SCIENTIFIC OPINION. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

EFSA Journal 2014;12(2):3563 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of malic acid and a mixture of sodium and calcium malate when used as technological additives 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 An application has been made for the re-authorisation of malic acid as a preservative in feed and an acidity regulator in feed for cats and dogs and for the authorisation of a mixture of sodium and calcium salts of malic acid to be used as an acidity regulator in feed for all animal species. Evidence from feeding studies with dogs, poultry, piglets, veal calves and ruminants showed that the highest typical use level of 1 600 mg malic acid/kg complete feedingstuffs (supplied as acid or salts) is safe for all animal species. Setting a maximum content was not considered as necessary. Malic acid and its sodium and calcium salts are permitted food additives without limit in the EU. The use of malic acid and its sodium and calcium salts in animal nutrition is safe for the consumer. Malic acid and its salts are considered as strongly irritant to the skin and mucosa and as a particular risk to the eyes. Exposure via inhalation for those handling the additives is also considered to present a risk. Malates are components of the diet of humans and animals and are rapidly and completely metabolised to CO 2. Consequently, their use in animal production would not pose a risk to the environment. Malic acid is used in food as a preservative. It is reasonable to expect that the effect seen in food will be observed in feed when it is used at comparable concentrations and under similar conditions. However, the FEEDAP Panel has reservations about the effectiveness of malic acid as a preservative in complete feedingstuffs with a moisture content of 12 %. The ability of malic acid and its salts to act as acidity regulators in feed has not been demonstrated. European Food Safety Authority, 2014 KEY WORDS malic acid, calcium malate, sodium malate, technological additive, preservative, acidity regulator 1 2 3 On request from the European Commission, Question No EFSA-Q-2010-01514, adopted on 29 January 2014. 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 Acknowledgement: The Panel wishes to thank the members of the Working Group on Organic Acids, including Andrew Chesson, Noel Dierick, Jurgen Gropp and Piet Western, 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), 2014. Scientific Opinion on the safety and efficacy of malic acid and a mixture of sodium and calcium malate when used as technological additives for all animal species. EFSA Journal 2014;12(2):3563, 18 pp. doi:10.2903/j.efsa.2014.3563 Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2014

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 application for the re-authorisation of DL-malic acid when used as a preservative in feed for all animal species and as an acidity regulator in feed for cats and dogs and for the authorisation of a mixture of the sodium and calcium salts of malic acid when used as an acidity regulator in feed for all animal species. No maximum contents are indicated, but typical use levels described by the applicant are around 1 600 mg/kg feed for the free acid and 500 to 2 000 mg/kg feed for the mixture of the salts. Both the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Scientific Committee for Food (SCF) concluded that there was clear evidence that both enantiomers of malic acid are readily metabolised by laboratory animals and humans and that there was no reason to distinguish between L-malic acid and DL-malic acid when considering their safe use in food. The Committee for Veterinary Medicinal Products (CVMP) also concluded that there was no reason to suggest that D-malic acid or the DL racemates should be regarded differently from the naturally occurring L-form. There is evidence from published feeding studies with dogs, poultry, piglets, veal calves and ruminants that the additive is safe when used in feed for all animal species at the highest typical use level of 1 600 mg malic acid/kg complete feedingstuffs, whether supplied as the free acid or its salts. The margin of safety is sufficiently high that setting a maximum content is not necessary. Malic acid and its sodium and calcium salts are permitted food additives in the EU that may be added directly to food intended for human consumption without any limitation. The use of malic acid and its sodium and calcium salts in animal nutrition is safe for the consumer. Malic acid and its salts are considered as strongly irritant to the skin and mucosa and as a particular risk to the eyes. Exposure via inhalation for those handling the additives is also considered to present a risk. Malates are normal constituents of the diet of humans and animals and, when ingested, are rapidly and completely metabolised to CO 2. The contribution of sodium or calcium, arising from the use of the salts of malic acid, to the background levels of these elements in the environment would be negligible. Consequently, the use of the additives in animal nutrition would not pose a risk to the environment. Malic acid is used in food as a preservative. It is reasonable to expect that the effect seen in food will be observed in feed when it is used at comparable concentrations and under similar conditions. However, the FEEDAP Panel has reservations about the effectiveness of malic acid as a preservative in complete feedingstuffs with a moisture content of 12 %. The ability of malic acid and its salts to act as acidity regulators in feed has not been demonstrated. EFSA Journal 2014;12(2):3563 2

TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 5 Assessment... 8 1. Introduction... 8 2. Characterisation... 8 2.1. Characterisation of the additives... 8 2.1.1. Malic acid... 8 2.1.2. Mixture of sodium and calcium malate... 9 2.2. Stability and homogeneity... 10 2.2.1. Storage conditions and shelf-life... 10 2.2.2. Stability in vitamin mineral premixtures... 10 2.2.3. Stability in feed... 10 2.2.4. Homogeneity... 11 2.3. Conditions of use... 11 2.4. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL). 11 3. Safety... 11 3.1. Safety for the target species... 11 3.1.1. Dogs... 11 3.1.2. Poultry... 12 3.1.3. Piglets... 12 3.1.4. Ruminants... 12 3.1.5. Veal calves... 13 3.1.6. Conclusion... 13 3.2. Safety for the consumer... 13 3.3. Safety for the user... 13 3.4. Safety for the environment... 13 4. Efficacy... 14 4.1. Preservation... 14 4.2. Acidity regulation... 14 Conclusions... 14 Documentation provided to EFSA... 14 References... 15 Appendix... 17 EFSA Journal 2014;12(2):3563 3

BACKGROUND Regulation (EC) No 1831/2003 4 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 and 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 ACIAC EEIG (Acids Authorisation Consortium European Economic Interest Grouping) 5 for authorisation/re-evaluation of the product malic acid, when used as a feed additive for all animal species (category: technological additive; functional group: preservative) and when used as a feed additive for cats and dogs (category: technological additive; functional group: acidity regulator) under the conditions mentioned in Table 1a, and for the product sodium and calcium malate when used as a feed additive for all animal species (category: technological additive; functional group: acidity regulator), under the conditions mentioned in Table 1b. 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 15 June 2011. Malic acid is currently authorised for use as preservative in feed for all animal species and categories and as acidity regulator in feed for cats and dogs without a time limit and without a maximum level. Sodium malate is presently authorised as acidity regulator in feed for cats and dogs without a time limit and without a maximum level. Calcium malate and the mixture of sodium and calcium malate are not authorised feed additives in the European Union. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) issued several opinions on malic acid and its sodium and calcium salts (JECFA, 1966, 1967, 1969, 1980). The Scientific Committee on Food (SCF) issued an opinion on malic acid and its salts (EC, 1991). The Committee For Veterinary Medicinal Products (CVMP) of the European Agency for Evaluation of Medicinal Products (EMEA) had delivered a report on malic acid (EMEA, 1997). The European Food Safety Authority (EFSA) issued an opinion on the safety and bioavailability of magnesium and zinc malate for use in food supplements and of calcium malate for use in food supplements, foods for particular nutritional uses, and foods intended for the general population (EFSA, 2006) and an opinion on calcium acetate, calcium pyruvate, calcium succinate, magnesium pyruvate magnesium succinate and potassium malate added for nutritional purposes to food supplements (EFSA, 2009). 4 5 6 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, 18.10.2003, p. 29. On 13/03/2013, EFSA was informed by the applicant that ACIAC EEIG was liquidated on 19/12/2012 and their rights as applicant were transferred to FEFANA asbl (EU Association of Specialty Feed Ingredients and their Mixtures). Avenue Louise, 130A, Box 1, 1050 Brussels, Belgium. Companies: Polynt S.p.A., Scanzorosciate, Italy; Norel S.A, Llica de Vall, Spain. EFSA Dossier reference: FAD-2010-0143. EFSA Journal 2014;12(2):3563 4

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 malic acid and sodium/calcium malate, when used under the conditions described in Tables 1a and 1b. EFSA Journal 2014;12(2):3563 5

Table 1a: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No Category(ies) of additive Functional group(s) of additive Malic Acid E 296 1. Technological additives a. Preservatives and, j. Acidity regulators Malic Acid Composition, description Description Chemical formula C 4 H 6 O 5 Purity criteria Assay Min. 99.5% by wt Water Min. 0.5% by wt Sulphated Ash Max. 0.02% by wt Fumaric acid Max. 1.0% by wt Maleic acid Max. 0.05% by wt Method of analysis HPLC (malate ion measured) Trade name Name of the holder of authorisation Not applicable Not applicable Species or category of animal All species and categories of animal Maximum Age All species and categories of animal Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs None specified None specified None specified Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring Specific conditions for use in complementary feedingstuffs Marker residue Other provisions and additional requirements for the labelling None specified None specified Not applicable Not applicable Maximum Residue Limit (MRL) 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 2014;12(2):3563 6

Table 1b: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No Category(ies) of additive Functional group(s) of additive Sodium Calcium Malate E 350(i) and E 352(i) 1. Technological additives j. Acidity regulators Composition, description Sodium Calcium Malate Chemical formula Mixture of C 4 H 4 O 5 Na 2 and C 4 H 4 O 4 Ca Description Purity criteria Malic acid Min. 60% by wt Sodium Max. 4% by wt Calcium Max. 20% by wt Arsenic Max. 3 mg/kg Lead Max. 5 mg/kg Iron Max. 5 mg/kg Method of analysis HPLC (malate ion measured) Trade name Name of the holder of authorisation Not applicable Not applicable Species or category of animal All animal species Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs None specified None specified None specified None specified Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring Specific conditions for use in complementary feedingstuffs Marker residue Other provisions and additional requirements for the labelling None specified None specified Not applicable Not applicable Maximum Residue Limit (MRL) 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 2014;12(2):3563 7

ASSESSMENT 1. Introduction Malate occurs in all living organisms as an intermediate in the citric acid cycle. It occurs in relatively high amounts in many fruits and vegetables. Malic acid has two stereoisomeric forms (L- and D- enantiomers), although only the L-isomer exists naturally. Malic acid, sodium malate and calcium malate are individually approved food additives in the EU 7 used for a variety of technological functions and they are also found in cosmetics 8 and in some pharmacological preparations. 9 DL-Malic acid (E 296) is presently listed in the EU Register of Feed Additives 10 as a preservative for use in feed for all animal species without restrictions and for use as an acidity regulator in feed for dogs and cats. Sodium malate (E 350) is also authorised, but only as an acidity regulator in feed for dogs and cats. The calcium salt is not currently authorised for any purpose as a feed additive in the European Union. The application under consideration is for the re-authorisation of DL-malic acid as a technological additive when used as a preservative in feed for all animal species and an acidity regulator in feed for cats and dogs as required by Regulation (EC) No 1831/2003. 11 An application for the authorisation of a mixture of the sodium and calcium salts of malic acid when used as a technological additive (functional group: acidity regulator) in feed for all animal species is also included. 2. Characterisation 2.1. Characterisation of the additives 2.1.1. Malic acid DL-Malic acid (hydroxybutanedioic acid, HO 2 CCH 2 CHOHCO 2 H, Chemical Abstracts Service (CAS) No 6915-15-7 (or 617-48-1), European Inventory of Existing Commercial chemical Substances EINECS No 210-514-9) is a white crystalline powder with a molecular weight of 134.09. It is readily soluble in water (558 g/l at 20 C) with a pk a1 of 3.4 and pk a2 of 5.5. The structural formula of malic acid is reported in Figure 1. Figure 1: Structural formula of malic acid Malic acid is synthesised by hydration of maleic anhydride under high temperature and pressure to form malic and fumaric acid. The precipitate of the less soluble fumaric acid is separated by centrifugation and the resulting solution is concentrated to form crystals of crude malic acid, which are separated, re-dissolved in water and passed through a decolourisation unit containing activated 7 European Parliament and Council Directive No 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners. OJ L 61, 18.3.1995, p. 1. 8 European Commission Cosmetic Ingredients Database (CosIng). Available online: http://ec.europa.eu/consumers/sectors/cosmetics/cosing/index_en.htm 9 European Pharmacopeia 5th edition. (European Treaty Series, No 50). Directorate for the Quality of Medicines of the Ph. Eur., Strasbourg, France. 10 Available online: http://ec.europa.eu/food/food/animalnutrition/feedadditives/registeradditives_en.htm 11 OJ L 268, 18.10.2003, p. 29. EFSA Journal 2014;12(2):3563 8

charcoal. After concentration of the solution, pure crystals of malic acid are formed, which are separated and dried. DL-Malic acid is manufactured for use as a food additive to meet the specification set by Commission Directive 2008/84/EC 12 of not less than 99.0 % (w/w). Analysis of five samples of the additive showed that this specification was exceeded in all cases (mean of 99.8 %) with trace amounts of water essentially accounting for the remainder. 13 Directive 2008/84/EC also sets maximum limits for the process and other contaminants including fumaric acid (1.0 % by weight), maleic acid (0.05 %), arsenic ( 3 mg/kg), lead ( 5 mg/kg) and mercury ( 1 mg/kg). Analytical data from the same five samples 14 showed that values for fumaric acid (mean 0.6 %) and maleic acid (0.016 %) were compliant with this specification. All values for arsenic, lead and mercury were < 1 mg/kg product and within the specification. Additionally, it was shown that the iron content of all five batches was < 5 mg/kg product. The particle size distributions of five commercial batches of malic acid, said to be representative of the material intended for use as an additive in feed, have been determined by laser diffraction. 15 Approximately 20 % of particles (by volume) were of inhalable size (< 100 μm diameter), 13 % had diameters of < 50 μm and ~ 6 % were respirable (< 10 μm diameter). No information on the dusting potential of malic acid was provided. 2.1.2. Mixture of sodium and calcium malate Calcium malate (calcium hydroxybutanedioic acid, Ca(C 2 H 4 O(COO) 2 ), CAS No 17482-42-7, molecular weight 172.15) and sodium malate (disodium salt of hydroxybutanedioic acid, Na 2 (C 2 H 4 O(COO) 2 ), CAS No 676-46-0, molecular weight 178.05 excluding water of hydration) are simple salts of malic acid. Both are white powders and, in contrast to the free acid, are poorly soluble in water. The mixture of the sodium and calcium salts, the subject of this assessment, is the result of the addition of sodium hydroxide and calcium oxide to DL-malic acid in sufficient quantities to ensure complete neutralisation. The feed specifications proposed by the applicant include an upper limit of 4 % sodium, equating to no more than 15 % sodium malate by weight, and 20 % calcium, equivalent to 86 % calcium malate by weight. Analysis of five commercial batches of the mixture showed a mean malic acid content of 62.0 % by weight (range 58.3 66.3 %), a mean sodium content of 2.8 % (range 2.7 3.0 %), a mean calcium content of 17.6 % (range 17.3 18.3 %) and a mean moisture content of 9.5 %. 16 Although these values comply with the proposed specification, they account for only 92 % of the total mass of the additive. Even taking into account the identified impurities, the sum of the identified components does not exceed 93 %. The applicant also identifies approximately 3 % insoluble material of unknown composition. As the starting material is malic acid of high purity, such insoluble material is most likely to be derived from the mineral precursor. Analysis of a further three batches of the mixture gave a mean value for malic acid of 64.0 %, a mean sodium content of 1.0 %, a mean calcium content of 18.8 %, a mean fumaric acid content of 0.16 % and a mean moisture content of 12.9 %. 17 This adds up to a mean content of 96.9 % for the identified components, with the insoluble material probably accounting for the remainder. X-ray diffraction 12 Commission Directive 2008/84/EC of 27 August 2008 laying down specific purity criteria on food additives other than colours and sweeteners. OJ L 253, 20.9.2008, p. 1. 13 Technical Dossier/Section II/Annex_II_1_Analytical Data. 14 Technical Dossier/Section II/Annex_II_1_Analytical Data. 15 Technical Dossier/Section II/Annex_II_1_Analytical Data. 16 Supplementary Information October 2013. 17 Supplementary Information February 2013/Annex_Qii_Malate_product description. EFSA Journal 2014;12(2):3563 9

analysis also showed the presence of calcium carbonate, presumably accounting for the insoluble fraction. 18 The additive is considered a simple admixture of the two salts and, as such, is not differentiated from the individual salts in chemical databases. Food additive legislation recognises the two salts individually but not the mixture and applies a very similar specification for both. Although the admixture does not meet the purity criteria applied by food additive legislation (~ 98 % by weight) to the individual components, the specifications set for impurities could apply. These are essentially the same set for the free acid, other than the inclusion of fluoride (< 30 mg/kg additive). Data from five batches of the mixture 19 (four batches for heavy metals and arsenic) showed clear compliance with these specifications. The applicant also analysed the batches for cadmium content, with values below 0.1 mg/kg additive. The particle size distribution of a single commercial batch of the mixture of sodium and calcium salts of malic acid in powder form was determined by laser diffraction. 20 All particles were found to have diameters of 34 μm or less with approximately 70 % (by volume) below 10 μm. The dusting potential as determined by the Stauber Heubach method was high at 15.2 g/m 3 (based on a single product batch). 21 2.2. Stability and homogeneity 22 2.2.1. Storage conditions and shelf-life The proposed shelf-life of malic acid is at least two years when stored in the appropriate packaging at ambient temperature in a dry, well-ventilated place away from direct sunlight. This proposal was supported by the analysis of three batches of malic acid stored for two years at 20 25 C. After the storage period, no losses were detected. A duplicate study with the mixture of salts similarly demonstrated a shelf-life of at least two years. 2.2.2. Stability in vitamin mineral premixtures Typical layer, piglet and ruminant vitamin mineral premixtures were formulated with either malic acid or the mixture of its sodium and calcium salts at inclusion levels equivalent to 1 %, 1 % and 1.5 % malic acid, respectively, on a final feed basis. All samples were stored at ambient temperature and analysed at the start of the study and after six months. No significant losses were observed over the test period. This confirms that malic acid and the mixture of its salts are stable in premixtures for at least six months. 2.2.3. Stability in feed Single batches of broiler, piglet and ruminant feeds were formulated as examples of the major applications of malic acid and its salts in animal nutrition. The inclusion levels of malic acid, or sodium/calcium malate, were equivalent to 1 % malic acid in each of the feeds. Both meal and pelleted forms of the broiler and piglet feeds were studied because both are available commercially, whereas, for the ruminant feed, only the pelleted form was monitored. No losses of malate, whether supplied as the free acid or as its salts, were recorded in either mash or pelleted feed after three months in storage at ambient temperatures. In most cases, a small increase was observed, probably because only the anion was monitored and additional malate was shown to be present in all feed materials. 18 Supplementary Information October 2013/Annex_X-ray_method_1 and Annex_X-ray_method_2. 19 Technical Dossier/Section II/Annex_II_1_Analytical Data. 20 Technical Dossier/Section II/Annex_II_1_Analytical Data. 21 Supplementary Information February 2013/Annex_Qv_Malate_Dusting potential. 22 Technical dossier/section II/Annex_II_3_Stability Data. EFSA Journal 2014;12(2):3563 10

2.2.4. Homogeneity Malic acid and sodium calcium malate for all animal species Ten sub-samples were collected from each of the five feeds used in the stability studies formulated with either malic acid or the mixture of its sodium and calcium salts at inclusion levels of 1 % (malic acid) additive. The malate content was measured in each sub-sample and the coefficient of variation (CV) was calculated for each form of the additive and each feed. The individual calculated CVs did not exceed 5 % and, when averaged across the five feeds, a value of 3.2 % was obtained for the malic acid-treated feeds and a value of 3.6 % was obtained for the mixture of salts. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) notes that the additive concentrations used in the stability and homogeneity studies in premixtures and feedingstuffs are higher than the indicated typical use level (see section 2.3). 2.3. Conditions of use Malic acid is intended for use as a preservative for all feedingstuffs and all animal species without restriction. The applicant indicates a typical use level of 1.5 to 1.6 g/kg feedingstuff, but the actual amount used may vary with respect to moisture content, ph and microbial load in the feed and the proposed duration of storage. The same use level would apply when malic acid is used as an acidity regulator in feed for dogs and cats. The admixture of sodium and calcium malate is intended for use only as an acidity regulator in feedstuffs for all animal species at an indicative level of 0.5 to 2.0 g/kg complete feed. 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 the malic acid and sodium calcium malate in animal feed. The Executive Summary of the EURL report can be found in Appendix A. 3. Safety Both the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 1980) and the Scientific Committee for Food (SCF) (EC, 1991) concluded that there was clear evidence that both enantiomers of malic acid are readily metabolised by laboratory animals and humans and that there was no reason to distinguish between L-malic acid and DL-malic acid when considering their safe use in food. The Committee for Veterinary Medicinal Products (CVMP) also concluded that there was no reason to suggest that D-malic acid or the DL racemates should be regarded differently from the naturally occurring L-form (EMEA, 1997). 3.1. Safety for the target species Although specific tolerance studies were not provided, a number of feeding studies have been provided by the applicant, which include concentrations of malate or the free acid higher than the highest proposed typical use level. Where the enantiomeric form of the acid was cited, this is indicated, otherwise the test item is described simply as malic acid. 3.1.1. Dogs A 104-week feeding study with DL-malic acid was conducted in beagle dogs (TRW/Hazleton Laboratories, 1971, as described in CIR, 2001). The dogs (four/sex/group) were provided diets supplemented with 0, 500, 5 000 or 50 000 mg/kg malic acid without any reported adverse effects. Body gains were reported to be normal for all animals and no significant changes in haematological, blood or urine parameters were observed. No significant lesions were detected upon macro- or microscopic examinations and no dose-dependent changes in absolute or relative organ weights were reported. As the highest dietary level of malic acid examined in this study is about 25-fold greater than EFSA Journal 2014;12(2):3563 11

the anticipated maximum use level of 1 600 mg/kg complete feedingstuff, no safety concerns would be anticipated in dogs. 3.1.2. Poultry An experiment was made to evaluate the effects of malic acid in a maize soybean meal diet on the growth performance of chickens for fattening (Biggs and Parsons, 2008). Male chickens were divided into three groups and fed the unsupplemented basal diet or diet enriched with 20 000 or 40 000 mg malic acid/kg diet for 21 days. Each group had five replicates with five birds/replicate, and malic acid at the inclusion rate of 40 000 mg/kg, but not 20 000 mg/kg, significantly decreased feed intake and thus the weight gain of chicks when compared with those fed the control diet. There were no significant changes in feed efficiency between the control and treatment groups. Although duration was short and the observations were limited to performance parameters, chickens appeared to tolerate malic acid at a concentration some 10-fold greater than the anticipated use level. 3.1.3. Piglets Six organic acids, including malic acid, were investigated for their efficacy in the control of postweaning diarrhoea syndrome of piglets under field conditions (Tsiloyiannis et al., 2001). This study was carried out on a commercial pig farm approximately four weeks after a diarrhoea outbreak. Groups of weaned piglets (mean weaning age of 25 days), were provided a basal diet or a basal diet supplemented with 12 000 mg malic acid/kg feed for a period of 28 days. There were four replicate groups of six male and six female piglets per treatment group. The clinical condition of the piglets was monitored daily and each piglet was weighed at weaning and on days 7, 14, 21 and 28 of the study. Feed conversion per pen was also determined. The severity of diarrhoea per pen was scored daily and the weekly diarrhoea score for each pen was calculated. Weekly diarrhoea scores and overall mortality rate in the malic acid group was significantly lower than in the control group. Piglets in the malic acid group were significantly heavier at 28 days than those in the control group. No adverse health effects were reported. The duration of the study was short and only performance parameters were considered. However, piglets appeared to tolerate malic acid at a concentration about eight-fold greater than the highest proposed use level. 3.1.4. Ruminants Three published studies with dairy cows designed to examine the effects of malic acid supplementation on milk yield and composition are reported. Malic acid was supplied to the cows at a calculated level of approximately 6 800 mg/kg complete feed (88 % dry matter) for 100 days (Kung et al., 1982), 10 700 mg/kg complete feed for 63 days (Wang et al., 2009) and 23 000 mg/kg complete feed for 42 days (Foley et al., 2009a). No significant effects on milk yield or composition compared with untreated controls were seen in any of the studies or on plasma levels of urea and glucose in the two studies where this was measured. In two 28-day experiments with beef cattle, reduced feed intakes were observed following the inclusion of DL-malic acid at 22 000 mg/kg complete feed, the lowest dose tested (Foley et al., 2009b). Dietary supplementation with DL-malic acid or the mixed salts of malic acid at a level of 3 500 mg/kg complete feed did not result in any significant changes in the performance of growing/finishing bull calves (Belgian Blue) over a 148-day period (Castillo et al., 2007). Performance parameters of average daily weight gain, daily feed intake and feed-to-gain ratio were unaffected by either product when compared with a control group fed unsupplemented feed. Martin et al. (1999) also performed three separate experiments to determine the effects of dietary supplementation of malic acid on feedlot cattle performance. The treatment levels of malic acid corresponded to use levels expressed on a complete feed basis of approximately 3 500 or 6 000 mg/kg (experiment 1), 4 300 or 8 800 mg/kg (experiment 2) or 10 200 mg/kg (experiment 3). The duration of the three experiments was 98, 52 and 133 days, respectively. No adverse effects of treatment on cattle performance, including dry matter intake, EFSA Journal 2014;12(2):3563 12

average daily weight gain and feed efficiency, were observed in comparison with animals fed the unsupplemented diet. The use of sodium/calcium malate as a feed additive in the diet of Merino lambs at concentrations of 3 300 or 6 600 mg/kg (highest concentration tested) complete feedingstuffs did not adversely affect feed intake and digestion, performance parameters or plasma concentrations of various metabolites including glucose, urea-nitrogen, cholesterol, triglycerides and total volatile fatty acids measured after 35 days (Carro et al., 2006). Although the studies described have some limitations (mostly, only zootechnical parameters were observed), overall they suggest that ruminants appear to tolerate malic acid at concentrations ranging between about 3- and 15-fold the highest proposed use level. 3.1.5. Veal calves A total of 16 individually housed veal calves (Pie Rouge de Campine) with an initial body weight of about 44 kg were allocated to two dietary treatments (six males and two female calves per treatment) and were fed a milk replacer treated with 0 or 20 g malic acid/kg feed for 95 days (De Vuyst et al., 1974). Total weight gain and feed-to-gain ratio were not significantly different between treatments. 3.1.6. Conclusion Considering all the studies provided, the FEEDAP Panel concludes that the additive is safe when used in feed for all animal species at the highest typical use level of 1 600 mg malic acid/kg complete feedingstuffs, whether supplied as the free acid or its salts. The margin of safety is sufficiently high that setting a maximum content is not necessary. 3.2. Safety for the consumer Malic acid is an intermediate in the citric acid cycle. Malic acid and its sodium and calcium salts are permitted food additives that may be added directly to food intended for human consumption without any limitation. JECFA (1980) established a group acceptable daily intake (ADI) as not specified for malic acid and its sodium, calcium and potassium salts. Subsequently the SCF (EC, 1991) agreed with the group ADI not specified set by JECFA. The CVMP (EMEA, 1997) studied the safety of use of malic acid and it sodium, calcium and potassium salts in veterinary medicines, and concluded that there was no need to set maximum residue levels (MRLs) to limit consumer exposure via residues in foods derived from treated animals. There are no conventional toxicological studies available and the above assessments were based on the well-established metabolic pathway and the observation that malic acid is ubiquitous in the diet. The FEEDAP Panel concludes that the use of malic acid and its sodium and calcium salts in animal nutrition is safe for the consumer. 3.3. Safety for the user Malic acid and its salts, like those of most carboxylic acids, are considered as strongly irritant to the skin and mucosa and as a particular risk to the eyes. In the absence of data on inhalation toxicity, inhalation of the additive should be considered as hazardous. Because of the particle size distribution of the additives and the high dusting potential of the malate salts, it is likely that handling the additives could result in a production of respirable dust that could present a risk to unprotected workers. 3.4. Safety for the environment Malates are normal components of the diet of humans and animals and, when ingested, they are rapidly and completely metabolised to CO 2. The contribution of sodium or calcium, arising from the use of the salts of malic acid, to the background levels of these elements in the environment would be EFSA Journal 2014;12(2):3563 13

negligible. Consequently, the FEEDAP Panel concludes that the use of malic acid or a mixture of its sodium and calcium salts in animal nutrition would not pose a risk to the environment. 4. Efficacy 4.1. Preservation Malic acid is an authorised food additive within the EU and elsewhere in the world and is used for a number of technical functions including that of a preservative. It is reasonable to expect that the effect in food will be observed in feed when it is used at comparable concentrations and under similar conditions. The FEEDAP Panel has reservations about the effectiveness of malic acid as a preservative in complete feedingstuffs with a moisture content of 12 %. However, it is recognised that, under practical conditions of storage, the moisture content of all or part of the feed may rise above this level. Under these circumstances, the additive could be effective in preventing or reducing deterioration. 4.2. Acidity regulation An in vitro study was conducted with a single feed of unknown composition or purpose to demonstrate the effect of malic acid and the mixture of its calcium and sodium salts on the ph and buffering capacity of the feed. Small samples of the feed (10 g) were dispersed in 100 ml water and treated with increasing volumes of 1 M hydrogen chloride (0 8 ml) 23 or 0.1 M sodium hydroxide (0 20 ml) 24 in the presence or absence of 0.15 % malic acid or 0.1 % of the malate salts. The presence of malic acid or malate did not have significant effects on the fall of ph accompanying increasing hydrogen chloride addition, or on the rise in ph following the various volumes of sodium hydroxide addition. The study does not demonstrate the capacity of the additives to act as acidity regulators in feedingstuffs. CONCLUSIONS There is evidence from published feeding studies with dogs, poultry, piglets, veal calves and ruminants that the additive is safe when used in feed for all animal species at the highest typical use level of 1 600 mg malic acid/kg complete feedingstuffs, whether supplied as the free acid or its salts. The margin of safety is sufficiently high that setting a maximum content is not necessary. The use of malic acid and its sodium and calcium salts in animal nutrition is safe for the consumer. Malic acid and its salts are considered as strongly irritant to the skin and mucosa and as a particular risk to the eyes. Exposure via inhalation for those handling the additives is also considered to present a risk. The use of malic acid and its sodium and calcium salts in animal nutrition is safe for the environment. Malic acid is considered effective as a feed preservative. However, the FEEDAP Panel has reservations about the effectiveness of malic acid as a preservative in complete feedingstuffs with a moisture content of 12 %. The ability of malic acid and its salts to act as acidity regulators in feed has not been demonstrated. DOCUMENTATION PROVIDED TO EFSA 1. Malic acid and sodium calcium malate for all animal species. November 2010. Submitted by FEFANA asbl. 2. Malic acid and sodium calcium malate for all animal species. Supplementary information. February 2012. Submitted by FEFANA asbl. 23 Supplementary Information July 2012/Annex_Qii_Malic acid and Malate_acidity reg_hcl. 24 Supplementary Information July 2012/Annex_Qii_Malic acid and Malate_acidity reg_naoh. EFSA Journal 2014;12(2):3563 14

3. Malic acid and sodium calcium malate for all animal species. Supplementary information. July 2012. Submitted by FEFANA asbl. 4. Malic acid and sodium calcium malate for all animal species. Supplementary information. February 2013. Submitted by FEFANA asbl. 5. Malic acid and sodium calcium malate for all animal species. Supplementary information. October 2013. Submitted by FEFANA asbl. 6. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for malic acid and sodium calcium malate. 7. Comments from Member States received through the ScienceNet. REFERENCES Biggs P and Parsons CM, 2008. The effects of several organic acids on growth performance, nutrient digestibilities, and caecal microbial populations in young chicks. Poultry Science, 87, 2581 2589. Carro MD, Ranilla MJ, Giráldez FJ and Mantecón AR, 2006. Effects of malate on diet digestibility, microbial protein synthesis, plasma metabolites, and performance of growing lambs fed a highconcentrate diet. Journal of Animal Science, 84, 405 410. Castillo C, Benedito JL, Pereira V, Vázquez P, López Alonso M, Méndez J and Hernádez J, 2007. Malic acid supplementation in growing/finishing feedlot bull calves: influence of chemical form on blood acid-base balance and productive performance. Animal Feed Science and Technology, 135, 222 235. CIR (Cosmetic Ingredient Review), 2001. Final Report on the safety assessment of malic acid and sodium malate. International Journal of Toxicology, 20, 47 55. De Vuyst A, Moreels A, Ska P, Arnould R and Romedenne J, 1974. De waarde van appelzuur in de voeding van mestkalveren. Landbouwtijdschrift, 27, 35 41. EFSA (European Food Safety Authority), 2006. Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission related to Calcium, Magnesium and Zinc Malate added for nutritional purposes to food supplements as sources for Calcium, Magnesium and Zinc and to Calcium Malate added for nutritional purposes to foods for particular nutritional uses and foods intended for the general population as source for Calcium. The EFSA Journal 2006, 391a,b,c,d, 1 6. EFSA (European Food Safety Authority), 2009. Scientific Opinion of the Panel on Food Additives and Nutrient Sources added to Food on calcium acetate, calcium pyruvate, calcium succinate, magnesium pyruvate magnesium succinate and potassium malate added for nutritional purposes to food supplements following a request from the European Commission. The EFSA Journal 2009, 1088, 1 25. EMEA (The European Agency for Evaluation of Medicinal Products), 1997. Malic acid: summary report Committee For Veterinary Medicinal Products (CVMP). Available online: http://www.ema.europa.eu/docs/en_gb/document_library/maximum_residue_limits_- _Report/2009/11/WC500014853.pdf European Commission (EC), 1991. Food-science and techniques. First series of food additives of various technological functions. Reports of the Scientific Committee for Food (SCF), 25th series. Available online: http://ec.europa.eu/food/fs/sc/scf/reports/scf_reports_25.pdf Foley PA, Kenny DA, Lovett DK, Callan JJ, Boland TM and O Mara FP, 2009a. Effect of DL-malic acid supplementation on feed intake, methane emissions, and performance of lactating dairy cows at pasture. Journal of Dairy Science, 92, 3258 3264. EFSA Journal 2014;12(2):3563 15

Foley PA, Kenny DA, Callan JJ, Boland TM and O Mara FP, 2009b. Effect of DL-malic acid supplementation on feed intake, methane emission, and rumen fermentation in beef cattle. Journal of Animal Science, 87, 1048 1057. JECFA (The Joint FAO/WHO Expert Committee on Food Additives), 1966. Specifications for the identity and purity of food additives and their toxicological evaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flour-treatment agents, acids, and bases. WHO Technical Report Series, 339. Available online: http://whqlibdoc.who.int/trs/who_trs_339.pdf JECFA (The Joint FAO/WHO Expert Committee on Food Additives), 1967. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers, stabilizers, flour-treatment agents, acids and bases. FAO Nutrition Meetings Report Series, 40 A,B,C. Available online: http://www.inchem.org/documents/jecfa/jecmono/40abcj01.htm JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1969. Specifications for the identity and purity of food additives and their toxicological evaluation: some colours, emulsifiers, stabilizers, anticaking agents and certain other substances. WHO Technical Report Series, 445. Available online: http://whqlibdoc.who.int/trs/who_trs_445.pdf JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1980. Evaluation of certain food additives. WHO Technical Report Series, 648. Available online: http://whqlibdoc.who.int/trs/who_trs_648.pdf Kung J Jr, Huber JT, Krummrey JD, Allison L and Cook RM, 1982. Influence of adding malic acid to dairy cattle rations on milk production, rumen volatile acids, digestibility and nitrogen utilisation. Journal of Dairy Science, 65, 1170 1174. Martin SA, Streeter MN, Nisbet DJ, Hill GM and Williams SE, 1999. Effects of DL-malate on ruminal metabolism and performance of cattle fed a high-concentrate diet. Journal of Animal Science, 77, 1008 1015. Tsiloyiannis VK, Kyriakis SC, Vlemmas J and Sarris K, 2001. The effect of organic acids on the control of porcine post-weaning diarrhoea. Research in Veterinary Science, 70, 287 293. Wang C, Liu Q, Yang W, Dong Q, Yang X, He D, Dong K and Huang Y, 2009. Effects of malic acid on feed intake, milk yield, milk components and metabolites in early lactaion Holstein dairy cows. Livestock Science, 124, 182 188. EFSA Journal 2014;12(2):3563 16

APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for malic acid and sodium calcium malate 25 In the current application authorisation is sought under article 10(2) for malic acid under the category of technological additives functional group 1a (preservatives) and 1j (acidity regulators), and under article 4(1) for sodium calcium malate under the category of technological additives functional group 1j (acidity regulators) according to the classification system of Annex I of Regulation (EC) No 1831/2003. According to the Applicant, malic acid is a white crystalline or granular solid. The feed additive has a minimum purity of 99.5%. According to the Applicant, sodium calcium malate is a mixture of sodium and calcium salts of malic acid and is a white crystalline or granular solid. The feed additive has a minimum purity of 60% expressed as malic acid content and a maximum sodium and calcium content of 4 % and 20%, respectively. Authorisation is sought for the use of malic acid as preservative for all categories and species and as acidity regulator for cats and dogs, while authorisation is sought for the use of sodium calcium malate for all categories and species. Both feed additives are intended to be mixed into premixtures and feedingstuffs. The Applicant suggested no minimum or maximum levels as in the previous legislation. For the quantification of malic acid in the feed additive, the EURL recommends for official control the method described in the European Pharmacopoeia Monograph 2080, based on potentiometrical, acid/base titration with 1 M sodium hydroxide. For the quantification of malic acid in premixtures and feedingstuff the Applicant proposed a method based on high performance liquid chromatography with refractive index or UV detection (HPLC- RI/UV); this method does not distinguish between malic acid and its salts. This HPLC- RI/UV method was further ring trial validated by five laboratories and a relative standard deviation for reproducibility (RSD R ) ranging from 4.3% to 7.5% was determined for premixtures and feedingstuffs containing 10 to 72 g malic acid/kg, respectively. The determination of sodium, calcium and malate is necessary for the characterisation of sodium calcium malate in feed additive. For the quantification of total sodium and total calcium in the product, the EURL recommends for official control the internationally agreed EN ISO 6869:2000 method, based on atomic absorption spectrometry. For the quantification of total malate in feed additive, premixtures and feedingstuffs the Applicant proposed the HPLC-UV/RI method mentioned above. Based on the performance characteristics presented, the EURL recommends for official control the ring trial validated ion-exchange HPLC-UV method to determine malic acid (expressed as total malic acid) in premixtures and feedingstuffs and sodium calcium malate (expressed as total malic acid) in feed additive, premixtures and feedingstuffs. 25 The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/sitecollectiondocuments/finrep-fad- 2010-0143.pdf EFSA Journal 2014;12(2):3563 17

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. EFSA Journal 2014;12(2):3563 18