Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- In vitro gene mutation study in bacteria: not mutagenic to S. thyphimurium TA 100, TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA (OECD 471, GLP, K, Rel.1 - LR dossier).


- In vitro Chromosome aberration test in Chinese hamster lung (CHL/IU): clastogenic in the presence and absence of metabolic activation (OECD 473, GLP, K, Rel.2).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro cytogenicity study in mammalian cells
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to item 10 of Table R.7.7-5 of the Chapter R.7a (Version 6.0 - July 2017), in case of negative Gene mutation test in bacteria, positive Cytogenicity test in vitro and negative Cytogenicity test in vivo, no further tests are required at Annexes VII, VIII, IX & X.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to the OECD TG 473 ( 1983) with the following deviations compared to the current version (2016): CPA used as the positive control without S9 in the 6-hrs exposure. This is not adequate as CPA requires metabolic activation. 200 cells scored (as per the original OECD TG 473) instead of 300 as currently required. Historical control data are missing.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
yes
Remarks:
CPA used as the positive control without S9 in the 6-hrs exposure. This is not adequate as CPA requires metabolic activation.
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: Glass container sealed with air replaced with nitrogen gas until use with storage at room temperature under light shielded conditions
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable for 4 hrs in DMSO at concentrations ranging from 156 μg/ml to 40.0 mg/ml. 
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHL/IU cells derived from Chinese hamsters obtained from the Research Resource Bank (JCRB)
- Suitability of cells: common cell line due to high sensitivity for chemical induced chromosomal aberration
- Normal cell cycle time (negative control): not reported

For cell lines:
- Absence of Mycoplasma contamination: not reported
- Number of passages if applicable: used within 10 passages after thawing
- Cell cycle length, doubling time or proliferation index : not reported
- Modal number of chromosomes: not reported
- Periodically checked for karyotype stability:not reported
- Periodically ‘cleansed’ of spontaneous mutants: not reported

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle, MEM culture solution with 10% metal calf serum. The MEM culture solution was prepared by dissolving 9.4 g of the Eagle MEM medium Nissui (i) powder (Nissui Pharmaceutical Co., Ltd.) in 1 L of distilled water and then sterilized by autoclave at 121°C for 15 minutes and adding 3000 mg of L-glutamine (sterilized, Nissui Pharmaceutical Co., Ltd.) and about 12.5 mL of 10% NaHCO3 solution. The MEM culture solution at a two-fold concentration was prepared by dissolving 9.4 g of the medium above in 500 mL of distilled water and following the same procedure as the MEM culture solution.
The 2×104 CHL/IU cells were inoculated in a dish (6 cm in diameter, Corning) containing 5 mL of the culture solution and incubated in a CO2 incubator (5% CO2) set at 37°C.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Kikkoman Corporation (lot No.: RAA-314, manufactured August 19994), purchased and stored in a deep freezer set at -80°C until use.
- method of preparation of S9 mix
S9: 3
20 mM HEPES (pH 7.2): 2
50 mM MgCl2: 1
330 mM KCl: 1
50 mM G-6-P: 1
40 mM NADP: 1
Distilled water: 1
Total 10 mL
- concentration or volume of S9 mix and S9 in the final culture medium: ratio 4:1:1 (cells : MEM culture solution : S9)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported
Test concentrations with justification for top dose:
- 6-hour treatment: -S9 and +S9: 0.05, 0.1, 0.2 mg/mL.
- Continuous treatment (24 and 48-hours): -S9: 0.005, 0.01, 0.02 mg/mL.
Maximum dose level limited by cytotoxicity (i.e. concentration with 60% growth inhibition).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: not reported

- Justification for percentage of solvent in the final culture medium: 0.5 % (v/v)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not reported
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6-hrs -S9 / 6-hrs + S9/ 24-hrs - S9 / 48-hrs -S9
- Harvest time after the end of treatment (sampling/recovery times): 24 or 48 hrs

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Colcemid, 01 µg/mL
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After incubation was complete, the culture solution was discarded, and then 10% formalin solution was added to fix the cells adhered to the dish. Staining with 0.1% crystal violet solution was performed after fixation.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Structural aberrations: 200 cells / group Polyploidy: 800 cells / group
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosome analysis was performed according to the classification method by the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen and Genome Society,1 and observations were performed for structural aberrations in chromosomal and chromatid patterns, including gaps, breakages, and exchange, as well as the presence or absence of polyploids.
- Determination of polyploidy: yes
- Determination of endoreplication: no


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g. relative total growth
Rationale for test conditions:
The maximum concentration is the one inhibiting growth inhibition by more than 50% (concentration with 60 % growth inhibition).
Evaluation criteria:
A positive result was defined as the observation of a significant difference for the two tests performed below. If a significant difference was not obtained in the trend test, the result was determined as a false positive. If the number of cells observed was less than 100 for structural aberration and less than 400 for polyploids, the result was considered indeterminable due to cytotoxicity.
Statistics:
The incidence of cells with chromosomal aberrations was compared between the background data for the solvent and the groups treated with the test substance by Fisher’s exact test with consideration for multiplicity and a familial significance level of 5% with reference to the method by Hayashi. If a significant difference was observed as a result of Fisher’s exact test, the Cochrane-Armitage test for trends (p < 0.05) was performed for dose dependency.
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
- 24-hours treatment (-S9): at 0.020 mg/mL (high concentration group). - 48-hours treatment (-S9): increase in polyploids at 0.020 mg/mL. - 6-hours treatment (+/- S9): at 0.10 and 0.20 mg/mL. Increase in polyploids in all treatment groups (+S9)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 0.20 mg/mL in the 4-hours -S9 group.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not reported
- Precipitation and time of the determination: none reported
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: none reported

STUDY RESULTS
- Concurrent vehicle negative and positive control data: cf. attached tables.
- Chromosome aberration test (CA) in mammalian cells: cf attached tables
In the groups with 24 hours of continuous treatment, a significant (p < 0.05) increase in the cells with structural chromosomal aberrations was observed in the high concentration group (0.020 mg/mL) with a significant difference also observed in the trend test. No other groups were observed with significant increases in the cells with structural aberrations. Meanwhile, no significant increase in polyploids was observed for any treatment groups. In the groups with 48 hours of treatment, no treatment groups showed a significant increase in the cells with structural aberration, while a significant (p < 0.05) increase in polyploids was observed in the high concentration group (0.020 mg/mL) with a significant difference also observed in the trend test.
In the groups with 6 hours of treatment without the S9 mix, the number of cells with structural aberrations increased significantly (p < 0.05) and in a dose-dependent manner, although the specified number of cells could not be analyzed for the high concentration group (0.20 mg/mL) due to cytotoxicity. With regard to polyploids, a significant (p < 0.05) increase was observed in the low concentration group (0.050 mg/mL); however, no dose dependency was observed. The induction of polyploids was not observed for any other treatment groups. The groups with 6 hours of treatment with the S9 mix also showed a dose-dependent and a significant (p < 0.05) increase in the cells with structural aberrations, along with dose-dependent and a significant (p < 0.05) increase in polyploids. The MC treatment group in continuous treatment and CPA treatment group in treatment with the S9 mix, which were used as positive controls, were found to induce cells with structural aberrations, such as chromatic exchange (cte) and chromatid breakage (ctb).

HISTORICAL CONTROL DATA
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: not reported
Conclusions:
Under the test conditions, the test item induced chromosomal aberrations in CH/IU cells in vitro.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration) performed according to the OECD Test Guideline No. 473 and in compliance with GLP, CHL/IU cell cultures were exposed to the test item diluted in DMSO under the following conditions:


- continuous 24-hr treatment, without S9: 0; 0.0050; 0.010 and 0;020 mg/mL;


- continuous 48-hr treatment, without S9: 0; 0.0050; 0.010 and 0;020 mg/mL;


- 6-hr treatment, without S9: 0; 0.050; 0.10 and 0.20 mg/mL;


- 6-hr treatment, with S9: 0; 0.050; 0.10 and 0.20 mg/mL.


 


 The test item was tested up to cytotoxic concentrations (> 50% growth inhibition).


 


As a result of continuous treatment of CHL/IU cells for 24 and 48 hours, a significant increase in the cells with structural chromosomal aberrations was observed in the high concentration group (0.020 mg/mL) with 24 hours of treatment with a significant difference also observed as a result of the trend test. In addition, a significant increase in polyploid cells was observed in the high concentration group (0.020 mg/mL) with 48 hours of treatment with a significant difference also observed as a result of the trend test. For the group treated for 6 hours with and without the S9 mix, there was a dose-dependent and significant increase in the cells with structural aberration. Furthermore, the group with the S9 mix showed significant increase in polyploid cells in a dose-dependent manner.


 


Positive controls induced the appropriate response.  


 


Under the test conditions, the test item induced chromosomal aberrations in CH/IU cells in vitro.


 


This study is classified as acceptable and satisfies the requirement for an in vitro cytogenetic mutagenicity assay. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

- In vivo Micronucleus test in mouse (oral: gavage): not clastogenic and not aneugenic (OECD 474, GLP, K, Rel.1)

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:
From 14 May 2021 to 11 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to the OECD TG 474 and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
GLP compliance:
yes (incl. QA statement)
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: Cool location (in a refrigerator, permissible window: 1°C to 10°C, measured values: 3°C to 8°C), light-shielded, airtight
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability of the test substance was confirmed as a result of stability confirmation performed by the study contractor after the completion of the animal study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Slightly soluble in water (17 g/L, 86°C)
- Reactivity of the test material with the incubation material used (e.g. plastic ware): none reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
Species:
mouse
Strain:
ICR
Remarks:
Crlj:CD1(ICR)SPF
Details on species / strain selection:
Mice are used widely in micronucleus tests, and the strain of mice used in this study was selected because of the properties being well known and the abundance of background documents.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 31.7 to 37.0 g for males and 22.9 to 25.5 g for females in the preliminary test, and 31.0 and 35.2 g for males in the main test.
- Assigned to test groups randomly: under following basis: The animals were stratified by body weight on the day of group allocation (day of first dose) and divided into groups so that the mean body weight in each group was as uniform as possible. Animal assignments were performed by a combination of the computerized block placement method and the random sampling method (necessary groups were formed by block placement method and the test groups and the animal numbers within the group were randomly allocated).
- Fasting period before study: none
- Housing: individually in plastic cages (W155×D245×H150 mm: Clea Japan, Inc.) with bedding (White Flake: Charles River Laboratories Japan, Inc.).
- Diet (e.g. ad libitum): free access to solid feed CRF-1 [Oriental Yeast Co., Ltd., lot No.: 100203 (preliminary test) and 100302 (main test)
- Water (e.g. ad libitum): free access (Gotemba City Waterworks, water bottle used)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C to 23°C for the preliminary test and 20°C to 23°C for the main test
- Humidity (%): 48% to 54% in the preliminary test and 48% to 56% in the main test
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: May 26, 2010 / To: June 25, 2010
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Since the test substance is a type 3 petroleum, corn oil was selected as it allows preparation of satisfactory solution for administration.
- Concentration of test material in vehicle: 12.5; 25; 50 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw (total volume administered)
- Lot/batch no. (if required): PEG0519
- Purity: for biochemistry
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each concentration was prepared by weighing the test substance and diluting by adding corn oil to make the specified volume.
- storage method: The amount required for each day was dispensed into brown glass bottles and stored in a cool location [in a refrigerator, permissible window: 1°C to 10°C, measured values: 4°C to 5°C (preliminary test), 3°C to 5°C (main test)], and then used within eight days of preparation.
- stability: The solutions at the concentration of 0.1 mg/mL and 200 mg/mL have been confirmed to be stable for up to eight days in a cool location (in a refrigerator) and 24 hours at room temperature according to the confirmation by BoZo Research Center Inc.
- confirmation of the concentration of the test solution: Each concentration of the test solution used in the main test was sampled in the amount of 10 mL each for the confirmation of concentration. The result showed that the percentage of concentration to the labeled value of each concentration was 103.6% to 105.0%, which was within ±10% when labeled value was set as 100, indicating no problems in the result.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Twice, at about 24 hours interval
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Preliminary and main test
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Preliminary and main test
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Preliminary and main test
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Preliminary test
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Preliminary test
No. of animals per sex per dose:
3M+3F for the preliminary test.
5M for the main study.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): This was selected according to the Toxicity Guidelines as MMC is used widely in micronucleus test and abundant background data are available.
- Route of administration: Oral (gavage)
- Doses / concentrations: 1 mg/kg bw/day - 0.1 mg/mL
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The specimen for micronucleus observation was prepared according to the method by Schmid. The mice applicable to the specified time after administration were euthanized by cervical dislocation, and then both femurs were extracted and amputated at both ends. Following this, the bone marrow cells were extracted into a centrifuge with about 0.1 to 0.2 mL of fetal calf serum (GIBCO BRL, lot No. 494548) using 1 mL disposable injection syringe and 23 G injection needle. The bone marrow cells and the fetal calf serum were mixed with this injection syringe and needle to make the cells loose, and then centrifugation (Tomy Industries, Co., Ltd., tabletop multi-tube centrifuge LC-220) was performed at 1000 rpm for 5 minutes. Following this, the supernatant was discarded, and the precipitate was mixed well in a mixer before smearing onto a slide glass (one slide of specimen was prepared for each of the left and right femur in each animal). The smeared specimen was air dried and fixed with methanol (Wako Pure Chemical Industries, Ltd., lot No.: KWP4454) for 3 minutes and then air dried again. For the preparation of specimens, the comparison table of the animal number and of the sample number was prepared, and the specimen was attached with labels displaying the study number, stage, sample number, type of test, and date of preparation (blinding).

METHOD OF ANALYSIS:
One slide glass with satisfactory smear conditions was selected based on the sample number as the observation is performed under blinded conditions. Fluorescent staining of the bone marrow smear sample with acridine orange and the observations were performed according to the method by Hayashi et al. A slide was placed on a cover glass with a small amount of 40 µg/mL acridine orange solution dropped beforehand, and the observation was performed at the total magnification factor of 600 using a fluorescent microscope (system biological microscope BX40: Olympus Corporation, universal reflected light fluorescence device BX-FLA: Olympus Corporation) with the transmission of excitation light near the wavelength of 489 nm and wavelength of 515 nm as the observation filter. The specimens were discarded after the completion of observations, and the specimens that were not used for observations were discarded after the observation of all specimens.
Evaluation criteria:
For each animal, the number of PCE in 200 total erythrocytes and the number of MNPCE in 2000 PCE were counted, and the incidences (%) were calculated. In addition, the mean and standard deviation were calculated for the number of MNPCE and incidence (%) and the number of PCE and incidence (%) in each group with the maximum and minimum values also calculated for the incidences (%).
Statistics:
Significance for the incidence of micronucleus was determined by first confirming that the incidence (%) of MNPCE in the negative and positive control groups are within the range of mean ± 3 SD of the respective background data at the laboratory, and then the negative control group and the test substance groups were compared by the Kastenbaum-Bowman test based on binomial distribution (significance level: one-sided 5%) and the Cochran-Armitage test for trends (significance level: two-sided 1% and 5%). For the incidence of PCE, the negative control group and each test substance group were compared by Bartlett’s test to investigate the equality in variance (significance level: two-sided 1%), and then Dunnett’s test (significance level: two-sided 1% and 5%) was performed as the variance was found to be equal.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: As a result of oral administration in male and female mice at the doses of 125, 250, 500, 1000, and 2000 mg/kg per day twice at the interval of about 24 hours in the preliminary study, all male and female animals in the 1000 and 2000 mg/kg per day groups were found to have died by the day after the second dose. Animals with decreasing tendencies in body weight were observed for all groups. Based on the above, the dose for the main test was set as follows: 500 mg/kg per day, which was the dose without deaths and was thought to be the maximum tolerable dose, was set as the high dose; this dose was divided by the common ratio of 2 to set the doses of 250 and 125 mg/kg per day, resulting in three doses.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity: No changes in general conditions were observed in the test substance groups, and no changes in generation conditions were observed in the negative control group and the positive control group. No abnormalities were found in the changes in the body weight of animals in the test substance groups or the positive control group in comparison with the negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): In the test substance groups, the incidence of MNPCE was found to be 0.09% ± 0.08% for the 125 mg/kg per day group, 0.09% ± 0.05% for the 250 mg/kg per day group, and 0.11% ± 0.02% for the 500 mg/kg per day group. Compared with 0.07% ± 0.08% in the negative control group, no statistically significant increase was observed for any test substance group, and no dose-dependent changes were observed. In addition, the incidence of PCE in 200 total erythrocytes for the test substance group showed no statistically significant changes compared with the negative control group. Meanwhile, the incidence of MNPCE in the positive control group was found to increase notably compared with the negative control group, and the incidence of MNPCE in the negative and positive control groups were within the range of mean ± 3 SD of the respective background data at the laboratory.
- Appropriateness of dose levels and route: yes, administration in the main test was performed at the high dose of 500 mg/kg per day, which was the dose without deaths in the preliminary test, as well as 250 and 125 mg/kg per day, as an oral dose twice at the interval of about 24 hours.
- Statistical evaluation: No statistically significant increase was observed for the incidence of MNPCE in the test substance groups.
- Other: the main test was performed for male animals only as no notable difference was observed in the onset of toxicity between the sexes.
Conclusions:
Under the test conditions, the test item did not induce chromosomal aberrations in the bone marrow of Crlj:CD1(ICR)SPF mice and is therefore not considered to be a clastogenic or aneugenic substance.
Executive summary:

In an in vivo mammalian cell cytogenetics assay (micronucleus) performed according to the OECD Test Guideline No. 474 and in compliance with GLP, the test item diluted in corn oil was administered orally to Crlj:CD1(ICR)SPF mice at the doses of 125, 250, and 500 mg/kg bw per day twice at the interval of about 24 hours, and then a bone marrow smear specimen was prepared at about 24 hours after the second dose. In addition, negative and positive control groups were with a single administration of corn oil and 1 mg/kg mitomycin C, respectively.


 


In the preliminary test performed to determine the doses, the test substance was administered orally to mice at the doses of 125, 250, 500, 1000, and 2000 mg/kg per day twice at the interval of about 24 hours, and as a result all male and female animals in the 1000 and 2000 mg/kg per day groups were found to have died by the day after the second dose. Animals with decreasing tendencies in body weight were observed for all groups.


Based on the result obtained, administration in the main test was performed at the high dose of 500 mg/kg per day, which was the dose without deaths in the preliminary test, as well as 250 and 125 mg/kg per day, as an oral dose twice at the interval of about 24 hours. The bone marrow was collected about 24 hours after the second dose. In addition, corn oil was administered at the same interval as the test substance for the negative control group, and 1 mg/kg mitomycin C was administered once for the positive control group. As a result of the test, no statistically significant increase was observed for the incidence of MNPCE in the test substance groups in comparison with the negative control group, and no dose-dependent changes were observed. In addition, the incidence of PCE in 200 total erythrocytes for the test substance group showed no statistically significant changes compared with the negative control group. Furthermore, the incidence of MNPCE in the negative and positive control groups were within the range of mean ± 3 SD of the respective background data at the laboratory; therefore, the test was thought to be conducted appropriately.


  


Under the test conditions, the test item did not induce chromosomal aberrations in the bone marrow of Crlj:CD1(ICR)SPF mice and is therefore not considered to be a clastogenic or aneugenic substance.


 


 This study is classified as acceptable and satisfies the requirement for an in vivo cytogenetic mutagenicity assay. 

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

Additional information

Table 7.6.1/1 : Summary of genotoxicity tests :


 










































Test n°



Test Guideline / Reliability



Focus



Strains  / cells tested



Metabolic activation



Test concentration



Statement



1 (1996)



Ames Test


(OECD 471, GLP)


K, rel.1



Gene mutation



S. thyphimurium


TA 1535,


TA 1537,


TA 98,


TA 100,


E.coli WP2 uvrA



-S9


+S9



Tested up to cytotoxic concentrations



-S9: not mutagenic


+ S9: not mutagenic



2 (1996)



CAT


(OECD 473, GLP)


K, rel.2



Chromosomal aberration



Chinese Hamster Lung cells



-S9


+S9



Tested up to cytotoxic concentrations



-S9: clastogenic


+ S9: clastogenic



3


(2011)



MNT


(OECD 474, GLP)


K, rel.1



Chromosomal aberration



Crlj :CD1(ICR) mice



NA



Tested up to the MTD



Not clastogenic, not aneugenic



Gene mutation Assay (Test n° 1)


A bacterial reverse mutation assay (Ames test) was performed with the substance (Test n°1). This study was used to conclude on the potential of the substance to induce gene mutation in bacteria.


No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains with any dose of test material, either in the presence or absence of metabolic activation. The test indicate that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test. 


 


Chromosomal aberration (Test n°2 and 3)


The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in Chinese hamster lung cells, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured Chinese hamster lung cells. Under the test conditions, there was a dose-dependent and significant increase in the cells with structural aberration. Furthermore, the group with the S9-mix showed significant increase in polyploid cells in a dose-dependent manner. The substance is therefore considered as positive for inducing chromosomal mutations in Chinese hamster lung cells under activation and non-activation conditions used in this assay.


The clastogenic and aneugenic potential of the substance was determined using an in vivo micronucleus assay in Crlj:CD1(ICR)SPF mice, which identify substances that cause micronuclei in erythroblasts sampled from bone marrow. Under the test conditions, the test item did not induce chromosomal aberrations in the bone marrow of Crlj:CD1(ICR)SPF mice and is therefore not considered to be a clastogenic or aneugenic substance.


Based on the higher degree of reliability of the in vivo over the in vitro studies in the existing dataset, the substance is considered negative for inducing chromosome aberrations.

Justification for classification or non-classification

Harmonised classification:


The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 


 


Self-classification: 


Based on the available information, no additional self-classification is proposed.