[No public or meaningful name is available]

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24-09-2001 to 24-01-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Well documented study report following a screening study and method to guideline with acceptable deviations according to the regulatory conclusion that the substance is hydrolytically stable under specific conditions. The study would not fulfil the EU Method C.7 or OECD TG 111 (2004) Tier 3 requirements.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The test followed a method in accordance with OECD TG 111 (hydrolysis as a function of pH) and/or EU Method C.7 - 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 (incl. QA statement)

Remarks:

inspected: November 2019 ; signature: August 2020

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: Duplicate vessels were performed per pH and sampling date, by single injection analysis.
Preliminary test (tier 1): all pH : 0 hours and 120 hours (as appropriate depending on results).
Definitive test (tier 2): The definitive test was conducted for at least two half-live periods, optimally until 90 % degradation is reached or 30 days, whichever is shorter. Samples were taken at test start (0 h) and at 10 to 12 spaced points, at each test temperature. See tables for further information. This included up to 554 hours at 20 °C temperature. All test item containing samples were stabilized immediately by liquid-liquid extraction (if possible, less than 30 min until start of analyses, but at least not more than 2.5% of the total study time) and analysed.
- Sampling method: In the preliminary test: after sampling, 0.75 mL cyclohexane and approx. 1.5 g sodium chloride were added to the whole sample (15 mL). Afterwards, the vessels were shaken for 2 min for liquid extraction. The organic layer was transferred into a fresh vial and dried with (water free) sodium sulphate, corresponding to an enrichment factor of 20. 0.1 mL of the dried extracts were diluted with 0.1 mL cyclohexane, corresponding to a dilution factor of 2 and a final enrichment factor of 10. In the definitive test: After sampling, 1.5 mL cyclohexane was added via syringe pierced through the septum of the test vessel to the whole sample (15 mL). Afterwards, the vessels were opened and resealed after addition of approx. 1.5 g sodium chloride. The vessels were shaken for 2 min for liquid extraction. The organic layer was transferred into a fresh vial and dried with (water free) sodium sulphate, corresponding to an enrichment factor of 10. Further dilution steps of the samples with cyclohexane. Dilution factors are documented in the full study report, as applicable. Within the definitive test, the time between sampling and analysing exceeded 30 min, but the test samples were stabilized via liquid extraction.
- Sampling methods for the volatile compounds, if any: Not applicable.
- Sampling intervals/times for pH measurements: Start prior to test item application at 20°C, 30°C and 50°C with a quality control check at room temperature (ca. 25°C)
- Sampling intervals/times for sterility check: In the definitive test : as transformation occurs the sterility of the test solutions was checked at the end of the test by colony forming units (CFU)-determination with Water Plate Count Agar from additional sample. By incubation at 36 ± 1 °C for 48 ± 4 h and at 22 ± 1 °C for 72 ± 4 h. In a deviation: determination of colony forming units the temperature was below the temperature range of 36 ± 1 °C due to technical reasons. This was considered to have had no impact on the conduct of the study. Prior to the study: buffer solutions were purged/degassed with nitrogen for 5 minutes and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilized by filtration through 0.2 µm. 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.): None reported.

Buffers:

- pH: Sterile buffer solutions with pH values : pH 4, pH 7 and pH 9
- Type and final molarity of buffer: See below, in accordance with the OECD TG 111 guideline.
- Composition of buffer: Buffer solutions were prepared from chemicals with analytical grade or better quality following the composition guidance given in KÜSTER-THIEL, Rechentafeln für die Chemische Analytik (edition not specified, in German), and/or the OECD TG 111 guideline. The buffers were prepared by direct weighing of the buffer components (nominal values) as below.
• pH 4 : 0.18 g of sodium hydroxide and 5.7555 g of mono potassium citrate were dissolved in 500 mL ultrapure water.
• pH 7: 3.854 g of ammonium acetate were dissolved in 500 mL ultrapure water.
• pH 9 : 0.426 g sodium hydroxide, 1.8638 g potassium chloride and 1.5458 g boric acid were dissolved in 500 mL ultrapure water.
Prior to the study: buffer solutions were purged/degassed with nitrogen for 5 minutes and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilized by filtration through 0.2 µm. Thereby ensuring sterility of the test system.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: (sterile) HPLC vials, volume: 20 mL
- Sterilisation method: Prior to the study: buffer solutions were purged/degassed with nitrogen for 5 minutes and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilized by filtration through 0.2 µm. Thereby ensuring sterility of the test system.
- Lighting: Not applicable. Photolytic effects were avoided by using opaque water baths.
- Measures taken to avoid photolytic effects: All solutions shielded from light. See above.
- Measures to exclude oxygen: Buffer solutions were purged/degassed with nitrogen for 5 minutes and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilized by filtration through 0.2 µm. Thereby ensuring sterility of the test system.- 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?: None reported. Co-solvent: 0.1% (v/v) of acetone, was utilised within the study.

TEST MEDIUM
- Volume used/treatment: See below for information on the test system
- Test container: (sterile) HPLC vials, volume: 20 mL
- Test volume: 15 mL
- Spiking solution: 100 µg/L in acetone
- Test concentration/solution: 100 ng/L, corresponding to half of the water solubility determined in previously conducted water solubility study, documented in the full study report.
- Co-solvent: 0.1% (v/v) of acetone
The test solutions were prepared at test start via spiking solution. The test item was dissolved in MTBE and diluted with acetone to 100 µg/L. 15 mL of sterile buffer solution were spiked with 0.015 mL of this dilution inside the test containers. After the vials were sealed, they were transferred into the thermostat. The time between test item application and transfer to thermostat did not exceed 30 min. Within the definitive test, the time between sampling and analysing exceeded 30 min, but the test samples were stabilized via liquid extraction.
- Kind and purity of water: Deionised or double distilled water (‘ultra-pure’ produced in the laboratory – documented in the full study report).
- Preparation of test medium: See above.
- Renewal of test solution: No.
- Identity and concentration of co-solvent: 0.1% v/v acetone
OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Dissolved oxygen: Minimised to extent possible.

Number of replicates:

Duplicates [per pH and sampling date] (double vessels, single injection)

Positive controls:

no

Remarks:

Reference item : test is not an OECD TG 111 guideline requirement

Negative controls:

no

Remarks:

However, for each test system, two blank samples were prepared in the same manner of the fortified samples without the spiking step and analysed

Preliminary study:

In the preliminary test (tier 1) at 50 °C: more than 10 % of the test item was degraded after 120 hours for each tested pH value (4, 7 and 9).
pH 4: 92.2% at 120 hours
pH 7: 92.0% at 120 hours
pH 9: 85.1% at 120 hours
The extent of hydrolysis after 120 hours (5 days) indicated further testing would be required to estimate the rate constant and half life.
Estimated half-life from tier 1 was pH 4, pH 7 and pH 9 : << 1 year

Test performance:

In the definitive test: Within the advanced test, the time between sampling and analysing exceeded 30 min, but the test samples were stabilized via liquid extraction.
The temperature was above the temperature range of 20 ± 0.5 °C in sum for 9 h (4h and 5 h). This was not considered to have impacted the conclusions of the study.
The definitive test was conducted with a test item concentration of 100 ng/L in buffer solutions at pH 4, 7 and 9 with 0.1% acetone as co-solvent at temperatures of 20, 30 and 50 °C, respectively. Samples were taken at test start (0 h) and at 10 to 12 spaced points until test end. Pure test system (buffer solution at the respective pH value with 0.1% acetone as co-solvent) was analysed at test start and test end and there was no analytical interference with the test item. All applicable validity criteria were considered to be met. For further information on results, see tables.

Transformation products:

not measured

Remarks:

No major transformation product could be detected (using the analytical method employed in the test).

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

- Formation and decline of each transformation product during test: Not reported.
- Pathways for transformation: Not reported.
- Other: Other information outside of the study is available, as to the theoretical transformation products (and/or that may have been analytically identified).

Remarks on result:

other: See tables.

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

10.8 d

Type:

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

Remarks on result:

other: based on the obtained data the pseudo first order reaction kinetics was deemed to be the best fit model for computation of kinetics data

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

7.72 d

Type:

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

Remarks on result:

other: based on the obtained data the pseudo first order reaction kinetics was deemed to be the best fit model for computation of kinetics data

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

9.45 d

Type:

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

Remarks on result:

other: based on the obtained data the pseudo first order reaction kinetics was deemed to be the best fit model for computation of kinetics data

Other kinetic parameters:

For all test conditions the ln concentration vs. time plots have regression graphs with slopes significantly non-zero. First order reaction kinetics were applied for data computation. A confirmation of pseudo first order reaction kinetics with coefficients of determination > 0.8 was achieved for pH 4 at 50°C, all test conditions at pH 7 and for pH 9 at 20 °C. For the test conditions pH 4 at 20°C and 30°C as well as pH 9 at 30°C and 50°C no clear confirmation of pseudo first order reaction kinetics with coefficients of determination > 0.8 was achieved. Nevertheless, based on the obtained data the pseudo first order reaction kinetics was deemed to be the best fit model for computation of kinetics data.

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). By use of appropriate buffers however pH was not continuously monitored throughout the study, sterility was examined by colony forming units (CFU)-determination with Water Plate Count Agar from additional samples of the definitive test by incubation.
- Anomalies or problems encountered (if yes): Based on the obtained data, reaction rate constants and half-lives were calculated for all test conditions. It was considered in the study, that the missing differences between the kinetics data of the different pH conditions indicated, that hydrolysis might not be the primary manner of test item transformation/degradation. Nevertheless, it was considered the obtained data was a good approximation for the stability of the test item in aqueous solution. See information as to the Procedural Recovery: Quality Controls (Definitive Test and Definitive Test Analytical Method) in tables. Results were corrected for recoveries, as applicable.

MAJOR TRANSFORMATION PRODUCTS
Not examined. No major transformation product could be detected (using the analytical method employed in the test).

MINOR TRANSFORMATION PRODUCTS
No examined. No transformation product could be detected (using the analytical method employed in the test).

MINERALISATION (distinguish between dark and irradiated samples)
Not examined. All testing completed in the dark or protected from light.

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: See information within “overall remarks, attachments” as to the modelled data from the primary hydrolysis data. Full tables of the primary data for each measured time-point, are available in the full study report.

Table 1. Results of Procedural Recovery: Quality Controls (Definitive Test and Definitive Test Analytical Method)

Day		pH 4		pH 7		pH 9
	Replicate	Calc. conc.	RR	Calc. conc.	RR	Calc. conc.	RR
		[ng/L]	[%]	[ng/L]	[%]	[ng/L]	[%]
1	1	8.43	84.3	8.21	82.1	9.21	92.1
	2	8.87	88.7	8.71	87.1	8.04	80.4
	3	10.4	104	9.30	93.0	10.0	100
2	1	3.58	35.8	11.4	114	8.26	82.6
	2	4.38	43.8	6.76	67.6	11.3	113
	3	8.72	87.2	5.85	58.5	6.75	67.5
3	1	4.71	47.1	5.48	54.8	6.19	61.9
	2	4.98	49.8	5.69	56.9	4.81	48.1
	3	5.14	51.4	4.60	46.0	7.89	78.9
4	1	6.41	64.1	6.97	69.7	8.13	81.3
	2	7.76	77.6	6.85	68.5	6.29	62.9
	3	7.14	71.4	6.32	63.2	7.16	71.6
7	1	10.4	104	9.66	96.6	9.40	94.0
	2	9.69	96.9	7.87	78.7	8.33	83.3
	3	9.59	95.9	8.64	86.4	8.91	89.1
11	1	10.8	108	9.45	94.5	9.76	97.6
	2	9.33	93.3	10.8	108	9.56	95.6
	3	7.42	74.2	9.30	93.0	6.08	60.8
23	1	6.14	61.4	6.09	60.9	7.42	74.2
	2	8.05	80.5	6.49	64.9	6.87	68.7
	3	6.68	66.8	7.21	72.1	6.98	69.8
24	1	8.16	81.6	8.33	83.3	8.75	87.5
	2	8.08	80.8	7.92	79.2	8.77	87.7
	3	7.83	78.3	7.89	78.9	7.57	75.7

Calc. conc. = Calculated concentration, enrichment factor of 10 taken into account

RR = Recovery rate regarding to the fortified concentration of the test item

Days associated with Dates are provided in the full study report.

 

Table 2. Reaction Rate Constants and Half-Lives with pH from individual tests

pH 4
	20 °C	30 °C	50 °C
Slope of regression graph	-2.18 x10^-3	-2.71 x10^-3	-3.42 x10^-2
Correlation factor [r2]	0.594	0.546	0.972
Reaction rate constant kobs [1/s]	6.05 x10^-7	7.52 x10^-7	9.49 x10^-6
Half-life T½ [h]	318	256	20.3
Confidence interval of half-life T½ [h]	213 to 650	151 to 575	18.2 to 22.5
Half-life T½ [d]	13.3	10.7	0.845
Confidence interval of half-life T½ [d]	8.88 to 27.1	6.29 to 24.0	0.758 to 0.938
pH 7
	20 °C	30 °C	50 °C
Slope of regression graph	-3.19 x10^-3	-3.85 x10^-3	-2.31 x10^-2
Correlation factor [r2]	0.865	0.948	0.948
Reaction rate constant kobs [1/s]	8.85 x10^-7	1.07 x10^-6	6.42 x10^-6
Half-life T½ [h]	217	180	30.0
Confidence interval of half-life T½ [h]	171 to 278	153 to 209	26.3 to 34.0
Half-life T½ [d]	9.06	7.51	1.25
Confidence interval of half-life T½ [d]	7.13 to 11.6	6.38 to 8.71	1.10 to 1.42
	pH 9
	20 °C	30 °C	50 °C
Slope of regression graph	-2.91 x10^-3	-2.79 x10^-3	-1.30 x10^-2
Correlation factor [r2]	0.814	0.702	0.797
Reaction rate constant kobs [1/s]	8.08 x10^-7	7.75 x10^-7	3.60 x10^-6
Half-life T½ [h]	238	248	53.5
Confidence interval of half-life T½ [h]	181 to 325	172 to 395	39.5 to 73.5
Half-life T½ [d]	9.93	10.3	2.23
Confidence interval of half-life T½ [d]	7.54 to 13.5	7.17 to 16.5	1.65 to 3.06

 

Table 3. Results of Arrhenius Calculations from individual tests

pH value	[°C]	-EA/R	ln A	EA [J * mol-1]
4	20	-9143	16.6	7.60 x10^4
	30
	50
7	20	-6562	8.23	5.46 x10^4
	30
	50
9	20	-5057	2.98	4.20 x10^4
	30
	50

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 it indicates that the substance is hydrolytically unstable under specific conditions.

Conclusions:

The substance was found in a tier 1 preliminary test to be unstable to hydrolysis in water at pH 4, pH 7 and pH 9 (t1/2: < 5 days) at 50 °C and therefore the equivalent half-life at 25°C: would be << 1 year. Subsequently, in a tier 2 definitive test: the substance was found to be unstable with half-lives determined by the Arrhenius equation as follows: pH 4 : 10.8 days, pH 7: 7.72 days and pH 9: 9.45 days.

Executive summary:

The test followed a method in accordance with OECD TG 111 and EU Method C.7 (abiotic degradation: hydrolysis as a function of pH) under GLP serving as both a screening study (tier 1) and rate constant determination (tier 2) for the hydrolysis properties of the item. The study was conducted with the test vessels protected from light. Buffer solutions were prepared from chemicals with analytical grade or better quality and were purged/degassed with nitrogen for 5 minutes and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilized by filtration through 0.2 µm. Thereby ensuring sterility of the test system. The test solutions were prepared at test start via spiking solution. The test item was dissolved in MTBE and diluted with acetone to 100 µg/L. 15 mL of sterile buffer solution were spiked with 0.015 mL of this dilution inside the test containers (HPLC vials, total volume: 20 mL). After the vials were sealed, they were transferred into the thermostat. The time between test item application and transfer to thermostat did not exceed 30 minutes. Within the definitive test, the time between sampling and analysing exceeded 30 minutes, but the test samples were stabilized via liquid extraction. The preliminary test was conducted with a test item concentration of 100 ng/L in buffer solutions at pH 4, 7 and 9 with 0.1% acetone as co-solvent at 50°C. For all pH values, a significant reduction (> 10%) of the test item concentration was observed in the preliminary test. The definitive test was conducted with a test item concentration of 100 ng/L in buffer solutions at pH 4, 7 and 9 with 0.1% acetone as co-solvent at temperatures of 20°C, 30°C and 50°C, respectively. Samples were taken at test start (0 hours) and at ten to twelve spaced points until test end. Pure test system (buffer solution at the respective pH value with 0.1% acetone as co-solvent) was analysed at test start and test end and there was no analytical interference with the test item. It was considered in the study, that the missing differences between the kinetics data of the different pH conditions indicated, that hydrolysis might not be the primary manner of test item transformation/degradation. Nevertheless, it was considered the obtained data was a good approximation for the stability of the test item in aqueous solution. Reaction rate constants, half-lives and activation energies were therefore calculated using information from the and analysed samples the Arrhenius equation. Based on a first order reaction kinetics, these were as follows: reaction rate constant (kobs) at 25°C [1/s]: pH 4 : 7.43 x10^-7, pH 7: 1.04 x10^-6 and pH 9: 8.49 x10^-7. The equivalent half-lives at 25°C: pH 4 : 10.8 days, pH 7: 7.72 days and pH 9: 9.45 days. Using a classification scheme of hydrolysis being ‘fast’ : t1/2 ≤ 2.4 hours , ‘moderate’ : 2.4 hours ≥ t1/2 and ≤ 30 days and ‘slow’ : t1/2 > 30 days, respectively. For all test conditions of pH 4, pH 7 and pH 9 at 25°C, the test item showed a moderate hydrolysis rate.

Description of key information

Hydrolysis: half-life for hydrolysis: pH 4 : 10.8 days, pH 7: 7.72 days and pH 9: 9.45, at 25 °C, 1 atm, OECD TG 111, 2020

Based on a first order reaction kinetics, these were as follows: reaction rate constant (kobs) at 25°C [1/s]: pH 4 : 7.43 x10^-7, pH 7: 1.04 x10^-6 and pH 9: 8.49 x10^-7

Key value for chemical safety assessment

Half-life for hydrolysis:

10.8 d

at the temperature of:

25 °C

Additional information

Key study : OECD TG 111, 2020 : The test followed a method in accordance with OECD TG 111 and EU Method C.7 (abiotic degradation: hydrolysis as a function of pH) under GLP serving as both a screening study (tier 1) and rate constant determination (tier 2) for the hydrolysis properties of the item. The study was conducted with the test vessels protected from light. Buffer solutions were prepared from chemicals with analytical grade or better quality and were purged/degassed with nitrogen for 5 minutes and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilized by filtration through 0.2 µm. Thereby ensuring sterility of the test system. The test solutions were prepared at test start via spiking solution. The test item was dissolved in MTBE and diluted with acetone to 100 µg/L. 15 mL of sterile buffer solution were spiked with 0.015 mL of this dilution inside the test containers (HPLC vials, total volume: 20 mL). After the vials were sealed, they were transferred into the thermostat. The time between test item application and transfer to thermostat did not exceed 30 minutes. Within the definitive test, the time between sampling and analysing exceeded 30 minutes, but the test samples were stabilized via liquid extraction. The preliminary test was conducted with a test item concentration of 100 ng/L in buffer solutions at pH 4, 7 and 9 with 0.1% acetone as co-solvent at 50°C. For all pH values, a significant reduction (> 10%) of the test item concentration was observed in the preliminary test. The definitive test was conducted with a test item concentration of 100 ng/L in buffer solutions at pH 4, 7 and 9 with 0.1% acetone as co-solvent at temperatures of 20°C, 30°C and 50°C, respectively. Samples were taken at test start (0 hours) and at ten to twelve spaced points until test end. Pure test system (buffer solution at the respective pH value with 0.1% acetone as co-solvent) was analysed at test start and test end and there was no analytical interference with the test item. It was considered in the study, that the missing differences between the kinetics data of the different pH conditions indicated, that hydrolysis might not be the primary manner of test item transformation/degradation. Nevertheless, it was considered the obtained data was a good approximation for the stability of the test item in aqueous solution. Reaction rate constants, half-lives and activation energies were therefore calculated using information from the and analysed samples the Arrhenius equation. Based on a first order reaction kinetics, these were as follows: reaction rate constant (kobs) at 25°C [1/s]: pH 4 : 7.43 x10^-7, pH 7: 1.04 x10^-6 and pH 9: 8.49 x10^-7. The equivalent half-lives at 25°C: pH 4 : 10.8 days, pH 7: 7.72 days and pH 9: 9.45 days. Using a classification scheme of hydrolysis being ‘fast’ : t1/2 ≤ 2.4 hours , ‘moderate’ : 2.4 hours ≥ t1/2 and ≤ 30 days and ‘slow’ : t1/2 > 30 days, respectively. For all test conditions of pH 4, pH 7 and pH 9 at 25°C, the test item showed a moderate hydrolysis rate.