Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
small deviation not considered to have influenced the integrity and validity of the study and its results
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
small deviation not considered to have influenced the integrity and validity of the study and its results
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
L-isoleucine
EC Number:
200-798-2
EC Name:
L-isoleucine
Cas Number:
73-32-5
Molecular formula:
C6H13NO2
IUPAC Name:
L-isoleucine
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: SPF-reared, Wistar-derived (Crl:[WI]WU BR)
- Source: Charles River Deutschiand, Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: mean body weights: 272 g (males) and 178 g (females)
- Fasting period before study: no data on fasting before study
- Housing (during observation period): macrolon cages with a stainless steel wire lid and wood shavings, 5 same-sex animals per cage
- Diet (e.g. ad libitum): commercially available rodent diet (Rat & Mouse No. 3 Breeding Diet RM3) from SDS Special Diets Services, Witham, England; ad libitum. During exposure the animals had no access to feed.
- Water (e.g. ad libitum): tap water suitable for human consumption; ad libitum. During exposure the animals had no access to water.
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): at least 30%, but slightly exceeded the 70% upper limit on a few occasions, due to wet cleaning of the animal room. On four occasions (not lasting longer than 1,5 hour), relative humidity exceeded the 70% upper limit which could not be attributed to room cleaning activities (maximum of 82.6% on 6 June 2005).
- Air changes (per hr): ca. 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light and 12-hour dark cycle

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks:
humidified compressed air
Details on inhalation exposure:
Exposure equipment

Animals were exposed to the test atmosphere in a nose-only inhalation chamber, a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. The inhalation chamber consisted of a cylindrical aluminium column, surrounded by a transparent cylinder. The column had a volume of ca. 50 litres and consisted of a top assembly with two mixing chambers, underneath a rodent tube section and the exhaust section at the bottom. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling, particle size analysis and measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from high to low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals' thorax to the nose is prevented. The unit was illuminated externally by normal laboratory TL-lighting.

Generation of the test atmosphere
Since the Mass Median Aerodynamic Diameter (MMAD) of the supplied test material as estimated during preliminary testing was larger (viz. >30 /µm) than the preferred range of 1-4 /µm, the original test material was mechanically processed (milled) to reduce the particle size before the start of the exposure. Portions of the test material were ground with a universal mill (IKA-Werke GmbH & Co, KG, type M20) 10 times for 30 seconds. In order to limit the increase in temperature of the test material, milling was intermitted at 30-second intervals and the inner wall of the grinding chamber was cooled with a stream of compressed air.
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing the milled test material to an eductor (Fox mini type 031, Fox Valve Development corp., Dover, NJ, USA) using a dry material feeder (Gericke GMD 60, Gericke AG, Regensburg, Switzerland), which was modified to enable a more even supply of the test material to the eductor. The eductor was placed at the top inlet of the exposure unit and was operated with humidified compressed air controlled by a pressure reducing valve at a constant pressure of 2.0 bar. The milled test material was delivered in a slip stream of ambient (humidified) air. The resulting aerosol was directed downward towards the animal noses. At the bottom of the unit the test atmosphere was exhausted (see Figure 1).
Before exposure, the air consumption of the eductor was established to be 35.92 L/min at the input pressure used (2.0 bar; measured in duplicate, results were 35.93 and 35.90 L/min, respectively).
During the generation of the test atmosphere, the setting of the feeder and the input pressure of the eductor were recorded at regular intervals (approximately each half hour). In this way, total air flow during exposure was monitored indirectly through the aerosol generation system. The mean air flow through the exposure unit was therefore 35.92 L/min.
The animals were placed in the exposure unit after stabilization of the test atmosphere. The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 43 minutes.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric analysis
Duration of exposure:
4 h
Concentrations:
Nominal concentration: 13.98 g/m3
Actual concentration: 5.41 +/- 20 g/m3
No. of animals per sex per dose:
5 males and 5 females
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: daily
- Frequency of weighing: on day 0, 7 and 14
- Necropsy of survivors performed: yes, examination for gross pathological changes, with particular reference to any changes in the respiratory tract

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 410 mg/m³ air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
None
Clinical signs:
other: Although observation of the rats was limited due to the stay in restraining tubes, a slightly decreased breathing rate was observed in all animals during the last three hours of the exposure period. An hour after exposure, moderate lethargy, moderate ble
Body weight:
Overall body weight gain in male and female animals was reduced during the first week after exposure, and was normal during the second week after exposure.
Gross pathology:
Macroscopic examination at necropsy revealed grey discoloured lungs in all animals. Small petechiae on one or two lung lobes were observed in one male and one female animal. However, the latter finding is part of common background pathology in rats of this strain and age, and is occasionally also seen in untreated animals (Slaoui et. al., 1998). Therefore, it is not considered to be treatment-related.

Applicant's summary and conclusion

Conclusions:
Since no mortality occurred, the 4-hour LC50 value of L-ISOLEUCINE in rats was larger than 5.41 g/m3 for both sexes.
Executive summary:

The acute inhalation toxicity of L-isoleucine was studied in one group of 5 maleand 5 female rats. This group was exposed nose-only for a single 4 -hour period to a test atmosphere containing L-isoleucine at a limit concentration of 5.41 ± 0.20 g/m3.The mass median aerodynamic diameter (MMAD) of the particles in the test atmosphere measured during exposure was 3.8 um. The distribution of particle sizes had a geometric standard deviation (gsd) of 2.4. Similar values were obtained during preliminary testing.

After exposure, the animals were kept for a 14-day observation period. All rats were necropsied on day 14 and examined for gross pathological changes.

A slightly decreased breathing rate was observed in all animals during the last three hours of exposure.

Shortly after exposure, four out of five female animals showed moderate lethargy, moderate blepharospasm and a slightly hunched appearance. No abnormalities were seen in the other female animal or in the male animals. During the 14-day observation

period, clinical abnormalities were not seen and no mortality occurred.

Overall body weight gain was reduced during the first week after exposure, and was normal during the second week after exposure.

At necropsy, macroscopic examination revealed grey discolouration of the lungs of all animals.

Since no mortality occurred, the 4 -hour LC50 value of L-isoleucine in rats was larger than 5.41 g/m3for both sexes.