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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03.03. - 21.08.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
Council regulation (EC) No 440/2008. Published in OJ No L 142/518 May 2008
Deviations:
yes
Remarks:
(see Any other information ...)
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
Adopted April 13, 2004
Deviations:
yes
Remarks:
(see Any other information ...)
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products:
Tier 2 - I: Each reaction solution was analyzed at time intervals that provide spaced data points between 10 % and 90 % hydrolysis of the test substance. The Tier 2 test was stopped after period of 30 days for all pH (4, 7 and 9).
There occurred deviation from the Study plan during the study. The sample solutions were not monitored at time intervals that provide a minimum of six spaced data points normally between 10% and 90% hydrolysis of the test substance. However, this interval had not any observable influence on hydrolysis.

- Sampling method:
Preparation of sample solutions – Tier 2
Approximately 10 mg of Reactive Blue 234 for Tier 2 – I (approximately 100 mg of Reactive Blue 234 for Tier 2 – II) was weighed into a 10 mL volumetric flask, 7 mL of buffer solution of certain pH (4.0, 7.0, or 9.0) was added and then dissolved in an ultrasonic bath for 5 minutes and diluted to volume (Stock Solution – ST.S).

ST.S1 for buffer solution of certain pH 9.0
ST.S2 for buffer solution of certain pH 7.0
ST.S3 for buffer solution of certain pH 4.0

0.5 mL of ST.S was pipetted into a 50 mL volumetric flask and replenished to the volume with a buffered solution of certain pH (4.0, 7.0, or 9.0) – in duplicate (Sample Solution SS). A nominal concentration of 10 mg/L was prepared for hydrolysis of Unstable Substance (Tier 2).
During the experimental part of hydrolysis, the volumetric flasks were placed in the thermostatic bath kept at a given temperature. At certain period of time, a portion of sample solution was transferred into a vial followed by a quick cooling to room temperature.

- Sampling intervals/times for pH measurements:
The actual pH value of each buffer was measured. The pH-meter calibrated to buffer solutions CertiPUR was used.

- Sampling intervals/times for sterility check:
Tier 2 - II: The sterility test was performed after the hydrolysis in a buffered solution of pH 4, pH 7 and pH 9. The buffer solutions of pH 7 were largely contaminated, this was the reason for repeating the test.
The similar test performed at pH 7 for all temperature was conducted in the way to check sterility over time of its running. Some contamination of growth microorganisms was observed as well. It was found that this contamination had no observable influence on hydrolysis.

- Sample storage conditions before analysis:
All sample solutions stored at ambient temperature are stable for minimum of 96 hours.
Change is 0.92 % for buffer pH 9, 3.66 % for buffer pH 7, resp. – 0.59 % for buffer pH 4. Stability fulfils the criterion of acceptability
Buffers:
The hydrolysis of test substance was examined at buffered solutions of selected pH value: 4, 7 and 9. The actual pH value at given temperature was measured using a calibrated pH meter. All prepared buffer solutions were sterilized in an autoclave (121 °C, 50 min) and purged by nitrogen.
Buffered solutions were prepared in accordance with the Method C.7 – Degradation – Abiotic Degradation: Hydrolysis as a Function of pH, Directive 92/69/EEC. Published in OJ No L 142/518 A, May 2008, appendix 3 Clark and Lubs.

Buffer pH 4.0
500 mL 0.1M potassium hydrogen phthalate + 4.0 mL 0.1 M NaOH / 1000 mL UPW
pH = 3.99 (24.6 °C)

Buffer pH 7.0
296 mL 0.1M NaOH + 500 mL 0.1 M potassium dihydrogen phosphate/ 1000 mL UPW
pH = 7.01 (24.6 °C)

Buffer pH 9.0
213 mL 0.1 M NaOH + 500 mL 0.1 M KCl in 0,1 M H3BO3 / 1000 mL UPW.
pH = 9.02 (24.6 °C)
Estimation method (if used):
The results of Tier 2 have shown the linear dependence of logarithm of concentration on time. The hydrolysis was considered to be described as a pseudo-first order reaction.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: volumetric flask, the body of each flask was wrapped in an aluminium foil and tightly closed
- Sterilisation method:
Hydrolysis of Unstable Substance (Tier 2) - I: All glassware, reagent-grade water and buffer solutions were sterilised in autoclave (121 °C, 23 min).

- Measures to exclude oxygen: oxygen is removed from a solution by purging with nitrogen

- Details on test procedure for unstable compounds:
see Test performance below

TEST MEDIUM
- Kind and purity of water: Ultrapure Water (UPW) – prepared in house by water purification system; Water purification system IWA 20iol, Watek

CALIBRATION SOLUTION
Approximately 10 mg of Reactive Blue 234 was weighed into a 10 mL volumetric flask, 7 mL of UPW was added and then dissolved in an ultrasonic bath for 5 minutes and diluted to volume (calibration stock solution - CSS).
This stock solution was diluted to three more concentrations with UPW (calibration solutions - CAL) in this manner:
Tier 1, Tier 2 -I
CS No.1: 0.05 mL CSS/10mL, nominal concentration of RB234 - 5 mg/L
CS No.2: 0.1 mL CSS/10mL, nominal concentration of RB234 - 10 mg/L
CS No.3: 0.2 mL CSS/10mL, nominal concentration of RB234 - 20 mg/L

Tier 2 -II
CS No.1: 0.05 mL CSS/10mL, nominal concentration of RB234 - 5 mg/L
CS No.2: 0.1 mL CSS/10mL, nominal concentration of RB234 - 10 mg/L
CS No.3: 0.5 mL CSS/10mL, nominal concentration of RB234 - 50 mg/L
CS No.4: 1.0 mL CSS/10mL, nominal concentration of RB234 - 100 mg/L

SAMPLE SOLUTION
Approximately 10 mg of Reactive Blue 234 for Tier 2 – I (approximately 100 mg of Reactive Blue 234 for Tier 2 – II) was weighed into a 10 mL volumetric flask, 7 mL of buffer solution of certain pH (4.0, 7.0, or 9.0) was added and then dissolved in an ultrasonic bath for 5 minutes and diluted to volume (Stock Solution – ST.S).
ST.S1 for buffer solution of certain pH 9.0
ST.S2 for buffer solution of certain pH 7.0
ST.S3 for buffer solution of certain pH 4.0
0.5 mL of ST.S was pipetted into a 50 mL volumetric flask and replenished to the volume with a buffered solution of certain pH (4.0, 7.0, or 9.0) – in duplicate (Sample Solution - SS). A nominal concentration of 10 mg/L was prepared for hydrolysis of Unstable Substance (Tier 2).
During the experimental part of hydrolysis, the volumetric flasks were placed in the thermostatic bath kept at a given temperature. At certain period of time, a portion of sample solution was transferred into a vial followed by a quick cooling to room temperature.


OTHER TEST CONDITIONS
- Adjustment of pH: The actual pH value of each buffer was measured. The pH-meter calibrated to buffer solutions CertiPUR was used.
- Dissolved oxygen: oxygen is removed from a solution by purging with nitrogen
Number of replicates:
2 replicates
Positive controls:
no
Negative controls:
no
Statistical methods:
For each pH value and temperature at which Tier 2 is performed, the linearity of data according to Eq. [7] is statistically examined:
If linearity is found sufficient, pseudo first-order rate constant kobs (Eq. [7]), half-life time (Eq. [6]) and the parameters of Arrhenius equation (Eq. [9]) are determined. The pseudo-first order rate constant of hydrolysis is estimated at 25°C (Eq. [8]). (see attached document Calculations)
Preliminary study:
The preliminary test (Tier 1) has shown that the test substance was hydrolysed by more than 10% in period of 4 days at pH 4, pH 7 and pH 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH.
Test performance:
Preliminary test (Tier 1)
The preliminary test was performed at 50 °C over the period of 4 days. A degree of hydrolysis (%) was calculated from the change in concentration. The test has shown that the test substance was hydrolyzed by more than 10 % in period of 4 days at pH 4, 7 and 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH and further experiments were conducted.
Hydrolysis of Unstable Substance (Tier 2) - I
Tier 2 was carried out at three temperatures 23, 35 and 50 °C and buffered solutions of pH 4, pH 7 and pH 9. The temperature of thermostatic bath was controlled during the whole test. The actual pH value of buffer solution at selected temperature was measured. Each reaction solution was analyzed at time intervals that provide spaced data points between 10 % and 90 % hydrolysis of the test substance. The Tier 2 test was stopped after period of 30 days for all pH (4, 7 and 9).
Hydrolysis of Unstable Substance (Tier 2) II
The sterility test was performed after the hydrolysis in a buffered solution of pH 4, pH 7 and pH 9. The buffer solutions of pH 7 were largely contaminated, this was the reason for repeating the test.
The similar test performed at pH 7 for all temperature was conducted in the way to check sterility over time of its running. Some contamination of growth microorganisms was observed as well. It was found that this contamination had no observable influence on hydrolysis.
Transformation products:
no
% Recovery:
48.1
pH:
4.06
Temp.:
50 °C
Duration:
4 d
Remarks on result:
other: Tier 1
% Recovery:
23.8
pH:
6.98
Temp.:
50 °C
Duration:
4 d
Remarks on result:
other: Tier 1
% Recovery:
24.6
pH:
8.85
Temp.:
50 °C
Duration:
4 d
Remarks on result:
other: Tier 1
Key result
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0.011 d-1
DT50:
64.868 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: The test substance was hydrolysed by more than 10% in period of 4 days at pH 4, pH 7 and pH 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH.
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.007 d-1
DT50:
102.08 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: The test substance was hydrolysed by more than 10% in period of 4 days at pH 4, pH 7 and pH 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH.
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.003 d-1
DT50:
240.92 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: The test substance was hydrolysed by more than 10% in period of 4 days at pH 4, pH 7 and pH 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH.
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
Validity criteria fulfilled:
yes
Conclusions:
see Executive summary
Any major hydrolysis product observed during the hydrolysis (at least those representing ≥ 10 % of the applied dose) should be identified by an appropriate analytical method. Any major hydrolysis product was not observed during the hydrolysis.
Executive summary:

The procedure was performed according to: Method C.7 – Degradation – Abiotic Degradation: Hydrolysis as a Function of pH, Council regulation (EC) No 440/2008. Published in OJ No L 142/518 May 2008 and OECD Test Guidline No.111, Hydrolysis as a function of pH, Adopted April 13, 2004.

The preliminary test (Tier 1) has shown that the test substance was hydrolysed by more than 10% in period of 4 days at pH 4, pH 7 and pH 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH.

The results of Tier 2 have shown the linear dependence of logarithm of concentration on time. The hydrolysis was considered to be described as a pseudo-first order reaction.

The half-life times of hydrolysis of test substance at given pH of buffered solution and certain temperature are presented in the following table 1:

Table 1: Summary of results of Tier 2

Conditions

Parameter

k (1 / den)

t(1/2) (den)

pH 4 :

1.0893E-02

6.3632E+01

 (6.9660E-03 - 1.4820E-02)

 (4.6771E+01 - 9.9504E+01)

pH 4 :

1.3793E-02

5.0254E+01

 (1.5186E-03 - 2.6067E-02)

 (2.6591E+01 - 4.5643E+02)

pH 4 :

3.6982E-02

1.8743E+01

 (2.1226E-02 - 5.2737E-02)

 (1.3143E+01 - 3.2656E+01)

pH 7 :

6.4522E-03

1.0743E+02

 (3.0023E-03 - 9.9021E-03)

 (7.0000E+01 - 2.3087E+02)

pH 7 :

9.0905E-03

7.6250E+01

 (5.1279E-03 - 1.3053E-02)

 (5.3102E+01 - 1.3517E+02)

pH 7 :

1.4805E-02

4.6817E+01

 (7.7695E-03 - 2.1841E-02)

 (3.1735E+01 - 8.9214E+01)

pH 9 :

2.4909E-03

2.7827E+02

 (1.6589E-03 - 3.3230E-03)

 (2.0859E+02 - 4.1785E+02)

pH 9 :

7.9196E-03

8.7523E+01

 (6.5177E-03 - 9.3215E-03)

 (7.4360E+01 - 1.0635E+02)

pH 9 :

5.7581E-02

1.2038E+01

 (4.2093E-02 - 7.3069E-02)

 (9.4862E+00 - 1.6467E+01)

 

 

 

 

 

 

 

 

A(pre-exponential factor)

E/R

pH 4

2.6142E+04

4.3858E+03

 (1.0799E-20 - 6.3282E+28)

 (-1.2939E+04 - 2.1710E+04)

pH 7

1.3504E+02

2.9510E+03

 (7.8005E-02 - 2.3378E+05)

 (6.5022E+02 - 5.2518E+03)

pH 9

5.5607E+13

1.1181E+04

 (2.0199E-09 - 1.5308E+36)

 (-4.7625E+03 - 2.7124E+04)

Table 2: Rate constant of hydrolysis of the test substance at given pH of buffered solution

Conditions

Arrhenius equation

pH 4

k = 2.6142E+04 · exp[-(4.3858E+03) / T]

pH 7

k = 1.3504E+02 * exp[-(2.9510E+03) / T]

pH 9

k = 5.5607E+13· exp[-(1.1181E+04) / T]

Table 3: Estimation of rate constants and half-life times at temperature of 25 °C

Conditions

k (1 / den)

t  (1/2) (den)

pH 4

1.0685E-02

6.4868E+01

 (1.2294E-04 - 9.2877E-01)

 (7.4631E-01 - 5.6383E+03)

pH 7

6.7905E-03

1.0208E+02

 (3.7530E-03 - 1.2286E-02)

 (5.6416E+01 - 1.8469E+02)

pH 9

2.8771E-03

2.4092E+02

 (4.7255E-05 - 1.7517E-01)

 (3.9570E+00 - 1.4668E+04)

Description of key information

The procedure was performed according to: Method C.7 – Degradation – Abiotic Degradation: Hydrolysis as a Function of pH, Council regulation (EC) No 440/2008. Published in OJ No L 142/518 May 2008 and OECD Test Guidline No.111, Hydrolysis as a function of pH, Adopted April 13, 2004.

The preliminary test (Tier 1) has shown that the test substance was hydrolysed by more than 10% in period of 4 days at pH 4, pH 7 and pH 9. Therefore the test substance was considered to be hydrolytically unstable at these values of pH.

The results of Tier 2 have shown the linear dependence of logarithm of concentration on time. The hydrolysis was considered to be described as a pseudo-first order reaction.

The half-life times of hydrolysis of test substance at given pH of buffered solution and certain temperature are presented in the table 1 in Robust Study Summary. The estimated half-life times at 25 °C are presented in the table 3.

Any major hydrolysis product observed during the hydrolysis (at least those representing ≥ 10 % of the applied dose) should be identified by an appropriate analytical method. Any major hydrolysis product was not observed during the hydrolysis.

Key value for chemical safety assessment

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

Additional information

Table 3: Estimation of rate constants and half-life times at temperature of 25 °C

Conditions

k (1 / den)

t  (1/2) (den)

pH 4

1.0685E-02

6.4868E+01

 (1.2294E-04 - 9.2877E-01)

 (7.4631E-01 - 5.6383E+03)

pH 7

6.7905E-03

1.0208E+02

 (3.7530E-03 - 1.2286E-02)

 (5.6416E+01 - 1.8469E+02)

pH 9

2.8771E-03

2.4092E+02

 (4.7255E-05 - 1.7517E-01)

 (3.9570E+00 - 1.4668E+04)