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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP. FOB and sperm analysis were not performed

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Pale yellow powder
- Analytical purity: 98.4 %
- Expiration date of the lot/batch: November 1997
- Storage condition of test material: 4°C in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 6 weeks
- Weight at study initiation: 151-195 g for the males, 120 - 146 g for the females
- Housing: in groups of five according to sex in metal cages with wire mesh floors
- Diet (e.g. ad libitum): a standard pelleted laboratory rodent diet (Special Diet Services Rat and Mouse Maintenance Diet) was offered ad libitum
- Water (e.g. ad libitum): drinking wate was offered ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 22.5° C
- Humidity (%): 31 to 71%
- Air changes (per hr): 19
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methylcellulose 1%
Details on oral exposure:
The test substance was formulated in 1 % aqueous methylcellulose (1 % MC).
The test substance was weighed out and ground using a mortar and pestle. A small amount of the vehicle was added and ground into the powder to form a smooth paste. Further amounts of the vehicle were added and mixed in. Once up to volume, the suspension was further mixed using a high shear homogeniser. Any samples were taken whilst the suspensions were being thoroughly mixed using a magnetic stirrer. Each concentration was made by direct dilution. Formulations were prepared freshly once a week.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Concentration analyses of formulations prepared for administration on day 1 and 22 were performed by the Department of Analytical Chemistry. Four 1 mL samples were collected at each dosage on each occasion for analysis. The analytical procedure was validated for TKA 40135 in 1% MC with respect to the specificity of the chromatographic analysis, linearity of detector response, precision of injection, limit of detection, method accuracy and precision. The mean results for the test formulations analysed during the toxicity study were within 5.5% of
nominal concenttations confirming the accuracy of formulation. The results confirm that the formulations were homogeneous and stable during ambient temperature storage for 2 days and refrigerated storage for 8 days, a period representing the maximum time from preparation to completion of dosing.
Duration of treatment / exposure:
28 days
Frequency of treatment:
One single oral dosage per day
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 150, 1000 mg/kg bw/day (group1, 2, 3 and 4)
Basis:
actual ingested
No. of animals per sex per dose:
In the control and the high dose group (0, 1000 mg/kg bw/day): ten animals per sex and group
15 and 150 mg/kg bw/day: five per sex and group
Control animals:
yes, concurrent no treatment
Details on study design:
DOSING
The dosage was selected on the basis of available toxicity data, including an acute oral toxicity study performed at the same facilities.
The test substance was administered by oral gavage to rats and rubber catheter at a dose volume of 10 mL/kg bw/day.
Control animals received the vehicle, 1 % MC, at the same dose volume.
Prior to dosing, the test substance formulations were mixed by inversion.
Subsequent mixing using a magnetic stirrer occurred for a period of at least 10 minutes before dosing commenced.

STUDY DESIGN
Following the 4 week treatment period, five male and five female animals from groups 1 and 4 and surviving animals of groups 2 and 3 were sacrificed (day 32 post-treatment sacrifice). These rats were dosed until the day prior to sacrifice. The remaining animals of groups 1 and 4 were retained for a 2 week post-treatment recovery observation period and were sacrificed on day 46; these rats received their last dose on Day 28.

Examinations

Observations and examinations performed and frequency:
MORTALITY AND CLINICAL SYMPTOMS OF TOXICITY
All animals were observed daily for signs of ill health, behavioural changes or symptoms of toxicity.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately mid-day.

BODY WEIGHT
All rats were weighed at the time of allocation to groups, prior to dosing on day 1 and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

WATER CONSUMPTION
Daily monitoring by visual appraisal was maintained throughout the dosing period.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Blood was withdrawn under light anaesthesia from the orbital sinus of five male and five female rats selected from the control and the high dose groups, and all rats of the low and the mid dose groups. These samples were collected after 4 weeks of treatment. Further removal of blood samples for re-analysis was carried out for individual animals. Investigations were carried out for remaining rats after the 2 week recovery period, for all individual parameters which had been statistically significant after the 4 weeks treatment period.

Following haematology parameters were considered:
Packed cell volume (PCV), Haemoglobin (Hb), Red cell count (RBC), Mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Total white blood cell count (WBC Total), Differential WBC count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelet count (Plt). Cell morphology also was considered with the most common morphological changes, such as
(anisocytosis (Anis), micro/macrocytosis (Micro/Macro), variation in colour (Var), hypo/hyperchromasia (Hypo/Hyper), left shift (LS), atypical/blast cells (Atyp/Blast) being recorded. Thrombotest (TT) and Activated Partial Thromboplastin Time (APTT) also were performed/measured.

Following clinical chemical parameters were considered:
Glucose (Gl), Total Protein, Albumin (Alb), Globulin (Glob), Albumin/Globulin ratio (A/G), Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP), Glutamic-pyravic transaminase (GPT), Glutamic-oxaloacetic transaminase (GOT), Gamma-glutamyltransferase (GT), Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic phosphorus (P), Cholesterol (Chol), Triglycerides (Triglyc).

URINALYSIS
Each rat was placed in a urine collecting cage overnight prior to collection of samples, without access to food or water. Overnight urine samples were collected after 4 weeks of treatment, and after the recovery period of 14 days.
Following parameters were considered:
Volume (Vol), pH, Specific Gravity (SG), Protein, Total reducing substances (TRS), Glucose (Gluc), Ketones (Ket), Bile pigments (Bile pigs), Urobilinogen (Uro-bi), Haem. pigments (Haem Pigs).
Microscopic examination of urine samples was carried out, the deposit was examined for the presence of Epithelial cells, Polymorphonuclear leucocytes, Mononuclear leucocytes, Erythrocytes, Organisms, Renal tubule casts, Sperm, Other abnormal constituents.
Sacrifice and pathology:
Any rat found dead was subjected to detailed macroscopic examination in an attempt to define the cause of death.
After 4 week of treatment and following clinical pathology investigations, surviving animals were selected for sacrifice by carbon dioxide asphyxiation and were subjected to complete gross pathology. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.
Remaining rats of the control and the high dose group were sacrificed after a treatment-free recovery period of 14 days and examined in a similar manner as described above.

ORGAN WEIGHT
The following organs from each animal were dissected free of fat and weighed: adrenals, liver, seminal vesicles, brain, ovaries, spleen, epididymides, prostate, testes, kidneys.

GROSS PATHOLOGY
The macroscopic appearance of the tissues of all rats was recorded and samples of the following tissues were preserved in adequate fixative: adrenals, aorta, brain, caecum, colon, duodenum, eyes, femur (for bone and marrow sections) with joint, Harderian gland, head (to preserve nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth,lacrymal gland and Zymbal's gland), heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (cervical and mesenteric), mammary glands, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, sternum, testes with epididymis, thymus (where present), thyroid and parathyroid, tongue, trachea, urinary bladder, uterus (with cervix), vagina, all gross lesions. From these tissues, the following were required for histopathological examination: adrenals, heart, kidneys, liver, spleen, testes including epididymis, all gross lesions. These tissues were embedded in paraffin and sectioned; the sections were stained with haematoxylin and eosin. A transverse section of each testis (left and right) and a full longitudinal section of each epididymis (left and right) were cut as near as possible to 2 µm and stained with Periodic Acid Schiff-haematoxylin. Microscopic examination of the testes was made with reference to the stages of the cycle of the seminiferous epithelium.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as basic experimental unit.
For bodyweight gains, organ weight and clinical pathology, iIf the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode was analysed by Fisher's exact test, followed by Mantel's test for a trend in proportions. Otherwise, Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was fried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. When more than two groups were being compared, this analysis was followed by Williams' test for a dose related response; for two group comparisons Student's t test was used.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. When more than two groups were being compared, this analysis was followed by the non-parametric equivalent of Williams' test; a non-parametric equivalent of Student's t test was used for two group comparisons.
Covariate analysis of organ weight data (with final bodyweight as covariate) was performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.
Significant differences between control animals and those treated with the test substance were expressed at the 5% {p <0.05 for t test analysis and p <0.05 for Williams' test analysis) or 1% {p <0.01 for t test analysis and p <0.01 for Williams' test analysis) level.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
There was no treatment-related mortality and/or clinical symptoms.
In fact, one female rat of the 1000 mg/kg bw/day group was found dead in week 1. Prior to death the animal had shown no signs of ill health, behavioural change or reaction to treatment. The macroscopic post-mortem examination revealed pale gelatinous material and clear fluid in the thoracic cavity as well as oesophagus perforation. The death of this animal was therefore considered accidental.

BODY WEIGHT AND WEIGHT GAIN
Overall, bodyweight was considered to be unaffected by treatment.
At week 4, bodyweight gains for male and female rats in all treated groups were comparable to controls.
During recovery, bodyweight gains for male and female animals of the high dosage group were lower than control but did not attain statistical significance.

FOOD CONSUMPTION
Overall, food consumption was similar to control for male and female rats in all treatment groups. Efficiency of food utilisation for both sexes in all treated groups was essentially similar to concurrent controls.
During the recovery period, food consumption for females of the 1000 mg/kg bw/day group was higher than control; for the males of this group, food consumption during recovery period was similar to control. Overall efficiency of food utilisation, during the recovery period, for males and females receiving 1000 mg/kg bw/day was lower in comparison with concurrent controls, reflecting the lower bodyweight gains noted for these animals.

WATER CONSUMPTION
Water consumption was found to be similar in all groups and therefore unaffected by treatment.

HAEMATOLOGY
There were no findings of toxicological importance.
In fact, at ending of the treatment period, a dose-related increase in neutrophil counts was reported for the males in all treated groups, with statistical significance being attained for the high dose group of 1000 mg/kg bw/day (2.97 x 10E+9/L versus 1.34 10E+9/L for control). However, these changes were considered to be of no toxicological importance in the absence of other corroborative changes indicative of an effect of treatment. Furthermore, statistically significantly higher than control mean corpuscular haemoglobin (MCH) was recorded for the males in all treated groups (20.4, 20 and 20.2 pg at 15, 150 and 1000 mg/kg bw /day, respectively, versus 19.1 for control). However, in the absence of a dosage relationship and as there was overlap between the groups a treatment related effect was considered unlikely. There were no differences from control for haematological parameters measured at the end of the recovery period.

CLINICAL CHEMISTRY
There were no findings considered to be of toxicological importance.
In fact, at ending of the treatment period, lower than control glutamic-oxaloacetic transaminase (GOT) levels were recorded for females in
all treated groups with statistical significance being achieved for the intermediate and the high dosage (respectively 57 and 56 mU/mL versus 66 for control). However, in the absence of other corroborative changes indicative of an effect of treatment, this was considered unlikely to be of toxicological importance.
For the males receiving 1000 mg/kg bw/day, gamma-glutamyltransferase (1 mU/mL versus 3 for control) and alkaline phosphatase levels (260 mU/mL versus 392 for control) were statistically significantly lower than control. However, in the absence of other changes indicative of an effect of treatment, these changes were considered of no toxicological importance. Albumin levels were marginally lower than control for males treated at 1000 mg/kg bw/day (2.8 g/dL versus 3 for control), but since the change was minor in nature and was unaccompanied by other changes indicative of a reaction to treatment, it was considered of no toxicological importance. Slightly lower than control calcium (5.6 mEq/L versus 5.9) and phosphorous levels (5.0 mEq/L versus 5.6) were further recorded for the males treated at 1000 mg/kg bw/day. However, these minor differences from control were considered not to be related to treatment.
There were no differences from control for biochemical parameters measured at the end of the recovery period.

URINALYSIS
There were no findings of toxicological importance
In fact, at ending of the treatment period, lower than control urinary volumes and associated increased urinary protein levels and specific gravity achieved statistical significance for male rats in all treated groups (urinary volume: 4.8, 5.7 and 3.5 mL at 15, 150 and 1000 mg/kg bw /day, respectively, versus 8.8 for control; protein: 380, 288 and 436 mg/dL at 15, 150 and 1000 mg/kg bw /day, respectively, versus 129 for control). In the absence of a clear dosage-relationship and as there were some particularly high control values for urinary volume a treatment related effect was considered unlikely.
Following the two week recovery period protein levels were higher than control for male rats of the high dosage group (235 mg/dL versus 135), but did not attain statistical significance. In the absence of changes at the treatment period indicative of an effect of treatment, this finding was considered coincidental and of no toxicological importance. There were no further differences from control in the urinary parameters measured at the end of the recovery period.

NECROPSY
Necropsy performed at the end of the treatment period and at the end of the recovery period revealed no gross abnormalities in any of the treated groups or controls.
Referring to organ weights, there were no differences from control in the organ weights measured at the end of the treatment period. After recovery, group mean (bodyweight adjusted) spleen weight was found to be statistically significantly higher for males treated at 1000 mg/kg/day compared to control (0.87 g versus 0.70). This was considered to be caused by a particularly high value for one animal only. In the absence of any treatment related effects on this organ at the end of treatment, the difference from control was considered to be incidental reflecting normal biological variation.
At the end of the treatment period, no treatment-related findings were reported in tissues subjected to histopathological examination. In fact, changes when they were observed were within the range of background histopathological findings typical for the age and strain of the laboratory-maintained rat, and of no toxicological relevance. Thus, no treatment-related findings were reported in rats sacrificed at termination and rats sacrificed after a recovery period of 2 weeks.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects were seen

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body weights gain, group mean values (g):

Week

Dosage (mg/kg bw/day)

Males

Females

0

15

150

1000

0

15

150

1000

Cumulative gain in g (week 0 to 4)

187

180

186

176

87

86

90

89

SD

28

13.4

41.6

27.7

14.8

18.7

3.3

17.6

as % of control

-

96

99

94

-

99

103

102

Cumulative gain in g after recovery

29

Not measured

Not measured

22

12

Not measured

Not measured

10

SD

8.9

-

-

2.9

6.4

-

-

4

as % of control

-

-

-

76

-

-

-

83

Food consumption, group mean values (g):

 

Week

Dosage (mg/kg bw/day)

Males

Females

0

15

150

1000

0

15

150

1000

Cumulative gain in g (week 0 to 4)

852

826

861

843

578

585

578

622

as % of control

-

97

101

99

-

101

100

108

Cumulative gain in g after recovery

440

Not measured

Not measured

425

307

Not measured

Not measured

387

as % of control

-

-

-

97

-

-

-

126

Applicant's summary and conclusion