Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2016 to 28 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2015
Deviations:
no
Principles of method if other than guideline:
For the control group and high dose group, there were additional animals that were dosed but not mated in order to draw comparison with the mating group about the general toxicity.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: Extremely pale yellow liquid
- Storage conditions of test material: Room temperature in the dark under nitrogen
Specific details on test material used for the study:
- Storage conditions of test material: In a dark cold place (actual values: 4 to 8 °C), in a sealed container, purged by nitrogen
- Stability under test conditions: There was no abnormality in the quality of the test material before using or remaining after the end of the administration in the characteristic test

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
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 are available.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks of age at receipt
- Weight at study initiation: The body weight range at the start of administration was 463 to 537 g (mean body weight 488 g) for males and 246 to 295 g (mean body weight 267 g) for females; values were within ± 20 % of the mean value of each sex.
- Housing: The animals were housed in plastic cages (W 440 × D 275 × H 180 mm) with bedding in groups of 2 animals of the same sex until the group allocation (however, when the number of females received were an odd number, 3 females were housed in the last cage). After group allocation, a divider was placed in each plastic cage and each animal was housed individually in each division. After the initial day of mating, males were housed individually in bracket-type stainless-steel wire-mesh cages (W 254 × D 350 × H 170 mm) and two animals (1 male and 1 female) were housed in the same cage during mating. From gestation day 0 until day 13 post-partum, dams were housed with their litters in plastic Econ cages (W 340 × D 400 × H 185 mm) with bedding.
- Diet: NMF (radiation-sterilised, Oriental Yeast Co., Ltd. For environmental enrichment, 7979C.CS certified/irradiated Diamond Twists (Envigo RMS, Inc.), ad libitum via stainless-steel feeders
- Water: ad libitum via an automatic water supply system or using water bottles
- Acclimation period: 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, behaviour and appearance of excrement once daily in the morning. Animals were weighed on the day of receipt and at the end of quarantine and once a week after the end of the quarantine, and the body weight gain from the previous measurement was calculated. In addition, detailed clinical observations (once on day 16 and 17 of acclimation in males and females, respectively), observation of oestrous 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 in the scrotum descent on the last day of acclimation in males were conducted.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C (actual values: 22 to 25 °C)
- Relative humidity: 50 ± 20 % (actual values: 42 to 49 %)
- Air changes: 10 to 15 times per hour
- Photoperiod: 12-hour light cycle (lighting: 07:00 to 19:00)

IN-LIFE DATES
From: 27 April 2016
To: 28 June 2016 (males and non-mated females); 06 July 2016 (pups); 07 July 2016 (dams)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
In accordance with the toxicity study guideline, the oral route was selected.
The method of administration was by gavage, which is routinely used for oral administration to rodents. Dose volume was set at 5.0 mL/kg body weight and the dosing formulation was administered by gavage using a stomach tube. Individual dose volume was calculated based on the animal’s most recent body weight. The animals in the control group received the vehicle only in the same manner. Administration was done between 08:38 and 12:14 except when it was done between 14:16 and 14:37 to animals which had not completed delivery in the morning and had completed it at the observation in the afternoon.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dose concentration, the requisite amount of the test material was weighed accurately in a beaker. Approximately 50 % of the specified volume of the vehicle was added little by little and the test material was dissolved completely in the vehicle by stirring using a magnetic stirrer. After confirming that dissolution was complete, the solution was transferred to a measuring cylinder. The beaker was washed several times with a small amount of the vehicle and the washings were also put into the measuring cylinder. The vehicle was added to the measuring cylinder to the specified volume to prepare the test solution at the specified concentration.
- Rate of preparation (frequency): Preparation was done for 7-day aliquots at maximum at a time, and the prepared solutions were put into brown glass bottles.
- Storage temperature: Stored in a cold place (in a refrigerator, acceptable values: 1 to 10 °C, measured values: 3 to 5 °C), and used for administration within the time for which stability had been verified (2.00 and 200 mg/mL for 8 days in a cold place (in a refrigerator, acceptable values 1 to 10 °C) and then for 24 hours at room temperature (acceptable values 1 to 30 °C)).

VEHICLE
- Concentration in vehicle: 20, 60 and 150 mg/mL
- Amount of vehicle (if gavage): Dose volume 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A total of two times, once in the initial administration and once in week 6 of administration, the test solution of each dose concentration to be used for administration was analysed for the test material. In addition, for the test solution used for the initial administration, it was confirmed before the start of administration. One fraction (10 mL) was collected from each concentration and analysed by GC method.
The percentage of the the nominal concentration ranged from 101.8 to 103.0 %. All concentrations were confirmed to be within the acceptable range [percentage of the nominal concentration is within 100.0 ± 10.0 %]. The analytical method validation was determined at the testing facility.

SUMMARY OF ANALYTICAL GC METHODS
The test material was used as the reference standard. For the preparation of measurement samples, samples were diluted in acetone with a dilution factor of 200, 600 and 1500 for the low, medium and high dose levels, respectively. Test samples were sampled by n = 1

GC SYSTEM AND CONDITIONS
- GC: HP6890N (Agilent Technologies Inc.)
- Injector: G2613A (Agilent Technologies Inc.)
- Autosampler tray: G2614A (Agilent Technologies Inc.)
- Data processor: GC ChemStation G2070AJ (Agilent Technologies Inc.)
- Column: DB-1 (0.32 mm I.D. × 30 m, film thickness 0.25 µm, Agilent Technologies Inc.)
- Carrier gas: Helium
- Flow mode: Constant flow mode
- Flow rate: 3.0 mL/min
- Injection port: Split injection port
- Split ratio: 10:1
- Injection port temperature: 250 °C
- Detection: Flame ionisation detector (FID)
- Detection temperature: 280 °C
- Flow rate of hydrogen: 45 mL/min
- Flow rate of air: 450 mL/min
- Flow rate of make-up gas (N2): 30 mL/min
- Temperature of column oven: 100 °C (Hold 0 min) to 150 °C (10 °C/min, Hold 0 min) to 250 °C (40 °C/min, Hold 3 min)
- Injection volume: 1 μL
Duration of treatment / exposure:
The length of the administration period was 42 days for males (14 days prior to mating and 28 days after the start of mating), 51 to 63 days for females in the mating group (14 days prior to mating and throughout the mating and gestation periods until day 13 of lactation), 40 days for the not delivered female and 42 days for females in the non-mated group. The recovery period was 14 days after the end of the administration during which administration was withdrawn.
Frequency of treatment:
Once every day (7 times/week)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
The number of animals per group was 12 animals of each sex for the mating group and 10 females each for the control group and high dose group for the non-mated group to compare it with the mating group about the general toxicity. For 5 males and 5 females in the control group and high dose group, the test material was withdrawn after the final administration and they were regarded as recovery animals.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral gavage toxicity range-finding study in rats at dose levels of 250, 500 and 1000 mg/kg bw/day. No death occurred in males and females at 1000 mg/kg bw/day, but decreased spontaneous movement and excoriation (antebrachial or crural region) in males and females, swelling of extremities in females and suppressed body weight gain associated with low values in food consumption in males were observed. In addition, males and females at 1000 mg/kg bw/day showed low values in the red blood cell count, haemoglobin and haematocrit, high values in reticulocyte count, AST and ALT and high values in spleen, lung and liver weight. Remarkable toxic change was not detected in males and females at 500 mg/kg/day. Therefore, considering that the length of the administration period was more than 3 times that of the dose range finding study, 750 mg/kg bw/day (which was the median of the high-dose and middle-dose) was selected as a highest dose and 300 and 100 mg/kg bw/day were selected as the middle and low doses, respectively.
- Rationale for animal assignment: The animals with favourable body weight gain that were selected 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 first day of administration.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs including mortality, external appearance, posture, behaviour and excrement 3 times a day, before dosing, immediately after and 1-3 hours after dosing during the administration period (but twice a day, before dosing and immediately after dosing, on the days for detailed clinical observation), and once a day (in the morning) during the recovery period. On the day of necropsy, clinical observation was performed once. The periodical in-the-hand observation of animals was done on day 1 and 2 of administration and thereafter as detailed clinical observation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observation was done for all animals. It was done once a week during the administration period and recovery period for males and non-mated females, and once a week during the pre-mating administration period and on the designated days during the mating, gestation and lactation periods for females in the mating group (on days 6 or 13 of mating period for non-copulated female, on GDs 1, 7, 14 and 20 for the copulated females and on LDs 6 and 13 for the females that delivered). The observation during the administration period was conducted after dosing. For the results of observation/examination of the detailed clinical observation and manipulative test, the data except numeric data were recorded using the scored evaluation method. For the above observations, examinations and measurements, animals were placed at random and the observer was restricted from access to information such as dose level (blind process).

- Detailed clinical observations: The animals were observed for the following: Posture, convulsion and abnormal behaviour in the home cage observation; ease of removal from cage, fur and skin condition, secretions in eyes and nose, exophthalmos, palpebral closure, visible mucosal membranes, autonomic nervous functions (lacrimation, salivation, piloerection, pupil size, abnormal respiration), and reactivity and vocalisation to handling in the in-the-hand observation; and arousal, convulsion, abnormal behaviour, stereotypy, gait, posture, grooming, number of rearings, excretions (number of defecations and urination) in the open field observation.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 1, 3, 6, 13 and 20 of acclimation. Males in the mating group and females in the non-mated group were weighed on days 1, 8, 15, 22, 29, 36 and 42 of administration and on the day of necropsy, animals in the recovery group on days 1, 8 and 14 of recovery and the day of necropsy additionally. Females in the mating group were weighed on days 1, 8, 15 and 22 of administration and on GDs 0, 7, 14 and 20 and on LDs 0, 4, 7 and 13 and the day of necropsy. Body weight was measured before dosing during the administration period; however, the animals that delivered in the afternoon were weighed after completion of parturition.
The body weight data on the day of necropsy of the female that had not delivered by GD 25 and dams with total litter loss during the lactation period was excluded from statistical analysis.

FOOD CONSUMPTION: Yes
- Time schedule: The amount of food remaining was measured for all animals; males in the mating group and females in the non-mated group on days 2, 8, 15, 30, 36 and 42 of administration, recovery group animals on days 2, 8 and 14 of recovery additionally, and females in the mating group on days 2, 8 and 15 of administration, GDs 1, 7, 14 and 20 and LDs 2, 4, 7 and 13. Daily food consumption of each animal was then calculated from the amount of the feed provided on the previous day. Measurements of the amounts of feed supplied and remainder were conducted before dosing during the administration period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The day after the final administration or the day following day 14 of recovery. Approximately 1 mL of blood was collected via the abdominal aorta into blood collecting tubes containing EDTA-2K. Blood samples (0.9 mL) collected with test tubes containing 3.8 % sodium citrate (1 volume to 9 volumes of blood) were centrifuged (approximately 3000 rpm, approximately 1600 × g, approximately 10 minutes) and plasma samples obtained were examined. Furthermore, May-Grünwald-Giemsa stained blood smears of all animals were prepared for microscopic examination; however, microscopic examination was not done since it was judged unnecessary.
- Anaesthetic used for blood collection: Yes, under isoflurane anaesthesia
- Animals fasted: Yes. All animals were fasted overnight (for 16 to 20 hours)
- How many animals: 5 males and 5 females in all dose groups were subjected to laparotomy. The not copulated females, the female that had not delivered by GD 25 and dams with total litter loss during the lactation period were also examined, but the data were excluded from statistical analysis.
- Parameters examined in the EDTA-2K blood samples: Red blood cell count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocyte count (RETIC), platelet count (PLT), white blood cell count (WBC) and differential white blood cell count (lymphocyte (LYMP), neutrophil (NEUT), eosinophil (EOS), basophil (BASO), monocyte (MONO) and large unstained cell (LUC)).
- Parameters examined in the sodium citrate blood samples: Prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples (approximately 6 mL) that were collected at the same time as for haematological examination were put into test tubes containing coagulation promoting agent (Venoject II-Autosep) and centrifuged (approximately 3000 rpm, approximately 1 600 × g, approximately 10 minutes) and the sera obtained were examined. Blood samples (approximately 2 mL) which were collected into heparinised test tubes (approximately 20 units of heparin per 1 mL blood) were centrifuged (approximately 3000 rpm, approximately 1600 × g, approximately 10 minutes) and the plasma obtained was examined.
- Animals fasted: Yes. All animals were fasted overnight (for 16 to 20 hours)
- How many animals: 5 males and 5 females in all dose groups were subjected to laparotomy. The not copulated females, the female that had not delivered by GD 25 and dams with total litter loss during the lactation period were also examined, but the data were excluded from statistical analysis.
- Parameters examined in sera: ALP, total bile acids (T-BA), total cholesterol (T-CHO), triglyceride (TG), total bilirubin (T-BIL), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRNN), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (P), total protein (TP), albumin (ALB) and A/G ratio (A/G).
- Parameters examined in the heparin blood samples: AST, ALT and γ-GTP.
- Measurement of blood hormones: For the purpose of the measurement of T4 and TSH, the part of serum samples (approximately 0.5 mL × 3) separated were preserved in a deep freezer at -80 °C. For the serum samples of males, dams and non-mated females obtained as described, and serum samples of pups (pups of 5 litter mainly conducted blood chemistry examination in the mating group) were examined for T4 and TSH. As abnormality was observed in a result of a measurement about males and pups on PND 13, it was measured for the non-mated female, dams on LD14, pups on PND 4 and recovery animals. Measurement was not done for the not copulated females, the animal which had not delivered by GD 25 and dams with total litter loss during the lactation period. The serum samples remaining after examination were reserved for use in the measurement of blood hormones.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was done in the final week of administration (days 36 and 37 in week 6 of administration) and in the final week of recovery (days 8 and 9 in week 2 of recovery). The examination during the administration period was conducted after dosing.
- Metabolism cages used for collection of urine: Yes. The 5 subject animals from each group and 5 animals of each sex in the control and high dose groups during the recovery period were placed in the cages with urine collectors.
- Animals fasted: 4-hour urine was collected under deprivation of feed but with free access to water and then 20-hour urine was collected with free access to feed and water.
- Parameters examined: pH, protein, ketones, glucose, occult blood, bilirubin, urobilinogen, specific gravity, colour, sediments and urine volume (4-hour and 20-hour).
The items from pH to sediments were examined and urine volume measured using the 4-hour urine, while urine volume were measured using the 20-hour urine obtained thereafter. One-day urine volume was calculated by totalling the 4- and 20-hour urine volume. The supernatant of the 20-hour urine collected by centrifugation was preserved in a deep freezer at -40 °C for possible examination, but supplemental analysis was not done.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The manipulative test and measurement of grip strength and motor activity were done for 5 males, 5 females in each mating group and 5 females in each non-mated group. They were done on LD 13 (day 51 to 54 of administration) during the administration period on females in the mating group, in the final week of administration [on day 38 and 39 (week 6) of administration for males and females, respectively] on the other animals and in the final week of the recovery period [day 10 and 11 (week 2) of recovery for males and females, respectively] for males and females in the control and high dose groups during the recovery period. For the measurement of motor activity, the process was not blind.
- Battery of functions tested: Animals were examined in a manipulative test for auditory response, approach response, touch response, tail pinch response, pupillary reflex and aerial righting reflex. Grip strength of the forelimb and hindlimb was measured using a CPU gauge MODEL-RX-5 (Aikoh Engineering Co., Ltd.). Measurement was done twice for both forelimbs and hind limbs and mean values were calculated. Motor activity was measured using an Experimental Animal Motor Activity Sensor NS-AS01 (NeuroScience Inc.). The length of measurement was 1 hour and values of 10-minute intervals and 0 to 60 minute value were recorded.

IMMUNOLOGY: No

OTHER: Vaginal smear examinations, observation of dams for delivery and nursing and observation of liveborn pups took place.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy
All animals alive on the day following the final administration or on the last day of the recovery period, after blood collection for haematology, blood chemistry and measurement of blood hormones, were sacrificed by exsanguination via the abdominal aorta under isoflurane anaesthesia.
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 not copulated females, the female that had not delivered by GD 25 and dams with total litter loss during the lactation period were also examined in same manner. For dams which delivered pups, the implantation sites were counted at necropsy.

- Organ Weight
The testes*, epididymides*, levator ani and bulbocavernosus muscle, cowper’s gland* and glans penis of all males in the mating group 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. However, the not copulated females, the female that had not delivered by GD 25 and dams with total litter loss during the lactation period were also examined, but the data were excluded from statistical analysis.

HISTOPATHOLOGY: Yes
The organs/tissues listed in the table below were removed from all animals and fixed in phosphate buffered 10 % formalin. At the time of fixation, phosphate buffered 10 % formalin was injected in the lung. The eyeballs and optic nerves were fixed in phosphate buffered 3 % glutaraldehyde/2.5 % formalin and the testis and epididymis fixed in Bouin’s solution, and these organs were preserved in phosphate buffered 10 % formalin.
For the five animals in each group (both sexes in mating group and non-mated female) on which blood sampling was conducted at the end of administration or recovery and not copulated animals (male and female), the female that had not delivered by GD 25 and dams with total litter loss during the lactation period, the subject organs/tissues were then embedded in paraffin, sections prepared and subjected to haematoxylin 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 not copulated animals, the undelivered female and dams with total litter loss. Bilateral organs were removed bilaterally and the one side were examined microscopically for the sciatic nerve, eyeball, adrenal, kidney, mammary gland (inguinal region), femur (including bone marrow) and femoral skeletal muscle; both sides were examined for the thyroid, parathyroid, testis, ovary, epididymis and seminal vesicles (including coagulating gland), uterine cervix and both uterine horns. For the one side of optical nerves and skin (inguinal region), H&E stained specimens were prepared but they were not examined microscopically. The sections of the liver, mammary gland (inguinal region), ovary and thymus from animals in the middle and low dose groups and recovery group were examined, as treatment-related changes were suspected in these organs. The examination was also extended to the femur (including bone marrow) of animals in the middle and low dose groups and recovery group as treatment-related changes were suspected; however, it was finally judged that there were no treatment-related changes in this organ.
Other examinations:
DETAILS ON MATING
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 was designated as GD 0. The number of days until copulation was counted with the first day of mating regarded as day 0. The length of the mating period for the same male and female was 14 days at maximum.
For the two females for which copulation was not confirmed (one in the control and one in the 750 mg/kg group) the last day of the mating period was tentatively designated as GD 0, and clinical observations, measurements of body weight and food consumption and administration were continued. Twenty-five days later, blood was collected under anaesthesia with isoflurane inhalation, and the animal was sacrificed by exsanguination via the abdominal aorta and subjected to pathological examinations. Since implantation was not confirmed in these females, they were judged to be not copulated females; therefore, the data of these animals after the last day of the mating period was excluded from statistical analysis except for copulation index.

VAGINAL SMEAR EXAMINATION
For all females in the mating group, a vaginal smear was collected every day in the morning from the start day of administration until successful copulation and observed microscopically. During the pre-mating administration period, the number of oestruses, mean number of days from oestrus to the next oestrus (oestrous cycle) and a ratio of the females which showed oestrous cycle abnormality for the females examined were determined using the vaginal smears consisting of many cornified epithelial cells as an index for oestruses. During the mating period, vaginal smears were examined for the presence of sperm and classified as pro-oestrus (P), oestrus (E), metoestrus (M) and dioestrus (D). Vaginal smear pictures on the day of necropsy were collected and classified as above.
For all females in the non-mated group, a vaginal smear on the day of necropsy was collected in the morning and classified as above, and results were assumed reference data.
Females whose vaginal smear showed a periodic change (in order of P, E, M, D, and next P) and had oestrous cycle of 4 to 5 days were regarded as normal.

DELIVERY AND NURSING
1) 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 units 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 behaviour such as gathering pups, nesting and lactation as indices.
The female in the 300 mg/kg group which had not delivered by the morning of GD 25 was sacrificed by exsanguination via the abdominal aorta under isoflurane anaesthesia and subjected to pathological examinations. As retention of conceptus was recognised in the uterus, it was judged that this female was pregnant and the numbers of corpora lutea and implantation were counted, and condition of conceptus was recorded. One dam in the control group and 6 dams in the 750 mg/kg group that had total litter loss during the lactation period were sacrificed by exsanguination via the abdominal aorta under isoflurane anaesthesia after blood collection and necropsied, and the numbers of implantation sites counted. Dams which delivered pups were subjected to pathological examinations on LD 14.

2) Observation of Liveborn Pups
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. The liveborns had anogenital distance (AGD, unit: 0.1 mm) measured using a slide gauge on PND 0. Liveborn pups (including pups with external abnormalities) 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 behaviour once daily and weighed individually on PNDs 0, 4, 7 and 13 and litter mean values of the body weight were calculated for male and female pups. For the pups that died during the lactation period, external surfaces were observed macroscopically and fixed and preserved in Bouin’s solution.
After observation and measurement on PND 4, the pups were culled randomly to 4 males and 4 females per litter as far as possible. For 5 litters (but 4 litters in the 300 mg/kg group) in the order of delivery of each group, blood (approximately 0.6 mL in total) was collected using a syringe via the abdominal aorta under isoflurane anaesthesia into non-treatment sample tubes from 2 or 3 culled pups selected randomly from each litter. However, the blood collection was not carried out in all litters in the 750 mg/kg group because the necessary numbers of culled pups were not provided. Blood samples were centrifuged (approximately 3000 rpm, approximately 1600 × g, approximately 10 minutes) and the sera obtained were pooled and preserved in a deep freezer at -80 °C for blood hormone measurement. The pups, after blood sampling, were sacrificed by exsanguination via the abdominal aorta under isoflurane anaesthesia with the other remaining pups, and were discarded.
All pups alive on PND 13 were sexed and male pups were examined for the number of nipple/areolae and the results were recorded. For 5 litters in the order of delivery of each group, blood (approximately 1 mL) was collected using a syringe via the abdominal aorta under isoflurane anaesthesia into non-treatment sample tubes from 1 pup in each sex selected randomly from each litter. Blood samples were centrifuged (approximately 3000 rpm, approximately 1600 × 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 for blood hormone measurement. The pups were sacrificed by exsanguination via the abdominal aorta under isoflurane anaesthesia with other remaining pups after blood sampling, detailed macroscopic examination was 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; the relative organ weight per 100 g body weight was calculated from the total value of these absolute organ weights and animal’s body weight at necropsy.
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
CAGE-SIDE OBSERVATIONS
1) Administration Period
In males, decreased spontaneous movement was observed at 750 mg/kg bw/day from day 6 to day 35 of administration and swelling in limbs from day 18 to day 38 of administration, both sporadically in 1 to 3 animals. One animal in the 300 mg/kg bw/day group (Animal No. 3010) showed haemorrhage in the oral cavity on day 6 of administration and pale skin from day 6 to day 11 of administration; however, they were considered to be changes unrelated to administration of the test material judging from the incidence of their occurrences.
In females in the mating groups, decreased spontaneous movement was observed sporadically in 1 to 4 females from day 5 to day 15 of administration and swelling of limbs in 1 female from day 0 to day 7 of gestation; however, there were no abnormalities during the lactation period. For 1 dam in the control group (Animal No.:1104) and 2 dams in the 750 mg/kg group (Animal Nos. 4106 and 4110), the whole litter died on day 1 of lactation.
In the females in the no-mating group, a decrease in spontaneous movement was recorded sporadically in 1 to 2 females from day 5 to day 15 of administration.

2) Recovery Period
There were no abnormalities in males or females in the recovery group of the 750 mg/kg bw/day group.

DETAILED CLINICAL OBSERVATION - HOME CAGE OBSERVATION
1) Administration Period
In males, 2/12 males in the 750 mg/kg bw/day group showed flattened posture in week 2 of administration.
In females in the mating group, 3/12 females in the 750 mg/kg bw/day group showed flattened posture in week 2 of administration. In dams, there were no effects from administration of the test material during the gestation or lactation period.
There were no abnormalities in females in the no-mating group.

2) Recovery Period
There were no abnormalities in males or females in the recovery group of the 750 mg/kg group.

DETAILED CLINICAL OBSERVATION - IN-THE-HAND OBSERVATION
1) Administration Period
In males, at the time of taking out from the cage and handling in week 2 of administration, there were 2/12 animals that showed only slight reaction and were atypically docile to the handling. In this group, salivation (slight) was observed in 3/12 animals in week 3 of administration and scratched wound (slight) in 2/12 and 1/12 animals in weeks 4 and 5 of administration, respectively. Otherwise 1 male in the 300 mg/kg bw/day group (Animal No.: 3010) showed pale skin (slight) in week 2 of administration; however, it was judged to be unrelated to administration of the test material since it was the animal that showed haemorrhage in the oral cavity and pale skin.
In females in the mating group, at the time of taking out from the cage and handling in week 2 of administration, there was 1 female that showed only slight reaction and was atypically docile to the handling among the 12 females in the 750 mg/kg group. In dams, there were no effects from administration of the test material during the gestation or lactation period.
In the females in the no-mating group, 1/10 females showed only slight reaction and was atypically docile to handling when it was taken out of the cage in week 2 of administration. In this group, salivation (slight) was observed in 1, 2 and 1 animal in weeks 3, 5 and 6 of administration.

2) Recovery Period
There were no abnormalities in males or females in the recovery group of the 750 mg/kg bw/day group.

DETAILED CLINICAL OBSERVATION - OPEN FIELD OBSERVATION
1) Administration Period
There were no abnormalities in males.
In females in the mating group, there were 3/12 females that showed slight abnormal gait (ataxic gait) in the 750 mg/kg group and the number of rearings in this group was significantly lower than in the control group. There were no effects from administration of the test material in dams during the gestation or lactation period.
In females in the non-mating group, 2/10 females in the 750 mg/kg bw/day group showed slight abnormal gait (ataxic gait) in week 1 of administration and the number of rearings in this group showed significantly low values compared to that of the control group in weeks 1 and 3 of administration.

2) Recovery Period
In males in the 750 mg/kg bw/day group, the number of rearings in week 1 of recovery was significantly lower than in the control group; however, it was judged to be an incidental significant difference since there were no abnormalities during the administration period or in week 2 of recovery.
In females in the 750 mg/kg bw/day group, there were no abnormalities.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in any dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Administration Period
In males, there were significantly low values in the body weight on day 22 of administration and in the body weight gain during the administration period in the 750 mg/kg bw/day group compared to those of the control group.
In females in the mating group, significantly low values in the body weight gain during the gestation period were recorded in the 750 mg/kg bw/day group compared to those of the control group. The body weight of the animals in this group was significantly lower than in the control group on day 4 of lactation; however, it was judged to be an incidental significant difference since it was a change only at one time point and the body weight gain during the lactation period was higher than in the control group.
In females in the no-mating group, there were significantly higher values in the body weight in the 750 mg/kg bw/day group on days 15, 22 and 36 of administration and body weight gain during the administration period; however, they were judged to be incidental changes since there were no such tendencies in males or in the females in the mating group.

2) Recovery Period
There were no significant differences in males or females in the 750 mg/kg bw/day group compared to those in the control group. The body weight gain of the males in the 750 mg/kg bw/day during the recovery period was higher than that of the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1) Administration Period
In males, the food consumption in the 750 mg/kg bw/day group on day 2 of administration was significantly lower than in the control group.
In females in the mating groups, significantly low values for food consumption compared to those of the control group were recorded on day 2 of administration, on day 20 of gestation and on days 2, 7 and 13 of lactation, and significantly low values for food consumption on days 2, 4, 7 and 13 of lactation and in the total food consumed during the lactation period in the 300 mg/kg group. Otherwise the food consumption in the 100 mg/kg bw/day group on day 2 of administration was significantly higher than in the control group; however, it was dose-unrelated change and thus judged to be incidental.
In females in the non-mating group, the food consumption of the animals in the 750 mg/kg bw/day group was significantly lower than in the control group on day 2 of administration.

2) Recovery Period
There were no significant differences in males or females in the 750 mg/kg bw/day group from those of the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
1) End of Administration Period
There were no changes that are considered to be related to administration of the test material in any animal.
In males, the platelet count in the 300 mg/kg bw/day group was significantly higher than in the control group; however, it was a dose-unrelated change and thus judged to be an incidental significant difference.
In females in the mating groups, the platelet count in the 750 mg/kg bw/day group was significantly lower than in the control group; however, it was judged to be an incidental change since there were no abnormalities in any other parameters in the coagulation system and the change was within the normal range of the historical background data of the test facility. In the 100 mg/kg bw/day group, the mean corpuscular haemoglobin concentration in the 100 mg/kg bw/day group was significantly lower than in the control group; however, it was judged to be incidental since it was a dose-unrelated change.
In females in the no-mating group, the mean corpuscular volume and the mean corpuscular haemoglobin were significantly higher and the reticulocyte count significantly lower in the 750 mg/kg bw/day group than those in the control group; however, they were judged to be incidental since there were no abnormalities in the red blood cell count or haemoglobin concentration and they were within the range of the normal range of the historical background data of the test facility.

2) End of Recovery Period
There were significantly low values in the platelet count and significant prolongation in the activated partial thromboplastin time in males and significant elongation in the prothrombin time in females in the 750 mg/kg bw/day group compared to those of the control group; however, they were judged to be incidental changes since there were no abnormalities at the end of the administration period and they were within the normal range of the historical background data of the test facility.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) End of Administration Period
In males, ALT, ALP, albumin and A/G ratio in the 750 mg/kg bw/day group were significantly higher than those of the control group and albumin was significantly higher in the 300 mg/kg bw/day group. In the 300 mg/kg bw/day group, a significantly lower value in chlorine was recorded in comparison with that in the control group; however, it was a dose-unrelated change and thus judged to be incidental.
In males, a significantly lower value in T4 compared to that of the control group was recorded in the 750 mg/kg bw/day; however, there were no abnormalities in TSH.
In females in the mating group, A/G ratio in the 750 mg/kg bw/daygroup was significantly higher than in the control group. Sodium was significantly higher in the 300 mg/kg bw/day group than in the control group; however, it was judged to be an incidental change since it was a dose-unrelated change.
In females in the no-mating group, significantly high values in ALP, albumin and A/G ratio compared to those in the control group were recorded at 750 mg/kg bw/day. At 750 mg/kg bw/day, the total cholesterol and inorganic phosphorus were significantly higher than in the control group; however, they were judged to be incidental since such changes were not recorded for males or for females in the mating group, and they were within the normal range of the historical background data of the test facility. Otherwise total bilirubin in the 750 mg/kg bw/day group was significantly lower than in the control group; however, since it was a low value, it was judged to be of no toxicological significance and an incidental change.
In females in the mating groups, there were no significant differences in T4 or TSH in any test material administration group compared to those of the control group.
In females in the non-mating group, a significantly low value in T4 compared to that of the control group was recorded in the 750 mg/kg bw/day group; however, there were no abnormalities in TSH.

2) End of Recovery Period
In males in the 750 mg/kg bw/day group, there were no significant differences from those in the control group in any test item.
In females in the 750 mg/kg bw/day group, a significantly high value in potassium was recorded in comparison with that in the control group; however, it was judged to be an incidental change since there were no abnormalities in any other electrolyte and the change was within the normal range of the historical background data of the test facility.
There were no significant differences in T4 or TSH from the control group in males or females in the 750 mg/kg bw/day group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
1) Final Week of Administration Period
There were no abnormalities in the qualitative items in any animal; however, the urine volume in males in the 750 mg/kg bw/day group was significantly higher than in the control group.

2) Final Week of Recovery Period
There were no abnormalities in the qualitative items of any animal, and there was no significant difference in the urine volume between the 750 mg/kg bw/day group and the control group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
MANIPULATIVE TEST
There were no effects from administration of the test material in the auditory response, approach response, touch response, tail pinch response, pupillary reflex or air-righting reflex.

GRIP STRENGTH
There were no effects from administration of the test material in any animal.

MOTOR ACTIVITY
There were no effects from administration of the test material in any animal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and any test material administration group in the weight of the thyroid gland or in the weight of the male reproductive organs.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological abnormalities that are considered to be related to administration of the test material in any animal. There were no changes that could cause no-delivery on day 25 of gestation or whole litter loss. In 1 female in the 300 mg/kg bw/day group (Animal No.: 3109) that had not delivered by day 25 of gestation, there was a conceptus including the foetus/placenta of 1 animal in the uterus; however, there were no other implantation sites. However, it was considered to be an incidental abnormality in delivery since there was no tendency toward decrease in the number of implantation sites in any other animal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The changes that were considered to be caused by administration of the test material were observed in the liver (males, females in the mating group and females in the no-mating group), mammary gland (females in the mating group), ovaries (females in the mating group) and in the thymus (females in the mating group).

- Liver
Eosinophilic granular changes in hepatocytes were observed in males, in females in the mating group and in females in the no-mating group at 750 mg/kg bw/day. This change was not observed in recovery animals.

- Mammary glands
Acinar involution was observed in females in the mating groups at 300 and 750 mg/kg bw/day. Since there was a possibility that the mammary gland during the lactation period could be degenerated by the decrease in the incidence of stimuli by lactation, the right side was also examined for confirmation for 7 females (Animal Nos.: 4101, 4102, 4103, 4109, 4111, 1104, and 3109). In the results, 2 animals (Animal Nos: 4101 and 4103) showed a difference between the right and left. Since this change was observed only in females in the mating group and the females in the recovery group had no copulation, the reversibility was unknown.

- Ovaries
The remaining gravid corpus luteum was observed in females in the mating group in the 300 and 750 mg/kg bw/day groups. This change was observed only in the females in the mating group to be necropsied on day 14 of lactation. Since it is a change in the gravid corpus luteum, the reversibility was not confirmed using the animals in the recovery group which were non-copulated females.

- Thymus
Atrophy of the thymus was observed in females in the 750 bw/day mg/kg mating group. The reversibility of this change was unclear since the change was only observed in females in the mating group and the recovery group included only no-mated animals.

There were other changes in addition to the above in various organs; however, they were considered to be incidental based on the incidence of their occurrences and their pathological nature.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
OESTROUS CYCLE
In the 750 mg/kg bw/day group, there was a tendency toward low values in the incidence of oestrus and a tendency toward elongation in the mean oestrous cycle and the percentage of animals showing abnormalities in the oestrous cycle was significantly higher than in the control group.
In the other items, there were no significant differences between the control group and any test material administration group in the index of animals with abnormal oestrous cycle, number of oestruses and the mean duration of cycles (oestrous cycle).

MATING AND FERTILITY
There were no significant differences between the control group and any test material administration group in the number of days until copulation, copulation index, insemination index or fertility index.

DELIVERY FINDINGS, DELIVERY AND NURSING CONDITION
In the 750 mg/kg group, there were 4 dams for which all pups died on day 0 of lactation (Animal Nos.: 4104, 4107, 4108 and 4112). In comparison with the control group, the post-implantation loss index in this group was significantly higher and the number of live-born pups and the live birth index significantly lower than in the control group. In the 100 mg/kg group, the gestation length in days was significantly shorter than that of the control group; however, it was judged to be incidental since it was a dose-unrelated change.
In the other items, gestation index, gestation length in days, number of implantation sites, post-implantation loss index, delivery index, number of live-born pups, live birth index and the index of external abnormalities, there were no significant differences between the control group and any test material administration group. In the delivery conditions of the pregnant animals, all dams except one in the 300 mg/kg group (Animal No.: 3109) delivered 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 behaviour of any dam.

ANOGENITAL DISTANCE (AGD) OF LIVEBORNS
In the normalized AGD of male and female pups in the 750 mg/kg bw/day group, there were significantly higher values compared to those in the control group. However, since there were no abnormalities in the measured values of AGD, in the sex ratio of pups, or in the numbers of papilla / areola of nipple, they were considered to be caused by the low body weight at the time of birth, and thus judged to be incidental changes having no toxicological significance. Otherwise, there were no significant differences between the control group and any test article administration group in the measured values of AGD or normalized AGD in male or female pups.

VIABILITY OF PUPS
A statistically significant low value in the viability index on day 4 after birth compared to that of the control group was recorded for the 300 and 750 mg/kg groups. Otherwise there were no statistically significant differences between the control group and any test material administration group in the viability index on day 4 or day 13 after birth.

SEX RATIO OF PUPS
There were no effects from administration of the test material on the sex ratio of the pups.

BODY WEIGHT OF PUPS
In the 750 mg/kg group, there were statistically significant low values in the body weight of male and female pups on days 0, 4, 7 and 13 after birth and in the body weight gain after birth in comparison with those in the control group. In the 300 mg/kg group, the body weight of male pups on day 4 after birth, body weight of male and female pups on day 7 and 13 after birth and the body weight gain after birth were significantly lower than in the control group. In the body weight of live-born pups in the 100 mg/kg group, there were no effects from administration of the test material.

NECROPSY OF DEAD PUPS
There were no gross pathological abnormalities in any pups that died.

THE NUMBER OF NIPPLES/AREOLAE OF MALE PUPS
There were no significant differences in the number of nipples / areolae of male pups between the control group and any test material administration group.

BLOOD HORMONE LEVELS IN PUPS
In the pups on day 4 and 13 after birth, there were no statistically significant differences in T4 or TSH in any test material administration group from those of the control group.

NECROPSY OF PUPS ON PND 13
There were no macroscopic abnormalities in any pups.

THYROID WEIGHT OF PUPS
There were no effects from administration of the test material in the weight of the thyroid glands in the male and female pups. In the 300 and 750 mg/kg groups, there were significantly low values than in the control group in the body weight of the male and female pups at the time of necropsy. The relative weight of the thyroid glands in male pups in the 750 mg/kg group was significantly higher than in the control group; however, it was considered to be an incidental significant difference accompanied by the low value in the body weight at the time of necropsy since it was a change only in the relative weight.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity, parental animals
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive-developmental toxicity
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no treatment-related effects
Dose descriptor:
NOAEL
Remarks:
reproductive-developmental toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reproductive function and reproductive performance
Dose descriptor:
NOAEL
Remarks:
toxicity to pups
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: viability

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
other: hepatobiliary and mammary gland
Organ:
liver
mammary gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for the repeated dose toxicity of the test material was 100 mg/kg for males and females.
Executive summary:

The repeated dose and reproductive-developmental toxicity of the test material was assessed in a screening study conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

The test material was dissolved in corn oil and administered orally by gavage at 100, 300 or 750 mg/kg bw/day to Sprague-Dawley strain SPF rats (12 males and 12 females for each group in the mating group, and 10 females each in the control group and 750 mg/kg group as the non-mated group) for 14 days before mating and throughout the mating period until the day before necropsy (42 days in total) to males, for 14 days before mating and during the mating and gestation periods until day 13 of lactation (51 to 63 days in total) to females in the mating group and for 42 days to the females in the non-mated group. In addition, for some animals in the 0 and 750 mg/kg bw/day groups (5 males in the mated group and 5 females in the non-mated group), a 14-day recovery period was provided after the 42-day administration to examine the reversibility of the toxic changes. The animals in the control group received the vehicle, corn oil, alone in the same manner.

No deaths occurred in any dose group and there were no changes suggestive of effects from administration of the test material in the manipulative tests, grip strength, motor activity, qualitative items in urinalysis, haematological examination, weight of the thyroid gland, in the reproductive organ weights in males or at necropsy. 

In the 750 mg/kg group, decreased spontaneous movement, flattened posture, lowered reactivity to handling, abnormal gait (ataxic gait) and decreased number of rearings as well as salivation (slight) were observed in males and females, swollen extremities and scratched wound (slight) in males, and swollen extremities in females in the mating group. In males in the 750 mg/kg bw/day group, a low value for food consumption on the day following the start of administration and suppressed body weight gain during the administration period were recorded, while the females in the mating groups showed low values for food consumption on the day following the start of administration, during the late gestation period and during the lactation period and suppressed body weight gain during the gestation period. A low value for food consumption on the day after the start of administration was also recorded for the females in the no-mating group at the same dose level. In the 750 mg/kg group, males showed a high value in urine volume, high values in ALT, ALP, albumin and A/G ratio, females in the mating group showed a high value in A/G ratio and the females in the no-mating group showed high values in ALP, albumin and A/G ratio. A low value in T4 was recorded for males in the 750 mg/kg bw/day group and females in the no-mating group at dose level of 750 mg/kg bw/day. Males and females in the 750 mg/kg bw/day group showed eosinophilic granular changes in the liver, and the females in the mating group in the 300 and 750 mg/kg groups showed involution of the acinus in the mammary glands (inguinal region), the remaining corpus luteum graviditatis in the ovaries, and atrophy of the thymus was observed in females in the 750 mg/kg bw/day mating group. Males in the 300 mg/kg bw/day group showed a high value in albumin. Among the above changes, the changes in the mammary glands (inguinal region), ovaries and thymus were observed only in females in the mating group and since the animals in the recovery group were not subjected to mating, the reversibility was unclear. The other changes were no longer observed or decreased, showing reversibility. 

Under the conditions of this study, it was judged that the NOAEL for the repeated dose toxicity of the test material was 100 mg/kg bw/day for males and females. It was judged that the NOAEL for the reproductive toxicity of the test material was 750 mg/kg bw/day for parent males and 100 mg/kg bw/day for parent females and pups.