Chromium tris((2-ethylhexanoate)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Chromium (3+) picolinate (CAS n° 14639-25-9, EC n° 477-680-4)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOG APPROACH
The read across follows Scenario 2 - Different compounds have qualitatively similar properties: properties of the target substance predicted to be quantitatively equal to those of the source substance or prediction based on a worst-case approach (as described in the 2017 Read-Across Assessment Framework document).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Chromium tris(2-ethylhexanoate) (EC 222-357-3, CAS 3444-17-5)
SOURCE: Chromium (3+) picolinate (CAS n° 14639-25-9, EC n° 477-680-4)

3. ANALOG APPROACH JUSTIFICATION
The target substance and the source substance are organic salts of Chromium (3+). The organic counter ion of the target and source substances are small (6-8 carbon) carboxylic acids of similar molecular weight and polarity. The biologically-reactive center of all the molecules would be predicted to be the Cr (3+).
4. DATA MATRIX
See Read Across document attached to CSR.

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Alkaline version of the in vivo Comet assay

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

other: CBA/Ca

Sex:

male

Details on test animals or test system and environmental conditions:

weight at study initiation: around 20g
age at study initiation: 7-8 weeks
source: Scanbur BK - Sweden
water: free access to tap water
diet: free access to standardized rodent pellet diet (B&K Universal, batch 3386)
acclimatisation: at least 7 days before the start of the study

Route of administration:

intraperitoneal

Vehicle:

Phosphate bufferd saline (PBS)

Details on exposure:

Chromium picolinate and the positive control (cyclophosphamide) were dissolved in phosphate buffered saline (PBS) and injected intraperitoneal. as freshly prepared solutions (0.01 ml/g b.wt.). Control animals received the same volume of vehicle only.

Duration of treatment / exposure:

one single injection, 3 individual experiments

Frequency of treatment:

one single injection

Post exposure period:

16 hours

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Remarks:

highest soluble dose

No. of animals per sex per dose:

vehcile: 3*4 male animals
chromium picolinate: 3*4 male animals
positie control: 3*2 male animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CAS n°50-18-0) was obtained from Sigma (USA) and administered via intraperitoneal route at 200 mg/ Kg bw.

Tissues and cell types examined:

Freshly isolated lymphocytes from whole blood and isolated nuclei from liver cells were examined.

Details of tissue and slide preparation:

Immediately after sacrifice, 0.5 ml blood was taken from each mouse by heart puncture using a heparinised syringe, and at the same time a small piece of the liver was cut out in a standardized way. The blood and liver samples were then immediately put on ice until further processed for the Comet assay which was performed the same day.
The slides were prepared from layers of cells mixed with agarose (low melting point grade fromfrom Iternational Biotechnologies, USA). Alkaline solutionwas used to lyse the cells at 4°C during 1 hour. After lysis, the slides were immediately transferred to an electrophoresis unit in which the supercoiled DNA was relaxed in the electrophoresis buffer at 4 °C for 40 min. The electrophoresis was performed at 4°C for 10 min. Immediately after the electrophoresis, the slides were neutralised, dried at room temperature and stored in a sealed box until the day of image analysis.
Ethidium bromide-stained nucleoids were examined at 500 x magnification in a fluorescence microscope. The microscope was attached to a black and white chargecoupled device video camera connected to a computer-based image analysis system. Fifty ‘comets’ per slide were randomly captured, avoiding the edges and damaged parts of the gel. Apparently dead cells (comets without distinct heads) and superimposed comets were not captured and analyzed.

Evaluation criteria:

The tail moment was used as an indicator of DNA damage expressing both the distance of migration and the relative amount of DNA in the tail after electrophoresis.

Statistics:

The 2-tailed Students t-test was used.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no evidence of Chromium picolinate-induced DNA damage in the peripheral lymphocytes or hepatocytes isolated from mice given a single intraperitoneal injection of 3 mg/kg b.wt. 16 h before sacrifice. Chromium picolinate did not affect the percentage of clouds on the slides.
The positive control cyclophosphamide increased the level of DNA damage both in the liver cells and in the lymphocytes, and this increase was observed without a concomitant increase in the number of clouds.

In vivo effect of chromium picolinate on the DNA damage migration in lymphocytes and hepatocytes 16hours after a single exposure:

Treatment	Doses (mg/Kg)	Lymphocytes			Hepatocytes
		Tail moment (mean +/- SEM)	Clouds (%)	(n)	Tail moment (mean +/- SEM)	Clouds (%)	(n)
Control (PBS)	0	58 +/- 0.9	1.4 +/- 0.3	24	18.6 +/- 3.3	3.1 +/- 1.0	24
Chromium picolinate	3	4.1 +/- 0.6	1.2 +/- 0.4	24	20.0 +/- 4.2	4.0 +/- 1.1	24
Cyclophosphamide	200	38.0 +/- 8.3 (P < 0.001)	0.7 +/- 03	12	50.2 +/- 14.3	2.2 +/- 06	12

Conclusions:

The potential genotoxicty of Chromium picolinate was investigated using the alkaline version of the Comet assay to evaluate primary DNA damage. There was no evidence of Chromium picolinate-induced DNA damage in the peripheral lymphocytes or hepatocytes isolated from mice given a single intraperitoneal injection of 3 mg/kg b.wt. 16 h before sacrifice.

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the potential genotoxicity of chromium picolinate in mammalian cells in vivo and in vitro

Author:

Andersson, A. et al.

Year:

2007

Bibliographic source:

Food and Chemical Toxicology 45 (2007), 1097-1106

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

one sampling time

GLP compliance:

not specified

Type of assay:

other: micronucleus assay to evaluate chromosomalt aberrations

Test material

Test material information

Constituent 1

Reference substance name:

Chromium (III) picolinate

Cas Number:

14639-25-9

Molecular formula:

C18H12CrN3O6

IUPAC Name:

Chromium (III) picolinate

Test material form:

not specified

Details on test material:

origin: ICN Biomedicals Inc. (Germany)

Test animals

Species:

mouse

Strain:

other: CBA/Ca

Sex:

male

Details on test animals or test system and environmental conditions:

weight at study initiation: around 20g
age at study initiation: 7-8 weeks
source: Scanbur BK - Sweden
water: free access to tap water
diet: free access to standardized rodent pellet diet (B&K Universal, batch 3386)
acclimatisation: at least 7 days before the start of the study

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Phosphate bufferd saline (PBS)

Details on exposure:

Chromium picolinate and the positive control (colchicine) were dissolved in phosphate buffered saline (PBS) and injected intraperitoneal as freshly prepared solutions (0.01 ml/g b.wt.). Control animals received the same volume of vehicle only.

Duration of treatment / exposure:

one single injection

Frequency of treatment:

one single injection

Post exposure period:

After 42 h, blood samples (50 ll/animal) were collected from the orbital plexus under light anaesthesia. This time point was chosen because it is known that it takes about 20 h before polychromatic erythrocytes formed in the bone marrow will appear in the blood.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.75 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1.5 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

3 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

vehcile: 4 male animals
chromium picolinate: 3 male animals per dose
positie control: 3 male animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Colchicine (CAS n° 64-86-8) was obtained from Sigma Aldrich (USA) and administered via intraperitoneal route at 1 mg/ Kg bw.

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes in the blood

Details of tissue and slide preparation:

Three parallel aliquots of 5 µL from each blood sample was layered on a 65% Percoll gradient (Amersham Pharmacia Biotech, Sweden) and centrifuged at 600 x g for 20 min. The erythrocytes collected in the pellet after the Percoll centrifugation were fixed in a solution of
glutaraldehyde and stored for 2–5 days at 4°C until further processed.
The glutaraldehyded fixed cells were stained in a PBS-buffer containing the fluorescent dyes Hoechst 33342 (Sigma/Aldrich, USA) and thiazole orange (Molecular probes, USA). After staining, the samples were analyzed on a FACSVantege SE flow cytometer (BD Immunocytometry systems, USA) equipped with an argon ion laser (Enterprise II, Coherent, USA).

Evaluation criteria:

The frequencies of immature (polychromatic) erythrocytes from peripheral blood with (fMNPCE) or without (fPCE) chromosomal material (micronuclei) was determined using a flow cytometer-based micronucleus assay. An increase in fMNPCE indicates genotoxicity; a decrease in fPCE indicates cytotoxicity.

Statistics:

The relative frequencies of the polychromatic erythrocytes with micronuclei and of the polychromatic erythrocytes without micronuclei were calculated and statistically analyzed using the 2-tailed Students t-test.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no evidence of clastogenic, aneugenic or cytotoxic effects in the erythrocytes isolated from whole blood from mice given a single
intraperitoneal injection of up to 3 mg/kg b.wt. of chromium picolinate, 42 h before sacrifice.
In contrast, the positive control colchicine (a well-established aneugen) increased the frequency of micronucleated polychromatic erythrocytes in peripheral blood.

Any other information on results incl. tables

In vivo effect of chromium picolinate on the frequency of polychromatic erythrocytes with micronuclei 42 h after a single exposure:

Treatment	Dose [mg/kg]	Frequency of MNPCE [‰] (mean ± SEM)	Frequency of PCE [%]
Control PBS)	0	1.00 ± 0.05	1.9 ± 0.4
Chromium picolinate	0.75	0.95 ± 0.08	2.4 ± 0.4
	1.5	0.94 ± 0.04	2.1 ± 0.3
	3.0	1.08 ± 0.13	2.2 ± 0.3
Colchicine	1.0	3.78 ± 0.46*	1.0 ± 0.4

MNPCE: polychromatic erythrocytes with micronuclei

PCE: polychromatic erythrocytes without micronuclei

* p < 0.001

Applicant's summary and conclusion

Conclusions:

The potential genotoxicty of Chromium picolinate was investigated using the flow cytomemeter-based micronucleus assay to evaluate chromosome aberrations. There was no evidence of clastogenic, aneugenic or cytotoxic effects in the erythrocytes isolated from whole blood from mice given a siingle injection of up to 3 mg/Kg bw 42 hours before sacrifice.