N,N''-(methylenedi-4,1-phenylene)bis[N'-octylurea]

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:

07 October, 2003 - 05 November 2003

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. As the suspension in ethanol showed white particles which may interfere with the scoring, the supernatant has been tested but the concentration of the substance is unknown. No information on the solubility of the test substance in ethanol has been reported. As the Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02), the study is considered reliable with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

As the suspension in ethanol showed white particles which may interfere with the scoring, the supernatant has been tested but the conc. of the substance is unknown. Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02).

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Deviations:

yes

Remarks:

As the suspension in ethanol showed white particles which may interfere with the scoring, the supernatant has been tested but the conc. of the substance is unknown. Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-060-3

EC Name:

-

Cas Number:

122886-55-9

Molecular formula:

C31H48N4O2

IUPAC Name:

3-octyl-1-[4-({4-[(octylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): KY-UN
- Stability under test conditions: not stated
- Storage condition of test material: at room temperature and protected from light

Method

Target gene:

- S. typhimurium: Histidine gene

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:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Preliminary test (without and with S9) TA98: 0.1, 1, 5, 10, 25 and 50 µL/plate
Main study 1 and 2: TA1535, TA1537, TA98, TA100 and TA102:
Without and with S9-mix: 3.13, 6.25, 12.5, 25 and 50 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test substance was insoluble in most vehicle used in this type of study (water, dimethylsulfoxide,
ethanol, acetone and tetrahydrofuran), an extract in ethanol was used for treatment.
The test item was suspended in the vehicle at a concentration of 50 mg/mL. This suspension was incubated for approximately 14 hours, at 37°C under shaking. Then the suspension was centrifuged at approximately 2500 rpm during 6 minutes. The supernatant was removed and used for treatment.
The preparations were made inunediately before use.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine 50 µg/plate in DMSO for TA1537

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene 0.5 µg/plate in DMSO for TA98

Remarks:

without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

other: Mitomycin C 0.5 µg/plate in distilled water for TA102

Remarks:

without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

other: sodium azide 1 µg/plate in DMSO for TA1535 and TA100

Remarks:

without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

other: 2-anthramino 2 µg/plate in DMSO for TA1535, TA1537, TA98, TA100 and 10 µg/plate in DMSO for TA102

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: first experiment: in agar (plate incorporation); second experiment with S9-mix: preincubation

DURATION
- Exposure duration: 48 to 72 hours

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

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies.

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

Evaluation criteria:

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As the suspension in ethanol showed white particles which may interfere with the scoring, the supernatant has been tested but the conc. of the substance is unknown. No information on the solubility of the test substance in ethanol has been reported.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 50 µL/plate

RANGE-FINDING/SCREENING STUDIES:
- A moderate toxicity was observed at the dose-level of 50 µL/plate of test item extract in ethanol/plate, in TA 98 strain without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 50 µL/plate.

Applicant's summary and conclusion

Conclusions:

An extract of KY-UN in ethanol was tested in the Salmonella typhimurium assay according to OECD 471 guideline and GLP principles. Based on the results KY-UN is not mutagenic.

Executive summary:

An extract of KY-UN in ethanol was tested in the Salmonella typhimurium assay in strains TA98, TA100, TA1535, TA1537 and TA102 with and without S9 -mix, according to OECD 471 guideline and GLP principles. As the suspension in ethanol showed white particles which may interfere with the scoring, the supernatant has been tested but the concentration of the substance is unknown. No information on the solubility of the test substance in ethanol has been reported. No precipitation and no toxicity was observed up to and including the top dose of 50 µL/plate. Based on the study results, KY-UN is not mutagenic. As the Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02), the study is considered reliable with restrictions.