3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Sarada, 2018)

The mutagenicity of the test material on the applied bacterial strains was judged negative.

Chromosome aberration (Ogura 2020)

The test material induced structural aberration and suspected to induce numerical aberration in Chinese hamster lung fibroblasts (CHL/IU cells).

Gene mutation study (waiver)

An in vitro gene mutation study in mammalian cells does not need to be conducted vecause a positive result was found in an in vitro cytogenicity study in mammalian cells. The study is therefore waived as scentifically unecessary according to REACH Annex XI as other information is available.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2018 - 13 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Stability: Not reactive to light nor air.
Solubility in water: less than 0.1 mg/mL
Solubility in DMSO: 50 mg/mL and more
Solubility in acetone: 100 mg/mL and more
Stability in solvent DMSO: no exothermic reaction nor generation of gas.
Storage conditions: Room temperature.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Source of TA100: The Division of Mutagenesis, National Institute of Hygienic Sciences, japan (The Division of Genetic and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences, Japan at present); April 15, 1982.
- Source of other TA strains: Dr. Ames, U.C. Berkley, CA, U.S.A.; January 20, 1988. Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 1987.

- Storage: 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 stores at -85 to -80 °C.

- Reason for choice of strains: These strains are very sensitive to mutagens and are the most commonly used in bacterial reverse mutation assays.

- Characterisation: As characteristic test, the number of viable cells, 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.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Source: Dr. Ames, U.C. Berkley, CA, U.S.A.; January 20, 1988. Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 1987.

- Storage: 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 stores at -85 to -80 °C.

- Reason for choice of strains: These strains are very sensitive to mutagens and are the most commonly used in bacterial reverse mutation assays.

- Characterisation: As characteristic test, the number of viable cells, 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.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 mix
- Source of S9: Oriental Yeast, Co., Ltd.
- Composition of S9 mix per 1 mL: Water (0.9 mL), S9 (0.1 mL), MgCl2 (8.0 μmol), KCl (33.0 μmol), Glucose-6-phosphate (5.0 μmol), NADPH (4.0 μmol), Na-Phosphate buffer, pH 7.4 (100.0 μmol).
- Concentration or volume of S9 mix in the final culture medium: 0.5 mL of S9 mix added to plates with metabolic activation.

Test concentrations with justification for top dose:

PRELIMINARY TEST
1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate.

MAIN TEST
2.4, 4.9, 10, 20, 39, 78 μg/plate (TA98, TA100 and TA1537 without metabolic activation)
10, 20, 39, 78, 156, 313 μg/plate (TA1535 without metabolic activation)
39, 78, 156, 313, 625, 1250 μg/plate (WP2 uvrA without metabolic activation)

39, 78, 156, 313, 625, 1250 μg/plate (TA98, TA100, TA1535 and WP2 uvrA with metabolic activation)
2.4, 4.9, 10, 20, 39, 78 μg/plate (TA1537 with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the solubility of the test material in water was < 0.1 mg/mL, the solubility test was performed with DMSO. The test material was dissolved at 50 mg/mL in DMSO, and neither exothermic nor generation of gas was observed. Therefore, DMSO was used as a solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191: 2 -Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine . 2HCL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2AA: 2 - Aminoanthracene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

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 the two main tests which were performed at the same doses.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: The preincubation method was used for all tests.
For tests 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.
For tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer.
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 was carried out equally.
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, 2.0mL 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.
All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies were counted.
Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

COUNTING PROCEDURE
The number of revertant colonies were counted visually due to the precipitate of the test material on the plates.
The revertant colonies of positive controls were counted with a colony counter.
- Model: Colony Analyzer CA-11
- Manufacturer: System Science Co., Ltf.
- Correction: Count loss correction
- Coefficient: 1- 100 colonies: x 1.11; 101 - 400 colonies: x 1.16; 401 - colonies: x 1.25

Evaluation criteria:

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 material was judged to be positive.

Statistics:

The results at each concentration were demonstrated with the mean and the standard deviation.

Key result

Species / strain:

S. typhimurium, other: TA100,m TA 1535, TA98, TA1537

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY STUDY
Growth inhibition by the test material was observed at:
> 78 μg/plate in S. typhimurium TA100 and TA98 without metabolic activation, and S. typhimurium TA1537 both with and without metabolic activation.
> 313 μg/plate in S. typhimurium 1535 without metabolic activation.
> 1250 μg/plate in E. coli WP2 uvrA both with and without metabolic activation and S. typhimurium TA100, TA 1535 and TA 98 with metabolic activation.
Precipitate of the test material on the plates was observed at > 78 μg/plate without metabolic activation and at > 1250 μg/plate with metabolic activation.

MAIN STUDY
- 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 the study was performed correctly.
- The precipitate of the test material on the plates was observed at 78 μg/plate and more without metabolic activation, and at 1250 μg/plate and more with metabolic activation.
- In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Remarks on result:

other: Mutagenicity was judged negative.

Conclusions:

Under the conditions of this study, the mutagenicity of the test material on the applied bacterial strains was judged negative.

Executive summary:

The mutagenicity potential of the test material was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA according to the standardised guidelines OECD 471, under GLP conditions. 

In this study, 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. 

It was therefore concluded the test material is not mutagenic to the applied bacterial strains, under the conditions of this assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2020 - 13 March 2020

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

The test material was treated as 100% in purity

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Fibroblasts

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: CHL/IU cells have been recommended in the methods described in “5. TEST METHOD”.

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Number of passages if applicable: 7 for the first cell growth inhibition test, 9 for the second cell growth inhibition test, 12 for the first chromosomal aberration test, 15 for the second chromosomal aberration test.
- Methods for maintenance in cell culture: Cells were suspended in medium Eagle’s minimum essential medium and 10 vol % heat-inactivated newborn calf serum including 10 vo l% DMSO and were frozen in liquid nitrogen for storage.
- Cell cycle length, doubling time or proliferation index: About 15 hours.
- Modal number of chromosomes: 25 per cell

Cells were suspended in Eagle's minimum essential medium and 10 vol% heat-inactivated newborn claf serum including 10 vol% DMSO and were frozen in liquid nitrogen.

CULTURE CONDITIONS
- CO2 concentration: 5 %
- Humidity: Under humid condition
- Temperature: 37 °C

MEDIUM
- Type and composition of media: L-Glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approximately 1.95 g/L) were added to Eagle’s minimum essential medium and basal medium (MEM) was prepared. This medium was then supplemented with 10 vol % heat-inactivated NBCS.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 Mix
- Components in 1mL of S9 mix: (S9, 0.3 mL; MgCl2, 5 μmol; KCl, 33 μmol; Glucose-6-phsophate, 5 μmol; NADP, 4 μmol; Na-Phosphoric acid buffering solution, HEPES (pH7.2), 4 μmol.
- Method of preparation of S9 mix: S9 mix was prepared just before use.
- Protein content of S9: 23.52 mg/mL and 24.89 mg/mL

Test concentrations with justification for top dose:

FIRST GROWTH INHIBITION TEST
- Without metabolic activation, with metabolic activation and 24 hours continuous treatment: 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

SECOND GROWTH INHIBITION TEST
- Without metabolic activation: 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125 and 250 μg/mL
- With metabolic activation: 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 μg/mL
- 24 hours continuous treatment: 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125 and 250 μg/mL

FIRST CHROMOSOMAL ABERRATION TEST
- Without metabolic activation: 7.81, 11.0, 15.6, 22.1, 31.3, 44.2, 62.5, 88.4, 125 and 250 μg/mL
- With metabolic activation: 7.81, 15.6, 31.3, 44.2, 62.5, 88.4, 125, 250, 500 and 1000 μg/mL
- 24 hours continuous treatment: 1.95, 3.91, 7.81, 9.29, 11.0, 13.1, 15.6, 22.1 and 31.3 μg/mL

SECOND CHROMOSOMAL ABERRATION TEST
- With metabolic activation: 3.75, 7.50, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 40.0 and 50.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was not suspended in distilled water at 20.0 mg/mL and soluble in DMSO at 200 mg/mL. The test material solution of 200 mg/mL prepared with DMSO was considered to be stable from the facts that there were no change in colour, exothermic reaction nor gas generation at room temperature within 2 hours after preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

The positive control item solutions were stored in a frozen place in a cell experimental room (permissible range: -80 °C or below) and thawed just before use, stored at room temperature and used within 2 hours after thawing.

Details on test system and experimental conditions:

CELL GROWTH INHIBITION TESTS
- One dish was used for each dose.
- Short-term treatments with and without metabolic activation of the test material at various concentrations were conducted, with treatment for 6 hours. The medium was then removed, rinsed and cultured for another 18 hours in fresh medium. For the 24-hour continuous treatment, the medium was removed from a pre-culture, and the cells were treated for 24 hours.
50 µL of a 10 µg/mL demecolcine solution was added to each dish at 2 hours before the end of the culture.
- At the start and the end of the treatment and at the end of the culture, precipitation of the test material, the colour change of the medium and the corrosion of the dishes were observed macroscopically.
- At the end of the culture, cells were suspended with 0.25 w/v% trypsin and diluted and the number of cells measured using a Particle counter/analyser (CDA-1000, Sysmex) and the cell growth rate compared with the negative control (Relative Cell Cound, RCC), Relative Population Doubling (RPD) and Relative Increase in Cell Count (RICC) were calculated according to the following:

PD = [log {(Number of cells at the end of the culture) / (Number of cells at the start of the treatment)}] / log 2

RPD (%) = (PD in test material group) / (PD in negative control group) × 100

RICC (%) = [Increase in number of cells in test material group {(the end of the culture) −
(the start of the treatment)}] / [Increase in number of cells in negative control group{(the end of the culture) − (the start of the treatment)}] × 100

MAIN TEST
- Procedures: The main test was carried out using the same procedure as in the cell growth inhibition tests.
Specimen preparation was carried out for the negative and the positive controls and all test material doses at which RPD was 30% or more. Two dishes were used for each dose. Two dishes were used for calculation of RPD and RICC. RPDs and RICCs for the positive controls were also calculated in all treatment methods.
- Specimen preparation: Leftover cells were collected by a centrifugation at 1000 rpm for 5 minutes and were treated hypotonically with 3 mL of 0.075 mol/L KCl at 37 °C for 15 minutes. After the hypotonic treatment, the cells were pre-fixed once with approximately 0.3 mL of a fixative solution (methanol : acetic acid = 3 : 1), and were completely fixed twice with 3 mL of fixative solutions. Then, the cell suspension was prepared with appropriate amount of a fixative solution, the suspension was dropped onto a glass slide. Four specimens per dose (two specimens per dish) were prepared. A specimen was dried and stained for about 15 minutes with 2 vol % Giemsa solution made with 1/15 mol/L phosphate buffer solution (pH 6.8).
- Dose levels: The dose levels of the test material were set on the basis of the results of the cell growth inhibition test. When RPD or RICC was less than 50%, it was judged that cytotoxicity is defined. The doses were set to obtain the dose which RPD or RICC was 40% or more and 50% or less.
In the first chromosomal aberration test without metabolic activation, the doses which RPD was 50% or less were obtained. However, the doses adequate for the evaluation of the chromosome aberration were not obtained with metabolic activation. Therefore, the specimen preparation was not carried out and the second chromosomal aberration test was carried out.

- Observations:
All specimens of the negative and positive controls were observed.
For the test material without metabolic activation, the lowest dose which RPD was 30% or more and 50% or less was selected as the maximum dose and the following doses were selected for observation: 7.81, 11.0 and 15.6 µg/mL
For the test material with metabolic activation, the lowest dose which RPD was 40% or more and 50% or less was selected as the maximum dose and the following doses were selected for observation: 20.0, 25.0 and 27.5 µg/mL
All specimens for observation including those of the negative and the positive control groups were coded and observed microscopically. Specimens of the 24 hours continuous treatment were not observed because the result was judged to be positive in the short-term treatment with metabolic activation.

- Structural aberration: Three hundred metaphase cells per dose (75 cells per specimen) containing 25 ± 2 chromosomes were observed. The total number of cells with structural aberrations excluding gaps and the number of aberrant cells in each aberration category were recorded. Gaps were defined as an achromatic region smaller than the width of one chromatid.
1) Chromatid break
2) Chromatid exchange
3) Chromosome break
4) Chromosome exchange (dicentric, ring and translocation)
5) Fragmentation

- Numerical aberration: The numbers of polyploid cells with triploid or more (38 or more chromosomes) and endoreduplicated cells among 300 metaphase cells per dose (75 cells per specimen) were recorded.

Evaluation criteria:

Regarding the frequencies of cells with chromosomal aberrations, the test material was judged to be negative if a) or b) was satisfied:
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 exhibits a statistically significant increase compared with the concurrent negative control.

Regarding the frequencies of cells with chromosomal aberrations, the test material was judged to be positive if cases of c) to e) or f) were fulfilled:
c) outside the distribution of the historical data of the negative control group,
d) there is a statistically significant increase compared with the concurrent negative control,
e) the increase of the frequencies of cells with chromosomal aberrations is dose-related,
f) both chromosomal aberration test and confirmation test are fulfilled in cases of c) and d).

Statistics:

Fisher’s exact test was performed in order to compare in the negative control group with positive control group and to compare in the negative control group with test material group when the frequencies of cells with chromosomal aberrations in each test material group were outside the distribution of the historical data of the negative control group. When the test material induced a statistically significant increase in the Fisher’s exact test, the dose-response relation was tested by the Cochran-Armitage trend test.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

FIRST CELL GROWTH INHIBITION TEST
In all treatment methods, the precipitation of the test material was observed at 250 µg/mL or more at the start of the treatment and all test material doses at the end of the treatment. With and without metabolic activation, the precipitation of the test material was obserbed at all test material doses at the end of the culture. The colour change of medium and the corrosion of the culture dish were not observed in all treatment methods.

SECOND CELL GROWTH INHIBITION TEST
With and without metabolic activation, the precipitation of the test material was observed at 250 µg/mL or more at the start of the treatment and at 31.3 µg/mL or more at the end of the treatment and at the end of the the culture. In 24 hours continuous treatment, at 250 µg/mL at the start of treatment and at 15.6 µg/mL or more at the end of treatment. The colour change of medium and the corrosion of the culture dish were not observed in all treatment methods.

FIRST CHROMOSOMAL ABERRATION TEST
> Without metabolic activation:
- The precipitation of the test material was observed at 250 μg/mL at the start of the treatment and at 22.1 μg/mL or more at the end of the treatment and the end of the culture. The colour change of medium and the corrosion of the culture dish were not observed.
- The frequency of cells with structural aberrations was within the historical data for the negative and positive control. A statistically significant increase was observed at both sides of 1 % level for the positive control. For the test material the frequencies were within the range of the historical data of the negative control. Therefore, it was judged to be negative.
- The frequency of cells with numerical aberrations was within the range of the historical data for the negative control. For the test material the frequencies were within the range of the historical data of the negative control. Therefore, it was judged to be negative.
> With metabolic activation:
- The precipitation of the test material was observed at 250 μg/mL or more at the start of the treatment and at 31.3 μg/mL or more at the end of the treatment and the end of the culture. The colour change of medium and the corrosion of the culture dish were not observed.
- 24 hour continuous treatment: The precipitation of the test material was observed at 15.6 μg/mL or more at the end of the treatment. The color change of medium and the corrosion of the culture dish were not observed.

SECOND CHROMOSOMAL ABERRATION TEST
> With metabolic activation:
- The precipitation of the test material was observed at 30.0 μg/mL or more at the end of the treatment and the end of the culture. The colour change of medium and the corrosion of the culture dish were not observed.
- The frequency of cells with structural aberrations was within the historical data for the negative and positive control. A statistically significant increase was observed at both sides of 1 % level for the positive control. For the test material at 25.0 and 27.5 μg/mL, the frequencies of structural aberrations were not within the range of the historical data of the negative control. The statistically significant increase was observed at 27.5 μg/mL compared with the concurrent negative control and the increase of the frequencies of cells with structural aberrations was dose-related. Therefore, it was judged to be positive.
- The frequency of cells with numerical aberrations was within the range of the historical data for the negative control. For the test material the frequencies of numerical aberrations were not within the range of the historical data of the negative control. The statistically significant increase was observed at 27.5 μg/mL compared with the concurrent negative control. However, the increase of the frequencies was not dose related.
- D20 was calculated to be 0.21 mg/mL for structural aberration.

Specimens of the 24 hour continuous treatment were not observed because the result was judged to be positive in the short-term treatment with metabolic activation.

Remarks on result:

other: The test material induced structural aberration and suspected to induce numerical aberration.

Summary of results of the second chromosomal aberration test (short-term with metabolic activation)

Dose (µg/mL)	Number of cells with structural chromosomal aberrations (frequency %)		Average Cell growth rate (%)	Average RPD (%)	Average RICC (%)	Number of cells with structural chromosomal aberrations (frequency %)
	Number of cells observed	Total number of cells with aberrations				Number of cells observed	Total number of cells with aberrations
Negative control (DMSO) 0	150 150 300	2 3 5 (1.7)	100	100	100	150 150 300	0 1 1 (0.3)
3.75	0	-	105.4	107.5	110.7	0	-
7.50	0		97.2	95.9	94.5	0
10.0	0		96.8	95.3	93.8	0
12.5	0		101.9	102.7	103.7	0
15.0	0		95.2	93.0	90.5	0
17.5	0		94.5	91.8	89.1	0
20.0	150 150 300	3 0 3 (1.0) #	93.1	89.8	86.4	150 150 300	0 5 5 (1.7)
22.5	0	-	88.8	83.0	77.9	0	-
25.0	150 150 300	6 5 11 (3.7) #	84.1	75.6	68.6	150 150 300	2 1 3 (1.0)
27.5	150 150 300	8 7 15 (5.0) #	68.1	45.8	37.2	150 150 300	5 3 8 (2.7)*
30.0	0	-	55.3	16.4	12.0	0	-
40.0	0		43.3	-21.8	-11.6	0
50.0	0		68.1	45.9	37.2	0
Positive control (CPA) 4	150 150 300	44 43 87 (29.0)**	74.5	58.4	49.9	150 150 300	0 1 1 (0.3)

The number of aberrant cells at each dish was shown at the first and the second lines. The total number of them was shown at the third line.

RPD: relative population doubling, RICC: relative increase in cell count, DMSO: dimethyl sulfoxide, CPA: cyclophosphamide monohydrate, n.s.: no specimens

The specimens at 3.75, 7.50, 10.0, 12.5, 15.0, 17.5, 22.5 and 50.0 μg/mL were not observed.

*: significant increase compared to negative control at p<0.05 [Fisher’s exact test]

#: the increase of the frequencies was dose-related at p<0.05 [Cochran–Armitage trend test]

 

Historical data in testing facility:

Negative control

The minimum range below 0 was shown “< 0”.

Treatment method		Frequency of cells with chromosomal aberrations (%)
		Structural aberration		Numerical aberration
		Mean	SD	Mean	SD
Short term treatment	Without metabolic activation	1.6	0.98	0.2	0.27
	With metabolic activation	0.9	0.47	0.1	0.18
24 hour continuous treatment		1.3	0.74	0.2	0.23
Treatment method		Range of frequency of cells with chromosomal aberrations (%, Values in parentheses indicate 95% control limit)
		Structural aberration		Numerical aberration
		Lower control limit	Upper control limit	Lower control limit	Upper control limit
Short term treatment	Without metabolic activation	< 0 (< 0)	3.8 (3.7)	< 0 (< 0)	1.1 (1.0)
	With metabolic activation	< 0 (< 0)	2.5 (2.5)	< 0 (< 0)	0.6 (0.6)
24 hour continuous treatment		< 0 (< 0)	3.3 (3.2)	< 0 (< 0)	0.9 (0.8)

 

Positive control

Treatment method		Substance	Dose (μg/mL)	Frequency of cells with chromosomal aberrations (%)
				Structural aberration
				Mean	SD
Short term treatment	Without metabolic activation	MMC	0.05	22.6	2.90
	With metabolic activation	CPA	4.00	26.1	3.85
24 hour continuous treatment		MMC	0.05	52.9	5.66
Treatment method		Range of frequency of cells with chromosomal aberrations (%, Values in parentheses indicate 95% control limit)
		Lower control limit		Upper control limit
Short term treatment	Without metabolic activation	14.4 (14.8)		30.8 (30.4)
	With metabolic activation	17.3 (17.7)		34.9 (34.5)
24 hour continuous treatment		40.3 (40.9)		65.5 (64.9)

The minimum range below 0 was shown “< 0”.

The latest 20 test data completed by December, 2019 were used.

Upper and lower control limits were calculated from number of cells with chromosomal aberrations using c chart.

The value was converted to the frequency from the number of cells with chromosomal aberrations.

Conclusions:

Under the conditions of this study the test material induced structural aberration and suspected to induce numerical aberration in Chinese hamster lung fibroblasts (CHL/IU cells).

Executive summary:

The ability of the test material to induce chromosomal aberrations was investigated using Chinese hamster lung fibroblasts (CHL/IU cells) in accordance with the standardised guideline OECD 473, under GLP condictions. 

In the chromosomal aberration test, the observation doses for evaluation were selected to be 7.81, 11.0 and 15.6 μg/mL in the short-term treatments without metabolic acvitation and 20.0, 25.0 and 27.5 μg/mL in the short-term treatments with metabolic activation.  As a result of the observation of specimens, in the short-term treatment without metabolic activation, the frequencies of cells with structural aberrations at all observation doses of the test material were within the range of the historical data of the negative control. In the short-term treatment with metabolic activation, the frequencies of cells with structural aberrations at 25.0 and 27.5 μg/mL were not within the range of the historical data of the negative control. The statistically significant increase was observed at 27.5 μg/mL compared with the concurrent negative control and the increase of the frequencies of cells with structural aberrations was dose-related. Therefore, structural aberration was judged to be positive. The frequencies of numerically aberrant cells at all observation doses of the test material in the short-term treatment without metabolic activation was within the range of the historical data of the negative control. In the short-term treatment with metabolic activation, the frequencies of numerically aberrant cells at all observation doses of the test material were not within the range of the historical data of the negative control. The statistically significant increase was observed at 27.5 μg/mL compared with the concurrent negative control. However, the increase of the frequencies of numerically aberrant cells was not dose-related.

Under the conditions of this study the test material induced structural aberration and suspected to induce numerical aberration in Chinese hamster lung fibroblasts (CHL/IU cells).

Reference 3

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:

An in vitro gene mutation study in mammalian cells does not need to be conducted vecause a positive result was found in an in vitro cytogenicity study in mammalian cells. The study is therefore waived as scentifically unecessary according to REACH Annex XI as other information is available.

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus Study (Kawamura 2021)

Under the conditions of this study, the test material did not induce micronuclei in mouse 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:

30 September 2020 - 11 November 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:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Stability: Measured by infrared (IR absorption spectra, confirmed to be stable during the test period.
Solubility: Water, 0.03 mg/L; acetone, 100 mg/mL; DMSO, 50 mg/mL

Treated as 100% purity.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Mouse is a species commonly used in this type of study and historical control data of this strain are available at the test facility.

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: Preliminary assay: males, 29.7 to 33.2 g; females, 25.7 to 29.0 g. Main assay: males, 30.5 to 34.4 g.
- Assigned to test groups randomly: Yes. Using a computer system (MiTOX, Mitsui E & S Systems Research Inc.), animals were assigned to test groups based on the body weight on the day of group assignment by the stratified random sampling method so that the group mean body weight was comparable among groups. In the main assay, group assignment (including the positive control group) was performed on the day before the start of administration in the negative control and test material groups, and individual body weight of males assigned to test groups ranged from 30.5 to 34.4 g, all of which were within ± 20 % of the mean (32.17 g).
- Housing: Animals were housed in groups of five or three during the quarantine and acclimatisation period, and housed by group after assignment in polycarbonate cages (320 W x 220 D x 135 H, mm), with bedding for experimental animals
- Diet: ad libitum.
- Water: Sapporo City tap water, ad libitum.
- Acclimation period: The period up to the day of group assignment, including the quarantine period (from the day of receipt, quarantine day 1, to quarantine day 6).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C ± 3 °C (actual range: preliminary assay and main assay; 21 - 23 °C).
- Humidity: 50 % ± 20 % (actual range: preliminary assay; 38 - 53 %, main assay; 44 - 58 %).
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial lighting for 12 h.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Japanese Pharmacopoeia olive oil
- Justification: Because the test material is hardly soluble in water.
- Concentration of test material in vehicle: Negative control article: 0; test material: 12.5, 25, 50, 100, 200 mg/mL
- Amount of vehicle: 10 mL/kg/time based on the body weight on each day of administration.
- Lot/batch no.: E03300

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation method: The amount of test material required for each dose was accurately weighed out into an agate mortal and pulverized with an agate pestle to mix with a small amount of the vehicle. When the mixture became fluidal, it was transferred to a graduated glass using a syringe. The mortar and pestle were rinsed with the vehicle several times until the rise appeared clear and the rinse was also transferred to the graduated glass. The content of the graduated glass was stirred with a stirrer to make a suspension. To the suspension, the vehicle was added to achieve the prescribed volume and the mixture was stirred to make a homogenous suspension.
- Doses (preliminary study): 125, 250, 500, 1000, 2000 mg/kg/day.
- Doses (main study): 500, 1000, 2000 mg/kg/day.
- Preparation frequency: Dosing suspensions were prepared twice, once for each of the preliminary assay and main assay, and used within two days under refrigerated storage.
- Storage conditions: Airtight and light-resistant glass containers and refrigerated (3.1 °C - 3.6 °C).
- Administration methods and routes: administered by oral gavage using a disposable syringe and a stomach tube into the stomach.

Duration of treatment / exposure:

2 days.

Frequency of treatment:

Twice within approximately 24 h-interval for the negative control article and the test material. Once within 24 h-interval for the positive control article (MMC).

Post exposure period:

On administration days 1 and 2, animals were observed for clinical signs prior to, immediately after and 4 h after administration. On the day of slide preparation animals were observed 21 to 22h (preliminary assay and main assay) after the final administration.
On the day of slide preparation, animals were weighed 21 to 22 h (preliminary assay and main assay) after the final administration with an electronic balance.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control article

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Test material

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Test material

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Test material

No. of animals per sex per dose:

Preliminary assay: 3 males and 3 females per dose
Main assay: 5 male animals per dose (negative control article, test material, positive control article).

Control animals:

yes

Positive control(s):

- Positive control: Mitomycin C (MMC)
- Route of administration: After disinfection of the right lower abdomen with 70 % ethanol swab, the dosing solution was injected into the abdominal cavity using a 25 G x 5/8" disposable injection needle and disposable syringe.
- Doses / concentrations: MMC was dissolved in the vehicle to obtain a concentration of 0.1 mg/mL. The positive control material solution was prepared at the time of use. One dose a day was administered (1 mg/kg/day).

Tissues and cell types examined:

- 4000 immature erythrocytes for each animal (2000 immature erythrocytes for each slide).
- 500 erythrocytes (250 erythrocytes for each side) for each animal in the main assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- In the preliminary assay, no toxic signs (clinical sign and effects on bone marrow) or effects on the body weight were observed in males or females in any dose group. Therefore, males were selected, and 2000 mg/kg/day was set as the highest dose, and two lower doses were selected in a twofold dilution series (3 doses in total) for the main assay.

TREATMENT AND SAMPLING TIMES:
- Slides were prepared 22 to 23 h after the final administration.

DETAILS OF SLIDE PREPARATION:
- Under anaesthesia with isoflurane, animals were euthanised by cervical dislocation and then bone marrow cells in the right and left femurs were flushed with foetal bovine serum.
- The cell suspension obtained was centrifuged at 150 x g (1000 rpm) to remove surplus serum, and a part of the cell suspension obtained was smeared onto a glass slide.
- The slide was air-dried at room temperature and then fixed with methanol.
- 4 slides were prepared for each animal during the main assay.
- After fixation with methanol, 2 slides per animal were coded by personnel other than those engaged in observation of the slides.

METHOD OF ANALYSIS:
- Each slide was stained with 0.005 % acridine orange staining solution, and then washed with 1/15 mol/L phosphate buffer (pH 6.8, instant phosphate buffer).
- A glass cover slip was placed on the slide, and sealed with enamel.
- The slides were observed with a fluorescence microscope (BX50: BX-FLA, Olympus Corporation) at a total magnification of 1000x.
- For the main assay, the incidence of micronucleated immature erythrocytes (incidence of micronuclei) was calculated from 4000 immature erythrocytes for each animal (2000 immature erythrocytes for each slide).
The proporation of immature erythrocytes was calculated from 250 erythrocytes for each animal in the preliminary assay or 500 erythrocytes (250 erythrocytes for each slide) for each animal in the main assay.

Evaluation criteria:

The validity of the test was verified when the mean incidence of micronuclei in the negative and positive control groups were within the range of ± 2 SD of the mean value of respective historical control data.

The result was determined positive when the incidence of micronuclei in any test material group was outside the range of mean ± 2 SD of the mean historical control value in the negative control group, and the incidence of micronuclei in the test material group was significantly higher than in the negative control group by the conditional binominal test in a dose-response manner.

It was also determined to exclude the dose of the test material at which the proportion of immature erythrocytes was less than 20 % of that in the negative control group from the evaluation, however this case did not occur in this study.

Statistics:

Body weight measurements:
- Group means and standard deviations of body weights were calculated and the following statistical analysis was performed using MiTOX.
- The body weights in the negative control and test material groups were analysed for homogeneity of variances by the Bartlett test. When variances were homogenous (p > 0.05), data were analysed by Dunnett's test. When variances were heterogenous (p < 0.05), data were analysed by Steel's test.
- Statistically significance level was less than 5 % for comparison with the control.

Incidence of micronuclei:
- The incidences of micronuclei in all test material groups were within ± 2 SD of the mean historical control value in the negative control group therefore statistical analysis was not performed.
- For the positive control group, these were analysed using the conditional binomial test (Test by the Kastenbaum an Bowman's table) to the negative control group.
- The test was conducted with an upper-tailed significance level of 5 % (represented as p > 0.05 and p > 0.01).

Proportion of immature erythrocytes:
- The following statistical analyses were formed using EXSUS (v. 8.1) and SAS (v. 9.3).
- The proportions were analysed by the Bartlett test. When variances were homogenous (p > 0.05) data were analysed by Dunnett's test, when variances were heterogenous ( p ≤ 0.05) data were analysed by Steel's test.
- The proportions were analysed by the F test (two tailed) for homogeneity. When variances were homogenous (p > 0.05) data were compared by Student's t-test, when variances were heterogenous ( p ≤ 0.05) data were compared by Aspin-Welch's t-test (two tailed).
- Statistically significance level was less than 5 % for comparison with the control.

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:

RESULTS OF PRELIMINARY TEST
- Dose range: 125, 250, 500, 1000 and 2000 mg/kg/day group.
- Clinical signs: No clinical signs were observed in males or females at any dose.
- Body weight: The mean body weights in males or females before the first administration, before the final administration and from 21 to 22 h after the final administration were similar among all doses.
- Proportion of immature erythrocytes: Similar to the negative control groups in all doses and there were no effects on bone marrow.

RESULTS OF DEFINITIVE STUDY
- Clinical sings: No clinical sings were observed in negative control group, at any dose, or the positive control group.
- Body weight: The mean body weights before the first administration, before the final administration and from 21 to 22 h after the final administration were similar to those in the negative control group with no statistically significant differences in all doses.
- The mean body weights from 21 to 22 h after administration in the positive control were similar to those before administration.

INCIDENCE OF MICRONUCLEI
- Negative control group: 0.12 % ± 0.07 % (mean ± SD, n = 5)
- 500 mg/kg/day: 0.10 % ± 0.06 %
- 1000 mg/kg/day: 0.08 % ± 0.04 %
- 2000 mg/kg/day: 0.07 % ± 0.05 %
- The values in the negative control group and test material groups were within the control values (± 2 SD of the mean value) of the historical control data of the test facility.
- Positive control group: 2.79 % ± 0.35 % (mean ± SD, n = 5), statistically significantly higher than in the negative control groups (p ≤ 0.01).
- The value in the positive control group was within the range of control values (± 2 SD of the mean value) of the historical control data of the test facility.

PROPORTION OF IMMATURE ERYTHROCYTES
- Negative control group: 53.2 % ± 2.8 % (mean ± SD, n = 5)
- 500 mg/kg/day: 52.0 % ± 3.9 %
- 1000 mg/kg/day: 55.8 % ± 6.0 %
- 2000 mg/kg/day: 55.7 % ± 8.4 %
- Positive control group: 57.1 % ± 4.2 % (mean ± SD, n = 5). Similar to that in negative control group with no statistically significant differences.

DISCUSSION
- The assay result was negative in the test material groups with no increase in the incidences of micronuclei.
- No effects of the test material on the body weights or clinical signs at any dose of the test material, or no inhibition of the bone marrow which is indicated by a decrease in the proportions of immature erythrocytes was observed.
- Though it is considered appropriate to set the high dose level that causes signs of toxicity, the 2-day successive administration of the doses used in this assay, 2000 mg/kg/day which is the upper limit dose of the single dose toxicity guideline (OECD 474), and 1000 and 500 mg/kg/day set with twofold dilution series, were likely to be sufficiently high doses to assay the potential to induce micronuclei.
- The mean incidence of micronuclei in the negative control group was within the range of the control values of the historical control data of the test facility.
- In the positive control group, the incidences of micronuclei clearly increased and the mean incidence was within the range of the control values of the historical control data of the test facility.
- The results indicated that the test system has sufficient sensitivity and that the potential of the test material to induce micronuclei was appropriately assessed.
- These results confirmed that the test material does not induce micronuclei in mouse bone marrow cells under the conditions of this assay.

Historical control data for micronucleus test (mice)

	Control data (Data: 2015.1 – 2020.9)		Positive control data (Date: 2015.1 – 2020.9)
			Mitomycin C 1 mg/kg
	% MNIE^1)	% IE^2)	% MNIE	% IE
No. of data	41	41	37	37
Mean	0.12	56.9	3.05	54.1
S.D.	0.03	2.7	0.43	4.0
Mean ± 2 S.D.	0.06 – 0.18	51.5 – 62.3	2.19 – 3.91	46.1 – 62.1
Maximum of group mean	0.17	61.8	4.11	61.8
Minimum of group mean	0.06	48.6	2.37	47.6

1) The incidence of micronucleated immature erythrocytes

2) The proportion of immature erythrocytes

Conclusions:

Under the conditions of this study, the test material did not induce micronuclei in mouse 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.

Based on the results of the preliminary assay using male and female mice, the test material was administered by oral gavage to male mice at 500, 1000, and 2000 mg/kg/day twice with approximately 24-h intervals. Japanese Pharmacopoeia olive oil was administered to the negative control group by the same method as in the test material groups, and Mitomycin C was intraperitoneally administered once to the positive control group. Bone marrow slides were prepared 22 to 23 h after the final administration.

No clinical signs were observed in the negative control, test material or positive control.

The mean body weights at each measurement time after administration in the test material groups were similar to those in the negative control group with no statistically significant differences.

The incidence of micronucleated immature erythrocytes (incidence of micronuclei) was 0.12 % ± 0.07 % (mean ± SD, n = 5) in the negative control group and the incidences in the 500, 1000 and 2000 mg/kg/day groups were 0.10 % ± 0.06 %, 0.08 % ± 0.04 % and 0.07 % ± 0.05 %, respectively, which were within the range of the control values of the historical control data of the test facility.

The incidence of micronuclei in the positive control group was 2.79 % ± 0.35 % (mean ± SD, n = 5), which was statistically higher than in the negative control group. This indicated that this assay was performed appropriately.

The proportion of immature erythrocytes in the negative control group was 53.2 % ± 2.8 % (mean ± SD, n = 5), while the proportions in the 500, 1000 and 2000 mg/kg/day groups were 52.0 % ± 3.9 %, 55.8 % ± 6.0 % and 55.7 % ± 8.4 %, respectively, which were similar with no statistically significant differences. Thus, bone-marrow toxicity was not induced by the test material.

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

- Genetic toxicity in vitro:

Bacterial reverse mutation assay (Sarada, 2018)

The mutagenicity potential of the test material was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, according to the standardised guidelines OECD 471, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In this study, 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. 

It was therefore concluded the test material is not mutagenic to the applied bacterial strains, under the conditions of this assay.

 

Chromosome aberration (Ogura, 2020)

The ability of the test material to induce chromosomal aberrations was investigated using Chinese hamster lung fibroblasts (CHL/IU cells) in accordance with the standardised guideline OECD 473, under GLP condictions. 

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

In the chromosomal aberration test, the observation doses for evaluation were selected to be 7.81, 11.0 and 15.6 μg/mL in the short-term treatments without metabolic acvitation and 20.0, 25.0 and 27.5 μg/mL in the short-term treatments with metabolic activation.  As a result of the observation of specimens, in the short-term treatment without metabolic activation, the frequencies of cells with structural aberrations at all observation doses of the test material were within the range of the historical data of the negative control. In the short-term treatment with metabolic activation, the frequencies of cells with structural aberrations at 25.0 and 27.5 μg/mL were not within the range of the historical data of the negative control. The statistically significant increase was observed at 27.5 μg/mL compared with the concurrent negative control and the increase of the frequencies of cells with structural aberrations was dose-related. Therefore, structural aberration was judged to be positive. The frequencies of numerically aberrant cells at all observation doses of the test material in the short-term treatment without metabolic activation was within the range of the historical data of the negative control. In the short-term treatment with metabolic activation, the frequencies of numerically aberrant cells at all observation doses of the test material were not within the range of the historical data of the negative control. The statistically significant increase was observed at 27.5 μg/mL compared with the concurrent negative control. However, the increase of the frequencies of numerically aberrant cells was not dose-related.

Under the conditions of this study the test material induced structural aberration and suspected to induce numerical aberration in Chinese hamster lung fibroblasts (CHL/IU cells).

 

- Genetic toxicity in vivo:

Mouse Micronucleus Study (Kawamura 2021)

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).

Based on the results of the preliminary assay using male and female mice, the test material was administered by oral gavage to male mice at 500, 1000, and 2000 mg/kg/day twice with approximately 24-h intervals. Japanese Pharmacopoeia olive oil was administered to the negative control group by the same method as in the test material groups, and Mitomycin C was intraperitoneally administered once to the positive control group. Bone marrow slides were prepared 22 to 23 h after the final administration.

No clinical signs were observed in the negative control, test material or positive control.

The mean body weights at each measurement time after administration in the test material groups were similar to those in the negative control group with no statistically significant differences.

The incidence of micronucleated immature erythrocytes (incidence of micronuclei) was 0.12 % ± 0.07 % (mean ± SD, n = 5) in the negative control group and the incidences in the 500, 1000 and 2000 mg/kg/day groups were 0.10 % ± 0.06 %, 0.08 % ± 0.04 % and 0.07 % ± 0.05 %, respectively, which were within the range of the control values of the historical control data of the test facility.

The incidence of micronuclei in the positive control group was 2.79 % ± 0.35 % (mean ± SD, n = 5), which was statistically higher than in the negative control group. This indicated that this assay was performed appropriately.

The proportion of immature erythrocytes in the negative control group was 53.2 % ± 2.8 % (mean ± SD, n = 5), while the proportions in the 500, 1000 and 2000 mg/kg/day groups were 52.0 % ± 3.9 %, 55.8 % ± 6.0 % and 55.7 % ± 8.4 %, respectively, which were similar with no statistically significant differences. Thus, bone-marrow toxicity was not induced by the test material.

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

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.