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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:
16 April to 23 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken at a GLP accredited laboratory to internationally accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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 Guidelines Relating to New Chemical Substances (Notification No. 700 of KANPOGYO, No. 1039 of YAKUHATSU, No. 1014 of 61KIKYOKU, December 5, 1986)
Deviations:
no
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): BPS-MAE
- Physical state: White powder
- Analytical purity: 99.2%
- Lot/batch No.: G130131
- Stability under test conditions: Confirmed stable
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Crj:CD (SD) IGS
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier.
- Age at study initiation: 6 weeks
- Weight at study initiation: males, 192.8 to 225.5 g; Females, 136.2 to 164.4 g.
- Fasting period before study: No
- Housing: 2 rats were housed together in a stainless-steel wire bottom cages (W260 x H200 x D380 mm).
- Diet: The rats were allowed free access to pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan).
- Water: The rats were provided with well water supplemented with sodium hypochlorite (approximately 2 ppm) ad libitum from an automatic dispenser.
- Acclimation period: males, 7 days; females, 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 27°C
- Humidity: 55 ± 10%
- Air changes: 13 to 15 air changes per hour.
- Photoperiod: 12-hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): 1 or 2 times a week.
- Mixing appropriate amounts with (Type of food): pellets, CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan
- Storage temperature of food: low temperature.

VEHICLE
- Justification for use and choice of vehicle: CMC is a commonly used vehicle.
- Concentration in vehicle: 4, 20 and 100 mg/ml
- Amount of vehicle (if gavage): 0.5% solution.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg
Basis:
other: Gavage
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
other: Gavage
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
other: Gavage
No. of animals per sex per dose:
Male: 12 animals at 0 mg/kg bw/day
Male: 6 animals at 40 mg/kg bw/day
Male: 6 animals at 200 mg/kg bw/day
Male: 6 animals at 1000 mg/kg bw/day
Female: 12 animals at 0 mg/kg bw/day
Female: 6 animals at 40 mg/kg bw/day
Female: 6 animals at 200 mg/kg bw/day
Female: 6 animals at 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The results of a former 7-Day Repeated Dose Toxicity Study of BPS-MAE in Rats [PPL Study No. P030158 (non-GLP), dose levels: 0, 50, 100, 500 and 1000 mg/kg, 3 males and females/group] did not reveal test substance-related changes in any test substance treatment group. Based on the above results, the high-dose level in this study was set at 1000 mg/kg, the maximum dose in the OECD testing
guidelines, and administration was expected to be possible for 28 days. The middle- and low-dose levels were set at 200 and 40 mglkg, respectively, as 5-fold dose decrements.

- Rationale for animal assignment (if not random): Group assignment was carried out by the stratified sequential randomization method on the basis of animal body weight on the day before the start of administration.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked were; During the recovery period, all the animals were observed for clinical signs and mortality
once a day.
(1) Functional observation (detailed clinical observation)
Detailed clinical observation was conducted once before the start of administration (the day of group assignment) and once weekly during the administration (4 hours after administration) and recovery periods. Observation of animals outside their home cages was conducted by investigators holding an animal in their hands and observing it, and this was followed by observation in an open field for 2-5 minutes.
A. Cage-side observation
Posture, convulsions, stereotypies, bizarre behavior, tremors
B. Handheld observation
Handling reactivity, abnormal vocalization, tremor, twitching, convulsion, respiration, salivation, lacrimation, pupil size, exophthalmos, ocular or nasal secretions, skin, piloerection, fur, mucous membranes, incontinent urination, muscle tone, body temperature
C. Open-field observation
Arousal, abnormal gait, stereotypies, bizarre behavior, ptosis, diarrhea, defecation, urination
(2) Sensory reactivity to stimuli
Six males and 6 females with low animal numbers in each group underwent the following tests in week 4 of administration and week 2 of the recovery period: auditory reactivity, visual reactivity, touch response, pain response, righting reflex, and pupil response.
(3) Grip strength measurement
Six males and 6 females with low animal numbers in each group underwent grip strength measurement in week 4 of administration and week 2 of the recovery period. The grip strengths of the foreleg and hind leg were measured twice each using a dynamometer (CPU gauge: Model 9502A, Aikoh Engineering Co., Ltd.), and the mean values were calculated.
(4) Motor activity measurement
Six males and 6 females with low animal numbers in each group underwent motor activity measurement in week 4 of administration and week 2 of the recovery period. The animals' motor activity in a polycarbonate cage (W265xH185xD425 mm) was measured individually using a movement analyzer (SCANET SV-10, Toyo Industry Co., Ltd.). Data were collected at 10-minute intervals with measurement duration of 1 hour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observation was conducted once before the start of administration (the day of group assignment) and once weekly during the administration (4 hours after administration) and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals' body weights were measured on the day of the start of administration (day 1), and once a week thereafter. In addition, their body weights were measured on the day of the final administration and at the end of the recovery period. Their final body weights were measured before necropsy on the days of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: When necropsy was conducted in the administration and recovery periods, about 2 mL of blood was collected from the posterior vena cava.
- Anaesthetic used for blood collection: Yes, sodium pentobarbital (30 mg!k:g),
- Animals fasted: Yes, for 18 hours or longer before blood sampling.
- How many animals: All animals sampled
- Parameters checked were; Leukocyte, Erythrocyte (RBC), Haemoglobin, Mean copuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet, Reticulocyte count, Differential leukocyte count, Prothrombin time and Activated partial prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: When necropsy was conducted in the administration and recovery periods, about 2 mL of blood was collected from the posterior vena cava.
- Animals fasted: Yes
- How many animals: All animals sampled
- Parameters checked in were;
Total protein (T.Protein) Biuret method
Albumin BeG method
A/G ratio Calculated from total protein and albumin
Total bilirubin (T.Bilirubin) Vanadate oxidation method
GOT UV-rate method
GPT UV-rate method
y-glutamyltranspeptidase (y-GTP) L-y~GIutamyl-3-hydroxymethyl-4-Nitroanilide reaction
Alkaline phosphatase (ALP) p-Nitrophenylphosphate reaction
Total cholesterol (T.Cholesterol) COD-HDAOS method
Triglycerides GPO-HDAOS method, glycerol-blanking method
Phospholipids Choline oxidase-DAOS method
Glucose Hexokinase-G-6-PDH method
Blood urea nitrogen (BUN) Urease-GLDH method
Creatinine Jaffe method
Inorganic phosphorus (IP) PNP-XOD method
Calcium (Ca) MXB method

URINALYSIS: Yes
- Time schedule for collection of urine: In week 4 of the administration period, 6 animals with small animal numbers in each group were housed individually in metabolic cages. Fresh urine samples were collected in the morning (before administration) under conditions of fasting and free access to water. Urinalysis was not carried out in week 2 of the recovery period, because the results in week 4 of the administration period revealed no test substance-related changes in any test substance administraition
group.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked were; pH, Protein, Glucose, Ketone bodyu, Bilirubin, Occult blood and Urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during detailed clinical observations,
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity, grip strength and motor activity.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; In the organ weight measurement, higher absolute and relative kidney weights were observed in the females in the 1000 mg/kg group, and these changes were considered to be related to administration of the test substance. They were considered to have little toxicological significance, however, since there was no histopathological change in the kidneys.
The higher absolute and relative kidney weights in the females in the 1000 mg/kg group had returned to normal at the end of the 14-day recovery period, suggesting good reversibility.
HISTOPATHOLOGY: Yes; Slight cellular infiltration of the lymphocytes in the prostate was observed in the males in the control group, slight focal fibrosis in the liver was observed in the females in the control group, and slight focal hemorrhage in the lungs was observed in the males in the 1000 mg/kg group. These findings were considered to be unrelated to administration of the test substance, however, since they were
observed in the control group or were spontaneous changes observed in normal rats.
Examination revealed no histopathlogical abnormality of the kidneys in the females in the 200 mg/kg group, in which a higher relative kidney weight was seen.

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):
effects observed, treatment-related
Food efficiency:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
Organ weight measurement revealed higher absolute and relative kidney weights in the females in the 1000 mg/kg group.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical observations:
There were no treatment-related deaths or other differences from control for other parameters measured, including clinical appearance, body weight, food and water consumption that were considered to be related to treatment.

There were no differences or abnormalities noted in the animals during the functional observation tests, and assessment of sensory reactivity to stimuli, grip strength and of motor activity, respectively.

Laboratory findings:
Hematology revealed statistically significant shortened activated partial thromboplastin time in males treated at 200 and 1000 mg/kg bw/d and in females at 40 mg/kg bw/d, in addition in males increased MCV and MCH at 40 mg/kg bw/d. At the end of recovery a lower basophils count was observed for the 1000 mg/kg bw/d males.

There were no test substance-related effects on clinical biochemistry and urinalysis data which could be considered of biological significance.

Effects in organs:
At 1000 mg/kg bw/d female rats showed an increase in both absolute and relative kidney weight when compared with controls. A higher relative kidney weight was observed in males at 200 mg/kg bw/d. At the end of recovery a higher absolute spleen weight and a lower relative kidney weight were observed in females at 1000 mg/kg bw/d.

No macroscopic or microscopic findings attributed to administration of the test substance were noted.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: clinical signs
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: clinical signs
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the above results, the no-observed-effect level (NOEL) under the conditions of this study was considered to be greater than 1000 mg/kg/day for males and 200 mg/kg/day for females. Good recovery from these changes was observed.