Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
(screening study)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 December 2018 to 12 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 December 2018 to 12 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Testing Methods Concerning New Chemical Substances
Version / remarks:
Notification No. 0331-7, PFSB, MHLW; No. 5 of March 29, 2011, MIB, METI; No.
110331009, EPB, MOE; dated March 31, 2011
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in toxicity studies using rodents, there is abundant historical data and a large number of animals are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks old at receipt, 9 weeks old at the start of administration
- Weight at receipt: 224.8 to 247.6 g (males), 162.3 to 188.7 g (females)
- Fasting period before study: No, but animals were fasted before scheduled necropsy for about 17 to 24.5 hours.
- Housing: Animals were housed 2 or 3 animals per cage before grouping. After gouping, animals were housed 1 male and 1 female per cage during the mating period, one dam and its litter per cage during the lactation period and one animal per cage for the other periods. For males and females except for copulated females and dams, animals were housed in hanging type stainless wire mesh cages (W × D × H: 226 × 346 × 198 mm). Copulated females were housed in polymethylpentene cages (W × D × H: 220 × 380 × 195 mm). Both types of cages were furnished with two types of nesting material (Paper Clean, Diamond Twists) and one kind of gnawing material (Diamond Twists) for the improvement of animal welfare.
- Diet: Pelleted diet, ad libitum (except during fresh urine collection and measurement of motor activity)
- Water: Well water admixed with NaClO, ad libitum (except during the measurement of motor activity)
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
Data for each lot of the diet were obtained from the supplier and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.
Water was analysed twice a year. The results were confirmed to be within the acceptable limits established by the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.1 to 23.7 °C
- Humidity: 51.4 to 72.6 %
- Air changes: 10 to 20 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Following correction for substance purity, a prescribed amount of the test material was was weighed into a beaker and an appropriate amount of the vehicle was added and stirred with a magnetic stirrer.
After confirming of dissolution, it was transferred to a measuring cylinder. The final volume was adjusted by adding a proper quantity of the vehicle to provide the required concentration of dosing solution. Further dilution provided lower concentration dosing solutions.
Dosing formulations were prepared once within 12 days (3- to 10-day interval). The dosing formulations after preparation were divided into glass vials for each dosing day and stored at room temperature (17.9°C to 22.4°C).

DOSE VOLUME:
Individual volume was calculated on the basis of the most recently measured body weights.
Details on mating procedure:
- M/F ratio per cage: The test males and females of each dose level were cohabited at one-on-one basis for 24 hours from Day 15 to the end of the mating.
- Proof of pregnancy: The vaginal smears were collected in the morning from the day after the initiation of mating, and copulation was confirmed by the presence of the vaginal plug or sperm in the smear sample. The day of successful mating was regarded as Day 0 of gestation (GD 0).
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC
Details on analytical verification of doses or concentrations:
At the first preparation, 10 mL each of the analytical samples was taken from the whole dosing formulation at each concentration and the test material concentration analysed by HPLC according to a previously validated method. Nominal test material concentrations of 12.5, 50 and 200 mg/mL provided mean measured concentrations of 12.68, 50.11 and 203.1 mg/mL, respectively and were within 101.4, 100.2, and 101.6% of nominal, respectively.
Test material formulations at 1 and 200 mg/mL were confirmed to be stable after storage for 12 days at room temperature.

High Performance Liquid Chromatography (HPLC) conditions:
Instrumentation: Prominence UFLC (Shimadzu Corporation)
Data processing: LabSolutions (Shimadzu Corporation)
Column: Ascentis Express RP-Amide (2.7 μm, 3.0 mm I.D. × 50 mm, Sigma-Aldrich Co.)
Column temperature: A constant temperature of about 40°C (set at 40°C)
Mobile phase:
Mobile phase A; water*
Mobile phase B; acetonitrile*
Linear gradient conditions and flow rate:
0.00 and 3.00 mins (55% A : 45% B) at a flow rate of 0.9 mL/min
3.01 and 12.00 mins (0% A : 100% B) at a flow rate of 1.5 mL/min
21.01 and 22.00 min (55% A : 45% B) at a flow rate of 0.9 mL/min
Analysis time: 22 minutes
Wavelength for detection: UV 200 nm
Injection volume: 10 μL
Auto-sampler set temperature: Room temperature (Not setting)
Needle wash solvent: Acetonitrile*
* sonicated under vacuum and degassed.
Duration of treatment / exposure:
Males: From 14 days before mating until the day before necropsy through the mating period (42 days in total)
Females: From 14 days before mating until Day 13 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery.
Recovery females (Satellite females), for 42 days without mating (as for males)
Frequency of treatment:
daily
Details on study schedule:
n/a
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
0 (corn oil control) and 1000 mg/kg bw: 7 males and 12 females (main dosing group) and an additional 5 males and 5 females (recovery group)
62.5 and 250 mg/kg bw: 12 males and 12 females (main dosing group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were selected following consideration of the effects observed in a 14-day study, during which 5 animals/sex/dose received oral gavage doses of test material at 0 (corn oil control), 110, 330, and 1000 mg/kg bw, for 14 consecutive days.
Following administration of test material, a lowering tendency of body weight was noted in males of the 1000 mg/kg group throughout the dosing period; however, a low value of body weight gain was transient. Haematology revealed anaemia in females of the 330 and 1000 mg/kg groups, and prolonged PT and APTT in males of the 1000 mg/kg group as an effect on the coagulation system.
At necropsy, enlargement of the liver was noted in both sexes of the 1000 mg/kg group and a high value of liver weight was noted in both sexes of the 330 and 1000 mg/kg groups. In the blood chemistry, a high value of ALAT was noted in both sexes of the 1000 mg/kg group, suggesting hepatic injury. The effects on protein/lipid metabolism were noted in males of the 1000 mg/kg group and females of the 110 mg/kg group and above. Moreover, whitish patch of mucosa in the forestomach was noted in 1 female of the 1000 mg/kg group, suggesting that the test material is irritating. Although various treatment-related changes were observed, none of the changes was severe. Therefore, the high dose in this study set at 1000 mg/kg, at which clear toxicity effects are expected to be seen. Lower doses were set at 250 and 62.5 mg/kg, with a common ratio of 4. A control group (0 mg/kg group) dosed with the vehicle (corn oil) alone was also established.
- Rationale for animal assignment: All animals except for the animals with abnormal estrous cycle (as determined during the quarantine and acclimation period) were used for grouping. Animals were assigned to groups by the stratified randomisation on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weights ± 20% (calculated for each sex) were used.
- Post-exposure recovery period in satellite groups:
For 5 males and 5 females (non-mating satellite females) each in the control and 1000 mg/kg groups, a 14-day recovery period was set after the end of the dosing period.
Positive control:
n/a
Parental animals: Observations and examinations:
The initial day of dosing was designated as Day 1, Day 1 to Day 7 as Week 1, period after Day 43 as the recovery period. For the test females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).

CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were observed twice a day (before dosing and after dosing) during the dosing period and once a day in the other periods.

DETAILED CLINICAL OBSERVATIONS: Yes (as part of functional observation battery)
- Time schedule: For all animals, detailed clinical observations were performed once before the start of dosing and once a week in the afternoon during the dosing and recovery periods (day of gestation or parturition was prescinded).
Detailed clinical observations included:
- Hand-held observation, during which animals were removed from the cage for the observation by grasping their trunk gently from behind. Their reactivity to handling, trauma, colour of skin, soiled fur, piloerection, secretion, salivation, lacrimation, exophthalmos, palpebral closure, colour of conjunctiva and pupil size were observed.
- Observation on open field, during which animals were placed in the center of the open field and observed quietly for one minute. Parameters observed included arousal, posture/body position, gait, tremor, convulsion, respiration, stereotypy, bizarre behaviour, urination, defecation and number of rearings.

OBSERVATIONS DURING DELIVERY AND LACTATION PERIODS
All copulated females were allowed natural delivery. The observation of delivery was conducted once daily (a.m. 9:00) from GD 21 to GD24. Females that delivered their litter completely by 9:00 a.m. were judged as “delivered” on the corresponding day (the delivery day was regarded as Day 0 of lactation [LD 0]). When delivery was completed at 9:00 or later, the following day was defined as LD 0. The delivered animals (dams) were allowed to nurse offspring until LD 13, and postpartum behaviour such as lactation, nesting and presence or absence of cannibalism was observed every day. One female (No. 691) not delivered even after 24 days after the copulation confirmation was determined as non-delivered females.

BODY WEIGHT: Yes
- Time schedule for examinations:
The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36 and 42. The recovery males were also weighed on Days 43, 50 and 56. The satellite females were weighed at the same frequency as the recovery males. The test females were weighed on Days 1, 8 and 15, once every 7 days after the initiation of cohabitation, on GDs 0, 7, 14 and 20, and on LDs 0, 4, 7 and 13.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured between Days 1 and 8, 8 and 15, 22 and 29, 29 and 36 and 36 and 41 for the test and recovery males, and between Days 43 and 50 and 50 and
55 for the recovery males. For the satellite females, it was measured between Days 1 and 8, 8 and 15, 15 and 22, 22 and 29, 29 and 36, 36 and 41, 43 and 50 and 50 and 55. Food consumption of the test females was measured at the same frequency as body weight measurement. However, food consumption was not measured for either sex during the mating period. After the completion of copulation, the measurement for males was started from the nearest measurement day. Gross weight of each feeder was weighed, and the mean daily food consumption for each period was expressed as the start day of each measurement.

WATER CONSUMPTION: No

HORMONE MEASUREMENT
Hormone (total T4) concentrations in rat plasma samples (parental animals [F0 male and F0 female], offspring [F1 offspring on PND 13], and Satellite female) were measured. A total of 80 samples (30 samples of F0 male, 20 samples of F1 offspring on PND 13, 20 samples of F0 female, and 10 samples of Satellite female) were assayed.
The plasma samples were stored frozen until analysis. The frozen plasma samples were thawed immediately before use, and then centrifuged (1710 × g for 10 minutes at set 4°C) to remove any aggregates in the plasma. The supernatant was used for assay.
An aliquot (60 μL) of the test sample or “standard solution (20 μL) (0, 25.5, 51.0, 102, 204, and 408 nmol/L) and PBS (phosphate buffered saline, 40 μL)” was pipetted into a tube coated with anti-T4 antibody. An aliquot (500 μL) of the tracer (125I-labelled T4) was added to the tube and the solution was mixed and shaken for 1 hour at room temperature. After the solution was aspirated, the remaining 125I radioactivity (cpm) in the tube was measured for 1 minute using the gamma counter.
A calibration curve was prepared with the mean cpm values of the duplicate assay for the standard solutions. The concentrations in the test samples were calculated using the calibration curve and the cpm value of the single assay for the test samples and corrected with the volume factor at the time of measurement. Hormone concentrations were calculated using the software (RiaSmart, PerkinElmer, Inc.) and Excel 2010 (Microsoft Corporation). The lower limit of quantification (LLOQ) was set at the lowest concentration of the standard solution (except for the zero concentration).

OTHERS:
The following were included as part of the repeated dose toxicity assessment: haematology, clinical chemistry, urinalysis, functional observational battery (including sensory activity, grip strength, motor activity).
Oestrous cyclicity (parental animals):
Vaginal smears were collected with swabs from all test females in the morning (same time every day) from the initial dosing day to the day of successful copulation to confirm the oestrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The oestrous cycle was classified into diestrus (D), proestrus (P), oestrus (E) and metestrus (M). The mean oestrous cycle (number of days from the estrous period to the next estrous period) and the number of the oestrous period during the test period were calculated.
Litter observations:
The day of birth was defined as “Postnatal day 0 (PND 0)” for offspring.

BODY WEIGHT
All live offspring were weighed individually on PNDs 0, 4, 7 and 13.

EXTERNAL EXAMINATION
All live offspring on PND 13, stillborn and dead offspring were observed for external anomaly. Based on the results, the following parameters were calculated.
- External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100
- External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On PND 4, the litter size was standardised to 8 (4/sex/litter) by random removal of offspring. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 regardless of the sex ratio. Litter with less than 8 offspring was maintained as is. The offspring culled at the litter size adjustment (offspring excluded from blood collection) were euthanised by overdose with pentobarbital sodium (undiluted solution, 0.1 to 0.5 mL/body) via an intraperitoneal injection and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- AGD (Anogenital distance)
Anogenital distance (AGD: distance between the anus and the genital node) was determined on PND 4 following adjustment of the number of offspring with a micrometer caliper. In order to correct variations in measured values due to weight differences among individuals, the AGI (anogenital index) divided by the cubic root of the body weight on the measurement day was also calculated.
- Nipple development
All live male offspring were observed for the appearance of nipples (or areolae) on PND 12. Based on the results, the following parameter was calculated.
Nipple development anomaly index (%): (Number of offspring with nipple development anomaly / Number of observed offspring) × 100
Postmortem examinations (parental animals):
TERMINAL PROCEDURES
All surviving animals were euthanised by exsanguination under anaesthesia and subjected to necropsy. In addition, the vaginal smears were collected from the females on LD 14 (necropsy day) in the morning and the stage of the oestrous cycle was examined under a microscope. At necropsy on LD 14, the ovaries and uterus were excised to examine for the numbers of implantation sites.
Non-delivered female (No. 691) was necropsied on GD 26. This animal was euthanised and necropsied in the same manner as the surviving animals.

GROSS PATHOLOGY: Yes
The following organs/tissues were collected and examined: Heart, thymus, spleen, mandibular lymph node, mesenteric lymph node, femur/bone marrow (femur), sternum/bone marrow (sternum), trachea, lung (including bronchus), stomach, duodenum, jejunum, ileum (including Payer’s patch), cecum, colon, rectum, liver, pancreas, submandibular gland, kidney, urinary bladder, testis, epididymis, prostate (ventral lobe), seminal vesicle (including dorsolateral lobe and coagulating gland), cowper’s gland, glans penis, ovary, uterus, vagina, pituitary, thyroid/parathyroid, adrenal, spinal cord (cervical), brain (cerebrum, cerebellum and medulla oblongata/pons), eyeball, femoral muscle/Sciatic nerve (femoral region), levator ani/bulbocavernosus muscle (LABC), and any other organs/tissues indicating gross lesion.

ORGAN WEIGHTS: Yes
After the scheduled necropsy, the organs listed above were weighed (absolute weight) and the ratios of the organ weights to body weight (relative weight) were calculated on the basis of the body weight measured on the day of necropsy. The measurement was conducted in 5 animals per group with the smallest animal numbers of the test males; all recovery males; all satellite females; 5 animals per group from the earlier parturition date with the smallest animal numbers in the test females. However, the testis and epididymis weights were measured in all males. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
After necropsy, the organs/tissues listed above were fixed in 10 vol% phosphate buffered formalin solution; however, the testes and epididymides were fixed in Bouin’s solution and the eyes were fixed in Davidson’s solution, and they were preserved in 10 vol% phosphate buffered formalin solution. As for the dead animal, all of the organs/tissues were preserved in 10 vol% phosphate buffered formalin solution. The organs/tissues of 5 animals per group with the smallest animal numbers of the test males and 5 animals per group from the earlier parturition date with the smallest animal numbers for the test females in the control and 1000 mg/kg groups collected at the end of the dosing period, one dead animal (No. 693) and gross lesion were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and then examined by microscopy.
As for the dead animal, the following organs could not be examined due to autolysis: mandibular lymph node, bone marrow (sternum and femur), trachea, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, submandibular gland, urinary bladder, ovary, uterus, vagina, pituitary, thyroid, adrenal, spinal cord and sciatic nerve.
According to these results, a change suspected to be attributable to the test material treatment was observed in the 1000 mg/kg group as follows: liver, pituitary, thyroid, adrenal, femur, spleen, stomach, cecum and kidney of both sexes and heart, sternum, thymus and lung of females. Therefore, the relevant organs/tissues of the relevant sex in the low and middle dose groups and all groups in the recovery study were additionally examined, according to the above criteria. Vacuolation was noted in the adrenal and kidney and foam cells were noted in the lung. To confirm whether the vacuole and foam cells are derived from lipid, these organs from one representative each in the 1000 mg/kg group (adrenal: No. 637, kidney: No. 693 and lung: No. 688) was subjected to Oil red O staining for microscopic examination.
Postmortem examinations (offspring):
The necropsy was performed on PND 13. The offspring subjected to blood sampling were necropsied after blood sampling by decapitation after anaesthetising by inhalation of isoflurane. Other animals were euthanised by exsanguination from the lateral iliac artery under anaesthesia with pentobarbital sodium (undiluted solution, 0.1 to 0.5 mL/body) via the intraperitoneal injection and necropsied. The thoracoabdominal organs/tissues were examined macroscopically.
As the results, an abnormal finding was noted in the liver of one offspring in one dam (offspring No. F04 in dam No. 698). Therefore, the organ with macroscopic lesion was collected and fixed and preserved in 10 vol% phosphate buffered formalin along with the same organs and tissues from one dam (No. 653) of the control group for comparison.
Furthermore, the thyroid glands of 1 male and 1 female (animals subjected to blood sampling for hormone level measurement) per litter were collected and fixed and preserved in 10 vol% phosphate buffered formalin. As for one dam (No. 667), the thyroid gland was collected from 2 females because there were no males in this litter.

BLOOD SAMPLING
The blood sampling was performed on PNDs 4 and 13 and conducted for the offspring from 5 dams per group from the earlier parturition date with the smallest animal numbers in the test females. On PND 4, the blood was collected from at least 1 culled animal of each sex in each litter. On PND 13, the blood sampling was performed on 1 animal of each sex in each litter. When a blood sample could not be obtained from either sex, it was obtained from 2 animals of the same sex in the litter.
The blood sampling was performed by decapitation after anesthetizing by inhalation of isoflurane. Approximately 0.4 mL of blood (including humor liquid, pooled blood per litter) was collected into a polypropylene tube containing heparin sodium. The blood was immediately cooled on ice and then promptly centrifuged (approximately 1870 × g, 10 min, 4°C) to obtain plasma (120 μL or more). The collected plasma was stored frozen.
Statistics:
Statistical analysis was performed with a computer system (tsPharma LabSite, Fujitsu Limited). However, a statistical analysis system EXSUS Version 8.1.0 (CAC Croit Corporation, statistical analysis software: SAS 9.4 [SAS Institute Japan Inc.]) was used for sex ratio. In all cases, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used. Analysis for concentration of total T4 was conducted according to Work Plan. The data of offspring were handled on a litter-basis.
Multiple comparison test:
For the following numeral data, mean values and standard deviations were calculated in each group. Bartlett’s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group.
Relevant parameters included: oestrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring and AGD.
The following items were tested with the chi-square test for comparison between the control and each test material group: copulation index, fertility index, gestation index and sex ratio
The following items were tested with the Wilcoxon’s rank sum test for comparison between the control and each test material group: delivery index, stillborn index, external anomaly index, external anomaly typing index, birth index, viability index on Days 4 and 13 and nipple development anomaly index.
Reproductive indices:
Based on the results of the vaginal smears, the following parameters were calculated:
- Days until copulation: Number of days from the start of mating to the detection of copulation
- Copulation index (%): (Number of animals with successful copulation / Number of animals cohabited) × 100
- Fertility index (%): (Number of pregnant animals / Number of females with successful copulation) × 100

Based on the findings following excisation of the ovaries and uterus at necropsy (on LD 14), the following parameters were calculated.
(As for non-delivered one female (No. 691), the presence of implantation was examined for the uterus at necropsy on GD 26. As a result, the implantation was confirmed macroscopically. Thus, this female was judged to be pregnant.)
- Gestation length: Days until completion of delivery from GD 0
- Gestation index (%): (Number of pregnant animals delivered live offspring / Number of pregnant animals) × 100
- Delivery index (%): (Number of delivered offspring / Number of implantations) × 100
Offspring viability indices:
The number of litter (numbers of live newborn and stillborn), sex and the presence or absence of external anomalies were examined on PND 0. Thereafter, mortality was observed daily. Based on the results, the following parameters were calculated. The stillborn and dead offspring before culling were discarded.
- Birth index (%): (Number of live offspring at birth / Number of implantations) × 100
- Stillborn index (%): (Number of stillborns / Total number of delivered offspring) × 100
- Viability index on Day 4 (%): (Number of live offspring on PND 4 / Number of live offspring at birth) × 100
- Viability index on Day 13 (%): (Number of live offspring on PND 13 / Number of live offspring after culling) × 100
- Sex ratio: (Number of male live offspring / Number of all live offspring)
- External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100
- External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted. Mass was observed in the axilla of one female (No. 698) treated with 1000 mg/kg on LD 4 and thereafter. However, it was judged that this change was not related to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female (No. 693) treated with 1000 mg/kg was found dead on LD 2. In this animal, reduced locomotor activity was observed the day before, but no other remarkable change was observed. A suppressed body weight gain was noted in this animal until the death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A low value of body weight was noted in males treated with 1000 mg/kg throughout the dosing and recovery periods (statistically significant compared to vehicle control).
A low value of body weight (statistically significant compared to vehicle control) was noted in females treated with 1000 mg/kg on GD 20.
In satellite females, no significant difference was noted between the control and 1000 mg/kg group throughout the dosing or recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A low value of food consumption (statistically significant compared to vehicle control) was noted in males treated with 1000 mg/kg during Day 1-8. Thereafter, food consumption in males treated with 1000 mg/kg was similar to or higher (during Day 29-36) than that of Controls throughout the dosing and recovery periods.
Prior to pairing, food consumption was low in females treated with 1000 mg/kg during Day 1-8 (statistically significant compared to vehicle control). Thereafter, food consumption was similar to Controls for females at all dose levels during the gestation and lactation periods.
In satellite females (after Day 8), food consumption was similar to or higher than (statistically significant compared to vehicle control) that of Controls throughout the dosing and recovery periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, some erythroid parameters at 1000 mg/kg were marginally and not statistically significantly lower compared to the concurrent vehicle control as follows: a low value of RBC count in females (93.9% of Controls), a lower value of haemoglobin concentration in males (94.9%) and females (93.6%), a lower value of haematocrit in males (93.3%) and females (94.3%).
At the end of the recovery period, lower values of RBC count, haemoglobin concentration and haematocrit and a higher value of reticulocyte ratio were noted in males treated with 1000 mg/kg (statistically significant compared to vehicle control).
Besides, at the end of the dosing period, shortening of PT and APTT was noted in females treated with 1000 mg/kg, the differences from the control attained the statistical significance. However, these were judged not to be treatment-related as the changes were slight compared to those of Controls and opposite to the toxicity effect. A statistically significant prolongation of PT was noted in males treated with 250 mg/kg, however, the change was not accompanied by a dose-dependency and considered not to be treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, treatment-related changes were noted as follows: higher values of albumin and A/G ratio in males treated with 1000 mg/kg; a higher value of ALAT in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a high value or higher tendency of total cholesterol and Ca in both sexes treated with 1000 mg/kg and a lower value of Na in males treated with 1000 mg/kg.
At the end of the recovery period, none of the above-mentioned changes was noted.
Besides, at the end of the dosing period, lower values of ALP and total bile acid were noted in females treated with 1000 mg/kg, attaining statistical significance. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
At the end of the recovery period, a lower value of glucose was noted in males treated with 1000 mg/kg as well as K in females treated with 1000 mg/kg. However, these were judged not to be treatment-related as the similar changes were not noted at the end of the dosing period and no related change was noted in any examination. Lower values of γGT, ALP and total bile acid were noted in males treated with 1000 mg/kg. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
During the dosing period, pH was biased toward the acidic side in males treated with 1000 mg/kg and significant difference was noted when compared with Controls. An increase of urine volume was noted in males treated with 1000 mg/kg.
During the recovery period, none of the above-mentioned changes was noted.
Besides, high values of Na, K and Cl were noted in males treated with 1000 mg/kg during the recovery period. However, these were judged not to be treatment-related as the changes, although statistically different, were slight compared to those of the control group, the similar changes were not noted during the dosing period or no related change was noted in the histopathology.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Detailed clinical observations: No treatment-related change was noted in any animal in the hand held observation or observation on the open field.
The following changes were noted in the number of rearing: a low value in males treated with 1000 mg/kg on Day 35, a high value in females treated with 250 mg/kg and above on Day 7 and a low value in females treated with 1000 mg/kg on Days 35 and 42, with the differences from the control attained the statistical significance. These were transient changes and no related changes were noted in the other parameters. Therefore, it was judged that the changes were not related to the test material treatment.
- Sensory reactivity to stimuli: Reactivity to stimuli was comparable in males and females at all dose levels and no abnormality was observed.
- Grip strength: No significant difference was noted in males or females between the control and test material groups.
- Motor activity: No treatment-related change was noted.
A low value of motor activity was noted between 20 and 30 minutes in females treated with 1000 mg/kg (statistically significant compared to vehicle control). However, this was judged not to be treatment-related as the moving pattern was normal and no related change was noted in the detailed clinical observations. As a change without dose-dependency and considered as not treatment-related, a high value of motor activity was noted between 40 and 50 minutes in females treated with 62.5 mg/kg (statistically significant compared to vehicle control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver:
- Hypertrophy of centrilobular hepatocytes was noted in 3/5 males and 1/5 females treated with 250 mg/kg (grade: minimal) and 5/5 males and 5/5 females treated with 1000 mg/kg (minimal or mild). After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
Pituitary:
- Minimal hypertrophy of basophilic cells in the anterior lobe was noted in 3/5 males treated with 250 mg/kg and 5/5 males and 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thyroid gland:
- Minimal hypertrophy of follicular cells was noted in 2/5 males treated with 250 mg/kg and 5/5 males and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Adrenal gland:
- Minimal vacuolation of cortical cells in the fascicular zone was noted in 1/5 females treated with 250 mg/kg and 4/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
- Minimal vacuolation of cortical cells in the glomerular zone was noted in 4/5 males treated with 250 mg/kg and 3/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- These vacuoles were positive for Oil red O staining.
Sternum:
- Minimal increase of trabecular bone was noted in 1/5 females treated with 250 mg/kg and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in all satellite females treated with 1000 mg/kg (minimal).
Femur:
- Minimal increase of trabecular bone was noted in 1/5 males and 1/5 females treated with 250 mg/kg and 4/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 2/5 males and 5/5 satellite females treated with 1000 mg/kg (minimal).
Spleen:
- Minimal erythrocytic extramedullary hematopoiesis was noted in 1/5 males and 1/5 females treated with 250 mg/kg and 1/5 males and 4/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 1/5 males and 1/5 satellite females treated with 1000 mg/kg (minimal).
Cecum:
- Minimal thickening of mucosa was noted in 1/5 males and 3/5 females treated with 250 mg/kg and 2/5 males and 4/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Kidney:
- Mild basophilic tubule was noted in 1/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
Stomach:
- Minimal mineralisation of mucosa in the glandular stomach was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal erosion in the limiting ridge was noted in 1/5 males treated with 250 mg/kg and 2/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal hyperkeratosis in the limiting ridge was noted in 3/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal parakeratosis in the limiting ridge was noted in 3/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal inflammatory cell infiltration in the submucosa of limiting ridge was noted in 2/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal vacuolation of squamous epithelium in the limiting ridge was noted in 1/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
Lung:
- Minimal accumulation of foam cells in the alveolus was noted in 1/5 females treated with 250 mg/kg and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Fine vacuoles of the foam cells were positive for Oil red O staining.
Heart:
- Minimal mineralisation of aortic wall was noted in 2/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thymus:
- Minimal atrophy was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.

> [Dead animal (No. 693) treated with 1000 mg/kg]
Thymus: Moderate atrophy was noted.
Spleen: Mild atrophy was noted.
Liver: Minimal hypertrophy of centrilobular hepatocytes was noted.
Kidney: Mild vacuolation of proximal tubular epithelium was noted. The vacuoles were positive for Oil red O staining.
Sternum: Minimal increase of trabecular bone was noted.
Femur: Minimal increase of trabecular bone was noted.

> Others:
Various histopathological changes were noted in both sexes of the control and test material groups. However, these changes were judged not to be treatment-related as they are observed occasionally in normal rats and there was no clear dose-dependency in the incidence.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Mass in the axilla noted in 1 female (No. 698) treated with 1000 mg/kg was adenocarcinoma of the mammary gland in the histopathology. Adenocarcinoma of mammary gland is known to occur infrequently in young mature rats and no gross abnormalities were noted in the mammary gland of any other animals, therefore, it was judged not to be treatment-related.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Hormone measurement:
In parental animals (F0 male), plasma total T4 concentrations of the test material-treated group (Group 1000 mg/kg) tended to be lower compared to those in the control group after the dosing period. In parental animals (F0 female), plasma total T4 concentrations of the test material-treated groups (Groups 62.5, 250, and 1000 mg/kg) were equivalent to those of the control groups. The plasma total T4 concentration of recovery groups (Group 1000 mg/kg) were equivalent to those of the control groups in F0 male and Satellite female.
All values in the quality control sample were within the range of 100 ± 25% of the nominal value, and variations in duplicate cpm of the standard solutions were within the range of acceptability. There were no abnormalities in the procedures for the determination or in the values of the test samples. The results were therefore considered acceptable.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant difference was noted in the mean oestrous cycle or count of oestrus, indicating no effect of the test material on prolongation or shortening of the oestrous cycle between the control and test material groups.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Mating: No significant difference was noted in the copulation index, duration of mating or fertility index between the control and test material groups.
- Observation during delivery and lactation periods: No abnormality was noted in the delivery or nursing. No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index between the control and test substance groups.
One female (No. 691) treated with 1000 mg/kg, which had only one implantation, did not deliver. However, this was judged not to be treatment-related due to its low incidence.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See 'Remarks'
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
other: effects on calcium metabolism and injury of the kidney, hepatic effects, effects on bone/lipid metabolism, and reaction to anaemia.
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormality was observed in any offspring following external examination.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No significant difference was noted in viability index on PNDs 4 or 13 between the control and test material groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant low value of body weight was noted in both sexes of the 1000 mg/kg group on PND 13.
Besides, a high value of body weight was noted in females of the 62.5 mg/kg group on PNDs 7 and 13. However, this was judged not to be treatment-related as there was no dose-dependency.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No significant difference was noted in males or females between the control and test material groups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No significant difference was noted in the anomaly index between the control and test material groups.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted in any offspring.
Whitish patch of the liver was noted in one female of the 1000 mg/kg group (offspring No. F04 in dam No. 698). However, this was judged not to be treatment-related due to its low incidence.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No significant difference was noted in the number of litter, live newborns and stillborn, birth index, sex ratio or viability index on PNDs 4 or 13 between the control and test material groups.

- Hormone concentration (total T4) analysis [Offspring on PND 13]
No significant difference was noted between the control and test material groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
(developmental toxicity)
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

- PARENTAL GENERATION

Summary of bodyweights, g (mean ± SD)

Day

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

0 (17)

62.5 (12)

250 (12)

1000 (17)

1

370.18 ± 11.31

368.34 ± 10.91

368.61 ± 13.93

367.61 ± 11.12

230.74 ± 10.20

231.73 ± 9.27

231.80 ± 12.70

233.05 ± 11.49

8

411.53 ± 17.39

403.28 ± 17.29

404.06 ± 15.94

386.56 ± 15.33**

248.94 ± 9.37

246.68 ± 11.24

247.23 ± 14.02

244.68 ± 10.47

15

440.40 ± 20.25

426.15 ± 26.46

426.96 ± 20.61

408.28 ± 17.71**

258.92 ± 13.60

256.73 ± 11.96

258.48 ± 15.67

255.48 ± 10.28

22

465.89 ± 20.21

453.23 ± 32.50

453.22 ± 26.16

425.06 ± 18.46**

263.62 ± 9.64

256.60 ± 17.43

29

495.29 ± 22.27

476.41 ± 38.61

476.37 ± 30.82

451.31 ± 20.10**

267.86 ± 7.71

269.38 ± 11.31

36

519.59 ± 22.65

497.98 ± 42.47

498.53 ± 33.35

469.81 ± 22.99**

280.20 ± 9.14

278.80 ± 17.93

42

536.88 ± 21.91

514.63 ± 39.88

511.93 ± 35.54

484.18 ± 25.06**

286.10 ± 9.73

284.80 ± 21.75

43

537.84 ± 24.80

479.94 ± 31.80*

284.56 ± 13.69

283.80 ± 17.71

50

553.80 ± 24.88

494.18 ± 34.65*

292.34 ± 9.20

291.18 ± 17.98

56

562.62 ± 24.74

511.18 ± 32.89*

296.32 ± 10.78

301.28 ± 19.97

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Results in itallics relate to 5 animals

Day 1 to 42; Treatment period, Day 43 to 56; Recovery period

Summary of female bodyweights, g (mean ± SD)

Day

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

Gestation period

0

266.18 ± 16.28

266.75 ± 13.71

266.59 ± 18.10

263.90 ± 13.36

7

301.64 ± 20.12

300.63 ± 16.86

298.92 ± 18.85

284.27 ± 16.47

14

336.97 ± 25.50

340.59 ± 21.19

342.07 ± 19.36

316.99 ± 19.42

20

419.53 ± 33.02

419.03 ± 32.66

425.38 ± 24.23

379.78 ± 38.42*

Lactation period

0

325.03 ± 30.56

329.48 ± 22.57

335.88 ± 19.11

303.72 ± 23.02

4

340.01 ± 31.55

343.58 ± 21.36

351.46 ± 20.97

322.94 ± 25.21

7

346.39 ± 31.44

348.66 ± 19.08

354.37 ± 24.07

336.30 ± 23.07

13

354.93 ± 33.29

361.38 ± 15.85

368.46 ± 19.88

359.01 ± 21.42

- OFFSPRING

Summary of bodyweights in offspring, g (mean ± SD)

Parameter

Sex

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (11)

Day 0

No of pups

Male

87

70

82

61

Female

80

86

84

76

Mean pup weight

Male

7.5 ± 0.5

7.8 ± 0.6 (11)

7.4 ± 0.7

6.8 ± 0.7

Female

7.0 ± 0.4

7.4 ± 0.8

7.1 ± 0.7

6.6 ± 0.7

Day 4

No of pups

Male

87

69

82

58

Female

80

85

84

70

Mean pup weight

Male

12.0 ± 1.0

12.9 ± 1.3 (11)

12.5 ± 1.6

11.2 ± 1.7 (10)

Female

11.2 ± 0.9

12.3 ± 1.8

11.8 ± 1.4

10.7 ± 1.4 (10)

Day 7

No of pups

Male

49

44

48

38

Female

47

46

48

42

Mean pup weight

Male

20.2 ± 1.8

21.9 ± 1.8 (11)

20.9 ± 2.0

18.9 ± 2.3 (10)

Female

18.6 ± 1.5

20.9 ± 2.2*

19.8 ± 2.0

17.5 ± 1.8 (10)

Day 13

No of pups

Male

49

44

48

38

Female

47

46

48

42

Mean pup weight

Male

37.3 ± 2.8

40.0 ± 2.7 (11)

38.0 ± 2.5

33.8 ± 2.5** (10)

Female

35.3 ± 2.2

38.4 ± 2.9**

37.2 ± 1.8

31.6 ± 2.5** (10)

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Number in parentheses indicates the number of litters.

Conclusions:
The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg due to decreased body weight of F1 offspring at 1000 mg/kg on PND 13.
Executive summary:

The reproductive toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 422 and the Japanese Testing Methods Concerning New Chemical Substances, and under GLP conditions.

During the study, the test material was repeatedly administered by oral gavage at 0 (control group), 62.5, 250 and 1000 mg/kg from 14 days before mating through mating for 42 days in males and satellite females, and from 14 days before mating through gestation and parturition until Day 13 of lactation in females to assess the repeated dose toxicity and reproductive and developmental toxicity. In addition, a 14-day recovery period was set for the control and 1000 mg/kg groups and the reversibility of the toxicity effect was assessed.

One dam treated with 1000 mg/kg was found dead on LD 2. In the dead animal, no other remarkable change was observed in the clinical observation. In the histopathology, the following changes were noted in this animal: moderate atrophy of the thymus, mild atrophy of the spleen, mild vacuolation of proximal tubular epithelium (positive for Oil red O staining, indicating that the vacuoles were lipid), minimal hypertrophy of centrilobular hepatocytes and minimal increase of trabecular bone in the sternum and femur. The cause of death remains unclear; however, it was possible that the death occurred due to overlapping effects of the test material and postpartum stress.

In the surviving animals, a lower body weight was noted in males treated with 1000 mg/kg throughout the dosing period, relative to the concurrent vehicle control. A lower body weight was noted in females treated with 1000 mg/kg on GD 20. Food consumption in both sexes treated with 1000 mg/kg was lower than the control during the first week of treatment.

Adaptive physiological responses attributable to the test material treatment were noted at 250 mg/kg and above and this is not considered to be adverse in nature. The related changes were noted as follows: enlargement of the liver in males treated with 1000 mg/kg; a higher liver weight in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a higher thyroid weight in females treated with 1000 mg/kg; minimal or mild hypertrophy of centrilobular hepatocytes with a tendency of increasing incidence and severity, and minimal hypertrophy of basophilic cells in the anterior lobe of the pituitary in both sexes treated with 250 mg/kg and above; minimal hypertrophy of follicular cells of the thyroid in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and a low total T4 level in males treated with 1000 mg/kg. However, the above-mentioned pathological changes in males treated with 250 mg/kg and above and females treated with 1000 mg/kg were judged to be toxic effects as the blood chemistry revealed the effects on protein/lipid metabolism in both sexes treated with 1000 mg/kg and increased ALAT in males treated with 250 mg/kg above and females treated with 1000 mg/kg. Increased calcium level was noted in both sexes treated with 1000 mg/kg, suggesting an association with hyperthyroidism.

Minimal increase of trabecular bone in the sternum and femur was noted in females treated with 250 mg/kg and above and both sexes treated with 250 mg/kg and above, respectively. The cause remains unclear.

Lipid accumulation in the cells or dysbolism of lipid was noted in the adrenal and alveolus of the lung; however, the cause remains unclear. Related changes were noted as follows. A high total cholesterol level was noted in both sexes treated with 1000 mg/kg. A higher adrenal weight was noted in females treated with 1000 mg/kg.

Histopathological examination revealed the following: minimal vacuolation of cortical cells in the fascicular zone of the adrenal in males treated with 1000 mg/kg and females treated with 250 mg/kg and above; minimal vacuolation of cortical cells in the glomerular zone of the adrenal in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and minimal accumulation of foam cells in the alveolus of the lung in females treated with 250 mg/kg and above. The vacuoles in the adrenal and fine vacuoles in the alveolus of the lung were positive for Oil red O staining. The changes suggesting the irritation of the test material were noted in the stomach and the cecum. In the stomach, the following changes were noted in the limiting ridge (all findings were of minimal severity): erosion in males treated with 250 mg/kg and above; hyperkeratosis, parakeratosis, inflammatory cell infiltration of submucosa and vacuolation of squamous epithelium in males treated with 1000 mg/kg. In the cecum, minimal thickening of mucosa was noted in both sexes treated with 250 mg/kg and above.

The damage to the tubular epithelium in the kidney was noted. In the urinalysis, pH was biased toward the acidic side and urine volume increased in males treated with 1000 mg/kg. In the blood chemistry, a low Na level was noted, but slightly, in males treated with 1000 mg/kg. A higher kidney weight was noted in both sexes treated with 1000 mg/kg. In the histopathology, mild basophilic tubule was noted in only one male treated with 1000 mg/kg.

Dysbolism of calcium was noted: a high calcium level was noted in both sexes treated with 1000 mg/kg in the blood chemistry and minimal mineralisation of aortic wall and mucosa in the glandular stomach was noted in females treated with 1000 mg/kg. Slight anaemia was noted in both sexes treated with 1000 mg/kg. In the spleen, minimal erythrocytic extramedullary hematopoiesis was noted in both sexes treated with 250 mg/kg and above, indicating a reaction to anaemia.

A lower thymus weight was noted in females treated with 1000 mg/kg. In the histopathology, minimal atrophy of the thymus was noted in one female treated with 1000 mg/kg. It was judged to be a secondary reaction associated with stress as this animal had a suppressed body weight gain.

In the recovery test, among the changes noted at the end of the dosing period, except for the changes noted in the sternum and femur, reversibility or a tendency to recover was observed.

In the sternum, minimal increase of trabecular bone was noted in satellite females treated with 1000 mg/kg and the incidence was similar to that of females treated with 1000 mg/kg at termination. In the femur, minimal increase of trabecular bone was noted in males for recovery test and satellite females treated with 1000 mg/kg. The incidence in males for recovery test was lower than that of males treated with 1000 mg/kg at termination, while the incidence in satellite females was similar to that of females treated with 1000 mg/kg at termination. These changes may be reversible if the recovery period is extended.

A lower value of body weight was noted in males for recovery test treated with 1000 mg/kg throughout the recovery period. However, body weight gains during the recovery period were similar to or higher than that of Controls, indicating reversibility.

A higher value of the liver weight was noted in satellite females treated with 1000 mg/kg. Minimal hypertrophy of centrilobular hepatocytes was noted in one satellite female treated with 1000 mg/kg and the incidence and severity were lower than those of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility.

In the adrenal gland, minimal vacuolation of cortical cells in the fascicular zone was noted in one satellite female treated with 1000 mg/kg. The incidence was lower than that of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility. Minimal vacuolation of cortical cells in the glomerular zone was not noted in any animal after recovery, indicating reversibility.

A higher value of the spleen weight was noted in males for recovery test treated with 1000 mg/kg. Minimal erythrocytic extramedullary haematopoiesis was noted in one male for recovery test and one satellite female treated with 1000 mg/kg. The incidence in satellite females was lower than that of females treated with 1000 mg/kg at termination. On the other hand, the incidence in males for recovery test was similar to that of males treated with 1000 mg/kg at termination and haematological examination also revealed anemia in males for recovery test. However, the severity was slight and increased hematopoiesis was noted, indicating a tendency to recover.

A higher kidney weight was noted in both sexes treated with 1000 mg/kg. However, this was judged not to be toxicologically significant as no related change was noted in any examination.

There were no effects on parental reproductive parameters.

In offspring, a low value of body weight was noted in both sexes at 1000 mg/kg on PND 13. However, since the viability was not affected, the toxic effect on offspring was judged not to be severe.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg bw/day.

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg bw/day due to decreased body weight of F1 offspring at 1000 mg/kg bw/day on PND 13.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 December 2018 to 12 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Testing Methods Concerning New Chemical Substances
Version / remarks:
Notification No. 0331-7, PFSB, MHLW; No. 5 of March 29, 2011, MIB, METI; No.
110331009, EPB, MOE; dated March 31, 2011
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in toxicity studies using rodents, there is abundant historical data and a large number of animals are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks old at receipt, 9 weeks old at the start of administration
- Weight at receipt: 224.8 to 247.6 g (males), 162.3 to 188.7 g (females)
- Fasting period before study: No, but animals were fasted before scheduled necropsy for about 17 to 24.5 hours.
- Housing: Animals were housed 2 or 3 animals per cage before grouping. After gouping, animals were housed 1 male and 1 female per cage during the mating period, one dam and its litter per cage during the lactation period and one animal per cage for the other periods. For males and females except for copulated females and dams, animals were housed in hanging type stainless wire mesh cages (W × D × H: 226 × 346 × 198 mm). Copulated females were housed in polymethylpentene cages (W × D × H: 220 × 380 × 195 mm). Both types of cages were furnished with two types of nesting material (Paper Clean, Diamond Twists) and one kind of gnawing material (Diamond Twists) for the improvement of animal welfare.
- Diet: Pelleted diet, ad libitum (except during fresh urine collection and measurement of motor activity)
- Water: Well water admixed with NaClO, ad libitum (except during the measurement of motor activity)
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
Data for each lot of the diet were obtained from the supplier and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.
Water was analysed twice a year. The results were confirmed to be within the acceptable limits established by the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.1 to 23.7 °C
- Humidity: 51.4 to 72.6 %
- Air changes: 10 to 20 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Details on route of administration:
The test material was administered orally using a disposable syringe attached to a gastric tube.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Following correction for substance purity, a prescribed amount of the test material was was weighed into a beaker and an appropriate amount of the vehicle was added and stirred with a magnetic stirrer.
After confirming of dissolution, it was transferred to a measuring cylinder. The final volume was adjusted by adding a proper quantity of the vehicle to provide the required concentration of dosing solution. Further dilution provided lower concentration dosing solutions.
Dosing formulations were prepared once within 12 days (3- to 10-day interval). The dosing formulations after preparation were divided into glass vials for each dosing day and stored at room temperature (17.9°C to 22.4°C).

DOSE VOLUME:
Individual volume was calculated on the basis of the most recently measured body weights.
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC
Details on analytical verification of doses or concentrations:
At the first preparation, 10 mL each of the analytical samples was taken from the whole dosing formulation at each concentration and the test material concentration analysed by HPLC according to a previously validated method. Nominal test material concentrations of 12.5, 50 and 200 mg/mL provided mean measured concentrations of 12.68, 50.11 and 203.1 mg/mL, respectively and were within 101.4, 100.2, and 101.6% of nominal, respectively.
Test material formulations at 1 and 200 mg/mL were confirmed to be stable after storage for 12 days at room temperature.

High Performance Liquid Chromatography (HPLC) conditions:
Instrumentation: Prominence UFLC (Shimadzu Corporation)
Data processing: LabSolutions (Shimadzu Corporation)
Column: Ascentis Express RP-Amide (2.7 μm, 3.0 mm I.D. × 50 mm, Sigma-Aldrich Co.)
Column temperature: A constant temperature of about 40°C (set at 40°C)
Mobile phase:
Mobile phase A; water*
Mobile phase B; acetonitrile*
Linear gradient conditions and flow rate:
0.00 and 3.00 mins (55% A : 45% B) at a flow rate of 0.9 mL/min
3.01 and 12.00 mins (0% A : 100% B) at a flow rate of 1.5 mL/min
21.01 and 22.00 min (55% A : 45% B) at a flow rate of 0.9 mL/min
Analysis time: 22 minutes
Wavelength for detection: UV 200 nm
Injection volume: 10 μL
Auto-sampler set temperature: Room temperature (Not setting)
Needle wash solvent: Acetonitrile*
* sonicated under vacuum and degassed.
Duration of treatment / exposure:
Males: From 14 days before mating until the day before necropsy through the mating period (42 days in total)
Females: From 14 days before mating until Day 13 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery.
Recovery females (Satellite females) For 42 days without mating (as for males)
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
0 (corn oil control) and 1000 mg/kg bw: 7 males and 12 females (main dosing group) and an additional 5 males and 5 females (recovery group)
62.5 and 250 mg/kg bw: 12 males and 12 females (main dosing group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected following consideration of the effects observed in a 14-day study, during which 5 animals/sex/dose received oral gavage doses of test material at 0 (corn oil control), 110, 330, and 1000 mg/kg bw, for 14 consecutive days.
Following administration of test material, a lowering tendency of body weight was noted in males of the 1000 mg/kg group throughout the dosing period; however, a low value of body weight gain was transient. Haematology revealed anaemia in females of the 330 and 1000 mg/kg groups, and prolonged PT and APTT in males of the 1000 mg/kg group as an effect on the coagulation system.
At necropsy, enlargement of the liver was noted in both sexes of the 1000 mg/kg group and a high value of liver weight was noted in both sexes of the 330 and 1000 mg/kg groups. In the blood chemistry, a high value of ALAT was noted in both sexes of the 1000 mg/kg group, suggesting hepatic injury. The effects on protein/lipid metabolism were noted in males of the 1000 mg/kg group and females of the 110 mg/kg group and above. Moreover, whitish patch of mucosa in the forestomach was noted in 1 female of the 1000 mg/kg group, suggesting that the test material is irritating. Although various treatment-related changes were observed, none of the changes was severe. Therefore, the high dose in this study set at 1000 mg/kg, at which clear toxicity effects are expected to be seen. Lower doses were set at 250 and 62.5 mg/kg, with a common ratio of 4. A control group (0 mg/kg group) dosed with the vehicle (corn oil) alone was also established.
- Rationale for animal assignment: All animals except for the animals with abnormal oestrous cycle (as determined during the quarantine and acclimation period) were used for grouping. Animals were assigned to groups by the stratified randomisation on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weights ± 20% (calculated for each sex) were used.
- Post-exposure recovery period in satellite groups: For 5 males and 5 females (non-mating satellite females) each in the control and 1000 mg/kg groups, a 14-day recovery period was set after the end of the dosing period.
Positive control:
n/a
Observations and examinations performed and frequency:
The initial day of dosing was designated as Day 1, Day 1 to Day 7 as Week 1, period after Day 43 as the recovery period. For the test females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).

CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before dosing and after dosing) during the dosing period and once a day in the other periods.

DETAILED CLINICAL OBSERVATIONS: Yes (as part of functional observation battery)
- Time schedule: For all animals, detailed clinical observations were performed once before the start of dosing and once a week in the afternoon during the dosing and recovery periods (day of gestation or parturition was prescinded).
Detailed clinical observations included:
- Hand-held observation, during which animals were removed from the cage for the observation by grasping their trunk gently from behind. Their reactivity to handling, trauma, colour of skin, soiled fur, piloerection, secretion, salivation, lacrimation, exophthalmos, palpebral closure, colour of conjunctiva and pupil size were observed.
- Observation on open field, during which animals were placed in the center of the open field and observed quietly for one minute. Parameters observed included arousal, posture/body position, gait, tremor, convulsion, respiration, stereotypy, bizarre behaviour, urination, defecation and number of rearings.

BODY WEIGHT: Yes
- Time schedule for examinations: The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36 and 42. The recovery males were also weighed on Days 43, 50 and 56. The satellite females were weighed at the same frequency as the recovery males. The test females were weighed on Days 1, 8 and 15, once every 7 days after the initiation of cohabitation, on GDs 0, 7, 14 and 20, and on LDs 0, 4, 7 and 13.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured between Days 1 and 8, 8 and 15, 22 and 29, 29 and 36 and 36 and 41 for the test and recovery males, and between Days 43 and 50 and 50 and
55 for the recovery males. For the satellite females, it was measured between Days 1 and 8, 8 and 15, 15 and 22, 22 and 29, 29 and 36, 36 and 41, 43 and 50 and 50 and 55. Food consumption of the test females was measured at the same frequency as body weight measurement. However, food consumption was not measured for either sex during the mating period. After the completion of copulation, the measurement for males was started from the nearest measurement day. Gross weight of each feeder was weighed, and the mean daily food consumption for each period was expressed as the start day of each measurement.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (The animals were anaesthetised with intraperitoneal injection of sodium pentobarbital and about 2.0 to 2.5 mL of blood was collected from the posterior vena cava).
- Animals fasted: Yes (All animals were fasted for 16 hours and above from the day before necropsy (actual fasting time: about 17 to 24.5 hours): from Day 42 for the test males, Day 56 for the recovery males and satellite females and LD 13 for the test females.)
- How many animals: Subjected animals were 5 animals per group with the smallest animal numbers for the test males, all recovery males, all satellite females and 5 animals per group from the earlier parturition date with the smallest animal numbers for the test females.
- Parameters checked included: Red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte ratio and count, platelets, white blood cell, differential leukocyte ratio and count, prothrombin time (PT), and activated partial thromboplastin time (APTT)
For examination of the coagulation system, 0.9 mL of blood was collected into a tube containing 0.1 mL of 3.2 w/v% trisodium citrate, and then plasma was obtained by centrifugation at 1870 × g for 15 minutes (set temperature: 4°C). For examination of the other items, the remaining blood was put into a blood container containing 2 mg of EDTA-2K as an anticoagulant.

CLINICAL CHEMISTRY: Yes
After blood sampling for the hematology, 3 to 5 mL of blood was collected from the posterior vena cava under anesthesia for blood chemistry. The blood was left standing at room temperature for about 60 minutes, and then serum was obtained by centrifugation at 1870 × g for 10 minutes (set temperature: 4°C). The serum samples were stored frozen in a deep freezer until examination.
- Parameters checked included: Total protein, albumin, A/G ratio, asparate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyltranspeptidase (γ-GT), alkaline phosphatase (ALP), total bilirubin, total bile acid, total cholesterol, triglycerides, glucose, urea nitrogen, creatinine, calcium (Ca), inorganic phosphorus, sodium (Na), potassium (K), and chloride (Cl).

URINALYSIS: Yes
Urinalysis was conducted for 5 males per group with the smallest animal numbers in the final dosing week (Day 41) by a reagent strip method. Fresh urine samples were collected using metabolic cages after 9:00 a.m. (before dosing) under fasting conditions (water was given).
According to these results, pH was biased to the acidic side, and significant change was noted; this was suspected to be treatment-related. Therefore, the urine sediment test (Day 41) and accumulated urine test (Day 42) during the dosing period and urinalysis during the recovery period (Days 55 and 56) were additionally performed. Urine samples were collected successively for about 24 hours. Diet was given after the fresh urine collection and water was given with a watering bottle during the urine collection. The pooled urine for measurement of electrolyte excretion collected during the dosing period was stored frozen until examination.
- Parameters checked included: pH, protein, glucose, ketone, occult blood, urine sediments, volume, specific gravity, sodium, potassium, chloride

FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule for examinations and dose groups that were examined: Functional tests and motor activity measurement were performed in 5 males per group with the smallest animal numbers once in the afternoon in the final week (Week 6) of the dosing period. For females, 5 dams from the nearer parturition date were selected from each group and performed once in the afternoon of the final week during the lactation period. These examinations were performed following the clinical observation after dosing during the dosing period.
According to these results, the functional tests and motor activity measurement were not performed during the recovery period since no treatment-related change was noted during the dosing period.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
TERMINAL PROCEDURES
All surviving animals were euthanised by exsanguination under anaesthesia and subjected to necropsy. In addition, the vaginal smears were collected from the females on LD 14 (necropsy day) in the morning and the stage of the oestrous cycle was examined under a microscope.
Non-delivered female (No. 691) was necropsied on GD 26. This animal was euthanised and necropsied in the same manner as the surviving animals.

GROSS PATHOLOGY: Yes
The following organs/tissues were collected and examined: Heart, thymus, spleen, mandibular lymph node, mesenteric lymph node, femur/bone marrow (femur), sternum/bone marrow (sternum), trachea, lung (including bronchus), stomach, duodenum, jejunum, ileum (including Payer’s patch), cecum, colon, rectum, liver, pancreas, submandibular gland, kidney, urinary bladder, testis, epididymis, prostate (ventral lobe), seminal vesicle (including dorsolateral lobe and coagulating gland), cowper’s gland, glans penis, ovary, uterus, vagina, pituitary, thyroid/parathyroid, adrenal, spinal cord (cervical), brain (cerebrum, cerebellum and medulla oblongata/pons), eyeball, femoral muscle/Sciatic nerve (femoral region), levator ani/bulbocavernosus muscle (LABC), and any other organs/tissues indicating gross lesion.

ORGAN WEIGHTS: Yes
After the scheduled necropsy, the organs listed above were weighed (absolute weight) and the ratios of the organ weights to body weight (relative weight) were calculated on the basis of the body weight measured on the day of necropsy. The measurement was conducted in 5 animals per group with the smallest animal numbers of the test males; all recovery males; all satellite females; 5 animals per group from the earlier parturition date with the smallest animal numbers in the test females. However, the testis and epididymis weights were measured in all males. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
After necropsy, the organs/tissues listed above were fixed in 10 vol% phosphate buffered formalin solution; however, the testes and epididymides were fixed in Bouin’s solution and the eyes were fixed in Davidson’s solution, and they were preserved in 10 vol% phosphate buffered formalin solution. As for the dead animal, all of the organs/tissues were preserved in 10 vol% phosphate buffered formalin solution. The organs/tissues of 5 animals per group with the smallest animal numbers of the test males and 5 animals per group from the earlier parturition date with the smallest animal numbers for the test females in the control and 1000 mg/kg groups collected at the end of the dosing period, one dead animal (No. 693) and gross lesion were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and then examined by microscopy.
As for the dead animal, the following organs could not be examined due to autolysis: mandibular lymph node, bone marrow (sternum and femur), trachea, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, submandibular gland, urinary bladder, ovary, uterus, vagina, pituitary, thyroid, adrenal, spinal cord and sciatic nerve.
According to these results, a change suspected to be attributable to the test material treatment was observed in the 1000 mg/kg group as follows: liver, pituitary, thyroid, adrenal, femur, spleen, stomach, cecum and kidney of both sexes and heart, sternum, thymus and lung of females. Therefore, the relevant organs/tissues of the relevant sex in the low and middle dose groups and all groups in the recovery study were additionally examined, according to the above criteria. Vacuolation was noted in the adrenal and kidney and foam cells were noted in the lung. To confirm whether the vacuole and foam cells are derived from lipid, these organs from one representative each in the 1000 mg/kg group (adrenal: No. 637, kidney: No. 693 and lung: No. 688) was subjected to Oil red O staining for microscopic examination.
Other examinations:
HORMONE MEASUREMENT
Hormone (total T4) concentrations in rat plasma samples (parental animals [F0 male and F0 female], offspring [F1 offspring on PND 13], and Satellite female) were measured. A total of 80 samples (30 samples of F0 male, 20 samples of F1 offspring on PND 13, 20 samples of F0 female, and 10 samples of Satellite female) were assayed.
The plasma samples were stored frozen until analysis. The frozen plasma samples were thawed immediately before use, and then centrifuged (1710 × g for 10 minutes at set 4°C) to remove any aggregates in the plasma. The supernatant was used for assay.
An aliquot (60 μL) of the test sample or “standard solution (20 μL) (0, 25.5, 51.0, 102, 204, and 408 nmol/L) and PBS (phosphate buffered saline, 40 μL)” was pipetted into a tube coated with anti-T4 antibody. An aliquot (500 μL) of the tracer (125I-labelled T4) was added to the tube and the solution was mixed and shaken for 1 hour at room temperature. After the solution was aspirated, the remaining 125I radioactivity (cpm) in the tube was measured for 1 minute using the gamma counter.
A calibration curve was prepared with the mean cpm values of the duplicate assay for the standard solutions. The concentrations in the test samples were calculated using the calibration curve and the cpm value of the single assay for the test samples and corrected with the volume factor at the time of measurement. Hormone concentrations were calculated using the software (RiaSmart, PerkinElmer, Inc.) and Excel 2010 (Microsoft Corporation). The lower limit of quantification (LLOQ) was set at the lowest concentration of the standard solution (except for the zero concentration).

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY
The following parameters were assessed: oestrous cycle, mating (days until copulation, copulation index, and fertility index), observation during delivery and lactation periods, examination after lactation (gestation length, gestation index, and delivery index).

OBSERVATIONS AND EXAMINATION OF OFFSPRING
The following items were examined: observation of offspring which included mortality, sex, presence or absence of external anomalies (birth index, sillborn index, viability index on Day 4, viability index on Day 13, sex ratio, external anomaly index, and external anomaly typing index), body weight, external examination (external anomaly index, and external anomaly typing index), anogenital distance, and nipple development.
Offspring necropsy was performed on PND 13. The following pathological examinations were conducted: blood sampling for hormone concentration (total T4) analysis.
Statistics:
Statistical analysis was performed with a computer system (tsPharma LabSite, Fujitsu Limited). However, a statistical analysis system EXSUS Version 8.1.0 (CAC Croit Corporation, statistical analysis software: SAS 9.4 [SAS Institute Japan Inc.]) was used for urinalysis (qualitative data: results from reagent strip method and urinary sediment observation) and sex ratio. In all cases, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used. Analysis for concentration of total T4 was conducted according to Work Plan.
When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group.
For results from reagent strip method and urinary sediment observation in urinalysis during the dosing period, Steel’s multiple comparison test was performed after the grades were converted into numeric values. During the recovery period, Aspin-Welch’s t test was performed in the same manner. The multiple comparison test was performed for the following repeated dose toxicity parameters: number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis (except for urine colour), hematology, blood chemistry, absolute and relative organ weight.
In the hormone concentration, data of F0 male and F0 female after dosing period and F1 offspring (PND 13) were tested by Bartlett's test for homogeneity of variance. When the variances were homogeneous, Dunnett's test was to be performed. When the variances were heterogeneous, the Steel test was to be performed. Data of F0 male and Satellite female after recovery period were tested by the F test for homogeneity of variance between the groups. When the variances were homogeneous, the Student's t-test was to be performed, and when the variances were heterogeneous, the Aspin & Welch t-test was performed.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted. Mass was observed in the axilla of one female (No. 698) treated with 1000 mg/kg on LD 4 and thereafter. However, it was judged that this change was not related to treatment with the test material.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female (No. 693) treated with 1000 mg/kg was found dead on LD 2. In this animal, reduced locomotor activity was observed the day before, but no other remarkable change was observed. A suppressed body weight gain was noted in this animal until the death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A low value of body weight was noted in males treated with 1000 mg/kg throughout the dosing and recovery periods (statistically significant compared to vehicle control).
A low value of body weight (statistically significant compared to vehicle control) was noted in females treated with 1000 mg/kg on GD 20.
In satellite females, no significant difference was noted between the control and 1000 mg/kg group throughout the dosing or recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A low value of food consumption (statistically significant compared to vehicle control) was noted in males treated with 1000 mg/kg during Day 1-8. Thereafter, food consumption in males treated with 1000 mg/kg was similar to or higher (during Day 29-36) than that of Controls throughout the dosing and recovery periods.
Prior to pairing, food consumption was low in females treated with 1000 mg/kg during Day 1-8 (statistically significant compared to vehicle control). Thereafter, food consumption was similar to Controls for females at all dose levels during the gestation and lactation periods.
In satellite females (after Day 8), food consumption was similar to or higher than (statistically significant compared to vehicle control) that of Controls throughout the dosing and recovery periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, some erythroid parameters at 1000 mg/kg were marginally and not statistically significantly lower compared to the concurrent vehicle control as follows: a low value of RBC count in females (93.9% of Controls), a lower value of haemoglobin concentration in males (94.9%) and females (93.6%), a lower value of haematocrit in males (93.3%) and females (94.3%).
At the end of the recovery period, lower values of RBC count, haemoglobin concentration and haematocrit and a higher value of reticulocyte ratio were noted in males treated with 1000 mg/kg (statistically significant compared to vehicle control).
Besides, at the end of the dosing period, shortening of PT and APTT was noted in females treated with 1000 mg/kg, the differences from the control attained the statistical significance. However, these were judged not to be treatment-related as the changes were slight compared to those of Controls and opposite to the toxicity effect. A statistically significant prolongation of PT was noted in males treated with 250 mg/kg, however, the change was not accompanied by a dose-dependency and considered not to be treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, treatment-related changes were noted as follows: higher values of albumin and A/G ratio in males treated with 1000 mg/kg; a higher value of ALAT in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a high value or higher tendency of total cholesterol and Ca in both sexes treated with 1000 mg/kg and a lower value of Na in males treated with 1000 mg/kg.
At the end of the recovery period, none of the above-mentioned changes was noted.
Besides, at the end of the dosing period, lower values of ALP and total bile acid were noted in females treated with 1000 mg/kg, attaining statistical significance. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
At the end of the recovery period, a lower value of glucose was noted in males treated with 1000 mg/kg as well as K in females treated with 1000 mg/kg. However, these were judged not to be treatment-related as the similar changes were not noted at the end of the dosing period and no related change was noted in any examination. Lower values of γGT, ALP and total bile acid were noted in males treated with 1000 mg/kg. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
During the dosing period, pH was biased toward the acidic side in males treated with 1000 mg/kg and significant difference was noted when compared with Controls. An increase of urine volume was noted in males treated with 1000 mg/kg.
During the recovery period, none of the above-mentioned changes was noted.
Besides, high values of Na, K and Cl were noted in males treated with 1000 mg/kg during the recovery period. However, these were judged not to be treatment-related as the changes, although statistically different, were slight compared to those of the control group, the similar changes were not noted during the dosing period or no related change was noted in the histopathology.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Detailed clinical observations: No treatment-related change was noted in any animal in the hand held observation or observation on the open field.
The following changes were noted in the number of rearing: a low value in males treated with 1000 mg/kg on Day 35, a high value in females treated with 250 mg/kg and above on Day 7 and a low value in females treated with 1000 mg/kg on Days 35 and 42, with the differences from the control attained the statistical significance. These were transient changes and no related changes were noted in the other parameters. Therefore, it was judged that the changes were not related to the test material treatment.
- Sensory reactivity to stimuli: Reactivity to stimuli was comparable in males and females at all dose levels and no abnormality was observed.
- Grip strength: No significant difference was noted in males or females between the control and test material groups.
- Motor activity: No treatment-related change was noted.
A low value of motor activity was noted between 20 and 30 minutes in females treated with 1000 mg/kg (statistically significant compared to vehicle control). However, this was judged not to be treatment-related as the moving pattern was normal and no related change was noted in the detailed clinical observations. As a change without dose-dependency and considered as not treatment-related, a high value of motor activity was noted between 40 and 50 minutes in females treated with 62.5 mg/kg (statistically significant compared to vehicle control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, treatment-related changes were noted as follows: a higher value or higher tendency of absolute and relative liver weights in males treated with 250 mg/kg and/or above and females treated with 1000 mg/kg; a higher value of relative kidneys weight in males treated with 250 mg/kg and above; higher values of absolute and relative kidneys and adrenals weights and a higher value of relative thyroids weight and lowering tendencies of absolute and relative thymus weights in females treated with 1000 mg/kg.
At the end of the recovery period, a higher value of absolute kidneys weight was noted in females treated with 1000 mg/kg and a higher value of relative kidneys weight was noted in both sexes treated with 1000 mg/kg. A higher value of relative spleen weight was noted in males treated with 1000 mg/kg. Higher values of absolute and relative liver weights were noted in females treated with 1000 mg/kg.
Besides, at the end of the dosing period, lower values of absolute seminal vesicle and levator ani muscle and bulbocavernosus muscle and a high value of relative brain weight were noted in males treated with 1000 mg/kg; these were judged to be due to a lowering tendency of the final body weight. As a change without dose-dependency and considered as not treatment-related, a low value of absolute seminal vesicle weight and a high value of relative levator ani muscle and bulbocavernosus muscle were noted in males treated with 62.5 mg/kg and males treated with 250 mg/kg, respectively. At the end of the recovery period, low values of absolute thymus and heart weights and a high value of relative epididymides weight were noted in males treated with 1000 mg/kg; these were judged to be due to the low value of final body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, enlargement of the liver was noted in 7/7 males treated with 1000 mg/kg.
At the end of the recovery period, no macroscopic change was observed in any animal.
In the dead animal (No. 693) treated with 1000 mg/kg, no macroscopic change was noted.
In non-delivered female (No. 691) treated with 1000 mg/kg, no macroscopic change was noted.
Besides, at the end of the dosing period, mass was noted subcutaneously in the axilla of one female (No. 698). However, this was judged not to be treatment-related. As a congenital abnormality, abnormal lobulation of the liver was noted in one female (No. 672).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver:
- Hypertrophy of centrilobular hepatocytes was noted in 3/5 males and 1/5 females treated with 250 mg/kg (grade: minimal) and 5/5 males and 5/5 females treated with 1000 mg/kg (minimal or mild). After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
Pituitary:
- Minimal hypertrophy of basophilic cells in the anterior lobe was noted in 3/5 males treated with 250 mg/kg and 5/5 males and 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thyroid gland:
- Minimal hypertrophy of follicular cells was noted in 2/5 males treated with 250 mg/kg and 5/5 males and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Adrenal gland:
- Minimal vacuolation of cortical cells in the fascicular zone was noted in 1/5 females treated with 250 mg/kg and 4/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
- Minimal vacuolation of cortical cells in the glomerular zone was noted in 4/5 males treated with 250 mg/kg and 3/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- These vacuoles were positive for Oil red O staining.
Sternum:
- Minimal increase of trabecular bone was noted in 1/5 females treated with 250 mg/kg and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in all satellite females treated with 1000 mg/kg (minimal).
Femur:
- Minimal increase of trabecular bone was noted in 1/5 males and 1/5 females treated with 250 mg/kg and 4/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 2/5 males and 5/5 satellite females treated with 1000 mg/kg (minimal).
Spleen:
- Minimal erythrocytic extramedullary hematopoiesis was noted in 1/5 males and 1/5 females treated with 250 mg/kg and 1/5 males and 4/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 1/5 males and 1/5 satellite females treated with 1000 mg/kg (minimal).
Cecum:
- Minimal thickening of mucosa was noted in 1/5 males and 3/5 females treated with 250 mg/kg and 2/5 males and 4/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Kidney:
- Mild basophilic tubule was noted in 1/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
Stomach:
- Minimal mineralisation of mucosa in the glandular stomach was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal erosion in the limiting ridge was noted in 1/5 males treated with 250 mg/kg and 2/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal hyperkeratosis in the limiting ridge was noted in 3/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal parakeratosis in the limiting ridge was noted in 3/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal inflammatory cell infiltration in the submucosa of limiting ridge was noted in 2/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal vacuolation of squamous epithelium in the limiting ridge was noted in 1/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
Lung:
- Minimal accumulation of foam cells in the alveolus was noted in 1/5 females treated with 250 mg/kg and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Fine vacuoles of the foam cells were positive for Oil red O staining.
Heart:
- Minimal mineralization of aortic wall was noted in 2/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thymus:
- Minimal atrophy was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.

> [Dead animal (No. 693) treated with 1000 mg/kg]
Thymus: Moderate atrophy was noted.
Spleen: Mild atrophy was noted.
Liver: Minimal hypertrophy of centrilobular hepatocytes was noted.
Kidney: Mild vacuolation of proximal tubular epithelium was noted. The vacuoles were positive for Oil red O staining.
Sternum: Minimal increase of trabecular bone was noted.
Femur: Minimal increase of trabecular bone was noted.

> Others:
Various histopathological changes were noted in both sexes of the control and test material groups. However, these changes were judged not to be treatment-related as they are observed occasionally in normal rats and there was no clear dose-dependency in the incidence.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Mass in the axilla noted in 1 female (No. 698) treated with 1000 mg/kg was adenocarcinoma of the mammary gland in the histopathology. Adenocarcinoma of mammary gland is known to occur infrequently in young mature rats [1] and no gross abnormalities were noted in the mammary gland of any other animals, therefore, it was judged not to be treatment-related.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Hormone measurement:
In parental animals (F0 male), plasma total T4 concentrations of the test material-treated group (Group 1000 mg/kg) tended to be lower compared to those in the control group after the dosing period. In offspring (F1 offspring [PND 13]) and parental animals (F0 female), plasma total T4 concentrations of the test material-treated groups (Groups 62.5, 250, and 1000 mg/kg) were equivalent to those of the control groups. The plasma total T4 concentration of recovery groups (Group 1000 mg/kg) were equivalent to those of the control groups in F0 male and Satellite female.
All values in the quality control sample were within the range of 100 ± 25% of the nominal value, and variations in duplicate cpm of the standard solutions were within the range of acceptability. There were no abnormalities in the procedures for the determination or in the values of the test samples. The results were therefore considered acceptable.

- Reproductive/Developmental toxicity:
There were no effects on parental reproductive parameters.
In offspring, a low value of body weight was noted in both sexes at 1000 mg/kg on PND 13. However, since the viability was not affected, the toxic effect on offspring was judged not to be severe.
Key result
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See 'Remarks'
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
other: effects on calcium metabolism and injury of the kidney, hepatic effects, effects on bone/lipid metabolism, and reaction to anaemia.
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Summary of bodyweights, g (mean ± SD)

Day

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

0 (17)

62.5 (12)

250 (12)

1000 (17)

1

370.18 ± 11.31

368.34 ± 10.91

368.61 ± 13.93

367.61 ± 11.12

230.74 ± 10.20

231.73 ± 9.27

231.80 ± 12.70

233.05 ± 11.49

8

411.53 ± 17.39

403.28 ± 17.29

404.06 ± 15.94

386.56 ± 15.33**

248.94 ± 9.37

246.68 ± 11.24

247.23 ± 14.02

244.68 ± 10.47

15

440.40 ± 20.25

426.15 ± 26.46

426.96 ± 20.61

408.28 ± 17.71**

258.92 ± 13.60

256.73 ± 11.96

258.48 ± 15.67

255.48 ± 10.28

22

465.89 ± 20.21

453.23 ± 32.50

453.22 ± 26.16

425.06 ± 18.46**

263.62 ± 9.64

256.60 ± 17.43

29

495.29 ± 22.27

476.41 ± 38.61

476.37 ± 30.82

451.31 ± 20.10**

267.86 ± 7.71

269.38 ± 11.31

36

519.59 ± 22.65

497.98 ± 42.47

498.53 ± 33.35

469.81 ± 22.99**

280.20 ± 9.14

278.80 ± 17.93

42

536.88 ± 21.91

514.63 ± 39.88

511.93 ± 35.54

484.18 ± 25.06**

286.10 ± 9.73

284.80 ± 21.75

43

537.84 ± 24.80

479.94 ± 31.80*

284.56 ± 13.69

283.80 ± 17.71

50

553.80 ± 24.88

494.18 ± 34.65*

292.34 ± 9.20

291.18 ± 17.98

56

562.62 ± 24.74

511.18 ± 32.89*

296.32 ± 10.78

301.28 ± 19.97

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Results in itallics relate to 5 animals

Day 1 to 42; Treatment period, Day 43 to 56; Recovery period

Summary of food consumption, g/animal/day (mean ± SD)

Day

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

0 (17)

62.5 (12)

250 (12)

1000 (17)

1

25.51 ± 1.65

25.59 ± 2.00

25.39 ± 2.27

22.99 ± 2.36*

17.71 ± 1.35

18.15 ± 1.56

17.88 ± 1.43

16.25 ± 1.75*

8

24.02 ± 1.79

22.73 ± 2.54

23.78 ± 1.96

24.26 ± 1.73

17.52 ± 1.94

17.48 ± 1.35

17.63 ± 1.74

18.44 ± 1.44

15

15.77 ± 1.63

18.85 ± 0.84**

22

23.89 ± 1.41

22.72 ± 2.38

24.28 ± 2.29

25.80 ± 1.90

15.50 ± 0.33

19.37 ± 1.05†

29

23.53 ± 1.32

22.44 ± 2.22

23.99 ± 1.82

25.82 ± 1.61**

15.63 ± 0.91

20.38 ± 1.45**

36

24.01 ± 1.18

22.90 ± 2.91

24.46 ± 1.81

25.54 ± 2.10

15.08 ± 1.07

19.78 ± 0.70**

43

27.51 ± 2.05

27.29 ± 2.59

17.98 ± 0.66

23.80 ± 3.66†

50

29.95 ± 2.71

29.97 ± 1.87

18.32 ± 0.41

23.02 ± 1.72†

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Significantly different from the control group ; † p < 0.01 (Steel test)

Results in itallics relate to 5 animals

Day 1 to 36; Treatment period, Day 43 to 50; Recovery period

The mean daily food consumption for each period is expressed as the start day of each measurement

Summary of selected haematology parameters (mean ± SD)

Study period

Parameter

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (5)

62.5 (5)

250 (5)

1000 (5)

0 (5)

62.5 (5)

250 (5)

1000 (5)

end of dosing period‡

Red blood cell count (10^6/µL)

8.536 ± 0.239

8.610 ± 0.327

8.408 ± 0.371

8.162 ± 0.481

7.042 ± 0.216

6.976 ± 0.284

7.048± 0.265

6.614 ± 0.371

Haemoglobin conc (g/dL)

15.18 ± 0.59

15.38 ± 0.43

15.16 ± 0.50

14.40 ± 0.86

14.46 ± 0.37

14.36 ± 0.50

14.16 ± 0.59

13.54 ± 0.77

Heamatocrit (%)

42.02 ± 1.85

41.82 ± 1.36

42.00 ± 2.01

39.20 ± 2.12

39.32 ± 1.26

38.74 ± 1.28

38.60 ± 1.59

37.08 ± 1.53

Reticulocyte (%)

3.630 ± 0.448

3.520 ± 0.430

3.606 ± 0.264

3.812 ± 0.551

5.120 ± 0.315

4.938 ± 0.643

4.836 ± 0.604

6.104 ± 1.176

PT (sec)

11.30 ± 0.47

12.74 ± 1.81

15.18 ± 2.21†

13.22 ± 2.77

8.74 ± 0.21

8.70 ± 0.07

8.74 ± 0.15

8.48 ± 0.16*

APTT (sec)

19.80 ± 2.11

21.32 ± 2.94

23.46 ± 2.19

22.76 ± 3.60

15.92 ± 0.42

16.02 ± 0.60

15.94 ± 0.61

14.50 ± 1.01*

end of recovery period (Day 57)

Red blood cell count (10^6/µL)

8.910 ± 0.447

7.976 ± 0.166**

7.704 ± 0.664

7.388 ± 0.521

Haemoglobin conc (g/dL)

15.80 ± 0.99

14.20 ± 0.50*

14.54 ± 1.04

14.10 ± 0.84

Heamatocrit (%)

42.40 ± 2.41

38.86 ± 0.77*

39.64 ± 2.44

38.92 ± 2.13

Reticulocyte (%)

3.554 ± 0.340

4.584 ± 0.717*

3.574 ± 1.174

4.022 ± 0.890

PT

11.90 ± 1.53

12.72 ± 1.03

8.94 ± 0.22

8.82 ± 0.11

APTT

20.88 ± 1.60

19.98 ± 2.44

16.12 ± 0.66

16.32 ± 0.56

 ‡ Day 43 (males), Day 14 lactation period (Females)

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Significantly different from the control group ; † p < 0.05 (Steel test)

Summary of selected blood chemistry parameters (mean ± SD)

Study period

Parameter

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (5)

62.5 (5)

250 (5)

1000 (5)

0 (5)

62.5 (5)

250 (5)

1000 (5)

end of dosing period‡

Albumin (g/dL)

3.74 ± 0.11

3.92 ± 0.18

3.92 ± 0.22

4.26± 0.18**

3.76 ± 0.25

3.60 ± 0.20

3.56 ± 0.19

3.74 ± 0.23

A/G ratio

2.318 ± 0.158

2.452 ± 0.188

2.898 ± 0.308

4.306 ± 0.880†

2.388 ± 0.452

2.258 ± 0.335

2.228 ± 0.125

2.466 ± 0.385

ALAT (U/L)

18.8 ± 1.3

21.2 ± 3.1

23.6 ± 2.5†

34.6 ± 6.6†

51.6 ± 17.0

44.2 ± 12.4

59.6 ± 10.2

78.0 ± 14.5*

γGT (U/L)

0.68 ± 0.23

0.48 ± 0.26

0.70 ± 0.20

0.72 ± 0.25

0.52 ± 0.24

0.74 ± 0.52

0.70 ± 0.19

0.74 ± 0.27

ALP (U/L)

324.6 ± 58.5

283.4 ± 48.3

272.0 ± 65.5

332.8 ± 84.1

352.2 ± 66.0

278.4 ± 102.4

353.0 ± 203.2

196.8 ± 27.0†

Total bile acid (µmol/L)

8.32 ± 2.24

9.72 ± 3.67

9.60 ± 4.87

11.06 ± 6.07

34.34 ± 16.69

22.82 ± 5.36

19.00 ± 7.44

14.14 ± 7.16*

Total cholesterol (mg/dL)

54.2 ± 13.3

51.6 ± 5.9

59.2 ± 9.9

74.0 ± 5.4**

81.0 ± 26.4

80.0 ± 16.4

76.8 ± 12.6

97.8 ± 22.7

Glucose (mg/dL)

115.0 ± 13.3

112.0 ± 13.6

106.4 ± 7.2

110.4 ± 9.6

03.0 ± 15.1

97.6 ± 5.8

95.2 ± 7.9

100.6 ± 11.3

Ca (mg/dL)

9.20 ± 0.37

9.26 ± 0.09

9.40 ± 0.20

10.06 ± 0.47†

9.56 ± 0.53

9.32 ± 0.11

9.50 ± 0.44

10.60 ± 0.62

Na (mmol/L)

144.9 ± 1.0

144.2 ± 1.4

144.8 ± 0.7

143.0 ± 1.1*

140.3 ± 2.2

141.3 ± 1.9

141.1 ± 1.4

140.6 ± 2.8

K (mmol/L)

4.31 ± 0.27

4.26 ± 0.14

4.16 ± 0.22

4.36 ± 0.08

4.11 ± 0.23

3.92 ± 0.25

4.10 ± 0.25

4.03 ± 0.30

end of recovery period (Day 57)

Albumin (g/dL)

3.66 ± 0.11

3.80 ± 0.19

4.46 ± 0.39

4.48 ± 0.39

A/G ratio

1.932 ± 0.122

2.092 ± 0.258

3.012 ± 0.136

2.838 ± 0.448

ALAT (U/L)

23.8 ± 4.0

29.0 ± 4.1

36.8 ± 28.0

22.8 ± 10.3

γGT (U/L)

0.84 ± 0.09

0.60 ± 0.20*

0.88 ± 0.19

0.84 ± 0.21

ALP (U/L)

274.2 ± 50.8

201.2 ± 30.7*

127.4 ± 36.7

114.2 ± 32.6

‡ Day 43 (males), Day 14 lactation period (Females)

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Significantly different from the control group ; † p < 0.05, †† p < 0.01 (Steel test)

Selected organ weights (mean ± SD)

Organ

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (5)

62.5 (5)

250 (5)

1000 (5)

0 (5)

62.5 (5)

250 (5)

1000 (5)

Terminal body weight (g)

518.72 ± 25.15

492.70 ± 59.11

477.96 ± 39.67

457.88 ± 22.25

339.56 ± 25.60

333.00 ± 12.50

334.06 ± 13.65

308.12 ± 7.99*

Brain - rel (%)

0.40 ± 0.01

0.43 ± 0.05

0.45 ± 0.02

0.46 ± 0.03*

0.58 ± 0.04

0.60 ± 0.04

0.60 ± 0.02

0.63 ± 0.04

Thyroids - rel (x10^-3%)

4.46 ± 0.54

4.49 ± 0.45

5.22 ± 1.43

0.46 ± 0.03*

4.79 ± 0.50

4.76 ± 0.54

5.24 ± 0.41

5.92 ± 0.60**

Liver - abs. (g)

13.410 ± 1.376

13.948 ± 0.148

15.324 ± 1.497

21.602 ± 1.271**

12.004 ± 1.192

11.488 ± 1.111

12.638 ± 0.808

15.270 ± 0.462**

Liver - rel. (%)

2.58 ± 0.22

2.82 ± 0.10

3.20 ± 0.07†

4.73 ± 0.43†

3.53 ± 0.21

3.44 ± 0.24

3.78 ± 0.13

4.96 ± 0.23**

Kidneys - abs (g)

3.154 ± 0.239

3.266 ± 0.396

3.380 ± 0.247

3.478 ± 0.219

2.214 ± 0.224

2.114 ± 0.090

2.292 ± 0.215

2.522 ± 0.153*

Kidneys - rel. (%)

0.61 ± 0.03

0.66 ± 0.04

0.71 ± 0.08*

0.76 ± 0.07**

0.65 ± 0.02

0.64 ± 0.03

0.69 ± 0.06

0.82 ± 0.04**

Adrenals - abs. (mg)

67.20 ± 6.00

60.78 ± 7.00

56.94 ± 12.12

60.50 ± 8.85

68.40 ± 4.24

69.78 ± 2.56

75.50 ± 6.07

81.04 ± 8.35**

Adrenals - rel. (x10^-3%)

12.94 ± 0.60

12.35 ± 0.70

11.99 ± 2.74

13.30 ± 2.45

20.27 ± 2.45

20.97 ± 0.97

22.61 ± 1.68

26.36 2) 3.29**

Seminal vesicle - abs (g)

2.394 ± 0.064

2.116 ± 0.092†

2.482 ± 0.465

1.982 ± 0.298†

LABC - abs. (g)

1.590 ± 0.174

1.432 ± 0.141

1.740 ± 0.098

1.254 ± 0.135**

LABC - rel. (%)

0.31 ± 0.05

0.29 ± 0.03

0.37 ± 0.03*

0.27 ± 0.03

 LABC: Levator ani muscle and Bulbocavernosus muscle

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Significantly different from the control group ; † p < 0.05 (Steel test)

Conclusions:
Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg based on occurrence of death, decreased body weight, effects on calcium metabolism and injury of the kidney occurring at 1000 mg/kg and hepatic effects, effects on bone/lipid metabolism, injury of stomach and cecum and reaction to anaemia occurring at 250 mg/kg and above.
Executive summary:

The repeated dose toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 422 and the Japanese Testing Methods Concerning New Chemical Substances, and under GLP conditions.

During the study, the test material was repeatedly administered by oral gavage at 0 (control group), 62.5, 250 and 1000 mg/kg from 14 days before mating through mating for 42 days in males and satellite females, and from 14 days before mating through gestation and parturition until Day 13 of lactation in females to assess the repeated dose toxicity and reproductive and developmental toxicity. In addition, a 14-day recovery period was set for the control and 1000 mg/kg groups and the reversibility of the toxicity effect was assessed.

One dam treated with 1000 mg/kg was found dead on LD 2. In the dead animal, no other remarkable change was observed in the clinical observation. In the histopathology, the following changes were noted in this animal: moderate atrophy of the thymus, mild atrophy of the spleen, mild vacuolation of proximal tubular epithelium (positive for Oil red O staining, indicating that the vacuoles were lipid), minimal hypertrophy of centrilobular hepatocytes and minimal increase of trabecular bone in the sternum and femur. The cause of death remains unclear; however, it was possible that the death occurred due to overlapping effects of the test material and postpartum stress.

In the surviving animals, a lower body weight was noted in males treated with 1000 mg/kg throughout the dosing period, relative to the concurrent vehicle control. A lower body weight was noted in females treated with 1000 mg/kg on GD 20. Food consumption in both sexes treated with 1000 mg/kg was lower than the control during the first week of treatment.

Adaptive physiological responses attributable to the test material treatment were noted at 250 mg/kg and above and this is not considered to be adverse in nature. The related changes were noted as follows: enlargement of the liver in males treated with 1000 mg/kg; a higher liver weight in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a higher thyroid weight in females treated with 1000 mg/kg; minimal or mild hypertrophy of centrilobular hepatocytes with a tendency of increasing incidence and severity, and minimal hypertrophy of basophilic cells in the anterior lobe of the pituitary in both sexes treated with 250 mg/kg and above; minimal hypertrophy of follicular cells of the thyroid in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and a low total T4 level in males treated with 1000 mg/kg. However, the above-mentioned pathological changes in males treated with 250 mg/kg and above and females treated with 1000 mg/kg were judged to be toxic effects as the blood chemistry revealed the effects on protein/lipid metabolism in both sexes treated with 1000 mg/kg and increased ALAT in males treated with 250 mg/kg above and females treated with 1000 mg/kg. Increased calcium level was noted in both sexes treated with 1000 mg/kg, suggesting an association with hyperthyroidism.

Minimal increase of trabecular bone in the sternum and femur was noted in females treated with 250 mg/kg and above and both sexes treated with 250 mg/kg and above, respectively. The cause remains unclear.

Lipid accumulation in the cells or dysbolism of lipid was noted in the adrenal and alveolus of the lung; however, the cause remains unclear. Related changes were noted as follows. A high total cholesterol level was noted in both sexes treated with 1000 mg/kg. A higher adrenal weight was noted in females treated with 1000 mg/kg.

Histopathological examination revealed the following: minimal vacuolation of cortical cells in the fascicular zone of the adrenal in males treated with 1000 mg/kg and females treated with 250 mg/kg and above; minimal vacuolation of cortical cells in the glomerular zone of the adrenal in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and minimal accumulation of foam cells in the alveolus of the lung in females treated with 250 mg/kg and above. The vacuoles in the adrenal and fine vacuoles in the alveolus of the lung were positive for Oil red O staining. The changes suggesting the irritation of the test material were noted in the stomach and the cecum. In the stomach, the following changes were noted in the limiting ridge (all findings were of minimal severity): erosion in males treated with 250 mg/kg and above; hyperkeratosis, parakeratosis, inflammatory cell infiltration of submucosa and vacuolation of squamous epithelium in males treated with 1000 mg/kg. In the cecum, minimal thickening of mucosa was noted in both sexes treated with 250 mg/kg and above.

The damage to the tubular epithelium in the kidney was noted. In the urinalysis, pH was biased toward the acidic side and urine volume increased in males treated with 1000 mg/kg. In the blood chemistry, a low Na level was noted, but slightly, in males treated with 1000 mg/kg. A higher kidney weight was noted in both sexes treated with 1000 mg/kg. In the histopathology, mild basophilic tubule was noted in only one male treated with 1000 mg/kg.

Dysbolism of calcium was noted: a high calcium level was noted in both sexes treated with 1000 mg/kg in the blood chemistry and minimal mineralisation of aortic wall and mucosa in the glandular stomach was noted in females treated with 1000 mg/kg. Slight anaemia was noted in both sexes treated with 1000 mg/kg. In the spleen, minimal erythrocytic extramedullary hematopoiesis was noted in both sexes treated with 250 mg/kg and above, indicating a reaction to anaemia.

A lower thymus weight was noted in females treated with 1000 mg/kg. In the histopathology, minimal atrophy of the thymus was noted in one female treated with 1000 mg/kg. It was judged to be a secondary reaction associated with stress as this animal had a suppressed body weight gain.

In the recovery test, among the changes noted at the end of the dosing period, except for the changes noted in the sternum and femur, reversibility or a tendency to recover was observed.

In the sternum, minimal increase of trabecular bone was noted in satellite females treated with 1000 mg/kg and the incidence was similar to that of females treated with 1000 mg/kg at termination. In the femur, minimal increase of trabecular bone was noted in males for recovery test and satellite females treated with 1000 mg/kg. The incidence in males for recovery test was lower than that of males treated with 1000 mg/kg at termination, while the incidence in satellite females was similar to that of females treated with 1000 mg/kg at termination. These changes may be reversible if the recovery period is extended.

A lower value of body weight was noted in males for recovery test treated with 1000 mg/kg throughout the recovery period. However, body weight gains during the recovery period were similar to or higher than that of Controls, indicating reversibility.

A higher value of the liver weight was noted in satellite females treated with 1000 mg/kg. Minimal hypertrophy of centrilobular hepatocytes was noted in one satellite female treated with 1000 mg/kg and the incidence and severity were lower than those of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility.

In the adrenal gland, minimal vacuolation of cortical cells in the fascicular zone was noted in one satellite female treated with 1000 mg/kg. The incidence was lower than that of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility. Minimal vacuolation of cortical cells in the glomerular zone was not noted in any animal after recovery, indicating reversibility.

A higher value of the spleen weight was noted in males for recovery test treated with 1000 mg/kg. Minimal erythrocytic extramedullary haematopoiesis was noted in one male for recovery test and one satellite female treated with 1000 mg/kg. The incidence in satellite females was lower than that of females treated with 1000 mg/kg at termination. On the other hand, the incidence in males for recovery test was similar to that of males treated with 1000 mg/kg at termination and haematological examination also revealed anemia in males for recovery test. However, the severity was slight and increased hematopoiesis was noted, indicating a tendency to recover.

A higher kidney weight was noted in both sexes treated with 1000 mg/kg. However, this was judged not to be toxicologically significant as no related change was noted in any examination.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg based on occurrence of death, decreased body weight, effects on calcium metabolism and injury of the kidney occurring at 1000 mg/kg and hepatic effects, effects on bone/lipid metabolism, injury of stomach and cecum and reaction to anaemia occurring at 250 mg/kg and above.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Testing Methods Concerning New Chemical Substances
Version / remarks:
Notification No. 0331-7, PFSB, MHLW; No. 5 of March 29, 2011, MIB, METI; No.
110331009, EPB, MOE; dated March 31, 2011
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane
EC Number:
236-502-3
EC Name:
2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane
Cas Number:
13410-58-7
Molecular formula:
C21H36O4
IUPAC Name:
2-({[4-(2-{4-[(oxiran-2-yl)methoxy]cyclohexyl}propan-2-yl)cyclohexyl]oxy}methyl)oxirane
Test material form:
liquid
Details on test material:
- Appearance: transparent liquid
- Storage conditions: Room temperature (actual temperature: 17.4°C to 23.4°C; permissible range: 1°C to 30°C), in a dark place, in a tight container
- Stability: Each lot of the test substance was confirmed to be stable during the experimental period

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks old at receipt, 9 weeks old at the start of administration
- Weight at receipt: 224.8 to 247.6 g (males), 162.3 to 188.7 g (females)
- Fasting period before study: No, but animals were fasted before scheduled necropsy for about 17 to 24.5 hours.
- Housing: Animals were housed 2 or 3 animals per cage before grouping. After gouping, animals were housed 1 male and 1 female per cage during the mating period, one dam and its litter per cage during the lactation period and one animal per cage for the other periods. For males and females except for copulated females and dams, animals were housed in hanging type stainless wire mesh cages (W × D × H: 226 × 346 × 198 mm). Copulated females were housed in polymethylpentene cages (W × D × H: 220 × 380 × 195 mm). Both types of cages were furnished with two types of nesting material (Paper Clean, Diamond Twists) and one kind of gnawing material (Diamond Twists) for the improvement of animal welfare.
- Diet: Pelleted diet, ad libitum (except during fresh urine collection and measurement of motor activity)
- Water: Well water admixed with NaClO, ad libitum (except during the measurement of motor activity)
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
Data for each lot of the diet were obtained from the supplier and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.
Water was analysed twice a year. The results were confirmed to be within the acceptable limits established by the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.1 to 23.7 °C
- Humidity: 51.4 to 72.6 %
- Air changes: 10 to 20 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Following correction for substance purity, a prescribed amount of the test material was was weighed into a beaker and an appropriate amount of the vehicle was added and stirred with a magnetic stirrer.
After confirming of dissolution, it was transferred to a measuring cylinder. The final volume was adjusted by adding a proper quantity of the vehicle to provide the required concentration of dosing solution. Further dilution provided lower concentration dosing solutions.
Dosing formulations were prepared once within 12 days (3- to 10-day interval). The dosing formulations after preparation were divided into glass vials for each dosing day and stored at room temperature (17.9°C to 22.4°C).

DOSE VOLUME:
Individual volume was calculated on the basis of the most recently measured body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the first preparation, 10 mL each of the analytical samples was taken from the whole dosing formulation at each concentration and the test material concentration analysed by HPLC according to a previously validated method. Nominal test material concentrations of 12.5, 50 and 200 mg/mL provided mean measured concentrations of 12.68, 50.11 and 203.1 mg/mL, respectively and were within 101.4, 100.2, and 101.6% of nominal, respectively.
Test material formulations at 1 and 200 mg/mL were confirmed to be stable after storage for 12 days at room temperature.

High Performance Liquid Chromatography (HPLC) conditions:
Instrumentation: Prominence UFLC (Shimadzu Corporation)
Data processing: LabSolutions (Shimadzu Corporation)
Column: Ascentis Express RP-Amide (2.7 μm, 3.0 mm I.D. × 50 mm, Sigma-Aldrich Co.)
Column temperature: A constant temperature of about 40°C (set at 40°C)
Mobile phase:
Mobile phase A; water*
Mobile phase B; acetonitrile*
Linear gradient conditions and flow rate:
0.00 and 3.00 mins (55% A : 45% B) at a flow rate of 0.9 mL/min
3.01 and 12.00 mins (0% A : 100% B) at a flow rate of 1.5 mL/min
21.01 and 22.00 min (55% A : 45% B) at a flow rate of 0.9 mL/min
Analysis time: 22 minutes
Wavelength for detection: UV 200 nm
Injection volume: 10 μL
Auto-sampler set temperature: Room temperature (Not setting)
Needle wash solvent: Acetonitrile*
* sonicated under vacuum and degassed.
Details on mating procedure:
- M/F ratio per cage: The test males and females of each dose level were cohabited at one-on-one basis for 24 hours from Day 15 to the end of the mating.
- Proof of pregnancy: The vaginal smears were collected in the morning from the day after the initiation of mating, and copulation was confirmed by the presence of the vaginal plug or sperm in the smear sample. The day of successful mating was regarded as Day 0 of gestation (GD 0).
Duration of treatment / exposure:
Males: From 14 days before mating until the day before necropsy through the mating period (42 days in total)
Females: From 14 days before mating until Day 13 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery.
Recovery females (Satellite females), for 42 days without mating (as for males)
Frequency of treatment:
daily
Duration of test:
Maternal animals were sacrificed on Lactation Day (LD) 13.
Recovery females were sacrificed on study Day 56.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
0 (corn oil control) and 1000 mg/kg bw: 12 females (main dosing group) and an additional 5 females (recovery group)
62.5 and 250 mg/kg bw: 12 females (main dosing group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were selected following consideration of the effects observed in a 14-day study, during which 5 animals/sex/dose received oral gavage doses of test material at 0 (corn oil control), 110, 330, and 1000 mg/kg bw, for 14 consecutive days.
Following administration of test material, a lowering tendency of body weight was noted in males of the 1000 mg/kg group throughout the dosing period; however, a low value of body weight gain was transient. Haematology revealed anaemia in females of the 330 and 1000 mg/kg groups, and prolonged PT and APTT in males of the 1000 mg/kg group as an effect on the coagulation system.
At necropsy, enlargement of the liver was noted in both sexes of the 1000 mg/kg group and a high value of liver weight was noted in both sexes of the 330 and 1000 mg/kg groups. In the blood chemistry, a high value of ALAT was noted in both sexes of the 1000 mg/kg group, suggesting hepatic injury. The effects on protein/lipid metabolism were noted in males of the 1000 mg/kg group and females of the 110 mg/kg group and above. Moreover, whitish patch of mucosa in the forestomach was noted in 1 female of the 1000 mg/kg group, suggesting that the test material is irritating. Although various treatment-related changes were observed, none of the changes was severe. Therefore, the high dose in this study set at 1000 mg/kg, at which clear toxicity effects are expected to be seen. Lower doses were set at 250 and 62.5 mg/kg, with a common ratio of 4. A control group (0 mg/kg group) dosed with the vehicle (corn oil) alone was also established.
- Rationale for animal assignment: All animals except for the animals with abnormal estrous cycle (as determined during the quarantine and acclimation period) were used for grouping. Animals were assigned to groups by the stratified randomisation on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weights ± 20% (calculated for each sex) were used.
- Post-exposure recovery period in satellite groups:
For 5 males and 5 females (non-mating satellite females) each in the control and 1000 mg/kg groups, a 14-day recovery period was set after the end of the dosing period.

Examinations

Maternal examinations:
The initial day of dosing was designated as Day 1, Day 1 to Day 7 as Week 1, period after Day 43 as the recovery period. For the test females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).

CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were observed twice a day (before dosing and after dosing) during the dosing period and once a day in the other periods.

DETAILED CLINICAL OBSERVATIONS: Yes (as part of functional observation battery)
- Time schedule: For all animals, detailed clinical observations were performed once before the start of dosing and once a week in the afternoon during the dosing and recovery periods (day of gestation or parturition was prescinded).
Detailed clinical observations included:
- Hand-held observation, during which animals were removed from the cage for the observation by grasping their trunk gently from behind. Their reactivity to handling, trauma, colour of skin, soiled fur, piloerection, secretion, salivation, lacrimation, exophthalmos, palpebral closure, colour of conjunctiva and pupil size were observed.
- Observation on open field, during which animals were placed in the center of the open field and observed quietly for one minute. Parameters observed included arousal, posture/body position, gait, tremor, convulsion, respiration, stereotypy, bizarre behaviour, urination, defecation and number of rearings.

OBSERVATIONS DURING DELIVERY
All copulated females were allowed natural delivery. The observation of delivery was conducted once daily (a.m. 9:00) from GD 21 to GD24. Females that delivered their litter completely by 9:00 a.m. were judged as “delivered” on the corresponding day (the delivery day was regarded as Day 0 of lactation [LD 0]). When delivery was completed at 9:00 or later, the following day was defined as LD 0. The delivered animals (dams) were allowed to nurse offspring until LD 13, and postpartum behaviour such as lactation, nesting and presence or absence of cannibalism was observed every day. One female (No. 691) not delivered even after 24 days after the copulation confirmation was determined as non-delivered females.

BODY WEIGHT: Yes
- Time schedule for examinations:
The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36 and 42. The recovery males were also weighed on Days 43, 50 and 56. The satellite females were weighed at the same frequency as the recovery males. The test females were weighed on Days 1, 8 and 15, once every 7 days after the initiation of cohabitation, on GDs 0, 7, 14 and 20, and on LDs 0, 4, 7 and 13.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured between Days 1 and 8, 8 and 15, 22 and 29, 29 and 36 and 36 and 41 for the test and recovery males, and between Days 43 and 50 and 50 and
55 for the recovery males. For the satellite females, it was measured between Days 1 and 8, 8 and 15, 15 and 22, 22 and 29, 29 and 36, 36 and 41, 43 and 50 and 50 and 55. Food consumption of the test females was measured at the same frequency as body weight measurement. However, food consumption was not measured for either sex during the mating period. After the completion of copulation, the measurement for males was started from the nearest measurement day. Gross weight of each feeder was weighed, and the mean daily food consumption for each period was expressed as the start day of each measurement.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
All surviving animals were euthanised by exsanguination under anaesthesia and subjected to necropsy. In addition, the vaginal smears were collected from the females on LD 14 (necropsy day) in the morning and the stage of the oestrous cycle was examined under a microscope.
Non-delivered female (No. 691) was necropsied on GD 26. This animal was euthanised and necropsied in the same manner as the surviving animals.
The following organs/tissues were collected, weighed and examined macroscopically and microscopically: Heart, thymus, spleen, mandibular lymph node, mesenteric lymph node, femur/bone marrow (femur), sternum/bone marrow (sternum), trachea, lung (including bronchus), stomach, duodenum, jejunum, ileum (including Payer’s patch), cecum, colon, rectum, liver, pancreas, submandibular gland, kidney, urinary bladder, testis, epididymis, prostate (ventral lobe), seminal vesicle (including dorsolateral lobe and coagulating gland), cowper’s gland, glans penis, ovary, uterus, vagina, pituitary, thyroid/parathyroid, adrenal, spinal cord (cervical), brain (cerebrum, cerebellum and medulla oblongata/pons), eyeball, femoral muscle/Sciatic nerve (femoral region), levator ani/bulbocavernosus muscle (LABC), and any other organs/tissues indicating gross lesion.

OTHER EXAMINATIONS
The following were included as part of the repeated dose toxicity assessment: haematology, clinical chemistry (including hormone (total T4) measurement), functional observational battery (including sensory activity, grip strength, motor activity).
The following were included as part of the reproductive toxicity screen: oestrus cycle, mating performance (days until copulation, copulation index, fertility index), gestation length, gestation index, delivery index, number of litters (numbers of live newborn and stillborn), sex, offspring mortality (calculation of: birth index, stillborn index, viability index, sex ratio), offspring bodyweight.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes (at necropsy on LD 14, the ovaries and uterus were excised)
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes
All live offspring on PND 13, stillborn and dead offspring were observed for external anomaly Aditionally, all live male offspring were observed for the appearance of nipples (or areolae) on PND 12.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The following items were tested with the Wilcoxon’s rank sum test for comparison between the control and each test material group: external anomaly index, external anomaly typing index, and nipple development anomaly index.
Indices:
Based on the findings of the external examinations, the following were calculated:
- External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100
- External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100
Based on the observed appearance of nipples, the following was calculated:
- Nipple development anomaly index (%): (Number of offspring with nipple development anomaly / Number of observed offspring) × 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted. Mass was observed in the axilla of one female (No. 698) treated with 1000 mg/kg on LD 4 and thereafter. However, it was judged that this change was not related to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female (No. 693) treated with 1000 mg/kg was found dead on LD 2. In this animal, reduced locomotor activity was observed the day before, but no other remarkable change was observed. A suppressed body weight gain was noted in this animal until the death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A low value of body weight (statistically significant compared to vehicle control) was noted in females treated with 1000 mg/kg on GD 20.
In satellite females, no significant difference was noted between the control and 1000 mg/kg group throughout the dosing or recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Prior to pairing, food consumption was low in females treated with 1000 mg/kg during Day 1-8 (statistically significant compared to vehicle control). Thereafter, food consumption was similar to Controls for females at all dose levels during the gestation and lactation periods.
In satellite females (after Day 8), food consumption was similar to or higher than (statistically significant compared to vehicle control) that of Controls throughout the dosing and recovery periods.
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):
At the end of the dosing period, some erythroid parameters at 1000 mg/kg were marginally and not statistically significantly lower compared to the concurrent vehicle control as follows: a low value of RBC count in females (93.9% of Controls), a lower value of haemoglobin concentration in females (93.6%), a lower value of haematocrit in females (94.3%).
Besides, at the end of the dosing period, shortening of PT and APTT was noted in females treated with 1000 mg/kg, the differences from the control attained the statistical significance. However, these were judged not to be treatment-related as the changes were slight compared to those of Controls and opposite to the toxicity effect.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing period, lower values of ALP and total bile acid were noted in females treated with 1000 mg/kg, attaining statistical significance. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
At the end of the recovery period, a lower value of K was noted in females treated with 1000 mg/kg. However, this was judged not to be treatment-related as the similar changes were not noted at the end of the dosing period and no related change was noted in any examination.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Detailed clinical observations: No treatment-related change was noted in any animal in the hand held observation or observation on the open field.
The following changes were noted in the number of rearing: a high value in females treated with 250 mg/kg and above on Day 7 and a low value in females treated with 1000 mg/kg on Days 35 and 42, with the differences from the control attained the statistical significance. These were transient changes and no related changes were noted in the other parameters. Therefore, it was judged that the changes were not related to the test material treatment.
- Sensory reactivity to stimuli: Reactivity to stimuli was comparable in females at all dose levels and no abnormality was observed.
- Grip strength: No significant difference was noted in females between the control and test material groups.
- Motor activity: No treatment-related change was noted.
A low value of motor activity was noted between 20 and 30 minutes in females treated with 1000 mg/kg (statistically significant compared to vehicle control). However, this was judged not to be treatment-related as the moving pattern was normal and no related change was noted in the detailed clinical observations. As a change without dose-dependency and considered as not treatment-related, a high value of motor activity was noted between 40 and 50 minutes in females treated with 62.5 mg/kg (statistically significant compared to vehicle control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, treatment-related changes were noted as follows: a higher value or higher tendency of absolute and relative liver weights in females treated with 1000 mg/kg; higher values of absolute and relative kidneys and adrenals weights and a higher value of relative thyroids weight and lowering tendencies of absolute and relative thymus weights in females treated with 1000 mg/kg. At the end of the recovery period, a higher value of absolute and relative kidneys weight was noted in females treated with 1000 mg/kg.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the recovery period, no macroscopic change was observed in any animal.
In the dead animal (No. 693) treated with 1000 mg/kg, no macroscopic change was noted.
In non-delivered female (No. 691) treated with 1000 mg/kg, no macroscopic change was noted.
Besides, at the end of the dosing period, mass was noted subcutaneously in the axilla of one female (No. 698). However, this was judged not to be treatment-related. As a congenital abnormality, abnormal lobulation of the liver was noted in one female (No. 672).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver:
- Hypertrophy of centrilobular hepatocytes was noted in 1/5 females treated with 250 mg/kg (grade: minimal) and 5/5 females treated with 1000 mg/kg (minimal or mild). After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
Pituitary:
- Minimal hypertrophy of basophilic cells in the anterior lobe was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thyroid gland:
- Minimal hypertrophy of follicular cells was noted in 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Adrenal gland:
- Minimal vacuolation of cortical cells in the fascicular zone was noted in 1/5 females treated with 250 mg/kgand 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
- Minimal vacuolation of cortical cells in the glomerular zone was noted in 5/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- These vacuoles were positive for Oil red O staining.
Sternum:
- Minimal increase of trabecular bone was noted in 1/5 females treated with 250 mg/kg and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in all satellite females treated with 1000 mg/kg (minimal).
Femur:
- Minimal increase of trabecular bone was noted in 1/5 females treated with 250 mg/kg and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 5/5 satellite females treated with 1000 mg/kg (minimal).
Spleen:
- Minimal erythrocytic extramedullary hematopoiesis was noted in 1/5 females treated with 250 mg/kg and 4/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 1/5 satellite females treated with 1000 mg/kg (minimal).
Cecum:
- Minimal thickening of mucosa was noted in 3/5 females treated with 250 mg/kg and 4/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Stomach:
- Minimal mineralisation of mucosa in the glandular stomach was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Lung:
- Minimal accumulation of foam cells in the alveolus was noted in 1/5 females treated with 250 mg/kg and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Fine vacuoles of the foam cells were positive for Oil red O staining.
Heart:
- Minimal mineralisation of aortic wall was noted in 2/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thymus:
- Minimal atrophy was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.

> [Dead animal (No. 693) treated with 1000 mg/kg]
Thymus: Moderate atrophy was noted.
Spleen: Mild atrophy was noted.
Liver: Minimal hypertrophy of centrilobular hepatocytes was noted.
Kidney: Mild vacuolation of proximal tubular epithelium was noted. The vacuoles were positive for Oil red O staining.
Sternum: Minimal increase of trabecular bone was noted.
Femur: Minimal increase of trabecular bone was noted.

> Others:
Various histopathological changes were noted in control and test material groups. However, these changes were judged not to be treatment-related as they are observed occasionally in normal rats and there was no clear dose-dependency in the incidence.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Mass in the axilla noted in 1 female (No. 698) treated with 1000 mg/kg was adenocarcinoma of the mammary gland in the histopathology. Adenocarcinoma of mammary gland is known to occur infrequently in young mature rats and no gross abnormalities were noted in the mammary gland of any other animals, therefore, it was judged not to be treatment-related.
Other effects:
no effects observed
Description (incidence and severity):
- Hormone measurement:
In parental animals (F0 female), plasma total T4 concentrations of the test material-treated groups (Groups 62.5, 250, and 1000 mg/kg) were equivalent to those of the control groups. The plasma total T4 concentration of recovery groups (Group 1000 mg/kg) were equivalent to those of the control groups in Satellite female.
All values in the quality control sample were within the range of 100 ± 25% of the nominal value, and variations in duplicate cpm of the standard solutions were within the range of acceptability. There were no abnormalities in the procedures for the determination or in the values of the test samples. The results were therefore considered acceptable.

- Oestrus cycle:
No significant difference was noted in the mean oestrous cycle or count of oestrus, indicating no effect of the test material on prolongation or shortening of the oestrous cycle between the control and test material groups.

- Mating:
No significant difference was noted in the copulation index, duration of mating or fertility index between the control and test material groups.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- Observation during delivery and lactation periods:
No abnormality was noted in the delivery or nursing. No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index between the control and test substance groups. One female (No. 691) treated with 1000 mg/kg, which had only one implantation, did not deliver. However, this was judged not to be treatment-related due to its low incidence.
No significant difference was noted in the number of litter, live newborns and stillborn, birth index, sex ratio or viability index on PNDs 4 or 13 between the control and test material groups.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: See 'Remarks'

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant low value of body weight was noted in both sexes of the 1000 mg/kg group on PND 13.
Besides, a high value of body weight was noted in females of the 62.5 mg/kg group on PNDs 7 and 13. However, this was judged not to be treatment-related as there was no dose-dependency.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No significant difference was noted in the number of litter, live newborns and stillborn, birth index, or viability index on PND 4 between the control and test material groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No significant difference was noted in the sex ratio between the control and test material groups.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant low value of body weight was noted in both sexes of the 1000 mg/kg group on PND 13.
Besides, a high value of body weight was noted in females of the 62.5 mg/kg group on PNDs 7 and 13. However, this was judged not to be treatment-related as there was no dose-dependency.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No significant difference was noted in the viability index on PND 13 between the control and test material groups.
External malformations:
no effects observed
Description (incidence and severity):
No abnormality was observed in any offspring.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Anogenital distance: No significant difference was noted in males or females between the control and test material groups.
- Nipple development in males on PND 12: No significant difference was noted in the anomaly index between the control and test material groups.
- Pathological examination: No treatment-related change was noted in any offspring.
Whitish patch of the liver was noted in one female of the 1000 mg/kg group (offspring No. F04 in dam No. 698). However, this was judged not to be treatment-related due to its low incidence.
- Hormone concentration (total T4) analysis: No significant difference was noted between the control and test material groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

- MATERNAL ANIMALS

Summary of female bodyweights, g (mean ± SD)

Day

Dose (mg/kg bw/day) (no. of animals)

0 (17)

62.5 (12)

250 (12)

1000 (17)

1

230.74 ± 10.20

231.73 ± 9.27

231.80 ± 12.70

233.05 ± 11.49

8

248.94 ± 9.37

246.68 ± 11.24

247.23 ± 14.02

244.68 ± 10.47

15

258.92 ± 13.60

256.73 ± 11.96

258.48 ± 15.67

255.48 ± 10.28

22

263.62 ± 9.64

256.60 ± 17.43

29

267.86 ± 7.71

269.38 ± 11.31

36

280.20 ± 9.14

278.80 ± 17.93

42

286.10 ± 9.73

284.80 ± 21.75

43

284.56 ± 13.69

283.80 ± 17.71

50

292.34 ± 9.20

291.18 ± 17.98

56

296.32 ± 10.78

301.28 ± 19.97

 Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Results in itallics relate to 5 animals

Day 1 to 42; Treatment period, Day 43 to 56; Recovery period

Summary of female bodyweights, g (mean ± SD)

Day

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

Gestation period

0

266.18 ± 16.28

266.75 ± 13.71

266.59 ± 18.10

263.90 ± 13.36

7

301.64 ± 20.12

300.63 ± 16.86

298.92 ± 18.85

284.27 ± 16.47

14

336.97 ± 25.50

340.59 ± 21.19

342.07 ± 19.36

316.99 ± 19.42

20

419.53 ± 33.02

419.03 ± 32.66

425.38 ± 24.23

379.78 ± 38.42*

Lactation period

0

325.03 ± 30.56

329.48 ± 22.57

335.88 ± 19.11

303.72 ± 23.02

4

340.01 ± 31.55

343.58 ± 21.36

351.46 ± 20.97

322.94 ± 25.21

7

346.39 ± 31.44

348.66 ± 19.08

354.37 ± 24.07

336.30 ± 23.07

13

354.93 ± 33.29

361.38 ± 15.85

368.46 ± 19.88

359.01 ± 21.42

- OFFSPRING

Summary of bodyweights in offspring, g (mean ± SD)

Parameter

Sex

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (11)

Day 0

No of pups

Male

87

70

82

61

Female

80

86

84

76

Mean pup weight

Male

7.5 ± 0.5

7.8 ± 0.6 (11)

7.4 ± 0.7

6.8 ± 0.7

Female

7.0 ± 0.4

7.4 ± 0.8

7.1 ± 0.7

6.6 ± 0.7

Day 4

No of pups

Male

87

69

82

58

Female

80

85

84

70

Mean pup weight

Male

12.0 ± 1.0

12.9 ± 1.3 (11)

12.5 ± 1.6

11.2 ± 1.7 (10)

Female

11.2 ± 0.9

12.3 ± 1.8

11.8 ± 1.4

10.7 ± 1.4 (10)

Day 7

No of pups

Male

49

44

48

38

Female

47

46

48

42

Mean pup weight

Male

20.2 ± 1.8

21.9 ± 1.8 (11)

20.9 ± 2.0

18.9 ± 2.3 (10)

Female

18.6 ± 1.5

20.9 ± 2.2*

19.8 ± 2.0

17.5 ± 1.8 (10)

Day 13

No of pups

Male

49

44

48

38

Female

47

46

48

42

Mean pup weight

Male

37.3 ± 2.8

40.0 ± 2.7 (11)

38.0 ± 2.5

33.8 ± 2.5** (10)

Female

35.3 ± 2.2

38.4 ± 2.9**

37.2 ± 1.8

31.6 ± 2.5** (10)

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Number in parentheses indicates the number of litters.

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg due to decreased body weight of F1 offspring at 1000 mg/kg on PND 13.
Executive summary:

The developmental toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 422 and the Japanese Testing Methods Concerning New Chemical Substances, and under GLP conditions.

During the study, the test material was repeatedly administered by oral gavage at 0 (control group), 62.5, 250 and 1000 mg/kg from 14 days before mating through mating for 42 days in males and satellite females, and from 14 days before mating through gestation and parturition until Day 13 of lactation in females to assess the repeated dose toxicity and reproductive and developmental toxicity. In addition, a 14-day recovery period was set for the control and 1000 mg/kg groups and the reversibility of the toxicity effect was assessed.

One dam treated with 1000 mg/kg was found dead on LD 2. In the dead animal, no other remarkable change was observed in the clinical observation. In the histopathology, the following changes were noted in this animal: moderate atrophy of the thymus, mild atrophy of the spleen, mild vacuolation of proximal tubular epithelium (positive for Oil red O staining, indicating that the vacuoles were lipid), minimal hypertrophy of centrilobular hepatocytes and minimal increase of trabecular bone in the sternum and femur. The cause of death remains unclear; however, it was possible that the death occurred due to overlapping effects of the test material and postpartum stress.

In the surviving animals, a lower body weight was noted in females treated with 1000 mg/kg on GD 20. Food consumption in females treated with 1000 mg/kg was lower than the control during the first week of treatment.

Adaptive physiological responses attributable to the test material treatment were noted at 250 mg/kg and above and this is not considered to be adverse in nature. The related changes were noted as follows: a higher liver weight in females treated with 1000 mg/kg; a higher thyroid weight in females treated with 1000 mg/kg; minimal or mild hypertrophy of centrilobular hepatocytes with a tendency of increasing incidence and severity, and minimal hypertrophy of basophilic cells in the anterior lobe of the pituitary in animals treated with 250 mg/kg and above; minimal hypertrophy of follicular cells of the thyroid in females treated with 1000 mg/kg. However, the above-mentioned pathological changes in females treated with 1000 mg/kg were judged to be toxic effects as the blood chemistry revealed the effects on protein/lipid metabolism in animals treated with 1000 mg/kg and increased ALAT in females treated with 1000 mg/kg. Increased calcium level was noted in females treated with 1000 mg/kg, suggesting an association with hyperthyroidism.

Minimal increase of trabecular bone in the sternum and femur was noted in females treated with 250 mg/kg and above. The cause remains unclear.

Lipid accumulation in the cells or dysbolism of lipid was noted in the adrenal and alveolus of the lung; however, the cause remains unclear. Related changes were noted as follows. A high total cholesterol level was noted in animals treated with 1000 mg/kg. A higher adrenal weight was noted in females treated with 1000 mg/kg.

Histopathological examination revealed the following: minimal vacuolation of cortical cells in the fascicular zone of the adrenal in females treated with 250 mg/kg and above; minimal vacuolation of cortical cells in the glomerular zone of the adrenal in females treated with 1000 mg/kg and minimal accumulation of foam cells in the alveolus of the lung in females treated with 250 mg/kg and above. The vacuoles in the adrenal and fine vacuoles in the alveolus of the lung were positive for Oil red O staining. The changes suggesting the irritation of the test material were noted in the stomach and the cecum. In the cecum, minimal thickening of mucosa was noted in animals treated with 250 mg/kg and above.

The damage to the tubular epithelium in the kidney was noted. A higher kidney weight was noted in animals treated with 1000 mg/kg.

Dysbolism of calcium was noted: a high calcium level was noted in animals treated with 1000 mg/kg in the blood chemistry and minimal mineralisation of aortic wall and mucosa in the glandular stomach was noted in females treated with 1000 mg/kg. Slight anaemia was noted in animals treated with 1000 mg/kg. In the spleen, minimal erythrocytic extramedullary hematopoiesis was noted in animals treated with 250 mg/kg and above, indicating a reaction to anaemia.

A lower thymus weight was noted in females treated with 1000 mg/kg. In the histopathology, minimal atrophy of the thymus was noted in one female treated with 1000 mg/kg. It was judged to be a secondary reaction associated with stress as this animal had a suppressed body weight gain.

In the recovery test, among the changes noted at the end of the dosing period, except for the changes noted in the sternum and femur, reversibility or a tendency to recover was observed.

There were no effects on parental reproductive parameters.

In offspring, a low value of body weight was noted in both sexes at 1000 mg/kg on PND 13. However, since the viability was not affected, the toxic effect on offspring was judged not to be severe.

No abnormality was noted in the delivery or nursing. No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index between the control and test substance groups. No significant difference was noted in the number of litter, live newborns and stillborn, birth index, sex ratio or viability index on PNDs 4 or 13 between the control and test material groups. No abnormality was observed in any offspring and no significant difference was noted in the anomaly index (concerning nipple development) between the control and test material groups.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg bw/day.

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg bw/day due to decreased body weight of F1 offspring at 1000 mg/kg bw/day on PND 13.