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Description of key information

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): L-isoleucine
- Physical state: white powder
- Analytical purity: 94.2%
- Lot/batch No.: 1
- Storage condition of test material: ambient temperature, dark
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: Wistar outbred rat; Crl:(WI) WU BR
- Source: Charles River, Germany
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: 164-168 g
- Fasting period before study: overnight and untill 4 hours after exposure
- Housing: max. 6 animals per macrolon cage
- Diet (e.g. ad libitum): standard laboratory diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%): 30-70%, occasionally up to 74.4% because of meteorological circumstances and/or wet cleaning of the animal room
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
maize oil
Details on oral exposure:
The study was carried out with treatment of two groups of 3 animals each with a dose level of 2000 mg/kg bw. The animals were dosed with a 10 mL/kg bw dose-volume of a 200 mg/mL suspension of the test substance in maize oil in order to obtain the 2000 mg/kg dose level. The exact amount of the test substance to be dosed was calculated for each animal individually and administered by means of a syringe, equipped with an oral gavage. Prior to dosing, the animals had fasted overnight. Approx. 4 hours after dosing, they had access to food again. The animals were observed for mortality up to 14 days after treatment.
Doses:
2000 mg/kg
No. of animals per sex per dose:
3 female animals
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: clinical signs within 1-4 hours after dosing, and daily afterwards
- Frequency of weighing: on days 0, 3, 7 and 14
- Necropsy of survivors performed: not applicable
- Other examinations performed: at the end of the observation period, all surviving animals were killed with carbon dioxide and examined for external changes. Next, the abdomen and the thorax of each animal was opened and examined for gross pathological changes.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality observed.
Clinical signs:
other: No clinical signs observed.
Gross pathology:
No treatment-related gross alterations observed.
Interpretation of results:
practically nontoxic
Conclusions:
L-isoleucine is not harmful if swallowed.
Executive summary:

A sample of L-isoleucine was tested for acute oral toxicity in an experiment with female rats (limit testing) according to OECD 423 and EU B.1tris Guidelines.

No mortality or clinical signs were observed after treatment with 2000 mg/kg bw dose level. Macroscopic examination of the surviving animals at the end of the observation period did not reveal any treatment-related gross alterations.

Since all animals survived the 2000 mg/kg bw dose level, the oral LD50 of L-isoleucine is considered to be higher than 2000 mg/kg bw. According to the EC-standards (Official Journal of the European Communities, L 110A, Volume 36, 4 May 1993), L-isoleucine is considered not harmful if swallowed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records
Reference
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
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
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
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
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
5 410 mg/m³ air

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No studies are available for acute toxicity via dermal route. In accordance with column 2 of REACH Annex VIII testing on acute toxicity by dermal route is only required if exposure of humans via this route is likely taking into account the physicochemical properties of the substance (such as vapour pressure, water solubility, volatility and lipophilicity).

In the case of L-isoleucine exposure via the dermal route is not considered to be of concern.

For acute toxicity via the oral route 3 studies are available. In the first study (key - Prinsen, 2005) a sample of L-isoleucine was tested for acute oral toxicity in an experiment with female rats (limit testing) according to OECD 423 and EU B.1tris Guidelines. No mortality or clinical signs were observed after treatment with 2000 mg/kg bw dose level. Macroscopic examination of the surviving animals at the end of the observation period did not reveal any treatment-related gross alterations. Since all animals survived the 2000 mg/kg bw dose level, the oral LD50 of L-isoleucine is considered to be higher than 2000 mg/kg bw.

In the second study (supporting - Davies and Halliday, 1971) rats were fed a maximum practical dose of 16g amino acid/kg bw and observed for 2 weeks after dosing. One female rat died within 42 hours of dosing and the autopsy revealed a pale liver. The autopsy of all other animals revealed no abnormalities. There were no signs of reaction to the treatment. Slight depression in bodyweight increase in treated animals during the first week. Bodyweight increases during the second week were normal compared to controls. As the oral administration of 16000 mg/kg bw L-isoleucine to rats did not lead to the death of the animals, the LD50 was found to be > 16000 mg/kg bw.

In a third study (supporting - BOZO Research Center, 2007) L-isoleucine was administered once to 5 male and 5 female rats at a dose level of 2000 mg/kg bw. No deaths occurred, and thus the lethal dose was estimated to be > 2000 mg/kg bw. No test article-related effects were found in the clinical signs, body weights or necropsy examinations of the treated animals. It was therefore concluded that the toxicity of L-isoleucine is extremely low.

For the acute toxicity via the inhalation route, one study is available wherein the OECD 403 was followed and thus one group of 5 male and 5 female rats were exposed nose-only for 4 hours to L-isoleucine. After exposure, the animals were observed for 14 days. A slight decrease in breathing rate was observed in all animals during the last three hours of exposure. Shortly after exposure, 4/5 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. Since no mortality occurred, the 4 -hour LC50 value of L-isoleucine in rats was larger than 5.41 g/m3 for both sexes.

Justification for classification or non-classification

For acute oral and inhalation toxicity the criteria for classification used here are shown in Table 3.1.1 in Part 3: Health hazards of the CLP Regulation No 1272/2008. Here, the LD/LC50 values are used as the acute toxicity estimates (ATE) and the substance can be allocated to one of the four toxicity categories . As the oral LD50 for L-isoleucine > 2000 mg/kg bw and the inhalation LD50 > 5 mg/L, L-isoleucine does not require to be classified according to the criteria of the CLP regulation.

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