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Diss Factsheets

Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jul 2019 - 04 Jun 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
[amino({5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl})methylidene]azanium chloride
EC Number:
811-931-4
Cas Number:
1350653-27-8
Molecular formula:
C14 H12 Cl F2 N5
IUPAC Name:
[amino({5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl})methylidene]azanium chloride
Test material form:
solid
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Details on sampling:
Preliminary Test (Tier 1)
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)
Sampling of the Test Samples: At pH 4, samplings were taken after
-20/40/50°C:0, 1, 4, 7, 10, 14, 21 and 30 d
At pH 7, samplings were taken after
-20/40°C: 0, 1, 4, 7, 10, 14, 21 and 30 d
-50°C: 0, 1, 2, 4, 7, 10, 14, 18 and 21 d
At pH 9, samplings were taken after
-20°C: 0, 2 h, 4 h, 24 h, 28 h, 2 d, 4 d, 7 d , 10 d, 14 d, 21 d and 30 d
-40°C: 0, 2 h, 4 h, 6 h, 8 h, 24 h, 2 d, 4 d and 7 d
-50°C: 0, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 2 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 1000 mL:
pH 4: 0.05 M acetate buffer
90 mL sodium acetate (0.1 M) was added to 410 mL acetic acid (0.1 M). The solution was filled up to 1000 mL with pure water. Acetic acid (0.16 M) was used for Tier 1.
pH 7: 0.05 M phosphate buffer
296 mL NaOH (0.1 M) was added to 500 mL potassium dihydrogenphosphate (0.1 M). The solution was filled up to 1000 mL with pure water.
pH 9: 0.05 M boric acid buffer
213 mL NaOH (0.1 M) was added to 500 mL of a solution of boric acid (0.1 M) in potassium chloride (0.1 M). The solution was filled up to 1000 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 ± 0.3°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 by filtration (0.2 µm) prior to application. The used glassware was sterilized using an autoclave (20 min at 121°C) prior to application.
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 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.
Preparation of the Blank Samples
Blank Samples: A blank sample for each pH 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 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 for Tier 1 and Tier 2. A commercially available dip slide kit was used for a total count of bacteria and to determine the total count of yeast and moulds.
Duration of testopen allclose all
Duration:
30 d
pH:
4
Temp.:
20 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
4
Temp.:
40 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
7
Temp.:
20 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
7
Temp.:
40 °C
Initial conc. measured:
10 mg/L
Duration:
21 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
10 mg/L
Duration:
30 d
pH:
9
Temp.:
20 °C
Initial conc. measured:
10 mg/L
Duration:
7 d
pH:
9
Temp.:
40 °C
Initial conc. measured:
10 mg/L
Duration:
2 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
10 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. Samples were incubated at three different pH-values and at one temperature. The test was carried out for 5 days. At the end of incubation, the recoveries were 80.2%, 47.7% and < LOQ (< 10%) 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 measured
Total recovery of test substance (in %)open allclose all
% Recovery:
79.4
pH:
4
Temp.:
20 °C
Duration:
30 d
% Recovery:
90.9
pH:
4
Temp.:
40 °C
Duration:
30 d
% Recovery:
80.1
pH:
4
Temp.:
50 °C
Duration:
30 d
% Recovery:
75
pH:
7
Temp.:
20 °C
Duration:
30 d
% Recovery:
43
pH:
7
Temp.:
40 °C
Duration:
30 d
% Recovery:
8.4
pH:
7
Temp.:
50 °C
Duration:
21 d
% Recovery:
13.3
pH:
9
Temp.:
20 °C
Duration:
30 d
% Recovery:
< 10
pH:
9
Temp.:
40 °C
Duration:
7 d
% Recovery:
< 10
pH:
9
Temp.:
50 °C
Duration:
2 d
Dissipation DT50 of parent compoundopen allclose all
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
0.005 d-1
DT50:
138 d
Type:
not specified
pH:
4
Temp.:
40 °C
Hydrolysis rate constant:
0.002 d-1
DT50:
460 h
Type:
not specified
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0.006 d-1
DT50:
113 h
Type:
not specified
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 d-1
DT50:
> 10 000 h
Type:
not specified
pH:
7
Temp.:
40 °C
Hydrolysis rate constant:
0.027 d-1
DT50:
25.8 d
Type:
not specified
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.187 d-1
DT50:
3.7 d
Type:
not specified
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0.003 d-1
DT50:
216 d
Type:
not specified
pH:
9
Temp.:
40 °C
Hydrolysis rate constant:
0.05 d-1
DT50:
139 d
Type:
not specified
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.166 d-1
DT50:
4.2 d
Type:
not specified
Details on results:
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.
pH4:
Samples were incubated at 20, 40 and 50°C. The test was carried out for 30 days for all pH levels. During incubation the concentration of the test item decreased. At the end of incubation recoveries were:
• 20°C: 79.4% of the applied concentration
• 40°C: 90.9% of the applied concentration
• 50°C: 80.1% of the applied concentration
Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature.
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 = 0.004 d-1 ;DT50 = 184.2 d
pH7:
Samples were incubated at 20, 40 and 50°C. The test was carried out for 21 days at 50°C, and for 30 days at 20 and 40°C. During incubation the concentration of the test item decreased. At the end of incubation recoveries were:
• 20°C: 75.0% of the applied concentration
• 40°C: 43.0% of the applied concentration
• 50°C: 8.4% of the applied concentration
Hydrolysis rate constants and corresponding DT50 and DT90 values for the test item were determined for each temperature. However, for samples at 20°C specific parameters could not be determined, as the recovery values were fluctuating significantly (reason unkonwn). For further calculations, those values were not taken into consideration.
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 = 0.001 d-1 ;DT50 = 604.5 d
pH9:
Samples were incubated at 20, 40 and 50°C. The test was first carried out for 2 days at 50°C, and for 7 days at 20 and 40°C. Based on the results, the incubation at 20°C was repeated and prolonged for 21 days. During incubation the concentration of the test item decreased. At the end of incubation recoveries were:
• 20°C: 13.3% of the applied concentration
• 40°C: < 10% of the applied concentration (< LOQ)
• 50°C: < 10% of the applied concentration (< LOQ)
Based on the visual assessment of the concentration variation over time within the kinetic evaluation, results after 8 days at 50°C were considered as outlier and were not used for further result evaluation. Nevertheless, a sufficient number of values was available 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.
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 = 0.007 d-1 ;DT50 = 105.1 d
The present study investigated the hydrolytic behaviour of Fluoroamidine Hydrochloride 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 first 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 79.4%, 90.9% and 80.1% of the applied concentration at 20, 40 and 50°C, respectively. At pH 7, the average recoveries were 75.0% of the applied concentration after 30 days at 20°C, 43.0% after 30 days at 40°C and 8.4% after 21 days at 50°C. At pH 9, the average recoveries were 24.5% after 21 days at 20°C and < 10% (< LOQ) after 7 days at 40°C and 2 days at 50°C.

Any other information on results incl. tables

Summary of Results:

pH 4 pH 7 pH 9
Temperature Reaction rate
constant (k)		 DT50 Temperature Reaction rate
constant (k)		 DT50 Temperature Reaction rate
constant (k)		 DT50
[°C] [d¯¹] [d] [°C] [d¯¹] [d] [°C] [h¯¹] [h]
20 0.005 138.0 20 2.63E-12 >10000 20 0.003 216.0
40 0.002 460.0 40 0.027 25.8 40 0.050 13.9
50 0.006 113.0 50 0.187 3.7 50 0.166 4.2

Table 1: Results of the Preliminary Test (Tier 1)

Incubation
period		 Replicate % Applied concentration
[h]   [% applied] [% applied],mean
pH 4
0 A 102.5 100.0
  B 97.5  
3 A 105.5 104.7
  B 104.0  
24 A 106.3 105.7
  B 105.1  
120 A 80.8 80.2
  B 79.6  
pH 7
0 A 99.3 100.0
  B 100.7  
3 A 97.5 96.8
  B 96.2  
24 A 90.8 89.7
  B 88.7  
120 A 40.9 47.7
  B 54.4  
pH 9
0 A 100.0 100.0
  B 100.0  
3 A 62.8 62.8
  B 62.8  
24 A <LOQ <LOQ
  B <LOQ  
120 A <LOQ <LOQ
  B <LOQ  
Result evaluation with values ≥ LOQ  

Table 2: Results of the Main Test at pH 4 - (Tier 2)

                 
20°C 40°C 50°C
Incubation
period		 % Applied concentration Incubation
period		 % Applied concentration Incubation
period		 % Applied concentration
[d] [% applied] [% applied],mean [d] [% applied] [% applied],mean [d] [% applied] [% applied],mean
0 93.7 100.0 0 100.5 100.0 0 93.7 100.0
  106.3     99.5     106.3  
1 92.8 93.9 1 91.4 90.4 1 96.4 92.8
  94.9     89.5     89.3  
4 99.9 96.8 4 97.9 102.1 4 94.4 92.4
  93.7     106.3     90.3  
7 83.3 83.7 7 92.5 98.3 7 82.9 83.6
  84.2     104.1     84.3  
10 91.5 89.9 10 100.9 102.3 10 96.9 97.8
  88.2     103.7     98.6  
14 92.7 91.4 14 92.5 93.8 14 85.8 85.8
  90.1     95.2     85.8  
21 91.2 92.8 21 100.6 99.7 21 84.1 83.6
  94.4     98.9     83.1  
30 82.8 79.4 30 93.4 90.9 30 80.3 80.1
  75.9     88.3     79.8  
                 

Table 3: Results of the Main Test at pH 7 - (Tier 2)

20°C 40°C 50°C
Incubation
period		 % Applied concentration Incubation
period		 % Applied concentration Incubation
period		 % Applied concentration
[d] [% applied] [% applied],mean [d] [% applied] [% applied],mean [d] [% applied] [% applied],mean
0 98.7 100.0 0 98.0 100.0 0 98.7 100.0
  101.3     102.0     101.3  
1 84.2 86.0 1 95.4 94.9 1 81.8 83.6
  87.8     94.5     85.4  
4 56.7 55.6 4 92.5 96.4 2 66.0 69.9
  54.5     100.3     73.9  
7 49.6 48.5 7 98.8 98.8 4 35.4 39.6
  47.4     98.8     43.8  
10 51.9 52.0 10 70.4 75.0 7 22.5 22.3
  52.0     79.5     22.1  
14 82.6 83.4 14 72.4 71.3 10 13.8 13.9
  84.1     70.1     14.0  
21 91.0 87.1 21 53.5 58.0 14 22.0 20.0
  83.2     62.4     18.1  
30 74.3 75.0 30 44.0 43.0 18 10.4 10.8
  75.6     42.0     11.2  
- - - - - - 21 8.2 8.4
  -     -     8.6  

Table 4: Results of the Main Test at pH 9 - (Tier 2)

20°C 40°C 50°C
Incubation
period		 % Applied concentration Incubation
period		 % Applied concentration Incubation
period		 % Applied concentration
[h] [% applied] [% applied],mean [h] [% applied] [% applied],mean [h] [% applied] [% applied],mean
0 103.0 100.0 0 123.2 100.0 0 103.0 100.0
  97.0     76.8     97.0  
2 87.3 95.9 2 90.0 89.5 1 84.5 91.0
  104.5     89.1     97.5  
4 93.1 93.8 4 77.6 78.8 2 74.8 69.7
  94.6     79.9     64.6  
24 88.4 87.2 6 77.6 77.2 3 60.2 60.2
  86.0     76.8     60.2  
28 80.0 84.5 8 68.6 67.2 4 53.0 53.8
  89.0     65.8     54.7  
48 77.8 79.8 24 27.1 27.1 6 38.2 36.9
  81.8     27.2     35.6  
96 61.7 63.8 48 11.1 11.2 8 50.9 50.9
  65.8     11.2     50.9  
168 48.2 48.9 96 <LOQ - 24 <LOQ -
  49.7     <LOQ     <LOQ  
240 44.3 42.7 168 <LOQ - 48 <LOQ -
  41.1     <LOQ     <LOQ  
336 34.9 35.1 - - - - - -
  35.2     -     -  
504 24.4 24.5 - - - - - -
  24.5     -     -  
720 13.3 13.3 - - - - - -
  13.3     -     -  

Kinetic Evaluation - Main Test (Tier 2)

           
           
pH     Temperature
    20°C 40°C 50°C
4 Kinetic Model SFO SFO SFO
c21 3.96 3.51 3.91
DT50 [d] 138.0 460.0 113.0
DT90 [d] 459.0 1530.0 376.0
k [d¯¹] 0.005 0.002 0.006
ln k   -5.294 -6.498 -5.096
DT50/DT90 values calculated using HPLC data
1: the minimum error at which Chi2 is passed at 5 % significance level  
           
           
           
pH     Temperature
    20°C 40°C 50°C
7 Kinetic Model SFO SFO SFO
c21 19.4 5.67 12.0
DT50 [d] >10000 25.8 3.7
DT90 [d] >10000 85.7 12.3
k [d¯¹] 2.63E-12 0.027 0.187
ln k   -26.664 -3.617 -1.676
DT50/DT90 values calculated using HPLC data
1: the minimum error at which Chi2 is passed at 5 % significance level  
20°C: will not be used for further calculations (considered as outlier)  
           
           
pH     Temperature
    20°C 40°C 50°C
9 Kinetic Model SFO SFO SFO
c21 4.72 4.51 4.68
DT50 [h] 216.0 13.9 4.2
DT90 [h] 717.0 46.1 13.9
k [h¯¹] 0.003 0.050 0.166
ln k   -5.741 -2.996 -1.796
DT50/DT90 values calculated using HPLC data
1: the minimum error at which Chi2 is passed at 5 % significance level  

Arrhenius Parameters

               
               
pH Temperature ln k 1/T -E/R E ln A A
  [°C]   [K-1]   [kJ/mol]   [d¯¹]
4 20 -5.294 3.41E-03 388 -3.2 -6.884 1.02E-03
40 -6.498 3.19E-03
50 -5.096 3.09E-03
k = hydrolysis rate constant            
E = activation energy [kJ/mol]            
T = absolute temperature [K]            
R = gas constant [8.314 J/mol*K]          
A = pre-exponential factor            
               
               
               
pH Temperature ln k 1/T -E/R E ln A A
  [°C]   [K-1]   [kJ/mol]   [d¯¹]
7 *     -19638 163.3 59.095 4.62E+25
40 -3.617 3.19E-03
50 -1.676 3.09E-03
k = hydrolysis rate constant            
E = activation energy [kJ/mol]            
T = absolute temperature [K]            
R = gas constant [8.314 J/mol*K]          
A = pre-exponential factor            
* Specific parameters could not be determined, as the recovery values were fluctuating significantly (reason unkonwn). For further calculations, those values were not taken into consideration.
               
               
pH Temperature ln k 1/T -E/R E ln A A
  [°C]   [K-1]   [kJ/mol]   [h¯¹]
9 20 -5.741 3.41E-03 -12481 103.8 36.840 9.99E+15
40 -2.996 3.19E-03
50 -1.796 3.09E-03
k = hydrolysis rate constant            
E = activation energy [kJ/mol]            
T = absolute temperature [K]            
R = gas constant [8.314 J/mol*K]          
A = pre-exponential factor            

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The present study investigated the hydrolytic behaviour of Fluoroamidine Hydrochloride 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 first 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 79.4%, 90.9% and 80.1% of the applied concentration at 20, 40 and 50°C, respectively. At pH 7, the average recoveries were 75.0% of the applied concentration after 30 days at 20°C, 43.0% after 30 days at 40°C and 8.4% after 21 days at 50°C. At pH 9, the average recoveries were 24.5% after 21 days at 20°C and < 10% (< LOQ) after 7 days at 40°C and 2 days at 50°C.
Executive summary:

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

Test Item: Fluoroamidine Hydrochloride

Guidelines/Recommendations: This study was based on the procedures indicated by the following -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 Fluoroamidine Hydrochloride 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: 10.05 and 10.04 mg/L (two individual replicates)

Main test (Tier 2): 20°C, 50°C: 10.01 and 10.22 mg/L (two individual replicates)

40°C: 10.03 and 10.13 mg/L (two individual replicates)

Results: The present study investigated the hydrolytic behaviour of Fluoroamidine Hydrochloride 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 first 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 79.4%, 90.9% and 80.1% of the applied concentration at 20, 40 and 50°C, respectively. At pH 7, the average recoveries were 75.0% of the applied concentration after 30 days at 20°C, 43.0% after 30 days at 40°C and 8.4% after 21 days at 50°C. At pH 9, the average recoveries were 24.5% after 21 days at 20°C and < 10% (< LOQ) after 7 days at 40°C and 2 days at 50°C.

pH 4 pH 7 pH 9
Temperature Reaction rate
constant (k)		 DT50 Temperature Reaction rate
constant (k)		 DT50 Temperature Reaction rate
constant (k)		 DT50
[°C] [d¯¹] [d] [°C] [d¯¹] [d] [°C] [h¯¹] [h]
20 0.005 138.0 20 2.63E-12 >10000 20 0.003 216.0
40 0.002 460.0 40 0.027 25.8 40 0.050 13.9
50 0.006 113.0 50 0.187 3.7 50 0.166 4.2

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