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Toxicological information

Genetic toxicity: in vivo

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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:
20. Jun. 2006 - 17. Aug. 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
460-100-9
EC Name:
-
Cas Number:
342573-75-5
Molecular formula:
Hill formula: C8H16N2O4S CAS formula: C6H11N2.C2H5O4S
IUPAC Name:
3-ethyl-1-methyl-1H-imidazol-3-ium ethyl sulfate

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system : NMRI BR mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EEC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany. In the micronucleus main test 5 male and 5 female mice were treated per sampling time in each treatment group. Young adult animals were selected (6-8 weeks old). The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mice were identified by a unique number on the tail written with a marker pen. The animals were allocated to treatment groups as they came to hand from the delivery boxes. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
Animal husbandry:
Conditions : The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of 21 ± 3°C (actual range 19.2 - 22.5°C) and a relative humidity of 30 - 70% (actual range 39 - 86%). Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the maximum level for humidity (with max. 16%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.
Accommodation : The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages (type Mil height: 14 cm) containing Woody Clean bedding (Woody-Clean type 3/4; TechnilabBMI BV, Someren, The Netherlands). Paper bedding was provided as nest material (TechniLabBMI BV). Certificates of analysis of bedding and paper were examined and then retained in the NOTOX archives. The acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet : The animals had free access to standard pelletedlaboratory animal diet (SM R/M .. Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany). Each batch was analysed for nutrients and contaminants on Rregular basis. Certificates of analysis were examined and then retained in the NOTOX archives.
Water : The animals had free access to tap-water. Certificates of analysis (performea quarterly) were examined and then retained in the NOTOX archives.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate´and a low dose of 1-Ethyl-3 Methyllmidazolium Ethylsulfate . The route of administration was chosen to maximize the chance of the test article reaching the target tissue.
The dosing volume was 1 0 ml/kg b.w ..
Duration of treatment / exposure:
24 - 48 h
Frequency of treatment:
once
Doses / concentrations
Remarks:
Doses / Concentrations:
95, 190, 375 mg/kg b.w.
Basis:
other: intraperitoneal injection
No. of animals per sex per dose:
5
Control animals:
yes

Examinations

Tissues and cell types examined:
polychromatic erythrocytes of bone marrow smears
Details of tissue and slide preparation:
Bone marrow of the groups treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate was sampled 24 or 48 hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen). The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether (Merck, Darmstadt, Germany) and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight.
Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMAtek slide stainer (Miles, Bayer Nederland BV). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in MicroMount (Klinipath) and mounted with a coverslip.
Evaluation criteria:
Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, onesided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated
polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Negative controls validity:
valid
Additional information on results:
Based on the results of the dose range finding study dose levels of 375, 190 and 95 mg/kg b.w. were selected as appropriate doses for the micronucleus main test. Five male and five female animals were used in each treatment group. Five additional male and female animals, treated with 375 mg 1-Ethyl-3 Methyl Imidazolium Ethylsulfate /kg b.w., were used to correct for possible deaths.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of 1-Ethyl-3 Methyl Imidazolium Ethylsulfate treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical negative control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met.

Any other information on results incl. tables

 

Group

Treatment

Dose [mg/kgb.w.]

Sampling time [hours]

Number of micronucleated polychromatic erythrocytes per 2000 [mean±S.D.]

Ratio polychromatic/

normochromatic

erythrocytes

(mean ± S.D.)

Males

A

Solvent control

0

24

0.6±0.9

0.98±0.03

B

TEGO

375

24

0.8±0.8

0.94±0.05

C

TEGO

375

48

1.2±0.4

0.92±0.06

D

TEGO

190

24

0.6±1.3

0.95±0.03

E

TEGO

95

24

0.4±0.5

0.94±0.02

F

CP

50

48

25.6±5.5

0.52±0.10

Females

A

Solvent control

0

24

1.4±1.5

0.95±0.03

B

TEGO

375

24

1.6±0.5

0.97±0.05

C

TEGO

375

48

0.8±0.8

0.97±0.02

D

TEGO

190

24

1.2±0.8

0.95±0.05

E

TEGO

95

24

0.8±0.8

0.93±0.05

F

CP

50

48

24.6±3.3

0.48±0.12

Solvent control = physiological saline

TEGO = 1-Ethyl-3 Methyl lmidazolium Ethylsulfate (TEGO IL IMES (PO-2006-63))

CP=Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that this test is valid and that 1-Ethyl-3 Methyl Imidazolium Ethylsulfate is not clastogenic in the micronucleus test under the experimental conditions described in this report.
Executive summary:

Micronucleus test in bone marrow cells of the mouse with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate.

1-Ethyl-3 Methyl Imidazolium Ethylsulfate was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines. Batch JA061 07023 of 1-Ethyl-3 Methyl Imidazolium Ethylsulfate was a light yellow liquid with a purity of ± 99.5%. The test substance was dissolved in physiological saline. Five male and five female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight (b.w.) of cyclophosphamide (CP), respectively. Animals were dosed with 1-Ethyl-3 Methyllmidazolium Ethylsulfate (TEGO IL IMES (PO-2006-63)) at 375 (two groups), 190 (one group), and 95 (one group) mg/kg b.w.. After dosing the animals of the dose level of 375 mg/kg b.w. showed the following toxic signs: quick breathing, convulsions, tremors, eyes closed, ataxia, hunched posture and rough coat. The animals of the dose levels of 190 and 95 mg/kg b.w. showed no abnormalities after dosing except for two male animals of the group treated with 190 mg/kg b.w. which were breathing quickly. Bone marrow of the groups treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate (TEGO IL IMES (PO-2006-63)) was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical negative control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met. The groups that were treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the negative controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the negative controls, demonstrating toxic effects on erythropoiesis. It is concluded that 1-Ethyl-3 Methyl Imidazolium Ethylsulfate is not clastogenic in the micronucleus test under the experimental conditions described in this report.