4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

Endpoint summary

Description of key information

Short-term repeated dose toxicity: oral

The test item was repeatedly administered by oral gavage at 0 (control group), 110, 330 and 1000 mg/kg to male and female Crl:CD(SD) rats for 28 days to assess the toxicological effects of test item and its reversibility by observing functional and morphological changes based on OECD 407.

As there was foam cells noted in the ileum, mesenteric lymph node and lung of males or females receiving 110 mg/kg or more, NOAEL could not be determined in this study. However, as the number of occurrence and the degree of the change decreased almost in proportion to lower dose of test item, the dose of 110 mg/kg was considered proximal to LOAEL.

Short-term repeated dose toxicity: Dermal and inhalation

Waived as s a reliable oral toxicity study is available.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 October 2016 and 29 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Testing Methods Concerning New Chemical Substances (Notification No. 0331-7, PFSB, MHLW; No. 5 of March 29, 2011, MIB, METI; No. 110331009, EPB, MOE; dated March 31, 2011)

Version / remarks:

30 MARCH 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name (abbreviation): THEMIS
CAS number: 159034-96-5
Lot number: 160823
Purity : 100.0%
Appearance at ambient temperature: Pale pink powder
Amount received: 538.75 g
Supplier: Ajinomoto Fine-Techno Co., Inc.
Storage conditions and area : Room temperature (actual temperature: l 7.8°C to 22.3°C; permissible range: 1°C to 30°C), shielded from light, in a tight container, Test Substance Storage Room A046

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Rationale for selection
This strain is widely used in toxicity studies using rodents, and there is abundant historical data and a large number of animals are available

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier (Breeding facility): Charles River Laboratories Japan, Inc. (Hino Breeding Center)
Age: At receipt: 5-week-old; At the start of administration: 6-week-old
Body weight range at receipt: Males: 116.8 to 134.7 g (permissible range: 100 to 150 g); Female: 89.3 to 110.7 g (permissible range: 70 to 120 g)

Quarantine and acclimation
(Upon animal receipt, species, strain, age, number and sex were confirmed and observation for clinical signs and external appearance and body weight measurement were performed. A quarantine period was set for 5 days and an acclimation period was set for 7 days (between animal receipt and on the day before administration). During these periods, all animals were observed once daily and checked for body weight gain from the animal receipt to the end of quarantine. In addition, detailed clinical observations were performed once before grouping. According to these results, abnormal finding was observed in the eye of 1 male (No. 1029).No abnormality was noted in the other animals.

Grouping
All animals except for 1 male (No. 1029) were used for grouping based on the results of the observation for clinical signs, detailed clinical observations and body weight measurements during the quarantine and acclimation periods. Animals were assigned to groups by the stratified randomization on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weights± 20% (calculated for each sex) were used for this study. Body weight range is shown below.
Males: 190.7 to 214.9 g (permissible range: 162.3 to 243.5 g)
Female: 130.4 to 154.5 g (permissible range: 114.5 to 171.7 g)

Identification of animals
1) Before grouping
Upon receipt, animals were identified by marking the ID No. (the last one or two figures of quarantine animal number) on the tail with a black oil-based felt tip pen, and a label (indicating the study number for computer system, quarantine animal number and sex was attached to the front of each cage. The quarantine animal numbers are shown below.
Males: 1001 to 1032
Females: 2001 to 2032

2) After grouping
A microchip was subcutaneously implanted into the back of each animal and they were identified using a microchip reader (DAS-6008, Bio Medic Data Systems, Inc.). In addition, a label indicating the study number, animal number, dose level and sex was attached to the front of each cage.

Handling of remaining animals
The remaining animals were euthanized by exsanguination under sodium pentobarbital anesthesia after the initiation of administration.

Animal management
Animal room
During the quarantine period: A212; After the quarantine period: A203

Environmental conditions
Room temperature
Actual temperature: 22.5°C to 24.2°C (permissible range: 20.0°C to 26.0°C)

Relative humidity
Actual humidity: 46.8% to 58.5% (permissible range: 35.0% to 75.0%)

Ventilation
10 to 20 times per hour
Lighting period
12 hours per day (7:00 to 19:00)

Animal accommodation
Cages
Hanging type stainless wire mesh cages (W x D x H: 226 x 346 x 198 mm)
Feeders
Made of stainless
Cage racks
Made of stainless, with an automatic water supply system
Sanitary trays under cages
Made of aluminum
Number of animals per cage
Before grouping: 2 animals/cage; After grouping: 1 animal/cage
Frequency of housing equipment exchange
(1)Cages and feeders
Once at the grouping and once within two weeks after the start of administration
(2)Cage racks
Once at the grouping and once within two weeks after the start of administration
(3) Sanitary trays under cages
Once at the grouping and once a week after the start of administration

Diet
Description
Radiation sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd.)

Feeding method
Diet was given ad libitum except during fresh urine collection and measurement of motor activity. Animals were fasted from the evening before scheduled necropsy for about 21 to 24 hours.

Analysis
Data for a lot (lot No. 160705) of the diet was obtained from Oriental Yeast Co., Ltd., and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.

Water
Description
Well water admixed with NaClO (free residual chlorine level: about 2 ppm)
Water supply method
Water was given ad libitum through an automatic water supply system except during measurement of motor activity. Water bottles were used during the collecting of urine sample.
Analysis
Water was analyzed twice a year at Nichigo Kyushu Co., Ltd. The results were confirmed to be within the acceptable limits established by the test facility.

Environmental enrichment
A resting board (made of stainless) was attached to the cage and two types of nesting material (Paper Clean, Japan SLC, Inc. and Diamond Twists, ENVIGO Co., Ltd. [former name: Harlan Laboratories Inc.] were put into the cage for improvement of animal welfare. The resting board was exchanged concurrently with the cages.

Route of administration:

oral: gavage

Details on route of administration:

Oral (by gavage)
The test substance was administered orally using a disposable syringe attached to a gastric tube. Dosing formulations for the test substance groups were stirred with a magnetic stirrer during administration.

Vehicle:

methylcellulose

Remarks:

0.5 w/v% methylcellulose (MC) solution

Details on oral exposure:

Frequency and period of administration
Once daily for 28 days, between 9:01 and 11:39 (permissible range: 9:00 to 15:00)

Rationale for administration conditions
In accordance with the guideline applied in this study

Dose level and its rationale
Dose levels were set at 110, 330 and 1OOO mg/kg.
As a result of a study entitled "A 7-Day Repeated Dose Oral Toxicity Study ofTHEMIS in Rats (Study No. P160404)" (dose levels: 0 [0.5 w/v% MC solution], 110, 330 and 1OOO mg/kg/day; 5 animals/sex/group) at the test facility, no toxicological significant change was noted. Accordingly, the high dose was set at 1OOO mg/kg, which is the maximum dose level in accordance with the guideline. Lower doses were set at 330 and 110 mg/kg. A control group (0 mg/kg group) dosed with the vehicle (0.5 w/v% MC solution) alone was also established.

Dose volume
10 mL/kg
Individual volume was calculated on the basis of the most recently measured body weights.

Preparation of dosing formulations
Preparation method and storage conditions
Dosing formulations were prepared once within 12 days based on the results of a study entitled "Analytical Method Validation for the Determination, and Homogeneity and Stability Test ofTHEMIS in 0.5 w/v% methylcellulose solution (Study No. P160402)" conducted at the test facility. The dosing formulations after preparation were divided into glass vials for each dosing day and stored in a cold place (actual temperature: 3.6°C to 6.2°C, permissible range: 1°C to 15°C, in a medical refrigerator in Dosing Formulation Storage Room A032) for which they were confirmed to remain stable.

Control dosing formulation
The required amount of the vehicle was put into clear glass vials.

(11, 33 and 100 mg/mL dosing formulations)
(1) A prescribed amount of the test substance was weighed and ground in an agate mortar.
(2) The vehicle was gradually added and mixed with a pestle. The suspension in the mortar was transferred to a measuring cylinder.
(3) The mortar and pestle were washed with the vehicle and the washing was transferred to the measuring cylinder.
(4) The final volume was adjusted by adding a proper quantity of the vehicle to required concentrations of 11, 33 and 100 mg/mL. The dosing formulations after preparation were mixed several times by end-over-end rotation and taken into brown glass vials.

Identification of dosing formulations
Vials containing the dosing formulations were Iabeled with the study number, dosing article name, dose level, test substance concentration, preparation date, name of the person preparing the dosing formulation, storage conditions and expiration period.

Handling of remaining dosing formulations
Remaining dosing formulations were discarded after the completion of administration on each day.

Homogeneity and stability confirmation
Test substance formulations at 5 and 100 mg/mL were confirmed to be stable and homogenous after storage for 13 days in a cold place and in a brown glass vial followed by 24 hours at room temperature, according to Analytical Method Validation for the Determination, and Homogeneity and Stability Test (Study No. P160402) conducted at the test facility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of the test substance concentration in the dosing formulation
At the first preparation, 40 mL each of the analytical samples was taken from one point of each layer (upper, middle and lower layers) of the whole dosing formulation at each concentration. Forty mL of the vehicle was taken for calculation of the test substance concentration and MC content in the vehicle was confirmed. Test substance concentration was analyzed according to the method validated in Analytical Method Validation for the Determination, and Homogeneity and Stability Test (Study No. P160402) conducted at the test facility.

Criteria
The relative standard deviation (RSD) of analytical values (concentration) in each layer should not be more than 10%, and measured concentration (mean) should be within 100±10% as the ratio to the nominal concentration.

Results
Results are shown in the following table.
Nominal concentration (m!!/mL) Measured concentration (Mean) (mg/mL) Ratio to the nominal concentration (%) RSD (%) Judgment
11 11.0 100.0 0.5 Passed
33 35.0 106.1 1.2 Passed
100 99.2 99.2 0.9 Passed

Confirmation of amount of MC in the vehicle (n=3)
(1) An aluminum dish was left standing at room temperature for 30 minutes and more and weighed (tare weight [AJ, g).
(2) Five mL of vehicle was accurately pipetted and transferred to the dish. The pipette was washed with a small amount of purified water and the washing was transferred to the dish.
(3) The dishes were dried in a vacuum drying oven at 100°C for 3 hours.
(4) The dishes were confirmed to be dried completely.
(5) The dishes were taken out from the oven and then left standing for 30 minutes in a glove box, in which the air was replaced with nitrogen gas.
(6) The dishes were weighed (dish+ remaining portion on the dish, dried weight [BJ, g).
(7) The amount of MC in the vehicle was calculated by the following equation.

Amount of MC in the vehicle (mean) [VJ, g = I([BJ-[A])/3

Measurement of test substance concentration in the dosing formulation (n=1 of upper, middle and lower layers)
(1) An aluminum dish was left standing at room temperature for 30 minutes and more and weighed (tare weight [CJ, g).
(2) Each 1 mL of the 33 and 100 mg/mL dosing formulations and 5 mL of the 11 mg/mL dosing formulation were accurately pipetted with stirring and transferred to the dish. The pipette was washed with a small amount of purified water and the washing was transferred to the dish.
(3) The dishes were dried in a vacuum drying oven at 100°C for 3 hours.
(4) The dishes were confirmed to be dried completely.
(5) The dishes were taken out from the oven and left standing for 30 minutes in a glove box, in which the air was replaced with nitrogen gas.
(6) The dishes were weighed (dish+ remaining portion on the dish, dried weight [DJ, g).
(7) The test substance concentration in the dosing formulation was calculated by the following equation.

Test substance concentration in the dosing formulation (mg/mL) = ([DJ - [C] - [VJ /5 x (pipetted volume (mL)) x 1000 I pipetted volume (mL)

Duration of treatment / exposure:

28 Days
Recovery period
Five males and 5 females each in the control and high dose groups were subjected to a recovery period of 14 days after the end of the dosing period.

Frequency of treatment:

Once daily

Dose / conc.:

110 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Low and Middle Dose: 5 Males, 5 Females
Control and High Dose: 10 Males, 10 Females

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to assess the toxic effects of test item and its reversibility by observing functional and morphological changes after a 28-day repeated oral administration of test item to rats.

The test item was repeatedly administered by oral gavage at 0 (control group), 110, 330 and 1000 mg/kg to male and female Crl:CD(SD) rats for 28 days to assess the toxicological effects of test item and its reversibility by observing functional and morphological changes. The control group was given the vehicle (0.5 w/v% methylcellulose solution) alone. Each group consisted of 5 males and 5 females. A 14-day recovery period (5 animals/sex/group) was set for the control and 1000 mg/kg groups.

Dose level and its rationale
As a result of a study entitled “A 7-Day Repeated Dose Oral Toxicity Study of THEMIS in Rats (Study No. P160404)” (dose levels: 0 [0.5 w/v% MC solution], 110, 330 and 1000 mg/kg/day; 5 animals/sex/group) at the test facility, no toxicological significant change was noted. Accordingly, the high dose was set at 1000 mg/kg, which is the maximum dose level in accordance with the guideline. Lower doses were set at 330 and 110 mg/kg. A control group (0 mg/kg group) dosed with the vehicle (0.5 w/v% MC solution) alone was also established.

Dose volume
10 mL/kg
Individual volume was calculated on the basis of the most recently measured body weights.

Observations and examinations performed and frequency:

Observations and measurements
The following parameters were examined. The first day of dosing was designated as Day 1, and Day 1 to 7 were designated as Week 1. Day 29 and later was designated as the recovery period.

Clinical observation
All animals were observed twice a day (before dosing and after dosing) during the dosing period and once a day in the other periods.

Functional observational battery
Examinations were conducted on a blind test basis and the animals and examination order were randomized except for motor activity measurement. No randomization was conducted for detailed clinical observations before the start of dosing. For all animals, the detailed clinical observations were performed once before the start of dosing (before grouping) and once a week in the afternoon during the dosing and recovery periods.
Functional tests and motor activity measurement were performed for all animals once in the afternoon in the final week (Week 4) of the dosing period. Detailed clinical observations, functional tests and motor activity measurement were performed following the clinical observation after dosing during the dosing period.
According to these results, the functional tests and motor activity measurement were not performed during the recovery period since no treatment-related change was noted during the dosing period.
Detailed clinical observations
(1) Hand held observation
Animals were removed from the cage for the observation by grasping their trunk gently from behind.
Parameters:
Reactivity to handling, trauma, color of skin, soiled fur, piloerection, secretion, salivation, lacrimation, exophthalmos, palpebral closure, color of conjunctiva and pupil size
(2) Observation on open field
The floor of an open field was wiped with a tightly squeezed wet cloth before examination. The cloth was washed every time of examination. Animals were placed in the center of the open field and observed quietly for one minute.
Parameters:
Arousal, posture/body position, gait, tremor, convulsion, respiration, stereotypy, bizarre behavior, urination, defecation and number of rearing

Functional tests
(1) Sensory reactivity to stimuli
Sensory reactivity to stimuli was observed on the open field.
Parameters:
Approach response, touch response, auditory response, righting reaction and tail pinch response

(2) Grip strength measurement
Grip strength was measured following sensory reactivity to stimuli. Grip strength was measured twice for both forelimbs and hindlimbs using a grip dynamometer (CPU gauge: Model 9502A, Aikoh Engineering Co., Ltd.), and a higher value was adopted.

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

Body weight measurement
All animals were weighed on Days 1, 8, 15, 22 and 28 during the dosing period and on Days 29, 36 and 42 during the recovery period. Measurement was performed before dosing during the dosing period. The final body weight was also measured on the day of scheduled necropsy.

Food consumption measurement
For all animals, gross weight of each feeder was weighed. The mean daily food consumption was calculated from Day 1 to 8, 8 to 15, 15 to 22 and 22 to 27 during the dosing period and from Day 29 to 36 and 36 to 41 during the recovery period. The mean daily food consumption for each period was expressed as the last day of each period.

Urinalysis
Urinalysis was conducted for 5 males and 5 females with the smallest animal numbers in the respective groups in the final week (Day 27) of the dosing period by the reagent strip method. Fresh urine samples were collected using metabolic cages after 8:00 a.m. (before dosing) under fasting conditions (water was given).
According to these results, no treatment-related changes were noted in any parameter. Therefore, urinary sediment and accumulated urine test during the dosing period and urinalysis during the recovery period were not performed.

Hematology
Blood sampling was conducted at the scheduled necropsy (Days 29 and 43). All animals were fasted for about 21 to 24 hours before blood sampling. The animals were anesthetized with intraperitoneal injection of sodium pentobarbital (30 mg/kg), and about 2.0 to 2.5 mL of blood was collected from the posterior vena cava. For examination of the coagulation system, 0.9 mL of blood was collected into a tube containing 0.1 mL of
3.8 w/v% trisodium citrate, and then plasma was obtained by centrifugation at 1870 x g for 15 minutes (set temperature: 4°C). For examination of the other items, the remaining blood was put into a blood container (SB-41, Sysmex Corporation) containing 2 mg ofEDTA-2K as an anticoagulant.

Blood chemistry
After blood sampling for the hematology at the scheduled necropsy on Days 29 and 43, 3 to 5 mL of blood was collected from the posterior vena cava under anesthesia for blood chemistry. The blood was left standing at room temperature for about 60 minutes, and then serum was obtained by centrifugation at 1870 x g for 10 minutes (set temperature: 4°C). The serum samples were stored frozen in a deep freezer (actual temperature: -81.8°C to -76.0°C, permissible range: -90°C to -65°C) until examination. The remaining samples were stored in a deep freezer and discarded by the completion of the study.

Storage of serum samples for hormone measurement
In case of serum hormone measurement, the remaining serum samples following the blood chemistry were taken, and they were frozen and stored in a deep freezer (permissible range: -90°C to -65°C). The serum samples were discarded since it was determined that measurement was not necessary based on the results from the various examinations in this study.

Sacrifice and pathology:

Pathological examination
Organs and tissues for examination
Organs/Tissues were collected and examined.
Necropsy
All animals were euthanized by exsanguination after blood sampling and all organs and tissues were immediately examined macroscopically.

Organ weight measurement
After necropsy, the organs were weighed (absolute weight), and the ratios of the organ weights to body weight (relative weight) were calculated on the basis of the body weight measured on the day of necropsy. Paired organs were weighed together.

Histopathology
After necropsy, the organs/tissues were fixed in 10 vol% phosphate buffered formalin solution; however, the testes and epididymides were fixed in Bouin's solution and the eyes were fixed in Davidson's solution, and they were preserved in 10 vol% phosphate buffered formalin solution. The organs/tissues of all males and all females in the control and 1000 mg/kg groups collected at the end of the dosing period were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and then examined by microscopy.
According to these results, changes suspected to be attributable to the test substance treatment were observed in the liver, duodenum, jejunum, ileum, spleen, mesenteric lymph node and lung of both sexes and heart of males in the 1000 mg/kg group.
Therefore, the additional examination was performed for the organs of males or femals in the other groups collected at the end of the dosing period and those of all groups collected at the end of the recovery period.

Statistics:

Statistical analysis was performed with a computer system (Provantis®, Instem LSS Limited).
For numeral data, mean values and standard deviations were calculated in each group. Bartlett's test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett's multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel's multiple comparison test was performed to compare with the control group. For urinalysis, Steel's multiple comparison test was performed after the grades were converted into numeric values. Fisher Exact test was used for the histopathological findings. In all cases, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality was observed in any animal throughout the dosing or recovery period.

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was noted in males or females between the control and test substance groups throughout the dosing or recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant difference was noted in males or females between the control and test substance groups throughout the dosing or recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicological change suspected to be attributable to the test substance was noted.
At the end of the dosing period, the following changes were noted: high values of MCHC in females of the 330 and 1000 mg/kg groups and high values of reticulocyte count (and (ratio) in females of the 1000 mg/kg group. However, these were judged not to be toxicologically significant as the changes were within normal range of the historical data of the test facility and no related change was noted in any examination. Shortening of PT was noted in males of the 1000 mg/kg group.
However, this was judged not to be toxicologically significant as the change was opposite to the toxicity effects. As a change without dose-dependency and considered as not treatment-related, high values of MCH and low values of large unstained cell ratio and eosinophil count were noted in females of the 110 mg/kg group.
At the end of the recovery period, high values of MCH and basophil ratio were noted in females of the 1000 mg/kg group.However, these were judged not to betreatment-related as the changes were within normal range of the historical data of the test facility and were not noted at the end of the dosing period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, high values of ASAT and ALAT was noted in females of the 1000 mg/kg group.
At the end of the recovery period, the above-mentioned changes were not noted.

Besides, at the end of the dosing period, the following changes were noted: low values of total bile acid in males of the 330 and 1000 mg/kg groups and females of the 1000 mg/kg group, high values of urea nitrogen in males of the 1000 mg/kg group and high values of inorganic phosphorus in males of the 1000 mg/kg group and females of the 330 and 1000 mg/kg groups. However, these were judged not to be toxicologically significant as the changes were within normal or almost normal range of the historical data of the test facility and no related change was noted in any examination. At the end of the recovery period, high values of ASAT and low values of ALP were noted in males and females of the 1000 mg/kg group, respectively. However, these were judged not to be treatment-related as the changes were within normal range of the historical data of the test facility and were not noted at the end of the dosing period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

In the qualitative analysis, no significant difference was noted in males or females between the control and test substance groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Tests
(I) Sensory reactivity to stimuli
Reactivity to stimuli was comparable in males and females of all groups and no abnormality was observed.

(2) Grip strength
No significant difference was noted in males or females between the control and test substance groups.

Motor activity
No significant difference was noted in males or females between the control and test substance groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the dosing period, high values of absolute and relative liver weight were noted in females of the 1000 mg/kg group.
At the end of the recovery period, high values of relative liver weight were noted in females of the 1000 mg/kg group.

Besides, as a change without dose-dependency and considered as not treatment-related, high values of absolute and relative spleen weights and absolute kidneys weight were noted in males of the 330 mg/kg group at the end of the dosing period.
At the end of the recovery period, low values of absolute brain weight were noted in females of the 1000 mg/kg group. However, this was judged not to be treatment-related as the changes were within normal range of the historical data of the test facility and were not noted at the end of the dosing period.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic change was observed in any animal

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the dosing period, the following changes were observed.
In the liver, fine vacuolation of hepatocytes was observed in 1 female (grade: minimal) of the 330 mg/kg group and 5 males (minimal) and 5 females (minimal: 1 female, mild: 4 females) of the 1000 mg/kg group. Appearance of foam cell was observed in the sinusoid in 3 males (minimal) and 3 females (minimal) of the 1000 mg/kg group.
In the duodenum, appearance of foam cell was observed in the lamina propria in 5 males (minimal) and 5 females (minimal) of the 1000 mg/kg group.
In the jejunum, appearance of foam cell was observed in the lamina propria in 5 males (minimal) and 4 females (minimal) of the 1000 mg/kg group.
In the ileum, appearance of foam cell was observed in the lamina propria in 4 males (minimal) and 1 female (minimal) of the 110 mg/kg group, 4 males (minimal: 2 males, mild: 2 males) and 5 females (minimal) of the 330 mg/kg group and 5 males (mild) and 4 females (minimal: 1 female, mild: 3 females) of the 1000 mg/kg group.
In the spleen, appearance of foam cell was observed in 5 males (minimal) and 5 females (minimal) of the 1000 mg/kg group.
In the mesenteric lymph node, appearance of foam cell was observed in I male (minimal) and 2 females (minimal) of the 110 mg/kg group, 3 males (minimal) and I female (minimal) of the 330 mg/kg group and 5 males (minimal: 3 males, mild: 2 males) and 4 females (minimal) of the 1000 mg/kg group.
In the lung, appearance of foam cell was observed in the alveolus in 1 female (minimal) of the 110 mg/kg group, 4 males (minimal) and 1 female (minimal) of the 330 mg/kg group and 5 males (minimal) and 3 females (minimal) of the 1000 mg/kg group.

At the end of the recovery period, the following changes were observed.
In the liver, fine vacuolation of hepatocytes was observed in 4 females (minimal) of the 1000 mg/kg group. Appearance of foam cell was observed in the sinusoid in 2 males (minimal) of the 1000 mg/kg group.
In the duodenum, appearance of foam cell was observed in the lamina propria in 1 male (minimal) and 2 females (minimal) of the 1000 mg/kg group.
In the jejunum, appearance of foam cell was observed in the lamina propria in 5 males (minimal) and 4 females (minimal) of the 1000 mg/kg group.
In the ileum, appearance of foam cell was observed in the lamina propria in 4 males (minimal) and 3 females (minimal) of the 1000 mg/kg group.
In the spleen, appearance of foam cell was observed in 5 males (minimal) and 3 females (minimal) of the 1000 mg/kg group.
In the mesenteric lymph node, appearance of foam cell was observed in 4 males (minimal) and 5 females (minimal) of the 1000 mg/kg group.
In the lung, appearance of foam cell was observed in the alveolus in 4 males (minimal) and 3 females (minimal) of the 1000 mg/kg group.

Besides, inflammatory cell infiltration was observed in the heart in 4 males of the 1000 mg/kg group at the end of the dosing period and suspected to be attributable to the test substance. Therefore, an additional examination was performed. As the results, microscopic image of the heart in these animals was within the range of physiological variation and this change was judged to be toxicologically insignificant.
Other various histopathological changes were noted in both sexes of each group. However, these changes were judged not to be treatment-related since they are observed occasionally in normal rats and there was no clear dose-dependency in the incidence.

Key result

Dose descriptor:

LOAEL

Effect level:

110 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

According to the results, as there was foam cells noted in the ileum, mesenteric lymph node and lung of males or females receiving 110 mg/kg or more, NOAEL could not be determined in this study. However, as the number of occurrence and the degree of the change decreased almost in proportion to lower dose of test item, the dose of 110 mg/kg was considered proximal to LOAEL.

Executive summary:

The test item was repeatedly administered by oral gavage at 0 (control group), 110, 330 and 1000 mg/kg to male and female Crl:CD(SD) rats for 28 days to assess the toxicological effects of test item and its reversibility by observing functional and morphological changes based on OECD 407. The control group was given the vehicle (0.5 w/v% methylcellulose solution) alone. Each group consisted of 5 males and 5 females. A 14-day recovery period (5 animals/sex/group) was set for the control and 1000 mg/kg groups.

 

In the blood chemistry, high values of ASAT and ALAT were noted in females of the 1000 mg/kg group.

 

In the organ weight, high values of absolute and relative liver weight were noted in females of the 1000 mg/kg group.

In the histopathology, vacuolation of hepatocytes was observed in females of the 330 mg/kg group and both sexes of the 1000 mg/kg group. Appearance of foam cells was observed in the sinusoid of the liver in both sexes of the 1000 mg/kg group.

Moreover, appearance of foam cells was observed in the spleen of both sexes of the 1000 mg/kg group, in the lamina propria of the intestine (duodenum,jejunum and ileum) in both sexes of the ll0, 330 or 1000 mg/kg group, in the mesenteric lymph node in both sexes of the 110 mg/kg group and above and in the lung in males or females of the ll0, 330 and 1000 mg/kg group.

 

In the recovery test, high values of relative liver weight were noted in females of the 1000 mg/kg group. The above-mentioned histopathological changes almost remained in the 1000 mg/kg group. Meanwhile, the number of occurrence of these findings and their severity were less than those at the end of the dosing period.

No treatment-related or toxicologically significant change was observed in the clinical observation, functional observational battery, body weight, food consumption, urinalysis,. hematology or necropsy.

 

According to the above results, as there was foam cells noted in the ileum, mesenteric lymph node and lung of males or females receiving 110 mg/kg or more, NOAEL could not be determined in this study. However, as the number of occurrence and the degree of the change decreased almost in proportion to lower dose of THEMIS, the dose of 110 mg/kg was considered proximal to LOAEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

110 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 (reliable without restriction)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

28d repeated oral: LOAEL: 110 mg/kg

Significant effects: Foam cells noted in the ileum, mesenteric lymph node and lung of males or females receiving 110 mg/kg or more

Classification:

The effects observed are outside of the scope for STOT classification. In the study, as there was no cell damage observed in any organ or tissue, while there was a tendency toward recovery without any aggravation of general condition among animals, the effects of the test substance on the general condition were considered negligible. At this stage where uncertainty in the dose findings below 1000mg/kg/bw is identified, there is no target organ which could be classified according to CLP criteria. Therefore, this substance should not to be classified for STOT-RE according to Regulation (EC) 1272/2008 Table 3.9.1 and 3.9.2.