2,4-Imidazolidinedione, 5-(2-methylpropylidene)-, (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

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-(2-methylpropylidene)imidazolidine-2,4-dione

EC Number:

700-726-4

Cas Number:

1369499-44-4

Molecular formula:

C7H10N2O2

IUPAC Name:

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

Method

Target gene:

his operon

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 : liver of male Wistar rats which received on 3 consecutive days doses of 80 mg/kg bw Phenobarbital (i.p.) plus 80 mg/kg bw β-Naphthoflavone (oral) and were killed 24 h after the last administration.
- method of preparation of S9 mix: S9 mix was prepared freshly before use. One part of S9 fraction was mixed with 9 parts of a cofactor solution resulting in the following mixture 10% S9 fraction, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM Na2HPO4/NaH2PO4 (ph 7.4), 8 mM MgCl2
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): enzymatic activity was checked on all strains with 2-aminofluorene and 2-aminoanthracene.

Test concentrations with justification for top dose:

50 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of bacteria and S9 activity

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Tester strain TA 98 without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Tester strains TA 100 and TA 1535 without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Tester strain TA 102 without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Tester strain TA 1537 without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminofluorene and 2-Aminoanthracene

Remarks:

Tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 with metabolic activation

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

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The following criteria must be met for the mutagenicity assay to be considered valid:
- 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 10E+08 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.

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 item. A test item 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, other: 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:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the plate incorporation test reduced background lawn was observed in the following cases:
5000 µg/plate; TA 98, TA 1535 and TA 1537; without S9 mix.
In the preincubation test reduced background lawn was detected in the following case:
5000 µg/plate; TA 1537 ; without S9 mix.
Additionally, precipitation was observed in the preincubation test in the following cases:
5000 µg/plate; TA 98, TA 100, TA 102, TA 1535 and TA 1537 ; with S9 mix
5000 µg/plate; TA 98, TA 100, TA 102, TA 1535 and TA 1537 ; without S9 mix

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
Under conditions tested, the test item did not induce gene mutations by base pair changes or frame-shifts in the genome of the tester strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102. Thus, the test item is considered non-mutagenic in this bacterial reverse mutation assay.