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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May 2021 - 07 June 2021
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 June 2020
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C13 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid
EC Number:
938-645-3
Cas Number:
1689515-39-6
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C13 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid
Test material form:
liquid
Details on test material:
- Physical appearance: clear yellow liquid
- Storage conditions: At room temperature

Method

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:
Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 254 (500 mg/kg body weight)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL; 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Test concentrations with justification for top dose:
FIRST EXPERIMENT: DIRECT PLATE ASSAY
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 were tested in triplicate. Based on the results of the dose-range finding test, the dose-range for the mutation assay with the tester strains, TA1535, TA1537 and TA98 was selected.
- TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (with and without metabolic activation).
- TA1535, TA1537 and TA98: 17, 52, 164, 512, 1600 and 5000 μg/plate. (with and without metabolic activation).
SECOND EXPERIMENT: PRE-INCUBATION ASSAY
Second Experiment: Pre-Incubation Assay
Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in all tester strains.
- TA1535, TA1537, TA100, TA98 and WP2uvrA: 17, 52, 164, 512, 1600 and 5000 μg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water

The test item was corrected for the water content (50.6%). A correction factor of 2 was used. A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in Milli-Q water.
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;2-aminoanthracene
Remarks:
For details on positive control substances, see Table 1 and Table 2 in "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) - Experiment 1; pre-incubation - Experiment 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Experiment 2 (pre-incubation assay): 30 ± 2 minutes
- Exposure duration/duration of treatment: 48 ± 4 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were evaluated.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The revertant colonies were counted automatically with the colony counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.
Rationale for test conditions:
according to guideline
Evaluation criteria:
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 times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three 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 times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three 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.

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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See explanation below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
FIRST EXPERIMENT
Precipitation:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

Cytotoxicity:
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed at any of the dose levels tested.

Mutagenicity:
In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item but with tester strain TA1537 in the absence of S9-mix, the lowest concentration of the test item induced a 3.7-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was within the historical control data range and not dose related and therefore considered not biologically relevant .

SECOND EXPERIMENT:
Precipitation:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

Cytotoxicity:
Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the absence and presence of S9-mix, where no toxicity was observed at any of the dose levels tested.

Mutagenicity:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item but with tester strain TA1537 in the presence of S9-mix, the test item induced a 3.0-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was within the historical control data range and not dose related and therefore considered not biologically relevant.

ACCEPTABILITY CRITERIA
All bacterial strains showed negative responses over the entire dose-range, i.e. no biologically relevant, dose-related increase in the number of revertants in two independently repeated experiments. The negative and 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.

Details on results and historical control data are included in the attachments.

Applicant's summary and conclusion

Conclusions:
Based on the results of an Ames test, performed according to OECD guideline 471 and in accordance with GLP principles, the test item is concluded to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

A bacterial reverse mutation test was performed according to OECD guideline 471 and in accordance with GLP principles. The negative and 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. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four 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 when tested up to and including the recommended top concentration. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.