4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2016 to 21 Nov 2019

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)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

Methylphenyl-U-14C and Oxoisoxazolidinyl-4,5-14C

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: See Table 1 in 'Any other information on materials and methods incl. tables'.
- Sampling method: Complete treated samples were removed at each sampling time.
- Collection of CO2 and volatile organics: NA
- Sampling intervals/times for pH measurements: pH was measured at all sampling intervals.
- Sampling intervals/times for sterility check: Sterility was confirmed at the Day 0 and study termination using agar plates
- Sample storage conditions before analysis: All samples were quantified on the day of sampling and analyzed within three days of sampling

Buffers:

- pH: 4,7 and 9 (see Table 2 in "Any other information on materials and methods incl.tables" )

Details on test conditions:

TEST SYSTEM
- Test vessel: Clear glass tubes (with glass stoppers) of 20 mL capacity
- Sterilisation method: Autoclave (solutions and equipment)
- Lighting: Dark
- Measures to exclude oxygen: Oxygen depleted by bubbling with nitrogen
- Traps for CO2 and organic volatiles: Sealed vessel, no collection of volatiles
- Is there any indication of the test material adsorbing to the walls of the test apparatus? Yes
- Preparation of test systems: To minimize the process of microbial degradation during incubation, the buffer solutions and glassware were autoclaved for approximately 30 min at 121°C, after which time the pH of the solutions was measured. Autoclaved material was stored on a sterile bench until use. All treatments were performed in a sterile bench under laminar flow conditions.

TEST MEDIUM
- Volume used per vessel: 20mL
- Identity and concentration of co-solvent: Acetonitrile, final concentration of 0.5 - 0.6% v/v in each test solution

OTHER CONDITIONS:
- Temperature: Test item samples were incubated in a thermo-regulated water bath at the desired temperature in the dark. The temperature was monitored throughout the incubation period using an electronic thermo logger.
- pH: The pH of a control was measured at each interval.

PREPARATION OF THE TEST SOLUTION: [Methylphenyl-U-14C]-test substance (Label position 1)
- Application Solution (pH 4 and pH 7): An aliquot of 0.05 mL of the stock solution (15.057 MBq 14C-test substance in 2 mL acetonitrile as provided by the sponsor) was diluted with acetonitrile to 20 mL. The radioactivity content of the solution was measured after preparing a dilution containing 100 µL in 10 mL of acetonitrile and measuring triplicate aliquots of 1000 µL by LSC. The total amount of radioactivity in the application solution was 582’500 dpm/mL or 2.06 mg/L.
- Application Solution (pH 9): Stock solution was prepared by dissolving all of the test item (4.26 mg = 20.1 MBq 14C-test substance) in 10 mL of acetonitrile. The radioactivity content of the solution was measured after preparing a dilution containing 100 µL in 100 mL of acetonitrile and measuring triplicate aliquots of 1000 µL by LSC. The total amount of radioactivity in the stock solution was 132’498’000 dpm/mL or 468 mg/L. An aliquot of 0.103 mLof the stock solution was diluted with acetonitrile to 20 mL. Subsequently, the radioactivity content was measured after preparing a dilution containing 0.1 mL of the stock solution in 20 mL of acetonitrile and measuring 3 aliquots of 1000 µL by liquid scintillation counting (LSC). The radioactivity content of the application solution was determined to be 2.66 mg test item/L, based on the measured radioactivity (753’400 dpm per 1 mL stock) and the specific activity of 4.72 MBq/mg.

PREPARATION OF THE TEST SOLUTION: [Oxoisoxazolidinyl-4,5-14C]-test substance (Label position 2)
A stock solution was prepared in a separate study by adding the test item (delivered in a glass bulb as a solution in 2 mL acetonitrile) to a 10 mL volumetric flask. Similar to the previous stock solution, the flask was filled to the mark by repeated rinsing the glass bulb with small volumes of acetonitrile. The stock solution was then transferred to a 20 mL glass vial and sealed with a septum cap. This stock solution was also used for the current study. The radioactivity in the stock solution was determined by combining 5 µL of the stock solution with 100 mL acetonitrile, followed by triplicate 1000 µL LSC measurements. Based on the measured mean amount of radioactivity, the stock solution contained 1’214’500’000 dpm (20.244 MBq).
An application solution was then prepared by adding 62 µL of the stock solution to a 20 mL volumetric flask and filled to the mark with acetonitrile. The radioactivity of the application solution was determined by preparing two dilutions containing 0.1 mL of the application solution in 20 mL of acetonitrile and measuring 3 aliquots of 1000 µL by LSC. The radioactivity content was determined to be 2.36 mg test item/L, based on the measured radioactivity (740’600 dpm per 1 mL application solution) and the specific activity of 5.24 MBq/mg.

APPLICATION OF 14C-TEST SUBSTANCE
- A stream of nitrogen passed through a sterile filter was directly introduced into the test solution for 30 minutes. The test solution was transferred to the test vessels using a sterile pipette. For pH 7 and pH 9 tests, the head space of the samples was not flushed by nitrogen, as new information from separately conducted anaerobic studies, indicated that incubation under nitrogen could have an effect on the degradation of the test item. All manipulations were performed on a sterile bench. The content of organic solvent in the aqueous phase was calculated to be up to 0.6% (v/v).

- MP-label: A volume of 0.12 mL of the application solution was transferred using a syringe directly to the test vessels containing 20 mL of sterile test solution. The head space above the applied test solution was filled with nitrogen. An aliquot of 120 µL application solution was separately diluted in 20 mL of acetonitrile and aliquots of this dilution were measured by LSC before and after application.

- OXO-label: A volume of 0.10 mL of the application solution was transferred using a syringe directly to the test vessels containing 20 mL of sterile test solution. The space above the applied test solution was filled with nitrogen. An aliquot of 120 µL application solution was separately diluted in 20 mL of acetonitrile and aliquots of this dilution were measured by LSC before and after application.

PRELIMINARY TEST
The hydrolysis of 14C-test substance (radio-labelled in three positions) at 0.020 mg/L to 0.022 mg/L was initially studied in sterile aqueous buffered solutions (acetonitrile cosolvent 0.1 to 0.6% v/v) at pH 4, pH 7 and pH 9 and incubated for 5 days in the dark at 50 °C. The purpose was to check for > 10% of degradation within 5 days as requested by the official guideline.

Duration:

29 d

pH:

4

Temp.:

25 °C

Initial conc. measured:

0.014 mg/L

Duration:

29 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.014 mg/L

Duration:

30 d

pH:

4

Temp.:

60 °C

Initial conc. measured:

0.013 mg/L

Duration:

30 d

pH:

4

Temp.:

70 °C

Initial conc. measured:

0.013 mg/L

Duration:

29 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.013 mg/L

Duration:

29 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.013 mg/L

Duration:

30 d

pH:

7

Temp.:

60 °C

Initial conc. measured:

0.014 mg/L

Duration:

30 d

pH:

7

Temp.:

70 °C

Initial conc. measured:

0.013 mg/L

Duration:

29 d

pH:

9

Temp.:

10 °C

Initial conc. measured:

0.012 mg/L

Duration:

29 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.012 mg/L

Duration:

29 d

pH:

9

Temp.:

35 °C

Initial conc. measured:

0.012 mg/L

Number of replicates:

2

Positive controls:

no

Negative controls:

yes

Statistical methods:

The rate of degradation of the test substance was estimated using CAKE v.3.2 software. The rate of degradation was estimated by fitting single first-order kinetics (SFO) to the data.

Preliminary study:

Preliminary results showed that the radiolabelled test substance (methylphenyl (MP), halophenyl (HP) and oxoisoxazolidinyl (OXO) labels) was considered hydrolytically stable at 50°C in pH 4 buffer, with the parent molecule representing 89.7 - 97.1% AR after 5 days of incubation. At pH 7 and 50°C, the radiolabelled test substance was slightly less stable (with the parent molecule representing 87.7 – 91.9% AR after 5 days of incubation) and at pH 9 and 50°C, the radiolabelled test substance represented < LOD (Limit Of Detection),

Transformation products:

yes

No.:

#1

No.:

#2

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

- pH 4
No significant (> 10% AR) degradates were observed at 25°C throughout the incubation period. At elevated temperatures, one significant degradate, M1, resulting from cleavage of the OXO ring, was observed in MP-treated samples, which reached maximums of 3.0%, 7.2% and 15.9% AR after 29-30 days incubation at 50, 60 and 70°C, respectively.
In the OXO label, amounts of radioactivity, equivalent to those of M1, remained in the aqueous phase after partitioning. Several attempts by HPLC and LC-MS were made to identify the radioactivity remaining in the water phase of the OXO label treated samples incubated at pH4 and elevated temperatures. However, it was not possible to identify any parent related signal below the corresponding radio peak because the radioactivity showed a retention time very close to the retention time of not retained components. Therefore, the presence of high concentrations of not retained buffer salts could have caused the suppression of the MS signal below the radioactive fraction. In addition, a possible extensive degradation process leading to a molecular structure without sufficiently ionizable substituents and with a molecular weight too low to be detected by MS, could explain, as well, the difficulty in identifying any parent related signal below the radiopeak. However, it could be shown by TLC analysis of fractionated samples that this radioactivity was multi-component and individual constituents were unlikely to reach amounts above 10% AR.

- pH 7
At 25°C, the most prominent degradate observed was M21, which was present in both labels and reached a maximum mean level of 9.2% AR by 25 DAT (OXO label). M1 was a minor degradate, reaching a maximum of 2.2% AR at 19 DAT (MP label only).
More significant degradation was observed at the elevated temperatures. Three degradates were observed at > 10% AR. M21 was present in both MP- and OXO treated samples. At 50°C, levels of M21 increased throughout the incubation period reaching a maximum of up to 57.0% AR by 21 – 29 DAT. At 60 and 70°C, M21 reached maximum amounts of 60.1 % AR (OXO label) and 63.1% AR (MP label) by 9 and 2 DAT, respectively, before declining to ≤ 23.7% and ≤ 2.7% AR, respectively. M6 was also present in both MP- and OXO treated samples. Its levels rose steadily throughout the incubation period, reaching maximum levels of 21.8 % AR (OXO label), 57.7% AR (MP label) and 77.3% AR (OXO label) at 50, 60 and 70°C, respectively, at the end of the incubation period (29 – 30 DAT). M2 was present in MP-treated samples only and reached up to 11.4% AR by the end of the incubation period at 70°C but was not detected at any significant level (≤1.0% AR) at the lower temperatures.
Three other degradates were identified that reached levels of < 10% AR in experiments conducted at 50, 60 and 70°C. M1, M5 and M14 were present at up to 7.6% (21 DAT, MP-label), 4.0% (15 DAT, MP-label) and 5.6% AR (3 DAT, OXO-label). No other degradates were observed that exceeded 10% AR.

- pH 9
Two degradates were observed at >10% AR. M21 was the most significant degradate observed in both labels across all three temperatures. At 10 and 25°C, SYN551203 increased steadily throughout the incubation period, reaching up to 83.5% AR (OXO label) by 31 DAT and up to 91.1% AR (OXO label) by 22 DAT, respectively. At 35°C, M21 was present to up to 94.1% AR (OXO label) by 36 HAT before declining to ca. 77% AR by the end of the incubation period (696 HAT). M14 was also observed at levels of >10% AR in both labels at 35°C, reaching up to 13.3% AR (MP label) by 696 HAT, but was less significant (≤4.9% AR) at 10 and 25°C.
No other degradate reached > 10% AR, including M1 (max 2.9% AR), M2 (max 3.3% AR), M5 (max 4.4% AR) and M6 (max 5.6% AR).

% Recovery:

97.7

St. dev.:

2.9

pH:

4

Temp.:

25 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

97.3

St. dev.:

2

pH:

4

Temp.:

25 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

96.9

St. dev.:

2.4

pH:

4

Temp.:

50 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

94.9

St. dev.:

2.4

pH:

4

Temp.:

50 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

96.6

St. dev.:

2.5

pH:

4

Temp.:

60 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

97.3

St. dev.:

3.3

pH:

4

Temp.:

60 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

96.2

St. dev.:

2.2

pH:

4

Temp.:

70 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

97.2

St. dev.:

3.9

pH:

4

Temp.:

70 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

97.1

St. dev.:

3.5

pH:

7

Temp.:

25 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

100

St. dev.:

2.5

pH:

7

Temp.:

25 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

97.5

St. dev.:

3.8

pH:

7

Temp.:

50 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

98.4

St. dev.:

3.7

pH:

7

Temp.:

50 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

97.4

St. dev.:

3

pH:

7

Temp.:

60 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

97.7

St. dev.:

3.8

pH:

7

Temp.:

60 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

98.9

St. dev.:

2.7

pH:

7

Temp.:

70 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

96.9

St. dev.:

4.2

pH:

7

Temp.:

70 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

97.7

St. dev.:

2.9

pH:

9

Temp.:

10 °C

Duration:

31 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

96.7

St. dev.:

2.2

pH:

9

Temp.:

10 °C

Duration:

31 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

100

St. dev.:

2.6

pH:

9

Temp.:

25 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

100

St. dev.:

2.1

pH:

9

Temp.:

25 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

% Recovery:

99.2

St. dev.:

2.4

pH:

9

Temp.:

35 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Methylphenyl label

% Recovery:

98.2

St. dev.:

2.3

pH:

9

Temp.:

35 °C

Duration:

29 d

Remarks on result:

other:

Remarks:

Oxoisoxazolidinyl label

Key result

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0.001 d-1

DT50:

536 d

Type:

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

Remarks on result:

other:

Remarks:

Calculated using the Arrhenius Equation

pH:

4

Temp.:

20 °C

Hydrolysis rate constant:

0 d-1

DT50:

2 102 d

Type:

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

Remarks on result:

other:

Remarks:

Calculated using using the Arrhenius Equation

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.001 d-1

DT50:

1 290 d

Type:

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

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.001 d-1

DT50:

759 d

Type:

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

pH:

4

Temp.:

60 °C

Hydrolysis rate constant:

0.002 d-1

DT50:

350 d

Type:

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

pH:

4

Temp.:

70 °C

Hydrolysis rate constant:

0.005 d-1

DT50:

140 d

Type:

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

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.003 d-1

DT50:

262 d

Type:

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

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.071 d-1

DT50:

9.81 d

Type:

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

pH:

7

Temp.:

60 °C

Hydrolysis rate constant:

0.221 d-1

DT50:

3.14 d

Type:

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

pH:

7

Temp.:

70 °C

Hydrolysis rate constant:

0.674 d-1

DT50:

1.03 d

Type:

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

pH:

9

Temp.:

10 °C

Hydrolysis rate constant:

0.128 d-1

DT50:

5.41 d

Type:

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

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.367 d-1

DT50:

1.9 d

Remarks on result:

other:

Remarks:

Calculated using the Arrhenius Equation

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.51 d-1

DT50:

1.36 d

Type:

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

pH:

9

Temp.:

35 °C

Hydrolysis rate constant:

1.991 d-1

DT50:

0.348 d

Type:

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

Other kinetic parameters:

See Table 3 in 'Any other information on results incl. tables'.

Details on results:

RADIOCHEMICAL PURITY
Prior to application, the radiochemical purity of 14C-labelled the test substance in the treatment solutions was determined by HPLC employing radio- and UV-detection to be > 96.62% for MP-label and > 97.42% for OXO-label. After staggered application the radiochemical purity was 97.1% for the MP-label and 98.3% for the OXO-label thus demonstrating the stability of the test item during the application process.
The homogeneity of the radioactive solution was determined by LSC measurement from application solutions prior to and after application showing standard deviations ranging from 0.1 ot 2.3%. Based on these results, the test item was considered to be homogeneously dissolved.

TREATMENT RATE
The radiochemical purity determined before each treatment proved the stability of the test item. The initial test item concentrations in the samples, are shown in the table below. The concentration of co-solvent did not exceed 0.6% v/v at all conditions.

pH
The pH was measured at all sampling intervals. No significant variation of pH was observed in the buffer solutions.

STERILITY
No colonies were detected on any of the nutrient agar plates for the preliminary tests and the main tests, whereas colonies were observed on the positive control plates, confirming the validity of the testing method.

MASS BALANCE
See Tabel 5 in 'Ant other information on results incl.tables'

RADIOACTIVE RESIDUES IN AQUEOUS SAMPLES
Several metabolites were detected during the definitive test. Most of them were observed in both labels. The metabolic pattern and amount of the metabolites formed was dependent on pH and temperature. The most predominant metabolites were, for both labels, M1, M2, M3 and for the MP label only, M4 and M5. All metabolites were identified by co-chromatography (HPLC and TLC) with authenticated reference items. Besides these metabolites, several radioactive fractions were observed, all in very minor amounts. One of these was M6.
- pH 4: In all pH 4 samples treated with the MP labelled test item, the majority of the radioactivity could be partitioned into an organic solvent with ≤ 0.8% of radioactivity remaining in the aqueous phase. In samples treated with the OXO labelled test item at pH 4, radioactivity remaining in the aqueous phase after partitioning represented up to 1.4% AR at 25°C, with the values increasing with incubation time. Corresponding results for 50, 60 and 70°C were ≤ 3.0% AR, ≤ 7% AR and ≤ 14.5% AR, respectively. Analysis of the organic phase after partitioning demonstrated that 14C-labelled test substance degraded slowly at pH 4 with (mean) levels declining from 93.4 - 98.4% AR at 0 DAT to 93.3 - 96.3% at 25°C, 92.3 - 94.8% at 50°C, 87.8 - 93.5% at 60°C and 78.1 - 86.6% at 70°C after 29 - 30 days of incubation. No significant (> 10% AR) degradates were observed at 25°C throughout the incubation period. At elevated temperatures, one significant degradate, M4, resulting from cleavage of the OXO ring, was observed in MP-treated samples, which reached maximums of 3.0%, 7.2% and 15.9% AR after 29 - 30 days incubation at 50, 60 and 70°C, respectively. In the OXO label, amounts of radioactivity, equivalent to those of M4, remained in the aqueous phase after partitioning. Several attempts by HPLC and LC-MS were made to identify the radioactivity remaining in the water phase of the OXO label treated samples incubated at pH4 and elevated temperatures. However, it was not possible to identify any parent related signal below the corresponding radio peak because the radioactivity showed a retention time very close to the retention time of not retained components. Therefore, the presence of high concentrations of not retained buffer salts could have caused the suppression of the MS signal below the radioactive fraction. In addition, a possible extensive degradation process leading to a molecular structure without sufficiently ionizable substituents and with a molecular weight too low to be detected by MS, could explain, as well, the difficulty in identifying any parent related signal below the radiopeak. However, it could be shown by TLC analysis of fractionated samples that this radioactivity was multi-component and individual constituents were unlikely to reach amounts above 10% AR.
- pH 7: Degradation of the test substance was quicker at pH 7 than pH 4. Levels of parent declined from 97.7 - 102.0% AR at 0 DAT to 88.0 - 94.1% at 25°C and 12.1 - 13.2% AR at 50°C after 29 days incubation. At 60°C, parent levels declined to 2.6 - 3.1% AR by 21 DAT and at 70°C to 6.7 - 7.5% by 4 DAT. At 25°C, the most prominent degradate observed was M21, which was present in both labels and reached a maximum mean level of 9.2% AR by 25 DAT (OXO label). M4 was a minor degradate, reaching a maximum of 2.2% AR at 19 DAT (MP label only). More significant degradation was observed at the elevated temperatures. Three degradates were observed at > 10% AR. M1 was present in both MP- and OXO treated samples. At 50°C, levels of M1 increased throughout the incubation period reaching a maximum of up to 57.0% AR by 21 - 29 DAT. At 60 and 70°C, M1 reached maximum amounts of 60.1 % AR (OXO label) and 63.1% AR (MP label) by 9 and 2 DAT, respectively, before declining to ≤ 23.7% and ≤ 2.7% AR, respectively. M2 was also present in both MP- and OXO treated samples. Its levels rose steadily throughout the incubation period, reaching maximum levels of 21.8 % AR (OXO label), 57.7% AR (MP label) and 77.3% AR (OXO label) at 50, 60 and 70°C, respectively, at the end of the incubation period (29 - 30 DAT). M5 was present in MP-treated samples only and reached up to 11.4% AR by the end of the incubation period at 70°C but was not detected at any significant level (≤ 1.0% AR) at the lower temperatures. Three other degradates were identified that reached levels of < 10% AR in experiments conducted at 50, 60 and 70°C. M3, M4 and M6 were present at up to 7.6% (21 DAT, MP-label), 4.0% (15 DAT, MP-label) and 5.6% AR (3 DAT, OXO-label). No other degradates were observed that exceeded 10% AR.
- pH 9: Degradation was significantly quicker at pH 9 than at pH 7 and pH 4. Levels of the test substance declined rapidly from 92.6 - 99.7% AR at 0 DAT to 5.2 - 5.4% AR by 31 DAT at 10°C, 5.1 - 6.8% AR by 8 DAT at 25°C and 2.4 - 9.9% AR by 2 DAT at 35°C. Two degradates were observed at > 10% AR. M1 was the most significant degradate observed in both labels across all three temperatures. At 10 and 25°C, M1 increased steadily throughout the incubation period, reaching up to 83.5% AR (OXO label) by 31 DAT and up to 91.1% AR (OXO label) by 22 DAT, respectively. At 35°C, M1 was present to up to 94.1% AR (OXO label) by 36 HAT before declining to ca. 77% AR by the end of the incubation period (696 HAT). M3 was also observed at levels of > 10% AR in both labels at 35°C, reaching up to 13.3% AR (MP label) by 696 HAT, but was less significant (≤ 4.9% AR) at 10 and 25°C. No other degradate reached >10% AR, including M2 (max 5.6% AR), M4 (max 2.9% AR), M5 (max 3.3% AR) and M6 (max 4.4% AR).

STRUCTURAL CONFIRMATION
The structure of the major metabolites were identified by co-chromatography with authenticated reference standards with at least two independent analytical methods. These standards were partially available in very limited amounts and not always throughout the entire study period. Therefore, the standards were used in selected samples only. Furthermore, it was not possible to entirely separate all standards in all analytical methods, mainly due to stability reasons under acidic conditions

DEGRADATION PATHWAY
This study demonstrated that the test substance is hydrolytically stable under acidic (pH 4) at temperatures up to 50°C. At temperatures of 60°C and 70°C the test substance degraded slowly leading to the formation of M1, M4 (radio-detectable only with the MP label due to the label position) and polar entities from the OXO ring. These were the only products observed upon hydrolysis of MP labelled the test substance at pH4. Furthermore, this study demonstrated that the test substance is hydrolytically unstable at pH7 and pH9, and degraded via ring opening of the oxoisoxazolidinyl moiety (OXO ring) resulting in the formation of M1. Further degradation leads to M2, M3, M4, M5 and M6.

HYDROLYTIC DT50 OF THE TEST SUBSTANCE
The rate of degradation in buffer solutions for the test substance was calculated by single firstorder (SFO) kinetics using software tool CAKE (version 3.2). Quantification data of the test substance from each replicate from both labels at each timepoint/temperature were used for the calculations. The SFO kinetic model describes the degradation of the test substance under all tested pH conditions and temperatures with χ-square values < 10, except for pH 9 and 35 °C, where it represents 18.2. The linear regression coefficient r2 is > 0.9 for pH 7 and 9 conditions except for pH 7 and 25°C. At pH 4 conditions, r2 ranged from 0.03 to 0.7. The overall goodness of the fit as concluded from a visual assessment of the plots was deemed sufficient in all cases. Additionally, the half-life for the pH 4, 7 and 9 buffer solutions at various temperatures was estimated by plotting the logarithm of the mean concentration vs. the number of days after incubation (linear regression, Excel). The slopes of the straight line plots were then used to calculate k and DT50 values. The results were consistent with the SFO results. DT50 values for the test substance in pH 4, pH 7 and pH 9 buffer solutions at 20 °C were calculated to be 2952, 536 and 1.9 days, respectively.

Table 3. Summary of DT50 Values

Buffer/ Temperature	SFO
	DT50 [days]	DT90 [days]	k	χ2	r2	Prob > t
pH 4
25 °C	1.29E+03	4.29E+03	5.36E-004	0.745	0.05926	0.1263
50 °C	759	2.52E+03	9.13E-004	1.00	0.08486	0.08333
60 °C	350	1.16E+03	0.001979	0.558	0.3271	0.001826
70 °C	140	464	0.004961	1.12	0.7041	1.96E-007
pH 7
25 °C	262	870	0.002648	0.731	0.3342	0.00152
50 °C	9.81	32.6	0.07065	2.03	0.9933	5.31E-024
60 °C	3.14	10.4	0.221	3.84	0.9895	4.83E-021
70 °C	1.03	3.41	0.6744	10	0.9873	4.70E-023
pH 9
10 °C	5.41	18	0.1282	9.51	0.9405	6.87E-015
25 °C	1.36	4.52	0.5097	8.26	0.9868	4.69E-029
35 °C	0.348	1.16	1.991	18.2	0.9605	5.47E-019

 

Table 4. Result of temperature and pH monitor

pH value	Test	Temperature MP-label	Temperature OXO-label
pH 4	25 °C	24.8 ± 0.1	24.7 ± 0.0
	50 °C	50.1 ± 0.1	49.7 ± 0.3
	60 °C	59.9 ± 0.1	59.2 ± 0.4
	70 °C	69.8 ± 0.3	69.5 ± 0.2
pH 7	25 °C	25.3 ± 0.1	25.3 ± 0.1
	50 °C	50.4 ± 0.1	50.4 ± 0.1
	60 °C	60.2 ± 0.3	60.2 ± 0.3
	70 °C	70.2 ± 0.3	70.2 ± 0.1
pH 9	10 °C	9.7 ± 0.0	9.7 ± 0.0
	25 °C	25.0 ± 0.0	25.0 ± 0.0
	35 °C	35.1 ± 0.1	35.1 ± 0.1
Verification of pH		pH 4 at 25°C to 70°C: range from 4.07 to 4.25. pH 7 at 25°C to 70°C: range from 7.11 to 7.17. pH 9 at 10°C to 35°C: range from 9.22 to 9.43.

Table 5. Summary of the respective mass balances

Total radioactivity	Sum of activity in the treated aqueous solutions.
Recovery at 0 DAT	pH 4: Range 92.8 to 100.6% of applied radioactivity - Average 97.0% pH 7: Range 97.3 to 105.9% of applied radioactivity - Average 99.9% pH 9: Range 94.4 to 102.3% of applied radioactivity - Average 97.6%
Overall recovery (all samples)	pH 4: Range 91.9 to 104.9% of applied radioactivity - Average 96.7% pH 7: Range 90.7 to 107.9% of applied radioactivity - Average 98.0% pH 9: Range 93.5 to 104.7% of applied radioactivity - Average 98.8%

Validity criteria fulfilled:

yes

Remarks:

See Validity criteria in 'Any other information on materials and methods incl. tables'

Conclusions:

The test substance was applied to sterile aqueous buffers at pH 4, 7 and 9 and incubated in the dark for up to 31 days.
At pH 4, the test substance was relatively stable with DT50 values of 1290, 759, 350 and 140 days at 25, 50, 60 and 70 °C, respectively. M1 was the only significant (> 10% AR) metabolite found in methylphenyl-treated samples, reaching maximum levels of < LOD, 3.0%, 7.2% and 15.9% AR at 25, 50, 60 and 70 °C, respectively. In the OXO label, amounts of radioactivity, equivalent to those of M1 in the MP label samples, remained in the aqueous phase after partitioning. This radioactivity was shown to be multicomponent by TLC with each individual constituent unlikely to amount to greater than 10% AR.
Degradation of the test substance was faster at pH 7 than pH 4. DT50 values of 262, 9.81, 3.14 and 1.03 days were obtained at 25, 50, 60 and 70°C, respectively. Three degradates were observed at > 10% AR, namely M1, M2 and M5 which reached up to 63.1%, 77.3% and 11.47% AR, respectively, depending on the incubation temperature.
Degradation at pH 9 was rapid resulting in DT50 values of 5.41, 1.36 and 0.348 days at 10, 25 and 35 °C, respectively. Two degradates were observed at > 10% AR, namely M1 and M3 which reached maximum levels of up to 94.1% AR and 13.3% AR, respectively, depending on the incubation temperature.
The lnk and 1/T values for each temperature were used to generate an Arrhenius plot which allowed for the estimation of the k and DT50 values at 25°C. For pH 4, 7 and 9, DT50 values at 20 °C were 2102 days, 536 days and 1.9 days, respectively.

Executive summary:

The hydrolysis of the substance was studied according to the OECD TG 111 and in compliance with GLP criteria. The 14C-labelled test substance (radio-labelled in three positions) at 0.020 mg/L to 0.022 mg/L was initially studied in sterile aqueous buffered solutions (acetonitrile cosolvent 0.1 to 0.6% v/v) at pH 4, pH 7 and pH 9 and incubated for 5 days in the dark at 50 °C.

Following these preliminary tests, hydrolysis of 14C-labelled test substance was further investigated at concentrations of 0.012 mg/L to 0.014 mg/L (acetonitrile cosolvent 0.5 to 0.6% v/v) at pH 4 at 25, 50, 60 and 70 °C, at pH 7 at 25, 50, 60 and 70 °C and at pH 9 at 10, 25 and 35 °C for up to 31 days. In each test, duplicate samples were taken for analysis at up to a maximum of 11 intervals during incubation. The aqueous solutions were either (a) partitioned with organic solvent followed by a concentration step (pH 4) or (b) after adding 5 mL of acetonitrile to the samples, concentrated under reduced pressure (pH 7 and pH 9 samples). The samples were analyzed by liquid scintillation counting (LSC) and high performance liquid chromatography (HPLC) with radio-detection. Selected samples were analysed by TLC.

Preliminary results showed that 14C-labelled test substance (methylphenyl (MP), halophenyl (HP) and oxoisoxazolidinyl (OXO) labels) was considered hydrolytically stable at 50 °C in pH 4 buffer, with the parent molecule representing 89.7 - 97.1% AR after 5 days of incubation. At pH 7 and 50 °C, 14C-labelled test substance was slightly less stable (with the parent molecule representing 87.7 – 91.9% AR after 5 days of incubation) and at pH 9 and 50 °C, 14C-labelled test substance represented < LOD (Limit Of Detection), < LOD and 5.4% AR after 5 days of incubation for MP, HP and OXO label, respectively. The total radioactivity balance for all labels ranged from 68.7 to 106.2% for the preliminary test. The low recovery of 68.7% was observed with the MP label at pH 9. Recoveries > 90% were obtained after method optimization in the definitive test. Following the preliminary test results, it was decided to perform an extended version of the definitive test including all pH values at up to four temperatures. The definitive test was performed with 14C-labelled test substance labelled in the methylphenylring (MP-label) and 14C-labelled test substance labelled in the oxoisoxazolidinyl ring (OXO label) as the preliminary experiments indicated that no additional information of metabolite formation was expected from the halophenyl label.

In the definitive test, the total radioactivity balance for both labels and all temperatures tested ranged from 91.9% to 104.9% of applied radioactivity (AR) at pH 4, from 90.7% to 107.9% at pH 7 and from 93.5% to 104.7% at pH 9 indicating no significant loss due to volatile radioactivity.

In pH 4 condition, the 14C-labelled test substance degraded slowly and elevated temperatures with (mean) levels ranging from 93.4 - 98.4% AR at 0 Days After Treatment (DAT) to 93.3 – 96.3% at 25 °C, 92.3 – 94.8% at 50 °C, 87.8 – 93.5% at 60 °C and 78.1 – 86.6% at 70 °C after 29 – 30 days incubation. No significant (> 10% AR) degradates were observed at 25 °C throughout the incubation period. At elevated temperatures, one significant degradate, M4, resulting from cleavage of the OXO ring, was observed in MP-treated samples, which reached maximums of 3.0%, 7.2% and 15.9% AR after 29 - 30 days incubation at 50, 60 and 70 °C, respectively.

In the OXO label, amounts of radioactivity, equivalent to those of M4, remained in the aqueous phase after partitioning. Several attemps were made to analyse the radioactivity remaining in the water phase of the OXO label treated samples incubated at pH 4 and elevated temperatures. It was not possible to identify the radioactivity, either by HPLC or LC-MS due to interference of the buffer salts in the highly concentrated samples, the radioactivity not being retained by the HPLC column nor the molecules ionizing sufficiently to get distinct MS signals. However, it could be shown by TLC analysis of fractionated samples that this radioactivity was multi-component and none of the individual constituents were likely to be above 10% AR.

In pH 7 condition, degradation of the test substance was quicker than it does in pH 4. Levels of parent declined from 97.7 - 102.0% AR at 0 DAT to 88.0 – 94.1% at 25 °C and 12.1 - 13.2% AR at 50 °C after 29 days incubation. At 60 °C, parent levels declined to 2.6 – 3.1% AR by 21 DAT and at 70 °C to 6.7 - 7.5% by 4 DAT. At 25 °C, the most prominent degradate observed was M1, which was present in both labels and reached a maximum mean level of 9.2% AR by 25 DAT (OXO label). M4 was a minor degradate, reaching a maximum of 2.2% AR at 19 DAT (MP label only). More significant degradation was observed at the elevated temperatures. Three degradates were observed at >10% AR. M1 was present in both MP- and OXO treated samples. At 50°C, levels of M1 increased throughout the incubation period reaching a maximum of up to 57.0% AR by 21 - 29 DAT. At 60 and 70°C, M1 reached maximum amounts of 60.1 % AR (OXO label) and 63.1% AR (MP label) by 9 and 2 DAT, respectively, before declining to ≤ 23.7% and ≤ 2.7% AR, respectively. M2 was also present in both MP- and OXO treated samples. Its levels rose steadily throughout the incubation period, reaching maximum levels of 21.8 % AR (OXO label), 57.7% AR (MP label) and 77.3% AR (OXO label) at 50, 60 and 70 °C, respectively, at the end of the incubation period (29 – 30 DAT). M5 was present in MP-treated samples only and reached up to 11.4% AR by the end of the incubation period at 70°C but was not detected at any significant level (≤ 1.0% AR) at the lower temperatures. Three other degradates were identified that reached levels of < 10% AR in experiments conducted at 50, 60 and 70 °C. M3, M4 and M6 were present at up to 7.6% (21 DAT, MP-label), 4.0% (15 DAT, MP-label) and 5.6% AR (3 DAT, OXO label), respectively. No other degradates were observed that exceeded 10% AR.

In pH 9 condition, degradation was significantly quicker than it does at pH 7 and pH 4. Levels of the test substance declined rapidly from 92.6 – 99.7% AR at 0 DAT to 5.2 – 5.4% AR by 31 DAT at 10 °C, 5.1 – 6.8% AR by 8 DAT at 25 °C and 2.4 – 9.9% AR by 2 DAT at 35 °C. Two degradates were observed at > 10% AR. M1 was the most significant degradate observed in both labels across all three temperatures. At 10 and 25°C, M1 increased steadily throughout the incubation period, reaching up to 83.5% AR (OXO label) by 31 DAT and up to 91.1% AR (OXO label) by 22 DAT, respectively. At 35 °C, M1 was present at up to 94.1% AR (OXO label) by 36 hours before declining to approximately 77% AR by the end of the incubation period (696 hours). M3 was also observed at levels of > 10% AR in both labels at 35 °C, reaching up to 13.3% AR (MP label) by 696 hours, but was less significant (≤ 4.9% AR) at 10 and 25°C. No other degradate reached > 10% AR, including M2 (max 5.6% AR), M4 (max 2.9% AR), M5 (max 3.3% AR) and M6 (max 4.4% AR).

 The degradation rate of the parent was also determined using non-linear regression. The results were consistent with the SFO results. The ln k and 1/T values for each temperature were used to generate an Arrhenius plot which allowed for the estimation of the k and DT50 values at 25 °C. For pH 4, 7 and 9, DT50 values at 20 °C were 2102 days, 536 days and 1.9 days, respectively. The temperatures remained constant throughout the incubation periods (9.7 ± 0.0 °C, 25.0 ± 0.4 °C, 35.1 ± 0.1 °C, 50.1 ± 0.7 °C, 59.9 ± 1.1 °C and 69.9 ± 0.6 °C). There was no significant variation in the pH values of the buffered solutions. The samples also remained sterile thoughout the study.

Description of key information

Hydrolytic DT50 at pH 7 at 20 °C = 536 days (calculated using the Arrhenius plot), OECD TG 111, Adam 2019

Key value for chemical safety assessment

Half-life for hydrolysis:

536 d

at the temperature of:

20 °C

Additional information

The hydrolysis of the substance was studied according to the OECD TG 111 and in compliance with GLP criteria. The 14C-test substance (radio-labelled in three positions) at 0.020 mg/L to 0.022 mg/L was initially studied in sterile aqueous buffered solutions (acetonitrile cosolvent 0.1 to 0.6% v/v) at pH 4, pH 7 and pH 9 and incubated for 5 days in the dark at 50 °C.
Following these preliminary tests, hydrolysis of 14C-test substance was further investigated at concentrations of 0.012 mg/L to 0.014 mg/L (acetonitrile cosolvent 0.5 to 0.6% v/v) at pH 4 at 25, 50, 60 and 70°C, at pH 7 at 25, 50, 60 and 70°C and at pH 9 at 10, 25 and 35° for up to 31 days. In each test, duplicate samples were taken for analysis at up to a maximum of 11 intervals during incubation. The aqueous solutions were either (a) partitioned with organic solvent followed by a concentration step (pH 4) or (b) after adding 5 mL of acetonitrile to the samples, concentrated under reduced pressure (pH 7 and pH 9 samples). The samples were analyzed by liquid scintillation counting (LSC) and high-performance liquid chromatography (HPLC) with radio-detection. Selected samples were analysed by TLC.
Preliminary results showed that 14C-test substance (methylphenyl (MP), halophenyl (HP) and oxoisoxazolidinyl (OXO) labels) was considered hydrolytically stable at 50°C in pH 4 buffer, with the parent molecule representing 89.7 - 97.1% AR after 5 days of incubation. At pH 7 and 50°C, 14C-test substance was slightly less stable (with the parent molecule representing 87.7 – 91.9% AR after 5 days of incubation) and at pH 9 and 50°C, 14C-test substance represented < LOD (Limit Of Detection), < LOD and 5.4% AR after 5 days of incubation for MP, HP and OXO label, respectively. The total radioactivity balance for all labels ranged from 68.7 to 106.2% for the preliminary test. The low recovery of 68.7% was observed with the MP label at pH 9. Recoveries > 90% were obtained after method optimization in the definitive test. Following the preliminary test results, it was decided to perform an extended version of the definitive test including all pH values at up to four temperatures. The definitive test was performed with 14C-test substance labelled in the methylphenylring (MP-label) and 14C-test substance labelled in the oxoisoxazolidinyl ring (OXO label) as the preliminary experiments indicated that no additional information of metabolite formation was expected from the halophenyl label.
In the definitive test, the total radioactivity balance for both labels and all temperatures tested ranged from 91.9% to 104.9% of applied radioactivity (AR) at pH 4, from 90.7% to 107.9% at pH 7 and from 93.5% to 104.7% at pH 9 indicating no significant loss due to volatile radioactivity.
In pH 4 condition, 14C-test substance degraded slowly at elevated temperatures with (mean) levels ranging from 93.4 - 98.4% AR at 0 Days After Treatment (DAT) to 93.3 – 96.3% at 25°C, 92.3 – 94.8% at 50°C, 87.8 – 93.5% at 60°C and 78.1 – 86.6% at 70°C after 29 – 30 days incubation. No significant (> 10% AR) degradates were observed at 25°C throughout the incubation period. At elevated temperatures, one significant degradation product, M1, resulting from cleavage of the OXO ring, was observed in MP-treated samples, which reached maximums of 3.0%, 7.2% and 15.9% AR after 29-30 days incubation at 50, 60 and 70°C, respectively.
In the OXO label, amounts of radioactivity, equivalent to those of M1, remained in the aqueous phase after partitioning. Several attempts were made to analyse the radioactivity remaining in the water phase of the OXO label treated samples incubated at pH 4 and elevated temperatures. It was not possible to identify the radioactivity, either by HPLC or LC-MS due to interference of the buffer salts in the highly concentrated samples, the radioactivity not being retained by the HPLC column nor the molecules ionizing sufficiently to get distinct MS signals. However, it could be shown by TLC analysis of fractionated samples that this radioactivity was multi-component and none of the individual constituents were likely to be above 10% AR.
In pH 7 condition, degradation of the test substance was quicker than it does in pH 4. Levels of parent declined from 97.7 – 102.0% AR at 0 DAT to 88.0 – 94.1% at 25°C and 12.1 - 13.2% AR at 50°C after 29 days incubation. At 60°C, parent levels declined to 2.6 – 3.1% AR by 21 DAT and at 70°C to 6.7 – 7.5% by 4 DAT. At 25°C, the most prominent degradation product observed was M21, which was present in both labels and reached a maximum mean level of 9.2% AR by 25 DAT (OXO label). M1 was a minor degradation product, reaching a maximum of 2.2% AR at 19 DAT (MP label only). More significant degradation was observed at the elevated temperatures. Three degradates were observed at >10% AR. M21 was present in both MP- and OXO treated samples. At 50°C, levels of M21 increased throughout the incubation period reaching a maximum of up to 57.0% AR by 21 – 29 DAT. At 60 and 70°C, M21 reached maximum amounts of 60.1 % AR (OXO label) and 63.1% AR (MP label) by 9 and 2 DAT, respectively, before declining to ≤ 23.7% and ≤ 2.7% AR, respectively. M6 was also present in both MP- and OXO treated samples. Its levels rose steadily throughout the incubation period, reaching maximum levels of 21.8 % AR (OXO label), 57.7% AR (MP label) and 77.3% AR (OXO label) at 50, 60 and 70°C, respectively, at the end of the incubation period (29 – 30 DAT). M2 was present in MP-treated samples only and reached up to 11.4% AR by the end of the incubation period at 70°C but was not detected at any significant level (≤ 1.0% AR) at the lower temperatures. Three other degradates were identified that reached levels of < 10% AR in experiments conducted at 50, 60 and 70°C. M1, M5 and M14 were present at up to 7.6% (21 DAT, MP-label), 4.0% (15 DAT, MP-label) and 5.6% AR (3 DAT, OXO label), respectively. No other degradates were observed that exceeded 10% AR.
In pH 9 condition, degradation was significantly quicker than it was at pH 7 and pH 4. Levels of the test substance declined rapidly from 92.6 – 99.7% AR at 0 DAT to 5.2 – 5.4% AR by 31 DAT at 10°C, 5.1 – 6.8% AR by 8 DAT at 25°C and 2.4 – 9.9% AR by 2 DAT at 35°C. Two degradates were observed at >10% AR. M21 was the most significant degradation product observed in both labels across all three temperatures. At 10 and 25°C, M21 increased steadily throughout the incubation period, reaching up to 83.5% AR (OXO label) by 31 DAT and up to 91.1% AR (OXO label) by 22 DAT, respectively. At 35°C, M21 was present at up to 94.1% AR (OXO label) by 36 hours before declining to approximately 77% AR by the end of the incubation period (696 hours). M14 was also observed at levels of >10% AR in both labels at 35°C, reaching up to 13.3% AR (MP label) by 696 hours, but was less significant (≤4.9% AR) at 10 and 25°C. No other degradation product reached >10% AR, including M1 (max 2.9% AR), M2 (max 3.3% AR), M5 (max 4.4% AR) and M6 (max 5.6% AR).
The degradation rate of the parent was also determined using non-linear regression. The results were consistent with the SFO results. The ln k and 1/T values for each temperature were used to generate an Arrhenius plot which allowed for the estimation of the k and DT50 values at 20 °C. For pH 4, 7 and 9, DT50 values at 20 °C were 2102 days, 536 days and 1.9 days, respectively. The temperatures remained constant throughout the incubation periods (9.7 ± 0.0 °C, 25.0 ± 0.4 °C, 35.1 ± 0.1 °C, 50.1 ± 0.7 °C, 59.9 ± 1.1 °C and 69.9 ± 0.6 °C). There was no significant variation in the pH values of the buffered solutions. The samples also remained sterile throughout the study.