o-xylene

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: Non-guideline animal experimental study, published in peer-reviewed literature, GLP status unknown, fully adequate for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Brain morphological investigations 8 weeks following exposure. Brainstem auditory-evoked responses used to determine auditory thresholds at different frequencies. The cochlea and organ of Corti examined by light and electron microscopy, respectively.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-xylene

EC Number:

203-396-5

EC Name:

p-xylene

Cas Number:

106-42-3

Molecular formula:

C8H10

IUPAC Name:

p-xylene

Details on test material:

Analytical purity: >99%
Source: Acros, Geel, Belgium

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa Credo, Domaine des Oncines, Saint-Germain-sur l’Arbresle, France
- Age at study initiation: 14 weeks
- Diet: UAR-Alimentation, Villemoisson, Epinay-sur-Orge, France; sterilized with γ-ray, ad libitum
- Water: Filtered tap water (pore size 0.3 µm) ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Humidity: 55 ± 5% %
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

TYPE OF EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 200 L stainless steel inhalation chambers designed to maintain a dynamic and adjustable airflow (10-30 m3/hr), maintained at negative pressure (2-3 mm H2O)
- System of generation: An additional airflow was bubbled through xylene and the output vapour was diluted with air to the required concentration before entering the exposure chamber.
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: 10-30 m3/hr

TEST ATMOSPHERE
- Brief description of analytical method used: p-xylene concentrations in the exposure chambers were continuously monitored using a gas liquid chromatograph.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved exposure concentrations were determined once during each 6 hr exposure period (sample collection on activated charcoal and analysis by gas chromatography).

Duration of treatment / exposure:

13 weeks followed by 8 week recovery period

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 450, 900, 1800 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 1954, 1908, 7817 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

16

Control animals:

yes, sham-exposed

Details on study design:

Study aim - to evaluate the potential ototoxicity of individual xylene isomers using electrophysiological methods in rats exposed by inhalation to three different concentrations 6 hr/day, 5 days/week for 13 weeks. Brainstem auditory evoked responses were used to determine auditory thresholds at different frequencies. A quantitative morphological study (histocochleogram) and scanning electron microscopy of the organ of Corti were carried out to determine whether there were any microscopic alterations.

Dose selection rationale - chosen on the basis of the results obtained in preliminary range-finding studies. The highest exposure concentration was chosen to produce a reduction in body weight gain of less than 10% and no mortality after four weeks of exposure.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes

NEUROPHYSICAL MEASUREMENTS: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: No data

MORPHOLOGY: Yes

Statistics:

Continuous, parametric variables - Bartlett's test for homogeneity of variances (Bartlett 1937) and by analysis of variance. If the analysis of variance was significant, individual mean comparisons were made with Scheffe's multiple range test to realize comparisons between any pair of groups. Non-parametric data (audiometric thresholds) - Kruskal-Wallis test (Kruskal & Wallis 1952; Gad & Weil 1986). The probability value of P<0.05 was used as the critical level of significance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: No mortality, all rats remained in good health.

BODY WEIGHT AND WEIGHT GAIN: No statistically significant differences.

NEUROPHYSICAL EXAMINATION: Increased auditory thresholds were observed at 2, 4, 8 and 16 kHz in rats exposed to 1800ppm. The auditory threshold shifts (35-38dB) did not reverse after 8 weeks of recovery. None of the electrophysiological parameters changed in the groups exposed to 450 or 900 ppm.

MORPHOLOGICAL STUDY: Exposure to 1800 ppm caused a loss of hair cells in the three rows of outer cells of the organ of Corti. The highest losses occurred in the third row and the lowest in the first row. The hair cell losses were observed in the apical and the upper and lower medium parts of the cochlea. Only the basal part of the cochlea, which transcribes the high frequencies, was spared. Exposure to 900 ppm caused much lower losses, and were observed mainly in the third row of outer hair cells. Occasional losses were observed in the second row of outer hair cells. These losses were most prominent in the area which transcribes the middle frequencies (5 to 25 kHz).

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

1 800 ppm

Sex:

male

Basis for effect level:

other: equivalent to 7817 mg/m3, no significant changes in bodyweight or bodyweight gain at highest dose tested

2

Dose descriptor:

NOAEC

Remarks:

ototoxicity

Effect level:

450 ppm

Sex:

male

Basis for effect level:

other: equivalent to 1954 mg/m3, increased auditory evoked response thresholds, structural / ultrastructural changes in the cochlea and organ of Corti at 900 ppm

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Increased auditory thresholds and a loss of outer hair cells from the organ of Corti were present in male rats 8 weeks after the cessation of sub-chronic exposure to p-xylene.

Executive summary:

Male Sprague-Dawley rats were exposed to p-xylene by inhalation (0, 450, 900 or 1800 ppm, 6 hr/day, 5 days/week for 13 weeks) and brainstem auditory-evoked responses determined 8 weeks after treatment ended. The cochlea and organ of Corti were also examined using light or electron microscopy, respectively. 900 and 1800 ppm p-xylene produced moderate to severe ototoxicity in rats highlighted by brainstem auditory evoked responses, increased electrophysiological auditory thresholds and histological analysis. The NOAEC was 450 ppm (1.95 mg/L).