Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

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:

From 28 May to 19 October 2018

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only the 4 highest dose levels (instead of 6mentioned in the study plan) could be tested with the TA 98 strain with S9 mix. This deviation was considered not to have compromised the validity or integrity of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

EC Number:

272-897-9

EC Name:

Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

Cas Number:

68919-40-4

Molecular formula:

R_C8 H14 N2 O4 Na With R=Cn H2n+1 n=8, 10, 12, 14, 16, 18

IUPAC Name:

disodium 3-({2-[N-(2-hydroxyethyl)hexadecanamido]ethyl}amino)propanoate 3-[(2-hexadecanamidoethyl)(2-hydroxyethyl)amino]propanoate

Test material form:

cream / paste

Specific details on test material used for the study:

- Appearance: Clear paste
- Storage condition: At room temperature
- Purity: 99.7%
- Correction factor: No correction factor

Method

Target gene:

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
. the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the
bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,. the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
. the plasmid pKM101 which increases chemical and spontaneous mutagenesis by enhancing error-prone DNA repair system was added to TA 98, TA 100 and TA 102 strains,
. in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

- source of S9: obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route
- method of preparation of S9 mix: induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function.
- Volume of S9 mix and S9 in the final culture medium: 0.5 mL

Test concentrations with justification for top dose:

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose levels to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.
The selected dose levels were:

1) Experiments without S9 mix:
- 20.58, 61.73, 185.2, 555.6, 1666.7 and 5000 µg/plate in the TA 102 strain in both mutagenicity experiments,
- 6.86, 20.58, 61.73, 185.2, 555.6 and 1666.7 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains in the first mutagenicity experiment,
- 2.29, 6.86, 20.58, 61.73, 185.2 and 555.6 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains in the second mutagenicity experiment.

2) Experiments with S9 mix
- 20.58, 61.73, 185.2, 555.6, 1666.7 and 5000 µg/plate in the TA 102 strain in both mutagenicity experiments,
- 6.86, 20.58, 61.73, 185.2, 555.6 and 1666.7 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains in both mutagenicity experiments.

Vehicle / solvent:

- Vehicle used: water for injections.
- Justification for choice of vehicle:According to available solubility data

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water for injections

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method. The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspensions (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 50°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The pre-incubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

DURATION
- Exposure duration: 48 to 72 hours of incubation at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of a decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
After 48 to 72 hours of incubation at 37°C, the number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK). Also, the thinning of the bacterial lawn and the presence of precipitate were evaluated.

- OTHER: Preliminary toxicity test: To assess the toxicity of the test item to the bacteria, six dose levels (one plate/dose level) were tested in the TA 100 and TA 102 strains with and without S9 mix as well as for the TA 98 strain without S9 mix, and four dose levels (one plate/dose level) were tested in the TA 98 strain with S9 mix.

Evaluation criteria:

The test item is considered to have shown mutagenic activity in this study if:
. a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
. and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
. neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observedat any of the tested dose levels,
. nor any evidence of a dose-response relationship is noted.

Results and discussion

Test results

Test results 1

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST :
The dose levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels ≥ 500 μg/plate in TA 98 strain with and without S9 mix, as well as in the TA 100 strain without S9 mix. A strong toxicity was also observed at dose levels ≥ 1000 μg/plate in the TA 100 strain with S9 mix and at 5000 μg/plate in the TA 102 strain without S9 mix.
The strong toxicity induced at dose levels ≥ 500 μg/plate in the TA 100 strain without S9 mix, at dose levels ≥ 1000 μg/plate in TA 98 without S9 mix and TA 100 with S9 mix and at dose levels ≥ 2500 μg/plate in TA 98 with S9 mix prevented the scoring of revertant colonies since non-revertant but surviving cells were observed (including microcolonies).
Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose levels to be used in the main experiments was based on the level of toxicity, according to the criteria specified in
the international guidelines.

CONTROLS: The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels for each strain and test condition. The study was therefore considered to be valid.

MUTAGENICITY EXPERIMENTS:
- Experiments without S9 mix:
A moderate to strong toxicity was noted at dose levels ≥ 185.2 µg/plate in the TA 1535, TA 1537 and TA 100 strains in both experiments and in the TA 98 strain in the second experiment, at dose levels ≥ 555.6 µg/plate in the TA 98 strain in the first experiment and at 5000 µg/plate in the TA 102 strain in both experiments. The strong toxicity induced at dose levels ≥ 555.6 µg/plate prevented the scoring of revertant colonies since non-revertant but surviving cells were observed (including microcolonies).

- Experiments with S9 mix:
A moderate to strong toxicity was noted at dose levels ≥ 555.6 µg/plate in the TA 1535, TA 1537 and TA 100 strains in both experiments and TA 98 strain in the second experiment, at dose levels ≥ 1666.7 µg/plate in the TA 98 strain in the first experiment, and at 5000 µg/plate in the TA 102 strain in the second experiment. The strong toxicity induced at dose levels ≥ 1666.7 µg/plate (excepted in TA 100 strain in the second experiment) prevented the scoring of revertant colonies (presence of microcolonies).

In both experiments, without and with S9 mix, the test item did not induce any noteworthy increase in the number of revertants, in any strains or experiments. These results met the criteria for a negative response.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test item Miranol C2M AA did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of metabolic activation.

Executive summary:

The potential of the test item, Miranol C2M AA, to induce reverse mutations in Salmonella typhimurium was investigated in bacterial reverse mutation test according to the OECD 471.

A preliminary toxicity test was performed to define the dose levels of Miranol C2M AA, dissolved in water for injections, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system.

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

Under the experimental conditions of this study, the test item Miranol C2M AA did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.