SCIENTIFIC OPINION. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3. European Food Safety Authority (EFSA), Parma, Italy

Transcription

1 SCIENTIFIC OPINION Scientific Opinion on the safety of ferrous ammonium phosphate as a source of iron added for nutritional purposes to foods for the general population (including food supplements) and to foods for particular nutritional uses 1 EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion on the safety of Ferrous Ammonium Phosphate (FAP) when added for nutritional purposes in foodstuffs for particular nutritional uses (PARNUTS) and foods intended for the general population (including food supplements) as a source of iron and on the bioavailability of iron from this source. FAP is stable at neutral ph in formulated foods. The bioavailability of iron from FAP was shown to be within the range of that from other iron salts used for fortification purposes, and specifically, less than that from ferrous sulphate and greater than that from ferric pyrophosphate. In intended food categories, FAP provides between 0.7 to 14 mg of iron per serving, which corresponds to 5 to 100% of the RDA for iron in adults. Studies evaluating the toxicity of FAP in experimental animals have not been conducted. FAP dissociates under the low ph conditions of the stomach in its components, thus releasing ferrous, ammonium and phosphate ions. Given the previous evaluations of ferrous, ammonium and phosphate salts as food additives and as nutrient sources by the SCF, EFSA and JECFA and that the available information on their toxicity did not identify toxicological effects, the Panel considers that additional toxicological data on FAP are not required. The Panel concludes that the use of FAP as a source of iron in PARNUTS and in foods intended for the general population (including food supplements), at the proposed use levels, is not of safety concern provided that established upper safety limits for iron are not exceeded. KEY WORDS Ferrous ammonium phosphate, FAP, CAS Registry Number , Iron(II) ammonium phosphate, Ammonium iron(ii) phosphate, Phosphoric acid ammonium iron(ii) salt (1:1:1). 1 On request from the European Commission, Question No EFSA-Q , adopted on 14 April Panel members : F. Aguilar, B. Dusemund, P. Galtier, J. Gilbert, D.M. Gott, S. Grilli, R. Gürtler, J. König, C. Lambré, J- C. Larsen, J-C. Leblanc, A. Mortensen, D. Parent-Massin, I. Pratt, I.M.C.M. Rietjens, I. Stankovic, P. Tobback, T. Verguieva, R.A. Woutersen. Correspondence: ans@efsa.europa.eu 3 Acknowledgements: The Panel wishes to thank the members of the Working Group A on Food Additives and Nutrient Sources of the ANS Panel for the preparation of this opinion: F. Aguilar, N. Bemrah, P. Galtier, J. Gilbert, S. Grilli, R. Gürtler, N-G. Ilbäck, C. Lambré, J.C. Larsen, J-C. Leblanc, A. Mortensen, I. Pratt, I. Stankovic, C. Tlustos. Suggested citation purposes: EFSA Panel on Food Additives and Nutrient Sources (ANS); Scientific Opinion on the safety of ferrous ammonium phosphate as a source of iron added for nutritional purposes to foods for the general population (including food supplements) and to foods for particular nutritional uses.. [26 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on the safety of Ferrous Ammonium Phosphate (FAP) when added for nutritional purposes in foodstuffs for particular nutritional uses (PARNUTS) and foods intended for the general population (including food supplements) as a source of iron and on the bioavailability of iron from this source. The present opinion deals with the safety of FAP when added for nutritional purposes in PARNUTS and foods intended for the general population (including food supplements) as a source of iron and on the bioavailability of iron from this source. The safety of iron itself in terms of amounts that may be consumed is outside the remit of this Panel. FAP is an inorganic salt with iron(ii), ammonium and phosphate ions in a 1:1:1 molar ratio. The content of iron(ii) is 22-30% (w/w). The Panel notes that the representative commercial FAP product stability study for 19 and 36 months storage under the recommended conditions did not adequately confirm the stability of ferrous ion against oxidation. From the stability studies in food and one in vitro solubility study under conditions mimicking the conditions in infant and adult stomachs, it can be deduced that FAP is stable at neutral ph in formulated foods but readily dissociates under the low ph conditions of the stomach, thus releasing the bioavailable ferrous ion. The Panel reviewed a randomised double-blind, crossover human bioavailability study of iron from FAP. The bioavailability of iron from FAP was shown to be within the range of that from other iron salts used for fortification purposes, and specifically, less than that from ferrous sulphate and greater than that from ferric pyrophosphate. The Panel notes that FAP is intended for use as a direct replacement for currently permitted iron forms in all PARNUTS food categories, with the exception of baby foods and infant formulae. The petitioner also proposed the use of FAP as a source of iron in fortified foods. The proposed conditions of use of FAP in intended food categories provides between 0.7 and 8.2 mg of iron per serving, which corresponds to between 5 and 58.6% of the Recommended Daily Amount (RDA) for iron in adults. FAP is also intended for use in food supplements as a direct replacement for other permitted sources of iron. One daily serving of FAP will not exceed the RDA for iron of 14 mg. The Panel calculated the total dietary intake of iron, phosphorus and ammonia from the consumption of food categories fortified with FAP and from other dietary sources in European countries. The Panel assumed a daily intake of a beverage serving and a serving of solid food both fortified with FAP at the maximum proposed use levels for beverages (27.2 mg FAP per serving) and solid foods (14 mg FAP per serving) provided by the petitioner in the technical dossier. The additional exposure to 12.4 mg iron/day from FAP (assuming 30% of iron content in a serving fortified with 41.2 mg FAP) would result in an anticipated total average exposure to iron ranging from 20.8 to 31.4 mg/day and at the high percentile in an anticipated exposure ranging from 25.2 to 43.3 mg/day for adults and an anticipated total average exposure ranging from 17.9 to 28.2 mg/day and at the high percentile in an anticipated exposure ranging from 20.7 to 39.4 mg/day for children. The Panel notes that the use levels of FAP proposed by the petitioner will provide 12 mg iron/day, which is below the guidance value for supplemental intake of iron of 17 mg/day recommended by the Expert Group on Vitamins and Minerals (EVM) in The proposed total exposure will not exceed the Provisional Maximum Tolerable Daily Intake (PMTDI) for iron established by Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1993, even in high consumers. 2

3 The Panel notes that an additional exposure to 22.7 mg phosphorus/day (assuming 55% of phosphorus content in a serving fortified with 41.2 mg FAP) would result in an anticipated total average exposure ranging from to mg/day and at the high percentile in an anticipated exposure ranging from to mg/day for adults and an anticipated total average exposure ranging from to mg/day and at the high percentile in an anticipated exposure ranging from to mg/day for children. These amounts of phosphate do not exceed the Tolerable Upper Intake Levels (ULs) defined by the Institute of Medicine (IOM) in The intake of 3.7 mg ammonia/day from FAP (assuming 9% of ammonia content in a serving fortified at 41.2 mg FAP) seems negligible compared to its endogenous production levels of 3000 mg/day in the colon. Studies evaluating the toxicity of FAP in experimental animals have not been conducted. In the study on the bioavailability of iron from FAP in humans, no gastrointestinal complaints or other adverse effects were reported following consumption of two servings of a milk product fortified with FAP, providing a total iron dose of 5 mg. FAP dissociates at low ph to its respective ferrous, ammonium and phosphate ions. Given the previous evaluations of ferrous, ammonium and phosphate salts as food additives and as nutrient sources by the Scientific Committee on Food (SCF), EFSA and JECFA and that the available information on their toxicity did not identify toxicological effects, the Panel considers that additional toxicological data on FAP are not required. The Panel notes that, at the proposed use levels, the corresponding exposure to iron from FAP does not exceed the guidance value for supplemental intake of iron of 17 mg /day recommended by the EVM. Likewise, the corresponding exposure to phosphorus from FAP does not exceed the ULs defined by the IOM and the ammonia exposure is negligible compared to its endogenous production level. Therefore, the Panel concludes that the use of FAP as a source of iron in PARNUTS and in foods intended for the general population (including food supplements), at the proposed use levels, is not of safety concern provided that established upper safety limits for iron are not exceeded. 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 4 Background as provided by the European Commission... 5 Terms of reference as provided by the European Commission... 5 Assessment Introduction Technical data Identity of the substance Specifications Manufacturing process Methods of analysis in food Stability, reaction and fate in foods Case of need and proposed uses Proposed use in foods for particular nutritional uses (PARNUTS) Proposed use in fortified foods Information on existing authorisations and evaluations Ferrous ammonium phosphate Iron Phosphorus (as phosphate) Ammonia (as ammonium ion) Exposure Iron Phosphorus Ammonia Biological and toxicological data Absorption, distribution, metabolism and excretion (ADME) Bioavailability of iron from FAP In vitro studies Human studies Bioavailability of iron from ferrous sulphate ADME of iron from ferrous salts ADME of ammonia from ammonium salts ADME of phosphorus from phosphate Toxicological data Discussion Conclusions Documentation provided to EFSA References Glossary and abbreviations

5 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The European Community legislation lists nutritional substances that may be used for nutritional purposes in certain categories of foods as sources of certain nutrients. The Commission has received a request for the evaluation of ferrous ammonium phosphate as a source of iron added for. The relevant Community legislative measures are: Commission Directive 2001/15/EC of 15 February 2001 on substances that may be added for specific nutritional purposes in food for particular nutritional uses 4. Regulation (EC) 1925/2006 of the European Parliament and of the Council on the addition of vitamins and minerals and of certain other substances to foods 5. Directive 2002/46/EC of the European Parliament and of the Council on the approximation of the laws of the Member States relating to food supplements 6. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002, the European Commission asks the European Food Safety Authority to: - provide a scientific opinion, based on its consideration of the safety and bioavailability of ferrous ammonium phosphate as source of iron added for nutritional purposes in foodstuffs for particular nutritional uses and foods intended for the general population (including food supplements). 4 OJ L 52, , p OJ L 404, , p OJ L 183, , p.51. 5

6 ASSESSMENT 1. Introduction The present opinion deals with the safety of Ferrous Ammonium Phosphate (FAP) when added for nutritional purposes in foodstuffs for particular nutritional uses (PARNUTS) and foods intended for the general population (including food supplements) as a source of iron and on the bioavailability of iron from this source. The safety of iron itself in terms of amounts that may be consumed is outside the remit of this Panel. 2. Technical data 2.1. Identity of the substance FAP is an inorganic salt with iron(ii), ammonium and phosphate ions in an equal molar ratio. The molecular formula of FAP described by the petitioner is FeNH 4 PO 4 XH 2 O, where X 1 (typically). The content of iron(ii) in the product is 22-30% (w/w). The CAS Registry Number of the anhydrous form is and the molecular weight is g/mol. Synonyms: Iron(II) ammonium phosphate, ammonium iron(ii) phosphate and phosphoric acid, ammonium iron(ii) salt (1:1:1) Specifications FAP is described as greyish-green fine powder, practically insoluble in water and soluble in dilute mineral acids. The ph of a 5% suspension in water is in the range of The content of iron(ii) is 22-30% (w/w), iron(iii) not more than 7% (w/w), and total iron not less than 28% (w/w). Specifications of purity for FAP as proposed by the petitioner and according to the Joint FAO/WHO Expert Committee on Food Additives (JECFA) specifications (JECFA, 2009) are presented in Table 1. Table 1: Chemical specifications for FAP as proposed by the petitioner and according to JECFA (2009). Specification parameter Specifications as proposed by the petitioner JECFA specifications (JECFA 2009) Iron(II) 22-30% (w/w) 24-30% Ammonia 5-9% (w/w) - Phosphate 49-55% (w/w) - Iron(III) < 7% (w/w) < 7% Total Iron > 28.0% (w/w) - Water < 3.0% (w/w) < 3% Sulphate < 0.2% (w/w) - Aluminium < 30 mg/kg - Arsenic < 3 mg/kg < 3 mg/kg Lead < 10 mg/kg < 2 mg/kg 6

7 Mercury - < 1 mg/kg Cadmium < 3 mg/kg < 1 mg/kg Nickel < 50 mg/kg - Fluoride < 50 mg/kg < 50 mg/kg Aerobic mesophilic count < cfu/g - The analyses of three commercial batches of FAP show that the material produced by the manufacturing process described below is consistent and complies with the proposed specifications. The average particle size (D50) of three commercial batches of FAP is 2.1 µm. The Panel notes that according to Commission Regulation (EC) No 629/2008 7, the maximum levels of lead, mercury and cadmium in food supplements, as sold, should be 3.0 mg/kg, 0.1 mg/kg and 1 mg/kg, respectively Manufacturing process The manufacturing process is briefly described. Iron powder, phosphoric acid and ammonia solution are combined in demineralised water to generate FAP. Any excess starting materials (e.g. unreacted iron powder) or insoluble salts are removed during the purification and spray drying (volatiles such as ammonia will evaporate) processes. The product is then milled into fine powder with the required particle size. The manufacturing site operates under the principles of Good Manufacturing Practice (GMP) and is certified by the German Health Authorities. The relevant side reaction that might potentially influence the bioavailability of iron is the oxidation of iron(ii) to iron(iii) during the manufacturing process. According to the petitioner, the iron(iii) formation is limited to low levels (maximum 7%) and is monitored by the Quality Control department Methods of analysis in food The level of total iron is used as a measure of the amount of FAP present in a fortified food product and can be determined either by colorimetric procedure as iron(ii) phenanthroline complex (limit of quantification approximately 1 mg/100 g) or by Atomic Absorption Spectroscopy (AAS) (limit of quantification approximately 0.5 mg/100 g). Both methods are well established procedures applicable to the range of foods that would be typically fortified with FAP such as milk or cereal-based products Stability, reaction and fate in foods The petitioner notes that significant oxidation does not occur when the FAP is stored under suitable conditions, but the Panel considers that this statement is not adequately confirmed by the supplied data. As representative stability data, the petitioner provided results for the contents of ammonia (NH 3 ), iron(ii), iron(iii) and total iron initially and after 19 and 36 months storage in original packaging. With respect to the contents of iron(ii) and iron(iii), the tested material did not comply with the proposed specifications (22-30% and less than 7%, respectively). Initially, the contents of iron(ii) and iron(iii) were 21.7% and 8.5%, respectively, while after 36 months storage, the content of iron(ii) decreased to 17.1% and the content of iron(iii) increased to 12.7%. 7 Commission Regulation (EC) No 629/2008 of 2 July 2008 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs. OJ L 173, , p

8 Accelerated stability experiments during one month storage at elevated temperatures (25-40 o C) and high humidity (60-75%) did not cause changes in the contents of iron(ii) and iron(iii) beyond the limits set in the proposed specifications. According to the data presented by the petitioner, FAP has high stability in formulated foods at neutral ph. Accelerated and real-time storage stability studies on a range of milk-based powders and cerealtype products indicate that products fortified with FAP are stable over prolonged periods (representative of shelf-life) (Nestlé, 2000). Addition of soluble iron salts to food products high in polyphenols such as tea, chocolate milk or banana-containing products can lead to an interaction between the iron and polyphenols in the food matrix. The resulting iron/phenol complex is an intense dark grey or black colour causing the product to become discoloured, particularly when a powdered beverage formulation is reconstituted with water or milk at high (boiling) temperatures. Experiments to evaluate discoloration both visually and by colorimetry have been performed in a range of beverages fortified with FAP under typical formulation and reconstitution conditions and compared with controls containing either no supplemental iron salts or the common fortificant, ferrous sulphate. In general, no colour changes were observed in tea, cocoa powders, or milk products under conditions typical of use. In all cases, except banana cereals, FAP was more effective than ferrous sulphate at retaining product colour and was often comparable in colour to the product without iron supplementation (Nestlé, 2000; Sher et al., 2001; Rekhif et al., 2002). The main concern relating to the possible instability and its effect on biological properties including nutrient value following fortification of products with FAP is the pro-oxidative effect. The addition of soluble iron sources to fat-containing products, in particular products high in unsaturated fatty acids, can result in lipid oxidation and lead to changes in the organoleptic and nutritional properties of the products. Accelerated oxidative stability studies have been conducted with fish oils (high in unsaturated fatty acids) fortified with iron salts. The time taken for the fish oil to undergo oxidation when fortified with FAP was comparable to the control (i.e. without iron fortification) and significantly slower than for oils fortified with ferrous sulphate (Nestlé, 2000; Sher et al., 2001; Rekhif et al., 2002). Lipid oxidation has also been monitored in a typical fat-containing infant formula product fortified with FAP. The extent of oxidation was measured by determining the levels of the volatile oxidation product hexanal in the headspace above the samples. The level of hexanal detected in the FAP product was comparable to the control without iron supplementation and far lower than the one containing iron(ii) sulphate (Nestlé, 2000; Rekhif et al., 2002) Case of need and proposed uses According to the petitioner, FAP is intended to be used to fortify a range of foods and beverages with a source of bioavailable iron in order to aid the prevention of iron deficiency. FAP has been developed as an alternative to other commonly used iron fortification salts as a source of iron that is highly stable at the neutral ph of most formulated foods but that readily dissociates under the low ph (2-3) conditions of the stomach to the iron(ii) ions. It is intended as a direct replacement for other permitted iron sources in products such as those containing polyphenols (e.g. tea, cocoa etc.) or those with a high fat content where the pro-oxidative effect of iron can occur and where the organoleptic properties of the finished food products are of a particular concern. 8

9 Proposed use in foods for particular nutritional uses (PARNUTS) FAP is intended for use as a direct replacement for currently permitted iron forms in all PARNUTS food categories, with the exception of baby foods and infant formulae (Council Directive 89/398/EEC 8 ). The petitioner states that under the intended conditions of use, the daily intake of iron from FAP would not exceed the levels anticipated through existing iron supplementation programmes. The levels of addition of FAP would be similar to other forms of ferrous iron currently approved for use in PARNUTS Proposed use in fortified foods The petitioner also proposes the use of FAP as a source of iron in fortified foods. The intended conditions of use of FAP in fortified foods, and the corresponding use levels of FAP for all typical examples of fortified food uses of FAP in the EU are summarised in Table 2. The use level of FAP for sport beverages, fortified cereal bars, and meal replacement bars provides 2.1 mg of iron per serving, which corresponds to 15% of the Recommended Daily Amount (RDA) for iron in adults. Table 2: Summary of the individual typical fortified food uses and use levels for FAP and the corresponding use levels for iron as proposed by the petitioner. Food category Beverages Fortified foods Milk and milk products Soups RTD: Ready-to-Drink. Proposed fooduse Serving size (g or ml) FAP Use level (mg/serving ) Use level (mg/100 g or ml) Iron Use level (mg/serving ) Powdered 15 a chocolate or malt beverages Sport beverages Fortified cereal bars Meal replacement 60 a bars Nutritionally complete supplement drinks, powdered and RTD Powdered milk beverages 30 (powder) 250 (RTD) (powder) 10.9 (RTD) Use level (mg/100 g or ml) (powder) 3.3 (RTD) 32.5 a Stock 2 a Cubes/Powders Instant noodles 30 a Council Directive of 3 May 1989 on the approximation of the laws of the Member States relating to foodstuffs intended for particular nutritional uses (89/398/EEC). OJ L 186, , p

10 a Provided by Nestlé and for use-levels adjusted for dilution where relevant. Proposed use in food supplements According to the petitioner, FAP is also intended for use in food supplements as a direct replacement for other sources of iron permitted for use in food supplements under Commission Regulation (EC) No 1170/ One daily serving of FAP from a food supplement product will not exceed the RDA for iron of 14 mg laid down under Directive 2008/100/EC 10 on nutrition labelling for foodstuffs. The form of food supplements containing FAP may include tablet, powder or beverage style formulations Information on existing authorisations and evaluations Ferrous ammonium phosphate The use of FAP as a source of iron in foods and food supplements is currently not permitted in the EU. In 2009, JECFA evaluated the safety of FAP for use in food fortification and prepared new specifications (JECFA, 2009, 2010). The newly available information on the toxicity of iron did not indicate a need to revise the Provisional Maximum Tolerable Daily Intake (PMTDI) for iron of 0.8 mg/kg bw. Consideration of the toxicity of ammonium and phosphate did not indicate a need to revise the JECFA s previous evaluations of these ions. JECFA concluded that FAP is acceptable for use as a source of iron for dietary fortification, provided that the total intake of iron does not exceed the PMTDI. Products, including FAP, that are intended to provide a source of additional iron should not be consumed by individuals with any type of iron storage disease, except under medical supervision (JECFA, 2010) Iron Iron is included in Annex I of Commission Regulation (EC) No 1170/2009 which lists the mineral substances which may be used in the manufacture of food supplements. The various iron sources are listed in Annexes II and III of this Regulation, as approved mineral substances which may be used in the manufacture of food supplements and which may be added to foods. JECFA established a PMTDI for iron of 0.8 mg/kg bw/day based on the safe long-term intake of ferrous supplements of 50 mg iron/day (for a 60 kg individual) (JECFA, 1983). In 1993, the Scientific Committee on Food (SCF) recommended daily intakes of iron of 6 mg and 4 mg for infants aged year and 1-3 years respectively, assuming 15% absorption of the daily intake. For adults, assuming 10% absorption of the daily intake, the recommended dietary iron intake has been estimated to be between 8 and 10 mg iron/day (SCF, 1993). The US Food and Nutrition Board (FNB) established a tolerable upper intake level (UL) of 45 mg iron/day for individuals aged 14 years and older, and 40 mg iron/day for younger age groups (IOM, 2001). 9 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. Official Journal of the European Union L 314, , p Commission Directive 2008/100/EC of 28 October 2008 amending Council Directive 94/496/EC on nutrition labelling for foodstuffs as regards recommended daily allowances, energy conversion factors and definition. Official Journal of the European Union L 385, , p

11 The Expert Group on Vitamins and Minerals (EVM) concluded that there were insufficient appropriate data to establish a Safe Upper Level for iron (EVM, 2003). It was also stated that for guidance purposes only, a supplemental intake of approximately 17 mg iron/day (equivalent to 0.28 mg/kg bw/day for a 60 kg adult) would not be expected to produce adverse effects in the majority of people (EVM, 2003). In 2004, the EFSA Scientific Panel on Dietetic Products, Nutrition and Allergies (NDA) concluded that the available data were insufficient to establish a UL for iron. Based on estimates of current iron intakes in European countries, the risk of adverse effects from a high iron intake from food sources, including fortified foods in some countries, but excluding supplements, is considered to be low for the population as a whole, except for those homozygous for hereditary haemochromatosis (up to 0.5% of the population). However, intake of iron from food supplements in men and postmenopausal women may increase the proportion of the population likely to develop biochemical indicators of high iron stores. Some groups at special risk for poor iron status, such as menstruating women or children, could benefit from additional iron intake and/or an improved availability of dietary iron (EFSA, 2004). In 2006, the EFSA Panel on Food Additives, Flavourings, Processing Aids and materials in Contact with Food (AFC) evaluated ferrous bisglycinate as a source of iron for use in the manufacturing of foods and in food supplements. The AFC Panel considered that the use of ferrous bisglycinate, as a source of iron in foods intended for general population, food supplements and PARNUTS including foods for infants and young children, meeting the specifications proposed, does not present a safety concern (EFSA, 2006). In 2009, EFSA s Panel on Food Additives and Nutrient Sources added to Food (ANS) evaluated the safety of ferrous phosphate and the bioavailability of iron from this source in food supplements. The Panel was of the opinion that, given the previous evaluations of ferrous iron, phosphoric acid and phosphates as food additives and as nutrient sources by the SCF, EFSA and JECFA, no additional toxicological evaluation of ferrous phosphate was required. Therefore the Panel concluded that the use of ferrous phosphate in food supplements as a source of iron is not of safety concern at the proposed use levels (EFSA, 2009b). In 2009, the Panel evaluated the safety of ferric sodium EDTA as a source of iron added for nutritional purposes to foods for the general population (including food supplements) and to PARNUTS. The Panel concluded that iron is bioavailable from ferric sodium EDTA and that the use of ferric sodium EDTA as a source of iron in food is of no safety concern as long as it does not lead to an exposure to EDTA above 1.9 mg EDTA/kg bw/day Phosphorus (as phosphate) Phosphorus is included in Annex I of Commission Regulation (EC) No 1170/2009 which lists the mineral substances which may be used in the manufacture of food supplements. The various phosphorus salts are listed in Annexes II and III of this Regulation, as approved mineral substances which may be used in the manufacture of food supplements and which may be added to foods. Phosphoric acid and a number of phosphate salts are authorised food additives in the EU (Directive 95/2/EC 11 ). For phosphorus, in 1993 the SCF established Population Reference Intakes (PRI) of 300 mg for children, 775 and 625 mg in males and females aged years respectively and 550 mg/day in adults (SCF, 1993). More recently, higher recommendations were established by the Institute of Medicine (IOM, 1997) and D-A-CH (2000) as follows: 700 mg/day for adults and up to 1250 mg/day 11 European Parliament and Council Directive No 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners. OJ L61, , p. 1 as amended 11

12 for adolescents. For the younger age groups, a factorial approach was used to calculate PRIs. Estimates of habitual dietary intakes for phosphorus in European countries are on average around mg/day ranging up to about 2600 mg/day. The contribution of food supplements to total daily phosphorous intake is low (EFSA 2005). Regarding phosphorus, the NDA Panel could not derive a UL but indicated that normal healthy individuals can tolerate phosphorus intakes without adverse systemic effects up to at least 3000 mg/day. However, in some individuals, mild gastrointestinal symptoms have been reported following exposure to supplemental intakes >750 mg phosphorus/day. However, the NDA Panel highlighted, that there is no evidence of adverse effects associated with the current dietary intakes of phosphorus in EU countries (EFSA, 2005). A group Maximum Tolerable Daily Intake (MTDI) for phosphorus from all food sources of 70 mg/kg bw was established at the 26 th meeting of JECFA (JECFA, 1982). The IOM established a UL for phosphorus in different population groups ranging from 3000 to 4000 mg/day (IOM, 1997). The EVM could not establish a Safe Upper Level for phosphorus but concluded that for guidance purposes only, a supplemental intake of 250 mg/day would not be expected to produce adverse effects, including mild gastrointestinal upset (EVM, 2002). In 2009, the Panel evaluated the safety of ferrous phosphate and the bioavailability of iron from this source in food supplements. The Panel was of the opinion that, given the previous evaluations of ferrous iron, phosphoric acid and phosphates as food additives and as nutrient sources by the SCF, EFSA and JECFA, no additional toxicological evaluation of ferrous phosphate was required (EFSA, 2009b) Ammonia (as ammonium ion) Ferric ammonium citrate and ammonium molybdate are listed in Annexes II and III of Commission Regulation (EC) No 1170/2009 as approved mineral substances which may be used as sources of iron and molybdenum, respectively in the manufacture of food supplements and which may be added to foods. Ammonium hydroxide and a number of ammonium salts are authorised food additives in the EU (Commission Directive 95/2/EC on food additives other than colours and sweeteners). In 1965, ammonia solution was evaluated as a food additive by JECFA and an Acceptable Daily Intake (ADI) not limited was established (JECFA, 1965). No restriction was placed on the intake of ammonium from ammonium salts, provided that the contribution from food is assessed and considered acceptable. Ammonia has been evaluated by the International Programme on Chemical Safety (IPCS, 1986). The IPCS concluded that ammonia does not present a direct threat to man except as a result of accidental exposure, particularly in industry. The World Health Organization (WHO) has assessed the safety of ammonia in its Guidelines for Drinking Water Quality (WHO, 2006). The WHO concluded that ammonia is not of direct safety concern for human health in the concentrations expected in drinking water, and therefore, a healthbased guideline was not derived. In 2008, EFSA s AFC Panel evaluated ammonia and two ammonia salts (ammonium chloride and ammonium hydrogen sulphide) as flavouring substances in the Flavouring Group Evaluation 46 (FGE.46). On the basis of the default Maximised Survey-derived Daily Intake (MSDI) approach the 12

13 AFC Panel concluded that the evaluated substances would not give rise to safety concerns at the estimated levels of intake arising from their use as flavouring substances (EFSA, 2009a) Exposure In the technical dossier, the petitioner provided information on the exposure to iron, phosphate and ammonium ion calculated from use levels, standard food servings and UK food consumption data. The Panel calculated the total dietary intake of iron, phosphorus and ammonia from the consumption of FAP from fortified food categories, assuming the daily intake of a beverage serving and a serving of solid food both fortified at the maximum proposed use levels for beverages (27.2 mg FAP per serving) and solid foods (14 mg FAP per serving) provided by the petitioner in the technical dossier and their intakes from other sources in the European countries Iron Foods particularly rich in iron are liver and offal, game and beef; cereals, cereal products and pulses also contain moderate to high levels. According to EFSA (2006) and Flynn et al. (2009), the average and high percentile iron intakes from food for adults in European countries vary from 8.4 mg/day in Irish females to 19 mg/day in German males and from 12.8 mg/day in Irish females to 30.9 mg/day Polish males, respectively. The Panel notes that the additional exposure of 12.4 mg iron/day (assuming 30% of iron content in a serving fortified with 41.2 mg FAP) would for adults result in an anticipated total average exposure ranging from 20.8 to 31.4 mg/day and at the high percentile in an anticipated exposure ranging from 25.2 to 43.3 mg/day (see Table 3). According to available data from the French food consumption survey (AFSSA, 2009) and Flynn et al. (2009), the average and high percentile iron intakes from food for children vary from 5.5 to 15.8 mg/day and from 8.3 to 27 mg/day, respectively (see Table 3). The Panel notes that the additional exposure of 12.4 mg iron/day (assuming 30% of iron content in a serving fortified at 41.2 mg FAP) would for children result in an anticipated total average exposure ranging from 17.9 to 28.2 mg/day and at the high percentile in an anticipated exposure ranging from 20.7 to 39.4 mg/day (see Table 3). Table 3: Summary information on iron intake and anticipated exposure to iron from FAP. Nutrient: Iron Recommended Intake -Adults Recommended Intake -Children Tolerable Upper Intake Level (UL) -Adults including pregnant and lactating women Average intake (mg/day) 8-10 (males) (females of reproductive age) 30 (pregnant women) 6 (0.5-1 year old) 4 (1-3 years old) 40 (below 14 years old) 45 (14 years old and older) High intake (95 th or 97.5 th percentile (mg/day) References SCF, 1993; IOM, 2001 SCF, 1993 Insufficient data EFSA,

14 Tolerable Upper Intake Level (UL) -Children Insufficient data EFSA, 2006 Intake range from food in Europe -Adults Intake range from food in Europe -Children (3-17 years old) Maximum proposed use level of FAP* per serving (beverage+solid food) Maximum use level of iron per serving assuming 30% iron in FAP Source : Ferrous Ammonium Phosphate EFSA, 2006 Flynn et al., 2009 AFSSA, 2009; Flynn et al., Technical dossier 12.4 Technical dossier Total anticipated exposure to iron from fortified foods and food intake for adults. Total anticipated exposure to iron from fortified foods and food intake 2 for children (3-17 years old). Calculation Panel Calculation Panel *FAP=Ferrous Ammonium Phosphate. 1 calculation based on maximum proposed use level of 12.4 mg iron/serving plus average dietary intake of mg/day and high dietary intake of mg/day for adults. 2 calculation based on maximum proposed use level of 8.2 mg iron/serving plus average dietary intake of mg/day and high dietary intake of mg/day for children. by by Phosphorus Phosphorus is widely found in foods as phosphates; foods especially rich in protein are usually high in phosphorus, such as dairy products ( mg/100 g), meats (200 mg/100 g), fish (200 mg/100 g) and grain products ( mg/100 g). Phosphoric acid (E 338) and phosphate salts (E 339-E 341, E 343, E 450-E 452) are also added as food additives in processed foods and in soft drinks as regulators and stabilisers. No specific data on the contribution of phosphorus-containing food additives to the total intake of phosphorus in the EU have been identified (EFSA, 2005). In the US, the contribution of phosphorus from phosphoruscontaining food additives is estimated to be 320 mg/day, i.e % of the total adult phosphorus daily intake (Calvo and Park, 1996). For the present evaluation, and regarding the difficulty to know the exact contribution of phosphorus from phosphorus-containing food additives in Europe, the Panel based the calculations on the reported figures assuming that food intake includes both sources (i.e. nutrients and food additives). Phosphorus is also present in drinking water, with a maximum allowed content of 2.2 mg/l (Council Directive 80/778/EEC 12 ). According to Flynn et al. (2009), the average and high percentile phosphorus intakes from food for adults in European countries vary from 1015 mg/day in Polish females to 1816 mg/day in Dutch males and from 1503 mg/day in Spanish females to 2709 mg/day in the German population, respectively. The Panel notes that an additional exposure to 22.7 mg phosphorus/day (assuming 55% of phosphorus content in a serving fortified with 41.2 mg FAP) would for adults result in an anticipated total average exposure ranging from to mg/day and at the high percentile in an anticipated exposure ranging from to mg/day (see Table 4). 12 Council Directive 80/778/EEC relating to the quality of water intended for human consumption. OJ L 229, , p

15 According to available data from Flynn et al. (2009), the average and high percentile phosphorus intakes from food for children vary from 920 to 1518 mg/day and from 1331 to 2571 mg/day, respectively. The Panel notes that the additional exposure of 22.7 mg phosphorus/day (assuming 55% of phosphorus content in a serving fortified with 41.2 mg FAP) would for children result in an anticipated total average exposure of to mg/day and at the high percentile in an anticipated exposure ranging from to mg/day (see Table 4). Table 4: Summary information on phosphorus intakes from food and food additives and anticipated exposure to phosphorus from FAP. Nutrient: Phosphorus Population Reference Intake -Adults Population Reference Intake -Children Tolerable Upper Intake Level (UL) -Adults including pregnant and lactating women Tolerable Upper Intake Level (UL) -Children Average intake (mg/day) (Pregnant women) 300 (6 months-3 years old) (11-17 years old males) (11-17 years old females) 4000 (males and females including lactating women) 3500 (pregnant women) 3000 (elderly males and females) Not established (0-12 months) 3000 (under 9 years old) 4000 (9 to 18 years old) High intake (95 th or 97.5 th in mg/day) References SCF, 1993 SCF, 1993 IOM, 2001 IOM, 2001 Intake range from food in Europe Flynn et al., Adults Intake range from food in Europe Flynn et al., Children (3-17 years old) Maximum proposed use-level of FAP* 41.2 Technical dossier per serving (beverage+solid food) Maximum use-level of phosphorus per serving assuming 55% phosphorus in 22.7 Technical dossier FAP Source: Ferrous Ammonium Phosphate Total anticipated exposure to phosphorus from fortified foods and food intake 1 Calculation by for Panel adults. Total anticipated exposure to phosphorus from fortified foods and food intake 2 Calculation by for Panel children (3-17 years old). *FAP=Ferrous Ammonium Phosphate 1 calculation based on maximum proposed use level of 22.7 mg phosphorus/serving plus average dietary intake of mg/day and high dietary intake of mg/day for adults 2 calculation based on maximum proposed use level of 22.7 mg phosphorus/serving plus average dietary intake of mg/day and high dietary intake of mg/day for children 15

16 Ammonia Ferrous ammonium phosphate as a source of iron added for Ammonia has been reported to occur naturally in certain foods: 235 mg/kg in fresh apple, 8130 mg/kg in barley, 8800 mg/kg in beetroot, up to mg/kg in cooked cabbage, 3970 mg/kg in carrot, 6376 mg/kg in cauliflower, mg/kg in celery leaves and/or stalks, mg/kg in tilsit cheese, 0.2 mg/kg in fried chicken, up to 820 mg/kg in coffee, mg/kg in sweet corn, up to 9 mg/kg in egg, up to 2928 mg/kg in fish (fatty, salted), mg/kg in hop oil, mg/kg in kale, mg/kg in lettuce, 1192 mg/kg in malt, 8450 mg/kg in radish (raw), 6340 mg/kg in rhubarb, 400 mg/kg in black tea, up to 40 mg/kg in red wine and up to 69 mg/kg in white wine (TNO, 2000). No data have been found on the dietary intake of ammonium from natural sources in Europe. Based on a US estimate, the average daily ammonia intake from six different ammonium salt additives in food has been calculated to be 18 mg (WHO, 1986). The intake of 3.7 mg ammonia/day from FAP (assuming 9% of ammonia content in a serving fortified at 41.2 mg FAP) seems negligible compared to its endogenous production levels. 3. Biological and toxicological data 3.1. Absorption, distribution, metabolism and excretion (ADME) Bioavailability of iron from FAP In vitro studies In in vitro solubility studies under conditions mimicking those in infant and adult stomachs (i.e., at 37 C and ph of 3.5 and 2.0, respectively), 95 to 100% of the total iron from FAP was released rapidly (Nestlé, 2000). In a subsequent in vitro solubility study, 67% of total iron from a diluted HCl solution, containing FAP, was released within 3 hours at 37 C and ph The solubility of ferrous sulphate and ferric pyrophosphate under similar conditions were 100 and 8% (w/w), respectively (Nestlé, 2007a) Human studies The bioavailability of iron from FAP, ferrous sulphate and ferric pyrophosphate was assessed in a study consisting of two randomised, double-blind, 2-period crossover trials conducted in parallel in which test meals containing FAP, ferrous sulphate, or ferric pyrophosphate were consumed by test subjects (38 healthy women, aged 18 to 30 years). Test meals consisted of 250 ml of reconstituted full cream instant milk powder supplemented with [ 57 Fe]-FAP, [ 58 Fe]-ferrous sulphate, or [ 57 Fe]-ferric pyrophosphate at a fortification level of 2.5 mg iron per serving. In the first trial, subjects consumed 2 servings of either FAP or ferrous sulphate test meals on Day 1 and then 2 servings of the other test meal on Day 2. In the second trial, subjects consumed 2 servings of either ferric pyrophosphate or ferrous sulphate test meals on Day 1 and then 2 servings of the other test meal on Day 2. Baseline measurements for haemoglobin, serum ferritin, and C-reactive protein were taken from blood on Day 1 before test meal administration to determine iron status. Blood was also taken on Day 16 of the study for the determination of isotope-labelled iron incorporation into erythrocytes. The results of the above study indicate that the geometric mean of fractional iron absorption, from ferrous sulphate, FAP, and ferric pyrophosphate were 10.4, 7.4, and 3.3%, respectively. Thus, the amounts of iron absorbed from the 5 mg provided by ferrous sulphate, FAP, and ferric pyrophosphate were 0.52 mg, 0.37 mg, and 0.17 mg, respectively. These differences in iron absorption between FAP and ferrous sulphate or FAP and ferric pyrophosphate were statistically significant; the absorption of iron from FAP was statistically significantly greater than that from ferric pyrophosphate (p<0.0001), 16

17 but statistically significantly less than that of ferrous sulphate (p=0.0002). The absorption values of the different iron formulations also correlated well with their respective solubility under simulated gastric conditions (hydrochloric acid solution, ph 1.93, 37 C). The solubility of ferrous sulphate, FAP, and ferric pyrophosphate under such conditions were 100, 67, and 8% w/w, respectively, after 3 hours. The three subjects suffering from cold/flu during the measurement period did not show different levels of iron absorption than the other tested persons. The impact of baseline plasma ferritin level on iron absorption was also investigated. Baseline ferritin concentrations had a statistically significant (p=0.0007) inverse impact on iron absorption, i.e. an increase of ferritin by 20 ng/ml led to a 5.3% decrease of iron absorption. The authors noted that the greater bioavailability of FAP compared to ferric pyrophosphate appears to be related to its greater solubility at gastric ph (Nestlé, 2007a and 2007b) Bioavailability of iron from ferrous sulphate The petitioner also provided data on the bioavailability of iron from ferrous sulphate. The rationale for this approach is that both ferrous sulphate and FAP are iron(ii) salts and in vivo data (Nestlé 2007a and 2007b) show that FAP exhibits only slightly lower iron bioavailability than ferrous sulphate. Iron absorption values from ferrous sulphate-fortified foods vary widely from less than 1 to 26% (Ashworth and March, 1973; Davidsson et al., 1997, 1998, 2001, 2002 and 2005; Fidler et al., 2003; Fomon et al., 1989; Hurrell, 2002; Layrisse et al., 2000; MacPhail et al., 1981; Martínez-Torres et al., 1979; Mendoza et al., 2001 and 2004; Pérez-Expósito et al., 2005; Rios et al., 1975; Stekel et al., 1986). The absorption of iron specifically from ferrous sulphate-fortified cereal-based foods and milk-based products is similar, ranging from 2 to 11%. These iron absorption values are consistent across all age groups (infants, children, adolescents, and adults). Higher iron absorption values are frequently the result of concomitant ascorbic acid supplementation of the food vehicle, while very low values are sometimes observed in foods with high polyphenol and/or phytic acid content. The iron absorption value of 10.4% from a ferrous sulphate fortified-milk product reported by Nestlé (Nestlé 2007a and 2007b) is consistent with the high-end range reported in the above studies. Therefore, the petitioner concluded that it can be expected that the absorption of iron from FAP-fortified cereal-based foods and milk-based products will be slightly less than 10%. As FAP following oral consumption dissociates to ferrous, ammonium and phosphate ions in the stomach, the Absorption, Distribution, Metabolism and Elimination (ADME) of the constituent ions are briefly discussed below ADME of iron from ferrous salts The absorption of iron in general is dependent on the physiological requirement for iron, which is largely dependent on the status of tissue iron stores as well as on the rate of erythropoiesis. The absorption of iron is also dependent on the source of iron and differs between heme and non-heme iron compounds. Heme iron is highly bioavailable and is absorbed approximately 2- to 3-fold more readily than non-heme iron. Non-heme iron can exist in ferrous (Fe 2+ ), ferric (Fe 3+ ) or elemental form. The absorption of non-heme iron requires prior solubilisation of the iron salts or elemental form in the upper gastrointestinal tract. As iron must be in the ferrous form for absorption, it is more readily absorbed from ferrous than from ferric salts, while elemental iron is absorbed more gradually due to the slow rate of solubilisation. The mechanism of absorption of non-heme iron into intestinal mucosal cells is not clearly established, but may involve the divalent metal transporting protein. The absorption of non-heme iron is also dependent on other dietary constituents. Such constituents can either facilitate (e.g. vitamin C, vitamin A, folic acid, organic acids from fruits, meat proteins) or inhibit (e.g. polyphenols, phytic acid, calcium, zinc) absorption of iron. Following absorption into intestinal mucosal cells, intracellular iron (with no distinction between heme and non-heme iron) is released into the blood stream where it is oxidised into the ferric form and 17