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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-06 to 2017-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-12 to 2017-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
storage at room temperature
Target gene:
please refer to Table 1
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver
- method of preparation of S9 mix : Salt solution for S9 Mix: NADP Na 7.66 g (final: 4 mM), D-glucose-6 phosphate Na 3.53 g (final: 5 mM), MgCl2 1.90 g (final: 8 mM), KCl 6.15 g (final: 33 mM), Ultrapure water ad 1000 mL, Sterilized by filtration through a 0.22 μm membrane filter.
- concentration or volume of S9 mix and S9 in the final culture medium: The complete S9 Mix was freshly prepared containing components as follows: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4: 500 mL, Rat liver homogenate (S9): 100 mL, Salt solution for S9 Mix: 400 mL, The S9 Mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.
Test concentrations with justification for top dose:
5000, 1600, 500, 160, 50, 16 and 5 μg/plate
The selection of the concentration range was based on the recommendations in OECD 471 Guideline. At the concentration choice the non-toxicity and the limited solubility of the test item was taken into consideration. Therefore, in this test the highest test item concentration was 5000 μg/plate. This top concentration is a precipitating concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: dimethyl sulfoxide (DMSO) was found as suitable vehicle for preparing the test item solutions
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, NPD (TA98); 9-Aminoacridine, 9AA (TA1537); 2-aminoanthracene, 2AA (Salmonella typhimurium TA98; TA100; TA1535; TA1537 and Escherichia coli WP2 uvrA with metabolic activation)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 1 (plus initial mutation test)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37ºC in a shaking incubator
- Exposure duration/duration of treatment: After preparation the plates were incubated at 37°C for about 48 hours.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Induction of revertant colonies
Evaluation criteria:
The colony numbers on the untreated control, vehicle control, positive control and the test plates were determined visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated:
Mutation Rate= Mean revertants at the test item (or control*) treatments/ Mean revertants of vehicle control
*: untreated, vehicle or positive control

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains in the concentration range of 5000-500 μg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases and additionally the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

RANGE-FINDING/SCREENING STUDIES
Based on the solubility test, the stock solution with a concentration of 25 mg/mL was prepared in DMSO (for test item suspension preparation, dissolution < 1 min. ultrasonic treatment was applied) and diluted in five steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000; 1600; 500; 160; 50; 16 and 5 μg/plate.
The revertant colony numbers of vehicle control plates in both strains with and without S9 were in line with the corresponding historical control data ranges. The increases of the positive control treatments showed a significant more than 3-fold increase in the number of revertant colonies of S. typhimurium TA98 and TA100, both in the presence and the absence of S9 and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control.

HISTORICAL CONTROL DATA
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases and the number of revertants mostly fell in the corresponding historical control ranges (or even were above the range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. In E. coli WP2 uvrA at the Methyl methanesulfonate (MMS) controls the obtained revertant colony numbers were above the corresponding historical control data ranges, without any effect on the results and conclusion of the study. The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
The colony and background lawn development was not affected in any case; all of the obtained revertant mean colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

Table 2:Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (µg/plate) Salmonella typhimurium tester strains Escherichia coli WP2uvrA
TA98 TA100 TA1535 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean values of revertants per plate Mutation rate (MR) Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR
Untreated Control 17.0 1.09 29.0 1.43 97.0 1.30 106.7 1.35 9.7 0.91 11.3 1.13 8.0 0.96 7.3 0.92 27.0 1.45 39.3 0.99
DMSO Control (100 µL) 15.0 1.00 23.7 1.00 - - 90.7 1.00 - - 15.3 1.00 8.3 1.00 7.3 1.00 - - 30.7 1.00
UltrapureWater Control - - - - 81.3 1.00 - - 11.0 1.00 - - - - - - 31.0 1.00 - -
DMSO Control (200µL) 15.7 1.00 20.3 1.00 74.3 1.00 79.0 1.00 10.7 1.00 10.0 1.00 8.3 1.00 8.0 1.00 18.7 1.00 39.7 1.00
5000 16.3 1.04 15.3 0.75 64.7 0.87 75.7 0.96 11.3 1.06 9.3 0.93 7 0.84 5.3 0.67 30 1.61 39.3 0.99
1600 13.3 0.85 15.3 0.75 56.0 0.75 72.7 0.92 11.0 1.03 13.7 1.37 6.7 0.80 7.3 0.92 24.3 1.30 34.7 0.87
500 13.0 0.83 22.7 1.11 63.3 0.85 72.7 0.92 11.3 1.06 10.7 1.07 8.0 0.96 7.0 0.88 27.3 1.46 42.0 1.06
160 13.3 0.85 22.3 1.10 68.0 0.91 79.3 1.00 10.7 1.00 8.3 0.83 11.7 1.40 5.3 0.67 29.7 1.59 38.7 0.97
50 17.0 1.09 24.7 1.21 70.7 0.95 68.3 0.86 11.7 1.09 11.7 1.17 7.7 0.92 9.0 1.13 31.0 1.66 38.3 0.97
16 17.3 1.11 22.0 1.08 65.0 0.87 68.3 0.86 8.7 0.81 9.0 0.90 8.7 1.04 10.3 1.29 26.0 1.39 33.0 0.83
5 21.3 1.36 13.7 0.67 59.7 0.80 82.0 1.04 9.3 0.88 9.0 0.90 10.3 1.24 9.0 1.13 20.3 1.09 34.0 0.86
NPD (4 µg) 284.3 18.96 - - - - - - - - - - - - - - - - - -
SAZ (2 µg) - - - - 1645.3 20.23 - - 1434.7 130.42 - - - - - - - - - -
9AA (50 µg) - - - - - - - - - - - - 520.7 62.48 - - - - - -
MMS (2 µL) - - - - - - - - - - - - - - - - 1570.7 50.67 - -
2AA (2 µg) - - 1928.0 81.46 - - 1384.0 15.26 - - 138.7 9.04 - - 88.0 12.00 - - - -
2AA (50 µg) - - - - - - - - - - - - - - - - - - 240.7 7.85

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: DMSO (200 μL) was applied as vehicle of the test item and DMSO (100 μL) was applied as vehicle of the positive control substances 9AA, NPD and 2AA. The ultrapure water (100 μL) was applied as vehicle of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (200 μL); the mutation rate of the 9AA, NPD and 2AA refers to the DMSO sample (100 μL). The mutation rate of MMS and SAZ refers to ultrapure water (100 μL).

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay following internationally accepted guidelines and recommendations: OECD 471, EU Method B13/14, EPA Guideline OPPTS 870.5100. The study was performed following GLP conditions.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved, suspended in dimethyl sulfoxide (DMSO). Due to the limited solubility of the test item, lower stock solution concentration and consequently higher treatment volumes (0.2 mL) were applied at the test item treatments.

Based on the results of the preliminary Concentration Range Finding Test (Informatory Toxicity Test) the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate.

The selection of the concentration range was based on the recommendations in OECD 471 Guideline. At the concentration choice the guideline criterion for soluble, non-toxic test compounds was taken into consideration where the recommended maximum test concentration is 5 mg/plate.

The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges (or even were above the range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains in the concentration range of 5000-500 μg/plate in the absence and presence of exogenous metabolic activation (±S9) following the plate incorporation and pre-incubation procedures.

In the performed Initial and Confirmatory Mutation Tests using plate incorporation and pre-incubation methods inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained revertant mean colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
bis[2,2,6,6-tetramethyl-1-(phenylsulfanyl)piperidin-4-yl] carbonate
EC Number:
827-277-8
Cas Number:
1771689-37-2
Molecular formula:
C31H44N2O3S2
IUPAC Name:
bis[2,2,6,6-tetramethyl-1-(phenylsulfanyl)piperidin-4-yl] carbonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
storage at room temperature

Method

Target gene:
please refer to Table 1
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver
- method of preparation of S9 mix : Salt solution for S9 Mix: NADP Na 7.66 g (final: 4 mM), D-glucose-6 phosphate Na 3.53 g (final: 5 mM), MgCl2 1.90 g (final: 8 mM), KCl 6.15 g (final: 33 mM), Ultrapure water ad 1000 mL, Sterilized by filtration through a 0.22 μm membrane filter.
- concentration or volume of S9 mix and S9 in the final culture medium: The complete S9 Mix was freshly prepared containing components as follows: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4: 500 mL, Rat liver homogenate (S9): 100 mL, Salt solution for S9 Mix: 400 mL, The S9 Mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.
Test concentrations with justification for top dose:
5000, 1600, 500, 160, 50, 16 and 5 μg/plate
The selection of the concentration range was based on the recommendations in OECD 471 Guideline. At the concentration choice the non-toxicity and the limited solubility of the test item was taken into consideration. Therefore, in this test the highest test item concentration was 5000 μg/plate. This top concentration is a precipitating concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: dimethyl sulfoxide (DMSO) was found as suitable vehicle for preparing the test item solutions
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, NPD (TA98); 9-Aminoacridine, 9AA (TA1537); 2-aminoanthracene, 2AA (Salmonella typhimurium TA98; TA100; TA1535; TA1537 and Escherichia coli WP2 uvrA with metabolic activation)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1 (plus confirmatory experiment (pre-incubation) )

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48h

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Induction of revertant colonies
Evaluation criteria:
The colony numbers on the untreated, vehicle control, positive control and the test plates were determined visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated:
Mutation Rate = Mean revertants at the test item (or control*) treatments / Mean revertants of vehicle control
*: untreated, vehicle or positve control

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains in the concentration range of 5000-500 μg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases and additionally the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

RANGE-FINDING/SCREENING STUDIES
Based on the solubility test, the stock solution with a concentration of 25 mg/mL was prepared in DMSO (for test item suspension preparation, dissolution < 1 min. ultrasonic treatment was applied) and diluted in five steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000; 1600; 500; 160; 50; 16 and 5 μg/plate.
The revertant colony numbers of vehicle control plates in both strains with and without S9 were in line with the corresponding historical control data ranges. The increases of the positive control treatments showed a significant more than 3-fold increase in the number of revertant colonies of S. typhimurium TA98 and TA100, both in the presence and the absence of S9 and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control.

HISTORICAL CONTROL DATA
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases and the number of revertants mostly fell in the corresponding historical control ranges (or even were above the range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. In E. coli WP2 uvrA at the Methyl methanesulfonate (MMS) controls the obtained revertant colony numbers were above the corresponding historical control data ranges, without any effect on the results and conclusion of the study. The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers and agreed with the actual historical control data ranges in all strains in both main experimental phases.
The colony and background lawn development was not affected in any case; all of the obtained revertant mean colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

Any other information on results incl. tables

Table 2: Summary Table of the Results if the Initial Mutation Test

Initial Mutation Test (PlateIncorporationTest)
Concentrations (µg/plate) Salmonella typhimurium tester strains Escherichia coli WP2uvrA
TA98 TA100 TA1535 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean values of revertants per plate Mutation rate (MR) Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR Mean MR
Untreated Control 20.3 1.09 18.3 0.95 82.0 1.03 119.0 1.23 10.0 0.91 10.3 0.84 6.3 1.19 9.3 1.04 34.7 1.08 46.3 1.39
DMSO Control (100 µL) 19.0 1.00 18.0 1.00 - - 84.0 1.00 - - 9.0 1.00 7.0 1.00 9.0 1.00 - - 38.0 1.00
Ultrapure Water Control - - - - 72.0 1.00 - - 9.0 1.00 - - - - - - 27.7 1.00 - -
DMSO Control (200 µL) 18.7 1.00 19.3 1.00 80.0 1.00 97.00 1.00 11.00 1.00 12.3 1.00 5.3 1.00 9.00 1.00 32.0 1.00 33.3 1.00
5000 19.7 1.05 28.3 1.47 102.0 1.28 103.0 1.06 15.7 1.42 13.7 1.11 5.0 0.94 9.3 1.04 31.3 0.98 35.7 1.07
1600 20.0 1.07 28.7 1.48 84.7 1.06 115.0 1.19 14.7 1.33 9.0 0.73 7.3 1.38 9.0 1.00 30.3 0.95 36.3 1.09
500 17.7 0.95 26.3 1.36 90.0 1.13 107.3 1.11 15.0 1.36 10.7 0.86 6.7 1.25 9.7 1.07 22.3 0.70 41.0 1.23
160 14.3 0.77 16.0 0.83 80.3 1.00 110.3 1.14 13.3 1.21 13.0 1.05 4.7 0.88 10.3 1.15 30.0 0.94 41.0 1.23
50 15.3 0.82 28.3 1.47 95.7 1.20 83.7 0.86 17.0 1.55 13.3 1.08 4.7 0.88 8.0 0.89 33.3 1.04 42.0 1.26
16 16.3 0.88 24.0 1.24 81.7 1.02 104.3 1.08 11.7 1.06 13.3 1.08 5.0 0.94 10.3 1.15 33.0 1.03 38.3 1.15
5 15.0 0.80 21.0 1.09 95.7 1.20 119.3 1.23 12.3 1.12 9.7 0.78 5.7 1.06 8.0 0.89 29.0 0.91 32.0 0.96
NPD (4 µg) 506.0 26.63 - - - - - - - - - - - - - - - - - -
SAZ (2 µg) - - - - 898.0 12.47 - - 1525.3 169.48 - - - - - - - - - -
9AA (50 µg) - - - - - - - - - - - - 666.0 95.14 - - - - - -
MMS (2 µL) - - - - - - - - - - - - - - - - 1685.3 60.92 - -
2AA (2 µg) - - 1053.3 58.52 - - 2349.3 27.97 - - 215.7 23.96 - - 136.0 15.11 - - - -
2AA (50 µg) - - - - - - - - - - - - - - - - - - 235.3 6.19

MR: Mutation Rate; NPD: 4 -Nitro-1,2 -phenylenediamine; SAZ: Sodium azide; 9AA: 9 -Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2 -aminoanthracene

Remarks: DMSO (200 μL) was applied as vehicle of the test item and DMSO (100 μL) was applied as vehicle of the positive control substances 9AA, NPD and 2AA. The ultrapure water (100 μL) was applied as vehicle of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (200 μL); the mutation rate of the 9AA, NPD and 2AA refers to the DMSO sample (100 μL). The mutation rate of MMS and SAZ refers to ultrapure water (100 μL).

Applicant's summary and conclusion

Conclusions:
No relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiment. Occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. To verify the results a following confirmatory mutation test (pre incubation method) was performed.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay following internationally accepted guidelines and recommendations: OECD 471, EU Method B13/14, EPA Guideline OPPTS 870.5100. The study was performed under GLP-conditions.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved, suspended in dimethyl sulfoxide (DMSO). Due to the limited solubility of the test item, lower stock solution concentration and consequently higher treatment volumes (0.2 mL) were applied at the test item treatments.

Based on the results of the preliminary Concentration Range Finding Test (Informatory Toxicity Test) the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate.

The selection of the concentration range was based on the recommendations in OECD 471 Guideline . At the concentration choice the guideline criterion for soluble, non-toxic test compounds was taken into consideration where the recommended maximum test concentration is 5 mg/plate.

The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges (or even were above the range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains in the concentration range of 5000-500 μg/plate in the absence and presence of exogenous metabolic activation (±S9) following the plate incorporation procedure.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. To verify the results a following confirmatory mutation test (pre incubation method) was performed. (see Key_Genetic toxicity in vitro: Ames test_pre incub._Vertesi, 2018b)