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Toxicology in Vitro 22 (2008) 819–826 www.elsevier.com/locate/toxinvit
Brief communication
Chromium picolinate does not produce chromosome damage q James R. Komorowski, Danielle Greenberg 1, Vijaya Juturu * Research and Development, Nutrition 21 Inc, 4 Manhattanville Road, Purchase, NY 10577, United States Received 19 November 2007; accepted 18 December 2007 Available online 25 December 2007
Abstract Chromium picolinate (CrPic) is used as a dietary supplement and has beneficial effects in reducing diabetes risk factors. The present study evaluated the cytogenetic effects of CrPic in bone marrow cells of Sprague–Dawley rats (5 animals/sex/group). Test animals were dosed orally with 33, 250 or 2000 mg/kg of CrPic, which corresponded to doses of 4.1, 30.8 and 246 mg/kg of chromium. The lowest dose of CrPic, 33 mg/kg is estimated to be the human equivalent for a 50 kg person (200 mcg Cr). The animals were dosed once, and sacrificed either 18 or 42 hours (h) later. The mitotic index was determined for each rat. Metaphase cells (50 or 100/rats) were examined for interstitial deletions, chromatid and chromosome gap, breaks or other anomalies. The average percentage of damaged cells at 18 h in vehicle treated males and females were 1.2% and 0.6%, respectively. The mean values at 18 h for doses of 33, 250 and 2000 mg/kg, were 0.4%, 0.8%, 0.4% for males and 0.6%, 0.2% and 0.6% for females, respectively. At 42 h, the mean values for vehicle treated males and females were 0.4% and 0.2%, respectively. For doses of 33, 250 and 2000 mg/kg at 42 h the average percent damage was 14%, 0.8% and 0.4% for males and 0.2%, 0.2% and 0.0% for females, respectively. None of these values were statistically increased compared to the vehicle controls. The positive control Cyclophosphamide (CPM) induced a significant increase in chromosomal damage at 18 h averaging 30% in males and 37% in females, respectively (p < 0.001). In the current study CrPic did not induce chromosomal damage in bone marrow cells at single doses of 33, 250 and 2000 mg/kg of body weight and thus there was no indication of any toxicity of CrPic. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Chromium; Chromium picolinate; Chromosomal damage; Cytogenic effects
1. Introduction Chromosomal aberration (CA) tests have been used in evaluating the clastogenicity of chemicals. Chromium Picolinate (CrPic, ChromaxÒ) is a dietary supplement that has been commercially available for the past two decades. It has been estimated that 10 million people took chromium picolinate containing supplements in 1998. Chromium is an essential trace mineral involved in carbohydrate, protein and lipid metabolism. Dietary chromium (Cr+3) is often lost during food handling and processing. Chromium bio-
q Abstract was presented at 42nd Annual Meeting of American College of Nutrition, Orlando, Florida, USA. * Corresponding author. Tel.: +1 914 701 4508; fax: +1 914 696 0860. E-mail address:
[email protected] (V. Juturu). 1 Currently working at Pepsi Cola Company, 700 Anderson Hill Road, Purchase, NY 10577, USA.
0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.12.007
availability is inhibited by other nutrients such as phytates, dietary fiber and other minerals. Chromium picolinate is one form of trivalent chromium that is available as a popular dietary supplement. It has been widely used in the United States for more than 10 years. It has been estimated that over 10 million people have taken CrPic containing supplements since 1998 (NBI, 2001). CrPic is absorbed to a greater extent than many other forms of dietary chromium (Anderson et al., 1997, 2004; Di Silvestro and Emily Dy., 2007). CrPic has shown its beneficial effects in reducing diabetes associated risk factors (Cefalu and Hu, 2004; Martin et al., 2006; Broadhurst and Domenico, 2006) and no toxicity effects were observed in these human studies or in in vivo and in vitro studies (Anderson et al., 1997; Cefalu et al., 1999; Campbell et al., 1997, 1999, 2004; Lindemann et al., 1995; Hagen et al., 2000; Shinde and Goyal, 2003; Sahin et al., 2007; IOM, 2004). More than 30 human clinical studies using doses of 200–1000 mcg Cr
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as CrPic per day for a period of 10 days to one year have reported no adverse events, no toxic effects and no negative interactions with drugs intended for the control of diabetes (Cefalu and Hu, 2004; Berner et al., 2004). Some of the in vitro CrPic genotoxicity studies raised concerns on its safety (Stearns et al., 1995, 2002; Hepburn et al., 2003). These reports include mutagenic and clastogenic activity in mammalian cells in vitro (Stearns et al., 1995, 2002) and induction of dominant lethal mutations in Drosophila (Hepburn et al., 2003). However, mutagenicity studies on CrPic have yielded contradictory results in Chinese hamster ovary (CHO) cells (Stearns et al., 1995, 2002) and mouse lymphoma cells (Whittaker et al., 2005), no mutagenicity in Salmonella typhimurium (Juturu and Komorowski, 2002; Whittaker et al., 2005), and no chromosomal aberrations in Chinese hamster ovary cells (CHO) (Slesinski et al., 2005; Gudi et al., 2005) and micro nucleus tests (Rhodes et al., 2004). Hepburn et al. (2003) concluded that their initial results showing DNA damage were not likely to occur in humans. The absence of genotoxicity findings from the in vivo studies (Rhodes et al., 2004) is consistent with the lack of genotoxicity reported in DNA damage studies in human volunteers given repeated doses of chromium tripicolinate (Kato et al., 1998). Chromium tripicolinate was also tested for in vitro chromosome damage potential in CHO cells in independent tests conducted with 4-h and 20-h exposure times in the absence of metabolic activation and with a 4-h exposure time in the presence of a rat-liver S9 homeogenate metabolic activation system. No significant cytotoxicity was observed at any dose level including the highest precipitating dose (Gudi et al., 2005). Anderson et al. (1997) found that chromium concentrations in the kidney and liver of two groups of rats fed either chromium tripicolinate or chromium chloride at concentrations up to 100 mg chromium per kg of diet for 140 days increased linearly with increasing dose over time. Animal studies on other trivalent chromium compounds have shown that oral exposures result in a wide tissue distribution (e.g., liver, kidneys, spleen, hair, heart, red blood cells, bone, and bone marrow), with the greatest increases in chromium concentration again occurring in the liver and kidneys (Agency for Toxic Substances and Disease Registry, 1998; U.S. Environmental Protection Agency, 1998). These findings are consistent with the results of Anderson et al. (1997). Animal studies, specifically those conducted with very high doses of CrPic have shown no evidence of toxicity (Anderson et al., 1997; Rhodes et al., 2004). The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by the test substance to the bone marrow cells of animals, usually rodents (Tice et al., 1994). Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour suppressor genes are involved in cancer in humans and experimental sys-
tems. Rodents are routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and processed. Other species and target tissues are not the subject of this method. This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species and among tissues (Adler, 1984; Preston et al., 1987; Richold et al., 1990). In light of the controversial reports, we evaluated the clastogenic potential of chromium picolinate (CrPic) based upon its ability to induce chromosome aberrations in bone marrow cells of rat in vivo model under standard OECD Guideline 473 and the International Conference on Harmonization ICH guidelines (1996 and 1997). This study was performed in compliance with the provisions of the Good Laboratory Practice (GLP) regulations.
2. Materials and methods 2.1. Animals Male and female Sprague–Dawley rats were obtained from Taconic Farms (Germantown, NY). Animals were quarantined for a minimum of 5 days prior to testing. All animals were asymptomatic and were released from quarantine prior to the start of the study. Animals were housed individually in suspended stainless steel cages with wire mesh bottom and front. Noncontact Cellu-DriTM bedding from Shepherd specialty papers (Kalamazoo, MI) was provided in dropping pans under the suspended cages. Female rats were 12 weeks old on the day of dosing. Male rats were 11 weeks on day of dosing. Prior to dosing, animals were assigned by sex to groups on the basis of body weight using a computer randomization program. Individual female weights ranged from 219 to 263 g, and individual male weights ranged from 349 to 394 g. Animals were housed in a limited access AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited facility. Fluorescent lighting was controlled to automatically provide approximately 12 h alternate light and dark cycles. Temperature and humidity were centrally controlled and recorded daily. The temperature readings ranged from 16–24 °C and humidity ranged between 41% and 50% relative humidity. Animals remained healthy during the period of study. Pellets of standard Purina Mills #5001 Rodent chow (St. Louis, MO) were provided ad libitum. There were no contaminants in the feed that were considered to have influenced the results of the study. Animals also had free access to tap water (Cambridge City tap water). The water used in the study met US Environmental Protection Agency (USEPA) drinking water standards and was monitored at least annually for levels of organophosphorus pesticides, metals, coliform bacteria, and other contaminants.
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2.2. Chromium picolinate (test article) The test article Chromium Picolinate (ChromaxÒ) was obtained from Nutrition 21, Inc, Purchase, NY The compound was a reddish powder and contained 12.3% chromium, based on weight (Fig. 1). Dose formulations were prepared on the day of dosing as suspensions in deionized water. A separate suspension was prepared for each of the three dose groups so that dosing volumes were constant at 10 mL/kg. 2.3. Negative and positive control article Deionized water prepared at the testing facility was used as a vehicle control. Cyclophosphamide (CPM, Lot No: 44 H-0486) purchased from Sigma Chemical Co. was used as a positive control.
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dosing while the positive control was sacrificed only at 18 h after dosing. The time required for uptake and metabolism of the test substance as well as its effect on cell-cycle kinetics can affect the optimum time for chromosome aberration detection. The first mitoses occur at 42 h. The optimal minimal time is 12–18 h. In the current study we used optimal minimal time 18 h and a maximum time 42 h to observe chromosomal aberrations. These doses of CrPic correspond to 4.1, 30.8 and 246 mg/kg of chromium (Cr) based on Cr content of 12.3% by weight. The low dose of CrPic was 33 mg/kg corresponds to the expected human dose of 200 mcg of Cr for a 50 kg person. Each dose of CrPic and vehicle and positive control groups consisted of 6 male and 6 female rats. Extra animals were used to optimize the quality of the slides from 5 animals, which were used for chromosomal analysis (Table 1).
2.4. Experimental design Chromium picolinate solutions and controls were administered once to animals orally via gavage. The dosing volumes for each animal were determined by the weight of the animal and the final necessary dose. The animals were dosed at a volume of 10 mL/kg. The positive control (Cyclophosphamide, CPM) was administered as a single oral dose approximately 18 h prior to sacrifice. The dosing volume for the positive control (CPM) article was also 10 mL/kg to provide a final dose of 30 mg/kg. Animals appeared clinically within normal limits after dosing and prior to sacrifice. The expected human exposure may indicate the need for a higher-dose level to be used in the test. Cr Pic dose levels were selected to cover a range from the maximum to little or no toxicity for human consumption. The CrPic was administered at 33, 250 and 2000 mg/kg; these animals were sacrificed at either 18 or 42 h after dosing. Vehicle animals were also sacrificed 18 and 42 h after O
N
N Cr3+
O
Approximately 2 h before sacrifice, animals were injected intraperitoneally with colchicines (1.5 mg/kg) to block dividing cells in metaphase. Animals were sacrificed by euthanasia with carbondioxide. A femur was excised from each animal, and hypotonic solution (0.03 M KCl, 0.01 M sodium citrate) was dripped through the femur to remove the bone marrow cells. Cells were centrifuged, resuspended in warm hypotonic solution and incubated at 37 °C for 20 min. Following treatment in hypotonic solution, cells were centrifuged and resuspended three times in fixative (methanol; glacial acetic acid at 3:1 ratio). Slides were air-dried and stained with 5% Giemsa for approximately 5 min. The mitotic index was determined from each animal by counting a minimum of 500 total cells. One hundred metaphase cells from each of 5 males and 5 females per group were analyzed for chromatid gap (TG), chromatid break (TB), chromosome gap (SG), chromosome break (SB), interstitial deletion (ID), triradial (TR), quadriradial (QR), dicentric chromosome (D), complex rearrangements
Table 1 Test groups: Males and female animals are divided into groupsa
O
O
2.5. Collection of bone marrow cells and chromosome analysis
Treatment
O O
N
Fig. 1. Chemical structure of chromium tripicolinate.
Chromium picolinate (CrPic) High Medium Low Vehicle control Positive control (Cyclophosphamide [CPM])
Dose mg/kg
2000 (246) 250 (30.8) 33 (4.1) – 30
Number of animals/sex sacrificed after dosing 18 h
42 h
5 5 5 5 5
5 5 5 5 0
a Route of administration: oral; frequency of administration: once. Chromium concentration (mcg) in parenthesis.
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(CR), ring (R), double minute chromosome (DM), cell with at least one pulverized chromosome (PU) and cells with greater than 10 aberrations (>10). Coordinates for positive cells were recorded. The number of chromosomes for analyzed cells was recorded. The mitotic index is used as a measure of cytotoxicity. The frequency of total aberrations per cell was calculated for each animal. Chromatid and chromosome gaps were not used for calculating the frequency of chromosomal aberrations since they are not thought to be biologically significant.
chromosome (PU) and cells with greater than 10 aberrations (>10)] when compared to vehicle controls (p < 0.001). The rats treated with CrPic presented no significant differences in mean mitotic index compared with the vehicle control group (Table 2). The mean total number of chromosomal aberrations in CrPic groups treated rats displayed no significant differences. The positive control, cyclophosphamide (CPM) at 30 mg/kg, induced a significant amount of chromosomal damage in both males (30%) and females (37%) that were statistically different from all CrPic doses and from the vehicle control (p < 0.001).
2.6. Statistical analysis
4. Discussion
Statistical analysis for the difference in the mean number of chromosomal aberrations and mean percent mitotic index between groups was determined by one-way ANOVA test. Mean values of each group were compared. Test articles not demonstrating a statistically significant increase in aberrations are concluded to be negative for potential to produce chromosome aberrations. Statistical analysis was performed following the methods outlined by Margolin et al. (1986).
Genotoxicity is only predictive of carcinogenic potential. The ultimate goal in mutagen screening program is to test the potential mutagens directly on human genetic material or to extrapolate to humans. The growing use of chromium picolinate (CrPic) as a nutritional supplement for reducing the risk of diabetes mellitus (DM) and dyslipidemia has resulted in heightened awareness of safety issues concerning chromium-containing supplements. CrPic was better absorbed and more bioavailable (Anderson et al., 1997; Anderson et al., 2004; Di Silvestro and Emily Dy., 2007) than other available commercial chromium supplements. The difference between trivalent form and hexavalent chromium, which is chromium in the 6+ oxidation states and is designated as chromium (VI), is critical. Cr VI is classified as carcinogenic to humans via inhalation (IARC., 1990; U.S. Environmental Protection Agency, 1998) while Cr III in trace amounts is essential for proper nutrition. The intermediates oxidation states of Cr are thought to be responsible for the adverse effects of the hexavalent from of Cr. The trivalent form in marked contrast does not enter the cell readily and is about 500–1000 times less toxic than is the hexavalent from of Cr (Costa, 2000; Cohen and Costa, 1998,2000). CrPic has shown several pharmacological effects and it has a potential mechanism of action in the insulin-signaling pathway (Wang et al., 2003; Li and Elmendorf, 2001; Chen et al., 2006). In the current study we assessed the safety of CrPic in an in vivo model. We found no indication of chromosomal damage by CrPic based on the observations of interstitial deletions, chromatid and chromosome gap or breaks of other anomalies. In a recent study, Rhodes et al. (2004) reported no adverse effects at a dose of 50,000 ppm corresponding to a daily dose of about 7500 mg/kg/day—an extremely high dose for an essential trace element being used in small doses as a nutritional supplement. In another study (National Toxicology Program, 2003), the numbers of micronuclei indicative of chromosome damage were assessed in 2000 polychromatic erythrocytes (PCE) per animal from bone marrow harvested 24 h following the last dose showed no significant or dose-related increases in micronucleated PCE from exposure to chromium tripicolinate (anhydrous). In the 90-day subchronic dosing study (Rhodes et al., 2004) with mice dosed with chromium
3. Results Chromium picolinate administration did not induce chromosomal aberrations in either male or female Sprague–Dawley rats following a single oral dose at any level tested. The mean percentages of cells with chromosomal damage for vehicle control males were 1.2% at the 18-h time point and 0.4% at the 42-h time point. Males dosed with CrPic had similar percentages of cells with chromosomal damage (Tables 1 and 2). At the 18-h time point, there were 0.4%, 0.8% and 0.4% cells with chromosomal damage for animals dosed with 33, 250 and 2000 mg/kg, respectively. At the 42-h time point, there were 1.4%, 0.8% and 0.4% cells with damage from animals dosed with 33, 250 and 2000 mg/kg, respectively. None of these values were statistically significant compared to the pooled vehicle control for males (average of 0.8%). In female rats treated with vehicle, the mean percentage of cells with chromosomal damage was 0.6% at the 18-h time point and 0.2% at the 42-h time point. There were no statistical differences (p < 0.05) in the chromosomal damage seen in female rats given CrPic. At the 18-h time point, the percentages of cells with chromosomal damage were 0.6%, 0.2% and 0.6% for animals dosed with 33, 250 and 2000 mg/kg, respectively. At the 42-h time point, the percentages of cells with chromosomal damage were 0.2%, 0.2% and 0.0%. In all CrPic-treated groups we found no differences in frequency of chromosomal aberrations [i.e., chromatid gap (TG), chromatid break (TB), chromosome gap (SG), chromosome break (SB), interstitial deletion (ID), triradial (TR), quadriradial (QR), dicentric chromosome (D), complex rearrangements (CR), ring (R), double minute chromosome (DM), cell with atleast one pulverized
Table 2 Mitotic index (MI), distribution of the different types of chromosomal aberrations (CA) treated with chromium picolinate Treatment
42 h Males NC CrPica 2000 250 33 Females NC CrPica 2000 250 33
TB
SG
SB
ID
TR
QR
DC
CR
R
DMC
% Cells with chromosome damage
1.62 ± 0.4
1.2 ± 0.5
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
1.2 ± 0.5
1.94 ± 0.3 1.6 ± 0.5 1.62 ± 0.4
0.0 ± 0.0 0.75 ± 0.5 0.33 ± 0.6
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.4 ± 0.3 0.3 ± 0.3 0.33 ± 0.3
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.4 ± 0.2 0.85 ± 0.4 0.3 ± 0.17
2.2 ± 0.2
0.6 ± 0.24
0.2 ± 0.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.6 ± 0.2
2.2 ± 0.27 2.28 ± 0.3 2.36 ± 0.4
0.2 ± 0.20 0.2 ± 0.20 0.2 ± 0.20
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.2 ± 0.2 0.0 ± 0.0 0.4 ± 0.2
0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.6 ± 0.4 0.2 ± 0.2 0.6 ± 0.4
2.36 ± 0.4
0.4 ± 0.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.4 ± 0.2
2.54 ± 0.3 3.2 ± 0.4 2.3 ± 0.9
0.4 ± 0.2 0.6 ± 0.4 1.3 ± 2.3
0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.0.3
0.0 ± 0.0 0.2 ± 0.4 1.7 ± 1.5
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.4 ± 0. 2 0. 8 ± 0. 4 1.5 ± 1.04
4.8 ± 0.3
0.0 ± 0.0
0.2 ± 0.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.2 ± 0.2
4.3 ± 0.2 4.6 ± 0.5 4.04 ± 0.4
0.0 ± 0.0 0.2 ± 0.2 0.2 ± 0.2
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.2 ± 0.2 0.2 ± 0.2 0.0 ± 0.0
J.R. Komorowski et al. / Toxicology in Vitro 22 (2008) 819–826
18 h Males NC CrPica 2000 250 33 Females NC CrPica 2000 250 33
MI (%)
NC: negative control; MI: mitotic index; TB: chromatid break; SG: chromosome gap; SB: chromosome break; ID: interstitial deletion; TR: triradial; QR: quadriradial; DC: dicentric chromosome; CR: complex rearrangements; R: ring; DMC: double minute chromosome; CrPic: chromium picolinate, mg/Kg; CPM: Cyclophosphamide 30 mg/Kg. a No significant observations of chromosomal aberrations in chromium picolinate treated animals.
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tripicolinate (monohydrate), the numbers of micronuclei in PCE were comparable and not statistically different in comparing the control and high-dose males or females. In the repeat-dosing test, male and female B6C3F1 mice were fed a diet containing chromium tripicolinate (monohydrate) for 90 days at doses of 80 ppm to 50,000 ppm in the diet. These doses are approximately equivalent to 12– 7500 mg/kg body weight, respectively, using default dietary dose estimates. Blood samples were collected on the last day of feeding and micronuclei were determined in 1000 peripheral blood polychromatic erythrocytes (PCE) per animal from 10 animals each in the control and 50,000ppm dose groups; 1000 normochromatic erythrocytes (NCE) per animal from 10 animals were evaluated in each of the control and dosed groups (Rhodes et al., 2004). MacGregor et al. (1990) reported that due to the kinetic characteristics of the PCE and NCE populations of erythrocytes, the micronucleus frequency in PCEs reflects damage that occurred within the past 48 h, while the NCE population reflects damage accumulated over the past month. No significant increases in NCE were seen in males at any dose level of the 90 day study indicating the absence of chromosomal damage from repeated ingestion of chromium tripicolinate (monohydrate). In females, the highest dose (approximately equivalent to 7500 mg chromium tripicolinate/kg/day), a marginally significant difference from control was observed in micronucleated NCE (p = 0.04). However, this dose greatly exceeds that that would result from dietary supplementation, and even exceeds the maximum doses at which any agent is required to be tested for regulatory approval by ICH and OECD testing guidelines (Tice et al., 1994). None of the other doses produced incidences of micronucleated NCE that were significantly different from the control values. In the current study, the results showed that CrPic did not produce increases in chromosome aberrations in vivo at any of the doses employed or in evaluations conducted in cells harvested from treated animals at 18 or 42 h postdosing. The differences in genotoxicity effects may be due to soluble vs. insoluble forms of the chemical, chemical is not well characterized thus there is a potential for spurious effects in such comparisons that may be related to differences in cytotoxity, or in more recent findings that impurities in samples may have amplified effects at precipitating doses (Slesinski et al., 2005). In recent human clinical trial, Finch et al. (2004) concluded that a daily oral administration of 600 mcg Cr, as CrPic, is safe and well tolerated with no clinically meaningful differences in adverse events (AEs), including sexual dysfunction and weight gain, or clinical indexes (CIs) in atypical depression patients as compared to placebo. In another study, liver and kidney function tests showed no significant changes in CrPic-treated volunteers (15 days to 9 months) (Campbell et al., 1999,2004) and no adverse or toxic effects were reported in 12-week treatment. In an animal study with STZ-induced diabetic rats (Shinde and Goyal, 2003) histopathological changes of kidney and liver
tissue from diabetic rats given CrPic for 6 weeks showed a decrease in the intensity and incidence of tubular and hepatocellular vacuolations, cellular infiltration, reduction in lesions associated with diabetic state and hypertrophy of hepatic cells and tubules in kidney sections. Shinde and Goyal (2003) clearly showed that there were no hepatotoxic or nephrotoxic effects detected following exposure to CrPic in STZ diabetes induced rats. Shinde and Goyal (2003) concluded that CrPic demonstrated significant anti-diabetic effects in diabetic rats and additionally improved renal and hepatic functions and morphology of the tissues resulting from the diabetic state. Mita et al. (2005) reported that renal Cr content and the recovery of renal Cr concentration after Cr supplementation were significantly lower in diabetic mice than in the non diabetic mice (p < 0.001). These observations suggest that CrPic supplementation improves insulin sensitivity and improves renal function by recovering renal Cr concentration. In addition, Seaborn and Stoecker (1989) reported that chromium supplementation increased bone chromium concentrations. Hence, the CrPic effect on bone marrow cells in determining the safety is appropriate to consider the outcome of results. In this study, CrPic, prepared under Good Manufacturing Practices (GMP) conditions to attain maximum test doses, was not clastogenic. In addition, no dose-related or time duration related increases in chromosome or chromatid aberrations were observed in CrPic-treated group. The study indicate a lack of genotoxic potential up to the limits required by FDA for the demonstration of safety (US Food and Drug Administration, 2004) and well beyond the exposure levels anticipated from use as a dietary supplement. 5. Conclusions Chromium picolinate was evaluated for its ability to induce chromosomal damage in male and female Sprague–Dawley rats. Animals were dosed once and sacrificed 18 or 42 h later. In conclusion, under the conditions of this study (conducted according to OECD and ICH guidelines), CrPic did not induce chromosomal damage in either males or females at either time point. These results add to the accumulating body of evidence that CrPic when produced under GMP conditions is not genotoxic in in vivo model. Acknowledgements The authors would like to acknowledge Dr. Herman S Lilja at TSI Mason laboratories. This study was conducted as a part of Nutrition 21, Inc. (Purchase, NY) safety tests for dietary supplements. The study was funded by Nutrition 21. The authors J.K. and V.J. are employees of Nutrition 21. References Adler, I.D., 1984. Cytogenetic tests in mammals. In: Venitt, S., Parry, J.M. (Eds.), Mutagenicity Testing: a Practical Approach. IRL Press, Oxford, Washington, DC, pp. 275–306.
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