2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-11-29 to 1996-03-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 4, 1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

EC Number:

235-546-0

EC Name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Cas Number:

12270-13-2

Molecular formula:

C19H23N4O2S.CH3O4S C20H26N4O6S2

IUPAC Name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Test material form:

liquid

Details on test material:

see below

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEDIA USED: MEM with 10% FCS (complete medium)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test concentrations were based on the results from a pre-test.
In the main study the following concentrations of the test article were evaluated:
Experiment 1:
without S9 mix: 1.0, 3.0, 10.0, 30.0, 60.0 and 100.0 µg/mL
with S9 mix: .0, 3.0, 10.0, 30.0, 100.0 and 300 µg/mL
Experiment II:
without S9 mix: 1.0, 3.0, 10.0, 30.0, 40.0 and 50.0 µg/mL
with S9 mix: 1.0, 3.0, 10.0, 20.0, 30.0 and 40.0 µg/mL

Vehicle / solvent:

- vehicle/solvent used: the test item was dissolved in aqua bidest. The final concentration of the vehicle in the culture medium did not exceed 10% v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

MEM medium

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9, final concentration 0.6 mg/mL

Controls 2

Untreated negative controls:

yes

Remarks:

MEM medium plus S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest plus S9 mix

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9, final concentration 3.85 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (short time exposure, Experiment I with and without metabolic activation)
- Expression time (cells in growth medium): 6-7 days (subcultured after 3 days)
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-16 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: one culture per test group (expression period), five flasks per culture per test group (selection period)

DETERMINATION OF CYTOTOXICITY
- Methods: Cloning efficiency

Rationale for test conditions:

according to OECD test guideline 476

Evaluation criteria:

A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article 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 significant response is described as follows: The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations.
The test article 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 spontaneous mutation rate in the range normally found (0 - 45 mutants per 10^6 cells) a concentration-related increase of the mutations within this range has to be discussed.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The study was performed in two independent main experiments, using identical procedures, with and without liver microsomal activation. Strong toxic effects occurred at concentrations of 30 µg/mL and above without and 10 µg/mL and above with metabolic activation. In the second experiment several closely spaced concentrations of the test article were chosen to cover the threshold of toxicity. No precipitation of the test article occurred up to the maximal concentration. In the presence and absence of metabolic activation the cloning efficiency of the cells was reduced down to almost zero at the highest concentrations tested. In the mass-cultures of the larger flasks these toxic effects were less striking. The range of the controls was not exceeded in any of the test groups with or without metabolic activation. Furthermore, there was no indication of any concentration dependent increase of the number of colonies below the threshold of biological relevance. In both experiments of this study (with and without S9 mix) the range of the negative controls was from 5.0 up to 17.5 mutant colonies per 10^6 cells; the range of the groups treated with the test article was from 3.0 up to 15.2 mutant colonies per 10^6 cells. EMS (0.6 mg/mL) and DMBA (3.85 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Any other information on results incl. tables

Table 1: Pre-test: Determination of Toxicity
Concentration µg/mL		extinction mean plus standard deviation		% of control
without S9
Blanc		0.163 +/- 0.021		/
Negative control		1.067 +/- 0.045		102.92
Solvent control		1.041 +/-0.063		100
30		0.853 +/- 0.055		78.52
50		0.604 +/- 0.085		50.21
100		0.439 +/- 0.029		31.39
300		0.307 +/- 0.021		16.34
500		0.210 +/- 0.009		5.31
1000		0.191 +/- 0.005		3.22
3000		0.193 +/- 0.006		3.35
5000		0.194 +/- 0.004		3.52
with S9 mix
Blanc		0.140 +/- 0.034		/
Negative control		1.177 +/- 0.043		101.87
Solvent control		1.158 +/- 0.042		100
30		1.129 +/- 0.063		97.12
50		0.903 +/- 0.175		74.95
100		0.322 +/- 0.018		17.85
300		0.538 +/- 0.015		39.12
500		0.464 +/- 0.049		31.84
1000		0.206 +/- 0.024		6.49
3000		0.187 +/- 0.004		4.66
5000		0.187 +/- 0.008		4.62

Applicant's summary and conclusion

Conclusions:

In this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

In a mammalian cell HPRT gene mutation assay, V79 cells cultured in vitro were exposed to the test item in aqua bidest at concentrations of 1.0, 3.0, 10.0, 30.0, 60.0 and 100.0 µg/mL in the absence of mammalian metabolic activation and at concentrations of 1.0, 3.0, 10.0, 30.0, 100.0 and 300 µg/mL in the presence of S9 (experiment I). Strong toxic effects occurred at concentrations of 30.0 µg/mL and above without and at 10.0 µg/mL and above with metabolic activation. Thus, in a second experiment several closely spaced concentrations of the test article were chosen to cover the threshold of toxicity (1.0, 3.0, 10.0, 30.0, 40.0 and 50.0 µg/mL (without S9) and 1.0, 3.0, 10.0, 20.0, 30.0 and 40.0 (with S9)). The following concentrations were evaluated for mutagenicity: 1.0, 3.0, 10.0 and 30.0 µg/mL in the presence and absence of mammalian metabolic activation (experiment I) and for experiment II concentrations of 1.0, 10.0, 40.0 and 50.0 µg/mL (without S9) and 1.0, 10.0, 30.0 and 40.0 µg/mL with metabolic activation. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.