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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 May 2018 to 06 August 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
2016
Deviations:
yes
Remarks:
See below
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
2004
Deviations:
yes
Remarks:
See below
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:
not specified
Principles of method if other than guideline:
Recovery results were outside the quality criteria set in the guidelines.
Sterility confirmation tests were not conducted at the end of the higher Tier test.
Proposed pathway of hydrolysis was not provided in the study report.
GLP compliance:
yes
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
TESTS CONDUCTED
Tier 1 (pH 4, 7 and 9) and Tier 2 (pH 7 and 9)

PREPARATION OF THE TEST SOLUTIONS
- Test item was spiked to sterile buffers using a test item solution in acetonitrile.
- Target concentration: 200 µg/L
- For each sampling time (see below), duplicate sterile vessels under vacuum were filled with 6 mL test solution.
- Blank buffers containing a similar content of co-solvent (acetonitrile) were treated similarly as the test samples taken at t=0.
- The vessels were placed in a temperature controlled environment in the dark.

SAMPLING DETAILS
- Samples for analysis were taken immediately after preparation (t=0), at t= 5 days (in case of Tier 1) or at several sampling points > t=0 (in case of Tier 2).
- Samples taken from test solutions with a temperature > 20°C were cooled to room temperature using running tap water prior to further treatment.

DETAILS ON PRETREATMENT PRIOR TO ANALYSIS
The samples were diluted in a 1:1 (v:v) ratio with acetonitrile and analyzed. If necessary, the samples were further diluted with 50/50 (v/v) acetonitrile/water (Tier 1) or with 50/50 (v/v) acetonitrile/buffer pH 7 or pH 9 (Tier 2) to obtain concentrations within the calibration range.

pH MEASUREMENTS
The pH of each of the test solutions (except for the blanks) was determined at least at the beginning and at the end of the test.
Buffers:
Buffer pH 4: Solution of 16.7% 0.01 M sodium acetate and 83.3% 0.01 M acetic acid in water.
Buffer pH 7: Solution of 0.01 M potassium di-hydrogenphosphate in water adjusted to pH 7 using 1 N sodium hydroxide.
Buffer pH 9: Solution of 0.01 M boric acid and 0.01 M potassium chloride in water adjusted to pH 9 using 1 N sodium hydroxide.

Details:
- Kind of water: tap water purified by a purification system (Milli-Q, Millipore)
- Sodium azide content: 0.0009% (w/v)

Details on test conditions:
TEST SYSTEM
- Sterilisation method: Each buffer was filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter (Whatman) and transferred into a sterile vessel.
- Measures to exclude oxygen: nitrogen gas was purged through the sterile buffers for 5 minutes.
- Measures taken to avoid photolytic effects: the test vessels were kept in the dark.

CO-SOLVENT
- The volume of the co-solvent was < 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume.

DETAILS TIER 1
- pH 4.0 at 50.0 ± 0.1°C
- pH 7.0 at 50.0 ± 0.1°C
- pH 9.0 at 50.0 ± 0.1°C

DETAILS TIER 2
- pH 6.9 - 7.1 at 19.7 ± 0.3°C, 50.0 ± 0.1°C and 60.1 ± 0.2°C
- pH 8.9- 9.0 at 19.9 ± 0.1°C, 50.0 ± 0.1°C and 60.2 ± 0.1°C




Duration:
312 h
pH:
7
Temp.:
20 °C
Initial conc. measured:
>= 0.092 - <= 0.094 mg/L
Remarks:
Tier 2. Duplicate test solutions.
Duration:
98.98 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
>= 0.127 - <= 0.133 mg/L
Remarks:
Tier 2. Duplicate test solutions.
Duration:
94.38 h
pH:
7
Temp.:
60 °C
Initial conc. measured:
>= 0.119 - <= 0.14 mg/L
Remarks:
Tier 2. Duplicate test solutions.
Duration:
165.38 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
>= 0.132 - <= 0.195 mg/L
Remarks:
Tier 2. Duplicate test solutions.
Duration:
192.35 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
>= 0.141 - <= 0.162 mg/L
Remarks:
Tier 2. Duplicate test solutions.
Duration:
22.1 h
pH:
9
Temp.:
60 °C
Initial conc. measured:
>= 0.074 - <= 0.118 mg/L
Remarks:
Tier 2. Duplicate test solutions.
Number of replicates:
Two
Positive controls:
no
Negative controls:
no
Preliminary study:
At pH 4, a decrease of substance concentration of < 10% was observed after 5 days. At pH 7 and pH 9, a decrease of substance concentration of ≥ 10% was observed after 5 days. See table in the field 'Any other information on results incl. tables'.
Test performance:
RECOVERIES
- Recovery is the concentration analysed at t=0 relative to the nominal concentration.
- The mean (n=2) recovery at each pH and temperature was calculated (see table below).
- Quality criteria: 90-110%

TIER 1
Mean recovery at pH 9 (71%)
Mean recovery at pH 4 (114%) and at pH 7 (53%). The low recovery at pH 7 might be an indication that other processes play a role besides or instead of hydrolysis. The high recovery obtained at pH 4 is not discussed in the study report.

TIER 2
Mean recoveries at pH 7 (46%, 65% and 65%): unacceptable
Mean recoveries at pH 9 (48%, 76% and 82%): unacceptable
These low values might be an indication that other processes play a role besides or instead of hydrolysis.
Transformation products:
not specified
Details on hydrolysis and appearance of transformation product(s):
no data
% Recovery:
46
pH:
7
Temp.:
20 °C
Duration:
0 h
Remarks on result:
other: Tier 2
% Recovery:
65
pH:
7
Temp.:
50 °C
Duration:
0 h
Remarks on result:
other: Tier 2
% Recovery:
65
pH:
7
Temp.:
60 °C
Duration:
0 h
Remarks on result:
other: Tier 2
% Recovery:
82
pH:
9
Temp.:
20 °C
Duration:
0 h
Remarks on result:
other: Tier 2
% Recovery:
76
pH:
9
Temp.:
50 °C
Duration:
0 h
Remarks on result:
other: Tier 2
% Recovery:
48
pH:
9
Temp.:
60 °C
Duration:
0 h
Remarks on result:
other: Tier 2
Key result
pH:
4
Temp.:
25 °C
DT50:
> 1 yr
Remarks on result:
hydrolytically stable based on preliminary test
Details on results:
The preliminary study indicated that the substance is hydrolytically stable at pH 4 (half-life time at 25°C is > 1 year) and no further study was performed at pH 4.

pH 7: a decrease of substance concentration of ≥ 10% was observed after 5 days and 50°C (Tier 1)
pH 7: no decrease of substance concentration of ≥ 10% was observed within 312.08 h and 20°C (Tier 2)
pH 7: a decrease of substance concentration of ≥ 10% was observed after 4.38 h and 50°C (Tier 2)
pH 7: a decrease of substance concentration of ≥ 10% was observed after 0.23 h and 60°C (Tier 2)

pH 9: a decrease of substance concentration of ≥ 10% was observed after 5 days and 50°C (Tier 1)
pH 9: a decrease of substance concentration of ≥ 10% was observed after 44.17 h and 20°C (Tier 2)
pH 9: a decrease of substance concentration of ≥ 10% was observed after 18.55 h and 50°C (Tier 2)
pH 9: a decrease of substance concentration of ≥ 10% was observed after 0.17 h and 60°C (Tier 2)

From the Tier 1 and Tier 2 studies with pH 7 and pH 9 it was concluded that substance concentration decreases in time. However, no linear Arrhenius equation could be constructed from the Tier 2 results. As the measured decrease in substance concentration was not consistent with a hydrolysis process it was assumed that other processes might play a role besides or instead of hydrolysis. As a consequence no rate constant (and half-life time) at 25°C could calculated for pH 7 and pH 9. No half-life time at 25°C could calculated for pH 7 and pH 9.

Table: Preliminary test at pH 4, pH 7 and pH 9 and 50°C

pH code

Sampling time

Analyzed concentration
[mg/L]

Degree of hydrolysis [%]

pH

Individual

Mean

pH 4

0 hours

0.224

 

 

4.1

 

 

0.232

 

 

4.1

 

5 days

0.205

10

9.5

4.1

 

 

0.208

8.8

 

4.1

pH 7

0 hours

0.109

 

 

7.1

 

 

0.103

 

 

7.0

 

5 days

0.0153

86

85

7.1

 

 

0.0159

85

 

7.1

pH 9

0 hours

0.149

 

 

9.0

 

 

0.134

 

 

9.0

 

5 days

0.0258

82

82

9.0

 

 

0.0257

82

 

9.0

Small responses at the retention time of the test item were detected in the chromatograms of the blank buffer solutions (Tier 1 and Tier 2). It was considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks.

Conclusions:
The substance is hydrolytically stable at pH 4 (half-life time at 25°C of > 1 year). The substance is not stable at pH 7 and pH 9. Tier 2 results indicated that other processes for substance decrease play a role besides or instead of hydrolysis. Half-life times at 25°C at pH 7 and pH 9 could therefore not be determined.
Executive summary:

Hydrolysis of the substance at pH values normally found in the environment (pH 4, 7 and 9) was investigated in a GLP-compliant study according to EC C.7, OECD 111 and EPA OPPTS 835.2120. The buffers used in the Tier 1 and Tier 2 tests were prepared in tap water purified by a Millipore purification system. A degree of hydrolysis of < 10% was observed at pH 4 after 5 days at 50°C (Tier 1). This corresponds with a half-life time at 25°C of > 1 year. The substance is concluded to be hydrolytically stable at pH 4. A decrease in concentration ≥ 10% was determined at pH 7 and pH 9 after 5 days and 50°C (Tier 1). The substance showed pseudo-first order kinetics in the Tier 2 test at pH 7 and pH 9. Plotting the logarithm of rate constant (kobs) versus the inverse of temperature (Arrhenius equation) however did not result in a linear relationship. As a consequence no rate constant (and half-life time) at 25°C could be calculated for pH 7 and pH 9. It was assumed in the study report that the measured decrease in substance concentration in the Tier 1/Tier 2 tests at both pH 7 and 9 was not consistent with a hydrolysis process; other processes might play a role besides or instead of hydrolysis. A proposed pathway of hydrolysis or of any other process was not provided in the study report. Recoveries of < 90% were determined for pH 7 and pH 9 and therefore not fulfilling the quality criteria.

Description of key information

The substance is hydrolytically stable at pH 4 (half-life time at 25°C of > 1 year).

The substance is not stable at pH 7 and at pH 9. No half-life times could be determined because other processes possibly play a role here besides or instead of hydrolysis.

Key value for chemical safety assessment

Additional information

Hydrolysis of the substance at pH values normally found in the environment (pH 4, 7 and 9) was investigated in a GLP-compliant study according to EC C.7, OECD 111 and EPA OPPTS 835.2120. The buffers used in the Tier 1 and Tier 2 tests were prepared in tap water purified by a Millipore purification system. A degree of hydrolysis of < 10% was observed at pH 4 after 5 days at 50°C (Tier 1). This corresponds with a half-life time at 25°C of > 1 year. The substance is concluded to be hydrolytically stable at pH 4. A decrease in concentration ≥ 10% was determined at pH 7 and pH 9 after 5 days and 50°C (Tier 1). The substance showed pseudo-first order kinetics in the Tier 2 test at pH 7 and pH 9. Plotting the logarithm of rate constant (kobs) versus the inverse of temperature (Arrhenius equation) however did not result in a linear relationship. As a consequence no rate constant (and half-life time) at 25°C could be calculated for pH 7 and pH 9. It was assumed in the study report that the measured decrease in substance concentration in the Tier 1/Tier 2 tests at both pH 7 and 9 was not consistent with a hydrolysis process; other processes might play a role besides or instead of hydrolysis. A proposed pathway of hydrolysis or of any other process was not provided in the study report. Recoveries of < 90% were determined for pH 7 and pH 9 and therefore not fulfilling the quality criteria.