Registration Dossier

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 well conducted and documented. However, study was not conducted according to a testing guideline or under the GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
Use of in vitro systems to generate test substance metabolites with detection by analytical methods.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid at room temperature.
Details on test material:
The test substance was (2,3-epoxypropyl)-3-carboxy-3,6-dimethyl heptanoate, a dominate isomer of 2,3-epoxypropyl neodecanoate. Pure (2,3-epoxypropyl)-3-carboxy-3,6-dimethyl heptanoate was synthized specifically for these metabolic studies.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Male C3H mice (~ 25 gm) were also used in the study. Fresh full thickness healthy human breast skin was obtained from patients undergoing mammoplastic reduction surgery in a regional hospital, MCA, Alkmaar, The Netherlands. Human liver and lung microsomes, prepared by standard methods, were purchased from the Human Cell Culture Center, Laurel, MD, USA. Fisher 344 rats (approximately 250 g) and male C3H mice (approximately 25 g) were obtained 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 period of 1 week were used as the source for skin, liver and lung tissue. A standard rodent diet and deionized water were supplied ad libitum.

Administration / exposure

Route of administration:
other: Test substance was administered in vitro to cell free preparations.
Vehicle:
DMSO
Details on exposure:
2,3-Epoxypropyl neodecanoate isomer was dissolved in DMSO at a concentration of 10 mM. After dissolution an equal volume of a 20 mM solution of 3H-GSH, freshly prepared in deoxygenated 100 mM phosphate buffer test substance isomer was dissolved in DMSO at a concentration of 10 mM. The mixture was kept at room temperature for 18 h and aliquots were analysed by H PLC. For enzyme activity cytosol was diluted with de-oxygenated phosphate buffer to the desired final protein concentration and incubated in a final volume of 50 pL at 37°C in 2.0 ml amber screw-cap vials with inhibitors using a shaking waterbath. After preincubation for about 3 mm, 45 uL 3H-GSH dissolved in deoxygenated phosphate buffer was added. After another minute of preincubation, the reaction was initiated by addition of 5 uL test substance isomer dissolved in DMSO. The final concentrations of 2,3-epoxypropyl neodecanoate isomer were 0, 0.041, 0.12, 0.37, 1.11, 3.33 and 10.0mM and the final concentration GSH was 10 mM (final volume 100 itI). Incubation was stopped by the addition of an equal volume of ice- cold methanol (kept on dry ice) and the precipitated protein pelleted (10 mm, 2500 g, 4°C). Aliquots of the supernatant were analysed by HPLC with on line radioactivity detection. To access hydrolysis of ester and epoxide functional groups lncubations were carried out with both microsomal and cytosolic fractions. The final concentrations of test substance isomer ranged from 0.041 to 50.0 mM. The maximum concentration of DMSO in the final incubation mixture was 5% v/v.




Duration and frequency of treatment / exposure:
For enzyme kinetics and hydrolysis the incubation times were 0.5, 1, 2, 5, 10, 15, 20 and 30 minutes.
Doses / concentrations
Remarks:
Doses / Concentrations:
The final concentrations of 2,3-epoxypropyl neodecanoate isomer for enzyme rate analysis were 0, 0.041, 0.12, 0.37, 1.11, 3.33 and 10.0mM. For assessment of epoxy and ester hydrolysis the final concentrations of the test substance isomer ranged from 0.041 to 50.0 mM.
No. of animals per sex per dose:
Study was conducted in vitro.
Control animals:
no
Positive control:
No data
Details on study design:
Tissues collected from rodents were processed to cytosol and microsomes. The animals were perfused in situ with ice-cold isotonic 0.05 M TRIS-buffered (pH 7.40) 0.15 M potassium chloride solution. Livers and lungs were excised, weighed and placed on ice while the skin was removed. The skin and lungs were minced with scissors and pulverised in liquid nitrogen using a hammer mill. Livers were only minced with scissors. Skin powder, lung powder and minced livers were homogenized in TRIS buffered (pH 7.40) isotonic KCI solution with six passes (1,100 rpm) of a teflon-glass homogenizer (Braun). Homogenates were centrifuged at 10,000 g at 4°C for 20 mm, the lipid layer was removed and the supernatant was centrifuged at 105,000 g at 4°C for 60 mm. 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 M TRIS buffer (pH 7.40) containing 0.25 M sucrose and 1 mM EDTA and centrifuged again at 105,000 g 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 M 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; there was no apparent loss of activity over this period. Protein concentrations of all skin subcellular preparations and lung and liver subcellular preparations of rodents were assayed using the modified micro-Lowry assay for total micro-protein (Sigma Chemical Ca). For enzyme kinetic analysis cytosol was diluted with de-oxygenated phosphate buffer to the desired final protein concentration and incubated in a final volume of 50 uL at 37°C in 2.0 ml amber screw-cap vials with inhibitors using a shaking waterbath. After preincubation for about 3 mm, 45 uL 3H-GSH dissolved in deoxygenated phosphate buffer was added. After another minute of preincubation, the reaction was initiated by addition of 5 uL test substance isomer dissolved in DMSO. Incubation was stopped by the addition of an equal volume of ice- cold methanol (kept on dry ice) and the precipitated protein pelleted (10 mm, 2500 g, 4°C). Aliquots of the supernatant were analysed by HPLC with on line radioactivity detection and fractions (0.5-1.0 ml) were collected for liquid scintillation counting. Linearity of the GSH conjugation with protein concentration was tested by incubation during 10 mm with varying amounts of liver cytosol protein. Linearity with time was investigated by incubation during 0.5, 1, 2, 5, 10, 15, 20 and 30 min. with a liver cytosol protein concentration of I mg/mL. To access the hydrolysis of the epoxide and ester finctional groups incubations were carried out with both microsomal and cytosolic fractions. Following pre-incubation with inhibitors, the reaction was started by addition of 5 uL test substance isomer dissolved in DMSO. The final concentrations of test substance isomer ranged from 0.041 to 50 mM. Linearity of the hydrolytic reactions with protein concentration was tested by incubation during 10 min. 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 min. with a liver microsome concentration of I mg protein/mL. For GC-MS analysis, the incubation was stopped by addition of 800 u1 ice-cold DCM containing and immediate mixing on a vortex mixer. For HPLC analysis, the incubation was stopped by addition of an equal volume of ice-cold methanol (kept on dry ice), mixing and precipitation of the protein by centrifugation (2400 g, 15 mm).

Details on dosing and sampling:
Dosing was conducted in vitro. The final concentrations of 2,3-epoxypropyl neodecanoate isomer for enzyme rate analysis were 0, 0.041, 0.12, 0.37, 1.11, 3.33 and 10.0mM. For assessment of epoxy and ester functional group hydrolysis the final concentrations of the test substance isomer ranged from 0.041 to 50.0 mM. For enzyme kinetics and functional group hydrolysis the incubation times were 0.5, 1, 2, 5, 10, 15, 20 and 30 minutes.
Statistics:
The formation of GSH conjugates or hydrolysis products in the incubation mixtures was fitted to the Michaelis-Menten equation, with correction for nonenzymatic GSH conjugation, using the linear least- squares approximation.

Results and discussion

Preliminary studies:
No data

Toxicokinetic / pharmacokinetic studies

Details on absorption:
No data, in vitro study.
Details on distribution in tissues:
No data, in vitro study.
Transfer into organs
Test no.:
#1
Transfer type:
other: No data from in vitro study.
Observation:
not determined
Remarks:
Data not available from in vitro study.
Details on excretion:
No data, in vitro study.
Toxicokinetic parameters
Test no.:
#1
Toxicokinetic parameters:
other: Enzymatic GSH conjugation with test substance isomer.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
When 2,3-epoxypropyl neodecanoate isomer was incubated with GSH in the presence of hepatic cytosol (1 mg protein/mi) a HPLC peak with the same retention time as a standard GSH-test substance isomer appeared within a few min. LC-MS analysis confirmed that the peak in both the intrinsic and enzymatic reactions was the GSH conjugate of 2,3-epoxypropyl neodecanoate isomer.

Any other information on results incl. tables

The GSH conjugation with 2,3 -epoxypropyl neodecanoate isomer followed Michaelis-Menten kinetics. The overall hydrolytic detoxification of the epoxide and ester functional groups is highly complex because the extent to which the enzymatic hydrolysis was catalysed by carboxylesterase (CE) or epoxide hydrolyase (EH) varied considerably between microsomes and cytosol and also between the various organs and among the three species. Hepatic microsomal CE activities were roughly equal in all three species, but dermal and pulmonary CE activities were an order of magnitude higher in rodents than in human. Hepatic cytosolic CE activities towards test substance isomer were similar in rats and humans and an order of magnitude lower in mice. CE activities in pulmonary cytosol were similar in rodents and 1-2 orders of magnitude lower in humans.

Microsomal EH activities were similar in the liver of the three species. Dermal as well as pulmonary microsomal EH activities were similar in rats and humans. In mice, dermal microsomal EH activity was too small to be determined but pulmonary EH was much higher than in either rat or human. Hepatic cytosolic EH activities were similar in rats and mice but an order of magnitude higher in mice. Mouse skin cytosolic EH activity was 2 orders of magnitude higher than rat skin and 1 order of magnitude higher than human skin. Rodent pulmonary cytosolic EH activities were similar and 1 order of magnitude higher than in humans.

An overall assessment of the enzymztic kinetic data suggest that in general the efficiency of the total enzymatic hydrolysis of 2,3 -epoxypropyl neodecanoate ester and epoxide functional groups decreases in the following order: mice> rats> humans.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
In all the in vitro systems derived from rat, mouse and human tissue the 2,3-epoxypropyl neodecanoate was rapidly detoxified by one of the most common detoxification pathways for epoxides, GSH conjugation. In vitro hepatic GSH conjugation was higher than pulmonary, and pulmonary GSH conjugation was higher than dermal GSH conjugation in all species. Overall, GSH conjugation in humans was low compared with rodents. Both in microsomal and cytosolic fractions the test substance isomer functional groups were rapidly hydrolysed. In general, mouse preparations were more efficient than rat, and rat preperations were more efficient than human. Also the hydrolytic capacity for detoxifying 2,3-epoxypropyl neodecanoate isomer outweighs the GSH conjugation reaction. Based on these in vitro data, an estimation was made of the in vivo clearance due to GSH conjugation and hydrolysis of the test substance isomer. In all species and organs, GSH conjugation plays only a minor role compared with CE and EH catalysed hydrolysis. The estimates also indicate that the in vivo clearances are roughly similar in rats and mice. The in vivo clearance by human tissues, however, is much slower than in rats and mice, especially in lung and skin being approximately an-order-of-magnitude lower.
Executive summary:

The findings from in vitro metabolism studies conducted with cell-free tissue perparations from human, rat and mouse, liver, lung and skin demonstrated that an isomer of 2,3 -epoxypropyl neodecanoate was rapidly detoxified. The predominate pathway of detoxication was epoxide hydrolase and carboxylesterase hydrolysis relative to glutathione conjugation. Estimation of in vivo clearance based on the in vitro kinetic data and scaling suggested that the human detoxication rate was approximately an-order-of-magnitude slower relative to rodents.