Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 - 15 September 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

EC Number:

275-085-2

EC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Cas Number:

70983-43-6

Molecular formula:

see structural formula below, for n=4 C13H24N2O3 HO.Na

IUPAC Name:

Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts

Test material form:

liquid

Remarks:

oily liquid

Details on test material:

Batch No.: 54542L16
Storage at room temperature, protected from light.

Method

Target gene:

histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) rat liver S9 mix

Test concentrations with justification for top dose:

Initial Mutation Test and Confirmatory Mutation Test:
-S9 Mix: 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate;
+S9 Mix: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.

For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate.
Test substances that are cytotoxic already below 5 mg/plate or 5 ml/plate should be tested up to a cytotoxic concentration.

Concentration Range Finding Test (Informatory Toxicity Tests):
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
The revertant colony numbers of vehicle control and positive control plates with and without S9 Mix were in line with the corresponding historical control data ranges in both tester strains.
In the performed Informatory Toxicity Test strong inhibitory, cytotoxic effect of the test item was observed in both examined strains at the concentrations of 5000 and 1600 µg/plate, without exogenous metabolic activation (-S9) and at 5000 µg/plate, with addition of metabolic activation (+S9). At these concentration levels the cytotoxicity was indicated by decreased revertant colony counts (absent revertant colonies or revertants below the corresponding historical control data and actual vehicle control ranges) and affected background lawn development: absent or reduced background lawn.
The colony and background lawn development was not affected in any further case; all of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix).

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: In the solubility test the test item behaviour was investigated in the applied test system. The test item was dissolved and further diluted in DMSO, accordingly. The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension. No precipitation was observed.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

Positive control concentration: 4 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control concentration: 2 µg/plate for TA100 and TA1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

positive control concentration: 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

posititve control concentration: 2 µL/plate for WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

Positive control with metabolic activation, concentration: 2 µg/plate for S. typhimurium strains; 50 µg/plate for E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: initial mutation test: in agar (plate incorporation); confirmatory test: in agar (plate incorporation) with preincubation

- Bacterial cultures: The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-13 hours in a 37°C Benchtop Incubator Shaker.

- Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The typical content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain 100 µL (containing aprox 10^9 CFU/ml)
phosphate buffer (pH: 7.4) or S9 mix 500 µL
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube.

- Exposure duration: incubated at 37°C for about 48 hours

- Preincubation period (in confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to allow direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, two mL of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates.

NUMBER OF REPLICATIONS: Triplicates

METABOLIC ACTIVATION SYSTEM: Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

VALIDITY CRITERIA
The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in
induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest doses tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.
- The test has to be included five analyzable concentrations (where the precipitate does not interfere with the scoring) and a minimum of three non-precipitated dose levels.

Evaluation criteria:

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.

Statistics:

Based on the evaluation criteria no statistical analysis was required.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Validity criteria:
All criteria for the validity of the performed experiments according to the OECD guideline were met.

- Controls :
In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in both main experimental phases, in all tester strains.


- Initial and Confirmatory Mutation Tests:
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were noticed. These increases did not show a clear dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.

The highest revertant colony number increases were observed in the Initial Mutation Test (Plate Incorporation Test) in Salmonella typhimurium TA1537 strain at 50 µg/plate, in the presence of metabolic activation and in the Confirmatory Mutation Test (Pre-Incubation Test) in Salmonella typhimurium TA1535 at 50 µg/plate, in the presence of metabolic activation (+S9). The mutation rate was 1.65 in both cases. This value remained in the range of the corresponding vehicle historical control data in both cases and additional concentration related increase in revertant colony counts was not noticed in any case. This mutation rate was furthermore far below the genotoxicological threshold for being positive at both strains.

In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed in all examined strains. The cytotoxicity was indicated by absent or decreased revertant colony counts (a lot of them below the corresponding historical control data ranges) and/or affected background lawn development: absent reduced or slightly reduced background lawn. The cytotoxicity results are summarized in Table 1. The table contains the unequivocal cytotoxicity results, only, where the obtained revertant colony numbers were below the vehicle (in some cases below the corresponding historical control data ranges), and/or affected background lawn development occurred. All of the further observed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system. In general, 160 µg/plate was considered as the lowest concentration showing cytotoxicity

In the performed experiments no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).,

Applicant's summary and conclusion

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 Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS Nr. 70983-43-6) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5-dihydro-3(hydroxyethyl), hydroxides, sodium salts (CAS Nr. 70983-43-6) was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

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/ß-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 in dimethyl sulfoxide (DMSO).  

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:  

-S9 Mix: 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate;

+S9 Mix: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.

The maximum test concentration was 1600 µg/plate (-S9), and 5000 µg/plate (+S9). The concentration choice the guideline criterion for soluble, cytotoxic test compounds was taken into consideration. Test items that are cytotoxic already below 5 mg/plate are tested up to a cytotoxic concentration.  

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

In the Initial and Confirmatory Mutation Tests, inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by absent or decreased revertant colony counts (a lot of them below the corresponding historical control data ranges) and/or affected background lawn development: absent, reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as the lowest concentration showing cytotoxicity.

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 fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Imidazolium compounds, 2-C4-8-alkyl-1-(2carboxyethyl)-4,5-dihydro-3-(hydroxyethyl), hydroxides, sodium salts 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 by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Imidazolium compounds, 2-C4-8-alkyl-1-(2-carboxyethyl)-4,5dihydro-3-(hydroxyethyl), hydroxides, sodium salts (CAS Nr. 70983-43-6) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.