Styrene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented report meeting basic scientific principles.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Reference 2

Reference Type:

secondary source

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:

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

Materials and methods

Principles of method if other than guideline:

A 13-week rat study was conducted in order to assess the neurotoxic potential of styrene.
Groups of 14 male Fischer 344 rats were exposed to 0, 50, 200 or 800 ppm styrene for 6 hours/day, 5 days/week.

GLP compliance:

no

Limit test:

no

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:

- Name of test material (as cited in study report): Styrene (vinyl benzene)
- Physical state: clear oily solvent
- Analytical purity: 99.86 % pure
- Impurities (identity and concentrations): no minor impurities > 0.1 % observed
- Lot/batch No.: MM010491
- Source: The Dow Chemical Company, Midland, MI, USA.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY, USA.
- Age at study initiation: approx. 6 weeks
- Weight at study initiation:
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (#5002)
- Water (e.g. ad libitum): municipal drinking water
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass 4.1 m3 Rochester-type exposure chambers (square with pyramidal top and bottom)
- Temperature, humidity, pressure in air chamber: The minimum and maximum chamber temrperature and relative humidity were recorded at the end of each exposure period.
- Air flow rate: 800 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Miran 1A infrared spectrometer

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration at 4 points within the animal breathing zone was within normal limits (<10% difference from the reference).
The concentiation of styrene in the chamber was monitored 1-2 times per hour with a Miran 1A infrared spectrometer.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 0.21, 0.85, 3.41 mg/L air
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 50, 200, 800 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

14

Control animals:

other: yes, concurrent air control

Details on study design:

- Dose selection rationale: auditory function had been shown to be damaged in rats exposed to 800 ppm styrene tor 14 hours/day, for 3 weeks.
Lower levels were set a 200 and 50 ppm to provide dose response information and to determine a NOEL for ototoxicity.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
The examination included specific observations on respiration, muscle tone, movement, tremor, skin and haircoat condition, salivation, lacrimation, urine staining and fecal staining.

BODY WEIGHT: Yes
- Time schedule for examinations: pre exposure, weekly

FOOD CONSUMPTION:
No data

FOOD EFFICIENCY:
No data

WATER CONSUMPTION:
No data

OPHTHALMOSCOPIC EXAMINATION:
No

HAEMATOLOGY:
No

CLINICAL CHEMISTRY:
No

URINALYSIS:
No

NEUROBEHAVIOURAL EXAMINATION: Yes, functional observational battery (FOB)
- Time schedule for examinations: pre exposure (-4), at one and two month exposure and three days post exposure.
- Dose groups that were examined: all dose groups
- Battery of functions tested: any unusual responses with respect to body position, activity level, coordination of movement, and gait; any unusual or bizarre behavior (including observations of salivation, abnormal movements or behaviour); hind limb grip strength performance test.

OTHER: POST EXPOSURE TESTS
Rats were evaluated for neurotoxicity during the first week post-exposure with a functional observational battery, by evoked potential testing of the visual, auditory and somatosensory systems, by conduction velocity evaluation of caudal nerves, and by a comprehensive neuropathologic examination (12 rats per group):
Conducted tests:
- Flash Evoked Potential (recorded from the visual cortex and from the cerebellum)
- Auditory Brainstem Response to Clicks (recorded from the electrode over the cerebellum)
- Auditory Brainstem Response to Tone Pips (recorded at 4, 8, 16 and 30 kHz from the electrode over the cerebellum)
- Somatosensory Evoked Potentials (recorded over the sensory cerebral cortex and cerebellar vermis)
- Caudal Nerve Action Potentials (recorded from the ventrolateral caudal nerves)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Neuropathology at terminal necropsy was performed on 8 rats per group after electrophysiological tests were carried out.
Cochleas were removed from 4 rats in the control and 200 ppm groups and 3 rats from the 800 ppm group and sections specially processed for histopathology.

Statistics:

F-max test, variance/covariance structure (pooled variance), ANOVA, MANCOVA, ANCOVA.

Results and discussion

Results of examinations

Details on results:

There were no exposure-related effects on mortality, bodyweight, functional observations, or grip strength and no treatment-related alterations in histopathological lesions in nerve tissues or limb muscles, other than in the auditory system.

Results of the examinations on the auditory systems:
Histopathological investigations revealed lesions in the organ of Corti of animals exposed to 800 ppm styrene (two tissue samples were examined).
These lesions were characterised as the loss of two outer hair cells per cross section from the upper basal turn, and the occasional absence of an outer hair cell from the lower middle turn. There were no alterations involving the inner hair cells, the Deiters’ cells or the pilar cells. The organ of Corti was not affected in animals exposed to 0 or 200 ppm styrene (no tissues were examined at 50 ppm).
There were no treatment-related alterations in somatosensory evoked potential from the sensory cortex or the cerebellum.
However in the 800 ppm group, auditory brainstem response (ABR) thresholds were elevated by approximately 40 dB at 16, 25 and 30 kHz.
Hair cell loss at 800 ppm occurred in the cochlea in areas that relate to mid – high frequency (15-30 kHz) hearing.

Several FEP (Flash Evoked Potential), CNAP (Caudal Nerve Action Potential) and ABR (Auditory Brainstem Response) parameters were altered in the highest dose group, but these changes could not be confirmed when the highest dose and control groups were re-tested approximately 1 week after the inital post exposure testing.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

for ototoxicity

Effect level:

0.85 mg/L air (nominal)

Sex:

male

Basis for effect level:

other: histopathological changes in the organ of Corti.

2

Dose descriptor:

LOAEC

Remarks:

for ototoxicity

Effect level:

3.41 mg/L air (nominal)

Sex:

male

Basis for effect level:

other: histopathological changes in the organ of Corti.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

In this study, the NOAEL for ototoxicity was 200 ppm, with evidence of damage and impairment in the auditory system at the next highest dose of 800 ppm.