Scientific Opinion on the safety and efficacy of anthranilate derivatives (chemical group 27) when used as flavourings for all animal species 1

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1 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of anthranilate derivatives (chemical group 27) 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 The chemical group 27 defined as anthranilate derivatives consists of two flavouring substances: methyl anthranilate and methyl-n-methylanthranilate. Both compounds are currently authorised for use as flavours in food, and are naturally found in various fruits and plants. The use of methyl anthranilate and methyl-nmethylanthranilate in avian species was found contra-indicated. The applicant s proposed use levels of 5 to 25 mg methylanthranilate/kg complete feed are safe in the other species with a margin of safety up to three. For methyl-n-methylanthranilate, the proposed upper use level of 5 mg/kg feed is not safe for piglets, therefore the highest feed concentration safe for all animals species other than avian species is 4 mg/kg complete feed. Considering the absent or very low margin of safety, the simultaneous administration of either substance in feed and water for drinking, and the simultaneous use of both compounds are not considered safe without a proportional reduction in their concentration. No residues of safety concern derived from methylanthranilic acid and methyl-n-methylanthranilic are to be expected in animal tissues or products. Consequently, the use of these two compounds in animal nutrition would not increase consumer exposure resulting from natural occurrence and food use. No data relevant to an assessment of user safety was made available. The applicant considers that both substances are irritants to the respiratory system, the eyes and the skin. The use of the anthranilate derivatives in animal nutrition is not expected to have a significant impact on the environment. Since the function of both compounds 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, anthranilate derivatives, chemical group 27, methyl anthranilate, methyl-nmethylanthranilate 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 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 anthranilate derivatives (chemical group 27) when used as flavourings for all animal species.. [13 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 methyl anthranilate and methyl-n-methylanthranilate when used as flavour compounds for all animal species. Both compounds belonging to chemical group 27 are currently authorised for use as flavours in food, and are naturally found in various fruits and essential oil plants. Because of the neurotoxic effects in birds, the use of methyl anthranilate and methyl-nmethylanthranilate as a flavour in avian species is contra-indicated. A calculation based on the lowest NOAEL for each compound showed that the applicant s proposed use levels of 5 to 25 mg methylanthranilate/kg complete feed are safe in the remaining species with a margin of safety up to three. For methyl-n-methylanthranilate, the proposed upper use level of 5 mg/kg feed is not safe for piglets, therefore the highest feed concentration safe for all animal species other than avian species is 4 mg/kg complete feed. Considering the absent or very low margin of safety, (i) the simultaneous administration of either substance in feed and water for drinking, and (ii) the simultaneous use of both compounds are not considered safe without a proportional reduction in their concentration. No residues of safety concern derived from methylanthranilic acid and methyl-n-methylanthranilic are to be expected in animal tissues or products. Consequently, the FEEDAP Panel considers that the use of these two compounds in animal nutrition would not increase consumer exposure resulting from natural occurrence and food use. No data relevant to an assessment of user safety was made available. The applicant considers that both substances are irritants to the respiratory system, the eyes and the skin. The use of the anthranilate derivatives in animal nutrition is not expected to have a significant impact on the environment. Since the function of both compounds 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 target 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 the substances methylanthranilate and methyl-n-methylanthranilate, 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 25 August The additives are 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). They have not been previously assessed by EFSA for this purpose. The substances of chemical group (CG) 27 have been previously assessed by JECFA (2006a) and EFSA s Scientific Panel on Food Contact Material, Enzymes, flavourings and Processing Aids (CEF) in 2008 as food flavourings. 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 methylanthranilate and methyl-n-methylanthranilate, 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 27 - Anthranilate derivatives: Methyl anthranilate Methyl N-methylanthranilate - 2. Sensory additives b) flavouring compounds Composition, description Chemical formula Description Purity criteria (if appropriate) Methyl anthranilate C 8 H 9 O 2 N 98% Methyl N-methylanthranilate C 9 H 11 O 2 N 98% Method of analysis (if appropriate) Gas Chromatography Mass Spectrometry (GC-MS) 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) 27 for flavouring substances is defined in Commission Regulation (EC) No 1565/2000 as anthranilate derivatives. The present application concerns methylanthranilate and methyl-n-methylanthranilate, which can be assigned to this CG. Both substances are natural components found in various fruits and essential oil plants. Both compounds have been evaluated by JECFA (2006a) and ADIs were set of 1.5 mg/kg bw for methyl anthranilate and 0.2 mg/kg bw for methyl-n-methylanthranilate based on a calculation of maximum human exposure. The CEF Panel (EFSA, 2008) assessed these compounds for use as food flavourings as part of Flavouring Group Evaluation 84 (FGE.84) and reached similar conclusions. 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 two compounds methylanthranilate and methyl-n-methylanthranilate 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 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 27 under application Flavouring Methylanthranilate Methyl-N-methylanthranilate CAS No Flavis No Structural formula Molecular formula C 8 H 9 O 2 N C 9 H 11 O 2 N Molecular weight Physical status Liquid Liquid Boiling point ( C) pka Log K ow Water solubility 1.86 g/l Insoluble 8 OJ L 133, , p. 1. 6

7 Methylanthranilate and methyl-n-methylanthranilate are produced by chemical synthesis. Typically synthesis is by methylation of anthranilic acid to produce methyl anthranilate. This can be further methylated with dimethyl sulphate to produce methyl-n-methylanthranilate. Alternatively, methyl-nmethylanthranilate can be produced by esterification of N-methylanthranilic acid. Data was provided on the batch to batch variation in five batches of each additive. 9 The content of the active substances were in the range of 99.4 to 100 % for methyl anthranilate and of 98.3 to 99.1 % for methyl-n-methylanthranilate. These values comply with JECFA specifications (JECFA, 2006b) of > 98 % for both compounds. Potential contaminants are considered as part of the product specification and when necessary 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 both 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, 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 delivery Conditions of use The applicant proposes the use of the two additives 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 methyl anthranilate and 5 and 25 mg/kg complete feed for methyl- N-methylanthranilate. 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 methyl anthranilate and methyl-n-methylantharanilate in animal feed. The Executive Summary of the EURL report can be found in the Appendix. 3. Safety The assessment of safety is based on the high use level proposed by the applicant (5 mg/kg complete feed for methyl anthranilate and 25 mg/kg complete feed for methyl-n-methylanthranilate) Safety for the target species Chemical formulations containing methyl anthranilate have been found to be effective bird aversion agents, acting as chemosensory repellents (Müller-Schwartze, 2009) by activating pain receptors associated with taste and smell (Kirifides et al., 2004). As a consequence, the FEEDAP Panel considers that the use of both compounds in avian species is contra-indicated. 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 in the EU (EFSA, 2008) are for methylanthranilate 686 and for 9 Technical dossier/section II/Annex 2.1 and Supplementary information May

8 methyl-n-methylanthranilate 51 µg/person/day. Table 3 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 4. Table 3: 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 Salmon Piglet Dairy cow Methylanthranilate Methyl-N-methylanthranilate * mbw: metabolic body weight (kg 0.75 ) for a 60 kg person = The data of Table 3 indicates that the intake of 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. The values available for methylanthranilate and methyl-n-methylanthranilate are 150 and 20 mg/kg bw/day, respectively. These are derived from 90-day studies in rats identified by JECFA (1968, 1980a) for methylanthranilate (based on effects in kidney and liver) and JECFA (1980a,b) for methyl-n-methylanthranilate (based on increased kidney weight). Subsequent reviews confirmed these values (JECFA, 2006a; EFSA, 2008). No more recent toxicological studies are available. Applying a safety factor of 100, the maximum safe intake of methylanthranilate for target animals is 1.5 mg/kg bw/day and of methyl-n-methylanthranilate 0.2 mg/kg bw/day. Table 4 shows the derived maximum safe feed concentrations of both flavourings for selected target species. Table 4: Maximum safe feed concentrations in selected target species for methylanthranilate and methyl-n-methylanthranilate Target animal Default settings BW (kg) FI (g/d) Maximum safe intake/feed concentration Methylanthranilate Methyl-N-methylanthranilate Intake (mg/d) mg/kg feed Intake (mg/d) Salmonids Veal calves (milk replacer) Cattle for fattening Pigs for fattening Sows Dairy Cows Piglets BW: body weight; FI: feed intake Conclusions on the safety for target species The use of both compounds in avian species is contra-indicated. mg/kg feed The FEEDAP Panel considers the proposed range of use levels of 5 to 25 mg methylanthranilate/kg complete feed as safe in the remaining species with a margin of safety up to three. For methyl-nmethylanthranilate, the proposed upper use level of 5 mg/kg feed is not safe for piglets, therefore the highest feed concentration safe for all animals species other than avian species is 4 mg/kg complete feed. 8

9 Considering the absent or very low margin of safety, (i) the simultaneous administration of either substance in feed and water for drinking, and (ii) the simultaneous use of both compounds are not considered safe without a proportional reduction in their concentration Safety for the consumer The safety for the consumer of both compounds used as food flavours has already been assessed by the CEF Panel (EFSA, 2008) and JECFA (2006a). Both compounds are presently authorised as food additives without limitations. As the intake of methylanthranilate and methyl-n-methylanthranilate by target animals exceeds by one to two orders of magnitude 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. No data have been supplied and none could be found following a search of the relevant literature. In its evaluation of anthranilate derivatives, JECFA identified that the anthranilic acid esters, methyl anthranilate and methyl-n-methylanthranilate, are expected to be readily absorbed, either unchanged or in hydrolysed form. Once absorbed, the unchanged esters are hydrolysed in the liver to their corresponding carboxylic acids (anthranilic acid and N-methylanthranilic acid). These anthranilic acids are then rapidly excreted in the urine (JECFA, 2006a). JECFA also stated that these substances are expected to be metabolised to innocuous products. No new data concerning the metabolic fate of both compounds have been published since the last JECFA assessment. When the safety for the human consumer of residues from tissues and products from exposed target animal species is concerned, the comparative metabolic fate of these compounds in the target animals and laboratory species has to be considered. In this context, a re-assessment of the studies examined by JECFA (JECFA, 2006a,c) and other published studies indicates that: i) the hydrolysis of the esters occurs to a considerable extent in pig jejunum preparations, ii) ester hydrolysis (by carboxyesterases) is performed efficiently by rat and pig liver homogenates, and by guinea-pig liver microsomes, iii) the N-demethylation of methyl-n-methylanthranilic occurs in the guinea-pig liver, iv) the end products excreted in the urine of the rat and rabbit are the corresponding free and conjugated (glucurono- and glycine) anthranilic acids, mainly hippuric acid conjugate in the human, v) in certain fish anthranilic acid administered by intramuscular injection is excreted in the urine unchanged and as anthranilic acid glucuronide (goosefish), unchanged and as anthranilic acid glycine conjugate (flounder) or essentially anthranilic acid glucuronide (dog-fish). These data indicate that the two anthranilate derivatives under assessment undergo a similar metabolic fate in mammals (including the human) and fish. It must be noted that the key enzymes of that metabolic pathway, carboxyesterases and N-dealkylases, are expressed in birds (Mulla et al., 1981 for the former, Foster and Khan, 1976 and Foster et al., 1980 for the latter). The metabolism of these anthranilate derivatives towards more hydrophilic compounds rapidly excreted in urine precludes their accumulation in tissues. It should be noted that anthranilic acid is an intermediate of tryptophan biosynthesis. Consequently, no residues of safety concern derived from either methylanthranilic acid or methyl-n-methylanthranilic acid are to be expected in animal tissues or products, and the use of these two compounds up to the highest doses safe for the target animals would not measurably increase consumer exposure and thus not contribute to the ADI Safety for the user No data on the safety for the user was provided. The applicant considers both additives in solid form are possible eye and respiratory irritants. This is reflected in the Material Safety Data Sheets provided by the applicant for methyl anthranilate and methyl-n-methylanthranilate, which indicates these hazards and recommends protective measures for those handling the products. 9

10 3.4. Safety for the environment The two anthranilate derivatives are natural compounds, which are absorbed and metabolised in the target animal species to free and conjugated (glucuronic acid, glycine) anthranilic acid. Based on the available data, anthranilic acid degrades rapidly both under aerobic and anaerobic conditions (Lu et al., 2002; Kameya et al., 1995). Long-term exposure is therefore not expected. The pka of anthranilic acid is 2.14, indicating that this compound will primarily exist in the environment in an anionic form. The potential for bioconcentration will therefore be low. Based on the available data, anthranilic acid has an acute toxicity to aquatic organism > 10 mg/l (Zhao et al., 1998). Overall, based on the fate and toxicological properties of anthranilic acid it is not expected that the use of the anthranilate derivatives in animal nutrition will have a significant impact on the environment. 4. Efficacy Methylanthranilate and methyl-n-methylanthranilate are used in food as flavourings. Since their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. CONCLUSIONS The use of methyl anthranilate and methyl-n-methylanthranilate in avian species is contra-indicated. The FEEDAP Panel considers the applicant s proposed range of use levels of 5 to 25 mg methylanthranilate/kg complete feed as safe in the remaining species with a margin of safety up to three. For methyl-n-methylanthranilate, the proposed upper use level of 5 mg/kg feed is not safe for piglets, therefore the highest feed concentration safe for all animals species other than avian species is 4 mg/kg complete feed. Considering the absent or very low margin of safety, (i) the simultaneous administration of either substance in feed and water for drinking, and (ii) the simultaneous use of both compounds are not considered safe without a proportional reduction in their concentration. No residues of safety concern derived from methylanthranilic acid and methyl-n-methylanthranilic are to be expected in animal tissues or products. Consequently, the FEEDAP Panel considers that the use of these two compounds in animal nutrition would not increase consumer exposure resulting from natural occurrence and food use. No data relevant to an assessment of user safety was made available. The applicant considers that both substances are irritants to the respiratory system, the eyes and the skin. The use of the anthranilate derivatives in animal nutrition is not expected to have a significant impact on the environment. Since the function of these additives in feed is essentially the same as that in food, no further demonstration of efficacy is considered necessary by the FEEDAP Panel. DOCUMENTATION PROVIDED TO EFSA 1. Chemically defined flavourings from Flavouring Group 27 - Anthranilate derivatives for all animal species and categories. July Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 2. Chemically defined flavourings from Flavouring Group 27 - Anthranilate derivatives for all animal species and categories. Supplementary information. May Submitted by Feed Flavourings Authorisation Consortium European Economic Interest Grouping (FFAC EEIG). 10

11 3. Chemically defined flavourings from Flavouring Group 27 - Anthranilate derivatives 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 methyl anthranilate and methyl-n-methylanthranilate. 5. Comments from Member States received through the ScienceNet. REFERENCES EFSA (European Food Safety Authority), Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (CEF) on Flavouring Group Evaluation 84 (FGE 84). Consideration of Anthranilate derivatives evaluated by JECFA (65 th meeting). The EFSA Journal, 856, Foster TS and Khan SU, Metabolism of atrazine by the chicken. Journal of Agricultural Food Chemistry, 24, Foster TS, Khan SU and Akhtar MH, Metabolism of deethylatrazine, deisopropylatrazine, and hydroxyatrazine by the soluble fraction (10500 g) from goose liver homogenates. Journal of Agricultural Food Chemistry, 28, JECFA (Joint FAO/WHO Expert Committee on Food Additives), Joint FAO/WHO Expert Committee on Food Additives. Report: WHO Technical Report Series, no JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1980a. Evaluation of certain food additives. Twenty-third report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, no. 648, Geneva. JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1980b. Toxicological evaluation of certain food additives. Twenty-third Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Toxicological monographs WHO Food Additives, No 14. 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 ( ). JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2006c. Safety evaluation of certain food additives. Sixty-fifth meeting of the Joint FAO/WHO Expert Committee on Food Additives. Food additive series (FAS)56. Kameya T, Mutayama T, Kitano M and Urano K, Testing and classification methods for the biodegradabilities of organic compounds under anaerobic conditions. The Science of the Total Environment, 170, Kirifides ML, Kurnellas MP, Clark L and Bryant BP, Calcium responses of chicken trigeminal ganglion neurons to methyl anthranilate and capsaicin. Journal of Experimental Biology, 207, Lu GH, Zhao YH, Yang SG and Cheng XJ, Quantitative structure-biodegradability relationships of substituted benzenes and their biodegradability in river water. Bulletin of Environmental Contamination and Toxicology, 69, Mulla MS, Mian LS and Kawecki JA, Distribution, transport, and fate of the insecticides malathion and parathion in the environment. Residue Reviews, 81,

12 Müller-Schwartze D, Sour Grapes: Methyl Antranilate as Feeding Repellent for Birds. In: Hands-On Chemical Ecology: Simple Fields and Laboratory Exercices, Part I, 13-17, Springer Science+Business Media, LCC Zhao YH, Ji GD, Cronin MTD and Dearden, QSAR study of the toxicity of benzoic acids to Vibrio fischeri, Daphnia magna and carp. The Science of the Total Environment, 216,

13 APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for methyl anthranilate and methyl N-methyl anthranilate 10 The Chemically Defined Flavourings - Group 27 (CDG27 - Anthranilate derivatives), in this application comprises two substances, 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%. 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 CDG27 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 CDG27 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 CDG27 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 CDG27 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 (Methyl N-methylanthranilate and Methyl anthranilate) 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. 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. 10 The full report is available on the EURL website pdf 13