1-Ethyl-3-methylimidazolium Diethyl Phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registration substance showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 97a, TA 102, and TA 1535. Test concentrations up to the limit concentration of 5 µL/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals-health effects (2nd edition): 471, 472 Bacterial Reverse Mutation Test (China environmental press)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot No.: 187-9-113-Y
Purity: >/=98%
light yellow liquid

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : from pretreated rat liver; purchased from Beijing Hongjie Technology Co. Ltd., China
- method of preparation of S9 mix: no data; purchase
- concentration or volume of S9 mix and S9 in the final culture medium : 8 mmol/L
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
histidine and biotin requirement test, ant-tetracycline test, ampicilin resistance test, crystal violet/UV sensitivity test, spontaneous back mutation test

Test concentrations with justification for top dose:

Experiment I:
0.05, 0.158, 0.5, 1.58 and 5 µL/plate
Experiment II:
0.008, 0.04, 0.2, 1 and 5 µL/plate
According to the pre test there was no cytotoxicty up to 5 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
recommended by guideline
- Justification for percentage of solvent in the final culture medium:
recommended by guideline

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: acridine orange, 2-aminofluorene, 1,8-dihydroxy-anthraquinone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 (e)8 / plate
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: n.a.
- Exposure duration/duration of treatment: 48 h
- Harvest time after the end of treatment (sampling/recovery times): n.a.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

Conditions were chosen based on the guideline and results of the pre test.

Evaluation criteria:

The test substance is considered to be mutagenic, if
- a dose-related increase occurs in revertant count with or without metabolic activation in one or more strains or
- the number of revertant colonies in one or more dose groups shows a reproducible increase
The test substance is considered to be not mutagenic, if no increase in the revertant number is observed.
Significant positive results do not require further validation. Negative or inconclusive results require a change in the test conditions (changed to 5 times dose spacing) in the experimental repetition.

Statistics:

not mandatory

Species / strain:

S. typhimurium TA 97

Remarks:

TA 97a

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Conclusions:

Based on the result of the present Ames Test the test substance is considered to be not mutagenic.

Executive summary:

The test item was investigated for its mutagenic potential in Salmonella th. according to the Ames method. The present study is conducted under GLP and the method applied equivalent to OECD 471.

The test substance was evaluated for mutagenicity in the bacterial reverse mutation test using the plate incorporation method with and without metabolic activation in Salmonella th. strain TA 97a, TA 98, TA 100, TA 102 and TA 1535.

In total 5 concentrations were selected (0.05, 0.158, 0.5, 1.58 and 5 µL/plate) and vehicle as well as a positive controls were run in parallel. In the absence and presence of S9 mix three plates were used for each treatment. Zhe plates were incubated for 48 h at 37°C after the substance was added. Colonies were counted and the mutagenic response evaluated. When the negative results were obtained, a second experiment with changed test condtions (5 times dose spacing; 0.008 to 5 µL/plate) was performed.

The results showed that under the present test conditons all the standards for trsting validity were satisfied. In this experiment, with and without metabolic activation no biologically relevant increase in the mutant count of TA97a, TA 98, TA 100, TA 102 and TA 1535 were reported.

Therefore the test substance is considered to be not mutagenic in the Ames test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans. 

Justification for classification or non-classification

The registration substance does not have to be not classified for mutagenicity since it did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation.