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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
The reliability of the source read-across study was established to be R2: comparable to guideline study acceptable for assessment
Justification for type of information:
Justification for read-across is detailed at section 13.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Guidelines for Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs
Version / remarks:
Ordinance N.1, Article N.24, September 11, 1989
Principles of method if other than guideline:
This study evaluated toxicological and behavioral effects of test item during a dosing study with male and female Sprague-Dawley rats. The substance was incorporated into a standard diet at doses equal to 1.25 %, 2.5 %, and 5.0 % (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: animals were 4-week-old when arrived to the testing laboratory; at the test start, they were 6-weeks-old.
- Source: Charles River Japan, Tokyo, Japan.
- Housing: animals were housed individually in conventional stainless steel hanging cages.
- Diet: standard diet, ad libitum.
- Water: ad libitum.
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 55 ± 10 %
- Photoperiod: 12 hours cycle dark/light from 7:00 to 19:00 p.m.

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
Control and test item-supplemented diets did not differ in colour and odour.
All diets were administered ad libitum.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
1.25 other: % w/w nominal in diet
Remarks:
Mean intake male: 841.2 ± 29.3 mg/kg/day; female 967.9 ± 46.5 mg/kg/day
Dose / conc.:
2.5 other: % w/w nominal in diet
Remarks:
Mean intake male: 1677.4 ± 69.4 mg/kg/day; female 197.2 ± 86.5 mg/kg/day
Dose / conc.:
5 other: % w/w nominal in diet
Remarks:
Mean intake male: 3356.6 ± 115.1 mg/kg/day; female 3986.3 ± 282.9 mg/kg/day
No. of animals per sex per dose:
12 rats per group
Recovry: 6 rats per group
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: selected rats were divided by a computer block-placement method into four separate groups, based on their weight, to eliminate the homogeneity of the group mean body weight.
- Recovery groups: following the treatment period, six rats from the control and high-dose groups were randomly selected for continuation in the recovery period.
- Post-exposure recovery period: 5 weeks.
- Diet administration: during the recovery, only a standard unsupplemented diet was provided.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS
All rats were observed twice daily (morning and afternoon) during the 13-week test article administration period, and once daily (morning) during the 5-week recovery period.

BODY WEIGHT
The rats were weighted twice a week at a specified time (9:00 AM to 12:00 noon).

FOOD CONSUMPTION AND COMPOUND INTAKE
24-hour diet intake was measured twice per week. Estimated intake of test item (mg/kg/day) for each week was calculated based on the mean consumption for 1 day for that week.

WATER CONSUMPTION AND COMPOUND INTAKE
Water intake was measured once per week.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmologic examinationwas performed prior to the start of administration. Animals in which abnormalities of the external appearance of the eyes, the anterior part, the vitreous body, and the fundus oculi of the eyes were observed using an ophthalmoscope were not included in the study. The ophthalmologic examination was repeated in week 13 of administration (six randomly selected rats from each group), and week 5 of recovery (all rats).

HAEMATOLOGY and BLOOD CHEMISTRY
Hematological examination was conducted from the blood samples collected on the day following the final administration (week 13), and at the end of recovery period from the rats deprived of food overnight prior to blood sample collection. Blood samples were collected from the abdominal aorta by laparotomy under ether anesthesia into blood collecting tubes containing an anticoaugulant (EDTA-2K). The following parameters were measured: red blood cell count (RBC), mean corpuscular volume (MCV) (ACL100), hemoglobin (Hb) (cyanmethemoglobin method; ACL100) to reticulocyte ratio (Brecher method), platelet and white blood cell count (ACL100), differential leucocyet count (May-Giemsa staining), prothrombine and activated partial thromboplastin time (PT and APTT) (automatic analyzer, Monarch), and fibrinogen (thromboplastin method; automatic analyzer, Monarch). Hematocrit and mean corpuscular hemoglobin were calculated from the abovemeasured parameters.

Additional plasma parameters (GOT, GPT, and lactate dehydrogenase [LDH]) were obtained from blood samples collected from the abdominal aorta into heparinized tubes. The serum parameters (total cholesterol, triglycerides, phospholipids, total bilirubin, blood glucose, urea nitrogen, creatine, uric acid, sodium, potassium, chloride, calcium, inorganic phosphorus, and total protein) were obtained from blood samples that were allowed to stand for 30 to 60 min, and thereafter centrifuged at 3000 rpm (10 min).

URINALYSIS
Urinalysis was conducted in all rats in weeks 5 and 13 of the administration period, and in week 5 of the recovery, as follows. Animals were placed in metabolic cages for 4 h, provided with water, and deprived of diet. Urine samples were collected.
Immediately thereafter, rats were given a diet, and further 20-h urine samples were collected while providing both diet and water. The following parameters were evaluated only from the 4-h samples: pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen, urine colour, and sedimentation (microscopic examination).
The following parameters were evaluated only from the 24-h samples: volume of urine (volumetry), specific gravity (refractometry), and electrolyte concentration.
Sacrifice and pathology:
GROSS PATHOLOGY
The rats were sacrificed by exsanguinations from the abdominal aorta and observed for any external malformations. The organs and tissues in the cephalic, thoratic, and abdominal cavities were examined macroscopically. The brain, pituitary, salivary, and thyroid glands, heart, lungs (including bronchia), liver, spleen, kidneys, adrenals, testes, prostate, ovaries, and uterus were excised and weighted. The relative organ weights were calculated using the animals’ fasting body weights.
All the organs listed above, plus spinal cord, sciatic nerve, thoratic aorta, trachea, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, thymus, mesenteric lymph nodes, cervical lymph nodes, epididymides, seminal vesicles, vagina, mammary glands, skin, eyes, optic nerve, harderian glands, sternum (bone marrow), femur (bone marrow), femoral muscle, and gross lesions were excised and fixed in phosphate-buffered formalin solution.

HISTOPATHOLOGY
Femoral bone marrow samples were collected at autopsy from all rats and May-Giemsa–stained specimen were prepared and examined microscopically.
After paraffin embedding, the excised organs and tissues were prepared for microscopic examination by sectioning and staining with hematoxylin and eosin. Representative samples were photographed.
Statistics:
Data were analyzed for homogeneity of variance using Bartlett’s test. Homogenous data observed at the level of 5 % (w/w) were analyzed using the parametric one-way analysis of variance (ANOVA), and the significance of differences was assessed using Scheffe’s method to compare the values between the control group and each amino acid–administered group. Heterogeneous data converted to rank-sum were analyzed using the Kruskal-Wallis nonparametric test. Any significant differences observed were further evaluated using the method of distribution free multiple comparison (Gad and Weil 1982). Means ± standard deviations (SD) are reported.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No signs related to administration of test article were observed during the administration period. Swelling of the hind limbs (tarsal joints) was observed in one female rat in the control group, gryposis of the upper jaw in one female rat in 1.25 % concentration group, fracture of the incisors in one male rat of both the control and the 5.0 % concentration group, and malocclusion in one male in the control group. All changes were incidental. No clinical signs were observed during the recovery period.
Mortality:
no mortality observed
Description (incidence):
No deaths were observed throughout the administration and recovery periods.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights in male and female rats in each concentration group were comparable to those recorded in controls, andno statistically significant difference was observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Administration-related changes in diet consumption were not observed. In thehigh dose, this included increased intake on day 42 in males and day 3 in females. However, those were temporary, dose-independent phenomena and the total consumption did not differ among the groups.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significant administration-related changes in water intake were recorded.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no abnormalities observed in any of the animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, a significant increase in the hemoglobin level was seen in females in the 5.0 % concentration group (controls, 14.7 ± 0.6 g/dl; 5.0 % group, 15.5 ± 0.6 g/dl).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in serum chlorides was observed in males of all concentrations groups (controls, 111.9 ± 0.7 mEq/l; 1.25 % group, 109.9 ± 1.1 mEq/l; 2.5 % group, 110.3 ± 1.5 mEq/l; 5.0 % group, 109.8 ± 1.3 mEq/l), and females of the 5.0 % concentration group (controls, 112.7 ± 1.2 mEq/l; 5.0 % group, 110.1 ± 1.2 mEq/l).
Additionally, a significant increase in total bilirubin was observed in females in the 2.5 % concentration group (controls, 0.09 ± 0.01 mg/dl; 2.5 % group, 0.11 ± 0.02 mg/dl), but the change was not observed in the 5.0 % concentration group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
A dose-dependent tendency for decreased pH was seen in both genders that received Lys diets. Males ingesting 5.0 % concentration diet were characterized by significantly higher urine volume (controls, 15.3 ± 5.8 ml/day; 5.0 % group, 21.3 ± 8.1 ml/day). This change was not observed at the end of the recovery period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At the end of the administration period, there were no treatment-related changes in absolute or relative organ weights. At the end of the recovery period, a significant increase in the absolute weight of the heart in males of the 5.0 % concentration group, a significant increase in unilateral weight of the kidneys in males and females in the 5.0 % concentration group, and a decrease in the absolute bilateral weight of the adrenals in males of the same group were seen. As the changes were not observed at the end of the administration period, they were considered to be accidental ones.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant treatment-related pathologies; minor changes were few and dose independent.
One female in the 5.0 % concentration group had dilated renal pelvis, and one female in the 2.5 % concentration group had slight hyaline casts. Weak retinal atrophy was observed in one male in the 5.0 % concentration group, and minor retinal degeneration in one female in both the control and the 2.5 % concentration group.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological alterations at the end of the administration and recovery periods were not related to the treatment. Slight focal myocarditis was observed in one male in each of the 1.25 % and 2.5 % concentration groups. Focal pneumonia was observed in one female in the 5.0 % concentration group. Mild erosion in the glandular stomach wall (macroscopic red spots in the glandular area) was seen in one or two females in each group, including the control group. Slight cellular infiltration of the lamina propria mucosae was observed in one female in the control group, and one male and one female in the 2.5% concentration group. Minor focal necrosis was detected in two females in the 1.25 % concentration group, and one male in the 5.0 % concentration group. A small cyst was found in the kidney in one female in the control group.
There were no abnormalities in other organs and tissues.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
3 357 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
NOAEL
Effect level:
3 986 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (13 wk) (rat male): 3357 mg/kg/day
NOAEL (13 wk) (rat female): 3986 mg/kg/day.
Executive summary:

Method

The evaluation of the Repeated dose toxicity by oral route was conducted according to Guidelines for Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Ordinance N.1, Article N.24, September 11, 1989) with male and female Sprague-Dawley rats. The test article was mixed into a standard diet at three concentrations (1.25, 2.5 and 5 %), and the diets were orally administered ad libitum to male and female rats for 13 continuous weeks. The administration period was followed by a 5-week recovery, during which only a standard diet was offered.

 

Observations

No deaths were observed throughout the administration and recovery periods. In male and female rats in each concentration group, treatment related changes were not observed in the clinical signs, body weights, diet consumption, water intake, ophthalmology, gross pathology, organ weights, or histology. These results agree with the previous chronic observation, in which the test item (3.0 %, w/w) effects were investigated in a 2-year ad libitum feeding paradigm (Anderson and Raiten 1992). Moreover, Yamori (1993) did not report any adverse effects of increasing the test article intakes by about 150 % from the requirement in stroke-prone, spontaneously hypertensive rats over a 25-week period. An increase in the level of hemoglobin in females of the 5.0 % concentration group was the main hematological change. However, this increase was within both the range of the concurrent and historical laboratory values observed in the experimental facility. The values of urea nitrogen and creatine, and the lack of histological abnormalities, indicate absence of renal dysfunction. A slight rise in urine volume was found in females in the 5.0% concentration group, but it was considered as an adaptive reaction to large increases in ingested chlorides.

 

Results

Based on the above data, the no-observedadverse- effect level (NOAEL) for the test article is estimated at 5.0 % for both genders (males 3.357 ± 0.115 g/kg/day; females, 3.986 ± 0.283 g/kg/ day). These relatively high NOAELs are consistent with human observations (Flodin 1997) that the test item is a safe and well-tolerated dietary substance for long-term use.