7-methyl-3-methyleneocta-1,6-diene

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

other: enzyme induction

Principles of method if other than guideline:

The induction of liver monooxygenases after myrcene exposure was investigated in rats.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Source: FIOCRUZ Central Animal House breeding stock, Brazil
Body weight: 200 g
The animals were housed in standard rat cages and were kept under controlled temperature (23 ± 1°C), humidity (~70%) and dark/light cycle (lights on from 10:00 to 22:00 h). A pelleted diet (Nuvital, Nuvilab, Curitiba, Brazil) and tap water were available ad libitum.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

No data

Duration and frequency of treatment / exposure:

1 or 3 days

Remarks:

Doses / Concentrations:
1000 mg/kg bw

No. of animals per sex per dose / concentration:

2 to 7 animals/enzyme activity

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

None

Preliminary studies:

None

Details on absorption:

No data

Details on distribution in tissues:

No data

Details on excretion:

No data

Metabolites identified:

not measured

Details on metabolites:

No data

Table 1: monooxygenase activities after myrcene administration for 1 to 3 days in Wistar female rats

	Rate of O-dealkylation (pmol resorufin/min per mg protein)				ECOD (pmol 7-hydroxycoumarin/min per mg protein)	P450 2B1/2B2   (OD units µg protein)
Treatment	MROD	EROD	PROD	BROD
Control	57.9 ± 21.7 (4)	61.6 ± 15.5 (4)	7.9 ± 3.2 (4)	14.1 ± 4.0 (4)	802.5 ± 220.0 (6)
Myrcene (1 day)	314.5 ± 141.7* (5)	92.4 13.0* (4)	182.8 ± 55.2* (4)	184.8 ± 67.2* (4)	1671.7 ± 499.3* (6)
Inducing factor	5.43	1.50	23.13	13.10	2.08
Control	44.2 ± 14.7 (4)	57.7 ± 6.3 (6)	7.3 ± 3.3 (5)	9.9 ± 1.0 (4)		0.34 ± 0.01 (2)
Myrcene (3 days)	142.5 ± 44.4* (6)	79.3 ± 33.1* (7)	166.3 ± 50.0*(6)	333.9 ± 97.7* (5)		2.80 ± 0.22* (3)
Inducing factor	3.22	1.37	22.78	33.73		8.24

Data are shown as means ± S.D. ( ) Number of animals used. Statistical comparisons between control and treated groups were made by Student's t-test (*P< 0.05). Substrates: methoxyresorufin (MROD), ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD).

Levels of P4502B1/2B2 were determined by immunoblotting with antibodies against P450 2B1.

The induction of CYP2B isoenzymes was confirmed by SDS-PAGE and immunoblotting.

Conclusions:

Myrcene is an inducer of CYP2B subfamily isoenzymes in the rat.

Executive summary:

The induction of liver monooxygenases after myrcene exposure was investigated in rats. Female Wistar rats were treated by gavage with myrcene (1000 mg/kg body weight) or corn oil (vehicle) for 1 or 3 consecutive days. Activities of ethoxycoumarin-O-deethylase (ECOD) and alkoxy-resorufin O-dealkylases (methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD) and benzyloxy-resorufin-O-dealkylation(BROD)) were determined fluorimetrically in the hepatic microsomal fraction. Exposure to myrcene, either for 1 or 3 days, produced marked (13 to 34-fold) increases in the activities of PROD and BROD and only minor changes in ECOD, EROD and MROD. Since pentoxyresorufin and benzyloxyresorufin are metabolized mainly by CYP2B isoenzymes, these results suggest that myrcene induces this phenobarbital-inducible P450 subfamily. The induction of CYP2B isoenzymes was confirmed by SDS-PAGE and immunoblotting.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

other: enzyme inhibition

Principles of method if other than guideline:

The inhibition of liver CYP2B1 monooxygenase after myrcene exposure was investigated in rats.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

female

Vehicle:

DMSO

Duration and frequency of treatment / exposure:

No data

Remarks:

Doses / Concentrations:
0.02 to 1 µM

No. of animals per sex per dose / concentration:

No data

Preliminary studies:

None

Details on absorption:

No data

Details on distribution in tissues:

No data

Details on excretion:

No data

Metabolites identified:

not measured

Details on metabolites:

None

Table 1: Inhibitory effects of beta-myrcene on microsomal PROD and EROD activities

	Beta-myrcene	Rate of O-dealkylation (pmoles of resorufin x mg protein x min^-1)	Percentage of activity	IC50(µM)
PROD	0	1889±319	100	0.14
	0.02	1209±62*	64
	0.2	853±98*	45
	1.0	269±33*	14
EROD	0	224±25	100	> 50.0
	0.2	208±12	92
	5.0	179±22*	79
	10.0	171±13*	76
	50.0	155±13*	69

Conclusions:

Myrcene is an inhibitor of PROD activity but not of EROD activity in vitro.

Executive summary:

The effect of myrcene on the activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP4501A1, and pentoxyresorufin-O-depenthylase (PROD), a selective marker for CYP2B1, was investigated in liver microsomes of untreated rats. Results revealed that myrcene had almost no effect on EROD (IC50> 50 μM), but produced a concentration-dependent inhibition of PROD activity (IC50 = 0.14 μM).The analysis of alterations produced by myrcene on PROD kinetic parameters (Lineweaver-Burk plot) suggested that inhibition is competitive (Ki = 0.14μM).

As induction of CYP2B1 by myrcene was demonstrated in another study (De Oliveira a, 1997), it can be concluded that myrcene is a substrate – and a competitive inhibitor – of this isoenzyme.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well described study giving reliable information on several terpenes in vivo metabolism in rabbit.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

Albino rabbits were orally administered test item and urine was collected for 3 days for identification of urinary metabolites.

GLP compliance:

no

Radiolabelling:

no

Species:

rabbit

Strain:

other: albino (Japanese White)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Miyamoto Jikken Dobutsu, Hiroshima, Japan
- Weight at study initiation: 2-3 kg
- Fasting period before study: for 2 days before experiment
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Oriental RC-4, ad libitum
- Water (e.g. ad libitum): ad libitum

Route of administration:

oral: gavage

Vehicle:

other: 100 mL water containing 0.1 g Tween 80

Details on exposure:

Rabbits were administered 20 mL solution through stomach tube followed by 20 mL water, corresponding to 400-700 mg/kg bw.

Duration and frequency of treatment / exposure:

Once

Remarks:

Doses / Concentrations:
400-700 mg/kg bw

No. of animals per sex per dose / concentration:

6

Control animals:

no

Positive control reference chemical:

None

Details on study design:

None

Details on dosing and sampling:

The urine was collected daily for 3 days after drug administration and stored at 0-5°C until time of analysis.

Extraction of urinary metabolites:
The urine was adjusted to pH 4.7 with acetate buffer and incubated with beta-glucuronidase-arylsulfatase (3 mL/1000 mL of the fresh urine) at 37°C for 48 h, followed by continuous ether extraction for 48 h. The ether extracts were washed with 5% NaHCO3 and 5% NaOH to remove the acidic and phenolic fractions, respectively, and dried (magnesium sulfate). Ether was evaporated under reduced pressure to give neutral metabolites. The neutral metabolites were chromatographed on a column containing 100 g of silicic acid (200 mesh). Elution was started with n-hexane, and n-hexane-ethyl acetate mixtures (95:5, 90:10, 85:15, 70:30, and 50:50) were used as subsequent eluents. The acidic metabolites were recovered from the sodium bicarbonate layer by acidification with 5% HCl, followed by ether extraction. The ether extracts were esterified with diazomethane in ether or with dimethyl sulfate in the presence of potassium carbonate in anhydrous acetone. These esters of the acidic metabolites also were chromatographed in the same manner as the neutral metabolites.

Identification of urinary metabolites:
Purification by silicic acid gave pure metabolites. When necessary, metabolites were isolated by preparative TLC or GLC. Structure determination or identification was based on spectral data and chemical transformations.

Statistics:

None

Preliminary studies:

None

Details on absorption:

None

Details on distribution in tissues:

None

Details on excretion:

None

Metabolites identified:

yes

Details on metabolites:

The main urinary metabolites from myrcene were myrcene-3(10)-glycol, uroterpenol, myrcene-1,2-glycol, 2-hydroxymyrcene-1-carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid.

None

Conclusions:

The main urinary metabolites of myrcene were myrcene-3(10)-glycol, uroterpenol, myrcene-1,2-glycol, 2-hydroxymyrcene-1-carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid.

Executive summary:

The biotransformation of myrcene was studied in albino rabbits orally administered at 400-700 mg/kg bw of myrcene in water with 0.1% Tween 80.

Urine was collected daily for 3 days and urinary metabolites were identified. In this study, the main urinary metabolites from myrcene were myrcene-3(10)-glycol, uroterpenol, myrcene-1,2-glycol, 2-hydroxymyrcene-1-carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

Isolation and identification of urine metabolites after daily oral administration of myrcene for 20 days in rats.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

other: IISc.

Sex:

male

Details on test animals or test system and environmental conditions:

- Bodyweight: 180-200g
After dosing, control and experimental rats were housed separately in metabolism cages with free access to food (animal food from Hindustan Lever, India) and water.

Route of administration:

oral: gavage

Vehicle:

other: 1% methyl cellulose solution

Details on exposure:

Control rats were given only the vehicle (4 mL/kg bw).

Duration and frequency of treatment / exposure:

20 days

Remarks:

Doses / Concentrations:
800 mg/kg bw/day

No. of animals per sex per dose / concentration:

No data

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

None

Details on study design:

No data

Details on dosing and sampling:

After dosing, control and experimental rats were housed separately in metabolism cages with free access to food (animal food from Hindustan Lever, India) and water. Urine was collected in bottles maintained at 0-4°C.

Extraction of urinary metabolites
Urines collected daily for 20 days following oral administration of beta-myrcene to rats were adjusted to pH3-4 with 1-M HCI and extracted three times with ether. The aqueous portion containing conjugated metabolites was then subjected to acid hydrolysis (pH 3-4, refluxed for 6 h) and extracted with ether. The ether extracts of the acidified urine and of the hydrolysed aqueous portion were separated into neutral and acidic fractions as described earlier (Chadha and Madyastha, 1984).

Thin-layer chromatography
TLC was carried out on silica gel G-coated plates (0.25 mm) developed with hexane-ethyl acetate as the solvent systems. The compounds on the chromatogram were visualized either by exposing the plates to I2 vapour or by spraying with 1% vanillin in 50% (v/v) H2SO4 followed by heating at 100°C for 5-10 min. The terpenoid compounds gave colours from pale blue to deep violet. Preparative TLC was carried out on silica gel G-coated plates (0.8 mm).

Gas chromatography
Analyses were carried out on a Chemito model 3800 instrument equipped with a hydrogen flame ionization detector. The chromatograph was fitted with a stainless-steel column containing 3% SE 30 on Chromosorb-W. N2 at a flow rate of 30 mL/min was used as the carrier gas. Methyl esters and neutral compounds were run at a column temperature of 140 and 160°C, respectively.

Spectra
NMR spectra were recorded either on a Varian T-60 or Bruker 270 MHz spectrometer. IR spectra were recorded on a Perkin Elmer Model 397 spectrophotometer. UV visible spectra were recorded with a Hitachi 557 double-beam double-wavelentgth spectrophotometer.

Statistics:

None

Preliminary studies:

None

Details on absorption:

None

Details on distribution in tissues:

None

Details on excretion:

None

Metabolites identified:

yes

Details on metabolites:

Metabolites isolated from the urine of rats after oral administration of beta-myrcene were: 10-hydroxylinalool, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid.

None

Conclusions:

Metabolites isolated from the urine of rats after oral administration of beta-myrcene were: 10-hydroxylinalool, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid.

Executive summary:

Urinary metabolites of myrcene were identified in rats orally administered at 800 mg/kg bw/d for 20 days. After dosing, experimental rats were housed separately in metabolism cages and urine was collected.

Metabolites isolated from the urine of rats after oral administration of beta-myrcene were: 10-hydroxylinalool, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid.

Description of key information

Metabolites isolated from the urine of rats after oral administration of myrcene were:

10-hydroxylinalool, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid.
The main urinary metabolites from myrcene were myrcene-3(10)-glycol, uroterpenol, myrcene-1,2-glycol, 2-hydroxymyrcene-1-carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid in rabbits.
Myrcene was found to be a substrate for CYP2B isoenzymes in rats.

Additional information

Myrcene has a molecular weight of 136 (less than 500) and log P > 4 (=5.29). Therefore, it can be well absorbed by dermal route, particularly in the stratum corneum.

Metabolites isolated from the urine of rats after oral administration of myrcene were identified: 10-hydroxylinalool, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid (Madyastha et al., 1987). Also, the main urinary metabolites from myrcene were myrcene-3(10)-glycol, uroterpenol, myrcene-1,2- glycol, 2-hydroxymyrcene-1-carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid in rabbits after oral exposure (Ishida et al., 1981).

Myrcene was found to be a CYP2B isoenzymes inducer in rats after oral exposure and was shown to be a potent competitive inhibitor of PROD activity (with pentoxyresorufin as substrate - selective marker for CYP2B1) in vitro in phenobarbital-induced liver microsomes (De-Oliveira et al., 1997 a,b). These results provide support that myrcene is a substrate for CYP2B1 isoenzyme.

Thus, myrcene is absorbed by oral route and is metabolised in the liver into water-soluble compounds eliminated in urine.

These data are supported by unpublished data by J. Webb at al., described in Delgado et al., 1993. In non-pregnant female rats orally administered 1.0 g/kg bw, blood levels as high as 14.1± 3.0μg/mL (peak value) were detected 60 min after a single oral dose. At this dose-level, myrcene plasmatic half-life was 285 min.

Discussion on bioaccumulation potential result:

Myrcene has a molecular weight of 136 (less than 500) and log P > 4 (=5.29). Therefore, it can be well absorbed by dermal route, particularly in the stratum corneum.

Metabolites isolated from the urine of rats after oral administration of beta-myrcene were identified: 10-hydroxylinalool, 7-methyl-3-methylene-oct-6-ene-1,2-diol, 1-hydroxymethyl-4-isopropenyl cyclohexanol, 10-carboxylinalool and 2-hydroxy-7-methyl-3-methylene-oct-6-enoic acid (Madyastha et al., 1987). Also, the main urinary metabolites from myrcene were myrcene-3(10)-glycol, uroterpenol, myrcene-1,2-glycol, 2-hydroxymyrcene-1-carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid in rabbits after oral exposure (Ishida et al., 1981).

Myrcene was found to be a CYP2B isoenzymes inducer in rats after repeated oral exposure and was shown to be a potent competitive inhibitor of PROD activity ( with pentoxyresorufin as substrate - selective marker for CYP2B1) in vitro in phenobarbital-induced liver microsomes (De-Oliveira et al., 1997 a,b). These results provide support that myrcene is a substrate for CYP2B1 isoenzymes.

Thus, myrcene is absorbed by oral route and is metabolised in the liver into water-soluble compounds eliminated in urine.

These data are supported by unpublished data by J. Webb at al., described in Delgado et al., 1993. In non-pregnant female rats orally administered 1.0 g/kg bw, blood levels as high as 14.1± 3.0μg/mL (peak value) were detected 60 min after a single oral dose. At this dose-level, myrcene plasmatic half-life was 285 min.