Scientific Opinion on the safety and efficacy of allylhydroxybenzenes (chemical group 18) when used as flavourings for all animal species 1

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1 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of allylhydroxybenzenes (chemical group 18) when used as flavourings for all animal species 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Eugenol, eugenyl acetate, 1-methoxy-4-(prop-1(trans)-enyl)benzene (trans-anethole) and 4-allyl-2,6- dimethoxyphenol are flavouring substances belonging to chemical group 18, defined as allylhydroxybenzenes. All four additives are currently authorised for use as flavours in food and are naturally found in various herbs and plants. The use of eugenol and eugenyl acetate in fish is contra-indicated. The range of use levels 5 25 mg/kg feed for eugenol and trans-anethole proposed by the applicant is safe for all animal species (other than fish), with a low margin of safety (2 to 6). For 4-allyl-2,6-dimethoxyphenol and eugenyl acetate, the proposed range of use levels 1 5 mg/kg feed is safe for all animal species (other than fish), with a higher margin of safety (10 to 30). The use of eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol as flavours in mammals up to the highest use levels proposed in feed is safe for the consumer. However, the lack of data on metabolism and residues in poultry precludes an assessment of consumer exposure from this source. No data on the safety for the user was provided. The applicant indicated possible effects of eugenol on the respiratory system. Eugenol and 4-allyl-2,6-dimethoxyphenol are also described as irritating to eyes and skin, and eugenyl acetate as a skin irritant. Both eugenol and trans-anethole may cause skin sensitisation. The impact on the environment from the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol in animal feed is expected to be low. Since eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is considered necessary. European Food Safety Authority, 2011 KEY WORDS Sensory additives, flavourings, allylhydroxybenzenes, chemical group 18, eugenol, eugenyl acetate, 1-methoxy- 4-(prop-1(trans)-enyl)benzene, trans-anethole, 4-allyl-2,6-dimethoxyphenol 1 On request from the European Commission, Question No EFSA-Q , adopted on 15 November Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Secundino López Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 The Panel wishes to thank the members of the Working Group on Feed Flavourings, including Paul Brantom, Joaquim Brufau, Gérard Pascal and Guido Rychen, for the preparatory work on this scientific opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on the safety and efficacy of allylhydroxybenzenes (chemical group 18) when used as flavourings for all animal species.. [14 pp.]. doi: /j.efsa Available online: European Food Safety Authority, 2011

2 SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of eugenol, eugenyl acetate, 1-methoxy-4-(prop-1(trans)-enyl)benzene (trans-anethole), and 4-allyl-2,6-dimethoxyphenol. All four additives, belonging to chemical group 18, are currently authorised for use as flavours in food, and are naturally found in various herbs and plants. Chemical formulations containing eugenol are used in water as an anaesthetic for fish and the FEEDAP Panel concludes that its use as a flavour in feed for fish is contra-indicated. Given the chemical similarity of the four compounds under assessment, comparable effects cannot be excluded. A calculation based on the lowest NOAEL for each compound showed that the range of use levels 5 25 mg/kg feed for eugenol and trans-anethole proposed by the applicant is safe for all animal species (other than fish), with a low margin of safety (2 to 6). For 4-allyl-2,6-dimethoxyphenol and eugenyl acetate, the proposed range of use levels 1 5 mg/kg feed is safe for all animal species (other than fish), with a higher margin of safety (10 to 30). The FEEDAP Panel considers that the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6- dimethoxyphenol as flavours in mammals up to the highest use levels proposed in feed is safe for the consumer. However, the lack of data on metabolism and residues in poultry precludes an assessment of consumer exposure from this source. No data on the safety for the user was provided. The applicant indicated possible effects of eugenol on the respiratory system. Eugenol and 4-allyl-2,6-dimethoxyphenol are also described as irritating to eyes and skin, and eugenyl acetate as a skin irritant. Both eugenol and trans-anethole may cause skin sensitisation. The impact on the environment from the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl- 2,6-dimethoxyphenol in animal feed is expected to be low. Since eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is considered necessary. 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Characterisation of the flavouring additives Stability and homogeneity Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 7 3. Safety Safety for the target species Conclusions on the safety for the target species Safety for the consumer Safety for the user Safety for the environment Efficacy Conclusions and recommendations Documentation provided to EFSA References Appendix

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7; in addition, Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without a time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from the Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG) 5 for authorisation of the substances eugenol, eugenyl acetate, 1-methoxy-4-(prop-1(trans)-enyl)benzene and 4-allyl-2,6-dimethoxyphenol, to be used as feed additives for all animal species (category: sensory additives; functional group: flavourings) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive) and under Article 10(2) (re-evaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 6 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 9 June The additives are listed as food and feed flavourings in the register of Flavouring substances (CD 217/1999) 7 and in the European Union Register of Feed Additives (Regulation (EC) No 1831/2003). They have not been previously assessed by EFSA for this purpose. The four substances of chemical group (CG) 18 have been previously assessed by JECFA (2000, 2001 and 2006a) and EFSA Scientific Panel on Food Contact Material, Enzymes, Flavourings and Processing Aids (CEF) (EFSA, 2008, 2009) as food flavourings and some of them (eugenol and eugenyl acetate) by the Council of Europe (CoE, 1992). TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animal(s), consumer, user and the environment and the efficacy of the active substances eugenol, eugenyl acetate, 1-methoxy-4-(prop-1(trans)-enyl)benzene and 4-allyl-2,6- dimethoxyphenol, when used under the conditions described in Table OJ L 268, , p. 29. Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG), Avenue Louise 130A, B-1050, Brussels, Belgium. EFSA Dossier reference: FAD OJ L 84, , p. 1. 4

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category(ies) of additive Functional group(s) of additive Chemically defined flavourings from Chemical Group 18 Allylhydroxbenzes: Eugenol 1-Methoxy-4- (prop-1(trans)-enyl)benzene 4-Allyl-2,6-dimethoxyphenol Eugenyl acetate - 2. Sensory additives b) flavouring compounds Eugenol Composition, description (CAS No ) 1-Methoxy-4- (prop-1(trans)- enyl)benzene (CAS No ) 4-Allyl-2,6-dimethoxyphenol (CAS No ) Eugenyl acetate (CAS No ) Chemical formula Description Purity criteria (if appropriate) Method of analysis (if appropriate) C 10 H 12 O 2 98% Gas Chromatography Mass Spectrometry (GC-MS) C 10 H 12 O 99% Gas Chromatography Mass Spectrometry (GC-MS) C 11 H 14 O 3 98% Gas Chromatography Mass Spectrometry (GC-MS) C 12 H 14 O 3 98% Gas Chromatography Mass Spectrometry (GC-MS) Trade name (if appropriate) - Name of the holder of authorisation (if appropriate) - Species or category of animal All species and categories Maximum Age Conditions of use Minimum content Maximum content mg or Units of activity or CFU/kg of complete feedingstuffs (select what applicable) Withdrawal period (if appropriate) Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) Marker residue Other provisions and additional requirements for the labelling - All feedingstuffs and water for drinking - - Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues

6 ASSESSMENT 1. Introduction The chemical group (CG) 18 for flavouring substances is defined in Commission Regulation (EC) No 1565/2000 as allylhydroxybenzenes. The present application concerns four compounds, eugenol, eugenyl acetate, 1-methoxy-4-(prop-1(trans)-enyl)benzene (also known as trans-anethole), and 4-allyl- 2,6-dimethoxyphenol, which can be assigned to this CG. All four are naturally found in various herbs and plants. The four compounds have been assessed by JECFA (2000, 2001 and 2006a) and were considered safe for use in food. Acceptable Daily Intake (ADI) values have been set for two of the four compounds (eugenol and trans-anethole). The EFSA Scientific Panel on Food Contact Material, Enzymes, Flavourings and Processing Aids (CEF) (EFSA, 2008, 2009) considered the same compounds for use as food flavourings and reached similar conclusions. It did not express an opinion on trans-anethole because it was not included in the evaluation programme (Regulation (EC) No 1565/2000). 8 They are currently listed in the European Union database of flavouring substances and as such authorised for use in food. A consortium of companies (FFAC) supplying flavours to the feed industry has requested authorisation for the use of the four compounds as additives to feed and water for drinking (category: sensory additives; functional group: flavouring compounds) for use in all animal species. Regulation (EC) No 429/ allows substances already approved for use in human food to be assessed with a more limited procedure than for other feed additives. However, the use of this procedure is always subject to the condition that the food safety assessment is relevant to the use in feed. 2. Characterisation 2.1. Characterisation of the flavouring additives The physico-chemical characteristics of the additives under application are summarised in Table 2. Table 2: Chemically defined flavourings from CG 18 under application Flavouring Eugenol Eugenyl acetate trans-anethol 4-Allyl-2,6- dimethoxyphenol CAS No FLAVIS No Structural formula Molecular formula C 10 H 12 O 2 C 12 H 14 O 3 C 10 H 12 O C 11 H 14 O 3 Molecular weight Physical status Liquid Solid Liquid Liquid Boiling point ( C) pk a Log K ow Water solubility Slightly soluble Insoluble Slightly soluble Insoluble 8 OJ L 180, , p OJ L 133, , p. 1. 6

7 Eugenol is typically extracted from cloves or clove oil and the acetate formed by acetylation with acetic anhydride. The compound trans-anethole may be produced by chemical synthesis by the esterification of p-cresol with methanol and subsequent condensation or by direct extraction from pine oil. 4-Allyl-2,6-dimethoxyphenol is produced by reacting 4-hydroxy-2,6-dimethoxybenzoic acid with allylbromide. Data was provided on the batch to batch variation in five batches of each additive (with the exception of 4-allyl-2,6-dimethoxyphenol, where only three batches were available because of low usage). 10 The content of the active substance exceeded the JECFA specifications (JECFA, 2006b) for eugenol, eugenyl acetate and trans-anethole (Table 3). The fourth compound failed to meet the JECFA specification of > 98 % but analysis showed that 2.2 % (range %) was 2,6-dimethoxyphenol derived from the synthetic precursor. Table 3: Identification of the substances and data on purity EU Register name JECFA specification % Assay % Average Range Eugenol > Eugenyl acetate > trans-anethole > Allyl-2,6-dimethoxyphenol > Potential contaminants are considered as part of the product specification and are monitored as part of the HACCP procedure required of all consortium members. The parameters considered include residual solvents, heavy metals and other undesirable substances Stability and homogeneity A shelf life of at least 24 months is given for all four compounds when stored in closed containers under recommended conditions (in a cool, dry place, away from heat). This assessment is made on the basis of compliance with the original specification after storage. Although no data is required for the stability of volatile additives in premixtures and feed, the use in water for drinking introduces other issues relating to the product stability, such as degradation due to microbial activity. As no data on the short-term stability of the additive in water for drinking were provided, the FEEDAP Panel is not in the position to evaluate this route of delivery Conditions of use The applicant proposes the use of the four additives in feed or water for drinking for all animal species and without withdrawal. The applicant proposes a normal use level of 5 mg/kg and a high use level of 25 mg/kg complete feed for eugenol and trans-anethole, and a normal use level of 1 mg/kg and a high use level of 5 mg/kg complete feed for 4-allyl-2,6-dimethoxyphenol and eugenyl acetate. No specific proposals are made for the dose used in water for drinking Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) EFSA has verified the EURL report as it relates to the methods used for the control of eugenol, 1- methoxy-4-(prop-1(trans)-enyl)benzene, 4-allyl-2,6-dimethoxyphenol and eugenyl acetate in animal feed. The Executive Summary of the EURL report is given in the Appendix. 10 Technical dossier/section II/Annex 2.1 and Supplementary information June

8 3. Safety The assessment of safety is based on the high use level proposed by the applicant (25 mg/kg complete feed for eugenol and trans-anethole and 5 mg/kg complete feed for 4-allyl-2,6-dimethoxyphenol and eugenyl acetate) Safety for the target species Chemical formulations containing eugenol are used in water as an anaesthetic for fish. In the absence of data on the correspondence between the exposure of fish via feed and aquatic exposure, the FEEDAP Panel considers it prudent to avoid the use of eugenol (and eugenyl acetate) as a flavouring agent in feed for fish. It cannot be excluded that the chemically-related compounds trans-anethole and 4-allyl-2,6-dimethoxyphenol demonstrate the same properties. The first approach to the safety assessment for target species takes account of the applied use levels in animal feed relative to the maximum reported exposure of humans on the basis of the metabolic body weight. The data for human exposure are 950, 19 and µg/person/day for eugenol, eugenyl acetate and 4-allyl-2,6-dimethoxyphenol, respectively. No human exposure data is available for transanethole. Table 4 summarises the result of the comparison with human exposure for selected target animals. The body weight of target animals is taken from the default values shown in Table 5. Table 4: Comparison of exposure of humans and target animals to the flavourings under application Flavouring Use level in feed (mg/kg) Human exposure µg/mbw (kg 075 )/day* Target animal exposure µg/mbw (kg 075 )/day Turkey Piglet Dairy cow Eugenol Eugenyl acetate trans-anethole Allyl-2,6-dimethoxyphenol * mbw: metabolic body weight (kg 0.75 ) for a 60 kg person = The data in Table 4 clearly indicate that the intake by the target animals exceeds that of humans resulting from the use in food. As a consequence, the safety for the target species at the feed concentration applied cannot be derived from the risk assessment for food use. As an alternative, the maximum feed concentration which can be considered as safe for the target animal can be derived from the lowest NOAEL available. A NOAEL of 300 mg/kg bw/day is available for both eugenol (JECFA, 2006a; EFSA, 2009) and trans-anethole (JECFA, 2000). The NOAEL for eugenol is derived from a 103-week study in rats (NTP, 1983) based on effects on body weight in females; that for trans-anethole, from a 90-day study in rat (Minnema, 1997, unpublished report) 11 based on elevated serum γ-glutamyl transferases. This NOAEL can be applied to eugenyl acetate. The FEEDAP Panel considers that in the absence of specific data, the same NOAEL can be used by default for 4-allyl-2,6-dimethoxyphenol. No more recent toxicological studies have been published that would challenge these conclusions. Consequently, the maximum safe intake of eugenol and other flavourings belonging to CG 18 is 3 mg/kg bw/day. Table 6 shows the maximum safe feed concentrations of eugenol for selected target species. 11 Technical dossier/supplementary information June

9 Table 5: Maximum safe feed concentrations in selected target species for the chemically defined flavourings from CG 18 under application Target animal Default settings Maximum safe intake/feed concentration BW (kg) FI (g/d) Intake (mg/d) mg/kg feed Veal calves (milk replacer) Cattle for fattening Pigs for fattening Sows Dairy Cows Turkeys for fattening Piglets Chickens for fattening Laying hens BW: body weight; FI: feed intake Conclusions on the safety for the target species The use of eugenol and eugenyl acetate in fish is contra-indicated. The FEEDAP Panel considers that the proposed range of use levels 5 25 mg eugenol/kg feed is safe for all animal species except fish, with a margin of safety in the range of 2 to 6. The same conclusion applies to trans-anethole. For 4-allyl-2,6-dimethoxyphenol and eugenyl acetate, the proposed range of use levels 1 5 mg/kg feed is safe for all animal species except fish with a higher margin of safety (10 to 30) Safety for the consumer The safety for the consumer of all four compounds in CG 18 used as food flavours has been already assessed by JECFA (2000, 2001 and 2006a), and all but trans-anethole by EFSA (2008 and 2009). All four compounds are presently authorised as food additives without limitations. As the intake of eugenol, eugenyl acetate and 4-allyl-2,6-dimethoxyphenol by target animals substantially exceeds that of humans resulting from use in food, the potential transfer of significant amounts of residues in edible tissues and products has to be considered, particularly for the two compounds with an ADI (eugenol 2.5 mg/kg bw and trans-anethole 2.0 mg/kg bw). Eugenol, eugenyl acetate and 4-allyl-2,6-dimethoxyphenol are rapidly absorbed in the gastrointestinal tract and undergo phase II conjugation in the liver in humans, pigs and rodents (JECFA, 2006a). The resulting glucuronide and sulfate conjugates are subsequently excreted in the urine. To a lesser extent, these compounds are metabolised to polar products, which are also conjugated and eliminated, primarily in the urine. Only small amounts (< 1 %) of eugenol are excreted unchanged. Eugenyl acetate is hydrolysed to eugenol and the corresponding carboxylic acid. The presence of a small amount of 2,6-dimethoxyphenol in 4-allyl-2,6-dimethoxyphenol does not introduce additional concerns. trans-anethole is mainly metabolised via O-demethylation and epoxidation of the side chain, followed by formation of diols in rodents and humans (JECFA, 2000). Some metabolites of trans-anethole have given rise to safety concern. The epoxide has been shown to be cytotoxic and hepatotoxic and genotoxic in some studies. However, after reviewing the available data, JECFA finally concluded in 2000 that trans-anethole was unlikely to be genotoxic in vivo and set an ADI of 2 mg/kg bw. Metabolic pathways of the four compounds are common to mammalian species but no data have been reported concerning the metabolic fate of the four substances in poultry. The efficient metabolism of the compounds in mammals and the subsequent rapid excretion of the metabolites preclude their accumulation in tissues and transfer to products. The use of the four compounds in mammals would not measurably increase consumer exposure and would not significantly contribute to the ADI set for 9

10 eugenol and trans-anethole. Consequently, the FEEDAP Panel considers that the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol as flavours in mammals up to the highest use levels proposed in feed is safe for the consumer. The lack of data on metabolism and residues in poultry precludes an assessment of consumer exposure from this source Safety for the user No data on the safety for the user was provided. The applicant indicated possible effects of eugenol on the respiratory system and proposed its classification as irritating to the respiratory system and potential sensitiser by inhalation. No respiratory effects were described in the Material Safety Data Sheets provided by the applicant for trans-anethole, 4-allyl-2,6-dimethoxyphenol or eugenyl acetate. Eugenol and 4-allyl-2,6-dimethoxyphenol are classified as irritating to eyes and skin, whereas eugenyl acetate is classified as irritating to skin only. Both eugenol and trans-anethole may cause skin sensitisation Safety for the environment Allylhydroxybenzenes are known to have a broad spectrum of activity against insects, mites, fungi, and nematodes (Isman, 2000). The acute toxicity available for aquatic species showed that L(E)C50 values are in the range 5 30 mg/l. As indicated in Section 3.3, all four compounds are significantly metabolised in the mammalian target species and only a limited percentage is excreted as the parent compound, although in potency the excreted glucuronide and sulfate conjugates of eugenol, eugenyl acetate and 4-allyl-2,6- dimethoxyphenol could be transformed back to the parent compound. Several studies indicated that this group of compounds are readily biodegradable and have a short half-life under field conditions (Rabenhorst, 1996; Environment Canada, 2010). The estimated bioconcentration factors for these compounds are all < 200 and therefore do not raise concern for bioaccumulation and secondary poisoning. In the absence of terrestrial toxicity studies relevant to assess exposure via spreading of manure, it is difficult to determine the exact impact on the environment. Though in view of the metabolism in the target species and the fast biodegradation, the impact on the environment from the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol in animal feed is expected to be low. 4. Efficacy Since eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS The use of eugenol and eugenyl acetate in fish is contra-indicated. The FEEDAP Panel concluded that the range of use levels 5 25 mg/kg feed for eugenol and transanethole proposed by the applicant is safe for all animal species with a low margin of safety (2 to 6). For 4-allyl-2,6-dimethoxyphenol and eugenyl acetate), the proposed range of use levels 1 5 mg/kg feed is safe for all animal species, with a higher margin of safety (10 to 30). The FEEDAP Panel considered that the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl- 2,6-dimethoxyphenol as flavours in mammals up to the highest use levels proposed in feed is safe for the consumer. However, the lack of data on metabolism and residues in poultry precluded an assessment of consumer exposure from this source. 10

11 No data on the safety for the user was provided. The applicant indicated possible effects of eugenol on the respiratory system. Eugenol and 4-allyl-2,6-dimethoxyphenol are also indicated as irritating to eyes and skin, and eugenyl acetate as a skin irritant. Both eugenol and trans-anethole may cause skin sensitisation. The impact on the environment from the use of eugenol, eugenyl acetate, trans-anethole and 4-allyl- 2,6-dimethoxyphenol in animal feed is expected to be low. Since eugenol, eugenyl acetate, trans-anethole and 4-allyl-2,6-dimethoxyphenol are used in food as flavourings, and their function in feed is essentially the same as that in food, no further demonstration of efficacy was considered necessary. RECOMMENDATIONS Given the chemical similarity of eugenol with trans-anethole and 4-allyl-2,6-dimethoxyphenol, the FEEDAP Panel recommends that the use of trans-anethole and 4-allyl-2,6-dimethoxyphenol should be considered contra-indicated in fish. DOCUMENTATION PROVIDED TO EFSA 1. Chemically defined flavourings from Flavouring Group 18 - Allylhydroxybenzenes for all animal species and categories. April Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 2. Chemically defined flavourings from Flavouring Group 18 - Allylhydroxybenzenes for all animal species and categories. Supplementary information. June Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 3. Chemically defined flavourings from Flavouring Group 18 - Allylhydroxybenzenes for all animal species and categories. Supplementary information. August Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 4. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for Eugenol, 1-Methoxy-4-(prop-1(trans)-enyl)benzene, 4-Allyl-2,6- dimethoxyphenol and Eugenyl acetate. 5. Comments from Member States received through the ScienceNet. REFERENCES CoE (Council of Europe), Flavouring substances and natural sources of flavourings. 4th Ed. vol. I. Chemically defined flavouring substances. Council of Europe, partial agreement in the social and public health field. Strasbourg. EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (CEF), Scientific Opinion on the Flavouring Group Evaluation 58 (FGE.58). Consideration of phenol derivatives evaluated by JECFA (55 th meeting) structurally related to ring substituted phenolic substances evaluated by EFSA in FGE.22 (2006) (Commission Regulation (EC) No 1565/2000 of 18 July 2000). The EFSA Journal, 711, EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (CEF), Scientific Opinion on Flavouring Group Evaluation 60 (FGE.60). Consideration of eugenol and related hydroxyallylbenzene derivatives evaluated by JECFA (65 th meeting) structurally related to ring-substituted phenolic substances evaluated by EFSA in FGE.22 (2006). The EFSA Journal, 965, Environment Canada, 2010, online. Screening Assessment for the Challenge. Benzene, 1,2- dimethoxy-4-(2-propenyl)- (Methyl eugenol). Available from 11

12 Isman MB, Plant essential oils for pest and disease management. Crop Protection, 19, JECFA (Joint FAO/WHO Expert Committee on Food Additives), Evaluation of certain food additives. Fifty-first meeting of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no Geneva, 9-18 June JECFA (Joint FAO/WHO Expert Committee on Food Additives), Evaluation of certain food additives and contaminants. Fifty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no Geneva, 6-15 June JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2006a. Evaluation of certain food additives. Sixty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no Geneva, 7-16 June JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2006b. Combined Compendium of food additive specifications - Joint FAO/WHO Expert Committee on Food Additives - All specifications monographs from the 1st to the 65th meeting ( ). NTP (National Toxicology Program), 1983, online. Toxicology and carcinogenesis studies of eugenol (CAS no ) in F344/N rats and B6C3F1 mice (feed studies). NTP-TR 223. Available from Rabenhorst J, Production of methoxyphenol type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp. Applied Microbiology and Biotechnology, 46,

13 APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Eugenol, 1-Methoxy-4-(prop-1(trans)- enyl)benzene, 4-Allyl-2,6-dimethoxyphenol and Eugenyl acetate 12 The Chemically Defined Flavourings - Group 18 - CDG18, allylhydroxybenzenes - in this application comprises four substances (Eugenol, 1-Methoxy-4-(prop-1(trans)-enyl)benzene, 4-Allyl-2,6- dimethoxyphenol and Eugenyl acetate) for which authorisation as feed additives is sought under the category "sensory additives", functional group 2(b) "flavouring compounds", according to the classification system of Annex I of Regulation (EC) No 1831/2003. In the current application submitted according to Article 4(1) and Article 10 (2) of Regulation (EC) No 1831/2003, the authorisation for all species and categories is requested. The flavouring compounds of interest have a purity of 98 to 99%. Mixtures of flavouring compounds are intended to be incorporated only into feedingstuffs or drinking water. The Applicant suggested no minimum or maximum levels for the different flavouring compounds in feedingstuffs. For the identification of volatile chemically defined flavouring compounds CDG18 in the feed additive, the Applicant submitted a qualitative multi-analyte gas-chromatography mass-spectrometry (GC-MS) method, using Retention Time Locking (RTL), which allows a close match of retention times on GC-MS. By making an adjustment to the inlet pressure, the retention times can be closely matched to those of a reference chromatogram. It is then possible to screen samples for the presence of target compounds using a mass spectral database of RTL spectra. The Applicant maintained two FLAVOR2 databases/libraries (for retention times and for MS spectra) containing data for more than 409 flavouring compounds. These libraries were provided to the EURL. The Applicant provided the typical chromatogram for the CDG18 of interest. In order to demonstrate the transferability of the proposed analytical method (relevant for the method verification), the Applicant prepared a model mixture of flavouring compounds on a solid carrier to be identified by two independent expert laboratories. This mixture contained twenty chemically defined flavourings belonging to twenty different chemical groups to represent the whole spectrum of compounds in use as feed flavourings with respect to their volatility and polarity. Both laboratories properly identified all the flavouring compounds in all the formulations. Since the substances of CDG18 are within the volatility and polarity range of the model mixture tested, the Applicant concluded that the proposed analytical method is suitable to determine qualitatively the presence of the substances from CDG18 in the mixture of flavouring compounds. Based on the satisfactory experimental evidence provided, the EURL recommends for official control for the qualitative identification in the feed additive of the individual (or mixture of) flavouring compounds of interest (Eugenol, 1-Methoxy-4-(prop-1(trans)-enyl)benzene, 4-Allyl-2,6- dimethoxyphenol and Eugenyl acetate) the GC-MS-RTL (Agilent specific) method submitted by the Applicant. As no experimental data were provided by the Applicant for the identification of the active substance(s) in feedingstuffs and water, no methods could be evaluated. Therefore the EURL is unable to recommend a method for the official control to identify the active substance(s) of interest in feedingstuffs or water. 12 The full report is available on the EURL website pdf 13

14 Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary. 14