Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well-documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to identify species differences in toxic metabolite (epoxide) formation and detoxification in rodents and humans. The in vitro metabolism of beta-chloroprene (2-chloro-1,3-butadiene, CD) was studied in liver microsomes of B6C3F1 mice, Fischer/244 and Wistar rats, Syrian hamsters and humans.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Beta-chloroprene (CD >99% purity) inhibited with phenothiazine and N-nitroso-diphenylamine was supplied by DuPont-Dow Elastomers L.L.C (LaPlace, LA). Special care was used for handling and storage because dimerization, oxidation and atuo-polymerization of liquid CD will occur if exposed to air at room temperature. The inhibitors were removed prior to the study by passage through activated alumina (D-37269, ICN Biomedicals, GmbH, Germany) under N2 atmosphere. The CD was collected in glass vials, sealed with silicone-Teflon-lined septa and stored in an air-tight can at -70°C. Opened vials were re-sealed with a blanket of N2. CD stored under these conditions has remained > 98% pure for 2+ years based on analysis by gas chromatography-flame ionization detection.
Radiolabelling:
no

Test animals

Species:
other: mice, rats or Golden Syrian hamsters
Strain:
other: male B6C3F1 mice, Fischer 344 rats, Wistar rats or Golden Syrian hamsters
Sex:
not specified
Details on test animals and environmental conditions:
PREPARATION OF MICROSOMES AND CYTOSOL
Microsomes and cytosol were prepared by differential centrifugation of livers pooled from male B6C3F1 mice, Fischer 344 rats, Wistar rats or Golden Syrian hamsters (Charles River Laboratories, Raleigh, NC). The cytosolic fraction was saved, and the microsomal pellet was washed with, and suspended in, 0.25M sucrose, 50 mM Tris, 5.4 mM EDTA, pH 7.4. The cytosolic and microsomal fractions were stored at < -70 °C. Protein concentrations were measured using the Bio-Rad Protein Assay kit and human liver microsomes were purchased as a mixed pool from 15 different individuals.

Administration / exposure

Details on exposure:
N/A
Duration and frequency of treatment / exposure:
N/A
Doses / concentrations
Remarks:
Doses / Concentrations:
N/A
No. of animals per sex per dose:
N/A
Positive control:
N/A
Details on study design:
SYNTHESIS, IDENTIFICATION AND METABOLISM OF CEO
SYNTHESIS OF CEO
The (1-chloroethenyl)oxirane (CEO, CAS 3132-77-2, bp 110 °C) was synthesised according to published methods [Rauleder et al., (1978); Rauleder et al., (1979)] by oxidation of 3,4-dichloro-1-buene with 3-chloro-benzenecarboperoxic acid to form the intermediate 3,4-dichloro-1-epoxybutane.
IDENTIFICATION OF CEO
Qualitative identification of CEO as an oxidative metabolite of CD was conducted using incubation methods similar to those published by Csanady et al., (1992).
METABOLISM OF CEO
Samples were analysed using the GC/MSD headspace method and the potential for species differences in the in-vitro metabolism of CEO was compared with liver microsomes from the five animals and liver cytosol from four rodent species.
HEADSPACE ANALYSIS OF CD AND CEO
The concentration of CEO in the headspace was quantified by GC/MSD.
LIQUID-TO-AIR PARTITIONING OF CD AND CEO IN LIVER MICROSOMAL SUSPENSIONS
Partition coefficients were determined using a modification of a described tonometry method [Eger, (1987), Lerman et al., (1985) and Lerman et al., (1986)].
Details on dosing and sampling:
N/A
Statistics:
Not performed.

Results and discussion

Preliminary studies:
N/A

Toxicokinetic / pharmacokinetic studies

Details on absorption:
N/A
Details on distribution in tissues:
N/A
Details on excretion:
N/A

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Microsomal oxidation of CD in the presence of NADP+, extraction with diethyl ether, and analysis by GC-mass selective detection (MSD) indicated that (1-chloroethenyl) oxirane (CEO) was an important metabolite of CD in the liver microsomal suspensions of all species studied. Other potential water-soluble oxidative metabolites may have been present.

Any other information on results incl. tables

This report is the first to identify CEO as an epoxide metabolite of CD. This finding extends the results of an earlier study showing that hepatic microsomes produced a volatile metabolite of CD suggested to be an epoxide [Bartsch et al., (1979)]. The oxidation of CD to CEO was evident in rodent and human liver microsomes and most likely involves catabolism by CYP 2E1, shown by nearly complete in-vitro inhibition with 4-MP. CYP 2E1 is an isozyme of cytochrome P450 oxidase involved in the metabolism of butadiene, isoprene and other small organic molecules. The inhibition of CD metabolism by 4 -MP is consistent with its use to inhibit the in-vivo uptake of 1,3 -butadiene [Medinsky et al., (1994)]. However, the in-vivo studies with 4 -MP and 1,3 -butadiene showed partial inhibition, most likely because of the activity of other sytochrome P450 isozymes such as CYP 2A6 [Duescher et al., 1994; Elfarra et al., (2001)]. Therefore it is possible that other cytochrome P450 isozymes in addition to CYP 2E1 will contribute to the metabolism of CD.

As part of metabolite identification, the relative amount of CEO present in microsomal incubations of each species was studies using 1 -butanol as an internal standard for extraction and GC-MSD analysis. Clearly all five species formed CEO (B6C3F1 mice, Fischer/344 rat liver microsomes > Wistar rat > human > hamsters).

In the current study, CEO was sufficiently volatile to be quantified in the headspace despite having a liquid to air partition coefficient of 58. A decline of CD was linked with the appearance of CEO in the vial headspace during an experiment using B6C3F1 mouse liver microsomes. The uptake of CD from the headspace is linked to CEO formation, but it is also possible that more polar metabolites of CD were formed but not detected by diethyl ether extraction or headspace analysis in the current study. An underlying assumption is that a 1 -to-1 stoichiometric relationship exists for conversion of the parent chemical to the oxidised metabolite, however this has not been confirmed by testing the use of a radiolabelled substrate.

Further metabolism of CEO was observed in liver microsomes, for example in mouse liver microsomes, the CEO concentration showed an initial increase over 10 min that was followed by a decline attributable to either epoxide hydrolase-mediated hydrolysis or further oxidative metabolism.

The interspecies differences in the susceptibility to CD-induced cancer between mice, rats and hamsters could be related to the balance of CEO formation and detoxification.

Generally, the preliminary in-vitro metabolism data presented here for mice, rats and humans were consistent with published observations for the initial metabolism of 1,3 -butadiene (BD) to epoxybutene (EB) [Csanady et al., (1992); Dahl and Henderson, (2000)]. BD is metabolised to EB and diepoxybutane (DEB) metabolites, and both epoxides exhibit species differences in the rate of reactions for hydrolysis by epoxide hydrolase and GSH conjugation by cytosolic GHS-S-transferase [Himmelstein et al., (1997)]. For EB, GSH conjugation was slower in humans than mice [Csanady et al., (1992)].

Isoprene, the 2-methyl analog of chloroprene and butadiene undergoes similar metabolism for which species differences in isoprene epoxidation also have been reported [Watson et al., (2001)]. It is not yet know if CEO can be metabolised to a diepoxide.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: Results suggest that metabolism may help explain species differences showing a greater sensitivity for CD-induced tumorigenicity in mice, for example, compared with hamsters.
The purpose of this study was to identify species differences in toxic metabolite (epoxide) formation and detoxification in rodents and humans. The in vitro metabolism of beta-chloroprene (2-chloro-1,3-butadiene, CD) was studied in liver microsomes of B6C3F1 mice, Fischer/244 and Wistar rats, Syrian hamsters and humans. Microsomal oxidation of CD in the presence of NADP+, extraction with diethyl ether, and analysis by GC-mass selective detection (MSD) indicated that (1-chloroethenyl) oxirane (CEO) was an important metabolite of CD in the liver microsomal suspensions of all species studied. Other potential water-soluble oxidative metabolites may have been present. The oxidation of CD was inhibited by 4-methyl pyrazole, an inhibitor of CYP 2E1. CEO was sufficiently volatile at 37 °C for vial headspace analysis using GC-MSD single ion monitoring (m/z = 39). CEO was synthesised and used to conduct partition measurements along with CD and further explore CEO metabolism in liver microsomes and cytosol. The liquid-to-air partition coefficients for CD and CEO in the microsomal suspensions were 0.7 and 58, respectively. Apparent species differences in the uptake of CEO by microsomal hydrolysis were hamster~human>rats>mice. Hydrolysis was inhibited by 1,1,1-trichloropropene oxide, a competitive inhibitor of epoxide hydrolase. A preliminary experiment indicated that the uptake of the CEP in liver cytosol by GSH conjugation was hamster>rats~mice (human cytosol not yet tested). In general, the results suggest that metabolism may help explain species differences showing a greater sensitivity for CD-induced tumorigenicity in mice, for example, compared with hamsters.
Executive summary:

The purpose of this study was to identify species differences in toxic metabolite (epoxide) formation and detoxification in rodents and humans. The in vitro metabolism of beta-chloroprene (2-chloro-1,3-butadiene, CD) was studied in liver microsomes of B6C3F1 mice, Fischer/244 and Wistar rats, Syrian hamsters and humans. Microsomal oxidation of CD in the presence of NADP+, extraction with diethyl ether, and analysis by GC-mass selective detection (MSD) indicated that (1-chloroethenyl) oxirane (CEO) was an important metabolite of CD in the liver microsomal suspensions of all species studied. Other potential water-soluble oxidative metabolites may have been present. The oxidation of CD was inhibited by 4-methyl pyrazole, an inhibitor of CYP 2E1. CEO was sufficiently volatile at 37 °C for vial headspace analysis using GC-MSD single ion monitoring (m/z = 39). CEO was synthesised and used to conduct partition measurements along with CD and further explore CEO metabolism in liver microsomes and cytosol. The liquid-to-air partition coefficients for CD and CEO in the microsomal suspensions were 0.7 and 58, respectively. Apparent species differences in the uptake of CEO by microsomal hydrolysis were hamster~human>rats>mice.  Hydrolysis was inhibited by 1,1,1-trichloropropene oxide, a competitive inhibitor of epoxide hydrolase. A preliminary experiment indicated that the uptake of the CEP in liver cytosol by GSH conjugation was hamster>rats~mice (human cytosol not yet tested). In general, the results suggest that metabolism may help explain species differences showing a greater sensitivity for CD-induced tumorigenicity in mice, for example, compared with hamsters.