Reaction mass of 6-(2-ethyl-6-methylanilino)-5-(4-{4-[ethyl(3-sulfobenzyl) amino] phenylazo} phenylazo)-4-hydroxynaphthalene-2-sulfonic acid, sodium and/or disodium salt(s) and 6-(2-ethyl-6-methylanilino)-5-(4-{4-[ethyl(4-sulfobenzyl) amino] phenylazo} phenylazo)-4-hydroxynaphthalene-2-sulfonic acid, sodium and/or disodium salt(s)

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Acid Navy RN-2682

IUPAC Name:

Acid Navy RN-2682

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 93.8 ... 3000 µg/ml
Concentration range in the main test (with metabolic activation): 46.9 ... 1500 µg/ml
Concentration range in the main test (without metabolic activation): 93.8 ... 3000 µg/ml
Concentration range in the main test (without metabolic activation): 11.7 ... 1500 µg/ml

Vehicle / solvent:

Deionised water.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - sqrt10. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used:
in a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/ slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. The cells were stained 24 hrs after start of treatment. Using a 400-fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cell number of the treatment groups is given as % cells in relation to the control.

Dose selection
The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/ml, 5 µL/ml or 10 mM,
whichever is the lowest, if formulation in an appropriate solvent is possible.
In the pre-test, 7150 µg/ml (similar active ingredient, purity: approx. 70 %) was applied as top concentration for treatment of the cultures. Test item concentrations between 55.9 and 7150 µg/ml (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 1787.5 µg/ml and above in the absence of S9 mix.
In addition, 4 hrs treatment with 893.8 µg/ml and above in the presence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 3000 µg/ml (with and without S9 mix) was chosen as top concentration in Experiment I.
Dose selection of Experiment II was also influenced by test item toxicity. In the range finding experiment, clearly reduced cell numbers were observed after 24 hrs exposure with 446.9 µg/ml and above. Therefore, 1500 µg/ml was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix, 1500 µg/ml was chosen as top treatment concentration with respect to the results obtained in Experiment I. Due to a technical error, the experimental part with S9 mix was repeated with the same top test item concentration.

Doses applied without S9 mix:
preparation interval exposure period exp. conc. in µg/ml
18 h 4 h I 93.8 - 3000
18 h 18 h II 11.7 - 1500
28 h 28 h II 11.7 - 1500

Doses appplied with S9 mix:
preparation interval exposure period exp. conc. in µg/ml
18 h 4 h I 93.8 - 3000
28 h 4 h II (repeated) 46.9 - 1500
28 h 4 h II 46.9 - 1500 (precipitation seen 4 h after start of treatment at 375, 750, 1500)

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 1787.5 µg/ml and above in the absence of S9 mix. In addition, 4 hrs treatment with 893.8 µg/ml and above in the presence of S9 mix induced strong toxic effects.
Considering the toxicity data of the pre-test, 3000 µg/ml (with and without S9 mix) was chosen as top concentration in Experiment I.

Dose selection of Experiment II was also influenced by test item toxicity. In the range finding experiment, clearly reduced cell numbers were observed after 24 hrs exposure with 446.9 µg/ml and above. Therefore, 1500 µg/ml was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix, 1500 µg/ml was chosen as top treatment concentration with respect to the results obtained in Experiment I. Due to a technical error, the experimental part with S9 mix was repeated with the same top test item concentration.

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II, in the presence of S9 mix, two statistically significant increases in the number of aberrant cells, excluding gaps were observed after treatment with 93.8 and 187.5 µg/ml (1.5 % and 4.0 %, respectively). Since both values were clearly within the range of laboratory historical control data (0.0 - 4.0 % aberrant cells, excluding gaps), this observation has to be regarded as biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Applicant's summary and conclusion

Conclusions:

Negative with and without metabolic activation.