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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Specific details on test material used for the study:
Stability in the solvent was analytically proved.
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 233 (224-246) g, females 162 (146-178) 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
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Administration volume: 5 mL/kg

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:
Before the start of treatment the suitability of the formulations was confirmed by the analysis of concentration and stability of dosage forms prepared in the same way as it was done in the study. The dosage forms prepared for analysis were analyzed shortly after preparation and 8 days thereafter. The analysis revealed that the test item was stable over this period within the defined limits. 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
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 developmental toxicity study after oral administration performed in female rats (see IUCLID-chapter 7.8.2)
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)

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
- Anaesthetic used for blood collection: Yes (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
- Anaesthetic used for blood collection: Yes (CO2/air)
The blood samples for determination of glucose concentrations were taken from the caudal vein of non-fasting, non-anesthetized animals.
- 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 group 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 kidneys and 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.
Details on results:
CLINICAL SIGNS AND MORTALITY
Survival was not influenced by the treatment with the test substance up to 1000 mg/kg.
The behavior and clinical appearance were not toxicologically relevantly changed by the test substance 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 showed no differences attributable to the treatment with the test substance 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 and concentrations of sodium and potassium in peripheral blood were not affected by the test substance at any dose. The concentrations of cholesterol, urea, protein and albumin in peripheral blood were increased in males at 1000 mg/kg and in females mostly starting at 100 mg/kg. Furthermore, the glucose concentration was decreased without dose dependence in females at 300 mg/kg and above. A higher concentration of bile acid in females at 1000 mg/kg is considered questionable as this parameter shows a considerable variation. Taken together, concentration changes of clinico-chemical parameters in males at 1000 mg/kg and in females mostly starting at 100 mg/kg are interpreted as adaptive and not as adverse effects.

NEUROBEHAVIOUR
No adverse effects are inferred from marginally to slight differences in number of rearing and motor/locomotor activities in males at 1000 mg/kg. Without clear dose dependence slight changes of motor/locomotor activities in males at 100 and 300 mg/kg are assessed as not biological relevant.

ORGAN WEIGHTS/ GROSS PATHOLOGY/ HISTOPATHOLOGY: NON-NEOPLASTIC
Thymus weights were increased in females at 1000 mg/kg (up to 47 %), a trend to higher weights could also be observed in this sex at 100 and 300 mg/kg. Without histopathological correlate a test substance-induced effect without toxicological relevance is considered. Liver weights were slightly increased in 1000 mg/kg males. At necropsy liver appeared swollen or enlarged in this group. These observations corresponded on the microscopic level with hepatocellular hypertrophy in conjunction with fat deposition in centrilobular liver cells in males at 1000 mg/kg. Liver histopathology was inconspicuous in females at any dose.
Gross and histopathological investigation as well as organ weight measurements did not give any indication of test substance-related functional or morphological alterations in the other organs examined.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: liver findings (hypertrophy and 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
Critical effects observed:
no

Taken together, concentration changes of clinico-chemical parameters in males at 1000 mg/kg and in females mostly starting at 100 mg/kg are interpreted as adaptive and not as adverse effects. However, due to hypertrophy together with fat deposition in males at 1000 mg/kg a mild test substance-induced adverse effect cannot be excluded in this group for males.

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, clinical appearance, body weight development, food and water intake as well as enzyme activities and electrolyte concentrations were not affected by the treatment in males and females up to 1000 mg/kg. No adverse effects are inferred from lowered number of rearing and higher motor/locomotor data in males at 1000 mg/kg since the differences to control were marginally and without statistical significance.

Hematological parameters were comparable to controls in both sexes up to 1000 mg/kg. Thymus weights were increased in females starting at 100 mg/kg. Without histopathological correlate no toxicologically relevant effect is considered. Concentrations of cholesterol, urea, protein and albumin were increased in peripheral blood in males at 1000 mg/kg and in females mostly starting at 100 mg/kg. Furthermore, glucose was decreased in females starting at 300 mg/kg. A higher concentration of bile acid in females at 1000 mg/kg is considered questionable as this parameter shows a considerable variation. Taken together, concentration changes of clinico-chemical parameters in males at 1000 mg/kg and in females mostly starting at 100 mg/kg are interpreted as adaptive and not as adverse effects.

Liver weights were slightly increased and at necropsy livers appeared swollen or enlarged in 1000 mg/kg males. These observations corresponded on the microscopic level with hepatocellular hypertrophy in conjunction with fat deposition in centrilobular liver cells in this group. Females were not affected. Due to morphological changes in the liver of males at 1000 mg/kg a mild test substance-induced adverse effect cannot be excluded in this group.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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
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

An oral Repeated Dose Toxicity study (28 days, OECD TG 407) revealed no mortalities, no effects on body weight development and food and water intake. Clinical appearance and behaviour was not toxicologically relevantly changed. There were no changes in hematological parameters, enzyme activities and concentrations of electrolytes. Concentration changes of clinico-chemical parameters in males at 1000 mg/kg and in females mostly starting at 100 mg/kg are interpreted as adaptive and not as adverse effects.

Liver weights were slightly increased and at necropsy livers appeared swollen or enlarged in 1000 mg/kg males. These observations corresponded on the microscopic level with hepatocellular hypertrophy in conjunction with fat deposition in centrilobular liver cells in this group. 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.

The NOAEL was 300 mg/kg bw for males due to liver findings and 1000 mg/kg bw for females.

Justification for classification or non-classification

No classification required for repeated dose toxicity according to Regulation (EC) No 1272/2008, Annex I.