Registration Dossier

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 Menthyl acetat rac. was investigated and the potential generation of Menthol was monitored. Therefore the test item Menthyl acetat rac. 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

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
no

Administration / exposure

Positive control:
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 Menthyl acetat rac. 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 Menthyl acetat rac. after 240 minutes was 71.5 % and 64.5 %, when 20 and 100 µM of Menthyl acetat rac., respectively, were applied to the hepatocytes.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The measured amount of Menthol was between 7 and 12 µg/mL when 20 µM Menthyl acetat rac. were applied. The range was 4-17 µg/mL when 100 µM Menthyl acetat rac. were applied. The amount of Menthol detected was similar throughout the incubation time from 0 to 240 minutes. Therefore there is no correlation between metabolized Menthyl acetat rac. and emerging Menthol. The amount of Menthol detected without hepatocytes was between 3-6 µg/mL.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: Menthylacetat rac. was significantly metabolized by hepatocytes of humans
Menthylacetat rac. was significantly metabolized by hepatocytes of humans, i.e. from 64.5 to 71.5 % 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 Menthyl acetat rac. Therefore the test item Menthyl acetat rac. 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 Menthyl acetat rac. was significantly metabolized by human hepatocytes in comparison to the buffer control without hepatocytes. The percentage of Menthyl acetat rac. remaining after 240 minutes was between 28.5% and 35.5 %. The measured amount of Menthol was between 4 and 17 µg/mL. The amount of Menthol detected was similar about the incubation time from 0 to 240 minutes. Therefore there is no correlation between metabolized Menthyl acetat rac. and emerging Menthol.