3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Test material information

Constituent 1

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, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation (administration): 42 +/- 1 day
- Mean weight at study initiation: males 188 g, females 138 g
- Housing: 5 animals per cage in H-Temp (PSU) cages
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: drinking water (from water bottles), ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
For each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. The test-substance preparations were mixed weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The HPLC analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The stability of the test substance in the diet over a period of up to 12 days at room
temperature was proven before the start of the study.

Duration of treatment / exposure:

91/ 92 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/ d
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 94.6, 285.7, 953.6 mg/kg bw/ d in males; 0, 98.8, 295.1 and 983.1 mg/kg bw/ d in females
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: By request of the sponsor, the following dose levels adjusted in the diet were selected for the present study:
1000 mg/kg body weight/day: as high dose, which is the limit dose according to the test guidelines
300 mg/kg body weight/day: as mid dose
100 mg/kg body weight/day: as low dose
The oral route was selected since this has been proven to be suitable for the detection of a
toxicological hazard.
- Post-exposure recovery period in satellite groups: no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

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

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE:
Group food consumption was determined weekly for each cage. The average food consumption/cage was used to estimate the mean food consumption in grams per animal and day. The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and food consumption.

FOOD EFFICIENCY:
Food efficiency (group means) was calculated based upon individual values for body weight and average food consumption for animals in each cage

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: d-1 and d90
- Dose groups that were examined: control and high dose animals

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: morning of d92/d93
- Anaesthetic used for blood collection: Yes: isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Hematology
Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes and Prothrombin time (Hepato Quick’s test) (HQT)
Clinical chemistry
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium(CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein
(TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS: Yes
- Time schedule for collection of urine: d91/d92
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes (FOB, MA)
- Time schedule for examinations: d84-d87
- Dose groups that were examined: all
- Battery of functions tested:
FOB: Home cage observations, Open field observations, Sensorimotor Tests/Reflexes
MA for 1 hour

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Organ weights
1. Anesthetized animals
2. Liver
3. Kidneys
4. Adrenal glands
5. Testes
6. Epididymides
7. Ovaries
8. Uterus
9. Spleen
10. Brain
11. Heart
12. Thymus
13. Thyroid glands

Organ/Tissue fixation
1. All gross lesions
2. Salivary glands (mandibular and sublingual glands)
3. Esophagus
4. Stomach (forestomach and glandular stomach)
5. Duodenum, jejunum and ileum
6. Cecum, colon and rectum
7. Liver
8. Pancreas
9. Brain
10. Pituitary gland
11. Sciatic nerve
12. Spinal cord (cervical, thoracic and lumbar cords)
13. Eyes with optic nerve
14. Adrenal glands
15. Thyroid glands
16. Parathyroid glands
17. Trachea
18. Lungs
19. Pharynx
20. Larynx
21. Nose (nasal cavity)
22. Aorta
23. Heart
24. Bone marrow (femur)
25. Lymph nodes (mesenteric and axillary lymph nodes)
26. Spleen
27. Thymus
28. Kidneys
29. Urinary bladder
30. Testes
31. Ovaries
32. Oviducts, uterus and vagina
33. Epididymides, prostate and seminal vesicle including coagulation gland
34. Female mammary gland
35. Skin
36. Skeletal muscle
37. Sternum with marrow
38. Femur with knee joint
39. Extraorbital lacrimal glands

HISTOPATHOLOGY: Yes
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table at "Any other information on materials".

Statistics:

Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means

Urinalysis, (except color, turbidity, volume and specific gravity): Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity, clinical pathology parameters, urine volume, urine specific gravity, organ weights: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study.

BODY WEIGHT AND WEIGHT GAIN
No substance-related significantly changes in body weight as well as body weight change were measured in treated animals when compared to controls.
The apparently lower body weight and body weight change in males of group 2 (300 mg/kg) were not dose dependent and thus considered to represent normal biological variability rather than a relation to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE
Variations of daily food consumption were observed in all groups including controls.
Differences between food consumption of control and treated groups did not display a consistent pattern. In addition, no significantly differences were observed. Therefore the observed changes were not considered to be related to treatment.
The approximate, mean daily test-substance intake in mg/kg body weight/day over the entire study period is shown in the following table:
Target dose Real mean daily test-substance intake
(mg/kg bw/day) (mg/kg bw/day)
Males Females
100 94.6 98.8
300 285.7 295.1
1000 953.6 983.1

FOOD EFFICIENCY
During numerous time intervals, food efficiency differed from controls (increased/ decreased values) in all treatment groups. However, as there was no consistent trend, these deviations were considered incidental.

WATER CONSUMPTION
No test-substance influence on water consumption was observed.

OPHTHALMOSCOPIC EXAMINATION
Cornea opacity of the right eye was observed in 1/10 males of group 3 (1000 mg/kg bw/day) on days 91 and 92. Due to the isolated occurrence this observation was assessed as spontaneous in nature and therefore not substance-related.

HAEMATOLOGY
No treatment-related changes in the hematological parameters including coagulation values were measured.
At the end of the study the prothrombin time (Hepato Quick’s time) was increased in females of the 300 mg/kg bw/day dose group. This increase was not dose-dependent. The mean of the prothrombin time was within the historical control range (26.3 - 37.9 seconds). Therefore, this change was regarded as incidental rather than treatment-related.
In females of the 300 and 1000 mg/kg bw/day dose groups the absolute eosinophil counts were marginally lower compared to controls, but within the historical control range (0.06 –0.12 G/L). Furthermore, this decrease was not accompanied by any change of the total white blood cell counts (WBC). Therefore, this change was regarded as incidental rather than treatment-related.

CLINICAL CHEMISTRY
No treatment-related, adverse changes in the clinical chemistry parameters were measured.
In males of the 1000 mg/kg bw/day dose group the triglyceride values were statistically significantly higher compared to controls. This was the only parameter in clinical pathology which was deviated in dosed males and furthermore, no treatment-related histophathological finding in the liver was observed. Therefore, these higher triglyceride values were regarded as treatment-related, but not adverse.

URINALYSIS
No treatment-related, adverse changes in the urinalyses parameters were observed.
In females of the 300 mg/kg bw/day dose group the urine volume was decreased and the urinary specific gravity was increased. Both parameters were not dose-dependently changed.
Moreover, a higher specific gravity is indicating an adaptive concentrating capacity of the kidneys during a reduced urine flow rate. Therefore, these deviations were regarded as non-adverse. Furthermore, as the changes were not dose related, a relation related to treatment is unlikely.
In 7 of 10 males of the 1000 mg/kg bw/day dose group – and in one male of the 300 mg/kg bw/day dose group – the urine was daffodil-like (intense yellow) discolored. The discoloration may be due to the excretion of the test article and/or its metabolite. Since no other changes in clinical pathology parameters were found. Therefore, this change of urine color was regarded as treatment-related, but not adverse.

NEUROBEHAVIOUR
FOB
Deviations from "zero values" were obtained in several animals. However, as all findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Besides this, the following examinations were carried out within the FOB and were assessed individually:
Home cage observations: No substance-related findings were observed.
Open field observations: No substance-related findings were observed.
Sensorimotor tests/reflexes: No substance-related findings were observed.
Quantitative parameters: No substance-related findings were observed.
MA
Regarding the overall motor activity, no significant deviations were observed.
Comparing the single intervals with the control groups, the isolated significantly decrease of interrupts in males of group 1 (100 mg/kg bw/day) at interval 10 with the lack of a doseresponse relationship is considered as spontaneous in nature and therefore not substance related.

ORGAN WEIGHTS, GROSS PATHOLOGY AND HISTOPATHOLOGY
No test item-related effects were seen on organ weights, and no macroscopic or histopathological changes considered to be test item-related were observed in the organs and tissues evaluated in this study.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 953.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to 983.1 mg/kg bw/d in females; no adverse effects observed up to the highest dose tested (1000 mg/kg bw/ d nominal)

Target system / organ toxicity

Critical effects observed:

not specified