Propanoic acid, 2-hydroxy-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester, (2S)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-10-08 to 2013-

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study performed according to scientific principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The stability of Frescolat® ML was investigated and the potential generation of Menthol was monitored. Therefore the test item Frescolat® ML was incubated with cryopreserved hepatocytes from humans, samples were taken after defined time points (0, 15, 60, 120 and 240 min) and the content of test item and potential metabolite was determined.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Menthyl lactate (Frescolat®)

IUPAC Name:

Menthyl lactate (Frescolat®)

Constituent 2

Reference substance name:

Methyl lactate

EC Number:

208-930-0

EC Name:

Methyl lactate

Cas Number:

547-64-8

IUPAC Name:

methyl 2-hydroxypropanoate

Constituent 3

Reference substance name:

Propanoic acid, 2-hydroxy-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester, (2S)-

EC Number:

612-179-8

Cas Number:

61597-98-6

Molecular formula:

C13H24O3

IUPAC Name:

Propanoic acid, 2-hydroxy-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester, (2S)-

Details on test material:

- Name of test material (as cited in study report): Frescolat®ML
- Storage condition of test material: Room temperature

Radiolabelling:

no

Administration / exposure

Positive control reference chemical:

1.0 µM naloxone (high metabolism) and 1.0 µM piroxicam (low metabolism) were added to the hepatocytes (0.5x10^6 cells/mL) to control the metabolic activity. Samples were taken after 0, 5, 15, 60, 120 and 240 min.

Details on study design:

At first stock solutions of the established concentrations of Frescolat®ML were prepared in TBME. The stock solutions were further diluted in Krebs-Henseleit (KH) buffer and applied to reaction vials. Afterwards the cryopreserved hepatocytes of humans were removed from the nitrogen tank and were thawed using Krebs-Henseleit buffer. After centrifugation the cells were resuspended in KH buffer to the final concentration of 1.0x10^6 cells/mL. The cryopreserved hepatocytes were applied to the test item or control solutions in the reaction vials leading to 0.5x10^6 cells/mL. 3 replicates were prepared for each concentration and the corresponding time points. The solutions within the vials were agitated at low speed. The vials were incubated at 37°C/5 % CO2 for a total incubation time of 4 hours. After the indicated time points (0, 15, 60, 120 and 240 min (T1 – T5)) the respective samples were stopped with 1.3 mL TBME. Samples were stored at -80°C immediately.

Details on dosing and sampling:

Two concentrations of the test item were used in the study (20 µM and 100 µM). 3 replicates were performed.

Results and discussion

Main ADME results

Type:

metabolism

Results:

The metabolized percentage of Frescolat®ML after 240 minutes was 97.4 % and 99.6 % when 20 and 100 µM of Frescolat®ML, respectively, were applied to the hepatocytes.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The measured amount of Menthol was between 16 and 27 µg/mL when 20 µM Frescolat® ML was applied. The range was 16-37 µg/mL when 100 µM Frescolat® ML was applied. The amount of Menthol detected was similar throughout the incubation time from 0 to 240 minutes. Therefore there is no correlation between metabolized Frescolat® ML and emerging Menthol. The amount of Menthol detected without hepatocytes was less than 0.5 µg/mL.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: Frescolat®ML was significantly metabolized by hepatocytes of humans
Frescolat®ML was significantly metabolized by hepatocytes of humans, i.e. from 97.4 to 99.6 % within 240 min.

Executive summary:

The liver is the prime organ in the metabolism of many xenobiotics and human hepatocytes are recognized as a suitable test system for the generation of human metabolites in vitro. The aim of the present study is to determine the metabolic activity of human hepatocytes towards the test item Frescolat^®ML. Therefore the test item Frescolat^®ML was incubated with cryopreserved hepatocytes from humans, samples were taken after defined time points (0, 15, 60, 120 and 240 min) and the content of test item and potential metabolite Menthol was determined. The test item Frescolat^®ML was significantly metabolized by human hepatocytes in comparison to the buffer control without hepatocytes. The percentage of Frescolat^®ML remaining after 240 minutes was between 0.4 % and 2.6 %. The measured amount of Menthol was between 16 and 37 µg/mL. The amount of Menthol detected was similar during the incubation time from 0 to 240 minutes. Therefore there is no correlation between metabolized Frescolat^®ML and emerging Menthol.