Reaction products of diazotized 2-amino-4-nitrophenol, coupled with resorcinol and subsequently coupled with diazotized 5-amino-4-hydroxynaphthalene-2,7-disulfonic acid and diazotiazed paranitroaniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

Since January 27, 1992 to May 29, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant with international guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

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:

Similar substance 2

IUPAC Name:

Similar substance 2

Method

Target gene:

no data

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

without S9 mix:               
0.30; 1.00; 2.50 mg/ml                 
with S9 mix:
0.10; 0.30; 1.00 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 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: MEM medium supplemented with 10 % fetal calf serum

DURATION
- Exposure duration: 18h, 28h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 5 x 10^4 - 1x 10^5

Evaluation criteria:

A test article is classified as mutagenic if it induces either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a concentration-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:

The test article precipitated in the culture medium (macroscopically visible) with concentrations of 1.00 mg/ml and higher (with and without S9 mix). A precipitation (microscopically visible) could be observed at concentrations of 0.10 mg/ml and higher (fixation interval 18 h, with and without S9 mix) and did not allow appropriate evaluating of metaphases after treatment with the highest concentration used (5.00 mg/ml). At fixation interval 28 h precipitation was found at concentrations of 0.30 mg/ml and higher (without S9 mix) and 1.00 mg/ml and higher (with S9 mix).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, the substance is considered to be non-mutagenic in this chromosomal aberration test.

Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Preparation of chromosomes was done 18 h (low, medium and high concentration rate), and 28 h (high concentration rate) after start of treatment with the test article which was dissolved in distilled water. The treatment interval was 4 h.

In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control culture where 25 metaphases were scored. The following concentrations were evaluated:

without S9 mix:               

18 h: 0.30; 1.00; 2.50 mg/ml        

28 h: 2.50 mg/ml             

with S9 mix:

18 h: 0.10; 0.30; 1.00 mg/ml

28 h: 1.00 mg/ml

 

As requested by the sponsor and according to recommendations in several guidelines the highest concentration of the test article administered was 5.00 mg/ml. A plating efficiency assay as indicator for toxicity response was performed in a former study of CCR (Project no: 131523).

A precipitation of the test article occured from concentrations of 0.10 mg/ml and did not allow appropriate evaluation of the highest concentration used (5.00 mg/ml). In the absence of S9 mix the mitotic index was reduced at both fixation intervals at the highest concentration scored (2.50 mg/ml). In presence of S9 mix at fixation interval 18 h a concentration dependend decrease of mitotic index was found at concentrations higher than 0.10 mg/ml. A steep increase in toxicity of the test article in the concentration range 1.00 - 2.50 mg/ml at both fixation intervals and in presence of S9 mix prevented the evaluation of cultures treated with concentrations higher than 1.00 mg/ml. There was no relevant increase in cells with structural aberrations after treatment with the test article at each fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.