2,4-Imidazolidinedione, 5-(phenylmethylene)-, (5Z)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jun - 19 Aug 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(5Z)-5-(phenylmethylidene)imidazolidine-2,4-dione

EC Number:

700-728-5

Cas Number:

74805-60-0

Molecular formula:

C10H8N2O2

IUPAC Name:

(5Z)-5-(phenylmethylidene)imidazolidine-2,4-dione

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction was sourced from RCC, Rossdorf, Germany. It was obtained from the livers of male Wistar rats which received on 3 consecutive days doses of 80 mg/kg bw Phenobarbital (i.p.) plus 80 mg/kg bw beta-Naphthoflavone (oral) and were killed 24 h after the last adminstration.
- method of preparation of S9 mix: One part of S9 fraction was mixed with 9 parts of a co-factor solution resulting in the following mixture: 10% S9 fraction, 22 mM KCl, 5 mM glucose-6-phosphate, 4 nM NADP, 100 mM Na2HPO4/Na2H2PO4 (ph 7.4), 8 mM MgCl2.
- concentration or volume of S9 mix and S9 in the final culture medium; 0.5 mL S9 mix in 2.7 mL final culture medium, corresponding to 1.85% S9.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility and activity of the preparation were checked in the S. typhimurium gene mutation assay regularly. Protein content, sterility and activity of the preparation in the S. typhimurium gene mutation assay were certified by the supplier.

Test concentrations with justification for top dose:

25-500 µg/plate for TA 98
50-1000 µg/plate for TA 1535, TA 1537, TA 100 and TA 102

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

Water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st main experiment: plate incorporation, 2nd main experiment: preincubation

DURATION
- 1st main experiment: 72 hours incubation
- 2nd main experiment: 30 min preincubation, 72 hours incubation

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

Criteria for determination of a valid test:
- In the solvent control, each tester strain culture must exhibit a characteristic mean number
of spontaneous revertants.
- To ensure that appropriate numbers of bacteria are plated, overnight culture titers must be
in excess of 108 bacteria/mL.
- The mean of each positive control must exhibit a significant increase in the number of
revertants over the mean value of the respective vehicle control. In case of no significant
increase in the number of revertants by addition of 2-Aminofluorene, a parallel significant
increase by addition of 2-Aminoanthracene will be regarded as sufficient and vice versa.
- Normally, at least four non-toxic dose levels are required to evaluate the assay data.
Exceptions from this requirement, however, may be justified.

Criteria for evaluation of test results:
For a test compound to be considered positive, it must (in two independent experiments)
cause at least a doubling in the mean revertants per plate of at least one tester strain. This
increase must be accompanied by a dose response towards increasing concentrations of the
test article. A test article that does not meet these criteria will be called non-mutagenic in
bacteria. Single increases in revertant frequencies, which are not dose-related and not
reproducible in two independent tests are considered non-relevant. If however these
increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate and above; TA98; without S9 mix at 5000 µ/plate; TA98; with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In a pre-experiment, the toxicity of the test item was determined with strains TA 98 and TA
100. Reduced background lawn was observed in the following cases:
- 500 - 5000 µg/plate; TA 98; without S9 mix.
- 5000 µg/plate; TA 98; with S9 mix.

Precipitation of the test item was observed in the following cases:
- 1600 - 5000 µg/plate; TA 98; with and without S9 mix.
- 1600 - 5000 µg/plate; TA 100; with and without S9 mix

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Under the conditions of this study, the test item did not induce gene mutations by base pair changes or frame-shifts in the genome of the tester strains used. Thus, the test item is considered non-mutagenic in this bacterial reverse mutation assay.