SCIENTIFIC OPINION Flavouring Group Evaluation 214: · PDF fileFlavouring Group Evaluation...

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The EFSA Journal (2009) 880, 1-27 © European Food Safety Authority SCIENTIFIC OPINION Flavouring Group Evaluation 214: alpha,beta-Unsaturated aldehydes and precursors from chemical subgroup 3.1 of FGE.19: Cinnamyl derivatives 1 Scientific Opinion of the Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) (Question No EFSA-Q-2008-760) 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 Flavouring Group Evaluation 214 (FGE.214) concerns 29 substances. The 29 substances correspond to subgroup 3.1 of FGE.19. Eleven of these substances are alpha,beta- unsaturated cinnamyl aldehydes [FL-no: 05.014, 05.039, 05.040, 05.041, 05.048, 05.050, 05.051, 1 For citation purposes: Scientific Opinion 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

Transcript of SCIENTIFIC OPINION Flavouring Group Evaluation 214: · PDF fileFlavouring Group Evaluation...

Page 1: SCIENTIFIC OPINION Flavouring Group Evaluation 214:  · PDF fileFlavouring Group Evaluation 214 (FGE.214) The EFSA Journal (2009) 880, 3-27 TABLE OF CONTENTS Panel Members

The EFSA Journal (2009) 880, 1-27

© European Food Safety Authority

SCIENTIFIC OPINION

Flavouring Group Evaluation 214: alpha,beta-Unsaturated aldehydes and precursors from chemical subgroup 3.1

of FGE.19: Cinnamyl derivatives1

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

(Question No EFSA-Q-2008-760)

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 Flavouring Group Evaluation 214 (FGE.214) concerns 29 substances. The 29 substances correspond to subgroup 3.1 of FGE.19. Eleven of these substances are alpha,beta-unsaturated cinnamyl aldehydes [FL-no: 05.014, 05.039, 05.040, 05.041, 05.048, 05.050, 05.051,

1 For citation purposes: Scientific Opinion 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

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05.118, 05.122, 05.154 and 05.155] and 18 are precursors for such aldehydes [FL-no: 02.017, 02.030, 06.013, 06.014, 09.018, 09.026, 09.053, 09.085, 09.090, 09.133, 09.306, 09.339, 09.459, 09.468, 09.470, 09.708, 09.739 and 09.780]. The configuration of the double bond in cinnamaldehyde [FL-no: 05.014] 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: 05.014], showing an ability to induce chromosomal damage in vitro and by the positive result obtained for 2-methoxycinnamaldehyde [FL-no: 05.048] 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: 05.048, 05.051, 05.118, 05.122, 05.154, 05.155 and 09.306], 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,beta- unsaturated 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.

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TABLE OF CONTENTS

Panel Members ................................................................................................................................................................. 1 Summary ........................................................................................................................................................................... 1 Key words:......................................................................................................................................................................... 2 Background ....................................................................................................................................................................... 4 Terms of Reference ........................................................................................................................................................... 5 Acknowledgements ........................................................................................................................................................... 5 Assessment ......................................................................................................................................................................... 5 

1.  Presentation of the Substances in the Flavouring Group Evaluation 214 ................................................................ 5 1.1.  Description ........................................................................................................................................................ 5 

2.  Toxicity .................................................................................................................................................................... 6 2.1.  (Q)SAR Predictions .......................................................................................................................................... 6 2.2.  Carcinogenicity Studies .................................................................................................................................... 6 2.3.  Genotoxicity Studies ......................................................................................................................................... 7 2.4.  Conclusion on Genotoxicity and Carcinogenicity ............................................................................................ 8 

3.  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) ........................................................................................................................................... 13 Table 3: (Q)SAR Predictions on Mutagenicity in Five Models for 11 Aldehydes from Subgroup 3.1 .................... 17 Table 5: Genotoxicity (in vitro) ...................................................................................................................................... 20 Table 6: Genotoxicity (in vivo) ....................................................................................................................................... 23 References........................................................................................................................................................................ 25 Abbreviations .................................................................................................................................................................. 27 

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Flavouring Group Evaluation 214 (FGE.214)

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BACKGROUND

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 Opinion 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, TOPKAT, 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 4.4) (EFSA, 2008b) should be further examined to determine whether evaluation through the Procedure is feasible. Corresponding to

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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 OF 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).

ACKNOWLEDGEMENTS

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 214

1.1. Description

The present Flavouring Group Evaluation 214 (FGE.214) 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: 05.014, 05.039, 05.040, 05.041, 05.048, 05.050, 05.051, 05.118, 05.122, 05.154 and 05.155] and 18 are precursors for such aldehydes [FL-no: 02.017, 02.030, 06.013, 06.014, 09.018, 09.026, 09.053, 09.085, 09.090, 09.133, 09.306, 09.339, 09.459, 09.468, 09.470, 09.708, 09.739 and 09.780]. The configuration of the double bond in cinnamaldehyde [FL-no: 05.014] 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: 05.014, 05.039, 05.040, 05.041,

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05.048, 05.050, 05.051, 05.118, 05.122, 05.154 and 05.155], 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: 05.014, 05.039, 05.040, 05.041, 05.048, 05.050, 05.051, 05.118, 05.122, 05.154 and 05.155]. The 11 aldehydes and their (Q)SAR predictions are shown in Table 3.

2. 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 (CHO), -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: 05.014], 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,beta-unsaturated double bonds, there are also two substances with similar structures ([FL-no: 05.154 and 05.122]) without double bond substituents for which an out of domain prediction was obtained.

Except for cinnamaldehyde [FL-no: 05.014], 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 CHO cells are inconclusive (See Table 3).

2.2. Carcinogenicity Studies

Two carcinogenicity studies are available for the substances in subgroup 3.1. For trans-cinnamaldehyde, 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: 05.014]

Groups of 50 male and 50 female B6C3F1 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

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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 B6C3F1 mice. The Panel agrees with this conclusion.

2.3. 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: 05.014] 19 in vitro studies (in total 27 tests) and four in vivo studies (five different endpoints) have been evaluated. Only 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 SOS-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: 05.050, 05.040, 05.041, 05.048 and 05.051] 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: 05.048].

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 LD50. In these animals, no DNA fragmentation was observed in hepatocytes and in stomach mucosal cells. Similarly, no increase in

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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: 05.050, 05.040 and 05.041] no indication of genotoxicity in vivo was obtained.

2.4. Conclusion on Genotoxicity and Carcinogenicity

Some concern could be raised by studies carried out with cinnamaldehyde [FL-no: 05.014], showing an ability to induce chromosomal damage in vitro, and by the positive result obtained for 2-methoxycinnamaldehyde [FL-no: 05.048] 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: 05.048, 05.051, 05.118, 05.122, 05.154, 05.155 and 09.306], like the unsubstituted cinnamyl derivatives, were not of concern with respect to genotoxicity.

3. Conclusions

The present Flavouring Group Evaluation 214 (FGE.214) concerns 29 substances corresponding to subgroup 3.1 of FGE.19. Eleven of these substances are alpha,beta-unsaturated cinnamyl aldehydes [FL-no: 05.014, 05.039, 05.040, 05.041, 05.048, 05.050, 05.051, 05.118, 05.122, 05.154 and 05.155] and the 18 others are precursors for such aldehydes [FL-no: 02.017, 02.030, 06.013, 06.014, 09.018, 09.026, 09.053, 09.085, 09.090, 09.133, 09.306, 09.339, 09.459, 09.468, 09.470, 09.708, 09.739 and 09.780].

The configuration of the double bond in cinnamaldehyde [FL-no: 05.014] 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: 05.014], showing an ability to induce chromosomal damage in vitro and by the positive result obtained for 2-methoxycinnamaldehyde [FL-no: 05.048] 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: 05.048, 05.051, 05.118, 05.122, 05.154, 05.155 and 09.306], 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,beta-unsaturated cinnamaldehyde-derivatives in FGE.214 (subgroup 3.1 of FGE.19) using the Procedure.

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TABLE 1: SPECIFICATION SUMMARY OF THE SUBSTANCES IN THE FLAVOURING GROUP EVALUATION 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

Phys.form Mol.formula Mol.weight

Solubility 1) Solubility in ethanol 2)

Boiling point, °C 3) Melting point, °C ID test Assay minimum

Refrac. Index 4) Spec.gravity 5)

02.017 647

Cinnamyl alcohol 6)

OH

2294 65 104-54-1

Solid C9H10O 134.18

Insoluble to slightly soluble Moderately soluble

258 30 IR 98 %

n.a. n.a.

02.030 674

alpha-Pentylcinnamyl alcohol 6) OH

2065 79 101-85-9

Liquid C14H20O 204.31

Insoluble Miscible

141 (7 hPa) IR 95 %

1.533-1.540 0.954-0.962

05.014 656

Cinnamaldehyde 6)

O

2286 102 104-55-2

Liquid C9H8O 132.16

Insoluble Miscible

248-250 IR 98 %

1.547-1.553 1.030-1.040

05.039 684

alpha-Butylcinnamaldehyde 6) O

2191 127 7492-44-6

Liquid C13H16O 188.27

Insoluble Miscible

265 MS 98 %

1.539-1.547 0.977-0.984

05.040 685

alpha-Pentylcinnamaldehyde 6) O

2061 128 122-40-7

Liquid C14H18O 202.30

Insoluble Miscible

284-287 IR 97 %

1.554-1.562 0.962-0.969

05.041 686

alpha-Hexylcinnamaldehyde 6) O

2569 129 101-86-0

Liquid C15H20O 216.32

Insoluble Miscible

174-175 (20hPa) IR 95 %

1.547-1.553 0.950-0.961

05.048 688

2-Methoxycinnamaldehyde 6)

O

O 3181 571 1504-74-1

Solid C10H10O2 162.19

Slightly soluble Miscible

160-161(16 hPa) 45-46 IR 94 %

n.a. n.a.

05.050 683

alpha-Methylcinnamaldehyde 6) O

2697 578 101-39-3

Liquid C10H10O 146.19

Insoluble Miscible

148 (35 hPa) IR 95 %

1.598-1.607 1.034-1.040

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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

Phys.form Mol.formula Mol.weight

Solubility 1) Solubility in ethanol 2)

Boiling point, °C 3) Melting point, °C ID test Assay minimum

Refrac. Index 4) Spec.gravity 5)

05.051 689

3-(4-Methoxyphenyl)-2-methylprop-2-enal 6) O O

3182 584 65405-67-6

Liquid C11H12O2 176.21

Insoluble Miscible

106-109(0.1hPa) MS 95 %

1.625-1.632 0.989-0.996

05.118 687

4-Methoxycinnamaldehyde 6)

O

O

3567 11919 1963-36-6

Solid C10H10O2 162.18

Insoluble Moderately soluble

277 57-58 IR 96 %

n.a. n.a.

05.122 682

p-Methylcinnamaldehyde 6)

O

3640 10352 1504-75-2

Solid C10H10O 146.19

Insoluble Moderately soluble

154 (33 hPa) 41 IR 95 %

n.a. n.a.

05.154

4-Hydroxy-3,5-dimethoxycinnamaldehyde 6)

O

O

O

OH

10341 4206-58-0

Solid C11H12O4 208.21

Practically insoluble or insoluble 1 ml in 1 ml

393 108 MS 95 %

n.a. n.a.

05.155

4-Hydroxy-3-methoxycinnamaldehyde 6)

O

O

OH

10342 458-36-6

Solid C10H10O3 178.19

Slightly soluble 1 ml in 1 ml

175 (7 hPa) 82 NMR 95 %

n.a. n.a.

06.013 681

alpha-Pentylcinnamaldehyde dimethyl acetal 6)

O O

2062 47 91-87-2

Liquid C16H24O2 248.36

Slightly soluble Miscible

300 IR 97 %

1.504-1.511 0.954-0.963

06.014 648

Cinnamaldehyde ethylene glycol acetal 6)

O

O

2287 48 5660-60-6

Liquid C11H12O2 176.22

Insoluble Miscible

265 NMR 90 %

1.561-1.570 1.095-1.103

09.018 650

Cinnamyl acetate 6) O

O

2293 208 103-54-8

Liquid C11H12O2 176.22

Insoluble Miscible

262-265 IR 98 %

1.539-1.544 1.047-1.054

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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

Phys.form Mol.formula Mol.weight

Solubility 1) Solubility in ethanol 2)

Boiling point, °C 3) Melting point, °C ID test Assay minimum

Refrac. Index 4) Spec.gravity 5)

09.026 677

alpha-Pentylcinnamyl acetate 6) O

O

2064 216 7493-78-9

Liquid C16H22O2 246.35

Miscible

291 IR 97 %

1.487-1.495 0.953-0.961

09.053 652

Cinnamyl butyrate 6) O

O

2296 279 103-61-7

Liquid C13H16O2 204.27

Insoluble Miscible

300 IR 98 %

1.525-1.530 1.010-1.020

09.085 649

Cinnamyl formate 6) O O

2299 352 104-65-4

Liquid C10H10O2 162.19

Insoluble Miscible

250 IR 95 %

1.550-1.556 1.075-1.082

09.090 676

alpha-Pentylcinnamyl formate 6) O O

2066 357 7493-79-0

Liquid C15H20O2 232.32

Miscible

277 IR 85 %

1.516-1.526 0.980-0.999

09.133 651

Cinnamyl propionate 6) O

O

2301 414 103-56-0

Liquid C12H14O2 190.24

Insoluble Miscible

289 IR 98 %

1.532-1.537 1.029-1.034

09.306

2-Methoxycinnamyl acetate

O

O

O 10752

Solid C12H14O3 206.24

Practically insoluble or insoluble 1 ml in 1 ml

318 50 MS 95 %

n.a. n.a.

09.339

Cinnamyl 2-methylcrotonate 6) O

O

61792-12-9

Liquid C14H16O2 216.28

Practically insoluble or insoluble 1 ml in 1 ml

340 18 MS 95 %

1.521-1.527 0.995-1.001

09.459 654

Cinnamyl isovalerate 6) O

O2302 454 140-27-2

Liquid C14H18O2 218.30

Insoluble Miscible

313 IR 95 %

1.517-1.524 0.990-0.996

09.468 678

alpha-Pentylcinnamyl isovalerate 6) O

O

2067 463 7493-80-3

Liquid C19H28O2 288.43

Insoluble Miscible

171 (5 hPa) IR 97 %

1.498-1.508 0.939-0.950

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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

Phys.form Mol.formula Mol.weight

Solubility 1) Solubility in ethanol 2)

Boiling point, °C 3) Melting point, °C ID test Assay minimum

Refrac. Index 4) Spec.gravity 5)

09.470 653

Cinnamyl isobutyrate 6) O

O

2297 496 103-59-3

Liquid C13H16O2 204.27

Insoluble Miscible

254 IR 96 %

1.520-1.528 1.005-1.014

09.708 655

Cinnamyl phenylacetate 6)

O

O

2300 235 7492-65-1

Liquid C17H16O2 252.31

Insoluble Miscible

333-335 IR 96 %

1.575-1.581 1.089-1.095

09.739 673

Cinnamyl cinnamate 6)

O

O

2298 332 122-69-0

Solid C18H16O2 264.32

Insoluble Moderately soluble

370 42 IR 95 %

n.a. n.a.

09.780 760

Cinnamyl benzoate 6)

O

O

743 5320-75-2

Solid C16H14O2 238.29

Insoluble Miscible

335 31 IR 98 %

n.a. n.a.

1) Solubility in water, if not otherwise stated. 2) Solubility in 95% ethanol, if not otherwise stated. 3) At 1013.25 hPa, if not otherwise stated. 4) At 20°C, if not otherwise stated. 5) At 25°C, if not otherwise stated. 6) Stereoisomeric composition not specified.

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TABLE 2: SUMMARY OF SAFETY EVALUATION APPLYING THE PROCEDURE (BASED ON INTAKES CALCULATED BY THE MSDI APPROACH) (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

Class 2) Evaluation procedure path 3)

Outcome on the named compound [4) or 5)]

05.048 688

2-Methoxycinnamaldehyde

O

O 0.49 71

Class I A3: Intake below threshold

4)

05.050 683

alpha-Methylcinnamaldehyde O

2.4 390

Class I A3: Intake below threshold

4)

05.051 689

3-(4-Methoxyphenyl)-2-methylprop-2-enal O O

0.012 0.05

Class I A3: Intake below threshold

4)

05.118 687

4-Methoxycinnamaldehyde O

O 0.037 0.01

Class I A3: Intake below threshold

4)

05.122 682

p-Methylcinnamaldehyde O

0.012 0.9

Class I A3: Intake below threshold

4)

09.018 650

Cinnamyl acetate O

O

180 300

Class I A3: Intake below threshold

4)

09.053 652

Cinnamyl butyrate O

O

2.6 2

Class I A3: Intake below threshold

4)

09.085 649

Cinnamyl formate O O 1.8 17

Class I A3: Intake below threshold

4)

09.133 651

Cinnamyl propionate O

O

3.7 25

Class I A3: Intake below threshold

4)

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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

Class 2) Evaluation procedure path 3)

Outcome on the named compound [4) or 5)]

09.459 654

Cinnamyl isovalerate O

O3.9 8

Class I A3: Intake below threshold

4)

09.470 653

Cinnamyl isobutyrate O

O11 22

Class I A3: Intake below threshold

4)

09.708 655

Cinnamyl phenylacetate

O

O

0.0024 1

Class I A3: Intake below threshold

4)

09.739 673

Cinnamyl cinnamate

O

O

1.3 36

Class I A3: Intake below threshold

4)

09.780 760

Cinnamyl benzoate

O

O

ND 1

Class I A3: Intake below threshold

4)

02.017 647

Cinnamyl alcohol

OH

1500 1900

Class I A3: Intake above threshold, A4: Not endogenous, A5: Adequate NOAEL exists

4)

05.014 656

Cinnamaldehyde

O

2100 59000

Class I A3: Intake above threshold, A4: Not endogenous, A5: Adequate NOAEL exists

4)

05.154

4-Hydroxy-3,5-dimethoxycinnamaldehyde

O

O

O

OH

0.085

Not evaluated by the JECFA.

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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

Class 2) Evaluation procedure path 3)

Outcome on the named compound [4) or 5)]

05.155

4-Hydroxy-3-methoxycinnamaldehyde

O

O

OH

0.19

Not evaluated by the JECFA.

09.306

2-Methoxycinnamyl acetate

O

O

O 0.011

Not evaluated by the JECFA.

09.339

Cinnamyl 2-methylcrotonate O

O

0.012

Not evaluated by the JECFA.

02.030 674

alpha-Pentylcinnamyl alcohol OH 3.3 1

Class II A3: Intake below threshold

4)

05.039 684

alpha-Butylcinnamaldehyde O 0.012 0.07

Class II A3: Intake below threshold

4)

05.040 685

alpha-Pentylcinnamaldehyde O 22 23

Class II A3: Intake below threshold

4)

05.041 686

alpha-Hexylcinnamaldehyde O 74 11

Class II A3: Intake below threshold

4)

06.013 681

alpha-Pentylcinnamaldehyde dimethyl acetal

O O

0.012 0.007

Class II A3: Intake below threshold

4)

09.026 677

alpha-Pentylcinnamyl acetate O

O

2.4 260

Class II A3: Intake below threshold

4)

09.090 676

alpha-Pentylcinnamyl formate O O 1.2 0.5

Class II A3: Intake below threshold

4)

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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

Class 2) Evaluation procedure path 3)

Outcome on the named compound [4) or 5)]

09.468 678

alpha-Pentylcinnamyl isovalerate O

O

0.012 0.5

Class II A3: Intake below threshold

4)

06.014 648

Cinnamaldehyde ethylene glycol acetal

O

O

590 0.007

Class III A3: Intake above threshold, A4: Not endogenous, A5: Adequate NOAEL exists

4)

1) 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: Class I = 1800, Class II = 540, Class III = 90 µg/person/day. 3) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot. 4) 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.

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TABLE 3: (Q)SAR PREDICTIONS ON MUTAGENICITY IN FIVE MODELS FOR 11 ALDEHYDES FROM SUBGROUP 3.1

FL-no JECFA-no

Sub- group

EU Register name Structural formula FEMA no CoE no CAS no

ISS Local Model Ames Test TA100

MultiCASE Ames test

MultiCASE Mouse lymphoma test

MultiCASE Chromosomal aberration test in CHO

MultiCASE Chromosomal aberration test in CHL

05.014 656

3.1 Cinnamaldehyde O

2286 102 104-55-2

POS NEG POS NEG POS

05.039 684

3.1 alpha-Butylcinnamaldehyde O

2191 127 7492-44-6

NEG NEG OD OD NEG

05.040 685

3.1 alpha-Pentylcinnamaldehyde O

2061 128 122-40-7

NEG NEG OD OD NEG

05.041 686

3.1 alpha-Hexylcinnamaldehyde O

2569 129 101-86-0

NEG NEG OD OD NEG

05.048 688

3.1 2-Methoxycinnamaldehyde

O

O 3181 571 1504-74-1

NEG NEG POS POS NEG

05.050 683

3.1 alpha-Methylcinnamaldehyde O

2697 578 101-39-3

NEG NEG OD OD NEG

05.051 689

3.1 3-(4-Methoxyphenyl)-2-methylprop-2-enal O O

3182 584 65405-67-6

NEG NEG OD OD NEG

05.122 682

3.1 p-Methylcinnamaldehyde O

3640 10352 1504-75-2

NEG NEG OD NEG NEG

05.154

3.1 4-Hydroxy-3,5-dimethoxycinnamaldehyde

O

O

O

OH

- 10341 4206-58-0 NEG NEG OD EQU NEG

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FL-no JECFA-no

Sub- group

EU Register name Structural formula FEMA no CoE no CAS no

ISS Local Model Ames Test TA100

MultiCASE Ames test

MultiCASE Mouse lymphoma test

MultiCASE Chromosomal aberration test in CHO

MultiCASE Chromosomal aberration test in CHL

05.155

3.1 4-Hydroxy-3-methoxycinnamaldehyde

O

O

OH

- 10342 458-36-6 NEG NEG POS EQU NEG

05.118 687

3.1 4-Methoxycinnamaldehyde O

O 3567 11919 1963-36-6

NEG NEG POS EQU NEG

Column 2: Structure group 3.1: cinnamyl derivatives. Column 6: Local model on aldehydes and ketones, Ames TA100 (NEG: Negative; POS: Positive; OD: Out of domain). Column 7: MultiCASE Ames test (OD: Out of domain; POS: Positive; NEG: Negative; EQU: Equivocal). Column 8: MultiCASE Mouse lymphoma test (OD: Out of domain; POS: Positive; NEG: Negative; EQU: Equivocal). Column 9: MultiCASE Chromosomal aberration in CHO (OD: Out of domain; POS: Positive; NEG: Negative; EQU: Equivocal). Column 10: MultiCASE Chromosomal aberration in CHL (OD: Out of domain; POS: Positive; NEG: Negative; EQU: Equivocal).

OD, 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.

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Table 4: Carcinogenicity Studies

Table 4: Carcinogenicity Studies Chemical Name [FL-no] Species; Sex

No./Group Route Dose levels Duration Results Reference Commentsa

Trans-cinnamaldehyde Rat; M, F 50

Oral 0, 50, 100 or 200 mg/kg bw/day

2 years There were no neoplasms or non-neoplastic lesions that were attributed to exposure to trans-cinnamaldehyde

(NTP, 2004l) Valid

Mice; M, F 50

Oral 0, 125, 270 or 550 mg/kg bw/day

2 years There were no neoplasms that were attributed to exposure to trans-cinnamaldehyde

(NTP, 2004l) Valid

a: Validity of carcinogenicity studies: Valid. Limited validity (e.g. if certain aspects are not in accordance with OECD 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. OECD) or current standards and/or inappropriate test system). Validity cannot be evaluated (e.g. insufficient documentation, short abstract only, too little experimental details provided).

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TABLE 5: GENOTOXICITY (IN VITRO)

Table 5: GENOTOXICITY (in vitro) Chemical Name [FL-no] Test System Test Object Concentration Reported Result Reference Comments e

Cinnamaldehyde [05.014] Reverse mutationc S. typhimurium TA1537, TA1538, TA98,

TA100, TA1535 600 µg/plate Negativea (Sekizawa &

Shibamoto, 1982)

Valid. Published non-GLP study with sufficient details; the result is considered valid.

Reverse mutation S. typhimurium TA1537, TA98, TA100, TA1535

10 mg/plate (10,000 µg/plate)

Negativea (Prival et al., 1982)

Validity cannot be evaluated. Published non-GLP study with insufficient documentation (no figures); the validity cannot be evaluated.

Reverse mutation S. typhimurium TA104 (with preincubation)

0.8 µmol (105 µg)

Negativea (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.

Reverse mutation S. typhimurium TA98, TA100 Up to 0.5 mg/plate (500 µg/plate)

Positivea,d (Ishidate et al., 1984)

Valid. According to current guidelines (in TA100 with and without metabolic activation)a,b

Reverse mutation S. typhimurium TA1537, TA92, TA94, TA98, TA100, TA1535 (with plate incorporation and preincubation)

500 µg/plate Negativea (Lijinsky & Andrews, 1980)

Valid.

Reverse mutation S. typhimurium TA98, TA100 (with plate incorporation and preincubation)

0.05 - 500 µg/plate Negativea (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)

Negativea (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 Negativea (Kato et al., 1989)

Validity cannot be evaluated. Abstract – limited data reported.

Reverse mutation S. typhimurium TA1537, TA98, TA100, TA1535 (with preincubation)

100 µg/plate Negativea (Mortelmans et al., 1986)

Valid.

Reverse mutation S. typhimurium TA100 (with preincubation)

5 µmol/plate (661 µg/plate)

Negativea (Neudecker et al., 1983)

Limited validity. Only in one strain.

Mutation E. coli WP2 uvrA 0.8 mg/plate (800 µg/plate)

Negativeb (Yoo, 1986)

Validity cannot be evaluated. Only in Japanese (insufficient documentation).

DNA repair B. subtilis M45 (rec-) 0.2 mg/disk (200 µg/disc)

Positiveb (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)

Positiveb (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)

Positivea (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 Negativeb (Oda et al., 1979)

Validity cannot be evaluated (relevance for evaluation of genotoxicity uncertain).

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Table 5: GENOTOXICITY (in vitro) Chemical Name [FL-no] Test System Test Object Concentration Reported Result Reference Comments e

Sister chromatid exchange

Chinese hamster ovary cells 33.3 µmol/L (4401 µg)

Negativeb (Sasaki et al., 1987)

Validity cannot be evaluated (relevance for evaluation of genotoxicity uncertain).

Chromosomal aberration

Chinese hamster fibroblasts Up to 0.015 mg/ml (15 µg/ml)

Positiveb (Ishidate et al., 1984)

Valid.

Cinnamaldehyde [05.014] (cont.) Chromosomal aberration

Chinese hamster B241 cells 20 nmol/L (2.6 µg)

Positiveb (Kasamaki & Urasawa, 1985)

Valid (unusual cell line).

Chromosomal aberration

Chinese hamster B241 cells 10 nmol/L (1.3 µg)

Positivea (Kasamaki et al., 1982)

Limited validity (limited documentation; results for only one test concentration reported; long incubation period of 24 hrs; unusual cell line).

Chromosomal aberration

Chinese hamster ovary cells 18.3 µg/ml 100 µg/ml

Negativeb Negativec

(Galloway et al., 1987)

Valid.

Sister chromatid exchange

Chinese hamster ovary cells

Up to 6.8 µg/ml Positiveb (Galloway et al., 1987)

Valid. Weakly positive without S9.

Sister chromatid exchange

Chinese hamster ovary cells

91.8 µg/ml Positivec (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 (16.5 mg/ml)

Positiveb (Eder et al., 1993)

Limited validity. Positive at cytotoxic levels.

SOS chromo ? ? Negative (Eder et al., 1993)

Limited validity. Results poorly reported – relevance questionable.

Mutation (HGPRT) Chinese hamster V79 cells 100 µmol/L (13 216 µg)

Negativeb (Fiorio & Bronzetti, 1994)

Insufficient validity (only one concentration and only without S9 tested).

Micronucleus formation

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.

alpha-Methylcinnamaldehyde [05.050] Reverse mutation S. typhimurium TA100(with preincubation)

4 µmol/plate (585 µg/plate)

Negativea (Neudecker et al., 1983)

Limited validity (only one strain tested).

Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 (with preincubation)

500 µg/plate Negativea (Mortelmans et al., 1986)

Valid.

Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

5 concentrations up to cytotoxicity, or max. 3600 µg/plate

Negativea (Wild et al., 1983)

Valid.

Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

5 concentrations up to cytotoxicity, or max. 3600 µg/plate

Negativea (Wild et al., 1983)

Valid.

alpha-Pentylcinnamaldehyde [05.040] Reverse mutation S. typhimurium TA97, TA102 (with preincubation)

1.0 mg/plate (1000 µg/plate)

Negativea (Fujita & Sasaki, 1987)

Validity cannot be evaluated.

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Table 5: GENOTOXICITY (in vitro) Chemical Name [FL-no] Test System Test Object Concentration Reported Result Reference Comments e

alpha-Hexylcinnamaldehyde [05.041] Reverse mutation S. typhimurium TA98, TA100, TA1535,

TA1537, TA1538 5 concentrations up to cytotoxicity, or max. 3600 µg/plate

Negativea (Wild et al., 1983)

Valid.

2-Methoxycinnamaldehyde [05.048] Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 (with preincubation)

666 µg/plate Positivea (Mortelmans et al., 1986)

Valid.

3-(4-Methoxyphenyl)-2-methylprop-2-enal [05.051]

Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

5 concentrations up to cytotoxicity, or max. 3600 µg/plate

Negativea (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 OECD 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. OECD) or current standards and/or inappropriate test system). Validity cannot be evaluated (e.g. insufficient documentation, short abstract only, too little experimental details provided).

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TABLE 6: GENOTOXICITY (IN VIVO)

Table 6: GENOTOXICITY (in vivo) Chemical Name [FL-no] Test System Test Object Route Dose Result Reference Comments aCinnamaldehyde [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 Oral (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 500 000 µg/kg bw Negative (Hayashi et al., 1984)

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 0-2 550 000 µg/kg bw Positive (Mereto et al., 1994)

Valid. After 2/3 hepatectomy. The highest dose cause 100% mortality. Relevance not clear.

Micronucleus formation Rat hepatocytes Gavage 0-1 650 000 µg/kg bw Positive (Mereto et al., 1994)

Valid. After 2/3 hepatectomy. The highest dose cause 100% mortality. Relevance not clear.

Micronucleus formation Mouse bone marrow Gavage 0-2 550 000 µg/kg bw Negative (Mereto et al., 1994)

Limited validity. PCE/NCE ratios were not affected, at the highest dose tested 100% lethality.

Micronucleus formation Rat bone marrow Gavage 0-1 650 000 µg/kg bw Negative (Mereto et al., 1994)

Limited validity. PCE/NCE ratios were not affected , at the highest dose tested 100 % lethality.

Nuclear anomalies Mouse forestomach mucosal cells

Gavage 0-2 550 000 µg/kg bw Negative (Mereto et al., 1994)

Validity cannot be evaluated (meaning of endpoint for genotoxicity is unclear, at the highest dose tested 100 % lethality).

Nuclear anomalies Rat forestomach mucosal cells

Gavage 0 - 1 650 000 µg/kg bw Positive (Mereto et al., 1994)

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.

DNA fragmentation Rat hepatocytes and gastric mucosal cells

Gavage 1 100 000 µg/kg bw Negative (Mereto et al., 1994)

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.

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alpha-Pentylcinnamaldehyde [05.040] 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).

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).

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 OECD 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. OECD) or current standards and/or inappropriate test system). Validity cannot be evaluated (e.g. insufficient documentation, short abstract only, too little experimental details provided).

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ABBREVIATIONS

CAS Chemical Abstract Service

CHL Chinese hamster lung cell(s)

CHO Chinese hamster ovary cell(s)

CoE Council of Europe

DNA Deoxyribonucleic acid

DTU-NFI Danish Technical University – National Food Institute

EC European Commission

EFSA The European Food Safety Authority

EU European Union

FAO Food and Agriculture Organization of the United Nations

FEMA Flavor and Extract Manufacturers Association

FGE Flavouring Group Evaluation

FLAVIS Flavour Information System database

GLP Good Laboratory Practise

ID Identity

IR Infrared spectroscopy

ISS Istituto Superiore di Sanita

JECFA The Joint FAO/WHO Expert Committee on Food Additives

LD50 Lethal dose, 50 %, median lethal dose

MS Mass spectrometry

MSDI Maximum Survey-derived Daily Intake

NMR Nuclear magnetic resonance

No number

NOAEL No observed adverse effect level

NTP National Toxicology Programme

OECD Organisation for Economic Co-operation and Development

PCE/NCE Polychromatic erythrocytes/normochromatic erythocytes

(Q)SAR (Quantitative) structure-activity relationship

SCE Sister chromatid exchange

SCF Scientific Committee on Food

WHO World Health Organisation