2,4-xylidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-02-11 to 1991-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to former version of OECD technical guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,4-xylidine

EC Number:

202-440-0

EC Name:

2,4-xylidine

Cas Number:

95-68-1

Molecular formula:

C8H11N

IUPAC Name:

2,4-dimethylaniline

Method

Target gene:

Chromosome aberration

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: CHO cell line, supplied by Toxicology Department Bayer AG, Dr. Lehn, D-5600 Wuppertal, Germany
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Study I/II (with and without S9 mix)
8h: 300; 500; 700; 900; 1000 µg/mL
24h: 30; 100; 300, 500; 700; 900; 1000 µg/mL
30h: 300; 500; 700; 900; 1000 µg/mL

Vehicle / solvent:

- DMSO (Merck, D-6100 Darmstadt, Germany)
- Purity >= 99.5%
- Final concentration in culture medium did not exceed 1% v/v

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Dissolved in nutrient medium, final concentration: 0.72mg/mL

Migrated to IUCLID6: Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Dissolved in nutrient medium, final concentration 4.20µg/mL

Migrated to IUCLID6: With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4hrs
- Expression time (cells in growth medium): 8, 24, 30h

SPINDLE INHIBITOR (cytogenetic assays): 5, 21 and 27h after start of the treatment colcemid was added to the cultures
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 per test group (100 per slide)


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Mutagenicity:
- A dose-related increase in number of structural chromosomal aberrations OR
- A reproducible significant positive response for at least one of these test points
Both, biological and statistical significance should be considered together

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of chi-square test

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Toxic effects were observed in both cytogenetic experiments evidenced by reductions of the mitotic indices after treatment at least in the samples scored treated with the top concentrations
- In both experiments, there were clonal effects in the CHS cell line revealing dicentric marker chromosomes distributed randomly in the treatment and control groups; therefore, dicentric chromosomes were recorded but not included in the calculation of the aberration rates
- Due to their random distribution this measure did not affect the validity of the study

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):
positive with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article 2,4-Xyldine induced structural chromosome aberration in the CHO cell line in the presence of metabolic activation and was non-mutagenic in the absence of S9 mix.

Executive summary:

Based on the results of this chromosome aberration study 2,4-Xylidine was non-mutagnic in the absence and mutagenic in the presence of metabolic activtion.