Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate

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:

key study

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:

Data published, GLP not specified

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Chromium (III) propionate

EC Number:

919-722-0

Cas Number:

85561-43-9

Molecular formula:

C9H15CrO6

IUPAC Name:

Chromium (III) propionate

Constituent 2

Reference substance name:

nitrate

Cas Number:

14797-55-8

Molecular formula:

NO3-

IUPAC Name:

nitrate

Specific details on test material used for the study:

Chromium (III) propionate cation (CrProp) in the form of its nitrate salt (chemical formula [Cr3O(O2CCH2CH3)6(H2O)3]+(NO3)- was synthesized in the laboratory of Department of Product Ecology, Poznan University of Economics. The contents of elemental Cr (20.5%) was determined by the AAS method (spectrometer AAS-3 with BC correction, Zeiss, Germany).

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Licensed Laboratory Animals Breeding Center (Poznan, Poland)
- Age at study initiation: 12 weeks
- Weight at study initiation: ca. 196 g
- Assigned to test groups randomly: no, according to similar mean body mass
- Housing: single cages
- Diet (e.g. ad libitum): ad libitum (Labofeed H)
- Water (e.g. ad libitum): ad libitum (deionized water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 55 - 60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

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

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): commercial diet for maintenance of adult rodents (Labofeed H)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

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 other: mg Cr(III)/kg diet

Remarks:

Cr(III) 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

Control animals:

yes, plain diet

Positive control(s):

Cr(VI) as K2Cr2O7 at a dose of 10 mg Cr(VI)/kg diet

Examinations

Tissues and cell types examined:

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. Rat peripheral blood lymphocytes (PBL) were obtained. The liver, kidneys, heart, spleen, pancreas and ovaries were harvested and weighed.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 4 week exposure via food, sampling after 12 h starvation period

DETAILS OF SLIDE PREPARATION: Rat peripheral blood lymphocytes (PBL) were separated by the standard method. The cells were suspended in the RPMI 1640 medium without L-glutamine and centrifuged over Gradisol L at 1200 rpm for 15 min. Next, centrifugation was performed twice at 700 rpm for 8 min. The PBL suspension (30 µl) was mixed with 70 µl of 1% low melting point agarose in the RPMI 1640 medium at 37 °C. The mixture was pipetted onto microscope slides previously pre-coated with
a layer of 1% normal agarose. The slides were immersed in lysis solution (2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris, 1% of freshly added Triton X-100, pH 10) for 1 h to remove proteins. Slides were prepared in duplicate.

METHOD OF ANALYSIS: The slides were then placed in a horizontal electrophoretic tank in cold buffer (4°C, 3 M NaOH, 1 mM Na2EDTA, pH 13) for 40 min to allow DNA unwinding. The electrophoresis was carried out in the same solution for 30 min (at 300 mA, 0.56 V/cm). Afterwards electrophoresis slides were removed from the tank, immersed in neutralization buffer (0.4 M Tris, pH 7.5), and stained with DAPI (2 µg/mL in distilled water).

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 examined

Positive controls validity:

valid

Additional information on results:

Average feed intake was similar across all 3 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 lower in the Cr(VI) group compared to the control and CrProp groups but not significantly so. Results are presented in Table 1.
The Cr(VI) 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.
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 (Table 1).

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:

Chromium(III) propionate complex was not genotoxic in the comet assay.

Executive summary:

The genotoxicity of a chromium(III) propionate complex was assessed in rat peripheral blood lymphocytes using the comet assay. Female adult Wistar rats (6 per group) were administered Cr(III) for 4 weeks in standard diet, provided ad libitum. Cr(III) was given at a dose of 1000 mg Cr(III)/kg diet (as [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]NO₃equivalent to 100 mg Cr/kg bw/day). Cr(III) treatment did not affect body weight gain, feeding efficiency or organ weights as compared to rats fed standard diet alone. Cr(III) treatment did not produce a positive result in the comet assay with lymphocytes, suggesting the compound is not genotoxic in rats.