Pentanediamide, N1,N5-dibutyl-2-[(2-ethyl-1-oxohexyl)amino]-, (2S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro mutagenicity studies have been conducted on the test material; an Ames test and a chromosome aberration test. Both gave negative results in the presence and absence of metabolic activation.

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:

27 February 2001 - 9 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 98: his D 3052; rfa-; UvrB-; R-factor: frame shift mutations
TA1535: his G 46; rfa- uvrb-: base pair substitutions
TA 100: his G 46; rfa; uvrb-; R-factor: base pair substitutions

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-Factor plasmid pKM 101 carries the ampicillin resistance marker.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

trp-; uvrA-: base pair substitutions and others

Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp*) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detescting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: On the day of the experiment, the test item was dissolved in ethanol (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see materials and methods for more information

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS:
For each strain and dose level including controls, three plates ware used.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described in the main test.
Toxicity of the test item results in a reduction in the number at spontaneous revertants or a clearing of the bacterial background lawn.

The pre-experiment is reported as main experiment I, if the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant topic effects were observed and 5000 µg/plate were chosen as maximal concentration.

The concentration range included two logarithmic decades. The following concentrations were tested.
Experiment l: 33, 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/plate

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
- A test item is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.

- A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutageoic in this system.

A biologically relevant response is described as follows:

- A test item is considered mutagenic if in the strains TA 98, TA100, and WP2uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.

- Also a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No statistical evaluation of the data is required

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 μg/plate and above and strain TA 98 with and without metabolic activation at 5000 μg/plate in experiment I.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
Weak toxic effects, evident as a reduction in the number of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 µg/plate and above and strain TA 98 with and without metabolic activation at 5000 µg/plate in experiment I.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EH at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coil reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

Weak toxic effects, evident as a reduction in the nurnber of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 µg/plate and above and strain TA 98 with and without metabolic activation at 5000 µg/plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EH at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 January 2006 - 31 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: The cell line of the Chinese hamster fibroblasts in lung (CHL/IU cells) was used.

Details on mammalian cell type (if applicable):

The CHL cell line was supplied by the Division of Mutagenesis, National Institute of Hygienic Sciences (present name: Division of Genetics and Mutagenesis, National Institute of Health Sciences) at October 6, 1987. The cells of the following passage number were used in this study.
First Cell growth inhibition test: 13 passages
Second Cell growth inhibition test: 17 passages

Chromosome aberration test
Short-term exposure experiments: 20 passages
Continuous exposure experiments: 22 passages

The cells were cultured every three to four days.

Cell Culture Medium
Eagle's MEM (GIBCO/ Lot No. 1272034) supplemented with 10% calf serum (HyClone/ Lot No. AMM17780, inactivation for 30 minutes at 56 °C) was used as cell culture medium.

Chromosome Number
The cells have a modal chromosome number of 25.

Growth Form
The cells grow in a monolayer on a plate and the doubling time is approximately 15.8 hours.

Cell Culture Conditions
The cell cultures were incubated using CO2 gas incubator (TE-HER, CP series CPD-172M: Hirasawa , INC.) under the conditions of a highly humidified atmosphere with 5% carbon dioxide at 37°C.

Cell Stock Conditions
Cells were stored in the deep temperature freezer (-81 to -77°C). 10 v/v% of Dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries, Ltd., JIS guaranteed reagent, purity: >99%, lot No. PKL4402) was added to the culture medium at the time of storage.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.120, 0.240, 0.480, 0.959 and 1.918 mg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:
In the results of the solubility test, the test substance was insoluble in physiological saline and could not be applied at concentration of 3.836 mg/mL ((prepared concentration was 767.2 mg/mL (added volume of test solution: 25 µL) and prepared concentration 383.6 mg/mL (added volume of test solution: 50 µL)) equivalent to 10mM in DMSO because it had not fluidity. Since this test substance could be suspended in 1% CMC-Na solution at concentration of 3.836 mg/mL (prepared concentration was 42.196 mg/mL) which was equivalent to 10 mM, therefore, 1% CMC-Na solution was selected as vehicle for the first cell growth inhibition test. The results of cell growth inhibition were not clear.
For this reason, the solubility test was performed again with DMSO as vehicle. Because the test substance was dissolved at 0.120 mg/mL (prepared concentration was 11.988 mg/mL (added volume of test solution: 50 µL)) and suspended at 1.918 mg/mL (prepared concentration was 191.8 mg/mL (added volume of test solution: 50 µL)) in the above test, the second cell growth inhibition test was performed using DMSO as vehicle. It was considered that the test substance suspended in DMSO was more effectively acted on the cells when it was compared to 1% CMC-Na solution, because the cell growth inhibition in DMSO was more clear. Thus, DMSO was selected as vehicle for chromosome aberration test. Evolution of gas, exothermic reaction, etc. in 1% CMC-Na solution and DMSO were not observed.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without S9 Mix: Mitomycin C (MMC); With S9 Mix: N-nitrosodimethylamine (DMN)

Remarks:

The positive controls selected for the experiments are the known clastogens and easily soluble in water.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preparation interval (cell harvest) of each group and experimental designs were as follows:

Experiment Exposure period Recovery Cell harvest
Short-term exposure experiment : Without S9 mix 6 hours 18 hours 24 hours
Short-term exposure experiment: With S9 mix 6 hours 18 hours 24 hours
Continuous exposure experiment: 24-hour exposure 24 hours - 24 hours
Continuous exposure experiment: 48-hour exposure 48 hours - 48 hours

NUMBER OF CELLS EVALUATED:
200 consecutive well-spread metaphases (100 metaphases per dish) obtained from each dose group were observed under the microscopes. The number of cells carrying structural chromosome aberrations and their aberration type were recorded.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. The number of polyploid cells was determined at the same time (cells carrying greater than 37 chromosomes including triploid were recorded as polyploidy).
- Determination of endoreplication: Yes
- Other:
The type of chromosome aberration was classified as follows:
Gaps are the unstained part on the vertical axis of the chromatid, whose width shows less than the width of the chromatid and the form is clear. Breaks are the unstained part on the vertical axis of the chromatid, whose width shows more than the width of the chromatid and the form is clear, or that the fragments deviate from the axis of the chromosome or chromatid. Exchanges are defined as the mutual exchange induced by two or more cuttings of a chromosome or chromatid. The other structure aberrations were defined as "others".

Structure aberrations
Chromatid break (ctb)
Chromatid exchange (cte) (quadriradial exchange, etc.)
Chromosome break (csb)
Chromosome exchange (cse) (dicentric, ring, etc.)
Others (fragmentation, frg)
Other chromosome aberration
Gap (g)
Numerical aberration
Polyploidy (polyploid and endoreduplication)

Evaluation criteria:

In accordance with evaluation criteria defined by Sofuni, et al, the following criteria were set to evaluate the frequency of aberrant cells in each concentration group. A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.

Negative (-) less than 5%
Equivocal (±) 5% or more, less than 10%
Positive (+) 10% or more

When more than 50 cells in metaphase per dish was not observed by the cytotoxicity, it was excluded from the judgment, and recorded as "TOX".
The short-term exposure experiments were performed first. When the result of the short-term exposure experiment was positive, the continuous exposure experiment wasn't to be performed. When the results of the short-term exposure experiment with and without S9 mix were negative, the continuous exposure experiment was to be performed. Furthermore, when the result of chromosome aberration test was positive but not definitely dose-dependent (the positive result was found only in a single dose group) or that was equivocal (±), a confirmatory test was to be performed including the same dose level which was positive. In the results of the confirmatory test, it was to be judged equivocal if the equivocal result was observed again, and to be judged positive if the positive result was induced without dose-dependence.

Statistics:

No statistical analysis was performed to the results.

Species / strain:

other: The cell line of the Chinese hamster fibroblasts in lung (CHL/IU cells) was used.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: In the results of solubility test, the test substance was insoluble to physiological saline, DMSO and 1% CMC-Na solution, therefore, it could be used only as suspension.
- Precipitation:
The precipitation of the test substance in the culture medium was observed at more than 0.120 mg/mL for the first cell growth inhibition test when 1% CMC-Na solution was used as vehicle. The precipitation in the second cell growth inhibition test and the chromosome aberration test was also observed at more than 0.240 mg/mL which used DMSO as vehicle. The adherence to a dish was observed at more than 0.480 mg/mL in 24 and 48-hour exposure in both the second cell growth inhibition test and chromosome aberration test, while the adherence was not observed in the first cell growth inhibition test.


RANGE-FINDING/SCREENING STUDIES:
The dose range in the chromosome aberration test was selected in accordance with the results of the cell growth inhibition test.

In the results of the first cell growth inhibition test, the cell growth inhibition was not clear, 1% CMC-Na solution was vehicle as the maximum concentration of 3.836 mg/mL which was equivalent to 10 mM. For this reason, the second cell growth inhibition was performed in the maximum concentration of 1.918 mg/mL which was selected because it was impossible to prepare the test substance in the concentration of 3.836 mg/mL into DMSO.
As a result, the concentration of 50% cell growth inhibition test was more than 1.918 rng/mL in the short-term exposure experiments both without and with S9 mix and continuous 24-hour exposure experiment, and at 1.285 mg/mL in continuous 48-hour exposure experiment. The adherence to a dish was observed at more than 0.480 mg/mL in 24 and 48-hour exposure in the second cell growth inhibition test, while the adherence was not noted in the first cell growth inhibition test. Therefore, it was considered that the test substance suspended in DMSO was more effectively acted on the cells when it was compared to 1% CMC-Na solution.
Based on the above results, the following doses were selected and DMSO was used as vehicle for the chromosome aberration test.

Short-term exposure experiments:
Without S9 mix; 0.120, 0.240, 0.480, 0.959 and 1.918 mg/mL (5 doses with a common ratio of 2)
With S9 mix; 0.120, 0.240, 0.480, 0.959 and 1.918 mg/mL (5 doses with a common ratio of 2)

Continuous exposure experiments:
24-hour exposure; 0.120, 0.240, 0.480, 0.959 and 1.918 mg/mL (5 doses with a common ratio of 2)
48-hour exposure; 0.120, 0.240, 0.480, 0.959 and 1.918 mg/mL (5 doses with a common ratio of 2)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In the results of the chromosome aberration test in both with and without S9 mix, the frequencies of cells carrying structural chromosome aberrations or polyploidy were less than 5% at any concentration.

In the positive (MMC) control group in short-term exposure experiment without S9 mix, the frequency of cells carrying structural chromosome aberrations was 17.5 %. In the positive (DMN) control group in the short-term exposure experiment with S9 mix, the frequency of cells carrying structural chromosome aberrations was 46.5 %. These results showed that the experiments were performed appropriately.

In the results of the chromosome aberration test in both 24 and 48-hour exposure experiments, the frequencies of cells carrying structural chromosome aberrations or polyploidy were less than 5% at any concentration.

In the positive (MMC) control groups in the first continuous exposure experiments, the frequencies of cells carrying structural chromosome aberrations in 24 and 48-hour exposure were 48.5 and 58.5%, respectively. These results showed that the experiments were performed appropriately.

Historical negative and positive control data were given.

Based on the above results, it is concluded that this test substance does not induce chromosome aberrations in CHL/IU cells.

In addition, the results of cell growth inhibition showed that there were some differences between the second cell growth inhibition test data and chromosome aberration test data after 24-hour exposure. It was considered that the difference was made in the staining degree of the test substance adhered to a dish in these tests.

Conclusions:

Under the test conditions, it is concluded that the test substance does not induce chromosome aberrations in the cell line of Chinese hamster fibroblast in lung (CHL/IU cells).

Executive summary:

This in vitro chromosome aberration test in cultured mammalian cells (CHL/IU cells) was performed to assess the potential of the test material to induce chromosome aberrations.

In the results of solubility test, the test substance was insoluble to physiological saline, DMSO and 1% CMC-Na solution, therefore, it could be used only as suspension.

The first cell growth inhibition test was performed using the maximum concentration of 3.836 mg/mL, equivalent to 10 mM, which was suspended in 1% CMC-Na solution as vehicle. The cell growth inhibition was not clear in this test. The precipitation of the test substance in culture medium was observed in more than 0.120 mg/mL, but the adhesion to a dish was not noted.

The second cell growth inhibition test was carried out by using DMSO as vehicle. DMSO was workable in the concentration of below 1.918 mg/mL, though it was impossible to prepare the test substance in the concentration of 3.836 mg/mL equivalent to 10 mM. Therefore, the test substance was suspended in DMSO up to 1.918 mg/mL and used for the second cell growth inhibition test. As a result, the concentrations of 50% cell growth inhibition were more than 1.918 mg/mL in the short-term exposure experiments both without and with a metabolic activation system (hereinafter referred to as "without S9 mix" and "with S9 mix") and the continuous 24-hour exposure experiment (hereinafter referred to as "24-hour exposure experiment"), and at 1.285 mg/mL in the continuous 48-hour exposure experiment (hereinafter referred to as "48-hour exposure experiment"). In addition, the precipitation of the test substance in culture medium was observed at more than 0.240 mg/mL, and the adherence to a dish was also observed at more than 0.480 mg/mL in 24-hour and 48-hour exposure experiments. From the above results, it was considered that the test substance suspended in DMSO was more effectively acted on the cells when it was compared to 1% CMC-Na solution.

Therefore, DMSO was used for the chromosome aberration test as vehicle. The maximum concentration of test substance in both short-term and continuous exposure experiments was 1.918 mg/mL, and 5 applied concentrations which were prepared with dilution by a common ratio of 2, were set up and examined.

In the results of the chromosome aberration test in both short-term and continuous exposure experiments, the frequencies of cells carrying structure chromosome aberrations and polyploidy were less than 5% at any concentration, so the results were judged negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Study

The test item was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

Weak toxic effects, evident as a reduction in the nurnber of revertants, occurred in the test strain TA 1537 (without metabolic activation) at 2500 µg/plate and above and strain TA 98 with and without metabolic activation at 5000 µg/plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EH at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Chromosome Aberration Study

This in vitro chromosome aberration test in cultured mammalian cells (CHL/IU cells) was performed to assess the potential of the test material to induce chromosome aberrations.

In the results of solubility test, the test substance was insoluble to physiological saline, DMSO and 1% CMC-Na solution, therefore, it could be used only as suspension.

The first cell growth inhibition test was performed using the maximum concentration of 3.836 mg/mL, equivalent to 10 mM, which was suspended in 1% CMC-Na solution as vehicle. The cell growth inhibition was not clear in this test. The precipitation of the test substance in culture medium was observed in more than 0.120 mg/mL, but the adhesion to a dish was not noted.

The second cell growth inhibition test was carried out by using DMSO as vehicle. DMSO was workable in the concentration of below 1.918 mg/mL, though it was impossible to prepare the test substance in the concentration of 3.836 mg/mL equivalent to 10 mM. Therefore, the test substance was suspended in DMSO up to 1.918 mg/mL and used for the second cell growth inhibition test. As a result, the concentrations of 50% cell growth inhibition were more than 1.918 mg/mL in the short-term exposure experiments both without and with a metabolic activation system (hereinafter referred to as "without S9 mix" and "with S9 mix") and the continuous 24-hour exposure experiment (hereinafter referred to as "24-hour exposure experiment"), and at 1.285 mg/mL in the continuous 48-hour exposure experiment (hereinafter referred to as "48-hour exposure experiment"). In addition, the precipitation of the test substance in culture medium was observed at more than 0.240 mg/mL, and the adherence to a dish was also observed at more than 0.480 mg/mL in 24-hour and 48-hour exposure experiments. From the above results, it was considered that the test substance suspended in DMSO was more effectively acted on the cells when it was compared to 1% CMC-Na solution.

Therefore, DMSO was used for the chromosome aberration test as vehicle. The maximum concentration of test substance in both short-term and continuous exposure experiments was 1.918 mg/mL, and 5 applied concentrations which were prepared with dilution by a common ratio of 2, were set up and examined.

In the results of the chromosome aberration test in both short-term and continuous exposure experiments, the frequencies of cells carrying structure chromosome aberrations and polyploidy were less than 5% at any concentration, so the results were judged negative.

Justification for classification or non-classification

In the absence of any positive in vitro genetic toxicity data, the registered substance does not require classification as mutagenic in accordance with the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.