Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
december 2010 - april 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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
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-C104
- Physical state: blue colored powder
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: five years from shipment day (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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 9 weeks
- Weight at receipt (8 weeks old): 251.6 to 280.8 g for males and 164.6 to 193.3 g for females
- Housing: stainless-steel cages, except during gestation and lactation period (polymethylpentene cages). One male and one female were housed in a cage during the mating period. One dam and its litter were housed in a cage during the lactation period. As for the other periods, animals were housed individually. Bedding material: satirized wood chip
- 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:9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8° to 23.9°C
- Humidity (%): 52.0 to 79.6%
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: January 7,2011 To: April 18, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once every 5 or 7 days and stored in a cool place shielded from light. Dose volume 10 mL/kg.
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:
males: from 14 days before mating until necropsy through the mating period (42 days).
females: from 14 days before mating until day 4 of lactation through the mating and pregnancy periods and delivery.
recovery females (satellite group): for 42 days without mating.
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:
Each test group consisted of 12 males (including 5 males for recovery test) and 12 females (5 females each were added for recovery test in the control and 1000 mg/kg bw/day groups).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results obtained in "Fourteen days repeated oral dose toxicity study of E-C104 in rats (No. P101038; dose levels 0, 100, 300 and 1000 mg/kg bw, with 5 rats/sex/dose). As no toxicologically relevant effects were observed, the highest dose was set at 1000 mg/kg bw/day as stated in the guideline and two lower doses were established.
- 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
- Time schedule for examinations: males were weighed on days 1, 8, 15, 22, 29, 36, 42 and 43. The recovery males were also weighed on days 50 and 56. The satellite females were weighed in the same manner as the recovery males. The test females were weighed on days 1, 8 and 15, once every 7 days after initiation of copulation, and days 0, 7, 14 and 20 of gestation and days 0 and 4 of lactation for females after parturition. The final body weight was also measured on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was measured between days 1 to 8, 8 to 15, 22 to 29, 29 to 36 and 36 to 40 for test and recovery males and between days 43 to 50 and 50 to 54 for recovery males only. For females it was measured between days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56 for the satellite females. Food consumption of the test females was measured at the same time as the body weights. Food consumption was not measured for either sex during the mating period. Gross weight of each feeder was weighed, and the main daily food consumption of each animal was calculated for each period.

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: 5 animals with smaller animal numbers for the test males, all animals for the recovery males and satellite females, and 5 animals from the earlier parturition date with small numbers for the test females.

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 40
- Metabolism cages used for collection of urine: No, fresh urine samples excreted spontaneously on a washed try 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 6
- Dose groups that were examined: 5/sex/dose group
- 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 data of offspring were handled on a litter-basis. The body weight and food consumption of the female not pregnant, as well as the body weight after initiation of mating in the females failed mating were excluded from evaluation.
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):
no substance related mortality; substance-mixed feces (dark blue color) were noted in all animals of all dose groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
no substance related mortality; substance-mixed feces (dark blue color) were noted in all animals of all dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In high dose females body weight was higher on day 0 of lactation compared to the control group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
only incidental higher or lower values compared to controls were observed, which are considered not toxicologically relevant
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
high dose females: non-significant increases in counts of platelet, neutrophil and lymphocyte; high dose recovery males: increase in reticulocyte number; recovery high dose females, increase in neutrophils and decrease in lymphocytes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
high dose males and females : decrease in total bile acids, in total cholesterol and in ALT; high dose males: decreased plasma glucose level; high dose females: decreased plasma triglyceride level.
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):
no treatment related effects in FOB tests observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no treatment related effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Blue coloration was observed in the GI tract, mesenteric, submaxillary, cervical and bronchial lymph nodes, lung and kidney. No reversibility in coloration in lymph nodes and kidney in mid-and high dose.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
appearance of pigment-laden macrophages in the stomach of high dose males and in GI-tract, lung and lymph nodes in males and females of mid and high dose groups
Histopathological findings: neoplastic:
no effects observed
Details on results:
HISTOPATHOLOGY: NON-NEOPLASTIC: piment-laden macrophages were noted in the GI-tract, lungs and lymph nodes of mid and high dose rats and in the stomach of high dose male rats. In the absence of cytopathy in organs and tissues, this observation is considered not toxicologically relevant

Effect levels

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased number globule leukocyte in glandular stomach (m), changes in platelet count, neutrophil and lymphocyte numbers, total cholesterol, plasma glucose and triglyceride levels.

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-C104 for 28 days followed by a 14-day recovery period (control and high dose only) increase in globule leukocyte in the glandular stomach was noted in high dose males. In high dose animals effects were observed on haematology (platelet count, numbers of neutrophils and lymphocytes, total cholesterol, plasma glucose and plasma triglyceride levels). Pigment-laden macrophages were noted in the stomach of high dose males and in the GI-tract, lungs and lymph nodes of mid and high dose rats, but the toxicological relevance is unclear. Based on the effects observed in the high dose group (globule leucocyte number, haematology and clin biochem) , the NOAEL in this study is set at 250 mg/kg bw/day.
Executive summary:

E-C104 was repeatedly administered to rats (7/sex/dose) by oral gavage at dose levels 0, 50, 250 and 1000 mg/kg bw/day from 14 days before mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until day 4 of lactation in females to determine the effects of repeated exposure and reproductive and developmental toxicity (OECD 422). Reversibility was studied in satellite groups for controls and high dose (5/sex/dose). As a result of the test substance administration, substance-mixed feces (dark blue color) was noted in all dose groups. Haematological examination showed increases in platelet count and numbers of neutrophils and lymphocytes. At the end of the recovery period, increase in reticulocyte number was observed in males and females showed increase in neutrophils and decrease in lymphocytes. Clinical biochemistry showed in high dose animals decreased levels of total bile acids, total cholesterol and ALT. In high dose males decreased plasma glucose levels were noted, whereas in high dose females a decreased plasma triglyceride level was noted. Gross pathology showed blue coloration in the GI contents, mesenteric, submaxillary, cervical and bronchial lymph nodes, lung and kidneys (with no reversibility in coloration in the lymph nodes and kidney in the mid- and high dose). Histopathological examination showed pigment-laden macrophages in the GI-tract, mesenteric lymph nodes and lungs in rats dosed at and above 250 mg/kg bw/day, but the toxicological relevance is unclear. No reproduction toxicity and no developmental toxicity was observed.