9beta,11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

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)

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

- Purity: 98.5 %

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

sampling schedule:
- pH4 (50 °C) [h]: 0, 24, 48, 120, 144
- pH7 (50 °C) [h]: 0, 24, 48, 120, 144, 192, 312
- pH9 (50 °C) [h]: 0, 0.3, 0.6, 1.0, 1.5, 2.6, 3.4, 4.0, 5.0, 6.0

- pH7 (70 °C) [h], test solution I: 0, 4.0, 7.9, 16.0, 24.0, 28.0, 30.0, 48.0, 52.1
- pH7 (70 °C) [h], test solution II: 0, 4.0, 8.0, 16.2, 24.0, 28.0, 30.3, 48.1, 51.75
- pH7 (80 °C) [h]: 0, 1.0, 2.25, 4.0, 6.0, 8.0, 16.0, 24.0, 27.0

- pH9 (60 °C) [h], test solution I: 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0
- pH9 (60 °C) [h], test solution II: 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0
- pH9 (70 °C) [h]: 0, 0.08, 0.17, 0.25, 0.33, 0.42, 0.5, 0.75, 1.0

Buffers:

Buffer solutions
- 0.001 M acetate buffer solution pH 4:
0.656 g = 0.008 mol sodium acetate p.a. Merck Art. 106268 (M = 82.03 g/mol) and
2.4 mL = 0.04 mol acetic acid 100 % p.a. Merck Art. 63 E were dissolved to 500 mL with double distilled water. The pH value was adjusted to 4 by adding hydrochloric acid (Titrisol Merck Art. 109973, 0.1 M)

- 0.001 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 were dissolved to 500 mL with double distilled water. 5 ml of the resulting solution were diluted to 500 ml with double distilled water. The pH value was adjusted to 7 by adding sodium hydroxide solution (Titrisol Merck Art. 109956, 1 M).

- 0.001 M borate buffer solution pH 9:
2.52 g = 0.012 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 500 mL with double distilled water. 5 ml of the resulting solution were diluted to 500 ml with double distilled water.

As the pH value of borate buffer solutions decreases with increasing temperature, the following pH values had to be adjusted at 25 °C to obtain a pH value of 9.00 at elevated temperatures. Sodium hydroxide solution (Titrisol Merck Art. 109956, 1 M) was used to adjust the pH values.
pH at 25 °C: 9.20, 9.28, 9.36 corresponds to pH= 9.00 at 50, 60, 70 °C, respectively

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:

The rate of hydrolysis in aqueous solutions buffered to pH values of 4, 7 and 9 was studied. Since the hydrolytic behaviour of the test substance was unknown, the preliminary test (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 (corresponding 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 pH 9 within 5 days at 50 ± 0.1 °C, additional hydrolysis experiment had to be conducted at pH 7, 80 °C and at pH 9, 60 °C.

Duration:

144 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.22 mg/L

Duration:

312 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.21 mg/L

Duration:

6 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.22 mg/L

Duration:

52 h

pH:

7

Temp.:

70 °C

Initial conc. measured:

0.22 mg/L

Duration:

27 h

pH:

7

Temp.:

80 °C

Initial conc. measured:

0.19 mg/L

Duration:

3 h

pH:

9

Temp.:

60 °C

Initial conc. measured:

0.25 mg/L

Duration:

1 h

pH:

9

Temp.:

70 °C

Initial conc. measured:

0.18 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:

In preliminary hydrolysis experiments a dependence of the rate of hydrolysis on the buffer concentration, especially at pH 7, had been found. Therefore all hydrolysis experiments were conducted using diluted (0.001 M instead of 0.05 M) buffer solutions.

Test performance:

I. Preparation of the test solutions:
- Test solutions, buffered to pH values of 4, 7 and 9, each with a test concentration of approx. 0.2 mg/L containing 1 % acetonitrile, were prepared by adding a stock solution in acetonitrile to the corresponding buffer solution which had been equilibrated at 50 °C for one hour followed by purging with nitrogen to remove dissolved oxygen.
- Since the test substance showed the tendency to be adsorbed on glass surfaces, every sample was prepared individually in 5 mL autosampler vials by adding 40 µL of the stock solution to 4 mL of the buffer solution. Thus no transfer steps were necessary.
- Afterwards, the vials were tightly closed with screw caps and three vials were prepared for each sampling time and each of the pH values 4, 7 and 9.
- All experiments were conducted using sterilized equipment and buffer solutions.

II. Procedures for incubation and sampling:
- The vials were incubated in a thermostatic water bath at 50 ± 0.1 °C
- The initial test concentration was measured in triplicate on the test solutions after work-up (see following)
- The contents of each vessel were completely transferred to a 10 mL volumetric flask
- The vials were rinsed several times with acetonitrile, the extracts combined and the flasks filled up to the mark with acetonitrile
- Complete recovery of the test substance was assured and the pH values in aliquots of the test solutions were measured in a separate vessel at the test temperature of 50 °C at the start of incubation and at each sampling time
- During the incubation of the test solutions possible photolytic degradation of the test substance 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, three vials from each of the hydrolysis solutions were removed from the thermostatic bath and the samples were worked up as described above and analyzed

Transformation products:

yes

No.:

#1

Details on hydrolysis and appearance of transformation product(s):

Epoxid-Valerat was hydrolyzed at pH 7 and 9 with a half-life of 297 and 4.8 days at 25 °C. As can be expected for the alkaline hydrolysis of esters, the corresponding alcohol, ZK 30526 (CAS 61618-89-1), is the hydrolysis product. This is consistent with the appearence of a peak at shorter retention t-imes in the HPLC diagrams. Due to the sterically favoured position of the ester group for cleavage, hydrolysis is relatively rapid at pH 9 and considerable degradation occurs even at pH 7.

% Recovery:

21

pH:

9

Temp.:

60 °C

Duration:

3 h

Remarks on result:

other: test solution (1)

% Recovery:

18.5

pH:

9

Temp.:

60 °C

Duration:

3 h

Remarks on result:

other: test solution (2)

% Recovery:

9.8

pH:

9

Temp.:

70 °C

Duration:

1 h

% Recovery:

3.6

pH:

7

Temp.:

70 °C

Duration:

52.1 h

Remarks on result:

other: Test solution (1)

% Recovery:

5.2

pH:

7

Temp.:

70 °C

Duration:

51.75 h

Remarks on result:

other: Test solution (2)

% Recovery:

7.8

pH:

7

Temp.:

80 °C

Duration:

27 h

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Remarks on result:

other: stable according to pretest

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

297 d

Type:

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

Remarks on result:

other: Arrhenius extrapolation

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.006 h-1

DT50:

4.8 d

Type:

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

Remarks on result:

other: Arrhenius extrapolation

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.178 h-1

DT50:

3.88 h

Type:

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

Remarks on result:

other: 95 % confidence intervals for t 1/2: 3.54-4.31 h

pH:

9

Temp.:

60 °C

Hydrolysis rate constant:

0.599 h-1

DT50:

1.16 h

Type:

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

Remarks on result:

other: 95 % confidence intervals for t 1/2: 1.10-1.22 h

pH:

9

Temp.:

70 °C

Hydrolysis rate constant:

1.873 h-1

DT50:

0.37 h

Type:

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

Remarks on result:

other: 95 % confidence intervals for t 1/2: 0.33-0.41 h

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.004 h-1

DT50:

187.3 h

Type:

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

Remarks on result:

other: 95 % confidence intervals for t 1/2: 172.9 - 204.3 h

pH:

7

Temp.:

70 °C

Hydrolysis rate constant:

0.046 h-1

DT50:

14.93 h

Type:

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

Remarks on result:

other: 95 % confidence intervals for t 1/2: 14.38-15.52 h

pH:

7

Temp.:

80 °C

Hydrolysis rate constant:

0.148 h-1

DT50:

4.69 h

Type:

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

Remarks on result:

other: 95 % confidence intervals for t 1/2: 4.43-4.99 h

Details on results:

The rate constants pH 7 and 9 at 25 °C were calculated by applying the Arrhenius equation.

Validity criteria fulfilled:

not specified

Conclusions:

The rate of hydrolysis of Epoxid-Valerat was determined at pH 4, 7 and 9 and the rate constants and half-lives at 25 °C were 4.8 days (pH 9), 297 days (pH 7) and > 1 year (pH 4). Thus Epoxid-Valerat is considered to be stable at pH 4 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). Less than 10 % of the test substance were degraded at pH 4 within 6 days at 50 ± 0.1 °C. Therefore no further testing was necessary at pH 4. More than 10 % of the test item was degraded at pH 7 and 9 within 5 days at 50 ± 0.1 °C. Therefore further hydrolysis experiments were necessary at these pH values. Hydrolysis experiments were conducted at two elevated temperatures with replication at the lower of those two temperatures in order to extrapolate from the experimentally determined hydrolysis rate constants at higher temperatures to the rate constant at 25 °C by using the Arrhenius equation and the experimental data. The rate of hydrolysis of Epoxid-Valerat was determined at pH 4, 7 and 9 and the rate constants and half-lives at 25 °C were 4.8 days (pH 9),  297 days (pH 7) and > 1 year (pH 4). According to the criterion in EC guideline C.7 Epoxid-Valerat is considered to be stable at pH 4 at 25 °C.

Description of key information

The rate of hydrolysis of Epoxid-Valerat was determined at pH 4, 7 and 9 and the rate constants and half-lives at 25 °C were 4.8 days (pH 9),  297 days (pH 7) and > 1 year (pH 4). According to the criterion in EC guideline C.7 Epoxid-Valerat is considered to be stable at pH 4 at 25 °C.

Key value for chemical safety assessment

Half-life for hydrolysis:

4.8 d

at the temperature of:

25 °C

Additional information

"Should read: 4.8 days at pH 9"