Disodium [N-(2-chlorophenyl)-2-[(2-hydroxy-5-nitrophenyl)azo]-3-oxobutyramidato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 22 July 1994 to 24 January 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, Annex (November 18, 1987) Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [N-(2-chlorophenyl)-2-[(2-hydroxy-5-nitrophenyl)azo]-3-oxobutyramidato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

EC Number:

276-649-0

EC Name:

Disodium [N-(2-chlorophenyl)-2-[(2-hydroxy-5-nitrophenyl)azo]-3-oxobutyramidato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Cas Number:

72403-66-8

Molecular formula:

C36H21ClCrN7Na2O12S

IUPAC Name:

disodium [N-(2-chlorophenyl)-2-[(2-hydroxy-5-nitrophenyl)azo]-3-oxobutyramidato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Test material form:

other: solid

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (hprt)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F 10 medium supplemented with 10% foetal calf serum and antibiotics
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

CYTOXICITY TEST
Range with metabolic activation:
500.0000 µg/ml
250.0000 µg/ml
125.0000 µg/ml
62.5000 µg/ml
31.2500 µg/ml
15.6250 µg/ml
7.8125 µg/ml
3.9063 µg/ml
1. 9531 µg/ml
0.9766 µg/ml
0.4883 µg/ml
0.2441 µg/ml

Range without metabolic activation:
500.0000 µg/ml
250.0000 µg/ml
125.0000 µg/ml
62.5000 µg/ml
31.2500 µg/ml
15.6250 µg/ml
7.8125 µg/ml
3.9063 µg/ml
1. 9531 µg/ml
0.9766 µg/ml
0.4883 µg/ml
0.2441 µg/ml

MUTAGENICITY TEST
Original experiment:
Range with metabolic activation:l
250.0000 µg/ml
83.3333 µg/ml
27.7778 µg/ml
9.2593 µg/ml

Range without metabolic activation:
100.0000 µg/ml
33.3333 µg/ml
11.1111 µg/ml
3.7037 µg/ml

Confirmatory experiment:
Range with metabolic activation:
300.0000 µg/ml
100.0000 µg/ml
33.3333 µg/ml
11.1111 µg/ml

Range without metabolic activation:
150.0000 µg/ml
50.0000 µg/ml
16.6667 µg/ml
5.5556 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

N-dimethylnitrosamine with metabolic activation; ethylmethanesulphonate without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours
- Expression time (cells in growth medium): from 7 to 8 days

SELECTION AGENT (mutation assays):
- 6-thioguanine

NUMBER OF REPLICATIONS:
- cultures were treated in duplicate with four test chemical concentrations, a positive and a negative control

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines: ARLETT, C.F., D.M. SMITH, M.H.L. Green, D.B. McGREGOR, G.M. CLARKE, J. COLE, J.C. ASQUITH (1990) Mammalian cell gene mutation assays based upon colony formation. In: Statistical Evaluation of Mutagenicity Test Data (ed Kirkland, D.J.) Cambridge University press, pp 67-101.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test with and without metabolic activation 12 concentrations of substance were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated by 2-fold intervals.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was seen during original experiment performed with and without metabolic activation.
No toxicity was seen during confirmatory experiment performed with and without metabolic activation.

Remarks on result:

other: strain/cell type: Chinese Hamster cell line V79

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Non mutagenic

Executive summary:

Method

The experiment was conducted in agreement to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test), EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture) and EEC Directive 87/302, Annex (November 18, 1987) Part B ( Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test).

The test was performed following the GLP.

The test article was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in DMSO.

A preliminary range finding test was run assessing cytotoxicity. The substance was tested at concentrations up to 500.0 µg/ml. Higher concentration could not be applied due to solubility limitations in the vehicle. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested. Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment.

Depending on the toxicity of the test compound 2.5-5.0x10E6 cells of passage 27 (original experiment) and passage 31 (confirmatory experiment) were plated in 30 ml growth medium into flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone.

In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 µl/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 µl/ml.

Cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).

The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype. At the end of the expression period the cultures were trypsinised pelleted, resuspended in fresh growth medium and counted.

The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 µg/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control).

After seven to eight days incubation at 37°C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.

In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The number of colonies which developed within these cultures reflected the viability at the end ofthe expression period (viability values).

Results

According to the acceptance criteria outlined in the above mentioned guidelines, in the presence and absence of metabolic activation, no relevant increase in mutant frequency was observed at any concentration level of substance tested in the original or the confirmatory experiment in comparison with the negative control.

In the two experiments performed in the presence of metabolic activation, statistically significant differences were obtained at two concentrations each. However, at none of these concentrations the number of normalized mutant clones differed by more than 20 compared to the respective control cultures.

Moreover, no concentration dependency was obvious and the effects were not reproducible (appeared at different concentrations in the two experiments). They are therefore considered to be purely fortuitous and not related to treatment with the test compound.

The positive controls induced a clear increase in mutant frequency.

Conclusion

Non mutagenic.