Scientific Opinion on the safety and efficacy of DL-methionyl-DL-methionine for all aquatic animal species 1

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1 EFSA Journal 2015;13(2):4012 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of DL-methionyl-DL-methionine for all aquatic 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 DL-Methionyl-DL-methionine is intended to replace supplemental methionine in feed for aquatic animal species. This dipeptide differs from free DL-methionine, particularly in its solubility in water. In the organism, DLmethionyl-DL-methionine will be cleaved to produce methionine. Since supplementing appropriate quantities of methionine to meet requirements is safe for the target species, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) extends this conclusion to the safety of DL-methionyl-DL-methionine for fish and crustaceans. The amino acid L-methionine originating from the metabolism of DL-methionyl-DL-methionine in the body is incorporated into the tissue proteins of aquatic animal species, without affecting their amino acid composition. There is no concern for the safety of consumers from the use of the additive under application in aquatic species nutrition. The product DL-methionyl-DL-methionine is not regarded as an irritant to the eyes and the results of an in vitro study indicated that it is unlikely to be an irritant to the skin. It is also considered likely not to be a sensitiser by the dermal route. Since there is no evidence of acute toxicity by the inhalation route it is not regarded as hazardous for the user. The use of DL-methionyl-DL-methionine as an additive to feed for aquatic animal species does not represent a risk to the environment. Based on in vitro and in vivo studies with rainbow trout, carp, tilapia and shrimp, the FEEDAP Panel concludes that the product, DL-methionyl-DL-methionine, is an efficacious source of the essential amino acid L-methionine for fish and crustaceans. European Food Safety Authority, 2015 KEY WORDS nutritional additive, amino acid, DL-methionyl-DL-methionine, methionine dipeptide, aquatic animal species, safety, efficacy 1 On request from the European Commission. Question No EFSA-Q , adopted on 27 January Panel members: Gabriele Aquilina, Vasileios Bampidis, Maria De Lourdes Bastos, Lucio Guido Costa, Gerhard Flachowsky, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Fernando Ramos, Derek Renshaw, Guido Rychen, Maria Saarela, Kristen Sejrsen, Patrick Van Beelen, Robert John Wallace and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Amino Acids, including Paul Brantom, Noël Dierick and Jürgen Gropp, for the preparatory work on this scientific opinion. Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Scientific Opinion on the safety and efficacy of DL-methionyl-DL-methionine for all aquatic animal species. EFSA Journal 2015;13(2):4012, 14 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

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 DL-methionyl-DL-methionine produced by chemical synthesis for all aquatic animals. The product DL-methionyl-DL-methionine is intended to replace supplemental methionine in feed for aquatic animal species. This dipeptide differs from free DL-methionine, particularly in its solubility in water. In the organism, dipeptide DL-methionyl-DL-methionine will be cleaved to produce methionine. Since supplementing appropriate quantities of methionine to meet requirements is safe for the target species, the FEEDAP Panel extends this conclusion to the safety of DL-methionyl-DL-methionine for fish and crustaceans. The amino acid L-methionine originating from the metabolism of DL-methionyl-DL-methionine in body is incorporated into the tissue proteins of aquatic animal species, without affecting their amino acid composition. There is no concern for the safety of consumers from the use of the additive under application in aquatic species nutrition. The product DL-methionyl-DL-methionine is not regarded as an irritant to the eyes and the results of an in vitro study indicated that it is unlikely to be an irritant to the skin. It is also considered likely not to be a sensitiser by the dermal route. Since there is no evidence of acute toxicity by the inhalation route it is not regarded as hazardous for the user. The use of DL-methionyl-DL-methionine as an additive to feed for aquatic animal species does not represent a risk to the environment. Based on in vitro and in vivo studies with rainbow trout, carp, tilapia and shrimp, the FEEDAP Panel concludes that the product DL-methionyl-DL-methionine is an efficacious source of the essential amino acid L-methionine for fish and crustaceans. EFSA Journal 2015;13(2):4012 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Background... 4 Terms of reference... 5 Assessment Introduction Characterisation Characterisation of the additive Characterisation of the active substance Physico-chemical properties Manufacturing process Stability and homogeneity Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 8 3. Safety Safety for the target species Digestion, metabolism Acute toxicity in laboratory rodents Conclusions on the safety for target species Safety for the consumer Conclusions on the safety for the consumer Safety for the user Conclusions on the safety for the user Safety for the environment Efficacy Conclusions from the in vitro studies Conclusions of the in vivo studies Conclusions on efficacy Post-market monitoring Conclusions Documentation provided to EFSA References Appendix A. Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for DL-Methionyl-DL-Methionine EFSA Journal 2015;13(2):4012 3

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. The European Commission received a request from the company Evonik Industries AG 5 for authorisation of the product DL-methionyl-DL-methionine, when used as feed additive for all aquatic animals (category: nutritional additive; functional group: amino acids, their salts and analogues) 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). 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 11 March The additive DL-methionyl-DL-methionine is a dipeptide of the amino acid D/L-methionine and is produced by chemical synthesis. DL-methionyl-DL-methionine has not been previously authorised as feed additive in the European Union (EU). The additives DL-methionine, DL-methionine sodium salt, hydroxy analogue of methionine, and calcium salt of methionine hydroxy analogue, when used as a feed additive for all animal species; isopropyl ester of methionine hydroxy analogue, DL-methionine technically pure protected with copolymer vinylpyridine/styrene, and DL-methionine protected with ethylcellulose when used as a feed additive for ruminants, are currently authorised for use for all animal species and ruminants, respectively, by Regulation (EU) No 469/ The EFSA FEEDAP Panel (2012) published a scientific opinion on DL-methionine, DL-methionine sodium salt, hydroxy analogue of methionine, calcium salt of methionine hydroxy analogue for all animal species; isopropyl ester of methionine hydroxy analogue and DL-methionine technically pure protected with copolymer vinylpyridine/styrene for dairy cows; DL-methionine technically pure protected with ethylcellulose for ruminants. The EFSA FEEDAP Panel (2013) published a scientific opinion on the safety and efficacy of L- methionine produced by Escherichia coli (KCCM 11252P and KCCM 11340P) for all animal species. According to Commission Directive 2006/141/EC amino acids as L-methionine may be used in the manufacture of infant formulae and follow-on formulae in order to satisfy the requirements on amino acids and other nitrogen compounds. 8 L-Methionine is currently authorised as flavouring substance in feed. It is included in the Community list of flavouring substances as FL. No Methionine is 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 Evonik Industries AG (Rodenbacher Chaussee 4, D-63457, Hanau, Germany). 6 EFSA Dossier reference: FAD Commission Implementing Regulation (EU) No 469/2013 of 22 May 2013 concerning the authorisation of DL-methionine, DL-methionine sodium salt, hydroxy analogue of methionine, calcium salt of hydroxy analogue of methionine, isopropyl ester of hydroxy analogue of methionine, DL-methionine protected with copolymer vinylpyridine/styrene and DLmethionine protected with ethylcellulose as feed additives. OJ L 136, , p Commission Directive 2006/141/EC on infant formulae and follow-on formulae, OJ L 401, , p. 1. EFSA Journal 2015;13(2):4012 4

5 registered as an ingredient for use in cosmetics as antistatic and for skin conditioning (Commission Decision 2006/257/EC). 9 DL-Methionine is described in the European Pharmacopoeia, monograph 01/2008:0624. Methionine does not require maximum residue levels in all food producing species when used as pharmacologically active substance. 10 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 animals, consumer, user and the environment and the efficacy of the product DLmethionyl-DL-methionine, produced by chemical synthesis, when used under the conditions described in Table 1. 9 Commission Decision of 9 February 2006 amending Decision 96/335/EC establishing an inventory and a common nomenclature of ingredients employed in cosmetic products. OJ L 97, , p Commission Regulation (EC) No 1931/1999, amending Annexes I, II and III of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. OJ L 240, , p. 3. EFSA Journal 2015;13(2):4012 5

6 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 DL-Methionyl-DL-Methionine (DL-Met-DL-Met) Nutritional Additives Amino Acids, Salts and Analogues Composition, description > 93.0 % DL-Methionyl-DL Methionine Description Chemical Purity criteria formula (if appropriate) C 10 H 20 N 2 O 3 S 2 > 93 % Met-Met < 3 % DL-Methionine Method of analysis (if appropriate) Reg(EC)No. 152/2009 Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Evonik Industries AG Species or category of animal all aquatic animal species Maximum Age Conditions of use Minimum content Maximum content mg/kg of complete feedingstuffs Withdrawal period (if appropriate) all ages not applicable not applicable not applicable 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 not applicable not applicable not applicable not applicable Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues not applicable not applicable not applicable not applicable EFSA Journal 2015;13(2):4012 6

7 ASSESSMENT 1. Introduction Modern fish feed formulation is characterised by an increasing use of plant proteins at the expense of fish meal and related materials because of their restricted availability and high price. The amino acid profile of protein-rich plants (oil seeds) is typically deficient in methionine compared with the animals requirements. Fish feed therefore requires to be supplemented with methionine to achieve an optimally balanced amino acid profile of the diet. DL-Methionyl-DL-methionine is intended to replace supplemental methionine and applied for use in feed for aquatic animal species without restrictions in maximum content or time of administration. This dipeptide differs from free DL-methionine, particularly in its solubility in water. This substance is currently not authorised for use as a nutritional additive in the European Union (EU). However, there is an authorisation for DL-methionine and its sodium salt produced by chemical synthesis, and for L-methionine produced by fermentation, for all animal species. 2. Characterisation Characterisation of the additive The additive contains by specification 93 % DL-methionyl-DL-methionine and 3 % DL-methionine on a dry matter basis. Other components do not exceed 4 % on a dry matter basis and are described in the technical dossier. 12 The specification is confirmed by analyses of five batches. 13 The content of DL-methionyl-DLmethionine in the product ranged from 94.9 to 99.9 %, whereas free DL-methionine ranged from 0.2 to 1.6 %. The water content was between 0.4 and 1.0 %. Residuals from the chemical synthesis were identified and ranged 0.3 to 3.9 %. Data provided for the content of heavy metals ( 0.01 mg cadmium, < 0.01 mg mercury and < 0.10 mg lead/kg) and arsenic (< mg/kg) in three batches did not give rise to safety concerns. 14 Dioxins were < 0.08 ng World Health Organization (WHO) polychlorinated dibenzodioxin/dibenzofuran (PCDD/F)-toxic equivalents (TEQ)/kg and dioxin-like polychlorinated biphenyls (PCBs) were < 0.01 ng/kg in three batches. Non-dioxin-like PCBs were in three batches 3.2 g PCB (International Council for the Exploration of the Sea (ICES)-6)/kg (ICES-6: sum of PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and PCB 180) Characterisation of the active substance DL-Methionyl-DL-methionine is a dipeptide of DL-methionine (International Union of Pure and Applied Chemistry (IUPAC) name 2-[(2-amino-4-methylsulfanylbutanoyl)amino]-4- methylsulfanylbutanoic acid, Chemical Abstracts Service (CAS) number ) and has the molecular formula C 10 H 20 N 2 O 3 S 2. Its molecular weight is g/mol. Its molecular structure is given in Figure This section has been amended following the confidentiality claims made by the applicant. 12 Technical dossier/section II Technical dossier/section II/Annexes II.2 and II Technical dossier/section II/Annex II Supplementary information October 2013/Annexes II.5 to II.8. EFSA Journal 2015;13(2):4012 7

8 Figure 1: Molecular structure of DL-methionyl-DL-methionine 2.3. Physico-chemical properties The additive is a white to brown crystalline powder. Its solubility in water is 21 g/l at 20 C, and its density is g/cm 3. Particle size distribution was measured in one batch of the additive by sieving; 58 % of the particles have a diameter < 100 m (w/w). The fraction of particles with a diameter 32 m represents 22 % 16. The dusting potential was determined in g/m 3 (three batches) and showed a potential for users to be exposed to respirable dust Manufacturing process DL-Methionyl-DL-methionine, the dipeptide of DL-methionine, is produced through chemical synthesis. The details are described in the dossier. The material safety data sheets for the chemicals used in the synthesis and for the product under assessment are provided Stability and homogeneity The stability studies provided showed that, under the conditions described, either no losses of DLmethionyl-DL-methionine or losses within acceptable limits occur. Regarding the capacity of the additive to distribute homogeneously in feed, the coefficient of variation was within acceptable limits Conditions of use DL-Methionyl-DL-methionine is proposed to be used as a nutritional additive for aquatic species without maximum content and other restrictions. The diets will be supplemented with the additive in order to meet the animals requirements for the sulphur-containing amino acids L-methionine and L- cysteine. It can be added directly to compound feed or via premixtures Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) The European Food Safety Authority (EFSA) has verified the EURL report as it relates to the methods used for the control of the active substance in animal feed. The Executive Summary of the EURL report can be found in Appendix A. 16 Technical dossier/section II/Annex II Supplementary information October 2013/Annex II Technical dossier/section II/Annexes II.6 to II.9. EFSA Journal 2015;13(2):4012 8

9 3. Safety 3.1. Safety for the target species Tolerance studies of essential amino acids such as methionine and its dipeptide cannot be designed in accordance with the protocols of conventional toxicity experiments because high dietary concentrations of a particular amino acid will result in amino acid imbalances with depression of feed intake and hence impaired performance. Consequently, tolerance studies are not required for amino acids naturally occurring in the proteins of animals or plants already authorised as feed additives (Regulation (EC) No 429/2008) Digestion, metabolism Gilbert et al. (2008) reviewed absorption and utilisation of the peptide and its implications for animal nutrition health. Regarding the fate of luminal protein digestion products, they stated that: The end products of stomach digestion of proteins enter the small intestine where pancreatic enzyme digestion (trypsin, chymotrypsin, elastase, etc.) begins the process of generating absorbable end products. Hydrolases are expressed on the brush border membrane of absorptive, epithelial cells serving to further digest luminal nutrients. The end products of digestion are absorbed by these epithelial cells in the intestine through the action of nutrient transporters located on the brush border membrane. Amino acids are transported into the enterocyte as free amino acids by a variety of amino acid transporters that vary in substrate specificity or as di- and tripeptides by the peptide transporter, PepT1. Expression of PepT1 is largely restricted to the small intestine in most species. There is sufficient evidence that amino acids as well as small peptides are absorbed in the intestine of different fish species (see Gropp et al., 1990; Schuhmacher et al., 1997; Dabrowski et al., 2003; Gilbert et al., 2008). It is assumed that the dipeptide DL-methionyl-DL-methionine follows the pathway described above. It will be absorbed in fish as DL-methionyl-DL-methionine and/or as methionine. Possible biochemical pathways of DL-methionyl-DL-methionine are described by Friedman and Gumbmann (1988). The dipeptide could be modified by inversion and racemisation (D-methionyl-Dmethionine to L-methionyl-D-methionine or D-methionyl-L-methionine), the dipeptides are hydrolysed, resulting in L-methionine (by hydrolysis) or D-methionine (by racemisation after hydrolysis). It is well established that D-enantiomer of methionine is enzymatically transformed (by oxidative deamination to α-ketoacid and its subsequent transamination) into L-methionine in mammals, birds and fish (Mello, 2003; Forster and Dominy, 2006) Acute toxicity in laboratory rodents The applicant provided two oral acute toxicity studies. In an oral acute toxicity study comparing DL-methionyl-DL-methionine, DL-methionine and L- methionine in rats, a dose of 10 g/kg body weight (bw) showed no signs of toxicity. 19 In mice, the oral acute lethal dose killing 50 % of the animals (LD 50 ) of DL-methionyl-DL-methionine, DL-methionine and L-methionine were all > 10 g/kg bw. The applicant indicated that the lowest oral dose causing toxic effects was 7.9 g/kg bw for DL-methionyl-DL-methionine, 6.3 g/kg bw for DLmethionine and 5 g/kg bw for L-methionine. 20 The symptoms were seen 5 10 minutes after administration and consisted of slight sedation and ataxia (from 7.9 g/kg bw) for 3 8 hours which subsequently resolved. No apparent lesions were found at necropsy Conclusions on the safety for target species When assessing, among others, DL-methionine for all animal species (EFSA FEEDAP Panel, 2012), the Panel concluded that: Supplementing appropriate amounts of methionine to meet requirements is 19 Technical dossier/section III/Reference III.3.5 and supplementary information October 2013/Section Technical dossier/section III/Reference III.3.6. EFSA Journal 2015;13(2):4012 9

10 safe for the target species. The earliest signs of a critical overdose are depressed feed intake and weight gain. The toxic levels of supplemented methionine vary and depend on the basal diet and its content of sulphur-containing amino acids. The FEEDAP Panel concludes that product DL-methionyl-DL-methionine is a safe source of essential amino acid L-methionine for aquatic species (fish and crustaceans) when supplemented in appropriate quantities into feed to meet the animal requirements Safety for the consumer Absorption, distribution, metabolism and excretion of methionine have already been extensively reviewed by the FEEDAP Panel in The consumer will be exposed to L-methionine, not to DL-methionyl-DL-methionine per se. There is no evidence that excess of supplementary methionine results in any accumulation in animal tissues. After absorption, methionine is mainly used for protein synthesis or is metabolised to homocysteine via S- adenosyl-methionine and S-adenosyl-homocysteine or transaminated to 3-methylthiopropionate and then to volatile sulphur compounds (methanethiol and/or hydrogen sulphide). Homocysteine can further be converted to cystathionine and then to cysteine or recycled into methionine using 5- methyltetrahydrofolate or betaine (from diet or choline oxidation) as a methyl donor. Thus, it is concluded that there will be no additional consumer exposure to methionine above the natural basal content of food products from animals fed with compound feed supplemented with the additive under application Conclusions on the safety for the consumer L-Methionine from DL-methionyl-DL-methionine is incorporated into the protein of tissues of fish and crustaceans, showing a constant amino acid pattern. Consequently, the use of the additive will not result in an increased content of methionine in tissues and products. Doses exceeding animal requirements will be catabolised and/or excreted Safety for the user 21 Studies on the safety for the user have been performed with the product under application. Dusting potential and particle size distribution show a potential for users to be exposed to respirable dust. The technical dossier contains sufficient information on the effects of the additive on the skin, eyes and the respiratory system to conclude on the safety for the user Conclusions on the safety for the user The product DL-methionyl-DL-methionine is not regarded as an irritant to the eyes and the results of an in vitro study indicated that it is unlikely to be an irritant to the skin. It is also considered likely not to be a sensitiser by the dermal route. Since there is no evidence of acute toxicity by the inhalation route, it is not regarded as hazardous for the user Safety for the environment DL-Methionyl-DL-methionine will initially be converted in the animals to L-methionine, which is a physiological and natural component of proteins in animals and plants. It is excreted not as L- methionine but mainly as ammonia in teleost fish and as sulphate and carbon dioxide. The use of DLmethionyl-DL-methionine in aquatic species would not lead to any localised increase in its concentration in the environment. It is concluded that the use of this product as a feed additive for aquatic species does not represent a risk to the environment. 21 This section has been amended following the confidentiality claims made by the applicant. EFSA Journal 2015;13(2):

11 4. Efficacy 22 DL-Methionyl-DL-methionine for all aquatic animals The enzymatic transformation of D- to L-methionine has been detected in food-producing animals but also in carnivorous and omnivorous fish, shrimp and prawns (Robinson et al., 1978; Schwarz et al., 1998; Kim et al., 1992; Sveier et al., 2001; Forster and Dominy, 2006). The applicant provided some in vitro and in vivo studies to demonstrate that (i) DL-methionyl-DLmethionine is hydrolysed in the gut to methionine and (ii) DL-methionyl-DL-methionine can contribute to meet the requirement for methionine in certain fish species and shrimp Conclusions from the in vitro studies There is clear evidence for the presence of enzymes that can cleave DL-methionyl-DL-methionine into methionine in tissues of rainbow trout, mirror carp and shrimp Conclusions of the in vivo studies The studies with Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and penaeid shrimp (Litopenaeus vannamei), demonstrate the potential of the dipeptide DL-methionyl-DLmethionine to substitute for the free amino acid DL-methionine as source of L-methionine Conclusions on efficacy The FEEDAP Panel concludes that DL-methionyl-DL-methionine can replace supplemental free amino acid methionine in feed for all aquatic animal species and therefore is an effective source of the essential amino acid L-methionine. This has been demonstrated by (i) in vitro studies with tissues of rainbow trout and carp, (ii) with preparations of tissues from rainbow trout, carp and shrimp and (iii) in in vivo studies on tilapia, trout and shrimp. 5. Post-market monitoring The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 23 and Good Manufacturing Practice. CONCLUSIONS The dipeptide DL-methionyl-DL-methionine will be cleaved in the organism to methionine. Since supplementing appropriate amounts of methionine to meet requirements is safe for the target species, the FEEDAP Panel extends this conclusion to the safety of DL-methionyl-DL-methionine for fish and crustaceans. The amino acid L-methionine originating from the metabolism of DL-methionyl-DL-methionine in the body is incorporated into the tissue proteins of aquatic animal species, without affecting their amino acid composition. There is no concern for the safety of consumers arising from the use of the additive under application in aquatic species nutrition. The product DL-methionyl-DL-methionine is not regarded as an irritant to the eyes and the results of an in vitro study indicated that it was unlikely to be an irritant to the skin. It is also considered likely not to be a sensitiser by the dermal route. Since there is no evidence of acute toxicity by the inhalation route it is not regarded as hazardous for the user. The use of DL-methionyl-DL-methionine as an additive to feed for aquatic animal species does not represent a risk to the environment. 22 This section has been amended following the confidentiality claims made by the applicant. 23 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, , p. 1. EFSA Journal 2015;13(2):

12 Based on in vitro and in vivo studies with rainbow trout, carp, tilapia and shrimp, the FEEDAP Panel concludes that the product DL-methionyl-DL-methionine is an efficacious source of essential amino acid L-methionine for fish and crustacean species. DOCUMENTATION PROVIDED TO EFSA 1. DL-methionyl-DL-methionine for all aquatic animals including crustaceae. November Submitted by Evonik Industries AG. 2. DL-methionyl-DL-methionine for all aquatic animals including crustaceae. Supplementary information. October Submitted by Evonik Industries AG. 3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for DL-Methionyl-DL-Methionine. 4. Comments from Member States received through the ScienceNet. REFERENCES Dabrowski K, Lee KJ and Rinchard J, The smallest vertebrate, teleost fish, can utilize synthetic dipeptide-based diets. Journal of Nutrition, 133, EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Scientific Opinion on DL-methionine, DL-methionine sodium salt, hydroxy analogue of methionine, calcium salt of methionine hydroxy analogue for all animal species; isopropyl ester of methionine hydroxy analogue and DL-methionine technically pure protected with copolymer vinylpyridine/styrene for dairy cows; DL-methionine technically pure protected with ethylcellulose for ruminants. EFSA Journal 2012;10(3):2623, 42 pp. doi: /j.efsa EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Scientific Opinion on the safety and efficacy of L-methionine produced by Escherichia coli (KCCM 11252P) and Escherichia coli (KCCM 1134P) for all animal species. EFSA Journal 2013;11(10):3428, 16 pp. doi: /j.efsa Forster IP and Dominy WG, Efficacy of three methionine sources in diets for Pacific white shrimp, Litopenaeus vannamei. Journal of the World Aquaculture Society, 37, Friedman M and Gumbmann MR, Nutritional value and safety of methionine derivatives, isomeric dipeptides and hydroxy analogs in mice. The Journal of Nutrition, 118, Gilbert, ER, Wong, EA and Webb KE Jr, Board invited review: peptide absorption and utilization: implications for animals nutrition and health. Journal of Animal Science, 86, Gropp J, Wagner HW, Schuhmacher A and Renz A, In-vitro Untersuchungen zum intestinalen Aminosäurentransport bei Regenbogenforellen (Salmo gairdneri Rich.). Journal of Animal Physiology and Animal Nutrition, 63, Kim Kl, Kayes TB and Amundson CH, Requirements for sulfur amino acids and utilization of D-methionine by rainbow trout (Oncorhynchus mykiss). Aquaculture, 101, Mello JPF, Amino acids as multifunctional molecules. In: Mello JPF (Ed) Amino acids in animal nutrition (second edition). p. 1 14, CABI Publishing, Wallingford, UK. NRC (National Research Council), Nutrient requirements of fish. The National Academies Press. Washington, DC, USA, Robinson EH, Allen OW Jr, Poe WR and Wilson RP, Utilization of dietary sulfur compounds by fingerling channel catfish: L-methionine, DL-methionine, methionine hydroxy analogue, taurine and inorganic sulfate. Journal of Nutrition, 108, EFSA Journal 2015;13(2):

13 Schuhmacher A, Wax C and Gropp JM, Plasma amino acids in rainbow trout (Oncorhynchus mykiss) fed intact protein and a crystalline amino acid diet. Journal of Aquaculture, 151, Schwarz JF, Kirchgessner M and Deuringer U, Studies on the methionine requirement of carp (Cyrprinus carpio L.). Aquaculture, 161, Sveier H, Nordas H, Berge GE and Lied E, Dietary inclusion of crystalline D and L-methionine: effects on growth, feed and protein utilisation, and digestibility in small and large Atlantic salmon (Salmon salar L.). Aquaculture Nutrition, 7, EFSA Journal 2015;13(2):

14 Appendix A. Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for DL-Methionyl-DL-Methionine 24 In the current application authorisation is sought under Articles 4(1) for DL-Methionyl-DL-ethionine, under the category/functional group 3(c) nutritional additives / amino acids, their salts and analogues according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of DL- Methionyl-DL-Methionine for all aquatic animal species. The feed additive is a white to cream powder having a minimum content of the active substance (Met-Met) of 93 % and a maximum DL-Methionine content of 3%. It is intended to be mixed either in premixtures or to be added directly to complete feedingstuffs. The Applicant suggested no minimum or maximum feed additive concentrations in feedingstuffs. For the quantification of DL-Methionyl-DL-Methionine in feed additive, premixtures and feedingstuffs the Applicant suggested at first the ring-trial validated Community method designed for the analysis of amino acids in premixtures and feedingstuffs. However, no experimental data were presented to demonstrate the applicability of the method to determine DL-Methionyl-DL-Methionine in the relevant matrices. Consequently, the Applicant was requested by the EURL to submit supplementary information, discussed hereafter. For the quantification of DL-Methionyl-DL-Methionine in feed additive a single-laboratory validated and further verified method based on Reversed Phase High Performance Liquid Chromatography coupled to photometric detection (RP-HPLC-UV) was submitted. The following performance characteristics were calculated by the EURL based on the experimental data provided: relative standard deviation for repeatability (RSDr) ranging from 0.50 to 0.92%; relative standard deviation for intermediate precision (RSDip) of 0.63%; and recovery rate (RRec) of 100.1%. Based on these satisfactory performance characteristics, the EURL recommends for official control, the singlelaboratory validated and further verified RP-HPLC-UV method to quantify DL-Methionyl-DL- Methionine in feed additive. For the quantification of DL-Methionyl-DL-Methionine in premixtures and feedingstuffs the Applicant submitted experimental data obtained applying the ring-trial validated Community method based on HPLC (or amino acid analyser) equipped with ion exchange column, post column derivatisation and photometric detection at 570 nm (IEC-UV). The Applicant applied the protocol for the determination of free (synthetic and natural) amino acids. The following performance characteristics were reported for premixtures: RSDr ranging from 5.2 to 5.4%; RSDip ranging from 4.0 to 4.3%; and RRec ranging from 89.1 to 101.9%; and for feedingstuffs: RSDr ranging from 1.6 to 4.4%; RSDip ranging from 1.9 to 4.0%; and RRec ranging from 89.8 to 90.8%. Based on these satisfactory performance characteristics, the EURL recommends for official control the ring-trial validated Community method, based on IEC-UV to quantify DL-Methionyl-DL-Methionine in premixtures and feedingstuffs. 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. 24 The full report is available on the EURL website: EFSA Journal 2015;13(2):