Exo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Principles of method if other than guideline:

- Principle of test: Study of metabolism of Borneol by the analysis of incubations of in vitro-prepared rat liver microsomes
- Short description of test conditions: see description below
- Parameters analysed / observed: Metabolites of Borneol in rat liver microsomes.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Center of Guangdong Province, P.R. China
- Age at study initiation: ca. 50 days
- Weight at study initiation: 230–250 g
- Housing: steel cages
- Diet (e.g. ad libitum): control diet
- Water (e.g. ad libitum): control diet
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22–26°C
- Humidity (%): 50–60%

Route of administration:

other: In vitro incubation with rat liver microsomes

Details on exposure:

A typical incubation mixture consisted of 2.5 mg/mL rat liver microsmal protein, 0.1M potassium phosphate buffer (pH 7.4), 1mM Nicotinamide-adenine dinucleotide phosphate (NADPH), and 325μM Borneol with a final volume of 1 mL. Borneol was dissolved in methanol (final concentration in the reaction medium of < 1.0%).
The reaction was initiated by the addition of the NADPH, and then the oxygen was quickly added with a syringe needle going into the middle of the mixtures for 40 s.
Control incubations were performed using inactivated microsomes, which were boiled in 90°C water for 45 min.
After incubation in a shaking water bath at 37°C for 30 min, the reaction was terminated by adding 2 mL ethyl acetate. The mixture was extracted for 10 min by shaking vigorously and then centrifuged at 15,000 × g for 10 min.
The organic phases were directly injected into the Gas chromatography (GC)–Mass spectrometry (MS) for analysis.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: rat liver microsomes
- Method type(s) for identification: GC-MS

Type:

metabolism

Results:

Four metabolites (M1, M2, M3, and M4) were observed in the incubation mixture, which were not present in the control incubations

Metabolites identified:

yes

Details on metabolites:

Borneol was rapidly metabolized to four metabolites:
M1 (m/z 152): molecular weight of two mass units less than Borneol. M1 was confirmed as camphor by comparison with the standard mass spectrum library (NIST library, 95% similarity).
M2 (m/z 122): molecular weight of 32 mass units less than Borneol. It was proposed that this is the de-methylated and de-hydrated metabolite of Borneol. Although, further investigation is required to positively identify this metabolite.
M3 (m/z 170) and M4 (m/z 170): molecular weight of 16 mass units more than Borneol. it was proposed that M3 and M4 are probably hydroxylated metabolite(s) of borneol, although these will require further study to be certain.

Conclusions:

Borneol was rapidly metabolized to four phase I metabolites in incubations with normal rat liver microsomes in the presence of NADPH.

Executive summary:

The metabolism of borneol was studied by the analysis of incubations of in vitro-prepared rat liver microsomes. Male Sprague-Dawley rats, aged approximately 50 days and weighing 230–250 g, were used for the study. A typical incubation mixture was performed in a shaking water bath at 37°C for 30 min and consisted of 2.5 mg/mL rat liver microsmal protein, 0.1M potassium phosphate buffer (pH 7.4), 1mM NADPH, and 325μM Bornel with a final volume of 1 mL. Gas chromatography (GC)–mass spectrometry (MS) method was developed for the identification of Borneol and its metabolites. Four phase I metabolites were detected: M1 (m/z 152) confirmed as camphor, M2 (m/z 122) proposed as the de-methylated and de-hydrated metabolite, M3 (m/z 170) and M4 (m/z 170) both proposed as the hydroxylated metabolites.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance DL-Borneol which shares the same functional groups with the substance Isoborneol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Metabolites identified:

yes

Details on metabolites:

Results based on read-across from analogue Borneol.
Borneol was rapidly metabolized to four metabolites:
M1 (m/z 152): molecular weight of two mass units less than Borneol. M1 was confirmed as camphor by comparison with the standard mass spectrum library (NIST library, 95% similarity).
M2 (m/z 122): molecular weight of 32 mass units less than Borneol. It was proposed that this is the de-methylated and de-hydrated metabolite of Borneol. Although, further investigation is required to positively identify this metabolite.
M3 (m/z 170) and M4 (m/z 170): molecular weight of 16 mass units more than Borneol. it was proposed that M3 and M4 are probably hydroxylated metabolite(s) of borneol, although these will require further study to be certain.

Conclusions:

Based on the read-across approach from the analogue DL-Borneol, Isoborneol was determined to be rapidly metabolized to four phase I metabolites in incubations with normal rat liver microsomes in the presence of NADPH.

Executive summary:

The metabolism of borneol was studied by the analysis of incubations of in vitro-prepared rat liver microsomes. Male Sprague-Dawley rats, aged approximately 50 days and weighing 230–250 g, were used for the study. A typical incubation mixture was performed in a shaking water bath at 37°C for 30 min and consisted of 2.5 mg/mL rat liver microsmal protein, 0.1M potassium phosphate buffer (pH 7.4), 1mM NADPH, and 325μM Bornel with a final volume of 1 mL. Gas chromatography (GC)–mass spectrometry (MS) method was developed for the identification of Borneol and its metabolites. The read-across was applied and based on results of this study, Isoborneol was determined to have four phase I metabolites: M1 (m/z 152) confirmed as camphor, M2 (m/z 122) proposed as the de-methylated and de-hydrated metabolite, M3 (m/z 170) and M4 (m/z 170) both proposed as the hydroxylated metabolites.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Objective of study:

metabolism

Principles of method if other than guideline:

- Principle of test: Study on the glucuronidation of Isoborneol by human embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein.
- Short description of test conditions: see description below
- Parameters analysed / observed: rate of glucuronidation.

GLP compliance:

not specified

Radiolabelling:

no

Species:

other: Human

Details on test animals or test system and environmental conditions:

embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein were used.

Route of administration:

other: In vitro incubation with embryonic kidney 293 cells

Details on exposure:

No data

Dose / conc.:

0.5 other: mM

Remarks:

Concentration of Isoborneol on incubation mixture

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Human embryonic kidney 293 cells

Type:

metabolism

Results:

The rate of glucuronidation of 0.5 mM Isoborneol by human embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein was 25 pmol/min/mg protein

Metabolites identified:

no

Conclusions:

The rate of glucuronidation of 0.5 mM Isoborneol by human embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein was 25 pmol/min/mg protein.

Executive summary:

The glucuronidation of Isoborneol by human embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein was investigated. The assays were performed with a concentration of Isoborneol of 0.5 mM. The rate of glucuronidation of Isoborneol was found to be 25 pmol/min/mg protein.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

other: sedative effects of fragrance compounds and essential oils

Principles of method if other than guideline:

- Principle of test: Influence of the sedative effects of several fragrance compounds and essential oils in motility of mice after their inhalation. In addition, serum samples of the mice were analyzed by GC-MS, GC-FTIR and GC-FID to identify and quantify potent compounds effective in increasing or decreasing the motility of mice by inhalation alone.
- Short description of test conditions: see description below
- Parameters analysed / observed: Effects on motility and concentration in blood samples of the fragrance compounds

GLP compliance:

not specified

Radiolabelling:

no

Species:

mouse

Strain:

Swiss

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 6-8 weeks and 6 month old
- Weight at study initiation: 28.5 g mean
- Housing: groups of four housed on a bedding of wood shavings in polycarbonate cages (Makrolon, type 11).
- Diet (e.g. ad libitum): Standardized pelleted food T 799 (Tagger, Graz, Austria); ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 60 ± 10%
- Air changes (per hr): 12-15 times per hour
- Photoperiod (hrs dark / hrs light): light-dark rhythm, 12:12 h

Route of administration:

inhalation

Vehicle:

unchanged (no vehicle)

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: mice were placed in light barrier cages and exposed to air containing isoborneol. Air was passed into the cage through a glass tube containing the test compound.

TEST ATMOSPHERE
- Brief description of analytical method used: charcoal tubes (120, NIOSH, Catalogue no. 226-01) supplied by Drager Company, Liibeck, Germany.
- Samples taken from breathing zone: yes

Duration and frequency of treatment / exposure:

1 h of exposure

Dose / conc.:

50 other: mg

Remarks:

Maximum dose (range of 20-50 mg)

No. of animals per sex per dose / concentration:

4

Control animals:

yes, concurrent no treatment

other: pretreated with 0.1% caffeine solution (0.5 mL, ip)

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled (delete / add / specify): serum
- Time and frequency of sampling: Blood samples were taken from the animals after 0, 30, 60 and 90 minutes of inhalation
- Other: Samples were taken by puncturing the retrobulbar venous plexus. Serum samples of the mice were analyzed by GC-MS, GC-FTIR and GC-FID chromatographic analysis.

Statistics:

Statistics were calculated with an Atari 1040 personal computer (WISTAT scientific statistic packageprogram). Significance was determined by the t test and F-test; the level of significance chosen for p to reject the null hypothesis was <0.05.

Type:

absorption

Results:

The serum isoborneol level after exposure to 20-50 mg of isoborneol was 0.36 ng/ml.

Metabolites identified:

no

Isoborneol seems to be an activator after exposure of 20 -50 mg in mice, increasing motility +46.90% of the untreated control animals (100% motility). In addition, isoborneol was able to reduce the motility (-11.23%) of the control animals pretreated with 0.1% caffeine solution (0.5 mL, ip).

Conclusions:

The concentration of isoborneol in blood serum of mice after exposure to doses of 20-50 mg was 0.36 ng/ml.

Executive summary:

A pharmacology study was performed on several fragrance compounds included isoborneol. Motility of animals and blood serum levels of isoborneol were measured in groups of four female outbred Swiss mice placed in light barrier cages and exposed to air containing 20-50 mg isoborneol. Blood samples were taken from the animals after 0, 30, 60 and 90 minute of inhalation by puncturing the retrobulbar venous plexus. Blood serum samples were analyzed by GC-MS, GC-FTIR and GC-FID chromatographic analysis. Isoborneol increased the motility +46.90% of the untreated control animals (100% motility) and reduced the motility (-11.23%) of the control animals pretreated with 0.1% caffeine solution (0.5 mL, ip). The blood serum isoborneol level after exposure to 20-50 mg of isoborneol was 0.36 ng/ml.

Description of key information

Weight of Evidence: Read-across aproach. Based on the read-across approach from the analogue DL-Borneol, Isoborneol was determined to be rapidly metabolized to four phase I metabolites in incubations with normal rat liver microsomes in the presence of NADPH.

Weight of Evidence: In vivo mice study on Isoborneol. The concentration of isoborneol in blood serum of mice after exposure to doses of 20-50 mg was 0.36 ng/ml.

Weight of Evidence: In vitro human study on Isoborneol. The rate of glucuronidation of 0.5 mM Isoborneol by human embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein was 25 pmol/min/mg protein.

Additional information

Weight of evidence. Read-across approach from experimental data on the analogue substance DL-Borneol: The metabolism of borneol was studied by the analysis of incubations of in vitro-prepared rat liver microsomes. Male Sprague-Dawley rats, aged approximately 50 days and weighing 230–250 g, were used for the study. A typical incubation mixture was performed in a shaking water bath at 37°C for 30 min and consisted of 2.5 mg/mL rat liver microsmal protein, 0.1M potassium phosphate buffer (pH 7.4), 1mM NADPH, and 325μM Bornel with a final volume of 1 mL. Gas chromatography (GC)–mass spectrometry (MS) method was developed for the identification of Borneol and its metabolites. The read-across was applied and based on results of this study, Isoborneol was determined to have four phase I metabolites: M1 (m/z 152) confirmed as camphor, M2 (m/z 122) proposed as the de-methylated and de-hydrated metabolite, M3 (m/z 170) and M4 (m/z 170) both proposed as the hydroxylated metabolites.

Weight of Evidence: In vivo mice study on Isoborneol. A pharmacology study was performed on several fragrance compounds included isoborneol. Motility of animals and blood serum levels of isoborneol were measured in groups of four female outbred Swiss mice placed in light barrier cages and exposed to air containing 20-50 mg isoborneol. Blood samples were taken from the animals after 0, 30, 60 and 90 minute of inhalation by puncturing the retrobulbar venous plexus. Blood serum samples were analyzed by GC-MS, GC-FTIR and GC-FID chromatographic analysis. Isoborneol increased the motility +46.90% of the untreated control animals (100% motility) and reduced the motility (-11.23%) of the control animals pretreated with 0.1% caffeine solution (0.5 mL, ip). The blood serum isoborneol level after exposure to 20-50 mg of isoborneol was 0.36 ng/ml.

Weight of Evidence: In vitro human study on Isoborneol. The glucuronidation of Isoborneol by human embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein was investigated. The assays were performed with a concentration of Isoborneol of 0.5 mM. The rate of glucuronidation of Isoborneol was found to be 25 pmol/min/mg protein.