1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Dec 2012 - 20 Jun 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2130 (Hydrolysis as a Function of pH and Temperature)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF New Test Guideline Annex No.2-6-1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
Test 1 (Pre-test, 50 °C ):
0, 0.25, 1.04, 2.25, 7, 14, 21 and 30 d at pH 4 and pH 7;
0, 0.04, 0.08, 0.17, 0.25, 1.04 and 2.04 d after treatment (DAT) corresponding to 1, 2, 4, 6, 25 and 49 h at pH 9.

Test 2 (Main test, 25 °C):
0, 1.04, 2.25, 7, 14, 21 and 30 d at pH 4 and pH 7;
0, 0.125, 0.25, 1.04, 2.25, 7, 14, 21 and 30 d at pH 9.

Test 3 (Optional test):
0, 1.04, 2.25, 7, 14, 21 and 30 d at pH 7;
0, 0.25, 1.04, 2.25, 7, 14, 21 and 30 d at pH 9.
- Sampling method: At each sampling interval for each pH, duplicate samples from the respective sample set were removed from the water baths or the walk-in climatic chamber. Before any processing the sterility checks of the test solution were performed. Afterwards, aliquots for LSC and HPLC/radiodetection were taken. Finally, the pH of the test solutions was determined.
- Sampling intervals/times for pH measurements: At each sampling interval in each test.
- Sampling intervals/times for sterility check: At each sampling interval in each test.
- Sampling Storage: The first analysis of the test solutions by LSC and the primary chromatographic method was usually done within two days after sampling. After analysis, the solutions were stored at < -18 °C in the dark.
- Other observation: The preliminary test (test 1, 50 °C) for pH 9 was terminated after 2 days, due to fast degradation of the test item (> 95% AR).

Buffers:

The tests were performed in solutions buffered at pH levels of 4, 7, and 9 (acetate, TRIS, and borate buffers):

- pH: 4
- Type and final molarity of buffer: Acetate, 0.01 M
- Composition of buffer: Stock solution pH 4 (0.04 M): 1.36 g CH3COONa x 3 H2O in a 250 mL volumetric flask which was made up to volume with ultrapure water (electrical conductivity: 0.055 μS/cm, TOC: 1 ppb). The buffer used for the test was prepared by diluting the buffer stock solution 1 + 3
(v+v) with ultrapure water to yield the final buffer concentration of 0.01 mol/L (0.01 M). The pH value of the acetate buffer was determined as 4.0.

- pH: 7
- Type and final molarity of buffer: TRIS, 0.01 M
- Composition of buffer: 1.22 g tris(hydroxymethyl)-aminomethane and 46.6 mL 0.1 M hydrochloric acid were added to a 1000 mL volumetric flask which was made up to volume with ultrapure water (electrical conductivity: 0.055 μS/cm, TOC: 1 ppb). The final buffer concentration was 0.01 mol/L (0.01 M). The pH value of the TRIS buffer was determined as 7.0.

- pH: 9
- Type and final molarity of buffer: Borate, 0.01 M
- Composition of buffer: Stock solution pH 9 (0.02 M): A 0.02 M buffer stock solution was prepared by weighing 0.62 g boric acid (H3BO3) and 0.75 g potassium chloride (KCl) in a 500 mL volumetric flask. After dissolving H3BO3 and KCl in 250 mL ultrapure water (electrical conductivity: 0.055 μS/cm, TOC: 1 ppb), 106 mL 0.04 M aqueous sodium hydroxide solution were added. Finally, the volumetric flask was made up to volume with ultrapure water. The buffer used for the test was prepared by diluting the buffer stock solution 1 + 1 (v+v) with ultrapure water to yield the final buffer concentration. The pH value of the borate buffer was determined as 9.0.

After preparation of the buffers, the oxygen content was depleted by a constant nitrogen flow for approx. 2 h until the oxygen concentration was ≤ 0.97 mg/L. Afterwards, the buffers were sterilized by autoclaving (20 min, 120 °C).

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 10 mL glass crimp-top vials closed by crimp caps with Teflon®-faced septa and further work was done in a laminar flow cabinet. Vials were sealed and placed afterwards in temperature-controlled water baths (25 and 50 °C) or a walk-in climatic chamber (20 °C) to maintain the targeted test temperatures.
- Sterilisation method: In order to establish sterile conditions all glassware and laboratory consumables were sterilized by autoclaving (20 min, 120 °C).
- Lighting: darkness
- Measures taken to avoid photolytic effects: Test was performed in the dark.
- Measures to exclude oxygen: Oxygen-depleted test solution was used; the oxygen content was depleted by a constant nitrogen flow for approx. 2 h until the oxygen concentration was ≤ 0.97 mg/L.
- Details of traps for volatile, if any: No traps for volatiles were used.
- If no traps were used, is the test system closed/open: used glass vials were sealed afterwards with Teflon®-faced septa
- Is there any indication of the test material adsorbing to the walls of the test apparatus? No

TEST MEDIUM
- Volume used/treatment: 5 mL
- Kind and purity of water: ultrapure water (electrical conductivity: 0.055 μS/cm, TOC: 1 ppb)
- Identity and concentration of co-solvent: test item was dissolved in 3.4 mL acetonitrile, resulting in a stock solution with a nominal concentration of 0.50 mg/mL (1.96 MBq/mL). The radioactivity content was determined by liquid scintillation counting (LSC) as 2.03 MBq/mL (equal to 0.52 mg/mL)..

OTHER TEST CONDITIONS
- Dissolved oxygen: after 2 h constant nitrogen flow oxygen concentration was ≤ 0.97 mg/L

Duration:

2 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

7

Temp.:

20 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

4

Temp.:

20 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

4

Temp.:

25 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

9

Temp.:

20 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.3 mg/L

Remarks:

measured concentration: 0.34 mg test item/L

Number of replicates:

Two replicates were removed from the water bath at each interval for each pH.

Positive controls:

no

Negative controls:

no

Statistical methods:

Non-linear regression analysis was used to determine the kinetic parameters (KinGUI 2), and linear regression analysis was used to determine the radioactivity detector response (Microsoft® Excel). Arithmetic means were used for all LS measurements. Values are presented as single values and as means, where applicable. Outlier rejection criteria were not used.

Preliminary study:

DT50 at pH4 and 50 °C = 10.9 d
DT50 at pH7 and 50 °C = 3.74 d
DT50 at pH9 and 50 °C = 0.04 d

Transformation products:

yes

No.:

#1

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

- Formation and decline of each transformation product during test: Hydrolysis of the test item was accompanied by the formation of one degradation product, with the maximum amount of 99.6% AR at DAT-30 (pH 9, optional test, 20 °C). The total unidentified residues amounted to a maximum of 3.3% AR and no single component exceeded 3.3% AR at any sampling interval of all conducted tests. One degradation product was identified with a maximum amount of 99.6% AR.
- Pathways for transformation: Intramolecular cyclisation, resulting in formation of test item degradation product.

% Recovery:

>= 92.5 - <= 98.2

pH:

4

Temp.:

50 °C

Duration:

30 d

% Recovery:

>= 92.5 - <= 100.3

pH:

7

Temp.:

50 °C

Duration:

30 d

% Recovery:

>= 93.2 - <= 101.1

pH:

9

Temp.:

50 °C

Duration:

30 d

% Recovery:

>= 92.7 - <= 95.8

pH:

4

Temp.:

25 °C

Duration:

30 d

% Recovery:

>= 92.2 - <= 96.9

pH:

7

Temp.:

25 °C

Duration:

30 d

% Recovery:

>= 98.7 - <= 103.1

pH:

9

Temp.:

25 °C

Duration:

30 d

% Recovery:

>= 93.7 - <= 101.2

pH:

4

Temp.:

20 °C

Duration:

30 d

% Recovery:

>= 93.4 - <= 96.7

pH:

7

Temp.:

20 °C

Duration:

30 d

% Recovery:

>= 97.1 - <= 103.9

pH:

9

Temp.:

20 °C

Duration:

30 d

pH:

4

Temp.:

25 °C

DT50:

287 d

Type:

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

Remarks on result:

other: Chi2 error: 953%

Remarks:

DT90 = 1.7 d

pH:

7

Temp.:

25 °C

DT50:

38.8 d

Type:

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

Remarks on result:

other: Chi2 error: 2.3%

Remarks:

DT 90 = 129 d

pH:

9

Temp.:

25 °C

DT50:

0.75 d

Type:

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

Remarks on result:

other: Chi2 error: 3.8%

Remarks:

DT 90 = 2.48 d

pH:

4

Temp.:

50 °C

DT50:

10.9 d

Type:

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

Remarks on result:

other: Chi2 error: 2.7%

Remarks:

DT90 = 36.0 d

pH:

7

Temp.:

50 °C

DT50:

3.74 d

Type:

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

Remarks on result:

other: Chi2 error: 3.1 %

Remarks:

DT90 = 12.4 d

pH:

9

Temp.:

50 °C

DT50:

0.04 d

Type:

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

Remarks on result:

other: Chi2 error: 5.5%

Remarks:

DT90 = 0.13 d

pH:

4

Temp.:

20 °C

DT50:

265 d

Type:

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

Remarks on result:

other: Chi2 error: 1.8%

Remarks:

DT90 = 882 d

pH:

7

Temp.:

20 °C

DT50:

58 d

Type:

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

Remarks on result:

other: Chi2: error: 1.7%

Remarks:

DT90 = 193 d

pH:

9

Temp.:

20 °C

DT50:

1.27 d

Type:

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

Remarks on result:

other: Chi2 error: 4.2%

Remarks:

DT90 = 4.21 d

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): No

MAJOR TRANSFORMATION PRODUCTS
At pH 4:
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:

preliminary test (test 1, 50 °C): During 30 d the amount of degradation product increased up to 74.5 % AR; the amount of test item in test solution decreased from 100.0 % AR at DAT-0 to 16.3 % AR at DAT 30
main test (test 2, 25 °C): During 30 d the amount of degradation product increased up to 6.2 % AR; the amount oftest item in test solution decreased from 100.0% AR at DAT-0 to 89.6 % AR at DAT 30
optional test (test 3, 20 °C): During 30 d the amount of degradation product increased up to 3.2 % AR; the amount of test item in test solution decreased from 100.0 % AR at DAT-0 to 91.5 % AR at DAT-30

At pH7:
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
preliminary test (test 1, 50 °C): During 30 d the amount of degradation product increased up to 92.3 % AR; the amount of test item in test solution decreased from 100.0 % AR at DAT-0 to 1.6 % AR at DAT 30
main test (test 2, 25 °C): During 30 d the amount of degradation product increased up to 38.4 % AR; the amount oftest item in test solution decreased from 100.0 % AR at DAT-0 to 56.5 % AR at DAT-30
optional test (test 3, 20 °C): During 30 d the amount of degradation product increased up to 26.7 % AR; the amount of test item in test solution decreased from 100.0 % AR at DAT-0 to 68.1 % AR at DAT-30

At pH9:
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
preliminary test (test 1, 50 °C): During 30 d the amount of degradation product increased up to 96.0 % AR; the amount of test item in test solution decreased from 97.2 % AR at DAT-0 to 2.9 % AR at DAT-2.04
main test (test 2, 25 °C): During 30 d the amount of degradation product increased up to 97.4 % AR; the amount of test item in test solution decreased from 97.2 % AR at DAT-0 to 1.8 % AR at DAT-30
optional test (test 3, 20 °C): During 30 d the amount of degradation product increased up to 99.6 % AR; the amount of test item in test solution decreased from 97.2 % AR at DAT-0 to 1.5 % AR at DAT-30

VOLATILIZATION (at end of study): Since the test systems were sealed and a loss in material balance was not observed, no attempt was made to trap volatiles.

UNIDENTIFIED RADIOACTIVITY (at end of study)
- at pH4, 7 and 9: The total unidentified residues amounted to a maximum of 3.3% AR and no single component exceeded 3.3% AR at any sampling interval of all conducted tests. The complete material balances found at the sampling intervals for all test conditions demonstrated that there was no significant loss of radioactivity from the test systems or during sample processing.

PATHWAYS OF HYDROLYSIS
- Description of pathwayS: yes
- Figures of chemical structures attached: yes

Conclusion:

The hydrolytic degradation of the test item strongly depended on the temperature and the pH conditions. The test item degraded rapidly at pH 9 and well at pH 7 at all temperatures in the laboratory. For pH 4, it was degraded rapidly at 50 °C and slowly at 20 and 25 °C. The calculated half-lives of the test item were between 0.04 (pH 9) and 10.9 (pH 4) days at 50 °C, between 0.75 (pH 7) and 287 (pH 4) days at 25 °C and between 1.27 (pH 9) and 265 (pH 4) days at 20 °C. One degradation product was identified with a maximum amount of 99.6% AR (pH 9) at 20 °C.

 

Validity criteria fulfilled:

yes

Description of key information

DT50 at pH4 = 10.9 - 287 days (OECD 111)

DT50 at pH7 = 3.74 - 58.0 days (OECD 111)

The test item degraded rapidly at pH 9 and well at pH 7 at all temperatures in the laboratory. For pH 4, it was degraded rapidly at 50 °C and slowly at 20 and 25 °C

Additional information

Hydrolysis of the test item as a function of pH was investigated according to OECD guideline 111 under GLP conditions (M-565616-01-1). The hydrolytic degradation was studied in sterile aqueous buffer solutions at pH 4, pH 7 and pH 9 in the dark with a nominal test concentration of 0.3 mg/L for a maximum of 30 days at 50.0, 25.0 and 20.0 °C. Radio labeled test item was used as test substance and the amounts of test item and its degradation products in the solutions were determined by liquid scintillation counting (LSC) and HPLC/radiodetection. Degradation products were identified by HPLC-MS(/MS) including accurate mass determination. One degradation product ≥ 10% was detected. The complete material balances found at all sampling intervals demonstrated that there was no significant loss of radioactivity from the test systems or during sample processing. The results show that the hydrolytic degradation of the test item under sterile conditions strongly depends on the temperature and the pH value. The DT50 values at pH 4 was 10.9 to 287 days and at pH 7 3.74 to 58.0 days. The test item degraded rapidly at pH 9 and well at pH 7 at all temperatures in the laboratory. Therefore, it can be concluded that hydrolysis of the test item contributes to the elimination of this compound from the aqueous environment.