Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
other: SIDS
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
SIDS for L-Glutamic acid
Author:
OECD
Year:
2013
Report date:
2013
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Specific details on test material used for the study:
- Name of test material (as cited in study report): L-Glutamic acid
- Molecular weight: 147.13
- Physical state: White powder
- Analytical purity: 100.1%
- Purity test date: 2011-03-24
- Lot/batch No.: BCBF3951V
- Expiration date of the lot/batch: 1 year after opening at room temperature
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: ORIENT BIO Co., Ltd. Korea
- Age at study initiation: 6 weeks
- Weight at study initiation: male 197.2 g-217.3 g, female 141.7 g-178.0 g
- Fasting period before study: no data
- Housing: Two or three rats were reared each wire cage [270(W)×500(D)×200(H)mm, Daejong] during experiment period
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Air changes (per hr): 10-15 times/h
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle of 150-300 Lux

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1% (w/v) Tween 80 in 1% CMC-Na
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
After weighing the test substance, it was suspended by 0.1% (w/v) Tween 80 in 1% CMC-Na so that the final concentration will be 6.25, 25 and 100 mg/mL as low, middle and high dose. The prepared test substance was continually suspended by vortexing until administration and the preparation of each group was done every day for 28 days.
VEHICLE
- Justification for use and choice of vehicle (if other than water): The preliminary preparation was conducted to select the vehicle for the test substance. The water, corn oil, 1% CMC-Na and 0.1% Tween 80 in 1% CMC-Na were tested for vehicle. As a result, the 0.1% Tween 80 in 1% CMC-Na was selected as vehicle because the test substance was not soluble but suspended evenly by the 0.1% Tween 80 in 1% CMC-Na compared with other vehicles tested.
- Concentration in vehicle: 0.1% (w/v) Tween 80 in 1% CMC-Na
- Amount of vehicle (if gavage): 10 mL
- Lot/batch no. (if required): MKBG3475V (Tween 80), 120M0216V / SLBB5612V (CMC-Na) 89L4K21 (distilled water)
Duration of treatment / exposure:
28 d
Frequency of treatment:
once a day, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Test group: 5, Recovery group (control and high dose): 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose, 1000 mg/kg was established according to the results of TBH-0787 as 7 days DRF (dose range finding) study and toxicity data. Middle and low dose was designed 250 and 62.5 mg/kg, respectively.
- Rationale for selecting satellite groups: Each 5 animals of high dose group and control group was designed for recovery group to durability, extensity and reversibility of toxic response for test substance.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day during acclimatization and recovery period / twice a day (before/after administration) during administration period such as clinical signs, toxicity signs and dead animals.- Cage side observations checked in table [No.1] and appendix [No.1] were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a weekBODY WEIGHT: Yes
- Time schedule for examinations: upon receipt and grouping, and once a week during administration period
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before grouping and at the last week of experiment
- Dose groups that were examined: control and high dose group
HAEMATOLOGY: Yes
- Time schedule for collection of blood: The animals were fasted over 18 hours and blood was collected from abdominal artery under anesthesia with isoflurane.
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.12] and appendix [No.12] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The animals were fasted over 18 hours and blood was collected from abdominal artery under anesthesia with isoflurane.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.13] and appendix [No.13] were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: at last week of administration
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.7] and appendix [No.7] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of administration
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 9)
HISTOPATHOLOGY: Yes (see table14) Internal organs were removed and fixed with 10% neutral formalin individually. The organs were liver, kidney, adrenal gland, heart, lung, brain, pituitary gland, spinal cord, spleen, seminal vesicle, coagulatory gland, testis, ovary, epididymis, prostate gland, uterus, vagina, tongue, trachea, esophagus, thymus, thyroid/parathyroid gland, stomach, duodenum, urinary bladder, small/large intestine, eye/harderian gland, skin, mammary gland, submandibular gland, skeletal muscle, sciatic nerve, pancreas, intestinal lymphnode, femur and sternum. But testis and epididymis were fixed with Bouin's solution and eyes fixed with Davidson's solution. Histopathological analysis was conducted for the control, high dose group and low dose group observed macroscopic lesion.
Other examinations:
Organ weight: After necropsy findings, absolute and relative (organ-to-body weight ratio) weights were measured in all survivors when they were sacrificed.
Statistics:
The functional assessments, body weight, food consumption, organ weight, hematological data and biochemical data were analyzed for homogeneity of variances using Levene's test. One way ANOVA analysis was performed to evaluate the significance of differences. If the variance was homogeneous and the significance of difference was confirmed, Scheffe's multiple comparison test was performed as a post-hoc test. If the variance was not homogeneous, the data were analyzed by procedure of Dunnett's T3 test. And the body weight, food consumption, organ weight, hematological data and biochemical data for recovery group were analyzed for homogeneity of variances using F test. If the variance was homogeneous, the datawere analyzed by procedure of Student t test. If the variance was not homogeneous, the data were analyzed by procedure of Mann-whitney test. All analyses analysis were performed using SPSS (Ver 19.0) program.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no dead animals and abnormal clinical signs related to administration of the test substance during the experimental period in both sexes.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistical differences compared to control group in both sexes.
Description (incidence and severity):
It was significantly increased (9.8%, p<0.05) in females of the 250 mg/kg group compared to the control group in 1 week after administration and significantly increased (4.1% in 2 weeks, 12.4% in 3 weeks, p<0.01) in males of the 250 mg/kg group compared to the control group in 2 and 3 weeks after administration. It was significantly decreased (15.3%, p<0.01) in females of the 1000 mg/kg group compared to the control group in 1 week after recovery. It was also significantly increased (10.4%, p<0.01) in males of the 1000 mg/kg group and significantly decreased (7%, p<0.01) in females of the 1000 mg/kg group compared to the control group in 2 weeks after administration.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no toxicological changes related to administration of the test substance.
Description (incidence and severity):
The hemoglobin in males of the 250 mg/kg bw/day group and total RBC in females of the 62.5 and 250 mg/kg bw/day group were significantly increased (6.5% in hemoglobin, 6.4% and 7% in total RBC of 62.5 and 250 mg/kg bw/day group, p<0.05) compared to control group at the end of administration.
The mean cell volume (MCV), mean corpuscular hemoglobin (MCH), count and percentage of reticulocyte and activated partial thromboplastin time (APTT) were significantly decreased (5% in MCV, 4.9% in MCH, 31% and 36.4% in count and percentage of reticulocyte, 16.8% in APTT, p<0.05) in females of the 1000 mg/kg bw/day group compared to control group.
Description (incidence and severity):
The total bilirubin was significantly increased (28.6%, p<0.05) in females of the 250 mg/kg bw/day group compared to control group at the end of administration. The total bilirubin, aspartate transaminase (AST), calcium and creatine kinase were significantly increased (40.0% in total bilirubin, 16.2% in AST, 2.0% in calcium, 35.7% in creatine kinase, p<0.05) in males of the 1000 mg/kg bw/day group.The blood urea nitrogen (BUN) was also significantly increased (20.4%) and triglycerides (TG), glucose, calcium and total bile acid (TBA) were significantly decreased (58.3% in TG, 19.5% in glucose, 4.0% in calcium, 70.6% in TBA, p<0.05 or p<0.01) in females of the 1000 mg/kg group compared to control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no toxicological changes related to administration of the test substance.
Description (incidence and severity):
As a result of sensory reactivity assessments and grip strength in the last week of administration, there were no abnormal effects and changes related to administration of the test substance. In the motor activity test, there were no abnormal changes related to administration of the test substance.
Description (incidence and severity):
In relative organ weight, the weight of prostate and seminal vesicle was significantly decreased (22%, p<0.05) in males of the 250 mg/kg bw/day group compared to control group at the end of administration. The weight of lung was significantly decreased (7.7%, p<0.05) in males of the 1000 mg/kg bw/day group compared to control group at the end of recovery.
Description (incidence and severity):
In test group, 1 diffuse dark red color change in left lateral lobe and dark red color change in right lobe of lung in females of the 62.5 mg/kg bw/day group and 1 enlarged submaxillary lymph nodes in females of the 1000 mg/kg bw/day group. In recovery group, 1 swollen lung and foamy materials in trachea in males of the control group, 1 dark red color change at margin of right lateral lobe of liver in females of the control group and 1 swollen lung and foamy materials in trachea/dark red color change in right lateral lobe of liver in females of the 1000 mg/kg group.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In results of histopathology for the main organs of control and high dose group, there were no lesions related to the test substance.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female

Applicant's summary and conclusion

Conclusions:
From the above results, there were no significant toxicological changes up to 1000 mg/kg bw/day. Therefore, the NOAEL of L-Glutamic acid was considered to be above 1000 mg/kg bw/day in both sexes.