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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:
17 February 2017 - 18 July 2017
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)
Version / remarks:
2004
Deviations:
yes
Remarks:
see field principles of method if other than guideline
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
(EC) No 440/2008
Deviations:
yes
Remarks:
see field principles of method if other than guideline
Principles of method if other than guideline:
Deviations:
-Two test vessels were not sterile at the end of the test in Tier 2, as shown by the results of the sterility test (pH 7, 15 °C and 25 °C). However, the kinetics of the hydrolysis resembled those of an abiotic process rather than of a biological process, as determined initial reaction rates were steady from test start on, which is not consistent with microbiological growth and concomitant test item degradation. The observed levelling off of the reaction rate after about 85 to 90% of hydrolysis at pH 7/25 °C is most likely due to an interfering equilibrium process between the three main test item components and their reaction products, which was also observed at pH 7/50 °C, where the test assays were sterile demonstrating that this process is most likely abiotically driven.

-The temperature could not be held at the required ±0.5 °C for all the test combinations of pH and temperature (see Table 16). Apart from pH 9/35 °C, the overall temperature mean over the duration of the respective test was however in the required range of ±0.5 °C. Therefore, no significant effects on the results obtained at these pH and temperature combinations are expected. At pH 9/35 °C, the temperature ranged from 35.1 to 37.3°C with a mean of 36.1 °C. In this case, the determined rate is most likely slightly faster than it would have been using the correct temperature range.
GLP compliance:
yes (incl. certificate)
Remarks:
The sterility tests were performed by a third laboratory in accordance with ISO/IEC 17025. These determinations are, hence, excluded from the statement of GLP-compliance.
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Sampling for each series of HPLC analysis were performed under sterile conditions (Bunsen burner, fume hood) as follows:
- Vessels were taken out of the thermostat cabinet
- Sampling: 5 ml were transferred with a sterilized pipette tip into a test tube (samples for HPLC do not need to be sterile, but the test vessels must remain sterile). Samples were not filtered.
- Replacing the nitrogen in the headspace of the test vessel and immediate closure with screw cap; vessels are brought back to the thermostat cabinet and put on the stirring device.
- Measurement of pH (precision 0.1 unit) with the remaining sample volume.

- Sampling intervals:
Tier 1 study: 5 min past test start to ensure complete dissolution of the test item and at regular intervals daily from day 1 to 5.

Tier 2 study:
For pH 7 at 15° and 25°C: at the test start, then after 74 h, 192 h, 262 h, 329 h, 433 h, 500 h, 601 h, 720 h.
For pH 7 / 50°C: set of duplicate test assays. Assay 1: at the test start, then after 2 h, 5 h, 8 h, 24 h, 29 h, 32 h, 48 h. / Assay 2: at the test start, then after 15 h, 20 h, 40 h.
For pH 9 / 15°C: set of duplicate test assays. Assay 1: at the test start, then after 15 h, 20 h / Assay 2: at the test start, then after 2 h, 5 h, 8 h, 12 h, 24 h, 35 h, 48 h.
For pH 9 / 25°C: at the test start, then after 0.5 h, 1 h, 1.4 h, 1.8 h, 2.7 h, 4 h, 5 h, 7.1 h, 7.8 h.
For pH 9 / 35°C: at the test start, then after 0.2 h, 0.4 h, 0.7 h, 0.9 h, 1.2 h, 1.4 h, 1.7 h, 2.3 h, 2.9 h, 3.6 h.

- Sample storage conditions before analysis:
Immediate HPLC measurement or freezing of the sample after sample preparation
Buffers:
pH 4 (Tier 1 only):
mixture of 0.05 M sodium acetate and 0.05 M acetic acid
19 ml 0.05 M sodium acetate and 81 ml 0.05 M acetic acid

pH 7 (Tier 1 and 2):
mixture of 0.1 M monopotassium phosphate and 0.1 N NaOH
29.63 ml 0.1 N NaOH + 50 ml 0.1 M monopotassium phosphate to 100 ml (means “Milli-Q water ad 100 ml") as described in the OECD guideline 111 for a temperature of 20 °C

pH 9 (Tier 1 and 2):
mixture of 0.1 M H3BO3 in 0.1 M KCl and 0.1 N NaOH
21.3 ml 0.1 N NaOH + 50 ml 0.1 M H3BO3 to 100 ml with 0.1 M KCl as described in the OECD guideline 111 for a temperature of 20 °C
Details on test conditions:
Study design
In the Tier 1 study, two test vessels (buffer with test item), as well as a blank vessel (buffer without test item) were prepared for each pH value (4, 7, 9), yielding six test vessels and three blank vessels in total. Potential hydrolysis of the test item at 50 °C was monitored over five days. Each replicate was sampled every day during the test.

In the Tier 2 study, the test item was incubated in buffered aqueous solutions at pH 7 at ca. 50, 25 and 15 °C and at pH 9 at ca. 35, 25 and 15 °C. These pH values were selected, as significant hydrolysis was observed in the Tier 1 study at these pH values. For each pH and temperature combination, at least two test vessels (buffer with test item), as well as a blank vessel (buffer without test item), were prepared. Potential hydrolysis of the test item was followed by HPLC analysis for a maximum of 30 days or until at least 90% hydrolysis was observed, with at least 6 sampling points regularly spread over the whole time-period.
At pH 7/50 °C and pH 9/15 °C, the time-span up to 90% of hydrolysis was about 48 hours. To regularly spread at least six sampling points over this time-period and to avoid nightshifts, one set of two test vessels and one blank vessel was prepared in the early morning and one set in the late afternoon (assay 1 and assay 2). For the evaluation, both data sets were successfully combined.

Test system and test conditions
In the Tier 1 study, the test item was incubated for 5 days at ca. 50 °C (in thermostat cabinet Heraeus T6060) in three different buffers at pH 4, 7 and 9 (as given in Table 1) in 100 ml vessels (Schott, Duran).

In the Tier 2 study, the test conditions were held as follows:
-Duration/contact time: 30 days maximum or until at least 90% hydrolysis was observed
-Temperature:
a) 50 ± 0.5 °C in the dark, at pH 7 only, controlled by a thermostat cabinet (Heraeus T6060 or WTC Binder)
b) 35 ± 0.5 °C in the dark, at pH 9 only, controlled by a thermostat cabinet (Heraeus T6060 or WTC Binder)
c) 25 ± 0.5 °C in the dark, both pH levels, controlled by a thermostat cabinet (WTW TS 606/4-i or 606/2-i)
d) 15 ± 0.5 °C in the dark, both pH levels, controlled by a thermostat cabinet (WTW TS 606/4-i or 606/2-i)
-Test buffers: see field buffers above
-Test vessels: 100 ml glass vessels with screw cap (Schott, Duran)


Preparation of the test
In the Tier 1 study, the procedure described below was followed for one temperature only (50 °C) at pH 4, 7 and 9 during five days with one sampling per day (but without sterility test at the end).

For the Tier 2 study, the procedure was the same, except that only pH 7 and 9 were further investigated, each at three different temperatures. Furthermore, the test was conducted for a maximum of 30 days or until at least 90% hydrolysis was observed. The test and blank vessels (including a magnetic rod for stirring) as well as the pipette tips used for sampling were sterilized by autoclaving before use. Preparation of the buffers was done in separate, large glass vessels. The respective buffers including the headspace were sparged with nitrogen for about thirty minutes to minimize reaction of the test item with oxygen. Then, the vessel was closed, and placed at the respective temperature for at least three hours to allow for temperature equilibration. Subsequently, the pH of the buffers was checked and corrected, before three times 100 ml of each buffer solution was filter-sterilized (0.45 µm aPES membrane, Rapid-Flow, Thermo Scientific) directly into the test and blank vessels. The test item was added to the test vessels from a stock solution (about 5000 mg/l test item in acetonitrile:water (1:1; v:v)) to yield a test item concentration of about 10 mg/l. The same amount of solvent without the test item was added to the blank vessels. Then the headspace of each test and blank vessel was filled with nitrogen, the vessels were closed, brought back into the thermostat cabinet (i.e. in the dark) at the respective temperature, and stirred. The pH of the test solutions was not adjusted after addition of the test item, as no significant changes are expected due to the high buffer capacity and the low test item concentrations.

The test item solutions were stirred in the respective thermostat cabinet for about five minutes to ensure complete dissolution of the test item before the first sampling (t0) for HPLC measurement was performed.
Hydrolysis of the test item was followed for a maximum incubation period of 30 days or until at least 90% hydrolysis was observed. A minimum of six spaced samplings between about 10% and 90% hydrolysis were performed per test replicate. The time of sampling was recorded. The temperature in the thermostat cabinet was recorded by a data logger several times per day.


Sterility test
At the end of the hydrolysis study, the number of colony forming units per millilitre (CFU/ml) was determined in the pooled replicate test vessels according to European Pharmacopoeia (Colony Forming Units; aerobic germs in R2A medium with incubation over 5 days at 30°C).
Positive controls:
no
Negative controls:
no
Statistical methods:
95% confidence intervals were calculated using the “Regression” module of the “Data Analysis” Tool of Excel, Microsoft Office 10.
Preliminary study:
The initial test item concentration ranged from 97% to 108% of the nominal concentration at pH 4 and 7, whereas it was only 58% at pH 9. This low recovery is due to rapid hydrolysis at pH 9/50 °C, as the first sample was taken after about five minutes past test start to ensure complete dissolution of the test item.
This preliminary test showed that the three main test item components are stable at pH 4, but hydrolyze readily at pH 7 and 9. Therefore a tier 2 study was performed.
Test performance:
Sterility control
To verify, whether the observed hydrolysis was not due to biological activity, sterility tests were conducted at the end of the test (in Tier 2 only). Since either none or up to 5 CFU/ml were found in the test vessels at pH 7/50 and pH 9/15, 25 and 50°C, it can be concluded that the hydrolysis observed in the corresponding test assays is solely due to abiotic processes. At pH 7, 20’000 and 32’000 CFU/ml were detected in the 15 °C and 25 °C test vessels respectively. However, the kinetics of the hydrolysis determined in the corresponding test assays resembled those of an abiotic process rather than of a biological process, as determined initial reaction rates were steady from test start on, which is not consistent with microbiological growth and concomitant test item degradation. The observed levelling off of the reaction rate after about 85% to 90% of hydrolysis at pH 7/25 °C is most likely due to an interfering equilibrium process between the three main test item components and their reaction products, which was also observed at pH 7/50 °C, where the test assays were sterile demonstrating that this process is most likely abiotically driven.

pH
The pH measurements after each sampling showed that the pH of the test solutions remained constant at ±0.1 unit of the target value..

Temperature
The temperature could not be held at the required ±0.5 °C for all of the test combinations of pH and temperature. Apart from pH 9/35 °C, the overall temperature mean over the duration of the respective test was however in the required range of ±0.5 °C. Therefore, no significant effects on the results obtained at these pH and temperature combinations are expected. At pH 9/35 °C, the temperature ranged from 35.1 to 37.3°C with a mean of 36.1 °C. In this case, the determined rate is most likely slightly faster than it would have been using the correct temperature range.
Transformation products:
not measured
Remarks:
the most likely transformation products are listed below
No.:
#1
No.:
#2
No.:
#3
Details on hydrolysis and appearance of transformation product(s):
Suggested pathways for transformation:

The most likely hydrolytic pathway of N-butylpthalimide is expected to be a hydrolysis to form 2-(Butylcarbamoyl)benzoic acid (see table identity of transformation products #1) in a first step and in a second step reaction to Phthalic acid and N-Butylamine.

The pathway should essentially be the same for N-propylphthalimide and N-sec-butylphthalimide, the only difference being the aliphatic rest on the amide (see table identity of transformation products #2 and #3). Also, the hydrolysis of N-propylphthalimide and N-butylphthalimide proceeds faster than the one of N-sec-butylpthalimide due to steric hindrance.

The first hydrolysis step is very likely to happen. The second step, however, may not take place, as the aliphatic amine is not considered to be a good leaving group.
Key result
pH:
4
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
N-propylphthalimide
Key result
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
481 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 461-503 h
Remarks:
N-propylphthalimide
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.004 h-1
DT50:
161 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 148-177 h
Remarks:
N-propylphthalimide
Key result
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.109 h-1
DT50:
6.4 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 5.6-7.3 h
Remarks:
N-propylphthalimide
Key result
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.113 h-1
DT50:
6.2 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 5.9-6.5 h
Remarks:
N-propylphthalimide
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.585 h-1
DT50:
1.2 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 1.1-1.3 h
Remarks:
N-propylphthalimide
Key result
pH:
9
Temp.:
35 °C
Hydrolysis rate constant:
1.51 h-1
DT50:
0.46 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 0.44-0.48 h
Remarks:
N-propylphthalimide
Key result
pH:
4
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
N-sec-butylphthalimide
Key result
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
823 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 775-878 h
Remarks:
N-sec-butylphthalimide
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.003 h-1
DT50:
242 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 237-247 h
Remarks:
N-sec-butylphthalimide
Key result
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.076 h-1
DT50:
9.1 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 8.5-9.8 h
Remarks:
N-sec-butylphthalimide
Key result
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.063 h-1
DT50:
10.9 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 10.8-11.1 h
Remarks:
N-sec-butylphthalimide
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.301 h-1
DT50:
2.3 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 2.2-2.4 h
Remarks:
N-sec-butylphthalimide
Key result
pH:
9
Temp.:
35 °C
Hydrolysis rate constant:
0.913 h-1
DT50:
0.76 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 0.72-0.80 h
Remarks:
N-sec-butylphthalimide
Key result
pH:
4
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Remarks:
N-butylphthalimide
Key result
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.001 h-1
DT50:
542 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 518-567 h
Remarks:
N-butylphthalimide
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.004 h-1
DT50:
176 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 162-192 h
Remarks:
N-butylphthalimide
Key result
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.101 h-1
DT50:
6.8 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 6.1-7.8 h
Remarks:
N-butylphthalimide
Key result
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.104 h-1
DT50:
6.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 6.4-6.9 h
Remarks:
N-butylphthalimide
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.475 h-1
DT50:
1.5 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 1.4-1.6 h
Remarks:
N-butylphthalimide
Key result
pH:
9
Temp.:
35 °C
Hydrolysis rate constant:
1.39 h-1
DT50:
0.5 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: 95% confidence interval for half-life: 0.47-0.53 h
Remarks:
N-butylphthalimide
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): see field "Test performance"

Results Tier 2 study
Initial concentrations/ recovery:
The initial concentration was about 10 mg/l test item corresponding to 1.35 mg/l N-propylphthal-imide, 3.08 mg/l N-sec-butylphthalimide and 5.55 mg/l N-butylphthalimide for all combinations of pH and temperature. The initially determined concentrations ranged from 91% to 100% of the nominal concentration and are thus in the required range of 90% to 110% recovery according to TG OECD 111.

Half-lives
- The Tier 1 study indicated that all three test item components were stable at pH 4, but readily hydrolysed at pH 7 and 9.
- According to the tier 2 study, half-lives at pH 7 and 15°C for the three components ranged between 481-823 h. Half-lives at pH 9 and 15°C for the three components ranged between 6.2-11 h.
- For all three test item components, the hydrolysis was strongly pH and temperature dependent.
- Overall, hydrolysis was faster at pH 9 than pH 7 and the higher the temperature was. At pH 7/15 and 25 °C, all half-lives obtained are higher than 150 hours. At pH 9/15 °C and pH 7/50 °C, half-lives were in the same order of magnitude with about 6 to 11 hours. At pH 9/25 and 35 °C half-lives obtained are <= 2.3 hours.

Discussion of results:

The test item components N-proplyphthalimide and N-butylphthalimide hydrolysed with comparable rates, whereas the hydrolysis of N-sec-butylphthalimide was significantly slower, which is most likely due to steric hindrance of the reactive sites at the carbonyl moieties by the branched alkyl moiety.

Since all plots of ln(Ct/C0) vs. time give a linear function (see attachment), it can be assumed that the hydrolysis of all three main test item components follows a pseudo-first order reaction.

At pH7/25 °C and pH 7/50°C only the initial six data points up to a hydrolysis percentage of about 90% were used for calculation, as the rate levelled off subsequently. This is most likely due to an interfering equilibrium process between the three main test item components and their reaction products. This observation was already made in the Tier 1 study and only occurred at pH 7, whereas at pH 9 the hydrolysis carried on at least until the three main test item components were no longer detectable in the HPLC chromatograms.

Validity criteria fulfilled:
yes
Remarks:
No validity criteria are given by TG OECD 111. The requirements which were not fulfilled are mentioned in the field "Test performance".
Conclusions:
The hydrolysis of the test item “Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide” was investigated in a tiered approach according to guideline OECD 111.
The Tier 1 study indicated that all three test item components were stable at pH 4, but readily hydrolysed at pH 7 and 9. According to the tier 2 study, half-lives at pH 7 and 15°C for the three components ranged between 481-823 h. Half-lives at pH 9 and 15°C for the three components ranged between 6.2-11 h.
Hydrolysis was demonstrated to be a possible pathway of degradation under environmentally relevant pH ranges of neutral to basic.
Executive summary:

The hydrolysis of the test item “Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide” was investigated in a tiered approach according to guideline OECD 111.

In the Tier 1 study, hydrolysis of the three main test item components was investigated over five days at ca. 50 °C and at pH 4, 7 and 9. The Tier 1 study indicated that all three test item components were stable at pH 4, but readily hydrolysed at pH 7 and 9.

In the Tier 2 study, the reaction rates and half-lives of the three main test item components were determined for the pH values, where the test item was unstable in the Tier 1 study. The test item was incubated at ca. 50, 25 and 15 °C and ca. 35, 25 and 15 °C in buffered aqueous solutions at pH 7 and 9, respectively. Monitoring times were set to encompass either a maximum of 30 days or about 10% to 90% of the hydrolysis, whichever came first.

For all three test item components, the hydrolysis was strongly pH and temperature dependent. Overall, hydrolysis was faster at pH 9 than pH 7 and the higher the temperature was. At pH 9/15 °C and pH 7/50 °C, hydrolysis rates and half-lives were in the same order of magnitude.

The test item components N-proplyphthalimide and N-butylphthalimide hydrolysed with comparable rates, whereas the hydrolysis of N-sec-butylphthalimide was significantly slower, which is most likely due to steric hindrance of the reactive sites at the carbonyl moieties by the branched alkyl moiety.

A summary of the obtained pseud-first order hydrolysis rates (kobs) and the corresponding half-lives is given below (minimum and maximum values of the three main components):

 pH Temp. °C   kobs [h-1]  half-live [h]
 4

50°C

no hydrolysis

 7

15

25

50

0.000842 – 0.00144

0.00286 – 0.0043

0.0761 – 0.109

481 – 823

161 – 242

6.4 - 9.1

  

9

15

25

35

0.0634 – 0.113

0.301 – 0.585

0.913 – 1.51

6.2 – 11

1.2 – 2.3

0.46 – 0.76

 

Description of key information

The hydrolysis of the test item “Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide” was investigated in a tiered approach according to guideline OECD 111.

In the Tier 1 study, hydrolysis of the three main test item components was investigated over five days at ca. 50 °C and at pH 4, 7 and 9. The Tier 1 study indicated that all three test item components were stable at pH 4, but readily hydrolysed at pH 7 and 9.

In the Tier 2 study, the reaction rates and half-lives of the three main test item components were determined for the pH values, where the test item was unstable in the Tier 1 study. The test item was incubated at ca. 50, 25 and 15 °C and ca. 35, 25 and 15 °C in buffered aqueous solutions at pH 7 and 9, respectively.

Hydrolysis was fastest at pH 9. Comparing hydrolysis of the three components at pH 9, 15°C the half-lives were in the same order of magnitude for all three components.

However, the test item components N-proplyphthalimide and N-butylphthalimide hydrolysed with comparable rates, whereas the hydrolysis of N-sec-butylphthalimide was significantly slower, which is most likely due to steric hindrance of the reactive sites at the carbonyl moieties by the branched alkyl moiety.

According to the tier 2 study, half-lives at pH 7 and 15°C for the three components ranged between 481-823 h. Half-lives at pH 9 and 15°C for the three components ranged between 6.2-11 h.

As key value for chemical safety assessment the value for the component with the longest half-life at pH 7 and 15°C was used: N-sec-butylphthalimide half-life: 823 h. The 95% confidence interval for the indicated half-life: 775-878 h

Key value for chemical safety assessment

Additional information