N,N''-(methylenedi-4,1-phenylene)bis[N'-cyclohexylurea];N-(4-[[4-[[(phenylamino)carbonyl]amino]phenylmethyl]phenyl]-N'-cyclohexylurea;reaction mass of: N,N''-(methylenedi-4,1-phenylene)bis[N'-phenylurea]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October, 1994 - 28 October, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles. The study was not conducted with E.coli and/or with S. typhimurium TA102, which have an AT base pair at the primary reversion site, as currently required. This study is therefore considered a Salmonella typhimurium reverse mutation assay only.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

423-070-8

EC Name:

-

Cas Number:

58890-25-8

Molecular formula:

not applicable (reaction mass)

IUPAC Name:

3-cyclohexyl-1-[4-({4-[(cyclohexylcarbamoyl)amino]phenyl}methyl)phenyl]urea; 3-cyclohexyl-1-[4-({4-[(phenylcarbamoyl)amino]phenyl}methyl)phenyl]urea; 3-phenyl-1-[4-({4-[(phenylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): KY-RB
- Description: White powder

Method

Target gene:

- S. typhimurium: Histidine gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
Experiment 2, all 4 strains:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: daunomycine 4 µg/plate in saline for TA98

Remarks:

without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine 60 µg/plate in saline for TA1537

Remarks:

without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: sodium azide 1 µg/plate in saline for TA1535

Remarks:

without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains (TA1535, TA1537: 5 µg/plate; TA98, TA100: 0.5 µg/plate)

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

An Ames test is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary range-finding test with strain TA100 or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two (2) times the concurrent control, with or without S9-mix.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) It induces at least a two-fold, dose related increase in the mean number of revertant colonies with respect to the number induced by the solvent control in any of the tester strains, either with or without S9-mix. However, any mean plate count of less than 20 is considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at the dose level of 3330 µg/plate and moderate precipitation was observed at the dose level of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 3330 µg/plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance KY-RB is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD 471 guideline (1983) and GLP principles.

Executive summary:

The genetic toxicity of KY-RB was assessed up to the precipitating concentration of 3330 µg/plate using S. typhimurium TA100, TA1535, TA98 and TA1537 strains, in accordance with OECD 471 guideline (1983) and according to GLP principles. All strains showed no cytotoxicity up to and including highest tested concentrations.

All bacterial strains showed negative responses with and without metabolic activation in two independently repeated experiments. The study was not conducted with E.coli and/or with S. typhimurium TA102, which have an AT base pair at the primary reversion site, as currently required. Based on the results of this study, the substance KY-RB is not mutagenic in the Salmonella typhimurium reverse mutation assay.