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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG 471): negative

In vitro chromosome aberration test (OECDTG 473): negative

Genemutation in mammalian cells (OECDTG 490): negative

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:

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.

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).

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

Target gene:

- S. typhimurium: 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 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.

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

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

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

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

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.

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.

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.

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:

19 November, 2003 - 14 January, 2004

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 no homogeneous suspension in ethanol could be obtained, 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

As no homogeneous suspension in ethanol could be obtained, the supernatant has been tested but the concentration of the substance is unknown. The Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Deviations:

yes

Remarks:

As no homogeneous suspension in ethanol could be obtained, the supernatant has been tested but the concentration of the substance is unknown. The Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02).

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 (one male and one female for each experiment) and collected into heparinized sterile tubes.
- Lymphocyte cultures
0.5 mL of heparinized whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and phytohemagglutinin.
- 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:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

First cytogenetic test:
With and without S9-mix, 3 h exposure time, 20 h harvest time: 3.75, 7.5 and 15 µL
Second cytogenetic test:
Without S9-mix, 20 h exposure; 20 h fixation: 3.75, 7.5 and 15 µL
Without S9-mix, 44 h exposure; 44 h fixation: 15 µL
With S9-mix, 3 h exposure; 20 h fixation: 3.75, 7.5 and 15 µL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test substance is not soluble in the vehicle usually (DMSO, culture medium) used in this test and no homogeneous suspension (to the naked eye) could be obtained in the vehicles usually used in this test.
Therefore, a test substance extract in ethanol was used for treatment and the dose-levels were expressed as µL of test substance extract in ethanol/5.5 mL culture medium. The highest technically achievable dose used for treatment was limited by the highest treatment volume usually used in the Laboratory for ethanol (15 µL into 5.5 mL culture medium).
The test substance 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:

other: mitomycin C (MMC) in distilled water, used at a final concentration of 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment)

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

other: cyclophosphamide (CPA), used at a final concentration of 12.5 or 25 µg/mL

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

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

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

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

Evaluation criteria:

Acceptance criteria:
This study was considered valid since the following criteria were met:
- the frequency of cells with structural chromosome aberration in the vehicle controls was consistent with the historical data,
- the frequency of cells with structural chromosome aberration in the positive controls was significantly higher than that of the controls and consistent with the historical data.

Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration 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 aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the x^2 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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

As no homogeneous suspension in ethanol could be obtained, 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.

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 15 µL test substance extract in ethanol

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of cells with structural chromosome aberration of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Conclusions:

A chromosome aberration study with an extract of KY-UN in ethanol was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that KY-UN is not clastogenic in human lymphocytes.

Executive summary:

An extract of KY-UN in ethanol was tested in a chromosome aberration study according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. As no homogeneous suspension in ethanol could be obtained, 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 15 µL into 5.5 mL culture medium.

Both in the presence and absence of S9 -mix the test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations.

Based on the study results, it is concluded that KY-UN is not clastogenic. As the Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02), the study is considered reliable with restrictions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2017 - 28 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

The test item precipitated in the exposure medium at concentrations of 12.5 μg/ml and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 50 μg/mL.
- Dose-range finding test (with and without S9; cytotoxicity test): 3.13, 6.3, 12.5, 25 and 50 μg/mL
- Experiment 1 (with and without S9) and experiment 2 (without S9): 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3 and 12.5 μg/mL.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: as recommended in OECD test guideline 490

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL

DURATION
- Exposure duration:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT: 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- test concentrations: 1 per test concentration
- positive control: 1
- solvent control: 2

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING STUDY
- The test item precipitated in the exposure medium at the test item concentration of 12.5 μg/mL and above.
- Both in the absence and presence of S9-mix, in the 3-hour exposure period, no toxicity in the relative suspension growth was observed up to a test item concentration of 50 μg/mL compared to the solvent control.
- In the 24-hour exposure period (without S9-mix), no toxicity in the relative suspension growth was observed up to a test item concentration of 50 μg/mL compared to the solvent control.

FIRST AND SECOND EXPERIMENT (Mutation experiments)
- The test item precipitated in the exposure medium at the test item concentration of 12.5 μg/mL.
- No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL DATA: see table 1 and table 2

ACCEPTABILITY CRITERIA
- The positive controls both produced significant increased in the mutation frequency, which was within the 95% control limits of the historical data range.
- The absolute cloning efficiency of the solvent controls (CEday2) was 108 and 115 (without S9) and 70 and 93 (with S9) in experiment 1 and 76 and 93 in experiment 2.
- The spontaneous mutation frequency in the solvent control was 99 and 87 (without S9) and 97 and 64 (with S9) per 10^6 survivors in experiment 1 and 76 and 85 per 10^6 survivors in experiment 2. These frequencies were within the 95% control limits of the historical data range.
- The suspension growth over the two-day expression period for cultures treated with ethanol was between 17 and 23 (3 hour treatment) and 57 and 70 (24 hour treatment).

Table 1Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	86	81	87
SD	23	26	28
n	220	202	273
Upper control limit (95% control limits)	135	135	145
Lower control limit (95% control limits)	37	28	28

SD = Standard deviation; n = Number of observations

Table 2Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	857	688	1710
SD	246	187	815
n	110	102	139
Upper control limit (95% control limits)	1425	1124	4214
Lower control limit (95% control limits)	289	253	-793

SD = Standard deviation; n = Number of observations

Distribution historical control data from experiments performed between January 2013 and November 2016. 

Conclusions:

KY-UN is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of KY-UN to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The test item was tested up to and including a dose of 12.5 μg/mL in the absence of S9 in a 3 and 24 hour treatment period and in the presence of S9 in a 3 hour treatment period.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test item precipitated at the highest dose level. No toxicity was observed up to and including the highest dose level, in the absence and in the presence of S9. The test item did not induce an increase in the mutation frequency. Therefore, KY-UN is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

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.

Chromosoom aberration test:

An extract of KY-UN in ethanol was tested in a chromosome aberration study according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. As no homogeneous suspension in ethanol could be obtained, 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 15 µL into 5.5 mL culture medium.

Both in the presence and absence of S9 -mix the test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations.

Based on the study results, it is concluded that KY-UN is not clastogenic. As the Competent Authority of France accepted the study in 2004 (notification no. 04-00-0000-02), the study is considered reliable with restrictions.

Mouse lymphoma test:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of KY-UN to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The test item was tested up to and including a dose of 12.5 μg/mL in the absence of S9 in a 3 and 24 hour treatment period and in the presence of S9 in a 3 hour treatment period.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test item precipitated at the highest dose level. No toxicity was observed up to and including the highest dose level, in the absence and in the presence of S9. The test item did not induce an increase in the mutation frequency. Therefore, KY-UN is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies, the substance KY-UN does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its amendments.