L-Glutamine, L-alanyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-06-03 to 1991-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

yes

Remarks:

only 2 doses tested

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1984

Deviations:

yes

Remarks:

only 2 doses tested

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

475-290-9

EC Name:

-

Cas Number:

39537-23-0

Molecular formula:

C8H15N3O4

IUPAC Name:

(2R)-2-[(2S)-2-aminopropanamido]-4-carbamoylbutanoic acid

Test animals

Species:

rat

Strain:

other: Bor:WISW (SPFCpb)

Details on species / strain selection:

The test system was selected based on international recommendations. The rat is the preferred rodent species for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co.KG, Borchen, Germany
- Age at study initiation: 7 weeks (males); 8 weeks (females)
- Weight at study initiation: 145 - 195 g (males); 135 - 166 g (females)
- Housing: individually in Macrolon cages, type II
- Diet: standart diet, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 (for short periods up to 25 °C)
- Humidity (%): 40 - 70 (for short periods up to 80%)
- Photoperiod: 6 a.m. - 6 p.m. CET artificial lighting; 6 p.m. - 6 a.m. CET natural light-dark-rhythm

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The solutions for administration with different substance concentrations according to the intended doses were prepared daily.

VEHICLE
- Test substance concentration in vehicle: 46.4 mg/mL (low-dose group, 215 mg/kg bw/day), 215 mg/mL (high-dose group, 1000 mg/kg bw/day)
- Administration volume: 4.64 mL/kg bw
- Purity: The tap water quality is regularly monitored by Stadtwerke Bielefeld (municipal water provider)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance was determined in aqueous samples at the beginning and at the end of the study by HPLC.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

215 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main study:
Control group: 5
Low-dose group (215 mg/kg bw/day): 5
HIgh-dose group (1000 mg/kg bw/day): 5

Satellite groups:
Control group 5
HIgh-dose group (1000 mg/kg bw/day): 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a previous dose finding study
- Post-exposure recovery period in satellite groups: 6 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Occurence of toxicity symptoms: daily
Mortality: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Reflexes: once a week, starting with pretest period
Examination of eyes, hearing and teeth: prior to first substance administration and in test week 4

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, starting with pretest period

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once a week, starting with pretest period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: test week 4
- How many animals: all animals
- Anaesthetic: CO2
- Parameters checked: Erythrocytes (RBC), hematocrit (Hct), hemoglobin (Hb), leucocytes (VBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), thrombocytes (Platelets), and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: test week 4
- How many animals: all animals
- Parameters checked: Alanine aminotransferase (ALAT), albumin, alkaline phosphatase (AKP), aspartate aminotransferase (ASAT), blood urea, calcium (Ca), chloride (Cl), cholinesterase (CHE), creatine kinase (CK), creatinine, gamma-glutamyltransferase (Gamma-GT), glucose, glutamate dehydrogenase (GLOB), inorganic phosphate (P), potassium (K), sodium (Na), total bilirubin (Tot.Bili), total cholesterol (Chol), total protein (Tot. Prot), and triglycerides (Triglyc)

URINALYSIS: Yes
- Time schedule for collection of urine: test week 4
- How many animals: all animals
- Parameters checked: Bilirubin, glucose, hemoglobin/erythrocytes, ketones, leucocytes, nitrite, osmolality, pH-value, protein, and urobilinogen. Microscopical examinations of the urine sediment were done in animals whose urine state showed pathological changes in hemoglobin/erythrocytes, leucocytes, and protein.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subjected to full gross necropsy, examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.
- Organs/tissues preserved: All gross lesions, adrenal glands (r/1), bone marrow (as present in sternum), bone marrow smear (from femur), brain (cerebrum, cerebellum, brain stem), cecum, colon, duodenum, heart, ileum, jejunum, kidneys (r/1), liver, lungs (including main bronchi), ovaries (r/1), rectum, spleen, stomach, and testes (r/1).
- Organ weights: Adrenals (r/1), brain, heart, kidneys (r/1), liver, ovaries (r/1), spleen, and testes (r/1).

HISTOPATHOLOGY: Yes

Statistics:

For food consumption, body weights, and organ weights the DUNNETT-Test was used.
For values of hematological and clinical chemistry examinations the DUNNET-Test was used in case of normal distribution, otherwise the STEEL-Test was employed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia in 1/5 control male and 2/5 control female animals (3 weeks after termination of treatment). Not considered to be related to the test substance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute organ weights showed no significant differences between the control and treatment groups. In relative organ weights minimal alterations of statistical significance in the weight of kidneys (left, decrease in low dose males week 5, slight increase in high dose females week 5) and testes (left, high dose males week 11) were recorded, but these marginal variations are well within the normal range of the species and they are considered to be incidental findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slight or marked alopecia in 2/5 animals and one rat each showed a reddish fluid in the urinary bladder, a yellowish nodule in the epididymis or a nodule in one horn of the uterus. All findings are considered as incidental changes known to occur in this species.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Only minimal changes in livers, kidneys, lungs, epididymis and stomach were observed. All findings were considered as spontaneous changes.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

1

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The administration of 215 and 1000 mg/kg bw/day test substance resulted in no observable effects. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 1000 mg/kg bw/day in the rat.