1-butylpyrrolidin-2-one

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-11 - 2017-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

OECD, 2004

Deviations:

yes

Remarks:

refer to 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

other: OCSPP 835.2120

Version / remarks:

U.S. EPA, 2008

Deviations:

yes

Remarks:

refer to 'Principles of method if other than guideline'

Principles of method if other than guideline:

Protocoll deviations
The protocol states that this test will be conducted for select pH values at 50 ± 0.5 ºC. The incubation temperatures dropped sometimes below 49.5 ºC.
Although temperature deviations extended for long durations throughout the study, the average temperature and minimum temperature during these deviations were all very close (≤ 0.6 ºC) to the accepted temperature range. Such a small discrepancy should not impact the rate of hydrolysis in any meaningful way. Therefore this deviation is expected to have minimal impact on the results or interpretation of this study.

The protocol states that this test will be conducted for select pH values at 50 ± 0.5 ºC. The incubation temperatures exceeded sometimes 50.5 ºC.The temperature deviation was brief, and the average temperature and maximum temperature during this deviation were all very close (≤ 0.3 ºC) to the accepted temperature range. Such a small discrepancy should not impact the rate of hydrolysis in any meaningful way. Therefore this deviation is expected to have minimal impact on the results or interpretation of the study.

GLP compliance:

yes

Specific details on test material used for the study:

stored at room temperaturein a dark, ventilated cabinet in the original container

Test Substance pH Determination
The pH was determined for a 1.0% by mass solution of the substance in purified reagent water. A test sample was prepared by aliquoting 210 μL of the test substance into 20.00 g of nitrogen-purged purified reagent water and mixing gently. The pH of the test sample was measured in triplicate using a Yellow Springs Instrument pH100 pH meter. The individual measurements, 5.07, 5.10, and 5.11, yielded an average pH of 5.09.

Radiolabelling:

no

Analytical monitoring:

not specified

Details on sampling:

Test System Sampling
Test samples were analyzed after 0 and 5 days of incubation. For each sampling interval, duplicate samples for each aqueous buffer solution were analyzed. An aliquot of each sample was diluted to a final volume of 10 mL with 1:4 acetonitrile:purified reagent water and mixed thoroughly. Samples were then analyzed by HPLC-UV to determine concentrations of TamiSolve NxG.

Buffers:

Aqueous pH Buffer Solutions
Aqueous pH buffers use during this study were prepared as described below:
pH 4: 410 mL of 0.01 M glacial acetic acid solution was combined with 90 mL of 0.01M sodium acetate trihydrate solution. The resultant buffer had a pH of 4.0 and required no adjustment.
pH 7: 98 mL of 0.02 M sodium phosphate monobasic dihydrate solution was combined with 152 mL of 0.02 M sodium phosphate dibasic dihydrate solution, and then diluted to 500 mL with purified reagent water. The resultant buffer had a pH of 7.2, and was adjusted to 7.0 with 1.0 N hydrochloric acid.
pH 9: 0.3108 g boric acid was added to 2.5 mL of 1.0 M sodium hydroxide solution and brought to a final volume of 500 mL with purified reagent water. The resultant buffer had a pH of 9.1 and was adjusted to 9.0 with 1.0 N hydrochloric acid.

Buffers were sterilized in an autoclave at approximately 121 °C and 15 psi for 90 minutes. Prior to dosing, buffers were purged with nitrogen to exclude oxygen. The pH of the sterile, nitrogen-purged buffer solutions was verified to be 4.0 ± 0.1, 7.0 ± 0.1, and 9.0 ± 0.1 prior to dosing.

Details on test conditions:

Standard Reagents
A 1:4 acetonitrile:purified reagent water solution was typically prepared by combining 200 mL of acetonitrile and 800 mL of purified reagent water.
A 0.1% phosphoric acid in purified reagent water mobile phase solution was typically prepared by adding 2.0 mL of phosphoric acid to 2000 mL of purified reagent water.
All aqueous solutions were prepared using purified reagent water generated from a Millipore water purification unit. All chemicals and solvents were at least reagent grade and were obtained from commercial sources.

Dosing Stock Preparation
A dosing stock was prepared by transferring 10 mL of the test substance to a 10-mL amber bottle and purging the headspace with nitrogen for approximately 30 seconds. The stock was sealed with an aluminum crimp cap with a PTFE-lined septum and stored refrigerated until use.

Dosing
A nominal test concentration of 1.0 mg/mL was selected for this study, which is less than one-half of the water solubility of TamiSolve NxG at 20 °C.
Each of the sterile aqueous buffers was dosed in bulk with a 104-μL aliquot of the dosing solution (8835A), diluted to 100 mL with the appropriate aqueous buffer, and inverted to mix. Individual samples were then prepared by aliquoting 10.0 mL of the bulk-dosed solution into sterile 10-mL amber vials.

Incubation
Two samples for each aqueous pH buffer solution were processed immediately after dosing for day 0 analyses. An additional four samples for each pH were incubated in the dark, in a thermostatic water bath set to 50.0 °C. Average incubation temperature was monitored using a HOBO data logger and temperature probe. The sterility was determined for the buffer solutions and for the hydrolysis samples on day 0 and day 5 of the test, respectively. Petrifilm aerobic count plates were used to determine sterility. At sampling, a portion of each of the test solutions was aseptically added to individual sterile plates and the plates were incubated for a minimum of two days at ambient temperature in the laboratory. The absence of microbial growth was used to indicate sterility.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.001 mg/L

Remarks:

nominal test concentration

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.001 mg/L

Remarks:

nominal test concentration

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.001 mg/L

Remarks:

nominal test concentration

Duration:

0 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.001 mg/L

Remarks:

nominal test concentration

Duration:

0 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.001 mg/L

Remarks:

nominal test concentration

Duration:

0 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.001 mg/L

Remarks:

nominal test concentration

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Statistical methods:

CALCULATIONS
Limits of Quantification
The nominal concentrations of the lowest and highest calibration standards were used as the instrument lower limit of quantification (LLQ) and upper limit of quantification (ULQ), respectively. These were multiplied by the lowest and highest dilution factors used for test samples and QC samples to determine the method lower limit of quantification (MLLQ) and method upper limit of quantification (MULQ), respectively.

Sample Concentration
A calibration curve was generated with each batch of sample analysis using the calibration standards. The calibration curve was generated in Laura using a weighted (1/x) linear regression to determine an association between TamiSolve NxG concentration and instrument response. The calibration curve was then applied in Laura to the results of each test sample and QC sample analysis to determine the diluted concentration of each sample. This result was then multiplied by the dilution factor for each sample in Excel to determine the calculated concentration.
In order for a batch of sample analysis to be considered acceptable, the following criteria were all met; (1) at least 80% of calibration standards analyzed yielded a recovery of 80.0 to 120% of nominal concentration and were used in the regression analysis, (2) the regression’s coefficient of determination (r2) was ≥ 0.990, (3) at least 66% of QC samples analyzed yielded a recovery of 80.0 to 120% of nominal concentration, and (4) all blank QC samples analyzed yielded a concentration < 50% of the MLLQ.

Preliminary study:

not specified

Test performance:

Test Conditions
The incubation temperature measurements were maintained between 48.9 and 49.8 °C for the duration of the test (see Protocol Deviations). The pH of the sterile buffer solutions, after dosing, was 4.0, 7.0, and 8.9 for the pH 4, pH 7, and pH 9 solutions, respectively. The sterility of the pH 4, pH 7, and pH 9 buffer solution samples at day 0 (initiation) and day 5 (termination) were checked for each pH value. All samples were confirmed to be sterile.

Transformation products:

no

% Recovery:

95.5

pH:

4

Temp.:

50 °C

Duration:

5 d

% Recovery:

96.9

pH:

7

Temp.:

50 °C

Duration:

5 d

% Recovery:

97.4

pH:

9

Temp.:

50 °C

Duration:

5 d

% Recovery:

98.9

pH:

4

Temp.:

50 °C

Duration:

0 d

% Recovery:

98.8

pH:

7

Temp.:

50 °C

Duration:

0 d

% Recovery:

99.7

pH:

9

Temp.:

50 °C

Duration:

0 d

Key result

Remarks on result:

other: see 'Remarks'

Remarks:

Hydrolysis rates were not calculated since less than 10% of the substance was hydrolyzed after five days.The substance is considered hydrolytically stable in pH 4, pH 7, and pH 9 aqueous solutions.

Limits of Quantification

The limits of quantification are summarized in the table below. Any results below the LLQ are denoted as “–” in tables. The limits of quantification are summarized in the table below. Any results below the LLQ are denoted as “–” in tables.

LLQ (μg/mL)	ULQ (μg/mL)	MLLQ (μg/mL)	MULQ (μg/mL)
5	50	20	2,000

Test Sample Recovery

Results for sample recovery are summarized in the table below:

Matrix	Average Day 0 Recovery (% of nominal)	Average Day 5 Recovery (% of nominal)
pH 4	98.9	95.5
pH 7	98.8	96.9
pH 9	99.7	97.4

Quality Control Sample Recovery

All QC results met acceptance criteria as outlined before.

Hydrolysis Rate

Hydrolysis rates were not calculated since less than 10% of the substance was hydrolyzed after five days.

Validity criteria fulfilled:

yes

Conclusions:

The substance is considered hydrolytically stable in pH 4, pH 7, and pH 9 aqueous solutions.

Executive summary:

The test procedures followed OCSPP 835.2120 (U.S. EPA, 2008) and OECD 111 (OECD, 2004) Guidelines and was conducted according to GLP.

The rate of hydrolysis of the substance was studied in three aqueous buffer solutions. For the Tier 1 test, samples consisted of 10.0-mL sterile pH 4, 7, and 9 buffer aliquots. The substance was applied at a nominal concentration of 1 mg/mL, and samples were incubated in the dark at 50 ± 0.5 °C. Samples were analyzed after 0 and 5 days of incubation. Samples were diluted with 1:4 acetonitrile:purified reagent water and analyzed by high-performance liquid chromatography with UV detection (HPLC-UV) for determination of concentrations. Less than 10% of the test substance was hydrolyzed after 5 days; therefore, no additional hydrolysis testing was performed.

The results are summarized in the table below:

Matrix	Test Day	Average Concentration (mg/mL)	Recovery (% of nominal)
pH 4	0	0.989	98.9
	5	0.955	95.5
pH 7	0	0.988	98.8
	5	0.969	96.9
pH 9	0	0.997	99.7
	5	0.974	97.4

Description of key information

The test procedures followed OCSPP 835.2120 (U.S. EPA, 2008) and OECD 111 (OECD, 2004) Guidelines and was conducted according to GLP.

The substance is considered hydrolytically stable in pH 4, pH 7, and pH 9 aqueous solutions.

Additional information

The rate of hydrolysis of the substance was studied in three aqueous buffer solutions. For the Tier 1 test, samples consisted of 10.0-mL sterile pH 4, 7, and 9 buffer aliquots. The substance was applied at a nominal concentration of 1 mg/mL, and samples were incubated in the dark at 50 ± 0.5 °C. Samples were analyzed after 0 and 5 days of incubation. Samples were diluted with 1:4 acetonitrile:purified reagent water and analyzed by high-performance liquid chromatography with UV detection (HPLC-UV) for determination of concentrations. Less than 10% of the test substance was hydrolyzed after 5 days; therefore, no additional hydrolysis testing was performed.

The results are summarized in the table below:

Matrix	Test Day	Average Concentration (mg/mL)	Recovery (% of nominal)
pH 4	0	0.989	98.9
	5	0.955	95.5
pH 7	0	0.988	98.8
	5	0.969	96.9
pH 9	0	0.997	99.7
	5	0.974	97.4