Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
december 2010-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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
E-BK105
IUPAC Name:
E-BK105
Details on test material:
- Name of test material (as cited in study report): E-BK105
- Physical state: black colored powder
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: five years from shipment (November 4, 2010)
- 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 or test system 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): 115.7 to 137.5 g for males and 93.1 to 115.2 g for females
- Housing: stainless-steel cages
- 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): 22.4° to 23.9°C
- Humidity (%): 51.2 to 62.1%
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: January 18, 2011 To: March 1, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once every 6 or 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, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5; for the control and 1000 mg/kg bw 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 days repeated dose toxicity study of E-BK105 in rats (No. P101032; dose levels 0, 100, 300 and 1000 mg/kg bw, with 5 rats/sex/dose). Low body weight gain or decreased body weight were noted on days 4 to 7 in 2 high dose males accompanied by low food intake. In addition, low values in the erythrocyte count and haematocrit and low values of potassium in females at and above 300 mg/kg bw/day, high MCHC values and low sodium values in high dose males and females, a high glucose value in high dose females, high adrenal weight and low seminal weight in high dose males. in addition, black feces were noted in males and females at and above 300 mg/kg bw/day. From these results the high dose level in the main study was set at 1000 mg/kg bw/day in which several toxicological changes were presumed to be induced, and subsequent doses were set at 250 and 50 mg/kg bw/day.
- 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 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
- All animals 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 the day of the scheduled necropsy.

FOOD CONSUMPTION:
- For all animals, gross weight of each feeder was weighed. 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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- 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
- 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.

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).
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%) for grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weight. 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 to compare each test substance group to the control group.
For Histopathological examination, the grades were converted to numerical values, and the Mann-Whitney U test was applied to compare the control and each test substance group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
from day 2 until 2 days after the final dosing black feces (test substance) were noted at 50 mg/kg bw/day (2m, 1f) and all rats in the higher dose groups
Mortality:
mortality observed, treatment-related
Description (incidence):
from day 2 until 2 days after the final dosing black feces (test substance) were noted at 50 mg/kg bw/day (2m, 1f) and all rats in the higher dose groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
in high dose females body weight was increased from day 22 onwards
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In high dose animals increased food consumption was observed on days 15 to 27 (males) and from day 8 in females
Food efficiency:
not examined
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):
in high dose animals increased numbers (abs and rel) of reticulocytes (stat. sign in males)
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Description (incidence and severity):
by means of reagent strip method
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):
High dose males showed an increased liver weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All animals showed black coloration of kidneys (and still present at the end of the recovery period in high dose rats (2m, 4f). In high dose animals black intestinal contents in the jejenum (1m), the ileum (2m, 2f) and the cecum (4m) were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Squamous cell hyperplasia in(proventricular border) in mid dose (1m, 1f) and all high dose rats; increased globule leukocyte (glandular stomach) in high dose (4m, 3f). Hypertrophy of kidney distal tubular epithelium in mid (2m, 1f) and all high dose rats.
Details on results:
CLINICAL SIGNS: the staining of the feces is considered to be attributable to the color of the test substance.

HAEMATOLOGY: observed shorter PT and APTT values in high dose males are considered not toxicologically relevant. Slight differences in blood cell counts at the end of the recovery period were observed in high dose rats; in the absence of effects during the dosing period, these effects are not considered for risk assesment.

CLINICAL CHEMISTRY: In high dose females increase in total cholesterol was observed at the end of the recovery period. In the abseence of a similar effect during the dosing period, this effect is not considered for risk assessment.

ORGAN WEIGHTS: Observed increased thymus weight at the end of the recovery period is considered not relevant for risk assessment, as this effect was not observed at the end of the dosing period.

HISTOPATHOLOGY: NON-NEOPLASTIC
Squamous cell hyperplasia in stomach (proventricular border) in mid dose (1m, 1f) and all high dose rats; increased globule leukocyte (glandular stomach) in high dose (4m, 3f). Hypertrophy of kidney distal tubular epithelium in mid (2m, 1f) and all high dose rats and hypertrophy of the kidney collecting duct in mid (1m) and high dose (5m, 4f) rats.
At the end of the recovery period, in high dose rats slight hypertrophy of the collecting duct epithelium was noted (4m, 3f), and slight hypertrophy of the distal tubular epithelium (3m, 1f). In the glandular stomach a slight increase of globule leucocyte in high dose rats (1m, 1f), and slight squamous cell hyperplasia was noted in the proventriculus border in 1 female.

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 rats orally exposed to 0, 50, 250 or 1000 mg/kg bw/day E-BK105 for 28 days followed by a 14-day recovery period (control and high dose only) slight squamous cell hyperplasia at the provericulus border of the stomach was observed at the end of the exposure period in mid and high dose animals, accompanied by an increase of globule leucocytes in the glandular stomach in high dose animals. In kidneys of mid and high dose rats slight hypertrophy of the distal tubular epithelium was observed, together with slight hypertrophy of the collecting tubule epithelium in mid dose males and in high dose males and females. In addition, low plasma sodium values (mid dose females and high dose males and females), low plasma potassium values (high dose females) and low values of plasma chloride (high dose males and females) were noted. At the end of the recovery period, histopathological changes were still present, as wel as decreased plasma sodium and chloride levels . Based on these effects, the NOAEL in this study is set at 50 mg/kg bw/day.
Executive summary:

E-BK105 was repeatedly administered by oral gavage at dose levels of 0, 50, 250 and 1000 mg/kg bw/day to male and female rats for 28 days to assess the toxicological effects. A 14 -day recovery period was set for the control and high dose group to study reversibility of effects. slight squamous cell hyperplasia at the provericulus border of the stomach was observed in mid and high dose animals, accompanied by an increase of globule leucocytes in the glandular stomach in high dose animals. In kidneys of mid and high dose rats slight hypertrophy of the distal tubular epithelium was observed, together with slight hypertrophy of the collecting tubule epithelium in mid dose males and and in high dose males and females. In addition, low plasma sodium values (mid dose females and high dose males and females), low plasma potassium values (high dose females) and low values of plasma chloride (high dose males and females) were noted. Decreased plasma sodium and chloride levels were still present at the end of the 14-day recovery period. Based on these effects, the NOAEL in this study is set at 50 mg/kg bw/day.