Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 -31 March 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white solid
- Lot/batch No.: 85-36
- Expiration date of the lot/batch: 18 December 1998
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
other: Wistar Crl:(WI)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 182-205 g (males), 156-174 g (females)
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were indivudually housed overnight in Macrolon plastic cages with sterilized sawdust (B.M.I. Helmond, the Netherlands) provided as bedding.
- Diet: Free access to standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium)
- Water: Free access to tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): app. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and placed on a magnetic stirrer during dosing. The test substance formulation in propylene glycol was stable for at least 4 hours. Adjustment was made for the specific gravity of the vehicle in all formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the test laboratory
- Concentration in vehicle: 1, 4 and 20%
- Amount of vehicle (if gavage): 5 mL/kg
- Specific gravity: 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analysed to check stability, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations). The formulations were stirred during the sampling. Duplicate samples for the determination of the concentrations and stability were taken at 50% height of the formulation. For the determination of the homogeneity, duplicate samples were taken at 90% height, at 50% height and at 10% height of the formulation. The samples were prepared for HPLC determination of the analytical concentrations. The actual concentrations were found to be 93-99% of nominal, which represents an acceptable level of accuracy. In addition the substance formulation propylene glycol was noted as stable for at least 4 hours and homogenous (95-104% relative to mean concentration).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200 and 1000 mg/kg bw/d
Basis:
other: nominal
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a range-finding study 3 rats/sex/group were administered 50, 200 and 1000 mg/kg bw/d for 5 days. No mortality was observed and no effect on the body weight or food consumption was noted. In 2 males in the 50 mg/kg bw/d group and 1 male in the 1000 mg/kg bw/d group, salivation was observed. The liver and kidney weight were normal and no gross pathological effects were observed. 1000 mg/kg bw is the recommended maximum dose according to OECD test guideline 422. Based on the results of the range-finding study, the same 3 doses were selected for the main study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality, once daily for detailed clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes. Observations were made outside the home cage in a standard arena.
- Time schedule: Once before the first exposure on day 1, and thereafter on day 8, 15, 22 and 28

BODY WEIGHT: Yes
- Time schedule for examinations: Once before the first exposure on day 1, and thereafter on day 8, 15, 22 and 28

FOOD CONSUMPTION AND COMPOUND INTAKE: food consumption was measured weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No, only a subjective appraisal was maintained during the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to necropsy, drawn from the retro-orbital sinus
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, overnight with a maximum of 20 hours
- How many animals: 5/sex/dose
- Parameters examined: Erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, red cell distribution width, total leucocyte count, differential leucocyte count (neutrophils, eosiniphils, basophils, lymphocytes, monocytes), prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 28, prior to necropsy
- Animals fasted: Yes, overnight with a maximum of 20 hours
- How many animals: 5/sex/dose
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, bilirubin (total), cholesterol (total), creatinine, glucose, urea, protein (total), albumin, alkaline phosphatase, potassium, sodium, calcium, chloride, phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 of the study for males and females
- Dose groups that were examined: All dose groups
- Battery of functions tested:
Standard functional observational battery (FOB) including grip strength measurements, auditory response, static righting reflex, pupillary reflex; and motor activity (MA), measured as number of movements per hour over a 12-hour period, with a computerised motor activity monitoring system (Pearson Technical Services, Debenham, Stowmarket, England). Each animal was tested individually.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were necropsied and the tissues/organs subjected to macroscopic examination
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY: Yes. Samples of the tissues/organs listed below were collected from all animals and fixed in a buffered solution. Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, as well as from the animal which died spontaneously, and all gross lesions of all animals were examined. Slides were stained with hematoxylin and eosin. Tissues mentioned between brackets were not examined as there were no signs of target organ involvement.
Adrenal glands, Aorta, Brain, Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland),(Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung - infused with formalin, Lymph nodes - mandibular - mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer’s patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord - cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions
Statistics:
The following statistical methods were used to analyse the data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one rank test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group rneans were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been
rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red staining of the fur, salivation, alopecia
Mortality:
mortality observed, treatment-related
Description (incidence):
Red staining of the fur, salivation, alopecia
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
non-adverse decrease in mean corpuscular hemoglobin count and partial thromboplastin time in males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
non-adverse, increased potassium levels in females
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One female receiving 50 mg/kg bw/d was found dead on day 28, although motor activity test results indicate the animal died on day 27, as the test started. Based on the clinical signs (hunched posture, laboured breathing) and findings during the necropsy, the death may have been related to mis-dosing.
Alopecia was noted in 2, 0, 2 and 1 males in the 0, 50, 200 and 1000 mg/kg bw/d groups, respectively. This is a normal observation in rats during repeated dose studies and is not a substance-related effect. Salivation was observed in 3, 3, 2 and 2 males, and in 1, 2, 3 and 2 females administered 0, 50, 200 and 1000 mg/kg bw/d, respectively. This effect is frequently noted in rats dosed by gavage, due ot the repeated use of the stomach tube, and is not considered to be substance-related. One male and 1 female in the 50 mg/kg bw/d group, and all (5) the females in the 1000 mg/kg bw/d group had red staining of the fur from week 2 or 3 onwards during the study period. It is a normal occurrence in rats that red liquid is discharged from the nose and eyes and is spread over the fur by grooming. During periods of stress or discomfort excretion of this liquid may be increased. The staining of the fur may also indicate a reduced grooming activity of the animals. The cause of the increased staining of the fur in the animals could not be established, but based on the lack of other clinical signs of discomfort and the lack of further neurological or pathological results, the effect is not considered to be a sign of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related effects on body weight and body weight gain during the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE
There was no difference in food intake between the control group and treatment groups during the study period.

HAEMATOLOGY
A slight, but statistically significant decrease in the mean corpuscular hemoglobin count in males administered 200 mg/kg bw/d, and in the partial thromboplastin time in males administered 50 and 200 mg/kg bw/d was observed. As the effects were not noted in the highest dose group, they are not likely to be treatment-related. No haematologic effects were seen in females.

CLINICAL CHEMISTRY
There were no statistically significant changes in clinical chemistry parameters in the males. Females administered 1000 mg/kg bw/d had a statistically significant increase in potassium levels (4.98 mmol/L) compared to the control group (4.05 mmol/L). The significance may be due to a low control group level, as the low- and mid-dose group levels were 4.77 and 4.38 mmol/L, respectively. As there were no similar effects in males and no related histopathological effects, this result is not considered to be of toxicological relevance.

NEUROBEHAVIOUR
The observation of one male in the control group showed statistically significant reduction in motor activity during the 12 hour monitoring was the only effect on the neurobehaviour parameters noted. No effects on the neurobehaviour was observed in male or female rats trated with the test substance in the functional observation tests (including grip strength and reflex testing) and motor activity tests.

ORGAN WEIGHTS
There were no treatment-related effects on the absolute or relative organ weights of the animals.

GROSS PATHOLOGY
In the female administered 50 mg/kg bw/d that died on day 27, haemorrhage in the lungs, a thickened limiting ridge in the stomach and discolouration of the mandibular lymph node was noted. These effects are most likely due to mis-dosing of the animal on day 27. The uterus of one female in the 1000 mg/kg bw/d group contained a watery fluid. This is related to a specific stage in the oestrous cycle and is therefore a normal finding. There were no treatment-related findings of the gross pathology examination in any of the other treated or control animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the female administered 50 mg/kg bw/d that died on day 27, haemorrhage in the lungs, a thickened limiting ridge in the stomach and coagulation of the mandibular lymph node was noted, confirming the results of the gross pathology examination. These effects are most likely due to mis-dosing of the animal on day 27.
There were no treatment-related histopathologic findings in any of the other animals.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects up to and including the highest dose level tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results presented in this report the No Observed Adverse Effect Level (NOAEL) for TS-2863 is considered to be 1000 mg/kg bw/d. The clinical signs were of minimal severity and no morphological or other functional changes were observed in the animals.
Executive summary:

A subacute 28 -day oral toxicity study was performed in 5 rats/dose/sex, administered TS-2863 by gavage, according to EU method B.7 and OECD guideline 407.

Based on the results of a 5-day dose range-finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 200 and 1000 mg/kg bw/d. The following parameters were evaluated: mortality; clinical signs daily; neurobehaviour (functional observation battery tests); body weight and food consumption weekly; hematologic and clinical chemistry parameters, clinical pathology and macroscopy at termination; organ weights; and histopathology on a selection of tissues.

RESULTS

50 mg/kg bw/d: No substance-related findings noted.

200 mg/kg bw/d: No substance-related findings noted.

1000 mg/kg bw/d: Red staining of the fur observed in females.

From the results presented in this report the No Observed Adverse Effect Level (NOAEL) for TS-2863 is considered to be 1000 mg/kg bw/d. There were no substance-related effects up to and including the highest dose level.