EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS)

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1 SCIENTIFIC PININ ADPTED: 26 April 2016 doi: /j.efsa Calcium phosphoryl oligosaccharides (Ps-Ca )asa source of calcium added for nutritional purposes to food, food supplements and foods for special medical purposes EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) Abstract Ps-Ca is a calcium salt of phosphoryl oligosaccharides (Ps) obtained from potato starch. Ps-Ca is stable at the temperature and ph values encountered for its intended use in chewing gum, hard confectionary, sugar-free soft candy and apple juice. Ps-Ca is highly soluble in water and readily dissociates into phosphorylated oligosaccharides and calcium in the gastrointestinal tract. Ps-Ca is expected to be absorbed, distributed and eliminated in a manner similar to other dietary sources of calcium and oligosaccharides. Calcium was bioavailable as revealed by the increase in plasma calcium concentrations after intra-gastric administration in rats. Ps are not acutely toxic and a no observed adverse effect level of 1,000 mg/kg body weight (bw) per day, the highest dose tested, was established from a 90-day oral toxicity study in rats. No evidence of genotoxicity was observed in a reverse bacterial mutation assay and an in vitro mammalian chromosome aberration test. verall, the Panel considered that the toxicity data provided in support of the current application were in line with the Tier 1 requirement of the Guidance for submission for food additive evaluations issued by the ANS Panel in 2012 and, in the absence of an indication for a toxic potential of Ps-Ca, there was no need for additional data. verall, the Panel concluded that calcium is bioavailable from Ps-Ca and although the toxicity database was too limited to derive an acceptable daily intake, there is no safety concern for its use as a source of calcium added for nutritional purposes to food, food supplements and foods for special medical purposes at the proposed use and use levels. The Panel noted that Ps-Ca would be a major contributor to the daily intake of calcium with respect to the overall average daily intake of calcium European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: Ps-Ca, calcium phosphoryl oligosaccharides, calcium, nutrient source, food supplements, foods for special medical purposes Requestor: European Commission Question number: EFSA-Q Correspondence: fip@efsa.europa.eu EFSA Journal 2016;14(6):4488

2 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Panel members: Fernando Aguilar, Riccardo Crebelli, Alessandro Di Domenico, Birgit Dusemund, Maria Jose Frutos, Pierre Galtier, David Gott, Ursula Gundert-Remy, Claude Lambre, Jean-Charles Leblanc, liver Lindtner, Peter Moldeus, Alicja Mortensen, Pasquale Mosesso, Dominique ParentMassin, Agneta skarsson, Ivan Stankovic, Ine Waalkens-Berendsen, Rudolf Antonius Woutersen, Matthew Wright and Maged Younes Acknowledgements: The Panel wishes to thank the members of the Working Group on Applications for the preparatory work on this scientific opinion and EFSA staff members: Paolo Colombo, Juho Lemmetyinen, Camilla Smeraldi and Alexandra Tard for the support provided to this scientific opinion. Suggested citation: EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food), Scientific opinion on calcium phosphoryl oligosaccharides (Ps-Ca ) as a source of calcium added for nutritional purposes to food, food supplements and foods for special medical purposes. EFSA Journal 2016;14(6):4488, 26 pp. doi: /j.efsa ISSN: European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union. 2 EFSA Journal 2016;14(6):4488

3 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Summary The European Commission asked the European Food Safety Authority (EFSA) to provide a scientific opinion on the safety of the proposed inclusion of calcium phosphoryl oligosaccharides (Ps-Ca )in Annex II to Regulation 1925/2006 as a mineral substance which may be added to food; in Annex II to Directive 2002/46/EC as an authorised substance which may be used in the manufacture of food supplements, and in the Annex to Commission Regulation (EC) No 953/2009 as a substance that may be added for specific nutritional purposes in foods for special medical purposes (FSMPs), and on the bioavailability of the mineral calcium from this source. The present opinion deals with the safety of a particular source of calcium, Ps-Ca, to be used in foods for the general population, food supplements and FSMP, and the bioavailability of calcium from this source. The safety of calcium itself, in terms of amounts that may be consumed, is outside the remit of this Panel. The Panel based its assessment on a dossier submitted by an applicant and containing data generated with a test material described as phosphoryl oligosaccharides containing 5% calcium from a single lot (PS-010) which the Panel considered relevant for the trade mark compound Ps-Ca and data on generic calcium salts of phosphoryl oligosaccharides (Ca-Ps). The latter were still considered relevant for the assessment of Ps-Ca. Ps-Ca is a calcium salt of phosphoryl oligosaccharides (Ps) made from potato starch which readily dissociates in aqueous solution to yield a bioavailable source of calcium. The applicant has proposed chemical and microbiological specifications for Ps-Ca and provided evidence from the analysis of seven independent, non-consecutive batches that the manufacture of Ps-Ca results in a consistent product conforming to the proposed product specifications. The applicant has produced a statement that the manufacture of Ps-Ca is conducted in compliance with the Food Safety System Certification (FSSC) and with current Good Manufacturing Practice (cgmp). The manufacturing process is controlled by Hazard Analysis Critical Control Points (HACCP) in accordance with Regulation (EC) No 852/2004 on the hygiene of foodstuffs. Ps-Ca is manufactured by first processing potato starch at an elevated temperature using heatresistant enzymes to generate a glucose syrup, which then undergoes a decolourisation step. The phosphorylated oligosaccharides are then isolated from the syrup using an ion exchange column and eluted using an aqueous solution of calcium chloride. The resulting Ps-Ca ingredient is then spraydried, filtered, packaged and tested for conformity to product specifications. Ps-Ca may be detected in its intended food matrices (including gum and apple juice) by highperformance liquid chromatography, atomic absorption spectrophotometry and/or inductively coupled plasma mass spectrometry. The heat and ph stability of Ps-Ca has been evaluated using the liberation of inorganic phosphate as a marker of degradation. Ps are stable at temperatures of up to approximately 90 C. Additionally, the results of the ph stability test indicate that Ps are stable ph range 2 11 (at 70 C) but starts to degrade at ph 12 (at a temperature of 100 C). The data indicate that Ps-Ca is expected to remain stable at the temperature and ph values typically encountered for its intended use in chewing gum, hard confectionary, sugar-free soft candy and apple juice. Ps-Ca is comprised of 5% (w/w) calcium and is intended for use in sugar-free chewing gum, sugar-free hard confectionery and sugar-free soft candy at concentrations of between 2.5% and 3.0%, and in selected fruit juices at a concentration ranging between 1.0% and 1.5%. The Panel considered that Ps-Ca is highly soluble; it readily dissociates into phosphorylated oligosaccharides and calcium in the gastrointestinal tract and is rapidly absorbed in a rat jejunum loop model. It is expected to be absorbed, distributed and eliminated in a manner similar to other dietary sources of calcium and oligosaccharides. Calcium was bioavailable as revealed by the increase in plasma calcium concentrations at h after intra-gastric administration in rats. No mortality was observed in male and female rats administered Ps at 2,000 mg/kg body weight, the highest dose tested. No adverse effects on toxicological parameters were observed in repeat dose oral toxicity studies in which rats were administered Ps at doses of up to 1,000 mg/kg body weight (bw) per day for 15, 28 or 90 days. A no observed adverse effect level of 1,000 mg/kg bw per day, the highest dose tested, was established based on the results of the 90-day oral toxicity study in rats. No evidence of genotoxicity was observed in a reverse bacterial mutation assay and an in vitro mammalian chromosome aberration test. Based on the negative results of the in vitro testing, no in vivo assays were deemed necessary by the ANS Panel, which considered that there was no concern for genotoxicity for Ps-Ca used as a nutrient source. 3 EFSA Journal 2016;14(6):4488

4 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium verall, the Panel considered that the toxicity data provided in support of the current application were in line with the Tier 1 requirement of Guidance for submission for food additive evaluations issued by the ANS Panel in 2012 and, in the absence of an indication for a toxic potential of Ps-Ca, there was no need for additional data to be provided. As calculated by the Panel, the mean exposure to Ps-Ca in the total population from its proposed use as a source of supplemental calcium added to foods for the general population, ranged from 2.0 mg/day in adults up to 4,739 mg/day in adolescents. High-level intakes ranged from 22.5 mg/day in children and adolescents up to 16,800 mg/day in adolescents. The corresponding mean intakes of calcium based on the proposed uses of Ps-Ca as a source of supplemental calcium in foods in the general population were calculated by the Panel, assuming that calcium corresponds to 5% weight of Ps-Ca as stated by the applicant. They ranged from 0.1 mg/day in adults up to 237 mg/day in adolescents. High-level intakes ranged from 1 mg/day in children and adolescents up to 840 mg/day in adolescents. Ps-Ca is also proposed for use in food supplements and/or (i.e. not for both at the same time) FSMPs at levels contributing to the daily intake of calcium of mg/day. The mean total intake of calcium from all the proposed uses of Ps-Ca in foods and the normal diet was calculated by the Panel adding the exposure estimates to calcium deriving from the proposed uses of Ps-Ca, to the mean calcium intake from the normal diet as reported in the dietary surveys considered by the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) in its opinion on dietary reference values for calcium. High-level intakes were calculated by adding the mean intake of calcium from the proposed uses of Ps-Ca to the highest value for calcium intake from the regular diet, for each age group using data from the dietary surveys considered by the NDA Panel in its 2015 opinion on calcium intake. A tolerable upper intake level (UL) of 2,500 mg of calcium per day from all sources was proposed for adults and for pregnant and lactating women by the Scientific Committee for Food in 2003 and later confirmed by the NDA Panel in The mean total intake of calcium from all the proposed uses of Ps-Ca and the normal diet would provide a daily intake of calcium ranging from 384 mg/day in infants up to 1,561 mg/day in adolescents. For adults and the elderly, the maximum estimated mean total intakes of calcium were 1,347 mg/day and 1,181 mg/day, respectively, i.e. below the UL of 2,500 mg/day of total calcium. For high-level exposure, the resulting intake of calcium from all sources would range from 742 mg/day in infants up to 2,546 mg/day in adolescents. For adults in the high-level group, the maximum estimated intake of total calcium was 2,414 mg/day, thus close to the UL of 2,500 mg/day total calcium. The estimated intake of calcium from all the proposed uses of Ps-Ca, in addition to the dietary intake, would therefore correspond to 33 54% and 31 47% of the UL for adults and the elderly at the mean intake. For high-level intake, the estimated intake of calcium from all the proposed uses of Ps-Ca in addition to the dietary intake would correspond to 52 97% and 50 81% of the UL for adults and the elderly, respectively. The Panel noted that Ps-Ca would be a major contributor to the daily intake of calcium with respect to the overall average daily intake of calcium. The Panel concluded that calcium is available from Ps-Ca. The toxicity database was too limited to derive an acceptable daily intake (ADI), but the results of the available studies did not give any indication for a toxic potential of Ps-Ca. Therefore, the Panel concluded that there is no safety concern for the use of Ps-Ca as a source of calcium added for nutritional purposes to food, food supplements and FSMP at the proposed use and use levels. The Panel recommended that the proposed specifications should include limits for individual toxic metals (arsenic, cadmium, lead and mercury) in order to ensure that Ps-Ca as a source of calcium for use in foods for the general population, food supplements and FSMPs will not be a significant source of exposure to those toxic elements in food. In the light of the manufacturing process applied, specifications for Ps-Ca should also include possible residual enzymes or proteins in the final product. The Panel also recommended that enzymes used in the manufacturing process of Ps-Ca should be authorised for use in food in the EU. 4 EFSA Journal 2016;14(6):4488

5 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Table of contents Abstract... 1 Summary Introduction Background and Terms of Reference as provided by the European Commission Background as provided by the European Commission Terms of Reference as provided by the European Commission Information on existing evaluations and authorisations Data and methodologies Data Methodologies Assessment Technical data Identity of the substance Specifications Manufacturing process Absence of genetically modified organisms Methods of analysis in food Stability of the substance and reaction and fate in food Fate of Ps-Ca in apple juice Proposed uses and use levels Use in foods for the general population according to Regulation (EC) No 1925/ Use in food supplements according to Directive 2002/46/EC Use in FSMP according to Commission Regulation (EC) No 953/ Exposure data Food consumption data used for the exposure assessment EFSA Comprehensive European Food Consumption Database Exposure estimates to calcium from the use of Ps-Ca as a nutrient source in foods Dietary intakes of calcium in the general population Exposure to calcium from the use of Ps-Ca as a nutrient source in food supplements or FSMPs Exposure assessment to calcium from the overall sources (proposed uses of Ps-Ca and from the regular diet) Uncertainty analysis Biological and toxicological data Absorption, distribution, metabolism and excretion Fate in model gastric fluid Absorption Distribution Excretion Bioavailability of calcium Acute toxicity Short-term and subchronic toxicity Genotoxicity In vitro In vivo Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Reproductive toxicity studies Developmental studies Hypersensitivity, allergenicity and food intolerance ther studies Human studies Discussion Conclusions Recommendations Documentation provided to EFSA References Abbreviations Appendix A Summary of the total estimated exposure to Ps-Ca as a source of calcium added to foods at the use levels proposed by the applicant EFSA Journal 2016;14(6):4488

6 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium 1. Introduction The present opinion deals with the safety of a particular source of calcium, Ps-Ca, to be used in foods for the general population, food supplements and foods for special medical purposes (FSMP), and the bioavailability of calcium from this source. The safety of calcium itself, in terms of amounts that may be consumed, is outside the remit of this Panel Background and Terms of Reference as provided by the European Commission Background as provided by the European Commission The European Union 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 calcium phosphoryl oligosaccharides (Ps-Ca ) as a source of calcium added for nutritional purposes to foods for the general population, food supplements and FSMP. The relevant Union legislative measures are: 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 1 ; Regulation (EC) No 1925/2006 of the European Parliament and of the Council on the addition of vitamins and minerals and of certain other substances to foods 2 ; Commission Regulation (EC) No 953/2009 on substances that may be added for specific nutritional purposes in foods for particular nutritional uses Terms of Reference as provided by the European Commission In accordance with Article 29 (1)(a) of Regulation (EC) No 178/2002 4, the European Commission asks the European Food Safety Authority (EFSA) to provide a scientific opinion based on its consideration of the safety and bioavailability of Ps-Ca as a source of calcium added for nutritional purposes to foods for the general population, food supplements and FSMP Information on existing evaluations and authorisations In 2011, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) published a scientific opinion on the substantiation of a health claim related to sugar-free chewing gum with calcium phosphoryl oligosaccharides and maintenance of tooth mineralisation (EFSA NDA Panel, 2011). Because no human studies were provided in the application, the Panel concluded that a cause and effect relationship was not sufficiently established over and above the established role of sugar-free chewing gum on the maintenance of tooth mineralisation. According to the applicant, Chewing gum containing Ps-Ca is authorised in Japan under the category Foods for Specified Health Uses (FSHU). The complex Ps-Ca is not currently authorised for use as a source of calcium in foods in any other jurisdiction and no evaluations on such substances have been performed by any scientific body. A tolerable upper intake level (UL) of 2,500 mg of calcium per day from all sources was proposed for adults and for pregnant and lactating women by the Scientific Committee on Food (SCF, 2003) and later confirmed by the NDA Panel (EFSA NDA Panel, 2012). An UL for infants, children or adolescents could be set neither by the SCF nor the NDA Panel due to the lack of data. 1 Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements J L 183, , p Regulation (EC) No 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of vitamins and minerals and of certain other substances to foods. J L 404, , p Commission Regulation (EC) No 953/2009 of 13 ctober 2009 on substances that may be added for specific nutritional purposes in foods for particular nutritional uses. J L 269, , p Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety J L 31, , p EFSA Journal 2016;14(6):4488

7 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium 2. Data and methodologies 2.1. Data The present evaluation is based on the data on Ps-Ca provided by the applicant in a dossier submitted in support of its application for the inclusion of the substance as a source of calcium in food, food supplements and FSMP (Documentation provided to EFSA n.1). The dossier submitted contained data generated with both the trademark substance and generic calcium salts of phosphoryl oligosaccharides (Ca-Ps). The latter were still considered relevant for the assessment of Ps-Ca. Data on dietary intake of calcium in the general population were extrapolated from the scientific opinion of the NDA Panel on dietary reference values for calcium (EFSA NDA Panel, 2015). The applicant has provided information on the UK NDNS rolling programme , which was used to calculate an exposure estimate to calcium from Ps-Ca. The Panel noted that the UK NDNS rolling programme data are already included in the EFSA Comprehensive Database (EFSA, 2011a) and therefore decided not to use these data. The applicant has also provided an estimate of the exposure to Ps-Ca from its proposed use as a supplemental source of calcium in the food categories ther confectionery without added sugar, Chewing gum without other sugar and Fruit juices as defined by Council Directive 2001/112/EC obtained using the Food Additives Intake Model (FAIM) template. 5 The Panel decided not to use the estimate of exposure generated from the FAIM tool as provided by the applicant because of missing data for the age group of infants and young children. Accordingly, the Panel performed a refined assessment, limited to the food categories in which Ps-Ca is proposed for use Methodologies The assessment was conducted in line with the principles described in the EFSA Guidance on transparency in the scientific aspects of risk assessment (EFSA Scientific Committee, 2009) and following the relevant existing Guidances from the EFSA Scientific Committee. The ANS Panel assessed the safety of Ps-Ca in line with the principles contained in the latest existing guidance on the safety evaluation of food additives, namely the Guidance for submission for food additive evaluations in 2012 (EFSA ANS Panel, 2012). The evaluation of bioavailability of the nutrient (calcium) from the source Ps-Ca was conducted in line with the principles contained in the Guidance on submissions for safety evaluation of nutrients or of other ingredients proposed for use in the manufacture of foods (SCF, 2001). Dietary exposure to Ps-Ca from the intended use as a source of calcium added to food and the relative calcium intake derived from this intended use was estimated using the food consumption data available within the EFSA Comprehensive European Food Consumption Database. An estimate of the total intake of calcium from all the proposed uses of Ps-Ca in foods and the normal diet was calculated by the Panel adding the exposure estimates to calcium deriving from the proposed uses of Ps-Ca, to the mean calcium intake from the normal diet for each of the dietary surveys considered by the NDA in its opinion on dietary reference values for calcium (EFSA NDA Panel, 2015) for each age group. High-level intakes were calculated by adding the mean intake of calcium from the proposed uses of Ps-Ca to the highest value for calcium intake from the regular diet, for each of the dietary surveys considered in the 2015 NDA Panel opinion on calcium intake, for each age group. The contribution of the calcium source from the intended use of Ps-Ca to the overall dietary intakes of calcium including natural sources was calculated (see Section 3.3). Uncertainties on the exposure assessment were identified and discussed with regard to their impact on the final exposure calculation. 3. Assessment 3.1. Technical data Identity of the substance According to the information provided in the dossier, Ps-Ca is a calcium salt of phosphoryl oligosaccharides (Ps) made from potato starch. 5 Available online: EFSA Journal 2016;14(6):4488

8 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Ps-Ca is a solid, white to off-white powder, soluble in water at approximately 70 g/100 ml. It has an indicative average molecular weight of 800 g/mol, with CAS no The Panel noted that, according to the SciFinder database (SciFinder, online), the CAS number indicated by the applicant corresponds to maltodextrin, dihydrogen phosphate, calcium salt. The Panel noted that according to the information available in the SciFinder database, the formula associated with the CAS number provided by the applicant is not specific and the CAS number cannot be unambiguously associated with the material which is the subject of this application. The P moiety contains between 3 and 6 a-1,4 linked glucose molecules (i.e. maltotriose, maltotetraose, maltopentose and maltohexose). Most of them carry one phosphate group, with some of the maltopentose and maltohexose molecules carrying two phosphate groups (Kamasaka et al., 1997). The average degree of polymerisation is approximately 5. From the phosphorous content of starch ( %), the phosphorous content of glucose monophosphate (12%) and the average degree of polymerisation of Ps, it follows that Ps represent approximately 1 5% of the native potato starch. Due to the use of calcium chloride in the isolation process of Ps, the final product contains % of calcium. According to the applicant, the salt readily dissociates in aqueous solution. The Panel noted that calcium would be available in the form of the cation (Ca 2+ ). The solubility of Ps-Ca was investigated by comparison with soluble and insoluble calcium compounds (To-o et al., 2003). Briefly, Ps-Ca, calcium chloride, calcium lactate, calcium carbonate or dibasic calcium phosphate was dissolved in a solution of distilled water, acetate buffer, phosphate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 0.1 M phosphate buffer (at ph 4.5 or 7.4) or 0.1 M carbonate buffer at 30 C and vigorously shaken for 30 s at 5-min intervals, for a total of 30 min. Solutions were filtered on a 0.45-lm membrane, and the calcium concentration of the filtrate was determined by the o-cresolphthalein complexation method with the calcium C-test (kit from Wako Pure Chemical Industries). The results indicated that Ps-Ca demonstrated high solubility comparable to that of calcium chloride (in the presence or absence of phosphate) over a wide ph range. Comparatively, the solubility of calcium lactate was approximately one-third that of Ps-Ca, and calcium carbonate and dibasic calcium phosphate demonstrated limited solubility in buffers, with the exception of calcium carbonate in the acetate buffer. The authors concluded that the results suggested Ps-Ca would be soluble in the small intestine when ingested. The structural formulae of the major Ps of Ps-Ca are presented in Figure 1. H P P H 6 2 -phosphoryl maltotriose P H H 6 3 -phosphoryl maltotriose P 6 3 -phosphoryl maltotetraose H H P 6 4 -phosphoryl maltopentaose P 3 3 -phosphoryl maltotetraose 3 4 -phosphoryl maltopentaose =P C-3 bound phosphate group P = C-6 bound phosphate group Calcium cation (Ca 2+ ) not included in the figure Figure 1: Structural formulae of the major phosphorylated oligosaccharides of Ps-Ca (Documentation provided to EFSA n.1) Ps-Ca can be identified by high-performance liquid chromatography (HPLC) with a refractive index detector using phosphoryl maltotriose calcium as a reference compound. Ps-Ca has been characterised within a 1% solution and also in a chewing gum matrix Specifications The specifications for Ps-Ca as proposed by the applicant are listed in Table EFSA Journal 2016;14(6):4488

9 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Table 1: Specifications for Ps-Ca as proposed by the applicant Specification parameter Specification value Analytical method Appearance White powder Sensory test Loss on drying 6.5 % 105 C, 4 h ph (1% solution) ph electrode Residue on ignition 19.0% 550 C treatment Saccharides 80.0% Phenol-sulphuric acid method rganic phosphorus 3.0% Molybdic acid Inorganic phosphorus 0.3% Molybdic acid Calcium % Colourimetric method Colouration 0.3% Determination of absorbance (10 Brix solution, ph 5.5) Turbidity 0.1 Determination of absorbance (10 Brix solution, ph 5.5) Arsenic 1 mg/kg Atomic absorption Heavy metals 10 mg/kg Sodium-sulphuric acid method Total aerobic plate counts < 100 CFU/g Standard agar plate Yeast and moulds 20 CFU/g PDA method Ps-Ca : calcium phosphoryl oligosaccharides; CFU: colony forming unit; PDA: potato dextrose agar. The results of seven non-consecutive batch analyses on Ps-Ca indicated that it is a consistent product compliant with the proposed specifications, including those established for microbiological and heavy metal contamination. The Panel noted that in contrast to other nutrient sources, the proposed specifications did not include limits for individual toxic metals (cadmium, lead and mercury). This is possibly due to the analytical method proposed, which can only give an estimate of the total metal content (expressed as lead), and therefore, in the Panel s opinion, is not fit for purpose Manufacturing process According to the applicant, Ps are isolated from an enzymatic hydrolysate of potato starch (i.e. from glucose syrup). The potato starch slurry is treated with heat-resistant a-amylase enzyme at an elevated temperature, leading to the gelatinisation of the starch and its liquefaction. The liquefied starch is further degraded with glucoamylase and pullulanase to yield glucose syrup. The glucose syrup is then passed through an ion-exchange column for the isolation of the anionic Ps. The column is washed and the Ps are eluted with an aqueous solution of calcium chloride. The resulting eluate is decolourised, spray-dried and passed through a sieve and magnetic filter to yield the final Ps-Ca as a dry, white to off-white powder. The following method of production of Ca-Ps is described in the literature (Kamasaka et al., 1995, 1997; To-o et al., 2002). Potato starch is suspended in a solution containing 6 mm NaCl, 2 mm CaCl and 3.0 U/mL of bacterial liquefying a-amylase (BLA) and gradually heated to 100 C to liquefy the starch. The solution is cooled and kept at 50 C for 1 h. Following, glucoamylase (600 U/mL) and pullulanase (200 U/mL) and BLA (300 U/mL are added and the reaction mixture is incubated at 40 C for 2 h. The amylolytic reaction is monitored by the iodine starch complex method and glucose oxidase method. The action of the amylolytic enzymes is hindered by phosphate groups linked to the glucose residues and therefore phosphorylated oligosaccharides remain as indigestible fragments in the reaction mixture. When the iodine starch colour reaction of the solution indicates to be negative, the solution is heated for 10 min at 100 C and treated over an anion-exchange column pre-equilibrated with a 20 mm acetate buffer (ph 4.5). Neutral sugars are washed out with the buffer, and phosphorylated oligosaccharides are eluted by the same buffer containing 0.5 M NaCl. The eluted phosphorylated oligosaccharides are precipitated with ethanol (finally 70%, v/v). To remove the salts, the ethanol precipitate is washed several times with 70% ethanol. Ca-Ps are prepared by neutralising the desalted P solution (ph 2.0) with Ca() 2 to ph 6.5. Ca-Ps are then freeze-dried. 9 EFSA Journal 2016;14(6):4488

10 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium According to the applicant, the manufacture of Ps-Ca is conducted in accordance with Good Manufacturing Practice (cgmp) and is controlled by Hazard Analysis Critical Control Points (HACCP) in accordance with Regulation (EC) No 852/ The Panel noted that the final product should be free of remaining enzyme activity and protein residues resulting from the manufacturing process Absence of genetically modified organisms The Panel noted that three enzymes are used in the degradation of starch during the manufacture of Ps-Ca. The a-amylase added to the potato starch slurry hydrolyses (1,4)-a-D-glucosidic linkages in starch polysaccharides. The glucoamylase and pullulanase subsequently added to the liquefied starch hydrolyse (1,4)- and (1,6)-a-D-glucosidic linkages at the non-reducing ends of polysaccharides, and the (1,6)-a-D-glucosidic linkages in pullulan, amylopectin and glycogen, respectively. The enzymes are manufactured by fermentation of genetically modified microorganisms that are not present in the enzyme preparations added to the starch slurry. According to the applicant, all three enzymes are food-grade; they meet the purity specifications established by the Joint FA/WH Expert Committee on Food Additives (JECFA) and Food Chemicals Codex, and they are intended to be submitted by the enzyme manufacturer for evaluation and authorisation under Regulation (EC) No 1331/ Methods of analysis in food Ps-Ca was analysed in chewing gum via HPLC using a size-exclusion and ligand-exchange chromatography column for carbohydrates with a refractive index detector. Ps-Ca is quantified using the reference standard phosphoryl maltotriose calcium. These methods have been reviewed and accepted by the Japanese National Institute of Health and Nutrition as standard methods to quantify Ps-Ca. Ps-Ca was detected in apple juice using atomic absorption spectrophotometry and inductively coupled plasma mass spectrometry (Mita et al., 2013). Calcium content was determined using atomic absorption spectrophotometry at a wavelength of nm with 1% hydrochloric acid and 5% strontium chloride solution. Inorganic phosphate concentration was determined using inductively coupled plasma mass spectrometry Stability of the substance and reaction and fate in food The heat and ph stability of Ps-Ca has been evaluated by the applicant using the liberation of inorganic phosphate as a marker of degradation. Ps are stable at temperatures of up to approximately 90 C. Additionally, the results of the ph stability test indicate that Ps are stable at ph range 2 11 (at 70 C) but start to degrade at ph 12 (at a temperature of 100 C). The applicant stated that Ps-Ca is expected to remain stable at the temperature and ph values typically encountered for its intended use in chewing gum, hard confectionary, sugar-free soft candy and apple juice. However, the Panel noted that, during the processing of confectionary, temperatures higher than those tested may be reached. Ps-Ca has also undergone batch stability testing. Analyses of three non-consecutive batches of Ps-Ca indicate that the ingredient is expected to remain stable for up to 3 years under normal storage conditions of 25 C (Documentation provided to EFSA n.1) Fate of Ps-Ca in apple juice The fate of powdered Ps-Ca in apple juice was evaluated by Mita et al. (2013). The calcium and inorganic phosphate concentration was determined using atomic absorption spectrophotometry and inductively coupled plasma mass spectrometry, respectively. The ph of the juice increased with increasing Ps-Ca concentration, with statistically significant changes observed at concentration of 1% and higher. The ionic calcium and inorganic phosphate concentrations increased in a linear trend with increasing Ps-Ca concentration. 6 Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. J L 139, , p Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings, J L 354, , p EFSA Journal 2016;14(6):4488

11 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium 3.2. Proposed uses and use levels Use in foods for the general population according to Regulation (EC) No 1925/2006 The applicant requests the inclusion of Ps-Ca as a source of dietary calcium among the authorised substances included in Annex II of Regulation (EC) No 1925/2006. Ps-Ca is comprised of 5% (w/w) calcium and is intended for use in sugar-free chewing gum, sugar-free hard confectionery and sugar-free soft candy at concentrations of between 2.5% and 3.0%, and in selected fruit juices at a concentration ranging between 1.0% and 1.5%. The proposal from the applicant for the use and use levels of Pos-Ca as a source of supplemental calcium in foods is presented in Table 2. Table 2: FCS category number Proposed use and use levels of Pos-Ca in foods and corresponding levels of calcium Food categories Use in food supplements according to Directive 2002/46/EC In addition to use in food, the applicant requests the inclusion of Ps-Ca as a source of calcium in food supplements among the authorised substances included in Annex II of Directive 2002/46/EC. The proposed use levels in food supplements are intended to provide mg/day of supplemental calcium, corresponding to an intended intake of g/day of Ps-Ca from the use in food supplements Use in FSMP according to Commission Regulation (EC) No 953/2009 The applicant is also proposing the use of Ps-Ca as an ingredient in FSMP at levels providing mg calcium/day, corresponding to an intended intake of g/day of Ps.Ca in individuals consuming the FSMP Exposure data Proposed use level of Ps-Ca (mg/kg) Food consumption data used for the exposure assessment Corresponding level of calcium (mg/kg) ther confectionery without added sugar 30,000 1, Chewing gum without added sugar 30,000 1, Fruit juices as defined by Council Directive 2001/112/EC 15, PS-Ca : calcium phosphoryl oligosaccharides; FCS: Food Classification System EFSA Comprehensive European Food Consumption Database Since 2010, the EFSA Comprehensive European Food Consumption Database (Comprehensive Database) has been populated with data on national food consumption at a detailed level. Competent authorities in the European countries provide EFSA with data on the level of food consumption by the individual consumer from the most recent national dietary survey in their country (cf. Guidance of EFSA on the Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment (EFSA, 2011a)). New consumption surveys recently 8 added in the Comprehensive database were also taken into account in this assessment. 9 The food consumption data gathered by EFSA were collected by different methodologies and thus direct country-to-country comparisons should be interpreted with caution. Depending on the food category and the level of detail used for exposure calculations, uncertainties could be introduced owing to possible subjects under-reporting and/or misreporting of the consumption amounts. Nevertheless, the EFSA Comprehensive Database represents the best available source of food consumption data across Europe at present. 8 Available online: 9 Available online: EFSA Journal 2016;14(6):4488

12 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Food consumption data from the population groups infants, toddlers, children, adolescents, adults and the elderly were used for the exposure assessment. For the present assessment, food consumption data were available from 33 different dietary surveys carried out in 19 European countries, as listed in Table 3. Table 3: Population groups considered for the exposure estimates of Ps-Ca Population Infants Toddlers Children (a) Adolescents Adults Age range From 4 months up to and including 11 months of age From 12 months up to and including 35 months of age From 36 months up to and including 9 years of age From 10 years up to and including 17 years of age From 18 years up to and including 64 years of age Countries with food consumption surveys covering more than 1 day Bulgaria, Denmark, Finland (b), Germany (b), Italy (b), UK (b) Belgium, Bulgaria, Denmark, Finland (b), Germany (b), Italy (b), the Netherlands, Spain, UK (b) Austria, Belgium, Bulgaria, Czech Republic, Denmark, Finland (b), France (b), Germany (b), Greece, Italy (b), Latvia, Netherlands (b), Spain, Sweden, UK (b) Austria, Belgium, Cyprus, Czech Republic, Denmark, Finland (b), France (b), Germany (b), Italy (b), Latvia, Spain, Sweden, UK (b) Austria, Belgium, Czech Republic, Denmark, Finland (b), France (b), Germany, Hungary, Ireland (b), Italy (b), Latvia, Netherlands (b), Romania, Spain, Sweden, UK (b) The elderly (a) 65 years of age and older Austria, Belgium, Denmark, Finland (b), France (b), Germany, Hungary, Ireland (b), Italy (b), Romania, Sweden (b),uk (b) Ps-Ca : calcium phosphoryl oligosaccharides. (a): The terms children and the elderly correspond respectively to other children and the merging of the categories elderly and very elderly in the EFSA guidance Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment (EFSA, 2011a). (b): Dietary surveys considered by the NDA Panel in its opinion on Dietary Reference Values for calcium (EFSA NDA Panel, 2015). Consumption records were codified according to the FoodEx classification system (EFSA, 2011b). Nomenclature from the FoodEx classification system has been linked to the FCS as presented in Annex II of Regulation (EC) No 1333/2008, part D, to perform exposure estimates. In practice, FoodEx food codes were matched to the FCS food categories Exposure estimates to calcium from the use of Ps-Ca as a nutrient source in foods The Panel performed a refined exposure assessment, limited to the food categories in which Ps-Ca is proposed for use: food categories ther confectionery without added sugar, Chewing gum without other sugar and Fruit juices. The estimated exposures to Ps-Ca, expressed as mg/day, from its use as a source of calcium in foods, at the proposed use levels, for all six population groups (min max across the dietary surveys) are reported in Table 4. Table 4: Estimated exposure (mg/day) Estimated exposures to Ps-Ca (mg/day per person) from its proposed use as a source of calcium in foods (number of surveys) and relative estimates of exposure to calcium from this source Infants Toddlers Children Adolescents Adults The elderly (4 11 months) (12 35 months) (3 9 years) (10 17 years) (18 64 years) ( 65 years) Exposure to Ps-Ca at the proposed use levels Mean (6) ,970 (10) 6.7 3,092 (18) 6.9 4,739 (17) 2.0 3,309 (17) ,005 (14) High level ,950 (5) 1,296 7,887 (8) ,813 (18) ,800 (17) ,000 (16) ,225 (14) 12 EFSA Journal 2016;14(6):4488

13 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Estimated exposure (mg/day) Infants Toddlers Children Adolescents Adults The elderly (4 11 months) (12 35 months) (3 9 years) (10 17 years) Because Ps-Ca is comprised of 5% (w/w) calcium, the corresponding estimated exposure to calcium (expressed as mg/day) from this source has also been calculated by the Panel and is reported as well in Table 4. The mean exposure to Ps-Ca in the total population, as calculated by the Panel (Table 4), ranged from 2.0 mg/day in adults up to 4,739 mg/day in adolescents. High-level intakes ranged from 22.5 mg/day in children and adolescents up to 16,800 mg/day in adolescents. The corresponding mean intakes of calcium based on the proposed uses of Ps-Ca as a source of supplemental calcium in foods in the total population were calculated by the Panel, assuming that calcium corresponds to 5% weight of Ps-Ca as stated by the applicant. They ranged from 0.1 mg/day in adults up to 237 mg/day in adolescents. High-level intakes ranged from 1 mg/day in children and adolescents up to 840 mg/day in adolescents. Detailed results by age class and survey are presented in Appendix A. The main food category contributing to the exposure to Ps-Ca was almost exclusively fruit juices (food category ) in infants, toddlers and the elderly population groups Dietary intakes of calcium in the general population The applicant has submitted information on population reference intake (PRI) values for calcium previously derived by the Scientific Committee for Food (SCF, 1993). The Panel, however, noted that a more recent scientific opinion has been issued by the NDA Panel (2015) and used their dietary reference values in order to estimate the contribution of calcium intake from the intended uses of Ps-Ca for the general population. Calcium intake in the general population, as recently estimated by the NDA Panel (2015) is presented in Table 5. The NDA Panel estimated the dietary intake of calcium using the EFSA Comprehensive European Food Consumption Database (EFSA, 2011a), according to the food classification and description system FoodEx2 (EFSA, 2011b). Data from 13 dietary surveys in nine European Union (EU) countries were used. The countries included Finland, France, Germany, Ireland, Italy, Latvia, the Netherlands, Sweden and the UK. The data covered all age groups, from infants to adults aged 75 years and older. Mean calcium intake ranged from 307 mg/day in infants (aged between 1 and 11 months, four surveys) to 1,273 mg/day in children aged 10 to < 18 years (six surveys). High-level intake of calcium ranged from 658 mg/day in infants (three surveys) to 2,258 mg/day in children aged 10 to < 18 years (six surveys) Exposure to calcium from the use of Ps-Ca as a nutrient source in food supplements or FSMPs The applicant anticipated an exposure to calcium from the use of Ps-Ca supplements or in FSMPs corresponding to mg/day. (18 64 years) either in food Exposure assessment to calcium from the overall sources (proposed uses of Ps-Ca and from the regular diet) Table 5 summarises the estimated total exposure to calcium from the proposed uses of Ps-Ca as a source of calcium added to foods and food supplements or FSMPs and in addition to the regular dietary intake in six population groups according to the use levels proposed by the applicant. ( 65 years) Corresponding calcium intake from the proposed use of Ps-Ca in foods (from EFSA database) Mean 2 22 (6) (10) (18) (17) (17) (14) High level 6 98 (5) (8) (18) (17) (16) (14) Ps-Ca : calcium phosphoryl oligosaccharides; EFSA: European Food Safety Authority EFSA Journal 2016;14(6):4488

14 Ps-Ca (calcium phosphorylated oligosaccharides) as a source of calcium Table 5: Summary of anticipated exposure to calcium from the proposed uses of Ps-Ca in foods, food supplements or FSMPs and from the regular diet (number of surveys) Estimated exposure (mg/day) Infants Toddlers Children Adolescents Adults The elderly (4 11 months) (12 35 months) (3 9 years) (10 17 years) (18 64 years) ( 65 years) A: Dietary intake of calcium (a) (EFSA NDA Panel, 2015) Mean (4) (5) (7) 675 1,273 (6) 730 1,122 (7) 690 1,017 (7) (b) High-level (P95) (3) 915 1,310 (4) 978 1,624 (7) ,258 (6) 1,193 2,188 (7) 1,151 1,814 (7) (b) B: Estimated calcium intake from the proposed use of Ps-Ca for fortification of foods (c) Mean 2 13 (4) (5) (7) (6) (8) (7) High level (P95) 6 52 (3) (4) (7) (6) (8) (7) C: Estimated total calcium intake from foods (general population) (A + B) Mean (d) , , , ,056 High level (e) ,356 1,049 1,658 1,2072,421 1,214 2,289 1,167 1,888 Contribution (%) of calcium from Ps-Ca (B) to total calcium intake from foods (C) Mean High level D: Estimated supplemental calcium intake from the proposed use of Ps-Ca either in food supplements or in FSMPs E: Estimated supplemental calcium intake from all proposed uses and use levels of Ps-Ca (B + D) Mean High level Tolerable upper level of calcium (EFSA NDA Panel, 2015) n.s. n.s. n.s. n.s. 2,500 F: Total calcium intake from all sources (C + D) Mean , , , , ,181 High-level ,481 1,125 1,784 1,282 2,546 1,289 2,414 1,242 2,013 Contribution (%) of calcium from Ps-Ca from all proposed uses (E) to total calcium intake from all sources (F) Mean High-level Ps-Ca : calcium phosphoryl oligosaccharides; n.s.: not set; FSMPs: foods for special medical purposes. (a): Although the classification into age groups used by the NDA Panel in its 2015 opinion was slightly different from the one used by the ANS Panel, it was assumed that estimates of calcium intake would still be comparable. The following age groups were used by the NDA Panel: < 1 year; 1 to < 3 years; 3 to < 10 years; 10 to < 18 years; 18 to < 65 years; 65 to < 75 years; and 75 years. (b): Data for the age group 65 to < 75 and 75 years combined. (c): Values from dietary surveys considered by the NDA Panel in its 2015 opinion. (d): Mean total intake of calcium from foods was estimated by adding the mean values from dietary intake to the mean intake of calcium from Ps-CA in foods. (e): High-level intake of calcium from foods was estimated by adding the highest value from dietary intake to the mean intake of calcium from Ps-CA in foods. The Panel estimated total intake of supplemental calcium deriving from all the proposed uses of Ps-Ca (in the selected food categories and in either food supplements or FSMP) from the dietary surveys considered in the 2015 NDA Panel opinion. The estimated mean intake of calcium from all the proposed uses of Ps-Ca (Scenario E) ranged from 77 mg/day in infants up to 288 mg/day in adolescents. High-level intake ranged from 81 mg/day in infants up to 598 mg/day in adolescents. The mean total intake of calcium from all the proposed uses of Ps-Ca and the normal diet was calculated by the Panel adding the exposure estimates to calcium as calculated above, to the average calcium intake from the normal diet as recently estimated by the NDA in its opinion on dietary reference values for calcium. High-level intakes were calculated by adding the mean intake of calcium from the proposed uses of Ps-Ca to the highest value for calcium intake from the regular diet, for each age group. Mean total intakes of calcium from all the proposed uses of Ps-Ca and the normal diet (scenario F) ranged from 384 mg/day in infants up to 1,561 mg/day in adolescents. For adults and the elderly, 14 EFSA Journal 2016;14(6):4488