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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:
2014-11-20 to 2015-01-15
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
As a preliminary study, this study did not follow a specific guideline and was designed to allow selection of appropriate dose levels for use in the
upcoming study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liqiud
Details on test material:
Characteristics: Slightly yellowish liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Strain: Crl:WI rats
- Age of animals: young adult rats, approximately 7 weeks at the onset of treatment.
- body weight: males: 305 - 341 g, females: 189 - 233 g
- Fasting period before study: 16 hours
- Diet: ad libitum, ssniff® SM R/M "Autoclavable complete diet for rats and mice , produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum
- Housing: group-housed, 2 or 3 animals/sex/cage
- Acclimatisation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 23.5° C
- Humidity (%): 24-57 %
- Illumination: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Air change: 15-20 air exchanges/hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on oral exposure:
The test item was formulated in the vehicle at concentrations of 25, 75 and 250 mg/mL for dose level of 100, 300 and 1000 mg/kg bw/day and
a dose volume of 4 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed.
Stability assessment was made by IR analysis, concentration and homogeneity by Xray fluorescence. All formulations were found to be in the
range of 96 to 108% of nominal concentration. All formulations were shown to be homogeneous. No test item was detected in the control samples.
Top, middle and bottom duplicate samples were taken, once, one set to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
other: very dry DMSO at a dose volume of 4 mL/kg bw
No. of animals per sex per dose:
5 male / 5 female,
Control animals:
yes, concurrent vehicle
Details on study design:
-Dose levels based on previous data obtained in an acute oral toxicity study (Acute Toxic Class Method, OECD 423, in which the test item was proven to be non-toxic after a single oral administration at the dose level of 2000 mg/kg bw/day).
- A control group was treated concurrently with the vehicle only (water-free DMSO).
- Determination of MTD was performed in a sequential manner.
- Starting with one male and female rat treated with vehicle (control), 100, 300 and 1000 mg/kg bw/day for three days.
- On Day 3, there were no signs of toxicity at any dose level, a further 4 rats were treated at same dose levels for at least 14-day.
- All animals were treated daily until all animals have received at least 14 days of consecutive treatment.

Positive control:
not necessary

Examinations

Observations and examinations performed and frequency:
Clinical chemistry:
- Clinical observations and mortality: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each
working day and once prior to necropsy).
- Body weight measurement: On the day of animal assignment to the study, prior to the first treatment and then daily, before necropsy (fasted).
- Food consumption measurement: The food was measured with precision of 1 g daily from Day -1 for each animal.
CLINICAL PATHOLOGY
Blood samples for clinical pathology evaluation (haematology and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia.
- Haematology:
RBC Red Blood Cell (erythrocyte) count, (1012/L) M/L
WBC White Blood Cell (leukocyte) count, (109/L) K/L
Hgb Haemoglobin concentration, (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
RDW Red Cell (erythrocyte) volume (%)
Plt Platelet (thrombocyte) count (109/L) K/L
MPV Mean Platelet Thrombocyte volume (fL)
RETIC % Reticulocyte count (%)
NE % Neutrophil (%)
LY % Lymphocyte (%)
MO % Monocyte (%)
BA % Basophil (%)
EO % Eosinophil (%)
LUC % Large Unstained Cells
-Clinical chemistry:
Glucose Blood sugar concentration (mmol/L)
T-BIL Total Bilirubin concentration (μmol/L
Urea Urea concentration (mmol/L)
Chol. Cholesterol concentration (mmol/L)
Creat. Creatinine concentration (μmol/L)
Tot. Prot. Total Protein concentration (g/L)
Alb. Albumin concentration (g/L)
AST/GOT Aspartate Aminotransferase activity (U/L)
ALT/GPT Alanine Aminotransferase activity (U/L)
ALKP Alkaline. Phosphatase – activity (U/L)
GGT Gamma Glutamyltransferase –activity (U/L)

Sacrifice and pathology:
PATHOLOGY EVALUATION
Gross necropsy was performed on all animals.
-Organ weight measurements
Brain
Epididymides
Heart
Kidneys
Liver
Prostate
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus
Uterus including cervix
Adrenals
Ovaries
Thyroids with parathyroids
Histopathology: No histopathology evaluation was required to be performed during this study.
Statistics:
Statistical analysis performed on numerical data as appropriate depending upon the effects observed and number of animals tested. The mean and
standard deviations values, the frequency of clinical observations, and necropsy findings were calculated as applicable. Statistical evaluation of data
as applicable was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups werechecked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) was made.If the obtained result was significant Duncan’s Multiple Range test were used to assess the significance of inter-group differences.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Details on results:
MORTALITY
There was no unscheduled mortality during the study.

CLINICAL OBSERVATIONS
There were no adverse clinical signs recorded during the study with test item administered daily for at least 14 days at dose levels of 100, 300 and 1000 mg/kg bw/day. Noisy respiration was observed in one Mid dose male animal on Days 1 and 2.

BODY WEIGHT MEASUREMENTS
There were no signs of toxicity on body weights or body weight gain during the study.
Minor differences to control were recorded in body weight or body weight gain, occasionally attaining statistical significance, which were without any
toxicological significance. For example, the body weight gain value was higher in Mid dose females between Days 0 and 1 (p<0.05) or was lower in
Mid dose females between Days 6 and 7 (p<0.05).

FOOD CONSUMPTION MEASUREMENTS
There were no signs of toxicity on food consumption.

CLINICAL PATHOLOGY
Haematology
Haematology parameters evaluated at the completion of the at least 14-day treatment period did not show any signs of toxicity. Variations were noted in a few parameters in the treated animals, on some occasions attaining statistical significance. In females, the Mean Corpuscular (erythrocyte)
Haemoglobin Concentration (MCHC) was statistically higher and the Reticulocyte count (Retic) was statistically lower than control in Mid dose group
(p<0.05). Based on the isolated incidence and lack of a consistent dose or gender response, these variations were regarded as incidental and without
signs of toxicity.
Clinical chemistry
There were no toxicologically relevant differences between the controls and treated groups in the clinical chemistry parameters evaluated at the
completion of the treatment period.
Decreases in Alanine Aminotransferase activity (ALT) were noted in males at Low and Mid dose levels, attaining statistical significance (p<0.05). The
mean Urea concentration (UREA), the Albumin concentration (Alb.) and consequently the Total Protein concentration (Tot.Prot) were slightly higher
(p<0.05) in the High dose males when compared to the control mean. As no consistent dose response was observed and the variations were
considered minor, these variations were regarded as not being toxicologically significant, or could not be clearly ascribed to test item administration.

PATHOLOGY EVALUATION
There were no test item-related macroscopic findings at necropsy following the at least 14-day treatment period.
During scheduled necropsy, small testes, epididymides and prostate in 1/5 Control male, small seminal vesicles/prostate in 1/5 Mid Dose male,
uterine dilatation in 1/5 Low Dose female were found. These changes can be considered as incidental.

ORGAN WEIGHT MEASUREMENTS
There were no clear toxicologically significant changes in organ weights noted after test item administration once a day for at least 14 days, at up to
and including 1000 mg/kg bw/day, evaluated at necropsy.
The absolute brain weight of the Low dose males was lower as compared to control attaining statistical significance (p<0.05). Compared to control
the body weight related ovary weight was lower with statistical significance (p<0.05) in High dose females. The results were ascribed to biological
variability or to individual, incidental changes and considered to be unrelated to treatment.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
haematology
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
In conclusion, test itemadministrated to Wistar rats at 100, 300 and 1000 mg/kg bw/day, daily for at least 14 consecutive days, invery dry DMSO at a dose volume of 4 mL/kg bw, was not associated with any overt adverse effects that could be clearly ascribed to test item administration.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no other informations

Applicant's summary and conclusion

Conclusions:
In conclusion, the test item administrated to Wistar rats at 100, 300 and 1000 mg/kg bw/day, daily for at least 14 consecutive days, in very dry DMSO at a dose volume of 4 mL/kg bw, was not associated with any overt adverse effects that could be clearly ascribed to test item administration.
Executive summary:

The objective of the study was to determine the Maximum Tolerated Dose (MTD) and to obtain preliminary information on the toxicity of VESTANAT EP-M 95 when administered to Wistar rats at 100, 300 and 1000 mg/kg bw/day via daily oral (gavage) administration for at least 14 days, in preparation for a subsequent 28-day study.

A control group was treated concurrently with the vehicle only (water-free DMSO).

Analysis of test item formulations for concentration and homogeneity was performed once during the treatment period, using an X-ray fluorescence method.

The examinations included clinical signs, mortality, body weights, food consumption, clinical pathology, gross pathology and organ weights.

There were no clinical signs or unscheduled mortality during the study.

There were no signs of toxicity observed on body weights or body weight gains during the study.

There was no effect of treatment on food consumption.

There were no test item related findings on the clinical pathology (haematologyand clinical chemistry) parameters evaluated at the completion of the treatment period. 

No test item-related changes were seen during gross necropsy at dose levels up to and including 1000 mg/kg bw/day.

There were no clear toxicologically significant changes in organ weights.

In conclusion, test item administrated to Wistar rats at 100, 300 and 1000 mg/kg bw/day, daily for at least 14 consecutive days, invery dry DMSO at a dose volume of 4 mL/kg bw, was not associated with any overt adverse effects that could be clearly ascribed to test item administration.