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Administrative data

Description of key information

The NOAEL for general, systemic toxicity of the test substance is 90 mg/kg bw/day, derived from an GLP OECD TG study in rats (subchronic, gavage). Due to severe effects in the Range Finding Studies, this was the highest possible dose to be tested. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2015 - ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on Sep 21, 1998
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on Aug 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han) from Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 33 ± 1 days when supplied, 42 ± 1 days at the start 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) 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: 9 days

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
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: 0.1, 0.3, and 0.9 g/100 ml, respectively in the 10, 30 and 90 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 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
Dose / conc.:
90 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a range finding study the planned repeated administration of 1-Methylimidazol over 14 days via gavage at a dose level of 125 mg/kg bw/d caused a significant body weight loss within three days (project no. 01R0492/11R088). The administration had been stopped to avoid the inevitable moribund state of the animals by continuing the administration. In a second range finding study the test substance was repeatedly administrated over 14 days at a dose level of 90 mg/kg bw/d (project no. 01R0492/11R136). At this dose level a transient slight body weight loss accompanied with piloerection was observed in all animals within the first three days of administration. In the OECD422 study (project no. 85R0492/11R130) 1-methylimidazole was administered daily to groups of 10 male and 10 female Wistar rats (F0) by gavage at doses of 10, 30 and 90 mg/kg bw/d. The food consumption of the high dose males was statistically significantly compared to the control at study day 7 (-11%). However, no other severe effects on food consumption or body weight/body weight change were observed. The test substance 1-methylimidazole is classified as Skin Corr. 1B;H314: Causes severe skin burns and eye damage according to EC/1272/2008; with a pH = 11.3 of an aqueous solution (100 g/L). The males were probably more susceptible to its corrosive property, because they received higher amounts of the basic aqueous solution, as these were calculated based on the body weight (10 mL/kg bw). In both range finding studies and the OECD422 study, the males were about 50 % heavier than the females. This effect seems to be crucial especially during the first days of the administration period.
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
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: 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 (90 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), 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 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. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their deaths 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 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 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 after hematoxylin and eosin (H&E) stain.
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.
Details on results:
CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study.
Salivation within 2 hours after application was observed in 8 males and 3 females of the HD group from study day 34 onwards, and plough nose-first into bedding in all males and 9 females of the HD group; both probably induced by local affection of the upper digestive tract after gavaging the corrosive test substance. No clinical findings were observed in males and females of the LD and MD groups.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes of mean body weights and mean body weight change values in both sexes were observed.
In males of the HD group, body weight was statistically significant decreased on study day 7 (-4.2%). Furthermore, body weight change value was significantly decreased on study day 7 (-22.4%).

FOOD CONSUMPTION
No test substance-related findings were observed.

WATER CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
No test substance-related findings were observed.

HAEMATOLOGY
No treatment-related, adverse changes among hematological parameters were observed.
In HD males relative reticulocyte counts were higher compared to controls. The values were slightly above the historical control range (reticulocytes 1.5-1.9 %). However, this increase reflects the capacity of the bone marrow to produce red blood cells and therefore, it was regarded as adaptive rather than adverse effect. Other measured red blood cell parameters were not changed in these individuals.

CLINICAL CHEMISTRY
No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period in HD males chloride and sodium levels were decreased and urea values were increased. However, sodium and chloride values were within historical control ranges (chloride 98.8-105.9 mmol/L, sodium 141.1-146.7 mmol/L) and therefore these alterations were regarded as incidental and not treatment-related. Urea levels were marginally above the historical control range (urea 4.38-5.88 mmol/L), but this was the only relevantly changed clinical chemistry parameter in these animals. Therefore, this change was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
In HD females chloride levels were decreased. The values were marginally below the historical control range (chloride 98.4-105.1 mmol/L). This was the only changed clinical chemistry parameter in these individuals. Therefore, the alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).

URINALYSIS
No treatment-related, adverse changes among urinalysis parameters were observed.
In HD males, urine volume was decreased and more transitional epithelial cells were found in the urine sediment. Lower urine volume per se was regarded as maybe treatment-related, but not adverse effect. The amount of transitional epithelial cells can also found in the urine sediment of control animals and therefore this finding was regarded as not treatment-related.

NEUROBEHAVIOUR
- Functional Observation Battery (Home cage observations, Open field observations, Sensorimotor tests/ reflexes, Quantitative parameters: Rearing/grip strength fore/hind limbs/landing foot-splay test): No test substance-related effects were observed.
- Motoractivity measurement: No test substance-related effects were observed.

ORGAN WEIGHTS
- Absolute organ weights: The statistically significant decrease in absolute adrenal glands (HD: 84%), ovaries (MD: 83%, HD: 86%) and spleen (MD: 88%, HD: 84%) in females was regarded to be unrelated to treatment. It was still within the historical control values and there was no histopathologic finding that could explain the weight decrease. All other mean absolute weight parameters did not show significant differences when compared to the control group.
- Relative organ weights: The statistically significant increase of relative liver weight in males and females was regarded to be treatment related: MD: 105%, HD: 113% (males); HD: 110% (females). The increase in relative heart weight of males of test group 1 (10 mg/kg bw/d) was thought to be incidental due to a missing dose-response relationship and no histopathologic findings in the heart of HD animals. All other mean relative weight parameters did not show significant differences when compared to the control group.

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:
In the liver, there was minimal to slight centrilobular hypertrophy in males and females of all test groups observed. LD, MD, HD: 5/10, 8/10, 10/10 in males; 0/10, 2/10, 6/10 in females. This finding was regarded to be treatment-related. As there were no correlating findings in clinical chemistry, these findings were regarded as non-adverse.
All other 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.
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse findings up to highest dose tested.
Critical effects observed:
not specified
Conclusions:
The administration of 1-Methylimidazol by gavage to male and female Wistar rats for 3 months caused no signs of systemic toxicity up to the highest dose level tested (90 mg/kg bw/d). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 90 mg/kg bw/d for male and female Wistar rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
90 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Sufficient due to GLP OECD TG studies.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a GLP OECD408 study, 1-Methylimidazol was administered by gavage to 10 male and female Wistar rats per dose levels of 0, 10, 30 and 90 mg/kg bw/d over a period of 3 months (2016).

Food consumption and body weight were determined weekly. The animals were examined at least daily for signs of toxicity or mortality and for any clinically abnormal signs before and within 2 h as well as within 5 h after treatment. Detailed clinical examinations were conducted prior to the start of the administration period and weekly thereafter. Further examinations were ophthalmology (before staring and at the end of the administration period), functional observational battery (FOB), measurement of motor activity (MA), clinicochemical and hematological examinations as well as urinalyses at the and of administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The stability of the test-substance preparations and their correct concentrations were confirmed. No test-substance-related, adverse findings were noted up to the highest dose group of 90 mg/kg bw/d. There were some treatment-related findings, considered to be non-adverse, but rather adaptive: In HD males relative reticulocyte counts and urea values were higher compared to controls and slightly above the historical control range. Furthermore, the urine volume was decreased, which was regarded as maybe treatment-related. In HD females chloride levels were decreased and the values were marginally below the historical control range.

A statistically significant increase of relative liver weights was observed in males and females of the mid and/or high dose group. Histopathology revealed a dose-dependent, minimal to slight centrilobular liver hypertrophy in males and females of all test groups. As there were no correlating findings in clinical chemistry, these findings were regarded as non-adverse.

The administration of 1-Methylimidazol by gavage to male and female Wistar rats for 3 months caused no signs of systemic toxicity up to the highest dose level tested (90 mg/kg bw/d). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 90 mg/kg bw/d for male and female Wistar rats.

Supportingly, in a GLP compliant combined oral repeated dose toxicity study and reproduction/developmental toxicity screening test according to OECD 422, 10 male and female Wistar rats (F0 animals) were gavaged with doses of 0, 10, 30 and 90 mg/kg bw/d in drinking water (2013). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as approximately 2 weeks of the lactation period.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Regarding clinical pathology, slight dysregulation in the liver cell metabolism of male and female rats of the HD group (90 mg/kg bw/d) could be assumed because of higher urea levels in both sexes, indicating an increased protein metabolism, as well as higher cholesterol levels in males. In addition, in HD males chloride levels were low whereas inorganic phosphate levels were high. In these animals, a slight functional effect on the kidneys cannot be excluded; this was confirmed by higher incidences of transitional epithelial cells as well as phosphate crystals in the urine. No test substance related findings were noted in any of the other parameters investigated in this study (mortality / viability, clinical signs, functional observations other than for locomotor activity, body weight, food consumption, haematology, macroscopy, organ weights and histopathology). All other findings recorded were considered to be incidental in nature and unrelated to treatment.

Thus, under the conditions of this screening test the NOAEL for general, systemic toxicity of the test substance was 30 mg/kg bw/d for the F0 parental animals based on the increased urea levels in both sexes at 90 mg/kg bw/d. Additionally, males exhibited signs of increased serum inorganic phosphate and cholesterol levels, as well as decreased serum chloride concentration and a higher incidence of both transitional epithelial cells and phosphate crystals in the urine at this dose.

All measures of reproductive performance or fertility were normal in both genders and at all doses. Therefore, no alterations to reproductive performance or fertility were identified in the parental rats. No effects on pup body weights or pathology at necropsy were observed; thus no substance-dependent developmental toxicity was identified at any dose.

The NOAEL for reproductive performance and fertility in the F0 parental rats is 90 mg/kg body weight/day, the highest dose tested. The NOAEL for developmental toxicity was 90 mg/kg body weight/day, the highest dose tested.

Prior to the OECD422 study two range findings studies were performed. In the first range finding study the planned repeated administration of 1-Methylimidazol over 14 days via gavage at a dose level of 125 mg/kg bw/d caused a significant body weight loss within three days. The administration had been stopped to avoid the inevitable moribund state of the animals by continuing the administration. In a second range finding study the test substance was repeatedly administrated over 14 days at a dose level of 90 mg/kg bw/d. At this dose level a transient slight body weight loss accompanied with piloerection was observed in all animals within the first three days of administration.

Justification for classification or non-classification

Based on the NOAEL of 90 mg/kg bw/day in a subchronic OECD TG study in rats, the test substance does not need to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.