Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the test item was not considered to be mutagenic in S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA in the presence and absence of phenobarbital & 5,6-benzoflavone-induced rat liver S9. (Bacterial reverse mutation test, Ministry of Health, Labour and Welfare, Japan (Ministry of Health, Labour and Welfare, Notification No. 77: September 1, 1988)/GLP).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 3, 2020 -March 25, 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: test material provided by sponsor:91112Y
- Purity.:92.4%;

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
Insoluble in water and dimethyl sulfoxide(DMSO) at 50mg/mL, soluble and stable in acetone.


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: Oriental Yeast Co., Ltd. (Lot No.20011711); Phenobarbital & 5,6-Benzoflavone-induced rat liver.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): sterile performed, the prepared S9 mix wa kept on an ice-bath until use(0.5mL/plate).
Test concentrations with justification for top dose:
Preliminary toxicity test: 4.88, 19.5, 78.1, 313, 1250, 5000 ug/plate.

Mutagenicity test 1 (+/-S9): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 ug/plate for TA 98 strain of-S9 mix,
0.610, 1.22, 2.44, 4.88, 9.77, 19.5, 39.1 ug/plate for TA 100, TA 1535, TA 1537 strains of -S9 mix,
19.5, 39.1, 78.1, 156.3. 312.5, 625 ug/plate for TA 98, TA 100, TA 1535, TA 1537 strains of + S9 mix and WP2urA strain of ±S9mix.

Mutagenicity test (-S9) 2: 2.44, 4.88, 9.77, 19.5, 39.1 ug/plate for TA 98 strain of-S9 mix,
0.610, 1.22, 2.44, 4.88, 9.77 ug/plate for TA 100, TA 1535, TA 1537 strains of -S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: According to the information provided by the study sponsor,the solubility of the test substance was insoluble in water and DMSO at 50mg/ml, soluble and stable in acetone at 100 mg/ml. From the above results, acetone was selected as a solvent for the test.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other:
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments:2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: (20 minutes at 37.0℃)
- Exposure duration/duration of treatment: 48h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: stereomicroscope(×40);
Evaluation criteria:
Determined as positive(+) if the revertant colony numbers of the test substance group increases two or more times than that of the negative control group and reproducibility or dose dependency was observed for the increase.
Statistics:
Statistical analysis were not used for evaluation of the resulis.
Species / strain:
S. typhimurium TA 100
Remarks:
Expt I & II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
156 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Expt 1 & II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
156 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Expt I & II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
156 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Expt I & II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
156 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Expt I&II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
156 µg/plate
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:
Preliminary toxicity test: Precipitations of the test item were observed at 313 µg/plate to above of ±S9mix .
Mutagenicity test 1 AND 2: Precipitations of the test item were observed at 156 µg/plate to above of ± S9mix, no cytotoxicity was observd in any strains of ± S9mix


RANGE-FINDING/SCREENING STUDIES (if applicable):
There was no increase in the number of revertant colonies for each strain compared to the negative controls, with or without metabolic activation. The positive controls of each strain showed marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain.


STUDY RESULTS
- Concurrent vehicle negative and positive control data: There was no increase in the number of revertant colonies for each strain compared to the negative controls, with or without metabolic activation. The positive controls of each strain showed marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain (Mutagenicity test 1 & 2).

Ames test:
- Signs of toxicity: none
- Individual plate counts: Yes
- Mean number of revertant colonies per plate and standard deviation: Mean and SD not provided.

Each strain confirmed the reproducibility, the numbers of revertant colonies in the negative and positive controls of each strain were within Mean ±3 standard deviation of the historical control data, contamination was not observed in the sterility test therefore, it was judged that the study met the validity criteria.

Conclusions:
In a reverse gene mutation assay in bacteria, the test item was considered non-mutagenic.
Executive summary:

In a reverse gene mutation assay in bacteria (USA-R-20053), strains of S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA were exposed to the test item (92.4%) in acetone at concentrations of 9.77 -313 µg/plate ± S9 in experiment 1 and 2. Both experiments were carried out via the pre-incubation method (20 mins at 37 °C) and metabolic activation was phenobarbital & 5,6-benzoflavone-induced rat liver S9.

In mutagenicity test 1 and 2, no cytotoxicity were observed , but precipitations of the test item were observed at 156 µg/plate to above of ± S9 mix for all strains.

The positive controls of each strain showed a marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain. There was no increase in the number of revertant colonies for each strain compared to the negative controls in test-item treated strains, with or without metabolic activation, in either experiment. Under the conditions of this study, the test item is considered non-mutagenic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) with the test item available.

In a reverse gene mutation assay in bacteria ( Bacterial reverse mutation test, OECD471/GLP), strains of S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA were exposed to the test item (92.4%) in acetone at concentrations of 9.77 -313 µg/plate ± S9 in experiment 1 and 2. Both experiments were carried out via the pre-incubation method (20 mins at 37 °C) and metabolic activation was phenobarbital & 5,6-benzoflavone-induced rat liver S9.

In mutagenicity test 1 and 2, no cytotoxicity were observed , but precipitations of the test item were observed at 156 µg/plate to above of ± S9 mix for all strains.

The positive controls of each strain showed a marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain. There was no increase in the number of revertant colonies for each strain compared to the negative controls in test-item treated strains, with or without metabolic activation, in either experiment. Under the conditions of this study, the test item is considered non-mutagenic.

Justification for classification or non-classification

Based on the available information in the dossier, the test item does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.