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Toxicological information

Genetic toxicity: in vitro

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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:
1996
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (no. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(2-hydroxyethyl)ammonium 7-{4-[4-(2-cyanoamino-4-hydroxy-6-oxidopyrimidin-5-ylazo)benzamido]-2-ethoxy-phenylazo}naphthalene-1,3-disulfonate
EC Number:
421-440-3
EC Name:
Tris(2-hydroxyethyl)ammonium 7-{4-[4-(2-cyanoamino-4-hydroxy-6-oxidopyrimidin-5-ylazo)benzamido]-2-ethoxy-phenylazo}naphthalene-1,3-disulfonate
Cas Number:
778583-04-3
Molecular formula:
Hill formula: C47 H66 N12 O19 S2 CAS formula: C29 H21 N9 O10 S2 x 3 C6 H15 NO3
IUPAC Name:
tris(tris(2-hydroxyethyl)azanium) 2-(cyanoamino)-5-{2-[4-({4-[2-(6,8-disulfonaphthalen-2-yl)diazen-1-yl]-2-methoxyphenyl}carbamoyl)phenyl]diazen-1-yl}-6-hydroxypyrimidin-4-olate

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium) supplemented with 10 % fetal calf serum (FCS).
- Properly maintained: yes (Cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 4.5
% carbon dioxide (95.5 % air))
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Concentration range in the main test (experiment I, with metabolic activation): 10-1000 µg/ml
Concentration range in the main test (experiment II, with metabolic activation): 10-3000 µg/ml
Concentration range in the main test (experiment I, without metabolic activation): 3-30 µg/ml
Concentration range in the main test (experiment II, without metabolic activation): 10-50 µg/ml
Vehicle / solvent:
- Deionised water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate, EMS (without metabolic activation); Cyclophosphamide, CPA (with metabolic activation)
Details on test system and experimental conditions:
- Exposure period (with metabolic activation): 4 hours - Exposure period (without metabolic activation): 18 and 28 hours - Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours
Evaluation criteria:
A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance should be considered together.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations: - Precipitation occured 4 hours after treatment at concentrations >= 100 µg/ml with S9-mix and at >= 30 µg/ml without 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):
positive with metabolic activation
negative without metabolic activation

As determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed.
Therefore, the substance is considered to be mutagenic in this chromosome aberration test.
Executive summary:

The substance, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 473, adopted May 26, 1983, „ In vitro Mammalian Cytogenetic Test" and EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29,1992.

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations.

Substantial reductions of the mitotic indices were observed in the absence of S9 mix, in experiment I after treatment with 10 and 30 µg/ml at preparation interval 18 hours. In the presence of S9 mix the mitotic indices were reduced after treatment with the highest evaluated concentrations (1000 µg/ml in experiment I and 3000 μg/ml in experiment II). In the presence of S9 mix, there were biologically relevant and statistically significant increases in cells carrying structural chromosome aberrations after treatment with 1000 μg/ml of the test article at fixation interval 28 h in experiment I as well as after treatment with 3000 μg/ml (18 h and 28 h fixation interval) in experiment II.

In the absence of S9 mix, no biologically relevant increase in cells carrying structural chromosome aberrations was observed.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells carrying structural chromosome aberrations.

In conclusion, as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the registration substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed.

Therefore, the substance is considered to be mutagenic in this chromosome aberration test.