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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 26 to August 12, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD test guidelines and GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): E-Y110
- Physical state: Orange powder, solid
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: July 7, 2015
- Storage condition of test material: Room temperature, in dark place

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Obtained from Japan Bioassay Research Center
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Obtained from Japan Bioassay Research Center
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from livers of 7 week old male SD rats treated with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Preliminary test: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate (+/-S9)
Main tests: 313, 625, 1250, 2500, 5000 µg/plate (+/-S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterilised distilled water (DW)
- Justification for choice of solvent/vehicle: the test substance is soluble at 100000 mg/l or more in water and from 35 mg/l to 200 mg/l in DMSO an d insoluble in acetone based on information from the sponsor. In the solvent selection test, the test substance was dissolved at 50 mg/ml in DW, increases in temperature, discoloration and foaming were not observed when the test substance was mixed with DW.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DW
Positive controls:
yes
Remarks:
in TA100, TA98 and WP2 uvrA
Positive control substance:
other: AF-2
Remarks:
without S9-mix: 0.01 µg/pl in TA100 and WP2 uvrA, 0.1 µg/pl in TA98
Negative solvent / vehicle controls:
yes
Remarks:
DW
Positive controls:
yes
Remarks:
in TA1535
Positive control substance:
sodium azide
Remarks:
Without S9-mix: 0.5 µg/plate

Migrated to IUCLID6: NaN3
Negative solvent / vehicle controls:
yes
Remarks:
DW
Positive controls:
yes
Remarks:
in TA1537
Positive control substance:
9-aminoacridine
Remarks:
Without S9-mix: 80 µg/plate

Migrated to IUCLID6: 9-AA
Negative solvent / vehicle controls:
yes
Remarks:
DW
Positive controls:
yes
Remarks:
in all strains with S9 mix
Positive control substance:
other: 2-AA
Remarks:
TA-100 1.0 µg/pl, TA1535 and TA1537 2.0 µg/pl, TA98 0.5 µg/pl, WP2 uvrA 10 µg/pl
Details on test system and experimental conditions:
METHOD OF APPLICATION:
preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hr

NUMBER OF REPLICATIONS:
1 plate/dose for the preliminary test, 3 plates/dose for each of the two main tests.

NUMBER OF CELLS EVALUATED:
All revertant colonies/plate were counted using an automated colony counter or manually.

DETERMINATION OF CYTOTOXICITY:
Precipitation was judged by observation of the plate surface macroscopically. No cytotoxicity was observed.
Evaluation criteria:
Mutagenicity is induced when a reproducible dose dependent increase in number of revertant colonies is present at >=2 times the number in the negative controls.
Main tests were accepted as valid if all the following criteria were satisfied:
- the negative control values and the positive control values are within the proper ranges calculated based on the historical data
- the postivie control values show clear positive responses in the respective test strains
- more than 4 doses show no microbial growth inhibition and more than 5 doses applicable to the evaluation
- result of the sterility test indicates that there is no bacterial contamination
- no plates became invalid for measurement due to contamination or other unexpected situations.
Reproducibility of the test result was confirmed in the preliminary and two main tests.
Statistics:
None required due to test results.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
- Other confounding effects: none known.

RANGE-FINDING/SCREENING STUDIES: The number of revertant colonies was less than twice the number of the respective negative controls. No microbial growth inhibition and precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values are within the proper ranges of the historical data.

Any other information on results incl. tables

See results attached: Table 1, 2 and 3.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

E-Y110 was considered not mutagenic in this bacterial reverse mutation test (OECD 471) with five strains with and without metabolic activation.
Executive summary:

Mutagenicity of E-Y110 was assessed in the bacterial reverse mutation test using five strains, Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA. The test was conducted by the pre-incubation method with and without S9 -mix. The tests were performed with doses up to 5000 µg/plate, based on a preliminary test. As a result in both main tests, the number of revertant colonies treated with E-Y110 was less than twice that treated with the negative control in any tester strain with or without S9 -mix. Microbial growth inhibition and precipitation was also not observed. From these results, it is concluded that E-Y110 has no mutagenic potential under the conditions of this study.