Registration Dossier

Administrative data

Description of key information

The NOAEL for systemic toxicity was considered to be 800 mg/kg/day for animals of both sexes.

Key value for chemical safety assessment

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:
30 August 2017 - 11 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control database. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ORIENTBIO INC., Republic of Korea
- Age at study initiation: male: 9 weeks; female: 9 weeks
- Weight at study initiation: male: 301–331 g; female: 198-237 g
- Fasting period before study: no
- Housing: Animals per cage: 1-2 (during the quarantine-acclimation period), 1 (during the dosing period), 1 male and 1 female (during the mating period), 1 female and neonates (during the lactation period); Stainless wire mesh cages, 260W×350D×210H (mm) and Polycarbonate cage 260W×420D×180H (mm)
- Diet: ad libitum, pelleted rodent chow (Teklad Certified Irradiated Global 18 % Protein Rodent Diet 2918C)
- Water: ad libitum, public tap water, filtered and irradiated by ultraviolet light
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the manufacturer, Envigo RMS, Inc. The results of feed analysis met the allowable standard of this facility.
Public tap water was filtered and irradiated by ultraviolet light. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year according to the Regulation of Quality Criteria for Potable Water and Test. The results of wateranalysis met the allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 26.7
- Humidity (%): 40.8 - 69.6
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The oral route was chosen to assess the toxicity by oral exposure of the test substance.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed using electronic balances, placed in a container, mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations. The dosing formulations were stored in a refrigerator (4.8–7.1 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: based on preliminary solubility test
- Concentration in vehicle: 0.5 and 500 mg/mL
- Amount of vehicle: 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations was conducted using a Gas Chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle layer of each dosing formulation prior to dosing and analyzed for verification of dose level concentration.
Duration of treatment / exposure:
Males of the main group were dosed for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 22 days of post-mating).
Males and females of the recovery groups were dosed for 49 days.
Females of the main group were dosed for 2 weeks prior to mating until Postpartum Day 13. Females showing no evidence of parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
once daily
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 12
Recovery groups: 6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 2-week repeated oral dose range finding study
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
No positive control was conducted.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: general condition, clinical signs, moribundity, mortality and females also signs of abortion and premature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing, once weekly for the dosing and recovery periods
- Detailed clinical observations included: skin, fur, eyes, mucous membranes, occurrence of secretion and excretion, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern etc.), changes in gait, posture and response to handling, and the presence of clonic or tonic movements, stereotypy (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: males of main group and males/females of recovery group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period and recovery period, the day prior to necropsy and on the day of necropsy (fasted body weights); females of main group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was recorded, individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 h
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, 18 h
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but free access to drinking water
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: few days before necropsy
- Dose groups that were examined: 6 males and 6 females were randomly selected from the main groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory reflex, corneal reflex, pupillary reflex, grip strength test, motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: pups: on PND 4 and 13; adults: at termination
- How many animals: at least two pups per litter (if available above culling target), all adult males and dams of the main group
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (Total T4) [ug/dL] and Thyroid stimulating hormone (TSH) [μUI/mL], Method: Chemiluminescent competitive immunoassay
Sacrifice and pathology:
Animals that died during the study period:
Necropsies were conducted on all animals found dead as soon as possible. When not feasible, they were stored under refrigeration until necropsy. Gross pathology findings were recorded and tissue samples were collected for histopathology.

GROSS PATHOLOGY: Yes. All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. Complete gross postmortem examinations were conducted on all animals including the external surfaces and internal organs.

Organ weights:
The organs listed in Table 4 were weighed from all males and females in the main groups and recovery groups.

HISTOPATHOLOGY: Yes, organs listed in Table 5 were prepared for histopathology for all animals of the main groups and all animals of the recovery groups. The testis and eyes with optic nerve will be fixed in Davidson’s fixative. All other tissues will be preserved in 10% neutral buffered formalin. The thyroids from all male and female pups per litter, sacrificed on PND 13 or shortly thereafter, will be preserved in 10% neutral buffered formalin.
Statistics:
The statistical analysis of this study were conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.).
For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights and birth and survival rates of pups, litter size, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters and organ weights, grip strength, motor activity, Bartlett’s test will be conducted to analyze for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test will be applied on homogeneous data, then, if significant (significance level: 0.05), Dunnett’s t-test will be applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test will be applied on heterogeneous data, then, if significant (significance level: 0.05), Steel’s test will be applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The data of sensory function, mating index, fertility index and other data associated with gestation will be analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery groups, Folded-F test will be employed to analyze for homogeneity of variance (significance level: 0.05, two-tailed). Student t-test will be applied for homogeneous data, but if overruled, Aspin-Welch t-test will be applied (significance levels: 0.05 and 0.01, two-tailed).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dead animals:
In the main group, one female in the 0 mg/kg/day group and 3 females in the 800 mg/kg/day group died. One dam in the 0 mg/kg/day group was found dead on PPD 1 after showing dystocia and delayed delivery. The dystocia observed at 0 mg/kg/day was considered to be incidental. Three pregnant females in the 800 mg/kg/day group were found dead before or during parturition. Salivation was observed in these three animals at 800 mg/kg/day. In the recovery group, one female in the 0 mg/kg/day group was found dead on Day 60 without any abnormal clinical signs. Since there were no deaths at 800 mg/kg/day in the recovery group, the death was considered as test substance-related reproductive toxicity.

Surviving animals:
All males of the main and recovery groups survived the duration of the study. In the main group, salivation was observed in four males and seven females at 800 mg/kg/day. In the recovery group, salivation was observed in five males and six females at 800 mg/kg/day. However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics. Mass in the chest was observed in one female at 50 mg/kg/day. The mass was microscopically identified as mammary gland adenocarcinoma. It was considered to be occurred spontaneously since it showed low incidence and no dose dependency. At 800 mg/kg/day, soft stool was temporarily observed in one male and one female in the main group and one male in the recovery group. It was considered to be little toxicological meaning or an individual difference since it showed low incidence. Stillbirth was observed in one female at 800 mg/kg/day. It was considered to be a test substance-related adverse effect.

No clinical signs were observed in males and females of the main and recovery groups in the detailed examinations conducted once a week.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the main group, one female in the 0 mg/kg/day group (PPD 1) and 3 females in the 800 mg/kg/day group (GD 21, GD 22, GD 23) died. In the recovery group, one female in the 0 mg/kg/day group (Day 60) was found dead. Since there were no deaths at 800 mg/kg/day in the recovery group, the death was considered as test substance-related reproductive toxicity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the main group, significant low values in body weight were noted in females at 800 mg/kg/day on GD 14 and GD 20 compared to the controls. It was considered to be a test substance-related reproductive toxicity since there was no significant adverse effect on the body weight at 800 mg/kg/day in the recovery group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant difference in food consumption was noted in males and females of the main and the recovery groups when compared to 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):
No effect was observed in any animal in the main and recovery groups. Other statistical significances were considered not to be test substance-related changes because they were small differences and the values were within the range of historical reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effect was observed in any animal in the main and recovery groups. There was an exception of high BUN and Crea values. It was considered to be incidental since it showed low incidence (only one female) and no microscopic lesion in the kidney of the female. Other statistical significances were considered not to be test substance-related changes because they were small differences and/or the values were within the range of historical reference data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the main and recovery groups, no effect was noted in urinalysis in males of any dosing group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on the auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main and recovery groups when compared to the control group. In the main group, there was no test substance-related effect in the grip strength test when compared to the control group. In the recovery groups, a statistically significant increase in the hindlimb grip strength was noted in females at 800 mg/kg/day. However, it had little toxicological significance because it was a small difference. In the main and recovery groups, there was some statistical significance in the spontaneous motor activity. However, it had little toxicological relevance because it showed small difference, temporary change or no dose-dependency.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A significant decrease in T4 was noted in males at 800 mg/kg/day. However, it was considered to have little toxicological meaning since there was no abnormal microscopic finding in the thymus of males.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The relative organ weights of some organs (the brain, heart and kidney) were statistically increased in males at 800 mg/kg/day. However, these changes were not considered to be test substance-related adverse effects since they were related to low fasted body weight at 800 mg/kg/day. Other statistical significances in the absolute and/or relative organ weights were considered not to be test substance-related effects because they were small differences and/or the values were within the range of historical reference data.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dead animals
Three dams were found dead at 800 mg/kg/day just before or during parturition. Among the three dams, only one dam showed small thymus and spleen. There were no abnormal macroscopic findings in the other dams. In addition, two females the 0 mg/kg/day group were found
dead. One dam died after dystocia and the other one female died during the recovery period. Macroscopic examination revealed multiple black foci in the glandular stomach and small thymus and spleen in one dam. However, no abnormal macroscopic findings were observed in the female of the recovery group.

Surviving animals (Terminal sacrifice):
Four dams whose pups were all dead showed small thymus (4/4) and spleen (2/4) at 800 mg/kg/day. Small thymus was also observed in two dams at 200 mg/kg/day. However, dams whose pups were survived at 800 mg/kg/day did not show any macroscopic changes. One non-delivered female in the 0 mg/kg/day group was revealed as non-pregnant animals, and there were no abnormal findings noted. A subcutaneous mass in the chest region was observed in one dam at 50 mg/kg/day. All other macroscopic findings showed a lack of dose-dependent relationship; were found at an isolated frequency, and were considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Dead animals:
There were no microscopic findings related to the test substance. However, it was considered that the test substance affected the deaths at 800 mg/kg/day since only dams died at 800 mg/kg/day before or during parturition. Slight to severe atrophy of the lymphoid organs (thymus and spleen) was noted in 2 dead dams at 800 mg/kg/day, and slight/moderate adrenocortical hypertrophy and minimal to moderate serous atrophy of the bone marrow (femur and sternum) were noted in 3 dead dams at 800 mg/kg/day. These findings were considered to be stress-related secondarily to exacerbated/moribund condition. Moderate suppurative inflammation in the spleen was observed in one dead dam at 800 mg/kg/day. However, this finding was deemed to be incidental, and not to be primary cause of death based on the severity of the finding. In addition, acute centrilobular necrosis in the liver was noted in one dead dam at 800 mg/kg/day. This finding was deemed to be not associated with the test substance since the finding was of isolated frequency and could be occurred as ischemicevent during agonal phase. In one dead dam at 0 mg/kg/day, there were severe atrophy of the lymphoid organs (thymus and spleen), slight adrenocortical hypertrophy and minimal erosion in the glandular stomach. This dam was found dead after dystocia, thus it was considered that dystocia contributed to those stress-related findings and deaths. Another dead female which died during the recovery period did not show any abnormal findings, and the cause of death could be not determined.

Surviving animals (Terminal sacrifice):
Microscopic examination did not reveal any target organ in either terminal or recovery groups. Severe thymic atrophy was noted in 4 dams at 800 mg/kg/day, and moderate or severe atrophy of white pulp in the spleen was found in 2 dams at 800 mg/kg/day. While the atrophic changes of the lymphoid organs were consistently observed in dams whose pups were all dead or in a dam on stillbirth, those were not seen in other dams at 800 mg/kg/day. Based on the distribution of the incidence, these findings were considered to be secondary to poor condition/stress associated with the test substance. Minimal/slight thymic atrophy was noted in 2 dams at 200 mg/kg/day. However, the cortex of the thymus in pregnant rat can be slightly atrophied, and the dams showed normal condition and normal lymphocyte relative count (referring to biochemical data). Therefore, it was considered to be not of toxicological significance.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Dead animals:
There were no microscopic findings related to the test substance.

Surviving animals (Terminal sacrifice):
Microscopic examination did not reveal any target organ in either terminal or recovery groups. The subcutaneous mass observed in one dam at 50 mg/kg/day corresponded to mammary gland adenocarcinoma. The tumors was considered to be not related to the test substance since the mammary tumor was found at an isolated frequency at 50 mg/kg/day and can occur spontaneously in female rats of this breed and age.
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effects observed were considered as test substance-related reproductive toxicity.
Key result
Critical effects observed:
no
Conclusions:
The NOAEL for systemic toxicity was considered to be 800 mg/kg/day for animals of both sexes.
Executive summary:

A combined repeated oral dose toxicity study with the reproduction/developmental toxicity screening test was performed to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic toxicity and reproduction/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential of the test substance when administered orally to male and female rats at dose levels of 0 (control), 50, 200 and 800 mg/kg/day. All males of the main and recovery groups survived the duration of the study. One dam in the main group and one female in the recovery group were found dead at 0 mg/kg/day. Three pregnant females of the main group were found dead at 800 mg/kg/day before or during parturition. Four dams whose pups were all dead were observed at 800 mg/kg/day. Stillbirth was observed in one female at 800 mg/kg/day. Low value in the body weight was noted in pregnant females at 800 mg/kg/day in the main groups. Atrophy of the lymphoid organs, adrenocortical hypertrophy and/or serous atrophy of the bone marrow were noted in three dead dams. These findings were considered to be stress-related secondarily to exacerbated/moribund condition. Thymic atrophy and/or atrophy of white pulp in the spleen were observed in dams whose pups were all dead at 800 mg/kg/day. Therefore, the NOAEL for systemic toxicity was considered to be 800 mg/kg/day for animals of both sexes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
800 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was conducted according to guideline and GLP.

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

Additional information

A combined repeated oral dose toxicity study with the reproduction/developmental toxicity screening test was performed to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic toxicity and reproduction/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential of the test substance when administered orally to male and female rats at dose levels of 0 (control), 50, 200 and 800 mg/kg/day. All males of the main and recovery groups survived the duration of the study. One dam in the main group and one female in the recovery group were found dead at 0 mg/kg/day. Three pregnant females of the main group were found dead at 800 mg/kg/day before or during parturition. Four dams whose pups were all dead were observed at 800 mg/kg/day. Stillbirth was observed in one female at 800 mg/kg/day. Low value in the body weight was noted in pregnant females at 800 mg/kg/day in the main groups. Atrophy of the lymphoid organs, adrenocortical hypertrophy and/or serous atrophy of the bone marrow were noted in three dead dams. These findings were considered to be stress-related secondarily to exacerbated/moribund condition. Thymic atrophy and/or atrophy of white pulp in the spleen were observed in dams whose pups were all dead at 800 mg/kg/day. Therefore, the NOAEL for systemic toxicity was considered to be 800 mg/kg/day for animals of both sexes.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776