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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:

- Source: Charles River WIGA GmbH, Sandhoferweg 7, D-97633 Sulzfeld, Germany

- Age at study initiation: 6-8 weeks

- Weight at study initiation: 150-166g males, 103-116g females

- Housing: Animals were housed individually in Makrolon type-3 cages, with standard softwood bedding

- Diet: pelleted standard Kilba 343 rat maintenance diet, ad libitum

- Water: community tap water (Geneva), ad libitum

- Acclimation period: 7-13 days


- Temperature (°C): 22 +/-3C

- Humidity (%): 30-70

- Air changes (per hr): 10-15

- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: none
Details on inhalation exposure:

- Exposure apparatus: Inhalation exposure was performed according to the method of Sachsse et al. (1973, 1976). The test article stream reached the animals noses through ports situated at different levels around the axis of the chamber. Each level had 8 ports and could be rotated, allowing close observation of all the animals without interruption of exposure.

- Exposure chamber volume: The internal active volume of the exposure chamberfor exposing 24 animals is ca. 0.7 litres.

- Method of holding animals in test chamber: The animals were confined separately in Makrolon tubes which were positioned radially around the exposure chamber.

- Source and rate of air: Mean airflow rate was 1.2 l air/minute per animal (group 1), 1.0 l air/minute per animal (group 2) and 0.8 l air/minute per animal (group 3). The time for the concentration to reach 99% of its ultimate value (T99) at an animal port is 7 seconds.

- Method of conditioning air: The flow past, nose only design of this exposure system is based upon the fluid dynamic modelling of the aerosol flow. It ensures a uniform test article distribution, provides a constant stream of 'fresh' test article to each animal, and precludes rebreathing the exhaled air.

- System of generating particulates/aerosols: The test article was placed as supplied in a glass flask feeding a nebulizer. The level of test article in the reservoir of the nebulizer was kept constant and was generated at room temperature. The test atmosphere generated by the nebulizer was diluted with compressed filtered air to achieve the concentrations required for this study and discharged into the exposure chamber with a target mass median aerodynamic diameter of 4 micrometres or less.

- Temperature, humidity, pressure in air chamber: Samples for the measurement of the test article concentration (analytical, gravimetric), oxygen concentration, relative humidity and temperature were collected from a port of the exposure chamber, directly from the feed tube delivering 'resh' test article to the animals nose. Therefore, all the measurements were iso axial and represented exactly what was delivered to the animals.


- Brief description of analytical method used: The analytical determinations of the test article in the test atmosphere were performed using test atmosphere samples collected in wash-bottles connected to the exhaust of the Gelman filter holder (used for gravimetric determinations). The volatile phase of the test atmosphere was passed into three wash bottles placed in series containing each 80ml of n-hexane cooled at -70C. The content of each wash bottle was transferred into 100ml volumetric flasks, the wash bottles were rinsed with hexane and the flasks were made up to 100ml with the rinsing. Aliquots of each wash bottle were put into appropriate sealed vials and sent for analysis at ambient temperature.

- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)

- MMAD (Mass median aerodynamic diameter): 4 micrometres or less.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
20.12 mg/l; 30.03 mg/l; 54.37 mg/l
No. of animals per sex per dose:
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: Clinical signs and mortality were noted during and following the exposures over a 15-day observation period. Body weights were recorded once during the acclimatisation period and then on test days 1 (prior to exposure), 2, 3, 4, 5, 9, 12 and 15. Food consumption was measured in the rats which survived the treatment once during acclimatisation period (over 5 days), then during 4 intervals following treatment (from days 1-6, 6-9, 9-12 and 12-15).

- Necropsy of survivors performed: yes

- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other: All macroscopical abnormalities following necropsy were recorded. The lungs, liver, spleen and thymus of all surviving animals were weighed before fixation.
The LOGIT-Model (LOGIT: A program for Dose-response Analysis, koshiver J. and Moore D., Computer Programs in Biomedicine, 10, 1979, 61-75) was used to calculate LC50.

Results and discussion

Effect levels
Key result
Dose descriptor:
Effect level:
36 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: (corresponding to approx. 2,975 ppm)
See Table 3.
Clinical signs:
other: Reduced food consumption in all groups. Hunched posture, stiff gait, ruffled fur in all groups; restlessness and/or excitement during exposure in all animals that died; tachypnea (mid dose), rales and/or head drop (some low and mid dose males). Following
Body weight:
All animals of the low and medium exposure groups as well as the surviving male of the high exposure group lost weight during the day of exposure (in a few cases during two to three days) following exposure. This reduction in body weight was slightly more pronounced in the medium than in the low exposure group. After 9 days the body weights of the surviving animals were again close to pre-exposure values. The growth rate until the end of the 15 day observation period was considered to remain slightly under normal values.
Gross pathology:
No macroscopic abnormalities were noted in any animals of the low exposure group. The lungs of all decedent animals of the medium and high exposure groups showed red discoloration. In addition, dark red or reddish foci were seen on the thymus of 1 male and 1 female of the medium exposure group, and of one male of the high exposure group. The mandibular lymph nodes of 1 high exposure male showed a reddish discolouration. Changes in the lungs are related to spontaneous death due to the treatment. Changes in the thymus and in the lymph nodes are considered to be incidental.
Other findings:
- Organ weights: All organ weight differences (if any) between groups were marginal. Trend of lung weight increase in both sexes, thymus weight decrease in mass, and spleen weight increase in both sexes. Data from a single animal were not considered as sufficient for accurate evaluation of organ weight changes at the high exposure level.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
An acute inhalation LC50 value of 36 mg/l/4h air was determined in both male and female rats in a reliable study conducted according to an appropriate protocol, and in compliance with GLP.