3-nitro-o-xylene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

restrictions: no historical control data; only 100 metaphases analyzed; no statistical evaluation; methods used for determination of cytotoxicity no longer recommended (PE assay) or not recommended for cell lines (mitotic index)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3-nitro-o-xylene

EC Number:

201-474-3

EC Name:

3-nitro-o-xylene

Cas Number:

83-41-0

Molecular formula:

C8H9NO2

IUPAC Name:

1,2-dimethyl-3-nitrobenzene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Charge 22

- Purity test date:
12.10.1987


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
4°C; light protected

- Solubility and stability of the test substance in the solvent/vehicle:
> 3 months in H20 and DMSO



TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment, the test article was dissolved in ethanol. The final
concentration of ethanol in the culture medium did not exceed 1 % v/v.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix according to Ames et al.

Test concentrations with justification for top dose:

without S9 mix:
7 h: 25; 50; 100; 150 µg/ml
18 h: 15; 100; 150; 200; 250**; 300** µg/ml
28 h: 150; 200; 250**; 300** µg/ml

with S9 mix:
7 h: 25; 50; 100; 150 µg/ml
18 h: 15; 100; 150; 200; 250**; 300** µg/ml
28 h: 150; 200; 250**; 300** µg/ml

**slight precipitation in the culture medium

These concentration ranges have been determined in a pre-experiment using the plating efficiency assay.

Treatment of the cells with 200 µg/ml in the presence of S9 mix reduced clearly the plating efficiency of the V79 cells. Higher concentration precipitated in the culture medium. Also the mitotic index was reduced after treatment with 150 and 200 µg/ml at fixation interval 18 h with and without S9 mix.

The following dose levels were were selected to evaluate metaphases for cytogenetic damage:

without S9 mix:
7 h: 100 µg/ml
18 h: 15; 100; 150 µg/ml
28 h: 150 µg/ml

with S9 mix:
7 h: 150 µg/ml
18 h: 15; 150; 200 µg/ml
28 h: 150 µg/ml

In the main-experiment in the absence of S9 mix at fixation intervals 7, 18 and 28 h 100 and 150 µg/ml were chosen as maxima concentrations because with higher concentrations not enough scorable cells could be found.

In the presence of S9 mix at fixation intervals 7 and 28 h cells after treatment with 150 µg/ml as maximum concentration could be scored for cytogenetic damage; at interval 18 h cells after treatment with 200 µg/ml could be evaluated as highest concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
ethanol

- Justification for choice of solvent/vehicle:
The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells):
Preparation of chromosomes was done 7 h (high dose), 18 h (low,medium and high dose), and 28 h (high dose) after start of treatment with the test article.

SPINDLE INHIBITOR (cytogenetic assays):
colcemid

STAIN (for cytogenetic assays):
V79

NUMBER OF REPLICATIONS:
2 per experiment in 2 experiments (I and II)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
after treatment with colcemid, the cells were treated on the slides in the chambers with hypotonic solution for 20 min at 37 °C. After incubation in the hypotonic solution the cells are fixed with 3:1 absolute methanol:glacial acetic acid. After fixation the cells were stained with aceto-orcein.


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
At least 100 well spread metaphases per slide


DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency index (colony forming ability); mitotic index

Evaluation criteria:

A test article is considered mutagenic in this assay if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test article which producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points is considered non-mutagenic in this assay.

Statistics:

According to the study report, a statistical evaluation of the results could not be performed because there was no adequate statistical procedure available.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid