1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo-1-piperidinyl)phenyl]-, ethyl ester

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 September 2016 to 23 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Reference to the validated analytical method used for the quantification of the hydrolysis samples.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- The buffer solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter (Whatman) and transferred into sterile vessels.
- To exclude oxygen, nitrogen gas was purged through the solutions for 5 minutes.
- The substance was spiked to the buffer solutions at a target concentration of 2 mg/L using a spiking solution in acetonitrile.
- The spiking volume was < 1% of the sample volume; nominal concentrations were not corrected for the spiking volume.

- For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment.

- Samples for analysis were taken immediately after preparation (t=0) and after 5 days (Tier 1)
- Samples for analysis were taken immediately after preparation (t=0) and at several sampling points after t=0 (Tier 2).
- Samples taken from solutions with a temperature > 20°C were cooled to room temperature using running tap water.

- Blank buffer solutions containing a similar content of blank spiking solution (acetonitrile) were treated similarly as the test samples and analysed at t=0.

- The pH of the test solutions (except for the blanks) was determined at each sampling point.

Buffers:

- Acetate buffer pH 4: aqueous solution of 16.7% 0.1 M sodium acetate and 83.3% 0.1 M acetic acid.
- Phosphate bffer pH 7: aqueous solution of 0.1 M potassium di-hydrogenphosphate adjusted to pH 7 using 10 N sodium hydroxide.
- Borate buffer pH 9: aqueous solution of 0.1 M boric acid and 0.1 M potassium chloride adjusted to pH 9 using 10 N sodium hydroxide.

- Type of water: tap water purified by a purification system

Details on test conditions:

Tier 1 preliminary test at pH 4, 7 and 9 and 49.9 ± 0.1°C.

Tier 2 main study at pH 7 and 9 and different temperatures.
Actual pH and temp.:
-pH 6.9 - 7.0; 19.9 ± 0.3°C, 50.1 ± 0.3°C and 59.8 ± 0.4°C
-pH 9.0 - 9.1; 19.9 ± 0.3°C, 50.3 ± 0.1°C and 60.0 ± 0.1°C

- All solutions containing the substance were protected from light.

Duration:

793.75 h

pH:

7

Temp.:

20 °C

Initial conc. measured:

1.9 mg/L

Remarks:

initial conc. of duplicate samples: 1.90 and 1.90 mg/L

Duration:

793.52 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

1.9 mg/L

Remarks:

initial conc. of duplicate samples: 1.91 and 1.92 mg/L

Duration:

793.3 h

pH:

7

Temp.:

60 °C

Initial conc. measured:

2 mg/L

Remarks:

initial conc. of duplicate samples: 1.93 and 1.98 mg/L

Duration:

510.37 h

pH:

9

Temp.:

20 °C

Initial conc. measured:

1.9 mg/L

Remarks:

initial conc. of duplicate samples: 1.86 and 1.87 mg/L

Duration:

22.23 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

1.9 mg/L

Remarks:

initial conc. of duplicate samples: 1.86 and 1.87 mg/L

Duration:

2.78 h

pH:

9

Temp.:

60 °C

Initial conc. measured:

2 mg/L

Remarks:

initial conc. of duplicate samples: 1.95 and 1.95 mg/L. Concentrations were also analysed at t=73.8 h; because the obtained logarithms relative concentrations were < 0 these results were not used for the calculations.

Number of replicates:

Two

Positive controls:

no

Negative controls:

no

Preliminary study:

A degree of hydrolysis of ≥ 10% was observed at pH 7 after 5 days. As no substance was measured after 5 days at pH 9, this suggest complete hydrolysis of the compound. According to the guideline, the higher Tier test was required to determine the half-life time of the substance at pH 7 and pH 9.

A degree of hydrolysis of < 10% was observed at pH 4 after 5 day (half-life time at 25°C is > 1 year). No further tests were required at pH 4.

Test performance:

RECOVERIES
- Recovery is the concentration analysed at t=0 relative to the nominal concentration.
- The mean recovery was calculated from duplicate samples.
- The mean recoveries at each pH and temperature (Tier 2) are shown below.
- The mean recoveries fell within the criterion range of 90-110%.
- Conclusion: the analytical method was adequate to support the hydrolysis study on the substance.
- Remark: The concentrations analysed in the test samples were not corrected for recovery.

Transformation products:

not specified

% Recovery:

95

pH:

7

Temp.:

20 °C

Duration:

0 h

% Recovery:

96

pH:

7

Temp.:

50 °C

Duration:

0 h

% Recovery:

98

pH:

7

Temp.:

60 °C

Duration:

0 h

% Recovery:

93

pH:

9

Temp.:

20 °C

Duration:

0 h

% Recovery:

93

pH:

9

Temp.:

50 °C

Duration:

0 h

% Recovery:

98

pH:

9

Temp.:

60 °C

Duration:

0 h

Key result

pH:

4

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

265 d

Remarks on result:

other: DT50 at 25°C from the Arrhenius equation for 50°C and 60°C

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.009 h-1

DT50:

3.2 d

Remarks on result:

other: DT50 at 25°C from the Arrhenius equation for 20°C, 50°C and 60°C

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.002 h-1

DT50:

13 d

Type:

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

pH:

7

Temp.:

60 °C

Hydrolysis rate constant:

0.006 h-1

DT50:

4.6 d

Type:

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

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.005 h-1

DT50:

6.2 d

Type:

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

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.171 h-1

DT50:

4.1 h

Type:

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

pH:

9

Temp.:

60 °C

Hydrolysis rate constant:

0.428 h-1

DT50:

1.6 h

Type:

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

Details on results:

No substance was detected in the blank buffer solutions.

Table: Preliminary test (Tier 1) results

pH code	Sampling time	Analysed concentration [mg/L]	Degree of hydrolysis [%]		Actual pH
			Individual	Mean
pH 4	0 hours	2.00			4.0
		2.01			4.0
	5 days	1.91	4.8	4.4	4.0
		1.93	3.9		4.0
pH 7	0 hours	1.99			7.1
		2.17			7.1
	5 days	1.45	30	30	7.0
		1.47	29		7.0
pH 9	0 hours	2.05			9.0
		2.13			9.0
	5 days	not detected	not applicable	not applicable	9.0
		not detected	not applicable		9.0

Validity criteria fulfilled:

yes

Conclusions:

Half-life time at 25°C and pH 4, pH 7 and pH 9 was determined to be > 1 year, 265 days and 3.2 days, respectively.

Executive summary:

The rate of hydrolysis of the substance at pH values normally found in the environment (pH 4-9) was determined in a GLP-compliant study according to EC C.7, OECD 111 and EPA OPPTS 835.2120.

In the preliminary Tier 1 study, a degree of hydrolysis of < 10% was observed at pH 4 after 5 days at 50°C. This corresponds with a half-life time at 25°C of > 1 year. The substance can be considered hydrolytically stable at pH 4. In the preliminary Tier 1 study, hydrolysis of > 10% was determined at pH 7 and pH 9 after 5 days and 50°C. The Tier 2 study was therefore conducted at pH 7 and pH 9. The substance was shown to degrade by a pseudo-first order behaviour at 50 and 60°C (pH 7) and at 20, 50 and 60°C (pH 9). No hydrolysis occurred at pH 7 and 20°C. The half-life time at 25°C were determined from the Arrhenius equation. The half-life time at 25°C and pH 7 was determined to be 265 days. The half-life time at 25°C and pH 9 was determined to be 3.2 days.

Description of key information

The half-life time of the substance at 25°C and pH 4, pH 7 and pH 9 was determined to be > 1 year, 265 days and 3.2 days, respectively.

Key value for chemical safety assessment

Half-life for hydrolysis:

265 d

at the temperature of:

25 °C