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Repeated dose toxicity: inhalation

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short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the O.E.C.D. test guideline 412 with GLP compliance.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Only two test concentrations were due to the test substance's limited vapor pressure.
GLP compliance:
Limit test:

Test material

Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.

Test animals

Fischer 344
Details on test animals and environmental conditions:
The Fisher rats were acquired from Charles River Breeding Laboratory, Kingston, NY and were six weeks of age. Prior to and after exposure, animals were singly housed in rooms designed to maintain adequate environmental conditions concerning temperature and relative humidity for the species under test and according to the test guideline. A 12-hour photoperiod was to be used. Water and Purina Certified Rodent Chow #5002 (Purina Mills Inc., St. Louis, MO) were available ad libitum for all animals except during exposure.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
clean air
Details on inhalation exposure:
Animals were singly housed and whole-body exposed to test substance vapor in stainless steel and glass one cubic meter Rochester-type inhalation chambers (90 cm wide x 90 cm high x 90 cm deep) under dynamic airflow conditions. Airflow was maintained at approximately 225 liters/minute.

Vapors of the test substance were generated using a glass J-tube method (Miller et al., 1980). Compressed clean air, heated with a flameless torch (FI-IT-4, Master Appliance, Racine, WI) to the minimum extent necessary, passed through the J-tube to volatilize the test material. The compressed air for the 4 ppm chamber was typically heated to 40 to 50° C just before entering the vaporization area. Chambers were controlled by a system designed to maintain temperature and relative humidity at approximately 22° C and 50% relative humidity.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The analytical concentration of the test substance the chamber was determined three times per exposure period by adsorption onto Chromosorb 102 sampling tubes (SKC, Inc., Eighty-Four, PA). The tubes were desorbed in a suitable solvent (98% CS2/ 2% acetone), and the extracts analyzed with a capillary column on a HP 5890 gas chromatograph (Hewlett Packard Co., Avondale, PA) that was calibrated with the test substance in CS2. The analytical system was checked by preparing a test substance spiked Chromosorb 102 tube on each exposure day.
Duration of treatment / exposure:
Six hours per day for four weeks.
Frequency of treatment:
Five days per week.
Doses / concentrations
Doses / Concentrations:
0.6 and 4 ppm of the test substance (4.0 and 26.8 mg/m3)
analytical conc.
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were observed daily in the afternoon post-exposure. A weekly individual clinical examination was performed on all rats. Following the completion of the exposure phase of the study blood samples for the accessment of hematological and clinical chemistry parameters were collected from the orbital sinus of rats anesthetized with methoxyflurane immediately prior to necropsy. Urine was collected from rats during the fourth week of exposure.

All surviving animals were necropsied the day following the last exposure to the test material. All animals were fasted overnight prior to the scheduled necropsy. Each animal was weighed, anesthetized with methoxyflurane and humanely euthanized. Weights of the brain, lungs, liver, kidneys, adrenals and testes were recorded from all animals at the scheduled sacrifice. All animals were examined for gross pathological alterations by a veterinary pathologist. A complete set of required tissues was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. A complete histopathological examination of the tissues was made from all animals in the control and highest exposure group. Tissues to be examined histopathologically were processed by conventional techniques, sectioned at approximately 6 microns, stained with hematoxylin and eosin and evaluated by light microscopy.
Positive control:


Observations and examinations performed and frequency:
SEE "Details on study design."
Sacrifice and pathology:
SEE "Details on study design."
All parameters examined statistically were first tested for equality of variance using Bartlett’s test. All parameters were subjected to appropriate parametric analysis as described below. In-life body weights were evaluated using a three-way analysis of variance (ANOVA) with the factors of sex, dose and time interval (Winer, 1971). Organ weights (absolute and relative except testes), terminal body weights and hematologic, clinical chemistry an urine specific gravity data were evaluated using a two-way (ANOVA) with the factors of sex and dose (Winer, 1971). Results for absolute and relative testes weights were analyzed using a one way ANOVA. If significant dose effects were determined in the one- way ANOVA, then separate doses were to be compared to controls using separate one-way ANOVA’s for each dose compared to control; a Bonferroni correction was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Based on clinical examination.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
No adverse effects reported.

Effect levels

Dose descriptor:
Effect level:
> 4 ppm
Based on:
test mat.
Basis for effect level:
other: No adverse effects reported.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

The NOEC from this study of 4 ppm was limited by the Maximum Achievable vapor concentration. Use of this data to establish systemic DNELs will result in ultra conservative values. The conduct of an oral rat 90-day O.E.C.D. 409 subchronic study as proposed in the Test Plan will provide higher test substance blood levels and a more definitive picture of potential adverse systemic effects.
Executive summary:

The test substance, 2,3-epoxypropyl o-tolyl ether was accessed for potential adverse effects in an O.E.C.D. test guideline no 412 four-week inhalation study in rats. Due to the low achievable vapor concentration of 4 ppm no adverse effects were reported.