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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
Study period:
From Jul. 18, 1988 to Jun. 8, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guideline, with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Government under the revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by Environmental Agency (No. 700), Ministry of Health or Welfare (No. 1039) and Ministry of International Trade and Industry (No.1014).
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG CH 4414 Fuellinsdorf/Switzerland.
- Age at study initiation: 7 weeks.
- Weight at study initiation: Males: 131 - 150 g; Females: 130 - 149 g.
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH 4132 Muttenz, Switzerland). Bedding was autoclaved at 120°C for 60 min. before use.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Seven days under laboratory conditions, after clinical health examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3°C.
- Humidity (%): 40-70%.
- Air changes (per hr): 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
At each dose level, the test article-vehicle mixture (w/w) was prepared by weighing both components in a glass beaker on a tared Mettler balance.
The test article was mixed into the vehicle using an homogenizer. Homogeneity of the test article in the vehicle was maintained before and during treatment using a magnetic stirrer.
Frequency:
The mixtures were prepared daily during the first 9 days of administration, based on the analysis of stability over 2 hours. They were prepared weekly thereafter (from August 2, 1988), when the results of stability analysis over 9 days were available. Stock mixtures were divided into daily aliquots and kept refrigerated in glass flasks (approximately 4°C) for no more than 8 days.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Provided in 5-day Oral Toxicity (Range-Finding, Gavage) Study in the Rat, RCC Project No. 207437.
- Amount of vehicle (if gavage): 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed at RCC UMWELTCHEMIE AG, CH 4452 Itingen, Switzerland according to a method supplied by the Sponsor. Samples for analysis of content, homogeneity and stability (during 2 hours and 8 days) of the test article mixture in vehicle at the lowest and highest dosage administered were taken from mixtures prepared prior to treatment start, and immediately dispatched to the analytical laboratory.
Samples for analysis of content and homogeneity were taken at all dose levels from the mixtures administered to the animals, during weeks 1 and 3 of treatment. An additional sampling for control analysis of content and homogeneity was performed after the end of the treatment.
All analyses showed homogeneity of the mixtures. The mixtures were stable in the range of administered concentrations during 8 days at least. The mean concentrations ranged between 93.2% and 99.7% at all measurements performed at the highest dose level (300 mg/kg bw). At the lowest dosage (10 mg/kg bw), the concentration was of 98.1% at measurement prior to study start whereas mean concentrations ranged between 74.7% and 79.5%, respectively 75.7% and 78.0% at the low and intermediate dosages in the mixtures administered to the animals during the study. The values obtained by analysis of the control mixtures prepared after treatment were consistent with those obtained by analysis of the administered mixtures. Therefore, these values were considered to reflect the concentrations effectively administered.
Duration of treatment / exposure:
28 days in males and 29 days in females.
Frequency of treatment:
Once daily, 7 days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 50, and 300 mg/kg body weight/day (m/f)
Basis:
actual ingested
No. of animals per sex per dose:
10/sex/group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dosage selection based upon following studies:

1) Acute Oral Toxicity Study in Rats, performed by CIBA-GEIGY AG, GU project no, 884013:
LD50 greater than 1000 mg/kg bw and lower than 2000 mg/kg bw.
A single dose of the test article was administered at 3 dose levels as follows: 500 mg/kg bw to 5 male rats, 1000 mg/kg bw to 5 male and 5 female rats, and 2000 mg/kg bw to 5 female rats. All females died at the highest dose level. No mortality occurred in males at the intermediate and lowest dose levels. In the 2000 mg/kg bw dosage group, a slight to severe reduction of spontaneous activity of the animals was observed. Edema in lungs and liquid in the thoracic cavity were observed in females from the highest dosage group at necropsy.

2) 5-Day Oral Toxicity (Range-Finding, Gavage) Study in the Rat, RCC Project No. 207437 (Ciba-Geigy Project No. 884139; Preliminary Results):
TK 13010/ZP was administered by gavage to 3 males and 3 females in each dosage group at dose levels of 0, 100, 300, and 900 mg/kg bw.
The following parameters were recorded: mortality, clinical signs, body weight, food consumption, organ weights (adrenals, heart, kidneys, liver, spleen, ovaries and testes) and macroscopic findings.
The following main changes were attributed to the treatment:
At 900 mg/kg bw: mortality of two out of three animals in each sex, with associated poor health condition preceding death; body weight and food consumption decrease in the surviving male and female animals; at necropsy, stomach distended with gas in most animals, and moderate liver weight Increase in surviving male and female rats.
At 300 mg/kg bw: a slight, transient decrease of food consumption in both sexes; a slight absolute and relative liver weight increase in both sexes.
At 100 mg/kg bw: a slight, transient decrease of food consumption in both sexes.
The following daily doses were selected:
10 mg/kg bw, expected to cause no observable effects.
50 mg/kg bw, expected to result in no or minimal effects,
300 mg/kg bw, expected to result in observable adverse
effects, but not to produce fatalities which would prevent a meaningful evaluation of the study.

- Post-exposure recovery period in satellite groups: 15 days in males and 14 days in females.

Positive control:
Not available.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule:
VIABILITY / MORTALITY: Mortality was recorded twice daily.
CLINICAL SIGNS: Clinical signs were assessed once daily. Descriptions of all abnormalities were recorded and subsequent progress was monitored.



BODY WEIGHT: Yes.
- Time schedule for examinations: The body weight of each animal was recorded weekly throughout the study.



OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations: During pretest (July 21, 1988), towards the end of the treatment (August 18, 1988) and during the last week of
the treatment-free (recovery) period (August 31, 1988).
- Dose groups that were examined: Ophthalmoscopic examination was performed on all surviving animals of the control group and of the highest dosage group

HAEMATOLOGY: Yes.
- Time schedule for collection of blood:
Blood sampling: August 22/23, 1988; September 6, 1988.
- Anaesthetic used for blood collection: Yes (light ether).
- Animals fasted: Yes.
- How many animals: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia.



CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood:
Blood sampling : August 22/23, 1988; September 6, 1988.
- Animals fasted: Yes.
- How many animals: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia.



URINALYSIS: Yes.
- Time schedule for collection of urine:
urine sampling : August 22/23, 1988; September 6, 1988.
- Metabolism cages used for collection of urine: No data.
- Animals fasted: No data.



NEUROBEHAVIOURAL EXAMINATION: No.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
HISTOPATHOLOGY: Yes.
Other examinations:
OTHER: The food consumption was recorded weekly throughout the study.
Statistics:
The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory 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 t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. 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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
lower in high dose group
Food efficiency:
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related mortalities occurred during the study.
One female from the control group died following blood sampling at the end of the treatment period. One female from group 4 died accidentally after 11 days of treatment. No treatment-related clinical signs were observed throughout the study.

BODY WEIGHT AND WEIGHT GAIN
The statistical analysis of body weight did not reveal differences between control values and values in any treated group in both sexes throughout the study. However, marginally lower mean body weight values were observed in males of group 4 when compared to control values. This marginal difference could be indicative of a treatment-related effect. A statistically significant decrease of mean body weight gain was observed in males and females of group 4 during the treatment. During the recovery period, the values were still lower, though statistically were not different from the control values, and showed a tendency to approach control values during the second week of this treatment-free period.

FOOD CONSUMPTION
In group 4, both absolute mean food consumption and mean food consumption per unit body weight showed a slight, statistically-significant decrease in males and in females during the first treatment week, when compared to the respective controls. From the second experimental week up to the end of the recovery period, the values were comparable to those of the controls, except for a slight, statistically significant increase in the mean food consumption per unit body weight in males during the recovery period.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related changes were observed either towards the end of the treatment period or during the second week of the recovery period.

HAEMATOLOGY
For hematological data no changes of toxicological significance were noted at termination of the treatment. However, a slight increase in the erythrocyte count, hemoglobin concentration, hematocrit, reticulocytes and nucleated erythrocytes (also increased in females) was noted for males of group 4. These changes were no longer observed at the end of the treatment-free recovery period.

CLINICAL CHEMISTRY
The assessment of biochemical data indicated the following effects at termination of the treatment for rats of group 4:
- slightly decreased glucose level for both sexes (for females not statistically significant),
- slightly increased creatinine level for both sexes,
- slightly decreased total cholesterol level for males and slightly increased level for females,
- slightly increased triglyceride level for females,
- slightly increased alkaline phosphatase activity for both sexes.
These changes were no longer observed at the end of the treatment-free recovery period.

URINALYSIS
Urinalysis data indicated no treatment-related changes.


ORGAN WEIGHTS
A slight increase of the mean liver weight and of the mean liver to body weight ratio was observed in both sexes in group 4. These weight changes were considered to be treatment-related. They were found to be reversible at the end of the treatment-free recovery period.

GROSS PATHOLOGY
Based on the histopathological examination, the few gross lesions noted in various organs in rats of all groups were considered to be spontaneous in nature and not related to the treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
A histological examination was performed on the adrenal glands, heart, kidneys, liver, lungs, ovaries, spleen and testes from rats of groups 1 and 4, as well as all gross lesions. Minor morphological alterations in the form of multifocal centrilobular hepatocellular hypertrophy were recorded in the liver of eight high dose group rats and one control rat at the end of the treatment period.
The marginal nature of this change, with severity degrees of minimal to slight, was regarded as an indicator of hepatic metabolic compensation at the highest dose level. This change was no longer in evidence following the fourteen day recovery period.

HISTORICAL CONTROL DATA (if applicable)
Other pathology findings were spontaneous in nature, the majority of which were within the normal range of background lesions for these ages and this strain of rat.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In the present study, the major changes which were considered treatment-related or which suggest a possible effect of the treatment, were confined to dose 4 (300 mg/kg bw). These changes are :

- a slight initial decrease in food consumption;

- a marginal to slight decrease of body weight and of body weight gain;

- a slight increase in the erythrocyte count, hemoglobin concentration, hematocrit, reticulocytes and nucleated erythrocytes (mainly in males); these findings may reflect hemopoietic compensation due to an increased oxygen demand required by tissue, particularly with regard to the liver.

- a slight decrease in the glucose level (both sexes) and in the total cholesterol level (males); a slight increase in creatinine level (both sexes), in the total cholesterol level (females), in the triglyceride level (females), and in the alkaline phosphatase activity (both sexes).

These findings were not considered of toxicological significance but may reflect adaptive changes due to an increased functional load. This was also supported by the morphological hepatocellular hypertrophy observed in the liver of most treated rats of group 4 at the end of the treatment.

- a slight increase of the mean liver weight and mean liver to body weight ratio, corresponding in most animals to a slight multifocal centrilobular hepatocellular hypertrophy; these findings, together with the changes recorded by the clinical laboratory investigations, indicated the liver as a potential target organ. All the above findings were no longer observable at the end of the 2-week recovery period except for thie mean body weight which did not correspond to the controls. This indicates a full recovery of the animals and shows the adaptive nature of the changes observed.

Based upon these results, the "no-observable-effect-level" when administered daily by gavage to rats during 4 consecutive weeks was 50 mg/kg bw in rats of both sexes.