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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:
01/2022 - 04/2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
Dose range finding study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Dose range finder
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11/2021 - 03/2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: 28d dose range finding study to serve for the dose selection of the upcoming 90-day Repeated Dose Toxicity GLP study (OECD TG 408)
- Short description of test conditions: test substance was administered to rats via oral gavage once daily for 7 days per week
- Parameters analysed / observed: mortality, clinical obseration, body weights, food consumption, clinical pathology (haematology, coagulation, clinical biochemistry), macroscopic examination, organ weights
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: 94180397V0
- Purity: 98.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (15-25℃, ≤70% relative humidity)
- Stability during storage: The test item was considered stable when stored under appropriate storage conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was heated to 60°C for no longer than 3 hours per day until it reached liquid phase. Formulations were prepared in clean glass containers. The appropriate amount liquid test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation.

FORM AS APPLIED IN THE TEST
- solved in destilled water
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 6-7 weeks old
- Weight at study initiation: Males: 181-205 g, females: 140-158 g
- Housing: Rodents were group-housed, up to 3 animals of the same sex and dose group/cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
The food was routinely analysed and was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-23.6℃
- Humidity (%): 30-63%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16.11.2021 To: 14.12.2021
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the selected vehicle (distilled water), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. Formulations were prepared fresh every day prior to administration to animals. The formulations were stirred with magnetic stirrer from the preparation until completion of each treatment.

VEHICLE
- Concentration in vehicle: 30 or 40 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of control (vehicle) and test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of the Test Facility. Representative samples of control (vehicle) and test item formulations were analysed at once during the study (during the first day during the treatment period).
Analysis of the formulations for concentration and/or homogeneity of test item was performed using a developed chromatographic HPLC-UV (High Performance Liquid Chromatography) method in the Analytical Department of the Test Facility. Documentation regarding stability of formulations is included in the report. Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration. Acceptance criteria of the homogeneity was that the RSD (Relative Standard Deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.

Results:
All test item formulations were shown to be homogeneous. The measured concentrations of the test item varied between 101% and 102% of the nominal concentrations. The relative standard deviation (RSD) was below 10% in each case. No test item was detected in the control sample. These results were considered suitable for the study purposes.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first treatment (Day 1) to allow for within-subject comparisons; daily during the treatment period, at approximately 2 hours after dosing, approximately the same time but considering the peak period of anticipated effects after dosing, if any were observed during the first days of treatment. When signs of toxicity were observed, animals were observed more frequently. Detailed clinical observation was also made on the last day of the study (Day 29), in the morning hours (am).

BODY WEIGHT: Yes
- Time schedule for examinations: first day of treatment, then at least twice weekly, including on Day 28 (last treatment day) and prior to necropsy (scheduled necropsy, on Day 29 (fasted); or at death, for animal found dead)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY / CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluation were collected immediately prior to the scheduled necropsy.
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: 5 per sex per dose
- Parameters checked in table 1 were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened, and the appearance of the tissues and organs were observed macroscopically. The stomach was washed out and the inner surface observed. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
The following organs were trimmed of fat and weighed in the surviving animals: kidneys, liver, spleen, brain, adrenals. Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated. As significant difference in size was observed of testis and epididymis, the individual weights of these organs were recorded.
On completion of the macroscopic examination the following tissues and organs were retained from the surviving animals: adrenal gland, kidney, liver, spleen, stomach. The testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

HISTOPATHOLOGY: No
Statistics:
At the Test Facility, the statistical evaluation of data was performed, as appropriate using the statistical program package of SAS 9.2 (when using Provantis).
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of (co)variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences, identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test, identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size is <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used, identifying differences of <0.05, <0.01 or <0.001 as appropriate.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the surviving animals the following clinical signs were observed: decreased activity in all Low and High dose males and females, heightened startle response in 3/5 High dose males and in 1/5 Low dose and 1/4 High dose females, hunched back in 1/5 Low dose and 4/5 High dose males and in 3/5 Low dose and 4/4 High dose females, increased salivation in 1/5 High dose males, piloerection in 3/5 Low dose and 5/5 High dose males and in 4/5 Low dose and 4/4 High dose females, intermittent tremors in 1/4 High dose female, noisy respiration in 1/4 High dose female. These clinical signs were considered to be treatment related.
Other observed clinical signs (alopecia, crust, excessive scratching, thin fur, red-brown staining, abnormal mucous membrane) were not considered to be test item related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One High dose female animal was found dead on Day 2 of the study. In this animal, slightly decreased activity and piloerection were recorded on Day 1. The cause of death could not be determined based on the available data.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, statistically significant, test item related decrease in body weight in the High dose group (approx. -21% compared to controls) and in body weight gain in Low and High dose groups (approx. -27% and -64%, respectively, compared to controls) were seen.
In females, slightly decreased body weight, body weight gain were recorded in a dose dependent manner, however without statistical significance.
High dose males and females showed an initial loss of body weight, which started to reverse after 8-10 days.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, statistically significantly decreased food consumption was seen in Low and High dose groups (approx. -14% and -31%, respectively, compared to controls).
In females, slightly decreased food consumption was recorded in a dose dependent manner, however without statistical significance.
These changes were considered as test item related.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slight, statistically significant decrease in haemoglobin in Low and High dose males and females and in haematocrit in Low and High dose males were observed.
Platelet count increased in Low and High dose males and females, with statistical significance in High dose males.
In females, relative reticulocyte values increased statistically significantly both in Low and High dose groups.
Other changes in the measured haematology parameters are considered to be incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Creatinine increased statistically significantly in Low and High dose males only, which is considered to be a test item related effect on kidney.
Decrease in albumin and total protein and the changes in phosphorous, sodium, potassium and chloride may correlate with the kidney effect or decreased food consumption observed in the test item treated groups.
Increased bile acid and cholesterol values in Low and High dose groups of both sexes together with increased liver weights indicate a possible hepatic effect of the test item.
Other changes were not considered as test item related.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weights of High dose males were statistically significantly lower (-22.7%) compared to controls; at the Low dose males terminal body weights were below control (-9.5%). Terminal body weight reduction in High dose females were smaller (-9.4%) and did not reach statistical significance.
Treatment-related effects were observed of increased adrenal weights in High dose males (+45% in males, and 21% in females without statistical significance, when adjusted for body weight). This can indicate non-specific stress.
Treatment-related effects were observed in the kidney weights in Low and High dose males and females. In High dose males, absolute kidney weights were larger related to body weights by 45.1% and in the Low dose by 33.1%. In High dose females, absolute kidney weights were larger related to body weights by 24.6% in Low dose by 21.5%.
Treatment-related effects were observed in the liver weights in Low and High dose males and females. In High dose males, liver weight related to body weights were larger by 24.8% and in Low dose males by 17.4 %. In High dose females, liver weights related to body weights were larger by 47.5% and in Low dose females by 33.0%.
There were no statistically significant differences among groups in the weights of spleen when compared to Controls in either sex.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related changes were observed in the testes and epididymis. Bilateral soft testes were observed in 1/5 High dose males. Multifocal white discolouration of the left testes was observed in 1/5 High dose males. Small left testes and soft left testes was observed in 2/5 and 3/5 Low dose males respectively. In right testes, small size and soft texture was observed in 1/5 and 2/5 Low dose males respectively. Small left epididymis was observed in 2/5 and 1/5 Low and High dose males respectively. Right epididymis was small in 1/5 each in Low and High dose males respectively.
All other observed changes were considered incidental or a common background.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Conclusions:
In conclusion, under the conditions of this 28-day study, test item related changes with a dose response were seen in Low and High dose males and/or females; death of one female and adverse clinical signs in most High dose animals, loss of body weight at the High dose, decreased body weight and food consumption in all groups, relatively minor changes in haematology, changes in clinical chemistry parameters mainly suggesting hepatic or renal effects, and increased liver and kidney weights. At necropsy changes in the testes and epididymis were noted. At the High dose (400 mg/kg bw/day) the Day 28 body weight was >20% below control and is considered to probably be above the maximum tolerated dose (MTD) for the 90-day study (OECD 408).
Executive summary:

The objective of this study was to assess the toxicity of the test item following a 28-day oral gavage administration in 2 dose levels in Hannover Wistar rats, as requested by the Sponsor. The study is designed to provide information on the systemic toxicity, to allow the Sponsor to make the dose selection of the upcoming 90-days study (OECD 408) with the same test item.


Table 2: Doses administered in the 28-day oral gavage dose range finding study
















































 


Gr. No.



 


Group Designation



 


Dose Levels



Dose Concentration



Dose volume



 


Animal Numbers



(mg/kg bw/day)



(mg/mL)



(mL/kg bw)



Male



Female



1



Control



0



0



10



5



5



2



Low dose



300



30



10



5



5



3



High dose



400



40



10



5



5



General clinical and mortality observations were performed twice daily, detailed clinical observation was performed at least daily during the study. Body weight was recorded on the first day of treatment, then at least twice weekly, and prior to necropsy. Animal food consumption was determined at regular intervals. At necropsy, samples were taken and processed for haematology, coagulation and clinical chemistry, weights of selected organs were measured, and any macroscopic changes were recorded. No histopathology was conducted in the study.


One High dose female animal was found dead on Day 2 of the study. The cause of death could not be determined based on the available data.
Test item related decreased activity, heightened startle response, hunched back, increased salivation, piloerection, intermittent tremors and noisy respiration were observed in Low and/or High dose males and females.
In High dose males and females, test item related decrease in body weight and food consumption was seen, which started to reverse after 8-10 days.
In haematology, decreased haemoglobin and haematocrit (by up to ~12%) and increased platelet values in Low and High dose animals, and increased relative reticulocytes in Low and High dose females were observed.
Clinical chemistry findings of increased creatinine correlated with the increased kidney weights, increased bile acid and cholesterol correlated with increased liver weights, suggesting a test item related effect on kidney and liver. Decreased albumin and total protein and changes in phosphorous, sodium, potassium and chloride may correlate with a kidney effect or the decreased food consumption observed in the test item treated groups.
Treatment related increased kidney and liver weights were seen in both sexes in the Low and High groups; test item related increase in adrenal weights in High dose males were also observed.
Necropsy revealed test item related macroscopic changes in the testes and epididymis in Low and High dose males.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023
Report date:
2023

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dimethylimidazole
EC Number:
217-101-2
EC Name:
1,2-dimethylimidazole
Cas Number:
1739-84-0
Molecular formula:
C5H8N2
IUPAC Name:
1,2-dimethyl-1H-imidazole
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 6-7 weeks old at start of the treatment
- Weight at study initiation: Males: 169-204 g, females: 128-159 g
- Housing: group-housed, up to 3 animals of the same sex and dose group/cage (Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum (ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 26.2°C
- Humidity (%): 26 - 70%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the selected vehicle (distilled water), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 6 days prior to administration to animals according to stability assessment results of the analytical method validation study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of control (vehicle) and test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of the Test Facility. Representative samples of control (vehicle) and test item formulations were analysed at four times during the study (on the first treatment day and then approximately every fourth week).
Analysis of the formulations for concentration and homogeneity of test item was performed using a validated analytical HPLC-UV (High Performance Liquid Chromatography with UV detection) method (Study code: 21/192-316AN) in the Analytical Department of the Test Facility. Documentation regarding stability of formulations is included in the report.
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the RSD (Relative Standard Deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
High dose: dose level was reduced to 200 mg/kg bw/d on day 71, based on mortality, clinical signs and body weight loss
No. of animals per sex per dose:
10 animals/group/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. This was preceded by an acute toxicity study in which the acute lethal dose was 800 mg/kg bw (1965) and an OECD 422 with a NOAEL of 150 mg/kg bw/day (2013). In the DRF (2023, Study Code: 21/192-101PE), groups of rats received the test compound at 0, 300 or 400 mg/kg bw/day for a period of 28 days. In males, decrease in body weight in the High dose group (approx. -21% compared to controls), and in body weight gain in Low and High dose groups (approx. -27% and -64%, respectively, compared to controls) were observed. High dose males and females showed an initial loss of body weight, which started to reverse after 8-10 days. Only slight adverse findings were seen at 300 mg/kg bw/day (BW loss 11%/10% in 1♂/1♀). Based on these results the dose level of 300 mg/kg bw/day was selected as suitable High dose level. In order to establish a dose relationship and a No Observed effect level, the doses of 100 and 300 mg/kg bw/day were chosen as Mid and Low dose. Based on the mortality, clinical signs and body weight loss observed in the High dose groups, the High dose level was reduced to 200 mg/kg bw/day from Day 71.
- The change in the dose level of the High dose group was implemented on Day 71. A two days dosing holiday was implemented in case of High dose males and females then a reduction in the dose level due to mortalities, clinical signs and body weight loss observed in the High dose groups. The dosing holiday and new dose level were set by the Sponsor’s Representative, in agreement with the Study Director.
A constant volume of 10 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, at approximately 2 hours after dosing,

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at prior to the first treatment on Day 1 (to allow for within-subject comparisons), then weekly and on the day of necropsy in the morning (am). These observations were made outside the home cage in a standard arena, at similar times as practical.
Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Any clinical sign noted during dosing or at any other occasions was recorded at the time seen.
The general and detailed clinical signs were reported together as a single summary of the number of animals per group affected for each observation, and the total number of occasions per group.
- Mortality: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Animals which showed clinical signs considered severe were isolated and/or sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights with precision of 1 g were recorded at randomisation (prior to start of treatment), then weekly, including on Day 90 (last treatment day) and prior to necropsy (fasted, on Day 91; or at death, for animals found dead and euthanized in extremis during the course of the study). The growth curve was prepared graphically, body weight gain was calculated weekly and as the total during the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g weekly (on body weight measurements day).
Daily food consumption was calculated for each interval.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic examination was conducted in all animals before treatment on Day -3/-2. Due to oversight, ophthalmoscopic examination was not performed in Week 13. Instead, eyes and optic nerves were examined microscopically from animals of all dose groups.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional Observational Battery and locomotor activity measurement was performed in the study. Assessment of any potential test item related neurotoxicity was performed during the last exposure week (on Day 88-90). Selected animals (five animals/sex/group) were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested.
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured and reported at the first instance.
Fore/hind grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled
back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity (arena, video system and SMART v. 2.5 software of Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into a 50 x 50 cm open-field for 1-hour observation time, DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

CLINICAL CHEMISTRY: Yes
- Time schedule: At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all surviving animals. Blood samples for haematology and for clinical biochemistry.
On the morning of scheduled necropsy, 1 blood sample was collected for thyroid hormone analysis from all animals by sublingual sampling during a period of up to about 2 hours, and 3 blood samples were collected for clinical pathology examination by heart puncture under terminal pentobarbital anaesthesia;
• one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood),
• one for blood clotting times (in tubes with sodium citrate as anticoagulant),
• one to obtain serum (in tubes with no anticoagulant) for clinical chemistry,
The tubes of blood collected at termination were handed to the Clinical Pathology group for processing and analysis.

- Coagulation: APTT Activated Partial Thromboplastin Time; PT Prothrombin Time

- Paramaters checked: Na+- Sodium; K+ - Potassium; Cl- - Chloride; Ca++ - Calcium; Phos. - Inorganic Phosphorus; TBIL - Total Bilirubin; AST/GOT - Aspartate Aminotransferase activity; ALT/GPT - Alanine Aminotransferase activity; GGT - Gamma Glutamyl Transferase activity; ALKP - Alkaline Phosphatase activity; Urea/BUN - Urea/Blood Urea Nitrogen; Crea. - Creatinine; GLU - Glucose; TRIG - Triglycerides; Chol. - Cholesterol; dHDL - High Density Lipoprotein; dLDL - Low Density Lipoprotein; BA - Bile Acid; TP - Total Protein; ALB - Albumin; A/G - Albumin/Globulin

HAEMATOLOGY: Yes
- Paramaters checked: RBC Red Blood Cell (erythrocyte) count; Hgb Haemoglobin conc.; Hct Haematocrit; MCV Mean Corpuscular (erythrocyte) Volume; MCH Mean Corpuscular (erythrocyte) Haemoglobin; MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration; RDW Red Cell Distribution Width (erythrocyte) volume; Plt Platelet (thrombocyte) count; MPV Mean Platelet (thrombocyte) volume; RETIC Reticulocyte; WBC White Blood Cell (leukocyte) count; NE % Neutrophil; LY % Lymphocyte; MO % Monocyte; BA % Basophil; EO % Eosinophil; LUC % Large Unstained Cells

URINALYSIS: Yes

OTHER:
Examination of Vaginal Smears:
- Prior to the necropsy (Day 91 or pre-terminal), the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Thyroid Hormone Analysis:
- Samples for all animals were assessed for T3, T4 and TSH levels.
- Blood samples were collected into tubes with no anticoagulant from an available vein (sublingual vein) so that all samples were taken during a period of up to about 2 hours, before necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
-Gross necropsy was performed on each animal irrespective of the date of death, including the animals found dead or euthanized pre-terminally in extremis. Clinical pathology samples were also collected. No organ weight measurement was performed, but tissue or organ samples were retained for those animals.
- Surviving animals were euthanized at termination on Day 91. After blood sampling, gross necropsy was performed on each animal. The animals were euthanized by exsanguination under pentobarbital anaesthesia.
- Gross findings; Adrenals; Animal identification; Aorta; Brain; Epididymides; Eye with the optic nerve; Oesophagus; Femur with marrow, Heart; Kidney; Large intestine; Extraorbital lachrymal gland; Lungs with bronchi; Lymph node; Ovaries; Oviduct; Pancreas; Pituitary gland; Prostate; Salivary gland (including mandibular, sublingual and parotid glands); Skeletal muscle (quadriceps); Small intestine; Spinal cord; Spleen; Sternum with marrow; Stomach; Testis; Thymus; Thyroid with parathyroid gland; Tongue; Trachea; Urinary bladder; Uterus; Sciatic nerve; Seminal vesicles with coagulating glands; Harderian gland; Skin, subcutis with mammary gland (inguinal); Vagina; Liver

No sperm analysis will be made.

Organ Weights: Brain; Epididymides; Heart; Kidneys; Liver; Prostate; Seminal vesicles with coagulating glands; Spleen; Testes; Thymus; Uterus including cervix; Adrenals; Ovaries; Thyroids with parathyroids; Pituitary

HISTOPATHOLOGY: Yes
- The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose) and any animals found dead or euthanized pre-terminally during the study. In addition, any organs or tissues with macroscopic abnormalities (except minor, common background changes) were subjected to histological examination from all groups.
As treatment-related microscopic findings were identified by the Study Pathologist, the affected tissue/organ from the lower dose level(s) were examined microscopically as appropriate, in order to establish the no effect level.
Statistics:
At the Test Facility, the statistical evaluation of data (labelled as † in the lists on the next pages) was performed with the program package SAS 9.2 (when using Provantis).
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of (co)variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences, identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test, identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size is <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used, identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
FOUND DEAD & PRE-TERMINAL EUTHANISED ANIMALS
test item related general clinical signs: decreased activity, hunched back, piloerection, increased salivation, noisy respiration, partially closed eyes, red discharge on nose/around eyes, scales, thin fur, fissure, alopecia, crust, wound, and/or oedema
TERMINAL EUTHANISED ANIMALS
Control animals– thin fur; alopecia
Low dose animals– crust; thin fur; scar and wound
Mid dose animals – thin fur; alopecia
High dose males – alopecia; abnormal skin colour; decreased activity; coloured discharge; partially closed eyes; thin fur; hunched back; hypersensitivity to touch; increased salivation; paresis; piloerection; red discharge; tonic convulsion; scales; scar; wound
High dose females – alopecia; decreased activity; coloured discharge (red, on both eyes and on the ventral area of the neck); partially closed eyes; thin fur; hunched back; hypersensitivity to touch; increased salivation; noisy respiration; piloerection; red discharge (on both eyes and on nose/snout); scales; scar; fissure (on forelimb); red liquid (in the oral cavity); oral plaque; oral ulcer ; oedema
None of the clinical changes in the Mid or Low dose groups were considered as treatment related. Treatment related clinical observations were limited to the High dose group. The effects in the High dose group were adverse and indicative of systemic toxicity; the observation of dermal adverse effects seen especially on the paws were considered to be a specific toxic effect. Treatment related but probably non-adverse systemic effects included salivation and noisy respiration.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of five unscheduled deaths were observed at the High dose level at 300 mg/kg bw/day or at 200 mg/kg bw/d; three rats (1 m / 2 f) were pre-terminally euthanized and two animals (1 m / 1 f) were found dead, indicating that the High dose exceeded the MTD.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item related lower body weight and body weight gain were observed in High dose males and females (terminal BW was -24% and -11%, BWG D1-71 was 46% and 40% below control for males and females, respectively). A slight trend for a decrease was also seen in Mid dose males and females and Low dose females, but without a dose response or without statistical significance so this differences were considered probably not treatment related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item related effect on food consumption was seen in High dose males and females (FC D1-71 was -17% and -10% for males and females compared to the control). In the Mid and Low groups there were no statistical differences and no convincingly persistent trends for food intake below the normal range.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Due to oversight, ophthalmoscopic examination was not performed in Week 13. Histopathology of eyes was performed, there were no test item related findings, therefore this deviation was considered to have no significant impact on the outcome of the study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Decreased red blood cell count, haematocrit and haemoglobin values, and increased platelet count in Mid and/or High dose males and females were seen. These were possibly related to the adverse dermal changes observed caused by systemic test item exposure.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In clinical chemistry, there were a range of changes in the inorganic parameters (increased phosphorus and creatinine) and urea, indicative of renal or hepatic effects, in the High and Mid dose groups, but most were within historical control ranges. Together with the histological information these changes were not considered sufficient to be clearly adverse. There was also evidence of changes to lipids (cholesterol and LDL) in the groups with significant reduced food intake and lower body weights. There were no histological changes indicative of adverse effects on lipid disposition, and none of the differences were large enough to be clearly adverse.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly decreased T3 concentration in Mid and High dose males and T4 concentration in High dose males was measured. In the absence of any histological thyroid changes or organ weight effects it is not a clear direct thyroid effect. Since the High dose had exceeded the MTD in this study and needed to be reduced, this finding is not considered as an indication of an adverse endocrine effect.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
During the modified Irwin test, hunched back and piloerection in High dose males and females were recorded. These changes were related to the general clinical condition of the High dose animals. All dose groups of males and females had a normal locomotor activity. Decreased grip strength in High dose males and in Mid and High dose females and decreased foot splay in Mid and High dose males were observed. Similarly reduced grip strength was associated with general systemic toxicity. The neuromuscular assessments did not indicate a clearly significant test item effect, the differences were ascribed to the general systemic toxicity of the test item.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weight changes mainly occure in High dose animals. Some of these were considered to be secondary to the reduced body weight: brain, heart, spleen, thyroid, prostate and semical vesicles. Decrease of organ weight of thymus, testis and epididymes were stress related respectively secundary to excessive general systemic toxicity effect of the test item. Test item related organ weight changes were observed in the following organs: Adrenal gland and liver weights were increased of both sexes at the High dose correlated with histological changes. Also kidney weights were statistically significant increased, but in the absence of histological changes this difference was most probably related to an adaptive, non-adverse effect of treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
FOUND DEAD ANIMAL
Test item-related findings were observed in the adrenal gland, testis and epididymis.
PRE-TERMINAL EUTHANASIA
Test item-related findings were observed in the adrenal gland, testis, epididymis and liver.
TERMINAL EUTHANASIA
Test item-related changes were observed in the testis and epididymis at the High dose level.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FOUND DEAD ANIMAL
- enlargement of the adrenal glands with minimal to mild bilateral diffuse increased of cortical vacuolation (zona fasciculata/reticularis)
- moderate bilateral diffuse tubular degeneration/atrophy with minimal multinucleated giant cells in the testis, minimal bilateral luminal reduced sperm content and cellular debris in the epididymis
PRE-TERMINAL EUTHANASIA
- minimal bilateral diffuse increased of cortical vacuolation (zona fasciculata/reticularis)
- small soft testes correlated with moderate bilateral diffuse tubular degeneration/atrophy
- changes in the epididymis and liver including minimal bilateral luminal reduced sperm content and cellular debris, and minimal centrilobular hypertrophy
TERMINAL EUTHANASIA
Minimal bilateral diffuse increased of cortical vacuolation (zona fasciculata/reticularis) in adrenals of High dose males and females, moderate to marked tubular degeneration/atrophy associated with minimal to moderate multinucleated giant cells in the testis and epididymis with minimal to mild reduced sperm content and cellular debris in High dose males and minimal centrilobular hypertrophy of the liver in High dose females were seen. These changes appeared to be caused by the test item. Track-down histopathology examination of adrenal gland, liver, testes and epididymis did not show any test item related changes. In addition, a number of changes appeared to be secondary to the relatively severe body weight effects and are considered to be stress or body weight related; decreased lymphocyte cellularity of cortex/medulla in the thymus, decreased white pulp cellularity in spleen, minimal to mild atrophy of prostate and seminal vesicles and minimal decreased cellularity of hematopoietic cells in sternal bone marrow.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Examination of Vaginal smears: There were no test item-related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
histopathology: non-neoplastic
mortality

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

The high dose of 300 mg/kg bw/ds was considered to have exceeded the MTD. This level was reduced to 200 mg/kg bw/d on study day 71 based on observed lethality effects. 


Details on all examinations can be found in the attached summary tables. 

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, test item related adverse clinical signs, including dermal changes, were observed in High dose groups, with mortality at the dose level of 300 mg/kg bw/day. Decreased body weight gain and food consumption were seen in High dose males and females, which improved when the High dose was reduced to 200 mg/kg bw/day. A range of test item related changes were observed in clinical pathology, organ weight and necropsy in Mid and High dose groups, most of which were considered as secondary to or associated with low body weights. Some test item related changes that may not be associated with low body weights were seen during microscopic evaluation. Track down histopathology of adrenal glands of both sexes, testis and epididymis of males and liver of females did not show test item related findings in Low and Mid dose groups, therefore the No Observed Adverse Effect Level (NOAEL) for 1,2-dimethylimidazole is considered to be the Mid dose level (100 mg/kg bw/day).
Executive summary:

The purpose of this OECD No. 408 study was to obtain information on the possible toxic effects of repeated (daily) oral gavage administration of the test item, 1,2-dimethylimidazole, for 90 days to male and female Hannover Wistar rats (10 animals/group/sex). A control group received the vehicle only (distilled water).
The dose levels 0, 30, 100, 300 mg/kg bw/day were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 300 mg/kg bw/day was selected as the High dose for this study, this level was reduced to 200 mg/kg bw/day on Day 71 based on observed lethality effects.


Analysis of test item formulations demonstrated, that no test item was detected in the control samples, and all formulations were stable, homogeneous and were within 100 ± 10% of nominal concentration.


The examinations included clinical signs, mortality, body weights, food consumption, neurological assessment (including landing foot splay, grip strength and motor activity assessment), vaginal smears, clinical pathology (including haematology, coagulation, urinalysis, thyroid hormone analysis), gross pathology, organ weights and histopathology. Full histopathology was performed in Group 1 (Control) and Group 4 (High dose), based on the results, microscopic examination of adrenal gland in Low and Mid dose males and females, testes and epididymis in Low and Mid dose males and liver in Low and Mid dose females was also made.


A total of three rats were pre-terminally euthanized and two animals were found dead during the study. Test item related general clinical signs of decreased activity, hunched back, piloerection, increased salivation, noisy respiration, partially closed eyes and red discharge on nose/around eyes were observed. Signs of dermal changes were seen that were also attributed to treatment such as scales, thin fur, fissure, alopecia, crust, wound, and/or oedema. Furthermore, test item related findings in adrenal, testis, epididymis and/or liver and secondary changes (due to body weight loss) in thymus, spleen, prostate, bone marrow, seminal vesicles or ovary were seen in these five animals (generally corresponding with findings at terminal necropsy).
In terminally euthanised High dose males and females, the following test item related adverse general clinical signs were recorded: decreased activity, hunched back, piloerection, noisy respiration, increased salivation, partially closed eyes and hypersensitivity to touch. Also dermal effects included alopecia, scar, wound, scale and oedema on the lip were observed. The clinical and dermal signs were generally restricted to the High dose group in both sexes, the occasional signs seen in Mid and Low group animals were considered as background findings.


Test item related lower body weight, body weight gain and food consumption were observed in High dose males and females, males were more affected; from about Day 43 the males failed to gain any weight until the dose level was reduced to 200 mg/kg bw/day (on Day 71 High dose male weight gain from Day 1 was 46% below control). A slight, dose dependent trend for a decrease was also seen in Mid dose males and females and Low dose females, without statistical significance for the overall weight gain.
During the modified Irwin test, hunched back and piloerection in High dose males and females and decreased spatial locomotion in High dose females were recorded. In locomotor activity evaluation, the overall total travelled distance (0-60 minutes) decreased in Mid and High dose males and females dose dependently. Decreased grip strength in High dose males and in Mid and High dose females and decreased foot splay in Mid and High dose males were observed. Similarly reduced grip strength was associated with general systemic toxicity. The neuromuscular assessments did not indicate a clearly significant test item effect, the differences were ascribed to the general systemic toxicity of the test item.


No test item related changes were seen in the oestrus cycle of females.
In haematology, decreased red blood cell count, haematocrit and haemoglobin values, and increased platelet count in Mid and/or High dose males and females were seen, these were possibly related to the adverse dermal changes observed (dry, squamous skin with scars and wounds, particularly on the paws, interdigitally in 1/8 case) caused by systemic test item exposure.
In clinical chemistry, there were a range of changes in the inorganic parameters and urea, indicative of renal or hepatic effects, in the High and Mid dose groups, but most were within historical control ranges. Together with the histological information these changes were not considered sufficient to be clearly adverse. There was also evidence of changes to lipids (cholesterol and LDL) in the groups with significant reduced food intake and lower body weights. There were no histological changes indicative of adverse effects on lipid disposition, and none of the differences were large enough to be clearly adverse.
Statistically significantly decreased T3 concentration in Mid and High dose males and T4 concentration in High dose males was measured. In the absence of any histological thyroid changes or organ weight effects it is not a clear direct thyroid effect. Since the High dose had exceeded the maximum tolerated dose (MTD) in this study and needed to be reduced, this finding is not considered as an indication of an adverse endocrine effect.


Test item related organ weight changes were observed in the following organs: decreased epididymis in High dose males, increased adrenal gland in High dose males and females, increased liver in all Low, Mid and High dose females, increased kidney in Mid and High dose males and females. Decreased thymus (an organ generally sensitive to stress), spleen (only males), prostate and seminal vesicles weights were also seen in High dose males and/or females, these changes were considered as secondary changes due to the body weight and general systemic toxicity effect of the test item; histopathology indicated no specific organ effects in these tissues.
Macroscopically, small testis and epididymis were observed in terminally euthanized High dose males, as test item-related changes; although these animals had a body weight effect beyond the normal MTD, the testis changes are not fully explained by the body weight at this dose level.
Microscopically, minimal bilateral diffuse increased of cortical vacuolation (zona fasciculata/reticularis) in adrenals of High dose males and females, moderate to marked tubular degeneration/atrophy associated with minimal to moderate multinucleated giant cells in the testis and epididymis with minimal to mild reduced sperm content and cellular debris in High dose males and minimal centrilobular hypertrophy of the liver in High dose females were seen. These changes appeared to be caused by the test item. In addition, a number of changes appeared to be secondary to the relatively severe body weight effects and are considered to be stress or body weight related; decreased lymphocyte cellularity of cortex/medulla in the thymus, decreased white pulp cellularity in spleen, minimal to mild atrophy of prostate and seminal vesicles and minimal decreased cellularity of hematopoietic cells in sternal bone marrow.