Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian Chromosomal Aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

415-070-1

EC Name:

-

Molecular formula:

C52H26CrN10Na5O22S4

IUPAC Name:

chromium(3+) pentasodium bis(6-[2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]-2-[2-(3-nitro-2-oxido-5-sulfonatophenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

-Experiment I
without S9 mix:
18 h: 3.0; 10.0; 30.0; 100.0; 300.0; 600.0 µg/ml
28 h: 30.0; 100.0; 300.0; 600.0 µg/ml
with S9 mix:
18 h : 10.0; 30.0; 100.0; 300.0; 600.0; 1000.0 µg/ml
28 h: 100.0; 300.0; 600.0; 1000.0 µg/ml
-Experiment II
with and without S9 mix:
18 h: 0.1; 0.3; 1.0; 3.0; 10.0; 30.0 µg/ml
28 h: 1.0; 3.0; 10.0; 30.0 µg/ml
According to the results from the pre-test 6 concentrations (18 h interval) were chosen to be applied in the chromosomal aberration assay. The highest concentration used in the pre-test (1000.0 µg/ml without S9 mix and 5000.0 µg/ml with S9 mix) was limited by a precipitation of the test substance in culture medium during incubation at 37° C and a strong colouring of the test medium. As strong toxic effects were observed after treatment with 600.0 µg/ml (without S9 mix) and 300,0 µg/ml (with S9 mix) and higher, the cytogenetic experiments were performed with the selected concentrations.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 Mix

Controls 2

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
-Cell density at seeding: 5 x 10^5 cells per flask were seeded in 15 ml of MEM (minimal essential medium) supplemented with 10 % fetal calf serum.

CELL CULTURES
Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank 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 cm^2 plastic flasks. The cells per flask were seeded in MEM supplemented with 10 % fetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in an atmosphere with 4.5 % carbon dioxide (95.5 % air).

PRE-TEST FOR TOXICITY
High density cultures (approx. 200,000 cells/slide) were treated with the test substance to simulate the conditions of the main experiment. Cell number and morphology were examined qualitatively 4 h and 24 h after the start of treatment (with metabolic activation: after washing with Saline G ) .

DURATION
The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. Per concentration duplicate cultures were used.

DETERMINATION OF CYTOTOXICITY
Toxic effects (low number of remaining/surviving cells, low metaphase number, partially combined with poor metaphase quality) were determined qualitatively for all test groups in both experiments. In the absence of S9 mix, in both experiments cultures after treatment with 30.0 µg/ml (18 h and 28 h) as highest concentration were evaluated for cytogenetic damage. In addition, 10.0 µg/ml and 3.0 µg/ml were evaluated at fixation interval 18 h. In the presence of S9 mix, in experiment I cultures treated with 100.0 µg/ml (18 and 28 h) in experiment II 10.0 µg/ml (18 and 28 h) as top concentrations were evaluated. Additionally, 10.0 and 30.0 ng/ml (exp. I) and 1.0 and 3.0 ng/ml (exp. II), respectively, were evaluated at fixation interval 18 h.

SEEDING OF THE CULTURES
Three days old exponentially growing stock cultures more than 50 % confluent were trypsinized at 37 °C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium 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 (per litre) with: NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHC03 350 mg.
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/1 EDTA. The cells were seeded into Quadriperm dishes which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 10^4 - 6 x 10^4 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT
-Exposure time 4 hours (with S9 mix):
In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h preparation interval) the culture medium was replaced with serum-free medium containing different concentrations of the test article and 50 µl/ml S9 mix. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as (per litre)with: NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HP04.7H20 290 mg, KH2PO4 150 mg (pH was adjusted to 7.2).
-Exposure time 18 and 28 hours (without S9 mix):
In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h preparation interval) the culture medium was replaced with complete medium (10 % FCS) containing different concentrations of the test article without S9 mix. This medium was not changed until preparation of the cells. All cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2 (95.5 % air).

PREPARATION OF THE CULTURES
15,5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 h later, 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 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After fixation the cells were stained with Giemsa.

ANALYSIS OF METAPHASE CELLS
Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To escribe a cytotoxic effect the mitotic Index (% cells in mitosis) was determined. In addition, the number of polyploid cells was scored (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test substance was classified as mutagenic if it induced reproducibly either a significant concentration-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 substance producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly 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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The concentration range of the test substance set up in the respective experiments were limited by a strong precipitation of the test substance in the test medium during incubation at 37° C and a strong colouring of the test medium. In the main experiments microscopically visible precipitation was found with concentrations starting at 10.0 µg/ml (without S9 mix) and with all concentration used in the presence of S9 mix (both experiments).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects shown by a reduction of mitotic indices and cell density could be observed in exp. I as well as in the pre-experiment in the absence of S9 mix after treatment with 600.0 µg/ml (and higher) at both fixation intervals, in the presence of S9 mix after treatment with 1000.0 µg/ml (18 h) and 600.0µg/ml and higher (28 h).
However, in both experiments lower concentrations were evaluated, where no reduction of mitotic Indices could be found up to the highest concentration evaluated. The evaluation of higher concentrations in the respective intervals were not appropriate due to the strong precipitation of the test substance.
- Other observations:
In both experiments, at both fixation intervals in the absence and presence of S9 mix the test substance did not increase the frequency of cells with aberrations to a biologically relevant extend. The aberration rates of the cells after treatment with the test substance (exp.I: 0.0 % - 2.5 %; exp. II: 0.0 % - 3.0 %) were in or near to the range of the solvent control values (exp. I: 0.0 % - 2.5 %; exp. II: 0.0 % - 2.0 %) and in the range of historical control data: 0.0 % - 4.0 %.
In exp. I, in the presence of S9 mix after treatment with 100.0 µg/ml (fixation interval 28 h) and in exp. II, in the absence of S9 mix after treatment with 3.0 µg/ml (fixation interval 18 h) the statistical evaluation (chi-square test) revealed a significant difference between the treatment groups (2.0 % and 3.0 % aberrant cells) versus the corresponding controls (0.0 %aberrant cells). However, taking into account the extremely low value of the corresponding control and our historical control data range, in this case the statistical significance can be regarded as being biologically not relevant. In both experiments, no biologically relevant increase in rate of polyploid metaphases (exp. I: 1.0 % - 4.5 %; exp. II: 1.5 % -4.5 %) as compared to the rates of the controls (exp. I: 1.0 % -2.5 %; exp. II: 1,0 % - 3.0 %) were found after treatment with the test substance.
POSITIVE CONTROLS
In both experiments, EMS (0.6 mg/ml) and CPA (0.93 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

Applicant's summary and conclusion

Conclusions:

The substance did not induce reproducibly structural chromosomal aberrations in the V79 Chinese hamster cell line.

Executive summary:

The test item was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments, according to the OECD 473 (1983) and the method B.10 of the EEC-Directive 92/69 EEC.The chromosomes were prepared 18 h and 28 h after start of treatment with the test item. The treatment interval was 4 h with metabolic activation (S9 Mix), 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 chromosomal aberrations. The test concentrations, 3 concentrations evaluated at 18 h and the highest evaluable concentration at 28 h, were with and without S9 Mix ranging up to 100 and 30 µg/ml (experiment I) and 10 and 30 µg/ml (experiment II), respectively. The concentration range was limited by a strong precipitation of the test substance in the test medium during incubation at concentrations starting at 10.0 µg/ml in the absence of S9 mix and with all concentrations set up in the presence of S9 mix. Toxic effects could not be found up to the highest concentration evaluated. The evaluation of higher concentrations in the respective intervals was not appropriate due to the strong precipitation of the test substance. In both independent experiments, there were no biologically relevant increases in cells with structural aberrations after treatment with the test substance at both fixation intervals either with or without S9 mix. In both experiments, no biologically relevant increase in the frequency of polyploid metaphases was found after treatment with the test substance compared to the frequencies of the controls. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

The substance did not induce structural chromosomal aberrations under the test condition. Therefore, the substance is considered to be non-mutagenic in this chromosome aberration test.