Trisodium 3-[(E)-(4-{[halo-{2-[(halo-[4-substituted-phenyl]aminoheteromonocyclyl) amino]propyl}amino)-heteromonocyclyl]amino}-5-sulfonato-1- naphthyl)diazenyl]-1,5-naphthalenedisulfonate

Genetic toxicity: in vitro

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environmental Agency

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Cytotest Cell Research GmbH, der gleichnamigen Firma, In den Leppsteinwiesen 19, 64380 Roßdorf

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40826/A
- Purity: Approx. 78%
- Lot/batch No.:TZ 5604 BOP 01/06
- Expiration date: 1 February 2011
- Storage conditions: At room temperature in the desiccator.
- Stability in solvent: 7 days in water, saline, PEG, and CMC at room temperature.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Without S9 mix: 40, 80, 160, 320, 640, 1280 µg/mL
- With S9 mix: 10, 20, 40, 80, 160, 320 µg/mL

Vehicle / solvent:

Deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

RANGE-FINDER
A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - square root 10. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used: In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. The cells were stained 24 hrs after start of treatment. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group).

EXPERIMENTAL PERFORMANCE
- Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTA-solution at 37°C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131 Karlsruhe). Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1E4 - 6E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).
- Exposure duration: The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
- Preparation of the cultures: Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs after the start of the treatment. The cells on the slides were treated 2.5 hrs later 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 a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
- Evaluation of Cell Numbers: For evaluation of cytotoxicity indicated by reduced cell numbers additional two cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
- Analysis of Metaphase Cells: Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetic) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates 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 describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

- Acceptability of the test: The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of our historical laboratory control data: 0.0 - 4.0 %.
b) The positive control substances should produce significant increases in the number of cells with structural, chromosome aberrations, which are within the range of the laboratory's historical control data.
- Evaluation of results: A test item is classified as non-clastogenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and/or no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if: the number of induced structural chromosome aberrations is not in the range of the historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
A test item can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together.

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:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY: In a range finding pre-test on toxicity cell numbers were scored 24 hrs after start of treatment as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after treatment with 156.3 µg/mL and above in the absence and presence of S9 mix. In addition, 24 hrs continuous treatment with 78.1 µg/mL and above in the absence of S9 mix induced strong toxic effects.

TEST-SPECIFIC CONFOUNDING FACTORS: In the pre-experiment, neither precipitation of the test item in culture medium nor a relevant influence of the test item on the pH value or osmolarity was observed (solvent control 265 mOsm, pH 7.3 versus 308 mOsm and pH 7.4 at 5000 µg/mL).

MAIN TEST: In this study, dose-related increases in the number of aberrant cells were observed at 80 to 320 µg/mL in the absence of S9 mix (3.0%, 11.5%, and 12.0% aberrant cells exclusive gaps, respectively) and at 40 to 160 µg/mL in the presence of S9 mix (2.0 %, 5.0 %, and 11.0 % aberrant cells exclusive gaps, respectively). The increase in the number of aberrant cells, exclusive gaps at 160 µg/mL and 320 µg/mL (11.5 % and 12.0 %, respectively) in the absence of S9 mix and at 160 µg/mL (11.0%) in the presence of S9 mix, were statistically significant when compared to the corresponding solvent controls (1.0 % and 2.5 %, respectively). The values at the two highest evaluated concentrations, in the absence and presence of S9 mix, clearly exceeded our historical control data range (0.0 - 4.0 % aberrant cells exclusive gaps). Therefore, the statistical significances and the dose-dependencies have to be regarded as being biologically relevant. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4 - 3.4 %) as compared to the rates of the solvent controls (2.3 - 3.3 %).

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main experiment, in the absence of S9 mix, toxic effects indicated by clearly reduced cell numbers of below 50 % of control were observed after 4 hrs treatment with 320 µg/mL (26.0 % of control). In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

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

Under the experimental conditions reported, the test substance induced structural chromosome aberrations in V79 cells (Chinese
hamster cell line) in the prensence or absence of S9 mix.

Executive summary:

In a GLP compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix. The exposure period was 4 hours with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. Dose selection of the main experiments was based on a pretest. In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In the absence and the presence of S9 mix, dose-related significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.