4-chloro-o-xylene

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:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 471 guideline; under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-o-xylene

EC Number:

210-438-6

EC Name:

4-chloro-o-xylene

Cas Number:

615-60-1

Molecular formula:

C8H9Cl

IUPAC Name:

4-chloro-1,2-dimethylbenzene

Details on test material:

- Name of test material (as cited in study report):4-chloro-o-xylene
- Physical state: Liquid
- Storage condition of test material: room temperature, protected from light, keeping container closed and away from ignition sources

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay: 6.7, 10, 33, 67, 333, 667, 1000, 3333 and 5000 micrograms/plate

Initial mutagenicity assay (B1):
Salmonella strains: 100, 333, 1000, 3333 and 5000 micrograms/plate (in the presence of S9)
Salmonella strains: 10, 33, 100, 333 and 1000 micrograms/plate (in the absence of S9)
E.coli strain: 10, 33, 100, 333 and 1000 micrograms/plate (in the presence and absence of S9)

First repeat mutagenicity assay (B2):
1.0, 3.3, 10, 33, 100, 333, 1000 and 3333 micrograms/plate

Second repeat mutagenicity assay (B3):
5.0, 15, 50, 150, 500, 1500 and 5000 micrograms/plate

Third repeat mutagenicity assay (B5)
5.0, 15, 50, 150, 500, 1500 and 5000 micrograms/plate

Independent repeat assay (B4):
5.0, 15, 50, 150, 500, 1500 and 5000 micrograms/plate

Repeat of independent repeat assay (B6):
5.0, 15, 50, 150, 500, 1500 and 5000 micrograms/plate

Vehicle / solvent:

DMSO was selected as the vehicle based on solubility of the test material and compatibility with the target cells.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: a-aminoanthracene; 2-nitrofluorene; sodium azide; 9-aminoacridine; methyl methanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): Plates were incubated at 37 +/- 2 degrees C for at least 48 hours.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: toxicity of the test material was determined by i) >50% reduction in the mean number of revertants per plate when compared to the mean vehicle control and this must be accompanied by a dose-dependent drop in the revertant count. ii) a moderate reduction in the background lawn.

OTHER: To establish the dose-range, a preliminary experiment was performed . Vehicle and ten doses (6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 micrograms/plate) of the test material were plated (single replicates) with overnight cultures of TA98, TA100, TA1537 and WP2 uvrA in the presence and absence of S9 mix.

Evaluation criteria:

For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test material. Data from the strains TA1535 and TA1537 were deemed positive if the increase in mean revertants at the peak dose response is equal to or greater than 3 times the mean vehicle control value. Data from the strains TA98, TA100 and WP2 uvrA were deemed positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

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

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Under the conditions of this study the test material did not induce gene mutations in the strains tested. The test material can therefore considered as non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The study was performed to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535 and TA1537 and E. Coli strain WP2uvrA, in both the presence and absence of S9 mix. The study was performed to GLP and was designed to the requirements of OECD 471. In a preliminary toxicity assay, the maximum dose tested was 5000 ug/plate. Based on the findings of the preliminary toxicity assay, the maximum doses tested in the mutagenicity assay 5000 ug/plate in the presence of S9 with all tester strains, 1000 ug/plate in the absence of S9 with Salmonella tester strains and 5000 ug/plate in the absence of S9 with E.col. Positive controls appropriate for each strain, in the presence and absence of S9 mix, were included. Due to excessive toxicity in some test conditions, portions of the assay were repeated. An independent repeat repeat assay was also performed up to a maximum dose of 5000 ug/plate. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of reverant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535 and TA1537 and E. Coli strain WP2uvrA in both the presence and absence of S9 mix.