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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:
17 Mar - 14 Apr 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

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
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Crl:(WI) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 185-208 g (males); 153-173 g (females)
- Fasting period before study: no
- Housing: the animals were housed in groups of 5 per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdust (B.M.I. Helmond, the Netherlands) provided as bedding.
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (optimal temperature)
- Humidity (%): 50 (optimal humidity level)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Mar 1998 To: 14 Apr 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and placed on a magnetic stirrer during dosing. Adjustment was made for the specific gravity of test substance and vehicle. The solution was shown to be stable for at least 4 hours.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle choice was based on trial formulations performed at the test laboratory
- Concentration in vehicle: 10, 40 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-analyses were performed to determine the concentration of the test material of all dosing solutions. The actual concentrations were shown to be 95-107% of the nominal concentration, as analysed in duplicate samples. The homogeneity of the low- and high concentration samples was measured in duplicate samples. The concentration as sampled at 10, 50 and 90% height was shown to be 97-106% of nominal values. A relative difference of -2.8 - 2.1% in the stability from t=o to t=4 hours was measured in duplicate samples.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
administered by gavage, calculated weekly according to body weight
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
administered by gavage, calculated weekly according to body weight
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
administered by gavage, calculated weekly according to body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a range-finding study was performed with 3 rats/sex/dose, administered 0, 50 200 and 1000 mg/kg bw/day for 5 days (report No. 227306). There was no mortality. No treatment-related clinical signs were observed, no effect on body weight or food consumption was noted, there were no macroscopic findings, and the liver and kidney weights were comparable to those of the control group. Therefore, the same dose levels were selected for the main study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed for mortality twice daily and for clinical signs once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on Day 8, 15, 22 and 28, detailed clinical observations were made outside the cage in a standard arena. Symptomes were recorded and graded according to a fixed scale.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on Day 1, 8, 15, 22 and 28 of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured weekly.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes; ether
- Animals fasted: Yes, overnight for a maximum of 20 hours, with free access to water
- How many animals: all the animals in the control and treatment groups
- Parameters checked: erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, red cell distribution width, total leucocyte count, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Animals fasted: Yes, overnight for a maximum of 20 hours, with free access to water
- How many animals: all the animals in the control and treatment groups
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, bilirubin, cholesterol, creatinine, glucose, urea, total protein, albumin, alkaline phosphatase, sodium, potassium, chloride, calcium, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of the treatment
- Dose groups that were examined: the control group and all treatment groups
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, activity test (based on hourly data per animal, using a computerised motor activity monitoring system)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All the animals in the control and treatment groups were necropsied and a description of all macroscopic abnormalities was recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
adrenal glands, aorta, brain, caecum, cervix, clitoral gland, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, female mammary glandarea, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), nasopharynx, oesophagus, ovaries, pancreas, Peyer's patches (jejunum, ileum, if detectable), pituitary gland, preputial gland, prostate, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, sceletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions.

The following organ weights were recorded from the surviving animals on the scheduled day of necropsy: adrenal glands, brain,
epididymides, heart, kidneys, liver, spleen, testes, thymus.

HISTOPATHOLOGY: Yes. The organ and tissue samples were processed, embedded and cut at a thickness of 2.4 micrometers and stained with haematoxylin and eosin. For the control and 1000 mg/kg bw/day groups, the following organs were histologically examined: adrenal glands, aorta, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), oesophagus, ovaries, pancreas, Peyer's patches (jejunum, ileum, if detectable), pituitary gland, preputial gland, prostate, rectum, sciatic nerve, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, trachea, urinary bladder, uterus. The organs and tissues that were preserved but not examined during histopathology did not show signs of toxicity or other effects. The observed gross lesions were examined in animals from all the groups.
Statistics:
The following statistical methods were used to analyse the 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 of the treated groups and the control groups for each sex. The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
50 mg/kg bw/day: females, salivation (non-adverse)
Mortality:
mortality observed, treatment-related
Description (incidence):
50 mg/kg bw/day: females, salivation (non-adverse)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: females, increase relative leucocyte level, decrease relative neutrophil level (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: males, increased chloride level, increased calcium level (non-adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study period. No treatment-related clinical signs were observed in any group. Salivation was observed on one or several days in 1/5 males and 1/5 females in the low-dose group, and 2/5 males and 1/5 females in the high-dose group (see Table 1). As there is no dose-response effect and no other clinical or neurological signs that indicate that the effect is treatment-related, this is considered to be an incidental finding, which is probably caused by the administration procedure. Incidental observations of alopecia, red staining, piloerection and scabs were made, involving one or several animals. These findings are considered to be within the expected range for rats of this age and strain and regularly occur during a subacute study.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in body weight or body weight gain were observed between the control and treatment groups.

FOOD CONSUMPTION
No significant differences in food consumption were observed between the control and treatment groups.

HAEMATOLOGY
In females of the high-dose group, a statistically significant increase in relative leucocyte level (app. 8%) and decrease in relative neutrophil level (app. 8%) was noted, compared with the control group values. These values are related to each other as a percentage of the total lymphocyte concentration, and a variation in one will necessarily affect one or several other specific lymphocyte values. As the changes are minimal, no effects were seen in males and no other haematological effects were noted, this effect may be treatment-related, but is considered not to be of toxicological relevance.

CLINICAL CHEMISTRY
In males of the high-dose group, the level of calcium and chloride was significantly increased. As these increases were around 5% compared with the control group levels and no similar effects were observed in the female groups they are not considered be of toxicological relevance. The statistically significant change in bilirubin level in mid-dose males is considered to be incidental, as no effect was seen in the highest dose level and the mean value was the same in all male groups.

NEUROBEHAVIOUR
1/5 females in the control group showed a statistically significant increase in motor activity. This is considered to be an incidental occurrence as no other animals exhibited the same behaviour. No treatment-related effects were observed.

ORGAN WEIGHTS
There were no statistically significant differences in absolute or relative organ weight in the treatment groups, compared to the control group.

GROSS PATHOLOGY
No treatment-related findings were reported. In 2/5 females in the mid-dose group watery fluid was observed in the uterus. This is a normal finding during a part of the estrus cycle in rats.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related histopathological findings. The incidence and severity of the observed lesions were similar in the control group and treatment groups and are considered to have occured spontaneously.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were observed up to and including the highest dose level

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Clinical signs; salivation observed as number of rats per group and days per rat

Group (mg/kg bw/day)

Control

50

200

1000

Males (No. - days observed)

0

1 (Day 9)

0

2 (Day 9 and Day 9, 27-28)

Females

0

1 (Day 27-28)

0

2 (both Day 26-28)

Table 2: Haematology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

50

200

1000

Control

50

200

1000

Neutrophils (1)1

0.093 ± 0.022

0.108 ± 0.049

0.116 ± 0.021

0.090 ± 0.047

0.138 ± 0.022

0.079 ± 0.031

0.115 ± 0.023

0.063 ± 0.018*

Lymphocytes (1)1

0.883 ± 0.026

0.862 ± 0.053

0.851 ± 0.040

0.872 ± 0.056

0.830 ± 0.027

0.891 ± 0.027

0.851 ± 0.029

0.908 ± 0.027*

WBC (G/L)

13.5 ± 3.2

12.5 ± 0.5

12.1 ± 2.4

13.6 ± 2.7

7.7 ± 1.9

8.0 ± 1.8

8.2 ± 3.6

7.8 ± 1.9

*Statistically significant (p < 0.05)

1relative part of total =1

 

Table 3: Clinical chemistry results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

50

200

1000

Control

50

200

1000

Calcium (mmol/L)

2.53 ± 0.04

2.52 ± 0.04

2.52 ± 0.02

2.44 ± 0.02*

2.49 ± 0.07

2.47 ± 0.06

2.52 ± 0.05

2.47 ± 0.05

Chloride (mmol/L)

101 ± 2

100 ± 1

99 ± 2

97 ± 1**

98 ± 2

100 ± 2

100 ± 1`

98 ± 2

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Applicant's summary and conclusion