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

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1 EFSA Journal 2013;11(2):3104 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of vitamin C (ascorbic acid, sodium ascorbate, calcium ascorbate, ascorbyl palmitate, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate) as a feed additive for all animal species based on a dossier submitted by DSM Nutritional Products Ltd 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2, 3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Vitamin C is essential for primates, guinea pigs and fish. Vitamin C, in the form of ascorbic acid and its calcium and sodium salts, ascorbyl palmitate, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate, is safe for all animal species. Setting a maximum content in feed and water for drinking is not considered necessary. Data on the vitamin C consumption of consumers are based on the levels of vitamin C in foodstuffs, including food of animal origin, produced in accordance with current EU legislation on the supplementation of feed with vitamin C. The exposure is far below the guidance level. Any potential contribution of the use of vitamin C in feed is therefore already considered in the above data. Consequently, the use of vitamin C in animal nutrition is not of concern for consumer safety. In the absence of inhalation toxicity studies it would be prudent to assume that inhalation of dust from the additives presents a health hazard to workers. Sodium calcium ascorbyl phosphate is not an irritant to skin and eyes and is unlikely to be a skin sensitiser. This conclusion is extrapolated to sodium ascorbyl phosphate. In the absence of data, ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate should be considered as irritant to skin and eyes and as dermal sensitisers. The supplementation of feed with vitamin C does not pose a risk to the environment. Ascorbic acid, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate are regarded as effective sources of vitamin C when added to feed or water for drinking. Since ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate are authorised for use as antioxidants in food and their function in feed is essentially the same as that in food, no further demonstration of efficacy is considered necessary. European Food Safety Authority, On request from the European Commission, Question No EFSA-Q , adopted on 30 January Revision 1 4 December 2013: minor editorial change. The change does not affect the overall conclusions of the scientific output. 2 Panel members: Gabriele Aquilina, Alex Bach, Vasileios Bampidis, Maria De Lourdes Bastos, Gerhard Flachowsky, Josep Gasa-Gasó, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Derek Renshaw, Guido Rychen, Maria Saarela, Kristen Sejrsen, Patrick Van Beelen, Robert John Wallace and Johannes Westendorf. One member of the Panel did not participate in the discussion on the subject referred to above because of potential conflicts of interest identified in accordance with the EFSA policy on declarations of interests. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Vitamins, including Georges Bories, Jürgen Gropp, Alberto Mantovani and the late Reinhard Kroker, 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 vitamin C (ascorbic acid, sodium ascorbate, calcium ascorbate, ascorbyl palmitate, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate) as a feed additive for all animal species based on a dossier submitted by DSM Nutritional Products Ltd. EFSA Journal 2013;11(2):3104. [36 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2013

2 KEY WORDS Nutritional additive, vitamin C, ascorbic acid, sodium ascorbate, calcium ascorbate, ascorbyl palmitate, ascorbyl phosphates EFSA Journal 2013;11(2):3104 2

3 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 vitamin C in the form of ascorbic acid, sodium ascorbate, calcium ascorbate, ascorbyl palmitate and sodium calcium ascorbyl phosphate. Ascorbic acid, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate are used as vitamins, intended for addition to feed. Ascorbic acid and sodium ascorbyl phosphate are also intended for use in water for drinking. Ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate are used as antioxidants. Vitamin C (formerly known as antiscorbutic vitamin) is essential for primates, guinea pigs and fish. It is involved in the synthesis of collagen and in incorporating iron into ferritin. It is required for neutrophil function and decreases circulating glucocorticoids. Vitamin C is thought to play a critical role in the immune response. Vitamin C, in the form of ascorbic acid, and its calcium and sodium salts, ascorbyl palmitate, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate, is safe for all animal species. Setting a maximum content in feed and water for drinking is not considered necessary. Data on the vitamin C consumption of consumers are based on the levels of vitamin C in foodstuffs, including food of animal origin, produced in accordance with current EU legislation on the supplementation of feed with vitamin C. The exposure is far below the guidance level. Any potential contribution of the use of vitamin C in feed is therefore already considered in the above data. Consequently, the use of vitamin C in animal nutrition is not of concern for consumer safety. The scarcity of data does not allow an extrapolation of the conclusions regarding the safety for the user to the different forms of vitamin C under application. The FEEDAP Panel concludes that in the absence of inhalation toxicity studies it would be prudent to assume that inhalation of dust from the additives presents a health hazard to workers and measures should be taken to minimise inhalation exposure. Sodium calcium ascorbyl phosphate is not an irritant to skin and eyes and is unlikely to be a skin sensitiser. This conclusion is extrapolated to sodium ascorbyl phosphate. In the absence of data, ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate should be considered to be irritant to skin and eyes and as dermal sensitisers. The supplementation of feed with vitamin C does not pose a risk to the environment. Owing to the long history of use and its established nutritional role in domestic animals, ascorbic acid, sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate are regarded as effective sources of vitamin C. This conclusion includes the use of ascorbic acid and sodium ascorbyl phosphate in water for drinking. Since ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate are authorised for use as antioxidants in food and their function in feed is essentially the same as that in food, no further demonstration of efficacy is necessary. The FEEDAP Panel made some recommendations concerning the nomenclature and the specifications of the substances under application. EFSA Journal 2013;11(2):3104 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 3 Table of contents... 4 Background... 6 Terms of reference... 7 Assessment Introduction Characterisation Manufacture of vitamin C Ascorbic acid (E300) Qualitative and quantitative composition Physical state Stability and homogeneity Formulated ascorbic acid additive Sodium ascorbate (E301) Qualitative and quantitative composition Physical state Stability and homogeneity Calcium ascorbate (E302) Qualitative and quantitative composition Physical state Stability and homogeneity Ascorbyl palmitate (E304) Qualitative and quantitative composition Physical state Stability and homogeneity Sodium calcium ascorbyl phosphate Qualitative and quantitative composition Physical state Stability and homogeneity Formulated additive Sodium ascorbyl phosphate Qualitative and quantitative composition Physical state Stability and homogeneity Physico-chemical incompatibilities in feed Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) Safety Safety for the target species Conclusions on the safety of vitamin C for the target species Safety for the consumer Absorption, distribution, metabolism and excretion (ADME) Toxicological studies Assessment of consumer safety Safety for the user Effects on the respiratory system Effects on the eyes and skin Photoallergenicity and skin sensitisation Conclusions on user safety Safety for the environment Efficacy EFSA Journal 2013;11(2):3104 4

5 5. Post-market monitoring Conclusions and recommendations Documentation provided to EFSA References Appendices Appendix A Appendix B EFSA Journal 2013;11(2):3104 5

6 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7; in addition Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from the company DSM nutritional Products Ltd 5 for (i) the re-evaluation and (ii) the authorisation of a new use (i.e. use in water for drinking) of vitamin C in the form of L-ascorbic acid and ascorbyl monophosphate calcium sodium salt, 6 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) and for (iii) the re-evaluation of authorisation of vitamin C in the form of L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate and 6-palmityl L-ascorbic acid, when used as a feed additive for all animal species (category: technological additive; functional group: antioxidant) under the conditions mentioned in Table 1a-f. 7 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. 8 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 20 April Vitamin C in the form of L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, 6-palmityl L- ascorbic acid and ascorbyl monophosphate calcium sodium salt has been authorised without time limit under Council Directive 70/524/EEC 10 for its use for all animal species as a nutritional additive and in the form of L-ascorbic acid sodium L-ascorbate, calcium L-ascorbate and 6-palmityl L-ascorbic acid as technological additive (antioxidant). The Scientific Panel on Dietetic Products, Nutrition and Allergies (NDA) expressed an opinion on the tolerable upper intake level of vitamin C (L-ascorbic acid, its calcium, potassium and sodium salts and L-ascorbyl-6-palmitate) (EFSA, 2004). The Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) issued an opinion on calcium ascorbate with a content of threonate for use as source of vitamin C in food supplements (EFSA, 2007). A statement on the safety of calcium ascorbate with a content of 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, , p DSM Nutritional Products Ltd, Switzerland, represented in the European Union by DSM Nutritional Products SP. Z.o.o., Tarczynska 113, Mszczonow Poland. 6 Sodium ascorbyl phosphate is considered by the applicant as the water-soluble form of ascorbyl monophosphate calcium sodium salt. 7 In July 2012 the applicant withdrew the following parts of the application: (i) the use of sodium L-ascorbate, calcium L- ascorbate and 6-palmityl L-ascorbic acid as nutritional additives and (ii) the use in water for drinking of ascorbyl monophosphate calcium sodium salt. 8 EFSA Dossier reference: FAD A new mandate was received in EFSA in March 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, , p. 1. EFSA Journal 2013;11(2):3104 6

7 threonate produced by a new manufacturing process as a source of vitamin C in food supplements has been published by EFSA in 2011 (EFSA, 2011a). The Panel on Food Additives and Nutrient Sources added to Food (ANS) issued an opinion on calcium ascorbate, magnesium ascorbate and zinc ascorbate added for nutritional purposes in food supplements (EFSA, 2009a), another opinion on the inability to assess the safety of vitamin C-enriched yeast for nutritional purposes as a source of vitamin C in food supplements and the bioavailability of vitamin C from this source, based on the supporting dossier (EFSA, 2009b) and another opinion on the use of sodium ascorbate as a food additive in vitamin D preparations intended to be used in formulae and weaning food for infants and young children (EFSA, 2010a). Ascorbates will be re-evaluated by the ANS Panel as part of the food additives re-evaluation programme specified under Regulation (EU) No 257/ This opinion is expected to be delivered by the end of Ascorbyl fatty acid esters will be re-evaluated by the ANS Panel as part of the food additives re-evaluation programme specified under Regulation (EU) No 257/2010. This opinion is expected to be delivered by the end of The NDA Panel expressed four opinions on the substantiation of several health claims related to Article 13(1) of Regulation (EC) No 1924/ (EFSA, 2009c, 2010b,c, 2011b). 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 vitamin C in the form of L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, 6-palmityl L-ascorbic acid and ascorbyl monophosphate calcium sodium salt and sodium ascorbyl phosphate, when used under the conditions described in Table 1a-f. 11 Commission Regulation (EU) No 257/2010 of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives. OJ L 80, , p Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 December 2006 on nutrition and health claims made on foods. OJ L 404, , p. 9. EFSA Journal 2013;11(2):3104 7

8 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 Vitamin C - L-Ascorbic acid E300 Nutritional; Technological Vitamins, provitamins and chemically well defined substances having a similar effect; Antioxidant Composition, description L-ascorbic acid C 6 H 8 O 6 Description Chemical formula Purity criteria min 99 % (on dry substance) Lead : max 2 mg/kg Arsenic : max 3 mg/kg Mercury: max 1 mg/kg Method of analysis Iodine titration (EurPh01/2008:253) Atomic absorption (EurPh01/2008/20223) Trade name Name of the holder of authorisation Species or category of animal all animal species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs 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 Vitamin C - L-ascorbic acid can be used as such or in a form of a preparation (to improve its stability), in premixtures, compound feeds and water for drinking None Not necessary due to the long history of use of the additive None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2013;11(2):3104 8

9 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 L-Ascorbate E301 Technological Antioxidant Composition, description Sodium L-ascorbate Description Chemical formula C 6 H 7 O 6 Na Purity criteria min 99 % (on dry substance) Lead : max 2 mg/kg Arsenic : max 3 mg/kg Mercury: max 1 mg/kg Method of analysis Iodine titration (EurPh01/2008:253) Atomic absorption (EurPh01/2008/20223) Trade name Name of the holder of authorisation Species or category of animal all animal species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs 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 Sodium L-ascorbate is used as such in premixtures, feed materials and compound feeds None None None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2013;11(2):3104 9

10 Table 1c: 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 Calcium L-Ascorbate E301 Technological Antioxidant Composition, description Calcium L-ascorbate Description Chemical formula C 12 H 14 O 12 Ca 2H 2 O Purity criteria min 99 % (on dry substance) Lead : max 2 mg/kg Arsenic : max 3 mg/kg Mercury: max 1 mg/kg Method of analysis Iodine titration (USP 32) Atomic absorption (FCC6) USP32 Atomic absorption (FCC6) Trade name Name of the holder of authorisation Species or category of animal all animal species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs 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 Calcium L-ascorbate is used as such in premixtures, feed materials and compound feeds None None None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2013;11(2):

11 Table 1d: 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 6-Palmityl- L-ascorbic acid E304 Technological Antioxidant Composition, description Description Chemical formula 6-Palmityl- L-ascorbic acid C 22 H 38 O 7 Purity criteria min 98 % Lead : max 2 mg/kg Arsenic : max 3 mg/kg Mercury: max 1 mg/kg Method of analysis Iodine titration (EurPh 01/2008:0807 Atomic absorption (EurPh 01/2008:20223) Trade name Name of the holder of authorisation Species or category of animal all animal species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs 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 6-Palmityl- L-ascorbic acid is used as such in premixtures, feed materials and compound feeds None None None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2013;11(2):

12 Table 1e: 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 ROVIMIX STAY-C 35 L-Ascorbic acid monophosphate calcium sodium salt Nutritional Vitamins, provitamins and chemically well defined substances having a similar effects Composition, description ROVIMIX STAY-C 35 L-Ascorbic monophosphate calcium sodium salt Description Chemical formula C 6 H 9 O 9 P Purity criteria min 35 % vitamin C equivalent Method of analysis HPLC Trade name (if appropriate) ROVIMIX STAY-C 35 Name of the holder of authorisation (if appropriate) DSM Nutritional Products Ltd Species or category of animal all animal species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs 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 Vitamin C - Ascorbyl monophosphate Calcium sodium salt is used as such or in the form of preparations in premixtures, compound feeds None Not applicable due to the long history of use. None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2013;11(2):

13 Table 1f: 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 STAY-C 50 L-Ascorbic acid monophosphate sodium salt Nutritional Vitamins, provitamins and chemically well defined substances having a similar effects Composition, description STAY-C 50 L-Ascorbic acid monophosphate sodium salt Description Chemical formula Purity criteria Method of analysis C 6 H 6 O 9 Na 3 P. 2H 2 O min 45 % vitamin C equivalent Heavy metals max 10 mg/kg HPLC Trade name (if appropriate) STAY-C 50 Name of the holder of authorisation (if appropriate) DSM Nutritional Products Ltd Species or category of animal all animal species and categories Maximum Age Conditions of use Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs 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 Vitamin C - Ascorbyl monophosphate sodium salt is used as such or in the form of preparations in premixtures, compound feeds and water for drinking None Not applicable due to the long history of use. None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or animal food products Maximum content in tissues EFSA Journal 2013;11(2):

14 ASSESSMENT This opinion is based on data provided by a single company involved in the production/distribution of vitamin C in the form of ascorbic acid, sodium ascorbate, calcium ascorbate, ascorbyl palmitate and ascorbyl phosphate. It should be recognised that these data cover only a fraction of existing additives containing vitamin C in different forms. 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 The term vitamin C is used as generic description of ascorbic acid (reduced form) and dehydroascorbic acid (oxidised form). Only the L-isomer of both forms has vitamin activity. Vitamin C is widely distributed and occurs in large quantities in feeds of plant origin that are most relevant for animal nutrition, particularly in green feeds and silages. Vitamin C is involved in the synthesis of collagen and in incorporating iron into ferritin. It is required for neutrophil function and decreases circulating glucocorticoids. Thus, vitamin C is thought to play a critical role in the immune response. It also reduces oxidised tocopherol to its active form in liver. The antioxidant property of vitamin C is also utilised when protection against oxidation of food or feed during manufacturing and storage is required. Vitamin C, in the form of L-ascorbic acid (hereafter referred to as ascorbic acid), sodium L-ascorbate (hereafter referred as sodium ascorbate), calcium L-ascorbate (hereafter referred to as calcium ascorbate), 6-palmityl-L-ascorbic acid (hereafter referred to as ascorbyl palmitate) and ascorbyl monophosphate calcium sodium salt (hereafter referred to sodium calcium ascorbyl phosphate), is included in the European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003 and foreseen for re-evaluation. It is authorised for use in all animal species, without a time limit, as a nutritional additive and, in the case of ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate, as a technological additive (antioxidant). No maximum levels of vitamin C in feeds are established in the EU. The applicant asks for the re-evaluation of the use of vitamin C in the form of ascorbic acid and sodium ascorbyl phosphate as nutritional additives to feed and water for drinking (new use). The sodium calcium ascorbyl phosphate is intended to be used as nutritional additive only in feed. The applicant is also seeking re-evaluation of the use of vitamin C in the form of ascorbic acid, sodium ascorbate, calcium ascorbate and 6-palmityl-L-ascorbic acid as a technological additive to feed for all animal species and categories. Ascorbic acid (E300), sodium ascorbate (E301), calcium ascorbate (E302) and ascorbyl palmitate (E304) are approved for food use as vitamins and antioxidants whereas sodium calcium ascorbyl phosphate and sodium ascorbyl phosphate are not approved for use in food (for details, see Appendix B). Ascorbic acid, sodium ascorbate, calcium ascorbate and ascorbyl palmitate are described in the European Pharmacopoeia (PhEur) as Monographs (MG) 0253, 1791, 1182 and Characterisation 2.1. Manufacture of vitamin C Sodium ascorbate is obtained by reacting methyl-d-sorbosonate (or ketogulonic acid methyl ester) with sodium carbonate. The precipitate is cooled, centrifuged and washed with methanol. The washed cake is dissolved in water and passed through a stripper to remove residual methanol. The sodium ascorbate solution is decolourised by passing it through activated carbon columns. Pure sodium ascorbate solution is vacuum concentrated and filtered. EFSA Journal 2013;11(2):

15 Ascorbic acid is obtained from sodium ascorbate by cation exchange. The solution is then purified by passing through anion exchange columns, decolourised on activated carbon, concentrated and crystallised. Calcium ascorbate is obtained by reacting ascorbic acid with calcium carbonate in water and ethanol, following which it is isolated dried, and then filled into containers. Ascorbyl palmitate is prepared by reaction of ascorbic acid with sulphuric acid followed by esterification with palmitic acid. Sodium calcium ascorbyl phosphate results from the reaction of ascorbic acid (alone or in combination with sodium ascorbate) with calcium hydroxide and sodium trimetaphosphate. After further purification (cation and anion exchange) followed by neutralisation, sodium ascorbyl phosphate is obtained. Both products are used in the formulation of additives Ascorbic acid (E300) Ascorbic acid (International Union of Pure and Applied Chemistry (IUPAC) name (5R)-[(1S)-1,2- dihydroxyethyl]-3,4-dihydroxyfuran-2(5h)-one; synonyms L-ascorbic acid, vitamin C, 3-oxo-Lgulofuranolactone, enol form) is identified by the Chemical Abstracts Service (CAS) number and the European Inventory of Existing Chemical Substances (EINECS) number The structural formula of ascorbic acid is shown in Figure 1. Figure 1: Structural formula of ascorbic acid The molecular formula of ascorbic acid is C 6 H 8 O 6 and its molecular weight is It has a melting point of C (with decomposition) and shows a density of approximately 1.65 g/cm 3. It is soluble in water (300 g/l at 20 C) and ethanol (20 g/l at 20 C). It has two pk a values: 4.2 and The ph of a 5 % (w/v) solution in water is Ascorbic acid occurs in the form of white or almost white crystals or crystalline powder and is almost odourless. Ascorbic acid is described in the European Pharmacopoeia (PhEur 7.0, 0253) as having a purity of % ascorbic acid, less than 0.2 % related substances (impurity E, oxalic acid), sulphated ash < 0.1 % and heavy metals (expressed as lead) < 10 mg/kg Qualitative and quantitative composition By specification, the active substance contains at least 99.0 % ascorbic acid. Analysis of five batches of the ascorbic acid under application showed an average ascorbic acid content of ± 0.1 %, compliant with PhEur The product also complied with the thresholds of PhEur for the amounts of substance-related impurities, sulphated ash and heavy metals, as demonstrated by the analysis of five batches. 9 Residual organic solvents conformed to VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products) 13 Technical dossier/section II/Annex EFSA Journal 2013;11(2):

16 thresholds (EMA, 2010). Heavy metals (lead and mercury) and arsenic levels complied with the relevant thresholds for food additives set by Regulation (EC) 231/ Physical state Three batches of ascorbic acid were analysed for particle size distribution by laser diffraction. The fraction of particles below 50 µm was between 1.0 and 2.0 % (v/v). The dusting potential, measured using the Stauber Heubach method, was 0.34 g/m 3, based on the average of three batches Stability and homogeneity Ascorbic acid (three batches, stored in aluminium bags) was demonstrated to have a shelf-life of 36 months at 25 ± 2 C. Under accelerated conditions at 40 ± 2 C the shelf-life was shown to be 12 months. 16 Ascorbic acid (three batches) was added to a premixture for chickens for fattening that also contained trace elements and choline chloride at a concentration of 25 g/kg. The samples were stored at 25 C for up for six months. Losses observed after three months were about 10 % of the initial ascorbic acid concentration; after six months the losses had increased up to about 32 %. 17 Ascorbic acid (three batches) was added to mash and pelleted feed for chickens for fattening at a level of 300 mg/kg feed. After storage for three months at 25 C, no losses were observed in the mash feed. Pelleting (90 C) decreased the initial vitamin C content to 82 %; recovery of ascorbic acid after three months storage of the pelleted feed was in the range %. 18 The stability of ascorbic acid in tap water (three batches) was measured when added at 0.5 g/l and kept for 48 hours at 25 C in a dark room. The retention of ascorbic acid was nearly complete. 19 Homogeneity of distribution was calculated on the basis of 15 samples from three batches of pelleted feed at a concentration 300 mg/kg. The coefficient of variation was 5 %. 14 Vitamin C in the form of ascorbic acid is highly soluble in water and therefore homogeneity in water for drinking need not be demonstrated Formulated ascorbic acid additive The applicant provided data on an additive containing 97.5 % active substance ascorbic acid and 2.5 % ethylcellulose. The composition was confirmed by the analysis of five batches (98.7 ± 0.2 % ascorbic acid). 20 Laser diffraction of one batch of the additive revealed that the fractions below 10, 50 and 100 µm amounted to 3 %, 12 % and 23 % (v/v), respectively. The dusting potential, as the average of three batches, was 0.37 g/m Sodium ascorbate (E301) Sodium ascorbate (IUPAC name 2,3-didehydro-L-threo-hexono-1,4-lactone sodium enolate; synonyms sodium L-ascorbate, vitamin C (sodium salt), L-ascorbic acid monosodium salt) is identified 14 Commission Regulation (EC) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council. OJ L , p Technical dossier/section II/Annex 2.24 and Supplementary information/july Technical dossier/section II/Annex 2.61 and Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex 2.26 and Supplementary information/july 2012/Annex i.2. EFSA Journal 2013;11(2):

17 by the CAS number and the EINECS number The structural formula of sodium ascorbate is shown in Figure 2. Figure 2: Structural formula of sodium ascorbate The molecular formula of sodium ascorbate is C 6 H 7 O 6 Na and its molecular weight is It has a melting point of 218 C (decomposes without melting) and shows a bulk density of approximately 0.86 g/cm 3. It is soluble in water (620 g/l at 25 C) and slightly soluble in ethanol. The ph of a 10 % (w/v) solution in water is Sodium ascorbate in the crystalline form is a white to yellowish, practically odourless, powder. Sodium ascorbate is described in the European Pharmacopoeia (PhEur 7.0, 1791) as having a purity of % sodium ascorbate in the dried substance, less than 0.3 % related substances (impurity E, oxalic acid), loss on drying < 0.25 %, sulphates < 150 mg/kg, copper < 5 mg/kg, iron < 2 mg/kg, nickel < 1 mg/kg and heavy metals (expressed as lead) < 10 mg/kg Qualitative and quantitative composition By specification, the active substance contains at least 99.0 % sodium ascorbate in the dried substance. Analysis of five batches of the sodium ascorbate under application showed an average sodium ascorbate content of ± 0.2 % in the dry product, compliant with PhEur The product also complied with the thresholds of PhEur for the amounts of substance-related impurities, loss on drying, sulphates, heavy metals (expressed as lead), copper, iron and nickel, as demonstrated by the analysis of five batches. 18 Residual organic solvents conformed to VICH thresholds. Heavy metals (lead and mercury) and arsenic levels complied with the relevant thresholds set for food additives by Regulation (EC) 231/ Physical state Three batches of sodium ascorbate were analysed for particle size distribution by laser diffraction. The fraction of particles below 10 µm amounted to 10 % (v/v) and the fractions below 50 and 100 µm to 44 % and 76 %, respectively. 24 The dusting potential, measured according to the Stauber Heubach method, was 2.06 g/m 3, based on the average of three batches Stability and homogeneity Sodium ascorbate (three batches, stored in aluminium bags) was demonstrated to have a shelf-life of 18 months at 25 ± 2 C. The shelf-life under accelerated conditions at 40 ± 2 C was shown to be six months. 26 Since the substance is used as an antioxidant only, the applicant did not provide data on the stability of the additive (maintenance of its effect) in feedingstuffs. 22 Technical dossier/section II/Annex OJ L , p Technical dossier/section II/Annexes Technical dossier/section II/Annexes 2.97 and Supplementary information/july Technical dossier/section II/Annex 2.63 and Annex EFSA Journal 2013;11(2):

18 2.4. Calcium ascorbate (E302) Vitamin C for all animal species Calcium ascorbate (chemical name calcium L-(+)-ascorbate dehydrate; synonyms calcium L-ascorbate, calcium ascorbate dehydrate, ascorbic acid (calcium salt), vitamin C (calcium salt), L-ascorbic acid monocalcium salt) is identified by the CAS number (as dehydrate) and the EINECS number The structural formula of calcium ascorbate acid is shown in Figure 3. Figure 3: Structural formula of calcium ascorbate The molecular formula of calcium ascorbate acid is C 12 H 14 O 12 Ca 2H 2 O and its molecular weight is It has a melting point of 165 C (with decomposition) and shows a bulk density of approximately 0.76 g/cm 3. It is soluble in water (about 500 g/l) and insoluble in methanol and ethanol. The ph of a 10 % (w/v) solution in water is Calcium ascorbate is a white to yellowish, practically odourless powder, with a slightly bitter taste. Calcium ascorbate is described in the European Pharmacopoeia (PhEur 7.0, 1182) has having a purity of % calcium ascorbate, loss on drying < 0.1 %, fluorides < 10 mg/kg, copper < 5 mg/kg, iron < 2 mg/kg and heavy metals (expressed as lead) < 20 mg/kg Qualitative and quantitative composition By specification, the active substance contains at least 99.0 % calcium ascorbate in the dried substance. Analysis of five batches of the calcium ascorbate under application showed an average calcium ascorbate content of 99.2 ± 0.1 % in the dry product (loss on drying %), compliant with PhEur The product also complied with the thresholds of PhEur for the amounts of substance-related impurities, loss on drying, fluorides, copper, iron and heavy metals (expressed as lead), as demonstrated by the analysis of five batches. 22 Residual organic solvents conformed to VICH thresholds. Heavy metals (lead and mercury) and arsenic complied with the relevant thresholds for food additives set by Regulation (EC) 231/ Physical state Three batches of calcium ascorbate were analysed for particle size distribution by laser diffraction. The proportion of particles with diameters of less than 10, 50 and 100 µm was 0 %, 3.0 %, and 37 % by volume, respectively. 29 The dusting potential, measured using the Stauber Heubach method, was 0.31 g/m 3, based on an average of three batches Stability and homogeneity Calcium ascorbate (three batches in composite bags) was demonstrated to have a shelf-life of 48 months at 25 ± 2 C. Shelf-life under accelerated conditions at 40 ± 2 C was shown to be 12 months Technical dossier/section II/Annex OJ L , p Technical dossier/section II/Annex 2.32 and Annex Technical dossier/section II/Annex 2.97 and Supplementary information/july Technical dossier/section II/Annex 2.65 and Annex EFSA Journal 2013;11(2):

19 Since the substance is used as an antioxidant only, the applicant did not provide data on the stability of the additive (maintenance of its effect) in feedingstuffs Ascorbyl palmitate (E304) Ascorbyl palmitate (IUPAC name 2,3-didehydro-L-threo-hexono-1,4-lactone-6-palmitate; chemical name L-ascorbyl-6-palmitate, synonym L-ascorbyl palmitate) is identified by the CAS number and the EINECS number The structural formula of ascorbyl palmitate is shown in Figure 4. Figure 4: Structural formula of ascorbyl palmitate The molecular formula of ascorbyl palmitate is C 22 H 38 O 7 and its molecular weight is It has a melting point of C and shows a bulk density of approximately 0.24 g/cm 3. Ascorbyl palmitate is practically insoluble in water ( 1.8 g/l at 20 C). It is soluble in ethanol and methanol (125 and 183 g/l, respectively, at 22 C) and in plant oils. Ascorbyl palmitate is a liposoluble ester of vitamin C. It is a white to yellowish white, odourless powder composed only of the active substance. Ascorbyl palmitate is described in the European Pharmacopoeia (PhEur 7.0, 0807) as having a purity of % in the dried substance, loss on drying < 1.0 %, sulphated ash < 0.1 % Qualitative and quantitative composition By specification, the active substance contains at least 98.0 % ascorbyl palmitate. Analysis of five batches of the ascorbyl palmitate under application showed an average ascorbyl palmitate content of 99.2 ± 0.3 % (loss on drying %), 32 compliant with PhEur The product also complied with the thresholds of PhEur for the amounts of substance-related impurities, sulphated ash and heavy metals, as demonstrated by the analysis of five batches. 19 Residual organic solvents conformed with VICH thresholds. Heavy metals (lead and mercury) and arsenic complied with the relevant thresholds set for food additives by Regulation (EC) 231/ Physical state Three batches of ascorbyl palmitate were analysed for particle size distribution by laser diffraction. The proportion of particles with diameters of less than 10, 50 and 100 µm was 14 %, 37 %, and 45 %, by volume, respectively. The dusting potential, measured using the Stauber Heubach method, was 2.65 g/m 3, based on an average of three batches Technical dossier/section II/Annex OJ L , p Technical dossier/section II/Annex 2. and Supplementary information/july EFSA Journal 2013;11(2):

20 Stability and homogeneity Vitamin C for all animal species Ascorbyl palmitate (three batches, stored in aluminium bags) was demonstrated to have a shelf-life of 36 months at 25 ± 2 C. Shelf-life under accelerated conditions at 40 ± 2 C was shown to be six months. 35 Since the substance is used as an antioxidant only, the applicant did not provide data on the stability of the additive (maintenance of its effect) in feedingstuffs Sodium calcium ascorbyl phosphate Sodium calcium ascorbyl phosphate (chemical name sodium-, calcium-l-ascorbyl phosphate) is not identified by a CAS number or an EINECS number. The structural formula of sodium calcium ascorbyl phosphate is shown in Figure 5. Figure 5: Structural formula of sodium calcium ascorbyl phosphate The empirical molecular formula of sodium calcium ascorbyl phosphate is C 6 H 6 O 9 P CaNa and it has a molecular weight of 316. It has a bulk density of approximately of approximately 0.64 g/cm 3. No additional information is available. Sodium calcium ascorbyl phosphate is beige, spray-dried powder consisting of the phosphorylated calcium/sodium salt of ascorbic acid. It is specified to contain at least 35 % ascorbic acid equivalent bound to phosphate Qualitative and quantitative composition Analysis of five batches of the additive showed an average ascorbic acid 2-monophosphate content of 34.9 ± 0.5 %, equivalent to 38.8 ± 0.7 % phosphorylated ascorbic acid. 36 A more detailed analysis of three other batches showed 33.6 % ascorbic acid monophosphate, 2.7 % ascorbic acid diphosphate and 0.8 % ascorbic acid triphosphate, the sum of which (37.1 %) is called by the applicant phosphorylated ascorbic acid. 37 During manufacturing, some inorganic compounds are formed as side-products. The concentration in the additive is about 16 % calcium hydroxide, 35 % sodium trimetaphosphate and approximately 13 % sodium hydroxide. Phosphorus content has been determined to be between 9.5 and 10.1 %, sodium between 13.5 and 13.7 % and calcium between 5.4 and 5.9 % (all data from three batches). 38 Heavy metals (lead, cadmium and mercury) and arsenic levels were < 1.0 mg/kg, as analysed in three batches Technical dossier/section II/Annex 2.70 and Annex Technical dossier/section II/Annex Supplementary information July 2012/Annex ii.7 38 Supplementary information July 2012/Annexes ii.1, ii.3 and ii Supplementary information July 2012/Annexes ii.2, ii.4 and ii.6. EFSA Journal 2013;11(2):

21 Physical state Vitamin C for all animal species Six batches of sodium calcium ascorbyl phosphate were analysed for particle size distribution by laser diffraction. The proportion of particles with diameters < 10, 50 and 100 µm was 18 %, 63 % and 84 % (v/v), respectively. 40 The dusting potential, measured using the Stauber Heubach method, was g/m 3, based on an average of three batches Stability and homogeneity Sodium calcium ascorbyl phosphate (three batches, stored in aluminium bags) was demonstrated to have a shelf-life of 24 months at 5 C and at 25 ± 2 C. 42 Stability studies in a premixture (two batches) and in mash and pelleted feed for chickens for fattening were carried out with the formulated additive (see Section 2.6.4). The additive was incorporated in a premixture containing trace elements and choline chloride at a concentration of 107 g/kg. Recovery of the intended initial concentration was 92 %; after three and six months storage at 25 C, recoveries of 99 % and 90 %, respectively, of the intended initial concentration were obtained. 43 Stability studies in feed were performed with concentrations of 0.57 g additive/kg feed. Conditioning (80 C) and pelleting (84 C) resulted in a nominal loss of 6 % of the phosphorylated ascorbic acid. Storage of the mash feed for three months at 25 C did not cause substantial losses. Pelleted feed showed recovery of 83 % after three months. 44 Homogeneity was calculated on the basis of six samples each of the three feed batches. The coefficient of variation was 2.5 % Formulated additive Sodium calcium ascorbyl phosphate is placed on the market by the applicant in the form of a formulated additive (Rovimix Stay-C 35) which consists of 99 % sodium calcium ascorbyl phosphate and 1 % silicon dioxide. It contains a minimum of 35 % phosphorylated ascorbic acid. Considering the structural formula, it is estimated that the additive contains about 22 % ascorbic acid Sodium ascorbyl phosphate Sodium ascorbyl phosphate (chemical name L-ascorbic acid 2-(dihydrogen phosphate) trisodium salt; synonyms vitamin C phosphate sodium salt, trisodium ascorbyl phosphate, L-ascorbic acid monophosphate trisodium salt) is identified by the CAS number and the EINECS number The structural formula of sodium ascorbyl phosphate is shown in Figure 6. Figure 6: Structural formula of sodium ascorbyl phosphate 40 Technical dossier/section II/Annex Technical dossier/section II/Annex 2.31 and Supplementary Information/July Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex EFSA Journal 2013;11(2):

22 The molecular formula of sodium ascorbyl phosphate is C 6 H 6 O 9 Na 3 P 2H 2 O and its molecular weight is It is a white to off-white odourless powder. It has a melting point of > 200 C (with decomposition) and shows a bulk density of approximately 0.25 g/cm 3. Sodium ascorbyl phosphate is soluble in water (789 g/l at 20 C). The ph of a 3 % (w/v) solution in water is Sodium ascorbyl phosphate is a white to off-white, odourless powder consisting of the phosphorylated sodium salt of ascorbic acid Qualitative and quantitative composition Analysis of five batches of the product showed an average sodium ascorbyl monophosphate content of 99.3 ± 0.1 %. 45 A more detailed analysis of three other batches confirmed the content of sodium ascorbyl monophosphate in the product to be 99.2 %, equivalent to 48.6 % ascorbic acid. 46 Phosphorus content was determined to be between 7.7 and 8.6 %, and sodium between 19.4 and 19.8 % (all data from three batches). Heavy metals (lead, cadmium and mercury) and arsenic levels were < 1.0 mg/kg as analysed in three batches Physical state Two sets of three batches each of sodium ascorbyl phosphate were analysed for particle size distribution (v/v) by laser diffraction. The results were highly inconsistent. Whereas almost all particles (99 %) in the first set of batches were shown to have a diameter < 10 µm, 48 the same fraction in the second set of batches amounted to only 6 %; 14 % of the particles were below 50 µm in the second set. The dusting potential, measured using the Stauber Heubach method, was g/m 3, based on an average of the three batches in the second set Stability and homogeneity Sodium ascorbyl phosphate (three batches, stored in aluminium bags) has been demonstrated to have a shelf-life of 36 months at 25 ± 2 C. 50 No data on the stability of the substance in premixtures and feedingstuffs were provided. Since phosphorylation of ascorbic acid at position 2 obviously protects vitamin C from destruction by oxidation, as shown for sodium calcium ascorbyl phosphate, a similar stability for sodium ascorbyl phosphate can be expected. To demonstrate the stability of sodium ascorbyl phosphate in water for drinking, the applicant provided a public Australian document of Industrial Chemicals Notification and Assessment (administered by the National Occupational Health and Safety Commission) of As reported in this document, sodium ascorbyl phosphate was stable (recoveries between 95 and 106 %) at ph 5 to 8 at ambient temperature and 8 C for 185 days. 51 Homogeneity studies in water for drinking are not necessary considering the high solubility of the product in water Physico-chemical incompatibilities in feed Ascorbic acid is sensitive to light, temperature and humidity. Its activity as an antioxidant may lead to further decomposition if oxidising agents are present in feed. No physico-chemical incompatibilities or interactions have been reported between vitamin C (different forms of ascorbic acid) and feed 45 Technical dossier/section II/Annex Supplementary information July 2012/Annexes ii.8, ii.10 and ii Supplementary information July 2012/Annexes ii.9, ii.11 and ii Technical dossier/section II/Annex Technical dossier/section II/Annex 2.97 and Supplementary Information/July Technical dossier/section II/ Annex Technical dossier/section II/Annex EFSA Journal 2013;11(2):