SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of folic acid as a feed additive
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
EFSA Journal 2012;10(5):2674 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of folic acid as a feed additive for all animal species1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT Folic acid, a synthetic folate compound, is converted in animals to biologically active folates. These are essential for DNA synthesis, repair and methylation, in particular nucleotide biosynthesis and remethylation of homocysteine. Oral administration routes of folic acid via feed or water for drinking are considered bioequivalent. Folic acid is safe for the target animals and there is no need to define a maximum content in feed. Population exposure to folic acid/folates in Europe is below the tolerable upper intake level (1 mg/adult/day and 200 µg/toddler/day). As folic acid supplementation of animal feedingstuffs is widespread and routine, exposure figures already contain the contribution from edible tissues and the products of animals fed folic acid- supplemented diets. Liver and milk folate is not influenced by dietary folic acid. The only variation to be expected is in the concentration of folates in eggs and meat. The potential exposure of adults and toddlers resulting from the consumption of eggs and meat from treated animals is, however, small and would be about 60 and 27 µg/person/day, respectively. The FEEDAP Panel concluded that the use of folic acid in animal nutrition is not of concern for the safety of consumers. In the absence of any information, the FEEDAP Panel considers it prudent to treat folic acid as an irritant to skin, eyes and the respiratory tract and as a sensitiser. Folates occur widely in nature (in green vegetables and certain fruits, e.g. citrus). Folic acid added to feed would be excreted as the physiological end-products of folate metabolism. The use of folic acid in animal nutrition does not pose a risk to the environment. Folic acid is regarded as an effective source of folate in animal nutrition. © European Food Safety Authority, 2012 KEY WORDS Nutritional additive, vitamins and provitamins, folic acid, folate, safety 1 On request from the European Commission, Question No EFSA-Q-2010–01280, adopted on 24 April 2012. 2 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 Water-soluble Vitamins, including Annette Schuhmacher, 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 folic acid for all animal species. EFSA Journal 2012;10(5):2674. [18 pp.] doi:10.2903/j.efsa.2012.2674. Available online: www.efsa.europa.eu/efsajournal © European Food Safety Authority, 2012
Folic acid for all animal species 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 folic acid an additive to feed and water for drinking for all animal species. Folic acid, a synthetic folate compound, is converted in animals to biologically active folates. They are essential for DNA synthesis, repair and methylation, in particular nucleotide biosynthesis and remethylation of homocysteine. Oral administration routes of folic acid via feed or water for drinking are considered bioequivalent. Folic acid is safe for the target animals and there is no need to define a maximum content in feed. Population exposure to folic acid/folates in Europe is below the tolerable upper intake level (1 mg/adult/day and 200 µg/toddler/day). As folic acid supplementation of animal feedingstuffs is widespread and routine, exposure figures already contain the contribution from edible tissues and the products of animals fed folic acid-supplemented diets. Liver and milk folate is not influenced by dietary folic acid. The only variation to be expected is in the concentration of folates in eggs and meat. The potential exposure of adults and toddlers resulting from the consumption of eggs and meat from treated animals is, however, small and would be about 60 and 27 µg/person/day, respectively. Consequently, the FEEDAP Panel concluded that the use of folic acid in animal nutrition is not of concern for the safety of consumers. In the absence of any information, the FEEDAP Panel considers it prudent to treat folic acid as an irritant to skin, eyes and the respiratory tract and as a sensitiser. Folates occur widely in nature (in green vegetables and certain fruits, e.g. citrus). Folic acid added to feed would be excreted as the physiological end-products of folate metabolism. The use of folic acid in animal nutrition does not pose a risk to the environment. Folic acid is regarded as an effective source of folate in animal nutrition. The FEEDAP Panel recommends the use of the specifications for folic acid according to PhEur (MG 0067) in terms of purity, substance-related impurities and other impurities (sulphated ash). EFSA Journal 2012;10(5):2674 2
Folic acid for all animal species TABLE OF CONTENTS Abstract .................................................................................................................................................... 1 Summary .................................................................................................................................................. 2 Table of contents ...................................................................................................................................... 3 Background .............................................................................................................................................. 4 Terms of reference.................................................................................................................................... 4 Assessment ............................................................................................................................................... 6 1. Introduction ..................................................................................................................................... 6 2. Characterisation ............................................................................................................................... 7 2.1. Characterisation of the active substance ................................................................................... 7 2.2. Stability and homogeneity ........................................................................................................ 8 2.2.1. Shelf life of the additive ..................................................................................................... 8 2.2.2. Stability of the additive when added to premixtures, feed and water for drinking ............. 8 2.2.3. Homogeneity ...................................................................................................................... 9 2.3. Physicochemical incompatibilities in feed ................................................................................ 9 2.4. Conditions of use ...................................................................................................................... 9 2.5. Evauation of the analytical methods by the European Union Reference Laboratory (EURL) . 9 3. Safety ............................................................................................................................................... 9 3.1. Safety for the target species ...................................................................................................... 9 3.2. Safety for the consumer .......................................................................................................... 10 3.2.1. Absorption, distribution, metabolism and excretion of folic acid .................................... 10 3.2.2. Toxicology ........................................................................................................................ 11 3.2.3. Assessment of consumer safety ........................................................................................ 11 3.2.4. Conclusions on consumer safety ...................................................................................... 11 3.3. Safety for the user ................................................................................................................... 12 3.3.1. Effects on the respiratory system...................................................................................... 12 3.3.2. Effects on the eyes and skin ............................................................................................. 12 3.3.3. Conclusions on user safety ............................................................................................... 12 3.4. Safety for the environment...................................................................................................... 12 4. Efficacy.......................................................................................................................................... 12 5. Post-market monitoring ................................................................................................................. 13 Conclusions and recommendations ........................................................................................................ 13 Documentation provided to EFSA ......................................................................................................... 13 References .............................................................................................................................................. 13 Appendix A ............................................................................................................................................ 16 Appendix B ............................................................................................................................................ 17 Appendix B ............................................................................................................................................ 17 EFSA Journal 2012;10(5):2674 3
Folic acid for all animal species BACKGROUND Regulation (EC) No 1831/20034 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 time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from the VITAC EEIG Vitamins Authorisation Consortium5 for (i) authorisation of a new use (i.e. use in water for drinking) and (ii)e re-evaluation of the product folic acid, when used as a feed additive for all animal species (category: nutritional additive; functional group: vitamins, provitamins and chemically well-defined substances having similar effect) 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 16 December 2010. Folic acid has been authorised without time limit under Council Directive 70/524/EEC7 for its use for all animal species as a nutritional additive. The Scientific Committee on Food expressed an opinion on the tolerable upper intake level of folate (EC, 2000). The Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food issued a scientific opinion related to calcium L-methylfolate (EFSA, 2004). The Panel on Dietetic Products, Nutrition and Allergies (NDA) issued two opinions on substantiation of several health claims related to folate pursuant to Article 13(1) of Regulation (EC) No 1924/2006 (EFSA, 2009a, 2010 and 2011). The EFSA Scientific Cooperation Working Group issued a report on Analysis of Risk and Benefits of Fortification of Food with Folic acid (EFSA, 2009b). 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 folic acid, when used under the conditions described in Table 1. 4 Regulation (EC) 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. 5 VITAC EEIG Vitamins Authorisation Consortium, Avenue Louise 130A, B-1050 Brussel, Belgium. Companies: Chr. Olesen nutrition A/S, Gentofte, Denmark; DSM Nutritional Products LTD., The Netherlands; Feed Additive Technologies Ltd., Genolier, Switzerland; ORFFA International BV, The Netherlands. 6 EFSA Dossier reference: FAD-2010–0197. 7 Commission List of the authorised additives in feedingstuffs published in application of Article 9t (b) of Council Directive 70/524/EEC concerning additives in feedingstuffs (2004/C 50/01). OJ C 50, 25.2.2004, p. 1. EFSA Journal 2012;10(5):2674 4
Folic acid for all animal species Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Folic acid Registration number/EC No/No - (if appropriate) Category of additive 3 / Nutritional Additives a / vitamins, pro-vitamins and chemically well-defined Functional group(s) of additive substances having similar effect Description Chemical Purity criteria Method of analysis Composition, description formula (if appropriate) (if appropriate) Min 95 % (on dry Eur. Pharma material) 01/2005/0067 Folic acid C19H19N7O6 Loss on drying : max Eur. Pharma 8.5 % 2005/20232 Trade name (if appropriate) Not appropriate Name of the holder of Not appropriate authorisation (if appropriate) Conditions of use Minimum content Maximum content Species or Withdrawal category of Maximum Age mg/kg of complete feedingstuffs, period animal supplementary feed (based on end feed) and (if appropriate) in water All animal species and - - - - categories Other provisions and additional requirements for the labelling The product can be put on the market either as active Specific conditions or restrictions for use substance or as a preparation. It is in both forms applicable (if appropriate) in feed and in premix. The preparation may be used for water application. Specific conditions or restrictions for - handling (if appropriate) Post market monitoring - (if appropriate) Specific conditions for use in - complementary feedingstuffs or water (if appropriate) EFSA Journal 2012;10(5):2674 5
Folic acid for all animal species ASSESSMENT This opinion is based in part on data provided by a consortium of companies involved in the production/distribution of folic acid. It should be recognised that these data cover a fraction of the existing additives containing folic acid. The application is for the active substance, and the composition of the additive formulations is not the subject of the application. The Panel has sought to use the data provided, together with data from other sources, to deliver an opinion. 1. Introduction Folate is the generic name for a number of compounds having similar activity. Folic acid is a synthetic folate compound used in feed and in food supplements and food fortification because of its stability and high bioavailability compared with natural folates, and it becomes biologically active after reduction. Natural (dietary) folates are mostly reduced folates, i.e. derivatives of tetrahydrofolate (THF), such as 5-methyl-THF, 5-formyl-THF and 5,10-methylene-THF, and exist mainly as pteroylglutamates, with up to nine additional glutamate molecules attached to the pteridine ring. THF, and its derivatives, is the biologically active form in the organism upon conversion of dietary folates to dihydrofolic acid in the liver. Folates are essential for DNA synthesis, repair and methylation, in particular nucleotide biosynthesis and remethylation of homocysteine; consequently, folates are especially critical for biological processes involving rapid cell proliferation, such as embryogenesis and haematopoiesis. THF compounds are also substrates in a number of single-carbon-transfer reactions relevant to mitochondrial function, such as glutathione biosynthesis. Vitamin B12 is necessary for THF’s function in methyl transfer. Folic acid is included in the European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003. It is authorised without a time limit in application of Article 9t (b) of Council Directive 70/524/EEC8 concerning additives in feedingstuffs (2004/C 50/01) for its use in all animal species as a nutritional additive. The applicant has asked for a re-evaluation of the use of folic acid as an additive to feed for all animal species and categories without restrictions for age, (withdrawal) time and content in feedingstuffs. The applicant is also seeking evaluation of a new use for folic acid in water for drinking. Folic acid in the form of pteroylmonoglutamic acid and calcium L-methylfolate is authorised for use in food (Regulation (EC) No 1925/2006,9 amended by Regulation (EC) No 1170/2009)10 and in food supplements (Directive 2002/46/EC, Annex II),11 and for addition for specific nutritional purposes in foods for particular nutritional uses (Regulation (EC) No 953/2009),12 to processed cereal-based foods and baby foods for infants and young children (Directive 2006/125/EC, Annex IV)13 and to infant formulae and follow-on formulae when reconstituted as instructed by the manufacturer (Directive 2006/141/EC, Annex III).14 Folic acid is also listed as a pharmacologically active substance in veterinary medicinal products, and it is not subject to maximum residue levels when used in food- 8 Commission List of the authorised additives in feedingstuffs published in application of Article 9t (b) of Council Directive 70/524/EEC concerning additives in feedingstuffs (2004/C 50/01). OJ C 50, 25.2.2004, p. 1. 9 Regulation (EC) No 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of vitamins and minerals and of certain other substances to foods. OJ L 404 30.12.2006, p. 26. 10 Commission Regulation (EC) No 1170/2009 of 30 November 2009 amending Directive 2002/46/EC of the European Parliament and of Council and Regulation (EC) No 1925/2006 of the European Parliament and of the Council as regards the lists of vitamin and minerals and their forms that can be added to foods, including food supplements. OJ L 314 1.12.2009, p. 36. 11 Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements. OJ L 183 12.7.2002, p. 51. 12 Commission Regulation (EC) 953/2009 of 13 October 2009 on substances that may added for specific nutritional purposes in foods for particular nutritional uses. OJ L 269, 14.10.2009, p. 9. 13 Commission Directive 2006/125 EC of 5 December 2006 on processed cereal-based foods and baby-foods for infants and young children. OJ L 339 6.12.2006, p. 16. 14 Commission Directive 2006/141 EC of 22 December 2006 on infant formulae and follow-on formulae and amending Directive 1999/21/EC. OJ L 401 30.12.2006, p. 1. EFSA Journal 2012;10(5):2674 6
Folic acid for all animal species producing animals (Commission Regulation (EC) No 37/2010).15 It is listed as an ingredient in cosmetic products as a skin-conditioning agent (Commission decision 2006/257/EEC).16 Folic acid is described in the European Pharmacopeia (PhEur) in Monograph (MG) 0067. 2. Characterisation 2.1. Characterisation of the active substance Folic acid (IUPAC name: (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6- yl)methyl]amino]benzoyl]amino]pentanedioic acid; synonyms: pteroyl-L-glutamic acid, vitamin B9, vitamin M, folacin, 2-amino-6-((p-((1,3-dicarboxypropyl)carbamoyl)aniline)methyl)-4-pteridinol; N- (4-(((2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl)amino)benzoyl)-L-glutamic acid) is identified by the CAS (Chemical Abstracts Service) number 59-30-3 and the EINECS (European Inventory of Existing Chemical Substances) number 200-419-0. The structural formula of folic acid is shown in Figure 1. Figure 1: Structural formula of folic acid. The molecular formula of folic acid is C19H19N7O6, and its molecular weight is 441.4. It has a melting point of 250 °C (decomposition), a density of 1.68 g/cm3. Folic acid is practically insoluble in water (1.6 mg/L at 25 C, up to about 1 % in boiling water (PubChem, NCBI)). However, folic acid recrystallised four times showed a solubility of 561 mg/L at pH 6 (Wu et al., 2010). It dissolves in diluted acids and in alkaline solutions. Folic acid is produced by chemical synthesis. In the first step, 4-nitrobenzoyl chloride reacts with monosodium L-glutamate to form N-4-nitrobenzoyl-L-glutamic acid, which undergoes catalytic hydrogenation to form N-4-aminobenzoyl-L-glutamic acid. The additive is synthesised by condensing N-4-aminobenzoyl-L-glutamic acid with 2,4,5-triamino-6-hydroxypyrimidine and 1,1,3- trichloroacetone. The product is purified by a multi-step process. The applicant provided a flow chart showing the generic synthetic process. To formulate the additive containing a minimum of 80 % folic acid, the active substance is dispersed into an aqueous solution containing maltodextrins as a carrier and spray dried. Folic acid is a yellowish to orange, crystalline and practically odourless and tasteless powder. PhEur MG 0067 requires a minimum of 96 % folic acid in the anhydrous substance. Analysis of 17 batches of the product from four producers showed an average content of folic acid of 98.2 ± 0.9 % in the dried substance within a narrow range of 97.4 ± 0.2 % to 99.4 ± 0.2 %. Water content was in the range of 6.9–8.3 % (PhEur 5.0–8.5 %).17 Folic acid complies with PhEur MG 0067 for both criteria. The amount of substance-related impurities (known as impurities A–F) and sulphated ash comply with the thresholds of PhEur, as demonstrated by the analysis of 10 batches. Heavy metals (expressed as 15 Commission Regulation (EU) 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. OJ L 15, 20.1.2010, p. 1. 16 Commission Decision 2006/257/EC of 9 February 2009 amending Decision 96/335/EC establishing an inventory and a common nomenclature of ingredients employed in cosmetic products. OJ L 97 5.04.2006, p. 1. 17 Technical dossier/Section II/annexes 2.3–2.6. EFSA Journal 2012;10(5):2674 7
Folic acid for all animal species lead) were < 20 mg/kg. Data for residual solvents (toluene, acetone and ethanol) indicated that VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products) thresholds are not exceeded. Control measures are in place.18 Three batches, each from a different company, were analysed for particle size distribution, determined using either laser diffraction analysis or a sieving method. The particle fraction below 50 µm was 80.1 % and 23.1 % (v/v) in the products analysed by laser diffraction, whereas the particle fraction below 53 µm was only 5.1 % in the product analysed by sieving. In addition, the particle size distribution of a preparation containing 80 % of folic acid (three batches) showed on average that 48 % of particles were smaller than 50 µm and 11 % had a particle size below 10 µm.19 One batch of the active substance and of a preparation containing 80 % folic acid showed a dusting potential of 0.52 and 0.6 g/m3, respectively.20 Another test on three batches of the preparation showed much higher dusting potential (an average of 10.7 g/kg, corresponding to 13.3 g/m3).18 2.2. Stability and homogeneity Folic acid as a dry, crystalline powder is relatively stable to heat and humidity but becomes unstable at a pH below 5.0 and upon exposure to light. 2.2.1. Shelf life of the additive Folic acid (three batches, stored in a climate chamber) was demonstrated to have a shelf life of 36 months at 25 C and 60 % relative humidity. Shelf-life under accelerated conditions (40 °C and 75 % relative humidity) was shown for six months (three batches) and for 24 months (three batches). The shelf life of a preparation containing 80 % folic acid was demonstrated in three batches for 24 months and in one batch for 36 months, both at 25 C and 60 % relative humidity.21 2.2.2. Stability of the additive when added to premixtures, feed and water for drinking The applicant provided information on the stability of three batches of a preparation containing 80 % folic acid when incorporated in a complete premixture for broilers containing trace elements and choline chloride and stored at 25 C and 60 % relative humidity. The recovery after mixing was 114 % of the intended value, and the retention after three and six months was 104 % and 113 %, respectively.20 However, according to published literature (Whitehead, 2002), the loss of folic acid from premixtures containing choline chloride and trace elements could be expected to be 15 % after one month and 57 % after six months. The stability of a preparation containing 80 % folic acid was tested in mash and pelleted feed for chickens for fattening during processing and storage for a period of three months. The added amount was 5 mg folic acid per kilogram of feed. Stability was not influenced by pelleting at 75 °C or by storage of processed feed for three months. Folic acid is sensitive to the temperatures applied during feed processing (e.g. expanding, conditioning and extrusion).22 18 Technical dossier/Section II/Annex 2.8. 19 Technical dossier/Section II/Annex 2.9. 20 Technical dossier/Supplementary information January 2012. 21 Technical dossier/Section II/Annex 2.33. 22 Technical dossier/Section II/Annex 2.35. EFSA Journal 2012;10(5):2674 8
Folic acid for all animal species Marchetti et al. (1999) studied the stability of folic acid in commercial fish feed. Pelleting reduced the initial concentration (6.36 mg/kg) by 36 %, extrusion by 52 %. No further loss occurred during storage for 90 days. The stability of a representative preparation containing a minimum of 80 % folic acid (three batches) was tested in water for drinking. The product was first diluted with glucose, and the dry blend was added to tap water. The final solution containing 5.0 mg folic acid/L was stored in the dark at 25 °C and tested after 24 and 48 hours. No significant loss was observed.23 2.2.3. Homogeneity Based on a statistical method (Jansen, 1992), the coefficient of variation (CV) for homogeneity of folic acid in poultry feed was calculated to be around 10.9 % and 7.6 % for the active substance (95 %) and for the preparation containing 80 % folic acid, respectively. However, this method has been developed to test the working accuracy of mixing equipment. The applicant provided a study to evaluate the homogeneous distribution of folic acid (80 % preparation, three batches) in water for drinking at a concentration of 5 mg folic inL water. For each batch, five independent samples were taken and the concentration of folic acid was measured. The CV of five different determinations ranged between 4.8 % and 9.2 %, with an average value of 6.6 %.24 The CV of 15 different determinations was 10.8 %. 2.3. Physicochemical incompatibilities in feed Besides choline chloride in premixtures, no other physicochemical incompatibilities or interactions have been reported between folic acid and feed materials, carriers, other approved additives or medicinal products when folic acid was added to premixtures and feed. 2.4. Conditions of use Folic acid is intended for use via feed and water for drinking in all animal species and categories without a maximum content or withdrawal period. Incorporation into complete feeds is recommended through premixture. When used in water for drinking it is recommended that the folic acid concentration amounts to only half that routinely used in complete feed. 2.5. 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 folic acid in animal feed. The executive summary of the EURL report is in Appendix A. 3. Safety According to Regulation (EC) No 429/2008,25 tolerance, metabolism and residue, and toxicological (concerning consumer safety) studies are not required for vitamins, provitamins and chemically defined substances having similar effects which are already authorised as feed additives under Directive 70/524/EEC and which do not have the potential to accumulate, which FEEDAP considers to be the case for folic acid. 3.1. Safety for the target species Requirements for folic acid (NRC, see McDowell, 2000) are in the range of 0.25–1.0 mg/kg feed for poultry, 0.3–1.3 mg/kg for pigs, 1–10 mg/kg for fish and 0.2–0.8 mg/kg for pets. Vitamin 23 Technical dossier/Section II/Annex 2.33 and supplementary information January 2012. 24 Technical dossier/Supplementary information January 2012. 25 Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, 22.5.2008, p. 1. EFSA Journal 2012;10(5):2674 9
Folic acid for all animal species supplementation of commercial compound feed is mostly oriented towards recommended allowances in the range of 1–3 mg/kg for pigs and poultry, 3–7 mg/kg for fish and 1 mg/kg feed for pets (AWT, 2002). A survey on vitamin supplementation of commercial feeds for pigs and poultry in Europe (Belgium, Denmark, Germany, Italy, Netherlands, Portugal, Spain, United Kingdom) identified a range of 0–18 mg folic acid/kg as the commercial usage level (Gropp, 1994). A range for piglets and pigs of 0–3 mg/kg feed was reported by Whittemore et al. (2002). Limited information is available concerning folic acid tolerance and toxicity in domestic animal species. Adverse effects following the ingestion of elevated amounts of folic acid have not been observed in animals, and the vitamin is generally regarded as non-toxic by the oral route (NRC, 1987; EMA, 1997). The FEEDAP Panel concluded that folic acid is safe for the target animals and there is no need to define a maximum content in feed. 3.2. Safety for the consumer 3.2.1. Absorption, distribution, metabolism and excretion of folic acid The majority of dietary folate is absorbed in the small intestine by active, carrier-dependent mechanisms, and also by passive diffusion. Ingested folic acid is enzymatically reduced and methylated in the intestinal lumen and the enterocytes. At high intake levels, folic acid may appear unmodified in the circulation and is exclusively taken up by the liver and either converted into one- carbon derivatives of THF prior to rapid release into bile or polyglutamated and incorporated into the hepatic folate pool. Folic acid bioavailability depends upon dihydrofolate reductase action in the liver. It is present in animal tissues and products as THF, mostly as methyl-THF, with unmetabolised folic acid accounting for less than 10 % (House et al., 2002; Hoey et al., 2009). Both free folate and folate degradation products are excreted in the bile (NRC, 1987). Increased dietary folic acid is reflected by plasma concentrations (McCann et al., 2004; Girard et al., 2005; Hebert et al., 2005; Bunchasak and Kachana, 2009; Hassanien et al, 2010;). Tissue uptake is carrier mediated through folate-binding proteins. About 50 % of the folate body store in humans and various animal species is considered to be present in the liver. Several studies have investigated the potential for enriching the folate content of foods of animal origin with high levels of folic acid supplementation in feed. The folate content of muscle was not significantly increased in pigs fed 10 mg/kg folic acid for 28 days when compared with unsupplemented controls (Giguère et al., 2008). Also, no significant increase compared with unsupplemented controls was observed in the liver of laying hens fed up to 10 mg/kg for up to 56 days (Bunchasak and Kachana, 2009; Tactacan et al., 2010) or in the thigh muscle and liver of chickens for fattening fed 2–32 mg/kg for 42 days: although a numerical increase was observed in breast muscle following supplementation with 2 mg folic acid/kg compared with the unsupplemented group (159 vs 101 μg/kg), no further increase was observed at higher doses (McCann et al., 2004). No increased excretion in milk was observed in cows supplemented with up to 6 mg/kg for 305 days (Girard et al., 2005); however, a significant increase in milk folates (+ 60 %) was observed at extremely high supplementation levels (120 mg/kg feed for 11 weeks) (Graulet et al., 2007). More studies are available concerning the effect of dietary folic acid on folate concentration in eggs. Although study protocols used variable feed concentrations and durations, the overall results indicate that feed supplementation with 2–8 mg folic acid/kg most efficiently increased the folate concentration in eggs (by about twofold; Hoey et al., 2009); higher dietary levels were less effective or did not even increase folate concentration further. Moreover, the effect of supplementation is markedly higher when eggs from unsupplemented hens have folate levels around 20 μg/egg than when they are ≥ 50 μg/egg (Herbert et al., 2005; Roth-Maier and Böhmer 2007; Hoey et al., 2009; Dickson et al., 2010; Tactacan et al., 2010). An example of a high proportional increase is provided by Dickson et al. (2010), whereby the administration of feed supplemented with 4 mg/kg for 11 28-day periods (the EFSA Journal 2012;10(5):2674 10
Folic acid for all animal species entire production cycle) led to folate concentrations in the range of 46.9–57.9 μg/egg: such concentrations represented an approximately two- to threefold increase compared with the controls (17.7–22.3 μg/egg, basal content 0.9 mg folate/kg feed). The authors of the studies suggest that deposition in tissues and eggs follows a saturation mechanism (McCann et al., 2004; Hebert et al., 2005; Hoey et al., 2009). 3.2.2. Toxicology Toxicological studies on folic acid have been assessed in the Scientific Committee on Food (SCF) opinion (EC, 2000) and in the EFSA Scientific Cooperation (ESCO) report analysing the risks and benefits of fortifying food with folic acid (EFSA, 2009b). Overall, folic acid is a substance with very low oral toxicity in mammals. 3.2.3. Assessment of consumer safety 3.2.3.1. Tolerable upper intake level (UL) The SCF (EC, 2000) defined an upper safe level of 1 mg/person/day for (synthetic) folic acid on the basis of findings in patients with pernicious anaemia (vitamin B12 deficiency) treated with high doses of folic acid. High doses of folic acid may ‘mask’ the anaemia caused by vitamin B12 deficiency, a condition frequent in the elderly; in turn, ‘masking’ the anaemia will favour the onset of irreversible neurological degeneration, the later and most serious outcome of vitamin B12 deficiency. The UL for folic acid defined by the SCF applies also to other (dietary) folates, taking into account their lower absorption. The SCF has also defined a UL for toddlers (1–3 years) of 200 µg/child/day. The FEEDAP Panel is aware of the concerns raised by some studies about the safety of folic acid fortification of food, either voluntary or mandatory, the latter being adopted in several countries outside the EU to reduce the incidence of neural tube defects and other malformations. These concerns address, in particular, the potential promotion of colorectal cancer and are based on findings from both experimental and human studies. An analysis of the risks and benefits of fortifying food with folic acid was carried out by an ESCO working group from EFSA: the report concluded that the data are currently insufficient to allow a full quantitative risk assessment. However, potential concerns about a tumour-promoting effect are solely related to the direct intake of folic acid added to foods; on the other hand, no such concerns have been identified with regard to folates, including THF and related compounds, present in foods of vegetable or animal origin (EFSA, 2009b). 3.2.3.2. )Consumer exposure Folates occur naturally in several sources of human and animal food, particularly in fresh vegetables, mushrooms and yeast, as well as green leaves, grasses and citrus fruit. Among foods of animal origin, liver and eggs, and especially egg yolk, are considered a good source. The ESCO working group (EFSA 2009b) considered folate intake data, based on national surveys of food consumption carried out in several European countries. The national estimates of average (mean or median) dietary folate intakes were 151–345 μg/day for adult men and 122–339 μg/day for adult women. These figures included consumption of foods voluntarily fortified with folic acid, which are widely available in many EU countries; mandatory fortification is not endorsed in Europe. When the consumption of supplements was considered, the overall (dietary folates plus folic acid) figures were 338–385 μg/day for men and 220–478 μg/day for women. 3.2.4. Conclusions on consumer safety As folic acid supplementation of animal feedingstuffs is widespread and routine, the FEEDAP Panel has reason to assume that the abovementioned population exposure figures already contain edible tissues and the products of animals fed folic acid-supplemented diets. The only variation expected in the concentration of folates in foodstuffs refers to eggs and meat, as folate levels in liver and milk are EFSA Journal 2012;10(5):2674 11
Folic acid for all animal species not influenced by dietary folic acid. In fact, the difference in consumer exposure to folates resulting from the consumption of eggs and meat from animals fed supplemented or unsupplemented diets is small and would be in the order of 60 µg/day in adults and 27 µg/day in toddlers, based on EU consumption figures for consumers only26 (following the guidance in EFSA, 2012). The details can be found in Appendix B. The FEEDAP Panel considers that the use of folic acid in animal nutrition is not a concern for the safety of consumers. 3.3. Safety for the user 3.3.1. Effects on the respiratory system Folic acid showed a highly variable fraction (5–80 %) of particles with diameter < 50 µm but a dusting potential of 0.5 g/m3. The additive as the formulated preparation (containing a minimum of 80 % of folic acid) showed a large variation in its dusting potential, i.e. 10.7 g/kg (corresponding to 13.3 g/m3, average of three batches) and 0.6 g/m3 (one batch). As no data on acute inhalation toxicity have been provided, and significant inhalation exposure cannot be ruled out, inhalation of dust is considered hazardous. 3.3.2. Effects on the eyes and skin No studies have been made available. The applicant reported that no adverse effects have been observed during occupational handling and that folic acid allergies are very rare in humans. However, the safety data sheets on folic acid indicate a potential for irritation of the skin, eyes and respiratory tract. 3.3.3. Conclusions on user safety In the absence of any information, the FEEDAP Panel considers it prudent to treat folic acid as an irritant to skin, eyes and the respiratory tract and as a sensitiser. 3.4. Safety for the environment Folates occur widely in nature (in green vegetables and certain fruits, e.g. citrus). Synthetic folic acid added to feed would be excreted as the physiological by-products of folate metabolism; thus, the use of synthetic folic acid in animal nutrition is not expected to substantially increase the concentration of folic acid and its metabolites in the environment. Therefore, the use of folic acid in animal nutrition does not pose a risk to the environment. 4. Efficacy According to Regulation (EC) No 429/2008, efficacy studies are not required for vitamins, provitamins and chemically well-defined substances having similar effects which are already authorised as feed additives. Owing to the long history of its use and its established nutritional role in domestic animals, folic acid is regarded as an effective source of folate. Folic acid has been used in animal nutrition worldwide for decades. Data on the requirements, allowances and recommendations for feed supplementation are easily accessible in standard literature for animal nutrition experts. 26 European Food Safety Authority, 2011. Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment. EFSA Journal 9(3):2097 [34 pp.]. doi:10.2903/j.efsa.2011.2097. Available online: www.efsa.europa.eu/efsajournal.htm EFSA Journal 2012;10(5):2674 12
Folic acid for all animal species 5. Post-market monitoring The FEEDAP Panel considers that there is no need for a specific post-market monitoring plan other than the requirements established in the Feed Hygiene Regulation27 and good manufacturing practice. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS Oral administration routes of folic acid via feed or water for drinking are considered bioequivalent. Folic acid is safe for the target animals and there is no need to define a maximum content in feed. The use of folic acid as an additive in animal nutrition is safe for consumers. In the absence of any information, the FEEDAP Panel considers it prudent to treat folic acid as an irritant to skin, eyes and the respiratory tract and as a sensitiser. The use of folic acid in animal nutrition does not pose a risk to the environment. Folic acid is regarded as an effective source of folate in animal nutrition. RECOMMENDATIONS The FEEDAP Panel recommends the use of the specifications for folic acid according to PhEur (MG 0067) in terms of purity, substance-related impurities and other impurities (sulphated ash). DOCUMENTATION PROVIDED TO EFSA 1. Folic acid as a feed additive for all animal species. November 2010. Submitted by VITAC EEIG Vitamins Authorisation Consortium. 2. Folic acid as a feed additive for all animal species. Supplementary information. January 2012. VITAC EEIG Vitamins Authorisation Consortium. 3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for folic acid. 4. Comments from Member States received through the ScienceNet. REFERENCES AWT (Arbeitsgemeinschaft für Wirkstoffe in der Tierernährung e.V.), 2002. Vitamins in Animal Nutrition. ISBN 3–86037–167–3. Bunchasak C and Kachana S, 2009. Dietary folate and vitamin B12 supplementation and consequent vitamin deposition in chicken eggs. Tropical Animal Health and Production, 41, 1538–1539. Dickson TM, Tactacan GB, Hebert K, Guenter W and House JD, 2010. Optimization of folate deposition in eggs through dietary supplementation of folic acid over the entire production cycle of Hy-Line W36, Hy-Line W98, and CV20 laying hens. The Journal of Applied Poultry Research, 19, 80–91. EC (European Commission), 2000. Opinion of the Scientific Committee on Food on the Tolerable Upper Intake of Folate. Available from: http://ec.europa.eu/food/fs/sc/scf/out80e_en.pdf 27 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, 8.2.2005, p. 1. EFSA Journal 2012;10(5):2674 13
Folic acid for all animal species EFSA (European Food Safety Authority), 2004. Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on Calcium L-methylfolate. The EFSA Journal, 135, 1–20. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2009a. Scientific opinion on substantiation of health claims related to folate and blood formation (ID 79), homocysteine metabolism (ID 80), energy-yielding metabolism (ID 90), function on the immune system (ID 91), function of blood vessel (ID 94, 175, 192), cell division (ID 193), and material tissue growth during pregnancy (ID 2882) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA Journal, 7(9): 1213, 22 pp. EFSA (European Food Safety Authority), 2009b. ESCO report prepared by the EFSA Scientific Cooperation Working Group on Analysis of Risks and Benefits of Fortification of Food with Folic Acid. Available from: http://www.efsa.europa.eu/en/scdocs/scdoc/3e.htm EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2010. Scientific opinion on the substantiation of a health claim related to folate and contribution to normal psychological functions (ID 81, 85, 86, 88), maintenance of normal vision (ID 83, 87), reduction of tiredness and fatigue (ID84), cell division (ID 195, 2881) and contribution to normal amino acid synthesis (ID 195, 2881) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA Journal, 8(10): 1760, 19 pp. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2011. Scientific opinion on the substantiation of a health claim related to folate and maintenance of normal blood pressure (ID 176) pursuant to Article 13 (1) of Regulation (EC) No 1924/2006. EFSA Journal, 8(6): 2221, 14 pp. EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP), 2012. Guidance for establishing the safety of additives for the consumer. EFSA Journal, 10(1): 2537, 12 pp. EMA (European Medicines Agency), 1997. Committee for Veterinary Medicinal Products: Folic Acid, Summary Report, September 1997. EMEA/MRL/262/97-Final. Available from: http://www.emea.europa.eu/docs/en_GB/document_library/Maximum_Residue_Limits_- _Report/2009/11/WC500014327.pdf Giguère A, Girard CL, and Matte JJ, 2008. Methionine, folic acid and vitamin B12 in growing- finishing pigs: impact on growth performance and meat quality. Archives of Animal Nutrition 62, 193–206. Girard CL, Lapierre H, Matte JJ and Lobley GE, 2005. Effects of dietary supplements of folic acid and rumen-protected methionine on lactational performance and folate metabolism of dairy cows. Journal of Dairy Science, 88, 660–670. Graulet B, Matte JJ, Desrochers A, Doepel L, Palin M-F and Girard CL, 2007. Effect of dietary supplement of folic acid and vitamin B12 on metabolism of dairy cows in early lactation. Journal of Dairy Science, 90, 3442–3455. Gropp JM, 1994. Vitamin fortification levels in European commercial poultry and swine diets. Proceedings of the Arkansas Nutrition Conference, Fayetteville, Arkansas, pp. 105–134. Hassanien HHM, Ismaeli ZSH and Yakout HM, 2010. Effect of dietary folic acid supplementation on laying hens productive performance and immunity. Egyptian Poultry Science Journal, 30, 713–729. Hebert K, House JD and Guenter W, 2005. Effect of dietary folic acid supplementation on egg folate content and the performance and folate status of two strains of laying hens. Poultry Science, 84, 1533–1538. EFSA Journal 2012;10(5):2674 14
Folic acid for all animal species Hoey L, McNulty H, McCann EM, McCracken KJ, Scott JM, Marc BB, Molloy AM, Graham C and Pentieva K, 2009. Laying hens can convert high doses of folic acid added to the feed into natural folates in eggs providing a novel source of food folate. British Journal of Nutrition, 1010, 206–212. Hommes OR and Obbens EAMT, 1973. Liver function and folate epilepsy in the rat. Journal of the Neurological Sciences, 20, 269–272. House JD, Braun K, Ballance DM, O’Connor CP and Guenter W, 2002. The enrichment of eggs with folic acid through supplementation of the laying hen diet. Poultry Science, 8, 1332–1337. Jansen HD, 1992. Mischtechnik im Futtermittelbetrieb. Anforderungen an Mischenlage, Arbeits- und Mischgenauigkeit. Die Mühle+ Mischfuttertechnik, 129, 265–270. Ly A, Lee H, Chen J, Sie KK, Renlund R, Medline A, Sohn KJ, Croxford R, Thompson LU and Kim YI, 2011. Effect of maternal and postweaning folic acid supplementation on mammary tumor risk in the offspring. Cancer Research, 71, 988–997. Marchetti M, Tossani N, Marchetti S and Bauce G, 1999. Stability of crystalline and coated vitamins during manufacture and storage of fish feeds. Aquaculture Nutrition, 5, 115–120. McCann MEE, McCracken KJ, Hoey L, Pentieva K, McNulty H and Scott J, 2004. Effect of dietary folic acid supplementation on the folate content of broiler chicken meat. British Poultry Science, 45(Suppl. 1), S65–S66. McDowell LR, 2000. Vitamins in Animal and Human Nutrition, 2nd edition. Ames, IA: Iowa State University Press. ISBN 0–8138–2630–6. NRC (National Research Council), 1987. Vitamin Tolerance of Animals. Washington, DC: National Academies Press, pp. 64–66. Roth-Maier DA and Böhmer BM 2007. Fortification of eggs with folic acid as a possible contribution to enhance the folic acid status of populations. International Journal for Vitamins and Nutrition Research, 77, 297–301. Tactacan GB, Jing M, Thiessen S, Rodriguez-Lecompte JC, O’Connor DL, Guenter W and House JD, 2010. Poultry Science, 89, 688–696. Threlfall G, Taylor DM and Buck AT, 1966. The effect of folic acid on growth and deoxyribonucleic acid synthesis in the rat kidney. Laboratory Investigation 15, 1477–1485. Whitehead CC, 2002. Vitamins in feedstuffs. In McNab JM and Boorman KN (eds), Poultry Feedstuffs. Supply, Composition and Nutritive Values. Poultry Science Symposium Series, Volume 26. Wallingford, UK: CABI Publishing, pp. 181–190. ISBN 0-85199-464-4. Whittemore CT, Close WH and Hazzledine MJ, 2002. The need for nutrient requirement standards for pigs. A report of the British Society of Animal Science nutritional standards working group: pigs. Pig News and Information, 23, 67N–74N. Wu Z, Li X, Hou C and Qian Y, 2010. Solubility of folic acid in water at pH values between 0 and 7 and temperatures (298.15, 303.15 and 313.15) K. Journal of Chemical and Engineering Data, 55, 3958–3961. EFSA Journal 2012;10(5):2674 15
Folic acid for all animal species APPENDIX A Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Folic Acid28 In the current application authorisation is sought under articles 4(1) and 10(2) for folic acid under the category/functional group 3(a) ‘nutritional additives’/‘vitamins, pro-vitamins and chemically well defined substances having similar effect’ according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of the feed additive for all animal species and categories. According to the applicant, folic acid is a yellowish to orange, crystalline powder with a minimum purity of 95 %. The feed additive is intended to be incorporated in feedingstuffs or complementary feedingstuffs through premixtures or directly in water. The applicant did not specify any maximum or minimum concentration of folic acid in feedingstuffs or water. For the identification and quantification of folic acid in the feed additive the applicant proposes the internationally recognised European Pharmacopoeia method (Ph.Eur. 6th edition, Monograph 0067), based on liquid chromatography coupled with UV detection at 280 nm (LC-UV). Even though no performance characteristics are provided, the EURL considers this method suitable to be used within the frame of official control. For the quantification of folic acid in premixtures the applicant proposed a single laboratory validated and further verified method, based on reversed phase high performance liquid chromatography (RP- HPLC) with UV at 280 nm. The following performance characteristics were reported: - a relative standard deviation for repeatability (RSDr) ranging from 0.3 to 2.2% - a relative standard deviation for intermediate precision (RSDip) ranging from 1.8 to 2.5% Based on these performance characteristics, the EURL recommends for official control the validated and further verified RP-HPLC-UV method to determine folic acid in premixtures within a concentration range between 200 mg and 200 g/kg. For the quantification of total FOLATES (including added folic acid) in feedingstuffs and water the applicant proposed the ring-trial validated method EN 14131 using a microbiological assay with Lactobacillus casei, subsp, rhamnosus (ATCC 7469) to extracted folates. The following performance characteristics were determined for folate concentrations ranging from 0.5 to 13 mg/kg: - RSDr ranging from 4.9 to 9.2 %, and - a relative standard deviation for reproducibility (RSDR) ranging from13.8 to 22.3 %. Even though the applicant did not provide any data for the quantification of folic acid in water the concentration range covered by the CEN method includes the recommended minimum concentration suggested by the applicant for water. Based on the performance characteristics presented the EURL recommends for official control the CEN ring-trial validated microbiological method (EN 14131) to determine total FOLATES (including added folic acid) in feedingstuffs and 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. 28 The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/SiteCollectionDocuments/FinRep-FAD- 2010–0197.pdf EFSA Journal 2012;10(5):2674 16
Folic acid for all animal species APPENDIX B Comparison of the exposure of consumers resulting from the consumption of eggs and meat from animals fed diets unsupplemented or supplemented with folic acid The calculation of the additional exposure of consumers to folates due to supplementation of the diets of animals with folic acid is based on: 1. the concentrations of residues in relevant tissues/products: only eggs and meat were considered, as liver and milk folate levels are not influenced by dietary folic acid, as described in section 3.2.1 2. folate concentration in eggs from hens supplemented with 2–8 mg folic acid per kilogram of feed compared with unsupplemented hens (122 vs 60 μg/100 g) according to Hoey et al. (2009) 3. folate concentration in meat (breast muscle) from broiler chicken supplemented with up to 16 mg folic acid/kg compared with the unsupplemented group (161 vs 101 μg/kg) according to McCann et al. (2004) 4. default values for daily food consumption are derived from the EFSA Comprehensive European Food Consumption Database and represent the high intake (95th percentile) of consumers only for relevant food items listed in the table for chronic intake by toddlers and adults.29 Table 2: Additional consumer daily exposure to folates resulting from the consumption of eggs and meat from animals fed supplemented or unsupplemented diets Unsupplemented diet Supplemented diet Chronic Concentration Exposure Concentration in Exposure intake(a) (g) in food (mg/kg) (µg) food (mg/kg) (µg) Toddlers(b) Eggs 35 0.60 21 1.22 43 (c) Meat 90 0.10 9 0.16 14 Total 30 57 Adults(d) Eggs 70 0.60 42 1.22 84 (c) Meat 290 0.10 29 0.16 46 Total 71 130 (a) Chronic intake is the 95th percentile of the distribution of average individual consumption levels (over the survey period) for consumers only from all available EU national surveys. (b) Toddlers: 1–3 years of age, 12 kg body weight. (c) Meat, including processed meat products. (d) Adults: 18–65 years of age, 60 kg body weight. 29 European Food Safety Authority, 2011. Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment. EFSA Journal 9(3): 2097 [34 pp.]. doi:10.2903/j.efsa.2011.2097. Available online: www.efsa.europa.eu/efsajournal.htm EFSA Journal 2012;10(5):2674 17
You can also read