Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23.09. - 14.03.2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP. Eyes were not examined histopathologically. Uterus weights were not recorded and not all recommended organs were examined histopathologically.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted July 27, 1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
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: RCC Ltd., Biotechnology and Animal Breeding Division, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation:
males: 130 -158 g (mean 145 g)
females: 112 -133 g (mean 123 g)
- Fasting period before study: No
- Housing: In groups of five per sex in Makrolon type-4 cages
- Diet: standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: 23.09. - 18.11.2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
PEG 300
Details on oral exposure:
REPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C) in glass beakers, protected from light. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 10, 30 or 100 mg/mL
- Amount of vehicle: dose volume of 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of homogeneity was performed by analysing samples of each dose group from three different segments (top, middle, bottom) of the respective mixing container. Evaluation of stability was performed by analysing samples of each dose group over the storage periods of two hours and seven days at storage conditions. Analytically determined test item concentrations were in good agreement with the nominal values. The test item was homogeneously distributed in the vehicle and was stable for at least seven days.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150 and 500 mg/kg bw/d (group 1, 2, 3 and 4, respectively)
Basis:
actual ingested
No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were based on the results of a non-GLP 5-day dose range-finding study in which the test item was administered by gavage to 2 rats per group and sex. One male and two females treated with 1000 mg/kg bw/d of the test item died spontaneously on the 4th or 5th day of treatment. Animals showed several clinical signs at 1000 mg/kg bw/d like slight to moderate emaciation, hunched posture or slightly ruffled fur e.g.
- Post-exposure recovery period in satellite groups: 14 days (control and high dose group animals only)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery). Observations for mortality/viability were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy.

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

FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 and after 6 weeks
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (18 hrs)
- How many animals: all animals
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, platelet (thrombocyte) count, reticulocyte count, reticulocyte maturity index, methemoglobin, total leukocyte count, differential leukocyte count, coagulation: thromboplastin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 and after 6 weeks
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Glucose, urea, creatinine, total bilirubin, cholesterol, total triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin, globulin, albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18-hour fasting period into a specimen vial.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume (18 hours), specific gravity (relative density), color, appearance, pH, nitrite, protein, glucose, ketones, urobilinogen, erythrocytes, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
- Dose groups that were examined: all animals
- Battery of functions tested:
Grip strength
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.
Locomotor activity
Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system. Animals were randomized and monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At autopsy the following weights were recorded from all animals: brain, heart, liver, thymus, kidneys, adrenals, spleen, testes and epididymides or ovaries

HISTOPATHOLOGY: Yes

Microscopical examinations:
Adrenal glands, bone marrow (femur), brain (cerebrum, cerebellum, brain stem), cecum, colon, duodenum, epididymides, heart, ileum (with Peyer's patches, jejunum (with Peyer's patches), kidneys, liver, lungs, lymph nodes (mesenteric, mandibular), ovaries, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid (incl. parathyroid gland), trachea, urinary bladder, uterus, vagina, gross lesions
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as clinical laboratory data :
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Student's t-test was applied to grip strength and locomotor activity data.
- Fisher's exact-test was applied to the macroscopic findings.

For clinical laboratory data, quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to Bartlett. Alternatively, if the variances were considered to be heterogenous (p<0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were then compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<0.05).

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy. No test item related clinical signs were noted.

BODY WEIGHT AND WEIGHT GAIN
No test item related differences in mean body weights or mean body weight gain were observed when compared with controls.

FOOD CONSUMPTION
The mean daily and relative food consumption of test item treated animals were comparable with those of the control animals.

HAEMATOLOGY
No test item related differences in parameters of hematology were noted in animals after four or six weeks when compared with controls.

CLINICAL CHEMISTRY
No test item related differences in parameters of clinical biochemistry were noted in animals after four or six weeks when compared with controls.

URINALYSIS
No test item related differences in parameters of urinalysis were noted in animals after four or six weeks when compared with controls.

NEUROBEHAVIOUR
Functional Observational Battery: No test item related clinical signs were noted in males or females (week 4).
Grip Strength: No test item related differences were seen in mean fore- or hindlimb grip strength.
Locomotor Activity: No test item related differences were seen in mean locomotor activity.

ORGAN WEIGHTS
The mean liver weights, liver to body weight- and liver to brain weight-ratios were increased in females at 500 mg/kg bw/d when compared with controls after four weeks of treatment. The mean kidney to body weight ratio was increased in females at 500 mg/kg bw/d and the mean kidney to body weight- and kidney to brain weight ratios were increased in females at 150 mg/kg bw/d when compared with controls after four weeks of treatment. These findings were considered to be test item related because a dose response relationship could be observed and these findings compare well with the described microscopic findings. However, they were fully reversible after four weeks treatment followed by a two-week recovery period. After four weeks of treatment followed by a two-week recovery period the mean kidney weight and kidney to brain weight ratio were increased in males when compared with controls. However no effects on absolute or relative kidney weights were observed in males treated at 500 mg/kg bw/d for four weeks. No other test item-related changes in organ weights, organ to body- or organ to brain weight ratios were noted.

GROSS PATHOLOGY and HISTOPATHOLOGY
In the cortex of the kidneys an increased incidence and grade of basophilic tubuli in test itemtreated animals at 500 mg/kg bw/d (group 4), especially in males of the recovery group was recorded. This finding may be a sign of an increased tubular regeneration after a preceeding tubular damage. The change was still recorded after a 14-day treatment-free recovery penod, and was regarded as an adverse effect in group 4 animals (especially in males). Adaptive effects were noted in stomach/ forestomach and liver.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were noted at 50 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion