1H-Pyrazolo[3,4-b]pyridine-3-carboxamide, 5-fluoro-1-[(2-fluorophenyl)methyl]-

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-03 to 2019-01-29

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

EC Number:

811-886-0

Cas Number:

1361232-73-6

Molecular formula:

C14 H10 F2 N4 O

IUPAC Name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

Test material form:

solid

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

Application
Stock/Application Solutions
Stock Solution of the Test Item: Two individual stock solutions (A and B) were prepared in methanol with a concentration of 996.6 mg/L and 1000.2 mg/L.
Application Solution of the Test Item: The stock solutions were used.
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.
Application Procedure: Preliminary test (Tier 1):
Test sample solutions were prepared in duplicate (A and B). Hereto 0.2 mL of the stock solutions (A and B) were pipetted to 50 mL of each buffer solution resulting in a nominal concentration of 4.0 mg/L.
Number of Samples: Preliminary test (Tier 1): 8 samples per pH level
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: Preliminary test (Tier 1): 1 sample per pH level

Course of the Study
Aqueous samples were incubated and samples were taken at specific time points.
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.

HPLC: Samples were diluted by a factor of 2 with acetonitrile to stabilise the samples during analysis.

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.
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) or heated in an oven (glassware) prior to application.

Details on test conditions:

Stock/Application Solutions
Stock Solution of the Test Item: Two individual stock solutions (A and B) were prepared in methanol with a concentration of 996.6 mg/L and 1000.2 mg/L.
Application Solution of the Test Item: The stock solutions were used.

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.
Application Procedure: Preliminary test (Tier 1):
Test sample solutions were prepared in duplicate (A and B). Hereto 0.2 mL of the stock solutions (A and B) were pipetted to 50 mL of each buffer solution resulting in a nominal concentration of 4.0 mg/L.
Number of Samples: Preliminary test (Tier 1): 8 samples per pH level

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: Preliminary test (Tier 1): 1 sample per pH level

Course of the Study
Aqueous samples were incubated and samples were taken at specific time points.
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.

Test Parameters
Temperature: The temperature was recorded continuously.
pH-Value: The pH of the sample solution was determined at each sampling point.
Sterility of the Test Solution: A sterility confirmation test was carried out during the preliminary test. A commercially available dip slide kit (TSA with TTC and Rose Bengal Chloramphenicol Agar, VWR Chemicals) was used for a total count of microorganisms and to determine the total count of yeast and moulds.

Duration of test

Duration of test 1

Duration:

120 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

3.756 mg/L

Remarks:

nominal 4.0 mg/L, % of nominal at 0h: 93.9%

Duration of test 2

Duration:

24 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

3.7 mg/L

Remarks:

nominal 4.0 mg/L, % of nominal at 0h: 92.5%

Duration of test 3

Duration:

1 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

3.732 mg/L

Remarks:

nominal 4.0 mg/L, % of nominal at 0h: 93.3%

Number of replicates:

Two samples of solutions of each pH value were taken at each sampling point.

Positive controls:

no

Negative controls:

no

Results and discussion

Preliminary study:

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 each tested pH-value of 4, 7 and 9. Recoveries (mean) were in the range of 98.8%-103.3% of the nominal applied concentration after 120 hours. Thus the test item can be stated as hydrolytically stable. The main test (Tier 2) was not performed.

Test performance:

The present study (Preliminary Test (Tier 1)) investigated the hydrolytic behaviour of Fluorpyridoamid in aqueous solutions buffered at pH 4, 7 and 9 and at one elevated temperature. Samples were incubated in the dark. The test was carried out for 5 days. At the end of incubation, 98.8%-103.3% of the nominal applied concentration were found and thus the test item can be stated as hydrolytically stable (t0.5 > 1 year at 25°C).

Transformation products:

no

Total recovery of test substance (in %)

Total recovery of test substance (in %) 1

% Recovery:

103.3

St. dev.:

0.4

pH:

4

Temp.:

50 °C

Duration:

120 h

Total recovery of test substance (in %) 2

% Recovery:

100.8

St. dev.:

1.4

pH:

7

Temp.:

50 °C

Duration:

120 h

Total recovery of test substance (in %) 3

% Recovery:

98.8

St. dev.:

3.8

pH:

9

Temp.:

50 °C

Duration:

120 h

Dissipation DT50 of parent compound

Key result

Temp.:

25 °C

Type:

not specified

Remarks on result:

other: At the end of incubation, 98.8%-103.3% of the nominal applied concentration were found and thus the test item can be stated as hydrolytically stable (t0.5 > 1 year at 25°C).

Details on results:

The present study investigated the hydrolytic behaviour of Fluorpyridoamid in aqueous solutions buffered at pH 4, 7 and 9 and at one elevated temperature. Samples were incubated in the dark. The test was carried out for 5 days. At the end of incubation, 98.8%-103.3% of the nominal applied concentration were found and thus the test item can be stated as hydrolytically stable (t0.5 > 1 year at 25°C).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Accuracy of the Applied Concentration: Immediately after application it should be in the range of 70-110% (preferably 90-110%) of the nominal. Immediately after application (0 h) in Preliminary test (Tier 1) 92.5%-93.9% of the nominal was found.

Conclusions:

The test item can be stated as hydrolytically stable (t0.5 > 1 year at 25°C).

Executive summary:

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

Test Item: Fluorpyridoamid

Guidelines/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 Fluorpyridoamid 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 ± 1.0 °C

Treatment Rate: Preliminary test (Tier 1): 4.0 mg/L (two individual replicates)

Results:

The present study investigated the hydrolytic behaviour of Fluorpyridoamid in aqueous solutions buffered at pH 4, 7 and 9 and at one elevated temperature. Samples were incubated in the dark. The test was carried out for 5 days. At the end of incubation, 98.8%-103.3% of the nominal applied concentration were found and thus the test item can be stated as hydrolytically stable (t0.5 > 1 year at 25°C).

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