1-(2-hydroxyethyl)imidazolidin-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-imidazolidone

EC Number:

204-436-4

EC Name:

2-imidazolidone

Cas Number:

120-93-4

IUPAC Name:

imidazolidin-2-one

Details on test material:

- Name of test material (as cited in study report): Ethylene Urea
- physical state, appearance: white solid
- Analytical purity: 90.1 g/100 g (+-0.1 g/100 g). Water: 11.2 g/100 g. Determined by 1H-NMR spectroscopy
- Lot/batch No.: 11-0041
- Storage condition of test material: room temperature
- Test substance No.: 08/0054-4

Method

Target gene:

HPRT locus in V79 cells

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver microsome preparations (Phenobarbital/ß-naphthoflavone induced S9 mix)

Test concentrations with justification for top dose:

30.9, 61.9, 123.8, 247.5, 495.0, 990.0 µg/mL

Vehicle / solvent:

deionised water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: Final concentration: 0.15 mg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Migrated to IUCLID6: Final concentration: 1.1 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.
- Exposure duration: The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

NUMBER OF REPLICATIONS/ NUMBER OF CELLS EVALUATED:
Approximately 1.5E+06 (single culture) and 5E+02 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. Three or four days after treatment 1.5E+06 cells per experimental point were sub-cultivated in medium. Following the expression time of 7 days five cell culture flasks were seeded with about 3 - 5E+05 cells each in medium containing 6-thioguanine. Two additional flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.

DETERMINATION OF CYTOTOXICITY
In a pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results

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: no relevant shift of pH up to the maximum concentration of the test item.
- Effects of osmolality: no relevant shift of osmolarity up to the maximum concentration of the test item.
- Precipitation: no precipitation occurred up to the highest concentration with and without metabolic activation following 4 and 24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 7.7 to 990 µg/mL (≈10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of mutant colonies per 10E+6 cells found in the solvent controls falls within the laboratory historical control data.

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