2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 19 January 2017. Experimental completion date: 27 May 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8)
Appearance/physical state: extremely pale yellow slightly viscous liquid
Batch: 1609501020
Purity: 94.5%
Expiry date: 05 October 2017
Storage conditions: approximately 4 °C, in the dark

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The sample solutions were taken from the waterbath at various times and the pH of each solution recorded.
Duplicate aliquots (A and B) of each sample solution were diluted by a factor of 5 using a diluent of acetonitrile containing 0.1% v/v methanesulfonic acid.

Buffers:

Buffer for pH 1.2: Potassium chloride (0.05 mol dm-3) hydrochloric acid (0.07 mol dm-3)
Buffer for pH 4: Citric acid (0.06 mol dm-3) sodium chloride (0.04 mol dm-3) sodium hydroxide (0.07 mol dm-3)
Buffer for pH 7: Disodium hydrogen orthophosphate (anhydrous) (0.03 mol dm-3) Potassium dihydrogen orthophosphate (0.02 mol dm-3) Sodium chloride (0.02 mol dm-3)

These solutions were subjected to ultrasonication and degassing with nitrogen to minimize dissolved oxgen content.

Details on test conditions:

Test System
The determination was carried out using a procedure designed to be compatible with Method C.7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.
The test system consisted of sterile buffer solutions at pH’s 1.2, 4, 7 and 9.

Preparation of the Test Solutions
Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 0.100 g/L in the three buffer solutions. A 1% co-solvent of acetonitrile was used to aid solubility.
The test solutions were split into individual vessels for each data point (such that two independent, separate vessels were removed for analysis at each incubation timepoint).
The solutions were shielded from light whilst maintained at the test temperature.

Preliminary Test
Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5 °C for a period of 120 hours for preliminary test 1.
A second repeat preliminary test (preliminary test 2) was also performed at pH 7 and pH 9 with the adaptation that the polymerization inhibitor 4-methoxyphenol was added to the buffer solutions at a nominal concentration of 2.0 g/L in an attempt to evaluate hydrolysis in isolation, should polymerization be influencing the behaviour observed.

Evaluation Directly at 25 °C
Results from the preliminary tests showed it was necessary to undertake further testing at pH 7 and pH 9, with solutions being maintained at 25.0 ± 0.5 °C for a period of at least 30 days.

Testing at pH 1.2 and 37 °C
Sample solutions at pH 1.2 were maintained at 37.0 ± 0.5 °C for a period of 24 hours to evaluate the behaviour of the test item under physiologically relevant (gastric) conditions.

Duration:

120 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.092 g/L

Remarks:

preliminary test

Duration:

32 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.107 g/L

Duration:

32 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.085 g/L

Duration:

24 h

pH:

1.2

Temp.:

37 °C

Initial conc. measured:

0.1 g/L

Duration:

120 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.101 g/L

Remarks:

preliminary test

Duration:

120 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.085 g/L

Remarks:

preliminary test

Number of replicates:

carried out in duplicate at all pH ranges

Positive controls:

no

Negative controls:

no

Preliminary study:

At pH 4
Less than 10% hydrolysis after 5 days at 50 °C, equivalent to a half-life greater than 1 year at 25 °C. No further testing required.

At pH 7 without polymerization inhibitor
The extent of the decrease in test item concentration after 120 hours and in particular the non-pseudo first order kinetics observed indicated that further testing was required, with the preliminary test being repeated with a polymerization inhibitor present in the test system.

At pH7 with polymerization inhibitor
The extent of the decrease in test item concentration after 120 hours confirmed non-pseudo first order kinetics remained despite modifying the test system; indicating that the Arrhenius plot method could not be employed to evaluate hydrolysis characteristics of the test item at environmentally relevant temperatures. Further testing was therefore performed evaluating the test item directly at 25 °C.

At pH9 without polymerization inhibitor
The extent of the decrease in test item concentration after 120 hours and in particular the non-pseudo first order kinetics observed indicated that further testing was required, with the preliminary test being repeated with a polymerization inhibitor present in the test system.

At pH9 with polymerization inhibtitor
The extent of the decrease in test item concentration after 120 hours confirmed non-pseudo first order kinetics remained despite modifying the test system; indicating that the Arrhenius plot method could not be employed to evaluate hydrolysis characteristics of the test item at environmentally relevant temperatures. Further testing was therefore performed evaluating the test item directly at 25 °C.

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on hydrolysis and appearance of transformation product(s):

Rapid Hydrolysis in Water
The Sponsor reported evidence of the test item hydrolysing rapidly in the presence of water, in which it was proposed that 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate hydrolysed to form 2-[(2-hydroxyethyl)amino]ethyl 2-methylacrylate and Cyclohexanone.
Further reaction of this primary hydrolysis product was then proposed as a two stage reaction involving initial hydrolysis of the ester functional group followed by a condensation reaction between the resulting secondary amine containing diol, and carboxylic acid, to yield N,N-bis(2-hydroxyethyl)-2-methylacrylamide.

Evaluation of this proposal was performed. During the determination of the water solubility of the test item, no parent test item was detected on analysis of any sample solutions, with the only peak detected being attributed to a highly soluble hydrolysis product. Although it was not possible to confirm absolute structures for individual species during HPLC-MS analysis of subsequent sample solutions prepared; the observed behaviour of the test item in water, as well as all analytical data generated, was in full support of this proposal.
Nuclear magnetic resonance (NMR) data generated by the Sponsor shows the absence of any parent compound in aqueous solutions, as well as further transformation of the initial dissolved products.

% Recovery:

98.3

pH:

4

Temp.:

50 °C

Duration:

120 h

% Recovery:

18.7

pH:

7

Temp.:

25 °C

Duration:

768 h

% Recovery:

89.9

pH:

9

Temp.:

25 °C

Duration:

768 h

% Recovery:

94.7

pH:

1.2

Temp.:

37 °C

Duration:

24 h

Key result

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

237 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

197 d

Type:

(pseudo-)first order (= half-life)

Details on results:

pH 7 at 25.0 ± 0.5 ºC
On reducing the incubation conditions to the environmentally relevant temperature of 25 °C, the decrease in test item concentration as a function of time largely fitted a pseudo-first order kinetics mechanism for at least two half-lives, i.e. a reduction of greater than 75% of the initial concentration. As a result of this, it has been possible to provide an estimation of the reaction kinetics from statistical analysis of the data across the first 480 hours (20 days) monitored. This resulted in a first order correlation coefficient (r) of -0.995.
kobs = 2.92 x 10-3 hour-1
= 8.12 x 10-7 second-1
t½ = 237 hours

pH 9 at 25.0 ± 0.5 ºC
On reducing the incubation conditions to the environmentally relevant temperature of 25 °C, the decrease in test item concentration as a function of time fitted a pseudo-first order kinetics mechanism for a period of approximately 30 days (albeit only an approximately 10% decrease in concentration, however the maximum permitted test period is limited within the regulatory method guidelines). As a result of this, it has been possible to provide an estimation of the reaction kinetics from statistical analysis of the data which resulted in a first order correlation coefficient (r) of -0.993.
kobs = 1.47 x 10-4 hour-1
= 4.08 x 10-8 second-1
t½ = 197 days

pH 1.2 at 37.0 ± 0.5 ºC
Under the physiologically relevant (gastric) conditions of pH 1.2 and an incubation temperature of 37 °C, no significant secondary hydrolysis (<10%) was observed over a period of 24 hours.

Discussion

The test item is described by the Sponsor as a methylacrylate monomer with a oxazolidine group, for which the potentially reactive hydroxyl and amine groups are temporarily blocked with cyclohexanone. Essentially spontaneous hydrolysis of the test item on contact with aqueous media was therefore proposed by the Sponsor, with the release of cyclohexanone and exposure of the hydroxyl and amine functional groups as detailed in the details on hydrolysis and appearance of transformation products section. This degree of reactivity was evaluated and supported under Envigo Study Number MK30PC.

As standard solutions within this study were also generated in aqueous media, in fact it can be concluded that hydrolysis of the parent test item was complete in all solutions prior to analysis even being possible; with the actual chemical species under quantification tentatively attributed to the primary hydrolysis product detailed in

details on hydrolysis and appearance of transformation products section

of this report. Critically this also presents the dissolved species originating from the test item on accidental release in the environment, due to the extremely rapid rate of the initial hydrolysis, and as such, the further transformation of this compound under environmentally and physiologically representative pH values and temperatures remain both relevant and valid for regulatory consideration.

The kinetics of the study at 25 °C have been determined to be consistent with that of an essentially pseudo-first order reaction. This was since the graphs of log10 concentration versus time were straight lines over a period of two half-lives at pH 7 and for the maximum permitted incubation time (30 days) at pH 9. However, it must be noted that the hydrolysis characteristics showed significant second order trends when evaluated at an elevated

temperature. For this reason, it was considered not to be possible to undertake testing at multiple temperatures and apply the Arrhenius relationship to the resulting plots and hence determinations were performed directly at the test temperature of 25 °C.

For the 25 °C tests, a timepoint was also taken for both pH at 720 hours but on availability of the analytical data it was strongly suspected that the pH 7 and pH 9 sample solutions were transposed in error at the time of analysis. On re-dilution and re-analysis of these sample solutions, this error was confirmed, however by this time the solutions had been removed from the waterbath for a significant period of time and were no longer considered either valid or representative of the relevant timepoint.

On plotting the logarithmically transformed concentration data for the pH 9, 25 °C test, two apparently erroneous data points were noted, specifically elevated concentrations at the initial timepoint and the 240 hour timepoint. In order to prevent excessive and inappropriate steerage of the correlation, these two data points were excluded from the final correlation fitted to the data as a whole. These exclusions were not considered to significantly impact on the validity of the estimated half-life and rate constant since seven valid data points remained, exceeding the guideline minimum requirement of six.

No significant peaks were observed at the approximate retention time of the test item on analysis of any matrix blank solutions.

Validity criteria fulfilled:

yes

Conclusions:

The test item was concluded to undergo essentially spontaneous hydrolysis on dissolution in aqueous media at pH 4, pH 7 and pH 9, with no parent test item being detected on analysis of even the initial timepoint samples. With reference to the proposed hydrolysis pathway detailed in details on hydrolysis and appearance of transformation products section, the primary hydrolysis product, which also presents the dissolved species originating from the test item on accidental release in the environment, then underwent further hydrolysis with results as follows:
pH Rate Constant (s-1) Estimated half life at 25 °C for second step of the hydrolysis pathway
4 Not applicable >1 year
7 8.12 x E-7 237 hours
9 4.08 x E-8 197 days

Under the physiologically relevant (gastric) conditions of pH 1.2 and an incubation temperature of 37 °C, no significant secondary hydrolysis (<10%) was observed over a period of 24 hours.

Executive summary:

The hydrolysis characteristics of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS 4203-89-8), as a function of pH, have been determined. Testing was carried out using a procedure designed to be compatible with Method C.7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.

The test item was concluded to undergo essentially spontaneous hydrolysis on dissolution in aqueous media at pH 4, pH 7 and pH 9, with no parent test item being detected on analysis of even the initial timepoint samples. With reference to the proposed hydrolysis pathway detailed in details on hydrolysis and appearance of transformation products section, the primary hydrolysis product, which also presents the dissolved species originating from the test item on accidental release in the environment, then underwent further hydrolysis with results as follows:

pH	Rate Constant (s^-1)	Estimated half life at 25 °C for second step of the hydrolysis pathway
4	not applicable	>1 year
7	8.12 x 10^-7	237 hours
9	4.08 x 10^-8	197 days

Under the physiologically relevant (gastric) conditions of pH 1.2 and an incubation temperature of 37 °C, no significant secondary hydrolysis (<10%) was observed over a period of 24 hours.

Description of key information

The test item was concluded to undergo essentially spontaneous hydrolysis on dissolution in aqueous media at pH 4, pH 7 and pH 9, with no parent test item being detected on analysis of even the initial timepoint samples.