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Introduction

Background to COM review

1. Chromium is a Group 6 metallic element which is ubiquitous in the environment where it generally occurs in the hexavalent or trivalent form. Chromium is an essential element, involved in carbohydrate metabolism. Chromium (III) picolinate is a food supplement which is widely available in the UK.

2. Chromium was one of the minerals recently reviewed by the Expert Group on Vitamins and Minerals (EVM) (http://www.foodstandards.gov.uk/multimedia/pdfs/vitmin2003.pdf). The EVM noted the reports of genotoxicity associated with chromium picolinate and excluded it from their recommendations for a Safe Upper Level for chromium. Following the publication of the EVM report, the Food Standards Agency advised that consumers should use other forms of trivalent chromium supplements.

Advice requested from COM

3. The Food Standards Agency has asked the COM to review the available information on the mutagenicity of trivalent chromium and specifically on chromium picolinate in order to support consumer advice and, if appropriate, to recommend what further studies would be required to draw definite conclusions. A US National Toxicology Program (NTP) carcinogenicity bioassay of chromium picolinate is underway but the final report is unlikely to be available for several years.

Public Health Issue

4. Hexavalent chromium compounds are established human carcinogens on the basis of both animal studies and epidemiological evidence of carcinogenicity (ie considered Group 1 carcinogens by the WHO International Agency for Research on Cancer (IARC) (http://monographs.iarc.fr/). Trivalent chromium compounds have lower toxicity, which is generally attributed to their lower solubility and to their very limited ability to cross cell membranes. Trivalent chromium compounds were considered by IARC to be not classifiable with regard to carcinogenicity in humans (ie Group 3).

5. Trivalent chromium may be the ultimate carcinogen responsible for the effect of hexavalent chromium since it is able to bind DNA directly. However, it is unclear whether the reduction of hexavalent to trivalent chromium and the subsequent oxidative damage, or the direct binding of trivalent chromium to DNA, or both is responsible.

Data considered by the COM

Chromium picolinate and other chromium compounds

6. Chromium picolinate contains trivalent chromium bonded to three molecules of picolinic acid. The formation of additional chromium co-ordination complexes with each molecule of picolinic acid through the lone pair of electrons present on the nitrogen aids the stability of the molecule. Picolinic acid is an isomer of niacin (vitamin B₃) and a minor metabolite of tryptophan metabolism. Unlike other trivalent chromium compounds, chromium picolinate is soluble in water at neutral pH.

Absorption, distribution, metabolism and excretion

7. The data on the absorption, distribution, metabolism and excretion (ADME) of chromium picolinate are limited. The available data from human volunteer studies and from experimental studies in rats suggest that the gastrointestinal absorption of chromium picolinate is significantly greater than of other forms of trivalent chromium and is comparable to that of hexavalent chromium.

8. In a recent in-vivo ADME study, rats were given a daily intravenous dose of radiolabelled chromium picolinate (⁵¹chromium or ³H- picolinate) for 14 days.¹ Retention of chromium was substantial, with daily urinary and faecal excretion of approximately 10% of the ⁵¹Cr at the beginning of the experiment, increasing to approximately 20% by the end. The majority of the radiolabel was found in the urine and when subject to column chromatography co-eluted with chromodulin. ³H-labelled material was largely excreted via the urine. At the end of the treatment period, both ⁵¹Cr and ³H labels were widely distributed in the tissues but were predominantly present in the liver, where the sub-cellular pattern of distribution differed to that in other tissues. Thus the absorption, distribution, metabolism and excretion of chromium picolinate is complex and includes some degree of dissociation.

Oxidative damage

9. Hexavalent chromium is readily reduced to trivalent chromium both in vitro and in vivo, resulting in oxidative and cytotoxic damage to cells. The evidence for oxidative damage in vivo, after treatment with chromium picolinate includes increased urinary excretion of 8-hydroxy-2' deoxyguanosine and increased lipid peroxidation in liver and kidney cells of rats.² Evidence of in vitro oxidative damage includes damage to mitochondria from CHO cells and lipid peroxidation in cultured macrophage J774A.1 cells.^3,4 However the only other in-vivo study available does not report any oxidative damage associated with chromium picolinate or with other forms of trivalent chromium.⁵ Overall, it can be concluded that chromium picolinate induces oxidative damage in vivo to a lesser extent than hexavalent chromium compounds. However, in-vivo oxidative damage associated with chromium picolinate treatment has been reported by only one research group, where the test material was synthesised in the laboratory.

Interactions with DNA

10. Trivalent chromium compounds are able to bind DNA and RNA in cell free systems. While some studies with chromium picolinate suggest little direct interaction with DNA,² Speetjens and colleagues,⁶ have shown a dose-dependent relaxation of supercoiled plasmid DNA to the circular nicked form by trivalent chromium picolinate in the presence of ascorbic acid and air. The authors noted that chromium picolinate was stable and thus could be incorporated into cells intact. They further speculated that ascorbate could reduce trivalent chromium to divalent chromium, which could then enter Fenton or Haber-Weiss reaction cycles to produce hydroxyl radicals leading to oxidative damage. Members considered that more direct evidence was required to confirm this suggestion.

11. Chromium picolinate induces DNA fragmentation in cultured J774A.1 murine macrophages.⁷

In vitro mutagenicity

12. Trivalent chromium compounds are negative in a large number of in-vitro bacterial mutagenicity tests. In contrast, positive results have been documented for hexavalent chromium compounds. An adequate study using chromium picolinate was conducted in Salmonella typhimurium strains as part of the US NTP. This study reported negative results.⁸ Other published bacterial tests with chromium picolinate have also yielded negative results.^9,10

13. Chromium picolinate has been reported to cause up to a 40 fold increase in mutation at the hprt locus in CHO AA8 cells in the absence of exogenous metabolic activation.¹¹ However the results were from a single, unusually long, treatment time of 48 hours. The chromium picolinate used had been synthesised by the testing laboratory and there was uncertainty regarding the nature and quantities of impurities in test material used.

14. In response to recommendations made by the COM in October 2003, the ability of chromum picolinate to cause hgprt mutations in vitro in CHO cells was tested using commercial grade material using a protocol adhering to International standards.¹² Chromium picolinate was negative in both S9 activated and non-activated assays. In order to fully replicate the work of Stearns and colleagues,¹¹ an additional experiment was conducted using a 48 hour incubation in the absence of S9 activation.¹³ This was also negative. Additional information on dosing solution analysis and historical control data were reported to the COM in October 2004. The Committee was satisfied regarding the identity of the test material and dosing solution analysis. Overall it was agreed that the data should be considered as indicating a negative result.

15. Positive results have been documented for both hexavalent and trivalent chromium compounds in in-vitro clastogenicity tests in mammalian cells. The significance of the results with trivalent compounds is uncertain as the evidence of mutagenicity was documented at high cytotoxic concentrations and under prolonged exposure conditions where it was considered that endocytic uptake of test material had occurred. In a study by Stearns and colleagues¹⁴ chromium picolinate was tested in both a solubilised and a particulate form and resulted in a dose-related increase in chromosomal aberrations in CHO AA8 cells following incubation for 24 hours in the absence of exogenous metabolic activation. The magnitude of the response (3-18 times control) reported clearly suggested a clastogenic effect. The Committee noted that the test material used had been synthesised in the testing laboratory and commented that there was uncertainty regarding the nature and quantities of impurities in the test material used in this assay. The Committee also noted that chromium picolinate would have been expected to be poorly soluble in the solvent used (acetone). Overall it was agreed that there was a need for independent replication of the study undertaken by Stearns before any conclusions could be reached. In response to recommendations made by the COM in October 2003, the ability of chromium picolinate to cause chromosome aberrations in CHO cells in vitro was tested and reported to be negative.¹⁵ The Committee considered that the submitted study had been adequately conducted according to internationally accepted guidelines but observed that there were limitations regarding this study (eg the wide historical positive control range for structural aberrations both in the absence and presence of exogenous metabolic activation). Additional information on dosing solution analysis and historical control data were reported to the COM in October 2004. The Committee was satisfied regarding the identity of the test material and dosing solution analysis. Overall it was agreed that although the committee noted some limitations in the study, the data should be considered as indicating a negative result.

In vivo mutagenicity

16. Chromium picolinate but not trivalent chromic chloride was active in a multi-generation Drosophila study where it was observed to delay pupation and decrease pupal viability. Further analysis indicated that chromium picolinate increased lethal mutations and dominant female sterility.¹⁶ It is not possible to extrapolate such data to in-vivo exposure in mammals.

17. Three in-vivo mutagenicity studies of chromium picolinate have been reported; two in rats and one in mice. No evidence of chromosomal damage was reported in a study in which rats were given an oral dose of up to 2,000 mg/kg bw. The data were published in abstract form^10,17 with further details being contained in the study report.¹⁸

18. No increase in micronuclei was reported in peripheral blood samples from F344 rats given gavage doses of up to 2,500 mg/kg chromium picolinate for 3 days.¹⁹ This study has been undertaken as part of the preliminary studies of the US NTP prior to commissioning carcinogenicity bioassays. The ratio of polychromatic to normochromatic erythrocytes was not altered in this study. However it is not possible to conclude that the bone-marrow has been exposed to chromium picolinate in this study since previous work²⁰ had suggested that trivalent chromium may be retained in the fatty tissue surrounding the bone-marrow. There are no comparable absorption data available for chromium picolinate. As part of the US NTP study, B6C3F1 mice were given diets containing up to 50,000 ppm chromium picolinate for 13 weeks and micronuclei in peripheral blood erythrocytes counted.²¹ There was no clear evidence for a mutagenic effect, although a statistical test for a dose-related trend had reported a positive finding in female mice (P=0.005). Pair-wise comparisons with control suggested no mutagenic effect. The NTP data have not yet been published in a peer-reviewed form.

Picolinic acid

19. An increase in hgprt mutations and chromosomal aberrations in the absence of exogenous metabolic activation was also documented in CHO AA8 cells.^11,12 These tests were undertaken concurrently with the tests using chromium picolinate described previously. Chromium picolinate was reported to be more mutagenic in the hgprt system than the equivalent concentrations of either free picolinate or trivalent chromic chloride. Similarly, the clastogenic effects of chromium picolinate were apparent at lower equivalent concentrations than those associated with free picolinate.

20. Picolinic acid was also studied in a multigeneration Drosophila study.¹⁶ Treatment with picolinic acid alone also increased the numbers of individuals arrested during pupation and reduced larval and adult viability. The significance of this with respect to the mutagenicity of picolinic acid is unclear. It is also not possible to extrapolate such data to in-vivo exposure in mammals.

Conclusions

21. The evaluation of the mutagenicity of chromium picolinate is complex and the available data are conflicting. Chromium picolinate has given positive results in some in-vitro mutagenicity tests. The mechanism by which this occurs is unclear. However, in these studies the test material had been synthesised in the laboratory concerned and an adequate specification was not available. Replication of the tests using commercial grade material in tests conducted to internationally accepted protocols gave negative results. The Committee expressed some reservations regarding the conduct of these studies (possible limitations in sensitivity) and in particular regarding the repeat in-vitro chromosome aberration study in CHO cells. However, overall it can be concluded that the balance of the data suggest that chromium picolinate should be regarded as not being mutagenic in vitro.

22. The available in-vivo tests in mammals with chromium picolinate are negative. In view of the negative in-vitro results with commercial grade chromium picolinate, there is no further requirement for in-vivo testing at the current time.

23. The ongoing US NTP carcinogenicity bioassays of chromium picolinate will provide important in-vivo data in the future. The in-life phase of the NTP bioassays is due to end in July 2004. These data should be considered when the full results of the bioassay are available.

December 2004
COM/04/S3

REFERENCES

1. Hepburn D D D and Vincent J B (2002). In Vivo Distribution of Chromium from Chromium Picolinate in Rats and Implications for the Safety of the Dietary Supplement. Chemical Research in Toxicology, 15, 93-100.

2. Hepburn D D D, Burney J M, Woski S A, Vincent J B (2003a). The Nutritional Supplement Chromium Picolinate Generates Oxidative DNA Damage and Peroxidised Lipids In Vivo. Polyhedron, 22, 455-463.

3. Manygoats K R, Yazzie M, Stearns D M (2002). Ultrastructural Damage in Chromium Picolinate-Treated Cells. Journal of Biology and Inorganic Chemistry, 7, 791-197.

4. Bagchi D, Bagchi M, Balmoori J, Ye X, Stohs S J (1997). Comparative Induction of Oxidative Stress in Cultures j774A.1 Macrophage Cells by Chromium Picolinate and Chromium Nicotinate. Research Communications in Molecular Pathology and Pharmacology, 97, 335-346.

5. Preuss H G, Grojec P L, Lieberman S, Anderson R A (1997). Effects of Different Chromium Compounds on Blood Pressure and Lipid Peroxidation in Spontaneously Hypertensive Rats. Clinical Nephrology, 47, 325-330.

6. Speetjens J K, Collins R A, Vincent J B, Woski S A (1999). The Nutritional Supplement Chromium (III) Tris (Picolinate) Cleaves DNA. Chemical Research in Toxicology, 12, 483-487.

7. Bagchi D, Stohs S J, Downs B W, Bagchi M, Preuss H G (2002). Cytotoxicity and Oxidative Mechanisms of Different forms of chromium. Mutation Research, 180, 5-22.

8. NTP, National Toxicology Program- Salmonella assay (unpublished results downloaded from NTP website).

9. Jutura V and Komorowski J R (2002). Antimutagenic activity of Chromium Picolinate in the Salmonella Assay, The Toxicologist, 44 (2) supp 1, Abstract 38.2, A59.

10. Esber H J, Moreno V, Loveday K S (1997). Evaluation of Chromium Picolinate in the Ames and the rat In Vivo Chromosomal Aberration. Mutation Research, 379, S89. [Abstract]

11. Stearns D M, Silveria S M, Wolf K K, Luke A M (2002). Chromium (III) trispicolinate is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese Hamster Ovary Cells, Mutation Research, 513, 135-142.

12. San R H C and Clarke J J (2004a). In vitro Mammalian Cell Gene Mutation (CHO/HGPRT) Test with an Independent Repeat Assay. Bioreliance Report no AA85MC.782001.BTL

13. San R H C and Clarke J J (2004b). In vitro Mammalian Cell Gene Mutation (CHO/HGPRT) Test with a 48-hour exposure. Bioreliance Report no AA85MC.782048.BTL

14. Stearns D M, Wise J P, Patierno S R, Wetterhan K E (1995). Chromium Picolinate Produces Chromosome Damage in Chinese Hamster Ovary Cells. FASEB J, 9, 1643-1645.

15. Gudi R and Rao M (2004a). In vitro Mammalian Chromosome Aberration Test. Bioreliance Report no AA85MC.331.BTL

16. Hepburn D D D, Xiao J, Bindom S, Vincent J B, O'Donell J (2003). Nutritional Supplement Chromium Picolinate Causes Sterility and Lethal Mutations in Drosophila melangoster. PNAS, 100, 3766-3771.

17. Greenberg D, Komorowski J R, Loveday K (1999). Rat Chromosomes are Unharmed by Orally Administered Chromium Picolinate. Journal of the American College of Nutrition, 18, 27.

18. Kiopes A L (1999). Evaluation of Chromium Picolinate in the Rat In Vivo Chromosomal Aberration Assay. Study Report no 30102. Published by Primedica Corporation Inc.

19. NTPb National Toxicology Program- Micronucleus assays (unpublished results downloaded from NTP website).

20. Kraintz L and Talmage R V (1952). Distribution of Radioactivity Following Intravenous Administration of Trivalent Chromium 51 in the Rat and Rabbit. Proceedings of the Society of Experimental Biology and Medicine, 81, 490-492.

21. NTPc National Toxicology Program- 13 week study (unpublished results downloaded from NTP website).

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