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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 2-Ethyl-4-Methyl-Imidazole
- Physical state: liquide, viscous/yellow to amber, clear
- Analytical purity: content: 97.9 g/100 g

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 32 ± 1 days (age when supplied)/ 42 ± 1 days (age at the beginning of the administration period)
- Housing: 5 animals per cage in H-Temp polysulfonate cages type 2000P supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Motor activity measurements were conducted in polycarbonate cages type III (floor area about 800 cm2). The cages and wire covers were supplied by TECNIPLAST, Hohenpeissenberg, Germany resp. by Ehret, Emmendingen, Germany.
- Diet (e.g. ad libitum): Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water (ad libitum)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test-substance preparations were produced at least weekly and stored at room temperature. The administration volume was 10 mL/kg body weight.

VEHICLE
- Concentration in vehicle: 0.25, 0.80 and 2.30 g/100ml, respectively int the 25, 80 and 230 mg/kg bw/d dose groups
- Amount of vehicle (if gavage):10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability of 2-Ethyl-4-Methyl-Imidazole in deionized water at room temperature for a period of 7 days was proven before the start of the administration period
- Concentration control analyses 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:
94 (male rats) and 95 days (female rats)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 80 and 230 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

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 and within 5 hours after the administration for any clinically abnormal signs. Abnormalities and changes were documented for each rat.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals in a standard arena (50 x 37.5 x 25 cm)
- DCO included (but were not limited to): 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.

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 (as representative value over 7 days) 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: The eyes of all animals were examined prior to the start of 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 (230 mg/kg bw/d) were examined for any changes using an ophthalmoscope

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 94 and 95 (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), prothrombin time (Hepato Quick’s test; HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 94 and 95 (start of administration period: day 0)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), Serum 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).

URINALYSIS: Yes
- Time schedule for collection of urine: day 93
- 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. One female (animal no. 76) of test group 3 was sacrificed in a moribund state. It was necropsied and assessed by gross pathology. The following organ 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 of the control and high dose animals 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 animal No. 76 were fixed in 4% neutral-buffered formaldehyde solution.
Statistics:
- Clinical observaton: body weight, 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. Rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity were analyzed by a 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.
- Clinical pathology: blood parameters were analyzed by a non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians. Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity were analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. Urine pH, volume and specific gravity were analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 hypothesis of equal medians.
- 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
Animal No. 76 (test group 3, 230 mg/kg bw/d) was sacrificed moribund on study day 89. In the macroscopical pathological assessment effects secondary to a gavage accident had been determined. Therefore, the moribund status of the animal No. 76 was not considered to be related to the test substance.
Animal No. 76 (test group 3, 230 mg/kg bw/d) showed general poor condition, moderate to severe, and a swelling in the axillary region.
Fur alopecia shoulder region was observed in 2 of 10 male (animal Nos. 32 and 33) and 2 of 10 female animals (animal Nos. 72 and 75) of test group 3 (230 mg/kg bw/d) as well as in 1 male animal (animal No. 29) of test group 2 (80 mg/kg bw/d). A reduce of the left testis was observed in animal 31 (test group 3, 230 mg/kg bw/d). These findings were considered as incidental, spontaneous in nature and not related to treatment.
Salivation after treatment from slight to moderate was observed in 9 of 10 males and all female animals of test group 3 (230 mg/kg bw/d) as well as in 8 of 10 male and 6 of 10 female animals of test group 2 (80 mg/kg bw/d). Plough nose first into bedding after treatment was observed in 6 of 10 male and in 9 of 10 female animals of test group 3 (230 mg/kg bw/d). From the temporary, short appearance immediately after treatment it was concluded that the findings of this paragraph were induced by a bad taste of the test substance or local affection of the upper digestive tract.
No adverse clinical findings were observed for male and female animals in test group 1 (25 mg/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN
Mean body weight of male animals in test group 3 (230 mg/kg bw/d) was significantly lower compared to the control group from day 56 onwards to the end of administration period with a maximal difference of -10.1 % on study day 84. The mean body weight of female animals in test group 3 (230 mg/kg bw/d) was slightly lower compared to the control group from study day 28 onwards. Thereby, a significant difference was observed only on study day 84 (-6.1%).
Mean body weight change values of male animals in test group 3 (230 mg/kg bw/d) were significantly lower from study day 49 onwards to the end of administration period with a maximal difference of -16 % on study day 84. The mean body weight change of female animals in test group 3 (230 mg/kg bw/d) was significantly lower only on study day 84 (-11%).

FOOD CONSUMPTION
No test substance-related findings were observed.

WATER CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings were observed.
All apparent findings were assessed as being incidental in nature since they occurred in the control group and did not show a dose-response relationship.

HAEMATOLOGY
At the end of the administration period in males of test group 3 (230 mg/kg bw/d) total white blood cell (WBC) counts (not statistically significantly) and absolute neutrophil counts were increased.
In males of test groups 1, 2 and 3 (25, 80 and 230 mg/kg bw/d) hemoglobin values were higher compared to controls and in males of test group 2 (80 mg/kg bw/d) platelet counts were increased. Both parameters were not dose-dependently changed. In males of test group 3 (230 mg/kg bw/d) relative reticulocyte counts were increased and in females of the same test group absolute large unstained cell (LUC) counts were higher compared to controls. However, both parameters were within historical control ranges (males relative reticulocytes 1.5-1.9 %; females absolute LUCs 0.01-0.02 Giga/L). Therefore, all in this paragraph mentioned alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
At the end of the administration period in rats of both sexes of test group 3 (230 mg/kg bw/d) urea (not statistically significantly in females), cholesterol and inorganic phosphate levels were increased and chloride values were decreased. Additionally, in females of the mentioned test group triglyceride levels were increased and albumin levels were decreased.
In male and female rats of test group 2 (80 mg/kg bw/d), cholesterol levels were already increased. In this test group this was the only relevantly altered clinical pathology parameter and therefore this change was regarded as treatment-related but non-adverse (ECETOC Technical Report No. 85, 2002).
In females of test groups 1, 2 and 3 (25, 80 and 230 mg/kg bw/d) aspartate aminotransferase (AST) activities were decreased and in males of test groups 1 and 2 (25 and 80 mg/kg bw/d) alanine aminotransferase (ALT) activities were increased. Both parameters were not dose dependently changed (for AST in females at least when medians were regarded) and the means were within historical control ranges (AST females 1.32-2.10 µkat/L; ALT males 0.53-0.87 µkat/L). In males of test group 3 total bilirubin levels were increased, in males of test groups 2 and 3 (80 and 230 mg/kg bw/d) potassium values were higher compared to controls and in males of test group 2 chloride levels were decreased. However, all these mentioned parameters were within historical control ranges (total bilirubin 0.56-2.76 µmol/L; potassium 4.39-5.00 mmol/L; chloride 98.8-105.9 mmol/L). Therefore, all mentioned alterations in this paragraph were regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
- Functional observational battery (FOB) (Home cage observations, open field observations and sensorimotor tests/reflexes): No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed. In test group 2 (80 mg/kg bw/d) and test group 3 (230 mg/kg bw/d) the values of food splay test in both sexes ranged from 10.9 to 11.5 cm and therefore decreased significant by -20.1 to -20.4% in males and by -14.3 to -14.5%. These findings reflect the normal range of biological variation inherent in the strain of rats used for this study. The respective values were within the range of the historical control data (study means 10.3 – 14.4 cm in males and 8.8 and 12.8 cm in females; PART III, Supplement). Therefore, this changes were regarded as incidental and not treatment-related.
- Motor activity measurement: Regarding the single intervals and overall motor activity, no test substance-related deviations to the control were noted for male and female animals of test groups 1-3 (25, 80 and 230 mg/kg bw/d).

ORGAN WEIGHTS
Absolute organ weights: when compared with the control group 0 (=100%), the following mean absolute weights were significantly increased or decreased in test group 3. Terminal body weight (HD: 89%) and spleen (HD: 79%) in male animals. Terminal body weight (HD: 95%), heart (HD: 91%) and Liver (HD: 111%) in female animals. 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 (=100%), the following mean relative organ weights were significantly increased or decreased in one test group. Kidneys (HD: 114%) and liver (HD: 113%) in male animals. Heart (MD: 91%) and liver (HD: 117%) in female animals.
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The terminal body weights were significantly decreased in males and females of test group 3 (230 mg/kg bw/day), resulting in decreased absolute spleen weights in test group 3 males and decreased absolute heart weights in test group 3 females. The decreased relative heart weight in test group 2 females was regarded as incidental as there was no dose-response or a histopathological correlate.
The increased relative liver and kidney weights in males of test group 3 were also related to the decreased terminal body weight (-11%) in this test group. The increased relative liver weight of test group 3 males (2.42%) was only slightly above historical controls (2.11-2.299%), whereas the absolute weight of test group 3 males (8.45 g) was within historical controls (7.611g – 8.493g) and the body weight (348.58 g) was below historical controls (352.840 - 386.520 g), therefore the increased relative weight was assumed to have been caused by the decreased terminal body weight. In addition, there were no histopathological correlates that explained all these weight changes. No findings correlated to the altered liver parameters in males of test group 3, which were observed in clinical chemistry. The increased absolute and relative liver weights in females of test group 3 were regarded to be treatment-related.

GROSS PATHOLOGY
All findings were single or few observations. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY
- Liver: Treatment-related findings were observed in the liver of females of test group 3. Minimal (7 animals) to slight (2 animals) centrilobular hypertrophy of hepatocytes was observed in 9/10 female animals of test group 3 (230 mg/kg bw/day) that correlated with increased liver weights. Mostly minimal alveolar histiocytosis in the lung occurred with higher incidence in test group 3 females. This incidence was the same as in male control animals; therefore, this finding was regarded as incidental in test group 3 female animals.All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.
- Decedent: Animal 76 (test group 3 female) showed a blood coagulum with admixed food material under the skin, which was considered secondary to a gavage accident.

Effect levels

Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Signs of systemic toxicity at a dose of 230 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The administration of 2-Ethyl-4-Methyl-Imidazole by gavage to male and female Wistar rats for 3 months caused signs of systemic toxicity at a dose level of 230 mg/kg bw/d.
Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 80 mg/kg bw/d for male and female Wistar rats.