(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

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:

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

Remarks:

GLP; 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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

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 (incl. QA statement)

Remarks:

inspected: February 2000; signature: April 2000

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
Preliminary test (tier 1): 0 hours, 24, 48, 54, 72, 96 and 120 hours (as appropriate depending on results).
Definitive test (tier 2): Not conducted based on the results of tier 1 (absence of significant: hydrolysis)
In both the tier 1 and tier 2 testing other time periods may have been measured (recorded in the primary data but not included in the presented results).
- Sampling method: Aliquots of the sample solutions were taken from the flasks at various times and the pH of each solution recorded. The concentrations were determined by Gas Chromatography (GC). Duplicate aliquots (100 mL) of each sample were extracted with three portions (3 x 25 mL) of dichloromethane, each extract being filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in methanol (10 mL). For standards: Duplicate standard solutions of test material were prepared in methanol at a nominal concentration of 30 mg/L.
- 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 subject to sonification and degassing with nitrogen. 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:

- Type and final molarity of buffer:
• pH 4 :
Potassium hydrogen phthalate: 0.005 mol dm^-3
• pH 7:
1. disodium hydrogen orthophosphate (anhydrous): 0.003 mol dm^-3
2. Potassium dihydrogen orthophosphate: 0.002 mol dm^-3
3. Sodium Chloride: 0.002 mol dm^-3
• pH 9 :
1. disodium tetraborate: 0.001 mol dm^-3
2. Sodium Chloride: 0.002 mol dm^-3

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 membrane filter and subject to sonification and degassing. Thereby ensuring sterility of the test system.
- Lighting: Not applicable.
- Measures taken to avoid photolytic effects: All solutions shielded from light.
- Measures to exclude oxygen: Ultrasonication and degassing with nitrogen of buffer solutions.
- 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. Silanised glassware was utilised during testing minimise adsorption.

TEST MEDIUM
- Volume used/treatment: Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 3.0x 10^-3 g/L in the three buffer solutions. Using a 0.8% v/v methanol co-solvent at pH 7 and pH 9 and a 0.8% v/v acetonitrile co-solvent used at pH 4.
- Kind and purity of water: Deionised or double distilled water.
- Preparation of test medium: Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 3.0x 10^-3 g/L in the three buffer solutions. Using a 0.8% v/v methanol co-solvent at pH 7 and pH 9 and a 0.8% v/v acetonitrile co-solvent used at pH 4. Aliquots of the sample solutions were taken from the flasks at various times and the pH of each solution recorded.
- Renewal of test solution: No.
- Identity and concentration of co-solvent: 0.8% v/v methanol co-solvent at pH 7 and pH 9 and a 0.8% v/v acetonitrile co-solvent used at pH 4.
OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Dissolved oxygen: Minimised to extent possible.

Number of replicates:

None (single vessel, double injection).

Positive controls:

no

Negative controls:

no

Preliminary study:

In the preliminary test (tier 1) at 50 °C:
pH 4: greater than 50% decline was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method.
pH 7: Half-life of > 120 hours. The extent of hydrolysis after 120 hours (5 days) indicated no further test at 40 °C would be required to estimate the rate constant and half life.
pH 9: Half-life of > 120 hours. The extent of hydrolysis after 120 hours (5 days) indicated no further test at 40 °C would be required to estimate the rate constant and half life.
In the definitive test (tier 2) at 40 °C: Not performed. Estimated half-life from tier 1 was pH 7: > 1 year and pH 9: > 1 year.

Test performance:

In the test at pH 4: greater than 50% decline was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method.
At pH 9: Due to an analytical concentration that was at 0 hours: inconsistently low in comparison with the pH 7 initial value and both pH 7 and pH 9: 120 hour values, the pH 9 initial concentration was been calculated 'as weighed' and corrected for the mean pH 9 observed recovery.

Transformation products:

not measured

% Recovery:

86.7

pH:

4

Temp.:

50 °C

Remarks on result:

other: analytical method recovery

% Recovery:

92.8

pH:

7

Temp.:

50 °C

Remarks on result:

other: analytical method recovery

% Recovery:

90.2

pH:

9

Temp.:

50 °C

Remarks on result:

other: analytical method recovery

pH:

4

Temp.:

50 °C

Remarks on result:

not determinable

pH:

7

Temp.:

50 °C

DT50:

> 5 d

Remarks on result:

hydrolytically stable based on preliminary test

pH:

9

Temp.:

50 °C

DT50:

> 5 d

Remarks on result:

hydrolytically stable based on preliminary test

pH:

4

Temp.:

25 °C

Type:

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

Remarks on result:

not determinable

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

Remarks on result:

hydrolytically stable based on preliminary test

pH:

9

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

Remarks on result:

hydrolytically stable based on preliminary test

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): In the test at pH 4: greater than 50% hydrolysis was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method ; at pH 9: Due to an analytical concentration that was at 0 hours: inconsistently low in comparison with the pH 7 initial value and both pH 7 and pH 9: 120 hour values, the pH 9 initial concentration was been calculated 'as weighed' and corrected for the mean pH 9 observed recovery.

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: greater than 50% decline was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method.
pH 7: Half-life of > 120 hours. The extent of hydrolysis after 120 hours (5 days) indicated no further test at 40 °C would be required to estimate the rate constant and half life.
pH 9: Half-life of > 120 hours. The extent of hydrolysis after 120 hours (5 days) indicated no further test at 40 °C would be required to estimate the rate constant and half life.
In the definitive test (tier 2) at 40 °C: Not performed. Estimated half-life from tier 1 was pH 7: > 1 year and pH 9: > 1 year.

In the test at pH 4: greater than 50% hydrolysis was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method.
At pH 9: Due to an analytical concentration that was at 0 hours: inconsistently low in comparison with the pH 7 initial value and both pH 7 and pH 9: 120 hour values, the pH 9 initial concentration was been calculated 'as weighed' and corrected for the mean pH 9 observed recovery.

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 stable under specific conditions.

Conclusions:

The substance was found to be stable to hydrolysis in water at pH 7 (t1/2: > 5 days) and pH 9 (t1/2: > 5 days) at 50 °C and therefore the equivalent half-life at 25°C: would be > 1 year. The substance was found to be unstable at pH 4; however this was considered to be oxidation and not hydrolysis based on structural assessment and consideration of mechanism. It was considered that the test item (oxidation) was outside the scope of the test method at pH 4.

Executive summary:

The test followed a method in accordance with 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. The pH 4, 7 and 9 buffer solutions were filtered through a 0.2 µm membrane filter and subject to sonification and degassing. Thereby ensuring sterility of the test system. Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 3.0 x 10^-3 g/L in the three buffer solutions using a 0.8% v/v methanol co-solvent at pH 7 and pH 9 and a 0.8% v/v acetonitrile co-solvent at pH 4. In the preliminary test (tier 1) sampling was performed at a minimum: 0 hours, 24, 48, 54, 72, 96 and 120 hours (as appropriate depending on results). Aliquots of the sample solutions were taken from the flasks at various times and the pH of each solution recorded. The concentrations were determined by Gas Chromatography (GC). Duplicate aliquots (100 mL) of each sample were extracted with three portions (3 x 25 mL) of dichloromethane, each extract being filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in methanol (10 mL). For standards: Duplicate standard solutions of test material were prepared in acetone at a nominal concentration of 30 mg/L. Based on the preliminary test results the definitive test (tier 2) test did not need to be performed. Mean procedural recoveries were pH 4: 86.7%, pH 7: 92.8% and pH 9: 90.2% and therefore the analytical system was validated. In the preliminary test (tier 1) at 50 °C at pH 4: greater than 50% decline was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method. The substance was found to be stable to hydrolysis in water at pH 7 (t1/2: > 5 days) and pH 9 (t1/2: > 5 days) at 50 °C and therefore the equivalent half-life at 25°C: would be > 1 year.

Description of key information

Hydrolysis: half-life for hydrolysis: pH 4: could not be determined (test item oxidises to carboxylic acid) ; pH 7: t1/2: > 1 year; pH 9: t1/2: > 1 year, at 25 °C, 1 atm, EU Method C.7, 2001

Additional information

Key study : EU Method C.7 , 2001 : The test followed a method in accordance with 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. The pH 4, 7 and 9 buffer solutions were filtered through a 0.2 µm membrane filter and subject to sonification and degassing. Thereby ensuring sterility of the test system. Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 3.0 x 10^-3 g/L in the three buffer solutions using a 0.8% v/v methanol co-solvent at pH 7 and pH 9 and a 0.8% v/v acetonitrile co-solvent at pH 4. In the preliminary test (tier 1) sampling was performed at a minimum: 0 hours, 24, 48, 54, 72, 96 and 120 hours (as appropriate depending on results). Aliquots of the sample solutions were taken from the flasks at various times and the pH of each solution recorded. The concentrations were determined by Gas Chromatography (GC). Duplicate aliquots (100 mL) of each sample were extracted with three portions (3 x 25 mL) of dichloromethane, each extract being filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in methanol (10 mL). For standards: Duplicate standard solutions of test material were prepared in acetone at a nominal concentration of 30 mg/L. Based on the preliminary test results the definitive test (tier 2) test did not need to be performed. Mean procedural recoveries were pH 4: 86.7%, pH 7: 92.8% and pH 9: 90.2% and therefore the analytical system was validated. In the preliminary test (tier 1) at 50 °C at pH 4: greater than 50% decline was observed after 48, 72, 96 and 120 hours. However, it was considered that the test item was oxidising to carboxylic acid within the test system despite sonication and vacuum degassing of the buffer solutions prior to purging with nitrogen gas. This also based on structural assessment and absence of chemical groups prone to hydrolysis and/or protection of the test system from light and/or the utilisation of closed system and silanised glassware. Oxidation was additionally only observed at pH 4. It was considered that the test item (oxidation) was outside the scope of the test method. The substance was found to be stable to hydrolysis in water at pH 7 (t1/2: > 5 days) and pH 9 (t1/2: > 5 days) at 50 °C and therefore the equivalent half-life at 25°C: would be > 1 year.