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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
For completeness reasons the study was added here, however, this is not a data requirement.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD 408
GLP compliance:
not specified
Test type:
other: 90-days oral repeated dose toxicity study
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: males 176 - 200 g, females 151 - 173 g
- Housing: individually in suspended, stainless-steel cages
- Diet (e.g. ad libitum): free acces to certified rodent diet sterilized by gamma irradiation (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan).
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 5 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h a day (from 7 a.m. to 7 p.m.)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
L-tyrosine suspensions of 20, 60 and 200 mg/mL were prepared once a day and mixed just prior to each administration.
Dose volume: 10 mL/kg
Doses:
200, 600 and 2000 mg/kg bw/d
No. of animals per sex per dose:
10
Control animals:
yes
Remarks:
vehicle controls
Details on study design:
- Duration of observation period following administration: 90 days
- Frequency of observations and weighing: day 1 and 2 and weekly thereafter
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, organ weights, histopathology, heamatology, clinical chemistry, ophthalmology, food and water consumption
Statistics:
Body weight, food consumption, water consumption, urinalysis (expect qualitative analysis), hematology, blood chemistry, and organ weight data were recorded using a MiTOX RDT system. Numerical data obtained during the study were used to calculate group mean values and standard deviations. Group variances for the appropriate parameters were compared using Bartlett's method (significant at p < 0.01 in two-tailed test). When the differences between group variances were not significant, Dunnett's multiple comparison method was applied to determine the significance of differences between the control group and each L-tyrosine-treated group (significant at p < 0.05 in two-tailed test) (Dunnett, 1955). If the Bartlett's test indicated significant differences between group variances for a given parameter, that parameter was compared among groups using the Steel's multiple comparison method for mean ranking (significant at p < 0.05 in two-tailed test).
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No animals died.
Clinical signs:
Crust formation was seen in the neck in a few animals in all groups including the control group, and was not considered to be treatment-related because a similar finding was also seen in the control group and its incidence did not increase in a dose-dependent manner.
Loss of fur in the forelimb that was bilaterally seen in one or two rats in the L-tyrosine treated groups, was considered to be incidental because this finding is historically quite common.
Body weight:
No differences between controls and treated groups.
Gross pathology:
Significant increases, or a tendency thereof, were found in absolute and relative weights of liver and kidneys in males and females at 2000 mg/kg bw/day. Decreased absolute adrenal weight (right side) was noted in females at 600 and 2000 mg/kg bw/day, but was considered to be incidental because it was seen unilaterally, was not associated with a changes in relative adrenal weight, and individual weight was within the range of variation in the control group.
Other findings:
- Stomach: Edema of the cornified layer at the limiting ridge (minimal to slight) was seen in 2 females at 600 mg/kg bw/day and in 3 males and 4 females at 2000 mg/kg bw/day. Edema of the cornified layer at the forestomach (minimal) was seen in 1 female at 2000 mg/kg bw/day.
- Liver: Centrilobular hypertrophy of hepatocytes (minimal) was seen in 2 males and 2 females at 2000 mg/kg bw/day.
- Kidney: Diffusely increased hyaline droplets were seen in the proximal tubules (minimal to moderate) in 9 males at 2000 mg/kg bw/day with higher grade and incidence than in the control.
Conclusions:
We can assume that the LD50 for males and females will be > 2000 mg/kg bw/d because after 90 days of dosing no animal died.
Executive summary:

In this current study the potential toxicity of L-tyrosine was evaluated in a 13-week repeated-dose oral toxicity study in rats according to OECD 408. The study was not GLP. L- tyrosine was administered by gavage to 4 groups each consisting of 10 males and 10 females. The doses were 0 (vehicle: water), 200, 600 or 2000 mg/kg bw/day.

No deaths occured during the administration period in any of the study groups. No L-tyrosine-related changes were observed in clinical signs, body weight, food and water consumption, ophthalmology or necropsy.

Some L-tyrosine-related changes were observed in the stomach, the liver and kidneys.

Additionally, significant changes were seen in biochemical parameters.

Cataract formation was not observed in this study at any dose level.

Even though this study is not an acute toxicity study, based on the above results, we can assume that the LD50 for males and females will be > 2000 mg/kg bw/d because after 90 days of dosing no animal died.

Data source

Reference
Reference Type:
publication
Title:
13-week repeated dose toxicity study of L-tyrosine in rats by daily oral administration
Author:
Yusuke Shibui, Yasuhiro Manabe, Terutaka Kodama, Akinori Gonsho
Year:
2016
Bibliographic source:
Food Chem Toxicol. 87:55-64

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: males 176 - 200 g, females 151 - 173 g
- Housing: individually in suspended, stainless-steel cages
- Diet (e.g. ad libitum): free acces to certified rodent diet sterilized by gamma irradiation (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan).
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 5 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h a day (from 7 a.m. to 7 p.m.)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
L-tyrosine suspensions of 20, 60 and 200 mg/mL were prepared once a day and mixed just prior to each administration.
Dose volume: 10 mL/kg
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Animal assignment: randomized complete block design with body weight stratification by MiTOX computer system (Mitsui Zosen Systems Research Inc., Chiba, Japan).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: clinical signs including appearance/posture, behavior, feces/urine, body surface, fluid secretion/excretion, and body temperature.

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 and 2, and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: day 2 and weekly thereafter

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: day 2 and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: day 86
- Procedure: The pupils were dilated with a mydriatic agent (Mydrin®-P ophthalmic solution, Santen Pharmaceutical Co., Ltd., Osaka), and the anterior segments and media were examined using a binocular indirect ophthalmoscope (OMEGA®, Heine Optotechnik GmbH & Co. KG, Herrsching, Germany) and a portable slit lamp (Kowa SL-15, Kowa Optimed Inc., Tokyo). The fundus was observed using the binocular indirect ophthalmoscope.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of the scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- Parameters checked: red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte count based on its percentage (RETIC), platelet count (PLT), and white blood cell count (WBC), white blood cell differential based on their percentage included neutrophils (NEUT), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), large unstained cells (LUC), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fbg), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (T-BIL), creatinine (CRE), blood urea nitrogen (BUN), triglycerides (TG), total choles- terol (T-CHO), phospholipids (PL), glucose (GLU), sodium ion (Na), potassium ion (K), chloride ion (CI), calcium (Ca), inorganic phos- phorus (IP), total protein (TP), and fractionation of protein including albumin/globulin ratio (A/G), albumin (ALB), a1 globulin (ALPHA1), a2 globulin (ALPHA2), beta globulin (BETA) and gamma globulin (GAMMA).

URINALYSIS: Yes
- Time schedule for collection of urine: day 85 for 4h, and night 85-86 for 22.5h
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: pH, glucose, protein, occult blood, ketones, bilirubin and urobilinogen; overnight urine samples: urinary volume (U. Vol), specific gravity, sodium ion (Na), potassium ion (K), and chloride ion (CI)
Sacrifice and pathology:
SACRIFICE:
- Time scheduled: day 92 or 93
- Animals fasted: Yes
- Method: exsanguination
- Anaesthetic: isoflurane

GROSS PATHOLOGY: Yes
- Type: complete gross pathological examination including the external appearance of the carcass, as well as organs and tissues in the abdominal, thoracic, and cranial cavities.
- Organs weighed: brain, pituitary, submaxillary gland (submandibular gland and sublingual gland), thymus, heart, liver, spleen, kidney, adrenal, testis, and ovary; paired organs were weighed individually except for the submaxillary glands
- Organ-to-body weight percentages were calculated.

HISTOPATHOLOGY: Yes
- Tissues or representative samples collected: brain, spinal cord, cervical lymph node, subman- dibular gland, sublingual gland, lacrimal gland (exorbital), eye (R/L), Harderian gland (R/L), trachea, lung (R/L), thymus, aorta, heart, liver, spleen, kidney (R/L), adrenal gland (R/L), tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, mesenteric lymph node, urinary bladder, testis (R/L), epididymis (R/ L), seminal vesicles, prostate, ovary (R/L), uterus, vagina, sternum with bone marrow, femur with bone marrow, skeletal muscle, sciatic nerve, pituitary, thyroid, parathyroid, skin, mammary gland, and gross lesions.
- Preservation: 10% neutral-buffered formalin except testes, eyes with Harderian glands which were preserved formaldehyde-acetic acid solution and glutaraldehyde-formaldehyde solution, respectively prior to 10% neutral-buffered formalin fixation.
- Embedding: paraffin
- Staining: hematoxylin and eosin
Statistics:
Body weight, food consumption, water consumption, urinalysis (expect qualitative analysis), hematology, blood chemistry, and organ weight data were recorded using a MiTOX RDT system. Numerical data obtained during the study were used to calculate group mean values and standard deviations. Group variances for the appropriate parameters were compared using Bartlett's method (significant at p < 0.01 in two-tailed test). When the differences between group variances were not significant, Dunnett's multiple comparison method was applied to determine the significance of differences between the control group and each L-tyrosine-treated group (significant at p < 0.05 in two-tailed test) (Dunnett, 1955). If the Bartlett's test indicated significant differences between group variances for a given parameter, that parameter was compared among groups using the Steel's multiple comparison method for mean ranking (significant at p < 0.05 in two-tailed test).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Crust formation was seen in the neck in a few animals in all groups including the control group, and was not considered to be treatment-related because a similar finding was also seen in the control group and its incidence did not increase in a dose-dependent manner.
Loss of fur in the forelimb that was bilaterally seen in one or two rats in the L-tyrosine treated groups, was considered to be incidental because this finding is historically quite common.
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):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A significant increase of RBC was noted in males at 2000 mg/kg bw/day. Decreased reticulocyte counts in females at 600 mg/kg bw/day group was considered to be incidental because of a lack of dose-dependency.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significant increases, or a tendency thereof, in ALT, AST, triglycerides, total cholesterol, phospholipids, potassium ion, calcium, total protein, and a1 globulin, were noted in males and females at 2000 mg/kg bw/day. Decreases of LDH and CPK observed in females at 200 and 600 mg/kg bw/day were considered to be incidental because of a lack of dose-dependency.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis showed an increasing tendency of urinary protein in males at 2000 mg/kg bw/day
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significant increases, or a tendency thereof, were found in absolute and relative weights of liver and kidneys in males and females at 2000 mg/kg bw/day. Decreased absolute adrenal weight (right side) was noted in females at 600 and 2000 mg/kg bw/day, but was considered to be incidental because it was seen unilaterally, was not associated with a changes in relative adrenal weight, and individual weight was within the range of variation in the control group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Description (incidence and severity):
- Stomach: Edema of the cornified layer at the limiting ridge (minimal to slight) was seen in 2 females at 600 mg/kg bw/day and in 3 males and 4 females at 2000 mg/kg bw/day. Edema of the cornified layer at the forestomach (minimal) was seen in 1 female at 2000 mg/kg bw/day.
- Liver: Centrilobular hypertrophy of hepatocytes (minimal) was seen in 2 males and 2 females at 2000 mg/kg bw/day.
- Kidney: Diffusely increased hyaline droplets were seen in the proximal tubules (minimal to moderate) in 9 males at 2000 mg/kg bw/day with higher grade and incidence than in the control.
Other findings observed in the L-tyrosine treated groups were considered to be those found in historical controls or incidental because of its low incidence and/or characteristics.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Applicant's summary and conclusion

Conclusions:
Based on effects, edema of the cornified layer at the limiting ridge or forestomach was seen in 600 mg/kg bw/day female group and in both sexes of 2000 mg/kg bw/day group, and in the liver, increased weight and hypertrophy of centrilobular hepatocytes were seen in both sexes at 2000 mg/kg bw/day, associated with slight increases in ALT and AST, the NOAEL of L-tyrosine in this 13- week repeated dose toxicity study was considered to be 600 for males and 200 mg/kg bw/day for females.
Executive summary:

In the current study the potential toxicity of L-tyrosine was evaluated in a 13-week repeated-dose oral toxicity study in rats according to OECD 408. The study was not GLP. L- tyrosine was administered by gavage to 4 groups each consisting of 10 males and 10 females. The doses were 0 (vehicle: water), 200, 600 or 2000 mg/kg bw/day.

No deaths occured during the administration period in any of the study groups. No L-tyrosine-related changes were observed in clinical signs, body weight, food and water consumption, ophthalmology or necropsy.

Some L-tyrosine-related changes were observed. In the stomach, edema of the cornified layer was seen at the limiting ridge or forestomach (minimal to slight) in females at 600 mg/kg bw/day and in both sexes at 2000 mg/kg bw/day. This change is often related to mucosal irritation. This might be a local irritant effect of L-tyrosine.

In the liver, significant increases, or a tendency thereof, in absolute and relative weights and minimal hypertrophy of centrilobular hepatocytes were seen in both sexes at 2000 mg/kg bw/ day. These changes were accompanied by significant increases, or a tendency thereof, in ALT and AST in both sexes at 2000 mg/kg bw/ day. Although the centrilobular hypertrophy of hepatocytes was assessed as minimal, the hepatic change was accompanied by slight increases in ALT and AST, these histological changes were considered as signs of mild hepatic toxicity. In addition, significant increases were noted in triglycerides, total cholesterol and phospholipids in both sexes at 2000 mg/kg bw/day.

Regarding the kidney morphology and function, increase in hyaline droplets in the proximal tubules and increased tendency of urinary protein were seen in males at 2000 mg/kg bw/day. Hyaline droplets are ususally observed in the proximal tubules in male rats, but in this experiment, the histological changes occurred in higher grades and incidences in 2000 mg/kg bw/day male group than in the control group. The hyaline droplets found in the proximal tubule is thought to indicate accumulation of protein reabsorbed from the primitive urine, and thus, we considered the increased tendency of urinary protein to be related to this histopathological finding. These changes were observed only in males, on the other hand, increases in absolute and relative kidney weights were observed in both sexes at 2000 mg/kg bw/day. The relationship between kidney weight and hyaline droplets in the proximal tubule or urinary protein remained unclear.

Additionally, significant increases, or tendency thereof, in potassium ion, calcium, total protein, and a1 globulin were seen in both sexes at 2000 mg/kg bw/day. Although the precise mechanisms were unclear, we could not deny the relation between these biochemical changes and histopathological findings in the liver and/or kidney. The increased RBC in males at 2000 mg/kg bw/day was not considered toxicologically significant due to the lack of changes in other RBC parameters and compound-related histopathologic findings in the bone mallow and spleen. Cataract formation was not observed in this study at any dose level.

Based on the above mentioned results and discussion, the no-observed-adverse-effect level (NOAEL) of L-tyrosine in this 13- week repeated dose toxicity study was considered to be 600 for males and 200 mg/kg bw/day for females.