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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 7, 2016 - September 22, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Tier I: Before the solutions were placed in the incubator, the samples of them were taken out, signed as “Day 0” samples. Consequently - after 1 day (24 hours) -the next samples were taken from the examined testing solutions (pH 4, 7 and 9, 50 ºC).
Tier II: The test was conducted until 90% hydrolysis was observed (pH 7 and 9, at 20 and 30 ºC).
Tier III: In order to identify the hydrolysis products the following sampling was performed after 1 hour of hidrolysis (pH 4 and 7, 20 ºC) and after 24 hours of hydrolysis (pH 9, 50 ºC).
- Sampling intervals/times for pH measurements: Two parallel samples for each pH values were prepared (six in total).
Buffers:
Buffers solutions from Avantor Performance Materials Poland S.A. at three values: pH 4.0; pH 7.0 and pH 9.0
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks: glass flasks fitted with glass stoppers
- Other equipment used: incubator and magnetic stirrer

TEST MEDIUM
- Preparation of test medium: Test item stock sample solution was prepared in acetone.10 mg of the test item was weighed and dissolved in 10 mL of acetone (1g/l). To each 79.2 ml of three buffer solutions (pH values: 4.0; 7.0 and 9.0) 0.8 ml of obtained stock DCC solution (1g/l) was added. The test item concentration of this stock solution was precisely determined before its application. The measured test item concentration in acetone was 8.37 mg/kg (8.37 mg/l). The concentrations in g/l were used for hydrolysis, using water density 1 kg/l.
Duration:
1 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
0.08 mg/L
Remarks:
Tier 1
Duration:
1 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
3.98 mg/L
Remarks:
Tier 1
Duration:
2.33 h
pH:
7
Temp.:
20 °C
Initial conc. measured:
7.72 mg/L
Remarks:
Tier 2
Duration:
1.58 h
pH:
7
Temp.:
30 °C
Initial conc. measured:
8.44 mg/L
Remarks:
Tier 2
Duration:
24 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
8.52 mg/L
Remarks:
Tier 1
Duration:
600 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
12.11 mg/L
Remarks:
Tier 2
Duration:
147 h
pH:
9
Temp.:
30 °C
Initial conc. measured:
10.82 mg/L
Remarks:
Tier 2
Number of replicates:
Preliminary test Tier 1 and test Tier 2: two replicates
Tier 3: no replicates.
Positive controls:
no
Negative controls:
no
Statistical methods:
The statistical calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.
Preliminary study:
Measurements in buffer solution at pH 4.0: the hydrolysis process was so fast that the determined concentration of the test substance at the start of test (0.08 mg/L) was about the LOD ( 0.06 mg/L). The DCC content on ‘’0’’ day was in fact determined after 0.25 hour of hydrolysis (it took as much as 0.25 hour from sampling of the solution). As this 0.25h hydrolysis underwent at ambient temperature about 20 0C, the rate constant at ambient temperature of 20ºC at pH 4 after 0.25 hwas calculated and found to be 18.6 h-1 and the halflife period t(0.5)= 0.04h. No further testing at pH 4 was conducted as there was no possibility to obtain at 20ºC any other loss results than 100%.

Measurements in buffer solution at pH 7.0: the preliminary test indicated that more than 99 % of the test substance has been hydrolyzed after one day and the test substance was considered to be hydrolytically unstable.

Measurements in buffer solution at pH 9.0: The determined rate constant after 1 day at 50ºC was equal to 0.045 h-1 and the half-life period is equal to 15.4h. Test was continued five more days and the determined rate constant after these 5 days at 50ºC was > 0.03 h-1 and the half-life period <23.1 h, so about 99 % of the test substance has been hydrolyzed after 5 days and the test substance was considered to be hydrolytically unstable.

On the basis of these results, Tier 2 was performed.
Test performance:
Measurement in buffer solution at pH 7.0 and 20ºC: the test was conducted up to 140 min as more than 90 % hydrolysis of the test substance was observed. The determined rate constant after 140 min (2.33 h) at 20ºC (calculated by regression analysis) was equal 1.26 h-1 and the half-life period was 0.55 h.

Measurement in buffer solution at pH 7.0 and 30ºC: the test was conducted up to 95 min as more than 90 % hydrolysis of the test substance was observed. The determined rate constant after 95 min (1.58 h) at 30ºC (calculated by regression analysis) was equal 2.40 h-1 and the half-life period was 0.289 h.

Measurement in buffer solution at pH 9.0 and 20ºC: the test was conducted up to 600h when more than 90 % hydrolysis of the test substance was observed. The determined rate constant after 600 h at 20ºC (calculated by regression analysis) was equal 4.12 x 10-3 h-1 and the half-life period was 168.2 h.

Measurement in buffer solution at pH 9.0 and 30ºC: the test was conducted up to 147 h when more than 90 % hydrolysis of the test substance was observed. The determined rate constant after 147 h at 30ºC (calculated by regression analysis) was equal 17.4 x 10-3 h-1 and the half-life period was 39.8 h.
Transformation products:
yes
Details on hydrolysis and appearance of transformation product(s):
The chromatograms of the solutions after hydrolysis revealed a presence of big peaks appeared at 6.6 min of retention time. The major hydrolysis product was identified as 1,3-Dicyclohexylurea.
% Recovery:
< 1
pH:
4
Temp.:
50 °C
Duration:
0.25 h
Remarks on result:
other: the hydrolysis process was so fast that the determined concentration at hte start of the test was about the LOD (0.06 mg/l)
% Recovery:
5.49
pH:
7
Temp.:
20 °C
Duration:
2.33 h
% Recovery:
2.87
pH:
7
Temp.:
30 °C
Duration:
1.58 h
% Recovery:
< 1
pH:
7
Temp.:
50 °C
Duration:
24 h
% Recovery:
7.16
pH:
9
Temp.:
20 °C
Duration:
600 h
% Recovery:
7.26
pH:
9
Temp.:
30 °C
Duration:
147 h
% Recovery:
34.41
pH:
9
Temp.:
50 °C
Duration:
24 h
% Recovery:
< 1.8
pH:
9
Temp.:
50 °C
Duration:
120 h
Key result
pH:
4
Temp.:
20 °C
Hydrolysis rate constant:
18.6 h-1
DT50:
0.04 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: experimental
Key result
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
1.26 h-1
DT50:
0.55 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: experimental
Key result
pH:
7
Temp.:
30 °C
Hydrolysis rate constant:
2.4 h-1
DT50:
0.29 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: experimental
Key result
pH:
9
Temp.:
20 °C
Hydrolysis rate constant:
0.041 h-1
DT50:
168.2 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: experimental
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.008 h-1
DT50:
87.2 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: calculated
Key result
pH:
9
Temp.:
30 °C
Hydrolysis rate constant:
0.017 h-1
DT50:
39.8 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: experimental
Key result
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.045 h-1
DT50:
15.4 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: experimental
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes, pH and temperature were set and maintaned at various fixed values.

MAJOR TRANSFORMATION PRODUCTS
- At pH4.1 after 1 hour incubation at 20ºC: 1,3-dicyclohexylurea
- At pH7.1 after 1 hour incubation and at 20ºC: 1,3-dicyclohexylurea
- At pH9.24 after 24 hours incubation and at 50ºC: 1,3-dicyclohexylurea

Preliminary test (Tier I)

Measurements in buffer solution at pH 4.0 at 50ºC

Time, days

0 (in fact 0,25h)

1

Concentration [mg/l]

1 flask

0.08

<LOD

2 flask

0.07

<LOD

pH

1 flask

4.00

4.02

2 flask

4.01

4.01

Average concentration [mg/l]

0.08

<LOD

Concentration loss [%]

99

>99

Measurements in buffer solution at pH 7.0 at 50ºC

Time, days

0 (in fact 0,25h)

1

Concentration [mg/l]

1 flask

4.30

<LOD

2 flask

3.66

<LOD

pH

1 flask

7.00

7.01

2 flask

7.00

7.00

Average concentration [mg/l]

3.98

<LOD

Concentration loss [%]

52.5

>99

LOD = 0.06 mg/l

Measurements in buffer solution at pH 9.0 at 50ºC

Time, days

0 (in fact 0,25h)

1

Concentration [mg/l]

1 flask

7.98

2.86

2 flask

9.05

2.89

pH

1 flask

9.00

9.02

2 flask

9.01

9.00

Average concentration [mg/l]

8.52

2.88

Concentration loss [%]

0.0

65.59

Measurements in buffer solution at pH 9.0 at 50ºC (5 days)

Time, days

0 (in fact 0,25h)

5

Concentration [mg/l]

1 flask

7.39

<= LOQ

2 flask

7.80

<= LOQ

pH

1 flask

9.02

9.01

2 flask

9.02

9.02

Average concentration [mg/l]

7.60

<= LOQ

Concentration loss [%]

30.2

>98.2

LOQ = 0.2 mg/l.

Tier II

Measurements in buffer solution at pH 7.0 and 20ºC

Time, min

0 (in fact 0,25h)

30

60

140

Concentration [mg/l]

1 flask

7.93

5.50

2.24

0.47

2 flask

7.50

4.34

1.80

0.40

pH

1 flask

7.03

7.01

7.01

7.02

2 flask

7.02

7.02

7.03

7.02

Average concentration [mg/l]

7.72

4.92

2.02

0.44

Concentration loss [%]

3.65

38.59

74.79

94.51

 

Measurements in buffer solution at pH 7.0 and 30ºC

Time, min

0 (in fact 0,25h)

45

95

Concentration [mg/l]

1 flask

9.85

2.05

0.37

2 flask

7.02

1.05

0.08

pH

1 flask

7.03

7.03

7.02

2 flask

7.04

7.04

7.04

Average concentration [mg/l]

8.44

1.55

0.23

Concentration loss [%]

0

80.65

97.13

 

Measurements in buffer solution at pH 9.0 and 20ºC

Time, h

0

24

26

49

98

169

264

432

600

Concentration [mg/l]

1 flask

11.78

12.20

10.41

8.74

7.79

5.67

3.28

1.84

0.70

2 flask

12.44

12.69

8.39

9.31

7.44

5.34

3.44

1.87

0.70

pH

1 flask

9.04

9.05

9.05

9.04

9.07

9.10

9.09

9.09

9.11

2 flask

9.03

9.04

9.08

9.05

9.08

9.11

9.09

9.10

9.10

Average concentration [mg/l]

12.11

12.45

9.40

9.03

7.62

5.51

3.36

1.86

0.70

Concentration loss [%]

0

0

3.89

7.67

22.09

43.66

65.64

80.98

92.84

 

Measurements in buffer solution at pH 9.0 and 30ºC

Time, h

0

3

5

24

27

53

72

147

Concentration [mg/l]

1 flask

10.77

10.10

9.29

6.48

5.71

4.20

3.15

0.73

2 flask

10.86

10.42

9.36

6.83

6.16

4.12

2.98

0.68

pH

1 flask

9.01

9.04

9.01

9.02

9.06

9.04

9.05

9.06

2 flask

9.01

9.03

9.02

9.04

9.04

9.04

9.04

9.03

Average concentration [mg/l]

10.82

10.26

9.33

6.66

5.94

4.16

3.07

0.71

Concentration loss [%]

0

0

4.60

31.90

39.26

57.46

68.61

92.74

Hydrolysis rate, half-life period and activation energy

 

pH 4

pH 7

pH 9

20ºC (experimental)

Kobs= 18.6 h-1         

 t0.5= 0.04 h

Kobs= 1.26 h-1         

 t0.5= 0.55 h

Kobs= 4.12x10-3h-1         

 t0.5= 168.2 h

25ºC (calculated)

__

__

Kobs= 7.95x10-3h-1         

 t0.5= 87.2 h

30ºC (experimental)

__

Kobs= 2.40 h-1         

 t0.5= 0.29 h

Kobs= 17.4x10-3h-1         

 t0.5= 39.8 h

50ºC (experimental)

__

__

Kobs= 45.0x10-3h-1         

 t0.5= 15.4 h

Activation energy (kJ/mol)

 

50.86

59.85

Validity criteria fulfilled:
yes
Remarks:
LOD=0.0627 mg/ml (>10% initial concentration); selectivity: no other peaks interfering with the substance peak; linearity: R2 > 0.99; precision RSD 9.51% (theorically < 13006%) and RSD for repeatibility was 2.03% (theorically < 7.99%)
Conclusions:
The substance was found to be hydrolitically unstable at pH 4.0 (20ºC) and pH 7.0 (20ºC and 30ºC) with half-life <12h, being more stable at ph 9.0 (20, 25, 30 and 50ºC) with half-life >12h. The major hydrolysis product was identified as 1,3-dicyclohexylurea.
Executive summary:

An Hydrolysis study was conducted on the test substance, according to OECD TG 111 (Hydrolysis as a function of pH), GLP Study. A validated GC method was used for determining the content of the test substance in the samples. This method was based on the separation of the individual components in the sample and the determination of their contents by comparison with the external calibration. A preliminary test (Tier 1) was conducted at pH 4.0, 7.0 and 9.0 and 50ºC, which showed that about 99% of the substance was hydrolyzed after 0.25 h at pH 4.0, after one day at pH 7.0 and after 5 days at pH 9.0, so further testing was necessary. The hydrolysis rate constant at 20ºC and pH 4 after 0.25 h was calculated to be 18.6 h-1 and the half-life period 0.04h. No further testing at pH 4 was conducted as it was not possible to obtain any loss results other than 100% at 20ºC. The determined rate constant after 1 day at 50ºC and pH 9.0 was found to be 0.045 h-1 and the half-life period 15.4h. Based on this data, a Tier 2 assay was then performed at two temperatures (20ºC and 30ºC) and two pH values (7.0 and 9.0) for determining the hydrolysis rate constant and the half-life period for each situation. The determined rate constant at pH 7 after 140 min (2.33 h) at 20ºC was 1.26 h-1 and the half-life period 0.55 h; at 30ºC the determined rate constant after 95 min (1.58 h) was 2.40 h-1 and the half-life period 0.289 h. The determined rate constant at pH 9.0 after 600 h at 20ºC 0 was found to be 4.12 x 10-3 h-1 and the half-life period 168.2 h; at 30ºC was 17.4 x 10-3 h-1 and the half-life period 39.8 h. The calculated rate constant at 25ºC was 7.95 x 10-3 h-1 and the half-life period 87.2h. A third assay was made for identifying the major hydrolysis product by coupling selective mass detector to a chromatograph. The major hydrolysis product was identified as 1,3-dicyclohexylurea. On the basis of these results, the substance was considered hydrolitically unstable at pH 4.0 (20ºC) and pH 7.0 (20ºC and 30ºC) with a half-life <12h, being more stable at pH 9.0 (20, 25, 30 and 50ºC) with half-life >12h.

Description of key information

Key study. Method according to OECD 111, GLP Study. The substance is hydrolytically unstable at pH 4.0 (20ºC) and pH 7.0 (20ºC and 30ºC), with a half-life <12h; it is more stable at pH 9.0 (20ºC, 25ºC, 30ºC and 50ºC). The hydrolysis rate constant at 25ºC and pH 9.0 is 0.00795 h-1, and the half-life period at 25ºC and pH 9.0 is 87.2 h. The main hydrolysis product of the test item is 1,3 -dicyclohexylurea.

Key value for chemical safety assessment

Half-life for hydrolysis:
87.2 h
at the temperature of:
25 °C

Additional information

A study on the hydrolysis of the test item as a function of pH was performed according to OECD TG 111 (GLP study), using a validated GC method. A preliminary test (Tier 1) conducted at 50ºC at three pH values showed that about 99% of the substance was hydrolysed after 0.25 h at pH 4.0, 1 day at pH 7.0 and 5 days at pH 9.0.

In subsequent tests (Tier 2), the hydrolysis rate at 20ºC and pH 4 was found to be 18.6 h-1, with a half-life = 0.04h; at 50ºC and pH 9.0, it was 0.045 h-1 and the half-life 15.4h; at 20ºC and pH 7.0, it was 1.26 h-1 and the half-life 0.55 h; at 30ºC and pH 7.0, it was 2.40 h-1 and the half-life 0.289 h; at 20ºC and pH 9.0, 4.12 E-3 h-1 and the half-life 168.2 h; at 30ºC and pH 9.0, 17.4 E-3 h-1 and the half-life 39.8 h. Based on this data, the calculated rate constant at 25ºC and pH 9.0 was 7.95 E-3 h-1 and the half-life 87.2h.

In a third assay (Tier 3) the main hydrolysis product of the test item was identified as 1,3-dicyclohexylurea.

On the basis of these results, the substance is hydrolytically unstable at pH 4.0 (20ºC) and pH 7.0 (20ºC and 30ºC) with a half-life <12h, being more stable at pH 9.0 (20, 25, 30 and 50ºC) with a half-life >12h.