Chromium (III) oxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity assessment of chromium(III) propionate complex in the rat model using the comet assay

Author:

Staniek, H., Kostrzewska-Poczekaj, M., Arndt, M., Szyfter, K. & Krejpcio, Z.

Year:

2010

Bibliographic source:

Food and Chemical Toxicology 48: 89 - 92.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was designed to assess the genotoxicty of the test substance in rat peripheral blood lymphocytes using the comet assay

GLP compliance:

not specified

Remarks:

not specified in the publication

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

chromium cation triaqua-µ3-oxo-µ-hexapropionatotrichromium(III)

IUPAC Name:

chromium cation triaqua-µ3-oxo-µ-hexapropionatotrichromium(III)

Test material form:

not specified

Details on test material:

The test substance was Chromium(III) propionate cation (CrProp; [Cr3O(O2CCH2CH3)6(H2O)3]+(NO3)-) in the form of its nitrate salt. The chemical was synthesised in the laboratory of Poznan University of Economics according to the method of Earnshaw et al (1966)*.

*Reference:
- Earnshaw, A., Figgis, B.N., Lewis, J., 1966. Chemistry of polynuclear compounds. Part VI. Magnetic properties of trimeric chromium and iron carboxylates. Journal of Chemistry Society A: Inorganic Physical Theoretical, 1656–1663.

Specific details on test material used for the study:

not specified

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

not specified

Sex:

female

Details on test animals or test system and environmental conditions:

12 week old female Wistar rats, obtained from the Licensed Laboratory Animals Breeding Centre (Poland). The rats were divided into 3 groups of 6 animals so that the mean body weight was similar in each group (~196g per animal). Rats were fed 'Labofeed H' ad libitum, and deionised water was provided ad libitum. The rats were housed in single cages, temperature was maintained at 19-22°C, ambient humidity was 55-60%, and artificial light was provided on a 12 hour light/dark cycle.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

DIET PREPARATION
The test substance was administered in a commercial diet for maintenance of adult rodents (Labofeed H). The diet was available ad libitum.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

ad libitum

Post exposure period:

No post exposure period; rats were sacrificed 12 hours after the end of the exposure period.

Doses / concentrations

Dose / conc.:

1 000 mg/kg diet

Remarks:

dose indicates mg Cr(III)/kg diet (given as [Cr3O(O2CCH2CH3)6(H2O)3]NO3, equivalent of 100 mg Cr/ kg body mass/day)

No. of animals per sex per dose:

Six female rats per dose

Control animals:

yes, plain diet

Positive control(s):

Cr(VI) as K2Cr2O7 (35.35 %Cr content) at a dose of 10 mg Cr(VI)/kg diet (equivalent of 1 mg Cr/kg body mass/day)
- Route of administration: oral: feed

Examinations

Tissues and cell types examined:

Slides were examined with an Axiophot fluorescence microscope (Opton, Germany) with IMAC-CCD S30 camera and ISIS 3 v 2.00 image analysis system (Meta-
Systems Hard- and Software, Altlussheim, Germany). The spontaneous strand breaks were measured as total comet length (increase in DNA migration). Average values were calculated for 50 comets per slide.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES / DETAILS OF SLIDE PREPARATION / METHOD OF ANALYSIS: :
At the end of the study after a 12 hour starvation period, rats were sacrifced by carbon dioxide asphyxiation, blood was collected into Li-heparinised tubes. The liver, kidneys, heart, spleen, pancreas and ovaries were harvested and weighed.
Rat peripheral blood lymphocytes were obtained from 10 individuals. The comet assay was conducted as follows: the cells were separated and suspended in RPMI 1640 medium without L-glutamine and centrifuged over Gradisol L at 1200 rpm for 15 minutes. Centrifugation was then performed twice at 700 rpm for 8 minutes. The suspension (30 µL) was mixed with 70 µL of 1% low melting point agarose in RPMI 1640 medium at 37°C. The mixture was pipetted onto microscope slides pre-coated with a layer of 1% normal agarose. The slides were immersed in lysi solution (2.5 M NaCl, 0.1 M Na2EDTA, 10mM Tris, 1% freshly added Triton X-100, pH 10) for 1 hour to remove proteins. The slides were then placed in a horizontal electrophoretic tank in cold buffer (4°C, 3 M NaOH, 1mM Na2EDTA, pH 13) for 40 minutes to allow DNA unwinding. The electrophoresis was carried out in the same solution for 30 minutes (at 300 mA, 0.56 V/cm). Slides were removed from the tank, immersed in neutralisation buffer (0.4M Tris, pH 7.5) and stained with DAPI (2 µg/mL in distilled water). Slides were prepared in duplicate.

OBSERVATIONS:
- feed intake was measured daily
- body mass gains were monitored weekly

Evaluation criteria:

Slides were examined with an Axiophot fluoresence microscope. The spontaneous strand breaks were measured as total comet length (increase in DNA migration). Average values were calculated for 50 comets per slide.

Statistics:

One-way ANOVA and Tukey's t-test.

Results and discussion

Test results

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY:
The mean comet length obtained from lymphocytes of rats exposed to Cr(VI) was significantly longer (by 27%) than the control group and the CrProp group.
Please also refer to the field "Any other information on results incl. tables" below.

OBSERVATIONS (findings of test item and positive control groups):
Average feed intake was similar across all experimental groups. Body weight gain was significantly lower (by 30%) in the Cr(VI) group compared to the CrProp and control groups. Feeding efficiency ratio (body weight gain (g) per 100 g diet) was insignificantly lower in the Cr(VI) group compared to the control and CrProp groups.
The Cr(VI)-treated rats had significantly lower spleen and pancreas weight (by 30.6% and 54.5%, respectively), increased heart weight (by 65.2%) as compared to the control group. CrProp did not affect organ weights.
There were no signs of toxicity with CrProp at a dose of 1000 mg Cr/kg diet.
Histological analyses did not show deleterious changes in liver and kidney tissue.
Please also refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1. Mean comet length and nutritional indices in rats fed Cr(III) or Cr(VI) in the diet for 4 weeks.

Index		Control group	Positive control Cr(VI) group	Test group Cr(III)
Comet length	Mean ± SD	57.76±0.51^a	73.50±2.19^b	59.08±1.09^a
	Median	57.25	73.92	58.58
Feed intake (g/day/rat)	Mean ± SD	17.6±0.5	17.7±0.63	18.5±0.5
Body weight gain (g/28 days)	Mean ± SD	9.5±3.0^b	7.5±2.7^a	107±2.8^b
Feeding efficiency ratio (g/bw/100 g of diet)	Mean ± SD	1.90±0.97	1.51±0.92	2.11±0.69

Letter subscripts indicate significance at p < 0.05.

Applicant's summary and conclusion

Conclusions:

CrProp (Cr(III)) was not genotoxic in the comet assay.