1H,5H-Pyrazolo(1,2-a)pyrazol-1-one, 2,3-diamino-6,7-dihydro-, dimethanesulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 September 2006 to 31 January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): R0054002C, synonym IMEXINE OBH
- Physical state: beige to yellow powder
- Purity: 99.7%
- Impurities (identity and concentrations): not indicated
- Purity test date: 20 July 2006
- Lot/batch No.: R0054002C 013 L 001, synonym 0604070001
- Expiration date of the lot/batch: 01 August 2007
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature and under nitrogen gas

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest Saint Isle, France
- Age at study initiation: 7 weeks old
- Weight at study initiation: 295 to 347g for males, 178 to 238 g for female
- Housing: suspended wire-mesh cages (43 x 21.5 x 18 cm) with two rats of the same sex and group in each cage. A metal tray containing autoclaved sawdust was placed under each cage.
- Diet (e.g. ad libitum): SsniffR/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water filtered with a 0.22 µm filter
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h


IN-LIFE DATES: From: 19 September 2006 To: 31 January 2007

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: purified water (obtained by reverse osmosis)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was administered as a solution in the vehicle. The test item was dissolved in the required quantity of vehicle in order to achieve the concentrations of 20, 60 and 200 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were prepared weekly under nitrogen atmosphere.


VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the treatment period, the concentration of samples taken from each dosage form (including control) prepared for use in weeks 1, 4, 8 and 13 was determined.
HPLC/UV

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 principal animals
+ 6 additional animals/sex (0 and 1000 mg/kg/day) for recovery period
+ 6 satellite animals/sex/group for toxicokinetic investigations

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of a previously 15 days toxicity study in rats (at 1000 mg/kg/day: abnormal reddish color of litter, minor variations in potassium and protein concentrations, slightly low urine pH with nitritesand increased urine color - but no histopathology findings).
- Rationale for animal assignment (if not random): competurized procedure, so that the average body weight of each group was similar
- Post-exposure recovery period in satellite groups: 4 weeks

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week


BODY WEIGHT: Yes
- Time schedule for examinations: twice before group allocations, on the first day of the treatment and then once a week


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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 AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the treatment period and at the end of the treatment period
- Dose groups that were examined: control and high-dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 12 (and recovery animals during week 17 for clotting parameters)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 12 (and recovery animals during week 17)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.2] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: during week 12 (and recovery animals during week 17)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once in week 11
- Dose groups that were examined: all
- Battery of functions tested: functional observation battery (reactivity or motor activity)

Sacrifice and pathology:

Macroscopic post-mortem examination: all principal animals and the satellite animal found dead
Microscopic examination: all tissues listed in table (N°4) for all animals from the control and the high-dose groups and animals that died or were sacrificed prematurely, and all macroscopic lesions of animals in the low and intermediate dose groups.

Other examinations:

None.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
At 1000 mg/kg/day: two males were prematurely killed or found dead. All animals showed red-orange colored litter and excessive salibation from week 1 or 2 until the end of the dosing period. In addition, yellow-orange colored urogenital region was sporadically observed during the dosing and recovery period. Loud breathing was noted in a few animals by the end of the dosing period, and in one male and one female during the recovery phase. This sign was also observed in one male and one female associated with other signs of poor clinical condition by week 6 or 8.
At 300 and 100 mg/kg/day: all animals showed red-orange colored litter from week 1 until the end of the dosing period.

HAEMATOLOGY
At 1000 mg/kg/day: males had slightly lower activated partial thromboplastin time when compared to controls., and females showed slightly hogher fibrinogen concentration. these changes were reversible.

CLINICAL CHEMISTRY
At all doses, males showed non dose-related slightly higher urea concentration.
At 1000 mg/kg/day: marginally low chloride (both sexes), creatinine (females) and protein (males) concentrations were noted. In males alkaline phosphatase activity was lower than controls. Theses changes were reversible.

URINALYSIS
At 1000 mg/kg/day, a reversible low urine pH was noted for both sex. Presence of proteins, nitrites, bilirubin and glucose were noted at 1000 and 300 mg/kg/day. Trace levels of proteins and nitrites were observed in the urine at 100 mg/kg/day. Marked urine color observed on all or almost all the animals treated at 1000 or 300 mg/kg/day and on approximately half of the animal treated at 100 mg/kg/day was considered to have possibly interfered with the results obtained for the presence of nitrites and bilirubin. All these changes were reversible.

NEUROBEHAVIOUR
At 1000 mg/kg/day: loud breathing and piloerection were noted in one female, slightly lower mean number of horizontal and rearing movements were noted in females.

Effect levels

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the findings noted at urinalysis, it was considered that the NOAEL was 300 mg/kg/day.

Executive summary:

The test item was administered by the oral route (gavage) to male and female Sprague-Dawley rats for 13 weeks at the dose-levels of 100, 300 and 1000 mg/kg/day in purified water. On completion of the dosing period, recovery animals were held for a 4 -week treatment-free period in order to evaluate the reversibility of any findings.

During the study, mean body weight, mean body weight change, mean food consumption , opthalmological examinations and histopathological examinations were not affected by the treatment.

At 1000 mg/kg/day: two males were found dead or prematurely sacrificed with signs of poor clinical condition. All animals showed red-orange colored litter and excessive salivation during all the dosing period, and sporadically yellow to orange urogenital region was noted. Loud breathing was noted in a few animals during the teratment or treatment-free period. In one male and one female this was associated with a number of clinical signs. At the detailed clinical observation (during the functionnal observation battery), one female had loud breathing and piloerection. At motor activity measurment, females showed a slightly lower mean number of horizontal and rearing movements than controls. Slightly low activated partial thromboplastin time (males) and slightly high fibrinogen concentration (females) were noted. Minimal changes of blood biochemistry of low toxicological significance were noted (urea, chloride, creatinine and protein concentration). Low urine pH and marked urine color were noted in all or almost all the animals. Minor reversible changes in urine parameters were considered to be of no toxicological significance.

At 300 mg/kg/day: one satellite male was found dead on day 11 without relevant clinical signs or necropsy findings. All animals showed red-orange colored litter during the whole dosing period. Slightly urea concentration was noted in males. Urinalysis revealed marked urine color and minor reversible changes in urine parameters, which were considered to be of no toxicological significance.

At 100 mg/kg/day:

All animals showed red-orange colored litter during the whole dosing period. Slightly urea concentration was noted in males. Urinalysis revealed marked urine color and minor reversible changes in urine parameters, which were considered to be of no toxicological significance.