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Description of key information

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening with the test item (CAS 101 -97 -3) the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity was considered to be 200 mg/kg bw/day.

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:
2017-01-05 to 2017-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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 (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc. 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks (Males) and 9 weeks (Females)
- Weight at study initiation: 349.4-425.3 g at the initiation of dosing (Males), 231.8-282.9 g at the initiation of dosing (Females)
- Fasting period before study: none
- Housing: during acclimation, pre-treatment, treatment and post-mating in stainless-steel cage (1 or 2 animals), during mating one male and one female animal in stainless-steel cage, during gestation (1 female) and lactation (1 female with pups) in poly sulfone cages
- Diet: Lab Diet® #5053 PMI Nutrition International, USA, ad libitum
- Water: ad libitum filtered, ultraviolet light-irradiated municipal tap water
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
Microbial monitoring for diet was performed at KIT and a certificate of analysis for the diet was provided by the supplier

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of dose formulations were conducted according to a validated GC method (KIT Study No.: G316052) in the test site. Samples for dose formulation analysis were transferred at room temperature. Results of dose formulations were 107.4, 102.3 and 95.6% at each dose levels of 50, 200 and 800 mg/kg. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
Duration of treatment / exposure:
Dosing of the males has begun 14 days prior to mating and was continued till the day prior to sacrifice (at least 28 days). Dosing of the females has begun 14 days prior to mating and was continued till lactation day (LD) 13. Animals in recovery group were not mated and were assigned to a 2-weeks recovery period after the completion of administration.
Frequency of treatment:
once a day at approximately the same time
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
No. of animals per sex per dose:
vehicle control: 18
50 and 500 mg/kg bw dose groups: 12
800 mg/kg bw dose group: 18
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range finding study
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was observed once a week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled sacrifice
- Anaesthetic used for blood collection: not applicable
- Animals fasted: No
- How many animals: all scheduled sacrifice animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled sacrifice
- Anaesthetic used for blood collection: not applicable
- Animals fasted: No
- How many animals: all scheduled sacrifice animals
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before scheduled sacrifice
- Dose groups that were examined: 6 males and 6 females, selected from each group and in all animals of the recovery group
- Battery of functions tested: approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity

ORGAN WEIGHTS:
Organs weighed at terminal and recovery sacrifice: see table 3 in the field 'any other information on material and methods incl. tables'


THYROID HORMONE ANALYSIS:
Blood samples were taken from all adult animals at termination from the caudal vena cava. Thyroid hormone (T4 and TSH) concentrations in the serum samples of the males were determined.

ESTROUS CYCLE:
A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle during the treatment period until mating was examined.
In addition, vaginal smear of sacrificed females were taken at termination to examine the stage of the estrus cycle and allow correlation with histopathology of female reproductive organs.

MATING:
Mating confirmation was checked every morning during the mating period. Mating was confirmed with vaginal plug(s) and/or sperm in the vaginal smear. Day 0 of pregnancy was defined as the day of mating confirmation. Pregnancy was confirmed by implantation sites on the uterus at sacrifice or parturition.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Complete necropsy was performed under the direct supervision of a veterinary pathologist. After blood sampling, the animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
Mean values and standard deviations were calculated in the final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima System or Statistical Analysis Systems. Data was considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data was analyzed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were analyzed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and the significance of inter-group differences between the control and treated groups were assessed using Dunn’s Rank Sum Test.
For comparing control group and recovery group, the data was analyzed for homogeneity for variance using F-test. Homogeneous data was analyzed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and significant difference between control and recovery group was assessed using Dunn’s Rank Sum Test.
Data presented as frequencies was analyzed by χ2-test followed by the Fisher's exact test where necessary.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males at 200 and 800 mg/kg bw, salivation was observed in 12 and 18 animals, respectively. In females at 200 and 800 mg/kg bw, salivation was observed in 3 and 18 animals, respectively. It was considered test item-related but not toxicologically relevant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not show a dose-response relationship.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item-related decrease in body weight gain (76% and 69% of control, respectively) was observed in males and females at 800 mg/kg bw during the treatment days 1 to 50.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in food consumption were observed in both sexes during the study.
Statistically significant changes in food consumption was found for a few time periods during the study, but were scattered over time and sex and thus were not considered test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease of platelet counts (PLT, down to 80% of control) was observed in both sexes at 800 mg/kg bw. Also, decrease but not statistically significant of absolute and relative reticulocyte counts (RETA and RET%, 87% and 90% controls, respectively) in males and significantly decrease of absolute and relative reticulocyte counts (83% and 83% controls, respectively) in females were observed at 800 mg/kg bw. Increase but not statistically significant of absolute monocyte counts (MONA, 1.50-fold over control) and a significant increase of relative monocyte counts (MON%, 1.56-fold over control) were observed in males at 800 mg/kg bw. And significant decreases of absolute and relative eosinophil counts (EOSA and EOS%, 43% and 50% of controls, respectively) were observed in males at 800 mg/kg bw. These changes were not considered adverse as there were no histopathological correlates and reversed after recovery period.
Other statistically significant changes were not considered test item-related, because these changes were minimal, lacked a dose-relationship, were not correlated with microscopic changes and/or were observed only in the recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase of alanine aminotransferase (ALT, 1.59-fold over control) was observed in males at 800 mg/kg bw and significant increase of total bilirubin (TBIL, 1.41-fold over control) was observed in females at 800 mg/kg bw. These changes were not considered adverse as there were no histopathological correlates and reversed after recovery period.
Other statistically significant changes were not considered test item-related, because these changes were minimal, lacked a dose-relationship, were not correlated with microscopic changes and/or were observed only in the recovery group.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in relative liver weight (1.11-fold over control) in males and absolute and relative liver weights (up to 1.14-folds over controls) in females were observed at 800 mg/kgbw. Significantly decreased absolute and relative adrenal glands weights (down to 74% of controls) in males and decrease but not significant of absolute and relative adrenal glands weights (down to 84% of controls) in females were observed. Significantly decreased absolute prostate and seminal vesicles weight (with coagulation gland, 83% of control) in males and increased absolute and relative heart weights (up to 1.11-fold over controls) in females were observed at 800 mg/kg bw. These changes were not considered adverse as there were no histopathological correlates and reversed after the recovery period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings observed during this study were considered to be incidental or spontaneous and of a nature that is commonly seen in SD rats at low incidence or severity.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No test item-related changes in functional behavior examination were observed in both sexes during the study.
No test item-related changes in motor activity examination were observed in both sexes during the study.
Statistically significant changes in females at 50 mg/kg bw regarding motor activity were not considered test item-related since there was no dose-dependence and the effects were not observed in males.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic findings (e.g. slight dilatation or mineralisation of renal tubules) observed during this study were considered to be incidental or spontaneous since they were infrequent, of low severity and similarly distributed among control and test item-treated groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related changes in thyroid hormone (T4) were observed in adult males.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Conclusions:
The No-Observed-Adverse-Effect Level (NOAEL) for general toxicity was considered to be 200 mg/kg/day.
Executive summary:

The test item, was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 50, 200 and 800 mg/kg with a dose volume of 2 mL/kg in corn oil as vehicle. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in recovery group at 0 and 800 mg/kg (6 animals per sex per group) were also administered but not mated, and then assigned to 2 weeks of recovery period after the completion of administration. No deaths or moribund animals occurred in any group throughout the study. Three females were subjected to unscheduled sacrifice on GD 27 because of non-parturition. There were no test item-related changes in the macroscopic and microscopic of unscheduled sacrificed animals. No test item-related change was observed in food consumption, functional behavior examination, motor activity examination, macroscopic and microscopic findings. A significant decrease in body weight gain (76% and 69% of control, respectively) was observed in males and females at 800 mg/kg during the treatment days 1 to 50. Changes in hematology, clinical chemistry and organ weights were not considered adverse since they were not correlated with microscopic findings and reversed after recovery period.

Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity was considered to be 200 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline study

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

Mode of Action Analysis / Human Relevance Framework

Additional information

Key study:

The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 50, 200 and 800 mg/kg with a dose volume of 2 mL/kg in corn oil as vehicle. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in recovery group at 0 and 800 mg/kg (6 animals per sex per group) were also administered but not mated, and then assigned to 2 weeks of recovery period after the completion of administration. No deaths or moribund animals occurred in any group throughout the study. Three females were subjected to unscheduled sacrifice on GD 27 because of non-parturition. There were no test item-related changes in the macroscopic and microscopic of unscheduled sacrificed animals. No test item-related change was observed in food consumption, functional behavior examination, motor activity examination, macroscopic and microscopic findings. A significant decrease in body weight gain (76% and 69% of control, respectively) was observed in males and females at 800 mg/kg during the treatment days 1 to 50. Changes in hematology, clinical chemistry and organ weights were not considered adverse since they were not correlated with microscopic findings and reversed after recovery period.

Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity was considered to be 200 mg/kg/day.

Supporting Study:

This dose range-finding study was conducted to evaluate the potential effects of the test item when administered by oral gavage to Sprague-Dawley rats for 2 weeks at dose levels of 0, 60, 250 and 1000 mg/kg bw/d. No deaths or moribund animals occurred in any groups throughout the study. Test item-related salivation in both sexes at 1000 mg/kg bw/d and in males at 250 mg/kg bw/d was observed but it was not considered to be adverse. A decrease in body weight gain was observed in males at 1000 mg/kg bw/d and in females at 250 and 1000 mg/kg bw/d. A statistically significant increase in absolute and relative liver weight was observed in males at 250 and 1000 mg/kg bw/d, respectively. Additionally, a significant increase in relative liver weight was observed in females at 1000 mg/kg bw/d. Significant decreases in absolute and relative thymus weight were observed in males at 1000 mg/kg bw/d. There were no test item-related changes in body weight, food consumption, hematology, clinical chemistry and macroscopic findings in any of the treatment groups during the study. In conclusion, a decrease in body weight gain and an increase in relative liver weight in both sexes were observed at 1000 mg/kg bw/d. In addition, a decrease in thymus weight was observed in males at 1000 mg/kg bw/d. Based on the results of this dose range-finding study, 800 mg/kg bw/d is selected as the high dose that is anticipated to produce minimal toxicity, and 200 and 50 mg/kg bw/d are selected as the middle and low dose for a definitive combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening in Sprague-Dawley rats.

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. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.