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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:
October 20, 2014 - October 30, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): L 131
- Appearance: Faint light-green powder

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with 5% (v/v) S9-mix) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with 5% (v/v) S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with 10% (v/v) S9-mix: 492, 878, 1568, 2800 and 5000 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: At concentrations of 0.17 mg/ml and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test substance concentrations were used within 3 hours after preparation.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

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
Vehicle 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
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of L 131 on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and no precipitate was observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative control values were within the laboratory historical control data ranges, except the response for TA100 in the absence of S9-mix, in the dose range finding study. The value (69) was below the limit of the range (76). As this mean plate count of 69 was just below the limit of the range, this had no effect on the results of the study.
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the second experiment in the absence of S9-mix. The value (1125) was above the limit of the range (998). Since the value was more than 3 times greater than the concurrent solvent control values, this had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cytotoxicity was only observed in tester strain TA98 in the absence of S9-mix at the highest tested concentration, in Experiment 1 only.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic activity of L 131 was evaluated in accordance with OECD 471 (1997) and according to GLP principles. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.
Executive summary:

The mutagenic activity of L 131 was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, both in the absence and presence of S9-mix. The substance was tested up to and including 5000 µg/plate in all tester strains. Adequate solvent and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.