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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:
1988-01-20 to 1988-05-05
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
no
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

Constituent 1
Chemical structure
Reference substance name:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
EC Number:
403-080-9
EC Name:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
Cas Number:
92484-48-5
Molecular formula:
C16 H17 N3 O4 S .Na
IUPAC Name:
sodium 3-(2H-1,2,3-benzotriazol-2-yl)-5-(butan-2-yl)-4-hydroxybenzene-1-sulfonate
Constituent 2
Reference substance name:
sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
IUPAC Name:
sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
Details on test material:
- Name of test material: sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate (FAT 75'309/A)
- Physical state: solid

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Without S9 mix:
7h: 110 µg/mL
18h: 8, 80, 110 µg/mL
28h: 110 µg/mL

With S9 mix:
7h: 130 µg/mL
18h: 10, 50, 120 µg/mL
28h: 130 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Seeding of the Cultures:
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes which contained microscopic slides ( 4 chambers per dish and test group). In each chamber 5 x 10E4 - 1 x 10E5 cells were given with regard to preparation time. The medium was MEM + 10 % FCS.

Treatment:
After 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, without S9 mix and with 20 µL/mL S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with "saline G". All incubations were done at 37 degree C in a humidified atmosphere with 4.5 % CO2.
Evaluation criteria:
The test article will be classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control. This can be confirmed by means of the non-parametric Mann-Whitney test.
A test article which produce no significant positive response at any test point will be regarded as non-mutagenic in this assay.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

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:
valid
Positive controls validity:
valid
Additional information on results:
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix after treatment with concentration of 110 µg/mL and 130 µg/mL, respectively, the colony forming ability was reduced to about 20 % as compared to the solvent controls. In the main experiment, at fixation intervals 7 and 18 hours, the mitotic index was reduced after treatment with the highest dose levels in the absence and presence of S9 mix, indicating that the test substance had cytotoxic properties. In the presence of S9 mix the test article increased significantly the frequency of cells with aberrations at any fixation interval. At fixation interval 18 h the enhancement of the aberration rates was dose dependent. Additionally, after treatment with 120 µg/mL (18 h) and 130 µg/mL (28 h) 3.25 % and 11.25 %, respectively, of the cells were carrying exchanges. In the absence of S9 mix only at fixation interval of 18 h a slight but significant enhancement of the aberration rate could be observed in the test group after treatment with 110 µg/mL. EMS (0.72 mg/mL) and CPA (1.40 µg/mL) were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.
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

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 V79 Chinese Hamster cell line.
Executive summary:

An in vitro chromosome aberration test was performed according to OECD Guideline 473 and EU method B.10. The test article sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese Hamster in vitro. 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:

7 h: 110 µg/mL

18 h: 8; 80; 110 µg/mL

28 h: 110.00 µg/mL

with S9 mix:

7 h: 130 µg/mL

18 h: 10; 50; 120 µg/mL

28 h: 130 µg/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 110 µg/mL (without S9 mix) and 130 µg/mL (with S9 mix) reduced clearly the plating efficiency of the V79 cells. THe highest concentrations caused a level of 18% survival (110 µg/mL; without S9 mix) and 67.1 % survival (130 µg/mL; with S9 mix). Also the mitotic index was reduced with the highest concentration at any fixation interval in the presence and absence of S9 mix. There were significant enhancements of cells with structural aberrations after treatment with the test article at any fixation interval with metabolic activation by S9 mix. Without metabolic activation only at fixation interval 18 h a significant enhancement of the aberration rate could be observed. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was concluded to be positive in the presence and absence of metabolic activation in the V79 cytogenetic assay.