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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
reproductive toxicity, other
Remarks:
reproductive-developmental toxicity
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
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
Justification for study design:
- Premating exposure duration for parental (P0) animals : 14 days prior to mating
- Basis for dose level selection : preliminary 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)
- Inclusion/exclusion of extension of Cohort 1B : not specified
- Termination time for F2 : not specified
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : not specified
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : not specified
- Route of administration : oral gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]: 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.
Specific details on test material used for the study:
Appearance: Blue powder
Purity: At least 85.5%
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
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.
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
Route of administration:
oral: gavage
Remarks on MMAD:
n/a
Vehicle:
CMC (carboxymethyl cellulose)
Details on 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
Details on mating procedure:
females were housed together overnight with males in the same group on a one-to-one basis in the cages of males. If vaginal plugs were observed or sperm was present in the vaginal smear the following morning, it was considered that copulation was confirmed and the day was designated as GD 0. At the time of mating, each male was transferred to a wire-mesh cage (W254 × D350 × H170 mm: Lead Engineering Co., Ltd.), and then each female placed in the same cage for co-housing. The number of days until copulation was counted with the starting day of mating regarded as day 0. The length of the mating period for the same male and female was 6 days at maximum.
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)
Details on study schedule:
not specified
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:
none
Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
During the pre-mating administration period, the number of estruses, mean number of days from estrus to the next estrus (estrous cycle) and a ratio of the females which showed estrous cycle abnormality for the females examined were determined using the vaginal smears consisting of many cornified epithelial cells as an index for estruses.
Sperm parameters (parental animals):
During the mating period, vaginal smears were examined for the presence of sperm and classified as proestrus (P), estrus (E), metestrus (M) and diestrus (D).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); The pups were culled randomly to 4 males and 4 females per litter as far as possible.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: anogenital distance, mortality and behavior, weight, blood hormone measurement, number of nipple/areolae, genitalia, including the external appearance and those in the thoracic and abdominal cavities, thyroid


GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on the day following the final administration or on the last day of the recovery period
- Maternal animals: All surviving animals on the day following the final administration or on the last day of the recovery period

GROSS NECROPSY
- Gross necropsy consisted of detailed macroscopic examination on the organs/tissues throughout the body of each animal, including the external appearance and those in the cephalic, thoracic and abdominal cavities

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13 days of age.
GROSS NECROPSY
- Gross necropsy consisted of detailed macroscopic examination on the organs/tissues throughout the body of each pup being careful about genitalia, including the external appearance and those in the thoracic and abdominal cavities
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Statistics:
group mean with standard deviation was calculated for each group, and subjected to Bartlett’s test for homogeneity of variance (level of significance: 0.01).
Reproductive indices:
The post-implantation loss index, delivery index, index of external abnormalities, livebirth index and viability index on PND 4 and 13 were analyzed by Wilcoxon’s rank-sum test (levels of significance: 0.05 and 0.01) after the group mean with standard deviation was calculated for each group.
The index of animals with abnormal estrous cycle, copulation index, insemination index, fertility index, gestation index and sex ratio were calculated from the number of animals with abnormal estrous cycle, number of copulated animals, number of males that impregnated females, number of pregnant females and number of females that delivered liveborns, number of male or female pups which were counted for each group, and then analyzed by Fisher’s exact test (the levels of significance: 0.05 and 0.01, two-tailed).
Offspring viability indices:
(No. of live pups on PND 13 / No. of pups culling on PND 4) × 100
Clinical signs:
no effects observed
Description (incidence and severity):
No deaths occurred in any group. 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
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not exmained
Mortality:
no mortality observed
Description (incidence):
none observed
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
Food efficiency:
not examined
Description (incidence and severity):
not exmained
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not exmained
Ophthalmological findings:
not examined
Description (incidence and severity):
not exmained
Haematological 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 males, 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 (Attached Data 5). In the other examination items, there were no statistically significant differences between the control group and any test article administration group. 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 the other examination items, there were no statistically significant differences between the control group and any test article administration group. 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.
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.
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):
not examined
Description (incidence and severity):
not exmained
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant differences between the control group and any test article administration group
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
Description (incidence and severity):
not specified
Other effects:
no effects observed
Description (incidence and severity):
There were no significant differences between the control group and any test article administration group in T4 or TSH in males in the mating group at the end of the administration period or in the pups on PND 13.
Reproductive function: oestrous cycle:
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).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
no statistically significant differences between the control group and any test article administration group in the insemination index or fertility index.
Reproductive performance:
no effects observed
Description (incidence and severity):
no statistically significant differences between the control group and any test article administration group in the number of days until copulation, copulation index, insemination index or fertility index.
no toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
not exmained P1
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
not exmained
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
not exmained
Reproductive performance:
not examined
Description (incidence and severity):
not exmained
not exmained
Clinical signs:
no effects observed
Description (incidence and severity):
No deaths occurred in any group.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not exmained
Mortality / viability:
no mortality observed
Description (incidence and severity):
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. In female pups in the 50 mg/kg group, a significantly high value was recorded in the body weight on PND 0; however, it was judged to be an incidentally significant difference since it was not dose-related.
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.
Food efficiency:
not examined
Description (incidence and severity):
not exmained
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not exmained
Ophthalmological findings:
not examined
Description (incidence and severity):
not exmained
Haematological findings:
no effects observed
Description (incidence and severity):
none observed
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.
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
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no changes that are considered to be related to administration of the test article.
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.
Histopathological findings:
no effects observed
Description (incidence and severity):
No animals showed abnormalities that are considered to be related to administration of the test article.
Other effects:
not examined
Description (incidence and severity):
not exmained
Behaviour (functional findings):
not examined
Description (incidence and severity):
not exmained
Developmental immunotoxicity:
not examined
Description (incidence and severity):
not exmained
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Clinical signs:
not examined
Key result
Reproductive effects observed:
no
Treatment related:
no
Conclusions:
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. 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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification