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90d repeated oral:

In this repeated dose 90-day oral toxicity study, varying degrees of toxicity effects were observed in male rats in the intermediate group and in females and males in the high group. Based on the results, the no observed effect concentration was estimated to be 100 mg/kg in female and 25 mg/kg in male rats. The NOAEL was estimated to be 10.26 mg/ (kg bw d) in female rats and 2.32 mg/ ( kg bw d) in male rats in accordance with the sample intake of rats.

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:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-03-04 to 2015-9-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
GLP compliance:
no
Remarks:
non GLP, but in compliance with China National Metrology Accreditation
Limit test:
no
Specific details on test material used for the study:
Batch No.: 141104
Purity: 95.4%
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zhejiang Center of Laboratory animals
- Quantity & Sex: 50 females (nulliparous and non-pregnant) and 50 males
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 104-149 g, not exceed ±10 per cent of the mean weight inner-group and not exceed ± 5 per cent of the mean weight between-groups.
- Housing: Suspended, wire bottom, stainless steel cages, 5 animals per cage by sex
- Diet (e.g. ad libitum): Conventional laboratory diets, ad libitum
- Water (e.g. ad libitum): Tap water by aseptic filtration, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±3 °C
- Humidity (%): 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours light / dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Vehicle: none.
Prior to the initiation of the study, the test substance was blended into the animal diet in sufficient quantities to achieve the nominal dosages.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg diet
Dose / conc.:
25 mg/kg diet
Dose / conc.:
100 mg/kg diet
Dose / conc.:
400 mg/kg diet
No. of animals per sex per dose:
Four groups: one control group, one low dose group, one Intermediate dose group, one high dose group, each group was 30, 20, 20, 30 animals with half males and females.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on acute oral LD50 values and the pre-test
- Rationale for animal assignment (if not random): randomly
- Rationale for selecting satellite groups: control group and high dose group
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
Mortality: Yes
- Time schedule: During exposure period and additional 14-day observation period

Clinic signs: Yes
- Time schedule: During exposure period and additional 14-day observation period

Ophthalmological examination: Yes
- Time schedule: Prior to the administration of the test substance and at the termination of the study

Sensory reactivity: Yes
- Time schedule: At the end of the exposure and additional 14-day observation period

Assessment of grip strength: Yes
- Time schedule: Each rat was measured 2 times in the thirteen exposure week and at the end of additional 14-day observation period

Assessment of motor activity: Yes
- Time schedule: At the end of 90-day exposure period or 14-day observation period

Body weight and body weight gain: Yes
- Time schedule: Body weights were recorded prior to the initiation of the study, weekly thereafter, and at death or sacrifice.

Food consumption: Yes
- Time schedule: at the end of the exposure period or 14-day observation period

Food efficiency:
- Time schedule: at the end of the exposure period or 14-day observation period

Sample intake: Yes
- Time schedule: Based on the food consumption per day, concentration of the diet, and average body weight weekly

Hematology: Yes
- Time schedule: At the end of 90 day exposure period or additional 14-day observation period
- Anaesthetic used for blood collection: Yes, 10% chloral hydrate
- Animals fasted: Yes, fasted overnight
- Parameters checked: white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), lymphocyte (LYM), granulocyte (GRA), monocyte (MID), eosinophil (EOS), basophil (BAS), red blood cell volume distribution width (RDW), platelet (PLT), prothrombin time (PT), activated partial thromboplastin time (APTT)

Clinical chemistry: Yes
- Time schedule: At the end of 90 day exposure period or additional 14-day observation period
- Animals fasted: Yes, fasted overnight
- Parameters checked: total bilirubin (BIL-T), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (CREA), glucose (GLU), potassium (K), natrium (Na), chlorine (Cl), A/G

Urinalyses: Yes
- Time schedule: At the end of 90 day exposure period or additional 14-day observation period
- Parameters checked: glucose (uGLU), bilirubin (uBIL), ketone (KET), occult blood (RBC), acidity (PH), protein (PRO), urobilinogen (uBG), nitrite (NIT), and leukocytes (LEU)

Gross necropsy: Yes

Organ Weight and Organ Coefficient: Yes
- Time schedule: At the end of 90 day exposure period or additional 14-day observation period

Histopathological examination: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals in the study was subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymis, uterus, ovaries, thymus, spleen, brain and heart of all animals were trimmed of any adherent tissue, and their wet weight taken as soon as possible.

HISTOPATHOLOGY: Yes
Tissues and organs (all gross lesion, brain, spinal cord, stomach, thyroid, thymus, small and large intestines, pancreas, liver, kidneys, adrenals, spleen, heart, trachea and lungs, gonads, uterus, accessory sex organs, prostate, urinary bladder, lymph nodes, peripheral nerve, bone marrow) samples from control and treated rats were cut into thin slices, and then fixed into 4% formaldehyde for more than one week. The tissues and organs were then processed through a graded series of ethanol and xylene, and embedded in paraffin. Organs and tissues sections were stained with hematoxylin and eosin for histopathological examination under a microscope and recorded the abnormal condition.
Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with the control group, the results of body weight and body weight gain of all treated group showed no statistical significance (p>0.05) at the end of 90-day exposure period or 14-day observation period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Compared with the control group, the results of food consumption per day of all treated group showed no statistical significance (p>0.05) at the end of the exposure period or 14-day observation period.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Cornpared with the control group, the results of food efficiency of all treated group showed no statistical significance (p>0.05) at the end of the exposure period or 14-day observation period.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For female rats, in the high group, PT was increased (p<0.05) at the end of 90-day exposure period; GRA was increased (p<0.05) at the end of 14-day observation period; RBC, HCT and LYM was decreased (p<0.05) at the end of 14-day observation period.
For male rats, in the high group, EOS, BAS was increased (p<0.05) at the end of 90-day exposure period.
The following indicators also changed, but there was no obvious dose-effect relationship or obvious biological significance: at the end of 90-day exposure period, PLT of female rats in the high group was increased; APTT of male rats in the low and intermediate group was increased; HCT and RDW of male rats in the intermediate group were decreased.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For female rats, Na+ was decreased in the high group at the end of 14-day observation period (p<0.05).
For male rats, Na+ and Cl- were increased in intermediate and high group at the end of 90-day exposure period (p<0.05).
The following indicators also changed, but there was no obvious dose-effect relationship or obvious biological significance: Na+ of the female rats in low and intermediate group was decreased; GLU and Cl- of female rats in intermediate group were increased. TP and ALB of female rats in the high group was increased. AST and ALT of male rats in the intermediate group were increased.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
For fernale and rnale rats, in each treated group (low, interrnediate, high), no significant differences were found in any pararneters cornpared with the control group (p>0.05) .
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The results of assessment of functional observations (hearing, vision, tactile, nociperception test and righting reflex, etc.) showed that there was no statistical significance in any treated groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
For female rats, compared with the control group, the coefficient of ovary in the high group was increased at the end of 90-day exposure period or 14-day observation period (p<0.05).
For male rats, the coefficient of kidney in the high group was decreased at 14-day observation period (p<0.05).
Gross pathological findings:
no effects observed
Description (incidence and severity):
The results of gross necropsy showed that no abnormal gross anatomy was found in each group.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormal changes were observed in the high dose group compared with the control group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Grip strength: At the end of 90-day exposure period, compared with the control group, grip strength of male rats in the high dose group was decreased (p<0.05). At the end of the additional 14-day observation period, compared with the control group, grip strength of female rats in the high dose group was decreased (p<0.05).
Motor activity: No statistical significance
Key result
Dose descriptor:
NOAEL
Effect level:
10.26 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
2.32 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
The no observed effect concentration was estimated to be 100 mg/kg in female and 25 mg/kg in male rats. The NOAEL was estimated to be 10.26 mg/ (kg bw d) in female rats and 2.32 mg/ ( kg bw d) in male rats in accordance with the sample intake of rats.
Executive summary:

The evaluation of subchronic oral tοxicities in male and female Sprague-Dawley (SD) rats was conducted on the test substance according to OECD Guideline 408.

Fifty SD rats per sex, 4-5 weeks old, were randomly assigned to four groups: one control group, one low dose group, one intermediate dose group, one high dose group, the number of animals was 30, 20, 20, 30 respectively with half males and females. The test substance was mixed in the diets used in the repeated dose 90-day oral toxicity study; the concentrations of the sample in the feed were 0, 25, 100 and 400 mg/kg for male and female rats. All test animals were exposed and observed for 90 days. Ten rats (five per sex per group) in the control and high groups (two additional groups) were observed for at least 14 days post treatment, for observation of reversibility or persistence of any toxic effects. Clinical signs, mortality, food consumption and body weight change were observed. When the study was terminated, assessment of grip strength, assessment of motor activity and functional observations, urinalyses, gross necropsy, hematology, clinical biochemistry and histopathology were carried out.

The results of repeated dose 90-day oral toxicity study showed as follow. Compared with the control group, at the end of 90-day exposure period, Na+ and Cl- of male rats in intermediate group were increased; The coefficient of ovary, PT of female rats in high group were increased; EOS, BAS, Na+, Cl- of male rats in high group were increased; grip strength of male rats in high group was decreased. At the end of the additional 14-day observation period, grip strength, RBC, HCT, LYM and Na+, of female rats in high group were decreased; the coefficient of ovary, GRA of female rats in high group were increased; the coefficient of kidney of male rats in high group was increased.

Compared with the control group, during 90-day exposure period and additional 14-day observation period, no abnormalities were observed in clinical signs, body weight and food consumption in all treated groups. There were no abnormal changes in each dose group for males and females on ophthalmological examination prior to the administration of the test substance and at the termination of the study. Compared with the control group, the results of assessment of functional observations (hearing, vision, tactile, nociperception test and righting reflex, etc.) and motor activity showed that there was no statistical significance in any treated groups. There was no statistical significance in any treated groups on gross anatomy. No significant histopathological changes were observed in the high group compared with the control group.

In this repeated dose 90-day oral toxicity study, varying degrees of toxicity effects were observed in male rats in the intermediate group and in females and males in the high group. Obvious toxicity effects were not observed in female rats in the intermediate group and male and female rats in the low group. In additional 14 days of observation, no delayed toxicity effects were found in male and female rats in the high group.

Based on the results, the no observed effect concentration was estimated to be 100 mg/kg in female and 25 mg/kg in male rats. The NOAEL was estimated to be 10.26 mg/ (kg bw d) in female rats and 2.32 mg/ ( kg bw d) in male rats in accordance with the sample intake of rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2.32 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline study, non GLP, but in compliance with China National Metrology Accreditation

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

90d repeated oral:

The evaluation of subchronic oral tοxicities in male and female Sprague-Dawley (SD) rats was conducted on the test substance according to OECD Guideline 408.

Fifty SD rats per sex, 4-5 weeks old, were randomly assigned to four groups: one control group, one low dose group, one intermediate dose group, one high dose group, the number of animals was 30, 20, 20, 30 respectively with half males and females. The test substance was mixed in the diets used in the repeated dose 90-day oral toxicity study; the concentrations of the sample in the feed were 0, 25, 100 and 400 mg/kg for male and female rats. All test animals were exposed and observed for 90 days. Ten rats (five per sex per group) in the control and high groups (two additional groups) were observed for at least 14 days post treatment, for observation of reversibility or persistence of any toxic effects. Clinical signs, mortality, food consumption and body weight change were observed. When the study was terminated, assessment of grip strength, assessment of motor activity and functional observations, urinalyses, gross necropsy, hematology, clinical biochemistry and histopathology were carried out.

The results of repeated dose 90-day oral toxicity study showed as follow. Compared with the control group, at the end of 90-day exposure period, Na+ and Cl- of male rats in intermediate group were increased; The coefficient of ovary, PT of female rats in high group were increased; EOS, BAS, Na+, Cl- of male rats in high group were increased; grip strength of male rats in high group was decreased. At the end of the additional 14-day observation period, grip strength, RBC, HCT, LYM and Na+, of female rats in high group were decreased; the coefficient of ovary, GRA of female rats in high group were increased; the coefficient of kidney of male rats in high group was increased.

Compared with the control group, during 90-day exposure period and additional 14-day observation period, no abnormalities were observed in clinical signs, body weight and food consumption in all treated groups. There were no abnormal changes in each dose group for males and females on ophthalmological examination prior to the administration of the test substance and at the termination of the study. Compared with the control group, the results of assessment of functional observations (hearing, vision, tactile, nociperception test and righting reflex, etc.) and motor activity showed that there was no statistical significance in any treated groups. There was no statistical significance in any treated groups on gross anatomy. No significant histopathological changes were observed in the high group compared with the control group.

In this repeated dose 90-day oral toxicity study, varying degrees of toxicity effects were observed in male rats in the intermediate group and in females and males in the high group. Obvious toxicity effects were not observed in female rats in the intermediate group and male and female rats in the low group. In additional 14 days of observation, no delayed toxicity effects were found in male and female rats in the high group.

Based on the results, the no observed effect concentration was estimated to be 100 mg/kg in female and 25 mg/kg in male rats. The NOAEL was estimated to be 10.26 mg/ (kg bw d) in female rats and 2.32 mg/ ( kg bw d) in male rats in accordance with the sample intake of rats.

Justification for classification or non-classification

90-day repeated-dose oral test:

In accordance with Regulation (EC) No. 1272/2008 Table 3.9.1 and 3.9.3, substances are classified as specific target organ toxicants following repeated exposure when substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans.

As no significant toxic effects observed in Repeated Dose 90-day Oral Toxicity Study, therefore this substance should not be classified for specific target organ toxicity - repeated exposure.