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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:
April 02, 1990 - November 08, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline 473 study; GLP study. In all experiments cells were treated for 4 hours, no continuous treatment was performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene
EC Number:
408-210-8
EC Name:
Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene
Cas Number:
124605-82-9
Molecular formula:
C33 H26 N8 O14 S4 .x Li .x Na
IUPAC Name:
dilithium(1+) disodium 5-amino-3-{2-[4-(2-{4-[2-(8-amino-1-hydroxy-3,6-disulfonaphthalen-2-yl)diazen-1-yl]-2-methylphenyl}diazen-1-yl)phenyl]diazen-1-yl}-4-hydroxynaphthalene-2,7-disulfonate
Details on test material:
- Physical state: dark blue solid
- Stability under test conditions: Pure: stable for 5 years. In solvent: stable for at least 48 hours in water, methanol, and acetone
- Storage condition of test material: room temperature

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
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment I:
without S9 mix:
7 h: 0.03; 0.1; 0.5; 1.0 mg/ml
18 h: 0.003; 0.01; 0.03; 0.1; 0.5; 1.0 mg/ml
28 h: 0.03; 0.1; 0.5; 1.0 mg/ml
with S9 mix:
7 h: 0.1; 0.5; 2.5; 5.0 mg/ml
18 h: 0.01; 0.03; 0.1; 0.5; 2.5; 5.0 mg/ml
28 h: 0.1; 0.5; 2.5; 5.0 mg/ml

Experiment II:
without S9 mix:
28 h: 0.03; 0.10; 0.25; 0.50; 0.80; 1.00 mg/ml


The following dose levels were evaluated:
without S9 mix:
7 h: 0.1 mg/ml
18 h: 0.01; 0.03; 0.1 mg/ml
28 h: 0.5 mg/ml

with S9 mix:
7 h: 0.1 mg/ml
18 h: 0.01; 0.03; 0.1 mg/ml
28 h: 0.1 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dissolved in culture medium without fetal calf serum.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
culture medium without fetal calf serum.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without S9-mix: Ethylmethanesulfonate, With S9-mix: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into Quadriperm dishes (Heraeus, D-6450 Hanau, GER) which contained microscopic slides. 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, either without S9 mix or with 20 µl/ml S9 mix. After 4 hours this medium was replaced with normal medium after rinsing twice with "saline G". 5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with giemsa.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per slide

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
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 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 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.
Statistics:
statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment on toxicity (colony forming ability) after treatment with concentrations higher than 0.05 (without S9 mix) and 0.001 mg/ml (with S9 mix) the colony forming ability was clearly reduced. In the test groups with S9 mix, the toxicity showed no dose dependency.
According to the results from this pre-experiment six concentrations (18 h interval) to be applied in the chromosomal aberration assay were chosen.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The mitotic index was at least slightly reduced after treatment with the highest concentration at fixation intervals 7 h (with and without S9 mix), 18 h (with S9 mix) and 28 h (without S9 mix) .
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Both, in the absence and presence of S9 mix the test article did not induce any biologically relevant increase of the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.0 % - 5.5 %) were in or near to the range of the control values; 1.0 % - 3.5 %.

In experiment I (28 h, without S9 mix), a relative high spontaneous aberration rate (4.5 %) were found with 2.0 % cells carrying exchanges (data not reported). To overcome possible incalculable effects, several experimental points (one control and 6 treatment groups) at this interval were repeated in an independent experiment. In this experiment a slight increase in the aberration rate was found as compared to the corresponding control. However, this increase was observed only in one of the two slides. In addition, the increase (5.5 %) was near to the control rate (3.5 %) and not statistically significant. Therefore, this effect was not regarded as biologically relevant. The positive controls showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

MUTAGENICITY DATA

Fixation Interval: 7 h % aberrant cells
article number of cells analyzed concentration mg/ml S9-Mix incl. Gaps excl. Gaps exchanges
solvent control 200 0 - 8 1 0
test article 200 0.1 - 3.5 1 0
solvent control 200 0 + 6.5 3 0
test article 200 0.1 + 7.5 3 1
Fixation Interval: 18 h % aberrant cells
article number of cells analyzed concentration mg/ml S9-Mix incl. Gaps excl. Gaps exchanges
solvent control 200 0 - 6.5 2.5 0.5
positive control EMS 200 0.72 - 11 11 6.5
test article 200 0.01 - 4 1.5 0
test article 200 0.03 - 6.5 3.5 1
test article 200 0.1 - 3.5 2.5 0
solvent control 200 0 + 5.5 1.5 0
positive control CPA 200 0.0014 + 23 19 10
test article 200 0.01 + 3 0 0
test article 200 0.03 + 5 0.5 0
test article 200 0.1 + 3.5 2 0.5
Fixation Interval: 28 h % aberrant cells
article number of cells analyzed concentration mg/ml S9-Mix incl. Gaps excl. Gaps exchanges
solvent control 200 0 - 5 3.5 0.5
test article 200 0.5 - 9 5.5 0.5
solvent control 200 0 + 3 2 0.5
test article 200 0.1 + 4 1.5 0.5

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.
Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose) and 28 h (high dose) after start of the treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosome aberrations.

The following dose levels were evaluated:

Without S9 mix:

7 h: 0.10 mg/ml

18 h: 0.01; 0.03; 0.10 mg/ml

28 h; 0.50 mg/ml

with S9 mix:

7 h: 0.10 mg/ml

18 h: 0.01; 0.03; 0.10 mg/ml

28 h: 0.10 mg/ml

Both, in the absence and presence of S9 mix the test article did not increase biologically relevantly the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.0 % - 5.5 %) were in or near to the range of the control values; 1.0 % - 3.5 %. In experiment I (28 h, without S9 mix), a relative high spontaneous aberration rate (4.5 %) were found with 2.0 % cells carrying exchanges (data not reported). To overcome possible incalculable effects, several experimental points (one control and 6 treatment groups) at this interval were repeated in an independent experiment. In this experiment a slight increase in the aberration rate was found as compared to the corresponding control. However, this increase was observed only in one of the two slides. In addition, the increase (5.5 %) was near to the control rate (3.5 %) and not statistically significant. Therefore, this effect was not regarded as biologically relevant. No relevant deviation from the control data was found after treatment with the test article regarding polyploid metaphases. EMS (0.72 mg/ml) and CPA (1.40 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V7 9 Chinese hamster cell line.