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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 11 June, 2012 to 14 Sep, 2012
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:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
(2004)
Deviations:
no
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:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken immediately after preparation (t=0) and at several sampling points after t=0. The samples taken after t=0 were cooled to room temperature using running tap water (except for the samples in the 20°C environment).
Buffers:
-Acetate buffer pH 4, 0.01 M: solution of 16.7% 0.01 M sodium acetate and 83.3% 0.01 M acetic acid.
-Phosphate buffer pH 7, 0.01 M: solution of 0.01 M potassium di-hydrogenphosphate adjusted to pH 7 using 1 N sodium hydroxide.
-Borate buffer pH 9, 0.01 M: solution of 0.01 M boric acid and 0.01 M potassium chloride adjusted to pH 9 using 1 N sodium hydroxide.

The buffers contain 0.0009% (w/v) sodium azide.
Details on test conditions:
- Tier 1 preliminary test was performed at pH 4, 7 and 9 at 49.9 ± 0.2°C.
- Tier 2 main study was performed at pH 4, 7 and 9 each at three different temperatures.
- The buffer solutions were filter-sterilised through a 0.2 µm filter and transferred into sterile vessels.
- To exclude oxygen, nitrogen gas was purged through the solution for 5 min.
- The test substance was spiked to the solutions at a target concentration of 100 µg/L using a spiking solution in acetonitrile.
- The spiking volume was < 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume.
- All solutions containing the test substance were protected from light.
- For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment.
- The pH of each test solution was determined at t=0 and at each sampling point after t=0.
- Initial conc. measured: see attached document (Tables 13 - 18 for pH 4, Tables 25 - 30 for pH 7 and Tables 37-42 for pH 9).
- Blank buffer solutions containing a similar content of blank spiking solution were treated similarly as the test samples and analysed at t=0. At pH 4 and pH 7, no test substance was detected in the blank buffer solutions. At pH 9, no test substance was detected in the blank buffer solutions at the temperatures of 20°C and 40°C but small signals for DPPA and DPHA were observed in the blank buffer solutions at the temperature of 50°C.
Duration:
769.5 h
pH:
4
Duration:
768.5 h
pH:
4
Duration:
724.5 h
pH:
4
Duration:
746.5 h
pH:
7
Duration:
643 h
pH:
7
Duration:
166 h
pH:
7
Duration:
335.5 h
pH:
9
Duration:
28 h
pH:
9
Duration:
22.8 h
pH:
9
Number of replicates:
Two
Positive controls:
no
Negative controls:
no
Preliminary study:
A degree of hydrolysis of ≥ 10% was observed at pH 4, pH 7 and pH 9 after 5 d at 50°C (based on both DPPA and DPHA responses). According to the guideline, the higher Tier test was required to determine the half-life time of the test substance.
Test performance:
RECOVERIES (TIER 2)
- Recovery= Concentration analysed at t=0 relative to the nominal concentration.
- Mean recovery ( mean of duplicate test samples): See Tables 19-20 (pH 4), Tables 31-32 (pH 7) and Tables 43-44 (pH 9) in the attached document.
- The mean recoveries measured for the test solutions at pH 4, pH 7 and pH 9 did fall within the acceptable range of 90-110% (DPPA and DPHA). It demonstrated that the analytical method was adequate to support the hydrolysis study on the test substance.

HYDROLYSIS RATE (TIER 2):
For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10% and 90% were plotted against time

pH 4: linear relationships were obtained at all temperatures indicating pseudo-first order kinetics (DPHA and DPPA).
pH 7: At all temperatures linear relationships were obtained for DPHA. For DPPA nonlinear relationships were obtained at 50°C and 60°C. This is because for this component two processes occur simultaneously: DPPA hydrolyses itself and DPPA is formed as a product from the hydrolysis of DPHA.
pH 9: At all temperatures linear relationships were obtained for DPHA and nonlinear relationships were obtained for DPPA.

For each temperature and pH, all logarithms of the relative concentrations were correlated with time using linear regression analysis (see Figures 7 - 12 in the attached document). Statistical parameters of the regression curves: See Tables 22-23 (pH 4), 33-34 (pH 7) and 45-46 (pH 9) in the attached document.

The half-life times and rate constants (of both components of the substance) were determined according to the model for pseudo-first order reactions. The Arrhenius equation was used to determine the rate constant and half-life time at 25°C. See Tables 23-24 (pH 4), Tables 35-36 (pH 7) and Tables 47-48 (pH 9) in the attached document.

Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
For DPPA, two processes occur simultaneously: DPPA hydrolyses itself and DPPA is formed as a product from the hydrolysis of DPHA. There may be an overestimated half-life time of DPPA since this component is also formed during the hydrolysis of DPPA.
Key result
pH:
4
Temp.:
25 °C
DT50:
ca. 201 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Based on the response of the constituent DPPA
Key result
pH:
7
Temp.:
25 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Based on the response of the constituent DPPA
Key result
pH:
9
Temp.:
25 °C
DT50:
ca. 47 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Based on the response of the constituent DPPA
Key result
pH:
4
Temp.:
25 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Based on the response of the constituent DPHA
Key result
pH:
7
Temp.:
25 °C
DT50:
ca. 81 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Based on the response of the constituent DPHA
Key result
pH:
9
Temp.:
25 °C
DT50:
ca. 29 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Based on the response of the constituent DPHA
Details on results:
Actual pH: 4.0-4.2, 7.0-7.1 and 9.0-9.1.

Preliminary test - Tier 1:

A degree of hydrolysis of ≥ 10% was observed at pH 4, pH 7 and pH 9 after 5 days for both DPPA and DPHA. According to the guideline, the higher Tier test was required to determine the half-life time of the test substance components. No test substance was detected in the blank buffer solutions. The mean recoveries of the of the test substance containing buffer solutions at t=0 fell within the criterion range of 90-110% for both DPPA and DPHA. It demonstrated that the analytical method was adequate to support the hydrolysis study on both components of the test substance.

Conclusions:
Under the study conditions: (a) Half-lives at 25°C and pH 4, 7 and 9 were determined to be 201 d, > 1 year and 47 h, respectively, when based on the response of the constituent DPPA (b) Half-lives at 25°C and pH 4, 7 and 9 were > 1 year, 81 d and 29 h, respectively, when based on the response of the constituent DPHA.
Executive summary:

A study was conducted to determine the rate of hydrolysis of the test substance, DPHA at pH values normally found in the environment (pH 4, 7 and 9) according to EU Method C.7, OECD Guideline 111 and EPA Guideline OPPTS 835.2120, in compliance with GLP. The hydrolysis rates of the test substance were measured at three different temperatures from which the hydrolysis rate at 25°C was determined. Measurements were based on the responses of the two major constituents, DPPA and DPHA. At pH 4, linear relationships were obtained at all temperatures, indicating pseudo-first order kinetics (for both DPHA and DPPA). At pH 7 and pH 9 at all temperatures, linear relationships were obtained for DPHA (pseudo-first order behaviour). For the constituent DPPA, non-linear relationships were obtained at 50 and 60°C for pH 7 and at all temperatures for pH 9. For each temperature and pH, all logarithms of the relative concentrations were correlated with time using linear regression analysis. Under the study conditions: (a) Half-lives at 25°C and pH 4, 7 and 9 were determined to be 201 d, > 1 year and 47 h, respectively, when based on the response of the constituent DPPA (b) Half-lives at 25°C and pH 4, 7 and 9 were > 1 year, 81 d and 29 h, respectively, when based on the response of the constituent DPHA (Lammers, 2012).

Description of key information

Based on the study results:(a) Half-lives at 25°C and pH 4, 7 and 9 were determined to be 201 d, > 1 year and 47 h, respectively, when based on the response of the constituent DPPA (b) Half-lives at 25°C and pH 4, 7 and 9 were > 1 year, 81 d and 29 h, respectively, when based on the response of the constituent DPHA. 

Key value for chemical safety assessment

Additional information

A study was conducted to determine the rate of hydrolysis of the test substance, DPHA at pH values normally found in the environment (pH 4, 7 and 9) according to EU Method C.7, OECD Guideline 111 and EPA Guideline OPPTS 835.2120, in compliance with GLP. The hydrolysis rates of the test substance were measured at three different temperatures from which the hydrolysis rate at 25°C was determined. Measurements were based on the responses of the two major constituents, DPPA and DPHA. At pH 4, linear relationships were obtained at all temperatures, indicating pseudo-first order kinetics (for both DPHA and DPPA). At pH 7 and pH 9 at all temperatures, linear relationships were obtained for DPHA (pseudo-first order behaviour). For the constituent DPPA, non-linear relationships were obtained at 50 and 60°C for pH 7 and at all temperatures for pH 9. For each temperature and pH, all logarithms of the relative concentrations were correlated with time using linear regression analysis. Under the study conditions: (a) Half-lives at 25°C and pH 4, 7 and 9 were determined to be 201 d, > 1 year and 47 h, respectively, when based on the response of the constituent DPPA (b) Half-lives at 25°C and pH 4, 7 and 9 were > 1 year, 81 d and 29 h, respectively, when based on the response of the constituent DPHA (Lammers, 2012).