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Endpoint:
direct observations: clinical cases, poisoning incidents and other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: Any kind of reliability rating is not considered to be applicable, since human studies/reports are not conducted/reported according to standardised guidelines.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reason / purpose:
reference to same study
Objective of study:
other: bioavailability
Qualifier:
no guideline followed
Principles of method if other than guideline:
In view of its function as an essential trace element, the bioavailability of Chromium(III) form the most important commercially used human oral Cr supplements was investigated by using radiolabelled compounds and whole-body-counting in rats.
GLP compliance:
not specified
Remarks:
not specified in the publication
Specific details on test material used for the study:
1) Chromium cloride
A solution of 51CrCl3, typical specific activity, 846 mCi/mg in 0.5 M HCl, was purchased from PerkinElmer Life and Analytical Sciences, Boston, USA. The 5 mCi-51Cr-activity of one delivered plastic flask was diluted with 500 µl of water.

SYNTHESIS OF CHROMIUM COMPOUNDS
1) Chromium picolinate
51CrPic3 x H2O was synthesized in a modified version according to Evans and Pouchnick (1993)*. Into a centrifugal filter device (Ultrafree-MC, 10.000 NMWL filter unit, low binding regenerated cellulose,Millipore) were pipetted 50 µL of an aqueous solution of picolinic acid (0.114 mM) and 240 µL of the diluted 51CrCl3-tracer solution. After mixing, 50 µL of an aqueous solution of CrCl3 (37 µM) were added and stirred. The mixture was incubated at 50 °C over night. After centrifugation at 5,000xg for 20 minutes, the reddish crystals remaining on the filter membrane were washed 3 times with 50 µL of ice-cold water. For the rat experiments, saturated aqueous solutions of 51CrPic3 (0.6 mM) were freshly prepared by adding 400 µL of water to the filter unit, incubation at 50 °C for 30 minutes, followed by centrifugation at 5,000xg for 20 minutes.

2) Chromium nicotinate
Into a centrifugal filter device (Ultrafree-MC, 10.000 NMWL filter unit, low binding regenerated cellulose, Millipore) were dissolved 14 mg of nicotinic acid in 50µL 0.1 mM NaOH. 240 µL of the diluted 51CrCl3-tracer and 40 µMol CrCl3 (=10 mg) in 50 µL of H2O were added. The tube was heated in a block thermostat at 57 °C over night. After centrifugation at room temperature at 5,000xg for 20 minutes, the blue-gray solid on the filter membrane was washed 3 times with 100 µL of ice-cold water.

3) 2) Chromium (D (or L)-phenylalaninate)3
(Modified procedure according to Yang et al. 2005)*. 10 mg (38 µmol) CrCl3 x 6H2O were dissolved in 200 µL of water in a 1.5 mL Eppendorf tubes. A 200 aliquot of a 51CrCl3-tracer solution (74 MBq) was added. 18.5 mg (=112 µMol) L or D-phenylalanine (M = 165.19 g/mol), respectively, was weighted in another Eppendorf tube and dissolved in 200 µL of water by heating to 80 °C in a thermo block. The 51CrCl3-solution was added and the mixture remained heated for 4 hours. After freeze-drying, the green-violet solid obtained was washed with 2 x 400 µL of
acetone. UV-spectroscopy in water showed identical absorption curves of both substances with peaks at 416 and 568 nm.

RADIOCHEMICAL PURITY
The radiochemical purity of the synthesized 51Cr compounds was tested by reverse phase HPLC (column Nucleosil C18 5U; eluent water/methanol, UV detection at 262 nM) using a method described by Olin et al. (1996)*. Following injection of CrPic3, the 51Cr-activity, found in peak at 4.14 min retention time, accounted for 95 % of the injected 51Cr-activity. From all other radiolabel chromium compounds, including Cr-Nic2, the 51Cr activity was found outside detectable peaks or was tightly bound to the column material and could eluated by a EDTA-buffer. This indicated that these more labile Cr-complexes disintegrated under the experimental conditions.

*References:
- Evans GW, Pouchnick DJ (1993) Composition and biological activity of chromium-pyridine carboxylate complexes. J Inorg Biochem 49:177–187.
- Yang X, Palanichamy K, Ontko AC, Rao MN, Fang CX, Ren J, Sreejayan N (2005) A newly synthetic chromium complexchromium(phenylalanine)3 improves insulin responsiveness
and reduces whole body glucose tolerance. FEBS Lett 579:1458–1464.
- Olin KL, Stearns DM, Armstrong WH, Keen CL (1996) Comparative retention/absorption of 51chromium from 51Cr chloride, 51Cr nicotinate and 51Cr picolinate in a rat model. Trace Elem Electrolytes 11:182–186.
Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany
- Fasting period: 4 - 6 hours before and 1 hour after the administration of chromium compounds
- Weight at study initiation: 200–300 g
- Housing (after administration): kept in cages of 3–4 rats except some experiments in which some rats were kept in individual metabolic cages over 2 days for a quantitative collection of urine and faeces.
- Diet: standard food in pellet diet (Altromin 1328)
- Water (ad libitum): tap water
Route of administration:
other: oral (gavage)
Vehicle:
water
Details on exposure:
Aqueous solutions were administered by gastric intubation or intraperitoneal injections.
Duration and frequency of treatment / exposure:
singel administration
Remarks:
6 - 12 µg Cr/rat
No. of animals per sex per dose:
3 to 12 female rats
Control animals:
not specified
Positive control:
not specified
Details on study design:
not specified
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces and tissues
Some rats were kept in individual metabolic cages over 2 days for a quantitative collection of urine and faeces.
The activity, measured immediately after administration of 51Cr, in the whole body was taken as the 100 % reference value. The 51Cr whole body retention were measured at given time points in the centre of a 200 cm long 4π-geometry whole body radioactivity detector with liquid organic scintillator in the energy range from 980 to 3,000 keV (Braunsfurth et al. 1977)*.
The biological half-life of 51Cr was calculated from a triple term exponential fit algorithm to the measured whole body retention of 51Cr values in a period of 1–100 days after the administration of 51Cr. The rats were sacrificed by exsanguination while under narcosis with Ketanest/Rompun. 51Cr activity in the excrements and tissues of rats was measured in the whole body counter or for longer sensitivity in a 3n X 3n NaJ detector (autogamma 5260, Canberra-Packard, Frankfurt, Germany).

*Reference:
- Braunsfurth JS, Gabbe EE, Heinrich HC (1977) Performance parameters of the Hamburg 4p whole body radioactivity detector. Phys Med Biol 22:1–17.
Statistics:
The mean 51Cr retention data R(t) from the whole-body counting were fitted by a compartment model with the condition A1 + A2 + A3 = 100 % for the 3
compartments:

R(t) = A1 x exp(-ln(2)/T1/.5 x t) + A2 x exp(-ln(2)/T2/0.5 x t) + A3 x exp(-ln(2)/T3/0.5 x t)

As start values for the fit software (Slide Write Plus 7.0), the data of Mertz et al. (1965)* for the retention of 51CrCl3 in rats were used. For the long-term half-life, only a lower and upper threshold could be fitted within the limited observation period. The lower threshold for the long half-life was found from a 2-compartment fit, while the upper threshold of T3/0.5 results from a 3-term fit with fixed parameters T1/0.5 and T2/0.5 from the 2-term fit and 5.9 days, respectively. With this upper threshold for T3/0.5, the final 3-compartment fit resulted in the 3 pool sizes and their half-lives of 51Cr-retention.
For comparison of whole-body-retention data between groups of rats, the Student t test was used. Differences between their mean values were regarded
as significant at p < 0.05.

*Reference:
- Mertz W, Roginski EE, Reba RC (1965) Biological activity and fate of trace quantities of intravenous chromium(III) in the rat. Am J Physiol 209:489–494.
Preliminary studies:
not specified
Toxicokinetic parameters:
other:
Remarks:
In rats, the apparent oral absorption of 51Cr(III) from Cr-picolinate, Cr-nicotinate, Cr-(D(or L)-phenylalaninate), Cr-proprionate, or Cr-chloride was generally low (0.04–0.24 %) in rats with slightly higher values for Cr-chloride and Cr-phenylalaninate.
Toxicokinetic parameters:
other:
Remarks:
Considering the rapid urinary excretion in rats, the true absorption of 51Cr was higher for CrPic3 (<1 %).
Metabolites identified:
not specified
Details on metabolites:
not specified
Bioaccessibility testing results:
not specified
Conclusions:
According to the current study, in rats, the apparent oral absorption of 51Cr(III) from chromium picolinate, chormium nicotinate, chromium (D (or L)-phenylalaninate), chromium proprionate, or chromium chloride was generally low (0.04–0.24 %) in rats with slightly higher values for chromium chloride and (D (or L)-chromium phenylalaninate). Considering the rapid urinary excretion in rats, the true absorption of 51Cr was higher for chromium picolinate (< 1 %).

Data source

Reference
Reference Type:
publication
Title:
Bioavailability of chromium(III)-supplements in rats and humans
Author:
Laschinsky, N. et al.
Year:
2012
Bibliographic source:
Biometals, DOI 10.1007/s10534-012-9571-5, published online 20 July 2012

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
basic toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In view of its function as an essential trace element, the bioavailability of Chromium(III) form the most important commercially used human oral Cr supplements was investigated by using radiolabelled compounds and whole-body-counting in humans.
GLP compliance:
not specified
Remarks:
not specified in the publication

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
SYNTHESIS OF CHROMIUM COMPOUNDS
1) Chromium picolinate
51CrPic3 x H2O was synthesized in a modified version according to Evans and Pouchnick (1993)*. Into a centrifugal filter device (Ultrafree-MC, 10.000 NMWL filter unit, low binding regenerated cellulose,Millipore) were pipetted 50 µL of an aqueous solution of picolinic acid (0.114 mM) and 240 µL of the diluted 51CrCl3-tracer solution. After mixing, 50 µL of an aqueous solution of CrCl3 (37 µM) were added and stirred. The mixture was incubated at 50 °C over night. After centrifugation at 5,000xg for 20 minutes, the reddish crystals remaining on the filter membrane were washed 3 times with 50 µL of ice-cold water. For the rat experiments, saturated aqueous solutions of 51CrPic3 (0.6 mM) were freshly prepared by adding 400 µL of water to the filter unit, incubation at 50 °C for 30 minutes, followed by centrifugation at 5,000xg for 20 minutes.

2) Chromium (D (or L)-phenylalaninate)3
(Modified procedure according to Yang et al. 2005)*. 10 mg (38 µmol) CrCl3 x 6H2O were dissolved in 200 µL of water in a 1.5 mL Eppendorf tubes. A 200 aliquot of a 51CrCl3-tracer solution (74 MBq) was added. 18.5 mg (=112 µMol) L or D-phenylalanine (M = 165.19 g/mol), respectively, was weighted in another Eppendorf tube and dissolved in 200 µL of water by heating to 80 °C in a thermo block. The 51CrCl3-solution was added and the mixture remained heated for 4 hours. After freeze-drying, the green-violet solid obtained was washed with 2 x 400 µL of
acetone. UV-spectroscopy in water showed identical absorption curves of both substances with peaks at 416 and 568 nm.

RADIOCHEMICAL PURITY
The radiochemical purity of the synthesized 51Cr compounds was tested by reverse phase HPLC (column Nucleosil C18 5U; eluent water/methanol, UV detection at 262 nM) using a method described by Olin et al. (1996)*. Following injection of CrPic3, the 51Cr-activity, found in peak at 4.14 min retention time, accounted for 95 % of the injected 51Cr-activity. From all other radiolabel chromium compounds, including Cr-Nic2, the 51Cr activity was found outside detectable peaks or was tightly bound to the column material and could eluated by a EDTA-buffer. This indicated that these more labile Cr-complexes disintegrated under the experimental conditions.

*References:
- Evans GW, Pouchnick DJ (1993) Composition and biological activity of chromium-pyridine carboxylate complexes. J Inorg Biochem 49:177–187.
- Yang X, Palanichamy K, Ontko AC, Rao MN, Fang CX, Ren J, Sreejayan N (2005) A newly synthetic chromium complexchromium(phenylalanine)3 improves insulin responsiveness
and reduces whole body glucose tolerance. FEBS Lett 579:1458–1464.
- Olin KL, Stearns DM, Armstrong WH, Keen CL (1996) Comparative retention/absorption of 51chromium from 51Cr chloride, 51Cr nicotinate and 51Cr picolinate in a rat model. Trace Elem Electrolytes 11:182–186.

Method

Type of population:
general
Subjects:
- Number of subjects exposed: 3 subjects
- Sex: male
- Age and weight: 56 (91 kg), 58 (87 kg), and 65 (65 kg) years, respectively
Ethical approval:
not specified
Route of exposure:
oral
Reason of exposure:
intentional
Exposure assessment:
estimated
Details on exposure:
Chromium picolinate and 4 weeks later Chromium (D-phenylalaninate)3 (400 µg Cr with 16 and 11 µCi 51Cr-activity) were administered orally as aqueous solution (100 mL) in three male volunteers.
Examinations:
The retained 51Cr activities were measured after 7 days in the whole-body counter. Urine was collected quantitatively for 48 hours and the 51Cr activities were measured in the whole body counter. The additional radiation burden from orally administered 51Cr (50 years equivalent dose) was between 0.01 and 0.02 mSv, which reflects < 1 % of the natural radiation burden of 2.4 mSv/year of a subject in Germany.
Medical treatment:
not specified

Results and discussion

Clinical signs:
not specified
Results of examinations:
Bioavailability of chromium picolinate and chromium (D-phenylalaninate)3 was analysed in human volunteers by intraindividual comparison. The apparent absorption (=Cr bioavailability) of 51Cr from both compounds was 0.8–1 % in humans. Most of the freshly absorbed chromium picolinate was rapidly excreted via urine in humans.
Effectivity of medical treatment:
not specified
Outcome of incidence:
not specified

Applicant's summary and conclusion

Conclusions:
The current study analyses the bioavailability of chromium picolinate and chromium (D-phenylalaninate) in human volunteers by intraindividual comparison. The apparent absorption (=Cr bioavailability) of 51Cr from both compounds was 0.8–1 % in humans. Most of the freshly absorbed chromium picolinate was rapidly excreted via urine in humans.