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Hydrolysis

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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24-Sep-2010-July 29, 2014
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Guideline GLP study. Conducted several order of magnitude above water solubility although this was not recognised until after the experiment had been conducted.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Remarks:
No significant guideline deviations were carried out. The length of the study did require study plan amendments as did several staff changes and changes in sampling regime etc.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
This report concerns all the data measured for Tiers I and II hydrolysis studies. Sampling was therefore conducted at multiple time points
throughout each of these studies. Measured data from each sample point is summarized in tables included under any other information on results. The sampling procedures was however as follows:

Tier I, At each sampling point , two tubes of the incubated 10 ml aliquots were taken out of the waterbath and extracted twice with each 2 mL of
hexane. 200 μL of the organic phase were transferred into a GC vial and 800 μL of hexane were added prior to GC analysis.

Tier II, At each sampling point, two tubes of the incubated 10 ml aliquots were taken out of the water bath, a small amount of sodium chloride was added in order to increase extraction efficiency and the solutions were extracted twice with each 2 mL of hexane. The united organic phases were
filled up to 5 mL with hexane. 200 μL of the organic phase were transferred into a GC vial andm800 μL of hexane were added prior to GC analysis

Tier III, Before and after incubation, aliquots of the worked up test solutions (as in the Tier I&II tests) at each pH value and temperature were analyzed by measuring the MS signal of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide and 3,3,5-trimethylcyclohexanone after GC separation of
the injected sample solution.
Buffers:
Buffer pH 4, Biphthalate J.T. Baker Art. No. 5657
Buffer pH 7, Phosphate J.T. Baker Art. No. 5656
Buffer pH 9, Borate J.T. Baker Art. No. 7145

All autoclaved prior to use.
Details on test conditions:
Tier I: According to the guideline, a preliminary hydrolysis test was performed at 50.0 °C ± 0.5 °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 and after 120 hours (corresponding to 5 days).For each pH value an aliquot of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide was dissolved in 100 mL of acetonitrile. 1 mL of this solution were transferred into a 10 mL
volumetric flask and filled with acetonitrile to obtain a stock solution. 3 mL of the stock solution were transferred into a 300 mL volumetric flask and
filled up to the mark with the respective buffer solution to prepare a sample solution. 10 mL aliquots were filled in test tubes in order to
perform a duplicate test (2 tubes were used at each sampling time) and incubated at 50.0 °C
± 0.5 °C.

Tier II: Due to the instability of the test item found in the preliminary test at 50.0 °C, further hydrolysis tests were performed at 20.0 °C, 30.0 °C and 50.0 °C in the buffered test solution at pH 4.0, pH 7.0 and pH 9.0. The test temperature was kept constant ±0.5 °C. To test for first order
behaviour the concentration of the test item in each test solution was determined immediately after preparation and at several time intervals.
For each temperature and pH value an aliquot of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide was dissolved in 100 mL of acetonitrile. 1 mL of this solution were transferred into a 10 mL volumetric flask and filled up with acetonitrile to obtain a stock solution. 3 mL of the stock
solution were transferred into a 300 mL volumetric flask and filled up to the mark with the respective buffer solution to prepare a sample solution. 10 mL aliquots were filled in test tubes in order to perform a duplicate test (2 tubes were used at each sampling time) and incubated at the respective
temperature.


TierIII: The potential main decomposition product of parent degradation in the test solutions is 3,3,5-trimethylcyclohexanone, which is also an
impurity of the test item. This substance was measured in the same manner as the tier II testing. Other potential degredation products tert-butanol, a
cetone, methane or carbon dioxide could not be measured with the analytical method in use and were not measured.
Number of replicates:
Analysis per time, temperature and pH point was always conducted in duplicate.
Positive controls:
not specified
Negative controls:
not specified
Statistical methods:
No data
Preliminary study:
The preliminary test (Tier 1) was performed with Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide at 50.0 °C ± 0.5 °C at each of pH 4.0, pH 7.0, and pH 9.0 in duplicate. The test solutions of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide were thermostated to 50.0 °C and analyzed at defined time intervals. Initial results demonstrated that the parent material was not stable at all tested pH. This was oringinally concluded to be due to hydrolysis. A Main
(Tier II) test was therefore conducted two ambient temperatures (20 °C and 30 °C) and 50 °C in order to calculate the rate constant (k25) and the half-life of
the hydrolysis at 25 °C.
Test performance:
No rate constant and half-life at pH 7.0 and pH 9.0 and 25 °C could be calculated by interpolation of the individual test temperatures. However, sufficiently reproducible values for half-lives and reaction rate constants were calculated for pH 7.0 and 9.0 assays at 20°C and so these are reported instead of the 25 °C calculated values. The half-life at pH 7.0 and 20 °C was calculated to be 96 hours (4.0 days) and at pH 9.0 and 20 °C to be 88 hours (3.7 days). The values for the elevated temperatures of 30 °C and 50 °C at pH 7.0 and pH 9.0 are not reproducible. The determination coefficients of linear regression were not sufficiently high at 30 °C at pH 7.0 (≥ 0.632) and pH 9.0 (≥ 0.100) and at 50 °C at pH 9.0 (≥ 0.518). Beyond that, the decrease at 30 °C was faster than at 50 °C for both pH values. The test item appears to undergo reactions which cannot be clearly explained at this time. However, as peroxides are known to thermally decompose even in aqueous solution at relatively low temperatures, it is possible that degradation rates determined above 20 °C were not strictly related to hydrolysis.
Transformation products:
yes
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
3,3,5-Trimethylcyclohexanone was the suspected primary breakdown product of the test substances. For this reason concentrations of this
substance were also measured throughout the studies at all pHs and temperatures. Further expected degradation products such as tert-butanol,
acetone, methane or carbon dioxide could not be identified by the GC method used. The concentration of this potential degradation product increased or remained similar with time depending on the pH and temperature. In brief, 3,3,5-trimethylcyclohexanone increased at all temperatures at pH 4.0, at 30 °C and 50°C but not at 20°C at pH 7.0 and at 50°C only at pH 9.0. This is a further indication that the reactions occurring at 20-30°C cannot be related to the reaction mechanisms at higher temperatures. The percent increase also varied between samples within the same pH and temperature.
Details on results:
No conclusive evidence of hydrolysis can be obtained from this data. At higher temperatures (i.e. 50°C) elements of degradation appear to take place but cannot be exclusively attributed to hydrolysis. At pH 4, increasing concentrations of the principal degradation product, trimethylcyclohexanone, at 20, 30 and 50°C were obsereved, consistent with the hypothesis that hydrolytical degradation was occurring at this low pH. However, as the experiment was performed at concentrations orders of magnitude above the true solubility, any results on parent compound loss with time are likely to be more related to emulsion stability than hydrolysis during this study. Therefore no quantitative determination of hydrolysis can be proposed and the available evidence from this study suggests that, if it occurs, hydrolysis will be limited to lower pHs.

Due to the uncertainties highlighted by this study additional testing on test substance solubility was commissioned which confirmed that the hydrolytical half-life was, at best, no less than 400 hours at pHs 7 to 8 and 20°C.

Due to the inconclusive results a series of additional analytical investigations involving solubility and degradation product testing were subsequently conducted to assist with interpretation of hydrolysis data as well as to find the optimum conditions for aquatic studies. The data from these investigations demonstrated that the water solubility of the test substance was approximately 2 μg/L (Harlan 2013 Report Number 41203654 and Harlan 2013 Report Number 41206896). These summaries can be found under section 4.8 water solubility. Therefore this hydrolysis study had actually been performed well above the water solubility level of the test substance (approximately 2 μg/L) and hydrolysis / degredation values measured during hydrolysis testing are likely to reflect emulsion stability data as well as aspects of thermal and possibly some hydrolytical degradation.

Validity criteria fulfilled:
no
Remarks:
validity is limited due to the testing being conducted above the solubility limit of the substance.
Conclusions:
This study does provide information regarding the behaviour of the test substance with changing pH and temperature. However due to the test
being conducted way in excess of the solubility limit and inconclusive results being observed it is not possible to attribute these effects to hydrolysis although there is some evidence that hydrolysis does occur at pH4. This study cannot be used to provide quantitative valuse of hydrolysis for this substance.
Executive summary:

Guideline Tier I, II and III hydrolysis studies were conducted in a GLP laboratory to relevant test guidelines. At this time the water solubility of the test substance was believed to be 0.5 mg/L and therefore the study appeared to meet the validity criteria. However, several problems occurred during the performance of the study including inexplicable differences in slope compared to temperature, substance loss without any significant increase in degradation product and inconsisitencies between starting concentrations prepard in the same way. Thus further studies were carried out to determine the true water solubility of the test substance which confirmed the tendancy for the material to form temporarily stable emulsions and demonsrated that the true water solubility was nearly two orders of magnitude lower than the concentrations used in the hydrolysis studies which therefore can lon longer be considered to comply with the test guideline. This study cannot be used to provide quantifiable evidence of hydrolysis. Nevertheless, as one of the major expected degradation products was observed to increase in certain cases this can be reviewed qualitatively:

Increase in the major degradation product, trimethylcyclohexanone, was not observed at pH 7 and 9 except at 50°C. The explanation for this is that thermal dgradation of the substance will occur (also in water) at 50°C but not at the lower temperature used in the studies and this cannot be considered to be hydrolysis in the true sense of the term and moreove cannot be extrapolated to environmentally relevant situations.

At pH4 trimethylcyclohexanone concentrations increased over the study at all temperatures used. Thus it is likely that the substance hydrolyses, perhaps even rapidly, at pH4, but as the concentration of the the test substance used was close to 100 µg/L while the solubility is two orders of magnitude lower, no quantitative determination of hydrolysis rate can be proposed.

Endpoint:
hydrolysis
Type of information:
experimental study
Remarks:
Study designed to adress existing contradictory data points
Adequacy of study:
key study
Study period:
October-November 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted to repeat doubtful result in existing data pH& and a single temperature
Justification for type of information:
On the basis of a higher water soubility measurment existing hydrolysis data was reviewed. Parent material decrease without formation of hydrolysis products was observed in a few cases (Stoob 2014). In order to clarify the status of the parent material a hydrolysis study was repeated at 20ºC in pH buffer to establish if data could be reproduced.
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
2004
Deviations:
yes
Remarks:
Modified for a particular temperature and pH
Principles of method if other than guideline:
There were four 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 sterilized also.
· A single test temperature of 20 °C was used. At the temperature of 50°C, as mentioned in OECD
111, the test substance is not thermally stable, as it is too close to the SADT (self accelerating
decomposition temperature) (MSDS, 2015).
· In addition to the standard OECD guideline buffer, demineralized water containing humic acid was
tested.
· Not all pH buffers as indicated in the OECD guideline were tested as these had already been
conducted in previous studies. This study focused on pH 7 to allow the calculation of an
environmentally relevant half-life for PBT assessment and compare/investigate existing conflicting
data (Stoob 2014).
GLP compliance:
no
Remarks:
Test conducted repeated conditions observed during previous GLP hydrolysis studies to establish if degradation was taking place.
Specific details on test material used for the study:
Chemical name: 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane
EC name: Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide
Trade name: Trigonox 29
CAS: 6731-36-8
Batch/Lot no.: 1504447036
Water solubility: 93 μg/L (Akzo Nobel, 2017)
Composition: see CoA (Annex 1)
Storage: 10°C

Certirficate of analysis included
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
The sterilised buffer pH 7 and demineralized water containing humic acid were transferred to volumetric
flaks and purged with nitrogen for at least 5 minutes.

A stock solution of the test substance was prepared in acetonitrile. This stock solution was spiked to
the volumetric flasks (purged with nitrogen) and filled up with the appropriate pH buffer or test media,
resulting in an initial concentration of 45 μg/L test substance. Subsequently 10 mL of the spiked test
solutions were transferred by pipette to multiple sterile glass test vials, 20 mL glass headspace vials
with PTFE seal screw tops. The vials were closed tightly and placed in a thermostatically controlled
water bath in the dark at a temperature of 20 ± 0.5°C. At the moment the test vials were placed in the
water bath, the first sample was taken and analyzed using the analytical method described in Annex 2.
Subsequent samples were taken on different time intervals and analyzed to determine the percentage
of hydrolysis. Samples were analyzed directly after sampling in order to prevent further hydrolysis. To
check the pH of the buffer solution and test media at the actual test temperature, a separate glass bottle
for each buffer/test solution was filled with an aliquot of the buffer/test solution, without test substance,
and placed in the thermostatically controlled water bath (at the test temperature). After equilibration with
the temperature of the water bath, the pH of this bottle was checked
Buffers:
7.0 pH 29.63 ml 0.1N Sodium hydroxide + 50 mL 0.1M mono Potassium phosphate
Estimation method (if used):
Arrhenius Relationship
Details on test conditions:
The test was performed for pH buffer 7 and demineralized water containing humic acid at a temperature
of 20 °C in the dark.
Duration:
99 d
pH:
7
Temp.:
20 °C
Initial conc. measured:
35.1 µg/L
Number of replicates:
8 samples taken over 99 days
Positive controls:
no
Negative controls:
no
Statistical methods:
Calculated according to guideline
Preliminary study:
N/A
Test performance:
N/A
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Hydrolysis products were scanned using GCMS in an effeort to identify any formed products. No distinguishable hydrolysis products were visible above the baseline.
pH:
7
Temp.:
20 °C
Hydrolysis rate constant:
0 h-1
DT50:
63 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other:
Remarks:
Hydrolytically stable
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): sampling without leaching solution leads to loss on glass. Samples were extracted from vials with hexane so no adsorbance loss could occur.

Results with reference substance:
N/A

Test substance recovery during study:

Sample name

 

Time

hrs

Concentration (Ct)

Hydrolysis

µg/L

%

pH 7 T=0

0

35.1

0.0

pH 7 T=24h

24

31.5

10.3

pH 7 T=96h

96

24.8

29.4

pH 7 T=8d

192

26.0

26.0

pH 7 T=18d

432

25.1

28.7

pH 7 T=55d

1320

16.1

54.3

pH 7 T=76d

1824

16.4

53.3

pH 7 T=99d

2376

7.3

79.2

Validity criteria fulfilled:
yes
Remarks:
Study applicable only in support of key endpoint and to address contradictory historical data
Conclusions:
Rapid hydrolysis in historical data observed under the same conditions as tested in this study are concluded not to have been caused by hydrolysis and likely by adsorption. The test material is not considered rapidly hydrolyzing but hydrolytically stable under these conditions . Some loss did occur without the detection of accompanying hydrolysis products being possible. Indicating some loss by hydrolysis cannot be excluded.
Executive summary:

Adapted form of guideline was followed and substance identification was acceptable. Reliable with restrictions to be used as supporting evidence together with other data.

Description of key information

Preliminary tests apparently showed rapid potential for hydrolysis but final studies were originally found to be of limited validity when the water solubility of the substance proved to be lower than expected. Reconfirmation of the water solubility resulted in a slightly higher figure due to loss by adsorption being poorly corrected for by contract labs. Hydrolysis studies was therfore re-assessed. Hydrolysis study Stoob (2014) showed some evidence of hydrolysis to expected degradation products at higher temperatures. At lower temperatures none of the studies provided conclusive information on hydrolysis as no hydrolyisis products could be detected when parent concentration decreased at lower temperatures. An environmentally relevant endpoint was chosen at 20ºC pH7 that had shown a decrease in parent concentrations Stoob (2014). This endpoint was repeated to see if the data could be reproduced (Dam 2017). Reductions in parent concentrations were also observed. However if the test vessels were extracted completely and the samples taken in such a way as not to loose the test material significantly higher concentrations of parent material were recovered by Dam 2017. Indicating that the apparent rapid removal in older studies was not due to hydrolysis. Some loss was observed by Dam 2017 but this data is considered to best account for the difficult test material properties and give the most realistic hydrolysis half-life under environmental conditions.

 

Key value for chemical safety assessment

Half-life for hydrolysis:
63 d
at the temperature of:
20 °C

Additional information

In early studies, it was believed that rapid hydrolysis of the parent substance had been observed. This was further tested in Harlan, (2010) however, results were conflicting and after multiple attempts the final test was abandoned in the light of new solubility data which showed that all hydrolysis studies had been performed well above the water solubility level of the test substance (then thought to be 2 µg/L) Further work during solubility studies (Mead, 2013; Mullee, 2013) clarified that the expected major degradation product, trimethylcyclohexanone, was present as an impurity throughout the study but did not increase. Therefore the substance is considered hydrolytically stable at pH7 -8.

Dam (2017) further clarified the problematic endpoints in stoob (2014) and demonstrated that at lower temperatures the test material was not degrading rapidly and that degradation at higher temperatures was likely not caused by hydrolysis. Loss did occur but not as rapidly as originally indicated in earlier studies. Nevertheless, as one of the major expected degradation products was observed to increase in certain cases the following information should be considered:

Increase in the major degradation product, trimethylcyclohexanone, was not observed at pH 7 and 9 except at 50°C. The explanation for this is that thermal degradation of the substance occurs (also in water) at 50°C but not at the lower temperature used in the studies and this cannot be considered to be hydrolysis in the true sense of the term and moreover cannot be extrapolated to environmentally relevant situations.

At pH4 trimethylcyclohexanone concentrations increased over the study at all temperatures used. Thus it is likely that the substance hydrolyses, perhaps even rapidly, at pH4 but not under environmentally relevant condictions. Data from (Dam2017) will therefore be used for the environmental risk assessment due to this being generated at what is considered to be the most accurate water solubility value and without the deficiencies of the other existing hydrolysis data.

Furthermore the lack of rapid biodegradation suggests hydrolytical stability. The current half life determination of 63 days also corresponds better with the existing biodegradation data.