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Description of key information

OECD Guideline, GLP-compliant 28-day repeated dose study via oral route. The animals (male/female) were administered with a single daily dose of the test substance by gavage for 28 days. Some effects were noted at the highest tested dose (1000 mg/kg), however, these were judged to be not toxicologically significant and therefore the NOEAL was set at 1000 mg/kg accordingly.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Oct 1988 - 07 Jun 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted in Oct 2008
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 158 - 177 g (males); 123 - 139 g (females)
- Housing: 2 rats of the same sex per cage were housed in polycarbonate cage (265W x 426D x 200Hmm, Tokiwa Kagaku Kikai Co., Ltd.) supplied with bedding (Beta chip, Charles River Japan, Inc.). The cages were stored in a steel cage rack (Tokiwa Kagaku Kikai Co., Ltd.) of 4 rows. A stainless feeder for pellet food (Tokiwa Kagaku Kikai Co., Ltd.) and a polycarbonate water-dispensing bottle (500 mL or 700 mL: Tokiwa Kagaku Kikai Co., Ltd.) were used.
- Diet: pellet food for laboratory animals (MF: Oriental Yeast Co., Ltd.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The defined amount of test substance was weighed, suspended in 0.5% sodium CMC solution, and stored in the refrigerator until just before the administration. The solution was prepared once a week.
The stability of the solution was confirmed to be 16 days in the testing laboratory.

VEHICLE
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 (main)
6 (recovery control and recovery high-dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of an acute oral toxicity study performed at the same testing facility (Study No. 8K258).
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals were observed for mortality, signs of toxicity and abnormal behaviour.
- Time schedule: Once daily

BODY WEIGHT: Yes, all animals
- Time schedule: body weights were recorded in all animals prior to administration and once per week thereafter.

FOOD CONSUMPTION: Yes, all animals
The weekly food consumption was estimated by the difference of the weight of the cages, which were weighed at the beginning and every week thereafter, and the mean daily food consumption was calculated according to the weekly consumption of each terms.

HAEMATOLOGY: Yes, the blood samples were taken from the caudal vein for the blood testing. As the anti-coagulant, 3.13% sodium citrate was used for prothrombin time and activated partial thromboplastin time, and EDTA-2k was used for the other parameters.
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: pentobarbital
- How many animals: Yes, all animals
- Parameters checked: erythrocytes, leukocytes, platelet count, hemoglobin concentration, hematocrit, white blood cell differential, reticulocyte count, prothrombin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular hemoglobin and mean hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- How many animals:
- Parameters checked: total protein, blood glucose, triglyceride, total cholesterol, urea nitrogen, creatinine, calcium, inorganic phosphorus, GOT, GPT, γ-GTP, ALP, A/G ratio, sodium, potassium and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 6 days before the sacrifice, fresh urine was collected from all animals. In addition, urine was collected for a defined period (15.5 or 17 h).
- Parameters checked for fresh urine: pH, occult blood, protein, glucose, ketone, bilirubin and urobilinogen
- Parameters checked for collected urine: urine volume, sodium, potassium, and chloride
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, after blood sample collection, all animals were sacrificed by exsanguination via ventral aorta dissection, and necropsy was performed and recorded.
- Organ weights were recorded for brain, liver, kidney, adrenal gland, testes or ovary

HISTOPATHOLOGY: Yes, the organs were collected from all animals and fixed in 10% neutral buffered formalin.
- Organs collected: brain, pituitary gland, eye, lung, stomach, thyroid gland including parathyroid gland, heart, liver, spleen, kidney, adrenal gland, bladder, bone marrow (femur), and testes or ovary.
- Organs checked: heart, liver, spleen, kidney, and adrenal gland from all animals from control and high-dose groups sacrificed at 28 days, and testis from one male from a high-dose group with abnormality observed on necropsy, were microscopically evaluated after the standard paraffin embedding, sectioning and H. E. staining.
Histological examination was not performed of the animals sacrificed after the recovery period.
Statistics:
When the equality hypothesis of the data was not rejected, the Student t-test was used, and the Welch t-test was used when the equality was rejected. For the qualitative urine testing and pathological testing data, the Armitage χ² test was used.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: A significant reduction of the body weight gain on Day 14 (5%), Day 21 (7%) and Day 28 (9%) was observed in the high-dose males when compared with the control group. In addition, the body weight was significantly reduced in the high-dose recovery group on Day 35 (15%) and Day 42 (14%) when compared with the control recovery group.
200 mg/kg bw/day: In females the body weight gain was significantly decreased on Day 21 (7%) and Day 28 (9%) when compared with the control females.

Please refer to Table 1 (under "Any other information on results incl. tables).
The reduced body weight was only minimal and considered to be caused by the reduced food consumption. Therefore, the body weight reduction was considered to be not toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: In males, the food intake was significantly reduced in the first week during treatment (7%) and in the last week during recovery (10%) when compared with the respective control group. A significant decrease (9 and 4%) was determined in females at 1 and 2 weeks after the administration, when compared with the control females.
200 mg/kg bw/day: On Day 21 and on Day 28, the food intake was significantly reduced (9% and 12%) in females when compared with the control females.
40 mg/kg bw/day: In males a 6% and a 7% reduction of the food intake was observed on Day 21 and Day 28, respectively.

Please refer to Table 2 (under "Any other information on results incl. tables).
The reduction of the food consumption was slight and therefore considered to be not toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: A significant decrease in the mean corpuscular volume (4%), mean corpuscular hemoglobin (4%), and lymphocyte count (7%), and a significant increase in segmented cells (125%) were seen in males, when compared with the control group. Haematocrit was significantly reduced (5%) in females, when compared with the control females.
40 mg/kg bw/day: A significant decrease in the mean corpuscular volume (4%) was seen in males, when compared with the control males.
Recovery 1000 mg/kg bw/day: A significant elongation in the prothrombin time (3%) and a significant decrease in monocyte count (66%) were seen in males, and a significant decrease in mean corpuscular volume (4%) was seen in females, when compared with the respective control animals, respectively.

These observed effects were minor and therefore considered to be incidental without toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: A significant increase in alkaline phosphatase (49%) and a significant decrease in total cholesterol (33%) in males, and a significant decrease in the A/G ratio (20%) in females were not noted when compared with the respective control group.
200 mg/kg bw/day: A significant decrease in calcium (2%) in males was noted when compared with the control males. In females, a significant decrease in creatinine (20%), trigryceride (55%, and total protein (6%) were noted, when compared with control females.
40 mg/kg bw/day: A significant decrease in calcium (2%) and a significant increase in the A/G ratio (12%) in males, when compared with the control males.
Recovery 1000 mg/kg bw/day: In males, a significant decrease in total cholesterol (16%), calcium (2%) and total protein (6%), respectively, and a significant increase in inorganic phosphorus (9%) and sodium (1%) were seen when compared with control males.

No correlation in the liver and kidney was observed at pathological and histopathological examination, respectively, therefore these effects were considered to be without toxicological relevance.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: In males a significant increase in urine protein, a significant acidic shift of urine pH, a significant change in occult blood, a significant decrease in sodium (88%), potassium (40%) and chloride (24%) were seen, when compared with the control. A significant decrease in ketone and sodium (50%), and a significant increase in urobilinogen was noted in females, when compared with the control.
40 mg/kg bw/day: A significant alkaline shift of urine pH in males and a significant decrease in ketone in females was observed, when compared with the control.
10 mg/kg bw/day: A significant change in urobilinogen and a significant decrease in urine volume (63%) were observed in males, when compared with the control males. In females, a significant alkaline shift of urine pH was observed.
Recovery 1000 mg/kg bw/day: A significant increase in urine protein, ketone, and bilirubin were seen in the female animals, when compared with control females.

Since no toxicologically relevant findings were observed in the kidney at the pathologcial and histopathological examination, the findings of the urinalysis were not considered to be toxicologically relevant.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: a significant decrease in the absolute weight of adrenal glands (11%), a significant increase in the relative liver weights (19%) and absolute ovaries weight (19%) in females, and a significant increase in the relative liver weights (12%) in males were determined when compared with the control animals.
200 mg/kg bw/day: a significant decrease in the absolute liver weights (16%) and in the relative liver weights (9%) and a significant increase in the relative kidney weights (7%) in males, and a significant decrease in the absolute liver weights (15%) and a significant increase in relative brain weights (11%) in females were noted when compared with the respective control animals.
40 mg/kg bw/day: a significant increase in the absolute adrenal glands weight (12%) and in the relative adrenal glands weight (18%) in males was determined when compared with the control animals.
Recovery 1000 mg/kg bw/day: A significant decrease in the absolute liver weights (19%) and absolute testes weights (8%) in males were determined when compared with the recovery control group. The relative brain (15%) weight was significantly increased in males when compared with control males.

Since the changes in absolute and relative organ weights were minor and/or without dose dependency and/or without correlation at pathological and histopathological examination, they were considered to be without toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
1000 mg/kg bw/day: a mild enlargement of the right testis was seen in 1/6 males, which was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Control: focal necrosis (slight, 1/6 males); extramedullary hematopoiesis (slight, 1/6 males and 1/6 females); eosinophilic body (slight; 1/6 males); cyst (slight, 1/6 males)
1000 mg/kg bw/day: extramedullary hematopoiesis (slight, 2/6 males)

These effects were minor and therefore considered to be incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the higehst tested dose level.
Key result
Critical effects observed:
no

Table 1: Body weights of males and females

Dose level (mg/kg bw/day)   Prior to treatment Day 7 Day 14 Day 21 Day 28 Day 35 (Recovery Day 7) Day 42 (Recovery Day 14)
Males
Control Mean 168 234 303 363 415 446 477
SD 3.5 9.3 14.7 23.9 32.2 46.1 52.5
n 12 12 12 12 12 6 6
40 Mean 170 234 298 353 397    
SD 4.8 4.5 14 21.1 25    
n 6 6 6 6 6    
200 Mean 168 232 294 344 387    
SD 5.5 11.5 16.2 26.6 33.6    
n 6 6 6 6 6    
1000 Mean 169 228 289 * 338 * 381 * 382 * 411 *
SD 4.3 8.5 13.4 23.3 31.1 26.8 27.4
n 12 12 12 12 12 6 6
Females
Control Mean 131 165 193 217 242 264 274
SD 5.7 8.4 11 12.4 14.5 6.1 14.4
n 12 12 12 12 12 6 6
40 Mean 131 162 188 214 269    
SD 5.7 10.5 11.4 10.7 13.1    
n 6 6 6 6 6    
200 Mean 130 161 186 203 * 221 **    
SD 4.7 7.3 7.1 14.2 11.6    
n 6 6 6 6 6    
1000 Mean 130 161 187 209 230 246 254
SD 4.3 7.7 9.4 13 13.7 19.9 20.2
n 12 12 12 12 12 6 6

Significantly different from control value, *p<0.05, **p<0.01

Table 2: Foord consumption (males and females)

Dose level (mg/kg bw/day)   Day 7 Day 14 Day 21 Day 28 Day 35 (Recovery Day 7) Day 42 (Recovery Day 14)
Males
Control Mean 23.3 27.3 30.1 30.6 30.4 30.1
SD 0.81 0.63 0.76 0.93 1.22 0.96
n 6 6 6 6 3 3
40 Mean 22.9 26.0 28.5 * 28.5 *    
SD 0.40 1.37 0.68 0.85    
n 3 3 3 3    
200 Mean 22.9 26.7 28.5 29.3    
SD 0.90 1.78 3.31 2.30    
n 3 3 3 3    
1000 Mean 21.8 * 27.1 29.5 31.4 27.7 27.0 *
SD 1.18 2.26 3.30 2.76 2.34 1.25
n 6 6 6 6 3 3
Females
Control Mean 17.3 18.4 20.1 21.1 22.0 21.8
SD 0.76 0.51 0.54 0.89 1.05 2.16
n 6 6 6 6 3 3
40 Mean 17.0 18.1 19.6 20.2    
SD 1.00 1.27 0.89 0.30    
n 3 3 3 3    
200 Mean 16.6 18.4 18.3 * 18.6 **    
SD 0.85 0.75 1.55 0.15    
n 3 3 3 3    
1000 Mean 15.8 ** 17.6 * 19.3 20.4 21.8 19.9
SD 0.77 0.55 1.13 0.77 1.36 1.67
n 6 6 6 6 3 3

Significantly different from control value, *p<0.05, **p<0.01

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on available repeated-dose data from 28-day repeated dose study and reproductive toxicity screening test (where general toxicity of the parental animals are assessed) both conducted via the oral route, the substance is does not warrant classification for specific target organ toxicity after repeated exposure (STOT RE). The NOAEL for both studies were determined as 1000 mg/kg/day and 250 mg/kg/day, respectively.