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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-04 - 2011-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on Jul 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

adopted on Aug 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus 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 (Lab test substance number: 06/0104-3)
- Physical state: liquid, yellowish clear
- Analytical purity: 98.65%
- Lot/batch No.: 100004P040
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed.
- Storage condition of test material: room temperature, under N2, protected against humidity
- Other: the test substance was homogeneous under the testing conditions on account of the high purity and was ensured by mixing before preparation of the test substance solutions.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:NMRI mice from Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: mean 28.5 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: single housing in Makrolon cages, type M II
- Diet: standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water: drinking water from bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS (fully air-conditioned rooms with central air conditioning)
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 50, 100 and 200 mg/ml (analytical values: 52.758, 102.077 and 194.327 mg/ml), respectively in the 500, 1000 and 2000 mg/kg bw test groups.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
- The determination of the concentrations in the vehicle was carried out by means of HPLC, and the homogeneity of the samples was confirmed indirectly based on this data.

Duration of treatment / exposure:

not applicable; the animals were treated once only

Frequency of treatment:

once

Post exposure period:

24 and 48 hours; the animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. in a range of 97% - 106% of the nominal concentration.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CCP) for clastogenicity and vincristine (VCR)
- Justification for choice of positive control(s): CCP for clastogenicity and VCR for spindle poison effects
- Route of administration: orally (CCP) and IP (VCR)
- Doses / concentrations: 20 (CCP) and 0.15 (VCR) mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow cells from the femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a pretest was conducted for dose selection

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
- After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
DETAILS OF SLIDE PREPARATION:
- The animals were anesthetized with isoflurane, sacrificed by cervical dislocation, and the two femora were then prepared by dissecting and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum.
- The suspension was mixed thoroughly, centrifuged, and the the precipitate was resuspended in fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.
- The slides were stained with eosin and methylene blue. After briefly rinsing in deionized water, the preparations were soaked in deionized and subsequently, stained with Giemsa solution.
- After rinsing twice in deionized water and clarifying in xylene, the preparations were then mounted in Corbit-Balsam for analysis.

METHOD OF ANALYSIS: In general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
- Number of polychromatic erythrocytes;
- Number of polychromatic erythrocytes containing micronuclei (for clastogenic or aneugenic effects);
- Number of normochromatic erythrocytes;
- Number of normochromatic erythrocytes containing micronuclei;
- Ratio of polychromatic to normochromatic erythrocytes (an alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects);
- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]; the size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).

Evaluation criteria:

- Acceptance criteria: the mouse micronucleus test is considered valid if (1) the quality of the slides allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs; (2) the ratio of PCEs/NCEs in the concurrent vehicle control animals is within the normal range for the animal strain selected; (3) the number of cells containing micronuclei in vehicle control animals is within the range of the historical vehicle control data both for PCEs and for NCEs; the two positive control substances induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.
- Assessment criteria: a finding is considered positive if (1) statistically significant and dose-related increase in the number of PCEs containing micronuclei is obtained; (2) the number of PCEs containing micronuclei exceeds both the concurrent vehicle control value and the range of the historical vehicle control data. A test substance is considered negative if the number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows: * p ≤ 0.05; ** p ≤ 0.01. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

The administration of the test substance (and of positive control substances) did not lead to any clinical signs of toxicity. A slight inhibition of erythropoiesis was detected at 2000 mg/kg bw at 24-hour sacrifice interval.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a pretest for the determination of the acute oral toxicity, the recommended highest dose of 2000 mg/kg bw was tolerated by all animals (male and female) without any signs of toxicity. Thus, only male animals were used for the cytogenetic investigations as requested by the current OECD Guideline. Therefore, a dose of 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg bw were administered as further doses.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
* The single oral administration of the vehicle deionized water led to 1.0‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.3‰ after the 48-hour sacrifice interval, respectively.
* The highest dose of 2000 mg/kg bw led to 0.7‰ polychromatic erythrocytes containing micronuclei after 24 hours and 1.0‰ after 48 hours.
* In the two lower dose groups, rates of micronuclei of 1.1‰ (1000 mg/kg group) and 0.7‰ (500 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.
* The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (18.9‰) in the number of polychromatic erythrocytes containing mainly small micronuclei, as expected.
* Vincristine sulfate, a spindle poison, also produced a statistically significant increase (31.7‰) in the number of polychromatic erythrocytes containing micronuclei, with a significant portion increase (11.7‰) attributable to large micronuclei.
* The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.

- Ratio of PCE/NCE (for Micronucleus assay) and appropriateness of dose levels and route: a slight change in the ratio of PCE/NCE was observed at 2000 mg/kg body weight at 24-hour preparation interval as indication for target organ toxicity. Therefore, it is shown that the test substance reached the blood / the bone marrow.

Any other information on results incl. tables

Table 1: Summary of the result – Induction of micronuclei in bone narrow cells

Test group	Sacrifice interval (hours)	Number of evaluated animals	Micronuclei in PCE		PCE 2000 erythrocytes#
			sum of small and large micronuclei (‰)	large micronuclei (‰)
VC (10 ml/kg bw)	24	5	1.0	0.0	1496
TS (500 mg/kg bw)	24	5	0.7	0.0	1322
TS (1000 mg/kg bw)	24	5	1.1	0.0	1327
TS (2000 mg/kg bw)	24	5	0.7	0.0	1284
CCP (20 mg/kg bw)	24	5	18.9**	2.3	1396
VCR (0.15 mg/kg bw)	24	5	31.7**	11.7*	1366
VC (10 ml/kg bw)	48	5	1.3	0.2	1393
TS (2000 mg/kg bw)	48	5	1.0	0.0	1314
PCE: polychromatic erythrocytes; VC: vehicle control; TS (test substance); CCP: cyclophosphamide; VCR: vincristine sulfate; *: p≤ 0.05; **: p≤ 0.01; #: calculated number of PCEs per 2000 erythrocytes (PCE + NCE) when scoring a sample of up to 10000 PCE per test group; NCE: normochromatic erythrocytes.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative