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Description of key information

In a subacute oral toxicity study the NOAEL was found to be 1000 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:
2016-11-08 to 2017-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 10 weeks (Males) and 12 weeks (Females)
- Weight at study initiation: 316.5-401.5 g at the initiation of dosing (Males)
248.4-314.9 g at the initiation of dosing (Females)
- Housing: 2 or one animal during acclimation, pre-treatment and treatment, 1 male and 1 female during mating period, 1 mated female during gestation
- Diet: ad libitum, standard rat and mouse pellet diet (Lot No.: MAR 28 162, MAY 24 162 and JUN 29 163, Lab Diet® #5053 PMI Nutrition International, USA; irradiated by gamma-ray
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-20 times/hour
- 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. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.

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:
Dose formulation analyses were conducted by GC according to a validated method in the test site.
Duration of treatment / exposure:
The control item or dose formulations were administered for 7 days/week. Dosing of the males was started 14 days prior to mating and continued through the day prior to sacrifice (at least 50 days). Dosing of the females was started 14 days prior to mating and continued through lactation day (LD) 13.
Frequency of treatment:
once a day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
vehicle control and high dose groups: 18 males, 18 females
100 and 300 mg/kg bw/day dose groups: 12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose level was selected based on the results of a repeated dose 2-week dose range-finding study.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

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

FOOD CONSUMPTION
- Time schedule: once per week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology were taken from 6 males and 6 females of each group prior to terminal sacrifice and recovery sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 6 males and 6 females
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology were taken from 6 males and 6 females of each group prior to terminal sacrifice and recovery sacrifice
- Animals fasted: No
- How many animals: 6 males and 6 females
- Parameters checked in table No. 2 were examined.

Thyroid Hormone (T4) Analysis:
Blood samples (approximately 0.6 mL, if possible) for thyroid hormone (T4) analysis were taken on post natal day 4 from at least two culled pups (one male and 1 female, if possible) per litter, on lactation day 13 from all dams and at least two pups) (one male and 1 female, if possible) per litter; and at termination from all adult males.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Other examinations:
Thyroid hormone (T4) concentration in the serum samples (Pup samples from PND 13 and male samples at termination) was analyzed based on the ELISA method. T4 concentration analysis with the ELISA method was conducted according to the following criteria; above 0.980 of R2 in calibration curve, within ±20% of relative error (RE) and coefficient of variation (CV) in each sample.
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.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weight. The litter size was used as the covariate. Litter data was statistically evaluated using the statistical unit as a litter.
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 300 and 1000 mg/kg bw/day, salivation was observed in 10 and 18 animals, respectively. In females at 300 and 1000 mg/kg, salivation was observed in 4 and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
In males and females at 1000 mg/kg bw/day, subdued behavior was observed in 14 and 7 animals, respectively and prone position was observed in 6 and 13 animals, respectively. These clinical signs occurred after dosing and then disappeared. In addition, there was no effect of test item on the body weight, food consumption, functional behavior, motor activity, macroscopic and microscopic findings. Therefore, they were not considered adverse.
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:
mortality observed, non-treatment-related
Description (incidence):
One male at 1000 mg/kg bw/day was found dead on treatment day 9. In the microscopic findings, slight chronic active inflammation, marked alveolar edema and mononuclear cell infiltration and moderate type II alveolar cell hypertrophy/hyperplasia in lung, moderate acute inflammation in trachea and slight lymphoid hyperplasia in spleen were observed. Changes in lung and trachea were considered the cause of death. But, these changes were not considered related to test item as there were no correlated microscopic changes in other animals in same treated group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes in body weight gains were found for a few time periods during the study, but were scattered over time, sex and showed no dose dependency and thus were not considered test item-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes in food consumption were found for a few time periods during the study, but did not correlated with body weight changes 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 decreased total red blood cell count (RBC, 93% of control) was observed in males at 1000 mg/kg bw/day. This change was not considered adverse since there were no histopathological correlates. Decreased RBC was fully 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 recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decreased blood urea nitrogen (BUN, 86% of control) in males at 1000 mg/kg bw/day, significant increased glucose (GLU, up to 1.36-folds over control) in females at 300 and 1000 mg/kg bw/day and significant increases of total bilirubin (TBIL, 1.25-fold over control), calcium (Ca, 1.09-fold over control) and inorganic phosphorus (IP, 1.23-fold over control) in females at 1000 mg/kg bw/day were observed. After the recovery period, decreased BUN was still observed in males at 1000 mg/kg bw/day (85% of control, statistically not significant) and all other changes were fully reversed. These changes were not considered adverse since there were no histopathological correlates and/or values were within the normal historical control ranges.
Other statistically significant change was not considered test item-related, because there was no dose-dependent and not correlated with microscopic changes.
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 absolute and relative liver weights (up to 1.18-folds over control) was observed in females at 1000 mg/kg bw/day. It was not considered adverse as there were no histopathological correlates. Increased liver weights were fully reversed after recovery period.
Other statistically significant changes were not considered test item-related, because they were not dose-dependent and did not correlated with microscopic changes and/or were observed only in recovery group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic findings 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 estrus cycle, precoital time or fertility were observed during the study. No test item-related changes in thyroid hormone (T4) were observed in adult males and pups on PND 13 during the study.
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: no adverse effects observed
Key result
Critical effects observed:
no
Conclusions:
The No-Observed-Adverse-Effect Level (NOAEL) for general toxicity effects was considered to be 1000 mg/kg/day.
Executive summary:

The study was conducted to evaluate the potential of the test substance to cause general systemic and reproductive/development toxicity. The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 100, 300 and 1000 mg/kg bw/day 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 the recovery group at 0 and 1000 mg/kg bw/day (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. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and conducted. In addition, reproductive/developmental observations including estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were conducted. Thyroid hormone (T4) level in blood was also analyzed for adult males and pups at sacrifice. In general systemic observations, test item-related salivation was observed in both sexes at 300 and 1000 mg/kg bw/day, however, it was not considered to have toxicological significance since it was considered to be attributed to the palatability of the test item. Subdued behavior and prone position were observed at 1000 mg/kg bw/day, however it was not considered adverse since this clinical sign occurred after dosing and then disappeared, and there were no effect of test item on body weight, food consumption, functional behavior, motor activity, macroscopic and microscopic findings. In hematology, decreased total red blood cell count (RBC, 93% of control) was observed in males at 1000 mg/kg bw/day. In clinical chemistry, decreased blood urea nitrogen (BUN, 86% of control) in males at 1000 mg/kg bw/day, increased glucose (GLU, up to 1.36-folds over control) in females at 300 and 1000 mg/kg bw/day and increases of total bilirubin (TBIL, 1.25-fold over control), calcium (Ca, 1.09-fold over control) and inorganic phosphorus (IP, 1.23-fold over control) in females at 1000 mg/kg bw/day were observed. In organ weight, an increase in absolute and relative liver weights (up to 1.18-folds over control) was observed in females at 1000 mg/kg bw/day. These changes were not considered adverse since they were not correlated with microscopic findings. In conclusion, no test item-related adverse systemic effects were observed up to 1000 mg/kg bw/day. Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity effects was considered to be at least 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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

Additional information

A study was conducted to evaluate the potential of the test substance to cause general systemic and reproductive/development toxicity after oral uptake. The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 100, 300 and 1000 mg/kg bw/day 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 1000 mg/kg bw/day (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. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and conducted. In addition, reproductive/developmental observations including estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were conducted. Thyroid hormone (T4) level in blood was also analyzed for adult males and pups at sacrifice. In general systemic observations, test item-related salivation was observed in both sexes at 300 and 1000 mg/kg bw/day, however, it was not considered to have toxicological significance since it was considered to be attributed to the palatability of the test item. Subdued behavior and prone position were observed at 1000 mg/kg bw/day , however it was not considered adverse since this clinical sign occurred after dosing and then disappeared, and there were no effect of test item on body weight, food consumption, functional behavior, motor activity, macroscopic and microscopic findings. In hematology, decreased total red blood cell count (RBC, 93% of control) was observed in males at 1000 mg/kg bw/day. In clinical chemistry, decreased blood urea nitrogen (BUN, 86% of control) in males at 1000 mg/kg, increased glucose (GLU, up to 1.36-folds over control) in females at 300 and 1000 mg/kg bw/day and increases of total bilirubin (TBIL, 1.25-fold over control), calcium (Ca, 1.09-fold over control) and inorganic phosphorus (IP, 1.23-fold over control) in females at 1000 mg/kg bw/day were observed. In organ weight, an increase in absolute and relative liver weights (up to 1.18-folds over control) was observed in females at 1000 mg/kg bw/day. These changes were not considered adverse since they were not correlated with microscopic findings.

In conclusion, no test item-related adverse systemic effects were observed up to 1000 mg/kg bw/day. Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity effects was considered to be at least 1000 mg/kg bw/day.

 

Justification for classification or non-classification

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 oral toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

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