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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-14 - 2011-04-20
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted on Sep 21, 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on Aug 1998
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): 1-Ethyl-3-methylimidazolium ethylsulphate (Lab test substance number: 06/0104-2)
- Physical state: liquid/ yellowish clear
- Analytical purity: 98.6 g/100 g
- Impurities (identity and concentrations): 1-methylimidazol (0.36 g/100 g), sulfate (< 0.01 g/100 g), water (0.04 g/100 g)
- Lot/batch No.: 100003p040
- 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; protect against humidity; protect against light
- Other: the test substance was homogeneous (visually)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han) from Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 35 ± 1 days when supplied, 42 ± 1 days at the start of the administration period
- Housing: 5 animals per cage in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Motor activity measurements were conducted in polycarbonate cages (floor area about 800 cm2) supplied by TECNIPLAST, Hohenpeißenberg, Germany
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water from water bottles; ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS (fully air-conditioned rooms in which central air conditioning)
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released manually. The test-substance preparations were produced at least once a week.

VEHICLE
- Concentration in vehicle: 1.0, 5.0 and 10.0 g/100 ml, respectively in the 100, 500 and 1000 mg/kg bw dose groups.
- Amount of vehicle (if gavage): 10 mL/kg bw

The means of the nominal concentrations of the samples taken at the beginning of the study were in a range of 100.8-103.7% of the nominal concentrations. The means of the nominal concentrations of the samples from test substance preparations prepared at the end of the study were in a range of 100.9-102.6% of the nominal concentrations. These results demonstrated the correctness of the concentrations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability of the test substance in drinking water at room temperature for a period of 7 days was demonstrated analytically before the start of the administration period;
- Concentration control analyses (HPLC) of the test-substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.
Duration of treatment / exposure:
91 (male rats) and 92 days (female rats)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 500 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the test substance properties, the following dose levels were selected for the present study: 1000 mg/kg bw/day as highest dose, 500 mg/kg bw/day as mid dose, and 100 mg/kg bw/day as low dose.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check for moribund and dead rats was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If rats were in a moribund state, they were sacrificed and necropsied. All rats were checked daily before and within 2 hours after the administration for any clinically abnormal signs. Abnormalities and changes were documented for each rat.
- Cage side observations included: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/ arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period, on study day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.

FOOD CONSUMPTION
- Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per rat and day.

WATER CONSUMPTION
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior and at the end of the administration period
- Dose groups that were examined: all prior to the administration period. At the end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 92 and 93 (start of administration period: day 0)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time (Hepato Quick’s test; HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 92 and 93 (start of administration period: day 0)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), sodium (NA), potassium (K), Chloride (Cl), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), magnesium (Mg).

URINALYSIS: Yes
- Time schedule for collection of urine: day 90
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color (turbidity), volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: functional observation battery (FOB; including home cage observation, open field observations and sensory motor tests reflexes) and motor activity assessment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; the animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The following weights were determined in all animals sacrificed on schedule: anesthetized animals, adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid glands, uterus with cervix

HISTOPATHOLOGY: Yes; the following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, Harderian glands, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina. The eyes with optic nerve of animals that have died or were sacrificed intercurrently were fixed in 4% buffered formaldehyde solution. Fixation was followed by histotechnical processing and examination by light microscopy.
Statistics:
- Clinical observations: body weight and body weight change were analyzed by a comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means. Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test and motor activity were analyzed by non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
- Clinical pathology: clinical pathology parameters, urine volume and urine specific gravity were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. Urinalysis, except color, turbidity, volume and specific gravity were analyzed by pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions
- Pathology: weight parameters were analyzed by Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- No rat died prematurely in the present study.
- Slight and moderate salivation after treatment was seen in 8 males (from study day 22 onwards) and 8 females (from study day 20 onwards) of test group 3 (1000 mg/kg bw/d). Likewise slight and moderate salivation after treatment was seen in 7 male rats (from study day 53 onwards) and 7 female rats (from study day 31 onwards) of test group 2 (500 mg/kg bw/d).
One female rat (of the high dose group) showed alopecia in the neck region from study day 56 onwards what was possibly caused by fixing the rat for administration. Thus, the finding was assessed as being spontaneous and not test substance-related.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes of body weight and body weight change were observed in any test group. Body weight change was significantly decreased in male animals of test group 1 (100 mg/kg bw/d) on study day 21 (-8%) but was assessed as being incidental and not related to treatment.

FOOD CONSUMPTION
No test substance-related effects on food consumption were obtained. The food consumption values observed for male rats of test groups 1-3 (100, 500 and 1000 mg/kg bw/d) between study day 90 and 91 were lower because of a very high mean value in the controls which was caused by food spilling in cage 1 (5 male animals) while the value of control cage 2 (5 male animals) was within the normal range.

WATER CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related findings.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalyses parameters were observed.
In the urine sediment of female rats of test group 1 (100 mg/kg bw/d) a higher incidence of crystals with unknown origin (tyrosin like crystals) was found. These crystals were not found dose-dependently and they were not accompanied by any other finding among the urine or blood parameters. Therefore, this finding was regarded as incidental and not treatment-related.

NEUROBEHAVIOUR
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
- Rearing was decreased by -55% in male animals of test group 3 (1000 mg/kg bw/d) and by -47% in males of test group 2 (500 mg/kg bw/d). However, both parameters were within the historical control range.
- Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female rats. Overall motor activity of male animals in test groups 2 and 3 (500 and 1000 mg/kg bw/d) as well as single interval 2 of male animals of test group 3 (1000 mg/kg bw/d) were decreased. All changes were assessed as being incidental as overall motor activity values were within the historical control range.

ORGAN WEIGHTS
- Absolute organ weights: when compared with the control group 0 (set to 100%), the following mean absolute weights were significantly increased in female animals: (1) heart (104% in the 100 mg/kg bw dose group, 97% in the 500 mg/kg bw dose group, and 106% in the 1000 mg/kg bw dose group) and ovaries (112% in the 100 mg/kg bw dose group, 100% in the 500 mg/kg bw dose group, and 116% in the 1000 mg/kg bw dose group). However, these findings were regarded as incidental as they did not show a clear dose response or histopathological correlates. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights: when compared with the control group 0 (set to 100%), relative liver weights (to body weight) was significantly increased in male animals (101% in the 100 mg/kg bw dose group, 104% in the 500 mg/kg bw dose group, and 112% in the 1000 mg/kg bw dose group). However, the liver weight change in male animals in test group 3 (1000 mg/kg bw/d) was regarded as treatment-related although a histopathological correlate could not be detected. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTORICAL CONTROL DATA (if applicable)
Historical control data were available and were used for the evaluation of the biological significance of the observed changes.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related, adverse findings were observed at clinical examinations, clinical pathology and pathology up to a dose level of 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion