Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Sarada (2016)

Under the conditions of this study, the test material was considered to be non-mutagenic in the bacterial reverse mutation assay.

Chromosome Aberration: Furukuma (2020)

Under the conditions of the study the test material is considered “equivocal” for the chromosome-aberration-inducing potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 July 2016 to 25 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test solution of the maximum concentration was prepared, which was fully stirred with mixer and dissolved after the test substance was weighed and the solvent was added. The lower doses were prepared by diluting stepwise from the test solution of the maximum concentration. The test was carried out with the weight converted by multiplying measured weight by 0.820 because the purity of the test substance was 82.0 wt %. Preparation of the test solution was carried out just prior to use under lamps with ultraviolet absorbent filter.

The amount of test substance and the volume of solvent used for the preparation of the test solution of the maximum concentration in each test were as follows.
Preliminary test [Prepared maximum concentration of test solution: 50 mg/mL]
- The amount of test substance 113.0 mg x 0.820 = 92.660 mg
- The volume of solvent (Water for injection) 1.853 mL

The first main test [Prepared maximum concentration of test solution: 50 mg/mL]
- The amount oftest substance 421.5 mg x 0.820 = 345.630 mg
- The volume of solvent (Water for injection) 6.913 mL

The second main test [Prepared maximum concentration of test solution: 50 mg/mL]
- The amount oftest substance 414.7 mg x 0.820 = 340.054 mg
- The volume of solvent (Water for injection) 6.801 mL
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: S. typhimurium TA100 was supplied by The Division of Mutagenesis, National Institute of Hygienic Sciences, Japan (The Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences, Japan at present).
Other S. typhimurium TA strains were supplied by Dr. Ames, U.C. Berkeley, CA, U.S.A.
E. coli WP2 uvr A was supplied by Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan.
- Methods for maintenance in cell culture if applicable: The bacterial suspension and DMSO (spectrophotometric grade) were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided into 0.3 mL aliquots, and then frozen and stored at -85 to -80 °C.
- Charecterisation: The number of viable cell, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.
- Pre-culture procedure: A bacterial suspension of each strain (20 μL of S. typhimurium TA strains, 5 μL of E. coli WP2 uvrA) was inoculated into an L-form culture tube (35 mL capacity) containing 10 mL of Nutrient Broth. This culture tube was left at 4 °C until starting incubation, and then incubated while shaking (100 rpm) in a water bath at 37 °C for 8 hours. After incubation, the optical density was measured and the number of viable cell was calculated by growth curve for each strain. The bacterial cultures were stored at room temperature until starting the test.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cultures were maintained in nutrient broth no. 2 supplied by Oxoid Limited. Top agar was bacto-agar (Difco Laboratories) and minimal glucose agar plates were Tesmedia AN (Oriental Yeast , Co. Ltd.).
- Properly maintained: Yes
Additional strain / cell type characteristics:
other: Base-pair substitution type: Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA; Frame-shift type: Salmonella typhimurium T A98 and TA 1537
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 1.2, 4.9, 20, 78, 313, 1 250 and 5 000 μg/plate.
Definitive test: 313, 625, 1250, 2 500 and 5 000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection.
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was soluble at 100 g/L and more in water. Therefore water for injection was used as solvent for preparation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Treated only with water that was used to prepare the test solution.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino Jacridine · 2HCl and 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation method
- Without metabolic activation: 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution.

- With metabolic activation: 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer.

DURATION
- Preincubation period: The mixture was pre-incubated in a water bath at 37 °C for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. And 0.1 mL of the positive control solution was carried out equally.
- Exposure duration: All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

NUMBER OF REPLICATIONS: One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in two main tests which were performed at the same doses.

- OTHER: For the sterility test, 0.1 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tube, 2.0 mL of top agar were then added to the tube, and the contents of each tube were poured over the surface of the minimal glucose agar plate. These operations were conducted under lamps with ultraviolet absorbent filter.
As top agar, the 0.5 mM biotin-0.5 mM L-histidine solution and the 0.5 mM L-tryptophan solution were added to the soft agar solution (0.6 % Agar and 0.5 % NaCl) by volume of 1/10, for the S. typhimurium TA strains and the E. coli strain, respectively.

- Counting procedure: The number of revertant colonies was counted with a colony counter.
Rationale for test conditions:
In the preliminary test, the growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation.
Therefore, as the highest dose level of the test substance in the main tests, the 5 000 μg/plate dose was selected for all strains both with and without metabolic activation. This highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.
Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results at each concentration were demonstrated with the mean and the standard deviation.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.
The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in historical data, indicating that this study was performed correctly.
From these results, mutagenicity of the test substance was judged negative. The growth inhibition of the test strains by the test substance was not observed. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation.
In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.
Conclusions:
Under the conditions of this study, the test material was considered to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals under GLP conditions.

Mutagenicity potential of the test material was assessed with Salmonella typhimurium TAl00, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in to main tests. The dose range for the main test was determined from the preliminary test using the following seven dose levels: 1.2, 4.9, 20, 78, 313, 1 250 and 5 000 μg/plate. In the preliminary test, the growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 5 000 μg/plate dose was selected for all strains both with and without metabolic activation. This highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

The main test concentrations were 313, 625, 1250, 2500 and 5 000 µg/plate. The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in historical data, indicating that this study was performed correctly.

Neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control, nor a dose-related response, was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

Under the conditions of this study, the test material was considered to be non-mutagenic in the bacterial reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November 2019 - 25 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.
- Stability under storage conditions: stable.
- Solubility in dimethylsulfoxide: insoluble at 200 mg/mL
- Solubility in acetone: ≤ 0.03 g/L
- Saline solubility: soluble at 20.0 mg/mL
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type: CHL/IU (a Chinese hamster lung fibroblast cell line).
- Source: Health Science Research Resources Bank, Japan Health Science Foundation.
- Suitability of cells: CHL/IU cells had relatively larger and fewer chromosomes, which were suitable for observation. There was also abundant background data on the effects of chemicals on CHL/IU cells.

CELL LINES
- Absence of Mycoplasma contamination: Mycoplasma contamination not detected.
- Number of passages: 5 at the time of receipt. Cells passaged 11 times in total were frozen for preservation. After thawing, cells whose passage number was 30 or less were used for the test.
- Cell doubling time: 14 hours.
- Modal number of chromosomes: 25

MEDIA USED
- Eagle’s MEM (SIGMA-ALDRICH Co. LLC.) supplemented with foetal bovine serum (Lot No. 1524128, Biological Industries) at a concentration of 10 %. Foetal bovine serum was inactivated at this facility.
- Culture dish: 60 mm Petri dish (effective culture area: 20.8 cm², Becton, Dickinson and Company).
- Culture temperature and CO2 concentration: 37 °C and 5 % (SCA-80C CO2 incubator, ASTEC, Ltd.)
- Preculture conditions: 2 x 10^4 cells were inoculated in the Petri dish containing 5 mL of medium and cultured for 3 days.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
- Source of S9: Oriental Yeast Co., Ltd. Lot No.: 19060703.
- Composition of S9 mix (1 mL): S9 (0.3 mL), MgCl2 (5 μmol), KCl (33 μmol), glucose-6-phosphate (5 μmol), NADP (4 μmol), HEPES (4 μmol) and purified water (0.7 mL).
- Amount of S9 added (final concentration): 5%
- Amount of S9 protein added (final concentration: 1.1 mg/mL
Test concentrations with justification for top dose:
SHORT TERM TREATMENT PROCESS
- Absence of a metabolic activation system: relative increase in cell counts (RICC) of < 50% was not observed at any dose. Therefore, 2.00 mg/mL was determined to be the maximum concentration and 3 concentrations (0.500, 1.00 and 2.00 mg/mL) were selected which corresponded to a geometric sequence with a common ratio of 2.
- Presence of a metabolic treatment process: growth inhibition of ≥ 50 % (100-RICC) was observed at a dose of 2.00 mg/mL. Therefore, 2.00 mg/mL was determined to be the maximum concentration and 6 concentrations (0.354, 0.500, 0.707, 1.00, 1.41 and 2.00 mg/mL) were selected which corresponded to a geometric sequence with a common ration of 2^1/2.

CONTINUOUS TREATMENT PROCESS
- Cell growth inhibition of ≥ 50 % (100-RICC) was observed at and above doses of 0.500 mg/mL. Therefore, the top dose was considered at the next highest dose level of 1.00 mg/mL with a common ratio of 2^1/3 with the following settings used: top dose of 0.794 mg/mL at 7 levels in total with a common ratio of 2^1/3 (0.199, 0.250, 0.315, 0.397, 0.500, 0.630 and 0.794 mg/mL).

- Cytotoxicity was observed in short-term treatment process in the presence of a metabolic activation system and continuous treatment process for 24 hours of the cell growth inhibition test, therefore the concentration of the test material solution was not analysed.
Vehicle / solvent:
- Saline (Water for injection, Lot No. K9E99, Otsuka Pharmaceutical Factory, Inc.) was used as a solvent to prepare the test material solutions.

- The molecular weight of the test material was 872.95, thus, the solubility of the test material was checked at the final concentration of 2.00 mg/mL in the media. The test material was soluble at 20.0 mg/mL in saline and insoluble at 200 mg/mL in DMSO therefore saline was selected as a solvent.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
SHORT TERM TREATMENT PROCESS
Cell growth inhibition preliminary test:
- Number of cultures per concentration: 1
- Number of cells in each dish: 2 x 10^4 cells
- Precultivation period: 3 days
- Test material solution: one test tube containing the culture medium was prepared for one dose level beforehand. Test tubes were added with 0.3 mL of test material solution and mixed with the medium (3 mL). Test tubes used for the test with metabolic activation were supplemented with 0.5 mL of S9 mix.
- Incubation period in CO2 incubator: 6 hours immediately after replacing medium in culture dishes with those in test tubes and 18 hours after dishes were checked for precipitation of test material, washed with PBS to remove test material and added with 5 mL of fresh medium.
- Observation of cells: after incubation cells were observed under the inverted phase-contrast microscope.
- Measurement of cytotoxicity: the relative increase in cell count (RICC) was calculated.

Chromosome aberration test:
- Number of cultures per concentration: 2
- Preculture and treatment with test material: same as cell growth inhibition test.

CONTINOUS TREATMENT PROCESS
Cell growth inhibition preliminary test:
- Number of cultures per concentration: 1
- Number of cells in each dish: 2 x 10^4 cells
- Precultivation period: 3 days
- Test material solution: one test tube containing the culture medium was prepared for one dose level beforehand. Test tubes were added with 0.5 mL of test material solution and mixed with the medium (5 mL).
- Incubation period in CO2 incubator: 24 hours immediately after replacing medium in culture dishes with those in test tubes.
- Observation of cells: after incubation cells were observed for precipitation of test material.
- Measurement of cytotoxicity: the relative increase in cell count (RICC) was calculated.

Chromosome aberration test:
- Number of cultures per concentration: 2
- Preculture and treatment with test material: same as cell growth inhibition test.

CHROMOSOME SPECIMEN PREPARATION
- Colcemid solution (GIBCO) was added to the culture plates at the final concentration of 0.2 μg/mL, 2 hours prior to the end of the incubation period.
- After treatment the culture medium in the dish was transferred to a centrifuge tube, the cells attached to the dish were detached using 0.25 % trypsin solution and added to the same centrifuge tube. Cells were centrifuged and the supernatant was removed.
- Cells were treated with 0.075 mol/L potassium chloride solution in a thermostat water bath at 37 °C for 15 minutes.
- A few drops of fixing solution (mixture of alcohol and acetic acid) were added for semi fixation of cells. Cells were centrifuged and the solution in the tube was replaced with fresh fixing solution. This was repeated until cells were sufficiently fixed.
- A cell suspension was prepared, put on the slide glass and air-dried. The chromosome specimen was stained with 2 % Giemsa solution.

METHOD OF COUNTING CELLS
- Cells were detached from the culture dish using 0.25 % trypsin solution and a cell suspension was prepared.
- A part of the cell suspension was stained with trypan blue and cells were counted using a cell counter (Bio-Rad Laboratories, Inc.).

OBSERVATION OF THE SPECIMENS OF CHROMOSOME ABERRATION TEST
- Blind method was used. Well-spread metaphase cells (300 cells per concentration) were examined under a microscope (600-1000 x).
- Chromosome structural aberration: structural aberrations were recorded (chromatid break, chromatid exchange, chromosome break, chromosome exchange, other fragmentation). A cell was counted as 1 aberrant cell if it had 1 or more aberrations of any type. The types of aberrations were also recorded.
- Chromosome numerical aberration: the number of cells was recorded when cells with chromosome doubling of haploid (triploid of hyperploid: called “poly”) or endoreduplication (called “end”) occurred.
- Gaps: chromatid and chromosome gaps (called “g”) were not included in the structural aberration. Achromatic regions smaller than the chromatid width in a coaxial line were considered gaps, whereas achromatic regions larger than the chromatid width were considered break-type aberrations.
Rationale for test conditions:
- Since the elongation of cell cycle was not observed at continuous treatment process for 24 hours, the cell growth inhibition test and chromosome aberration test using the continuous treatment process for 48 hours were not carried out.
- As the modal number of chromosomes in the CHL/IU cells was 25, cells with a chromosome number of 25 ± 2 were regarded as the object of the analysis of chromosome structural aberration.
Evaluation criteria:
ACCEPTANCE CRITERIA
The assay was considered valid if the frequency of chromosomes with structural aberration or numerical aberration met the following criteria with the control values of the mean of two plates. In case a deviation occurs from the following criteria, the treatment group was regarded as invalid and a retest was performed.
1) The chromosome aberration of the negative control is inside the control limits based on the laboratory historical negative control data.
2) The structural aberration of the positive control is inside the range based on the laboratory historical positive control data, and it should be statistically significant compared to the concurrent negative control.

POSITIVE RESULTS
- All of the following criteria must were fulfilled:
1) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) The increase is dose-related when evaluated with the trend test.
3) All of the results are outside the control limits based on the laboratory historical negative control data.

NEGATIVE RESULTS
- All of the following criteria were fulfilled:
1) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) There is no concentration-related increase when evaluated with the trend test.
3) All of the results are inside the control limits based on the laboratory historical negative control data.

In case the results were neither clearly negative nor clearly positive an analysis of additional cells or a repeat experiment using possibly modified experimental conditions was performed. If even after further investigation, a conclusion of positive or negative result could not be made, the test material response was concluded to be equivocal.
Statistics:
- The test concentrations were compared with the negative control by use of Fisher’s exact test (two sides, p < 0.05 is significant., GraphPad Prism 8 Software).
- The trend analyses for dose-related were performed by use of Chochran-Armitage trend test (two sides, p < 0.05 is significant., GraphPad Prism 8 Software).
- The control limits based on the laboratory historical negative control data were < 95 % of Poisson distribution.
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
SHORT TERM TREATMENT PROCESS
Cell growth inhibition test:
- In the presence and absence of a metabolic activation system, a cell growth inhibition test, 2.00 mg/mL was determined to be the maximum concentration, and was performed within the dose range of 0.0313 to 2.00 mg/mL at 7 dose levels using a common ratio of 2.
- Absence of a metabolic activation system: RICC of < 50 % was not observed at any dose. Precipitation of the test material was not observed at any dose in the initiation or completion of treatment.
- Presence of a metabolic activation system: RICC of < 50 % was not observed at dose of 2.00 mg/mL. Precipitation of the test material was not observed at any dose in the initiation or completion of treatment.

Chromosome aberration test:
- Absence of a metabolic activation system: RICC of < 50 % was not observed at any dose therefore chromosome analysis was performed at 3 doses of 0.500, 1.00 and 2.00 mg/mL.
- Frequency of both chromosome structural aberration and chromosome numerical aberration exhibited no significant different compared with concurrent negative control by use of Fisher’s exact test, and no significant trend by use of Cochran-Armitage trend test.
- Presence of a metabolic activation system: cell growth inhibition of 55 ± 5 % (100-RICC) was observed at a dose of 1.41 mg/mL, therefore chromosome analysis was performed at 3 doses of 0.7070, 1.00 and 1.41 mg/mL.
- Both chromosome structural aberration and chromosome numerical aberration were not observed to increase above concurrent negative control at any dose, therefore statistical analysis was not performed.

CONTINUOUS TREATMENT PROCESS
Cell growth inhibition test:
- In the 24 hours treatment process, a cell growth inhibition test, 2.00 was determined to be the maximum concetrnation, and was performed with the dose range of 0.0313 to 2.00 mg/mL at 7 dose levels using a common ratio of 2.
- RICC of < 50 % was observed at and above doses of 0.500 mg/mL.
- Precipitation of the test material was not observed at any dose in the initiation or completion of treatment.

Chromosome aberration test:
- Cell growth inhibition of 55 ± 5 % (100-RICC) was observed at doses of 0.397 and 0.500 mg/mL, therefore chromosome analysis was performed at 3 doses including doses at which little cytotoxicity was observed, 0.199, 0.315 and 0.500 mg/mL.
- The frequency of chromosome structural aberration exhibited significant difference compared with concurrent negative control at doses of 0.199, 0.315 and 0.500 mg/mL by use of Fisher’s exact test.
- The chromosome structural aberration exhibited no significant trend by use of Cochran-Armitage trend test, therefore the result was equivocal.
- The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose, therefore statistical analysis was not performed.
- The result of chromosome structural aberration was equivocal therefore a confirmation test was performed within the dose range of 0.125 to 0.794 mg/mL at 9 levels using a common ratio of 2^1/3.

CONFIRMATION TEST
- Cell growth inhibition of 55 ± 5 % (100-RICC) was observed at doses of 0.397 and 0.500 mg/mL, therefore chromosome analysis was performed at 3 doses including doses at which little cytotoxicity was observed, 0.125, 0.250 and 0.500 mg/mL.
- The frequency of chromosome structural aberration exhibited significant difference compared with concurrent negative control at doses of 0.125 and 0.250 mg/mL by use of Fisher’s exact test.
- The chromosome structural aberration exhibited no significant trend by use of Cochran-Armitage trend test, therefore the result was equivocal.
- The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose, therefore statistical analysis was not performed.
- The reproducibility of the chromosome structural aberration was confirmed, the result was equivocal.

HISTORICAL NEGATIVE CONTROL DATA
- Short term treatment process: the frequency of chromosome structural aberrations and numerical aberrations at all of the results were inside the control limits based on the laboratory historical negative control data.
- Continuous treatment process: the frequency of chromosome structural aberrations at doses of 0.199 0.315 and 0.500 mg/mL (chromosome aberration test and at doses of 0.125 and 0.250 mg/mL (confirmation test) were outside the control limits based on the laboratory historical negative control data.
- The frequency of chromosome numerical aberrations at all of the results were inside the control limits based on the laboratory historical negative control data.
Remarks on result:
other: Equivocal

Results of chromosome aberration test (- S9 mix, short term treatment process)

Treatment time (h)

Dose (mg/mL)

Number of chromosome structural aberrations and classification

Number of chromosome numerical aberrations

Cells

ctb

cte

csb

cse

others

Total (%)

gap

Cells

poly

others

Total (%)

RICC (%)

6-18

Negative control (saline)

150

0

1

0

0

0

1

1

150

0

0

0

100

150

0

0

0

0

0

0

0

151

1

0

1

100

300

0

1

0

0

0

1 (0.3)

1

301

1

0

1 (0.3)

100

0.500

150

1

0

0

0

0

1

1

151

1

0

1

104

150

0

0

0

0

0

0

1

150

0

0

0

84

300

1

0

0

0

0

1 (0.3)

2

301

1

0

1 (0.3)

94

1.00

150

1

0

0

0

0

1

0

151

1

0

1

89

150

1

0

0

0

0

1

0

150

0

0

0

85

300

2

0

0

0

0

2 (0.7)

0

301

1

0

1 (0.3)

88

2.00

150

1

1

0

0

0

2

0

151

1

0

1

84

150

0

0

0

0

0

0

0

151

1

0

1

90

300

1

1

0

0

0

2 (0.7)

0

302

2

0

2 (0.7)

88

Positive control (MMC) 0.07 μg/mL

150

150

300

9

12

21

8

9

17

0

0

0

0

0

0

0

0

0

16

21

37

(12.3)**

0

0

0

150

150

300

0

0

0

0

0

0

0

0

0 (0.0)

/

** Significant

Results of chromosome aberration test (+ S9 mix, short term treatment process)

Treatment time (h)

Dose (mg/mL)

Number of chromosome structural aberrations and classification

Number of chromosome numerical aberrations

Cells

ctb

cte

csb

cse

others

Total (%)

gap

Cells

poly

others

Total (%)

RICC (%)

6-18

Negative control (saline)

150

2

0

0

0

0

2

1

151

1

0

1

100

150

2

1

0

0

0

2

0

151

1

0

1

100

300

4

1

0

0

0

4 (1.3)

1

302

2

0

2 (0.7)

100

0.354

 #

 

 

 

 

 

 

 

 #

 

 

 

67

 #

 

 

 

 

 

 

 

 

 

 

83

 #

 

 

 

 

 

 

 

 

 

 

75

0.500

 

 

 

 

 

 

 

 

 

 

66

 

 

 

 

 

 

 

 #

 

 

 

80

 #

 

 

 

 

 

 

 

 

 

 

73

0.707

150

1

0

0

0

0

1

0

150

0

0

0

90

150

1

2

0

0

0

2

0

151

1

0

1

74

300

2

2

0

0

0

3 (1.0)

0

301

1

0

1 (0.3)

81

1.00

150

0

2

0

0

0

2

0

150

0

0

0

59

150

0

2

0

0

0

2

1

151

1

0

1

58

300

0

4

0

0

0

4 (1.3)

1

301

1

0

1 (0.3)

59

1.41

150

0

0

0

0

0

0

2

150

0

0

0

40

150

1

0

0

0

0

1

0

152

2

0

2

44

300

1

0

0

0

0

1 (0.3)

2

302

2

0

2 (0.7)

42

2.00

 #

 

 

 

 

 

 

 

 

 

 

16

 

 

 

 

 

 

 

 #

 

 

 

9

 

 

 

 

 

 

 

 

 

 

12

Positive control

(BP) 8 μg/mL

150

5

14

0

0

0

17

0

152

2

0

2

/

150

3

17

0

0

0

19

0

150

0

0

0

300

8

31

0

0

0

36 (12.0)**

0

302

2

0

2 (0.7)

# Cells not observed, ** Significant

Results of chromosome aberration test 24-hour continuous treatment process)

Treatment time (h)

Dose (mg/mL)

Number of chromosome structural aberrations and classification

Number of chromosome numerical aberrations

Cells

ctb

cte

csb

cse

others

Total (%)

gap

Cells

poly

others

Total (%)

RICC (%)

24-0

Negative control (saline)

150

1

0

0

0

0

1

0

150

0

0

0

100

150

0

0

0

0

0

0

0

150

0

0

0

100

300

1

0

0

0

0

1 (0.3)

0

300

0

0

0 (0.0)

100

0.199

150

4

1

0

0

0

5

1

150

0

0

0

69

150

8

1

0

0

0

9

2

150

0

0

0

68

300

12

2

0

0

0

14 (4.7)**

3

300

0

0

0 (0.0)

69

0.250

 #

 

 

 

 

 

 

 

 #

 

 

 

61

 

 

 

 

 

 

 

 

 

 

51

 #

 

 

 

 

 

 

 

 #

 

 

 

56

0.315

150

6

0

0

0

0

6

2

150

0

0

0

56

150

5

0

0

0

0

5

0

150

0

0

0

55

300

11

0

0

0

0

11 (3.7)**

2

300

0

0

0 (0.0)

55

0.397

 

 

 

 

 

 

 

 #

 

 

 

56

 

 

 

 

 

 

 

 

 

 

45

 

 

 

 

 

 

 

 

 

 

50

0.500

150

4

1

0

0

0

5

1

150

0

0

0

53

150

5

0

0

0

0

5

0

150

0

0

0

42

300

9

1

0

0

0

10 (3.3)**

1

300

0

0

0 (0.0)

47

0.630

 

 

 

 

 

 

 

 #

 

 

 

41

 

 

 

 

 

 

 

 

 

 

37

 

 

 

 

 

 

 

 #

 

 

 

39

0.794

 

 

 

 

 

 

 

 

 

 

29

 

 

 

 

 

 

 

 

 

 

29

 

 

 

 

 

 

 

 

 

 

29

Positive control (MMC) 0.03 μg/mL

150

12

9

0

0

0

20

0

150

0

0

0

/

150

7

12

0

0

0

19

0

150

0

0

0

300

19

21

0

0

0

39 (13.0)**

0

300

0

0

0 (0.0)

 # Cells not observed, ** Significant

Conclusions:
Under the conditions of the study the test material is considered “equivocal” for the chromosome-aberration-inducing potential.
Executive summary:

The chromosome-aberration-inducing potential of the test material was investigated in accordance with the standardised guidelines OECD 473, under GLP conditions.

The frequency of chromosome structural aberration exhibited no significant difference compared with concurrent negative control by use of Fisher's exact test and exhibited no significant trend by use of Cochran-Armitage trend test in any of the tests using short-term treatment process in the absence of a metabolic activation system.

The chromosome structural aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the presence of a metabolic activation system, therefore statistical analysis was not performed.

The frequency of chromosome structural aberration exhibited significant difference compared with concurrent negative control at doses of 0.199, 0.315 and 0.500 mg/mL (chromosome aberration test), and at doses of 0.125 and 0.250 mg/mL (confirmation test), by use of Fisher's exact test in all of the tests using continuous treatment process for 24 hours. However, it exhibited no significant trend by use of Cochran-Armitage trend test in any of the tests using continuous treatment process for 24 hours. The reproducibility was confirmed by a confirmation test.

The frequency of chromosome numerical aberration exhibited no significant difference compared with concurrent negative control by use of Fisher's exact test and exhibited no significant trend by use of Cochran-Armitage trend test in any of the tests using short-term treatment process in the absence of a metabolic activation system.

The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the presence of a metabolic activation system and continuous treatment process for 24 hours, therefore statistical analysis was not performed.

Since elongation of the cell cycle was not observed at continuous treatment process for 24 hours, the cell growth inhibition test and chromosome aberration test using continuous treatment process for 48 hours were not carried out.

Results from both the positive and negative controls under all treatment conditions were within the acceptable ranges that confirmed the validity of the tests, indicating that these tests were carried out appropriately.

Under the conditions of the study the test material is considered “equivocal” for the chromosome-aberration-inducing potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Vivo Micronucleus Assay: Ogura (2020)

Under the conditions of the study the test material did not induce micronuclei in mice bone marrow cells.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2020 - 18 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was put into a tight container and stored at room temperature of the test item storage room (permissible range: from 10 to 30 °C)
- Stability under storage conditions: stable.
- Solubility in dimethylsulfoxide: ≤ 439 g/L
- Solubility in acetone: ≤ 0.03 g/L
- Water solubility: ≥ 250 mg/mL (confirmed in a fourteen day repeated dose oral toxicity study in rats)
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Mice are widely used in micronucleus tests because the observation of the micronuclei is easy and the historical data is abundant.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks.
- Weight at study initiation: Male: 31.6-37.1 g, Female: 25.2-26.8 g
- Assigned to test groups randomly: yes, under following basis: after acclimation, healthy mice were allocated by a body weight-stratified random sampling method on the day before administration.
- Housing: Animals were housed 5 or less by cage (before allocation) and 3 animals or less by cage (after allocation) in polycarbonate cages with flat bottom (210 W x 320 D x 130 H mm).
- Diet: ad libitum
- Water: ad libitum (Hita City water supply, chlorinated at about 3 to 5 ppm by adding sodium hypochlorite (Purelox))
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 23.1 – 24.3 °C
- Humidity: 51.1 – 63.4 %
- Air changes: 10 - 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
- Vehicle: distilled water.
- Justification for choice of vehicle: The test material was soluble in water at 250 mg/mL and the test material solution of 250 mg/mL prepared with water was considered to be stable as there was no change in colour until 19 hours after preparation.
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle: 10 mL/kg based on the body weight on the administration day
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test material (2.765 g) was weighed and dissolved in distilled water using a laboratory mixer and ultrasonicator to make a 10 mL solution of 200 mg/mL test material solution. This solution was serially diluted with distilled water to prepare 100 and 50.0 mg/mL test material solutions, using a magnetic stirrer.
- The test material solutions were prepared on each administration day, stored in a cold place and used within 19 hours of preparation.
Duration of treatment / exposure:
2 days.
Frequency of treatment:
Test material solution: 2 doses within a 24 hour interval.
Negative control: 2 doses within a 24 hour interval.
Positive control (MMC): 1 dose within a 24 hour interval during the second administration of the test material.
Post exposure period:
Animals were observed frequently until 1 hour after each administration. In addition, the animals were observed at 1 day after each administration. Body weights were measured once a day for 3 days (from the first administration day to the day after the second administration) using an electronic balance (Sartorius). The specimens of the negative and the positive control groups, and all 3 doses in the test material group were observed microscopically (x 1000) in a blinded manner.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
500 mg/kg/day: 5 male animals.
1000 mg/kg/day: 5 male animals.
2000 mg/kg/day: 5 male animals and 3 female animals.
Control animals:
yes
Positive control(s):
- Positive control: Mitomycin C (MMS)
- Justification for choice of positive control: MMC is listed as a positive control material in the test method described in “5. TEST METHOD”, and historical data has been accumulated in the testing facility.
- Route of administration: intraperitoneal injection.
- Doses / concentrations: MMC (2 mg) was weighed, added into distilled water, and mixed with a laboratory mixer to make a 10 mL solution of 0.2 mg/mL MMC solution. The positive control material solution was treated in the safety cabinet. One dose a day was administered (2 mg/kg/day).
Tissues and cell types examined:
4000 polychromatic erythrocytes (PCE) per animal and 500 erythrocytes (TE) per animal.
Details of tissue and slide preparation:
TIMING OF PREPARATION
- Specimens were prepared 24 hours after the second administration in the test material groups.

DETAILS OF SLIDE PREPARATION
- The animals were euthanised by a cervical dislocation.
- The femur was removed and the bone marrow cells were collected with approximately 0.8 mL of a heat-inactivated foetal bovine serum into a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes.
- A small amount of the cell suspension was smeared on a glass slide.
- The smears were air-dried and fixed with methanol.
- The specimens were stained with 3 vol % Giemsa solution prepared with 1/15 mol/L phosphate buffer solution (pH 6.8) and treated with 0.004 w/v % citrate aqueous solution.
- The specimens of the negative and positive control groups, and all three doses in the test material group were observed. Slide numbers were allocated randomly.

METHOD OF ANALYSIS
- The frequency of micronucleated polychromatic erythrocytes (MNPCE/PCE) was calculated.
- The ratio of PCE to TE (PCE/TE) was calculated.
Evaluation criteria:
EVALUATION CRITERIA
- The test was considered as negative if either a) or b) were satisfied for the MNPCE/PCE in the test material dose:
a) all results are inside the distribution of the historical data of the negative control group.
b) outside the distribution of the historical data of the negative control group, but none of the doses of the test material exhibit a statistically significant increase compared with the concurrent negative control.
- The test was considered to be positive is all c), d), e) and f) were fulfilled:
c) outside the distribution of the historical data of the negative control group.
d) the doses of the test material exhibit a statistically significant increase compared with the concurrent negative control.
e) the increase of the MNPCE/PCE is dose-related.
f) both the micronucleus test and confirmation test are fulfilled in case of c) and d).

ACCEPTABLE CRITERIA
- The means of the MNPCE/PCE in the concurrent negative control are “under control” in the quality control method used for the control chart in the historical data.
- The means of the MNPCE/PCE in the concurrent positive control are “under control” in the quality control method used for the control chart in the historical data. Concurrent positive controls produce a statistically significant increase compared with the concurrent negative control.
- In the test material group, the PCE/TE is 20 % or more compared with that of the negative control group at all observation doses.
Statistics:
MNPCE/PCE
- The Conditional Binomial test (Kastenbaum and Bowman) was conducted to compare the MNCPE/OCE in each dose of the test material group and the positive control group with that in the negative control group.
- Upper tailed significance levels of 5 % and 1 %.
- A significant different was not obtained.

PCE/TE
- Statistical tool, “StatLight” was used for all tests.
- All the data except for the positive control group was tested by Bartlett’s test for homogeneity of variance. Since the variances were homogenous the William’s test was conducted.
- Since there were no significant differences within the William’s test the Dunnett’s test was performed to compare the mean in the negative control group with that in each dose of the test material group.
- The comparisons were tested by the F test for homogeneity of variance between the groups.
- Since the results of the F test showed homogeneity, Student’s t test was performed to compare the mean in the positive control group with that in the negative control group.
- Significance levels: 5 % for Bartlett’s test and F test. Both sides of 5 % for Williams test, and both sides of 5 % or 1 % for other tests.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS/ OBSERVATIONS
- No abnormalities were observed at any doses of the test material group, the negative control group or the positive control group.
- No abnormal changes were noted in the body weight of the animals when compared with the negative control group in all doses of the test material group until the day after the second administration.

MNPCE/PCE
- Mean result in negative control group: 0.085 %
- Mean result in the positive control group: 7.195 %
- Mean result at 500 mg/kg/day: 0.035 %
- Mean result at 1000 mg/kg/day: 0.055 %
- Mean result at 2000 mg/kg/day: 0.060 %
- At all doses in the test material group the results were within the range of the historical data of the negative control.
- There was no significant increase in MNPCE/PCE in any doses of the test material group compared with the negative control group at 5 % level.
- At the positive control group, a statistically significant increase was observed at 1 % level.

PCE/TE
- Mean result in the negative control group: 47.0 %
- Mean result in the positive control group: 38.3 %
- At the positive control group a statistically significant decrease was observed at 5 % level, compared with the negative control group.
- Mean result at 500 mg/kg/day: 47.2 %
- Mean result at 1000 mg/kg/day: 47.4 %
- Mean result at 2000 mg/kg/day: 49.2 %

DISCUSSION
- The means of MPCE/PCE in the negative and positive control groups were within the range of the respective historical data in the testing facility.
- In the test material group the PCE/TE was 20 % or more compared with the negative control group at all observation doses therefore it was confirmed that the test was conduced appropriately.
- The means of the MNPCE/PCE of the test material group at all observation doses were within the range of the historical data of the negative control. Therefore, the results were judged to be negative.
- No statistically significant differences were observed in the PCE/TE test at any doses of the test material group compared to the negative control group therefore the exposure of the test material to bone marrow cells was not proven.
- The potential to induce micronuclei of the test material could be evaluated adequately as 2000 mg/kg/day, which was the maximum dose in the test method, was set in the present test.
Conclusions:
Under the conditions of the study the test material did not induce micronuclei in mice bone marrow cells.
Executive summary:

The ability of the test material to induce micronuclei in mice was investigated in accordance with the standardised guidelines OECD 474, under GLP conditions.

During the study, the test material was administered to 7 week old CD-1 mice twice in a 24-hour interval by oral gavage. The highest dose for administration was determined to be 2000 mg/kg/day from the results of the acute oral toxicity study in rats, and two lower doses of 1000 and 500 mg/kg/day were set based on a geometric ratio of 2. Five males were used for each dose level. The test material was administered to three females at a dose of 2000 mg/kg/day to evaluate sex differences in the maximum tolerance dose (MTD). Negative and positive control groups were set. As the negative control item, distilled water was administered at 10 mL/kg twice in a 24-hour interval by oral gavage. The positive control, Mitomycin C was administered once, intraperitoneally at 2 mg/kg/day. There was no mortality at the dose of 2000 mg/kg/day in both male and female mice, therefore it was estimated that there were no sex differences and the MTD determined by the death of animals was 2000 mg/kg/day or more. Therefore, the three doses were set for male mice in the micronucleus test. The frequencies of the micronucleated polychromatic erythrocytes to polychromatic erythrocytes (MNPCE/PCE) in bone marrow cells and the ratio of PCE to total erythrocytes (PCE/TE) were examined.

The means of MNPCE/PCE in all observation doses of the test material group were within the range of the historical data of the negative control. Therefore, the results were judged to be negative. As such it was concluded that the test material did not induce micronuclei in mice bone marrow cells under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames: Sarada (2016)

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals under GLP conditions.The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Mutagenicity potential of the test material was assessed with Salmonella typhimurium TAl00, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in to main tests. The dose range for the main test was determined from the preliminary test using the following seven dose levels: 1.2, 4.9, 20, 78, 313, 1 250 and 5 000 μg/plate. In the preliminary test, the growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 5 000 μg/plate dose was selected for all strains both with and without metabolic activation. This highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

The main test concentrations were 313, 625, 1250, 2500 and 5000 µg/plate. The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in historical data, indicating that this study was performed correctly.

Neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control, nor a dose-related response, was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

Under the conditions of this study, the test material was considered to be non-mutagenic in the bacterial reverse mutation assay.

Chromosome Aberration: Furukuma (2020)

The chromosome-aberration-inducing potential of the test material was investigated in accordance with the standardised guidelines OECD 473, under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The frequency of chromosome structural aberration exhibited no significant difference compared with concurrent negative control by use of Fisher's exact test and exhibited no significant trend by use of Cochran-Armitage trend test in any of the tests using short-term treatment process in the absence of a metabolic activation system.

The chromosome structural aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the presence of a metabolic activation system, therefore statistical analysis was not performed.

The frequency of chromosome structural aberration exhibited significant difference compared with concurrent negative control at doses of 0.199, 0.315 and 0.500 mg/mL (chromosome aberration test), and at doses of 0.125 and 0.250 mg/mL (confirmation test), by use of Fisher's exact test in all of the tests using continuous treatment process for 24 hours. However, it exhibited no significant trend by use of Cochran-Armitage trend test in any of the tests using continuous treatment process for 24 hours. The reproducibility was confirmed by a confirmation test.

The frequency of chromosome numerical aberration exhibited no significant difference compared with concurrent negative control by use of Fisher's exact test and exhibited no significant trend by use of Cochran-Armitage trend test in any of the tests using short-term treatment process in the absence of a metabolic activation system.

The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the presence of a metabolic activation system and continuous treatment process for 24 hours, therefore statistical analysis was not performed.

Since elongation of the cell cycle was not observed at continuous treatment process for 24 hours, the cell growth inhibition test and chromosome aberration test using continuous treatment process for 48 hours were not carried out.

Results from both the positive and negative controls under all treatment conditions were within the acceptable ranges that confirmed the validity of the tests, indicating that these tests were carried out appropriately.

Under the conditions of the study the test material is considered “equivocal” for the chromosome-aberration-inducing potential.

In Vivo Micronucleus Assay: Ogura (2020)

The ability of the test material to induce micronuclei in mice was investigated in accordance with the standardised guidelines OECD 474, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the test material was administered to 7 week old CD-1 mice twice in a 24-hour interval by oral gavage. The highest dose for administration was determined to be 2000 mg/kg/day from the results of the acute oral toxicity study in rats, and two lower doses of 1000 and 500 mg/kg/day were set based on a geometric ratio of 2. Five males were used for each dose level. The test material was administered to three females at a dose of 2000 mg/kg/day to evaluate sex differences in the maximum tolerance dose (MTD). Negative and positive control groups were set. As the negative control item, distilled water was administered at 10 mL/kg twice in a 24-hour interval by oral gavage. The positive control, Mitomycin C was administered once, intraperitoneally at 2 mg/kg/day. There was no mortality at the dose of 2000 mg/kg/day in both male and female mice, therefore it was estimated that there were no sex differences and the MTD determined by the death of animals was 2000 mg/kg/day or more. Therefore, the three doses were set for male mice in the micronucleus test. The frequencies of the micronucleated polychromatic erythrocytes to polychromatic erythrocytes (MNPCE/PCE) in bone marrow cells and the ratio of PCE to total erythrocytes (PCE/TE) were examined.

The means of MNPCE/PCE in all observation doses of the test material group were within the range of the historical data of the negative control. Therefore, the results were judged to be negative. As such it was concluded that the test material did not induce micronuclei in mice bone marrow cells under the conditions of this study.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.