Registration Dossier

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:
2019-09-19 - 2020-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of N-[(5-methyl-1H-pyrazol-1-yl)methyl]acetamide AND N-[(3-methyl-1H-pyrazol-1-yl)methyl]acetamide
EC Number:
700-208-8
Molecular formula:
C7H11N3O
IUPAC Name:
Reaction mass of N-[(5-methyl-1H-pyrazol-1-yl)methyl]acetamide AND N-[(3-methyl-1H-pyrazol-1-yl)methyl]acetamide
Test material form:
solid: crystalline
Details on test material:
Name P70/05
Appearance white to yellow powder
Composition N-[(3(5)-Methyl-1 H-pyrazol-1 -yl)-methyl]acetamide (3:5 =66:34)

Test animals

Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld,Germany

Number and sex of animals: 80 animals (40 male and 40 female rats)
In addition, 8 spare animals (4 male and 4 female animals) were available for possible replacement during the adaptation period.

Body weight (at 1st dosing)
Males: 222.4 g - 258.8 g
Females: 193.5 g - 232.1 g

Age of animals(at 1st dosing)
Males: 47 days
Females: 56 days

Selection of species: Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Identification of animals: After randomisation, each rat received a continuos number on the tail, either by tattoo or marker Additionally, the animal cages were labelled with study number, animal ID number, sex, type of study, route of administration and treatment group.

Adaptation period: 6 days (males) or 8 days (females)

Diet:
A certified commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany; see Appendix 2: 'Composition of the Diet') served as food. This food was offered ad libitum. Food residue was removed and weighed.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL.

Housing:
The animals were kept in collectives of up to 3 animals in MAKROLON cages (type III plus or type IV, as appropriate) with a minimum basal surface of approximately 850 cm2 and a height of 18 cm at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 10% (maximum range). Deviations from the maximum range caused for example during cleaning procedures were dealt with in SOPs.
The light intensity was <60 lux in the cage (during the day). Rooms had a 12 hour light and a 12-hour dark cycle.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.

Environmental enrichment:
The animals receive one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
Octagon-shaped red-tinted huts (polycarbonate) are placed in the cages to offer the animals a resting and hiding place.

Drinking water:
Tap water was offered ad libitum.
Drinking water is examined according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on Drinking Water 2001] by the Hamburger Wasserwerke, 20539 Hamburg, Germany, at least four times a year (see Appendix 2: 'Limitation for Contaminants in the Drinking Water').
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trink-wasserverordnung 2001.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of administration Oral, via gavage.

Frequency of administration Daily for a 90-day period.
Vehicle:
other: hydroxylpropyl methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared once daily.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg b.w. The administration formulations were continuously stirred throughout the entire administration procedure to ensure homogeneity.
The dose of the test item was adapted to the animals' body weights daily up to and including test week 6, thereafter weekly.
The control animals received the vehicle at a constant volume orally once daily in the same way.
In addition, the stability, homogeneity and concentration of the test item formulations were monitored.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC/UV detection
The analytical method applied was validated by LPT with regard to linearity of the calibration curve as well as accuracy, precision, stability, specificity and sensitivity.
The results of the validation confirmed that the method employed is suitable for the determination and quantification of N-[(3(5)-Methyl-1H-pyrazol-1-yl)methyl]-acetamide (MPA) in 0.5% aqueous hydroxylpropyl methylcellulose gel formulations.
Duration of treatment / exposure:
90-days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Group size / Dose levels:
The animals were allocated to the test groups based on body weight by means of a computer generated randomisation program (Provantis® Integrated Preclinical Software, version 10.2.1, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom).
Duration of study:
- 6 adaptation days (males)
- 8 adaptation days (females)
- 91 test days
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
Clinical signs:
The animals were observed individually at least once daily, preferably at the same time each day, for any signs of behavioural changes, reaction to treatment, or illness.
Any signs of illness or reaction to treatment were recorded for each individual animal. Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
Animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 a.m. with a final check performed at approximately 4.00 p.m.
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals. In test week 13 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time of day, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, 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) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

Neurological screening:
In test week 13 (before any blood sampling for laboratory examinations, approximately 1-2 hours after the administration), screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli based on GAD ), as well as the assessment of grip strength (MEYER ) and motor activity assessment were conducted in all animals outside the home cage as described below.

Observational screening:
Righting reflex:
The animal was grasped by its tail and flipped in the air (approx. 60 cm) above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet, in this case zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature:
An electronic probe thermometer (with a blunt probe) was used to take a rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
Salivation:
The animals were observed for discharge of clear fluid from mouth, most frequently seen as beads of moisture on lips in rats. Normal state is to see none, in which case a zero (0) was recorded. If present, a plus sign (+) was recorded.
Startle response
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, in which case a zero (0) was recorded. If there was no response, a plus sign (+) was recorded.
Respiration
While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing
Rats are normally obligatory nose-breathers. Each animal was observed, whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded. Mouth breathing was documented by a plus sign (+).
Urination
When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyurea) with 3 being normal.
Convulsions
If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.
Pilo-erection
The fur of the animal's back was observed, whether it was raised or elevated. In the normal animal no pilo-erection should be observed and a score of zero (0) was recorded. If pilo-erection was present, a plus sign (+) was recorded.
Diarrhoea
In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. Normal state is for there to be none (0), in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
Pupil size
It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.

Pupil response:
The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign was recorded.
Lacrimation:
The animal was observed for the secretion and discharge of tears. In rats the tears contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded. If a discharge was present, a plus sign was recorded.
Impaired gait:
The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).
Stereotypy:
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns, occurring out of context and with an abnormal high frequency). These were graded on a scale of 0 (absent = normal) to 5 (marked).
Toe pinch:
The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.
Tail pinch:
The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale of 1 to 5 with 3 being the normal response.
Wire manoeuvre:
The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score of from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
Hind leg splay:
Using an ink pad, the hind paws were marked with ink. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured.
Positional passivity:
The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
Tremors:
Periods of continued fine movements, usually starting in the limbs (and perhaps limited to them). The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
Positive geotropism
The animal was placed on the inclined (at an angle of approx. 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face 'up-hill', in which case a score of zero (0) was recorded. If this did not occur, a negative sign was recorded.
Limb rotation:
One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly in-creased resistance or rigidity (5), with 3 being normal.
Auditory function:
Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+). If there was no response a zero (0) was recorded.

Functional tests:
Grip strength:
Prior to testing, the gauge (Chatillon, Model DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus was adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge had been zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. The animal continued to be pulled along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.

Locomotor activity:
The motility was measured using the TSE InfraMot system . The infrared sensor was mounted on top of the home cage and any movements were measured for the duration of 12 minutes by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.

Mortality:
Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately 4:00 p.m.
None of the animals died or had to be sacrificed prematurely.

Body weight:
The body weight of each rat was determined and recorded at the time of group allocation, daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, thereafter weekly throughout the experimental period. Weekly values are stated in the report.

Food and drinking water consumption:
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. A mean amount consumed per animal was calculated by dividing the amount of food removed from the cage by the number of animals housed in the cage. The residue was discarded. The report includes weekly mean values of the individual animals.
Drinking water consumption was monitored by daily visual appraisal throughout the study.

Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples collected were divided into tubes as follows:
EDTA anticoagulant (whole blood) for haematological investigations
Citrate anticoagulant (plasma) for coagulation tests
Li-Heparin anticoagulant (plasma) for clinical chemistry tests
Serum for thyroid hormone determination

Blood samples for haematology, coagulation and clinical chemistry were taken at the following times:
•At the end of test week 13
(before necropsy on test day 91) All animals

Haematology:
The parameters listed below were determined:
Haemoglobin content (HGB)
Erythrocytes (RBC)
Leucocytes (WBC)
Reticulocytes (Reti)
Platelets (PLT)
Haematocrit value (HCT)
Differential blood count (relative)9
Differential blood count (absolute)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin
(MCH)
Mean corpuscular haemoglobin
concentration (MCHC)
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

Coagulation parameters:
The parameters listed below were determined:
Prothrombin time (PT)
Activated partial
thromboplastin time
(aPTT)

Clinical chemistry:
The parameters listed below were determined:
Albumin
Bilirubin (total)
Bile acids
Cholesterol (total)
HDL Cholesterol
LDL Cholesterol
Creatinine
Glucose
Protein (total)
Urea (in blood)
Calcium
Chloride
Potassium
Sodium
Alanine amino-transferase (ALAT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT)
Lactate dehydrogenase (LDH)

Thyroid hormone (T3, T4, TSH) determination
In order to obtain approximately 2 x 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight on the following occasions:
• At the end of test week 13
(before necropsy on test day 91) All animals
The T3, T4 and TSH ELISA analyses were performed by using commercial kits by IBL International GmbH (Hamburg, Germany) and a Tecan Sunrise microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany).

Ophthalmological and auditory examinations
Examinations were performed on all animals before start of dosing and at the end of the dosing and recovery period.
The eyes were examined with a HEINE ophthalmoscope.
After examination of the pupillary reflex, mydriasis was produced by instillation of STULLN® eye drops onto the cornea. The following ocular structures were then examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.
Sacrifice and pathology:
Necropsy and organ weights:
On test day 91, the animals were dissected following a randomisation scheme.
At necropsy, the oestrus cycle of all females was determined by taking vaginal smears. These observations provided information regarding the stage of oestrus cycle at the time of humane killing and assisted the histological evaluation of oestrogen sensitive tissues.
The wet weights of the following organs of all main study and recovery animals were determined before fixation:
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Ovary (2)
Pituitary
Spleen
Testicle (2)
Thymus
Thyroid (1)
Uterus (including cervix)
As a whole: Prostate and seminal vesicles with coagulating glands

Organ preservation
The following organs or parts of organs with the exception of the eyes and testicles of all main study and recovery animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution and the testicles in modified Davidson's solution for optimum fixation.
Adrenal gland (2)
Aorta abdominalis
Bone (os femoris with joint)
Bone marrow (os femoris)
Brain (3 levels: cerebrum, cerebellum,
medulla/pons)
Epididymis (2)
Eye with optic nerve
Gross lesions observed
Heart (left and right ventricle, septum)
Intestine, large (colon, rectum)
Intestine, small (duodenum, jejunum, ileum,
including Peyer´s patches), Swiss roll
method
Kidney and ureter (2)
Liver (2 lobes)
Lungs (with mainstem bronchi and
bronchioles (preserved by inflation with
fixative and then immersion))
Lymph node (1, cervical)
Lymph node (1, mesenteric)
Mammary gland (male and female)
Muscle (skeletal, leg) Nerve (sciatic)
Oesophagus
Ovary and oviducts (2)
Pancreas
Pituitary
Prostate and seminal vesicles with
coagulating glands
Salivary glands (mandibular, parotid,
sublingual)
Skin (left flank)
Spinal cord (3 sections: cervical, mid-
thoracic, lumbar)
Spleen
Stomach
Testis (2)
Thymus
Thyroid (2, including parathyroids)
Tissue masses or tumours
(including regional lymph nodes)
Trachea (including larynx)
Urinary bladder
Uterus (including cervix)
Vagina
The organs of all group 1 and 4 animals were examined histologically after preparation of haematoxylin-eosin stained paraffin sections. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined histopahologically.

Due to findings in group 4 animals, the following organs of animals of the intermediate groups 2 and 3 were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining:
Liver
Thyroid glands
Kidney (left & right)
Adrenal glands
Statistics:
Data for toxicology and pathology were captured, as far as possible; using the departmental computerized systems (Provantis® Integrated Preclinical Software, version 10.2.1, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item groups 2 to 4 were compared to the control group 1.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
None of the male and female rats treated orally with 100, 300 or 1000 mg N ((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day p.o. for 90 days revealed any changes of behaviour or external appearance.None of the animals treated orally with 100, 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.
Mortality:
no mortality observed
Description (incidence):
None of the male and female rats treated with 100, 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day p.o. for 91 days died or had to be sacrificed prematurely.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slightly reduced body weight was noted in a dose-related way for the male and female animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days. The body weight gain was reduced accordingly for the females at 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day and for the males at 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the relative food consumption of the male and female animals treated orally with 100, 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days compared to the control group during the 90 day treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination did not reveal any changes of the eyes and the optic region in the animals treated with 100, 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days at the end of the treatment period on test day 91.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A dose-related increase in the number of white blood cells, reticulocytes, neutrophilic granulocytes, lymphocytes, monocytes, large unstained cells and basophilic granulocytes and a decrease of red blood cells and eosinophilic granulocytes was noted for the male and female animals treated orally with 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days compared to the control group.
No test item-related influence was observed for the haemoglobin content (HGB), the number of platelets (PLT), the percentage of reticulocytes (Reti), the haematocrit value (HCT), the prothrombin time (PT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A dose related increase was noted in the total cholesterol, the HDL cholesterol, the LDL cholesterol and the bile acid values for the male and female animals treated orally with 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days compared to the control group. Furthermore, the plasma activity of the alanine aminotransferase (ALAT) was increased in a dose related way. A slight tendency for an increase in cholesteroland bile acid levels and ALAT plasma activity was noted for the animals treated with 100 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days, primarily for the females, however, the changes were classified as not noteworthy and, hence, as not toxicologically adverse.
No test item-related influence was noted for the plasma levels of albumin, bilirubin (total), creatinine, glucose, protein (total), urea, calcium, chloride, potassium and sodium. Further, the plasma activity of alkaline phosphatase (aP), aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) were not influenced.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological screening performed at the end of the treatment period in test week 13 (1 to 2 hours after the administration) did not reveal any test item-related influence in the male and female rats treated orally with 100, 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A test item-related increase in absolute and relative organ weights were noted in the kidneys, liver and thyroid of the male and female animals treated with 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days compared to the control group. A slight tendency for an increase in kidney weights was noted for the female animals treated with 100 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days, however, the changes were classified as not noteworthy and, hence, as not toxicologically adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic inspection at necropsy did not reveal any test item-related changes in the organs or tissues of the animals treated with 100, 300 or 1000 mg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/kg b.w./day for 90 days at terminal sacrifice on test day 91.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the livers of male and female rats in the 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w.-treated group, an increased incidence and severity of centrilobular hepatocellular hypertrophy was observed in a dose-related way. This hepatocellular hypertrophy causes an increase in liver weight in animals of the group treated with 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w. Hepatocellular hypertrophy without clear signs of degeneration in general is considered a non-adverse adaptive response; however, the presence of significantly increased circulating lipid parameters (especially cholesterol, HDL, LDL, and bile acids) cannot completely exonerate the liver from its possible role in these potential adverse changes.
In the thyroid glands of several males and females treated with 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w. a multifocal vacuolar mild change of follicular epithelial cells was recognized.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
No test item-related influence was noted on any of the thyroid hormone levels of the male and female rats treated orally with 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days compared to the control group at the end of the treatment period on test day 91 and for the T4 serum levels of the intermediate and high dose groups. Changes in thyroid hormone serum levels (T3 and TSH) were noted for the animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days compared to the control group at the end of the treatment period on test day 91. For details see table 1 under "additional information on results"

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
kidney
liver
thyroid gland
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

Test item-related changes in body weight compared to the control group 1 in %

Test day

Group 3:

300mg/kg b.w./day

Group 4:

1000mg/kg b.w./day

males

females

males

females

1

-0.6

-0.1

-0.4

-1.0

8

-4.7*

-2.0

-5.7*

-3.8

15

-4.6

-2.2

-8.1*

-3.8

22

-4.1

-1.7

-7.4

-3.2

29

-1.2

-3.6

-5.2

-4.8

36

-2.0

-3.0

-7.6

-4.0

43

-2.7

-5.4

-8.4

-6.3

50

-2.2

-5.9

-7.6

-5.7

57

-2.2

-5.0

-9.4

-6.5

64

-2.3

-5.9

-8.6

-9.2*

71

-3.1

-7.3

-9.1

-9.2*

78

-3.9

-10.2**

-10.0

-8.9*

85

-5.0

-6.5

-11.9

-8.7*

90

-3.7

-7.1

-13.6**

-9.6*

*          statistically significant at p ≤ 0.05

**        statistically significant at p ≤ 0.01

Test item-related changes in thyroid hormone levels compared to the control group 1 in %

Test day

91

Group 2:

100mg/kg b.w./day

Group 3:

300mg/kg b.w./day

Group 4:

1000mg/kg b.w./day

males

females

males

females

males

females

T3 concentration

+3

+3

+22*

+17

+5

+28*

TSH concentration

+17

-25

+34

-31

+21*

+158**

*          statistically significant at p ≤ 0.05

**        statistically significant at p ≤ 0.01

Test item-related changes in haematological parameters compared to the control group 1 in %

Parameter

Group 3:

300mg/kg b.w./day

Group 4:

1000mg/kg b.w./day

males

females

males

females

RBC

-5*

none

-6**

-6**

WBC

+10

+15

+34*

+42*

Reti

+12

+21

+11

+46**

Neut (rel.)

-6

-26

+4

-16

Neut (abs.)

none

-9

+36

+28

Lym (rel.)

none

+5

none

none

Lym (abs.)

+12

+19

+33

+44*

Mono (rel.)

none

+10

+21

+41

Mono (abs.)

+9

+25

+56**

+85**

LUC (rel.)

+8

+11

+32

+31

LUC (abs.)

+18

+27

+78*

+83

Baso (rel.)

-9

+10

-7

+17

Baso (abs.)

none

+33

+23

+83**

Eos (rel.)

+11

-38

-31

-64

Eos (abs.)

+16

-17

-14

-43*

*          statistically significant at p ≤ 0.05

**        statistically significant at p ≤ 0.01

none:   change below ±5% compared to the control group

Statistically significant changes in haematological parameters unrelated to the test item

Parameter

 

Increase ­

Decrease¯

Group/

Sex

Test day

 

Statistical significance

Reason

 

HGB

¯

4 f

91

p ≤ 0.05

A

RBC

¯

2 m

91

p ≤ 0.05

A

Reti

­

4 f

91

p ≤ 0.05

A

PLT

­

4 f

91

p ≤ 0.05

A, B

HCT

¯

4 f

91

p ≤ 0.05

A, B

PT

¯

4 m

91

p ≤ 0.05

A

MCV

­

4 m

91

p ≤ 0.01

A

MCH

­

4 m

91

p ≤ 0.01

A

m:       male

f:         female

A:        the slight alteration in comparison to control animals is without any biological relevance

B:        lacking dose-dependency

Test item-related changes in clinical chemistry parameters compared to the control group 1 in %

Test day

Group 2:

100mg/kg b.w./day

Group 3:

300mg/kg b.w./day

Group 4:

1000mg/kg b.w./day

males

females

males

females

males

females

Bile acids

-33

+12

none

+38

+287**

+123

Cholesterol

(total)

none

+23

+37*

+56**

+97**

+75**

HDL Cholesterol

none

+22

+36**

+56**

+94**

+71**

LDL Cholesterol

+33

+35

+96**

+88**

+159**

+57*

ALAT

none

+25

none

+46

+159**

+106**

*          statistically significant at p ≤ 0.05

**        statistically significant at p ≤ 0.01

none:   change below ±10% compared to the control group

Statistically significant changes in clinical chemistry parameters unrelated to the test item

Parameter

 

Increase ­

Decrease¯

Group/

Sex

Test day

 

Statistical significance

Reason

 

Bilirubin (total)

­

¯

4 m

2 f

91

p ≤ 0.05

p ≤ 0.05

A

A, B

Chloride

¯

¯

4 m

4 f

91

p ≤ 0.01

p ≤ 0.05

A

A

Potassium

¯

¯

3 m

4 m

91

p ≤ 0.05

A

m:       male

f:         female

A:        the slight alteration in comparison to control animals is without any biological relevance

B:        lacking dose-dependency

Test item-related changes in relative and absolute organ weights compared to the control group 1 in %

Organ

Group 2:

100mg/kg b.w./day

Group 3:

300mg/kg b.w./day

Group 4:

1000mg/kg b.w./day

males

females

males

females

males

females

Relative organ weight

Kidney (left)

none

+15*

+12*

+12**

+36**

+25**

Kidney (right)

none

+17*

+11

+15**

+31**

+25**

Liver

none

none

+9

+18*

+50**

+49*

Thyroid/

Parathyroid (left)

none

none

none

+41*

+24

+61**

Absolute organ weight

Kidney (left)

none

+12

+9

none

+17*

+10

Kidney (right)

none

+13

+7

+6

+13

+9

Liver

none

none

+5

+9

+28**

+31**

Thyroid/

Parathyroid (left)

none

none

none

+29

+5

+41*

m:       male

f:         female

A:        the slight alteration in comparison to control animals is without any biological relevance

B:        lacking dose-dependency

Statistically significant changes in organ weights unrelated to the test item

Organ

 

Increase ­

Decrease¯

Group/

Sex

Test day

 

Statistical significance

Reason

 

Relative organ weights

Brain

­

4 m

91

p ≤ 0.01

A

Testis, left

­

4 m

91

p ≤ 0.01

A

Testis, right

­

4 m

91

p ≤ 0.01

A

Ovary, right

¯

4 f

91

p ≤ 0.05

A, C

Absolute organ weights

Epididymis, right

¯

4 m

91

p ≤ 0.01

A

Prostate and

seminal vesicle

¯

4 m

91

p ≤ 0.05

A

Ovary, right

¯

4 f

91

p ≤ 0.01

A, C

m:       male

f:         female

A:        the slight alteration in comparison to control animals is without any biological relevance

C:        effect is due to the relative low or high value observed for the control group

Stage of the oestrus cycle at necropsy

Stage of estrus at necropsy

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Proestrus

3 of 10

-

3 of 10

2 of 10

Estrus

-

3 of 10

-

3 of 10

Metestrus

5 of 10

2 0f 10

3 of 10

3 of 10

Diestrus

2 of 10

5 of 10

4 of 10

2 of 10

Applicant's summary and conclusion

Conclusions:
Based on the findings in this 90-day oral repeated dose toxicity study in rats, the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day when given by daily oral administration via gavage for 90 consecutive days.
Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity of N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA) when given to rats by oral administration via gavage daily for 90 days.

Rats were treated with 100, 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl) methyl]-acetamide (MPA)/kg b.w./day.

None of the animals died or had to be sacrificed prematurely.

No test item-related changes were observed for the behaviour, external appearance, faeces, the detailed clinical observations, neurological screening parameters, the food and drinking water consumption, the eyes or optic region, the auditory acuity and at macroscopic inspection at necropsy.

A slightly reduced body weight was noted in a dose-related way for the male and female animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days. The body weight gain was reduced accordingly for the females at 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day and for the males at 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day.

A dose-related increase in the number of white blood cells, reticulocytes, neutrophilic granulocytes, lymphocytes, monocytes, large unstained cells and basophilic granulocytes and a decrease of red blood cells and eosinophilic granulocytes was noted for the male and female animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days compared to the control group.

A dose-related increase was noted in the total cholesterol, the HDL cholesterol, the LDL cholesterol and the bile acid values for the male and female animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days. Furthermore, the plasma activity of the alanine aminotransferase (ALAT) was increased in a dose-related way. A slight tendency for an increase in cholesterol and bile acid levels and ALAT plasma activity was noted for the animals treated with 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days, primarily for the females.

An increase in T3 and TSH serum levels was noted for the animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days compared to the control group at the end of the treatment period on test day 91. The changes were particularly pronounced in the female animals treated orally with 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days.

There was no influence on the stages of the oestrus cycle at necropsy.

A test item-related increase in absolute and relative organ weights was noted in the kidneys, liver and thyroid of the male and female animals treated orally with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days compared to the control group. A slight tendency for an increase in kidney weights was noted for the female animals treated with 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day for 90 days.

In the kidneys of males treated with 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w. a clearly increased incidence and severity of multifocal tubular basophilia was observed. This multifocal tubular basophilia, which is considered to be an adverse change was accompanied by an increased incidence and severity of hyaline droplet accumulation within the proximal convoluted tubules in males of these groups. Possibly these changes are related to the increase in mean kidney weight in males treated with 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w.

In the livers of male and female rats in the 300 or 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w.-treated group, an increased incidence and severity of centrilobular hepatocellular hypertrophy was observed in a dose-related way. This hepatocellular hypertrophy caused an increase in liver weight in animals of the group treated with 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w.

In the thyroid glands of several males and females treated with 1000 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w. a multifocal vacuolar change of follicular epithelial cells was recognized.

The No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day when given by daily oral administration via gavage for 90 consecutive days.

The findings observed in the animals treated with 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day (increased biochemical parameters - cholesterol, bile acids and ALAT - and kidney weights in the females) were classified as not noteworthy and, hence, as not toxicologically adverse, as the marginal findings observed provided no evidence for substance-related organ toxicity during the histopathological evaluation. In addition, most of the changes at the low dose level of 100 mg N-[(3(5)-Methyl-1H-Pyrazol-1-yl)methyl]-acetamide (MPA)/kg b.w./day were within the range of background data for control animals of this strain and age.