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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
March 5, 1990 - September 26, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD Guideline 407, GLP study. No neurobehavioural examination performed. Please refer to IUCLID section 13 for read across justification.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

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:
yes
Remarks:
, no neurobehavioural examination performed
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to guideline
Guideline:
other: Japanese Chemical Substance Law (1987)
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
refer to IUCLID section 13
IUPAC Name:
refer to IUCLID section 13

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., CH-4414 Fuellinsdorf
- Age at study initiation: Males: 6 weeks, Females: 8 weeks
- Weight at study initiation: Males 159 - 175 g, Females 160 - 178 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba no. 343, Batches 64/90, 67/90 and 68/90 rat maintenance diet ('Kliba', Klingentalmuehle AG, CH-4303 Kaiseraugst) ad libitum.
- Water: Community tap water from Itingen was available ad libitum
- Acclimation period: Seven days under laboratory conditions, after veterinary 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 hours artificial fluorescent light, 12 hours dark, music and light period

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: bi-distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared balance and the vehicle, bi-distilled water, was added. Weight/volume dilutions were prepared daily prior to application using a homogenizer and kept homogeneous during application with a magnetic stirrer.

VEHICLE
- Amount of vehicle: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations, homogeneity and stability of the test article/vehicle mixtures were determined during the acclimatization period. Further samples for analysis were taken during week 3 of the test and subsequently analyzed.
UV/VIS spectrophotometry: The mean concentrations found were in the range from 100.0 % to 108.1 % of the nominal concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 20, 100, 500 mg/kg bw (group 1, 2, 3 and 4)
Basis:
actual ingested
No. of animals per sex per dose:
10 males, 10 females (groups 1 and 4)
5 males, 5 females (groups 2 and 3)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon data from acute and subacute studies, especially an acute oral toxicity study and a subacute 5-day range-finding study.
- Post-exposure recovery period: 5 males and 5 females of groups 1 and 4 (control and high dose) were kept untreated for an additional 2 weeks and then sacrificed.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality were recorded once daily. Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION:
The food consumption was recorded once during the acclimatization period and weekly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment and a second time on the recovery individuals of groups 1 and 4 at termination of the recovery period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes approximately 18 hours
- How many animals: all
- Parameters checked: Erythrocyte count (RBC), Hemoglobin (HB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELETS), Reticulocyte count (RETIC.), Nucleated erythrocytes normoblasts (NEN), Heinz bodies (HEINZ-BOD.), Methemoglobin (MET-HB), Total leukocyte count (WBC), Differential leukocyte Count (Diff. WBC Count), Red cell morphology, Thromboplastin time (=Prothrombin time) (PT), Activated partial thromboplastin time (APTT),

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Animals fasted: Yes approximately 18 hours
- How many animals: all
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total (BILL T.), Cholesterol, total (CHOLEST. T.), Triglycerides (TRIGL.), Phospholipids (PHOS. LIPID), Aspartate aminotransferase (ASAT/GOT), Alanine aminotransferase (ALAT/GPT), Lactate dehydrogenase (LDH), Creatine kinase (CK), Alkaline phosphatase (ALP), Gamma-glutamyltransferase (G-GT), Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein, total

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 and 6 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Urine was collected during the 18-hour fasting period
- Parameters checked: Volume (18-hour), Specific gravity SPEC. GRAV.), pH, Color, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen (UROBILI.), Urine Sediment SED. MICRO.).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
ORGAN WEIGHTS: The following organ weights were recorded on the scheduled dates of necropsy and their organ to body weight as well as organ to brain weight ratios determined: Brain; Pituitary gland; Heart; Thyroid gland; Liver; Kidneys; Adrenals; Spleen; Testes; Ovaries.

GROSS PATHOLOGY: Yes, samples of the following tissues and organs were collected from all animals at necropsy and fixed: Adrenals; Aorta; Brain; Cecum; Colon; Duodenum; Epididymides; Esophagus; Eyes with optic nerve and Harderian gland; Female mammary gland area; Femur including joint; Heart; Ileum; Jejunum; Kidneys; Larynx; Lacrimal gland, extraorbital; Liver; Lung infused with formalin; Lymph nodes, mandibular, mesenteric; Nasopharynx; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary gland, mandibular, sublingual; Seminal vesicles; Sciatic nerve; Skeletal muscle; Skin; Spinal cord, cervical, midthoracic, lumbar; Spleen; Sternum with marrow; Stomach; Testes; Thymus; Thyroid gland; Tongue; Trachea; Urinary bladder infused with formalin; Uterus with uterine cervix; Gross lesions.

HISTOPATHOLOGY: Yes, the following organ and tissue samples were analyzed: Adrenals, Heart, Kidneys, Liver, Spleen and Stomach collected at terminal sacrifice from the animals of the control and high-dose groups were examined by a pathologist. The same applied to all Gross lesions. Upon detection of treatment related morphologic changes in the livers and kidneys of high dose animals histological examination of the same organs was extended to all dose groups.
Statistics:
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 for each sex.

The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.

The exact Fisher-test was applied to the overall ophthalmoscopy data.

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

Details on results:
CLINICAL SIGNS AND MORTALITY
No death occurred prior to the scheduled times of termination and necropsy.
No clinical signs were found in groups 1, 2 and 3 (0, 20, 100 mg/kg, respectively) throughout the study.
Individuals of group 4 (500 mg/kg) showed the following clinical signs: in all individuals blue discoloration of the feces on treatment day 2. One of ten males displayed slight sedation on treatment day 2. In one of ten females slight sedation from approximately 1 hour until 3 days after treatment start (=day 4) concomitant with slight emaciation from treatment day 2 to 4 and slightly ruffled fur during most of the treatment period. Slight emaciation was also found in another female individual on treatment day 8. In one main test male and two recovery females slight and/or moderate alopecia of various body parts mainly during the latter half of the treatment period, and in one of these females also throughout the recovery period. Some of these findings, e.g. sedation and emaciation may reflect a general discomfort of the corresponding rat individuals. However, they were regarded to give no clear indication of toxicity, because they occurred in only few of the high dose individuals.

BODY WEIGHT AND WEIGHT GAIN
The body weight did not appear to be markedly affected by the treatment with test article, although from recovery day 8 until termination high dose females gained statistically significantly less weight than control females did.

FOOD CONSUMPTION:
The absolute and relative food consumption rates of females of group 4 (500 mg/kg) were significantly lower than the corresponding controls during treatment days 1 to 8, but had increased to levels above control by treatment days 15 to 22. During the latter period the relative food consumption rates of both sexes of group 4 were statistically significantly higher than their controls. These findings and the fact that the rates of food consumption declined in all groups from pretest to treatment days 1 to 8 may reflect a process of adaptation to the administration of test article, but were considered to be toxicologically non-significant.

OPHTHALMOSCOPIC EXAMINATION
A degenerated cornea was found in the right eye of one control female towards the end of the recovery period. No other abnormalities occurred.

HAEMATOLOGY
The assessment of hematological data indicated no changes of toxicological significance neither at termination of the treatment nor at the end of the treatment free recovery period. However, a slight increase in the reticulocyte count and slight polychromatophilia was noted for female rats of group 4 after 4 weeks of treatment. This may be interpreted as indicating a demand for new erythrocytes and a competent bone marrow. In addition, a slight increase in the methemoglobin concentration was noted for both sexes of group 4 after 4 weeks. This was not considered to be an effect of treatment, but more likely a false positive related to spectral interference of the test article with absorbance measurements. High absorbance at red wavelengths for blue dyes may cause an abnormal increase in the methemoglobin reading. All of the changes noted were found to be reversed at termination of the treatment-free recovery period. All other statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

CLINICAL CHEMISTRY
For biochemical data the following effects were noted for rats of group 4 after 4 weeks of treatment:
- slightly increased urea concentration for both sexes;
- slightly increased bilirubin concentration for males;
- slightly increased triglyceride and phospholipid concentration for both sexes;
- slightly decreased sodium concentration for males.
- slightly increased calcium concentration for females.
The changes noted were considered to be of metabolic nature possibly due to changes in the liver and kidney. These findings were found to be reversed at termination of the treatment-free recovery period. All other statistical differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

URINALYSIS
Urinalysis data indicated a slightly higher pH value, slight proteinuria and ketonuria, moderate to marked bilirubinuria and marked urobilinogenuria for both sexes of group 4 after 4 weeks of treatment. Furthermore, both sexes of group 3 indicated a light brown and both sexes of group 4 a violet urine discoloration. These changes were no longer observed at termination of the treatment-free recovery period. It is assumed that these findings are not treatment-related, but rather false positives related to the blue nature of the test article, which gave a highly pigmented urine sample at the highest dose level, and thereby causing a masking effect which interfered with the test procedure. All other differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation.

ORGAN WEIGHTS
After 28 days of treatment with 500 mg/kg/day (group 4) the liver, kidney and adrenal weights were statistically significantly higher than the corresponding controls in both sexes, and the spleen weights in males. In recovery individuals most of the above organ weights and ratios had returned to levels similar to their controls after the treatment-free 14-day recovery period. However, the liver to body weight ratio of recovery females was still statistically significantly higher than control. No other significant differences of organ weights or their ratios from control occurred

GROSS PATHOLOGY
Gastro-intestinal discoloration was apparent in one male group 2 (20 mg/kg) and 10 group 4 (5 male; 5 female) main test animals. There were no other treatment related findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no findings which equated with the macroscopic discoloration of the gastro-intestinal tract.
The livers from five group 4 main test (2 male; 3 female) and recovery (3 male; 2 female) animals showed a slight increase in mononuclear cell infiltration, particularly around the portal triads. These cells were histiocytic in appearance, having foamy pink cytoplasm. There was an increase in the number of animals showing moderate hyaline droplet degeneration in groups 3 (100 mg/kg) and 4 (500 mg/kg) male main test kidneys (3 and 4 animals respectively, compared to one group 1 (recovery) animal), which was not apparent in the recovery animals. The remaining findings were considered to be of a spontaneous nature consistent with the age and strain of animal.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: at the next higher dose, liver and kidney weights were increased, increases in the severity of periportal mononuclear cell infiltration in the liver, increase in the number of males showing moderate hyaline droplet degeneration in the kidneys.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Based upon the results obtained in this study, the "no-adverse-effect-level" of the test substance is 100 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days.

 

This conclusion is mainly based on the following changes in high dose rats when compared to controls treated with the vehicle only:

- Absolute and/or relative weights of liver, kidneys and adrenals statistically significantly higher than their controls in both sexes and of the spleen in males at termination of the treatment period.

- Slight increases in the severity of periportal mononuclear cell infiltation in the liver of both sexes at termination of the treatment and of the recovery period.

- An increase in the number of males showing moderate hyaline droplet degeneration in the kidneys at termination of the treatment period.

- A slight increase in the reticulocyte count and slight polychromatophilia for females at termination of the treatment period.

- The following changes of blood biochemistry parameters at termination of the treatment period:

Slightly increased urea concentration for both sexes.

Slightly increased bilirubin concentration for males.

Slightly increased triglyceride and phospholipid concentration for both sexes.

Slightly decreased sodium concentration for males.

Slightly increased calcium concentration for females.

 

The liver was identified as a potential target organ because of the increases in liver weight in the severity of periportal mononuclear cell infiltration and in reticulocyte counts, the kidney because of the increases in kidney weight and in the number of males showing moderate hyaline droplet degeneration in the kidneys. The changes in the blood biochemistry parameters listed above were considered to be of metabolic nature possibly due to changes in the liver and kidneys. A further potential target organ may be the spleen, because of the increases in spleen weight in high dose males and the increases in reticulocytes in high dose females.

Except for the histopathology findings in the liver and the liver to body weight ratio in females, the above findings had returned to normal in recovery individuals of group 4 (500 mg/kg) by the end of the treatment-free 14-day recovery period. Therefore, the "no-adverse effect level" was set at 100 mg/kg, despite the finding of moderate hyaline degeneration in the kidneys of males of group 3 (100 mg/kg) at termination of treatment

Applicant's summary and conclusion

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