Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 21 - September 30, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
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: Reddish brown powder
- Storage condition of test material: Room temperature (actual temperature: 19.3 to 22.7°C), dark place
- Stability under storage conditions: Not indicated

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories Japan, Inc. (Hino breeding center)
- Age at study initiation: Young adult animals (9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (200.6-229.7g).
- Housing: Individual 1 animal per cage in labeled Stainless-steel cages (226x346x198mm).
- Diet (e.g. ad libitum): Free access to pellet diet (CRF-1, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): Free access to well water.
- Acclimation period: The acclimatization period was 6 days for experiement 1 and 8 days for experiment 2.
- Health inspection: During the acclimation period, the animals were examined for clinical signs once daily and body weights twice to confirm their health status.

Data for each lot of diet generated by Japan Food Research Laboratories was provided by Oriental Yeast Co., Ltd., and the contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.
The water was analyzed every 6 months at Nichigo Kyushu Co., Ltd. in compliance with the Water Quality Standard (Waterworks Law) of MHLW, Japan.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.6 – 24.6
- Humidity (%): 44.9 - 61.3
All housing equipment was sterilized by autoclaving after washing with water. The floor of the animal room was cleaned and wiped every day with a disinfectant-soaked mop. NaClO was used as the disinfectants on every day.
- Air changes (per hr): 10 to 20 per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 03, 2010 to August 19, 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:

GAVAGE METHOD: disposable syringe

Frequency: single dosage, on Day 1.

VEHICLE
- Justification for choice of vehicle: No data.

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg (25 mL/kg) body weight.

DOSAGE PREPARATION: The test substance was weighed and transferred to a measuring cylinder. Vehicle of about 80% of the preparation volume was added to the measuring cylinder and dissolved by ultrasonic irradiation. The solution was added up to the preparation volume and mixed upside down. The dosing solution was transferred into a brown glass container.

Doses:
2000 mg/kg body weight

No. of animals per sex per dose:
6 (2 groups of three females in a stepwise manner (experiment 1 and 2))
Control animals:
no
Details on study design:
Animals were fasted for approximately 18 to 19 hours from the evening on the day before the administration until 3 hours after administration of the test substance. Water was available ad libitum.

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality and clinical signs: Before dosing (Day 1) and at 30 minutes, 1, 3 and 5 hours postdose on the day of the administration and once daily for 14 days thereafter.
Body weights: All animals were subjected to the body weight measurement just before the administration (Day 1) and on Days 4, 8 and 15. Moreover, the body weight gain between each measurement day was calculated.
- Necropsy of survivors performed: At the end of the 15-day observation period, all animals were euthanized by exsanguination from the external iliac artery under intraperitoneal sodium pentobarbital (30 mg/kg) anesthesia and were examined macroscopically.
- Other examinations performed: none
Statistics:
No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No death was noted in any animal.
Clinical signs:
Test substance-mixed feaces was observed in all animals from 0.5 or 1 hour on day 1 (administration day) to Day 3 or 4 and chromaturia (reddish brown) was observed in all animals from Day 2 to Day 3 or 4 in experiment 1 and 2. Furthermore, soiled periproctal was observed in 1 animal on Day 3 and 4 in experiment 1. The test substance is a reddish brown-powder, therefore, the feaces and chromaturia were attributed to the test substance. In addition, soiled periproctal observed in 1 animal in experiment 1 was possibly a secondary effect associated with the feaces and chromaturia.
Body weight:
Although a decrease in the body weight was noted in 1 animal on day 4in experiment 1, an increase was observed thereafter. No abnormalities were noted in body weight gain in the remaining 2 animals in experiment 1 and any animal in experiment 2.
Gross pathology:
No abnormalities were noted in any animal.
Other findings:
None.

Any other information on results incl. tables

Deviations from the protocol:

In the protocol, the animals were to be fasted for approximately 18 hours predose, however, the animals were actually fasted for approximately 19 hours in experiment 1. The result of experiment 1 was comparable to that of experiment 2, in which the animals were fasted for approximately 18 hours. Therefore, the prolongation of the fasting time (1 hour) was considered to have no effect on the study result.

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an acute oral toxicity study performed according to OECD 423 and GLP, the LD50 value of E-BW102 in female rats was established to exceed 2000 mg/kg body weight.
According to Regulation (EC) No. 1272/2008, E-BW102 needs not to be classified for acute oral toxicity.