Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
january 2011 - june 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: Testing methods concerning new chemical substances (Notification No 1121002, PFSB, MHLW; No 2 of November 13, 2003, MIB, METI; No 031121002, EPB, MOE; dated November 21, 2003
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): E-BW102
- Physical state: red brown colored powder
- Stability under test conditions: the test substance was confirmed to be stable during the experimental period based on infrared absorption spectra
- Storage condition of test material: room temperature (permissible range 1°-30°C) in an air-tight container in a dark place

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. (Hino Breeding Center)
- Age at study initiation: 6 weeks
- Weight at receipt (5 weeks old): 118.7 to 131.7 g for males and 92.1 to 108.2 g for females
- Housing: stainless-steel cages, one animal per cage
- Diet: ad libitum autoclave-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd)
- Water: ad libitum well water admixed with NaClO (about 2 ppm)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5° to 25.1°C
- Humidity (%): 44.1 to 58.5%
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: January 27,2011 To: April 26, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepeared once every 5 to 7 days and stored in a cool place shielded from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dosing solution at each concentration were analysed. Analytical method validation included linearity, repeatability, specificity, within-run precision and stability testing. All concentrations were within 100 ± 10% as the ratio to the nominal concentration; substance solutions were conformed to be stable after storage in a cool place and shielded from light for 8 days and following 6 hours at room temperature.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250, 500, 1000 (500), 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5, with for the control and 1000 mg/kg bw/day groups an additional 5/sex for a 14 day recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results obtained in "Seven-day repeated dose toxicity study of E-BW102 in rats, study No P101034, dose levels 0, 100, 300 and 1000 mg/kg bw/day with 5 rats/sex/dose). Low body weight was noted in males in the high dose group on day 7. One male of the high dose group showed BW decrease and a low food consumption on days 4 to 7. Moreover, a low value of the total protein was noted in high dose females, increased values of A/G ratio were noted in 300 and 1000 mg/kg bw/day females, and low values or its tendencies of sodium and potassium were noted in high dose males and females. A low potassium value was also noted in 300 mg/kg bw/day females. In addition, a low seminal vesicle weight in high dose males and high adrenal weight in high dose females was observed. In high dose animals test substance mixed feces colored reddish brown were noted, and reddish brown contents in the stomach were noted in high dose males. Based on these results, the high dose in the main study was set at 1000 mg/kg bw/day at which no mortality was observed and toxicity was expected, and lower doses were set at 250 and 50 mg/kg bw/day.
However, the high dose for males was changed to 500 mg/kg bw/day based on the mortality on day 26 (a total of 5 animals died; this group is referred to as 1000/500 mg/kg bw/day). In addition, as a suficient number of animals for evaluation of recovery could not be obtained, all animals were subjected to necropsy at the end of the dosing period. Accordingly, the control 2 and the 500 mg/kg bw/day groups were additionally established for males.

- Post-exposure recovery period in satellite groups: 14 days for controls and 500 mg/kg bw/day groups.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during the dosing period; once a day during recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before dosing; once a week until week 6

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on days 1, 8, 15, 22 and 28 during the dosing period and on days 29, 36 and 42 during the recovery period. The final body weight was also measured on teh day of the scheduled necropsy.

FOOD CONSUMPTION:
- For all animals, gross weight of each feeder was weiged. Mean daily food consumption values during days 1 to 8, 8 to 15, 15 to 22, 22 to 27, 29 to 36 and 36 to 41 were calculated.

HAEMATOLOGY: Yes, according to guideline
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, sodium pentobarbital (i.p., 30 mg/kg bw)
- Animals fasted: Yes, at least 18 hours
- How many animals: all animals at the scheduled necropsy

CLINICAL CHEMISTRY: Yes, according to guideline
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, at least 18 hours
- How many animals: same as for haematology

URINALYSIS: Yes
- Time schedule for collection of urine: day 27
- Metabolism cages used for collection of urine: No, fresh urine samples excreted spontaneously on a washed tray under the cage were collected.
- Animals fasted: Yes
- Parameters checked: PH, protein, glucose, ketone body and occult blood were determined by means of reagent strip method. Urinary sediment was tested with fresh urine (24-hour pooled urine samples): volume, color, osmotic pressure, specific gravity, sodium, potassium, chloride

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to guideline
HISTOPATHOLOGY: Yes, according to guideline
Statistics:
Statistical analysis was performed with computer system, MiTOX-PPL with significant levels of 1% and 5% (two-tailed).
For grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weight, the group mean and standard deviation of the following items were calculated and homogeneity of the variance was tested by the Bartlett's test (significant level 5%). When the variance was homogeneous, the Dunnett's multiple comparison was applied, and when it was not homogeneous, the Steel's multiple comparison was applied for control group and each test substance group.
For Histopathological examination, the grades were converted to numerical values, and the Mann-Whitney U test was applied to compare between the control and each test substance group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mortality at 1000 mg/kg bw/day: 5 males; at and above 250 mg/kg bw/day: test substance mixed feces from day 2 onwards
Mortality:
mortality observed, treatment-related
Description (incidence):
mortality at 1000 mg/kg bw/day: 5 males; at and above 250 mg/kg bw/day: test substance mixed feces from day 2 onwards
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose males: decreased bw gain from day 8 to 22; mid dose males showed increased bw gain from day 22 onwards.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
low values were noted in males that died; high values were noted in mid dose males from days 15 to 27 and in males of the 500 mg/kg bw/day group on days 8 to 27
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
high dose: changes in reticulocyte (m, f) after 4 and 6 weeks; changes in neutrophils and lymphocyte numbers (m, f) after 4 weeks and 6 weeks (m); in high dose females recovery group: changes in erythrocyte number, Hb and MCV.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
high dose females: increased A/G ratio; high dose males (1000/500 and 500): decreased plasma Na, K and Cl; Mid and high dose females decreased plasma Na, K and Cl; high dose recovery females: decreased plasma tot prot, Alb and glucose
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
in males (all dose groups); low values of protein; in the 1000 and 1000/500 groups: increased volume, low osmolality and spec gravity, color change (pale yellow); K and Cl increased in females. Mid dose males: decreased osmolality and spec gravidity
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
in FOB
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
mid and high dose rats: increased liver weight (abs., m, f; rel only in high dose), increased adrenal weight (abs., m, f; rel m and high dose f); high dose males: decreased prostate weight (abs., rel); after recovery period decreased spleen weight (m)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
in dead animals: dilatation of stomach (3m), reddish-brown contents in stomach to ileum (3m), reddish-brown contents in cecum (2m), reddish-brown contents in colon (1m); enlargement adrenals (4m)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
changes were noted in the stomach, jejenum, ileum, mesenteric lymph nodes, kidneys and adrenals of males and females, in the duodenum of males and in the spleen of females.
Details on results:
CLINICAL SIGNS AND MORTALITY: 5 high dose males died between days 8 to 25. These males showed decreased food consumption and body weight gain.
The staining of the feces is considered to be attributable to the color of the test substance.

FOOD CONSUMPTION: dereased food intake in males who died. Increased food intake was observed in mid dose males (days 15-27) and males dosed 500 mg/kg bw/day (days 8-27)

HAEMATOLOGY: In high dose males (1000/500 and 500) and high dose females increased levels of abs and rel reticulocyte were observed. At the end of the recovery period a decrease in reticulocyte (abs and rel) was observed in males dosed 500 mg/kg bw/day. In males dosed 500 mg/kg bw/day and in high dose females, neutrophil numbers (abs and rel) were increased and rel lymphocyte numbers were decreased. Neutrophil numbers were still decreased in males at the end of the recovery period. In high dose females, increases in erythrocyte number and Hb and decrease in MCV were observed at the end of the recovery (but not during the dosing period).

ORGAN WEIGHTS: In mid and high dose males and females absolute liver weight was increased, whereas relative liver weight was increased in high dose rats. Absolute adrenal weight was increased in mid and high dose males and females; relative adrenal weight was increased in mid and high dose males and in high dose females.

HISTOPATHOLOGY: NON-NEOPLASTIC
Stomach: at and above 250 mg/kg bw/day hyperplasia of the squamous epithelium of the limiting ridge of forestomach was observed, with increased severity in high dose females. In high dose females and in males dosed at and above 250 mg/kg bw/day increase in the globule leukocyte was noted.
Duodenum: in one high dose male necrosis of the mucosal epithelium was observed.
Jejenum: in all dose groups pigment-laden macrophages were noted.
Ileum: In high dose males (1000/500 mg/kg bw/day) and high dose females pigment-laden macrophages in the lamina propria and Peyer's patches were noted.
Mesenteric lymph node: pigment-laden macrophages were observed in females of all dose groups and in males of dose groups at and above 250 mg/kg bw/day.
Kidneys: In males and females dosed at and above 250 mg/kg bw/day hypertrophy of the epithelial cells in the distal renal tubule was observed and hypertrophy of the epithelial cells in the collecting tubule was noted in high dose females and in males dosed at and above 250 mg/kg bw/day.
Adrenals: In males of the 500 mg/kg bw/day group and above and in high dose females vacuolation of the zona fasciculata cell of the cortex was increased, with increased severity in males dosed 1000/500 mg/kg bw/day. In males of the 1000/500 mg/kg bw/day group hypertrophy of the zona glomerulosa and fasciculata cells of the cortex was observed, including observation of severe hypertrophy of the glomerulosa cells.
Spleen: in high dose females an increase of extramedullary hematopoiesis was noted.

At the end of the recovery period:
Jejenum: pigment-laden macrophages in the lamina propria were observed in high dose rats with an incidence comparable to the incidence observed at the end of the dosing period.
Mesenteric lymph node: pigment-laden macrophages were observed in high dose rats with an incidence comparable to the incidence observed at the end of the dosing period.
Adrenals: Vacuolation of the zona fasciculata cells of the cortex was increased in high dose animals, with an incidence comparable to that observed at the end of the dosing period.

Dead animals: Hyperplasia of the squamous epithelium of the limiting ridge of the forestomach (2 males), pigment-laden macrophages in mesenteric lymph nodes (5 males), hypertrophy of the distal and collecting tubule epithelial cell in the kidneys (5 males), hypertrophy of the zona glomerulosa cell of the adrenal cortex (3 males), atrophy of the thymus and splenic white pulp and a decrease of vacuolation of the zona fasciculata cell of the adrenal cortex were noted.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
in an OECD 407 study, rats were orally exposed to E-BW102. Mortality was observed in the high dose male group (1000 mg/kg bw/day group). In surviving animals, at and above 250 mg/kg bw/day, hyperplasia of the squamous epithelium of the limiting ridge of the forestomach, increased number of globule leukocyte in the glandular stomach and hypertrophy of the epithelial cells in the distal renal tubule and collecting tubule was observed in males and females. Prostate weight of males dosed 1000/500 mg/kg bw/day was decreased, and at and above 250 mg/kg bw/day increases in liver and adrenal weight was observed in males and females. Plasma Na, Cl and K were decreased in high dose rats (500 mg/kg bw/day and above) with decreased plasma Na in females at and above 250 mg/kg bw/day. Numbers of reticulocyte and neutrophils were increased, and relative lymphocyte number was decreased in high dose rats. Based on effects observed at and above 250 mg/kg bw/day, the NOAEL in this study is set at 50 mg/kg bw/day.
Executive summary:

E-BW102 was administered to rats (5/sex/dose) by oral gavage at dose levels of 0, 50, 250 and 1000 mg/kg bw/day during 28 days to assess the toxicological effects. A 14 -day recovery period (5/sex) was set for the control and high dose groups to study reversibility of effects. As 5 high dose group males died by day 26, the high dose was changed to 500 mg/kg bw/day for males on day 26 and thereafter (1000/500 mg/kg bw/day group). For males, another control group and a 500 mg/kg bw/day dose group were included.

In males that died, body weight gain and food consumption was decreased. Necropsy of the high dose group males that died, histopathological observations included atrophy of thymus and splenic white pulp, decreased vacuolation of the zona fasciculata cell of the adrenal cortex, hyperplasia of the squamous cell epithelium of the limiting ridge of the forestomach, pigment-laden macrophages in mesenteric lymph nodes and hypertrophy in kidneys (epithelial cells in the distal tubule and collecting tubule and adrenals (zona glomerulosa cells of the cortex).

In surviving animals, at and above 250 mg/kg bw/day, hyperplasia of the squamous epithelium of the limiting ridge of the forestomach, increased number of globule leukocyte in the glandular stomach and hypertrophy of the epithelial cells in the distal renal tubule and collecting tubule was observed in males and females. Prostate weight of males dosed 1000/500 mg/kg bw/day was decreased, and at and above 250 mg/kg bw/day increases in liver and adrenal weight was observed in males and females. Plasma Na, Cl and K were decreased in high dose rats (500 mg/kg bw/day and above) with decreased plasma Na in females at and above 250 mg/kg bw/day. Numbers of reticulocyte and neutrophils were increased, and relative lymphocyte number was decreased in high dose rats. Based on these observations, the NOAEL in this study is 50 mg/kg bw/day.