Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 27 November 2009 to 3 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP. Preliminary study for a 90-day toxicity test.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
preliminary study: 14 day exposure, 5 animals per group
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bioagri Laboratorios - DF / BRAZIL
- Age at study initiation: 8 weeks old
- Weight at study initiation: 257.1g ± 12.7g for males and 169.4g ± 9.8g for females
- Fasting period before study: /
- Housing: up to 3 animals/sex/cage in polypropylene rodent cages (41x34x19cm) with wire lids on top and bedding material (autoclaved wood shavings)
- Diet: Nuvilab CR-1 for rodents (ad libitum)
- Water: filtered drinking water (ad libitum)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.2-24.6°C
- Humidity: 44.1-69%
- Air changes: 10-20 times per hour
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: From 1 December 2009 to 22 December 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test suspensions were prepared under inert atmosphere. The test item was homogenized before use. Before preparation, the vehicle was degassed by sonication for 15 minutes, then saturated with inert gas, and kept under inert atmosphere for 15 minutes. Each suspension was stirred and dispensed into individual containers properly identified. The prepared doses were stored for no longer than 2 hours at room temperature before administration to the animals. Test suspensions were stirred continuously during the administration period to maintain homogeneity.

VEHICLE
- Concentration in vehicle: 10 to 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analysis of the test item in the vehicle was performed during the study:
Suspensions were prepared at the lowest (10 mg/mL) and highest (100 mg/mL) concentrations. Unique samples were taken on the day of preparation and after 4 and 9 days storage between +2°C and +8°C, under inert gas and protected from light. All samples were analyzed for test item levels.
On each occasion, the mean concentration was determined and compared to the nominal value. Acceptance criteria at each time-point: mean concentration = nominal value ± 10%. All values were within the acceptable range.

Concentration of the test item in prepared suspensions was verified during the study at each new preparation (i.e. each day of treatment).
Acceptance criteria at each time-point: mean concentration = nominal value ± 10%. All values were within the acceptable range.
Duration of treatment / exposure:
14 days
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle), 100, 200/300, 1000 mg/kg bw/day
Basis:
other: nominal
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a previous acute toxicity study by the oral route where the maximum dose tested, 500 mg/kg/day, did not modify body weight and did not induce clinical signs or macroscopic changes.
- Rationale for animal assignment: randomisation

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on Saturday, Sunday and public holidays
- Cage side observations: morbidity and or mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the initiation of treatment and once weekly during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: at the end of the acclimation period and before randomization, and then twice weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
> Red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count
> Total white blood cell count, differential white blood cell counts, lymphocytes, band neutrophils, segmented neutrophils, eosinophils, basophils, monocytes
> Prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked: glucose, blood urea nitrogen, creatinine, total protein, globulin, albumin, albumin/globulin ratio, calcium, total cholesterol, sodium, potassium, alkaline phosphatase, aspartate aminotransferase, alanine, aminotransferase.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, density, pH, smell, appearance (clarity), color, nitrites, protein, glucose, ketones, urobilinogen, bilirubin, occult blood, leukocytes, erythrocytes, epithelial cells, bacteria, mucus, crystals

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Weighed organs: adrenals (right and left), brain, heart, kidneys (right and left), liver, spleen, thymus, thyroids (with parathyroid), testes (right/left), epididymis (right/left), ovaries (right/left), uterus

HISTOPATHOLOGY: Yes
Hitopathological examination was performed on all tissues collected and preserved from all animals of every groups.
The following organs and tissues were collected and preserved for histopathological preparation in 10% neutral buffered formalin:
- Common organs: adrenal glands (right/left), bone marrow (sternum, femur), brain, esophagus, stomach, intestine, eyes, heart/aorta, kidneys (right/left), liver, lungs (right/left), lymph nodes, pancreas, peripheral nerves (sciatic), pituitary, salivary glands (right/left), skeletal muscle (bicep femoris), skin, spinal cord, spleen, thyroid/parathyroid, thymus (or remnant), trachea, urinary bladder
- Males: testes (right/left), epididymis (right/left), bulbourethral gland (right/left), preputial gland (right/left), prostate, seminal vesicle
- Females: ovaries (right/left), mammary gland, uterus, vagina
Statistics:
According to the results of test for normality, statistical analyses used were either parametric (ANOVA followed by Dunnett’s test) or non-parametric (Wilcoxon, Krushkal-Wallis) tests. Fisher’s Exact Test applied to the incidence of clinical observations and macroscopic lesions, where appropriate. The level of significance was set at p<0.05, and the statistical program used is SAS Sofltware (v.8).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Group 4 – 1000 mg/kg/day
- 5/5 females and 1/5 male were found dead on study day 2;
- 4/5 males were sacrificed on study day 4 due to severe clinical signs;
- All animals found dead on study day 2 (all females and 1 male) showed congestion of the lungs and liver at macroscopic examination;
- In males sacrificed on study day 4, the main macroscopic findings were darkening muscles of the abdominal and thoracic cavity, diaphragm and cervical region;
- Microscopic examination showed:
> mild to severe congestion of the majority of the organs (brain, adrenal gland, stomach, heart, liver, lungs, kidneys, small intestine and large intestine) in both sexes, mild congestion of salivary gland, pancreas, ovary, uterus and thymus in females;
> Slight to mild Zenker hyaline degeneration in several evaluated skeleta muscles in both sexes;
> Mild kidney nephrosis in both sexes;
> Emphysema in females.

Group 3 – 200 mg/kg day
- 1/5 female was sacrificed on study day 4 due to severe clinical signs;
- Body weight loss was observed in males during day 11 and 14;
- Lower body weight gain was observed in females which was associated with a statistically significant lower food consumption during the first week;
- Statistically significant higher platelets levels were observed in males (dose-related) and females (not dose-related);
- Higher APTT were observed in males and females (possibly related to higher platelet level);
- Lower total proteins (statistically significant), globulin, albumin and urea nitrogen (not statistically significant) levels were observed in females;
- A trend for lower total proteins and globulin levels were observed in males;
- Higher cholesterol levels were observed in males and females;
- Coloration of the urine was observed in males and females;
- Higher urine bilirubin, urobilinogen and occult blood levels were observed in males and females
- Higher absolute and relative liver weights were observed in males and females;
- Higher absolute and relative bilateral adrenal weights were observed in males;
- Lower absolute and relative ovarian weights were observed in females;
- Microscopic examination showed:
> Slight to severe congestion of the majority of organs (heart, liver and kidneys in both sexes, stomach and lungs in males and ovaries in females);
> Slight to mild Zenker hyaline degeneration of many evaluated skeletal muscles (eyes, esophagus, heart, skin and rear leg) in both sexes;
> Mild kidney nephrosis in both sexes;
> Emphysema in males.

Group 2 – 100 mg/kg/day

- Lower body weight gain was observed in males (statistically significant) and females (not statistically significant) which was associated with a decrease in food consumption during first week for females;
- Statistically significant higher platelets levels were observed in males and females;
- Higher APTT were observed in males and females;
- Higher cholesterol levels were observed in females;
- Coloration of the urine was observed in males and females;
- Higher urine bilirubin, urobilinogen and occult blood levels were observed in males and females;
- Higher absolute and relative liver weights were observed in females.
- Microscopic examination showed:
> Slight to mild congestion of the heart and kidneys in both sexes and the liver in males;
> Slight nephrosis of kidneys in both sexes;
- Slight Zenker Hyaline degeneration of many evaluated skeletal muscles (eyes, esophagus, lumbar vertebra and rear leg) in both sexes.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

On the basis of the results obtained in the present study, doses selected for the 13-week oral repeated toxicity study with the test item in Wistar rats were 10, 30 and 100 mg/kg bw/day.

Applicant's summary and conclusion