1,6-hexanediyl-bis(2-(2-(1-ethylpentyl)-3-oxazolidinyl)ethyl)carbamate

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-24

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:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Publication no. L 383 A, November 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2110 (Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on test conditions:

Preparation of the stock solution 1:
About 1.5 g of the test item were exactly weighed into a 10 mL measuring flask, dissolved in acetonitrile and filled to the mark with acetonitrile.

Preparation of the test solution 1:
333 μL of stock solution 1 were added to 100 mL of acetonitrile : water (1:1), giving a mixture with a concentration of 0.8 mmol/L.
After adding the stock solution 1, 20 mL of the received solution were extracted with 2 mL diethylether, after extraction on a mechanical shaker and centrifugation the organic layer was dried with sodium sulphate and analysed by GC-MS. The acetonitrile-water phase was investigated by HPLC-MS.

Preparation of the test solution 2:
333 μL of stock solution 1 were added to 100 mL of demineralized water, giving a mixture with a concentration of 0.8 mmol/L.
After adding the stock solution 1, an immediate clouding of the solution took place. The precipitate covered the flask wall and was dissolved in 10 mL acetonitrile after decanting the aqueous phase. The acetonitrile phase was investigated by GC-MS and the decanted aqueous phase was investigated by HPLC-MS.

Number of replicates:

No data available.

Negative controls:

not specified

Preliminary study:

Results HPLC-MS:
In both aqueous phases two signals were detected, the first signal with Rt = 3.5 min and a molecular peak of m/z = 216 and a second signal with Rt = 7.8 min and a molecular peak of m/z = 379. As the ionization was performed in the positive mode, the molecular mass of the first component is: Mw = 215, corresponding to the minor component 2-(1-Ethylpentyl)-3-(2-hydroxyethyl)-oxazolidine. The molecular mass of the second component is: Mw = 378, corresponding to the main hydrolysis product 1,6 hexanediyl-bis-carbamic acid-bis-2-amino((2-hydroxy)-ethyl)-ethylester. These two components were detected in the stock solution (hydrolysis of the main product already takes place during chromatography with an eluent consisting of water / acetonitrile) and in the aqueous phases after adding stock solution 1 to water / acetonitrile (1/1) or to demineralised water.

Results GC-MS:
GC-MS investigation of the diethylether extract (from test mixture 1) and the precipitate (from text mixture 2) , dissolved in acetonitrile only showed one major signal with a retention time of 7.9 min, identified by comparison with the Wiley 275.L mass spectra data base as 2-Ethyl-hexanale, the first hydrolysis product. Substances at Rt 14.1 and 22.0 min are artefacts.

Transformation products:

yes

No.:

#1

No.:

#2

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

According to the detection of the hydrolysis products, the following hydrolysis process takes place:
1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate (test item) -> 2 2-ethylhexanal + 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester

Key result

Remarks on result:

not determinable

Other kinetic parameters:

Due to the fast hydrolysis no further tests for the hydrolysis were required.

Details on results:

The 2-Ethyl-hexanale is insoluble in water and forms droplets at the flask walls (test mixture 2). It is soluble in organic medium (diethylether and acetonitrile) and was therefore detected by GC-MS.
According to EPI-Suites WSKOW v 1.40 (calculation program of the EPA for water solubilities) the water solubility of the second hydrolysis product 1,6 hexanediyl-bis-carbamic acid-bis-2-amino((2-hydroxy)-ethyl)-ethylester is 15.8 g/L. This product was therefore detected by HPLC-MS in the aqueous phase. Hydrolysis takes place immediately after contact with water. No test item 1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate could be detected.
Due to the fast hydrolysis no further tests for the hydrolysis were required.

Validity criteria fulfilled:

yes

Conclusions:

The hydrolysis of the test item (1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate) was investigated using HPLC-MS and GC-MS. Measurements revealed a fast hydrolysis of the test item. The hydrolysis degradation products of the test item were identified qualitatively. The first hydrolysis product was determined to be 2-ethylhexanal. The second hydrolysis degradation product was identified on the basis of its molecular weight. The second, more likely hydrolysis product, which is in good consistency with the experimentally determined molecular weight, is 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester.

Executive summary:

The hydrolysis of the test item (1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate) and the identification of the decomposition products were assessed in accordance with the OECD guideline 111 and the EU method C.7.

The test item immediately hydrolysed in water and the formed hydrolysis products are partly insoluble in water as well. To investigate the hydrolysis process a stock solution of the test item in acetonitrile was prepared. This solution was added to a mixture of acetonitrile : water (1:1) and to water resulting in test mixtures of 0.8 mmol/L test item in acetonitrile : water (1:1) and in water. The acetonitrile / water mixture was investigated by HPLC-MS and its diethylether extract was investigated by GC-MS.

After adding the acetonitrile stock solution to water, an immediate precipitation was observed. An organic extract of the mixture was investigated by GC-MS and the aqueous phase was investigated by HPLC-MS. By GC-MS analysis of both extracts only one signal was detected, identified by comparison with the Wiley 275.L mass spectra data base as 2-Ethyl-hexanale, the first hydrolysis product. The HPLC-MS analysis of both aqueous mixtures showed two signals, one of the minor component 2-(1-Ethylpentyl)-3-(2-hydroxyethyl)-oxazolidine and of the hydrolysis product 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester. The 2 -ethylhexanal is insoluble in water and forms droplets at the flask walls. It is soluble inorganic medium (diethylether and acetonitrile) and was therefore detected by GC-MS. Hydrolysis takes place immediately after contact with water. No test item 1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate could be detected.

Additional remark:

In the course of the GC-MS analysis in the present hydrolysis study the first hydrolysis product was clearly identified as 2-ethyl-hexanale. The other, second hydrolysis product was detected using HPLC-MS. For the second hydrolysis product the molecular weight (Mw) was reported to be 378 g/mol. However, the postulated hydrolysis reaction considering these hydrolysis products seems to be not correct, e.g. in particular regarding the completeness and the mass balance in chemical reactions, i.e. the postulated chemical structure of the second hydrolysis product is not consistent with the experimentally determined molecular weight (378 g/mol). Thus, considering 2-ethyl-hexanale as one of the hydrolysis degradation product, the other hydrolysis degradation product, which is more likely, is 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester with a calculated molecular weight of 376.5 g/mol.

Reference 2

Endpoint:

hydrolysis

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2011-01-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No GLP and guideline study with read-across substance, but detailed and well-described analytical method available and the study was conducted in equivalence conditions to good laboratory praxis. Read-across from a similar product to show mechanistic aspects of hydrolysis.

Principles of method if other than guideline:

Hydrolysis tests at pH 4, 7 and 9 using FIMS (Flow Injection Mass Spectrometry) were performed. The hydrolysis tests indicated, how fast and completely the test item was degraded in presence of water (pH 4, 7 and 9), and if the hydrolysis reaction was acid-catalyzed. Therefore, short- and long-term measurements were performed.

GLP compliance:

yes

Remarks:

the study was conducted in equivalence conditions to good laboratory praxis

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The sample solution (100 μg/mL, dissolved in acetonitrile) was injected by a 500 μL syringe directly into the mass spectrometer at 10 μL/min (APCI, positive ion-mode).
- Instrument: LTQ-Orbitrap XL
- Resolution: 60000
- Tune: APCILF10_10-507_267.1.LTQTune

Buffers:

- Buffer at pH = 4, 7, 9

Details on test conditions:

HPLC-APCI-MS measurements:
Test item (non-hydrolysed sample):
For the HPLC-APCI-MS measurements test test item was dissolved and diluted in methanol (100 μg/mL).
- Column: Hypersil ODS (C18) 3u, 150 x 4.6 mm, Alltech
- Solvent: D: 1 mM NH4HCOO in MeOH
- Flow rate: 800 μL/min.
- Injection volume: 10 μL
- Instrument: LTQ-Orbitrap XL
- Resolution: R = 60000
- Detection: APCI, pos. Ion Mode, Full scan [m/z: 80-1200]
- Tune-Method: APCIHF500_10-692_ACN.LTQTune

Test item (hydrolysed sample):
For the HPLC-APCI-MS measurements the test item was dissolved in ACN, diluted and hydrolysed in 0.1 % TFA in water : 0.1 % TFA in ACN = 80:20 (10 μg/mL).
- Column: Synergi 4u Hydro-RP 80A, 75 x 4.6 mm (Phenomenex)
- Solvents: A: 0.1 % trifluoroacetic acid (TFA) in water; B: 0.1 % trifluoroacetic acid (TFA) in ACN
- Gradient: [min] / % A / % D
0.0 / 95 / 5
1.0 / 95 / 5
6.0 / 0 / 100
8.0 / 0 / 100
8.5 / 95 / 5
12.0 / 95 / 5
- Flow rate: 600 μL/min
- Injection volume: 10 μL
- Instrument: LTQ-Orbitrap XL (high resolution mass spectrometer)
- Resolution: R = 60000
- Detection: APCI, positive ion mode, full scan [m/z: 80-1200]

Duration:

0.017 min

pH:

4

Initial conc. measured:

100 other: µg/mL

Duration:

0.017 min

pH:

7

Initial conc. measured:

100 other: µg/mL

Duration:

0.017 min

pH:

9

Initial conc. measured:

100 other: µg/mL

Duration:

2 min

pH:

4

Initial conc. measured:

1 000 other: μg/mL

Duration:

18 h

pH:

4

Initial conc. measured:

1 000 other: μg/mL

Duration:

2 min

pH:

7

Initial conc. measured:

1 000 other: μg/mL

Duration:

18 h

pH:

7

Initial conc. measured:

1 000 other: μg/mL

Duration:

2 min

pH:

9

Initial conc. measured:

1 000 other: μg/mL

Duration:

18 h

pH:

9

Initial conc. measured:

1 000 other: μg/mL

Positive controls:

not specified

Negative controls:

not specified

Test performance:

FIMS:
The mass spectrum of the test substance sample showed the expected mass at m/z = 487.34811. Based on the accurate mass measured, the chemical formula of C24H46O6N4 and 4 ring double bond equivalents (RDB’s: 2 double bonds and two rings) were calculated for the neutral molecule.
The mass spectrum of the main component of the test substance (MS/MS of m/z = 487.35) showed two fragments at m/z = 415.29150 and 372.24933.

Transformation products:

yes

No.:

#1

Details on results:

The short term measurements indicated that the test substance hydrolysed faster at pH 4 than at pH 7 and 9.
The long term measurements indicated that the hydrolysis reaction occured at pH 4, 7 and 9 within a few minutes.
The compounds (partially hydrolysed test substance; [MH+] = 433.3 and fully hydrolysed test substance, [MH+] = 379.3) and isobutyraldehyde (CAS 78-84-2) were formed during the hydrolysis reaction of the test item. Furthermore, fragments of fully hydrolysed tets substance with m/z = 274.2 and m/z = 143.1 were observed.

HPLC-APCI-MS results:
- Test item (non-hydrolysed sample):
The HPLC-MS measurements showed, that only a very small amount of hydrolysed products are present in test substance. Due to its very low hydrolysis stability, a water free mobile phase has to be used to separate the test substance properly. Under these conditions no chromatographic method could be developed to separate the main compound from other compounds except its hydrolysis products. Therefore, the test substance was completely hydrolysed and a chromatographic method was developed to separate the hydrolysed main compound from its potential by-products.

- Test item (hydrolysed sample)
The mass spectrum of the fully hydrolysed test substance showed the expected mass at m/z = 379.25360 (corresponding to the fully hydrolysed test item). Based on the accurate mass measured, the chemical formula of C16H34O6N4 and 2 ring double bond equivalents (RDB’s: 2 double bonds) were calculated for the neutral molecule.
Based on the extracted ion chromatograms no mono and non-hydrolysed test substance could be observed. Traces of diethanolamine (m/z = 106.1) were found.

- FIMS data showed that at pH = 4 after 2 minutes and 18 hours no signals of the test substance could be detected. Thus the hydrolysis half-live at pH = 4 was considered to be < 2 minutes.

- FIMS data showed that at pH = 7 after 2 minutes and 18 hours no signals of the test substance could be detected. Thus the hydrolysis half-live at pH = 7 was considered to be < 2 minutes.

- FIMS data showed that at pH = 9 after 18 hours no signals of the test substance could be detected. Thus the hydrolysis half-live at pH = 9 was considered to be < 18 h.

Conclusions:

The hydrolysis of the structurally analogue (read-across) substance (bis[2-[2-(2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexane-1,2-diylbiscarbamate) at pH = 4, 7 and 9 was investigated using FIMS (Flow Injection Mass Spectrometry). Short- and long-term measurements show that the test substance hydrolysed faster at pH 4 than at pH 7 and 9 and that the hydrolysis reaction occured at pH 4, 7 and 9 within a few minutes.

Executive summary:

The hydrolysis of the structurally analogue (read-across) substance (bis[2-[2-(2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexane-1,2-diylbiscarbamate) at pH = 4, 7 and 9 (at room temperature) was investigated using FIMS (Flow Injection Mass Spectrometry). Short- and long-term measurements show that the analogue substance hydrolysed faster at pH 4 than at pH 7 and 9 and that the hydrolysis reaction occured at pH 4, 7 and 9 within a few minutes. The compounds (partially hydrolysed test substance; [MH+] = 433.3 and fully hydrolysed test substance, [MH+] = 379.3) and isobutyraldehyde (CAS 78-84-2) were formed during the hydrolysis reaction of the test item. Furthermore, fragments of fully hydrolysed tets substance with m/z = 274.2 and m/z = 143.1 were observed.

FIMS data showed that at pH = 4 and 7 after 2 minutes and 18 hours no signals of the read-across substance could be detected. Thus, the hydrolysis half-lives at pH = 4 and 7 were considered to be < 2 minues. At pH = 9 after 18 hours no signals of the test substance could be detected. Thus the hydrolysis half-live at pH = 9 was considered to be < 18 h.

Description of key information

The hydrolysis of the test item (1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate) was investigated using HPLC-MS and GC-MS. Measurements revealed a fast hydrolysis of the test item. The hydrolysis degradation products of the test item were identified qualitatively. The first hydrolysis product was determined to be 2-ethylhexanal. The second hydrolysis degradation product was identified on the basis of its molecular weight. The second, more likely hydrolysis product, which is in good consistency with the experimentally determined molecular weight, is 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester.

Additional information

The hydrolysis of the test item (1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate) and the identification of the decomposition products were assessed in accordance with the OECD guideline 111 and the EU method C.7.

The test item immediately hydrolysed in water and the formed hydrolysis products are partly insoluble in water as well. To investigate the hydrolysis process a stock solution of the test item in acetonitrile was prepared. This solution was added to a mixture of acetonitrile : water (1:1) and to water resulting in test mixtures of 0.8 mmol/L test item in acetonitrile : water (1:1) and in water. The acetonitrile / water mixture was investigated by HPLC-MS and its diethylether extract was investigated by GC-MS.

After adding the acetonitrile stock solution to water, an immediate precipitation was observed. An organic extract of the mixture was investigated by GC-MS and the aqueous phase was investigated by HPLC-MS. By GC-MS analysis of both extracts only one signal was detected, identified by comparison with the Wiley 275.L mass spectra data base as 2-Ethyl-hexanale, the first hydrolysis product. The HPLC-MS analysis of both aqueous mixtures showed two signals, one of the minor component 2-(1-Ethylpentyl)-3-(2-hydroxyethyl)-oxazolidine and of the hydrolysis product 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester. The 2-ethylhexanal is insoluble in water and forms droplets at the flask walls. It is soluble inorganic medium (diethylether and acetonitrile) and was therefore detected by GC-MS. Hydrolysis takes place immediately after contact with water. No test item 1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate could be detected.

 

Additional remark:

In the course of the GC-MS analysis in the present hydrolysis study the first hydrolysis product was clearly identified as 2-ethyl-hexanale. The other, second hydrolysis product was detected using HPLC-MS. For the second hydrolysis product the molecular weight (Mw) was reported to be 378 g/mol. However, the postulated hydrolysis reaction considering these hydrolysis products seems to be not correct, e.g. in particular regarding the completeness and the mass balance in chemical reactions, i.e. the postulated chemical structure of the second hydrolysis product is not consistent with the experimentally determined molecular weight (378 g/mol). Thus, considering 2-ethyl-hexanale as one of the hydrolysis degradation product, the other hydrolysis degradation product, which is more likely, is 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester with a calculated molecular weight of 376.5 g/mol.

 

Considering the results form a hydrolysis study (supporting study) with the analogue substance (bis[2-[2-(2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexane-1,2-diylbiscarbamate) the second, more likely hydrolysis product which is formed in the course of the hydrolysis of 1,6-hexanediyl-bis(2-[2-{1-ethylpentyl}-3-oxazolidinyl]ethyl) carbamate is 1,6 hexanediyl-bis-carbamic acid bis(N-hydroxyethyl-2-aminoethyl) ester. This further confirms the assumption above.