2-chlorotoluene

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:

From 18 February 1982 to 28 June 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Colony sizing not reported

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

2-chlorotoluene

EC Number:

202-424-3

EC Name:

2-chlorotoluene

Cas Number:

95-49-8

Molecular formula:

C7H7Cl

IUPAC Name:

1-chloro-2-methylbenzene

Test material form:

liquid

Details on test material:

purity not specified, or relating to pure substance in case of QSAR

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: adult, male Sprague Dawley ratsm, Aroclor 1254 induced
- method of preparation of S9 mix: prepared according to method of the laboratory
- concentration or volume of S9 mix and S9 in the final culture medium: 0.3 nl/10mL

Test concentrations with justification for top dose:

without metabilit activation:
Experiment 1: 1.95, 3.91, 7.81, 15.6, 31.3 nl/ml
Experiment 2: 7.5, 10, 15, 20, 30, 40 nl/ml

with metabolic activation:
Experiment 1: 3.91, 7.81, 15.6, 31.3, 62.5 nl/ml
Experiment 2: 10, 15, 20, 30, 40, 60 nl/ml

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate; dimethylnitrosamine

Remarks:

ethylmethane sulfonate is used as positive control substance for assay without metabolic activation
dimethylnitrosamine is used as positive control substance for assay with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: 4 (trial 3 was terminated due to contamination)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 - 3 days
- Selection time: 3 x 10E 6 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 days incubation.
- Method used: agar
- selective agent: -bromo-2' deoxyuridine (BrdU), selectin medium contains 100 µg/ml BrdU

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Relative cytotoxicities is expressed as the reduction in growth compared to the growth of untreated cells. This is used to select seven to ten doses in the range of 0 to 50-90% reduction in the 24-hour growth.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective). The ratio of resistant colonies to total viable cell number is the mutant frequency.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: without: 40 nl/ml; with: 60 nl/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Without metabolic activation , the test material was cytotoxic at 62.5 nl/ml. Five treatments from 1.95 nl/ml to 31.3 nl/ml were therefore chosen for mutant analysis in an attempt to obtain a wide range of toxic action. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 31.7x10 E-6. However, only moderate toxicities were induced (percent relative growths, 62.1% to 39.9%). Since it is preferable to include in the evaluation results from treatments that induced high toxicities (percent relative growths, 10% to 15%), another assay was performed.
In the second experiment without metabolic activation, the test material was assayed from 7.5 nl/ml to 40 nl/ml with small increments between concentrations. The assayed treatments induced percent relative growths ranging from 58.3% to 19.3% which included moderate toxicites. A sharp toxicity curve prevented analysis of highly toxic treatments; the next highest concentration (60 nl/ml) was lethal. In order for a treatment to be considered mutagenic in this assay a mutant frequency exceeding 38.5x10 E-6 was required and the assayed treatments induced mutant frequencies ranging from 10.9x10E- 6 to 23.7x10E- 6 . Since no evidence for mutagenic activity activity was observed, the test material was considered nonmutagenic without activation at oncentrations that approached lethality.
In the presence of metabolic activation, the test material was assayed from 10 nl/ml to 60 nl/ml. The minimum criterion for mutagenicity in this assay was a mutant frequency exceeding 52.4x 10E-6 . None of the assayed treatments induced this level of mutant action. The observed toxicities ranged from nondetectable to moderate (percent relative growths, 127.1% to 23.8%). High toxicities could not be analyzed because of a sharp toxicity curve (80 nl/ml was excessively toxic).
The test material was therefore considered nonmutagenic with activation at treatments that approached excessive toxicity. In the assays used in this evaluation, the average cloning efficiencies for the solvent and untreated negative controls varied from 80.1% and 84.4% without activation to 76.8% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of
the background.
For details on results see illustration below.

Any other information on results incl. tables

The test material, orthochlorotoluene, did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The test material was assayed up to 40 µl/ml without activation and 60 nl/ml with activation, and nondetectable to moderate toxicities were induced. Highly toxic treatments were not available for analysis because of a sharp toxicity curve. None of the assayed treatments induced a significant increase in the background. The test material is therefore considered inactive with and without metabolic activation in the mouse lymphoma forward mutation assay at concentrations that approached excessive toxicity.

Applicant's summary and conclusion

Conclusions:

In an mammalian cell gene mutation assay performed similar to OECD test guideline 476 with mouse lymphoma L5178Y cells under GLP conditions, the test substance was negative in two separate experiments with and without metabolic activation.

Executive summary:

2-chlorotoluene was tested in the mouse lymphoma assay with and without metabolic activation (Aroclor-induced rat liver S9 mix). Two separate experiments with concentrations of  0, 1.95, 3.91, 7.81, 15.6, 31.3 nl/ml (experiment 1 and 0, 7.5, 10, 15, 20, 30, 40 nl/ml (experiment 2, both without metabilit activation) and 0, 3.91, 7.81, 15.6, 31.3, 62.5 nl/ml (experiment 1) or  0, 10, 15, 20, 30, 40, 60 nl/ml (experiment 2, both with metabolic activation) were performed.

All experiments were negative (with and without metabolic activation), the test material, orthochlorotoluene, did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The test material was assayed up to 40 µl/ml without activation and 60 nl/ml with activation, and nondetectable to moderate toxicities were induced. Highly toxic treatments were not available for analysis because of a sharp toxicity curve. None of the assayed treatments induced a significant increase in the background. The test material is therefore considered inactive with and without metabolic activation in the mouse lymphoma forward mutation assay at concentrations that approached excessive toxicity.