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

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:

November 06, 2002 - December 12, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study has been performed according to OECD and/or EC guidelines and according to GLP principles. Deviations: - The highest recommended dose-level (5000 µg/plate) was not tested as the test item was not soluble in the vehicle usually used in this test and no homogeneous suspension could be obtained in the vehicle usually used in this test, the suspension in ethanol shows white particles which may interfere with the scoring. Therefore, a test item extract in ethanol was tested and the dose-levels were expressed as µL of test item extract in ethanol. - The negative control values were not within the laboratory historical control data ranges (first experiment: TA 1535 -/+S9, TA 98 -/+S9, TA 100 -/+S9 and TA 102 +S9; second experiment: TA 1537 -S9, TA 98 +S9, TA 100 +S9 and TA 102 +S9) (historical profile between 20-03-2001 and 15-02-2002). These minor deviations were not considered to have compromised the validity or integrity of the 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:

yes

Remarks:

the dose-level (5000 µg/plate) was not tested as the test item was not soluble and no homogeneous suspension could be obtained in the vehicle usually used in this test. Therefore, a test item extract in ethanol was used for treatment.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

the dose-level (5000 µg/plate) was not tested as the test item was not soluble and no homogeneous suspension could be obtained in the vehicle usually used in this test. Therefore, a test item extract in ethanol was used for treatment.

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:

Hill formula: C31H48N4O2 CAS formula: C31H48N4O2

IUPAC Name:

N,N’’-(methylenedi-4,1-phenylene)bis(N’-octylurea)

Constituent 2

Reference substance name:

N-{4-[(4-{[(octadecylamino)carbonyl]amino}phenyl)methyl]phenyl}-N’-octylurea

Cas Number:

117328-86-6

Molecular formula:

Hill formula: C41H68N4O2 CAS formula: C41H68N4O2

IUPAC Name:

N-{4-[(4-{[(octadecylamino)carbonyl]amino}phenyl)methyl]phenyl}-N’-octylurea

Constituent 3

Reference substance name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-690-3

EC Name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

43136-14-7

Molecular formula:

Hill formula: C51H88N4O2 CAS formula: C51H88N4O2

IUPAC Name:

N,N’’-(methylenedi-4,1-phenylene)bis(N’-octadecylurea)

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): KY-EU
- Appearance: white powder
- Storage condition of test material: in darkness at room temperature

Method

Target gene:

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 with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test:
Without and with S9-mix:
S. typhimurium TA 98 , TA 100 and TA 102: 0.1, 1, 5, 10, 25 and 50 µL of test item extract in ethanol/plate

Mutagenicity experiment 1 and 2:
Without and with S9-mix:
S. typhimurium TA 98 , TA 100, TA 1535, TA 1537 and TA 102: 3.13, 6.25, 12.5, 25 and 50 µL of test item extract in ethanol/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item being insoluble in most vehicle used in this kind of study (water, dimethylsulfoxide, ethanol, acetone and tetrahydrofuran), an extract in ethanol was tested. The dose-levels were expressed as µL of test item extract in ethanol.

The test item was suspended in ethanol 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 immediately before use.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 1 µg/plate in dimethylsulfoxide for TA 100 and TA 1535

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Migrated to IUCLID6: 50 µg/plate in dimethylsulfoxide for TA 1537

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Migrated to IUCLID6: 0.5 µg/plate in dimethylsulfoxide for TA 98

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Migrated to IUCLID6: 0.5 µg/plate in distilled water for TA 102

Controls 5

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine 2 µg/plate in dimethylsulfoxide for TA 98, TA 100, TA 1535 and TA 1537

Remarks:

with S9

Controls 6

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine 10 µg/plate in dimethylsulfoxide for TA 102

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: The preliminary test, both experiments without S9 mix and the first experiment with S9 mix: in agar (plate incorporation);the second experiment with S9: preincubation.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: Preliminary test: one plate/dose-level, two independent main experiments: three plates/dose-level.

NUMBER OF CELLS EVALUATED: Not indicated

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

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

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.

Statistics:

Not performed.

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

Vehicle controls validity:

other: see "additional information on results"

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the preliminary toxicity test, no precipitate was observed in the petri plates when scoring the revertants at all dose-levels.

RANGE-FINDING/SCREENING STUDIES:
- No noteworthy toxicity was noted towards the three strains used, with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The strain-specific positive control values were within the laboratory historical control data ranges.
- The negative control values were not within the laboratory historical control data ranges (first experiment: TA 1535 -/+S9, TA 98 -/+S9, TA 100 -/+S9 and TA 102 +S9; second experiment: TA 1537 -S9, TA 98 +S9, TA 100 +S9 and TA 102 +S9) (historical profile between 20-03-2001 and 15-02-2002). These minor deviations were not considered to have compromised the validity or integrity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was noted towards all the strains used, both with and without S9 mix.

Any other information on results incl. tables

Tables with results and historical control data see the attached background material.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

KY-EU was tested in the Salmonella typhimurium reverse mutation assay with TA 1535, TA 1537, TA 100, TA 98 and TA 102 according to OECD Guideline 471 and GLP principles.
KY-EU did not induce a dose-related increase in the number of revertant colonies in each of the five strains both with and without S9 mix, to concentrations up to and including the highest concentration tested due to precipitation of the test substance. These results were confirmed in a independently repeated experiment.