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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:
07 September 1998 to 19 October 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

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

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): T-9601
- Physical state: Powder
- Analytical purity: 94.7
- Lot/batch No.: HI-295
- Expiration date of the lot/batch: May 2001
- Stability under test conditions: is guaranteed for 15 days in the vehicle
- Storage condition of test material: darkness at approximately 5C in a refrigerator

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: HARLAN WINKLEMANN, Gartenstr. 27, 33178 Borchen, SPF Breeding colony
- Age at study initiation: approx 5 - 6 weeks
- Weight at study initiation: Males - 106.3g, Females - 105.5g
- Housing: fully air-conditioned rooms in malrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ssniff R/M-H (V 1534) ad libitum, execpt for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles ad libitum, execpt for the periof in which the animals were kept in diuresis cages
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sesame oil DAB 10
Details on oral exposure:
Preparation of the test compound

Dose Concentration Volume applied Vehicle
(mg/kg b.w.) in % (w/v) (ml/kg b.w.)
0.0 0.00 5 sesame oil DAB 10
62.5 1.25 5 sesame oil DAB 10
250.0 5.00 5 sesame oil DAB 10
1000.0 20.00 5 sesame oil DAB 10

T-9601 was suspended in the stated concentrations in sesame oil DAB 10 at the following dates:
1. September 07, 1998
2. September 21, 1998
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determination of the solubility, the stability and the homogeneous distribution of the test substance in oleum sesami was undertaken. Test substance and vehicle were homogenized with ultrasonic energy and yielded suspensions.

Abstract of the analytical method Sample preparation

Three samples (A.B.C) from each test substance vehicle mixture were taken : One sample from the top, a second sample from the middle and a third sample from the bottom of the containers with the test substance vehicle mixtures. All samples were diluted with acetonitrile.

Chromatography
The determination of the test substance was performed by spectro-photometric detection (X= 449 nm) after HPLC-separation on a reverse phase column.

Evaluation of the results
The evaluation of the results has been performed by the method of the external standard. A solution of the test substance in acetonitrile was used as the external standard.
The achieved concentration, stability and the homogeneous distribution of T-9601 in oleum sesami for 4 hours and 15 days at room temperature were confirmed as acceptable in the range of 80 to 120 %.

Remark: For technical reasons 0.964 instead of 1 mg/ml and 250.5 instead of 250 mg/ml was tested. This minor discrepancies fall within the standard deviation of the analytical method and are not considered to have influenced the stability and homogeneity of the spiked vehicle.
Duration of treatment / exposure:
29 days, 28 applications
Frequency of treatment:
7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 62.5 mg/kg bw/day
Male: 5 animals at 250 mg/kg bw/day
Male: 10 animals at 1000 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 62.5 mg/kg bw/day
Female: 5 animals at 250 mg/kg bw/day
Female: 10 animals at 1000 mg/kg bw/day
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Acute oral toxicty testing of the substance in the rat yielded a median lethal dose (LD50) greater than 2000 mg/kg body weight in both males and females. Based on these results the substance was tested in the study at the dose levels of 0.0, 62.5, 250 and 1000 mg/kg body weight per day.
- Rationale for animal assignment (if not random): The rat has proven to be a suitable species for subacute oral toxicity testing with many different substances and is the species of choice according to the international guidelines.
- Post-exposure recovery period in satellite groups: 14 days


Groups of male and female rats received T-9601 by orai gavage at dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day for 28 days. On day 29 five males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 days.
Behavior and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly.
Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth.
Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period.
Hematological examinations and clinical chemistry were carried at the end of the treatment period and after the recovery period.
Urine analysis was carried out at the end of the treatment period and after the recovery period.
During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.
Body weights, hematological and clinical chemistry data, urine data {volume, specific weight), absolute and relative organ weights, neurotaxicological measurements (motor activity, forelimb and hindlimb grip strength) were analyzed with the aid of a statistical program to show differences compared to the controls.
Positive control:
Not conducted

Examinations

Observations and examinations performed and frequency:
Behaviour and state of health
The behaviour and general health condition of the animals were observed once daily.

Neurotoxicological examinations
Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Each animal was assessed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, salivation, nasal discharge, piloerection, pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, tremor, and any other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination was also evaluated.

At the termination of the study sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive) was evaluated including startle reflex (click response), response to approach with the finger to the nose of the animal, and righting reflex. The presence and absence of pupillary constriction was assessed using a pen flash¬light directed into the eye. Assessments of motor function were performed including measurement of motor activity, and forelimb and hind limb grip strength. The animals were evaluated for motor activity during a 60-minute period in an 16-station auto¬mated motor activity monitoring device (FMI, Fohr Medical Instruments GmbH). Activity counts were recorded by the interruption of photocells in 3-minute-intervals to give a total of 20 intervals. Fore- and hind limb grip strength were measured by a strain gauge device (FMI, Fohr Medical Instruments GmbH).

Body weight
The body weights of all animals were determined before the start of the study and then twice weekly throughout the study.

Food and water consumption
Food consumption was determined continuously (2 times per week) and is given in the results as food consumption / animal over a 24 hour period
Water consumption was determined once weekly over a period of 16 hours (from approx. 3.00 p.m. to 7.00 a.m.)

Hematological investigations
At the termination of the study and after the recovery period, hematological examinations were performed on all animals without previous withdrawal of food. Blood samples were taken from the retrobulbar venous plexus in narcosis ((intraperitoneal injection of 67 + 6,7 mg/kg body weight Ketamine-Hydrochloride + Xylazine). In order to prevent systematic errors, blood sampling was conducted in a randomized order (randomization table No. 98.0803 and 98.0804). Parameters determined are listed under any other information.

Clinical chemistry
After blood sampling for hematological testing, the animals were killed by section of the vena cava cranialis in deep narcosis and exsanguinated. In order to prevent sys¬tematic errors, exsanguination was conducted in a randomized order . Parameters determined are listed under any other information.

Urine analysis
Urine analysis was performed on all animals a few days before termination of the study and a few day before the end of the recovery period. For this purpose, the urine was collected by using metabolism cages (overnight from day 25 to day 26 as well as from day 39 to day 40). Food and water were withdrawn during this period. Parameters determined are listed under any other information.
Sacrifice and pathology:
Necropsy and macroscopic examination
After exsanguination, all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

All abnormal Findings were recorded.

Endotracheal Fixation of the Lungs: The lungs, including part of the trachea, were removed. The lungs were then fixed endotracheally with a formalin solution using a needle inserted into the trachea. The instillation pressure was between 20 and 30 cm water column. Following completion of the endotracheal fixation the lungs were fixed, together with the other organs, in formalin solution.

Organ weights
The following organs were weighed and the organ to body weight ratios calculated:
Heart, Testes, Liver, Epididymides, Kidneys, Thymus, Adrenal glands, Brain, Spleen

Histopathology
The following tissues or organs (or pieces of them) were preserved in a suitable fixative and processed for histopathological investigations:
Heart, Lungs, Liver, Spleen, Kidneys, Stomach, Jejunum, Colon, Brain, Thymus, Trachea, Thyroid glands with parathyroid glands, Testes, Epididymides, Ovaries, Uterus, Urinary bladder, Lymph nodes iliac, Lymph nodes mandibular, Adrenal glands, Prostrate gland, Bone marrow (sternum), N. ischiadicus, Spinal cord (cervical), macroscopic findings.
Other examinations:
No other examinations.
Statistics:
The following parameters were compared statistically with the control group values at the level of significance p = 0.05:
Body weights at the designated measurement times
Hematological data
Clinical chemistry parameters
Urine analysis (Volume and specific weight)
Absolute organ weights and organ to body weight ratios
Neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength)
Evaluation was performed by l/S Research and Preclinical Development, Hoechst Marion Roussel Deutschland GmbH, with the aid of a program package for the evaluation of toxicological studies. The calculation methods used are referred to on the computer printouts.

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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly decreased red blood cell counts- females, intermediate & high dose group. Hemoglobin and hematocrit levels slightly decreased - females high dose group. Males from the high dose group showed slightly increased white cell counts.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urine was discolored dark yellow in both sexes from the high dose group
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weights were slightly increased in males from the intermediate and high dose group. These changes were no longer observed at the end of the recovery period.
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
No deaths were encountered in any of the three dosed groups or in the control group. Also behavior and state of health remained unaffected by the administration of the test compound.
Opacity of the refracting media of the eyes and changes of the oral mucosa were not observed.

BODY WEIGHT AND WEIGHT GAIN
Body weight development remained unaffected by the administration of the test compound throughout the study. Body weights were statistically significantly decreased in females from the high dose group on day 1 of the study. As this change was present before the first dosing, a compound-related effect can be ruled out.

FOOD CONSUMPTION
Food consumption remained unaffected by the administration of the test compound throughout the study

WATER CONSUMPTION
Water consumption remained unaffected by the administration of the test compound throughout the study

HAEMATOLOGY
Final value: Statistical evaluation revealed decreased erythrocyte counts, hemoglobin and hematocrit values in females from the high dose group as well as increased leukocyte counts in males from the high dose group. Erythrocyte counts were also statistically significantly decreased in females from the intermediate dose group.
Recovery value: There were no statistically significant changes

CLINICAL CHEMISTRY
Final value: In all cases, where statistically significant changes occurred (increases in potassium values in males from the intermediate and high dose group; increase in calcium values in males from the high dose group; increase in chloride values in males from the intermediate and high dose group and in females from the high dose group, increase in phosphorus levels in males from the high dose group, increase in glucose levels in males from the intermediate and high dose group and in females from all dose groups; increases in urea values in males from the high dose group, decreases in urea values in females from the high dose group; increases in cholesterol values in males from the high dose group; increase in sodium values in females from the high dose group; decreases in triglyceride levels in females from all dose groups; decrease in GPT activity in females from the high dose group) the changes were minor, showed no dependency, or occurred in one sex only. Therefore, a compound-related effect is not evident.

Recovery value: GGT activities were slightly, but statistically significantly increased in males. Females showed statistically significant increases in calcium and uric acid values. As these parameters were not altered at the end of the treatment period, a compound-related effect is not evident.

URINALYSIS
Urine was discolored dark yellow at the end of the treatment period. This change was no longer observed at the end of the recovery period. No other treatment-related changes were detected by urine analysis.
Examination of the sediment did not reveal any abnormalities.

ORGAN WEIGHTS
Final value: Statistical evaluation revealed increases in absolute liver weights in males from the high dose group and increased relative liver weight in males from the intermediate and high dose group. Additionally absolute and relative adrenal weights were decreased to a statistically significant degree in males from the high dose group.
Recovery value: Relative heart weights were increased in males. As heart weights were not affected at the end of the treatment period, a compound-related effect is not evident,

GROSS PATHOLOGY
Yellow discoloration of adipose tissue was observed at necropsy in all dose groups. This finding was reversible by the end of the recovery period in males, but still present in females.

HISTOPATHOLOGY
Histopathological examinations did not reveal any compound-related effect.

Effect levels

Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
With regard to the present study the 'No Observed Adverse Effect Level" (NOAEL) is 62.5 mg/kg body weight per day. However, no clear toxic effects were observed at the dose levels of 250 and 1000 mg/kg per day.
Executive summary:

Study data conducted to EEC- Guideline B.7 and OECD Guideline 407 in compliance with GLP.

In conclusion, slight decreases in erythrocyte counts, hemoglobin and hematocrit val­ues were observed in females after repeated oral administration of T-9601 at the daily dose of 1000 mg/kg body weight. Males showed slightly increased liver weights. Slight decreases in erythrocyte counts (females) and slightly increased liver weights (males) were also observed at the daily dose of 250 mg/kg body weight. A compound-related effect cannot be excluded. However, the changes were minor and there were no histopathological correlates for anemia or liver damage. Likewise, clinical chemistry parameters were inconspicuous, and histopathological examination did not reveal any compound-related effect.

No compound-related adverse effects were observed at the dose level of 62.5 mg/kg body weight per day.

The discoloration of adipose tissue observed at all dose levels is considered not to be of toxicological relevance.

With regard to the present study the 'No Observed Adverse Effect Level" (NOAEL) is 62.5 mg/kg body weight per day. However, no clear toxic effects were observed at the dose levels of 250 and 1000 mg/kg per day.