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

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:

2001-04-04 to 2001-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

1-{5-[(dimethylcarbamoyl)amino]-2-methylphenyl}-3,3-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

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N"(4-Methyl-m-phenylen)bis(N',N'­dimethylharnstoff)
- Substance type: organic
- Physical state: colourless solid
- Analytical purity: 100 %
- Purity test date: 01 March 2000
- Lot/batch No.: 0232 07
- Expiration date of the lot/batch: March 2003
- Storage condition of test material: store dry in closed containers
- Other: stable at dry storage conditions for at least 2 years

Method

Target gene:

Gene involved in histidine synthesis.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n.a.

Additional strain / cell type characteristics:

other: deep rough (rfa) mutation; uvrB gene deletion (BER)

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Experiments I and II: 31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-NOPD

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation;

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant count through the mean values of the solvent control.

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (range of spontaneaus reversion frequencies-historical control- data range):
TA 1535: 5-30
TA 1537: 4-32
TA 98: 18-63
TA 100: 79-197
TA 102: 173-396
-corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement over the control plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

TDI-Urone has no mutagenic potential under the conditions of this study.

Executive summary:

The test item TDI-Urone was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre­ incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 100, TA 98 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I and II: 31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg//plate

No toxic effects of the test item (indicated by a reduction of the background lawn or by a reduction of the spontaneaus rate) were observed in both experiments with or without metabolic activation.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with TDI-Urone at any concentration level, either in the presence or absence of metabolic activation (S9-mix) in experiment I and II. There was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance.

Therefore, TDI-Urone is not considered to be mutagenic in bacterial cells.