Scientific Opinion on the safety of the complexation product of sodium tartrate and iron(iii) chloride as a food additive 1

Transcription

1 EFSA Journal 2015;13(1):3980 SCIENTIFIC OPINION Scientific Opinion on the safety of the complexation product of sodium tartrate and iron(iii) chloride as a food additive 1 EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The complexation product of sodium tartrates and iron(iii) chloride (Fe mta) is proposed for use as an anticaking agent, only in salt or its substitutes, with a maximum use level of 106 mg Fe mta/kg salt. Fe mta can be expected to dissociate into its constituent iron(iii) and tartrate components upon ingestion. Studies in rats demonstrated that up to 90 % of ingested DL-tartrate or tartaric acid were absorbed, studies in humans suggested that only 20 % of an ingested dose of tartaric acid were absorbed. There are no ADME (absorption, distribution, metabolism and excretion) data for meso-tartrate. From a 90-day rat study, the lowest calculated BMDLs (Benchmark Dose Level) for Fe mta were: 75 mg/kg body weight (bw) per day for males (BMDL 05 for serum bile acids) and 267 mg/kg bw per day for females (BMDL 10 for goblet cells hyperplasia). Several in vitro studies showed that there is no safety concern for genotoxicity. No reprotoxicity and developmental toxicity was reported, however no study was specifically designed for teratogenicity. No long-term or carcinogenicity studies were available. The Panel concluded that the toxicity database was insufficient to establish an ADI (Acceptable Daily Intake) and calculated MoS (Margin of Safety) by comparing the highest intake of Fe mta of mg/kg bw per day for children at the 97.5 percentile with the lowest BMDLs. The resulting MoS were 815 and for males and females, respectively. Owing to the conservative assumptions included in the exposure assessment, the Panel concluded that there is no safety concern for the single condition of use and use level of Fe mta proposed. The Panel noted that this evaluation was based on a limited toxicity database, for a single use resulting in a very low exposure, and therefore concluded that any extension of use and/or use level of Fe mta, would require a new risk assessment. European Food Safety Authority, 2015 KEY WORDS food additive, anti-caking agent, complexation product of sodium tartrates and iron(iii) chloride, Fe mta 1 On request from the European Commission, Question No EFSA-Q , adopted on 9 December Panel members: Fernando Aguilar, Riccardo Crebelli, Alessandro Di Domenico, Birgit Dusemund, Maria Jose Frutos, Pierre Galtier, David Gott, Ursula Gundert-Remy, Claude Lambré, Jean-Charles Leblanc, Oliver Lindtner, Peter Moldeus, Alicja Mortensen, Pasquale Mosesso, Dominique Parent-Massin, Agneta Oskarsson, Ivan Stankovic, Ine Waalkens- Berendsen, Rudolf Antonius Woutersen, Matthew Wright, Maged Younes. Correspondence: fip@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the former Working Group "A" Food Additives and Nutrient Sources ( ) and the members of the Standing Working Group on Applications: Maria-José Frutos, David Gott, Lieve Herman, Claude Lambré, Jean-Charles Leblanc, Peter Moldeus, Alicja Mortensen, Ivan Stankovic, Paul Tobback, Ine Waaalkens-Berendsen, Rudolf Antonius Woutersen and Matthew Wright for the preparatory work on this scientific opinion and EFSA staff: Paolo Colombo, Ana Rincon, and Camilla Smeraldi 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 the safety of the complexation product of sodium tartrate and iron(iii) chloride as a food additive. EFSA Journal 2015;13(1):3980, 30 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

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 an opinion based on a dossier presented by the applicant. According to the applicant, the complexation product of sodium tartrates and iron(iii) (Fe mta) is proposed for use, only in salt or its substitutes, as an alternative to the currently permitted anti-caking agents, with a maximum use level of 106 mg Fe mta/kg salt. Fe mta is a mixture of sodium tartrates [DL- and meso-tartrates] with iron(iii) chloride. The proportion of each constituent comprising the mixture corresponds to 1.5 moles of tartrates (total), meso-tartrate represents approximately 65 % of the total tartrate content. The Panel noted that although there were no ADME (absorption, distribution, metabolism and excretion) data for Fe mta, the complexation product can be expected to dissociate into its constituents iron(iii) and tartrate, upon ingestion. This was supported by the weak Fe/tartrate interactions identified by electrospray mass spectrometry, where only the individual species were identified. Therefore, in addition to the data available for the Fe mta as a compound, the Panel considered appropriate to take also into consideration the available biological and toxicological data on its individual constituents for its safety assessment, as reported in the dossier. Studies in rats demonstrated that a large proportion (55 to 90 %) of ingested DL-tartrate or tartaric acid was absorbed. The Panel noted that the retention of tartrates in the kidneys was attributed to precipitation of the poorly soluble calcium DL-tartrate in the renal tubules. The Panel noted that retention of tartrates was observed until the end of the study period (8 days after dosing), but that the actual duration of this retention was not determined. However, the Panel also noted that the metabolic fate of tartrates in humans and rats may differ substantially. Based on the excretion in the urine, the results of the initial studies in humans suggested that 20 % of an ingested dose of tartaric acid was absorbed from the gastrointestinal tract (Underhill et al., 1931a; Finkle, 1933). These results have been further supported by Chadwick et al. (1978), who demonstrated that 12 % of an oral dose of [ 14 C] tartrate was recovered unchanged in the urine of human subjects, while 46 % of the ingested radioactivity was recovered as expired CO 2. Unabsorbed tartrate likely undergoes metabolism by the intestinal microflora, which would account for the high proportion of radioactivity recovered as exhaled CO 2 (Underhill et al., 1931a; Finkle, 1933). The Panel noted that there are no ADME data for the meso-tartrate components of Fe mta. Recent acute, sub-chronic (90-day) and reprotoxicity and developmental toxicity studies in rats, performed under Good Laboratory practices (GLP) and based on Organisation for Economic Cooperation and Development (OECD) guidelines were available. The test material met the requirements of the specifications proposed by the applicant. The oral LD 50 for Fe Mta was > mg/kg body weight (bw). From the 90-day study, the authors determined no-observed adverse effect levels (NOAELs) of 500 and mg Fe mta /kg bw per day for adverse local effects (inflammatory/hyperplastic lesions in the colon and secondary haematological effects) and systemic effects (increased kidney, liver, and spleen weights and clinical biochemistry changes), respectively, in Wistar rats. The Panel did not agree with this conclusion and considered the lowest dose tested of 500 mg/kg bw per day as a lowest observed adverse effect level (LOAEL). A benchmark dose (BMD) analysis was conducted for each endpoint where systemic effects were reported. The lowest BMDLs FOR Fe mta for the various effects that were considered as adverse by the Panel were: 75 mg/kg bw per day for males (BMDL 05 for serum bile acids) and 267 mg /kg bw per day for females (BMDL 10 for goblet cells hyperplasia). EFSA Journal 2015;13(1):3980 2

3 Even at the highest dose tested (2 000 mg/kg bw per day of Fe mta), no signs of reprotoxicity or developmental toxicity : indices of mating, fertility, and conception, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care and early postnatal pup development (pup mortality, clinical signs, body weight, and macroscopic findings), were reported. The Panel noted that there was no specific study available for the evaluation of the teratogenic potential of Fe mta. Several well-conducted in vitro studies showed that there is no safety concern about the genotoxicity of Fe mta. Fe mta did not cause skin irritation or sensitisation. Data on salt intakes indicate that the mean daily intakes of salt by the European population are in the range of 7 to 12 g per day. Based on these estimates, the maximum exposure by an individual to Fe mta from its proposed use in salt could be mg/kg bw per day (mean for children) when considering that Fe mta is present in all the salt consumed. Due to the absence of data for high consumers from the children and adolescents populations, the Panel estimated the 97.5 percentile of intake from the mean by applying the factor of 2. As a result, the highest intake of Fe mta would be mg/kg bw per day for children at the 97.5 percentile. Given that not all salt consumed will contain Fe mta at the maximum proposed use level and recognising the limitations in survey data results, these values were considered by the Panel an over-estimate of the actual consumptions. The Panel noted that tartrates (apart from meso-tartrate), iron and the impurity oxalic acid are natural components of the diet. At the maximum level of proposed use of the anti-caking agent in salt or its substitutes, the levels of exposure to Fe mta will not make a significant contribution to the current intakes of tartrates (total), iron or oxalic acid (impurity) from other sources in the diet. The Panel concluded that the toxicity database was insufficient to establish an acceptable daily intake (ADI) for Fe mta and therefore calculated a Margin of Safety (MoS) by comparing the highest intake of Fe mta of mg/kg bw per day for children at the 97.5 percentile with the lowest BMDL for Fe mta for adverse effects in males (75 mg/kg bw per day for males), and females (267 mg/kg bw per day). The resulting MoS were 815 and for males and females, respectively. Owing to the conservative assumptions included in the exposure assessment, these values were considered by the Panel as not giving reason for safety concern for the single condition of use and use level of 106 mg Fe mta/kg salt as an anti-caking agent proposed by the applicant. The Panel also noted that this evaluation of a single use was based on a limited toxicity database and concluded that any extension of use and/or use level, would require a new risk assessment of this compound, including investigations of all the effects for which BMDLs were calculated. EFSA Journal 2015;13(1):3980 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 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 Proposed specifications Manufacturing process Methods of analysis in food Reaction and fate in food Case of need and proposed uses Information on existing authorisations and evaluations Fe mta Tartaric acid Iron Exposure Food consumption data used for exposure assessment Exposure to Fe mta from its use as food additive Exposure to tartaric acid and its salts, and iron, via other sources Dietary exposure to contaminants Uncertainty analysis Biological and toxicological data Absorption, distribution, metabolism and excretion Fe mta Tartaric acid Iron Toxicological data of Fe mta Acute oral toxicity Short-term and subchronic toxicity Genotoxicity Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Other studies Toxicological data for oxalic acid Discussion Conclusions Documentation provided to EFSA References Abbreviations EFSA Journal 2015;13(1):3980 4

5 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The use of food additives is regulated under the European Parliament and Council Regulation (EC) 1333/2008 on Food additives 4. Only food additives that are included in the Union list, in particular in Annex II to that regulation, may be placed on the market and used in foods under the conditions of use specified therein. An application has been introduced for the authorisation of the use of Iron(III) meso-tartrate as instant-release and anti-caking agent in salt and its substitutes. This new additive can be used as an alternative for the anti-caking agents that are currently permitted such as calcium salts of ferrocyanide and various silicates. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION The European Commission asks the European Food Safety Authority to give an opinion about the safety of the use of Iron(III) meso-tartrate anti-caking agent in salt and its substitutes in accordance with regulation (EC) 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. 4 Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. OJ L 354, EFSA Journal 2015;13(1):3980 5

6 ASSESSMENT 1. Introduction Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to food (ANS) was asked to deliver an opinion based on the safety of the complexation product of sodium tartrates and iron(iii) chloride (Fe mta) when used as a food additive. 2. Technical data 2.1. Identity of the substance Fe mta is described by the applicant as a mixture of sodium tartrates [D-, L-, and meso-tartrates] with iron(iii) chloride. The proportion of each constituent comprising the complexation product mixture corresponds to 1.5 moles of tartrates (total), the meso-tartrate content represents approximately 65 % of this total tartrate content. The identity of the tartrates of Fe mta is presented in Table 1. Table 1: chloride. Identity of the tartrates of the complexation product of sodium tartrates and iron(iii) Tartrate CAS Number Molecular formula Structure Molecular weight (g/mol) D(-)-Tartaric acid, disodium salt unknown C 4 H 4 O 6 Na L(+)-Tartaric disodium salt acid, C 4 H 4 O 6 Na meso-tartaric acid, disodium salt unknown C 4 H 4 O 6 Na According to the applicant, the common name is: complexation product of sodium tartrates and iron(iii) chloride, and the chemical name is iron(iii) complexation product of D(+)-, L(-)- and meso- 2,3 dihydroxibutanedioc acids. The chemical formula proposed by the applicant is Fe(OH) 2 C 4 H 4 O 6 Na and the molar mass is g/mol. The applicant has applied for the CAS number to be assigned to the complexation product (Supplementary information submitted by the applicant, 2013). It is described as a dark green aqueous solution (ca. 35 % w/w) with a density of g/cm 3 (20 0 C) and a ph The applicant indicated that Fe mta is highly soluble in water. EFSA Journal 2015;13(1):3980 6

7 The applicant has provided evidence by negative ion electrospray ionisation mass spectrometry (ESI-MS) technique, that due to weak Fe/tartrate interactions only individual constituents of Fe mta can be identified (Application dossier, 2012) Proposed specifications The Fe mta is manufactured as an aqueous solution (ca. 35 % w/w). The specifications proposed by the applicant are presented in Table 2. Table 2: Specifications for Fe mta as proposed by the applicant (Supplementary information submitted by the applicant, 2014) Parameter Proposed specification Description Identification Assay: meso-tartrate D(-)- and L(+)-tartrate Iron(III) Purity: Chloride Sodium Arsenic Lead Mercury Oxalate Dark green aqueous solution typically comprising ca. 35 % by weight complexation product (a) Positive tests for tartrate and iron ph of a 35 % aqueous solution of complexation products between 3.5 and 3.9 > 28 %, expressed as the anion on dry basis > 10 %, expressed as the anion on dry basis > 8 %, expressed as the anion on dry basis < 25 % on dry basis < 23 % on dry basis < 3 mg/kg < 2 mg/kg < 1 mg/kg < 1.3 % expressed as oxalate on dry basis (a): Prepared as an aqueous solution, not less than 60 % water. Microbiological specifications were not considered necessary on the basis that microbial growth would not be sustained under the highly alkaline conditions of the manufacturing process or intended use as an anti-caking agent in salt Manufacturing process Fe mta is manufactured in a two-step process, involving the isomerisation of L-tartrate to an equilibrium mixture of D-, L- and meso-tartrates followed by addition of iron(iii) chloride. All raw materials used in the manufacturing process are food or pharmaceutical grade. The only by-product of potential concern from a toxicological perspective is oxalate, and its levels are limited to no more than mg/kg in the ca. 35 % w/w aqueous solution of the final complexation product (equivalent to a maximum content of 1.5 % on a dried basis, expressed as oxalic acid) Methods of analysis in food Fe mta may be identified by Raman spectroscopy (solution of the complexation product at ph 3.8) (Supplementary information submitted by the applicant, 2014). According to the applicant, the presence of Fe mta in foods may be determined by measurement of the constituents, iron(iii) and tartrate (total and meso-tartrate), using spectrophotometry and HPLC. Confirmation of the identity and concentration of the complexation products is performed in the diluted solution immediately prior to addition to the salt or its substitute, to which it is added. EFSA Journal 2015;13(1):3980 7

8 2.5. Reaction and fate in food Complexation product of sodium tartrate and iron(iii) chloride as a food additive Experimental results provided by the applicant showed that sunlight (UV), ph and concentration are factors that influence the stability of Fe mta. In sunlight (UV light), solutions of Fe mta both at high (Fe = 4.2 wt %) and low concentration (Fe = 0.58 wt %) were not stable. Precipitation occurred within a few days or weeks depending on the concentration and ph. Fe mta solutions were relatively more stable at high concentration and ph. The interaction of tartaric acid and iron(iii) is strongly affected by sunlight. The photoreaction involves reduction of iron(iii) to iron(ii) and concomitant oxidation of tartaric acid. Clark et al. (2007) studied the impact of storage conditions of tartaric acid solutions in the presence of trace levels of iron(iii) and proposed a pathway for the degradation of tartaric acid via photochemical and Fenton chemistry mechanisms (Figure 1). A number of instable intermediate species are formed in the solution that degrade to simpler acids. Figure 1: Proposed degradation of tartaric acid (from Clark et al., 2007) According to the applicant the same degradation processes would be anticipated to occur in solutions of Fe mta and would apply generally to D-, L- and meso-tartrates. The resulting crystals were grinded and analysed by X-ray powder diffraction (XRPD). According to the applicant these crystals might be iron(ii)-tartrate trihydrate [Fe(OHCHCOO) 2 3H 2 O], the solubility in water of iron(ii) complexation products with tartrates is relatively slow and super saturation is reached over time. Any small amounts of crystals of iron(ii) tartrates, which may form are removed by filtration prior to distribution (Supplementary information submitted by the applicant, 2013). In complete dark at room temperature (around 20 C) at high (Fe = 4.2 wt %) and low concentration (Fe = 0.58 wt %), Fe mta was stable when stored up to 3 months; ph had little influence and concentration had no effect on the stability of the solution. The applicant also mentioned that some solid particles appeared in the concentrated solution of Fe mta after storage for a few days, the composition of these particles was unknown. EFSA Journal 2015;13(1):3980 8

9 2.6. Case of need and proposed uses Complexation product of sodium tartrate and iron(iii) chloride as a food additive According to the applicant, Fe mta is proposed to be used as an anti-caking agent, only in salt and its substitutes, with a use level of 106 mg Fe mta/kg salt Information on existing authorisations and evaluations Fe mta Fe mta has not been previously evaluated by other Committees Tartaric acid L(+)-tartaric acid (E 334), monosodium tartrate (E 335i) and disodium tartrate (E335ii) are authorised food additives in accordance with Annex II to Regulation (EC) No 1333/2008 on food additives. The safety of L- and DL-tartaric acids and their sodium and potassium salts have been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (JECFA, 1974a, 1974b, 1977, 1978). Specifically, L-tartaric acid was evaluated for its use as a flavouring agent, sequestrant, emulsifier, acid, and synergist for antioxidants, and disodium L-tartaric acid was evaluated for its use as sequestrant and stabiliser in meat products and sausage casings (JECFA, 1974a, 1974b). JECFA allocated a group ADI of 0 to 30 mg/kg body weight (bw) per day for L-tartaric acid and its sodium, potassium, and potassium/sodium salts [expressed as L-tartaric acid] (JECFA, 1974a, 1974b), which was maintained at the 21 st meeting in 1977 (JECFA 1977, 1978). The ADI was based on the results of a long-term (2-year) feeding study in rats (JECFA, 1974a, 1974b). A second long-term (104 days) feeding study on monosodium L(+)-tartrate in rats (Hunter et al., 1977) was considered by JECFA at its 21 st meeting in The no observed-effect level (NOEL) was the highest dose tested in the study and the application of a 100-fold safety factor reflected the established ADI of 30 mg/kg bw per day (JECFA, 1978). Due to insufficient data and apparent effects on the kidneys at high dose level, the ADI of 30 mg/kg bw per day was not considered by JECFA to apply to DL-tartrate. In 1991, the Scientific Committee for Food (SCF) evaluated the safety of L- and DL-tartaric acids and their sodium, potassium, and potassium sodium salts for use as food additives with technological functions, including in weaning foods (SCF, 1991a,b). The SCF concurred with the group ADI of 30 mg/kg bw per day for L-tartaric acid and its salts previously established by JECFA. However, the Committee concluded that at present the data were insufficient to set an ADI for DL-tartaric acid and it salts Iron JECFA has evaluated the safety of iron compounds, with the latest evaluations at the 68 th meeting in 2007 and the 71 st meeting in 2009, as part of the evaluations of sodium iron(iii) ethylene diamine tetraacetate (JECFA, 2008a, 2008b) and ferrous ammonium phosphate (JECFA, 2010a, 2010b), respectively. These latest evaluations took into consideration toxicological data on iron and iron compounds not previously evaluated. In these assessments, JECFA established and maintained a provisional maximum tolerable daily intake (PMTDI) of 0.8 mg/kg body weight/day for all sources of iron with the exception of iron oxides used as colouring agents, or for iron supplementation during pregnancy, lactation, or for special clinical requirements. The SCF and the US Institute of Medicine (IOM) have established recommended dietary allowances (RDAs) of 8 to 18 mg iron/day for healthy adults, and the IOM and Health Canada have set recommended upper limit intakes of 45 mg iron/day (0.75 mg iron/kg body weight for a 60 kg individual) (SCF, 1993; IOM, 2001). EFSA Journal 2015;13(1):3980 9

10 In addition, six different Iron(III) salts are classified as generally recognised as safe (GRAS) for direct addition to foods as nutrient supplements, with no limitations on their use other than current Good Manufacturing Practices Exposure Food consumption data used for exposure assessment In 2005 EFSA considered salt intake by the EU population as part of opinions on the tolerable upper intake levels for sodium and chloride, respectively (EFSA, 2005a, 2005b). Mean daily intakes of salt were estimated to range from 8 to 11 g per day, with around 10 to 15 % arising from natural occurring sodium or chloride in unprocessed foods, and around 10 to 15 % from discretionary salt added during cooking and at the table. In addition to EU-wide population estimates, values for average (mean) salt intake on an individual country basis are available. Intake data collated from a number of sources relating to individual country populations are summarised in Table 3. Differences between reported salt intakes cannot be assumed to arise from consumption patterns only but may also be the results of alternative survey methodologies and data collection methods (i.e., urinary sample analysis vs. questionnaire). These data are more recent than that on which EFSA based its mean daily intake estimates for salt in 2005, but the values reported by this selection of European countries fall within approximately the same range as the previous assessment (mean values 7 to 12 g compared to 7 to 11 g in 2005). Table 3: Reported mean salt consumption by various European countries Country Mean Salt Consumption Year (a) Reference Males: 9 to 11 g/day Denmark 2009 WASH, 2009 [Denmark] Females: 7 to 8 g/day Finland Males: 9.3 g/day National Salt Initiatives, 2009 [Finland] 2007 Females: 6.8 g/day Norway 10 g/day (total) National Salt Initiatives, 2009 [Norway] 2007 Processed foods: ca. 7.5 g/day Sweden Males (11-20 yr): 11.7 g/day 2006 WASH, 2009 [Sweden] France 8.5 g/day 2006 National Salt Initiatives, 2009 [France] United Kingdom Switzerland Males: 9.7 g/day Females: 7.7 g/day to 10 g/day Males: 10.6 g/day Females: 8.1 g/day 2009 NCSR, 2008 National Salt Initiatives, 2009 [United Kingdom] National Salt Initiatives, 2009 [Switzerland]; Salt Strategy (FOPH, 2009) Lithuania 8 to 10 g/day National Salt Initiatives, 2009 [Lithuania] Poland 12 g/day (total) Table salt: 8 g/day Sodium in foods: 4 g/day 2008 WASH, 2009 [Poland] (a): Year of survey provided where known, in some instances the year of the report is provided where the actual date of data collection not known Although, the data presented in Table 3 provide a general insight into the consumption patterns of the European population, the results do not consider individual population groups or high-level users. Dietary studies tend to overestimate true intakes of sodium, and therefore to assess the intake of sodium, it is recommended that measurements of sodium excretion are made. However, only the UK adult survey (NDNS, 2008) used 24-hour urinary excretion to provide data on salt intakes in adults, with no other surveys identified by the applicant reporting sodium intake using sodium excretion as a status parameter. Data were identified in children and adolescents from a number of different surveys across Europe but these did not include sodium excretion as a nutritional status. Taking these EFSA Journal 2015;13(1):

11 limitations into account, the available intake data for adults from the UK survey and the combined results of the various surveys reporting children and adolescent intakes are summarised in Table 4. Table 4: Estimated intakes of salt by different population groups based on sodium intakes from various surveys within Europe Population groups Mean intake High level intake Reference (g/person/day) (97.5th percentile; g/person/day) Children Lambert et al., 2004 Adolescents Lambert et al., 2004 Adults Female (19-64 yr) NCSR, 2008 (urinary Na excretion analysis) Male (19-64 yr) NCSR, 2008 (urinary Na excretion analysis) Exposure to Fe mta from its use as food additive Exposure to Fe mta from its proposed use as a food additive has been calculated using the levels proposed by the applicant combined with salt consumption data reported in the literature. However, the Panel noted that its estimates should be considered as being conservative as it is assumed that all dietary salt consumed contained the anti-caking complexation product of sodium tartrates with Iron(III) chloride added at the proposed use levels, this is likely to overestimate Fe mta consumption by up to ten fold. Table 5 summarises the estimated exposure to Fe mta from its use as a food additive as well as exposure expressed as individual components of the complexation product. Table 5: Summary of anticipated exposure to Fe mta and its major constituents from its use as a food additive based on the use levels proposed by the applicant by different population groups Population group Salt intake g/day Intake of Fe mta and equivalent intake of its major constituents from estimated daily intake of salt (mg/person/day) Fe mta meso-tartaric Total tartaric Iron(III) (106 mg/kg salt) acid acid (12 mg/kg salt) (33 mg/kg salt) (47 mg/kg salt) mg/person/day Mean P97.5 Mean P97.5 Mean P97.5 Mean P97.5 Mean P97.5 Children Adolescents Adults g/kg bw per mg/kg bw per day day Children Adolescents Adults Based on the intake estimates of salt in the EU, the maximum exposure of an individual to Fe mta from its proposed use in salt could be up to mg/kg bw per day (mean for children). Due to the absence of data for high consumers from the children and adolescents populations, the Panel agreed to apply an uncertainty exposure factor of 2 from the mean to get the high exposure (97.5 percentile). As a result, the highest intake of Fe mta would be mg/kg bw per day for children at the 97.5 EFSA Journal 2015;13(1):

12 percentile. The estimated individual (adults) exposure would be up to 1.76 mg Fe mta/person/day, comprising 0.55 mg meso-tartaric acid, 0.78 mg total tartaric acid and 0.20 mg iron Exposure to tartaric acid and its salts, and iron, via other sources Via regular diet Tartaric acid and its salts The EU population is exposed to L-tartaric acids and its salts from their widespread use as food additives and also from their natural occurrence in fruit and fruit-derived products (e.g. juices). There is also a limited history of use of a mixture of acids including DL- and meso-tartaric acids as a flavouring agent. Examples of the occurrence of tartrates in general foods for the EU population are provided in Table 6. No overall intake levels by the EU population were estimated in Table 6, however, given the extensive use of tartaric acid and its salts in usual food products, together with its natural occurrence in fruit and fruit products, exposure to L-tartaric acid from these sources will far exceed the total tartaric acid intake from the proposed use of the complexation product of sodium tartrates with Iron(III) chloride Iron(specifically Iron(III)). Iron is a normal component of the diet with a RDA of 14 mg set by Commission Directive 2008/100/EC 5. Typical sources of iron in the diet are summarised in Table 7. Under the worst case scenario of all salt consumed by the EU population containing the complexation product of sodium tartrates with Iron(III) chloride as an anti-caking agent at 12 mg/kg salt, the total exposure by a person consuming 16.6 g of salt per day would be 0.20 mg/ person/day which is not significant compared to normal exposure levels from nutrient sources. 5 Commission Directive 2008/100/EC of 28 October 2008 amending Council Directive 90/496/EEC on nutrition labelling for foodstuffs as regards recommended daily allowances, energy conversion factors and definitions. OJ L 285, , p EFSA Journal 2015;13(1):

13 Table 6: Examples of exposure to tartaric acid and its salts by the EU population from general foods Food Additive Source Role/formation Content History of use References Variety of functions including emulsifiers/stabilisers, chelating agents, ph adjustment agents, E 334 L(+)-Tartaric acid basis E 335 Sodium tartrates preservatives, flavour enhancers and Sodium salts of L(+)- modifiers, flour and baking additives, tartaric acid: (i) Monosodium tartrate anti-caking agents, firming agents and min. 99 % on anhydrous (ii) Disodium tartrate humectants. Examples of food products basis E 336 Potassium tartrates use include: Potassium salts of L(+)- (i) (ii) E 337 E 354 Monopotassium tartrate Dipotassium tartrate Sodium potassium tartrate Calcium tartrate ph control agent in acidified milk, jams and jellies, grape-flavoured beverages and canned fruits, or wine. Preservative for meat carcasses (1-3 %). Monopotassium tartrate anti-caking agent and leavening agent (cream of tartar) in baked goods, candy, crackers, confectionery and frostings. Dipotassium tartrate used as stabiliser in wine. Sodium potassium tartrate acts as ph control and sequestrant in cheeses and jams. Sodium tartrate functions acts as ph control agent in fats, oils and jams and as an emulsifier in cheese L(+)-tartaric acid: min % on anhydrous tartaric acid (min. 98 % and 99 % on anhydrous basis for mono- and dibasic salts, respectively) Sodium potassium salt of L(+)-tartaric acid (min. 99 % on anhydrous basis) Calcium salt of L(+)- tartaric acid (min. 98 %) Directive 95/2/EC: Annex I (Food Additives Generally Permitted for Use in Foodstuffs) all additives permitted quantum satis; Annex II (Foodstuffs in which a Limited Number of Additives of Annex I may be Used) tartaric acid permitted in cocoa and chocolate products (max. 0.5 %), potassium tartrate permitted in grape juice (quantum satis); tartaric acid and its sodium salts permitted in extra jam and jelly, jam, jellies and marmalade (quantum satis); tartaric acid and its sodium and potassium salts permitted in canned and bottled fruit and vegetables (quantum satis); Annex VI Part 3 (Food Additives Permitted in Processed Cereal-Based Foods and Baby Foods for Infants and Young Children in Good Health tartaric acid and its sodium, potassium and calcium salts (not E 337) permitted in biscuits and rusks (5 g/kg as a residue) Directive 95/2/EC 6 Food Additive Handbook (Section I.14; Smith and Hong-Shum, 2011) Commission Directive 2008/84/EC 7 6 Directive 1995/2/EC of the European Parliament and of the Council of 20 February 1995 on food additives other than colours and sweeteners. OJ L 61, , pp Commission Directive 2008/84/EC of 27 August 2008 laying down specific purity criteria on food additives other than colours and sweeteners, OJ L 253, , EFSA Journal 2015;13(1):

14 Flavouring agent: L-, D-, DL-, meso-tartaric acid Fruit acid Source Role/formation Content History of use References L(+)-tartaric acid and its potassium, calcium and magnesium salts Flavouring agent Occurs widely in fruit as acid or its salts, including in grapes and other berries; also present in tamarind Mixture of the different isomers of tartaric acid Naturally occurring fruit acid (note the D- enantiomer whilst not as readily isolated in nature has been isolated from some micro-organisms such as A. niger) Flavouring Number: CoE Number: 18 FEMA Number: 3044 Natural constituent of plants at varying low levels with other fruit acids Identified in some fruit juices, particularly grape and pomegranate (levels of 0.8 and 3.4 mg/l reported) Flavour Ingredients (Burdock, 2009) McNair, 1936; Ehling and Cole, 2011 EFSA Journal 2015;13(1):

15 Table 7: Examples of exposure to iron salts by the EU population from general foods Source (a) Role/formation Content History of use References Iron(III) salts permitted under Commission Regulation No 258/97 Source of iron in fortified foods and Ferric ammonium citrate food supplements Ferric sodium diphosphate Ferric diphosphate Ferric saccharate Range of iron(ii) salts and also elemental iron also permitted Ferric EDTA Natural occurrence in foods Foods rich in iron: Liver, beef, game and offal Foods high/moderate in iron: Cereals, cereal products and pulses Foods with lower levels of iron: Milk, dairy products, pork, poultry, green vegetables Typically manufactured as pure salts (ca. 95 % on dried basis) Naturally occurring sources of iron Recommended Daily Allowance (RDA) of 14 mg laid down by Commission Directive 2008/100/EC: Food supplements typically deliver 100 % of the RDA Fortified foods must provide 15 % of the RDA to make the claim source of Exposure by the general population to iron from food sources reported to range from mean values of 10 to 22 mg/person/day to 97.5 th percentile values of 16 to 41 mg/person/day across a number of different EU countries including Austria, Germany, Netherlands, Sweden and the UK Commission Directive 2008/100/EC 8 Commission Regulation (EC) No 258/97 9 Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the Tolerable Upper Intake Level of Iron (EFSA, 2004) (a): Iron(III) salts are general not widely used as food additives but there are examples of iron(ii) salts: E 535 ferrous gluconate and E 585 ferrous lactate re permitted for use in olives darkened by oxidation (max. 150 mg/kg expressed as iron); as discussed in Section II.G, sodium, potassium and calcium ferrocyanide (E 535, E 536 and E 538) are also permitted for use under specified conditions 8 Commission Directive 2008/100/EC of 28 October 2008 amending Council Directive 90/496/EEC on nutrition labelling for foodstuffs as regards recommended daily allowances, energy conversion factors and definitions. OJ L 285, , p Regulation (EC) No 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients OJ L 43, , p EFSA Journal 2015;13(1):

16 Dietary exposure to contaminants Dietary exposure to oxalic acid Complexation product of sodium tartrate and iron(iii) chloride as a food additive Under the worst case scenario where all salt consumed by the EU population contained Fe mta as an anti-caking agent at 106 mg/kg salt, the total oxalic acid exposure would not exceed mg/kg by a person consuming 12 g of salt per day Uncertainty analysis According to the EFSA guidance related to uncertainties in dietary exposure assessment (EFSA, 2007), the following sources of uncertainties have been considered and summarised in Table 8: Table 8: Qualitative evaluation of influence of uncertainties Sources of uncertainties Direction (a) Consumption data: different methodologies / representativeness / under +/- reporting / misreporting / no portion size standard Extrapolation from food consumption survey of few days to estimate + chronic exposure Exposure model: exposure calculations based on the maximum proposed + use levels applied to all dietary salt intake (a): + = uncertainty with potential to cause over-estimation of exposure; - = uncertainty with potential to cause underestimation of exposure. The Panel noted that overall, uncertainties in exposure assessment tend to its overestimation. 3. Biological and toxicological data Similar to the iron complexes of other dicarboxylic acids, on digestion, Fe mta likely dissociates into its respective Iron(III) and tartrate components. Therefore, in addition to the biological and toxicological data available for the Fe mta as a compound, the Panel considered appropriate to take also into consideration the available data on its individual constituents in its safety assessment as provided in the application dossier. The Panel noted that there were no such data available for mesotartrate Absorption, distribution, metabolism and excretion Fe mta Although there were no specific ADME (absorption, distribution, metabolism and excretion) data available for Fe mta, the toxicokinetic behaviour of Fe mta can be based on its physical and chemical properties. Fe mta is highly soluble in water and its solubility was therefore not considered a rate-limiting factor in its absorption from the gastrointestinal tract Tartaric acid Animal data The majority of the investigations on the metabolic fate of tartaric acid have been conducted in rats. Underhill et al. (1931b) reported that 68 to 99 % of an ingested dose of tartaric acid was recovered intact in the urine of rats, rabbits and dogs, and excretion rates were similar following oral and subcutaneous administration. These results indicated that tartrate was nearly completely absorbed following oral administration in these species and did not undergo substantial metabolism. In contrast, only 27 % of an orally administered dose was recovered in the urine of guinea pigs, while nearly EFSA Journal 2015;13(1):

17 100 % of a subcutaneously administered dose was recovered in the urine. No tartrate was detected in the faeces. Following absorption, from several studies (Down et al., 1977), there is evidence to suggest that tartrates distribute to the kidneys and bones in male CFY rats. In the first investigation, according to the authors, groups of 10 CFY rats received doses of 2.73 g/kg bw per day monosodium ( 14 C) L(+) tartrate or ( 14 C) DL(-) tartrate for 7 consecutive days. The ( 14 C) L(+) tartrate and ( 14 C) DL(-) tartrate were administered by oral intubation and after the final administration the animals were killed at regular intervals over the following 12 days. The first animal was killed immediately after administration of the final dose. Prior to killing, blood samples were withdrawn by cardiac puncture and liver, kidney, and bone samples were collected. In a second investigation, groups of 8 CFY rats received 2.73 g/kg body weight/day of monosodium ( 14 C) L(+) tartrate or ( 14 C) DL(-) tartrate by oral intubation for a period of 7 days. All the animals were killed 6 hours after administration of the final dose and the livers and kidneys were removed. In a third investigation whole body autoradiography of CFY rats was completed up to 8 days after administration of the same dose of monosodium ( 14 C) L(+) tartrate and ( 14 C) DL(-) tartrate. After administration of monosodium ( 14 C) L(+) tartrate, radioactivity peaked in all tissues 1 hour after administration. Monosodium ( 14 C) L(+) tartrate was cleared from the plasma biphasically with the first phase exhibiting a half-life of 3 hours and the second phase exhibiting a half-life of 53 hours. Peak concentrations of radioactivity following monosodium ( 14 C) L(+) tartrate administration mirrored those observed in the plasma. Whole body autoradiography following monosodium ( 14 C) L(+) tartrate administration revealed that 3 hours after the last dose, radioactivity was mainly present in the gastrointestinal tract, liver, kidneys, and bones. After 24 hours, radioactivity was only detected in the bones, and persisted up to 192 hours post-dosing. Peak concentrations of monosodium ( 14 C) DL(-) tartrate in whole blood and plasma were observed 3 hours after the last administration. Monosodium ( 14 C) DL(-) tartrate was cleared from the plasma biphasically with half-lives of 15 and 58 hours, respectively. The peak concentrations of radioactivity following monosodium ( 14 C) DL(-) tartrate administration mirrored those observed in the plasma. Whole body autoradiography following monosodium ( 14 C) DL(-) tartrate administration revealed that 3 hours after the last dose, radioactivity was mainly present in the gastrointestinal tract, liver, kidneys, and bones. Aggregates of radioactive particles were detected in the cortex and medulla of the kidneys and these subsisted until at least 192 hours post-dosing. Within 96 hours of the last dose, detected radioactivity in the kidneys and bones of rats administered ( 14 C) DL(-) tartrate was twice the detected level in rats administered ( 14 C) L(+) tartrate. Retention of tartrates in the kidneys was attributed to precipitation of the poorly soluble calcium DL-tartrate in the renal tubules. In the kidneys, histological examinations revealed crystalluria following ( 14 C) DL(-) tartrate administration but none were observed following ( 14 C) L(+) tartrate administration. The Panel noted that retention of tartrates were observed during all the study period (8 days after dosing) but that the actual duration of this retention was not determined.. The retention of radioactivity in the bones was attributed to the metal-sequestering properties of tartaric acid. Chadwick et al. (1978) reported that up to 60 % of an orally administered dose of [ 14 C]-DL-tartrate was absorbed into the systemic circulation in rats (sex and strain not reported), as evidenced by the excretion of the radiolabelled compound in the urine. In the study conducted by Chadwick et al. (1978) an additional 21.8 % of the administered dose was detected in expired air within 6 hours of administration. Within 48 hours after administration of 400 mg/kg bw radiolabelled monosodium [ 14 C]-L-tartrate to 3 male and 3 female CFY rats by oral intubation 70.1, 13.6, and 15.6 % of the administered radioactivity was detected in the urine, faeces, and expired air, respectively (Chasseaud et al., 1977). When the same dose was administered by intravenous injection, excretion in the urine, faeces, and expired air accounted for 81.3, 0.9, and 7.5 % of the administered dose, respectively. In order to investigate the impact of bacterial metabolism on the elimination of [ 14 C]-DL-tartrate, the authors also administered an equivalent dose by intracaecal injection. Following this exposure, 66.6 % EFSA Journal 2015;13(1):

18 of the administered dose was recovered as expired CO 2, while only 1.4 % of the administered dose was recovered in the urine from this route. Gry and Larsen (1978) reported that larger fractions of 1000 mg/kg bw dose of radiolabelled D(-) and L(+)-tartrate administered by gavage were recovered in the urine of fasted male and female Wistar rats as compared to fasted female Danish Landrace pigs and fasted female guinea pigs. In the male and female Wistar rats (n=5/sex/group) the percentages of D(-) and L(+)-tartrate recovered in the urine were 52.1 and 72.9 % of the administered doses, respectively, compared to 26 and 33 % of the administered doses, respectively, in female Danish Landrace pigs (n=3/group) and 5.4 and 3.6 % of the administered doses, respectively, in female guinea pigs (n=11 or 12/group). The authors observed similar rates of recovery for the D(-) and L(+) forms of tartrate in the Danish Landrace pigs and the guinea pigs. In rats, a significantly larger proportion of the L(+) tartrate than of D(-) tartrate, was recovered in the urine Human data The metabolic fate of tartrates in humans and rats (and other species) may differ substantially (see Figure 2). The original research into the metabolism of tartaric acid in humans was conducted by Underhill et al. (1931a), who determined that tartaric acid was not well absorbed following oral administration. Based on the excretion in the urine, the results of the initial studies in humans suggested that as little as 20 % of an ingested dose of tartaric acid was absorbed from the gastrointestinal tract (Underhill et al., 1931a; Finkle, 1933). These results have been supported by Chadwick et al. (1978), who demonstrated that 12 % of an oral dose of [ 14 C]-DL-tartrate was recovered unchanged in the urine of human subjects, while 46 % of the ingested radioactivity was recovered as expired CO2. In contrast, 63.8 % of an intravenously administered dose of [14C]-DLtartrate was recovered in the urine of a single human subject, while 18 % was excreted as 14 CO2. Negligible amounts of radiolabelled tartrate were recovered in the faeces following oral administration. Figure 2: Metabolic fate of orally ingested tartrate in humans and rats. Numbers are presented as per cent of the ingested dose (modified from Chadwick et al., 1978) Iron Iron absorption occurs in the proximal small intestine, and absorption and transfer into the systemic circulation involve regulated iron uptake carrier proteins on the apical and basal surfaces of enterocytes (SACN, 2010, Gantz, 2013). Non-heme iron is transported into the enterocytes by the divalent metal transporter 1 (DMT1), while the mechanism of heme iron absorption is unclear (SACN, 2010). In foods, non-heme iron is present as iron salts that dissociate upon ingestion. Absorption of iron is dependent on its dissolution and subsequent reduction to the ferrous form (Fe 2+ ) (EFSA, 2004). EFSA Journal 2015;13(1):

19 3.2. Toxicological data of Fe mta Acute oral toxicity Complexation product of sodium tartrate and iron(iii) chloride as a food additive An acute oral toxicity study of Fe mta was submitted in support of the application (Van Otterdijk, 2010a, unpublished). The study was conducted under Good Laboratory Practice (GLP) and was based on the OECD Test Guideline (TG) 423 (2001), the test material met the specifications proposed by the applicant. Two groups of female Wistar rats (3/group) were administered a single dose of mg Fe mta /kg bw by gavage and then observed for 15 days. Parameters evaluated included mortality, body weights and clinical signs. All animals were killed and subjected to necropsy at the end of the observation period. No mortalities were reported. All animals presented hunched posture and piloerection on day 1; no significant changes in body weight gain were reported and no macroscopic abnormalities were detected upon necropsy. The oral LD 50 of Fe mta was determined by the authors to be >2 000 mg/kg bw Short-term and subchronic toxicity A repeated-dose study combined with a reproductive/developmental toxicity study was submitted in support of the application (van Otterdijk, 2011, unpublished). The study was performed under GLP and based on OECD TG 408 (1998), TG 421 (1995) and TG 422 (1996).The test material met the specifications proposed by the applicant. Wistar Han rats (10/sex/group) were administered by gavage at doses of Fe mta of 0 (control), 500, 1000 or 2000 mg/kg bw per day. Males were administered for 78 days prior to mating and during mating, for a total of 90 to 91 days. Females were administered for 78 days prior to mating, during mating and gestation, and for at least 4 days during lactation, for a total of 104 to 109 days. Parameters assessed included mortality, clinical signs, functional observations (auditory function, papillary reflex, static righting reflex, and grip strength), locomotors activity, body weight, food consumption, ophthalmology, haematology and clinical biochemistry. All animals were killed on the day after the final administration day and their organs were removed, weighed, and examined macroscopically. Selected organs and tissues were examined microscopically. After statistical analysis, any difference was considered significant when p>0.05. No compound-related mortalities occurred during the study period. There were no statistically significant differences between treated and control groups with respect to functional observation tests, ophthalmology or food consumption and no clinical signs of toxicity were reported during the study period. Salivation was observed immediately after dosing in most treated animals; this effect was considered to be a physiological response to the unpalatability of the test compound rather than an indication of toxicity. Red staining of the nose, rales and chromodacryorrhea (porphyrin staining around the eyes) were observed in single animals in all treated groups and occurred within historical control ranges. These observations were therefore not considered to be toxicologically significant by the authors. Compared to their respective controls, females from the high dose group exhibited significantly lower total movement and all treated females exhibited significantly lower ambulation counts. Given the transient nature of these effects, they were considered not to be of toxicological significance. Body weights in males in the mid- and high-dose groups were lower than control group weights from study weeks 8 and 3 (respectively) onwards and eventually achieved statistical significance. Transient reduced body weights were observed in females in the high-dose group in study week 5 and in the mid-dose group on day 4 of lactation. These changes in body weight were slight and/or transient and were not considered to be toxicologically significant by the authors. Dark-coloured intestinal contents were observed at necropsy in most females in the low-dose group and all females in the mid- and high-dose groups, but none of the males. These findings were attributed by the authors to the dark green colour and staining properties of the test material and were not considered by the authors to be toxicologically significant. The Panel noted that this observation may reflect a difference between males and females in the absorption of the compound. Other EFSA Journal 2015;13(1):