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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is published in a peer-reviewed jouranl without GLP compliance.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000
Report date:
1999

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Metabolic inactivation of the test substances in vitro by human rat and mouse lung and liver subcellular fractions.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-epoxypropyl o-tolyl ether
EC Number:
218-645-3
EC Name:
2,3-epoxypropyl o-tolyl ether
Cas Number:
2210-79-9
Molecular formula:
C10H12O2
IUPAC Name:
2-[(2-methylphenoxy)methyl]oxirane
Constituent 2
Reference substance name:
Oxirane, 2-[(2-methylphenoxy)methyl]-
IUPAC Name:
Oxirane, 2-[(2-methylphenoxy)methyl]-
Test material form:
other: Liquid at room temperature.
Details on test material:
The test substance, 2,3-epoxypropyl o-tolyl ether also known as o-Cresyl glycidyl ether (o-CGE) was synthesized from the appropriate alcohol and epichlorohydrin (ECH). The corresponding 14C-labelled o-CGE was synthesized from the alcohol and [U-’4C]-ECH, diluted with non-labelled ECH to a specific activity of 0.2 mCi/mmol. Purity was > 92%.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
Male Fisher 344 rats (~ 250 gm) and male C3H mice (~ 25 gm) were from the Central Laboratories for the Blood Banks (CLB) (Amsterdam, The Netherlands). Animals were kept on a 12 h light/dark cycle in humidity- (60 ± 15% relative humidity) and temperature- (22 ± 2 °C) controlled mass-air-displacement rooms and following an acclimation of 1 week were used as the source for rodent skin, liver and lung tissue. A standard rodent diet (VRF-I, Broekman lnstituut (Charles River Labaratories), Someren, The Netherlands) and de-ionized water were supplied ad libitum.

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
acetone
Details on exposure:
The test substance, 0-CGE was dissolved in acetone, aliquots were transferred into 1.5 ml vials and the acetone was carefully evaporated with a gentle stream of nitrogen. Phosphate buffer (0.1 M, pH 7.40), containing L-Asp-L-Phe as internal standard, was deoxygenated with He for 10 min. GHS was dissolved in the deoxygenated phosphate buffer. Linearity of the GSH conjugation with protein concentration was tested by incubation during 10 mm with varying amounts of liver cytosol protein ranging from 0.2 to 5.0mg protein/mI, Linearity with time was investigated by incubation during 0.5, 1, 2, 5, 10, 15,20 and 30 mm with a liver cytosol protein concentration of I mg/ml.

To follow Epoxide hydrolase (hydrolysis) activity incubation was carried out with both microsomal and cytosolic fractions. Following pre-incubation of the subcellular fractions, the reaction was started by addition of o-CGE dissolved in DMS0. For HPLC analysis, the incubation was stopped by addition of an equal volume of cold methanol (kept on dry ice), mixing and precipitation of the denaturated protein by centrifugation (2400g, 15 mm). Linearity of the hydrolytic reactions with protein concentration was tested by incubation during 10 mm with varying amounts of liver microsomal protein. Linearity with time was investigated by incubation during 0.5, 1,2, 5, 10, 15, 20 and 30 mm with a liver microsonle concentration of I mg protein/mi.
Duration and frequency of treatment / exposure:
One time only for up to 30 minutes.
Doses / concentrations
Remarks:
Doses / Concentrations:
0,0.041,0.12,0.37,1.11,3.33 and 10.0 mn.
No. of animals per sex per dose / concentration:
No data
Control animals:
no
Positive control reference chemical:
No
Details on study design:
Rodent subcelluar tissue fractions were prepared following anaesthetized with sodium pentobarbital, the abdomen was opened and the abdominal
aorta cut. The animals were subsequently perfused in situ with ice-cold isotonic 0.05 M TRIS-buffered (pH 7.40) 0.15 at potassium chloride solution. Livers and lungs were excized, weighed and placed on ice. The lungs were minced with scissors and pulverised in liquid nitrogen using a hammer mill (6700 Freezer/Mill, Glen Creston, Inc., Stanmore, UK). Livers were only minced with scissors. Lung powder and minced livers were homogenized in TRIS-buffered (pH 7,40) isotonic KCI solution with six passes (1100 rpm) of a Teflon—glass homogenizer (Braun). Homogenates were centrifuged at 10 000g at 4°C for 20 mm, the lipid layer was removed and the supernatant was centrifuged at 105 000g at 4°C for 60 miii. The supernatant (cytosolic fraction) was stored frozen at —80 °C. The pellet was dispersed with four passes of a Teflon—glass homogenizer in 0.05 at TRIS buffer (pH 7.40) containing 0.25 is sucrose and 1 mM EDTA and centrifuged again at 105 000g at 4°C for 60 mm. The supernatant was discarded and the pellet resuspended with four passes of a Teflon—glass homogenizer in 0.1 is potassium phosphate buffer (pH 7.40) containing 0.25 M sucrose (microsomal fraction). The microsomes were stored at —80 °C, Subcellular fractions were used within 6 months of preparation.
Protein concentrations of all lung and liver subcellular preparations of rodents were assayed using the modified micro-Lowry assay for total micro-protein. Human liver and lung aubcellular preparations were assayed for total micro-protein using the Coomassie blue method (all Sigma).

Human liver and lung microsomes, prepared by standard differential centrifugation methods (Boogaard et al. 1996), were from the Human Cell Culture Center (Laurel, MD, USA).
Details on dosing and sampling:
No data, study was in vitro.
Statistics:
Linear regression analysis using the least squares approximation was performed as needed.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Kinetic constant for Glutathione conjugation
Organ Species Vmax
Liver Human 84.5
Rat 61.3
Mouse 150
Lung Human 50
Rat 29
Mouse 29.5

Oxidized glutathione and GSH congugated o-CGE were detected. The glutathione-S-transferase conjugation of o-CGE was linera for approximately 15 minutes under the conditions of the study. GSH conjugation in lung cytosol was lower that in liver cytosol.

Kinetic constant for Epoxide Hydrolase Hydrolysis
Organ Fraction Species Vmax
Liver cytosol Human 24.5
Rat 3.9
Mouse 16.8
microsomes Human 133
Rat 30.6
Mouse 12.0

Lung cytosol Human 1.0
Rat 1.0
Mouse 1.6
microsomes Human 15.2
Rat 5.6
Mouse 29.7

The data suggest relatively low Epoxide hydrolase activity in human and lug cytosol. Generally, microsomal hydrolysis was substantially greater thancytosol fraction hydrolysis. Human liver microsome Epoxide hydrolase activity was approximately an order-of-magnitude greater than rodent derive Epoxide hydrolase. However, mouse lung Epoxide hydrolase was approximatelt two-fold more active than human Epoxide hydrolase.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Generally, conjucation of o-CGE (2,3-epoxypropyl o-tolyl ether ) to glutathione by Glutathione-S-transferase by rodent and human subcellular cytosol fractions from the liver and lung was similar. Hydrolysis of o-CGE by Epoxide hydrolase was the most efficient means of detoxification. Human liver Epoxide hydrolase was approximately 4-10-fold more efficient than rodent Epoxide hydrolase in the hydrolysis of o-CGE. Mouse lung Epoxide hydrolase was approximately 2-fold more efficient compared to human lung Epoxide hydrolase. Generally, rat Epoxide hydrolase was the least efficient in the detoxification of o-CGE.
Executive summary:

The detoxification of o -CGE (2,3 -epoxypropyl o-tolyl ether) was assessed in vitro in human, rat and mouse subcellular cytosolic and microsome fractions. The rate of o-CGE conjugation to glutathione by glutathione-S-trasnferase and of hydrolysis by Epoxide hydrolase was determined. Generally, conjucation of o-CGE (2,3-epoxypropyl o-tolyl ether ) to glutathione by Gultathione-S-transferase by rodent and human subcellular cytosol fractions from the liver and lung was similar. Hydrolysis of o-CGE by Epoxide hydrolase was the most efficient means of detoxification. Human liver Epoxide hydrolase was approximately 4-10-fold more efficient than rodent Epoxide hydrolase in the hydrolysis of o-CGE. Mouse lung Epoxide hydrolase was approximately 2-fold more efficient compared to human lung Epoxide hydrolase. Generally, rat Epoxide hydrolase was the least efficient in the detoxification of o-CGE.