p-benzoquinone dioxime

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-05-2018 to 14-09-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Deviations:

no

Principles of method if other than guideline:

The test followed a method in accordance with EU Method C.7 and equivalent or similar to OECD TG 111 (hydrolysis as a function of pH) - as a screening study (tier 1) for the hydrolysis properties of the test substance and/or determination of hydrolysis rate constants (tier 2). Specific identification of hydrolysis products (tier 3) was not performed.

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
Preliminary test (tier 1): 0 hours, 2.4 and 120 hours (as appropriate depending on results).
Definitive test (tier 2): Not applicable. A main study at pH 9 was not performed. The test item undergoes self-reactivity/precipitation under the conditions of the study at pH 9. Therefore it is technically not possible to conduct further hydrolysis testing at this pH. This was demonstrated by a solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours.
- Sampling method: Test samples were analyzed by single injection. No pretreatment. Samples were cooled to room temperature using running tap water and analysed. In the preliminary test matrix-matched calibration was applied. Injecting solutions containing buffer pH 9, however, chromatography was disturbed. Therefore the sample preparation procedure was modified and verified analyzing quality control (QC) samples in buffers pH 4, 7 and 9 at a target concentration of 1 or 100 mg/L. Subsamples of 50 or 100 µL were diluted with acetate buffer pH 4, 0.1 M in a ratio of at least 1/9 (v/v) to obtain concentrations within the calibration range. Repeating the preliminary test at pH 9 subsamples of 100 µL were diluted with acetate buffer pH 4, 0.1 M in a ratio of 1/9 (v/v) and analyzed. In case of precipitation, the content of the sample tube was quantitatively transferred to a glass vessel and diluted with acetate buffer pH 4, 0.1 M in a ratio of 1/9 (v/v).
- Sampling methods for the volatile compounds, if any: Not applicable.
- Sampling intervals/times for pH measurements: Not reported.
- Sampling intervals/times for sterility check: The buffer solutions were filtered through a 0.2 µm membrane filter and and degassing. Thereby ensuring sterility of the test system.
- Sample storage conditions before analysis: On the autosampler prior to analysis.
- Other observation, if any (e.g.: precipitation, color change etc.): Precipitation was observed in test vessels (not samples) as described at pH 9.

Buffers:

- pH: 4
- Type and final molarity of buffer: Acetate buffer pH 4, 0.1 M
- Composition of buffer: 16.7% 0.1 M sodium acetate in water and 83.3% 0.1 M acetic acid in water. Buffer contained 0.0009% (w/v) sodium azide

- pH: 7
- Type and final molarity of buffer: Phosphate buffer pH 7, 0.1 M
- Composition of buffer: 0.1 M potassium di-hydrogen-phosphate in water adjusted to pH 7 using 10N sodium hydroxide. Buffer contained 0.0009% (w/v) sodium azide

- pH: 9
- Type and final molarity of buffer: Phosphate buffer pH 7, 0.1 M
- Composition of buffer: 0.1 M boric acid in water and 0.1 M potassium chloride in water adjusted to pH 9 using 10N sodium hydroxide. Buffer contained 0.0009% (w/v) sodium azide

Note: In the preliminary test matrix-matched calibration was applied. Injecting solutions containing buffer pH 9 indicated chromatography was disturbed. Therefore the sample preparation procedure was modified and verified analyzing quality control (QC) samples in buffers pH 4, 7 and 9 at a target concentration of 1 or 100 mg/L. Subsamples of 50 or 100 µL were diluted with acetate buffer pH 4, 0.1 M in a ratio of at least 1/9 (v/v) to obtain concentrations within the calibration range. Repeating the preliminary test at pH 9 subsamples of 100 µL were diluted with acetate buffer pH 4, 0.1 M in a ratio of 1/9 (v/v) and analyzed. In case of precipitation, the content of the sample tube was quantitatively transferred to a glass vessel and diluted with acetate buffer pH 4, 0.1 M in a ratio of 1/9 (v/v).

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: stoppered glass flasks
- Sterilisation method: The buffer solutions were filtered through a 0.2 µm FP 30/0.2 CA-S filter and subject to degassing, to exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. Thereby ensuring sterility of the test system.
- Lighting: Not applicable.
- Measures taken to avoid photolytic effects: All solutions were placed in the dark.
- Measures to exclude oxygen: Degassing with nitrogen gas for 5 minutes. Closed vessels.
- Details on test procedure for unstable compounds: Not applicable.
- Details of traps for volatile, if any: Not applicable (closed system).
- If no traps were used, is the test system closed/open: Closed system.
- Is there any indication of the test material adsorbing to the walls of the test apparatus?: Not reported.
TEST MEDIUM
- Volume used/treatment: For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 49.9°C  0.2°C for pH 4 and 7 and at 50.0°C  0.1°C for pH 9.
- Kind and purity of water: Milli-Q RO Water
- Preparation of test medium: The buffer solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. The test item was spiked to the solutions at a target concentration of 2 mg/L (preliminary test at pH4, pH 7 and pH 9) or 20 mg/L (repeat of the preliminary test at pH 9) using a spiking solution in methanol. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 49.9°C ± 0.2°C for pH 4 and 7 and at 50.0°C ± 0.1°C for pH 9. The spiking volume was < 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume.
- Renewal of test solution: No.
- Identity and concentration of co-solvent: Methanol. Stock solutions of the test item were prepared in methanol at concentrations of 1000 and 2000 mg/L. Spiking solutions were made up from a stock solution. Spiking volume was < 1% of the sample volume.
OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Dissolved oxygen: Minimised.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

2 mg/L

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

2 mg/L

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

>= 2 - <= 20 mg/L

Remarks:

20 mg/L was used in the repeat experiment

Number of replicates:

None (duplicate vessels, single injection).

Positive controls:

no

Negative controls:

no

Preliminary study:

In the preliminary test (tier 1) at 50 °C:
pH 4: < 10 % hydrolysis was observed after 5 dayss. Which is equivalent to a half-life > 1 year at 25 °C.
pH 7: < 10 % hydrolysis was observed after 5 dayss. Which is equivalent to a half-life > 1 year at 25 °C.
pH 9: During the preliminary test at pH 9, brown undissolved material was observed in the sample tubes after 5 days. The test item was fully soluble at pH 9 at the concentration level of the hydrolysis preliminary test (determined within the study report: solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours). As solubility issues were ruled out, it was considered that the test item underwent self-reactivity/precipitation under the conditions of the study at pH 9. Therefore, it was technically not possible to conduct further hydrolysis testing at pH 9.

Test performance:

No unusual findings were reported in the study.

Transformation products:

not measured

pH:

4

Temp.:

50 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

50 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

50 °C

Remarks on result:

not determinable

Remarks:

test item undergoes self-reactivity/precipitation at pH 9. Therefore it is not technically possible to conduct further hydrolysis testing

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes. (No indications in the study of any anomalies). Yes, by use of appropriate buffers however pH was not continuously monitored throughout the study, sterility was not examined.
- Anomalies or problems encountered (if yes): At pH 9, during the preliminary test at pH 9, brown undissolved material was observed in the sample tubes after 5 days. The test item was fully soluble at pH 9 at the concentration level of the hydrolysis preliminary test (determined within the study report: solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours). As solubility issues were ruled out, it was considered that the test item underwent self-reactivity/precipitation under the conditions of the study at pH 9. Therefore, it was technically not possible to conduct further hydrolysis testing at pH 9.

MAJOR TRANSFORMATION PRODUCTS
Not examined.

MINOR TRANSFORMATION PRODUCTS
No examined.

MINERALISATION (distinguish between dark and irradiated samples)
Not examined.

INDICATION OF UNSTABLE TRANSFORMATION PRODUCTS:
Not examined.

VOLATILIZATION (at end of study)
Not examined.

UNIDENTIFIED RADIOACTIVITY (at end of study)
Not examined.

PATHWAYS OF HYDROLYSIS
Not examined.

SUPPLEMENTARY EXPERIMENT (if any): RESULTS:
In the preliminary test (tier 1) at 50 °C:
pH 4: < 10 % hydrolysis was observed after 5 dayss. Which is equivalent to a half-life > 1 year at 25 °C.
pH 7: < 10 % hydrolysis was observed after 5 dayss. Which is equivalent to a half-life > 1 year at 25 °C.
pH 9: During the preliminary test at pH 9, brown undissolved material was observed in the sample tubes after 5 days. The test item was fully soluble at pH 9 at the concentration level of the hydrolysis preliminary test (determined within the study report: solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours). As solubility issues were ruled out, it was considered that the test item underwent self-reactivity/precipitation under the conditions of the study at pH 9. Therefore, it was technically not possible to conduct further hydrolysis testing at pH 9.

Validity criteria fulfilled:

yes

Remarks:

The study meets the tier 1 and tier 2 validity criteria. This is limited as detailed in 'Rationale for reliability incl. deficiencies'. The study is reliable as indicates that the substance is hydrolytically unstable under specific conditions.

Conclusions:

The substance was found to be stable to hydrolysis in water at pH 4 (t1/2: > 1 year) and pH 7 (t1/2: > 1 year). The test item underwent self-reactivity/precipitation under the conditions of the study at pH 9. Therefore it is technically not possible to conduct further hydrolysis testing at this pH. This was demonstrated by a solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours.

Executive summary:

The test followed a method in accordance with EU Method C.7 and OECD TG 111 for abiotic degradation: hydrolysis as a function of pH under GLP. The study served as both a preliminary screening study (tier 1) and rate constant determination (tier 2) for the hydrolysis properties of the item, where applicable. The study was conducted with the test vessels protected from light. The pH 4, 7 and 9 buffer solutions were filtered through a 0.2 µm membrane filter and subject to degassing. Thereby ensuring sterility of the test system. The test item was spiked to the solutions at a target concentration of 2 mg/L (preliminary test at pH4, pH 7 and pH 9) or 20 mg/L (repeat of the preliminary test at pH 9) using a spiking solution in methanol. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 49.9°C ± 0.2°C for pH 4 and 7 and at 50.0°C ± 0.1°C for pH 9. The spiking volume was < 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume. Samples were taken in the Preliminary test (tier 1): 0 hours, 2.4 and 120 hours and concentrations determined by Ultra Performance Liquid Chromatography (UPLC) by single injection. The mean recoveries of the procedural recovery samples containing test item fell within the criterion of 70 − 110%. From the method validation: samples were not corrected for recoveries. It demonstrated that the analytical method was adequate for the determination of the test item concentration in the test samples. In the preliminary test: The mean recoveries of the of the test item fell in the range of 90-110% at pH 7 and 9. Whereas, pH 4, at t=0 slightly exceeded the criterion range of 90-110% (i.e. 114%). It was, however, considered that this slight deviation did not hamper the conclusion that no significant hydrolysis was observed at this pH. In the preliminary test (tier 1) at 50 °C at pH 4 and pH 7: less than 10% hydrolysis was observed after 5 days. Which is equivalent to a half-life > 1 year at 25 °C. A main study at pH 9 was not performed. The test item undergoes self-reactivity/precipitation under the conditions of the study at pH 9. At pH 9, during the preliminary test at pH 9, brown undissolved material was observed in the sample tubes after 5 days. The test item was fully soluble at pH 9 at the concentration level of the hydrolysis preliminary test (determined within the study report: solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours). As solubility issues were ruled out, it was considered that the test item underwent self-reactivity/precipitation under the conditions of the study at pH 9. Therefore, it was technically not possible to conduct further hydrolysis testing at pH 9. Applicant assessment indicates: The test item is hydrolytically stable at pH 4 to 7 with equivalent half-life > 1 year at 25 °C. At pH 9 the test item undergoes self-reactivity/precipitation and it is not technically possible to conduct further testing. The self-reactivity/precipitation was not the result of hydrolysis products.

Description of key information

Hydrolysis: half-life for hydrolysis: pH 4 : t1/2: > 1 year ; pH 7: t1/2: > 1 year ; pH 9: t1/2: not technically possible to conduct due to self-reactivity/precipitation, at 25 °C, 1 atm, EU Method C.7, 2018

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

Key study : EU Method C.7 , 2018 : The test followed a method in accordance with EU Method C.7 and OECD TG 111 for abiotic degradation: hydrolysis as a function of pH under GLP. The study served as both a preliminary screening study (tier 1) and rate constant determination (tier 2) for the hydrolysis properties of the item, where applicable. The study was conducted with the test vessels protected from light. The pH 4, 7 and 9 buffer solutions were filtered through a 0.2 µm membrane filter and subject to degassing. Thereby ensuring sterility of the test system. The test item was spiked to the solutions at a target concentration of 2 mg/L (preliminary test at pH4, pH 7 and pH 9) or 20 mg/L (repeat of the preliminary test at pH 9) using a spiking solution in methanol. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 49.9°C ± 0.2°C for pH 4 and 7 and at 50.0°C ± 0.1°C for pH 9. The spiking volume was < 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume. Samples were taken in the Preliminary test (tier 1): 0 hours, 2.4 and 120 hours and concentrations determined by Ultra Performance Liquid Chromatography (UPLC) by single injection. The mean recoveries of the procedural recovery samples containing test item fell within the criterion of 70 − 110%. From the method validation: samples were not corrected for recoveries. It demonstrated that the analytical method was adequate for the determination of the test item concentration in the test samples. In the preliminary test: The mean recoveries of the of the test item fell in the range of 90-110% at pH 7 and 9. Whereas, pH 4, at t=0 slightly exceeded the criterion range of 90-110% (i.e. 114%). It was, however, considered that this slight deviation did not hamper the conclusion that no significant hydrolysis was observed at this pH. In the preliminary test (tier 1) at 50 °C at pH 4 and pH 7: less than 10% hydrolysis was observed after 5 days. Which is equivalent to a half-life > 1 year at 25 °C. A main study at pH 9 was not performed. The test item undergoes self-reactivity/precipitation under the conditions of the study at pH 9. At pH 9, during the preliminary test at pH 9, brown undissolved material was observed in the sample tubes after 5 days. The test item was fully soluble at pH 9 at the concentration level of the hydrolysis preliminary test (determined within the study report: solubility test conducted at pH 9 where the test item was soluble in the range of 20.8 – 27.0 mg/L over a period of 72 hours). As solubility issues were ruled out, it was considered that the test item underwent self-reactivity/precipitation under the conditions of the study at pH 9. Therefore, it was technically not possible to conduct further hydrolysis testing at pH 9. Applicant assessment indicates: The test item is hydrolytically stable at pH 4 to 7 with equivalent half-life > 1 year at 25 °C. At pH 9 the test item undergoes self-reactivity/precipitation and it is not technically possible to conduct further testing. The self-reactivity/precipitation was not the result of hydrolysis products.

 

Weight of Evidence: conclusion:

The substance appears hydrolytically stable at all pH ranges with t1/2 > 1 year at 25 °C. At pH 9 there is apparent self-reactivity/precipitation which occurs within a 5 day period which was not the result of hydrolysis reactions. Further testing for hydrolysis was deemed not technically feasible.