Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
other: S typhimurium; TA98, TA 100, TA 1535, TA 1537 and E.Coli WP2uvrA.
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of Sprague-Dawley rats treated with Aroclor.
Test concentrations with justification for top dose:
Doses of 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate used in all tests

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Standard vehicle for tests of this type in which the substance formed a clear colourless solution.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 TA1537 without metabolic activation, 2-aminoanthracene TA1535, Ta1537, TA98, TA100 and E.Coli WP2uvrA with metabolic activation
Details on test system and experimental conditions:
Test substance preparation:
A solubility test was performed based on visual assessment. The test item formed a clear colorless solution in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves until the test item had completely dissolved. To protect the test item from light, amber-colored glassware or tubes wrapped in tin-foil were used for test item preparations. Test item concentrations were used within approximately 2 hours after preparation. Any residual volumes were discarded.

Dose range finder:
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was 5000 μg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

First experiment: Direct plate assay:
The dose-range finding study with two tester strains was reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Second experiment: Pre-incubation assay:
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony counting:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Rationale for test conditions:
Standard as per OECD guidelines
Evaluation criteria:
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item 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 concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in 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 or TA98 is greater than three (3) times the concurrent 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 follow up experiment.
Statistics:
No statistical analysis required

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
First assay (Direct plate assay):
Precipitate: In the tester strains TA100 and WP2uvrA, precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period. In tester strain WP2uvrA, in the absence of S9-mix, precipitation at the end of the incubation period was also observed at the concentration of 1600 μg/plate.

In the tester strains, TA1535, TA1537 and TA98, precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 μg/plate and above at the end of the incubation period. In tester strain TA1537, in the presence of S9-mix, precipitation at the end of the incubation period was only observed at the concentration of 5000 μg/plate.

Toxicity: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1537 and TA98 in the presence of S9-mix at the highest dose level tested. In tester strain TA1537, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 5000 μg/plate in the presence of S9-mix. Due to the low mean number of revertant colonies in the solvent control, the mean number of 2 revertants is not considered to be a toxic effect.

Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second assay (Pre-incubation assay):
Precipitate: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate.

Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strain TA1537 in the presence of S9-mix at the highest dose level tested.

Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Any other information on results incl. tables

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative control values were within the laboratory historical control data ranges, except the response for TA1537 in the absence of S9-mix in the first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (2 revertant colonies) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered to be not affected. 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.

Applicant's summary and conclusion

Conclusions:
In conclusion, 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.