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Toxicological information

Specific investigations: other studies

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Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline, non-GLP, animal experimental study, published in peer reviewed literature, fully adequate for assessment of ototoxicity but not other repeat-dose end-points.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1984

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The rats were exposed to toluene at different concentrations and for varying exposure times. Hearing loss was assessed by behavioural and electrophysiological methods.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: inhalation

Test material

Constituent 1
Details on test material:
Industrial grade toluene was purchased from Van Waters and Rogers. The material contained over 99% toluene, with less than 1.0% contamination by other hydrocarbons, including benzene (0.1%), octanes, and xylenes as measured by gas chromatography-mass spectroscopy.

Test animals

Species:
rat
Strain:
other: Fischer
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Madison, WI, USA (Experiment 1-3), Simonsen Laboratories, Gilroy, CA, USA (Experiments 4-7).
- Age at arrival: 23 or 35 days
- Weight at study initiation: not reported
- Housing: 2-3 rats/cage in standard plastic rat cages or directly in exposure chambers (12/cage).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 23±1°C
- Humidity: 40-60%
- Photoperiod: 14 hrs dark / 10 hrs light

IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4-chamber system constructed of clear Plexiglas, the interior of each chamber measured 50x50x25 cm and had a volume of 62.5 L.
- System of generation: Toluene concentration in each chamber was controlled by bubbling filtered air through the solvent and diluting it with the main air flow (15 - 20 L/min). Control rats were exposed to filtered air, at the same flow rate.

TEST ATMOSPHERE
- Brief description of analytical method used: monitored daily by gas chromatography
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored daily by gas chromatography but no detail provided of analytical concentrations and refers to target concentrations only
Duration of treatment / exposure:
Variety of durations from 4-14 h/day or intermittent
Frequency of treatment:
Variety from single exposure to 7d/week for up to 16 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Expt 1:0, 400/850, 700/1000, 100/1000 ppm (1507/3203, 2638/3768, 377/3768 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Expt 2: 0, 1000, 2000, 4000 ppm (0, 3768, 7537, 15074 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Expt 3: 0, 1500, 3000 ppm (0, 5653, 11306 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Expt 4: 0, 1000 ppm (0, 3768 mg/m3); Expt 5: 0, 1500, 2000, 4000 ppm (0, 5625, 7500, 15000 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Expts 6 and 7: 0, 2000, 4000 ppm (0, 7537, 15074 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, sham-exposed

Examinations

Examinations:
Test methodology included:
Multisensory conditioned avoidance response (CAR): Rats learn to pull or climb a pole suspended from the ceiling of the test chamber to avoid an electric shock through the grill floor. Shocks are preceded by an increase in light intensity, a non-aversive shock or a sound. All rats were trained on all three stimuli which were presented randomly (one per trial) during each session. A response during the warning signal ended the trial and was scored as a successful avoidance. A response during the aversive shock was scored as an escape. Absence of any response was scored as a failure. training was in 3 phases. First rats were trained in a 30 trial session to escape aversive shock presented randomly and 20 seconds duration in the absence of a response). Then the rats were given four daily 60 trial sessions to learn to avoid the shock with each of the warning stimuli. The warning stimulus lasted 10 seconds and shock 20 seconds. During the third phase, the rats were given several 80 trial sessions in which the warning stimulus was reduced to 5 seconds and aversive shock reduced to 10 seconds.

Behavioural audiometry: Auditory response thresholds were estimated by giving the rats a series of test sessions during which only the warning used was the sound. Each session consisted of 60 trials, 10 at each of five tone intensities and 10 blank trials. The different tone intensities and blank trials were presented randomly and only one frequency (4, 8, 12, 16 or 20 kHz) was tested in a given 60 trial session. The auditory response threshold was defined as the estimated intensity at which 50% avoidance was expected for each rat at each frequency.

Electrophysiological audiometry: Brainstem auditory-evoked responses (BAERs) were recorded from sub-dural scalp electrodes in awake or anaesthetised, restrained rats. The BAERs were elicited by tone pips of 4, 8 and 16 kHz and 25 to 95 dB in 10 dB steps.

Results and discussion

Details on results:
Seven experiments examined concentration and exposure parameters necessary to cause toluene-induced ototoxicity. Hearing loss, measured by behavioural and electrophysiological methods, was observed after as few as 2 weeks. 3000 ppm (11306 mg/m3) 4 h/day for 9 weeks is a NOAEC for ototoxicity.

Any other information on results incl. tables

Hearing loss, measured by behavioural and electrophysiological methods, was repeatedly observed after as few as 2 weeks of exposure to 1000 ppm toluene for 14 hours per day, but lower concentrations (400 and 700 ppm) were without effect even after 16 weeks of exposure. Three-day exposures to 1500 ppm for 14 hours per day or 2000 ppm for 8 hours per day were ototoxic, whereas single exposures to 4000 ppm for 4 hours or 2000 ppm for 8 hours were without effect. Intermittent exposure to 3000 ppm for 30 minutes every hour for 8 hours per day caused hearing loss within 2 weeks, but a similar exposure schedule for 4 hours per day was ineffective even after 9 weeks.

Exposure Schedule

LOAEC

NOAEC

14 h/day, 16 weeks

1000 ppm

700 ppm

8 h/day, 3 days

2000 ppm

 

Intermittent 8 h/day, 2 weeks

3000 ppm

 

Intermittent 4 h/day 9 weeks

 

3000 ppm

Applicant's summary and conclusion

Conclusions:
Toluene is ototoxic in rats at relatively intense schedules of exposure. The toluene concentration and the duration of exposure must be above certain levels before hearing loss will occur.
Executive summary:

The relationship between toluene concentration, exposure pattern, and hearing loss in rats was investigated in a series of experiments in male Fischer rats. Hearing loss was measured by conditioned avoidance response to tone frequencies of 4, 8, 12, 16 or 20 kHz, and brainstem auditory-evoked response to tone pips of 4, 8, and 16 kHz.

Two weeks of exposure to 1000 ppm (3768 mg/m3) toluene 14 h/day caused hearing loss. Lower concentrations (400 (1507 mg/m3) and 700 ppm (2638 mg/m3)) were without effect even after 16 weeks of exposure. Three-day exposures to 1500 ppm (5653 mg/m3) for 14 h/day or to 2000 ppm (7537 mg/m3) for 8 h/day were ototoxic. Single exposures to 4000 ppm (15074 mg/m3) for 4 h or to 2000 ppm for 8 h were without effect. Intermittent exposure to 3000 ppm (11306 mg/m3) for 30 minutes every hour for 8 h/day caused hearing loss within 2 weeks, but a similar exposure schedule for 4 h/day was ineffective even after 9 weeks. Seven experiments with young male Fischer rats were done to examine concentration and exposure parameters necessary and sufficient to cause toluene-induced ototoxicity. Hearing loss, measured by behavioural and electrophysiological methods, was observed after as few as 2 weeks.

It is concluded that 3000 ppm (11306 mg/m3) 4h/day for 9 weeks is a NOAEC for ototoxicity.