Calcium 4,5-dichloro-2-[[4,5-dihydro-3-methyl-5-oxo-1-(3-sulphonatophenyl)-1H-pyrazol-4-yl]azo]benzenesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read across substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,5-dichloro-2-[[4,5-dihydro-3-methyl-5-oxo-1-(3-sulphonatophenyl)-1H-pyrazol-4-yl]azo]benzenesulphonate

EC Number:

265-633-9

EC Name:

Disodium 4,5-dichloro-2-[[4,5-dihydro-3-methyl-5-oxo-1-(3-sulphonatophenyl)-1H-pyrazol-4-yl]azo]benzenesulphonate

Cas Number:

65212-76-2

Molecular formula:

C16H12Cl2N4O7S2.2Na

IUPAC Name:

disodium 4,5-dichloro-2-{[3-methyl-5-oxo-1-(3-sulfonatophenyl)-4,5-dihydro-1H-pyrazol-4-yl]diazenyl}benzenesulfonate

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S-9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

1st experiment:
4 hours exposure, 18 hours harvest time, without S-9 mix - 0; 62.5; 125; 250; 500 µg/ml;
4 hours exposure, 18 hours harvest time, with S-9 mix 0; 62.5; 125; 250; 500 µg/ml.
2nd experiment: For clarification/substantiation of the results of the 1st experiment, the following doses were analyzed:
4 hours exposure, 18 hours harvest time, with S-9 mix 0; 25; 50; 75; 100; 125; 150 µg/ml.

Vehicle / solvent:

Due to the limited solubility af the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Without metabolic activation, 350 µg ethyl methanesulfonate/mI culture medium added in a volume of 1.0 ml; with metabolic activation (S-9 mix), 0.5 µg cyclophosphamide/ml culture medium added in a volume of 1.0 ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 14 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg Colcemid/ml culture medium
STAIN (for cytogenetic assays): solution of Giemsa and Titrisol

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative cell growth (cell counts)

OTHER EXAMINATIONS:
- Determination of polyploidy: Changes in the number of ohromosomes by whole chromosome sets. A special form of polyploid cells are endoreduplications.
- Other: Aneuploidy (Metaphases with absent (hypoploid) or additional (hyperploid) chromosomes); Endopolyploidy (Tetraploid metaphases with diplo-chromosomes [products of endomitotic chromosome reduplication])

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible significant increase in the number of structural chromosomal aberrations. The proportion of aberrations exceeded both the concurrent negative oontrol range and the negative historical control range.
A test substance is generally considered nonclastogenic in this test system if: There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies. The aberration frequencies were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF AG).
The proportion of metaphases with aberrations was calculated for each group.
A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equai proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are significant, labeis (* p <0 0.5, ** p < 0.01) are printed in the tables.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

According to the results of the determination of the mitotic index, a suppression of the mitotic activity was observed after the addition of a metabolizing system from about 50 µ/ml onward. However, a dose-dependency was not demonstrated.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1st experiment:
After an exposure time of 4 hours without metabolic activation and a harvest time of 18 hours, clastogenicity was not observed.
After the addition of S-9 mix, an increase in structurally damaged metaphases was demonstrated at the two lowest doses of 62.5 and 125.0 µg/mI. In the higher dose groups, i.e. 250.0 and 500.0 µg/mI, the number of aberrant metaphases was within the historical negative control range.

2nd experiment:
For further clarification/substantiation of these findings a 2nd experiment was carried out under the same experimental conditions, but selecting closer doses and analyzing additional dose levels to check the dose-response.
According to the findings of this study, there was a slight increase in the number of chromosomally damaged cells from about 50 µg/ml onward with a maximum response at about 75 -100 µg/ml and a slight decrease at 125.0 - 150.0 µg/ml.
Even if statisticaily not significant in all cases, the values for aberrant metaphases excl. gaps and/or for cells containing exchanges exceeds not only the range of the concurrent negative control but also the range of the historical negative control data base.
The decrease in the aberration rate observed at doses > 125.0 µg/ml may be explained by a limited or reduced availibility of the test substance in the culture due to increasing precipitation in the higher dose groups.

Any other information on results incl. tables

After an exposure time of 4 hours and a harvest time of 18 hours, the test substance led to a biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps with an increased amount of exchanges after the addition of a metabolizing system in two experiments performed independently of each other. Therefore, a further experiment including additional exposure and sampling times, as required by current guidelines, was not examined. No increase in the number of cells containing numerical chromosomal aberrations was demonstrated. The increase in the frequencies of chromosomal aberrations induced by the positive control agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test substance is a chromosome-damaging (clastogenic) agent under in vitro conditions using V79 cells.