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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:
other: Test performed according to GLP and internationally accepted study protocols. No deviations from protocol.
Qualifier:
according to
Guideline:
other: Revision of Guidelines for Single and Repeated Administration Toxicity Studies (notification n°88 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan, August 1993.
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Japan
- Strain: Sprague-Dawley SPF [Crj:CD (SD)]
- Age at study initiation: 5 weeks
- Weight at study initiation: 164-186g (males), 120-144g (females)
- Fasting period before study: overnight, approx. 16 hours
- Housing: per 2 in stainless steel wire mesh cages (W254 x D350 x H170 mm)
- Diet (e.g. ad libitum): ad libitum, irradiation sterilized CRF-1, Oriental Yeast Co., Ltd
- Water (e.g. ad libitum): ad libitum, Gotemba Municipal Water Works, via automatic water supply system
- Acclimation period: 7 days (quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): 15 to 20 per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 22/09/1997 To 13/10/1997
Route of administration:
oral: gavage
Vehicle:
other: gummi arabicum pulveratum + water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 5% w/v

MAXIMUM DOSE VOLUME APPLIED: 20 mL/ kg bw

DOSAGE PREPARATION: The requisite amounts of test article and gummi arabicum pulveratum (5% w/v in the final concentration) were weighed accurately. The test article was mixed with gummi arabicum pulveratum with agate morter (approx. 5 minutes) while adding water (for injection) to the proper quantity (Water for injection: JP, Otsuka Pharmaceutical Factory Inc., Lot n° 7D72), and test suspensions of the prescribed concentrations were prepared.

Preparation of test solutions was done at the time of use.

Control animals were administered 5 %w/v gummi arabicum solution.
Doses:
0, 500, 1000 and 2000 mg/kg bw
No. of animals per sex per dose:
6 males and 6 females per dose
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: frequently for the first 6 hours after dosing and once daily thereafter
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
The lethal dose was estimated based on the cumulative mortaility for 14 days after administration. Body weights were subjected to multiple tests shown in a diagram available in the test report (Bartlett's Test, F-test, One-way analysis of variance, Kruskal-Wallis' Rank-Sum test, et cetera).
Preliminary study:
Prior to the main study, a preliminary study was conducted using 3 animals of each sex during the quarantine/acclimatization period. The dose level was 2000 mg/kg bw, in accordance with the toxicity study guidelines. No deaths occurred at this dose level. Based on these results, 2000 mg/kg bw was set as a high dose level and 2 other dose levels were set at 1000 and 500 mg/kg bw (i.e. ratio 2).
Sex:
male/female
Dose descriptor:
LD0
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No deaths occurred in either sex in any dose group. The lethal dose was estimated more than 2000 mg/kg bw.
Clinical signs:
No abnormalities were observed in any animals.
Body weight:
The body weight changes in both sexes in all dose groups were comparable to the control group.
Gross pathology:
No abnormalities were found in the external appearance or the tissue/organs in the cranial, thoracic and abdominal cavities in any of the animals.
Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: Japan
Conclusions:
When L-leucine was administered once by gavage to rats, the toxicity was suggested to be very low.
Executive summary:

A single dos toxicity study of L-leucine by oral administration was conducted in Sprague-Dawley rats [Crj:CD(SD), 6 animals / sex / dose] at dose levels of 0 (5% gummi arabicum solution), 500, 1000 and 2000 mg/kg bw. No deaths occured in either sex in any dose group. No abnormal clinical signs were observed in any animals. The body weight changes of both sexes in all dose groups were comparable to the control group. The necropsy reveales no abnormalities in any animal.

The toxicity of L-leucine upon single oral administration was found to be very low. The lethal dose was estimated > 2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
16 000 mg/kg bw
Quality of whole database:
All available studies (including the study on read-across substance L-valine) are in line with each other, i.e. no mortalities occurred in any of the experiments. The LD50 is therefore > 16000 mg/kg bw.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three studies are available that assess the acute oral toxicity of L-leucine. The key study (Mochizuki, 1997) is carried out according to GLP and an internationally accepted protocol. No deaths occurred at the maximum tested dose of 2000 mg/kg bw. Similarly, no mortality was observed in any of the 2 supporting studies in which the doses ranged from 2000 to 16000 mg/kg bw

 

As a consequence it was concluded that L-leucine does not exert acute toxicity following oral administration of a 2000 mg/kg bw dose.

As described above, sufficient test results for L-leucine are available to exclude acute oral toxicity of the substance. Nevertheless, read-across to a test results available for structural analogue L-valine is added to the dossier in order to substantiate the applicability of the read-across approach.

Read-across between L-valine and L-leucine is deemed justified based on a comparison of the main factors driving oral, inhalatory and dermal absorption and toxicity: molecular weight, log Kow, water solubility, vapour pressure and chemical reactivity (functional groups).

- both are essential amino acids that are in the zwitterion state at physiological pH

- the chemical structure differs only in that L-leucine has one extra methylene group in the aliphatic side chain

- the substances both have a high water solubility: 23 g/L for LEU, 58 g/L for VAL

- the molecular weights of the substances are very close together: 131 g/mol for LEU and 117 g/mol for VAL

- the substances both have a low log Kow value: -1.59 for LEU (calculated by EpiWin QSAR), -2.08 for VAL (calculated by EpiWin QSAR)

- the substances both have a low vapour pressure: 7.35E-07 Pa for LEU (calculated by EpiWin QSAR), 1.22E-06 Pa for LEU (calculated by EpiWin QSAR)

 

As the chemical structure and reactivity, and the relevant physicochemical parameters of L-leucine and L-valine are almost identical, it can be concluded that read-across for oral, inhalatory and dermal absorption and toxicity information between those 2 substances is acceptable.

The test available for L-valine is carried out according to OECD guidelines and reveals no acute oral toxicity at the tested concentrations (2000 mg/kg bw). This is in agreement with the results obtained from the acute oral toxicity testing performed on L-leucine.

As L-leucine has a low vapour pressure and 99.45% of the particles are > 100 µm, the inhalatory route is not considered a relevant exposure route for this substance.

Dermal absorption of L-leucine can be excluded based on its low lipophilicity, which is expressed by its high water solubility (23000 mg/L) and low log Kow value (-1.59).


Justification for selection of acute toxicity – oral endpoint
GLP study according to internationally accepted guideline.

Justification for classification or non-classification

No effects of acute toxicity were observed upon oral administration of L-leucine up to the maximum tested dose of 2000 mg/kg bw. As a consequence, classification of L-leucine for oral acute toxicity is not required in accordance with the criteria described in Annex I to regulation 1272/2008 (CLP) and Annex VI to Directive 67/548 (DSD).