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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:
2019-04-18 - 2020-01-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
Sample preparation: At each test point approx. 1.5 mL of the test solution were taken and measured directly or otherwise frozen with liquid nitrogen. Plastic vials were used.
Buffers:
pH: 4
Composition of buffer: Citric acid/NaOH/NaCl

pH: 7
Composition of buffer: KH2PO4/Na2HPO4

pH: 9
Composition of buffer: Na2B4O7/HCl
Details on test conditions:
Calibration
The test item was used as standard compound for the calibration with a stipulated overall content of 100 % (CoA). The calibration was done on every measurement day before and after the test samples.

Preparation of standard solutions used for calibration
Approx. 51.0 mg of the test item were dissolved in dichloromethane and filled up with dichloromethane to the mark in a 50 mL volumetric flask to prepare a stock solution of 1019.4 mg/L. Thereafter, 0.49 mL of the stock solution was added to acetonitrile in a 50 mL volumetric flask and filled up to the mark.
This gives a solution containing 9.99 mg/L test substance.
Defined volumes of this solution were diluted with acetonitrile to obtain nine standard solutions in the range of 0.25 to 25.0 µg/L. These solutions were used to calibrate the HPLC-system and the calibration results were applied to the experiments.
The concentration of the lowest calibration solution used was employed as the limit of quantification (0.25 µg/L).

Preparation of the solution used for verification of the calibration
The recovery and repeatability were checked during the experiment by analyzing independent solutions for verification of the calibration on every day of analysis between the sample solutions (QC-standard).
The QC-standard was prepared analogous to the calibration solutions at a concentration of 5 µg/L.
The HPLC chromatograms of the control solutions show that the test item gives a well-resolved peak.

Preparation of a test item stock solution
A stock solution of the test item was prepared by weighing 22.8 mg of the test item (assay 100.0 %) in a 100 mL volumetric flask. The flask was then filled up to the mark with acetone, resulting in a concentration of 228.0 mg/L.
A second stock solution of the test item was prepared by weighing 24.5 mg of the test item (assay 100.0 %) in a 100 mL volumetric flask. The flask was then filled up to the mark with acetone, resulting in a concentration of 245.0 mg/L.

Preparation of the hydrolysis test solutions
2.19 mL of the test item stock solution were diluted to 50 mL with acetone. The resulting solution was then diluted by the factor of 5 with acetone to obtain a test item solution with a final concentration of 1.997 mg/L (L1).
2.04 mL of the second test item stock solution were diluted to 50 mL with acetone. The resulting solution was then diluted by the factor of 5 with acetone to obtain a test item solution with a final concentration of 1.999 mg/L (L2).
The final hydrolysis test solutions were prepared by adding 3 mL of the diluted solution (L1 / L2) to 297 mL of the respective buffer solution resulting in final concentrations of 19.973 / 19.992 µg/L.
For determination of the starting value (T0), the fresh prepared hydrolysis test solution was transferred into a sample vial. At each subsequent test point individual sample solutions were prepared in the same way.

Analysis of the hydrolysis sample solutions
The concentration of the test item and the proposed hydrolysis product in all sample solutions was determined by HPLC-MS/MS at defined time intervals.

Calculation of hydrolysis
Hydrolysis was observed and calculated by determination of the concentration of the test item in the test solutions. In addition, the concentration of the hydrolysis product was monitored over time in each test solution.
Calculation was done using an external standardization method and a calibration curve. In an external standard calibration method, the absolute analyte response is plotted against the analyte concentration to create the calibration curve. The resulting calibration curve is applied and the amount of analyte present is determined.
To verify the calibration the recovery rate of the QC-standard was calculated.

pH determination
The pH determination of the hydrolysis test solutions was performed with pH-meter with single-rod glass electrode.

Sterility test
A plate count test was conducted at the end the hydrolysis tests.

Solubility and test concentration
The test item is not soluble in water in sufficient quantities (water solubility < 0.53 µg/L). Therefore, an organic solvent has to be used to obtain an adequate dissolution in the different buffer solutions. According to the guideline the organic solvent does not exceed 1 % (v/v).
The tests were conducted at an initial concentration of approx. 20 µg/L. Clear solutions were obtained which fulfills the requirements of OECD 111 allowing the use of low amounts of water miscible solvents (max. 1 % v/v) for adequate dissolution of the test item.
Duration:
409 h
pH:
4
Temp.:
10 °C
Initial conc. measured:
14.4 µg/L
Duration:
148 h
pH:
4
Temp.:
20 °C
Initial conc. measured:
15 µg/L
Duration:
24 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
16.1 µg/L
Duration:
763 h
pH:
7
Temp.:
20 °C
Initial conc. measured:
11.9 µg/L
Duration:
529 h
pH:
7
Temp.:
30 °C
Initial conc. measured:
16.4 µg/L
Duration:
125 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
15.7 µg/L
Duration:
763 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
12.5 µg/L
Duration:
596 h
pH:
9
Temp.:
30 °C
Initial conc. measured:
9.11 µg/L
Duration:
240 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
15.4 µg/L
Number of replicates:
Experiments were performed once.
Positive controls:
no
Negative controls:
no
Statistical methods:
Calibration
Correlation
A good correlation between the injected amount of the test item and the detector response was observed in the concentration range 0.25 to 25.0 µg/L. Nine concentrations were measured and the correlation coefficient (r) was for every calibration (each day a new one) greater than 0.99.
Limit of quantitation
The limit of quantitation (LOQ) for the test item is defined as the lowest concentration used for calibration. The LOQ is therefore 0.25 µg/L.

Sensitivity
Regarding the chromatogram of the lowest concentration of 0.25 µg/L used for calibration, the analytical method is sufficiently sensitive to quantify test item concentrations down to 10 % or less of the initial concentration used in the hydrolysis experiment.
No residues of test item were detected in blank values of untreated control samples.

Recovery and Repeatability
The recovery and repeatability of the analytical method were checked during the hydrolysis study by analyzing independent control solutions (QC-standards) of the test item at a concentration of 5 µg/L. The control solution was analyzed on the days of application before, between and after the sample solutions
Results of the solutions for verification of the calibration show stability of the chromatographic system. Recoveries of the test item in the range from 92 % to 125 % (mean 105 %) and a relative standard deviation (RSD) of 1.83 % indicate a satisfying repeatability and precision of the method applied to quantify the test item concentrations at the respective level over the test duration.
Preliminary study:
Based on previous data (Cizek, 2012) hydrolytic unstability of the substance was known.
FIRST PRELIMINARY TEST
Before the study was initiated, solubility pre-tests of the test item were performed at three different concentration levels in buffer solutions. One individual sample was prepared for each concentration (10, 20 and 40 µg/L) and the concentration of the test item in the test solutions was determined by HPLC-MS/MS applying the analytical method described above.
The test item was found to be soluble in demineralized water including 1 % acetone as organic solvent additive with a solubility of at least 40 µg/L.
According to OECD Guideline 111 the concentration of the test item should not exceed 0.01 mol/L or half of the saturation concentration. Consequently, the test item was applied in buffer solutions (including 1 % acetone) with a concentration of 20 µg/L.
A clear solution was obtained which fulfills the requirements of OECD 111 allowing the use of low amounts of water miscible solvents (max. 1 % v/v) for adequate dissolution of the test item.
In compliance with OECD 111 Guideline no further solubility test with organic solvent additives > 1% v/v was performed.

SECOND PRELIMINARY TEST
To have the opportunity to measure the test samples preferably together, freezing of the test samples was tested.
It has to be ruled out that the test item precipitates due to the freezing which would lead to wrong concentrations.
Therefore the test solutions from FIRST PRELIMINARY TEST of the concentrations of 10 and 20 µg/L each were a) frozen directly, b) diluted 1:1 with acetone and c) diluted 1:1 with acetonitrile and afterwards frozen in with liquid nitrogen.
The frozen samples were kept frozen until the next day and then defrosted right before measurement. The recoveries were determined and all were acceptable. Because the LOQ raises by dilution and a new source of error by dilution occurs, the freezing of the undiluted sample solution was chosen for the main test.
Test performance:
Neither unusual observations nor deviations from test procedure as well as no other information affecting results is reported.
Transformation products:
yes
Remarks:
based on theoretical considerations and available data from previous studies
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
Degradation product #1:
- RT: approx. 1.29 min
- MW: 177
- Fragments: 178.1 [M+H]+ → 133.0
- Chemical name: 2,6-diisopropylaniline (DIPA) (CAS 24544-04-5)

The development of the degradation product was screened in all test solutions (counts) via HPLC-MS/MS.
% Recovery:
6.5
pH:
4
Temp.:
10 °C
Duration:
409 h
% Recovery:
3.3
pH:
4
Temp.:
20 °C
Duration:
148 h
% Recovery:
5.4
pH:
4
Temp.:
50 °C
Duration:
24 h
% Recovery:
8.4
pH:
7
Temp.:
20 °C
Duration:
763 h
% Recovery:
2.9
pH:
7
Temp.:
30 °C
Duration:
529 h
% Recovery:
8.9
pH:
7
Temp.:
50 °C
Duration:
125 h
% Recovery:
15.2
pH:
9
Temp.:
20 °C
Duration:
763 h
% Recovery:
6.1
pH:
9
Temp.:
30 °C
Duration:
596 h
% Recovery:
6.7
pH:
9
Temp.:
50 °C
Duration:
240 h
pH:
4
Temp.:
10 °C
Hydrolysis rate constant:
0 s-1
DT50:
102.4 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
32.9 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
5.79 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0 s-1
DT50:
28.36 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: calculated based on measured temperatures for this pH
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
222.5 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
30 °C
Hydrolysis rate constant:
0 s-1
DT50:
104.5 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
36.3 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0 s-1
DT50:
153.62 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: calculated based on measured temperatures for this pH
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0 s-1
DT50:
227.6 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
30 °C
Hydrolysis rate constant:
0 s-1
DT50:
156.4 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
64.12 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0 s-1
DT50:
186.31 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: calculated based on measured temperatures for this pH
Other kinetic parameters:
No further parameters were observed.
Details on results:
Sterility test
A plate count test was conducted at the end the hydrolysis tests. All tests were negative; therefore biotic degradation can be excluded for all tested solutions.

pH, temperature, and other experimental conditions maintained throughout the study: Yes
Anomalies or problems encountered: None reported.

pH 4:
The overall degradation of the test item observed at 10 °C and pH 4 is approx. 93.5 % after 409 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.
The overall degradation of the test item observed at 20 °C and pH 4 is approx. 96.7 % after 148 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.
The overall degradation of the test item observed at 50 °C and pH 4 is approx. 94.6 % after 24 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.

pH 7:
The overall degradation of the test item observed at 20 °C and pH 7 is approx. 91.6 % after 763 hours (32 days). According to the OECD TG 111 the test was terminated after a degradation of > 90 %.
The overall degradation of the test item observed at 30 °C and pH 7 is approx. 97.1 % after 529 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.
The overall degradation of the test item observed at 50 °C and pH 7 is approx. 91.1 % after 125 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.

pH 9:
The overall degradation of the test item observed at 20 °C and pH 9 is approx. 84.8 % after 763 hours (32 days). According to the OECD TG 111 the test was terminated after a degradation of > 90 %.
The overall degradation of the test item observed at 30 °C and pH 9 is approx. 93.9 % after 596 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.
The overall degradation of the test item observed at 50 °C and pH 9 is approx. 93.3 % after 240 hours. According to the OECD TG 111 the test was terminated after a degradation of > 90 %.

In all tested samples the degradation product was observed.
Validity criteria fulfilled:
yes
Remarks:
Quality criteria passed
Conclusions:
For the test item abiotic degradation > 90 % was observed for all measured test solutions at pH 4, 7, 9 and temperatures up to 50 °C. As degradation product 2,6-Diisopropylaniline was detected and its qualitative increase monitored over the time.
Executive summary:

The tests were performed based on OECD Guidelines for Testing of Chemicals, Section 1 – Physical-Chemical Properties, OECD 111, Council Regulation (EC) No 440/2008, Guideline Part C – Methods for the Determination of Ecotoxicity, C.7. “Abiotic Degradation: Hydrolysis as a Function of pH” in a GLP-study.

As the test item was expected to hydrolysis in aqueous media the hydrolysis behavior was investigated at different temperatures and pH values in demineralized water up to a degradation of > 90 %. The stability was monitored by HPLC analysis with MS/MS-detection.

The concentration of the test item in the hydrolysis test solutions was determined by HPLC-MS/MS at defined time intervals. In addition, the development of the proposed degradation product was screened in all test solutions (counts).

According to OECD 111 Guideline a sterility test was conducted at the end of the hydrolysis tests. No microbes (colonies) were found in all test solutions. Therefore, biotic degradation can be excluded for all tested solutions.

Due to the poor water solubility of the test item, a test concentration of approx. 20 μg/L (including 1 % acetone as organic solvent additive) had to be applied for adequate dissolution. In addition to the test item itself the mass trace of the proposed degradation product 2,6-Diisopropylaniline was screened. The counts were recorded without external calibration.

Preliminary tests

Test concentration

Because the test item is not soluble in water in sufficient quantities (water solubility < 0.53 μg/L, an organic solvent has to be used to obtain an adequate dissolution in the different buffer solutions. According to the guideline the organic solvent does not exceed 1 % (v/v).

Before the hydrolysis study itself was initiated, solubility pre-tests of the test item were performed at three different concentration levels in buffer solutions. One individual sample was prepared for each concentration (10, 20 and 40 μg/L) and the concentration of the test item in the test solutions was determined by HPLC-MS/MS.

The test item was found to be soluble in demineralized water including 1 % acetone as organic solvent additive with a solubility of at least 40 μg/L.

According to OECD Guideline 111 the concentration of the test item should not exceed 0.01 mol/L or half of the saturation concentration. Consequently, the test item was applied in buffer solutions (including 1 % acetone) with a concentration of 20 μg/L. Clear solutions were obtained which fulfills the requirements of OECD 111 allowing the use of low amounts of water miscible solvents (max. 1 % v/v) for adequate dissolution of the test item.

Freezing of test samples

To have the opportunity to measure the test samples preferably together, freezing of the test samples was tested.

It has to be ruled out that the test item precipitates due to the freezing which would lead to wrong concentrations.

The test solutions from the solubility test of the concentrations of 10 and 20 μg/L each were a) frozen directly, b) diluted 1:1 with acetone and c) diluted 1:1 with acetonitrile and afterwards frozen in with liquid nitrogen.

The frozen samples were kept frozen until the next day and then defrosted right before measurement. The recoveries after measurement were acceptable. Because the LOQ raises by dilution and a new source of error by dilution occurs, the freezing of the undiluted sample solution was chosen for the main test.

Analytical result

For the test item abiotic degradation > 90 % was observed for all measured test solutions.

The tests were performed in buffer solutions at pH values of 4, 7 and 9 at different temperatures.

For pH 4 the temperatures of 10, 20 and 50 °C were investigated. For pH 7 and pH 9 it was 20, 30 and 50 °C.

Calculated hydrolysis rate at 25 °C

Based on the measurements at different temperatures the correlating hydrolysis rate and the Half-Life time at 25 °C was calculated according to the Arrhenius equation:

 Calculated hydrolysis of the test item at 25 °C

 pH 4

 pH 7

 pH 9

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 28.36

 6.79 • 10-8

 153.62

 1.25 • 10-6

 186.31

 1.03 • 10-6

 Correlation coefficient

 0.99121

 0.99757

 0.99785

pH 4

The test item is shown to be hydrolytically unstable at 10, 20 and 50 °C for pH value 4. The concentration of the test item after the last measurements was < 10% of the initial value. Therefore the test item is considered to be hydrolytically unstable within the tested parameters.

The degradation of the test item can be described by first order kinetics. Half-life times and hydrolysis rates were calculated:

 Calculated hydrolysis of the test item at 25 °C

10 °C

20 °C

 50 °C

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 102.35

 1.88 • 10 -6

 32.93

 5.85 • 10-6

 5.79

 3.33 • 10 -5

pH 7

The test item is shown to be hydrolytically unstable at 20, 30 and 50 °C for pH value 7. The concentration of the test item after the last measurement was < 10% of the initial value. Therefore the test item is considered to be hydrolytically unstable within the tested parameters.

The degradation of the test item can be described by first order kinetics.

Half-life times and hydrolysis rates were calculated:

 Calculated hydrolysis of the test item at 25 °C

20 °C

30 °C

 50 °C

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 222.46

 8.66 • 10 -7

 104.50

 1.84 • 10-6

 36.309

 5.30 • 10-6

pH 9

The test item is shown to be hydrolytically unstable at 20, 30 and 50 °C for pH value 9. The concentration of the test item after the last measurement was < 10% of the initial value. Therefore the test item is considered to be hydrolytically unstable within the tested parameters.

The degradation of the test item can be described by first order kinetics. Half-life times and hydrolysis rates were calculated:

 Calculated hydrolysis of the test item at 25 °C

20 °C

30 °C

 50 °C

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 227.56

 8.46 • 10-7

 156.38

 1.23 • 10-6

 64.13

 3.00 • 10-6

As degradation product 2,6-Diisopropylaniline was detected and its qualitative increase monitored over the time.

Description of key information

The tests were performed based on OECD Guidelines for Testing of Chemicals, Section 1 – Physical-Chemical Properties, OECD 111, Council Regulation (EC) No 440/2008, Guideline Part C – Methods for the Determination of Ecotoxicity, C.7. “Abiotic Degradation: Hydrolysis as a Function of pH” in a GLP-study.

As the test item was expected to hydrolyse in aqueous media the hydrolysis behavior was investigated at different temperatures and pH values in demineralized water up to a degradation of > 90 %. The stability was monitored by HPLC analysis with MS/MS-detection.

The concentration of the test item in the hydrolysis test solutions was determined by HPLC-MS/MS at defined time intervals. In addition, the development of the proposed degradation product was screened in all test solutions (counts).

According to OECD 111 Guideline a sterility test was conducted at the end of the hydrolysis tests. No microbes (colonies) were found in all test solutions. Therefore, biotic degradation can be excluded for all tested solutions.

Due to the poor water solubility of the test item, a test concentration of approx. 20 μg/L (including 1 % acetone as organic solvent additive) had to be applied for adequate dissolution. In addition to the test item itself the mass trace of the proposed degradation product 2,6-Diisopropylaniline was screened. The counts were recorded without external calibration.

Preliminary tests

Test concentration

Because the test item is not soluble in water in sufficient quantities (water solubility < 0.53 μg/L, an organic solvent has to be used to obtain an adequate dissolution in the different buffer solutions. According to the guideline the organic solvent does not exceed 1 % (v/v).

Before the hydrolysis study itself was initiated, solubility pre-tests of the test item were performed at three different concentration levels in buffer solutions. One individual sample was prepared for each concentration (10, 20 and 40 μg/L) and the concentration of the test item in the test solutions was determined by HPLC-MS/MS.

The test item was found to be soluble in demineralized water including 1 % acetone as organic solvent additive with a solubility of at least 40 μg/L.

According to OECD Guideline 111 the concentration of the test item should not exceed 0.01 mol/L or half of the saturation concentration. Consequently, the test item was applied in buffer solutions (including 1 % acetone) with a concentration of 20 μg/L. Clear solutions were obtained which fulfills the requirements of OECD 111 allowing the use of low amounts of water miscible solvents (max. 1 % v/v) for adequate dissolution of the test item.

Freezing of test samples

To have the opportunity to measure the test samples preferably together, freezing of the test samples was tested.

It has to be ruled out that the test item precipitates due to the freezing which would lead to wrong concentrations.

The test solutions from the solubility test of the concentrations of 10 and 20 μg/L each were a) frozen directly, b) diluted 1:1 with acetone and c) diluted 1:1 with acetonitrile and afterwards frozen in with liquid nitrogen.

The frozen samples were kept frozen until the next day and then defrosted right before measurement. The recoveries after measurement were acceptable. Because the LOQ raises by dilution and a new source of error by dilution occurs, the freezing of the undiluted sample solution was chosen for the main test.

Analytical result

For the test item abiotic degradation > 90 % was observed for all measured test solutions.

The tests were performed in buffer solutions at pH values of 4, 7 and 9 at different temperatures.

For pH 4 the temperatures of 10, 20 and 50 °C were investigated. For pH 7 and pH 9 it was 20, 30 and 50 °C.

Calculated hydrolysis rate at 25 °C

Based on the measurements at different temperatures the correlating hydrolysis rate and the Half-Life time at 25 °C was calculated according to the Arrhenius equation:

 Calculated hydrolysis of the test item at 25 °C

 pH 4

 pH 7

 pH 9

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 28.36

 6.79 • 10-8

 153.62

 1.25 • 10-6

 186.31

 1.03 • 10-6

 Correlation coefficient

 0.99121

 0.99757

 0.99785

pH 4

The test item is shown to be hydrolytically unstable at 10, 20 and 50 °C for pH value 4. The concentration of the test item after the last measurements was < 10% of the initial value. Therefore the test item is considered to be hydrolytically unstable within the tested parameters.

The degradation of the test item can be described by first order kinetics. Half-life times and hydrolysis rates were calculated:

 Calculated hydrolysis of the test item at 25 °C

10 °C

20 °C

 50 °C

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 102.35

 1.88 • 10-6

 32.93

 5.85 • 10-6

 5.79

 3.33 • 10-5

pH 7

The test item is shown to be hydrolytically unstable at 20, 30 and 50 °C for pH value 7. The concentration of the test item after the last measurement was < 10% of the initial value. Therefore the test item is considered to be hydrolytically unstable within the tested parameters.

The degradation of the test item can be described by first order kinetics.

Half-life times and hydrolysis rates were calculated:

 Calculated hydrolysis of the test item at 25 °C

20 °C

30 °C

 50 °C

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 222.46

 8.66 • 10-7

 104.50

 1.84 • 10-6

 36.309

 5.30 • 10-6

pH 9

The test item is shown to be hydrolytically unstable at 20, 30 and 50 °C for pH value 9. The concentration of the test item after the last measurement was < 10% of the initial value. Therefore the test item is considered to be hydrolytically unstable within the tested parameters.

The degradation of the test item can be described by first order kinetics. Half-life times and hydrolysis rates were calculated:

 Calculated hydrolysis of the test item at 25 °C

20 °C

30 °C

 50 °C

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Half-life-time [h]

 Hydrolysis rate constant k [1/s]

 Test item

 227.56

 8.46 • 10-7

 156.38

 1.23 • 10-6

 64.13

 3.00 • 10-6

As degradation product 2,6-Diisopropylaniline was detected and its qualitative increase monitored over the time.

Key value for chemical safety assessment

Half-life for hydrolysis:
153.62 h
at the temperature of:
25 °C

Additional information

The above mentioned value for the chemical safety assessment was calculated at a pH-value of 7, which is the most appropriate pH-value due to environmental conditions.

The above mentioned hydrolysis study was performed after the final decision of ECHA on a compliance check, which was issued on 01 August 2017. The study is in line with the provided supporting information (Cizek, 2012 and EFSA 2010). The rate of hydrolysis increases with decreasing pH and temperature. 2,6-Diisopropylaniline is the hydrolyisis product. Only the determined half-lives are lower in the new key study than in the supporting study (Cicek, 2012). This can be explained by the improved analytical method and the enhanced test-design, which becomes necessary because of the very low water solubility (< 0.53 µg/L - Holzaht-Grimme, 2019). To achieve a stable and measureable concentration for hydrolysis-testing in water it was necessary to work with an organic solvent additive. This is described in the preliminary work on the developement of a suitable analytical method for the determination of the hydrolysis properties, where amongst others some solvents were tested for their applicability, because it was observed that the sample builds adducts with some solvents like for example methanol (Ranz, 2020).