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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:
3 February 2009 to 19 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ink BH 11 M
-Chemical Name: Mixture of partial ammonium and sodium salt of 6-(2-{5-[3-methyl-2,7-dioxo-1-(3-Sulfobenzoyl)-3,7-dihydro-2H-naptho [1,2,3-de] quinolin-6-ylamino]-2,4-disulfoanilino}-2-oxoethyl amino) hexanoic acid
- Physical state: dark red powder
- Analytical purity: 87.3%
- Impurities (identity and concentrations): water 7.1wt%, By-product 1 3.4 wt%, By-product 2 1.3 wt%, By-product 3 0.5 wt%, unidentified organic impurities 0.4 wt%, Inorganic impurities <0.1 wt %
- Lot/batch No.: MB-1-A
- Storage condition of test material: Room temperature in dark
- Other: routine safety and hygienic procedures were sufficient to ensure personnel health and safety; dyestuff for inkjet printing ink

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- ECACC: 86041102
- Lot. No: 05F013
- Supplier: ECACC (European Collection of Cells Culture)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and beta-naphthoflavone (BNF) induced rat liver S9
Test concentrations with justification for top dose:
312.5 ug/mL
625.0 ug/mL
1250.0 ug/mL
2500.0 ug/mL
5000.0 ug/mL
Vehicle / solvent:
DME medium (Dulbecco's Modified Eagle's)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DME Medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulphonate (without metabolic activation) and N-Nitrosodimethylamine (with metabolic activation)
Details on test system and experimental conditions:
In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in DME medium. Cells were seeded into 92 x 17 mm dishes (for tissue cultures quality TC sterile) at 5x10^5 cells each and were incubated for 24 hours in DME medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 μl/ml) and were incubated at 37°C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME medium containing 10 % foetal bovine serum. Evaluation of cell number was performed at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). Another group of cells was treated for 20 hours without metabolic and for 3 hours with metabolic activation, the cell number evaluation was performed at 28 hours. The cell numbers of the treatment groups were noted as % of cells in relation to the solvent control. The results of the tests were used to select the concentrations of test item for the Chromosome Aberration Assays.

Experimental Design Cytogenetic Experiment (Experiment A)
This experiment consisted of a 3-hour treatment, following by harvest at 20 hours from the beginning of treatment. The test item was dissolved in DME for the treatment (stock formulation: 25 mg/ml). The appropriate amount of this stock formulation was diluted with medium to obtain the examination concentrations. Duplicate cultures were used at each concentration and the negative control culture as well as the positive controls for treatment without and with S9 mix. 5 x 10^5 cells/dish were set up for each group. After 24 hours the culture medium of exponentially growing cell cultures were replaced with DME medium containing 5 % foetal bovine and the test item. The exposure period was 3 hours at 37°C. After the exposure periods the cells were washed with DME medium and covered with growth medium. Sampling was made at 20 hours approximately 1.5 normal cell cycles from the beginning of treatment. For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 10^5 cells/dish were set up.

Experimental Design Cytogenetic Experiment (Experiment B)
This experiment consisted of 3 and 20-hour treatments, following by harvest at 28 hours from the beginning of treatment. The test item was dissolved in DME for the treatment (stock formulation: 25 mg/ml). The appropriate amount of this stock formulation was diluted with medium to obtain
the examination concentrations. In the Cytogenetic Experiment B the exposure period without metabolic activation was 20 hours and with metabolic activation was 3 hours. After the exposure periods the cells were washed with DME medium and covered with growth medium. Sampling was made at approximately 2 normal cell cycles (28 hours) from the beginning of treatment to cover a potential mitotic delay. Experiment B, as experiment A, included a concurrent S9 non-activated and S9 activated positive control. For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 10^5 cells/dish was set up.
Evaluation criteria:
The Chromosome Aberration Assay is considered valid if following criteria are met:
- the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data.
- the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.
Treatment of results
- The percentage of cells with structural chromosome aberration(s) was evaluated.
- Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
- Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
- Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
- Individual culture data were summarised in tabular form.
- There were not equivocal results in this study.
Interpretation of Results
The test item is regarded as non-clastogenic if:
- the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
- and/or no significant increase in the number of metaphases with structural chromosome aberration is observed
A test item is classified as clastogenic if it meets the following criteria:
- increase in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data)
- the increase is reproducible between replicate cultures and between tests (when the treatment conditions are the same)
- the increase is statistically significant
Both, biological and statistical significance should be considered together.
Statistics:
For statistical analysis, Fisher exact test was utilised. The parameters evaluated for statistical analysis were as follows: number of cells with aberration (with and without 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:
5000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Ink BH11 M did not induce an increase in the number of cells with aberrations without gaps at any examine concentration, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationship was noted.

No biologically relevant increase in the rate of polypolid and endoreduplicated metaphases was found after treatment with the different concentrations of Ink BH11 M.

In the control group, the percentage of cells with structural aberrations without gap was less than 5% proving the suitability of the cell line used.

Positive controls Ethylmethane sulphonate (0.4 and 1.0 uL/mL) and N-nitrosodimethylamine (1.0 uL/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations. The studies are considered valid.
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):
negative with and without metabolic activation

Ink BH11 M tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Ink BH11 M is considered not clastogenic in this system.
Executive summary:

INK BH11 M was tested in an in vitro cytogenetics assay using Chinese hamster lung cells, in accordance with OECD Guideline No. 473. The results of the experiment indicate that INK BH11 M was unable to induce chromosome aberrations in Chinese hamster lung cells when tested up to cytotoxic concentrations in either the absence or presence of S9. INK BH11 M is considered not clastogenic in this system.