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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 to 30 June, 2020
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 1404301
- Expiration date of the lot/batch: 08.04.2024

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25 °C)
- Stability under storage conditions: stable
Analytical monitoring:
yes
Buffers:
• Buffer Solution of pH 4.0 ± 0.1: An aliquot of 410 mL of 0.2 M acetic acid solution was mixed with 90 mL of 0.2 M sodium acetate solution in a 2000 mL volumetric flask. The contents were diluted to volume with Milli-Q water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be in the range, 4.02 to 3.99 on various occasions of preparation.
• Buffer Solution of pH 7.0 ± 0.1: An aliquot of 195 mL of 0.2 M monobasic sodium phosphate solution and 305 mL of 0.2 M dibasic sodium phosphate solution were transferred into a 2000 mL volumetric flask. The contents were diluted to volume with Milli-Q water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be in the range of 7.01 to 6.99 on various occasions of preparation.
• Buffer Solution of pH 9.0 ± 0.1: An aliquot of 1000 mL of 0.1 M boric acid solution was transferred into a 2000 mL volumetric flask and the volume was made up to the mark using Milli-Q water. The pH of the resulting buffer solution was adjusted to 9.0 with 50 % v/v NaOH solution using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be in the range, 9.02 to 9.00 on various occasions of preparation.
Note: All the buffer solutions were sonicated for 5 minutes and sterilized by passing it through a sterilized 0.2 µm filters.
Duration:
5 d
pH:
4
Temp.:
50 °C
Duration:
5 d
pH:
7
Temp.:
50 °C
Duration:
5 d
pH:
9
Temp.:
50 °C
Preliminary study:
The Tier 1 test results revealed that the hydrolysis of analyte after 5 days of incubation at 50 ± 0.5 °C was 55.08, 73.73 and 79.83 % at pH 4.0, 7.0 and pH 9.0, respectively. The test item, FAT 36038/J was observed to be hydrolytically unstable after five days of incubation 50±0.5 °C in all the three buffers.
Transformation products:
no
pH:
4
Temp.:
50 °C
DT50:
< 1 yr
Type:
not specified
Remarks on result:
other: found to be hydrolytically unstable in the pre study, however owing to very low water solubility (<0.8 µg/L), tier 2 not undertaken
pH:
7
Temp.:
50 °C
DT50:
< 1 yr
Type:
not specified
Remarks on result:
other: found to be hydrolytically unstable in the pre study, however owing to very low water solubility (<0.8 µg/L), tier 2 not undertaken
pH:
9
Temp.:
50 °C
DT50:
< 1 yr
Type:
not specified
Remarks on result:
other: found to be hydrolytically unstable in the pre study, however owing to very low water solubility (<0.8 µg/L), tier 2 not undertaken

Method Validation


The analytical method for the determination of a.i.concentration in buffers of pH 4.0, 7.0 and 9.0 was validated by establishing linearity, range, accuracy, precision, specificity, limit of detection and limit of quantification.


 


Detector Linearity


A series of working standard solutions of concentration 0.03 to 2.00 µg/mL were analyzed in triplicate by HPLC. The detector response was found linear in this concentration range with a correlation coefficient (r) of 1.0000.


 


Limit of Detection (LOD) for the Method


The minimum quantity of the standard of 0.02 µg/mL, was detected by the HPLC with signal to noise ratio of 4.06.


 


Limit of Quantification (LOQ) of the Method


The lowest concentration of the reference standard of 0.03 µg/mL analysed using the method being validated. Further, at this concentration the percent relative standard deviation (%RSD) of the peak area of five measurements was 2.92 % which was less than the value of 18.17 % obtained using extended Horwitz equation. The signal to noise ratio of the analyte peak at this concentration was 10.27.


 


Accuracy, Precision and Specificity


Accuracy as mean per cent recovery of analyte was found to be 102.53, 101.36, 103.33 and 95.97, 96.69, 102.48 for low and high doses of pH 4.0, pH 7.0 and pH 9.0, respectively. This was considered acceptable as the obtained mean percent recovery was within 90 to110.


 


The precision as % rsd of the method for analysis of the analyte was found to be 1.82, 1.00 and 1.82 for low dose of pH 4.0, pH 7.0 and pH 9.0, respectively. This was considered acceptable as the obtained %rsd <15.16 based on %rsd <2 (1 - 0.5 log C) x 0.67, where C is the percent concentration of the active ingredient in the test item as a decimal fraction.


 


The precision as % rsd of the method for analysis of the item was found to be1.02, 1.94 and 0.92 for high dose of pH 4.0, pH 7.0 and pH 9.0, respectively. This was considered acceptable as the obtained %rsd <10.72 based on % rsd <2 (1 - 0.5Iog C)   x 0.67, where C is the percent concentration of the active ingredient in the test item as a decimal fraction.


 


Specificity of the method was considered acceptable since, the control sample (unfortified) did not show any interference at the retention time of the analyte.


 


Determination of Solubility of FAT 36038/J in 1 % Co-solvent (Acetonitrile)


The overall mean solubility of the FAT 36038/J in double distilled water containing 1% co-solvent (Acetonitrile) was determined to be 2.653± 0.03 µg/mL.


 


Hydrolysis at 50±0.5 °C (Tier 1)


The test samples in pH 4.0, 7.0, and 9.0 buffer solutions were subjected to hydrolysis at 50±0.5 °C. Duplicate samples were analysed along with respective control samples for analyte concentration on day 0 and 5 days of post treatment.


 


Note: A slight precipitation was observed in all the buffer solutions at end of 5 days incubation period at 50 ±0.5 °C.


 


Hydrolysis at pH 4.0:


The mean analyte concentration in the sterile pH 4.0 buffer samples was 1.18 µg/mL at 0 and 5 days of post treatment, respectively. The hydrolysis of the analyte after 5 days of incubation at pH 4.0 at 50 ± 0. 5 °C was 55.08 % (<10 %).


 


Hydrolysis at pH 7.0:


The mean analyte concentration in the sterile pH 7.0 buffer samples was 1.18 µg/mL at 0 and 5 days of post treatment, respectively. The hydrolysis of the analyte after 5 days of incubation at pH 7.0 at 50 ± 0. 5°C was 73.73 % (<10 %).


 


Hydrolysis at pH 9.0:


The mean analyte concentration in the sterile pH 9.0 buffer samples was 1.19 µg/mL at 0 and 5 days of post treatment, respectively.  


The hydrolysis of the analyte after 5 days of incubation at pH 9.0 at 50 ± 0.5 °C was 79.83 % (<10 %).


The Tier 1 test results revealed that the hydrolysis of analyte after 5 days of incubation at 50 ± 0.5 °C was 55.08, 73.73 and 79.83 % at pH 4.0, 7.0 and pH 9.0, respectively.


 


pH Measurements


The pH of the samples prepared using buffers solutions of pH 4.0, 7.0 and 9.0 were measured as follows:


 






































pH



Day 0



Day 5



R1



R2



R1



R2



4.0



4.02



4.01



4.00



3.99



7.0



7.02



6.99



7.01



7.00



9.0



9.01



9.00



9.02



8.99



 


Temperature Record


The mean maximum and m1mmum temperature of water bath maintained at 50 ± 0.5 °C during the preliminary test was 50.2 °C and 49.8 °C, respectively.


 


Sterility Monitoring


Sterility was checked at start and end of the hydrolysis experiment for each of the test systems. All the samples were found to be sterile, showing no counts of bacteria.

Validity criteria fulfilled:
yes
Conclusions:
FAT 36038/J was observed to be hydrolytically unstable after five days of incubation 50±0.5 °C in all the three buffers.
Executive summary:

The study Hydrolysis as a function of pH of FAT 36038/J was carried out as per OECD 111 and OPPTS 835.2120. Analyte at nominal concentration of about 1 ug/mL in sterile buffer solutions of pH 4.0, 7.0 and 9.0 was incubated for 5 days at 50±0.5°C in the preliminary test. Hydrolysis reactions were monitored by analysing the analyte concentration at set intervals using an in-house developed and validated HPLC method. The Tier 1 test results revealed that the hydrolysis of analyte after 5 days of incubation at 50 ± 0.5°C was 55.08, 73.73 and 79.83 % at pH 4.0, 7.0 and pH 9.0, respectively. The test item, FAT 36038/J was observed to be hydrolytically unstable after five days of incubation 50±0.5°C in all the three buffers. The solubility of the test item is very low (negligibly small) in water. The analytically viable concentration in water for the Tier 1 test could only be achieved with the addition of 1 % (v/v) co-solvent. Further, the test item as per the Tier 1 results is hydrolytically unstable, Hence, the requirement of determination of rate of degradation of the test item under Tier 2 is not required.

Description of key information

FAT 36038/J was observed to be hydrolytically unstable after five days of incubation 50 ± 0.5 °C in all the three buffers.

Key value for chemical safety assessment

Additional information

The study Hydrolysis as a function of pH of FAT 36038/J TE was carried out as per OECD 111 and OPPTS 835.2120. Analyte at nominal concentration of about 1 ug/mL in sterile buffer solutions of pH 4.0, 7.0 and 9.0 was incubated for 5 days at 50±0.5°C in the preliminary test. Hydrolysis reactions were monitored by analysing the analyte concentration at set intervals using an in-house developed and validated HPLC method.

The Tier 1 test results revealed that the hydrolysis of analyte after 5 days of incubation at 50 ± 0.5 °C was 55.08, 73.73 and 79.83% at pH 4.0, 7.0 and pH 9.0, respectively. The test item, FAT 36038/J TE was observed to be hydrolytically unstable after five days of incubation 50±0.5 °C in all the three buffers.The solubility of the test item is very low (negligibly small) in water. The analytically viable concentration in water for the Tier 1 test could only be achieved with the addition of 1% (v/v) co-solvent. Further, the test item as per the Tier 1 results is hydrolytically unstable, Hence, the requirement of determination of rate of degradation of the test item under Tier 2 is not required.

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