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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - Nov 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(2008)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Stability in the solvent was analytically proved.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain Wistar (Hsd Cpb:WU)
- Source: Harlan Nederland, AD Horst, Netherlands
- Age at delivery of animals: 6 weeks
- Weight at study initiation: males 222 (210-238) g, females 173 (164-183) g
- Housing: from tattooing to necropsy in groups with 2 or 3 animals per cage in Makrolon cages type IV
- Diet and water: ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 55
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Administration volume: 5 mL/kg bw

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared as needed and taking into account the analytically determined stability.

VEHICLE
polyethylene glycol 400
- Justification for use and choice of vehicle (if other than water): The test item gave a solution in vehicle for which stability was analytically verified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Content checks on formulations (including controls) were determined twice during the study.
The samples were quantified by reversed phase (C18) HPLC with UV-detection (DAD, wavelength 200 nm). Standard solutions of the authentic test item were used for calibration.
Duration of treatment / exposure:
28 days (males), 29 days (females)
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
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:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: according to results obtained in a previous dose toleration study (female rats, oral administration) performed within a developmental toxicity study (see IUCLID-chapter 7.8.2)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for Morbidity and Mortality twice daily (once daily on weekends and public holidays); General Clinical Observations (in-cage) daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Open Field Observation weekly (once before the start of treatment);
Animals were placed into a standard arena (open field) for behavioral observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behaviour, breathing and excretory products were assessed. Findings and abnormalities were recorded either using a coding system or uncoded.

BODY WEIGHT: Yes
- Time schedule: just prior to first treatment and daily thereafter until last weighing immediately before necropsy.

FOOD AND WATER INTAKE: Yes
- Time schedule for examinations: weekly
On the basis of this data the following was calculated:
- for each interval: mean daily food intake per animal, mean daily food intake per kg body weight;
- for the total period: measurement of mean food intake per animal and day, mean food intake per kg body weight and day;
- cumulative food intake per animal and cumulative food intake per kg body weight.
Comparable calculations were done for the water intake.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
The blood samples were collected in the morning from the retro-orbital venous plexus of non-fasting animals anesthetized with or CO2/air.
- How many animals: all dose groups and controls
- Parameters examined: Differential blood count, erythrocyte morphology, erythrocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, hemoglobin concentration, hematocrit, leucocyte count, reticulocyte count, thrombocyte count, thromboplastin time (Hepato-Quick).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
The blood samples for determination of glucose concentrations were taken from the caudal vein of non-fasting, non-anesthetized animals. The blood samples used for determinating the other parameters were collected in the morning from the retro-orbital venous plexus of non-fasting animals anesthetized with or CO2/air.
- How many animals: all dose groups and controls
- Parameters examined: Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, cholesterol, creatinine, total protein, urea, glucose, gall acids, potassium, sodium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational Battery on day 24 (males) and day 25 (females); Motor/Locomotor Activity on day 24 (males) and day 25 (females).
- Dose groups that were examined: all dose groups and controls
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls)
Animals were sacrificed at day 29 (males) and day 30 (females) by exsanguination under deep diethyl ether anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. The following organs of the animals killed at the end of the treatment were weighed before fixation: Brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), epididymides (both), testes (both), seminal vesicles with coagulation gland, prostate, ovaries (both) and uterus. The organ weights are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organs and tissues were fixed and histopathologically evaluated for the control and highest dose group:
Abnormalities, Adrenal glands, Aorta, Brain (cerebrum, cerebellum, brain stem), Epididymides, Esophagus, Eyes, Eyelids, Exorbital lacrimal glands, Femur (with joint), Harderian glands, Head (with skull cap), Nasal Cavity, Heart, Intestine, Peyer's patches, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Kidneys, Larynx, Liver, Lungs, Lymph nodes, mandibular, Lymph nodes, mesenteric, Lymph nodes, popliteal, Optic nerves, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulation glands), Skeletal muscle (thigh), Skin (mammary region), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow , Stomach, Testes, Thymus, Thyroid glands (with parathyroids), Tongue, Trachea, Ureters, Urethra, Urinary bladder, Uterus (with cervix), Vagina, Zymbal's glands, Physical identifier.
Deviating from this the livers from all dose groups were evaluated histopathologically.

Statistics:
Statistical evaluations on body and organ weight data were done using the Dunnett-test in connection with a variance analysis. Evaluating clinical pathology parameters an analyses of variance followed by a Dunnett test, an adjusted Welch test or a Kruskal-Wallis test was performed. For all these tests SAS® routines were used.
All variables that were not dichotomous were described by sex, dose group and time point using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
For the statistical evaluation of samples drawn from continuously distributed random variables three types of statistical tests were used. The choice of the test being a function of prior knowledge obtained in former studies. Provided that the variables in question were approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared more likely, a p value adjusted Welch test was applied. If the evidence based on experience with historical data indicated, that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate.
With respect to data collected in the functional observational battery categorical variables were analyzed with a repeated measures analysis of variance followed by a one-way analysis of variance using the SAS procedure PROC CATMOD. As part of the one-way analysis, contrasts were performed to compare the results of each treated group with those of the control group. The logic of the analysis plan for continuous variables was analogous to that of the categorical variables, but using the SAS procedure PROC GLM.
Statistics of MA/LMA will be generated with an evaluation step of the SPADER (=Safety Pharmacology Automated Data Evaluation and Reporting) application.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
Survival was not affected by the treatment with the test substance up to 1000 mg/kg.
The behaviour and clinical appearance were not toxicologically relevantly changed in both sexes up to 1000 mg/kg.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was not toxicologically relevantly changed at any dose.

FOOD AND WATER INTAKE
Food and water intake were not toxicologically relevantly changed in both sexes up to 1000 mg/kg.

HAEMATOLOGY
Red and white blood parameters as well as blood coagulation were not toxicologically relevantly changed in any dose group.

CLINICAL CHEMISTRY
Enzyme activities, substrates and electrolytes in peripheral blood were not affected by the test substance up to 1000 mg/kg.

NEUROBEHAVIOUR
FOB did not reveal any test substance-induced toxicologically relevant findings in males and females up to 1000 mg/kg. Motor and locomotor activities did not reveal any indications of a neurotoxicological potential of the test substance in males and females up to 1000 mg/kg.

ORGAN WEIGHTS
Liver weights were slightly increased in males at 1000 mg/kg. Females were not affected.

GROSS PATHOLOGY
Necropsy revealed distinct lobulation and surface changes in livers of males at 1000 mg/kg. Females were not affected. Gross pathological investigation did not give any indication of test substance-related functional or morphological alterations in the other organs examined.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological evaluation revealed centrilobular vacuolation together with eosinophilia in males at 1000 mg/kg. The vacuolation was proved as hepatocellular fat deposition and might indicate an (adverse) influence on fat metabolism. Females were not affected.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: slightly increased liver weights, distinct lobulation and surface changes of liver at necropsy and histopathological correlate (hepatocellular fat deposition)
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: no toxicological relevant changes within all investigations

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Executive summary:

An oral subacute dose study according to OECD TG 407 with the test item administered by gavage as formulation in polyethylene glycol was conducted using 5 male and 5 female Wistar rats per dose group and doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw per day.

Survival was not affected by the treatment up to the highest dose. Clinical and functional observations as well as motor activity measurements did not reveal any indications of test substance-induced effects. Body weight development as well as food and water intake was not affected by the test substance at any dose. Hematological and clinico-chemical parameters were not toxicologically relevantly changed. In males liver weights were slightly increased and necropsy revealed distinct lobulation and surface changes at 1000 mg/kg. These morphological observations corresponded to centrilobular vacuolation together with eosinophilia in this group. The vacuolation was proved as hepatocellular fat deposition and might indicate an (adverse) influence on fat metabolism. Females were not affected. Gross and histopathological investigation did not give any indication of test substance-related functional or morphological alterations in the other organs examined.

Therefore the no-observed-adverse-effect level (NOAEL) was 300 mg/kg body weight in males based on liver findings and 1000 mg/kg body weight in females.