4'-aminoazobenzene-4-sulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Data source

Reference

Reference Type:

secondary source

Title:

Gene mutation toxicity study of the test chemical

Author:

European Commission

Year:

2008

Bibliographic source:

SCCS

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD guideline 487 and 473

Version / remarks:

Draft

Principles of method if other than guideline:

In vitro mammalian chromosome aberration test was carried out using Chinese hamster V79 cells.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride

EC Number:

269-944-0

EC Name:

[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride

Cas Number:

68391-32-2

Molecular formula:

ClN5O3

IUPAC Name:

8-[(4-amino-3-nitrophenyl)diazenyl]-7-hydroxy-N,N,N-trimethylnaphthalen-2-aminium chloride

Constituent 2

Reference substance name:

(8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride

IUPAC Name:

(8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride

Test material form:

other: Dark brown powder

Details on test material:

- Name of test material : (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride
- Molecular formula : C19H20N5O3.Cl
- Molecular weight : 401.852 g/mol
- Smiles notation :[N+](c1cc(\N=N\c2c3cc([N+](C)(C)C)ccc3ccc2O)ccc1N)([O])=O.[ClH]
- InChl : 1S/C19H19N5O3.ClH/c1-24(2,3)14-7-4-12-5-9-18(25)19(15(12)11-14)22-21-13-6-8-16(20)17(10-13)23(26)27;/h4-11H,1-3H3,(H2-,20,21,22,25);1H
- Structural formula attached as image file (if other than submission substance): No data available
- Substance type: Organic
- Physical state: Solid
- Analytical purity: No data available
- Impurities (identity and concentrations): No data available
- Composition of test material, percentage of components: No data available
- Isomers composition: No data available
- Purity test date: No data available
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available
- Radiochemical purity (if radiolabelling): No data available
- Specific activity (if radiolabelling): No data available
- Locations of the label (if radiolabelling): No data available
- Expiration date of radiochemical substance (if radiolabelling): No data available
- Stability under test conditions: No data available
- Storage condition of test material: No data available
- Other: No data available

Method

Target gene:

No data available

Species / strain

Species / strain / cell type:

other: Chinese hamster V79 cells

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction from phenobarbital/βnaphthoflavone-induced rats was used as exogenous metabolic activation system.

Test concentrations with justification for top dose:

Experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix

Experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix

Experiment IB: 31.3, 62.5, and 125.0 without S9-mix

Experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix

Experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix

Experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix

Experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The test chemical was soluble in Deionised water

Controls

Untreated negative controls:

yes

Remarks:

as per OECD guideline

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

as per OECD guideline

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration:
Experiment I (without & with S9-mix): 4 hr
Experiment II (with S9-mix): 4 hr
Experiment II (without S9-mix): 20 hrs

- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: For assessment of cytotoxicity a XTT test was performed

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding.

Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture.

For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test.

Rationale for test conditions:

No data available

Evaluation criteria:

The cell line was observed for micronucleated cells

Statistics:

No data available

Results and discussion

Test results

Species / strain:

other: Chinese hamster V79 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, In experiment IIA, in the absence and the presence of S9-mix, a statistically significant increase in the number of micronucleated cells exceeding the range of the historical control data was observed at the highest doses (512.5 and 1025 μg/ml, respectively). These concentrations were strongly cytotoxic indicated as by cell numbers of 7.9% and 12.9% of control, respectively.

Due to the steep dose-toxicity curve a repeat experiment, designated experiment IIB, was performed with narrower dilution steps to prove if the genotoxicity observed could have been an artefact induced by general test item toxicity. In the absence of S9-mix, at a cytotoxic level of about 40% of control the number of micronucleated cells (2.05% and 2.00%) slightly exceeded the historical control data range (0.0 – 1.8% micronucleated cells). Therefore, the test item was regarded as non-genotoxic in the absence of metabolic activation.

In the presence of S9-mix, at cytotoxic test item levels and associated with precipitation from doses equal or exceeding 200 μg/ml, the number of micronucleated cells (2.68% and 2.33%) slightly exceeded the range of the historical control data (0.0 – 1.8% micronucleated cells). Due to the high value of the respective solvent control (1.80% micronucleated cells), these two slight increases have to be regarded as biologically irrelevant.

The observations of experiment IIA in the absence and the presence of metabolic activation were not confirmed in the repeat experiment IIB with narrower dilution steps. Therefore, it has to be considered that the findings in both parts of experiment IIA were artefacts induced by general test item toxicity.

Other: In all experiments clear toxic effects indicated by reduced cell numbers below 40% of control were observed at least at the highest concentrations scored after treatment with test chemical except in experiment IB in the absence of S9-mix. In experiment IA, in the absence of S9-mix, a statistically significant but non-dose-related increase in the rate of micronucleated cells was observed at the lowest and highest dose. The values of highest dose were at the laboratory’s control data range (0.0 – 1.8% micronucleated cells). Concerning the lowest dose, in the confirmatory experiment IB this finding was not confirmed. Consequently, the positive finding was considered not biologically relevant. In experiment IA, in the presence of S9-mix no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce an increase in micronucleated cells in the V79 cell line in the presence and absence of S9 metabolic activation system and thus, the test was considered to be negative.

Executive summary:

In vitro mammalian chromosome aberration test was carried out using Chinese hamster V79 cells to determine the mutagenic nature of the test chemical.

 

Different conc. of test chemical was used in the various experiment. They are-

Experiment IA:128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix

Experiment IA:128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix

Experiment IB:31.3, 62.5, and 125.0 without S9-mix

Experiment IIA:128.1, 256.3 and 512.5 μg/ml without S9-mix

Experiment IIA:128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix

Experiment IIB:100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix

Experiment IIB:100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix

 

Deionised water was used as a vehicle.

 

Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system.

 

A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test.

 

On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by Basic Brown 17 toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding.

 

The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture.

 

For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test.

 

Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells in the V79 cell line in the presence and absence of S9 metabolic activation system and thus, the test was considered to be negative.