Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 05 January 2017 to 09 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The Joint Notification Yakusyoku 0331 No. 8 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, H23·03·29 seikyoku No. 6 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, kanpoki No. 110331010 of the Environmental Policy Bureau, Ministry of the Environment (March 31, 2011) and the Joint Notification Yakusyoku 0331 No. 7 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare , H23·03·29 seikyoku No. 5 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, kanpoki No. 110331009 of the Environmental Policy Bureau, Ministry of the Environment (March 31, 2011).
Deviations:
no
Qualifier:
according to
Guideline:
other: JAPAN: Industrial Safety and Health Law
Version / remarks:
The Notifications of Ministry of the Labour, No. 76 (September 1, 1988) and No. 13 (revised March 29, 2000) and the Notifications of Ministry of the Labour, No. 77 (September 1, 1988) and No. 67 (revised June 2, 1997).
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Principles of Good Laboratory Practice (as revised in 1997) and OECD Guideline for Testing of Chemicals (21st July 1997), 471: Bacterial Reverse Mutation Test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
-Name of test substance (as cited in study report): Kurimate
-Lot No. MB10-55
-Purity: >99%
-CAS No. 1864727-96-7
-Molecular weight: 184.23
-Appearance at ordinary temperature: Colorless liquid
-Stability: Stable at room temperature
-Storage condition: Room temperature

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E.coli strain)
Species / strainopen allclose all
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Dose-determination test: 4.88, 19.5, 78.1, 313, 1250 and 5000 μg/plate
Mutagenicity test 1 and 2: 156, 313, 625, 1250, 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: the test substance was not soluble in water at 50 mg/mL, but was soluble in DMSO at 50 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene (2-AA), 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Remarks:
+S9: 2-AA (1 µg/plate, TA100; 2 µg/plate, TA1535, TA1537; 10 µg/plate, WP2uvrA; 0.5 µg/plate, TA98) -S9: AF-2 (0.01 µg/plate, TA100, WP2uvrA; 0.1 µg/plate, TA98); 9-AA (80 µg/plate, TA1537); NaN3 (0.5 µg/plate, TA1535)
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation method

DURATION
- Pre-incubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: A dose-determination test was carried out in a single plate, and mutagenicity tests were carried out in triplicate plates for each dose.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition and test substance precipitation
Evaluation criteria:
When the test substance induces a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the negative control and the increase is reproducible, the test substance is judged positive in this test system.
Statistics:
Any statistical method was not used for the data analysis.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above dose levels of 2500 μg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated 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:
at and above dose levels of 2500 μg/plate without metabolic activation, and at 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated 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
Remarks:
at and above dose levels of 2500 μg/plate without metabolic activation, and at 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated 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 and above dose levels of 2500 μg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated 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:
cytotoxicity
Remarks:
at and above dose levels of 2500 μg/plate without metabolic activation, and at 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRECIPITATION: precipitation on the plates was not observed.
RANGE-FINDING/SCREENING STUDIES: growth inhibition by the test substance was observed in all strains with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:
Throughout the tests, Kurimate did not induce any increases in the number of revertant colonies to at least twice as that of the negative control for any of the bacterial strains.
The test substance was concluded to be non-mutagenic under the conditions of this test.