Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

The relative humidity ranged between 30-73 % instead of 30-70 %. The initial body weight of one males at start of treatment exceeded the upper limit of 45 g (45.4 g). These deviations do not affect the validity of the experiment.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronuclues Assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

EC Number:

255-527-0

EC Name:

Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Cas Number:

41741-86-0

Molecular formula:

C34 H24 Cr N8 O6 .Na

IUPAC Name:

Sodium;chromium(3+);2-[(3-methyl-5-oxido-1-phenylpyrazol-4-yl)diazenyl]benzoate

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 40.9g (SD ± 4.1 g) females mean value 26.3 g (SD ± 2.8 g)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: yes, 18h
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-73
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionised water

Duration of treatment / exposure:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively

Frequency of treatment:

once

Post exposure period:

The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test item.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

24 h sampling time

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

24 h sampling time

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

24 h sampling time

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

48 h sampling time

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with. At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

ruffled fur, eylid closure and reduction of spontaneus activity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Sumimary of Micronucleus Test Results

Test group	dose level (mg/kg bw)	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythocytes
vehicle	0	24	0.06	0-2	994
test item	500	24	0.09	0-5	974
test item	1000	24	0.045	0-2	1021
test item	2000	24	0.06	0-4	1040
positive control	40	24	1.01	12-32	1024
test item	200	48	0.095	0-4	1070

Applicant's summary and conclusion

Conclusions:

During the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 ml/kg body weight, 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg body weight. 48 h preparation interval: 2000 mg/kg body weight. The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg body weight. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.