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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to internationally accepted protocol. Study examines a very complete set of parameters and is well documented.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Guidelines for Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Ordinance N.1, Article N.24; 1989)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Lot/batch No.: 301FKB7

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4 weeks
- Housing: individually in stainless steel hanging cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 10%
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was mixed into standard diet (CRF-1; Oriental Yeast, Tokyo, Japan).

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with standard diet CRF-1; Oriental Yeast, Tokyo, Japan
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
treatment: during 13 weeks
recovery: 5 weeks
Frequency of treatment:
daily during 13 week treatment period
no treatment during recovery period
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1.25% w/w
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.50% w/w
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5.00% w/w
Basis:
nominal in diet
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, plain diet
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon) during treatment, once daily (morning) during recovery.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week, at a specified time (9:00-12:00).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Twenty-four hour diet intake was measured twice per week.
- Estimated intake of test substance (mg/kg/d) for each week was calculated from the mean consumption for 1 day for that week.

WATER CONSUMPTION: Yes
- Twenty-four hour water intake was measured once per week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment, at week 13 (6 animals per dose group) and at the end of the recovery period (all animals).
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day following the final administration and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes: ether
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters checked: red blood cell count (RBC), mean corpuscular volume (MCV), hemoglobin (Hb), hemoglobin to reticulocyte ratio, platelet and white blood cell count, differential leucocyte count, prothrombin and activated partial thromboplastin time (PT and APTT) and fibrinogen. Hematocrit and mean corpuscular hemoglobin were calculated from the above-measured parameters. Additional plasma parameters (GOT, GPT, and lactate dehydrogenase [LDH]) were obtained from blood samples collected from the abdominal aorta into tubes containing heparin.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked: total cholesterol, triglycerides, phospho-lipids, total bilirubin, blood glucose, urea nitrogen, creatine, uric acid, sodium, potassium, chloride, calcium, inorganic phosphorus, and total protein

URINALYSIS: Yes
- Time schedule for collection of urine: in weeks 5 and 13 of the treatment period and week 5 of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: partly: animals were fasted during the first 4 hours. Urine samples were collected after 4h. Subsequently the animals were provided with food again and a second urine sample was collected 20h later.
- The following parameters were evaluated only from the 4-h samples: pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen, urine color, and sedimentation
- The following parameters were evaluated only from the 24-h samples: volume of urine (volumetry), specific gravity (refractometry), and electrolyte concentration.

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Femoral bone marrow samples were collected at autopsy from all rats and May-Giemsa-stained specimen were prepared and examined microscopically. The rats were sacrificed by exsan- guinations from the abdominal aorta and observed for any exter- nal malformations. Then, the organs and tissues in the cephalic, thoratic, and abdominal cavities were examined macroscopically.
The brain, pituitary, salivary, and thyroid glands, heart, lungs (including bronchia), liver, spleen, kidneys, adrenals, testes, prostate, ovaries, and uterus were excised and weighted. The rel- ative organ weights were calculated from the animals' fasting body weights.

HISTOPATHOLOGY: Yes
All the organs listed above, plus spinal cord, sciatic nerve, thoratic aorta, trachea, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, thymus, mesenteric lymph nodes, cervical lymph nodes, epi- didymides, seminal vesicles, vagina, mammary glands, skin, eyes, optic nerve, Harderian glands, sternum (bone marrow), femur (bone marrow), femoral muscle, and gross lesions were excised and fixed in phosphate-buffered formalin solution. Af- ter paraffin embedding, the excised organs and tissues were prepared for microscopic examination by sectioning and staining with hematoxylin and eosin. Representative samples were photographed.
Statistics:
Data were analyzed for homogeneity of variance using Bartlett's test. Homogenous data observed at the level of 5% (w/w) were analyzed using the parametric one-way analysis of variance (ANOVA), and the significance of differences was assessed using Scheffe's method to compare the values between the control group and each amino acid-administered group. Heterogeneous data converted to rank-sum were analyzed using the Kruskal-Wallis nonparametric test. Any significant differences observed were further evaluated using the method of distribution free multiple comparison (Gad and Weil 1982).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths and no signs related to administration of the amino acid Leu were observed during the administration period.Swelling of the hindlimbs was found in one female in the control group. Mal-occlusion of the incisors was observed in two males (control group, 1.25% Leu-ingesting group). All above changes were incidental. No clinical signs were observed during recovery period.

BODY WEIGHT AND WEIGHT GAIN
Body weights of males and females in all tested concentration groups during the administration and re- covery periods were comparable to those recorded in controls (Table 1).

FOOD CONSUMPTION, WATER CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The diet consumption of males fed 2.5% diet increased singularly on days 45 and 59 ofthe administration. No other sig- nificant changes were observed in the total diet consumption at the end of the administration period. Similarly, no group differ- ences in diet intake were observed during the recovery period, and no water intake changes were found throughout the study.

OPHTHALMOSCOPIC EXAMINATION
Opacity in the anterior portion (cornea) of the left eye was observed in one male in each ofthe 1.25% and 5.0% concentra- tion groups, and abnormal running of vessels (retina) was found in one male in the control group at the end of the administration period.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Examinations at the end of the administration period did not reveal any significant administration-related changes in hemato- logical parameters and blood chemistry. A significant decrease in myeloblasts was found in females in the 1.25% concentration group (controls, 0.2% ± 0.1%; 1.25% group, 0.1% ± 0.1%), and a significantly high value of M/E ratio (controls, 1.2% ± 0.3%; 2.5% group, 1.5% ± 0.2%) was seen in females in the
2.5% concentration group. Those changes were not reproduced at the end of the recovery period.
At the end of the recovery period, a significant increase in the level of inorganic phosphorus (controls, 5.9 ± 0.4 mg/dl; 5.0% group, 6.7 ± 0.3 nmg/dl) and albumin ratio (controls, 43.3% ± 1.4%; 5.0% group, 45.3% ± 1.6%) and a significantly low value of al-globulin ratio (controls, 22.2% ± 2.8%; 5.0% group, 18.9% ± 2.1 %) were noted in males in the 5.0% concen- tration group. In females of this concentration group, significant increases in the levels of creatine (controls, 0.59 ± 0.04 mg/dl; 5.0% group, 0.64 ± 0.03 mg/dl) and total protein (controls, 6.5% ± 0.11 g/dl; 5.0% group, 7.1 ± 0.5 g/dl) were observed. As none of the above changes was seen at the end of the admin- istration period, they were thought to be incidental.

URINALYSIS
No intergroup differences were observed in the quantitative and qualitative parameters.

ORGAN WEIGHTS
See tables.

GROSS PATHOLOGY AND HISTOPATHOLOGY
A small number of abnormalities in two organs (kidney and stomach) at the end ofthe administration period were judged to be incidental. At the end ofthe administration period, absolute organ weights recorded are depicted in Tables 3A (males) and 3B (females). A significant increase in the adrenal gland weight was recorded in the 2.5% concentration group; however, the increase was recoverable and dose independent. At the end ofthe recovery period, there were significant increases in the absolute weight of the pituitary (controls, 15.1 ± 1.8 mg; 5.0% group 17.9 ± 1.1 mg) in females in the 5.0% concentration groups. Because these changes were not observed at the end ofthe administration period, they were considered incidental.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 other: %
Based on:
other: nominal concentration test material in diet
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at any tested dose level.
Dose descriptor:
NOAEL
Effect level:
3 330 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed at any tested dose level.
Dose descriptor:
NOAEL
Effect level:
3 840 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed at any tested dose level.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

Body weight gain, males (g)

Body weight gain, females (g)

 

Administration

Recovery

Administration

Recovery

Standard diet

381.5 ±49.4

41.7 ± 13.7

187.4 ±60.3

21.5 ± 14

+ Leu (1.25%)

399.8 ± 50.4

 

189.7 ±38.5

 

+ Leu (2.50%)

427.7 ± 32.3

 

185.7 ±33.9

 

+ Leu (5.00%)

402.6 ± 55.0

43.8 ± 8.7

181.0 ±26.8

14.7 ±5.9

Means ± SD of 12 rats (Administration) and 6 rats (Recovery) are shown.

 

Average intake (mg/kg/day)

 

Males

Females

Standard diet

783.0 ± 22.9

944.0 ±46.1

+ Leu (1.25%)

832.6 ± 30.6

961.0 ±64.2

+ Leu (2.50%)

1660.2 ± 39.5

1904.8 ±67.0

+ Leu (5.00%)

3332.9 ±101.0

3835.2 ± 257.0

Note.Means± SD of ]2rats are shown.

Absolute body and organ weights @ week 13 in male rats

 

Body weight

(g)

Brain

(g)

Pituitary

(mg)

Salivary gland

(both) (mg)

Standard diet

539.7 ±48.9

2.18 ±0.08

14.5 ±1.5

807±79

+ Leu (1.25%)

531.8 ±38.2

2.13 ±0.08

13.8 ±1.1

803±108

+ Leu (2.50%)

530.3 ±45.2

2.12 ±0.09

14.2 ±1.1

817±108

+ Leu (5.00%)

521.4 ±57.1

2.12 ±0.09

14.5 ±1.5

806±77

 

Thymus

(mg)

Heart

(g)

Lung

(g)

Liver

(g)

Standard diet

318 ±74 

1.55 ±0.11

1.58 ±0.11

13.88±1.62

+ Leu (1.25%)

249±74

1.56 ±0.15

1.64 ±0.12

13.84±1.54

+ Leu (2.50%)

293±63

1.51 ±0.17

1.60 ±0.13

13.23±1.74

+ Leu (5.00%)

263±72

1.53 ±0.16

L57 ±0.10

13.52±2.31

 

Spleen

(g)

Kidneys

(both) (g)

Adrenal glands

(both) (mg)

Testis

(both) (g)

Prostate

(g)

Standard diet

0.80±0.13

3.25±0.21

66±6

3.51±0.15

1.31±0.21

+ Leu (1.25%)

0.77±0.11

3.25±0.28

64±9

3.38±0.23

1.27±0.25

+ Leu (2.50%)

0.80±0.14

3.29±0.46

63±10

3.36±0.28

1.40±0.22

+ Leu (5.00%)

0.82±0.17

3.27±0.39

69±12

3.42±0.27

1.24±0.28

Note.Means± SD of ]2rats are shown.

Absolute body and organ weights @ week 13 in female rats

 

Body weight

(g)

Brain

(g)

Pituitary

(mg)

Salivary gland

(both) (mg)

Standard diet

317.1 ±73.0

1,97 ±0.07

16.0 ±2.6

537±60

+ Leu (1.25%)

326.3 ±45.0

2.03 ± 0.09

16.1 ±3.4

513±25

+ Leu (2.50%)

321.8 ±38.6 2

2.01 ± 0.05

15.6 ±3.0

554±78

+ Leu (5.00%)

314.7 ±32.7.

1.95 ±0.06

14.1 ±2

513±61

 

Thymus

(mg)

Heart

(g)

Lung

(g)

Liver

(g)

Standard diet

272±122

1.02 ±0.10

1.20 ±0,10

7,97±1,39

+ Leu (1.25%)

247±46

1.07 ±0.14

1,20 ±0,09

8,34±L53

+ Leu (2.50%)

236±47

1,05 ±0,09

1,22 ±0,08

8.27±1,05

+ Leu (5.00%)

264±45

1.04 ±0.08

1,15 ±0,08

7.59 ±0,60

 

Spleen

(g)

Kidneys

(both) (g)

Adrenal glands

(both) (mg)

Ovary

(both) (g)

Uterus

(mg)

Standard diet

0.60± 0.10

2.02 ±0.21

75±12

93.6 ±18.5

617 ±135

+ Leu (1.25%)

0.57± 0.10

2.08±0.18

79±9

85.6±21.7

622 ±129

+ Leu (2.50%)

0.59± 0.11

2.14±0.18

84±10*

100.9±16,7

593 ±110

+ Leu (5.00%)

0.52± 0.08

2.02±0.16

75±9

90.0±16,3

595 ±104

Note.Means± SD of ]2rats are shown.

*p < .05from control group.

 

Applicant's summary and conclusion

Conclusions:
From this 90d repeated dose toxicity study with rats it was estimated that the No Observed Adverse Effect Level for L-leucine is at least 3330 mg/kg bw/d.
Executive summary:

This study evaluated toxicologicat and behavioral effects of L-Ieucine (Leu) during a 90d repeated dose study with male and female Sprague-Dawley rats. The amino acids was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (wlw). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals.

No significant, dose-related effects on body weight were found in rats fed a Leu-supplemented diet. We estimate the no-observed- adverse-effect level (NOAEL) for Leu at 5.0% for both genders (males, 3.333 ± 0.101 g/kg/day: females, 3.835 ± 0.257 g/kg/day).