Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 2017 - 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
November 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: crystalline
Details on test material:
Appearance: white crystalline powder
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
Stability at higher temperatures: stable, maximum temperature: 40 °C, maximum duration: 2 days
Solubility in water: 167.13 g/L (20°C)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
5% rat liver S9-mix induced by Aroclor 1254
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
5% rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) 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 S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: A solubility test in water was performed where the test item was dissolved in Milli-Q water.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
other: 2-nitrofluorene
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191 2.5 µg/plate in DMSO for TA1537 (for the 1st experiment only)
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and conditions:
METHOD OF APPLICATION: first experiment: direct plate; second experiment: pre-incubation

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments, a direct plate assay and a pre-incubation assay, were conducted.

NUMBER OF CELLS EVALUATED: 10E8 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 were determined both macroscopically and microscopically by using a dissecting microscope.

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 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a repeat 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 independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed at the start or at the end of the incubation period in both experiments.

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 and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except for the response for TA100 (absence of S9-mix) in the first
experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY (in both experiments):
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In strain TA1537 (presence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed. However, since no doserelationship was observed this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

ADDITIONAL INFORMATION ON MUTAGENICITY:
First experiment (direct plate assay): no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Second experiment (pre-incubation assay): no biologically relevant increase in the number of revertants was observed upon treatment with the test item under all conditions tested. In tester strain TA1537 (absence of S9-mix), a 7.5-fold increase in the number of revertant colonies compared to the solvent control was observed at 512 μg/plate. The three numbers of revertant colonies observed at this concentration were: 5, 3 and 83. The 83 value is not only very different from the two other values obtained at 512 μg/plate, but is also not in range with all other numbers of revertant colonies at all other tested concentrations. This observation of an outlier, so far beyond the historical control data range and the two other values of the triplicate, is a rare phenomenon and is probably linked to an incidental fluctuation in the number of revertant colonies. The absence of any observed dose effect is confirming that this 83 value is an outlier. Therefore, the 7.5 times increase of revertant colonies observed at 512 μg/plate is considered to be related to a specific outlier and to be not biologically relevant.

Any other information on results incl. tables

In this study, the vehicle and positive controls were within the historical data, the strains were tested up to a dose of 5 mg/plate. Therefore, the validity criteria were met and the study was considered acceptable.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 and GLP principles, the test substance was found not to be mutagenic with or without metabolic activation when tested up to 500 µg/plate.