[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 NOV 2020 - 08 FEB 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Studies Chemicals, Chapter 5 Health impact test field, Section 22 Genotoxicity (Bacterial Reverse Mutation Test)

Version / remarks:

Notification No. 2020-46, National Institute of Environmental Research, Republic of Korea (Nov. 3,
2020)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on Jul. 21, 1997, corrected on Jun. 26, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

bis(2,2,6,6-tetramethyl-1-(1,3-benzothiazol-2-ylsulfanyl)piperidin-4-yl)carbonate

Cas Number:

2311845-49-3

Molecular formula:

C33H42N4O3S4

IUPAC Name:

bis(2,2,6,6-tetramethyl-1-(1,3-benzothiazol-2-ylsulfanyl)piperidin-4-yl)carbonate

Test material form:

solid: particulate/powder

Method

Target gene:

his and trp (please refer to any other information on materials and methods incl. tables)

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

pKM101

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Male rat, Sprague Dawley, Molecular Toxicology, Inc.
- method of preparation of S9 mix: S9 Mix was prepared by mixing the S9 fraction and S9 cofactor (cofactor I) with S9 fraction of 5 %. The prepared S9 Mix was kept cool condition before treatment. S9 Mix was treated with 0.5 mL/plate. Enzyme activity was confirmed by mutagenesis of 2AA and B[a]P.
- concentration or volume of S9 mix and S9 in the final culture medium: final concentration 5%

Test concentrations with justification for top dose:

5.1, 15.4, 46.3, 138.9, 416.7, 1250 μg/plate
Based on results in the concentration range finding study, highest concentration in the main study was set 1250 μg/plate in all strains both in the presence and absence of the metabolic activation system. It was serially diluted to consist of six concentration levels by the common ratio of 3.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: This vehicle has no toxic effect on the test systems and the metabolic activation system. Also results of vehicle test, when test substance was
prepared in sterile distilled water, precipitation was observed. In case of DMSO, test substance was suspended. However, no precipitation was observed. Therefore, the vehicle was chosen as DMSO.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene (2AA), N-Methyl-N’-nitro-Nnitrosoguanidine (MNNG)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 (main study, confirmatory study)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation): 0.5 mL of S9 Mix, 0.1 mL of the test substances and 0.1 mL of bacterial culture were added in a sterile test tube. In case of absence of metabolic activation system, 0.5 mL of 0.2 M Sodium Phosphate Buffer (pH 7.4) was added instead of S9 Mix. The negative control group was treated with 0.1 mL of vehicle.
And, the positive control group was treated with 0.1 mL of positive control substance. Each prepared tube was added 2 mL of top agar and mixed evenly using vortex mixer. After mixed, poured into a minimal nutrient plate medium (MNA) and applied evenly.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: After treatment was finished, turned the plate upside down and incubated above 48 hours in a 37 °C incubator.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Each plate was observed on the basis of the background lawn pattern of the negative control group using a stereomicroscope, and the thickness and colony pattern of the background lawn are closely observed and used as a cytotoxicity index of the test substance

METHODS FOR MEASUREMENTS OF GENOTOXICIY
After incubated, number of revertant in the plates were counted using an oil pen.
The average and standard deviation of the number of revertant obtained from the plate of each concentration group and the number of revertant were expressed with integers.

Rationale for test conditions:

In order to confirm the reproducibility of the main study, a confirmatory study was performed using the same concentrations and method as in the main study.

Evaluation criteria:

If the test substance met the following criteria, it was judged to be negative.
- When the mean number of colonies in the test substance-treated group did not show a
concentration-dependent increase and was less than 2 times the number of negative control
colonies.

If the test substance was met all the following criteria, it was judged as positive.
Regardless of the application of the metabolic activation system to the test substance exposure, the test substance was judged to induce a return mutation in the bacteria when the following results were obtained in at least one strain. At this time, the biological relevance was considered.
- When the mean number of colonies in the test substance-treated group will be shown a
concentration-dependent increase and will be more than twice as large as the number of colonies
in the negative control group.
- When the mean number of colonies in the treatment of the test substance is 2.0 times higher than the number of colonies in the negative control group, and when reproducibility is observed as a result of the confirmation test.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Remarks:

pKM101

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 specified

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

with: 2-Aminoanthracene without: N-Methyl-N-nitro-N-nitrosoguanidine

Test results 2

Species / strain:

S. typhimurium TA 1537

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 specified

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

with: 2-Aminoanthracene without: 9-Aminoacridine

Test results 3

Species / strain:

S. typhimurium TA 1535

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 specified

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

with: 2-Aminoanthracene without: Sodium azide

Test results 4

Species / strain:

S. typhimurium TA 100

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 specified

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

with: 2-Aminoanthracene without: Sodium azide

Test results 5

Species / strain:

S. typhimurium TA 98

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 specified

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

with: Benzo[a]pyrene without: 2-Nitrofluorene

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
In this study, turbidity was observed at 313, 1250 and 5000 μg/plate in the treatment mixture. When colony counting, precipitation was observed at 313, 1250 and 5000 μg/plate in the plates. Count of colony at 5000 µg/plate was impossible due to precipitation of test substance.
Thin background lawn and reduction of colony determining cytotoxicity were not observed. The background lawn was in normal condition. There were no increases in mean number of revertants at all concentrations in all strains both in the presence and absence of the metabolic activation system. Therefore, the highest concentration in the main test was set to 1250 μg/plate, including the concentration at which precipitation of the test substance was observed, excluding the concentration at which counting was impossible.

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

Ames test:
- Signs of toxicity: There were no increases in mean number of revertants at all concentrations in all strains both in the presence and absence of the metabolic activation system.
- Mean number of revertant colonies per plate and standard deviation: see attached tables

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data):
The mean number of revertant in the negative and positive control group was evaluated to be within the range of historical control data, see attached tables

Applicant's summary and conclusion

Conclusions:

All validity criteria of this test were fulfilled. No increases in mean number of revertant were observed at all concentrations in all strains both in the presence and absence of the metabolic activation system. In the positive control group, the number of revertant increased more than twice compared to the negative control group. In conclusion, the test substance was considered as not having reverse mutagenic potential on bacterial strains of S. typhimurium and E. coli under the present study conditions.

Executive summary:

The bacterial reverse mutation test was performed according to OECD guideline 471 under GLP compliance. This study was conducted in order to evaluate the mutagenic potential of the test substance in the Bacterial Reverse Mutation Test using Salmonella typhimurium strains (S. typhimurium TA98, TA100, TA1535 and TA 1537) and Escherichia coli strain (E. coli WP2 uvrA) in the presence and absence of an exogenous
metabolic activation system (S9 mix).

A concentration range finding study was conducted to determine the concentration of the test substance prior to the main study. The confirmatory study was conducted to confirm the reproducibility of the results of the main study.

The highest concentration of the test substance in the concentration range finding study was selected as 5000 μg/plate and serially diluted to consist of six concentration levels by the common ratio of 4.
In the concentration range finding study, precipitation was observed at 313, 1250 and 5000 μg/plate. Count of revertant at 5000 μg/plate was impossible due to heavy precipitation of the test substance. There were no cytotoxicity and no increases in mean number of revertants at all concentrations in all strains both in the presence and absence of the metabolic activation system.

According to results of concentration range finding study, the highest concentration in the main study and confirmatory study was selected as 1250 μg/plate and serially diluted to consist of six concentration levels by the common ratio of 3. In the main and confirmatory study, precipitation was observed at 416.7 and 1250 μg/plate. There were no cytotoxicity and no increases in mean number of revertants at all concentrations in all strains both in the presence and absence of the metabolic activation system.
In the positive control group, increases of mean number of revertant was observed more than twice compared to the negative control group. All validity criteria of this test were fulfilled. 

In conclusion, the test substance was considered as not having reverse mutagenic potential on bacterial strains of S. typhimurium and E. coli under the present study conditions.