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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - September 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to OECD 407 (2008) and GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (2003) of Manufacturing Industries Bureau, METI & NO. 031121002 of Environmental Health Department, MOE (2003)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): B508
- Substance type: Blue powder
- Physical state: solid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Hino breeding center, Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 134.1-154.9 g for males and 118.4-141.5 for females
- Housing: individually in stainless steel cage with wire-mesh floor
- Diet (e.g. ad libitum): MF pelleted diet ad libitum
- Water (e.g. ad libitum): chlorinated tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3-23.7
- Humidity (%): 51.2-63.3
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 15 To: September 26, 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 w/v% methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount was ground to a fine powder and mixed with the vehicle to prepare teh 10.0 w.v% for the highest dose group by stirring with a magnetic stirrer. The lower concentrations of 2.5 and 0.5 w/v% were diluted from the 10.0 w/v% solution. The solutions were prepared once per 6-10 days and stored in the dark in a refrigerator (not further specified).

VEHICLE
- Justification for use and choice of vehicle (if other than water): not indicated
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): Metolose SM100 lot no. 7035212, Shin-Etsu Chemical Industries
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in 10.0, 2.5 and 0.5 w/v% dose formulations of first and final preparation was within 92.0 to 108% of the nominal concentration as determined by HPLC with UV-detection at 345 nm.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in a 14-d range-finding study rats (number not specified) were exposed to 25, 250, 500 and 1000 mg/kg bw/d; no adverse effects attributable to the test substance wre noted in males and females of all dose groups.
- Post-exposure recovery period in satellite groups: an additional 14-day recovery group in control and at 1000 mg/kg bw/d

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times a day during the dosing period; twice daily during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before dosing, and once weekly at 2-5 hours after dosing during dosing and recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the test and on day 1, 3, 8, 12, 17, 21, 26 and 28 of dosing and on days 1, 5, 10 and 14 of recovery

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at completion of dosing period/recovery period
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes
- How many animals: all
- Parameters examined were those according to OECD 407, 2008.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at completion of dosing period/recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters examined were those according to OECD 407, 2008, except that bilirubin instead of bile acids were measured. However, as this was not required by OECD 407 (1995), the valid guideline at the time of the test, this is not considered a deviation of the guideline.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the dosing period/recovery period; urinary sediment was not examined at the end of recovery as no abnormalities were noted at the end of dosing
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were according to OECD 407, 2008.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 of dosing period for reflex test and grip strength and locomotor activity; as locomotor activity showed effects in males, also males from the recovery group were tested in week 2
- Dose groups that were examined: week 4 of dosing: all animals; week 2 of recovery: males from control and 1000 mg/kg bw/d
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to OECD 407, 2008
Organ weights: according to OECD 407, 2008, except that prostate + seminal vesicles with coagulating glands were not weighed. However, as this was not required by OECD 407 (1995), the valid guideline at the time of the test, this is not considered a deviation of the guideline.
HISTOPATHOLOGY: Yes, according to OECD 407, 2008
Other examinations:
none
Statistics:
Data regarding body weights, food intakes, haematological and clinical chemistry parameters, urine volume and urine osmotic pressure, organ weights, grip strength and locomotor activity count were analyzed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, the difference between the vehicle control group and each of the treatment groups was analyzed by Dunnett's test. If the variances were not homogeneous, the difference between the vehicle control group and each of the treatment groups was analyzed by the nonparametric Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred. No adverse effects were seen at all doses. Dark indigo stool was continuously observed in all treated animals during the dosing period and up to day 4 of the recovery period.

BODY WEIGHT AND WEIGHT GAIN
No effects observed.

FOOD CONSUMPTION
No effects observed.

HAEMATOLOGY
Red blood cell count was statistically significantly decreased in males at 50 and 1000 mg/kg bw/d (both 94% of control) at the end of dosing. The ratio of eosinophils was statistcally significantly in males at 1000 mg/kg bw/d (185% of control) and activated partial thromboplastin time was statistically significantly extended in females at 1000 mg/kg bw/d (105% of control) at the end of the recovery period. As these changes were slight, not seen in the other sex and within the range of historical data in our lab (specified in the report), these effects are not considered to be toxicologically relevant for the test substance.

CLINICAL CHEMISTRY
Glucose was statistically significantly decreased in males at 250 and 1000 mg/kg bw/d at the end of dosing (85 and 86% of control, resp.) and in males at 1000 mg/kg bw/d at the end of recovery (90% of control). Total protein was statistically significantly increased in females at 1000 mg/kg bw/d at the end of dosing (107% of control). Total bilirubin was statistically significantly decreased in females at 1000 mg/kg bw/d at the end of recovery (78% of control). As these changes were slight, no correlating histopathological effects were observed, changes not seen in the other sex and within the range of historical data in our lab (specified in report), these effects are not considered to be toxicologically relevant.

URINALYSIS
No abnormalities observed at all dose levels.

NEUROBEHAVIOUR
No abnormalities for reflex behaviour and grip strength were noted. The locomotor activity was statistically significantly decreased in the 10-20 min interval in males at 1000 mg/kg bw/d However, because there were no abnormalities at other intervals and the total of 60 minutes, these changes were not considered to be attributable to administration of the test substance.

ORGAN WEIGHTS
A statistically significant increase in relative heart weight of males at 250 and 1000 mg/kg bw/d ((both 109% of control) and relative kidney weight of males at 1000 mg/kg bw/d (111% of control) at the end of dosing was observed. As these changes were not accompanied by changes in absolute weight, no histopathological effects were observed and the changes were within the historical control data, they were not considered to be toxicologically relevant.

GROSS PATHOLOGY
No treatment-related adverse effects observed. Dark-blueish contents of the intestinal tract in animals at 250 and 1000 mg/kg bw/d were seen due to the blue colour of the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related effects observed.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at highest dose tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In the absence of treatment-related adverse effects, the NOAEL of B508 is 1000 mg/kg bw/d after oral dosing for 28 days.
Executive summary:

Rats were given 50, 250 or 1000 mg/kg bw/d of B508 in 0.5 w/v% methylcellylose by gavage according to OECD 407 (2008) and GLP principles.No mortality and no adverse clinical signs occurred. No effects were observed on body weight, food consumption, functional behaviour, haematology and clinical chemistry parameters, urinalysis, organ weights, gross- and histopathology. Therefore, the NOAEL was established to be >= 1000 mg/kg bw/d.