Registration Dossier

Diss Factsheets

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Report in a recognised journal and performed according to a guideline that is used for food additives.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives released by the Ministry of Health, Labour and Welfare /Japan (MHLW).
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Aspartic acid
EC Number:
200-291-6
EC Name:
Aspartic acid
Cas Number:
56-84-8
Molecular formula:
C4H7NO4
IUPAC Name:
aspartic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Names: L-aspartic acid; L-Asp
Lot No.: 0010663004
purity: 100.2 %, by a neutralizing titration method
Supplier: Ajinomoto Co., Inc. (Kanagawa, Japan).

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
Specific pathogen-free Fischer 344 (F344/DuCrlCrlj) rats were purchased at 5 weeks of age from Charles River Japan Inc. (Kanagawa, Japan) and acclimatized on the control diet for 1 week before the experimentation.
The rats were housed individually in stainless steel cages; kept under the controlled conditions of temperature (22–24 °C), relative humidity (50–
60 %) and ventilation (more than 10 times/hour) with a 12-hour light/dark cycle; and allowed free access to food and drinking water throughout both acclimation and experimental periods.
After confirming normal health status at the end of the acclimation period, 50 rats of each sex were randomly allocated to five groups each consisting of 10 rats, given the control and experimental diets for 90 days.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: none
Details on oral exposure:
L-ASP was admixed into a modified AIN-93G powder diet (Oriental Yeast Co. Ltd., Tokyo, Japan) to give concentrations of 0% (control), 0.05% (corresponding to the human intake level) (Supli-market, 2007), 1.25%, 2.5% or 5.0% every 4 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The L-Asp content in all experimental diets was analyzed at their preparation, with actual values of 0.56 ± 0.06, 12.71 ± 0.13, 24.90 ± 0.82 and 51.31 ± 0.75 g/kg diet for the 0.05%, 1.25%, 2.50% and 5.0% doses, respectively.
After keeping the 0.05% and 5.0% diets for 15 or 30 days at 4 °C, the contents of L-Asp were found to be well stable with values of 0.54 and 51.34 g/kg diet, respectively.
Duration of treatment / exposure:
90 days.
Frequency of treatment:
Continuous in the feed.
Doses / concentrations
Remarks:
Doses / Concentrations:
0% (control), 0.05% (corresponding to the human intake level), 1.25%, 2.5% or 5.0%
Basis:
nominal in diet
No. of animals per sex per dose:
10 males and 10 females per dosage group.
Control animals:
yes, concurrent no treatment
Details on study design:
Comments on the concentrations used: L-ASP was admixed into diet at concentrations of 0 %, 0.05 %, 1.25 %, 2.5 % or 5.0 %. It should be noted that the 0.05 %-group was set to assess effects of a human-relevant dose and thus should be considered an additional group to the standard three-doses (with ratios of 2) for safety assessment study. In fact, the dose 0.05 % is 25-fold lower than its immediate upper dose of 1.25 %.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
During the experimental period, the rats were observed daily, and clinical signs and mortality (if any) were recorded.
Body weights, food and water intakes were monitored weekly.

At the end of the experimental period of 90 days, all rats were deprived of food (but not water) overnight, and fresh urine samples were obtained to be used in the urinalysis of urobilinogen, occult blood, bilirubin, ketone, glucose, protein, pH and nitrous acid by a test paper. All rats were then anesthetized by ether and sacrificed by exsanguination after collecting blood samples via the abdominal aorta.

Hematological examination was carried out using an automatic analyzer (Sysmex KX-21NV) for red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit level (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC) and platelet count (PLT). Differential counts of leukocytes were made by light microscopical observation of smear specimens stained with a routine May– Glünwald–Giemsa protocol.

Serum biochemistry determination was performed with a Toshiba automatic analyzer (TBA-120FR) for levels of total protein (TP), albumin (ALB), albumin/globulin ratio (A/G), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), total bilirubin (T-BIL), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), sodium (Na), potassium (K), chlorine (Cl) and calcium (Ca).
Sacrifice and pathology:
At the terminal sacrifice, complete necropsies were performed on all animals. For each animal, the body weight was determined, and gross observations were made.
The brain, thyroids (with parathyroids), heart, spleen, liver, adrenals, kidneys, testes, ovaries and uterus were then excised, and their absolute and relative weights were determined. These organs as well as the pituitary gland, eyes, harderian gland, thymus, nasal cavity, trachea, lungs (including bronchi, fixed by inflation with fixative), salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, pancreas, urinary bladder, skin with mammary gland, skeletal muscle, epididymides, seminal vesicle, prostate, preputials, oviducts, vagina, lymph nodes (submandibular and mesenteric), thoracic aorta, sciatic nerve, spinal cord (cervical, mid-thoracic and lumbar), bone (femur and sternum) and bone marrow, Zymbal’s gland and all gross lesions of each animal were fixed in 10% neutrally buffered formalin.
Paraffin-embedded or frozen sections were then routinely prepared.
All organs were histopathologically examined by using paraffin-embedded sections stained with a routine hematoxylin and eosin (HE) protocol. Detection of possible lipid deposits in the liver was carried out on frozen sections stained with an Oil red O protocol. Immunohistochemical examination for the expression of a2u-globulin was performed on paraffin-embedded kidney sections of 0% and 5.0% male and female groups by a streptavidin horseradish peroxidase technique with diaminobenzidine as the chromogen, using an anti-rat a2u-globulin antibody (R&D Systems, Inc., Minnesota, USA) as the primary antibody.
Statistics:
For numerical data such as body and organ weights as well as hematological and serological outcomes, equality of means between the control and each treated group values was assessed by Bartlett’s test. Homogeneity of variance was then analyzed by a one-way analysis of variance, and finally differences between control and each treated group values were evaluated by Dunnett’s (when sample numbers were equal) or Scheffe’s (when sample numbers were unequal) post hoc multiple comparison tests. If the Bartlett’s test was significant, on the other hand, data were subjected to the Kruskal–Wallis test and the Dunnett’s or Scheffe’s type rank sum tests.
For contingent data such as incidences of histopathological lesions and positive cases of urinalysis, differences between the control and each treated group values were evaluated by the Fisher’s exact probability test.
Inter-group differences were considered statistically significant when p-values less than 0.05 were obtained.

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:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
increased
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No rats died or became moribund until the end of the experiment.
No treatment-related clinical signs were observed in the control or treated rats during the study.
BODY WEIGHT AND WEIGHT GAIN
There were no significant differences in average body weights between rats in the control group and those in any of the treated groups during the study, for both sexes.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The average daily food intake was in similar ranges among groups.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The average daily water intakes of the 2.5% or greater males and of the 1.25% or greater females were also significantly increased.
HAEMATOLOGY
In male rats, RBC values of the 1.25% and 5.0% groups, and HCT for the 1.25% or greater groups were significantly higher than the control group values. Values for MCH of the 0.05%, 1.25% and 5.0% groups, and MCHC of 0.05% or greater groups were significantly lower than the control group values. In females values of MCH and MCHC of the 5.0% group were also significantly lowered. Most of the statistically significant values were in the range of the historic control values. No apparent changes were observed in the other items, when compared with the control rats. Morphological findings and white blood cell differential counts showed no significant adverse effects in any of the treated groups.
CLINICAL CHEMISTRY
Serum biochemistry demonstrated values for T-CHO in the 2.5% or greater male groups and the 5.0% females, and TG for the 0.05% or greater male groups and of the 1.25% or greater female groups to be significantly lower than those of the control group. Values for BUN of the 2.5% or greater males and of the 5.0% females, CRE of the 2.5% or greater males and of the 1.25% or greater females, UA of the 2.5% male and of the 1.25% or greater females, and K of the 2.5% or greater females were significantly lower than the control group values. Most of the statistically significant values, except for TG, were in the range of the historic control values. Values for TG of the 2.5% or less males were higher than the historic control values. There were also some sporadic changes in the other measurements. Values for TP, GLU, T-BIL, ALP and Ca of males and females showed no significant changes in any of the treated groups.
URINALYSIS
In male rats, incidences of positive bilirubin in the 5.0% group, and ketone and protein in the 1.25% or greater groups were significantly higher than the control group value. In female rats, incidences of positive ketone in the 1.25% and 2.5% group, and protein in the 2.5% or greater groups were also significantly elevated. No apparent changes were observed in the other urinalysis, when compared with the control rats.
ORGAN WEIGHTS
In male rats, the only significant changes were increased relative weights of the heart in the 1.25% group and the kidneys in the 5.0% group relative to the control group values. There were no apparent changes in female rats.
GROSS PATHOLOGY
No treatment-related macroscopic changes were observed in any organs of either sex.
HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver, there were no treatment-related changes. Accumulation of lipid in hepatocytes was not apparent in HE-stained specimens but was clearly detected by the Oil red O staining. There was no variation between control and treated animals. In the kidneys, regenerative renal tubules with tubular dilation were more frequently observed in male rats of the 2.5% or greater groups than in the control group. In such animals, albeit without statistical significance, inflammatory cell infiltration was observed more frequently than in the control rats. The a2u-globulin immunohistochemistry for the kidney sections demonstrated positive granular signals in animals of both sexes, but much more markedly in males than in females, and in both the treated (with the highest dose of 5.0%) and control rats. Importantly, however, there were neither quantitative nor qualitative differences in the outcome for a2u-globulin between the control and treated rats. In the salivary glands, acinar cell hypertrophy of the submandibular and parotid glands were more frequently observed in rats of both sexes of the 5.0% and 2.5% or greater groups, respectively, than in the control groups. This acinar cell hypertrophy was a diffuse change affecting the whole glands, with serous acinar cells featuring granular cytoplasm and pyknotic nuclei locating in the basal area. Acinar cell hypertrophy was not apparent in sublingual glands or minor salivary glands in any of the animals. In the other organs, there were no apparent treatment- related histopathological changes.
HISTOPATHOLOGY: NEOPLASTIC (if applicable)
None.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
12 500 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical chemistry; urinalysis; histopathology. Kidney and possibly salivary gland adverse effects.
Dose descriptor:
NOAEL
Effect level:
697 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The NOAEL in % in diet is converted to mg/kg bw/d.
Dose descriptor:
NOAEL
Effect level:
715 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The NOAEL in % in diet is converted to mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

In the present study using young adult rats, no signs or symptoms suggesting neurotoxicity of L-aspartic (L-Asp) acid were observed, and thus a specific neurotoxicity test was not employed. It is suggested, therefore, that neurotoxicity of the amino acid may be limited not only to the species level of rodent but also the age of the newborn. For the same reason, specific ophthalmologic or immunotoxicity tests were not conducted.

In the present study, L-aspartic acid caused toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Regarding the renal toxicity, while changes in urinalysis and serum biochemistry were observed in animals given 1.25% or higher doses of L-Asp, kidney histopathology showed toxic changes only in male rats given 2.5% or higher.

In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

Applicant's summary and conclusion

Conclusions:
The NOAEL for L-aspartic acid is 1.25 % in the diet of rats (697 mg/kg body weight/day for males and 715 mg/kg body weight/day for females).
Executive summary:

A subchronic oral toxicity study of L-aspartic acid (L-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days.

Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups.

The present results indicate that L-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of L-Asp.

In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.