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Toxicological information

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

Data source

Reference
Reference Type:
publication
Title:
Ototoxicity in rats exposed to ethylbenzene and to two technical xylene vapours for 13 weeks
Author:
Gagnaire, F, Langlais, C, Grossman, S and Wild, P
Year:
2007
Bibliographic source:
Arch Toxicol 81, 127-143

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
8 weeks after exposure brainstem auditory-evoked responses were evaluated.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: inhalation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Composition of samples used:
Mixed xylene no. 1: 20% o-xylene, 20% p-xylene, 40% m-xylene, 20% ethylbenzene
Mixed xylene no. 2: 30% o-xylene, 10% p-xylene, 50% m-xylene, 10% ethylbenzene
- Source: xylenes from Acros, Geel, Belgium; ethyl benzene from Aldrich, Steinheim, Germany
- Purity: 99% (for all)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, Saint-Germain-sur-l'Arbresle, France
- Age at study initiation: approx. 15 wk
- Housing: individual, polypropylene cages with woodchip bedding
- Diet : UAR-Alimentation, Epinay-sur Orge, France, sterilised by gamma irradiation, ad libitum
- Water : filtered tap water, ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Humidity: 50-60%
- Photoperiod (lights on): 07.00 - 19.00 hr

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
TYPE OF EXPOSURE: Whole body
CHAMBER DESCRIPTION
- Exposure apparatus: 200 L stainless steel inhalation chambers, maintained at slight negative pressure
- Method of holding animals in test chamber: not stated (individual housing presumed)
- Source and rate of air: filtered room air
- Air flow rate: 4-30 m^3/hr, dynamic and adjustable
- Other: ambient sound levels in the chambers were < 66 dB SPL.

TEST ATMOSPHERE
- Generation: concentrated vapour (ethyl benzene, xylenes) was diluted to the required concentration with air prior to entering the exposure chamber
- Analysis: exposure levels monitored continuously using a gas liquid chromatograph, with GC-FID analysis of ethylbenzene and xylene isomer concentrations once every 6 hr.

SAMPLE COMPOSITION
Mixed xylene no. 1: 20% o-xylene, 20% p-xylene, 20% ethylbenzene, 40% m-xylene
Mixed xylene no. 2: 30% o-xylene, 10% p-xylene, 10% ethylbenzene, 50% m-xylene

EXPOSURES
Mixed xylene no. 1: 0, 250, 500, 1000 or 2000 ppm
Mixed xylene no. 2: 0, 250, 500, 1000 or 2000 ppm
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of chamber air were collected (activated charcoal) once during each 6 hr exposure period and analysed by chromatography (GC-FID, polyethylene glycol coated Supelcowax-10 column) with toluene used as internal standard. The results were used to calibrate for GLC device employed for continuous monitoring of ethyl benzene / xylene levels within the chamber.
Duration of treatment / exposure:
6 hr/d
Frequency of treatment:
6 d/wk for 13 wk
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, 1000 and 2000 ppm for two xylene mixtures
Basis:
nominal conc.
No. of animals per sex per dose:
14
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection: exposure concentrations based on results of preliminary range-finding studies, and designed to result in a slight (<10%) reduction in body weight following 4 wk treatment.
- Recovery period: 8 wk after final exposure
- Surgical procedures: 3 silver electrodes implanted into the skull under general anaesthesia (active electrode placed over the inferior colliculus; reference electrode posterior to bregma and right of the midline; ground electrode over the nasal bone) and fixed in place with dental acrylic. This was followed by a recovery period of at least 7 days.

Examinations

Examinations:
Observations and examinations performed and frequency
- Brainstem auditory-evoked response: measurements made using a Racia computerized system following 4, 8 and 13 wk exposure and again at the end of an 8 wk recovery period (study wk 21). Responses recorded with a bandpass of 110 Hz-3.2 kHz with the averaged response recorded after 1,024 clicks (stimulus rate = 10 clicks/s, in 5-dB steps). Audiometric thresholds estimated at 2, 4, 8 and 16 kHz by visual inspection.
- Morphological examination: at end of recovery period, animals fixed (paraformaldehyde/gluteraldehyde) via cardiac perfusion and the organ of Corti and basilar membrane removed, fixed (buffered 1% osmium tetroxide) and examined (apparently without staining) using light microscopy. Cytocochleograms (total cell count) were constructed with the frequency coordinates superimposed on the length coordinates of the organ of Corti. Four left and four right cochleas from eight rats per group (including controls) were processed in this manner.

Results and discussion

Details on results:
An irreversible, functional deficit in hearing (assessed from auditory evoked response) was apparent in rats exposed to two samples of mixed xylene, with a NOAEC of 500 ppm in one instance and a NOAEC of 1000 ppm in the other. Microscopic examination of the organ of Corti and basilar membrane demonstrated clear reductions in hair cells with a LOAEC of 250 ppm and a NOAEC of 500 ppm for the two samples, respectively. The calculated theoretical concentration of mixed xylene causing a 50% loss of hair cells (EC50) was 1114 ppm - 1636 ppm, respectively.

Any other information on results incl. tables

MIXED XYLENE No. 1

- General condition: no exposure-related mortality, and animals in all five groups remained in good health throughout the study.

- Body weight: no statistically significant differences.

- Auditory evoked response: thresholds were increased at all four frequencies from wk 4 onwards in animals exposed to 2000 ppm or 1000 ppm mixed xylene no. 1. The effect was greatest increase in the 2000 ppm group where deficits of 35 dB at 2 kHz, 39 dB at 4 kHz,42 dB at 8 kHz and 48 dB at 16 kHz were recorded at the end of the exposure period with no recovery after 8 wk. Thresholds in the 1000 ppm were increased non-significantly by 13 to 19 dB. No shift was observed in the controls or the groups exposed to 250 or 500 ppm mixed xylene no. 1.

- Morphology: exposure to 2000 ppm mixed xylene resulted in an almost complete loss of hair cells in the three rows of the OHC (greatest in row 3) with an approx. 6% loss of inner hair cells (IHC) also. Pronounced loss of OHC was also apparent at 1000 ppm (approx. 50% reduction in cells of the third row) but no of IHC. Exposure to 250 and 500 ppm caused significant losses (up to 60% in the 5-8-kHz region) in the third row of the OHC in one animal from each group. There was no significant hair cell loss in the controls. 250 ppm mixed xylene no.1 was therefore considered a LOAEL by the study authors. The calculated EC50 was 1114 ppm.

 

MIXED XYLENE No. 2

- General condition: there was no exposure-related mortality and animals in all five groups remained in good health throughout the study.

- Body weight: no statistically significant differences.

- Auditory evoked response: thresholds were higher from wk 4 at all four frequencies in animals exposed to 2000 ppm, increasing slightly over the course of the study to reach 15 dB at 2 kHz, 17 dB at 4 kHz, 19 dB at 8 kHz and 22 dB at 16 kHz by wk 13. There was no recovery following 8 wk with no exposure. No threshold shifts were present in the controls or in animals exposed to 250, 500 or 1000 ppm mixed xylene no. 2.

- Morphology: exposure to 2000 ppm mixed xylene no. 2 lead to hair cell loss in the three rows of the OHC which was greatest in row 3 (average loss approx. 65%). Four high dose animals also showed substantial IHC losses in the medium part of the organ of Corti. Exposure to 1000 ppm caused OHC losses but no loss of IHC. There was no significant hair cell loss in the controls or in the 250 ppm or 500 ppm groups. The calculated EC50 was 1636 ppm.

Mean measured exposure concentrations (GC-FID):

(Table based on Gagnaire F et al, 2007, Arch Toxicol 81, 127-143, Table 1)

 

Mixture 1

Mixture 2

Target (ppm)

250

500

1000

2000

250

500

1000

2000

EB

51

101

200

402

25

50

101

200

o-xylene

50

100

198

399

75

150

302

598

m-xylene

101

201

399

802

125

250

503

995

p-xylene

51

101

201

405

25

51

102

202

There was irrerversible loss of hair cells in the organ of Corti and basiliar membrane for mixed xylene sample 1 at 250 ppm and for mixed xylene sample 2 at 500 ppm.

Applicant's summary and conclusion

Conclusions:
An irreversible, functional deficit in hearing (as assessed from auditory evoked response) was apparent in male rats exposed to two samples of mixed xylene, with a NOAEC of 500 ppm in one instance and a NOAEC of 1000 ppm in the other. Microscopic examination of the organ of Corti and basilar membrane demonstrated clear reductions in hair cells with a LOAEC of 250 ppm and a NOAEC of 500 ppm for the two samples, respectively. The calculated theoretical concentration of mixed xylene causing a 50% loss of hair cells (EC50) was 1114 ppm - 1636 ppm, respectively.
Executive summary:

Mixed xylene no. 1: 20% o-xylene, 20% p-xylene, 20% ethylbenzene, 40% m-xylene

Mixed xylene no. 2: 30% o-xylene, 10% p-xylene, 10% ethylbenzene, 50% m-xylene

Brainstem auditory-evoked responses were recorded following 4, 8 and 13 wk of exposure and again after an 8 wk recovery period. Cytocochleograms (total hair cell counts) were made at the end of the recovery period. The results demonstrated an irreversible, functional loss of hearing (as assessed from auditory evoked response) in male rats with a NOAEC of 500 ppm for mixed xylene no. 1 and a NOAEC of 1000 ppm for mixed xylene no. 2. Microscopic examination of the organ of Corti and basilar membrane demonstrated clear reductions in hair cells with a LOAEC of 250 ppm and a NOAEC of 500 ppm for the two samples, respectively. The calculated theoretical concentration of mixed xylene causing a 50% loss of hair cells (EC50) was 1114 ppm for mixed xylene no. 1 and 1636 ppm for mixed xylene no. 2. The TLAELs calculated from the 95, 99 and 99.9% statistical upper confidence limits are 242, 254 and 273 ppm respectively or mixed xylene 1 and 417, 446 and 469 ppm respectively for mixed xylene 2.