3-methyl-1-vinyl-1H-imidazolium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results indicated, that the test substance (59.3% analytical purity) was not mutagen in the bacterial reverse mutation assay (Ames test). In additon, the test article analogue (approx. 100% act. ingr.) 3-methyl-1-vinyl-1 H-imidazolium methyl sulfate is considered to be not mutagen in the HPRT test and not clastogen in the Chromosome Aberration test.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-05-10 and 2000-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-conform study in accordance to OECD guideline and EU method.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

S9-mix from rat liver induced by Aroclor 1254

Test concentrations with justification for top dose:

0; 34; 170; 850 ; 4250 and 8500 µg/plate

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was selected as the vehicle .

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9: 2-aminoanthracene (TA1535, TA100, TA1537, TA98); without S9: N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, TA100), 4-nitro-o-phenylendiamine (TA98), 9-aminoacridine (TA1537), 4-nitroquinoline-N-oxide (E. coli WP2 uvrA)

Details on test system and experimental conditions:

A) METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3 plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

B) METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3 plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The substance is considered to be positive if the following criteria are met:
- dose-related and reproducible increase in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

The test substance is generally considered nonmutagenic if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic was occasionally observed depending on the strain and test conditions from about 4250 - 8500 µg/plate .

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Remarks:

A weak bacteriotoxic was occasionally observed depending on the test conditions from about 4250 - 8500 µg/plate .

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found.


RANGE-FINDING/SCREENING STUDIES: no

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A weak bacteriotoxic (slight decrease in the number of his+ or trp+ revertants and/or slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 4250 - 8500 µg/plate .

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The following tables show the revertants per plate (mean from three plates).

Table 1: Without S9 mix

Dose/plate	TA 1535		TA 100		TA1537		TA98		E .coli WP2 uvrA
	SPT	PIT	SPT	PIT	SPT	PIT	SPT	PIT	SPT	PIT
Solvent control (water)	18	17	109	113	9	9	30	25	35	27
34 µg	16	16	103	105	10	9	26	27	32	26
170 µg	16	14	107	97	9	7	27	27	36	24
850 µg	12	14	83	95	9	9	23	21	37	24
4250 µg	15	12	60	93	8	7	24	19	29	29
8500 µg	10	7	68	80	7	6	20	14	31	24
Pos. contr.
MNNG (5 µg)	532	613	538	763
AAC (100 µg)					520	526
NOPD (10 µg)							827	597
4-NQO (5 µg)									737	496

Table 2: With S9 mix

Dose µg/plate	TA 1535		TA 100		TA1537		TA98		E .coli WP2 uvrA
	SPT	PIT	SPT	PIT	SPT	PIT	SPT	PIT	SPT	PIT
Solvent control (water)	17	17	117	117	9	12	35	35	37	36
34	18	17	131	104	10	14	30	35	38	36
170	20	17	121	108	11	12	23	33	39	31
850	21	17	113	103	10	11	22	33	35	28
4250	18	16	109	94	9	12	26	27	38	29
8500	19	11	98	85	8	8	22	29	40
Pos. contr.
2-AA (2.5 µg)	120	115	642	532	95	95	640	562
2-AA (60 µg)									210	208

SPT: Standard plate test

PIT: Preincubation test

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test

In a bacterial reverse mutation assay (Ames test), the test substance (59.3% purity) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of five bacterial strains (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). Two independent experiments were performed, a standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). 34 - 8500 µg/plate of the test substance were added. During the experiment, no precipitation of the test substance was found. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 4250 - 8500 µg/plate. The results indicated no increase in the number of his+ or trp+ revertants in the standard plate test or in the preincubation test either without S9 -mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

HPRT test

The study was performed to investigate the potential of the test substance analogue 3-Methyl-1-vinyl-1 H-imidazolium methyl sulfate (99.97% purity) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration in the pre-test was 2220 μg/mL equal to a molar concentration of about 10 mM. The test item was dissolved in deionised water. The concentration range of the main experiments was limited by cytotoxic effects of the test item. The highest tested concentrations of the main experiments was 1110.0 µg/mL (with S9 mix) and 555.0 µg/mL (without S9 mix). No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 3-Methyl-1-vinyl-1 H-imidazolium methyl sulfate is considered to be non-mutagenic in this HPRT assay.

Chromosome Aberration test

The test item analogue 3-Methyl-1-vinyl-1 H-imidazolium methyl sulfate (99.98% purity), dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in four independent experiments. The following study design was performed:

a) without S9 mix

- Experiment IA: 4 h (exposure period), 14 h (recovery), 18 h (preparation interval)

- Experiment IIA & IIB: 18 h (exposure period), 18 h (preparation interval)

- Experiment IIA: 28 h (exposure period) & 28 h (preparation interval)

b) with S9 mix

- Experiment IA & IB: 4 h (exposure period), 14 h (recovery), 18 h (preparation interval)

- Experiment IIA & IIB: 4 h (exposure period), 24 h (recovery), 28 h (preparation interval).

In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive controls in Experiment IIB after 18 hours continuous treatment and Experiment IIA after 28 hours continuous treatment without metabolic activation, where only 50 metaphases were evaluated.

A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item analogue. The highest applied concentration (2202.0 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight if the test item analogue.

In Experiment IA and IB in the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA after 28 hours continuous treatment in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration. After 18 hours continuous treatment without S9 mix and pulse treatment with S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, in the presence of S9 mix the cell number was markedly reduced below 60 % of control. In Experiment IIB after 18 hours continuous treatment without S9 mix it was not possible to evaluate concentrations in a cytotoxic range, however, the mitotic index was reduced below 60 % of control. In the presence of S9 mix only a moderate reduction in cell number could be observed, but these concentrations were not evaluable due to low metaphase number and quality. In Experiment IA one single increase in chromosomal aberrations was observed in the presence of S9 mix after treatment with 550.5 µg/mL (8.5 % aberrant cells, excluding gaps). The value was statistically significant and exceeded the range of the historical control data (0.0 - 4.0 % aberrant cells, excluding gaps). In the absence of S9 mix no clastogenicity was observed. In the confirmatory experiment IB with S9 mix one single value (3.5 % aberrant cells, excluding gaps) was statistically significant. This value is in the range of the historical solvent control data (0.0 -4.0 % aberrant cells, excluding gaps) and can therefore be regarded as biologically irrelevant. Thus, the positive finding of Experiment IA could not be confirmed. In Experiment IIA and IIB no clastogenicity was observed. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, test substance analogue is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or precipitating or the highest evaluable concentrations.

Justification for selection of genetic toxicity endpoint
GLP-conform study in accordance to OECD guideline and EU method.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be genotoxic under Regulation (EC) No 1272/2008,as amended for the sixth time in Directive EC 605/2014.