(1R,2R,4S)-1,3,3-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: Analysis of the metabolism of the test item in human liver microsomes
- Short description of test conditions: see description below
- Parameters analysed / observed: Identification of metabolites of the test item and kinetic analysis

GLP compliance:

not specified

Radiolabelling:

no

Species:

other: Human

Sex:

not specified

Details on test animals or test system and environmental conditions:

Human liver microsomes were obtained from Gentest Corporation (Woburn, MA, USA) (HG23, HG03, HH18, HG88, HG74, HG06, HK37, HH13, HG64, HH47) and were stored at -80ºC.
Recombinant CYPs1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 expressed in microsomes of Trichoplusia ni cells infected with a baculovirus containing human P450 cDNA were obtained from Gentest Co. (Woburn, MA, USA).

Route of administration:

other: In vitro incubation with human liver microsomes

Details on exposure:

Biotransformation of (+)-fenchol by P450 enzymes were determined as follows:
Standard reaction mixtures contained human liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 µM (+)-fenchol in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1.
Incubations were carried out at 37ºC for 30 min and terminated by adding 1.5 ml of dichloromethane. Formation of oxidation increased linearly with incubation time up to 30 min. The mixtures were mixed vigorously and the extracts (organic layer) collected by centrifugation at 3000 rpm for 5 min.
The organic phase was transferred to an insert for analysis by gas chromatography-mass spectrometry

Details on dosing and sampling:

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

Statistics:

Kinetic parameters for (+)-fenchol metabolite formation by microsomes were estimated using a computer program (KaleidaGraph program from Synergy Software, Reading, PA, USA) designed for non-linear regression analysis of the hyperbolic Michaelis–Menten equation. Substrate concentrations used for the analysis of metabolism of (+)-fenchol were 5, 10, 50, 100, 200, 500 µM

Type:

metabolism

Results:

(+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. CYP2A6 was identified as the major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes.

Metabolites identified:

yes

Details on metabolites:

Metabolite: Fenchone.
Kinetic analysis of (+)-fenchol oxidation catalysed by liver microsomes derived from human sample HG03 and recombinant CYP2A6 showed that the apparent Km values were 0.04 and 0.09mM, and the Vmax values were 0.29 and 6.96 nmol min-1 nmol-1 P450, respectively (See Table 1 below).
CYP2A6 is suggested to be the principal CYP enzyme in catalysing the oxidation in (+)-fenchol by human liver microsome.

Table 1. Kinetic analysis of the oxidation by human liver microsomes and recombinant P450 enzymes

expressed in Trichoplusia ni cells

Oxidation of (+)-Fenchol
	Fenchone
Enzyme source	Km (1)	Vmax (2)	Vmax/km (3)
HG03	0.04	0.29	7.25
CYP2A6	0.09	6.96	77.3

*Substrate concentrations used were 10, 50, 100, 200, 500, 750, 1000 µM.

(1) mM

(2) nmol/min/nmol P450

(3) nM-1 min-1.

Conclusions:

The test item was oxidized to fenchone by human liver microsomal P450 enzymes, with CYP2A6 identified as the major enzyme involved.

Executive summary:

The metabolism of the test item was studied by the analysis of incubations of in vitro-prepared human liver microsomes. Incubations were carried out at 37ºC for 30 min and contained human liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 mM test item in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting

of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1. The biotransformation of the test item was investigated by gas chromatography-mass spectrometry (GC-MS). Under these conditions, the test item was found to be oxidized to fenchone, with CYP2A6 identified as the major enzyme involved.

Reference 2

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: Analysis of the metabolism of the test item in rat liver microsomes
- Short description of test conditions: see description below
- Parameters analysed / observed: Identification of metabolites of the test item and kinetic analysis

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: Nihon Clea Co.(Osaka, Japan)
- Weight at study initiation: ca. 200 g
- Preparation: β-naphthoflavone and isosafrole (50mg kg-1, daily for 3 days), phenobarbital (80mgkg-1, daily for 3 days), or pregenolone-16-carbonitrile (PCN,100mgkg-1, daily for 3 days). Ethanol was given to rats in drinking water for 6 days at a final concentration of 15% (v/v). Rats were starved overnight before being killed, and liver microsomes were prepared as previously described and suspended in 10mM Tris-HCl buffer (pH 7.4) containing 1.0mM EDTA and 20% glycerol (v/v) (Guengerich 1994;
Shimada et al. 2002a).

Route of administration:

other: In vitro incubation with rat liver microsomes

Details on exposure:

Biotransformation of (+)-fenchol by P450 enzymes were determined as follows:
Standard reaction mixtures contained rat liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 µM (+)-fenchol in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1.
Incubations were carried out at 37ºC for 30 min and terminated by adding 1.5 ml of dichloromethane. Formation of oxidation increased linearly with incubation time up to 30 min. The mixtures were mixed vigorously and the extracts (organic layer) collected by centrifugation at 3000 rpm for 5 min.
The organic phase was transferred to an insert for analysis by gas chromatography-mass spectrometry

Details on dosing and sampling:

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

Statistics:

Kinetic parameters for (+)-fenchol metabolite formation by microsomes were estimated using a computer program (KaleidaGraph program from Synergy Software, Reading, PA, USA) designed for non-linear regression analysis of the hyperbolic Michaelis–Menten equation. Substrate concentrations used for the analysis of metabolism of (+)-fenchol were 5, 10, 50, 100, 200, 500 µM

Type:

metabolism

Results:

(+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. CYP2B1 was identified as the major enzyme involved in the oxidation of (+)-fenchol by liver microsomes of PB-treated rats.

Metabolites identified:

yes

Details on metabolites:

Metabolites: Fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol.
Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min-1 nmol-1 P450, respectively (See Table 1 below).
CYP2B1 is suggested to be the principal CYP enzyme in catalysing the oxidation in (+)-fenchol by liver microsomes of PB-treated rats.

Table 1. Kinetic analysis of the oxidation by rat liver microsomes and recombinant P450 enzymes

expressed in Trichoplusia ni cells

Oxidation of (+)-Fenchol
	Fenchone			6-Exo-Hydroxyfenchol			10-Hydroxyfenchol
Enzyme source	Km (1)	Vmax (2)	Vmax/km (3)	Km (1)	Vmax (2)	Vmax/km (3)	Km (1)	Vmax (2)	Vmax/km (3)
PB-treated	0.12	0.82	6.83	0.07	1.43	20.4	0.07	3.29	47.0
CYP2B1	0.06	2.94	49.0	0.03	6.1	203	0.03	13.8	460

*Substrate concentrations used were 10, 50, 100, 200, 500, 750, 1000 µM.

(1) mM

(2) nmol/min/nmol P450

(3) nM-1 min-1.

Conclusions:

The test item was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats, with CYP2B1 identified as the major enzyme involved.

Executive summary:

The metabolism of the test item was studied by the analysis of incubations of in vitro liver microsomes of phenobarbital-treated rats. Incubations were carried out at 37ºC for 30 min and contained rat liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 mM Fenchol in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1. The biotransformation of the test item was investigated by gas chromatography-mass spectrometry (GC-MS). Under these conditions, the test item was found to be oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol, with CYP2B1 identified as the major enzyme involved.

Reference 3

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:

toxicokinetics

Principles of method if other than guideline:

- Principle of test: The effect of several aglycones including fenchol on UDP-glucuronosyltransferase specific activitiy in male Gunn rats
- Short description of test conditions: see description below
- Parameters analysed / observed: UDP-glucuronosyltransferase activity

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

other: Gunn rats

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA-CREDO (Saint-Germain sur l'abresle, France)
- Weight at study initiation: 200 g
- Diet (e.g. ad libitum): ad libitum (UAR, Villemoisson/Orge)
- Water (e.g. ad libitum): ad libitum

Route of administration:

other: In vitro incubation with rat liver microsomes

Details on exposure:

Each animal was treated by intraperitoneal injection with phenobarbital (80 mg/kg) dissolved in 0.9% NaCl (w/v) daily for 4 days. The corresponding controls received saline solution only. Animals were decapitated 24 hr after the last treatment.
Microsomal suspensions were activated with Triton X-100 and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and test item.

Dose / conc.:

0.25 other: mM

Remarks:

Concentration of Fenchol on the microsomal solution

No. of animals per sex per dose / concentration:

4

Control animals:

yes

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): liver microsomes
- Other: UDP-glucuronosyltransferase activities in rat liver microsomes were measured by a modification of Mulder and Van Doorn (1975) method. Each run (twenty-nine assays) took 10min, and readings at 340nm were recorded every 20 sec. The specific activity was calculated by linear regression. Each value is given as a mean of four determinations.

Type:

metabolism

Results:

The activity of UDP-glucuronosyltransferase was enhanced 1.4 times over the NaCl control.

Metabolites identified:

no

Conclusions:

The activity of UDP-glucuronosyltransferase of 0.25 mM test item in liver microsomes of phenobarbital-treated Gunn rats was enhanced 1.4 times over the control group.

Executive summary:

The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male Gunn rats. Four rats received intraperitoneal doses of 80 mg/kg phenobarbital (PB) in 0.9% NaCl daily for 4 days. The control animals received 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose and the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.25 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.4 times over the NaCl control.

Reference 4

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:

toxicokinetics

Principles of method if other than guideline:

- Principle of test: The effect of several aglycones including fenchol on UDP-glucuronosyltransferase specific activitiy in male guinea pigs.
- Short description of test conditions: see description below
- Parameters analysed / observed: UDP-glucuronosyltransferase activity

GLP compliance:

not specified

Radiolabelling:

no

Species:

guinea pig

Strain:

other: "tricolores"

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre d'Elevage d'Animaux de Laboratoire, Ardenay, France
- Weight at study initiation: 350-400 g

Route of administration:

other: In vitro incubation with guinea pigs liver microsomes

Details on exposure:

Each animal was treated by intraperitoneal injection with phenobarbital (20-40 mg/kg bw) for 9 days (total dose, 320 mg/kg bw). The corresponding controls received saline solution only (0.9% NaCl (w/v)). Animals were decapitated 24 hr after the last treatment.
Microsomal suspensions were activated with Triton X-100 and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and test item.

Dose / conc.:

0.175 other: mM

Remarks:

Concentration of Fenchol on the microsomal solution

No. of animals per sex per dose / concentration:

4

Control animals:

yes

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): liver microsomes
- Other: UDP-glucuronosyltransferase activities in liver microsomes were measured by a modification of Mulder and Van Doorn (1975) method. Each run (twenty-nine assays) took 10min, and readings at 340nm were recorded every 20 sec. The specific activity was calculated by linear regression. Each value is given as a mean of four determinations.

Type:

metabolism

Results:

The activity of UDP-glucuronosyltransferase was enhanced 1.2 times over the NaCl control.

Metabolites identified:

no

Conclusions:

The activity of UDP-glucuronosyltransferase of 0.175 mM test item in liver microsomes of phenobarbital-treated guinea pigs was enhanced 1.2 times over the control group.

Executive summary:

The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male guinea pigs. Four animals received intraperitoneal doses of 20-40 mg/kg bw phenobarbital (PB) daily for 9 days. The control animals received a saline solution 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose and the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.175 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.2 times over the NaCl control.

Reference 5

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:

toxicokinetics

Principles of method if other than guideline:

- Principle of test: The effect of several aglycones including fenchol on UDP-glucuronosyltransferase specific activitiy in male Wistar rats
- Short description of test conditions: see description below
- Parameters analysed / observed: UDP-glucuronosyltransferase activity

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA-CREDO (Saint-Germain sur l'abresle, France)
- Weight at study initiation: 200 g
- Diet (e.g. ad libitum): ad libitum (UAR, Villemoisson/Orge)
- Water (e.g. ad libitum): ad libitum

Route of administration:

other: In vitro incubation with rat liver microsomes

Details on exposure:

One group of animals was treated by intraperitoneal injection with phenobarbital (80 mg/kg) dissolved in 0.9% NaCl (w/v) daily for 4 days. The corresponding controls received saline solution only. Animals were decapitated 24 hr after the last treatment. Another group of Wistar rats received 3-methylcholanthrene in corn oil, as a single injection of 80 mg/kg on the first day and these animals were killed on day 5. Corresponding controls received corn oil only.
Microsomal suspensions were activated with Triton X-100 and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and test item.

Dose / conc.:

0.25 other: mM

Remarks:

Concentration of Fenchol on the microsomal solution

No. of animals per sex per dose / concentration:

4

Control animals:

yes

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): liver microsomes
- Other: UDP-glucuronosyltransferase activities in rat liver microsomes were measured by a modification of Mulder and Van Doorn (1975) method. Each run (twenty-nine assays) took 10min, and readings at 340nm were recorded every 20 sec. The specific activity was calculated by linear regression. Each value is given as a mean of four determinations.

Type:

metabolism

Results:

The activity of UDP-glucuronosyltransferase was enhanced 1.2 times over the NaCl control when induced with phenobarbital.

Type:

metabolism

Results:

The activity of UDP-glucuronosyltransferase was the same as the corn oil control when induced with 3-methylcholanthrene.

Metabolites identified:

no

Conclusions:

The activity of UDP-glucuronosyltransferase of 0.25 mM test item in liver microsomes of induced wistar rats compared to controls was enhanced 1.2 times for phenobarbital whereas showed no difference for 3-methylcholanthrene.

Executive summary:

The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male Wistar rats. Four rats received intraperitoneal doses of 80 mg/kg phenobarbital (PB) in 0.9% NaCl daily for 4 days and corresponding control animals received 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose. Another group of 4 rats received 3-methylcholanthrene in corn oil, as a single injection of 80 mg/kg on the first day and these animals were killed on day 5. The orresponding controls received corn oil only. After sacrifice of animals the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.25 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.2 times for phenobarbital whereas showed no difference for 3-methylcholanthrene compared to controls.

Reference 6

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 fenchol which shares the same functional groups with the substance d-alpha fenchol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Type:

metabolism

Results:

Based on results with analogue fenchol, the activity of UDP-glucuronosyltransferase of d-alpha fenchol is expected to be enhanced 1.2 times over the NaCl control.

Metabolites identified:

no

Conclusions:

Based on the read-across approach from the analogue fenchol, the activity of UDP-glucuronosyltransferase of 0.175 mM of d-alpha fenchol in liver microsomes of phenobarbital-treated guinea pigs is expected to be enhanced 1.2 times over the control group.

Executive summary:

The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male guinea pigs. Four animals received intraperitoneal doses of 20-40 mg/kg bw phenobarbital (PB) daily for 9 days. The control animals received a saline solution 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose and the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.175 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.2 times over the NaCl control. The read-across was applied and the activity of UDP-glucuronosyltransferase of 0.175 mM of d-alpha fenchol is expected to be enhanced 1.2 times over the control group.

Reference 7

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 fenchol which shares the same functional groups with the substance d-alpha fenchol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Type:

metabolism

Results:

Based on results with analogue fenchol, the activity of UDP-glucuronosyltransferase in d-alpha fenchol is expected to be enhanced 1.4 times over the NaCl control.

Metabolites identified:

no

Conclusions:

Based on the read-across approach from the analogue fenchol, the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol in liver microsomes of phenobarbital-treated Gunn rats is expected to be enhanced 1.4 times over the control group.

Executive summary:

The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male Gunn rats. Four rats received intraperitoneal doses of 80 mg/kg phenobarbital (PB) in 0.9% NaCl daily for 4 days. The control animals received 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose and the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.25 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.4 times over the NaCl control. The read-across was applied and the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol is expected to be enhanced 1.4 times over the control group.

Reference 8

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 fenchol which shares the same functional groups with the substance d-alpha fenchol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Type:

metabolism

Results:

Based on results with analogue fenchol, the activity of UDP-glucuronosyltransferase of d-alpha fenchol is expected to be enhanced 1.2 times over the NaCl control when induced with phenobarbital.

Type:

metabolism

Results:

Based on results with analogue fenchol, the activity of UDP-glucuronosyltransferase for d-alpha fenchol is expected to be the same as the corn oil control when induced with 3-methylcholanthrene.

Metabolites identified:

no

Conclusions:

Based on the read-across approach from the analogue fenchol, the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol in liver microsomes of induced wistar rats is expected to be enhanced 1.2 times for phenobarbital and be the same for 3-methylcholanthrene compared to controls.

Executive summary:

The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male Wistar rats. Four rats received intraperitoneal doses of 80 mg/kg phenobarbital (PB) in 0.9% NaCl daily for 4 days and corresponding control animals received 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose. Another group of 4 rats received 3-methylcholanthrene in corn oil, as a single injection of 80 mg/kg on the first day and these animals were killed on day 5. The orresponding controls received corn oil only. After sacrifice of animals the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.25 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.2 times for phenobarbital whereas showed no difference for 3-methylcholanthrene compared to controls. The read-across was applied and the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol is expected to be enhanced 1.2 times for phenobarbital and be the same for 3-methylcholanthrene compared to controls.

Description of key information

Weight of Evidence: In vitro human study on (+)-Fenchol. The test item was oxidized to fenchone by human liver microsomal P450 enzymes, with CYP2A6 identified as the major enzyme involved.

Weight of Evidence: In vitro rat study on (+)-Fenchol. The test item was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats, with CYP2B1 identified as the major enzyme involved.

Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue fenchol, the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol in liver microsomes of phenobarbital-treated Gunn rats is expected to be enhanced 1.4 times over the control group.

Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue fenchol, the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol in liver microsomes of induced Wistar rats is expected to be enhanced 1.2 times for phenobarbital and be the same for 3-methylcholanthrene compared to controls.

Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue fenchol, the activity of UDP-glucuronosyltransferase of 0.175 mM of d-alpha fenchol in liver microsomes of phenobarbital-treated guinea pigs is expected to be enhanced 1.2 times over the control group.

Additional information

Weight of evidence: In vitro human study on (+)-Fenchol. The metabolism of the test item was studied by the analysis of incubations of in vitro-prepared human liver microsomes. Incubations were carried out at 37ºC for 30 min and contained human liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 mM test item in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1. The biotransformation of the test item was investigated by gas chromatography-mass spectrometry (GC-MS). Under these conditions, the test item was found to be oxidized to fenchone, with CYP2A6 identified as the major enzyme involved.

Weight of evidence: In vitro rat study on (+)-Fenchol. The metabolism of the test item was studied by the analysis of incubations of in vitro liver microsomes of phenobarbital-treated rats. Incubations were carried out at 37ºC for 30 min and contained rat liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 mM test item in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1. The biotransformation of the test item was investigated by gas chromatography-mass spectrometry (GC-MS). Under these conditions, the test item was found to be oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol, with CYP2B1 identified as the major enzyme involved.

Weight of Evidence: Read-across approach. The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male Gunn rats. Four rats received intraperitoneal doses of 80 mg/kg phenobarbital (PB) in 0.9% NaCl daily for 4 days. The control animals received 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose and the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.25 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.4 times over the NaCl control. The read-across was applied and the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol is expected to be enhanced 1.4 times over the control group.

Weight of Evidence: Read-across approach. The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male Wistar rats. Four rats received intraperitoneal doses of 80 mg/kg phenobarbital (PB) in 0.9% NaCl daily for 4 days and corresponding control animals received 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose. Another group of 4 rats received 3-methylcholanthrene in corn oil, as a single injection of 80 mg/kg on the first day and these animals were killed on day 5. The orresponding controls received corn oil only. After sacrifice of animals the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.25 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.2 times for phenobarbital whereas showed no difference for 3-methylcholanthrene compared to controls. The read-across was applied and the activity of UDP-glucuronosyltransferase of 0.25 mM of d-alpha fenchol is expected to be enhanced 1.2 times for phenobarbital and be the same for 3-methylcholanthrene compared to controls

Weight of Evidence: Read-across approach. The effect of the test item on UDP-glucuronosyltransferase specific activities was measured in male guinea pigs. Four animals received intraperitoneal doses of 20-40 mg/kg bw phenobarbital (PB) daily for 9 days. The control animals received a saline solution 0.9% NaCl alone. The animals were sacrificed 24 h after the last dose and the liver microsomes were prepared. Microsomal suspensions were activated with Triton X-100 and and incubated in water, Mulder and van Doorn medium (UDP-glucuronic acid, pyruvate kinase, phosphoenolpyruvate, NADH, lactate dehydrogenase, Tris–HCL buffer, MgCl2) and 0.175 mM fenchyl alcohol. UDP-glucuronosyltransferase activities were measured by a modification of Mulder and Van Doorn (1975) method. The activity was enhanced 1.2 times over the NaCl control. The read-across was applied and the activity of UDP-glucuronosyltransferase of 0.175 mM of d-alpha fenchol is expected to be enhanced 1.2 times over the control group.