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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Testing was conducted between 09 March 2011 and June 23 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
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:
Prior to the tests, the buffer solutions were sterilized for 30 minutes at 120 °C in an autoclave. Nitrogen was passed through the buffer solutions at room temperature to reduce the oxygen in the solution.

The hydrolysis was carried out in flasks, which were stoppered or sealed with an inert material (e. g. glass).

Preliminary Test (Tier 1)
According to the guideline, a preliminary hydrolysis test was performed at 50 °C at pH 4.0, pH 7.0 and pH 9.0, each. Aliquots of each test solution were analyzed at the beginning, after 2.4 hours, after 48 hours and after 120 hours using the analytical method as described later in this report. The results after 2.4 hours and 48 hours are not reported, but are part of the raw data.

The buffer solutions used for the test were filtered sterile (0.2 µm, PES). A spike solution of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide was prepared by dissolving an aliquot of 25.48 mg of test item in 100 mL of methanol. Nine times 1 mL of the spike solution was dissolved in 99 mL of the respective buffer solution to prepare a test solution of 15.29 ng/mL. This is corresponding to three samples at each pH value in order to perform a triplicate test.

Hydrolysis of unstable substances (Tier 2)
Due to the instability of the test item found in the preliminary test at 50 °C, further hydrolysis tests were performed at 20 °C, 37 °C and 50 °C in the buffered test solution at pH 4.0, pH 7.0 and pH 9.0. The test temperature was observed to stay constant. To test for first order behavior the concentration of the test item in each test solution was determined immediately after preparation and at time intervals, which provide a minimum of six spaced data points between approximately 10% and approximately 90% of hydrolysis of the test item. A sterility test was conducted after the tests at each pH and temperature and was found to be negative.


Test at 20 °C and pH of 4.0, 7.0 and 9.0
An aliquot of 26.09 mg of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide were dissolved in 100 mL of methanol to prepare a spike solution. Nine times 1 mL of the spike solution was dissolved in 99 mL of the respective buffer solution to prepare a test solution of 15.13 ng/mL. This is corresponding to three samples at each pH value in order to perform a triplicate test.


Test at 37 °C and pH of 4.0, 7.0 and 9.0
An aliquot of 25.95 mg of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide were dissolved in 100 mL of methanol to prepare a spike solution. Nine times 1 mL of the spike solution was dissolved in 99 mL of the respective buffer solution to prepare a test solution of 15.05 ng/mL. This is corresponding to three samples at each pH value in order to perform a triplicate test.


Test at 50 °C and pH of 4.0, 7.0 and 9.0
An aliquot of 24.58 mg of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide were dissolved in 100 mL of methanol to prepare a spike solution. Eight times 1 mL of the spike solution was dissolved in 99 mL of the respective buffer solution to prepare a test solution of 14.99 ng/mL. This is corresponding to three samples at pH value of 4.0 and 9.0 and two samples at pH 7.0 in order to perform at least a duplicate test.


Identification of hydrolysis products (Tier 3)
A scan was made of the relevant retention times using GC-MS. The test item was dissolved once in methanol and once in methanol and water and was compared with methanol. No additional peaks of hydrolysis products could be identified for these two mixtures. Therefore, identification of hydrolysis products is not possible.

Details are not reported here, but are part of the raw data.


Standard Solutions
For example (tier 1): 20.8 mg of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide were dissolved in 100 mL of n-Hexane to prepare a stock solution of 208.0 µg/mL Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide. Calculated volumes of this stock solution were diluted with n-Hexane to obtain six standard solutions in the range from 0.0104 µg/mL to 0.156 µg/mL Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide. These standard solutions were used to calibrate the GC-MS-system.

A second set of standard solutions was prepared accordingly and was also used for the calibration of the test item. During and at the end of the test the stability and accuracy of the calibration solution was confirmed analyzing the first calibration solution.


Sample Preparation
Before and after incubation at the respective test temperatures, 5 mL of the sample solutions was taken and extracted with 1 mL of n-Hexane. An aliquot of the n-Hexane phase was collected for analysis.

Aliquots of the test solutions at each pH value were analyzed by measuring the MS signal of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (m/z = 111) after GC separation of the injected sample solution.
Buffers:
Buffer pH 4, Biphthalate Fluka Art. No. 82566
Buffer pH 7, Phosphate Fluka Art. No. 82571
Buffer pH 9, Borate Fluka Art. No. 33648

387.6 mL of the buffer pH 7 were diluted up to 1 L with water and adjusted to pH 7.02 with phosphoric acid.

Prior to the tests, the buffer solutions were sterilized for 30 minutes at 120 °C in an autoclave. Nitrogen was passed through the buffer solutions at room temperature to reduce the oxygen in the solution.
Duration:
8 h
pH:
4
Temp.:
20 °C
Initial conc. measured:
0.09 mg/L
Duration:
7.5 h
pH:
4
Temp.:
37 °C
Initial conc. measured:
0.06 mg/L
Duration:
1.5 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
ca. 0.06 - 0.07 mg/L
Duration:
8 h
pH:
7
Temp.:
20 °C
Initial conc. measured:
0.09 mg/L
Duration:
7.5 h
pH:
7
Temp.:
37 °C
Initial conc. measured:
ca. 0.07 - 0.08 mg/L
Duration:
1.5 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
0.07 mg/L
Duration:
8 h
pH:
9
Temp.:
20 °C
Initial conc. measured:
0.09 mg/L
Duration:
7.5 h
pH:
9
Temp.:
37 °C
Initial conc. measured:
ca. 0.06 - 0.08 mg/L
Duration:
1.5 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
0.07 mg/L
Number of replicates:
Three samples at each pH value and temperature were taken at each sampling time.
Positive controls:
no
Negative controls:
yes
Remarks:
All sterility tests were determined to be negative.
Statistical methods:
Not specified.
Preliminary study:
The hydrolysis preliminary test was performed with Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at 50.0 °C at each of pH 4.0, pH 7.0, and pH 9.0 in triplicate. The test solutions of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide were thermostated to 50.0 °C and analyzed in time intervals.

A typical chromatogram of a standard solution is given in Figure 1 (see attached). Typical chromatograms of the test solutions after incubation at pH 4.0, pH 7.0 and pH 9.0 are given in Figure 2 to Figure 4 (see attached). An example of a calibration curve is given in Figure 5 (attached). The calibration data of test item standards are given in Table 25 (attached). The r² fit was 0.9962 (optimum 1.0000). This reflects the linearity of the GC-MS system within the calibration range of 0.0104 µg/mL to 0.156 µg/mL of the test item.

The concentrations of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide found in the test solutions before and after incubation times of 5 days are given in the table given in the section below. The tabulated values represent rounded results, which were obtained by calculation using the exact raw data.

EXAMPLE: The mean peak heights relating to the standard and sample solutions are shown in table 6.2 (please see remarks on results including tables and figures section).
EXAMPLE:
Transformation products:
not specified
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0.259 h-1
DT50:
2.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: slope=-6243; y-axis intercept=16.6; r=-0.991
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.257 h-1
DT50:
2.7 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: slope = -6756; y-axis intercept = 21.3; r = -0.994
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.244 h-1
DT50:
2.8 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: slope = -7300, y-axis intercept = 23.1, r = -0.991
Other kinetic parameters:
None.

Results According to the Preliminary Test

 

 

Incubation Time at 50.0 °C ± 0.5 °C

 

0 hours

5 days

 

Initial concentration measured

Concentration
measured

Hydrolyze Reaction

 

[μg/mL]

[μg/mL]

[%]

pH 4.0

0.013

0.000*

96

0.013

0.000*

98

0.013

0.000*

95

pH 7.0

0.015

0.000*

99

0.012

0.000*

98

0.014

0.000*

99

pH 9.0

0.016

0.000*

97

0.014

0.000*

97

0.016

0.000*

97

 

*          was found to be below the lowest point of calibration ( c = 0.01 µg/mL)

The following tables and figures are all attached to the IUCLID document

Main Test (Tier 2)

Hydrolysis at pH 4.0

The half-life time of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at pH 4.0 is presented in the following table:

 

20 °C

37 °C

50 °C

Half life time [hours]

3.8

1.1

0.5

 

 

Hydrolysis at pH 7.0

 

The half-life time of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at pH 7.0 is presented in the following table:

 

20 °C

37 °C

50 °C

Half life time [hours]

3.9

1.2

0.4

 

 

Hydrolysis at pH 9.0

 The half-life time of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at pH 9.0 is presented in the following table:

 

20 °C

37 °C

50 °C

Half life time [hours]

4.4

1.1

0.5

 

 

Evaluation of the Half-Life Time at 25 °C

The half-life time of the hydrolysis reaction of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at 25 °C was calculated to be:

 

pH 4.0

pH 7.0

pH 9.0

Half life time [hours]

2.7

2.7

2.8

 


Validity criteria fulfilled:
yes
Conclusions:
Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at pH 4.0 has a half-life time of 2.7 hours at 25 °C. Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at pH 7.0 has a half-life time of 2.7 hours at 25 °C. Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide at pH 9.0 has a half-life time of 2.8 hours at 25 °C.
Executive summary:

The hydrolysis pre-test for the test item was performed at 50.0 °C ± 0.5 °C at each of pH 4.0, pH 7.0 and pH 9.0.

 

Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide was not stable at pH 4.0, pH 7.0 and at pH 9.0, therefore further testing was performed in order to calculate the rate constant (k25) and the half-life time of the hydrolysis at pH 4.0, pH 7.0 and pH 9.0 at 25 °C. The results are summarized below (the values typed in italics were calculated using the Arrhenius equation):

 

pH

Duration

Tempe-rature

Reaction rate constant

k

Reaction rate constant

k

Half-life time

t 1/2

 

[h]

[°C]

[1/hours]

[1/s]

[hours]

4.0

-

25

2.59 x 10-1

7.19 x 10-5

2.7

8.00

20

1.84 x10-1*

5.12 x10-5

3.8

7.50

37

5.67 x10-1*

1.58 x10-4

1.1

1.50

50

1.34 x100*

3.72 x10-4

0.5

7.0

-

25

2.57 x 10-1

7.15 x 10-5

2.7

8.00

20

1.80x10-1*

4.99x10-5

3.9

7.50

37

5.83x10-1*

1.62x10-4

1.2

1.50

50

1.58x100**

4.39x10-4

0.4

9.0

-

25

2.44 x 10-1

6.78 x 10-5

2.8

8.00

20

1.63x10-1*

4.52x10-5

4.4

7.50

37

6.29x10-1*

1.75x10-4

1.1

1.50

50

1.64x100*

4.56x10-4

0.5

 

*        The tabulated value is the mean of three samples.

**       The tabulated value is the mean of two samples.

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-12-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is a preliminary test 50°C in which significant degradation was observed. The definitive test is missing and therefore the results need to be treated with care.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data
Radiolabelling:
no
Analytical monitoring:
not specified
Details on sampling:
- Sampling intervals for the parent/transformation products: 0, 4.5, and 24 hours
- Sampling method: Samples were taken at the different time intervals, and transferred to 25 ml volumetric flasks. The vials were then rinsed with 10 ml acetonitrile to extract potentially adsorbed test substance from the test walls and transferred to the same 25 ml volumetric flask. The volumetric flasks were then filled up to the volume of 25 mL with acetonitrile and analysed using LC-MS/MS.
- Sampling intervals/times for pH measurements: 0, 4.5, 24 hours
- Sampling intervals/times for sterility check: no data
- Sample storage conditions before analysis: no data
Buffers:
- pH: 4, 7, 9
- Composition of buffer: A stock solution of the test substance was prepared in acetonitrile at a concentration of 544 mg/L. Subsequently a dilution was made in acetonitrile at a concentration of 7.5 mg/L. An aliquot of this stock solution was added to the buffer solutions to reach the final test substance concentration of 75 µg/L. The pH of each buffer solution was checked by measuring the pH in a separated glass vessel which had been equilibrated in the 50ºC water bath.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 10 mL glass vials
- Sterilisation method: no data
- Lighting: no data
- Measures taken to avoid photolytic effects: no data
- Measures to exclude oxygen: no data
- Details on test procedure for unstable compounds: not applicable
- If no traps were used, is the test system closed
- Is there any indication of the test material adsorbing to the walls of the test apparatus? No

TEST MEDIUM
- Volume used/treatment 25 mL
- Kind and purity of water: no data
- Preparation of test medium: A stock solution of the test substance was prepared in acetonitrile at a concentration of 544 mg/L. Subsequently a dilution was made in acetonitrile at a concentration of 7.5 mg/L. An aliquot of this stock solution was added to the buffer solutions to reach the final test substance concentration of 75 µg/L. The pH of each buffer solution was checked by measuring the pH in a separated glass vessel which had been equilibrated in the 50ºC water bath. Samples were taken at the different time intervals, and transferred to 25 ml volumetric flasks. The vials were then rinsed with 10 ml acetonitrile to extract potentially adsorbed test substance from the test walls and transferred to the same 25 ml volumetric flask. The volumetric flasks were then filled up to the volume of 25 mL with acetonitrile and analysed using LC-MS/MS.
- Renewal of test solution: no data
- Identity and concentration of co-solvent: not applicable
OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Dissolved oxygen: no data
Duration:
0 h
pH:
4
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
0 h
pH:
7
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
0 h
pH:
9
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
4.5 h
pH:
4
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
4.5 h
pH:
7
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
4.5 h
pH:
9
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
24 h
pH:
4
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
24 h
pH:
7
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Duration:
24 h
pH:
9
Temp.:
50
Initial conc. measured:
ca. 75 µg/L
Number of replicates:
2 replicates per pH concentration at each sampling time.
Positive controls:
not specified
Negative controls:
not specified
Statistical methods:
no data
Preliminary study:
no data
Test performance:
no data
Transformation products:
no
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
no data
Remarks on result:
other: The hydrolysis route is not known. The half-life can be tentatively determined as between 4 and 13 hours at 50°C and extrapolated to 44.1 -143 hours at all three pHs at 20°C using the Arrhenius equation.
Other kinetic parameters:
None

Table 1: Results of hydrolysis test conducted by AkzoNobel:

Sample

Nominal

 conc.
(µg/L)

T

 
(hrs)

Measured

conc.

(µg/L)

pH 4

AVG

conc.
(µg/L)

pH 4

Measured

conc.
(µg/L)

pH 7

AVG


(µg/L)

pH 7

Measured

conc.
(µg/L)

pH 9

AVG

conc.
(µg/L)

pH 9

I

75

0

39.1

 

42.0

 

43.0

 

II

75

0

41.1

40.1

42.7

42.4

41.2

42.1

I

75

4.5

15.7

 

12.8

 

17.3

 

II

75

4.5

12.4

14.1

14.3

13.5

19.4

18.3

I

75

24

4.9

 

7.2

 

7.5

 

II

75

24

5.8

5.4

5.9

6.5

7.2

7.3

Table 2: Results of hydrolysis test conducted by DuPont:

Sample

Nominal

 conc.
(µg/L)

T

 
(hrs)

Measured

conc.
 (µg/L)

Measured

conc.
(µg/L)

Measured

conc.
(µg/L)

pH 4

pH 7

pH 9

I

50

0

44.5

59.6

30.7

I

50

1

39.5

42.9

29.4

I

50

2

35.1

43.3

28.7

I

I

50

50

3

24

38.9

1.6

41.8

1.8

27.7

2.5

Validity criteria fulfilled:
not specified
Conclusions:
Hydrolysis of the substance was assessed under GLP, following OECD guideline 111. As the possible hydrolysis product TBA (tertiary-butyl alcohol) could not be detected no confirmation of the increase of this product in 24 hours could be given. It is likely that Trigonox 101 decomposes rapidly in aqueous samples but the hydrolysis route is not known (yet).Therefore these results could theoretically be considered as hydrolysis of the test substance. The half-life can be tentatively determined as between 4 and 13 hours at 50°C and extrapolated to 44.1 -143 hours at all three pHs at 20°C using the Arrhenius equation.
Executive summary:

The results of both studies (AkzoNobel and DuPont) showed a significant decomposition of Trigonox 101 after 24 hours.

As the possible hydrolysis product TBA (tertiary-butyl alcohol) could not be detected no confirmation of the increase of this product in 24 hours could be given.

It was plausible that the substance would decompose pretty quickly in aqueous samples but as the hydrolysis route is not known (yet) the degradation cannot be specified as hydrolysis but only as decomposition. However this study was based on the requirements of the preliminary part of the hydrolysis study as described in OECD guideline 111. Therefore these results could theoretically be considered as hydrolysis of the test substance. The half-life can be tentatively determined as between 4 and 13 hours at 50°C and extrapolated to 44.1 -143 hours at all three pHs at 20°C using the Arrhenius equation.

Description of key information

An OECD 111 study is available with a half life of 2.7 at pH 7.

Key value for chemical safety assessment

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

Additional information

Three studies evaluating the hydrolysis of Trigonox 101 are available. The first two are screening studies; the third study is the definitive study.

The screening studies (Vos, 2010; Powley, 2008) were performed under GLP according to OECD guideline 111. In both studies, significant decomposition of Trigonox 101 occurred within 24 hours at pH 4, 7 and 9. As the possible hydrolysis product TBA (tertiary- butyl alcohol) could not be detected no confirmation of the increase of this product in 24 hours could be given. Although these are only the screening studies performed at one temperature (50 °C), the results are consistent as the test substance is expected to decompose rapidly in aqueous samples but the hydrolysis route is not yet known. These results can be considered as hydrolysis of the test substance. The half- life was tentatively determined as between 4 and 13 hours at 50 °C and extrapolated to 44.1 - 143 hours at all three pHs at 20 °C using the Arrhenius equation.

In the definitive study (Habeck, 2011), performed under GLP and following OECD Guideline 111 and EU Method C.7, the results showed a half- life time between 2.7 and 2.8 hours for all pHs (4, 7 and 9) at 25 °C. For risk assessment purposes the half life at pH 7 is selected (2.7 hours).