N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction - Reproduction/Developmental Toxicity Screening

The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day in both sexes.

The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg/day in parental animals and offspring.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 22, 2016 to August 30, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guideline for Testing of Chemicals “Reproduction/Developmental Toxicity Screening Test” (No. 421, July 28, 2015)

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

This strain is widely used in reproduction/developmental toxicity studies using rodents, and there is abundant historical data and a large number of animals are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number and sex of animals purchased: 52 males and 60 females
Supplier (Breeding facility): Charles River Laboratories Japan, Inc. (Hino Breeding Center)

Age
At receipt: 7-week-old
At the start of administration: 10-week-old
Body weight range at receipt
222.9 to 246.1 g (males, permissible range: 180 to 260 g)
148.8 to 178.5 g (females, permissible range: 130 to 200 g)

Quarantine and acclimatization
Upon receipt, the species, strain, age, number, and sex were checked. Furthermore, observation of clinical signs, external appearance, and measurement of body weight were performed. The quarantine and acclimatization periods (from animal receipt to the day before the start of administration) were for 20 days, including the quarantine period for 6 days after receipt. All animals were observed once daily and confirmed for individual body weight gain. Female vaginal smears were collected with a swab for 10 days during acclimation from the end of quarantine, and the estrous cycle was observed under a microscope stained with Giemsa. As a result, trauma and small testis were observed in 1 male each (quarantine animal Nos. 1024 and 1052, respectively). In the other animals, there were no abnormalities in the clinical observation or body weight measurement. As for the estrous cycle examinations, 5-day cycles in 2 females (quarantine animal Nos. 2021 and 2054) were observed.

Identification of animals during quarantine and acclimatization periods
Upon receipt, animals were identified by marking the identification number (the last 1 or 2 digits of the quarantine animal number) on the tail with a black oil-based felt tip pen, and a label indicating the study number (computer registration number), quarantine animal number, and sex was attached to the front of each cage.
Quarantine animal number: Males: Nos. 1001 to 1052, females: Nos. 2001 to 2060

Group assignment
On the day before the start of administration for both sexes, 2 males with small testis or trauma and 2 females showing 5-day estrous cycles were excluded from the group assignment on the basis of the results of clinical observation and estrous cycle examination.
The other animals were assigned to each group by the stratified randomization on the basis of the body weights to obtain almost the same mean body weights for each group (Computer system; Provantis®, Instem LSS Limited). The animals weighing within ± 20% of the mean body weights (calculated for each sex) were used for this study.
Body weight range is shown below.
Males: 359.8 to 449.3 g (permissible range: 327.3 to 490.9 g)
Females: 218.8 to 252.4 g (permissible range: 188.0 to 282.0 g)
Identification of animals after group assignment: The animals were identified by ear tags inscribed with an animal number, and a label indicating the study number (computer registration number), animal number, dose level, and sex was attached to the front of each cage.
Handling of remaining animals: Remaining animals were excluded from the study 2 days after the start of dosing, and then euthanized by exsanguination from the lateral iliac artery under anesthesia by intravenous injection of pentobarbital sodium.

Animal management
Animal room: A203

Environmental conditions
Room temperature: Actual range: 23.3°C to 24.8°C (permissible range: 20.0°C to 26.0°C)
Relative humidity: Actual range: 44.3% to 60.9% (permissible range: 35.0% to 75.0%)
Ventilation: 10 to 20 times per hour
Lighting period: 7:00 to 19:00 light and 19:00 to 7:00 dark cycles

Housing equipment
Cages
For males and for females except during the gestation and lactation periods: Stainless cages (W × D × H: 226 × 346 × 198 mm)
For females during the gestation and lactation periods: Polymethylpentene cages (W × D × H: 220 × 380 × 195 mm)
Cage racks and feeders: Stainless
Sanitary tray under cages: Aluminum
Watering bottle: For F0 females during the gestation and lactation periods: Polymethylpentene

Environmental enrichment
Equipment for animal enrichment such as toys (alumina ball) and nesting materials [Paper clean (Japan SLC, Inc.)], and rest board was placed in the stainless cages. In addition, nesting materials [Paper clean (Japan SLC, Inc.) and Palsoft (Oriental Yeast Co., Ltd.)] were placed within the polymethylpentene cages (including bedding). Toys and rest board were exchanged once every two weeks or more frequently (7- to 14- day interval). Nesting materials were placed once a week or more frequently (4- to 7- day interval) when used in the stainless cages and concurrently with the cage exchange when used in the polymethylpentene cages.

Number of animals per cage
Before group assignment: 2 animals/cage
After group assignment:
Mating period; 1 male and 1 female/cage
Lactation period; 1 litter/cage
Other period; 1 animal/cage

Frequency of housing equipment exchange
Cage racks: Once every four weeks or more frequently (1- to 28- day interval)
Stainless cages (including board) and feeders: Once every two weeks or more frequently (7- to 14- day interval)
Polymethylpentene cages: Once a week or more frequently (6- to 7- day interval), but no exchange on Day 21 of gestation to Day 4 of lactation
Lids for polymethylpentene cages (served as feeder): Once every two weeks (14- day interval), but no exchange on Day 21 of gestation to Day 4 of lactation
Sanitary trays under cages: Once a week or more frequently (4- to 7- day interval) and every day for males during the mating period
Watering bottles: Once every 4 days or more frequently (1- to 4- day interval)
Cleaning and sanitation: All housing equipment was sterilized by autoclaving after water washing.

Bedding
Specification: Wood chip sterilized with autoclave (White Flake, Charles River Laboratories Japan, Inc.)
Lot number: 15.12.17
Frequency of exchange: Bedding in the polymethylpentene cage was exchanged along with the cage.
Analysis: Contaminants in the bedding were periodically obtained from the supplier and it was confirmed to be within the acceptable range stipulated by the test facility based on the analysis data obtained from the supplier (Charles River Laboratories Japan, Inc.).

Diet
Description: Pellet diet for experimental animals (CRF-1: Oriental Yeast Co., Ltd., radiation sterilized)
Lot numbers: 160212, 160307, and 160407
Method of feeding: Ad libitum
Analysis: The data for each lot (lot Nos. 160212, 160307, and 160407) of diet were obtained from Oriental Yeast Co., Ltd., and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.

Water
Description: Well water
Method of sanitization: Admixed with sodium hypochlorite (free residual chlorine concentration: about 2 ppm)
Method of water supply: Ad libitum. Automatic water supply system was used for males and females except during gestation and lactation periods. Watering bottles were used for females during gestation and lactation periods.
Analysis: The water was analyzed twice a year at Nichigo Kyushu Co., Ltd. From the analytical data, it was confirmed that the quality of the water meets the specifications of the test facility.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Vehicle
Name (manufacturer): Olive oil (NIKKO Pharmaceutical Co., Ltd.)
Lot number: 245156
Amount received: 25 L

Storage conditions and location
Room temperature (actual temperature: 17.0°C to 22.5°C, permissible range: 1°C to 30°C), Test Substance Storage Room A046

Preparation of dosing formulations
Preparation method, frequency, and storage conditions
Based on the results of another study entitled “Stability Study of SH-0850, Homogeneity, Stability, and Concentration Determination Study of Test Substance Solution (Study No. X18-1025)” [2], dosing formulations were prepared once every 7 days (permissible range: 13 days). Dosing formulations were divided into glass vials after preparation for each dosing day, and stored in a cold place (actual temperature: 3.6°C to 6.0°C, permissible range: 2°C to 10°C, medical refrigerator in Dosing Formulation Storage Room A032) up to the established stability period.

(Control dosing formulation)
The required amount of the vehicle was taken into clear glass vials for each dosing day.

(10, 30, and 100 mg/mL dosing formulations)
(1) A prescribed amount of the test substance was weighed and ground in an agate mortar.
(2) The vehicle was gradually added and mixed with a pestle.
(3) The suspension in the mortar was transferred to a measuring cylinder.
(4) The mortar and pestle were washed with the vehicle, and the washing was transferred to the measuring cylinder.
(5) The final volume was adjusted by adding an appropriate quantity of the vehicle to the required concentration of 100 mg/mL.
(6) This dosing formulation was diluted with the vehicle to make concentrations of 30 and 10 mg/mL.
(7) The dosing formulations were divided to brown glass vials for each dosing day.

Identification of dosing formulations
The glass vials containing dosing formulations were labeled with the study number, dosing formulation name, dose level, test substance concentration, preparation date, and name of the person preparing the formulations, storage conditions, and expiration period.

Handling of remaining dosing formulations
The remaining dosing formulations were discarded on each dosing day.

Details on mating procedure:

Mating
The males and females of each dose level were cohabited at one-on-one basis for 24 hours from Day 15 up to 14 days. The vaginal smears were collected in the morning from the day after the initiation of mating, and mating was confirmed with the vaginal plug or sperm in the smear sample. The day of successful mating was regarded as Day 0 of gestation (GD 0).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Confirmation of concentration
Upon the first preparation, the concentrations of the test substance in dosing formulations were confirmed at the test facility. Twenty mL of each dosing formulation was taken for the analysis from the upper, middle, and lower layers of the whole dosing formulation.
These formulations were pipetted (each layer, n=1) while stirring with a magnetic stirrer, and analyzed by the validated analytical method.

Confirmed concentration
10, 30, and 100 mg/mL

HPLC conditions
High performance liquid chromatograph: Prominence UFLC (Shimadzu Corporation)
Detector: RID-10A (Shimadzu Corporation)
Data processing software: LC solution (Shimadzu Corporation)
Column: L-Column ODS (5μm, 4.6 mm I.D. × 150 mm; Chemicals Evaluation and Research Institute, Japan)
Column temperature: A constant temperature of about 40°C (set at 40°C)
Autosampler set temperature: 15°C
Mobile phase : A mixture of methanol/ water (1:1, v/v)
Flow rate : 1 mL/min
Polarity: +
Injection volume: 20 μL
Injector wash solvent: A mixture of methanol/ water (1:1, v/v)
Analysis time: 7 min

Duration of treatment / exposure:

Males
From 14 days before mating (Days 1 to 15) until the day before necropsy through the mating period (35 days in total).

Females
From 14 days before mating (Days 1 to 15) until Day 12 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery. Non-delivery females were maintained until the day before necropsy.

Frequency of treatment:

Once daily between 9:06 and 11:57 (permissible range: 9:00 and 15:00)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 animals/dose (12 male/12 female)

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for selection of administration conditions
In accordance with the test guideline applied in this study.

Reason for selection of the dose levels
The dose levels were set based upon the results of the study entitled “A 28-Day Repeated Dose Oral Toxicity Study of SH-0850 in Rats (Study No. B11-1025, dose levels; 0, 100, 300, and 1000 mg/kg/day)” [1]. According to the results, there was no change related to the test substance treatment observed in either sex. Accordingly, the high dose level was set at 1000 mg/kg/day, which is the critical dose in OECD test guideline, and the middle and low dose levels were set at 300 and 100 mg/kg/day by a common ratio of approximately 3 (3 doses in total). The control group (0 mg/kg/day) was given the vehicle (olive oil) only.

Positive control:

Not specified

Parental animals: Observations and examinations:

The following items were examined. The initial day of dosing was designated as Day 1, and Days 1 to 7 as Week 1, in addition, before the mating period was designated as Days 1 to 15. For the females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).

Clinical observation
All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.

Body weight
Males were weighed on Days 1, 8, 15, 22, 29, and 36. Females were weighed on Days 1, 8, and 15 before the mating period, GDs 0, 7, 14, and 20 during the gestation period, and LDs 0, 4, 7, and 13 during the lactation period. Non-mated females were weighted on Day 22. The data were treated as referential data and shown in only Appendix.

Food consumption
Males were weighed on Days 2, 8, 15, and 36. Females were weighed on Days 2, 8, and 15 before the mating period, GDs 1, 7, 14, and 20 during the gestation period, and LDs 1, 4, 7, and 13 during the lactation period. No measurements were carried out during the mating period. A feeder containing the diet was weighed and set in the animal cage in the morning (after completion of delivery on that day). On the morning of the day following feeding, the remaining diet was weighed to calculate the food consumption for each day. The day of measurement of the remaining diet was designated as the day of measurement of food consumption.

Observation during the delivery and lactation
All copulated females were allowed natural delivery. The observation of delivery was conducted once daily (a.m. 9:00) from GDs 21 to 24. Females that delivered their litter completely by 9:00 a.m. were judged as “delivered” on the corresponding day (the delivery day was regarded as LD 0), and when delivery was completed at 9:00 or later, the following day was defined as LD 0. Five females (Nos. 68, 79, 86, 91, and 96) that did not deliver by 24 days after copulation were regarded as “non-delivered”. For nursing, the dams were observed once a day for maternal behavior, including lactation, nest building, and cannibalism, until LD 13.

Hormone concentration (T4) analysis
Only males were subjected to the parental animal measurement. The measurements were conducted at the test site for the parental animals.

Blood sampling
Animals for blood sampling and sampling time point
The blood was sampled upon necropsy (males, Day 36; females, LD13) for males and females. Non-delivery females were not subjected to blood sampling.

Blood sampling procedures
The animals were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg). Approximately 0.8 mL of blood was drawn from the abdominal portion of vena cava into a syringe containing heparin sodium. The blood was transferred to a test tube, immediately cooled on ice, and then promptly centrifuged (approximately 1870 × g, 10 min, 4°C) to obtain plasma (240 μL or more). The collected plasma was stored frozen in a deep freezer (actual range: -80.1°C to -71.5°C, approximately -80°C, permissible range, -90°C to -65°C) until shipment to the test site.

Oestrous cyclicity (parental animals):

Estrous cycle
Vaginal smears were collected with swabs from all females in the morning (same time every day) from the initial dosing day to the day of successful copulation to confirm the estrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M).

Litter observations:

The delivery day in F0 dams was defined as “Postnatal day 0 (PND 0)” for F1 offspring.
The number of delivered offspring (numbers of live offspring and stillborns), sex, and the presences or absences of external anomalies were examined on PND 0. Thereafter, clinical signs and mortality were observed daily. Based on the results, the following parameters were calculated. Stillborn and offspring found dead were observed for external anomaly. The stillborn and dead offspring before standardization of litter size were discarded.

Body weight
All live offspring were weighed individually on PNDs 0, 4, 7, and 13.

External examination
All live offspring were observed for external anomaly including that in the oral cavity on PND 13.

Standardization of litter size
On PND 4, the litter size was standardized to 8 (4/sex/litter) by random removal of F1 offspring. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 regardless of the sex ratio. Litter with less than 8 offspring was maintained as is. The offspring culled at the litter size adjustment (offspring excluded from blood collection in Section 6.12.1.2) were euthanized by overdose with pentobarbital sodium (undiluted solution, 0.05 to 0.1 mL/body) via an intraperitoneal injection and discarded. Offspring not culled after standardization were allocated as follows.

AGD (Anogenital distance)
The anogenital distance (AGD: distance between the anus and the genital node) was determined on PND 4 following adjustment of the number of offspring with a micrometer caliper (manufactured by Mitutoyo). The relative AGD value divided by the cubic root of the body weight on the measurement day was also calculated.

Nipple development
All live male offspring were observed for the appearance of nipples (or areolae) on PND 12. Based on the results, the following parameters were calculated.

Hormone concentration (T4) analysis
The offspring were subjected to the measurement on PND 13 only. No measurements were conducted on PND 4 in any offspring. The measurements were conducted at the test site for the offspring.

Blood sampling
Animals for blood sampling and sampling time point
The blood sampling was performed on PNDs 4 and 13. On PND 4, the blood was drawn from at least 1 culled animal of each sex in each litter. However, the blood sampling was not performed when there were less than 2 culled animals for blood sampling. On PND 13, the blood sampling was performed on 1 animal of each sex in each litter.
When a blood sample could not be obtained from either sex, it was obtained from 2 animals of the same sex in the litter.

Blood sampling procedures
The blood sampling was performed by decapitation after anesthetizing by inhalation of isoflurane. Approximately 0.4 mL of blood (including body fluid) was drawn into a polypropylene container containing heparin sodium. The blood was immediately cooled on ice and then promptly centrifuged (approximately 1870 × g, 10 min, 4°C) to obtain plasma (120 μL or more). The collected plasma was stored frozen in a deep freezer (actual range: -80.1°C to -71.5°C, approximately -80°C; permissible range, -90°C to -65°C) until shipment to the test site.

Postmortem examinations (parental animals):

Organs and tissues for examination
The organs/tissues listed in the following table were collected and examined.
Thyroid (including parathyroid); Testis; Epididymis; Seminal vesicle (including dorsolateral and coagulating gland); Prostate (ventral); Levator ani/bulbocavernosus muscle (LABC); Cowper’s gland; Glans penis; Ovary; Uterus; Other gross lesions

Necropsy
The male (Day 36) and female (LD 13) necropsy were conducted one day after the administration period. The non-delivery females (Nos. 68, 79, 86, 91, and 96) necropsy was conducted from 24 days after copulation. According to the results, the 5 females without delivery were not pregnant (refer to Section 6.10.3). In addition, the vaginal smears were collected from the females on LD 13 (necropsy day) in the morning and the stage of the estrous cycle was examined under a microscope. The animals were euthanized by exsanguination from the lateral iliac artery after blood sampling at the end of the administration period for the blood sampling animals (not conducted for blood sampling in the non-delivery females). The thoracoabdominal organs/tissues were examined macroscopically.

Organ weight
After necropsy, organs were weighed (absolute weight), and the ratio of organ weight to body weight (relative weight) was calculated based on the body weight on the day of necropsy. Paired organs were measured together.

Histopathology
The organs and tissues were fixed in 10 vol% phosphate buffered formalin. The testes and epididymides were pre-fixed in Bouin’s solution, and then preserved in 10 vol% phosphate buffered formalin. Examination was conducted for the control and high dose groups. The organs and tissues of these animals or the ovary of non-pregnant females were embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined microscopically. As a result, no changes attributable to the test substance treatment were observed in any organs/tissues of the 1000 mg/kg group. Therefore, the additional examination was not performed.

Examination after lactation
Dams were subjected to necropsy on LD 13, and the ovaries and uterus were excised to examine for the number of implantation sites. Non-delivered females (Nos. 68, 79, 86, 91, and 96) were also examined for the uterus in the same manner on GD 24. The uterus of females for which implantation was not confirmed macroscopically was soaked in 10 vol% ammonium sulfide solution. As a result, the implantation was not confirmed macroscopically and thus, these females were judged to be non-pregnant. Based on the results, the following parameters were calculated

Postmortem examinations (offspring):

Necropsy
The necropsy was performed on PND 13. The offspring subjected to blood sampling were necropsied after blood sampling. Other animals were euthanized by exsanguination from the lateral iliac artery under anesthesia with pentobarbital sodium (undiluted solution, 0.1 to 0.5 mL/body) via the intraperitoneal injection and necropsied. The thoracoabdominal organs/tissues were examined macroscopically. Furthermore, the thyroid glands of 1 male and 1 female (animals subjected to blood sampling for hormone level measurement) were collected and fixed and preserved in 10 vol% phosphate buffered formalin.

Statistics:

Statistical analysis was performed for the following data, except for the sex ratio, using a computer system (Provantis®, Instem LSS Limited). SAS 9.1.3 (SAS Institute Japan Ltd.) was used for the sex ratio. In any case, two-tailed test was used and levels of p<0.01 and p<0.05 were considered significant. The mean and standard deviation were calculated and homogeneity of variance was tested by Bartlett’s method (p<0.05).
When the groups were accepted as homogeneous, Dunnett’s multiple comparison test was used for comparison of the groups of data. When the groups of data were found to be heterogeneous by Bartlett’s test, Steel’s multiple comparison test was conducted. For non-parametric data such as incidences, Wilcoxon's rank sum test was performed between the control and treated groups. Other non-parametric data was analyzed by chi-square test.
The data of offspring were analyzed on the basis of litter mean values. Moreover, the body weight and food consumption of non-pregnant females were excluded from the evaluation.

Multiple comparison test
Body weights, food consumption, absolute and relative organ weights, estrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes), and AGD

Chi-square test
Copulation index, fertility index, gestation index, delivery index, and sex ratio

Wilcoxon’s rank sum test
Stillborn index, external anomaly index, external anomaly typing index, live birth index, viability index on Day 4, viability index on Day 13, and nipple development anomaly index

Reproductive indices:

Days until copulation: Number of days from the start of mating to the detection of copulation

Copulation index (%):
(Number of animals with successful copulation / Number of animals cohabited) × 100

Fertility index (%):
(Number of pregnant animals / Number of females with successful copulation) × 100

Gestation length: Days until completion of delivery from GD 0

Gestation index (%):
(Number of pregnant animals delivered with live offspring / Number of pregnant animals) × 100

Delivery index (%):
(Total number of offspring at birth / Number of implantations) × 100

Offspring viability indices:

Live birth index (%):
(Number of live offspring at birth / Number of implantations) × 100

Stillborn index (%):
(Number of stillborns / Total number of delivered offspring) × 100

Viability index on Day 4 (%):
(Number of live offspring on PND 4 / Number of live offspring at birth) × 100

Viability index on Day 13 (%):
(Number of live offspring on PND 13 / Number of live offspring after culling) × 100

Sex ratio:
(Number of male live offspring / Number of female live offspring)

External anomaly index (%):
(Number of offspring with external anomaly / Number of observed offspring) × 100

External anomaly typing index (%):
(Number of offspring with external anomaly by each type / Number of observed offspring) × 100

External anomaly index (%):
(Number of offspring with external anomaly / Number of observed offspring) × 100

External anomaly typing index (%):
(Number of offspring with external anomaly by each type / Number of observed offspring) × 100

Nipple development anomaly index (%):
(Number of offspring with nipple development anomaly/Number of observed offspring) × 100

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs were observed in either sex of any group throughout the dosing period, including the gestation and lactation periods of pregnant females.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No death occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed between the control group and any treated group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant difference was observed between the control group and any treated group.

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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in any animal.
As a change in the control group, focal atrophy of seminiferous tubule was observed in 1 male. No histopathological change was observed in the ovary of the non-pregnant animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The plasma T4 concentration of DAIGUARD-850-treated groups (Groups low-, middle-, and high-dose) was equivalent to that of the control group in parental animals [F0, male] There was no notable change in the plasma T4 concentration attributable to the test substance.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related changes in estrous cycle were observed in females of any treated group.
In 1 female (No. 60) of the control group irregular estrous cycle (7-day cycle) was observed. However, no statistically significant change was observed in the mean estrous cycle or number of estrus between the control group and any treated group.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

In the 300 mg/kg group, a higher value for days until copulation was observed; however, it was not considered to be treatment-related because there was no dose-dependency.
No statistically significant difference was observed in the copulation index or fertility index between the control group and any treated group. Accordingly, the copulation indices were 100%, 100%, 100%, and 100% for the control, 100, 300, and 1000 mg/kg groups, respectively, and the fertility indices were 100%, 91.7%, 83.3%, and 83.3% for the control, 100, 300, and 1000 mg/kg groups, respectively.
No statistically significant difference was observed in the gestation length, number of implantation sites, delivery index, or gestation index between the control group and any treated group. Moreover, no abnormality was observed at delivery nor any abnormality was observed in the nursing conditions of the dams.

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No effects observed

Clinical signs:

no effects observed

Description (incidence and severity):

No external abnormalities were observed in any offspring of any group on PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No statistically significant difference was observed in number of live offspring at birth, birth index, sex ratio, number of stillborn at birth, stillborn index, or viability index on Day 4 or 13 between the control group and any treated group. No external anomaly was observed in any offspring.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed between the control group and any treated group.

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

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No statistically significant difference was observed in the plasma AGD value between the control group and any treated group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in nipple development anomaly index.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were observed in any sex of any group.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The plasma T4 concentration of DAIGUARD-850-treated groups (Groups low-, middle-, and high-dose) was equivalent to that of the control group in offspring [F1, PND 13]. There was no notable change in the plasma T4 concentration attributable to the test substance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No effects observed

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Full summary and individual results tables can be found attached under background information.

Conclusions:

DAIGUARD-850 was repeatedly administered by oral gavage at dose levels of 0 (control group), 100, 300, and 1000 mg/kg from 14 days before mating through mating for 35 days in males, and 14 days before mating through gestation and delivery until Day 12 of lactation in females to determine reproductive and developmental toxicity of the observed changes.

Repeated dose toxicity
No death occurred during the study. No changes related to the test substance treatment were observed in the clinical signs, body weight, food consumption, necropsy, organ weight, or histopathological examination in males or females or plasma T4 concentrations in males.

Reproductive/Developmental toxicity
In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, or number of implantation sites, delivery index, or delivery or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, viability index on Day 4, or viability index on Day 13, nor in the external examination, anogenital distance (AGD), nipple development, plasma T4 concentrations on PND 13, or body weight.

NOAEL
From the above results, no-observed-adverse-effect level (NOAEL) in the effects on parental animals and reproductive toxicity was judged as follows.

Effects on parental animals
The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day in both sexes under the conditions of this study.

Reproductive/Developmental toxicity
The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg/day in parental animals and offspring under the conditions of this study.

Executive summary:

DAIGUARD-850 was repeatedly administered by oral gavage at dose levels of 0 (control group), 100, 300, and 1000 mg/kg from 14 days before mating through mating for 35 days in males, and 14 days before mating through gestation and delivery until Day 12 of lactation in females to determine the reproductive and developmental toxicity of the observed changes. Each test group consisted of 12 males and 12 females. The control group received the vehicle (olive oil) only.

 

Effects on parental animals

No death occurred during the study.

No changes related to the test substance treatment were observed in the clinical signs, body weight, food consumption, necropsy, organ weight, or histopathological examination in males or females or plasma T4 concentrations in males.

 

Reproductive/Developmental toxicity

In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of implantation sites, delivery index, or delivery or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, viability index on Day 4, or viability index on Day 13, nor in the external examination, anogenital distance (AGD), nipple development, plasma T4 concentrations on PND 13, or body weight.

 

NOAEL

From the above results, no-observed-adverse-effect level (NOAEL) in the effects on parental animals and reproductive toxicity was judged as follows.

 

Effects on parental animals

The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day in both sexes under the conditions of this study.

 

Reproductive/Developmental toxicity

The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg/day in parental animals and offspring under the conditions of this study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction - Reproduction/Developmental toxicity screening test

DAIGUARD-850 was repeatedly administered by oral gavage at dose levels of 0 (control group), 100, 300, and 1000 mg/kg from 14 days before mating through mating for 35 days in males, and 14 days before mating through gestation and delivery until Day 12 of lactation in females to determine the reproductive and developmental toxicity of the observed changes. Each test group consisted of 12 males and 12 females. The control group received the vehicle (olive oil) only.

 

Effects on parental animals

No death occurred during the study.

No changes related to the test substance treatment were observed in the clinical signs, body weight, food consumption, necropsy, organ weight, or histopathological examination in males or females or plasma T4 concentrations in males.

 

Reproductive/Developmental toxicity

In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of implantation sites, delivery index, or delivery or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, viability index on Day 4, or viability index on Day 13, nor in the external examination, anogenital distance (AGD), nipple development, plasma T4 concentrations on PND 13, or body weight.

 

NOAEL

From the above results, no-observed-adverse-effect level (NOAEL) in the effects on parental animals and reproductive toxicity was judged as follows.

The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day in both sexes under the conditions of this study.

The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg/day in parental animals and offspring under the conditions of this study.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available