamyl derivatives 1 Scientific pinion of the Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) (Question No EFSA-Q ) Adopted on 27 November 2008 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. SUMMARY The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (the Panel) was asked to provide scientific advise for 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 asked to evaluate flavouring substances using the Procedure as referred to in the Commission Regulation (EC) No 1565/2000. The present concerns 29 substances. The 29 substances correspond to subgroup 3.1 of FGE.19. Eleven of these substances are alpha,betaunsaturated cinnamyl aldehydes [FL-no: , , , , , , , 1 For citation purposes: Scientific pinion of the Panel on Food Contact Materials, Flavourings and Processing Aids (CEF) on a request from European Commission on Flavouring Group Evaluation 214: alpha,beta-unsaturated aldehydes and precursors from chemical subgroup 3.1 of FGE.19: Cinnamyl derivatives. The EFSA Journal (2009) 880, 1-27 European Food Safety Authority

2 05.118, , and ] and 18 are precursors for such aldehydes [FL-no: , , , , , , , , , , , , , , , , and ]. The configuration of the double bond in cinnamaldehyde [FL-no: ] has not been specified. However, the substance is anticipated to contain more than 97 % trans-cinnamaldehyde. 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 unsubstituted cinnamyl derivatives, were not of concern with respect to genotoxicity. The Panel concluded that the data available do not preclude an evaluation of the alpha,betaunsaturated cinnamaldehyde-derivatives in FGE.214 (subgroup 3.1 of FGE.19) using the Procedure. Key words: alpha,beta-unsaturated aldehydes, cinnamyl aldehydes, cinnamyl derivatives, flavouring substances, safety evaluation. The EFSA Journal (2009) 880, 2-27

3 TABLE F CNTENTS Panel Members... 1 Summary... 1 Key words:... 2 Background... 4 Terms of Reference... 5 Acknowledgements... 5 Assessment Presentation of the Substances in the Flavouring Group Evaluation Description Toxicity (Q)SAR Predictions Carcinogenicity Studies Genotoxicity Studies Conclusion on Genotoxicity and Carcinogenicity Conclusions... 8 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 214 (JECFA, 2001b)... 9 Table 2: Summary of Safety Evaluation Applying the Procedure (Based on Intakes Calculated by the MSDI Approach) (JECFA, 2001b) Table 3: (Q)SAR Predictions on Mutagenicity in Five Models for 11 Aldehydes from Subgroup Table 5: Genotoxicity (in vitro) Table 6: Genotoxicity (in vivo) References Abbreviations The EFSA Journal (2009) 880, 3-27

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 2008/478/EC (EC, 2008a). 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). For the submission of data by the manufacturer, deadlines have been established by Commission Regulation (EC) No 622/2002 (EC, 2002b). After the completion of the evaluation programme the community list of flavouring substances for use in or on foods in the EU shall be adopted (Article 5 (1) of Regulation (EC) No 2232/96) (EC, 1996). Flavouring Group Evaluation 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 aldehyde and ketone structures were considered by the Panel to be structural alerts for genotoxicity. The Panel noted that there were limited genotoxicity data on these flavouring substances but that positive genotoxicity studies were identified for some substances in the group. 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 considering a number of models (DEREKfW, TPKAT, DTU- NFI MultiCASE Models and ISS Local Models (Gry et al., 2007)). The Panel noted that for most of these models internal and external validation has been performed, but considered that the outcome of these validations was not always extensive enough to appreciate the validity of the predictions of these models for these alpha,beta-unsaturated carbonyls. Therefore, the Panel considered it inappropriate to totally rely on (Q)SAR predictions at this point in time and decided not to take substances through the Procedure based on negative (Q)SAR predictions only. 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. (EFSA, 2008b) should be further examined to determine whether evaluation through the Procedure is feasible. Corresponding to The EFSA Journal (2009) 880, 4-27

5 these 11 subgroups 11 Flavouring Group Evaluations (FGEs) were established (FGE.201, 202, 203, 210, 212, 213, 214, 216, 217, 218 and 220). If the Panel concludes for any substances in these 11 FGEs that they cannot be evaluated using the Procedure then it has to be decided if there is a safety concern for certain substances or if additional data are required in order to finalise the evaluation. If the Panel concludes that a genotoxic potential can be ruled out for the substances they will be merged with structurally related substances in other FGEs and evaluated using the Procedure. TERMS F REFERENCE European Food Safety Authority (EFSA) is requested to carry out a risk assessment on flavouring substances prior to their authorisation and inclusion in a community list according to Commission Regulation (EC) No 1565/2000 (EC, 2000a). ACKNWLEDGEMENTS European Food Safety Authority wishes to thank the members of the Working Groups on Flavourings for the preparation of this opinion: 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. ASSESSMENT 1. Presentation of the Substances in the Flavouring Group Evaluation Description The present concerns 29 substances, which are listed in Table 1. The 29 substances correspond to subgroup 3.1 of FGE.19 (EFSA, 2008b). Eleven of these substances are alpha,beta-unsaturated cinnamyl aldehydes [FL-no: , , , , , , , , , and ] and 18 are precursors for such aldehydes [FL-no: , , , , , , , , , , , , , , , , and ]. The configuration of the double bond in cinnamaldehyde [FL-no: ] has not been specified. However, the substance is anticipated to contain more than 97 % trans-cinnamaldehyde (Eilerman, 2001). A summary of their current evaluation status by the JECFA is given in Table 2 (JECFA, 2001b). The alpha,beta-unsaturated aldehyde and ketone structures are considered by the Panel to be structural alerts for genotoxicity (EFSA, 2008b). Accordingly, the available data on genotoxic or carcinogenic acitivity for the 11 aldehydes in FGE.214 [FL-no: , , , , The EFSA Journal (2009) 880, 5-27

6 05.048, , , , , and ], corresponding to the 29 substances in FGE.214, will be considered in this FGE. The Panel has also taken into consideration the outcome of the predictions from five selected (Q)SAR models (Benigni & Netzeva, 2007a; Gry et al., 2007; Nikolov et al., 2007) on the 11 aldehydes [FL-no: , , , , , , , , , and ]. The 11 aldehydes and their (Q)SAR predictions are shown in Table Toxicity 2.1. (Q)SAR Predictions In Table 3 the outcomes of the (Q)SAR predictions for possible genotoxic activity in five in vitro (Q)SAR models (ISS Local Model-Ames test, DTU-NFI MultiCASE-Ames test, -Chromosomal aberration test in Chinese hamster ovary cells (CH), -Chromosomal aberration test in Chinese hamster lung cells (CHL), and -Mouse lymphoma test) are presented. Negative predictions were obtained for all substances in models for bacterial gene mutations, except for cinnamaldehyde [FL-no: ], for which a positive result was generated by the ISS Local Model. The same result was obtained with the MultiCASE model for Chromosomal aberrations in Chinese Hamster Lung (CHL) cells. The predictions for genotoxicity in the DTU-NFI MultiCASE Mouse lymphoma assays are either positive (four cases) or out of domain (seven cases). Although positive predictions were obtained for four substances which all have unsubstituted alpha,betaunsaturated double bonds, there are also two substances with similar structures ([FL-no: and ]) without double bond substituents for which an out of domain prediction was obtained. Except for cinnamaldehyde [FL-no: ], no concern for genotoxicity is indicated from the two models for Ames mutagenicity and for the model for Chromosomal aberrations in CHL cells. The predictions for genotoxicity in the Mouse lymphoma assay and Chromosomal aberrations in CH cells are inconclusive (See Table 3) Carcinogenicity Studies Two carcinogenicity studies are available for the substances in subgroup 3.1. For transcinnamaldehyde, a carcinogenicity study has been carried out in mice and rats (Hooth et al., 2004; NTP, 2004l). This is the main geometrical isomer anticipated in cinnamaldehyde [FL-no: ] Groups of 50 male and 50 female B6C3F 1 mice were fed diets containing 1,000, 2,100 or 4,100 mg/kg feed microencapsulated trans-cinnamaldehyde for two years. Additional groups of 50 male and 50 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1,000, 2,100 or 4,100 mg/kg feed delivered average daily doses of approximately 125, 270 or 550 mg/kg bw to males and females. Survival of males in the 2,100 mg/kg group was less than that of the vehicle control group. Mean body weights of males and females in 2,100 and 4,100 mg/kg groups were generally less than those of the vehicle controls throughout the study, and mean body weights of males in 1,000 mg/kg group were less after week 74. Feed consumption by exposed mice was similar to that by the vehicle controls. The incidences of olfactory epithelial pigmentation in males in 4,100 mg/kg group and in The EFSA Journal (2009) 880, 6-27

7 females in 2,100 and 4,100 groups were significantly greater than those in vehicle controls. There were no neoplasms that were attributed to exposure to trans-cinnamaldehyde (NTP, 2004l). Groups of 50 male and 50 female F344/N rats were fed diets containing 1,000, 2,100 or 4,100 mg/kg feed microencapsulated trans-cinnamaldehyde for two years. Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1,000, 2,100 or 4,100 mg/kg feed delivered average daily doses of approximately 50, 100 or 200 mg/kg bw to males and females. Survival of males in the 4,100 mg/kg group was greater than that of the vehicle controls. Mean body weights of males and females in 4,100 mg/kg group were generally less than those of the vehicle controls throughout the study. Feed consumption by males in 2,100 and 4,100 mg/kg groups and females in 4,100 mg/kg group was less than that by the vehicle controls at the beginning and end of the study. There were no neoplasms or non-neoplastic lesions that were attributed to exposure to trans-cinnamaldehyde (NTP, 2004l). Conclusion on carcinogenicity: According to the NTP, under the conditions of the two-year feed studies there was no evidence of carcinogenic activity of trans-cinnamaldehyde in male and female F344/W rats and male and fenale B6C3F 1 mice. The Panel agrees with this conclusion Genotoxicity Studies In subgroup 3.1 there are studies available for six of the substances. Validation and study results are presented in Table 5 and 6. For cinnamaldehyde [FL-no: ] 19 in vitro studies (in total 27 tests) and four in vivo studies (five different endpoints) 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 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 chromatid exchanges (SCE), 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 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 The EFSA Journal (2009) 880, 7-27

8 micronuclei frequency was observed in bone marrow cells from these animals. In another valid in vivo bone marrow assay with intraperitoneal injection no increase in bone marrow micronuclei formation was observed either. From the few studies available with other substances [FL-no: , and ] no indication of genotoxicity in vivo was obtained 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 unsubstituted cinnamyl derivatives, were not of concern with respect to genotoxicity. 3. Conclusions The present concerns 29 substances corresponding to subgroup 3.1 of FGE.19. Eleven of these substances are alpha,beta-unsaturated cinnamyl aldehydes [FL-no: , , , , , , , , , and ] and the 18 others are precursors for such aldehydes [FL-no: , , , , , , , , , , , , , , , , and ]. The configuration of the double bond in cinnamaldehyde [FL-no: ] has not been specified. However, the substance is anticipated to contain more than 97 % trans-cinnamaldehyde. 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 unsubstituted cinnamyl derivatives, were not of concern with respect to genotoxicity. The Panel concluded that the data available do not preclude an evaluation of the alpha,betaunsaturated cinnamaldehyde-derivatives in FGE.214 (subgroup 3.1 of FGE.19) using the Procedure. The EFSA Journal (2009) 880, 8-27

9 TABLE 1: SPECIFICATIN SUMMARY F THE SUBSTANCES IN THE FLAVURING GRUP EVALUATIN 214 (JECFA, 2001B) Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 214 (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula FEMA no CoE no CAS no Cinnamyl alcohol 6) alpha-pentylcinnamyl alcohol 6) Cinnamaldehyde 6) alpha-butylcinnamaldehyde 6) alpha-pentylcinnamaldehyde 6) alpha-hexylcinnamaldehyde 6) 2-Methoxycinnamaldehyde 6) alpha-methylcinnamaldehyde 6) H H Phys.form Mol.formula Mol.weight Solid C 9 H C 14 H C 9 H C 13 H C 14 H C 15 H Solid C 10 H C 10 H Solubility 1) Solubility in ethanol 2) to slightly soluble Moderately soluble Slightly soluble Boiling point, C 3) Melting point, C ID test Assay minimum % 141 (7 hpa) 95 % % 265 MS 98 % % (20hPa) 95 % (16 hpa) % 148 (35 hpa) 95 % Refrac. Index Spec.gravity 5) The EFSA Journal (2009) 880, 9-27

10 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 214 (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula FEMA no CoE no CAS no 3-(4-Methoxyphenyl)-2-methylprop-2-enal 6) 4-Methoxycinnamaldehyde 6) p-methylcinnamaldehyde 6) Hydroxy-3,5-dimethoxycinnamaldehyde 6) H Phys.form Mol.formula Mol.weight C 11 H Solid C 10 H Solid C 10 H Solid C 11 H Solubility 1) Solubility in ethanol 2) Moderately soluble Moderately soluble Practically insoluble or insoluble 1 ml in 1 ml Boiling point, C 3) Melting point, C ID test Assay minimum (0.1hPa) MS 95 % % 154 (33 hpa) % MS 95 % Refrac. Index Spec.gravity 5) Hydroxy-3-methoxycinnamaldehyde 6) H Solid C 10 H Slightly soluble 1 ml in 1 ml 175 (7 hpa) 82 NMR 95 % alpha-pentylcinnamaldehyde dimethyl acetal 6) C 16 H Slightly soluble % Cinnamaldehyde ethylene glycol acetal 6) Cinnamyl acetate 6) C 11 H C 11 H NMR 90 % % The EFSA Journal (2009) 880, 10-27

11 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 214 (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula FEMA no CoE no CAS no alpha-pentylcinnamyl acetate 6) Phys.form Mol.formula Mol.weight C 16 H Solubility 1) Solubility in ethanol 2) Boiling point, C 3) Melting point, C ID test Assay minimum % Refrac. Index Spec.gravity 5) Cinnamyl butyrate 6) Cinnamyl formate 6) alpha-pentylcinnamyl formate 6) Cinnamyl propionate 6) Methoxycinnamyl acetate C 13 H C 10 H C 15 H C 12 H Solid C 12 H Practically insoluble or insoluble 1 ml in 1 ml % % % % MS 95 % Cinnamyl 2-methylcrotonate 6) Cinnamyl isovalerate 6) C 14 H C 14 H Practically insoluble or insoluble 1 ml in 1 ml MS 95 % % alpha-pentylcinnamyl isovalerate 6) C 19 H (5 hpa) 97 % The EFSA Journal (2009) 880, 11-27

12 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 214 (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula FEMA no CoE no CAS no Cinnamyl isobutyrate 6) Cinnamyl phenylacetate 6) Phys.form Mol.formula Mol.weight C 13 H C 17 H Solubility 1) Solubility in ethanol 2) Boiling point, C 3) Melting point, C ID test Assay minimum % % Refrac. Index Spec.gravity 5) Cinnamyl cinnamate 6) Cinnamyl benzoate 6) Solid C 18 H Solid C 16 H Moderately soluble % % 1) Solubility in water, if not otherwise stated. 2) Solubility in 95% ethanol, if not otherwise stated. 3) At hpa, if not otherwise stated. At 20 C, if not otherwise stated. 5) At 25 C, if not otherwise stated. 6) Stereoisomeric composition not specified. The EFSA Journal (2009) 880, 12-27

13 TABLE 2: SUMMARY F SAFETY EVALUATIN APPLYING THE PRCEDURE (BASED N INTAKES CALCULATED BY THE MSDI APPRACH) (JECFA, 2001B) Table 2: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula MSDI 1) (μg/capita/day) EU USA 2-Methoxycinnamaldehyde Class 2) Evaluation procedure path 3) utcome on the named compound [ or 5)] alpha-methylcinnamaldehyde (4-Methoxyphenyl)-2-methylprop-2-enal Methoxycinnamaldehyde p-methylcinnamaldehyde Cinnamyl acetate Cinnamyl butyrate Cinnamyl formate Cinnamyl propionate The EFSA Journal (2009) 880, 13-27

14 Table 2: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula MSDI 1) (μg/capita/day) EU USA Cinnamyl isovalerate Class 2) Evaluation procedure path 3) utcome on the named compound [ or 5)] Cinnamyl isobutyrate Cinnamyl phenylacetate Cinnamyl cinnamate Cinnamyl benzoate ND Cinnamyl alcohol Cinnamaldehyde Hydroxy-3,5-dimethoxycinnamaldehyde H A3: Intake above threshold, A4: Not endogenous, A5: Adequate NAEL exists A3: Intake above threshold, A4: Not endogenous, A5: Adequate NAEL exists Not evaluated by the JECFA. H The EFSA Journal (2009) 880, 14-27

15 Table 2: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula MSDI 1) (μg/capita/day) EU USA Hydroxy-3-methoxycinnamaldehyde Class 2) Evaluation procedure path 3) utcome on the named compound [ or 5)] 0.19 Not evaluated by the JECFA. H Methoxycinnamyl acetate Not evaluated by the JECFA Cinnamyl 2-methylcrotonate Not evaluated by the JECFA alpha-pentylcinnamyl alcohol H I alpha-butylcinnamaldehyde I alpha-pentylcinnamaldehyde I alpha-hexylcinnamaldehyde I alpha-pentylcinnamaldehyde dimethyl acetal I alpha-pentylcinnamyl acetate I alpha-pentylcinnamyl formate I The EFSA Journal (2009) 880, 15-27

16 Table 2: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) (JECFA, 2001b) FL-no JECFA-no EU Register name Structural formula MSDI 1) (μg/capita/day) EU USA alpha-pentylcinnamyl isovalerate Class 2) Evaluation procedure path 3) I utcome on the named compound [ or 5)] Cinnamaldehyde ethylene glycol acetal ) EU MSDI: Amount added to food as flavour in (kg / year) x 10E9 / (0.1 x population in Europe (= 375 x 10E6) x 0.6 x 365) = µg/capita/day. 2) Thresholds of concern: = 1800, I = 540, II = 90 µg/person/day. 3) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot. No safety concern based on intake calculated by the MSDI approach of the named compound. 5) Data must be available on the substance or closely related substances to perform a safety evaluation. II A3: Intake above threshold, A4: Not endogenous, A5: Adequate NAEL exists The EFSA Journal (2009) 880, 16-27

17 TABLE 3: (Q)SAR PREDICTINS N MUTAGENICITY IN FIVE MDELS FR 11 ALDEHYDES FRM SUBGRUP 3.1 FL-no JECFA-no Subgroup EU Register name Structural formula FEMA no CoE no CAS no 3.1 Cinnamaldehyde alpha-butylcinnamaldehyde alpha-pentylcinnamaldehyde alpha-hexylcinnamaldehyde Methoxycinnamaldehyde ISS Local Model MultiCASE Ames Test TA100 Ames test MultiCASE MultiCASE Chromosomal Mouse lymphoma aberration test in test CH MultiCASE Chromosomal aberration test in CHL PS NEG PS NEG PS NEG NEG D D NEG NEG NEG D D NEG NEG NEG D D NEG NEG NEG PS PS NEG alpha-methylcinnamaldehyde NEG NEG D D NEG (4-Methoxyphenyl)-2-methylprop-2-enal NEG NEG D D NEG p-methylcinnamaldehyde Hydroxy-3,5-dimethoxycinnamaldehyde H NEG NEG D NEG NEG NEG NEG D EQU NEG The EFSA Journal (2009) 880, 17-27

18 FL-no JECFA-no Subgroup EU Register name Structural formula FEMA no CoE no CAS no Hydroxy-3-methoxycinnamaldehyde Methoxycinnamaldehyde H Column 2: Structure group 3.1: cinnamyl derivatives. Column 6: Local model on aldehydes and ketones, Ames TA100 (NEG: Negative; PS: Positive; D: ut of domain). Column 7: MultiCASE Ames test (D: ut of domain; PS: Positive; NEG: Negative; EQU: Equivocal). Column 8: MultiCASE Mouse lymphoma test (D: ut of domain; PS: Positive; NEG: Negative; EQU: Equivocal). Column 9: MultiCASE Chromosomal aberration in CH (D: ut of domain; PS: Positive; NEG: Negative; EQU: Equivocal). Column 10: MultiCASE Chromosomal aberration in CHL (D: ut of domain; PS: Positive; NEG: Negative; EQU: Equivocal). ISS Local Model MultiCASE Ames Test TA100 Ames test MultiCASE MultiCASE Chromosomal Mouse lymphoma aberration test in test CH MultiCASE Chromosomal aberration test in CHL NEG NEG PS EQU NEG NEG NEG PS EQU NEG D, out of applicability domain: not matching the range of conditions where a reliable prediction can be obtained in this model. These conditions may be physicochemical, structural, biological, etc. The EFSA Journal (2009) 880, 18-27

19 Table 4: Carcinogenicity Studies Table 4: Carcinogenicity Studies Chemical Name [FL-no] Trans-cinnamaldehyde Species; Sex No./Group Rat; M, F 50 Mice; M, F 50 Route Dose levels Duration Results Reference Comments a ral 0, 50, 100 or 200 mg/kg bw/day ral 0, 125, 270 or 550 mg/kg bw/day 2 years There were no neoplasms or nonneoplastic lesions that were attributed to exposure to transcinnamaldehyde 2 years There were no neoplasms that were attributed to exposure to transcinnamaldehyde a: Validity of carcinogenicity studies: Valid. Limited validity (e.g. if certain aspects are not in accordance with ECD guidelines or current standards and / or limited documentation). Insufficient Insufficient validity (e.g. if main aspects are not in accordance with any recognised guidelines (e.g. ECD) or current standards and/or inappropriate test system). Validity cannot be evaluated (e.g. insufficient documentation, short abstract only, too little experimental details provided). (NTP, 2004l) (NTP, 2004l) Valid Valid The EFSA Journal (2009) 880, 19-27

20 TABLE 5: GENTXICITY (IN VITR) Table 5: GENTXICITY (in vitro) Chemical Name [FL-no] Test System Test bject Concentration Reported Result Reference Comments e Reverse mutation c S. typhimurium TA1537, TA1538, TA98, 600 µg/plate Negative a (Sekizawa & Valid. Published non-glp study with sufficient details; the Cinnamaldehyde [05.014] TA100, TA1535 Shibamoto, 1982) result is considered valid. Reverse mutation Reverse mutation S. typhimurium TA1537, TA98, TA100, TA1535 S. typhimurium TA104 (with preincubation) 10 mg/plate (10,000 µg/plate) 0.8 µmol (105 µg) Reverse mutation S. typhimurium TA98, TA100 Up to 0.5 mg/plate (500 µg/plate) Reverse mutation S. typhimurium TA1537, TA92, TA94, TA98, TA100, TA1535 (with plate incorporation and preincubation) Negative a (Prival et al., 1982) Validity cannot be evaluated. Published non-glp study with insufficient documentation (no figures); the validity cannot be evaluated. Negative a (Marnett et al., 1985a) Valid. Published non-glp study carried out with only one strain and only without S9; however, for the purpose og the study, the result is considered valid. Positive a,d (Ishidate et al., 198 Valid. According to current guidelines (in TA100 with and without metabolic activation) a,b 500 µg/plate Negative a (Lijinsky & Andrews, 1980) Valid. Reverse mutation S. typhimurium TA98, TA100 (with plate incorporation and preincubation) µg/plate Negative a (Kasamaki et al., 1982) Limited validity (only two strains tested). Reverse mutation S. typhimurium TA97, TA98, TA100 (with preincubation) 1 mg/ml (1000 µg/ml) Negative a (Azizan & Blevins, 1995) Validity cannot be evaluated. Published non-glp study with insufficient documentation (no figures); the validity cannot be evaluated. Reverse mutation S. typhimurium TA98, TA100, TA104 (with preincubation) Not reported Negative a (Kato et al., 1989) Validity cannot be evaluated. Abstract limited data reported. Reverse mutation S. typhimurium TA1537, TA98, TA100, 100 µg/plate Negative a (Mortelmans et al., Valid. TA1535 (with preincubation) 1986) Reverse mutation S. typhimurium TA100 (with 5 µmol/plate Negative a (Neudecker et al., Limited validity. nly in one strain. preincubation) (661 µg/plate) 1983) Mutation E. coli WP2 uvra 0.8 mg/plate (800 µg/plate) Negative b (Yoo, 1986) Validity cannot be evaluated. nly in Japanese (insufficient documentation). DNA repair B. subtilis M45 (rec-) 0.2 mg/disk (200 µg/disc) Positive b (Sekizawa & Shibamoto, 1982) Insufficient validity. The test system used is considered inappropriate, not relevant for the evaluation. DNA repair B. subtilis M45 (rec-) and H17 (rec+) 10 µl/disc (10,500 µg/disc) Positive b (Yoo, 1986) Insufficient validity. Rec assay not considered relevant for evaluation of genotoxicity. DNA repair B. subtilis M45 (rec-) and H17 (rec+) 10 µl/disc (10,500 µg/disc) Positive a (Kuroda et al., 1984a) Insufficient valitidy ( Rec assay not considered relevant for evaluation of genotoxicity). DNA repair B. subtilis M45 (rec-) and H17 (rec+) 21 µg/disc Negative b (da et al., 1979) Validity cannot be evaluated (relevance for evaluation of genotoxicity uncertain). The EFSA Journal (2009) 880, 20-27

21 Table 5: GENTXICITY (in vitro) Chemical Name [FL-no] Test System Test bject Concentration Reported Result Reference Comments e Sister chromatid exchange Chinese hamster ovary cells 33.3 µmol/l (4401 µg) Negative b (Sasaki et al., 1987) Validity cannot be evaluated (relevance for evaluation of genotoxicity uncertain). Chromosomal Chinese hamster fibroblasts Up to mg/ml Positive b (Ishidate et al., 198 Valid. aberration (15 µg/ml) Cinnamaldehyde [05.014] (cont.) Chromosomal aberration Chinese hamster B241 cells 20 nmol/l (2.6 µg) Positive b (Kasamaki & Urasawa, 1985) Valid (unusual cell line). Chromosomal aberration Chromosomal aberration Sister chromatid exchange Sister chromatid exchange Chinese hamster B241 cells 10 nmol/l (1.3 µg) Positive a (Kasamaki et al., 1982) Limited validity (limited documentation; results for only one test concentration reported; long incubation period of 24 hrs; unusual cell line). (Galloway et al., 1987) Valid. Chinese hamster ovary cells 18.3 µg/ml Negative b 100 µg/ml Negative c Chinese hamster ovary cells Up to 6.8 µg/ml Positive b (Galloway et al., 1987) Valid. Weakly positive without S9. Chinese hamster ovary cells 91.8 µg/ml Positive c (Galloway et al., 1987) Valid, however, the result (obtained in the presence of S9) is considered equivocal. DNA strand breaks Mouse L1210 lymphoma cells 500 µmol / 4 ml Positive b (Eder et al., 1993) Limited validity. Positive at cytotoxic levels. (16.5 mg/ml) SS chromo?? Negative (Eder et al., 1993) Limited validity. Results poorly reported relevance questionable. Mutation (HGPRT) Chinese hamster V79 cells 100 µmol/l ( µg) Negative b (Fiorio & Bronzetti, 199 Insufficient validity (only one concentration and only without S9 tested). Micronucleus formation alpha-methylcinnamaldehyde [05.050] Reverse mutation S. typhimurium TA100(with preincubation) Hep-G2 cells 500 µg/ml Positive (Sanyal et al., 1997) Limited validity. Published non-glp study, not according with standard, conventional guidelines; the moderate increase observed only at the highest concentration is considered of limited validity. 4 µmol/plate (585 µg/plate) Negative a (Neudecker et al., 1983) Limited validity (only one strain tested). Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 (with preincubation) 500 µg/plate Negative a (Mortelmans et al., 1986) Valid. Reverse mutation Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 alpha-pentylcinnamaldehyde [05.040] Reverse mutation S. typhimurium TA97, TA102 (with preincubation) 5 concentrations up to cytotoxicity, or max µg/plate 5 concentrations up to cytotoxicity, or max µg/plate 1.0 mg/plate (1000 µg/plate) Negative a (Wild et al., 1983) Valid. Negative a (Wild et al., 1983) Valid. Negative a (Fujita & Sasaki, 1987) Validity cannot be evaluated. The EFSA Journal (2009) 880, 21-27

22 Table 5: GENTXICITY (in vitro) Chemical Name [FL-no] Test System Test bject Concentration Reported Result Reference Comments e alpha-hexylcinnamaldehyde [05.041] Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA Methoxycinnamaldehyde [05.048] Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 (with preincubation) 5 concentrations up to cytotoxicity, or max µg/plate Negative a (Wild et al., 1983) Valid. 666 µg/plate Positive a (Mortelmans et al., Valid. 1986) 3-(4-Methoxyphenyl)-2-methylprop-2-enal [05.051] Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA concentrations up to cytotoxicity, or max µg/plate Negative a (Wild et al., 1983) Valid. a: With and without metabolic activation. b: Without metabolic activation.. c: With metabolic activation. d: Positive results in strain TA100 only. e: Validity of genotoxicity studies: Valid. Limited validity (e.g. if certain aspects are not in accordance with ECD guidelines or current standards and / or limited documentation). Insufficient Insufficient validity (e.g. if main aspects are not in accordance with any recognised guidelines (e.g. ECD) or current standards and/or inappropriate test system). Validity cannot be evaluated (e.g. insufficient documentation, short abstract only, too little experimental details provided). The EFSA Journal (2009) 880, 22-27

23 TABLE 6: GENTXICITY (IN VIV) Table 6: GENTXICITY (in vivo) Chemical Name [FL-no] Test System Test bject Route Dose Result Reference Comments a Cinnamaldehyde [05.014] Sex-linked recessive lethal mutation Drosophila melanogaster 800 mg/kg od diet (800 µg/g) Negative (Woodruff et al., 1985) Limited validity. Published study carried out within NTP. The moderate increase observed only at highest doses by injection and not by feeding is considered of limited validity. Unscheduled DNA synthesis Rat hepatocytes ral (gavage) 0, 50, 200, 1000 mg/kg bw Negative (Mirsalis et al., 1989) Valid. According to current guidelines. Micronucleus formation Mouse bone marrow cells Intraperitoneal µg/kg bw Negative (Hayashi et al., 198 Valid. Published non-glp pilot study with too few animals but positive for controls. It should be considered together with Hayashi et al. (1988). Taking into account the results of both studies, the final result is considered valid. Micronucleus formation Mouse hepatocytes Gavage µg/kg bw Positive (Mereto et al., 199 Micronucleus formation Rat hepatocytes Gavage µg/kg bw Positive (Mereto et al., 199 Micronucleus formation Mouse bone marrow Gavage µg/kg bw Negative (Mereto et al., 199 Micronucleus formation Rat bone marrow Gavage µg/kg bw Negative (Mereto et al., 199 Valid. After 2/3 hepatectomy. The highest dose cause 100% mortality. Relevance not clear. Valid. After 2/3 hepatectomy. The highest dose cause 100% mortality. Relevance not clear. Limited validity. PCE/NCE ratios were not affected, at the highest dose tested 100% lethality. Limited validity. PCE/NCE ratios were not affected, at the highest dose tested 100 % lethality. Nuclear anomalies Mouse forestomach mucosal cells Gavage µg/kg bw Negative (Mereto et al., 199 Validity cannot be evaluated (meaning of endpoint for genotoxicity is unclear, at the highest dose tested 100 % lethality). Nuclear anomalies DNA fragmentation Rat forestomach mucosal cells Rat hepatocytes and gastric mucosal cells Gavage µg/kg bw Positive (Mereto et al., 199 Gavage µg/kg bw Negative (Mereto et al., 199 Validity cannot be evaluated. Mainly karyorrhexis and pyknosis which are signs of cytotoxicity. The meaning of this endpoint for genotoxicity is questionable, at the highest dose tested 100 % lethality. Valid. Alkaline elution assay. alpha-methylcinnamaldehyde [05.050] Sex-linked recessive lethal mutation D. melanogaster Feed 5 mm Negative (Wild et al., 1983) Limited validity (limited reporting, test system considered of limited relevance). Micronucleus formation Mouse, bone marrow Intraperitoneal 146, 292, 438 mg/kg bw Negative (Wild et al., 1983) Limited validity (only analysis at one time point; no PCE/NCE ratio reported. The EFSA Journal (2009) 880, 23-27

24 alpha-pentylcinnamaldehyde [05.040] alpha-hexylcinnamaldehyde [05.041] Sex-linked recessive lethal mutation D. melanogaster Feed 10 mm Negative (Wild et al., 1983) Limited validity (limited reporting, test system considered of limited relevance). Micronucleus formation Mouse, bone marrow Intraperitoneal 405, 809, 1313 mg/kg bw Negative (Wild et al., 1983) Limited validity (only analysis at one time point; no PCE/NCE ratio reported lethality at highest dose level). Sex-linked recessive lethal D. melanogaster Feed 10 mm Negative (Wild et al., 1983) Limited validity (limited reporting, test system considered of mutation limited relevance). Mouse, bone marrow Intraperitoneal 324, 540, 756 mg/kg bw Negative (Wild et al., 1983) Limited validity (only analysis at one time point; no PCE/NCE ratio reported). a: Validity of genotoxicity studies: Valid. Limited validity (e.g. if certain aspects are not in accordance with ECD guidelines or current standards and / or limited documentation). Insufficient validity (e.g. if main aspects are not in accordance with any recognised guidelines (e.g. ECD) or current standards and/or inappropriate test system). Validity cannot be evaluated (e.g. insufficient documentation, short abstract only, too little experimental details provided). The EFSA Journal (2009) 880, 24-27

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