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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test:

The test item was judged to have no gene mutation inducibility (negative) under the conditions of this study.

Chromosomal aberration test:

The test substance did not induce chromosomal aberrations under the present test conditions.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2014-05-28 to 2014-07-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 10181
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:
S9 Mix
Test concentrations with justification for top dose:
To set the dose levels for the main tests, the 100 mg/mL solution was diluted to 4 times at a common ratio of 4 and a total of 5 dose levels selected (19.5, 78.1, 313, 1250 and 5000 μg/plate) in the dose-finding test.
In the dose-finding test, precipitation of the test article on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration on the plate was not observed at any dose levels irrespective of the presence/absence of metabolic activation. Growth inhibition was observed at 78.1 μg/plate or more in S. typhimurium TA1537 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA 98, TA 100 and TA 1535 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains with or without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: Based on the information of the sponsor, since the test article was insoluble in water, DMSO and acetone, the solubility test was conducted with DMF and 1,4-dioxane. As the result, DMF was used as the vehicle in this study because it was soluble at 100 mg/mL, and 1,4-dioxane was insoluble at 100 mg/mL.
Untreated negative controls:
yes
Remarks:
DMF
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
positive control for TA 100, TA 98, TA 1537 with metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
positive control for TA 1535 without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Remarks:
positive control for TA 100, TA 98, WP2 uvrA with metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)- aminopropylamino]acridine.2HCl (ICR-191)
Remarks:
positive control for TA 1537 without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
positive control for TA 1535, WP2 uvrA with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes

Number of Plates: 2 plates at each dose level in the dose-finding test, 3 in the two main tests.
Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies compared to that of spontaneous revertant colonies (the negative control value), dose-response and reproducibility were noted, or even if no clear dose-response was observed but there were at least two-fold increase in the number of revertant colonies in comparison with that of spontaneous revertant colonies and reproducibility in the two main tests, the test article was judged to be positive.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
Dose-finding test:
- Observetion: Precipitation of the test article on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration on the plate was not observed at any dose levels irrespective of the presence/absence of metabolic activation. Growth inhibition was observed at 78.1 μg/plate or more in S. typhimurium TA1537 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA98, TA100 and TA1535 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains with or without metabolic activation.

First main test and second main test:
- Observetion: In both the two main tests, neither precipitation nor coloration of the test article on the plate was observed at any dose levels irrespective of the presence/absence of metabolic activation. Growth inhibition was observed at 39.1μg/plate or more in S. typhimurium TA1537 without metabolic activation, and at 156 μg/plate or more in S. typhimurium TA1535 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA98 and TA100 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains irrespective of the presence/absence of metabolic activation.
Remarks on result:
other: Growth inhibition was observed at 156 μg/plate or more
Conclusions:
The test item was judged to have no gene mutation inducibility (negative) under the conditions of this study.
Executive summary:

In order to examine the gene mutation inducibility of the test item, a reverse mutation assay was conducted in Salmonella typhimurium (hereinafter referred to as S. typhimurium) TA 100, TA 1535, TA 98 and TA 1537, and Escherichia coli (hereinafter referred to as E. coli) WP2 uvrA with or without metabolic activation by the pre-incubation method according to OECD Guideline 471.

N,N-dimethylformamide (hereinafter referred to as DMF) was used as the vehicle for the test article. A dose-finding test was conducted with dose levels between 19.5 and 5000 μg/plate to set the dose levels for the main test. From the result of the dose-finding test, the minimum dose which showed growth inhibition was selected as the maximum dose for the main test, and the main tests were conducted at 6 dose levels between 2.44 and 78.1 μg/plate in the S. typimurium TA 1537 without metabolic activation, and between 9.77 and 313 μg/plate in the S. typimurium TA 98, TA 100 and TA 1535 without metabolic activation, and between 39.1 and 1250 μg/plate in the E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation. The main test was conducted twice at the same dose levels.

Precipitation on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration by the test article was not observed at any dose levels irrespective of the presence/absence of metabolic activation.

In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 39.1 μg/plate or more in S. typhimurium TA 1537 without metabolic activation, and at 156 μg/plate or more in S. typhimurium TA 1535 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA 98 and TA 100 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.

In the two main tests, there was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response in any strains irrespective of the presence/absence of metabolic activation.

In conclusion, the test item was judged to have no gene mutation inducibility (negative) under the conditions of this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-09-11 to 2017-11-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 101Z4
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung fibroblasts (CHL/IU cells)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Health Science Research Resources Bank, Japan Health Sciences Foundation
- Doubling time: about 15 hours
- Modal number of chromosomes: 25 per cell

Culture condition
- Incubator: CO2 incubator
- Temperature: 37 °C
- Humidity: under humid condition
- CO2 concentration: 5%

Subculture
- Culture vessel: 90-mm diameter Petri dish
- Frequency of passage: twice a week
- Passage number of cells: 6 for cell growth inhibition test; 16 for chromosomal aberration test
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Cell growth inhibition test: 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL
Chromosomal aberration test:
- Short - term treatment
- without S9 mix: 15.0, 35.0, 55.0, 60.0, 62.0, 65.0, 70.0, 75.0 and 80.0 μg/mL (commom diffeence: 2.5, 5 or 20)
- with S9 mix: 35.0, 45.0, 55.0, 65.0, 75.0, 85.0, 95.0, 105 and 115 μg/mL (commom diffeence: 10)
- 24 hours continuous treatment: 20.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0 and 65.0 μg/mL (commom diffeence: 5 or 10)
Vehicle / solvent:
- Vehicle(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of vehicle: The test substance was not suspended in distilled water at 20.0 mg/mL and in DMSO and acetone at 200 mg/mL. The test substance was suspended in 0.5 w/v% CMC solution and 0.5 w/v% MC solution at 20.0 mg/mL. These suspensions were considered to be stable from the facts on account of neither change in color, exothermic reaction nor gas generation at room temperature under a yellow light until 2 hours after preparation. The state of the suspension in 0.5 w/v% CMC solution at 20.0 mg/mL was betler than that in 0.5 w/v% MC solution. Therefore, 0.5 w/v% CMC solution was selected as a vehicle.
Untreated negative controls:
yes
Remarks:
0.5 w/v% CMC solution
Positive controls:
yes
Positive control substance:
mitomycin C
other: Cyclophosphamide monohydrate (CPA)
Details on test system and experimental conditions:
Chromosomal Aberration Test
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 3 days
- Exposure duration: 6 hours for the short - term treatment, treated for 24 hours in the 24 hours continuous treatment
- Expression time (cells in growth medium): 18 hours for the short - term treatment
OBSERVATION
Dose for observation:
- short - term treatment: 60.0, 65.0 and 75.0 μg/mL (Without S9 mix); 55.0, 75.0 and 95.0 μg/mL (With S9 mix)
- 24 hours continuous treatment: 40.0, 50.0 and 60.0 μg/mL

Structural aberration: Three hundred metaphase cells per dose (75 cells per specimen) containing 25 ± 2 chromosomes were observed using a microscope. The total number of cells with structural aberrations and the number of aberrant cells in each aberration category were recorded.
Numerical aberration: The numbers of polyploid cells with triploid or more (38 or more chromosomes) and endoreduplicated cells among 300 metaphase cells per dose (75 cells per specimen) were recorded.

Cell growth inhibition test
- Setting dose of test substance: 31.3, 62.5, 125, 500, 1000, 2000 /mL in short - term treatment without or with S9 mix and 24 hours continuous treatment
- Observation: check the presence or absence of mitotic metaphase cells, and the frequency of the cells with chromosomal aberrations was calculated by observed 50 metaphase cells per dose
Evaluation criteria:
The acceptability criteria was considered to be negative, which were either in a) or in b):
a) all results are inside the distribution ofthe historical data ofthe negative control group,
b) outside the distribution of the historical data of the negative control group, but none of the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control.

Providing that all acceptability criteria in case of c) to e) or f) were fulfilled, a test substance was considered to be positive:
c) outside the distribution of the historical data of the negative control group,
d) the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control,
e) the increase of the frequencies of cells with chromosomal aberrations is dose-related,
f) both chromosomal aberration test and confirmation test are fulfilled in case of c) and d).
Statistics:
Fisher’s exact test was performed in order to compare in the negative control group with positive control group.
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung fibroblasts (CHL/IU cells)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cell Growth Inhibition Test:
In all treatment methods, the precipitation of the test substance was observed at all doses of the substance at the start and at the end of the treatment. In "-S9 mix" and "+S9 mix", the precipitation of the test substance was observed at 500 μg/mL or more at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed in all treatment methods.

Chromosomal Aberration Test:
Both the short - term treatment and 24 hours continuous treatment, the frequencies of cells with structural aberrations and numerically aberrant cells at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control, the precipitation of the test substance was observed at all doses of test substance at the start and at the end of the treatment, the color change of the medium and the corrosion of the culture dish were not observed.
Conclusions:
The test substance did not induce chromosomal aberrations under the present test conditions.
Executive summary:

The ability of the test item to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cells) according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).

The Chinese hamster lung fibroblasts were exposured to the test solutions of different concentrations in the absence and presence of metabolic activation. The study includes two tests, one is the cell growth inhibition test and the other is the chromosomal aberration test. The number of cells with structural aberrations and numerical aberrations were observed and recorded.

The frequencies of cells with structural aberrations and numerically aberrant cells at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control. The precipitation of the test substance was observed at all doses of test substance at the start and at the end of the treatment, the color change of the medium and the corrosion of the culture dish were not observed. Therefore, structural aberration and numerical aberration were judged to be negative.

Consequently, the test substance did not induce chromosomal aberrations under the present test conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation test:

In order to examine the gene mutation inducibility of the test item, a reverse mutation assay was conducted in Salmonella typhimurium (hereinafter referred to as S. typhimurium) TA 100, TA 1535, TA 98 and TA 1537, and Escherichia coli (hereinafter referred to as E. coli) WP2 uvrA with or without metabolic activation by the pre-incubation method according to OECD Guideline 471.

N,N-dimethylformamide (hereinafter referred to as DMF) was used as the vehicle for the test article. A dose-finding test was conducted with dose levels between 19.5 and 5000 μg/plate to set the dose levels for the main test. From the result of the dose-finding test, the minimum dose which showed growth inhibition was selected as the maximum dose for the main test, and the main tests were conducted at 6 dose levels between 2.44 and 78.1 μg/plate in the S. typimurium TA 1537 without metabolic activation, and between 9.77 and 313 μg/plate in the S. typimurium TA 98, TA 100 and TA 1535 without metabolic activation, and between 39.1 and 1250 μg/plate in the E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation. The main test was conducted twice at the same dose levels.

Precipitation on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration by the test article was not observed at any dose levels irrespective of the presence/absence of metabolic activation.

In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 39.1 μg/plate or more in S. typhimurium TA 1537 without metabolic activation, and at 156 μg/plate or more in S. typhimurium TA 1535 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA 98 and TA 100 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains

with metabolic activation.

In the two main tests, there was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response in any strains irrespective of the presence/absence of metabolic activation.

In conclusion, the test item was judged to have no gene mutation inducibility (negative) under the conditions of this study.

Chromosomal aberration test:

The ability of the test item to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cells) according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).

The Chinese hamster lung fibroblasts were exposured to the test solutions of different concentrations in the absence and presence of metabolic activation. The study includes two tests, one is the cell growth inhibition test and the other is the chromosomal aberration test. The number of cells with structural aberrations and numerical aberrations were observed and recorded.

The frequencies of cells with structural aberrations and numerically aberrant cells at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control. The precipitation of the test substance was observed at all doses of test substance at the start and at the end of the treatment, the color change of the medium and the corrosion of the culture dish were not observed. Therefore, structural aberration and numerical aberration were judged to be negative.

Consequently, the test substance did not induce chromosomal aberrations under the present test conditions.

Justification for classification or non-classification

Negative result both in Ames study and In vitro chromosome aberration study.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, this substance should not be classified as mutagen.