Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

226-103-2

EC Name:

N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

5280-68-2

Molecular formula:

C33H27ClN4O6

IUPAC Name:

N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-{[2-methoxy-5-(phenylcarbamoyl)phenyl]diazenyl}-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:supplemented MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital/beta-naphthoflavone pretreated Wistar rats

Test concentrations with justification for top dose:

without S9 mix:
Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml
Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:
Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Not all test groups were evaluated (for evaluation points see executive summary)

Vehicle / solvent:

- DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 h, experiment II: 24 h
- Expression time (cells in growth medium): 24 h
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3+7+8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 experiments, treatments performed in duplicate, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: colonies with more than 50 cells

DETERMINATION OF CYTOTOXICITY
- Method: colony formation

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontanous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontanous mutation rate in the range normally found (0.5-31.8 mutants per 10 to the power of 6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into considration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from 10.7 µg/ml with and without metabolic activation in both experiments.

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed with concentrations ranging from 1.3 to 170.0 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test substance did not increase the mutant frequency at the HPRT locus in Chinese hamster V79 cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476. Chinese hamster V79 cells were treated with the test substance in the following concentrations:

without S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Marked in bold: cultures which were not continued.

In experiment I and IA the treatment period was 4 h with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with a treatment time of 24 h.

A precipitation of the test substance was noted in both experiments at 10.7 and 170 µg/ml with and without metabolic activation. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.