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Description of key information

In an OECD 422 study, toxicity was noted at 300 mg/kg, and was characterized by mortality (five animals in total) and various clinical signs.
In an OECD 408 study, slight non adverse toxicity was noted at 150 mg/kg. This was considered as the NOAEL.

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2021 - December 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague Dawley [SD], SPF grade
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co., Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: Males: 313.78 to 373.62 g; Females: 181.01 to 233.13 g
- Fasting period before study: no
- Housing: Animals were group housed (up to 3 animals of the same sex and same dosing group together) in solid bottom cages (cage size 43×30×20 cm3), with corncob bedding, with a water bottle (except during the FOB test where the dedicated animals were housed individually).
- Diet (e.g. ad libitum): rodent feed ad libitum; There were no known contaminants (including heavy metals and pesticides) present in the diet expected to interfere with the test results.
- Water (e.g. ad libitum): reverse-osmosis purified and chlorinated water by a water bottle ad libitum; No contaminants were present at levels that would interfere with the outcome of the study.
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 25
- Humidity (%): 25.8 to 69.9 (The humidity was occasionally out of the target range 40-70%. However, all these excursions lasted less than 0.25 hours.)
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-01-05 To: 2021-04-29
Route of administration:
oral: gavage
Details on route of administration:
Oral administration by intubation is the recommended administration in OECD TG 408.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% (w/v) CMC-Na in purified water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was added to 70% of the final volume of vehicle while stirring. The formulation was stirred until a homogenous formulation was formed by visual inspection. The formation was brought to its final volume. The formulations intended for dosing were stirred at room temperature for more than 30 minutes before dosing and continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water. Previous experimental studies have shown that dose preparations are adequately prepared using 1% CMC.
- Concentration in vehicle: 1% in purified water
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): c2030035
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Triplicate samples (one for analysis, the other two for backup) were collected from the middle layer of the mid-dose formulations and control formulations for the first and last preparations. For the low- and high-dose formulations, mean results of the homogeneity results were used as concentration verification and no additional samples were collected.

Homogeneity was determined in this study for the low- and high-dose test item formulations in the first, week 4 and last preparations. For homogeneity analysis, triplicate samples (one for analysis, the other two for backup) were collected from the top, middle, and bottom layer of formulations.

The concentrations of the test item in dosing formulations were within 98.7% to 117.7% of nominal values.
Homogeneity analysis of the low- and high-dose dosing formulations was performed. The relative standard deviations (RSD) of top, middle, and bottom of samples were within 1.3% to 8.1%
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
10
+5 for recovery in the control and high dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the available OECD 422 study (Combined repeated dose and reproduction/developmental screening in rats, 29 days for males, 43-49 days for females, gavage, CMC as vehicle). Treatment with the test material by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg body weight/day revealed parental toxicity and developmental toxicity at 300 mg/kg body weight/day. Toxicity was noted at 300 mg/kg, and was characterized by mortality (five animals in total), clinical signs, reduced body weights, changes in haematology parameters indicative of slight anemia, macroscopic findings and microscopic findings of the thymus, stomach, skeletal muscle and mesenteric lymph node. Based on these results, the No Observed Adverse Effect Level (NOAEL) was 100 mg/kg/day for both parental toxicity and developmental toxicity. Therefore, 150 mg/kg/day was selected as the highest dose level for this study.

- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight
- Rationale for selecting satellite groups: Assessment of recovery
- Post-exposure recovery period in satellite groups: 14 days

- Dose range finding studies: No, use of available OECD 422 and its dose range-finding study
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily from D-2 to end of recovery phase. Cage side observation was not conducted when a detailed observation was scheduled.
Details in attached document.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during acclimation, once before dosing on Day 0, once weekly thereafter, at necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and day of necropsy.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimation, once at the end of the 90-d period, once at the end of the recovery phase
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior necropsy
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes
- How many animals: All
Details in attached document.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior necropsy
- Animals fasted: Yes
- How many animals: All
Details in attached document.

SERUM HORMONES: Yes
- Time of blood sample collection: Just prior necropsy
- Animals fasted: Yes
- How many animals: All
Details in attached document.

URINALYSIS: Yes
- Time schedule for collection of urine: Just prior necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
Details in attached document.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during acclimation, once in last week of the dosing phase, once during the recovery period
- Dose groups that were examined: All
Details in attached document.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Details in attached document.

HISTOPATHOLOGY: Yes
Details in attached document.
Optional endpoint(s):
Optional endpoints: Yes
- Estrus Cycle Evaluation: Determination of the stages of estrus cycle at necropsy; all females
- Sperm analysis: sperm motility, morphology, and count for 5 males per group in the dosing groups and all males of the recovery groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted at ≤ 35 mg/kg/day.
Slight salivations were observed in both sexes at 75 and 150 mg/kg/day from Days 33 to the end of the dosing phase (10 males and 2 females at 75 mg/kg/day, and 14 males and 15 females at 150 mg/kg/day).
All other clinical signs observed (coat soiled, alopecia, scab, ear abnormality, abnormal testes, mass, and material around mouth) were considered incidental and unrelated to the administration of the test item, as they were of the types seen in untreated rats in this laboratory, isolated occurrences, also observed in the control group, and/or were not dose dependent.
Mortality:
no mortality observed
Description (incidence):
There were no moribund or found dead animals during the course of this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in body weights were noted at ≤ 75 mg/kg/day.
A test-item related body weight decrease down to 5% was observed in males at 150 mg/kg/day during the first dosing week. Recovery was achieved by the following week. This change was related with the decrease in food consumption observed the same week.

All other differences noted in body weights or body weight changes, including those that were statistically significant, were considered normal variations or incidental and unrelated to the test item because they were small in magnitude and/or not dose dependent.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the first dosing week, test item-related significant decreases in food consumption were noted in males down to 23.3% at 150 mg/kg/day. Those change were recovered by the following week.

All other findings, including those that were statistically significant, in the pretest, dosing and recovery phases were considered normal variations or incidental and unrelated to the test item because they were small in magnitude and/or not dose dependent.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no test-item related ophthalmologic changes during the dosing and recovery phase examinations. All ocular findings were considered common background or spontaneous findings consistent with the age and strain of this species.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing phase, when compared with the control group, decreased group mean values of RBC (Erythrocyte count), HGB (Hemoglobin), and HCT (Hematocrit) were noted in males and/or females at ≥75 mg/kg/day, and increased numbers of RET (Reticulocytes, absolute and percent) were noted in males at ≥75 mg/kg/day.
At the end of the recovery phase, all of the changes at 150 mg/kg/day were resolved and the numbers were comparable to those of the control group. All of those changes were considered related to the test item and non-adverse based on the reversibility and magnitude, and/or within the historical control ranges of this laboratory.

All other differences observed in hematological parameters, including those that were statistically significant, were considered incidental and unrelated to the test item because they were small in magnitude, not dose-related, and/or within the historical control ranges of this laboratory.

There were no test-item related coagulation changes during the dosing and recovery phase examinations.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test-item related serum chemistry changes during the dosing and recovery phase examinations. All differences observed in serum chemistry parameters, including those that were statistically significant, were considered incidental and unrelated to the test item because they were small in magnitude, not dose-related, and/or within the historical control ranges of this laboratory.
Endocrine findings:
no effects observed
Description (incidence and severity):
No test item-related changes of T3 and T4 were observed in this study.
No test item-related changes of TSH at ≤ 75 mg/kg/day. When compared with control group, TSH decreased in male rats treated at 150 mg/kg/day. This effect was considered non-adverse due to small in magnitude.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test-item related urinalysis changes during the dosing and recovery phase examinations.
All differences observed in urinalysis were considered incidental and not related to test item administration because they were small in magnitude, were not dose related, and/or were within the historical control ranges for this laboratory.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Statistical analyses were performed on all FOB data including the quantitative parameter assessment (forelimb grip strength, hindlimb grip strength, hindlimb foot splay and rectal temperature). There were no test item-related effects on any of the FOB parameters. All changes were not considered to be test item-related as they were of small magnitude, comparable to the concurrent control values, or without dose dependency.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related organ weight changes in any treatment groups at the end of dosing and recovery phases.
All intergroup differences in organ weights from the dosing and recovery phases were not considered test item-related based on the absence of a dose response, the absence of microscopic correlates, and because they were within normal biological variations in the rats of this age.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related macroscopic changes in any treatment groups at the end of the dosing and recovery phases.
All macroscopic changes were not considered test item-related as they were considered common background or spontaneous findings consistent with the age and strain of this species
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in any treatment groups at the end of the dosing phase and recovery phases.
All microscopic findings were considered common background or spontaneous findings consistent with the age and strain of this species.
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related sperm motility, morphology, and count findings in any treatment groups at the end of the dosing phase and recovery phases.
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
neuropathology
ophthalmological examination
organ weights and organ / body weight ratios
serum/plasma biochemistry
serum/plasma hormone analyses
sperm measures
urinalysis
Critical effects observed:
no
Conclusions:
Single daily oral administration of the test item to male and female Sprague Dawley rats at 35, 75, and 150 mg/kg/day for up to 90 days with a subsequent 2 week recovery period was well tolerated. Minor toxicity was noted at ≥ 75 mg/kg/day during the dosing phase and was characterized by salivation, slight decreased body weights and food consumption, changes in hematology parameters indicative of slight anemia. All changes were recovered by the end of the 14-day recovery phase. Consequently, the no observed adverse effect level (NOAEL) was considered to be 150 mg/kg/day (the highest dose tested) for both sexes.
Executive summary:

The purpose of this study was to determine the potential toxicity of the test item when given once daily by oral gavage to rats for up to 90 days and to assess the reversibility, persistence, or delayed occurrence of toxic effects following a 2‑week recovery period following the OECD TG 408 and GLP principles.


Rats were randomly assigned to 4 groups with 10 sex/group (low and mid doses) or 15 sex/group (control and high dose). They were administered the control formulation 1% (w/v) CMC-Na in purified water or the test item at doses of 35, 75, or 150 mg/kg/day once daily via oral gavage for up to 90 days. The dosing volume was 10 mL/kg. Animals were approximately 7 to 9 weeks old at dosing initiation and weighed from 313.78 to 373.62 g for males and 181.01 to 233.13 g for females, respectively. Following completion of the dosing phase, five animals/sex in the control and high dose groups were assigned for a 14-day recovery and were necropsied on Day 105.


Criteria for evaluation included viability (mortality/moribundity), clinical observations, body weight, food consumption, estrus cycle, ophthalmology, functional observational battery, clinical pathology (hematology, serum chemistry, coagulation, and urinalysis), serum hormone (TSH, T3 and T4), gross (necropsy) evaluation, organ weights, histopathological evaluation and sperm analysis.


The concentrations of the test item in dosing formulations were within 104.0% to 115.3% of nominal values.


All animals survived to scheduled termination. Slight salivation was observed in both sexes at 75 and 150 mg/kg/day from Day 33 to the end of the dosing phase. A test item-related bodyweight decrease down to 5% was observed in males during the first dosing week with a recovery by the following dosing period. It was considered related to the food consumption decrease observed concomitantly. In the first dosing week, test item-related significant decreases in food consumption were noted in males down to 23.3% at 150 mg/kg/day. Those changes were recovered by the following week and were considered as not adverse due to the small magnitude and the lack of a dose response.


At the end of the dosing phase, when compared with the control group, decreased group mean values of RBC (Erythrocyte count), HGB (Hemoglobin), and HCT (Hematocrit) were noted in males and/or females at ≥75 mg/kg/day. Increased numbers of RET (Reticulocytes, absolute and percent) were noted in males at ≥75 mg/kg/day. At the end of the recovery phase, all of the changes were resolved and results were comparable to that of the control group.


There were no test item-related changes and/or adverse effects in estrus cycle, ophthalmology, functional observational battery, serum hormones (T3 and T4; TSH), clinical pathology (serum chemistry, coagulation, and urinalysis), organ weights, gross histopathology examinations, and sperm analysis at the end of the dosing and recovery phases.


In conclusion, single daily oral administration of the test item to male and female Sprague Dawley rats at 35, 75, and 150 mg/kg/day for up to 90 days with a subsequent 2‑week recovery period was well tolerated.  Minor toxicity was noted at ≥ 75 mg/kg/day during the dosing phase, and was characterized by slight salivation, slight decreased body weights and food consumption, changes in hematology parameters indicative of slight anemia. All changes were recovered by the end of the 14-day recovery phase. Consequently, the no observed adverse effect level (NOAEL) was considered to be 150 mg/kg/day (the highest dose tested) for both sexes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

Justification for classification or non-classification

Not Classified. - Based on available data and/or professional judgment, the classification criteria are not met.