Ethylbenzene

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to GLP and sufficient data is available for interpretation of results.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Reference 2

Reference Type:

publication

Title:

Mechanism of Ethylbenzene-Induced Mouse-Specific Lung Tumor: Metabolism of Ethylbenzene by Rat, Mouse and Human Liver and Lung Microsomes

Author:

Sagir, S.A.; Rick, D.L.; McClymont, E.L.; Zhang, F.; Bartels, M.J.; and Bus, J.S.

Year:

2009

Bibliographic source:

Tox. Sci. 107(2): 352-366

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Ethylbenzene was incubated with liver and lung microsomes to measure the formation of 1-phenylethanol (1-PE), acetophenone, 2,5-ethylquinone, and 3,4-ethylquinone. The latter two reactive metabolites were monitored via a glutathione (GSH) trapping technique.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethylbenzene

EC Number:

202-849-4

EC Name:

Ethylbenzene

Cas Number:

100-41-4

Molecular formula:

C8H10

IUPAC Name:

ethylbenzene

Details on test material:

- Name of test material (as cited in study report): Ethylbenzene
- Analytical purity: 99.8%
- Lot/batch No.: 01353MC

Radiolabelling:

not specified

Test animals

Species:

other: microsomes from liver and lung tissues from male F344 rats, male B6C3F1 mice and humans (mixed gender and race)

Details on test animals or test system and environmental conditions:

Mouse liver and lung microsomes were prepared from the pool of 126 and 100 animals, respectively. Human liver microsomes were from the pool of 50 individuals of mixed gender, race (Caucasian, African, and Hispanic) and age (6 to 78 years old with most of them between 30 and 50 years), 31 ofthem died from cerebrovascular stroke, 12 from head trauma, 5 from anoxia, 1 from myocardial infarction, and 1 from aortic aneurysm. Human lungmicrosomes of non-smokers were prepared from the pool of 4 individuals of mixed gender (3males, 1 female), 1 died from stroke, 1 from interacranial hemorrhage, 1 from drug overdose, and 1 from motor vehicle accident.
Microsomal samples were received in small aliquots (0.5 mL containing 5 or 10 mg protein) in eppendorf tubes on dry ice and immediately stored at –80 °C. Each microsomal preparation was analyzed by the supplier for at least CYP1A1/1A2 activity by the rate of metabolism of 7-ethoxyresorufin O-dealkylation (EROD assay). The samples were reanalyzed for CYP1A1/1A2 activity by EROD assay using a microplate fluorometric method. The 1A1/1A2 activity of human lung microsomes was about 7% of the levels measured in rat lung microsomes, and was 4-fold lower than what has been reported for nonsmoker human lung microsomes prepared from fresh tissues obtained within 15 minutes of lobectomy. The 1A1/1A2 activity of mouse lung microsomes was 16-fold higher than rat lung. The 1A1/1A2 activity in the liver microsomes was in the order of mouse > rat >> human

Administration / exposure

Route of administration:

other:

Vehicle:

not specified

Details on exposure:

Microsomal Metabolism of 2- and 4-Ethylphenol - One concentration of 2- or 4-ethylphenol (1 mM final concentration) was incubated for 30 minutes at 37 °C with mouse, rat and human lung and liver microsomes (1 mg protein/mL 0.1 M phosphate buffer, cofactors, pH 7.4) in 24-mL gas-tight glass vials. Each treatment (substrate/tissue type) was comprised of three replicates. Two types of control incubations were also conducted. One control type contained all components (0.1M phosphate buffer, NADPH, microsomes), but no substrate; the second control was conducted with substrate and microsomes, but no NADPH. After the completion of the incubations, samples were analyzed for the loss of 2- and 4-ethylphenol, as well as formation of ethylhydroquinone and ethylcatechol by high performance liquid chromatography with ultraviolet detection (HPLC/UV).
As a basis of comparison with the preliminary ethylbenzene metabolism study, mouse liver and/or lung microsomes were incubated with 750 μg of ethylbenzene delivered to 1 mL of the incubation medium using propylene glycol (PG) as vehicle (or, 7 mM EB in test system). After completion of 30 minute incubations with ethylbenzene, samples from the current study were analyzed for formation of 1-phenylethanol, 2EP, 4EP (as done in the preliminary study), as well as ethylhydroquinone (EHQ) and ethylcatechol (ECat).
Rate of Microsomal Metabolism of Ethylbenzene to 1-Phenylethanol, Acetophenone, Ethylhydroquinone and Ethylcatechol - Probe incubations of ethylbenzene with mouse liver microsomes were conducted (at a single substrate concentration) to assess the linearity of the rates of formation of 1-phenylethanol, acetophenone, 2,5-ethylquinone, and 3,4-ethylquinone over time. Mouse liver microsomes were incubated with 2 mM ethylbenzene (and 1mg protein/mL 0.1M phosphate buffer, pH 7.4, 1 mM NADPH, 10 mM glutathione) in glass vials. Incubations were conducted for 10, 15, 30, 60, and 90 minutes (two replicate vessels per incubation time).
The kinetics of 1-PE and AcPh (major metabolites of EB) as well as kinetics of 2,5- and 3,4-ethylquinone (potential reactive metabolites of ethylbenzene) formation were conducted. Liver and lung microsomes of three species (rat, mouse, human) were incubated in triplicate at 37 °C with ethylbenzene at six concentrations. The substrate concentrations were 7.0, 3.5, 1.8, 0.9, 0.45, and 0.22 mM ethylbenzene. The incubation time was 30 min, based on the results of the time-course experiments. Six separate PG-based solutions of ethylbenzene were prepared such that the ethylbenzene, for all treatment levels, was delivered in a volume of 10 μL.
Three types of control incubations were conducted. Control Type I contained all components (0.1 M phosphate buffer, NADPH, microsomes, glutathione, 10 μL PG), but no ethylbenzene; Control Type II was conducted with incubates containing 0.45, 1.8, or 7.0 mM ethylbenzene (single replicate for each [of six] microsome types), glutathione (GSH) and microsomes, but no NADPH; Control Type III contained all components (with 0.45 mM ethylbenzene), but no microsomes.

No. of animals per sex per dose / concentration:

Not applicable

Control animals:

no

Positive control reference chemical:

not applicable

Details on study design:

Further to description under details of exposure - Following the 30 min incubations, 0.2 mL of a so-called “kill solution” was injected through the septum of each incubation vial (i.e., vials remained sealed from the point of mixing components through incubation period, and until after kill solution was added). The kill solution was comprised of 2% formic acid (to reduce pH), 5% ascorbic acid (anti-oxidant), and a mixture of internal standards in a solution of 20/80 H2O/acetonitrile. In addition to controlling pH and reducing potential for oxidation, the kill solution served the following purposes: a) stop the metabolic reactions; b) enhance the solubility of ethylbenzene prior to analysis due to the addition of acetonitrile (as substrate concentrations in treatments of 1.8 mM and above may have exceeded the water solubility of ethylbenzene), c) and internal standards of volatile analytes would control for the loss those analytes during sample preparation in addition to controlling for the variability in MS response during analysis. The internal standards contained in the kill solution were d10-ethylbenzene, d5-1-PE, and d5-AcPh (with “dx” signifying the number of deuteriums substituted in the molecular structure), at a concentration of 100 μg/mL, each. After the addition of the kill solution, the vials were briefly chilled by placing them in an ice bath. Then the vials were opened, and to remove the precipitated protein and other solids, the samples were filtered through Whatman (Florham Park, New Jersey) 13-mm ZC PTFE syringe filters (0.2 μ pore) and collected in autosampler vials. The syringes used were 1-mL plastic body.
The collected filtrate was subdivided for the two separate assays, with each preparation completed as follows. For GC/MS analysis of ethylbenzene, 1-PE, and AcPh, an 0.3-mL aliquot was transferred to a 1-dram vial and extracted with 0.6 mL of CS2 (by shaking for 30 min on a flatbed shaker). The bottom layer (CS2) was transferred to a 1-dram vial containing ~100 mg MgSO4 to remove absorbed water from the CS2. The “dried” CS2 was then decanted to an autosampler vial for analysis by GC/MS.
In the preparation for analysis of reactive metabolites (e.g., 2EP-GSH and 4EP-GSH), a 0.3-mL aliquot of the initial filtrate was transferred to an autosampler vial to which was added a laboratory-synthesized internal standard (d4-4EP-GSH).
Samples were stored at –80 °C while awaiting analysis.

Details on dosing and sampling:

Following the incubation and after addition of a so called kill solution, each solution was processed for two types of analysis. The first analysis, by gas chromatography with mass spectrometry (GC/MS), was conducted for the determination of ethylbenzene and the major (volatile) metabolites, 1-PE and AcPh. The second analysis utilized a glutathione trapping technique (which is the reason that 10 mM GSH was added to the incubation solutions) as previously employed for the in vitro metabolism of 4-vinylphenol to hydroquinone and catechol. The GSH conjugates were analyzed by high performance liquid chromatography with multiple reaction monitoring mass spectrometry (HPLC/MRM/MS).

Statistics:

Descriptive statistics (i.e., mean ± standard deviation) for the depletion of parent ethylbenzene and formation of metabolites were calculated using Microsoft Excel® spreadsheets in full precision mode (15 digits of accuracy). The rates of metabolism were calculated using standard methods (e.g., Eadie-Hofstee, Lineweaver-Burk plots).

Results and discussion

Preliminary studies:

Not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Molar conversion of ethylbenzene to 1-phenylethanol (1-PE), acetophenone, 2,5-ethylquinone, and 3,4-ethylquinone varied quite broadly depending on microsome species/tissue and substrate concentration. None of the metabolites were formed at detectable levels in incubations with human lung microsomes. Alkyl-hydroxylated metabolites (1-phenylethanol, acetophenone) were formed at much higher levels than the ring-hydroxylated metabolites (catechols, hydroquinones, quinones). The highest levels of ring-hydroxylated metabolites were formed in incubations with mouse lung microsomes, a finding consistent with the a role for cytotoxic, ring-oxidized metabolites in the mouse lung specific toxicity of ethylbenzene.

Any other information on results incl. tables

Table 1: Conversion of 2-Ethylphenol to Ethylhydroquinone after Incubation with Liver and Lung Microsomes from Mouse, Rat, and Human Tissues

Table 2: Conversion of 4-Ethylphenol to Ethylcatechol after Incubation with Liver and Lung Microsomes from Mouse, Rat, and Human Tissues

Table 3: Results of Time-Course Experiments to Assess Linearity of Metabolite Formation (conversion of EB to 1-Phenylethanol and Acetophenone

Table 4: Results of Time-Course Experiments to Assess Linearity of MetaboliteFormation (conversion of EB to 2,5- and 3,4-ethylquinone via GSH trap

Table 5: Percent Conversion of Metabolites Formed in Incubations of Ethylbenzene with Liver Microsomes

Table 6: Percent Conversion of Metabolites Formed in Incubations of Ethylbenzene with Lung Microsomes

Table 7. Km and Vmax Values Derived for Metabolites whose Rates of Conversion Increase with Increasing Substrate Concentration: Values for Formation of 1 -Phenylethanol and Acetophenone (Top), and 2EP-GSH and 4EP-GSH (Bottom)

Table 8: Maximum and Minimum Rates of Formation (μmole/mg protein/minute) for Metabolites whose Rates of Conversion Decrease with Increasing Substrate Concentration

Applicant's summary and conclusion

Conclusions:

It was concluded that although ring oxidised metabolites accounted for a relatively small fraction of overall ethylbenzene metabolism, its selective elevation in mouse lung microsomes was consistent with the mode of action attributing preferential formation of lung derived cytotoxic, ring-oxidized metabolites as driving the mouse lung specific toxicity of ethylbenzene.

Executive summary:

Ethylbenzene was incubated at concentrations ranging from 0.22 to 7 mM, with liver and lung microsomes of mouse, rat and human to measure the formation of 1-phenylethanol (1-PE), acetophenone, 2,5-ethylquinone, and 3,4-ethylquinone. The latter two reactive metabolites were monitored via a glutathione (GSH) trapping technique.

Molar conversion to the four metabolites varied quite broadly depending on microsome species/tissue and substrate concentration. None of the metabolites were formed at detectable levels in incubations with human lung microsomes. Alkyl-hydroxylated metabolites (1-phenylethanol, acetophenone) were formed at much higher levels than the ring-hydroxylated metabolites (catechols, hydroquinones, quinones). Molar conversion to the major metabolite, 1-PE, ranged from 1% (rat lung at 7mM ethylbenzene) to 58% (mouse lung at 0.22 mM ethylbenzene). This was equivalent to the formation of 0.09 μmole 1-PE by rat lung and 0.13 μmole by mouse lung; a difference of ~2-fold. The mass of 1-PE increased with increasing substrate levels, although the percent conversion (relative to starting substrate concentration) decreased. There was more 1-PE formed in mouse lung tissue incubations than in incubations with mouse liver microsomes.

Levels of 1-PE formed in incubations with rat liver and lung microsomes were similar. The metabolism of ethylbenzene to 1-PE, ranked according to species, was mouse > rat > human. 1-PE was formed at a level that was roughly an order of magnitude greater than acetophenone.

In a previous study in which ethylbenzene was incubated with liver and lung microsomes, very little aromatic-oxidation to either 2-ethylphenol (2EP) or 4 -ethylphenol (4EP) was detected. It was surmised that the low levels of the mono-hydroxylated aromatic metabolites may have been due to further rapid oxidation to the di-hydroxylated catechol and quinone metabolites. It should be noted that in the earlier study, the GSH trapping technique used in this current study to afford greater sensitivity to quinones formed via catechols and hydroquinones was not employed. To investigate the potential for further oxidation, high concentrations of 2EP and 4EP were incubated with microsomes, and the formation of ethylcatechol (ECat) and ethylhydroquinone (EHQ) monitored. Conversion from the mono- to the dihydroxylated aromatics did occur, with molar conversion of 2EP to EHQ ranging from 6 to 9% in liver microsomes of the three species (mouse[8.9] > human[7.1] > rat[6.4]) and from 0.1 to 18% in lung microsomes (mouse[17.7] > rat[5.8] > human[0.1]). Conversion of 4EP to ECat ranged from 2 to 4% in liver microsomes (mouse[3.6] > human[2.1] ~ rat[2.0]) and from 0.3 to 7% in lung microsomes (mouse[7.1]>rat[1.4]>human[0.3]). In order to trap the reactive metabolites formed from 2EP and 4EP (i.e.,

the quinones derived from catechols and hydroquinones), experiments were conducted after adding excess GSH to each incubate.

Percent conversion of ethylbenzene to ring-hydroxylated metabolites was much lower than what was observed for the alky-hydroxylated metabolites, ranging from 0.0001% (4EP-GSH; rat lung) to 0.6% (2EP-GSH; mouse lung). 2EP-GSH concentrations were typically 10-fold higher than 4EP-GSH. At lower substrate concentrations, more 2EP-GSH formed during incubations with lung microsomes than liver microsomes, for both rats and mice. More 2EP-GSH was formed in incubations with mouse liver microsomes than in incubations with liver microsomes from rat and human.

The highest levels of ring-hydroxylated metabolites were formed in incubations with mouse lung microsomes. Interestingly, both mouse and rat lung microsomes (and to a lesser extent, mouse liver microsomes) exhibited decreasing amounts of ring-oxidized metabolite formation with increasing concentrations of ethylbenzene. This suggests the possibility of cytochrome P450 suicide inhibition by reactive ring-oxidized metabolite(s). The possible suicide inhibition appears to be isozyme-specific in that generation of alkyl-oxidized metabolites was not similarly decreased with increasing ethylbenzene substrate concentrations. This observation is consistent with the hypothesis that reactive ring-oxidized metabolites are likely formed by cytochrome P450 2F2, while alkyl-oxidized metabolite formation is mediated predominantly through cytochrome P450 2E1.

Although ring-oxidized metabolites accounted for a relatively small fraction of overall ethylbenzene metabolism, its selective elevation in mouse lung microsomes is nonetheless consistent with the hypothesized mode of action attributing preferential formation of lung-derived cytotoxic, ring-oxidized metabolites as driving the mouse lung specific toxicity of ethylbenzene.