Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

EC Number:

230-303-5

EC Name:

Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

Cas Number:

7023-61-2

Molecular formula:

C18H13ClN2O6S.Ca

IUPAC Name:

calcium 4-[(5-chloro-4-methyl-2-sulfonatophenyl)diazenyl]-3-hydroxy-2-naphthoate

Test material form:

solid: nanoform

Details on test material:

- Identity: TK10334
- Aggregate state at room temperature: solid
- Colour: red
- Stability in solvent: Not indicated by the sponsor
- Storage: room temperature
- Expiration Date: January 30, 2009
- Batch No.: non-surface treated PR48:2 (bill no. 247 2793 396)
- Content: 99.4 %

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.

Specific details on test material used for the study:

- Identity: TKA10334
- Aggregate state at room temperature: solid
- Colour: red
- Stability in solvent: Not indicated by the sponsor
- Storage: room temperature
- Expiration Date: January 30, 2009
- Batch No.: non-surface treated PR48:2
- Content: 99.4 %

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I
Exposure 4h, preparation interval 18h, with and without S9-mix: 0.6, 1.3, 2.5, 5, 10 and 20 microgramm/plate

Experiment II
Exposure 18h, preparation interval 18h, without S9-mix: 2.8, 6.6, 11.3, 22.5, 45.0, 90, 180 and 360 microgramm/plate
Exposure 28h, preparation interval 28h, without S9-mix: 2.8, 6.6, 11.3, 22.5, 45.0, 90, 180 and 360 microgramm/plate
Exposure 4h, preparation interval 28h, with S9-mix: 0.7, 1.4, 2.8, 6.6, 11.3 and 22.5 microgramm/plate

Vehicle / solvent:

deionized water (suspension)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: ethylmethane sulfonate

Details on test system and experimental conditions:

On the day of the experiment (immediately before treatment), the test item was suspended in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

A pre-test on cell grovth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item (2). Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - ViO. 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). The cell number of the treatment groups is given as % cells in relation to the control. The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 microL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible. With respect to the ability to formulate a homogeneous suspension of TKA 10334, in the pretest, 720 microgramm/mL was applied as top concentration for treatment of the cultures. Test item concentrations between 5.6 and 720 microgramm/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item, after 4 hrs treatment, was observed at 5.6 microgramm/mL and above. Since no relevant toxicity was observed up to the highest applied and/or scorable concentration in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. Therefore, for Experiment I, in the absence and the presence of S9 mix, 20 microgramm/mL was chosen as top treatment concentration. Dose selection of Experiment II, was also influenced by test item toxicity. In the range finding experiment, cleariy reduced cell numbers were observed, after 24 hrs exposure, with 180 microgramm/mL and above. Therefore, 360 pg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix, 22.5 microgramm/mL was chosen as top treatment concentration with respect to the results obtained in Experiment I.

Evaluation criteria:

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 the laboratory's historical control data range: 0.0 - 4.0 % aberrant cells, excluding gaps.
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.

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding 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 laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps); and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed. Statistical significance was confirmed by means of the Fisher's exact test (9) (p < 0.05).

However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not cleariy met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid, a test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical control data range (0.0 - 5.2 % polyploid cells).

Statistics:

not applicable

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 applicable

Positive controls validity:

valid

Additional information on results:

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 5.6 and 720 microgramm/mL were applied. In the absence of S9 mix, no toxic effects were observed up to the highest applied concentration. In the presence of S9 mix, no toxic effects could be observed up to the highest scorable concentration. In contrast, 24 hrs continuous treatment with 180 microgramm/mL and above in the absence of S9 mix induced strong toxic effects.

In the pre-experiment, in the absence and presence of S9 mix, precipitation of the test item in culture medium was observed after treatment with 5.6 microgramm/mL and above. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 277 mOsm, pH 7.5 versus 274 mOsm and pH 7.5 at 720 microgramm/mL).

In Experiment I, in the absence and presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 10 microgramm/mL. In Experiment II, in the absence and presence of S9 mix, precipitation occurred after treatment 5.6 microgramm/mL and above.

In Experiment I, in the absence and presence of S9 mix, and in Experiment II, in the presence of S9 mix, no toxic effects indicated by reduced mitotic indices and/or reduced cell numbers of below 50 % of control were observed after treatment up to the highest applied test item concentration. In Experiment II, in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, being far in excess of test item precipitation. In both experiments, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory's historical control data range: 0.0-4.0 % aberrant cells, excluding gaps. In Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, the number of aberrant cells, excluding gaps, was increased in a dose-related manner. Since all values were cleariy within the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps), this observation has to be regarded as biologically irrelevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4-3.1 %) as compared to the rates of the solvent controls (2.2 - 3.0 %).

In both experiments, either EMS (500 or 900 microgramm/mL) or CPA (1.4 or 2.0 microgramm/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.