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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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.

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.

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

Target gene:

Histidine gene

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.

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

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

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

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

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 26, 2002 - February 26, 2003

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. Deviation: - The highest recommended dose-level 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. Therefore, a test item extract in ethanol was tested and the dose-levels were expressed as µL of test item extract in ethanol into 5.5 mL treatment medium.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

the highest recommended dose-level was not tested as the test item was not soluble in the vehicle 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.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

the highest recommended dose-level was not tested as the test item was not soluble in the vehicle 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:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples: human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.
To prepare each culture, 0.5 mL of heparinised whole blood was to 5 mL of RPMI 1640 medium, supplemented with 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 mix

Test concentrations with justification for top dose:

No preliminary cytotoxicity test was performed.

The highest technically achievable dose used for treatment was limited by the highest treatment volume used in the laboratory for ethanol (15 μL into 5.5 mL culture medium).

Cytogenetic assay 1, the following dose levels were selected for scoring of chromosome aberrations:
With and without S9-mix : 3.75, 7.5 and 15 μL of ethanol extract/5.5 mL culture medium (3-hour treatment, 20 hour harvest).

Cytogenetic assay 2, the following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix : 3.75, 7.5 and 15 μL of ethanol extract/5.5 mL culture medium (20 hour treatment, 20 hour harvest).
Without S9-mix : 15 μL of ethanol extract/5.5 mL culture medium (44 hour treatment, 44 hour harvest).

With S9-mix : 3.75, 7.5 and 15 μL of ethanol extract/5.5 mL culture medium (3 hour treatment, 20 hour harvest).
With S9-mix : 15 μL of ethanol extract/5.5 mL culture medium (3 hour treatment, 44 hour harvest).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item is not soluble in the vehicles usually used in this test and no homogeneous suspension could be obtained in the vehicles in the vehicles usually used in this test. The suspension obtained in ethanol was the one giving more satisfactory results: adequate mixture between vehicle and test item predisposing to better extraction in the vehicle. Therefore, a test item extract in ethanol was used for treatment and all the dose-levels were expressed as µL of test item extract in ethanol into 5.5 mL treatment medium.

The test item was suspended in ethanol at a concentration of 100 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.

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Migrated to IUCLID6: final concentration of 3 μg/mL (3 hours of treatment) or 0.2 μg/mL (continuous treatment)

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: final concentration of 50 μg/mL and 25 μg/mL (the dose level which gave a satisfactory response in terms of quality and quantity of metaphases and extent of chromosomal damage was selected for analysis)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 3 h (with and without S9 mix), 20 h and 44 h (without S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h and 44 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 200 metaphases/dose, 100/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (1000 cells examined)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using Chi-square test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No data

RANGE-FINDING/SCREENING STUDIES: No preliminary cytotoxicity test was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with the historical data. The frequency of cells with structural chromosome aberrations in the positive controls was significant higher than that of the controls and consistent with the historical data.

OTHER EXAMINATIONS:
No effects of KY-EU on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Tables of the cytogenetic assays and the historical control data, see attached background material.

Conclusions:

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

A chromosome aberration study with KY-EU was performed according to OECD 473 guideline and GLP principles, with cultured peripheral human lymphocytes in two independent experiments. Based on the observed absence of increase in the frequency of cells with structural chromosomal aberrations, it is concluded that KY-EU is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 3, 2012 ~ Oct 25, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD test guidelines and GLP principles. No confirmation experiment for the negative results has been performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

No confirmation experiment for the negative results has been performed.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, induced by Phenobarbital (PB) and 5,6-benzoflavone (BF)

Test concentrations with justification for top dose:

Dose range finding study:
Short time (3h S9-) (12.5, 25.0, 50.0, 100, 200) µL/20mL
Short time (3h S9+) (12.5, 25.0, 50.0, 100, 200) µL/20mL
Continuous (24h S9-) (12.5, 25.0, 50.0, 100, 200) µL/20mL

Main Study
Short time S9- ( 50.0, 25.0, 12.5, 6.25, 3.13) µL/20mL
Short time S9+ (100, 50.0, 25.0, 12.5, 6.25) µL/20mL
Continuous S9- (120, 60.0, 30.0, 15.0, 7.50) µL/20mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the test substance was not dissolved or uniformly suspended in water for injection, 0.5% carboxymethylcellulose sodium salt solution, acetone and tetrahydrofuran but uniformly suspended in DMSO. However, the test substance in DMSO was not fluid.
Therefore, ethanol was selected as the extraction solvent for this study based on the information provided by the sponsor.

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs (short time), 24 hrs (continuous)
- Expression time (cells in growth medium): 1 day
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) (final concentration of 3 μg/mL)

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY :
- Method: cloning efficiency, relative total growth

Evaluation criteria:

- The mutation frequency in the test substance groups should be statistically increased as compared to the negative control group at one or more.
- The mutation frequency should be increased dose dependently

Statistics:

Statistical analysis was performed using SAS Program (version 9.2, SAS Institute Inc.,U.S.A.). The Mutation frequency was analyzed utilizing Dunnett’s test for multiple comparisons (significance level: 0.05 and 0.01, two-tailed).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Based on the results of this study, the test substance, KY-EU, did not show mutagenic potential in the mammalian cell gene mutation test system using L5178Y/TK+/- mouse lymphoma cells under the conditions of this study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The analysis of the mutant frequency was evaluated up to 100 μL/20 mL culture medium and 25 μL/20 mL culture medium in the short time treatment with and without metabolic activation, respectively, and 30 μL/20 mL culture medium in the continuous treatment without metabolic activation.

The dose levels of 50 μL/20 mL culture medium in the short time treatment without metabolic activation and 60 and 120 μL/20 mL culture medium in the continuous treatment without metabolic activation were not retained for analysis because of an excessive cytotoxicity.

As a result, the mutant frequency (MF) for the test substance groups was not statistically significantly increased when compared to that for the negative control group in the short time treatment with and without metabolic activation and in the continuous treatment without metabolic activation (Dunnett’s test, p≥0.05).

In the positive control group, the MF was statistically significantly increased when compared to that for the negative control group (short time treatment without metabolic activation and continuous treatment without metabolic activation: Dunnett’s test, p<0.01,short time treatrment with metabolic activation: Dunnett’s test, p<0.05).

Conclusions:

Interpretation of results (migrated information):
negative

KY-EU did not show mutagenic potential in the mammalian cell gene mutation test system using L5178Y/TK+/- mouse lymphoma cells with and without metabolic activation.

Executive summary:

The mouse lymphoma assay with KY-EU was conducted according to OECD 476 guideline and GLP principles.

The mutant frequency (MF) for the test substance groups was not statistically significantly increased when compared to that for the negative control group in the short time treatment with and without metabolic activation and in the continuous treatment without metabolic activation (Dunnett’s test, p≥0.05).

Based on the results KY-EU did not show mutagenic potential in the mammalian cell gene mutation test system using L5178Y/TK+/- mouse lymphoma cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

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.

Chromosome aberration test:

A chromosome aberration study with KY-EU was performed according to OECD 473 guideline and GLP principles, with cultured peripheral human lymphocytes in two independent experiments. Based on the observed absence of increase in the frequency of cells with structural chromosomal aberrations, it is concluded that KY-EU is not clastogenic in human lymphocytes.

Mouse lymphoma assay:

The mouse lymphoma assay with KY-EU was conducted according to OECD 476 guideline and GLP principles.

The mutant frequency (MF) for the test substance groups was not statistically significantly increased when compared to that for the negative control group in the short time treatment with and without metabolic activation and in the continuous treatment without metabolic activation (Dunnett’s test, p≥0.05).

Based on the results KY-EU did not show mutagenic potential in the mammalian cell gene mutation test system using L5178Y/TK+/- mouse lymphoma cells with and without metabolic activation.

Justification for selection of genetic toxicity endpoint
No study was selected, since all three in vitro studies were negative.

Short description of key information:
Three in vitro tests were performed (AMES test OECD 471, chromosome aberration test OECD 473 and MLA assay OECD 476). KY-EU was shown to be negative with and without metabolic activation in all three tests.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, KY-EU is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.