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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-09-11 to 2002-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats CrIGIxBrIHan
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 d
- Weight at study initiation: males 146.1 - 165.4 g(group mean: 154.9 g); females 116.5 - 131.8 g(group mean: 124.7 g.)
- Housing: single
- Cage type: DK III stainless steel wire mesh cages (Becker & Co., Castrop-Rauxel, Germany.)
- Diet (ad libitum): ground Kliba maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland.)
- Water (ad libitum): demineralized drinking water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
DOSING SOLUTIONS AND EXPERIMENTAL PROCEDURE:
For each concentration, the test substance was weighed out in a beaker, transferred to a 10-litre can, and demineralized water was added to the desired volume. Subsequently, the preparations were mixed with a magnetic stirrer for about 5 minutes. The mixtures were prepared twice a week.
The test substance was administered daily in demineralized water for about 3 months. Control animals received demineralized water only . At the end of the 3-month administration period all animals were sacrificed (withdrawal of food for about 16 - 20 hours).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out as a separate study. The stability of the test substance in water over a period of up to 96 days at room temperature was tested prior to the start of the study with a comparable batch. Concentration control analyses of the test substance preparations were performed with samples of all concentrations at the start and at the end of the administration period. Homogeneity analyses of the test substance preparations were not necessary as the preparations were aqueous solutions.
Duration of treatment / exposure:
3 months
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Dose / conc.:
200 ppm
Remarks:
14 mg/kg bw/day in males; 21 mg/kg bw/day in females
Dose / conc.:
1 000 ppm
Remarks:
64 mg/kg bw/day in males; 91 mg/kg bw/day in females
Dose / conc.:
5 000 ppm
Remarks:
285 mg/kg bw/day in males; 339 mg/kg bw/day in females
No. of animals per sex per dose:
10
Control animals:
yes
Positive control:
None

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS:
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further clinical examinations were carried out daily.

BODY WEIGHT:
Individual body weights were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and water consumption.

OPHTHALMOSCOPIC EXAMINATION:
One day prior to the start of the administration period the eyes of the animals of the all dose groups were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic (Pharma Stulln GmbH, Stulln, Germany). On day 91, only animals of the high dose group and controls were examined.

HAEMATOLOGY, CLINICAL CHEMISTRY:
Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anesthesia for examination of haematological and clinical chemical parameters. Following parameters were considered.

- Haematological parameters:
Leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and differential blood count.

- Clinical chemical parameters:
Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol and magnesium.

URINALYSIS:
The individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence.
The following examinations were carried out:
Volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and sediment.

SPERM PARAMETERS:
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals. The following parameters were determined:
Sperm motility, sperm morphology, sperm head count (cauda epididymis) and sperm head count (testis).

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery (FOB) was performed in all animals at the end of the administration period. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotortests as well as reflex tests. Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System. During the measurement the animals were kept in Polycarbonate cages with absorbent material and received no food and no water.
Sacrifice and pathology:
At study termination, the animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied, assessed by gross pathology, and organs were weighed. Tissues were collected and fixed for further histopathological examinations.

ORGAN WEIGHTS:
Liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart and thymus.

HISTOPATHOLOGY:
The following organs were fixed in 4 % formaldehyde solution:
All gross lesions, salivary glands (Glandula mandibularis and Glandula sublingualis), esophagus, stomach (forestomach and glandular stomach), duodenum/jejunum/ileum, cecum/colon/rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cord), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose (nasal cavities), aorta, heart, bone marrow (femur ), lymph nodes (mandibular and mesenteric), spleen, thymus, kidneys, urinary bladder, oviducts/uterus/vagina, prostate gland, seminal vesicles, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint and extraorbital lacrimal glands. The left testis and the left epididymis as well as both ovaries were fixed in Bouin's solution and embedded in paraplast, thereafter.
Statistics:
Means and standard deviations of each test group were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of the animals in each test group. Statistical analyses were performed using the Kruskal-Wallis test. The Wilcoxon test was used additionally for the hypothesis of equal medians.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY:
Neither mortalities nor clinical symptoms of toxicity were observed. Appearance and behavior of the animals showed no treatment-related changes.

BODY WEIGHT AND WEIGHT GAIN:
Body weight was significantly reduced in high dose males. Body weight change values of this group were significantly reduced during the entire treatment period. This was assessed as being treatment-related. No significant effects were seen in females.

FOOD CONSUMPTION:
Food consumption was significantly reduced in high dose males as well as in high dose females during the entire treatment period. The values were up to 13.3 % (males) and 18.9 % (females) below controls. This was assessed as being treatment-related.

FOOD EFFICIENCY:
Food efficiency was significantly reduced in high dose males and in mid dose males. Due to the isolated occurrence and the lack of a clear dose-response relationship, this was assessed as being incidental.

WATER CONSUMPTION:
Water consumption was significantly reduced in high dose males and females during the entire treatment period . The values were up to 35.7 % (males)and 50.4 % (females) below controls. Also in mid dose males and females, water consumption was reduced with statistical significance several days in males or most of the days in females, the values being up 23.7 % (males) and 27.1 % (females) below controls. This was assessed as being treatment-related .

OPHTHALMOSCOPIC EXAMINATION:
No substance-related effects were obtained. All findings were spontaneous in nature and equally distributed between treated animals and controls.

HAEMATOLOGY:
There are no treatment-related changes in the hematological parameters measured.

CLINICAL CHEMISTRY:
Compound-related differences in serum enzyme activities were not evident at any dose level in either males or females. Blood chemistry examinations revealed significantly increased urea concentrations in the serum of the high dose animals of either sex. No treatment-related changes were found in the other blood chemistry parameters examined.

URINALYSIS:
At the end of the study high dose males and females produced decreased amounts of urine with increased specific gravity. In the other urine parameters no treatment-related findings were observed.

ESTROUS CYCLE DETERMINATION:
In the estrus cycle determinations conducted from day 63 to day 91, no substance-related effects were obtained.

SPERM ANALYSIS:
There are no treatment-related changes in the sperm parameters measured.

NEUROBEHAVIOUR:
No substance-related effects were observed.

ORGAN WEIGHTS:
In female rats, the mean weight of the kidneys was significantly increased in the high dose group. The mean weight of the heart was significantly decreased in female rats of the mid dose group. In the high dose group, the mean heart weight of female rats was also significantly decreased, however, only slightly and without a dose-response relationship. In males of the high dose group, the mean weights of brain and adrenal glands were significantly increased in males of the high dose group. The other mean absolute weight parameters did not show significant differences when compared to the control group.

GROSS PATHOLOGY:
Only a few gross lesions were noted in the glandular stomach (erosion/ulcer), epididymides (abscess), ovaries (cyst) and skin (sparse hair). With one exception (sparse hair in two control females) these gross lesions occurred only once per group, with no indication of a relationship to treatment. They were hence all interpreted to have developed spontaneously and unrelated to treatment.

HISTOPATHOLOGY:
With the exception of the areas of sparse hair in the skin of two control female rats and of a low dose male rat, all gross lesions could be correlated with a meaningful histopathologic correlate. However, regardless of whether or not they had a microscopic correlate, all the gross lesions were considered to have developed spontaneously and to be unrelated to treatment. Histopathology failed to correlate the significantly increased mean absolute (high dose group) and relative kidney weights (mid and high dose groups) of female rats with meaningful histologic finding. A treatment-related effect could, however, not be excluded, especially for the high dose group. Further, no histologic correlate was obtained for the significantly increased mean relative weights of brain and adrenal glands in males of the high dose group. These mean weight increases were regarded to be incidental in nature and were most likely related to the slight although not significant decrease of the mean terminal body weight. Finally, also no morphologic correlate was obtained for the significantly decreased absolute heart weight noted in females of the high dose group. This was also most likely related to the slightly although not significantly decreased mean terminal body weight. This assumption was supported by the value of the mean relative heart weight that was almost identical in the high dose group as compared to the control group. The same mechanism was assumed for the significantly decreased mean absolute heart weight noticed in females of the mid dose group, as also the mean terminal body weight was slightly although not significantly decreased. Moreover, there was no indication of a dose-response relationship for the recorded mean absolute and relative weight deviations. All microscopic findings recorded were either single observations, or they were recorded at a low incidence, or they occurred in control animals only, or at comparable incidence and graded severity in control and high dose males and/or females.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
ca. 14.1 - <= 21.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 64.1 - <= 91 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
ca. 285.2 - <= 338.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion