5-[[4-chloro-6-[(3-sulphophenyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

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:

June 20th, 1988 to June 23th, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

5-[[4-chloro-6-[(3-sulphophenyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

EC Number:

279-015-1

EC Name:

5-[[4-chloro-6-[(3-sulphophenyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Cas Number:

78952-61-1

Molecular formula:

C27H22ClN7O16S5.xNa

IUPAC Name:

sodium 5-({4-chloro-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Red 198

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

Colchicine

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

200, 1100, 2000 µg/ml

Vehicle / solvent:

water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

Evaluation criteria:

-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's 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:

ambiguous

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

Additional information on results:

Solubility and toxicity
In a preliminary experiment Reactive Red 198 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 Reactive Red 198 was not toxic to the V79 cells in the absence of metabolic activation (S9-mix). Also 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 2000 µg/ml at 6, 18 and 28 h, and 18 hours after treatment additionally with 1100 and 200 µg/mL, both with and without S9-mix.

Mutagenicity
The test substance Reactive Red 198 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.
A reproducible enhancement in the number of phases with aberrations, but only inclusive gaps over the range of the solvent control was found at the concentration of 2000 µg/mL at 28h after treatment without metabolic activation (S9-mix). A statistical increase of aberrations inclusive and exclusive gaps was observed at the same dose level and preparation time. The significant enhancement of aberrations exclusive gaps is of no genotoxic relevance, because of the low level (=0%) in the solvent control. The types of aberrations in the 28h interval induced preliminary consisted of iso-breaks, iso-fragments, minutes and exchanges.

Applicant's summary and conclusion

Conclusions:

In conclusion Reactive Red 198 does not significantly induce clastogenic effects in V79 Chinese hamster cells in the presence and in the absence of a metabolic activation system, under the experimental conditions described.

Executive summary:

The test substance Reactive Red 198 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 no significant cytotoxic effect (reduction of plating efficiency) without metabolic activation up to the limit of solubility (2000 µg/mL). No cytotoxic effect was also 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 200, 1100, 2000 µg/mL were used, in the absence and in the presence of S9-mix metabolic activation.

The test compound induced a significant increase in the number of phases with aberrations inclusive gaps, and in the number of aberrations inclusive and exclusive gaps 28h after treatment with 2000 µg/mL without S9-mix. At the other dose levels the test compound did not induce a significant increase of phases with aberrations. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments.

Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Reactive Red 198 does indicate a slight mutagenic effect (=aberrations) in V79 Chinese hamster cell s in the absence of a metabolic activation system. This effect was considered as not sufficient to evaluate the compound as distinct genotoxic agent. In the presence of S9-mix no chromosome mutations were observed under the experimental conditions described.