Styrene

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP study)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

review article or handbook

Title:

European risk assessment report, Styrene CAS No. 100-42-5, EINECS No. 202-851-5, Draft for submission to SCHER, November 2007.

Author:

European Union

Year:

2007

Bibliographic source:

Styrene CAS No. 100-42-5, EINECS No. 202-851-5, Draft for submission to SCHER, November 2007

Materials and methods

Objective of study:

metabolism

Principles of method if other than guideline:

In vitro study on the characterisation of reactive metabolites (not measurable in vivo due to the rapid metabolism) of styrene, arising via the initial hydroxylation to 4-Vinylphenol, in the lung and/or liver tissue of mouse, rat and human donors.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Styrene

EC Number:

202-851-5

EC Name:

Styrene

Cas Number:

100-42-5

Molecular formula:

C8H8

IUPAC Name:

ethenylbenzene

Specific details on test material used for the study:

Non-radiolabelled chemicals
- Name of test material (as cited in study report): 4-vinylphenol
- Analytical purity: 100 %
- Lot/batch No.: 10902

- Name of test material (as cited in study report): styrene
- Analytical purity: 99.4 %
- Lot/batch No.: 17129EA

Radiolabelled chemicals:
- Name of test material (as cited in study report): radiolabeled 4VP
- Lot/batch No.: 170-095-004
- Radiochemical purity (if radiolabelling): >98 %
- Specific activity (if radiolabelling): 4 mCi/mmol
- Locations of the label (if radiolabelling): ring-U

- Name of test material (as cited in study report): radiolabeled styrene
- Lot/batch No.: 170-134-004
- Radiochemical purity (if radiolabelling): 100 %
- Specific activity (if radiolabelling): 4 mCi/mmol
- Locations of the label (if radiolabelling): ring-U

Radiolabelling:

yes

Remarks:

14C-styrene and 14C-4-vinylphenol

Test animals

Species:

other: human, mouse, rat

Strain:

other: CD1 mice, sprague-dawley rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratory (Portage, Michigan, USA)
- Mean age at the time of tissue collection: mice: 13.3 weeks, rats 11.4 weaks
HUMAN TISSUE
Microsomes from liver or lung tissue of human donors (mixed gender) were obtained from Xenotech LLC (Lenexa, Kansas, USA).

Administration / exposure

Route of administration:

other: in vitro incubation in 0.1 M phosphate buffer (pH 7.4)

Vehicle:

propylene glycol

Details on exposure:

Microsomes of liver and lung tissue obtained from male CD1 mice, male SD rats and human donors (50 and 4 donors of mixed genders for liver and lung samples respectively) were incubated with 0.01, 0.1 or 1.0 mM styrene or with 0.05 or 0.5 mM 4-VP. Selected mouse styrene or 4-VP incubations were used to determine the metabolic production of CO2. In addition, selected styrene incubations were analysed for the production of 4-VP and selected 4-VP lung microsomal incubations were performed in the presence of excess glutathione (GSH) in order to trap reactive 4-VP metabolites.

Duration and frequency of treatment / exposure:

METABOLITE IDENTIFICATION:
Microsomal incubations were performed for 30 minutes at 37ºC, with 0.5-0.9 mM NADPH added.
DETERMINATION OF METABOLIC CO2 PRODUCTION:
Microsomal incubations with addition of KOH prior to substrate addition
TRAPPING OF REACTIVE METABOLITES
Addition of 5-9 mM GSH prior to substrate addition

Doses / concentrations

Remarks:

Doses / Concentrations:
0.01, 0.1 or 1.0 mM styrene
0.05 or 0.5 mM 4-Vinylphenol

No. of animals per sex per dose / concentration:

not applicable

Control animals:

other: not applicable

Positive control reference chemical:

Selected control samples were prepared by omitting either substrate or NADPH cofactor.

Details on dosing and sampling:

IN VITRO METABOLITE CHARACTERISATION STUDIES
- Tissues sampled: Lungs and liver of mice and rat; Microsomes from liver or lung tissue of human donors.
- Method type(s) for identification: GC-MS, reversed-phase LC-ESI/MS/MS (Liquid Chrormtography/Mass Spectrometry)

Results and discussion

Preliminary studies:

not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Recovery of radioactivity from incubations of [14C]-4VP ranged from 83.4 - 95.1 %.
Recovery of radioactivity from incubations of [14C]-styrene ranged from 68.2 - 86.6 % and 86.2 - 97.4 % for liver and lung, respectively.

Details on distribution in tissues:

not applicable

Details on excretion:

not applicable

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Incubations of 4-VP with mouse lung microsomes produced two major and several minor reactive metabolites. By incubating these mouse lung microsomes with 4-VP in the presence of excess GSH, the two major metabolites of 4-VP were isolated. These were identified as the GSH conjugates of the side-chain epoxide and the ring-hydroxylated (4-VP hydroquinone) derivatives of 4-VP. The rate of formation of these two 4-VP downstream products was then determined from incubations of styrene with lung microsomes from mouse, rat and human donors in the presence of excess GSH. Relative formation rates for both 4-VP derivatives were highest in mouse lung microsomes (123.35 and 0.325 pmoles/min/mg of 4-VP epoxide GSH conjugate and 4-VP hydroquinone GSH conjugate respectively). Yields of 4-VP epoxide and 4-VP hydroquinone respectively were 79% (97.2 pmoles/min/mg) and 14% (0.045 pmoles/min/mg) lower in rat lung microsomes compared to mouse lung microsomes. Incubates from human lung microsomes contained only 5% (6.4 pmoles/min/mg) of 4-VP epoxide and 1.5% (0.005 pmoles/min/mg) of 4-VP hydroquinone concentrations measured in mouse lung.

Any other information on results incl. tables

No significant production of CO₂ was detected following incubation of styrene or 4-VP with microsomes of mouse liver or lung tissue indicating that the liver or lung are not the primary site of aromatic ring cleavage of styrene in the mouse. Substantial species differences were observed in the rate of styrene metabolism in both liver and lung microsomes, with the mouse having the highest styrene conversion (76-80%) compared to the rat (18-29%) and the human donors (0-26%). Following incubation with styrene, only trace levels of 4-VP were found in lung microsomes of both mice (0-0.015% of substrate) and rats (0-0.019%). No measurable 4-VP was detected in human lung microsome incubations. No detectable 4-VP was found in liver microsomes of either mice, rats or humans. These results suggest that conversion of styrene to 4-VP is a minor route of styrene metabolism in vitro and that the small amount of 4-VP produced is subsequently rapidly metabolised.

In human lung the reactive downstream metabolites of 4 -VP are produced to a lesser extent than in rats and much less than in mice lungs. Some caution needs to be exercised, as these are in vitro studies; however, at present, it is not technically possible to perform this kind of study in vivo due to the extreme reactivity of these downstream metabolites.