2-methylenepropane-1,3-diyl diacetate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction: According to the results of this study, a No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 12.5 mg/kg based on occurrence of moribundity and death, a decrease in body weight, an increase in food consumption, and injury to the liver, stomach, and duodenum at 50 or 200 (100) mg/kg. The NOAEL for reproductive and developmental toxicity was judged to be 200 mg/kg since no treatment-related change was noted at any dose level.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Initiation of the experiment (Initiation of administration): May 10, 2019
Completion of experiment (Histopathology): October 1, 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name (Abbreviation): MPDAc
CAS number: 3775-29-9
Lot number: 20160308-88
Purity: 99.0%
Appearance at ambient temperature: Colorless liquid
Reactivity, etc.: Photoreactive and volatile

Species:

rat

Strain:

other: 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:

Supplier (Breeding facility)
Charles River Laboratories Japan, Inc. (Hino Breeding Center)

Age
At receipt: 7-week-old
At the start of administration: 9-week-old

Body weight range at receipt
212.7 to 243.5 g (males, permissible range: 190 to 270 g)
157.2 to 184.7 g (females, permissible range: 140 to 210 g)

Quarantine and acclimation
Upon animal receipt, species, strain, age, number, and sex were confirmed and observation for clinical signs and external appearance and body weight measurement were performed. A quarantine period was set for 5 days and an acclimation period was set for 14 days (between the day of animal receipt and the day before the start of administration). During these periods, all animals were observed once daily and were checked for body weight gain from the animal receipt to the end of quarantine. In addition, detailed clinical observations were performed once before grouping. As for females, vaginal smears were collected with a swab for 10 days from the end of quarantine period to the day before the initiation of dosing, and the estrous cycle was observed. According to these results, an irregular estrous cycle was noted in 3 females (Nos. 2022, 2023, and 2056).

Grouping
All animals except for the animals with abnormal estrous cycle were used for grouping. Grouping was conducted on the day before the initial administration. Animals were assigned to groups by the stratified randomization on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weight ± 20% (calculated for each sex) were used for this study. Body weight range is shown below.
Males: 339.9 to 400.6 g (permissible range: 297.5 to 446.3 g)
Females: 204.4 to 246.8 g (permissible range: 183.0 to 274.5 g)

Identification of animals
Before grouping
Upon receipt, animals were identified by marking the ID No. (the last one or two figures of quarantine animal number) on the tail with a black oil-based felt tip pen, and a label indicating the study number, quarantine animal number, and sex was attached to the front of each cage. The quarantine animal numbers are shown below.
Males: 1001 to 1053
Females: 2001 to 2064
After grouping
The animals were identified by ear tags inscribed with animal number, and a label indicating the study number, animal number, dose level, and sex was attached to the front of each cage.
Offspring
Offspring were identified by plantar tattooing on the limbs on postnatal day 4.

Handling of remaining animals
The remaining animals were euthanized under over-anesthesia by intravenous
administration of thiopental sodium after the initiation of administration.

Animal management
Environmental conditions
Room temperature
Actual range: 23.1°C to 25.3°C (permissible range: 20.0°C to 26.0°C)
Relative humidity
Actual range: 43.2% to 72.0% (permissible range: 35.0% to 75.0%)
Ventilation
10 to 20 times per hour
Ventilation frequency was measured twice a year at the test facility. The results were confirmed to be within the acceptable limits established by the test facility.
Lighting period
12 hours per day (7:00 to 19:00)
Housing equipment
Cage racks
Stainless
Sterilization method: autoclave sterilization
Frequency of exchange: once or more every four weeks (actual frequency: 7- to 27-day interval)
Cages
(1) For males and females except during gestation and lactation periods: Hanging type stainless wire mesh cages (W × D × H: 226 × 346 × 198 mm) Sterilization method: autoclave sterilization
Frequency of exchange: once or more every two weeks (actual frequency: 7- to 14-day interval)
(2) For females during gestation and lactation periods: Polymethylpentene cages (W × D × H: 220 × 380 × 195 mm) Sterilization method: autoclave sterilization Frequency of exchange: once or more a week (Days 0, 7, 14 and 20 of gestation, and Days 4, 7 and 11 of lactation)
Cage lids used both as lids and feeders for polymethylpentene cages
Stainless
Sterilization method: autoclave sterilization
Frequency of exchange: once or more every two weeks (Days 0 and 14 of gestation, and Day 4 of lactation)
Feeder for wire mesh cages
Stainless
Sterilization method: autoclave sterilization
Frequency of exchange: once or more every two weeks (actual frequency: 7- to 14-day interval)
Polymethylpentene
Sterilization method: autoclave sterilization
Exchange: once or more every four days (actual frequency: 1- to 4-day interval)
Water supply system for wire mesh cages
Automatic water supply system attached to the rack
Sanitary trays for wire mesh cages
Aluminum
Sterilization method: autoclave sterilization
Exchange: once or more every seven days (actual frequency: 1- to 7-day interval)
During the dosing period, chromaturia was noted in 1 female in the high dose group. To confirm its properties, the sanitary tray was exchanged on the day when the abnormal urine was noted.
Number of animals per cage
Before grouping: 2 or 3 animals per cage
After grouping: One 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
Diet
Description
Radiation-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd.)
Feeding method
Diet was given ad libitum except during fresh urine collection and measurement of motor activity. Animals were fasted before scheduled necropsy for 16 hours or more (actual fasting time: about 19 to 24 hours).
Water
Description
Well water admixed with NaClO (free residual chlorine level: about 2 ppm)
Water supply method
Water was given ad libitum except during the measurement of motor activity. Automatic water supply system was used for males and females except during the gestation and lactation periods. Watering bottles were used for females during the gestation and lactation periods and males during the collecting of urine sample.
Bedding
Description
Autoclave-sterilized wood chip (White Flake, Charles River Laboratories Japan, Inc.)
Frequency of bedding exchange
Bedding placed in the polymethylpentene cages was exchanged concurrently with the cages.
Environmental enrichment
The following materials were put into the cages for improvement of animal welfare. Wire mesh cages: two types of nesting material (Paper Clean [Japan SLC, Inc.], Diamond Twists [ENVIGO]), one kind of gnawing material (Diamond Twists), and a resting board
Polymethylpentene cages:
one type of nesting material (Paper Clean) and one kind of gnawing material (Diamond Twists)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance was administered orally using a disposable syringe attached to a gastric tube.

Details on mating procedure:

The test males and females of each dose level were cohabited at one-on-one basis for 24 hours on Day 15 up to 14 days. 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). Based on the results, the following parameters were calculated.
(1) Days until copulation:
Number of days from the start of mating to the detection of copulation
(2) Copulation index (%):
(Number of animals with successful copulation / Number of animals cohabited) × 100
(3) Fertility index (%):
(Number of pregnant animals / Number of females with successful copulation) × 100

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of the test substance concentration and homogeneity in dosing formulation
At the first preparation, 10 mL each of the analytical samples was taken from one point of each layer (upper, middle, and lower layers, n=1 in each layer) of the whole dosing formulation at each concentration. The test substance concentration in the dosing formulations was analyzed according to the method validated in analytical method validation for the determination and stability study (study No. P180490) conducted at the test facility
The relative standard deviation (RSD) of analytical values (concentration) in each layer
should not be more than 10%, and measured concentration (mean) should be within
100 ± 10% as the ratio to the nominal concentration.
Nominal concentration 20 mg/mL; measured concentration 20.11 mg/mL; RSD 1.3 %
Nominal concentration 5 mg/mL; measured concentration 5.019 mg/mL; RSD 0.6 %
Nominal concentration 1.25 mg/mL; measured concentration 1.249 mg/mL; RSD 1.1 %

Duration of treatment / exposure:

Administration period
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
- Females in the high dose group was kept dosing until Day 39 (during the gestation or lactation period).
- One non-delivered female was kept dosing until the day before necropsy.

Frequency of treatment:

Frequency of administration: Once daily, between 8:01 and 12:43 (permissible range: 8:00 to 15:00)

Details on study schedule:

Animal receipt: April 25, 2019
Initiation of the experiment (Initiation of administration): May 10, 2019
Mating (Cohabitant): May 24, 2019
Parturition: June 16 to 29, 2019
Necropsy of males after the administration period: June 21, 2019
Necropsy of dams: June 30 to July 13, 2019
Necropsy after the recovery period: July 5, 2019
Completion of experiment (Histopathology): October 1, 201

Dose / conc.:

12.5 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12.5 mg/kg: 12 males 12 females
50 mg/kg: 12 males, 12 females
200 mg/kg: 7 males 12 females

Control animals:

yes, concurrent no treatment

Details on study design:

Dose level and its rationale
Dose levels were set at 12.5, 50, and 200 mg/kg.
As a result of an acute oral toxicity study [1] (two of six animals treated with 300 mg/kg died.), the dose levels were set at 10, 30, and 100 mg/kg in a dose-finding study entitled “A 14-Day Repeated Dose Oral Toxicity Study of MPDAc in Rats (Study No. P180491)” conducted at the test facility. No clear toxicological change was noted in the 100 mg/kg group. According to the results, the high dose in this study was set at 200 mg/kg, at which some toxicity effect was expected to be seen. Lower doses were set at 50 and 12.5 mg/kg. A control group (0 mg/kg group) dosed with the vehicle (corn oil) alone was also be established.

Dose volume
- 10 mL/kg
Individual volume was calculated on the basis of the most recently measured body weights.
- As for test females treated with 200 mg/kg, dose volume was reduced to 5 mL/kg on GD 20 and thereafter (June 16, 2019 and thereafter)

Parental animals: Observations and examinations:

The following items were examined. For the notation of the day, the initial day of dosing was Day 1, Day 1 to Day 7 was Week 1, and the period after Day 43 was the recovery period. For the test females, the day of successful copulation and the day of delivery were designated as Day 0 of gestation (GD 0) and Day 0 of lactation (LD 0), respectively.
Examination for test females treated with 200 (100) mg/kg
Death occurred in 4/12 test females treated with 200 mg/kg during the gestation period. Three of these animals died on GD 21. Most of the deaths occurred during the late gestation period. Since the test females treated with 200 mg/kg were considered to be at risk of death, the dose level and volume for the test females treated with 200 mg/kg were reduced to 100 mg/kg and from 10 mL/kg to 5 mL/kg, respectively, on GD 20 and thereafter (June 16, 2019 and thereafter*). However, one more test female died the next day (LD 1); it was judged that no further administration was possible, and the administration was discontinued (on Day 39, during the gestation or lactation period). Dose level for the test females is expressed as 200 (100) mg/kg.
As for all the test females treated with 200 (100) mg/kg including a non-pregnant female,
hematology, blood chemistry, pathology, and hormone concentration measurement were
conducted. These animals were not fasted. Non-delivered animals were anesthetized with thiopental sodium via the tail vein. The data obtained from these examinations were used as reference data.
*: The animals copulated on the third day after initiation of mating were GD 20 as of June 16, 2019 (pregnancy No. 3). Therefore, the dose volume as of June 16, 2019 was 5 mL/kg for animals with pregnancy No. 1 to 3, and 10 mL/kg for animals with pregnancy No. 4 and later.

Clinical observation
During the dosing period, all animals were observed at the following time points:
- Day 1 to 19; twice a day (before dosing and after dosing)
- Day 20 to 22; four or five times a day to confirm the peak time when salivation was noted (before dosing, just after dosing, 30 minutes, 2 hours, 3 hours, or 4 hours after dosing)
- Day 23 and thereafter; three times a day (before dosing, just after dosing, and 2 to 3 hours
after dosing During the recovery period, all animals were observed once a day.

Functional observation battery
Examinations were not conducted on a blind test basis. 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). 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. These examinations were conducted once in the afternoon of the final week during the lactation period. During the dosing period, the above examinations were performed following the clinical observation after dosing until Day 21 and after 2 to 3 hours after dosing on Day 28 and thereafter.
According to these results, the functional tests and motor activity measurement were not performed during the recovery period because no treatment-related change was noted during the dosing period.

Detailed clinical observations
(1) Hand held observation
Animals were removed from the cage for the observation by grasping their trunk gently from behind.
Parameters: Reactivity to handling, trauma, color of skin, soiled fur, piloerection, secretion, salivation, lacrimation, exophthalmos, palpebral closure, color of conjunctiva, and pupil size
(2) Observation on open field
Animals were placed in the center of the open field and observed quietly for one minute.
Parameters: Arousal, posture/body position, gait, tremor, convulsion, respiration, stereotypy, bizarre behavior, urination, defecation, and number of rearing

Functional tests
(1) Sensory reactivity to stimuli
Sensory reactivity to stimuli was observed on the open field.
Parameters: Approach response, touch response, auditory response, righting reaction, and tail pinch response
(2) Grip strength measurement
Grip strength was measured following sensory reactivity to stimuli. Grip strength was measured twice for both forelimbs and hindlimbs using a grip dynamometer (CPU gauge: MODEL 9502A, Aikoh Engineering Co., Ltd.), and the higher value was adopted.

Motor activity measurement
For measurement of motor activity, animals were acclimated to cages (polymethylpentene, W × D × H: 220 × 380 × 195 mm) following the clinical observation 2 to 3 hours after dosing. Animals were transferred to new cages at the time of measurement (no diet or water was given). The motor activity was recorded individually using a motor activity-measuring device (SCANET SV-40, Melquest Ltd.). Data were calculated every 10 minutes from the start of measurement, while a total of 1 hour was calculated.

Body weight measurement
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 in 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 measurement
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.

Urinalysis in males
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, no statistically significant difference was noted in any parameter in the qualitative test. However, in the clinical observation, abnormal urine was noted in 2 females of the high dose group; in the qualitative test, protein (3+) and ketone body (2+) were found in 1 male and 2 males of the high dose group, respectively. Therefore, additional tests (urine sediments test and pooled urine test) were performed. The urine sediment test (Day 41) was performed using the fresh urine samples. Urine samples were collected successively for about 24 hours (pooled urine). Diet was given after the fresh urine collection and water was given with a watering bottle during the urine collection. The pooled urine samples were examined for the other parameters.
Moreover, the three types of the examination were conducted for all individuals in the final week (Day 55) of the recovery period.
The pooled urine samples after the measurement were stored in a deep freezer and the remaining samples were discarded by the completion of the study.

Hematology
All animals were fasted for about 16 hours and above (actual fasting time: about 19 to 24 hours) from Day 42 for the test males, Day 56 for the recovery males and satellite females, and LD 13 for the test females. 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.
The animals were anesthetized with intraperitoneal injection of thiopental sodium and about 2.0 to 2.5 mL of blood was collected from the posterior vena cava. 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 (SB-41, Sysmex Corporation) containing 2 mg of EDTA-2K as an anticoagulant.

Blood chemistry
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 (actual temperature: -82.8°C to -75.3°C, permissible range: -90°C to -65°C) until examination. The remaining samples were stored in a deep freezer and discarded by the completion of the study.
Since the value for ASAT was over the instrumental maximum range (1600 U/L) in 1 female (No. 598), ASAT for the animal was re-examined after 2-fold dilution and the re-examined value was adopted.

Oestrous cyclicity (parental animals):

Vaginal smears were collected with a swab 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 estrous phase. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous phase was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of days from the estrus to the next estrus) and the number of the estrus during the test period were calculated. The data obtained during the mating period were treated as referential data.

Litter observations:

The observation of delivery was conducted once daily from GD 21 to GD 24(observation was made as appropriate between 7:00 a.m. and 16:00). Females that delivered their litter completely by 9:00 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 day 13 postpartum (PND 13), and postpartum behavior such as lactation, nesting, and presence or absence of cannibalism
was observed every day.
One female (No. 554) that did not deliver even after 24 days after the copulation confirmation was determined as non-delivered female

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. The data of stillborn and dead offspring were stored as raw data; the results are not included in this report.
(1) External anomaly index (%):
(Number of offspring with external anomaly / Number of observed offspring) × 100
(2) External anomaly typing index (%):
(Number of offspring with external anomaly by each type / Number of observed
offspring) × 100

Standardization of litter size
On PND 4, the litter size was standardized 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 euthanized by overdose with thiopental sodium (undiluted solution, 0.1 to 0.5 mL/body) via an
intraperitoneal injection and discarded.

AGD (Anogenital distance)
Anogenital distance (AGD: distance between the anus and the genital node) was determined with a micrometer caliper (Mitutoyo Corporation) on PND 4 following adjustment of the number of offspring. 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):

Observation and measurement for repeated dose toxicity
Necropsy
All surviving animals were euthanized by exsanguination under anesthesia 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 estrous cycle was examined under a microscope.
One non-delivered female (No. 554, see Section 6.11.2) was necropsied on GD 26. This animal was euthanized and necropsied in the same manner as the above-mentioned surviving animals.
Organ weight measurement
After the scheduled necropsy, organs 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, and 5 animals per group from the earlier parturition date with the smallest animal numbers in the test females. The testis and epididymis weights were measured in all males. Paired organs were weighed together.

Histopathology
After necropsy, the organs/tissues were fixed and preserved in 10 vol% phosphate buffered formalin solution; however, the testes and epididymides were fixed in Bouin’s solution and the eyeballs were fixed in Davidson’s solution, and they were preserved in 10 vol% phosphate buffered formalin solution. The organs/tissues of dead animals were fixed in 10 vol% phosphate buffered formalin solution.
The organs/tissues collected from the animals and all gross lesions were embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and then examined by microscopy. The animals for the microscopy were as follows: 5 test males per group with the smallest animal numbers in the control and high dose groups and 5 dams per group from the earlier parturition date with the smallest animal numbers in the control and middle dose groups collected at the end of the dosing period, a moribund animal, and dead animals. Furthermore, as for the testis, the spermatogenic cycle was examined for all males using PAS-stained specimens, and the presence or absence of abnormalities at each stage (stages I to XIV) was examined.
As for the dead animals, the following organs could not be examined due to autolysis: duodenum, jejunum, ileum cecum, colon, rectum, urinary bladder, and eyeball in 5 animals; spleen, bone marrow (femur and sternum), stomach, kidney, and thyroid in 4 animals; trachea and adrenal in 3 animals; thymus and mesenteric lymph node in 2 animals; lung, pancreas, uterus, pituitary, sciatic nerve, brain, and spinal cord in each 1 animal.
According to these results, a change suspected to be attributable to the test substance treatment was noted in the liver and stomach of both sexes, duodenum of males, and thymus of females. Therefore, the relevant organs/tissues of the relevant sex in the low and/or middle dose groups and all groups in the recovery test were additionally examined, according to the above criteria.
At the necropsy, small testis and epididymis were noted bilaterally in 1 male treated with 200 mg/kg; microscopically, abnormal findings were noted in these organs. Therefore, these changes had been judged to be treatment related and examined for all males. As the results, microscopic abnormal findings were not considered to be treatment-related.

Organs and tissues for examination
Organs/tissues listed below 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)
Levator ani/bulbocavernosus muscle (LABC)
Cowper’s gland
Glans penis
Ovary
Uterus
Vagina
Pituitary
Thyroid/Parathyroid
Adrenal
Spinal cord (cervical)
Brain (cerebrum, cerebellum and medulla oblongata/pons)
Femoral muscle/Sciatic nerve (femoral region)
Eyeball
Mammary gland (males)
Gross lesion

Reproductive and developmental toxicity
Necropsy
The necropsy was performed on PND 13. The offspring subjected to blood sampling was necropsied after blood sampling by decapitation after anesthetizing by inhalation of isoflurane. Other animals were euthanized by exsanguination under over-anesthesia with thiopental sodium via the intraperitoneal injection and necropsied. The thoracoabdominal organs/tissues were examined macroscopically. Furthermore, the thyroid glands of 1 male and 1 female per litter (animals subjected to blood sampling for hormone level measurement) were collected and fixed and preserved in 10 vol% phosphate buffered formalin.

Hormone concentration (total T4 and TSH) analysis
Only test males were subjected to the parental animal measurement. The offspring was subjected to the measurement on PND 13 only. The measurements were conducted at the test site for the test males and offspring (see Section 11.3). According to the results, a change suspected to be attributable to the test substance treatment was noted in the test males and offspring on PND 13 in the high dose group. Therefore, additional measurement was performed for the test females, satellite females, and offspring on PND 4.

Blood sampling
Blood samples were collected at approximately the same set time after 12:00 not to affect the results. When this was not possible, the blood collection time was confirmed to be unbiased among the groups. Also, animals before blood collection were kept separate from those immediately before blood collection. In other words, the necropsy room is divided into two areas. Anesthesia was conducted in one area where the animals were left to rest (resting room), while the blood sampling was conducted in the other area (blood sampling room).

Parental animals
(1) Animals for blood sampling and sampling time point
The blood was sampled from all males and females upon necropsy. The blood sampling for a moribund animal was conducted. One non-delivered female was not subjected to blood sampling.
(2) Blood sampling procedures
The animals were anesthetized by intraperitoneal injection of thiopental sodium. Approximately 0.8 mL of blood was collected into a syringe containing heparin sodium from the posterior vena cava. The blood was transferred to a test tube, immediately cooled on ice and then promptly centrifuged (approximately 1870 × g, 10 min, 4°C) to obtain plasma (320 μL or more). The collected plasma was stored frozen in a deep freezer (actual range: -80.3°C to -70.9°C, permissible range: -90°C to -65°C) until shipment to the test site. The plasma sample tubes were labeled with the study number, animal number, sex, and timing of blood sampling.

Handling of dead/moribund animals during the study
Dead animals were weighed and necropsied as soon as possible after death and the cause of death was searched. A moribund animal was weighed; blood was collected for various tests), and then euthanized, necropsied, and measured for organ weights. For dead animals that were pregnant, the uterus was examined, and the embryo or fetus were fixed and preserved in 10 vol% phosphate buffered formalin. Histopathology for the dead/moribund animals was performed
Offspring from the dead/moribund animals was discarded before culling and fixed and preserved in 10 vol% phosphate buffered formalin for one litter after culling. Live offspring was euthanized by overdose with thiopental sodium via an intraperitoneal injection.

Postmortem examinations (offspring):

Blood samples
(1) Animals for blood sampling and sampling time point
The blood sampling was performed on PNDs 4 and 13 and conducted for the offspring from all dams. On PND 4, the blood was collected from 2 culled offspring or more (at least 1 male and 1 female) in each litter. On PND 13, the blood was sampled from 1 animal of each sex in each litter (see Section 6.12.4). When a blood sample could not be obtained from either sex, it was obtained from 2 animals or more of the same sex in the litter. As for 1 dam with only one offspring (dam No. 586), the blood sampling was conducted.
(2) Blood sampling procedures
The blood sampling was performed by decapitation after anesthetizing by inhalation of isoflurane. Approximately 0.8 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 (320 μL or more). The collected plasma was stored frozen in a deep freezer (actual range: -80.3°C to -70.9°C, permissible range: -90°C to -65°C) until shipment to the test site. The plasma sample tubes were labeled with the study number, animal number (attached “-F1” following animal No.) and timing of blood collection.
As for one sample obtained from Dam No. 586 on PND 4, plasma volume was insufficient (about 270 μL), but no re-measurement was performed, therefore the measurement was not affected.

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 the following items: urinalysis (qualitative data: results from reagent strip method and urine 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 and TSH was conducted according to work plan.
The data of offspring were handled on a litter-basis. The body weight and food consumption of one non-pregnant female after confirmation of copulation were excluded from the evaluation. The body weight and food consumption of dead pregnant animals were analyzed until the day before necropsy.

Please see any other information on materials and methods section for further information

Reproductive indices:

At necropsy on LD 14, the ovaries and uterus were excised to examine the number of implantation sites. As for one non-delivered female, the presence of implantation was examined for the uterus at necropsy on GD 26. Based on the results, the following parameters were calculated.
(1) Gestation length:
Days until completion of delivery from GD 0
(2) Gestation index (%):
(Number of pregnant animals delivered live offspring / Number of pregnant animals) × 100
(3) Delivery index (%):
(Number of delivered offspring / Number of implantations) × 100

Offspring viability indices:

Observation of offspring
The number of litter (numbers of live newborn and stillborn), sex, and the presences or absences 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. The dead offspring after culling was fixed and preserved in 10 vol% phosphate buffered formalin solution.
(1) Birth index (%):
(Number of live offspring at birth / Number of implantations) × 100
(2) Stillborn index (%):
(Number of stillborns / Number of delivered offspring) × 100
(3) Viability index on Day 4 (%):
(Number of live offspring on PND 4 / Number of live offspring at birth) × 100
(4) Viability index on Day 13 (%):
(Number of live offspring on PND 13 / Number of live offspring after culling) × 100
(5) Sex ratio:
(Number of male live offspring / Number of male and female live offspring)
(6) External anomaly index (%):
(Number of offspring with external anomaly / Number of observed offspring) × 100
(7) External anomaly typing index (%):
(Number of offspring with external anomaly by each type / Number of observed
offspring) × 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the dosing period, salivation was noted in 10 males and 1 satellite female treated with 200 mg/kg. This finding appeared just after dosing and disappeared by 4 hours after dosing in 1 to 9 animals per day on Days 20 to 42. Moreover, 1 male (No. 539) treated with 200 mg/kg showed a decrease in locomotor activity and soiled fur on the face on Days 19 and 20 and soiled fur on the anus on Days 19 to 22.
During the recovery period, none of the above-mentioned changes were noted in any
animal.
Other than the above, chromaturia like hematuria was noted in 1 satellite female treated with 200 mg/kg (No. 657) on Days 3 and 4 during the dosing period. This was not judged to be toxicologically significant as described above. A mass was noted in the abdomen of 1 female (No. 573) treated with 12.5 mg/kg on LD 0 and thereafter. However, it was not considered to be treatment-related because there was no dose-dependency.

Detailed clinical observations
During the dosing period, slight or moderate salivation was noted in males treated with 200 mg/kg and females treated with 200 (100) mg/kg in the hand held observation; 5 males (Nos. 537, 538, 542, 544, and 548) and 1 female (No. 591) on Day 21, 4 males (Nos. 537, 538, 542, and 548) on Day 28, 3 males (Nos. 537, 542, and 544) and 1 female (No. 594) on Day 35, and 2 males (Nos. 543 and 544) on Day 42. Moreover, soiled fur was noted on the anus of 1 male (No. 539) treated with 200 mg/kg on Day 21.
During the recovery period, none of the above-mentioned changes were noted in any animal.
Other than the above, during the dosing period, statistically significant increases in the number of rearing were noted in males treated with 12.5 mg/kg on Days 21 and 35. However, these were not considered to be treatment-related because there was no dose-dependency.
During the recovery period, a statistically increase in the number of rearing was noted in males treated with 200 mg/kg on Day 49. However, this was not considered to be treatment-related because it was slight and no consistent tendency was noted during the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 50 mg/kg group, 1 female (No. 575) was euthanized moribund on LD 6. This animal showed a decrease in body weight on LD 6 (26.0% decrease compared to the body weight on LD 0) and a remarkable decrease in mean food consumption from LD 0 to LD 4.
In the 200 (100) mg/kg group, 5 females (Nos. 588, 592, 594, 595, and 596) died during the gestation or lactation period. One of the animals (No. 595) was found dead on GD 2. Three animals (Nos. 592, 594, and 596) were found dead on GD 21. One of these animals (No. 594) showed salivation on GDs 7 to 9, and 19 and decrease in locomotor activity on GD 20. Another animal (No. 588) showed salivation on LD 0 and was found dead on LD 1. These animals showed a decrease in body weight at their deaths (0.2 to 3.1% decrease compared to the body weight on the last measurement day). Since death occurred frequently in the test females treated with 200 (100) mg/kg, it was judged that no further administration for the surviving animals was possible and the administration was discontinued on Day 39 (during the gestation or lactation period).
Salivation was noted in 2 surviving females; on GDs 3-5 and 20 in No. 591 and GDs 19-21 in No. 598. The surviving animals showed an increase or decrease in body weight on the day of
discontinuation of administration (18.5% decrease to 8.6% increase compared to the body weight on the last measurement day). As for animals underwent discontinuation of administration on the delivery day, a body weight loss of 20% or more was noted between GD 20 and LD 0, which was similar to that of the controls.
Other than the above, chromaturia like hematuria was noted in 1 female treated with 200 (100) mg/kg (No. 594) on GD 20. However, this was not judged to be
toxicologically significant because the change was noted transiently and no related
change was noted in any examination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the dosing period, statistically significant decreases in body weights were noted
in males treated with 200 mg/kg on Days 29 to 43.
During the recovery period, no significant difference was noted between the control and
200 mg/kg groups

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the dosing period, statistically significant increases in food consumption were
noted in males treated with 200 mg/kg on Day 8 and thereafter and females treated with
200 (100) mg/kg on Day 8.
During the recovery period, no significant difference was noted between the control and
200 mg/kg groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

One female euthanized moribund treated with 50 mg/kg on LD 6 (No. 575)
No remarkable change was noted in any parameter.
Females treated with 200 (100) mg/kg
Remarkable changes were noted as follows.
No. 587: Low values of RBC count, HGB, HCT, and reticulocyte count, and high values of WBC count and lymphocyte count
No. 590: Low values of RBC count, HGB, HCT, and reticulocyte count and ratio
No. 591: Low values of reticulocyte count and ratio
No. 593: Low values of reticulocyte count and ratio and neutrophil count and ratio, and a high value of lymphocyte ratio
No. 597: Low values of RBC count, HGB, HCT, and reticulocyte count and ratio
No. 598: Low values of RBC count, HGB, HCT, reticulocyte count and ratio, and platelet count

Animals for scheduled euthanasia
At the end of the dosing period, a statistically significant decrease in platelet count and statistically significant increases in monocyte count and ratio were noted in males treated with 200 mg/kg.
At the end of the recovery period, none of the above-mentioned changes were noted. Other than the above, at the end of the dosing period, statistically significant differences were noted: high values of MCV and MCH in males treated with 50 mg/kg, and a low value of neutrophil ratio and a high value of lymphocyte count in females treated with 12.5 mg/kg. However, these were not considered to be treatment-related because there was no dose-dependency.
At the end of the recovery period, statistically significant differences were noted: low values of HGB, HCT, MCV, and MCH and a high value of reticulocyte count in males treated with 200 mg/kg and a high value of RBC and a low value of reticulocyte ratio in females treated with 200 mg/kg. However, these were not judged to be treatment-related because individual values in this group were similar to those in the control group or did not deviate significantly from the range in the control group, and the similar changes were not noted at the end of the dosing period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

One female euthanized moribund treated with 50 mg/kg on LD 6 (No. 575)
No remarkable change was noted in any parameter.
Females treated with 200 (100) mg/kg
Remarkable changes were noted as follows.
No. 593: High values of glucose, Ca, and K
No. 597: Low values of total protein, albumin, and A/G ratio
No. 598: Low values of total protein, albumin, and A/G ratio, and high values of ASAT, ALAT, and γGT

Animals for scheduled euthanasia
At the end of the dosing period, statistically significant increases in ALAT, γGT, total bilirubin, and total bile acid and statistically significant decreases in albumin, glucose, and Na were noted in males treated with 200 mg/kg. Individually, remarkable high values of ASAT and ALP were noted in 3 males and 1 male compared to those of the control, respectively.
At the end of the recovery period, none of the above-mentioned changes were noted.
Other than the above, at the end of the dosing period, a statistically significant increase in Ca was noted in males treated with 200 mg/kg. However, this was not judged to be treatment-related because individual values in this group were similar to those in the control group and no related change was noted in any examination.
At the end of the recovery period, a statistically significant decrease in γGT was noted in males treated with 200 mg/kg. However, this was not judged to be treatment-related because no related change was noted in any other parameter and the change was opposite to the toxicity effect.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

During the dosing period, statistically significant increases in Na and Cl were noted in males treated with 200 mg/kg.
During the recovery period, none of the above-mentioned changes was noted.
Other than the above, protein (3+) was noted in 1 male treated with 200 mg/kg. However, this was not judged to be treatment-related because the change was noted only in 1 male and no microscopic change was noted in the kidney.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

(1) Sensory reactivity to stimuli
Reactivity to stimuli was comparable in males and females at all dose levels and no abnormality was observed.
(2) Grip strength
No treatment-related change was noted.
As a change without dose-dependency and considered as not treatment-related, a statistically significant decrease in forelimb grip strength was noted in females treated with 12.5 mg/kg.

Motor activity
No treatment-related change was noted.
A statistically significant increase in motor activity was noted in males treated with 200 mg/kg between 0 and 10 minutes. However, this was not judged to be treatment-related because their moving pattern was normal and no significant difference was noted in total motor activity.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

One female euthanized moribund treated with 50 mg/kg on LD 6 (No. 575)
Treatment-related changes were noted in the liver, stomach, and thymus as follows.
Liver:
- Moderate necrosis of the centrilobular hepatocytes
- Mild hemorrhage
- Mild centrilobular inflammatory cell infiltration
Stomach:
- Minimal erosion of the forestomach
- Minimal hyperkeratosis of the forestomach
- Minimal hyperplasia of the squamous cells in the forestomach
- Minimal edema of the submucosa in the forestomach
Thymus:
- Mild atrophy
Females treated with 200 (100) mg/kg
Dead animals (Nos. 588, 592, 594, 595, and 596)
Treatment-related changes were noted in the liver, stomach, and thymus as follows.
Liver:
- Moderate or marked necrosis of the centrilobular hepatocytes; 5 females
- Mild or moderate hemorrhage; 5 females
- Mild centrilobular inflammatory cell infiltration; 4 females
Stomach:
- Minimal erosion of the forestomach; 1 female
Thymus:
- Moderate atrophy; 1 female
Discoloration of the spleen was noted in 1 female at the necropsy, but microscopic
examination was not conducted.
Surviving animals
Treatment-related changes were noted in the liver and thymus as follows.
Liver:
- Minimal, mild or moderate necrosis of the centrilobular hepatocytes; 4 females
- Minimal or mild hemorrhage; 2 females
- Minimal or mild centrilobular inflammatory cell infiltration; 3 females
- Minimal brown pigment deposition of the macrophage; 3 females
- Minimal regenerative hyperplasia; 1 female
Thymus:
- Minimal atrophy; 1 female
Animals for scheduled euthanasia
Treatment-related changes were noted in the liver, stomach, and duodenum.
At the end of the dosing period
Liver:
- Necrosis of the centrilobular hepatocytes in males treated with 200 mg/kg
- Hemorrhage in males treated with 200 mg/kg
- Centrilobular inflammatory cell infiltration in males treated with 200 mg/kg
- Brown pigment deposition of the macrophage in males treated with 200 mg/kg
- Regenerative hyperplasia in males treated with 200 mg/kg
Stomach:
- Hyperkeratosis of the forestomach in males treated with 200 mg/kg
- Hyperplasia of the squamous cells in the forestomach in males treated with 200 mg/kg
- Edema of the submucosa in the forestomach in males treated with 200 mg/kg
Duodenum:
- Neutrophil infiltration in the lamina propria in males treated with 200 mg/kg
Examination of spermatogenic cycle using PAS-stained specimens:
- No abnormality was noted in any animal at any stage (stages I to XIV).
At the end of the recovery period
Liver:
- Brown pigment deposition of the macrophage in males and females treated with
200 mg/kg
- Centrilobular fibrosis in a male treated with 200 mg/kg
Others
- Various histopathological changes were noted in both sexes of the control and test substance groups. However, these changes were not judged to be treatment-related as they are observed occasionally in normal rats and there was no clear dose-dependency in the incidence.
- Small testis noted in 1 male treated with 200 mg/kg (No. 539) at the necropsy was histopathological necrosis. This was not judged to be treatment-related because the change was noted only in 1 male and no effect on the testis weight was noted in any other males. In this male, macroscopic small epididymis and decrease in sperm in the lumen of epididymis were also noted; these were considered to be secondary changes associated with the testicular necrosis.
- Subcutaneous mass noted in 1 female treated with 12.5 mg/kg (No. 573) at the clinical observation and necropsy was adenocarcinoma in the histopathology and not judged to be treatment-related.

Description (incidence and severity):

Hormone concentration (total T4) analysis
One female euthanized moribund treated with 50 mg/kg on LD 6 (No. 575)
No marked change was noted in total T4 or TSH.
Females treated with 200 (100) mg/kg
No marked change was noted in total T4 or TSH.
Animals for scheduled euthanasia
At the end of the dosing period
A statistically significant decrease in total T4 was noted in males treated with 200 mg/kg.
At the end of the recovery period
The above-mentioned change was not noted.
Others
At the end of the recovery period, statistically significant differences were noted: a high value of total T4 and a low value of TSH were noted in the satellite females treated with 200 mg/kg. However, these were not judged to be treatment-related because the individual values were similar to those in the controls and no related change was noted in any examination.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related change was noted.
Irregular estrous cycle was noted in 2 females treated with 200 (100) mg/kg (Nos. 588 and 595). However, this was not judged to be treatment-related because the incidence of change was low and the copulation and conception were confirmed in these females. A statistically significant shortening of the mean estrous cycle was noted in females treated with 12.5 mg/kg; however, this was not judged to be treatment-related because there was no dose-dependency.

Description (incidence and severity):

Mating
No statistically significant difference was noted in the copulation index, days until copulation, or fertility index between the control and test substance groups.
As for 1 female treated with 200 (100) mg/kg (No. 593), copulation was confirmed; however, this female did not become pregnant. Macroscopically, no abnormal finding was noted in the ovaries of this female or the testes and epididymides of its male counterpart (No. 543). Although the cause of non-pregnancy was not clear, it was not considered to be treatment-related but spontaneous for its incidence.

Observation during delivery and lactation periods
In one moribund female (No. 575) treated with 50 mg/kg, no abnormal nursing behavior was noted. However, milk spot was not noted in any offspring on PND 4 or 5.
No statistically significant difference was noted in the gestation length, number of implantations, delivery index, gestation index, number of newborns, number of live newborns, stillborn index, birth index, sex ratio, or viability index on PND 4 or 13 between the control and test substance groups.
Other than the above, one female (No. 554) in the control group, which had only one implantation (placenta), did not deliver.
Females treated with 200 (100) mg/kg (Nos. 588, 590, and 598)
Abnormality was noted as follows.
No. 588: Loss of retrieving was noted on LD 0. In any offspring, milk spot was not noted and hypothermia was noted on PND 0.
No. 598: Although no abnormal nursing behavior was noted, milk spot was not noted in any offspring on PND 0.

Dose descriptor:

NOAEL

Effect level:

12.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

Clinical signs:

no effects observed

Description (incidence and severity):

In one moribund female (No. 575) treated with 50 mg/kg, no abnormality was noted in any offspring.
No statistically significant difference was noted in the anomaly index between the control and test substance groups on PND 0 or 13.
Offspring from dams treated with 200 (100) mg/kg (Nos. 588, 590, and 598)
No abnormality was noted in any offspring.

Description (incidence and severity):

In one moribund female (No. 575) treated with 50 mg/kg, no remarkable change was noted in the body weight on PND 0. Thereafter, a decreased body weight or suppressed body weight gain was noted on PNDs 4 and 6.
No statistically significant difference was noted between the control and test substance groups

Offspring from dams treated with 200 (100) mg/kg (Nos. 588, 590, and 598)
Remarkable changes were noted as follows.
A suppressed body weight gain was noted in all offspring of 1 dam (No. 588) on PND 1.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

In one moribund female (No. 575) treated with 50 mg/kg, no remarkable change was noted in any offspring in AGD or AGI.
No statistically significant difference was noted in AGD or AGI between the control and test substance groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in the anomaly index between the control and test substance groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic finding was noted in any offspring

Other effects:

no effects observed

Description (incidence and severity):

Hormone concentration (total T4) analysis
No treatment-related change was noted in total T4 or TSH.
On PND 0, individual offspring values of total T4 from 4 of 5 dams were below the lower limit of quantification (B.L.Q) and another offspring value was low compared to that of the controls on PND 4. Moreover, a low value of TSH was noted in these offspring compared to that of the controls on PND 4. These changes were considered to be unique to this time point (PND 0) for the following reason: blood sampling for these dams was conducted immediately after delivery; it has been reported that total T4 in neonatal rat is hardly detected at birth and TSH reaches the same level as adult rats after 10 days of birth [2].

Offspring for scheduled euthanasia
No treatment-related change was noted in any parameter.
A statistically significant increase in TSH was noted in offspring on PND 13. However, this was not judged to be treatment-related because the individual values were similar to those in the controls.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

Key result

Reproductive effects observed:

no

Conclusions:

According to the results of this study, a No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 12.5 mg/kg based on occurrence of moribundity and death, a decrease in body weight, an increase in food consumption, and injury to the liver, stomach, and duodenum at 50 or 200 (100) mg/kg. The NOAEL for reproductive and developmental toxicity was judged to be 200 mg/kg since no treatment-related change was noted at any dose level.

Executive summary:

MPDAc was repeatedly administered by oral gavage at 0 (control group), 12.5, 50, and 200 mg/kg from 14 days before mating through mating for 42 days in males, from 14 days before mating through gestation and parturition until Day 13 of lactation in females, and for 42 days without mating in satellite 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 200 mg/kg groups and the reversibility of the toxicity effect was assessed.
Deaths occurred in 4/12 test females treated with 200 mg/kg during the gestation period. Since the remaining test females treated with 200 mg/kg were considered at risk of deaths, the dose level and volume for the test females were reduced to 100 mg/kg and from 10 mL/kg to 5 mL/kg, respectively. Thereafter, since one more test female died during the lactation period, it was judged that no further administration was possible, and the administration was discontinued. Dose level for the test females is expressed as 200 (100) mg/kg.

Repeated dose toxicity
Moribund female treated with 50 mg/kg and females treated with 200 (100) mg/kg
One female treated with 50 mg/kg was euthanized moribund and 5 females treated with 200 (100) mg/kg died during the late gestation period or early lactation period. These animals showed body weight loss. Surviving females treated with 200 (100) mg/kg showed an increase or decrease in body weight on the day of discontinuation of administration. In some of these animals, deterioration of general condition was noted. Abnormal nursing behavior was noted in 1 dam treated with 200 (100) mg/kg and no milk spot and a decreased body weight or suppressed body weight gain were noted in their offspring. These changes were limited to the moribund and dead animals; therefore, the changes were judged to be caused by deterioration of general condition.
Hepatic injury was noted in almost all females treated with 50 or 200 (100) mg/kg
described below. The cause of death/moribundity may be deterioration of general
condition due to the liver injury and overlapping effects of the test substance and
postpartum stress.
At the necropsy, dark reddish patch, discoloration, or whitish patch was noted in the liver in a few females treated with 50 or 200 (100) mg/kg and an increase in the liver weight was noted in almost all surviving females treated with 200 (100) mg/kg. Microscopically, necrosis of the centrilobular hepatocytes, hemorrhage, and centrilobular inflammatory cell infiltration, brown pigment deposition of the macrophage, and regenerative hyperplasia were noted in the liver of almost all females treated with 50 or 200 (100) mg/kg; these changes were seen similarly in males for scheduled euthanasia but more severe than those at terminal necropsy. In the hematology, decreases in RBC count, HGB, HCT, reticulocyte count and/or ratio, and platelet count, increases in WBC count and lymphocyte count or ratio, and decreases in neutrophil count and ratio were noted in females treated with 200 (100) mg/kg. In the blood chemistry, increases in ASAT, ALAT, γGT, glucose, Ca, and K and decreases in total protein, albumin, and A/G ratio were noted in females treated with 200 (100) mg/kg.
The irritating effect of test substance was noted in the stomach. In the stomach of the moribund female treated with 50 mg/kg, whitish patch of the mucosa was noted in the forestomach at the necropsy, and histopathology revealed erosion, hyperkeratosis, hyperplasia of the squamous cells, and edema of the submucosa. In the dead and surviving females treated with 200 (100) mg/kg, salivation was noted in the clinical observation and detailed clinical observations. In a dead female treated with 200 (100) mg/kg, dark reddish patch of the mucosa in the glandular stomach was noted at the necropsy and erosion of the forestomach was noted in the histopathology.
As a secondary response to the stress, small thymus, a decrease in the thymus weight, or an increase in adrenals weight was noted at the necropsy and atrophy of the thymus was noted in the histopathology in females treated with 200 (100) mg/kg.
Other than the above, discoloration of the spleen was noted in 1 dead animal; however, histopathological examination was not conducted due to autolysis. Therefore, the detail remains unclear.

Animals for scheduled euthanasia
Salivation was noted transiently in males and a satellite female treated with 200 mg/kg on Days 20 to 42. Moreover, 1 male treated with 200 mg/kg showed a decrease in locomotor activity and soiled fur on the face on Days 19 and 20 and soiled fur on the anus on Days 19 to 22.
A decrease in body weight was noted in males treated with 200 mg/kg on Days 29 to 43.
Increases in food consumption were noted in males treated with 200 mg/kg on Day 8 and thereafter and females treated with 200 (100) mg/kg on Day 8.
In the urinalysis, excretion of Na and Cl was noted in males treated with 200 mg/kg.
In the hematology, a decrease in platelet count and increases in monocyte count and ratio were noted in males treated with 200 mg/kg.
In the blood chemistry, increases in ASAT, ALAT, γGT, ALP, total bilirubin, and total bile acid and decreases in albumin, glucose, and Na were noted in males treated with 200 mg/kg.

Reproductive/Developmental toxicity
In the parental animals, no treatment-related change was noted in any examination; the estrous cycle, copulation index, fertility index, days until copulation, gestation length, number of implantations, delivery index, gestation index, or parturition/maternal behavior.
In the offspring, no treatment-related change was noted in any examination; number of newborns, number of live newborns, stillborn index, birth index, sex ratio, viability index, external examination, body weight, anogenital distance, nipple development, necropsy or plasma total T4 concentration.

NOAEL
According to the results of this study, a No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 12.5 mg/kg based on occurrence of moribundity and death, a decrease in body weight, an increase in food consumption, and injury to the liver, stomach, and duodenum at 50 or 200 (100) mg/kg.

The NOAEL for reproductive and developmental toxicity was judged to be 200 mg/kg because no treatment-related change was noted at any dose level.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

2

Species:

rat

Quality of whole database:

Reliability 1