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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November 2017 to 08 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Incubated test solutions were run on a HPLC system to determine if any hydrolysis had occurred. A 5 mL volume of sample at pH 4 and 7 was extracted into
dichloromethane. The dichloromethane was transferred to a 7 mL vial and evaporated to dryness. An equal volume of mobile phase was added to the vial for
analysis. At pH 9, the sample could not be extracted and was diluted directly into methanol (0.45 mL sample and 4.5 mL methanol).
- 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.
Buffers:
The Buffers were prepared as follows:
- 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)

- The pH of each solution was checked prior to use. Buffer solutions containing the test material are sterilised by passing sample through a sterilised 0.2 μm cellulose acetate syringe filter prior to incubation at test temperature.
Details on test conditions:
SOLUTION PREPARATION
- Test Solutions were prepared by weighing approximately 0.06 g of test material into a 125 mL Erlenmeyer flask. The buffer (50 mL) was added to each flask and the solution was sonicated for 15 minutes to aid dissolution. Due to low solubility of the test material, a suspension was used rather than an aqueous half saturation solution. All solutions were cloudy after sonication. For pH 4 and 7, the supernatant was decanted and diluted by half. The solution at pH 9 was used undiluted. All pH’s were filtered through a 0.2 μm cellulose acetate filter to sterilise and the headspace blown off with nitrogen to limit the presence of oxygen. The solutions were not purged with nitrogen.
- 50 mL buffer was added for each solution.
- Incubated test solutions were run on a HPLC system to determine if any hydrolysis had occurred. A 5 mL volume of sample at pH 4 and 7 was extracted into dichloromethane. The dichloromethane was transferred to a 7 mL vial and evaporated to dryness. An equal volume of mobile phase was added to the vial for analysis. At pH 9, the sample could not be extracted and was diluted directly into methanol (0.45 mL sample and 4.5 mL methanol).
- 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.
Duration:
5 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
100 other: %
Duration:
5 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
100 other: %
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
100 other: %
Number of replicates:
2
Positive controls:
no
Negative controls:
no
Preliminary study:
The test material concentrations after five days incubation at 50°C were:
- pH 4: 95.6 % (main peak @ 4.0 min.)
- pH 7: 100 % (main peak @ 4.0 min.)
- pH 9: 100 % (main peak @ 3.5 min.)

- There were no extraneous peaks in HPLC chromatogram

There was no other evidence of hydrolysis

- No hydrolysis occurred at pH 4, 7 or 9, therefore no further work was required
Test performance:
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 all 3 pH’s and Tier 2 was not required.
Transformation products:
not specified
Key result
pH:
4
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
7
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
9
Temp.:
50 °C
Remarks on result:
hydrolytically stable based on preliminary test
Details on results:
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 all 3 pH’s and Tier 2 was not required.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test material was 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.

Samples of the test material at pH 4, 7 and 9 were incubated at 50°C for five days. Incubated test solutions were run on a HPLC system to determine if any hydrolysis had occurred. A 5 mL volume of sample at pH 4 and 7 was extracted into dichloromethane. The dichloromethane was transferred to a 7 mL vial and evaporated to dryness. An equal volume of mobile phase was added to the vial for analysis. At pH 9, the sample could not be extracted and was diluted directly into methanol (0.45 mL sample and 4.5 mL methanol).

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.

The test material concentrations after five days incubation at 50°C were: pH 4: 95.6 % (main peak at 4.0 min.), pH 7: 100% (main peak at 4.0 min.) and pH 9: 100% (main peak at 3.5 min.).

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 all 3 pH’s and Tier 2 was not required.

Under the conditions of this study, the test material was hydrolytically stable at pH 4, 7 and 9.

Description of key information

Under the conditions of this study, the test material was hydrolytically stable at pH 4, 7 and 9.

Key value for chemical safety assessment

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).

Samples of the test material at pH 4, 7 and 9 were incubated at 50°C for five days. Incubated test solutions were run on a HPLC system to determine if any hydrolysis had occurred. A 5 mL volume of sample at pH 4 and 7 was extracted into dichloromethane. The dichloromethane was transferred to a 7 mL vial and evaporated to dryness. An equal volume of mobile phase was added to the vial for analysis. At pH 9, the sample could not be extracted and was diluted directly into methanol (0.45 mL sample and 4.5 mL methanol).

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.

The test material concentrations after five days incubation at 50°C were: pH 4: 95.6 % (main peak at 4.0 min.), pH 7: 100% (main peak at 4.0 min.) and pH 9: 100% (main peak at 3.5 min.).

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 all 3 pH’s and Tier 2 was not required.

Under the conditions of this study, the test material was hydrolytically stable at pH 4, 7 and 9.