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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 9 to June 29 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Section 4 of the Guidelines for the Testing of Chemicals: Health Effects (ministry of environmental protection of People's Republic of China)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted Sept. 1998
Deviations:
yes
Remarks:
limited documentation (no raw data or results from individual animals were included in the study report), no food consumption measured, no detailed clinical observations, no ophthalmological examination
GLP compliance:
not specified
Remarks:
study was performed in China and it is not clear whether it was performed according to GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat was the preferred rodent species for this study and the SD strain was chosen due to its sensitivity against the known chemicals and uniformity in genetic properties.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shanghai SLAC Laboratory Animal Co., Ltd.
- Age at study initiation: not specified
- Weight at study initiation: 110 g to 140 g
- Fasting period before study: no
- Housing: 2 per polycarbonate cage
- Diet: powdered diet supplied by Suzhou Shuangshi Laboratory Animal Feed Co., Ltd., study report does not specify if feed was provided ad libitum
- Water: ad libitum
- Acclimation period: five days

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Prescribed amount of the test substance was weighed with a balance, and then diluted with corn oil to the specified concentrations.

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of an acute oral toxicity and repeated dose 28-day oral toxicity study.
- Rationale for selecting satellite groups: additional high dose and control group were assigned to assess the effects of a 14-day recovery period
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified
- Cage side observations: general clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: not reported
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not reported

FOOD EFFICIENCY:
- Food efficiency in percent is given in the results, no further description how these were dervived are given in the report

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period (after 90 days)
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: all the animals in the control and treatment groups
- Parameters checked: red blood cell (RBC), hemoglobin (Hb) content, white bood cell (WBC), leukocyte diffierential count (the rate of neurocytes (NEU%), the rate of lymphocytes (LYM%), the rate of monocytes (MONO), the rate of basophils (BASO%), the rate of eosinophils (EOS%)), platelet (PLT) counts, plasma prothrombin time (PT) and activated partial thromboplatin (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholesterol total (CHOL), bilirubin total (TBIL), glucose(GLU), blood urea nitrogen (BUN), creatinine (CREA), cholinesterase (CHE), total protein (TP), albumin (ALB), Na, K, Cl, Ca and P ions

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of administration period
- Metabolism cages used for collection of urine: not specified
- Animals fasted: not specified
- Parameters checked: specific gravity, color, pH, protein contents, blood/blood cells, glucose concentration

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: All the rats were subjected to a full gross necropsy and macroscopically observed in detail.
The following organs were weighed: brain, heart, lung, liver, spleen, kidney, adrenal gland, testis or ovary and thymus

HISTOPATHOLOGY: Yes: brain, heart, lung, liver, thymus, spleen, kidney, adrenal gland, testis or ovary, vervus ischiadicus, cervical, spinal cord, thyroid, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, mesenteric lymph node, prostate, epididymides, urinary bladder and uterus
Statistics:
Data were analyzed by ANOVA, and the additional high and control dosage group were analyzed by t-test using SPSS10.0.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared with the control group, the body weight gain of male rats in the high dose group was significantly decreased from the first (-6.4%) to the sixth (-8.6%) week and the eighth (-8.7%) to the thirteenth (-10.0%) week (p<0.05). The body weight gain of male rats in the high dose recovery group was significantly decreased during the entire treatment and recovery period, compared with the recovery control group (-4.5% in the first week up to -10.8%) in the fifteenth week. No effects were noted on body weight (gain) in the females of any treatment group, compared with the control group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
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):
The hematology analysis showed that red blood cell count of male rats in the high dose group was significant lower than in control group (-7.2%). The red blood cell counts were not significantly different in the high dose recovery group compared to the control recovery group, showing that this effect is reversible and thus probably not adverse. No historical reference data are given in the report but comparing the values for the red blood cell count in male rats of the high dose group (8.35 x 10E12/L) with historical reference data from male Wistar rats* ((7.37 - 9.25 x 10E12/L; age 19 - 21 weeks) the red blood cell counts of male rats in the high dose group falls within this historical range supporting that this effect is not of toxicological relevance. Basophil levels of female rats in the high dose recovery group were significantly lower than in the control group (-38%). Since the overall white blood cell count is not affected this effect is likely to be incidental and not treatment-related. Furthermore, the basophil levels (0.42%) fall in the range of historical reference values of female Wistar rats* (0 - 2%; age 19 - 21 weeks).

*technical bulletin: Baseline Hematology and Clinical Chemistry Values for Charles River Wistar Rats - (CRL:(WI)BR) as a Function of Sex and Age (http://www.criver.com/files/pdfs/rms/wistar-rats/rm_rm_r_hematology_crl_wi_br_sex_age.aspx)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Female rats: The cholinesterase level was significantly decreased (-33.7% in high dose group, -20.4% in high dose recovery group) in the high dose group and high dose recovery group compared to the respective control groups. However, no correlation in liver weights or histopathology was found in female rats. But in male rats effects on liver weights and histopathology was observed which indicates a treatment-related effect but cannot clearly be defined as toxicological relevant.
The calcium level was significantly increased (+7.6% in the high dose group, +6.5% in the high dose recovery group) in the high dose group and high dose recovery group compared with the respective control groups. This effect is considered to be incidental since it is only a minimal change. The blood urea nitrogen (BUN, +27.4%) was significantly increased and the cholesterol levels (-15.2%) were significantly decreased in the high dose recovery group compared to the control recovery group. This effect is considered to be not toxicological relevant since it only occurred in the recovery group. Chloride was significantly increased in the high dose group (+2.6%) but not in the high dose recovery group, thus indicating a reversible effect.
Male rats: Aspartate aminotransferase was significantly decreased (-14.3% in mid dose, 14.7% in high dose group), and cholinesterase was significantly increased (+48.3% in mid dose, +78.4% in high dose group) in the mid- and high dose groups, compared with the respective control groups. For both these effects, no significant difference was observed between the recovery treatment group and control group, indicating the effect was reversible. Creatinine was significantly increased (+26.7%) in the mid dose group, compared with the control group. As no similar effect was noted in the high dose group, this is considered to be incidental.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male rat each in the high and the control group showed a positive reaction in the protein measurement of the urine. This effect is considered to be incidental and not treatment-related since it was seen in the high dose group as well as in the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: The liver weight (-22.1%) was significantly reduced in the high dose recovery group compared to the control group. The relative liver weight was significantly increased for the high dose group (+14.2%) and significantly reduced for the high dose recovery group (-13.2%) compared to the respective control groups. The effects observed in the liver are considered to be treatment-related.
The relative organ weight of the brain was significantly higher in the high dose (+11.8%) and high dose recovery group (+12.1%) than in the respective control groups. Relative heart (+11.1%) and kidney (+17.1%) weight increases were observed in the high dose group of male rats compared to the control group.
Relative organ weight increase in testes are observed in the high (+12.1%) and mid (+10.3%) dose group and the high dose recovery group (+15.8%) of male rats compared to the respective control group. Since the body weight was significantly reduced in male rats of the high dose group and the organ weights did not change these effects are secondary due to the reduced body weight.
The spleen weight (-22.7%) and the relative spleen weight (-16.7%) was significantly reduced in the high dose recovery group. The effect seen in the spleen is considered to be incidental since it is only observed in the recovery group.

Females: In the mid dose group (150 mg/kg bw/day) for female rats the kidney weight was significantly lower (-10.3%) than for the control group. No effects were observed in the recovery group or in the relative kidney weight, therefore this effects is considered incidental.
The relative organ weight of adrenal gland was significantly higher (+26%) in the mid dose group of female rats compared to the control group. Since no dose-response was observed this effect is considered incidental.
The ovary weight (+45.5%) and the relative ovary weight (+25%) of female rats in high dose recovery group were significantly increased compared to the control recovery group. Since this effect was only observed in the recovery group this effect is considered incidental.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following histopatholgogical findings were observed: Two case in females of epithelial cells degeneration of kidney tubules, seven cases in males rats of infiltration of inflammatory cells in portal area and fatty degeneration of liver cells. The effects observed in the kidney cannot clearly be defined as toxicologically relevant or treatment-related, due to limited information in the study report. The histopathologic findings in the liver are probably treatment-related since they are found in a high number of male rats (7/10) in the high dose group.
No overt abnormality was found in the middle, low and recovery groups.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: urinary and hepatobiliary
Organ:
liver
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Oral application of 1000, 150 and 25 mg/kg bw/day test substance for 90-days to male and female Sprague-Dawley rats resulted in a significant body weight decrease in male rats of the high dose group, organ to body weight change in the liver of the high dose group in male rats and histopathology changes in liver in male rats and kidney in female rats of the high dose group. Based on these results a NOAEL is set to 150 mg/kg bw/day.