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Specific investigations: other studies

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Administrative data

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-GLP, non-guideline study, published in peer-reviewed literature acceptable for assessment of specific target organ toxicity (ototoxicity)

Data source

Reference
Reference Type:
publication
Title:
Low-level toluene disrupts auditory function in guinea pigs
Author:
McWilliams ML, Chen G, Fechter LD
Year:
2000
Bibliographic source:
Toxicol. Appl. Pharmacol., 167, 18-29

Materials and methods

Principles of method if other than guideline:
The effects of toluene on the auditory system of the guinea pig after 1 and 4 weeks of exposure were evaluated. Auditory function was assessed using distortion product otoacoustic emissions (DPOAE) with subsequent measurement of succinate dehydrogenase (SDH) staining density in hair cells using surface preparations.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: inhalation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No details reported

Test animals

Species:
guinea pig
Strain:
other: pigmented
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kuiper Rabbit Ranch or bred from animals obtained from Kuiper Rabbit Ranch at the University of Oklahoma Health Sciences Center (OUHSC) main animal facility.
- Age at study initiation: approximately 60 days
- Weight at study initiation: no details reported
- Diet: ad libitum (except during exposures)
- Water: ad libitum (except during exposures)
- Acclimation period: no details reported

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod: 12 hrs dark / 12hrs 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: custom-built glass chamber (0.3 m diameter X 0.6 m long)
- Method of holding animals in test chamber: wire-mesh cage within exposure chamber
- Air change rate: 16 air changes/hour
- Sound level in exposure chamber: 60 dB(A) and was below 60 dBlin for octave bands centered above 2 kHz with air supply flowing through the exposure chamber

TEST ATMOSPHERE
- Brief description of analytical method used: toluene concentration was monitored continuously using an infrared vapour analyzer.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
8 h/day
Frequency of treatment:
5 days/week for 1 or 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 ppm (0, 942, 1884, 3768 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
8 in total
Control animals:
yes, sham-exposed

Examinations

Examinations:
Observations and examinations performed and frequency
CAGE SIDE OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

CUBIC DISTORTION PRODUCT OTOACOUSTIC EMISSION (CDP): The CDP was measured in all guinea pigs for six primary frequency pairs between 6 and 24 kHz. The size of the CDP was maximized by using an F2:F1 ratio of 1.28. For input/output function measurements, the intensity of the primaries was varied in 5-dB steps with the sound level of F1 (L1 = 50 to 80 dB SPL) and the level of F2 (L2 = L1-10). Recordings were averaged for 1.97 s. The guinea pigs were anaesthetised prior to CDP measurements.
- Time schedule: Before first exposure and within 5 minutes following final exposure. For measurement of CDP recovery, guinea pigs were allowed 3 days without exposure before CDP measurement

Sacrifice and pathology
EVALUATION OF LOSS / DAMAGE TO HAIR CELLS AS DETERMINED BY SUCCINYL DEHYDROGENASE (SDH) STAINING DENSITY: Histological sections of the cochlea were made in two guinea pigs from each exposure condition endpoint (250, 500, and 1000 ppm 1 week; 500 and 1000 ppm 1 week plus 3-day recovery; 500 and 1000 ppm 4 weeks; and 500 and 1000 ppm 4 weeks plus 3-day recovery). The animals were decapitated while deeply anaesthetized after CDP recording. Both the round and oval windows were opened and the apex of the cochlea was drilled open to facilitate perfusion. The cochleae were perfused with SDH incubative solution and then fixed with 10% formalin for at least 2 days. After fixation, the cochleae were decalcified. The cochlea were micro-dissected and surface preparations of the organ of Corti were evaluated to determine SDH staining density (via computer-generated density readings) in sensory cells in subjects taken from each exposure group. SDH staining density was plotted as a function of location along the basilar membrane to permit a comparison between functional impairment and SDH staining density. Hair cell loss was quantified by counting of all visibly present cells within each section.

Other examinations
CUBIC DISTORTION PRODUCT OTOACOUSTIC EMISSION (CDP). measurement of the CDP-gram and input/output functions were made using two sound sources for generation of primary tones (F1 and F2) coupled to a low noise microphone. The CDP was measured in all guinea pigs for 6 primary frequency pairs between 6 and 24Hz. Recordings were averaged for 1.97 seconds.

Statistics
CDP amplitude data are analyzed by a split-plot factorial ANOVA. Treatment (control vs toluene groups) is analyzed between subjects while tone frequency and time relative to toluene exposure serve as the repeated measures within subjects. Post hoc analysis was performed by Scheffe's multiple-comparison test.

Results and discussion

Details on results:
1000 ppm (3768 mg/m3) for 8 h/day, 5 days/week for up to 4 weeks was the NOAEC for permanent hearing loss in guinea pigs.

Any other information on results incl. tables

Temporary disruption of auditory function in guinea pigs is seen in animals exposed to 250, 500, and 1000 ppm toluene for 8 h/day, 5 days/week for 1 and 4 weeks. Concentrations as low as 250 ppm toluene were able to disrupt auditory function acutely in the guinea pig, and 500 and 1000 ppm toluene produced greater acute dysfunction. SDH staining suggests that reduced enzyme activity in the mid frequency region of the cochlea occurs acutely following toluene exposure. Although the auditory dysfunction progressed between 1 and 4 weeks of exposure, a permanent loss did not develop and hair cell death was not seen.

Applicant's summary and conclusion

Conclusions:
Temporary disruption of auditory function in guinea pigs is seen in animals exposed to 250, 500, and 1000 ppm toluene for 8 h/day, 5 day/week for 1 and 4 weeks which recovered within 3 days of terminating exposure. Permanent hearing loss did not develop and hair cell death was not seen even at 1000 ppm. NOAEC for hair cell death was 1000 ppm (3768 mg/m3) for 8 h/day, 5 days/week for 4 weeks.
Executive summary:

Auditory dysfunction was assessed by cubic distortion product otoacoustic emission (CDP) and histology of hair cells in guinea pigs exposed to 250, 500 or 1000 ppm toluene for 8 h/day, 5 day/week for 1 and 4 weeks. All concentrations of toluene were able to temporarily disrupt auditory function acutely in the guinea pig, and 500 and 1000 ppm toluene produced greater acute dysfunction. Although the auditory dysfunction progressed between 1 and 4 weeks of exposure, recovery was seen in all animals within 3 days. A permanent loss did not develop and hair cell death was not seen. 1000 ppm (3768 mg/m3) for 8 h/day, 5 days/week for up to 4 weeks was the NOAEC for permanent hearing loss in guinea pigs.