3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2020 to 24 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2020 to 24 December 2020

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat is an animal species commonly used in this type of study, and this strain was selected because of its stable reproductive performance and historical control data available at the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks of age for males and females
- Weight at receipt: 276 to 319 g for males, 193 to 224 g for females
- Housing: Metal bracket-type cages (300 W × 410 D × 200 H mm) with wire mesh floors. For females with successful copulation, small cage trays with bedding for experimental animals were used from gestation day (GD) 17 to lactation day (LD) 13. Number of animals per cage were two during the quarantine and acclimatization period, 1 after group assignment, 1 male and 1 female during the mating period, and 1 litter after parturition.
- Diet: Supplied freely from metal feeders. However, all animals were fasted from early evening on the day before necropsy. Animals were additionally fasted overnight (16 to 20 hours) prior to blood sampling for clinical biochemistry.
- Water: Supplied freely from the automatic watering system or water containers.

QUARANTINE AND ACCLIMATISATION
- Acclimation period: from the day of receipt to the day of group assignment, including the quarantine period (from the day of receipt (quarantine day 1) to quarantine day 6).
- Method: Animals were observed for general conditions once daily, and weighed at the time of receipt, on quarantine day 6, and on the day of group assignment. No abnormalities were found in general conditions in any animal other than 1 female that showed reddish urine.
- Oestrous cycle examination: Females were examined for oestrous cyclicity by the smear method for 14 days from the day after the end of quarantine to the day of group assignment. No abnormalities were observed in any female other than 1 female that showed abnormal oestrous cyclicity.

DETAILS OF FOOD AND WATER QUALITY
- Food: The diet of the lots used was analysed for contaminants that may affect the study. The analytical results of all items were within the acceptable ranges, which were specified in the SOP of the test facility.
- Water: Samples of drinking water were collected at the end (animal room No. 301) of the same piping system as that in the animal room used in this study on July 2, 2020 and January 5, 2021 and analysed for contaminants that may affect the study. The analytical results of all items were within the acceptable ranges, which were specified in the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C (actual range 19 to 24 °C)
- Humidity: 50 ± 20 % (actual range 41 to 57 %)
- Ventilation: 10 to 15 times per hour
- Photoperiod: Artificial lighting for 12 hours (from 8:00 to 20:00)

IN-LIFE DATES: 15 September 2020 to 13 October 2020 (males and females in satellite group, necropsy at the end of administration).

Route of administration:

oral: gavage

Details on route of administration:

A dosing suspension was administered into the stomach by oral gavage using a disposable gastric tube and a glass syringe. Individual dose volume was calculated based on the body weight on the day closest to the administration day.

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- The amount of the test material required for each dose was accurately weighed out into an agate mortar and pulverized with an agate pestle to mix with a small amount of the vehicle (control article). When the mixture became fluidal, it was transferred to a graduated glass using a syringe. The mortar was rinsed with the vehicle several times until the rinse appeared clear and the rinse was also transferred to the graduated glass. The content of the graduated glass was stirred with a stirrer to make a suspension. To the suspension, the vehicle was added to achieve the prescribed volume, and the mixture was stirred to make a homogeneous suspension. For calculation of the amount of the test material required to prepare the dosing suspensions, correction for the purity of the test material was not conducted.
- Preparation of dosing suspensions was performed 7 times at intervals of 7 to 11 days during the study and the dosing suspensions were used within the verified stability period (13 days after preparation).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because the test material is hardly soluble in water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

> Stability of the test material during the administration period was confirmed by measuring infrared (IR) absorption spectra according to the KBr method using a fourier transform infrared spectrophotometer (FT-IR, Thermo Fisher Scientific K.K.) before the start and after the end of the administration period.

> Stability of dosing suspensions: For the 2.50 and 250 mg/mL dosing suspensions, the stability of the test material in the dosing suspensions for 16 days after preparation (the day of preparation was designated as the starting day [Day 0]) under refrigeration was certified in a study previously conducted by the sponsor.

> Analysis of test material preparations for concentration was performed using HPLC:
- Analytical method: HPLC
- Analytical instruments:
Alliance separation module: e2695, Waters Corporation
UV-VIS Detector: 2489, Waters Corporation
Data processor: Empower 3, Waters Corporation
Electronic balance: GH-202, A & D Co., Ltd.
Pipet: mLINE, Sartorius

- Analytical conditions:
Column: Cadenza CD-C18, 3 μm, 4.6 mm ID × 150 mm, Imtakt Corporation
Mobile phase: Acetonitrile / ultrapure water (65:35)
Rinse solution for autosampler: Acetonitrile / ultrapure water (65:35)
Flow rate: 1.0 mL/minute
Wavelength: 258 nm
Column temperature: 40 °C
Autosampler temperature: 15 °C
Injection volume: 5 μL
Run time: 8 minutes

- Results of concentration analysis: Dosing suspensions of all concentrations were analysed for concentrations of the test material at the first preparation and at the final preparation for males. The results showed that the contents ranged from 99.6 % to 105.7 % and the RSD ranged from 0.1 % to 1.4 % in the 25, 75, and 250 mg/mL dosing suspensions, which met the acceptance criteria (content, 100.0 % ± 10.0 %; RSD, 5.0 % or less).

- Results of homogeneity analysis: At the first preparation, the 25 and 250 mg/mL dosing suspensions were analysed for homogeneity of the test material. The results showed that the RSDs were 1.0 % and 0.6 % in the 25 and 250 mg/mL dosing suspensions, respectively, which met the acceptance criteria (RSD, 5.0 % or less).

Duration of treatment / exposure:

Males; for 28 days from 14 days before the start of mating. Females; for 14 days before the start of mating and during the mating period until successful copulation. Females with successful copulation; during gestation through day 13 after parturition (LD 13). A female with no evidence of parturition until day 25 after successful copulation; until GD 25. An unsuccessfully mated female; until 23 days after the end of the mating period. For 28 days for the satellite group.

Frequency of treatment:

Once daily on successive days.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control (Main Study group)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low Dose (Main Study group)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Middle Dose (Main Study group)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose (Main Study group)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control (Satellite group, test group for necropsy at the end of administration)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control (Satellite group, test group for necropsy at the end of recovery)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose (Satellite group, test group for necropsy at the end of administration)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose (Satellite group, test group for necropsy at the end of recovery)

No. of animals per sex per dose:

12 each of males and females in the main study group; 5 each of males and females in the satellite group (the males was selected from the main study group)

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELETION RATIONALE
- In the previous 14-day repeated-dose oral toxicity study in rats conducted by the sponsor, (dose levels: 0, 50, 200, 500, and 1000 mg/kg/day; 3 male and 3 female Crl:CD(SD) rats per group), significantly higher relative liver weights and hepatic hypertrophy were observed in males in the 1000 mg/kg group. No toxic changes were seen in either males or females at doses of 500 mg/kg or less. Based on the results, for the present study, 1000 mg/kg/day, which is the limit dose in the test guidelines, was selected as the highest dose level, and 300 and 100 mg/kg/day were selected as the middle and low dose levels, respectively, by dividing the high dose level by a common ratio of approximately 3.

ANIMAL ASSIGNMENT
- Using a computer system (MiTOX, Mitsui E & S Systems Research Inc.), 48 males and 68 females were selected and assigned to test groups based on the body weight on the day of group assignment (day before the start of administration) by the stratified random sampling method so that mean body weight was comparable among groups. For females, the mean body weight in the main study group was comparable to that in the satellite group. The body weight ranges at the group assignment were 366 to 448 g in males and 245 to 287 g in females, which were within ± 20 % of the mean body weight (408.4 g in males and 264.2 g in females).

OTHER
- Post-exposure recovery period in satellite groups: Un-dosed for 14 days after the end of the administration period.
- Mating procedure: Mating pairs were cohabited continuously from the evening of the first day of mating until confirmation of copulation.

OBSERVATION, MEASUREMENT AND EXAMINATION DAYS
- The first day of administration was designated as administration day 1 (Day 1), and the day after administration day 28 as recovery day 1 (Recovery Day 1). Males in the main study group and males and females in the satellite group were necropsied on the day after administration day 28 (Day 29) or on the day after recovery day 14 (Recovery Day 15). For females in the main study group, the day of successful copulation was designated as gestation day 0 (GD 0), the day of completion of parturition by 9:00 as lactation day 0 (LD 0), and the day of necropsy as Day 14 after parturition.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From Day 1 to the day of necropsy. Twice daily (before and after dosing) during the administration period, twice daily (in the morning and afternoon) in the recovery period, and once on the day of necropsy.
- Method: Individual animals were observed for mortality, external appearance, behaviour, etc. Any abnormalities found were recorded with the times of onset and disappearance.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males; before the start of administration and on Days 7, 14, 21, and 28 (Main study group and Satellite group), and Recovery Days 7 and 14 (Satellite group); Females; before the start of administration and on Days 7, 14, 21, 28, 35, 42, and 49 (Main study group and Satellite group), and Recovery Days 7 and 14 (Satellite group).
Observations included:
- Body position/posture, respiratory pattern, tremor/convulsion, stereotypes (rolling/repetitive circling), and bizarre behaviour (self-biting) were observed from outside the home cage.
- Ease of removal, ease of handling, muscle tone, piloerection, fur condition, appearance of skin, eyes and eyeballs, and mucous membranes, pupil size, lacrimation, salivation, and other secretions or excretions were observed when taking out of the cage.
- Gait, co-ordination of movement, reactivity to environmental stimuli, searching (sniffing and standing), excretions (urination and defecation), stereotypes (excessive grooming and unusual head movement), bizarre behaviour (walking backwards and vocalization), and aggression were observed in an open field.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females of the satellite group; before dosing on Days 1, 4, 7, 14, 21, and 28, and on Recovery Days 1, 7, and 14, and the day of necropsy. Females in the main study group; before dosing on Days 1, 4, 7, and 14, before dosing on GDs 0, 7, 14, and 20, and before dosing on LDs 0, 4, 7, and 13, and on the day of necropsy (Day 14 after parturition). For a female with no evidence of parturition, body weight before dosing on day 26 after successful copulation (day of necropsy) was recorded. For an unsuccessfully mated female, body weight before dosing on Days 21, 28, 35, 42, and 49, and on the day of necropsy (the next day of Day 51) was recorded.
- Body weight was measured with an electronic balance (GX- 2000, A & D Co., Ltd.)

FOOD CONSUMPTION: Yes
- Time period: On the same days as body weight measurement, except the weeks during mating and the day of necropsy.
- Method: The amount of food unconsumed and/or that supplied were measured on each day of measurement with an electronic balance (GX-2000, A & D Co., Ltd.). Food consumption of females during the lactation period was expressed as the total amount of food consumed by maternal animals and their offspring.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals fasted overnight (16 to 20 hours) from early evening.
- How many animals: Males in the main study group; 5 males not used in the functional observations in each group and selected in ascending order of animal numbers. Females in the main study group; the same 5 females as those used in the functional observations in each group. In the satellite group; all 5 males and 5 females.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected at necropsy.
- Animals fasted: Yes. Animals fasted overnight (16 to 20 hours) from early evening.
- How many animals: [1] to [20]; The same 5 animals per group as those used in the haematological examination in each group [21]; Serum for measurement was collected from all animals and was measured in all males in the main study group. Because no changes were observed in T4 concentrations in males in the main study group and postnatal-day-13 offspring, the serum T4 concentrations were not measured for females in the main study group and males and females in the satellite group.
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Males; administration week 4 (Main study group and Satellite group), and recovery week 2 (Satellite group). Females; administration week 4 and recovery week 2 (Satellite group).
- Metabolism cages used for collection of urine: Yes (urine collected for approximately 3 hours from immediately after dosing, for approximately 21 hours)
- Animals fasted: No
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In the administration week 4 (Day 23) and for males in the main study group and females in the satellite group. On LD 13 for females in the main study group. Because there were no changes indicative of effects of the test material in any parameter in the administration week 4, the observations in the recovery week 2 was not performed.
- Dose groups that were examined: Five males with body weight around the central value of the population were selected from each main study group based on the body weight on Day 21 except animals that were during mating. 5 females with surviving pups on LD 13 selected in the order of earlier parturition date in each main study group and 5 males and 5 females in each satellite group.
Battery of functions tested:
- Sensory and motor reactivity to visual, touch, auditory, pain, and proprioceptive stimuli, and air-righting reflex were examined on a desk.
- Grip strength was measured using CPU gage 3 times each for the forelimbs and for the hindlimbs, and recorded as an integer in grams.
- Motor activity was measured using an automatic measuring system. Each animal was monitored for one session of 60 minutes consisting of six 10-minute intervals (0–10, 10–20, 20–30, 30–40, 40–50, and 50–60 minutes) and the sum of the six intervals, after the above procedures

IMMUNOLOGY: No

OTHER:
- Oestrous cycle examination
- Reproductive performance
- Observation of parturition and nursing behaviour
- Clinical observation of offspring, measurements and necropsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Timing: Males in the main study group and females in the satellite group [those for necropsy at the end of administration]; Day 29 (the day after Day 28). Females in the main study group; on Day 14 after parturition. The female with no evidence of parturition, on day 26 after successful copulation. The unsuccessfully mated female, 24 days after the end of the mating period (the day after Day 51). Males and females in the satellite group [those for necropsy at the end of recovery]; on Recovery Day 15 (the day after Recovery Day 14).
- Method: After observation of external appearance and blood collection from the abdominal aorta under anesthesia with isoflurane, animals were euthanised by exsanguination, and then all organs and tissues were macroscopically observed.
- For females in the main study group, the number of implantations was counted. The organs and tissues described below were fixed and preserved in 10 % neutral buffered formalin. The lung was fixed after infusion with the fixative through the trachea. The eyeballs and harderian glands were fixed and preserved in Davidson’s fixative and the testes and epididymides were fixed in Bouin’s solution and preserved in 70 % ethanol. For bilateral organs, both sides of them, in principle, were fixed and preserved.
- Organs: Brain (cerebrum, cerebellum, and pons), spinal cord, pituitary gland, thymus, thyroids, parathyroids, adrenals, spleen, heart, oesophagus, stomach, duodenum, jejunum, ileum (with Peyer’s patches), cecum, colon, rectum, liver, pancreas, submandibular glands, trachea, lungs and bronchus, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles (with coagulating glands), ovaries, uterus (horn and cervix), vagina, eyeballs and harderian glands, mammary gland (right abdomen), femur (with bone marrow, right), mesenteric lymph nodes, submandibular lymph node, skeletal muscle (biceps femoris, right), and sciatic nerve (right), and gross lesions (with a border to the normal tissue).

ORGAN WEIGHTS: Yes
- Method: The organs shown below were weighed with an electronic balance (GR-200, A & D Co., Ltd.). Bilateral organs were measured together. The thyroids, pituitary gland, and seminal vesicles were weighed after fixation.
- Organs: Brain, heart, liver, kidneys, testes, epididymides, and seminal vesicles (with coagulating glands); (g). Pituitary glands, thyroids (with parathyroids), spleen, thymus, adrenals, prostate, ovaries, and uterus; (mg).
- Relative organ weight was calculated from the absolute organ weight and the body weight on the day of necropsy.

HISTOPATHOLOGY: Yes
- All organs and tissues of all animals in the control and high dose groups in the main study group were microscopically examined. There were no changes indicative of effects of the test material; therefore, microscopic examination was not performed for the animals in the low and middle dose groups in the main study group or satellite group. A pair of animals that was unsuccessfully mated, a male whose mate was non-pregnant, and the non-pregnant female were also examined.
- The gross regions (the kidneys of animal No. 10310, the incisors of animal No. 10304, and a subcutaneous mass of animal No. 50458) were also microscopically examined.
- Method: Embedded in paraffin wax, thin-sectioned, and stained with hematoxylin and eosin, and microscopically examined.

Statistics:

- The analyses were performed using the computer system (MiTOX).
- For the main study group, group means and standard deviations were analysed by the Bartlett test for homogeneity of variances, followed by the one-way analysis of variance when variances were homogeneous (p ≥ 0.05), or by the Kruskal-Wallis test when variances were heterogeneous (p < 0.05). When a significant difference was detected (p < 0.1) by the one-way analysis of variance, Dunnett’s test was performed to detect statistical differences between the test material administered groups and their corresponding controls. When a significant difference was detected (p < 0.1) by the Kruskal-Wallis test, Steel’s test was performed to detect statistical differences between the test material-administered groups and their corresponding controls. Detailed clinical observations, reactivity to environmental stimuli, and results of urinalysis except urine volume for the main study group were analysed by the Kruskal-Wallis test. When a significant difference was detected (p < 0.1) by the Kruskal-Wallis test, Steel’s test was performed to detect statistical differences between the test material-administered groups and their corresponding controls.
- For the satellite group, group means and standard deviations were analysed for homogeneity of variances by the F test. When variances were homogeneous (p > 0.05), Student’s t-test was used to detect any statistically significant difference between the control and high-dose groups. When variances were heterogeneous (p < 0.05), the Welch test was used. Detailed clinical observation, reactivity to environmental stimuli, and results of urinalysis except urine volume for the satellite group were analysed by the Wilcoxon rank sum test to detect any statistically significant difference between the control and high-dose groups.
- Statistically significance level was less than 5 % for comparison with the control group.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In males in the control and 100 mg/kg groups, no abnormal findings were observed in any animal throughout the administration period. In the 300 mg/kg group, crushing of the upper incisors and reddish urine were observed in 1 animal each, on Days 2 to 29 and Days 8 to 14, respectively. These were considered incidental because there was no dose-dependence. In the 1000 mg/kg group, no abnormal findings were observed in any animal.
- In females in the control, and 100 and 300 mg/kg groups, no abnormal findings were observed in any animal throughout the administration period. In the 1000 mg/kg group, crushing of the upper incisors was observed in 1 animal on GDs 13 to 17. A subcutaneous mass in the right thoracic region was observed in 1 animal on LDs 7 to 14. These were considered incidental because of a single occurrence.
- In the satellite group, no abnormal findings were observed in any female rat throughout the administration period.
- No significant differences were observed in any parameter of detailed clinical observation in males or females in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were observed in any parameter of detailed clinical observation in the 1000 mg/kg group.

RECOVERY PERIOD
- No abnormal findings were observed in any males or females.
- No significant differences were observed in any parameter of detailed clinical observation in males or females in the 1000 mg/kg group compared with the control group.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred in any test group during the administration period. No deaths occurred in any test group during the recovery period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In males, no significant differences were noted in body weight or body weight gain during the administration period in any test material-administered group compared with that in the control group.
- In females, no significant differences were noted in body weight or body weight gain in the pre-mating and gestation periods in any test material-administered group compared with that in the control group. In the lactation period, body weight on LD 13 was significantly lower in the 100 mg/kg group. However, this was considered incidental because there was no dose-dependence or reduction of food consumption.
- In the female satellite group, no significant differences were noted in body weight or body weight gain in the 1000 mg/kg group.

RECOVERY PERIOD
- No significant differences were noted in body weight or body weight gain of males or females in the 1000 mg/kg group compared with that in the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In males, no significant differences were noted in food consumption during the administration period in any test material-administered group compared with that in the control group.
- In females, no significant difference was noted in food consumption in the premating and lactation periods in any test material-administered group compared with that in the control group. In the gestation period, food consumption on GD 7 was significantly lower in the 100 mg/kg group. However, this was considered incidental because there was no dose-dependence or weight loss.
- In the female satellite group, no significant differences were noted in food consumption in the 1000 mg/kg group.

RECOVERY PERIOD
- No significant differences were noted in food consumption of males or females in the 1000 mg/kg group compared with that in the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- No significant differences were noted in males or females in any haematology biomarker in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were noted in any haematology biomarker in the 1000 mg/kg group.

AT THE END OF RECOVERY PERIOD
- No significant differences were noted in males or females in any haematology biomarker in the 1000 mg/kg group compared with the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In males, no significant differences were noted in any biochemistry biomarker in any test material-administered group compared with the control group.
- In females, no significant differences were noted in any biochemistry biomarker in the 300 or 1000 mg/kg group compared with the control group. Total cholesterol (TCho) and albumin were significantly lower and α1-globulin (α1-G) was significantly higher in the 100 mg/kg group. However, these were considered incidental because there was no dose-dependence.
- In the female satellite group, no significant differences were noted in any biochemistry biomarker in the 1000 mg/kg group.

AT THE END OF RECOVERY PERIOD
- In males, γ-glutamyl transpeptidase (γ-GTP), total cholesterol (T-Cho), and triglyceride (TG) were significantly lower and urea nitrogen (UN) was significantly higher in the 1000 mg/kg group compared with those in the control group.
- In females, creatinine (Crea) was significantly lower and inorganic phosphorus (IP) was significantly higher in the 1000 mg/kg group. However, these were considered unrelated to the test material administration because of the following reasons:
1) these findings were not observed at the end of the administration period;
2) lower values of γ-GTP, T-Cho, TG, and Crea are clinically insignificant; and/or
3) individual values were within the range of historical control data in the test facility (0.0 to 0.8 IU/L for γ-GTP, 35 to 100 mg/dL for T-Cho, and 7.3 to 16.2 mg/dL for UN in males; 0.25 to 0.46 mg/dL for Crea and 3.4 to 7.0 mg/dL for IP in females.

- Serum T4 concentration
AT THE END OF ADMINISTRATION PERIOD
No significant differences were noted in serum T4 concentrations of males in any test material-administered group compared with those in the control group.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

ADMINISTRATION WEEK 4
- No significant differences were noted in any urinalysis biomarker of males or females in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were noted in any urinalysis biomarker in the 1000 mg/kg group.

RECOVERY WEEK 2
- No significant differences were noted in any urinalysis biomarker of males or females in the 1000 mg/kg group compared with the control group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION WEEK 4
- No significant differences were observed in any parameter of functional observations in males in any test material-administered group compared with the control group.
- In the female satellite group, motor activity was significantly higher (40–50 minutes) in the 1000 mg/kg group, which was considered an incidental change because no significant difference was noted in the total count (0–60 minutes) and their individual values were within the range of historical control data in the test facility (0 to 553 for 40–50 minutes; 313 to 4324 for 0–60 minutes).
- No significant differences were noted in the other parameters between the high dose and control groups.

LACTATION DAY 13
- No significant differences were observed in any parameter of functional observations in any test material-administered group compared with the control group.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In males, both absolute and relative kidney weights in the 300 mg/kg group were significantly higher compared with those in the control group. However, this was considered unrelated to the test material administration because there was no dose dependence.
- No significant differences were noted in any organ weight measured in the 100 or 1000 mg/kg group compared with in the control group.
- In females, the absolute heart weight in the 100 mg/kg group was significantly lower compared with that in the control group. However, this was considered incidental because of no dose-dependence. No significant differences were noted in any organ weight measured in the 300 or 1000 mg/kg group compared with the control group.
- In the female satellite group, both absolute and relative thymus and pituitary gland weights were significantly lower in the 1000 mg/kg group compared with those in the control group. The relative liver weight was also significantly lower. However, these were considered unrelated to the test material administration because of the following reasons:
1) no histopathological changes related to the test material administration were observed in these organs; and
2) individual values were within the range of historical control data in the test facility (127 to 784 mg for absolute thymus weight; 44.10 to 270.34 mg/100 g BW for relative thymus weight; 10.1 to 23.2 mg for absolute pituitary gland weight; 2.89 to 8.50 mg/100 g BW for relative pituitary gland weight; and 2.08 to 2.89 g/100 g BW for relative liver weight)

AT THE END OF RECOVERY PERIOD
- No significant differences were noted in any organ weight measured of males in the 1000 mg/kg group compared with the control group.
- In females, both absolute and relative ovary weights, relative heart weight, and relative spleen weight were significantly higher in the 1000 mg/kg group. However, these were considered accidental changes because of the following reasons:
1) these were changes that were not observed at the end of the administration period; and
2) individual values were within the range of historical control data in the test facility (58 to 146 mg for absolute ovary weight; 17.75 to 59.59 mg/100 g BW for relative ovary weight; 0.27 to 0.48 g/100 g BW for relative heart weight; and 134.36 to 257.41 mg/100 g BW for relative spleen weight).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In males in the control, 100, and 1000 mg/kg groups, there were no macroscopic abnormal findings in any animal. In the 300 mg/kg group, 1 animal showed crushing of the upper incisors and another showed dilatation of renal pelvis (bilateral).
- In females in the control, 100, and 300 mg/kg groups, there were no macroscopic abnormal findings in any animal. In the 1000 mg/kg group, 1 animal showed a subcutaneous mass in the right thoracic skin.
- In the female satellite group, there were no macroscopic abnormal findings in any animal.

AT THE END OF RECOVERY PERIOD
- In both of males and females, there were no macroscopic abnormal findings in any animal.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the males necropsied on Day 29 in the 1000 mg/kg group, inflammatory cell infiltration of the liver (slight), focal inflammatory cell infiltration of the heart (slight), focal basophilic change of the renal tubule (mild), interstitial inflammation of the prostate (mild), and remnant of the ultimobranchial body in the thyroid (slight) were observed each in 1 or 2 animals. Corresponding to the macroscopic lesion in the 300 mg/kg group (dilatation of renal pelvis), dilatation of renal pelvis (mild) was observed.

In the females necropsied on Day 14 after parturition in the 1000 mg/kg group, findings including remnant of the ultimobranchial body in the thyroid (slight) and dysplasia of the retina in the eyeball (slight) were observed each in 1 animal. In the pairs of infertile animals in the 300 mg/kg group, focal basophilic change of the renal tubule (slight) was noted in 1 male and inflammatory cell infiltration of the urinary bladder (slight) and remnant of the epithelial cells in the thymus (slight) were noted in 1 female. Corresponding to the macroscopic lesion (crushing of the upper incisors), necrosis and fibrosis of the dental pulp (moderate) and inflammation of the gingiva (mild) were observed. None of these were considered to be causes of infertility.

In the satellite females necropsied on Day 29 in the 1000 mg/kg group, inflammatory cell infiltration of the liver (slight), inflammatory cell infiltration of the renal pelvic mucosa (slight), remnant of the epithelial cells in the thymus (slight), and remnant of the ultimobranchial body in the thyroid (slight) were observed each in 1 or 3 animals.

All abnormal findings observed were considered unrelated to the test material administration because of the following reasons:
1) there was no significant difference in their frequencies;
2) no enhancement of their grades compared with the control group; and/or
3) they have been known to be spontaneous

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the females necropsied on Day 14 after parturition in the 1000 mg/kg group: Corresponding to the macroscopic lesion in the 1000 mg/kg group (a subcutaneous mass), adenocarcinoma was observed in the thoracic skin.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No changes considered to be attributed to the test material administration were observed in the incidence of abnormal oestrous cycles, length of oestrous cycle, number of oestrus, copulation index, fertility index, number of days required for copulation, number of implantations, delivery index, gestation length, gestation index, or numbers of offspring delivered, live new-borns, and dead new-borns of parental animals in any test material administered
group.

For offspring, no changes considered to be attributed to the test material administration were observed in the sex ratio, viability index on PND 0, 4, or 13, general conditions, incidence of external anomalies, male and female body weight, anogenital distance, nipple retention of males, serum T4 concentrations of postnatal-day-13 offspring, or necropsy up to a dose of 1000 mg/kg/day.

Details on results:

No changes considered to be attributed to the test material administration were observed in general conditions, detailed clinical observations, functional observations, body weight, body weight gain, food consumption, or urinalysis during the administration period in male or female rats in any test material-administered group. There were no changes considered to be attributed to the test material administration in haematology, biochemistry including serum T4 concentrations of males, necropsy, organ weight, or histopathology up to a dose of 1000 mg/kg/day.

In the satellite group during the recovery period, no delayed effects were observed in any item examined in male or female animals in the 1000 mg/kg group.

Based on the above results, the no-observed-adverse-effect level (NOAEL) of the test material for repeated dose toxicity is considered 1000 mg/kg/day in both sexes under the present study conditions.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose level tested.

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of the study the NOAEL for repeated dose toxicity of the test material in adults was considered to be 1000 mg/kg/day. No adverse effects related to test material administration were observed. As such, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity.

Executive summary:

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guideline OECD 422, under GLP conditions. A combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening test in rats was conducted to investigate the potential effects of
repeated administration of the test material on the general toxicity, reproductive performance, and development of the offspring. Each group consisted of 12 male and 12 female Crl:CD(SD) rats, which were orally administered the test material at doses of 0 (control: olive oil), 100, 300, and 1000 mg/kg/day to males for totally 28 days from 14 days before the start of mating, or to females before and during the mating and gestation periods and up to lactation day 13 (for 55 days at most). For the 0 (control) and 1000 mg/kg groups, 5 males per group were selected to investigate the recovery for 14 days after administration; and 10 females per group were separately set (non-mating group), and 5 of them were necropsied at the end of 28-day administration, and the other 5 were investigated for recovery for 14 days.

 

No adverse effects related to the test material administration were observed in general conditions, detailed clinical observations, functional observations, body weight, body weight gain, food consumption, urinalysis, heamatology, biochemistry including serum T4 concentrations of males, necropsy, organ weight, or histopathology in male and female animals up to a dose of 1000 mg/kg/day.  Therefore, the no-observed-adverse-effect level (NOAEL) of the test material for repeated dose toxicity is considered 1000 mg/kg/day in both sexes under the present study conditions.

 

No adverse effects related to the test material administration were observed; in the reproductive performance of parental animals including oestrous cyclicity, and copulation, gestation, fertility, and delivery indices; and in the examination of their offspring including sex ratio, viability indices, general conditions, morphology, body weight, anogenital distance, nipple retention of males, serum T4 concentrations of postnatal-day-13 offspring, and necropsy up to a dose of 1000 mg/kg/day. Therefore, the NOAEL of the test material is considered 1000 mg/kg/day for reproductive performance of parental animals and for development of offspring under the present study conditions.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2020 to 24 December 2020

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:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks of age for males and females
- Weight at receipt: 276 to 319 g for males, 193 to 224 g for females
- Housing: Metal bracket-type cages (300 W × 410 D × 200 H mm) with wire mesh floors. For females with successful copulation, small cage trays with bedding for experimental animals were used from gestation day (GD) 17 to lactation day (LD) 13. Number of animals per cage were two during the quarantine and acclimatization period, 1 after group assignment, 1 male and 1 female during the mating period, and 1 litter after parturition.
- Diet: Supplied freely from metal feeders. However, all animals were fasted from early evening on the day before necropsy. Animals were additionally fasted overnight (16 to 20 hours) prior to blood sampling for clinical biochemistry.
- Water: Supplied freely from the automatic watering system or water containers.

QUARANTINE AND ACCLIMATISATION
- Acclimation period: from the day of receipt to the day of group assignment, including the quarantine period (from the day of receipt (quarantine day 1) to quarantine day 6).
- Method: Animals were observed for general conditions once daily, and weighed at the time of receipt, on quarantine day 6, and on the day of group assignment. No abnormalities were found in general conditions in any animal other than 1 female that showed reddish urine.
- Oestrous cycle examination: Females were examined for oestrous cyclicity by the smear method for 14 days from the day after the end of quarantine to the day of group assignment. No abnormalities were observed in any female other than 1 female that showed abnormal oestrous cyclicity.

DETAILS OF FOOD AND WATER QUALITY
- Food: The diet of the lots used was analysed for contaminants that may affect the study. The analytical results of all items were within the acceptable ranges, which were specified in the SOP of the test facility.
- Water: Samples of drinking water were collected at the end (animal room No. 301) of the same piping system as that in the animal room used in this study on July 2, 2020 and January 5, 2021 and analysed for contaminants that may affect the study. The analytical results of all items were within the acceptable ranges, which were specified in the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C (actual range 19 to 24 °C)
- Humidity: 50 ± 20 % (actual range 41 to 57 %)
- Ventilation: 10 to 15 times per hour
- Photoperiod: Artificial lighting for 12 hours (from 8:00 to 20:00)

IN-LIFE DATES: 15 September 2020 to 4 November 2020 (start of necropsy of dams)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- The amount of the test material required for each dose was accurately weighed out into an agate mortar and pulverized with an agate pestle to mix with a small amount of the vehicle (control article). When the mixture became fluidal, it was transferred to a graduated glass using a syringe. The mortar was rinsed with the vehicle several times until the rinse appeared clear and the rinse was also transferred to the graduated glass. The content of the graduated glass was stirred with a stirrer to make a suspension. To the suspension, the vehicle was added to achieve the prescribed volume, and the mixture was stirred to make a homogeneous suspension. For calculation of the amount of the test material required to prepare the dosing suspensions, correction for the purity of the test material was not conducted.
- Preparation of dosing suspensions was performed 7 times at intervals of 7 to 11 days during the study and the dosing suspensions were used within the verified stability period (13 days after preparation).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because the test material is hardly soluble in water.

DETAILS ON ROUTE OF ADMINISTRATION
A dosing suspension was administered into the stomach by oral gavage using a disposable gastric tube and a glass syringe. Individual dose volume was calculated based on the body weight on the day closest to the administration day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

> Stability of the test material during the administration period was confirmed by measuring infrared (IR) absorption spectra according to the KBr method using a fourier transform infrared spectrophotometer (FT-IR, Thermo Fisher Scientific K.K.) before the start and after the end of the administration period.

> Stability of dosing suspensions: For the 2.50 and 250 mg/mL dosing suspensions, the stability of the test material in the dosing suspensions for 16 days after preparation (the day of preparation was designated as the starting day [Day 0]) under refrigeration was certified in a study previously conducted at the sponsor.

> Analysis of test material preparations for concentration was performed using HPLC:
- Analytical method: HPLC
- Analytical instruments:
Alliance separation module: e2695, Waters Corporation
UV-VIS Detector: 2489, Waters Corporation
Data processor: Empower 3, Waters Corporation
Electronic balance: GH-202, A & D Co., Ltd.
Pipet: mLINE, Sartorius

- Analytical conditions:
Column: Cadenza CD-C18, 3 μm, 4.6 mm ID × 150 mm, Imtakt Corporation
Mobile phase: Acetonitrile / ultrapure water (65:35)
Rinse solution for autosampler: Acetonitrile / ultrapure water (65:35)
Flow rate: 1.0 mL/minute
Wavelength: 258 nm
Column temperature: 40 °C
Autosampler temperature: 15 °C
Injection volume: 5 μL
Run time: 8 minutes

- Results of concentration analysis: Dosing suspensions of all concentrations were analysed for concentrations of the test material at the first preparation and at the final preparation for males. The results showed that the contents ranged from 99.6 % to 105.7 % and the RSD ranged from 0.1 % to 1.4 % in the 25, 75, and 250 mg/mL dosing suspensions, which met the acceptance criteria (content, 100.0 % ± 10.0 %; RSD, 5.0 % or less)

- Results of homogeneity analysis: At the first preparation, the 25 and 250 mg/mL dosing suspensions were analysed for homogeneity of the test material. The results showed that the RSDs were 1.0 % and 0.6 % in the 25 and 250 mg/mL dosing suspensions, respectively, which met the acceptance criteria (RSD, 5.0 % or less)

Details on mating procedure:

Mating pairs were cohabited continuously from the evening of the first day of mating until confirmation of copulation. Successful copulation was confirmed by the presence of vaginal plugs in the vagina or fallen on the cage trays, or sperm in vaginal smears. The day was designated as GD 0. For a pair of the middle dose group, successful copulation was not confirmed during the 2-week mating period. All the other pairs were confirmed to have successful copulation by Mating day 5. The number of days from the start of cohabitation to successful copulation was counted.

Pregnancy was confirmed by the occurrence of parturition or by the presence of implantation sites in the uterus at necropsy. An animal was judged to be nonpregnant because no intrauterine implantation sites were found at necropsy.

Duration of treatment / exposure:

Males; for 28 days from 14 days before the start of mating. Females; for 14 days before the start of mating and during the mating period until successful copulation. Females with successful copulation; during gestation through day 13 after parturition (LD 13). A female with no evidence of parturition until day 25 after successful copulation; until GD 25. An unsuccessfully mated female; until 23 days after the end of the mating period. For 28 days for the satellite group.

Frequency of treatment:

Once daily on successive days.

Duration of test:

Males; for 28 days from 14 days before the start of mating. Females; for 14 days before the start of mating and during the mating period until successful copulation. Females with successful copulation; during gestation through day 13 after parturition (LD 13). A female with no evidence of parturition until day 25 after successful copulation; until GD 25. An unsuccessfully mated female; until 23 days after the end of the mating period. For 28 days for the satellite group.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Main Study group)

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose (Main Study group)

Dose / conc.:

300 mg/kg bw/day

Remarks:

Middle Dose (Main Study group)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose (Main Study group)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Satellite group, test group for necropsy at the end of administration)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Satellite group, test group for necropsy at the end of recovery)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose (Satellite group, test group for necropsy at the end of administration)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose (Satellite group, test group for necropsy at the end of recovery)

No. of animals per sex per dose:

12 each of males and females in the main study group; 5 each of males and females in the satellite group (the males was selected from the main study group)

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELETION RATIONALE
- In the previous 14-day repeated-dose oral toxicity study in rats conducted by the sponsor, (dose levels: 0, 50, 200, 500, and 1000 mg/kg/day; 3 male and 3 female Crl:CD(SD) rats per group), significantly higher relative liver weights and hepatic hypertrophy were observed in males in the 1000 mg/kg group. No toxic changes were seen in either males or females at doses of 500 mg/kg or less. Based on the results, for the present study, 1000 mg/kg/day, which is the limit dose in the test guidelines, was selected as the highest dose level, and 300 and 100 mg/kg/day were selected as the middle and low dose levels, respectively, by dividing the high dose level by a common ratio of approximately 3.

ANIMAL ASSIGNMENT
- Using a computer system (MiTOX, Mitsui E & S Systems Research Inc.), 48 males and 68 females were selected and assigned to test groups based on the body weight on the day of group assignment (day before the start of administration) by the stratified random sampling method so that mean body weight was comparable among groups. For females, the mean body weight in the main study group was comparable to that in the satellite group. The body weight ranges at the group assignment were 366 to 448 g in males and 245 to 287 g in females, which were within ± 20 % of the mean body weight (408.4 g in males and 264.2 g in females).

OTHER
- Post-exposure recovery period in satellite groups: Un-dosed for 14 days after the end of the administration period.

OBSERVATION, MEASUREMENT AND EXAMINATION DAYS
- The first day of administration was designated as administration day 1 (Day 1), and the day after administration day 28 as recovery day 1 (Recovery Day 1). Males in the main study group and males and females in the satellite group were necropsied on the day after administration day 28 (Day 29) or on the day after recovery day 14 (Recovery Day 15). For females in the main study group, the day of successful copulation was designated as gestation day 0 (GD 0), the day of completion of parturition by 9:00 as lactation day 0 (LD 0), and the day of necropsy as Day 14 after parturition.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From Day 1 to the day of necropsy. Twice daily (before and after dosing) during the administration period, twice daily (in the morning and afternoon) in the recovery period, and once on the day of necropsy.
- Method: Individual animals were observed for mortality, external appearance, behaviour, etc. Any abnormalities found were recorded with the times of onset and disappearance.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Females; before the start of administration and on Days 7, 14, 21, 28, 35, 42, and 49 (Main study group and Satellite group), and Recovery Days 7 and 14 (Satellite group).
Observations included:
- Body position/posture, respiratory pattern, tremor/convulsion, stereotypes (rolling/repetitive circling), and bizarre behaviour (self-biting) were observed from outside the home cage.
- Ease of removal, ease of handling, muscle tone, piloerection, fur condition, appearance of skin, eyes and eyeballs, and mucous membranes, pupil size, lacrimation, salivation, and other secretions or excretions were observed when taking out of the cage.
- Gait, co-ordination of movement, reactivity to environmental stimuli, searching (sniffing and standing), excretions (urination and defecation), stereotypes (excessive grooming and unusual head movement), bizarre behaviour (walking backwards and vocalization), and aggression were observed in an open field.

BODY WEIGHT: Yes
- Time schedule for examinations: Females of the satellite group; before dosing on Days 1, 4, 7, 14, 21, and 28, and on Recovery Days 1, 7, and 14, and the day of necropsy. Females in the main study group; before dosing on Days 1, 4, 7, and 14, before dosing on GDs 0, 7, 14, and 20, and before dosing on LDs 0, 4, 7, and 13, and on the day of necropsy (Day 14 after parturition). For a female with no evidence of parturition, body weight before dosing on day 26 after successful copulation (day of necropsy) was recorded. For an unsuccessfully mated female, body weight before dosing on Days 21, 28, 35, 42, and 49, and on the day of necropsy (the next day of Day 51) was recorded.
- Body weight was measured with an electronic balance (GX- 2000, A & D Co., Ltd.)

FOOD CONSUMPTION: Yes
- Time period: On the same days as body weight measurement, except the weeks during mating and the day of necropsy.
- Method: The amount of food unconsumed and/or that supplied were measured on each day of measurement with an electronic balance (GX-2000, A & D Co., Ltd.). Food consumption of females during the lactation period was expressed as the total amount of food consumed by maternal animals and their offspring.

POST-MORTEM EXAMINATIONS: Yes
- Timing: Females in the main study group; on Day 14 after parturition. The female with no evidence of parturition, on day 26 after successful copulation. The unsuccessfully mated female, 24 days after the end of the mating period (the day after Day 51). Females in the satellite group [those for necropsy at the end of recovery]; on Recovery Day 15 (the day after Recovery Day 14).
- Sacrifice: After observation of external appearance and blood collection from the abdominal aorta under anesthesia with isoflurane, animals were euthanised by exsanguination, and then all organs and tissues were macroscopically observed.
- Organs: Brain (cerebrum, cerebellum, and pons), spinal cord, pituitary gland, thymus, thyroids, parathyroids, adrenals, spleen, heart, esophagus, stomach, duodenum, jejunum, ileum (with Peyer’s patches), cecum, colon, rectum, liver, pancreas, submandibular glands, trachea, lungs and bronchus, kidneys, urinary bladder, ovaries, uterus (horn and cervix), vagina, eyeballs and harderian glands, mammary gland (right abdomen), femur (with bone marrow, right), mesenteric lymph nodes, submandibular lymph node, skeletal muscle (biceps femoris, right), and sciatic nerve (right), and gross lesions (with a border to the normal tissue).

OTHER: Yes
- Observation of parturition and nursing behaviour. All females with successful copulation were observed for signs of parturition at least 3 times (at 9:00, 13:00 and 17:00) daily from GDs 21 to 25. All pregnant females were spontaneously delivered. Females holding their offspring under the abdomen to nurse at observation at 9:00 were considered to have completed parturition, and the day was designated as LD 0 (postnatal day 0 [PND 0]). For females that had completed parturition by 13:00 or 17:00, the following day was defined as LD 0. Parturition resulting in birth of one or more live pups was considered normal. Gestation length was expressed as the number of days from GD 0 to LD 0.

- Observation of offspring: All offspring on PND 0 were observed for viability and external appearance. The numbers of live and dead newborns were counted for each litter, which were totalled to serve as the number of offspring delivered. The delivery index and the incidence of live offspring with external anomalies were calculated per litter, and incidence of dams with offspring showing external anomalies were calculated for each group.

- Sex ratio of offspring: All offspring on PND 0 were sexed by external observation of the length between the anus and genitalia. The sex ratios were calculated for each group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals fasted overnight (16 to 20 hours) from early evening.
- How many animals: Females in the main study group; the same 5 females as those used in the functional observations in each group. In the satellite group; all 5 males and 5 females.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected at necropsy.
- Animals fasted: Yes. Animals fasted overnight (16 to 20 hours) from early evening.
- How many animals: [1] to [20]; The same 5 animals per group as those used in the haematological examination in each group [21];
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Females; administration week 4 and recovery week 2 (Satellite group).
- Metabolism cages used for collection of urine: Yes (urine collected for approximately 3 hours from immediately after dosing, for approximately 21 hours)
- Animals fasted: No
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In the administration week 4 (Day 23) and for females in the satellite group. On LD 13 for females in the main study group. Because there were no changes indicative of effects of the test material in any parameter in the administration week 4, the observations in the recovery week 2 was not performed.
- Dose groups that were examined: 5 females with surviving pups on LD 13 selected in the order of earlier parturition date in each main study group and 5 males and 5 females in each satellite group.
Battery of functions tested:
- Sensory and motor reactivity to visual, touch, auditory, pain, and proprioceptive stimuli, and air-righting reflex were examined on a desk.
- Grip strength was measured using CPU gage 3 times each for the forelimbs and for the hindlimbs, and recorded as an integer in grams.
- Motor activity was measured using an automatic measuring system. Each animal was monitored for one session of 60 minutes consisting of six 10-minute intervals (0–10, 10–20, 20–30, 30–40, 40–50, and 50–60 minutes) and the sum of the six intervals, after the above procedures

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: Oestrus cycle data

Blood sampling:

- Plasma: Yes. Blood was collected from the abdominal aorta. One blood sample was treated with 3.8 % sodium citrate, and approximately 1 mL of the treated blood (0.1 mL of 3.8 % sodium citrate solution + 0.9 mL of blood) was centrifuged (approximately 2000 × g, 4 °C, 10 minutes) to obtain plasma, which was used for calculation of prothrombin time (PT) and activated partial thromboplastin time (APTT)
- Serum: Yes. Serum for measurement was collected from all animals. Because no changes were observed in T4 concentrations in males in the main study group and postnatal-day-13 offspring, the serum T4 concentrations were not measured for females in the main study group and females in the satellite group.
- Volume collected: Not specified
- Other: blood was collected at necropsy.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

Statistics:

- The analyses were performed using the computer system (MiTOX).
- For the main study group, group means and standard deviations were analysed by the Bartlett test for homogeneity of variances, followed by the one-way analysis of variance when variances were homogeneous (p ≥ 0.05), or by the Kruskal-Wallis test when variances were heterogeneous (p < 0.05). When a significant difference was detected (p < 0.1) by the one-way analysis of variance, Dunnett’s test was performed to detect statistical differences between the test material administered groups and their corresponding controls. When a significant difference was detected (p < 0.1) by the Kruskal-Wallis test, Steel’s test was performed to detect statistical differences between the test material-administered groups and their corresponding controls. In the offspring data, litter averages were used as the unit for statistical evaluation. The incidence of abnormal oestrous cycles, copulation index, fertility index, gestation index, sex ratio of live offspring, sex ratio of offspring, and incidence of dams with offspring showing external anomalies, and results of histopathology were analysed by Fisher's exact probability test.
- For the satellite group, group means and standard deviations were analysed for homogeneity of variances by the F test. When variances were homogeneous (p > 0.05), Student’s t-test was used to detect any statistically significant difference between the control and high-dose groups. When variances were heterogeneous (p < 0.05), the Welch test was used.
- Statistically significance level was less than 5 % for comparison with the control group.

Indices:

See 'Any other information on materials and methods incl. tables'

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In females in the control, and 100 and 300 mg/kg groups, no abnormal findings were observed in any animal throughout the administration period. In the 1000 mg/kg group, crushing of the upper incisors was observed in 1 animal on GDs 13 to 17. A subcutaneous mass in the right thoracic region was observed in 1 animal on LDs 7 to 14. These were considered incidental because of a single occurrence.
- In the satellite group, no abnormal findings were observed in any female rat throughout the administration period.
- No significant differences were observed in any parameter of detailed clinical observation in females in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were observed in any parameter of detailed clinical observation in the 1000 mg/kg group.

RECOVERY PERIOD
- No abnormal findings were observed in females.
- No significant differences were observed in any parameter of detailed clinical observation in females in the 1000 mg/kg group compared with the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deaths occurred in any test group during the administration period. No deaths occurred in any test group during the recovery period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In females, no significant differences were noted in body weight or body weight gain in the pre-mating and gestation periods in any test material-administered group compared with that in the control group. In the lactation period, body weight on LD 13 was significantly lower in the 100 mg/kg group. However, this was considered incidental because there was no dose-dependence or reduction of food consumption.
- In the female satellite group, no significant differences were noted in body weight or body weight gain in the 1000 mg/kg group.

RECOVERY PERIOD
- No significant differences were noted in body weight or body weight gain of females in the 1000 mg/kg group compared with that in the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In females, no significant difference was noted in food consumption in the premating and lactation periods in any test material-administered group compared with that in the control group. In the gestation period, food consumption on GD 7 was significantly lower in the 100 mg/kg group. However, this was considered incidental because there was no dose-dependence or weight loss.
- In the female satellite group, no significant differences were noted in food consumption in the 1000 mg/kg group.

RECOVERY PERIOD
- No significant differences were noted in food consumption of females in the 1000 mg/kg group compared with that in the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- No significant differences were noted in females in any haematology biomarker in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were noted in any haematology biomarker in the 1000 mg/kg group.

AT THE END OF RECOVERY PERIOD
- No significant differences were noted in females in any haematology biomarker in the 1000 mg/kg group compared with the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In females, no significant differences were noted in any biochemistry biomarker in the 300 or 1000 mg/kg group compared with the control group. Total cholesterol (TCho) and albumin were significantly lower and α1-globulin (α1-G) was significantly higher in the 100 mg/kg group. However, these were considered incidental because there was no dose-dependence.
- In the female satellite group, no significant differences were noted in any biochemistry biomarker in the 1000 mg/kg group.

AT THE END OF RECOVERY PERIOD
- In females, creatinine (Crea) was significantly lower and inorganic phosphorus (IP) was significantly higher in the 1000 mg/kg group. However, these were considered unrelated to the test material administration because of the following reasons:
1) these findings were not observed at the end of the administration period;
2) lower values of γ-GTP, T-Cho, TG, and Crea are clinically insignificant; and/or
3) individual values were within the range of historical control data in the test facility (0.25 to 0.46 mg/dL for Crea and 3.4 to 7.0 mg/dL for IP in females

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

ADMINISTRATION WEEK 4
- No significant differences were noted in any urinalysis biomarker of females in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were noted in any urinalysis biomarker in the 1000 mg/kg group.

RECOVERY WEEK 2
- No significant differences were noted in any urinalysis biomarker of females in the 1000 mg/kg group compared with the control group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION WEEK 4
- In the female satellite group, motor activity was significantly higher (40–50 minutes) in the 1000 mg/kg group, which was considered an incidental change because no significant difference was noted in the total count (0–60 minutes) and their individual values were within the range of historical control data in the test facility (0 to 553 for 40–50 minutes; 313 to 4324 for 0–60 minutes).
- No significant differences were noted in the other parameters between the high dose and control groups.

LACTATION DAY 13
- No significant differences were observed in any parameter of functional observations in any test material-administered group compared with the control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In females, the absolute heart weight in the 100 mg/kg group was significantly lower compared with that in the control group. However, this was considered incidental because of no dose-dependence. No significant differences were noted in any organ weight measured in the 300 or 1000 mg/kg group compared with the control group.
- In the female satellite group, both absolute and relative thymus and pituitary gland weights were significantly lower in the 1000 mg/kg group compared with those in the control group. The relative liver weight was also significantly lower. However, these were considered unrelated to the test material administration because of the following reasons:
1) no histopathological changes related to the test material administration were observed in these organs; and
2) individual values were within the range of historical control data in the test facility (127 to 784 mg for absolute thymus weight; 44.10 to 270.34 mg/100 g BW for relative thymus weight; 10.1 to 23.2 mg for absolute pituitary gland weight; 2.89 to 8.50 mg/100 g BW for relative pituitary gland weight; and 2.08 to 2.89 g/100 g BW for relative liver weight)

AT THE END OF RECOVERY PERIOD
- In female, both absolute and relative ovary weights, relative heart weight, and relative spleen weight were significantly higher in the 1000 mg/kg group. However, these were considered accidental changes because of the following reasons:
1) these were changes that were not observed at the end of the administration period; and
2) individual values were within the range of historical control data in the test facility (58 to 146 mg for absolute ovary weight; 17.75 to 59.59 mg/100 g BW for relative ovary weight; 0.27 to 0.48 g/100 g BW for relative heart weight; and 134.36 to 257.41 mg/100 g BW for relative spleen weight).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In females in the control, 100, and 300 mg/kg groups, there were no macroscopic abnormal findings in any animal. In the 1000 mg/kg group, 1 animal showed a subcutaneous mass in the right thoracic skin.
- In the female satellite group, there were no macroscopic abnormal findings in any
animal.

AT THE END OF RECOVERY PERIOD
- In females, there were no macroscopic abnormal findings in any animal.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the females necropsied on Day 14 after parturition in the 1000 mg/kg group, findings including remnant of the ultimobranchial body in the thyroid (slight) and dysplasia of the retina in the eyeball (slight) were observed each in 1 animal. In the pairs of infertile animals in the 300 mg/kg group, focal basophilic change of the renal tubule (slight) was noted in 1 male and inflammatory cell infiltration of the urinary bladder (slight) and remnant of the epithelial cells in the thymus (slight) were noted in 1 female. Corresponding to the macroscopic lesion (crushing of the upper incisors), necrosis and fibrosis of the dental pulp (moderate) and inflammation of the gingiva (mild) were observed. None of these were considered to be causes of infertility.

In the satellite females necropsied on Day 29 in the 1000 mg/kg group, inflammatory cell infiltration of the liver (slight), inflammatory cell infiltration of the renal pelvic mucosa (slight), remnant of the epithelial cells in the thymus (slight), and remnant of the ultimobranchial body in the thyroid (slight) were observed each in 1 or 3 animals.

All abnormal findings observed were considered unrelated to the test material administration because of the following reasons:
1) there was no significant difference in their frequencies;
2) no enhancement of their grades compared with the control group; and/or
3) they have been known to be spontaneous

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the females necropsied on Day 14 after parturition in the 1000 mg/kg group: Corresponding to the macroscopic lesion in the 1000 mg/kg group (a subcutaneous mass), adenocarcinoma was observed in the thoracic skin.

Other effects:

no effects observed

Description (incidence and severity):

Oestrus cycle: No significant differences were noted in the incidence of abnormal oestrous cycles, length of oestrous cycle, or number of oestrus between the test material-administered groups and control group.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not specified

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No significant differences were noted in males or females in the copulation index, fertility index, or the number of days required for copulation between the test material administered groups and control group.

Details on maternal toxic effects:

No changes considered to be attributed to the test material administration were observed in the incidence of abnormal oestrous cycles, length of oestrous cycle, number of oestrus, copulation index, fertility index, number of days required for copulation, number of implantations, delivery index, gestation length, gestation index, or numbers of offspring delivered, live new-borns, and dead new-borns of parental animals in any test material administered group.
Normal parturition was observed in all pregnant females. No significant differences were noted in the number of implantations, delivery index, gestation length, gestation index, numbers of offspring delivered, live newborns, and dead newborns, sex ratio of live offspring, or sex ratio of offspring in any test material-administered group compared with those in the control group.
Based on the above results, the NOAEL of the test material is considered 1000 mg/kg/day for systemic toxicity and reproductive performance of maternal animals.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Highest dose level tested.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in body weight of male or female offspring from PNDs 0 to 13 in any test material-administered group compared with that in the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant differences were noted in sex ratio of live offspring, or sex ratio of offspring in any test material-administered group compared with those in the control group.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no significant differences in AGD and AGD per cube root of body weight ratio in any test material-administered group compared with those in the control group.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

In clinical observation of offspring, there were no abnormal findings in any test material-administered group.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Details on embryotoxic / teratogenic effects:

Normal parturition was observed in all pregnant females. No significant differences were noted in the number of implantations, delivery index, gestation length, gestation index, numbers of offspring delivered, live newborns, and dead newborns, sex ratio of live offspring, or sex ratio of offspring in any test material-administered group compared with those in the control group. No external anomalies were observed in any live offspring on PND 0.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose level tested.

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Conclusions:

Under the conditions of the study the NOAEL for developmental toxicity of the test material in adults was considered to be 1000 mg/kg/day. No adverse effects related to test material administration were observed. As such, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity.

Executive summary:

The developmental toxicity of the test material was investigated in accordance with the standardised guideline OECD 422, under GLP conditions. A combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening test in rats was conducted to investigate the potential effects of repeated administration of the test material on the general toxicity, reproductive performance, and development of the offspring. Each group consisted of 12 male and 12 female Crl:CD(SD) rats, which were orally administered the test material at doses of 0 (control: olive oil), 100, 300, and 1000 mg/kg/day to males for totally 28 days from 14 days before the start of mating, or to females before and during the mating and gestation periods and up to lactation day 13 (for 55 days at most). For the 0 (control) and 1000 mg/kg groups, 5 males per group were selected to investigate the recovery for 14 days after administration; and 10 females per group were separately set (non-mating group), and 5 of them were necropsied at the end of 28-day administration, and the other 5 were investigated for recovery for 14 days.

No adverse effects related to the test material administration were observed in general conditions, detailed clinical observations, functional observations, body weight, body weight gain, food consumption, urinalysis, hematology, biochemistry including serum T4 concentrations of males, necropsy, organ weight, or histopathology in male and female animals up to a dose of 1000 mg/kg/day.  Therefore, the no-observed-adverse-effect level (NOAEL) of the test material for repeated dose toxicity is considered 1000 mg/kg/day in both sexes under the present study conditions.

No adverse effects related to the test material administration were observed; in the reproductive performance of parental animals including estrous cyclicity, and copulation, gestation, fertility, and delivery indices; and in the examination of their offspring including sex ratio, viability indices, general conditions, morphology, body weight, anogenital distance, nipple retention of males, serum T4 concentrations of postnatal-day-13 offspring, and necropsy up to a dose of 1000 mg/kg/day. Therefore, the NOAEL of the test material is considered 1000 mg/kg/day for reproductive performance of parental animals and for development of offspring under the present study conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Cas Number:

2102522-55-2

Molecular formula:

C20H18N2O4S

IUPAC Name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

Appearance (at room temperature): Off-white powder
Storage conditions: Room temperature (actual range 21.0 to 24.3 °C)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is an animal species commonly used in this type of study, and this strain was selected because of its stable reproductive performance and historical control data available at the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks of age for males and females
- Weight at receipt: 276 to 319 g for males, 193 to 224 g for females
- Housing: Metal bracket-type cages (300 W × 410 D × 200 H mm) with wire mesh floors. For females with successful copulation, small cage trays with bedding for experimental animals were used from gestation day (GD) 17 to lactation day (LD) 13. Number of animals per cage were two during the quarantine and acclimatization period, 1 after group assignment, 1 male and 1 female during the mating period, and 1 litter after parturition.
- Diet: Supplied freely from metal feeders. However, all animals were fasted from early evening on the day before necropsy. Animals were additionally fasted overnight (16 to 20 hours) prior to blood sampling for clinical biochemistry.
- Water: Supplied freely from the automatic watering system or water containers.

QUARANTINE AND ACCLIMATISATION
- Acclimation period: from the day of receipt to the day of group assignment, including the quarantine period (from the day of receipt (quarantine day 1) to quarantine day 6).
- Method: Animals were observed for general conditions once daily, and weighed at the time of receipt, on quarantine day 6, and on the day of group assignment. No abnormalities were found in general conditions in any animal other than 1 female that showed reddish urine.
- Oestrous cycle examination: Females were examined for oestrous cyclicity by the smear method for 14 days from the day after the end of quarantine to the day of group assignment. No abnormalities were observed in any female other than 1 female that showed abnormal oestrous cyclicity.

DETAILS OF FOOD AND WATER QUALITY
- Food: The diet of the lots used was analysed for contaminants that may affect the study. The analytical results of all items were within the acceptable ranges, which were specified in the SOP of the test facility.
- Water: Samples of drinking water were collected at the end (animal room No. 301) of the same piping system as that in the animal room used in this study on July 2, 2020 and January 5, 2021 and analysed for contaminants that may affect the study. The analytical results of all items were within the acceptable ranges, which were specified in the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C (actual range 19 to 24 °C)
- Humidity: 50 ± 20 % (actual range 41 to 57 %)
- Ventilation: 10 to 15 times per hour
- Photoperiod: Artificial lighting for 12 hours (from 8:00 to 20:00)

IN-LIFE DATES: 15 September 2020 to 4 November 2020 (start of necropsy of dams)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- The amount of the test material required for each dose was accurately weighed out into an agate mortar and pulverized with an agate pestle to mix with a small amount of the vehicle (control article). When the mixture became fluidal, it was transferred to a graduated glass using a syringe. The mortar was rinsed with the vehicle several times until the rinse appeared clear and the rinse was also transferred to the graduated glass. The content of the graduated glass was stirred with a stirrer to make a suspension. To the suspension, the vehicle was added to achieve the prescribed volume, and the mixture was stirred to make a homogeneous suspension. For calculation of the amount of the test material required to prepare the dosing suspensions, correction for the purity of the test material was not conducted.
- Preparation of dosing suspensions was performed 7 times at intervals of 7 to 11 days during the study and the dosing suspensions were used within the verified stability period (13 days after preparation).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because the test material is hardly soluble in water.

DETAILS ON ROUTE OF ADMINISTRATION
A dosing suspension was administered into the stomach by oral gavage using a disposable gastric tube and a glass syringe. Individual dose volume was calculated based on the body weight on the day closest to the administration day.

Details on mating procedure:

Mating pairs were cohabited continuously from the evening of the first day of mating until confirmation of copulation. Successful copulation was confirmed by the presence of vaginal plugs in the vagina or fallen on the cage trays, or sperm in vaginal smears. The day was designated as GD 0. For a pair of the middle dose group, successful copulation was not confirmed during the 2-week mating period. All the other pairs were confirmed to have successful copulation by Mating day 5. The number of days from the start of cohabitation to successful copulation was counted.

Pregnancy was confirmed by the occurrence of parturition or by the presence of implantation sites in the uterus at necropsy. An animal was judged to be nonpregnant because no intrauterine implantation sites were found at necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

> Stability of the test material during the administration period was confirmed by measuring infrared (IR) absorption spectra according to the KBr method using a fourier transform infrared spectrophotometer (FT-IR, Thermo Fisher Scientific K.K.) before the start and after the end of the administration period.

> Stability of dosing suspensions: For the 2.50 and 250 mg/mL dosing suspensions, the stability of the test material in the dosing suspensions for 16 days after preparation (the day of preparation was designated as the starting day [Day 0]) under refrigeration was certified in a study previously conducted at the sponsor.

> Analysis of test material preparations for concentration was performed using HPLC:
- Analytical method: HPLC
- Analytical instruments:
Alliance separation module: e2695, Waters Corporation
UV-VIS Detector: 2489, Waters Corporation
Data processor: Empower 3, Waters Corporation
Electronic balance: GH-202, A & D Co., Ltd.
Pipet: mLINE, Sartorius

- Analytical conditions:
Column: Cadenza CD-C18, 3 μm, 4.6 mm ID × 150 mm, Imtakt Corporation
Mobile phase: Acetonitrile / ultrapure water (65:35)
Rinse solution for autosampler: Acetonitrile / ultrapure water (65:35)
Flow rate: 1.0 mL/minute
Wavelength: 258 nm
Column temperature: 40 °C
Autosampler temperature: 15 °C
Injection volume: 5 μL
Run time: 8 minutes

- Results of concentration analysis: Dosing suspensions of all concentrations were analysed for concentrations of the test material at the first preparation and at the final preparation for males. The results showed that the contents ranged from 99.6 % to 105.7 % and the RSD ranged from 0.1 % to 1.4 % in the 25, 75, and 250 mg/mL dosing suspensions, which met the acceptance criteria (content, 100.0 % ± 10.0 %; RSD, 5.0 % or less).

- Results of homogeneity analysis: At the first preparation, the 25 and 250 mg/mL dosing suspensions were analysed for homogeneity of the test material. The results showed that the RSDs were 1.0 % and 0.6 % in the 25 and 250 mg/mL dosing suspensions, respectively, which met the acceptance criteria (RSD, 5.0 % or less).

Duration of treatment / exposure:

Males; for 28 days from 14 days before the start of mating. Females; for 14 days before the start of mating and during the mating period until successful copulation. Females with successful copulation; during gestation through day 13 after parturition (LD 13). A female with no evidence of parturition until day 25 after successful copulation; until GD 25. An unsuccessfully mated female; until 23 days after the end of the mating period. For 28 days for the satellite group.

Frequency of treatment:

Once daily on successive days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Main Study group)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose (Main Study group)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Middle Dose(Main Study group)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose (Main Study group)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Satellite group, test group for necropsy at the end of administration)

Doses / concentrations 6

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Satellite group, test group for necropsy at the end of recovery)

Doses / concentrations 7

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose (Satellite group, test group for necropsy at the end of administration)

Doses / concentrations 8

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose (Satellite group, test group for necropsy at the end of recovery)

No. of animals per sex per dose:

12 each of males and females in the main study group; 5 each of males and females in the satellite group (the males was selected from the main study group)

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELETION RATIONALE
- In the previous 14-day repeated-dose oral toxicity study in rats conducted by the sponsor, (dose levels: 0, 50, 200, 500, and 1000 mg/kg/day; 3 male and 3 female Crl:CD(SD) rats per group), significantly higher relative liver weights and hepatic hypertrophy were observed in males in the 1000 mg/kg group. No toxic changes were seen in either males or females at doses of 500 mg/kg or less. Based on the results, for the present study, 1000 mg/kg/day, which is the limit dose in the test guidelines, was selected as the highest dose level, and 300 and 100 mg/kg/day were selected as the middle and low dose levels, respectively, by dividing the high dose level by a common ratio of approximately 3.

ANIMAL ASSIGNMENT
- Using a computer system (MiTOX, Mitsui E & S Systems Research Inc.), 48 males and 68 females were selected and assigned to test groups based on the body weight on the day of group assignment (day before the start of administration) by the stratified random sampling method so that mean body weight was comparable among groups. For females, the mean body weight in the main study group was comparable to that in the satellite group. The body weight ranges at the group assignment were 366 to 448 g in males and 245 to 287 g in females, which were within ± 20 % of the mean body weight (408.4 g in males and 264.2 g in females).

OTHER
- Post-exposure recovery period in satellite groups: Un-dosed for 14 days after the end of the administration period.

OBSERVATION, MEASUREMENT AND EXAMINATION DAYS
- The first day of administration was designated as administration day 1 (Day 1), and the day after administration day 28 as recovery day 1 (Recovery Day 1). Males in the main study group and males and females in the satellite group were necropsied on the day after administration day 28 (Day 29) or on the day after recovery day 14 (Recovery Day 15). For females in the main study group, the day of successful copulation was designated as gestation day 0 (GD 0), the day of completion of parturition by 9:00 as lactation day 0 (LD 0), and the day of necropsy as Day 14 after parturition.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From Day 1 to the day of necropsy. Twice daily (before and after dosing) during the administration period, twice daily (in the morning and afternoon) in the recovery period, and once on the day of necropsy.
- Method: Individual animals were observed for mortality, external appearance, behaviour, etc. Any abnormalities found were recorded with the times of onset and disappearance.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males; before the start of administration and on Days 7, 14, 21, and 28 (Main study group and Satellite group), and Recovery Days 7 and 14 (Satellite group); Females; before the start of administration and on Days 7, 14, 21, 28, 35, 42, and 49 (Main study group and Satellite group), and Recovery Days 7 and 14 (Satellite group).
Observations included:
- Body position/posture, respiratory pattern, tremor/convulsion, stereotypes (rolling/repetitive circling), and bizarre behaviour (self-biting) were observed from outside the home cage.
- Ease of removal, ease of handling, muscle tone, piloerection, fur condition, appearance of skin, eyes and eyeballs, and mucous membranes, pupil size, lacrimation, salivation, and other secretions or excretions were observed when taking out of the cage.
- Gait, co-ordination of movement, reactivity to environmental stimuli, searching (sniffing and standing), excretions (urination and defecation), stereotypes (excessive grooming and unusual head movement), bizarre behaviour (walking backwards and vocalization), and aggression were observed in an open field.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females of the satellite group; before dosing on Days 1, 4, 7, 14, 21, and 28, and on Recovery Days 1, 7, and 14, and the day of necropsy. Females in the main study group; before dosing on Days 1, 4, 7, and 14, before dosing on GDs 0, 7, 14, and 20, and before dosing on LDs 0, 4, 7, and 13, and on the day of necropsy (Day 14 after parturition). For a female with no evidence of parturition, body weight before dosing on day 26 after successful copulation (day of necropsy) was recorded. For an unsuccessfully mated female, body weight before dosing on Days 21, 28, 35, 42, and 49, and on the day of necropsy (the next day of Day 51) was recorded.
- Body weight was measured with an electronic balance (GX- 2000, A & D Co., Ltd.)

FOOD CONSUMPTION: Yes
- Time period: On the same days as body weight measurement, except the weeks during mating and the day of necropsy.
- Method: The amount of food unconsumed and/or that supplied were measured on each day of measurement with an electronic balance (GX-2000, A & D Co., Ltd.). Food consumption of females during the lactation period was expressed as the total amount of food consumed by maternal animals and their offspring.

OTHER: Yes
- Observation of parturition and nursing behaviour. All females with successful copulation were observed for signs of parturition at least 3 times (at 9:00, 13:00 and 17:00) daily from GDs 21 to 25. All pregnant females were spontaneously delivered. Females holding their offspring under the abdomen to nurse at observation at 9:00 were considered to have completed parturition, and the day was designated as LD 0 (postnatal day 0 [PND 0]). For females that had completed parturition by 13:00 or 17:00, the following day was defined as LD 0. Parturition resulting in birth of one or more live pups was considered normal. Gestation length was expressed as the number of days from GD 0 to LD 0.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals fasted overnight (16 to 20 hours) from early evening.
- How many animals: Males in the main study group; 5 males not used in the functional observations in each group and selected in ascending order of animal numbers. Females in the main study group; the same 5 females as those used in the functional observations in each group. In the satellite group; all 5 males and 5 females.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected at necropsy.
- Animals fasted: Yes. Animals fasted overnight (16 to 20 hours) from early evening.
- How many animals: [1] to [20]; The same 5 animals per group as those used in the haematological examination in each group [21]; Serum for measurement was collected from all animals and was measured in all males in the main study group. Because no changes were observed in T4 concentrations in males in the main study group and postnatal-day-13 offspring, the serum T4 concentrations were not measured for females in the main study group and males and females in the satellite group.
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Males; administration week 4 (Main study group and Satellite group), and recovery week 2 (Satellite group). Females; administration week 4 and recovery week 2 (Satellite group).
- Metabolism cages used for collection of urine: Yes (urine collected for approximately 3 hours from immediately after dosing, for approximately 21 hours)
- Animals fasted: No
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In the administration week 4 (Day 23) and for males in the main study group and females in the satellite group. On LD 13 for females in the main study group. Because there were no changes indicative of effects of the test material in any parameter in the administration week 4, the observations in the recovery week 2 was not performed.
- Dose groups that were examined: Five males with body weight around the central value of the population were selected from each main study group based on the body weight on Day 21 except animals that were during mating. 5 females with surviving pups on LD 13 selected in the order of earlier parturition date in each main study group and 5 males and 5 females in each satellite group.
Battery of functions tested:
- Sensory and motor reactivity to visual, touch, auditory, pain, and proprioceptive stimuli, and air-righting reflex were examined on a desk.
- Grip strength was measured using CPU gage 3 times each for the forelimbs and for the hindlimbs, and recorded as an integer in grams.
- Motor activity was measured using an automatic measuring system. Each animal was monitored for one session of 60 minutes consisting of six 10-minute intervals (0–10, 10–20, 20–30, 30–40, 40–50, and 50–60 minutes) and the sum of the six intervals, after the above procedures

Oestrous cyclicity (parental animals):

Period: From the first day of administration to the day of successful copulation. Until the last day of the 2-week mating period for the unsuccessfully mated female.
Method: Vaginal smears were stained with Giemsa solution and examined with a light microscope to determine the stage of oestrous cycle.
Determination: Females showing repetition of a 4-, 5-, or 6-day oestrous cycle (proestrus, estrus, metestrus, and diestrus) were determined to be normal. During 14 days from Days 1 to 14, length of oestrous cycle, number of estrus, and the number of animals with abnormal oestrous cycle were calculated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: On PND 4
- The size of each litter was reduced to 8 offspring by random selection; if possible, 4 males and 4 females. When the number of male or female offspring in a litter was less than 4, partial standardization was done (e.g. 3 males and 5 females). When the total number of offspring in a litter was 8 or less, all offspring were nursed. Offspring not selected were euthanised by decapitation for those used for blood collection, or by an intraperitoneal injection of a 60 mg/mL pentobarbital sodium solution for the others.

PARAMETERS EXAMINED
- Observation of offspring: All offspring on PND 0 were observed for viability and external appearance. The numbers of live and dead newborns were counted for each litter, which were totalled to serve as the number of offspring delivered. The delivery index and the incidence of live offspring with external anomalies were calculated per litter, and incidence of dams with offspring showing external anomalies were calculated for each group.
- Sex ratio of offspring: All offspring on PND 0 were sexed by external observation of the length between the anus and genitalia. The sex ratios were calculated for each group.
- Offspring were observed for general conditions once daily. Offspring found dead were necropsied on the day of discovery, and the whole bodies were fixed and preserved in 10 % neutral buffered formalin.
- Body weight of offspring was individually measured with an electronic balance (GX-2000, A & D Co., Ltd.) and recorded in increments of 0.1 g. Litter means of body weights were calculated by sex.
- The length between the anus and genitalia of individual offspring was measured and recorded. In addition, the individual anogenital distance (AGD) was divided by the cube root of its body weight. Litter means were calculated by sex.
- The number of nipples of individual male offspring was counted. Litter means were calculated.
- The serum T4 concentrations of offspring on PND 13 were measured by ELISA method (2 times measurements). Because there were no effects on the results of parental males or postnatal-day-13 offspring, the serum T4 concentrations of postnatal-day-4 offspring were not measured.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
- Timing: Males in the main study group and females in the satellite group [those for necropsy at the end of administration]; Day 29 (the day after Day 28). Females in the main study group; on Day 14 after parturition. The female with no evidence of parturition, on day 26 after successful copulation. The unsuccessfully mated female, 24 days after the end of the mating period (the day after Day 51). Males and females in the satellite group [those for necropsy at the end of recovery]; on Recovery Day 15 (the day after Recovery Day 14).
- Method: After observation of external appearance and blood collection from the abdominal aorta under anesthesia with isoflurane, animals were euthanised by exsanguination, and then all organs and tissues were macroscopically observed.
- For females in the main study group, the number of implantations was counted. The organs and tissues described below were fixed and preserved in 10 % neutral buffered formalin. The lung was fixed after infusion with the fixative through the trachea. The eyeballs and harderian glands were fixed and preserved in Davidson’s fixative and the testes and epididymides were fixed in Bouin’s solution and preserved in 70 % ethanol. For bilateral organs, both sides of them, in principle, were fixed and preserved.
- Organs: Brain (cerebrum, cerebellum, and pons), spinal cord, pituitary gland, thymus, thyroids, parathyroids, adrenals, spleen, heart, oesophagus, stomach, duodenum, jejunum, ileum (with Peyer’s patches), cecum, colon, rectum, liver, pancreas, submandibular glands, trachea, lungs and bronchus, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles (with coagulating glands), ovaries, uterus (horn and cervix), vagina, eyeballs and harderian glands, mammary gland (right abdomen), femur (with bone marrow, right), mesenteric lymph nodes, submandibular lymph node, skeletal muscle (biceps femoris, right), and sciatic nerve (right), and
gross lesions (with a border to the normal tissue).

ORGAN WEIGHTS
- Method: The organs shown below were weighed with an electronic balance (GR-200, A & D Co., Ltd.). Bilateral organs were measured together. The thyroids, pituitary gland, and seminal vesicles were weighed after fixation.
- Organs: Brain, heart, liver, kidneys, testes, epididymides, and seminal vesicles (with coagulating glands); (g). Pituitary glands, thyroids (with parathyroids), spleen, thymus, adrenals, prostate, ovaries, and uterus; (mg).
- Relative organ weight was calculated from the absolute organ weight and the body weight on the day of necropsy.

HISTOPATHOLOGY
- All organs and tissues of all animals in the control and high dose groups in the main study group were microscopically examined. There were no changes indicative of effects of the test material; therefore, microscopic examination was not performed for the animals in the low and middle dose groups in the main study group or satellite group. A pair of animals that was unsuccessfully mated, a male whose mate was nonpregnant, and the non-pregnant female were also examined.
- The gross regions (the kidneys of animal No. 10310, the incisors of animal No. 10304, and a subcutaneous mass of animal No. 50458) were also microscopically examined.
- Method: Embedded in paraffin wax, thin-sectioned, and stained with hematoxylin and eosin, and microscopically examined.

Postmortem examinations (offspring):

GROSS NECROPSY
- Animals were observed for external appearance (including the oral cavity), and those for blood collection were euthanised by decapitation, and the others were euthanized by an intraperitoneal injection of a 60 mg/mL pentobarbital sodium solution. All organs and tissues were macroscopically observed. The thyroids of 1 male and 1 female per litter were fixed and preserved in 10 % neutral buffered formalin.

Statistics:

- The analyses were performed using the computer system (MiTOX).
- For the main study group, group means and standard deviations were analysed by the Bartlett test for homogeneity of variances, followed by the one-way analysis of variance when variances were homogeneous (p ≥ 0.05), or by the Kruskal-Wallis test when variances were heterogeneous (p < 0.05). When a significant difference was detected (p < 0.1) by the one-way analysis of variance, Dunnett’s test was performed to detect statistical differences between the test material administered groups and their corresponding controls. When a significant difference was detected (p < 0.1) by the Kruskal-Wallis test, Steel’s test was performed to detect statistical differences between the test material-administered groups and their corresponding controls. In the offspring data, litter averages were used as the unit for statistical evaluation. The incidence of abnormal oestrous cycles, copulation index, fertility index, gestation index, sex ratio of live offspring, sex ratio of offspring, and incidence of dams with offspring showing external anomalies, and results of histopathology were analysed by Fisher's exact probability test.
- For the satellite group, group means and standard deviations were analysed for homogeneity of variances by the F test. When variances were homogeneous (p > 0.05), Student’s t-test was used to detect any statistically significant difference between the control and high-dose groups. When variances were heterogeneous (p < 0.05), the Welch test was used.
- Statistically significance level was less than 5 % for comparison with the control group.

Reproductive indices:

See 'Any other information on materials and methods incl. tables'

Offspring viability indices:

See 'Any other information on materials and methods incl. tables'

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In males in the control and 100 mg/kg groups, no abnormal findings were observed in any animal throughout the administration period. In the 300 mg/kg group, crushing of the upper incisors and reddish urine were observed in 1 animal each, on Days 2 to 29 and Days 8 to 14, respectively. These were considered incidental because there was no dose-dependence. In the 1000 mg/kg group, no abnormal findings were observed in any animal.
- In females in the control, and 100 and 300 mg/kg groups, no abnormal findings were observed in any animal throughout the administration period. In the 1000 mg/kg group, crushing of the upper incisors was observed in 1 animal on GDs 13 to 17. A subcutaneous mass in the right thoracic region was observed in 1 animal on LDs 7 to 14. These were considered incidental because of a single occurrence.
- In the satellite group, no abnormal findings were observed in any female rat throughout the administration period.
- No significant differences were observed in any parameter of detailed clinical observation in males or females in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were observed in any parameter of detailed clinical observation in the 1000 mg/kg group.

RECOVERY PERIOD
- No abnormal findings were observed in any males or females.
- No significant differences were observed in any parameter of detailed clinical observation in males or females in the 1000 mg/kg group compared with the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deaths occurred in any test group during the administration period. No deaths occurred in any test group during the recovery period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In males, no significant differences were noted in body weight or body weight gain during the administration period in any test material-administered group compared with that in the control group.
- In females, no significant differences were noted in body weight or body weight gain in the pre-mating and gestation periods in any test material-administered group compared with that in the control group. In the lactation period, body weight on LD 13 was significantly lower in the 100 mg/kg group. However, this was considered incidental because there was no dose-dependence or reduction of food consumption.
- In the female satellite group, no significant differences were noted in body weight or body weight gain in the 1000 mg/kg group.

RECOVERY PERIOD
- No significant differences were noted in body weight or body weight gain of males or females in the 1000 mg/kg group compared with that in the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION PERIOD
- In males, no significant differences were noted in food consumption during the administration period in any test material-administered group compared with that in the control group.
- In females, no significant difference was noted in food consumption in the premating and lactation periods in any test material-administered group compared with that in the control group. In the gestation period, food consumption on GD 7 was significantly lower in the 100 mg/kg group. However, this was considered incidental because there was no dose-dependence or weight loss.
- In the female satellite group, no significant differences were noted in food consumption in the 1000 mg/kg group.

RECOVERY PERIOD
- No significant differences were noted in food consumption of males or females in the 1000 mg/kg group compared with that in the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- No significant differences were noted in males or females in any haematology biomarker in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were noted in any haematology biomarker in the 1000 mg/kg group.

AT THE END OF RECOVERY PERIOD
- No significant differences were noted in males or females in any haematology biomarker in the 1000 mg/kg group compared with the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

AT THE END OF ADMINISTRATION PERIOD
- In males, no significant differences were noted in any biochemistry biomarker in any test material-administered group compared with the control group.
- In females, no significant differences were noted in any biochemistry biomarker in the 300 or 1000 mg/kg group compared with the control group. Total cholesterol (TCho) and albumin were significantly lower and α1-globulin (α1-G) was significantly higher in the 100 mg/kg group. However, these were considered incidental because there was no dose-dependence.
- In the female satellite group, no significant differences were noted in any biochemistry biomarker in the 1000 mg/kg group.

AT THE END OF RECOVERY PERIOD
- In males, γ-glutamyl transpeptidase (γ-GTP), total cholesterol (T-Cho), and triglyceride (TG) were significantly lower and urea nitrogen (UN) was significantly higher in the 1000 mg/kg group compared with those in the control group.
- In females, creatinine (Crea) was significantly lower and inorganic phosphorus (IP) was significantly higher in the 1000 mg/kg group. However, these were considered unrelated to the test material administration because of the following reasons:
1) these findings were not observed at the end of the administration period;
2) lower values of γ-GTP, T-Cho, TG, and Crea are clinically insignificant; and/or
3) individual values were within the range of historical control data in the test facility (0.0 to 0.8 IU/L for γ-GTP, 35 to 100 mg/dL for T-Cho, and 7.3 to 16.2 mg/dL for UN in males; 0.25 to 0.46 mg/dL for Crea and 3.4 to 7.0 mg/dL for IP in females

- Serum T4 concentration
AT THE END OF ADMINISTRATION PERIOD
No significant differences were noted in serum T4 concentrations of males in any test material-administered group compared with those in the control group.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

ADMINISTRATION WEEK 4
- No significant differences were noted in any urinalysis biomarker of males or females in any test material-administered group compared with the control group.
- In the female satellite group, no significant differences were noted in any urinalysis biomarker in the 1000 mg/kg group.

RECOVERY WEEK 2
- No significant differences were noted in any urinalysis biomarker of males or females in the 1000 mg/kg group compared with the control group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

ADMINISTRATION WEEK 4
- No significant differences were observed in any parameter of functional observations in males in any test material-administered group compared with the control group.
- In the female satellite group, motor activity was significantly higher (40–50 minutes) in the 1000 mg/kg group, which was considered an incidental change because no significant difference was noted in the total count (0–60 minutes) and their individual values were within the range of historical control data in the test facility (0 to 553 for 40–50 minutes; 313 to 4324 for 0–60 minutes).
- No significant differences were noted in the other parameters between the high dose and control groups.

LACTATION DAY 13
- No significant differences were observed in any parameter of functional observations in any test material-administered group compared with the control group.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the males necropsied on Day 29 in the 1000 mg/kg group, inflammatory cell infiltration of the liver (slight), focal inflammatory cell infiltration of the heart (slight), focal basophilic change of the renal tubule (mild), interstitial inflammation of the prostate (mild), and remnant of the ultimobranchial body in the thyroid (slight) were observed each in 1 or 2 animals. Corresponding to the macroscopic lesion in the 300 mg/kg group (dilatation of renal pelvis), dilatation of renal pelvis (mild) was observed.

In the females necropsied on Day 14 after parturition in the 1000 mg/kg group, findings including remnant of the ultimobranchial body in the thyroid (slight) and dysplasia of the retina in the eyeball (slight) were observed each in 1 animal. In the pairs of infertile animals in the 300 mg/kg group, focal basophilic change of the renal tubule (slight) was noted in 1 male and inflammatory cell infiltration of the urinary bladder (slight) and remnant of the epithelial cells in the thymus (slight) were noted in 1 female. Corresponding to the macroscopic lesion (crushing of the upper incisors), necrosis and fibrosis of the dental pulp (moderate) and inflammation of the gingiva (mild) were observed. None of these were considered to be causes of infertility.

In the satellite females necropsied on Day 29 in the 1000 mg/kg group, inflammatory cell infiltration of the liver (slight), inflammatory cell infiltration of the renal pelvic mucosa (slight), remnant of the epithelial cells in the thymus (slight), and remnant of the ultimobranchial body in the thyroid (slight) were observed each in 1 or 3 animals.

All abnormal findings observed were considered unrelated to the test material administration because of the following reasons:
1) there was no significant difference in their frequencies;
2) no enhancement of their grades compared with the control group; and/or
3) they have been known to be spontaneous

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the females necropsied on Day 14 after parturition in the 1000 mg/kg group: Corresponding to the macroscopic lesion in the 1000 mg/kg group (a subcutaneous mass), adenocarcinoma was observed in the thoracic skin.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significant differences were noted in the incidence of abnormal oestrous cycles, length of oestrous cycle, or number of oestrus between the test material-administered groups and control group.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences were noted in males or females in the copulation index, fertility index, or the number of days required for copulation between the test material administered groups and control group.

Details on results (P0)

Normal parturition was observed in all pregnant females. No significant differences were noted in the number of implantations, delivery index, gestation length, gestation index, numbers of offspring delivered, live newborns, and dead newborns, sex ratio of live offspring, or sex ratio of offspring in any test article-administered group compared with those in the control group. No external anomalies were observed in any live offspring on PND 0.

No changes considered to be attributed to the test material administration were observed in the incidence of abnormal oestrous cycles, length of oestrous cycle, number of oestrus, copulation index, fertility index, number of days required for copulation, number of implantations, delivery index, gestation length, gestation index, or numbers of offspring delivered, live new-borns, and dead new-borns of parental animals in any test material administered group.

Based on the above results, the NOAEL of the test material is considered 1000 mg/kg/day for reproductive performance of parental animals.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

In clinical observation of offspring, there were no abnormal findings in any test material-administered group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Death or missing (possibly cannibalised by maternal animals) was sporadically observed in all test groups including the control group from PNDs 0 to 13. These occurred at low incidence and no significant differences were noted in the viability indices on PNDs 0, 4, and 13 between the test material-administered groups and control group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in body weight of male or female offspring from PNDs 0 to 13 in any test material-administered group compared with that in the control group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant differences were noted in serum T4 concentrations of postnatal-day-13 offspring in any test material-administered group compared with those in the control group.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no significant differences in AGD and AGD per cube root of body weight ratio in any test material-administered group compared with those in the control group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in any male offspring.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormalities were observed in any offspring found dead during PNDs 0 to 13 in any test material-administered group. In the control group, 1 dead offspring showed acephaly and absent anus on PND 0. In postnatal-day-13 offspring, no abnormalities were observed in any test material administered group.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

For offspring, no changes considered to be attributed to the test material administration were observed in the sex ratio, viability index on PND 0, 4, or 13, general conditions, incidence of external anomalies, male and female body weight, anogenital distance, nipple retention of males, serum T4 concentrations of postnatal-day-13 offspring, or necropsy up to a dose of 1000 mg/kg/day.

Based on the above results, the NOAEL of the test material is considered 1000 mg/kg/day for development of offspring under the present study conditions.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose level tested.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the NOAEL for reproductive toxicity of the test material in adults was considered to be 1000 mg/kg/day. No adverse effects related to test material administration were observed. As such, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity.

Executive summary:

The reproductive toxicity of the test material was investigated in accordance with the standardised guideline OECD 422, under GLP conditions. A combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening test in rats was conducted to investigate the potential effects of repeated administration of the test material on the general toxicity, reproductive performance, and development of the offspring. Each group consisted of 12 male and 12 female Crl:CD(SD) rats, which were orally administered the test material at doses of 0 (control: olive oil), 100, 300, and 1000 mg/kg/day to males for totally 28 days from 14 days before the start of mating, or to females before and during the mating and gestation periods and up to lactation day 13 (for 55 days at most). For the 0 (control) and 1000 mg/kg groups, 5 males per group were selected to investigate the recovery for 14 days after administration; and 10 females per group were separately set (non-mating group), and 5 of them were necropsied at the end of 28-day administration, and the other 5 were investigated for recovery for 14 days.

No adverse effects related to the test material administration were observed in general conditions, detailed clinical observations, functional observations, body weight, body weight gain, food consumption, urinalysis, hematology, biochemistry including serum T4 concentrations of males, necropsy, organ weight, or histopathology in male and female animals up to a dose of 1000 mg/kg/day.  Therefore, the no-observed-adverse-effect level (NOAEL) of the test material for repeated dose toxicity is considered 1000 mg/kg/day in both sexes under the present study conditions.

No adverse effects related to the test material administration were observed; in the reproductive performance of parental animals including estrous cyclicity, and copulation, gestation, fertility, and delivery indices; and in the examination of their offspring including sex ratio, viability indices, general conditions, morphology, body weight, anogenital distance, nipple retention of males, serum T4 concentrations of postnatal-day-13 offspring, and necropsy up to a dose of 1000 mg/kg/day. Therefore, the NOAEL of the test material is considered 1000 mg/kg/day for reproductive performance of parental animals and for development of offspring under the present study conditions.