Y-513

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 1, 2014 - May 21, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) (July 27, 1995)

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures in this study essentially conformed to the following guidelines.

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Y-513
- Physical state: Yellow powder
- Storage condition of test material: Room temperature (actual values: 17.2 to 24°C)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: At least 10 weeks
- Weight at study initiation (rats of 10 weeks): 397-479 g (males); 248-286 g (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed individually in bracket-type stainless-steel wire-mesh cages, except during mating.
From gestation day 17 until day 4 post-partum, dams were housed with their litters in plastic Econ cages.
- Diet: Free access to pelleted diet NMF(radiation-sterilized), from Oriental Yeast Co., Ltd.
- Water: Free access to tap water.
- Acclimation period: 25 days (including 3 days quarantined)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 – 24
- Humidity (%): 37 - 53
- Air changes (per hr): approx 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: December 24, 2014 - February 15, 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Gum arabic

Details on exposure:

Preparation of vehicle: A requisite amount of gum arabic (Lot nr. PDQ6904, Wako Pure Chemical Industries, Ltd.) was dissolved in water for injection to prepare 5.0 w/v% solution. The vehicle was prepared on each day the test suspensions were prepared.

Preparation of formulation: Requisite amount of the test article to prepare the test suspension for each dose level was weighed, put into a porcelain mortar and suspended in the vehicle, which was added little by little. After transfer to a measuring cylinder, the vehicle was added to make the specified volume and concentration. Then the suspension was mixed well by inverting the cylinder several times. The test suspensions were prepared at once every 2, 4, 5 or 7 days.

Storage conditions of formulations: Dosing formulations (including the dosing solution for the control group) were divided into one-day aliquots, put into brown glass bottles and stored in a cold place (in a refrigerator, acceptable temperature: 1 to 10°C; actual values: 3 to 6°C) and used by day 7 (day of preparation: day 0).

Stability: Y-513 in test suspensions within the range of 5 to 100 mg/mL were stable after 8 days in a cold place (1-10°C) followed by 24 hours at room temperature in a brown glass bottle.

Dose volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Once before the start of administration and once in week 4 of administration the test suspension of each dose concentration to be used for administration was analyzed. Three fractions (10 mL, each from the upper, middle and lower layers) were collected from each concentration and analyzed by HPLC.

The measured concentration of Y-513 to the nominal concentration for each dose concentration was between 95.5 and 106.0% (acceptable range: within 100.0 ± 10.0% of the nominal concentration), and coefficient of variation was not more than 3.3% (acceptable range: not more than 10.0%), both of them were within the acceptable range.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group in the cages of males);
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, and/or or by the appearance of an vaginal plug. This day was designated day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing one female (dosed at 50 mg/ kg bw/ day) who had not shown evidence of mating was separated from her male. Clinical observations, measurements of body weight and food consumption and administration were continued. Twenty-five days later, the animal was sacrificed by exsanguination via the abdominal aorta under anesthesia with isoflurane inhalation and subjected to pathological examinations.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40 to 53 days, i.e. during 2 weeks prior to mating, during mating (minimal 1 day), during post-coitum (minimal 3 weeks), and during at least 4 days of lactation.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 28 days
Females: 40-53 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previous repeated dose 28-day oral toxicity study in rats with a recovery period of 14 days (Study Code: B11-0846, Chemicals Evaluation and Research Institute, 2008), administration of Y-513 at the dose level of 1000 mg/kg bw/day was associated with no general toxicological effects on males and females. And there were no abnormalities in the recovery test. Therefore, in this study, 1000 mg/kg bw/ day, which corresponds to the recommended highest dose in the Test Guideline, was selected as the highest dose, and 250 and 50 mg/kg bw/ day were selected as the middle and low doses.

Parturition:
The females were allowed to litter normally. The day of birth was regarded as day 0 post-partum.

Selection of animals for selected measurements:
Only the organs in the control group and the high dose group were subjected to microscopic examination (for bilateral organs, organs of both sides).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS
- All animals were observed 3 times daily (before dosing, immediately after dosing, 1-3 hours after dosing) for clinical signs such as abnormalities in external appearance, appearance of excrement, nutritional condition, posture and behavior. Periodical in-the-hand observation during the gestation period was done on days 0, 7, 14 and 20 of gestation.

BODY WEIGHT
- Males and females were weighed on days 1, 4, 8, 11, 15, 18, 22 and 25 of administration and on the day of necropsy. Females were additionally weighed on gestation days 0, 4, 7, 11, 14, 17 and 20 and on days 0 and 4 post-partum.

FOOD CONSUMPTION
For males and females, food consumption was measured on days 2, 4, 8, 11 and 15 of administration during the pre-mating administration period. Additionally, for females, food consumption was measured also for gestation days 1, 4, 7, 11, 14, 17 and 20 as well as days 2 and 4 post-partum. One day’s food consumption was calculated as the difference from the amount supplied on the previous day. Both the amounts supplied and left uneaten were measured before the dosing of the day during the administration period, and in the morning on day 4 post-partum.

FOOD EFFICIENCY
No

WATER CONSUMPTION
No

OPHTHALMOSCOPIC EXAMINATION
No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day of birth: Number and sex of pups, stillbirths, live births, presence or absence of external abnomalies, body weight.
Daily: Mortality
Day 4: external abnormalies, sex, body weight.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
- For body weight, body weight gain (males: day 1 of administration to the day of necropsy, females: day 1 to day 15 of administration, gestation day 0 to 20, day 0 to 4 post-partum), food consumption, number of estruses, estrous cycle, number of days until copulation, length of gestation period, number of corpora lutea, number of implantation sites, number of liveborns, sex ratio (days 0 and 4 post-partum) and organ weight, 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).
-Homogeneous data were analyzed by Dunnett’s test (levels of significance: 0.05 and 0.01, two-tailed).
- The implantation index, stillbirth index, index of external abnormalities, livebirth index and viability index on postnatal day 4 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 copulation index, fertility (insemination) index and delivery index were calculated from the number of copulated animals, number of males that impregnated females, number of pregnant females and number of females that delivered liveborns 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).

Indices:

Reproductive indices; For each group, the following calculations were performed:
Copulation index (%) = (No. of copulated animals / No. of animals housed together) × 100
Fertility (insemination) index (%) = [No. of pregnant females (No. of males that impregnate females) / No. of females (males) copulated] × 100
Delivery index (%) = (No. of females which delivered liveborns / No. of pregnant females) × 100
Implantation index (%) = (No. of implantation sites / No. of corpora lutea) × 100

Offspring indices:
Stillbirth index (%) = (No. of stillborns / No. of liveborns + No. of stillborns) × 100
Index of external abnormalities (%) = (No. of liveborns with external abnormalities / No. of liveborns) × 100
Live birth index (%) = (No. of liveborns / No. of implantation sites) × 100
Viability index on day 4 post-partum (%) = (No. of live pups on day 4 post-partum / No. of liveborns) × 100
Sex ratio of liveborns on day 0 post-partum = (No. of male liveborns / No. of liveborns)
Sex ratio of live pups on day 4 post-partum = (No. of male live pups on day 4 post-partum / No. of live pups on day 4 post-partum)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study.

CLINICAL SIGNS
Yellow-colored feces were observed in all animals dosed at 250 and 1000 mg/kg bw/ day. In one female dosed at 250 mg/ kg bw/ day a mass in the inguinal region was observed from gestation day 20 to the end of the experiment. This was an incidental finding and not expected to be substance-related.

BODY WEIGHTS
No significant differences were noted in the body weight or body weight gains of the mothers during the administration period between the control group and any dosage group.

FOOD CONSUMPTION
In females, incidental high values in food consumptions were noted compared to controls (on day 15 of administration in the 50 mg/kg bw/ day group and on lactation day 2 in the 1000 mg/kg bw/ day), this was not found to be substance-related.

MACROSCOPIC EXAMINATION
One female dosed at 250 mg/kg bw had a subcutaneous nodule.

MICROSCOPIC EXAMINATION
No effects were seen in the reproductive organs of females in dosed groups compared to the control groups.

REPRODUCTIVE DATA/ GESTATION
The estrous cycle of dosed females during pre-mating period did not differ from the estrous cycle of the control animals. All copulated females (except for 2 females in the control group which were found to be non-pregnant) delivered their pups normally in each group and no significant differences were noted in the delivery index, gestation length, implantation index, stillborn index, live birth index, numbers of corpora lutea, implantation sites or liveborns between the control group and any dosage group. There were no abnormalities in nursing condition in any of the dams.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

SEX RATIO PUPS

The sex ratio of liveborns at birth and day 4 was low for the rats exposed to 50 mg/kg bw/ day (0.41 for both time points), but as this was not seen at higher doses (ratio's of 0.49 and 0.56 (at birth) , 0.49 and 0.55 (day 4) for resp. 250 and 1000 mg/ kg bw/ day) this was found to be incidental and not related to the test substance exposure.

MORTALITY PUPS

The mean number of liveborn pups and the live birth index did not differ between control and treatment groups. No significant differences were noted in the viability index on day 4 post-partum between the control group and any dosage group.

CLINICAL SIGNS

No clinical signs were noted in the pups.

BODY WEIGHT PUPS

No significant differences were noted in body weight on day 0 and day 4 post-partum in males or females between the control group and any dosage group.

MACROSCOPY PUPS

There were no abnormalities at birth and on day 4 in the external appearance of liveborns in any group.

Applicant's summary and conclusion

Conclusions:

In an oral repro-developmental toxicity screening study, performed according to OECD guideline 421, the maternal and developmental NOAEL were derived to be at least 1000 mg/kg bw/day, based on no adverse effects seen at the highest dose tested.

Executive summary:

An oral repro-developmental toxicity screening study was performed according to OECD guideline 421 and GLP principles. Test substance Y-513 was administered to male and female rats at 50, 250 and 1000 mg/kg bw/ day for 14 days before mating and throughout the mating period and throughout the gestation period until day 3 post-partum for females (40 -53 days in total). Accuracy and homogeneity of formulations were confirmed by analyses. No mortality occurred during the study. No clinical signs were noted and the test substance had no effect on body weight gain and/ or food consumption of the animals. No differences were noted between control and exposed groups during gross macroscopic investigation. All copulated females (except for 2 females in the control group which were found to be non-pregnant) delivered their pups normally in each group and no significant differences were noted in the delivery index, gestation length, implantation index, stillborn index, live birth index, numbers of corpora lutea, implantation sites or live-borns between the control group and any dosage group. There were no abnormalities in nursing condition in any of the dams. The mean number of live-born pups, the live birth index did not differ between control and treatment groups. The test substance did not influence the sex ratio of the pups. There were no abnormalities at birth and on day 4 in the external appearance of liveborns in any group.

Based on the outcome of this study, both the maternal and the developmental NOAEL were found to be at least 1000 mg/ kg bw/ day, based on no adverse effects seen at this dose level.