Thiophanate-methyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

The substance was tested for mutagenic activity in a study similar to OECD TG 471 with the bacterial strains S. typhimurium TA98, TA100,TA1535, TA1537, TA1538 and E.coli WP2 uvrA with and without metabolic activation in two independent studies. No increase of the number of revertants per plate was observed.

Chromosome Aberration Test

The test substance is considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both the metabolic activation and non-activation conditions in a study similar to OECD TG 473.

HPRT test

In a study similar to OECD TG 476 the substance was not genotoxic in mammalian cells in the absence or presence of S9 mix.

Micronucleus test

In a study similar to OECD TG 487 the substance was able to induce micronuclei in cultured human peripheral blood lymphocytes. The effect was only seen following treatment in the absence of S-9 and the micronuclei induced were formed predominantly from whole chromosomes, that is as a result of aneuploidy.

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:

March 8, 1990 - March 19, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EPA OPP 88-2

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : 9000 xg supernatant prepared from Sprague-Dawley adult male rat liver induced by phenobarbital and 5,6-benzoflavone was purchased from ORIENTAL YEAST CO., LTD.

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1 µg/plate (with and without S9 Mix)
The limit concentration required in the guideline was used for the tests.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

without S9 mix, TA100, TA98, WPuvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,2-benzpyrene

Remarks:

with S9 mix, TA100, TA98, TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix, TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix, TA1535, WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with S9 mix, TA1537

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2x10E9 cells/mL
- Test substance added: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 65 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

If mean number of revertants on triplicate plates of the test substance exceed two-fold compared to mean number of revertants on solvent control, and its dose relative appearance and reproducibility are observed, the test substance is judged a mutagen.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 625 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1250 µg/plate without S9 mix

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test compound precipitated at higher concentrations (final concentrations : 5000, 2500, 1250, 625 µg/plate) with and without presence of S9 mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see attachments

- Signs of toxicity : In the case of metabolic activation test, the test compound did not inhibit any bacterial growth. In the test without metabolic activation, the test substance showed inhibition of the bacterial growth at concentration of 1250 µg/plate or higher. E. coli was not inhibited at all.

- Individual plate counts : see attachments

- Mean number of revertant colonies per plate and standard deviation : see attachments

HISTORICAL CONTROL DATA
- no data

Conclusions:

The substance was tested for mutagenic activity in the Ames-test with the bacterial strains S. typhimurium TA98, TA100,TA1535, TA1537, TA1538 and E.coli WP2 uvrA with and without metabolic activation in two independent studies. No increase of the number of revertants per plate was observed.

Executive summary:

A study similar to OECD TG 471 was performed using Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and Escherichia coli (WP2 uvrA) with and without metabolic activation. This study consisted of two independent preincubation tests. Dimethylsulfoxide was used as solvent for preparation of the test substance. Eight concentrations of the test substance (5000, 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1 µg/plate) were used in the tests. The limit concentration required in the guideline was used as the highest concentration. No increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any concentration, either with or without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 18, 1988 - August 26, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

yes

Remarks:

A single harvest time was included only. According to the study report, this time had been found appropriate on the basis of the range finding test.

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: a permanent cell line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco
- Suitability of cells: proven
- Normal cell cycle time (negative control): 12 to 14 hours
- Modal number of chromosomes: 21

MEDIA USED
- McCoy's 5a culture medium which was supplemented with 10% fetal calf serum (FCS), 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% C02 in air

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 fraction was derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254 to induce the mixed function oxidase enzymes which are capable of metabolizing chemicals to more active forms.

Test concentrations with justification for top dose:

without metabolic activiation: 100, 200, 300 and 400 µg/mL
with metabolic activation: 250, 500, 750, 1000 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metablic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metablic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.2 x 10E6 cells /10 mL complete medium
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 17.25 hours (-S9 Mix) / 2 hours (+S9 Mix)
- Harvest time after the end of treatment: 20 hours after treatment

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: 0.1 µg/mL Colcemid® was incubated for 2.5 hours
- Methods of slide preparation and staining technique used including the stain used: The slides were stained in pH 6.8 buffered 5% Giemsa solution for the analysis of chromosomal aberrations.
- Criteria for scoring chromosome aberrations: Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 (range 19-23) were analyzed. One hundred cells, if possible, from each duplicate culture at four dose levels of the test article and from each of the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations. At least 25 cells were analyzed for chromosomal aberrations from one of the positive control culture. For control of bias, all slides except for the positive controls were coded prior to analysis. Cells with aberrations, but not gaps, were recorded on the data sheets by the microscope stage location.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival

Evaluation criteria:

The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e., complex aberrations may have more significance than simple breaks.
Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks.

Statistics:

Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). All factors as stated previously were taken into account and the final evaluation of the test article was based upon scientific judgement.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: A precipitate was visible at the time of harvest at 400 µg/mL under a 100 x magnification in the test with metabolic activation. A precipitate was visible at the time of dosing at 750 and 1000 µg/mL and was also visible at a magnification of 100 x at the time of harvest.

RANGE-FINDING/SCREENING STUDIES
Rangefinding Assay Without Metabolic Activation
A precipitate was visible at the time of dosing at 335 and 1000 µg/mL. Complete cellular toxicity was observed at 1000 µg/mL. Unhealthy cellular monolayer with reductions in visible mitotic cells were observed at 100 and 335 µg/mL. Cell cycle kinetics were evaluated at 3.35, 10.0, 33.5, 100, and 335 µg/mL. A significant delay in cell cycle was observed at 100 and 335 µg/mL.
Rangefinding Assay With Metabolic Activation
A precipitate was visible at the time of dosing at 335 and 1000 µg/mL. Unhealthy cellular monolayer and reductions in visible mitotic cells were observed at 335 and 1000 µg/mL. There was no significant toxicity at 100 µg/mL. Cell cycle kinetics were evaluated at 10.0, 33.5, 100, 335, and 1000 µg/mL. A significant delay in cell cycle was observed at 335 and 1000 µg/mL. A 20 hour delayed harvest was selected for the aberrations assay testing a dose range of 250, 500, 750, and 1000 µg/mL.

STUDY RESULTS
Without S9-Mix: Floating cellular debris, an unhealthy cell monolayer, a 43% reduction in the cell monolayer confluence and reduction in visible mitotic cells was observed at 400 µg/mL. Floating cellular debris, reduction in the number of visible mitotic cells, and slight reductions in the cell monolayer confluence were observed at 200 and 300 µg/mL. Results were analysed at 100, 200, 300, and 400 µg/mL. No significant increase in chromosomally aberrant cells was observed at the concentrations analysed. The test article is considered negative for inducing chromosomal aberrations under non-activation conditions.

With S9-Mix: An unhealthy cellular monolayer was observed at 1000 µg/mL. Results were evaluated at 250, 500, 750, and 1000 µg/mL. A significant increase in chromosomally aberrant cells was not observed at any of the doses analysed. The successful activation of the metabolic system is illustrated by the increased incidence of chromosomally aberrant cells in the cultures induced with cyclophosphamide, the positive control agent. The test article is considered negative for inducing chromosomal aberrations under conditions of metabolic activation.

HISTORICAL CONTROL DATA
- not available

Conclusions:

The test article is considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both the metabolic activation and non-activation conditions of this assay.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test item to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. The test article was dissolved in dimethyl sulfoxide at a stock concentration of 100 mg/mL. Complete toxicity was observed at 1000 µg/mL with significant delays in cell cycle at 100 and 335 µg/mL in the range-finding assay without activation. Significant delays in cell cycle were observed at 335 and 1000 µg/mL in the range-finding assay with metabolic activation. A 20 hour extended fixation was selected for the aberrations assays with and without activation. Duplicate cultures of CHO cells were incubated with 70.0 to 400 µg/mL of the test article in the non-activation assay and with 250 to 1000 µg/mL of the test material in the assay with activation. No significant increase in chromosomally aberrant cells was observed in either assay at any of the dose levels. The test article is considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both non-activation and activation conditions of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1984 - November 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Remarks:

When the study was performed, GLP was not compulsory. However, a statement of accordance to the principles of GLP is included in the report.

Type of assay:

bacterial forward mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese Hamster V79 cells, University of Trieste
- Suitability of cells: proven

For cell lines:
- Absence of Mycoplasma contamination: yes

MEDIA USED
- Dulbecco's MEM medium supplemented with 10% Foetal Calf Serum. Cells were incubated at 37°C in a 5% CO2 atmosphere (100% humidity)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
Two batches of S9 tissue homogenate, prepared from pools of 15 and 5 rat livers after mixed induction with Phenobarbitone and Betanaphthoflavone, were used in the study. The batches had protein contents of 35.0 and 35.2 mg/mL, and amino pyrine demethylase activities of 1.35 and 1.83 µM/mg liver.

- method of preparation of S9 mix
The pooled livers are washed three times in 0.15M KCL, and then weighed. The livers are then chopped finely, and homogenised in 0.15M KCL (3 mL homogenising solution: 1 mg liver) using an Ultraturrax homogeniser. The homogenate is centrifuged at 9000g for 10 minutes, and the supernatant is collected and distributed to sterile vials for storage at -80°C. The pellet, consisting of cell debris, nuclei and mitochondria, is discarded.

- quality controls of S9: The S9 tissue homogenates produced acceptable responses with the indirect mutagens 2-Aminoanthracene and Benzo(a)pyrene in Ames tests, with tester strain TA 100.

Test concentrations with justification for top dose:

100, 50, 25, 12.5 and 6.25 µg/mL (in the presence and absence of metabolic activation)
The top dose for the main assay was selected based on the results of a cytotoxicity assay.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The cultures are treated with the test or control solutions for three hours.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48, 96 and 168 h
- Selection time: After fourteen days incubation, the plates are stained with Giemsa and the number of clones are counted.
- selective agent is used : 6-thioguanine, 15 µg/mL

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

In this assay a test substance is considered mutagenic if a reproducible dose related increase in mutation frequency is observed and the induced frequencies are five fold greater than control values.

Statistics:

The results of the gene mutation assays were analysed by Analysis of Variance .

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was found to be soluble in DMSO at a maximum concentration of 10 mg/mL . When a 50 µL aliquot of this solution was added to 4.95 mL of minimal DMEM, a clear solution of 100 µg/mL was formed.
- Precipitation and time of the determination: no evidence of precipitation

RANGE-FINDING/SCREENING STUDIES: In the preliminary cytotoxicity assay, the test substance was assayed at a maximum dose of 100 µg/mL and eight ower doses spaced at 2 fold intervals. The test substance showed slight toxicity in the absence of S9 metabolism, but was not toxic over this dose range in the presence of S9. On the basis of these results, the top dose for the main assay was selected as 100 µg/mL.

STUDY RESULTS
Survival was assessed immediately after treatment in both experiments. The results show that 100 µg/mL reduced survival to values of 54% and 93% in the absence and presence of S9 mix respectively. In the second experiment the top dose reduced survival to 57% in the absence of S9 mix and 63% in its presence.
No reproducible five-fold increases in mutant numbers or mutation frequency (compared with solvent controls) were observed at any dose level of the test substance, in the absence or presence of S9 metabolism. Positive responses were obtained with the positive control treatments.

HISTORICAL CONTROL DATA
Not available

Conclusions:

The substance was not genotoxic in the present gene mutation assay in mammalian cells.

Executive summary:

The test item was examined for mutagenic activity in a study similar to OECD TG 476 (HPRT) in Chinese hamster V79 cells after in vitro treatment with and without a metabolic activation system (S9 Mix). Based on a preliminary cytotoxicity assay, two independent assays for mutation to 6-thioguanine resistance were performed using concentrations of 100, 50, 25, 12.5 and 6.25 μg/mL. No reproducible five-fold increases in mutant numbers or mutation frequency were observed at any dose level of the test substance, in the absence or presence of S9 metabolism. Positive responses were obtained with the positive control treatments.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1995 - October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: lymphocytes
- Suitability of cells: proven

For lymphocytes:
- Sex, age and number of blood donors: One healthy, non-smoking human volunteer (male)
- Whether whole blood or separated lymphocytes were used: whole bood
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: Phytohaemagglutinin

MEDIA USED
- Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
mammalian liver post-mitochondrial fraction (S-9)

- method of preparation of S9 mix
Male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, Annapolis, Maryland, USA.

- concentration or volume of S9 mix and S9 in the final culture medium
The final concentration of liver homogenate in the test system was 2%.

- quality controls of S9
Each batch was checked by the manufacturer for sterility, protein content (minimum 32 mg/mL), ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufln-O-dealkylase activities).

Test concentrations with justification for top dose:

The test item concentration ranges were 8.4 – 200 μg/mL (13 concentrations) without metabolic activation system and 150 – 700 μg/mL (12 concentrations) with metabolic activation system. The highest concentrations were selected on the basis of cytotoxicity.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Treatment media remained on cultures treated in the absence of S-9 until sampling, that is, 20 hours after the beginning of treatment. Cultures were treated in the presence of S-9 for 3 hours only. They
were then pelleted (200 x 'g', 10 minutes), washed twice with sterile saline, and resuspended in fresh medium containing foetal calf serum, gentamycin and Cytochalasin B. Cultures were incubated for a further 17 hours before harvesting.

FOR MICRONUCLEUS:
- Cytokinesis blocked method was used for micronucleus assay: Cytochalasin B made up in DMSO, was added (0.1 mL/culture) to the cultures to give a final concentration of 3 µg/mL.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Binucleate cells were to be only included in the analysis if all of the following morphological criteria were met:
1) the cytoplasm remained essentially intact, and
2) the daughter nuclei were of approximately equal size, and
3) any micronuclei present were separate in the cytoplasm or only just touching the main nucleus.
4) daughter nuclei contained multiple centromere signals.
Following Giemsa staining, micronuclei are seen as dark purple staining bodies no more than half the size of the main nuclei and lying separate in the cytoplasm.
One thousand binucleate cells from each replicate (2000 per dose level) were analysed for micronuclei.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes:
Any positive effects apparent following analysis of the data in the micronucleus assay were to be further investigated using FISH.

Approximately 15 µL of pre-warmed (37°C) hybridization solution (formamide, dextran sulphate, SSC) was placed directly to the probe coverslip (Chromoprobe™-M, for all human centromeres labelled with fluorescein, Supplier: Cytocell Limited, Oxon, UK) at 37°C. This was then placed over the appropriate region of the slide and sealed with rubber cement. Slides were then incubated at 37°C for approximately 5 minutes. The target DNA and probe were denatured by placing the slides on a hot plate at 77°C for 5 minutes. Slides were then hybridized in a moist chamber in the dark, at 37°C, overnight. Slides were drained of excess fluid and a 10 µL aliquot of propidium iodide-antifade solution added to the labelled area. Slides were coverslipped and sealed. Slides will then be stored in the dark for at least 10 minutes prior to visualization under fluorescence microscopy using a filter for FITC. The labelling of all human centromeres was confirmed by examination of five metaphase cells from the parallel colchicine-treated cultures for metaphases analysis.

Slide preparation (micronucleus analysis)
A few drops of suspension were gently spread onto clean, dry microscope slides. Fresh fixative was then added to the remaining suspension and the tubes returned to the refrigerator for FISH. After the slides had dried on a warm plate and allowed to cool, the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa stain pH 6.8 buffer (6). The slides were then rinsed and mounted with coverslips.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Ratio (binucleate/mononucleate plus binucleate)

Evaluation criteria:

A test chemical was to be considered to have clear clastogenic potential in this assay if:
1) a statistically significant increase in the proportion of cells with micronuclei occurred at one or more concentrations, and
2) the incidence of micronucleated cells at such data points exceeded the normal range.

A test chemical which induces frequencies of binucleate cells with micronuclei was to be considered as having:
1) a clear aneugenic and clastogenic potential, if the frequencies of centromere negative and positive micronuclei in binucleate cells are both statistically significant different from frequencies observed in negative control cultures, or
2) a clear aneugenic potential, if a statistically significant increase in centromere positive micronuclei in binucleate cells is observed and centromere negative micronuclei are not statistically increased when compared with frequencies observed in negative control cultures, or
3) a clear clastogenic potential, if a statistically significant increase in centromere negative micronuclei in binucleate cells is observed and centromere positive micronuclei are not statistically increased when compared with frequencies observed in negative control cultures.
The above criteria differentiate between any aneugenic or clastogenic potential.

Statistics:

The proportions of micronucleated cells in each replicate were used to establish acceptable homogeneity between replicates by means of a binomial dispersion test.
The proportion of cells with micronuclei for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p <0.05 were accepted as significant.
The proportions of centromere positive and negative micronucleated cells in each replicate was used to establish acceptable homogeneity between replicates by means of a binomial dispersion test.
The proportion of cells with centromere positive and negative micronuclei for each treatment condition was then compared with the proportion in negative controls by using Fisher's exact test. Probability values of p <0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
Cultures treated with the substance in the absence of S-9 had frequencies of cells with micronuclei which were significantly greater than those seen in concurrent negative controls at all dose levels analysed.
Cultures treated with the substance in the presence of S-9 had frequencies of cells with micronuclei which were similar to those seen in concurrent negative controls. A statistically significant increase in cells with micronuclei was apparent at the highest dose level analysed. The effect, however, was attributable to an increase in a single replicate and as numbers in both replicates fell within the normal range it was considered to be of questionable biological significance.

FISH analysis
Cultures treated with the test item in the absence of S-9 were analysed to determine the origin of the micronuclei observed. Cultures were seen to have frequencies of centromere-positive micronuclei which were significantly greater than those seen in concurrent negative controls at all dose levels analysed. A statistically significant increase in centromere-negative micronuclei at the lowest dose level was considered unlikely to be biologically important because the increase was small, was not seen at higher doses and was attributable to effects in a single replicate.

HISTORICAL CONTROL DATA
see attachment

Conclusions:

It is concluded that the substance was able to induce micronuclei in cultured human peripheral blood lymphocytes. The effect was only seen following treatment in the absence of S-9 and the micronuclei induced were formed predominantly from whole chromosomes, that is as a result of aneuploidy.

Executive summary:

The substance was tested for its clastogenic and aneugenic potential in an in vitro micronucleus assay (similar to OECD 487) using human lymphocyte cultures. Treatments were performed both in the absence and presence of a metabolic activation system. The test item concentration ranges were 8.4 – 200 μg/mL (13 concentrations) without metabolic activation system and 150 – 700 μg/mL (12 concentrations) with metabolic activation system. The highest concentrations were selected on the basis of cytotoxicity. The highest concentrations chosen for analysis were 47.5 and 550 μg/mL, which induced approximately 61 and 60 % reduction in the ratio of binucleate to mono- plus binucleate cells in the absence and presence of mammalian metabolic activation, respectively. Micronuclei were analysed by means of fluorescence in situ hybridisation (FISH) technique to distinguish between aneugenic (micronuclei with whole chromosomes, assessed by the presence of centromeres) and clastogenic effects (micronuclei with chromosome fragments, assessed by the lack of centromeres).

Treatment of cultures with the test item in the absence of a metabolic activation system resulted in micronucleus frequencies which were statistically significantly greater than those noted in concurrent negative controls at all concentrations and which fell outside the historical negative control range. Treatment in the presence of a metabolic activation system resulted in micronucleus frequencies similar to those seen in concurrent negative controls. A statistically significant increase in cells with micronuclei was apparent at the highest concentration analysed, but the effect was attributable to an increase in a single replicate. FISH analysis of positive samples indicated that the majority of the micronuclei induced contained one or more centromeres, and were thus formed from whole chromosomes rather than from chromosomal fragments.

With metabolic activation, there was no increase in the incidence of cells with micronuclei up to 500 µg/mL. At the highest concentration analysed (550 µg/mL), an increase was observed but this was due to a single replicate only (13 cells with micronuclei in one culture versus one only in the other).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus tests

In a study according to OECD TG 474 the test substance has shown clear evidence of weak genotoxic activity in the mouse micronucleus test in male and female B6D2F1 mice.

On the other hand, no clastogenicity in bone marrow cells of Crl:CD1(ICR) male mice was observed in a further study according OECD TG 474.

A spermatid micronucleus test in male mice according to OECD 474 was also negative.

In vivo Chromosome Aberration tests

The substance did not induce any chromosomal aberrations in mouse bone marrow in vivo up to and including the limit dose of 2000 mg/kg bw in a study according to OECD TG 475.

In a study according OECD TG 483 the substance did not induce any structural and/or numerical chromosomal aberrations in mouse spermatogonia in vivo up to and including the limit dose of 2000 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 1998 - 12 January 1999

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:

February 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: B6D2F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: 40 days
- Weight at study initiation: 20 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: each group was kept, with the sexes separated, in plastic disposable cages
- Diet: ad libitum, RM1(E)SQC standard laboratory pelleted rodent diet (Special Diet Services
- Water: ad libitum, tap water
- Acclimation period: 5 to 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 48 - 58
- Air changes (per hr): controlled environment
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Methylcellulose
- Justification for choice of solvent/vehicle: MC was selected since it provides homogeneously suspended condition.
- Amount of vehicle: 20 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were freshly prepared on the morning of the test (using identical methods for each phase of the test) and were diluted to the concentrations shown overleaf in aqueous 1% methylcellulose, obtained from Courtaulds, batch number T70654. All animals in all groups were dosed with the standard volume of 20 mL/kg bodyweight. All groups were treated orally by intragastric gavage.

Duration of treatment / exposure:

24 h or 48 h

Frequency of treatment:

once

Post exposure period:

no

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 (per group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Carbendazim (Methyl 1H-benzimidazol-2-ylcarbamate)
- Justification for choice of positive control(s): The test substance is metabolised to carbendazim in vivo, a known spindle poison which is positive in the micronucleus test following oral administration.
- Route of administration: oral, gavage
- Doses / concentrations: prepared as a suspension in aqueous 1 % methylcellulose (details as above) at a concentration of 50 mg/mL just prior to administration.

Tissues and cell types examined:

bone marrow of femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
From the results obtained in the preliminary toxicity study, dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for the micronucleus test.

TREATMENT AND SAMPLING TIMES:
Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Five males and five females from each group were sacrificed 24 and 48 hours after dosing, with the exception that animals in the positive control group were sampled at the 24 hour time point only. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone.

DETAILS OF SLIDE PREPARATION:
The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. Eight smears were made from each animal to allow conventional staining (using Giemsa) and optional centromere-specific staining for determining the mechanism of formation of micronuclei. The prepared smears were fixed in methanol (> 10 minutes) then allowed to air-dry before storing in a dust-free environment. Smears for conventional analysis were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of QUIT'S improved R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined.

OTHER: Centromeric staining
One unstained reserve slide from each animal in the positive control (carbendazim) and high level treatment group sampled at the 24 hour point and a total of ten slides from 9 animals treated with a chromosome-breaking agent (Mitomycin C) were stained and analysed. Slides from animals treated orally with Mitomycin C at 12 mg/kg and sampled 24 hours later were surplus reserve slides produced during the course of a separate parallel micronucleus test employing CD1 outbred mice, therefore no additional animal experimentation was required to produce the slides. Slides were encoded then stained with the mouse major satellite pan-centromeric probe, which was supplied conjugated to a fluorescent dye (FITC) to allow detection.
Only the hybridised region of each smear was examined using a fluorescence microscope. Micronucleated immature erythrocytes were identified by propidium iodide staining using a green excitation filter and a red barrier filter. Identified micronuclei were checked for the presence of an FITC-stained centromere using a blue excitation filter with a green barrier filter under a 100x oil immersion objective. Centromere-positive micronuclei contained a relatively large (compared with non-specific background staining) yellow-green slightly diffuse signal which was rounded or elongated in shape and lying in the same position and plane as the micronucleus.

THE PROPORTION OF IMMATURE ERYTHROCYTES
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time; a decrease at the 24 hour sampling time is not necessarily expected because of the relatively long transition time of erythroid cells [late normoblast -> immature erythrocyte (approximately 6 hours); mature erythrocyte (approximately 30 hours)]

Evaluation criteria:

The presence of a high proportion of centromere-positive micronuclei is indicative of chromosome-lagging and, therefore spindle damage. The presence of a low proportion of centromere-positive micronuclei is indicative of chromosome-breaking activity for the test substance. The presence of a high proportion of small micronuclei is indicative of a chromosome-breaking agent, while the presence of a high proportion of large micronuclei implies the presence of whole chromosomes and is indicative of aneugenic activity associated with spindle poisons.

Statistics:

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter-group comparisons (Je the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects (see next section) on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Key result

Sex:

male/female

Genotoxicity:

positive

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
- Dose range: 500, 1000, 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No mortalities or clinical signs of reaction were obtained at any time after administration of the standard limit dose (2000 mg/kg) of the test substance during this preliminary study.
- Harvest times: 1, 3, 6, 12 and 24 hours after treatment (plasma levels), 48 h (toxicity)
- Other: The substance appears to have been rapidly absorbed and systemically distributed before being cleared to reach very low levels between 12 to 24 hours after treatment; clearance was slowest in the high level group. Substantial amounts of the metabolite (carbendazim) were also seen in all groups treated with the test substance. In general the levels of carbendazim were lower but parallel to the detected levels of parent.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: No mortalities and no clinical signs of reaction were seen in any group at any time point in the micronucleus test.

- Induction of micronuclei: Animals treated with test item showed small but highly statistically significant dose-related increases in the number of micronucleated immature erythrocytes at both sampling times [P<0.001] with several mean values lying outside the laboratory historical control range while results for some individual animals lay at the extreme end of the historical control range. Carbendazim caused large, highly significant increases [P<0.001] in the frequency of micronucleated immature erythrocytes.

Centromeric staining
Carbendazim induced the expected high proportion (68%) of micronuclei containing centromeres, while Mitomycin C caused a low proportion of centromere-positive micronuclei (24%). The substance appeared to cause an intermediate proportion of centromere-positive micronuclei (34%).

Size analysis
Carbendazim induced a high proportion of large micronuclei while Mitomycin C induced mainly small micronuclei (mean sizes 40.1 and 25.4 units respectively). The substance appeared to produce micronuclei of intermediate size distribution (mean size 31.9 units)

- Proportion of immature erythrocytes:
A statistically significant dose-related decrease in the proportion of immature erythrocytes was obtained for animals treated with Thiophanate-methyl and sampled at the 24 hour time point [P<0.01]. Since this decrease was only very slight and was not seen at the later sampling time, it is possible that the apparent decrease was the result of chance variation rather than being evidence of treatment-related toxicity. Carbendazim caused a small but statistically significant decrease in the proportion [P<0.001]. It should be noted that cytotoxic compounds such as carbendazim only cause a small decrease in this proportion at the 24 hour sampling time because of the lag caused by erythrocyte maturation, although a much larger decrease would be expected if a later sampling time had been used.

- Appropriateness of dose levels and route: The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.

Conclusions:

The test substance has shown clear evidence of weak genotoxic activity in the mouse micronucleus test. Qualitative analysis of micronuclei to determine whether the substance operates primarily by causing spindle dysfunction in a similar manner to carbendazim (and would, therefore, be expected to exhibit a threshold dose for activity) were inconclusive. This was because of the large difference in effective dose levels of the two compounds in this study.

Executive summary:

A study according OECD TG 474 was conducted. Male and female B6D2F1 mice were treated by single oral gavage with the test item at dose levels of 500, 1000 or 2000 mg/kg bw. A concurrent negative control group received the vehicle only, while a positive control (PC) group was treated with carbendazim at 1000 mg/kg bw. Bone marrow smears were obtained each from 5 mice/sex/dose group at 24 and at 48 hours after dosing with the exception that mice in the PC group were sampled at the 24 hour time-point only.

A preliminary toxicity test had previously shown that a dose of 2000 mg/kg, the standard limit dose for the micronucleus test, was tolerated; this level was therefore selected as an appropriate maximum for use in this study. Serum analysis of mice treated with the substance in the course of this preliminary study showed that the test item was absorbed and systemically distributed following oral administration, so that significant bone marrow exposure could reasonably be expected. In addition, serum analysis showed that a proportion was metabolized to carbendazim (a known spindle-poison with aneugenic activity).

One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

Mice treated with the test substance showed a small dose-related and highly statistically significant increase in the frequency of micronucleated immature erythrocytes at both sampling times. Increases obtained at the low and intermediate dose levels were very small, with group mean values falling just outside the laboratory historical control range for mice.

A statistically significant dose-related decrease in the proportion of immature erythrocytes was obtained for animals treated with the substance and sampled at the 24 hour time point; since this decrease was only very slight and was not seen at the later sampling time, it is possible that the apparent decrease was the result of chance variation rather than being treatment-related. The positive control compound, carbendazim, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes together with a significant decrease in the proportion of immature erythrocytes.

Centromere-specific staining and size analysis of micronuclei in bone marrow smears prepared from these animals subsequently confirmed that carbendazim produced micronuclei predominantly from lagging chromosomes with the remainder being formed from chromosome fragments. In comparison, a smaller proportion of the micronuclei induced by the substance was apparently formed from lagging chromosomes. However, this difference in proportion could relate to the much lower rate of induction of micronuclei by the test item rather than indicating any fundamental difference in mechanism of activity between the two compounds.

The test substance has been shown to induce low numbers of micronucleated cells in the bone marrow of mice. Qualitative analysis of micronuclei to determine whether the substance operates primarily by causing spindle dysfunction in a similar manner to carbendazim (and would, therefore, be expected to exhibit a threshold dose for activity) was inconclusive. This was because of the large difference in effective dose levels of the two compounds with the test substance inducing much lower numbers of micronuclei than carbendazim at comparable dose levels.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15, 2018 - February 9, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

other: B6D2F1/Crl

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER LABORATORIES JAPAN, INC. (Hino, Japan)
- Age at study initiation: 9 weeks
- Weight at study initiation: 24.89-28.35 g
- Assigned to test groups randomly:yes
- Fasting period before study: no
- Housing: Polycarbonate cage for mice, a mouse per cage
- Diet: ad libitum, Laboratory animal diet, CRF-1 pellets
- Water: ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 ± 0.3
- Humidity (%): 56.6 ± 0.9
- Air changes (per hr): 8-35
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 1% methyl cellulose aqueous solution
- Justification for choice of solvent/vehicle: Since the test substance can be prepared in a homogeneous suspension, the vehicle (1% methyl cellulose aqueous solution) was selected.
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and pulverized in a mortar aided pestle, and suspended gently in 1% methyl cellulose aqueous solution. The suspension was ultrasonicated immediately before administration.

Duration of treatment / exposure:

24 h or 48 h

Frequency of treatment:

once

Post exposure period:

no

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C (MMC)
- Justification for choice of positive control: MMC is widely used as a positive control in the chromosome aberration tests and represented as an example in OECD Guidelines for the Testing of chemicals, TG-475.
- Route of administration: Intraperitoneal administration
- Doses / concentrations: 4 mg/kg bw

Tissues and cell types examined:

bone marrow from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the acute oral toxicity study in male mice, the LD50 had been determined to be 3514 mg/kg bw, thus, a dose of 2000 mg/kg bw was used as the limit dose of the guidelines, and three dose levels (500, 1000 and 2000 mg/kg bw) with a common ratio of 2 were selected for this study.

TREATMENT AND SAMPLING TIMES:
At 24 or 48 hours after dosing, the animals were sacrificed by exsanguination from the abdominal aorta under isoflurane inhalation anesthesia, and both femurs were removed. Three hours before the sacrifice, animals were administered colchicine at 4 mg/kg bw by intraperitoneally for cell division arrest.

DETAILS OF SLIDE PREPARATION:
1)Both femurs dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the head of the femur removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total
volume of 2.5 mL of isotonic solution. The cell suspensions were centrifuged for 10 min at 150 g.
2) After discarding the supernatant, pellet was treated with 4 mL of the hypotonic solution and gently mixed. The cell suspensions were shaken in a water bath for 20 min at 80 rpm at 37°C.
3) 2) were added 0.5 mL of the Carnoy fluid and gently mixed, and then centrifuged.
4) After discarding the supernatant, pellet was added 5 mL of the Carnoy fluid and pipetted.
5) Stored overnight in refrigerator.
6) The cells were pipetted and centrifuged. After discarding the supernatant, pellet was added 5 mL of the Carnoy fluid and pipetted, and then cells were incubated on ice for 30 min.
7) 6) was repeated.
8) The cells were pipetted and centrifuged, and then the supernatant discarded.
9) The pellets were resuspended with a small amount of the Carnoy fluid, and cells were dropped on slides with two drops per slide.
10) Each slide was air-dried.

The slides were stained with 2% (v/v) Giemsa dye solution for 15 min. After washing with water, the slides were washed with 0.15% (v/v) Acetic acid solution and water again, and then air-dried.

METHOD OF ANALYSIS:
All slides were coded and observed under blind. For the cytotoxic effect on bone marrow cells, more than 1000 cells per each animal were examined and recorded using microscope, and the mitotic index was determined from the
incidence of mitotic cells. For the metaphase analysis, at least 200 metaphases of well-spread chromosomes with 40 ± 2 were observed using microscope.

Evaluation criteria:

Structural aberration: A cell with one or more the following structural chromosome aberration(s) was recorded as a structural chromosomal aberrant cell.
Chromatid break (ctb)
Chromatid exchange (cte)
Chromosome break (csb)
Chromosome exchange (dicentric, ring, etc.) (cse)
Others (cells with greater than eight aberrations, pulverized cells
and pulverized chromosomes)
A gap was defined as an achromatic region of a single chromatid that was narrower than the width of the chromatid. Gaps were recorded in distinction from theother aberrations and not included in the structural aberration assessment.
Numerical aberration: A polyploid cell (a mode number with kinetochores was more than 76 (including endoreduplicated cells)) was recorded as a numerical
chromosomal aberrant cell.
The result was judged to be positive in cases where a statistically significant increase in the frequency of cells with structural or numerical aberration in the test substance treatment group compared to the vehicle control value was observed with dose-dependency.

Statistics:

Wilcoxon rank sum test (significant level was 5%) and Cochran-Armitage trend test (significant level was 5%, two sided) was performed for frequencies of aberrant cells in the treatment groups. Wilcoxon rank sum test was conducted between the negative control and each treatment group. Cochran-Armitage trend test was conducted for the treatment groups including the negative control. Both of the tests were performed using JMP. For positive control, Wilcoxon rank sum test was only performed compared with the negative control.

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 DEFINITIVE STUDY
No clinically observable signs of toxicity were detected throughout the study. No dose related change of body weights was recorded in any groups.

At 24 h after dosing:
Cytotoxicity
The mitotic indices (MI) were 2.21% (1.59-3.30%) in the negative control, and 2.68% (1.83-3.38%), 2.93% (1.79-3.80%), or 3.42% (1.40-4.40%) in 500, 1000, or 2000 mg/kg bw treatment group respectively. There was no effect on the MI caused by the treatment.
The MI in the positive control was 2.01% (0.70-3.03%), that was a lower value compared to the negative control.

Frequency of structurally aberrant cells
Frequencies of structurally aberrant cells were 2.00 ± 0.79% in the negative control, and 1.80 ± 0.91%, 1.60 ± 0.65%, or 1.40 ± 0.74% in 500, 1000, or 2000 mg/kg bw treatment group respectively. For the treatment groups, there was no statistically significant increase nor dose-response in the frequency of aberrant cells at any dose levels. Frequency of structural aberrant cells in the positive control was 16.30 ± 1.89%, and there was a statistically significant increase (p < 0.01) compared to the negative control.

Frequency of polyploidy
Frequencies of polyploidy were 0.40 ± 0.64% in the negative control, and 0.20 ± 0.27%, 0.40 ± 0.41%, or 0% in 500, 1000, or 2000 mg/kg bw treatment group respectively, and 1.37 ± 1.11% in the positive control. For the treatment groups, there was no statistically significant increase nor dose-response in the frequency of aberrant cells at any dose levels.

At 48 h after dosing:
Cytotoxicity
The MI were 3.57% (2.92-4.30%) in the negative control, and 4.18% (2.99-5.40%) in 2000 mg/kg bw treatment group. There was no effect on the MI caused by the treatment.

Frequency of structurally aberrant cells
Frequencies of structurally aberrant cells were 1.60 ± 0.55% in the negative control, and 1.70 ±1.10% in 2000 mg/kg bw treatment group. For the treatment group, there was no statistically significant increase in the frequency of aberrant cells as compared to the vehicle control.

Frequency of polyploidy
Frequencies of polyploidy were 0.40 ± 0.41% in the negative control, and 0.40 ± 0.64% in 2000 mg/kg bw treatment group. For the treatment group, there was no statistically significant increase in the frequency of aberrant cells as compared to the vehicle control.

The range of frequency of chromosomal aberrant cells in the testing facility is 0-4.00% for structural aberration, and 0-1.96% for numerical aberration, therefore the frequency of chromosomal aberrant cells in the negative control in this study was within the
historical control data. The frequency of chromosomal aberrant cells in the positive control in this study was significantly higher values compared to the concurrent negative control, and was within the historical control data. Thus, this study was considered to be valid.

Conclusions:

The substance did not induce any chromosomal aberrations in mouse bone marrow in vivo up to and including the limit dose of 2000 mg/kg bw.

Executive summary:

The mammalian bone marrow chromosomal aberration test according OECD TG 474 in B6D2F1/Crl male mice was performed to examine bone marrow genotoxicity of the substance. Six male mice (9 weeks old at the start of dosing) per group were dosed the substance in 1% methyl cellulose aqueous solution at 0 (negative control group), 500, 1000 or 2000 mg/kg bw once orally by gavage, and mitomycin C in saline as positive control at 4 mg/kg bw once by intraperitoneal injection. Bone marrow from both femurs was removed 24 or 48 hours after administration for all groups. Three hours before sacrifice, animals were administered colchicine at 4 mg/kg bw intraperitoneally for cell division arrest. Bone marrow was collected and slides were prepared and stained. Bone marrow in five mice per group was examined for the chromosomal damage.

There was no increase of bone marrow cells with structural and/or numerical chromosomal aberration by test item treatment. On the other hand, a statistically significant increase of cells with structural chromosomal aberration was observed in the positive control group. The substance did not induce any structural and/or numerical chromosomal aberrations in mouse bone marrow.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 23,2017 - April 5,2017

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:

September 26,2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Remarks:

Cr:CD1

Details on species / strain selection:

Mice are widely used in micronucleus studies, and the characteristics of the strain of mice used in this study are well-known with ample historical control data. the results of the various toxicity studies, this test substance showed no clear sex differences. Therefore, this study was conducted using only males.

Sex:

male

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: 33.1 to 38.7 g (mean: 35.4 g)
- Assigned to test groups:
Animals were assigned to groups in such a way that the group mean body weight was comparable as far as possible. Animal assignment was conducted through a combination of the block placement and random sampling methods using a computer (the requisite groups were established by the block placement method, and test groups and individual numbers within each group were assigned at random).
- Fasting period before study: no
- Housing: housed individually in plastic cages
- Diet: ad libitum, pelleted diet CR-LPF (radiation-sterilized: Oriental Yeast Co., Ltd.)
- Water: ad libitum, tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 41 - 46
- Air changes (per hr): 10 to 15 times
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Methylcellulose 400
- Justification for choice of solvent/vehicle: MC was selected since it provides homogeneously suspended condition.
- Concentration of test material in vehicle: 0.1 mg/mL
- Amount of vehicle: 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed for each dose concentration, suspended in the vehicle in an agate mortar, diluted to the specified volume, and then dispensed into brown glass bottles. The test formulations were prepared at the time of use and not stored.

Duration of treatment / exposure:

24 h

Frequency of treatment:

twice

Post exposure period:

no

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Justification for choice of positive control(s): MMC was selected since it is used widely in micronucleus tests, is known to induce micronucleus in mouse bone marrow cells, and ample background data are available.
- Route of administration: intraperitoneal
- Doses / concentrations: 1 mg/kg bw

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The high dose level was set at 2000 mg/kg, the limit dose by the Guidelines, and the middle and low dose levels at 1000 mg/kg and 500 mg/kg, respectively, using a common ratio of 2.

TREATMENT AND SAMPLING TIMES:
A total of 5 groups were provided by adding the negative control group in which animals received the vehicle and the positive control group in which animals received Mitomycin C. The bone marrow was collected approximately 24 hours after the second admimstration. Mice were euthanized by cervical dislocation under isoflurane anesthesia and the femurs of both sides were removed and both ends of the femurs were cut off. Then, bone marrow cells were flushed out into a centrifuge tube containing a small amount (ca. 0.1-0.2 mL) of fetal bovine serum using a 1 mL disposable syringe with a 23 G injection needle

DETAILS OF SLIDE PREPARATION:
The supernatant was a little discarded, and the precipitate was agitated by using a mixer and smeared on a glass slide on which the sample number was written (one smear specimen was prepared from the femur on each side of the animal). The smeared cells were air-dried, fixed with methanol for 3 minutes, and air-dried again.
One glass slide that showed satisfactory smear condition was selected based on the sample number in sequence for each animal, which was assigned for the purpose of observation by a blind method, and was put on a cover glass on which a small amount of 40 µg/mL acridine orange solution had been placed. The glass slide was observed at the magnification of 600-fold with a fluorescent microscope that has exciting light at wavelength of approximately 490 nm and an observation filter which transmits the light of wavelength at 515 nm or longer.

METHOD OF ANALYSIS:
For each animal, the number and proportion (%) of polychromatic erythrocytes (hereinafter referred to as PCE) per 500 erythrocytes and the number and proportion (%) of micronucleated polychromatic erythrocytes (hereinafter refeired to as MNPCE) per 4000 PCE was determined.

Statistics:

Fisher's Exact test

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 DEFINITIVE STUDY
- Clinical signs: There were no clinical signs in any animal. There were no treatment-related changes either in the test substance or positive control group
- Induction of micronuclei:
The proportions of MNPCE (micronucleated polychromatic erythrocytes) to 4000 PCE (polychromatic erythrocytes) were 0.13 ± 0.06%, 0.17 ± 0.05% and 0.14 ± 0.05% in the 500, 1000 and 2000 mg/kg groups, respectively, and they were not significantly different from the negative control value (0.14 ± 0.08%) and there was no dose-dependent increase. The incidence of PCE in 500 total eiythrocytes in each test substance group did not show any statistically significant change when compared to that in the negative control group. The incidence of MNPCE was 3.30 ± 0.36% in the positive control group showed a statistically significant (p<0.05) increase when compared to that in the negative control group. In the positive and negative control groups, the incidences of MNPCE were within the range of the mean ± 1.96 S.D. of the respective historical control data in the test facility.

Conclusions:

No clastogenicity in bone marrow cells of Crl:CD1(ICR) male mice was observed.

Executive summary:

In order to clarify the clastogenicity potential of the test item, a micronucleus test according OECD TG 474 with bone marrow cells was conducted in Crl:CD1 (ICR) male mice.The high dose level of was set at 2000 mg/kg bw, and a total of 3 dose levels including 1000 and 500 mg/kg bw were provided using a common ratio of 2. The test substance was administered orally twice at an interval of approximately 24 hours. At approximately 24 hours after the second administration, bone marrow smear specimens were prepared and observed. In addition, a negative control group in which the animals received 1 % methylcellulose solution and a positive control group in which animals received mitomycin C once intraperitoneally at 1 mg/kg were provided. At the specified time after administration, bone marrow smear specimens were prepared and observed. Each group consisted of 5 males.

In the results, the incidence of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in each test substance group did not show any statistically significant increase when compared to that in the negative control group, and there was no dose-dependent increase.The incidence of polychromatic erythrocytes in a total of 500 erythrocytes showed no statistically significant change in any test substance group in comparison with that of the negative control group.The incidence of micronucleated polychromatic erythrocytes in the positive control group was significantly (p<0.05) increase than that in the negative control group. In addition, this study was conducted appropriately because the incidences of micronucleated polychromatic erythrocytes in the positive control group and negative control group were within the range of the mean ± 1.96 S.D. of the respective historical control data in the test facility. On the basis of the above results, it was judged that the substance had no clastogenicity in the bone marrow cells in Crl:CD1 (ICR) male mice under the conditions of this study.

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2, 2016 - December 20, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

mammalian germ cell chromosome aberration test

Species:

mouse

Strain:

ICR

Remarks:

Crl:CD-1(ICR)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER LABORATORIES JAPAN, INC.
- Age at study initiation: 8 weeks
- Weight at study initiation: 29.53-36.95 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Polycarbonate cage for mice, 1-3 mice per cage (When the intense fighting was observed, the animals were housed in isolation as needed.)
- Diet: ad libitum, Laboratory animal diet, CRF-1 pellets, Oriental Yeast Co., Ltd., Tokyo
- Water: ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 ± 0.1
- Humidity (%): 57.4 ± 0.9
- Air changes (per hr): 8-35
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 1% methyl cellulose aqueous solution
- Justification for choice of solvent/vehicle: Since the test substance can be prepared in a homogeneous suspension, the vehicle (1% methyl cellulose aqueous solution) was selected.
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and pulverized in a mortar aided pestle, and suspended gently in 1% methyl cellulose aqueous solution. The suspension was ultrasonicated immediately before administration. The dose volume was 10 mL/kg bw.

Duration of treatment / exposure:

24 h or 48 h

Frequency of treatment:

once

Post exposure period:

no

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C (MMC)
- Justification for choice of positive control: MMC is widely used as a positive control in the chromosome aberration tests and represented as an example in OECD Guidelines for the Testing of chemicals, TG-475.
- Route of administration: Intraperitoneal administration
- Doses / concentrations: 4 mg/kg bw

Tissues and cell types examined:

testes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the acute oral toxicity study in male mice, the LD50 had been determined to be 3514 mg/kg bw; thus, a dose of 2000 mg/kg bw was used as the limit dose of the guidelines, and three dose levels (500, 1000 and 2000 mg/kg bw) with a common ratio of 2 were selected for this study.

TREATMENT AND SAMPLING TIMES::
At 24 or 48 hours after dosing, the animals were sacrificed by exsanguination from the abdominal aorta under isoflurane inhalation anesthesia, and both testes were removed. Five hours before the sacrifice, animals were administered colchicine at 5 mg/kg bw by intraperitoneally for cell division arrest.

DETAILS OF SLIDE PREPARATION:
1) Testes were washed in 5 mL of the TIM.
2) Testes weights were individually measured using an electronic scale.
3) The tunica albuginea was carefully removed and the seminiferous tubules were divided into 10 equal parts in 5 mL of the TIM.
4) 3) and 5.1 mL of the collagenase solution were mixed in a 100 mL glass flask.
5) The flasks were shaken in a water bath for 15 min at 80 rpm at 37°C.
6) The cell suspensions were filtered (mesh size: 100 µm, Falcon cell-strainer, Corning Inc.) and centrifuged for 10 min at 150 g.
7) After discarding the supernatant, pellet was washed with 2 mL of the isotonic solution. Centrifuged for 10 min at 150 g.
8) After discarding the supernatant, pellet was treated with 4 mL of the hypotonic solution for 15 min at ambient temperature.
9) 8) were added 0.5 mL of the Carnoy fluid and gently mixed.
10) 6) was repeated.
11) After discarding the supernatant, pellet was added 5 mL of the Carnoy fluid and pipetted.
12) Stored overnight in refrigerator.
13) The cells were pipetted and centrifuged. After discarding the supernatant, pellet was added 5 mL of the Carnoy fluid and pipetted, and then cells were incubated on ice for 10 min.
14) 13) was repeated.
15) The cells were pipetted and centrifuged, and then the supernatant discarded.
16) The pellets were resuspended with a small amount of the Carnoy fluid, and cells were dropped on slides with two drops per slide.
17) Each slide was air-dried

The slides were stained with 2% (v/v) Giemsa dye solution for 15 min. After washing with water, the slides were washed with 0.15% (v/v) Acetic acid solution and water again, and then air-dried.

METHOD OF ANALYSIS:
All slides were coded and observed under blind. For the cytotoxic effect on spermatogonia, more than 1000 cells per each animal were examined and recorded using microscope, therefore determined the mitotic index (a frequency of metaphase).
For the metaphase analysis, at least 200 metaphases of well-spread chromosomes with 40 ± 2 were observed using microscope.

Evaluation criteria:

Structural aberration: A cell with one or more the following structural chromosome aberration(s) was recorded as a structural chromosomal aberrant cell.
Chromatid break (ctb)
Chromatid exchange (cte)
Chromosome break (csb)
Chromosome exchange (dicentric, ring, etc.) (cse)
Others (cells with greater than eight aberrations, pulverized cells and pulverized chromosomes)
A gap was defined as an achromatic region of a single chromatid that was narrower than the width of the chromatid. Gaps were recorded in distinction from theother aberrations and not included in the structural aberration assessment.
Numerical aberration: A polyploid cell (a mode number with kinetochores was more than 76 (including endoreduplicated cells)) was recorded as a numerical chromosomal aberrant cell.
The result was judged to be positive in cases where a statistically significant increase in the frequency of cells with structural or numerical aberration in the test substance treatment group compared to the vehicle control value was observed with dose-dependency.

Statistics:

Wilcoxon rank sum test (significant level was 5%) and Cochran-Armitage trend test (significant level was 5%, two sided) was performed for frequencies of aberrant cells in the treatment groups. Wilcoxon rank sum test was conducted between the negative control and each treatment group. Cochran-Armitage trend test was conducted for the treatment groups including the negative control. Both of the tests were performed using JMP. For positive control, Wilcoxon rank sum test was only performed compared with the negative control.

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 DEFINITIVE STUDY
-Clinical Observation: No clinically observable signs of toxicity were detected throughout the study. In two negative control animals of 24 h sampling group abdominal crust, considered to be caused by fighting, was observed. No dose related change of body weights was recorded in any groups. No dose related change of testes weights was recorded in any groups.

- Metaphases Analysis

At 24 h after dosing
Cytotoxicity
The mitotic indices (MI) were 0.62% (0.30-0.90%) in the negative control, and 0.58% (0.40-0.80%), 0.76% (0.60-0.90%), or 1.02% (0.30-1.30%) in 500, 1000, or 2000 mg/kg bw treatment group respectively. There was no effect on the MI caused by the treatment. The MI in the positive control was 0.36% (0.10-0.50%), that was a lower value compared to the negative control.

Frequency of structurally aberrant cells
Frequencies of structurally aberrant cells were 0% in the negative control, and 0.10 ±0.22%, 0.20 ± 0.27%, or 0% in 500, 1000, or 2000 mg/kg bw treatment group respectively. For treatment groups, there was no statistically significant increase nor dose-response in
the frequency of aberrant cells at any dose levels. Frequency of structural aberrant cells in the positive control was 2.80 ± 1.30%, and there was a statistically significant increase (p < 0.01) compared to the negative control.

At 48 h after dosing
Cytotoxicity
The MI were 0.72% (0.30-1.00%) in the negative control, and 0.38% (0.20-0.60%, percent of negative control: approx. 53%) in 2000 mg/kg bw treatment group; that was a lower value compared to the negative control. However, this cytotoxicity was considered to be acceptable for metaphase analysis evaluation, since the percent of negative control was not below 50%. On the other hand, the MI in the positive control was 0.20% (0.09-0.30%), that was a clearly lower value compared to the negative control.

Frequency of structurally aberrant cells
Frequencies of structurally aberrant cells were 0.30 ± 0.27% in the negative control, and 0.10 ± 0.22% in 2000 mg/kg bw treatment group. For the treatment group, the frequency of aberrant cells was lower as compared to the negative control group. Frequency of structural aberrant cells in the positive control was 2.70 ± 1.15%, and there was a statistically significant increase (p < 0.05) compared to the negative control.

Frequency of polyploidy
Frequencies of polyploidy were 10.29 ± 1.55% in the negative control, 11.32 ± 1.62% in 2000 mg/kg bw treatment group, and 9.09 ± 3.10% in the positive control. For the treatment group, there was no statistically significant increase in the frequency of aberrant cells as compared to the vehicle control.

The range of frequency of chromosomal aberrant cells in the testing facility is 0-1.00% at both 24 and 48 h after dosing, therefore the frequency of chromosomal aberrant cells in the negative control in this study was within the historical control data. The frequency of chromosomal aberrant cells in the positive control in this study was showed significantly higher values compared to the concurrent negative control at any sampling points. Thus, this study was considered to be valid.

Conclusions:

The substance did not induce any structural and/or numerical chromosomal aberrations in mouse spermatogonia in vivo up to and including the limit dose of 2000 mg/kg bw.

Executive summary:

The mammalian spermatogonial chromosomal aberration test in Crl:CD-1(ICR) male mice was performed according to OECD TG 483 to examine germ cell genotoxicity of the test item. Six male mice (8 weeks old at the start of dosing) per group were dosed the substance in 1% methyl cellulose aqueous solution at 0 (negative control group), 500, 1000 or 2000 mg/kg bw in 24 hours after dosing group, and at 0 or 2000 mg/kg bw in 48 hours after dosing group once orally by gavage, furthermore, were administered mitomycin C in distilled water as positive control at 2 mg/kg bw once by intraperitoneal injection. Testes were removed 24 or 48 hours after administration for all groups. Five hours before the sacrifice, animals were administered colchicine at 5 mg/kg bw intraperitoneally for cell division arrest. Spermatogonia were collected following collagenase treatment and slides were prepared and stained. Spermatogonia in five mice per group were examined for the chromosomal damage. There was no increase of spermatogonial cells with structural and/or numerical chromosomal aberration by test item treatment. On the other hand, a statistically significant increase of cells with structural chromosomal aberration was observed in the positive control groups at each sampling point. The test item did not induce any structural and/or numerical chromosomal aberrations in mouse spermatogonia.

Reference 5

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2017 - March 2017

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:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: spermatid micronucleus test

Species:

mouse

Strain:

ICR

Remarks:

Crl:CD-1(ICR),SPF

Details on species / strain selection:

A representative mammal used widely in toxicity studies. Mice are the standard species in in vivo genotoxicity testing.

Sex:

male

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:
For 24 h after dosing group: 8 weeks old
For 48 h after dosing group: 8 or 9 weeks old
For 72 h after dosing group: 9 or 10 weeks old
For 14 days after dosing group: 9 weeks old

- Weight at study initiation:
For 24 h after dosing group: 30.31-38.43 g
For 48 h after dosing group: 31.03-37.68 g
First dosed group: 31.03-36.82 g
Last dosed group: 31.47-37.68 g
For 72 h after dosing group: 31.56-40.94 g
First dosed group: 31.76-39.09 g
Last dosed group: 31.56-40.94 g
For 14 days after dosing group: 30.04-38.58 g

- Assigned to test groups:
Simple random sampling. The body weight range at allocation was within ± 20% of mean.

- Fasting period before study: no

- Housing: housed individually in Polycarbonate cage for mice

- Diet: ad libitum, Laboratory animal diet, CRF-1 pellets

- Water: ad libitum, tap water

- Acclimation period:
For 24 h after dosing group: 6 days
For 48 h after dosing group: 5 days
For 72 h and 14 days after dosing groups: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 45 - 57
- Air changes (per hr): 8 to 38 times
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: 1% methyl cellulose aqueous solution
- Justification for choice of solvent/vehicle: Since the test substance can be prepared in a homogeneous suspension, the vehicle (1% methyl cellulose aqueous solution) was selected.
- Concentration of test material in vehicle: please refer to table 1 in "any other information on materials and methods"
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and pulverized in a mortar aided pestle, and suspended gently in 1% methyl cellulose aqueous solution (please refer to table 1 in "any other information on materials and methods"). The suspension was ultrasonicated immediately before administration.
Since the stability of 0.5, 1.0, 2.0 and 4.0 mg/mL test item formulation in 1% methylcellulose in a refrigerator (2-8°C) for 7 days had been verified, formulations were prepared three times in total at intervals not exceeding 7 days.

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

no

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C (MMC)
- Justification for choice of positive control: MMC is widely used as a positive control for clastogenicity in genotoxicity studies (structural aberrations).
- Route of administration: intraperitoneal
- Doses / concentrations: Please refer to table 1 in "any other information on materials and methods"

Colchicine (COL)
- Justification for choice of positive control: COL is a well-known spindle poison. In this study, COL was used for detecting the induction of micronucleated spermatids by tubulin polymerization inhibition (numerical aberrations).
- Route of administration: intraperitoneal
- Doses / concentrations: Please refer to table 1 in "any other information on materials and methods"

Tissues and cell types examined:

testes and peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the acute oral toxicity study in male mice, the LD50 of the test item had been determined to be 3514 mg/kg bw; thus, a dose of 2000 mg/kg bw was used as the limit dose of the reference guideline, and three dose levels (500, 1000 and 2000 mg/kg bw) with a common ratio of 2 were selected for this study.

TREATMENT AND SAMPLING TIMES:
Please refer to table 1 in "any other information on materials and methods"

Testis sampling
24 h, 48 h, 72 h and 14 days after administration, the animals were sacrificed by exsanguination from the abdominal aorta under isoflurane inhalation anesthesia, and both testes were removed.

Blood sampling
48 h and 72 h after administration, the animals were wormed up, and blood samples (approx.10 µL) were collected from caudal vein for observation slide, and then, blood samples (approx. 0.3 mL) were collected again from orbital venous plexus for measurement of erythrocytes count under isoflurane inhalation anesthesia, except for the COL administered group. EDTA(K2) was used for blood sample of erythrocytes count measurement as anticoagulant reagent.

DETAILS OF SLIDE PREPARATION:
For spermatid micronucleus test
1) Testes were washed in 5 mL of the isotonic solution.
2) Testes weights were individually measured using an electronic scale.
3) The tunica albuginea was carefully removed and the seminiferous tubules were squeezed with curved forceps to achieve their mechanical disruption in isotonic solution for 2 min.
4) The suspension was filtered (mesh size: 100 um, Falcon cell-strainer, Corning Inc.) and centrifuged at 150 xg for 10 min.
5) After discarding the supernatant, the cell pellet was resuspended with 2 mL of the isotonic solution and then centrifuged at 150 xg for 10 min.
6) After discarding the supernatant, pellet was added 4 mL of the isotonic solution and gently mixed.
7) The cell pellet was added 0.5 mL of the Carnoy fluid and gently mixed and then centrifuged at 150 xg for 10 min.
8) After discarding the supernatant, pellet was added 5 mL of the Carnoy fluid and pipetted.
9) The suspension was stored overnight or longer in refrigerator.
10) 1 mL of stored cells suspension was centrifuged, and then the supernatant discarded.
11) The final cell pellet was resuspended in the appropriate amount of the fixative for slide preparation, and then stored in refrigerator for at least 4 days.
12) The stored cell suspension was dropped on glass microscope slides with two drops per slide.
13) Each slide was air-dried.
14) Slides were fixed in ethanol and allowed to air-dry.
15) Slides were stained in SYBR® GOLD solution (diluted 5000 fold with TE buffer) for 30 minutes.
16) Slides were washed in purified water.
17) Slides were stored at room temperature until dry, and then coded.

For erythrocyte micronucleus test
Immediately after blood collection, the blood samples were put on acridine orange coated slides with one drop per slide. The slides were immediately covered with a cover glass, and then coded.

METHOD OF ANALYSIS:
Observation for spermatid micronucleus test
Coded slides were examined by fluorescence microscopy under blind conditions, and 4000 spermatids per animal were examined for the presence of micronuclei (MN-SM). Frequency of micronucleated spermatids (%MN-SM) were calculated. Spermatids, which had following features: small round cell, round nucleus with evident nucleolus, a little cytoplasm, and monotone pattern like marble, were judged to be early spermatids.

Observation for erythrocyte micronucleus test
Ratio of reticulocytes
For anti-coagulated blood sample, the ratio (%RET) of the reticulocyte count (RET) to the total red blood cell count (RBC) was calculated by the following method, and used as a toxicity index for bone marrow:
- Red blood cell count (RBC) reticulocyte count (RET): Fully automated hematology analyzer XT-2000i (Sysmex Corporation, Tokyo)
- Reticulocyte ratio (RET%): Calculation

Observation for erythrocyte micronucleus test
Coded slides were examined by fluorescence microscopy under blind, and 4000 polychromatic erythrocytes (PCE: type reticulocyte) per animal were examined for the presence of micronuclei (MN-PCE). Frequency of micronucleated PCE (%MN-PCE) was calculated.

Evaluation criteria:

Judgment for frequency of micronucleated spermatids
The result was judged to be positive in cases where a statistically significant increase in %MN-SM in the test substance treatment group compared to the vehicle control value was observed with dose-dependency.

Judgment for frequency of micronucleated PCE
The result was judged to be positive in cases where a statistically significant increase in %MN-PCE in the test substance treatment group compared to the vehicle control value was observed with dose-dependency.

Statistics:

All the statistical analysis tests were performed using JMP (Ver. 12, SAS Institute Japan). For body weights, testis weights, MN-SM, %MN-SM, RBC, RET, %RET, MN-PCE and %MN-PCE, the mean and the standard deviation (S.D.) of each group were calculated.

Statistical analysis for frequency of micronucleated spermatids
Wilcoxon rank sum test (significant level was 5%) and Cochran-Armitage trend test (significant level was 5%, two sided) was performed for the treatment groups. Wilcoxon rank sum test was conducted between the negative control and each treatment group. Cochran-Armitage trend test was conducted for the treatment groups including the negative control. For positive control, Wilcoxon rank sum test was only performed compared with the negative control.

Statistical analysis for frequency of reticulocytes
Since the variance was homogeneous in Bartlett's test (significant level was 5%, two sided), Dunnett's test was conducted between the negative control group and the test substance (significant level was 5%, two sided). For positive control, since the variance was not homogeneous in F-test (significant level was
5%), the Welch's t-test9 was conducted.

Statistical analysis for frequency of micronucleated PCE
Wilcoxon rank sum test (significant level was 5%) and Cochran-Armitage trend test (significant level was 5%, two sided) was performed for the treatment groups. Wilcoxon rank sum test was conducted between the negative control and each treatment group. Cochran-Armitage trend test was conducted for the treatment groups including the negative control. For positive control, Wilcoxon rank sum test was only performed compared with the negative control.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: spermatids 24 h after dosing

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: spermatids 48 h after dosing

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: spermatids 72 h after dosing

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: spermatids 14 days after dosing

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: PCE 48 h after dosing

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: PCE 72 h after dosing

Table 2 summary of results: spermatids

Group	Dose (mg/kg bw)	Micronucleus incidence (%) After dosing
		24 h	48 h	72 h	14 d
Control	0	0. 13	0.09	0.09	0.09
TM	500	0.10	0.07	0.11	0.09
	1000	0.08	0.09	0.10	0.08
	2000	0.11	0.10	0.08	0.09
COL	1	-	-	0.27*	-
MMC	2	-	0.29*	-	0.32*

P<0.05 (Wilcoxon rank sum test).

TM: test item, COL: colchicine, MMC: mitomycin C

Table 3 summary of results: PCE

 

Group	Dose (mg/kg bw)	Micronucleus incidence (%) After dosing
		48 h	72 h
Control	0	0.24	0.27
TM	500	0.21	0.27
	1000	0.23	0.25
	2000	0.21	0.16*
MMC	2	2.97*	-

*P<0.05 (Wilcoxon rank sum test).

TM: test item, COL: colchicine, MMC: mitomycin C

Conclusions:

Based on the results of this OECD 474 compliant study in male mice it is concluded that the test item did not show any evidence of causing an increase in the induction of micronucleated spermatids and micronucleated polychromatophilic erythrocytes when administered orally by gavage at 2000 mg/kg bw at any exposure times in this in vivo test procedure.

Executive summary:

The spermatid micronucleus test in Crl:CD-1(ICR) male mice was performed to examine germ cell genotoxicity of the test item. Five male mice (8-10 weeks old at the time of dosing) per group for each sampling time were administered test item at 0 (negative control group), 500, 1000 or 2000 mg/kg bw (limit dose) once orally by gavage. Five male mice per group for each sampling time were administered mitomycin C (MMC) at 2 mg/kg bw or colchicine (COL) at 1 mg/kg bw, as positive control, once by intraperitoneal injection. Animals were observed clinically at least once a day. Body weights were measured before dosing and sampling. At necropsy, blood samples were collected from the caudal vein 48 and 72 h after administration and slides were prepared and stained for the assessment of erythrocytes. Testes were removed under anesthesia 24 h, 48 h, 72 h, and 14 days after administration for negative control and test item, 48 h and 14 days after administration for MMC, and 72 h after administration for COL respectively. The left and right testes of each animal were weighed, and then spermatids were gently isolated by pressing the testis in isotonic solution and slides were prepared and stained for the assessment of spermatids. The frequency of micronucleated spermatids or micronucleated polychromatophilic erythrocytes was examined in 4000 spermatids or polychromatophilic erythrocytes per dose group and timepoint by means of fluorescence microscopy.

There was no mortality, clinical finding, and toxicologically relevant change of body weight and testis weights in test item treated groups as compare to the negative control. There was no statistically significant increase in the frequency of micronucleated spermatids and micronucleated polychromatophilic erythrocytes by test item treatment at any exposure times. On the other hand, the positive controls caused statistically significant increases in the frequency of micronucleated spermatids and micronucleated polychromatophilic erythrocytes at all exposure times. It is concluded that the test item did not show any evidence of causing an increase in the induction of micronucleated spermatids and micronucleated polychromatophilic erythrocytes in male CD-1 mice when administered orally by gavage at 2000 mg/kg bw at any exposure times in this in vivo test procedure.

Additional information

In vitro

Ames Test

A study similar to OECD TG 471 was performed using Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and Escherichia coli (WP2 uvrA) with and without metabolic activation. This study consisted of two independent preincubation tests. Dimethylsulfoxide was used as solvent for preparation of the test substance. Eight concentrations of the test substance (5000, 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1 µg/plate) were used in the tests. The limit concentration required in the guideline was used as the highest concentration. No increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any concentration, either with or without metabolic activation.

In vitro chromosome Aberration

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. The test article was dissolved in dimethyl sulfoxide at a stock concentration of 100 mg/mL. Complete toxicity was observed at 1000 µg/mL with significant delays in cell cycle at 100 and 335 µg/mL in the range-finding assay without activation. Significant delays in cell cycle were observed at 335 and 1000 µg/mL in the range-finding assay with metabolic activation. A 20 hour extended fixation was selected for the aberrations assays with and without activation. Duplicate cultures of CHO cells were incubated with 70.0 to 400 µg/mL of the test article in the non-activation assay and with 250 to 1000 µg/mL of the test material in the assay with activation. No significant increase in chromosomally aberrant cells was observed in either assay at any of the dose levels. The test article is considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells under both non-activation and activation conditions of this assay.

HPRT test

The test substance was examined for mutagenic activity in a study similar to OECD TG 476 (HPRT) in Chinese hamster V79 cells after in vitro treatment with and without a metabolic activation system (S9 Mix). Based on a preliminary cytotoxicity assay, two independent assays for mutation to 6-thioguanine resistance were performed using concentrations of 100, 50, 25, 12.5 and 6.25 μg/mL. No reproducible five-fold increases in mutant numbers or mutation frequency were observed at any dose level of the test substance, in the absence or presence of S9 metabolism. Positive responses were obtained with the positive control treatments.

In vitro Micronucleus

The substance was tested for its clastogenic and aneugenic potential in an in vitro micronucleus assay (similar to OECD 487) using human lymphocyte cultures. Treatments were performed both in the absence and presence of a metabolic activation system. The test substance concentration ranges were 8.4 – 200 μg/mL (13 concentrations) without metabolic activation system and 150 – 700 μg/mL (12 concentrations) with metabolic activation system. The highest concentrations were selected on the basis of cytotoxicity. The highest concentrations chosen for analysis were 47.5 and 550 μg/mL, which induced approximately 61 and 60 % reduction in the ratio of binucleate to mono- plus binucleate cells in the absence and presence of mammalian metabolic activation, respectively. Micronuclei were analysed by means of fluorescence in situ hybridisation (FISH) technique to distinguish between aneugenic (micronuclei with whole chromosomes, assessed by the presence of centromeres) and clastogenic effects (micronuclei with chromosome fragments, assessed by the lack of centromeres).

Treatment of cultures with the test substance in the absence of a metabolic activation system resulted in micronucleus frequencies which were statistically significantly greater than those noted in concurrent negative controls at all concentrations and which fell outside the historical negative control range. Treatment in the presence of a metabolic activation system resulted in micronucleus frequencies similar to those seen in concurrent negative controls. A statistically significant increase in cells with micronuclei was apparent at the highest concentration analysed, but the effect was attributable to an increase in a single replicate. FISH analysis of positive samples indicated that the majority of the micronuclei induced contained one or more centromeres, and were thus formed from whole chromosomes rather than from chromosomal fragments.

With metabolic activation, there was no increase in the incidence of cells with micronuclei up to 500 µg/mL. At the highest concentration analysed (550 µg/mL), an increase was observed but this was due to a single replicate only (13 cells with micronuclei in one culture versus one only in the other).

In vivo

In vivo Micronucleus tests

A study according OECD TG 474 was conducted. Male and female B6D2F1 mice were treated by single oral gavage with the test substance at dose levels of 500, 1000 or 2000 mg/kg bw. A concurrent negative control group received the vehicle only, while a positive control (PC) group was treated with carbendazim at 1000 mg/kg bw. Bone marrow smears were obtained each from 5 mice/sex/dose group at 24 and at 48 hours after dosing with the exception that mice in the PC group were sampled at the 24 hour time-point only.

A preliminary toxicity test had previously shown that a dose of 2000 mg/kg, the standard limit dose for the micronucleus test, was tolerated; this level was therefore selected as an appropriate maximum for use in this study. Serum analysis of mice treated with the substance in the course of this preliminary study showed that the test substance was absorbed and systemically distributed following oral administration, so that significant bone marrow exposure could reasonably be expected. In addition, serum analysis showed that a proportion was metabolized to carbendazim (a known spindle-poison with aneugenic activity).

One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

Mice treated with the test substance showed a small dose-related and highly statistically significant increase in the frequency of micronucleated immature erythrocytes at both sampling times. Increases obtained at the low and intermediate dose levels were very small, with group mean values falling just outside the laboratory historical control range for mice.

A statistically significant dose-related decrease in the proportion of immature erythrocytes was obtained for animals treated with the substance and sampled at the 24 hour time point; since this decrease was only very slight and was not seen at the later sampling time, it is possible that the apparent decrease was the result of chance variation rather than being treatment-related. The positive control compound, carbendazim, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes together with a significant decrease in the proportion of immature erythrocytes.

Centromere-specific staining and size analysis of micronuclei in bone marrow smears prepared from these animals subsequently confirmed that carbendazim produced micronuclei predominantly from lagging chromosomes with the remainder being formed from chromosome fragments. In comparison, a smaller proportion of the micronuclei induced by the substance was apparently formed from lagging chromosomes. However, this difference in proportion could relate to the much lower rate of induction of micronuclei by the test substance rather than indicating any fundamental difference in mechanism of activity between the two compounds.

The test substance has been shown to induce low numbers of micronucleated cells in the bone marrow of mice. Qualitative analysis of micronuclei to determine whether the substance operates primarily by causing spindle dysfunction in a similar manner to carbendazim (and would, therefore, be expected to exhibit a threshold dose for activity) was inconclusive because of the large difference in effective dose levels of the two compounds with test substance inducing much lower numbers of micronuclei than carbendazim at comparable dose levels.

A further micronucleus test according OECD TG 474 with bone marrow cells was conducted in Crl:CD1 (ICR) male mice. The high dose level of was set at 2000 mg/kg bw, and a total of 3 dose levels including 1000 and 500 mg/kg bw were provided using a common ratio of 2. The test substance was administered orally twice at an interval of approximately 24 hours. At approximately 24 hours after the second administration, bone marrow smear specimens were prepared and observed. In addition, a negative control group in which the animals received 1 % methylcellulose solution and a positive control group in which animals received mitomycin C once intraperitoneally at 1 mg/kg were provided. At the specified time after administration, bone marrow smear specimens were prepared and observed. Each group consisted of 5 males.

In the results, the incidence of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in each test substance group did not show any statistically significant increase when compared to that in the negative control group, and there was no dose-dependent increase.The incidence of polychromatic erythrocytes in a total of 500 erythrocytes showed no statistically significant change in any test substance group in comparison with that of the negative control group.The incidence of micronucleated polychromatic erythrocytes in the positive control group was significantly (p<0.05) increase than that in the negative control group. In addition, this study was conducted appropriately because the incidences of micronucleated polychromatic erythrocytes in the positive control group and negative control group were within the range of the mean ± 1.96 S.D. of the respective historical control data in the test facility. On the basis of the above results, it was judged that the substance had no clastogenicity in the bone marrow cells in Crl:CD1 (ICR) male mice under the conditions of this study.

A spermatid micronucleus test in Crl:CD-1(ICR) male mice was performed to examine germ cell genotoxicity of the test item. Five male mice (8-10 weeks old at the time of dosing) per group for each sampling time were administered test item at 0 (negative control group), 500, 1000 or 2000 mg/kg bw (limit dose) once orally by gavage. Five male mice per group for each sampling time were administered mitomycin C (MMC) at 2 mg/kg bw or colchicine (COL) at 1 mg/kg bw, as positive control, once by intraperitoneal injection. Animals were observed clinically at least once a day. Body weights were measured before dosing and sampling. At necropsy, blood samples were collected from the caudal vein 48 and 72 h after administration and slides were prepared and stained for the assessment of erythrocytes. Testes were removed under anesthesia 24 h, 48 h, 72 h, and 14 days after administration for negative control and test item, 48 h and 14 days after administration for MMC, and 72 h after administration for COL respectively. The left and right testes of each animal were weighed, and then spermatids were gently isolated by pressing the testis in isotonic solution and slides were prepared and stained for the assessment of spermatids. The frequency of micronucleated spermatids or micronucleated polychromatophilic erythrocytes was examined in 4000 spermatids or polychromatophilic erythrocytes per dose group and timepoint by means of fluorescence microscopy.

There was no mortality, clinical finding, and toxicologically relevant change of body weight and testis weights in test item treated groups as compare to the negative control. There was no statistically significant increase in the frequency of micronucleated spermatids and micronucleated polychromatophilic erythrocytes by test item treatment at any exposure times. On the other hand, the positive controls caused statistically significant increases in the frequency of micronucleated spermatids and micronucleated polychromatophilic erythrocytes at all exposure times. It is concluded that the test item did not show any evidence of causing an increase in the induction of micronucleated spermatids and micronucleated polychromatophilic erythrocytes in male CD-1 mice when administered orally by gavage at 2000 mg/kg bw at any exposure times in this in vivo test procedure.

In vivo Chromosome Aberration tests

A mammalian bone marrow chromosomal aberration test according OECD TG 474 in B6D2F1/Crl male mice was performed to examine bone marrow genotoxicity of the substance. Six male mice (9 weeks old at the start of dosing) per group were dosed the substance in 1% methyl cellulose aqueous solution at 0 (negative control group), 500, 1000 or 2000 mg/kg bw once orally by gavage, and mitomycin C in saline as positive control at 4 mg/kg bw once by intraperitoneal injection. Bone marrow from both femurs was removed 24 or 48 hours after administration for all groups. Three hours before sacrifice, animals were administered colchicine at 4 mg/kg bw intraperitoneally for cell division arrest. Bone marrow was collected and slides were prepared and stained. Bone marrow in five mice per group was examined for the chromosomal damage.

There was no increase of bone marrow cells with structural and/or numerical chromosomal aberration by substance treatment. On the other hand, a statistically significant increase of cells with structural chromosomal aberration was observed in the positive control group. The substance did not induce any structural and/or numerical chromosomal aberrations in mouse bone marrow.

A mammalian spermatogonial chromosomal aberration test in Crl:CD-1(ICR) male mice was performed according to OECD TG 483 to examine germ cell genotoxicity of the test substance. Six male mice (8 weeks old at the start of dosing) per group were dosed the substance in 1% methyl cellulose aqueous solution at 0 (negative control group), 500, 1000 or 2000 mg/kg bw in 24 hours after dosing group, and at 0 or 2000 mg/kg bw in 48 hours after dosing group once orally by gavage, furthermore, were administered mitomycin C in distilled water as positive control at 2 mg/kg bw once by intraperitoneal injection. Testes were removed 24 or 48 hours after administration for all groups. Five hours before the sacrifice, animals were administered colchicine at 5 mg/kg bw intraperitoneally for cell division arrest. Spermatogonia were collected following collagenase treatment and slides were prepared and stained. Spermatogonia in five mice per group were examined for the chromosomal damage. There was no increase of spermatogonial cells with structural and/or numerical chromosomal aberration by substance treatment. On the other hand, a statistically significant increase of cells with structural chromosomal aberration was observed in the positive control groups at each sampling point. The test substance did not induce any structural and/or numerical chromosomal aberrations in mouse spermatogonia.

Conclusions

The substance is harmonized classified for Muta. 2 (H341). The experimental results suggest that for the mutagenicity of the test item an aneugenic mechanism is prevalent while clastogenicity is not demonstrated. However, the reported in vivo aneugenic effects were only observed for a single mouse strain and in somatic cells only. The result was not confirmed in a different strain. The available in vivo studies in germ cells did not demonstrate any genotoxic effect.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test item is harmonized classified according to Annex VI of Regulation (EC) No 1272/2008 for mutagenicity Cat.2 (H341), as amended for the seventeenth time in Regulation (EU) 2021/849.