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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
Pyridinyl label (radiochemical purity: 98.3%)
Analytical monitoring:
yes
Details on sampling:
50°C experiment: Aliquots (25 ml) of pH 4, 7, and 9 buffer solutions were transferred to amber hypovials (Pierce) using a 25 ml glass bulb-pipette. Eight vials were prepared for the pH 4 and pH 7. 20 vials were required for pH 9. For buffer catalysis, 25 ml of Romil water was transferred, into four amber vials. All the vials were sealed using septa, capped and autoclaved for 20 minutes.

25°C experiment: Aliquots (25 ml) of each buffer solution were transferred to amber hypovials (Pierce) using a 25 ml glass bulb-pipette. Thirteen vials were prepared for each pH. For buffer catalysis, 25 ml of Romil water was transferred, into four amber vials. All the vials were sealed using septa, capped autoclaved for 20 minutes.

The radiolabelled stock solution was prepared by dissolving the radiochemical in acetonitrile, to give a concentration of approximately 714.4 µg/mL. The initial purities of the test solutions were assessed using thin layer chromatography (TLC) in up to three solvent systems. The radiochemical stock solution was sterilised immediately prior to application by pasing the solution through a sterile 0.2 µm Anotop 10 microfilter (Whatman).

All glass syringes were sterilised by immersion overnight in a solution of 7:3 ethanol:water prior to use.

For the 50°C experiment, three vials of each pH (one for sterility, two for day 0 analysis) were removed. The remaining vials were placed in a water bath maintained at a temperature of 50 ± 1°C for up to 32 days.
For the 25°C experiment, three vials of each pH (one for sterility, two for day 0 analysis) were removed. The remaining vials were placed in a water bath maintained at a temperature of 25 ± 1°C for up to 32 days.
Buffers:
pH 4 acetate buffer: 41.0 mL 0.01 M acetic acid + 9.0 mL 0.01 M sodium acetate
pH 5 acetate buffer: 14.8 mL 0.01 M acetic acid + 35.2 mL 0.01 M sodium acetate
pH 7 acetate buffer: 399 mL 0.01 M sodium acetate trihydrate + 1.0 mL 0.01 M acetic acid
pH 9 borate buffer: 0.01 M sodium tetraborate

High purity bottled water was used for the preparation of all buffer solutions.
Details on test conditions:
Buffer solution sterility: The sterility of a sample of each hydrolysis buffer solution was checked by dispensing 0.5 ml aliquots of each buffer solution on to plates containing a nutrient agar medium. The plates were then maintained at 20°C ± 2°C for a period of 7 days prior to inspection. Control plates were prepared at each time point and exposed to the environment of the laminar flow cabinet without the addition of sample in order to provide an indication of background levels of contamination. All sterility checks were carried out in a laminar flow cabinet using aseptic techniques.

Buffer catalysis experiments (25 and 50°C)
In order to demonstrate that the hydrolysis observed was not due to chemical reaction with the components of the buffer solutions, the following experiment was performed. 36 µL of the radiochemical was added to eight vials containing pure water by injecting directly through the septa with a sterilised 100 µL glass syringe. This resulted in a final radiolabeled test substance concentration of approximately 1 µg/mL. Four vials were placed in each of the water baths, which were set at temperatures of 25 and 50°C, respectively, for 32 days.
Duration:
32 d
Remarks:
The final concentration of the parent compound in the buffer vials was 1.07 µg/mL
Test performance:
Hydrolysis in the buffer catalysis solutions (i.e., pure water only) was very similar to the pH 7 experiments representing 94.6 and 97.2%, for the 50°C and 25°C experiments, respectively. This result showed that the hydrolysis observed in the buffered solutions was not due to chemical breakdown caused by the presence of the buffering agents.

The buffer solution sterility results showed that the sterility of the test system was maintained throughout the hydrolysis period. It can therefore be concluded that any breakdown of the test substance observed in this study was not microbially mediated.
Transformation products:
yes
No.:
#1
No.:
#2
Details on hydrolysis and appearance of transformation product(s):
Two major products were identified at levels up to 32.1 and 37.9%
% Recovery:
101.5
pH:
4
Temp.:
50 °C
Duration:
32 d
Remarks on result:
other: Recoveries ranged from 96.8 to 106.3%
% Recovery:
101.5
pH:
7
Temp.:
50 °C
Duration:
32 d
Remarks on result:
other: Recoveries ranged from 96.8 to 106.3%
% Recovery:
25.5
pH:
9
Temp.:
50 °C
Duration:
32 d
% Recovery:
100
St. dev.:
5
Temp.:
25 °C
Duration:
32 d
Remarks on result:
other: At pH 5, 7, and 9
Key result
pH:
9
Temp.:
50 °C
DT50:
360 h
Type:
other: first order
Details on results:
Major hydrolysis products of the test substance at pH 9 (50°C) are (E)-2-{2-[6-9(trifluromethyl)pyridin-2-yloxymethyl]phenyl}-3-methoxyacrylate and 2-{2-[6-9(trifluromethyl)pyridin-2-yloxymethyl]phenyl}acetic acid.
Validity criteria fulfilled:
yes
Conclusions:
The study demonstrates that under normal environmental conditions, the test substance will be stable to hydrolysis.
Executive summary:

This hydrolysis study was performed to conform to the EPA guidelines, Subdivision N, Section 161-1 and the Official Journal Of the European Commission Legislation (L 383 A : Method C7).

The test substance, labelled in the pyridinyl ring, was introduced into sealed amber vials containing sterile buffer solutions to achieve a concentration of 1.07 µg/mL. The vials were incubated at 50°C (pH 4, 7 and 9) and 25°C (pH 5, 7, and 9) for up to 32 days, in the absence of light and under sterile conditions were maintained throughout the study.

The vials incubated at 50°C were removed from the incubator for analysis on days 0 and 6, (with additional samples at 15, 20, 25, and 32 days for the pH 9 experiment), while samples analysis was carried out on days 0, 6, 15, 20, 25, and 32 for the 25°C hydrolysis.

At 50°C, no significant (i.e., <10% degradation of applied radioactivity) hydrolysis occured at pH 4 or 7. At pH 9, however, the half-life was calculated as 360 hours (i.e., 15 days). Two hydrolysis products were found above 10% of the applied radioactivity and were identified up to 35 and 40% of applied. At 25°C, no significant hydrolysis occurred (i.e., <10% degradation of applied radioactivity), at any pH.

The study demonstrates that under normal environmental conditions, the test substance will be stable to hydrolysis.

Description of key information

No significant hydrolytic degradation was observed to occur in pH buffers 5, 7 or 9 at 25°C. 

Key value for chemical safety assessment

Additional information