Scientific Opinion on the safety and efficacy of propenylhydroxybenzenes (chemical group 17) when used as flavourings for all animal species 1

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1 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of propenylhydroxybenzenes (chemical group 17) 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 Isoeugenol is a flavouring compound belonging to chemical group 17, defined as propenylhydroxybenzenes. The additive is currently authorised for use as flavour in food and is naturally found in various herbs and plants. The use of isoeugenol as a flavour in feed for fish and other aquatic species is contra-indicated. The range of use levels of 1 to 5 mg/kg feed proposed by the applicant for isoeugenol is safe for all animal species (except fish) with a margin of safety in the range of 1 to 4. Considering the low margin of safety, the simultaneous administration of isoeugenol in feed and water for drinking for poultry should be avoided. The use of isoeugenol as flavour in non-dairy 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 all other species and categories precludes an assessment of consumer exposure from these sources. No data on the safety for the user was provided. Based on the information available in the literature, the FEEDAP Panel considered isoeugenol as an irritant to the respiratory system, skin and eyes, and as a skin and respiratory sensitiser. The use of the additive in animal nutrition at the levels proposed is expected to be safe for the environment. Since isoeugenol is used in food as flavouring, and its function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. European Food Safety Authority, 2012 KEY WORDS Sensory additives, flavourings, propenylhydroxybenzenes, chemical group 17, isoeugenol 1 On request the European Commission, Question No EFSA-Q , adopted on 14 December 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 Acknowledgement: 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 propenylhydroxybenzenes (chemical group 17) when used as flavourings for all animal species.. [15 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2012

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 isoeugenol when used as a flavour compound for all animal species. The compound belonging to chemical group 17 is currently authorised for use as flavour in food, and is naturally found in various herbs and plants. Chemical formulations containing isoeugenol are used in water as an anaesthetic for fish, crustaceans and amphibians. In the absence of data on the correspondence between the exposure of fish via feed and the aquatic exposure, the FEEDAP Panel considered it prudent to exclude the use of isoeugenol as a flavouring agent in feed for fish and other aquatic species. The range of use levels of 1 to 5 mg/kg feed proposed by the applicant for isoeugenol is safe for all animal species (other than fish) with a margin of safety in the range of 1 to 4. Considering the low margin of safety, the simultaneous administration of isoeugenol in feed and water for drinking for poultry should be avoided. The FEEDAP Panel considered that the use of isoeugenol as flavour in non-dairy mammals up to the highest use levels proposed in feed is safe for the consumer. The lack of data on metabolism in all other species and categories precluded an assessment of consumer exposure from these sources. No data on the safety for the user was provided. Based on the information available in the literature, the FEEDAP Panel considered isoeugenol as an irritant to the respiratory system, skin and eyes, and as a skin and respiratory sensitiser. The use of this additive in animal nutrition at the levels proposed is expected to be safe for the environment. Since isoeugenol is used in food as flavouring, and its function in feed is essentially the same as that in food, no further demonstration of efficacy is 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 additive Stability and homogeneity Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) Safety Safety for the target species Conclusions on the safety for species Safety for the consumer Safety for the user Safety for the environment Efficacy Conclusions 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 isoeugenol, to be used as a feed additive 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 14 September The additive is listed as food and feed flavourings in the register of flavouring substances (Commission Decision 1999/217/EC) 7 and in the European Union Register of Feed Additives (Regulation (EC) No 1831/2003). It has not been previously assessed by EFSA for this purpose. Isoeugenol has been previously assessed by JECFA (2004a) and EFSA (2010; 2011a) as food flavouring and by EMEA (2008a; 2008b) and EMA (2011). 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 substance isoeugenol, when used under the conditions described in Table 1. 4 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, , p Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG), Avenue Louise 130A, B-1050, Brussels, Belgium. 6 EFSA Dossier reference: FAD Commission Decision of 23 February 1999 adopting a register of flavouring substances used in or on foodstuffs drawn up in application of Regulation (EC) No 2232/96 of the European Parliament and of the Council of 28 October 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 Chemical defined flavourings from Chemical Group 17: Isoeugenol - 2. Sensory additives b) flavouring compounds Composition, description Isoeugenol (CAS-No ) Description Chemical formula Purity criteria (if appropriate) C 10 H 12 O 2 99% Method of analysis (if appropriate) 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 Withdrawal mg or Units of activity or CFU/kg of complete feedingstuffs (select what applicable) 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, as part of a premixture only - - 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) 17 for flavouring substances is defined in Commission Regulation (EC) No 1565/ as propenylhydroxybenzenes. The present application concerns one compound, isoeugenol, which can be assigned to this CG. The substance is naturally found in various herbs and plants. The substance has been assessed by JECFA (2004) and was considered safe for use in food without limit and no Acceptable Daily Intake (ADI) has been set. Subsequently, the EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (CEF) considered the same compound for use as food flavouring in Flavouring Group Evaluation 81 (FGE.81) and as supporting substance in FGE.30 (EFSA 2010; 2011a). The CEF Panel concluded that the available data on the substance as well as the structure of the substance does not give rise to safety concern with respect to genotoxicity but expressed reservations (on the composition of the stereoisomeric mixture). Currently, isoeugenol is listed in the European Union database of flavouring substances and as such authorised for use in food. The European Medicines Agency (EMA) recently reviewed the toxicological information on isoeugenol, set an ADI and recommended an MRL for salmon (EMA, 2011). A consortium of companies (FFAC) supplying flavours to the feed industry has requested authorisation for the use of isoeugenol as additive 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 additive The physico-chemical characteristics of the additive under application are summarised in Table 2. 8 Commission Regulation (EC) No 1565/2000 of 18 July 2000 laying down the measures necessary for the adoption of an evaluation programme in application of Regulation (EC) No 2232/96 of the European Parliament and of the Council. OJ L 180, , p Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, , p. 1. 6

7 Table 2: Chemically defined flavouring from CG 17 under application Flavouring Isoeugenol CAS No Flavis No Structural formula Molecular formula C 10 H 12 O 2 Molecular weight Physical status Viscous liquid Boiling point ( C) 266 pka 9.88 Log K ow 3.04 Water solubility 0.81 g/l 10 Isoeugenol is produced by chemical synthesis. Typically several routes of synthesis are available and described in the dossier. 11 Isoeugenol is produced by chemical synthesis, e.g. by alkaline isomerisation of eugenol obtained from essential oils. Data was provided on the batch to batch variation in five batches of the additive. 12 The content of the active substance was in the range of 99.3 to 99.7 %. These values comply with JECFA specifications of > 99 % for isoeugenol (JECFA, 2006), on which the assessment for food safety was made. Potential contaminants are considered as part of the product specification and are monitored as part of the HACCP procedure applied by 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 the chemical under consideration when stored in closed containers under recommended conditions (in a cool and dry place). 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 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 comment on this route of administration Conditions of use The applicant proposes the use of the additive in feed or water for drinking for all animal species and without withdrawal time. The applicant proposes a normal use level of 1 mg/kg and a high use level of 5 mg/kg complete feed for isoeugenol. No specific proposals are made for the dose used in water for drinking. 10 Givaudan, 2003 according to HERA, Technical dossier/section II. 12 Technical dossier/section II/annex 2.1 and Supplementary information May

8 2.4. 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 isoeugenol in animal feed. The Executive Summary of the EURL report can be found in the Appendix. 3. Safety Chemical formulations containing isoeugenol are used in water as an anaesthetic for fish, crustaceans and amphibians. In the absence of data on the correspondence between the exposure of fish via feed and the aquatic exposure, the FEEDAP Panel considers it prudent to exclude the use of isoeugenol as a flavouring agent in feed for fish and other aquatic species. The assessment of safety is based on the high use level proposed by the applicant (5 mg isoeugenol/kg complete feed) Safety for the target species The approach to the safety assessment for the target species takes account of the use levels in animal feed, relative to those used in human food. To avoid unnecessary testing, the data used in the consumer safety assessment are used to provide an indication of safety for the target species. Any difference in metabolism of the substance is also considered in this evaluation along with any evidence for specific toxicity which might be relevant to the proposed target species. Compared with an estimated human exposure of 117 µg/person/day (equivalent to 5.4 µg/kg metabolic body weight (= kg 0.75 )/day) 13 the calculated intake by the target species (turkeys 313, piglets 526 and dairy cows 777 µg/kg 0.75 ) from the proposed maximum feed concentration of 5 mg exceeds that of humans resulting from use in food. As a consequence, the safety for the target species at the feed concentration supplied 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 animals is derived from the lowest NOAEL available for isoeugenol. A NOAEL of 150 mg/kg bw/day was identified in a three-month study in rat (ten males/ten females) based on increased hyperplasia in liver (NTP, 2010). Based on a two-year carcinogenicity study in rat and mice (NTP, 2010), EMA derived a LOAEL of 75 mg/kg bw in male mice based on liver adenomas and carcinomas observed at all doses (EMA, 2011). These tumours were not considered relevant to human exposure (EFSA, 2011a). However, the argumentation of the CEF Panel may not be applicable to target animal safety. The FEEDAP Panel considers that the maximum safe intake of isoeugenol for the target animals is 0.4 mg/kg bw/day (rounded from 0.375) applying a safety factor of 100 plus an additional safety factor of 2 to the LOAEL. Table 3 shows the maximum safe feed concentrations of isoeugenol for the target species. 13 See also Guidance for the preparation of dossiers for sensory additives 8

9 Table 3: Maximum safe feed concentrations of isoeugenol in selected target species 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 Dog Cat BW: body weight; FI: feed intake Conclusions on the safety for species The FEEDAP Panel considers the proposed range of use levels of 1 to 5 mg isoeugenol/kg feed as safe for all animal species except fish with a margin of safety in the range of 1 to 4. Considering the low margin of safety, the simultaneous administration of isoeugenol in feed and water for drinking for poultry should be avoided Safety for the consumer The safety for the consumer of isoeugenol has already been assessed by JECFA (2004) and EFSA (2010; 2011a). The CEF Panel concluded that the available data on the substance as well as the structure of the substance do not give rise to safety concern with respect to genotoxicity. Isoeugenol is presently authorised as a food additive without limitations. As the intake of isoeugenol by target animals substantially exceeds that of humans resulting from the use in food, the potential transfer of significant amounts of residues in edible tissues and products has to be considered, particularly considering the ADI recently set by EMA for isoeugenol (75 µg/kg bw or 4500 µg/person, obtained by applying an overall uncertainty factor of 1000 to the LOAEL of 75 mg/kg bw/day). Taking into account the established ADI of 75 µg/kg bw, an estimated per capita daily consumption of 300 g of fish and an average human body weight of 60 kg, EMA recommended also an MRL of 6 mg/kg for fin fish (muscle+skin) (EMA, 2011). A study on the biotransformation of isoeugenol in the rat following oral administration (single dose) of [ 14 C]-isoeugenol (156 mg/kg bw) showed that more than 85 % of the administered dose was excreted within 72 hours in the urine, predominantly as sulfate and glucuronide metabolites. No parent isoeugenol was detected in the blood at any of the time points analysed (LOD = mg/l). Less than 0.25 % of the radiolabel remained in selected tissues (Badger et al., 2002). Single administration toxicokinetic studies of isoeugenol in male and female rats and mice were conducted via intravenous and oral gavage routes (different oral doses comprised between 17 and 140 mg isoeugenol/kg bw). Absorption of isoeugenol following oral exposure was rapid in both species and sexes, maximum blood levels occurring within 2 to 20 minutes for all dose groups; large secondary peaks observed during the terminal phase of the elimination (240 minutes) indicated enterohepatic circulation. Bioavailability ranged from % in rats and % in mice. Isoeugenol was eliminated from the systemic circulation rapidly and extensively. Toxicokinetic data for both species indicated that the limited bioavailability was the result of extensive first-pass metabolism. There was no evidence of saturation in either rats or mice (NTP, 2010). Based on studies performed with other 1-propenylbenzenes (iso-allylbenzenes) than isoeugenol, the predominant paths for metabolic activation of this chemical group involve side-chain oxidation of the 9

10 double bond to form the corresponding 1,2-oxide, hydroxylation of the terminal methyl group to form the corresponding 3-hydroxypropenylbenzene and O-dealkylation; phase II takes place leading to sulfo- and glucurono- conjugates (NTP, 2010). The metabolic routes and toxicokinetic parameters described for isoeugenol in mammals are similar to those already assessed for compounds from the closely related chemical family 2-propenylbenzene (allylbenzene) (EFSA, 2011b). No data are available concerning the metabolic fate of isoeugenol in poultry and more generally birds. Where fish is concerned, an MRL has been established for isoeugenol in the flesh, due to considerable amounts of residues resulting from the use of this substance as anesthetics through balneation. It should be noted that this route of exposure implies very active absorption through the gills and distribution in the body by-passing first-pass metabolism: [ 14 C]- isoeugenol was rapidly absorbed and distributed (82 % of total residues in muscle) but also rapidly eliminated from the plasma and muscle+skin (1.4 and 1.9 hours depletion half-lives, respectively) at the end of the exposure period (EMA, 2011). Taking into account: i) the major difference in the metabolic fate of isoeugenol between mammals and fish, ii) the efficient metabolism of isoeugenol in laboratory mammals and the subsequent rapid excretion of the hydrophilic metabolites produced, the FEEDAP Panel considers that the presence of significant amounts of isoeugenol and derived metabolites in tissues of other mammals is unlikely and that the use of this compound as flavouring in the conditions described would not significantly increase consumer exposure and contribute to the ADI set for isoeugenol. Consequently, the FEEDAP Panel considers that the use of isoeugenol as flavour in non-dairy mammals up to the highest use levels proposed in feed is safe for the consumer. The lack of data on metabolism in all other species and categories precludes an assessment of consumer exposure from these sources Safety for the user No data on the safety for the user was provided. The applicant indicated possible effects of isoeugenol on the respiratory system, eyes and skin and proposed its classification as irritating to the respiratory system, eyes and skin and as a potential skin sensitiser. Little information is available on the irritancy of isoeugenol to skin and eyes ( The skin sensitisation potential of isoeugenol is well documented in the literature (Hilton et al., 1996; Bertrand et al., 1997; Takeyoshi et al., 2008). Contact allergy to isoeugenol has been reported by several authors (Tanaka et al., 2004; White et al., 1999; White et al., 2007). Based on the information available in the literature, the FEEDAP Panel considers isoeugenol as an irritant to the respiratory system, skin and eyes, and as a skin and respiratory sensitiser Safety for the environment The plant essential oils isoeugenol belong to the chemical class of allylhydroxybenzenes which are known to have a broad spectrum of activity against insects, mites, fungi and nematodes (Isman, 2000). As worst case estimate the concentration of isoeugenol in soil and water is calculated using the high use level proposed by the applicant of 5 mg/kg complete feed and assuming that the additive is excreted 100 % as parent compound. Using the screening methodology of the technical guidance (EFSA, 2008), the resulting PEC soil values for the different target species varies from 23 to 90 µg/kg dw. The PEC surfacewater values will vary from 0.6 to 2.5 µg/l, using an estimated K OC value of 675 (KOCWIN). The available acute toxicity studies for aquatic species show that the standard species tested are equally sensitive with L(E)C 50 values for algae (72 hours), daphnia (48 hours) and fish (96 hours) of 10.4, 7.5 and 5.1 mg/l, respectively. The NOEC for algae is 4.8 mg/l. The chronic NOEC of isoeugenol for D. magna is 0.4 mg/l (Clearwater et al., 2008). These values are very similar to the 10

11 QSAR predictions for phenols using the EPIWIN software. Based on the experimental data, the PNEC aquatic of isoeugenol would be 8 µg/l applying an assessment factor of 50. In the absence of toxicity data, the PNEC for soil is estimated with the equilibrium partition method using the PNEC aquatic and the estimated K OC value, resulting in a value of 105 µg/kg dw. By comparing the calculated PEC and PNEC values, a risk for soil and surface water is not expected. As indicated in Section 3.2, isoeugenol is strongly metabolised in the mammalian target species and only a limited percentage is excreted as parent compound, although in potency the excreted glucuronide and sulfate conjugates could be transformed back to the parent compound. The fast degradation observed in a ready test (Givaudan, 1996) indicates however that the compound will not be persistent in the environment. Therefore, the FEEDAP concludes that the use of this additive at the levels proposed is safe for the environment. 4. Efficacy Since isoeugenol is used in food as flavouring, and its function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. CONCLUSIONS The use of isoeugenol as a feed additive for fish and other aquatic species is contra-indicated. The FEEDAP Panel considers that the range of use levels of 1 to 5 mg/kg feed proposed by the applicant for isoeugenol is safe for all animal species (other than fish) with a margin of safety in the range of 1 to 4. Considering the low margin of safety, the simultaneous administration of isoeugenol in feed and water for drinking for poultry should be avoided. The FEEDAP Panel considers that the use of isoeugenol as flavour in non-dairy mammals up to the highest use levels proposed in feed is safe for the consumer. The lack of data on metabolism in all other species and categories precludes an assessment of consumer exposure from these sources. No data on the safety for the user was provided. Based on the information available in the literature, the FEEDAP Panel considers isoeugenol as an irritant to the respiratory system, skin and eyes, and as a skin and respiratory sensitiser. The use of this additive at the levels proposed is expected to be safe for the environment. Since isoeugenol is used in food as flavouring, and its function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. DOCUMENTATION PROVIDED TO EFSA 1. Chemically defined flavourings from Flavouring Group 17 - Propenylhydroxybenzenes for all animal species and categories. August Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 2. Chemically defined flavourings from Flavouring Group 17 - Propenylhydroxybenzenes for all animal species and categories. Supplementary information. May Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 3. Chemically defined flavourings from Flavouring Group 17 - Propenylhydroxybenzenes for all animal species and categories. Supplementary information. October Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 11

12 4. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for isoeugenol. REFERENCES Badger DA, Smith RL, Bao J, Kuester RK and Sipes IG, Disposition and metabolism of isoeugenol in the male Fischer 344 rat. Food Chemical Toxicology, 40, Bertrand F, Basketter DA, Roberts DW and Lepoittevin JP, Skin sensitization to eugenol and isoeugenol in mice: possible metabolic pathways involving ortho-quinone and quinone methide intermediates. Chemical Research in Toxicology, 10, Clearwater SJ, Hickey CW and Martin ML, 2008, onlnie. Overview of potential piscicides and molluscicides for controlling aquatic pest species in New Zealand. Science for conservation 283. Published by Science & Technical Publishing, Department of Conservation, Wellington, New Zealand, ISBN Available from EFSA (European Food Safety Authority), Technical Guidance of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP Panel) for assessing the safety of feed additives for the environment. The EFSA Journal, 842, EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (CEF), Scientific Opinion on Flavouring Group Evaluation 81 (FGE.81). Consideration of hydroxypropenylbenzenes evaluated by JECFA (61 st meeting) structurally related to 2-methoxy-4- (prop-1-enyl)phenyl 3- methylbutyrate from chemical group 17 evaluated by EFSA in FGE.30. EFSA Journal, 8(11):1899, 25 pp. EFSA Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (CEF), 2011a. Scientific Opinion on Flavouring Group Evaluation 30, Revision 1 (FGE.30Rev1). 4-Prop-1-enylphenol and 2-methoxy-4-(prop-1-enyl)phenyl 3-methylbutyrate from chemical group 17. EFSA Journal, 9(5):1991, 41 pp. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2011b. Scientific opinion on the safety and efficacy of allylhydroxybenzenes (chemical group 18) when used as flavourings for all animal species. EFSA Journal, 9(12):2440, 14 pp. EMEA, 2008a. Summary opinion of the Committee for Medicinal Products for Veterinary Use on the establishment of maximum residue limits. Isoeugenol. EMEA/CVMP/286527/2008. London. EMEA, 2008b. Summary opinion of the Committee for Medicinal Products for Veterinary Use on the establishment of maximum residue limits. Isoeugenol. EMEA/CVMP/506119/2008. London. EMA (European Medicines Agencies), 2011, online. Committee for Medicinal Products for Veterinary use. European public MRL assessment report (EPMAR). Isoeugenol in fish. EMA/CVMP/405186/2010. Available from _Report/2011/05/WC pdf Givaudan SA, Ready biodegradability of isoeugenol according to OECD Guideline No. 301 F. Givaudan SA. HERA, 2005, online. Human and Environmental Risk Assessment on ingredients of Household Cleaning Products. Isoeugenol, 4-hydroxy-3-methoxy-1-propen-1-yl benzene, CAS Available from HERA%20Isoeugenol%20(corrections%20May%202005).pdf Hilton J, Dearman RJ, Fielding I, Basketter DA and Kimber I, Evaluation of the sensitizing potential of eugenol and isoeugenol in mice and guinea pigs. Journal of Applied Toxicology, 16, Isman MB, Plant essential oils for pest and disease management. Crop Protection, 19,

13 JECFA, Evaluation of certain food additives. Sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no Rome, June JECFA, 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), 2010, online. Toxicology and Carcinogenesis Studies of Isoeugenol (CAS No ) in F344/N Rats and B6C3F1 Mice (Gavage Studies). TR 551, NIH Publication No Available from Tanaka S, Royds C, Buckley D, Basketter DA, Goossens A, Bruze M, Svedman C, Menne T, Johansen JD, White IR and McFadden JP, Contact allergy to isoeugenol and its derivatives: problems with allergen substitution. Contact Dermatitis, 51, Takeyoshi M, Iida K, Suzuki K and Yamazaki S, Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test. Journal of Applied Toxicology, 28, White IR, Johansen JD, Arnau EG, Lepoittevin JP, Rastogi S, Bruze M, Andersen KE, Frosch PJ, Goossens A and Menné T, Isoeugenol is an important contact allergen: can it be safely replaced by isoeugenyl acetate? Contact Dermatitis, 41, White JM, White IR, Glendinning A, Fleming J, Jefferies D, Basketter DA, McFadden JP and Buckley DA, Frequency of allergic contact dermatitis to isoeugenol is increasing: a review of 3636 patients tested from 2001 to The British Journal of Dermatology, 157,

14 APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Isoeugenol 14 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 for the following two applications: - Chemically Defined Flavourings - Group 12 (Maltol derivatives and ketodioxane derivatives, FAD ) and - Chemically Defined Flavourings - Group 17 (Propenylhydroxybenzenes Isoeugenol, FAD ). In the current applications 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. Maltol and Isoeugenol have a minimum purity of 98% and 99%, respectively. Maltol and Isoeugenol 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 Maltol and Isoeugenol 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 CRL. The Applicant provided the typical chromatogram for Maltol and Isoeugenol. 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 Maltol and Isoeugenol 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 Maltol and Isoeugenol in the feed additive. Based on the satisfactory experimental evidence provided, the CRL recommends for official control for the qualitative identification of Maltol derivatives and ketodioxane derivatives, [FAD ] or Propenylhydroxybenzenes Isoeugenol, [FAD ]) in the feed additive 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 substances in feedingstuffs and water, no methods could be evaluated. Therefore the CRL is unable to recommend a method for the official control to identify Maltol derivatives and ketodioxane derivatives, [FAD ] and Propenylhydroxybenzenes Isoeugenol, [FAD ]) in feedingstuffs or water. 14 The full report is available on the EURL website: pdf 14

15 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. 15