Pregna-1,4-diene-3,20-dione, 6-fluoro-11-hydroxy-16-methyl-21-[(1-oxopentyl)oxy]-, (6.alpha.,11.beta.,16.alpha.)-

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct - 13 Nov 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Radiolabelling:

no

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

Procedures for incubation and sampling:
- Aliquots of the test solutions were filled into sterilized 5 mL autosampler vials which were tightly closed with screw caps
- The vials were incubated in a thermostatic water bath at the corresponding test temperature and the temperature was kept constant within ± 0.1 °C
- Prior to incubation the initial concentration of the test item was measured on the test solutions of pH 4, 7 and 9
- Possible photolytic degradation of the test substance during the incubation was prevented by exclusion of light from the hydrolysis solutions by using a thermostatic bath made of stainless steel with a metal cover
- At each sampling time two vials from each of the hydrolysis solutions were removed from the thermostatic bath and aliquots of each vessel were analyzed by HPLC
- samples were taken at appropiate intervals

Buffers:

Buffer solutions
0.05 M acetate buffer solution pH 4:
-0.657 g = 0.008 mol sodium acetate p.a. Merck Art. 106268 (M = 82.03 g/mol) and 2.4 mL = 0.042 mol acetic acid 100 % p.a. Merck Art. 100063 E (M = 60.05 g/mol, density = 1.05 g/mL) were dissolved to 1000 mL with double distilled water
- pH value was adjusted to 4 by adding acetic acid

0.05 M phosphate buffer solution pH 7:
- 4.34 g = 0.031 mol disodium hydrogen phosphate Merck Art. 106566 (M = 141.96 g/mol) and 2.64 g = 0.019 mol potassium dihydrogen phosphate Merck Art. 105108 (M = 136.09 g/mol) were dissolved to 1000 mL with double distilled water
- pH value was adjusted to 7 by adding dilute phosphoric acid (10 % v/v)

0.05 M borate buffer solution pH 9:
- 2.52 g = 0.0125 mol disodium tetraborate Merck Art. 106309 (M = 201.22 g/mol) and 46 mL = 0.0046 mol hydrochloric acid Titrisol Merck Art. 109973 (0.1 M) were dissolved to 1000 mL with double distilled water
As the pH value of borate buffer solutions decreases with increasing temperature, the pH value had to be adjusted to 9.20 at 25 °C in order to obtain a pH value of 9.00 at 50 °C. For the experiment at 60 °C, the pH value had to be adjusted to 9.28 at 25 °C. The pH value was adjusted with 0.1 M sodium hydroxide solution.

- buffer solutions for calibration of pH meter:
standard buffer solutions pH 4 (Art. 1.09475), pH 7 (Art. 1.09477) and pH 9 (Art. 1.09476), Merck

Estimation method (if used):

The rate constant at 25 °C was calculated by using the Arrhenius equation and the experimental data.

Details on test conditions:

Preparation of the test solutions
- Test solutions with concentrations of approx. 0.5 mg/L in 0.05 M buffer solutions pH 4, 7 and 9 with 1 % acetonitrile were prepared by adding a stock solution of ZK 26232 in acetonitrile to the corresponding buffer solution in vials
- Vials and septa were pre-rinsed with the solutions, the rinsing solutions discarded and the produre was repeated in order to avoid adsorption on
surfaces
- All buffer solutions had been purged with nitrogen to remove dissolved oxygen
- Experiments were conducted using sterilized equipment and buffer solutions

Duration:

171.4 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.47 mg/L

Duration:

548.9 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.47 mg/L

Duration:

7.6 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.46 mg/L

Duration:

72.7 h

pH:

7

Temp.:

80 °C

Initial conc. measured:

0.47 mg/L

Duration:

4 h

pH:

9

Temp.:

60 °C

Initial conc. measured:

0.47 mg/L

Statistical methods:

The software (KIM version 1.0) used for this calculation is based on TopFit 2.0, a multi purpose kinetic data analysis program

Preliminary study:

Since more than 10 % of the test substance were degraded at pH 7 and 9 within 5 days at 50 °C, hydrolysis experiments at elevated temperatures were necessary for both pH values. Therefore additional experiments were conducted at pH 7, 80 °C and at pH 9, 60 °C.

Transformation products:

yes

No.:

#1

No.:

#2

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Remarks on result:

other: stable according to pre-test

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

1.3 yr

Remarks on result:

other: Arrhenius extrapolation

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.005 h-1

DT50:

5.8 d

Remarks on result:

other: Arrhenius extrapolation

Details on results:

The hydrolysis rates increased with pH, indicating a base-catalized ester hydrolysis resulting in the products Fluocortolone and valeric acid.

Sampling times and measured concentrations of the test substance in the hydrolysis solutions at pH 4, 50 °C

 

time [hours]	concentration (mean) [mg/L]	standard deviation of mean concentration [mg/L]	concentration (mean) in % of initial concentration
0.0 27.5 98.7 150.1 171.4	0.471 0.472 0.470 0.472 0.502	0.003 0.003 0.002 0.002 0.001	100.0 100.2 99.8 100.2 106.6

 

 

Sampling times and measured concentrations of the test substance in the hydrolysis solutions at pH 7, 50 °C

 

time [hours]	concentration (mean) [mg/L]	standard deviation of mean concentration [mg/L]	concentration (mean) in % of initial concentration
0.0 48.7 120.9 194.3 292.3 357.8 453.7 548.9	0.472 0.442 0.394 0.379 0.303 0.277 0.239 0.211	0.002 0.001 0.002 0.002 0.002 0.002 0.003 0.001	100.0 93.6 83.4 80.3 64.2 58.7 50.6 44.7

 

 

Sampling times and measured concentrations of the test substance in the hydrolysis solutions at pH 9, 50 °C

 

time [hours]	concentration (mean) [mg/L]	standard deviation of mean concentration [mg/L]	concentration (mean) in % of initial concentration
0.0 1.6 2.4 3.1 5.5 6.6 7.6	0.461 0.357 0.309 0.284 0.185 0.155 0.133	0.006 - 0.002 0.002 0.001 0.001 0.002	100.0 77.4 67.0 61.6 40.1 33.6 28.9

 

 

Sampling times and measured concentrations of the test substance in the hydrolysis solutions at pH 7, 80 °C

 

time [hours]	concentration (mean) [mg/L]	standard deviation of mean concentration [mg/L]	concentration (mean) in % of initial concentration
0.0 4.2 7.5 24.9 30.9 48.2 55.5 72.7	0.472 0.398 0.363 0.184 0.149 0.077 0.058 0.031	0.002 0.002 0.007 0.002 0.002 0.002 0.002 0.001	100.0 84.3 76.9 39.0 31.6 16.3 12.3 6.6

 

 

Sampling times and measured concentrations of the test substance in the hydrolysis solutions at pH 9, 60 °C

 

time [hours]	concentration (mean) [mg/L]	standard deviation of mean concentration [mg/L]	concentration (mean) in % of initial concentration
0.0 0.3 0.7 1.0 1.5 2.0 2.5 3.0 4.0	0.470 0.406 0.311 0.268 0.200 0.147 0.112 0.083 0.045	0.004 - - - - - - - -	100.0 86.4 66.2 57.0 42.6 31.3 23.8 17.7 9.6

 

Validity criteria for the hydrolysis study:

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
The analytical method used will depend on the nature of the substance and must be sufficiently precise and sensitive to detect a reduction of 10 % of the initial concentration.	Analysis via HPLC is sensitive enough to detect a reduction of 10% of the initial concentration.	Yes
A sufficient number of samples (not less than four) should be chosen to cover the range 20 to 70 % of hydrolysis to test for pseudo-first order behaviour at the specified pH values.	pH 4 (50°C): 5 measurements pH 7 (50°C): 8 measurements pH 9 (50°C): 7 measurements	Yes
The temperature, pH value, buffer composition and a table of all concentration-time data points shall be stated.	The temperature, pH value, buffer composition and a table of all concentration-time data points is stated for each test.	Yes

Validity criteria fulfilled:

yes

Conclusions:

The rate of hydrolysis was determined at pH 4, 7 and 9 and the half-lives at 25 °C were in the range between 5.8 days (pH 9) and higher than one year (pH 4, 7). Thus, Fluocortolon-21-Valerat is considered to be stable at pH 4 and 7 at 25 °C.

Executive summary:

The rate of hydrolysis in aqueous solutions buffered to pH values of 4, 7 and 9 was studied following the EU Guideline C.7. Since the hydrolytic behaviour of the test substance was unknown, the preliminary test defined by the guideline (incubation at 50 ± 0.1 °C) was performed at pH 4, 7 and 9 to assess whether more than 50 % of the test substance are degraded within 2.4 hours at 50 °C (corr. to a half-life of less than 24 hours at 25 °C) or if the test substance can be considered hydrolytically stable (less than 10 % hydrolytic degradation within 5 days at 50 °C corresponding to t½ > 1 year at 25 °C). Since more than 10 % of the test substance were degraded at pH 7 and 9 within 5 days at 50 ± 0.1 °C, additional hydrolysis experiments were conducted at pH 7, 80 °C and at pH 9, 60 °C. Half-lives at 25 °C were in the range between 5.8 days (pH 9) and higher than one year (pH 4, 7). Thus, Fluocortolon-21-Valerat is considered to be stable at pH 4 and 7 at 25 °C.

The hydrolysis rates increased with pH, indicating a base-catalized ester hydrolysis resulting in the products Fluocortolone and valeric acid.

Description of key information

According to EC guideline C.7 the rate of hydrolysis of Fluocortolon-21-Valerat was determined at pH 4, 7 and 9 and the half-lives at 25 °C were in the range between 5.8 days (pH 9) and higher than one year (pH 4 and 7). Thus, Fluocortolon-21-Valerat is considered to be stable at pH 4 and 7, 25 °C.

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

"Should read: > 1 yr at pH 7"

 

The hydrolysis rates increased with pH, indicating a base-catalized ester hydrolysis resulting in the products Fluocortolone and valeric acid.