4-chloro-2,5-dimethoxyaniline

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

Study period:

1988-03-09 to 1988-06-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed GLP-study following previous OECD TG, no colony sizing performedm evaluation criteria not given

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Remarks:

GLP compliance statement included in full study report.

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-2,5-dimethoxyaniline

EC Number:

228-782-0

EC Name:

4-chloro-2,5-dimethoxyaniline

Cas Number:

6358-64-1

Molecular formula:

C8H10ClNO2

IUPAC Name:

4-chloro-2,5-dimethoxyaniline

Method

Target gene:

HGPRT: Hypoxanthine-guanine phosphoribosyl transferase

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Rat-S9)

Test concentrations with justification for top dose:

Without metabolic activation: 37.5, 75, 150, 300 µg/ml
With metabolic activation: 0.25, 0.5, 1.0, 1.25, 1.5 µg/ml

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Mutagenicity test:
- Single cell suspension of two days old exponentially growing cultures (more than 50 % confluent, trypsinated)
- Day 1: Subculturing of a exponentially growing culture
a) About 400 cells in 25 cm^2 flasks with 5 ml medium for determination of the plating efficiency; in duplicate for each experimental point.
b) 6x10^5 - 1x10^6 cells in 175 cm^2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with test substance in the presence and absence of S9-Mix for 4 hours.
Day 5: Subculturing of b) in 175 cm^2 flasks
Day 8: Fixation and staining of the colonies in a)-flasks for the determination of the plating efficiency.
Day 9: Subculturing of b) in five 80 cm^2 flasks with culture medium containing 6-thioguanine: Mutant selection (about 400000 cells/flask); subculturing of b) in two 25 cm^2 flasks for plating efficiency (about 500 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.

- All incubations were done at 37 °C and 5% CO2.
- Staining was done with 10 % methylene blue in 0.01 n KOH solution.
- Only colonies with more than 50 cells were counted.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

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

Executive summary:

The test substance Aminochlorhydrochinondimethylether was examined for mutagenic activity in V79 Chinese hamster cells with two independent experiments. The induction of 6 -thioguanine resistant mutants after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance did not produce any significant cytotoxic effect without S9-mix up to the limit of solubility (300 µg/ml) and a significant cytotoxic effect at 2 µg/ml with metabolic activation.

For mutagenicity testing two independent experiments with and without metaboluc activation (S9-mix) were performed.

In the absence of S9 metabolic activation dose levels of 37.5, 75, 150, 300 µg/ml and in the presence of S9 metabolic activation dose levels of 0.25, 0.5, 1.0, 1.25, 1.5 µg/ml were used for mutant selection in the main experiments.

The test compound did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Aminochlorhydrochinondimethylehter does not induce gene mutations in the HGPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.