N,N''-(methyl-m-phenylene)bis[N',N'-dimethylurea]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-01 to 2012-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

EC Number:

241-523-6

EC Name:

N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

Cas Number:

17526-94-2

Molecular formula:

C13H20N4O2

IUPAC Name:

3-[3-(dimethylcarbamoylamino)-4-methylphenyl]-1,1-dimethylurea

impurity 1

Reference substance name:

3-[3-(dimethylcarbamoylamino)-2-methylphenyl]-1,1-dimethylurea

Cas Number:

17607-23-7

Molecular formula:

C13H20N4O2

IUPAC Name:

3-[3-(dimethylcarbamoylamino)-2-methylphenyl]-1,1-dimethylurea

impurity 2

Reference substance name:

unknown

IUPAC Name:

unknown

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid

Method

Target gene:

n.a.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank´s salts, glutamine and Hepes (25 mM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

With respect to the molecular weight and the purity (95 %) of the test item, 2782.0 µg/mL of TDI-Urone was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 10.9 and 2782.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
Test concentrations: 10.9, 21.7, 43.5, 86.9, 173.9, 347.8, 695.5, 1391.0, 2782.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

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
- Preincubation period: no preincubation
- Exposure duration: Experiment I 4 hours; experiment II: with S9: 4 hours; without S9: 18 hours
- Expression time (cells in growth medium): 18 hours
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): 15.5 hours

SELECTION AGENT (mutation assays): n.a.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): no stain

NUMBER OF REPLICATIONS: 1.5 replications

NUMBER OF CELLS EVALUATED: 100 metaphases per culture are eveluated for structural chromosomal aberrations

DETERMINATION OF CYTOTOXICITY: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype)
- Determination of endoreplication: the number of endomitotic cells evaluated at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases)

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data and
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: At the selected doses (maximum 2782.0 µg/ml) no influence on solubility, pH value, or osmolarity was detected.
- Evaporation from medium: no
- Water solubility: the test item is watewr soluble
- Precipitation: No precipitation of the test item was observed
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item,the solubility during exposure and changes in osmolarity and pH value at experimental conditions. At the selected dose no influence on solubility, pH value, or osmolarity was detected.The experimental conditions in this pre-experiment were identical to those required and described below for the mutagenicity assay.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test item is not cytotoxic

Applicant's summary and conclusion

Conclusions:

TDI-Urone did not induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro, when tested up to the highest required concentration under conditions of this study.

Executive summary:

In a chromosome aberration assay V79 Chinese hamster cells cultured in vitro were exposed to TDI-Urone, which was dissolved in deionized water, at concentrations of 10.9, 21.7, 43.5, 86.9, 173.9, 347.8, 695.5, 1391.0, 2782.0 µg/ml in the presence and absence of mammalian metabolic activation (S9).

The highest treatment concentration in this study, 2782.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity (95 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. The positive controls did induce the appropriate response. There was no evidence of induced structural chromosomal aberrations in comparison to the controls. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.0 % aberrant cells, excluding gaps) were slightly above the range of the solvent control values (1.0 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenicity study in mammalian cells. Therefore, TDI-Urone is not considered to be clastogenic.