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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
The administration volume was 5 ml/kg body weight. The formulations were prepared as needed taking into account the analytically determined stability. For the preparation of the formulations the content of test item of 100% was taken for calculation.
The test item was administered as a solution in the vehicle. The formulations were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of treatment the stability of the formulations was analytically confirmed. For these analyses dosage forms were prepared in the same way as done in the study.
- Stability: The dosage forms prepared for analysis were analyzed shortly after preparation (2 hours) and 8 days thereafter. The analysis revealed that the test item was stable over this period within the defined limits.
- Content checks: For this purpose the test substance concentrations in formulations (including controls) given to the animals were determined two times during the study.
Duration of treatment / exposure:
29 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

In a two-week pilot study 3 animals per sex and dose received 0, 100, 300 and 1000 mg/kg test substance. In-life data, necropsy, organ weight measurements and histopathology (stomach only) did not show any treatment-related changes up to 300 mg/kg. One female treated with 1000 mg/kg showed body weight reduction and macroscopical changes such as changed consistency of the cecum and dodenum and discolored lungs. These changes were suspected to be test substance-related. Histopathology done in the stomach of control and high dose animals revealed no test substance-related changes. 1000 mg/kg bw was considered to be the MTD in longer studies.
Dose selection was decided with 100, 300, and 1000 mg/kg bw and day for the main study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes (incl. Open Field Observations (OFO))
- Time schedule: once before start and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once, day 28
- How many animals: all dose groups incl. controls

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once, day 28
- How many animals: all dose groups incl. controls

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: Functional Observational Battery (FOB); Motor Activity (MA)
- Time schedule for examinations: FOB: once, day 26-27; MA: once, day 21-22
- Dose groups that were examined: all dose groups incl. controls
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls)

ORGAN Weights:
Adrenals, brain, epididymides, heart, kidneys, Liver, Ovaries, Spleen, Testes, Thymus, uterus, prostate, seminal vesicle with coagulation glands

Fixed organs:
Adrenals, aorta, brain (cerebrum, cerebellum, ponslmedulla), epididymides, esophagus, eyes, eyelids, extraorbital lachrymal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular, bronchial/hilus, and mesenteric), lung, mamma, optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary and muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroids (including parathyroid glands), tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, Zymbal’s glands and all organs or tissues with macroscopic findings.

HISTOPATHOLOGY: Yes (high dose group and controls)
- Microscopic: Adrenals, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, eyes, femur, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, lung, ovaries, oviducts, prostate, sciatic nerve, skeletal muscle (thigh), spinal cord (cervical, thoracic, lumbar), sternum with bone marrow, testes, seminal vesicles (incl. coagulating glands), stomach, trachea and thyroids glands, urinary bladder, uterus with uterine cervix, vagina and all organs or tissues with macroscopic findings.

HISTOPATHOLOGY: Yes (all dose groups and controls)
-Microscopic: liver, lymph nodes, spleen, thymus

Statistics:
Statistical tests on body weights and weight gain as well as on absolute organ weights were done using the Dunnett Exact Homogeneous Test. For relative organ weights the Dunnett Exact Homogeneous Test after log. Transformation was used.
If primary food and water intake data were recorded, the calculated food/water intake per animal was evaluated using adjusted Mann-Whitney U-test.
The Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log. Transformation or the Bonferroni/Mann-Whitney U-test was used for clinical pathology parameters.
Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit. These included measures of general tendency (mean, median) and general variability (standard deviation, minimum, maximum) as appropriate.
For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett’s tests were performed.

Heteroscedastic normally distributed variables were analysed using appropriately adjusted Dunnett’s tests, using Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log-transformation of the original values.

If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
one 1000 mg/kg bw male was sacrificed in moribund state showing clinical symptoms of poor general condition; all other animals did not show clinical signs
Mortality:
mortality observed, treatment-related
Description (incidence):
one 1000 mg/kg bw male was sacrificed in moribund state showing clinical symptoms of poor general condition; all other animals did not show clinical signs
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:

One 1000 mg/kg bw male was sacrificed in moribund state showing clinical symptoms of poor general condition; all other animals did not show clinical signs

GROSS PATHOLOGY:

Gross pathology revealed no relevant changes up to 300 mg/kg bw. At 1000 mg/kg the followiing changes were found:
Two males (including the unscheduled death) demonstrated dilatation, change in contents, change in consistency, and gas-filled intestinal tract. In addition, an increase in mucous cells and focal inflammation in the glandular stomach were evident in some males and females.

HISTOPATHOLOGY:

Hypertrophy (only males) and so called cobble-stone pattern (both sexes) of liver cells were found.
Two males revealed slight lymphocyte necrosis in the thymic cortex and the paracortex of the mesenteric lymph node (minimal).
In one male follicular cell hypertrophy of the thyroid gland was noticed. As the change was an isolated finding scored as minimal and in this male T3, T4 and TSH plasma levels were not remarkably changed a toxicological relevance is no assumed.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: effects on the gastro-intestinal tract, liver, thymus and lymph nodes at 1000 mg/kg bw and day; NOAEL = NOEL

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In a repeated dose oral toxicity study following OECD TG 407 the test substance was administered to 5 Wistar rats per sex at daily doses of 0, 100, 300, 1000 mg/kg bw for 4 weeks. Survival and clinical appearance were not affected up to 300 mg/kg in males and up to 1000 mg/kg in females. One 1000 mg/kg bw male was sacrificed in moribund state showing clinical symptoms of poor general condition, with gas-filled and dilated gastro-intestinal tract. Body weight development, food and water intakes, functional observations and motor activity measurements as well as hematology, clinical chemistry and organ weights revealed no toxicologically relevant changes up to and including 1000 mg/kg bw.

At 1000 mg/kg two males (including the unscheduled death) demonstrated dilatation, change in contents, change in consistency, and gas-filled gastro-intestinal tract. In addition, an increase in mucous cells and focal inflammation in the glandular stomach became evident in some males and females. Hypertrophy (only males) and so called cobble-stone pattern (both sexes) of liver cells were found. Two males revealed slight lymphocyte necrosis in the thymic cortex and the paracortex of the mesenteric lymph node (minimal).

The NOAEL (NOEL) was determined with 300 mg/kg bw and day for male and female rats in this study based on effects on the gastro-intestinal tract, liver, thymus and lymph nodes seen at 1000 mg/kg bw.