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Hydrolysis

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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:
EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Duration:
5 d
pH:
4
Temp.:
49 °C
Duration:
5 d
pH:
7
Temp.:
49 °C
Duration:
5 d
pH:
9
Temp.:
49 °C
Transformation products:
not specified
Remarks:
Only the sample stored at pH 7 showed any hydrolysis products.
Key result
Remarks on result:
hydrolytically stable based on preliminary test
Details on results:
Samples were stored for 5 days at 49°C, in a buffer solution at pH 4, pH 7 and pH 9. Hydrolysis was determined by identifying related free acids using GC/MS. Only the sample stored at pH 7 showed any hydrolysis products.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, 1% hydrolysis occurred at pH 7 and no hydrolys was noted at pH 4 or pH 9 after 5 days at 49°C.
Executive summary:

A study was conducted to determine the hydrolysis of the test substance according to EPA OPPTS Method 835.2120, in compliance with GLP. Under the study conditions, 1% hydrolysis occurred at pH 7 and no hydrolys was noted at pH 4 or pH 9 after 5 days at 49°C (Probst, 2010).

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 03, 2009 to September 23, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
The test substance was supplied by the Sponsor and was stored at <-5 °C. The radiolabeled test substance was purified at Ricerca Biosciences prior to use in the study because its radiochemical purity had decreased to less than 95%. The radiochemical purity of the test substance is the value obtained from HPLC-RAD analysis of the substance after repurification at Ricerca Biosciences, LLC. The radiochemical purity of the purified test substance was found to be 95.4%.
Radiolabelling:
yes
Analytical monitoring:
yes
Details on sampling:
APPLICATION OF TEST SUBSTANCE
Separate glass 100-mL volumetric flasks were filled nearly to volume with sterile pH 4 buffer (0.01 M), sterile pH 7 buffer (0.01 M) or sterile pH 9 buffer (0.01 M). To each volumetric flask was added 460 μL of the aqueous [14C]test substance dosing solution, the volumetric flasks were filled to volume and inverted to mix. Quantitation of the radioactivity in the test solutions was performed by LSC (triplicate 30 μL aliquots).
Individual test samples were prepared by separately transferring 4 mL of the pH 4, pH 7 and pH 9 buffers into sterile 4 mL amber vials using a sterile transfer pipette. The test samples were prepared using aseptic techniques in a biological fume hood. Each test vial was sealed with a sterile Teflon-lined screw cap and placed in the constant temperature bath in the dark at 50 ± 0.5 °C until removed for analysis.

SAMPLING INTERVALS
Duplicate pH 4, pH 7 and pH 9 buffer samples were analyzed immediately after the test substance was placed into the test vessels (Time 0). In addition to time 0, sampling intervals for each pH buffer system for the hydrolysis testing conducted at 50 ± 0.5 °C included 4 hours, 1 day, 3 days, 5 days.

STERILITY MEASUREMENTS
Sterility was determined at study initiation in the Day 0 samples and at the final sampling interval (Day 5) for each of the pH 4, pH 7 and pH 9 buffer test systems. Petrifilm aerobic count plates (3M Health Care, St Paul, MN) were used to determine sterility. At sampling, approximately 0.2 mL of each of the test solutions was aseptically added to individual sterile plates. The petrifilm plates were incubated for at least 2 days at ambient temperature in the laboratory. The absence of microbial growth demonstrated sterility.

PH MEASUREMENTS
The pH of the sterile pH 4, pH 7 and pH 9 buffers and prepared test solutions were measured and documented on Day 0. The pH of test samples at each pH was again measured at final sampling interval (Day 5).

SAMPLE STORAGE CONDITIONS
All samples were analyzed for material balance on the sampling day. Samples were then stored frozen until HPLC analysis after which time the remainder was stored frozen at <-5 ºC. All samples were analyzed within 6 days of sampling. Replicate 2 of the Day 3, pH 4 sample was reanalyzed after 15 days of frozen storage.

Buffers:
Preparation of sterile buffers
Buffer solutions were prepared at concentrations of 0.01 M. Just prior to buffer preparation HPLC grade water used in buffers was bubbled with nitrogen gas for ca. 5 minutes to remove dissolved oxygen. Following preparation buffers were filter sterilized by passing through a 0.2-µm filter. The pH and sterility of the buffers were verified using a Thermo Orion pH meter, Model 250 A+ (Thermo Electron Corporation., Beverly, MA). Preparation of the pH 4, 7, and 9 buffers used in the study are described below. All sterile work was done in a Forma Scientific Biological Safety Cabinet Model 1184 (Forma Scientific Inc., Marietta, OH).

pH 4 Buffer
The 0.01 M pH 4.0 buffer solution was prepared by combining 410 mL of 0.01 M acetic acid solution with 90 mL of 0.01 M sodium acetate solution. The final pH adjustment was made with NaOH as necessary to a pH of 4.0 ± 0.1.
• The 0.01 M acetic acid solution was prepared by adding 0.300 g of glacial acetic acid to a 500-mL volumetric flask and diluting to volume with HPLC grade water.
• The 0.01 M sodium acetate solution was prepared by adding 0.410 g of sodium acetate to a 500-mL volumetric flask and diluting to volume with HPLC grade water.

pH 7 Buffer
The 0.01 M pH 7.0 buffer solution was prepared by adding 195 mL of 0.02 M sodium phosphate (monobasic) and 305 mL of 0.02 M sodium phosphate (dibasic) to a 1000-mL volumetric flask. The contents were diluted to volume with HPLC grade water to produce a 0.01 M pH 7.0 (± 0.1) phosphate buffer solution. The final pH adjustment was made with NaOH as necessary to a pH of 7.0 ± 0.1.
• The 0.02 M sodium phosphate monobasic solution was prepared by adding 1.56 g of sodium phosphate monobasic (dihydrate) to a 500-mL volumetric flask and diluting to volume with HPLC grade water.
• The 0.02 M sodium phosphate dibasic solution was prepared by adding 1.42 g of sodium phosphate dibasic (anhydrous) to a 500-mL volumetric flask and diluting to volume with HPLC grade water.

pH 9 Buffer
A 0.5 M boric acid solution was prepared by adding 30.9 g of boric acid (61.83 g/mole) to a 1000-mL volumetric flask and diluting it to volume with HPLC grade water. Twenty mL of the 0.5 M boric acid solution was transferred to a 1000-mL volumetric flask and diluted to volume with HPLC grade water. This 0.01 M boric acid solution was adjusted to pH 9 with NaOH as necessary to produce a 0.01 M pH 9.0 (± 0.1) borate buffer solution.
Details on test conditions:
RATIONALE FOR TEST SOLUTION CONCENTRATION
The target test solution concentration of 1 mg/L (less than 0.01 M) was chosen because it was considered to be a typical concentration that might be observed if it entered an aquatic system under normal agricultural usage. Preliminary experiments were conducted to verify that [14C]test substance is completely soluble in water at 25°C at 1 mg/L.

DESCRIPTION OF EXPERIMENTAL SET-UP
Each test system was sterilized in a steam sterilizer (Tuttnaur-Brinkman Autoclave) for 30 minutes at 121°C prior to use in this study. Individual test systems were prepared in duplicate in 4 mL amber glass vials containing [14C] test substance. For duplicate hydrolysis samples, each test vessel contained 4 mL of sterile pH 4, 7 or 9 buffer containing [14C]test substance at a concentration of approximately 1.0 mg/mL. The test vessels were capped with Teflonlined screw caps and stored in the dark in a NESLAB GP-500 constant temperature bath at 50 ± 0.5 °C until removed for analysis.

TEMPERATAURE
The test systems were maintained in a NESLAB GP-500 Constant Temperature Bath (Portsmouth) at 50 ± 0.5 °C throughout the duration of the study. The
temperature was continuously monitored and recorded every 15 minutes.

PREPARATION OF TEST SUBSTANCE DOSING SOLUTION
A stock solution of the test substance was prepared for use in preparation of the dosing solution by dissolving [14C]test substance in acetonitrile. The dosing solution was prepared by concentrating a portion of the [14C]test substance stock solution to dryness and reconstituting the [14C]test substance in water. HPLC-grade solvents were used for the preparation of the solution.
Duration:
5 d
pH:
4
Temp.:
40 °C
Initial conc. measured:
0.969 mg/L
Duration:
5 d
pH:
7
Temp.:
40 °C
Initial conc. measured:
0.982 mg/L
Duration:
5 d
pH:
9
Temp.:
40 °C
Initial conc. measured:
0.955 mg/L
Number of replicates:
The actual concentration of [14C]test substance in the test vessels upon dosing was separately determined for each pH as the average of LSC values from the duplicate time.
Positive controls:
yes
Negative controls:
yes
Preliminary study:
Adsorption of the test substance to glass was monitored in the glass containers to be used in the study. Recovery of 14C from overnight incubation ranged from 100.6-101.7%. No adsorption to glass was observed in any of the test solutions at the nominal solution concentration of 1 mg/L used in the preliminary hydrolysis study.
Test performance:
Individual unknown degradates were present at levels less than 1.25% AR. These materials were also present in the Day 0 samples. Identification of unknowns was outside of the scope of this study.
Transformation products:
no
Details on hydrolysis and appearance of transformation product(s):
The test substance was found to be hydrolytically stable at 50 ºC; therefore no hydrolysis degradation pathway was determined.
The average percentage of test substance in solution at all three pHs ranged from 97.55% to 97.59% at time 0 and from 96.47% to 98.74% after five days. These results show that no hydrolysis occurred and demonstrate that test substance is hydrolytically stable in pH 4, pH 7 and pH 9 at 50 ºC.
pH:
4
Temp.:
50 °C
Duration:
5 d
Remarks on result:
other: >98.9<101
pH:
7
Temp.:
50 °C
Duration:
5 d
Remarks on result:
other: >97.4<100.5
pH:
9
Temp.:
50 °C
Duration:
5 d
Remarks on result:
other: >98.7<102.9
Key result
Remarks on result:
hydrolytically stable based on preliminary test
Details on results:
VERIFICATION OF STERILITY AND PH
Sterility of samples was confirmed for pH 4, pH 7, and pH 9 at Day 0 and at the final sampling interval (Day 5). All samples tested were confirmed to be sterile.
The pH of the buffer systems and samples was maintained at ± 0.1 for the pH 4, pH 7 and pH 9 test systems. The pH of the 0.01 M sterile pH 4 buffer was 3.97 at study initiation and 3.98 in the hydrolysis samples on Day 5. The pH of the 0.01 M sterile pH 7 buffer was 7.05 at study initiation and 7.07 in the hydrolysis samples on Day 5. The pH of the 0.01 M sterile pH 9 buffer was 8.93 at study initiation and 8.95 in the hydrolysis samples on Day 5.

VERIFICATION OF CHROMATOGRAPHIC PROCEDURES
A reference substance was analyzed by HPLC to verify proper column and instrument operation prior to sample analysis. A reference substance was co-injected with all of the samples analyzed by HPLC. Recovery of 14C from the HPLC instrument was determined for all hydrolysis samples. Samples were injected and HPLC effluent collected and analyzed by LSC. The average recovery for all analyses was 94.3%. These results confirm the quantitative recovery of radioactivity applied to the HPLC column.

DISTRIBUTION AND COMPOSITION OF RESIDUES

Sterile pH 4 Buffer
The average amount of [14C]test substance in the hydrolysis samples remained stable with an average of 97.59% AR at Day 0 and 98.55% AR at Day 5.

Sterile pH 7 Buffer
The average amount of [14C]test substance in the hydrolysis samples remained stable with an average of 97.55% AR at Day 0 and 96.47% AR at Day 5.

Sterile pH 9 Buffer
The average amount of [14C]test substance in the hydrolysis samples remained stable with an average of 97.57% AR at Day 0 and 98.74% AR at Day 5.
Results with reference substance:
The identities of test substance residues in pH 4, pH 7 and pH 9 hydrolysis samples were determined by HPLC co-injection with non-radiolabeled authentic reference standard.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, no significant degradation was observed in the preliminary study after 5 days at pH 4, pH 7 and pH 9 at 50 °C. Therefore, no additional experiments were required. On the basis of the results from studies conducted at 50°C, it can be concluded that aqueous abiotic hydrolysis will not be a significant route of elimination of the test substance from the environment.
Executive summary:

A study was conducted to determine the rate and route of hydrolysis of [14C] radiolabeled test substance in sterile pH 4, pH 7 and pH 9 buffer according to OECD Guideline 111 and EPA OPPTS Method 835.2120, in compliance with GLP. The substance was applied to pH 4 (0.01 M acetate), pH 7 (0.01 M phosphate) and pH 9 (0.01 M borate) sterile aqueous buffer solutions at concentrations of 0.969 μg/mL (0.969 ppm), 0.982 μg/mL (0.982 ppm) and 0.955 μg/mL (0.955 ppm), respectively. The samples were incubated for 5 days at 50 ± 0.5ºC in the dark. At selected intervals, duplicate samples were analyzed by liquid scintillation counting (LSC) to determine the concentration of radioactivity in solution. Samples were then analyzed directly by high performance liquid chromatography with radiochemical flow detection (HPLC/RAD) to determine the distribution of radioactive residues. The material balance of radioactivity for the sterile pH 4, pH 7 and pH 9 buffer hydrolysis samples throughout the study ranged from 97.4-102.9 percent of the applied radioactivity (%AR). Under the study conditions, no significant degradation was observed in the preliminary study after 5 days at pH 4, pH 7 and pH 9 at 50°C. Therefore, no additional experiments were required. On the basis of the results from studies conducted at 50°C, it can be concluded that aqueous abiotic hydrolysis will not be a significant route of elimination of the test substance from the environment (Grommes, 2010).

Description of key information

A study was conducted to determine the hydrolysis of the test substance according to EPA OPPTS Method 835.2120, in compliance with GLP. Under the study conditions, 1% hydrolysis occurred at pH 7 and no hydrolys was noted at pH 4 or pH 9 after 5 days at 49°C (Probst, 2010).

Key value for chemical safety assessment

Additional information