Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Period: February 10, 2017 to September 5, 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
please see any other information on materials and methods section
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
484-490-5
EC Name:
-
Molecular formula:
Hill formula:C21 H12 N2 O6 S Sr 5/2(H2O) CAS formula: C21 H14 N2 O6 S . Sr
IUPAC Name:
484-490-5
Test material form:
solid: particulate/powder
Details on test material:
Name: R507-2
Lot number: 161116
Appearance: Red powder
Purity: Not less than 97.4%
Date of expiry: November 4, 2021
Storage conditions: Protected from light, exclusion of moisture, ordinary temperature (actual temperature: 18 to 25°C), sealing
Storage place: Test article storage room, test article preparation room in the Second Laboratory Building and reference standard storage place in Analysis Department, Gotemba Laboratory
Handling precautions: Take care not to touch the skin and wear appropriate personal protective equipment such as protective glasses (goggle type), protective gloves, protective masks, protective clothing and so on.
Stability: There was no abnormality in the quality of the test article before using or remaining after the end of administration in the characteristic test.
Archive sample: Approximately 1 g of the test article for the archive sample was preserved at the test facility.
Disposition of the remaining test article: Except for the archive sample, the test article remaining after the end of animal experiment and analyses of this study was returned to the Sponsor.
Specific details on test material used for the study:
Name: R507-2
Lot number: 161116
Appearance: Red powder
Purity: Not less than 97.4 %
Date of expiry: November 4, 2021
Storage conditions: Protected from light, exclusion of moisture, ordinary temperature (actual temperature: 18 to 25 °C), sealing
Storage place: Test article storage room, test article preparation room in the Second Laboratory Building and reference standard storage place in Analysis Department, Gotemba Laboratory
Handling precautions: Take care not to touch the skin and wear appropriate personal protective equipment such as protective glasses (goggle type), protective gloves, protective masks, protective clothing and so on.
Stability: There was no abnormality in the quality of the test article before using or remaining after the end of administration in the characteristic test.
Archive sample: Approximately 1 g of the test article for the archive sample was preserved at the test facility.
Disposition of the remaining test article: Except for the archive sample, the test article remaining after the end of animal experiment and analyses of this study was returned to the Sponsor.

Test animals

Species:
rat
Strain:
other: SPF rats Crl: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 available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test Animals
Sixty (60) male and 60 female Sprague-Dawley strain SPF rats [Crl:CD(SD), Atsugi Breeding Center, Charles River Laboratories Japan, Inc.] were received at 8 weeks of age. The animals were quarantined for 3 days (the day of receipt: Day 1) and acclimated for 20 days. During the quarantine and acclimation periods, animals were observed for general condition such as external appearance, nutritional condition, posture, behavior and appearance of excrement once daily in the morning. Animals were weighed on the day of receipt, at the end of quarantine, and once a week after the end of quarantine, and the body weight gain from the previous measurement calculated. In addition, observation of estrous cycle for more than 14 days after the end of the quarantine period in females and examination of the balanic pattern and palpation of the testes descent in the scrotum on the final day of acclimation in males were conducted. In the observation and examination during the quarantine and acclimation periods, there were
2 males with the small testes and 3 females with abnormalities in estrous cycle. There were no abnormalities in body weight development in any animal. Based on the results of the observations and examinations described above, 48 healthy males with no abnormalities in the testis by palpation and sexually matured judging from the balanic pattern and 48 healthy females with estrus of 4-5 day intervals were selected. The body weight range at the start of administration was 417 to 488 g (mean body weight: 447 g) for males and 225 to 276 g (mean body weight: 249 g) for females, and they were within ± 20 % of the mean value for each sex. After group allocation, the remaining 12 males (including 2 males with small testes) and 3 females with abnormalities in estrous cycle were excluded from the study and transferred to the Animal Care Division (date of transfer: March 7, 2017). The remaining 9 females were kept in the study as untreated females for mating in case of death of females until the end of the
mating period, but they were finally transferred to the Animal Care Division (date of transfer: March 28, 2017) without being used for mating.

Animal Husbandry
Animals were housed in an animal room (Room No. 910) which was controlled to maintain the temperature at 23 ± 3 °C (actual values: 23 to 24 °C), the relative humidity at 50 ± 20 % (actual values: 42 to 52 %), air ventilation at 10 to 15 times per hour, and a 12-hour light cycle (lighting: 07:00 to 19:00). The animals were housed in plastic cages (W 440 × D 275 × H 180 mm: Hanyu-Seimitsu, Co., Ltd.) with bedding (ALPHA-dri, Shepherd Specialty Papers, Inc., Lot Nos. 07116, 09116 and 11116) in groups of 2 animals of the same sex until the group allocation. After group allocation, a divider was placed in each plastic cage and each animal was housed individually in each division. After the initiation of mating, males were housed individually in plastic cages with bedding same as above, and one male and one female housed together during mating. From gestation day 0 until day 13 post-partum, dams were housed with their litters in plastic cages with bedding same as above. The animals were allowed free access to pelleted
diet CRF-1 (radiation-sterilized, Oriental Yeast Co., Ltd., Lot Nos.: 160907, 161007 and 161209, via stainless-steel feeders) and to tap water (Gotemba Municipal Water: via an automatic water supply system). During the period of the animal experiment, a toy (stainless-steel stick which was hung from the cover of the cage) was installed and each animal received 7979C.CS certified/irradiated Diamond Twists (Envigo RMS, Inc., Lot No.: 7979C-082316P) at least once a week as enrichment.

Contaminants in Feed, Drinking Water, Bedding and Environmental Enrichment
Analysis of contaminants in the feed was performed for each lot by Eurofins Food and Product Testing. Analysis of contaminants in the bedding was performed for the lot used by N•P Analytical Laboratories. Periodic analysis of the tap water was carried out on a quarterly basis according to the Waterworks Law by Shibaura Semtek Co., Ltd. Analysis of contaminants in the environmental enrichment was performed for the lot used by Envigo RMS, Inc. The results obtained from the above facilities were filed after verifying that there were no contaminants that could adversely affect the results of the study.

Group Allocation
The animals with favorable body weight gain that were selected by the method described above. Test Animals were ranked according to their body weight on the day of grouping and assigned to the requisite number of groups by the 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 initial day of administration.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 5 w/v% gum arabic solution
Details on exposure:
Preparation of Vehicle
The requisite amount of gum arabic (Wako Pure Chemical Industries, Ltd., Lot No. LKK1889) was dissolved in water for injection (Japanese Pharmacopoeia, Otsuka Pharmaceutical Factory Inc., Lot Nos. 6H73, 6I83, 6I91) to prepare 5.0 w/v % solution. The amount of vehicle for use for 7 days at maximum was prepared at a time, stored in a cold place (in a refrigerator, acceptable temperature: 2 to 8 ºC, actual values: 3.6 to 7.1 ºC) and used within 10 days after preparation. Before handling of the test article on the day of preparation, the requisite amount of the vehicle for use as the dosing solution for the control group was separated and put into brown glass bottles.

Preparation of Test Suspensions
The requisite amount of 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. The suspension was transferred to a measuring cylinder after checking the suspension condition. The mortar and pestle were washed with a small amount of vehicle and the washing was also put into the measuring cylinder. Then, 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 once every 2, 5, 7 or 8 days.

Storage of Dosing 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: 2 to 8 °C; actual values: 2 to 6 °C), and used by day 8 (day of preparation: day 0). Dosing formulations were returned to room temperature before use.

Stability and Homogeneity of Test Suspensions
In the test facility, R507-2 in test suspensions (vehicle: 5 w/v % gum arabic solution) within the range from 5 to 100 mg/mL have been confirmed to be stable and homogeneous in a brown glass bottle after storage 8 days in a cold place (in a refrigerator, acceptable temperature: 1 to 10°C) and then 24 hours at room temperature (acceptable temperature: 1 to 30 °C), and these could be homogeneously re-suspended by stirring after storage.

Administration
The method of administration was oral administration by gavage, which is routinely used for oral administration to rodents. Dose volume was set at 10.0 mL/kg body weight and the dosing formulation was administered by gavage using a stomach tube. Administration was done between 09:18 and 12:19 except when it was done between 14:21 and 14:25 to animals which had not completed delivery in the morning and had completed it at the observation in the afternoon. The animals in the control group received the vehicle only in the same manner. Individual dose volume was calculated based on the animal’s most recent body weight. The dosing formulations were used while stirring at administration.
Details on mating procedure:
After the end of the pre-mating administration period, 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 designated as GD 0. The number of days until copulation was counted with the initial day of mating regarded as day 0. The length of the mating period for the same male and female was 4 days at maximum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of Concentration and Homogeneity of Test Suspensions
A total of twice, 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 for R507-2. Three fractions (10-mL each from the upper, middle and lower layers) were collected from each concentration and analyzed by HPLC. In the results, the measured concentration of R507-2 to the nominal concentration for each dose concentration was between 100.2 and 104.0 % (acceptable range: within 100.0 ± 10.0 % of the nominal concentration), and coefficient of variation (C.V.) not more than 2.9 % (acceptable range: not more than 10.0 %), both of them were within the acceptable range. The analytical method validation was determined at Gotemba Laboratory, BoZo Research Center, Inc. Analytical methods are shown briefly in the following.

Analyte: R507-2
Reference Standard: A portion of the test article was subdivided and used as the reference standard.

Preparation of Sample Solutions
the sample solutions were prepared before use. The sample solutions were sampled by n=1 while stirring with the stirrer. In the first dilution, the test formulations were dissolved in a water bath set at 50.0 °C and by sonication before making the final volume.

HPLC System
HPLC: 2695 Separation module (Waters Corporation)
Detector: 2487 Dual λ UV/VIS detector (Waters Corporation)
Data processor: Empower (Waters Corporation)

HPLC Conditions
Column: Inertsil ODS-3 (2.1 mm I.D. × 150 mm, particle size 5 μm, GL Sciences, Inc.)
Set temperature of column heater: 40 °C
Mobile phase: Mixture of 30 mmol/L ammonium acetate solution/acetonitrile (7:3)
Needle washer: Mixture of water/acetonitrile (7:3)
Flow rate: 0.3 mL/min
Detection: UV (518 nm)
Injection volume: 10 μL
Set temperature of autosampler: Room temperature
Analysis time: 10 minutes

Measurement Assurance
Re-analysis
In the analysis of dosing formulation for the first administration (date of analysis: February 28, 2017), measurement was stopped because the peak shape of the standard solution was different from that obtained from the pre-injection. After the investigation of the cause, the measurement was re-started.

Various factors that might have affected the reliability of data
In the analysis of dosing formulation for the first administration, the peak shape of the standard solution was different from that obtained from the pre-injection. The cause was examined, but not understood. The peak shape did not change to better but the reproducibility of peak area was good. It was judged that the phenomenon would not affect the reliability of the data and the measurement was re-started. And therefore, there were no factors that might have affected the reliability of data. All the results of system suitability met the acceptance criteria.
Duration of treatment / exposure:
The administration period was from 2 weeks before the initial day of mating, and through the mating period to the day before necropsy for males (29 days in total), or through the mating and gestation period until lactation day 13 for females (51 to 54 days in total).
Frequency of treatment:
The frequency of administration was once every day (7 times/week)
Details on study schedule:
Start of Study: February 10, 2017
Receipt of Test Article: January 18, 2017
Retrieval of Test Article: February 20, 2017
Animal Receipt: February 15, 2017
Start of Administration: March 7, 2017
Start of Mating: March 21, 2017
Necropsy for Males: April 5, 2017
Start of Parturition: April 13, 2017
Start of Necropsy for Pups: April 26, 2017
Start of Necropsy for Dams: April 27, 2017
End of Animal Experiment: April 30, 2017
End of Examination: July 5, 2017
End of Study: September 5, 2017
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 males and 12 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Selection of Dose Levels
In a previous repeated dose 28-day oral toxicity study in rats (Study Code: NOTOX Project 486324, NOTOX B.V.), no deaths occurred in either sex in the 1000 mg/kg/day group 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/day, which corresponds to the recommended highest dose in the toxicity study guideline, was selected as the highest dose, and 250 and 50 mg/kg/day selected as the middle and low doses, respectively.

Examinations

Parental animals: Observations and examinations:
Observation and Examination
Study days were designated as follows:
Initial day of administration: Day 1 of administration
Day 1 to 7 of administration: Week 1 of administration
The day copulation occurred: Gestation day (hereinafter referred to as GD) 0
The last day of delivery: Lactation day (hereinafter referred to as LD) 0
The day of birth: Postnatal day (hereinafter referred to as PND) 0
The following observations and examinations were done on the specified days.

Clinical Observation
All animals were observed for clinical signs including mortality, external appearance, excrement, posture and behavior 3 times a day, before dosing, immediately after and 1-3 hours after dosing during the administration period. On the day of necropsy, clinical observation was performed once before carrying the animals from the animal room for necropsy. In this study, colored feces (red) were observed in the 50, 250 and 1000 mg/kg groups. However, the changes in color of feces could not be determined in the observation during the mating due to the housing conditions. Periodical in-the-hand observation was carried out at a frequency of 1 to 2 times a week for animals excluding lactating animals. However, for pregnant animals, that was done on GDs 0, 7, 14 and 20.

Body Weight
All animals were weighed on days 1, 3, 8, 15 and 20 of acclimation. Males were weighed on days 1, 4, 8, 11, 15, 18, 22, 25 and 29 of administration and on the day of necropsy. Females were weighed on days 1, 4, 8, 11 and 15 of administration, GDs 0, 4, 7, 11, 14, 17 and 20, LDs 0, 4, 7 and 13 and the day of necropsy. Besides, the body weight of the uncopulated female was measured on day 18 of administration during the mating period. Body weight was measured between 07:21 and 10:53 (before dosing during the administration period) and before carrying the animals from the animal room for necropsy on the day of necropsy. However, the animals that delivered in the afternoon were weighed after completion of parturition (13:43 to 16:34). The body weight data on the day of necropsy of the female that had not delivered by GD 25, and the body weight data of the females during the mating period (Day 18 of administration) were excluded from statistical analysis.

Food Consumption
The amount of food remaining was measured for all animals: males on days 2, 4, 8, 11 and 15 of administration, and females on days 2, 4, 8, 11 and 15 of administration, GDs 1, 4, 7, 11, 14, 17 and 20 and LDs 2, 4, 7 and 13. Daily food consumption of each animal was then calculated by subtracting from the amount of the feed provided on the previous day. Measurements of the amounts of feed supplied and remainder were conducted between 09:24 and 11:01 (before dosing during the administration period).

Observation of Dams
Females that had copulated were observed for completion of parturition twice a day, in the morning and afternoon (until 17:00), from GD 21 until the morning of GD 25. The length of gestation was determined in the unit of 0.5 day. After completion of parturition, dams were observed for the presence or absence of abnormalities in parturition such as untreated placenta or amnion. All dams were allowed to nurse their own pups for 13 days after delivery, and they were observed for nursing condition using maternal behavior such as gathering pups, nesting and lactation as indices. The females in the control, 50 and 250 mg/kg groups which had not delivered by the morning of GD 25 (Animal Nos.: 1101, 2102, 3108 and 3109) were sacrificed by exsanguination via the abdominal aorta under isoflurane anesthesia and subjected to pathological examinations. The data of three females (Animal Nos.: 1101, 3108 and 3109) during the gestation period were excluded from the study results since they had no implantations in the examination for pregnancy and were judged to be non-pregnant. Because implantation sites were recognized in the uterus of one female (Animal No.: 2102), it was judged that this female was pregnant and the numbers of implantation and corpora lutea were counted. Dams which delivered pups were subjected to pathological examinations after blood sampling for blood hormone measurement on LD 14.

Measurement of Blood Hormones
Sample Collection
All mature animals (except for the animal which had not delivered by GD 25) were fasted overnight (for 16 to 21 hours) from the day of the final administration. Then, all males and females in all dose groups were subjected to laparotomy under isoflurane anesthesia and approximately 4 mL of blood was collected via the abdominal aorta and put into blood collecting tubes containing coagulation promoting agent (Venoject II-Autosep, Terumo Corporation, Japan). Blood samples were centrifuged (at approximately 3,000 rpm, at approximately 1,600 × g, for approximately 10 minutes), and the sera were obtained. For the purpose of the measurement of T4, part of serum samples (approximately 0.5 mL × 3) separated were preserved in a deep freezer at −80 °C (acceptable range: −70 °C or lower).
Sending Samples
For the serum samples of males and dams obtained as described above and serum samples of pups (PND 4 and 13) obtained as described below, the serum was packed with dry ice in a frozen state and sent to the test site (to the Principal Investigator).
Samples for Measurement
Blood hormone (T4) concentration was measured for males and pups on PND 13. Because no abnormality was observed in the result of the measurement of males and pups on PND 13, it was not measured for the dams on LD14 or pups on PND 4. Measurement was not done for the animal which had not delivered by GD 25.
Measurement of Hormone (T4) Concentration
The measurement of the T4 concentrations in rat sera obtained in this study was carried out at the test site by the RIA method
Oestrous cyclicity (parental animals):
For all females, a vaginal smear was collected every day in the morning from the initial day of administration until successful copulation and observed microscopically. During the pre-mating administration period, the number of estruses, mean number of days from estrus to the next estrus (estrous cycle) and the 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. 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). Vaginal smear pictures on the day of necropsy were collected and classified as above. Females whose vaginal smear showed a periodic change (in order of P, E, M, D, and next P) and had estrous cycle of 4 to 5 days were regarded as normal. Classification results of vaginal smear during the mating period and on the day of necropsy were taken as reference data and excluded from summation.
Litter observations:
On the day of birth, the numbers of liveborns and stillborns were recorded. Both liveborns and stillborns were observed for external abnormalities (including the oral cavity) and sexed (wherever possible). The stillborns were fixed and preserved in Bouin’s solution. For the liveborns, anogenital distance (AGD, unit: 0.1 mm) was measured using a slide gage on PND 0. Liveborn pups were fed by their dams until PND 13, with the day of birth regarded as PND 0. During the lactation period, the pups were observed for mortality and behavior once daily and weighed individually on PNDs 0, 4, 7 and 13, and litter mean values of the body weight calculated for male and female pups. For the pups that died during the lactation period, external surfaces were observed macroscopically, and they were fixed and preserved in Bouin’s solution. After observation and measurement on PND 4, litter size was adjusted to 8 pups (4 males and 4 females as far as possible) by random selection. For all litters, blood (approximately 0.6 mL in
total) was collected using a syringe via the abdominal aorta under isoflurane anesthesia into non-treated sample tubes from 1 to 3 culled pups selected randomly in each litter. Blood samples were centrifuged (at approximately 3,000 rpm, at approximately 1,600 × g, and for approximately 10 minutes), and the sera obtained were pooled and preserved in a deep freezer at −80 °C (acceptable range: −70 °C or lower) for blood hormone measurement. The pups, after blood sampling, were sacrificed by exsanguination via the abdominal aorta under isoflurane anesthesia with the other remaining pups, and were discarded. All pups alive on PND 13 were sexed and male pups examined for the number of nipple/areolae, and the results recorded. For all litters, blood (approximately 1 mL) was collected using a syringe via the abdominal aorta under isoflurane anesthesia, and put into non-treated sample tubes from 1 pup of each sex selected randomly from each litter. Blood samples were centrifuged (approximately 3,000 rpm,
approximately 1,600 × g, approximately 10 minutes), and the sera obtained were pooled and separated into sample tubes (approximately 0.3 mL × 3), and then preserved in a deep freezer at −80 °C (acceptable range: −70 °C or lower) for blood hormone measurement. The pups were sacrificed by exsanguination via the abdominal aorta under isoflurane anesthesia with other remaining pups after blood sampling, and detailed macroscopic examination conducted 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. The thyroid from 1 pup of each sex for each litter of all dams were removed and fixed by phosphate buffered 10 % formalin, and then preserved after weighing. For the thyroid, the right and left organs were weighed separately, and the relative organ weight per 100 g body weight was calculated from these absolute organ weights and animal’s body weight at necropsy. The absolute and relative weight of thyroid was evaluated for the total value on both left and right sides.
Postmortem examinations (parental animals):
Necropsy
All animals alive on the day following the final administration, after blood collection for the animals for measurement of blood hormones, were sacrificed by exsanguination via the abdominal aorta under isoflurane anesthesia, detailed macroscopic examination was conducted on the organs/tissues throughout the body of each animal, including the external appearance and those in the cephalic, thoracic and abdominal cavities, and the results were recorded. The female that had not delivered by GD 25 was also examined in same manner. For dams which delivered pups, the implantation sites were counted at necropsy.

Organ Weight
The testes, epididymides, prostate, seminal vesicle (including coagulating gland), levator ani and bulbocavernosus muscle, cowper’s gland and glans penis of all males and the ovary and uterus of all females were weighed. The thyroid (including parathyroid) of all animals were weighed. The relative organ weight per 100 g body weight was calculated from these absolute organ weights and animal’s body weight at necropsy. For the bilateral organs that were marked with, the right and left organs were weighed separately, but evaluation was conducted on the total of both sides. The female that had not delivered by GD 25 was also examined, but the data were excluded from statistical analysis.

Histopathology
The organs/tissues listed (see any other information on materials and methods) were removed from all mature animals and fixed in phosphate buffered 10 % formalin. The testis and epididymis were fixed in Bouin’s solution, and these organs were preserved in phosphate buffered 10 % formalin. For all animals in each group at the end of administration and females that had not delivered by GD 25, the subject organs/tissues were then embedded in paraffin, sections prepared and subjected to hematoxylin and eosin (H&E) staining. Microscopic examination was done first for all the subject organs/tissues in the control group and high dose group in the necropsy group at the end of the administration period and for the undelivered female and males for which the mating partner was infertile. Bilateral organs were removed bilaterally and both sides were examined microscopically for the thyroid, parathyroid, testis, ovary, epididymis, seminal vesicles (including coagulating gland), uterine cervix and both uterine horns.

Postmortem examinations (offspring):
See Litter observations
Statistics:
The differences between the control group and each dosage group were analyzed for statistical significance by the procedure described below. A computer (B-TOX system, Nikko Tsushin Co., Ltd.) was used for the statistical analysis. For body weight, body weight gain (males: day 1 to day 29 of administration, females: day 1 to day 15 of administration, GD 0 to 20, LD 0 to 13, pups: PND 0 to 13), food consumption (including of total during the pre-mating administration, gestation or lactation period), number of estruses, estrous cycle, number of days until copulation, gestation length in days, number of implantation sites, number of liveborns, AGD, number of nipple/areolae and organ weight (including body weight at necropsy), 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). Heterogeneous data were analyzed by Steel's test (levels of significance: 0.05 and 0.01, two-tailed). 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).
Reproductive indices:
Index of animals with abnormal estrous cycle (%) = (No. of animals with abnormal estrous cycle / No. of animals examined) × 100
Copulation index (%) = (No. of copulated animals / No. of mated animals) × 100
Insemination index (%) = (No. of males which impregnated females / No. of copulated males) × 100
Fertility index (%) = (No. of pregnant females / No. of copulated females) × 100
Gestation index (%) = (No. of females which delivered liveborns / No. of pregnant females) ×100
Gestation length in days (Day) = No. of days from GD 0 to a delivered day
Post-implantation loss index (%) = [(No. of implantation sites - No. of liveborns) / No. of implantation sites) × 100
Delivery index (%) = (No. of delivered pups / No. of implantation sites) × 100
Offspring viability indices:
Index of external abnormalities (%) = (No. of liveborn pups with external abnormalities / No. of liveborns) × 100
Live birth index (%) = (No. of liveborns / No. of delivered pups) × 100
Viability index on PND 4 (%) = (No. of live pups on PND 4 / No. of liveborns) × 100
Viability index on PND 13 (%) = (No. of live pups on PND 13 / No. of pups culling on PND 4) × 100
Sex ratio of liveborns on PND 0 (%) = (No. of male liveborns / No. of liveborns) × 100
Sex ratio of live pups on PND 4 (%) = (No. of male live pups on PND 4 / No. of live pups on PND 4) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The males and females in the 50, 250 and 1000 mg/kg groups showed colored feces (red) from the day following the start of administration throughout the administration period including the gestation and lactation periods. However, it was a change reflecting the excretion of the test article and thus
considered to be of no toxicological significance. Except for these changes, there were no abnormalities in general condition in any group.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in any group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males and females, no significant differences were noted in the body weight or body weight gains during the administration period between the control group and any test article administration group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, no significant differences were noted in the food consumption during the pre-mating administration period between the control group and any test article administration group.
In females, food consumption showed a significantly low value in comparison with that in the control group on GD 11 in the 1000 mg/kg group. However, since this change in food consumption of females was transient and slight, it was judged to be of no toxicological significance.
Description (incidence and severity):
There were no significant differences between the control group and any test article administration group in T4 in males at the end of the administration period or in the pups on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Granulomatous inflammation with foreign body was found in the lung of a female in the 1000 mg/kg group (Animal No.: 4103), who had macroscopically dark red focus in the lung. This change was considered to be an incidental change caused by aspiration of the test article and no test article-related toxicological effects. Other lesions were judged not to be treatment-related changes from the incidence of their occurrences or histopathological characteristics.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Delivery Findings, Delivery and Nursing Condition
There were no statistically significant differences between the control group and any test article administration group in the gestation length in days, gestation index, number of implantation sites, post-implantation loss index, delivery index, live birth index, number of liveborn pups, number of stillborn pups or the index of external abnormalities. In the delivery condition of the pregnant animals, all females except 1 female in the 50 mg/kg group (Animal No.: 2102) delivered during the period from GD 21 to 22, and the treatment of the placenta and amnion was normal. In the lactation condition, there were no abnormalities in the gathering of pups, nesting or lactation behavior in any dam. For the undelivered animal (Animal No.: 2102), it was considered to be unrelated to administration of the test article since
there were no histological abnormalities suggestive of the causes of the change in the female, the incidence was dose-unrelated, and the incidence was only one and thus low.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant differences were noted in the index of animals with abnormal estrous cycle, in the number of estruses or estrous cycle during the pre-mating administration period between the
control group and any test article administration group.
Reproductive performance:
no effects observed
Description (incidence and severity):
All pairs had successful copulation. No significant differences were noted in the number of days until copulation, copulation index or fertility (insemination) index between the control
group and any test article administration group.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
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
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
reproductive function (oestrous cycle)
reproductive performance
other: Blood hormone

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For the undelivered animal (Animal No.: 2102), it was considered to be unrelated to administration of the test article since there were no histological abnormalities suggestive of the causes of the change in the female, the incidence was dose-unrelated, and the incidence was only one and thus low.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences were noted in body weight from PND 0 to 13 in males or females between the control group and any test article administration group.
There were no externally gross abnormalities in any pups that died.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In the weight of the thyroid in male and female pups, 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):
There were no gross pathological changes in any pups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital Distance (AGD) of Liveborns
There were no statistically significant differences between the control group and any test article administration group in the AGD of male or female pups.

Viability of Pups
In the viability index on PND 4 and 13, there were no significant differences between the control group and any test article administration group.

Sex Ratio of Pups
There were no changes that are considered to be related to administration of the test article. There was a statistically significant low value in the sex ratio of the liveborn pups on PND 0 in
the 250 mg/kg group in comparison with that of the control group; however, it was judged to be an incidental difference since it was not a dose-related change.

The Number of Nipple/Areolae of Male Pups
There were no significant differences between the control group and any test article administration group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Discussion

R507-2 was administered orally by gavage at 0 (control group: 5.0 w/v % gum arabic solution), 50, 250 and 1000 mg/kg/day to groups of Sprague-Dawley strain SPF rats [Crl:CD(SD)] each consisting of 12 males and 12 females for 14 days before mating throughout the mating period until the day before necropsy to males (29 days in total), for 14 days before mating and during the mating and gestation periods until LD13 to females (51 to 54 days in total) in order to evaluate the effects of R507-2 on males and females, especially reproductive performance such as gonadal function, mating behavioral, conception, development of the conceptus and parturition in rats.

No deaths occurred in any dose group. In the 50, 250 and 1000 mg/kg groups, all males and females showed colored feces (red) from the day following the start of administration throughout the administration period; however, it was a change reflecting the excretion of the test article and thus considered to be of no toxicological significance. No test article-related toxicological effects were noted in the body weight or food consumption, hormones in blood (T4), or in organ weights (thyroid and reproductive organs) in any test article administration group. In the histopathological examinations, granulomatous inflammation with foreign body was found in the lung of 1 female in the 1000 mg/kg group, which was macroscopically consistent with the dark red focus. This foreign body was considered to be the test article because it showed the same color tone as the test article, and this change was considered to be an incidental change caused by aspiration of the test article. Therefore, there were no test article-related toxicological effects in the gross or histopathological findings in any test article administration group.

There were no test article related changes in the parameters relating to the reproductive functions (estrous cycle, mating data, or delivery data) and in the organs of the reproductive system in any test article administration group, and thus it was judged that there were no toxic effects in the copulation, insemination or fertility of males or females or on the maintenance of gestation or delivery of dams even in the 1000 mg/kg group.

Since there were no test article related changes in any examination item of liveborns (AGD, number of nipple/areolae, sex ratio, viability index, body weight, hormones in blood (T4), macroscopic observations and thyroid weight), it was judged that there were no toxic effects on the intra-uterine development, growth, differentiation or viability of pups.

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 R507-2 under the conditions of this study. Therefore, it was judged that the no observed adverse effect level (NOAEL) of the test article by repeated administrations was 1000 mg/kg for both males and females. The NOAEL for the reproductive and developmental toxicity was judged to be 1000 mg/kg for male parent animals, dams and pups.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, no observed adverse effect levels (NOAEL) for the general toxicity in males and females and for the reproductive and developmental toxicity were estimated to be 1000 mg/kg/day.
Executive summary:

R507-2 was administered orally by gavage to groups of Sprague-Dawley strain SPF rats [Crl:CD(SD)], each group consisting of 12 males and 12 females at dose levels of 50, 250 and 1000 mg/kg once daily for 14 days before mating and throughout the mating period until the day before necropsy for males (29 days in total), and throughout the gestation period until day 13 post-partum for females (51 to 54 days in total), in order to evaluate the effects of R507-2 in males and females especially on reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition in rats. The animals in the control group received 5.0 w/v % gum arabic solution, the vehicle for R507-2, alone in the same manner.

Effects on Males and Females

No deaths occurred in males or females in any group. Colored feces (red) which derived from the test article were observed in all males and females of the 50, 250 and 1000 mg/kg groups. However, no test article-related toxicological effects were noted in the body weight or food consumption, blood hormone (T4), organ weights in any test article administration group and there were no test article-related abnormalities in the gross pathological findings or histopathological findings in any test article administration group.

Effects on Reproductive Performance

No test article-related changes were noted in the estrous cycle, number of days until copulation, copulated index or fertility (insemination) index in any test article administration group. In addition, histopathological examination revealed no test article-related changes in the organs of the reproductive system such as testis, epididymis, prostate, seminal vesicle, ovary, uterus and vagina. There were no abnormalities in delivery or nursing conditions of dams.

Effects on Pups

No test article-related changes were noted in the body weight, blood hormone (T4), thyroid weight, anogenital distance, number of nipple/areolae, sex ratio or viability index and there were no abnormalities in the external appearance.

As described above, under the conditions of this study, no observed adverse effect levels (NOAEL) for the general toxicity in males and females and for the reproductive and

developmental toxicity were estimated to be 1000 mg/kg/day.