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

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September to 09 June 2010

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

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes

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: yellow powder
- Purity: 99.8%
- Impurities (identity and concentrations): not indicated
- Purity test date: 22 October 2007
- Lot/batch No.: R0054002C 013 L 001, synonym 0604070001
- Expiration date of the lot/batch: 01 February 2008
- Stability under test conditions: not indicated
- Storage condition of test material: at +4°C, protected from light, humidity and under nitrogen gas

Study design

Analytical monitoring:

yes

Details on sampling:

An aliquot of each sample was injected after a 4 to 12.5-fold dilution in water containing 0.01% of sodium dithionite.

Buffers:

Buffer pH = 4.0 (MERCK, Titrisol, ref 1.09884, batch No. 0C550629),
Buffer pH = 7.0 (MERCK, Titrisol, ref 1.09887, batch No. 0C539031),
Buffer pH = 9.0 (MERCK, Titrisol, ref 1.09889, batch No. 0C536926).
Sodium hydroxide 1N, Normadose (VWR, ref 32 066.606),
Deionized water, prepared at CIT using a Milli-Q apparatus (Millipore),
Acetonitrile for HPLC (Carlo Erba, ref. 412412),
Ammonium acetate (Aldrich, ref 238074-500g),
Sodium dithionite (Acros, ref. S/3800/53),
Boric acid (Acros), ref. 423485000,
Potassium chloride ACS (Sigma), ref P3911-500G

Preparation of buffer pH = 9.0 with potassium chloride and boric acid
Solvent A: potassium chloride (7.456 g) was transferred into a 1000-mL volumetric flask and made up to volume with water.
Solvent B: potassium chloride (6.183 g) was transferred into a 1000-mL volumetric flask and made up to volume with solvent A.

Buffer solution pH = 9.0 (solvent B/sodium hydroxide 0.1 M) (500 mL/213 mL)

Details on test conditions:

Buffer solutions at pH 4.0, 7.0 and 9.0 were prepared. The pH of each buffer solution was checked before use and was degassed 5 minutes (using nitrogen).
An aliquot of test item was introduced in a volumetric flask and each flask was made to volume with respectively buffer pH 4.0, 7.0 and 9.0. The solutions obtained were transferred in a test bottle protected from light. The tests were not performed under sterile conditions.

An aliquot of test item (25 to 50 mg) was introduced in a volumetric flask (50 to 100 mL) and made to volume with buffer pH 4.0, 7.0 and 9.0, respectively.
A sample of the prepared solution was analyzed in duplicate after a 12.5-fold dilution in dilution solvent* (see § Analytical Procedure) in order to determine the initial concentration of the test item.
All buffer solutions were placed in an oven at specified temperature (the temperature was recorded during the test).
At each sampling time, samples of each prepared solution were diluted (4 to 12.5-fold), and injected in chromatographic system (see § 2.3 Analytical Procedure), the pH value of the solutions was measured and the visual appearance of the solutions noted.

*dilution solvent: water with 0.01% of sodium dithionite.

Number of replicates:

Test 1, pH 4.0 at 50°C: 5 replicates
Test 1, pH 7.0 at 50°C: 5 replicates
Test 1, pH 9.0 at 50°C: 5 replicates
Test 2, pH 7.0 at 50°C: 5 replicates
Test 3, pH 4.0 at 65°C: 4 replicates
First test 3, pH 7.0 at 65°C: 4 replicates
Test 3, pH 9.0 at 65°C: 3 replicates
Second Test 3, pH 7.0 at 65 °C: 3 replicates
Test3, pH 4.0 at 79°C: 4 replicates
First Test 3, pH 7.0 at 79°C: 3 replicates
Test 3, pH 9.0 at 79°C: 3 replicates
Second test 3, pH 7.0 at 80°C: 3 replicates

Results and discussion

Preliminary study:

Resulting solutions at the beginning of the test were colorless.
No significant variation of pH was observed for any buffer along the test. Resulting solutions at the end of the test were orangey (for pH = 4.0), yellow (for pH = 7.0) and yellowish (for pH = 9.0).

pH 4.0, pH 7.0 and pH 9.0
The results showed that after 5 days, IMEXINE OBH was hydrolyzed in the conditions of pH tested at 50 ± 1°C.
In accordance with the guideline, the investigations were continued since more than 10% after 5 days, but less than 50% after 2.4 hours, was hydrolyzed.
Test 1 was performed in order to determine the nature of the reaction.

Test performance:

The study was performed in accordance with study plan No. 32584 PDA with the following deviations from the agreed study plan:
• during test 1 at 50°C, the observed temperature was lower than 50°C (approximately 48.5°C) for approximately 10 minutes,
• during test 3 at 65°C for pH= 4.0 and 7.0, three sampling times were performed instead of two sampling times,
• during test 3 at 79°C for pH = 4.0, three sampling times were performed instead of two sampling times,
• during test 3 at 79°C, the observed temperature was lower than 78°C (approximately 77.5°C) for a few minutes,
• during test 3 at 80°C, the observed temperature was lower than 79°C (approximately 78.5°C) for a few minutes,
• the buffer solutions were bubbled with helium instead of only nitrogen.

These minor deviations were not considered to have compromised the validity or integrity of the study.

Transformation products:

not measured

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.05 h-1

DT50:

ca. 13.2 h

Type:

(pseudo-)first order (= half-life)

Dissipation DT50 of parent compound 2

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.82 h-1

DT50:

ca. 8.45 h

Type:

(pseudo-)first order (= half-life)

Dissipation DT50 of parent compound 3

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.03 h-1

DT50:

ca. 25.1 h

Type:

(pseudo-)first order (= half-life)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

pH 4.0
Following advanced test results, IMEXINE OBH can be considered as hydrolytically unstable with:
Kobs = 0.0526 h-1 and t1/2 = 13.2 h at 25°C (estimation).

pH 7.0
Following advanced test results (set of tests performed twice, second set results presented as the extrapolation at 25°C was more reliable), IMEXINE OBH can be considered as hydrolytically unstable with:
Kobs = 0.0820 h-1 and t1/2 = 8.45 h at 25°C (estimation).

pH 9.0
Following advanced test results, IMEXINE OBH can be considered as hydrolytically unstable with:
Kobs = 0.0276 h-1 and t1/2 = 25.1 h at 25°C (estimation).