2-(3-(4-amino-9,10-dihydro-3-sulpho-9,10-dioxoanthracen-4-yl)aminobenzenesulphonyl)vinyl) disodium sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

92/69/EWG, B.10 (Säuger zytogenetischer in vitro-Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-(3-(4-amino-9,10-dihydro-3-sulpho-9,10-dioxoanthracen-4-yl)aminobenzenesulphonyl)vinyl) disodium sulphate

EC Number:

219-949-9

EC Name:

2-(3-(4-amino-9,10-dihydro-3-sulpho-9,10-dioxoanthracen-4-yl)aminobenzenesulphonyl)vinyl) disodium sulphate

Cas Number:

2580-78-1

Molecular formula:

C22H18N2O11S3.2Na C22H18N2Na2O11S3

IUPAC Name:

disodium 1-amino-9,10-dioxo-4-[(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Blue 19

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver

Test concentrations with justification for top dose:

Preparation time 18 h:
with S9: 500, 1000, 2000 µg/mL
without S9: 125, 250, 500 µg/mL


Preparation time 6 and 28 h:
with S9: 2000 µg/mL
without S9: 500 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Analysis of metaphases
After the slides had been coded, 100 metaphases per experimental group were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22+/- 1 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: gap (G), break (8), fragment (F), minute (M), deletion (D), exchanges including intrachanges (Ex), dicentrics (Di), chromosome disintegration (CD) and ring formation (RI). In addition, metaphases with 5 and more aberrations were classified separately as multiple aberrations (rnA).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared with the results from the dose group and the positive control at each preparation time.

Evaluation criteria:

The evaluation of the results was performed as follows:
- The test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one
of the concentrations tested . The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven
by adequate biometry (Binomial statistic with Fisher's exact test).
- The test substance is classified as mutagenic if there is a reproducible concentration related increase in the aberration rate.
- The test substance is classified as not mutagenic when it tests negatively both with and without metabolic activation.

Statistics:

Fisher - Exact test

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
In a preliminary experiment the test substance was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 2000 µg/ml.
The cytotoxicity experiment proved that the test substance was toxic to the V79 cells in the absence of metabolic activation (S9-mix) from 500 µg/ml up to the limit of solubility of 2000 µg/ml. And in the presence of metabolic activation (S9-mix) no indication of toxicity was observed up to the limit of solubility.
On the basis of these results the preparation of chromosomes was done after 2 h treatment with 500 µg/ml at 6, 18 and 28 hand 18 hours after treatment with 125 and 250 µg/ml without S9-mix; as also with 2000 µg/ml at 6, 18 and 28 hours and18 hours after treatment with 500 and 1000 µg/ml with S9-mix.
A cytotoxic effect was observed as a decrease of the mitotic index in all dose groups at the preparation times 6 and 18 hours after treatment. No decrease of the mitotic index was observed at the preparation time 28 hours.

Mutagenicity
The test substance was assessed for its mutagenic potential in vitro in the chromosome-aberration-test with two independent cell cultures without metabolic activation and two independent cell cultures with metabolic activation.
No relevant reproducible enhancement of the chromosome aberration rate over the range of the negative control was found with any of the concentrations used, neither with, nor without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
The results lead to the conclusion that the test compound is not mutagenic in the chromosome aberration.

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be non-mutagenic in this chromosome aberration assay

Executive summary:

The test substance was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect without metabolic activation from 500 µg/ml up to the limit of solubility (2000 µg/ml). No cytotoxic effect was observed with metabolic activation up to the limit of solubility. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 µg/ml) were used.

For main experiment dose levels of 125, 250 and 500 µg/ml were used in the absence and 500, 1000 and 2000 µg/ml in the presence of 59-mix metabolic activation.

The test compound did not induce a significant increase in the number of chromosome aberrations at any preparation time and dose level of the test substance. But a cytotoxic effect of the compound was observed in the rnain experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome mutations (= aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.