1,3-diethyldiphenylurea

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:

3.4.2012 - 20.9.2012

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:

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

Deviations:

yes

Remarks:

See Overell remarks

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diethyldiphenylurea

EC Number:

201-645-2

EC Name:

1,3-diethyldiphenylurea

Cas Number:

85-98-3

Molecular formula:

C17H20N2O

IUPAC Name:

1,3-diethyl-1,3-diphenylurea

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Centralit
- Physical state: white solid crystalline
- Analytical purity: 99.9 % (w/w)
- Lot/batch No.: 0500906
- Expiration date of the lot/batch: 16.9.2016
- Stability under test conditions: stable
- Storage condition of test material: in dry room at the temperature < 40°C

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640 with L-Glutamine
PHA-M (Phytohaemagglutinin M)
Foetal Bovine Serum

- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

500, 250, and 100 μg/mL (3h)
600, 500, 250, 100 and 50 μg/mL (24h) - concentrations of 500, 250, 100 μg/mL were used for analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was not soluble in culture medium.

Controls

Untreated negative controls:

yes

Remarks:

untreated culture

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (without metabolic activation), DMSO+S9 (with metabolic activation)

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: thiotepa

Remarks:

thiotepa (without metabolic activation), CPA+S9 (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: diluted in DMSO

DURATION
- Exposure duration: 3h, 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution
STAIN (for cytogenetic assays): Giemsa-Romanowski solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Cytotoxic effect of the test substance is was indicated by relative mitotic index. If the mitotic index is reduced more than 50 %, it will refer to cytotoxicity.
Mutagenic potential is indicated by increasing of number of chromosome aberrations in comparison with the negative solvent control (two-fold increase rule) and/or by dependence of increasing number of aberrations on dose (dose-response relationship).

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentration 5000, 2500, 1000, 800 and 700 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 with metabolic activation
negative without metabolic activation

Under the above-described experimental design, the test substance Centralit was non mutagenic for the human peripheral blood lymphocytes in both experiments without and with metabolic activation.

Executive summary:

The In Vitro Mammalian Chromosome Aberration Test assayed the test substance Centralit for the mutagenicity. The performed test was based on EU Method B.10, Mutagenicity – In Vitro Mammalian Chromosome Aberration Test, which is analogous to the OECD Test Guideline No. 473.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was diluted in dimethylsulfoxide and assayed in doses of 50 - 5000µg/mL, which were applied to cultures in volume of 20µL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Centralit was non mutagenic for the human peripheral blood lymphocytes in both experiments without and with metabolic activation.