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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics, other
Remarks:
hydrolysis assay
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Objective of study:
other: hydrolysis
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
An in vitro hydrolysis test in different media with and without enzymolysis was conducted with the test item. The test media were as follows: simulated gastric juice, simulated intestinal fluid, and rat liver homogenate. In addition, the test substance was also tested in distilled water without enzymolysis. Chemical composition of the substance before and after hydrolysis test procedure was determined by extraction with addition of an internal standard substance and following GC with FID. Extraction was made by liquid-liquid extraction in a modified device described by Kutscher and Stendel with pentane/ethylether 2:1.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]ethan-1-ol
EC Number:
701-039-2
Cas Number:
156324-78-6
Molecular formula:
C13H24O4
IUPAC Name:
2-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]ethan-1-ol
Radiolabelling:
no

Test animals

Details on test animals or test system and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Administration / exposure

Route of administration:
other: in vitro hydrolysis study in different media
Vehicle:
propylene glycol
Duration and frequency of treatment / exposure:
4 hours
Doses / concentrationsopen allclose all
Dose / conc.:
122 other: mg
Remarks:
hydrolysis test with simulated gastric juice or simulated intestinal fluid
Dose / conc.:
20 other: mg
Remarks:
hydrolysis test with rat liver homogenate
No. of animals per sex per dose / concentration:
not applicable
Positive control reference chemical:
not applicable
Details on study design:
not specified
Details on dosing and sampling:
GENERAL INFORMATION ON HYDROLYSIS PROCEDURE
- hydrolysis test was conducted in different media (with and without enzymolysis): simulated gastric juice, simulated intestinal fluid, and rat liver homogenate
- hydrolysis procedure for each medium with and without enzymolysis was conducted twice.
- hydrolysis of the test item without enzymolysis was also determined in distilled water (untreated batch).
- chemical composition of the substance before and after hydrolysis test procedure has been determined by extraction with addition of an internal standard substance and following GC with FID equipped with a 60 m x 0.32 mm internal diameter DB-1 column and a 60 m x 0.32 mm i.d. WAX column.
- extraction was made by liquid-liquid extraction in a modified device described by Kutscher and Stendel with pentane/ethylether 2:1.

HYDROLYSIS WITH SIMULATED GASTRIC JUICE OR SIMULATED INTESTINAL JUICE
- test medium (simulated gastric juice): pepsin solved in buffer I (sodium chloride, hydrochlorid acid conc. and distilled water (pH = 1.1))
- hydrolysis test with simulated gastric juice: 1.22 g of a 10 % solution of the test item in the vehicle (= 122 mg test item) has been solved in test medium 3:1 and given in a flask.

- test medium (simulated intestinal fluid): pancreatin solved in buffer II (dibasic potassium phosphate, distilled water, sodium hydroxide solution (pH = 7.5))
- hydrolysis test with simulated intestinal juice: 1.22 g of a 10 % solution of the test item in the vehicle (= 122 mg test item) has been solved in test medium 4:1 and given in a flask.

The following applies to both hydrolysis tests:
- flask content has been stirred at 37 - 38 °C for four hours and kept in the refrigerator over night (16 hours).
- menthon has been added as an internal standard and distilled water.
- extraction was made by liquid-liquid extraction for 8 hours with pentane/ether (2:1).
- extract was dried with sodium sulfate and concentrated to 1 mL.

HYDROLYSIS WITH LIVER HOMOGENATE
- test medium: masculine rat liver was homogenized in Sörensen buffer solution (0.1 M; pH = 7.6) and cooled down (4 °C).
- 0.200 g of a 10 % solution of the test item in the vehicle (= 20 mg test item) was incubated with test medium (5:1) at 37 °C for 4 hours.
- then trichloroacetic acid (20 %) was added to inactivate the enzymes and the batch has been frozen until GC process.
- menthon was added as an internal standard and distilled water
- extraction was made by liquid-liquid extraction for 8 hours with pentane/ether (2:1)
- extract was dried with sodium sulfate and concentrated to 1 mL.

CONCENTRATED SUBSTANCE
- 1.29 g of a 10 % solution of the test item in the vehicle (= 129 mg test item) was solved in distilled water and extracted in the same as described above without enzymolysis.
Statistics:
not specified

Results and discussion

Preliminary studies:
not applicable
Main ADME results
Type:
other: hydrolysis
Results:
The incubation with simulated gastric juice and intestinal fluid did not modify the test item, whereas the incubation with rat liver homogenate did hydrolyse the menthol from the test item almost completely (theoretical value 12.7 mg).

Toxicokinetic / pharmacokinetic studies

Details on absorption:
not measured
Details on distribution in tissues:
not measured
Details on excretion:
not measured

Metabolite characterisation studies

Metabolites identified:
not measured

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
not measured

Any other information on results incl. tables

As hydrolysis product menthol was to be expected. In the tests the final content of menthol was measured via the relation to the internal standard. Each test was done twice. The tests without enzymolysis and the test with distilled water were used for control.

 

Untreated batch (mg)

Inactive* simulated gastric juice (pH 1) (mg)

Active simulated gastric juice (pepsin pH 1) (mg)

Inactive* intestinal fluid (pH 7.5) (mg)

Active simulated intestinal fluid (pancreatin pH 7.5) (mg)

Inactivated rat liver homogenate (pH 7.6) (mg)

Rat liver homogenate (pH 7.6) (mg)

Menthol

2.0

2.0

2.2

3.2

1.4

0.2

10.7

2.4

4.1

1.2

5.3

1.8

0.2

11.6

* inactive means without enzyme addition

Applicant's summary and conclusion

Conclusions:
The incubation with simulated gastric juice and intestinal fluid did not modify the test item, whereas the incubation with rat liver homogenate did hydrolyse the menthol from the test item almost completely (theoretical value 12.7 mg).
Executive summary:

An in vitro hydrolysis test in different media with and without enzymolysis was conducted with the test item. The test media were as follows: simulated gastric juice, simulated intestinal fluid, and rat liver homogenate. In addition, the test substance was also tested in distilled water without enzymolysis. Chemical composition of the substance before and after hydrolysis test procedure was determined by extraction with addition of an internal standard substance and following GC with FID. Extraction was made by liquid-liquid extraction in a modified device described by Kutscher and Stendel with pentane/ethylether 2:1.

The results showed that the incubation with simulated gastric juice and intestinal fluid did not modify the test item, whereas the incubation with rat liver homogenate did hydrolyse the menthol from the test item almost completely (theoretical value 12.7 mg).