Scientific Opinion on the re-evaluation of gold (E 175) as a food additive 1

EFSA Journal 2016;14(1):4362 ABSTRACT SCIENTIFIC OPINION Scientific Opinion on the re-evaluation of gold (E 175) 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 The present opinion deals with the re-evaluation of the safety of gold (E 175) when used as a food additive. Gold (E 175) was previously evaluated by the Scientific Committee on Food (SCF) in 1975. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has not reviewed gold due to lack of data. None of the Committees established an acceptable daily intake (ADI). The Panel noted the limited data on absorption, distribution, metabolism and excretion (ADME) of elemental gold and the absence of toxicological data on gold used as a food additive (E 175) and considered the data to be too limited to perform a risk assessment for E 175. Elemental gold has a very low solubility and thus the systemic availability and effects are expected to be low. In contact with tissues ionic gold can be released from elemental gold and a high local concentration of gold can be reached at the site of contact. The Panel noted that no data on subchronic, chronic toxicity and genotoxicity of elemental gold were available. The Panel concluded that, despite the absence of toxicity data, but taking into account the low solubility of elemental gold, systemic availability and thus systemic effects of elemental gold would not be expected. The Panel recommended that the specifications for gold (E 175) should include the mean particle size and particle size distribution (± SD), as well as the percentage (in number) of particles in the nanoscale (with at least one dimension below 100 nm), present in the powder form of gold (E 175). The methodology applied should comply with the EFSA Guidance document. Exposure estimates of gold (E 175) reached up to 1.32 µg/kg body weight (bw)/day in the maximum level exposure assessment scenario and up to 0.33 µg/kg bw/day in the refined, non-brand-loyal, exposure scenario. European Food Safety Authority, 2016 KEY WORDS gold, E 175, food additive, CAS No 7440-57-5, CI 77480 1 On request from the European Commission, Question No EFSA-Q-2011-00345, adopted on 9 December 2015. 2 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, Agneta Oskarsson, Dominique Parent-Massin, Ivan Stankovic, Ine Waalkens- Berendsen, Rudolf Antonius Woutersen, Matthew Wright and Maged Younes. Correspondence: fip@efsa.europa.eu 3 Acknowledgements: The Panel wishes to thank the members of the Standing Working Group on the re-evaluation of food colours: Fernando Aguilar, Riccardo Crebelli, Alessandro Di Domenico, Maria Jose Frutos, Pierre Galtier, David Gott, Claude Lambré, Jean-Charles Leblanc, Agneta Oskarsson, Jeanne Stadler, Paul Tobback, Ine Waalkens-Berendsen and Rudolf Antonius Woutersen, for the preparatory work on this scientific opinion and EFSA staff members: Alexandra Tard and Stavroula Tasiopoulou for the support provided to this scientific opinion. The ANS Panel wishes to acknowledge all European competent institutions and other organisations that provided data for this scientific output. Suggested citation: EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food), 2016. Scientific Opinion on the re-evaluation of gold (E 175) as a food additive. EFSA Journal 2016;14(1):4362, 43 pp. doi:10.2903/j.efsa.2016.4362 Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2016

SUMMARY Following a request from the European Commission (EC), the Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion re-evaluating the safety of gold (E 175) when used as food colour. The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations and additional literature that has become available since then. No new toxicological or biological information was submitted to the Panel for the re-evaluation of gold (E 175) following European Food Safety Authority (EFSA) public calls for data. However, information on manufacturing processes was submitted. To assist in identifying any emerging issue or any information relevant for the risk assessment, EFSA outsourced a contract to deliver an updated literature review on toxicological endpoints, dietary exposure and occurrence levels of gold (E 175), which covered the period up to the end of 2014. Further update has been performed by the Panel. Gold (E 175) is authorised as a food additive in the European Union (EU) in accordance with Annex II to Regulation (EC) No 1333/2008, and specific purity criteria have been defined in Commission Regulation (EU) No 231/2012. The Scientific Committee on Food (SCF) has previously evaluated gold (E 175) and found the available data inadequate, but accepted the continued use of gold for external colouring and decoration of foods only (SCF, 1975). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has not reviewed gold because of lack of data on toxicity, purity and of the exact nature of the gold used on or in foods (JECFA, 1978; 1984; 2001). None of the committees established an acceptable daily intake (ADI). The common oxidation states of gold are +1 and +3, in addition to its metallic ground state (valence number 0), which is the chemical form of gold as a food additive (E 175). According to a manufacturer of gold as a food additive, the dimension of the gold powder used for the food additive is approximately 1 mm. The Panel noted that data on elemental gold are inadequate for risk assessment. Data on toxicity of gold nanoparticles and gold(i) complexes have been published. Considering the possibility of the presence of minute amounts of gold nanoparticles in the food additive as a consequence of the manufacturing process of gold powder, the Panel found it relevant to consider the data on toxicity of nanoparticles of gold as supportive evidence. Although data on nanoparticulate gold or gold(i) complexes were not of direct relevance for the risk assessment of elemental gold as a food additive, these data were reviewed by the Panel as indicative of potential health hazard. In vivo data on kinetics of metallic gold are very limited, but systemic absorption of gold is presumed to be very low due to the low solubility of elemental gold. However, ionic gold released from metallic gold in contact with tissues and cells, and a local high concentration of gold can be reached at the site of contact. A low uptake and a high retention of gold nanoparticles have been demonstrated in experimental animals. In vitro data and results from experimental animals demonstrate that cellular internalisation and gastrointestinal uptake of gold nanoparticles depends on particle size and physicochemical characteristics of the particles. The Panel noted that no data on subchronic, chronic toxicity or genotoxicity of elemental gold are available. Gold nanoparticles of different size and shape have been tested in several in vitro genotoxicity assays, and to a more limited extent also in vivo. In vitro, gold nanoparticles were positive in studies performed in mammalian cells, where induction of DNA strand breaks, micronuclei, chromosomal aberrations and aneuploidy was observed, together with the expression of markers of oxidative stress. EFSA Journal 2016;14(1):4362 2

Less conclusive results were obtained in mammals in vivo: positive in two studies using the intraperitoneal route of administration, which however is considered not relevant for oral risk assessment, and negative in an oral study in rats, in which however no evidence of exposure of the target tissue was provided. Overall, the Panel concluded that the available data do not allow evaluation of the genotoxic hazard associated to the use of gold as a food additive. No studies on reproductive or developmental toxicity of elemental gold were available. It has been reported that gold, used in medicines or dentistry, could elicit hypersensitivity reactions and effects such as lichen planus and skin eruption. However, the Panel noted that this exposure was not representative of the oral intake of gold used as a food additive, and therefore were not relevant to the risk assessment of E 175. There are a few case reports on induction of a lichen planus skin eruption following ingestion in the form of a gold-containing liqueur. Monovalent gold complexes have been used extensively in the treatment of rheumatoid arthritis. One of the medical drugs, auranofin (1-thio-β-D-glucopyranosatotriethyl phosphine gold-2,3,4,6- tetraacetate), is administered perorally. The Panel noted that the toxicokinetics of auranofin is not clarified, but there are findings indicating that orally administered auranofin is rapidly degraded in the gastrointestinal tract before absorption, and gold is released. The main target organs for toxicity of auranofin were the gastrointestinal tract, skin and kidney. Auranofin caused fetal abdominal anomalies in rabbits, but not in rats. The Panel considered that the studies with auranofin provided supplemental information about hazards due to exposure to gold but considered that these results were not directly applicable to the risk assessment of gold as a food additive. For the maximum level exposure assessment, mean estimates ranged from <0.01 to 0.33 µg/kg body weight (bw)/day across all population groups. Estimates based on the high percentile (95th percentile) ranged from 0 to 1.32 µg/kg bw/day across all population groups. For the refined estimated exposure scenario, in the brand-loyal scenario, mean exposure to gold (E 175) from its use as a food additive ranged from < 0.01 µg/kg bw/day for infants and the elderly to 0.31 µg/kg bw/day in children. The high exposure to gold (E 175) ranged from 0 µg/kg bw/day for infants to 1.3 µg/kg bw/day in children. In the non-brand-loyal scenario, mean exposure to gold (E 175) ranged from < 0.01 µg/kg bw/day for infants, adults and the elderly to 0.08 µg/kg bw/day in children. The high exposure ranged from 0 µg/kg bw/day for infants to 0.33 µg/kg bw/day in children. The Panel concluded that, despite the absence of toxicity data, but taking into account the low solubility of elemental gold, systemic availability and thus systemic effects of elemental gold would not be expected. The Panel recommended that the specifications for gold (E 175) should include the mean particle size and particle size distribution (± SD), as well as the percentage (in number) of particles in the nanoscale (with at least one dimension between 0 and 100 nm), present in the powder form of gold (E 175) used as a food additive. The methodology applied should comply with the EFSA Guidance document (EFSA Scientific Committee, 2011), e.g. scanning electron microscopy (SEM) or transmission electron microscopy (TEM). EFSA Journal 2016;14(1):4362 3

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... 6 1. Introduction... 6 2. Technical data... 6 2.1. Identity of the substance... 6 2.2. Specifications... 7 2.3. Manufacturing process... 7 2.4. Methods of analysis in food... 8 2.5. Reaction and fate in food... 8 2.6. Case of need and proposed uses... 8 2.7. Reported use levels or data on analytical levels of gold (E 175) in food... 8 2.8. Information on existing authorisations and evaluations... 9 2.9. Exposure assessment... 10 2.9.1. Food consumption data used for exposure assessment... 10 2.9.2. Exposure to gold (E 175) from its use as a food additive... 11 2.9.3. Main food categories contributing to exposure to gold (E 175)... 13 2.9.4. Uncertainty analysis... 14 2.9.5. Dietary occurrence from sources other than the food additive... 15 3. Biological and toxicological data... 15 3.1. Absorption, distribution, metabolism and excretion (ADME)... 16 3.1.1. Animals... 16 3.1.2. Humans... 17 3.1.3. In vitro studies... 18 3.2. Toxicological data... 19 3.2.1. Genotoxicity... 19 3.2.2. Chronic toxicity and carcinogenicity... 22 3.2.3. Reproductive and developmental toxicity... 22 3.2.4. Immunotoxicity, hypersensitivity/allergenicity and intolerance... 22 3.2.5. Other studies... 23 4. Discussion... 27 5. Conclusions... 30 6. Recommendations... 30 Documentation provided to EFSA... 30 References... 30 Appendices... 36 Appendix A. Summary of the reported use levels provided by industry and concentration levels of gold (E 175) (mg/kg or mg/l) used in the exposure scenarios... 36 Appendix B. Summary of total estimated exposure to gold (E 175) from its use as a food additive for the maximum level exposure scenario and the refined exposure assessment scenarios per population group and survey: mean and high level (µg/kg bw/day)... 37 Appendix C. References of the studies with capped material considered by the Panel... 40 Abbreviations... 42 EFSA Journal 2016;14(1):4362 4

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Regulation (EC) No 1333/2008 4 of the European Parliament and of the Council on food additives requires that food additives are subject to a safety evaluation by the European Food Safety Authority (EFSA) before they are permitted for use in the European Union (EU). In addition, it is foreseen that food additives must be kept under continuous observation and must be re-evaluated by EFSA. For this purpose, a programme for the re-evaluation of food additives that were already permitted in the European Union before 20 January 2009 has been set up under Regulation (EU) No 257/2010. 5 This Regulation also foresees that food additives are re-evaluated whenever necessary in the light of changing conditions of use and new scientific information. For efficiency and practical purposes, the re-evaluation should, as far as possible, be conducted by group of food additives according to the main functional class to which they belong. The order of priorities for the re-evaluation of the currently approved food additives should be set on the basis of the following criteria: the time since the last evaluation of a food additive by the Scientific Committee on Food (SCF) or by EFSA, the availability of new scientific evidence, the extent of use of a food additive in food and the human exposure to the food additive taking also into account the outcome of the Report from the Commission on Dietary Food Additive Intake in the EU 6 of 2001. The report Food additives in Europe 2000 7 submitted by the Nordic Council of Ministers to the Commission, provides additional information for the prioritisation of additives for re-evaluation. As colours were among the first additives to be evaluated, these food additives should be re-evaluated with the highest priority. In 2003, the Commission already requested EFSA to start a systematic re-evaluation of authorised food additives. However, as a result of adoption of Regulation (EU) 257/2010, the 2003 Terms of References are replaced by those below. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION The Commission asks EFSA to re-evaluate the safety of food additives already permitted in the Union before 2009 and to issue scientific opinions on these additives, taking especially into account the priorities, procedures and deadlines that are enshrined in the Regulation (EU) No 257/2010 of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives. 4 Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives, OJ L 354, 31.12.2008, p. 16. 5 Commission Regulation (EU) No 257/2010 of 25 March 2010 setting up the programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives, OJ L 80, 26.03.2010, p. 19. 6 Report from the Commission on Dietary Food Additive Intake in the European Union, Brussels, 1 October 2001, COM (2001) 542 final. 7 Food Additives in Europe 2000, Status of safety assessments of food additives presently permitted in the EU, Nordic Council of Ministers. TemaNord 2002:560. EFSA Journal 2016;14(1):4362 5

ASSESSMENT 1. Introduction The present opinion deals with the re-evaluation of the safety of gold (E 175) when used as a food additive. Gold (E 175), authorised as a food colour in the EU, has previously been evaluated by the EU Scientific Committee for Food (SCF) in 1975 (SCF, 1975), and was on the agenda of the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) in 1977, 1984 and 2001 (JECFA, 1978; 1984; 2001). The Panel on Food Additives and Nutrient Sources added to Food (ANS) was not provided with a newly submitted dossier and based its evaluation on previous evaluations, additional literature that became available since then and the data available following public calls for data. 8,9,10,11 To assist in identifying any emerging issue or any information relevant for the risk assessment, EFSA outsourced a contract to deliver an updated literature review on toxicological endpoints, dietary exposure and occurrence levels of gold (E 175), which covered the period up to the end of 2014. Further update has been performed by the Panel. 2. Technical data 2.1. Identity of the substance Gold (E 175) is a colouring agent and a chemical element (Au) with atomic number 79. It has an atomic weight of 197.0. The Chemical Abstract Service (CAS) Registry Number is 7440-57-5, the International Numbering System (INS) number is 175, the European Inventory of Existing Commercial chemical Substances (EINECS) number is 231-165-9 and the Colour Index number is 77480. According to Commission Regulation (EU) No 231/2012, 12 its chemical name is gold. Gold occurs as gold-coloured powder or tiny sheets. It has a melting point of 1,064.3 C and a density of 19.3 g/cm 3 (SciFinder software, 2013). Synonyms include: Aurum and Pigment metal 3. Gold is a soft, yellow metal with the highest ductility and malleability of any element. It is also a stable and chemically unreactive metal that does not oxidise and is inert towards most chemicals. In addition to the elemental form (Au 0 or Au(0)), two oxidation states of gold are common (Au + or Au(I) and Au 3+ or Au(III)). Gold salts are not stable in aqueous solutions but tend to precipitate as elemental gold. However, coordination complexes of gold are stable. Gold(I) is stabilised by ligands, such as sulphur and phosphorous donor ligands, and gold(iii) by oxygen and nitrogen donors. In the presence of reducing agents or absence of donor ligands, both gold(i) and (III) are easily reduced to gold(0) (Cotton et al., 1999; Butterman and Amey, 2005; Merchant, 1998). 8 Call for scientific data on food colours to support re-evaluation of all food colours authorised under the EU legislation. Published: 8 December 2006. Available online: http://www.efsa.europa.eu/en/dataclosed/call/afc061208.htm 9 Call for scientific data on silver (E 174) and gold (E 175), used as food colours. Published: 24 February 2011. Available online: http://www.efsa.europa.eu/en/dataclosed/call/ans110224.htm 10 Call for food additives usage level and/or concentration data in food and beverages intended for human consumption. Published: 9 March 2014. Available from: http://www.efsa.europa.eu/en/dataclosed/call/datex140310.htm 11 Call for scientific data on selected food additives permitted in the EU. Published: 24 March 2014. Available online: http://www.efsa.europa.eu/en/dataclosed/call/140324.htm 12 Commission Regulation (EU) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council. OJ L 83, 22.3.2012, p. 1 295. EFSA Journal 2016;14(1):4362 6

2.2. Specifications Re-evaluation of gold (E 175) as a food additive Specifications for gold (E 175) have been defined in Commission Regulation (EU) No 231/2012 on specifications for food additives (Table 1). JECFA has not prepared specifications for gold. Table 1: Specifications for gold (E 175) according to Commission Regulation (EU) No 231/2012 Assay Description Identification Purity Silver Copper Commission Regulation (EU) No 231/2012 Content not less than 90% Au Gold-coloured powder or tiny sheets No information Not more than 7%, after complete dissolution Not more than 4%, after complete dissolution The Panel noted that according to the EU specifications for gold, impurities of silver and copper are accepted up to 7% and 4%, respectively. The Panel noted that gold for use as a food colour can be obtained in a much higher purity grade (99.99%), according to one manufacturer (see Section 2.3). The Panel considered that a description of the particle size distribution should be included in the specifications. In particular, information should be provided on the mean particle size and on the particle size distribution (± SD). This would allow further assessment of the relevance of the studies, which would be conducted to evaluate the possible biological effects of gold (E 175) as a food additive. Information became available to EFSA concerning particle size distribution from one producer of gold (E 175). Information was provided on particle size distribution, measured by laser diffraction, which did not reveal any presence of nanosized particles (Documentation provided to EFSA n.3). The Panel however noted that the presence of nanoparticles cannot be excluded because according to EFSA guidance (EFSA Scientific Committee, 2011), at least two methods are required, one of which based on electron microscopy. 2.3. Manufacturing process Neither the SCF nor JECFA have provided any information concerning the manufacture of gold. New data were submitted following a public call for data. Gold is a rare element; however, it is widely distributed in nature in the elemental form present in arsenides, sulphides, and especially tellurides, and often alloyed with other elements, usually silver. The metal is extracted by treatment with cyanide solutions in the presence of air (cyanidation), and precipitated by addition of zinc (Cotton et al., 1999; Kirk-Othmer, 2005) or isolated by amalgamation with mercury (Butterman and Amey, 2005). The metal can be purified by electrodeposition to a very high purity grade ( 99.99%; total impurities 100 mg/kg); 99.999% pure gold has mainly electronics applications, but is in general too soft for other uses. For the latter, various gold alloys have been developed. According to the limited information provided by industry as a response to EFSA s call for data (Documentation provided to EFSA n.5), gold used as a food additive (with the chemical composition of 961.5 Au and 38.5 Ag ((Documentation provided to EFSA n.5) is produced from pure 24- carat gold with a minimum certified grade of gold of 99.99%. Production starts from thin sheets, with approximate thickness some tenth of a micrometre. These sheets are reduced by a milling process to commercial sizes purchased by the customer. In case of production of powder, crumbs or flakes, the metal sheets are cut by a mechanical mill until the right size is obtained. The size is controlled by EFSA Journal 2016;14(1):4362 7

means of different grids that are mounted on the mill. These grids are characterised by a defined dimension/space of mesh. The dimension/granulometry of the powder, crumbs and flakes is approximately 1, 4 and 30 mm, respectively. In case of production of squared leaves, the metal sheets are cut manually with a knife. In case of production of unsquared leaf, the sheets are extracted from the packs without any further cutting operation. To eliminate potential bacterial contamination, the gold is heated to not less than 100 C for at least 120 s ((Documentation provided to EFSA n.5). The Panel noted that the response to EFSA s call for data was limited to only one manufacturer of gold used as food additive, and considered that a manufacturing process concerning a specific case may not be representative of the market situation. 2.4. Methods of analysis in food Determination of gold in food (human milk, infant formulas, mixed food samples) has been described by use of inductively coupled plasma sector-field mass spectrometry (ICP-SFMS) (Prohaska et al., 2000; Krachler et al., 2000; Wittsiepe et al., 2003) or by inductively coupled plasma mass spectrometry (ICP-MS) (Ysart et al., 1999). 2.5. Reaction and fate in food According to the information provided by the industry, the appropriate conditions of storage of edible gold would be a temperature between 15 and 25 C, and degree of moisture under 50%. The shelf life is stated as being 10 years from the date of packaging ((Documentation provided to EFSA n.5). No information concerning reaction and fate of gold in food has been identified. 2.6. Case of need and proposed uses Maximum levels of gold (E 175) have been defined in Annex II to Regulation (EC) No 1333/2008 13 on food additives. These levels are referred to by the Panel as maximum permitted levels (MPLs) in this document. Currently, gold (E 175) is an authorised food additive in the EU used at quantum satis (QS) in the external coating of confectionery, decoration of chocolates and in liqueurs (Table 2). Table 2: MPLs of gold (E 175) in foods according to Annex II to Regulation (EC) No 1333/2008 FCS category no. FCS food category Restrictions/exceptions MPL (mg/l or mg/kg as appropriate) 05.2 Other confectionery including breath Only external coating freshening microsweets of confectionery Quantum satis 05.4 Decorations, coatings and fillings, except fruitbased fillings covered by category 04.2.4 chocolates Only decoration of Quantum satis 14.2.6 Spirit drinks as defined in Regulation (EC) No 110/2008 Only liqueurs Quantum satis FCS: Food Categorisation System (food nomenclature) presented in the Annex II to Regulation (EC) No 1333/2008. 2.7. Reported use levels or data on analytical levels of gold (E 175) in food Most food additives in the EU are authorised at a specific MPL. However, a food additive may be used at a lower level than the MPL. Therefore, information on actual use levels is required for performing a more realistic exposure assessment, especially for those food additives for which no MPL is set and which are authorised according to QS. 13 Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. OJ L 354, 31.12.2008, p 16 33. EFSA Journal 2016;14(1):4362 8

In 2011, EFSA launched a public call 14 for scientific data on gold (E 175) used as a food colour, to support the re-evaluation of gold (E 175) authorised under the EU legislation. Among other information, information on the human exposure to the food additive from the different types of food where it is permitted (e.g. consumption pattern and uses, actual use levels and maximum use levels, frequency of consumption and other factors influencing exposure) was requested. In response to this public call, few usage data on gold (E 175) in confectionery were submitted to EFSA by one data provider (Documentation provided to EFSA n.4) (Appendix A). In addition, in the framework of Commission Regulation (EU) No 257/2010 15 setting up a programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council, EFSA launched a public call 16 for food additives usage level and/or concentration data in food and beverages. Data on gold (E 175) were requested from relevant stakeholders. European food manufacturers, national food authorities, research institutions, academics, food business operators and any other interested stakeholders were invited to submit usage and/or concentration data on gold (E 175) in foods. No information concerning actual use levels or analytical levels of gold (E 175) in food were submitted in response to this call for data. According to the Mintel GNDP database, 17 in Europe, gold (E 175) is used in liqueurs (added in the form of flakes). It is also used as external coating of chocolate products and in sugar confectionary for the decoration of cakes and other sweets. Information on the percentage of gold (E 175) in these foods could also be retrieved on the packaging (below 1%). 2.8. Information on existing authorisations and evaluations Gold, used as a food additive, has previously been evaluated by the SCF in 1975 (SCF, 1975). The full SCF statement reads as follows: No specification was available to the Committee. The Committee did not establish an acceptable daily intake (ADI) because of the inadequacy of available biological information, but felt able to accept the use of this colour for surface colouring and decoration of food only, without further investigation. No references were given. The presently permitted use in liquors was thus not endorsed by the SCF. Gold was on the agenda of JECFA in 1977 (JECFA, 1978). The Committee did not review any studies, but concluded: In view of the rare use of gold as a colourant and the lack of knowledge of the exact nature of the gold used on or in foods, no specification was prepared. No data were available on the toxicity of elemental gold. In view of the very small amounts likely to be ingested by an individual, the Committee did not consider that the use of gold would present a hazard. No monograph was prepared. In 1984, JECFA concluded that: The Committee was unable to prepare specifications for gold and silver because the data on their purity were inadequate (JECFA, 1984). In 2001, JECFA stated that the latest evaluation was in 1977 and that no ADI was allocated. In the comments it was noted: Very limited use. Not considered to present a hazard (JECFA, 2001). 14 Call for scientific data on silver (E 174) and gold (E 175), used as food colours. Published: 24 February 2011. Available online: http://www.efsa.europa.eu/it/dataclosed/call/ans110224.htm 15 Commission Regulation (EU) No 257/2010 of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives. OJ L 80, 26.3.2010, p. 19 27. 16 Call for food additives usage level and/or concentration data in food and beverages intended for human consumption. Published: 10 March 2014. Available online: http://www.efsa.europa.eu/en/dataclosed/call/datex140310.htm 17 Mintel Global New Products Database (http://www.mintel.com/global-new-products-database). Accessed on 17/09/2015. EFSA Journal 2016;14(1):4362 9

2.9. Exposure assessment 2.9.1. Food consumption data used for exposure assessment 2.9.1.1 EFSA Comprehensive European Food Consumption Database Since 2010, the EFSA Comprehensive European Food Consumption Database (Comprehensive Database) has been populated with national data on 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 added in the Comprehensive database were also taken into account in this assessment. 18,19 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 underreporting by subjects 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. Food consumption data were available for the exposure assessment for the following population groups: infants, toddlers, children, adolescents, adults and the elderly. For the present assessment, food consumption data were available from 33 different dietary surveys carried out in 19 European countries (Table 3). Table 3: Population groups considered for the exposure estimates of gold (E 175) Population Infants 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 Countries with food consumption surveys covering more than one day Bulgaria, Denmark, Finland, Germany, Italy, UK Toddlers Belgium, Bulgaria, Denmark, Finland, Germany, Italy, Netherlands, Spain, UK Children (a) Austria, Belgium, Bulgaria, Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Latvia, Netherlands, Spain, Sweden, UK Adolescents From 10 years up to and Austria, Belgium, Cyprus, Czech Republic, Denmark, including 17 years of age Finland, France, Germany, Italy, Latvia, Spain, Sweden, UK Adults From 18 years up to and Austria, Belgium, Czech Republic, Denmark, Finland, including 64 years of age France, Germany, Hungary, Ireland, Italy, Latvia, Netherlands, Romania, Spain, Sweden, UK The elderly (a) From 65 years of age and Austria, Belgium, Denmark, Finland, France, Germany, older Hungary, Ireland, Italy, Romania, Sweden, UK (a): The terms children and the elderly correspond, respectively, to other children and the merge of elderly and very elderly in the Guidance of EFSA on the Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment (EFSA, 2011a). Consumption records were codified according to the FoodEx classification system (EFSA, 2011b). Nomenclature from the FoodEx classification system has been linked to the Food Classification System (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. 18 Available online: http://www.efsa.europa.eu/en/datexfoodcdb/datexfooddb.htm 19 Available online: http://www.efsa.europa.eu/en/press/news/150428.htm EFSA Journal 2016;14(1):4362 10

2.9.1.2 Food categories selected for the exposure assessment of gold (E 175) Re-evaluation of gold (E 175) as a food additive The food categories in which the use of gold (E 175) is authorised were selected from the nomenclature of the EFSA Comprehensive Database (FoodEx classification system food codes), at the most detailed level possible (up to FoodEx level 4) (EFSA, 2011b). For liqueurs, following the public calls for data, no use levels were reported. However, in Russell et al., 1997, concentration of gold flakes is given, as five 750-mL bottles of liqueur containing gold flakes were analysed. The concentration ranged between 8 and 17 mg per bottle. Considering that the metallic flakes were 75% gold by weight, the levels taken into account in the current exposure assessment are 8 17 mg gold/l. Within the Mintel GNDP database, gold (E 175) was found to be also used in alcoholic drinks with an alcohol content of 37.5%. On the basis of this information, the Panel decided to consider these alcoholic drinks in the exposure assessment, in addition to liqueurs (which according to the legislation have a minimum alcoholic content of 15%). Therefore, alcoholic drinks which according to the FoodEx nomenclature are classified as spirits within the Comprehensive Database were also taken into account. Gold (E 175) is authorised in the two other food categories: Other confectionery including breath freshening microsweets (FCS 05.2) and decorations, coatings and fillings (FCS 05.4) as coating. According to the Mintel GNDP database, gold (E 175) is only used at a low level on the coating (below 1%). This percentage was applied to the consumption data for cocoa products and confectionary to retrieve consumption of the food additive. 2.9.2. Exposure to gold (E 175) from its use as a food additive The Panel estimated chronic exposure to gold (E 175) for the following population groups: infants, toddlers, children, adolescents, adults and the elderly. Dietary exposure to gold (E 175) was calculated by multiplying gold (E 175) concentrations for each food category (Appendix A) with their respective consumption amount per kilogram of body weight for each individual in the Comprehensive Database. The exposure per food category was subsequently added to derive an individual total exposure per day. These exposure estimates were averaged over the number of survey days, resulting in an individual average exposure per day for the survey period. Dietary surveys with only one day per subject were excluded, as they are considered not adequate to assess repeated exposure. This was carried out for all individuals per survey and per population group, resulting in distributions of individual exposure per survey and population group (Table 3). Based on these distributions, the mean and 95th percentile of exposure were calculated per survey for the total population and per population group. High percentile exposure was only calculated for those population groups where the sample size was sufficiently large to allow calculation of the 95th percentile of exposure (EFSA, 2011a). Therefore, in the present assessment, high levels of exposure for infants from Italy and for toddlers from Belgium, Italy and Spain were not included. Thus, for the present assessment, food consumption data were available from 33 different dietary surveys carried out in 19 European countries (Table 3). Exposure assessment to gold (E 175) was carried out by the ANS Panel based on (1) maximum use levels provided to EFSA (defined as the maximum level exposure assessment scenario) and (2) reported use levels (defined as the refined exposure assessment scenario) as provided by industry. These two scenarios are discussed in detail below. 2.9.2.1 Maximum level exposure assessment scenario The regulatory maximum level exposure assessment scenario is based on the MPLs as set in Annex II to Regulation (EC) No 1333/2008. As gold (E 175) is authorised according to QS in all food categories, a maximum level exposure assessment scenario was estimated based on the maximum reported use levels provided by industry, as described in the EFSA Conceptual framework (EFSA ANS Panel, 2014). EFSA Journal 2016;14(1):4362 11

The Panel considers the exposure estimates derived following this scenario as the most conservative, as it is assumed that the consumer will be continuously (over a lifetime) exposed to gold (E 175) present in food at the maximum reported use levels. 2.9.2.2 Refined exposure assessment scenario The refined exposure assessment scenario is based on use levels reported by industry. This exposure scenario can consider only food categories for which the above data were available to the Panel. Appendix A summarises the concentration levels of gold (E 175) used in the refined exposure assessment scenario. Based on the available dataset, the Panel calculated two refined exposure estimates based on different model populations: 1. The brand-loyal consumer scenario: It was assumed that a consumer is exposed long-term to gold (E 175) present at the maximum reported use level for one food category. This exposure estimate is calculated as follows: Combining food consumption with the maximum of the reported use levels for the main contributing food category at the individual level. Using the mean of the typical reported use levels for the remaining food categories. 2. The non-brand-loyal consumer scenario: It was assumed that a consumer is exposed long-term to gold (E 175) present at the mean reported use levels in food. This exposure estimate is calculated using the mean of the typical reported use levels for all food categories. 2.9.2.3 Anticipated exposure to gold (E 175) Table 4 summarises the estimated exposure to gold (E 175) from its use as a food additive in six population groups (Table 3) according to the different exposure scenarios (Section 2.9.2.2). Detailed results per population group and survey are presented in Appendix B. Table 4: Summary of anticipated exposure to gold (E 175) from its use as a food additive in the maximum level exposure assessment scenario and in the refined exposure scenarios, in six population groups (minimum maximum across the dietary surveys in µg/kg bw/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) Maximum level exposure assessment scenario Mean < 0.01 0.02 0.01 0.26 0.04 0.33 0.03 0.24 0.01 0.09 < 0.01 0.05 High level 0 0.11 0.05 0.76 0.17 1.32 0.12 0.93 0.06 0.50 0.03 0.21 Refined exposure scenario Brand-loyal scenario Mean < 0.01 0.02 0.01 0.25 0.04 0.31 0.03 0.23 0.01 0.09 < 0.01 0.04 High level 0 0.10 0.05 0.65 0.16 1.30 0.12 0.93 0.05 0.47 0.03 0.18 Non-brand-loyal scenario Mean < 0.01 0.01 < 0.01 0.07 0.01 0.08 0.01 0.06 < 0.01 0.02 < 0.01 0.02 High level 0 0.03 0.01 0.19 0.04 0.33 0.03 0.23 0.02 0.13 <0.01 0.08 EFSA Journal 2016;14(1):4362 12

2.9.3. Main food categories contributing to exposure to gold (E 175) Re-evaluation of gold (E 175) as a food additive Table 5: Main food categories contributing to exposure to gold (E 175) using maximum use levels (> 5% to the total mean exposure) and number of surveys in which each food category is contributing FCS category no. 05.2 05.4 14.2.6 FCS food category Other confectionery including breath freshening microsweets Decorations, coatings and fillings, except fruitbased fillings covered by category 04.2.4 only decorations of chocolates Spirit drinks as defined in Regulation (EC) No 110/2008 only liqueurs Infants Toddlers Children Adolescents Adults 8.0 41.9 (4) 58.1 100 (6) Range of % contribution to the total exposure (number of surveys) (a) 8.8 58.5 (10) 41.5 91.2 (10) 7.7 84.7 (18) 15.3 92.3 (18) - - - 5.5 89.3 (17) 10.7 94.5 (17) 8.2 (1) 8.6 77.5 (16) 20.4 83.3 (17) 7.1 47.2 (2) The elderly 12.1 72.9 (13) 18.8 61.1 (14) 8.3 51.3 (6) (a): The total number of surveys may be greater than the total number of countries as listed in Table 3, as some countries submitted more than one survey for a specific population. Table 6: Main food categories contributing to exposure to gold (E 175) using the brand-loyal refined exposure scenario (> 5% to the total mean exposure) and number of surveys in which each food category is contributing FCS category no. 05.2 05.4 14.2.6 FCS food category Other confectionery including breath freshening microsweets Decorations, coatings and fillings, except fruitbased fillings covered by category 04.2.4 only decorations of chocolates Spirit drinks as defined in Regulation (EC) No 110/2008 only liqueurs Infants Toddlers Children Adolescents Adults 8.0 41.9 (4) 58.1 100 (6) Range of % contribution to the total exposure (number of surveys) (a) 6.0 57.9 (10) 42.1 94.0 (10) 6.4 89.3 (18) 10.7 93.6 (18) - - - 6.0 92.4 (16) 7.6 96.4 (17) 5.1 11.1 (2) 7.5 80.2 (16) 17.7 84.5 (17) 7.0 49.1 (14) The elderly 10.1 74.4 (12) 17.2 61.4 (14) 8.3 55.9 (13) (a): The total number of surveys may be greater than the total number of countries as listed in Table 3, as some countries submitted more than one survey for a specific population. EFSA Journal 2016;14(1):4362 13

Table 7: Main food categories contributing to exposure to gold (E 175) using the non-brand-loyal refined exposure scenario (> 5% to the total mean exposure) and number of surveys in which each food category is contributing FCS category no. 05.2 05.4 14.2.6 FCS food category Other confectionery including breath freshening microsweets Decorations, coatings and fillings, except fruitbased fillings covered by category 04.2.4 only decorations of chocolates Spirit drinks as defined in Regulation (EC) No 110/2008 only liqueurs Infants Toddlers Children Adolescents Adults 8.0 41.9 (4) 58.1 100 (6) Range of % contribution to the total exposure (number of surveys) (a) 8.8 58.5 (10) 41.5 91.2 (10) 7.7 84.7 (18) 15.3 92.3 (18) - - - 5.4 89.3 (17) 10.7 93.5 (17) 7.4 18.3 (4) 7.0 76.1 (16) 20.1 80.4 (17) 7.5 62.7 (15) The elderly 9.5 68.0 (12) 17.5 58.1 (14) 14.5 66.5 (13) (a): The total number of surveys may be greater than the total number of countries as listed in Table 3, as some countries submitted more than one survey for a specific population. 2.9.4. Uncertainty analysis Uncertainties in the exposure assessment of gold (E 175) have been discussed above. In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2007), the following sources of uncertainties have been considered and are summarised in Table 8. Table 8: Qualitative evaluation of influence of uncertainties on the dietary exposure estimate Sources of uncertainties Direction (a) Consumption data: different methodologies/representativeness/under +/ reporting/misreporting/no portion size standard Use of data from food consumption survey of a few days to estimate long-term (chronic) + exposure for high percentiles (95th percentile) Correspondence of reported use levels to the food items in the EFSA Comprehensive Food +/ Consumption Database: uncertainties to which precise types of food the levels refer to Concentration data: - levels considered applicable for all items within the entire food category, + - no reported use levels for liqueurs, use of literature data +/ Maximum level exposure assessment scenario: - food categories authorised at the highest level reported + Refined exposure assessment scenarios: - exposure calculations based on one maximum and one mean level (reported +/ use from industries) Uncertainty in possible national differences in use levels of food categories +/ (a):uncertainties with potential to cause overestimation of exposure are indicated by + ; uncertainties with potential to cause underestimation of exposure are indicated by. Overall, the Panel considered that the uncertainties identified would, in general, result in an overestimation of the real exposure to gold (E 175) as a food additive in European countries for the maximum level exposure scenario and for the refined exposure scenario, if it is considered that the food additive may not be used in all foods of the food categories for which it is authorised. EFSA Journal 2016;14(1):4362 14

2.9.5. Dietary occurrence from sources other than the food additive Re-evaluation of gold (E 175) as a food additive Gold has been detected in ready-to-drink infant formulae in the range < 0.05 0.20 μg/kg, and in human milk at concentrations in the range from 0.1 2.1 μg/kg (Prohaska et al., 2000). Gold concentrations in mussels (Mytilus edulis) and in mackerel (Pneumatophorus japonicus) have been reported to be 2 38 and 0.12 μg/kg dry weight, respectively (Eisler, 2004a). Wittsiepe et al. investigated gold intake from food (sources not specified) in German children by using duplicate portion sampling. The gold intake was reported to be in the range of < 0.015 2.6 μg/kg bw/week (median 0.068 μg/kg bw/week) (Wittsiepe et al., 2003). No influence of food consumption patterns was found on the dietary intake of gold. Similar intake of gold was reported by Ysart et al. (1999), with a mean and upper range in adults of 0.001 and 0.002 mg/day, respectively (corresponding to 0.1 and 0.2 μg/kg bw/week, respectively). Thus, the daily mean intake of gold from the regular diet can be estimated to be in the range from 0.01 µg/kg bw/day to 0.02 µg/kg bw/day. Dietary exposure from the use of gold as a food additive and from the regular diet would lead to a mean intake for children of 0.03 0.10 µg/kg bw/day (non-brand-loyal scenario). On average, dietary exposure from the food additive would represent around 30% of the total exposure (from both the food additive and the regular diet). Oral exposure to metallic gold(0) may occur from its use as a dental restoration material and from the use in pharmaceuticals. Monovalent organogold complexes with sulphur and phosphor ligands are used in the treatment of rheumatoid arthritis. Intake of gold is also reported from traditional Ayurvedic medicines in India (Merchant, 1998; Butterman and Amey, 2005). 3. Biological and toxicological data The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations and literature that became available since then. No new toxicological or biological information was submitted to the Panel for the re-evaluation of gold following EFSA public calls for data. A literature search was conducted on the most commonly available databases for toxicological and biological information (PubMed, Science Direct, Toxline and Web of Knowledge) to cover recent published literature on gold. Data on toxicology of elemental gold are very limited. Some biological data on metallic gold(0) are available from its use in dental restorations. In contrast, monovalent gold complexes have been extensively studied due to their use in the treatment of rheumatoid arthritis, the so-called chrysotherapy, since the early 1930s (see reviews by Merchant, 1998; Eisler, 2003; Aitio et al., 2015). The Panel noted that data on monovalent gold complexes may be of some relevance for hazard assessment of elemental gold as a food additive, and data on gold complexes are presented in Section 3.2.5. Gold nanoparticles are widely investigated for different biomedical applications, as a drug delivery system, therapeutic agent in cancer and for imaging and diagnostics (see reviews by Fadeel and Garcia-Bennett, 2010; Kunzmann et al., 2011; Thakor et al., 2011; Arvizo et al., 2012; Gerber et al., 2014; Zhang, 2015). According to a manufacturer of gold as a food additive, the dimension of the gold powder used for the food additive is approximately 1 mm. There are no reports on the presence of nanoparticles of gold in the food additive. However, the Panel decided to consider also data on gold as nanoparticles in this risk assessment due to the possibility of presence of minute amounts of gold nanoparticles in the food additive. The Panel noted that studies on gold nanoparticles are mostly done with particles capped with surface-bound molecules, such as polyethylene glycol (PEG) to prevent aggregation of particles and avoid macrophage recognition and phagocytosis (Kunzmann et al., 2011). Studies with nanoparticles consisting of only elemental gold, which would be more relevant for risk assessment of gold as a food additive, are almost lacking. In this opinion only data with non-capped nanoparticles are included. However, when corresponding capped nanoparticles have been studied in the same experiments, also those data are included. Data on capped nanoparticles are referenced in Appendix C. EFSA Journal 2016;14(1):4362 15