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

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1 SCIENTIFIC OPINION Scientific Opinion on the safety of trivalent chromium as a nutrient added for nutritional purposes to foodstuffs for particular nutritional uses and foods intended for the general (including food supplements) 1 EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The Panel on Food Additives and Nutrient Sources added to Food delivers a scientific opinion re-evaluating the safety of chromium(iii) as a nutrient added to foods for particular nutritional uses (PARNUTS) and foods intended for the general (including food supplements). The Panel concluded that following oral administration, trivalent chromium is poorly absorbed. The results of in vitro bacterial mutagenicity assays have been consistently negative. The results of other genotoxicity studies are conflicting. The Panel also evaluated new in vitro and in vivo studies on the genotoxicity of chromium(iii) compounds which were not covered by previous evaluations. The Panel concluded that in very large amounts, certain trivalent chromium compounds are cytotoxic and have been shown to cause chromosomal damage. The Panel also evaluated the data on longterm toxicity and carcinogenicity for chromium(iii). Based on the facts i) that maximum intake levels of up to 250 µg/day for supplemental intake as suggested by the WHO would be in the same order of magnitude as the exposure resulting from normal dietary intake, ii) that in vitro, at high concentrations, chromium(iii) might cause DNA damage, iii) that this DNA damage is not reflected in in vivo genotoxicity assays performed according to standard OECD protocols and in NTP studies, iv) that chromium(iii) is not carcinogenic, v) that there is a large margin of safety of 4 to 5 orders of magnitude between a daily intake of 250 µg/day, equivalent to 4.1 µg/kg bw/day for a 60 kg person, and the NOAEL calculated for chromium(iii) from the NOAEL for chromium(iii) picolinate in the long-term NTP studies in mice and rats,, the Panel concluded that the safety of chromium(iii) as a nutrient added to PARNUTS and foods intended for the general (including food supplements) is not of concern, provided that the intake of chromium(iii) from these sources does not exceed 250 µg/day, the value established by the WHO for supplemental intake of chromium that should not be exceeded. European Food Safety Authority, 2010 KEY WORDS Trivalent chromium, nutrient, food supplements, foods for particular nutritional uses (PARNUTS) 1 On request from the European Commission, Question No EFSA-Q , adopted on 10 November Panel members: F. Aguilar, B. Dusemund, P. Galtier, J. Gilbert, D.M. Gott, S. Grilli, R. Gürtler, J. König, C. Lambré, J-C. Larsen, J-C. Leblanc, A. Mortensen, D. Parent-Massin, I. Pratt, I.M.C.M. Rietjens, I. Stankovic, P. Tobback, T. Verguieva, R.A. Woutersen. Correspondence: ans@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group B on Food Additives and Nutrient Sources added to Food: M. Bakker, D. Boskou, B. Dusemund, D. Gott, T. Hallas-Møller, A. Hearty, J. König, D. Marzin, D. Parent-Massin, I.M.C.M. Rietjens, G.J.A. Speijers, P. Tobback, T. Verguieva, R.A. Woutersen for the preparatory work on this scientific opinion. Suggested citation: EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the safety of trivalent chromium as a nutrient added for nutritional purposes to foodstuffs for particular nutritional uses and foods intended for the general (including food supplements).. [46 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Safety of trivalent chromium as a nutrient added for nutritional Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to Food was asked to deliver a scientific opinion re-evaluating the safety of chromium(iii) as a nutrient added to food for particular nutritional uses (PARNUTS) and foods intended for the general (including food supplements). In previous opinions the Panel noted that recent reviews and evaluations of chromium(iii) point at conflicting outcomes of genotoxicity assays and report diverging views and conclusions on the consequences of this genotoxicity issue for the ultimate safety assessment of chromium(iii). Therefore, the present opinion focuses especially on the genotoxicity of chromium(iii), the consequences of this genotoxicity issue for the ultimate safety assessment of chromium(iii), and on any new data on this issue that have emerged since previous evaluations. The Scientific Committee on Food (SCF) issued an opinion on the Tolerable Upper Intake Level (UL) of chromium(iii) and concluded that limited data from subchronic, chronic and reproductive toxicity studies on soluble chromium(iii) salts and the available human data do not give clear information on the dose-response relationships, and that therefore a UL could not be derived. The US Food and Nutrition Board also concluded that the data from animal and human studies are insufficient to establish a UL for soluble chromium(iii) salts (IOM, 2001). The UK Expert Group on Vitamins and Minerals (EVM) also concluded that overall there are insufficient data from human and animal studies to derive a safe upper level for chromium. However, in the opinion of the EVM, a total daily intake of about 0.15 mg/kg bw/day chromium(iii) (or 10 mg/person/day) would be expected to be without adverse health effects. The World Health Organization (WHO) considered that supplementation with chromium should not exceed 250 μg/day. The bioavailability and toxicity of chromium compounds has been reviewed by several institutions. Following oral administration, chromium(iii) was reported to be very poorly absorbed via the gastrointestinal tract (0.4 to 2.8%) in both rats and humans. Some studies reveal that there can be differences in the bioavailability and tissue levels of chromium resulting from intake of different forms of chromium compounds, but these differences are small and the overall bioavailability of chromium from all these sources is low. The Panel concluded that following oral administration, chromium(iii) is poorly absorbed. The present opinion presents an overview of the genotoxicity studies on chromium(iii) picolinate in particular. While the results of in vitro bacterial mutagenicity assays have been consistently negative, the results of in vitro chromosomal aberration studies using Chinese hamster ovary cells are conflicting. The evaluation of the Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) of the UK, who evaluated the genotoxicity of chromium(iii) picolinate in 2004, led to the overall conclusion that the balance of the data in the in vitro genotoxicity assays suggest that chromium(iii) picolinate is negative with respect to genotoxicity. The conclusions have been supported by the recent work conducted by the US National Toxicology Programme (NTP), in which further in vitro and in vivo assessment result in a confirmation of a lack of mutagenic potential for chromium(iii) picolinate monohydrate in the presence and absence of metabolic activation. The UK Food Standards Agency (FSA) subsequently revised its opinion on the consumption of chromium(iii) picolinate, and released a statement indicating there is no need to avoid chromium(iii) picolinate. 2

3 The Panel also evaluated additional studies on the genotoxicity of chromium(iii) compounds, which were not covered by the COM evaluation. These studies covered new in vitro and in vivo genotoxicity studies. The Panel noted that although these new studies gave some equivocal results, two studies may be considered to be the most essential. These include a study with chromium(iii) picolinate in the bone marrow of Sprague-Dawley rats performed according to OECD guideline 475. Single oral doses up to 2000 mg/kg bw chromium picolinate did not induce chromosomal aberrations in cells derived from male or female rats at any of the concentrations tested in comparison to the control. They also include the NTP study reporting an in vivo micronucleus assay, in which male rats were given orally chromium picolinate (anhydrous) at doses up to 2500 mg/kg bw, and mice were given chromium picolinate monohydrate in the feed at doses up to mg/kg diet, both for 13 weeks. Chromium picolinate (anhydrous) did not produce chromosomal damage as determined by the absence of micronuclei in the in vivo rat micronucleus assay. Chromium picolinate monohydrate was negative in the male mice. The weak increases in the presence of micronuclei in female mice were considered equivocal findings as the anhydrous form did not show this effect at all. The Panel concluded that in very large amounts, certain chromium(iii) compounds are cytotoxic and have been shown to cause chromosomal damage. The authors of these studies and others have suggested that the chromosomal toxicity of high concentrations of chromium(iii) compounds may be due to a pro-oxidant action that can be suppressed with antioxidants. The Panel noted that results of the new genotoxicity studies suggest that in vitro, at high concentrations, chromium(iii) picolinate might cause DNA damage. The Panel also noted that this conclusion would be applicable to other chromium(iii) compounds. The Panel also evaluated the data on long-term toxicity and carcinogenicity for chromium(iii). The carcinogenicity study conducted by the NTP in mice demonstrated that there were no adverse effects associated with the dietary administration of chromium(iii) picolinate monohydrate at levels as high as mg/kg diet for a period of 2 years, equivalent to a No-Observed-Adverse-Effect Level (NOAEL) of 6100 mg/kg bw/day chromium(iii) picolinate monohydrate and 727 mg chromium(iii)/kg bw/day. The administration of mg/kg diet chromium(iii) picolinate monohydrate in the diet of rats for 2 years induced a statistically significant increase in the incidence of preputial neoplasms as compared to control and historical data. Although this effect did not rise above historical levels in the high dose group for rats ( mg/kg diet) the NTP considered this result equivocal evidence of carcinogenicity in male rats. The NTP concluded that under the conditions of these 2-year feeding studies, there was equivocal evidence of carcinogenic activity of chromium(iii) picolinate in male rats based on an increase in the incidence of preputial gland adenomas. There was no evidence of carcinogenic activity due to chromium(iii) picolinate in female rats or in male or female mice. The incidences of these adenomas were not dose-related and did not occur consistently across species, and not across sexes. Therefore, the Panel concluded that this benign lesion is not related to treatment and that the NOAEL in the rat study was 2400 mg/kg bw/day chromium(iii) picolinate monohydrate, equivalent to 300 mg chromium(iii)/kg bw/day. In addition to the carcinogenicity study conducted by the NTP, studies using other chromium(iii) compounds (e.g., chromic oxide, chromium acetate) have been performed in laboratory animals (i.e., mice and rats), and these data indicated that lifetime dietary supplementation with up to 2 g/kg bw/day chromium did not produce carcinogenicity (Schroeder et al., 1964, 1965; Ivankovic and Preussmann, 1975). 3

4 The Panel concluded that chromium(iii) is not carcinogenic. Dietary intake of chromium can be considered separately from its potential use in fortified foods, supplements and PARNUTs, and also based on exposure from their combined uses including background intake from the diet. In previous opinions, proposed use levels of chromium sources in fortified foods and PARNUTS have been provided. In relation to fortified foods, it has been proposed to use chromium in fortified foods for the general at levels typically up to 30% of the Recommended Daily Allowance (RDA) for chromium of 40 µg per serving (equivalent to 12 µg of chromium per serving). In relation to PARNUTS, chromium has been proposed for use at levels that will typically not exceed 300 µg/serving of chromium in PARNUTS foods. Data from the UK indicate that chromium is typically consumed from food supplement products at levels of up to 600 µg/day. Data from 2003 on the estimated dietary intakes of chromium from all foods sources in the UK indicate that chromium intakes from food range up to 170 µg/day, with mean consumption levels of 100 µg/day. Data from other EU countries suggest that chromium intake from foods ranges from µg/day for mean intakes, and that an intake of up to 580 µg/day may occur. Two scenarios of combined exposure to chromium were examined. The first scenario related to chromium exposure from its supplemental intake only (i.e. from food supplements, fortified foods and PARNUTS) and for this, typical exposure was found to be mg/day (0.015 mg/kg bw/day), with an upper intake of 1.54 mg/day (0.026 mg/kg bw/day). The second scenario related to scenario 1 plus the additional exposure from the proposed intake from foods; for this, the typical intake ranged from mg/day ( mg/kg bw/day), with an upper intake of 2.12 mg /day (0.035 mg/kg bw/day). The Panel noted that an intake of 1.54 mg chromium/day from food supplements, fortified foods and PARNUTS would be 2.7 to 25 times higher than what could be present in the diet. In judging the safety of chromium(iii) as a nutrient added to food for particular nutritional uses and foods intended for the general (including food supplements) the Panel noted that a Tolerable Upper limit for chromium is not available. The Panel also noted that both the limit of 1 mg chromium/day proposed by the SCF, and of 250 μg chromium/day for supplementation proposed by WHO are based on studies that were not designed to test the safety of chromium. The Panel also noted that an intake of 250 μg chromium/day from supplementation would be in the range of intake of chromium from the regular diet. Therefore, the Panel concluded that until more is known about chromium, the value set by WHO seems most adequate to limit the intake of chromium from foods for particular nutritional uses and foods intended for the general (including food supplements). Based on this consideration and the facts: i) that maximum intake levels of up to 250 μg/day for supplemental intake as suggested by the WHO would be in the same order of magnitude as what would result from normal dietary intake, ii) iii) iv) that in vitro, at high concentrations, chromium(iii) picolinate might cause DNA damage, and that this also applies to other sources of chromium(iii), that this DNA damage is not reflected in in vivo genotoxicity assays performed according to standard OECD protocols and in NTP studies, that chromium(iii) is not carcinogenic, 4

5 v) that there is a large margin of safety of 4-5 orders of magnitude between a daily intake of 250 μg/day, amounting to 4.1 μg/kg bw/day for a 60 kg person) and the NOAEL from the long-term NTP studies of 6100 mg/kg bw/day chromium(iii) picolinate monohydrate, equivalent to 727 mg chromium(iii)/kg bw/day in mice and of 2400 mg/kg bw/day chromium(iii) picolinate monohydrate equivalent to 300 mg chromium(iii)/kg bw/day in rats, the Panel concluded that the safety of chromium(iii) as a nutrient added to PARNUTS and foods intended for the general (including food supplements) is not of concern, provided that the intake of chromium(iii) from these sources does not exceed 250 μg/day, the value established by the WHO for supplemental intake of chromium that should not be exceeded. The Panel noted that specifications for sources of chromium(iii) to be added to food including food supplements should ensure that levels of chromium(vi) which is a genotoxic carcinogen are as low as possible and at least are such that they result in an adequate margin of exposure (EFSA, 2005). 5

6 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 6 Background as provided by the European Commission... 7 Terms of reference as provided by the European Commission... 7 Assessment Introduction Technical data Identity of the substance Specifications Manufacturing process Methods of analysis in food Stability, reaction and fate in foods Case of need and proposed uses Information on existing authorisations and evaluations Exposure Biological and toxicological data Bioavailability Toxicological data Acute oral toxicity Short-term and subchronic toxicity Genotoxicity Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Hypersensitivity Discussion References Glossary/abbreviations

7 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The Scientific Committee on Food (SCF) published in April an opinion on the tolerable upper intake level of trivalent chromium (Cr(III)). The limited data from studies did not give enough information to derive a tolerable upper level. However, it was concluded that in a number of limited human studies, there was no evidence of adverse effects associated with supplementary intake of chromium up to a dose of 1 mg chromium/day, which is well below the dietary intake of trivalent chromium in Europe. Under Regulation 953/2009 5, Directive 2002/46/EC 6 and Regulation (EC) No 1925/ chromium(iii) chloride and chromium(iii) sulphate and their hexahydrates can be used as sources of chromium(iii) in the manufacture of foodstuffs for particular nutritional uses and foods intended for the general (including food supplements). Recently, new sources of chromium have been assessed by the Panel on food additives and nutrient sources added to food (ANS). In its opinion concerning a mixture of chromium di- and tri-nicotinate as a source of chromium, ANS stated in its conclusions the following: "the Panel notes that recent reviews and evaluation of chromium(iii) (Eastmond et al., 2008; Levina and Lay, 2008) point at conflicting outcomes of genotoxicity assays and report diverging views and conclusions on the consequences of this genotoxicity issue for the ultimate assessment of chromium(iii). The Panel is aware that given this situation, the safety of chromium(iii) might need to be re-evaluated in light of the recent reviews and evaluations". Considering these concerns, the Commission would ask EFSA to re-evaluate the safety of chromium(iii) taking into account the latest scientific evidence. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In view of the above and in accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002 8, the Commission requests EFSA to give an opinion on: The safety of chromium(iii) as a nutrient added to foodstuffs for particular nutritional uses and foods intended for the general (including food supplements) Commission Regulation (EC) No 953/2009 of 13 October 2009 on substances that may be added for specific nutritional purposes in foods for particular nutritional uses OJ L 269, , p Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements. OJ L 183, , p Regulation EC N o 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of vitamins and minerals and of certain other substances to foods. OJ L 404, , p Regulation (EC) No 178/2002 of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L31, , p.1. 7

8 ASSESSMENT 1. Introduction The present opinion re-evaluates the safety of chromium(iii) as a nutrient added to food for particular nutritional uses (PARNUTS) and foods intended for the general (including food supplements). The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations and additional literature that became available since then. The Panel noted that not all original studies on which previous evaluations were based were available for re-evaluation by the Panel. In previous opinions (EFSA, 2009a,b,c), the Panel noted that recent reviews and evaluations of chromium(iii) (Eastmond et al., 2008; Levina and Lay, 2008) point at conflicting outcomes of genotoxicity assays and report diverging views and conclusions on the consequences of this genotoxicity issue for the ultimate safety assessment of chromium(iii). The Panel was aware that the safety of chromium(iii) might need to be re-evaluated in light of these recent reviews and evaluations. Therefore, the present opinion focuses especially on the genotoxicity of chromium(iii), the consequences of this genotoxicity issue for the ultimate safety assessment of chromium(iii), and any new data on this issue that have emerged since previous evaluations. The establishment of a Tolerable Upper Intake Level (UL) of chromium(iii) is outside the remit of this Panel. 2. Technical data 2.1. Identity of the substance Chromium exists in many chemical valence states, but the trivalent form is most abundant in nature, most stable, and also the form that is found in foods (Norseth, 1981; SCF, 2003). It is found in microgram quantities throughout the food supply, primarily in fruits, vegetables and grain products (SCF, 2003) Specifications The Panel noted that chromium (III) may contain hexavalent chromium (chromium(vi)) and that chromium(vi) has been classified by the International Agency for Research on Cancer (IARC) to be in Group 1, being carcinogenic to humans (IARC, 1990). Therefore, the Panel noted that specifications for sources of chromium(iii) to be added to food, including food supplements, should ensure that levels of chromium(vi), which is a genotoxic carcinogen, are as low as possible and at least are such that they result in an adequate margin of exposure (EFSA, 2005) Manufacturing process The manufacturing process will vary with the chromium(iii) source of interest Methods of analysis in food The analytical method for the quantification of total chromium in food is the Association of Analytical Communities (AOAC) Official Method (Inductively Coupled Plasma-Atomic Emission 8

9 Spectrometric method) (ICP-AES). Liquid Chromatography-Mass Spectrometry (LC-MS) and High Performance Liquid Chromatography (HPLC) can also be applied. Furthermore, Graphite Furnace- Atomic Absorption Spectrometry (GF-AAS) can be used to determine the chromium content in foods (Miller-Ihli, 1996). These methods do not discriminate between chromium(iii) and chromium(vi) Stability, reaction and fate in foods The stability, reaction and fate in foods will vary with the chromium(iii) source of interest Case of need and proposed uses In previous opinions, proposed use levels of chromium sources in PARNUTS and in food supplements amounted to levels that would provide 200 mg of chromium/day (EFSA, 2008, 2009c), to levels up to 30% of the EU Recommended Daily Allowance (RDA) for chromium(iii) of 40 µg set by Commission Directive 2008/100/EC 9, being equivalent to 12 µg chromium per serving (EFSA, 2010), or to levels of μg chromium/day for adults (EFSA, 2009b), or to a level of 600 µg chromium/day (EFSA, 2009a). A maximum single dose was proposed to provide 100 μg chromium (EFSA, 2009c). Chromium(III) is proposed for use in fortified foods for general consumption in order to supplement dietary intakes of chromium. The highest typical fortification level of chromium(iii) is reported to be at 30% of the RDA per serving, which is equivalent to 12 µg of chromium per serving. Assuming 3 servings a day, this would amount to 36 µg chromium(iii) per day. Chromium(III) is proposed for use in PARNUTS foods, where the use levels will vary depending on the nutritional requirements of the intended consumer, but generally not exceed 300 µg chromium(iii)/serving of the mineral. Assuming 3 servings a day, this would amount to 900 µg chromium(iii) per day Information on existing authorisations and evaluations A number of EU Member States and the Institute of Medicine (IOM) in the US have established RDAs or equivalent intake values for chromium and these are summarised in Table 1. Based on these RDAs for adults, infants aged 6 to 12 months, and children of 1 to 3 or 4 years, in 2003 the SCF derived a nutrition Reference Labelling Value (RLV) of 40 µg chromium(iii) for adults and 20 µg for infants of 6 to 12 months and children of 1 to 4 years (SCF, 2003a). On the basis of the SCF Opinion, the European Commission has recently updated the Community legislation and established an RDA of 40 µg chromium for the nutritional labelling of foodstuffs (Commission Directive 2008/100/EC). Table 1: Recommended daily intakes of chromium for adults, infants and children from different EU Member States and the US Origin (Reference) Chromium Intakes (µg/day) 9 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. 9. 9

10 Adults Infants 6-12 months Children 1-3 or 4 years France (Martin, 2001) 65 (males), 55 (females) 25 Germany, Austria, Switzerland (D-A-CH Referenzwerte, 2000) 30 to to 60 United Kingdom (COMA, 1991) Above 25 1 to 13 United States (IOM, 2001) 35 (males), 25 (females) 11 European Commission/Scientific Committee on Food (Commission Directive 2008/100/EC; SCF, 2003a) Recommended daily allowance for adults for labelling purposes The UK Committee on Medical Aspects of Food Policy (COMA) calculated from balance studies a theoretical requirement for adults of 23 μg chromium/day by using regression equations, and concluded that a safe and adequate level of intake lies above 25 μg/day chromium for adults and between 0.1 and 1.0 μg chromium/kg bw/day for children and adolescents (COMA, 1991). It was also noted by the Committee that no adverse effects were observed for intakes ranging between 1000 to 2000 μg/day chromium(iii). The World Health Organization (WHO) considered that supplementation with chromium should not exceed the level of 250 μg/day (WHO, 1996). The opinion states the following: The available data are too limited to make it possible to determine the basal and normative requirements for chromium. However, glucose-intolerant subjects consuming less than 20 µg of chromium/day for 14 weeks responded to chromium supplementation (i.e. glucose metabolism variables improved); healthy subjects did not. Intakes of 24.5 µg and 37 µg of chromium/day were apparently adequate to maintain health in elderly people. This suggests that, for chromium, the average basal requirements is less than 20 µg/day, and that the mean intake to meet basal needs may be close to 25 µg/day. It is reasonable to derive a normative estimate of the requirement for chromium since there is evidence of a storage reserve of this element. A 30% increase above the basal requirement might be appropriate. Thus, the minimum mean intake likely to meet normative needs for chromium might be approximately 33 µg/day. The relative non-toxic nature of chromium as found in food indicates that the tolerable limit for chromium is quite high. Findings that supplements of µg chromium/day, in addition to the usual dietary intake, can in some cases reverse hypoglycaemia and impaired glucose tolerance, and improve both circulating insulin levels and the lipid profile, suggest that the upper limit of the safe range of mean intakes could be above 250 µg/day. However, until more is known about chromium, it seems appropriate that supplementation of this element should not exceed this amount. The Societies for Nutrition of Germany (DGE), Austria (ÖGE) and Switzerland (SGE), jointly established an adequate daily intake of µg chromium/day for adults (D-A-CH, 2000). Although in 2003 the EVM expressed concerns regarding the safety of chromium(iii) picolinate in supplements, and the UK Food Standards Agency (FSA) advised consumers to avoid this source of the mineral, in December 2004 this advice was withdrawn following evaluation of available data by the Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM, 2004). The Committee concluded that, overall, the balance of data indicated that chromium(iii) picolinate intakes are not associated with a risk of genotoxicity (COM, 2004). The FSA stated that there is no reason for people wishing to supplement their diet with chromium to avoid chromium(iii) picolinate containing products (FSA, 2004). The FSA advised that most people are able to obtain the chromium required for normal health from a healthy, balanced diet. 10

11 In 2003, the SCF issued an Opinion on the Tolerable Upper Intake Level of Trivalent Chromium (SCF, 2003b). The Committee stated: In a number of limited human studies, there was no evidence of adverse effects associated with supplementary intake of chromium up to a dose of 1 mg chromium/day. The dietary intake of trivalent chromium in European countries is well below these doses. Thus this value of 1 mg chromium/day was based on the observation that in a number of limited human studies, there was no evidence of adverse effects associated with supplementary intake of chromium up to a dose of 1 mg chromium/day. The SCF also stated that adequate human data on trivalent chromium are limited, and that these trials were mainly studies of efficacy and not designed to find potential toxic effects (SCF, 2003b). Furthermore, the SCF was not able to derive a Tolerable Upper Intake Level (UL) for chromium because available human data and the data from studies on subchronic, chronic and reproductive toxicity in experimental animals with soluble chromium(iii) salts did not provide clear information on the dose-response relationships (SCF, 2003b). Similarly, the US Food and Nutrition Board concluded that the data from animal and human studies were insufficient to establish a UL for soluble chromium(iii) salts (IOM, 2001). The Food and Nutrition Board derived Adequate Intakes (AI) for chromium for different age groups, e.g. 35 μg/day and 25 μg/day for 19 to 50 year old men and women, respectively (IOM, 2001). The EVM (2003) also concluded that overall there are insufficient data from human and animal studies to derive a safe upper level for chromium: Overall, there are insufficient data from human or animal studies to derive a Safe Upper Level for chromium, although oral toxicity of poorly absorbed trivalent chromium appears to be low. The study by Anderson (1997b) indicated that 15 mg/kg bw/day chromium (as chromium chloride) was not associated with adverse effects in rats. Based on this study, and allowing uncertainty factors of 10 for inter-species variation and 10 for inter-individual variation, a total daily intake of about 0.15 mg/kg bw/day (or 10 mg/person) would be expected to be without adverse health effects. EFSA evaluated the use of chromium(iii) picolinate as a source of chromium in 2009 (EFSA, 2009a). It was concluded that the use of picolinate as a source of chromium(iii) in food supplements could amount to use levels of 600 μg chromium/day, and that this amount of chromium would be above the level of 250 μg chromium/day considered by the WHO as a value for supplementation that should not be exceeded. The Panel also indicated that although the amount of picolinate that would be consumed as a result of the proposed uses (4300 μg picolinate/day) would be safe, the Panel could not conclude that these uses of chromium(iii) picolinate are of no safety concern. In 2009, EFSA also evaluated the use of chromium nicotinates as a source of chromium (EFSA, 2009b). It was concluded that the simultaneous use of nicotinate as a source of chromium(iii) in both PARNUTS and in food supplements, both at use levels up to 200 μg chromium/day, could amount to use levels of 400 μg chromium/day and that this amount of chromium would be above the level of 250 μg chromium/day considered by the WHO as a value for supplementation that should not be exceeded. Although the amount of nicotinate that would be consumed as a result of these proposed uses would be safe, the Panel could not conclude that these uses of chromium(iii) nicotinate are of no safety concern. In addition, EFSA evaluated the use of chromium(iii) lactate as a source of chromium (EFSA, 2009c), and concluded that the use of chromium(iii) lactate, at the proposed use levels, is of no safety concern provided that the maximum level of chromium supplementation identified by the WHO is not exceeded. Furthermore, in these previous opinions (EFSA 2009a, b, c) the Panel noted that recent reviews and evaluations of chromium(iii) (Eastmond et al., 2008; Levina and Lay, 2008) point at conflicting outcomes of genotoxicity assays and report diverging views and conclusions on the consequences of 11

12 this genotoxicity issue for the ultimate safety assessment of chromium(iii). The Panel was aware that given this situation, the safety of chromium(iii) might need to be re-evaluated in light of these recent reviews and evaluations. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) evaluated the safety and efficacy of chromium methionine as feed additive (EFSA, 2009d). This Panel concluded that the toxicology of chromium(iii) was not yet fully clarified, and that available data indicated that chromium(iii) is much less toxic than chromium(vi). The FEEDAP Panel noted that that the most recent available literature and the carcinogenicity studies in rats and mice indicated that chromium(iii) may be a genotoxic compound under in vivo conditions. The FEEDAP Panel considered it prudent to avoid any additional exposure of the consumers resulting from the use of supplementary Cr in animal nutrition. Chromium(III) chloride and chromium(iii) sulphate are permitted for use in food supplements in accordance with Annex II of Directive 2002/46/EC 10 on the approximation of the laws of the Member States relating to food supplements, and for addition to foods as a mineral substance in accordance with Annex II of Regulation (EC) No 1925/2006 on the addition of vitamins and minerals and of certain other substances to foods, as well as in Regulation 953/2009 on substances that may be added for specific nutritional purposes in PARNUTS. Although the intakes of chromium from food supplements are not limited at EU level by these Directives, a number of Member States have issued recommendations for chromium intakes from supplementation. For example, the UK recommends that food intakes of 10 mg chromium/day from foods and food supplements are unlikely to cause any harm (FSA, 2008) and in Germany, the Federal Institute for Risk Assessment (BfR) recommends that dietary supplementation is restricted to a level of 60 µg chromium/day (BfR, 2004). The BfR report also states that insufficient information is available to assess the safety of chromium(iii) picolinate for use in food supplements, but that the submission of further data should be encouraged (BfR, 2004). In the US, chromium(iii) picolinate is affirmed as Generally Recognized as Safe (GRAS) for use in functional foods and beverages up to daily intakes of 600 µg of chromium(iii). According to the IARC metallic chromium and chromium(iii) compounds are not classifiable as to their carcinogenicity to humans (Group 3) (IARC, 1990) Exposure Exposure from food supplements Data from the UK indicate that chromium is typically consumed from food supplement products at levels of up to 600 µg/day. In general, chromium supplements are currently available in capsules and in tablets, with typical doses in the range of 50 to 200 μg elemental chromium/day and up to 1000 μg chromium/day, and are available in various forms (e.g., chromium(iii) picolinate, chromium polynicotinate, chromium chloride, and high-chromium yeast). Exposure from fortified foods Chromium(III) is proposed for use in fortified foods for general consumption in order to supplement dietary intakes of chromium. In the UK for example, the mean consumption of chromium from all food sources was estimated to be 100 µg/day; this is more than 8-fold greater than the proposed 10 Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements. OJ L 183, , p

13 fortification level of chromium(iii) at 30% of the RDA per serving, which is equivalent to 12 µg of chromium per serving. Assuming 3 servings a day, intake would amount to 36 µg chromium per day. Exposure from PARNUTS Chromium(III) is proposed for use in PARNUTS foods where the use levels will vary depending on the nutritional requirements of the intended consumer but generally not exceed 300 µg chromium(iii)/serving. These levels of chromium intake fall well within the ranges consumed by the general in the EU from all sources, including food supplements. Assuming 3 servings a day intake would amount to 900 µg chromium(iii)per day. Current exposure to chromium from dietary sources Chromium(III) occurs naturally in food. Fruit, vegetables and grains are all natural sources of dietary chromium; contributing factors affecting chromium concentrations are the growing conditions, processing, handling and fortification of food (Anderson et al., 1997a; Berner et al., 2004). The chromium content of foods is not included in existing food composition databases. Therefore, the intake can only be assessed using total diets or duplicate portion techniques. In the UK a total diet study (TDS) showed that the highest concentration of chromium was found in meat products (230 µg/kg), followed by oils and fats (170 µg/kg), bread (150 µg/kg), nuts and miscellaneous cereals (140 µg/kg), fish, sugar and preserves (130 µg/kg) (EVM, 2002). The 1997 UK TDS reported that the mean chromium exposure was 100 µg/day and 170 µg/day at the 97.5 th percentile. The French TDS of 2001 indicated a mean exposure of 77 µg/day for adults (aged >15 years old) and 68 µg/day for children (aged 3 14 years old) and, at the 97.5 th percentile, an exposure of 126 µg/day for adults and 124 µg/day for children (Leblanc, 2004). In duplicate diets in Germany, Sweden and Spain, mean chromium intakes varied from 61 to 160 µg/day (SCF, 2003). In the UK EVM Report, maximum total chromium exposure was estimated to be 770 µg/day (dietary intake excluding water: 170 µg/day (97.5 th percentile), water: up to 2 µg (assuming consumption of 2 litres of water/day at a maximum UK concentration < 1 µg/l) and supplements: up to 600 µg/day) (EVM, 2003). In 2003, the SCF consolidated the estimated exposure to chromium from dietary sources for a number of EU Member States and the US, and a summary of the data is provided in Table 2 (SCF, 2003b). The EVM (2003) reported that in the UK the highest levels of chromium were found in processed meats (230 µg/kg), whole-grain products (bread and miscellaneous cereals 130 to 140 µg/kg), and nuts (140 µg/kg), and that levels were low in staple foods. No licensed medicines were found to contain chromium but it was present with other nutrients in a number of supplements that might only be sold under the supervision of a pharmacist for use in malabsorptive states, conditions leading to hypoproteinaemia and perioperative nutritional support, at levels up to 200 µg. Multivitamin and mineral food supplements were also sources of chromium at levels of up to 600 µg. Chromium is present naturally in drinking water, and the Codex Standard for natural mineral waters sets a limit on 0.05 mg/l as total chromium (Codex Alimentarius Commission, 1997). There were no potentially high-intake groups identified by any of the exposure assessments. Table 2: Estimated dietary intakes of chromium from food sources Country (Reference) Germany (D-A-CH Referenzwerte, 2000) Chromium Intakes (µg/day) Range Mean - 61 (males), 84 (females) 13

14 UK (EVM, 2003) Safety of trivalent chromium as a nutrient added for nutritional Up to 170 (food) Up to 600 (supplements) Up to 2 (drinking water) Sweden (Abdulla et al., 1989) 50 to Spain (Garcia et al., 2001) to 205 US (IOM, 2001) (NHANES, ; US DHHS, 2003) 22 to 48 (males), 13 to 36 (females) 3.2 to 100 (males), 4.4 to 127 (females) (males), 25 (females) 29.5 (males), 30.0 (females) Exposure to chromium from foods alone in EU adults was reported to range from µg /day, with an upper intake of 580 µg/day. Combined exposure Two scenarios of combined exposure to chromium were examined (Table 3). The first scenario related to chromium exposure from its supplemental intake only (i.e. from food supplements, fortified foods and PARNUTS), and for this, typical exposure was found to be mg/day (0.015 mg/kg bw/day), with an upper intake of 1.54 mg/day (0.026 mg/kg bw/day). The second scenario related to scenario 1 plus the additional exposure from the proposed intake from foods, and for this, the typical intake ranged from mg/day ( mg/kg bw/day), with an upper intake of 2.12 mg/day (0.035 mg/kg bw/day). Table 3: Summary information on chromium intake and anticipated exposure to chromium from its potential use in fortified foods, food supplements and in PARNUTS Nutrient: chromium Intake from foods alone (for EU adults) Typical intake (mg /day) Upper intake (mg /day) Intake from supplements alone Assumed intake from fortified foods alone (per serving) (assume 3 servings/day) Assumed intake from PARNUTS 0.90 (assume (per serving) alone servings/day) Scenarios of combined exposure to chromium from chromium picolinate 2 1. Total anticipated exposure to chromium from supplemental intake (i.e. food supplements + fortified foods + PARNUTS) (mg /kg bw/day for an adult of 60kg) 3. Total anticipated exposure to chromium from food intake, fortified foods, supplements and PARNUTS (mg/kg bw/day for an adult of 60kg) Typical intake (mg /day) (0.015) ( ) Upper intake (mg /day) 1.54 (0.026) 2.12 (0.035) Reference D-A-CH, 2000, EVM, 2002, Abdulla et al., 1989, Barbera et al., 1989, Garcia et al., 2001 Technical dossier EVM, 2003 EFSA, 2009 EFSA, 2009 Calculation by the Panel Calculation by the Panel 1 Derived from data from the UK on chromium content in supplements (EVM, 2003), where it states that It is also present in a number of multivitamin and mineral food supplements at levels up to 0.6 mg. 14

15 2 Calculations are based on proposed food intakes of an average of µg/day and high level intakes of up to 580 µg/day. At the average level, it is assumed 1 serving/day of fortified foods and/or PARNUTS are consumed, while at the high intake, it is assumed 3 servings/day are consumed. 3. Biological and toxicological data Several reviews have included evaluations on the bioavailability and safety of chromium(iii) (IPCS, 1988; IARC, 1980; WHO, 1996; EPA, 1998; ATSDR, 2000; IOM, 2001; EVM, 2002, 2003; SCF, 2003). In previous opinions (EFSA 2009a,b,c) the Panel noted that recent reviews and evaluations of chromium(iii) (Eastmond et al., 2008; Levina and Lay, 2008) point at conflicting outcomes of genotoxicity assays and report diverging views and conclusions on the consequences of this genotoxicity issue for the ultimate safety assessment of chromium(iii). The Panel was aware that given this situation, the safety of chromium(iii) might need to be re-evaluated in light of these recent reviews and evaluations. Therefore, the present opinion focuses especially on the genotoxicity of chromium(iii), the consequences of this genotoxicity issue for the ultimate safety assessment of chromium(iii), and any new data that have emerged on this issue since previous evaluations Bioavailability Following oral administration, chromium(iii) was reported to be very poorly absorbed via the gastrointestinal tract (0.4 to 2.8%) in both rats (Conn et al., 1932; Olin et al., 1994; Polansky et al., 1993) and humans (Anderson and Kozlovsky, 1985; Gargas et al., 1994; Olin et al., 1994; Gammelgaard et al., 1999; ATSDR, 2000). Some studies have revealed that there can be differences in the bioavailability and tissue levels of chromium resulting from intake of different forms of chromium compounds (Olin et al., 1994; US Patent ; 1993), but these differences are small and the overall bioavailability of chromium from all these sources is low. The mechanism of absorption is still unclear but it appears to involve processes other than passive diffusion (EVM, 2003). In general, following absorption, chromium(iii) does not enter blood cells, but rather competes for one of the binding sites on the iron-transport protein transferrin, from where it can be transferred to lowmolecular-weight chromium, or chromodulin (Sun et al., 2000; Vincent, 2000a,b; EVM, 2003) and transported to the liver, a process partly regulated by insulin (Clodfelder et al., 2001). Once absorbed, chromium may also be complexed with other compounds, such as nicotinic acid (ATSDR, 2000). In humans, long-term storage occurs particularly in the liver, spleen, kidneys, bone and several other organs. A similar pattern is seen in rats with incorporation in the kidneys and testes in addition to the liver, spleen, brain, and bone (FNB, 2001; Tandon et al., 1979). Most chromium(iii) is cleared rapidly from the blood and excreted in the urine with small amounts also being lost in perspiration, bile and faeces (Anderson et al., 1997b; Gargas et al. 1994; Jeejeebhoy, 1999; ATSDR, 2000; IOM, 2001; Hepburn and Vincent, 2002). Chromium(III) is removed from the tissues at a slower rate (ATSDR, 2000). Urinary chromium excretion reflects the dietary chromium intake in a dose-dependent manner (Kumpulainen, 1992; Uusitupa et al., 1992). Altogether, the Panel noted that the bioavailability of inorganic chromium(iii) from food sources and food supplements is generally very low (0.1-2%) (SCF, 2003) Toxicological data The toxicity of chromium compounds has been reviewed by several institutions (IPCS, 1988; IARC, 1980; WHO, 1996; EPA, 1998; ATSDR, 2000; IOM, 2001; EVM, 2002, 2003; SCF, 2003). 15

16 Oral exposure to chromium(iii) or chromium(vi) compounds resulted in adverse intestinal, hepatic, renal, immunological, neurological developmental and reproductive effects. Chromium(III) appears less toxic than chromium(vi) (EVM, 2003) with chronic intakes of up to 750 mg/kg bw/day not being associated with adverse effects. Both forms of chromium have been reported to reduce fertility, fetal weight and crown length and increase post-implantation in mice at higher doses (EVM, 2003) Acute oral toxicity Table 4 presents some oral LD 50 values for different forms of chromium(iii). Table 4: Reported oral LD 50 values for chromium(iii) complexes Species Form of chromium(iii) LD 50 (mg/kg bw) Equivalent dose of chromium(iii)(mg/kg bw) Reference Rat Chromium acetate Smyth et al., 1969 Chromium nitrate Rat Chromium nitrate 1540 (male) 200 (male) Vernot et al., (female) 183 (female) Short-term and subchronic toxicity In 2003, both the SCF and the EVM noted that there were limited data from subchronic studies on the toxicity of soluble chromium(iii) salts. The NTP conducted a 90-day toxicity study in both rats and mice in which animals were administered diets containing chromium picolinate monohydrate (NTP, 2008). In this study diets comprising 0, 80, 240, 2000, , or mg of chromium picolinate monohydrate/kg diet were administered to male and female F344/N rats and B6C3F1 mice, with 10 animals/sex consuming each diet. The animals were 6-weeks old at baseline. Based on the feed consumption, the administered doses of chromium picolinate monohydrate were reported to be 0, 7, 20, 160, 800, or 4240 mg/kg bw/day for male rats and 0, 6, 20, 160, 780, or 4250 mg/kg bw/day for female rats, respectively, for the different concentrations of chromium picolinate in the diets. In mice, consumption of the different chromium picolinate concentrations from the diets resulted in chromium picolinate monohydrate doses of 0, 17, 50, 450, 2300, or mg/kg bw/day in male mice, and 0, 14, 40, 370, 1775, and 9140 mg/kg bw/day in female mice, respectively. During the experimental period, feed intake was monitored weekly and all animals were subject to clinical examinations on a weekly basis. Blood samples were collected for haematological and clinical chemistry analyses on days 3 and 21 of the experimental period, as well as at the end of the experimental period. During the last 12 days of the experimental period, samples of sperm were collected from the male rats and mice for an assessment of sperm motility, while vaginal cytology samples were collected from the female animals. A necropsy was performed on all core animals at the end of the experimental period at which time, heart, right kidney, liver, lung, right testis, and thymus weights were recorded. The animals in the control and high dose ( mg/kg diet) groups were subjected to histopathological examinations in which the adrenal, clitoral, Harderian, mammary, parathyroid, pituitary, preputial, salivary, and prostate glands, bones, brain, oesophagus, eyes, gallbladder (mice only), heart, large intestine, kidney, liver, lung, lymph nodes, nose, ovaries, pancreas, skin, spleen, stomach, testis, thymus, trachea, urinary bladder, and uterus were examined (NTP, 2008). Similar results were observed in the subchronic toxicity studies conducted in mice and rats. No deaths were recorded and no significant differences were observed between the final mean body weights, 16