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Toxicological information

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 May 23, 1990 to January 28, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD/EU guidelines and according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(1981)
Deviations:
yes
Remarks:
(29-day treatment period for males and 30-day for females)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(1984)
Deviations:
yes
Remarks:
(29-day treatment period for males and 30-day for females)
Qualifier:
according to
Guideline:
other: Requirements of the Japanese Government under the revised Chemical Substance Law (1987) according to the Notification of December 9, 1986 by EA, Environmental Agency (No. 700), MHW, Ministry of Health and Welfare (No. 1039) and MITI
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: Organic
- Physical state: Yellow liquid
- Analytical purity: 94.4% active ingredient
- Expiration date of the lot/batch: May 1992 under storage conditions specified
- Storage condition of test material: In the original container, at room temperature, protected from light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as cited in the report: Albino Wistar Rat, HanIbm:WIST, outbred, SPF quality
- Source: BRL, Biological Research Laboratory Ltd., CH-4414 Fuellinsdorf, Switzerland, SPF quality
- Age at study initiation: males: 6 weeks
- Weight at study initiation: males: 130-150 g, females: 110-129 g
- Fasting: the animals were fasted for approximately 18 hours before scheduled necropsy, but water was provided
- Housing: individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel")
- Diet: pelleted standard Kliba no. 343 Batch No. 67/90 rat maintenance diet ("Kliba", Klingentalmuehle AG, Kaiseraugst, Switzerland) ad libitum
- Water: community tap water from Geneva was available ad libitum
- Acclimation period: 6 days under laboratory conditions, after clinical health examination
- Drinking water was analysed (bacteriological and chemical assays as well as contaminant analysis) and a chemical analysis of feed (assay for contaminants) was performed. Results of analyses are included in the report of the consecutive subacute study (RCC Project 267524) performed with the same stock of the test article.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark
- Music was broadcasted during the major part of the light period

IN-LIFE DATES:
- Delivery of animals: May 23, 1990
- Acclimation/Pretest: May 23 to 28, 1990
- Administration: May 29 to June 26 (males)/27 (females), 1990
- Recovery: June 27(males)/28 (females), 1990
- Termination: July 10, 1990

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Distilled water containing 4% Sodium Carboxymethylcellulose (CMC; Fluka AG, CH-9470 Buchs, Switzerland) and 0.2% Tween 80 (Fluka AG, CH-9470 Buchs, Switzerland)
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.The mixtures were prepared daily prior each application

DOSE VOLUME:
- 10 mL/kg bw
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 5 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. For technical reasons, an additional sample was taken for analysis in group 2 (5 mg/kg bw) during week 2.
About 2-3 g of each test article-vehicle was mixed with bidistilled water and treated in an ultrasonic bath. This sample solution was quantitatively transferred to a volumetric flask with acetonitrile. Then this flask was filled to volume with acetonitrile. If necessary, this sample solution was diluted with acetonitrile to yield a concentration within the calibration range and then 50 µL were analyzed by HPLC.
Stock solutions of the test item in acetonitrile in the range from 0.100 mg/mL to 1.053 mg/mL were prepared. The various standard solutions were prepared by respective dilution of these stock solutions with acetonitrile to yield concentrations in the range from 1.98 µg/mL to 100 µg/mL. These standard solutions were used to calibrate the HPLC.

Typical HPLC conditions and IR spectrum: see "Any other information about material and methods incl. tables"

The mean concentrations found were in the range
- from 102.3 % to 143.7 % of the normal concentration for Group 2 (5 mgkg bw/day)
- from 91.3 % to 102.4 % of the normal concentration for Group 3 (50 mg/kg bw/day)
- from 92.8 % to 1O1.4 % of the normal concentration for Group 4 (250 mg/kg bw/day)
The homogeneity varied in the range from -13 % to +9 % of the mean concentrations.
The test substance is stable in distilled water with 4 % CMC and 0.2 % Tween 80 at room temperature over a period of 5 days.

Prior to the start of the experiment, a sample of the test article was taken from the stock for identification by analysis to a method provided by the Sponsor. Analyses were performed at RCC UMWELTCHEMIE, CH 4452 Itingen, Switzerland.
Duration of treatment / exposure:
29 days for males and 30 days for females + 14 days of recovery for both sexes
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 50, 250 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
The study design included one main group and one recovery group, for a total of 10 male and 10 female rats at each dose level. From the 10 rats per sex in each dosage group, 5 were designated for sacrifice at the end of the teatment and 5 were designated for sacrifice after a 2-week treatment-free (recovery) period.
Control animals:
yes, concurrent vehicle
Details on study design:
The dosage selection was based upon a 5-day oral range-finding study with rat (see RCC 267682).In this study, the test item, mixed with sesame oil, was administered to 3 male and 3 female rats per dosage group, at dose levels of 50, 200 and 1000 mg/kg bw. No deaths occurred during the study. Apathy and stiff gait were recorded in both sexes at the highest dose level. Body weight and food consumption notably decreased in males and females treated with 1000 mg/kg bw; food consumption was also reduced in females dosed at 200 mg/kg bw. A dose-related increase of liver weight was observed in both sexes at all dose levels. At necropsy, at the highest dose level, the liver was found to be macroscopically enlarged in both sexes and erosion of gastric mucosa was seen in some males and females.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Daily
Cage side observations checked were: Mortality and abnormalities

BODY WEIGHT: Yes
Time schedule for examinations: weekly (using a Mettler balance)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Time schedule: weekly
Food Consumption per animal and per day = (Food consumption measured over the consumption interval) / (Total number of consumption days for all animals). The food consumption is calculated per cage and per food consumption interval. It expresses the average food consumed per animal and per day for each cage, and the observed food interval.

OPHTHALMOSCOPIC EXAMINATION: Yes
Performed on all animals during pretest (May 28, 1990) and on all animals of the control group and of the highest dosage group towards the end of the treatment (June 25, 1990) and during the last week of the treatment-free (recovery) period (July 4, 1990). A description of all abnormalities was recorded. Approximately 10 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen, Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Bifocal Type Miroflex Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwill, Switzerland).

HAEMATOLOGY: Yes
Time schedule for collection of blood: after 4 weeks (males: June 27, 1990, females: June 28, 1990) and after recovery (July 10, 1990)
Anaesthetic used for blood collection: Yes (light ether anesthesia)
Anticoagulants: EDTA-K2 (hematology), Sodium citrate, 3.8% (coagulation, 1:10)
Animals fasted: Yes (18 hours), but water was provided
How many animals: all animals
Parameters checked: erythrocyte count and morphology, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, Heinz bodies, methemoglobin, total leukocyte cand differential leukocyte count, cell classification, coagulation (thromboplastin time, activated partial thromboplastin time).

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: after 4 weeks (males: June 27, 1990, females: June 28, 1990) and after recovery (July 10, 1990)
Animals fasted: Yes (18 hours), but water was provided
Anticoagulants: Lithium heparin (140 USP Units)
How many animals: all animals
Parameters checked: glucose, urea, creatinine, bilirubin total, cholesterol total, triglycerides, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total, globulin, albumin/globulin ratio
Blood samples were collected between 6h30 and 8h30 to reduce biological variations caused by circadian rhythms and were drawn from the retro-orbital plexus.

URINALYSIS: Yes
Time schedule for collection of urine: after 4 weeks (males: June 27, 1990, females: June 28, 1990) and after recovery (July 10, 1990)
Metabolism cages used for collection of urine: Yes
Animals fasted: Yes (18 hours), water ad libitum
Parameters checked: volume (18-hour), specific gravity, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Necropsy:
All animals were necropsied and descriptions of all abnormalities were recorded.
They were fasted for 18 hours at least before necropsy but water was provided.
Necropsies were performed by experienced prosectors under the supervision of a pathologist.
All animals were anesthetized by intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
Necropsy dates: at the end of the treatment period (allocation group A): males: June 27, 1990; females: June 28, 1990
at the end of the recovery period (allocation group B): males and females: July 10, 1990

Organ weights:
The following organ weights were recorded on the scheduled dates of necropsy: adrenal glands, heart, kidneys, liver, ovaries, spleen, testes

Organ sampling:
Sampling :
Tissue and organ samples for histopathology, were collected from all animals at necropsy and fixed in a 4% neutral phosphate buffered formaldehyde solution: adrenal glands, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, extraorbital lacrymal glands, eyes with optic nerves, femur including joint, harderian glands, heart, ileum, jejunum, kidneys, larynx, liver, lungs (infused with formalin), lymph nodes (mandibular, mesenteric), mammary gland area, nasopharynx, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle (thigh region), skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid gland with parathyroid glands, tongue, trachea, urinary bladder (infused with formalin), uterus, vagina, gross lesions.

Sampling for electron microscopy:
At terminal sacrifice at the end of the treatment and recovery periods, representative samples of liver (a few mm3) were quickly removed after organ weighing from 2 surviving animals per sex with consecutive identification numbers in the control group and the highest dosage group (allocation groups A and B). Samples were cut into slices (about 1 mm thick), covered with a few drops of fixative and chopped into blocks of about 1 mm3. These blocks were immediately transferred into vials containing 2-3 mL glutaraldehyde 3% in 1.0 M phosphate buffer, pH 7.4, for fixation at approximately 4°C for a few hours.

Histotechnology for light microscopy:
In both allocation groups A and B, tissue samples of adrenal glands, heart, kidneys, liver, lungs, ovaries, spleen, testes and thyroid gland from rats of the control and high doses, as well as liver and thyroid gland from low and mid dose animals, and all gross lesions, were trimmed, processed, embedded in paraffin wax, sectioned at a nominal thickness of 2-4 microns and stained with hematoxylin and eosin.

Histotechnology for electron microscopy:
The fixed tissue blocks were washed in 0.1 M phosphate buffer (pH 7.4), post-fixed with 1% OsO4, dehydrated in graded acetone, and embedded in epon.

Histopathology:
Slides of the organs listed under "Histotechnology for light microscopy" were examined by a veterinary pathologist. Histologic alterations were described according to their distribution, severity and morphologic character.

Statistics:
The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data:
Unvariate 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.
Groups means were calculated for continuous data and medians were calculated for discrete data (scores).

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs and no mortality occurred throughout the study.
One male from group 2 (5 mg/kg bw/day) showed enlarged eyes during the last two treatment weeks. Injury of the right eye was recorded in one male from group 2 and in one male from group 3 (50 mg/kg bw/day) during the recovery period, due to retro-orbital blood sampling at the end of the treatment period.

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.
The marginally (statistically not significantly lower) mean body weight gain value in males of group 4 (250 mg/kg bw/day) at the end of the recovery period was attributed to the atypical growth of one animal (no. 40) and was considered to be of no relevance for the study.

FOOD CONSUMPTION AND COMPOUND INTAKE
Mean values of consumption and food consumption to body weight ratio in all treated groups were similar to those of the controls throughout the study.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related ocular changes were observed by ophthalmoscopic examination performed towards the end of treatment nor towards the end of the recovery period.

HAEMATOLOGY
The assessment of the hematological data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.
All differences in the results of the clinical laboratory data were considered to be incidental and of normal biological variation for rats of this strain and age.

CLINICAL CHEMISTRY
The assessment of the clinical biochemical data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.
All differences in the results of the clinical laboratory data were considered to be incidental and of normal biological variation for rats of this strain and age.

URINALYSIS
The assessment of the urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.
All differences in the results of the clinical laboratory data were considered to be incidental and of normal biological variation for rats of this strain and age.

ORGAN WEIGHTS
A slight to moderate increase of the mean liver weight and of the mean liver to body weight ratio was observed in both sexes on group 4 (250 mg/kg bw/day) at the end of the treatment period. These weight changes were considered to be treatment-related. They were found to be reversible at the end of the treatment-free recovery period.
In all treated animals of both sexes, all organ weights and organ to body weight ratios were comparable to those of the controls, at the end of the treatment and at the end of the treatment-free recovery period. Especially spleen weight decrease in males of group 4, which achieved a level of statistical significance at the end of the recovery period, was considered to have arisen fortuitously and to be of no experimental relevance.

MACROSCOPIC FINDINGS
No treatment-related macroscopic alterations were observed at necropsy, either at the end of the treatment or at the end of the recovery period.
The following incidental changes were observed at the end of the recovery period:
- right eye damaged during blood collection in one male and one female (from groups 3 and 4 respectively)
- reddish foci on thymus in 3 females (from groups 2, 3 and 4)
- reddish coloration of urinary bladder in one male (from group 4)
- pelvic dilatation of kidney in one male (from group 4)

MICROSCOPIC FINDINGS:
Slight hepatic centrilobular parenchymal hypertrophy was seen in high dose main test animals. This phenomenon was not seen in high dose recovery group animals or in any low or mid dose group animals.
Thyroid follicular cell hypertrophy was present at all dose levels in main test animals. The severity was dose-related and greater in males than females (with females being unaffected at the low dose level). Regression of the lesion was essentially complete after 2 weeks with just 1 high dose male being very slightly affected from the recovery groups.
The small number of other findings recorded are within the normal range of background alterations which may be seen in untreated rats of this age and strain.

Effect levels

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion