Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The NOAEL for the reproductive toxicity was determined to be 750 mg/kg for male parent males and 100 mg/kg for parent females and pups.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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
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
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
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)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1:1 during mating
- Length of cohabitation: 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. The length of the mating period for the same male and female was 14 days at maximum.
- Proof of pregnancy: 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.
- After successful mating each pregnant female was caged (how): individually and then with the litter
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)
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
Parental animals: Observations and examinations:
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.

OTHER:
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 (with lower animal numbers, but 5 females in the order of delivery for the mating groups) 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 (with lower animal numbers, but 5 females in the order of delivery for the mating groups) 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.
Oestrous cyclicity (parental animals):
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.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight and epididymis weight
Litter observations:
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.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
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
Reproductive indices:
Using the following equations, index of animals with abnormal oestrous cycle, copulation index, insemination index, fertility index and gestation index were calculated for each group.
Gestation length in days and post-implantation loss index were calculated for each dam (litter).

- Index of animals with abnormal oestrous cycle (%) = (No. of animals with abnormal oestrous 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
Offspring viability indices:
Using the following equations, sex ratio of PND 0 and 4 were calculated for each group.
Delivery index, index of external abnormalities, live birth index and viability index on PND 4 and 13 were calculated for each dam (litter).
For the body weight of liveborns, mean values for each dam were calculated separately for males and females. For AGD (measured values and after normalised using the cube root of body weight of pups on PND 0), mean values for each dam were calculated separately for males and females. For the number of nipple/areolae, mean values for each dam were calculated.

- Delivery index (%) = (No. of delivered pups / No. of implantation sites) × 100
- Index of external abnormalities (%) = (No. of delivered pups with external abnormalities / No. of delivered pups) × 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 delivered pups on PND 0 (%) = (No. of male delivered pups / No. of delivered pups) × 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
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.
Dermal irritation (if dermal study):
not examined
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 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 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 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 group compared to those in the control group. The body weight gain of the males in the 750 mg/kg 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/daygroup 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/daygroup 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, non-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
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
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).
Reproductive function: sperm measures:
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 male reproductive organs.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
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 bw/day 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 bw/day 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 bw/day 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.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive 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
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
other: hepatobiliary and mammary gland
Organ:
liver
mammary gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
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 bw/day 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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 750 mg/kg bw/day 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 bw/day 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 bw/day group, there were no effects from administration of the test material.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
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.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
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.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no significant differences in the number of nipples / areolae of male pups between the control group and any test material administration group.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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 bw/day 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 bw/day 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.
Gross pathological findings:
no effects observed
Description (incidence and severity):
NECROPSY OF DEAD PUPS
There were no gross pathological abnormalities in any pups that died.

NECROPSY OF PUPS ON PND 13
There were no macroscopic abnormalities in any pups.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
SEX RATIO OF PUPS
There were no effects from administration of the test material on the sex ratio of the pups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
The NOAEL for the reproductive toxicity was 750 mg/kg bw/day for male parent males and 100 mg/kg bw/day for parent females and pups.
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/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 bw/day 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 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 nw/day group, some clinical signs were observed as were effects on food consumption, body weight gain, urinalysis and clinical chemistry.

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 bw/day 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. 

In the 750 mg/kg bw/day group, the following changes were observed: a tendency toward a decrease in the oestrous count and a tendency toward elongation in the mean sex cycle (oestrous cycle), an increase in the percentage of females showing abnormalities in the sex cycle, an increase in the percentage of deaths after implantation, decreases in the number and percentage of live-born pups and the viability index on day 4 after birth, low body weight of male and female pups at the time of birth and suppressed body weight gain thereafter. In the 300 mg/kg bw/day group, a decrease in the viability index on day 4 after birth and suppressed body weight gain of male and female pups were observed. There were no changes suggestive of effects from administration of the test material in the number of days until copulation, copulation index, insemination index, or fertility index or in the histopathological examination of reproductive glands and accessory sex organs of males. There were no changes suggestive of effects from administration of the test material in the length of the gestation period, gestation index, number of implantation sites, delivery index, AGD or sex ratio and there were no abnormalities in the lactation condition. There were no effects from administration of the test material in the viability index on day 13 after birth, number of mammary papilla or areola papillaris, or the weight of the thyroid gland and there were no abnormalities in the external appearance of live-born pups or the pups that died. 

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was conducted under GLP conditions in accordance with the standardised guideline OECD 422. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch et al. (1997) and the quality of the database is therefore considered to be good.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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/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 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, some clinical signs were observed as were effects on food consumption, body weight gain, urinalysis and clinical chemistry.

Males and females in the 750 mg/kg 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 mating group. Males in the 300 mg/kg 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. 

In the 750 mg/kg group, the following changes were observed: a tendency toward a decrease in the oestrous count and a tendency toward elongation in the mean sex cycle (oestrous cycle), an increase in the percentage of females showing abnormalities in the sex cycle, an increase in the percentage of deaths after implantation, decreases in the number and percentage of live-born pups and the viability index on day 4 after birth, low body weight of male and female pups at the time of birth and suppressed body weight gain thereafter. In the 300 mg/kg group, a decrease in the viability index on day 4 after birth and suppressed body weight gain of male and female pups were observed. There were no changes suggestive of effects from administration of the test material in the number of days until copulation, copulation index, insemination index, or fertility index or in the histopathological examination of reproductive glands and accessory sex organs of males. There were no changes suggestive of effects from administration of the test material in the length of the gestation period, gestation index, number of implantation sites, delivery index, AGD or sex ratio and there were no abnormalities in the lactation condition. There were no effects from administration of the test material in the viability index on day 13 after birth, number of mammary papilla or areola papillaris, or the weight of the thyroid gland and there were no abnormalities in the external appearance of live-born pups or the pups that died. 

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 for males and females. It was judged that the NOAEL for the reproductive toxicity of the test material was 750 mg/kg for parent males and 100 mg/kg for parent females and pups.

Effects on developmental toxicity

Description of key information

The maternal and developmental NOAELs were determined to be 125 and > 250 mg/kg/day, respectively.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: International Flavors & Fragrances (Union Beach, New Jersey, USA), Lot No. 413118

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Weight at study initiation: on the day after arrical at the testing facility: males: 262-356 g, females: 188-224 g
- Housing: individual housing, except during the mating period when ech pair of male and females was housed in the male rat’s cage

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared weekly from bulk materials.

VEHICLE
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 103K0107 (Sigma Aldrich)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for the test item content.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 / 1
- Proof of pregnancy: vaginal sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
11 days, from day 7 to day 17 of gestation (GD7 - GD17)
Frequency of treatment:
daily
Duration of test:
21 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
62 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages for this study were selected on the basis of a range finding study, in which 125, 250, 500, or 1000 mg/kg/day were administered daily on DGs 7 to 17. Adverse clinical signs occurred at 500 and 1000 mg/kg/day, and body weight gains and feed consumption were reduced during the entire dosage period at dosages of 250 mg/kg/day and higher.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included check for viability, clinical signs, abortions and premature deliveries

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the start of the study and daily during the dosage and post-dosage periods

FOOD CONSUMPTION: Yes
- Time schedule: GDs 0, 7, 10, 12, 15, 18 and 21

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
Data generated during the course of study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized, and/or statistically analysed, using the above Systems in conjunction with Microsoft Excel (Microsoft Office 97, version SR-2) and/or The SAS System (version 6.12). Clinical Observation and other proportion data were analyzed using the variance test for homogeneity of the binomial distribution (Snedecor and Cochrati 1967). Continuous data were analyzed using Bartlett's test of homogeneity of variances (Sokal and Rohlf 1969) and the analysis of variance (Snedecor and Cochran 1967), when appropriate. Dunnett's test (Dnnnett 1955) was used to idendfy Statistical significance of differences among individual groups. If the analysis of variance was not appropriate, the Kruskal-Wallis test (Sokal and Rohlf 1969) or Dunn's method of multiple comparisons (Dunn 1964) was used to identify the Statistical significance of differences among the individual groups. If there were greater than 75%, Fisher's exact test (Siegel 1956) was used to analyze the data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only clinical signs related to the test item included significantly increased (p < .01) incidences of a clear, red or yellow perioral substance and/or red perivaginal substance in the 250 mg/kg/day dosage group. Excess salivation occurred in all dosage groups, but was most common at 250 mg/kg bw/d.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were significantly reduced (p < .01) at 250 mg/kg/day on DGs 7 to 10, whereas for the entire dosage period, body weight gain at 62, 125, and 250 mg/kg/day was 100.8%, 102.5%, and 92.4% of the control value, respectively. During the post-dosage period (DGs 18 to 21), body weight gains in all dosage groups were comparable to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption and body weight gains were reduced at 250 mg/kg/day (Table 1). Compared to controls, mean absolute (g/day) and relative (g/kg/day) feed consumption were significantly reduced (p < .01) at 250 mg/kg/day for the entire dosage period (DGs 7 to 18), whereas at 125 mg/kg/day a significant reduction was only noted on DGs 10 to 12. For the entire dosage period, absolute feed consumption at 62, 125, and 250 mg/kg/day was 97.9%, 96.8%, and 88.4% of the control value, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross changes attributable to the test item were observed at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Three dead fetuses were observed in one litter from the 250 mg/kg/day dosage group. This observation was considered unrelated to the test item because the incidence was not statistically significant and no dead fetuses were observed in the range-finding study.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Abnormalities:
not examined
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
A significant increase (p < .05) in the percentage of live male fetuses was noted in the 125 mg/kg/day dosage group, as compared to the control group value. This finding was considered unrelated to the test item because the finding was not dose dependent and the value was within the range observed historically at the Testing Facility.
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
All fetuses appeared normal at external examination
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was only one skeletal malformaiton: a fetus in the 62 mg/kg/day dosage group had fusion of one or more ribs (right, 7th and 8th ribs medially to distally, and 9th and l0th distally), as well as skeletal variations in skull bones, ribs and sternum.
All skeletal alterations (malformations or variations) in the fetuses were considered unrelated to the test item because (1) neither the fetal nor litter incidences were dose-dependent; (2) the incidences did not significantly differ from the vehicle control group values; and/or (3) the incidences were within the ranges observed historically at the Testing Facility.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal soft tissue alterations included; an irregularly shaped brain in one 62 mg/kg/day fetus, and folded retinas, a variation usually associated with tissue processing, in 0, 2, 1, and 2 fetuses in 0,2, 1, and 2 litters in the four respective dosage groups. There were no additional alterations in these fetuses.
All soft tissue alterations (malformations or variations) in the fetuses were considered unrelated to the test item because (1) neither the fetal nor litter incidences were dose-dependent; (2) the incidences did not significantly differ from the vehicle control group values; and/or (3) the incidences were within the ranges observed historically at the Testing Facility.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: rib
visceral/soft tissue: central nervous system
visceral/soft tissue: eye
Key result
Developmental effects observed:
no
Conclusions:
The maternal and developmental NOAELs were determined to be 125 and > 250 mg/kg/day, respectively.
Executive summary:

The test item was evaluated for its developmental toxicity in a study equivalent to OECD guideline 414 in Sprague-Dawley rats (25/group; cesarean-sectioning identified 21 to 25 pregnant rats/group). Oral dosages of 0 (corn oil), 62, 125, or 250 mg/kg/day were administered by gavage on days 7 through 17 of gestation (GDs 7 through 17). Rats were observed for viability, clinical signs, body weights, and feed consumption. Necropsy and cesarean sectioning occurred on GD 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. Analysis of dosage preparations verified calculated dosages ±10%. No deaths occurred. Excessive salivation occurred in all groups, but the incidence was increased at 250 mg/kg/day. The 250 mg/kg/day dosage also was associated with a significant increase in the incidences of a clear, red or yellow perioral and/or red perivaginal substance and significant reductions in mean feed consumption and body weight gains (11.6% and 7.4%, respectively) during the entire dosage period. No gross changes attributable to the test item were observed at necropsy. Cesarean section or litter parameters, as well as fetal alterations, were not affected by the test item at 250 mg/kg/day or either of the lower dosages tested. Based on these data, maternal and developmental no-observable-effect levels (NOAELs) of 125 and > 250 mg/kg/day, respectively, were established for the test item. lt is concluded that the test item is not a developmental toxicant in rats under the conditions of this study and dosing regimen.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was conducted equivalent to OECD guideline 414. It was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch et al. (1997). The quality of the database is considered to be good.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test item was evaluated for its developmental toxicity in a study equivalent to OECD guideline 414 in Sprague-Dawley rats (25/group; cesarean-sectioning identified 21 to 25 pregnant rats/group). Oral dosages of 0 (corn oil), 62, 125, or 250 mg/kg/day were administered by gavage on days 7 through 17 of gestation (GDs 7 through 17). Rats were observed for viability, clinical signs, body weights, and feed consumption. Necropsy and cesarean sectioning occurred on GD 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. Analysis of dosage preparations verified calculated dosages ±10%. No deaths occurred. Excessive salivation occurred in all groups, but the incidence was increased at 250 mg/kg/day. The 250 mg/kg/day dosage also was associated with a significant increase in the incidences of a clear, red or yellow perioral and/or red perivaginal substance and significant reductions in mean feed consumption and body weight gains (11.6% and 7.4%, respectively) during the entire dosage period. No gross changes attributable to the test item were observed at necropsy. Cesarean section or litter parameters, as well as fetal alterations, were not affected by the test item at 250 mg/kg/day or either of the lower dosages tested. Based on these data, maternal and developmental NOAELs of 125 and > 250 mg/kg/day, respectively, were established for the test item. lt is concluded that the test item is not a developmental toxicant in rats under the conditions of this study and dosing regimen.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

In an available developmental toxicity study equivalent to OECD 414 no indication for developmental toxicity was found up to the top dose of 250 mg/kg bw/day at which systemic toxic effects as reduced food consumption and body weight development in the dams were noted.

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 several effects were noted (e.g. oestrus cycle disturbances, increased death after implantation, reduced number and percentage of live-born pups, reduced viability index on day 4 after birth, lower body weight gain of pups) were noted at the top dose of 750 mg/kg bw/day. In addition at both 300 and 750 mg/kg bw/day involution of the acinus in the mammary glands and remainder of gravid corpus luteum were seen. The effects were observed in the presence of moderate maternal toxicity (e.g. clinical signs of toxicity, reduced body weight gain during gestation (-16%)) at the top dose of 750 mg/kg bw/day.

In summary, there is some evidence from the OECD 422 study that the test item might cause adverse effects on reproduction. Thus, the substance is classified for toxicity to reproduction Category 2 (H361) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.