2-Propenoic acid, ethyl ester, reaction products with mixed substituted alkyl esters of phosphorodithioic acid and propylene oxide

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 December 2017 to 20 February 2018

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:

2004

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Buffers:

pH 4
- 0.05M acetic acid adjusted with NaOH to pH 4
- pH = 4.01

pH 7
- 500 mL 0.1M KH2PO4 + 296.3 mL 0.1N NaOH in 1 litre adjusted to pH 7 with HCl or NaOH
- pH = 6.99

pH 9
- 500 mL 0.1M H3BO3 in 0.1M KCl + 213 mL 0.1N NaOH in 1 litre adjusted to pH 9 with HCl
- pH = 9.04 and 9.00

Details on test conditions:

PRELIMINARY TEST (TIER 1)
- Solution Preparation: The buffers were purged for 5 minutes with nitrogen to remove oxygen then 75 mL of each buffer was syringe filtered through a 0.2 μm syringe filter for sterility into an Erlenmeyer flask. A 0.7 mL volume of the methanolic stock solution (17.26 mg/mL) of the test material was then spiked into each buffer. The final solution in buffer is at a lower concentration than the water solubility of the test material. The solutions formed cloudy suspensions due to the presence of the buffers. Due to low solubility of the test material, these suspension were used rather than an aqueous half saturated solution. Samples were prepared in duplicate.
- 75 mL of pH 4 buffer was prepared with a final concentration of 161.9 mg/L test material.
- 75 mL of pH 7 buffer was prepared with a final concentration of 161.9 mg/L test material.
- 75 mL of pH 9 buffer was prepared with a final concentration of 161.9 mg/L test material.
- Buffers were filtered through 0.2 μm syringe filter prior to spiking of the test material. The sample solution was pipetted using a sterile pipet tip into 15 mL test tubes preconditioned overnight at 105 °C. Tubes were purged with nitrogen and sealed with Teflon lined caps.
- The test solutions were prepared December 13, 2017 and incubated at 50 °C for five days.
- Incubated test solutions were run on a HPLC system to determine if any hydrolysis had occurred. Ten mL aliquots of each sample (pH 4, 7 and 9) were evaporated to dryness in a 250 mL round bottom flask. A 5 mL aliquot of methanol was added to the flask. The content of the flask was sonicated to aid dissolution followed by syringe filtration through a 0.45 μm syringe filter to remove particulate into a 7 mL vial. A 100 μL aliquot was injected onto the HPLC.
- A significant decrease in any chromatographic peak area would indicate breakdown of the test material. The chromatograms were also examined for the appearance of additional peaks that would provide confirmation that hydrolysis had occurred.

DEFINITIVE TEST (TIER 2)
- Tier 2 was conducted at three temperatures (25 °C, 37.5 °C and 50 °C) and one pH (pH 9).
- Solution Preparation: Buffers were prepared then purged with nitrogen for 5 minutes to remove oxygen and then sterilised by passing thorough a 0.2 μm syringe filter. Test solutions were prepared by spiking 4.5 mL of the methanolic stock solution (24.89 mg/mL) of the test material into each buffer. The solution formed a cloudy suspension. Due to low solubility of the test material, a suspension was used rather than an aqueous half saturated solution.
- 450 mL of pH 9 buffer was prepared with a final concentration of 249 mg/L test material.
- Buffer was filtered through a sterilised 0.2 μm syringe filter into a sterile beaker. The methanolic stock solution was spiked in. 10 mL of this solution was transferred to 15 mL round bottom test tubes preconditioned overnight at 105 °C using a sterile pipette tip. Tubes purged with nitrogen and sealed with Teflon lined caps.
- Opaque incubators were used to preclude photolytic effects. Two temperature controlled ovens and a water bath were used as incubators to control temperature at 25 °C, 37.5 °C and 50 °C. For 25 °C, the samples were initially placed in a Napco 6100 Water Jacketed Incubator. The samples were removed after 2 days and moved to New Brunswick Scientific Co. Inc. incubator for the remainder of the test.
- Individual replicate test samples were prepared at each pH and temperature (six time points in duplicate for a total of 12 samples at each pH and temperature). A blank pH 9 buffer sample was included at each time point.
- An initial concentration was determined from the HPLC analysis at the initial time point (i.e. time 0). At appropriate time intervals (the method specifies a minimum of 6 time points falling between 10 and 90% hydrolysis), samples were removed from the incubator for analysis.
- Sample Analysis: Samples were analysed using the same method as in Tier 1 except the entire contents (10 mL) of each test tube were analysed at each time point by transferring contents to a 250 mL round bottom flask followed by rota-evaporation. A 5 mL aliquot of methanol was added to the dried flask. The content of the flask was sonicated to aid dissolution followed by syringe filtration through a 0.45 μm syringe filter to remove particulate into a 7 mL vial. A 100 μL aliquot was injected onto the HPLC system.

Duration:

21 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

249 mg/L

Number of replicates:

Individual replicate test samples were prepared at each pH and temperature (six time points in duplicate for a total of 12 samples at each pH and temperature).

Positive controls:

no

Negative controls:

yes

Remarks:

A blank pH 9 buffer sample was included at each time point.

Preliminary study:

Preliminary Test (Tier 1)
- It was difficult to determine if hydrolysis had occurred due to the nature of the test material. The test material is a UVCB substance with a number of constituents. In the HPLC run these are represented by a number of peaks with no single peak greater than 15 % of the total area counts. The t = 0 duplicate data agreed within 15 % but there was variability in the data at t = 5 days (greater than 30 % in some cases). The data were normalised to the main peak of the run to help determine if hydrolysis had occurred.

- pH 4 @ 50 °C: Hydrolysis not detected. No new peaks formed. Peak profile remained unchanged.
- pH 7 @ 50 °C: Hydrolysis not detected. No new peaks formed. Peak profile remained unchanged.
- pH 9 @ 50 °C: Possible hydrolysis. Peak profile changed with larger peaks earlier in the run.
- Other evidence of hydrolysis? None
- Conclusion: No hydrolysis occurring at pH 4 and 7. Possible hydrolysis at pH 9
- Further work required? Yes, at pH 9

- A greater than 10 % drop in peak area indicates the test material is hydrolytically unstable at 50 °C over a five day period and Tier 2 should be performed.
- The test material was hydrolytically stable at pH 4 and 7. Tier 2 was required at pH 9.
- Further method development work was done outside of the formal test to determine if the test material was hydrolysable. The test material was subjected to both acid (pH 2) and base (pH 12) forced hydrolysis at 80 °C for 60 minutes. After this treatment the test material showed no signs of acid hydrolysis but did show base hydrolysis in a similar pattern to the pH 9 Tier 1 hydrolysis. This confirmed that the pH 9 Tier 2 test should proceed.

Transformation products:

no

Details on hydrolysis and appearance of transformation product(s):

HYDROLYSIS (TIER 2) OBSERVATIONS
- The test material was composed of several peaks ranging from 0.9 min to 13 minutes. In the following sections of this report, the individual compounds of interest are referred to by their retention times.
- The profile of the test material in pH 9 buffer did not change over the course of the experiment. There was a size reduction is some of the peaks but it was considered insignificant when compared to the overall profile.

HYDROLYSIS (TIER 2) RESULTS
- The experiment was conducted over a 21-day period. As there was no hydrolysis at pH 9 / 50 °C, the test was also stopped at 37.5 °C and 25 °C after 21 days. As hydrolysis is a temperature dependent process, only the 50 °C results are discussed in detail here.
- All peaks were monitored for degradation with the following exceptions. The early eluting peaks between 0 and 1.06 minutes were not included in the analysis because they were unreliable due to blank interferences. Also any peak that was less than 5 % of the total was excluded.
- The peaks at 1.32, 2.14, 2.50, 4.24 and 12.93 min show no signs of hydrolysis. The difference between area counts at the start and end of the test was less than 10 %. Peaks at 1.21, 1.53 and 5.20 minutes initially showed signs of hydrolysis (lower area counts at T=End v.s. T=0) but when the amount hydrolysed is calculated and compared to the total chromatogram area counts, it only accounts for 5.2%. Data from the 185 hour time-point was excluded from the results. Data for some of the peaks at this time point seemed to indicate hydrolysis but hydrolysis was not shown for the same peaks before this time point (41.5, 65.4 and 137.1 hours) and at the remaining time points (305 and 496.8 hours). The overall profile of the test material remained unchanged over the course of the experiment.

pH:

9

Temp.:

25 °C

Remarks on result:

other: The test material is hydrolytically stable

pH:

9

Temp.:

37.5 °C

Remarks on result:

other: The test material is hydrolytically stable

pH:

9

Temp.:

50 °C

Remarks on result:

other: The test material is hydrolytically stable

Details on results:

Based on the above information the test material is hydrolytically stable at pH 9 at 25, 37.5 and 50 °C in OECD Test No. 111 Tier 2.
Based on OECD Test No. 111, the test material is hydrolytically stable at pH 4, 7 and 9.

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of this study, the test material is hydrolytically stable at pH 9 at 25, 37.5 and 50 °C in OECD Test No. 111 Tier 2. Based on OECD Test No. 111, the test material is hydrolytically stable at pH 4, 7 and 9.

Executive summary:

The hydrolysis of the test material was investigated in accordance with the standardised guideline OECD 111, under GLP conditions.

The preliminary test was performed at pH 4, 7 and 9 at 50 °C for five days. The test material was hydrolytically stable at pH 4 and 7. Tier 2 was required at pH 9.

Tier 2 was conducted at three temperatures (25 °C, 37.5 °C and 50 °C) and one pH (pH 9). The test material was composed of several peaks ranging from 0.9 min to 13 minutes. The profile of the test material in pH 9 buffer did not change over the course of the experiment. There was a size reduction is some of the peaks but it was considered insignificant when compared to the overall profile. The experiment was conducted over a 21-day period. As there was no hydrolysis at pH 9 / 50 °C, the test was also stopped at 37.5 °C and 25 °C after 21 days.

Under the conditions of this study, the test material is hydrolytically stable at pH 9 at 25, 37.5 and 50 °C in OECD Test No. 111 Tier 2. Based on OECD Test No. 111, the test material is hydrolytically stable at pH 4, 7 and 9.

Description of key information

Under the conditions of the study, the test material is hydrolytically stable at pH 9 at 25, 37.5 and 50 °C in OECD Test No. 111 Tier 2. Based on OECD Test No. 111, the test material is hydrolytically stable at pH 4, 7 and 9.

Additional information

The hydrolysis of the test material was investigated in accordance with the standardised guideline OECD 111, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The preliminary test was performed at pH 4, 7 and 9 at 50 °C for five days. The test material was hydrolytically stable at pH 4 and 7. Tier 2 was required at pH 9.

Tier 2 was conducted at three temperatures (25 °C, 37.5 °C and 50 °C) and one pH (pH 9). The test material was composed of several peaks ranging from 0.9 min to 13 minutes. The profile of the test material in pH 9 buffer did not change over the course of the experiment. There was a size reduction is some of the peaks but it was considered insignificant when compared to the overall profile. The experiment was conducted over a 21-day period. As there was no hydrolysis at pH 9 / 50 °C, the test was also stopped at 37.5 °C and 25 °C after 21 days.

Under the conditions of this study, the test material is hydrolytically stable at pH 9 at 25, 37.5 and 50 °C in OECD Test No. 111 Tier 2. Based on OECD Test No. 111, the test material is hydrolytically stable at pH 4, 7 and 9.