3,5-bis(difluoromethyl)-1H-pyrazole

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May - 29 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted in 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents

Version / remarks:

adopted in 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,5-bis(difluoromethyl)-1H-pyrazole

EC Number:

814-072-3

Cas Number:

77614-79-0

Molecular formula:

C5H4F4N2

IUPAC Name:

3,5-bis(difluoromethyl)-1H-pyrazole

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days (treatment) and 14 days (recovery period)

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

12 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 (main study)
10 (recovery groups, control and high dose group)

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were checked at least twice daily during the treatment period, once daily during the recovery period.
- Cage side observations included mortality and evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment and once weekly thereafter (during the treatmend and recovery period).

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to treatment and once weekly thereafter until scheduled necropsy.

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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: During Week 4 of treatment and Week 1 of recovery.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment phase and at the end of the recovery phase.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: Hematocrit (Hct), hemoglobin concentration (Hb), erythrocyte count (RBC), absolute reticulocyte count (Retic), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), mean cell volume (MCV), red cell distribution width (RDW), total leucocyte count (WBC), differential leucocyte count:, neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC), platelet count (Plt), prothrombin time (PT), activated partial thromboplastin time (APTT) and clauss fibrinogen concentration (Fib).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment phase and at the end of the recovery phase.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Bili), urea, creatinine (Creat), glucose (Gluc), total cholesterol (Chol), triglycerides (Trig), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (Phos), total protein (Total Prot), albumin (Alb), thyroid hormones T3 and T4 and thyroid stimulating hormone (TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatment.
- Dose groups that were examined: In the low and mid dose group and on all recovery phase animals (control and high dose group).
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE: Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS PATHOLOGY: Yes. All main study and recovery phase animals were subjected to a detailed necropsy. A full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

- Organ weights: Adrenals, brain, epididymides, heart, kidney, liver, ovaries, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus and uterus with cervix.

- Tissues examined: Abnormalities, adrenals, brain, cecum, colon, duodenum, epididymides, eyes, femur and marrow, heart, ileum, jejunum, kidneys, liver, lungs, lymphnodes (mesenteric and left axillary), ovaries, Peyer's patches, prostate, rectum, sciatic nerves (from only one animal), seminal vesicles with coagulating glands, skeletal muscle (from only one animal), spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), spleen, sternum and bone marrow, stomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus with cervix and vagina.

HISTOPATHOLOGY: Yes. Histopathology on all tissues listed below was performed for all animals that prematurely died and for all control and high dose group animals of the main study. From animals of the low and mid dose group, only abnormalities were given particular attention.
- Tissues examined by light microscopy: Abnormalities, adrenals, brain, cecum, colon, duodenum, epididymides, eyes, femur and marrow, heart, ileum, jejunum, kidneys, liver, lungs, lymphnodes (mesenteric and left axillary), ovaries, Peyer's patches, prostate, rectum, sciatic nerves (from only one animal), seminal vesicles with coagulating glands, skeletal muscle (from only one animal), spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), spleen, sternum and bone marrow, stomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus with cervix and vagina.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: No clinical signs of toxicity were observed.
5 mg/kg bw/day: Piloerection in 10/10 males and decreased activity in 1/10 males. The effects were limited to Day 1 but considered treatment-related. No such effects were noted for the females.
12 mg/kg bw/day: Piloerection in 18/20 males and 16/20 females and decreased activity in 14/20 males and 16/20 females. The effects were observed only on study Day 1 and confined predominantly to approximately one to two hours after dose administration. The findings were considered treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

2 mg/kg bw/day: 1/10 females died during blood sample collection for haematology/clinical chemistry on the day of scheduled necropsy. The death was not attributed to treatment.
5 mg/kg bw/day: No mortality was observed.
12 mg/kg bw/day: 1/20 males was found dead after dose administration on study Day 8. In the absence of any macroscopic and histopathological findings, the death was considered not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: Mean body weight gain was unaffected by treatment.
5 mg/kg bw/day: Statistically significant decrease in mean body weight gain in females (-29%) when compared to control animals. No such effect was noted for males.
12 mg/kg bw/day: Statistically significant decrease in mean body weight gain in males (-21%) and females (-27%) when compared to control animals. The finding was in line with reduced food consumption in both sexes, but fully reversible after 2 weeks of recovery. The effect was considered treatment-related but non-adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: Food consumption was not affected by treatment.
5 mg/kg bw/day: Statistically significant decrease in males (-8%) when compared to control animals. No such effect was noted for females.
12 mg/kg bw/day: Statistically significant decrease in males (-7%) and females (-9%) when compared to control animals. The effect was in line with reduced body weight gain but reversible after 2 weeks of recovery. The effect was considered treatment-related but non-adverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: Statistically significantly increased in males (+19%) and females (+11%) when compared to control animals.
5 mg/kg bw/day: Statistically significantly increased in males (+36%) and females (+48%) when compared to control animals.
12 mg/kg bw/day: Statistically significantly increased in males (+46%) and females (+60%) when compared to control animals. The effect was dose-related but fully reversible after 2 weeks of recovery.
The finding among all dose groups was clearly attributed to treatment. An effect on water balance was assumed by the authors, which is also in line with slight hemoconcentration and corresponding findings in haematology.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males there was a statistically significant increase in white blood cell count (9.08 ± 2.14, 8.02 ± 1.78 and 7.76 ± 1.55 x 10E9/L at the low, mid and high dose vs. 5.95 ± 1.26 x 10E9/L in control), in basophil count (0.07 ± 0.03, 0.07 ± 0.04 and 0.05 ± 0.02 x 10E9/L at the low, mid and high dose vs. 0.03 ± 0.09 x 10E9/L in control) and in lymphocyte count (7.12 ± 1.8, 6.54 ± 1.6 and 5.64 ± 1.53 x 10E9/L at the low, mid and high dose vs. 4.65 ± 1.08 x 10E9/L in control) observed for all dose groups, but without a clear dose-response relationship. Further, there was a statistically significant increase in reticulocytes (0.18 ± 0.023 vs. 0.15 ± 0.022 x 10E12/L in control) and in monocytes (0.20 ± 0.04 vs. 0.12 ± 0.03 x 10E9/L in control) at the high dose group only. The increases in reticulocytes, white blood cells, lymphocytes and basophils were still observed with statistical significance at the end of the recovery period. A statistically significant decrease was noted in prothrombin time (18.5 ± 1.46 vs. 19.6 ± 0.93 sec in control), which was fully reversible after 2 weeks of recovery.
In females there was a statistically significant increase in haematocrit (0.468 ± 0.013, 0.476 ± 0.017 and 0.463 ± 0.012 L/L at the low, mid and high dose vs. 0.451 ± 0.01 L/L in control), haemoglobin (15.0 ± 0.47, 15.4 ± 0.49 and 15.0 ± 0.37 g/dL at the low, mid and high dose vs. 14.3 ± 0.40 g/dL in control), red blood cell count (8.25 ± 0.22, 8.38 ± 0.35 and 8.18 ± 0.42 x 10E12/L at the low, mid and high dose vs. 7.81 ± 0.28 x 10E12/L in control) and reticulocyte count (0.165 ± 0.019, 0.184 ± 0.02 and 0.167 ± 0.025 x 10E12/L at the low, mid and high dose vs. 0.143 ± 0.022 x 10E12/L in control) observed for all dose groups, but without a clear dose-response relationship. There was a statistically significant increase in eosinophils (0.10 ± 0.025 and 0.09 ± 0.059 x 10E9/L at the mid and high dose vs. 0.05 ± 0.016 x 10E9/L in control) and a statistically significant increase in monocytes (0.14 ± 0.068 and 0.10 ± 0.029 x 10E9/L at the mid and high dose vs. 0.08 ± 0.021 x 10E9/L in control) for the mid and high dose group. In addition, there was a statistically significant increase in mean cell haemoglobin concentration (32.4 ± 0.60 vs. 31.8 ± 0.40 g/dL in control) in the high dose group. A statistically significant decrease was noted for the prothrombin time (18.7 ± 0.67, 18.0 ± 0.85 and 18.1 ± 0.58 sec at the low, mid and high dose vs. 20.8 ± 0.46 sec in control) among all dose groups. Changes in white blood cell count, lymphocyte and basophil count which were only observed in the low and mid dose group were not considered treatment-related. The increases in haematocrit, haemoglobin concentration, red blood cell distribution width as well as in activated partial thrombin time and the decrease in prothrombin time were still observed with statistical significance at the end of the 2-weeks recovery period. The increases in red blood cell count, reticulocytes, mean cell haemoglobin concentration, eosinophils and monocytes were fully reversible after 2 weeks of recovery.
All findings in males and females that were consistently observed at all dose levels or showing a clear dose-response relationship were considered treatment-related but of no toxicological relevance. The erythrocyte findings in females were explained by a slight hemoconcentration that again was attributed to an effect on water balance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males there was a statistically significant and dose-related decrease in urea observed at all dose levels (7.03 ± 0.81, 6.65 ± 1.47 and 6.32 ± 1.60 mmol/L at the low, mid and high dose vs. 8.47 ± 1.34 mmol/L in control). A statistically significant increase was observed in phosphorus (2.37 ± 0.14, 2.26 ± 0.10 and 2.23 ± 0.20 mmol/L at the low, mid and high dose vs. 2.08 ± 0.18 mmol/L in control) for all dose levels, but without a clear dose-response relationship. A statistically significant decrease in glucose (6.46 ± 1.61 and 7.31 ± 1.43 mmol/L at the mid and high dose vs. 8.34 ± 1.24 mmol/L in control) was observed at the mid and high dose level and a statistically significant decrease in creatinine (25 ± 2.9 µmol/L vs. 29 ± 2.8 µmol/L in control) was observed at the high dose level. All findings were related to treatment. Statistically significant alterations were also observed for sodium (140 ± 1.8, 150 ± 1.0 and 141 ± 2.2 mmol/L at the mid and high dose vs. 147 ± 2.0 mmol/L in control) and chloride chloride (100.8 ± 1.67, 107.9 ± 1.25 and 100.9 ± 1.37 mmol/L at the low, mid and high dose vs. 104.8 ± 2.41 mmol/L in control) for all dose levels, but without a dose-response relationship the changes were not attributed to treatment. The statistically significant decreases in urea and glucose and the statistically significant increase in inorganic phosphorus were still evident after 2 weeks of recovery, whereas all other findings showed recovery. The findings were considered treatment-related but non-adverse.
In females there was a statistically significant increase in inorganic phosphorus (2.25 ± 0.23, 2.14 ± 0.21 and 1.97 ± 0.33 mmol/L at the low, mid and high dose vs. 1.54 ± 0.18 mmol/L in control) at all dose levels, but without a clear dose-response relationship. A statistically significant increase was also noted for total cholesterol (2.47 ± 0.11 and 2.33 ± 0.36 mmol/L at the mid and high dose vs. 1.99 ± 0.46 mmol/L in control) and potassium (4.57 ± 0.78 and 4.88 ± 0.85 mmol/L at the mid and high dose vs. 3.60 ± 0.31 mmol/L in control) at the mid and high dose group and for calcium at the high dose group (2.81 ± 0.09 vs. 2.66 ± 0.08 mmol/L in control). Statistically significantly decreased for all dose groups was chloride (105.2 ± 1.0, 105.2 ± 2.0 and 105.3 ± 1.4 mmol/L at the low, mid and high dose vs. 107.5 ± 1.24 mmol/L in control). In addition, a statistically significant decrease was noted for creatinine at the high dose group only (31 ± 4.3 vs. 38 ± 2.8 µmol/L in control). All effects were fully reversible after 2 weeks of recovery. The findings were considered treatment-related but non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: No abnormalities were observed.
5 mg/kg bw/day: Reduced absolute and relative thymus weight (statistically not significant) in females (abs.: 0.302 ± 0.05 vs. 0.358 ± 0.06 g in control; rel.: 0.176 ± 0.03 vs. 0.198 ± 0.04% in control).
12 mg/kg bw/day: Reduced absolute and relative thymus weight (statistically not significant) in males (abs.: 0.374 ± 0.09 g vs. 0.406 ± 0.08 g in control; rel.: 0.148 ± 0.04% vs. 0.155 ± 0.03% in control) and females (abs.: 0.314 ± 0.05 vs. 0.358 ± 0.06 g in control; rel.; 0.176 ± 0.03 vs. 0.198 ± 0.04% in control) and reduced absolute and relative ovary weight (statistically not significant) in females (abs.: 0.078 ± 0.01 vs. 0.089 ± 0.03 g in control; rel.: 0.044 ± 0.007 vs. 0.05 ± 0.017% in control).
The findings were dose-dependent and attributed to treatment but considered minor as they fully recovered during the 2-week recovery period and as neither gross pathological nor histopathological findings were noted.

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

2 mg/kg bw/day: Statistically significantly decreased hind limp grop strength in males (0.41 ± 0.05 kg) when compared to control animals (0.51 ± 0.07 kg). As no dose response relationship was evident, the effect was considered not treatment-related.
5 mg/kg bw/day: Statistically significantly decreased motor activity in males (low beam level 517.8 ± 155.7 vs. 733.4 ± 293.2 in control). The effect was only transient, within historical control range data and therefore considered non-adverse.
12 mg/kg bw/day: Statistically significantly increased fore limp grip strengh in males (1.09 ± 0.08 kg) when compared to control animals (0.98 ± 0.12 kg). The effect was considered non-adverse and of no toxicological relevance. In addition, motor activity was statistically significantly decreased in males (high beam level 112.4 ± 56.5 vs. 274.6 ± 90.9 in control; low beam level 432.7 ± 158.0 vs. 733.4 ± 293.2 in control) and in females (high beam level 195.1 ± 70.5 vs. 449.2 ± 127.8 in control; low beam level 653.7 ± 166.7 vs. 1051.1 ± 307.2 in control). Decreased motor activity was still evident in both sexes by the end of the recovery period (males: high beam level 196.7 ± 63.3 vs. 239.8 ± 68.3 in control, low beam level 577.0 ± 126.7 vs. 685.1 ± 199.8; females: high beam level 198.9 ± 62.2 vs. 360.4 ± 162.7 in control, low beam level 550.6 ± 169.5 vs. 802.1 ± 290.2 in control), but to a lesser extent and with statistical significance in females only. The findings in motor activity were considered adverse and of toxicological relevance.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

5 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

food consumption and compound intake

neuropathology

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the test substance caused non-specific toxic responses (reduced body weight and food intake) or stress-related responses, reduced motoractivity and a suspected non-adverse effect in water balance. Several non-adverse findings in hematology and clinical chemistry were still present at the end of the recovery phase. Based on the experimental results, the NOAEL for systemic toxicity was 2 mg/kg bw/day in male and female rats.