Sodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[3-[(1-oxopropyl)amino]phenyl]amino]anthracene-2-sulphonate

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:

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

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[3-[(1-oxopropyl)amino]phenyl]amino]anthracene-2-sulphonate

EC Number:

289-550-2

EC Name:

Sodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[3-[(1-oxopropyl)amino]phenyl]amino]anthracene-2-sulphonate

Cas Number:

89923-62-6

Molecular formula:

C23H19N3O6S.Na

IUPAC Name:

sodium 1-amino-9,10-dioxo-4-[(3-propanamidophenyl)amino]-9,10-dihydroanthracene-2-sulfonate

Test material form:

other: solid

Details on test material:

None

Specific details on test material used for the study:

Name: FAT 20'242/B
Batch No.: FC-83/5T
Aggregate State at RT: Solid
Molecular Weight: 487.45
Purity: cf. Analytical Certificate in the sponsor file
Analysis: cf. Analytical Certificate in the sponsor file
Stability: Pure: stable for 24 months. In solvent: > 8 hours in H20, ethanol, acetone, DMSO and DMF
Storage: light protected
Expiration Date: March 1993

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of C C R allowing the repeated use of the same cell culture batch in experiments. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37°C in 80 cm2 plastic flasks (GREINER, D-7440 Nürtingen, F.R.G.). Seeding is done with about 5 x 10s cells per flask in 15 ml of MEM medium supplemented with 10 % fetal calf serum (FCS; SEROMED, D-1000 Berlin). The cells are subcultured twice weekly. The cell cultures are incubated at 37°C and 4.5 % carbon dioxide atmosphere.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from the liver of old male Wistar rats.

Test concentrations with justification for top dose:

1)Pre-experiment for toxicity:
without S9 mix:
7 h: 25; 50; 100; 150 µg/ml
18 h: 2.5; 10; 25; 50; 100; 150 µg/ml
28 h: 25; 50; 100; 150 µg/ml

with S9 mix:
7 h: 100; 200; 300; 400 µg/ml
18 h: 10; 50; 100; 200; 300; 400 µg/ml
28 h: 100; 200; 300; 400 µg/ml

2)Experiment:
Without S9 mix:
7 h: 50 µg/ml
18 h: 2.5; 25; 50 µg/ml
28 h: 150 µg/ml

With S9 mix:
7 h: 100 µg/ml
18 h: 10; 100; 200 µg/ml
28 h: 400 µg/ml

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in culture medium without fetal calf serum.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate: without metabolic activation; Cyclophosphamide: With metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plastic flask 25 cm3

NUMBER OF REPLICATIONS: 4 per dose

NUMBER OF CELLS EVALUATED: Determination of the mitotic index by scoring 1000 cells of each slide

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Analysis of metaphase cells:
Evaluation of the slides were performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.

A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Solvent control versus fixation interval S9 mix p-value

Test group 50.0 µg 7 h - n.t.
" 100 µg 7 h + n.t.
" 2.5 µg 18 h - n.t.
" 25 µg 18 h - n.t.
" 50 µg 18 h - n.t.
" 10 µg 18 h + n.t.
" 100 µg 18 h + n.t.
" 200 µg 18 h + 0.0147*
" 150 µg 28 h - 0.0000*
" 400 µg 28 h + 0.0000*

n.t. = Not tested
* Aberration rate is statistically significant higher than the control rate.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None

Any other information on results incl. tables

Dose Selection:

In the pre-experiment for 'toxicity the colony forming ability of the V79 cells was reduced to about 20% after treatment with 150.0 and 400.0 µg/ml*, respectively, in the absence and presence of S9 mix.

In the main experiment, in the absence of S9 mix at fixation intervals 7 and 18 h with concentrations higher than 50 µg/ml no metaphases could be found. At fixation interval 28 h cells after treatment with 150 µg/ml as maximum concentration were evaluated. In the presence of S9 mix after treatment with concentrations higher than 100 µg/ml (fixation interval 7 h) and 200 µg/ml (fixation interval 18 h) not enough scorable cells could be found. But at fixation interval 28 h. 400 µg/ml as maximum dose level could be evaluated for cytogenetic damage.

With the highest dose level applied in the absence and presence of S9 mix the mitotic index was clearly suppressed.

Applicant's summary and conclusion

Conclusions:

FAT 20242/B induced structural chromosome aberrations in the V79 Chinese Hamster cell line.

Executive summary:

The test article FAT 20'242/B was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese Hamster in vitro in accordance with OECD Guideline 473.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated:

without S9 mix: with S9 mix:

7 h

18 h

28 h

50.0 ug/ml 7 h: 100.0 ug/ml

2.5; 25.0; 50.0 ug/ml 18 h: 10.0; 100.0; 200.0 ug/ml

150.0 ug/ml 28 h: 400.0 ug/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment of the cells with 150.0 and 400.0 ug/ml, respectively, reduced clearly the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment with the highest concentrations at all fixation intervals in the presence and absence of S9 mix.

There were relevant enhancements of cells with structural aberrations after treatment with the highest dose levels at fixation intervals 18 and 28 h with and without metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

CONCLUSION:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V7 9 Chinese Hamster cell line. Therefore, FAT 20'242/B is considered to be clastogenic in this chromosomal aberration test.