Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 February 2016 - 5 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Storage conditions: Room temperature (actual values: 18.1 to 24°C)
Appearance: Blue powder
Purity: At least 85.5%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Sprague-Dawley strain SPF rats [Crl:CD(SD), Atsugi Breeding Center, Charles River Laboratories Japan, Inc.]
Sex:
male/female
Details on test animals and environmental conditions:
Rats were selected as the toxicity study guidelines require testing in rodents. The strain of rats employed in this study was chosen since it is widely used in general toxicity studies and reproductive/developmental toxicity studies, its characteristics are well known, and ample background data are available.
- Source: Atsugi Breeding Center
- Females (if applicable) nulliparous and non-pregnant: [no]
- Age at study initiation: 12 weeks
- Weight at study initiation: 403 to 463 g (mean body weight: 425 g) for males and 227 to 285 g (mean body weight: 253 g) for females,

- Housing: plastic cages in groups of 2
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD QUALITY: NMF (radiation-sterilized, Oriental Yeast Co., Ltd., Lot Nos.: 150910, 151016 and 151125

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 to 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Dose volume was set at 10.0 mL/kg body weight and the dosing formulation was administered by gavage using a stomach tube
Vehicle:
methylcellulose
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: test article was weighed, suspended in the vehicler, and the vehicle added to make the specified volume to prepare the 5, 25 and 100 mg/mL test suspensions (low, middle and high-dose formulations). The test suspensions were prepared at the time of use and not preserved.
- VEHICLE : methylcellulose
- Concentration in vehicle: 5, 25 and 100 mg/mL
- Amount of vehicle (if gavage): 10.0 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
spectrophotometry.
Duration of treatment / exposure:
42 days for males (14 days prior to mating and 28 days after the start of mating), 50 to 56 days for females in the mating group (14 days prior to mating and throughout the mating and gestation periods until day 13 of lactation), 42 days for the not delivered female (Animal No.: 2102) and 42 days for females in the non-mated group
Frequency of treatment:
once every day (7 times/week)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 animals of each sex for the mating group and 10 females each for the control group and high dose group for the non-mated group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of the previously conducted study, “A 14-day oral gavage toxicity study of B331 in rats (dose range finding study), dose levels: 50, 250 and 1000 mg/kg/day, Study No.: C-B765”. In that study, no deaths occurred and there were no toxicological effects from administration of the test article on any examination item in any animal. Therefore, in this study, 1000 mg/kg, which corresponds to the recommended highest dose in the Toxicity Test Guideline, was selected as the highest dose like the dose range finding study, and 250 and 50 mg/kg were selected as the middle and low doses, respectively, divided by 4 or 5.

- Rationale for animal assignment (if not random): block placement method using a computer so that group mean body weight was comparable among groups. Test groups and individual animal numbers were assigned at random. Group allocation was done on the day before the first day of administration
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
clinical signs including mortality, external appearance, posture, behavior and excrement 3 times a day, before dosing, immediately after and 1-3 hours after dosing during the administration period (but twice a day, before dosing and immediately after dosing, on the days for detailed clinical observation), and once a day (in the morning) during the recovery period. On the day of necropsy, clinical observation was performed once before carrying the animals from the animal room for necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: days 1, 3, 6, 13 and 20 of acclimation. Males in the mating group and females in the non-mated group were weighed on days 1, 8, 15, 22, 29, 36 and 42 of administration and on the day of necropsy, these animals in the recovery group on days 1, 8 and 14 of recovery and the day of necropsy additionally. Females in the mating group were weighed on days 1, 8 and 15 of administration and on GDs 0, 7, 14 and 20 and on LDs 0, 4, 7 and 13 and the day 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: The amount of food remaining was measured for all animals

HAEMATOLOGY: Yes (table 2)
- Anaesthetic used for blood collection: Yes: isoflurane
- Animals fasted: Yes: overnight (for 16 to 20 hours)
- How many animals: all animals

CLINICAL CHEMISTRY: Yes (table 3)
- Time schedule for collection of blood: final administration or from the day 14 of recovery
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes (table 1)
- final week of administration (days 36 and 37 in week 6 of administration) and in the final week of recovery (days 8 and 9 in week 2 of recovery). The examination during the administration period was conducted after dosing.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes 4 hour samples but free access to water. 20-hour urine was collected with free access to feed and water

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once a week
- Dose groups that were examined: all
- Battery of functions tested: grip strength and motor activity:

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
Vaginal Smear Examination, Mating, Delivery and Nursing,
Statistics:
Bartlett’s test for homogeneity of variance, Dunnett’s test, Steel test, F test, Student's t-test, Aspin-Welch's t-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The males and females in the 250 and 1000 mg/kg group showed colored feces (blue) from the day following the start of administration throughout the administration period including the gestation and lactation periods.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in any group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes that are considered to be related to administration of the test article during the administration period including the gestation and lactation periods. Females in the 50 mg/kg group showed a significantly high value in the body weight gain during the lactation period; however, it was not a dose-dependent change and thus judged to be an incidental significant difference.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes that are considered to be related to administration of the test article during the administration period including the gestation and lactation periods.
The females in the 50 mg/kg mating group showed significantly high values in the food consumption on day 2 of administration, on LD 13 and in the total amount of food consumption during the pre-mating administration period; however, they were judged to be incidental significant changes since they are not dose-related. The females in the non-mated group in the 1000 mg/kg group showed a significantly low value on day 42 of administration; however, it was judged to be an incidental change since it was a change only at one time point, there were no abnormalities in the total amount of food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
no changes that are considered to be related to administration of the test article.
statistically significant low values in the mean corpuscular hemoglobin concentration (MCHC) were recorded at 50 mg/kg and above, but they were judged to be incidental since there were no abnormalities in any other erythrocyte parameters and they were within the normal range of the historical background data of the test facility.
In females in the mating group, there was a significantly high value in the monocyte count (MONO) in the differential white blood cell count at 50 mg/kg; however, it was not a dose-related change and thus judged to be an incidental significant difference.
In females in the non-mated group, a significantly low value in the reticulocyte count (RETIC) was recorded in the 1000 mg/kg group; however, it was judged to be an incidental significant difference since there were no abnormalities in any other erythrocyte parameter and the change was within the normal range of the historical background data of the test facility
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no changes that are considered to be related to administration of the test article. In females in the mating group, a significantly low value in total bilirubin (T-BIL) was recorded in the 50 mg/kg group; however, it was not a dose-related change and thus judged to be an incidental significant difference. In females in the non-mated group, a significantly low value in the ALP was recorded in the 1000 mg/kg group; however, it was judged to be an incidental significant difference since the change was within the normal range of the historical background data of the test facility
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no animals that showed abnormalities in the qualitative items, and there were no statistically significant differences in the urine volume between the control group and the any test article administration group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes that are considered to be related to administration of the test article in the auditory response, approach response, touch response, tail pinch response, pupillary reflex or aerial righting reflex in any animal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In the weight of the thyroid and in the weight of the reproductive organs in males, there were no statistically significant differences between the control group and any test article administration group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No animals showed abnormalities that are considered to be related to administration of the test article.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no changes that are considered to be related to administration of the test article. Males in the 250 mg/kg group showed a significantly low value at 20-30 minutes, but it was judged to be an incidental significant difference since it was not a dose-related change.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No animals showed abnormalities that are considered to be related to administration of the test article.
As a result of the examination of the stomach in the control and 1000 mg/kg groups, erosion/ulcer in the glandular stomach was observed in 2/5 males at 1000 mg/kg. For the incidence of this change, there was no clear difference between the control group and the 1000 mg/kg group in females. However, in order to clarify its relationship to administration of the test article, examination was done for the 50 and 250 mg/kg groups and the recovery groups. In the results, since it became clear that the incidence of the occurrence of this change had no
dose-relationship, the change was judged finally to be incidental. In the undelivered female in the 50 mg/kg groups (Animal No.: 2102) on GD 25, hemorrhage and cell infiltration of the endometrium were observed at the left uterine horn. These changes were considered to be due to the exfoliation of conceptus, and the reason of non-delivery was unclear.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and any test article administration group in:
- the index of animals with abnormal estrous cycle, in the number of estruses or sexual cycle (estrous cycle).
- Mating and Fertility
- Delivery Findings, Delivery and nursing condition
- Anogenital Distance (AGD) of Liveborns
- Viability of Pups
- Sex Ratio of Pups
- Body Weight of Pups
- Necropsy of Dead Pups
- The Number of Nipple/Areolae of Male Pups
- Necropsy of Pups on PND 13
- Thyroid Weight of Pups
Details on results:
Based on these results, there were no toxic changes that are considered to be caused by administration of the test article by repeated oral administrations of B331 under the conditions of this study.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
repeated administrations
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
neuropathology
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive developmental toxicity
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results, there were no toxic changes that are considered to be caused by administration of the test article by repeated oral administrations of B331 under the conditions of this study. Therefore, it was judged that the no observed adverse effect levels (NOAEL) of the test article by repeated administrations was 1000 mg/kg for both males and females. The NOAEL for the reproductive developmental toxicity was judged to be 1000 mg/kg for male parent animals, dams and pups.