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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-29 June 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted by a recognised facility in compliance with GLP to a recent regulatory test guideline.
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
Preliminary Test
Preliminary test samples were prepared in scintillation vials at a nominal concentration of 100 ug/L.
Samples were prepared by fortifying 20 mL samples ofpH 4,7 and 9 buffer solutions using a Reofos 65 stock solution prepared in acetone. Three samples of each solution were taken for immediate analysis and three samples of each were placed into an incubator set at 50°C for a period of five days. On the fifth day, the samples were removed from the incubator and analyzed for Reofos 65.

Definitive Test
Definitive test samples in pH 7 and 9 buffer solutions were prepared in the same manner. Samples were prepared to allow for a single replicate to be maintained at 15"C, and two to be maintained at 25°C. At each interval, samples were removed from their respective incubator for Reofos 65 analysis.
Buffers:
The study was conducted using sterile solutions buffered at pH 4, 7 and 9. Acetate (pH 4), phosphate (pH 7) and borate (pH 9) buffers were used. Aqueous buffer solutions (1 or 2 liters each) were prepared using HPLC grade water and aqueous solutions of salts, acids and bases as follows:

pH 4 buffer - For each liter prepared, 16.691 grams of acetic acid and 4.102 grams of sodium acetate were added to a 1-L volumetric flask containing some HPLC-grade water and shaken to dissolve. The flask was brought to volume with water.

pH 7 buffer - For each liter prepared, 6.805 grams of potassium phosphate and 1.185 grams of sodium hydroxide were added to a 1-L volumetric flask containing some HPLC-grade water and shaken to dissolve. The flask was brought to volume with water.

pH 9 buffer - For each liter prepared, 0.852 grams of sodium hydroxide, 3.092 grams of boric acid and 3.728 grams of potassium chloride were added to a I-L volumetric flask containing some HPLC-grade water and shaken to dissolve. The flask was brought to volume with water.

The pH of each buffer solution was measured using a pH meter with an accuracy of 0.01 pH unit. Prior to use, the resulting pH-adjusted buffer solutions were filtered through 0.2-pm filters.
Estimation method (if used):
Not used.
Details on test conditions:
A stock solution of Reofos 65 was prepared by accurately weighing 0.1002 g of the test substance on an analytical balance. The test substance was transferred to 100-mL volumetric flask, and brought to volume using acetone. The primary stock solution contained 1.00 mg/ml of Reofos 65. The primary stock was diluted in acetone to produce a 0.100 mg/mL stock solution. The 0.100 mg/mL stock solution was used to fortify the quality control samples and prepare calibration standards in acetone.

Reagents and Solvents
All solvents were HPLC grade and all reagents were ACS reagent grade or better.

Matrix Fortification Samples
For the preliminary study, matrix fortification samples in each of the pH 4,7, and 9 buffer matrices were prepared and analyzed concurrently with the Day 0 and 5 hydrolysis samples. The matrix fortifications were each prepared at a nominal concentration of 100 µg/L. The measured Reofos 65 concentration for the pH 4,7 and 9 buffer matrix fortifications ranged from 80.3-1 09 µg/L yielding an overall mean recovery of 96.6% _+ 13.2 (RSD = 13.7%).

For the definitive study, matrix fortification samples in pH 7 and pH 9 buffer solutions were prepared and analyzed concurrently with hydrolysis samples at each interval. The matrix fortifications were each prepared at a nominal concentration of 100 pg/L. The measured Reofos 65 concentration for the pH 7 buffer matrix fortifications ranged from 96.5-1 15 µg/L yielding an overall mean recovery of 108% + 5.23 (RSD = 4.85%). A representative chromatogram of a matrix fortification for the pH 7 buffer is presented in Figure 9. The measured Reofos 65 concentration for the pH 9 buffer matrix fortifications ranged from 10 1-1 26 µg/L yielding an overall mean recovery of 109% 6.72 (RSD = 6.17%).
Duration:
30 d
pH:
7
Initial conc. measured:
107 µg/L
Duration:
30 d
pH:
9
Initial conc. measured:
105 µg/L
Number of replicates:
Preliminary study: 1
Main study: 3
Positive controls:
no
Negative controls:
no
Statistical methods:
The rate of hydrolysis was determined for samples prepared in pH 7 and 9 buffers maintained at 15°C and 25°C by measuring the decrease in concentration versus time. The hydrolysis rate constant was calculated assuming pseudo first order kinetics. Linear regression analysis was performed using the following equation:

Ln (Ct) = -kt + ln(C0)


Where k = degradation rate constant
Ct = concentration at time t (mean of replicates if applicable) expressed as percent of the
applied concentration remaining.
t = time
C0 = initial concentration (t = 0) expressed as 100%


The half-life of Reofos 65 in pH 7 and 9 buffer at 15°C and 25°C was calculated using the following
equation and the analytical results from linear regression analysis:

t1/2 = Ln(2) / k

Where tl/2 = degradation half-life (in days)
k = degradation rate constant
Preliminary study:
The hydrolytic stability of Reofos 65 for the preliminary study was evaluated at pH 4 (acetate buffer), pH 7 (phosphate buffer) and pH 9 (borate buffer) at 50°C over a five day period (Day 0 and Day 5). Samples were prepared at a nominal concentration of 100 ug/L, were collected and analyzed on Days 0 and 5.
Results from these analyses demonstrated that Reofos 65 was stable at pH 4, but resulted in degradation at pH 7 and pH 9.
Test performance:
The test was performed in accordance with the testing laboratory in house protocols, and no deviations were noted.

For the preliminary study, the hydrolytic stability of Reofos 65 was evaluated at pH 4,7 and 9, at 50°C over a five day period. Degradation of Reofos 65 was observed at pH 7 and 9.

For the definitive study, the hydrolytic stability of Reofos 65 was evaluated at pH 7 and pH 9, at 15°C and 25°C, over a 30-day period. Degradation of Reofos 65 at pH 7 was observed at both temperatures resulting in calculated half-lives of 42.6 days at 15°C and 18.5 days at 25°C. Degradation of Reofos 65 at pH 9 was observed at both temperatures resulting in calculated half-lives of 16.5 days at 15°C and 6.05 days at 25°C. These data clearly indicate the hydrolysis rate of Reofos 65 is both pH and temperature dependent.
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Not applicable.
% Recovery:
31.3
pH:
7
Temp.:
25
Duration:
30 d
% Recovery:
61.7
pH:
7
Temp.:
15
Duration:
30 d
% Recovery:
54
pH:
9
Temp.:
15
Duration:
30 d
% Recovery:
22.2
pH:
9
Temp.:
25
Duration:
30 d
pH:
7
Temp.:
15 °C
DT50:
42.6 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Rate constant not reported
pH:
7
Temp.:
25 °C
DT50:
18.5 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Rate constant not reported
pH:
9
Temp.:
15 °C
DT50:
16.5 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Calculated half life. Rate constant not reported.
pH:
9
Temp.:
25 °C
DT50:
6.05 d
Remarks on result:
other: Calculated half life. Rate constant not reported.
Other kinetic parameters:
None
Details on results:
For the preliminary study, the hydrolytic stability of Reofos 65 was evaluated at pH 4,7 and 9, at 50°C over a five day period. Degradation of Reofos 65 was observed at pH 7 and 9.

For the definitive study, the hydrolytic stability of Reofos 65 was evaluated at pH 7 and pH 9, at 15°C and 25°C, over a 30-day period. Degradation of Reofos 65 at pH 7 was observed at both temperatures resulting in calculated half-lives of 42.6 days at 15°C and 18.5 days at 25°C. Degradation of Reofos 65 at pH 9 was observed at both temperatures resulting in calculated half-lives of 16.5 days at 15°C and 6.05 days at 25°C. These data clearly indicate the hydrolysis rate of Reofos 65 is both pH and temperature dependent.

Preliminary study

 

The hydrolytic stability of Reofos 65 for the preliminary study was evaluated at pH 4 (acetate buffer), pH 7 (phosphate buffer) and pH 9 (borate buffer) at 50°C over a five day period (Day 0 and Day 5). Samples were prepared at a nominal concentration of 100 yglL, were collected and analyzed on Days 0 and 5. Results from these analyses demonstrated that Reofos 65 was stable at pH 4, but resulted in degradation at pH 7 and pH 9.

 

Transformation products

not measured

 

Total recovery of test substance (in %)

%Recovery

St. dev.

pH

Temp.

Duration

95.8

2.08

4

50 °C

5 d

56.2

1.47

7

50 °C

5 d

8.45

1.08

9

50 °C

5 d

 

Dissipation half-life of parent compound

pH

Temp.

Hydrolysis rate constant

Half-life

St. dev.

Type

Remarks (e.g. regression equation, r², DT90)

7

15 °C

 

42.65 d

 

 

 

7

25 °C

 

18.5 d

 

 

 

9

15 °C

 

16.5 d

 

 

 

9

25 °C

 

6.05 d

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
The substance is hydrolytically unstable at both increasing pH and increasing temperature. The substance is considered to hydrolyse with a half life of 18.5 days under ambient temperature and pH conditions.
Executive summary:

The hydrolysis of the substance has been assessed at pH 4, 7 and 9 in accordance with the OECD 111 test guideline using GC/MS to assess test concentrations under each condition. The substance was assessed initially at all three pH at elevated temperature over a 5 day period and it was determined that the no hydrolysis occurred at pH4 but the substance hydrolysed at pH7 and 9.

These latter pH's were assessed at 15 and 25 C and increased hydrolysis occurred at higher temperature as compared to the lower temperature. The substance is therefore considered to be hydrolytically unstable at both increasing pH and increasing tempertaure and is considered to have a half life of 18.5 days at ambient temperature and pH.

 

Description of key information

Hydrolysis as a function of pH

Key value for chemical safety assessment

Half-life for hydrolysis:
18.5 d
at the temperature of:
25 °C

Additional information

The hydrolysis of the substance has been assessed at pH 4, 7 and 9 in accordance with the OECD 111 test guideline using GC/MS to assess test concentrations under each condition. The substance was assessed initially at all three pH at elevated temperature over a 5 day period and it was determined that the no hydrolysis occurred at pH 4 but the substance hydrolysed at pH 7 and 9.

These latter pH's were assessed at 15 and 25 C and increased hydrolysis occurred at higher temperature as compared to the lower temperature. The substance is therefore considered to be hydrolytically unstable at both increasing pH and increasing tempertaure and is considered to have a half life of 18.5 days at ambient temperature and pH.