Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Guidelines for Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Ordinance N.1, Article N.24, September 11, 1989).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Tokyo, Japan
- Age at study initiation: 4 weeks upon arrival, 6 weeks upon begin of treatment
- Housing: conventional stainless steel hauging cages (Lead Engineering, Tokyo, Japan)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
according to guideline

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: feed
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): standard CRF-1 diet (Oriental Yeast, Tokyo, Japan)

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed was selected to gain preclinical saftey information after oral intake by humans
- Concentration in vehicle: 1.25 %, 2.50 %, 5.00 %

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 d
Frequency of treatment:
The test substance was administered at constant concentrations in the feed for 13 consecutive weeks. The testing animals were allowed free access to food (and drinking water).

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 % (control)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.25 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.5 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5 %
Basis:
nominal in diet
No. of animals per sex per dose:
12
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Safety information after oral intake of high concentrations.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon) during 13-week application period, once daily (morning) during 5-week recovery period

BODY WEIGHT: Yes. The rats were weighed twice per week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Measured twice per week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All animals prior to start of the study. 6 animals per dose in week 13 of administration. All animals after recovery period.
- 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: Red blood cell count (RBC), mean corpuscular volume (MCV), hemoglobin (Hb), Hb to reticulocyte ratio, platelet and white blood cell count, differential leucocyte count, prothrombin and activated partial thromboplastin time (PT and APTT), fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day following the final administration and at the end of the recovery period.
- Animals fasted: Yes, overnight
- How many animals: All.
- Parameters: AST (aspartate aminotransferase), ALT (alanine aminotransferase), LDH (lactate dehydrogenase), sera parameters (total cholesterol, triglycerides, phospholipids, total bilirubin, blood glucose, urea nitrogen, creatine, uric acid, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein).

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 5 and 13 of adminsitration period, and in week 5 of the recovery
- Collection times: after 4h, again after 20 h
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, deprived of food in case of 4-h-period. No fasting in case of additional 20-h-period.
- Parameters: pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen, urine color, sedimentation. Additionally from 24-h samples: volume of urine (volumetry), specific gravity (refractometry), and electrolyte concentration.

NEUROBEHAVIOURAL EXAMINATION: no data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Femoral bone marrow samples were collected at necropsy from all rats and May-Giemsa-stained specimen were prepared and examined microscopically. The rats were euthanized by exsanguinations via the abdominal aorta and examined grossly for any external abnormalities. Then, the organs and tissues in the cranial, thoracic, and abdominal cavities were examined grossly.
The brain, pituitary, salivary, and thyroid glands, heart, lungs (including bronchia), liver, spieen, kidneys, adrenals, testes, prostate, ovaries, and uterus were excised and weighted. All the organs listed above, plus spinal cord, sciatic nerve, aorta, trachea, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum,
colon, rectum, pancreas, thymus, mesenteric lymph nodes, cervical lymph nodes, epididymides, 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. Afterparaffin embedding, the excised argans and tissues were prepared for microscopic examination by sectioning and staining with hematoxylin and eosin.
Statistics:
Data were analyzed for homogeneity of variance using Bartlett's test. Homogenous data observed at the Ievel 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Mortality:
no mortality observed
Description (incidence):
treatment-related changes were not observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Infrequent changes were witnessed in the blood chemistry in the 2.5% and 5.0% (w/w) groups. All of those changes were determined toxicologically insignificant.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Infrequent changes were witnessed in the urinalysis in the 2.5% and 5.0% (w/w) groups. All of those changes were determined toxicologically insignificant.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
treatment-related changes were not observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs or mortality.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related changes in body weight.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No treatment-related changes in diet consumption.

FOOD EFFICIENCY
No effects reported.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related changes in ophtalmology.

HAEMATOLOGY
No significant administration-related changes.

CLINICAL CHEMISTRY
No significant administration-related changes.

URINALYSIS
No significant administration-related changes.

ORGAN WEIGHTS
No treatment-related changes in organ weights.

GROSS PATHOLOGY
No treatment-related changes in gross pathology.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related changes in histopathology.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
833 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: 1.25 % dose level
Dose descriptor:
NOAEL
Effect level:
964 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: 1.25 % dose level

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A sub-chronic oral toxicity guideline study of L-glutamine with rats was conducted for 90 days. The feeding of L-glutamine was not associated with overt signs of toxicity. Infrequent changes were witnessed in the urinalysis and blood chemistry in the 2.5% and 5.0% (w/w) groups. All of those changes were determined toxicologically insignificant. No effects of the administration were observed in the 1.25% (w/w) group. Therefore, the definitive toxic Ievel for L-glutamine was determined to be above 5.0% (w/w), and the no-observed-adverse-effect Ievel (NOAEL) was estirnated at 1.25% (w/w) for both genders (males 0.83 ± 0.01 g/kg/day; females, 0.96 ± 0.06 g/kg/day).