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Environmental fate & pathways

Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2004 to 6 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes
Details on sampling:
During each sampling event, an aliquot of each test system was transferred to a microcentrifgue tube using a glass disposable pipette. The samples were centrifuged for approximately 5 minutes at room temperature at approximately 6000 rpm. An aliquot of the supernatant and centrifuge tube was noted. In addition, an aliquot of each test system was transferred to a 5 mL vial using a glass disposable pipette. The appearance of the aliquot was noted and the pH measured.

For the preliminary test, approximately 1 mL of 50:50 acetonitrile:2-propanol (ACN/IPA) was added to the remaining supernatant and 'sediment'. A portion of this solution was transferred to an autosampler vial for analysis using HPLC/UV.

For Test 3, the remaining supernatant was removed using a glass disposable pipette and 1 mL of 50:50 ACN/IPA was added to the 'sediment'. A portion of this solution was transferred to an autosampler vial for analysis using HPLC/UV.
Buffers:
pH: 4
- Type and final molarity of buffer: 0.01 M sodium acetate
- Composition of buffer: Approximately 0.8 g anhydrous sodium acetate (CH3CO2Na) was dissolved for each litre of distilled water and pH adjusted to approximately 4.0 with glacial acetic acid.

pH: 7
- Type and final molarity of buffer: 0.02 M Phosphate buffer
- Composition of buffer: Approximately 1.4 g potassium phosphate monobasic anhydrous (KH2PO4) and 1.7 g potassium phosphate dibasic anhydrous (K2HPO4) were dissolved per litre of distilled water.

pH: 9
- Type and final molarity of buffer: 0.025 M sodium borate buffer
- Composition of buffer: Approximately 9.5 g sodium borate decahydrate (Na2B4O7.10H2O) was dissolved in 1.0 L of distilled water and pH adjusted to approximately 9.0 with 1M HCL.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 250 mL Erlenmeyer flasks
- Sterilisation method: All flasks were autoclaved prior to use
- Other: The Erlenmeyer flasks were wrapped in parafilm and placed in a temperature cotrolled water bath. Test systems (water baths) were monitored using a liquid in glass partial-emersion thermometer. Room temperature was monitored using a liquid glass thermometer.

TEST MEDIUM
- Volume used/treatment 200 mL
- Identity and concentration of co-solvent: 0.5 % DMF
Duration:
24.7 h
pH:
9
Positive controls:
no
Negative controls:
no
Statistical methods:
CALCULATIONS
Test substance concentration:
x = y - b / m

where:
y = peak area of principal test substance peak
m = slope
x = test substance concentration (mg/L)
b = y-intercept

Percent hydrolyzed (change in concentration):
Percent hydrolysis = ([t0] - [tn] / [t0]) x 100

where:
[t0] = concentration of test system at time 0 hours
[tn] = concentration of test system at time n hours
Preliminary study:
The preliminary test and test 1 were originally started without pre-treatment of the samples. What appeared to be hydrolysis was later determined to be test substance precipitating out of solution.
Transformation products:
not measured
Key result
pH:
4
Remarks on result:
not determinable
Key result
pH:
7
Remarks on result:
not determinable
Key result
pH:
9
Remarks on result:
not determinable

The preliminary test and test 1 were originally started without pre-treatment of the samples. What appeared to be hydrolysis was later determined to be test substance precipitating out of solution.

The change in concentration results for the test substance are listed below:

Table 1: pH 4 buffer preliminary test

Time (hours)   pH  Supernatant concentration (mg/L)  Sediment concentration (mg/L)
 0.0  4.11  4.50  1.31
 2.4  4.14  <0.5  2.63
 120  4.00  ND  2.40

ND = no test substance peak detected; instrument detection limit = 0.25 mg/L

Table 2: pH 7 buffer preliminary test

Time (hours)   pH  Supernatant concentration (mg/L)  Sediment concentration (mg/L)
 0.0  6.95  3.51  0.635
 2.4  6.95  <0.5  5.30
 120  6.90  <0.5  3.47

Table 3: pH 9 buffer preliminary test

 Time (hours)  pH  Supernatant concentration (mg/L)  Sediment concentration (mg/L)
 0.0  9.04  5.71  6.07
 2.4  8.94  3.85  <0.5
 120  8.99  ND  1.33

ND = no test substance peak detected; instrument detection limit = 0.25 mg/L

The instrument quantitation limit was based on the lowest standard analysed (0.5 mg/L)

For each test system, the concentration of test substance in supernatant decreased over time. The sediment concentrations are estimates (the exact volume of 50:50 ACN/IPA added was not measured using a pipette. Furthermore, the entire volume of supernatant was not removed before adding the 50:50 ACN/IPA. A sediment concentration that is larger than the corresponding supernatant concentration clearly indicates that some test substance was centrifuged to the bottom/sides of the centrifuge tube. The sediment concentration is greater than the supernatant concentration after 0 hours for pH 4 and 7 indicating that the test substance is not undergoing hydrolysis but precipitating out of solution. No further testing was possible.

The pH 9 preliminary test did not indicate that at time 2.4 hours that the test substance was precipitating out of solution although some test substance did appear to be precipitating out at 120 hours. It was decided to continue with test 3 for pH 9.

Table 4: pH 9 buffer test 3

 Time (hours)  pH  Supernatant concentration (mg/L)  Sediment concentration (mg/L)
 0.0  8.95  2.87  0.823
 1.0  8.97  4.53  1.27
 2.0  8.96  4.20  1.49
 3.0  8.97  3.90  2.46
 4.0  8.97  3.28  2.81
 5.0  8.96  3.24  2.61
 6.0  8.92  2.39  3.18
 24.7  8.98  0.792  3.82

For the pH 9 test system (test 3), the concentration of the test substance in the supernatant decreases over time with the exception of time 0 hour. The sediment concentrations are a better quantitation of true sediment concentration since the exact volume of 50:50 ACN/IPA added was measured using a pipette and the entire volume of supernatant was removed before adding the 50:50 ACN/IPA. The sediment concentration showed a trend of increasing concentration indicating that the test substance was not undergoing hydrolysis but precipitating out of solutions over time. No further testing was possible.

Throughout the preliminary tests and test 3, the test system aliquots were clear and colourless although a slight beam was observed using a Tyndall lamp. All centrifuged samples had clear and colourless supernatant with no visible precipitates.

Validity criteria fulfilled:
not applicable
Conclusions:
Under the conditions of the test, the potential of the test substance to undergo hydrolytic degradation in an aquatic environment could not be determined due to the limited solubility of the test substance in pH 4, 7 and 9 buffers using a 0.5 % co-solvent (DMF).
Executive summary:

In a GLP compliant hydrolysis study conducted in line with OECD Guideline 111 and EU Method C.7, the hydrolysis of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was investigated. The potential of the test substance to undergo hydrolytic degradation could not be determined due to the limited solubility of the substance in pH 4, 7 and 9 buffers.

Description of key information

Key study:- Hoffmann (2004d) 'Abiotic degradation: hydrolysis as a function of pH' conducted in line with OECD Guideline 111 and EU Method C.7. It was not possible to determine the hydrolytic degradation of the test substance due to the limited solubility of the substance in pH 4, 7 and 9 buffers.

Key value for chemical safety assessment

Additional information

The key study (Hoffmann, 2004d) was performed in compliance with GLP and to OECD Guideline 111 and EU Method C.7 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with accepted standardised guidelines and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). It was not possible to determine the hydrolytic degradation of the test substance due to the limited solubility of the substance in pH 4, 7 and 9 buffers.