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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:
From March 19 to November 28, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted May 26, 1983
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]
EC Number:
240-521-2
EC Name:
Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]
Cas Number:
16470-24-9
Molecular formula:
C40H44N12Na4O16S4
IUPAC Name:
tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[bis(2-hydroxyethyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELL DULTURES
Large stocks of the V79 cell line (supplied by LMP, D-6100 Darmstadt) were stored in liquid nitrogen in the cell bank of the laboratory allowing the repeated use of the same cell culture batch in experiments.
Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. Seeding was done with about 5 x 10^5 cells per flask in 15 ml of MEM (minimal essential medium) supplemented with 10 % fetal calf serum.
The cells were 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:
Aroclor 1254-induced rat liver S9-mix
Test concentrations with justification for top dose:
Experiment I (with and without S9 mix)
7 h: 1.0, 2.0, 3.5, 5.0 mg/ml
18 h: 0.1, 0.3, 1.0, 2.0, 3.5, 5.0 mg/ml
28 h: 1.0, 2.0, 3.5, 5.0 mg/ml

- Experiment II (with S9 mix):
7 h: 0.1, 0.3, 1.0, 2.0, 3.5, 5.0 mg/ml
Vehicle / solvent:
- Vehicle/solvent used: none
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 0.72 mg/ml = 5.76 mM dissolved in nutrient medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 1.40 µg/ml = 5.00 µM dissolved in nutrient medium
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 preparation
The S9 liver microsomal fraction was obtained from the liver of 8 - 12 weeks old male Wistar rats, strain WU which received a single i.p. injection of 500 mg/kg
b.w. Aroclor 1254 in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCI and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. In this experiment the protein concentration was 34.6 mg/ml in exp. I; in exp. II the protein concentration was 30.3 mg/ml.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/ml in the cultures. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT FOR TOXICITY
The toxicity of the test article was determined in a pre-experiment in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this pre-experiment were the same as for the experiment.
Toxicity of the test article was evidenced by a reduction in plating efficiency.

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 Ca-Mg-free salt solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose 1000 mg
NaHCO3 350 mg
The cells were seeded into Quadriperm dishes which contained microscopic slides (2 chambers per dish and test group). In each chamber 5 x 10^4 - 1 x 10^5 cells were seeded 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, either without S9 mix or with 20 µl/ml S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with "saline G".
The "saline G" solution is composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose 1100 mg .
Na2HPO4.7H20 mg
KHZPO4 150 mg
ph was adjusted to 7.2
All incubations were done at 37 °C in a humidified atmosphere with 4.5 % CO2.

PREPARATION of the CULTURES
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.

ANALYSIS of METHAPHASE CELLS
Evaluation of the slides was 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 numbers of 22 ± 1were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) was scored.

NUMBER OF REPLICATIONS: two slides per group.

ACCEPTABILITY of the ASSAY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % - 4.00 % ,
b) the positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.
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.
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
Remarks:
Treatment with 1.0 (without S9 mix) 3.0 (with and without S9 mix) and 5.0 mg/ml (with and without S9 mix) reduced the plating efficiency of the V7 9 cells. Also the mitotic index was at least slightly reduced after treatment with the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix the colony forming ability was reduced with 1.0 mg/ml and higher (without S9 mix) and 3.0 mg/ml and higher (with S9 mix). Higher concentrations than 5.0 mg/ml were not applied.

FIRST EXPERIMENT
In the absence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.5 % - 2.0 %) were in or near to the range of the control values: 0.0 % - 1.5 %. At fixation interval 7 h, in one test group (1.0 mg/ml) the statistical analysis revealed a significant result. This effect was due to the randomly extremely low control rate (0.0 %). As the aberration rate of the test group (2.0 %) is near to the control range of this study and within our historical control data (0.0 - 4.0 %) the statistical result is not regarded as biologically relevant.
In the presence of S9 mix, at fixation interval 7 h the aberration rate (10.0 %) was distinctly increased after treatment with 5.0 mg/ml as compared to the corresponding· solvent control (1.5 %). This result could not be confirmed as in this test group only one slide could be scored due to low metaphase number on the other slide. In addition, the metaphase quality was relatively poor so that an accurate scoring was partially inhibited. Similar observations were made on the slides treated with lower dose levels 1.0; 2.0 and 3.5 mg/ml on which not enough scorable cells could be found.

SECOND EXPERIMENT
A second experiment was performed to clarify the effects observed in the first experiment. In this experiment II, there was no incidence of an increased aberration rate after treatment with 5.0 mq/ml. On both slides enough scorable cells with good spreading quality could be scored.

Therefore, the finding observed in experiment I at fixation interval 7 h (with S9 mix) is not regarded as biologically relevant. At fixation intervals 18 and 28 h (in experiment I, with S9 mix) the aberration rates (0.5 - 2.5 %) were not biologically relevantly increased as compared to the solvent controls (1.0 %).
In the occurence of polyploid metaphases evaluation no relevant deviation from the control data was found after treatment with the test article.

POSITIVE CONTROLS
EMS (0.72 mg/ml) and CPA (1.40 ug/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Schedule Test group (mg/ml)   Genotoxicity Cytotoxicity
S9 mix aberrant cells (%)
Experiment Fixation interval incl gaps excl. Gaps with exchanges Mean of polyploid cells Mitotic index (%)
1 7 h control - 2.00 0.00 0.00 0.5 3.6
1.0 - 3.50 2.00 0.00 0.5 2.0
control + 3.50 1.50 0.00 2.5 7.8
5.0 + 17.00 10.00 0.00 1.5 1.9
18 h control - 3.00 1.50 0.50 0.5 15.2
EMS (0.72) - 15.50 11.50 8.00 1.5 11.2
0.3 - 2.00 1.00 0.00 1.0 9.1
2.0 - 2.00 0.50 0.00 1.0 11.4
5.0 - 6.00 1.50 0.50 0.0 6.0
control + 1.50 1.00 0.00 1.5 12.2
CPA (1.4 µg/ml) + 39.00 38.00 20.00 1.5 5.3
0.3 + 3.50 1.00 0.00 2.0 12.8
2 + 6.50 2.50 2.50 1.5 12.3
5 + 4.00 0.50 0.00 0.5 14.1
28 h control - 1.50 0.50 0.00 1.5 6.6
5.0 - 3.50 1.00 0.00 0.5 5.6
control + 2.00 1.00 0.00 0.0 13.5
5.0 + 4.00 1.50 0.50 1.0 9.8
2 7 h control + 0.50 0.50 0.00 2.0 7.5
5.0 + 2.00 1.00 1.00 1.5 7.9

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test article did not induce structural chromosome aberrations in the V79 Chinese Hamster cell line.
Executive summary:

Methods

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:

Experiment I both with and without S9 mix:

7 h: 1.0 mg/ml

18 h: 0.3; 2.0; 5.0 mg/ml

28 h: 5.0 mg/ml

Experiment II with S9 mix:

7 h: 5.0 mg/ml

In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix the colony forming ability was reduced with 1.0 mg/ml and higher (without S9 mix) and 3.0 mg/ml and higher (with S9 mix). Higher concentrations than 5.0 mg/ml were not applied.

Also, in the cytogenetic experiments the mitotic index was reduced at least after treatment with the highest dose level at fixation intervals 7 and 18 h (in the absence of S9 mix) and 28 h (in the absence and presence of S9 mix).

Results

In the rirst experiment in the absence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. At fixation interval 7 h, in one test group (1.0 mg/ml) the statistical analysis revealed a significant result. This effect was due to the randomly extremely low control rate. As the aberration rate of the test group is near to the control range of this study and within our historical control data the statistical result is not regarded as biologically relevant.

In the presence of S9 mix, at fixation interval 7 h the aberration rate was distinctly increased after treatment with 5.0 mg/ml as compared to the corresponding· solvent control. This result could not be confirmed. In addition, the metaphase quality was relatively poor so that an accurate scoring was partially inhibited. Similar observations were made on the slides treated with lower dose levels on which not enough scorable cells could be found.

A second experiment was performed to clarify the effects observed in the first experiment. In this experiment II, there was no incidence of an increased aberration rate after treatment with 5.0 mq/ml. On both slides enough scorable cells with good spreading quality could be scored.

Therefore, the finding observed in experiment I is not regarded as biologically relevant. At fixation intervals 18 and 28 the aberration rates were not biologically relevantly increased as compared to the solvent controls.

In the occurence of polyploid metaphases evaluation no relevant deviation from the control data was found after treatment with the test article.

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.