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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 October 2000 - 30 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
: No effects on reliability of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
: OECD GLP (1997)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Physical state: Orthorhombic crystal
- Purity: 98.2%
- Impurities: No data
- Lot/batch No.: GH01
- Manufacture: Tokyo Chemical Industry Co., Ltd.
- Stability under test conditions: Stable during the test period (Purity: before the test; 98.2%, after the test; 98.1%)
- Storage condition of test material: in cool, light-blocking, and air-tight conditions

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: male 191-221 g; female 146-175 g
- Housing: steinless-steel bracket cage for rat use
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24°C
- Humidity (%): 36-62%
- Air changes (per hr): ≧ 10 times/hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: water for injection listed in the Japanese Pharmacopoeia (producted by Fuso Pharmaceutical Industries,Ltd.)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
During the dosing period, beta-alanine was daily dissolved with water for injection to make concentrations of 4, 20 and 100 mg/mL, and the prepared solutions were administered by gavage within 6 hours. These solutions containing the vehicle of the control group were administered by gavage at the constant volume of 10 mL/kg body weight at all dose levels.

VEHICLE
- Concentration in vehicle: 4, 20, 100 mg/mL
- Amount of vehicle: 10 mL/kg in the vehicle control group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test substance in vehicle, and the given concentrations were confirmed by HPLC analysis on the day before the dosing and in the preparation of the dosing solutions.
At 6 hours after preparation of the dosing solutions, the residual percentage of beta-alanine were 98.0% and 99.8%, in the low and high dose levels, respectively, at room temperature.
The concentrations at the upper, middle, and low layers of the containers were within the range of 96.3 and 103% of nominal concentrations of all dose groups, confirming the homogenity of the test substance in the solutions of all dose groups.
The measured concentrations of each dose levels were within the range of 95.4 and 99.8% of nominal concentrations.
Duration of treatment / exposure:
Dosing period: 28 days
Recovery period: 14 days
Frequency of treatment:
Once in the morning.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
vehicle control group: 6 males and 6 females (dosing period); 6 males and 6 females (recovery period)
40 mg/kg group: 6 males and 6 females (dosing period)
200 mg/kg group: 6 males and 6 females (dosing period)
1000 mg/kg group: 6 males and 6 females (dosing period); 6 males and 6 females (recovery period)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A 7-day range-finding study in the same strain rats was performed at three dose levels of 10, 100 and 1000 mg/kg/day. As a result, the highest dose level caused no treatment-related effects on body weights, clinical signs, hematology, blood biochemistry, and organ weights. The highest dose, therefore, was chosen at 1000 mg/kg/day (the highest dose level on the test guideline). The levels of 200 and 40 mg/kg/day were chosen as a middle and lowest doses, respectively.
- Rationale for selecting satellite groups: According to the OECD Test Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day before and after dosing during the dosing period, and once a day during the recovery period. At twice a day, morbidity and mortality were observed.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once the day before the dosing, and once a week during the dosing and recovery periods.


BODY WEIGHT: Yes
- Time schedule for examinations: Once before dosing in the starting day of the dosing period, once a week during the dosing and recovery periods, and once at necropsy.


FOOD CONSUMPTION:
- Food consumption of each animal were measured once the day before the dosing, once a week during the dosing and recovery periods.


FOOD EFFICIENCY: No data


WATER CONSUMPTION: No data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once the day before the dosing, once at week 4 of the dosing period, and once week 2 of the recovery period.
- Dose groups that were examined: All animals in all groups.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the dosing and recovery periods.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes. For about 20 hours.
- How many animals: All animals in all groups.
- Parameters examined: RBC, WBC, Ht, Hb, Platelet, MCH, MCV, MCHC, differential blood count, PT, and APTT.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the dosing and recovery periods.
- Animals fasted: Yes
- How many animals: All animals in all groups.
- Parameters examined: AST, ALT, ALP, gamma-GTP, ChE, total protein, albumin, A/G ratio, glucose, total cholesterol, triglyceride, total bilirubin, BUN, creatinine, inorganic phosphorus, Ca, Na, K, and chlorine.


URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the dosing and recovery periods.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. A maximum of 20 hours.
- Parameters examined: Occult blood, ketone body, glucose, protein, pH, urobilinogen, billirubin, urine volume, specific gravity, and color.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At week 4 of dosing.
- Dose groups that were examined: All animals in all groups.
- Battery of functions tested: Sensory activity, grip strength, and motor activity.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Detailed gross pathology concerning external surface of the body, all orifices, and cranial, thoracic and abdominal cavities and their contents were conducted at the end of dosing and recovery periods.


HISTOPATHOLOGY: Yes
The following tissues were preserved in 10% neutral buffered formalin solution.
Brain (cerebrum, cerebellum and pons), heart, thymus, thyroid, trachea and lungs, stomach, duodenum, small and large intestine (including Peyer's patches), liver, kidneys, adrenals, spleen, urinary bladder, bone and bone marrow (sternal bone and femur including joint), spinal cord (cervical, mid-thoracic and lumbar), sciatic nerve, testes, prostate, seminal vesicle, epididymis, ovaries, vagina, mandibular and mesenteric lymph nodes.
Other examinations:
Organ weights:
Brain, thymus, heart, liver, spleen, kidneys, adrenals, testes and epididymis were weighed.
Statistics:
In the 28 day dosing groups, all data excepting qualitative data were analyzed by Bartlett’s equal variance test and when equal variance was assumed, a one-way analysis of variance was performed. When a significant difference was found, the difference was tested by a Dunnett’s paired comparison test between the control and the respective test substance groups. When equal variance was not assumed, a Kruskal-Wallis H test was performed. When a significant difference was found, the difference was tested by a Dunnett’s paired comparison test between the control and the respective test substance groups. In the recovery groups, the data obtained were analyzed by F test and when equal variance was assumed, a Student’s t test was performed. When equal variance was not assumed, a Aspin-Welch’s t test was performed between the control and the respective test substance groups.

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):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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
No deaths occurred during the dosing and recovery periods.
A mass in the subcutis of the left inguinal region was observed in one female of the 1000 mg/kg/day recovery group after the day 14 of recovery.



BODY WEIGHT AND WEIGHT GAIN
There were no differences between the dosing and control groups during the dosing and recovery periods.



FOOD CONSUMPTION
There were no differences between the dosing and control groups during the dosing and recovery periods.



OPHTHALMOSCOPIC EXAMINATION
There were no differences before the beginning of dosing, at week 4 of the dosing period, and week 2 of the recovery period.



HAEMATOLOGY
There were no differences between the dosing and control groups during the dosing and recovery periods.



CLINICAL CHEMISTRY
At the end of the dosing period, there were no differences concerning parameters of the clinical chemistry between the dosing and control groups.
At the end of the recovery period, a significant decrease of chlorine was detected in females of the 1000 mg/kg group compared to the control group.
This change was not due to the test substance-treatment, because this change was not found at the end of the dosing period, and was not unaccompanied by other changes of other electrolytes.



URINALYSIS
At the end of the dosing period, the number of males showing urinary protein positive (+) in the 1000 mg/kg group was more than that in the control group.
These increases of urinary protein were considered not to be toxicological significance, because the histogical changes were not found in kidneys.



NEUROBEHAVIOUR
At the neurotoxicological observation, there were no differences between the dosing and control groups during the dosing and recovery periods. Also, there were no abnormalities of the neuro-function in rats on week 4 of the dosing period.



ORGAN WEIGHTS (Table 1 and 2)
At the end of the dosing period, the relative liver weights of females in the 200 and 1000 mg/kg dosing groups were significantly higher than those in the control group. These weight changes, however, was considered to be due to the physiological variations. The reason is that this relative liver weights were within the background data (2.94 ± 0.18%), and histopathological changes of the liver were not found in the same group, absolute weights of the liver did not significantly differente from that of the control group, the blood biochemical values were not suggestive of changes of liver function, and these changes were not found in males of the dosing groups.
At the end of the recovery period, there were no differences between the organ weights of both sexes in the control and 1000 mg/kg groups.



GROSS PATHOLOGY: (see Table 3 and 4.)
At the end of the dosing period, small testes and small epididymides were found bilaterally in one male of the control group.
At the end of the recovery period, one male of the control group showed enlargment of right kidney and rough surface, yellow gray plaque, atrophy, and cyst of left kidney. A yellow white mass (20 x 20 x 10 mm) was found at subcutis of left inguinal resion in one male of the 1000 mg/kg group.




HISTOPATHOLOGY: (see Table 5-7.)
At the end of the dosing period, histopathologically severe atrophy of seminiferous tubule and disappearance of spermatogonia were observed in one male animal showing small testes at necropsy at control group. Slight ectopic thymic tissue in the thyroids were found in two males at control group.
At the end of the recovery period, in the control group, one male having abnormal findings right and left kidneys at necropsy showed severe atrophy of renal tubule, moderate mononuclear cell infiltration, and severe fibrosis in interstitial tissue of the kidneys. Also, very slight or slight mononuclear cell infiltrations in livers and slight mononuclear cell infiltrations in prostates were found in the control and dosing groups.




NEOPLASTIC: (see Table 8.)
At the end of the recovery period, a yellow-white mass (20 × 20 × 10 mm) was found in the subcutis of the left inguinal region in one female of the 1000 mg/kg group. Histopathologically, this mass was adenocarcinoma in the mammary gland, and was considered spontaneous, because this mass appeared too early 6 weeks after beginning of dosing. Spontaneous mammary adenocarcinoma had been reported in 10 weeks old SD rats in a published literature*.
* Oishi Y., Yoshikawa K., Suzuki J., Makino N., Hasa K., Yamauchi K., Tsubura A. (1995) A spontaneous mammary adenocarcinoma was detected in a 10-wk-old. Tocicl.Pathol. 23, 694-700.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Beta-Alanine had no toxicological effects on males and females SD rats received 1000 mg/kg. The NOEL and NOAEL of both sexes were considered to be 1000 mg/kg.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Absolute and relative organ weights of male rats treated orally with beta-alanine for 28 days.

― 28-day of treatment―

Dose           (mg/kg) 0 40 200 1000
Final body weight (g) 342±18 351±23 346±29 340±37
Brain                (mg) 2.05±0.10 2.08±0.07 2.09±0.09 2.04±0.13
               (Relative) 0.60±0.04 0.60±0.03 0.60±0.04 0.60±0.06
Thymus           (mg) 479.8±65.3 566.2±143.8 541.3±84.1 431.7±102.5
               (Relative) 140.6±19.6 161.1±37.6 156.6±21.8 127.7±31.9
Heart                 (mg) 1.24±0.09 1.25±0.14 1.27±0.09 1.14±0.09
               (Relative) 0.36±0.03 0.36±0.03 0.37±0.01 0.34±0.02
Liver                (mg) 10.03±1.24 10.59±1.40 10.34±1.46 10.08±1.29
             (Relative) 2.93±0.23 3.02±0.25 2.98±0.17 2.96±0.10
Spleen             (mg) 727±94 747±111 738±99 662±89
                 (Relative) 213±27 213±27 214±30 195±10
Kidney (R)      (mg) 1.29±0.14 1.29±0.07 1.29±0.16 1.30±0.17
              (Relative) 0.38±0.04 0.37±0.03 0.37±0.02 0.38±0.03
Kidney (L)        (mg) 1.34±0.14 1.32±0.09 1.36±0.19 1.35±0.14
              (Relative) 0.39±0.04 0.38±0.03 0.39±0.03 0.40±0.02
Adrenal (R)      (mg) 22.0±3.9 22.4±2.0 25.0±3.5 22.7±3.0
                (Relative) 6.4±0.9 6.4±0.7 7.2±0.9 6.7±1.0
Adrenal (L)        (mg) 25.5±3.1 24.7±3.1 25.4±2.8 24.5±3.2
                (Relative) 7.5±0.6 7.1±1.1 7.4±0.7 7.2±1.1
Testis (R)          (mg) 1.27±0.45 1.50±0.11 1.49±0.09 1.49±0.12
                (Relative) 0.38±0.13 0.43±0.03 0.44±0.04 0.44±0.06
Testis (L)          (mg) 1.28±0.44 1.49±0.11 1.47±0.07 1.49±0.12
                (Relative) 0.38±0.13 0.43±0.03 0.43±0.04 0.44±0.05
Epididymis (R)   (mg) 0.39±0.11 0.42±0.05 0.43±0.04 0.44±0.03
               (Relative) 0.11±0.03 0.12±0.01 0.13±0.02 0.13±0.02
Epididymis (L)  (mg) 0.37±0.10 0.42±0.06 0.42±0.04 0.43±0.04
                (Relative) 0.11±0.03 0.12±0.02 0.12±0.02 0.13±0.02

Relative : Relative organ weight (g or mg/100 g final body weight)                   (Mean ± S.D.)

Table 2. Absolute and relative organ weights of female rats treated orally with beta-alanine for 28 days.

― 28-day of treatment―

Dose           (mg/kg) 0 40 200 1000
Final body weight (g) 212±16 214±11 211± 15 204± 20
Brain                (mg) 1.91±0.08 1.91±0.05 1.94±0.07 1.94±0.11
              (Relative) 0.90±0.06 0.90±0.04 0.92±0.06 0.96±0.06
Thymus             (mg) 412.0±75.2 429.3±131.0 374.0±73.3 366.4±125.5
                (Relative) 193.5±24.3 199.1±52.4 176.3±27.3 176.8±47.0
Heart               (mg) 0.76±0.06 0.76±0.07 0.77±0.08 0.74±0.09
               (Relative) 0.36±0.01 0.36±0.03 0.36±0.03 0.36±0.02
Liver                (mg) 5.90±0.41 6.25±0.38 6.34±0.61 6.21±0.63
                (Relative) 2.79±0.14 2.92±0.09 3.00±0.11* 3.04±0.15**
Spleen              (mg) 481±56 531±78 472±62 485±150
               (Relative) 227±18 248±29 224±32 235±59
Kidney (R)       (mg) 0.83±0.08 0.81±0.07 0.85±0.05 0.88±0.11
               (Relative) 0.39±0.02 0.38±0.03 0.40±0.00 0.43±0.03
Kidney (L)        (mg) 0.82±0.08 0.82±0.06 0.84±0.06 0.87±0.11
               (Relative) 0.39±0.03 0.38±0.02 0.40±0.01 0.43±0.03
Adrenal (R)       (mg) 32.8±4.0 30.5±3.0 32.9±1.5 33.0±3.2
              (Relative) 15.5±2.0 14.3±1.4 15.6±1.5 16.3±2.6
Adrenal (L)       (mg) 33.4±3.3 32.8±3.8 35.0±3.6 34.9±5.4
               (Relative) 15.8±1.7 15.4±2.0 16.7±2.3 17.2±3.1

Relative : Relative organ weight (g or mg/100 g final body weight)                  (Mean ± S.D.)

* : Significantly different from control, p < 0.05

** : Significantly different from control, p < 0.01

Table 3. Necropsy of male rats treated orally with beta-alanine for 28 days.

― 14-day of recovery ―

Dose                              (mg/kg) 0 1000
No. of animals 6 6
Kidney:
cyst 1 0
atrophy 1 0
yellow gray plaque 1 0
rough surface 1 0
enlargement 1 0

Table 4. Necropsy of female rats treated orally with beta-alanine for 28 days.

― 14-day of recovery ―

Dose                             (mg/kg) 0 1000
No. of animals 6 6
Subcutis (left inguinal region):      
mass 0 1

Table 5. Histopathology of male rats treated orally with beta-alanine for 28 days.

― 28-day of treatment ―

Dose                              (mg/kg) 0 1000
No. of animals 6 6
Thyroid:      
ectopic thymic tissue 2 0
Liver:    
mononuclear cell infiltration 6 5
Testis:    
atrophy of seminiferous tubule 1 0
disappear of spermatogonia 1 0
increased Leydig cell 1 0
Prostste:      
mononuclear cell infiltration 1 2

Table 6. Histopathology of male rats treated orally with beta-alanine for 28 days.

― 14-day of recovery ―

Dose                              (mg/kg) 0 1000
No. of animals 1#  
 Kidney:    /  
atrophy of renal tubule 1  
mononuclear cell infiltration 1  
fibrosis in interstitial tissue 1  

#: Animal with gross pathological finding

/: not examined.

Table 7. Histopathology of female rats treated orally with beta-alanine for 28 days.

― 28-day of recovery ―

Dose                              (mg/kg) 0 1000
No. of animals 6 6
Liver:      
mononuclear cell infiltration 6 5

Table 8. Histopathology of female rats treated orally with beta-alanine for 28 days.

― 14-day of recovery ―

Dose                              (mg/kg) 0 1000
No. of animals   1#
  Subcutaneous mass:  /    
adenocarcinoma (mammary gland)   1

# : Animal with gross pathological finding

/: not examined.

Applicant's summary and conclusion

Conclusions:
No effects of beta-alanine on rats receiving the maximum dose of 1000 mg/kg were observed. The NOEL and NOAEL of this study are considered to be 1000 mg/kg in both male and female rats.
Executive summary:

A 28-day repeated dose oral toxicity study of beta-alanine was conducted using male and female Crj:CD(SD)IGS rats according to OECD TG 407 under GLP conditions. 40, 200, and 1000 mg/kg (6 rats/sex/group) of beta-alanine, dissolved in water for injection, were administered by gavage 7 days a week for 28 days, followed by 14-day recovery period. During the dosing and recovery periods, no deaths occurred, and no treatment-related changes were observed in clinical signs, neurotoxicological observation, functional tests, body weights, food consumption, ophthalmoscopy, urinalysis, hematology, blood biochemistry, and organ weights. At the end of the recovery period, a yellow-white mass (20 × 20 × 10 mm) was found in the subcutis of the left inguinal region in one female of the 1000 mg/kg group. Histopathologically, this mass was adenocarcinoma in the mammary gland, and was considered spontaneous. In summary, no effects of beta-alanine on male and female SD rats receiving the maximum dose of 1000 mg/kg were observed. The NOEL and NOAEL of this study are considered to be 1000 mg/kg in both male and female rats.