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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-C104
- Physical state: Blue 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: in the solvent selection test, the test substance 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, 2 plates/dose for negative and positive controls

NUMBER OF CELLS EVALUATED:
>10E8 viable cells were counted. 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
Remarks:
and precipitating at 313 µg/pl or more with S9 mix
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
Remarks:
and precipitating at 313 µg/pl or more with S9 mix
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from 313 µg/plate onwards the test substance precipated when strains were exposed to S9-mix. Without S9-mix, no precipitation was observed.
- 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 was observed, and precipitation was observed in S9 treated strains from 313 µg/plate onwards.

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-C104 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-C104 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-C104 was less than twice that treated with the negative control in any tester strain with or without S9 -mix. Microbial growth inhibition was also not observed, while precipitation of the test substance was observed from 313 µg/plate onwards with S9 -mix. From these results, it is concluded that E-C104 has no mutagenic potential under the conditions of this study.