Registration Dossier

Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-22 to 2020-02-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: EU method, OECD , GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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

Constituent 1
Chemical structure
Reference substance name:
2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidine-4,5,6-triamine
EC Number:
811-934-0
Cas Number:
1350653-30-3
Molecular formula:
C17 H14 F2 N8
IUPAC Name:
2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidine-4,5,6-triamine
Test material form:
solid
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Details on sampling:
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:For all pH levels: 0, 3, 24, 120 h
At each sampling point, samples were taken in duplicate.
Main Test (Tier 2)
Test Duration: The incubation was terminated after 30 d independently from pH or temperature.
Sampling of the Test Samples: For all pH and temperature levels, samplings were taken after 0, 1, 3, 6, 10, 14, 21 and 30 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. 1000 and 2000 mL solutions were prepared. Example mixture protocols are given in the following for 2000 mL:
pH 4:
0.05 M acetate buffer
180 mL sodium acetate (0.16 M and 0.10 M for Tier 1 and Tier 2 respectively) were added to 820 mL acetic acid (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) were 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) were 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:
Application
Stock/Application Solutions
Stock Solution of the Test Item: Two individual stock solutions (A and B) were prepared in ACN with a concentration of about 1.0 g/L for both solutions.
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 (Tier 1), 100 mL and 200 mL (Tier 2). Aliquots of the corresponding solution were then transferred to the test vessels for incubation. Each replicate (A and B) was prepared individually.
Application Procedure (per replicate):

Test phase Tier 1:
Temperature [°C]: 50
pH: 4, 7 and 9
Volume stock solution [mL]: 0.09
Total sample volume [mL]: 50
Test item concentration [mg/L]: 1.8

Test phase Tier 2:
Temperature [°C]: 20 and 50
pH: 4, 7 and 9
Volume stock solution [mL]: 0.36
Total sample volume [mL]: 200
Test item concentration [mg/L]: 1.8

Test phase Tier 3:
Temperature [°C]: 40
pH: 4, 7 and 9
Volume stock solution [mL]: 0.18
Total sample volume [mL]: 100
Test item concentration [mg/L]: 1.8

Number of Samples:
The following total number of samples was taken during incubation:
Preliminary Test (Tier 1): 8 per pH level
Main Test (Tier 2): 16 per pH level and temperature
Preparation of the Blank Samples
Blank Samples: An example blank sample for each pH level was prepared and consisted of the buffer solution without application of the test item.
Number of Samples: One blank sample of each buffer solution was prepared.

Test Conditions
Temperature:
Preliminary test (Tier 1): 50°C ± 0.3°C
Main test (Tier 2): 20°C ± 0.3°C, 40°C ± 0.1°C, 50°C ± 0.3°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: Degassed buffer solutions were sterilized through 0.2 µm filters prior to application. The used glassware was sterilized using an autoclave (20 min at 121°C) prior to application.
Test Units
Test Vessels: Glass flasks (hermetically closed) were used for the test.
Duration of testopen allclose all
Duration:
30 d
pH:
4
Temp.:
20
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
4
Temp.:
40
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
4
Temp.:
50
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
7
Temp.:
20
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
7
Temp.:
40
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
7
Temp.:
50
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
9
Temp.:
20
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
9
Temp.:
40
Initial conc. measured:
1.8 mg/L
Duration:
30 d
pH:
9
Temp.:
50
Initial conc. measured:
1.8 mg/L
Number of replicates:
Two samples of solutions of each pH value at each test temperature were taken at each sampling point.
Positive controls:
no
Negative controls:
no

Results and discussion

Preliminary study:
Values given are averages obtained from duplicate samples. Hydrolysis parameters were based on the applied concentration at 0 h, which means that all recoveries refer to the mean value at 0 h.
pH 4, 7 and 9:
Samples were incubated at three different pH-values and at one temperature (50°C). The test was carried out for 5 days. At the end of the incubation, the recoveries were 69.9%, 59.3% and 55.0% of the applied concentration, for pH 4, 7 and 9 respectively. Thus the test item was expected hydrolytically unstable at all three pH levels. The main test (Tier 2) was performed at pH 4, 7 and 9.
Transformation products:
not specified
Total recovery of test substance (in %)open allclose all
% Recovery:
84
pH:
4
Temp.:
20 °C
Duration:
30 d
% Recovery:
54
pH:
4
Temp.:
40 °C
Duration:
30 d
% Recovery:
55
pH:
4
Temp.:
50 °C
Duration:
30 d
% Recovery:
73
pH:
7
Temp.:
20 °C
Duration:
30 d
% Recovery:
34
pH:
7
Temp.:
40 °C
Duration:
30 d
% Recovery:
11
pH:
7
Temp.:
50 °C
Duration:
30 d
% Recovery:
58
pH:
9
Temp.:
20 °C
Duration:
30 d
% Recovery:
56
pH:
9
Temp.:
40 °C
Duration:
30 d
% Recovery:
25
pH:
9
Temp.:
50 °C
Duration:
30 d
Dissipation DT50 of parent compoundopen allclose all
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0.003 d-1
DT50:
200 d
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
40 °C
Hydrolysis rate constant:
0.023 d-1
DT50:
29.8 d
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0.019 d-1
DT50:
35.8 d
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0.012 d-1
DT50:
59.4 d
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.064 d-1
DT50:
10.8 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
40 °C
Hydrolysis rate constant:
0.044 d-1
DT50:
15.8 d
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0.011 d-1
DT50:
65.5 d
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
40 °C
Hydrolysis rate constant:
0.022 d-1
DT50:
31.2 d
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.031 d-1
DT50:
22.5 d
Type:
(pseudo-)first order (= half-life)
Details on results:
Test Parameters and Conditions
Temperature:
Preliminary test (Tier 1): 50°C ± 0.3°C
Main test (Tier 2): 20°C ± 0.3°C, 40°C ± 0.1°C, 50°C ± 0.3°C
pH-Value:
4.0 (pH 4), 7.0 (pH 7), 9.0 (pH 9)
Main test (Tier 2):
4.0-4.1 (pH 4), 7.0-7.1 (pH 7), 9.0-9.1 (pH 9)
Sterility of the Test Solution:
Sterility of the samples at pH 4, 7 (20°C and 50°C) and 9 was confirmed since no colonies were observed.
For pH 7 (40°C), potential contamination was observed (104 CFU/mL of bacteria were found → slight growth). However, since the results at 40°C were in accordance to the results at 20°C and 50°C it was concluded that degradation was based solely on hydrolysis. Thus, the observed contamination (source unknown) did not have an impact on the hydrolysis behaviour of the test item and therefore it was considered irrelevant.

Results of the Preliminary Test (Tier 1)
Values given are averages obtained from duplicate samples. Hydrolysis parameters were based on the applied concentration at 0 h, which means that all recoveries refer to the mean value at 0 h.

pH 4, 7 and 9:
Samples were incubated at three different pH-values and at one temperature (50°C). The test was carried out for 5 days. At the end of the incubation, the recoveries were 69.9%, 59.3% and 55.0% of the applied concentration, for pH 4, 7 and 9 respectively. Thus the test item was expected hydrolytically unstable at all three pH levels. The main test (Tier 2) was performed at pH 4, 7 and 9.
Results of the Main Test (Tier 2)
Values given are averages obtained from duplicate samples. Hydrolysis parameters were based on the applied concentration at 0 h, which means that all recoveries refer to the mean value at 0 h.
pH 4:
Samples were incubated at 20°C, 40°C and 50°C. The test was carried out for 30 days for each temperature. During incubation the concentration of the test item decreased. At the end of incubation recoveries were 83.7%, 53.8% and 55.2% of the applied concentration.
Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature:
k [d-1] DT50 [d] DT90 [d]
20°C 0.003 200.0 664
40°C 0.023 29.8 98.8
50°C 0.019 35.8 119.0

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 = 0.006 d-1 ;DT50 = 123.0 d
pH 7:
Samples were incubated at 20°C, 40°C and 50°C. The test was carried out for 30 days for each temperature. During incubation the concentration of the test item decreased. At the end of incubation recoveries were 73.0%, 34.2% and 10.9% of the applied concentration.
Results after 1 d and 3 d at 40°C were not used for further result evaluation as they were not in accordance to the following results and thus considered as outlier. However, a sufficient number of values were used for an accurate determination of the hydrolysis parameters.
“Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature:

k [d-1] DT50 [d] DT90 [d]
20°C 0.012 59.4 197.0
40°C 0.044 15.8 52.3
50°C 0.064 10.8 35.8

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 = 0.016 d-1 ;DT50 = 42.1 d
pH 9:
Samples were incubated at 20°C, 40°C and 50°C. The test was carried out for 30 days for each temperature. During incubation the concentration of the test item decreased. At the end of incubation recoveries were 58.2%, 55.5% and 25.2% of the applied concentration.
Results after 1 d and 3 d at 40°C were not used for further result evaluation as they were not in accordance to the following results and thus considered as outlier. However, a sufficient number of values were used for an accurate determination of the hydrolysis parameters.
Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature:

k [d-1] DT50 [d] DT90 [d]
20°C 0.011 65.5 218.0
40°C 0.022 31.2 104.0
50°C 0.031 22.5 74.7

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 = 0.013 d-1 ;DT50 = 53.9 d

Summary and Conclusion
The present study investigated the hydrolytic behaviour of Fluortrisamin in aqueous solutions buffered at pH 4, 7 and 9 and at three temperatures. Samples were incubated in the dark.
A preliminary test (Tier 1) was carried out for 5 days and one elevated temperature. The test item was expected hydrolytically unstable at all three pH levels, and the main test (Tier 2) was performed accordingly.
At pH 4, the average recoveries after 30 days were 83.7%, 53.8% and 55.2% of applied concentration at 20, 40 and 50°C respectively. At pH 7, the average recoveries after 30 days were 73.0%, 34.2% and 10.9% of applied concentration at 20, 40 and 50°C respectively. At pH 9, the average recoveries after 30 days were 58.2%, 55.5% and 25.2% of applied concentration at 20, 40 and 50°C respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The present study investigated the hydrolytic behaviour of Fluorotrisamine in aqueous solutions buffered at pH 4, 7 and 9 and at three temperatures. Samples were incubated in the dark.
A preliminary test (Tier 1) was carried out for 5 days and one elevated temperature. The test item was expected hydrolytically unstable at all three pH levels, and the main test (Tier 2) was performed accordingly.
At pH 4, the average recoveries after 30 days were 83.7%, 53.8% and 55.2% of applied concentration at 20, 40 and 50°C respectively. At pH 7, the average recoveries after 30 days were 73.0%, 34.2% and 10.9% of applied concentration at 20, 40 and 50°C respectively. At pH 9, the average recoveries after 30 days were 58.2%, 55.5% and 25.2% of applied concentration at 20, 40 and 50°C respectively.
Executive summary:

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

Test Item: Fluortrisamin

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 Fluortrisamin 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 ± 0.3°C
  • Main test (Tier 2): 20°C ± 0.3°C, 40°C ± 0.1°C, 50°C ± 0.3°C

Treatment Rate: Preliminary test (Tier 1):

  • 50°C:1.80 to 1.81 mg/L (two individual replicates)

Main test (Tier 2):

  • 20°C, 50°C: 1.79 and 1.83 mg/L (two individual replicates)
  • 40°C: 1.80 and 1.81 mg/L (two individual replicates)

 

Results:

The present study investigated the hydrolytic behaviour of Fluortrisamin in aqueous solutions buffered at pH 4, 7 and 9 and at three temperatures. Samples were incubated in the dark.

A preliminary test (Tier 1) was carried out for 5 days and one elevated temperature. The test item was expected hydrolytically unstable at all three pH levels, and the main test (Tier 2) was performed accordingly.

At pH 4, the average recoveries after 30 days were 83.7%, 53.8% and 55.2% of applied concentration at 20, 40 and 50°C respectively. At pH 7, the average recoveries after 30 days were 73.0%, 34.2% and 10.9% of applied concentration at 20, 40 and 50°C respectively. At pH 9, the average recoveries after 30 days were 58.2%, 55.5% and 25.2% of applied concentration at 20, 40 and 50°C respectively.

pH 4:

Temperature [°C]           "Reaction rate constant (k)" [d-¹]      DT50 [d]

20                                  0.003                                                200.0

40                                  0.023                                                29.8

50                                  0.019                                                35.8

pH 7:

Temperature [°C]           "Reaction rate constant (k)" [d-¹]      DT50 [d]

20                                  0.012                                                59.4

40                                  0.044                                                15.8

50                                  0.064                                                10.8

pH 9:

Temperature [°C]           "Reaction rate constant (k)" [d-¹]      DT50 [d]

20                                  0.011                                                65.5

40                                  0.022                                                31.2

50                                  0.031                                                22.5

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