1-chloro-2,5-dimethoxybenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-05-04 to 1994-05-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed GLP-study following a former OECD guideline 471 version.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

former version of OECD 471, without 5th strain (E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102)

Deviations:

no

Remarks:

deviations related to the former version

Principles of method if other than guideline:

Testing was done without 5th strain (E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-chloro-2,5-dimethoxybenzene

EC Number:

218-267-9

EC Name:

1-chloro-2,5-dimethoxybenzene

Cas Number:

2100-42-7

Molecular formula:

C8H9ClO2

IUPAC Name:

2-chloro-1,4-dimethoxybenzene

Details on test material:

- Name of test material (as cited in study report): Chlorhydrochinondimethylether

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix, rat, treatment: Aroclor1254

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500, 5000 µg per plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium-azide, 9-Aminoacridine, 2-Nitrofluorene, 2-Aminoanthracene

Details on test system and experimental conditions:

BACTERIA
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The suitable amount of bacteria in the cell suspension was checked by nephelometry. For inoculation, stock cultures which are stored at approx. -80°C, were used. The compound was tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537.

TOXICITY EXPERIMENTS AND DOSE RANGE FINDING
The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was evaluated microscopically.
In combination with the second experiment, toxicity testing was performed as follows:
0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10^6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

MUTAGENICITY TEST
Top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6 % (wlv) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution.
The following ingredients were added (in order) to 2 ml of molten top agar at approx. 45 °C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml S-9 Mix (if required) or buffer
After mixing, the liquid was poured into a petridish with minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his' revertants) were counted.
Two independent experiments were performed.

Evaluation criteria:

A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic to most strains at 2500µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

STERILITY CHECKS AND CONTROL PLATES
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the testmaterial and S-9 Mix sterility check plates. Control plates (background control and positive controls) gavethe expected number of colonies.

SOLUBILITY
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 microgram/plate.

TOXICITY
The test compound was tested at doses of 4 to 5000 microgram/plate and proved to be toxic to most of thebacterial strains at a dose of 2500 microgram/plate and above.

Thinning of the bacterial lawn and a reduction in the number of colonies have been observed at this dose.
Therefore 5000 microgram/plate was chosen as the highest dose in the second experiment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Summarizing, it can be stated thatChlorhydrochinondimethylether is not mutagenic in these bacterialtest systems neither with nor without exogenous metabolic activation at the dose levels investigated.

Executive summary:

Chlorhydrochinondimethylether was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537and TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing systemderived from rat liver homogenate. The test substance was dissolved in DMSO and a dose range of6 different doses from 4 microgram/plate to 5000 microgram/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be toxic to most of the bacterial strains at a dose of 2500 microgram/plate and above.

5000 microgram/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dosedependent increase in the number of revertants in any of the bacterial strains. Also in the presence of ametabolic activation system, treatment of the cells with

Chlorhydrochinondimethylether did not result inrelevant increases in the number of revertant colonies.

Summarizing, it can be stated thatChlorhydrochinondimethylether is not mutagenic in these bacterialtest systems neither with nor without exogenous metabolic activation at the dose levels investigated.