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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.02.2004 to 08.04.2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP, preparatory study only, preliminary and main test were not performed because adsorption would have caused an unacceptable error.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl (2R,5R)-5-hydroxy-1,3-oxathiolane-2-carboxylate
EC Number:
604-569-1
Cas Number:
147126-62-3
Molecular formula:
C14H24O4S
IUPAC Name:
(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl (2R,5R)-5-hydroxy-1,3-oxathiolane-2-carboxylate
Test material form:
solid: compact

Study design

Analytical monitoring:
yes
Details on sampling:
Aqueous solutions in acetonitrile with buffers were prepared in 1.8 mL glass crimp-top vials with vortexing ~ 5 s.
Buffers:
Solutions:
pH 4 Buffer: An acetate buffer was prepared by first dissolving 1.16 mL of glacial acetic acid in 1.000 L of deionized water solution (solution A; 0.02 M). Solution B was prepared by dissolving 2.72 g of sodium acetate trihydrate in 1,000 L of deionized water solution (0.02 M). Solution A (41.0 mL) and solution B (9.0 mL) were mixed. The pH of this buffer before use (4.0) was measured with a pH meter.

pH 7 Buffer: MOPS free acid (20.93 g) was dissolved in 2.00 L of deionized water solution (0.05 M MOPS). The solution was titrated to pH 7.0 with approx. 20 mL of 1.00 N aqueous NaOH. The pH of this buffer before use was 6.9.

pH 9 Buffer: Ethanolamine (1.22 g) was dissolved in approx. 900 mL of deionized water. The solution was titrated to pH 9.0 with 32 mL of 1.0 M HCI, and made up to 1,000 mL with deionized water (0.02 M amine). The pH of this buffer before use was 9.0.

Aq. H3P04 (4.3 mM): H3P04 (250 µL) was dissolved in 1.000 L of deionized water solution (DIW).
Details on test conditions:
Objective:
Investigate detectability of OSME by HPLC in buffer solutions containing 1% acetonitrile co-solvent.

Perparation:
SkOS1: OSME solution (1.003 mg/mL) in acetonitrile: OSME (10.03 mg) was dissolved in 10.00 mL of acetonitrile solution in a volumetric flask. Approx. 1 mL was aliquotted to a 1.8 mL glass crimp-top vial labelled SkOS1.

OSME ( ~ 100 mg) was ground to "fine powder" in mortar and pestle and ~ 10 mg were added to a 15 mL polypropylene centrifuge tube labelled K.

Aqueous solutions in buffer or DIW with 1% acetonitrile co-solvent were prepared in 15 mL polypropylene centrifuge tubes.

Solution K was mixed (end-over-end; ~ 1 rev/s) for ~ 2 h (~ 22 °C). The pH of solution K was measured with a pH strip.
Solution K (~ 3 mL) was filtered (0.45 µm) into a single-use 5 mL glass test tube.
The filtered K solution was mixed with solvents in 1.8 mL glass crimp-top vials, using a pipettor. Blanks were prepared.
The time at end of mixing was recorded.
The Test Solutions and SkOS1 were analyzed by HPLC.
Duration of testopen allclose all
Duration:
2 h
pH:
4
Temp.:
22
Initial conc. measured:
>= 802 - <= 993 mg/L
Duration:
2 h
pH:
9
Temp.:
22
Initial conc. measured:
>= 802 - <= 993 mg/L
Number of replicates:
Four replicants with substance, Buffer solution and SkOS1 solution and one blank with 1,000 µL acetonitrile and buffer solution performed at pH 4 and pH 9 too. Mixing time was 2 hours reported.

Results and discussion

Preliminary study:
A preparatory study was conducted at room temperature in which near-saturated solutions of the test substance in mixtures of buffer / acetonitrile (1%) co-solvent were analysed by HPLC to investigate the extent of adsorption to glass as a function of co-solvent concentration. The pH 9 solution showed the presence of presumably the acid hydrolysis product, whereas this was essentially absent in the pH 4 and pH 7 solutions. In another experiment it was confirmed that the hydrolysis was relatively minor in pH 4 buffer / acetonitrile solutions compared to pH 9 buffer / acetonitrile solutions over a period of 6 h at ~ 22 °C.

From this result it was concluded by the test institute that the observed absence of the test substance in the pH 4 and pH 7 solutions (containing 1% acetonitrile) was due to adsorption to vial glass. In the pH 9 solutions (containing 1% acetonitrile) the presence of presumably the acid hydrolysis product indicated that hydrolysis occured; adsorption may have also occured in this pH 9 solution.

The preliminary and the main test, as foreseen in OECD test guideline 111, were not conducted because it was judged by the test institute that adsorption would cause an unacceptable error.
Transformation products:
yes
Details on hydrolysis and appearance of transformation product(s):
1. The alcohol hydrolysis product has a much lower calculated water solubility (1.5 g/L at 25 °C) than the acid hydrolysis product (1,000 g/L at
25 °C) and was, therefore, expected to have a greater retention time than the acid hydrolysis product.

2. If OSME was comprised of a single isomer, then only one alcohol hydrolysis product HPLC peak was expected. However, HPLC analysis of acetonitrile solutions of OSME showed the presence of a minor (17 area%) peak just before the major peak, which may have been a different stereo- or regio-isomer of OSME. This was supported by the fact that, like the major OSME peak, the minor peak also decreased in size as the volume percent of pH 9 buffer increased (from 1% to 20%). If this was the case, i.e. that two isomers of OSME were present, then two isomers of the alcohol hydrolysis product might also be expected, which could account for the later peaks at 9. 7 min and 11.2 min.
Other kinetic parameters:
not reported
Details on results:
The test substance OSME is an ester, and, based on the general hydrolysis of carboxylic acid esters (Morrison, 1983, page 832), OSME is expected to undergo the hydrolysis reaction to give the "acid" and "alcohol" hydrolysis products.

In experiment # 2243 938 a near-saturated solution of OSME in water (mixing time = 2 h; pH= 5-6) containing 1% acetonitrile was diluted (1/1) in buffers (pH 4, pH 7, and pH 9; all containing 1% acetonitrile). The buffer solutions were analyzed by HPLC within 6 h of preparation. No OSME was detected in the pH 9 solution, and only traces of OSME were detected in the pH 4 and pH 7 solutions. A fairly large "early" peak was observed in the pH 9 solution, which was absent in the blank and was presumably the acid hydrolysis product. These results were consistent with:

1. rapid hydrolysis of OSME in pH 9 buffer, and
2. extensive adsorption of OSME to glass in the pH 4 and pH 7 buffers.

Note that the adsorption of OSME to glass in aqueous solution might be expected given its relatively low calculated water solubility of 0.07 g/L at 25 °C.

This apparent hydrolysis of OSME in pH 9 buffer and lack of hydrolysis in pH 4 buffer was confirmed in another experiment (# 384 1222) in which OSME solutions in acetonitrile (0.9 mg/mL) were spiked with pH 4 and pH 9 buffers to give buffer concentrations of 1%, 5%, 10%, and 20%. The solutions were analysed by HPLC within 6 h of preparation, during which time they were at ~ 22 "C. The pH 9 solutions showed the appearance of 3 new peaks which increased in size as the concentration of buffer increased. This was accompanied by a decrease in OSME peak size. These new peaks were observed to a much lesser extent or not at all in the analogous series of pH 4 solutions (Figure 6), indicating that OSME hydrolysis was relatively minor in the pH 4 solutions compared to the pH 9 solutions.

Based on the expected hydrolysis reaction of OSME, these new peaks of the pH 9 solutions were assigned as follows. The early peak at 1.0 min was assigned as the acid hydrolysis product because this compound has a relatively high water solubility (calculated at 1,000 g/L at 25 °C for neutral form), and was, therefore, expected to have a low HPLC retention time under the reversephase conditions employed.

The later new peaks at 9.7 min and 11.2 min were assigned as isomers of the alcohol hydrolysis product for the following reasons:

1. The alcohol hydrolysis product has a much lower calculated water solubility (1.5 g/L at 25 °C) than the acid hydrolysis product (1,000 g/L at 25 °C) and was, therefore, expected to have a greater retention time than the acid hydrolysis product.

2. If OSME was comprised of a single isomer, then only one alcohol hydrolysis product HPLC peak was expected. However, HPLC analysis of acetonitrile solutions of OSME showed the presence of a minor (17 area%) peak just before the major OSME peak, which may have been a different stereo- or regio-isomer of OSME. This was supported by the fact that, like the major OSME peak, the minor peak also decreased in size as the volume percent of pH 9 buffer increased (from 1% to 20%). If this was the case, i.e. that two isomers of OSME were present, then two isomers of the alcohol hydrolysis product might also be expected, which could account for the later peaks at 9. 7 min and 11.2 min.

Applicant's summary and conclusion

Conclusions:
In these experiments a near-saturated solution of OSME in water containing 1% acetonitrile was diluted (1/1 v/v) in pH 4, pH 7, and pH 9 buffer
solutions, also containing 1% acetonitrile co-solvent. Either no OSME or traces of OSME were detected by HPLC in these buffer solutions. The pH 9
solution showed the presence of presumably the acid hydrolysis product of OSME, whereas this was essentially absent in the pH 4 and pH 7
solutions. in another experiment it was confirmed that the hydrolysis of OSME was relatively minor in pH 4 buffer / acetonitrile solutions compared to pH 9 buffer / acetonitrile solutions over a period of 6 h at ~ 22 °C. This was consistent with the general observation that the hydrolysis of carboxylic acid esters is faster under basic conditions than under acid conditions (Mabey, 1978).

lt should be noted that homogeneaus conditions are required for the accurate determination of the hydrolysis rate. The observed adsorption of
OSME would introduce a heterogeneaus component to the hydrolysis reaction which would likely reduce the hydrolysis rate by effectively lowering
the concentration of OSME in solution. Therefore, given that the OECD hydrolysis test allows a maximum of 1% organic cosolvent (OECD Guideline 111, 1993), it was judged that the extensive adsorption of OSME observed at this low co-solvent concentration would introduce unacceptable error into the OECD hydrolysis test results. The OECD hydrolysis test was, therefore, not conducted.
Executive summary:

In these experiments a near-saturated solution of OSME in water containing 1% acetonitrile was diluted (1/1 v/v) in pH 4, pH 7, and pH 9 buffer solutions, also containing 1% acetonitrile co-solvent. Either no OSME or traces of OSME were detected by HPLC in these buffer solutions. The pH 9 solution showed the presence of presumably the acid hydrolysis product of OSME, whereas this was essentially absent in the pH 4 and pH 7 solutions. in another experiment it was confirmed that the hydrolysis of OSME was relatively minor in pH 4 buffer / acetonitrile solutions compared to pH 9 buffer / acetonitrile solutions over a period of 6 h at ~ 22 °C. This was consistent with the general observation that the hydrolysis of carboxylic acid esters is faster under basic conditions than under acid conditions (Mabey, 1978).

lt should be noted that homogeneaus conditions are required for the accurate determination of the hydrolysis rate. The observed adsorption of OSME would introduce a heterogeneaus component to the hydrolysis reaction which would likely reduce the hydrolysis rate by effectively lowering the concentration of OSME in solution. Therefore, given that the OECD hydrolysis test allows a maximum of 1% organic cosolvent (OECD Guideline 111, 1993), it was judged that the extensive adsorption of OSME observed at this low co-solvent concentration would introduce unacceptable error into the OECD hydrolysis test results. The OECD hydrolysis test was, therefore, not conducted.

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