Trisodium bis[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxynaphthalene-1-sulphonato(3-)]chromate(3-)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

other: 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

Remarks:

Study performed according to the GLP Principles. Justification for Read Across is detailed in the endpoint summary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02b

IUPAC Name:

Similar Substance 02b

Test animals

Species:

mouse

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: non-consanguinous OF-1 albino mice.
- Source: mice originating from an SPF colony (IFFA-CREDO, L'Arbresle, France).
- Weight at study initiation: 25 g
- Housing: animals were housed 5 of the same sex per cage in Makrolon type III cages.
- Diet: ad libitum, aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson/Orge, France.
- Water: ad libitum.
- Quarantine period: quarantine period of 1 week at test laboratory.

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

Mice receiving one intragastric intubation (1500 mg/kg) using 0.5 ml of a solution at 75 mg/ml per 25 g body weight.

Doses / concentrations

Remarks:

Doses / Concentrations:
1500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Five male and five female mice per group. The groups were: three test item-treated animals, one negative control and one positive control.

Control animals:

other: Negative control (distilled water): 0.5 ml - animals sacrificed at 44 hours

Positive control(s):

N,N',N"-triethylenethiophosphoramide
- Source: Lederle Laboratories Ltd.
- Concentration administered: 20 mg/kg
- Necropsy: sacrificed at 44 hours

Examinations

Tissues and cell types examined:

Mouse bone marrow smears.

Details of tissue and slide preparation:

Animals were sacrificed at regular intervals after treatment: one group at 20, one group at 44 and one group at 68 hours.

DETAILS OF SLIDE PREPARATION
After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe, and the cells were centrifuged at 120 x g for 5 minutes. The supernatant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed drying in air. The following day smears were stained with Giemsa (1:6 in water) and mounted after drying with a coverslip.

ESTIMATION OF NUMBERS OF MICRONUCLEI
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells). The latter are stained blue by Giemsa for around 24 h after the expulsion of the erythroblast nucleus: the colouration is probably due to traces of RNA remaining in these cells.

The proportion of polychromatic erythrocytes containing one or more micronuclei was compared with the total number of polychromatic erythrocytes, and statistical comparisons were made between these ratios for the different groups. A minimum of 500 polychromatic erythrocytes were counted per smear (two smears per animal).

In each smear an evaluation was made of the numbers of nucleated cells and the two types of erythrocytes (normochromatics and polychromatics) were counted up to a total of 2000 erythrocytes per animal. This was done in order to gain information on the mode of action of the test compound in bone marrow cells, and also to identify possible artifacts.
All slides were given coded labels and were microscopically analysed without knowledge of their treatment groups.

Statistics:

A complete statistical analysis using BMPD computer programme 7D was performed.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE FINDING
First a preliminary range finding test was carried out by treating three groups of three male mice at concentrations of 3000, 2000 and 1000 mg/kg, the dose of 3000 mg/kg being the limit of solubility of the compound. Only one death occurred at the dose of 3000 mg/kg and a mouse was in a poor state at the dose of 2000 mg/kg. Therefore a dose of 1500 mg/kg was chosen to perform the micronucleus test.

GENERAL APPEARANCE OF THE SMEARS
At low magnification of the microscope no noticeable differences in bone marrow nucleated cells were observed between animals treated with the test item and negative controls.
In the positive control group (Thio-TEPA) decreased numbers of bone marrow nucleated cells were noted.

TEST RESULTS
There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 1500 mg/kg compared to negative control animals.
In animals treated with positive control there was a statistically significant increased number of micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with the positive control.
The only difference for this ratio observed between groups is a decrease in animals receiving the test item after 68 h compared to those sacrificed after 20 h. There is no statistically significant difference between controls and animals treated with the test item.

Any other information on results incl. tables

NUMBERS OF OBSERVED MICRONUCLEI AND RATIO OF POLYCHROMATIC TO NORMOCHROMATIC ERYTHROCYTES.

The percentage of polychromatic erythrocytes carrying one or more micronuclei, compared with the total number of polychromatic erythrocytes, was as follows:

Group	Dose mg/kg	Time of sacrifice	Micronucleous erythrocytes (%)
			Mean	S.D.
Negative control	0	44	0.32	0.17
Test material	1500	20	0.38	0.15
Test material	1500	44	0.4	0.16
Test material	1500	68	0.24	0.14
Positive control	20	44	21.3	8.25

S.D. = Standard Deviation

The proportion of polychromatic erythrocytes compared with the total number of erythrocytes was as follows:

Group	Dose mg/kg	Time of sacrifice	Polychromatic erythrocytes (%)
			Mean	S.D.
Negative control	0	44	58.1	6.3
Test material	1500	20	62.5	5.3
Test material	1500	44	55.3	8.9
Test material	1500	68	47.5	11.2
Positive control	20	44	13.8	7.0

S.D. = Standard Deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
No mutagenic effect was observed in bone marrow smears.

Executive summary:

The test compound was assayed for mutagenicity using the micronucleus test (OECD and EEC protocols). The compound was administered orally to mice at a concentration of 1500 mg/kg of a solution at the maximal concentration of 75 mg/ml.

No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg showed pronounced evidence of mutagenicity 44 h after administration. Thio-TEPA shows that the strain of mouse used is sensitive to mutagens and that such products can be detected using the micronucleus test.

Conclusion

No mutagenic effect was observed in bone marrow smears.