1H-Pyrazole-3-carboxylic acid, 5-amino-1-[(2-fluorophenyl)methyl]-, ethyl ester

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

201-12-03 to 2019-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

adopted April 13, 2004.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 5-amino-1-[(2-fluorophenyl)methyl]-1H-pyrazole-3-carboxylate

EC Number:

805-733-7

Cas Number:

256504-39-9

Molecular formula:

C13H14FN3 O2

IUPAC Name:

ethyl 5-amino-1-[(2-fluorophenyl)methyl]-1H-pyrazole-3-carboxylate

Test material form:

solid

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

Preliminary Test (Tier 1)
Test Duration: The incubation was terminated after 5 days.
Sampling of the Test Samples: pH 4, 7 and 9: 0, 3, 24, 120 h
At each sampling point, samples were taken in duplicate.

Main Test (Tier 2)
Test Duration: The incubation at pH 9 was terminated after 30 d (20°C) and 10 d (40°C and 50°C).
Sampling of the Test Samples:
20°C: 0 h, 6 h, 1 d, 2 d, 4 d, 7 d, 10 d, 16 d, 23 d, 30 d
40°C: 0 h, 3 h, 6 h, 9 h, 1 d, 2 d, 3 d, 4 d, 8 d, 10 d
50°C: 0 h, 3 h, 6 h, 9 h, 1 d, 2 d, 3 d, 4 d, 7 d, 10 d
At each sampling point, samples were taken in duplicate.

Buffers:

Sterile aqueous solutions buffered at pH 4, 7 and 9.
The pH of each buffer solution was measured with a calibrated pH meter.
pH 4: 0.05 M acetate buffer
820 mL acetic acid (0.1 M) was added to 180 mL sodium acetate (0.1 M). The solution was filled up to 2000 mL with pure water.
pH 7: 0.05 M phosphate buffer
592 mL NaOH (0.1 M) was added to 1000 mL potassium dihydrogenphosphate (0.1 M). The solution was filled up to 2000 mL with pure water.
pH 9: 0.05 M boric acid buffer
426 mL NaOH (0.1 M) was added to 1000 mL of a solution of boric acid (0.1 M) in potassium chloride (0.1 M). The solution was filled up to 2000 mL with pure water.

Details on test conditions:

Test Units
Test Vessels: Glass flasks (hermetically closed) were used for the test.
Test Conditions
Temperature:
Preliminary test (Tier 1): 50°C ± 0.2°C
Main test (Tier 2): 50°C ± 0.1°C; 40°C ± 0.6°C; 20°C ± 1.5°C
Light Conditions: The samples were incubated in the dark.
Anoxic Conditions: To avoid oxidation the buffer solutions were purged with inert gas (nitrogen) prior to sterilisation.
Sterile Conditions: Degased buffer solutions and the used glassware were sterilised using an autoclave (20 min at 121°C) prior to application or heated in a oven (180°C, > 3 h) prior to application.
Application
Stock/Application Solutions
Stock Solution of the Test Item:
Two individual stock solutions (A and B) were prepared in acetonitrile with a concentration of 5.0 g/L.
Application Solution of the Test Item: The stock solutions were used for application.
Application of the Test Samples
The final concentration of the test item in the aqueous phase was below 0.01 M or half of its water solubility and the content of organic was < 1% v/v. The sample solutions were prepared with a volume of 50 mL and 200 mL and aliquots were transferred to the test vessels for incubation. Each replicate (A and B) was prepared individually.
Application procedure (per replicate):
Test phase/pH/Volume stock solution/Total sample volume/Test item concentration
-/-/[µL]/[mL]/[mg/L]
Tier 1/4, 7 and 9/500/50/50
Tier 2/9/2000/200/50
Number of Samples:
The following total number of samples was prepared:
Preliminary test (Tier 1): 16 per pH
Main test (Tier 2): 20 per temperature
Preparation of the Blank Samples
Blank Samples: A blank sample for each pH was prepared which consisted of the buffer solution without application of the test item.
Number of Samples: One blank sample of each buffer solution was prepared.
Course of the Study
Aqueous samples were incubated and samples were taken at specific time points.

Duration of test

Duration of test 1

Duration:

30 d

pH:

9

Temp.:

20 °C

Initial conc. measured:

50.7 mg/L

Duration of test 2

Duration:

72 h

pH:

9

Temp.:

40 °C

Initial conc. measured:

50.4 mg/L

Duration of test 3

Duration:

10 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

50.2 mg/L

Number of replicates:

At each sampling point, samples were taken in duplicate.
Test Duration: The incubation at pH 9 was terminated after 30 d (20°C) and 10 d (40°C and 50°C).
Sampling of the Test Samples:
20°C: 0 h, 6 h, 1 d, 2 d, 4 d, 7 d, 10 d, 16 d, 23 d, 30 d
40°C: 0 h, 3 h, 6 h, 9 h, 1 d, 2 d, 3 d, 4 d, 8 d, 10 d
50°C: 0 h, 3 h, 6 h, 9 h, 1 d, 2 d, 3 d, 4 d, 7 d, 10 d

Positive controls:

no

Negative controls:

no

Statistical methods:

Calculations:
Examples of data evaluation are shown in the
Result Evaluation
Degradation Time (DT50 and DT90):
Kinetic analysis and calculation of DT50 and DT90 values were performed using Cake (Version 3.2, Tessella).
Arrhenius Equation:
A plot of ln k versus 1/T gives a straight line with a slope of –E/R. The activation energy (E) was calculated by regression analysis.
ln k=(-E)/(R∙T)+ln A
k = rate constant, measured at different temperatures
E = activation energy [kJ/mol]
T = absolute temperature [K]
R = gas constant [8.314 J/mol*K]

DT50 or DT90 values were calculated by applying appropriate kinetic model calculations (non-GLP).
Presented Numerical Values:
All calculations were performed by electronic devices and software with varying decimal places. Numerical values were generally displayed with a smaller degree of precision (number of digits) and rounding differences may be observed in the results tables.

Results and discussion

Preliminary study:

Results of the Preliminary Test (Tier 1)
Values given are averages obtained from duplicate samples.
pH 4, 7 and 9:
Samples were incubated at three different pH-values and at one temperature. The test was carried out for 5 days. During incubation the concentration of the test item remained stable at pH 4 and 7 (recoveries: 104.1 and 99.2% of the nominal applied concentration, respectively). In case of pH 9 recoveries were < LOQ after 5 days. Thus the test item can be stated as hydrolytically stable at pH 4 and 7 and hydrolytically unstable at pH 9. The main test (Tier 2) was performed at pH 9.

Test performance:

1) Concerning (Study Plan): 2.2.3 Test Conditions
According to Study Plan: The temperatures will be maintained to at least ± 0.5°C.
Deviation to Study Plan: Main test (Tier 2): 40°C ± 0.6°C; 20°C ± 1.5°C
Reason for Deviation: Technical reason during sample placement (40°C) and incubation (20°C).
Presumed Effect on the Study: None. For the samples incubated at 40°C the deviation was of minor extent and only for a short period. For the samples at 20°C the devation was for a longer period of time but of minor extent as well (18 days, temp. range 18.5-18.7°C; 12 days, temp. range: 19.6-19.8°C). Incubation at 20°C (mean: 19.2°C) was considered acceptable.

2) Concerning (Study Plan): 2.4.2 Sampling Schedule
According to Study Plan: Main Test (Tier 2): Each test will be performed until 90% hydrolysis of the test item will be observed or for 30 days whichever will come first.
Deviation to Study Plan: At the end of incubation (10 days) of pH 9 at 40°C, 13.3% of the nominal applied concentration was found which corresponds to a hydrolysis of 86.7%.
Reason for Deviation: The test at pH 9, 40°C was not performed until 90% hydrolysis of the test item was observed or for 30 days.
Presumed Effect on the Study: None. The incubation period was sufficiently long to determine the DT50 value and the degradation pattern unequivocally.

Transformation products:

not measured

Total recovery of test substance (in %)

Total recovery of test substance (in %) 1

% Recovery:

61.7

St. dev.:

1

pH:

9

Temp.:

20 °C

Duration:

30 d

Total recovery of test substance (in %) 2

% Recovery:

13.3

St. dev.:

4

pH:

9

Temp.:

40 °C

Duration:

10 d

Total recovery of test substance (in %) 3

% Recovery:

< 10

St. dev.:

0

pH:

9

Temp.:

50 °C

Duration:

10 d

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.017 d-1

DT50:

40.3 d

Type:

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

Dissipation DT50 of parent compound 2

pH:

9

Temp.:

40 °C

Hydrolysis rate constant:

0.205 d-1

DT50:

3.4 d

Type:

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

Dissipation DT50 of parent compound 3

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.65 d-1

DT50:

1.1 d

Type:

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

Details on results:

Results of the Main Test (Tier 2)
Values given are averages obtained from duplicate samples.
pH 9:
Samples were incubated at 20, 40 and 50°C. The test was carried out for 30 days (20°C) and 10 days (40/50°C). During incubation the concentration of the test item decreased. At the end of incubation recoveries were 61.7 and 13.3% of the nominal applied concentration and < LOQ (10% of the nominal applied concentration) at 20, 40 and 50°C, respectively.
Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature:
20°C: k = 0.0172 d-1 ;DT50 = 40.3 d; DT90 = 134.0 d
40°C: k = 0.2049 d-1 ;DT50 = 3.4 d; DT90 = 11.2 d
50°C: k = 0.6495 d-1 ;DT50 = 1.1 d; DT90 = 3.6 d
The hydrolysis rate constant and the corresponding DT50 value was calculated for a temperature of 25°C based on the arrhenius parameters:
25°C: k = 0.033 d-1 ;DT50 = 21.0 d
Summary and Conclusion
The test item was found to be hydrolytically stable at pH 4 and 7 (DT50 > 1 year at 25°C) and hydrolytically unstable at pH 9.
The main test (Tier 2) was performed at pH 9. At the end of incubation, recoveries (mean) were 61.7 and 13.3% of the nominal applied concentration and < LOQ (10% of the nominal applied concentration) at 20, 40 and 50°C, respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

, Accuracy of the Applied Concentration: 0 h recoveries of the samples: Preliminary test (Tier 1): 96.9-100.8% of the nominal applied concentration. Main test (Tier 2): 100.4-101.4% of the nominal applied concentration.

Conclusions:

The test item was found to be hydrolytically stable at pH 4 and 7 (DT50 > 1 year at 25°C) and hydrolytically unstable at pH 9.
The main test (Tier 2) was performed at pH 9. At the end of incubation, recoveries (mean) were 61.7 and 13.3% of the nominal applied concentration and < LOQ (10% of the nominal applied concentration) at 20, 40 and 50°C, respectively.

Executive summary:

Title: Aminopyrazol: Hydrolysis as a Function of pH [OECD 111]

Test Item: Aminopyrazol

Guidelines/Recommendations:

This study was based on the procedures indicated by the following internationally accepted guidelines and recommendations:

OECD Guideline for Testing of Chemicals No. 111: "Hydrolysis as a function of pH", adopted April 13, 2004.

GLP: Yes (certified laboratory)

Purpose: The purpose of the study was to determine the rate of hydrolysis of Aminopyrazol at different environmentally relevant pH-values and at different temperatures.

Test Setup: Test Vessels:

Glass flasks were used for the test.

Aqueous Solution:

Sterile aqueous solutions buffered at pH 4, 7 and 9.

Test Conditions:

In the dark at, Preliminary test (Tier 1): 50°C ± 0.2°C

Main test (Tier 2): 50°C ± 0.1°C; 40°C ± 0.6°C; 20°C ± 1.5°C

Treatment Rate: 50 mg/L (two individual replicates)

Results:

The test item was found to be hydrolytically stable at pH 4 and 7 (DT50 > 1 year at 25°C) and hydrolytically unstable at pH 9.

The main test (Tier 2) was performed at pH 9. At the end of incubation, recoveries (mean) were 61.7 and 13.3% of the nominal applied concentration and < LOQ (10% of the nominal applied concentration) at 20, 40 and 50°C, respectively.

Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature:

20°C: k = 0.0172 d-1 ;DT50 = 40.3 d; DT90 = 134.0 d

40°C: k = 0.2049 d-1 ;DT50 = 3.4 d; DT90 = 11.2 d

50°C: k = 0.6495 d-1 ;DT50 = 1.1 d; DT90 = 3.6 d

The hydrolysis rate constant and the corresponding DT50 value was calculated for a temperature of 25°C based on the arrhenius parameters:

25°C: k = 0.033 d-1 ;DT50 = 21.0 d

This study is classified acceptable and satisfies the guideline requirements for hydrolysis studies.