Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information
No adverse effects were observed in a pre-natal developmental toxicity study with rats.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-documented non-GLP study according to internationally accepted protocol (Japanese). No deviations from protocol that might hamper the validity of the study. The test substance was applied from day 7-17. According to OECD TG414, the test substance should be administered at least from implantation, i.e. day 5 in the rat.
Qualifier:
according to
Guideline:
other: Revision of Guidelines for Reproductive and Developmental Toxicity Studies of Drugs, Notification No 316 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan, 1997
Deviations:
yes
Remarks:
minor deviations of temperature and humidity that were not judged to have affected the reliability of the study
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley [Crj : CD (SD) IGS]
- Source: Atsugi Breeding Center, Charles River Japan, Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: measured, but data not available intranslated study report
- Fasting period before study: no data
- Housing: bracket-type stainless steel wire mesh cages (WxDxH: 365x305x175 mm)
- Diet (e.g. ad libitum): ad libitum, pellet diet NMR (Oriental Yeast Co. Ltd.)
- Water (e.g. ad libitum): ad libitum, tap water (Gotemba City Water) via carbonate bottles
- Acclimation period: 1 week (quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 40-74%
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Requisite amount of carboxymethylcellulose was weighed, dissolved in water for injection and diluted to a concentration of 0.3% w/v. The vehicle was prepared at the time of use and used for preparation of test suspension or administration within 12 hours of preparation.
For each dose concentration, requisite amount of the test article was weighed, suspended in 0.3% w/v carboxymethylcellulose solution and diluted to the concentrations of 3 and 10% w/v. The test suspensions were prepared at the time of use and used for administration within 6 hours of preparation.

VEHICLE
- Concentration in vehicle: 3 or 10% w/v.
- Amount of vehicle (if gavage): dose volume: 10 mL/kg bw
- Lot/batch no. (if required): Lot N° 2115
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were housed overnight with male rats on a one-to-one basis. If the presence of a vaginal plug was observed in the vagina or sperm was present in the vaginal smear the following morning, it was considered that copulation was confirmed and that day was designated as day 0 of gestation. At the time of mating, females were 11 or 12 weeks of age, and males were 12 or 13 weeks of age.
Duration of treatment / exposure:
11 days: from day 7 to day 17 of gestation, which corresponds to the period from implantation to closure of the hard palate.
Frequency of treatment:
daily, 7 days per week
Duration of test:
Dams were sacrified on day 20 of gestation.
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the high dose was set at 1000 mg/kg bw, which exceeds the clinical dose level and which is thought to be the maximum administrable dose level for oral administration to rats. The low dose level was set at 300 mg/kg bw, approximately one-third of the high dose level.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before, immediately after and 2 hours after dosing during administration period; once every morning outside the exposure period
- Cage side observations: general condition, abnormalities in external appearance, excrement, nutritional condition, posture and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 4, 7-18 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- For all females, the amount of food left uneaten was measured on days 1, 4, 8, 11, 14, 18 and 20 of gestation and daily food consumption was calculated from the differences between the amount of food supplied on the previous day and the amount of food left uneaten.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Dams were sacrificed by exsanguination from abdominal aorta/vena cava under ether anesthesia in the afternoon of day 20 of gestation and major organs and tissues in the thoracic and abdominal cavities were observed macroscopically.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
For pregnant animals the ovary and uterus were removed and examined for the number of corpora lutea, the number of live fetuses, and numbers of embryo-fetal deaths (implantation sites, resorbed embryos, placentaln remnant, early macerated fetuses and dead fetuses). The placentae of live fetuses were observed macroscopically for any abnormalities and weighed individually. Litter mean of the placental weight was calculated for each sex.
Fetal examinations:
- External examinations: Yes: all per litter
Live fetuses of all females were observed for external malformations and sexed by distance from the anuys to the genital papilla. Individual body weight was measuredand litter mean was calculated for each sex. Fetuses with external abnormalities were photographed and fixed and preserved in phosphate buffered 10% formalin.

- Soft tissue examinations: Yes: ca. half per litter
For each litter approx. half the live fetuses ecept those with external abnormalities were fixed in Bouin's solution. For the control group and high dose group, internal orgnas were examined for visceral abnormalities / variations by Wilson's free-hand razor method for the cephalic cavity and by Nishimura's microdissection method for the thoracic and abdominal caivities. Representative samples of visceral abnormalities were photographed.

- Skeletal examinations: Yes: ca. half per litter
All fetuses except those with external abnormalities and those for visceral examination were fixed in 70% alcohol and subjected to alizarin red S staining by a modified Dawson's method to prepare clear skeletal specimens. For the control group and high dose group, skeletal specimens were examined with a stereoscopic microscope for skeletal abnormalities / variations and examined for the progress of ossification by counting the numbers of ossified metacarpi, metatarsi and sacral and caudal vertebrae. Since a significantly low value in the index of the ossified 5th sternebrae was observed in the high dose group, similar examination was done on the low dose group.

- Head examinations: No data
Statistics:
Body weight, food consumption, number of corpora lutea, number of implantations, placental weight, number of live fetuses and fetal body weight were subjected to one-way analysis of variance by Bartlett's test (the level of significance: 1%, two-tailed). Homogeneous data were analyzed by Dunnett's test (the levels of significance: 5% and 1% two-tailed) and heterogeneous data by Steel's test (the levels of significance: 5% and 1%, two-tailed). The implantation index, index of embryo-fetal deaths, index of external abnormalities, index of skeletal variations and index of ossified sternebrae were analyzed by
Steel's test (the levels of significance: 5% and 1%, two-tailed).
In thee analyses of the data of visceral examination and skeletal examination between the control group and the high dose group, the numbers of ossified metacarpi, metatarsi, and sacral and caudal vertebrae were analyzed by F-test for homogeneity of variance (the level of significance: 5% one-tailed), and homogeneous data were analyzed by student's t-test (the levels of significance: 5% and l%, two-tailed) and heterogeneous data by Aspin-Welch's t-test (the levels of significance: 5% and 1%, two-tailed). The index of visceral abnormalities, index of visceral variations, index of skeletal variations, and index of ossified sternebrae were analyzed by Wilcoxon's rank-sum test (the levels of significance: 5% and l%, two-tailed).
The sex ratio was analyzed by chi-square test with Yates' continuity correction (the levels of significance: 5% and l%, two-tailed) after calculating the sum of live male fetuses and live female fetuses for each group.
For the implantation index, placenta weight, body weight of live fetuses, index of embryo-fetal deaths, index of external abnormalities, index of visceral abnormalities, index of visceral variations, index of skeletal variations, number of ossified bones and index of ossified sternebrae, litter mean values or indices were the unit of samples at each test.
Indices:
- implantation index
- index of embryo-fetal eaths
- index of external abnormalities
- index of visceral abnormalities / variations
- index of ossified sternebrae
- index of skeletal abnormalities
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical signs: no deaths occurred in the control group or any dose group and there were no changes in general condition.

Body weight: in the 300 and 1000 mg/kg bw dose groups, body weight tended to be higher than in the control group, and body weight gain during the administration period (day 7-18 of gestation) was significantly higher than in the control group.

Food consumption: In the 1000 mg/kg bw group, food consumption from day 14 to 18 of gestation was significantly higher than in the control group. In the 300 mg/kg bw group, food consumption was comparable with that of the control group on all measurement days.

Gross pathology findings: No macroscopic abnormalities were observed in any major tissues/organs in the thoracic or abdominal cavity in any dams in the control group or in any dose group.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Cesarion section findings: There were no significant differences between the control group and any dose group in the number of corpora lutea, number of implantations, implantation index, index of embryo-fetal deaths, number of live fetuses or placental weight of live fetuses. There were no macroscopic abnormalities in the placenta of live fetuses in any group.

Findings in the observations of live fetuses at Cesarian section:
There were no significant differences between the control group and any dose group in sex ratio or body weight of live fetuses. In the observations for exernal abnormalities, omphalocele was observed in 1 fetus in the control group, while it was not observed in any dose group.

Visceral examination of live fetuses: The number of fetuses with visceral abnormalities was 1 each in 5 dams in the control group and a total of 3 fetuses in 2 dams in the 1000 mg/kg bw dose group. The abnormalities observed were ventricular septal defect and double azygos vein. However, there were no significant differences in the incidence of any abnormalities between the 2 groups. The number of fetuses with visceral variations was a total of 15 fetuses in 10 dams in the control group and a total of 10 fetuses in 7 dams in the 1000 mg/kg bw group. The variations observed were thymic remnant in the neck, dilatation of renal pelvis, convoluted ureter and left umbilical artery. However, there were no significant differences in the incidence of any variations between the 2 groups.

Skeletal examinations of live fetuses: There were no fetuses with skeletal abnormalities in the control group or in any dose group. The number of fetuses with skeletal variations was a total of 11 fetuses in 6 dams in the control group, a total of 13 fetuses in 7 dams in the low dose group and a total of 15 fetuses in 12 dams in the high dose group. The variations observed were cervical rib and 14th rib. However, there were no significant differences in the incidence of any variations between the control group and any dose group.
In the progress of ossification, comparison between the control group and the high dose group revealed that the index of ossified 5th sternebrae in the 1000 mg/kg bw group was significantly lower. However, comparison among 3 groups including the 300 mg/kg bw group revealed no significant differences in the index of ossified sternebrae, number of ossified metacarpi, number of ossified metatarsi or the number of ossified sacral and caudal vertebrae between the control group and any dose group.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Administration of L-leucine at 1000 mg/kg bw caused no adverse effects on pregnant rats or any embryo-fetal development under the conditions of this study.
Executive summary:

L-leucine was administered to groups of female rats that had copulated. Each group consisted of 20 females, and the dose levels were 0, 300 and 1000 mg/kg bw. The rats were administered the test substance via gavage as a solution in aqueous carboxymethylcellulose, daily from day 7 to day 17 of gestation, which corresponds to the period from implantation to closure of the hard palate. Both the effect on the dams as effects on embryo-fetal development were examined.

The study revealed no adverse effects. The NOAEL was therefore set at 1000 mg/kg bw.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

L-leucine was administered to groups of female rats that had copulated. Each group consisted of 20 females, and the dose levels were 0, 300 and 1000 mg/kg bw. The rats were administered the test substance via gavage as a solution in aqueous carboxymethylcellulose, daily from day 7 to day 17 of gestation, which corresponds to the period from implantation to closure of the hard palate. Both the effect on the dams as effects on embryo-fetal development were examined.

The study revealed no adverse effects. The NOAEL was therefore set at 1000 mg/kg bw.

Justification for classification or non-classification

No adverse effects of dams and foeti were observed upon oral administration of L-leucine to dams during days 7 -17 of gestation at a max. dose of 1000 mg/kg bw/d.

As a consequence, classification of L-leucine for toxic effects to reproduction 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).