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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-08-22 to 2019-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
refer to section "Principles of method if other than quideline"
Principles of method if other than guideline:
There were three modifications to OECD guideline 111:
- No thymol was added to the buffer solutions as, based on microbiological experience, the addition of thymol as a disinfectant is not necessary because the buffer solutions were sterilised also.
- A single test temperature of 20 °C was used together with a single pH of 7. The aim of the study was weight of evidence of similar behaviour. A reference material was therefore, also tested in parallel. In addition, at the temperature of 50 °C, as mentioned in OECD 111 was not tested, due to the test substance being not entirely stable at raised temperatures.
- Only Tier 2 of the guideline was conducted.
GLP compliance:
no
Radiolabelling:
no
Analytical monitoring:
yes
Remarks:
GC-MS
Details on sampling:
Subsequent samples were taken on different time intervals and analysed to determine the percentage of hydrolysis. Samples were analysed directly after sampling in order to prevent further hydrolysis.
Buffers:
- pH: 7
Details on test conditions:
TEST SYSTEM
- Sterilisation method: autoclaving at 121 °C for 20 minutes for the buffer solutions and at 180 °C for 30 minutes for the glassware
- Lighting: dark
- Measures taken to avoid photolytic effects: complete darkness
- Is there any indication of the test material adsorbing to the walls of the test apparatus? yes

OTHER TEST CONDITIONS
- Adjustment of pH: none
Duration:
30 d
pH:
7
Temp.:
20 °C
Remarks:
100 µL in 10 mL buffer solution
Number of replicates:
2
Positive controls:
no
Negative controls:
no
Preliminary study:
No preliminary test was performed, as there is already existing information on the hydrolysis of the test substance.
Transformation products:
not measured
% Recovery:
66
pH:
7
Temp.:
20 °C
Duration:
30 d
Key result
pH:
7
Temp.:
20 °C
DT50:
149 d
pH:
7
Temp.:
12 °C
DT50:
282.6 d
Remarks on result:
other: calculation based on the determined DT50 at 20 °C
Details on results:
Hydrolysis of unstable substance (Tier 2)
The hydrolysis test was performed at pH 7 in duplicate at a temperature of 20 °C in the dark. The pH value at the start of the test was measured to be 7.0. The temperature during the test varied between 20.0 and 20.2 °C. It was observed that on average 66 % of the test substance could be recovered from the nominally spike concentration when performing total extraction of the test vessel. In comparison to 88 % hydrolysis at pH 7 and 25 °C (Reference: supporting-001-Hydrolysis-2013).
It was concluded that the test substance has a half-life of 149 days and therefore, may be concluded to be hydrolytically stable according to the test guideline. There is still however a loss in recovery as the test proceeds. Some hydrolysis or the efficiency of the extraction procedure could be possible explanation for this. It is, however, clear that the hydrolysis half-life of the parent material is significantly longer than indicated in the original study, 64 h at pH 7 and 25 °C, largely due to adsorption of the test material.
Results with reference substance:
For comparison the reference substance, 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane was tested in parallel. The reference substance is structural analogue of the test substance. In a series of different Tier 1 and Tier 3 hydrolysis studies was determined that significant hydrolysis was not taking place and adsorption was the main reason for the initial hydrolysis observations of the reference substance. The reference substance was tested in parallel under the same test conditions as the test substance. The hylf-live of the reference substance was determined to be 49 days.

Table 1: Calculation half-lives, pH 7 (average values of duplicate test)

Substance

Temperature / °C

Slope

Kobs

t1/2 / days

Test substance

20

0.0021

0.0048 1/hrs

149

Ref. substance

20

0.0064

0.0146 1/hrs

49

 

Table 2: Results of test substance - 1,1-Di(tert-butylperoxy)cyclohexane at pH 7

 

 

Replicate I

Replicate II

Sample

Time days

Concentration

(Ct) / µg/L

Mass
balance*

%

Hydrolysis %

Concentration

(Ct) / µg/L

Mass
balance*

%

Hydrolysis %

pH 7 T=0

0

648.9

86.5

0

687.2

91.6

0

pH 7 T=3

3

535.4

71.4

17.5

546.3

72.8

20.5

pH 7 T=6

6

584.3

77.9

10.0

576.7

76.9

16.1

pH 7 T=8

8

528.7

70.5

18.5

543.4

72.4

20.9

pH7T=10

10

505.2

67.4

22.1

498.4

66.5

27.5

pH7T=15

15

550.3

73.4

15.2

576.2

76.8

16.2

pH 7 T=20

20

554.6

74.0

14.5

585.1

78.0

14.9

pH 7 T=24

24

538.7

71.8

17.0

541.2

72.2

21.2

pH 7 T=30

30

469.5

62.6

27.7

525.5

70.1

23.5

* mass balance is calculated as the % of test substance still available compared to nominally spiked concentration.

 

Table 3: Results of reference substance – 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane at pH 7

 

 

Replicate I

Replicate II

Sample

Time
days

Concentration

(Ct) / µg/L

Mass
balance*

%

Hydrolysis %

Concentration

(Ct) / µg/L

Mass
balance*

%

Hydrolysis %

pH 7 T=0

0

34.7

90.1

0

36.7

95.4

0

pH 7 T=3

3

24.6

63.8

29.2

23.4

60.9

36.2

pH 7 T=6

6

18.0

46.7

48.2

18.7

48.5

49.2

pH 7 T=8

8

23.8

61.8

31.4

25.0

64.8

32.0

pH7T=10

10

17.1

44.5

50.6

18.5

48.1

49.6

pH7T=15

15

6.3#

16.4#

81.8#

12.7#

33.0#

65.5#

pH 7 T=20

20

15.2

39.4

56.3

21.3

55.4

41.9

pH 7 T=24

24

18.4

47.7

47.1

17.7

45.9

51.9

pH 7 T=30

30

17.6

45.8

49.2

20.8

54.0

43.4

# The data was considered to be an outlier and therefore, not used as data point in the calculations

Validity criteria fulfilled:
yes
Conclusions:
In the current study, the test material was concluded to be hydrolytically stable and strongly adsorbing with a half-life of 149 days.
Executive summary:

The hydrolysis of the test item was assessed in accordance with the OECD 111, Tier 2, with some modifications. The objective of this study was to determine if the test substance, 1,1-Di(tert-butylperoxy)cyclohexane would hydrolyse at an single environmentally relevant pH value, using a suitable analytical method. As a reference substance the structural analogue 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane was tested in parallel under the same test conditions. Sterilised aqueous buffer solution at pH 7 was spiked with the test substance and reference substance and incubated (20 °C) in the dark under controlled laboratory conditions. After appropriate time intervals, buffer solutions were analysed for the amount of test and reference substance still available. It was observed that on average 66 and 50 % of the test and reference substance, respectively, could be recovered from the nominally spike concentration when performing total extraction of the test vessel. The test material and the reference material were concluded to have half-lives of 149 and 49 days, respectively and therefore, may both be concluded to be hydrolytically stable according to the test guideline. There is still however a loss in recovery with both materials as the test proceeds. Some hydrolysis or the efficiency of the extraction procedure could be possible explanation for this as well as largely due to adsorption of the test material.

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-11-28 to 2012-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Buffers:
- pH: 4, 7, and 9
Composition of buffer:
-Buffer solution pH = 4: 1 mL 0.2 M sodium hydroxide and 125 mL 0.2 M potassium hydrogen phthalate were diluted to 500 mL with ultra-pure water.
- Buffer solution pH = 7: 74 mL 0.2 M sodium hydroxide and 125 mL 0.2 M potassium dihydrogen phosphate were diluted to 500 mL with ultra-pure water.
- Buffer solution pH = 9: 54 mL 0.2 M sodium hydroxide and 125 mL 0.2 M boric acid and potassium chloride were diluted to 500 mL with ultra-pure water.
Details on test conditions:
TEST SYSTEM AND CONDITIONS:
The hydrolysis was examined at three different pH values (pH = 4, 7 and 9) in the dark.
Temperature: 5 °C ± 0.5 °C, 15 °C ± 0.5 °C and 25 °C ± 0.5 °C. (Based on the results a fast hydrolysis reaction of cyclohexylidenebis[tert-butyl] peroxide was supposed, therefore for the main test lower test temperatures were used then those suggested in the guideline.)
Light and oxygen: The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects. Nitrogen was bubbled into the water before the preparation of the solutions in order to exclude oxygen. All glassware, water and buffer solution were sterilised.
The sterile buffer solutions were prepared using reagent grade chemicals and sterile ultra-pure water.
The pH of each buffer solution was checked with a calibrated pH meter.
The samples were analysed directly after preparation.
Sterility confirmation tests were performed at each pH level at the end of the hydrolysis experiments.
For each temperature at least seven data points were taken so that the degrees of hydrolysis are in the range of 10 to 90 %.

Sample preparation for identification of transformation products (tier 3 study):
- Identification of hydrolysis products using HPLC-UV detection: The samples were stored at 15 °C for 236 hours (pH 4, 7 and 9) and at 5 °C for 328 hours (pH 7 and 9). After the storage period samples were measured by HPLC method using UV detection.
Reference solutions of tert-butyl hydroperoxide were prepared in acetonitrile and they were analysed by an HPLC-UV method.
Composition of the mobile phase: acetonitrile : water : phosphoric acid (70 : 30 : 0.05 (v/v/v)).

- Identification of hydrolysis products using GC-FID method:
The samples were stored for 284 hours at temperature 15 °C (pH= 4, 7, 9). After the storage period samples were measured directly.
Reference stock solution: 1 mg/mL cyclohexanone in acetonitrile.
Duration:
376 h
pH:
4
Temp.:
5 °C
Initial conc. measured:
48.4 mg/L
Duration:
284 h
pH:
4
Temp.:
15 °C
Initial conc. measured:
46.1 mg/L
Duration:
96 h
pH:
4
Temp.:
25 °C
Initial conc. measured:
48.8 mg/L
Duration:
376 h
pH:
7
Temp.:
5 °C
Initial conc. measured:
49.4 mg/L
Duration:
284 h
pH:
7
Temp.:
15 °C
Initial conc. measured:
49.1 mg/L
Duration:
192 h
pH:
7
Temp.:
25 °C
Initial conc. measured:
49.4 mg/L
Duration:
376 h
pH:
9
Temp.:
5 °C
Initial conc. measured:
51.6 mg/L
Duration:
284 h
pH:
9
Temp.:
15 °C
Initial conc. measured:
50.6 mg/L
Duration:
234 h
pH:
9
Temp.:
25 °C
Initial conc. measured:
46.5 mg/L
Number of replicates:
Two samples were analysed at the start of the study at each pH value. Two separate tubes were analysed at each analytical occasion of hydrolysis test. One sample was taken from the control vessel.
Positive controls:
no
Negative controls:
yes
Remarks:
buffer solution at pH 4, 7, 9
Statistical methods:
The cyclohexylidenebis[tert-butyl] peroxide concentrations were calculated applying the calibration equations (linear regression curves).
For the calculation of the half-lives of reactions at the different pH values the ln-transformed data of cyclohexylidenebis[tert-butyl] peroxide concentrations were plotted against time. Linear regression lines were fitted on the data. The rate constant and the half-lives of the reactions were obtained from the slope of this linear regression lines.
Calculation of the half-lives of reactions at 20 °C: Applying Arrhenius equation the rate constant and half-lives at pH = 4, 7 and 9 were calculated at 20 °C. The ln-transformed data of rate constants at three temperatures of the hydrolysis test were plotted against 1/T (T= t+273.15 °C). A line was fitted on the data and the rate constant at 20 °C was calculated from the constants and slopes of linear regression lines.
Preliminary study:
In the course of the preliminary test the test substance proved to be hydrolytically unstable at pH 4, pH 7 and pH 9. The hydrolysis rate of the test item was found to be more than 90 % after 5 days at 50 °C.
Test performance:
Based on the results a fast hydrolysis reaction of cyclohexylidenebis[tert-butyl] peroxide was supposed, therefore for the main test lower test temperatures were used then those suggested in the guideline.
Transformation products:
yes
No.:
#1
No.:
#2
Details on hydrolysis and appearance of transformation product(s):
Pathway of transformation (reaction equation): cyclohexylidenebis[tert-butyl] peroxide + water -> 2 tert-butyl hydroperoxide + cyclohexanon
% Recovery:
6
pH:
4
Temp.:
5 °C
Duration:
376 h
% Recovery:
4
pH:
4
Temp.:
15 °C
Duration:
284 h
% Recovery:
8
pH:
4
Temp.:
25 °C
Duration:
96 h
% Recovery:
25
pH:
7
Temp.:
5 °C
Duration:
376 h
% Recovery:
16
pH:
7
Temp.:
15 °C
Duration:
284 h
% Recovery:
12
pH:
7
Temp.:
25 °C
Duration:
192 h
% Recovery:
17
pH:
9
Temp.:
5 °C
Duration:
376 h
% Recovery:
17
pH:
9
Temp.:
15 °C
Duration:
284 h
% Recovery:
18
pH:
9
Temp.:
25 °C
Duration:
234 h
pH:
4
Temp.:
5 °C
Hydrolysis rate constant:
0.007 h-1
DT50:
100 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
15 °C
Hydrolysis rate constant:
0.01 h-1
DT50:
67 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0.026 h-1
DT50:
27 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
5 °C
Hydrolysis rate constant:
0.004 h-1
DT50:
193 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.007 h-1
DT50:
100 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.011 h-1
DT50:
64 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
5 °C
Hydrolysis rate constant:
0.004 h-1
DT50:
155 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.006 h-1
DT50:
116 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.007 h-1
DT50:
101 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
7
Temp.:
12 °C
Hydrolysis rate constant:
0.005 h-1
DT50:
127.1 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: The half-life and the hydrolysis rate constant at pH=7 and 12 °C (environmentally relevant temperture) were calculated on the basis of the half-life and the hydrolysis rate constant at pH7 and 15°C (100 h, 0.0069 h-1).
Other kinetic parameters:
Applying Arrhenius equation the rate constant and half-lives at pH = 4, 7 and 9 were calculated at 20 °C:
- Temperature: 20 °C, pH = 4: rate constant: 0.0171h-1; half-life: 40 hours,
- Temperature: 20 °C, pH = 7: rate constant:0.0086 h-1; half-life: 80 hours,
- Temperature: 20 °C, pH = 9: rate constant:0.0064 h-1; half-life: 109 hours
Details on results:
Applying Arrhenius equation the rate constant and half-lives at pH = 4, 7 and 9 were calculated at 20 °C.
Results of the sterility confirmation test: There were no microorganisms detected by the sterility confirmation tests.

Tier 3 results (Identification of transformation products):

On the basis of retention times both potential hydrolysis products (tert-butyl hydroperoxide and cyclohexanon) were identified successfully in stored buffer solutions. Tert-butyl hydroperoxide was identified in all stored buffer solutions (pH = 4, 7, 9) using an HPLC-UV method; cyclohexanone was detected by a GC-FID method in stored solutions at pH = 4.

Validity criteria fulfilled:
yes
Conclusions:
The hydrolysis of cyclohexylidenebis[tert-butyl] peroxide in different aqueous buffer solutions (pH = 4, 7 and 9) at 5, 15 and 25 °C was investigated using HPLC-UV. Measurements revealed a fast hydrolysis of cyclohexylidenebis[tert-butyl] peroxide (half-live: 27 hours) at pH = 4 and 25 °C. The degradation products of the hydrolysis of cyclohexylidenebis[tert-butyl] peroxide are tert-butyl hydroperoxide and cyclohexanon.
Executive summary:

The hydrolysis of cyclohexylidenebis[tert-butyl] peroxide and the identification of the decomposition products was assessed in accordance with the OECD guideline 111 and the EU method C.7. The hydrolysis of cyclohexylidenebis[tert-butyl] peroxide in different aqueous buffer solutions (pH = 4, 7 and 9) at 5, 15 and 25 °C was investigated using HPLC-UV. Measurements revealed a fast hydrolysis of the test substance (half-life: 27 hours) at pH = 4 and 25 °C. Applying Arrhenius equation the rate constant and half-lives at pH = 4, 7 and 9 were calculated at 20 °C. The half-life at pH = 4, 7 and 9 at 20 °C are 40 hours, 80 hours and 109 hours, respectively. It could be shown that the hydrolysis of cyclohexylidenebis[tert-butyl] peroxide was dependent from the pH and the temperature of the aqueous media. The degradation products of cyclohexylidenebis[tert-butyl] peroxide (tert-butyl hydroperoxide and cyclohexanon) were successfully identified on the basis of their retention times using HPLC-UV and GC-FID.

Description of key information

The test substance was determined to be hydrolytically stable at 20 °C and pH 7 in a Tier 2 study. The half-life was estimated to be 149 days. The half-life of the test item at environmental temperature (12 °C) was calculated to be 282.6 d.

Key value for chemical safety assessment

Half-life for hydrolysis:
282.6 d
at the temperature of:
12 °C

Additional information

The hydrolysis of the test item was assessed in accordance with the OECD 111, Tier 2, with some modifications. The objective of this study was to determine if the test substance, 1,1-Di(tert-butylperoxy)cyclohexane would hydrolyse at an single environmentally relevant pH value, using a suitable analytical method. As a reference substance the structural analogue 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane was tested in parallel under the same test conditions. Sterilised aqueous buffer solution at pH 7 was spiked with the test substance and reference substance and incubated (20 °C) in the dark under controlled laboratory conditions. After appropriate time intervals, buffer solutions were analysed for the amount of test and reference substance still available. It was observed that on average 66 and 50 % of the test and reference substance, respectively, could be recovered from the nominally spike concentration when performing total extraction of the test vessel. The test material and the reference material were concluded to have half-lives of 149 and 49 days, respectively and therefore, may both be concluded to be hydrolytically stable according to the test guideline. There is still however a loss in recovery with both materials as the test proceeds. Some hydrolysis or the efficiency of the extraction procedure could be possible explanation for this as well as largely due to adsorption of the test material. The half-life of the test item at environmental temperature (12 °C) was calculated to be 282.6 d.


 


 


In a previos study, the hydrolysis of cyclohexylidenebis[tert-butyl] peroxide and the identification of the decomposition products was assessed in accordance with the OECD guideline 111 and the EU method C.7. The hydrolysis of cyclohexylidenebis[tert-butyl] peroxide in different aqueous buffer solutions (pH = 4, 7 and 9) at 5, 15 and 25 °C was investigated using HPLC-UV. Measurements revealed hydrolysis of the test substance (half-life: 27 hours) at pH = 4 and 25 °C. Applying Arrhenius equation the rate constant and half-lives at pH = 4, 7 and 9 were calculated at 20 °C. The half-life at pH = 4, 7 and 9 at 20 °C are 40 hours, 80 hours and 109 hours, respectively. It could be shown that the hydrolysis of cyclohexylidenebis[tert-butyl] peroxide was dependent from the pH and the temperature of the aqueous media. The degradation products of cyclohexylidenebis[tert-butyl] peroxide (tert-butyl hydroperoxide and cyclohexanon) were successfully identified on the basis of their retention times using HPLC-UV and GC-FID. For chemical safety assessment the half-life and the hydrolysis rate constant at the environmentally relevant temperture (12 °C) were calculated to be 127.1 h and 0.00545 h-1, respectively.


 


In conclusion, the obtained results on the hydrolysis in the old study are mainly due to the adsoption of the test substance to the walls of the vessels, and not taking this information into account the conclusion that the test substance is not hydrolytically stable was taken. In the modified Tier 2 study, more time and effort was applied to avoid the adsorption of the test substance to the test vessels as much as possible, therefore, on average around 66 % of the spiked test item could be recoved and the test item was, therefore, determined as hydrolytically stable with a half-life of 149 days at 20 °C and pH 7. The half-life of the test item at environmental temperature (12 °C) was calculated to be 282.6 d.