L-alanine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: study design and conduct and animal welfare guidelines (Japanese Ministry of Health and Welfare, 1996, 1998, 1999).

Deviations:

not specified

GLP compliance:

yes

Remarks:

(Japanese Ministry of Health and Welfare, 1997)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

L-alanyl-L-glutamine

IUPAC Name:

L-alanyl-L-glutamine

Constituent 2

Reference substance name:

39537-23-0

EC Number:

609-717-9

Cas Number:

39537-23-0

IUPAC Name:

39537-23-0

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: males: 187 - 217 g; females: 143 - 170 g
- Caging: Stainless steel cages
- Diet (e.g. ad libitum): basal diet (powdered CRF-1; Oriental Yeast Co., Ltd., Lot No. 050908)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days quarantine/acclimatisation period before study begin

ENVIRONMENTAL CONDITIONS
- according to guideline

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: basal diet

Details on oral exposure:

The 50 rats of each sex were randomly divided into 5 groups, each having 10 rats/sex to ensure heterogeneity of body weights.
One group received basal diet (powdered CRF-1; Oriental Yeast Co., Ltd., Lot No.050908) and served as the controls and 3 others received basal diet containing either 1, 3, or 5% (w/w) of L-AG (Lot No. 050001). A fifth group received a comparison substance [5% L-AG prepared by a different method from the test article (Lot No. K950001)].

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

Food and water were provided ad libitum

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0% (w/w) (control)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1% (w/w) (Lot No. 050001)
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
3% (w/w) (Lot No. 050001)
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
5% (w/w) (Lot No. 050001)
Basis:
nominal in diet

Doses / concentrations 5

Remarks:

Doses / Concentrations:
5% (w/w) (Lot No. K950001) (comparison substance)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dosages were established on the basis of a 14-day range finding study.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: random
- Section schedule rationale (if not random): all animals

Examinations

Observations and examinations performed and frequency:

GENERAL CONDITION OF THE RATS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on dosing days following overnight fasting

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: Yes
- 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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: acclimation period and week 13
- Dose groups that were examined: all
Ophthalmic examinations were performed on all animals during the acclimation period and in 5 rats/sex/group in week 13. Eyes were examined using a direct ophthalmoscope (BXa-13 model: NEITZ Instruments Co., Ltd.).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (90days)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: all
At necropsy, blood was collected from all rats for determination of routine haematological (RBC, Hb, Ht, MCV, MCH, MCHC, reticulocyte percentage, platelet count, WBC, differential WBC percentage, PT, APTT and fibrinogen) parameters. EDTA-2K (SB-41, Sysmex Corporation) was used as the anticoagulant for the haematological analyses, 3.8% sodium citrate for determination of prothrombin time, activated partial thromboplastin time and fibrinogen.
The haematology (Coulter Counter T890, Beckman Coulter, Inc.; Coagulometer ACL 100, Instrumentation Laboratory) was conducted using automated instruments and/or test kits.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (90 days)
- Animals fasted: No data
- How many animals: all
At necropsy, blood was collected from all rats for determination of clinical chemistry parameters (AST, ALT, LDH, cGTP, AP, total cholesterol, triglycerides, phospholipids, total bilirubin, glucose, BUN, creatinine, Na, K, Cl, Ca, P, total protein, albumin, A/G ratio, and protein fractions). Venoject II-Autosep (Terumo Corporation) or sodium heparin (Ajinomoto Pharma Co., Ltd.) were used in the clinical chemistry evaluations.
The clinical chemistry assessments (Clinical Laboratory System TBA-120FR, Toshiba Corporation; Automatic Electrophoresis Apparatus CLINISCAN SA-V, Helena Laboratories) were conducted using automated instruments and/or test kits.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
Urinalysis (pH, protein, ketones, glucose, occult blood, bilirubin, urobilinogen, color, urinary sediment, volume, osmolality, Na, K, Cl, and water intake) was performed in week 13 (days 88–89) of administration.
The urinalysis (AUTION MINITM AM-4290, Arkray, Inc.; Automatic Osmometer AUTO & STAT OM-6030, Arkray, Inc.; Automatic Electrolyte Analyzer PVA-aII, Analytical Instrument, Inc.) was conducted using automated instruments and/or test kits.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Animals were killed by exsanguination from the abdominal aorta following blood collection. Necropsy included an external examination and opening of the cephalic, thoracic and abdominal cavities for observation of all major organs. The absolute and relative weights of the adrenals, brain, pituitary, thyroid, thymus, spleen, heart, lung, salivary glands, liver, kidney, testis, prostate, seminal vesicle, ovary and uterus were recorded/ calculated. Sections of these tissues, in addition to spinal cord, sciatic nerve, eyes, optic nerves, harderian glands, parathyroids, mandibular lymph node, mesenteric lymph node, thoracic aorta, trachea, tongue, oesophagus, stomach, duodenum, ileum, jejunum, cecum, colon, rectum, submandibular and sublingual glands, pancreas, urinary bladder, epididymides, vagina, oviduct, mammary gland, sternum, femur, femoral skeletal muscle, skin, nasal cavity and Zymbal’s glands, were preserved and stained with haematoxylin and eosin (H& E). The listed tissues were examined microscopically in each of the 5% L-AG groups and in the controls. In addition, given findings in the treated animals, sections of testis and epididymides were also evaluated for each of the 1% and 3% L-AG dose groups.

Statistics:

Body weight, food consumption, feed efficiency, quantitative urinalysis data, organ weight data, and the results of the haematological and clinical chemistry evaluations were subject to calculation of the group mean with standard deviations. For the control group and the test article (L-AG Lot No. 050001) groups, data were analyzed for homogeneity of variance by Bartlett’s test (p = 1%, two-tailed). Homogenous data were then evaluated by Dunnett’s test (p = 1% and 5%, two-tailed) while heterogeneous data were analyzed through the Dunnett-type mean rank test (p = 1% and 5%, two-tailed). For the comparative substance group (5% L-AG Lot No. K950001), homogeneity of variance was established through the Ftest (p = 5%, one-tailed) and group means were evaluated by Student’s t-test for homogenous data and by the Aspin-Welch’s t-test for heterogeneous data (p = 1% and 5%; two-tailed).

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

Food efficiency:

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

Behaviour (functional findings):

not examined

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
There were no deaths or clinical symptoms of toxicity over the 90-day treatment period.

BODY WEIGHT AND WEIGHT GAIN
There was no evidence of a treatment-related adverse effect on body weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There was no evidence of a treatment-related adverse effect on feed consumption.

FOOD EFFICIENCY
There was no evidence of a treatment-related adverse effect on feed efficiency.

OPHTALMOSCOPIC EXAMINATION
There was no evidence of a treatment-related adverse effect on the results of the ophthalmological assessments.

HAEMATOLOGY
In the comparative L-AG group (produced via a different method than the test article), the MCHC was significantly elevated in comparison to the controls. The change was not considered to be of toxicological significance since the magnitude of change was small, did not show dose-response relationships, and did not occur in the females.

CLINICAL CHEMISTRY
Clinical chemistry analyses revealed a significantly higher value for AST in the low-dose (634.5 mg/kg bw/day) males and an increased total cholesterol concentration in the high-dose (3129 mg/kg bw/day) males. None of the changes were considered to be of toxicological significance since the magnitude of change was small, did not show dose-response relationships, and did not occur in the females.

URINALYSIS
There were no differences in the results of the urinalysis tests.

ORGAN WEIGHTS
A few sporadic statistically significant differences between treated and control groups were identified for both absolute and relative organ weights. The absolute weight of the thymus was reduced in males of the 534.5 and 3129 mg/kg bw/day L-AG groups. An increased absolute weight of the heart was recorded in the females of the 3632 mg/kg bw/day comparative L-AG group while an increased absolute weight of the adrenal (paired) was reported in the low-dose L-AG males. Changes in relative organ weights included decreased thymus weight in the males of the low- and high-dose L-AG groups and increased adrenal weight in the low-dose males. None of these organ weight differences were concluded to be biologically significant since they did not show a dose-response, occurred only in one sex, were minor in nature, and did not correlate with any relevant histopathological changes.

GROSS PATHOLOGY
There were no abnormal findings.
The macroscopic examinations revealed no treatment related changes in either sex or treatment group.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no abnormal findings.

HISTOPATHOLOGY: NEOPLASTIC
Finding of one or more of seminiferous tubular atrophy, degeneration of the spermatogenic cells, vacuolization of the Sertoli cells in the testis, with associated hypospermia, or intraductal cell debris in the epididymis of 3/10 males of the high-dose group (3129 mg/kg bw/day). These findings were of the same nature and severity of normally occurring spontaneous changes observed in this age and strain of rat. In all cases the findings were graded as either ‘‘minimal’’ or ‘‘mild’’. The testing laboratory noted average incidence rates for seminiferous tubule atrophy of 3.54% (21/593) in 19-week-old rats with a range of 0–20%. Severity was concentrated in the minimal to mild range, however, a few of the historical controls were reported to show moderate (2/593) or severe atrophy (1/593) of the seminiferous tubules (Sugimura, personal communication). Similarly, both the incidence and severity of ‘‘degeneration of spermatogenic cell’’ and ‘‘vacuolization of Sertoli cell’’ in the high-dose group were within that reported in 19-week-old rats at the testing laboratory. In fact, the incidence of each of these observations ranged up to 60% and 40%, respectively. Also, the findings in the epididymis that follow from the testicular findings (i.e., they occurred in the same 3 rats) are likewise similar in incidence and severity to that found in controls from the same laboratory. Finally, it has been noted in the literature that there is a significant incidence of testicular lesions, including testicular degeneration/atrophy, in young Sprague–Dawley rats (i.e., from 13-week studies), with overall mean control incidence rates of 2.5% and 9.4% in oral and inhalation studies, with corresponding ranges of 0.0 to 5.9% and 2.5–26.7% (Lee et al., 1993). Historical control ranges can vary significantly depending upon the source of the rats and on the perspective of the reviewing pathologist.
Overall, there was no clear dose-response relationship that could be established between L-AG treatment and the testicular changes since none of these changes were observed in the mid-dose group (1860 mg/kg bw/day) or in the comparative L-AG group also dosed at 5.0% in the diet (3151 mg/kg bw/day). In addition, the changes noted in the present study were at the upper end of the historical control range for the age and strain of rat used at the laboratory.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

3 129 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

3 601 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A subchronic oral toxicity guideline study of L-alanyl-L-glutamine (L-AG) with rats was conducted for 90 days. No treatment-related significant or toxicologiocally relevant findings were observed.
The no observed adverse effect level (NOAEL) was considered to be 3129 mg/kg bw/day (5.0% in the diet) in males and 3601 mg/kg bw/day (5.0% in the diet) in females.