1H-Imidazolium, 3-ethyl-1-methyl-, ethyl sulfate (1:1)

Genetic toxicity: in vitro

Administrative data

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:

August 2010 - November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): 1-Ethyl-3-methylimidazolium ethylsulphate
- Physical state: liquid
- Analytical purity: Approx. 99 area-%
- Lot/batch No.: lot. 100003p040
- Stability under test conditions: guaranteed until 2012-02-28
- Storage condition of test material: Room temperature (dry storage)
- Other: appearance: yellowish, clear

Method

Target gene:

histidine (S. typhimurium) and tryptophane (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin)
- Properly maintained: yes
- Periodically checked for deep rough character (rfa); UV sensitivity (delta uvrB); ampicillin resistance (R factor plasmid). Histidine auxotrophy via the spontaneous rate in each experiment.

Additional strain / cell type characteristics:

other: histidine auxothroph ( his-)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan)
- Properly maintained: yes
- Periodically checked for UV sensitivity. Tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

Additional strain / cell type characteristics:

other: tryptophan auxotroph (trp-)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

- 1rst experiment (SPT = standard plate test: plate incorporation method according to Ames et al. 1975):
0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate; +/- S9 mix; 3 plates per dosis and control
- 2nd experiment (PIT = preincubation test described by Yahagi et al. 1977 + Matsushima et al. 1980):
0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate, +/- S9 mix, 3 plates per dosis and control

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility in water

Controls

Untreated negative controls:

other: sterility control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: for +/- S9 mix, for S. typhimurium + E. coli; details see below

Details on test system and experimental conditions:

DETAILS ON POSITIVE CONTROLS:
1) with S9 mix:
2-aminoanthracene (2-AA): 2.5 μg/plate for S. typhimurium, 60 μg/plate for E. coli
2) without S9 mix:
- N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): 5 μg/plate for TA 1535, TA 100;
- 4-nitro-o-phenylenediamine (NOPD): 10 μg/plate for strain TA 98
- 9-aminoacridine (AAC): 100 μg/plate for strain TA 1537
- 4-nitroquinoline-N-oxide (4-NQO): 5 μg/plate for E. coli

METHOD OF APPLICATION: in agar (plate incorporation) + preincubation

DURATION
- Preincubation period: 20 min (Experiment 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 - 72 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

MUTAGENICITY:
- mean number of revertant colonies per plate and standard deviations for all groups
- in general: 5 doses of the test substance with max. of 5 mg/plate
- triplicates

TITER: determination only in experiments with s9 mix for negative control and the 2 highest doses

TOXICITY: detection for all groups via decrease of number of revertants, clearing of diminution of the background lawn, reduction in the titer

SOLUBILITY: recording precipitation; if not interfering with colony scoring, max. dose (5 mg/plate) is generally selected and analyzed


ACCEPTANCE:
valid, if
- number of revertant colonies in negative control within range of historical negative control data for each tester strain
- no indication of bacterial contamination in sterility control
- positive control substances +/- S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or above
- titer of viable bacteria was ≥ 10exp8/mL

ASSESSMENT:
- mutagenic in this test, if 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 - or + S9 mix
- non-mutagenic in this test, if number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two independent experiments

Results and discussion

Test results

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:

no cytotoxicity

Remarks:

up to the max. dose (5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

other: sterility control: no contamination

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:
Results of all groups were within the range of the historical negative control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test and in the preincubation assay up to the highest required concentration.

Remarks on result:

other: other: all strains + E. coli WP2 uvrA

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

HISTORICAL NEGATIVE CONTROL DATA: revertants/plate (min.-max.)

Exp.	vehicle	S9 mix	TA1535	TA 100	TA 1537	TA 98	WP2 uvrA
SPT	water	-	10-48	76-144	4-16	17-53	24-60
		+	10-35	81-155	5-17	21-55	24-121
PIT	water	-	9-25	75-150	5-16	18-48	21-56
		+	9-25	80-151	5-16	17-49	23-70

RESULTS FOR TEST SUBSTANCE: mean of revertants/plate

		TA1535		TA 100		TA 1537		TA 98		WP2 uvrA
Exp.	Dosis (µg/plate)	-S9 mix	+ S9 mix	-S9 mix	+ S9 mix	-S9 mix	+ S9 mix	-S9 mix	+ S9 mix	-S9 mix	+ S9 mix
SIT	0	23	28	89	90	8	10	25	29	47	49
	33	23	25	85	87	7	7	22	26	45	45
	100	20	24	93	90	8	10	25	27	49	48
	333	21	27	85	92	7	10	22	27	49	48
	1000	23	33	79	91	7	9	23	25	53	48
	2500	22	26	100	90	7	8	21	30	38	55
	5000	23	22	86	83	9	10	21	26	53	55
PIT	0	30	30	103	114	9	10	34	40	49	46
	33	31	28	104	114	13	10	31	41	50	43
	100	36	36	100	120	9	13	37	40	48	50
	333	37	33	103	124	8	8	38	39	49	49
	1000	34	30	103	105	10	11	28	39	48	47
	2500	33	30	104	102	10	12	34	43	43	46
	5000	24	28	98	106	9	8	40	39	51	34

SPT: Standard Plate Test

PIT: Preincubation Test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions chosen, it is concluded that 1-Ethyl-3-methylimidazolium ethylsulphate is not a mutagenic test substance in the bacterial reverse mutation test (Ames test) in the absence and the presence of metabolic activation.

Executive summary:

In a GLP OECD471 study (BASF SE 2010, 40M0104/06M004), 1-Ethyl-3-methylimidazolium ethylsulphate was tested with S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA +/- S9 mix in 2 independent experiments (standard plate test and preincubation assay) with doses from 33 - 5000 µg/plate. No toxicity was observed in any of the groups. The results of the negative and the positive controls were valid. 1-Ethyl-3-methylimidazolium ethylsulphate did not lead to a relevant increase in the number of revertant colonies +/- S9 mix.

Thus, under the experimental conditions chosen, it is concluded that 1-Ethyl-3-methylimidazolium ethylsulphate is not a mutagenic test substance in the bacterial reverse mutation test (Ames test) in the absence and the presence of metabolic activation.