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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011-12-07 and 2012-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-conform study according to OECD guideline, EU method and US EPA guideline; for read-across justification, please refer to section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1-vinyl-1H-imidazolium methyl sulphate

EC Number:

247-832-2

EC Name:

3-methyl-1-vinyl-1H-imidazolium methyl sulphate

Cas Number:

26591-72-0

Molecular formula:

C6H9N2.CH3O4S

IUPAC Name:

3-methyl-1-vinyl-2,3-dihydro-1H-imidazol-1-ium methyl sulfate

Test material form:

other: solid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM), supplemented with penicillin/streptomycin (100 U/mL/100 Rg/mL) and 10 % (v/v) fetal bovine serum (FBS)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

WITHOUT S9 mix:
Experiment IA: 550.5, 1101.0, 2202.0 µg/mL
Experiment IIA: 68.8, 137.5, 275.3 µg/mL
Experiment IIB: 350.0, 400.0, 450.0 µg/mL

WITH S9 mix:
Experiment IA: 275.3, 550.5, 1101.0, 2202.0 µg/mL
Experiment IB: 412.9, 481.7, 550.5, 688.2, 1101.0 µg/mL
Experiment IIA: 34.4, 68.8, 550.9, 1101.0 µg/mL
Experiment IIB: 275.0, 550.0, 825.0, 1375.0 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: water
- Justification for choice of vehicle/solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: EMS; ethylmethane sulfonate; with S9: CPA; cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h, 18 h and 28 h
- Expression time (cells in growth medium): After 4 hours treatment cells were cultured in complete medium containing 10% (v/v) FBS for 14 hours. In the case of 18 and 28 hours exposure the medium was not changed until preparation of the cells.

SPINDLE INHIBITOR: Colcemid (0.2 µg/mL)
STAIN: with Giemsa

NUMBER OF REPLICATIONS: four independent experiments (in duplicate)

NUMBER OF CELLS EVALUATED: 100 metaphases per culture were evaluated for structural chromosome aberrations, 100 metaphases per culture were evaluated fo 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.
DETERMINATION OF CYTOTOXICITY
- Method: cell growth, mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data and

- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory historical control data.

Statistics:

Fisher’s exact test (p < 0.05)

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

depending on the treatment

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none (based on pre-experiment)
- Effects of osmolality: none (based on pre-experiment)
- Precipitation: Precipitation was observed. For further infomration refer to Table 1 and Table 2 "Any other information on results".

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment IA and IB (with and without S9 mix) no cytoxicity was observed up to the highest applied concentration. In experiment IIA (without S9 mix) after 28 hours continuous treatment cytotoxicity was observed at the highest concentration. After 18 hours continuous treatment without S9 mix and pulse treatment with S9 mix concentrations showing clear cytotoxicity. 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the chromosome aberration study

Table 1: without S9 mix

Exp.	Preparation interval	Dose level [µg/mL]	Polyploid cells (%)	Endomitotic cells (%)	Cell numbers in % of control	Mitotic indices in % of control	Incl. gaps*	Aberrant cells in % excl. gaps*	With exchanges
Exposure period 4 hours without S9 mix
IA	18 hrs	Solvent control^1	3.7	0.0	100.0	100.0	1.5	1.5	0.5
		Positive control^2	n.d.	n.d.	n.d.	58.6	21.5	21.0 (s)	14.0
		550.5	2.5	0.0	107.4	98.7	0.5	0.0	0.0
		1101.0	2.9	0.0	93.0	97.8	2.5	2.5	0.5
		2202.0	3.1	0.0	87.9	93.9	4.0	4.0	0.5
Exposure period 18 hours without S9 mix
IIA	18 hrs	Solvent control^1	1.9	0.0	100.0	100.0	2.5	2.5	0.0
		Positive control^4	n.d.	n.d.	n.d.	83.3	28.5	28.5 (s)	8.5
		68.8	1.7	0.0	70.3	81.4	3.0	3.0	0.5
		137.6	2.3	0.0	93.3	76.5	2.0	2.0	0.0
		275.3	1.7	0.0	62.5	90.2	3.5	3.0	0.0
IIB	18 hrs	Solvent control^1	2.3	0.0	100.0	100.0	2.5	2.5	0.5
		Positive control^3#	n.d.	n.d.	n.d.	63.9	43.0	43.0 (s)	9.0
		350.0	3.3	0.0	79.9	91.6	3.5	3.5	0.5
		400.0	2.1	0.0	98.0	97.5	3.0	2.5	0.0
		450.0	2.7	0.0	93.1	57.4	1.5	1.5	0.0
Exposure period 28 hours without S9 mix
IIA	28 hrs	Solvent control^1	2.6	0.0	100.0	100.0	2.5	2.5	1.0
		Positive control^4#	n.d.	n.d.	n.d.	66.8	44.0	44.0 (s)	19.0
		68.8	2.3	0.0	119.6	105.0	2.5	1.5	0.0
		137.6	1.9	0.0	97.1	83.5	1.5	1.5	0.0
		275.3	1.7	0.0	52.4	107.5	2.5	2.0	0.0

* Including cells carrying exchanges

#Evaluation of 50 metaphases per culture

n.d. Not determined

(S)Aberration frequency statistically significant higher than corresponding control values

1 Deionised water 10.0 % (v/v)

2 EMS 1000.0 µg/mL

3 EMS 600.0 µg/mL

4 EMS 500.0 µg/mL

Table 2: with S9 mix

Exp.	Preparation interval	Dose level [µg/mL]	Polyploid cells (%)	Endomitotic cells (%)	Cell numbers in % of control	Mitotic indices in % of control	Incl. gaps*	Aberrant cells in % excl. gaps*	With exchanges
Exposure period 4 hours with S9 mix
IA	18 hrs	Solvent control^1	4.8	0.8	100.0	100.0	6.0	4.0	1.5
		Positive control^2	n.d.	n.d.	n.d.	67.0	14.0	12.5 (s)	3.0
		275.3	2.2	0.3	79.3	106.9	2.5	2.5	1.5
		550.5	3.5	0.3	80.9	98.6	8.5	8.5 (s)	1.5
		1101.0	3.7	0.3	77.4	96.4	3.0	2.0	0.0
		2202.0	3.7	0.3	120.5	102.2	2.5	2.5	0.5
IB	18 hrs	Solvent control^1	4.4	1.3	100.0	100.0	1.0	0.5	0.0
		Positive control^2	n.d.	n.d.	n.d.	90.6	10.0	10.0 (s)	3.5
		412.9	3.2	0.3	77.5	95.7	4.0	2.5	2.0
		481.7^P	3.8	0.5	121.4	103.7	3.5	3.5 (s)	2.0
		550.5^P	3.3	0.4	106.0	94.0	0.5	0.5	0.0
		688.2^P	2.4	0.7	86.3	100.3	1.5	0.5	0.0
		1101.0^P	3.2	0.2	77.8	89.0	2.0	1.0	0.0
Exposure period 4 hours with S9 mix
IIA	28 hrs	Solvent control^1	2.4	0.0	100.0	100.0	2.5	2.5	0.5
		Positive control^3	n.d.	n.d.	n.d.	106.5	18.5	18.5 (s)	6.5
		34.4	2.4	0.0	114.9	96.3	3.5	3.0	0.0
		68.8^P	2.6	0.0	76.9	107.8	3.5	3.5	0.5
		550.9^P	1.7	0.0	91.3	113.3	1.0	1.0	0.5
		1101.0^P	2.0	0.1	56.4	82.3	1.0	1.0	0.5
IIB	28 hrs	Solvent control^1	4.1	0.0	100.0	100.0	1.5	0.5	0.0
		Positive control^3	n.d.	n.d.	n.d.	91.9	10.0	10.0 (s)	2.0
		275.0	2.6	0.0	96.4	101.6	1.5	1.5	0.5
		550.0	1.3	0.0	78.6	100.0	1.5	1.5	0.0
		825.0^P	1.4	0.0	81.2	105.2	2.5	2.0	0.5
		1375.0^P	2.1	0.0	94.3	97.7	1.5	1.0	0.5

 

* Including cells carrying exchanges

P Precipitation occurred at the end of treatment

n.d. Not determined

(S) Aberration frequency statistically significant higher than corresponding control values

1 Deionised water 10.0 % (v/v)

2 CPA 1.4 Rg/mL

3 CPA 2.0 Rg/mL