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-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.
Cross-reference
Reason / purpose:
reference to same study

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-Y110
- Physical state: orange colored powder
- 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): 261.1 to 294.4 g for males and 170.4 to 204.8 g for females
- Housing: stainless-steel cages, except during gestation and lactation period for females (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: 7 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 5, 2011 To: March 2, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once every 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:
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-Y110 in rats (No. P101036; 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 satelite 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
- 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
- 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 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 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; chromaturia (yellow) were noted in high dose animals from day 2 until final dosing day (males) or until two days after final dosing day (females).
Mortality:
mortality observed, treatment-related
Description (incidence):
no substance related mortality; chromaturia (yellow) were noted in high dose animals from day 2 until final dosing day (males) or until two days after final dosing day (females).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
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 males an increase in eosinophil ratio was observed at the end of the dosing period. In high dose recovery males, a decrease in lymphocyte ratio was observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
in males of the recovery group, increased levels of ALP and total bile acids were observed; in females of the recovery group, increased levels of AST, ALT and total bile acids were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
by means of reagent strip method
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In high dose females motor activity, as determined by FOB, was increased
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period: in high dose animals orange pigment was observed in the granulocyte of the villus of the duodenum (1 male), in the villus of the jejenum (3m, 2f) and in the villus of the ileum (1m, 2f).
Histopathological findings: neoplastic:
no effects observed
Details on results:
Haematology:
In high dose males of the recovery group, a decrease in lymphocyte ratio was observed. As no significant effect was observed in the leucocyte count, and this effect was not observed at the end of the dosing period, it is not considered toxicologically relevant.

Clinical Biochemistry: In high dose males of the recovery group increased levels of ALP and total bile acids were observed; in high dose females of the recovery group increased levels of AST, ALP and total bile acids were observed. As these effects were confined to the end of the recovery period only, these effects are 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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the observed increase in motor activity in high dose females, increased eosinophil ratio in high dose males and in some high dose animals orange pigment in granulocytes of villus of the duodenum, villus of the jejenum and in the villus of the ileum, the NOAEL for repeated dose toxicity is set at 250 mg/kg bw/day.
Executive summary:

E-Y110 was repeatedly administered by oral gavage at dose levels of 0, 50, 250 and 1000 mg/kg bw/day to rats to determine the effects of repeated toxicity and reproductive and developmental toxicity as well as reversibility of changes. Males and satellite females received the test substance from day 14 before mating through mating for 42 days, whereas females received the test substance 14 days before mating, through gestation and parturition until day 4 of lactation. Repeated dose toxicity was confined to the high dose group. In high dose females an increase in motor activity was observed in the FOB, and in high dose males an increase in eosinophil ratio. In some high dose animals, histopathological examination of the GI tract showed orange pigment in granulocytes of villus of the duodenum, villus of the jejenum and in villus of the ileum. Based on these observations, the NOAEL for repeated dose toxicity is set at 250 mg/kg bw/day.