Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

publication

Title:

Quantification of 1,8-cineole and of its metabolites in humans using stable isotope dilution assays

Author:

Horst K. and Rychlik M.

Year:

2010

Bibliographic source:

Mol. Nutr. Food Res. 54;1515-1529

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The aim of the current study was to identify and quantify cineole metabolites in humans after the intake of food-relevant doses.
The protocol of the study was approved by the Ethics Committee of the Faculty of Medicine of the Technische Universit.at Munchen (1996/07).

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

1,8-Cineol

IUPAC Name:

1,8-Cineol

Test material form:

solid

Details on test material:

404g of tea = 1017µg of 1,8-cineole

Specific details on test material used for the study:

Dried sage (6.4 g) was weighed into a tea filter and was brewed with 600mL of boiling water.
After letting it steep for 15 min in a capped bottle, the filter was removed.

Quantification of 1.8 cineole in tea infusion:
Aliquots of the tea infusion were weight in capped tubes, cooled to room temperature and the internal standard (IS) [9-2H3]-1,8-cineole (22.5 mg, 143 mmol) was added as ethereal solution. After stirring for 1 h, the tea was extracted with dichloromethane. The concentration of 1,8-cineole was 2.5270.11 mg/kg, quantified by relative area counts of analyte (A) and IS in their mass traces m/z5137 and m/z5140, respectively, using the linear equation y50.9926x10.0906 (y5area(IS)/area(A); x5n(IS)/n(A)), which was determined by analyzing definite mixtures of analyte and IS.

Radiolabelling:

no

Test animals

Species:

other: human

Details on species / strain selection:

For wash out, the volunteer (female, 26 years old, body mass index 19.2) used toothpaste devoid from terpenes and avoided spices, herbs and fruits and other foods and cosmetics containing 1,8-cineole during 3 days prior to the study.

Sex:

female

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

On an empty stomach, the volunteer drank the tea (404 g=1017 µg 1,8-cineole) within 10 min

Duration and frequency of treatment / exposure:

One ingestion during 10 minutes.

Doses / concentrations

Dose / conc.:

19 other: µg/kg bw

Remarks:

1.02 mg 1,8-cineole (19 µg/kg bw)

No. of animals per sex per dose / concentration:

only one female

Control animals:

no

Positive control reference chemical:

not relevant

Details on study design:

The volunteer drank the tea (containing 1.8-cineole) within 10 min and her urine and blood were collected for analysis. Blank samples from urine and blood were collected as controls before consumption of the sage tea.

Details on dosing and sampling:

Urine was collected at 2, 5, 7, 10, 17, 21, 28, 32, 35, 44, 50, 53, 60 and 69 h after consumption. Samples were split into aliquots and stored at -70°C until analysis.
venous blood samples were taken after 0.75, 1.7, 3.25, 6.75 and 24 h. Plasma and red blood cells were separated by centrifugation (41C, 3000 rpm, 15 min) and stored in aliquots at –70°C before analysis.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on excretion:

In contrast to blood, hydroxy-1,8-cineoles were abundant in urine showing highest contents during the first 2 h. In accordance with the plasma levels, 2-hydroxycineole showed highest contents in urine followed by its 9-isomer.
However, in contrast to the plasma contents, 3-hydroxycineole was more abundant in urine than the 7-isomer.
Summing up the urinary excretion over 10 h, 52.5% of the 1,8-cineole dose was identified as metabolites, of which 2-hydroxycineole, the 9- isomer, the 3-isomer and the 7-isomer accounted for 20.9%, 17.2%, 10.6% and 3.8%, respectively. After 10 h, only traces of metabolites could be detected.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

After consumption of 1.02 mg 1,8-cineole (19 mg/kg bw), the metabolites 2-hydroxy-1,8-cineole, 3-hydroxy-1,8-cineole, 7-hydroxy-1,8-cineole, and 9-hydroxy-1,8-cineole along with their parent compound were detectable in the blood plasma of the volunteer under study after liberation from their glucuronides.
All compounds peaked in plasma after 0.75 h with 2-hydroxycineole being the predominant metabolite at a plasma concentration of 86 nmol/L followed by the 9-hydroxy isomer at a plasma concentration of 33nmol/L. The 7- and the 3-isomer were detectable, but their plasma concentrations were below their LOQ.

Any other information on results incl. tables

The metabolism was found to occur very fast within the first hour after consumption and gave rise to four hydroxycineols, of which the 7-isomer was identified for the first time in humans.

Applicant's summary and conclusion

Conclusions:

2-hydroxy-1,8-cineole is the main metabolite of 1,8-cineole in humans.
3-hydroxy-1,8-cineole, 7-hydroxy-1,8-cineole, and 9-hydroxy-1,8-cineole have also been identified as metabolites in plasma and urine.

Executive summary:

The metabolism of 1,8-cineole after ingestion of sage tea was studied. After application of the tea, metabolites 2-hydroxy-1,8-cineole, 3-hydroxy-1,8-cineole, 9-hydroxy-1,8-cineole and, for the first time in humans, 7-hydroxy-1,8-cineole were identified in plasma and urine of one volunteer. For quantitation of these metabolites and the parent compound, stable isotope dilution assays were developed after synthesis of [2H3]-1,8-cineole, [9/10-2H3]-2-hydroxy-1,8-cineole and [13C,2H2]-9-hydroxy-1,8-cineole as internal standards. Using these standards, 1,8-cineole was quantified by solid phase microextraction GC-MS and the hydroxyl-1,8-cineoles by LC-MS/MS after deconjugation in blood and urine of the volunteer. After consumption of 1.02 mg of 1,8-cineole (19 mg/kg bw), the hydroxycineoles along with their parent compound were detectable in the blood plasma of the volunteer under study after liberation from their glucuronides with 2-hydroxycineole being the predominant metabolite at a maximum plasma concentration of 86 nmol/L followed by the 9-hydroxy isomer at a maximum plasma concentration of 33 nmol/L. Parent compound 1,8-cineole showed a low maximum plasma concentration of 19 nmol/L. In urine, 2-hydroxycineole also showed highest contents followed by its 9-isomer. Summing up the urinary excretion over 10 h, 2-hydroxycineole, the 9-isomer, the 3-isomer and the 7-isomer accounted for 20.9, 17.2, 10.6 and 3.8% of the cineole dose, respectively.